Originally published in 2012; updated May 2018.
Daniel A. Kluepfel & Lani Yakabe, USDA-ARS Crops Pathology and Genetics Research Unit, Dept. of Plant Pathology, Univ. of CA. Davis; and Janine Hasey, UCCE Farm Advisor, Sutter, Yuba, Colusa Counties
The Paradox hybrids (Juglans hindsii x Juglans regia) commonly used as walnut rootstocks are highly susceptible to Crown Gall caused by the common soil-borne bacterium Agrobacterium tumefaciens. Here we present information from our quest to develop a robust comprehensive approach to crown gall prevention. These areas include:
- Pre-plant fumigation/chemical control
- Long term soil survival of A. tumefaciens
- Use of “clean” black walnut seeds in Paradox hybrid rootstock production
- Contamination of graft wood and cutting tools
- Identification of novel crown gall resistant rootstocks
Pre-plant fumigation*: We investigated the direct effect of soil fumigants on A. tumefaciens populations in native field soil brought into the laboratory. The Methyl Bromide (MeBr) alternatives, Vapam, Telone® C-35, and Telone® C-35 followed by an additional application of chloropicrin, all reduced soil populations of A. tumefaciens. Notably, 1,3-dichloropropene (Telone® II) alone was not effective at controlling A. tumefaciens. The addition of chloropicrin to 1,3-dichloropropene in Telone® C-35 dramatically reduced A. tumefaciens populations in soil, but not in buried gall tissue. Chloropicrin applied after Telone® C-35 treatment was needed to reduce A. tumefaciens populations in buried gall material. Based on our laboratory data, Telone® C-35 is an effective preplant alternative to MeBr for the control of A. tumefaciens in soil. In sites with a history of high crown gall incidence, fumigation with Telone® C-35 plus chloropicrin, combined with extensive gall removal from the soil, should be considered.
Long-term Agrobacterium survival*: Agrobacterium tumefaciens has the ability to survive for years in soil in the absence of a plant host. For example, we documented A. tumefaciens survival for at least 2.5 years in orchard soil and at least 1.5 years in non-irrigated fallow soil. In addition, the A. tumefaciens strain we introduced in the orchard soil, and reisolated 2 years later, retained the ability to cause crown gall. Given these data, a short-term fallow rotation may be of limited value in reducing crown gall.
Use of “clean” black walnut seeds for Paradox hybrid rootstock production*: We detected A. tumefaciens on Paradox seeds collected from the orchard floor after shaking. Interestingly, the longer seeds remained on the orchard floor prior to collection, the greater the percentage of A. tumefaciens contaminated seeds were discovered. We now hypothesize that when these contaminated seeds are planted in fumigated soil, which contains a compromised native microbial community unable to suppress A. tumefaciens, crown gall incidence is elevated. This occurs because the pathogen experiences reduced competition and is ready to infect the seedling upon emergence from the germinating seed. A cost effective way to reduce crown gall incidence is to limit or eliminate contact of Paradox seeds with the ground prior to planting in fumigated soil. This could be accomplished using a catching frame or even shaking the mother trees on tarps that have been spread on the ground prior to shaking. The key is to eliminate soil contact by Paradox seeds prior to planting in fumigated soil to reduce crown gall incidence.
Contaminated grafting tools and graft wood*: We demonstrated the importance of grafting tool sanitation in crown gall prevention during production of grafted walnut trees on seedling Paradox hybrid rootstock. When sanitation measures are not followed, Paradox seedlings can develop galls at the graft union or bleeding wounds. This implicated not only the involvement of improperly sanitized grafting and cutting tools, but also potential contamination of graft wood with Agrobacterium. Grafting tools and graft wood should never be left on the ground where they can become contaminated with A. tumefaciens.
Bleach, a standard sanitizing agent is an effective disinfectant. However, it is corrosive and rapidly inactivated by dissolved or suspended solids such as organic matter, which are common in field situations. Surfactants/detergents are potentially effective alternatives for the control of microorganisms in environments with high levels of organic matter. We have shown that surfactants, known as quaternary ammonium compounds, effectively reduced populations of A. tumefaciens in solutions and on solid surfaces. The detergents, benzalkonium chloride (BC), Cetyl trimethylammonium bromide, (CTAB) and Physan 20 rapidly reduced populations of A. tumefaciens. More importantly, BC and CTAB activity was only reduced by 16% in the presence of organic material that reduced bleach efficacy by 64%. In our laboratory trials, these detergents dramatically reduced bacterial contamination on cutting blade surfaces resulting in lower gall formation on grafted test plants. These materials are also less phytotoxic than bleach.
It is also important to remember crown galls can harbor large populations of the crown gall pathogen. Therefore, when conducting any gall removal operation, it is important to disinfect cutting tools after use and properly dispose all gall material after removal. Before using any disinfectant, be certain they are registered for that use in California.
Host Resistance: The best form of disease control is the identification and development of disease resistant hosts. Our walnut rootstock improvement team has made significant advancements in the identification of walnut genotypes that exhibit resistance/tolerance to key soil-borne pathogens including Phytophthora, Armillaria (oak root fungus), lesion nematodes and A. tumefaciens (crown gall). In particular, Texas black walnuts (Juglans microcarpa) have been found to exhibit elevated resistance to several of these key pathogens. By crossing Texas black walnuts with English walnuts (J. regia), we have generated hybrids that continue to exhibit tolerance to crown gall and lesion nematodes. These novel experimental hybrids are being examined under various field conditions. The clonal Paradox rootstock RX1, as compared to VX211 and Vlach, has shown medium-low resistance to crown gall in the field and in screening trials.
Overall Prevention Strategy*: Based on our laboratory-based research and field observations, we developed a series of suggestions we feel will aid in the battle against crown gall. These include:
- Eliminate exposure of Paradox seeds and graft wood to soil prior to planting or grafting/budding.
- Do not place grafting or pruning equipment on the ground.
- Surface sterilize grafting tools frequently.
- Limit time between nursery/cold storage pick up and planting and keep stock cool prior to planting.
- Fumigate with Telone® C-35 or Telone® C-35 followed by chloropicrin in crown gall infested sites.
- Limit wounding of plant material.
- Avoid planting too deep.
- Avoid mounding soil up on newly planted trees.
- Keep crown of tree as dry as possible; Agrobacterium is favored by wet environments.
- Do not rely on short-term fallow rotations (e.g. <2 yrs.) to control Agrobacterium tumefaciens.
*These Crown Gall management “Points to Consider” are based on experimental laboratory, greenhouse and /or field research and observations. For answers to your crown gall prevention questions, consult your local UCCE Farm Advisor.