Moderate temperature fluctuations rapidly reduce the viability of Ralstonia solanacearum race 3, biovar 2, in infected geranium, tomato, and potato plants

Abstract

Most Ralstonia solanacearum strains are tropical plant pathogens, but race 3, biovar 2 (R3bv2), strains can cause bacterial wilt in temperate zones or tropical highlands where other strains cannot. R3bv2 is a quarantine pathogen in North America and Europe because of its potential to damage the potato industry in cooler climates. However, R3bv2 will not become established if it cannot survive temperate winters. Previous experiments showed that in water at 4°C, R3bv2 does not survive as long as native U.S. strains, but R3bv2 remains viable longer than U.S. strains in potato tubers at 4°C. To further investigate the effects of temperature on this high-concern pathogen, we assessed the ability of R3bv2 and a native U.S. strain to survive typical temperate winter temperature cycles of 2 days at 5°C followed by 2 days at -10°C. We measured pathogen survival in infected tomato and geranium plants, in infected potato tubers, and in sterile water. The population sizes of both strains declined rapidly under these conditions in all three plant hosts and in sterile water, and no culturable R. solanacearum cells were detected after five to seven temperature cycles in plant tissue. The fluctuations played a critical role in loss of bacterial viability, since at a constant temperature of -20°C, both strains could survive in infected geranium tissue for at least 6 months. These results suggest that even when sheltered in infected plant tissue, R3bv2 is unlikely to survive the temperature fluctuations typical of a northern temperate winter.

FIG. 1.

Ralstonia solanacearum R3bv2 had reduced virulence following long-term incubation in geranium tissue at −20°C. Young Bonny Best tomato plants were inoculated through a cut petiole with 250 to 350 CFU of R3bv2 strain UW551 either taken from water stock (closed triangles) or recovered from infected geranium tissue incubated at −20°C for 6 to 7 months (closed circles). Plants were rated daily on a disease index scale of 0 to 4, where 0 indicated healthy plants and 4 indicated 75 to 100% wilted plants. Each point represents the average disease index for four independent experiments, consisting of 16 plants per treatment; treatments differed by repeated-measures ANOVA (P < 0.001).

FIG. 2.

In infected host tissue, Ralstonia solanacearum populations declined rapidly following fluctuating temperature cycles of 48 h at 5°C and then 48 h at −10°C. Closed triangles represent the mean population sizes of R3bv2 strain UW551, and open squares represent endemic U.S. strain K60. Mean bacterial population sizes were measured by dilution plating: chopped stems of symptomatic R. solanacearum-inoculated geranium plants (A), chopped stems of wilting R. solanacearum-inoculated tomato plants (B), or cores of potato minitubers inoculated with ∼2 × 10⁹ CFU of the relevant R. solanacearum strain (C). Infected plant material was sampled after each 4-day temperature cycle. The experiment was repeated six times, with three technical replicates per biological replicate (A) or three times with three technical replicates per biological replicate (B, C). The dashed line represents the detection limit. Bars represent the standard errors of the means.

FIG. 3.

In sterile water, Ralstonia solanacearum populations declined following fluctuating temperature cycles of 48 h at 5°C and then 48 h at −10°C. Closed triangles represent the mean population sizes of R3bv2 strain UW551, and open squares represent endemic U.S. strain K60. Bacterial population size was determined after every cycle by dilution plating. The experiment was repeated three times, with three technical replicates per biological replicate. The dashed line represents the detection limit. Bars represent the standard errors of the means.