Fermentation: An Unreliable Seed Treatment for Bacterial Fruit Blotch of Watermelon.

Acidovorax citrulli is a seedborne pathogen that causes bacterial fruit blotch (BFB), a global threat to watermelon production. Treating watermelon seeds to eliminate A. citrulli is a critical component of BFB management, and several strategies have been evaluated to mitigate the impact of the disease. In China, watermelon seed producers routinely incubate seeds in watermelon juice (fermentation) to reduce the risk of seed infection by A. citrulli and seedling transmission of BFB. However, there has been limited effort to evaluate the efficacy of fermentation in mitigating A. citrulli seed infection. The current study showed that fermented watermelon fruit juice could inhibit A. citrulli population growth and demonstrated that the low pH conditions, not the temperature dynamic, generated during fermentation might play a major role in A. citrulli growth inhibition and could induce the viable but nonculturable (VBNC) state in A. citrulli. We developed an effective method that was based on propidium monoazide PCR to detect viable A. citrulli cells under low pH conditions or in fermented watermelon fruit juice. We also provided evidence that VBNC A. citrulli cells induced by fermented watermelon fruit juice could not be resuscitated and did not retain their virulence on watermelon seedlings. However, VBNC A. citrulli cells could be resuscitated in Luria-Bertani medium. Based on these observations, we conclude that fermentation in watermelon fruit juice may not be an effective seed treatment for BFB because it may increase the seed infection by A. citrulli.

Pectobacterium atrosepticum KDPG aldolase, Eda, participates in the Entner-Doudoroff pathway and independently inhibits expression of virulence determinants.

Pectobacterium carotovorum has an incomplete Entner-Doudoroff (ED) pathway, including enzyme 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda) but lacking phosphogluconate dehydratase (Edd), while P. atrosepticum (Pba) has a complete pathway. To understand the role of the ED pathway in Pectobacterium infection, mutants of these two key enzymes, Δeda and Δedd, were constructed in Pba SCRI1039. Δeda exhibited significant decreased virulence on potato tubers and colonization in planta and was greatly attenuated in pectinase activity and the ability to use pectin breakdown products, including polygalacturonic acid (PGA) and galacturonic acid. These reduced phenotypes were restored following complementation with an external vector expressing eda. Quantitative reverse transcription PCR analysis revealed that expression of the pectinase genes pelA, pelC, pehN, pelW, and pmeB in Δeda cultured in pyruvate, with or without PGA, was significantly reduced compared to the wild type, while genes for virulence regulators (kdgR, hexR, hexA, and rsmA) remained unchanged. However, Δedd showed similar phenotypes to the wild type. To our knowledge, this is the first demonstration that disruption of eda has a feedback effect on inhibiting pectin degradation and that Eda is involved in building the arsenal of pectinases needed during infection by Pectobacterium.

The Ribosomal Protein RplY Is Required for Pectobacterium carotovorum Virulence and Is Induced by Zantedeschia elliotiana Extract.

Pectobacterium carotovorum subsp. carotovorum strain PccS1, a bacterial pathogen causing soft rot disease of Zantedeschia elliotiana (colored calla), was investigated for virulence genes induced by the host plant. Using a promoter-trap transposon (mariner), we obtained 500 transposon mutants showing kanamycin resistance dependent on extract of Z. elliotiana. One of these mutants, PM86, exhibited attenuated virulence on both Z. elliotiana and Brassica rapa subsp. pekinensis. The growth of PM86 was also reduced in minimal medium (MM), and the reduction was restored by adding plant extract to the MM. The gene containing the insertion site was identified as rplY. The deletion mutant ΔrplY, exhibited reduced virulence, motility and plant cell wall-degrading enzyme production but not biofilm formation. Analysis of gene expression and reporter fusions revealed that the rplY gene in PccS1 is up-regulated at both the transcriptional and the translational levels in the presence of plant extract. Our results suggest that rplY is induced by Z. elliotiana extract and is crucial for virulence in P. carotovorum subsp. carotovorum.

AsnB, regulated by diffusible signal factor and global regulator Clp, is involved in aspartate metabolism, resistance to oxidative stress and virulence in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak in rice, which is a destructive disease worldwide. Xoc virulence factors are regulated by diffusible signal factor (DSF) and the global regulator Clp. In this study, we have demonstrated that asnB (XOC_3054), encoding an asparagine synthetase, is a novel virulence-related gene regulated by both DSF and Clp in Xoc. A sequence analysis revealed that AsnB is highly conserved in Xanthomonas. An asnB mutation in Xoc dramatically impaired pathogen virulence and growth rate in host rice, but did not affect the ability to trigger the hypersensitive response in nonhost (plant) tobacco. Compared with the wild-type strain, the asnB deletion mutant was unable to grow in basic MMX (-) medium (a minimal medium without ammonium sulphate as the nitrogen source) with or without 10 tested nitrogen sources, except asparagine. The disruption of asnB impaired pathogen resistance to oxidative stress and reduced the transcriptional expression of oxyR, katA and katG, which encode three important proteins responsible for hydrogen peroxide (H(2)O(2)) sensing and detoxification in Xanthomonas in the presence of H(2)O(2), and nine important known Xoc virulence-related genes in plant cell-mimicking medium. Furthermore, the asnB mutation did not affect extracellular protease activity, extracellular polysaccharide production, motility or chemotaxis. Taken together, our results demonstrate the role of asnB in Xanthomonas for the first time.

Zinc uptake regulator (zur) gene involved in zinc homeostasis and virulence of Xanthomonas oryzae pv. oryzae in rice.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. In order to understand the gene of zinc uptake regulator (zur) involved in virulence of the pathogen in rice, we generated a mutant OSZRM by homologous suicide plasmid integration. The mutant failed to grow in NYGB medium supplemented with Zn(2+) or Fe(3+) at a concentration of 500 muM or 6 mM, whereas the wild-type strain grew well at the same conditions. The zur mutant was hypersensitive to hydrogen peroxide and exhibited reduction catalase activity and the production of extracellular polysaccharide (EPS). Interestingly, the mutant showed a reduction in virulence on rice but still kept triggering hypersensitive response (HR) in tobacco. When the mutant was complemented with the zur gene, the response was recovered to wild-type. These results suggested that zur gene is a functional member of the Zur regulator family that controls zinc and iron homeostasis, oxidative stress, and EPS production, which is necessary for virulence in X. oryzae pv. oryzae.