*2.6. Inhibition of Zoospore Release from P. viticola Zoosporangia by KOF112*

As KOF112 inhibited the mycelial growth of *B. cinerea*, *C. gloeosporioides*, and *P. infestans* (Figure 1), mycelial disks of *B. cinerea* and *C. gloeosporioides* were used as inocula in the bioassays for biocontrol activity of KOF112 against gray mold and anthracnose, respectively (Figures 6 and 7). On the other hand, we used *P. viticola* zoosporangia as inoculum in the bioassay for biocontrol activity of KOF112 against downy mildew (Figure 5). Zoosporangia release many zoospores after inoculation, and the zoospores penetrate leaves through stomata [22]. Microscopic observation was performed to evaluate the effect of KOF112 on the early infection behaviors of *P. viticola* zoosporangia and zoospores. Empty zoosporangia were observed in the case of zoosporangia not treated or treated with SCD within 24 h after the treatment, whereas a large number of zoosporangia treated with KOF112 still had zoospores inside them after 24 h (Figure 8A). Interestingly, KOF112 seemed to surround zoosporangia that did not release zoospores (Figure 8A). Approximately 50% of zoosporangia not treated or treated with SCD released zoospores 24 h after the treatment, whereas approximately 24% of zoosporangia treated with KOF112 released zoospores after the same treatment period (Figure 8B). On the other hand, approximately 45% of zoospores germinated in spore suspension not treated or treated with 10% SCD after incubation for 20 h, whereas approximately 50% of zoospores germinated in the presence of KOF112 (Figure 8C). *Agrobacterium* sp. isolate CHB3 exhibited no effect on both zoospore release from zoosporangia and zoospore germination.

**Figure 8.** KOF112 inhibits zoospore release from zoosporangia but not zoospore germination. (**A**) Zoosporangia at 0, 3, and 24 h after preparation. Bar, 20 μm. (**B**) Zoospore release. The rates of

zoospore release from zoosporangia were calculated as described in Materials and Methods. Crosses (×) indicate means of eight independent preparations. \* *p* < 0.05 compared with control and SCD. (**C**) Zoospore gemination. The rates of zoospore germination were calculated as described in Materials and Methods. Crosses (×) indicate means of three independent preparations. Control, untreated. SCD, treated with 10% SCD. KOF112, treated with <sup>1</sup> <sup>×</sup> 108 cfu/mL KOF112. Agro, treated with 1 <sup>×</sup> 10<sup>8</sup> cfu/mL Agrobacterium sp. isolate CHB3, used as a control isolate with no antifungal activity.

These results suggest that KOF112 inhibits zoospore release from *P. viticola* zoosporangia but not zoospore germination.

#### *2.7. KOF112 Induces Plant Defense Response in Grapevine*

The possibility that KOF112 induced plant defense response in grapevine was evaluated. We selected genes encoding class IV chitinase and β-1,3-glucanase as indicators of plant defense response, because chitinase and β-1,3-glucanase gene expression is induced through jasmonic acid (JA) and salicylic acid (SA)-dependent defense pathways, respectively. KOF112 drastically upregulated the expression of a gene-encoding class IV chitinase in grape leaf disks 48 h after KOF112 treatment, compared with untreated control and SCD treatment (Figure 9). The transcripts of β-1,3-glucanase gene were also increased 24 and 48 h after KOF112 treatment compared with untreated control and SCD treatment (Figure 9). *Agrobacterium* sp. isolate CHB3 did not upregulate the transcription of both genes.

**Figure 9.** Transcription profiles of genes encoding class IV chitinase and β-1,3-glucanase in grape leaves treated with KOF112. Transcription levels of the genes in grape leaf disks 0, 24, and 48 h after KOF112 treatment were estimated by real-time RT-PCR. Data were calculated as gene expression relative to ubiquitin gene expression. Bars indicate means ± standard deviations of three independent experiments with three leaf disks. \* *p* < 0.05 compared with control and SCD. Control, untreated. SCD, treated with 10% SCD. KOF112, treated with <sup>1</sup> <sup>×</sup> 108 cfu/mL KOF112. *Agro*, treated with <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>8</sup> cfu/mL *Agrobacterium* sp. isolate CHB3, used as a control isolate with no antifungal activity.

These results suggest that KOF112 also works as a biotic elicitor and induces plant defense response in grapevine through both JA- and SA-dependent defense pathways.

#### **3. Discussion**

Grapes are one of the most important fruits cultivated worldwide. However, their high susceptibility to pre- and post-harvest pathogens has resulted in significant economic losses. The main diseases in viticulture are gray mold [23], ripe rot [24], and downy mildew [25]. As a biological control agent, KOF112 is able to suppress those three diseases. Although the disease-suppressing effect of biological control agents is weaker than that of chemical fungicides, the application of KOF112 having antagonistic activities toward a wide range of phytopathogenic fungi, including *Ascomycetes* and *Oomycetes*, may contribute to reducing

the frequency of chemical fungicide application in viticulture, as well as to inhibiting the development of chemical fungicide resistance. The supernatants of biological control agents could be used as a new biostimulant in sustainable agriculture [26]. The supernatant of *B. subtilis* GLB191, which contains surfactin and fengycin, exerted a direct antifungal effect and induced plant defense response, thereby contributing to protection against grape downy mildew [17]. Future laboratory and field trials are necessary to evaluate whether the supernatant of KOF112 can be used as a biostimulant against gray mold, ripe rot, and downy mildew, and to identify the optimum conditions, including the dose of KOF112 and the timing of KOF112 application, to ensure that the combination treatment of KOF112 and chemical fungicides works effectively in viticulture.

Most commercially available biological control agents are *Bacillus*, which produces a large number of antibiotics [27,28]. *B. velezensis* FZB42 (former name *B. amyloliquefaciens* subsp. *plantarum*) [29] is used commercially as a biological control agent in agriculture [30] and produces such metabolites as peptides and polyketides, having antifungal, antibacterial, and nematocidal activities [31]. However, doubtful evidence related to the direct antifungal activity of FZB42 metabolites against competing plant pathogens has been presented. The concentrations of the metabolites produced by FZB42 in plants [32] were relatively low and/or undetectable, with the exception of surfactin. On the other hand, FZB42 compensated changes in microbial community structure caused by pathogens and helped plant-associated *Bacilli* contribute to plant protection [33]. Consequently, the sublethal concentrations of cyclic lipopeptides produced by *Bacilli* triggered plant defense response systemically.

Cyclic lipopeptides were found to enhance plant defense response to phytopathogenic fungi [34,35]. Surfactin and fengycin protected grape against downy mildew by exerting a direct antifungal effect and inducing plant defense response [17]. Cyclic lipopeptides activated plant defense response through distinct defense pathways [36]. Mycosubtilin (iturin family) activated both JA- and SA-dependent defense pathways, whereas surfactin mainly induced an SA-dependent plant defense response [36]. In the present study, gene expression analysis suggested that KOF112 induced plant defense response in grapevine through both JA- and SA-dependent defense pathways. Thus, enhancing plant defense response by antifungal metabolites produced by biological control agents is one of the mechanisms for protecting plants. Future studies employing liquid chromatographytandem mass spectrometry analysis would identify the cyclic lipopeptides produced by KOF112 and reveal which cyclic lipopeptides contribute to the protective effect of KOF112 against fungal diseases.

In this study, we found that KOF112 also induced plant defense response in grapevine. Chitinase inhibited infection by *B. cinerea* [37] and *C. gloeosporioides* [38], whereas β-1,3 glucanase exerted direct antimicrobial activity against *B. cinerea* [39], *C. gloeosporioides* [40], and *P. viticola* [41]. Genome sequencing was attempted to clarify the biocontrol activity of the biological control agents [42]. Comparative genome analysis of KOF112 and FZB42 demonstrated that KOF112 might produce fewer antimicrobial peptides and polyketides than FZB42. The direct inhibition of phytopathogenic fungal mycelial growth in vitro is an indirect indication that KOF112 produces some antibiotics. Although the inhibition of zoospore release from *P. viticola* zoosporangia by KOF112 may be an interesting mechanism underlying the biocontrol activity against grape downy mildew, the probable antibiotic production by KOF112 may confer enhanced plant defense response in the same way as FZB42. Further studies employing the genomic analysis of KOF112 for exploring genes encoding bioactive substances, as well as the qualitative and quantitative analyses of antibiotics produced by KOF112, would reveal the main mechanism underlying plant disease control by KOF112. Because we were unable to analyze the biocontrol activity of KOF112 by field trials in vineyards, we could not verify whether our laboratory experiments using leaf disks support our hypothesis of increased plant resistance. We need to conduct field trials in vineyards to verify our hypothesis that KOF112 also works as a biotic elicitor in pest management strategies.

Grapevines are a rich source of potential biological control agents for fungal and oomycete pathogens. Bacterial isolates collected from endophytic and epiphytic communities living in grapevine leaves inhibited *B. cinerea* and *P. infestans* mycelial growth [43]. The colonization of biological control agents *in planta* is one of the driving forces for protecting plants against diseases. The suppressive activities of biological control agents against phytopathogenic fungi are influenced by the capacity of those agents to colonize in planta [20,44]. Endophytes are considered potential biological control agents because of their colonization ability. In plant root endospheres, high motility as well as enhanced plant cell-wall degradation and reactive oxygen-species-scavenging abilities seem to be important traits for successful endophytic colonization [45]. Because endophytic bacteria play a role in the resistance to biotic and abiotic stresses as well as plant growth and development, manipulating endophytic bacteria would help us develop novel and innovative techniques for improving agricultural production. In fact, plants have unique endophytes, some of which confer resistance to the plants themselves [46]. In this study, we focused on endophytic bacteria and isolated antifungal endophyte KOF112 from grapevine shoot xylem. It is reasonable to assume that endophytes show greater affinity for plants than soil microorganisms. Although KOF112 can induce plant defense response in grapevine, we cannot present concrete results of KOF112 colonization in grapevine at the moment. Further investigation by scanning electron microscopy would reveal whether KOF112 colonizes foliar-sprayed leaves. If KOF112 can colonize well in and/or on grapevine, KOF112 would have an edge over commercial biological fungicides that are generally considered to have a short life span on grapevine. Future studies involving field trials of KOF112 application to bunches and leaves and/or KOF112 injection into grapevine shoot xylems would reveal whether KOF112 colonization in grapevine results in the optimal biocontrol activity of endophytic KOF112 against fungal diseases.
