Biol Fertil Soils (2003) 38:367–376 DOI 10.1007/s00374-003-0669-3
ORIGINAL PAPER
Annemie Elsen · Raf Beeterens · Rony Swennen · Dirk De Waele
Effects of an arbuscular mycorrhizal fungus and two plant-parasitic nematodes on Musa genotypes differing in root morphology Received: 14 February 2003 / Accepted: 28 July 2003 / Published online: 3 September 2003 Springer-Verlag 2003
Abstract In this study, the effect of an arbuscular mycorrhizal fungus (AMF) and two migratory endoparasitic nematodes on Musa plant growth, including the root system, were examined. In addition, the AMF-nematode interaction was studied. Seven Musa genotypes with different root systems were selected. Based on their relative mycorrhizal dependency, two genotypes (Calcutta 4 and Obino l’Ewai) were selected for AMF-nematode interaction studies. The experiments were performed under greenhouse conditions. Mycorrhization with Glomus mosseae resulted in a significantly better plant growth even in the presence of nematodes. The effect of AMF on the root system was genotype-dependent and seemed to be related to the relative mycorrhizal dependency of the genotype. The nematodes also affected the root system, decreasing branching. Nematode population densities were significantly reduced in the presence of AMF, except for Pratylenchus coffeae in Obino l’Ewai. In the root system, it appeared that the decreased branching caused by the nematodes was counterbalanced by the increased branching caused by the AMF. Keywords Glomus mosseae · Musa spp. (banana) · Pratylenchus coffeae · Radopholus similis · Root morphology
Introduction Roots function both as a support system and as the nutrient uptake organ of plants. Root morphology changes in response to the soil environment to minimise the metabolic cost of maintaining the root system. In response to nutrient-limiting conditions, plants may increase root fineness, root/shoot ratio or the number or length of the A. Elsen ()) · R. Beeterens · R. Swennen · D. De Waele Laboratory of Tropical Crop Improvement, Katholieke Universiteit Leuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium e-mail:
[email protected] Fax: +32-16-321993
root hairs (Hetrick 1991). Each of these adaptations involves a different metabolic cost. Mycorrhizae are another alternative to such changes. Several studies have shown that arbuscular mycorrhizal fungus (AMF) colonisation can influence the root morphology of host plants (Berta et al. 1993; Tisserant et al. 1992; Yano et al. 1996). Most plants infected with AMF develop a denser root system, with a higher number of shorter primary roots of a greater diameter (Berta et al. 1993; Jaizme-Vega et al. 1994). In most plants, the branching of roots increases after AMF colonisation (Jaizme-Vega et al. 1994; Schellenbaum et al. 1991; Tisserant et al. 1992). However, less branched root systems have been observed in mycorrhizal plants of Andropogon gerardii (Hetrick et al. 1988) and Gossypium hirsutum (Price et al. 1989). Different mechanisms have been suggested for the alteration of root systems following AMF colonisation (Berta et al. 1993). From a physiological point of view, it is well recognised that AMF can increase nutrient absorption, especially elements with low mobility in the soil such as P. Other studies have reported changes in phytohormone balance in association with AMF (Allen et al. 1980; 1982; Dannenberg et al. 1992). Anatomically, depression of meristematic activity of mycorrhizal root apices have been found (Berta et al. 1993). Arbuscular mycorrhizal fungi are able to alter the rooting strategy of plants not only in response to soil fertility but also in response to micro-organisms present in the rhizosphere (Hetrick 1991). The rhizosphere comprises both beneficial and pathogenic micro-organisms. Migratory endoparasitic nematodes are pathogens infecting roots of many different hosts. Especially in the tropics, they can cause high yield losses (Sasser and Freckman 1987). Infected roots become necrotic, thereby inhibiting nutrient and water uptake and, like in banana and plantain, weakening the anchorage of the plant. Until now, the most commonly used approach to control nematodes is the use of chemicals. However, nematicides are expensive and toxic both for the user and the
368
environment. The application of AMF could be an alternative nematode management strategy. In Musa, there are indications that genotypes that are more resistant to migratory endoparasitic nematodes have a larger and denser root system than the susceptible genotypes (Blomme 2000; Stoffelen 2000). Until now, only one study has reported on the effect of AMF on root system morphology of Musa plantlets (Jaizme-Vega et al. 1994). The most important modifications in root morphology induced in Musa plants by AMF are increased branching and increased total root length, which lead to a denser rooting pattern. The influence of AMF on the root system of Musa genotypes differing in root morphology and the influence of the AMF/nematode interaction on the root system and vice versa have never been studied. In this study, we examined if AMF can be used to counter nematode effects by inducing a larger and denser root system in Musa. The first objective of this study was to investigate whether the effect of AMF on the root system differed for Musa genotypes with initially different root systems. Therefore a selection of genotypes was made according to the classification by Swennen et al. (1986), based on the contribution of secondary and tertiary root length to the total root length. The second objective was to study the interaction between AMF and two migratory endoparasitic nematodes, Radopholus similis (Cobb) Thorne and Pratylenchus coffeae (Zimmermann) Filipjev and Schuurmans Stekhoven on two Musa genotypes with a different root system and selected for their different response to AMF (high and low mycorrhizal dependency). The influence of the AMF on the root system and the influence of the altered root system on nematode reproduction were studied. Since biological diversity among R. similis populations has been reported (Elbadri et al. 2001; Fallas et al. 1995), two R. similis populations differing in pathogenicity were included in the study.
Materials and methods Biological materials Musa tissue culture plants were obtained from the International Musa germplasm collection at the INIBAP Transit Centre (ITC), K. U. Leuven, Belgium (Table 1). The selected genotypes were chosen
Table 1 Musa genotypes used in this study, and their root morphology
for their different root morphology (Stoffelen 2000; Swennen et al. 1986). The plant material was proliferated, regenerated and rooted in test tubes on Murashige and Skoog medium including vitamins, 30 g/l sugar, 10 mg/l ascorbic acid and 2 g/l Gelrite with pH 6.2 (Banerjee and De Langhe 1985). The plants were grown in culture rooms at 27€1C with continuous light. The G. mosseae (Nicol. and Gerd.) Gerd. and Trappe isolate used in this study was originally recovered from Pome banana (Musa AAB, Pome group) grown on a biological farm in Los Realejos, Tenerife, Canary Islands, Spain. This native isolate was maintained and propagated in sorghum pot cultures (Ferguson and Woodhead 1982). Glamus mosseae has been chosen because this species is a highly effective symbiont of Cavendish (Musa spp., AAA group) genotypes (Declerck et al. 1995). Two R. similis populations differing in pathogenicity were selected: a highly pathogenic population from Uganda and a less pathogenic population from Indonesia (Elbadri et al. 2001; Fallas et al. 1995). Both populations were originally isolated from Musa roots. The P. coffeae population was isolated from Musa roots in Ghana and maintained under monoxenic conditions on carrot discs at 27€1C in the dark. Prior to inoculation, the nematodes were collected using the Baermann funnel technique (Hooper 1990). Experimental design In the first two experiments (experiments 1 and 2), the micropropagated Musa plants were transplanted after deflasking to germination trays (604010 cm3) for early mycorrhizal colonisation during the hardening phase (mycorrhization). The substrate consisted of a sterilised 2:1 peat:quartz sand mixture. Arbuscular mycorrhizal inoculum consisted of 1.5 kg rhizosphere soil from 6month-old sorghum pot cultures of G. mosseae containing spores, hyphae and heavily infected root fragments (Jaizme-Vega et al. 1997) and it was spread as a layer between two layers of substrate. The plants not receiving mycorrhizal inoculum received 1.5 kg rhizosphere soil from 6-month-old sorghum pot cultures that were not colonised by AMF. Ten plants were considered per treatment. A period of 6 weeks allowed the establishment of the mycorrhizal symbiosis, then eight plants per treatment were transplanted to 1liter containers filled with sterilised 2:1 peat:quartz sand substrate. For each plant, 1 g Osmocote, a slow release fertiliser, was added to the substrate. During the experiments, the plants were maintained in the greenhouse at an ambient temperature of 20–27C, with a 12-h photoperiod (170–190 PAR) and a relative humidity of 50–70%. In the four experiments where interactions between AMF and Musa nematodes were studied (experiments 3–6), the selected genotypes were mycorrhized as described. In the treatments with nematodes, the plants were inoculated with 1,000 vermiform (juvenile and adult) nematodes per plant, 8 weeks after planting (2 weeks after transplant to individual containers). After nematode inoculation, the plants were kept in the greenhouse for 8 (when inoculated with R. similis; i.e. in experiments 3 and 5) or 10 weeks (when inoculated with P. coffeae; i.e. in experiments 4 and 6), in order to allow nematode reproduction.
Accession name
ITC numbera
Genome, subspecies/group
Root systemb
Calcutta 4 Grande Naine Igitsiri Kayinja Mbwazirume Obino l’Ewai
0249 1256 0081 0084 0109
AA, burmannicoides AAA, Cavendish AAA, Mutika/Lujugira ABB, Pisang Awak AAA, Mutika/Lujugira AAB, Plantain
Pisang Lilin
1400
AA, malaccensis derived
High contribution of tertiary roots High contribution of tertiary roots High contribution of tertiary roots High contribution of secondary roots High contribution of tertiary roots Equal contribution of secondary and tertiary roots High contribution of tertiary roots
a
ITC number = accession number at the International Musa germplasm collection at the INIBAP Transit Centre, K. U. Leuven b Classification as proposed by Swennen et al. (1986) and confirmed by Stoffelen (2000)
369 Assessment of variables
Statistical analysis
In the first two experiments (experiments 1 and 2), plant growth was assessed 16 weeks after planting. Plant height, number of leaves, foliar surface, fresh and dry shoot weight and fresh root weight were measured for each plant. The foliar surface was determined using a planimeter. The dry shoot weight was determined after drying the leaves, pseudostem and corm for 72 h in an oven at 70C. The relative mycorrhizal dependency (RMD) was determined by expressing the difference between the dry weight of the mycorrhized plant and the average dry weight of the non-mycorrhized plant as a percentage of the dry weight of the mycorrhized plant (Plenchette et al. 1983). Root growth was given by the total root weight, the weight of the primary roots, weight of the secondary and tertiary roots and weight of the in vitro roots. The roots present at time of deflasking (i.e. beginning of the experiment) and thus formed under in vitro conditions are considered as in vitro roots. For assessing the mycorrhizal colonisation, secondary and tertiary root samples were collected and stained with 0.05% trypan blue in lactic acid modified as described by Koske and Gemma (1989). Twenty 1-cm fine root segments per plant were mounted on slides and observed under a light microscope. The frequency of AMF colonisation (F%) was calculated as the percentage of root segments colonised by either hyphae, arbuscules or vesicles. In addition, the intensity of colonisation (I%), that is the abundance of hyphae, arbuscules and vesicles in each mycorrhizal root segment, was estimated (Plenchette and Morel 1996). In the four experiments where interactions between AMF and nematodes were studied (experiments 3–6), plant growth and mycorrhizal colonisation were assessed as described. In the treatments inoculated with nematodes, nematode damage and nematode reproduction were assessed. At the end of each experiment, the percentage root necrosis was measured by scoring five 10-cm longitudinally sliced functional primary roots (Speijer and De Waele 1997). The nematodes in the roots were extracted from the root system using a maceration-sieving technique. The reproductive potential of the nematodes (total number of nematodes per plant, total number of nematodes per root unit) was determined.
The data were analysed with the STATISTICA package (Statsoft Inc. 1997). Two factors were studied: genotype and presence/ absence of AMF in the first two experiments (experiments 1 and 2) and presence/absence of AMF and presence/absence of nematodes in the four following experiments (experiments 3–6). Data that were normally distributed and had homogeneous variances were subjected to ANOVA (Analysis of Variance). All plant parameters were analysed by two-way ANOVA, while the mycorrhizal data and nematode data were analysed by one-way ANOVA. Prior to analysis, nematode reproduction data were log(x+1) transformed. Data for root necrosis, frequencies and intensities of mycorrhizal colonisation were arcsin(x/100) transformed. Means were separated by the Tukey test (P 0.05). The relative proportions of the in vitro, primary and, secondary and tertiary roots were compared in a Chi-square test (P 0.05).
Table 2 Influence of Glomus mosseae on plant growth of four Musa genotypes differing in root morphology, 16 weeks after planting (n =6–8; experiments 1 and 2). Means are separated using the Tukey test (P 0.05). FSW Fresh shoot weight, DSW dry shoot weight, SH shoot height, FRW fresh root weight, 1st root first order root, 2nd + 3rd root second and third order roots
FSW (g) Experiment 1 without AMF Grande Naine Calcutta 4 Kayinja Obino l’Ewai with AMF Grande Naine Calcutta 4 Kayinja Obino l’Ewai c
Experiment 2 without AMF Grande Naine Igitsiri Mbwazirume Pisang Lilin with AMF Grande Naine Igitsiri Mbwazirume Pisang Lilin c
a b c
DSW (g)
Results Relative mycorrhizal dependency of genotypes differing in root morphology (experiment 1 and 2) Seven genotypes were tested in two parallel experiments with Grande Naine as a reference. Overall, the root colonisation with G. mosseae had a beneficial effect on plant growth. The mycorrhized plants had a higher fresh and dry shoot weight, were taller, had a larger foliar surface and had a higher fresh root weight (Table 2). Differences in plant growth were not only genotypedependent but also parameter-dependent. The presence of AMF increased the total fresh root weight by increasing the root weight of the first order SH (cm)
Foliar surface (cm2)
FRW (g)
1st root weight
2nd + 3rd root weight
In vitro root weight
Ba A B A
1.3 1.3 1.4 0.7
ab a a a
9 10 10 11
A A AB B
249 246 210 184
B AB AB A
16.9 18.3 22.0 15.7
A A B A
4.7 5.8 6.2 6.0
AB B B A
11.7 12.4 15.4 8.9
A A B A
0.4 0.2 0.5 0.8
28 B 22 A 28 B 24 A ***
3.8 2.5 3.4 2.8
d b cd bc
12 A 12 A 13 AB 15 B ***
454 402 405 374 ***
B AB AB A
39.3 36.2 46.3 37.2 ***
A A B A
10.7 10.9 12.5 7.7 ***
AB B B A
28.5 25.3 33.2 29.5 ***
A A B A
0.1 A 0A 0.6 B 0 AB N.S.
16 8 12 11
b a ab ab
1.8 0.7 0.9 0.9
b a a a
10 11 11 12
A B AB B
245 97 128 165
b a a a
12.6 5.1 5.4 10.0
a a a a
5.4 1.8 2.5 4.9
bc a ab abc
6.7 3.0 2.6 4.8
ab a a ab
0.53 0.29 0.26 0.29
abc a a a
39 28 31 23
e cd d c
4.8 3.9 3.0 3.0
e d c c
15 A 18 B 16 AB 16 B ***
477 398 362 262
d c c b
45.1 29.1 23.5 23.7
c b b b
13.2 5.6 6.2 10.5
d bc c d
31.5 21.5 17.1 11.7
e d cd bc
0.44 2.01 0.16 1.53
ab c a bc
15 13 15 10
A A B AB
Capital letters indicate a main effect of the treatment genotype at P 0.05 Small letters indicate an interaction effect of both treatments at P 0.05 *, **, *** indicate a main effect of the treatment mycorrhiza at P 0.05, 0.01 and 0.001, respectively
370 Table 3 Root colonisation by Glomus mosseae and relative mycorrhizal dependency of four Musa genotypes differing in root morphology, 16 weeks after planting (n =8; experiments 1 and 2). Prior to statistical analysis data were arcsin (x/100) transformed. [Same letter in same column indicates no significant difference according to the Tukey test (P 0.05).] F% frequency of mycorrhization, I% intensity of mycorrhization, RMD relative mycorrhizal dependency Genotype Experiment 1 Grande Naine Calcutta 4 Kayinja Obino l’Ewai Experiment 2 Grande Naine Igitsiri Mbwazirume Pisang Lilin
Fig. 1 Effect of Glomus mosseae (AMF) on the proportional root weight of primary (1st), secondary (2nd) and tertiary (3rd) and in vitro roots of four different Musa genotypes 16 weeks after planting (I experiment 1, II experiment 2)
roots and of the secondary and tertiary order roots. In contrast, the root weight of the in vitro roots was not altered by the presence of AMF, except for Igitsiri and Pisang Lilin. The number of primary roots tended to increase when AMF were present. In general, the AMF had a positive influence on the root growth, but the impact differed among genotypes. Figure 1 summarises the proportional weight of the different root types. After 16 weeks, the proportion of in vitro roots was very small in both the mycorrhizal and non-mycorrhizal treatments. The proportion of secondary and tertiary roots was larger than the proportion of primary roots, but some variation among genotypes was observed. The non-mycorrhized Pisang Lilin and Mbwazirume had the largest proportion of primary roots (49% and 46%, respectively), while the mycorrhized Igitsiri had the smallest proportion of primary roots (19%). The presence of AMF did not influence the root proportions of Calcutta 4, Kayinja and Pisang Lilin (Fig. 1). For Grande Naine, the results were not very clear. In the first experiment, the AMF did not influence the root proportions, while in the second experiment, the
F%
I%
RMD (%)
50 61 56 54
a a a a
17 20 21 15
a a a a
66 50 58 75
bc a ab c
72 70 50 33
b b a a
22 16 14 13
b ab a a
62 83 70 70
a c b b
presence of the AMF significantly decreased the primary root weight proportionally. For Obino l’Ewai, Igitsiri and Mbwazirume, the AMF significantly increased branching of the root system as indicated by a proportional decrease of the primary root weight in favour of an increase of the secondary and tertiary root weight. Mycorrhization with G. mosseae was successful in both experiments, as indicated by a high frequency (F%) and an acceptable intensity (I%) of mycorrhization (Table 3). The mycorrhization in Grande Naine is comparable in both experiments. Based on the relative mycorrhizal dependency, Obino l’Ewai and Calcutta 4 were selected for further interaction studies: Obino l’Ewai as a plantain with a high RMD and Calcutta 4, a parent in Musa breeding programs (Swennen and Vuylsteke 1993; Vuylsteke et al. 1993) with a low RMD. Interaction of G. mosseae and Radopholus similis in Obino l’Ewai (Musa AAB group; experiment 3) At the end of experiment 3 (i.e. 16 weeks after planting) the mycorrhized plants had a better shoot growth (Table 4). In both non-mycorrhizal and mycorrhizal treatments, the plants inoculated with the Indonesian R. similis population showed a significantly (P 0.05) better plant growth compared to the plants inoculated with the Ugandan R. similis population. In general, the presence of AMF increased the root weight significantly (P 0.05). The effect of G. mosseae and R. similis (Ugandan and Indonesian population) on the proportions of the primary, secondary and tertiary and in vitro roots is given in Fig. 2. No significant changes in the relative proportions of the roots were found. The presence of the AMF reduced significantly (P 0.05) the nematode population density for both R. similis populations (Table 5). The root necrosis, caused by the nematodes, was significantly (P 0.05) higher when
371 test (P 0.05). FSW Fresh shoot weight, DSW dry shoot weight, SH shoot height, FRW fresh root weight, 1st root first order root, 2nd + 3rd root second and third order roots
Table 4 Influence of Radopholus similis (rs) and Glomus mosseae (AMF) on the plant growth of Obino l’Ewai, 16 weeks after planting (n =8; experiment 3). Means are separated using the Tukey FSW (g) + + + b a b
AMF/ AMF/+ AMF/+ AMF/ AMF/+ AMF/+
rs rs rs rs rs rs
Uganda Indonesia Uganda Indonesia
14.1 10.0 18.5 22.7 22.5 29.8 ***
Aa A B A A B
DSW (g)
SH (cm)
1.2 AB 0.8 A 1.5 B 2.3 AB 2.0 A 2.7 B ***
13.3 10.5 14.6 16.3 15.6 17.9 ***
AB A B AB A B
Foliar surface (cm2)
FRW (g)
1st root weight
2nd + 3rd root weight
In vitro root weight
288 196 358 494 453 574 ***
9.5 4.2 10.0 17.6 16.3 20.1 ***
4.2 B 2.3 A 4.5 B 6.1 A 6.9 A 8.7 B ***
4.7 1.4 4.8 10.8 7.6 10.4 ***
0.6 0.5 0.7 0.7 1.7 1.3 **
AB A B AB A B
AB A B AB A B
B A B B A B
Capital letters indicate a main effect of the treatment nematode at P 0.05 *, **, *** indicate a main effect of the treatment mycorrhiza (AMF) at P 0.05, 0.01 and 0.001, respectively
Fig. 2 Effect of Glomus mosseae (AMF) and Radopholus similis (rs) or Pratylenchus coffeae (pc) on the proportional root weight of primary (1st), secondary (2nd) and tertiary (3rd) and in vitro roots
Table 5 Density of Radopholus similis (rs) in the root system of Obino l’Ewai and the mycorrhizal colonisation of Glomus mosseae (AMF) in the roots, 16 weeks after planting (n =8; experiment 3). Prior to statistical analysis, mycorrhizal data and root necrosis were arcsin (x/100) and nematode counts were log (x+1) transformed. Means are separated using the Tukey test (P 0.05). F% Frequency of mycorrhization, I% intensity of mycorrhization
+ + + c
a b c
AMF/ AMF/+ AMF/+ AMF/ AMF/+ AMF/+
rs rs rs rs rs rs
Uganda Indonesia Uganda Indonesia
of Obino l’Ewai (graphs on the left) and Calcutta 4 (graphs on the right) 16 (when inoculated with R. similis) or 18 weeks (when inoculated with P. coffeae) after planting
R.s. per gram roots
Root necrosis (%)
F%
I%
1,336 218 314 74 ***
41 19 44 27 **
53 bb 31 a 58 c
13 12 12 N.S.
Ba A B A
B A B A
Capital letters indicate a main effect of the treatment nematode population at P 0.05 Small letters indicate differences in mycorrhization at P 0.05 *, **, *** indicate a main effect of the treatment mycorrhiza (AMF) at P 0.05, 0.01 and 0.001, respectively
372 Table 6 Influence of Pratylenchus coffeae (pc) and Glomus mosseae (AMF) on the plant growth of Obino l’Ewai, 18 weeks after planting (n =8; experiment 4). Means are separated using the Tukey test (P 0.05). FSW Fresh shoot weight, DSW dry shoot weight, SH shoot height, FRW fresh root weight, 1st root first order root, 2nd + 3rd root second and third order roots
+ + b
AMF/ AMF/+ AMF/ AMF/+
pc pc pc pc
FSW (g)
DSW (g)
SH (cm)
Foliar surface (cm2)
FRW (g)
1st root weight
2nd + 3rd root weight
In vitro root weight
7.0 7.3 15.1 14.3 ***
0.4 0.4 1.2 1.1 ***
12.4 13.1 14.1 15.4 **
155 127 303 299 ***
4.6 3.7 11.5 10.8 **
1.2 1.5 3.8 3.5 ***
2.8 Ba 1.5 A 7.5 B 6.6 A ***
0.6 0.7 0.2 0.7 N.S.
a b
Capital letters indicate a main effect of the treatment nematode at P 0.05 *, **, *** indicate a main effect of the treatment mycorrhiza (AMF) at P 0.05, 0.01 and 0.001, respectively
Table 7 Density of Pratylenchus coffeae (pc) in the root system of Obino l’Ewai and the mycorrhizal colonisation of Glomus mosseae (AMF) in the roots, 18 weeks after planting (n =8; experiment 4).
+ + a a
AMF/ AMF/+ AMF/ AMF/+
pc pc pc pc
Prior to statistical analysis, mycorrhizal data and root necrosis were arcsin (x/100) and nematode counts were log (x+1) transformed. F% Frequency of mycorrhization, I% intensity of mycorrhization
P.c. per gram roots
Root necrosis (%)
F%
I%
100 74 N.S.
20 38 ***
89 89 N.S.
17 15 N.S.
*, **, *** indicate a significant difference according to the Tukey test at P 0.05, 0.01 and 0.001, respectively
Obino l’Ewai was mycorrhized. The differences in pathogenicity between the R. similis populations were confirmed. In the treatment with joint inoculation of the symbiont and Ugandan R. similis population, G. mosseae achieved a lower frequency of mycorrhizal colonisation (Table 5). However, the intensity of this mycorrhizal colonisation was not influenced by the presence/absence of the R. similis populations. Interaction of G. mosseae and Pratylenchus coffeae in Obino l’Ewai (Musa AAB group; experiment 4) At the end of experiment 4 (i.e. 18 weeks after planting), plants of all mycorrhizal treatments had significantly (P 0.05) greater fresh and dry shoot weight than plants without mycorrhiza (Table 6). The mycorrhized plants were significantly (P 0.05) taller and had a greater foliar surface than non-mycorrhized plants. For all these parameters, the absence/presence of the nematodes did not influence the results. The AMF had a positive influence on the root growth, while the nematodes had no significant (P 0.05) effect, except for the root weight of the secondary and tertiary roots (Table 6). Figure 2 summarises the proportional weight of the different root types. In the non-mycorrhized plants, the presence of the nematodes caused a significant decrease of the proportion of secondary and tertiary root weight in favour of an increase of the proportion of primary root weight. In the mycorrhized plants, no shift in proportions of root weight were observed when inoculated with P. coffeae. In the absence of nematodes, the proportion of secondary and tertiary root weight remained the same while the in vitro root weight decreased proportionally in
favour of the primary root weight. When inoculated with P. coffeae, the AMF significantly increased branching of the root system. The nematode population density (i.e. P. coffeae per gram roots) was not influenced by the presence of AMF (Table 7). Therefore, the total P. coffeae population significantly (P 0.05) increased in the presence of AMF because the mycorrhized plants had a considerably higher root weight. Moreover, the root necrosis caused by the nematodes was significantly (P 0.05) higher in the mycorrhized Obino l’Ewai plants. The nematode infection had no effect on the establishment of the AMF as indicated by the F% and I%. Interaction of G. mosseae and R. similis in Calcutta 4 (Musa AA group; experiment 5) At the end of experiment 5 (i.e. 16 weeks after planting), mycorrhization increased shoot height significantly (P 0.05) when inoculated with R. similis (Ugandan population; Table 8). The presence of R. similis (Ugandan or Indonesian population) had no influence on the plant growth. Figure 2 summarises the proportional weight of the different root types. The presence of R. similis (Ugandan and Indonesian population) did not influence the branching of the root system. Mycorrhizal colonisation significantly increased the proportion of secondary and tertiary roots in control plants and plants infected with R. similis (Ugandan population). For R. similis (Indonesian population), no significant effect of the nematode on the root morphology was observed.
373 Table 8 Influence of Radopholus similis (rs) and Glomus mosseae (AMF) on the plant growth of Calcutta 4, 16 weeks after planting (n =8; experiment 5). Means are separated using the Tukey test (P FSW (g) + + + c
a b c
AMF/ AMF/+ AMF/+ AMF/ AMF/+ AMF/+
rs rs rs rs rs rs
Uganda Indonesia Uganda Indonesia
8.3 2.9 11.6 15.2 15.7 15.6
ab a b b b b
DSW (g)
SH (cm)
0.8 0.3 1.2 1.5 1.7 1.6
10.7 8.1 12.4 15.7 14.2 15.0 ***
ab a b b b b
0.05). FSW Fresh shoot weight, DSW dry shoot weight, SH shoot height, FRW fresh root weight, 1st root first order root, 2nd + 3rd root second and third order roots
Foliar surface (cm2) Ba A B B A B
185 72 286 353 366 341
abb a b b b b
FRW (g)
1st root weight
2nd + 3rd root weight
In vitro root weight
5.4 1.6 7.6 13.0 12.6 8.6
2.5 0.6 3.2 4.2 5.3 4.2
2.5 0.6 3.9 8.5 6.8 3.9
0.4 0.4 0.6 0.3 0.6 0.5 n.s.
ab a bc c c bc
ab a b b b b
ab a ab c bc ab
Capital letters indicate a main effect of the treatment nematode at P 0.05 Small letters indicate an interaction effect of both treatments at P 0.05 *, **, *** indicate a main effect of the treatment mycorrhiza at P 0.05, 0.01 and 0.001, respectively
Table 9 Density of Radopholus similis (rs) in the root system of Calcutta 4 and the mycorrhizal colonisation of Glomus mosseae (AMF) in the roots, 16 weeks after planting (n =8; experiment 5). Prior to statistical analysis, mycorrhizal data and root necrosis were arcsin (x/100) and nematode counts were log (x+1) transformed. Means are separated using the Tukey test (P 0.05). F% Frequency of mycorrhization, I% intensity of mycorrhization
Table 10 Influence of Pratylenchus coffeae (pc) and Glomus mosseae (AMF) on the plant growth of Calcutta 4, 18 weeks after planting (n =8; experiment 6). Means are separated using the Tukey test (P 0.05). FSW Fresh shoot weight, DSW dry shoot weight, SH shoot height, FRW fresh root weight, 1st root first order root, 2nd + 3rd root second and third order roots
+ + + b
AMF/ AMF/+ AMF/+ AMF/ AMF/+ AMF/+
rs rs rs rs rs rs
Uganda Indonesia Uganda Indonesia
R.s. per gram roots
Root necrosis (%)
F%
I%
252 171 142 79 *
24 B 22 A 34 B 19 A N.S.
39 40 33 N.S.
11 11 12 N.S.
Ba A B A
a b
Capital letters indicate a main effect of the treatment nematode population at P 0.05 *, **, *** indicate a main effect of the treatment mycorrhiza (AMF) at P 0.05, 0.01 and 0.001, respectively
FSW (g) + + b a b
AMF/ AMF/+ AMF/ AMF/+
pc pc pc pc
5.0 4.0 12.4 9.3
aa a c b
DSW (g)
SH (cm)
Foliar surface (cm2)
FRW (g)
1st root weight
2nd + 3rd root weight
In vitro root weight
0.3 0.3 1.2 1.6
10.8 10.6 15.4 17.1 ***
88 74 309 341 ***
2.3 2.1 9.5 8.6 ***
0.6 0.8 2.2 2.5 ***
1.6 1.1 6.4 5.3 ***
0.1 0.2 0.9 0.8 ***
a a b c
Small letters indicate an interaction effect of both treatments at P 0.05 *, **, *** indicate a main effect of the treatment mycorrhiza at P 0.05, 0.01 and 0.001, respectively
The presence/absence of AMF did not influence the root damage, while infection with R. similis (Indonesian population) resulted in significantly (P 0.05) less root necrosis compared to infection with R. similis (Ugandan population; Table 9). Glamus mosseae significantly (P 0.05) suppressed the density of both the Ugandan and Indonesian population. As in experiment 3, the pathogenicity of the nematode populations was confirmed. The presence of the nematodes did not influence the mycorrhizal colonisation.
Interaction of G. mosseae and P. coffeae in Calcutta 4 (Musa AA group; experiment 6) At the end of experiment 6 (i.e. 18 weeks after planting), plants of all mycorrhizal treatments had a significantly (P 0.05) higher fresh and dry shoot weight (Table 10). The mycorrhized plants inoculated with nematodes had a significantly (P 0.05) lower fresh shoot weight and a significantly (P 0.05) higher dry shoot weight compared to the mycorrhized plants without nematodes. Plants of all mycorrhizal treatments were taller, had more leaves and a higher foliar surface. For these parameters, the presence/absence of nematodes had no influence on the shoot growth. In general, the presence of AMF had a positive influence on root growth, while the presence of P. coffeae did not influence root growth at all (Table 10).
374 Table 11 Density of Pratylenchus coffeae (pc) in the root system of Calcutta 4 and the mycorrhizal colonisation of Glomus mosseae (AMF) in the roots, 18 weeks after planting (n =8; experiment 6).
+ +
a a
AMF/ AMF/+ AMF/ AMF/+
pc pc pc pc
Prior to statistical analysis, mycorrhizal data and root necrosis were arcsin (x/100) and nematode counts were log (x+1) transformed. F% Frequency of mycorrhization, I% intensity of mycorrhization
P.c. per gram roots
Root necrosis (%)
F%
I%
234 57 ***
18 35 **
64 48 N.S.
13 12 N.S.
*, **, *** indicate a significant difference according to the Tukey test at P 0.05, 0.01 and 0.001, respectively
The effect of G. mosseae and P. coffeae on the proportion of root weight for all different root types is given in Fig. 2. The presence of P. coffeae significantly reduced branching in the non-mycorrhizal treatments but not in the mycorrhizal treatments. The presence of AMF did not alter the proportions of the different root types. The presence of AMF significantly (P 0.05) reduced the nematode population density (i.e. P. coffeae per gram roots; Table 11). However, due to the significantly (P 0.05) larger root system of the mycorrhized Calcutta 4, the total nematode population was not reduced by AMF. In the presence of AMF, P. coffeae caused significantly (P 0.05) more root necrosis. The mycorrhization of the root systems was successful, but the low intensity indicates the early stage of mycorrhization (Table 11). The presence of nematodes did not influence the mycorrhizal colonisation.
Discussion Each of the seven Musa genotypes included in our study responded positively to inoculation with G. mosseae. Growth response was similar for all parameters. Both root and shoot development benefit from the mycorrhization, corroborating observations of Blomme (2000). Our findings are similar to the results obtained by Jaizme-Vega and Azcon (1995), Pinochet et al. (1997) and Yano-Melo et al. (1999) on Musa species with different Glomus species and under different experimental conditions. Our study confirms the beneficial effect of early mycorrhization in the initial phases of plant growth. However, the magnitude of the response varied among genotypes. A great variation in dependency on mycorrhizal colonisation has previously been observed among Musa spp. (AAA group; Declerck et al. 1995). In our study, RMD also differed among genotypes: the lowest RMD was observed for Calcutta 4 and the highest RMD for Obino l’Ewai (in the first experiment) and Igitsiri (in the second experiment). Although mycorrhizal colonisation was successful in all seven Musa genotypes, some differences were observed. Thus a high RMD was not necessarily related to a good mycorrhizal colonisation. This confirms previous studies where infectivity (i.e. F%) of G. mosseae was not related to effectivity (i.e. RMD; Declerck et al. 1995; Jaizme-Vega and Azcon 1995).
The presence of R. similis or P. coffeae did not influence plant growth. No negative influence of these nematodes was observed either in shoot or in root development. In Musa, R. similis and M. javanica have been previously reported not to influence plant growth in an early stage of infection (Pinochet et al. 1997; Umesh et al. 1988). These root pathogens are destructive to the root system, hampering water and nutrient uptake. Normally, this results in a poorer growth of the infected plant, but under these experimental conditions, time of analysis is probably too early to detect these negative effects on plant growth. Glamus mosseae reduced nematode population buildup for both nematode species, regardless of the pathogenicity of the population. In both genotypes, the presence of AMF increased root necrosis significantly thereby apparently decreasing the tolerance. Only in Calcutta 4 inoculated with R. similis, did the presence of AMF have no influence on root necrosis. Many reports have demonstrated a decrease in nematode population development resulting in an increased resistance and/or tolerance for R. similis and Pratylenchus spp. (Camprubi et al. 1993; Smith and Kaplan 1988; Vaast et al. 1998). Previous studies in Musa reported a suppressive effect of AMF on the reproduction of R. similis and P. coffeae (Umesh et al. 1988). However, it remains difficult to explain the increased root necrosis. In this study, the effect of G. mosseae on the development of the primary roots was ambiguous. For most genotypes, the number of primary roots was higher in the mycorrhized plants. In M. acuminata, Jaizme-Vega et al. (1994) observed an increased number of primary roots and a lower primary root length in presence of AMF. In another endomycorrhizal system in which another monocotyl, i.e. Allium porrum, was involved, similar observations were made (Berta et al. 1990, 1993). Based on our results it appears that the effect of the AMF on the branching of the root system is genotypedependent. Genotypes with a proportionally high secondary and tertiary root weight (like Calcutta 4 and Kayinja) have a rather low mycorrhizal dependency and a high fresh root weight. These findings corroborate the study by Declerck et al. (1995). In addition, the AMF has no influence on the branching of the roots of genotypes with this type of root system. Therefore, it appears that genotypes with a well-developed root system (large, wellbranched root system, good development of root hairs)
375
have no need to establish a mycorrhizal symbiosis. Genotypes with a proportionally high root weight of primary roots (like Obino l’Ewai, Igitsiri, Mbwazirume or Pisang Lilin) have a high to medium mycorrhizal dependency and the presence of AMF increases the branching. In this study, the genotypes representing this type of root system (i.e. high proportion of primary roots) have been cultivated for a long time and are known for their poor anchorage and root growth. It is possible that these genotypes use the AMF symbiosis as a strategy to improve their root growth and the development of their root system. It is generally assumed that a denser root system has a greater absorbing power than an elongate one. Therefore, not only the network of extraradical mycelium with its absorbing power and explorative functions, but also the type of root system could improve the beneficial growth of mycorrhized plants. Moreover, a very branched root system is particularly useful for Musa, as they are easily uprooted by strong winds, especially when the root system is weakened by the presence of nematodes. The presence of a highly pathogenic R. similis or a P. coffeae population influenced the root system of both Obino l’Ewai and Calcutta 4 by reduced branching. Thus the proportion of primary roots increased in mycorrhized plants. However, the total root weight was not affected by the nematodes. The tested R. similis population with a low pathogenicity did not influence the root branching. In Musa, Stoffelen (2000) showed that nematodes can infect all root types, including in vitro roots, but they have a preference for the primary roots. In that study, 70–90% of the nematodes were extracted from the primary roots, but no reduction of primary root weight was observed. However, the nematode infection caused a significant reduction of the secondary and tertiary root weight (Stoffelen 2000). Our findings confirm this for Obino l’Ewai, but for Calcutta 4 no significant reduction was observed. In our study, the effect of the interaction between AMF and nematodes on the root morphology was studied for the first time. The analysis is rather complex since there are many factors involved. The three factors genotype, AMF and nematode influence the root system, making conclusions very difficult. All together, there is no net effect, since the reduced branching caused by the nematodes is counterbalanced by the increased branching caused by AMF. This could be a possible strategy to reduce the negative impact of nematode infection. Acknowledgements The authors would like to thank Dr. M. Jaizme-Vega for providing the Glomus mosseae isolate and Jo Reynders and Wim Dillemans for their technical assistance. This research was financed by a grant from the Katholieke Universiteit Leuven (OT/99/20).
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