In this study, bacterial biocontrol agents against the phytopathogen Lasiodiplodia theobromae were screened from Thua Nao, a Thai fermented soybean product. A total of 170 bacterial strains were isolated and initially screened for their antagonistic activity by dual culture test. Of these, 39 isolates were able to inhibit the fungal growth showing the percentage of inhibition ranging from 25.0–67.5. Among them, the isolate TN79 was selected as a potential antagonistic strain for further study. For this, the bacterial strain TN79 was cultured on nutrient agar for 2 weeks and its crude extracts were prepared using phosphate buffÍer pH 7.0. The bacterial crude extracts prepared were active and could inhibit all four fungal strains of L. theobromae. The optimum pH for antifungal activity of the extracts was 7. In addition, the extracts were also active when exposed to the UV light (254 nm) up to 1 hour and to proteinase K treatment (1 mg/ml). The bacterial strain TN79 was then characterized in terms of their phenotypic and genotypic properties including morphology, biochemical profiles, and rRNA gene sequence. Based on this analysis, the bacterium TN79 was closely related to Bacillus velezensis.
Adesemoye, A. O., Mayorquin, J. S., Wang, D. H., Twizeyimana, M., Lynch, S. C. and Eskalen, A. (2014): Identification of species of Botryosphaeriaceae causing bot gummosis in citrus in California. Plant Dis. 98, 55–61.
Ali, H. and Nadarajah, K. (2014): Evaluating the efficacy of Trichoderma spp and Bacillus subtilis as biocontrol agents against Magnaporthe grisea in rice. Australian J. Crop Sci. 8, 1324–1335.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990): Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
Amin, A., Khan, M. A., Ehsanullah, M., Haroon, U., Azam, S. M. F. and Hameed, A. (2012): Production of peptide antibiotics by Bacillus sp. GU 057 indigenously isolated from saline soil. Brazilian J. Microbiol. 43, 1340–1346.
Cavaglieri, L., Orlando, J., Rodríguez, M. I., Chulze, S. and Etcheverry, M. (2005): Biocontrol of Bacillus subtilis against Fusarium verticillioides in vitro and at the maize root level. Res. Microbiol. 156, 748–754.
Chantawannakul, P., Onchaeroen, A., Klanbut, K., Chukeatorite, E. and Lumyong, S. (2002): Characterization of proteases of Bacillus subtilis strain 38 isolated from traditionally fermented soybean in Northern Thailand. ScienceAsia 28, 241–245.
Che, J., Liu, B., Ruan, C., Tang, J. and Huang, D. (2015): Biocontrol of Lasiodiplodia theobromae, which causes black spot disease of harvested wax apple fruit, using a strain of Brevibacillus brevis FJAT-0809-GLX. Crop Prot. 67, 178–183.
Coutinho, I. B. L., Freire, F. C. O., Lima, C. S., Lima, J. S., Gonçalves, F. J. T., Machado, A. R., Silva, A. M. S. and Cardoso, J. E. (2017): Diversity of genus Lasiodiplodia associated with perennial tropical fruit plants in northeastern Brazil. Plant Pathol. 66, 90–104.
Dereeper, A., Audic, S., Claverie, J. M. and Blanc, G. (2010): BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol. Biol., 10, 8.
MacFaddin, J. F. (2000): Biochemical tests for identification of medical bacteria. Lippincott Williams and Wilkins, Philadelphia, USA, 928 p.
Marchesi, J. R., Sato, T., Weightman, A. J., Martin, T. A., Fry, J. C., Hiom, S. J. and Wade, W. G. (1998): Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl. Env. Microbiol. 64, 795–799.
Mascarenhas, P., Behere, A., Sharma, A. and Padwal-Desai, S. R. (1995): Post-harvest spoilage of mango (Mangifera indica) by Botryodiplodia theobromae. Mycol. Res. 100, 27–30.
Mbenoun, M., Momo, Z. E. H., Samuels, G., Nsouga, A. F. and Nyasse, S. (2008): Dieback due to Lasiodiplodia theobromae, a new constraint to cocoa production in Cameroon. Plant Pathol. 57, 381.
Melnick, R. L., Zidack, N. K., Bailey, B. A., Maximova, S. N., Guiltinan, M. and Backman, P. A. (2008): Bacterial endophytes: Bacillus spp. from annual crops as potential biological control agents of black pod rot of cacao. Biol. Control 46, 46–56.
Mohamed, H. and Saad, A. (2009): The biocontrol of postharvest disease (Botryodiplodia theobromae) of guava (Psidium guajava L.) by the application of yeast strains. Postharvest Biol. Technol. 53, 123–130.
Mortuza, M. G. and Ilag, L. L. (1999): Potential for biocontrol of Lasiodiplodia theobromae (Pat.) Griff. and Maubl. in banana fruits by Trichoderma species. Biol. Control 15, 235–240.
Netto, M. S. B., Assuncao, I. P. and Lima, G. S. A. (2014): Species of Lasiodiplodia associated with papaya stem-end rot in Brasil. Fungal Div. 67, 127–141.
Petchkongkaew, A., Taillandier, P., Gasaluck, P. and Lebrihi, A. (2008): Isolation of Bacillus spp. from Thai fermented soybean (Thua-nao): screening for aflatoxin B1 and ochratoxin A detoxification. J. Appl. Microbiol. 104, 1495–1502.
Priest, F. G., Goodfellow, M., Shute, L. A. and Berkeley, R. C. W. (1987): Bacillus amylolique faciens sp. nov., nom. rev. Int. J. Syst. Bacteriol. 37, 69–71.
Ramarathnam, R., Bo, S., Chen, Y., Fernando, W. G., Xuewen, G. and de Kievit, T. (2007): Molecular and biochemical detection of fengycin- and bacillomycin D-producing Bacillus spp., antagonistic to fungal pathogens of canola and wheat. Can. J. Microbiol. 53, 901–911.
Rooney, A. P., Price, N. P. C., Ehrhardt, J. L., Swezey, J. L. and Bannan, J. D. (2009): Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov. Int. J. Syst. Evol. Microbiol. 59, 2429–2436.
Sajitha, K. L., Florence, E. J. M. and Dev, S. A. (2014): Screening of bacterial biocontrols against sapstain fungus (Lasiodiplodia theobromae Pat.) of rubberwood (Hevea brasiliensis Muell.Arg.). Res. Microbiol. 165, 541–548.
Sambrook, J. and Green, M. R. (2012): Molecular Cloning: a Laboratory Manual. 4th ed. Cold Spring Harbor Laboratory Press, New York, 545 p.
Sansinenea, E. and Ortiz, A. (2011): Secondary metabolites of soil Bacillus spp. Biotechnol. Lett. 33, 1523–1538.
Thangavelu, R., Sangeetha, G. and Mustaffa, M. M. (2007): Cross-infection potential of crown rot pathogen (Lasiodiplodia theobromae) isolates and their management using potential native bioagents in banana. Australas. Plant Pathol. 36, 595–605.