Elsevier

Crop Protection

Volume 146, August 2021, 105685
Crop Protection

Efficacy of a biological control agent Rhizobium vitis ARK-1 against Virginia R. Vitis isolates, and relative relationship among Japanese and Virginia R. vitis isolates

https://doi.org/10.1016/j.cropro.2021.105685Get rights and content

Abstract

Non-tumorigenic Rhizobium (syn. Agrobacterium) vitis strain ARK-1 (ARK-1) has been shown to reduce crown gall in grapevine (Vitis vinifera) seedlings when co-inoculated with Japanese tumorigenic isolates of R. vitis. The objectives of this study were to test ARK-1's efficacy as a biological control agent against R. vitis isolated from grapevines in Virginia, and to examine genetic diversity of R. vitis found in Virginia, USA and Japan. ARK-1 was co-inoculated into wine grape trunks (V. vinifera cv. ‘Cabernet Sauvignon’) with a tumorigenic R. vitis isolate at a 1:1 cell ratio (~108 cell/ml). A total of four R. vitis isolates were tested individually. Compared to treatments of a tumorigenic isolate alone, ARK-1 co-inoculation significantly decreased (P ≤ 0.05) the mean probability of gall formation in all cases, and the mean gall diameter in all, but one case (isolate ACME15). The average reduction was 90% and 92% in the mean probability of gall formation and mean gall diameter, respectively. We conducted exploratory data visualization with distance-based clustering and correspondence analysis using data from ISSR- and rep-PCR, which showed separation of Virginia and Japanese isolates and placement of ARK-1 near to Virginia isolates. These results suggest that ARK-1 is a good candidate for biocontrol of grapevine crown gall in the mid-Atlantic region of the USA.

Introduction

Crown gall of grapevine is a devastating bacterial disease caused by Rhizobium vitis, previously known as Agrobacterium vitis (Kuykendall et al., 2001). This pathogen enters the grapevine through wounds from a variety of causes, such as winter injury, mechanical damages, grafting (Burr and Otten, 1999). R. vitis causes crown gall by transferring the T-DNA region of the tumor-inducing bacterial plasmid (Ti-plasmid) to the host cell, which subsequently integrates into the plant host genome to cause gall formation (Chilton et al., 1977; Gelvin, 2012; Pitzschke and Hirt, 2010). When galls invade the vascular tissue, it can result in vine death (Gohlke and Deeken, 2014; Wächter et al., 2003). In this paper, R. vitis isolates that carry the Ti-plasmid and are capable of causing crown gall will be referred to as “tumorigenic”.

Currently, cultural management strategies, such as hilling of the graft union during the winter, are the predominant methods used for management of crown gall. However, many of these strategies are not feasible or sustainable due to their cost and labor requirement (Burr et al., 1998). Appropriate cultivar and site selection are also recommended, but relatively cold tolerant cultivars, e.g., French-American hybrids, may not be popular in the market, and many growers have very limited options for the site. Chemical options are also very limited and often treat only the symptoms. For example, Gallex (AgBioChem, Inc., Los Molinos, CA) aids removal of galls, but since R. vitis is a systemic pathogen, it will not remove the pathogen from the infected vine.

The history of investigation for viable biological control agents for crown gall goes back to the early 1970s (New and Kerr, 1972). Among potential biological agents against crown gall, two agents have been extensively studied. Rhizobium rhizogenes (previously A. radiobacter biovar 2) (Velázquez et al., 2010) strain K84 produces an antimicrobial agrocin 84 that is antagonistic to sensitive strains of Rhizobium (Kerr, 1980; Reader et al., 2005). K84 is can prevent of crown gall in stone fruit (Prunus spp.), apples (Malus spp.), and other fruit and nut trees; however, K84 is ineffective at reducing crown gall in grapevine caused by R. vitis (Burr et al., 1998; Kawaguchi and Inoue, 2012a). Another effective biological control agent is a non-tumorigenic R. vitis strain F2/5, which reduces crown gall by stimulating the hypersensitive defense response in grapevine (Gall et al., 1994; Kaewnum et al., 2012; Zheng et al., 2003; Zheng and Burr, 2016). The hypersensitive response also induces necrosis of grapevine tissue, which can lead to vine mortality (Bazzi et al., 1999). Derivatives of F2/5 that do not induce necrosis have been developed and patented (Ryder and Jones, 1990).

Recently in Japan, a new strain of R. vitis, designated as ARK-1 by Kawaguchi, was recovered from ‘Pione’ grapevine (V. hybrid ‘Kyoho’ x ‘Muscat of Alexandria’) (Kawaguchi and Inoue, 2012a; Yamada and Sato, 2016). ARK-1 neither carries the Ti-plasmid nor causes disease symptoms (Kawaguchi and Inoue, 2012a). When ARK-1 was inoculated onto grapevine seedlings together with isolates that carry the Ti-plasmid (tumorigenic isolates), gall incidence decreased by approximately 90% without causing necrosis to grapevines (Kawaguchi and Inoue, 2012a). ARK-1 slows the population growth of tumorigenic R. vitis isolates at inoculation sites (Kawaguchi 2014) and suppresses Ti-plasmid gene expression (virA, virD2, virE2, and virG) critical to plant cell transformation by R. vitis (Kawaguchi, 2015; Kawaguchi et al., 2019), and ARK-1 potentially activates host plant defenses limiting successful infection (Kawaguchi and Noutoshi, 2020). Although ARK-1 seems to show great potential as a biological agent against crown gall, these studies have investigated the efficacy of ARK-1 only against Japanese isolates of R. vitis. Also, we have limited knowledge on the genomic information on ARK-1. Therefore, the objectives of this study are to examine the effect of ARK-1 in reducing gall formation by co-inoculating ARK-1 and a tumorigenic isolate, which were isolated from grapevines in Virginia, USA, in grapevine trunks, and also to investigate genetic variability of R. vitis isolates from Virginia and Japan in relation to ARK-1.

Section snippets

Isolation of R. vitis from Virginia vineyards

Virginia isolates of R. vitis were obtained from grapevines in five geographically distant vineyards in 2015 (Table 1). Vines with visible gall tissues, some are older (i.e., black and dry), and others are new (i.e., green to white in the center and moist), were collected. Gall tissue was cut into pieces, surface sterilized with 70% ethanol for 2 min and rinsed twice with sterile distilled water. The tissue was then soaked in 5 ml of sterile distilled water for 30 min in a sterile 15 ml tube to

Gall inhibition in grapevine

The mean probability of gall formation per treatment varied from 0.00 to 0.84 (Fig. 3a). The effect of the interaction between treatment and isolate on the mean probability of gall formation per treatment was significant (χ2 = 13.9, P < 0.001). Isolates HNVR15 and ZEME15 inoculated by itself resulted in significantly more gall formation (P ≤ 0.05) than the other two isolates. ARK-1 co-inoculation significantly reduced (P ≤ 0.05) the mean probability of gall formation with all four isolates. The

Discussion

Our study confirms that the levels of reduction provided by the ARK-1 treatment against four Virginia isolates are similar to the previous findings with Japanese table grapes. We challenged ARK-1 with a simultaneous 1:1 cell ratio co-inoculation of grapevine with ARK-1 and a single tumorigenic R. vitis isolate, which resulted in an average of 89.6% reduction for the mean probability of gall formation and 91.9% reduction for the mean gall diameter. The results were comparable to a previous study

Funding

This work was supported by the Virginia Wine Board [2016, 2017, and 2018] and the USDA/NIFA Hatch grant [project number VA-160112].

Author contributions

Wong (investigation and original draft), Kawaguchi (conceptualization, investigation and original draft), Nita (conceptualization, data analysis, original draft, funding acquisition, review, and editing).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Ms. Akiko Mangan and Ms. Morgan Gannon for technical help they provided. We also thank VA grape growers for submitting grape vines, Kumiai Chemical and Okayama prefectural government for providing ARK-1, and Drs. Boris Vinatzer and Anton Baudoin, and Ms. Elizabeth Bush for reviewing this manuscript.

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