Impact of biological amendments on Agrobacterium tumefaciens survival in soil
Introduction
California produces approximately 99% of the English walnuts (Juglans regia) grown in the United States and 54% of worldwide production (Ramos, 1998). In California, nearly 70% of the J. regia trees are grown on the hybrid rootstock Paradox (J. regia × J. hindsii; Hasey, personal communication), which imparts increased vigor across a wide-range of soil types and is tolerant to several Phythophthora species (Flint, 2003). However, Paradox is highly susceptible to infection by Agrobacterium tumefaciens, which causes crown gall (CG) (Agrios, 2005, Ramos, 1998). In nurseries, losses to CG can be extensive because each galled plant and adjacent symptomless plants, which also may be infected, are usually culled (Epstein et al., 2008). Crown gall incidence can be as high as 90% in orchards grown on Paradox rootstocks (Kluepfel, unpublished).
Crown gall may occur in walnut once A. tumefaciens gains entry through a plant wound (Agrios, 2005). While these infections can occur anytime a tree is injured, infestation of the hybrid seed by A. tumefaciens prior to planting in the nursery also plays an important role in future CG development (Yakabe et al., 2014). During harvest, hybrid seeds are collected from the orchard floor where they come in contact with soilborne populations of A. tumefaciens. The bacteria may reside in/on the seed husk until planted, where it then multiplies in the soil surrounding the seed. Fumigated soils, which contain a severely impacted soil microbial community, offer limited competition to A. tumefaciens introduced into these environments, resulting in an elevated abundance of A. tumefaciens and increased CG incidence (Yakabe et al., 2010). We hypothesize that increasing microbial diversity in the soil through addition of commercially available microbial mixtures or vermicompost will provide greater competition for A. tumefaciens and sufficiently reduce its abundance and ultimately limit infection of emerging walnut seedlings.
Currently, many walnut nurseries perform pre-plant fumigation with methyl bromide (MeBr) or other soil fumigants to control nematodes, weeds, and plant pathogens, including A. tumefaciens (Ramos, 1998). Though A. tumefaciens is susceptible to MeBr fumigation, inconsistent CG control is often observed under nursery conditions in MeBr fumigated soils (Flint, 2003, Harper and Belnap, 2001, Yakabe et al., 2010). This inconsistent CG control may arise from fumigation-induced modifications to the indigenous soil community (Ibekwe, 2004, Ibekwe et al., 2001, Stromberger et al., 2005), which could result in a microbial profile with a reduced capacity to limit growth of introduced pathogens (Yakabe et al., 2010). Fumigation with MeBr or MeBr alternatives such as 1,3-dichloropropene or chloropicrin, significantly reduced microbial activity (Dungan et al., 2003) and altered community composition (Ibekwe et al., 2001, Klose et al., 2006) four weeks after fumigation. These altered microbial communities appear to facilitate increased abundance and re-colonization of introduced nematodes (Yeates and van der Meulen, 1996) and fungal pathogens such as Fusarium oxysporum (Marois et al., 1983). This hypothesis also was proposed by Yakabe et al. (2014) to explain the elevated population levels reached by A. tumefaciens introduced to soils previously fumigated with MeBr.
Increasing soil microbial diversity and activity provides competition for the limited number of available soil resources, thereby reducing the abundance of targeted pathogens (Johnson, 2010, van Elsas et al., 2012). Biocontrol approaches to control soilborne pathogens typically take one of two routes: (1) addition of specific microbial species selected for their ability to suppress targeted parthogens (Fravel, 2005, Raaijmakers et al., 2002) or (2) addition of complex microbial communities contained in compost or commercially available microbial fermentation mixtures.
Compost application has provided consistent disease suppression for a number of pathogens (Hoitink et al., 1997), including Pythium spp. (Darby et al., 2006), Phytophthora spp. (Andrivon, 1994), and Verticillium dahlia (Termorshuizen et al., 2006). Standard compost is generated through biotic and abiotic decomposition of solid animal or plant waste. Vermicompost, however, is generated through the addition of earthworms to the standard decomposition processes (Dominguez, 2004, Dominguez et al., 2010). Earthworms alter the microbial community structure and diversity of the resulting compost after passing the organic matter through their gut (Fernandez-Gomez et al., 2011, Fernandez-Gomez et al., 2012). It is in the earthworm gut where the resident microbial community affects these changes and contributes to the enhanced microbial diversity and nutritional quality of the earthworm cast (Vivas et al., 2009). In addition, simple movement of the worm through the casts and soil provides aeration, thereby further stimulating microbial activity (Aira et al., 2006, Aira et al., 2008). These processes significantly increase bacterial abundance (Dominguez et al., 2010), diversity (Fracchia et al., 2006, Jack et al., 2011, Sen and Chandra, 2009, Vivas et al., 2009), and activity (Aira and Dominguez, 2011) compared to standard compost.
Studies examining disease suppression using vermicompost have reported promising results for the control of several diseases (Jack et al., 2011, Rivera et al., 2004, Stover, 2009). For example, application of vermicompost significantly reduced infection of tomato seedlings by Phytophthora nicotianae (Szczech and Smolinska, 2001), root-rot/wilt disease incidence in the medicinal plant Coleu forskohlii (Singh et al., 2012a), and seedling damping-off disease in cucumber (Scheuerell et al., 2005). The disease suppression ability of vermicompost is thought to occur due to the enhanced diversity of the microbial community that also may contain elevated populations of known disease-suppressing bacteria. For example, vermicomposts are often enriched in Streptomyces spp., which have strong anti-fungal characteristics (Fernandez-Gomez et al., 2012, Yasir et al., 2009), and when isolated, reduced Fusarium wilt incidence in chickpeas by 45–76% (Gopalakrishnan et al., 2011).
Here we examined the impact of two commercial microbial fermentation mixtures and commercially produced vermicompost on A. tumefaciens survival in soil. While pre-plant soil fumigation is used to manage CG, its primary purpose in walnut nurseries is to control weeds and plant parasitic nematodes. Since nursery operators in California must manage multiple pathogens simultaneously while complying with a zero tolerance for nematodes, they are unlikely to suspend all soil-fumigation practices. Therefore, a viable biologically based A. tumefaciens management strategy would, mostly likely, need to be complimentary to some form of soil fumigation. Toward this end, we determined the efficacy of vermicompost and two brands of commercial soil amendments to limit A. tumefaciens multiplication and persistence in both fumigated and non-fumigated soils. While numerous studies have examined the suppressive effect of vermicompost on soilborne eukaryotic plant pathogens (Jack, 2011, Scheuerell et al., 2005, Singh et al., 2012b, Szczech and Smolinska, 2001), we present data that illustrates the ability of vermicompost amendments to enhance microbial diversity that facilitates suppression of A. tumefaciens.
We also examined the effect of fumigation and vermicompost amendment on the soil microbial community composition using a culture-independent next-generation sequencing approach, which eliminates biases associated with cultivation. This level of resolution also allowed us to identify members of the microbial community associated with observed pathogen and disease suppression phenotypes. These discoveries open the door to developing a wider range of disease suppression techniques for seedling nursery production with the possibility of reducing dependency on soil fumigation.
Section snippets
Soil treatments
Soil was collected from fallow fields within the experimental walnut orchard at the UC Davis Armstrong Field Station in Davis, CA. The soil has a fine silty mixed texture and was collected from 20 to 30 cm depth in January 2012–March 2013. All soil was stored at room temperature in sealed plastic bags for one week until use.
Fumigated soils were treated with Telone-C35 (Tri-Form 35, TriCal, Inc., California, USA) at the label rate of 527 lbs acre−1 or MeBr 98% (Cardinal Chemical Corporation,
Fumigation effects
A. tumefaciens 186r effectively re-colonized MeBr and Telone-C35 fumigated soils and dropped close to detection limits in native field soil (Fig. 1). A. tumefaciens population means significantly increased from 1.7 × 106 CFU g−1 soil to 2.2 × 106 CFU g−1 soil in MeBr-fumigated soils and 1.6 × 106 CFU g−1 soil to 2.5 × 106 CFU g−1 soil in Telone-C35 fumigated soils during the 28-day incubation period (Fig. 1). Similar increases were seen in the qPCR data of Tip6 gene abundance, with mean gene abundance
Discussion
While fumigation with either Telone-C35 or MeBr reduces abundance of A. tumefaciens present in the soil (Yakabe et al., 2010), it also significantly decreased microbial diversity and altered the microbial community composition (Fig. 3, Fig. 4). Our current results were consistent with results reported by Yakabe et al. (2010), which confirmed the consistency of our bioassay system. This facilitates useful comparisons across experiments separated temporally. Yakabe et al. (2014) reported the
Acknowledgments
We thank Ali E. McClean, Rachel F. Greenhut, and Luis S. Curiel for their technical assistance. We also thank Jack Chambers of Terra Vesco for supplying vermicompost and Mike Stanghellini and Matt Gillis of Tri-Cal for soil fumigation assistance. This work was partly funded by the CDFA Fruit, Nut and Grapevine Improvement Advisory Board and USDA–ARS CRIS 5306-22000-015-00D.
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