​Efficacy of Bioagents against Sclerotinia Rot of Chickpea Incited by Sclerotinia sclerotiorum

Stem rot is also known as Sclerotinia wilt or white mold, caused by Sclerotinia sclerotiorum is a serious disease of chickpea. It infects all the economically important food and feeds legumes (Pratt and Knight, 1984). This fungus has a wide host range and has a worldwide distribution on numerous crops (Purdy, 1979: Boland and Hall, 1994). It is one of the deastructive pathogens associated with the root rot/wilt complex of chickpea and its occurrence is increasing in both incidence and severity on chickpea grown in the Mediterranean region (Anonymous, 1996).
       
The initial infection occurs in the late winter or early spring, and the fungal mycelia grow within and between plants. Patches like symptoms of dead plant parts enlarge and coalesce through spring and cause major losses in stands (Bolton et al., 2006). The fungus produces many black fleshy structures called Sclerotia, which survive from one cropping season to the next. Over-wintered Sclerotia may germinate during the summer or may remain dormant for many years (Adams and Ayers, 1979). The etiology, biology and epidemiology of the fungus had been studied extensively by several workers (Philips, 1987; Purdy, 1979; Roberts et al., 1982).
               
Cultivation of resistant varieties is the ideal and feasible control of the disease and no resistant varieties against this disease have been identified so far. Erect type cultivars can better withstand the disease and management can also minimize the crop losses. Stable resistance could not be achieved due to the prevalence of virulent isolates of S. sclerotiorum (Sharma et al., 2002). Management of Sclerotinia rot of chickpea through chemical alone is less effective as S. sclerotiorum having a broad host range and survives in soil for long periods in the form of Sclerotia. The Sclerotia will survive up to ten years even in the absence of the host plants and beneath prevailing dry soil conditions. The use of host plant resistance is the most economical strategy for the management of Sclerotinia rot in chickpea. Apart from the plant resistance, biological control of stem rot of chickpea is emerging as a new approach, because it is environmentally safe biopesticide and effective. To keeping in mind, the present investigation was carried out to manage this pathogen by used fungal and bacterial bioagents.

The antagonistic potential of four bioagents i.e. Trichoderma harzianum (Th-BKN), Trichoderma viride (Tv-BKN), Pseudomonas fluorescens (Pf-BKN) and Bacillus subtilis (Bs- BKN) against S. sclerotiorum was determined under laboratory conditions.
 
In vitro evaluation of fungal antagonists
 
Dual inoculation test
  
For evaluation of the antagonistic potential of Trichoderma spp. Dual inoculation technique was followed to ascertain the antagonistic capacity of T. harzianum and T. viride. One mycelial disc (5 mm diameter) each of the pathogen and antagonist was kept on the surface of potato dextrose agar (PDA) medium at 5 cm apart in Petri dishes. The inoculated Petri dishes were incubated at 26±1°C for 7 days. Three replications were kept for each fungal antagonist i.e. T. harzianum and T. viride. In the case of control, the Petri dishes were inoculated with mycelial discs of the test pathogen only. The mycelial growth of the test pathogen was measured after 7 days of inoculation. The inhibition of mycelial growth of the pathogen was calculated using the following formula as suggested by Bliss, 1934.
 
Where,                                                   
I  = Per cent inhibition
C = Mycelial growth of S. sclerotiorum in control (mm) 
T = Mycelial growth of S. sclerotiorum in the presence of antagonist (mm)
 
In vitro evaluation of bacterial antagonists
 
Paper disc inoculation method
 
Stock culture of B. subtilis was streaked on Nutrient Agar Media and stock culture of P. fluorescens was streaked on Pseudomonas Agar Fluorescens (PAF) slants and incubated at 27±1°C for 48 hours. Ten milliliters of sterilized distilled water were added to each slant containing the fresh colony of bacterial antagonist and suspension was prepared by scrapping the bacterial growth with the help of a sterilized inoculating needle. The suspension was then transferred to sterile Petri dishes. Sterilized filter paper discs (5 mm diameter) were dipped in the bacterial suspension. Four such inoculated discs were placed in opposite directions on the surface of Potato Dextrose Agar Media in Petri dishes. Mycelial discs (5 mm diameter) were taken from the periphery of actively growing culture of Sclerotinia sclerotiorum raised on Potato Dextrose Agar Medium was placed at the center of Petri dishes containing the inoculated paper discs. In the control, Petri dishes were inoculated with mycelial discs of the test pathogen only. Three replications were kept for each treatment. The inoculated Petri dishes having P. fluorescens and B. subtilis, discs were incubated at 27±1°C. Mycelial growth was recorded after 7 days of incubation. The inhibition of mycelial growth by the bacterial antagonist was calculated by using the following formula as suggested by Bliss, 1934.
 
Where, 
I  = Per cent inhibition.
C = Mycelial growth of S. sclerotiorum in control (mm)
T = Mycelial growth of S. sclerotiorum in the presence of antagonist (mm).

The efficacy of different fungal and bacterial bioagents was tested against S. sclerotiorum of chickpea by using different methods under in vitro conditions and the results obtained are presented below.
 
In vitro evaluation of fungal antagonists
 
Two native fungal bioagents T. harzianum (Th-BKN) and T. viride (Tv-BKN) isolated from chickpea rhizosphere were evaluated to ascertain the antagonistic potentiality against S. sclerotiorum by dual culture method. The results (Table 1, Plate 1 and Fig 1) revealed that both the fungal bioagents significantly inhibited the mycelial growth of S. sclerotiorum. The minimum mycelial growth of pathogen was recorded in T. harzianum (Th-BKN) (26.57 mm) followed by T. viride (Tv-BKN) (35.10 mm) as compared to control (90.00 mm). Both the fungal bioagents significantly inhibited the mycelia growth of the test pathogen as compared to control but maximum per  cent growth inhibition of pathogen was shown in T. harzianum (Th-BKN) (70.48%) followed by T. viride (Tv-BKN) (61.00%) after 7 days of incubation.
 

 

 

      
In vitro evaluation of bacterial antagonists
 
The efficacy of two native isolates bacterial bioagents viz., P. fluorescens (Pf-BKN) and B. subtilis (Bs-BKN) were evaluated against S. sclerotiorum by using the paper disc inoculation method. The results revealed that the highest mycelia growth of S. sclerotiorum was suppressed by P. fluorescens (Pf-BKN) (56.20 mm) followed by B. subtilis (Bs-BKN) (59.30 mm) as compared to control (90 mm). Both the bacterial bioagents significantly reduced mycelia growth and were found superior in controlling S. sclerotiorum. The results showed that the P. fluorescens (Pf-BKN) found relatively more inhibitory and gave maximum (37.56%) mycelial growth inhibition of S. sclerotiorum followed by B. subtilis (Bs-BKN) (34.11%) after 7 days of incubation. It was observed that fungal bioagents were competitively more effective than bacterial bioagents (Table 1, Plate 1 and Fig 1).
               
In the present experiment, the Antagonistic potential of two fungal and two bacterial bioagents was evaluated against S. sclerotiorum in vitro employing dual inoculation and paper disc inoculation methods. The fungal antagonists T. harzianum (Th-BKN) and T. viride (Tv-BKN) and two bacterial antagonists P. fluorescens (Pf-BKN) and B. subtilis (Bs-BKN) significantly inhibited the mycelia growth of isolates of S. sclerotiorum in vitro. The data showed that the obtained results revealed that all tested bioagents drastically reduced the linear growth of S. sclerotiorum. The antagonistic fungi Trichoderma harzianum (70.48%) showed superior inhibitory effect against the growth of pathogenic fungi as compared to bacterial isolates B. subtilis (34.11%) and P. fluorescens (37.56%), as the antagonistic fungi had a greater significant effect on the retardation of growth compared with the bacterial bioagents. The highest inhibitory effect was recorded for B. subtilis (Table 1). This indicated that T. harzianum and P. fluorescens are potential bioagents in controlling the stem rot of chickpea. Similar observations were reported by earlier workers Sharma et al.,(1999) found that Trichoderma harzianum caused maximum inhibition (75%) of mycelial growth followed by A. cylindrospora and T. viride. Similar results were also reported by Sharma (1994). Kuar et al., (2003) also tested the Trichoderma isolate viz., T. harzianum (Th 38) and T. viride (Tv 34) by dual culture against S. sclerotiorum and observed that T. harzianum (Th 38) completely parasitized the pathogen. Cuong et al., (2007) evaluated the antagonistic potential of Trichoderma harzianum and observed the maximum inhibition of mycelia growth of the pathogen in vitro. Javeria et al., (2014) also reported that maximum inhibition was found by T. harzianum causing 70.82% inhibition of the mycelial growth of the S. sclerotiorum. Sharma et al., (2016) evaluated the antagonistic potential of viz., Bacillus subtilis, Trichoderma harzianum, T. viride etc. and they recorded the maximum inhibition was found by T. viride 69.8% and minimum mycelial growth inhibition 42.2% was recorded in Bacillus subtilis.

The fungal antagonist T. harzianum (Th-BKN) and T. viride (Tv-BKN) and bacterial antagonist i.e. P. fiuorescens (Pf-BKN) and B. subtilis (Bs-BKN) significantly suppressed the mycelial growth of S. sclerotiorum isolate in vitro.