*Article* **Isolation and Characterization of Endophyte** *Bacillus velezensis* **KOF112 from Grapevine Shoot Xylem as Biological Control Agent for Fungal Diseases**

**Kazuhiro Hamaoka, Yoshinao Aoki and Shunji Suzuki \***

Laboratory of Fruit Genetic Engineering, The Institute of Enology and Viticulture, University of Yamanashi, Kofu 400-0005, Japan; g19dia03@yamanashi.ac.jp (K.H.); yaoki@yamanashi.ac.jp (Y.A.)

**\*** Correspondence: suzukis@yamanashi.ac.jp; Tel.: +81-55-220-8394

**Abstract:** As the use of chemical fungicides has raised environmental concerns, biological control agents have attracted interest as an alternative to chemical fungicides for plant-disease control. In this study, we attempted to explore biological control agents for three fungal phytopathogens causing downy mildew, gray mold, and ripe rot in grapevines, which are derived from shoot xylem of grapevines. KOF112, which was isolated from the Japanese indigenous wine grape *Vitis* sp. cv. Koshu, inhibited mycelial growth of *Botrytis cinerea*, *Colletotrichum gloeosporioides*, and *Phytophthora infestans*. The KOF112-inhibited mycelial tips were swollen or ruptured, suggesting that KOF112 produces antifungal substances. Analysis of the 16S rDNA sequence revealed that KOF112 is a strain of *Bacillus velezensis*. Comparative genome analysis indicated significant differences in the synthesis of non-ribosomal synthesized antimicrobial peptides and polyketides between KOF112 and the antagonistic *B. velezensis* FZB42. KOF112 showed biocontrol activities against gray mold caused by *B. cinerea*, anthracnose by *C. gloeosporioides*, and downy mildew by *Plasmopara viticola*. In the KOF112–*P. viticola* interaction, KOF112 inhibited zoospore release from *P. viticola* zoosporangia but not zoospore germination. In addition, KOF112 drastically upregulated the expression of genes encoding class IV chitinase and β-1,3-glucanase in grape leaves, suggesting that KOF112 also works as a biotic elicitor in grapevine. Because it is considered that endophytic KOF112 can colonize well in and/or on grapevine, KOF112 may contribute to pest-management strategies in viticulture and potentially reduce the frequency of chemical fungicide application.

**Keywords:** *Bacillus velezensis*; downy mildew; gray mold; ripe rot; PR protein; zoosporangia
