Chapter 2: Susceptibility and tolerance of rice hybrids to insect herbivores: ..... (light and dark grey for 3 and 4, respectively), and bold letters are used to highlight ...... Larsson S (1989) Stressful times for the plant stress: insect performance ..... Xu W, Virmani SS, Hernandez JE, Sebastian LS, Redoña ED, Li Z (2002) Genetic.
An assessment of herbivore susceptibility and tolerance in hybrid rice: contrasting responses to three herbivorous insects in three-line varieties
by Eduardo Crisol Martínez
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A thesis submitted as external candidate as partial fulfillment of the requirements for the degree of Master in Science
University College Cork (UCC) School of Biological, Earth and Environmental Sciences Head of school: Prof. John O‟Halloran
Table of contents Declaration .................................................................................................................. ix List of Abbreviations ................................................................................................... x Acknowledgements ....................................................................................................xii Abstract .....................................................................................................................xiii Chapter 1: Varietal tolerance to herbivores: an inherent advantage of hybrid rice? ............................................................................................................................. 1 1.1. Introduction ....................................................................................................... 2 1.2. Is hybrid rice inherently more susceptible than inbreds to planthoppers? ........ 9 1.3. Is hybrid rice inherently more susceptible than inbreds to stemborers? ......... 14 1.4. Hybrid vigour and tolerance to planthoppers and stemborers ........................ 17 1.5. Definitions and dynamics of herbivory tolerance ........................................... 21 1.6. Phenotyping for tolerance to planthoppers and stemborers ............................ 28 1.7. Ecological consequences of tolerance at field and landscape levels .............. 34 1.8. Introduction to this thesis ................................................................................ 37 Chapter 2: Susceptibility and tolerance of rice hybrids to insect herbivores: an experimental analysis of three-line hybrids and their parental lines............. 39 2.1. Introduction ..................................................................................................... 40 2.2. Materials and Methods .................................................................................... 46 2.2.1. Plant materials .......................................................................................... 46 2.2.2. The herbivores ......................................................................................... 47 2.2.3. Experiments ............................................................................................. 48 2.2.3.1. Field experiment ............................................................................... 48 2.2.3.2. Greenhouse experiment..................................................................... 49 2.2.4. Statistical Analyses .................................................................................. 51 2.3. Results ............................................................................................................. 53 2.3.1. Agronomic traits ...................................................................................... 53 2.3.1.1. Greenhouse studies ........................................................................... 53 2.3.1.2 Agronomic studies (field) .................................................................. 53 2.3.2. Plant resistance and tolerance to insect attack in the greenhouse study .. 58 2.3.2.1. Plant mortality ................................................................................... 58 2.3.2.2. Insect responses across plant types (insect biomass) ........................ 60 2.3.2.3. Dry weight of infested plant yield components ................................ 61 2.3.2.4. Partitioning of biomass into individual yield components................ 64
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2.3.2.5. Reduction of dry weight components (g) per mg of insect dry weight ........................................................................................................................ 67 2.3.2.6. Percentage reduction of plant dry weight components per mg insect dry weight ...................................................................................................... 70 2.4. Discussion ....................................................................................................... 72 Chapter 3: Research on planthopper-rice interactions: contrasting effects of experimental conditions on hybrid and inbred rice.............................................. 82 3.1. Introduction ..................................................................................................... 83 3.2. Materials and methods .................................................................................... 86 3.2.1. Plant materials .......................................................................................... 86 3.2.2.The herbivore ............................................................................................ 87 3.2.3. Plant growth parameters .......................................................................... 88 3.2.4. Experiment 1: Effects of pot size and fertiliser on plant fitness .............. 90 3.2.5. Experiment 2: Effects of insect cages on plant fitness............................. 90 3.2.6. Experiment 3: Effects of pot size and fertiliser on insect and host-plant biomass .............................................................................................................. 91 3.2.7. Data analyses ........................................................................................... 92 3.3. Results ............................................................................................................. 93 3.3.1. Choice of plant growth parameters .......................................................... 93 3.3.2. Experiment 1: Effects of pot size and fertiliser on plant fitness .............. 97 3.3.3. Experiment 2: Effects of insect cages on plant fitness........................... 100 3.3.4. Experiment 3: Effects of pot size and fertiliser on insect and host-plant biomass ............................................................................................................ 103 3.4. Discussion ..................................................................................................... 107 Chapter 4: General discussion .............................................................................. 116 4.1. Introduction ................................................................................................... 117 4.2. Are F1 hybrid rice varieties more susceptible and/or tolerant of pests than inbred rice? ........................................................................................................... 118 4.3. Experimental design: a review of the present study, and suggestions for comparing results from greenhouse and field experiments. ................................ 120 4.4. Future research .............................................................................................. 122 References ................................................................................................................ 124
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List of Figures Chapter 1 Fig. 1.1: The three-line hybrid system (shaded symbols indicate male-sterility) ........ 3 Fig. 1.2: Schematic representation of (A) heterosis and (B) heterobeltiosis using planthopper resistance as an example of a heritable trait. P1 and P2 = parent 1 and 2 respectively, F1 = hybrid; broken line = mid-parent value. ......................................... 4 Fig. 1.3: Adoption of hybrid rice by Chinese and Vietnamese farmers (redrawn from Cheng et al., 2007 and Duc Vien & Duong Nga, 2007) .............................................. 6 Fig. 1.4: Higher fitness of white-backed planthopper on hybrid compared to inbred rice varieties from published and unpublished studies: (A) oviposition, (B) female longevity and (C) incidence of white-backed planthopper on hybrid and inbred varieties in Vietnam (Thanh et al., 2007); (D) Comparison of white-backed planthopper survival on Chinese hybrid variety SY-63 H, the CMS-line ZS-97 A, and the restorer line MH-63 R. Open bars indicate the reduction in plant biomass resulting from 1mg of planthopper production and shaded bars indicate biomass productivity of white-backed planthoppers (from Sogawa et al., 2009); and (E) incidence of white-backed planthopper on hybrid (SY-63 H) and inbred (IR64) rice at CNRRI in 2010 under moderate (100Kg/ha – open bars) and high (250Kg/ha – shaded bars) nitrogen (Hu et al., unpublished). ......................................................... 12 Fig. 1.5: Association between resistance and tolerance (A) according to Painter (1951) and (B) as revised by Smith (2005) ................................................................ 21 Fig. 1.6: Graphical depiction of tolerance: Relationship between rice plant biomass reduction (tolerance) and Spodoptera frugiperda larval weight (resistance) among five different plant introductions (symbols)(redrawn from Lye & Smith, 1988). ..... 30 Chapter 2 Fig. 2.1: Mean (± SEM) yield (i) (filled grains dry weight) (ii) biomass accumulation (vegetative dry weight, (iii) reproductive dry weight, and (iv) number of tillers of hybrid and inbred varieties during the 2010 WS field experiment. Values were obtained from healthy non-damaged plants. Solid circles are hybrids, open circles are restorers, solid triangles are maintainers and open triangles are CMS (A) lines. Reproductive dry weight value refers to the added dry weight of both filled and unfilled grains. ........................................................................................................... 57 Fig. 2.2: Mean (± SEM) mortality of hybrid, restorer (R lines), maintainer (B lines) and CMS (A lines) rice lines under four treatments (uninfested (control)) (i), brown planthopper-infested (ii), white-backed planthopper-infested (iii), and yellow stemborer-infested (iv) under low (open bars) and high (solid bars) nitrogen fertiliser regimes. The same letters in parentheses next to each treatment indicate no significant differences among treatments following Tukey‟s test results. ................. 59
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Fig. 2.3: Mean (± SEM) dry weight (mg) of brown planthopper, white-backed planthopper and yellow stemborer per g plant dry weight (i, ii, iii respectively) and per plant (iv, v, vi respectively) for hybrid, restorer (R lines), maintainer (B lines) and CMS (A lines) plants under low (open bars) and high (solid bars) nitrogen fertiliser regimes. The same letters above bars indicate no significant differences between plant types, and the same letters in parentheses next to each treatment indicate no significant difference between insects following Tukey‟s test results. ... 61 Fig. 2.4: Mean (± SEM) dry weight of filled grains, above-ground plant parts and roots of hybrid, restorer (R lines), maintainer (B lines) and CMS (A lines) plants (including dead plants) infested with brown planthopper (i, iv, vii), white-backed planthopper (ii, v, viii), and yellow stemborer (iii, vi, ix), under low (open bars) and high (solid bars) nitrogen fertiliser regimes. Data from uninfested plants are presented (x, xi, xii) for visual comparison (see text). The same letters within a row above bars indicate no significant difference between groups for plant parts, and same letters in parenthesis next to each treatment indicate no significant difference between insect treatments following Tukey‟s test results. Note that the CMS (A) line is sterile and produces no or few filled grains. .......................................................... 63 Fig. 2.5: Mean biomass of filled grains (striped bars), above-ground biomass (open) and roots (solid) dry weight as a percentage of the total biomass in four plant types (H = hybrid, R = restorer, B = maintainer, and A = CMS) under low and high nitrogen fertiliser regimes (N1 and N2 in brackets, respectively) for four treatments brown planthopper-infested (i), white-backed-infested (iii), yellow stemborerinfested (v), and uninfested plants (vii). Total biomass (± SEM) of each plant type under each nitrogen regime, for each treatment (ii, iv, vi, viii, respectively) is shown at the end. The same letters above the dotted lines indicate homogeneous groups for plant parts, and bold letters in parenthesis next to each treatment indicate homogeneous groups between insect treatments for each biomass part (filled grains, above-ground vegetative, roots respectively) following Tukey‟s test results. ........... 66 Fig. 2.6: Mean (± SEM) reduction of filled grain, above-ground, and root dry weights (g) per mg dry weight of brown planthopper (i, iv, vii), white-backed planthopper (ii, v, viii), and yellow stemborer (iii, vi, ix), for hybrid, restorer (R lines), maintainer (B lines) and CMS (A lines) rice varieties growing under low (open bars) and high (solid bars) nitrogen fertiliser regimes. The CMS (A) line is sterile and produced no or few filled grains, and therefore, the results were not plotted. ....................................................................................................................... 69 Fig. 2.7: Mean (± SEM) percentage reduction of filled grain, above-ground, and root dry weights (g) per mg dry weight of brown planthopper (i, iv, vii), white-backed planthopper (ii, v, viii), and yellow stemborer (iii, vi, ix), for hybrid, restorer (R lines), maintainer (B lines) and CMS (A lines) rice varieties growing under low (open bars) and high (solid bars) nitrogen fertiliser regimes. .................................... 72 Chapter 3 Fig. 3.1: Effects of three pot sizes on mean (± SEM) of seven growth parameters in hybrid (i, v, ix, xiii, xvii, xxi), restorer (ii, vi, x, xiv, xviii, xxii) , maintainer (iii, vii, xi, xv, xix, xxiii) and CMS (iv, viii, x, xvi, xx) rice lines under low (open circles)
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and high (solid circles) nitrogen fertiliser regimes. CMS lines consistently produced a small number of filled grain. ................................................................................... 99 Fig. 3.2: Effects of mylar insect caging on mean (± SEM) of seven growth parameters in hybrid (i, v, ix, xiii, xvii, xxi, xxv, xxviii ), restorer (ii, vi, x, xiv, xviii, xxii, xvi, xxix), maintainer (iii, vii, xi, xv, xix, xxiii, xxvii, xxx) and CMS ( iv, viii, x, xvi, xx, xxiv) rice lines under low (open circles) and high (solid circles) nitrogen fertiliser regimes. CMS lines consistently produced a small number of filled grain. .................................................................................................................................. 102 Fig. 3.3: Effects of pot size on the mean (± SEM) biomass density of brown planthoppers reared on hybrid (i), restorer (ii), maintainer (iii), and CMS (iv) rice lines under low (open circles) and high (solid circles) nitrogen fertiliser regimes. 104 Fig. 3.4: Effects of pot size on mean (± SEM) of five growth parameters on brown planthopper-infested (broken lines) and control (solid lines) hybrid (i, v, ix, xiii, xvii ), restorer (ii, vi, x, xiv, xviii), maintainer (iii, vii, xi, xv, xix) and CMS (iv, viii, x, xvi, xx ) rice lines. Plants were grown in mylar insect cages under low (open symbols) and high (solid symbols) nitrogen fertiliser regimes. ............................... 106
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List of Tables Chapter 1 Table 1.1: Some examples of hybrid rice susceptibility to different insects ............... 8 Chapter 2 Table 2.1: The seven three-line hybrid sets from the IRRI hybrid rice breeding programme that were used in the present study. Hybrids are indicated with their respective parental lines ............................................................................................. 47 Table 2.2: Effects of growth conditions on plant fitness parameters per plant (mean ± SEM) including F-values. Parameters are from uninfested plants from an experiment carried out in the IRRI greenhouse during the WS of 2009....................................... 55 Table 2.3: Plant growth parameters with F-values after repeated measure GLM for hybrid and inbred plants during the first ten weeks after transplanting. Data are from the field experiment carried out in the WS of 2010 ................................................... 56 Table 2.4: Factors affecting plant mortality in the greenhouse experiment............... 59 Table 2.5: Factors affecting the absolute and relative insect biomass (dry weight) in the greenhouse experiment ........................................................................................ 60 Table 2.6: Factors affecting biomass of plants in the greenhouse experiment .......... 63 Table 2.7: Factors affecting the partitioning of biomass in plants from the greenhouse experiment .............................................................................................. 65 Table 2.8: Factors affecting the absolute reduction (g) of three plant parts per mg of insect .......................................................................................................................... 68 Table 2.9: Factors affecting the percentage reduction of dry weight per mg insect dry weight in the greenhouse experiment ........................................................................ 71 Chapter 3 Table 3.1: The seven three-line hybrid sets from the IRRI hybrid rice breeding programme that were used in the present study. Hybrids are indicated with their respective parental lines ............................................................................................. 87 Table 3.2: Incidence of significant Spearman‟s correlations (P ≤ 0.05) between each of 28 measured parameters for 4 plant types (hybrid, restorer, maintainer and CMSline). Significant correlations are indicated for potted plants grown at low (above diagonal) and high (below diagonal) nitrogen regimes in a greenhouse at IRRI during the 2009 WS. Numbers within the Table refer to the number of plant types which showed a significant correlation. Shading indicates the highest number of
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significant correlations (light and dark grey for 3 and 4, respectively), and bold letters are used to highlight recommended parameters .............................................. 95 Table 3.3: Incidence of significant Spearman‟s correlations (P ≤ 0.05) between each of 18 measured parameters for 4 plant types (hybrid, restorer, maintainer and CMSline). Numbers within the Table refer to the number of plant types which showed a significant correlation. Significant correlations are indicated for ifield plots at IRRI during the 2010 DS. Shading indicates the highest number of significant correlations (light and dark grey for 3 and 4, respectively), and bold letters are used to highlight recommended parameters. ......................................................................................... 96 Table 3.4: F values from ANOVAs from experiment 1 to examine the effects of plant growth conditions on seven rice plant parameters ............................................ 98 Table 3.5: F values from ANOVAs from experiment 2 to examine the effects of plant growth conditions on seven rice plant parameters .......................................... 101 Table 3.6: F values from ANOVA from experiment 3 to examine the effects of plant growth conditions on brown planthopper population biomass ................................ 103 Table 3.7: F values from ANCOVAs (Plant height, number of tillers and SPADvalue) and MANCOVA (vegetative and roots dry weights) from experiment 3 to examine the effects of plant growth conditions and presence/absence of brown planthopper on measured growth parameters of rice ............................................... 105
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Declaration
The thesis submitted here is my own work and has not been submitted for another degree, either at University College Cork or elsewhere.
Eduardo Crisol Martínez
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List of Abbreviations ADB: Asian Development Bank. A-line: Seed-parent, or CMS line, a sterile inbred used in the three-line hybrid system. B-line: Maintainer, one of the inbreds used in the three-line hybrid system. BPH: Brown planthopper (Nilaparvata lugens [Stål]). CCH: Compensatory Continuum Hypothesis. CMS: Cytoplasmic male sterile. CMS-D: CMS line originated from F7 population of (Dissi D52 × 37) × Ai-JiaoNan-Te. CMS-G: CMS line originated from rice line Gambianka. CMS-ID: CMS line originated from rice line Indonesia Paddy 6. CNRRI: Chinese National Rice Research Institute. CO2: Carbon dioxide. CSISA: Cereal System Initiative for South East Asia. DNA: Deoxyribonucleic acid. DS: Dry season. DW: Dry weight ESMS: Environmentally sensitive male sterile. FAO: Food and agriculture organization. FW: Fresh weight. INTAFOHR: International Task Force of Hybrid Rice.
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IPM: Integrated pest management. IRRI: International Rice Research Institute. MSST: Modified seedbox screening test. PCR: Polymerase chain reaction. PGMS: Photosensitive genetic male sterile. R-line: Pollen parent, also called restorer, one of the inbreds used in the three-line hybrid system. SES: Standard evaluation scores. SPAD: Soil plant analytical development. SPSS: Statistical package for the social sciences. SSST: Standard seedbox screening test. SST: genetic markers TGMS: Thermosensitive genetic male sterile. TN1: Taichung native 1 variety. WA-CMS: Wild abortive cytoplasmic male sterile. WBPH: White-backed planthopper (Sogatella furcifera [Horvath]). WS: Wet season. YSB: Yellow stemborer (Scirpophaga incertulas [Walker])
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Acknowledgements I would like to sincerely thank Dr. Finbarr Horgan, who has been, since we started to plan the research at the International Rice Research Institute (IRRI), not only my supervisor of this thesis, but also a mentor, and a good friend. Without his follow up, support and advice, this work would have never been finished. The work I present hereby is the result of almost two years of effort, in which many professionals (whom now I can count also as friends), mainly from IRRI have participated; thanks to all the staff of the Host Plant Resistance Group at the Crop and Environmental Sciences Division of IRRI. Additionally, I would also like to dedicate the work to my family, and more specially to my parents; they have always been there for me, and even though they were far away when I was doing the research, I felt somehow their presence next to me all the time, and they filled me with positive energy and faith. Last but equally important, I would like to include here all the people that I have met through this journey, and who somehow have contributed to lift me up whenever doubts, fear, or sadness faced me. All of these persons have turned me into a better professional, but more important, a better man.
Thank you all.
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Abstract Several reports indicate higher population densities of brown planthopper (BPH) (Nilaparvata lugens [Stål]), white-backed planthopper (WBPH) (Sogatella furcifera [Horváth]) and yellow stemborer (YSB) (Scirpophaga incertulas [Walker]) on hybrid than on inbred rice lines throughout Asia: however, few studies have compared hybrid lines and their corresponding parental lines for their interactions with these insect herbivores. In the present study, three-line hybrids and their parental lines were used to examine the interactions between insects (BPH, WBPH, and YSB) and rice lines and to assess whether hybrid varieties are more susceptible to and more tolerant of insect herbivores than are their parental inbred lines. Hybrid susceptibility is predicted based on suggested trade-offs between plant resource allocations to defence, reproduction (yield in rice) and growth and according to the Plant Vigour Hypothesis. However, the results from this study indicated that insect biomass per plant weight was generally lower on hybrids than on inbreds, suggesting that the hybrids were in fact less susceptible than the inbreds. Furthermore, according to the Compensatory Continuum Hypothesis, larger plants and plants under higher resource levels will be more tolerant of insect damage. The results from this study are in agreement with these predictions: In general hybrids had lower reductions in biomass and yield compared to inbreds when standardized for insect pressures. Throughout the course of the study, a series of methodological difficulties were encountered. These are described and recommendations made to help improve research methods. In particular the effects of pot size on the outcome of herbivory
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experiments with hybrids and inbreds were examined. Pots and insect cages had major effects on several aspects of rice plant growth and often affected hybrids more than inbred varieties thereby potentially confounding results from greenhouse experiments. Recommendations are made to limit nitrogen, restrict experiments to younger plant phenological stages and increase pot sizes in such studies.
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Chapter 1
Varietal tolerance to herbivores: an inherent advantage of hybrid rice?
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1.1. Introduction Hybrid rice was first developed in China in the 1960s when wild abortive (WA) – cytoplasmic male sterility (CMS) was discovered as a mutation in a single rice plant and incorporated into the Chinese National Rice Research Institute‟s (CNRRI) breeding programme (Virmani, 1994). Male sterility is necessary for the commercial exploitation of hybrid crops and the discovery of WA-CMS opened the way for a rapid development of hybrid rice technology, hybrid exploitation, and commercialization (Yuan, 1994; Tripp et al., 2010). Cytoplasmic male sterility is caused by an interaction between nuclear genes and genetic factors (undetermined sterility-inducing factors) present in the cytoplasm that result in the failure of pollen production without affecting female fertility (Dalmacio et al., 1995). These cytoplasmic genetic sterility factors can include rearranged mitochondrial DNA that produces protein products which interfere with normal pollen development (Newton, 1988). Male-sterile rice lines, other than the WA-CMS, have since been identified and adopted in breeding programmes, either as part of three-line (also known as the CMS system) or two-line hybrid systems (Lu et al., 1994; Yuan et al., 1994; Dalmacio et al., 1995). The CMS system is still the most popular system employed in the production of hybrid rice (Virmani et al., 2002). The CMS hybrid method requires three parental lines (Figure 1.1): the A line or seed-parent (the CMS line), the B line or maintainer, and the restorer (R) line or pollen parent. Hybrid seed is produced by allowing the A and R lines to cross pollinate. Because the A line is male sterile it cannot self-pollinate so that any seed produced on the A plants must be hybrid. The A and B lines have the same nuclear
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genome but different cytoplasmic genomes (i.e. they are alloplasmic). The B line is required to pollinate the A line in order to maintain a supply of A line seed.
Fig. 1.1: The three-line hybrid system (shaded symbols indicate male-sterility)
Restorer lines unrelated to the A (or B) line are selected based on desirable agronomic traits, but they must have an ability to combine with A lines such that this results in hybrid vigour above the mean performance of the B and R lines, i.e., positive heterosis (Virmani, 1994). Where hybrid performance is better than the mid-parental performance, this is referred to as heterosis or hybrid vigour. On rare occasions, the hybrid may surpass both parents for a given trait. This is known as heterobeltiosis (Figure 1.2)(Virmani, 1994). Hybrid rice breeding is constrained by the selection of parental lines with R and A line compatibility, whereby the R line must restore fertility. Restorer and A lines are also constrained by their combining ability. Furthermore, hybrid improvement in general is constrained by the limited availability of malesterile lines (Cheng et al., 2007; Peng et al., 2009).
3
The two-line hybrid system has been developed as an alternative to the threeline CMS-system. In the two-line hybrid system, male-sterility is controlled by one
Fig. 1.2: Schematic representation of (A) heterosis and (B) heterobeltiosis using planthopper resistance as an example of a heritable trait. P1 and P2 = parent 1 and 2 respectively, F1 = hybrid; broken line = mid-parent value.
or more pairs of recessive nuclear genes that determine hyper-sensitivity to environmental conditions with resulting male sterility. Photosensitive (PGMS) and thermosensitive (TGMS) genetic male sterile lines have been developed in China (Lu et al., 1994) and other countries (Virmani et al., 2003; Singh et al., 2011). The two-line system has a number of advantages over the three-line system: Maintainer lines are not needed in the two-line system since the PGMS and TGMS respond to ambient light and temperature regimes, respectively; therefore, by altering light or temperature, the degree of male sterility can be controlled so that seed of the A line can be produced readily. This feature increases the ease with which the PGMS and TGMS genes can be transferred to other rice lines and broadens the choice of
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suitable parents with adequate restorer and combination abilities. Furthermore, there are no negative effects due to the sterile cytoplasm and reliance on a single, unique WA-cytoplasm is avoided (Lu et al., 1994; Yuan, 1994). Nevertheless, relative to the CMS-system, the two-line system has not been widely adopted (Virmani et al., 2003; Cheng et al., 2007). Since the initial release of the first commercial hybrid rice varieties in China in 1976, the percentage of the area of irrigated rice fields producing hybrid varieties has increased to over 60% in China (ca. 15 million ha) and over 3% in other Asian countries (Figure 1.3) (Tripp et al., 2010). Hybrid rice technology has been rapidly adopted by farmers and promoted by both public and private institutes with claims of higher yields (up to 30%, but usually 15-20%) compared to the highest yielding inbred lines (Yuan, 1994; Cheng et al., 2007). The yield advantage of hybrid over inbred rice is attributed to heterosis for yield. A coordinated hybrid rice programme in China that links public institutes and private companies may have accelerated adoption of hybrids in that country (Mao et al., 2006; Tripp et al., 2010). Meanwhile, efficient marketing and an efficient supply chain have guaranteed the expansion of hybrids outside China, particularly to Vietnam, which is currently the second largest producer of hybrid rice in the world. The greatest emphasis on developing hybrid rice outside China has been in India and Vietnam, with 3% and 8% of the rice area in these countries now dedicated to hybrid rice, respectively (Vien & Nga, 2009; Tripp et al., 2010). Meanwhile, many multinational corporations have initiated or dedicated their rice breeding programmes to hybrid development because hybrids are more lucrative than inbred varieties since farmers depend on a continuing supply of commercially produced F1
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seeds since collecting farm-saved F2 seed is not appropriate (Tripp et al., 2010). Together, these factors are likely to lead to maintenance or expansion of the rice hybrid area in Asia. 18000000 16000000
China Vietnam
Area ha2
14000000 12000000 10000000 8000000 6000000 4000000 2000000 0 1975
1980
1985
1990
1995
2000
2005
Year Fig. 1.3: Adoption of hybrid rice by Chinese and Vietnamese farmers (redrawn from Cheng et al., 2007 and Duc Vien & Duong Nga, 2007)
Although hybrids have demonstrated higher yields than inbreds from a range of studies (Yuan, 1994; Cheng et al., 2007), there is often a general belief that hybrids are inherently more susceptible than inbred varieties to insect pests (see Table 1.1) and diseases (Mew et al., 1988). Furthermore, hybrids require generally higher fertiliser inputs than do inbred lines to guarantee their high yields (Van Pham et al., 2003; Kumar & Prasad, 2004). Together with higher seed and plant protection costs compared to inbred varieties, this can reduce the economic advantage to farmers gained from any yield increases (Tripp et al., 2010). A perceived higher susceptibility to biotic stresses in hybrids together with increased supplier-client communication (during purchase of F1 seed) is believed to underlie a higher
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pesticide use associated with hybrid compared to inbred rice varieties (Cheng, 2009; Sogawa et al., 2009). Numerous studies have indicated that higher fertiliser use is associated with stemborer fitness and damage (Jiang & Cheng, 2003 and references therein). Furthermore, indiscriminate insecticide use coupled with high levels of insecticideresistance among planthoppers (Sun et al., 1996; Nagata et al., 2002) has been associated with planthopper outbreaks and hopperburn (senescense of the rice plant as a result of insect feeding) (Gallagher et al., 1994; Heinrichs, 1994; Cuong et al., 1997; Visarto et al., 2001; Yin et al., 2008) while different susceptibilities of planthopper species to insecticides could result in shifts in the composition of planthopper assemblages, as have been noted over the past 20 years between brown planthopper (BPH) (Nilaparvata lugens [Stål]), and white-backed planthopper (WBPH) (Sogatella furcifera [Horváth]) in China (Matsumura et al., 2008; Cheng, 2009). Therefore, the question remains: are hybrids more susceptible to planthoppers by virtue of their particular plant growth patterns and physiology, or because of unknown factors associated with CMS sterility? Alternatively, are recent planthopper outbreaks, in particular outbreaks of the WBPH, and increased densities of stemborers associated with normal farmer practices when producing hybrid rice crops, coupled with a direct agrochemical marketing mechanism created through the F1 seed requirement? Or is hybrid rice suffering from bad press only because of poor selection of CMS or restorer lines, and inadvertent breeding for planthopper and disease susceptibility?
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Table 1.1: Some examples of hybrid rice susceptibility to different insects Hybrids evaluated for Inbred lines used in Evidence for higher susceptibility herbivore susceptibility comparisons of hybrids over inbred rice Sogatella furcifera (WBPH) Shanyou 6 Commercial inbred Reproduction 2.6-3.9 times varieties higher Nanyou 2 Commercial inbred Higher populations and outbreak varieties Chinese hybrids (not Local varieties in Increased frequency of identified) previous seasons outbreaks Shanyou 2, Shanyou 6, Local varieties in Increased damage (1600 ha Weiyou 6 previous seasons infested) Unknown Chinese Local varieties in Increased frequency of hybrid varieties previous seasons outbreaks Shanyou 6 Local varieties in Population densities 8-38 times previous seasons higher Shanyou 6, Shanyou Local varieties in 63, Weiyou 35 previous seasons Higher populations Chinese hybrids (not identified) Shanyou 63
Shanyou 6, Shanyou 28 Chilo suppressalis (SSB) Chinese hybrids (not identified) Li-Ming A
Local varieties in previous seasons Zhenshan 97A, Minghui 63 (parental lines) Commercial inbred varieties Commercial inbred varieties Commercial inbred varieties
Scirpophaga incertulas (YSB) Chinese hybrids (not Commercial inbred identified) varieties Other stemborer species Chinese hybrids (not Commercial inbred identified) varieties Hanyou Xiangchen, Commercial inbred Mingyou 55, Bayou varieties 161 *in Sogawa et al., 2009 **in Mew et al., 1988
153,000 ha of winter-spring rice destroyed in outbreak Higher densities of nymphs and macropterous immigrants, and more efficient conversion of plant biomass to insect biomass Populations were 2 (Shanyou 6) and 11 (Shanyou 28) times higher More frequent and severe infestations 20-35% higher adult and larval densities, and 107-170% higher egg masses
Source
Huang et al., 1985* Tan, 1987* Tang et al., 1998 Feng & Huang, 1983* Lin, 1994* Ruan, 1983* Yu et al., 1991*; Shi & Lei, 1992* Thanh et al., 2001* Sogawa et al., 2003b* Cai & Zhong, 1980** Anonymous, 1979** Tan et al., 1983**
Higher damage, survival rates, larval and pupal weights, and number of egg masses
Chieng, 1985
Higher populations
Liu, 1979
Higher damage due to 'whiteheads'
Zhu et al., 2007
It is beyond the scope of this thesis to identify specific hybrid rice farm management practices that may be associated with perceived vulnerability of hybrid rice to planthoppers and other insects (an indication of such factors is, however, presented in Table 1.1); Furthermore, it is important to bear in mind that the social
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and cultural context of hybrid rice production bears heavily on the outcome of herbivore-hybrid rice interactions in farmers‟ fields and can decouple experimental results gained from laboratories, greenhouses or experimental field stations from the realities of the rural communities and farmers‟ fields. This makes it difficult to predict the outcome of genetic improvement of hybrids for resistance to insects and diseases. In this thesis the concept of tolerance is introduced and the merits and drawbacks of insect tolerance in rice production are clearly defined. Tolerance is distinguished from resistance and increased attention to issues of tolerance in future rice breeding programmes are called for, indicating how tolerance can be measured in screening and experimental studies. Finally a series of hypotheses are presented to explain the susceptibility of hybrid rice to insects based on insect-rice interactions alone and recommendations made for improved hybrid rice management based on a better understanding of these interactions.
1.2. Is hybrid rice inherently more susceptible than inbreds to planthoppers? With expansion in the area dedicated to hybrid rice production in China, there has been a noted increase in planthopper incidence and outbreaks (Cheng, 2009). Although studies have indicated that hybrid varieties are more susceptible to BPH than are inbred varieties (Sogawa et al., 2003a; Chen et al., 2005; Faiz et al., 2007; Thanh et al., 2007), it is their apparent hyper-susceptibility to WBPH that has raised most concern. One commonly observed phenomenon during the course of hybrid expansion has been the shift from BPH-dominated planthopper assemblages to assemblages that are progressively dominated by WBPH (Mew et al., 1988; Sogawa, 1991; Thanh et al., 2007; Vien et al., 2007). Sogawa et al. (2009) reviewed
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the historical evidence for this shift in China whereby the frequency of WBPH outbreaks was positively correlated with the area under hybrid rice between 1980 and 1990 in Guangdong, Hunan and Guangxi provinces. The first observations of high WBPH densities in China were on the F1 variety Shanyou 6 in Zhejiang Province in the late 1970s and early 1980s, where WBPH populations were between 8 and 38 times higher on Shanyou 6 than on inbred varieties (Ruan, 1983 in Sogawa et al., 2009). Furthermore WBPH had demonstrated higher reproductive rates on hybrids compared to inbreds (Huang et al., 1985; Yu et al., 1991; Shi & Lei, 1992). Chinese hybrid rice was first introduced into the Red River Delta in Vietnam in the early 1990s and rapidly expanded to cover 70-80% of the rice area in the region. Ten years later, in 2000, WBPH outbreaks had occurred over 153,000 ha of Chinese hybrid rice in the Delta (Thanh et al., 2001, 2007). Comparing WBPH fitness on the highly susceptible hybrid Shanyou 63 with its CMS line Zhenshan 97A and restorer Minghui 63, Sogawa et al. (2009) indicated that susceptibility was significantly higher in the hybrid variety than in the restorer line, but that the susceptibility was similar between the hybrid and the near-isogenic CMS parent, suggesting that high or hyper-susceptibility to WBPH was bestowed through the CMS line containing the WA-CMS trait (Sogawa et al., 2003b). Sogawa et al. (2009) have shown that the hybrid variety Shanyou 63 has higher amino acid content in its phloem. Phloem amino acid content in rice, in particular asparagine, has been directly related to increased BPH fitness on rice plants (Sogawa & Pathak, 1970). The rapid expansion of rice area under hybrid rice has also increased the vulnerability of inbred varieties when large hopper populations build up and migrate
10
to adjacent fields of inbred varieties, such as occurred in central China where WBPH invaded japonica varieties on a massive scale (Sogawa et al., 2009). Since Sogawa‟s pioneering work (first published in English in 2009), numerous other studies have indicated that Shanyou varieties tend to promote WBPH populations (Mew et al., 1988; Thanh et al., 2007; Crisol et al., unpublished) and that WBPH populations are higher in hybrid rice fields than in inbred rice fields (Thanh et al., 2007; Hu et al., unpublished) (Figure 1.4). As mentioned above, the limited availability of CMS lines among Chinese hybrids has resulted in a predominance of varieties that share a narrow WA-CMS lineage (about 40% of all hybrids in China); other lineages with extensive coverage (ca 40% combined) include ID-CMS (originated from rice line Indonesia paddy 6), G (originated from rice line Gambianka) and D (originated from an F1 population of (Dissi D52 × 37) × Ai-Jiao-Nan-Te) (Cheng et al., 2007). This extremely narrow CMS line parental diversity combined with a limited occurrence of compatible restorer lines leads to a correspondingly low genetic diversity among hybrids, and because of their extensive planting, a low diversity of rice in Chinese and North Vietnamese rice fields in particular. This predominance of WA-CMS progeny, suggests that there may currently be a high regional susceptibility of rice to WBPH in China and North Vietnam. The introduction of resistance through restorer lines with dominant hopper-resistant genes is possible and has been achieved in the past (e.g. Bph1 from IR26 introduced to Nanyou 6 and Weiyou 6 [Mew et al., 1988]). However, the hyper-susceptibility of Shanyou varieties and the WA-CMS line could undermine the effects of WBPH-resistance genes, depending on the mechanisms underlying susceptibility, which are currently unknown.
11
A link between the genetic divergence (distance) between parental lines and consequent heterosis for yield in F1 hybrids has been sought in a number of
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Fig. 1.4: Higher fitness of white-backed planthopper on hybrid compared to inbred rice varieties from published and unpublished studies: (A) oviposition, (B) female longevity and (C) incidence of whitebacked planthopper on hybrid and inbred varieties in Vietnam (Thanh et al., 2007); (D) Comparison of white-backed planthopper survival on Chinese hybrid variety SY-63 H, the CMS-line ZS-97 A, and the restorer line MH-63 R. Open bars indicate the reduction in plant biomass resulting from 1mg of planthopper production and shaded bars indicate biomass productivity of white-backed planthoppers (from Sogawa et al., 2009); and (E) incidence of white-backed planthopper on hybrid (SY-63 H) and inbred (IR64) rice at CNRRI in 2010 under moderate (100Kg/ha – open bars) and high (250Kg/ha – shaded bars) nitrogen (Hu et al., unpublished).
experimental studies; however, evidence for the relationship is not always conclusive and correlations are often weak (Zhang et al., 1995; Kwon et al., 2002; Xu et al., 2002 but see Xiao et al., 1996) although this may be related to the low
12
resolution of genetic markers (SST) employed in studies of rice genetic diversity (Xu et al., 2002) or inappropriate statistical procedures (Piepho, 2005). Nevertheless, it is commonly held that increased genetic distance between parents results in improved hybrids (Singh et al., 2011), although there may be some threshold above which it becomes difficult to achieve successful restoration of fertility. Breeding programmes have therefore attempted to increase the diversity of their parental lines and optimize the genetic distance between male-sterile and restorer parents (Peng et al., 1999; Singh et al., 2011). In particular, the development of two-line hybrids with environmentally sensitive male sterile (ESMS)-parents, such as PGMS and TGMS, reduces the requirement for a maintainer line and may promote greater genetic diversity among hybrids in the future. A greater overall genetic diversity among hybrids will have consequent positive effects on field and regional susceptibility to planthoppers, since rice diversity at a landscape level may act as a barrier to planthopper invasion (e.g. Claridge & Den Hollander, 1982). In general, rice breeding programmes tend to restrict genetic diversity. For example, in India, of 29 rice varieties of hybrid origin released in Kerala during 1966-95, only 37 ancestors were used directly or indirectly in their development; of these, ten ancestors alone contributed 74.14% of the genetic base. Similarly, the cytoplasmic diversity was also limited, as 41.38% of the varieties could be traced back maternally to the same ancestor, PTB 10 (Thekkancheera), and thus probably carried its cytoplasm (Shivkumar et al., 1998). Moreover, the CMS-breeding funnel, whereby all varieties pass through the closely related CMS-parents, will greatly reduce the genetic distance between hybrid varieties (Li et al., 1999; Peng et al., 1999).
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Susceptibility is not necessarily a direct feature of the CMS line. Among Shanyou and WA-CMS related varieties there is hyper-susceptibility to WBPH that may or may not be cytoplasmically inherited from the CMS line (Sogawa et al., 2009). However, in a study by Faiz et al. (2007) using four IRRI CMS lines, there was no evidence that susceptibility was associated with the wild abortive cytoplasm, but was more likely the result of nuclear gene interactions. Recent studies indicate that hybrids bred at IRRI were generally no more susceptible than their inbred parents to WBPH or BPH (Horgan personal communication). This suggests that hybrid rice varieties per se are not inherently more susceptible to planthoppers when compared to inbred varieties, but that the predominance of certain CMS lines (susceptible to planthopper attack) in national breeding programs, particularly in China and the consequent low genetic variability among hybrids has increased hybrid vulnerability to hoppers. The introduction of BPH and WBPH resistance genes and augmentation of genetic diversity, particularly among male parent lines, should be included in future hybrid rice breeding programmes. However, the genetic and physiological mechanisms of resistance in hybrids will need to be assessed in conjunction with hybrid vigour and the subsequent potential for superior herbivory-tolerance of hybrids to ricehoppers. As we will see later, the effects of plant tolerance in rice ecosystems are still poorly understood.
1.3. Is hybrid rice inherently more susceptible than inbreds to stemborers? According to Zhu et al. (2007), the large-scale planting of hybrid varieties has been one of the principal factors associated with increasing stemborer densities
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in Chinese rice fields since the 1990s. Furthermore, in India, rice yield losses due to stemborers have also increased as the area planted with high-yielding hybrid varieties has increased (Muralidharan & Pasalu, 2006). By 1979, stemborer populations had increased by 3400% in China, only five years after hybrids were first commercialized in that country (Mew et al., 1988). Mew et al. (1988) suggested that the introduction of hybrids in China to regions with early rice planting produced an extended availability of rice plants with a short or no fallow period, allowing stemborer populations to build up to higher-than-normal levels. This might suggest that population build-up was a general phenomenon occurring on both hybrids and inbreds at a regional scale. Similarly, Zhu et al., (2007) suggested that stemborertolerant hybrids that are widely grown in the Yangtze Delta allowed populations to build-up, thereby increasing the risk of damage to other types of rice. However, direct evidence for this hypothesis is lacking. Several comparative studies from China have directly reported higher incidences of stemborers on hybrid compared to inbred rice varieties (Liu, 1979; Wu, 1984; Chieng, 1985; Tian & Li, 1994; Tu et al., 2000). In a study by Chieng (1985), there were 107-110% more yellow stemborer (YSB)(Scirpophaga incertulas [Walker]) egg masses on hybrid than on inbred rice with 7-17 times as many YSB egg masses on the hybrids, and also, survival rates and pupal weights were higher on hybrid rice compared to inbreds. Similarly, Tan et al. (1983) found 20-35% higher densities of adult striped stemborer (SSB)(Chilo suppressalis [Walker]), and 107170% more egg masses on hybrids compared to inbreds. Many of these reports were based on field surveys that potentially confounded the effects of crop management (particularly fertiliser regimes) and plant type.
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Hybrid rice normally receives higher fertiliser applications than inbred varieties (Cheng, 2009), and several studies have indicated that stemborers and other lepidopterans have higher survival, growth rates and biomass on plants with high nitrogen applications and/or with application of inorganic fertilisers (Dhandapani et al., 1990; Gill et al., 1993; Gines et al., 1994; Jiang & Cheng, 2003; Huang et al., 2010; Crisol et al., unpublished data; Romena & Horgan, unpublished data): Huang et al. (2010) showed that applications of common chemical fertilisers enhanced the incidence of SSB on hybrid rice, as compared with treatments with slow-release urea and organic manures. Additionally, Dhandapani et al. (1990) showed a higher percentage of both deadhearts and whiteheads caused by YSB when inorganic fertilisers were applied, as compared with green manure applications. Increased nitrogen applications in particular (especially when synthetic fertilisers with high levels of soluble nitrogen are used) can reduce the concentration of secondary compounds in plant tissue (Rühmann et al., 2002). Many of these secondary compounds are directly related to defenses against plant herbivores (Swain, 1977; Herms & Mattson, 1992). Organic fertilisers have also been shown to improve YSB resistance by increasing silicon, phenol and tannin contents in the inbred rice variety TKM6 (Usha Rani et al., 2009). Certain morphological and physiological features of hybrid varieties have been implicated in their apparently greater susceptibility to stemborers. Chieng (1985) found that survival of both SSB and YSB was higher, and larvae and pupae were larger, when larvae were fed on hybrid rice varieties compared with inbred varieties. The author suggested that large, thick stems, abundant foliage, and long maturity of the hybrids increased their susceptibility to stemborers. Furthermore,
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Zhu et al. (2007) suggested that a longer heading period in hybrid rice (e.g. Hanyou xiangchen, Mingyou 55 and Bayou 161) together with a large stem diameter and inner antrum and high nitrogen:carbon ratios increased stemborer fitness and the susceptibility of hybrid rice compared to inbred rice varieties. The results of field and laboratory studies are therefore generally inconclusive as regards the vulnerability of hybrid rice to stemborers. Studies suggest that hybrid rice varieties may be more susceptible to stemborers because of their morphology and physiology, both of which are directly related to hybrid growth rates and vigour; however, these characteristics have seldom been compared in controlled experiments and have not been directly tested as factors that increase hybrid susceptibility to stemborers. In many cases, management practices associated with hybrid rice production are likely to have a greater effect on stemborer populations than has plant morphology and/or physiology. Hybrid susceptibility to stemborers at the landscape scale is therefore likely to result from the interaction of several plant traits with normal hybrid management, and particularly with fertiliser applications.
1.4. Hybrid vigour and tolerance to planthoppers and stemborers Examples of heterosis for planthopper resistance are rare. This is partially due to the inability of researchers to acquire parental lines for evaluation since these are often restricted for distribution, especially where the hybrids are commercialized. Nevertheless, heterosis for BPH resistance has been shown for 3 of 11 tested IRRI hybrids (Cohen et al., 2003); however, in general, this resistance was weak (ca 15% mortality of nymphs) and resistance stability as plants aged was not evaluated in the
17
study. In contrast, Sogawa et al. (2009) demonstrated clear heterosis for susceptibility to WBPH in Shanyou 63. Interestingly, both these studies indicated clear reductions in damage ratings for hybrids compared to the most susceptible parents. This damage reduction is the direct consequence of a higher tolerance of hybrids to planthopper feeding supported through greater biomass accumulation or plant vigour; however, high plant biomass can also directly lead to higher planthopper populations. Explanations for the higher yields achieved with hybrids compared to inbred lines has been sought through a number of studies (Peng et al., 1999; Bueno & Lafarge, 2009; Wu, 2009; Bueno et al., 2010). Evidence suggests that superior yield is related to comparatively large sink (i.e. panicles and grains) and source (i.e. leaf and stem biomass) sizes. Three main hypotheses have been proposed to explain the genetic basis of heterosis: The dominance hypothesis states that heterosis is due to the accumulation of favourable dominant genes in a hybrid derived from two new parents. The overdominance hypothesis states that heterozygotes (Aa) are more vigorous and productive than either of the homozygotes (AA or aa). The pseudooverdominance hypothesis states that a number of different loci linked in repulsion phase and contained in a small chromosomal region are complemented by the presence of superior alleles in the hybrid, producing a better phenotype (Birchler et al., 2006). Heterosis for some traits may also be due to synergistic effects between the cytoplasm of the maternal parent and the nuclear component of the paternal parent (Ellison & Burton, 2009). Rice tolerance, including overcompensation, to stemborer damage has been demonstrated in several laboratory and field studies (Rubia et al., 1989; Litsinger,
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1991, 1993; Rubia et al., 1996a; Bandong & Litsinger, 2005) and rice plants have been shown to compensate for remarkably high levels of damage (30-60%) without any yield reduction (Rubia & de Vries, 1990; Ibrahim & Singh, 1992). However, it is apparent that tolerance levels are dependent on plant phenology and the time of stemborer attack. Damage to vegetative stages and during early tillering (known as „deadheart‟) rarely causes yield loss because plants can compensate by modifying tiller production and function (i.e. whether these will eventually become vegetative or reproductive), whereas damage to reproductive tillers occurring at late crop stages (known as whitehead) is generally thought to reduce yield (Pathak, 1968; Dale, 1994; Pathak & Khan, 1994; Reji et al., 2008 but see Islam & Karim, 1997). A number of studies have shown evidence for higher tolerance to stemborers in hybrid compared to inbred rice (Luo, 1987; Gines et al., 1994; Xu et al., 2007). Tan et al. (1983) have shown that the indica hybrid Li-Ming A had both higher larval counts and higher tolerance during both the heading and ripening stage, compared with the indica rice Dong-Ting. Xu et al. (2007) examined the physiological responses to stemborer feeding in the hybrid rice varieties Shanyou 63 and Liangyoupeijiu. They indicated that higher root activity and an enhanced ability to absorb potassium was associated with higher damage compensation in both hybrid varieties when compared with the japonica inbred Xiushui 11. Luo (1987) showed higher compensation to attack by SSB through increased head and grain weight, in the hybrid variety Weiyou 35 as compared with the inbred Xiang-Ai-Zao 10. Similarly the hybrid IR64616H showed significant compensation for whitehead damage compared with IR72, by producing 30-50% heavier productive panicles
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(Gines et al., 1994). Higher fertiliser applications to hybrid rice would further increase their tolerance to herbivore damage, but would tend to reduce resistance. Tolerance to herbivory is rarely evaluated in rice breeding programmes, and tolerance as a concept in the study of insect-plant interactions is often poorly understood or confused. Much of this confusion can be attributed to adherence to the original definitions of host plant resistance as set out by Painter (1951). Painter (1951) was one of the first and most influential contributors to the application of host plant resistance for agriculture. He defined resistance as the relative amount of heritable qualities possessed by a plant which influence the ultimate degree of damage caused by insects in the field. Under this definition, he identified three principal mechanisms that reduce insect damage as antixenosis (non-preference), antibiosis (reduced performance) and tolerance (improved plant survival or compensation for damage). These „mechanisms‟ were later described as functional categories by Maxwell & Jennings (1980) and functional modalities of resistance by Smith (2005). These latter authors clearly distinguished „resistance‟, where insect fitness is reduced either through antibiosis or antixenosis, from „tolerance‟, the ability of a plant to suffer herbivore damage without a corresponding reduction in its own fitness (Figure 1.5). This conceptual separation of resistance and tolerance has largely been accepted by evolutionary ecologists; however, in agricultural sciences, Painter‟s original definition has largely been maintained (e.g. Panda, 1979; Panda & Khush, 1995; Carozzi & Koziel, 1997; Sadasivam & Thayumanaran, 2003; Dhaliwal & Singh, 2005; Singh & Singh, 2005; Narayanasamy, 2010). Tolerance can produce very different evolutionary and ecological dynamics between plants and herbivores than occur with plant resistance (Tiffin & Inouye, 2000). Therefore separation of the
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concepts can greatly improve our understanding of the evolution of plant defences, the maintenance of field durability, the evolution of herbivore virulence, and can improve rice breeding strategies for efficient pest management.
Fig. 1.5: Association between resistance and tolerance (A) according to Painter (1951) and (B) as revised by Smith (2005)
1.5. Definitions and dynamics of tolerance to herbivory Tolerance to herbivory has been overlooked by ecologists and agriculturalists alike until relatively recently; however, for evolutionary ecologists the concept has now become a key issue in understanding the evolution of host-plant defences, and several papers have reviewed the theory and consequences of tolerance for
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herbivore-plant interactions (Rosenthal & Kotanen, 1994; Strauss & Agrawal, 1999; Simms, 2000; Stowe et al., 2000; Núñez-Farfán et al., 2007). Tolerance has been studied less in agricultural literature though several studies present evidence of tolerance, including some from empirical studies with rice pests (Rubia et al., 1996a; Rubia-Sanchez et al., 1999). There are several definitions of tolerance (see Strauss & Agrawal, 1999); however, in this thesis, that of Stowe et al. (2000) will be used, where tolerance is defined as the reaction norm (i.e. the shape of the response curve) of fitness across a damage gradient; This definition has a number of advantages over previous definitions: 1) tolerance can be treated as a phenotypically plastic trait; 2) it suggests that the reactions to damage can be complex and that there are gradients of tolerance, therefore incorporating special cases, such as overcompensation; 3) it allows environmental variables to be incorporated and to influence the fitness response. Furthermore, for phenotyping purposes, it indicates that tolerance is best measured using two or more damage or resource levels. The idea of tolerance as a response that varies according to levels of environmental stress prevents any oversimplification of its dynamics and effects. Previously, plants had been regarded as allocating resources between reproduction, growth, defences and maintenance, such that allocation away from any of these components is regarded as an allocation cost that can often be empirically measured. This conceptual frame suggested that rapidly growing or vigorous plants allocate resources away from defences, thereby increasing their vulnerability to pests and diseases (Herms & Mattson, 1992). However, there is still little evidence that vigorous plants (plants with a high relative growth rate) are less resistant to insects. Vigorous plants are often regarded as being more tolerant to damage because of their
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capacity to compensate for damage (but see Weis et al., 2000), suggesting that tolerance might be an inherent property of rapidly growing plants, that tolerance might be antagonistic (see Fineblum & Rausher, 1995) to resistance (i.e. high levels of tolerance would result in low levels of resistance and vice versa) and that tolerance is determined by resource availability and therefore, by the plant‟s environmental conditions, particularly as regards water and nutrients. These environmental influences on tolerance predict that a plant‟s ability to tolerate damage is greater in resource-rich environments (e.g. Maschinski & Whitham‟s (1989) Compensatory Continuum Hypothesis) (CCH). These ideas might suggest that tolerance is a feature of growth and maintenance only, with the absence of specialized genes. However, it is now apparent that plants express genes for tolerance that are independent of resistance genes or normal growth and maintenance genes: For example, feeding by the aphid Diuraphis noxia populations on resistant wheat triggers a mechanism that maintains photosynthetic activity due to the expression of transcripts that allowed the resistant plants to cope with the stress (Botha et al., 2006). In susceptible plants, this expression of transcripts does not occur, suggesting that plant genes controlling tolerance are differentially expressed in „resistant‟ and „susceptible‟ plants (Botha et al., 2006). Therefore, since both resistance and tolerance are adaptive, they bear fitness costs for the plant. Nevertheless, evidence is accumulating that plants can allocate resources simultaneously to both resistance and tolerance (Smith, 2005). Van der Meijden et al. (1988) first considered the possibility that tolerance and resistance might be negatively genetically correlated and therefore evolve antagonistically (which confounds the resource allocation idea). This is largely
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intuitive since as resistance increases, damage declines and thus the fitness advantage of tolerance also declines. This could arise from pleiotropic effects of genes involved in an allocation trade-off between resistance and tolerance, or because of linkage disequilibrium produced by correlated selection on these traits; however, the latter is less likely, since it would need closely linked genes for resistance and tolerance in repulsion. A few studies have detected negative genetic correlations between resistance and tolerance across species or within populations, including negative correlations between tolerance and constitutive or induced defences (Strauss & Agrawal, 1999 and references therein). A recent meta-analysis by Leimu and Koricheva (2006) of 31 ecological and agricultural studies found that the sign of the relationship between tolerance and resistance differed depending on the type of plant studied. Furthermore, tolerance and resistance tended to be positively correlated in crops but negatively correlated in wild plants. Among the few studies that have examined the fitness costs of tolerance to herbivory, all have indicated significant costs in terms of biomass or seed and fruit production (Strauss & Agrawal, 1999; Núñez-Farfán et al., 2007). However, independent studies have found positive correlations between tolerance to different herbivore species in the same plants, suggesting that tolerance mechanisms are general plant responses rather than the more costly species-specific responses (Leimu & Koricheva, 2006). But what are these mechanisms? Tiffin (2000) indicates that there are two major categories of tolerance mechanisms, these are „active‟ (induced) and „passive‟ (constitutive). Induced mechanisms include physiological responses to damage such as the increase in leaf level photosynthetic activity or compensatory growth (e.g.
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McNaughton, 1979; Crawley, 1983; Whitham et al., 1991; Rosenthal & Kotanen, 1994; Strauss & Agrawal, 1999). In general, compensatory growth and activation of dormant meristems following removal or damage of vegetative or floral meristems is perhaps the most widely recognized tolerance mechanism in plants in general (e.g. Inouye, 1982; Paige & Whitham, 1987; Prins & Verkaar, 1989; Doak, 1991; Bergelson & Purrington, 1995; Mabry & Wayne, 1997); There are numerous examples of these responses to stemborer damage in rice (Halteren, 1979; Soejitno, 1979; Tian, 1981; Akinsola, 1984; Luo, 1987; Viajante & Heinrichs, 1987; Yambao et al., 1993; Islam & Karim, 1999). Compensatory growth is facilitated by an increase in the photosynthetic rate of leaves adjacent to those that are directly damaged with translocation of assimilates from injured tillers to healthy tillers (Rubia et al., 1996a). Reductions in plant height may also support the production of new tillers (Rico, 1982). Ultimately, a plant‟s strategy may be to escape damage by producing a higher number of seed. In rice, the production of more and/or heavier grains per panicle has been noted following stemborer and planthopper attack (Akinsola, 1984; Luo, 1987; Islam, 1990; Islam & Karim, 1997) although some of these observations could be related to changes in phenology, a common mechanism of tolerance (Tiffin, 2000). “Passive” or constitutive tolerance mechanisms are expressed throughout the plant‟s growth and are inherent to the plant‟s morphology and growth patterns. These include differential resource allocations to various plant parts including rootshoot ratio (Chapin & McNaughton, 1989), stem number (or stem rigidity) (Rosenthal & Welter, 1995), and the proportion of photosynthetic surface (Bazzaz, 1979). For example, seeds with larger amounts of stored reserves may be more
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tolerant of partial cotyledon removal than smaller seeds (Danckwerts & Gordon, 1987; Van der Heyden & Stock, 1996) and rice plants with high above-ground biomass may be more tolerant that those with high root biomass. This constitutive tolerance varies as plants grow and develop: Plants may be most tolerant during later, sometimes reproductive stages, when their biomass is greatest. For rice this is likely to be at or after the maximum tillering stage. Compelling evidence that tolerance is adaptive and is a specific response to herbivore damage comes from observed differences between mechanical damage and insect-induced damage (Tiffin & Inouye, 2000 and references therein). This further indicates that, during phenotyping studies, simulation of insect damage by mechanical removal of leaves, shoots or other plant parts is not feasible. Induced tolerance mechanisms in particular suggest that plants can respond to specific elicitors generated during insect feeding in much the same way as occurs with induced resistance to pests (see Poveda et al., 2010). Such induced tolerance or resistance mechanisms are responses to specific elicitors derived from the source of the damage that differ according to the nature of the damage or the specific insectfeeding guild (Korth & Dixon, 1997). The idea of tolerance as a variable reaction that depends on resource availability links well with the Plant Vigour Hypothesis (PVH) (Price, 1991). The PVH of herbivore attack proposes that plant modules (such as tillers) which are growing vigorously, or have grown vigorously to become relatively large in a population of modules, are more attractive to certain kinds of herbivores. Plants growing in relatively nutrient-rich and high-light conditions grow rapidly and are heavily attacked by herbivores. Price (1991) presented numerous qualitative
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examples of vigorous plants attracting and enduring higher levels of damage. More recently, Cornelissen & Fernandes (2008) conducted a meta-analysis of 71 published studies to examine evidence for the PVH. They showed that sap-suckers (Homoptera, Hemiptera) in particular, increase in abundance (up to 75%) on more vigorous plants or modules. This is presumably due to increased assimilates (qualitative and quantitative measures) in the phloem and has direct consequences for planthoppers on vigorously growing rice varieties. According to the CCH (Maschinski & Whitham, 1989), which states that tolerance of herbivory should be greater in high resource or low competition conditions, vigorous plants should better tolerate higher incidences of herbivores than less vigorous plants. In effect, according to these two hypotheses, as herbivory increases, herbivory pressure (the effect of the herbivore on plant fitness) could remain the same, or even decrease. Under certain circumstances, plant fitness may actually increase when the plant is attacked by herbivores. This is referred to as overcompensation, and is common among plant-insect relationships (Agrawal, 2000; Poveda et al., 2010). In rice, overcompensation has been noted as increased seed production by hybrids and inbreds following WBPH attack. Presumably, the extra grain results from a shift from root biomass to accumulation of above-ground biomass, thereby increasing the source size. Moreover, feeding damage in economically non-important sinks could result in yield benefits where resources are redistributed to the economically important sinks.
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1.6. Phenotyping for tolerance to planthoppers and stemborers Rice breeders have included components of host-plant resistance to insects in their breeding programmes for over 50 years. Among the best known examples of resistant rice varieties are those produced in response to BPH outbreaks that occurred on IR8 in the 1970s (Sogawa & Pathak, 1970; Gallagher et al., 1994; Cuong et al., 1997; Khush & Virk, 2005). Resistant varieties with the Bph1, bph2 and Bph3 genes (uppercase indicates a dominant gene, lowercase a recessive gene) were sequentially developed during the 1970s and 1980s with apparent, but shortterm field successes (Alam & Cohen, 1998). Resistance to stemborers in rice has been less well documented; however, several commercial varieties are classified as resistant or moderately resistant to stemborer damage (Philippine Department of Agriculture personel communication). Research on rice resistance to stemborers has, however, declined substantially in the last 20 years (Du, 2008). Nevertheless, many breeding programmes routinely screen rice developmental lines either as recurrent inbred materials (F1-F6) or as elite breeding materials for their resistance to planthoppers and stemborers (Horgan, personal communication). The methods involved in such screening are necessarily simple and with a quick turnover time in order to process large numbers of rice plants. Screening protocols for resistance have consequently been criticized for imprecision in their results, difficulty in interpretation of the meaning of results, and/or a lack of applicability to the field (Kaneda et al., 1981; Panda & Heinrichs, 1983; Velusamy et al., 1986). Screening for rice tolerance to herbivore damage is seldom conducted during varietal development or even after the release of new varieties, and in general receives little attention in agricultural research (see above). Furthermore, some
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protocols for resistance screening confound resistance and tolerance. An example of this is resistance screening using the standard (SSST) and modified seedbox screening tests (MSST). These tests are used to determine field resistance to planthoppers. In the SSST, seeds of susceptible checks and test varieties (generally >15) are sown in lines in a seedbox (ca. 60 × 40 × 10 cm) and infested at seven days (SSST) or 20 days (MSST) after sowing (normally using eight second instar nymphs per seedling) (Horgan, 2009). In both tests, when susceptible checks are killed, plants are scored for damage. Researchers sometimes attribute „moderate resistance‟ to varieties with mid-damage scores. However, such varieties may actually have experienced equal rates of insect attack as susceptible varieties, but demonstrated less pronounced attack symptoms (Velusamy et al., 1986; Horgan, 2009). Screening protocols that monitor the damage caused by insects rather than directly measuring the fitness of the insects will always confound resistance with tolerance. Early attempts to distinguish tolerance from resistance were based on plotting measures of the intensity of herbivore attack against reductions in components of plant biomass (Figure 1.6) (Lye & Smith, 1988). A series of formulae have been developed for use on different cereal crops to assess their tolerance to insect herbivores (e.g. Schweissing & Wilde, 1979; Morgan et al., 1980; Panda & Heinrichs, 1983). These formulae had a number of common features. They included a calculation of the difference between infested and uninfested plants, either in terms of biomass (dry weight) or plant size (e.g. leaf area), and an estimate of damage, usually determined from a seedbox evaluation method. These estimates were rather crude given that visually assessed damage ratings were used to describe the pressure of insect attack. This was later rectified by Bramel-Cox et al. (1986) and improved
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by Dixon & Kindlmann (1990) and others who, among other improvements, incorporated direct measures of insect pressure and expressed plant biomass reduction as a proportion of total plant biomass to allow comparisons between different plant genotypes. Tolerance to aphids has been assessed using the SPADbased chlorophyll loss index. This index uses a SPAD chlorophyll meter to assess aphid-induced chlorophyll loss in leaves. The method attempts to standardize insect pressure by confining the aphids within a plastic ring on the leaf surface for a measured period of time. The loss in chlorophyll is then measured from the area that
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Larval weight (mg)
Fig. 1.6: Graphical depiction of tolerance: Relationship between rice plant biomass reduction (tolerance) and Spodoptera frugiperda larval weight (resistance) among five different plant introductions (symbols)(redrawn from Lye & Smith, 1988).
was enclosed by the ring (Girma et al., 1998; Deol et al., 2001). Smith (2005) gives a good synopsis of these early tolerance indices with full details of the parameters required to calculate each. Unlike experiments that assess plant tolerance to mechanical damage or abiotic stresses, the assessment of tolerance to insect damage is complicated by an 30
inability to standardize the levels of the stress. For example, experiments that mechanically remove leaves or leaf area can carefully control damage levels, but insect damage depends on the plant‟s induced and constitutive levels of resistance as well as the individual and population behaviours of the test insects. For example, although SPAD-based measurements suggest a standard aphid pressure that should be applied (Girma et al., 1998), aphids will probe, feed or salivate to different extents depending on the favourability of the host. Leaf-chewers may also react differently to the host plant‟s resistance factors, but the damage they inflict can be carefully measured using a variety of simple techniques (Litsinger, 1991 and references therein). Stemborer damage is less easily observed than damage from leaf-chewers, since the larvae feed entirely within the stem. Similarly, planthopper damage can only be assessed indirectly because rates and quantities of phloem feeding are difficult to assess (Velusamy & Heinrichs, 1986; Padgham & Woodhead, 1988). Because of this, several studies assess tolerance by comparing the slopes of regressions between herbivore pressure measured in absolute (i.e. dry weight of hoppers, number of stemborers, etc) or relative terms (i.e. damage to tissues, leafarea loss, etc) and plant responses across experimental replicates (Weis et al., 2000; Rubia-Sanchez et al., 2003). Plant responses can also be expressed in absolute (i.e. weight loss) or relative terms (percentage weight loss). However, Wise & Carr (2008) caution that the variables on the x- and y-axes in such regressions should be either both additive or both multiplicative. Care should also be taken when transforming data to meet requirements of normality and homogeneity of variances for parametric statistical analyses because certain transformations will convert scales
31
from additive to multiplicative. Transformations also become problematic when there is a zero response of the plant or overcompensation (such that the sign of the response changes) as these types of data cannot be adequately transformed without losing relatedness between observations. Because insect responses are unpredictable and phenotyping studies generally do not test the relative resistance of plant genotypes across a gradient of environmental conditions, problems can arise regarding the correct infestation levels to which the plants should be subjected. If the infestation levels are too high, or the insects left too long on the plant, then intraspecific competition can occur between individuals in the herbivore population (Litsinger, 1991). Where insects compete with each other, then the effects of competition can confound assessment of resistance and/or tolerance and the insect population may affect itself more than it affects the plant. Herbivore pressure should therefore be standardized to plant biomass. However, whether it is standardized to the biomass of the control (its potential host) or infested plant (its actual host), the comparative reduction in plant biomass across genotypes (biomass of infested – biomass of control) will determine the apparent fitness of the insect. Across plants with different levels of tolerance, this will lead to inaccuracies in assessing the insect response to the plant and prevents the accurate assessment of tolerance. The relative magnitude of intraspecific competition on test plants can be compared using a competition index calculated as the difference between the biomass of the insect per dry weight of infested and control plants. To avoid complications arising from high levels of intraspecific competition between insects, the herbivore pressure should be minimized. However, the pressure cannot be so low
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that it produces negligible plant responses. To optimize insect pressure for best results, pre-test experiments with different insect densities could be conducted to assess potential carrying capacities of the rice plants for the test insects. In the specific case of assessing tolerance in cereal crops such as rice, tolerance is best measured in terms of the stability of grain yields. This necessarily requires that plants be maintained through to harvest. It then becomes a key issue as to when the insect pressure should be applied. However, this will largely be determined by the objective of the experiments and the simulation of events in nature (i.e. the natural periods of maximum herbivore pressure). In summary, tolerance should be measured under different environmental conditions. At least one of these conditions is the presence or absence of insects. Therefore, screening for tolerance, unlike screening for resistance, requires a set of control plants. This will increase the cost and effort required to complete the screening. Because tolerance depends on environmental conditions, particularly nutrient availability, and on plant ontogeny, screening is best conducted to simultaneously assess the stability of tolerance as the magnitude difference between infested and control plants under different conditions. Care should be taken to avoid intraspecific competition within the populations of test insects, and plants should be maintained and evaluated at harvest because it is yield tolerance that is of most interest to rice breeders. These considerations will further increase screening costs. In the case of hybrid rice, comparisons with inbred varieties will need to take special care of plant growth conditions as hybrid and inbred plants may react differently to pot confinement and insect cages. As a rule, large pots should be used where plant tolerance is assessed at harvest.
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1.7. Ecological consequences of tolerance at field and landscape levels Inbred rice varieties with relatively high tolerance have been adopted by farmers and are used widely in Asia. However, this has often occurred without farmers‟ knowledge of the occurrence or consequences of herbivore tolerance. For example, IR36, which has noted high tolerance to planthopper damage (Velusamy et al., 1986) has remained one of the most popular varieties in the Philippines for decades (Khush & Virk, 2005). Similarly, many hybrid rice varieties are likely to have high levels of herbivore tolerance, and these levels may be enhanced where farmers apply high amounts of fertiliser (e.g. Tan et al., 1983). Farmers who adopt hybrid varieties are often informed of the susceptibility of hybrids to herbivory, but are rarely aware of tolerance traits in hybrids. This often drives farmers to apply higher amounts of chemical insecticides to hybrid rice than they would normally apply to inbred varieties (Cheng, 2009). In fact some hybrid rice seeds are distributed together with fertilisers and insecticides as part of a „package‟ management kit (Heong - IRRI, personal communication). Strategies for an integrated pest management for hybrid rice are frequently overlooked, as these are outweighed by fears of hyper-susceptibility (Cheng, 2009). A better understanding by farmers and agronomists alike of the nature of tolerance (which can cause increases in pest population size) could improve pest management, reduce insecticide use and abate pest outbreaks associated with hybrid rice varieties. The knowledge and practice of integrated pest management in general may be declining in South and South East Asia. This may partly result from discontinuance of IPM-education campaign activities, such as the Food and Agriculture Organization‟s Farmer Field Schools in Indonesia and the IRRI-
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supported Three Reductions-Three Gains initiative in the Mekong Delta with a concomitant increase in pesticide production and advertising in the region (Escalada et al., 2009). IPM relied on a loose understanding of damage and economic thresholds for the major pest species of rice, above which economic losses would be expected (Litsinger, 1991). In spite of this, few published threshold levels were ever available, particularly in the form of variety-specific thresholds. Furthermore, damage thresholds would be expected to vary between different rice varieties depending on their levels of herbivore tolerance and on the specific management conditions (particularly as regards fertiliser applications). But to my knowledge, no indication of higher threshold levels for tolerant rice varieties, and/or for hybrid rice has been proposed. Furthermore, evidence suggests that farmers are generally unaware of the herbivore resistance levels for the varieties they grow and do not normally adjust their crop management practices according to indicated resistance levels in their chosen variety (Widawski et al., 1998). Considering that hybrid rice may be more tolerant of insect damage, it is probable that these varieties require less, and not more, insecticide-based protection than do inbred rice varieties. The main disadvantage of hybrid tolerance to herbivory is that, in situations where planthopper or stemborer populations are not regulated by natural enemies, then the populations can build-up to higher levels in the landscape than would normally occur on susceptible, low-tolerance, varieties. This is because in susceptible varieties with low tolerance, herbivores reach the carrying capacity of the crop at a lower population density. In the case of planthoppers, this can result in hopperburn and consequent planthopper population decline at relatively low densities. Because population density is often directly related to the extent of the
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outbreak area (Wallner, 1985), then low-tolerance varieties effectively contain the outbreak. In contrast, tolerant varieties will allow populations to build up to comparatively higher densities, potentially resulting in larger outbreaks. This effect can be exacerbated where multi-season cropping is practiced. Where adjacent varieties have quantative resistance traits, insects at higher population densities can often overcome the resistance. Normally planthoppers and stemborers are effectively regulated by natural enemies (Heong, 2009). A range of spiders feed on planthoppers and stemborers. Crickets and small hemipterans such as Cyrtorhinus lividipennis are efficient predators of the eggs and small nymphs of planthoppers and several parasitoid wasps cause high mortality of planthopper and stemborer eggs (Kenmore et al., 1984; Reissig et al., 1986). Consequently, planthopper outbreaks have been related to the overuse of insecticides and the disturbance of pest population regulation by natural enemies (Gallagher et al., 1994; Heinrichs, 1994; Heong, 2009). In conditions of good crop management with adherence to the principles of IPM, hybrid tolerance to herbivores should represent a valuable tool that not only cushions the effects of low density herbivore populations, but, in the case of overcompensation, might also benefit from low levels of insect attack (e.g. Poveda et al., 2010). One difficulty that must be overcome is the possibility that tolerant varieties may promote the spread of planthopper-vectored viral diseases such as ragged stunt and grassy stunt (Cabauatan et al., 2009). Under such circumstances, rice varieties with some resistance to either viruses or planthoppers, but with tolerance to other insects might be advantageous. On the basis of these observations it is suggested that rice breeders should pay increased attention to the benefits of tolerance and that agronomists will need to
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develop improved strategies for an environmentally friendly, economically viable and sustainable management of hybrid rice varieties.
1.8. Introduction to this thesis This thesis examines aspects of tolerance to three important insect pests of rice in South and South East Asia, these are BPH, WBPH and YSB. In particular the work examines the hypothesis that hybrid rice varieties will be more tolerant of insect damage than are inbred varieties (Chapter 2). Comparisons of hybrid rice varieties with inbred varieties have been difficult because of the lack of availability of parental lines. In this study, seven three-line hybrid varieties were available, each with their respective inbred parental lines. Thus a total of 28 lines could be included in the research. In Chapter 2, all 28 lines were grown in a greenhouse under two fertiliser regimes at IRRI in the Philippines and infested with either BPH, WBPH or YSB with a set of control plants maintained to examine plant tolerance to herbivory. The same lines were grown in replicated blocks in the field to document their growth patterns and yield. Research that compares hybrids and inbreds must acknowledge the physiological differences between the two plant types. These differences will determine constraints on the experimental conditions required to conduct comparative studies. In Chapter 3, an analysis of the effects of experimental conditions on the comparative growth patterns of hybrid and inbred varieties is described. Plant type interactions with pot size, fertiliser regimes, and the presence/absence of BPH are highlighted and recommendations made to improve
37
methodologies for comparative studies of tolerance. Chapter 4 summarizes the main findings from this research and indicates directions for future research.
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Chapter 2
Susceptibility and tolerance of rice hybrids to insect herbivores: an experimental analysis of three-line hybrids and their parental lines
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2.1. Introduction Positive heterosis has been described as “the biological phenomenon in which an F1 hybrid of two genetically dissimilar parents shows increased vigour at least over the mid-parent value” (Shull, 1908 in Virmani, 1994). The term “hybrid vigour” is used as a synonym for heterosis to describe the physiological advantage of hybrids over parental or inbred plants. Situations where the value of a hybrid trait exceeds that of the better parent are referred to as heterobeltiosis (Fanseco & Peterson, 1968). Heterosis and heterobeltiosis have been observed, applied, and exploited in several crops for centuries but the genetic basis for such advantages in hybrids is not fully understood (Virmani, 1994; Duvick, 2001; Birchler et al., 2006; Semel et al., 2006). Three main models are used to explain hybrid vigour. These are the dominance model, the overdominance model, and the pseudo-overdominance model (Birchler et al., 2006). Of these, the first is still the most commonly accepted. The dominance model hypothesizes that in F1 hybrids, superior alleles at each locus will complement inferior ones, resulting in a phenotype that exhibits generally superior traits (Birchler et al., 2006). Nevertheless, in inbred crops like rice, superior alleles related to yield are likely present even in the inbred parents. Jones (1926) first reported heterosis of rice hybrids over their parental lines; however, it took more than 50 years for an agricultural application for heterosis to be developed in rice. In 1975, a team of Chinese scientists discovered the first wild rice plant with abortive pollen. This was to become the first example in rice of Cytoplasmic Male Sterility (CMS) which served as a basis for developing the first three-line hybrid rice variety and allowed China to trigger the commercial production of hybrid rice (Virmani, 1994). Since then, hybrid rice technology has
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been exploiting heterosis to increase rice production. This is because hybrid rice is reported to have a 15-20% yield advantage over the most popular inbred varieties (Xangsayasane et al., 2010). By 1990, 50% of the national harvested rice area in China (ca. 15 millions ha) was planted with hybrid varieties (Yuan, 2003). In the same year, during a session of the International Rice Commission, the Food and Agriculture Organization (FAO) and its member countries were recommended to promote the spread and development of hybrid technology for increased food security. In 1996, together with the International Rice Research Institute (IRRI) and other organizations, FAO created the International Task Force of Hybrid Rice (INTAFOHR) and since then, a programme entitled “Sustaining Food Security in Asia through the Development of Hybrid Rice Technology” has been promoting the cultivation of hybrid rice throughout Asia. By 2004, hybrid rice was commercially cultivated on about 1.5 million ha in seven Asian countries outside China (Bangladesh, India, Indonesia, Myanmar, the Philippines, Sri Lanka, and Vietnam). With the support of the FAO, hybrid rice areas in Sri Lanka and Indonesia have increased from 10,000 ha in 2006 to 80,000 ha in 2008 (Nguyen, 2010). Hybrid rice production has, however, faced a number of difficulties in SouthEast Asia. Among these are reports concerning increases in the frequency and severity of insect pest outbreaks on hybrid compared to inbred rice (Chen et al., 2005; Cheng, 2009). This has put into question the sustainability of hybrid rice production, particularly if, as has been suggested, hybrids are generally susceptible or hyper-susceptible to insect attack (Mew et al., 1988; Sogawa, 1991). Huang et al. (1985) and Tan (1987) found either enhanced development or higher survival of hemipteran and lepidopteran pests on hybrid over inbred varieties. Moreover,
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Sogawa et al. (2003) indicated that Chinese hybrid rice is highly susceptible to two planthoppers, the brown planthopper (BPH), Nilaparvata lugens (Stål), and whitebacked planthopper (WBPH), Sogatella furcifera (Horvath), and three pyralid moths, including the yellow stemborer (YSB), Scirpophaga incertulas (Walker). Sogawa et al. (2003) suggest that Chinese hybrid rice lacks any resistance to these insects. It is now generally accepted among rice entomologists that hybrid susceptibility to insect herbivores is due to several factors including the narrow genetic background of hybrids derived from breeding programmes that use a restricted pool of CMS (predominantly Minghui 63, susceptible to BPH and WBPH) and restorer lines (mainly IR36 and IR64, both susceptible to WBPH) (Fang et al., 2004; Mao et al., 2006; Cheng, 2009; Peng et al., 2009; Sogawa et al., 2009). Resistance to planthoppers in inbred rice varieties has received considerable research attention over the past 50 years and currently a large number of major resistance genes have been identified from Oryza sativa and a range of wild rice species (Brar et al., 2009). Despite the large number of available genes, many of which have associated PCR-based markers that can aid in marker-assisted selection, the underlying mechanisms of resistance are poorly understood, and few genes have been deliberately transferred to elite lines through rice breeding programmes (Horgan, 2009). Only four resistance genes (Bph1, bph2, Bph3, and bph4) are known to have been consciously bred into commercial rice varieties, and planthoppers have adapted to overcome these genes in most of Asia (Jena & Kim, 2010). Recently, the Bph14 and Bph15 genes (synonymous with Wbph7 and Wbph8) have been successfully transferred to Chinese hybrid rice varieties that are currently grown in China and Vietnam (Jena & Kim, 2010). However, the vast majority of hybrid rice
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varieties still do not possess major planthopper resistance genes. According to the Plant Vigour Hypothesis (PVH), which holds that vigorously growing plants, or large plants, are more favourable for certain kinds of herbivores (Price 1991), rice varieties without major resistance genes may be expected to be more susceptible than inbred varieties. In the case of the YSB, inbred breeding lines exhibiting moderate levels of resistance have been identified through conventional breeding methods, but without the high levels of antibiosis-based resistance that were achieved with homopteran pests such as planthoppers (Heinrichs, 1986). Bhattacharya et al. (2006) pointed out that a good level of resistance against YSB has been generally rare in rice germplasm, and Alinia et al. (1999) and Khanna & Raina (2002) indicated that this has limited the use of conventional breeding for insect resistance against stemborers. Hybrid varieties may be more susceptible to YSB and other stemborers because of their faster growth rates and a higher biomass accumulation that results in relatively large plants with thick stems, which are suitable for stemborer larvae (Chieng, 1985). Because most hybrid and inbred varieties are susceptible to YSB, moth control has been mainly achieved using chemical insecticides (Bandong & Litsinger, 2005). Research on pest tolerance as an additional defence strategy (which may or may not be linked to pest resistance) did not gain much attention until the early 1990´s, when it became a prevalent concept among plant evolutionary ecologists (Baucom & De Roode, 2011). Although there have been substantial controversies about its terminology and evaluation, tolerance is generally defined as a plant‟s ability to maintain fitness despite herbivore damage (Weis et al., 2000). Although
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both resistance and tolerance have the same outcome (i.e. they preserve the plant‟s fitness), they may have entirely different consequences for the ecology and evolution of hosts and pests: while resistance has negative effects on insect fitness, tolerance does not. Therefore, tolerance allows pest populations to grow (Baucom & de Roode, 2011). In contrast to monogenic host resistance, tolerance is more durable because it does not impose selection pressure on herbivores (Rosenthal & Kotanen, 1994). Several examples in the literature have documented examples of rice tolerance to BPH (Ho et al., 1982; Panda & Heinrichs, 1983; Jung-Tsung et al., 1986) and WBPH damage (Yu et al., 1990; Nalini & Gunathilagaraj, 1994). All of these published examples are from inbred varieties such as Triveni and Utri Rajapan and the mechanisms of tolerance in rice to planthoppers have not been further elucidated. Furthermore, a number of authors have documented tolerance of inbred or hybrid varieties to stemborers and other lepidopteran herbivores (Rubia et al., 1989, 1996a; Islam & Karim, 1997); but, to my knowledge, a systematic comparison of tolerance levels in hybrid and inbred varieties has not been carried out. Tolerance has been frequently associated with high plant vigour; for example, plants grown under high nutrient and light conditions often continue to grow rapidly despite heavy attacks by insect herbivores (Coley, 1983; Coley et al., 1985; Coley & Aide, 1990). Observations such as these support the Compensatory Continuum Hypothesis (CCH) which holds that a plant‟s tolerance to herbivory should be greater in high-resource, low-competition, or otherwise benign environments (Masckinski & Whitham, 1989). Based on these ideas, hybrid vigour could be expected to contribute to a relatively high herbivory tolerance in hybrid
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compared to inbred rice varieties. For example, Sogawa et al. (2003) suggested that the vigorous growth of rice hybrids might be a factor that enhances the fecundity of planthoppers. However, studies showing relatively enhanced plant growth in hybrids following planthopper attack are rare. Because tolerance has been related to growth rates, plant tolerance can furthermore be expected to change in response to different environmental resource levels. Although there is no consensus as to whether higher resource levels lead to greater or lower tolerance (Wise & Abrahamson, 2007), empirical evidence often supports the CCH. This hypothesis has not been tested among hybrid and inbred rice varieties in spite of the interest generated by reports of hybrid hyper-susceptibility to herbivores and the comparative disadvantages that hybrids are suggested to have. Having highlighted the importance of rice hybrids to modern agriculture and their suggested field susceptibility, and with the general aim of understanding the principles of tolerance and its potential relation to plant physiology and the environment, the present study was initiated with the following objectives: i)
to examine the relative responses of three of the principal insect pests of rice in Asia when feeding on hybrid rice varieties and their inbred parental lines;
ii)
to evaluate levels of plant tolerance against BPH, WBPH and YSB damage in a set of IRRI rice hybrids and their inbred parental lines; and
iii)
to examine the effects of resource availability on the dynamics of herbivorerice interactions for hybrid and inbred rice varieties. In contrast to previous studies of hybrid and inbred rice that have compared
unrelated hybrid and inbred lines (Cheng et al., 2007; Bueno & Lafarge, 2009; Xangsayasane et al., 2010), the present study, to facilitate comparisons, examines
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genetically related lines from three-line hybrids, including rice parental lines available at IRRI to compare the effects of plant type (hybrid or inbred) on insectrice interactions. The hypothesis, in consonance with both the CCH and the PVH, was that hybrids, because of their higher biomass and growth rates (Bueno & Lafarge, 2009) would support higher insect populations, but would also have a higher tolerance to insect damage as compared to inbred lines, and that both parameters (insect fitness and plant tolerance) would increase under high nutrient conditions, especially among hybrid varieties.
2.2. Materials and Methods 2.2.1. Plant materials Seven hybrid varieties and their parental lines were selected from a three-line hybrid rice breeding programme at the International Rice Research Institute (IRRI) in Laguna, The Philippines. Lines were selected to avoid repetition of parental lines. Only one of the R-lines (IR60819-34-2R) was repeated (Table 2.1); however, the repeated line was from a distinct batch and breeding programme and showed slight differences in growth and survival traits (data not shown). The parental lines in a three-line system are the Cytoplasmic Male Sterile, or CMS (A line), the maintainer (B line), and the restorer line (R line). Hybrid seed is produced by crossing the A and R lines. The A line cannot self-fertilise so any seed produced on A plants must be F1 by cross pollination with the R line. The B line is used to pollinate the A line to maintain a supply of A line seed. A and B lines are alloplasmic, having the same nuclear genome but differing in their cytoplasmic genomes (Virmani, 1994).
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Table 2.1: The seven three-line hybrid sets from the IRRI hybrid rice breeding programme that were used in the present study. Hybrids are indicated with their respective parental lines
Hybrid Restorer (R line) Maintainer (B line) CMS (A line) IR 82396 H IR46 R IR 80156 B IR 80156 A IR 82391 H IR60819-34-2 R* IR 79156 B IR 79156 A IR 84714 H IR60819-34-2 R* IR 80559 B IR 80559 A IR 81954 H IR72889-46-3-2-1 R IR 70369 B IR 70369 A IR 80637 H IR73013-95-1-3-2 R IR 73328 B IR 73328 A IR 82385 H IR73717-46-1-3-3 R IR 79125 B IR 79125 A IR 82363 H SRT 3 R IR 68897 B IR 68897 A Each three-line set (hybrids with their corresponding parental-lines) is considered as a 3-line block. Note: * repeated restorers (R lines); see text.
Two experiments were carried out using these 28 lines; the first, a greenhouse experiment conducted during the wet season (WS) of 2009, and the second a field experiment during the WS of 2010.
2.2.2. The herbivores Brown and white-backed planthoppers used here were derived from the „Laguna‟ colonies reared at IRRI. Laguna is a province in southern Luzon Island. The BPH colony was initiated with >500 adults collected from rice paddies in Laguna during 2004. The WBPH colony was initiated with hoppers collected in IRRI fields during 2009. Both colonies were maintained in wire mesh cages of 120×60×60 cm (H×W×L) under shaded greenhouse conditions (temperatures ranged from 25 to 45°C) with periodic introgression of wild-caught individuals. The colonies were maintained on Taichung Native 1 (TN1), which is a highly susceptible rice variety. Feeding plants were generally >30 days old and were changed every three days. Yellow stemborer adults were collected from several farmer fields in Laguna. These were brought to the greenhouse and confined on TN1 until egg masses were
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laid (three days). Eggs were monitored until neonates emerged, and plants were infested with larvae for up to six hours after emergence, but usually < 1 hour.
2.2.3. Experiments 2.2.3.1. Field experiment During the 2010 WS, seeds were planted at the IRRI experimental farm. For the field experiment, plants from each accession (Table 2.1) were seeded on 16 June in saturated garden soil in the greenhouse and transplanted, 21 days later, individually to plots of 100 plants (10×10 plants) with a plant spacing of 25×25 cm in a replicated randomized block design with five replicate blocks, each block containing the complete set of accessions (a total of 2800 plants per block). Plants received N-P-K fertiliser (100-40-2) equivalent to 150 kg of ammonium sulphate per ha. Fertiliser was applied at three different stages during crop growth: one third on the day prior to transplanting, one third during tillering, and one third at panicle initiation. Each accession was monitored for biomass accumulation and yield by destructively sampling plants at two-week intervals during the first ten weeks after transplanting.Samples consisted of a single healthy plant per block (avoiding obviously damaged plants, and plants which were next to a gap produced during previous sampling) which was arbitrarily selected and cut at the base. The samples were placed in plastic sleeve bags and returned to the laboratory where they were washed and then divided into vegetative parts (leaves and tillers) and grains (filled and unfilled). Each of these parts was placed in a paper bag and dried at 60ºC until a constant weight (generally 3 days) was obtained. Plant height and number of tillers
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(recorded as alive or dead) were also recorded. Chlorophyll content was measured in SPAD units, using a SPAD meter (Minolta Camera Company). The final sample for each accession was taken when ca. 85% of the total of panicles in each plot were mature and grain filling appeared complete. Individual plant yield (dry biomass of filled grain from a single arbitrarily selected plant) was recorded. All plant parts were weighed on a precision balance with 0.1 mg sensitivity immediately after they were oven-dried.
2.2.3.2. Greenhouse experiment Plants were grown under greenhouse conditions in the WS of 2009. Greenhouse temperatures ranged from 25 to 40ºC during the course of the experiment. Plants were seeded on 7 August in saturated, homogenized garden soil in the greenhouse, and transplanted to size-6 pots (15.0×9.5×15.5 cm [rim diameter × height × base diameter]) one seedling per pot, at between ten and 11 days after sowing. Plants were grown under two nitrogen regimes: N1 (no nitrogen added), and N2 (the equivalent of 150 kg of ammonium sulphate per hectare extrapolated to each pot volume). For the N2 regime, two-thirds of the total amount of fertiliser was applied one day before transplanting, and one-third at 2 to 3 days before insects were released. Pots were maintained in water-filled trays to prevent ants and rodents from reaching the plants, to maintain air humidity and to cool the pots. In order to avoid fertiliser losses to water in the surrounding trays, pots were sealed (i.e. no holes at the pot base). The rice plants were watered daily to avoid drought stress. Plants received no insecticide or fungicide treatments during the experiments.
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Thirty days after sowing, each potted test plant was enclosed with a cylindrical plastic cage (122.5 × 11.5 cm; length × diameter) with nylon mesh side windows (23 × 14 cm; length × width) and a nylon top. Each potted plant represented a replicate, and the potted plants (CMS, maintainer, restorer-lines and hybrid(s) for each three-line hybrid group) were arranged in a replicated randomized block design with six replicate blocks. Each block was maintained in a separate greenhouse compartment and consisted of plants under eight treatments, that is BPH, WBPH- and YSB-infested plants, and uninfested controls under the N1 regime, and BPH-, WBPH- and YSB-infested plants, and uninfested controls under the N2 regime. BPH- or WBPH-infested plants were each infested with two recently emerged gravid females (BPH or WBPH, respectively) at 30 days after sowing; YSB-infested plants were infested with six recently emerged neonate stemborers at 30 days after sowing. Insects were allowed to feed on the plants for one complete generation, after which time (when F1 gravid females were observed) the plants were evaluated for damage and the insects were removed and weighed. For BPH and WBPH, nymphs that had emerged from eggs already on the plants during the first evaluation were allowed to emerge and were collected as they emerged about 7 to 10 days later. Insects, once collected, were immediately oven-dried at 55 to 60º C for 2 days, and weighed on a precision balance with 0.001mg sensitivity. After the insects were removed, the plants were returned to the greenhouse and tended until harvest. To avoid potential contamination from escaped planthoppers the plastic cages remained in place until harvest. Plants were harvested following the same standards of the final sampling in the field experiment. At harvest, plant height, number of tillers (recorded as alive or
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dead), and total number of grains (recorded as filled and unfilled) were recorded. Each harvested plant was carefully washed, and divided into roots, vegetative part (leaves and tillers), unfilled, and filled grains. Each of these parts was placed in a separate paper bag, and immediately dried in an oven (3 days at 60ºC), and weighed on a precision balance with 0.1 mg sensitivity. Plants which had died during the experiment were also carefully washed, separated into different plant parts (when possible), dried, weighed and recorded with an approximate date of death.
2.2.4. Statistical analyses Agronomic parameters from uninfested plants from the greenhouse experiment were individually analyzed using ANOVA, with plant type, nitrogen regime, and 3-line block (see Table 2.1) as independent factors; henceforth, all these factors were included in all the analyses of data from the greenhouse experiment, except in the analysis of growth parameters of the field plants (Table 2.3 Figure 2.1). Growth parameters (number of tillers and above-ground biomass) for field-grown plants during the first ten weeks after transplanting were analysed using a repeated measures GLM. For the analyses, above ground biomass and tillers included both the vegetative and reproductive parts. Yield was analyzed using ANOVA. In these analyses, the 3-line block effect was omitted because of limited membership of each block and consequent insufficient degrees of freedom. Both plant mortality during the greenhouse experiment and resistance across plant types (insect biomass) were analyzed using ANOVA, using insect species as an additional independent factor in the insect biomass analyses. Absolute dry weight components of infested plants, and partitioning of biomass were both analyzed by
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MANOVA considering root, above-ground vegetative parts and filled grain dry weight as dependent variables, and insect species as an additional independent factor. Both absolute and percentage reduction of plant dry weight components per milligram of insect were analyzed using MANCOVA, including root, above-ground vegetative parts and filled grains as dependent variables, insect species as an additional independent factor, and insect dry weight as a covariate. Residuals were plotted following each parametric analysis and were found to be homogeneous and normally distributed. Where data could not be adequately transformed to meet requirements for homogeneity and normality, the data were ranked, and nonparametric analyses performed. Transformations are indicated in the results tables; however, non-transformed means (with their corresponding SEM) are presented in all tables and figures to facilitate comprehension of the results. All the data was analyzed using SPSS version 17. Filled grain dry weights of the CMS lines were not taken into consideration during the statistical analyses of partitioning of biomass (of plants from the greenhouse experiment). Subsample effects (variability between replicate blocks) were not considered: block replicates were intended only to improve the precission of parameter estimates. Dead infested and control plants on which no insect was found during sampling were considered to have died either due to fertiliser toxicity or natural causes and therefore were not included in any statistical analysis. Plants with an insect biomass below 2.5 mg but with severe damage symptoms were not included in the analyses of both absolute and percentage reduction of dry weight components per
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milligram of insect. These plants were regarded as having suffered precocious early mortality or stress due to fertiliser toxicity or other factors. Both the CMS and maintainer lines (alloplasmic lines that have the same nuclear genotype) were included in comparative analyses. This allowed an improved understanding of the restrictions on resource allocation and consequences for tolerance.
2.3. Results 2.3.1. Agronomic traits 2.3.1.1. Greenhouse studies Uninfested hybrid plants had similar above-ground vegetative and root biomass as the restorer lines, while the maintainer lines had lower values for the same parameters (Table 2.2). The above-ground vegetative biomass of CMS lines was significantly higher than that of maintainers or restorer lines, and was often higher than that of hybrids. Plant height, total tiller number, and 1000-filled grain weight did not differ significantly between plant types. Hybrid lines produced significantly higher numbers of unfilled grains and significantly lower numbers of filled grains when compared with either restorer or maintainer lines. All plant types responded significantly and similarly to the addition of nitrogen, increasing values of all recorded parameters, except „1000-grain weight‟.
2.3.1.2 Agronomic studies (field) Plant biomass increased linearly over the first ten weeks of the sampling period and was higher for hybrid varieties than inbreds throughout sampling (Figure
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2.1). There was a significant interaction term because of a slowing in growth rate of the CMS- and restorer lines at the outside of plant maturity (formation of reproductive structures)(Table 2.3). All plant types showed a slowing of growth between the third and fourth sampling date at about the time of booting (Figure 2.1). Tiller number changed over the sampling period in a non-linear fashion; however, there was no plant type effect on tiller number during the sampling period and no interaction between the factors (Table 2.3). Similar tiller numbers at each growth stage indicates that the higher biomass of the hybrids was mainly due to larger tillers. The time of grain maturity and harvest varied between lines, in general the maintainer lines and hybrids reached harvest earlier; however harvest time for the CMS lines was difficult to determine because of grain infertility. Nevertheless, four of seven restorer lines had a late harvest date during which time they continued to fill grains. The highest-yielding hybrid (IR82385) also continued to gain panicle weight before its late harvest (Figure 2.1). At the same time the sterile CMS lines underwent profuse tillering (Figure 2.1). Though hybrids produced heavier panicles and a higher yield, this was not significantly different from the yield estimates for the fertile inbred lines (Figure 2.1).
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Table 2.2: Effects of growth conditions on plant fitness parameters per plant (mean ± SEM) including F-values. Parameters are from uninfested plants from an experiment carried out in the IRRI greenhouse during the WS of 2009. Dry weight (g) 1000 filled Above-ground Plant type N (kg/ha) Df Plant height (cm) Total tillers Vegetative Roots Unfilled grains Filled grains grains weight (g) vegetative Hybrid 0 98.30 ± 2.54 2.48 ± 0.11 6.96 ± 0.38bc 5.12 ± 0.42b 0.47 ± 0.03b 0.35 ± 0.04b 1.37 ± 0.15a 23.24 ± 0.31 150 110.3 ± 1.23 3.97 ± 0.24 11.7 ± 0.43 8.46 ± 0.66 0.71 ± 0.06 0.65 ± 0.06 2.34 ± 0.30 22.99 ± 0.47 Restorer (R) 0 96.46 ± 1.69 2.55 ± 0.17 7.24 ± 0.35b 5.11 ± 0.29b 0.55 ± 0.02b 0.16 ± 0.02a 1.87 ± 0.13b 23.26 ± 0.74 150 102.65 ± 1.63 3.98 ± 0.16 10.7 ± 0.41 7.52 ± 0.32 0.67 ± 0.05 0.29 ± 0.05 2.73 ± 0.24 22.72 ± 0.89 Maintainer (B) 0 99.98 ± 3.68 2.55 ± 0.12 6.11 ± 0.38a 4.06 ± 0.32a 0.43 ± 0.04a 0.19 ± 0.05a 1.74 ± 0.05b 22.43 ± 1.09 150 105.73 ± 4.06 3.78 ± 0.24 9.35 ± 0.53 5.94 ± 0.51 0.49 ± 0.05 0.31 ± 0.07 2.90 ± 0.17 20.96 ± 0.56 CMS (A) 0 97.02 ± 3.93 2.33 ± 0.19 8.38 ± 0.40c 7.29 ± 0.42c 0.54 ± 0.03b 150 106.10 ± 3.73 3.87 ± 0.19 12.01 ± 0.51 10.43 ± 0.50 0.73 ± 0.08 Plant type1 3 1.313ns 0.348ns 13.134*** 29.550*** 6.207*** 16.848*** 9.169*** 3.197ns Nitrogen regime (N) 1 21.841*** 110.407*** 172.033*** 85.180*** 22.239*** 17.486*** 94.140*** 1.953ns 3-line block 6 4.648*** 0.372ns 1.959ns 2.410* 1.551ns 1.253ns 7.152*** 2.243ns Plant type × N 3 0.685ns 0.325ns 1.137ns 0.563ns 1.460ns 0.30ns 0.911ns 0.434ns 1 Df error 42 42 42 42 42 30 30 30 Transformation None log log log log rank rank log
* = (P≤ 0.05); ** (P≤ 0.01); *** = (P≤ 0.001 Note: Within columns, and among plant types, two values (included on the first row corresponding to each plant type) followed by the same letter are not significantly different at the 5% level using the Tukey Test. 1: Filled and unfilled grains dry weights and 1000-filled grain weights of CMS (A) lines were not taken in consideration in the statistical analyses.
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Table 2.3: Plant growth parameters (in bold letters) with F-values after repeated measure GLM for hybrid and inbred plants during the first ten weeks after transplanting. Data are from the field experiment carried out in the WS of 2010 Sources of variation Wilk’s λ1 Df F-values Plant dry weight2 Sampling period (SP) 1239.824***(4,21) SP × Plant type 4.304***(12,55.852) Plant type 3 10.492*** Total tiller number Sampling period (SP) 73.575***(4,21) 1.704ns(12,55.852) SP × Plant type Plant type 3 0.339ns Error df 24 Transformation none *** = (P≤ 0.001) 1: Wilks' λ is a test statistic used in multivariate analysis of variance (MANOVA) to test whether there are differences between the means of identified groups of subjects on a combination of dependent variables. Wilks' λ performs, in the multivariate setting, with a combination of dependent variables, the same role as the F-test performs in one-way analysis of variance. 2: Plant dry weight includes above-ground vegetative and reproductive (both filled and unfilled grains) dry weights.
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Fig. 2.1: Mean (± SEM) yield (i) (filled grains dry weight) (ii) biomass accumulation (vegetative dry weight, (iii) reproductive dry weight, and (iv) number of tillers of hybrid and inbred varieties during the 2010 WS field experiment. Values were obtained from healthy non-damaged plants. Solid circles are hybrids, open circles are restorers, solid triangles are maintainers and open triangles are CMS (A) lines. Reproductive dry weight value refers to the added dry weight of both filled and unfilled grains.
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2.3.2. Plant resistance to and tolerance of insect attack in the greenhouse study 2.3.2.1. Plant mortality Plant mortality during the greenhouse experiment was generally high among infested plants, indicating an overall high susceptibility and low tolerance to each of the three insect herbivores among the varieties (Table 2.4, Figure 2.2). However, there was generally lower mortality of WBPH-infested plants. The highest plant mortality occurred in plants infested with BPH, although, without a significant effect of plant type, the closely related CMS and maintainer lines had the highest levels of mortality (Figure 2.2). Fertiliser did not significantly affect mortality; however, there were two significant interactions between N and the other main factors (i.e. [insect × nitrogen], and [insect × plant type × nitrogen]). The significant [insect × nitrogen] interaction occurred because plants under both the control and BPH treatments showed increased overall mortality under the N2 regime, while YSB-infested plants showed a general decrease in mortality under the same fertiliser regime (Table 2.4, Figure 2.2). Additionally, the significant effect of the [insect × plant type × nitrogen] interaction was the result of a decrease in plant mortality under WBPH treatment for both hybrid and restorer lines under the N2 regime, while more CMS and maintainer line plants died under the same regime (Table 2.4, Figure 2.2).
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Table 2.4: Factors affecting plant mortality in the greenhouse experiment
Sources of variation Treatment Plant type Nitrogen regime (N) 3-line block Plant type × N Treatment × N Treatment × plant type Treatment × plant type × N Error df Transformation
Df 3 3 1 6 3 3 9 9
F-values 107.882*** 2.573ns 0.310ns 0.630ns 1.769ns 8.121*** 0.582ns 2.290* 186 rank
* = (P≤ 0.05); *** = (P≤ 0.001)
Fig. 2.2: Mean (± SEM) mortality of hybrid, restorer (R lines), maintainer (B lines) and CMS (A lines) rice lines under four treatments (uninfested (control)) (i), brown planthopper-infested (ii), white-backed planthopper-infested (iii), and yellow stemborer-infested (iv) under low (open bars) and high (solid bars) nitrogen fertiliser regimes. The same letters in parentheses next to each treatment indicate no significant differences among treatments following Tukey‟s test results.
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2.3.2.2. Insect responses across plant types (insect biomass) Plant type did not have a significant effect on either absolute insect biomass or the biomass standardized to plant weight(Table 2.5). Generally, the high nitrogen treatment led to a significant increase in insect dry weight. There was a significant [insect × plant type] interaction for the absolute insect biomass because of higher YSB biomass compared to WBPH biomass on hybrids, but similar biomass values for these two insects on the other plant types (Table 6, Figure 3). The average dry weight of total BPH per plant and per unit plant weight was higher than for the other two insects (Table 2.5, Figure 2.3). These absolute insect biomass differences among species occurred because of the generally higher number of insects found for both BPH and WBPH, and heavier individuals of YSB.
Table 2.5: Factors affecting the absolute and relative insect biomass (dry weight) in the greenhouse experiment
Dry weight (DW) of insect (mg)/ plant Sources of variation Df F value Insect species 2 249.231*** Plant type 3 2.653ns Nitrogen regime (N) 1 95.874*** 3-line block 6 1.641ns Plant type × N 3 0.709ns Insect × N 2 2.607ns Insect × plant type 6 3.151** Insect × plant type × N 6 0.368ns Df Error 138 Transformation rank * = P≤ 0.05; **= P≤ 0.01; *** = P≤ 0.001
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DW insect (mg)/ above-ground DW plant (g) F value 176.457*** 0.936ns 4.322* 1.399ns 2.581ns 0.269ns 1.351ns 1.140ns 138 rank
Fig. 2.3: Mean (± SEM) dry weight (mg) of brown planthopper, white-backed planthopper and yellow stemborer per g plant dry weight (i, ii, iii respectively) and per plant (iv, v, vi respectively) for hybrid, restorer (R lines), maintainer (B lines) and CMS (A lines) plants under low (open bars) and high (solid bars) nitrogen fertiliser regimes. The same letters above bars indicate no significant differences between plant types, and the same letters in parentheses next to each treatment indicate no significant difference between insects following Tukey‟s test results.
2.3.2.3. Biomass partitioning in infested plants The dry weight of filled grain, above-ground plant parts and roots differed according to the species of insect that attacked the plant, the plant type, and the level
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of nitrogen applied to the plant (Table 2.6, Figure 2.4). WBPH-infested plants had the highest weight of filled grain, above-ground vegetative biomass, and root biomass (Figure 2.4). The lowest filled grain dry weights occurred on BPH-infested plants, while the lowest root dry weights were on both BPH- and YSB-infested plants. Root biomass of infested plants was similar to that of uninfested plants. However, root biomass differed between plant types: Maintainer lines showed the lowest root biomass values, as compared with the similar values that hybrid, CMS and restorer lines had (Table 2.6, Figure 2.4). The dry weights of filled grain and above-ground vegetative biomass of BPH- and YSB-infested plants were both severely reduced compared to those of uninfested plants (Figure 2.4). The restorer lines had the highest filled-grain dry weight, while the CMS lines had the highest values of above-ground dry weight. Nitrogen had an overall significant positive effect on the three dry-weight components (Table 2.6, Figure 4). Overall, significant [plant type × nitrogen] interactions resulted from large effects of nitrogen addition to hybrid and CMS lines but only minor effects on the other plant types (Table 2.6, Figure 2.4). A significant [insect × plant type] interaction occurred because of the comparatively higher filled grain dry weight attained by both WBPH-infested maintainer- and hybrid lines (Table 2.6, Figure 2.4).
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Table 2.6: Factors affecting biomass of plants in the greenhouse experiment Source of variation Wilk's λ1 Df F-values Filled grain dry Above-ground Root dry weight (mg) dry weight (mg) weight (mg) Insect species 0.433***(6,272) 2 32.777*** 59.875*** 17.594*** Plant type 0.084***(9,331.139) 3 137.352*** 89.187*** 23.964*** Nitrogen regime (N) 0.403***(3,136) 1 40.718*** 201.512*** 63.395*** 3-line block 0.747***(18,385.151) 6 2.258* 1.838* 3.988*** Insect × N 0.970ns (6,272) 2 0.768ns 0.509ns 0.306ns Plant type × N 0.774***(9,331.139) 3 5.695*** 6.624*** 2.485ns Insect × plant type 0.703***(18,385.151) 6 5.409*** 2.160* 1.390ns Insect × plant type × N 0.892ns(18,385.151) 6 0.185ns 1.960ns 0.722ns Error df 138 138 138 Transformation rank rank rank * = (P≤ 0.05); ** (P≤ 0.01); *** = (P≤ 0.001) 1: Wilks' λ is a test statistic used in multivariate analysis of variance (MANOVA) to test whether there are differences between the means of identified groups of subjects on a combination of dependent variables. Wilks' λ performs, in the multivariate setting, with a combination of dependent variables, the same role as the F-test performs in one-way analysis of variance.
Fig. 2.4: Mean (± SEM) dry weight of filled grains, above-ground plant parts and roots of hybrid, restorer (R lines), maintainer (B lines) and CMS (A lines) plants (including dead plants) infested with brown planthopper (i, iv, vii), white-backed planthopper (ii, v, viii), and yellow stemborer (iii, vi, ix), under low (open bars) and high (solid bars) nitrogen fertiliser regimes. Data from uninfested plants are presented (x, xi, xii) for visual comparison (see text). The same letters within a row above bars indicate no significant difference between groups for plant parts, and same letters in parenthesis next to each treatment indicate no significant difference between insect treatments following Tukey‟s test results. Note that the CMS (A) line is sterile and produces no or few filled grains.
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2.3.2.4. Partitioning of biomass in experimental plants Figure 2.5 indicates the relative proportions of biomass represented by each of the three main component parts (filled grain, above-ground vegetative, and root biomass). The relative proportions of biomass among the three components were significantly affected by the insect species that attacked the plants, by plant type and by the nitrogen regime (Table 2.7). Furthermore, most two-way interactions were significant (Table 2.7). Plants were larger when grown under high nitrogen conditions regardless of whether they were attacked by insects or not, and regardless of insect species (Figure 2.5). However, feeding by YSB resulted in significantly higher, and feeding by BPH significantly lower filled grain dry weight compared to the control or WBPH-infested plants. There was a significant shift from aboveground vegetative biomass to grain in YSB-infested plants and a reduction in grain weight in BPH-infested plants with higher above-ground vegetative and root biomass compared to uninfested control plants and either YSB- or WBPH-infested plants (Table 2.7, Figure 2.5). Overall, insect attack led to a general increase in partitioning to root biomass and a general decrease in partitioning to above-ground biomass. There was also an increase in partitioning to grain biomass in YSB-infested plants, a decrease in BPH-infested plants, and no change in WBPH-infested plants compared to the controls (Table 2.7, Figure 2.5).
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Table 2.7: Factors affecting the partitioning of biomass in plants from the greenhouse experiment Source of variation Df F-values Wilk’s λ1 Filled grain Above-ground Roots biomass as biomass as as %-age of %-age of %-age of total total total Treatment (insects and control) 0.153***(9,447.958) 3 40.985*** 33.662*** 152.357*** Plant type 0.209***(9,447.958) 3 193.355*** 175.266*** 4.141** Nitrogen regime(N) 0.870***(3,184) 1 1.468ns 6.829** 15.838*** 3-line block 0.829**(18,520.916) 6 3.168** 1.428ns 3.233** Treatment × N 0.879**(9,447.958) 3 1.210ns 0.733ns 5.158** Plant type × N 0.965ns(9,447.958) 3 0.562ns 0.833ns 0.468ns Treatment × plant type 0.636***(27,538.017) 9 6.436*** 5.283*** 1.290ns Treatment × plant type × N 0.897ns(27,538.017) 9 0.657ns 0.872ns 0.553ns Df error 186 186 186 Transformation rank rank rank ns = (P≥ 0.05); * = (P≤ 0.05); ** (P≤ 0.01); *** = (P≤ 0.001) 1: Wilks' λ is a statistic test used in multivariate analysis of variance (MANOVA) to test whether there are differences between the means of identified groups of subjects on a combination of dependent variables. Wilks' λ performs, in the multivariate setting, with a combination of dependent variables, the same role as the F-test performs in one-way analysis of variance.
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Fig. 2.5: Mean biomass of filled grains (striped bars), above-ground biomass (open) and roots (solid) dry weight as a percentage of the total biomass in four plant types (H = hybrid, R = restorer, B = maintainer, and A = CMS) under low and high nitrogen fertiliser regimes (N1 and N2 in brackets, respectively) for four treatments - brown planthopper-infested (i), white-backed-infested (iii), yellow stemborer-infested (v), and uninfested plants (vii). Total biomass (± SEM) of each plant type under each nitrogen regime, for each treatment (ii, iv, vi, viii, respectively) is shown at the end. The same letters above the dotted lines indicate homogeneous groups for plant parts, and bold letters in parenthesis next to each treatment indicate homogeneous groups between insect treatments for each biomass part (filled grains, above-ground vegetative, roots respectively) following Tukey‟s test results.
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Nitrogen affected the three biomass components differently. It generally increased the proportion of above-ground biomass, while it tended to decrease the root biomass proportion, and had no significant effect on the filled grain biomass proportion (Table 2.7, Figure 2.5). There was a significant [insect × nitrogen] interaction, because of a greater decrease of root biomass in the N2 regime compared to the N1 regime in YSB-infested plants, but not for the other treatments (Table 8, Figure 5). There were significant [insect × plant type] interactions for both the filled grains and above-ground vegetative dry weight components because of the maintainer lines. For the filled grain biomass proportion, the maintainer lines had the highest values, except for BPH-infested plants, which had the lowest values, as compared with the restorer and hybrid lines. In the above-ground biomass proportion, the maintainer lines had the lowest values compared with both restorer and hybrid lines in all treatments, except on BPH-infested plants, which had the highest values.
2.3.2.5. Reduction of dry weight components (g) per mg of insect dry weight The species of insect feeding on the plants had a significant effect on the reduction of filled grain and root dry weights per unit insect dry weight; however, the nature of the effects differed markedly between insect species. WBPH-infested plants tended to have a smaller reduction in filled grain dry weight per mg of insect weight compared with plants infested with the other insects, while YSB-infested plants had the largest reduction of both above-ground vegetative and root dry weight per mg insect weight (Table 2.8, Figure 2.6). Generally, hybrid lines showed smaller
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reductions in filled grain dry weight per mg of insect dry weight than did the other lines, producing a significant plant type effect. Furthermore, insect attack produced significantly greater losses to both filled grain and root biomass on both BPH- and YSB-infested plants, but smaller losses when infested with WBPH. The covariate „insect dry weight‟ had a significant effect on the reduction in all three of the dry weight components. There was a significant [insect × nitrogen] interaction on the reduction of the three dry weight components because, while WBPH-infested plants tended to have minor biomass reductions under the N2 regime, the biomass of BPH- and YSB-infested plants tended to have major reductions under the same nitrogen regime(Table 2.8, Figure 2.6).
Table 2.8: Factors affecting the absolute reduction (g) of three plant parts per mg of insect
Source of variation
Wilk’s λ1
Df F-values Filled grains2
Above-ground vegetative 2.730ns 0.122ns 9.639*** 0.587ns 15.627*** 7.807*** 0.683ns 0.843ns 0.540ns 101 rank
Roots
2 6.650** Insect species 0.801***(6,198) 9.605*** 2 8.116*** Plant type 0.826**(6,198) 0.809ns Nitrogen regime (N) 0.893***(3,99) 1 6.240* 1.766ns 6 3.744*** 3-line block 0.741*(18,280.5) 0.543ns 1 5.777* Insect dry weight 0.854***(3,99) 5.471* 2 3.659* Insect × N 0.844**(6,198) 3.118* Plant type × N 0.939ns(6,198) 2 0.252ns 2.644ns Insect × plant type 0.910ns(12,262.221) 4 0.835ns 0.203ns Insect × plant type × N 0.940ns(12,262.221) 4 0.439ns 0.919ns Df error 101 101 Transformation rank rank * = (P≤ 0.05); ** (P≤ 0.01); *** = (P≤ 0.001) 1: Wilks' λ is a test statistic used in multivariate analysis of variance (MANOVA) to test whether there are differences between the means of identified groups of subjects on a combination of dependent variables. Wilks' λ performs, in the multivariate setting, with a combination of dependent variables, the same role as the F-test performs in one-way analysis of variance. 2: Filled grains dry weight of CMS (A) lines were not taken in consideration in the statistical analyses.
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Fig. 2.6: Mean (± SEM) reduction of filled grain, above-ground, and root dry weights (g) per mg dry weight of brown planthopper (i, iv, vii), white-backed planthopper (ii, v, viii), and yellow stemborer (iii, vi, ix), for hybrid, restorer (R lines), maintainer (B lines) and CMS (A lines) rice varieties growing under low (open bars) and high (solid bars) nitrogen fertiliser regimes. The CMS (A) line is sterile and produced no or few filled grains, and therefore, the results were not plotted.
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2.3.2.6. Percentage reduction of plant dry weight components per mg insect dry weight Changes in biomass (percentage reductions) following insect infestation differed between the three major biomass components according to the species and overall biomass (intensity of attack) of the insects (Table 2.9, Figure 2.7). For all components, the YSB-infested plants tended to have the largest reductions compared with plants infested with the other two insects, while the WBPH-infested plants tended to show the lowest percentage reduction of both filled grain and root dry weights (Table 2.9, Figure 2.7). The covariate „insect dry weight‟ also had a significant effect on all dry weight components. In general, hybrids tended to have smaller losses of filled grain dry weight compared to the other plant types, which explains the significant effect of plant type. Nitrogen regime had no effect on percentage reduction in plant biomass. However, there was a significant [insect × nitrogen] interaction for all three dry weight components. For percentage reduction of both filled grain and above-ground vegetative dry weight per mg insect weight, the interaction was due to smaller overall reductions under the N2 regime when plants were attacked by WBPH or YSB, but larger reductions when plants were attacked by BPH. BPH-infested plants had a greater loss of root dry weight per mg insect weight under the N2 regime; however, the opposite was found in YSB-infested plants with smaller losses under the N2 regime (Table 2.9, Figure 2.7).
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Table 2.9: Factors affecting the percentage reduction of dry weight per mg insect dry weight in the greenhouse experiment Source of variation Df F-values Wilk’s λ1
2 2 1 6 1 2 2 4 4
Filled grains 8.361*** 6.501** 1.096ns 2.748* 9.351** 4.280* 0.254ns 1.020ns 0.623ns 101 rank
Above-ground 4.772** 1.115ns 0.000ns 0.337ns 23.244*** 16.356*** 0.341ns 0.241ns 0.675ns 101 rank
Roots 8.802*** 1.673ns 0.832ns 1.100ns 5.689* 4.496* 1.022ns 0.456ns 0.434ns 101 rank
Insect species 0.787***(6,198) Plant type 0.871*(6,198) Nitrogen regime (N) 0.979ns(3,99) 3-line block 0.798ns(18,280.5) Insect dry weight 0.804***(3,99) Insect × N 0.736***(6,198) Plant type × N 0.971ns(6,198) Insect × plant type 0.918ns(12,262.221) Insect × plant type × N 0.951ns(12,262.221) Df error Transformation * = (P≤ 0.05); *** = (P≤ 0.001) 1: Wilks' λ is a test statistic used in multivariate analysis of variance (MANOVA) to test whether there are differences between the means of identified groups of subjects on a combination of dependent variables. Wilks' λ performs, in the multivariate setting, with a combination of dependent variables, the same role as the F-test performs in one-way analysis of variance. 2: Filled grains dry weight of CMS (A) lines were not taken in consideration in the statistical analyses.
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Fig. 2.7: Mean (± SEM) percentage reduction of filled grain, above-ground, and root dry weights (g) per mg dry weight of brown planthopper (i, iv, vii), white-backed planthopper (ii, v, viii), and yellow stemborer (iii, vi, ix), for hybrid, restorer (R lines), maintainer (B lines) and CMS (A lines) rice varieties growing under low (open bars) and high (solid bars) nitrogen fertiliser regimes.
2.4. Discussion Several authors have reported on the susceptibility or hyper-susceptibility of hybrid rice to planthoppers (Sogawa, 1991; Sogawa et al., 2003; Cheng, 2009) and stemborers (Chieng, 1985; Mew et al., 1988). It is difficult to experimentally compare susceptibilities among hybrid and inbred rice varieties because of limited
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access to parental lines. Nevertheless, at IRRI, access to rice hybrids and their inbred parental lines is open to the public and this facilitated our comparative study. A series of three-line hybrid sets, representing 28 distinct lines, was available for research and allowed us to make a general comparison of plant types (hybrid, fertile inbred and sterile inbred lines). In the present study, several indicators were used to examine plant tolerance (e.g. dry weight of plant yield components [Table 2.6, Figure 2.4], partitioning of biomass into individual yield components [Table 2.7, Figure 2.5], and both absolute [Table 2.8, Figure 2.6] and standardized [Table 2.9, Figure 2.7] reductions of dry weight components (g per mg insect dry weight)), plant resistance, as a measurement of the antibiosis component (e.g. insect biomass [Table 2.5, Figure 2.3]), or the effects of both components together (e.g. plant mortality [Table 2.4, Figure 2.2]). The results obtained are not in agreement with the hypothetical hybrid hypersusceptibility to insects. We did not find hybrid varieties to be inherently more susceptible to three common insect herbivores than the other three plant types. Different levels of hybrid tolerance and antibiosis, and distinct plant responses to insect attack were found when hybrid rice was attacked by different insects including overcompensation for filled grain dry weight when hybrids were infested with WBPH. Major differences were observed in the pathology of the three insect species and their effects on the host plants. Despite both planthoppers being sap-feeders and having similar life-histories, behaviours and morphologies, BPH populations grew faster than WBPH and different plant physiological responses were observed in plants infested with these two species. BPH tended to cause reductions of both filled grain and above-ground vegetative dry matter, while WBPH had severe effects on
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above-ground vegetative dry weight, but had generally little influence on grain yield. Interestingly, nitrogen produced an enormous increment in the BPH population causing high plant mortality, compared with the other two insects. In YSB-infested plants, while the damage per unit weight of insect was reflected in yield losses, fertiliser increased the tolerance of all plant types and reduced the YSB damage. Nitrogen played a key role in the insect-plant interactions and responses. High fertiliser levels tended to increase BPH, WBPH and YSB populations. Nitrogen also promoted the growth of all non-infested plants. Higher levels of tolerance against WBPH and YSB occurred under high nitrogen regimes. Therefore, our study is in agreement with the CCH for both WBPH and YSB-infested plants, but not for BPH-infested plants. In the present study, there were many examples of “hybrid vigour” or “positive heterosis” in hybrid rice. Some of these have been reported previously by other authors (Cheng et al., 2007; Bueno & Lafarge, 2009; Xangsayasane et al., 2010). In the greenhouse experiment, hybrid lines had higher root biomass and tended to have higher 1000-grain weight and plant height than the inbred lines (Table 2.2), whereas in the field experiment, hybrids had higher plant biomass, and tended to have higher panicle and filled grain dry weights(Figure 2.1, Table 2.3). Although results from greenhouse plants were obtained in artificial growth conditions, there were similarities with the field experiment foryield-related components (i.e. 1000-grain weight, panicles and filled grain dry weights). The high unfilled grain weight of hybrid lines in the greenhouse experiment was mainly due to growth-restrictions in pots (Tschaplinski & Blake, 1985).
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In this study, susceptibility of hybrids to insect attack was not higher when compared with the inbred lines. Both the absolute and proportional dry weights of BPH were found to be higher under both nitrogen regimes, as compared with the other two insects, indicating a faster population build-up of the BPH. In general, nitrogen tended to increase the planthopper biomass per unit plant dry weight. This is in agreement with results from a previous study (Lu et al., 2004). Higher BPH dry weight (although non-significantly different), along with higher above-ground dry weight, was found in the CMS lines compared with the other lines. The results partially agree with Cornelissen & Fernandes (2008) who showed that sap-suckers (Homoptera, Hemiptera) increase in abundance (up to 75%) on more vigorous plants or modules. This is presumably due to increased assimilate concentration in the phloem and has direct consequences for planthoppers on vigorously growing rice varieties. According to Cagampang et al. (1974) and Denno (1985), heavy feeding by BPH promotes proteolysis and high increment of amino acid content, which potentially improves insect fitness. Both absolute and proportional dry weights of YSB and WBPH were not significantly higher on hybrids than in the other plant types. These results do not agree with Sogawa et al. (2009), who suggested that susceptibility to planthoppers (especially to WBPH) was inherited from the Chinese hybrid variety Shanyou 63. The same author reported that during the 1990‟s, Shanyou 63 represented 40% of the total hybrid area in China. Planthopper outbreaks occurred throughout South China during the same period (Hu et al., 1992; Tang et al., 1998). The CMS lines used here were likewise decended from the WACMS from China. Therefore, the results obtained in the present study suggest that
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hybrid rice lines per se are not inherently more susceptible to planthoppers when compared to their parental inbred lines. Noted field susceptibilities of hybrid rice may therefore result more from specific hybrid-management decisions rather than inherent susceptibilities. Recent reports have indicated high insecticide resistance among planthoppers (Krishnaiah et al., 2006; Yang, 2007; Li et al., 2008). This indicates an excessive use of insecticide in Asian rice fields. Planthopper outbreaks are generally related to insecticide overuse (Gallagher et al., 1994; Heinrichs, 1994; Heong, 2009) while high fertiliser use can also be associated with increased planthopper fitness and plant damage, as shown in this study. In summary, environmental and management factors cannot be separated from variety or plant type when associating hybrid adoption with the recent increased incidences of rice pests in Asia. Regarding the susceptibility of hybrids to stemborers, contrasting results have been found. Liu (1979), Wu (1984), Chieng (1985), Tian & Li (1994), and Tu et al. (2000) have suggested a high abundance of stemborers in hybrid as compared with inbred rice varieties in the field, and Zhu et al. (2007) have related the widespread adoption of hybrid varieties with increasing stemborer densities in China. The nature of our greenhouse study meant that only the antibiosis resistance could be studied; therefore, direct comparisons with field studies cannot be made. The study found the highest stemborer biomass (insect weight/unit plant weight) occurred on hybrid lines under the lower nitrogen regime. Morphological aspects of hybrid varieties have been associated with their hypothetical stemborer susceptibility including large stem diameters and abundant foliage (Chieng, 1985; Zhu et al., 2007). Although this study did not evaluate tiller thickness, results from the field experiment showed that
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hybrids, although with no differences on tiller number compared with the other plant types, had higher plant-biomass,suggesting that stem diameter and/or leaf area were greater in hybrid lines than in inbreds. In the greenhouse experiment, the tiller biomass of hybrid and restorer lines was similar, and in CMS lines the tiller biomass was generally the highest under both nitrogen regimes (data not shown). However, due to the stress promoted by the limited pot size on tillering, the results from the field may be more relevant. The results from this study contrast with those from several authors who found higher fitness and biomass of stemborers on plants under high fertilizer regimes (Gill et al., 1993; Gines et al., 1994; Jiang & Cheng, 2003; Huang et al., 2010); however, those authors did not standardize the incidence of stemborers against plant biomass. Plant mortality was often correlated with the density of insects per plant; BPH-infested plants caused the highest mortality of plants under the high nitrogen regime, while YSB-infested plants caused lower mortality under the same regime. WBPH-infested plants showed a different pattern: while both hybrid and restorer lines had lower plant mortality under high, as compared with low nitrogen, both the CMS and maintainer lines had higher mortality under high nitrogen. Our results for BPH are in agreement with Lu et al. (2004), who found that nitrogen generally increased BPH-related plant mortality, and suggest that fertiliser favours development of this insect. In YSB-infested plants, fertiliser had a positive effect on tolerance to stemborer damage, since mortality of plants tended to decrease; similar results were found by Wale et al. (2006). Hybrid, CMS and maintainer lines showed higher mortality of uninfested plants under the N2 regime. Liao et al. (1994) and Poletto (2008) found higher plant mortality on rice treated with ammonium, as
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compared with plants treated with mixed forms of nitrogen (i.e. nitrates and ammonium). The results of the present study indicate a low level of fertiliser toxicity in the plants. Several differences were found in the reduction of plant biomass caused by the two planthopper species. Most interestingly, low levels of WBPH infestation caused some over-compensation of grain yield in hybrids, but not in the other plant types. Although Cohen et al. (2003) and Sogawa et al. (2009) obtained contrasting results regarding resistance to planthoppers, they showed clear reductions in damage ratings for hybrids compared to the most susceptible parents. This damage reduction is likely the consequence of a higher tolerance of hybrids to planthopper feeding; Differences in the interactions between BPH and WBPH and their host plants are highly apparent from the present study. Several studies indicate that planthopper assemblage structure in rice fields has altered in recent years. Rice yield losses were principally caused by BPH during the 1960s and the 1970s, while during the 1980s and 1990s there has been a shift towards WBPH-dominated assemblages (Mew et al., 1988; Thanh et al., 2007; Vien et al., 2007; Cheng, 2009). Sogawa et al. (2009) have suggested that the limited number of CMS lines used in Chinese hybrids has resulted in a predominance of varieties that share a narrow WA-CMS lineage and that these have a high susceptibility, but also a high tolerance to WBPH leading to large regional increases in WBPH densities. YSB-infested plants showed similar biomass values to BPH-infested plants in both above-ground vegetative and root dry weight. However, YSB-infested plants had a higher tolerance in terms of filled grain dry weight. But the fertiliser regime had a profound effect on the outcome of the interactions between YSB and the
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different plant types. Under both nitrogen regimes, hybrids appeared to be the most tolerant plant type as indicated by lower mortality of plants. Xu et al. (2007) examined the physiological responses to stemborer feeding in the hybrid rice varieties Shanyou 63 and Liangyoupeijiu. They indicated that higher root activity and an enhanced ability to absorb potassium were associated with higher damage compensation in both hybrid varieties when compared with the japonica inbred Xiushui 11. In the present study, the presence of hybrid lines tolerant under the N2 regime supports this hypothesis. A reported mechanism of compensation to stemborer damage is increased plant tillering (Rubia et al., 1996a); we did not observe this phenomenon, possibly due to the limited pot size used in the experiments. However, based on results from Rubia et al. (1996a) it is likely that damage might have promoted the production of new tillers, and the translocation of assimilates to these new healthy tillers, resulting in either larger panicles, or a larger number of smaller panicles and hence, higher yield tolerance compared to BPH. The two hypothetical mechanisms of tolerance described here are based on biomass allocation changes, and tend to be highly influenced by nitrogen addition. Therefore, adequate management of fertilisers, especially regarding timing, dosage, and type (organic fertilisers improved resistance against YSB [Usha Rani et al., 2009]) might be key components in protecting plants against stemborer-induced yield loss. A number of studies have shown evidence for higher tolerance to stemborers in hybrid compared to inbred rice (Luo, 1987; Gines et al., 1994; Xu et al., 2007). Luo (1987) showed higher compensation to attack by C. suppressalis (Walker), through increased head and grain weight, in the hybrid rice Wei-You 35 as compared with the inbred Xiang-Ai-Zao 10. Similarly the hybrid IR64616H showed
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significant compensation for whitehead (i.e. death of the growing panicle caused by YSB attack [Pathak, 1968]) damage compared with IR72, by producing 30-50% heavier productive panicles (Gines et al., 1994). Yield losses caused by high densities of stemborers in hybrid rice fields are often influenced by inadequate management of the varieties. Farmers are not aware of the tolerance of hybrid lines and generally do not know the capacity for compensation against “deadheart” (death of the growing shoot caused by YSB attack [Pathak, 1968]) (Rubia et al., 1989; Yambao et al., 1993); moreover, farmers often do not adhere to recommended insecticide application thresholds (Rubia et al., 1996b). In conclusion, no indicators of an inherent hybrid rice susceptibility to BPH or WBPH were found in this study. However, comparatively higher susceptibility to YSB was found on hybrid lines, when these were compared with inbred lines. Markedly different responses among the three insect species were observed. Comparatively higher densities of BPH than in the other insects developed on all infested plant types. Additionally, increments of plant damage per milligram of BPH biomass were higher when nitrogen fertiliser was applied. Overall, hybrid tolerance to the three insects was observed; however, it was not generally related to hybrid vigour. Tolerant hybrid lines re-allocated resources between root, vegetative and reproductive biomass to compensate for insect damage. Nitrogen increased tolerance in both hybrid and restorer lines to WBPH and YSB, while it decreased tolerance to BPH. Furthermore, overcompensation for grain yield occurred on WBPH-infested hybrids. Highlighting this high level of tolerance to WBPH in hybrid lines, and considering that it may also occur under field conditions, WBPH should not be a significant problem for hybrid rice. In addition, high pest densities reported recently
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in hybrid rice fields might be a consequence of a poor selection of maintainer and/or restorer lines (e.g. parental lines containing low genetic diversity, and/or a certain level of insect susceptibility) or an inadequate management of the hybrid varieties, but susceptibility is not inherent to hybrids.
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Chapter 3
Research on planthopper-rice interactions: contrasting effects of experimental conditions on hybrid and inbred rice
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3.1. Introduction Research into plant-insect interactions is often conducted in controlled environments (e.g. incubators, growth chambers, greenhouses or screenhouses). In such facilities, plants are typically grown in artificial or semi-artificial soil or nutrient media and in pots of different shapes and sizes. Researchers are faced with striking a balance between space limitations due to the normally high cost of growth facilities, and the requirement to maximize replications and increase the number of experimental factors (and treatments) to obtain the best results from their studies. These constraints may lead researchers to choose the most convenient rather than the optimum pot type, such that plant growth becomes confined by the spatial limits of the pot. Such effects can go unnoticed unless potted plants are compared with those produced under optimal, field or natural conditions (Weih & Nordh, 2005). Where plants with distinct physiologies are regarded as treatments in an experiment, then interactions between plant types and growth conditions can confound results. Pot experiments can quickly and, at a relatively low cost, generate large amounts of information about plant physiological processes; however, the utility of this information in predicting the field performance of plants is seldom considered (Weih & Nordh, 2005). Pot size and shape can affect the growth, reproduction, architecture, and allocation of resources in plants, often in an unpredictable manner. Considerable research has shown that pots can have a limiting effect on overall plant growth (Ray & Sinclair, 1998), affecting a number of physiological processes including nutrient efficiency (Huang et al., 1996) and the partitioning of photosynthates toward alternative sinks, (McConnaughay et al., 1993). The physical restriction of root growth can lead to a reduction in whole-plant growth,
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development and performance (McConnaughay & Bazzaz, 1991). Furthermore, plants may undergo water stress and premature senescence when root growth is physically restricted by small pot size for a prolonged time (Tschaplinski & Blake, 1985). The physiological effects of insect herbivory on plants have been well documented from both field (Bergelson & Crawley, 1992; Mulder & Ruess, 1998) and potted plants (Cohen et al., 2003; Suri & Singh, 2011); but discrepancies between results from the field and greenhouse (potted plants) are often reported (Service & Rose, 1985; Willis et al., 1998; Tiffin & Rausher, 1999). Normally, cages are used to contain herbivores in such studies. Cages will also impose a physiological stress on plants to a greater or lesser degree, depending on the cage construction. Cages create microclimatic conditions that directly influence insect fitness (Isichaikul et al., 1994) but microclimates might also indirectly affect insect fitness through primary effects on plant physiology. For example, Dewar (1977) observed that unnatural growth of fungi inside cages, enhanced by either high honeydew excretion and/or high relative humidity, affected both insect and plant fitness. Additionally, he highlighted the risk of disturbance to gravid females when cages are used, because the cages inhibited or delayed oviposition. Planthoppers have recently re-emerged as a considerable pest to rice production is Asia. Two planthopper species, the brown planthopper, Nilaparvata lugens (Stål) and the white-backed planthopper, Sogatella frucifera (Holváth), are particularly problematic. Of these, the recent widescale outbreaks of the latter have been associated with the increasing adoption of hybrid rice by Asian farmers,
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particularly in China (Sogawa et al., 2003; Cheng, 2009; Sogawa et al., 2009). This has raised the question of whether hybrid rice is inherently more susceptible to the white-backed planthopper than are inbreds, or whether the association between hybrids and the insect are due to other, indirect factors (Mew et al., 1988; Sogawa, 1991; Sogawa et al., 2009). Comparative studies of hybrid and inbred rice varieties (either hybrid and parental lines or physiologically similar lines) could help resolve the issue. The physiology of hybrid plants is often considerably different from that of inbred plants. In particular, hybrids have a noted greater response to nutrient inputs (Van Pham et al., 2003; Kumar & Prasad, 2004; Bueno & Lafarge, 2009; Lafarge & Bueno, 2009; Bueno et al., 2010). Comparative studies that use pots and cages to restrict insects to plants may risk imposing different levels of physiological stress on plants of different types, thereby hindering the interpretation of results and masking true differences between plant types. The present study is concerned with the precision and validity of experiments that assess the performance of planthoppers on rice varieties with different physiological characteristics. The objectives of the study were: i)
to identify parameters that are suitable for describing plant responses to
different growth conditions for both hybrid and inbred rice varieties; ii)
to examine the growth and yield responses of hybrid and inbred rice in pots
of different sizes and under different nitrogen regimes; iii)
to examine the effects of insect caging on growth and yield parameters of
hybrid and inbred rice; and iv)
to examine the effects of pot size and fertiliser regime on the growth of insect
populations as mediated through the plant.
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Throughout the study, hybrid and parental lines from a series of three-line hybrid sets were used to assess the effects of experimental conditions. The results are discussed in the light of improving methodologies for the study of insect-plant interactions and, in particular, to improve studies of planthopper-rice interactions that compare varieties with distinct growth patterns or distinct physiological responses to resource levels.
3.2. Materials and methods 3.2.1. Plant materials A set of eight rice hybrids each with their corresponding parental lines (malesterile [cytoplasmic male sterility-CMS] line, maintainer and restorer) obtained from a three-line hybrid-rice breeding programme at the International Rice Research Institute (IRRI) at Los Baños, Laguna (Philippines), were used in the experiments (Table 3.1). The hybrid seed production system is known as „three-line‟ because it requires three parental lines to produce the F1 hybrids. In the system, the inbred CMS line (A line) is multiplied by crossing the sterile plants with a fertile inbred alloplasmic maintainer line (B line) with the same nuclear genotype. The third line, the restorer (R line), is also an inbred line, but with different nuclear genotype to the CMS or maintainer line, possessing a single dominant fertility-restoring gene. When crossed with the CMS line, the restorer line restores fertility in the derived hybrid (Virmani, 1994). Seed was planted under greenhouse conditions in experiments (i.e. experiments 1, 2, and 3) conducted between 2009 and 2011. Greenhouse temperatures ranged from 25 to 40ºC during the course of the experiments.
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Greenhouse experiments (i.e. experiments 1 and 2) in 2009 and 2010 were conducted during the wet season (WS), whereas experiments (i.e. experiment 3) in 2011 were conducted during the dry season (DS). Greenhouse plants were sown in plastic pots (see 3.2.4.) using sterilized and homogenized garden soil as a growth medium (soil nutrient analysis was not conducted) and with added fertiliser. Pots were maintained in water-filled trays to prevent ants and rodents from reaching the plants, to maintain high air humidity and to cool the pots. Holes in the pot base were sealed to prevent fertiliser leaching, and plants were watered daily to avoid any drought stress. Plants received no insecticide or fungicide treatments during the experiments.
Table 3.1: The seven three-line hybrid sets from the IRRI hybrid rice breeding programme that were used in the present study. Hybrids are indicated with their respective parental lines Hybrid Restorer (R line) Maintainer (B line) CMS (A line) IR 82396 H IR46 R IR 80156 B IR 80156 A IR 82391 H IR60819-34-2 IR 79156 B IR 79156 A IR 84714 H1 IR60819-34-2 R1 IR 80559 B1 IR 80559 A1 IR 85471 H2 IR60819-34-2 R2 IR 80564 B2 IR 80564 A2 IR 81954 H IR72889-46-3-2-1 R IR 70369 B IR 70369 A IR 80637 H IR73013-95-1-3-2 R IR 73328 B IR 73328 A IR 82385 H1 IR73717-46-1-3-3 R1 IR 79125 B1 IR 79125 A1 IR 82363 H SRT 3 R IR 68897 B IR 68897 A 1: Lines not used in experiment 3 2: Lines used only in experiment 3 and to obtain the plant growth parameter
3.2.2.The herbivore Brown planthoppers were from the „Laguna‟ colony reared at IRRI. This colony was initiated with >500 adults collected from rice paddies at Laguna during 2004. The colony was maintained in wire mesh cages of 120×60×60 cm (H×W×L) under shaded greenhouse conditions (temperatures ranged from 25 to 45°C) with periodic introgression of wild-caught individuals. The colony was maintained on
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Taichung Native 1 (TN1) rice plants. Feeding plants were generally >30 days old and planthopper colonies were moved to fresh plants every 3 days.
3.2.3. Plant growth parameters Seed was planted on 7 August 2009 and transplanted to size 6 (see 3.2.4.) pots (one seedling per pot) between 10 and 11 days after sowing. Thirty days after sowing, each plant was enclosed in a cylindrical plastic insect-cage (122.5 × 11.5 cm; length × diameter) with two nylon mesh side windows (23 × 14 cm; length × width) and a nylon mesh top (henceforth, “type 1” cage). The cage protected plants from insect herbivores. Plants were grown under two fertiliser regimes: i) no fertiliser added and ii) fertiliser equivalent to 150 kg ammonium sulphate [(NH4)2 SO4]/ha extrapolated to pot volume and surface area (henceforth N1 and N2, respectively). For the N2 regime, two-thirds of the fertiliser was applied one day before transplanting, and one-third was applied 2 to 3 days before infestation. Plants of each accession were arranged in a completely randomized block design with six replicate blocks. Seeds from each accession (Table 3.1) were planted at the IRRI experimental farm on 16 June in saturated garden soil in the greenhouse and transplanted, 21 days later, individually to plots of 100 plants (10×10 plants) with a plant spacing of 25×25cm in a completely randomized block design with five blocks, each block containing the complete set of accessions. Plants received N-P-K fertiliser (100-402) until fulfilling the equivalent amount of 150 kg of ammonium sulphate [(NH 4)2 SO4]/ha, applied equally on three occassions during crop growth (one-third on the
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day prior to transplanting, one-third during tillering, and one-third at panicle initiation). Plants were destructively sampled at grain maturity. Twenty eight separate parameters (see 3.3.1.) were recorded after sampling, except for SPAD-values, which were recorded at harvest point using a SPAD meter (Minolta Camera Company). Later, plants were removed from the pots and the roots carefully washed under running water to remove adhering soil. The number of tillers (vegetative and reproductive) was recorded, root length and plant height were measured using a meter ruler, and the panicles were removed and manually threshed. The numbers of filled and unfilled grain were counted and grain was weighed both fresh and dry. Plants were divided into roots, vegetative parts (leaves and tillers), unfilled, and filled grains. Each part was placed in a separate paper bag and dried in a forced draught oven at 60°C for three days. Plant biomass was recorded using a precision balance with 0.1mg sensitivity. Field data were recorded at harvest. Plants were harvested at grain maturity (the date of which varied between accessions). A single plant, without apparent damage due to insects or disease, was collected at random from the centre of each plot. After sampling, plant height, number of tillers (alive and dead), and total number of grains (filled and unfilled) were recorded. Each plant was divided into vegetative (leaves and tillers) and reproductive (panicles) parts, and eighteen plant growth parameters (see 3.3.1.) were measured as indicated above. For field-grown plants, the plants were cut at the base during sampling, so that root characteristics were not recorded.
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3.2.4. Experiment 1: Effects of pot size and fertiliser on plant fitness Rice seed from the accessions (Table 3.1) was soaked on 8 September 2010, sown the next day, and transplanted to the pots 7 days later. Three different pot sizes were chosen based on their availability from suppliers: i) size 3: 13.0×7.5×11.5cm; ii) size 6: 15.0×9.5×15.5cm; and iii) size 10: 25.0×16.0×25.0cm (all measures indicate rim diameter × height × base diameter). All the pots had a truncated cone shape and the holes at the bottom of the pots were sealed with plastic resin to avoid leaching of fertiliser. Plants were grown under two fertiliser regimes, N1 and N2 (see above), with N2 receiving two-thirds of the amount at 7 days after sowing and onethird 37 days after sowing. Accessions belonging to the same plant type were considered as replicates with the experiment arranged as a completely randomized design. Each three-line set (hybrids with their corresponding parental-lines) is considered as a 3-line block. Plants were harvested at grain maturity and plant fitness parameters were recorded as indicated in 3.2.3. Eight parameters (plant height, total tillers, dry weight of vegetative, roots, unfilled and filled grains, 1000-grain weight and SPAD values) were recorded based on their utility in optimally describing hybrid and inbred architecture and yield (see 3.3.1.).
3.2.5. Experiment 2: Effects of insect cages on plant fitness Rice seed from the accessions (Table 3.1) was soaked on 8 September 2010, sown the next day, and transplanted 7 days later to size 6 pots. Pots were either caged or uncaged. Caged pots had the rice plants covered with type 1 insect cages as described above (3.2.4.). Plants received two nitrogen regimes (N1 and N2) as indicated above. Plants were destructively harvested at grain maturity and plant
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fitness parameters recorded as indicated above (3.2.4.). The eight parameters used in experiment 1 were also recorded for this experiment.
3.2.6. Experiment 3: Effects of pot size and fertiliser on insect and host-plant biomass Rice seed from six three-line hybrids (Table 3.1) was soaked on 2 March 2011, sown the next day, and transplanted 7 days later to size 6 or size 10 pots. Pots were caged using mylar-cages of two sizes. Type 1 cage (see 3.2.1.) was used with size 6 pots. A second cage (type 2) with dimensions 122.5 × 20.0 cm (length × diameter), with nylon mesh side windows (26 × 17 cm, length × width) was used with size 10 pots. Plants received two nitrogen regimes as indicated above (3.2.1.) with N2 receiving two-thirds of the nitrogen 7 days after sowing and one-third 3 days before infestation with brown planthoppers (32 days after sowing). Plants were infested 35 days after sowing with two gravid females per plant. A set of control plants for each of the accessions was maintained without planthoppers. The brown planthopper populations were allowed to build-up over 21 days after which the plants and insects were destructively sampled; before sampling, chlorophyll content (SPAD value) was determined. All planthoppers on each plant (no eggs were present on the plants at sampling time) were oven-dried at 60º C for 2-3 days, weighed on a precision balance with 0.001mg sensitivity, and recorded. Plants were sampled and fitness measures recorded as indicated above. Because the plants were not yet reproductive, only five parameters (plant height, number of tillers, SPAD values, and both vegetative and roots dry weights) were recorded.
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3.2.7. Data analyses In order to determine the effect of pot size, insect caging and nitrogen, ANOVAs were performed on each of the selected parameters. To determine the effect of pot size on insect fitness, ANCOVA was used with total plant dry weight as a covariate, and MANOVA was used to calculate the effects of nitrogen, pot size, the presence/absence of planthoppers, plant type, and 3-line block effects on the above-ground and root dry weights of the rice plants. Residuals were plotted following each analysis to assess their homogeneity and normality, and to determine optimal transformations. Transformations are indicated for each parameter with the results; however, untransformed means and standard errors are presented in the results tables and figures. A total of 28 fitness parameters were recorded after harvest of plants grown in the greenhouse (Table 3.2). Because of the different physiologies of the various plant types, fitness parameters need to be selected that adequately describe growth and development across plant types. Consequently, it is important to identify independent parameters which are poorly correlated across plant types, in order to avoid redundancy and to optimize sampling effort. Spearman‟s pairwise rank correlations were applied for all plant growth parameters because many of the parameters could not be adequately transformed to ensure homogeneity and normality of residuals. The frequency of significant correlations across each plant type was noted to determine redundancy among descriptive parameters. Parameters with infrequent significant correlations were noted. Key parameters were chosen based on their ability to adequately describe plant fitness for each of the four plant types under different nitrogen regimes. All analyses were conducted using SPSS Version 17.
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3.3. Results 3.3.1. Choice of plant growth parameters Eight of the recorded plant parameters obtained a high number of significant correlations with eight or more other parameters for all four plant types and at both nitrogen levels (Table 2). Of these, two parameters „dry weight of unfilled grain‟, and „vegetative dry weight‟ were chosen as adequately representing the yield/reproductive plant structures and vegetative plant structure respectively without losing power of comparison. „Root dry weight‟ was moderately correlated with other parameters for all four plant types; but was included as a key trait that indicates the plants physiological response to nutrient and other environmental stresses. „Days to harvest‟, „plant height‟, „ number of live tillers‟ and „dry weight of filled grains‟ had a low number of significant correlations with most other parameters and are therefore maintained as traits that give unique information on the physiological and architectural responses of the plants to their environment (Table 3.2). Finally the „chlorophyll content (in SPADS)‟, although not considered in detail in the present study because plants were entering pre-harvest senescence, was maintained as a further unique parameter that is directly related to the plant‟s physiological state. SPAD values were poorly correlated with other traits in the present study (Table 3.2). Within plant types, parameter values were poorly correlated for the two nitrogen regimes; however „dry weights of unfilled grains‟ at different nitrogen regimes were correlated for both CMS and maintainer lines, whereas „dry weight of filled grains‟ at different nitrogen regimes were correlated for hybrid and restorer lines (Table 3.2). Based on these analyses, we used the following eight parameters that adequately capture information on plant growth
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responses for different plant types and under different nitrogen regimes: dry weight of unfilled grain, dry weight of filled grain, vegetative dry weight, root dry weight, plant height, number of live tillers, days to harvest and SPAD-value. However, in most cases we did not evaluate the effects of experimental conditions on „days to harvest‟ or „SPAD-value‟ because harvesting was not strictly related to any measurable plant state (i.e. 80% grain filling, etc.) and was often conducted after plant senescence. SPAD-values were compared where plants were evaluated at the vegetative stage (i.e. experiment 3). Also, in experiments 1 and 2, we included a further parameter „1000-grain weight‟ as a potential source of variation underlying differences in plant yield. Of 18 parameters recorded after harvest of field-grown plants, eight parameters were each highly correlated with other parameters (≥ 8 significant correlations for all four plant types)(Table 3.3). These included „vegetative dry weight‟ and „number of live tillers‟ which were included as key parameters from the greenhouse study. „Rachis dry weight‟ and „dry weight of leaves‟ were the most frequently correlated parameters for all four plant types; however, these parameters were each highly correlated with two or more of the eight parameters chosen from the greenhouse study, such that plant growth was adequately described without including these further parameters (dry weights of rachis and leaves) (Table 3.3). Therefore, the eight parameters chosen from the greenhouse study were maintained as adequate descriptors based on their ability to concisely represent the field data. The 1000-grain weight was included for the reasons indicated above.
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Table 3.2: Incidence of significant Spearman’s correlations (P ≤ 0.05) between each of 28 measured parameters for 4 plant types (hybrid, restorer, maintainer and CMS-line). Significant correlations are indicated for potted plants grown at low (above diagonal) and high (below diagonal) nitrogen regimes in a greenhouse at IRRI during the 2009 WS. Numbers within the Table refer to the number of plant types which showed a significant correlation. Shading indicates the highest number of significant correlations (light and dark grey for 3 and 4, respectively), and bold letters are used to highlight recommended parameters Parameters maximum Number of no of significant A B C D E F G H I J K L M N O P Q R S T U V W X Y Z A A significant correlations A B correlations between N1 under N1 and N2 and N2 Days until harvest A X 0 2 0 1 1 0 2 2 1 2 2 2 1 2 3 3 1 1 1 0 1 0 1 1 1 1 1 Plant height (cm) B 1 X 0 0 0 3 0 1 0 3 0 2 0 0 0 3 3 4 2 4 4 4 0 1 3 4 3 3 1 2 Root Length (cm) C 0 0 X 1 0 0 0 2 1 0 1 1 1 1 2 0 0 1 0 1 0 0 0 0 0 0 0 0 Number of vegetative tillers D 0 1 0 X 4 2 4 3 1 1 0 1 0 0 0 1 1 0 0 0 1 0 0 0 0 1 0 0 2 Number of dead vegetative tillers E 1 1 0 4 X 1 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 2 Number of reproductive tillers F 0 0 0 0 0 X 3 4 4 0 1 1 1 0 1 0 0 0 2 0 0 1 3 1 2 3 3 1 2 1 Total number of living tillers G 0 1 0 4 4 3 X 0 0 2 0 1 0 0 0 1 1 0 0 0 1 1 0 2 1 1 2 0 2 1 Number of panicles H 2 0 0 0 0 4 1 X 4 0 4 4 4 0 4 0 1 2 2 2 1 0 3 1 2 1 2 1 5 1 Total panicle length (cm) I 2 0 0 0 0 4 3 4 X 0 4 4 4 4 4 0 0 3 2 3 4 0 4 0 0 0 1 1 8 Number of filled grains1 J 2 2 0 0 0 1 0 2 1 X 1 2 1 1 0 4 4 0 0 0 3 1 2 1 0 1 0 3 1 Number of unfilled grains K 2 1 0 0 1 3 1 4 4 2 X 4 4 4 4 1 1 4 4 4 4 4 3 1 0 3 1 1 9 2 Total number of grains L 1 3 0 1 1 3 1 3 4 1 4 X 4 4 4 1 1 4 4 4 4 4 4 0 0 3 2 1 10 3 Dry weight of unfilled grain (g) M 2 1 0 0 1 3 1 4 4 2 4 4 X 4 4 1 1 4 4 4 4 4 3 1 0 3 1 1 9 2 Dry weight of panicles (g) N 1 2 0 1 1 3 2 1 4 1 3 4 3 X 4 1 2 3 2 3 3 2 1 0 0 0 0 0 3 1 Dry weight of rachis (g) O 2 2 0 0 0 3 2 4 4 1 4 4 4 4 X 1 1 4 4 4 4 4 4 1 0 3 1 2 11 1 Fresh weigh of filled grains (g)1 P 2 2 1 1 0 1 0 2 1 3 2 1 2 1 1 X 4 0 0 0 3 2 1 0 0 1 0 3 1 Dry weight of filled grains (g)1 Q 2 3 1 1 0 1 0 2 1 3 2 1 2 1 1 3 X 0 0 0 3 2 1 0 0 1 0 3 2 Dry weight of stems (g) R 2 2 0 2 2 2 2 3 3 1 4 4 4 2 4 1 1 X 4 4 4 4 4 2 1 4 4 2 10 Dry weight of leaves (g) S 0 2 0 2 3 3 3 3 3 2 4 4 4 2 4 1 1 4 X 4 4 4 4 0 1 3 0 0 9 Total vegetative dry weight (g) T 2 2 0 2 3 3 3 3 3 1 4 4 4 2 4 1 1 4 4 X 4 4 4 2 1 4 3 2 9 1 Above-ground dry weight (g) U 0 4 0 2 2 3 3 2 4 1 4 4 4 4 4 2 2 4 4 4 X 4 4 1 1 4 1 4 13 Dry weight of roots (g) V 0 3 1 2 2 0 2 0 1 0 2 4 3 1 4 1 1 4 4 4 4 X 3 2 2 4 4 2 7 Leaf Area Index (cm2) W 2 1 1 2 2 3 3 2 2 1 3 3 3 2 3 0 0 3 4 3 4 3 X 0 0 3 0 2 2 1 Dry weight of 3cm-stem portion (g) X 0 0 0 0 1 0 0 0 0 0 1 0 1 0 0 0 0 1 1 1 1 1 1 X 4 2 4 2 1 Area of 3 cm stem (cm2) Y 0 1 1 0 1 0 0 0 1 1 1 0 1 0 1 1 1 2 2 2 2 1 2 4 X 1 2 1 1 Dr weight of main stem (g) Z 2 3 0 0 2 1 1 1 1 1 4 4 4 0 4 1 1 4 4 4 4 4 2 1 2 X 4 2 5 1 Dry weight of 1cm-stem section (g) AA 0 2 0 0 0 0 0 1 1 0 1 0 1 0 1 0 0 4 2 4 4 3 1 2 2 4 X 0 2 SPAD value AB 0 2 0 0 1 0 1 0 1 2 0 1 0 0 0 2 2 2 2 2 4 2 1 0 2 1 1 X 1
1: The CMS lines consistently produced small number of filled grains
Table 3.3: Incidence of significant Spearman’s correlations (P ≤ 0.05) between each of 18 measured parameters for 4 plant types (hybrid, restorer, maintainer and CMS-line). Numbers within the Table refer to the number of plant types which showed a significant correlation. Significant correlations are indicated for ifield plots at IRRI during the 2010 DS. Shading indicates the highest number of significant correlations (light and dark grey for 3 and 4, respectively), and bold letters are used to highlight recommended parameters. Parameter
Code
A
B
D
F
G
H
J
K
M
N
O
P
Q
R
S
T
U
AB
Total
Days to harvest
A
X
3
1
2
2
3
3
4
2
2
4
2
2
2
3
3
1
1
2
Plant height (cm)
B
X
1
1
2
0
1
3
1
1
1
1
1
3
3
3
2
0
0
Number of vegetative tillers
D
X
1
3
0
0
1
1
0
1
0
0
1
1
1
0
0
0
Number of reproductive tillers
F
X
4
4
3
4
4
4
4
3
3
3
4
4
4
0
9
Total number of living tillers
G
X
4
4
4
4
4
4
3
3
3
4
4
4
0
10
Number of panicles
H
X
3
4
4
4
4
3
3
3
4
4
4
0
9
X
2
2
3
3
4
4
3
4
3
3
0
3
X
4
3
4
2
2
3
3
3
3
0
6
X
3
4
2
2
3
3
3
3
0
5
X
4
3
3
4
4
4
4
1
8
X
3
3
4
4
4
4
0
11
X
4
3
4
3
3
0
3
X
3
4
3
3
0
3
X
4
4
4
1
5
X
4
4
1
11
X
4
1
8
X
0
8
X
0
Number of filled
grains1
J
Number of unfilled grains
K
Dry weight of unfilled grains (g) 1
M
Dry weight of panicles (g)
N
Dry weight of rachis (g)
O
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(g)1
Fresh weight of filled grains
P
Dry weight of filled grains (g)1
Q
Dry weight of stems (g)
R
Dry weight of leaves (g)
S
Vegetative dry weight (g)
T
Above-ground dry weight (g)
U
SPAD value
AB
1: CMS lines consistently produced a small number of filled grains
3.3.2. Experiment 1: Effects of pot size and fertiliser on plant fitness Pot size affected almost all recorded parameters: Fitness parameters recorded from plants grown in the largest pots were generally about two orders of magnitude greater than those grown in the next pot size (medium) (Figure 3.1, Table 3.4). The higher fertiliser regime increased tiller number, biomass of roots, above-ground vegetative plant structures, and filled grains. However there was a significant interaction between the effects of pot size and fertiliser on the total number of tillers, and the biomass of roots, above-ground vegetative biomass and unfilled grains (Table 3.4) because the effect of the fertiliser was not apparent for plants grown in small- and medium-sized pots, but only in large pots (Figure 3.1). Growth parameters were generally affected by plant type (except tiller number)(Figure 3.1, Table 3.4). There was a significant interaction between pot-size and plant type for vegetative plant structures and dry weight of unfilled grain (Figure 3.1) due to similar vegetative biomass in small and medium pots for all plant types, but higher weights for the CMS lines when grown in the large pots (Figure 3.1). There were no other significant interactions (Table 3.4). There was a significant 3-line block effect for four of the six parameters (Table 3.4).
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Table 3.4: F values from ANOVAs from experiment 1 to examine the effects of plant growth conditions on seven rice plant parameters
Sources of variation
Df2
Plant height (cm)
Total tillers
Dry weight (g) Vegetative 36.802*** 1284.265*** 3.978*** 30.156*** 0.457ns 9.011*** 2.667* 0.892ns 137 log
Root 9.456*** 361.991*** 4.743*** 3.455* 0.872ns 7.475*** 1.762ns 0.521ns 137 log
Unfilled grain 0.470ns 439.495*** 9.533*** 91.508*** 0.22ns 3.567* 2.570* 0.764ns 137 rank
Filled grain2 7.735** 527.528*** 0.877ns 4.404* 0.203ns 2.072ns 0.783ns 1.601ns 102 rank
Nitrogen regime (N) 1 1.156ns 29.613*** Pot size 2 204.163*** 765.787*** 3-line block 6 10.706*** 2.891** Plant type 3 7.394*** 2.214ns N × plant type 3 0.161ns 0.290ns N × pot size 2 0.428ns 3.644* Pot size × plant type 6 0.876ns 0.206ns N × pot size × plant type 6 0.491ns 0.661ns Error df 137 137 Transformation none log * = P≤ 0.05; ** = P≤ 0.01; *** = P≤ 0.001 2: Dry weights of filled grains and 1000 filled grains fresh weight of CMS lines were not taken in consideration in the analyses because CMS lines are sterile.
1000-grain fresh weight(g)2 0.840ns 0.058ns 12.986*** 19.333*** 0.335ns 0.227ns 0.567ns 0.555ns 100 rank
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Fig. 3.1: Effects of three pot sizes on mean (± SEM) of seven growth parameters in hybrid (i, v, ix, xiii, xvii, xxi), restorer (ii, vi, x, xiv, xviii, xxii) , maintainer (iii, vii, xi, xv, xix, xxiii) and CMS (iv, viii, x, xvi, xx) rice lines under low (open circles) and high (solid circles) nitrogen fertiliser regimes. CMS lines consistently produced a small number of filled grain.
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3.3.3. Experiment 2: Effects of insect cages on plant fitness Growing plants in an insect cage increased plant height and the dry weight of unfilled grains, but decreased tiller number, biomass of vegetative plant structures, root biomass, and dry weight of filled grains (Figure 3.2, Table 3.5). Fertiliser regime had no effect on any measured parameter (Table 3.5). Growth parameters were generally affected by plant type (except plant height and filled grains)(Figure 3.2, Table 3.5). There was a significant [cage × plant type] interaction for filled and unfilled grain because the cage had no effect on unfilled grain in the CMS lines but increased the weight of unfilled grain in the other plant types. This caused a significant reduction in the yield (dry weight of filled grains) of hybrids, which, under higher nitrogen conditions, was partially explained by smaller grains (Figure 3.2, Table 3.5).
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Table 3.5: F values from ANOVAs from experiment 2 to examine the effects of plant growth conditions on seven rice plant parameters
Sources of variation
Df
Plant height (cm) 0.440ns 60.633*** 7.397*** 1.332ns 0.401ns 0.231ns 0.812ns 0.750ns 90 none
Total tillers
Dry weight (g) Vegetative 0.595ns 4.793* 3.629*** 7.698*** 0.252ns 0.222ns 1.458ns 0.407ns 90 log
Roots 0.007ns 15.996*** 2.316* 3.969** 0.498ns 3.313ns 0.632ns 1.501ns 90 log
Nitrogen regime (N) 1 3.435ns Caging 1 10.814*** 3-line block 6 3.662*** Plant type 3 4.497*** N × plant type 3 0.214ns N × caging 1 0.175ns Caging × plant type 3 0.880ns N × caging × plant type 3 0.717ns Error df 90 Transformation log * = P≤ 0.05; ** = P≤ 0.01; *** = P≤ 0.001 2: Dry weights of filled grains and 1000 filled grains fresh weight of CMS lines were not taken in consideration in the analyses
Unfilled grains 0.096ns 52.697*** 6.316*** 51.439*** 0.315ns 0.767ns 8.111*** 0.246ns 90 log
Filled grains2 0.727ns 191.733*** 2.131ns 2.360ns 0.268ns 0.447ns 5.426** 1.116ns 84 rank
1000-grain fresh weight (g)2 2.571ns 4.851* 7.227*** 3.773* 1.134ns 0.956ns 3.588* 0.513ns 84 none
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Fig. 3.2: Effects of mylar insect caging on mean (± SEM) of seven growth parameters in hybrid (i, v, ix, xiii, xvii, xxi, xxv, xxviii ), restorer (ii, vi, x, xiv, xviii, xxii, xvi, xxix), maintainer (iii, vii, xi, xv, xix, xxiii, xxvii, xxx) and CMS ( iv, viii, x, xvi, xx, xxiv) rice lines under low (open circles) and high (solid circles) nitrogen fertiliser regimes. CMS lines consistently produced a small number of filled grain.
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3.3.4. Experiment 3: Effects of pot size and fertiliser on insect and host-plant biomass Total brown planthopper biomass per plant (BPH density) was affected by nitrogen fertiliser; plants tended to have higher BPH densities under lower nitrogen levels (Figure 3.3, Table 3.6). There was a significant covariate (total plant biomass) and 3-line block effect on BPH density. Pot size and plant type did not affect BPH density. There was a significant [nitrogen × pot size] interaction because BPHdensity was higher under the low nitrogen regime only in medium pots (Figure 3.3, Table 3.6). There was also a significant [nitrogen × pot size × plant type] interaction, because the restorer lines had higher BPH-densities under low nitrogen regimes in medium pots, but not in large pots whereas the opposite trend occurred with the maintainer lines (Figure 3.3, Table 3.6).
Table 3.6: F values from ANOVA from experiment 3 to examine the effects of plant growth conditions on brown planthopper population biomass
Source of variation
Df
Nitrogen regime (N) Pot size Plant type 3-line block Total dry weight (covariate) N × plant type Pot size × plant type N × pot size N × pot size × plant type Error df Transformation * = P≤ 0.05; *** = P≤ 0.001
1 1 3 5 1 3 3 1 3
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Planthopper density (mg/g plant-1) 4.376* 0.366ns 1.555ns 3.001* 20.767*** 0.555ns 0.984ns 4.547* 3.041* 74 log
Fig. 3.3: Effect of pot size on the mean (± SEM) biomass density of brown planthoppers reared on hybrid (i), restorer (ii), maintainer (iii), and CMS (iv) rice lines under low (open circles) and high (solid circles) nitrogen fertiliser regimes.
All measured plant parameters (plant height, number of live tillers, vegetative biomass, root biomass and SPAD-values) were significantly affected by nitrogen regime and pot size (Figure 3.4, Table 3.7). Plant type and the presence/absence of BPH had a significant effect on above-ground and on root biomass as well as SPADvalues, but did not affect plant height or tiller number (Figure 3.4, Table 3.7). There was a significant [nitrogen × plant type] interaction on tiller number, because, under low nitrogen conditions, hybrids had lower tiller numbers than did the other plant types, but at high nitrogen levels tillering was similar across all four plant types (Figure 3.4, Table 3.7). The same interaction occurred with above-ground vegetative
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Table 3.7: F values from ANCOVAs (Plant height, number of tillers and SPAD-value) and MANCOVA (vegetative and roots dry weights) from experiment 3 to examine the effects of plant growth conditions and presence/absence of brown planthopper on measured growth parameters of rice
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Sources of variation
Df
Plant height (cm)
Number of tillers
SPAD-value
Dry weight (g)
Nitrogen regime (N) Pot size Hoppers Plant type 3-line block Hoppers x plant type N × plant type Pot size × plant type N × hoppers Pot size × hoppers N × pot size N × plant type × hoppers Pot size × hoppers × plant type N × plant type × pot size N × pot size × hoppers N × Plant type × pot size × hoppers Error df Transformation
1 1 1 3 5 3 3 3 1 1 1 3 3 3 1 3
89.484*** 317.440*** 0.801ns 1.918ns 9.515*** 1.474ns 1.971ns 3.346* 0.000ns 0.306ns 0.533ns 0.546ns 0.841ns 0.418ns 0.572ns 1.237ns 155 none
307.709*** 258.816*** 0.035ns 1.208ns 7.200*** 1.079ns 3.561* 4.303** 0.691ns 2.797ns 1.490ns 0.461ns 0.548ns 1.830ns 0.332ns 1.583ns 155 log
50.385*** 184.388*** 7.271** 11.816*** 4.514*** 0.694ns 0.808ns 10.065*** 1.568ns 1.803ns 1.927ns 0.823ns 0.490ns 0.307ns 1.483ns 0.562ns 155 none
Vegetative 160.514*** 239.948*** 7.310** 4.353** 0.884ns 1.059ns 3.081* 1.466ns 0.043ns 0.368ns 1.323ns 0.488ns 0.724ns 2.308ns 1.270ns 0.182ns 155 log
Roots 31.102*** 20.518*** 29.875*** 4.509*** 0.410ns 1.188ns 1.144ns 0.167ns 1.337ns 1.723ns 0.248ns 1.079ns 1.096ns 0.843ns 0.518ns 0.748ns 155 log
Wilk’s λ2 0.446***(2,154) 0.309***(2,154) 0.821***(2,154) 0.910*(6,308) 0.964ns(10,308) 0.972ns(6,308) 0.933ns(6,308) 0.960ns(6,308) 0.986ns(2,154) 0.987ns(2,154) 0.990ns(2,154) 0.958ns(6,308) 0.978ns(6,308) 0.948ns(6,308) 0.992ns(2,154) 0.968ns(6,308)
* = P≤ 0.05; ** = P≤ 0.01; *** = P≤ 0.001 2: Wilks' λ is a test statistic used in multivariate analysis of variance (MANOVA) to test whether there are differences between the means of identified groups of subjects on a combination of dependent variables. Wilks' λ performs, in the multivariate setting, with a combination of dependent variables, the same role as the F-test performs in one-way analysis of variance.
Fig. 3.4: Effects of pot size on mean (± SEM) of five growth parameters on brown planthopperinfested (broken lines) and control (solid lines) hybrid (i, v, ix, xiii, xvii ), restorer (ii, vi, x, xiv, xviii), maintainer (iii, vii, xi, xv, xix) and CMS (iv, viii, x, xvi, xx ) rice lines. Plants were grown in mylar insect cages under low (open symbols) and high (solid symbols) nitrogen fertiliser regimes.
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biomass, because, under the high-nitrogen regime, hybrids produced a higher aboveground biomass; this was mainly because of fewer but larger tillers in hybrid lines compared with the other plant types. The [pot size × plant type] interaction was significant for plant height and number of tillers because the maintainer lines were generally taller and had higher tillering than other plant types in large pots, but not in medium pots (Figure 3.4, Table 3.7). There was also a [pot size × plant type] interaction for root biomass, because of the greater root biomass of the hybrid and CMS lines in large pots, but similar root biomass of the restorer and maintainer lines when grown in pots of either size (Figure 3.4, Table 3.7).
3.4. Discussion The widespread adoption of hybrid rice has been proposed as a means of supplying world food demands in the twenty-first century (Virmani, 1994; Peng et al., 1999; Cheng et al., 2007; Zhang, 2007; Wu, 2009). A number of comparative studies have indicated that hybrid rice varieties often attain higher yields than the best inbred varieties (Peng et al., 1999; Van Pham et al., 2003). However, there are concerns over the environmental costs of such production. This is because of perceived higher requirements of hybrid varieties for chemical fertilisers and pesticides (Kumar & Prasad, 2004; Sogawa et al., 2009). Hybrid rice varieties are more responsive to nutrient inputs than are inbred varieties (Peng et al., 2003; Kumar & Prasad, 2004; Bueno et al., 2010) and higher incidences of herbivore damage and disease in hybrid compared to inbred varieties has been reported from a number of studies (Mew et al., 1988; Sogawa et al., 2009). While increased herbivore damage could result from management decisions associated with hybrid
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rice production (e.g. higher agrochemical inputs), there is also evidence that hybrid rice may be inherently more susceptible than inbreds to certain insect herbivores, including planthoppers and stemborers (Mew et al., 1988; Sogawa et al., 2009). This susceptibility is thought to derive from the CMS line used in most hybrid rice breeding (Mew et al., 1988; Faiz et al., 2007; Sogawa et al., 2009). However, it is difficult to experimentally compare susceptibilities among hybrid and inbred rice varieties, because of limited access to parental lines, which are often regarded as trade secrets. At IRRI, access to rice hybrids and their inbred parental lines is open to the public and this facilitated our comparative study. Furthermore, a series of three-line hybrid sets, representing 28 distinct lines, was available for research and allowed us to make a more general comparison of plant types (hybrids, inbreds and sterile lines) than is normally possible (e.g. Sogawa et al., 2009). Our results indicate that careful attention should be paid to the experimental conditions under which comparisons of hybrid and inbred rice varieties are made. Several significant interactions between experimental variables and plant type were detected, which, in more limited experiments, such as comparisons of varieties that each represent a different plant type, could lead to confounding results and erroneous interpretations. Comparative and time-series studies of rice growth and yield normally record a range of plant fitness parameters. However, in most cases, these studies compare plant growth stages within a single variety, or compare varieties with similar physiologies. Often fitness parameters are chosen because they specifically address the focus of the comparative research (e.g. roots in drought studies, leaf nitrogen content in fertiliser studies etc.) or are related to economically important traits (1000-grain weight, grain yield, etc.). Where varieties with apparent physiological
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differences are compared, such as hybrid and inbred varieties, fitness parameters should be chosen which adequately describe growth and development across plant types. Therefore, independent parameters that are poorly correlated across plant types should be recorded to avoid redundancy. In the present study we identified eight parameters that are suitable for comparative studies between hybrid and inbred rice varieties. These are days to harvest, plant height, tiller number, vegetative above-ground dry weight, unfilled and filled grain dry weights, root dry weight, and SPAD values. These parameters were generally unrelated to any other measured parameter and gave unique information in support of comparisons. Of the eight chosen parameters, two were highly correlated with a number of other fitness parameters for all four plant types. However, these were maintained to optimally describe the biomass allocation to vegetative and reproductive structures across rice varieties. Because of the high costs associated with research and, in particular, data capture, we propose the above eight parameters as being suitable for comparative studies aimed at determining plant responses to herbivores in different rice varieties. In the present study, pot size was observed to affect all (except 1000- grain weight) recorded plant parameters. Most parameters followed the pattern of higher values for higher pot volumes. These results are in agreement with Ray & Sinclair (1998), Thomas & Strain (1991), and Balali et al. (2008), who observed higher yields in larger soil volumes, and Zhao et al. (2009) who indicated that the pot limited the elongation of roots and ultimately reduced the shoot dry weight of rice plants. The significant [pot size × nitrogen] interaction for above-ground vegetative, root and unfilled grain dry weights in the present study clearly indicated that the effects of fertiliser are not apparent in plants growing in small and medium pots.
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Moreover, it is important to highlight that for the experiment to determine the effects of insect cages on plant fitness, medium sized pots were used in both caged and noncaged plants, and nitrogen levels had no significant effect on any plant parameters. By restricting the soil volume available to roots, individual roots of the same plant are placed in close proximity, resulting in between-root competition for the uptake of nutrients (Fusseder & Kraus, 1986). Richards & Rowe (1977) and Hameed et al. (1987) indicated that the architecture and morphology of a confined root system results in less efficient water uptake and translocation. Restricted root systems may also differ in the production of growth hormones (Richards & Rowe, 1977; Carmi & Heuer, 1981). Additionally, the altered morphology and physiology of roots grown in confined spaces can lead to increased metabolic costs for the plant (McConnaughay & Bazzaz, 1991). Furthermore, Masle & Passioura (1987) and Masle (1992) suggested that root signals may exert strong control over stomatal behavior and plant development because of increased mechanical impedance by the soil on root elongation. Based on these facts, and taking into consideration the results from our study we encourage the use of large pot sizes in studies where inbred and hybrid rice plants need to be grown until harvest. In this study, the effects of pot shape were not examined on plant growth. All the pots used had a truncated cone shape, but they also differed in height and width, which could also have affected our results to some extent. For example, in a study by McConnaughay et al. (1993) the responses of plants of two species, Abutilon theophrasti and Setaria faberii, with different root-types to elevated CO2 differed according to pot shape but were independent of pot volume or nutrient content. Plants grown in the same volume but in short, wide pots had greater root and shoot
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biomass than plants growing in tall and thin pots. Furthermore, in a study aimed at optimizing the production of rice in greenhouses, 9 cm square pots resulted in a higher rice yield when compared with 12.5 cm diameter round pots in spite of lower volumes of soil in the latter (Eddy & Hahn, 2008). Additional sources of stress are imposed on plants when plant-insect studies are conducted in cages. Mylar cages are popular among rice entomologists because they are cheap and relatively easy to manage. Furthermore, mylar cages allow researchers to see the insects on the plant and monitor their population growth, life stages or mortality. Mylar cages are used extensively at rice research departments in universities and government institutes in India, Vietnam, Indonesia and the Philippines (Horgan, personal communication). The effect of mylar insect cages on plant growth and yield parameters of hybrid and inbred rice lines was investigated. It was seen that the insect cages consistently reduced tiller number, vegetative, root and filled grain dry weight, and increased plant height and unfilled grain dry weight; increased height and reduced tiller number are effects often associated with etiolation, a plant stress related to lack of light, on the other hand, light conditions in the greenhouse were optimal, and the experiment was carried during the DS, when solar radiation tends to be high. Therefore, some other physiological stresses may have promoted these effects. Additionally, there was a significant [cage × plant type] interaction for filled grain dry weight; the mylar cages reduced the grain yield of the hybrid varieties to a greater extent than for the inbred varieties. This additional stress, that is apparently more severe for hybrids, should be adequately considered when comparing host plant resistance and tolerance to herbivory among different plant types. Cage effects on plant architecture (e.g. plant height, tiller number, and
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dry weights) will consequently affect insect responses in bioassays and should be considered before extrapolating from greenhouse studies to field plants. We did not take into consideration the microclimatic conditions inside the plastic insect cages. Mochida (1964), Dyck et al. (1979) and Nair et al. (1980), among other authors, have indicated that temperature, light, rainfall and relative humidity greatly influence the occurrence of brown planthoppers in the field. Furthermore, Bae & Pathak (1970) showed detrimental effects of temperatures above 30º C on egg hatching, nymphal survival and adult longevity of brown planthoppers. Isichaikul & Ichikawa (1993) showed that brown planthopper nymphs living on rice plants selectively choose areas with relative humidities above 90% as their preferred microhabitat. Isichaikul et al. (1994) showed the high mortality and the inability of brown planthopper nymphs to moult in micro-environments of around 70% relative humidity. Considering these observations, it is likely that the microenvironment inside the cages was most stressful for the planthoppers during times of direct sunlight and at the highest temperatures. As plants gain biomass and volume, planthoppers might better find suitable microhabitats for their normal development. The analysis of the effects of pot size and fertiliser regime on the growth of insect populations as mediated through the plant showed that brown planthopper dry weight (mg) per gram of total plant dry weight (BPH-density) tended to be higher in medium pots as compared with large ones, and also higher in the lower nitrogen regime. Considering that, for the lines studied here, plants grown in medium pots had higher stress levels than plants grown in large pots, these results agree with the Plant Stress Hypothesis (White, 1984), which holds that stressed plants will be better
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hosts for herbivores, and generally contradicts the PVH (Price, 1991) which states that herbivores will perform better on vigorous plants. According to Larsson (1989), phloem feeders (such as planthoppers) should prefer stressed plants, in comparison with other insect feeding guilds (leaf-miners and leaf-chewers); however, some authors (Minkenberg & Ottenheim, 1990; Jauset et al., 1998; Inbar et al., 2001) have contradicted this, in favour of the PVH. Inbar et al. (2001) demonstrated a negative correlation between plant vigour and chemical defences, and Lu et al. (2004), Villareal et al. (2004)) and Lu (2007) observed higher fitness of planthoppers feeding on high-nitrogen rice plants when compared with low-nitrogen rice plants. In the present study, the maintainer and restorer lines tended to show opposite patterns; the maintainers had higher BPH-densities under the higher nitrogen regime for pots of a medium size, but restorers had higher BPH-densities under the lower nitrogen regime in the larger pots. It is interesting to highlight that the CMS lines showed a similar pattern to the alloplasmic maintainer lines in this aspect. Unlike pot size and cages, in experiment 3 nitrogen tended to affect all plant parameters, with the effect being observed in both medium and large pots. Furthermore, none of the parameters measured were significantly affected by the [pot size × nitrogen] interaction in experiment 3 (Table 3.7). This indicates that experiments involving young or immature plants harvested before the reproductive stages do not require the large pots recommended when experiments are continued to harvest stage. However, there were significant [plant type × nitrogen] interactions (for above-ground vegetative dry weight and tiller number), and [plant type × pot size] interactions (for plant height, root dry weight, and tiller number) for several parameters. The results indicate that at low nitrogen levels, hybrids in large pots
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have relatively poor tillering. Tiller number increased in hybrids under the higher nitrogen regime, to approach similar numbers as in the other plant types. However, the hybrid tillers were larger than in the other plant types as indicated by a significantly higher above-ground vegetative biomass under high nitrogen in large pots. The significant [pot size × plant type] interactions were due largely to the magnitude increase in plant height and root dry weight in the hybrid and CMS lines compared to the restorers and maintainers, and the relatively lower tillering of hybrids in the experiment, particularly under low nitrogen conditions. Heinrichs (1988) has cautioned that plant stress is promoted by many factors, including temperature, light, fertiliser, and water, and may determine insect responses. Water was a key factor of the brown planthopper response during the work carried out by Litsinger et al. (1987) who observed that brown planthopper outbreaks were rare in non-irrigated upland rice fields. Therefore, special attention should be paid to the proper irrigation of potted plants; I suggest observing the plants carefully and always keeping an adequate level of moisture in the soil, since, in general, soil dries faster in smaller pots than in large pots (especially in greenhouses) and this could lead to an additional source of stress for the plants. For example, Townend & Dickinson (1995) showed that the rate of soil drying was up to 25 times faster for plants in pots of different but small volumes (0.19, 0.55 and 1.90 dm3) in comparison with plants growing in large-volume boxes (0.25 m3). Soil conditions (structure, type, texture) will also affect rates of drying in pots. Root temperature has been shown to affect the rate of water absorption (Szaniawski & Strain, 1991), root initiation, root growth (Nambiar et al., 1979, Vapaavuori et al., 1992), shoot growth, and the nitrogen concentration of plants
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(Vapaavuori et al., 1992). Townend & Dickinson (1995) observed that temperatures in smaller pots followed air temperature closely, whilst larger pots were slightly buffered against changes in temperature. Generally, resistance and tolerance studies in the literature have targeted the above-ground plant part as the only factor affecting plant-insect interactions; however, roots are severely affected by growth conditions and these will certainly affect above-ground plant-insect responses. In conclusion, it is recommended that care should be exercised in choosing the size of pots for greenhouse experiments, particularly when rice plants of different types (e.g. genotypes, physiologies, phenologies, vigour) are to be compared. The known physiological differences between hybrids and inbreds restrict the choice of pot sizes to larger pots, particularly if experiments are to be conducted through to grain-filling and harvest. Medium pots, though not optimal, were adequate in many cases for comparative studies that ceased before the booting stage (indicative of the plant entering a reproductive phase). Hybrids, in particular, were sensitive to small pots thus confounding the results of comparative studies in greenhouses. Furthermore, for experiments that require yield data, smaller pots diminished any apparent plant response to different levels of nitrogen fertilisers. Therefore, it is suggested, where possible, that future insect studies limit the duration of experiments to avoid entering the reproductive stages of the plants, and to use large pots. Confining insects to rice plants using mylar cages will stress the plants and often reduce yield. The effects of mylar cages can be different for different plant types as indicated here for yield parameters, where hybrids produced fewer and smaller grains and fewer grain in cages than inbred rice under similar conditions.
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Chapter 4
General discussion
116
4.1. Introduction The brown planthopper (BPH) (Nilaparvata lugens [Stål]), the white-backed planthopper (WBPH) (Sogatella furcifera [Horváth]) and the yellow stemborer (YSB) (Scirpophaga incertulas [Walker]) are important rice pests in Asia (Karban & Chen, 2007). The widespread adoption of hybrid rice varieties with perceived high susceptibility to BPH, WBPH and YSB has been linked to high densities of these three herbivore species in recent years (Mew et al., 1988; Sogawa, 1991). Because WBPH outbreaks have been reported in hybrid rice fields throughout Asia and densities of WBPH are generally higher than densities of BPH on hybrid rice, hybrid rice has been referred to as “hyper-susceptible” to WBPH (Sogawa et al., 2009). However, previous studies that classified hybrids as inherently susceptible, or hypersusceptible, to insects, have had a number of weaknesses: Some previous studies compared hybrids with genetically unrelated inbred varieties (Yu et al., 1991; Liu et al., 2003). Other studies have compared data from fields of hybrid and inbred rice without controlling for farm management (Tan, 1987; Zhu et al., 2007). Some of the most cited evidence comes from studies that compared hybrids with their inbred parental lines, but did not replicate the varieties, or did not include a range of varieties, and cannot therefore make generalizations about the system (Sogawa et al., 2009). Whereas considerable attention has been focused on rice resistance against insect herbivores, few studies have examined rice tolerance. Based on the hybrid vigour (heterosis) or superior physiological features of the F1 hybrids when compared with their parental lines, this research project predicted that hybrids would express higher tolerance than inbred lines; furthermore, based on the Compensatory
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Continuum Hypothesis (CCH) (Maschinski & Whitham, 1989) it was hypothesized that, under higher fertiliser regimes, tolerance would increase. The objectives of the experiments described in the present work were to test the hypothesis that hybrid rice varieties are intrinsically more susceptible and/or tolerant to BPH, WBPH and YSB attack than are inbreds, and to examine the potential links between tolerance and hybrid vigour, by comparing several three-line F1 hybrid lines with their corresponding inbred parental (i.e. CMS, maintainer, and restorer) lines, under different fertiliser regimes. Because of low grain yields achieved during pot experiments conducted in Chapter 2, a study was conducted to examine the effects of the growth conditions (i.e. pot size, the use of mylar insectcage and fertiliser level) on plant physiology, insect fitness, and insect-plant interactions.
4.2. Are F1 hybrid rice varieties more susceptible and/or tolerant of pests than inbred rice? The hybrid lines examined in Chapter 2 were not hyper-susceptible to WBPH, nor were the hybrids significantly more susceptible to BPH, WBPH or YSB when compared with their parental lines. Despite the similar nature of BPH and WBPH, we observed different responses by these two planthoppers species to plant type and environment. BPH had higher population densities, caused greater damage per unit insect weight, and caused greater plant mortality than WBPH. Yellow stemborer caused the greatest plant damage per unit insect weight, although it caused lower plant mortality than BPH. Brown planthopper-infested plants failed to compensate for pest attack and had comparatively lower grain yields than the
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uninfested controls. On the other hand, both YSB- and WBPH-infested plants compensated for pest damage by re-allocating resources to below- and above-ground biomass in a similar manner. Whereas the above-ground vegetative biomass of hybrid varieties was, in general negatively affected by insect feeding, grain biomass tended to be comparatively less affected, probably because of the re-allocation of root biomass by infested plants. Hybrid lines tended to express higher levels of tolerance than inbreds (in terms of economic yield) through a more effective biomass re-allocation between roots, vegetative and reproductive parts. Moreover, a low level of overcompensation for grain yield was observed on WBPH-infested hybrid plants. Interestingly, a study conducted by Xu et al. (2007) indicated that stemborer-infested hybrid lines showed increased root activity and an enhanced ability to absorb nutrients. These and other tolerance mechanisms (e.g. increased tillering, or reallocation of plant assimilates [Rubia et al., 1996]) in stemborer-infested plants may explain tolerance in hybrid lines from the present work; however, further research is necessary to elucidate plant tolerance responses and to define their mechanisms. Nitrogen had a notable effect on the rice plants, the insects, and their interactions. The addition of nitrogen generally increased grain, vegetative, and root biomass of both infested and uninfested plants. It increased mortality of BPHinfested plants, while it decreased mortality of YSB-infested plants. The addition of nitrogen also increased planthopper biomass density (weight of hoppers/weight of plants) indicating that planthoppers benefitted when feeding on plants treated with additional nitrogen. Furthermore, the addition of nitrogen increased grain and aboveground vegetative biomass losses caused by BPH, while damage caused by both WBPH and YSB was reduced under added N conditions. Greater tolerance was
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attained under high nitrogen regimes as indicated by lower reductions in plant biomass per unit insect biomass. However, the effect differed according to insect species. Only the results from experiments with WBPH- and YSB- infested plants were in agreement with the CCH. Brown planthopper appears to preempt available nutrients before the plant can convert these to biomass.
4.3. Experimental design: a review of the present study, and suggestions for comparing results from greenhouse and field experiments. There has been little previous work on rice tolerance to herbivore attack.. This made it difficult to foresee potential problems during experimental design, or to define indicators or measurements of plant tolerance. For the experiments described in Chapter 2, the best indicator of plant tolerance was the percentage reduction, relative to a control, of plant biomass per mg insect biomass – which assumed a linear relationship. Furthermore, there were limitations on measuring plant resistance since, because of the confinement of the insects inside cages, antixenosis could not be examined. Therefore, the use of insect biomass per unit plant weight was an indicator of antibiotic resistance only, making it difficult to rigorously examine the PVH. In field experiments, antixenosis could have a large effect on determining planthopper densities. According to the PVH, insects would be more attracted to vigorous plants growing under high fertiliser regimes. Nevertheless, in the no-choice experiments that we carried out throughout Chapter 2, our main goal was to compare tolerance across different plant types. During the experiments in Chapter 2, plant mortality was often high, particularly among BPH-infested plants, so that a significant number of plants were
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excluded from some analyses (such as partitioning of biomass). Related to this fact, it was observed that some of the infested plants approached or reached the plant‟s carrying capacity for hopper densities. Intra-specific comparisons among insect species used to define plant tolerance or resistance indicators were not affected by this; however, insect inter-specific comparisons were biased towards insects that reached the plant‟s carrying capacity at higher densities. Future studies should avoid intraspecific competition in order to allow interspecific comparisons. Regarding the effects of growth conditions on plant physiology, potted hybrid lines produced lower-than-expected grain yields in experiments conducted in Chapter 2, having higher numbers of unfilled grains when compared with their parental lines. This did not occur during the field trials. Resources appeared to be more limited for hybrids where plants were grown in pots. The results from Chapter 3 on the effects of growth conditions on insects (BPH) and plants, revealed a number of significant interactions between experimental variables and plant types. The main finding was that the two nitrogen levels used in the experiments did not affect plants growing in medium or small pots. On the other hand, hybrid lines in particular, growing in large pots under low nitrogen regimes had poor tillering, although hybrid tillers were larger than those on the other plant types. Mylar insect cages significantly reduced tiller number, vegetative, root and filled grain dry weight, and increased plant height and unfilled grain dry weight. The reduction of filled grain in hybrid lines was more severe than in inbred lines. Rice plants growing in medium-sized pots, when sampled before reproductive stages, were significantly affected by nitrogen addition. These findings indicate that plants with different physiologies can be affected differently by growth conditions. This
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should be considered when planning experiments that compare hybrid and inbred rice varieties. Large pots are recommended in such studies.
4.4. Future research Hybrid rice has been suspected of increasing the incidence and severity of insect outbreaks in Asia (Sogawa et al., 2003). However, the results from this study indicate that hybrids are not inherently more susceptible to three of the major rice pests in Asia. Hybrids were generally more resistant than their parental lines and were also more tolerant of damage. Additionally, Chapter 3 highlighted the importance of growth conditions, and how these significantly affected different plant physiologies and produced [resource/growth condition × plant type] interactions. Therefore, how can we relate results from both greenhouse and field studies? On one hand, experiments that are carried out in the field reflect more closely the growing conditions occurring during a normal rice crop. On the other hand, the complexity of crop-environment interactions makes it difficult to define indicators of insect resistance or tolerance. Experiments carried out under artificial growth conditions obtain better indicators free of background “noise”. Optimizing growth conditions in controlled experiments will improve resistance and tolerance indicators and avoid confounding results that arise because of [resource/growth condition × plant type] interactions. In the present study, comparisons were made between F1 hybrids and their parental inbred lines. This is a rather unique situation because of the free access to IRRI‟s hybrid breeding materials. This is not possible with commercial hybrid varieties. Future comparative studies should allow for genetic distances between rice lines by addressing the genetic relatedness of the experimental populations: An
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experiment including a set of completely unrelated hybrid and inbred lines, where a phylogenetic tree is compared against insect responses to the varieties could link the genetic background of the lines with their levels of resistance and/or tolerance, and determine whether genetics or physiology underlies observed differences between insect responses to hybrid and inbred rice. Further research should promote a better understanding of mechanisms of resistance and tolerance. A strategy towards adequate deployment and management of resistant hybrid varieties must be encouraged: Resistant genes are limited, and a number of them have been already broken down in the field (Gallagher et al., 1994). In this way, hybrid tolerance may be a tool to reduce, in a sustainable manner, insect pressure in hybrid lines, extending the utility of the resistance genes. With adequate management, tolerance can help to reduce chemical inputs, and reduce rice vulnerability to pest outbreaks. Reinforcing links between research centres and extension services, offering adequate information and support to farmers, would be of great benefit for both farmers and the environment. Farmer‟s perception of the hybrids as high yielding but susceptible to insect damage is probably biased and promotes the use of pesticides. Under this scenario, and given the high frequency of resistance of many insect pests to current pesticides (Sun et al., 1996; Nagata et al., 2002), hybrid tolerance could aggravate the situation by allowing higher densities of pests to build up. Issues related to hybrid rice susceptibility and tolerance, and the interactions between the two, deserve further attention to better manage the insect pests that have now plagued Asian rice farmers for several decades.
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