Isolation and Molecular Identification of Endophytic Bacillus from the Roots of Carpinus Orientalis in Mazandarn and Semnan Provinces

Because of possessing a myriad of biological features useful in the biocontrol area, Bacillus species have been in the forefront of studies in this vast yet expanding area of research. Given the preservation of biodiversity and sustainability of the Hyrcanian ecosystems are of utmost importance ecologically and environmentally proper management of this ecosystem warrants serious attention and care. Studies on the microbiome and identification of the species living as endophytes and commensals are by itself a vast area of research in this regard. The present study was aimed at the identification and preliminary biological characterization of the Bacillus strains living as endophyte in the roots of the oriental hornbeam (Carpinus orientalis) growing in Mazandaran and Semnan provinces.

Samples of oriental hornbeam roots were randomly collected from the mountain site of southern Mazandaran and northern Semnan in the Spring 2018. Root segments were surface-disinfected, washed in sterile distilled water (SDW) and crushed in drops of SDW in sterile mortar and pestle. Isolation of bacteria were done following spreading drops of the suspension on plates of tryptic soy agar (TSA) and 3-4 days incubation at 28˚C. The isolates were characterized phenotypically and their protein profiles were determined by electrophoresis on polyacrylamide gel. Diversity of the isolates were assayed by comparing DNA fragment pattern on agarose gels after their amplification by REP-, BOX- and IS-50 PCR. A representative isolate from each of the four DNA profile groups was selected and a 1500bp fragment of the 16S rDNA of each amplified by PCR and sequenced. The nucleotide sequences were aligned and compared with those of a collection of Bacillus species deposited in GenBank using the BLASTn program. A phylogenetic tree of the isolates was constructed using the Maximum-Likelihood method. The ability of the isolates to produce IAA, hydrogen cyanide, protease and biofilm was evaluated.

Thirty Bacillus isolates were recovered from the roots of hornbeam on TSA medium. Based on their phenotypic and molecular characteristics the isolates were differentiated into four groups. Comparison of the nucleotide sequences of their 16S rDNA with the sequences retrieved from GenBank indicated affiliation of the isolates with Bacillus thuringensis, B. cereus, B. subtilis, and B. velezensis among which Bacillus thuringiensis was the most prevalent and B. subtilis the most populated group. All isolates produced indole acetic acid at 5 to 85 mgl- with B. thuringiensis producing the highest and B. velezensis the lowest concentration of the hormone. The majority of isolates were capable of producing lipase, gelatinase, lecithinase, protease, and amylase. Isolates of B. cereus and B. thuringiensis were considerably more proteolytic among all. Producing of biofilm was confined to the isolates of B. velezensis.

The possession of and the diversity in the capacity of producing various biologically beneficial biochemical and enzymes and of having other attributes of value in biocontrol, makes the Bacillus spp. understudy as potentially capable bacteria in the promotion of plant growth and in the biological control of pathogens. Isolates of B. thuringiensis and B. cereus appeared to be superior among the four species studies. This is the first report on the identification and partial characterization of the potentially beneficial Bacillus species living as endophyte in the root tissues of oriental hornbeam in Iran.