KB Medium Research Articles

Effect of Acetic Acid on Biofilm Formation in Paracidovorax citrulli, Causal Agent of Bacterial Fruit Blotch.

The unique tissue structure of pathogenic bacteria biofilm plays an important role in its pathogenicity and bactericide resistance. Inhibition or destruction of biofilm formation of pathogenic bacteria is of great significance for the control of plant bacterial diseases. In this study, Paracidovorax citrulli was inoculated into KB medium containing acetic acid, and after shaking at 28°C and 55 r/min for 48 h, it was found that the content of extracellular polysaccharide, extracellular protein and extracellular DNA (eDNA) decreased with the increase of acetic acid concentration, which resulted in the decrease of biofilm formation, it is not even possible to form biofilms on plastic slides. When the final concentration of acetic acid in the culture medium was greater than or equal to 0.5 mg/mL, there was no biofilm on the plastic slides. Therefore, the use of acetic acid as an inhibitor of P. citrulli has a good potential for control of bacterial fruit blotch.

First Report of Bacterial Blight of Pennycress Caused by Xanthomonas campestris in Wisconsin.

In May 2023, pennycress (Thlaspi arvense, L.) lines undergoing seed production in the Walnut Street Greenhouse at the University of Wisconsin-Madison displayed symptoms of chlorosis and black necrotic leaf spots (Fig. S1-A). Lesions eventually enlarged to 1-2 cm in diameter, became necrotic, and coalesced to cover a substantial portion of leaves. Symptoms were observed in ~30% of the pennycress lines adversely affecting overall growth and reproduction. Symptomatic leaves were surface sterilized for 30 seconds in 0.75% sodium hypochlorite, rinsed in sterile deionized water, and bacteria were isolated using three-phase streaking of symptomatic tissue onto KB medium (King et al., 1954). Single colonies of three isolates (creamy white to yellow) from this initial isolation were streaked onto KB medium to obtain pure cultures. Individual colonies were transferred for growth overnight in nutrient broth (Difco) and an equal amount of the broth was added to 30% glycerol in deionized (di) water and stored at -80 °C. To validate Koch's Postulates, bacteria were grown from these stocks on Yeast Dextrose Calcium Carbonate medium (Wilson et al., 1967) and were used to inoculate 5-week-old pennycress plants in the greenhouse. The bacteria were grown for 48 hours at 26°C, suspended in 300 ml of 0.05 M PBS buffer (pH=7.2) for inoculum preparation. Plants were inoculated with three bacterial isolates (approx. 108 CFU/ml) by piercing the mid veins or hydathodes with a sterilized toothpick dipped in the suspension. Inoculated plants were then enclosed in clear plastic bags for 24-48 hours and maintained in the greenhouse at a constant temperature of 26°C with a 16-hour photoperiod. After seven days, water-soaked lesions appeared on the inoculated leaves, eventually developing into the characteristic black spots (Fig. S1-B). DNA from the original isolates was extracted, and 16S PCR and sequencing of the positive bands was done. The negative control only produced brown spots at the site of inoculation (Fig. S1-C). The primer sequences were as follows: 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT (Eden et al., 1991; Weisburg et al., 1991). A BLAST analysis showed that the isolates had an E value of 0.0 to the genus Xanthomonas as well as 100% identity. Amplification and sequencing of the bacterium using gyrB amplicons revealed a 99-100% pairwise match with Xc. To enhance taxonomy resolution and confirm the identity of these isolates, the complete genomes of three samples were sequenced using NextSeq2000 Illumina platform (NCBI bioproject ID PRJNA1040293). Average Nucleotide Identity (ANI) analysis was conducted with representative strains from the Xc species (Dubrow et al., 2022), using PanExplorer (Dereeper et al., 2020) featuring integrated FastANI module (Jain et al., 2018). The isolates genomes exhibited over 98% identity and clustered with that of Xc pv. incanae and Xc pv. barbarae (Fig S2). Further work will be required to identify the pathovar of Xc identified in this study through phenotypic host range assay. This marks the first documented case of Xc in pennycress in the Midwestern US. Given the potential use of pennycress as a cover crop in the region, further investigations are warranted to assess its economic impact on production and develop management strategies.

Occurrence of Pseudomonas cichorii Causing Midrib Rot of Lettuce Under Hydroponic Cultivation in Chile

In Chile, lettuce is the vegetable that has increased in cultivated area the most in recent years, reaching 8,309 ha. The Coquimbo Region contributes the most to this growth in production with 3,284 ha in 2022 (ODEPA 2023). Most lettuce is grown under open field conditions, but there is significant production in greenhouses and an increase in hydroponic production systems (INIA 2017). During April to June 2021 and 2022 in the Coquimbo Region, butterhead-type lettuce seedlings (Lactuca sativa) cv. Neil, cultivated under a hydroponic system, showed severe brown to black lesions in the leaves and midrib (Figure S1). To determine the etiology of this problem, samples of diseased plants were taken. Pieces of symptomatic tissue were macerated, and the extract was spread on nutrient agar (NA) and on King's B medium (KB) and incubated at 23°C for 48 h. The bacterial colonies observed were predominantly circular, creamy-white in color with irregular margins and fluorescent in KB medium. Isolates were gram-negative strictly aerobic. LOPAT test (Lelliot et al. 1966) results of two selected isolates were: levan production (-), oxidase reaction (+), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+), which corresponds to the profile of Pseudomonas cichorii. Molecular identification was performed through amplification and sequencing of the 16S rRNA (GenBank Accessions No. OR540674 to OR540675), gyrB and rpoD genes (Hwang et al. 2005; Sarkar and Guttmann 2004) (GenBank Accessions No. OR558279 to OR558282). BLAST analysis of the 16S rRNA gene of the isolates resulted in a match with a 99.86% identity with P. cichorii type strain ATCC 10857 (NR_112070.1). BLAST analysis of gyrB and rpoD resulted in a match with a 100% (630/630 bp) and >99% (546/550 bp) identity respectively, with strains of P. cichorii. Five six-month-old lettuce plants cv. Desert Storm were pricked in the midrib with a toothpick smeared with a fresh colony grown on KB medium. Seven days after inoculation, the plants showed dark brown, watery lesions, characteristic of damage caused by P. cichorii (Figure S1). Bacteria were isolated again from the inoculated plants and were identified as P. cichorii using LOPAT and molecular identification techniques. Midrib rot caused by P. cichorii was reported as an emerging disease of greenhouse-grown lettuce by Cottyn et al. (2009). In Chile, P. cichorii was previously described affecting nectarine fruits (Pinto de Torres and Carreño Ibañez 1983) and reported as a pathogen of lettuce among others horticultural crops by Servicio Agrícola y Ganadero of the Government of Chile (Acuña 2008), but this is the first report of P. cichorii affecting hydroponic lettuce plants in Chile. These results will be the basis of future studies to evaluate the origin of the infection, the potential dissemination, and the implementation of disease management to avoid the damage caused by this bacterium in hydroponic systems in this crop of growing importance in Chile.

Economical Production of Phenazine-1-carboxylic Acid from Glycerol by Pseudomonas chlororaphis Using Cost-Effective Minimal Medium.

Phenazine compounds are widely used in agricultural control and the medicine industry due to their high inhibitory activity against pathogens and antitumor activity. The green and sustainable method of synthesizing phenazine compounds through microbial fermentation often requires a complex culture medium containing tryptone and yeast extract, and its cost is relatively high, which greatly limits the large-scale industrial production of phenazine compounds by fermentation. The aim of this study was to develop a cost-effective minimal medium for the efficient synthesis of phenazine compounds by Pseudomonas chlororaphis. Through testing the minimum medium commonly used by Pseudomonas, an ME medium for P. chlororaphis with a high production of phenazine compounds was obtained. Then, the components of the ME medium and the other medium were compared and replaced to verify the beneficial promoting effect of Fe2+ and NH4+ on phenazine compounds. A cost-effective general defined medium (GDM) using glycerol as the sole carbon source was obtained by optimizing the composition of the ME medium. Using the GDM, the production of phenazine compounds by P. chlororaphis reached 1073.5 mg/L, which was 1.3 times that achieved using a complex medium, while the cost of the GDM was only 10% that of a complex medium (e.g., the KB medium). Finally, by engineering the glycerol metabolic pathway, the titer of phenazine-1-carboxylic acid reached the highest level achieved using a minimum medium so far. This work demonstrates how we systematically analyzed and optimized the composition of the medium and integrated a metabolic engineering method to obtain the most cost-effective fermentation strategy.

First report of Pseudomonas mediterranea causing tomato pith necrosis in Croatia.

In May 2021, a tomato producer reported an occurrence of a disease unknown so far in a greenhouse near Split, Croatia. About 30% of plants (cultivar Signora) have been affected. Symptoms resembled tomato pith necrosis, bacterial disease caused by Pseudomonas corrugata, known to occur sporadically in tomato greenhouse production in Croatia. Leaves on plants developed interveinal chlorosis, followed by necrosis and leaf collapse. When main stems were longitudinally cut, brown, disintegrated and water-soaked partly hollow pith was evident. Severely affected plants wilted. With suspicion on presence of P. corrugata, bacteria were isolated from surface-sterilized pith tissue of two tomato plants by plating onto sucrose peptone agar (SPA) and King's B medium (KB). Colonies recovered were cream-colored on SPA and non-florescent on KB. Two isolates, assigned as 1-KB and 3A, were first identified by amplification of internal transcribed spacer (ITS1) between16S rRNA and 23S rRNA using primers D21 and D22 (Manceau and Horvais 1997). The 550-bp PCR products obtained were purified and sequenced. Subsequent BLAST search showed the sequences to have 100% identity with the strain DSM 16733 isolated from tomato in Italy (Accession No. LT629790.1) and 99.77% identity with the strain SM664-12 isolated from tomato in USA (Acc. No. KC405207.1) of Pseudomonas mediterranea from NCBI. ITS sequence for one isolate 3A was deposited in GenBank under the Accession No. OP765279.1. Further identification was performed by using species-specific primers PC1/1-PC1/2 for P. mediterranea (Catara et al. 2000, 2002). Amplification of 600 bp DNA fragment confirmed the identity of isolates 1-KB and 3A as P. mediterranea. For this region sequence of isolate 3A was deposited in GenBank under the Acc. No. OP068273.1. Pathogenicity was assessed on tomato plants (cultivar Moneymaker) grown in pots in bio-chamber. Plants were grown at 25/20 °C 12h/12h dark/light regime until 8-leaves stage (BBCH 18). P. mediterranea isolate 3A was used for the inoculation. Inoculum was prepared from the isolate grown on KB medium for 48 h and suspended in sterile distilled water (concentration of 109 CFU mL-1) by dilution plate counts. Ten plants were inoculated with 10 µl of bacterial suspension injected into the stem with a syringe. Five control plants were inoculated with sterile distilled water. After 40 days of plant growth, symptoms were visible on all plants inoculated with P. mediterranea isolate 3A. Although no wilting was observed and all plants were alive, chlorosis was observed on upper leaves, chlorosis and necrosis on middle leaves, while basal leaves wilted. Longitudinal cross-sections of stems revealed brownish pith tissue with longitudinal watery pits spreading from inoculation points (Fig.S1). Symptoms were not observed on control plants. Bacterium was reisolated from three plants showing the most severe symptoms and proved to be identical to the original using species-specific primer pair PC1/1-PC1/2. To our knowledge, this is the first confirmation of P. mediterranea causing tomato pith necrosis in Croatia. Tomato pith necrosis caused by P. mediterranea may become significant bacterial disease of greenhouse tomato in Croatia.

NtrC Contributes to Nitrogen Utilization, Stress Tolerance, and Virulence in Acidovorax citrulli

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, severely damages watermelon, melon, and other cucurbit crops worldwide. Nitrogen, one of the most important limiting elements in the environment, is necessary for the growth and reproduction of bacteria. As a nitrogen-regulating gene, ntrC plays an important role in maintaining bacterial nitrogen utilization and biological nitrogen fixation. However, the role of ntrC has not been determined for A. citrulli. In this study, we constructed a ntrC deletion mutant and a corresponding complementary strain in the background of the A. citrulli wild-type strain, Aac5. Through phenotype assays and qRT-PCR analysis, we investigated the role of ntrC in A. citrulli in nitrogen utilization, stress tolerance, and virulence against watermelon seedlings. Our results showed that the A. citrulli Aac5 ntrC deletion mutant lost the ability to utilize nitrate. The ntrC mutant strain also exhibited significantly decreased virulence, in vitro growth, in vivo colonization ability, swimming motility, and twitching motility. In contrast, it displayed significantly enhanced biofilm formation and tolerance to stress induced by oxygen, high salt, and copper ions. The qRT-PCR results showed that the nitrate utilization gene nasS; the Type III secretion system-related genes hrpE, hrpX, and hrcJ; and the pili-related gene pilA were significantly downregulated in the ntrC deletion mutant. The nitrate utilization gene nasT, and the flagellum-related genes flhD, flhC, fliA, and fliC were significantly upregulated in the ntrC deletion mutant. The expression levels of ntrC gene in the MMX-q and XVM2 media were significantly higher than in the KB medium. These results suggest that the ntrC gene plays a pivotal role in the nitrogen utilization, stress tolerance, and virulence of A. citrulli.

A putative multi-sensor hybrid histidine kinase, BarA Ac , inhibits the expression of the type III secretion system regulator HrpG in Acidovorax citrulli.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, severely damages watermelon, melon, and other cucurbit crops worldwide. Although many virulence determinants have been identified in A. citrulli, including swimming motility, twitching motility, biofilm formation, and the type III secretion system (T3SS), research on their regulation is lacking. To study virulence regulation mechanisms, we found a putative histidine kinase BarA Ac that may be related to the T3SS regulator HrpG in A. citrulli. We deleted and characterized barAAc (Aave_2063) in A. citrulli Aac5 strain. Compared to the wild-type Aac5, virulence and early proliferation of barAAc mutant in host watermelon cotyledons were significantly increased, and induction of hypersensitive response in non-host tobacco was accelerated, while biofilm formation and swimming motility were significantly reduced. In addition, the transcriptomic analysis revealed that the expression of many T3SS-related genes was upregulated in the ΔbarAAc deletion mutant when cultured in KB medium. Meanwhile, the ΔbarAAc deletion mutant showed increased accumulation of the T3SS regulator HrpG in KB medium, which may account for the increased deployment of T3SS. This suggests that the putative histidine kinase BarA Ac is able to repress the T3SS expression by inhibiting HrpG in the KB medium, which appears to be important for rational energy allocation. In summary, our research provides further understanding of the regulatory network of A. citrulli virulence.

First report of black rot caused by Xanthomonas nasturtii on watercress in Spain and Portugal

Xanthomonas nasturtii was first identified as the cause of black rot of watercress (Nasturtium officinale) on plants grown in Florida, USA (Vicente et al., 2017). Similar symptoms had been reported earlier in Hawaii (McHugh & Constantinides, 2004) and an unidentified Xanthomonas sp. was isolated in Portugal in 2003 from wild watercress (Cruz et al., 2017). Since 2017, watercress crops in southern Spain have occasionally exhibited small yellow leaf lesions around the hydathodes, leaf spots, V-shaped leaf lesions, wilt, distortion and senescence (Fig. 1). Symptoms were more frequent during mild and humid periods from November to February, reducing yields by up to 60% and leading to rejection of some crops (Lascelles, 2019). In 2021, symptoms were seen on watercress produced in Portugal and sold in the UK. Isolations were made from watercress leaves showing typical symptoms between 2017 and 2021 by excising and macerating small tissue fragments in sterile water and plating on King's medium B (KB). Seed produced in Spain was tested using a method based on the International Seed Testing Association method for detection of Xanthomonas campestris pv. campestris. Mixtures of bacterial colonies were observed on the plates after incubation for 48–72 hours at 28°C and colonies resembling Xanthomonas (yellow, mucoid) were sub-cultured. Isolates were purified and stored at -76°C in nutrient broth with 150 g/l glycerol (Vicente et al., 2017) and/or at -80°C in Protect® (Protect System, UK). In contrast to the type strain (NCPPB 4600) from Florida, isolates from Spain and Portugal did not cause browning of the KB medium (Fig. 2). Pathogenicity was tested on plants of three watercress accessions and Savoy cabbage cv. Wirosa F1 (Bejo Zaden B.V., The Netherlands) by stabbing with an insect pin charged with bacteria (Vicente et al., 2017). The Xanthomonas-like isolates were pathogenic to all three watercress accessions (Fig. 3) whereas no symptoms were observed on cabbage. To complete Koch's postulates, re-isolations were made from symptomatic watercress leaves inoculated with isolates WHRI 10015A (from Spain) and 10049A (from Portugal). Isolates with the same morphological characteristics were obtained. Fatty acid profiling (Weller et al., 2000) was performed on isolates grown on trypticase soy agar plates at 28°C for 48 hours; comparisons with libraries that did not include X. nasturtii, indicated the highest matches of 0.38 to 0.68 with X. axonopodis and X. campestris. DNA was extracted as outlined in Vicente et al. (2017). The partial 16S rRNA gene was amplified and sequenced for isolates from Spain and confirmed their identity as a Xanthomonas species. The partial gyrB gene was amplified and sequenced for selected isolates according to Parkinson et al. (2007) (Table 1). The phylogeny (Fig. 4) confirmed that isolates from Spain and Portugal belong to X. nasturtii and shows that there is some genetic diversity amongst them. This is the first report of X. nasturtii on watercress crops in Europe. We suspect that black rot of watercress may be widespread in production areas of the Iberian Peninsula and, as the crop is grown in water beds, the pathogen is likely to spread relatively easily. Based on detection in a watercress seed lot produced in Spain, the pathogen may be seed borne and therefore it is important to test watercress seed to avoid introducing the disease into clean beds and new areas/countries. This work was funded by Royalcress S.A., The Watercress Company, UKRI, Defra and the Scottish Government.

Hcp of the Type VI Secretion System (T6SS) in Acidovorax citrulli Group II Strain Aac5 Has a Dual Role as a Core Structural Protein and an Effector Protein in Colonization, Growth Ability, Competition, Biofilm Formation, and Ferric Iron Absorption.

A type VI secretion system (T6SS) gene cluster has been reported in Acidovorax citrulli. Research on the activation conditions, functions, and the interactions between key elements in A. citrulli T6SS is lacking. Hcp (Hemolysin co-regulated protein) is both a structural protein and a secretion protein of T6SS, which makes it a special element. The aims of this study were to determine the role of Hcp and its activated conditions to reveal the functions of T6SS. In virulence and colonization assays of hcp deletion mutant strain Δhcp, tssm (type VI secretion system membrane subunit) deletion mutant strain Δtssm and double mutant ΔhcpΔtssm, population growth was affected but not virulence after injection of cotyledons and seed-to-seedling transmission on watermelon. The population growth of Δhcp and Δtssm were lower than A. citrulli wild type strain Aac5 of A. citrulli group II at early stage but higher at a later stage. Deletion of hcp also affected growth ability in different culture media, and the decline stage of Δhcp was delayed in KB medium. Biofilm formation ability of Δhcp, Δtssm and ΔhcpΔtssm was lower than Aac5 with competition by prey bacteria but higher in KB and M9-Fe3+ medium. Deletion of hcp reduced the competition and survival ability of Aac5. Based on the results of Western blotting and qRT-PCR analyses, Hcp is activated by cell density, competition, ferric irons, and the host plant. The expression levels of genes related to bacterial secretion systems, protein export, and several other pathways, were significantly changed in the Δhcp mutant compared to Aac5 when T6SS was activated at high cell density. Based on transcriptome data, we found that a few candidate effectors need further identification. The phenotypes, activated conditions and transcriptome data all supported the conclusion that although there is only one T6SS gene cluster present in the A. citrulli group II strain Aac5, it related to multiple biological processes, including colonization, growth ability, competition and biofilm formation.

An in vitro bioassay for evaluating the virulence of Pseudomonas savastanoi pv savastanoi isolates on olive

Pseudomonas savastanoi pv. savastanoi causes olive knot disease that afflicts olive trees globally. A sensitive and reproducible in vitro pathogenicity bioassay using detached leaf segments was developed after isolation of Psav on KB medium amended with vancomycin, cephalexin and cycloheximide. Symptoms were clear under 20X magnification after 10 days. This bioassay will facilitate rapid screening of olive germplasm collection for resistance to Psav.

First Report of Leaf Blight of Adenophora triphylla var. japonica caused by Pseudomonas viridiflava in Korea.

Severe disease with leaf spots and necrotic symptoms were observed in Adenophora triphylla var. japonica (Regel) Hara (A. triphylla) during the survey in July 2020 on a field in Andong, Gyeongbuk province, Korea. It is a highly valued medicinal plant used to treat various diseases, including cough, cancer, and obesity. The infected plants initially showed spots with halo lesions, at later stages, enlarged and spread to the leaves, which the lesions becoming yellowing and chlorotic (Fig. 1). In some areas, disease incidence was up to 15% of the plants. The symptomatic samples were collected from A. triphylla and cut into 4 to 5 mm squares, surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed three times, and macerated in sterile distilled water (SDW). They were spread onto nutrient agar (NA) plates and incubated at 28°C for 3 days. The representative bacterial strains selected for identification showed fluorescent colonies on King's medium B (KB). Fifteen isolates from independent samples were subjected to biochemical and pathogenicity tests. The isolates induced a hypersensitive reaction in tobacco leaves, gave a reaction in the anaerobe respiratory test, and were negative for levan, oxidase, arginine dihydrolase, gelatin hydrolysis, aesculin hydrolysis, and starch hydrolysis. The isolated strains presented the following LOPAT profile: - - + - +. The Biolog GN2 microplate and the Release 4.20 system putatively found the isolate to exhibit 93% similarity with the bacterium, Pseudomonas viridiflava. Likewise, analysis of FAME profiles using the Microbial identification system (Sherlock version 3.1) also characterized the representative bacterial strain as P. viridiflava with 87% similarity. The genomic DNA of the isolate was extracted, and the 16S rDNA sequence was amplified with a universal bacterial primer set (27F and 1492R). The sequence was submitted to GenBank under the accession number MT975233. BLASTn analysis yielded 99.79% identity with P. viridiflava strain RT228.1b (accession no. AY604846.1) and 99.72% similarity with P. viridiflava KNOX249.1b strain (accession no. AY604848.1). Phylogenetic dendrogram constructed from the comparative analysis of 16S rDNA gene sequences showing the relationship between P. viridiflava GYUN274 and related Pseudomonas species (Fig. 2). Pathogenicity tests were conducted three times on seedling of A. triphylla by spraying 50 ml of bacterial suspensions of a 24-h culture in KB medium (108 CFU/ml). The leaves inoculated with SDW alone did not develop symptoms; however, the plants treated with isolated bacterial suspensions developed halo and blight symptoms similar to those observed in the field 7 days post-inoculation. Finally, Koch's postulates were verified by re-isolating P. viridiflava from all symptomatic tissues and determined to be morphologically identical to the original isolates. To our knowledge, this is the first report of leaf blight disease of A. triphylla caused by P. viridiflava in Korea. Based on the observed symptoms, and identification by morphological characteristics, molecular data, and pathogenicity against the host plant, the proper control measures can be identified in future studies.

Identification of new arylamine N-acetyltransferases and enhancing 2-acetamidophenol production in Pseudomonas chlororaphis HT66

Background2-Acetamidophenol (AAP) is an aromatic compound with the potential for antifungal, anti-inflammatory, antitumor, anti-platelet, and anti-arthritic activities. Due to the biosynthesis of AAP is not yet fully understood, AAP is mainly produced by chemical synthesis. Currently, metabolic engineering of natural microbial pathway to produce valuable aromatic compound has remarkable advantages and exhibits attractive potential. Thus, it is of paramount importance to develop a dominant strain to produce AAP by elucidating the AAP biosynthesis pathway.ResultIn this study, the active aromatic compound AAP was first purified and identified in gene phzB disruption strain HT66ΔphzB, which was derived from Pseudomonas chlororaphis HT66. The titer of AAP in the strain HT66ΔphzB was 236.89 mg/L. Then, the genes involved in AAP biosynthesis were determined. Through the deletion of genes phzF, Nat and trpE, AAP was confirmed to have the same biosynthesis route as phenazine-1-carboxylic (PCA). Moreover, a new arylamine N-acetyltransferases (NATs) was identified and proved to be the key enzyme required for generating AAP by in vitro assay. P. chlororaphis P3, a chemical mutagenesis mutant strain of HT66, has been demonstrated to have a robust ability to produce antimicrobial phenazines. Therefore, genetic engineering, precursor addition, and culture optimization strategies were used to enhance AAP production in P. chlororaphis P3. The inactivation of phzB in P3 increased AAP production by 92.4%. Disrupting the phenazine negative regulatory genes lon and rsmE and blocking the competitive pathway gene pykA in P3 increased AAP production 2.08-fold, which also confirmed that AAP has the same biosynthesis route as PCA. Furthermore, adding 2-amidophenol to the KB medium increased AAP production by 64.6%, which suggested that 2-amidophenol is the precursor of AAP. Finally, by adding 5 mM 2-amidophenol and 2 mM Fe3+ to the KB medium, the production of AAP reached 1209.58 mg/L in the engineered strain P3ΔphzBΔlonΔpykAΔrsmE using a shaking-flask culture. This is the highest microbial-based AAP production achieved to date.ConclusionIn conclusion, this study clarified the biosynthesis process of AAP in Pseudomonas and provided a promising host for industrial-scale biosynthesis of AAP from renewable resources.

First Report of Pseudomonas viridiflava Causing Bacterial Blight on Globe Artichoke in Turkey

The production of globe artichoke (Cynara scolymus L.), which is a major part of Mediterranean cuisine and packs an abundance of nutritional value, has been increasing over recent decades in Turkey. In the spring of 2018, globe artichoke (cv. Sakiz) plants with blight symptoms on outer bracts, dark brown sunken lesion on inner sides, red-brown areas on margins, and small and malformed buds were found in artichoke-growing areas in the Karaburun district of Izmir, Turkey. In some areas, disease incidence was up to 5% of the plants. Several buds in harvest stage exhibiting symptoms were collected from 30-ha artichoke production fields that have heavy soil texture and humid weather conditions and processed to identify the causal agent on 15 artichoke samples. Symptomatic outer bracts showing characteristics of bacterial blight symptoms as well as spines were surface sterilized with 1% NaOCl, rinsed in sterilized distilled water (SDW), and cultured on King’s medium B (KB) for 24 h at 28°C. After incubation, a predominant bacterium was isolated from all samples. Fifteen isolates from independent samples were subjected to biochemical and pathogenicity tests. Round, flat, and blue-green translucent colonies fluorescing under 360-nm UV light when grown on KB medium were observed in all samples taken. The isolates caused soft rot of potato tubers, induced a hypersensitive reaction in tobacco leaves, gave a reaction in the anaerobe respiratory test, and were negative for levan, oxidase, arginine dihydrolase, gelatin hydrolysis, aesculin hydrolysis, and starch hydrolysis. The isolated strains presented the following LOPAT profile: – – + – +. Acid was produced from mannitol, erythritol glycerol, D-sorbitol, D-mannose, and inositol but not from L-tyrosine, D-cellobiose, D-arabinose, and D-trehalose, all of which are typical characteristics of Pseudomonas viridiflava (Lelliott et al. 1966; Schaad et al. 2001). The bacterial identity was further confirmed by amplifying the 16S rRNA gene using the universal bacterial primers 8F and 1492R (Galkiewicz and Kellogg 2008) and gyrase subunit B (gyrB) gene using primers gyrB-Rps/gyrB-Fps (Sawada et al. 1999). The sequences were submitted to GenBank under accession numbers MH763694 for 16S and MK546431 for gyrB. BLASTn analysis of the 1,042-bp amplicon for 16S and 545-bp amplicon for gyrB indicated that it had 99% similarity with P. viridiflava strain 4G1113 (accession no. MG972916) and 100% similarity with P. viridiflava ATCC 13223 strain (accession no. KY612187), respectively. Pathogenicity tests were conducted twice on outer bracts and main stems of artichoke cultivar Sakiz by inoculating 20 µl of bacterial suspension of a 24-h culture in KB medium (1.9 × 10⁹ CFU/ml in SDW). The inoculated plants were placed in a transparent box for 7 days and kept at 25°C and 70% relative humidity. The bract leaves inoculated with only SDW did not develop symptoms; however, the bracts treated with isolated bacterial suspension developed blight symptoms similar to those observed in the field, 7 days postinoculation. The stems inoculated with the isolate also exhibited soft rot symptoms, and the same bacterium was consistently reisolated from both bracts and stems but not from parts of negative control plants. Reisolated bacteria exhibited the same biochemical profile with our isolate, thereby fulfilling Koch’s postulates. P. viridiflava causing a bacterial blight of artichoke bracts was previously reported in Greece (Sarris et al. 2012). To our knowledge, it is the first report of this bacterium causing bacterial blight on leaves and soft rot on stems of globe artichoke in Turkey. Given the availability of climatic conditions, the potential of disease spreading can cause a serious reduction in yield and marketability of this vegetable, which has increasing popularity owing to having high nutritional value, leading to substantial economic losses.

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.

Screening of Cellulase and Pectinase by Using <i>Pseudomonas fluorescence</i> and <i>Bacillus subtilis</i>

A study was conducted to determine the Production of cellulase and pectinase enzyme by using Plant growth promoting rhizobacteria like Pseudomonas fluorescence and Bacillus subtilis. These to micro organism are isolated by serial dilution method. One gram of soil sample was diluted in to 10 ml of sterile distilled water and 1 ml of sample solution was serially diluted in 9ml of sterile water up to 10 dilution. Each sample from dilution 10-5 and 10-6 were taken and streaked in to KB and NA medium and incubate at 24 hrs. After 24 hrs Pseudomonas fluorescence and Bacillus subtilis was observed in the medium of KB and NA medium. Both the culture was sub cultured and maintain in the same for the further work. CMCase medium was prepared and sterilized by autoclave for 121 °C for 15 minutes after sterilization these medium contain petriplate was streaked by bacteria and incubates for 48h after incubation period a clear halo zone was produced by these bacteria among these bacteria Pseudomonas fluorescence are able to produce high amount of cellulose compare to Bacillussubtilis. Pectin agar medium was prepared and sterilized by autoclave for 121 °C for 15 minutes after sterilization these medium contain petriplate was streaked by bacteria incubates for 48h after incubation period a clear halo zone was produced by these bacteria, among these bacteria Pseudomonas fluorescence are able to produce high amount of Pectinase compare to Bacillus subtilis. Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria that colonize plant roots and enhance plant growth by a wide variety of mechanisms.

Screening of Cellulase and Pectinase by Using <i>Pseudomonas fluorescence</i> and <i>Bacillus subtilis</i>

A study was conducted to determine the Production of cellulase and pectinase enzyme by using Plant growth promoting rhizobacteria like Pseudomonas fluorescence and Bacillus subtilis. These to micro organism are isolated by serial dilution method. One gram of soil sample was diluted in to 10 ml of sterile distilled water and 1 ml of sample solution was serially diluted in 9ml of sterile water up to 10 dilution. Each sample from dilution 10-5 and 10-6 were taken and streaked in to KB and NA medium and incubate at 24 hrs. After 24 hrs Pseudomonas fluorescence and Bacillus subtilis was observed in the medium of KB and NA medium. Both the culture was sub cultured and maintain in the same for the further work. CMCase medium was prepared and sterilized by autoclave for 121 °C for 15 minutes after sterilization these medium contain petriplate was streaked by bacteria and incubates for 48h after incubation period a clear halo zone was produced by these bacteria among these bacteria Pseudomonas fluorescence are able to produce high amount of cellulose compare to Bacillussubtilis. Pectin agar medium was prepared and sterilized by autoclave for 121 °C for 15 minutes after sterilization these medium contain petriplate was streaked by bacteria incubates for 48h after incubation period a clear halo zone was produced by these bacteria, among these bacteria Pseudomonas fluorescence are able to produce high amount of Pectinase compare to Bacillus subtilis. Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria that colonize plant roots and enhance plant growth by a wide variety of mechanisms.

오이 노균병의 생물적 방제를 위한 Bacillus amyloliquefaciens CC110균주 선발

Pseudoperonospora cubensis에 의한 오이 노균병의 생물적 방제를 위하여 오이 재배단지의 오이 식물체 126균주를 분리하였다. 그 중에서 잎 절편 생물검정 통하여 5균주를 선발하였다. CC110균주는 오이노균병 예방과 치료효과 검정에서 전혀 병이 발생하지 않았지만 무처리는 15.0~18% 노균병이 발생하였다. CC110균주는 gyrB gene sequence 분석에 의하여 Bacillus amyloliquefaciens subsp. plantarum로 동정하였다. CC110균주를 TSB배지에서 배양한 배양액은 LB, NB와 KB 배지에서 배양한 배양액보다 오이 노균병 발생이 적어 효과적이었다. CC110균주를 TSB배지에서 배양한 배양액의 원액, 2배와 5배 희석한 처리구에서 0%, 3.0%와 8.0% 그리고 배양여액을 원액, 2배와 5배 희석한 처리구에서 0%, 4.0%와 7.0% 노균병이 발생하였고, 무처리는 21.0% 발생하였다. 오이 유묘검정에서 CC110균주를 TSB배지에서 배양한 배양액은 오이 노균병이 35.0% 발생하였고 무처리는 82.0% 발생하였다. 비닐하우스 포장에서 오이 노균병에 대하여 B. amyloliquefaciens CC110균주를 5일 간격 4회 처리가 7일 간격 3회 처리보다 방제효과가 높았다. In order to select antagonists for biological control of downy mildew of cucumber, 126 bacteria were isolated from cucumber plants collected from several locations in Korea. Among them, Five isolates were selected as potential biocontrol agents of cucumber downy mildew using a leaf disc bioassay method. In preventive and curative effect tests, the isolate CC110 was found to be effective to control downy mildew on cucumber showing diseased area by 0% whereas that of control was 15.0~18.0%. A bacterium isolate CC110 was identified as Bacillus amyloliquefaciens subsp. plantarum based on phylogenetic analysis using gyrB gene sequence. The culture liquid of isolate CC110 in TSB media were more effective for the control of the disease than those cultured in LB, NB, and KB media in leaf disk bioassay. when undiluted liquid, two-fold, five-fold diluted culture broth, and undiluted liquid, two-fold, five-fold diluted filtrate of isolate CC110 in TSB media were treated, diseased area of cucumber powdery mildew were 0%, 3.0%, 8.0%, 0%, 4.0% and 7.0%, respectively, whereas diseased area in the control was 21.0%. In the cucumber seedling tests, when the culture broth of isolate CC110 in TSB media was treated, diseased area were 35.0%, whereas that of control was 82.0%. When B. amyloliquefaciens CC110 was treated four times at five-day interval in the vinylhouse test, the control effect of cucumber downy mildew was higher than that treated three at seven-day interval.

Bacillus amyloliquefaciens M27에 의한 오이 흰가루병의 생물적 방제

흰가루병균(Podosphaera fusca)에 의한 오이 흰가루병의 생물적 방제를 위하여 검정한 길항세균 B. amyloliquefaciens M27 균주는 4.0% 흰가루병이 발생하였고 무처리는 80.5% 발생하였다. MH와 LB배지에서 배양한 B. amyloliquefaciens M27 균주의 배양여액은 TSB, NB와 KB배지에서 배양한 배양여액보다 오이 흰가루병 방제효과가 우수하였다. M27 균주를 LB배지에서 배양한 배양여액 2, 5, 10, 20, 50배와 100배 처리한 경우 오이 흰가루병이 0%, 0%, 0%, 1.3%, 3.1%, 5.0%와 33.3%를 나타낸 반면에 무처리는 60.0%를 나타내었다. 7월, 10월과 12월에 오이 흰가루병이 발생할 때 LB배지에서 배양한 M27균주의 배양여액를 10배로 처리한 결과 88.9~98.9% 방제효과를 나타났다. 이와 같은 결과에서 B. amyloliquefaciens M27 균주의 배양여액은 오이 흰가루병에 방제에 매우 효과적이었다. Bacillus amyloliquefaciens M27 was isolated from the cotton-waste compost for cultivation of oyster mushroom (Pleurotus ostreatus). B. amyloliquefaciens M27 is a biocontrol agent with antagonistic activities against a wide range of fungal pathogens. The aim of this work was to evaluate the possibility of exploiting antagonistic bacteria, B. amyloliquefaciens M27, in the biological control of the cucumber powdery mildew fungus, Podosphaera fusca. In greenhouse tests, the isolate was found to be very effective to control powdery mildew on cucumber leaves showing 4.0% diseased area, whereas diseased area in the control was 80.5%. The filtrate of the isolate cultured on MH and LB media were more effective for control of the disease than those cultured on TSB, NB, and KB media. When two, five, ten, 20, 50 and 100-fold diluted culture broth of isolate on LB media were treated, disease areas were 0%, 0%, 0%, 1.3%, 3.1% and 5.0%, respectively, whereas diseased area in the control was 60.0%. The filtrate of the isolate cultured on LB media was treated to cucumber plants on July, October and December just before the outbreak of the powdery mildew occurred. When 10-fold diluted filtrate of the isolate was treated, control efficacy was 88.9~98.9% in the treated seasons. The results showed that the culture filtrate of B. amyloliquefaciens M27 was very effective to control powdery mildew of cucumber.

First Report of Bacterial Wilt in Mandevilla (= Dipladenia) splendens 'Red Riding Hood' in the United States Caused by Ralstonia solanacearum Biovar 3.

In November of 2007, 6-inch rooted cuttings of Mandevilla (= Dipladenia) splendens 'Red Riding Hood' were submitted from a greenhouse in Indiana to the Purdue Plant and Pest Diagnostic Lab. Plants exhibited leaf dieback, wilting, and reduced top growth. Microscopic observation revealed no fungal structures within the roots, stems, and leaves; however bacterial streaming was observed from the cut edge of stem and root tissue using ×100 magnification with phase contrast. A Ralstonia solanacearum ImmunoStrip test (Agdia Inc., Elkhart, IN) was used to determine that the samples (roots and stem) were positive for R. solanacearum, the causal agent of southern wilt. A bacterial suspension was prepared from infected tissue and streaked onto King's Medium B (KB). Gram-negative, nonfluorescent, oxidase-positive bacteria were consistently isolated from the diseased tissues and determined as R. solanacearum by BIOLOG (Hayward, CA) carbohydrate utilization. A culture of R. solanacearum and infected plant material were submitted to USDA APHIS PPQ as per select agent protocol. (3) CPHST NPGBL generated pure cultures and together with submitted plant materials they tested positive for R. solanacearum using Agdia ImmunoStrips. Culture and plant material tested positive for R. solanacearum and negative for biovar 2 using the Fegan conventional PCR (1) and the Central Science Lab (CSL, York, UK) real-time PCR (4). Pure cultures were determined to be negative for biovar 2 but positive for biovar 3 using the biovar carbohydrate utilization plate assay (2). On the basis of these results, the bacteria were identified as R. solanacearum biovar 3 and not as the select agent R. solanacearum race 3 biovar 2. Koch's postulates confirmed pathogenicity of the isolated bacteria on tomato, a susceptible host. Three 6-week-old plants were mechanically inoculated with a bacterial suspension of approximately 1 × 108 CFU/ml prepared from cultures grown on KB for 2 days at 28°C. Inoculum (0.1ml of bacterial suspension) was injected into stem axils with a 22-gauge hypodermic needle. Three 6-week-old control plants were inoculated with sterile water. Plants were kept at 24°C with supplemental 400W high-pressure sodium light. Within 5 days, all three inoculated plants exhibited wilt symptoms. No symptoms were observed in control plants. Bacteria were reisolated from symptomatic plants on KB medium as described above, and gram negative, nonfluorescent, oxidase-positive bacteria were obtained. Reisolated strains were identical to R. solanacearum using BIOLOG carbohydrate utilization testing, confirming the causal agent of the disease. Personal correspondence with other diagnosticians also confirms the presence of R. solanacearum biovar 3 in Mandevilla in Ohio, Michigan, and Minnesota. To our knowledge, this is the first documented report in the world of R. solanacearum biovar 3 on Mandevilla.

OCCURRENCE AND PATHOGENICITY OF PSEUDOMONAS FLUORESCENS AND P. PUTIDA ON TOMATO PLANTS IN ITALY

SUMMARY During 2006-2008, a survey conducted in soilless and soil-grown tomato crops in eastern and southeastern Sicily (Italy) disclosed the presence of widespread leaf chlorosis and necrosis associated with yellow, pink or brown discolouration of vascular and pith tissues. Bacteria were always isolated from symptomatic tissues on KB and NA media. A total of 158 isolates showed the LOPAT characters of group Va (-+-+-) and group Vb (++-+-) and 21 of these strains were identified as different biovars of Pseudomonas fluorescens and P. putida. Koch’s postulates showed that 42 fluorescent Pseudomonas isolates are able to induce vascular discolouration of tomato plants but not leaf chlorosis and necrosis. The interactions of vascular infections and abiotic stresses were studied in growth chamber experiments. Differences in virulence were found among P. fluorescens biovar I and P. putida bv. A strains on tomato plants grown under saline stress conditions. Both bacteria were able to induce leaf chlorosis, necrosis or death of tomato plants under saline stress conditions.