Extracellular proteases from Streptomyces phaeopurpureus ExPro138 inhibit spore adhesion, germination and appressorium formation in Colletotrichum coccodes

AbstractAim: To study the antifungal mechanism of proteases from Streptomycesphaeopurpureus strain ExPro138 towards Colletotrichum coccodes and toevaluate its utilization as biofungicide.Methods and Results: We screened proteolytic Streptomyces strains from theyam rhizosphere with antifungal activity. Forty proteolytic Streptomyces wereisolated, among which eleven isolates showed gelatinolytic activity andantagonistic activity on C. coccodes. Of the 11 isolates, protease preparationfrom an isolate designated ExPro138 showed antifungal activity. 16S rDNAsequence analysis of the strain showed 99% similarity with Streptomycesphaeopurepureus (EU841588.1). Zymography analysis of the ExPro138 culturefiltrate revealed that the strain produced several extracellular proteases. Theprotease preparation inhibited spore germination, spore adhesion topolystyrene surface and appressorium formation. Microscopic study of theinteraction between ExPro138 and C. coccodes revealed that ExPro138 wasmycoparasitic on C. coccodes. The protease preparation also reducedanthracnose incidence on tomato fruits compared with untreated control.Conclusion: This study demonstrates possibility of utilizing antifungalproteases derived from antagonistic microbes as biofungicide.Significance and Impact of the Study: Microbial proteases having the abilityto inhibit spore adhesion and appressorium formation could be used tosuppress infection establishment by foliar fungal pathogens at the initial stagesof the infection process.IntroductionColletotrichum is one of the most economically damaginggroups of phytopathogenic fungi that cause anthracnoseon a number of crop plants (Kleemann et al. 2008). Theconidia of Colletotrichum species are dispersed by rain-drop splashes and adhere to the aerial parts of the hostplant to initiate infection (Hughes et al. 1999). Colletotri-chum spores have extracellular matrix (ECM) on theirspore coat (Hutchison et al. 2002), which is essential forconidial attachment to host surface. The attached conidiaform appressorium to establish infection (Sanogo et al.2003). Colletotrichum coccodes causes anthracnose intomato and induces disease symptoms in ripe fruits andmortality in seedlings (Redman and Rodriguez 2002).The disease symptoms can be seen as dark, sunken andcircular lesions; as the disease progresses, black dots maybe visible on the inner side of the lesions in tomato fruits(Chapin et al. 2006). These symptoms cause rejection ofthe tomato by the consumers (Chapin et al. 2006). Tem-perature, wetness, inoculum density and relative humidityare important factors that lead to infection by C. coccodes