Phytophthora Foot Rot Pathogen of Black Pepper (Piper nigrum L.): Isolation, Characterisation and Evaluation of Streptomyces spp. as Potential Biocontrol Agent

Jin Chi, Kong (2023) Phytophthora Foot Rot Pathogen of Black Pepper (Piper nigrum L.): Isolation, Characterisation and Evaluation of Streptomyces spp. as Potential Biocontrol Agent. Masters thesis, Universiti Malaysia Sarawak.

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Abstract

Sarawak contributes 98% to Malaysian black pepper (Piper nigrum) production. However, its production has been challenged by foot rot, a deadly disease caused by Phytophthora capsici. Thus, it is important to have a better understanding of this pathogen’s characteristics for controlling or preventing the disease. Synthetic fungicides have been used to control the disease, but could not curb it entirely. In addition, repeated use of the chemicals may be hazardous to the environment and consumers’ health, cause chemical resistance in pathogen and influence the diversity of non-target soil microorganisms. Hence, there is a need to explore alternative control method which is more environmentally friendly and sustainable. Biological control using soil Streptomyces spp. can be an option. Streptomyces share the same niche with the pathogen and have been known to produce various antifungal metabolites. However, there is limited scientific literature about their antagonistic ability on P. capsici in Malaysia, especially in Sarawak. This study was conducted to i) isolate and characterise P. capsici isolates from black pepper foot rot samples and confirm their pathogenicity; ii) isolate Streptomyces spp. from soil and evaluate their antagonistic effect on P. capsici using dual culture method; and iii) characterise the potential Streptomyces isolates, which showed antagonistic ability toward P. capsici. Diseased plant materials were collected from black pepper farms in Julau district for isolation, characterisations (i.e., morphology, pathogenicity, radial mycelial growth and virulence) and molecular identification of the pathogen. Soil samples from within and border region of black pepper farms were collected to isolate Streptomyces to assess their antagonistic ability against P. capsici in vitro, thereafter potential Streptomyces isolates were characterised morphologically and identified to species. In this study, three isolates of Phytophthora sp. (I4, I5 and E3) were isolated from the diseased plant tissues collected from Nanga Ijit and Nanga Merurun farms, characterised and identified as P. capsici by morphological and molecular approaches. The three isolates were confirmed as the causal agents of the disease using Koch’s postulates. Formation of oogonia and amphigynous antheridia by pairing the isolates suggested the presence of two mating types. Variations in colony morphology and radial mycelial growth on CMA, 20% CV8A, PDA and ISP-2 were observed for I5 and E3, but not for I4. Isolate I5 exhibited similar virulence as I4 did but was more aggressive than E3. Forty-one (41) Streptomyces isolates demonstrated mixed antagonistic effect, i.e., antagonism zone, clear zone and percentage of mycelial growth inhibition toward P. capsici. Among them, only seven (7) isolates consistently exhibited antagonistic effect on I4 and E3. Total 15 isolates were selected as potential antagonists, and 16S rRNA sequence identified them into five Streptomyces species (Streptomyces jiujiangensis, S. corchorusii, S. bambusae, S. bungoensis and S. roseicoloratus). Phylogenetic analysis of the Streptomyces spp. indicated that clear sub-clustering according to species was not observed in Maximum Likelihood tree. Colony and spore chains morphology of the 15 Streptomyces isolates exhibited some variations on ISP-2. This study provides useful information regarding characteristics of the P. capsici and in vitro antagonistic ability of Streptomyces spp. which could help to develop sustainable action plans to control black pepper foot rot disease in Sarawak.

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