SR9092 - Studies on the Epidemiology of Erwinia Soft-Rot and Their Control in Ornamental Crops

Researcher:

Dr. Theo J. Blom, Dept. of Plant Ag., University of Guelph

Objectives:

1. To evaluate the risks associated with closed sub-irrigation systems in the dispersal of Erwinia spp.
   

2. To develop and/or adapt molecular tools to conduct ecological and epidemiological experiments.
   

3. To select and screen potential biological control agents (BCA) against E. carotovora subsp. Carotovora.
   

4. To explore the use of environmentally sound techniques to control Erwinia spp.

Expected Benefits:

1. Better understanding of the plant-pathogen complex and the development of grower friendly control strategies that will effectively manage this pathogen will increase the profitability of the industry and encourage more growers to adopt environmentally sound closed sub-irrigation systems.
   

2. The knowledge obtained from this research will help other sectors of the greenhouse industry as well, namely vegetable producers, where this plant pathogen also causes significant losses.
   

Summary of Research Results:

The number of E.c.c. bacteria found in commercial greenhouses using recirculating subirrigation systems varied between 0 and 800 cfu / mL depending on the crop with a mean of about 100 cfu /mL. Further studies showed that population densities of 100 cfu (bacterial cells)/mL of nutrient solution caused approximately a 10% disease on calla lilies.

The use of selective antibiotics (erythromycin) and a different carbon source (pectin) was used to detect Erwinia species with pectinolytic activity. These characteristics led us to believe that we are selectively isolating Erwinia carotovora. DNA samples were isolated from various known species/subspecies of Erwinia for use in polymerase chain reaction (PCR). Fragments specific to the 16S-23S ribosomal intergenic spacer region (ITS) were amplified via PCR using published primer sequences and visualized by running products on agarose gel. Preliminary results showed that there were sequence differences in the 16S-23S region between some Erwinia species and subspecies.

One experiment using Trichoderma hamatum as a biological control agent (applied 3 times) against E.c.c. did not prove to be very effective. The positive control treatment (plants artificially inoculated with E.c.c.) also resulted in low mortality.

Efforts were also placed on the isolation, selection, and purification of bacteriophages (= bacterial viruses). Liquid and solid samples were obtained from commercial operations in the Niagara Region. Samples were processed for isolation of bacteriophages. Molecular characterization of the phages with restriction endonuclease digestions (RFLP) indicated that two distinct types (Group 1 and 2) of bacteriophages had been isolated. The selected group 1 and group 2 phages were unable to produce plaques on non- E. carotovora subsp. carotovora hosts.

Phage titers were found to be stable in fertilizer solutions made with sterile reverse osmosis water, but not with non-sterile tap water. Phages did not survive in calla tuber pre-plant treatment solutions.

Biological control of E.c.c. with bacteriophages was assessed in a fertilizer solution, on the surface of plugs of calla tuber tissue in a laboratory and under greenhouse conditions. Selected bacteriophage isolates from groups 1 and 2 were assayed for their ability to reduce E.c.c. cfu density in calla fertilizer solution. Significant reductions of E.c.c. density were not achieved by any of the tested phage isolates. This appeared to be due to the presence of Fe-EDTA. In fertilizer solution without Fe-EDTA, 3 bacteriophages (Ercca3-3, Ercca4-1 and Ercca4-2) at a MOI of 100 (=ratio of bacteriophages and E.c.c. bacteria) significantly reduced cfu density of E.c.c. Only phage isolates from group 1 (Ercca1-3, Ercca2-1, Ercca3-3) exhibited significant (P = 0.05) levels of biological control activity on tuber plugs. Group 2 phage isolates, and a treatment consisting of a mixture of three group 1 and two group 2 phages, did not exhibit significant biological control activity.

In a greenhouse experiment with higher concentrations of E.c.c. (1 x 10⁵ cfu/mL), but with the same MOI (100:1) the results were very promising. Although the percent disease of the positive control (tubers with E.c.c. only) was higher than in a previous experiment E.c.c. (1 x 10³ cfu/mL), the percent diseased tubers decreased by at least 40% compared to that of the control. The mixture of the 4 bacteriophages (Ercca 1-2, Ercca 1-3, Ercca 2-5 and Ercca 4-2) applied 2 times was also effective in reducing the percentage of diseased tubers.

Greenhouse and laboratory studies showed that phosphorous played an important role in the maceration of calla tissue by E.c.c. In a greenhouse experiment, soft-rot incidence was significantly increased when superphosphate was added to the soil-less mix (51%) in comparison to regular soil-less mix (no superphosphate added) (31%). No effect on mortality was observed when phosphorous was added to the nutrient solution. In the laboratory, tubers sprayed with a solution of E.c.c. and mono potassium phosphate (KH₂PO₄) were macerated faster and in greater numbers than those sprayed with only E.c.c., KH₂PO₄ or water. Moreover, it was found that cell degrading (pectinolytic) enzyme activity of E.c.c was increased in the presence of phosphorous.