Endophytes are microorganisms isolated from sterilized plant organs that could constitute convenient biological control agents in the fight against several plant diseases [42]. Numerous researches have illustrated the potential of endophytic bacteria in the control of phytopathogens. Paul et al. [43] showed that endophytic bacteria, isolated from chili pepper plants (Capsicum annum L.) belonged to Pseudomonas, Bacillus and Burkholderia have antifungal properties against Alternaria panax, Botrytis cinerea, Colletrotichum acutatum, Fusarium oxysporum and Phytophtora capsici. In the present study, evaluated biological control properties of select endophytic bacterium from the strawberry plant against C. nymphaeae under in vitro, in vivo, and greenhouse conditions. Bacterial strain was identified using phenotypic, biochemical properties and molecular phylogenetic analysis of the 16S rDNA gene sequences. Based on the results of this research, DM12 strain is considered as biological control agents against C. nymphaeae and was able to control strawberry anthracnose under in vitro, in vivo and greenhouse conditions. A perfect perception of the mode of action of bacterial strain has a great impact in development of successful biological control agent. In this study, types of biological control mechanisms of DM12 strain against C. nymphaeae was investigated. The mechanisms of bacterial endophytes in controlling of phytopathogens depends on their competence. There are different mechanisms such as producing antifungal metabolites, siderophores, lytic enzymes, competing for nutrition and habitat and also, induce systemic resistance [44]. Hydrolytic enzymes such as chitinase and protease produced by bacterial strains have long been reported of being able to cut the glycosidic and peptide bonds of fungal pathogens [45]. Research represented that Serratia marcescens was able to produce of chitinase that lysis of hyphal tips of Sclerotium rolfsii [46]. Also, pectinase production by bacterial endophytes facilitates colonization of plants and via releasing cell wall oligosaccharides and active pectic fragments of plant cell walls elicit defense reactions in plants [47, 7]. It was shown that, pectinases from Penicillium oxalicum BZH-2002 induced local protection against Cladosporium cucumerinum on cucumber [48]. Siderophore is an iron-healing compound that has an important role in the biological control of plant diseases. It has been represented that siderophores produced by Bacillus subtilis inhibited disease of F. oxysporium f. sp. ciceri and Macrophomina phaseolina in chickpea [49]. Based on the results, production of chitinase, protease, pectinase and siderophore by DM12 strain was observed. Phytohormones of microbial origin promote plant growth, tolerance to abiotic stresses and resistance to pathogens. On the other hand, biological control agents enhance availability of nutrients by fixing nitrogen and solubilizing phosphate [50]. Findings indicate that DM12 strain in this study was able to secret of IAA and GAs and capable to phosphate solubilization.
Bacillus species able to produce lipopeptides that are antibiotic compounds and have antifungal activity and stimulate defense pathways in plants [51, 52]. It was reported B. subtilis strain S499 produce surfactins, iturins and fengycins that suppresses B. cinerea on apple [51]. Also, B. amyloliquefaciens PPCB004 inhibited of postharvest fungal pathogen growth by producing various lipopeptides [53]. The bacterial strain in this study was able to secret surfactin.
In this research the ability of production of antifungal compounds by DM12 strain in vitro conditions was evaluated. It has indicated that cell free supernatant from B. atrophaeus reduced mycelial growth and conidial germination of C. gloeosporioides [54]. Also has reported that ethyl acetate extract of B. subtilis suppressed mycelial growth of several fungal pathogens [55]. Endophytic bacteria are capable to secret compounds that expose antifungal activity against pathogens, stimulate systemic resistance and inhibit the mycelial growth and spore germination of pathogen [56]. Antifungal metabolites was secreted by DM12 strain and GC-MS analysis identified these compounds that inhibit growth of C. nymphaeae. Among the compounds identified in this study was eicosane which previous studies reported that this compound has antifungal properties [57, 58]. Ahsan et al. [59] showed that extracted eicosane produced by Streptomyces strain KX852460 controlled R. solani in tobacco. Furthermore, Geng et al. [60] investigated antifungal activity of essential oil of bitter almond that 21 different components including, eicosane, mesitylene, dodecane, tetradecane and hexadecane identified. Also, DM12 strain suppress the mycelia growth of fungal pathogen in dual culture test which can be due to the production of lytic enzymes and volatile and non-volatile metabolites. It was reported that, B. subtilis produce antifungal metabolites that reduced the mycelial growth of Gaeumannomyces graminis var. tritici in wheat [61]. In addition to, an antifungal protein secreted by B. licheniformis decreased the growth of Aspergillus niger, Magnaporthe oryzae and R. solani [62]. Research has indicated that, culture filtrate of B. subtilis suppress conidial germination and mycelial growth of Sclerotinia sclerotiorum in rapeseed [63].
Fruit decay and disease severity of anthracnose under in vivo and greenhouse conditions were suppress significantly by strain DM12. These findings can due to the colonization of plant tissues, occupy of ecological niche, competence for the acquisition of nutrition, secretion of enzymes (protease, pectinase and chitinase) and antifungal compounds by this strain and induce of defense pathways [64]. Previous findings showed that the antifungal protein extracted from B. amyloliquefaciens inhibited growth of fungal pathogen and fruit decay in loquats [65].
Also, in this study, was represented that each treatment including, endophytic bacterium, fungal pathogen each alone and endophytic bacterium and fungal pathogen together, have a different effect on metabolite profile of strawberry seedlings and production of different compounds were increased in endophytic bacterium alone and endophytic bacterium and fungal pathogen together treatments in comparison with the control. Research has shown that R. solani affects the metabolic profile of soybean, and more chemical compounds were detected in interaction with the pathogen than in controls [40]. Previous study was indicated that, 2-3-butanediol accumulates in infected plant and acts as a signal for induce resistant on host [66]. Also, changes in the profile of volatile metabolites of tomato fruit were observed as a result of A. alternata infection [67]. Research has shown that VOCs increased resistance of Arabidopsis against the necrotrophic fungal pathogen B. cinerea and represents that VOCs may induce disease resistance [68]. Pandey et al. [69] indicated that, the capsule endophyte Acinetobacter upregulated the expression of the key genes for the production of antifungal and antimicrobial alkaloids.
Thereby, endophytic bacteria, have wide range of antimicrobial and antifungal metabolites, enzymes and surfactants that play important roles in plant growth promotion and inhibit phytopathogens by colonizing the surface or inner of different parts of the plants [70]. In our study, bacterial strain produce several lytic enzymes, lipopeptides and phytohormones that have biological control properties. Also, metabolic profile of strawberry seedlings in various interactions were different. As a result, it has been demonstrated that the synergistic effects of lytic enzymes and antifungal metabolites produced by DM12 strain give a higher level of biological control efficiency against C. nymphaeae [56].