Screening of chitinase-producing rhizosphere actinomycetes and their genetic diversity

Wahyudi AT, Fitriansyah NG, Amri MF, Priyanto JA, Nawangsih AA. 2021. Screening of chitinase-producing rhizosphere actinomycetes and their genetic diversity. Biodiversitas 22: 4186-4192. Soil microbial community, especially rhizosphere actinomycetes, plays an important role in protecting plants from phytopathogenic fungi by producing various agroactive compounds, including mycolytic enzymes particularly chitinase. The objective of this study was to investigate chitinase activity and analyze the diversity of family 19 chitinase genes of actinomycetes isolated from maize and soybean rhizosphere. Of fivety actinimycetes, Seventeen isolates exhibited chitinolytic activities and formed a hydrolytic zone around the colony with chitinolytic index ranging from 0.49±0.01 to 2.15±0.69, qualitatively tested in 0.3% chitin agar medium. Six selected isolates (ARJ 36, ARJ 81, ARJ 15, ARK 17, ARK 143, ARK 103) showed chitinase activities ranging from 0.157±0.04 to 0.440±0.09 U/mL, based on chitinolytic index. The production of chitinase was confirmed by the presence of family 19 chitinase encoding genes from Streptomyces sp. Some conserved regions and essential amino acid residues were also detected. This study suggests that chitinolytic actinomycetes isolated from maize and soybean rhizosphere can be studied further as biological control candidates for controlling phytopathogenic fungi.

Chitinase from a Novel Strain ofSerratia marcescensJPP1 for Biocontrol of Aflatoxin: Molecular Characterization and Production Optimization Using Response Surface Methodology

Chitinase is one of the most important mycolytic enzymes with industrial significance, and produced by a number of organisms. A chitinase producing isolateSerratia marcescensJPP1 was obtained from peanut hulls in Jiangsu Province, China, and exhibited antagonistic activity against aflatoxins. In this study, we describe the optimization of medium composition with increased production of chitinase for the selected bacteria using statistical methods: Plackett-Burman design was applied to find the key ingredients, and central composite design of response surface methodology was used to optimize the levels of key ingredients for the best yield of chitinase. Maximum chitinase production was predicted to be 23.09 U/mL for a 2.1-fold increase in medium containing 12.70 g/L colloidal chitin, 7.34 g/L glucose, 5.00 g/L peptone, 1.32 g/L (NH4)2SO4, 0.7 g/L K2HPO4, and 0.5 g/L MgSO4·7H2O. Polymerase chain reaction (PCR) amplification of the JPP1 chitinase gene was performed and obtained a 1,789 bp nucleotide sequence; its open reading frame encoded a protein of 499 amino acids named as ChiBjp.

Selection and breeding of shiitake strains resistant to Trichoderma spp.

Bedlogs of shiitake, Lentinula edodes (Berk.) Pegler, are often attacked by Trichoderma spp. such as T. harzianum Rifai and T. polysporum Rifai, which produce antifungal substances and mycolytic enzymes. However, shiitake is more or less able to reject Trichoderma attacks. Shiitake cultures possess at least five straight-chain alcohols with double and triple bonds, which act as antifungal substances. Trichoderma inoculation tests for shiitake cultures showed that the resistance of shiitake in a wood-powder medium correlated positively with that in bedlogs when T. harzianum was used. Screening assays using the wood-powder medium showed some highly resistant and susceptible strains among 164 different wild shiitake strains. Monokaryons derived from basidiospores of a highly resistant strain, TMI-407, had high rejecting abilities compared with those from a susceptible strain, TMI-1161. Crossing between monokaryons from the two strains revealed that resistance was dominant. In general, these resistant mycelia produced much more antifungal substances than did susceptible mycelia. Key words: Lentinula edodes, shiitake, mushroom cultivation, resistance to Trichoderma spp., selection method, antagonism.