Fluorescent Pigment Production Research Articles

Rarity of Pseudomonas agarici on Edible Mushrooms Associated with Susceptibility to Biological Competition.

Taxonomically diverse Pseudomonas species induce bacterial blotch of edible mushrooms around the world. Pseudomonas tolaasii, [Pseudomonas gingeri], and Pseudomonas agarici are dominant mycopathogenic pseudomonads in mushroom production farms. In this study, among 216 mycopathogenic bacterial strains isolated from edible mushrooms in Iran, 96 strains were identified as Pseudomonas spp., while only three strains were preliminarily identified as P. agarici. Multilocus sequence analysis showed that only one strain (FH2) authentically belonged to P. agarici, while the other two strains either belonged to [P. gingeri] or represented a unique phylogenetic clade. The three strains also differed from each other in phenotypic characteristics e.g., production of fluorescent pigment and the reaction to tolaasin produced by P. tolaasii. Pathogenicity assays under controlled environment showed that the symptoms induced by authentic P. agarici were far less severe than those caused by the predominant species P. tolaasii. Furthermore, co-inoculation of P. agarici with three bacterial pathogens that are prevalent in Iran on mushroom caps i.e., P. tolaasii, Ewingella americana and Mycetocola sp. resulted in the development of combined symptoms representing characteristics of both pathogens. Antibiosis assay showed that tolaasin-producing strains of P. tolaasii could inhibit the growth of P. agarici, while tolaasin-negative strains of the same species were unable to do so. This led us to the hypothesis that the inhibitory effect of P. tolaasii on P. agarici is driven by tolaasin production in the former species. This inhibitory effect also associated with the rarity of P. agarici in natural conditions.

Morpho-physiological characterization of bacterial grain rot in rice seed from Gowa Regency, South Sulawesi

Bacterial Grain Rot caused by Burkholderia glumae is an important disease of rice. In South Sulawesi, this seed-borne pathogen has been detected in several places. There were some reports from the farmers regarding rice yield loss due to this disease. This study aimed to isolate and characterize morpho-physiologically bacterium causing grain rot of rice in the main rice-producing areas of Gowa Regency, South Sulawesi. Samples were obtained from 10 districts. Several grains of rice were isolated and grown in King’s B medium. Morphological characteristics were determined by the color, form, edge, and elevation of colonies. KOH solubility, catalase test, anaerobic growth, toxoflavin production assay, production of fluorescent pigment, and colony color on YDC medium were performed to determine physiological characteristics. 1 isolate from each area was found which has similar morphological traits to B. glumae. Colonies that were yellow, yellowish white, creamy white, and grayish white, with a circular form, entire edge, and convex elevation. The result of physiological characterization showed that all isolates tested positive for KOH solubility and catalase test as well. All isolates showed negative reactions for anaerobic growth, production of fluorescent pigment, and colony color on YDC medium. 5 isolates were able to produce yellow pigment (toxoflavin), so it can be concluded that all of the isolates that have been characterized morpho-physiologically are presumably confirmed as B. glumae.

Biocontrol adeptness of bacterial endophytes antagonistic to Colletotrichum spp. causing Colletotrichum leaf disease in rubber (Hevea brasiliensis) and harnessing its plant growth-promoting traits

Colletotrichum Leaf Disease (CLD) is one of the major leaf diseases which significantly affect growth and yield of natural rubber (NR). Development of eco-friendly disease management strategies is the need of the hour as various commercial fungicides are banned due to their detrimental effect on environment. The present study characterized antagonistic activities and plant growth promoting properties of five bacterial endophytes, isolated from rubber leaves and roots, against Colletotrichum acutatum and C. gloeosporioides, causing CLD in rubber. In the dual culture test, Bacillus sp. and Stenotrophomonas sp. showed 62 and 60 per cent inhibition of radial growth (PIRG). With the culture filtrate, Stenotrophomonas sp. (RLR1), Bacillus sp. (RRF1), B. subtilis (RLF1), Ochrobactrum anthropi (RLF3), and Burkholderia lata (RLF15) recorded suppression of growth up to 100%. These endophytic bacterial strains were identified based on biochemical characteristics and through 16S rRNA gene sequencing. The isolates were also tested for twenty five plant growth promoting traits. All five isolates responded positively for cellulase production, siderophore production, citrate utilization, and oxidase production. However, all five strains responded negatively to chitinase, urease, phosphate solubilisation, hydrogen sulphide production, and fluorescent pigment production. These results confirmed that the five identified bacterial isolates possessing growth-promoting characteristics have the potential to be used as biocontrol agents against Colletotrichum spp., a major devastating pathogen of rubber.

Bacterial community associated with canker disease from sweet cherry orchards of central valley of Chile presents high resistance to copper

The Chilean sweet cherry (Prunus avium (L.) L.) industry became the leading exporter worldwide. The bacterial canker is the most significant disease causing major economic losses. Pseudomonas syringae pv. syringae (Pss) is the only related pathovar recognized in Chile and it is mainly controlled with Cu-based antimicrobial compounds (CBAC). Soil contamination and the decreasing efficacy of CBACs by the emergence of Cu-resistant bacterial strains threaten the long- term sustainability of sweet cherry production. This study aimed at characterizing the bacterial community associated with canker infection injuries by assessing Cu resistance in sweet cherry orchards in the O’Higgins Region of Chile. Eighty bacteria isolates were obtained from tissue that presented bacterial canker symptoms from cherry trees. We assessed the production of fluorescent pigments in Fe-deficient media, presence of the housekeeping genes rpoD and cts, and presence of syringomycin-producing genes syrb and syrD. Their pathogenic ability was evaluated on immature sweet cherry fruits and Cu resistance was determined as the minimum inhibitory concentration (MIC) using CuSO4. Only five isolates were Cu-susceptible (MIC < 0.8 mM), while 75 isolates exhibited different levels of Cu resistance (MIC > 0.8 mM). At least one gene of the copABCD operon/or and its regulatory genes were detected in 17 isolates, suggesting that most isolates likely have different mechanisms of Cu tolerance. Six isolates were identified as Pss, presenting different degrees of Cu resistance, but all presented at least one feature of the copABCD operon. A bacterial community that presents high Cu-resistance, probably under control of diverse genetic mechanisms, decreases the efficacy of CBAC.

Identification and Characterization of Antagonistic Bacteria from Coffee Plant Litter

Nowadays, the use of antagonistic bacteria is widely applied in agriculture and used as biological control. A previous study which is yet to be identified based on physiological and biochemical characterizations discovered these organisms present in coffee plant litter could control fusarium wilt disease. Therefore, this study is a follow-up analysis that aims to identify coffee litter antagonistic bacteria with the ability to control fusarium wilt disease. The investigation techniques used were biochemical tests comprising of endospore staining, yellow colony growth test on media, bacterial purification and screening, macroscopic and microscopic characterization, Gram, the solubility of 3% KOH, fermentative oxidative, fluorescent pigment production, catalase, hypersensitivity, nitrogen-fixing, and phosphate solvent test. The results showed that antagonistic bacteria, including &lt;em&gt;Bacillus mycoides, Clostridium, Pseudomonas, Erwinia, &lt;/em&gt;and &lt;em&gt;Alcaligenes&lt;/em&gt; sp. were identified based on their character traits. This study represents new information on the bacteria types from coffee litter to control pathogenic fungi as an environmentally friendly biological control agent.

The diversity of Pseudomonas species isolated from fish farms in Turkey

The prevalence of Pseudomonas species in freshwater fish and water from aquaculture farms in Turkey was determined on a monthly basis, in the period covering 2013–2017 and two seasons of 2018. The farms included in the study were located in six different regions of Turkey. A total of 90 Pseudomonas strains were isolated from water and diseased and healthy fish, and were classified under 20 species. The phenotypic characterization of the strains was based on oxidase and catalase activities, haemolysis, tolerance to temperature and sodium chloride, the production of fluorescent pigments and antibiotic resistance spectra. The phylogenetic identification of the 90 isolates was performed by a 4-gene multilocus sequence analysis, based on the partial sequencing of the 16S rRNA, gyrB, rpoB and rpoD genes. This is the first report on the isolation of several Pseudomonas species, namely, P. brenneri, P. defensor, P. haemolytica, P. lactis, P. lundensis, P. lurida, P. mandelii, P. meridiana, P. migulae, P. proteolytica, P. simiae, and P. weihenstephanensis, from freshwater salmonid fish. Two of these species, P. haemolytica and P. lactis have been isolated for the first time from fish farms, an environment quite different from their original isolation source, raw milk. Furthermore, seven putative new Pseudomonas species were isolated from water and farmed rainbow trout. During the 2013 to 2018 period, several Pseudomonas species were detected to have spread from the Aegean and Central Anatolia regions to the Eastern Anatolia and Black Sea regions.

CHARACTERIZATION OF BACTERIAL ISOLATES FOR SUSTAINABLE RICE BLAST CONTROL

ABSTRACT Rice blast (Magnaporthe oryzae) limits rice (Oryza sativa) grain yields worldwide. The objective of this investigation was to morphologically, biochemically, and molecularly characterize six bacterial isolates, BRM 32109, BRM 32110, BRM 32111, BRM 32112, BRM 32113, and BRM 32114, and to determine their potential as antagonists to M. oryzae. Morphological characterization was based on colony formation and color, Gram staining, and fluorescent pigment production. Biochemical studies were based on cellulase, chitinase, phosphatase, indoleacetic acid, and siderophore production, as well as biofilm formation. The molecular identification used specific primers for PCR amplification of the 16S rRNA region, followed by sequencing. The antagonism studies involved three experiments, which had randomized designs. Two of them were conducted in laboratory conditions, pairing bacterial colonies and M. oryzae, using bacterial filtrates, and the third was conducted in greenhouse conditions. BRM 32111 and BRM 32112 were identified as Pseudomonas sp., BRM 32113 as Burkholderia sp., BRM 32114 as Serratia sp., and BRM 32110 and BRM 32109 as Bacillus spp. BRM 32112, BRM 32111, and BRM 32113 inhibited the colony of M. oryzae by 68%, 65%, and 48%, respectively. The bacterial suspensions of the BRM 32111, BRM 32112, and BRM 3212 filtrates suppressed leaf blast by 81.0, 79.2, and 66.3%, respectively. BRM 32111 and BRM 32112 were determined to be antagonists of M. oryzae and were found to solubilize phosphate, produce siderophores and cellulose, form biofilms, and suppress leaf blast. These isolates should be further investigated as potential biological control agents for leaf blast control.

Quorum Sensing Inhibitors against Chromobacterium violaceum CV026 Derived from an Actinomycete Metabolite Library.

Quorum sensing (QS) is a microbial signaling system that regulates the expression of many virulence genes. Herein, we studied five compounds-No. 1: (E)-2-methyl-3- (4-nitro-phenyl)-acrylaldehyde; No. 29-2: pimprinine [5-(1H-indol-3-yl)-2-methyloxazole]; No. 48: (2E,4E)-2-methyl-5-phenyl-2,4-pentadienoic acid; No. 74: (3E,5E)-5-methyl-6-(4-nitrophenyl)-hexa-3,5-dien-2-ol; and No. 130: methyphenazine-1-carboxylate-derived from an actinomycete metabolite library. These compounds were confirmed to be QS inhibitors that reduced violacein production in Chromobacterium violaceum CV026. Additionally, compounds No. 1, No. 74, and No. 130 significantly reduced fluorescent pigment production in Pseudomonas aeruginosa ATCC 27853.

Non rhizobial endophytic bacteria from chickpea (Cicer arietinum L.) tissues and their antagonistic traits

Bacteria that colonize plant tissues other than rhizobia and are beneficial for plant growth referred to non rhizobial plant growth-promoting endophytic bacteria (PGPEB). This study was designed to assay the biocontrol activity of plant growth promoting endophytic bacterial isolates those found positive for P. solubilization, ACC deaminase, Indole acetic acid and Gibberelic acid production. These bacterial isolates were obtained from chickpea (Cicer arietinum L.) tissues (roots and nodules). In a previous study a total of 263 non rhizobial endophytic bacterial isolates were isolated. Out of 263 isolates, 64.5% and 34.5% were Gram positive and negative, respectively. Further for biochemical characterization, catalase, oxidase, citrate utilization, nitrate reduction, methyl red and Voges Proskauer’s tests, were performed. On the basis of P solubilization, ACC deaminase, Indole acetic acid and Gibberelic acid production 75 potential isolates were selected and screened for their biocontrol activity viz. (production of cell wall degrading enzymes, production of HCN and fluorescent pigment). Out of 75 isolates, only 29 isolates produced cellulase, 64 isolates were able to produce protease and 28 were positive for both cellulose and protease. Of 75 endophytic isolates 12 isolates (7 from root tissue and 5 from nodules tissue, respectively) were positive for HCN production and 16 isolates were found to be fluorescent pigment producer under µv ligh. As chemical fertilizers and pesticides have detrimental effects on the environment. So these bacterial endophytic isolates will be used not only as a biofertilizer because of their plant growth promotional activities but also used as an alternative of synthetic chemicals for control of several plant diseases.

Pseudomonas hutmensis sp. nov., a New Fluorescent Member of Pseudomonas putida Group.

A rod-shaped, Gram-negative and aerobic bacterium, strain XWS2, was isolated from rhizosphere soil of a camphor tree in Hubei University of Chinese Medicine Huangjiahu Campus, Wuhan, China. Cells grew at 4-37°C (optimum 28°C), pH 5.0-9.0 (optimum 7.0) and with 0-5% NaCl (optimum 1%). Colonies growing on tryptone soybean agar are round, beige in color and approximately 2mm in diameter after 24h incubation at 28°C. Pellicle formation during liquid culture and strong fluorescent pigment production on King's B medium are typical features of strain XWS2. The genome of XWS2 is 6,170,117bp, containing 5682 predicted genes and 4770 genes are functionally annotated. Phylogenetic analysis based on 16S rRNA sequence showed that strain XWS2 formed an independent branch within the Pseudomonas putida group, with P. putida NBRC 14164T (99.86% similarity) and P. alkylphenolica KL28T (99.36% similarity) as the most closely related type strains. Genome sequence analysis based on average nucleotide identity and digital DNA-DNA hybridization are below the threshold values for species delineation. Phenotypic characteristics, physiological and biochemical tests also supported the strain represents a separate novel species within the Pseudomonas genus. The name Pseudomonas hutmensis sp. nov. is proposed, with type strain XWS2T (= CCTCC AB 2018189 T = KACC 19898T).

Characterization of Ralstonia solanacearum (Smith) Race1, Causing Bacterial Wilt of Brinjal

Ralstonia solanacearum (Smith) Yabuuchi et al. is one of the most severe pathogen of Solanaceous crops with a wide host range. Ralstonia solanacearum race 1 is responsible for causing bacterial wilt in brinjal. The pathogenic strains were isolated from wilted brinjal plants and isolated on triphenyl tetrazolium chloride (TTC) medium. The strains were confirmed by proving pathogenicity on brinjal plants. Phenotypic characterization revealed that out of five media tested TTC medium was an excellent source for growth and extracellular polysaccaride production (EPS) of R. solanacearum. Upon biochemical analysis the R. solanacearum strains showed positive reaction for catalase, KOH, citrate utilization and acid production while negative results were observed for Gram staining, starch hydrolysis and fluorescent pigment production. The biovar of the strain was identified by utilization of disaccharides (cellobiose, maltose and lactose) and sugar alcohols (dulcitol, mannitol and sorbitol). The strains of R. solanacearum were confirmed to belong to Race 1 based on symptom production on different hosts and hypersensitive response on tobacco plant. Therefore, it is confirmed that R. solanacearum race 1 causing bacterial wilt of brinjal belongs to biovar III. PCR amplification of ITS region of the strains of R. solanacearum was done and sequenced [MH481663 (ERY1) and MH481664 (VBN1)].

Exploitation of endophytic Pseudomonas sp. for plant growth promotion and colonization in rice

The present investigation was carried out to exploit bacterial endophytes associated with root and leaf tissue of rice plant for plant growth promotion (PGP) and colonization study in vitro. Total 10 endophytic bacterial isolates (Pseudomonas sp.) were evaluate for PGP traits like P solubilization, production of Indole acetic acid (IAA), siderophore, ACC deaminase, protease, cellulase, fluorescent pigment, urease and denitrification activity. Out of 10 endophytic bacteria 30 %, 60 %, 20 %, 70 %, 10 % and 10 % were positive for siderophore, protease, cellulase, fluorescent pigment, urease and denitrification respectively. Maximum IAA production was recorded with isolate LRBLE7 (18.8 ?gml-1) followed by LRBRE4 (16.0 ?gml-1) and maximum P-solubilization was recorded with isolate LRBRE4 (5.8 mg 100 ml-1) followed by LRBLE7 (4.4 mg 100 ml-1). ACC deaminase production was recorded with isolate LRBLE6 (O.D=0.352 nm) followed by LRBRE5 (O.D=0.324nm). Three potential isolates (LRBRE4, LRBRE6 and LRBLE7) were selected on the basis of multiple PGP traits and were subjected to colonization study of rice seedling in vitro. Potential bacterial isolates can be exploited for improving growth and productivity in rice under sustainable management system.

PM 7/128 (1) Xanthomonas axonopodis pv. allii

Specific scopeThis Standard describes a diagnostic protocol for Xanthomonas axonopodis pv. allii.This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2016‐09.

Detection of antimicrobial traits in fluorescent pseudomonads and molecular characterization of an antibiotic pyoluteorin.

Thirty isolates of fluorescent pseudomonads were obtained from rhizosphere of different crops in Raichur, India. The fluorescent pseudomonad strains were characterized in vitro for biochemical traits, antimicrobial traits, and pyoluteorin antibiotic production. All the isolates that showed fluorescent pigment production under UV light were rod shaped, Gram negative, positive for oxidase, catalase and citrate utilization tests, and negative for indole test. Out of 30 isolates, 07 isolates were positive for HCN production, 15 isolates were positive for H2S production, and all the isolates were positive for siderophore production. Among all the isolates, RFP-22 showed the maximum percent inhibition of mycelium (46.66 %) of Rhizoctonia solani, the pathogen, and the remaining isolates showed the moderate to least inhibition of mycelium growth of R. solani. The 16S rRNA analysis confirmed that the antibiotic positive isolates belonged to genus Pseudomonas. The amplification of 779 bp region in isolates RFP- 4 and RFP-19 corresponded to pyoluteorin antibiotic-coding pltB gene. Further characterization of pyoluteorin antibiotic through TLC and TOF–MS analysis confirmed the presence of pyoluteorin at 274.26 (g/mol) peak and 2.10 min retention time. Biochemical and molecular analyses confirmed the antagonism of Pseudomonas and isolate through pyoluteorin production.

Isolation and characterization of Pseudomonas syringae pv. syringae which induce leaf spot on walnut

In 2012, a leaf spot disease on walnut seedlings was observed in Hamedan province of Iran. The spots were necrotic with yellow halos. Symptomatic samples were collected and suspected bacterial agent was isolated on nutrient agar medium. Phenotypic characteristics such as production of fluorescent pigment on KB medium, LOPAT test and utilization of various carbon sources, revealed that the strains were Pseudomonas syringae pv. syringae. All tested strains were pathogenic on peach, plum and walnut seedling. To investigate the genetic diversity among the strains, rep-PCR using BOX and ERIC primers was performed. Results showed high similarity among the strains from the same region, while variation was found among those from different areas and they were divided into two groups based on geographic regions. The phylogenetic analysis based on 16S rRNA, rpoD and gyrB sequences showed that representative isolates were closely related to P. syringae pv. syringae. To the best of our knowledge, this is the first report of the presence of P. syringae pv. syringae as a causal agent of walnut seedlings leaf spot in Iran and worldwide.

Biochemical and molecular characterization of DAPG-producing plant growth-promoting rhizobacteria (PGPR) of groundnut (Arachis hypogaea L.)

Plant growth-promoting rhizobacteria (PGPR) thrive in the rhizosphere of plants and play a beneficial role in plant growth, and development along with biocontrol activities. The present study was undertaken with the aim of developing rhizobacterial inoculants for groundnut for enhancement of growth and yield and suppression of major soil-borne fungal diseases caused by Sclerotium rolfsii (stem rot) and Aspergillus niger (collar rot). Out of a total of 154 rhizobacterial isolates obtained from groundnut rhizosphere, 78 isolates were selected on the basis of in vitro antifungal activities against three major soil-borne fungal pathogens of groundnut, i.e. Aspergillus niger, Aspergillus flavus and Sclerotium rolfsii. The selected isolates were further screened for the production of 2,4-Diacetylphloroglucinol (2,4-DAPG) by the gene specific PCR amplification of phlD gene. A total of 11 rhizobacterial isolates were found to have DAPG-producing genes and selected for further studies. Gene specific primers were also used for characterization of the isolates for plant growth-promoting and biocontrol traits. The qualitative and quantitative estimation of the various attributes of the isolates were also carried out. Majority of the isolates showed production of IAA, siderophores and fluorescent pigments. The DAPG-producing rhizobacterial isolates have great potential as bio-inoculants for groundnut crop for suppressing soil-borne fungal pathogens and to enhance growth and yield.

Spectral Diversity and Regulation of Coral Fluorescence in a Mesophotic Reef Habitat in the Red Sea.

The phenomenon of coral fluorescence in mesophotic reefs, although well described for shallow waters, remains largely unstudied. We found that representatives of many scleractinian species are brightly fluorescent at depths of 50–60 m at the Interuniversity Institute for Marine Sciences (IUI) reef in Eilat, Israel. Some of these fluorescent species have distribution maxima at mesophotic depths (40–100 m). Several individuals from these depths displayed yellow or orange-red fluorescence, the latter being essentially absent in corals from the shallowest parts of this reef. We demonstrate experimentally that in some cases the production of fluorescent pigments is independent of the exposure to light; while in others, the fluorescence signature is altered or lost when the animals are kept in darkness. Furthermore, we show that green-to-red photoconversion of fluorescent pigments mediated by short-wavelength light can occur also at depths where ultraviolet wavelengths are absent from the underwater light field. Intraspecific colour polymorphisms regarding the colour of the tissue fluorescence, common among shallow water corals, were also observed for mesophotic species. Our results suggest that fluorescent pigments in mesophotic reefs fulfil a distinct biological function and offer promising application potential for coral-reef monitoring and biomedical imaging.

First Report of Bacterial Leaf Spot Disease Caused by Pseudomonas syringae pv. syringae on Panax notoginseng.

Panax notoginseng is a species that produces a rare type of Chinese herbal medicine and is cultivated primarily in Yunnan Province. P. notoginseng has a 3-year-long crop cycle before harvest. A new bacterial disease was observed on P. notoginseng plants in the Wenshan Mountain area of Yunnan in 2012. The disease affected primarily leaves. Symptoms were circular or irregular brown leaf spots, surrounded by a yellow halo, located on the edges of the leaves. Eight creamy white pigmented, rounded strains were isolated consistently from leaf spots on Luria-Bertani agar (LB) medium, incubated at 28°C. Three strains (SQYB-1, SQYB-2, SQYB-3) of eight isolates were prepared for further study. Three isolates were purified and characterized: all were gram-negative, rod-shaped, motile, aerobic, non-spore forming, and negative for oxidase, potato soft rot, arginine dehydrolase, presence of tyrosinase and urease, nitrate, and fluorescent pigment production. Moreover, they were positive for levan production, presence of catalase, and for tobacco hypersensitivity. All three strains isolated were identified as Pseudomonas syringae pv. syringae (Pss) based on morphology, metabolic profile (Biolog Microbial ID System), and 16S rDNA sequence analysis (1). The metabolic similarity index between experiment strain SQYB-1 and a type of strain Pss was 0.619. The primers of 16S rDNA sequence amplification were 27F/1492R. Before sequencing, we cloned the PCR products. There was 99% homology in 16S rDNA sequences between one isolate, SQYB-1 (NCBI Accession No. JX876901) and Pss (HQ840766), supporting the identification of the P. notoginseng strains as Pss (3). For Koch's postulates, 10 surface-disinfected young leaves on three plants were inoculated with SQYB-1 isolates by spraying a phosphate-buffered saline cell suspension (3.0 × 108 CFU/ml) onto the leaves (4). Controls were inoculated similarly with sterile phosphate-buffered saline. Plants were covered with polyethylene bags for 24 h at 25°C and then transferred to a greenhouse. Circular or irregular brown spots were observed on all bacteria-inoculated leaves within 9 to 14 days after inoculation. No symptoms were observed on controls. Bacteria reisolated from inoculated leaves were identical to the original isolates as determined by the methods described above. The present study indicated that isolate SQYB-1 could independently infect P. notoginseng leaves, which was different from the finding of Luo et al. concerning involvement of Pss in root rot (2). To our knowledge, this is the first report of Pseudomonas syringae pv. syringae causing bacterial leaf spot on P. notoginseng in China.

A new medium for the detection of fluorescent pigment production by pseudomonads

Two media (King’s B [KB] and CSGA) commonly used for the detection of fluorescent pigment by Pseudomonas spp. were compared to a new medium proposed in this study, PGS agar. Thirty‐nine strains of 10 different species of Pseudomonas from several geographic regions were screened. The efficacies of these media were examined under several conditions, including the addition of iron‐binding substances or supplementation with extra iron. The medium developed, which included an iron‐binding agent, was the most permissive for production of fluorescent pigments when compared to KB and CSGA. Thirty‐seven of the 39 pseudomonad strains screened were highly fluorescent on this new medium compared to 15 and 16 strains, respectively, on KB or CSGA. The optimal composition of the medium per litre was Bacto peptone 10 g, gelatin 20 g, sucrose 20 g, agar 15 g, dipotassium hydrogen phosphate 1 g, magnesium sulphate heptahydrate 1 g and conalbumin 2 g. Protocol validation tests performed through an intra‐laboratory study in comparison to KB demonstrated the effectiveness of the new PGS medium.

First Report of Pseudomonas cichorii Associated with Leaf Spot on Soybean in South Korea.

Unusual symptoms were observed on 5% of the soybean (Glycine max (L.) Merill) plants in commercial fields in South Korea in September 2008 and 2009. The lesions were at first water soaked, then enlarged and turned dark brown or black, often with concentric white rings and sometimes surrounded by a bright yellow halo. Most lesions were roughly circular to irregular. Six bacteria were isolated on tryptic soy agar (TSA) media from plants of soybean cv. Daewon. The isolate JBC1 formed colonies that were whitish to greenish, circular with convex elevation, and unable to grow anaerobically or at 37°C. Cells of the isolate were rod shaped with polar multitrichous flagella. The isolate was positive for the following characteristics: production of fluorescent and diffusible pigment on King's medium B, production of oxidase, ability to rot potato, and utilization of l-arabinose, d-aspartate, citrate, galactose, glucose, meso-inositol, mannitol, and meso-tartrate. However, the isolate was negative in the following tests: formation of yellow colonies on peptone sucrose agar and yeast extract dextrose calcium carbonate agar media, levan formation, production of nondiffusible pigment, urease and arginine dihydrolase, hydrolysis of starch, nitrate reduction, and utilization of d-arabinose, benzoate, geraniol, l-rhamnose, d-sorbitol, sucrose, and trehalose (1). The isolates elicited a clear hypersensitive reaction in tobacco leaves. The carbon source oxidation pattern analyzed by Biolog (Hayward, CA) GN database indicated that the isolate belonged to Pseudomonas cichorii (Swingle 1925) Stapp 1928. DNA was isolated with a commercial genomic DNA extraction kit (Solgent, Daejeon, South Korea) and the 16S rDNA was amplified using universal 27F and 1492R primers. The 1,367-bp amplicon was sequenced (GenBank Accession No. JF951725) and had 100% sequence identity with P. cichorii Accession No. EF101976.1. Phylogenetic analysis based on 1,367 bp of the 16S rDNA sequence also revealed that isolate JBC1 was closely related to P. cichorii. Healthy soybean plants of cv. Jangyeop were spray inoculated with 20 ml of a 108 suspension prepared from a 2-day-old culture grown on TSA. Healthy plants sprayed with just water served as noninoculated checks. Typical disease symptoms that were observed in the field developed on leaves of soybean plants 3 days after spray inoculation, while the check plants remained symptomless. The bacteria reisolated from inoculated plants were confirmed to be identical to the original strain by 16S rDNA analysis and the Biolog test. Inoculation on tomato, watermelon, melon, and oriental melon plants resulted in dark brown or black lesions forming on leaf margins and tips, which is different from those on soybean plants (2,3). With paprika and eggplant, necrotic spots with concentric white rings developed on the leaves of each plant. We propose leaf spot as the name for this disease on soybean. To our knowledge, this is the first report of leaf spot on soybean caused by P. cichorii, which is becoming a more important pathogen in subtropical regions and greenhouses (4).