Wheat Antixenosis, Antibiosis, and Tolerance to Infestation by ...

ECOLOGY AND BEHAVIOR

Wheat Antixenosis, Antibiosis, and Tolerance to Infestation by Delphacodes kuscheli (Hemiptera: Delphacidae), a Vector of “Mal de Rıó Cuarto” in Argentina M. E. BRENTASSI,1,2 C. CORRALES,3 J. W. SNAPE,4 A.F.G. DIXON,5

AND

A. M. CASTRO3,6,7

J. Econ. Entomol. 102(5): 1801Ð1807 (2009)

ABSTRACT “Mal de Rõó Cuarto,” is the most important virus disease of corn, Zea mays L., in Argentina. It is caused by the Mal de Rõó Cuarto virus (family Reoviridae, genus Fijivirus. MRCV), which is a persistent virus transmitted by Delphacodes kuscheli (Fennah 1955) (Hemiptera: Delphacidae). Because corn is not a natural host of D. kuscheli, it has little protection from this pest. In contrast, wheat, Triticum aestivum L., is one of the main hosts of this vector and a reservoir of MRCV. The aim of this work was to identify genes involved in antixenosis, antibiosis, and tolerance of infestation by D. kuscheli in wheat, which might be used to reduce the population level of this vector on corn. A set of recombinant dihaploid (RDH) lines for chromosome 6A derived from the F1 cross between ÔChinese SpringÕ (CS) ⫻ ÔChinese Spring (Synthetic 6A)Õ (S6A) substitution line, was used for mapping. The S6A parental line is resistant to the MRCV vector. Antixenosis, antibiosis, and tolerance were evaluated using conventional tests in controlled environmental conditions. Most of the RDH and S6A showed higher levels of antixenosis against D. kuscheli than the parental line CS. The RDH lines showed highly signiÞcant antibiosis in terms of the duration of Þrst, third, and Þfth nymphal instars, developmental time (days), survival and fecundity. There were highly signiÞcant differences in the tolerance to D. kuscheli based on the chlorophyll content of the Þrst and second leaves, foliar area, and aboveground fresh and dry weights. The duration of the Þfth nymphal instar and the developmental period were signiÞcantly associated with Xgwm1017 marker loci, located at 48 cM on 6AL. Another quantitative trait locus accounting for the variation in chlorophyll content of the Þrst leaf was associated with the interval between loci Xgwm459 and Xgwm334a, located in the telomeric region of the 6AS chromosome arm. The alleles with positive effects came from S6A. Antibiotic resistance of RDH could be useful for controlling the population increase of the MRCV vector on wheat, because prolonging the duration of development increases the period between two subsequent generations, so reducing the abundance of infective populations colonizing corn. KEY WORDS Delphacodes kuscheli, wheat, antibiosis, antixenosis, tolerance

The “Mal de Rõó Cuarto” (MRC) is the most important virus disease of corn, Zea mays L., in Argentina. The Mal de Rõó Cuarto virus (family Reoviridae, genus Fijivirus. MRCV) belongs to serogroup II of the genus Fijivirus (Arneodo et al. 2002) and is a persistent virus (Nome et al. 1981, Milne et al. 1983, Uyeda and Milne 1995, Diste´ fano et al. 2003). The 1 Department Entomological Sciences, Faculty of Natural Cs and Museum, UNLP, 1900-La Plata, Argentina. 2 Comisio ´ n de Investigaciones Cientõ´Þcas de Buenos Aires (CICBA), 1900-La Plata, Buenos Aires, Argentina. 3 Department Biological Sciences, Faculty Agricultural Cs., UNLP CC31, 1900-La Plata, Argentina. 4 John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom. 5 School of Biological Sciences, University of East Anglia, Norwich NR4 7TY, United Kingdom; and Institute of Systems Biology and Ecology AS CR, Na Sadkach 7, 37005 Ceske Budejovice, Czech Republic. 6 Consejo Nacional de Investigaciones Cientõ´Þcas y Tecnolo ´ gicas (CONICET), San Jose´ , Costa Rica. 7 Corresponding author, e-mail: [email protected].

worst epidemic in Argentina occurred in 1996 Ð1997, with yield losses estimated at US$120 million (Lenardon et al. 1999). The planthopper Delphacodes kuscheli (Fennah 1955) (Hemiptera: Delphacidae) is the main vector of MRCV (Remes Lenicov et al. 1985). The delphacids Delphacodes haywardi (Muir) and Toya propinqua (Fieber) also are recorded as vectors but have only a minor role in the epidemiology of the disease (Presello et al. 1997, Vela´ zquez et al. 2003). D. kuscheli is a native species widely distributed in Argentina particularly between 32⬚ and 35⬚ S (Remes Lenicov and Virla 1999). Its biology, life cycle on different hosts (Remes Lenicov et al. 1991b, Virla and Remes Lenicov 1991), feeding behavior (Brentassi 2004), reproductive behavior (Costamagna et al. 1998, Brentassi and Remes Lenicov 1999), and ecology and natural enemies (De Santis et al. 1988, Remes Lenicov et al. 1991a, Liljesthro¨ m and Virla 2004) have been studied.

0022-0493/09/1801Ð1807$04.00/0 䉷 2009 Entomological Society of America

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JOURNAL OF ECONOMIC ENTOMOLOGY

The infective populations of D. kuscheli develop on oat, Avena sativa L.; wheat, Triticum aestivum L.; barley, Hordeum vulgare L.; and several wild grasses (Remes Lenicov and Virla 1999). In contrast, maize does not host the vector, so virus transmission occurs when macropterous adults migrate from the winter hosts and feed on corn plants (Remes Lenicov et al. 1991b, Ornaghi et al. 1993). The vector of MRCV can be controlled with pesticides and manipulation of sowing dates (March et al. 1997). Most of D. kuscheli hosts are reservoirs of MRCV (Ornaghi 1993, March 2004, Laguna et al. 2002). Nevertheless, their resistance to infestation by D. kuscheli has scarcely been studied. Antixenotic and antibiotic resistance to D. kuscheli of oat cultivars, carrying resistance to greenbug, Schizaphis graminum (Rondani), has been reported previously (Costamagna et al. 2005). Wheat, which is the main host of D. kuscheli, is widely grown in the areas of Argentina where this vector is present (Costamagna et al. 1998), and this cereal is the main reservoir of MRCV (Rodrõ´guez Pardina et al. 1998, Arneodo et al. 2002). The management of wheat defenses could aid to control the intrinsic rate of population increase of the vector. Although there are no studies on the resistance to delphacids in wheat, ÔChinese Spring (Synthetic 6A)Õ (S6A) is reported to show antixenotic resistance to greenbug and Russian wheat aphid, Diuraphis noxia (Mordvilko) (Castro et al. 2005). Moreover, several genes governing tolerance to stress-induced hormones (ethylene, abscisic acid [ABA], jasmonic acid, and salicylic acid) are located on the 6A chromosome of the same mapping population (Castro et al. 2008). The aim of this work was to analyze in wheat the category of mechanisms of antixenosis, antibiosis, and tolerance of infestation by D. kuscheli in a set of double haploid recombinant lines segregating for chromosome 6A. Materials and Methods Plant Materials. A mapping population of double haploid recombinant (RDH) lines was developed for chromosome 6A from the F1 of the cross between ÔChinese SpringÕ (CS) ⫻ CS S6A by the maize cross technique (Castro et al. 2005). CS 6A is a single chromosome substitution line, in which chromosome 6A of ÔSyntheticÕ (Syn, donor parent) replaces its CS (receptor parent) homolog. The S6A parental line is resistant to the MRCV vector and CS is susceptible. The resistance against D. kuscheli of 63 of these RDH lines was tested. The RDH lines were developed at the IPK Gatersleben and John Innes Centre (BBSRC), Norwich, United Kingdom). The RDH lines, Syn, and both parents (CS and S6A) were grown in pots containing fertile soil and maintained under natural conditions until used in the trials. Insect Population. D. kuscheli populations were collected in oat Þelds in Rõó Cuarto (Co´ rdoba Province, Argentina), an area where MRC is endemic. The planthoppers were reared on ÔBordenave RanquelinaÕ

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Fig. 1. Antibiosis test. (A) Cylindrical cages where Þrst instar nymphs were conÞned and allowed to develop into adults. (B) Cage where the insect couples were isolated for mating.

barely and ÔSuregrainÕ oat seedlings, under controlled conditions (24 ⫾ 2⬚C, 60 Ð70% humidity, and a photoperiod of 16:8 [L:D] h). To avoid previous host effects the insects were transferred and maintained for two generations on each assayed genotype before starting the trials. Resistance Tests. Antixenosis was assessed by allowing D. kuscheli a free-choice among plants at a similar growth stage (second fully expanded leaf), following Castro et al. (2005). One seed of each parent (CS and S6A), of Syn, and of every recombinant line were planted singly in pots (66 pots) and randomly distributed in a circle, with the apical portion (1.5 cm in length) of the second leaf of each plant directed toward the center of the circle. A glass dish (35 cm in diameter, 10 cm in height) was inverted over the leaves. We released 330 adult planthoppers (1Ð3 d old), equivalent to Þve insects per plant (representing a natural level of infestation), with the aid of an aspirator, through a hole in the center of the bottom of the glass dish. To avoid the direction of light inßuencing plant selection by the planthoppers, the assay was carried out in the dark. The number of adults on each plant was carefully recorded 24 h later without disturbing the insects, while the plants remained in the circle. One seedling of every genotype randomly distributed represented one replicate and the assay consisted of 20 replicates. Antibiosis was assessed using seedlings (at third fully expanded leaf) grown individually in 100-ml pots. To avoid insect migration, two Þrst instar nymphs (emerged from the leaves of plants where adults were reared in the laboratory), were conÞned in cylindrical cages (7 cm in height), enclosing the ligulae zone of the third leaf on every plant (Fig. 1), and allowed to develop into adults. The nymphs were observed daily,

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BRENTASSI ET AL.: WHEAT RESISTANCE AGAINST D. kuscheli

Fig. 2. Dissected leaf where the number of eggs was recorded and counting using a stereoscopic microscope (42⫻).

and the duration of each instar (NI, NII, NIII, NIV, and NV), time from birth to the onset of reproduction (days), length of adult life (A), longevity (L), and survival (S) were recorded. S was the percentage of nymphs reared on each genotype that reached adulthood. For mating, males reared in other replicates on the same plant genotype were used. The insect couples, grown in the same plant genotype, were isolated in a cylinder (20 cm in height) with a Þne mesh covering the top (Fig. 1). At least 20 replicates (20 couples of insects) were used for every RDH line, Syn, and parental lines. Total fecundity (TF ⫽ total no. of eggs per adult) and longevity (L) were used as measures of reproductive performance. TF was recorded by dissecting the leaves and counting the eggs using a stereoscopic microscope (Fig. 2). Four pots of each genotype, containing 15 pregerminated seeds of the same line, were used for tolerance test. At the second fully expanded leaf stage every plant in half of the pots was infested with 1Ð2d-old macropterous females and males, at a rate of three insects per plant. The plants in the remaining pots were kept uninfested as controls. The trial consisted of two replicated blocks of 132 pots, one pot per genotype and treatment (infested and controls). At the onset of infestation every pot was covered with a cylinder (20 cm in height) with a Þne mesh covering at the top. The number of planthoppers per plant was recorded every other day, and more insects were added if necessary to keep the density constant. The foliar area (FA), aerial fresh weight (AFW), aerial dry weight (ADW), and chlorophyll content of Þrst (SL1) and second leaves (SL2) were evaluated

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twice: at the onset of infestation (0 d) and 15 d later, when susceptible plants were ⬎50% chlorotic. The differences between both recordings in the infested and control plants were calculated to assess the tolerance level. Chlorophyll content was determined using a hand-held chlorophyll meter (SPAD-502 Minolta, Milton Keynes, Buckinghamshire, United Kingdom). The data were tested for normality and the root square transformation was used for the number of eggs per adult. The data were analyzed using PROC GLM (SAS Institute 1998), and the least signiÞcant difference (LSD) test was used to compare the differences between means. Genetic Analysis. The construction of the map is described in Castro et al. (2005). QTL mapping of the traits was performed using QGENE software (Nelson 1997) and the program QTL Cafe´ (http://www. biosciences.bham.ac.uk/labs/kearsey) by using single marker analysis of variance (ANOVA), marker regression, and interval mapping using the means of the traits from ANOVA analyses.

Results Antixenosis. There were signiÞcantly different levels of antixenosis (F ⫽ 1.43; df ⫽ 65, 1,254; P ⫽ 0.0157). The level of antixenosis shown by the S6A and the RDH lines were signiÞcantly different from that of CS and Syn (Table 1). The level of antixenosis shown by 52 RDH lines was similar to that of the S6A line (Fig. 3). One RDH line showed a signiÞcantly lower mean value of antixenosis than S6A. Another six RDH lines were similar to CS and only one RDH line was as highly preferred as Syn (Fig. 3). Antibiosis. There were highly signiÞcant differences (P ⱕ 0.001) in most of the measures of antibiotic resistance (developmental time [d; days], NI, NIII, NV, A, TF) (df ⫽ 65, 1,225; d: F ⫽ 5.44, P ⱖ 0.0001; NI: F ⫽ 6.33, P ⱖ 0.0001; NIII: F ⫽ 1.85, P ⱖ 0.0001; NV: F ⫽ 2.47, P ⱖ 0.0001; A: F ⫽ 1.95, P ⱖ 0.0001; and TF: F ⫽ 26.7, P ⱖ 0.0001). The insects reared on Syn had a signiÞcantly longer developmental time (d) than those reared on CS and the S6A line. These differences were based on the signiÞcantly longer duration of NV recorded for the insects reared on Syn (Table 1). The planthoppers reared on 49 recombinant lines did not

Table 1. Mean values and the LSDs for levels of antixenosis (number of insects per plant) and antibiosis against D. kuscheli shown by Chinese Spring (CS), Synthetic (Syn), S6A, and 63 RDH lines Line

Antixenosis

CS Syn S6A RDH LSD

4b 6.5c 1.83a 2a 0.94

Antibiosis d

NI

NII

NIII

NIV

NV

A

L

S

TF

22.16b 28.2a 22.8b 23.04b 2.28

4.69a 5.00a 4.14b 4.06b 0.51

4a 3.78ab 3.25b 3.76ab 0.59

3.10b 3.88a 3.33ab 3.64ab 0.55

4.13a 4.50a 4.50a 4.43a 0.80

5.83b 10.60a 6.40b 7.47b 1.60

16.60a 9.2b 17.2a 15.06a 3.82

38.62a 37.4a 39.3a 38.24a 3.20

0.36a 0.38a 0.36a 0.49a 0.20

36a 0b 41a 19b 20.38

See text for meaning of abbreviations. Values within a column that share the same letters are not signiÞcantly different (P ⫽ 0.05).

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Fig. 3. Distribution of the number of adults of D. kuscheli on double haploid recombinant substitution (RDH) lines. The mean parental values for CS, S6A, Syn, and RDH lines are indicated by arrows.

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differ signiÞcantly in developmental time from those reared on S6A or CS, but those reared on eleven RDH lines had a signiÞcantly longer d, similar in length to that of insects reared on Syn (Fig. 4A). The planthoppers reared on three RDH lines had signiÞcantly shorter developmental times than those reared on the susceptible parent CS (Fig. 4A). There were no signiÞcant differences in the duration of the NII and NIV instars of the insects reared on CS, Syn, and S6A (Table 1). However, the duration of NV was signiÞcantly longer for the planthoppers reared on Syn than those reared on CS and S6A (Table 1). Moreover, the duration of NV for those reared on 25 RDH lines was signiÞcantly longer, with levels of antibiosis similar to that recorded for the Syn parent. The duration of the adult stage (A) of insects reared on CS and S6A did no differ signiÞcantly, however, it did for those reared on Syn, CS, and S6A (Table 1). The shortest adult stage was recorded on 21 RDH

Fig. 4. Distribution on different double haploid recombinant (RDH) lines of duration of development (d) (A), length of adult life (B), longevity (C), survival (D), and fecundity (E). The mean values for CS, Syn, S6A, and RDH are indicated by arrows.

October 2009 Table 2. Line CS Syn S6A RDH LSD


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Mean values and the LSD in the tolerance of infestation by D. kuscheli shown by CS, Syn, S6A, and RDH lines SL1

SL2

FA

AFW

ADW

Control

Infested

Control

Infested

Control

Infested

Control

Infested

13.07bc 31.72a 12.25c 15.47b

6.15d 30.92a 7.22d 11.57c

13.97c 32.0a 14.95c 13.54cd

12.10cd 25.72b 10.15d 11.41cd

10.56a 5.60b 11.10a 11.95a

9.27ab 6.33b 5.65b 7.85b

0.26a 0.19ab 0.24a 0.28a

0.19ab 0.21a 0.11b 0.16b

3.12

3.65

3.95

0.09

Control

Infested

0.032a 0.031a 0.023b 0.024b 0.029ab 0.029ab 0.036a 0.032a 0.0065

See text for meaning of abbreviations. Values within a trait that share the same letters are not signiÞcantly different (P ⫽ 0.05).

lines, which differed from that of adults reared on CS and S6A (Fig. 4B). The high level of antibiosis shown by these lines is reßected in the shorter adult stage. Longevity showed signiÞcant differences (F ⫽ 1.42; df ⫽ 65, 1,225; P ⫽ 0.0175). Longevity was similar on CS, Syn, and S6A and that of those insects reared on 19 RDH lines was signiÞcantly shorter than that recorded on Syn (Fig. 4C). Insect survival (S) was similar on Syn, CS, and S6A (Table 1). Nevertheless, on eight RDH lines survival was signiÞcantly lower than that recorded on the parental lines (Fig. 4D). On another eight RDH lines survival was signiÞcantly longer than that recorded on Syn and parental plants. The total fecundity (TF) of insects reared on Syn differed signiÞcantly from that of insects reared on both parental lines (Table 1). No eggs were laid by females reared on Syn and on 39 RDH lines, affected by plant antibiosis (Fig. 4E). The insects reared on another eight RDH lines had TFs that did not differ from those recorded on CS and S6A. Only on three RDH lines TF was signiÞcantly greater than that recorded on CS or S6A (Fig. 4E). Tolerance. There were highly signiÞcant differences (P ⱖ 0.0001) between genotypes, treatments, and in the interaction in most of the traits (SL1, SL2, FA, and AFW) of tolerance to infestation by D. kuscheli. There were highly signiÞcant differences between genotypes and in the interaction of the dry weight; however, there were no signiÞcant differences between treatments. Chlorophyll content of the Þrst (F ⫽ 1.80; df ⫽ 65, 131; P ⱖ 0.0001) and second leaves (F ⫽ 1.61; df ⫽ 65, 131; P ⱖ 0.0001) differed signiÞcantly in CS and Syn (Table 2). The highest chlorophyll contents were recorded for Syn in both the control and infested plants. The mean values for S6A and the RDH lines were similar to those recorded for the control and infested plants of the CS parental line. Infestation did not reduce signiÞcantly the chlorophyll content on the second leaf of CS and of the RDH lines (Table 2); however, Syn and S6A had a signiÞcantly lower chlorophyll content than their controls. Only Þve RDH lines showed similar values of chlorophyll content to those recorded for Syn. There were no signiÞcant differences in the FA between the control and infested plants of CS and Syn plants, but infested S6A and RDH had a signiÞcantly lower FA (Table 2). Plant tolerance to infestation by

D. kuscheli, in terms of aerial fresh weight, did not differ signiÞcantly except for S6A- and the RDH-infested plants (Table 2). Syn had a signiÞcantly lower dry weight compared with CS and to the RDH in both, infested and control plants. The aerial dry weight was no signiÞcantly reduced by infestation (Table 2). Genetic Analysis. Antixenosis was not associated with the 6A marker loci. There was signiÞcant association between the duration of the Þfth nymphal instar and S6A marker loci. One quantitative trait locus (QTL) was signiÞcantly associated with the Xgwm1017 marker locus, which is located at 48 cM (additive effect, ⫺1.732), with the positive allele provided by Syn (Fig. 5A). Similarly, the duration of development was associated with the same marker loci (additive effect, ⫺2.032) (Fig. 3A). The difference in chlorophyll content of the Þrst leaf of infested and control plants was signiÞcantly associated with the interval between marker loci Xgwm459 and Xgwm334a at 8 cM (additive effect, ⫺0.9876), with positive effect provided by Syn (Fig. 5B). The FA, AFW, and ADW were not signiÞcantly associated with the 6A marker loci. Discussion This study is the Þrst to report a biological and genetic analysis of antixenosis, antibiosis and tolerance of wheat to infestation by D. kuscheli. The substitution line S6A was signiÞcantly less preferred than CS and Syn. However, genetic analysis showed that antixenosis of wheat is not signiÞcantly associated with 6A molecular markers. Nevertheless, 37 RDH lines showed a level of antixenosis similar to the S6A line. It is possible that the level of antixenosis to D. kuscheli of the S6A and 37 RDH lines could be due to a regulator gene on chromosome 6A, which determines the expression of other genes in the CS genome. Recently, nine QTLs, associated with plant tolerance to stress-induced hormones, were mapped to chromosome 6A of these RDH lines (Castro et al. 2008). These genes possibly determine the expression of other defense genes (Yamaguchi-Shinosaki and Shinosaki 2006) and might account for the antixenosis recorded against D. kuscheli. Antibiotic resistance against D. kuscheli takes the form of a longer developmental time (d), shorter adult life (A), and reduced fecundity. A QTL associated with marker loci Xgwm1017, accounts for the increase

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Fig. 5. QTL interval mapping proÞles for antibiosis to D. kuscheli in terms of duration of Þfth nymphal instar and duration of development on the RDH lines (A) and for tolerance in terms of chlorophyll content of the Þrst leaf of infested plants (B). Arrows mark most likely position of the QTL on chromosome 6A.

in duration of insect development. On the same region of the 6AL chromosome, there are two QTLs that determine most of the variation recorded in wheat foliar area and dry weight, when plants are treated with ethylene and ABA (Castro et al. 2008). Probably, planthopper infestation switches on the same genes that enable plants to tolerate the effects of stressinduced hormones, and it is the effects of theses genes that constrain the development of D. kuscheli. Although signiÞcantly fewer offspring were produced on Syn and the 39 RDH lines, the genes responsible for this are not on the 6A chromosome. Probably the genes of S6A interacted with other genes in the CS genome and this resulted in the reduction in fecundity. Tolerance of this planthopper, evaluated by means of plant growth trait differences, indicated signiÞcant differences between infested and control plants for the SL1, SL2, FA, and AFW. D. kuscheli did not affect wheat aerial dry weight and most of the traits evaluated were not signiÞcantly associated with any marker loci, except chlorophyll content of the Þrst leaf. The variation in chlorophyll content was associated with the interval between loci Xgwm459 and Xgwm334a, located in the telomeric region on the short arm of 6A. Seven QTLs, triggered by stress-induced hormones, were associated with the same interval, suggesting that this interval may contain a gene(s) with the same or similar functions (Castro et al. 2008). Similarly, genes for antixenosis against greenbug and Russian wheat aphid biotype 2 have been mapped on the 6AS chromosome arm of the same RDH lines (Castro et al. 2005). The telomeric region of the 6A chromosome probably carries genes that enable the plant to tolerate biotic stresses. Nonetheless, the Þrst report of resistance against D. kuscheli in oats indicates an absence of antixenosis and antibiosis in commercial greenbugresistant oat cultivars (Costamagna et al. 2005). Stress signaling pathways are not independent, ABA, ethylene, jasmonic acid, and salicylic acid are produced by plants under stress, and when exogenously applied, they switch on a number of genes that respond to environmental or biotic stress (Yamaguchi-Shinosaki

and Shinosaki 2006). Nevertheless, the role of hormones in the regulation of stress-responsive gene expression is uncertain (Nakashima and Yamaguchi-Shinosaki 2005). Some genes can be switched on by both biotic stress and exogenously applied hormones. Other genes are switched on only by biotic factors or hormones (Yamaguchi-Shinosaki and Shinosaki 2006). The RDH lines tested in the current study showed variability in antixenosis, antibiosis, and tolerance of infestation by D. kuscheli. The genes determining antibiotic resistance to D. kuscheli may provide a way of reducing delphacid populations on wheat and in conjunction with an integrated pest management of the MRC vector, could be used to decrease the intrinsic rate of population increase of D. kuscheli. Acknowledgment This work was supported by Consejo Nacional de Investigaciones Cientõ´Þcas y Te´ cnicas of the Repu´ blica Argentina.

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