Introduction

Groundnut or peanut (Arachis hypogaea L.), is a very important legume crop of tropical and sub tropical areas of the world, described in 1753 by Linnaeus (Pattee and Young, 1982). In India, groundnut occupies 35 per cent of the total cropped area under oilseeds and accounts for 40 per cent of total oilseeds production (Anonymous, 2014). On an average, groundnut seed contains 45 per cent of oil and 26 per cent of protein and its kernels are relished either as snack, roasted or salted or raw form or also in the form of peanut butter. Obviously, poor soil fertility, abiotic and biotic stress factors limit the growth of groundnut crop and yield in many ways. Among biotic stresses, groundnut is attacked by many fungal, bacterial and viral pathogens. Collar rot caused by Aspergillus niger van Teighem is one of the most important disease of groundnut which is more extensive in the kharif than the rabi/summer seasons and causes more damage in sandy loam and medium black soil. Annual world yield loss caused by collar rot  is more than 10 per cent (Pande and Rao, 2000) and is more prevalent in soils with low moisture content and high temperature, approximately 30ºC (Kishore et al., 2007). Several attempts have been made to control collar rot disease in groundnut by chemical means as seed dressers and foliar sprays application by various workers from time to time. Fungicides used prior to the mid 1950s were not effective against A. niger. In recent years, several fungicides have been reported to control the disease with varying degree of success (Desai and Bagwan, 2005). Shivpuri et al. (2011) evaluated four seed dressers against collar rot disease (Aspergillus niger van Teighem) of groundnut under field condition during Kharif 2008 to 2010. All seed treatments were found significantly superior over check in reducing pre and post emergence rot, increasing root and shoot length and pod yield. Vitavax Power @ 3.0 g/kg seed showed minimum incidence of pre and post emergence mortality i.e. 11.80% and 4.50%, respectively. Devi and Prasad (2009) used captan seed treatment as 2g/kg and reported the lowest disease incidence (64.7%) and higher germination (80.7%) under pot culture experiment   compared to control exhibiting disease incidence and germination per cent as 97.7 and 75.3 per cent, respectively. Limited work has been reported in literature on management of this disease through the use of bio-agents and botanicals. Devi and Prasad (2009) reported that antagonistic nature of Trichoderma spp. in vitro against A. niger. The per cent inhibition of the pathogen was 68.0 per cent by T. viride followed by T. harzianum (54.0%) and Pseudomonas fluorescens (34.0%) and also seed treatment with T. viride @ 4g/kg in pot culture experiment, showed higher germination, disease incidence, shoot length, root length and dry matter production of 88.5 per cent, 72.3 35.3cm, 26.3 and 1.8g, respectively. Gajera et al. (2011) evaluated the twelve isolates of three Trichoderma strains in vitro against the collar rot. It was observed that T. viride 60 inhibited maximum (86.2%) growth of test fungus. Karthikeyan (1996) ascertained that soil application of the antagonist T. viride @ 500 g inoculum/20 sq.m, showed minimum disease incidence of 2.32 per cent as compared to control disease incidence of 10.40 per cent. Organic amendments are known to favour native antagonists and suppress soil and seed borne disease (Mayakrishnan, 1990; Alice, 1994). Since the pathogen is very difficult to manage due to its soil or seed borne nature and wide host range, therefore the present study was carried out with the objective to evaluation of different fungicides, bio-agent and botanicals for the control of Collar rot of groundnut caused by Aspergillus niger under in vitro and screen house conditions.

Materials and Methods

Efficacy of different Fungicides and bio-agents against A. niger in vitro

Evaluation of fungicides/Pesticides

The Efficacy of six fungicides viz., thiram, captan, carbendazim, carboxin, hexaconazole, propiconazole, chlorpyriphos (Insecticide) and pendimethalin (herbicide) on the growth of Aspergillus niger were tested under in vitro conditions using the standard procedure of poison food technique as given by Mayer (1962). Stock solution of each pesticide was prepared in double strength i.e. 50, 100, 200, 500 and 1000 ppm by dissolving weighed or measured quantity of pesticide in a measured volume of sterilized water. The double strength potato dextrose agar (PDA) medium was also prepared and sterilized at 15 lb pressure for 20 minutes. An equal volume of chemical solution and PDA was mixed in a sterilized conical flask and poured aseptically in the Petri plates. After solidification of medium, each Petri plate was centrally inoculated with 5 mm disc of fungus taken from 7 days old culture of A. niger with the help of sterilized cork borer and incubated at 28±1^ºC. Suitable controls were maintained for each chemical. Four replications of each pesticide were maintained and CRD was followed. Colony diameter of the fungus of each treatment along with control was measured (mm) and recorded after every 24 hours, till the test fungus occupied the full Petri plate in the controlled treatment. The per cent inhibition of mycelial growth over control was calculated by following formula given by Vincent (1947).

(C-T)

Growth inhibition (%) = ––––––– × 100

C

Where,

I = Per cent inhibition of mycelium growth

C = Radial growth of A. niger mycelium in control

T = Radial growth of A. niger mycelium in treatment

Evaluation of bio-agent

The antagonistic effect of three bio-agents viz., Trichoderma harzianum, Trichoderma viride and Pseudomonas fluorescens on the growth of A. niger was tested using dual culture technique (Morton and Strouble, 1955). The fungus was cultured on the PDA while the bacteria were cultured on NDA but the antagonistic effects of the bio-agents were tested on PDA. Fifteen ml of liquefied PDA medium was poured into sterile Petri plates and allowed to solidify. From seven days old culture of A. niger, 5 mm mycelial disc was cut from the margin of the actively growing colony with a sterile cork borer and placed near the periphery, on one side of the PDA plate while an antagonistic fungi were placed on the other side of the PDA plate just opposite to the first disc i.e. at an angle of 180º. Similarly, antagonistic bacteria obtained from three days old culture were streaked five cm long on the PDA medium at the two cm mark from the periphery of the Petri-dish. Simultaneously, five mm mycelial disc of A. niger were cut from the margin of the actively growing colony with a sterile cork borer and placed near the periphery on opposite side of the bacterial streak i.e. at an angle of 90º. All the plates were incubated at 28 ± 1 ºC for five days. Each treatment replicated four times as CRD and appropriate controls were maintained. The extent of antagonistic activity by fungal and bacterial antagonists was recorded on fifth day by measuring the growth of A. niger in dual culture plate and control plate. The per cent inhibition of A. niger was calculated as suggested by Vincent (1947) as described earlier for pesticides evaluation in vitro. The best treatments of both the fungicides and bio-agent evaluated in vitro were also tested for their efficacy under screen house conditions.

Efficacy of promising seed treating fungicides and bio-agents under screen house conditions

The antagonistic effect of the promising fungicides and bio-agents evaluated in vitro were further evaluated under the screen house conditions. These fungicides were: propiconazole (1ml/kg seeds), carbendazim (2g/ kg seeds) and captan  (3g/ kg seeds) While the bio-agents were: Trichoderma viride (20g/kg soil) and Trichoderma harzianum (20g/kg soil). Soils were pre-inoculated with inoculum of A. niger in the earthen pots of 12” diameter. For the fungicides, seeds of each genotypes viz., MH-4, MH-21, M-522, and HNG-10 were treated 72 hours prior to planting. For the bio-agents Trichoderma viride, and T. harzianum, they were initially grown on sterilized wheat medium by inoculating with 7 days old mycelium and incubated for 10 days at 28 ± 1 ºC to allow maximum establishment of the fungus mycelium. Prior to planting of seeds of each genotypes viz., MH-4, MH-21, M-522, and HNG-10, soils were inoculated with the respective bio-agents (20g/ kg soil).Ten seeds were planted in each pot and for each treatment four replications were maintained as CRD. Untreated seeds sown in pre-inoculated soil only with A. niger served as control. The observation was recorded 30 days after sowing (DAS) in terms of disease incidence.

Efficacy of different botanicals under screen house conditions

For screen house experiments earthen pots were filled with sterilized sandy loam soil at the rate of 1 kg/pot. Four pots of each genotype (MH-4, MH-21, M-522, and HNG-10) with each treatment of powdered botanicals @ 20g/kg soil namely neem cake and mustard cake. Five seeds were planted in each pot and for each treatment four replications were maintained as CRD and each were inoculated with spore suspension (10⁶/ml with the help of haemocytometer). Inoculated seeds sown in pots without botanicals served as control.  Per cent disease incidence was recorded 30 DAS.

Results and Discussion

Efficacy of different Fungicides and bio-agents against A. niger in vitro

Evaluation of fungicides/Pesticides

Efficacy of fungicides was tested in vitro under laboratory conditions for the per cent mycelial growth inhibition of A. niger. The data in Table 1 clearly revealed that propiconazole, carbendazim and carboxin completely inhibited the mycelial growth up to 100 per cent at 200, 500 and 1000 ppm concentration, respectively as comparable with 86.94, 88.05 and 59.96 per cent at their respective 100, 200 and 500 ppm. Captan and thiram were found very less effective as they inhibited 81.11 and 72.77 per cent of fungal growth, respectively at higher concentration of 1000 ppm. Hexaconazole (fungicide), pendimethalin (herbicide) and chlorpyriphos (insecticide) were also not found effective in inhibiting one per cent of the mycelial growth even at 1000 ppm.

Table 1: Effect of different pesticides on per cent mycelial growth inhibition of A. niger in vitro

*Mean of four replications. Figure in parenthesis indicate angular transformed values.

Shekhawat et al. (1986) reported almost similar result as completely inhibition of the mycelial inhibition in vitro at 1500 ppm by carbendazim and carboxin. Captan and thiram were found very less effective as they inhibited 81.11 and 72.77 per cent of fungal growth, respectively at higher concentration of 1000 ppm, while fungicide hexaconazole, herbicide pendimethalin and insecticide chlorpyriphos have failed to inhibit the mycelial growth even at 1000 ppm. Gupta et al. (1974) found that in vitro studies growth of A. niger was completely inhibited at higher concentration of vitavax at 1500 ppm, captan and thiram at 2,000 ppm. No report for fungicide hexaconazole, herbicide pendimethalin and insecticide chlorpyriphos has been put forward as an effective pesticide against A. niger.

Evaluation of bio-agent in vitro

Three bio-agents namely Trichoderma viride, T. harzianum and Pseudomonas fluroscence were tested for their inhibition of mycelial growth of A. niger in vitro (Table 2). T. viride showed maximum antifungal activity with 78.32 per cent inhibition of mycelial growth of A. niger followed by T. harzianum with 72.50 per cent inhibition of radial growth, while the bacterial bio-agent P. fluroscence inhibited 23.80 per cent of the mycelial growth of the test fungus over control.

Table 2: Effect of different bio-agents on per cent mycelial inhibition of A. niger in vitro

*Mean of four replications, Figures in Parenthesis indicate angular transformation values.

These above results are in agreement with Gajera et. al. (2011) who demonstrated that twelve isolates of three Trichoderma strains in vitro against the collar rot, maximum inhibition of pathogen by T. viride followed by T. harzianum. Devi and Prasad (2009) also found similar result in which the per cent inhibition of the pathogen was maximum by T. viride followed by T. harzianum and very less effect of Pseudomonas fluorescens on inhibition of the pathogen in vitro. The parasitic behavior of Trichoderma was recorded to be of a necrotrophic or destructive type (Barnet and Binder, 1978; Transmo and Dennis, 1978).

Efficacy of promising seed treating fungicides and bio-agents on inhibition of A. niger under screen house conditions

Treated seeds with fungicides showed significantly less disease incidence for collar rot (Table 3). Fungicides were superior to bio-agents as fungicide treated seeds significantly controlled the collar rot disease. Maximum control was shown by propiconazole, controlling 87.50 per cent disease in genotype HNG-10 and 84.21 per cent in M-522.  On an average, carbendazim reduced disease incidence by 67.18 per cent and captan by 61.17 per cent. In case of Bio-agents, on an average, T. viride showed maximum control of the disease by 39.12 per cent followed by T. harzianum (28.40%).

Table 3: Efficacy of promising seed treating fungicides and bio- agents on inhibition of A. niger under screen house condition after 30 days of sowing

*Mean of four replications, the values in parenthesis are angular transformation.

Similar results were obtained by Karthikeyan (1996) the lowest disease incidence in which, he, reported carbendazim (2g/kg seed) was used as seed treatments as compared to disease incidence in control. Devi and Prasad (2009) also found same results, when applied captan as seed treatment @ 2g/kg seed under pot culture experiment.

Efficacy of different botanicals under screen house conditions

Among the botanicals, neem cake powder @ 20g/kg soil significantly controlled the disease up to 32.53 per cent followed by mustard cake powder @ 20g/kg soil which controlled the disease up to 23.97 per cent as comparison to untreated control (Table 4).

Table 4: Efficacy of different botanicals under screen house conditions after 30 days of sowing

Neem cake powder @ 20 g/kg soil, mustard cake powder @ 20 g/kg soil,  *Mean of four replications. Figures in Parenthesis indicate angular transformation values.

Organic amendments are recorded to favour native antagonists and suppress soil and seed borne diseases (Mayakrishnan, 1990; Alice, 1994). Similarly, Karthikeyan (1996) observed that the organic amendment with neem cake was highly effective in reducing disease incidence of collar rot of groundnut. Mustard cake was not found effective against collar rot disease in the present study.

Conclusion

It is concluded from the study, that the Trichoderma viride and propoiconazole showed their maximum antifungal activity in vitro and under screen house conditions and are needed to be tested under field conditions, so that they can be incorporated in integrated disease management strategies against this pathogen.

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