Bio-management of Cucumber Wilt Complex Caused by Root-knot Nematode, Meloidogyne incognita and Fusarium oxysporum f. sp. cucumerinum in Polyhouse under Protected Cultivation

A polyhouse study was conducted to determine the potential of biocontrol agents viz. Trichoderma viride, Pseudomonas fluorescence, Purpureocillium lilacinum (Paecilomyces lilacinus) @ 0.3 and 0.5 g/kg soil and liquid formulation of bioagents, (T. viride + P. fluorescence + P. lilacinus) @ 0.5 and 1 ml/ kg soil, against root-knot nematode, Meloidogyne incognita and Fusarium oxysporum f. sp. cucumerinum disease complex on cucumber. Three main treatments, viz., nematode alone, fungus alone and both inoculated simultaneously were taken. Chemical checks with Bavistin @ 2 g/ l water and carbofuran @ 0.1 mg/ kg soil as well as untreated check were also maintained. Fungus was grown on sand maize meal medium. Soil was autoclaved and infested with root-knot nematode (1000 J2/ kg soil) and fungus (50 g/ kg soil). The bio-agents were mixed with the potted soil treatment wise. A waiting period of three days was given for multiplication of bioagents on the organic matter before sowing. All the treatments significantly improved the plant growth parameter, viz., shoot length (SL), root length (RL), fresh shoot weight (FSW), fresh root weight (FRW), dry shoot weight (DSW) and dry root weight (DRW) as compared to untreated check. However, maximum improvement in plant growth parameter was recorded in case of carbofuran @ 0.1 mg /kg soil followed by higher dose liquid formulation of bioagents. Bavistin was least effective among all the treatments against root-knot nematode, Meloidogyne incognita and Fusarium oxysporum f. sp. cucumerinum disease complex.

In India, growing of horticultural crops in polyhouses under protected cultivation is becoming very popular among the farmers throughout the country.Large numbers of polyhouses are being erected in Haryana under the ages of National Horticulture Mission to grow short duration crops.Cucumber (Cucumis sativus L.) is a widely cultivated plant in the gourd family, Cucurbitaceae.It is a creeping vine that bears cylindrical fruits that are used as culinary vegetables.There are three main varieties of cucumber: slicing, pickling, and burp less.Within these varieties, several different cultivars have emerged.The cucumber is originally from South Asia, but now grows on most continents.Cucumber is an edible cucurbit popular throughout the world due to a good source of vitamins, minerals, fiber and roughages.It having crisps texture and taste.Cucumber is truly a versatile vegetable because of wide range of uses from salads to pickles and digestive aids to beauty products.the caloric and nutritional value of cucumber is very low but it is a primary source of vitamins, mineral and fiber for human body (Keoprapari,1997).The annual production of cucumber in India is 698000 MT from 45000 ha area with productivity of 15.5 per ha only during 2012-13 (Anonymous, 2014).Polyhouse cultivation involves intensive cultivation of crops, optimum use of fertilizers and frequent use of irrigation, but continuous growing of the same crop with high day temperature and relative humidity within the greenhouse, polyhouse and low tunnel along with poor plant hygienic conditions inside and outside the greenhouse increase problem of soil borne pests and diseases including plant parasitic nematodes (Minuto et al., 2006) which results in the availability of ideal conditions for the growth and multiplication of these pests.
Under polyhouse cultivation crops, are attacked by a number of pests and diseases including nematodes which interfere with the successful cultivation under protected conditions.Among the nematodes, root-knot nematode (Meloidogyne spp.) is the most damaging under polyhouse conditions, parasitizing almost all the polyhouses crops.The damage becomes very severe in association with fungi.Though, yield loss due to this nematode is difficult to predict, approximate yield loss due to this nematode has been predicted by many authors in various crops.Another important biotic stress to which the crop exposed is the fungus, Fusarium oxysporum f. sp.cucumerinum.Considering the soil health, environmental safety and the long term hazards posed by the indiscriminate use of pesticides, bioagents promise to be the next best alternative for nematode management.With this aim, a study was conducted in a polyhouse to test the efficacy of certain easily available bioagents (P.lilacinus, T. viride and P. fluorescence) against Meloidogyne incognita and Fusarium oxysporum f. sp.cucumerinum disease complex on cucumber.

MATERIALS AND METHODS
Experiment was conducted in polyhouse (26.7±3) °C, 73.5±11%Relative Humidity and 0.918 kPa) in earthen pots (18 cm diameter) filled with a mixture of autoclaved sandy loam soil (sand 70%, silt 22% and clay 8%, pH 7.5).Autoclaved soil would be infested with root-knot nematode @ 1000 J 2 /kg soil and fungus (50 g/kg soil) as per the treatment.The experiment was conducted in pots (1 kg capacity) containing infested soil.Inoculum of root knot nematode was obtained from the nematode infected cucumber at farmer's field in Hisar Haryana, India.Root knot nematode females collected from the cucumber roots were processed for perineal pattern to confirm the species of root knot nematode associated with the plant.The pure culture was prepared in steamed sterilized soil in pots and Inoculum was used for experimentation.
Pure culture of F. oxysporum isolated from the infested plants during random survey of polyhouses was maintained on PDA (Potato Dextrose Agar) in petriplates at (27±5) °C in order to mass-produce pure culture of the Fungus was grown on sand maize meal medium (700gm sand + maize meal 300gm + 150ml distilled water).The flasks were incubated in a BOD (Biological Oxygen Demand) incubator at a temperature of (27±1) °C for 15 days.During incubation, the flasks were shaken three times in a day, to ensure proper growth of the fungal mycelium on the sand maize meal medium.
Root-knot nematode and fungus was also inoculated carefully adding the homogenous suspension of the two pathogens at the root zone of the plants, as per treatment.Each pot would be infested with root-knot nematode (1000 J 2 /kg soil) and fungus (50g /kg soil) and treated with (carbofuran at 1 mg a.i./kg soil, Bavistin at 1 mg a.i./kg soil, Trichoderma viride, Pseudomonas fluorescence, Purpureocillium lilacinum (Paecilomyces lilacinus) @ 0.3 and 0.5 g/kg soil and liquid formulation of bioagents, (T.viride + P. fluorescence + P. lilacinus) @ 0.5 and 1 ml/ kg soil were incorporated to the potted soil as per treatment.Also waiting period of three days was given for multiplication of bioagents on the organic matter before sowing.After seven days each pot would be sown with cucumber cv Sania @ 5 seeds as per treatment and also maintain untreated check.Uninoculated pots and nematode + fungus inoculated pots served as controls.One plant per pot was retained after 30 days.Each treatment was replicated four times in a completely randomized block design during the PATIL et al.: STUDY OF BIO-MANAGEMENT OF CUCUMBER WILT months of April to June, 2015 in the polyhouse under protected conditions and watered daily so that each pot as per requirement.
Evaluations were performed 60 days after sowing.Measurements were made on the plant growth parameters (shoot length, fresh and dry shoot and root weight) observations were made on the root population of nematode viz., Number of galls per plant, Number of egg masses per plant, Number of eggs per egg mass, Final nematode population per pot.Nematode population in soil was processed as per the sieving method of Cobb´s sieving and decanting technique followed by Modified Baermann´s funnel technique for estimation of nematode population in soil.Per cent wilt incidence due to fungus was assessed using number of wilt infected plants /total number of plants taken for observation.

Statistical analysis
Data were analysed using analysis of variance (ANOVA).Treatment means were compared and critical differences (CD) was calculated at P=0.05 to test for significant differences between treatments (T)

RESULTS
Data indicated that shoot length in all the treatments was significantly better over untreated inoculated checks viz., nematode alone (87.5 cm), fungus alone (85.6 cm) and nematode + fungus simultaneously (83.9 cm).Among the various treatments, maximum shoot length was observed in liquid formulation of bio-agents (T.viride + P. fluorescence + P. lilacinus) @ 15 ml per kg soil (151.1 cm), followed by Paecilomyces lilacinus @ 0.5 g per kg soil (145.7 cm) irrespective of whether nematode inoculated individually or concomitantly.However, in plants inoculated with nematode alone, shoot length was maximum in case of liquid formulation of bio-agents (155.8 cm), followed by P .lilacinus(151.7 cm) as compared to untreated inoculated check (87.5 cm).Plants inoculated with fungus alone, shoot length was maximum in case of liquid formulation of bio-agents (149.6 cm), followed by Trichoderma viride @ 0.5 g per kg soil (143.9 cm) as compared to untreated inoculated check (85.6 cm).Plants inoculated with nematode and fungus concomitantly, shoot length was maximum in case of liquid formulation of bio-agents (147.9 cm), followed by P. lilacinus (146.9 cm) as compared to untreated inoculated check (83.9 cm).In general, shoot length was significantly less in all the treatments compared to untreated uninoculated check irrespective of whether inoculated individually or concomitantly both pathogens.Maximum reduction in shoot length was observed in the presence of nematode and fungus followed by fungus alone while minimum in case of nematode alone.
Fresh shoot weight in all the treatments was significantly better over untreated inoculated checks viz., nematode alone (23.2 g), fungus alone (23.4 g) and nematode + fungus simultaneously (22.90 g).Among the various treatments, maximum fresh shoot weight was observed in liquid formulation of bio-agents (T.viride + P. fluorescence + P. lilacinus) @ 15 ml per kg soil (59.1 g), followed by Paecilomyces lilacinus @ 0.5 g per kg soil (55.7 g) irrespective of whether nematode or fungus inoculated individually or concomitantly.However, in plants inoculated with nematode alone, fresh shoot weight was maximum in case of liquid formulation of bio-agents (64.7 g), followed by P. lilacinus @ 0.5 g per kg soil (64.7 g) as compared to untreated inoculated check (23.2 g).Plants inoculated with fungus alone, fresh shoot weight was maximum in case of liquid formulation (57.2 g), followed by T. viride @ 0.5 g per kg soil (54.1 g) as compared to untreated inoculated check (23.4).Plants inoculated with nematode and fungus concomitantly, fresh shoot weight was maximum in case of liquid formulation of bio-agents (55.2 g), followed by P. lilacinus (5.6 g) as compared to untreated inoculated check.In general, fresh shoot weight was significantly less in all the treatments compared to untreated uninoculated check irrespective of whether inoculated individually or concomitantly with nematode and fungus.Maximum reduction in fresh shoot weight was observed in the presence of both nematode and fungus followed by fungus alone and minimum in case of nematode alone.
Dry root weight in all the treatments was significantly better over untreated inoculated checks viz., nematode alone (2.19 g), fungus alone (2.00 g ) and nematode + fungus simultaneously (1.76 g).Among the various treatments, maximum dry root weight was observed in liquid formulation of bio-agents (T.viride + P. fluorescence + P. PATIL   lilacinus) @ 15 ml per kg soil (6.57g), followed by Paecilomyces lilacinus @ 0.5 g per kg soil (5.56 g) irrespective of whether nematode or fungus inoculated individually or concomitantly.However, in plants inoculated with nematode alone, dry root weight was maximum in case of liquid formulation of bio-agents (7.27 g), followed by P. lilacinus (6.7 g) as compared to untreated inoculated check  (2.19 g).Plants inoculated with fungus alone, dry root weight was maximum in case of liquid formulation of bio-agents (6.70 g), followed by T. viride (6.30 g) as compared to untreated inoculated check (2.0 g).Plants inoculated with nematode and fungus concomitantly, dry root weight was maximum in case of liquid formulation of bioagents (5.78 g), followed by P. lilacinus (4.99 g)  whether inoculated individually or concomitantly with nematode and fungus.Number of galls per plant in all the treatments was significantly reduced over untreated inoculated checks viz., nematode alone (313) and nematode + fungus simultaneously (307).Among the various treatments, minimum number of galls per plant was observed in liquid formulation of bio-agents (T.viride + P. fluorescence + P. lilacinus) @ 15 ml per kg soil (156), followed by Paecilomyces lilacinus @ 0.5 g per kg soil (164) irrespective of whether nematode inoculated individually or concomitantly.However, in plants inoculated with nematode alone, number of galls per plant was minimum in case of liquid formulation of bio-agents (160), followed by P. lilacinus (167) as compared to untreated inoculated check (313).Plants inoculated with nematode and fungus concomitantly, number of galls per plant was minimum in case of liquid formulation of bio-agents (153), followed by P. lilacinus (161) as compared to untreated inoculated check (307).Maximum reduction in number of galls per plant was observed in the presence of both nematode and fungus followed by nematode alone.
Nematode population J2/200 cc soil in all the treatments was significantly reduced over untreated inoculated checks viz., nematode alone (644) and nematode + fungus simultaneously (637).Among the various treatments, minimum final nematode population J 2 /200 cc soil was observed in liquid formulation of bio-agents (T.viride + P. fluorescence + P. lilacinus) @ 15 ml per kg soil (171), followed by Paecilomyces lilacinus @ 0.5 g per kg soil (178) irrespective of whether nematode inoculated individually or concomitantly.However, in plants inoculated with nematode alone, final nematode population J 2 /200 cc soil was minimum in case of liquid formulation of bio-agents (175), followed by P. lilacinus (168) as compared to untreated inoculated check (644).Plants inoculated with nematode and fungus concomitantly, final nematode population J 2 /200 cc soil was minimum in case of liquid formulation of bio-agents (180), followed by P. lilacinus (176) as compared to untreated inoculated check (637).Maximum reduction in nematode population J 2 /200 cc soil was observed in the presence of both nematode and fungus followed by nematode alone.
In general, all the treatments significantly reduced incidence of nematode and fungus concomitantly on cucumber as compared to untreated inoculated check.Data were recorded 15 and 30 days after sowing.At 15 days after sowing, disease incidence was minimum (15 %) in case of soil treated with liquid formulation of bio-agents (T.viride + P. fluorescence + P. lilacinus) @ 15 ml per kg soil or P. lilacinus) @ 0.5 g per kg soil as compared to untreated inoculated check (65 %).At 30 days after sowing, disease incidence was minimum (15 %) in case of soil treated with liquid formulation of bio-agents followed by 20 % in case of P. lilacinus) @ 0.5 g per kg soil as compared to untreated inoculated check (85 %).

DISCUSSION
Cucumber is highly susceptible to M. incognita and 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 indicated 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;Wright, 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).Abuzar (2003) found similar effectiveness of carbofuran in suppressing M. incognita on Abelmoschus esculetus.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.
Bavistin as an important control measure against F. oxysporum (Prasad et al., 2000; Haseeb and Shukla, 2002;Abuzar, 2003, Haseeb et at., 2006).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 may be applied.It is clear from the results that besides chemicals A. indica seed powder were sufficiently effective against both the pathogens, this may be due to presence of active principles and toxic chemicals in A. indica cake (Abuzar and Haseeb, 2009;Abuzar and Haseeb, 2010).Initial investigations on antagonistic rhizobacteria against root-knot nematodes; include work by Kloepper et al. (1992).P. fluorescens was found not only effective against M. incognita but also against wilt causing fungi.Results show that the Meloidogyne incognita-Fusarium oxysporum disease complex can cause severe yield losses in V. radiata as in other crops.Although chemicals viz.carbofuran and Bavistin showed a significant effect in increase of growth parameters and in suppression of the disease complex, these can be replaced to some extent by A. indica neem cake avoid the hazards of chemicals.

Table 1 .
et al.: STUDY OF BIO-MANAGEMENT OF CUCUMBER WILT Effect of soil treatment with bio-agents on shoot length (cm) of cucumber infested with M. incognita and fungus

Table 2 .
Effect of soil treatment with bio-agents on dry shoot weight (g) of cucumber infested with M. incognita and fungus

Table 3 .
Effect of soil treatment with bio-agents on dry root weight (g) of cucumber infested with M. incognita and fungus

Table 4 .
Effect of soil treatment with bio-agents on number of galls/plant of cucumber infested with M. incognita and fungus

Table 5 .
Effect of soil treatment with bio-agents on number of egg masses/plant of cucumber infested with M. incognita and fungus

Table 6 .
Effect of soil treatment with bio-agents on final nematode population/200 cc soil of cucumber infested with M. incognita and fungus Maximum reduction in dry root weight was observed in the presence of nematode and fungus followed by fungus alone and minimum in case of nematode alone.In general, dry root weight was significantly lesser in all the treatments compared to untreated uninoculated check irrespective of PATIL et al.: STUDY OF BIO-MANAGEMENT OF CUCUMBER WILT

Table 7 .
Effect of soil treatment with bio-agents on fungus incidence (%) of cucumber infested with M. incognita and fungus

Table 8 .
Effect of soil treatment with bio-agents on nematode + fungus incidence (%) of cucumber infested with M. incognita and fungus