Potential of fungal and bacterial biocontrol agent under in vitro conditions against Macrophomina phaseolina causing pigeonpea stem canker

Introduction

Pigeonpea [

Cajanus cajan (L.) Mill spaugh] is one of the major legume crops grown in the tropics and sub tropics which accounts for about 5 per cent of world legume production with India being the largest producer. It is hardy, widely adopted and drought tolerant crop with a large temporal variation (90-300 days) for maturity. The crop is able to fix atmospheric nitrogen in the soil thereby increases the soil fertility (Smitha et al. , 2015) [22] . However, production of pigeonpea in the Indian subcontinent and other countries in Asia is severely

diseases such as Fusarium wilt, Phytophthora blight, powdery mildew, dry root rot, stem canker and sterility mosaic. Among these Macrophomina phaseolina (Tassi) Goid., which causes stem canker is becoming severe in most of the pigeonpea growing regions of India (Bajpal et al. , 1999) [2] . M. phaseolina , which causes stem canker in a wide range of hosts is one of the most destructive plant pathogens in the tropics and subtropics. Disease development is favoured by high temperature (30-35 C) followed by moisture stress and a good source of inoculum (Dhingra and Sinclair, 1973a) [4] .

Pigeonpea stem canker caused by

Macrophomina phaseolina (Tassi) Goid. is becoming very severe in many parts of pigeonpea growing regions in the country. Macrophomina is primarily a soil and seed-borne fungal pathogen that incites the disease by producing microsclerotia/pycnidia (Farr et al. , 1995 and Pun et al. , 1998) [6,19] . It has a wide host range of more than 500 species from 75 families with heterogeneous host specificity i.e. the ability to infect monocots as well as dicots and can exhibit non-uniform distribution in the soil (Mayek-Perez et al. , 2001 and Su et al. , 2001) [16,23] . In India, the first report of stem canker of pigeonpea occurrence was reported by Kannaiyan et al. (1979) [10] in part of Eastern Uttar Pradesh (Varanasi and Mirzapur districts). Recently, the stem canker incited by M. phaseolina has emerged as one of the important pathogens of different agricultural crops, including pigeonpea (Kaur et al., 2012b) [12] . Macrophomina Stem Canker disease (MSC), caused by M. phaseolina is potentially a serious disease in pigeonpea that occurs from flowers to physiological maturity. The disease also occurs in states of Bihar, Madhya Pradesh, Maharashtra, Orissa, Uttar Pradesh, Andhra Pradesh, Tamil Nadu, Gujarat and Rajasthan (Kannaiyan et al., 1981) [11] . The disease is also reported from Australia, Myanmar, Sri Lanka, Vietnam and Zambia (Nene et al. , 1996) [17] . The fungus produces necrotic lesions on the stem and girdles the plant at the base leading to premature flower drop, drying of branches that eventually results in total witling and the death of the plant. It causes huge economic losses ranging from 10-100 percent (Smitha et al., 2015) [22] . The decline in pigeonpea production was recorded in Maharashtra and Karnataka during 2023 and 2024 due to high incidences of Macrophomina stem canker. None of the cultivated cultivars could withstand the disease overcome. The climate change has been influencing this lesser-known Macrophomina stem canker disease into major epidemic. The dry and warm climate favourable to the disease development are frequently witnessed in the recent three years. In view of the severe outbreak of this disease, new investigation into the management became necessary to curtail the disease and its spread. The current study was conducted to know the efficacy of different antagonist against the pathogen for its effective control under in vitro.

The results of the study were anticipated to draw the strategies for the lab and field management of the disease.

Materials and Methods

Sevan fungal antagonists viz., Trichoderma asperallum, T. harzianum, T. lignorum, T. virens, Metarhizium anisopliae, Aspergillus niger and two Bacterial antagonists Bacillus subtilis and Pseudomonas fluorescens was evaluated in vitro against M. phaseolina, applying Dual culture technique (Arora and Upadhay, 1978) [1] . Seven days old cultures of the test bioagents and the test pathogen ( M. phaseolina) grown on agar media was used for further study. The culture disc (5 mm) of the test pathogen and bioagent were cut out with sterilized cork borer, from a week-old culture. Then two culture discs, one each of the test pathogen and bioagent was placed aseptically at equidistance and exactly opposite with each other on solidified PDA medium in Petri plates and plates were incubated at 28+2 C. Three replications were maintained. PDA plates inoculated only with culture disc of the test pathogen was maintained as untreated control. Observations on linear colony growth/diameter (mm) of the test pathogen and the test bioagent was recorded at an interval of 24 hrs of incubation and continued up to seven days or till the untreated control plates were fully covered with mycelial growth of the test pathogen. Based on cumulative data, per cent mycelial growth inhibition of the test pathogen with the test bioagents, over untreated control was calculated by applying following formula (Arora and Upadhay, 1978) [1] .

Details of experiment

Colony growth in control plate-Colony growth in intersecting plate Per cent Growth Inhibition =

x 100 Colony growth in control plate

Results and Discussion

The results obtained on mycelial growth and inhibition of M. phaseolina with seven fungal and two bacterial antagonists were represented in Table 1. Results (PLATE1, Table 1 and Fig. 1) revealed that all of the bioagents evaluated exhibited fungistatic action against M. phaseolina cuasing stem canker of pigeonpea and significantly inhibited its growth, over untreated control. However, Trichoderma harzianum was found most effective with significantly least colony growth of the pathogen (02.44 mm) and its significantly highest inhibition (97.29 %) which was followed by Aspergilus flavus (05.41 mm and 93.99%), A. niger (06.37 mm and 92.92 %), Bacillus s ubtilis (11.34 mm and 87.40 %), Pseudomonas fluorescens (13.43 mm and 85.08 %), T. asperellum (14.32 mm and 84.09 %), T. lingorum (19.57 mm and 78.26 %), Metarhizhium anisopliae (36.12 mm and 59.87 %) and T. koningii (58.89 mm and 34.57%), respectively of the p athogens colony growth and its inhibition. Thus, the bioagents viz., Trichoderma harzianum , Aspergilus flavus , A. niger, Bacillus subtilis, Pseudomonas fluorescens and T. asperellum were found most potential antagonists against M. phaseolina causing stem canker of pigeonpea. Thus, all of the nine bioagents evaluated by using dual culture technique were found fungistatic against M. phaseolina and significantly inhibited its mycelial growth, over untreated control. However, the bioagents found most effective in the order of merit were Trichoderma harzianum , Aspergilus flavus , A. niger, Bacillus subtilis , Pseudomonas fluorescens , T. asperellum , T. lingorum, Metarhizhium anisopliae and T. koningii , respectively of the

ts inhibition. These results are confirmed with earlier workers Lokesha and Benagi (2007) [14] reported that T. harzianum , T. viride and P. fluorescens significantly inhibited the mycelial growth of M. phaseolina causing dry root rot of pigeonpea (74-76%) by using Dual culture technique. Swamy et al. , (2018) among the bio-agents tested T. harzianum (Th-R) was found more effective as compared to other bio-control agents and inhibited maximum fungal growth (41.86%) of M. phaseolina causing stem canker of pigeonpea. The results are similar with Tandel (2004) [25] reported in vitro interaction study of known antagonist by dual culture method and studied strong antagonistic effect on M. phaseolina causing leaf blight of green gram with T. viride, T. harzianum , B. subtilis, T. longibrachyatum , T. koningii and Chaetomium globosum . Kumar and Kelaiya (2021) [13] , Gajera et al. (2012) [8] who studied in the laboratory experiment effect of Trichoderma spp. against M.

phaseolina . The highest growth inhibition of test fungus was recorded by bioagents T. koningi (74.3%) followed by T. harzianum (61.4%). The results of this study are in harmony with Biswas and Sen, (2000) [3] studied Trichoderma spp. was to be a potential antagonist against M. phaseolina through colony interaction. Sreedevi et al. (2011) stated that all five Trichoderma spp. were antagonistic against M. phaseolina by using dual culture technique. Elham et al. (2016) [5] reported that all three isolates of T. harzianum significantly inhibited the growth of M. phaseolina . These earlier reports lend support to the present findings. A multiplicity of mechanisms involving mycoparasitism, antibiosis, lysis and hyphal interference could be attributed to the reduction in the mycelial growth of M. phaseolina . Gireesha et al. , (2025) [9] studied in in vivo experiments, it was found that, out of the bioagents tested, T. harzianum was the most effective in inhibiting maximum mycelial growth of M. phaseolina followed by T. viride (67.72%) and P. fluorescens (61.41%). The specific activities of cell wall degrading enzymes such

-1, 3 glucanase, protease and cellulase were also recorded (Silva et al. , 2004) [21] . Rathore et al., (2020) [20] . Parmar and Patel (2020) [18] confirmed T. asperellum , T. harzianum, T. viride, T. longibacter , T. koningii bioagents inhibit the growth of M. phaseolina in vitro. Lakhran and Ahir (2022) [15] also reported the effectiveness of T. viride, B. subtilis and P. fluorescence on radial growth of M. phaseolina . Gadde et al., (2023a) [7] Bio-controls tested Trichoderma asperellum showed maximum mycelial inhibition (81.06%) while Pseudomonas fluorescence and Bacillus subtilis showed zero percent inhibition against M. phaseolina causing stem blight in pigeonpea. Indra et al. , (2024) [10] conformity with four fungal antagonists viz., T. asperellum , T. harzianum , T. virens and T. koningiopsis , three bacterial antagonists viz., Bacillus licheniformis , B. amyloliquefaciens and B. subtilis were effective against M. phaseolina causing root rot in mulberry. Mycelial growth inhibition of M. phaseolina by Trichoderma spp. may be attributed to the secretion of extracellular cell wall degrading enzymes such as chitinase -1, 3-glucanase, cellulose and lectin etc., production of secondary metabolites such as glioviridin, viridian and gliotoxin, also various mechanisms such as competition, lysis, antibiosis, production of volatile/non-volatile substances.

Conclusions

All the seven fungal and two bacterial antagonists tested, exhibited significant mycelial growth inhibition of M. phaseolina . However, Trichoderma harzianum was found most effective with significantly least colony growth of the pathogen and its significantly highest inhibition which was followed by Aspergilus flavus , A. niger, Bacillus subtilis , Pseudomonas fluorescens , T. asperellum , T. lingorum, Metarhizhium anisopliae and T. koningii respectively of the

Acknowledgement

AcknowledgmentThe authors are grateful to Department of Plant Pathology, College of Agriculture, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani for providing facilities to conduct this research. The authors are also grateful to all those who helped for providing useful information during in vitro studies.

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