A novel technique for treating seeds with biocontrol agents for the sustainable management of bacterial fruit blotch of watermelon

View the project final report

• 2014 annual report

Commodities

• Fruits: melons
• Vegetables: cucurbits

Practices

• Crop Production: biological inoculants
• Pest Management: biological control

Bacterial fruit blotch (BFB) of cucurbits is caused by the gram negative bacterium Acidovorax citrulli. Since the initial devastating outbreak on watermelon in 1980s, BFB has occurred natural on many cucurbits including melon, pumpkin, and cucumber. BFB causes direct economic losses by reducing fruit yield and seed quality since the pathogen is seedborne and seed transmitted. Presently there are no commercially available sources of BFB resistance. Current BFB management practices include foliar applied antimicrobial chemicals and exclusion of the pathogen. Even though these practices are routinely used, BFB outbreaks continue to occur sporadically with significant economic consequences. This is primarily because seeds are the most important source of primary inoculum and the pathogen can survive for long periods inside the seed, where it is protected from externally applied chemical seed treatments. The proposed research will evaluate a novel biocontrol seed treatment approach based on inoculation of female watermelon flowers with a nonpathogenic A. citrulli strain (AAC00-1ΔhrcC) to limit seed-to-seedling transmission of BFB. Application of AAC00-1ΔhrcC to the stigmas of female watermelon flowers results its deposition in the embryo of seeds where it is proximal to resident pathogenic A. citrulli populations. As the seeds germinate, AAC00-1ΔhrcC rapidly colonizes the seedling tissues and through direct competition, restricts A. citrulli population growth. We propose to optimize the stigma inoculation technique to attain maximum seed inoculum load and BFB control. This approach will yield an environmentally sound and sustainable management strategy by which seed producers can reduce BFB outbreaks in US watermelon production.

The main objective of this study is to optimize a flower inoculation technique for efficiently delivering biocontrol agents into watermelon seeds. The specific objectives include:

1. To optimize the concentration of flower stigma inoculum required to maximize seed inoculation with AAC00-1ΔhrcC .
   1.  Forty watermelon plants (cv. Crimson Sweet) will be established in three-gallon pots under standard greenhouse conditions. At anthesis, female watermelon stigmas will be hand pollinated and then inoculated with the non-pathogenic strain, AAC00-1ΔhrcC. A micropipette will be used to deliver 10 µl of cell suspension to each stigma. Bacterial concentrations will include 0, 10⁷, and 10⁸, and 10⁹ CFU/stigma (10 plants per treatment). Flowers will be allowed to develop into mature fruit (35-40 days after pollination); harvested and stored at 5°C until seed extraction. Fruit will be surface sterilized and seed will be extracted manually. Seed from each fruit will be maintained as a separate lot without fermentation; air-dried for 24-48 h at room temperature and stored at room temperature. Using a A. citrulli-specific quantitative real-time PCR assay, the percentage of infested seeds per lot and the concentration of bacteria per seed will be determined. Subsequently, seed germination percentage and the rate of seed germination will be determined for each seed lot.
2. To determine the efficacy of flower inoculation with AAC00-1ΔhrcC in reducing seed-to-seedling transmission of BFB.
   1. Seeds (n = 100 seeds/lot) from each lot will be inoculated with 10⁶ CFU/ml A. citrulli AAC00-1 (wild type) by vacuum infiltration, air-dried for 24 h and planted into germination boxes. Seeds will be allowed to germinate and grow for 15 days at 100% RH and 28°C. Each day the percentage of seedlings displaying BFB symptoms will be recorded. The effects of flower inoculation with different concentrations of AAC00-1ΔhrcC on BFB seedling transmission and seed physiology will be determined. This experiment will be conducted twice.
   2. A watermelon (cv. Crimson Sweet) field plot will be established at the Vidalia Onion and Vegetable Research Farm in Reidsville GA. At anthesis, female watermelon flowers will be hand pollinated and the stigmas will be inoculated with AAC00-1ΔhrcC and sterile water as a control. The optimal concentration of biocontrol bacteria determined in objective 1a will be used for field inoculations. A micropipette will be used to deliver 10 µl of cell suspension per stigma and flowers will be allowed to develop into mature fruit. After harvest, seeds will be allowed to ferment in fruit pulp for 3 days and subsequently washed and dried according to commercial seed production practices. The levels of biocontrol agents in the seed will be determined by a A. citrulli-specific quantitative real-time PCR assay. Seed samples will be vacuum-infiltrated with 10⁶ CFU/ml of AAC 00-1, air dried and planted in germination boxes at 28°C and 100% RH for 15 days. BFB seedling transmission and germination will be recorded daily.