Detection of Latent Infections of Ralstonia solanacearum Race 3 Biovar 2 in Geranium.

Ralstonia solanacearum race 3 biovar 2 is a regulated quarantine pathogen that infects solanaceous hosts such as potato as well as geranium, where it causes either bacterial wilt (also known as Southern Wilt) or a symptomless latent infection. Geranium growers and government regulators need reliable detection methods to identify infected plant material before it is exported. We previously found that R. solanacearum-infected geranium plants can shed millions of bacteria in effluent water that flows from pots. We tested a nondestructive sampling method wherein effluent water from infected plants grown under commercial conditions was both dilution plated and filter concentrated for real-time polymerase chain reaction (PCR). Under field conditions in Guatemala, effluent shedding of infected geranium plants was highly variable. Comprehensive growth chamber studies confirmed that latently infected and mildly symptomatic geranium plants often but not invariably shed detectable numbers of bacteria in their effluent. At the peak of bacterial shedding, just under 90% of infected plants shed detectable bacteria whereas, at the lowest point, 44% shed detectable numbers of pathogen cells. Bacterial shedding peaked several weeks after inoculation regardless of whether plants were symptomatic or latently infected. Bacterial stem population sizes did not correlate with either effluent population sizes or disease index rating. Finally, we found that the effluent from geranium plants grown in volcanic rock scoria medium contains inhibitors that reduce the effectiveness of real-time PCR detection methods.

Behavior of Ralstonia solanacearum Race 3 Biovar 2 During Latent and Active Infection of Geranium.

ABSTRACT Southern wilt of geraniums (Pelargonium hortorum), caused by the soilborne bacterium Ralstonia solanacearum race 3 biovar 2 (R3bv2), has inflicted significant economic losses when geranium cuttings latently infected with this quarantine pest were imported into the United States. Little is known about the interaction between R. solanacearum and this ornamental host. Using UW551, a virulent R3bv2 geranium isolate from a Kenyan geranium, we characterized development of Southern wilt disease and R3bv2 latent infection on geranium plants. Following soil inoculation, between 12 and 26% of plants became latently infected, carrying average bacterial populations of 4.8 x 10(8) CFU/g of crown tissue in the absence of visible symptoms. Such latently infected plants shed an average of 1.3 x 105 CFU/ml in soil run-off water, suggesting a non-destructive means of testing pools of asymptomatic plants. Similarly, symptomatic plants shed 2 x 10(6) CFU/ml of run-off water. A few hundred R. solanacearum cells introduced directly into geranium stems resulted in death of almost all inoculated plants. However, no disease transmission was detected after contact between wounded leaves. Increasing temperatures to 28 degrees C for 2 weeks did not convert all latently infected plants to active disease, although disease development was temperature dependent. Holding plants at 4 degrees C for 48 h, a routine practice during geranium cutting shipment, did not increase frequency of latent infections. R. solanacearum cells were distributed unevenly in the stems and leaves of both symptomatic and latently infected plants, meaning that random leaf sampling is an unreliable testing method. UW551 also caused potato brown rot and bacterial wilt of tomato, surpassing race 1 strain K60 in virulence on tomato at the relatively cool temperature of 24 degrees C.

Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efflux pumps and contribute to bacterial wilt virulence.

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>10(7) CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds.

Using the Ralstonia solanacearum Tat secretome to identify bacterial wilt virulence factors.

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.