**1. Introduction**

The production of Koshu wine in Japan started in 1874 in Yamanashi Prefecture. *Vitis* sp. cv. Koshu, a hybrid of *Vitis vinifera* L. and *V. davidii* Foex, is an indigenous wine grape in Japan [1]. Koshu was introduced from Europe to Japan through the Silk Road, and crossed with the Chinese wild species *V. davidii* en route to Japan. Koshu was recognized as a wine grape cultivar in 2010 by the International Organization for Vine and Wine, and was registered in *Vitis* International Variety Catalogue by Julius Kühn-Institut-Bundesforschungsinstitut für Kulturpflanzen (https://www.vivc.de/, accessed on 28 April 2021). At present, Koshu is one of the most widely cultivated wine grapes in Japan and one of the most important cultivars for white-wine making in Japan [2]. Whole genome analysis demonstrated that Koshu is susceptible to phytopathogenic attack as a result of deletions in genes associated with pathogen response, such as hypersensitive response [3]. For example, Koshu is more susceptible to downy mildew, which is caused by *Plasmopara viticola* (Berk and M.A. Curtis; Berl and De Toni), than other *V. vinifera* cultivars [4]. In addition, the humid climate of Japan, caused by the prolonged concentration of extremely moist airstreams over Japan as a result of global warming [5], has contributed to damaging Koshu grapevines by phytopathogenic fungal diseases. The high nighttime temperature

**Citation:** Hamaoka, K.; Aoki, Y.; Suzuki, S. Isolation and Characterization of Endophyte *Bacillus velezensis* KOF112 from Grapevine Shoot Xylem as Biological Control Agent for Fungal Diseases. *Plants* **2021**, *10*, 1815. https:// doi.org/10.3390/plants10091815

Academic Editors: Carlos Agustí-Brisach and Eugenio Llorens

Received: 11 May 2021 Accepted: 27 August 2021 Published: 31 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

resulting from the acceleration of global warming has promoted downy mildew infection in grapevines [6]. Thus, fungal disease control has become a subject of serious concern in viticulture for Koshu wine making.

A simple strategy against fungal disease is the use of chemical fungicides. However, there are two main problems with regard to the application of chemical fungicides. One is environmental pollution, and the other is the emergence of fungal phytopathogen populations resistant to the chemical fungicides. In particular, the latter has plagued vine growers in Japan, making it extremely difficult to control fungal diseases in grapevines. *P. viticola* is a high-risk phytopathogen that easily acquires chemical fungicide resistance [7]. Resistant genes, conferring resistance to quinone outside inhibitor and carboxylic acid amide, were detected in *P. viticola* populations in Japanese vineyards in 2010 [8] and in 2015 [9], respectively. Fungicide-resistant *Colletotrichum gloeosporioides* (Penzig) Penzig and Saccardo, which causes grape ripe rot, and *Botrytis cinerea* Pers. ex Fr., which causes grape gray mold, have already emerged in Japanese vineyards [10,11]. It is for these reasons that alternatives to chemical fungicides are attracting the interest of scientific communities.

One of the alternative disease control strategies to chemical fungicides is biological control using biofungicides. Biological control agents in biofungicides are isolated from nature, and are largely microorganisms [12]. A vast number of microorganisms isolated from nature have been identified as candidates for biological control agents in biofungicides [13,14]. Some microorganisms have been developed and launched as biofungicides in viticulture. For example, *Bacillus subtilis* QST-713 (product name, Serenade®) is available on the market, and is used to control gray mold in viticulture [15]. The introduction of biofungicide application in viticulture is expected to reduce the frequency of chemical fungicide application and to alleviate increasing environmental concerns on chemical fungicides and the emergence of resistance to the chemical fungicides in fungal phytopathogens.

Cyclic lipopeptides produced by biological control agents have received considerable attention as one of the tools for disease control in plants, because some cyclic lipopeptides also function as elicitors in plants as well as antimicrobial metabolites [16–18]. For example, fengycin and surfactin secreted by *B. subtilis* GLB191 contribute to protection against grape downy mildew by directly inhibiting *P. viticola* and inducing plant defense response [17]. Iturin A induces defense response in plants depending on its structure [19]. The cyclization of the seven amino acids and the β-hydroxy fatty acid chain of iturin A are required for the induction of plant defense response. Although evidence of how cyclic lipopeptides trigger plant defense response is lacking, microorganisms showing bifunctional activity against phytopathogens may be an innovative biological control agent in viticulture.

The objective of this study was to clarify the possibility of using endophytic bacteria as biological control agents in biofungicides used in viticulture. The colonization efficiency of biological control agents in biofungicides on/in plant tissues affects their antagonistic activities toward fungal phytopathogens [20]. In this study, we explored grapevine endophytic bacteria possessing in vitro antagonistic activities toward three fungal phytopathogens, *B. cinerea*, *C. gloeosporioides*, and *Phytophthora infestans* (a substitute of *P. viticola*), and isolated endophytic *Bacillus velezensis* KOF112 from the shoot xylem of Koshu grapevine. KOF112 showed in vivo biocontrol activities against gray mold caused by *B. cinerea*, anthracnose by *C. gloeosporioides*, and downy mildew by *P. viticola*. In addition, foliar application of KOF112 induced plant defense response in grapevine.
