Chemotaxis Is Required for Virulence and Competitive Fitness of the Bacterial Wilt Pathogen Ralstonia solanacearum

Abstract

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.Ralstonia solanacearum, a soilborne gram-negative bacterium, causes bacterial wilt disease in many important crop plants, including potato, tobacco, tomato, banana, and peanut plants. The pathogen causes severe losses worldwide due to its wide geographic distribution and unusually broad host range, which spans more than 50 plant families. R. solanacearum is metabolically versatile, surviving and thriving in such diverse habitats as water, soil, and latently infected plants (23). The bacterium normally invades plant roots from the soil through wounds or natural openings, colonizes the intercellular space of the root cortex and vascular parenchyma, and eventually enters the xylem vessel and spreads up into the stem and leaves, where the pathogen cell density commonly surpasses 10 9 CFU/g of host tissue (33, 55). Affected plants suffer chlorosis, stunting, wilting, and usually die rapidly. Many factors contribute to bacterial ...

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“…3). Our data show that root exudates significantly affect microbial abundance in soil, as reported elsewhere [31]. Given that members of the Bacillus genus exhibit PGP activities [24,32], we used 13k/19k to co-inoculate the seeded substrates.…”

Bioassay-guided isolation of a novel chemoattractant for Ralstonia solanacearum in tomato root exudates

“…The semisolid agar plate method developed by Okon et al (1980) was modified as described by Yao and Allen (2006). R. solanacearum was grown overnight in CPG medium (Hendrick and Sequeira 1984) at 28 °C on a rotary shaker (90 rpm).…”

“…R. solanacearum was grown overnight in CPG medium (Hendrick and Sequeira 1984) at 28 °C on a rotary shaker (90 rpm). The cultures were centrifuged at 4700×g for 10 min, and the pelleted bacteria were washed twice with sterile chemotaxis buffer (Yao and Allen 2006). The bacteria were then resuspended in the same buffer to an OD 600 of 2.0.…”

“…Additionally, root exudates from tomato (i.e., a host plant) produced stronger positive chemotactic activity than root exudates from rice (i.e., a nonhost plant). The chemoattractants in the tomato root exudates were determined to be heat stable and unaffected by proteinase K (Yao and Allen 2006). A subsequent analysis of the amino acids, organic acids, and sugars indicated that several amino acids, including aspartic acid, proline, and glutamine, are potentially strong chemoattractants.…”

Comparative structural bioinformatics analysis of Bacillus amyloliquefaciens chemotaxis proteins within Bacillus subtilis group

“…Thus, this complex system of chemotaxis is absolutely important for survival of microorganisms in their habitats. For example, chemotaxis has been shown to be critical for plant colonization of both pathogenic (Yao and Allen 2006) and symbiotic plant-associated microbes (Van de Broek et al 1998). Plant colonization is a multifaceted process which requires bacteria to respond to the complex chemical environment of the plant rhizosphere, in particular to detect and resist plant defence systems, as well as the ability to sense attractants and initiate growth on the plant surface.…”