Antagonistic activity and molecular characterization of mechanism of action of Bacillus amyloliquefaciens EU07 isolate against Phytophthora citrophthora, the causative agent of citrus brown rot and stem gummosis disease

The value of agriculture in the economy of countries has been increasing day by day, because it provides raw materials for industry as well as supply the world's food needs. Citrus plants, with an important place in the Rutaceae family, are not only used in purpose of nutritional needs, but also in cosmetics, industry and medicine. Citrus diseases are one of themajor problem in citrus production. Brown rot and stem gum disease caused by omycetes P. citropthora causes economical losses on citrus fruits. Chemical spray are generally used to control of the disease. But these methods are not sufficient, they cause residue issues and environmental pollution. Therefore, the importance of biological methods is getting attention. In the present study, the biological control potential of B. amyloliquefaciens EU07 against the P. citrophthora. In current studies the antagonistic effect of EU07 on the pathogen was investigated bu dual culture assays in vitro. EU07 has shown suppresion on mycelial growth by 69.2% in vitro assays at he end of 14-days of inoculation In further studies the effect major enzymes, produced by strain EU07 have been investigated whether they have role in this inhibiton observed on mycelial growth of P. citrophthora. Therefore to observe this competition both microorganisms have been performed in same liquid culture by giving equal chance for growth in terms of appropriate carbons sources. Culture filtrate of EU07 was was used for the enzymatic activity after by ethanol precipitation. EU07 showed 1.61 U fold higher protease activity when it faces with the pathogen compared to alone performance in the culture medium. Minor extracellular protease Vpr detected in the genome of strain EU07 showed high similarity to S8 family serine proteases of B. amyloliquefaciens species according to BLAST comparison through NCBI. These activity has been associated with the possible interaction between protease activity and RXLR effector protein of the pathogen. The computional analysis carried out by the help of HADDOCK server indicated to have binding possibility by means of the Van der Waals Energy with 48.2 +/- 14.9 kJ/mol. These data proved that EU07 is able to inhibit the pathogen due to higher protease activity. In future studies, the inhibitory potential of EU07 will be investigated more and detailed and as a biological control agent for the control of the fungi belonging to other Oomycetes group.