Biol Fertil Soils (2003) 38:367–376DOI 10.1007/s00374-003-0669-3

O R I G I N A L P A P E R

Annemie Elsen · Raf Beeterens · Rony Swennen ·Dirk De Waele

Effects of an arbuscular mycorrhizal fungusand two plant-parasitic nematodes on Musa genotypes differingin root morphologyReceived: 14 February 2003 / Accepted: 28 July 2003 / Published online: 3 September 2003� Springer-Verlag 2003

Abstract In this study, the effect of an arbuscularmycorrhizal fungus (AMF) and two migratory endopar-asitic nematodes on Musa plant growth, including the rootsystem, were examined. In addition, the AMF-nematodeinteraction was studied. Seven Musa genotypes withdifferent root systems were selected. Based on theirrelative mycorrhizal dependency, two genotypes (Calcut-ta 4 and Obino l’Ewai) were selected for AMF-nematodeinteraction studies. The experiments were performedunder greenhouse conditions. Mycorrhization with Glo-mus mosseae resulted in a significantly better plantgrowth even in the presence of nematodes. The effect ofAMF on the root system was genotype-dependent andseemed to be related to the relative mycorrhizal depen-dency of the genotype. The nematodes also affected theroot system, decreasing branching. Nematode populationdensities were significantly reduced in the presence ofAMF, except for Pratylenchus coffeae in Obino l’Ewai. Inthe root system, it appeared that the decreased branchingcaused by the nematodes was counterbalanced by theincreased branching caused by the AMF.

Keywords Glomus mosseae · Musa spp. (banana) ·Pratylenchus coffeae · Radopholus similis · Rootmorphology

Introduction

Roots function both as a support system and as thenutrient uptake organ of plants. Root morphology changesin response to the soil environment to minimise themetabolic cost of maintaining the root system. In responseto nutrient-limiting conditions, plants may increase rootfineness, root/shoot ratio or the number or length of the

root hairs (Hetrick 1991). Each of these adaptationsinvolves a different metabolic cost. Mycorrhizae areanother alternative to such changes.

Several studies have shown that arbuscular mycor-rhizal fungus (AMF) colonisation can influence the rootmorphology of host plants (Berta et al. 1993; Tisserant etal. 1992; Yano et al. 1996). Most plants infected withAMF develop a denser root system, with a higher numberof shorter primary roots of a greater diameter (Berta et al.1993; Jaizme-Vega et al. 1994). In most plants, thebranching of roots increases after AMF colonisation(Jaizme-Vega et al. 1994; Schellenbaum et al. 1991;Tisserant et al. 1992). However, less branched rootsystems have been observed in mycorrhizal plants ofAndropogon gerardii (Hetrick et al. 1988) and Gossypiumhirsutum (Price et al. 1989).

Different mechanisms have been suggested for thealteration of root systems following AMF colonisation(Berta et al. 1993). From a physiological point of view, itis well recognised that AMF can increase nutrientabsorption, especially elements with low mobility in thesoil such as P. Other studies have reported changes inphytohormone balance in association with AMF (Allen etal. 1980; 1982; Dannenberg et al. 1992). Anatomically,depression of meristematic activity of mycorrhizal rootapices have been found (Berta et al. 1993).

Arbuscular mycorrhizal fungi are able to alter therooting strategy of plants not only in response to soilfertility but also in response to micro-organisms present inthe rhizosphere (Hetrick 1991). The rhizosphere com-prises both beneficial and pathogenic micro-organisms.Migratory endoparasitic nematodes are pathogens infect-ing roots of many different hosts. Especially in thetropics, they can cause high yield losses (Sasser andFreckman 1987). Infected roots become necrotic, therebyinhibiting nutrient and water uptake and, like in bananaand plantain, weakening the anchorage of the plant. Untilnow, the most commonly used approach to controlnematodes is the use of chemicals. However, nematicidesare expensive and toxic both for the user and the

A. Elsen ()) · R. Beeterens · R. Swennen · D. De WaeleLaboratory of Tropical Crop Improvement,Katholieke Universiteit Leuven,Kasteelpark Arenberg 13, 3001 Leuven, Belgiume-mail: annemie.elsen@agr.kuleuven.ac.beFax: +32-16-321993

environment. The application of AMF could be analternative nematode management strategy.

In Musa, there are indications that genotypes that aremore resistant to migratory endoparasitic nematodes havea larger and denser root system than the susceptiblegenotypes (Blomme 2000; Stoffelen 2000). Until now,only one study has reported on the effect of AMF on rootsystem morphology of Musa plantlets (Jaizme-Vega et al.1994). The most important modifications in root mor-phology induced in Musa plants by AMF are increasedbranching and increased total root length, which lead to adenser rooting pattern. The influence of AMF on the rootsystem of Musa genotypes differing in root morphologyand the influence of the AMF/nematode interaction on theroot system and vice versa have never been studied.

In this study, we examined if AMF can be used tocounter nematode effects by inducing a larger and denserroot system in Musa. The first objective of this study wasto investigate whether the effect of AMF on the rootsystem differed for Musa genotypes with initially differ-ent root systems. Therefore a selection of genotypes wasmade according to the classification by Swennen et al.(1986), based on the contribution of secondary andtertiary root length to the total root length. The secondobjective was to study the interaction between AMF andtwo migratory endoparasitic nematodes, Radopholussimilis (Cobb) Thorne and Pratylenchus coffeae (Zim-mermann) Filipjev and Schuurmans Stekhoven on twoMusa genotypes with a different root system and selectedfor their different response to AMF (high and lowmycorrhizal dependency). The influence of the AMF onthe root system and the influence of the altered rootsystem on nematode reproduction were studied. Sincebiological diversity among R. similis populations has beenreported (Elbadri et al. 2001; Fallas et al. 1995), two R.similis populations differing in pathogenicity were in-cluded in the study.

Materials and methods

Biological materials

Musa tissue culture plants were obtained from the InternationalMusa germplasm collection at the INIBAP Transit Centre (ITC), K.U. Leuven, Belgium (Table 1). The selected genotypes were chosen

for their different root morphology (Stoffelen 2000; Swennen et al.1986). The plant material was proliferated, regenerated and rootedin 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 culturerooms at 27€1�C with continuous light.

The G. mosseae (Nicol. and Gerd.) Gerd. and Trappe isolateused in this study was originally recovered from Pome banana(Musa AAB, Pome group) grown on a biological farm in LosRealejos, Tenerife, Canary Islands, Spain. This native isolate wasmaintained and propagated in sorghum pot cultures (Ferguson andWoodhead 1982). Glamus mosseae has been chosen because thisspecies is a highly effective symbiont of Cavendish (Musa spp.,AAA group) genotypes (Declerck et al. 1995).

Two R. similis populations differing in pathogenicity wereselected: a highly pathogenic population from Uganda and a lesspathogenic population from Indonesia (Elbadri et al. 2001; Fallas etal. 1995). Both populations were originally isolated from Musaroots. The P. coffeae population was isolated from Musa roots inGhana and maintained under monoxenic conditions on carrot discsat 27€1�C in the dark. Prior to inoculation, the nematodes werecollected using the Baermann funnel technique (Hooper 1990).

Experimental design

In the first two experiments (experiments 1 and 2), the microprop-agated Musa plants were transplanted after deflasking to germina-tion trays (60�40�10 cm3) for early mycorrhizal colonisationduring the hardening phase (mycorrhization). The substrateconsisted of a sterilised 2:1 peat:quartz sand mixture. Arbuscularmycorrhizal inoculum consisted of 1.5 kg rhizosphere soil from 6-month-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 kgrhizosphere soil from 6-month-old sorghum pot cultures that werenot colonised by AMF. Ten plants were considered per treatment. Aperiod of 6 weeks allowed the establishment of the mycorrhizalsymbiosis, then eight plants per treatment were transplanted to 1-liter containers filled with sterilised 2:1 peat:quartz sand substrate.For each plant, 1 g Osmocote, a slow release fertiliser, was added tothe substrate. During the experiments, the plants were maintained inthe greenhouse at an ambient temperature of 20–27�C, with a 12-hphotoperiod (170–190 PAR) and a relative humidity of 50–70%.

In the four experiments where interactions between AMF andMusa nematodes were studied (experiments 3–6), the selectedgenotypes were mycorrhized as described. In the treatments withnematodes, 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 nematodeinoculation, the plants were kept in the greenhouse for 8 (wheninoculated 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), inorder to allow nematode reproduction.

Table 1 Musa genotypes usedin this study, and their rootmorphology

Accession name ITC numbera Genome, subspecies/group Root systemb

Calcutta 4 0249 AA, burmannicoides High contribution of tertiary rootsGrande Naine 1256 AAA, Cavendish High contribution of tertiary rootsIgitsiri 0081 AAA, Mutika/Lujugira High contribution of tertiary rootsKayinja � ABB, Pisang Awak High contribution of secondary rootsMbwazirume 0084 AAA, Mutika/Lujugira High contribution of tertiary rootsObino l’Ewai 0109 AAB, Plantain Equal contribution of secondary and

tertiary rootsPisang Lilin 1400 AA, malaccensis derived High contribution of tertiary roots

a ITC number = accession number at the International Musa germplasm collection at the INIBAPTransit Centre, K. U. Leuvenb Classification as proposed by Swennen et al. (1986) and confirmed by Stoffelen (2000)

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Assessment of variables

In the first two experiments (experiments 1 and 2), plant growthwas assessed 16 weeks after planting. Plant height, number ofleaves, foliar surface, fresh and dry shoot weight and fresh rootweight were measured for each plant. The foliar surface wasdetermined using a planimeter. The dry shoot weight wasdetermined after drying the leaves, pseudostem and corm for72 h in an oven at 70�C. The relative mycorrhizal dependency(RMD) was determined by expressing the difference between thedry weight of the mycorrhized plant and the average dry weight ofthe non-mycorrhized plant as a percentage of the dry weight of themycorrhized plant (Plenchette et al. 1983).

Root growth was given by the total root weight, the weight ofthe primary roots, weight of the secondary and tertiary roots andweight of the in vitro roots. The roots present at time of deflasking(i.e. beginning of the experiment) and thus formed under in vitroconditions are considered as in vitro roots. For assessing themycorrhizal colonisation, secondary and tertiary root samples werecollected and stained with 0.05% trypan blue in lactic acid modifiedas described by Koske and Gemma (1989). Twenty 1-cm fine rootsegments per plant were mounted on slides and observed under alight microscope. The frequency of AMF colonisation (F%) wascalculated as the percentage of root segments colonised by eitherhyphae, arbuscules or vesicles. In addition, the intensity ofcolonisation (I%), that is the abundance of hyphae, arbusculesand vesicles in each mycorrhizal root segment, was estimated(Plenchette and Morel 1996).

In the four experiments where interactions between AMF andnematodes were studied (experiments 3–6), plant growth andmycorrhizal colonisation were assessed as described. In thetreatments inoculated with nematodes, nematode damage andnematode reproduction were assessed. At the end of each exper-iment, the percentage root necrosis was measured by scoring five10-cm longitudinally sliced functional primary roots (Speijer andDe Waele 1997). The nematodes in the roots were extracted fromthe root system using a maceration-sieving technique. The repro-ductive potential of the nematodes (total number of nematodes perplant, total number of nematodes per root unit) was determined.

Statistical analysis

The data were analysed with the STATISTICA package (StatsoftInc. 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 nematodesin the four following experiments (experiments 3–6). Data thatwere normally distributed and had homogeneous variances weresubjected to ANOVA (Analysis of Variance).

All plant parameters were analysed by two-way ANOVA, whilethe mycorrhizal data and nematode data were analysed by one-wayANOVA. Prior to analysis, nematode reproduction data werelog(x+1) transformed. Data for root necrosis, frequencies andintensities of mycorrhizal colonisation were arcsin(x/100) trans-formed. Means were separated by the Tukey test (P �0.05). Therelative proportions of the in vitro, primary and, secondary andtertiary roots were compared in a Chi-square test (P � 0.05).

Results

Relative mycorrhizal dependency of genotypes differingin root morphology (experiment 1 and 2)

Seven genotypes were tested in two parallel experimentswith Grande Naine as a reference. Overall, the rootcolonisation with G. mosseae had a beneficial effect onplant growth. The mycorrhized plants had a higher freshand dry shoot weight, were taller, had a larger foliarsurface and had a higher fresh root weight (Table 2).Differences in plant growth were not only genotype-dependent but also parameter-dependent.

The presence of AMF increased the total fresh rootweight by increasing the root weight of the first order

Table 2 Influence of Glomusmosseae on plant growth of fourMusa genotypes differing inroot morphology, 16 weeks af-ter planting (n =6–8; experi-ments 1 and 2). Means areseparated using the Tukey test(P � 0.05). FSW Fresh shootweight, DSW dry shoot weight,SH shoot height, FRW freshroot weight, 1st root first orderroot, 2nd + 3rd root second andthird order roots

FSW(g)

DSW(g)

SH(cm)

Foliarsurface

FRW(g)

1st rootweight

2nd + 3rdrootweight

In vitrorootweight(cm2)

Experiment 1

without AMF

Grande Naine 15 Ba 1.3 ab 9 A 249 B 16.9 A 4.7 AB 11.7 A 0.4 ACalcutta 4 13 A 1.3 a 10 A 246 AB 18.3 A 5.8 B 12.4 A 0.2 AKayinja 15 B 1.4 a 10 AB 210 AB 22.0 B 6.2 B 15.4 B 0.5 BObino l’Ewai 10 A 0.7 a 11 B 184 A 15.7 A 6.0 A 8.9 A 0.8 AB

with AMF

Grande Naine 28 B 3.8 d 12 A 454 B 39.3 A 10.7 AB 28.5 A 0.1 ACalcutta 4 22 A 2.5 b 12 A 402 AB 36.2 A 10.9 B 25.3 A 0 AKayinja 28 B 3.4 cd 13 AB 405 AB 46.3 B 12.5 B 33.2 B 0.6 BObino l’Ewai 24 A 2.8 bc 15 B 374 A 37.2 A 7.7 A 29.5 A 0 ABc *** *** *** *** *** *** N.S.

Experiment 2

without AMF

Grande Naine 16 b 1.8 b 10 A 245 b 12.6 a 5.4 bc 6.7 ab 0.53 abcIgitsiri 8 a 0.7 a 11 B 97 a 5.1 a 1.8 a 3.0 a 0.29 aMbwazirume 12 ab 0.9 a 11 AB 128 a 5.4 a 2.5 ab 2.6 a 0.26 aPisang Lilin 11 ab 0.9 a 12 B 165 a 10.0 a 4.9 abc 4.8 ab 0.29 a

with AMF

Grande Naine 39 e 4.8 e 15 A 477 d 45.1 c 13.2 d 31.5 e 0.44 abIgitsiri 28 cd 3.9 d 18 B 398 c 29.1 b 5.6 bc 21.5 d 2.01 cMbwazirume 31 d 3.0 c 16 AB 362 c 23.5 b 6.2 c 17.1 cd 0.16 aPisang Lilin 23 c 3.0 c 16 B 262 b 23.7 b 10.5 d 11.7 bc 1.53 bcc ***

a Capital letters indicate a main effect of the treatment genotype at P � 0.05b Small letters indicate an interaction effect of both treatments at P � 0.05c *, **, *** indicate a main effect of the treatment mycorrhiza at P � 0.05, 0.01 and 0.001, respectively

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roots and of the secondary and tertiary order roots. Incontrast, the root weight of the in vitro roots was notaltered by the presence of AMF, except for Igitsiri andPisang Lilin. The number of primary roots tended toincrease when AMF were present. In general, the AMFhad a positive influence on the root growth, but the impactdiffered among genotypes.

Figure 1 summarises the proportional weight of thedifferent root types. After 16 weeks, the proportion of invitro roots was very small in both the mycorrhizal andnon-mycorrhizal treatments. The proportion of secondaryand tertiary roots was larger than the proportion ofprimary roots, but some variation among genotypes wasobserved. The non-mycorrhized Pisang Lilin and Mb-wazirume had the largest proportion of primary roots(49% and 46%, respectively), while the mycorrhizedIgitsiri had the smallest proportion of primary roots(19%). The presence of AMF did not influence the rootproportions of Calcutta 4, Kayinja and Pisang Lilin(Fig. 1). For Grande Naine, the results were not veryclear. In the first experiment, the AMF did not influencethe root proportions, while in the second experiment, the

presence of the AMF significantly decreased the primaryroot weight proportionally. For Obino l’Ewai, Igitsiri andMbwazirume, the AMF significantly increased branchingof the root system as indicated by a proportional decreaseof the primary root weight in favour of an increase of thesecondary and tertiary root weight. Mycorrhization withG. mosseae was successful in both experiments, asindicated by a high frequency (F%) and an acceptableintensity (I%) of mycorrhization (Table 3). The mycor-rhization in Grande Naine is comparable in both exper-iments. Based on the relative mycorrhizal dependency,Obino l’Ewai and Calcutta 4 were selected for furtherinteraction studies: Obino l’Ewai as a plantain with a highRMD 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 similisin 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 mycorrhizaltreatments, the plants inoculated with the Indonesian R.similis population showed a significantly (P � 0.05)better plant growth compared to the plants inoculatedwith the Ugandan R. similis population. In general, thepresence of AMF increased the root weight significantly(P � 0.05).

The effect of G. mosseae and R. similis (Ugandan andIndonesian 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 theroots were found.

The presence of the AMF reduced significantly (P �0.05) the nematode population density for both R. similispopulations (Table 5). The root necrosis, caused by thenematodes, was significantly (P � 0.05) higher when

Fig. 1 Effect of Glomus mosseae (AMF) on the proportional rootweight of primary (1st), secondary (2nd) and tertiary (3rd) and invitro roots of four different Musa genotypes 16 weeks after planting(I experiment 1, II experiment 2)

Table 3 Root colonisation by Glomus mosseae and relativemycorrhizal dependency of four Musa genotypes differing in rootmorphology, 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 differenceaccording to the Tukey test (P � 0.05).] F% frequency ofmycorrhization, I% intensity of mycorrhization, RMD relativemycorrhizal dependency

Genotype F% I% RMD (%)

Experiment 1

Grande Naine 50 a 17 a 66 bcCalcutta 4 61 a 20 a 50 aKayinja 56 a 21 a 58 abObino l’Ewai 54 a 15 a 75 c

Experiment 2

Grande Naine 72 b 22 b 62 aIgitsiri 70 b 16 ab 83 cMbwazirume 50 a 14 a 70 bPisang Lilin 33 a 13 a 70 b

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Table 4 Influence of Radopholus similis (rs) and Glomus mosseae(AMF) on the plant growth of Obino l’Ewai, 16 weeks afterplanting (n =8; experiment 3). 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 DSW SH Foliar surface FRW 1st rootweight

2nd + 3rdroot weight

In vitro rootweight(g) (g) (cm) (cm2) (g)

� AMF/� rs 14.1 Aa 1.2 AB 13.3 AB 288 AB 9.5 AB 4.2 B 4.7 B 0.6� AMF/+ rs Uganda 10.0 A 0.8 A 10.5 A 196 A 4.2 A 2.3 A 1.4 A 0.5� AMF/+ rs Indonesia 18.5 B 1.5 B 14.6 B 358 B 10.0 B 4.5 B 4.8 B 0.7+ AMF/� rs 22.7 A 2.3 AB 16.3 AB 494 AB 17.6 AB 6.1 A 10.8 B 0.7+ AMF/+ rs Uganda 22.5 A 2.0 A 15.6 A 453 A 16.3 A 6.9 A 7.6 A 1.7+ AMF/+ rs Indonesia 29.8 B 2.7 B 17.9 B 574 B 20.1 B 8.7 B 10.4 B 1.3b *** *** *** *** *** *** *** **

a Capital letters indicate a main effect of the treatment nematode at P � 0.05b *, **, *** 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 ofprimary (1st), secondary (2nd) and tertiary (3rd) and in vitro roots

of Obino l’Ewai (graphs on the left) and Calcutta 4 (graphs on theright) 16 (when inoculated with R. similis) or 18 weeks (wheninoculated with P. coffeae) after planting

Table 5 Density of Radopholussimilis (rs) in the root system ofObino l’Ewai and the mycor-rhizal colonisation of Glomusmosseae (AMF) in the roots,16 weeks after planting (n =8;experiment 3). Prior to statisti-cal analysis, mycorrhizal dataand root necrosis were arcsin(x/100) and nematode countswere log (x+1) transformed.Means are separated using theTukey test (P � 0.05). F%Frequency of mycorrhization,I% intensity of mycorrhization

R.s. per gram roots Root necrosis (%) F% I%

� AMF/� rs � � � �� AMF/+ rs Uganda 1,336 Ba 41 B � �� AMF/+ rs Indonesia 218 A 19 A � �+ AMF/� rs � � 53 bb 13+ AMF/+ rs Uganda 314 B 44 B 31 a 12+ AMF/+ rs Indonesia 74 A 27 A 58 c 12c *** ** N.S.

a Capital letters indicate a main effect of the treatment nematode population at P � 0.05b Small letters indicate differences in mycorrhization at P � 0.05c *, **, *** indicate a main effect of the treatment mycorrhiza (AMF) at P � 0.05, 0.01 and 0.001,respectively

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Obino l’Ewai was mycorrhized. The differences inpathogenicity between the R. similis populations wereconfirmed. In the treatment with joint inoculation of thesymbiont and Ugandan R. similis population, G. mosseaeachieved a lower frequency of mycorrhizal colonisation(Table 5). However, the intensity of this mycorrhizalcolonisation was not influenced by the presence/absenceof the R. similis populations.

Interaction of G. mosseae and Pratylenchus coffeae inObino 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 plantswithout mycorrhiza (Table 6). The mycorrhized plantswere significantly (P � 0.05) taller and had a greaterfoliar surface than non-mycorrhized plants. For all theseparameters, the absence/presence of the nematodes didnot influence the results. The AMF had a positiveinfluence on the root growth, while the nematodes hadno significant (P � 0.05) effect, except for the root weightof the secondary and tertiary roots (Table 6).

Figure 2 summarises the proportional weight of thedifferent root types. In the non-mycorrhized plants, thepresence of the nematodes caused a significant decreaseof the proportion of secondary and tertiary root weight infavour of an increase of the proportion of primary rootweight. In the mycorrhized plants, no shift in proportionsof root weight were observed when inoculated with P.coffeae. In the absence of nematodes, the proportion ofsecondary and tertiary root weight remained the samewhile the in vitro root weight decreased proportionally in

favour of the primary root weight. When inoculated withP. coffeae, the AMF significantly increased branching ofthe root system.

The nematode population density (i.e. P. coffeae pergram roots) was not influenced by the presence of AMF(Table 7). Therefore, the total P. coffeae populationsignificantly (P � 0.05) increased in the presence of AMFbecause the mycorrhized plants had a considerably higherroot weight. Moreover, the root necrosis caused by thenematodes was significantly (P � 0.05) higher in themycorrhized Obino l’Ewai plants. The nematode infec-tion had no effect on the establishment of the AMF asindicated 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 popula-tion; Table 8). The presence of R. similis (Ugandan orIndonesian population) had no influence on the plantgrowth.

Figure 2 summarises the proportional weight of thedifferent root types. The presence of R. similis (Ugandanand Indonesian population) did not influence the branch-ing of the root system. Mycorrhizal colonisation signif-icantly increased the proportion of secondary and tertiaryroots in control plants and plants infected with R. similis(Ugandan population). For R. similis (Indonesian popu-lation), no significant effect of the nematode on the rootmorphology was observed.

Table 6 Influence of Praty-lenchus coffeae (pc) and Glo-mus mosseae (AMF) on theplant growth of Obino l’Ewai,18 weeks after planting (n =8;experiment 4). Means are sepa-rated using the Tukey test (P �0.05). FSW Fresh shoot weight,DSW dry shoot weight, SHshoot height, FRW fresh rootweight, 1st root first order root,2nd + 3rd root second and thirdorder roots

FSW DSW SH Foliar surface FRW 1st rootweight

2nd + 3rdrootweight

In vitroroot weight(g) (g) (cm) (cm2) (g)

� AMF/� pc 7.0 0.4 12.4 155 4.6 1.2 2.8 Ba 0.6� AMF/+ pc 7.3 0.4 13.1 127 3.7 1.5 1.5 A 0.7+ AMF/� pc 15.1 1.2 14.1 303 11.5 3.8 7.5 B 0.2+ AMF/+ pc 14.3 1.1 15.4 299 10.8 3.5 6.6 A 0.7b *** *** ** *** ** *** *** N.S.

a Capital letters indicate a main effect of the treatment nematode at P � 0.05b *, **, *** 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 ofObino l’Ewai and the mycorrhizal colonisation of Glomus mosseae(AMF) in the roots, 18 weeks after planting (n =8; experiment 4).

Prior to statistical analysis, mycorrhizal data and root necrosis werearcsin (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%

� AMF/� pc � � � �� AMF/+ pc 100 20 � �+ AMF/� pc � � 89 17+ AMF/+ pc 74 38 89 15a N.S. *** N.S. N.S.

a *, **, *** indicate a significant difference according to the Tukey test at P � 0.05, 0.01 and 0.001, respectively

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The presence/absence of AMF did not influence theroot damage, while infection with R. similis (Indonesianpopulation) resulted in significantly (P � 0.05) less rootnecrosis compared to infection with R. similis (Ugandanpopulation; Table 9). Glamus mosseae significantly (P �0.05) suppressed the density of both the Ugandan andIndonesian population. As in experiment 3, the pathoge-nicity of the nematode populations was confirmed. Thepresence of the nematodes did not influence the mycor-rhizal 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). Themycorrhized plants inoculated with nematodes had asignificantly (P � 0.05) lower fresh shoot weight and asignificantly (P � 0.05) higher dry shoot weight com-pared to the mycorrhized plants without nematodes.Plants of all mycorrhizal treatments were taller, had moreleaves and a higher foliar surface. For these parameters,the presence/absence of nematodes had no influence onthe shoot growth. In general, the presence of AMF had apositive influence on root growth, while the presence ofP. coffeae did not influence root growth at all (Table 10).

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 �

0.05). FSW Fresh shoot weight, DSW dry shoot weight, SH shootheight, FRW fresh root weight, 1st root first order root, 2nd + 3rdroot second and third order roots

FSW DSW SH Foliar surface FRW 1st rootweight

2nd + 3rd rootweight

In vitro rootweight(g) (g) (cm) (cm2) (g)

� AMF/� rs 8.3 ab 0.8 ab 10.7 Ba 185 abb 5.4 ab 2.5 ab 2.5 ab 0.4� AMF/+ rs Uganda 2.9 a 0.3 a 8.1 A 72 a 1.6 a 0.6 a 0.6 a 0.4� AMF/+ rs Indonesia 11.6 b 1.2 b 12.4 B 286 b 7.6 bc 3.2 b 3.9 ab 0.6+ AMF/� rs 15.2 b 1.5 b 15.7 B 353 b 13.0 c 4.2 b 8.5 c 0.3+ AMF/+ rs Uganda 15.7 b 1.7 b 14.2 A 366 b 12.6 c 5.3 b 6.8 bc 0.6+ AMF/+ rs Indonesia 15.6 b 1.6 b 15.0 B 341 b 8.6 bc 4.2 b 3.9 ab 0.5c *** n.s.

a Capital letters indicate a main effect of the treatment nematode at P � 0.05b Small letters indicate an interaction effect of both treatments at P � 0.05c *, **, *** indicate a main effect of the treatment mycorrhiza at P � 0.05, 0.01 and 0.001, respectively

Table 9 Density of Radopholussimilis (rs) in the root system ofCalcutta 4 and the mycorrhizalcolonisation of Glomus mosseae(AMF) in the roots, 16 weeksafter planting (n =8; experiment5). Prior to statistical analysis,mycorrhizal data and root ne-crosis were arcsin (x/100) andnematode counts were log (x+1)transformed. Means are sepa-rated using the Tukey test (P �0.05). F% Frequency of myc-orrhization, I% intensity ofmycorrhization

R.s. per gram roots Root necrosis (%) F% I%

� AMF/� rs � � � �� AMF/+ rs Uganda 252 Ba 24 B � �� AMF/+ rs Indonesia 171 A 22 A � �+ AMF/� rs � � 39 11+ AMF/+ rs Uganda 142 B 34 B 40 11+ AMF/+ rs Indonesia 79 A 19 A 33 12b * N.S. N.S. N.S.

a Capital letters indicate a main effect of the treatment nematode population at P � 0.05b *, **, *** indicate a main effect of the treatment mycorrhiza (AMF) at P � 0.05, 0.01 and 0.001,respectively

Table 10 Influence of Praty-lenchus coffeae (pc) and Glo-mus mosseae (AMF) on theplant growth of Calcutta 4,18 weeks after planting (n =8;experiment 6). Means are sepa-rated using the Tukey test (P �0.05). FSW Fresh shoot weight,DSW dry shoot weight, SHshoot height, FRW fresh rootweight, 1st root first order root,2nd + 3rd root second and thirdorder roots

FSW DSW SH Foliar surface FRW 1st rootweight

2nd + 3rdrootweight

In vitrorootweight

(g) (g) (cm) (cm2) (g)

� AMF/� pc 5.0 aa 0.3 a 10.8 88 2.3 0.6 1.6 0.1� AMF/+ pc 4.0 a 0.3 a 10.6 74 2.1 0.8 1.1 0.2+ AMF/� pc 12.4 c 1.2 b 15.4 309 9.5 2.2 6.4 0.9+ AMF/+ pc 9.3 b 1.6 c 17.1 341 8.6 2.5 5.3 0.8b *** *** *** *** *** ***

a Small letters indicate an interaction effect of both treatments at P � 0.05b *, **, *** indicate a main effect of the treatment mycorrhiza at P � 0.05, 0.01 and 0.001, respectively

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The effect of G. mosseae and P. coffeae on theproportion of root weight for all different root types isgiven in Fig. 2. The presence of P. coffeae significantlyreduced branching in the non-mycorrhizal treatments butnot in the mycorrhizal treatments. The presence of AMFdid not alter the proportions of the different root types.

The presence of AMF significantly (P � 0.05) reducedthe nematode population density (i.e. P. coffeae per gramroots; 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 theroot systems was successful, but the low intensityindicates the early stage of mycorrhization (Table 11).The presence of nematodes did not influence the mycor-rhizal colonisation.

Discussion

Each of the seven Musa genotypes included in our studyresponded positively to inoculation with G. mosseae.Growth response was similar for all parameters. Both rootand shoot development benefit from the mycorrhization,corroborating observations of Blomme (2000). Our find-ings are similar to the results obtained by Jaizme-Vegaand Azcon (1995), Pinochet et al. (1997) and Yano-Meloet al. (1999) on Musa species with different Glomusspecies and under different experimental conditions. Ourstudy confirms the beneficial effect of early mycorrhiza-tion in the initial phases of plant growth. However, themagnitude of the response varied among genotypes.

A great variation in dependency on mycorrhizalcolonisation has previously been observed among Musaspp. (AAA group; Declerck et al. 1995). In our study,RMD also differed among genotypes: the lowest RMDwas observed for Calcutta 4 and the highest RMD forObino l’Ewai (in the first experiment) and Igitsiri (in thesecond experiment). Although mycorrhizal colonisationwas successful in all seven Musa genotypes, somedifferences were observed. Thus a high RMD was notnecessarily 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 notinfluence plant growth. No negative influence of thesenematodes was observed either in shoot or in rootdevelopment. In Musa, R. similis and M. javanica havebeen previously reported not to influence plant growth inan early stage of infection (Pinochet et al. 1997; Umesh etal. 1988). These root pathogens are destructive to the rootsystem, hampering water and nutrient uptake. Normally,this results in a poorer growth of the infected plant, butunder these experimental conditions, time of analysis isprobably too early to detect these negative effects on plantgrowth.

Glamus mosseae reduced nematode population build-up for both nematode species, regardless of the pathoge-nicity of the population. In both genotypes, the presenceof AMF increased root necrosis significantly therebyapparently decreasing the tolerance. Only in Calcutta 4inoculated with R. similis, did the presence of AMF haveno influence on root necrosis. Many reports havedemonstrated a decrease in nematode population devel-opment resulting in an increased resistance and/or toler-ance 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 effectof AMF on the reproduction of R. similis and P. coffeae(Umesh et al. 1988). However, it remains difficult toexplain the increased root necrosis.

In this study, the effect of G. mosseae on thedevelopment of the primary roots was ambiguous. Formost genotypes, the number of primary roots was higherin the mycorrhized plants. In M. acuminata, Jaizme-Vegaet al. (1994) observed an increased number of primaryroots and a lower primary root length in presence ofAMF. In another endomycorrhizal system in whichanother 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 theAMF on the branching of the root system is genotype-dependent. Genotypes with a proportionally high second-ary and tertiary root weight (like Calcutta 4 and Kayinja)have a rather low mycorrhizal dependency and a highfresh root weight. These findings corroborate the study byDeclerck et al. (1995). In addition, the AMF has noinfluence on the branching of the roots of genotypes withthis type of root system. Therefore, it appears thatgenotypes with a well-developed root system (large, well-branched root system, good development of root hairs)

Table 11 Density of Pratylenchus coffeae (pc) in the root systemof Calcutta 4 and the mycorrhizal colonisation of Glomus mosseae(AMF) in the roots, 18 weeks after planting (n =8; experiment 6).

Prior to statistical analysis, mycorrhizal data and root necrosis werearcsin (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%

� AMF/� pc � � � �� AMF/+ pc 234 18 � �+ AMF/� pc � � 64 13+ AMF/+ pc 57 35 48 12a *** ** N.S. N.S.

a *, **, *** indicate a significant difference according to the Tukey test at P � 0.05, 0.01 and 0.001, respectively

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have no need to establish a mycorrhizal symbiosis.Genotypes with a proportionally high root weight ofprimary roots (like Obino l’Ewai, Igitsiri, Mbwazirume orPisang Lilin) have a high to medium mycorrhizaldependency and the presence of AMF increases thebranching. In this study, the genotypes representing thistype of root system (i.e. high proportion of primary roots)have been cultivated for a long time and are known fortheir poor anchorage and root growth. It is possible thatthese genotypes use the AMF symbiosis as a strategy toimprove their root growth and the development of theirroot system.

It is generally assumed that a denser root system has agreater absorbing power than an elongate one. Therefore,not only the network of extraradical mycelium with itsabsorbing power and explorative functions, but also thetype of root system could improve the beneficial growthof mycorrhized plants. Moreover, a very branched rootsystem is particularly useful for Musa, as they are easilyuprooted by strong winds, especially when the rootsystem 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 bothObino l’Ewai and Calcutta 4 by reduced branching. Thusthe proportion of primary roots increased in mycorrhizedplants. However, the total root weight was not affected bythe nematodes. The tested R. similis population with a lowpathogenicity did not influence the root branching. InMusa, Stoffelen (2000) showed that nematodes can infectall root types, including in vitro roots, but they have apreference for the primary roots. In that study, 70–90% ofthe nematodes were extracted from the primary roots, butno reduction of primary root weight was observed.However, the nematode infection caused a significantreduction of the secondary and tertiary root weight(Stoffelen 2000). Our findings confirm this for Obinol’Ewai, but for Calcutta 4 no significant reduction wasobserved.

In our study, the effect of the interaction betweenAMF and nematodes on the root morphology wasstudied for the first time. The analysis is rather complexsince there are many factors involved. The three factorsgenotype, AMF and nematode influence the rootsystem, making conclusions very difficult. All together,there is no net effect, since the reduced branchingcaused by the nematodes is counterbalanced by theincreased branching caused by AMF. This could be apossible strategy to reduce the negative impact ofnematode infection.

Acknowledgements The authors would like to thank Dr. M.Jaizme-Vega for providing the Glomus mosseae isolate and JoReynders and Wim Dillemans for their technical assistance. Thisresearch was financed by a grant from the Katholieke UniversiteitLeuven (OT/99/20).

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