Biocontrol Potentiality of Active Ingredients from Endophytic Bacillus subtilis Isolated from Alhagi pseudalhagi Desv on Maize Spot Diseases

Abstract

Two endophytic bacterial strains, XJAS-AB-13 and XJAS-AB-11, with broad antifungal activity against maize spot pathogens Exserohilum turcicum and Bipolaris maydis, were screened from the Alhagi pseudalhagi Desv, by using agar diffusion method. Both of them were identified as Bacillus subtilis according to the physiological and biochemical properties, as well as the molecular analysis based on 16S rDNA sequence. The inhibitory activity of broth cultured XJAS-AB-13 and XJAS-AB-11, measured in vivo, and the result showed that the diseases inhibition efficiency of XJAS-AB-13 and XJAS-AB-11 broth on E. turcicum and B. maydis reached 63.33%, 45.0% and 23.33%, 58.34% respectively. Infection by different diseases caused an increase in total amount of the protein in general, where the increment generated by the Exserohilum turcicum was greater than Bipolaris maydis. Defense enzymes activity of Superoxide dismutase (SOD), however, declined sharply compare to the control group, while the others, catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX), were increasing. Through extraction and silica-gel chromatography, two different monomers, XJAS-B and XJAS-G, were isolated from the ethyl acetate extraction of the culture broth of XJAS-AB-11, and identified according to 1 HNMR and 13 CNMR spectral data as well as ESI-MS molecular weight analysis as cyclo-(D-leucyl-trans-4-hydroxy-L-proline) [(3R,7R,8aS)-7-hydroxy- 3-isobutylhexahydropyrrolo [1,2-a] pyrazine-1,4-dione] and 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen- 4-one. The preliminary comparative analysis on XJAS-AB-11 Naringenin-chalcone synthase (CHS) gene also indicated their high homology with some prokaryotic and eukaryotic CHS amino acid sequences. In addition, the amino acid sequence of XJAS-AB-11 CHS gene has showed similar amino acid composition with those of Streptomyces griseus, Arachis hypogaea and Gerbera hybrida at the active site.