Biocontrol activity of Trichoderma viride, Trichoderma harzianum, Bacillus subtilis and Pseudomonas fluorescens (in vitro) against Bipolaris oryzae, causal agent of rice brown spot disease

Author: Badavath Kishore, Anshu Kumar*, Pravesh Shivakoty and Venu Emmadi

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Abstract

Rice is one of the crops that farmers cultivate on a regular basis, and it is a essential cereal grain for everyone. Diseases are one of the major causes which hampered the plant produce. Rice brown spot is a devastating disease of rice which is caused by Bipolaris oryzae and accounts for huge yield losses, despite the fact there are limited management practices available. Biocontrol agents offered environment friendly management of diseases and they are currently preferred over the usage of chemicals. In this view, dual culture technique was employed with biocontrol agents (Trichoderma viride, T. harzianum, Pseudomonas fluorescens and Bacillus subtilis) against Bipolaris oryzae. In vitro studies indicated that of all the biocontrol agents utilised in this investigation, Trichoderma viride was shown to be the most antagonistic. Trichoderma viride (61.95%) was the most effective at inhibiting mycelial growth, followed by Trichoderma harzianum (59.78%). Among the bacterial biological control, Bacillus subtilis had the highest percent inhibition (45.52%), followed by Pseudomonas fluorescence (39.05%). This study may stimulate other researchers to evaluate different biocontrol agents against Bipolaris oryzae in order to better manage the disease.

Keywords

Brown spot, Biopolaris oryzae, Biocontrol agents, in vitro

Conclusion

In the present study, among fungal biocontrol agent, Trichoderma viride perform exceptionally well in controlling the brown spot of rice under in vitro condition. Microorganisms are naturally active in suppressing pathogens by micoparasitism, antibiosis, or the formation of pathogen-inhibiting volatile chemicals. According to the findings of this study, Bacillus subtilis emerged as a promising candidate for suppressing rice brown spot in vitro. The result concludes that indigenous species of Trichoderma and Bacillus strain has a great possibility of becoming a viable antagonist against Bipoaris oryzae. Biocontrol agents can be our new pillar in disease management strategies and their mechanism of action, compatibility with other components of integrated disease management and their efficiency in field are all key topics that need to be thoroughly researched. These findings could pave the way for the agriculture industry to adopt more ecologically friendly farming practices.

References

INTRODUCTION Rice (Oryza sativa L.) is the most widely cultivated crop in the world, and it is a staple cereal food for millions of people. Bipolaris oryzae (teleomorph=Cochliobolus miyabeanus) causes Brown spot of rice and considered as one of the most important disease of rice throughout the world (Khalili et al., 2012; Reddy et al., 2010; Savary et al., 2000). It is responsible for a drop of 26 to 52 percent grain yield (Chakrabarti, 2001). In India, the catastrophic Bengal famine of 1942-43 was the result of this disease which accounts for 50-90 per cent yield loss and death of over 4 million people (Chakrabarti 2001; Padmanabhan, 1973). Bipolaris oryzae causes symptoms of dark brown spots, elongated to circular in shape which may reach upto 1 cm or more in length. Leaf sheaths and coleoptiles exhibit similar symptoms. The pathogen infects inflorescence which results in failure of grain development and establishes a major problem during the commercial production of rice. Chemicals are often applied for the management of brown spot disease but their use is known to have negative consequences in environment. They are creating vulnerable conditions for beneficial soil microorganisms. Besides this, they increased plant protection costs which directly affect farmer’s livelihood. Treatments with fungicides are also inconsistent due to the complexity of soil ecology and year–to–year variations in climate conditions. Biocontrol agents are now gaining popularity among phytopathologists as a new alternative for the control of various plant diseases. Biocontrol agents are safe, long lasting and ecofriendly. The fungal and bacterial antagonists have been shown to promote the growth of various crops (Singh et al., 2012). Biocontrol agents could be less harmful to the environment than synthetic fungicides. Bacillus, Pseudomonas and Trichoderma were the most commonly employed fungal and bacterial biocontrol agents against diverse plant diseases (Nakkeeran et al., 2005; Saravanakumar et al., 2007). Mycoparasitism and antagonism are the two main weapon used by Trichoderma spp. for the control of fungal disease (Singh et al., 2005) Several Trichoderma species have been demonstrated to have antagonistic activity against wide range of pathogens (Dutta and Das, 2002; Das et al., 2006; Dutta et al., 1999; Dutta et al., 2008) Moreover, Trichoderma viride (T. viride) and Trichoderma harzianum (T. harzianum) are two well known widely used Trichoderma species. Biswas et al. (2010) found that application of Trichoderma viride or T. harzianum reduced the disease upto 70%. Bacterial biocontrol agents have been extensively utilized in plant disease management because it poses a range of biological control mechanism against different pathogens. They are associated with secondary metabolites production in agroecosystem and act as a potential antagonist against number of fungal pathogens. Biological control agents such as Pseudomonas fluorescens and Bacillus subtillis are commonly utilised. In previous studies mixture of Pseudomonas fluorescens and Bacillus subtillis are found effective against bacterial blight of cotton caused by Xanthomonas axonopodis pv. Malvacearum (Salaheddin et al., 2010). Talc based formulation of Pseudomonas fluorescens have been reported to suppress Bipolaris oryzae (Joshi et al., 2007). In future, there will be transition of disease management strategies from chemicals to biocontrol agents, necessitating the study of different antagonists as next-generation alternatives. Therefore, the efficiency of two fungal biocontrol agents, Trichoderma viride and Trichoderma harzianum, as well as two bacterial antagonists, Pseudomonas fluorescens and Bacillus subtillis, in suppressing rice brown spot disease was investigated under in vitro condition. MATERIALS AND METHODS A. Isolation of pathogen Isolates of Bipolaris oryzae were collected from diseased rice crops at the Bidhan Chandra Krishi Viswavidyalaya, Nadia, West Bengal. The leaves with typical brown spot symptoms induced by Bipolaris oryzae were cut with a sharp and sterilised blade, placed in paper bags, and brought to the laboratory. The necrotic patches of diseased leave along with some healthy tissue were cut into small pieces inside the laminar air flow and surfaces sterilized by dipping in 1 percent sodium hypochlorite for 20-30 seconds and were washed by sterilized water for three times. The cut pieces were plated in petri plate containing sterilize potato dextrose agar medium (PDA) (Riker and Riker, 1936) and kept in BOD incubator at 25±2°C for 3-4 days so that the fungi get ideal condition for growth. Purified culture were maintained in PDA slants and stored at 4°C. B. In Vitro evaluation of biocontrol agents against Bipolaris oryzae Efficacy of local biocontrol agent Trichoderma harzianum, T. viride, Bacillus subtilis and Pseudomonas fluorescens were tested on Potato Dextrose Agar against B. oryzae by dual culture technique. The local strain of biocontrol agents was collected from Department of Plant Pathology, B.C.K.V. Dual culture technique was carried out by picking a 5 mm disc from actively growing mycelium of Bipolaris oryzae with the help of sterilized cork borer and placed at one side of the Petri plate. After this, a 5 mm disc of fungal bioccontrol agents (Trichoderma harzianum or T. viride) was inoculated at opposite side of the previously inoculated test pathogen in the same plate (Dennis and Webster, 1971). Similarly, for bacterial biocontrol agent, Bacillus subtilis and Pseudomonas fluorescens, a 5 mm diameter disc was taken from 7 days old culture of B. oryzae and placed on one end of the Petri dish. Afterwards, bacterial antagonists were streaked with the help of inoculation needle on the opposite side of inoculated B. oryzae. Control plates were also maintained in PDA plates. These plates were incubated at 28 ±1°C temperature for 5 days. Completely Randomized Design (CRD) with three replications was taken to validate the result and the percentage inhibition of pathogen mycelial growth was calculated using the formula below (Vincent, 1927). RESULT AND DISCUSSION A. In vitro evaluation of fungal bio-control agents against Bipolaris oryzae causing brown spot of rice Trichoderma are mostly found in rhizosphere of plant. They are excellent candidate of biocontrol agents and effectives against seed and soil borne fungus, root rot, stem rot, wilt, and blight and also to some extent control nematodes. The unique nature of Trichoderma, being non-pathogenic to plants is because of its ability to produce a number of substances that cause localized or systemic resistance responses in plants. They produce antibiotics and cell wall degrading enzyme like chitinases, β-1, 3-Glucanases etc. Trichoderma are known for their ability to parasitize other fungi, a process which is called as mycoparasitism and cause lysis of the pathogens (Majumdar et al., 1996). In this study, both Trichoderma species significantly reduced the mycelial growth of the test pathogen, Bipolaris oryzae. Trichoderma viride was found to be the most antagonistic biocontrol agent of all the biocontrol agents used in this study (Table 1). It shows highest inhibition in radial growth of the pathogen (61.95%) followed by T. harzianum (59.78%) (Fig. 1 and 2). This result is in alignment with the earlier experiment conducted by Harish et al., (2008) in which Trichoderma viride (Tv2) was found to be significantly effective in inhibiting the pathogen's mycelial growth (62.92%) and spore germination (77.03%), followed by Trichoderma harzianum (Th5) and Trichoderma reesei (Tr3). Similar result was obtained by Manimegalai et al. (2011) which showed an inhibition of 67.9 % with T. viride. Bipolaris oryzae is effectively suppressed (upto 61.72%) by T. viride (Kumar et al., 2016). Another species of Trichoderma, T. harzianum render significant inhibition (48%) against Bipolaris oryzae (Abdel-Fattah et al., 2007). Singh et al. (2021) demonstrated that when T. harzianum was exposed to dual culture bioassays, it inhibited the growth of F. oxysporum (66%). Trichoderma asperellum showed potential in managing a range of fungal diseases and their antagonistic nature aid in enhancing medicinal plant productivity and quality. Trichoderma are opportunistic and avirulent plant symbionts capable of establishing strong colonization with root surfaces (Harman et al., 2004). The plant proteome and metabolism are significantly altered as a result of these root microbe interactions. Further, it was very well studied that Trichoderma developed over the pathogen, causing hyphal coiling, hyphal abnormalities and hyphae lysis (Malathi, 1996). The potential of T. viride on B. oryzae is primarily due to the production of lytic metabolites and through mechanism of mycoparasitism. B. Evaluation of bacterial bio-control agents against brown spot of rice Bacillus species posses a wide range of antimicrobial activities and they are used as anti-fungal agents (Milner et al., 1995). Bacillus subtilis has a profound antagonistic relationship with B. oryzae (Sarala et al., 2004). The data showed in the Table 2 revealed the antagonistic effect of selected bacterial biocontrol agents against Bipolaris oryzae. Both the pathogens cause reduction in radial growth of the pathogen. The percent inhibition over control ranges from 45.52% and 39.05%. Maximum percent inhibition was observed by Bacillus subtilis (45.522%) followed by Pseudomonas fluorescens (39.05%) (Fig. 2 and 3). In a study it was found that Bacillus amyloliquefaciens (BS5) exerted mycelial growth inhibition (76.66%) against Bipolaris oryzae (Prabhukarthikeyan et al., 2019). Bacillus species (B. subtilis, B. polymyxa, B. brevis, B. licheniformis, B. circulans, B. cereus etc) are active producer of antibiotics (Yilmaza et al., 2006). In previous studies, P. fluorescens was found to be antagonistic to B. oryzae, with the highest percent inhibition (75.22%), followed by S. marcescens (72.78%) and B. subtilis (70.56 %) (Sanjeevkumar et al., 2016). Alagesaboopathi and Selvankumar (2011) showed antagonistic effect of P. fluorescens against a wide range of fungal pathogen including Bipolaris oryzae, Rhizoctonia solani, Fusarium oxysporum, Alternaria brassica, Cochliobolus lunatus, Aspergillus niger and Trichothecium reseum. It was found that seed treatment with Pseudomonas spp. has lessen the disease severity upto 70% or more in brown spot (Joshi et al., 2007; Ludwig et al., 2009). Pseudomonas are potent producer of antibiotics such as 2, 4-DAPG, pyrrolnitrin and phenazine which enables it to work as a strong antagonistic agent.

How to cite this article

Badavath Kishore, Anshu Kumar, Pravesh Shivakoty and Venu Emmadi (2022). Biocontrol activity of Trichoderma viride, Trichoderma harzianum, Bacillus subtilis and Pseudomonas fluorescens (in vitro) against Bipolaris oryzae, causal agent of rice brown spot disease. Biological Forum – An International Journal, 14(1): 1698-1703.