Testing for Bacterial Fruit Blight in Cucurbit Seed

- Andrew Lyon

Bacterial fruit blotch, Acidovorax avenae subsp. citrulli (BFB), is an infectious seed borne disease that can cause up to 90 percent loss of marketable yield in some cucurbit (watermelon, melon, squash varieties, etc.) fields. Even low levels of infection can cause significant crop loss. There are no effective eradication treatments, which means exclusion is the best method of disease control. Since there are also no resistant commercial varieties, BFB management depends on growers using pathogen-free seeds and seedlings.

It is paramount for seed companies to test their seed for BFB before sale, and most governments require inspection for import/export. All levels of seed should be tested: foundation, stock and commercial seed. Phytosanitary field inspections are not enough to detect infected plants as the symptoms that form can be indistinguishable from other foliar disease. At the end of the day it is important to know that lawsuits filed by growers who received infected seed can run into the millions

Multiple methods are available to test for BFB. At Eurofins BDI we use both the grow-out and seed wash PCR methods. Both of these methods are validated by the National Seed Health System and we are ISO17025 accredited to perform them.

The seed wash PCR test is an indirect method to detect the presence of Acidovorax, but it gives no information on pathogens virulence or viability. Therefore seed wash results must be validated by a greenhouse grow-out to determine the biological state of the pathogen.

Greenhouse grow-outs are the most reliable method for detection with the lowest risk for false-positives. A representative sample of seeds per production lot is visually inspected after 18 days of growth in high humidity for BFB symptoms. Eurofins BDI has 13,000-sq.ft. of greenhouse space in Longmont, Colorado, plus 5,000-sq-ft. in Gilroy, California. Positive controls of each crop species are planted in every greenhouse. This includes a melon control, a watermelon control and a squash control. Seeds inoculated with A-016 – our proprietary strain – are planted in every greenhouse to ensure proper disease expression conditions, as well as ensure that contamination cannot occur

Symptomatic tissue is collected and small pieces are placed into a micro tube for immunostrip testing. Sample DNA will then be extracted and run through a qPCR reaction for detection of bacterial presence. Suspect tissue will then be plated on selective media to isolate the bacteria into pure culture. We finalize testing by reintroducing the cultured suspect bacteria into a healthy host plant. To confirm a positive finding we must see symptoms concurrent with BFB, and be able to isolate Acidiovorax citrulli from the artificially infected host.

With over 20 years’ experience in detecting BFB, Eurofins BDI is confident that the grow-out test with rigorous conformational steps is the best method of prevention of BFB. Combined with the additional seed-wash/qPCR method we aim to ensure that seed sold to growers is free from this destructive pathogen, and seed producers can rest easy knowing they are selling quality seed.