First report in South Korea of a bacterial leaf blight of oats caused by Pseudomonas salomonii.

In March 2020, approximately 20% of leaves in a commercial oat field growing Avena sativa (cv. Samhan) in Jeongeup, Korea (35.3859°, 126.5607°), displayed leaf blight in the seedling stage of development. The lesions observed in 2020 were of a yellow discoloration that spread from the leaf tip downward with minimal brown delineation (Fig. S1A). These symptoms differed from the haloes delineated at the edges by extensive brown necrosis caused by Pseudomonas coronofaciens (Kim 2020a; Fig. S1C). To isolate the causal agent, 3-cm-long pieces of symptomatic leaves from ten different oat plants were disinfected by submersion in 70% ethanol for 5 min followed by immersion in 1% sodium hypochlorite for 5 min and rinsing extensively with sterile distilled water. The air-dried segments were transferred intact to nutrient agar, and only one colony type (yellow-colored, wet, with a shiny convex surface) was observed. After single colony isolation, three isolates from different 2020 field-grown diseased leaves, termed 2007, 2009, and 2011, were selected at random. The isolates produced fluorescent siderophores on King's medium B and triggered a hypersensitive response (HR) when infiltrated into tobacco (cv. Xanthi) leaves (Fig. S1D). Multilocus sequence typing analysis with four housekeeping genes was used for taxonomic identification of these isolates (Maiden et al. 1998). The 16S rRNA sequences were amplified with the 27F/1492R universal primers (Weisburg et al. 1991). The primers for three housekeeping genes were designed using genome sequence of P. coronaficiens X-1 causing halo blight disease in Korea (NZ_CP050260.1, Kim, 2020b). The products obtained had sizes of 640 bp for gltA (using primers F: 5'-CCT GGT AGC CAA GAT GCC GAC-3'; R: 5'-CAA AGA TCA CGG TGA ACA TGC TGG-3'), 710 bp for gyrB (with primers F: 5'-TCG GCA GCC GAG GTC ATC ATG AC-3'; R: 5'-TTG TCT TTG GTC TGC GAG CTG AA-3'), and 870 bp for rpoD ( with primers F: 5'-GTG AAG GCG AAA TCG AAA TCG-3'; R: 5'-CCG ATG TTG CCT TCC TGG ATC AG-3'). The concatenated sequences (2,353 bp/2,376 bp) had 99% identity with the gene sequences from P. salomonii type strain (AY091528.1, NZ_FNOX01000003, LC486864.1, LC486849.1), but lesser identity (90%) with P. coronafaciens (Fig. S2). Pathogenicity of the Korean isolates was confirmed by fulfilling Koch's postulates using leaves of 2 week-old greenhouse-grown 'Samhan' seedlings. Plants (n=50) were sprayed with 108 cfu/ml bacterial suspensions in water or with sterile water as controls. The plants were incubated for a week at 23 °C in 100% relative humidity under a 10 h light/14 h dark photoperiod. Five days after bacterial inoculation, yellow discoloration appeared at the leaf tips which progressed downward with time (Fig. S1B). Three bacterial isolates extracted from yellowed, inoculated leaves had 16S rRNA gene sequences identical to that of P. salomonii Korean isolates, 2007, 2009 and 2011, and they caused the anticipated symptoms when inoculated into oat leaves. These findings indicate that P. salomonii should be added to the potential pathogens of oats grown in Korea. Understanding whether spring weather conditions (warmth and humidity) boost this oat disease will help devise disease alert systems for farmers (Anderson 2004; Chakraborty 2005).

Biocontrol Efficacy of Formulated Pseudomonas chlororaphis O6 against Plant Diseases and Root-Knot Nematodes.

Commercial biocontrol of microbial plant diseases and plant pests, such as nematodes, requires field-effective formulations. The isolate Pseudomonas chlororaphis O6 is a Gram-negative bacterium that controls microbial plant pathogens both directly and indirectly. This bacterium also has nematocidal activity. In this study, we report on the efficacy of a wettable powder-type formulation of P. chlororaphis O6. Culturable bacteria in the formulated product were retained at above 1 × 108 colony forming units/g after storage of the powder at 25 °C for six months. Foliar application of the diluted formulated product controlled leaf blight and gray mold in tomato. The product also displayed preventative and curative controls for root-knot nematode (Meloidogyne spp.) in tomato. Under laboratory conditions and for commercially grown melon, the control was at levels comparable to that of a standard commercial chemical nematicide. The results indicated that the wettable powder formulation product of P. chlororaphis O6 can be used for control of plant microbial pathogens and root-knot nematodes.

Biological Control Potential of Bacillus amyloliquefaciens KB3 Isolated from the Feces of Allomyrina dichotoma Larvae.

Most biocontrol agents for plant diseases have been isolated from sources such as soils and plants. As an alternative source, we examined the feces of tertiary larvae of the herbivorous rhino beetle, Allomyrina dichotoma for presence of biocontrol-active microbes. The initial screen was performed to detect antifungal activity against two common fungal plant pathogens. The strain with strongest antifungal activity was identified as Bacillus amyloliquefaciens KB3. The inhibitory activity of this strain correlated with lipopeptide productions, including iturin A and surfactin. Production of these surfactants in the KB3 isolate varied with the culture phase and growth medium used. In planta biocontrol activities of cell-free culture filtrates of KB3 were similar to those of the commercial biocontrol agent, B. subtilis QST-713. These results support the presence of microbes with the potential to inhibit fungal growth, such as plant pathogens, in diverse ecological niches.

The dctA gene of Pseudomonas chlororaphis O6 is under RpoN control and is required for effective root colonization and induction of systemic resistance.

Transcription from the dctA gene, which encodes an organic acid transporter in the root-colonizing bacterium Pseudomonas chlororaphis O6, is under complex regulatory control. Promoter sequence analysis revealed an RpoN binding site. The regulation of transcript accumulation by the level of ammonium ions in the growth medium confirmed RpoN regulation, even in the presence of glucose. A dctA mutant colonized tobacco roots to a lesser extent than the wild-type mutant during early seedling development. Colonization by the dctA mutant, as compared to the wild type, also reduced the level of systemically induced resistance against the soft rot pathogen Erwinia carotovora SCC1. We ascribe this reduced colonization to the inability of the mutant to utilize certain organic acid components in the root exudates. The dctA mutant failed to grow on succinate and fumarate, and showed reduced growth on malate. All altered properties of the mutant were complemented by the full-length dctA gene. We propose that organic acids in root exudates may provide important nutrient sources for the beneficial root-colonizing pseudomonad.

Transcriptional regulation and mutational analysis of a dctA gene encoding an organic acid transporter protein from Pseudomonas chlororaphis O6.

A dctA gene encoding a protein with identity to a C(4)-dicarboxylic acid/H(+) symporter was cloned from a beneficial root colonizer, Pseudomonas chlororaphis O6 (PcO6). Expression of the dctA gene was induced in minimal medium by several organic acids and was repressed by glucose. Highest expression was observed in early-logarithmic (log) cells grown on fumarate, acetate or succinate with decline as cells approached late-log growth phase. The dctA transcript accumulated weakly when cells were grown on malate, but strong expression was observed with benzoate. Expression of the dctA transcript was repressed in early-log cells upon addition of glucose to fumarate, but was detected as the cell culture aged. A dctA-deficient mutant of PcO6, constructed by marker exchange mutagenesis, did not grow on minimal medium containing succinate, benzoate, acetate or fumarate and growth on malate was delayed. The dctA mutant and wild-type grew equally on citrate, glucose, fructose, sucrose or inositol. We conclude that the transporter protein encoded by dctA is essential for utilization of certain organic acids and its expression is controlled by the availability of sugars.