by Sally Colby

Dr. Jill Calabro, research and science programs director for AmericanHort, recently moderated a discussion on Ralstonia solanacearum race 3 biovar 2 (R3bv2).

“The last find of this bacterial pathogen in the U.S. was 16 years ago in 2004, and it was eradicated then,” said Calabro. “R3bv2 is considered a federal quarantine pest, as well as a select agent, which means the federal government considers it could be used as an agent of agricultural bioterrorism. Any positive find must be treated very seriously.”

Dr. Caitilyn Allen, University of Wisconsin-Madison, discussed the interactions between Ralstonia and its hosts, looking for the traits Ralstonia needs to cause disease followed by studying plant responses. Part of the research was funded by the USDA-ARS Floriculture and Nursery Research Initiative.

Allen explained that bacterial wilt diseases are a group of diseases caused by a set of closely related bacteria that belong to the Ralstonia solanacearum species complex.

“These bacteria infect plants’ water-transporting xylem vessels,” said Allen. “Plants are sometimes just stunted, but more often show yellowing and wilting and they die.” Allen added that bacteria in the Ralstonia solanacearum complex include many different strains, with thousands of genetically distinct strains worldwide. Bacterial wilt is most often a problem in tropical and sub-tropical areas, and hosts include bananas, tomatoes, peanuts, cloves and tobacco. Potatoes are also affected, which Allen said is a concern.

In potatoes, the disease manifests as brown rot. This can be complicated to diagnose because a different strain of Ralstonia is well-established in the U.S. and causes problems in crops including tomatoes, tobacco, eggplant and blueberries. Although R3bv2 is a quarantine pest in the U.S., Canada and Europe, there are native strains that aren’t considered quarantine pests. Allen noted that the management challenge is distinguishing between R3bv2 strains of high concern and endemic, established strains.

“The most common symptom is wilting,” said Allen. “Sometimes it’s unilateral – one side is showing symptoms. One thing that happens with geraniums is V-shaped yellow lesions or streaks on the leaves.” Unfortunately, geraniums show this symptom in response to other abiotic and biotic problems. Rolled and wilted leaves are more specific signs, with upward rolling as symptoms first appear and complete collapse of leaves downward when symptoms are more advanced, along with browning of vascular tissue. “Another sign is creamy white bacterial ooze if you pinch an infected stem or more often if you place stems in water,” said Allen. “After 10 minutes, white threads of bacteria and ooze flow out of the stems. You can do this in the field.”

However, these detection methods don’t positively diagnose Ralstonia – they only indicate suspect cases. Allen said the organism grows slowly in culture, especially compared to other fungi and bacteria. “In latently infected plants, the pathogen might be in low numbers or might not be distributed evenly around the plant,” she said. “If you sample a leaf or stem on one side, it might be clean and indicate no infection, but the other side might have a stem with a large concentration of bacteria.”

Ralstonia can be diagnosed via PCR (polymerase chain reaction), a DNA-based assay. However, the test relies on an enzyme that can be inhibited by chemicals in plants and soil, so this diagnostic method is difficult.

Allen said a good diagnostic tool is ImmunoStrip®, which works like a pregnancy test with a band indicating positive results. This tool works well in the field, but it’s expensive. A tester samples the crown of the plant in a latently infected plant, and sensitivity is at about 10,000 cells, so if a plant is wilting, the test should detect a positive.

Matt Royer, associate deputy administrator of field operations for plant protection quarantine with USDA-APHIS, explained that the 2003-04 Ralstonia outbreak led to aggressive eradication efforts. “There were two million plants destroyed in 2003 and 2004,” said Royer. “The 2003 outbreak, the source was Kenya, and in 2004 the source was Guatemala.” He added that the response to those outbreaks was to strengthen off-shore programs, including a certification requirement. “We codified elements for a minimum sanitation protocol,” he said, “and had registration and certification of production sites.”

Royer explained the 2020 response, which began April 21, when USDA confirmed detection of Ralstonia in a single variety of geranium plants in a Michigan greenhouse. “There were at least 200 or more plants showing Ralstonia-like symptoms,” said Royer. “We took immediate action to contain and eradicate from that facility. We received the customer shipping list from Ball, which was helpful in identifying the 288 greenhouses in 39 states.”

The initial target plant identified as a concern was Fantasia Pink Flare geraniums, although the USDA looked at exposure of other hosts and non-host plants to identify, remove and destroy such plants.

Royer explained the basic regulatory steps, starting with contacting the facility and letting them know they’d received a suspect variety and would be visited by federal and state inspectors. “We set up a date to get more information first-hand,” said Royer. “We issued a document and set of instructions, including the most important – to hold all Fantasia Pink Flare plants for destruction.” Geraniums and other hosts received between specific shipping dates (October 2019 – April 2020) were also held, as well as any other plant material that may have been exposed directly in shipping or since being received.

As he explained the epidemiology of Ralstonia, Royer emphasized the fact that it spreads easily in water, and that some cultural practices lead to spread. “We’re trying to use that information when visiting facilities,” he said. “Again, the objective is to destroy plants and potting soil and plant containers.”

The USDA maintains a list of approved means of plant material destruction including incineration, steam sterilization or deposition in an approved landfill. “All areas need to be disinfected, including irrigation systems, irrigation ponds, soil in outdoor holding areas and any contaminated equipment,” said Royer. “We are also on the lookout for other plants that show signs of Ralstonia infection. If they’re negative, the plants are free to move. If positive, plants may not be sold.” Royer added that in many cases, additional testing is needed to confirm R3bv2, but the objective is to release facilities as soon as possible.

Five labs can officially test for Ralstonia, including the University of Florida, Michigan State University, Cornell University, California Department of Food and Agriculture and Kansas State University. The final confirmation of race and biovar is done at the Beltsville, MD, lab.

Royer said they learned from 2003 and 2004 that drip irrigation is generally less likely to spread Ralstonia than sub-irrigation. In facilities that have hanging baskets with R3bv2, all material below must be destroyed. Good sanitary practices are essential in stopping the spread, and tools should be disinfected between plants when pinching, budding, cutting and trimming.

“Keep an eye on your plants,” said Royer. “Scout often for signs of disease. There are other diseases plants can get in a nursey setting. Work with Extension, trade associations, regulators. Read up on information available and look at best practices to minimize the risk of spreading R3bv2.”