Studies on the management of root-knot nematode, Meloidogyne incognita-wilt fungus, Fusarium oxysporum disease complex of green gram, Vigna radiata cv ML-1108

Studies on the management of root-knot nematode, Meloidogyne incognita -wilt fungus, Fusarium oxysporum disease complex of green gram, Vigna radiata cv ML-1108 first-author †E-mail:profakhtar2002@yahoo.co.in †E-mail:profakhtar2002@yahoo.co.in profakhtar2002@yahoo.co.in Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh-202002, India Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh-202002, India Studies were conducted under pot conditions to determine the comparative efficacy of carbofuran at 1 mg a.i./kg soil, bavistin at 1 mg a.i./kg soil, neem ( Azadirachta indica ) seed powder at 50 mg/kg soil, green mould ( Trichoderma harzianum ) at 50.0 ml/kg soil, rhizobacteria ( Pseudomonas fluorescens ) at 50.0 ml/kg soil against root-knot nematode, Meloidogyne incognita –wilt fungus, Fusarium oxysporum disease complex on green gram, Vigna radiata cv ML-1108. All the treatments significantly improved the growth of the plants as compared to untreated inoculated plants. Analysis of data showed that carbofuran and A. indica seed powder increased plant growth and yield significantly more in comparison to bavistin and P. fluorescens . Carbofuran was highly effective against nematode, bavistin against fungus, A. indica seed powder against both the pathogens and both the bioagents were moderately effective against both the pathogens. Meloidogyne incognita Fusarium oxysporum Disease complex Management Vigna radiata INTRODUCTION 1 Green gram ( Vigna radiata ) is an important short duration pulse crop. Due to its high nutritive value, it is grown throughout the tropical countries of South and Southeast Asia, particularly in India (Vavilov, 1951 1951 ). Among various pests and diseases, nematodes-fungus disease complex particularly of Meloidogyne incognita and Fusarium oxysporum poses a great problem to the cultivation of pulse crops by inflicting severe yield losses (Perveen et al., 1999 1999 ; De et al., 2000 2000 ; Mahapatra and Swain, 2001 2001 ). The root-knot nematodes ( Meloidogyne spp.) are sedentary endoparasites and are among the most damaging agricultural pests, attacking a wide range of crops including green gram (Sikora and Greco, 1993 1993 ). The infection starts with root penetration of second stage juveniles (J2) hatched in soil from eggs encapsulated in egg masses laid by the females on the infected roots (Barker et al., 1985 1985 ). Wilt fungus ( Fusarium spp.), deutromycetous fungus, causes wilting of the infected plant that leads to death. Yield loss due to this fungus had been reported on various crops (Agrios, 1983 1983 ). Management of disease complexes appears to less straightforward than one might anticipate. The most obvious solution is to use chemical methods to control one of the interacting organisms and thus prevent the disease complex from occurring. However, it is fundamental to have prior knowledge of the interaction involved, as even low densities of fungi or nematode can result in a disease complex of significant importance (Bowers et al., 1996 1996 ). Carbofuran belonging to the carbamates group is a non-fumigant and had been reported to cure root-knot nematodes on various crops (Butool et al., 1998 1998 ). It mainly affects nematode neuromuscular activity by inhibiting the enzyme acetyl cholinesterase (Evans, 1973 1973 ; Wright, 1981 1981 ). Similarly, bavistin (benzimidazole group) has already been proved to cure fungi belongs to deutromycotina, particularly Fusarium spp. by inhibiting mitosis that ultimately causes the disorganization of the fine structure of fungal cells in plants (Howard and Aist, 1977 1977 ). Besides chemicals, various workers suggested other control measures in view of the need to replace highly toxic and potentially polluting chemicals used to control plant parasitic nematodes and fungi, with less dangerous chemicals or preferably with biological control agents and botanicals (Oostendrop and Sikora, 1989 1989 ). Some species of the genus Trichoderma have been used as biocontrol agents against soilborne phytopathogenic fungal pathogens (Chet, 1987 1987 ). These fungi may also promote plant growth (Inbar et al., 1994 1994 ). Several attempts have also been made to use Trichoderma spp. to control plant parasitic nematodes. Windham et al.( 1989 1989 ) reported reduced egg production in the root-knot nematode, M. arenaria following soil treatments with T. harzianum (T-12) and T. koningii (T-8) preparations. Besides fungi, bacteria especially rhizospheric bacteria have also been used for biological control of soil borne plant pathogens. Fluorescent Pseudomonas spp. is among the most effective rhizospheric bacteria in reducing soil–borne diseases in disease suppressive soils (Weller, 1988 1988 ). These bacteria can antagonize soil-borne pathogens through various mechanisms e.g. bacterial siderophores inhibit plant pathogens through competition for iron, antibiotics suppress competing microorganisms, and chitinases and glucanases lyse microbial cells (Bakker et al., 1991 1991 ). Along with chemical and biomanagement practices for the management of nematodes and fungi, bioactive products from neem ( A. indica ) have been proved economic and ecofriendly approach due to the presence of an array of complex compounds, triterpenes, or more specifically limonoids (Kraus, 1995 1995 ; Musabyimana and Saxena, 1999 1999 ). Considerable work has been carried out on the pathogenic potential and management of M. incognita - F. oxysporum disease complex on different pulse crops, but information on pathogenic potential of F. oxysporum and that to with M. incognita on V. radiata have not so far been reported and that is why F. oxysporum has not been considered as a major pathogen of V. radiata , but during survey of Aligarh district of Uttar Pradesh in India we observed severe symptoms of F. oxysporum on V. radiata (Haseeb et al., 2005 2005 ). Therefore, the present study was carried out to determine the comparative efficacy of chemicals, A. indica seed powder and microbial antagonists against M. incognita - F. oxysporum disease complex on V. radiata cv ML-1108. MATERIALS AND METHODS 1 Preparation of inoculum of M. incognita and F. oxysporum 2 A single egg mass of the M. incognita picked by hand with fine forceps from infected eggplant root was surface sterilized in 1:500 ( V / V ) aqueous solution of “chlorax” (sodium hypochlorite) for 5 min. It was then transferred to a small coarse sieve lined with tissue paper, placed beforehand in a petriplate containing sufficient amount of water. The petriplates were incubated at room temperature (27±5) °C for 5 d (den Ouden, 1958 1958 ). Seedlings of eggplant raised in autoclaved soil, were inoculated with the progeny of the single egg mass in order to get regular supply of the inoculum for the experiments. Pure culture of F. oxysporum isolated from the infested plants from the field was maintained on PDA (Potato Dextrose Agar) in petriplates at (27±5) °C in order to mass-produce pure culture of the fungus to be transferred to flasks containing sorghum seeds soaked overnight in 5% ( m / V ) sucrose solution. The flasks were incubated in a BOD (Biological Oxygen Demand) incubator at a temperature of (27±1) °C for 10 d. During incubation, the flasks were shaken three times in a day, to ensure proper growth of the fungal mycelium on the sorghum seeds. Spore suspension was made from the colonized sorghum seeds so that final CFU (colony forming units) of F. oxysporum was maintained at 10 8 CFU/ml. Preparation of inoculum of T. harzianum and P. fluorescens and procurement of A. indica seed powder 2 Pure culture of T. harzianum isolated from the experimental fields of Aligarh Muslim University, was maintained on PDA in petriplates at (27±5) °C. Healthy sorghum seeds were soaked in 5% ( m / V ) sucrose solution for 16 h and then strained and placed into 500 ml conical flasks to give 200 cm 3 of sorghum seeds/flask. Flasks with sorghum seeds were plugged with cotton and sterilized by autoclaving for 30 min at 1 kg/cm² pressure. The conical flasks containing sterilized sorghum seeds were inoculated with 1 cm-diameter PDA discs punched from the periphery of actively growing 5 d-old culture of T. harzianum and then placed in an incubator at (27±1) °C and the fungi were allowed to grow with periodic shaking of the flasks, so that the surface of all sorghum seeds was colonized and CFU reached above to 10 8 CFU/g culture. Pure culture of P. fluorescens isolated from the same experimental fields. The culture tubes, each containing 10 ml King’s ‘B’ (Broth) were autoclaved for 30 min at 1 kg/cm² pressure. After the culture tubes were cooled, each tube was inoculated with a single colony of P. fluorescens strain Pf-1 from pure bacterial culture maintained on King’s ‘B’ agar. The culture tubes were then placed in a BOD incubator for 48 h at (30±1) °C for the multiplication of P. fluorescens. For mass production, one-liter conical flasks containing 500 ml King’s ‘B’ (Broth) were autoclaved at the same pressure and time as mentioned above. After the flasks were cooled, each flask was inoculated with 1.0 ml of P. fluorescens cultured broth. The flasks were kept at (30±1) °C in the BOD incubator for 120 h and were shaken two times a day. Inoculum culture was mixed with talc at ratio of 1:4, and then the amount of talc was adjusted so that the final CFU of P. fluorescens was maintained at 2×10 8 CFU/ml. Whole seeds or dehulled seeds of A. indica were first dried in sunlight for 24 h and then in oven at 70 °C for 1 h, were manually pounded in a large steel mortar with pestle to produce a fine neem seed powder. Powdered form was used to allow easy decomposition of A. indica seeds in soil. Experimental procedure 2 Experiment was conducted in polyhouse ((26.7±3) °C, 73.5%±11% Relative Humidity and 0.918 kPa) in 21 earthen pots (18 cm top diameter) filled with a mixture of autoclaved sandy loam soil (sand 70%, silt 22% and clay 8%, pH 7.5) and compost (4:1). One seed was sown initially and the treatments (carbofuran at 1 mg a.i./kg soil, bavistin at 1 mg a.i./kg soil, A. indica seed powder at 50 mg/kg soil, T. harzianum at 50 ml/kg soil and P. fluorescens at 50 ml/kg soil) were applied at the time of sowing of seeds. Six pots were untreated. Each treatment was replicated 3 times in a completely randomized block design and watered daily so that each pot got about 500 ml water. After 4 d i.e. at two-leaf stage of seedlings all the pots except the 3 untreated pots were inoculated with 2000 freshly hatched second stage juveniles of M. incognita and 2 ml spore suspension of F. oxysporum into 1 cm holes around the base of the plant which were then filled with soil. Uninoculated pots and nematode+fungus inoculated pots served as controls. Recording of data 2 Sixty days after inoculation the plants were uprooted and root gall indices were determined on a scale 0~4, where 0=no infection or root galling, 1=slight infection (1%~25%), 2=moderate infection (26%~50%), 3=severe infection (51%~75%) and 4=very severe infection (76%~100%) (Taylor and Sasser, 1978 1978 ). In order to determine the extent of F. oxysporum infection washed roots of inoculated plants were cut into 1.0 cm pieces, then treated with 10% KOH solution and finally kept at 90 °C for 1 h. These root segments were washed again with distilled water, then acidified and stained with trypan blue (0.5% ( V / V ) in lactophenol) as described by Philips and Hayman ( 1970 1970 ). Five stained pieces of each taproot were mounted on slides in lactophenol and presence of mycelium of the fungus was estimated. The root infection was calculated by measuring the infected portion in relation to total length of root pieces (Biermann and Lindermann, 1981 1981 ). Fresh and dry root and shoot weights, seed and husk weight were obtained. Final nematode population in the entire soil volume was extracted by Cobb’s sieving and decanting technique along with Baermann funnel and in roots by macerating 5 g root tissues in a Warring blender (Southey, 1986 1986 ), and counted as per the procedure suggested by Doncaster ( 1962 1962 ). Data were analyzed by analysis of variance (Cochran and Cox, 1957 1957 ) and significant differences among treatments were tested by the least significant difference test (LSD) at probability levels of 5% (LSD 0.05 ) and 1% (LSD 0.01 ). RESULTS 1 Regular observations showed that no treatment affected seed germination except A. indica seed powder at 50 mg/kg soil, where the germination of seeds was delayed by 4 d as compared to other treatments, this was also observed earlier by Chakrabarti et al.( 2001 2001 ). Data (Table 1 1 ) shows that the effect of various treatments on all the growth and yield parameters viz. shoot length, fresh and dry weights of shoot and root, seed and husk weights were mostly significant ( P ≤0.05) as compared to untreated inoculated plants. However, no significant differences were found in shoot height of plants treated with bavistin (24.1 cm) and P. fluorescens (22.6 cm). Maximum increase in all the growth and yield parameters was found in carbofuran treated plants followed by A. indica seed powder, T. harzianum , bavistin and P. fluorescens treated plants as compared to untreated inoculated plants. Each value is an average of three replicates; Values in parenthesis represent percent reduction over uninoculated control 2 Treatments 2 Shoot height (cm) 2 Fresh weight (g) 2 Dry weight (g) 2 Seed weight (g) 2 Husk weight (g) Root Shoot Root Shoot Control 30.8 7.5 27.5 2.50 8.21 8.50 3.33 Nematode+fungus 22.1 (28.3) 4.8 (36.2) 18.6 (32.3) 1.62 (35.3) 5.63 (31.4) 5.34 (37.1) 2.12 (36.2) Nematode+fungus+carbofuran 29.2 (5.2) 7.0 (6.8) 25.9 (5.8) 2.36 (5.4) 7.85 (4.3) 7.88 (7.2) 3.13 (5.9) Nematode+fungus+neem seed powder 27.8 (9.7) 6.7 (10.2) 24.8 (9.8) 2.26 (9.7) 7.50 (8.7) 7.62 (10.4) 3.02 (9.4) Nematode+fungus+ T. harzianum 26.3 (14.6) 6.4 (15.1) 22.7 (17.3) 2.14 (14.5) 6.85 (16.5) 7.12 (16.2) 2.85 (14.3) Nematode+fungus+bavistin 24.1 (21.8) 6.0 (20.1) 22.1 (19.7) 2.06 (21.4) 6.54 (20.4) 6.77 (20.4) 2.66 (20.1) Nematode+fungus+ P. fluorescens 23.8 (22.6) 5.4 (22.8) 21.9 (20.5) 1.91 (23.5) 6.46 (21.3) 6.66 (21.7) 2.65 (20.5) LSD 0.05 2.5 0.6 2.0 0.22 0.61 0.70 0.26 LSD 0.01 3.6 0.9 2.8 0.30 0.85 0.98 0.37 The suppressive effect of the treatments on nematode population both in roots and soil was highly significant as compared to untreated inoculated plants, but no differences were observed in soil populations of nematode of plants grown in carbofuran (500) and A. indica seed powder (500) treated pots. The reproduction rate of M. incognita was significantly suppressed by all the treatments as compared to untreated inoculated plants. Similarly all the treatments were found to be highly effective in their ability to reduce root-knot index (RKI) when compared with untreated plants. Data presented in Table 2 2 shows that the greatest suppression of nematode reproduction i.e. reproduction factor (Rf) and root-knot index was achieved by application of carbofuran ( Rf =0.46, RKI =0.25) followed by A. indica seed powder ( Rf =0.99, RKI =0.50), T. harzianum ( Rf =1.27, RKI =1.50), P. fluorescens ( Rf =1.38, RKI =1.75) and bavistin ( Rf =1.50, RKI =2.25) respectively as compared to controls ( Rf =1.74, RKI =3.00). Bavistin was highly effective in suppression of taproot colonization by fungus (8% root colonized) followed by neem seed powder (30%), T. harzianum (35%), P. fluorescens (65%) and carbofuran (75%) respectively. Each value is an average of three replicates; Pi : Initial population 2 Treatments 3 Final nematode population (Pf) 2 Reproduction factor Rf = Pf / Pi 2 Root-knot index ( RKI ) 2 Disease index (%) Total root 1 kg soil Total Control – – – – – – Nematode+fungus 1488 2000 3488 1.74 3.00 95 Nematode+fungus+carbofuran 420 500 920 0.46 0.25 75 Nematode+fungus+neem seed powder 1474 500 1974 0.99 0.50 30 Nematode+fungus+ T. harzianum 1536 1000 2536 1.27 1.50 2.25 8 Nematode+fungus+bavistin 1508 1500 3008 1.50 2.25 8 Nematode+fungus+ P. fluorescens 1512 1250 2762 1.38 1.75 65 LSD 0.05 144.1 121.9 258.4 0.11 0.14 6.0 LSD 0.01 204.9 173.3 367.3 0.15 0.20 8.5 DISCUSSION 1 V. radiata is highly susceptible to M. incognita - F. oxysporum disease complex as indicated by severity in root-knot development, nematode population densities, root colonization by fungus and plant growth suppression in the inoculated controls. Our results indicate that carbofuran is most effective among the treatments in improving plant growth and reducing M. incognita population densities in soil. Carbofuran impairs nematode neuromuscular activity by inhibiting the function of the enzyme acetyl cholinesterase resulting in reduced movement and ability of invasion and multiplication (Evans, 1973 1973 ; Wright, 1981 1981 ). The nematodes may also be killed while feeding on root tissues by the systemic action of these nematicides when they are absorbed by the plant roots and translocated in the plant system (van Berkum and Hoestra, 1979 1979 ). Butool et al.( 1998 1998 ) found similar effectiveness of carbofuran in suppressing M. incognita on Hyoscyamus muticus . Bavistin was found most effective in controlling root colonization by fungus. It inhibits the nuclear division of fungi by inactivating the spindle, which is composed of microtubules. Various scientists have also been reported, bavistin as an important control measure against F. oxysporum (Etebarian, 1992 1992 ; Prasad et al., 2000 2000 ; Haseeb and Shukla, 2002 2002 ). To maintain a low inoculum load by continuous application of systemic fungicide alone is not practical for the control of wilt disease. To cope with this, A. indica seed powder and other bioagents may be applied. It is clear from the results that besides chemicals A. indica seed powder was sufficiently effective against both the pathogens, this may be due to presence of active principles and toxic chemicals in A. indica seed powder (Singh and Sitaramaiah, 1970 1970 ). Results showed significant suppression of both M. incognita and F. oxysporum by T. harzianum . The possible mechanism involved in Trichoderma antagonism had been studied intensively in terms of antibiotic and enzyme production as hyphal interactions (Dennis and Webster, 1971 1971 ; Elad et al., 1982 1982 ). Production of chitinases may have direct significance in the parasitism of Trichoderma on F. oxysporum as these enzymes function by breaking down the polysaccharides, chitin and β-glucan that are responsible for the rigidity of fungal cell walls thereby destroying cell wall integrity (Howell, 2003 2003 ). Significant suppression of nematode multiplication by P. fluorescens was due to its capability of altering root exudates, which could alter nematode behavior and suppress nematode population in root system (Oostendrop and Sikora, 1989 1989 ). Initial investigations on antagonistic rhizobacteria against root-knot nematodes, include work by Kloepper et al.( 1992 1992 ). P. fluorescens was found not only effective against M. incognita but also against wilt causing fungi. This finding accords with the results of Vidhyasekaran and Muthamilan ( 1995 1995 ). 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