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Interactions between arbuscularmycorrhizae and Fusarium oxysporumf. sp. ciceris: effects on fungaldevelopment, seedling growth and wiltdisease suppression in Cicer arietinumL.Ashok Shuklaab, Keerti Dehariyaa, Deepak Vyasa & Anuradha Jhab

a Department of Botany, Dr. Hari Singh Gour Central University,Sagar, Indiab National Research Centre for Agroforestry, Jhansi, IndiaPublished online: 17 Feb 2014.

To cite this article: Ashok Shukla, Keerti Dehariya, Deepak Vyas & Anuradha Jha , Archives OfPhytopathology And Plant Protection (2014): Interactions between arbuscular mycorrhizae andFusarium oxysporum f. sp. ciceris: effects on fungal development, seedling growth and wiltdisease suppression in Cicer arietinum L., Archives Of Phytopathology And Plant Protection, DOI:10.1080/03235408.2014.884831

To link to this article: http://dx.doi.org/10.1080/03235408.2014.884831

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RESEARCH ARTICLE

Interactions between arbuscular mycorrhizae and Fusariumoxysporum f. sp. ciceris: effects on fungal development, seedlinggrowth and wilt disease suppression in Cicer arietinum L.

Ashok Shuklaa,b*, Keerti Dehariyaa, Deepak Vyasa and Anuradha Jhab

aDepartment of Botany, Dr. Hari Singh Gour Central University, Sagar, India;bNational Research Centre for Agroforestry, Jhansi, India

(Received 8 January 2014; accepted 9 January 2014)

The purpose of present study was to develop a management strategy based on a timeeffective inoculation of arbuscular mycorrhizal fungi (AMF) to mitigate the yieldlosses of Cicer arietinum L. due to Fusarium oxysporum f. sp. ciceris (Foc). Theinteractions between AMF (mycorrhizal consortium; Myc) and Foc were studied inthree separate experiments in two successive years (2011 and 2012). In particular,we investigated: the effect of Myc on population density of Foc, the effect of Foc onmycorrhisation (root colonisation index and AMF spore density/50 g sand) and theinteractive effects of Myc and Foc on growth, phosphorus (P) content and diseaseseverity index of C. arietinum. Results suggested that pre-inoculating plants withAMF (Myc + Foc) considerably reduced Foc population density, while combined(Myc/Foc) and early inoculation of AMF (Myc + Foc) increased mycorrhisation,growth and P content of plants. Combined and early inoculation of AMF reduceddisease severity index up to 68 and 89.5%, respectively. Thus, the results suggestedthat soil pretreated with AMF acted as bioprotectant against the Fusarium. In conclu-sion, Myc should be inoculated before transplantation of crop seedlings to the fields.However, extrapolation of the results to the real field conditions should be done withcaution because of differences in growth conditions and substrate used in presentstudy i.e. net house and sand, respectively.

Keywords: arbuscular mycorrhizal fungi; bioprotection; chickpea; Fusarium; wiltdisease

Introduction

Cicer arietinum L. (chickpea) is an important legume crop and has become an integralpart of a sustainable production system. India has variable trends of chickpea productionthat have impacted on production over the years. It is being grown in almost 6.93 mil-lion ha which contribute 62% of global production (Shakya et al. 2008). MadhyaPradesh is a leading chickpea producing state among India and contributed to the 39%of the total production. In the recent past, farmers of this region have reported that itsproduction has declined (Singh et al. 2010a). Several biotic and abiotic factors can beresponsible for the destruction of agricultural economy, where plant diseases play adirect role (Akhtar & Siddiqui 2008). Chickpea is susceptible to a relatively largenumber of pathogens. Among these pathogens, Fusarium, Verticillium and Rhizoctonia

*Corresponding author. Email: ashokshukla06@gmail.com

© 2014 Taylor & Francis

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are predominant. Endemic occurrence of wilt disease caused by Fusarium oxysporum f.sp. ciceris [Foc (Padwick)] Matuo and K. Sato is relevant (Akhtar & Siddiqui 2007).

Fusarium is a soil-borne fungus which survives through chlamydospores in seedsand dead plant debris. The fungus infects through roots and penetrates into the vascularsystem, causing wilt and substantially reducing yields. Since Foc can survive in soil forseveral years, the use of fungicide to control the disease is impractical. Moreover, abusein pesticide deployment has favoured the development of resistant pathogens (Zeng2006). In order to prevent disease and protect the crop against pathogen, an alternativeto the chemical control is needed to mitigate the losses due to Fusarium (Borrero et al.2004). Currently, bioprotection is a well-documented process and has becomeincreasingly crucial, and in several cases, complementary or even replacing the chemicalcounterparts (Singh & Vyas 2009).

Arbuscular mycorrhizal fungi (AMF) have enormous potential for use as bioprotectant(Karagiannidis et al. 2002; Xavier & Boyetchko 2004). AMF-mediated bioprotection hasbeen exploited and accepted as a key practice for disease control (Garmendia et al. 2005;Garcia-Garrido 2009). According to Sikes (2010), the bioprotective effect of AMFdepends upon various factors such as host genotype, AMF species involved and theefficiency of root colonisation. Furthermore, several researchers have suggested thatestablishment of AMF in plant roots reduces the damage caused by pathogens, andimproves plant resistance to the biotic stresses (Garmendia et al. 2004; Vierheilig & Bago,2005), but the underlying mechanisms of bioprotection remain unknown (Pozo & Azcón-Aguilar 2007). According to Pozo et al. (2009), several mechanisms such as plant nutri-tion; damage compensation; competition for colonisation sites and photosynthates;changes in root architecture and rhizosphere microbial populations; and activation of plantdefence mechanisms, may play roles in plant protection by AMF. These mechanisms arebased on research using experimental systems that vary between researchers; hence, theresults not allowing final conclusions (Singh et al. 2010b). McAllister et al. (1994) sug-gested that the method and the time of inoculation with AMF can be important factors.Thus, the present study was carried out to develop a management strategy with timeeffective AMF (mycorrhizal consortium; Myc) inoculation to mitigate the yield losses ofchickpea due to Fusarium wilt. The interactions between Myc and pathogen (Foc) werestudied in three separate experiments with individual and combined inoculations of Mycand Foc. During the course of study, the effect of Myc on population density i.e. colonyforming unit (CFU) of Foc, the effect of Foc on mycorrhisation (root colonisation indexand AMF spore density/50 g sand), and the interactive effects of Myc and Foc on growth,phosphorus (P) content and disease severity index of chickpea were investigated.

Materials and methods

Present study was carried out in two successive years i.e. 2011 and 2012, under nethouse conditions at the Department of Botany, Dr. Hari Singh Gour Central University,Sagar (23°10′–24°27′ N latitude and 78°4′–79°21′ E longitude). Sagar is situated atNorth of Tropic of Cancer on an average altitude of 580 m above mean sea level. Theregion lies in agro-ecoregion 10; central highlands, hot sub-humid ecoregion withmedium and deep black soils (I5C3).

Biological materials

Seeds of C. arietinum (cv. JG 62) were provided by National Research Centre for Agro-forestry (NRCAF), Jhansi. These were surface sterilised in 0.1% sodium hypochlorite

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for 2 min, washed several times in distilled water and germinated on sterilised sand. Theselected cultivar is very common among the farmers and is being grown in this region.

Myc comprised of equal parts in weight of three indigenous AMF species, namelyFunneliformis mosseae (Nicolson and Gerd.) Walker and Schubler, Glomus fasciculatumThaxter and Rhizophagus irregularis (Blaszk., Wubet, Renker and Buscot) Walker andSchubler were used in all experiments which consisted of sand with chopped rootbits, spores and extramatrical mycelia. All these species were isolated locally andpropagated as pure culture on Zea mays L. for 5 months in sterilised sand. F. mosseae,G. fasciculatum and R. irregularis colonised 61, 74 and 52% of the root length ofZ. mays, respectively, as assessed microscopically.

A wilt causing virulent strain of Foc, isolated from heavily infected chickpea fieldsof the region was used in this study. The purified culture of Foc grown on potato dex-trose agar (PDA) was multiplied on sand maize flour medium. Fifteen grams of maizeflour and 85 g autoclaved sand were thoroughly mixed in 250 mL flasks (100 g/flask)and autoclaved at 15 Lbs for 30 min. Then, each flask was inoculated with pure cultureof Foc and incubated at 25 ± 2 °C for 2 weeks.

Pathogenicity of Foc inoculum

The pathogenicity of Foc was confirmed by sick soil inoculation technique (100 g inoc-ulum/3 kg sterilised sand) in four plastic pots (size: 24 × 16 cm). Each pot was seededwith five seeds of susceptible cultivar, JG 111 (provided by NRCAF, Jhansi). All thepots were watered as and when required and kept under net house conditions. Theobservation on wilt incidence was recorded after 15 d of sowing up to wilting (resultsnot presented in this communication). Re-isolation of fungus was done from diseasedplants, showing wilting symptoms. The fungus growth obtained was transferred on PDAslants for comparison with original Foc culture, and identified on the basis of morpho-logical and cultural characteristics (Nelson et al. 1983).

Treatments, inoculations and planting

Fifteen grams of Myc (average 96 spores/50 g sand) and Foc inocula (average 1.5 × 103

CFU/15 g sand) were applied in substrate cavity (3 kg sterilised sand/pot). In presentstudy, individual (Myc or Foc alone) and combined inoculations [Myc/Foc (co-inocula-tion), Myc + Foc (pretreated with Myc then after 1 week Foc was introduced) and Foc +Myc (pretreated with Foc then after 1 week Myc was introduced)], were performed.Individual inoculations (Myc or Foc) were done by introducing either Myc or Foc aloneat the time of planting, while combined inoculation (Myc/Foc) was carried out by simul-taneous inoculation of Myc and Foc in pots at planting. In Myc + Foc and Foc +Myctreated pots, sand around the root system was carefully removed after 1 week of Myc orFoc inoculation; and then, inoculum of Foc or Myc was poured in and sand wasreplaced back. Pots filled with sterilised sand served as a control in all the experiments.

Pre-germinated seedlings (grown on sterilised sand in plastic trays; size: 45 × 30 cm)of similar size were transplanted to the holes made in substrate cavity. The pots werecompletely randomised on net house benches and watered daily. Half strengthHoagland’s solution in de-ionised water was applied at weekly intervals. Thecomposition of the solution was: 0.03 g/L KH2PO4, 0.51 g/L KNO3, 0.246 g/LCa(NO3)2, 0.245 g/L MgSO4.7H2O, 1.43 g/L H3BO3, 0.91 g/L MnCl2.7H2O, 0.11 g/LZnSO4.5H2O, 0.04 g/L CuSO4.5H2O and 0.04 g/L H2MoO4.

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Experimental trials

Experiment 1: effect of Myc on population density of Foc

The following treatments with five replications each were included in this study: Focalone, Foc +Myc, Myc + Foc, Myc/Foc and un-inoculated (control). To evaluate theeffect of Myc on Foc population density, ~15 g sand were collected from each pot (25pots) 4, 8 and 12 weeks after inoculation (WAI). Sampling was done by removing toplayer of sand with a sterilised spoon from three different sides (approximately 5 g fromeach side) of the seedling. Collected samples were mixed gently to make a compositesample and 1 g sample was taken for further study. Then, a 10-fold aqueous dilution ser-ies (10−1–10−4) was prepared. One millilitre of each dilution was pipetted on Petridishes containing Fusarium-selective medium i.e. Komada’s medium (Komada 1975,1976) and incubated at 25 ± 2 °C for 2 weeks. Colonies developed on medium werecounted in suitable dilution (i.e. 10−2) and multiplied by dilution factor. The number ofCFU was expressed per g of dry sand.

Experiment 2: effect of Foc on mycorrhisation

In this study, the effect of Foc on mycorrhisation (root colonisation index and AMFspore density/50 g sand) was investigated. The trial consisted of five treatments viz.Myc alone, Myc + Foc, Myc/Foc, Foc +Myc and un-inoculated (control) which werereplicated five times (total 25 pots). Root and rhizosphere sand samples were taken fromeach pot 4, 8 and 12WAI, for observations. Fine roots were cleared with 10% KOHand stained with acid fuchsin (0.01% in lacto-glycerol) following the method ofKormanik et al. (1980). Colonisation in cleared root parts was determined with a micro-scope (Nikon Eclipse 400) at × 100 using the grid line intersect method (Giovannetti &Mosse 1980). Rhizospheres of each pot were sampled (after removing top sand layer)from five different sides (20 g per side) of growing seedling, which were mixedaltogether to make a composite sample. Then, ~50 g samples were used to enumeratethe AMF spore density (Gerdemann & Nicolson 1963). Collected samples were taken ina substantial amount of water (1 L) and decanted through a series of sieves (mesh sizes:250, 150 and 53 μ). Sievings were individually collected in separate jars. For observa-tion under a stereomicroscope (Nikon SMZ 800), the sievings were transferred on to agridded Petri dish (11 cm). Then, we isolated the AMF spores (Gerdemann & Nicolson1963) and calculated the spore density (number of spores/50 g sand).

Experiment 3: interactive effects of Myc and Foc on growth, P content and diseaseseverity index

The trial consisted of six treatments viz., Foc alone, Foc +Myc, Myc alone, Myc + Foc,Myc/Foc and un-inoculated (control), and each treatment was replicated five times (total30 pots). Seedlings were harvested after 12 weeks growth (one per replicate), and obser-vations on shoot length (cm) and fresh weight (g)/plant were taken by standard meth-ods. For dry weight (g)/plant estimation, harvested samples were dried in sun; and then,in hot air oven at 70 °C. The dried samples were ground to pass through 1 mm sieve,and stored in polyethylene bags (composite of all plant parts) for P analysis. P contentof plant was determined using vanado-molybdo phosphoric yellow colour method(Jackson 1973) with a UV–Vis spectrophotometer (Halo DB 20, Double Beam, made inAustralia) at 420 nm, and expressed in mg/plant on the basis of dry weight.

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To study the interactive effects of Myc and Foc on wilt disease severity index inC. arietinum (cv. JG 111), similar set of experiments were carried out with aforesaidtreatments (i.e. six) in two successive years i.e. 2011 and 2012. All the treatments werereplicated three times where one plant was maintained per pot, and observations weretaken after 1 month of inoculation at 7 d intervals up to complete wilting of the seed-lings. The severity of disease on individual plants were rated on a scale from 0 to 4point scale; where 0 = 0% (no wilting), 1 = 1–33%, 2 = 34–66%, 3 = 67–100% and 4 =dead plant. The disease severity index at the time of harvesting is being presented here.

Statistical analysis

Per cent root colonisation data were arcsine transformed to normalise their distribution.For each factor analysed, the means of the different treatments were compared andranked using Fischer F-test (p < 0.05). All the graphs were prepared by using statisticalpackage SYSTAT version 12.

Results and discussion

Experiment 1: effect of Myc on population density of Foc

In both the studied years, the effect of Myc on Foc population density changed overtime. In 2011, differences were significant (F-ratio 1051.427; p < 0.01) among the treat-ments, at 4WAI (Figure 1). Least CFU count (90.0 ± 4.12/g sand) was recorded in potsinfested with Foc alone. It was highest in pots pretreated with Foc (110.8 ± 3.96/g sand).However, after 8 weeks growth, values were highest in pots infested with Foc alone(73.2 ± 4.82/g sand) and least in pots pretreated with Foc (59.2 ± 2.28/g sand). After 12weeks, CFU was highest in Foc inoculated pots (61.6 ± 5.32/g sand), followed by com-bined inoculation (45.6 ± 7.02/g sand) and least in pots where Myc was introduced 1week earlier to Foc (Myc + Foc; 27.2 ± 1.92/g sand). Data on Foc population density inthe year 2012 are also presented in the same figure. At 4WAI, minimum CFU was

Figure 1. Effect of Myc on CFU of Foc in rhizosphere of chickpea cv. JG 62 in the years 2011and 2012, after 4, 8 and 12 weeks of inoculation. Foc: individual inoculation, Foc +Myc: pre-treated with Foc then after 1 week Myc was introduced, Myc + Foc: pretreated with Myc thenafter 1 week Foc was introduced, Myc/Foc: combined inoculation of Myc and Foc, Un: un-inocu-lated (control); WAI: weeks after inoculation; Bars represent standard error of the difference inmeans (SED), n = 5.

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recorded in Myc pretreated pots (85.5 ± 4.74/g sand). During subsequent periods, i.e. 8and 12WAI, similar trends were noticed as in the year 2011.

Thus, results suggested that initially Foc population density increased but laterdecreased with the age of the plant. This could be due to the quick colonisation of rhi-zosphere by extra-radical mycelium of AMF which create competitive environment forspace and resources for the pathogen (Brimner & Boland 2003). AMF can reduce theabundance of pathogenic fungi up to some extent by direct (interference competition)and indirect (exploitation competition) interactions (Wehner et al. 2010). Graham (2001)suggested that mycorrhizal fungi and pathogens frequently share same rhizospherewhere pathogens often have competitive advantage because of their saprophytic ability,but later on the situation changes as AMF got established in plant roots and preventpenetration of pathogens (Caron et al. 1986). Further, as per observed results, Foc popu-lation density reduced significantly when mycorrhiza got established in pots (at 12WAI). Whipps (2004) postulated that establishment of AMF reduces the population ofpathogen and its effectiveness depends upon AMF species involved, as well as the plantspecies (Linderman 2000). According to Linderman (1988) and Gryndler (2000), mycor-rhiza inoculated plants have specific modifications in their rhizosphere (i.e. mycorrhizo-sphere) where mycorrhiza produce substances which are capable of interfering withmicro-organisms (Lioussanne et al. 2008). The direct putative effects, different rootarchitecture and altered physiology due to mycorrhizal establishment can also explainthe quantitative changes in microbial populations (Filion et al. 1999; Toljander et al.2007; Finlay 2008; Vierheilig et al. 2008).

Experiment 2: effect of Foc on mycorrhisation

Early inoculation of Foc significantly (p < 0.01) reduced root colonisation index (arc-sine-transformed value of colonisation index) and spore density at 4, 8 and 12WAI, inboth the studied years (Figure 2). In 2011, highest colonisation index was recorded inpots infested with Myc alone, followed by Myc/Foc and Myc + Foc, at all the samplingdates. At 4 and 8WAI, differences in values of colonisation index were significant (p <0.01) among the mycorrhiza inoculated pots (i.e. Myc, Myc/Foc and Myc + Foc) butlater (12WAI) such differences were comparable between Myc/Foc and Myc + Foc.Similarly, significantly higher spore density was recorded in pots infested with Mycalone, followed by Myc + Foc, at all sampling dates. Data on mycorrhisation in year2012 are also presented in the same figure. At 4WAI, root colonisation index (36.38 ±0.85) and spore density (53.0 ± 4.6/50 g sand) were significantly higher in Myc aloneinoculated pots, but in subsequent stages i.e. 8 and 12WAI, root colonisation (43.21 ±1.35 and 45.41 ± 0.84, respectively) and spore density (60.5 ± 8.3/50 g sand and 79.0 ±5.6/50 g sand, respectively) were maximum in Myc pretreated pots. In general, the my-corrhisation was higher in the year 2012 as compared to the data obtained in the year2011.

Thus, the results suggested that Foc did not reduce mycorrhisation when AMF waspreviously established. Establishment of mycorrhiza in plant roots induces the synthesisof phytoalexin and the extent of its production is positively correlated with the degreeof AMF colonisation (Zeng 2006). According to Whipps (2004), several mechanisms ofmycorrhizal establishment might be operative simultaneously; the most important ofthese is exclusion which seems to be a simple case of competition for space andresources (Azcon-Aguilar & Barea 1996). Once mycorrhizal fungi colonise the rootcells, pathogen is excluded from that cell, and as a consequence, the most effective

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control against pathogen is achieved (Matsubara et al. 2001; Sylvia & Chellemi 2001).As we already mentioned that in present study, consortium of AMF genera was used asmycorrhizal representative which could be the reason of higher root colonisation andspore density. According to Wehner et al. (2010), increased richness of AMF taxa canbe responsible for extensive mycorrhisation as compared to individual AMF inoculant.Jansa et al. (2008) postulated that consortium of mycorrhiza having multiple AMF spe-cies vary in competitive ability. Our results also suggested that the sequence of AMFinoculation might be an important factor. Hence, we deduced that pre-transplant inocula-tion with mycorrhizal fungi can be a way to give efficient strains an immediate spatialadvantage over the indigenous fungi which have to compete for root space. Earlier, wealso observed that pre-inoculation of AMF have greater potential as biocontrol agent inLycopersicon esculentum (Singh et al. 2010c). Sylvia and Chellemi (2001) alsosuggested that an effective control can be achieved when colonisation by AMF tookplace before attack of the pathogens.

Experiment 3: interactive effects of Myc and Foc on plant growth, P content anddisease severity index

In the year 2011, significant reductions in growth and P content of plants were observedafter inoculating Foc (individual inoculation). However, the recorded values were

Figure 2. Interactions between Myc and Foc, and their effects on root colonisation index andAMF spore density/50 g sand in rhizosphere of chickpea cv. JG 62 in the years 2011 and 2012,after 4, 8 and 12 weeks of inoculation. Foc +Myc: pretreated with Foc then after 1 week Myc wasintroduced, Myc: individual inoculation, Myc + Foc: pretreated with Myc then after 1 week Focwas introduced, Myc/Foc: combined inoculation of Myc and Foc, Un: un-inoculated (control);WAI: weeks after inoculation; TCI: arcsine-transformed value of colonisation index; Barsrepresent standard error of the difference in means (SED), n = 5.

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comparable with un-inoculated (control) plants (Figure 3). On the other hand, theinoculation of Myc was found to be responsible for higher growth and P content.Significantly higher shoot length (42.8 ± 6.6 cm), fresh weight (36.41 ± 5.12 g/plant), dryweight (20.33 ± 3.57 g/plant) and P content (13.776 ± 2.333 mg/plant) were recorded inMyc alone inoculated pots. Data depicted in Figure 3 also suggested that growth and Pcontent were slightly higher in pots where Myc was introduced 1 week earlier to Foc(8.389 ± 1.314 mg/plant) as compared to co-inoculated pots (8.177 ± 1.356 mg/plant).However, earlier inoculation of Foc (Foc +Myc) reduced the plant growth. Almost

Figure 3. Interactions between Myc and Foc, and their effects on shoot length (cm), fresh weight(g)/plant, dry weight (g)/plant and phosphorus (P) content (mg)/plant of chickpea cv. JG 62 in theyears 2011 and 2012. Foc: individual inoculation, Foc +Myc: pretreated with Foc then after 1week Myc was introduced, Myc: individual inoculation, Myc + Foc: pretreated with Myc then after1 week Foc was introduced, Myc/Foc: combined inoculation of Myc and Foc, Un: un-inoculated(control); Bars represent standard error of the difference in means (SED), n = 5.

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similar trends were noticed in the second year also (2012). Further, the data on diseaseseverity index in chickpea in both the studied years are presented in Figure 4. In 2011,the index was found maximum in pots treated with Foc alone (3.8), followed by Foc +Myc (2.4), Myc/Foc (1.2) and Myc + Foc (0.4). No symptoms were recorded in potsinfested with Myc alone and un-inoculated control. More or less same trend wasrecorded in the second year in all the treatments. In both the studied years, differencesamong the treatments were significant (p < 0.01).

Thus, the results suggested that the interactions between Myc and Foc were depend-ing upon the sequences in which chickpea plants become colonised. Individual inocula-tion of Foc not only significantly reduced growth and P content of the plants but alsoexhibited the maximum severity of wilt disease, which could be a direct consequence ofthe action of pathogen (McAllister et al. 1994). Reductions in growth and P content ofchickpea seedlings were also observed in pots where Foc was inoculated 1 week earlierto Myc (Foc +Myc) or co-inoculated with Myc (Myc/Foc). Thus, we deduced thatactivities of Foc directly affected the symbiotic relationship. This could be due to thedevelopment of a less favourable environment for AMF. Further, the severity of diseasewas highest in pots infested with Foc alone, while early inoculation of Myc (Myc +Foc) and combined inoculation of Myc and Foc (Myc/Foc) reduced the severity of wiltup to 68 and 89.5%, respectively. Our results corroborated with the findings of earlierworkers. Ocampo (1993) postulated that AMF protect plants against the pathogeniceffect when inoculated simultaneously, while Khaosaad et al. (2007) suggested that pre-colonisation of plant roots by AMF suppress the activities of soil-borne fungal patho-gens, and this can be explained on the basis of release of volatile substances by AMF

Figure 4. Interactions between Myc and Foc, and their effects on disease severity index ofchickpea cv. JG 111 in the years 2011 and 2012. Foc: individual inoculation, Foc +Myc: pre-treated with Foc then after 1 week Myc was introduced, Myc: individual inoculation, Myc + Foc:pretreated with Myc then after 1 week Foc was introduced, Myc/Foc: combined inoculation ofMyc and Foc, Un: un-inoculated (control); Bars represent standard error of the difference inmeans (SED), n = 5.

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(Zeng 2006). Lingua et al. (2002) suggested that AMF-inoculated plants accumulatemore phenolic compounds which correspond to the reduction in the development ofpathogens.

Further, our results suggested that AMF inoculation increased plant growth.Improvement in plant growth upon AMF inoculation is mainly due to the enhancementof mineral nutrient status of plants (Shukla et al. 2012a). Smith and Smith (2012) sug-gested that plants with thriving mycorrhizal root systems are better able to grow/survivein comparison to their un-inoculated counterparts. The results also suggested thatinoculation of Myc increased P content of the plants which are in good accord with ourearlier work (Shukla et al. 2012b). According to Clark and Zeto (2000), mycorrhizafungi provide mechanical power to the plants by increasing nutrient transportation.Several researchers have suggested that improved nutritional status of AMF-inoculatedplants make them more tolerant against pathogens (Linderman 1994; Bodker et al.1998; Karagiannidis et al. 2002). Nutritional effect of AM symbiosis is only one amongthe several aspects of mycorrhizal effects on the pathogens (Trotta et al. 1996;Vierheilig et al. 2008). Moreover, in present study, we used Myc which consisted ofequal amounts of three different AMF genera, and this could be the reason of highergrowth and P content. Wehner et al. (2010) compared the effectiveness of single andmultiple AMF species on plant protection and pointed out that the assemblages ofmultiple AMF species exhibits greater potential against pathogens than a single AMFspecies.

Conclusions

The results clearly indicated that pre-inoculation of plants by mycorrhizal fungi can bea potential tool or practice. Early establishment of Foc reduced mycorrhisation as wellas plant growth up to some extent. On the other hand, the inoculation of Mycincreased growth and P uptake which makes plant more tolerant against pathogeniceffect of Foc. Hence, it may be stated that inoculation of mycorrhizal fungi should bedone prior to the transplantation of crop seedlings to the fields. However, extrapolationof the results to the real field conditions should be done with caution because of dif-ferences in growth condition and substrate used in the present study i.e. net house andsand, respectively. The information generated from this study can form the basis offurther field/microplot experiments involving time effective mycorrhizal inoculation tomitigate the losses due to Fusarium wilt of chickpea. Moreover, under field conditions,AMF species are surrounded by complex microbial communities particularly mycor-rhiza helper bacteria (MHB; Frey-Klett et al. 2007) which modulate AM symbiosisand can protect plants against the pathogenic effect of Fusarium. Thus, the consortiumof different mycorrhizal species along with important MHB (species of Bacillus, Pseu-domonas etc.) must be explored and exploited in future to obtain better results underfield conditions.

AcknowledgementsThis manuscript is based upon work supported by the University Grants Commission (UGC),New Delhi, India. Ashok Shukla gratefully acknowledges UGC-Dr D.S. Kothari Post DoctoralFellowship Scheme [Sanction No. 4-2/2006(BSR)/13-255/2008(BSR)], Government of Indiafor providing financial support. The authors are thankful to Head, Department of Botany, Dr HariSingh Gour Central University, Sagar, India for facilitating the research program and constantencouragement during the study.

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