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). References: (1) B. Cottyn et al. Syst. Appl. Microbiol. 32:211, 2009. (2) A. Obradovic et al. Plant Dis. 86:443, 2002. (3) E. Pauwelyn et al. J. Phytopathol. 159:298, 2011. (4) S. M. Walkil et al. Nigeria J. Appl. Biosci. 38:2540, 2011.

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First Report of Bacterial Leaf Spot Caused by Pseudomonas cichorii on Schefflera arboricola in Turkey

Plant Disease ◽

10.1094/pdis-93-8-0848b ◽

2009 ◽

Vol 93 (8) ◽

pp. 848-848

Author(s):

Y. Aysan ◽

M. Mirik ◽

F. Sahin

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Acid Methyl Ester ◽

Identification System ◽

Original Strain ◽

Bacterial Leaf Spot ◽

Pseudomonas Cichorii ◽

First Report ◽

Leaf Spots ◽

Schefflera Arboricola

In late winter and spring of 2006 and 2008, leaf spots with yellow halos were observed on dwarf schefflera (Schefflera arboricola cvs. Gold Capella, Trinette, and Green Gold) that were grown as potted plants in two commercial ornamental greenhouses in Adana and Mersin, Turkey. Average disease incidence was assessed as 10% during the term of the study. Isolations were made from leaf spots symptoms on King's medium B. Bacteria consistently isolated from diseased tissues formed green fluorescent colonies on the medium. Ten representative bacterial strains were examined and found to be gram negative, rod shaped, and aerobic, levan, pectolytic, and arginine dihydrolase negative, and oxidase positive. They all induced a hypersensitive response in tobacco (Nicotiana tabacum cv. Samsun). All strains were identified as Pseudomonas cichorii with similarity indices of 79 to 99% based on fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE). Pathogenicity of the strains was confirmed on five dwarf schefflera plants by leaf tissue infiltration with bacterial suspensions (107 CFU ml–1) in sterile distilled water. P. cichorii NCPPB 3802 and sterile water were used as positive and negative controls, respectively. The same symptoms as those observed in the commercial greenhouses were observed on dwarf schefflera leaves within 12 to 15 days after inoculation. The bacteria were reisolated from the inoculated plants and identified as the same as the original strain by conventional tests and FAME analysis. Negative control plants remained disease free. Occurrence of bacterial leaf spot caused by P. cichorii on vegetable crops in Turkey (1,3) and dwarf schefflera in other countries (2) has been reported previously, but to our knowledge, this is the first report of the observation of P. cichorii on dwarf schefflera in Turkey. References: (1) Y. Aysan et al. Plant Pathol. 52:782. 2003. (2) A. R. Chase and D. D. Brunk. Plant Dis. 68:73, 1984. (3) F. Sahin et al. Acta Hortic. 695:93, 2005.

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First Report of Alternaria Leaf Spot Caused by Alternaria sp. on Highbush Blueberry (Vaccinium corymbosum) in South Korea

Plant Disease ◽

10.1094/pdis-04-14-0344-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1434-1434

Author(s):

J.-H. Kwon ◽

D.-W. Kang ◽

M.-G. Cheon ◽

J. Kim

Keyword(s):

South Korea ◽

Leaf Spot ◽

Disease Incidence ◽

Vaccinium Corymbosum ◽

Its Rdna ◽

Universal Primers ◽

Conidial Suspension ◽

First Report ◽

Representative Isolate ◽

Alternaria Sp

In South Korea, the culture, production, and consumption of blueberry (Vaccinium corymbosum) have increased rapidly over the past 10 years. In June and July 2012, blueberry plants with leaf spots (~10% of disease incidence) were sampled from a blueberry orchard in Jinju, South Korea. Leaf symptoms included small (1 to 5 mm in diameter) brown spots that were circular to irregular in shape. The spots expanded and fused into irregularly shaped, large lesions with distinct dark, brownish-red borders. The leaves with severe infection dropped early. A fungus was recovered consistently from sections of surface-disinfested (1% NaOCl) symptomatic leaf tissue after transfer onto water agar and sub-culture on PDA at 25°C. Fungal colonies were dark olive and produced loose, aerial hyphae on the culture surfaces. Conidia, which had 3 to 6 transverse septa, 1 to 2 longitudinal septa, and sometimes also a few oblique septa, were pale brown to golden brown, ellipsoid to ovoid, obclavate to obpyriform, and 16 to 42 × 7 to 16 μm (n = 50). Conidiophores were pale to mid-brown, solitary or fasciculate, and 28 to 116 × 3 to 5 μm (n = 50). The species was placed in the Alternaria alternata group (1). To confirm the identity of the fungus, the complete internal transcribed spacer (ITS) rDNA region of a representative isolate, AAVC-01, was amplified using ITS1 and ITS4 primers (2). The DNA products were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and the resulting pOR13 plasmid was sequenced using universal primers. The resulting 570-bp sequence was deposited in GenBank (Accession No. KJ636460). Comparison of ITS rDNA sequences with other Alternaria spp. using ClustalX showed ≥99% similarity with the sequences of A. alternata causing blight on Jatropha curcas (JQ660842) from Mexico and Cajannus cajan (JQ074093) from India, citrus black rot (AF404664) from South Africa, and other Alternaria species, including A. tenuissima (WAC13639) (3), A. lini (Y17071), and A. longipes (AF267137). Two base substitutions, C to T at positions 345 and 426, were found in the 570-bp amplicon. Phylogenetic analysis revealed that the present Alternaria sp. infecting blueberry grouped separately from A. tenuissima and A. alternata reported from other hosts. A representative isolate of the pathogen was used to inoculate V. corymbosum Northland leaves for pathogenicity testing. A conidial suspension (2 × 104 conidia/ml) from a single spore culture and 0.025% Tween was spot inoculated onto 30 leaves, ranging from recently emerged to oldest, of 2-year-old V. corymbosum Northland plants. Ten leaves were treated with sterilized distilled water and 0.025% Tween as a control. The plants were kept in a moist chamber with >90% relative humidity at 25°C for 48 h and then moved to a greenhouse. After 15 days, leaf spot symptoms similar to those observed in the field developed on the inoculated leaves, whereas the control plants remained asymptomatic. The causal fungus was re-isolated from the lesions of the inoculated plants to fulfill Koch's postulates. To our knowledge, this is the first report of Alternaria sp. on V. corymbosum in South Korea. References: (1) E. G. Simmons. Page 1797 in: Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands, 2007. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (3) M. P. You et al. Plant Dis. 98:423, 2014.

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First report of Diaporthe ueckerae causing stem canker on soybean (Glycine max L.) in Colombia

Plant Disease ◽

10.1094/pdis-04-21-0718-pdn ◽

2021 ◽

Author(s):

Nathali López-Cardona ◽

YUDY ALEJANDRA GUEVARA ◽

Lederson Gañán-Betancur ◽

Carol Viviana Amaya Gomez

Keyword(s):

Management Strategies ◽

Elongation Factor ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Stem Canker ◽

Growth Stages ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Glycine Max L ◽

Soybean Plants

In October 2018, soybean plants displaying elongated black to reddish-brown lesions on stems were observed in a field planted to the cv. BRS Serena in the locality of Puerto López (Meta, Colombia), with 20% incidence of diseased plants. Symptomatic stems were collected from five plants, and small pieces (∼5 mm2) were surface sterilized, plated on potato dextrose agar (PDA) and incubated for 2 weeks at 25°C in darkness. Three fungal isolates with similar morphology were obtained, i.e., by subculturing single hyphal tips, and their colonies on PDA were grayish-white, fluffy, with aerial mycelium, dark colored substrate mycelium, and produced circular black stroma. Pycnidia were globose, black, occurred as clusters, embedded in tissue, erumpent at maturity, with an elongated neck, and often had yellowish conidial cirrus extruding from the ostiole. Alpha conidia were observed for all isolates after 30 days growth on sterile soybean stem pieces (5 cm) on water agar, under 25ºC and 12 h light/12h darkness photoperiod. Alpha conidia (n = 50) measured 6.0 – 7.0 µm (6.4 ± 0.4 µm) × 2.0 – 3.0 µm (2.5± 0.4 µm), were aseptate, hyaline, smooth, ellipsoidal, often biguttulate, with subtruncate base. Beta conidia were not observed. Observed morphological characteristics of these isolates were similar to those reported in Diaporthe spp. by Udayanga et al. (2015). DNA from each fungal isolate was used to sequence the internal transcribed spacer region (ITS), and the translation elongation factor 1-α (TEF1) gene, using the primer pairs ITS5/ITS4 (White et al. 1990) and EF1-728F/EF1- 986R (Carbone & Kohn, 1999), respectively. Results from an NCBI-BLASTn, revealed that the ITS sequences of the three isolates (GenBank accessions MW566593 to MW566595) had 98% (581/584 bp) identity with D. miriciae strain BRIP 54736j (NR_147535.1), whereas the TEF1 sequences (GenBank accessions MW597410 to MW597412) had 97 to 100% (330-339/339 bp) identity with D. ueckerae strain FAU656 (KJ590747). The species Diaporthe miriciae R.G. Shivas, S.M. Thomps. & Y.P. Tan, and Diaporthe ueckerae Udayanga & Castl. are synonymous, with the latter taking the nomenclature priority (Gao et al. 2016). According to a multilocus phylogenetic analysis, by maximum likelihood, the three isolates clustered together in a clade with reference type strains of D. ueckerae (Udayanga et al. 2015). Soybean plants cv. BRS Serena (growth stages V3 to V4) were used to verify the pathogenicity of each isolate using a toothpick inoculation method (Mena et al. 2020). A single toothpick colonized by D. ueckerae was inserted directly into the stem of each plant (10 plants per isolate) approximately 1 cm below the first trifoliate node. Noncolonized sterile toothpicks, inserted in 10 soybean plants served as the non-inoculated control. Plants were arbitrarily distributed inside a glasshouse, and incubated at high relative humidity (>90% HR). After 15 days, inoculated plants showed elongated reddish-brown necrosis at the inoculated sites, that were similar to symptoms observed in the field. Non-inoculated control plants were asymptomatic. Fungal cultures recovered from symptomatic stems were morphologically identical to the original isolates. This is the first report of soybean stem canker caused by D. ueckerae in Colombia. Due to the economic importance of this disease elsewhere (Backman et al. 1985; Mena et al. 2020), further research on disease management strategies to mitigate potential crop losses is warranted.

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First Report of Epicoccum layuense Causing Leaf Brown Spot on Camellia oleifera in Hefei, China

Plant Disease ◽

10.1094/pdis-08-21-1726-pdn ◽

2022 ◽

Author(s):

Xiang Xie ◽

Shiqiang Zhang ◽

Qingjie Yu ◽

Xinye Li ◽

Yongsheng Liu ◽

...

Keyword(s):

Leaf Spot ◽

Brown Spot ◽

Likelihood Method ◽

Leaf Spot Disease ◽

Original Strain ◽

Conidial Suspension ◽

Camellia Oleifera ◽

Sterile Water ◽

Beta Tubulin ◽

First Report

Camellia oleifera, a major tree species for producing edible oil, is originated in China. Its oil is also called ‘‘eastern olive oil’’ with high economic value due to richness in a variety of healthy fatty acids (Lin et al. 218). However, leaves are susceptible to leaf spot disease (Zhu et al. 2014). In May 2021, we found circular to irregular reddish-brown lesions, 4-11 mm in diameter, near the leaf veins or leaf edges on 30%-50% leaves of 1/3 oil tea trees in a garden of Hefei City, Anhui Province, China (East longitude 117.27, North latitude 31.86) (Figure S1 A). To isolate the causal agents, symptomatic leaves were cut from the junction of diseased and healthy tissues (5X5 mm) and treated with 70 % alcohol for 30 secs and 1 % NaClO for 5 min, and subsequently inoculated onto PDA medium for culture. After 3 days, hyphal tips were transferred to PDA. Eventually, five isolates were obtained. Then the isolates were cultured on PDA at 25°C for 7 days and the mycelia appeared yellow with a white edge and secreted a large amount of orange-red material to the PDA (Figure S1 B and C). Twenty days later, the mycelium appeared reddish-brown, and sub-circular (3-10 mm) raised white or yellow mycelium was commonly seen on the Petri dish, and black particles were occasionally seen. Meanwhile, the colonies on the PDA produced abundant conidia. Microscopy revealed that conidia were globular to pyriform, dark, verrucose, and multicellular with 14.2 to 25.3 μm (=19.34 μm, n = 30) diameter (Figure S1 D). The morphological characteristics of mycelial and conidia from these isolates are similar to that of Epicoccum layuense (Chen et al.2020). To further determine the species classification of the isolates, DNA was extracted from 7-day-old mycelia cultures and the PCR-amplified fragments were sequenced for internal transcribed spacer (ITS), beta-tubulin and 28S large subunit ribosomal RNA (LSU) gene regions ITS1/ITS4, Bt2a/Bt2b and LR0R/LR5, followed by sequencing and molecular phylogenetic analysis of the sequences analysis (White et al. 1990; Glass and Donaldson 1995; Vilgalys and Hester 1990). Sequence analysis revealed that ITS, beta-tubulin, and LSU divided these isolates into two groups. The isolates AAU-NCY1 and AAU-NCY2, representing the first group (AAU-NCY1 and AAU-NCY5) and the second group (AAU-NCY2, AAU-NCY3 and AAU-NCY4), respectively, were used for further studies. Based on BLASTn analysis, the ITS sequences of AAU-NCY1 (MZ477250) and AAU-NCY2 (MZ477251) showed 100 and 99.6% identity with E. layuense accessions MN396393 and KY742108, respectively. And, the beta-tubulin sequences (MZ552310; MZ552311) showed 99.03 and 99.35% identity with E. layuense accessions MN397247 and MN397248, respectively. Consistently, their LSU (MZ477254; MZ477255) showed 99.88 and 99.77% identity with E. layuense accessions MN328724 and MN396395, respectively. Phylogenetic trees were built by maximum likelihood method (1,000 replicates) using MEGA v.6.0 based on the concatenated sequences of ITS, beta-tubulin and LSU (Figure S2). Phylogenetic tree analysis confirmed that AAU-NCY1 and AAU-NCY2 are closely clustered with E. layuense stains (Figure S2). To test the pathogenicity, conidial suspension of AAU-NCY2 (106 spores/mL) was prepared and sterile water was used as the control. Twelve healthy leaves (six for each treatment) on C. oleifera tree were punched with sterile needle (0.8-1mm), the sterile water or spore suspension was added dropwise at the pinhole respectively (Figure S1 E and F). The experiment was repeated three times. By ten-day post inoculation, the leaves infected by the conidia gradually developed reddish-brown necrotic spots that were similar to those observed in the garden, while the control leaves remained asymptomatic (Figure S1 G and H). DNA sequences derived from the strain re-isolated from the infected leaves was identical to that of the original strain. E. layuense has been reported to cause leaf spot on C. sinensis (Chen et al. 2020), and similar pathogenic phenotypes were reported on Weigela florida (Tian et al. 2021) and Prunus x yedoensis Matsumura in Korea ( Han et al. 2021). To our knowledge, this is the first report of E. layuense causing leaf spot on C. oleifera in Hefei, China.

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First Report of Leaf Spot Caused by Pseudomonas cichorii on Coreopsis lanceolata in Italy

Plant Disease ◽

10.1094/pdis-93-9-0967a ◽

2009 ◽

Vol 93 (9) ◽

pp. 967-967 ◽

Cited By ~ 1

Author(s):

A. Garibaldi ◽

G. Gilardi ◽

C. Moretti ◽

M. L. Gullino

Keyword(s):

Leaf Spot ◽

Casein Hydrolysate ◽

Its Region ◽

Soft Rot ◽

Chrysanthemum Morifolium ◽

Luria Bertani ◽

Nutrient Broth ◽

Bacterial Leaf Spot ◽

Pseudomonas Cichorii ◽

First Report

Coreopsis lanceolata L. (Compositae), an ornamental species grown in parks and gardens, is very much appreciated for its long-lasting flowering period. In August of 2008, pot-grown plants with necrotic leaf lesions were observed in a commercial nursery located near Biella (northern Italy). Lesions were present, especially along the margin of basal leaves, and sometimes had a chlorotic halo. On infected leaves, dark brown necrosis developed. Leaf stalks were sometimes affected. In many cases, the leaves, especially those at collar level, were withered. Of 1,500 plants, 15% were infected by the disease. Microscopic examination did not reveal any fungal structures within the lesions. Small fragments of tissue from 30 affected leaves were macerated for 15 min in casein hydrolysate and 0.1-ml aliquots of the resulting suspension were spread onto Luria Bertani agar (LB) and potato dextrose agar (PDA). Plates were maintained at 22 ± 1°C for 48 h. No fungi were isolated from the leaf spots on LB or PDA. Colonies similar to those of Pseudomonas spp. were consistently isolated on LB. Colonies were fluorescent on King's medium B, levan negative, oxidase positive, potato soft rot negative, arginine dihydrolase negative, and tobacco hypersensitivity positive (LOPAT test). The bacterial colonies were identified as Pseudomonas cichorii (2). The internal transcribed spacer (ITS) region of rDNA was amplified using primers 27F and 1492R and sequenced (GenBank Accession No. FJ534557). BLAST analysis (1) of the 998-bp segment showed a 98% homology with the sequence of P. cichorii. The pathogenicity of one isolate was tested twice by growing the bacterium in nutrient broth shake cultures for 48 h at 20 ± 1°C. The suspension was centrifuged, the cell pellet resuspended in sterile water to a concentration of 107 CFU/ml, and 30 4-month-old healthy coreopsis plants were sprayed with the inoculum. The same number of plants was sprayed with sterile nutrient broth as a control. After inoculation, plants were covered with plastic bags for 48 h and placed in a growth chamber at 20 ± 1°C. Five days after inoculation, lesions similar to those seen in the field were observed on all plants inoculated with the bacterium, but not on the controls. Ten days later, 40% of the leaves were withered. Isolations were made from the lesion margins on LB and the resulting bacterial colonies were again identified as P. cichorii. The pathogen caused the same symptoms also on plants of Dendranthema frutescens (cv. Camilla), Chrysanthemum morifolium (cvs. Eleonora and Captiva), and an Osteospermum sp. (cv. Wild side) when artificially inoculated with the pathogen with the same methodology. The same bacterial leaf spot caused by P. cichorii was observed in 2005 in other nurseries in the same area on Phlox paniculata (3). To our knowledge, this is the first report of bacterial leaf spot caused by P. cichorii on C. lanceolata in Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) H. Bergey et al. Bergey's Manual on Determinative Bacteriology. Williams and Wilkins, Baltimore, MD, 1994. (3) A. Garibaldi et al. Plant Dis. 89:912, 2005.

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First Report of a Leaf Spot Disease of Tobacco Caused by Pseudomonas cichorii in China

Plant Disease ◽

10.1094/pdis-10-21-2158-pdn ◽

2021 ◽

Author(s):

Dayu Lan ◽

Fangling Shu ◽

Yanhui Lu ◽

Anfa Shou ◽

Wei Lin ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Leaf Spot ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Pseudomonas Cichorii ◽

Koch’S Postulates ◽

First Report ◽

Initial Symptoms ◽

Wide Range ◽

Koch's Postulates

Tobacco (Nicotiana tabacum L.), one of the chief commercial crops, is wildly cultivated worldwide. In June 2020 and 2021, an unknown bacterial leaf spot on tobacco was found in Hezhou and Hechi City, Guangxi, China. 30% of the tobacco were affected and the rate of diseased leaves reached about 10% in the field under high temperature and rainstorm. The disease mainly damaged the middle and top leaves of tobacco plants at vigorous growing stage. The initial symptoms were water-soaked spots on the frontal half of a leaf, and then expanded into circular to irregular spots with a yellow halo at the edge. The spots mostly appeared dark brown at high air humidity, while yellow brown at low humidity and exhibited a concentric pattern. In severe cases, the lesions coalesced and the whole leaf was densely covered with lesions, resulting in the loss of baking value. A bacterium was consistently isolated from diseased leaf tissues on nutrient agar (NA). Growth on NA was predominantly grayish white circular bacterial colonies with smooth margins, and the bacterium is rod-shaped, gram-negative and fluorescent on King’s B medium. Seven isolates (ND04A-ND04C and ZSXF02-ZSXF05) were selected for molecular identification and pathogenicity tests. Genomic DNA of the bacterium was extracted and the housekeeping gene of cts (encoding citrate synthase) was amplified with the primers cts-Fs/cts-Rs (forward primer cts-Fs: 5’-CCCGTCGAGCTGCCAATWCTGA-3’; reverse primer cts-Rs: 5’-ATCTCGCACGGSGTRTTGAACATC-3’) (Berge et al. 2014; Sarkar et al. 2004). 409-bp cts gene sequences were deposited in the GenBank database for seven isolates (accession no. OK105110-OK105116). Sequence of seven isolates shared 100% identity with several Pseudomonas cichorii strains within the GenBank database (accession no. KY940268 and KY940271), and the phylogenetic tree of cts genes of the seven isolates clustered with the phylogroup 11 of Pseudomonas syringae (accession no. KJ877799 and KJ878111), which was classified as P.cichorii. To satisfy Koch’s postulates, a pathogenicity test was tested by using a needle to dip a suspension of the bacterium (108 CFU/ml) and pricking three holes in the tobacco leaf. The control plants leaves were needled with sterile water. Each tobacco plant was inoculated with three leaves, and the test was repeated three times. All plants were placed in transparent plastic boxes and incubated in a greenhouse at 25 ± 3°C. The water-soaked spots appeared 24h after inoculation and quickly expanded through leaf veins. Three days after inoculation, all the inoculated leaves showed symptoms similar to those observed in the field. Control plants remained healthy. Only P. cichorii was successfully re-isolated from the lesions, confirming Koch’s postulates. Pseudomonas cichorii can infect eggplant, lettuce, tomatoand other crops, and has a wide range of hosts (Timilsina et al. 2017; Ullah et al. 2015). To our knowledge, this is the first report of P. cichorii causing leaf spot on tobacco in China.

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First Report of Bacterial Leaf Spot of Cucumber Caused by Pseudomonas cichorii in China

Plant Disease ◽

10.1094/pdis-02-18-0267-pdn ◽

2019 ◽

Vol 103 (1) ◽

pp. 147

Author(s):

X. Y. Fu ◽

R. Y. Zhang ◽

Z. Q. Tan ◽

T. Liu ◽

Z. Q. Peng

Keyword(s):

Leaf Spot ◽

Bacterial Leaf Spot ◽

Pseudomonas Cichorii ◽

First Report

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First Report of Pseudomonas cichorii Causing Leaf Spot of Vegetable Sponge Gourd in China

Plant Disease ◽

10.1094/pdis-03-13-0312-pdn ◽

2014 ◽

Vol 98 (1) ◽

pp. 153-153 ◽

Cited By ~ 3

Author(s):

B. J. Li ◽

H. L. Li ◽

Y. X. Shi ◽

X. W. Xie

Keyword(s):

Leaf Spot ◽

Pathogenic Bacteria ◽

Identification System ◽

Pathogenicity Test ◽

Distilled Water ◽

Pseudomonas Cichorii ◽

First Report ◽

Microbial Identification ◽

Pectolytic Activity ◽

Sponge Gourd

A suspect bacterial leaf spot on vegetable sponge gourd (Luffa cylindrical (L.) Roem.) was found in a commercial greenhouse in Pi County, Chengdu City, Sichuan Province, China, in February 2011. Approximately 20 to 30% of plants were affected, causing serious economic loss. Symptoms occurred only on seedlings and consisted of water-soaked, irregularly shaped, black lesions on the surface and margins of cotyledons. A bacterium was consistently isolated on nutrient agar from diseased leaf tissues that had been surface disinfected in 70% ethyl alcohol for 30 s. The bacterium produced small gray colonies with smooth margins, was gram negative, fluoresced on King's B medium, and showed pectolytic activity when inoculated on potato slices. The partial sequences of 16SrRNA gene (1,377 bp) of the bacterium (GenBank Accession No. KC762217), amplified by using universal PCR primers 16SF (5′-AGAGTTTGATCCTGGCTCAG-3′) and 16SR (5′-GGTTACCTTGTTACGACTT-3′), shared 100% similarity with that of Pseudomonas cichorii (GenBank Accession No. HM190228). The vegetable sponge gourd isolate was also identified by using the Biolog Microbial Identification System (version 4.2, Biolog Inc., Hayward, CA) as P. cichorii with the following characteristics (1): negative for arginine dihydrolase, gelatin liquefaction, and N2 production. Positive reactions were obtained in tests for catalase, oxidase, potato rot, utilization of melibiose, and mannitol. Tests were negative for utilization of sucrose, trehalose, D-arabinose, raffinose, cellobiose, and rhamnose. A pathogenicity test was conducted on 4-week-old vegetable sponge gourd plants by spray-inoculation with 108 CFU/ml sterile distilled water on the leaves of 15 vegetable sponge gourd plants and by needle puncture on the stems of 15 other plants with P. cichorii, respectively. Control plants were misted with sterile distilled water or punctured on the stem with a clean needle. Plants were placed in a greenhouse maintained at 28 ± 2°C with relative humidity of 80 to 85%. Symptoms, the same as seen on the original diseased plants, developed after 7 to 10 days on inoculated plants. Control plants remained healthy. The bacterium was readily re-isolated from inoculated plants and identified as P. cichorii using P. cichorii-specific primer hrpla/hrp2a (1). To our knowledge, this is the first report of P. cichorii causing disease on commercially grown vegetable sponge gourd in China. This new finding will provide the basis for developing resources for diagnostics and management, including screening varieties for resistance. References: (1) S. Mazurier et al. J. FEMS Microbiol. Ecol. 49:455, 2004. (2) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. APS Press, St. Paul, MN, 2001.

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First Report of a Leaf Spot of Radermachera sinica in China Caused by Bacillus megaterium

Plant Disease ◽

10.1094/pdis-05-14-0469-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1425-1425 ◽

Cited By ~ 3

Author(s):

Y. L. Li ◽

Z. Zhou ◽

Y. C. Yuan ◽

J. R. Ye

Keyword(s):

16S Rdna ◽

Bacillus Megaterium ◽

Leaf Spot ◽

Disease Incidence ◽

Bacterial Suspension ◽

Distilled Water ◽

Biochemical Tests ◽

16S Rdna Gene ◽

First Report ◽

Rdna Gene

Radermachera sinica is widely planted as an ornamental plant in homes, offices, and malls in China. A leaf spot of R. sinica occurred in Luoyang, China, from 2013 to 2014. Lesions mostly occurred in wounds and were irregular with light brown centers and purple borders. One or more lesions on a leaf sometimes covered the entire blade. Eighty plants were surveyed in Luoyang, with disease incidence of 17%. Five millimeter pieces from the borders of lesions were surface-disinfected with 75% ethanol for 30 s, 1% sodium hypochlorite for 5 min, washed three times in sterilized distilled water, placed on nutrient agar (NA) medium at 25°C in darkness, and incubated for 24 to 48 h. Four white, round, smooth, and shiny colonies were selected for further identification. All strains were gram-positive, aerobic rods with many peritrichous flagella, and could grow in medium containing 5% NaCl. The strains were positive for catalase, starch hydrolysis, liquefaction of gelatin, reduction of nitrate, acid production from glucose, mannitol, maltose, lactose, xylose, and pectinose. The strains were positive for phenylalanine deaminase, decomposition of tyrosine, and utilization of citrate. The strains were identified by biochemical tests as Bacillus megaterium (1). To confirm pathogenicity, the strains were grown on NA for 48 h and suspended in sterile distilled water to produce a suspension with a final concentration of 108 CFU/ml. Healthy leaves of biennial R. sinica plants were sterilized with 75% ethanol and washed three times with sterilized distilled water. Fresh wounds were made with a sterile needle on the healthy leaves. Each of four strains was tested by spray inoculation with a bacterial suspension on three leaves. Sterile distilled water was used as negative control. Plants were enclosed in plastic bags and placed in a growth chamber at 28°C with 80% relative humidity. After 5 days, water-soaked lesions were observed. Two weeks later, lesions 4 mm in diameter turned light brown with purple borders, and most of lesions occurred in puncture wounds. Symptoms similar to those observed on field plants developed on all inoculated leaves, while no symptoms appeared on the control leaves. B. megaterium was re-isolated from the lesions of inoculated leaves, but not from the control leaves. To confirm the bacterial identification, PCR was performed on the 16S rDNA gene with P1/P2 (P1: CAGAGTTTGATCCTGGCT, P2: AGGAGGTGATCCAGCCGCA) (2) and 1,463 bp of the 16S rDNA gene (GenBank Accession No. KJ789369) showed 100% sequence identity to B. megaterium DSM 319 (NC_014103.1). To our knowledge, this is the first report of a leaf spot of R. sinica caused by B. megaterium in China as well as anywhere in the world. References: (1) P. Vos et al. Bergey's Manual of Systematic Bacteriology. Vol 3: The Firmicutes. Springer, 2009. (2) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.

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