First Report of Leaf Blight on Saposhnikovia divaricata by Pseudomonas viridiflava in Gansu, China

Saposhnikovia divaricata (Turcz) Schischk, a perennial plant in the Umbelliferae, is widely cultivated in north China. As a traditional Chinese medicine, it can be used to cure colds and rheumatism (1). During disease surveys on medicinal plants in August 2010, a bacterial leaf blight was discovered with a general incidence of 40 to 60% on S. divaricata farms in Longxi, Weiyuan County in Gansu China. In young plants, tiny yellow-white points were visible on the backs of the leaves. They then expanded to 2- to 3-mm oil-soaked lesions; leaves appeared crimped and deformed. Later the leaves shriveled; black-brown oil-soaked lesions appeared on the vein and the tissue around it; and black streaks appeared on the stems. Ten diseased leaf and stem tissues were cut into 4- to 5-mm squares, surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed three times, and macerated for 5 min in sterilized distilled water. They were then streaked onto nutrient agar (NA) medium and incubated at 28°C for 3 days. Colonies on NA were round, smooth, translucent, and yellowish green. They were Gram negative and induced a hypersensitive response on tobacco (Nicotiana tabacum L.) leaves. The strain was positive for gelatin, catalase, oxidase, and utilization of glucose and saccharose. Pathogenicity tests were performed by spraying bacterial suspension containing 107 CFU/ml on six leaves of three healthy potted S. divaricata plants and injecting it into another six leaves on three plants. Plants inoculated with sterile distilled water alone served as controls. They were placed in a growth chamber at 25°C and bagged for 24 h to maintain >95% humidity. Thirty-six hours after inoculation, the inoculated leaves appeared water-soaked; 10 days later, the symptoms were apparent on leaves and the plant wilted. The negative control appeared normal. Finally, Koch's postulates were verified by re-isolating P. viridiflava from the leaves with typical blight. The genomic DNA of the isolate was extracted, and the partial 16S rDNA sequence was amplified with a universal bacterial primer set (27f and 1492r) (2). The sequence was deposited in GenBank as KM030291. BLAST search yielded 99% identity with P. viridiflava strains, including the strains KNOX209 (AY604847), RMX3.1b (AY574911), ME3.1b (AY574909), and UASWS0038 (AY919300). Based on the symptoms, colony morphology, biochemical tests, and 16S rDNA sequence identity, the pathogen was identified as P. viridiflava. To our knowledge, this is the first report of leaf blight of S. divaricata by P. viridiflava in Gansu province of China. In Jilin province, the same disease was reported in 2008 (3). The impact of P. viridiflava on S. divaricata production is not yet known. References: (1) Committee of China Pharmacopoeia. Pharmacop. People's Repub. 1:102, 2005. (2) C. Morenol et al. Microbiology 148:1233, 2002. (3) W. Xue. Dissertation. Jilin Agric. Univ. 1, 2008.

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First Report of Streptomyces galilaeus Associated with Common Scab in China

Plant Disease ◽

10.1094/pdis-07-13-0699-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 683-683 ◽

Cited By ~ 4

Author(s):

F. L. Guo ◽

H. Y. Zhang ◽

X. M. Yu ◽

W. Q. Zhao ◽

D. Q. Liu ◽

...

Keyword(s):

16S Rdna ◽

Common Scab ◽

Potato Scab ◽

Rdna Sequence ◽

Malt Extract ◽

16S Rdna Sequence ◽

First Report ◽

Carbon And Nitrogen ◽

Physiological Characterization ◽

Extract Agar

During a survey of potato scab pathogens in China from 2003 to 2012, a new pathogen was found in Shanxi and Neimenggu provinces. The incidence was approximately 20% of all recovered strains. The lesions caused by the pathogen were slightly raised and similar to those caused by Streptomyces scabies (3). Lesions were excised (approximately 10 mm3) from 40 infected tubers, surface-disinfested with 0.3% NaOCl for 30 s, rinsed in sterile water three times, cut into 5 mm3, then sliced into 1-mm pieces, and plated on water agar amended with ampicillin (50 μg/ml). Plates were incubated at 28°C in the dark for 4 days. The spores of Streptomyces sp. strains growing from the tuber pieces were collected from single bacterial colonies and cultured on oatmeal agar. To fulfill Koch's postulates, one strain, CPS-2, was grown at 28°C for 10 days and the spores were washed from the plates as inoculum. One hundred milliliters of inoculum (1 × 105 CFU/ml) was mixed with autoclaved soil and vermiculite (1:1) in each pot (15 cm in diameter). Cut tubers were planted in the pots (potato cv. Favorita, one plant per pot, five replicates) and grown under greenhouse conditions (22 ± 5°C). Typical common scab symptoms consisting of small, brown, raised lesions developed on potato tubers 12 weeks after planting. The same strain was re-isolated from the lesions of the new scabby tubers. Non-inoculated plants, treated as described above, but without strain CPS-2, remained healthy. The CPS-2 strain was identified based on morphological and physiological characterization and 16S rDNA sequence. On yeast-malt extract agar, the test strain produced grayish-white aerial hypha, reddish brown substrate mycelium and pigments, and loose spiral spore chains. Spores were smooth and were 0.8 to 0.9 × 1.1 to 1.2 μm in size (diameter and length). The ability of the strain to use single sources of carbon and nitrogen was verified according to the International Streptomyces project (4). The strain grew in media supplemented with L-arabinose, D-fructose, D-glucose, rhamnose, raffinose, meso-inositol, sucrose, and D-xylose, but not D-mannitol. It used L-hydroxyproline, L-methionine, and L-histidine, and produced melanin on tyrosine and peptone yeast extract agar. The strain did not grow at a pH less than 5.0 and was sensitive to streptomycin (20 μg/ml), phenol (0.1%), and crystal violet (0.5 μg/mL), but not to penicillin (10 IU/ml). The strain also produced hydrogen sulfide. The biological characteristics of strain CPS-2 were in accord with Streptomyces galilaeus. CPS-2 produced thaxtomin A in oatmeal liquid medium and the txt AB gene fragment was successfully amplified using specific primers (2). The 16S rDNA sequence of CPS-2 was amplified by PCR with primers 16S1-F: 5′-CATTCACGGAGAGTTTGATCC-3′ and 16S1-R: 5′-AGAAAGGAGGTGATCCAGCC-3′ (1) and sequenced. A BLAST search of the 16S rDNA sequence for CPS-2 was conducted using the NCBI GenBank database, resulting in 99.8% similarity to S. galilaeus (NR_040857). The 16S rDNA sequence for CPS-2 (1,388 bp) was deposited in GenBank (AY621378). To our knowledge, this is the first report of S. galilaeus causing common scab of potato in China. References: (1) R. A. Bukhalid et al. Appl. Environ. Microbiol. 68:738, 2002. (2) R. Flores-González et al. Plant Pathol. 57:162, 2008. (3) D. H. Lambert and R. Loria. Int. J. Syst. Bacteriol. 39:387, 1989. (4) E. B. Shirling and D. Gottlieb. Int. J. Syst. Bacteriol. 16:313, 1966.

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First Report of Crown Gall, Caused by Agrobacterium tumefaciens on Soapberry (Sapindus delavayi) in China

Plant Disease ◽

10.1094/pdis-10-12-0988-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 685-685

Author(s):

Y. J. Wang ◽

Y. Y. He ◽

Z. Xie ◽

L. Q. Zhang

Keyword(s):

Agrobacterium Tumefaciens ◽

16S Rdna ◽

Crown Gall ◽

Maximum Parsimony Analysis ◽

Bacterial Suspension ◽

Identification System ◽

Distilled Water ◽

Bacterial Identification ◽

Poor Growth ◽

First Report

Soapberry (Sapindus delavayi (Franch.) Radlk.,) plants are widely grown as shade trees in the subtropical to tropical regions of China. In July 2011, large, aerial galls were observed on the above-ground trunks of 5-year-old soapberry plants in two commercial nursery gardens located in Zhejiang Province. Disease incidence was estimated to be 75%. The galls varied in weight from 2 to 24 g and in texture from soft and spongy to hard, and in some cases, the galls completely girdled the trunk. The trees with galls exhibited poor growth compared with healthy trees. Isolations from the grinded and macerated galls yielded nearly pure white, circular, and glistening bacterial colonies on Roy Sauer medium (2). Six random colonies from different galls were selected for bacterial identification, and showed the same morphological, physiological, and biochemical characters and 16S rDNA sequences. All six isolates (isolate SD01 to SD06) were gram negative, rod-shaped bacteria. Carbon source utilization testing with the Biolog GN Bacterial Identification System (version 3.50) confirmed the bacteria as Agrobacterium tumefaciens with a similarity of 0.90. The most-parsimonious tree from the maximum parsimony analysis (PHYLIP package, version 3.68, 500 replicates) of bacterial 16S rDNA gene sequences showed that A. tumefaciens SD01 (GenBank Accession No. JX997939) clustered phylogenetically most closely (99.5% similarity) with A. tumefaciens C58 (AE007870.2). Pathogenicity was confirmed by injecting 3- to 5-week old tomato and sunflower plants and 2-year-old soapberry with approximately 5 μl of the bacterial suspension (108 CFU/ml) in sterile, distilled water. Sterile distilled water was used as a negative control. Ten plants of each treatment were inoculated. Inoculated plants were then transferred to a greenhouse at 25°C. Typical tumors developed at the inoculation sites on tomatoes and sunflower plants 3 weeks after inoculation and on soapberry 6 weeks after inoculation. No symptoms were observed on the control plants. The bacteria that were readily reisolated from the inoculated plants exhibited the same morphological, physiological characters and 16S rDNA sequence as the original culture and were confirmed as A. tumefaciens, fulfilling Koch's postulates. A. tumefaciens is endemic to China and has a very wide host range (1). However, crown gall of soapberry has never been found in China and other countries. To our knowledge, this is the first report of A. tumefaciens on soapberry plants in China. References: (1) M. A. Escobar and A. M. Dandekar. Trends Plant Sci. 8:380, 2003. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, 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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First Report of Group 16SrXII Phytoplasma Causing Stolbur Disease in Potato Plants in the Eastern and Southern Anatolia Regions of Turkey

Plant Disease ◽

10.1094/pdis-06-10-0439 ◽

2010 ◽

Vol 94 (11) ◽

pp. 1374-1374 ◽

Cited By ~ 6

Author(s):

S. Eroglu ◽

H. Ozbek ◽

F. Sahin

Keyword(s):

16S Rdna ◽

Rdna Sequence ◽

Universal Primers ◽

Effective Control ◽

Diseased Plant ◽

Eastern Anatolia ◽

Universal Primer ◽

16S Rdna Sequence ◽

First Report ◽

Potato Plants

In recent years, a stolbur-like disease has had devastating effects on the yield and marketable quality of potato production in Erzurum (Eastern Anatolia) and Akcakale-Sanliurfa (Southern Anatolia) regions of Turkey. Potato plants exhibited several different symptoms including stunting, upward rolling of the top leaves along with reddish or purplish coloration, chlorosis, shortened internodes, swollen nodes, proliferated axillary buds, aerial tubers, and early plant decline. An extensive survey from 2003 to 2010 was performed and diseased plant samples were collected. Total genomic DNAs were isolated from the leaf mid-veins of the six different symptomatic and two symptomless plants selected. Nested-PCRs, carried out by using phytoplasma-universal primer pair P1/P7 followed by R16F2n/R16R2 (2), amplified 16S rDNA fragments (F2nR2) from only templates derived from symptomatic plants. F2nR2 PCR products from two independent symptomatic plants were cloned and sequenced from both directions with M13 universal primers. The obtained 16S rDNA sequence (GenBank Accession No HM485579) was subjected to virtual restriction fragment length polymorphism (RFLP) analysis using iphyclassifier software (3). Results indicated that the phytoplasma, here identified in association with potato plants, shared best sequence identity (99%) with members of subgroup 16SrXII-A (e.g., GenBank Accession No. EU010006). Moreover, collective RFLP pattern of potato-associated phytoplasma differed from digestion profiles of previously described 16SrXII subgroups, sharing best similarity coefficient (0.94) with the reference phytoplasma strain of subgroup 16SrXII-A (GenBank Accession No. AJ964960). Thus, it was confirmed that potato-associated phytoplasma represents a new 16SrXII subgroup (16SrXII-N). Furthermore, a new primer set (PatsecF/PatsecR) was designed for priming specific PCR-amplification of potato-associated phytoplasma 16S rDNA sequence. PCR reaction was successfully used for specifically detecting stolbur phytoplasma in infected potato plants. The use of this method may help to determine possible alternative hosts and vectors of potato phytoplasma, which is important for development of an integrated management strategy for effective control of this disease in the future. Presence of potato stolbur diseases in the Eastern Anatolia Region of Turkey has previously been reported (1). To our knowledge, this is the first report of occurrence of a 16SrXII group phytoplasma causing potato stolbur diseases caused in the Eastern and Southern Anatolia regions of Turkey. References: (1) A. Citir. J. Turk. Phytopathol. 14:53, 1985. (2) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3)Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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First Report of Bacterial Soft Rot of Horn Lian (Typhonium giganteum) Caused by Pectobacterium carotovorum subsp. carotovorum in Jilin Province of China

Plant Disease ◽

10.1094/pdis-03-14-0329-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1268-1268 ◽

Cited By ~ 1

Author(s):

J. Gao ◽

N. Nan ◽

Y. N. Liu ◽

B. H. Lu ◽

W. Y. Xia ◽

...

Keyword(s):

Cell Suspension ◽

16S Rdna ◽

Soft Rot ◽

Rdna Sequence ◽

Jilin Province ◽

Pectobacterium Carotovorum ◽

Bacterial Soft Rot ◽

16S Rdna Sequence ◽

First Report ◽

Carotovorum Subsp

Horn lian (Typhonium giganteum) is a perennial herb of the family Aracea and is commonly used for expelling phlegm and as an antispasmodic treatment. In August 2012, horn lian grown in Changchun, Jilin Province of China, exhibited soft rot disease with ~60% incidence and experienced great losses. Water-soaked and dark green lesions on leaves expanded along main veins. Semitransparent, water-soaked, and sunken lesions on stems expanded rapidly and caused the whole plant to collapse with a foul smell. Nine representative strains were isolated from infected leaves and stems on nutrient agar (NA) medium after 36 h incubation at 28°C (1). Colonies were round, shiny, grayish white, and convex on NA medium. All strains were gram-negative, non-fluorescent on King's B medium (KB), facultatively anaerobic, motile with three to six peritrichous flagella (observed by electron transmission microscope), positive for catalase and pectolytic activity test on potato slices, but negative for oxidase, urease, and lecithinase. Strains grew at 37°C and in yeast salts broth medium containing 5% NaCl. They also liquefied gelatin and reduced nitrate, but did not reduce sucrose. Strains were also negative for starch hydrolysis, malonate utilization, gas production from glucose and indole. Results were variable for the Voges-Proskauer test. The strains utilized sucrose, arabinose, fructose, D-galactose, D-glucose, inositol, lactose, D-mannose, D-mannitol, melibiose, rhamnose, salicin, trehalose, maltose, raffinose, glycerol, D-xylose, and cellobiose as carbon sources, but not melezitose, α-CH3-D-gluconate, sorbitol, or dulcitol. Species identity was confirmed by molecular characterization of one of the nine strains, DJL1-2. DNA GC content indicated by high performance liquid chromatography (HPLC) was 51.7%. The 16S rDNA sequence (KC07897) of DJL1-2 showed 99% identity to that of a Pectobacterium carotovorum subsp. carotovorum (Pcc) strain (CP001657) and the sequence of the 16S-23S rDNA spacer region (KJ623257) was 93% similar to that of another known strain of Pcc (CP003776). As a result, the strains were identified as Pcc (2). Pathogenicity of the nine strains was evaluated by spraying 1 ml of bacterial cell suspension (108 CFU/ml) onto healthy leaves and injecting 0.1 ml of cell suspension into stems of 3-year-old horn lian plants with a sterile pipette tip. Three seedlings were used for each strain and sterilized water served as negative controls. Pcc SMG-2 reference strain (from milk thistle) was also inoculated into horn lian leaves and stems. Inoculated plants were covered with plastic bags for 24 h in a greenhouse at 28 to 30°C. After 72 h, water-soaked lesions similar to the naturally infected plants were observed on leaves and stems inoculated by the nine isolated strains and Pcc SMG-2, while negative control plants remained symptomless. Biochemical tests and 16S rDNA sequence analysis confirmed that the re-isolated bacteria were Pcc. To our knowledge, this is the first report of Pcc causing bacterial soft rot of horn lian in Changchun, Jilin Province, China. References: (1) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press, 1998. (2) N. W. Schaad, et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. American Phytopathological Society, St. Paul, MN, 2001.

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First Report of Herbaspirillum rubrisubalbicans Causing Mottled Stripe Disease on Sugarcane in China

Plant Disease ◽

10.1094/pdis-94-3-0379b ◽

2010 ◽

Vol 94 (3) ◽

pp. 379-379 ◽

Cited By ~ 3

Author(s):

ZQ. Tan ◽

R. Men ◽

RY. Zhang ◽

Z. Huang

Keyword(s):

16S Rdna ◽

Uv Light ◽

Rdna Sequence ◽

Universal Primers ◽

Bacterial Cells ◽

Bacterial Strains ◽

Potato Dextrose Agar Medium ◽

16S Rdna Sequence ◽

First Report ◽

Herbaspirillum Rubrisubalbicans

Narrow, red stripes were observed on leaves and sheaths of sugarcane in 2007 in DanZhou County of Hainan Province and XuWen County of GuangDong Province, China. Stripes were parallel to the leaf veins. Some stripes were short (2 to 10 cm) and some were >1 m long, extending from the base of leaves. Width of the stripes was 2 to 4 mm. Symptoms varied with the cultivar. Cv. Taiwang 25, which was the most affected, exhibited red stripes and stalk death from the apex. Cvs. Taiwang 26 and Guang Dong 00236 were slightly affected with only red stripes. Symptoms on cv. Taiwang 22 were mottled stripes. Severe losses were observed in the infected fields that were planted with cv. Taiwang 25, but there were no obvious losses in fields planted with the other three cultivars. Isolations were made from 10 individual plants from different cultivars and provinces that had red stripes, two of which also had apex death. Five independent bacterial isolates were obtained from tissue showing the red stripe symptoms on potato dextrose agar medium. The percentage of positive samples was 50%. No bacteria were obtained from necrotic apex tissue. Bacterial cells were 0.92 to 1.55 × 0.20 to 0.22 μm slightly curved rods that were motile with one to two polar flagella. Colonies on nutrient agar were 2 to 3 mm in diameter, circular, smooth, entire, and milky white. Colonies on King's medium B were nonfluorescent under 365-nm UV light. Five bacterial strains were inoculated by injecting bacterial suspensions (1 × 108 CFU/ml) into the base of the leaves of 6-month-old cv. Taiwang 25 plants (1). Red stripes appeared 7 to 10 days after inoculation and bacteria were reisolated. The reisolated bacteria were identical to the original strains in colony morphology and 16S rDNA sequence. A hypersensitive response appeared within 24 h when 1 × 108 CFU/ml bacteria suspensions were infiltrated into tobacco leaves. Approximately 1,000-bp DNA fragments were amplified with universal primers UP1 (5′-TACGTGCCAGCAGCCGCGGTAATA-3′) and UP2 (5′-AGTAAGGAGGGTATCCAACCGCA-3′) (3). Primers UP1 and UP2 are complementary to nucleotide sequence 509 to 523 and 1541 to 1522, respectively, of the Escherichia coli 16S rDNA gene. The fragment amplified by these primers was approximately 1,032 bp. The 16S rDNA sequences of the five strains were deposited in GenBank as Accession Nos. GQ476791–5. They all shared 99% nucleotide identity with the type strain of Herbaspirillum rubrisubalbicans (GenBank No. AJ238356.1). All five strains were identified as H. rubrisubalbicans on the basis of 16S rDNA sequence and pathogenicity to sugarcane, and the disease was identified as mottled stripe disease (2). Since we were not able to isolate bacteria from necrotic apex tissue, this symptom on cv. Taiwang 25 may not be related to the H. rubrisubalbicans infection. To our knowledge, this is the first report of mottled stripe disease in China. References: (1) H. M. A. EI-Komy et al. Folia Microbiol. 48:787, 2003. (2) A. S. Saumtally et al. A Guide to Sugarcane Diseases. P. Rott et al., eds. CIRAD and ISSCT, Montpellier, France, 2000. (3) Yan Zhi Yong et al. Chin. J. Epidemiol. 24:296, 2003.

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First Report of Pantoea sp. Induced Soft Rot Disease of Pleurotus eryngii in Korea

Plant Disease ◽

10.1094/pd-91-0109a ◽

2007 ◽

Vol 91 (1) ◽

pp. 109-109 ◽

Cited By ~ 9

Author(s):

M. K. Kim ◽

J. S. Ryu ◽

Y. H. Lee ◽

H. D. Yun

Keyword(s):

16S Rdna ◽

Water Drop ◽

Soft Rot ◽

Pleurotus Eryngii ◽

Rdna Sequence ◽

Pantoea Ananatis ◽

16S Rdna Sequence ◽

First Report ◽

Soft Rot Disease ◽

Rot Disease

The king oyster mushroom, Pleurotus eryngii, has become a popular crop because of its unique flavor and texture and is cultivated in many areas in Korea. In 2003, symptoms of water-soaked lesions and soft rot in the stipes and pileus of cultivated P. eryngii was observed in Jinju, Korea. Diseased tissue was plated on nutrient media. Dominate colonies were yellow, convex, circular with smooth margins, and had a shiny texture. Computer analysis of the data gathered, using the API kit (50CHE, bioMérieux, Marcy-l'Etoile, France), showed that the strain belongs to the Enterobacteriaceae. Although the API system did not give an exact identification, the metabolic profile of the bacterial strain closely resembled the database profile of Pantoea sp. (positive for acid production from the fermentation of d-fructose, d-galactose, d-glucose, d-trehalose, and d-ribose and negative for oxidase, urease, pectate, and thiosulfate). The 16S rDNA sequence of the bacterium was determined (GenBank Accession No. AY530796). When compared with those in GenBank, the bacterium was determined to belong to the Enterobacteriaceae family of the Gammaproteobacteria, and the highest degree of sequence similarity was found to be with Pantoea ananatis strain BD 588 (97.4%) and Pantoea ananatis strain Pna 97-1 (97.3%). In the phylogenetic tree, the bacterium clearly related to the Pantoea lineage, as evidenced by the high bootstrap value. A BLAST search with 16S rDNA sequence of the bacterium supported the API results that the isolate belongs to a species of Pantoea. Pathogenicity tests of this new Pantoea isolate were carried out with bacterial suspensions (approximately 1 × 106 CFU/ml) that were grown for 24 h in Luria-Bertani broth cultures. These were used to inoculate directly on the mycelia of P. eryngii that had been cultivated for 35 days in a plastic bottle. The water and broth were also inoculated to another set of bottles as a control experiment. Inoculated bottles were incubated in a cultivation room at 16 to 17°C with relative humidity between 80 and 95%. Early symptoms of the disease included a dark brown water drop that developed on hypha and primordium of the mushrooms after 5 to 7 days. After 13 days, water-soaked lesions developed on the stipes and pileus, and the normal growth of the mushrooms was inhibited. An offensive odor then developed along with a severe soft rot that was similar to the disease symptoms observed under natural conditions. Mushrooms in control bottles did not develop symptoms. Koch's postulates were fulfilled by isolating bacteria from typical lesions from inoculated mushrooms that were identical to the inoculated strain in colony morphology and biochemical characteristics. Pantoea ananatis was first reported as a pathogen of pineapple fruit causing brown rot (3). Several bacterial diseases, such as brown blotch on cultivated mushrooms by Pseudomonas tolaasii (2) and bacterial soft rot on winter mushroom by Erwinia carotovora subsp. Carotovora, causing severe damage to mushrooms are known (1). However, no Pantoea sp. induced disease of edible mushroom has been previously reported. To our knowledge, this is the first report of soft rot disease on P. eryngii caused by Pantoea sp. References: (1) H. Okamoto et al. Ann. Phytopathol. Soc. Jpn. 65:460. 1999. (2) S. G. Paine. Ann. Appl. Biol. 5:206. 1919. (3) F. B. Serrano. Philipp. J. Sci. 36:271, 1928.

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First report of bacterial leaf blight caused by Xanthomonas hortorum pv. carotae on carrots in Spain

Plant Disease ◽

10.1094/pdis-11-20-2493-pdn ◽

2021 ◽

Author(s):

José Luis Palomo Gómez ◽

Maria Shima ◽

Adela Monterde ◽

Inmaculada Navarro ◽

Silvia Barbé ◽

...

Keyword(s):

Sequence Analysis ◽

Xanthomonas Campestris ◽

Negative Control ◽

Leaf Blight ◽

Bacterial Leaf Blight ◽

Pcr Analysis ◽

Bacterial Suspension ◽

Rrna Gene ◽

Distilled Water ◽

First Report

In September 2019, symptoms resembling those of bacterial leaf blight were observed on carrot plants (Daucus carota L. subsp. sativus Hoffm.) cv. Romance cultivated in commercial plots in Chañe (Segovia), Spain. Symptoms were observed in two plots surveyed representing three hectares, with an incidence greater than 90%, and also in some plots in other nearby municipalities sown with the same batch of seeds. The lesions observed at the ends of the leaves were initially yellow that develop dark brown to black with chlorotic halos on leaflets that turned necrotic. Yellow, Xanthomonas-like colonies were isolated onto YPGA medium (Ridé 1969) from leaf lesions. Two bacterial isolates were selected and confirmed by real-time PCR using a specific primer set for Xanthomonas hortorum pv. carotae (Temple et al. 2013). All isolates were gram-negative, aerobic rods positive for catalase, able of hydrolyzing casein and aesculin and growing at 2% NaCl, while were negative for oxidase and urease tests. Sequences of 16S rRNA gene showed 100% similarity with Xanthomonas campestris, X. arboricola, X. gardneri, X. cynarae strains (GenBank accession numbers: MW077507.1 and MW077508.1 for the isolates CRD19-206.3 and CRD19-206.4, respectively). However, the resulting phylogeny of multilocus sequence analysis (MLSA) of a concatenation of the housekeeping genes atpD, dnaK, and efp (Bui Thi Ngoc et al. 2010), by using neighbour-joining trees generated with 500 bootstrap replicates, grouped the two isolates with the X. hortorum pv. carotae M081 strain (Kimbrel et al. 2011) (GenBank accession numbers: MW161270 and MW161271 for atpD for the two isolates, respectively; MW161268 and MW161269 for dnaK; MW161272 and MW161273 for efp). A pairwise identity analysis revealed a 100% identity between all three isolates. Pathogenicity of the isolates was tested by spray inoculation (Christianson et al. 2015) with a bacterial suspension (108 CFU/ml) prepared in sterile distilled water at 3 to 4 true-leaf stage (six plants per isolate). Sterile distilled water was used as negative control. The inoculated plants were incubated in a growth chamber (25°C and 95% relative humidity [RH]) for 72 h, and then transferred to a greenhouse at 24 to 28°C and 65% RH. Characteristic leaf blight symptoms developed on inoculated carrot plants, while no symptoms were observed on the negative control plants 20 days after inoculation. The bacterium was re-isolated from symptomatic tissue and the identity confirmed through PCR analysis. Based on PCR, morphological and phenotypic tests, sequence analysis, and pathogenicity assays, the isolates were identified as X. hortorum pv. carotae. To our knowledge, this is the first report of bacterial leaf blight of carrot caused by X. hortorum pv. carotae in Spain, and the first molecular and pathological characterization. It is important to early detect this pathogen and take suitable measures to prevent its spread, since it could cause yield losses for a locally important crop such as carrot.

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First Report of New Bacterial Leaf streak of Rice Caused by Pantoea ananatis in China

Plant Disease ◽

10.1094/pdis-11-21-2423-pdn ◽

2022 ◽

Author(s):

Xinhua Ding ◽

Chongchong Lu ◽

Mingxia Hao ◽

Lingguang Kong ◽

Lulu Wang ◽

...

Keyword(s):

16S Rdna ◽

Shandong Province ◽

Bacterial Suspension ◽

Distilled Water ◽

Bacterial Leaf Streak ◽

Accession Number ◽

Pantoea Ananatis ◽

First Report ◽

Rice Leaves ◽

Dark Green

Rice (Oryza sativa L.) is the largest grain crop, accounting for about 40 % of the total grain production in China. In mid-July 2021, bacterial leaf streak-like disease emerged in rice varieties Chunyou584 and Yongyou2604 in Linyi city, Shandong Province, China. Disease incidences of the disease ranged from 80% to 90% in the surveyed fields. Infected rice leaves displayed dark green to yellowish-brown water-soaked thin streaks, and a large amount of beaded yellow oozes were observed on the lesions. After drying, there were gelatinous granules that were not easy to fall off and spread between leaf veins (Fig.S1A). According to the field symptoms of this disease, it was preliminarily suspected to be rice bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc), which is a guaranteed disease in China. To isolate the causal agent, leaf discs (~1 cm2) of diseased leaves were collected from the margins of the lesions, surface sterilized and ground into pieces in sterile double distilled water. The 10-3, 10-4 and 10-5 dilutions were spread onto peptone sugar agar (PSA) and incubated at 28°C for 36 hours. Yellow mucous bacterial colonies were consistently obtained on PSA medium. To identify the pathogen, fragments of the 16S rDNA, leuS and rpoB were amplified and sequenced using the primers previously reported (Yu et al. 2021). Three strains (LY01, LY02 and LY03) showed identical colony morphology and LY01 was used for further analyses. Sequence analyses showed that the fragments of 16S rDNA (955 bp, GenBank accession number: OK261898), leuS (755 bp, GenBank accession number: OK298387) and rpoB (926 bp, GenBank accession number: OK298388) of strain LY01 shared 99.16%, 99.46% and 100% similarities with those of Pantoea ananatis TZ39 (GenBank accession numbers: CP081342.1 for 16S rDNA, MW981338.1 for leuS and MW981344.1 for rpoB), respectively, which suggest the pathogenic bacterial strain LY01 isolated is P. ananatis. In addition, the single colony of P. ananatis LY01 was shown as Fig. S2B. Furthermore, pathogenicity tests were also performed according to the following steps. Bacterial suspension at OD600=0.1 was inoculated into eight rice leaves of four healthy rice plants (Chunyou 584) at 25-33°C and 60%-80% relative humidity in the field using a clipping method (Yang et al. 2020) or spraying methods, and sterile distilled water was as negative control. The clipped leaves (Fig. S1B) and spray-inoculated leaves (Fig. S1C) showed dark green water-soaked streaks at 14 days after inoculation, respectively, which showed similar symptoms with those samples collected from the fields (Fig. S1A). On contrary, the control rice leaves remained healthy and symptomless (Fig. S2A). The bacterium was re-isolated in the inoculated rice leaves and the re-isolated bacterial isolates, which was confirmed by sequencing 16S rDNA, leuS and rpoB, incited the same symptoms as in fields, which fulfills Koch’s postulates. In the past decade, P. ananatis was reported to result in grain discoloration and leaf blight in China (Yan et al. 2010; Xue et al. 2020, Yu et al. 2021), which could result in 40% - 60% yield losses. To our best knowledge, this is the first report of the bacterial leaf streak-likely disease occurred in Shandong Province caused by P. ananatis, so we named it as Pantoea leaf streak of rice. Although P. ananatis was also reported in Zhejiang province and Jiangxi province, which caused leaf streak lesions on rice, the disease symptoms are completely different from those of Pantoea leaf streak of rice. To the best of our knowledge, this is the first report of Pantoea leaf streak of rice caused by P. ananatis. This study provides sloid evidence that Pantoea leaf streak of rice in Eastern China can be caused by the new pathogen, P. ananatis, rather than Xoc as traditionally assumed. Disease development and quarantine of the new Pantoea leaf streak of rice disease caused by P. ananatis on rice need more attention in the near future.

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

Plant Disease ◽

10.1094/pdis-11-12-1047-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 685-685 ◽

Cited By ~ 2

Author(s):

L.-H. Zhou ◽

Y. Han ◽

G.-H. Ji ◽

Z.-S. Wang ◽

F. Liu

Keyword(s):

16S Rdna ◽

Pseudomonas Syringae ◽

Leaf Spot ◽

Panax Notoginseng ◽

Rdna Sequence ◽

Bacterial Leaf Spot ◽

16S Rdna Sequence ◽

First Report ◽

Leaf Spots ◽

Phosphate Buffered Saline

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. References: (1) M. R. Gillings et al. PNAS 12:102, 2005. (2) W. F. Luo et al. J. Yunnan Agric. Univ. 14:123, 1999 (in Chinese). (3) C. L. Oliver et al. Plant Dis. 4:96, 2012. (4) H. Ornek et al. New Dis. Rep. 13:40, 2006.

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