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
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.
References (47)
- et al.
Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis
Cell
(1977) - et al.
Correspondence analysis as a tool in fungal taxonomy
Syst. Appl. Microbiol.
(1998) - et al.
Opines in crown gall tumours induced by biotype 3 isolates of Agrobacterium tumefaciens
Physiol. Mol. Plant Pathol.
(1988) - et al.
Analysis of core genes supports the reclassification of strains Agrobacterium radiobacter K84 and Agrobacterium tumefaciens AKE10 into the species Rhizobium rhizogenes
Syst. Appl. Microbiol.
(2010) - et al.
Applicability of rep-PCR genomic fingerprinting to molecular discrimination of members of the genera Phaeoacremonium and Phaeomoniella
Plant Pathol.
(2004) - et al.
Biological control of Agrobacterium vitis using non-tumorigenic agrobacteria
Vitis
(1999) - (1973)
- et al.
Crown gall of grape: biology of Agrobacterium vitis and the development of disease control strategies
Plant Dis.
(1998) - et al.
Crown gall of grape: biology and disease management
Annu. Rev. Phytopathol.
(1999) - et al.
Biological control of grape crown gall with non-tumorigenic Agrobacterium vitis strain F2/5
Am. J. Enol. Vitic.
(1994)
Measures of the amount of ecologic association between species
Ecology
Biological control of grape crown gall with non-tumorigenic Agrobacterium vitis strain F2/5
Am. J. Enol. Vitic.
Traversing the cell: Agrobacterium T-DNA’s journey to the host genome
Front. Plant Sci.
Plant responses to Agrobacterium tumefaciens and crown gall development
Front. Plant Sci.
A host-specific biological control of grape crown gall by Agrobacterium vitis strain F2/5: its regulation and population dynamics
Phytopathology
Biological control agent Agrobacterium vitis strain ARK-1 suppresses expression of the virD2 and virE2 genes in tumorigenic A. vitis
Eur. J. Plant Pathol.
Reduction in pathogen populations at grapevine wound sites is associated with the mechanism underlying the biological control of crown gall by Rhizobium vitis strain ARK-1
Microb. Environ.
Genetic diversity of Rhizobium vitis strains in Japan based on multilocus sequence analysis using the sequences of pyrG, recA and rpoD
J Gen Plant Pathol
Biological control of crown gall on grapevine and root colonization by nonpathogenic Rhizobium vitis strain ARK-1
Microb. Environ.
New antagonistic strains of non-pathogenic Agrobacterium vitis to control grapevine crown gall
J. Phytopathol.
New antagonistic strains of non-pathogenic Agrobacterium vitis to control grapevine crown gall
J. Phytopathol.
Evaluation of the nonpathogenic Agrobacterium vitis strain ARK-1 for crown gall control in diverse plant species
Plant Dis.
Biological control for grapevine crown gall using nonpathogenic Rhizobium vitis strain ARK-1
Proc. Japan Acad. Ser. B Phys. Biol. Sci.
Cited by (5)
Risk assessment of inferior growth and death of grapevines due to crown gall
2022, European Journal of Plant PathologyInsight into inducing disease resistance with Allorhizobium vitis strain ARK-1, a biological control agent against grapevine crown gall disease
2022, European Journal of Plant Pathology