Biocontrol of Fusarium Wilt of Chickpea using Arbuscular ... - Caryologia

CARYOLOGIA

Vol. 63, no. 4: 349-353, 2010

Biocontrol of Fusarium Wilt of Chickpea using Arbuscular Mycorrhizal Fungi and Rhizobium leguminosorum Biovar. Pradeep Kumar Singh*, Meenakshi Singh and Deepak Vyas Lab of Microbial Technology & Plant Pathology, Department of Botany Dr. H.S. Gour University Sagar, Madhya Pradesh, INDIA

Abstract — Present study deals with the biocontrol of Fusarium wilt of chickpea using arbuscular mycorrhizal fungi (AMF) Glomus hoi (Gh), Glomus fasciculatum (Gf) and Rhizobium leguminosorum Biovar. (Rl), which are the important members of rhizosphere and biological control agents, were examined on both the patho- system of Fusarium oxysporum f. sp. ciceris (Foc) and chickpea (Cicer arietinum). The colonization and nodulation of two biocontrol agents exhibited differences as a result of reciprocal interactions of these items as well as the effect of the Foc. Nodulation of Rl particularly decreased in triple inoculation. In addition, colonization of AMF significantly decreased in treatment of Foc+AMF than control, AMF. It was determined that single biological control agents inoculations were more effective than dual inoculations (AMF+Rl). When the morphological parameters of chickpea were considered, all of the morphological values were decreased in treatments which present Foc. Beside this all biological control agent increased total contents of P and N in treated plants compared to controls. Key words: Arbuscular Mycorrhizal Fungi, Biocontrol, Chickpea, Rhizobium leguminosorum.

INTRODUCTION Arbuscular mycorrhizal fungi and rhizobia are two important plant symbionts. They play a key role in natural ecosystems and influence plant productivity, plant nutrition and plant resistance (DEMIR and AKKOPRU 2007). Mycorrhiza benefits the host through mobilization of P from non-labile sources, whereas Rhizobium fixes N2 (DOR et al. 1997; SCHEUBLIN and VANDER HEIJDEN 2006). The well known activities of nitrogen-fixing bacteria improving the bioavailability of the major plant nutrients N and P, are very much enhanced in the rhizosphere of mycorrhizal plants where synergistic interactions of such organism with mycorrhizal fungi have been demonstrated (BAREA et al. 2002). A great deal of work has been carried out on the tripartite symbiosis legume- mycorrhiza-

*Corresponding Author: e-mail: [email protected]

Rhizobium (AZCON-AGUILAR and BAREA 1992; BAREA et al. 2000, 2002). The inoculation of mycorrhizal fungi has been shown to improve nodulation and N-fixation. Since soil borne pathogens, as well as symbionts share common habitat and show differential influence on the growth of the host plant, major interest has been focused on the relevance of AMF and rhizobia in the control of soil borne pathogens (DOR et al. 1997). As it is known that these pathogens are typically difficult to control through conventional fungicide applications. AMF have potential to reduce disease caused by fungal pathogens i.e. Phytopthora , Sclerotinia, Rhizoctonia, Pythium, Verticillium and Aphanomyces (AZCON-AGUILAR and BAREA 1998: DEMIR and AKKOPRU 2007; AYSAN and DEMIR 2009). However, the effect of AMF and Rhizobium on plant disease, nutrient uptake and rhizosphere microbial biomass and their activities are very difficult to generalize because the interactions involving arbuscular mycorrhiza, root rot fungi and Rhizobium vary with the microbial species and plant cultivars (DAR et al. 1997).

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The present study was aimed to assess the roles of AMF and Rhizobium in the biological control of the Fusarium wilt of chickpea. MATERIAL AND METHODS Present study was conducted at Department of Botany, Dr. H.S.Gour University, Sagar, Madhya Pradesh, India in 2006 and 2007. Chickpea seeds (Cicer arietinum cv. ICC 11322) were surface sterilized in 21% sodium hypochlorite, rinsed 3-4 times in sterile distilled water and sown in plastic pots (18×22cm.) containing a sterile mixture of soil and sand (3:1 v/v). The following treatments with 5 replications each were included in the study (1) G. hoi (2) G. fasciculatum (3) Rhizobium leguminosarum (Rl) (4) Fusarium oxysporum f. sp. ciceris (Foc) (5) Gh+Rl (6) Gh+Foc (7) Gh+Rl+Foc (8) Gf+Rl (9) Gf+Rl+Foc (10) Rl +Foc (11) Uninoculated control. All pots were placed in a growth chamber under standard conditions (14) h light (25-27°C, relative humidity). Plants were watered twice a week with deionized water and 100ml of nutrient solution (containing 720mg MgSO4.7H2O, 12.2 mg KH2PO4, 295mg Ca(NO3 )2.4H2O, 240 mg KNO3, 0.75 mg H3BO3, 0.001 mg CuSO4.5H2O, 4.3mg Fe Na EDTA and 0.00017mg NaMoO4.2H20, modified from VOSATKA and GRYNDLER (1999) was applied three times into each pot during the experiment 15 weeks. Inoculations - Glomus hoi (Gh) and G. fasciculatum (Gf) inoculations were carried by placing 2 gram soil inoculums, containing 60 spores/gram soil and 50 spores/gram soil respectively, per pot

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where the chickpea seeds were to be sown. Rhizobium grown in yeast extract mannitol both was applied to seeds, containing 3.9×106 cells/gram. The seeds were sown immediately. Control plants did not receive any inoculum. For Foc inoculation, the wheat grains were boiled in tap water for 45 minutes. The boiled grains were put into 250 ml glass bottles up to half length of the bottles and sterilized in an autoclave at 121°C for 45 minutes. A 5 mm disc agar covered with the mycelium of Foc was placed into bottles containing the sterilized wheat grains. The bottles were incubated at 24°C for 4 weeks. After the Foc has fully covered the grains surfaces with the mycelium the grains were then dried at room temperature. In addition 25 spores (wheat grains) of Foc were added to soil. Five grams of sterilized wheat grains were added to control pots (IRSHAD and ONOGUR 2002). Plant growth and nutrient uptake - At the end of the experiment, chickpea plants were harvested 15 weeks after seed sowing. Plant roots were separated dried 70°C (48h) and weighed. Plants were analysed for N and P for standard methods (KOCAR 1984) and nutrient uptake recorded. Determination of disease severity caused by Foc, root colonization by AMF and nodulation by Rhizobium - The disease development on each chickpea plant grown in growth chamber for 15 weeks was rated by using the following scale: 0, no symptoms; 1, pathogen development on the basal stem, no symptoms in the plant; 2, symptoms in plant tissues, plant is still living, 3 plant died. Chickpea roots were dyed in order to determined the existence of Gh and Gf by a modified method of PHILLIPS and HAYMAN (1970)

TABLE 1 — Plant growth of the chickpea inoculated with G. hoi (Gh), G. fasciculatum (Gf), R. leguminosarum (Rl) and Fusarium oxysporum f. sp. ciceris (Foc) alone or in combination. Treatments Control Gh Gf Rl Foc Gh+Rl Gf+Rl Gh+Foc Gf+Foc Rl+Foc Gh+Rl+Foc Gf+Rl+Foc

Plant Height (cm/plant) 31.30c 34.80b 35.60ba 34.12b 23.33ed 36.70a 36.24a 21.31f 23.59ed 30.90c 27.50d 29.62de

Fresh weight (gm/plant) Shoot Root 8.95b 1.22d 10.86ba 2.14cb 11.96ba 2.56b 8.12cb 3.42ba 5.46cd 1.60cbd 12.09a 3.72a 12.00a 3.86a 5.36cd 1.26cbd 5.63d 1.32dc 9.12b 1.61c 8.45cb 1.54d 8.90b 1.60c

Dry weight (gm/plant) Shoot Root 6.20c 0.70f 7.14b 1.23cd 7.24ba 1.40b 6.00c 1.50b 4.06d 0.86fe 7.36a 1.76a 7.30a 1.70a 4.02d 0.75f 4.12dc 0.79f 6.21c 0.84e 6.08dc 0.80f 6.14dc 0..81fe

Values are the means of five replicates. Means in the same column followed by the same letter are not significantly different according to the Duncan’s multiple range test.

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BIOCONTROL OF FUSARIUM WILT OF CHICKPEA USING ARBUSCULAR MYCORRHIZAL FUNGI …

and the colonization were determined by the Grid- Intersect method (GIOVANETTI and MOSSE 1980). The number and activity of nodules were recorded at the time of plant harvest. Statistical Analysis - The data were subject to analysis of variance and means compared using Duncan’s multiple range tests. RESULTS Plant growth and nutrient uptake - Inoculation with arbuscular mycorrhizal fungi (Gh and Gf) and Rl significantly increased shoot fresh and dry weights, respectively. However dry and fresh weight of roots were found significantly reduced in control plants (untreated plants) than those infected with Foc or inoculated with Gh, Gf or Rl either alone or in combination (Table 1). On the other hand, when inoculated with Gh+Rl showed significantly increased height. Data depicted in Table 2 shows impacts of the biological control agents on the P and N contents, fresh and dry weights of shoots and roots of chickpea in the presence of Foc. It was recorded that Mycorrhizal plants acquired more P in comparison to the control, even in the presence of Foc (Table 2). Results revealed that the inoculations of GH+Rl+Foc and Gh whose shoot and root contents 72.5 and 80.8% higher respectively, in comparison to control (Table 2). The N content of shoot in the single application of Rl was higher than those in single or dual applications of the AMF (Table 2). The dual inoculations of Gh and Rl had significant effect on the N content of roots (Table 2).

The numbers of nodules, AMF root colonization and disease severity - Data on percent root colonization by the Gh and Gf and by Rl, nodules number are presented in Table (3). Result reveals that that Foc alone significantly reduces the percent root colonization of AMF and nodule number of Rl compared with non treated control and the Foc treatments accompanied with the biological control agents (Gh+Rl+Foc and Gf+Rl+Foc) also reduced the colonization and levels of arbuscular mycorrhizal fungi and nodule number of Rl between 14.2-27.2%. Percent efficacy of the mycorrhizal fungi and Rl combinations with both single and dual inoculations against Foc are seen in Table 3. While the single inoculation of rhizosphere members inhibited Foc at the rate 10.3-24%, its dual inoculations increased disease severity 4.9% and 13.4%. Result reveals that Foc causes detrimental effect resulting reduction in root colonization and reduction in AMF and Rl treated plant respectively. DISCUSSION Pathogenic infection of Foc reduced growth and nutrient uptake in the chickpea, whereas inoculation with AMF or Rl, reduced pathogenicity. The decreased severity of disease and improved plant growth and nutrient uptake, expressed that the chickpea inoculated by AMF or Rl is somewhat resistant to the root pathogen Foc. Previous reports revealed that mycorrhizal plants offer increased tolerance to fungal root pathogen (ZAMBOLIN and SCHENCK 1983; JALALI et al. 1983; AKKOPRU

TABLE 2 — Nutrient uptake of the chickpea inoculated with Gh, Gf, Rl and Foc alone or in combination Nutrient Uptake Treatments Control Gh Gf Rl Foc Gh+Rl Gf+Rl Gh+Foc Gf+Foc Rl+Foc Gh+Rl+Foc Gf+Rl+Foc

P (%) Shoot 488.93f 1369.26cd 1296.78cbd 666.69fe 1196.59cd 1158.03d 1434.79cb 1486.07b 1736.56a 1264.13cbd 1779.10a 775.78e

N (%) Root 157.96e 824.84a 561.63bca 506.98bdc 369.57bedc 639.40ba 596.40ba 591.93ba 442.55bdc 298.36edc 789.7aa 263.26ed

Shoot 1.36g 1.82edf 1.98edc 2.68a 2.44ab 1.71egf 1.50gf 1.97edc 1.40g 2.27bac 2.23bdc 2.48ba

Root 0.78c 0.89b 0.90b 0.84b 0.76dc 1.05a 0.70d 0.87b 0.57fe 0.50f 0.91b 0.58e


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and DEMIR 2005; SINGH 2008) and bacterization of legume seeds/seedlings with Rhizobium significantly reduced some root diseases caused by soil born fungal pathogens (CHAKRABORTY and PURKAYOSTHA 1984; CHAKRABORTY and CHAKRABORTY 1989; DAR et al. 1997). It seems that enhanced plant growth improved nutrient assimilation and possibly a physical barrier have probably imparted altered resistance to the plants (DAR et al. 1997; SINGH et al. 2010), as the disease incidence was reduced in plants inoculated with AMF and Rl. Since the role of altered root exudates , changes in rhizosphere microbial activities and biochemical antagonism through phytolaexin and rhizobiotoxin production as mechanisms of disease tolerance induced by AMF and Rl ( SINGH 2008; SINGH et al.2010) cannot conduct here. The most important symbionts of rhizosphere are AMF and Rhizobium have already been known for their stimulating (SUBBA RAO et al. 1986; CHAMPARAT 1990; EDWARDS et al. 1998) or incubating (SCHEUBLIN and VANDER HEIJDIN 2006) effects on each other or on the growth of plant pathogens. Our results are in accord with them. In dual inoculations caused decreased in colonization and nodulation in test plants. These inhibiting effects could be due to the secretion of antimicrobial substances (WALLEY and GEMIDA, 1997). The colonization of AMF and nodule numbers of Rl was also significantly reduced in the presence of Foc. It is not clear how AMF root colonization or Rl nodulation is affected by soil borne pathogen (JOHANSSON et al. 2004). However it has been hypothesized that these effects may be related to the species and varieties of micro-organisms and the conditions in the rhizosphere (ANJAIR et al. 2003).

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Legumes being endowed with the unique ability to utilize the vast reservoir of atmospheric nitrogen with the help of symbiotic N2 fixing bacterium Rhizobium. Other soil microorganisms such as AMF have been credited for beneficial effects on plants. AMF absorbs P and translocates it to the roots (CHAMPARAT 1990). Similar synergistic effect of Rl and AMF has been recorded in chickpea. It is also well documented that inoculating plants with AMF can stimulate nodulation and N2 fixation by legumes (CHAMPARAT, 1990). Some workers showed that in P efficient soils, N2 fixation in several legumes inoculated with appropriate Rhizobium strain depended much on P, which could be supplied by AMF infection (POWELL, 1976; MANJUNATH and BAGYARAJ 1984). The results reported also were confirmed in this study. The present study concluded that suitable combinations of AMF and Rhizobium bacteria may increase the plant growth and resistance to pathogens. In future studies, therefore more detailed investigations in variance pathogens and interaction between the microorganisms and the test plants are needed to develop much more efficient biocontrol of the related diseases. REFERENCES AKKOPRU A. and DEMIR S., 2005 — Biological control of Fusarium wilts in tomato caused by Fusarium oxysporum f. sp. lycopersici by AMF Glomus intraradices and same rhizobacteria. Journal of Phytopathology, 153: 544-550. ANJAIR V., CORNELIS P. and KOEDOM N., 2003 — Effect of genotype and root colonization in biological con-

TABLE 3 — The number of nodules, AMF root colonization and disease severity of common chickpea plants inoculated with Gh, Gf, Rl and Foc alone or in combinations. Treatments Control Gh Gf Rl Foc Gh+Rl Gf+Rl Gh+Foc Gf+Foc Rl+Foc Gh+Rl+Foc Gf+Rl+Foc

No. of nodules – – – 8.45a – 6.45ab 6.00b – – 4.31c 5.96b 5.72cb

AMF Root colonization (%) – 75.40ba 82.90a – – 45.42d 52.32dc 36.94e 43.20ed – 56.72c 64.94b

Disease Severity (%) – – – – 62.00ba – – 40.00d 36.70ed 46.00dc 67.00a 63.90ba


BIOCONTROL OF FUSARIUM WILT OF CHICKPEA USING ARBUSCULAR MYCORRHIZAL FUNGI …

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