USDA, Canada Collaborate on Fusarium Wilt

Family warfare has broken out in the Fusarium clan. While some members of this fungal family are beneficial, others are harmful. Researchers may soon prove that when it comes to Fusarium, like family, you can't live with them—or without them.

A pathogenic strain of Fusarium oxysporum, causes Fusarium wilt, a disease that afflicts many crops such as watermelon, muskmelon, and basil but is a bigger problem for tomato growers. Methyl bromide was used to keep this pathogen at bay, but now that methyl bromide is being phased out due to concerns about ozone depletion, other alternatives are under investigation.

Researchers at USDA and Canada's equivalent—Agriculture and Agri-Food Canada (AAFC)—are collaborating on studies that pit Fusarium species against one another. Pathogenic F. oxysporum was controlled with varying degrees of success by several harmless members of the Fusarium family.

Deborah R. Fravel, a plant pathologist at ARS' Biocontrol of Plant Disease Laboratory in Beltsville, Maryland, and George Lazarovits, a research scientist and team leader at the Southern Crop Protection and Food Research Center in Ontario, Canada, are working together to find a workable biocontrol solution. Biocontrol is the use of one organism to control another.

"The Beltsville group under Dr. Fravel's guidance was heading in a parallel direction with our work, and collaboration was obvious," Lazarovits says.

Just to get to this point, however, took plenty of groundwork. Fravel was sent to Florida to obtain soil samples from tomato fields. "We screened 450 microbes and found another Fusarium that helped control wilt," explains Dr. Fravel.

Into the Fields

In 1997, field plots were set up at the USDA Beltsville Agricultural Research Center to test selected biocontrol agents for control of Fusarium wilt in tomatoes. The agents chosen reduced Fusarium wilt in greenhouse tests: isolates of F. oxysporum (CS–20) and F. solani (CS–1), and a commercial biocontrol agent, SoilGard, containing Gliocladium virens strain G1–21. These were tested alone and in various combinations. In 1998, another combination treatment, consisting of a fungus (G. virens strain G1–3) and a bacterium (Burkholderia vietnamiensis strain Bc–F), was also tested.

Tomato plants were grown in soilless-mix plug trays (98 cells/tray) in the greenhouse for about 6 weeks before transplanting to the field. Beneficial Fusarium isolates were placed in the plug trays as liquid inoculum (5 ml of 106 colony-forming units/ml of suspension/cell) at the time of seeding and again 1 week before transplanting. SoilGard granules were worked into the soilless mix at a rate of 2 g/L before seeding. The combination of dry fungus and bacterium was applied with a sticker as a seed treatment before planting and as a liquid 1 week before transplanting.

Plots were set up as single rows on 1.5-m centers, with 24 plants per 15-m row in 1997 and 12 plants per 7.6-m row in 1998. Pathogen inoculum of a race 1 isolate of F. oxysporum f. sp. lycopersci was incorporated into rows (2 kg/row of liquid culture) 1 day prior to transplanting in both years. In 1998, an additional 100 ml of inoculum was added to each transplant hole at the time of planting. Tomatoes were transplanted by hand in the fields, and overhead irrigation was used when necessary throughout the growing season. At the end of the season, stem sections from all plants were taken. Stem surfaces was sterilized and placed in agar plates on a Fusarium-selective medium to determine the incidence of plants systemically infected with the pathogen.

In 1997, very little disease developed, and none of the treatments showed any differences. Because no disease was noted, yield data were not taken that year.

But in 1998, biocontrol treatments containing F. oxysporum isolate CS–20 (CS–20, CS–1 + CS–20, and SG+CS–20) and the fungus plus bacterium treatment (G+B) significantly reduced the incidence of disease. Of the treatments, only CS–20 and G+B treatments showed significant effects on yield, with increased total weight, number of fruits, and average weight/fruit, compared with a pathogen control. The CS–20 and G+B treatments resulted in increased total weight of 34.3 percent and 37.7 percent, respectively, over the pathogen control. SoilGard and CS–1 alone showed no significant effect on disease incidence or yield.

The study shows that beneficial Fusarium strains can reduce tomato wilt and increase yield. Now researchers must figure out how the mechanisms of biocontrol work. Some beneficial strains work by competing with the pathogenic strains for nutrients and space. CS–20 seems to pump up the tomato plants' natural defenses against pathogens, a reaction called "induced systemic resistance."

Back to the Lab

ARS is testing several species of Fusarium for effectiveness in controlling Fusarium wilt, but one Fusarium cannot be easily distinguished from another. "We developed genetically tagged Fusarium isolates that can be readily tracked in the soil and on the plant," says Lazarovits. "The tags allow us to quantitatively recover the organisms from the environment they are introduced into."

Dr. Jian Bao, a molecular biologist who previously worked with Dr. Lazarovits and now works with Dr. Fravel, has provided data and beneficial strains of Fusarium—research tools Dr. Fravel's lab would otherwise have had to produce and work that could have taken a significant amount of research time.

Bao has developed a set of flourescent genetic tags for the beneficial Fusarium strains and another for the pathogenic strains. He will use these tags to determine where each strain resides in the plant.

Fravel asserts the collaboration is working well. "The labs have different strengths that complement each other," says Dr. Fravel. "Dr. Lazarovits' lab has done a lot of work in the molecular biology area. Our lab didn't have that level of expertise readily available."

Fravel continues, "ARS' strength is its extensive research in the biocontrol agents' mechanisms of action and the inoculum studies it's conducted."

"Most researchers have concentrated mainly on screening organisms for efficacy," says Lazarovits. "We need to do more on developing the tools needed to evaluate what happens to the organism in the environment we place it into."

Future Research

Using genetic tags, Fravel hopes to determine where the various Fusarium strains in one plant reside. "Researchers have worked on the supposition that beneficial Fusarium resides in the root," says Fravel. "Unfortunately, no one really looked."

Finding DNA fingerprints for all of the 350 Fusarium isolates found in the soil samples is a future goal, according to Fravel. This would enable researchers to quickly and accurately distinguish the good strains from the bad.

Last Updated: May 4, 2000