Dusit Athinuwat

Identification of an Operon, Pil-Chp, That Controls Twitching Motility and Virulence in Xylella fastidiosa

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierces disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

Xanthomonas axonopodis pv. glycines Soybean Cultivar Virulence Specificity Is Determined by avrBs3 Homolog avrXg1

Three races of Xanthomonas axonopodis pv. glycines were identified on pustule disease resistant and susceptible soybean cultivars based on virulence phenotype. For race 3, an avrBs3 homolog, avrXg1 was identified that conferred resistance expressed as a hypersensitive response on resistant cultivar Williams 82. Mutations in two predicted functional domains of avrXg1 resulted in gained virulence on Williams 82 and an increase in bacterial population number on susceptible cultivars. Expression of avrXg1 in race 1, that is predicted to confer a nonspecific HR, led to virulence on susceptible cultivars Spencer and PI 520733. Expression of avrXg1 in race 2, that is predicted of carrying avrBs3-like genes, resulted in gained virulence and fitness of pathogen on both resistant and susceptible cultivars. The results demonstrate multifunctions for avrXg1 dependent on pathogen and plant genetic backgrounds.

Characterization of the Xylella fastidiosa PD1671 Gene Encoding Degenerate c-di-GMP GGDEF/EAL Domains, and Its Role in the Development of Pierce’s Disease

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce’s disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.

The FTIR spectroscopy investigation of the cellular components of cassava after sensitization with plant growth promoting rhizobacteria, Bacillus subtilis CaSUT007

To evaluate the response of cassava stakes to plant growth promoting rhizobacteria, Bacillus subtilis CaSUT007, the changes in cellular compositions and phytohormone were investigated using the fourier transform infrared (FTIR) and high-performance liquid chromatography (HPLC) approach. The objective of this study was to test the hypothesis that CaSUT007 stimulates production of plant cellular components and phytohormone involved in metabolism and growth development mechanisms. Cassava stake treated with CaSUT007 or with sterile distilled water were germinated in sterile soil, after incubation for 28 days, CaSUT007 treated cassava stakes had more lateral root, longer roots, shoot length and greater biomass than the control which enhanced more than 1.3 fold of the cassavas phytohormone as indole-3-acetic acid content of non-treated control. We also focused on plant cellular composition and cassava stake tissues from the two treatments were harvested for FTIR analysis. FTIR analyses revealed that higher accumulated of lipid in response to the strain CaSUT007. The cassava stake treated with the beneficial bacteria B. subtilis strain CaSUT007 showed the higher content of the lipid content as (shown in the spectral regions of CH stretching and CH bending mode associated with cell membrane structure lipids) when compared with those of the cassava stake treated with distilled water. Our results initially demonstrated that CaSUT007 can enhance plant growth under greenhouse conditions by direct stimulation of plant lipid and phytohormone as indole-3-acetic acid production.

Foliar application of systemic acquired resistance (SAR) inducers for controlling grape anthracnose caused by Sphaceloma ampelinum de Bary in Thailand

Chitosan and benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) are active-elicitors that induce protection in grapevine against several diseases. In this study, treatment of grapevine with chitosan and BTH provided protection to anthracnose, caused by Sphaceloma ampelinum. Chitosan and BTH treatment also increased chitinase, s-1,3-glucanase and peroxidase activities levels in leaves over non-treated plants. Differential accumulation of these traits was more rapid and pronounced when chitosan and BTH treated plants were infected with S. ampelinum; this pattern indicating priming. The induced resistance by chitosan and BTH was also associated with increased production of salicylic acid (SA) in grapevine leaves, suggesting that SA-dependent signaling pathways are systemically triggered by these compounds. Apart from proteins with defense-related function, most of the proteins induced by chitosan and BTH were involved in defense mechanism, reflecting the strong direct positive effect that chitosan and BTH has on grapevine tolerance to anthracnose disease infection. Keywords: Anthracnose, grapevine, induce resistance, systemic acquired resistance (SAR) biochemical markers, Sphaceloma ampelinum African Journal of Biotechnology Vol. 12(33), pp. 5148-5156

The antagonistic activity of Trichoderma virens strain TvSUT10 against cassava stem rot in Thailand

In this current study, the beneficial filamentous fungi, Trichoderma virens , isolated from cassava field were investigated for antagonistic mode of action against Lasiodiplodia theobromae , the causal agent of cassava stem rot in Thailand. In vitro screening using the dual culture technique was undertaken to assess the potential of these Trichoderma isolates. Our results indicated that fifteen isolates of T. virens were collected from various areas of cassava field in Nakhon Ratchasima, Thailand. The T. virens isolate, TvSUT10, was the most effective isolate and inhibited L. theobromae mycelial growth by 84.12%, due to the antagonistic mechanism. Moreover, Trichoderma β-1,3-glucanase activity was determined, the result revealed that the highest activity was recorded in strain of T. virens TvSUT10 (25.7 U/ml). In addition, in the greenhouse experiment, the application of the TvSUT10 as a conidial suspension reduced the stem rot disease severity of cassava caused by 53%. The results indicated that the T. virens strain TvSUT10 has initial modes of action of biological control to protect cassava crop against L. theobromae infections in cassava. Key words : Trichoderma , cassava stem rot disease, growth inhibition, cassava, biocontrol.

Potential complications when developing gene deletion clones in Xylella fastidiosa

BackgroundThe Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. The Temecula 1 strain infects grapevines and induces Pierce′s disease, which causes symptoms such as scorching on leaves, cluster collapse, and eventual plant death. In order to understand the pathogenesis of X. fastidiosa, researchers routinely perform gene deletion studies and select mutants via antibiotic markers.MethodsSite-directed pilJ mutant of X. fastidiosa were generated and selected on antibiotic media. Mutant cultures were assessed by PCR to determine if they were composed of purely transformant cells or included mixtures of non-transformants cells. Then pure pilJ mutant and wildtype cells were mixed in PD2 medium and following incubation and exposure to kanamycin were assessed by PCR for presence of mutant and wildtype populations.ResultsWe have discovered that when creating clones of targeted mutants of X. fastidiosa Temecula 1 with selection on antibiotic plates, X. fastidiosa lacking the gene deletion often persist in association with targeted mutant cells. We believe this phenomenon is due to spontaneous antibiotic resistance and/or X. fastidiosa characteristically forming aggregates that can be comprised of transformed and non-transformed cells. A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells. After repeated transfer and storage the non-transformed cells became the dominant clone present.ConclusionsWe have discovered that special precautions are warranted when developing a targeted gene mutation in X. fastidiosa because colonies that arise following transformation and selection are often comprised of transformed and non-transformed cells. Following transfer and storage the cells can consist primarily of the non-transformed strain. As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

Hemin transported protein of Xanthomonas axonopodis pv. glycines functions on leaf colonization and virulence on soybean

Xanthomonas axonopodis pv. glycines (Xag) causes bacterial pustule disease on soybean. This bacterium is present worldwide around hot and humid growing regions such as Southeast Asia. To understand if the gene coding for hemin transport protein (hem) is involved in virulence of the pathogen in soybean, we generated a hem mutant in Xag by overlapping PCR mutagenesis. Disruption of hem significantly reduced the population size and the disease incidence when sprayed on soybean but not when injected directly to soybean. The hem mutant caused the hypersensitive response induction on tobacco as an Xag wildtype. Interestingly, the hem expression was also reduced when the Xag wildtype grow in planta. The hemin transporter protein involved in the production of extracellular polysaccharide, biofilm formation, motility and attachment but not for extracellular enzymes. This confirmed that epiphytic fitness of Xag strongly required hem functions. These results suggest that hem gene is essential for virulence of Xag on soybean during the infection process.

Efficiency of new plant growth promoting rhizobacteria on corn diseases control

The efficacy of two plant growth promoting rhizobacteria (PGPR) strains including Bacillus subtilis TU-Orga1 and Pseudomonas fluorescens TU-Orga2 obtained from rice rhizosphere against naturally occurring diseases as well as their capacity to improve crop yield of sweet corn cv. Insee2 was studied. TU-Orga1 was significantly greater in inhibition of Acidovorax avenae subsp. avenae (Aaa) and Erwinia chrysanthemi pv. zeae (Ecz), caused bacterial leaf streak and stalk rot of corn, respectively by antibiosis. Greenhouse experiments using TU-Orga1 and TU-Orga2 treatments increased highest salicylic acid accumulation in corn leaves with 7.85 and 6.98 mg g-1 fresh weight, respectively to protect Ecz infection. Each PGPR strain was single applied in the field through seed treatment and 3-foliar-spray-intervals at 14, 21, and 28 days after planting. Two PGPR treatments resulted in reduced severity of all diseases and increased yields when compared with the control treatment. There were differences among the treatments in that the highest level of disease suppression of bacterial stalk rot resulted with treatments TU-Orga1 (P = 0.05), whereas TU-Orga2 showed the highest level of disease suppression of bacterial leaf streak and sugarcane mosaic virus and provided significantly greater marketable yield increases than the other treatments. This illustrates the potential of these new biocontrol agents to suppress multiple diseases. They could become a component of an integrated program or an organic farming for corn disease management. Key words: Plant growth promoting rhizobacteria (PGPR), Acidovorax avenae subsp. avenae, Erwinia chrysanthemi pv. zeae, SCMV, systemic acquired resistant.