Assessment of the Phenotypic and Genotypic Diversity of Endophytic Bacillus Strains Isolated from the Roots of Hornbeam (Carpinus betulus L.) in Mazandaran and Golestan Provinces, Iran

Document Type : Research article

Authors

1 Ph.D. Student of Phytopathogenic Prokaryotes, Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

2 Corresponding author, Prof., Department of Plant Protection, Faculty of Crop Sciences, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

3 Associate Prof., Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

10.22092/ijfpr.2023.363172.2114

Abstract

Background and objectives: The beneficial characteristics of endophytic bacteria in general and environmental safety features of Bacillus spp. in particular, have made them potentially efficacious bacteria for plant growth promotion as well as plant defense elicitation against biotic and abiotic stresses. Given that the preservation of biodiversity and sustainability of the Hyrcanian forests in Iran is of utmost importance ecologically and environmentally, proper management of this ecosystem demands considerable attention and care. Studying the microbiome structure and activities, including determination of microorganisms living as endophytes or commensals can be regarded as the starting steps in this context. This study was aimed at the molecular identification and physiological characterization of endophytic Bacillus isolates recovered from the roots of hornbeam (Carpinus betulus L.) trees in some different locations of Hyrcanian forests.
Methodology: Samples of hornbeam roots were randomly collected from Mazandaran and Golestan provinces, Iran, in the early spring of 2020. Root segments were surface-disinfected, washed in sterile distilled water (SDW) and crushed in drops of SDW using a sterile mortar and pestle. Isolation of bacteria was done by spreading drops of the suspension on plates of tryptic soy agar (TSA). Single colonies were selected after 3-4 days of incubation at 28˚C and restreaked on TSA to obtain pure cultures. Initial identification of the isolates was based on colony morphology, Gram-stain reaction, position of endospores, and production of catalase and oxidase. The isolates were characterized phenotypically and their diversity was assessed by comparing their DNA fragment patterns following their amplification by REP-, BOX- and IS50-PCR and electrophoresis on agarose gels. The data were converted into a binary matrix using TotalLab TL120 software, where the digits 1/0 represented the presence/absence of a DNA band. The similarity matrix and clustering were generated by the use of NTSYS 2.02e software based on the unweighted pair group method with arithmetic average (UPGMA) clustering algorithm. A representative isolate was selected for each of the DNA profile groups and a fragment of the 16S rRNA and HSP60 genes of each was amplified by PCR. PCR products were sequenced by Microsynth company (Switzerland) through the Sanger sequencing method. The nucleotide sequences were aligned and compared with those of a collection of Bacillus species retreived from GenBank using the BLASTn program. Phylogenetic trees were constructed using the Maximum-Likelihood and Neighbor-Joining methods with 1000 bootstraps in MEGA X software package. Antibacterial activity of Bacillus isolates against Brenneria roseae, the causal agent of hornbeam wetwood disease, was investigated using sterilized blank paper disk impregnated with extracts prepared in dichloromethane. The isolates were assessed for their capacity to produce Indole acetic acid (IAA), hydrogen cyanide, protease, and biofilm.
Results: Thirty bacterial strains were isolated from hornbeam roots on TSA medium and subsequently characterized. The absence of bacterial colonies on the TSA plates inoculated with the final rinse water from the uncrushed bark fragments indicated the effectiveness of the surface disinfection procedure. The isolates were grouped into three clusters based on their phenotypic and genotypic characteristics. Comparison of the nucleotide sequences of the 16S rDNA and HSP60 with the sequences obtained from GenBank (NCBI) led to the identification of Bacillus thuringiensis, B. subtilis, and B. mycoides as the species constituting the three clusters, with B. thuringiensis being the predominant group. B. subtilis was the most populated and B. mycoides the least encountered species. Phylogenetic analysis based on the HSP60 gene sequence proved to be a reliable approach for determining the Bacillus species. IAA production level varied from eight to 75 mg/L among the species, with B. mycoides isolates producing the highest and B. subtilis the lowest levels of IAA. Most of the isolates had the capability to produce lipase, gelatinase, lecithinase, protease, and amylase. The isolates of B. thuringiensis produced higher levels of protease than the rest of the isolates. Biofilm production was particularly prominent in isolates of B. subtilis. The dichloromethane extract of B. subtilis isolate rAqa2 exhibited the highest growth inhibition (18.3 ± 0.25mm in diameter) of Brenneria roseae.
Conclusion: A high level of genotypic and phenotypic heterogeneity existed among the endophyte isolates recovered from the roots of C. betulus, despite their isolation from a common source species. This could be partly justified by viewing the Hyrcanian forests as a mixed-species ecosystem and a source of microbial consortia that could protect plants against stresses. This study is the first investigation on the molecular identification and characterization of endophytic Bacillus isolates from hornbeam roots in Iran.

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References

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