First Report of Group 16SrXII Phytoplasma Causing Stolbur Disease in Potato Plants in the Eastern and Southern Anatolia Regions of Turkey

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

Plant Disease ◽

10.1094/pdis-06-14-0641-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 281-281 ◽

Cited By ~ 1

Author(s):

Y. Wang ◽

C. Y. Zeng ◽

X. R. Chen ◽

C. D. Yang

Keyword(s):

16S Rdna ◽

Leaf Blight ◽

Gansu Province ◽

Rdna Sequence ◽

Bacterial Suspension ◽

Distilled Water ◽

16S Rdna Sequence ◽

First Report ◽

Saposhnikovia Divaricata ◽

The Impact

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 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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A Phytoplasma Closely Related to the Pigeon Pea Witches'-Broom Phytoplasma (16Sr IX) Is Associated with Citrus Huanglongbing Symptoms in the State of São Paulo, Brazil

Phytopathology ◽

10.1094/phyto-98-9-0977 ◽

2008 ◽

Vol 98 (9) ◽

pp. 977-984 ◽

Cited By ~ 54

Author(s):

D. C. Teixeira ◽

N. A. Wulff ◽

E. C. Martins ◽

E. W. Kitajima ◽

R. Bassanezi ◽

...

Keyword(s):

16S Rdna ◽

External Source ◽

Pigeon Pea ◽

Sao Paulo ◽

Rdna Sequence ◽

Universal Primers ◽

Insect Vector ◽

São Paulo ◽

16S Rdna Sequence ◽

Pcr Products

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of São Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and ‘Candidatus Liberibacter asiaticus’. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.

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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 Syringa oblata and S. reticulata Leafroll Disease in China

Plant Disease ◽

10.1094/pdis-93-3-0322c ◽

2009 ◽

Vol 93 (3) ◽

pp. 322-322

Author(s):

Z. N. Li ◽

H. Min ◽

Y. Yan ◽

Z. Zhao ◽

W. J. Wu ◽

...

Keyword(s):

16S Rdna ◽

Rflp Analysis ◽

Universal Primers ◽

University Campus ◽

Sequence Comparisons ◽

16S Rdna Sequence ◽

First Report ◽

Pcr Products ◽

Leafroll Disease ◽

Total Dna

Syringa oblata is an important ornamental tree widely grown in China. In September of 2008, S. oblata plants exhibiting symptoms of leafroll and yellowing were found in a garden on the Northwest A&F University campus. Samples were collected from this site. Total DNA was extracted from 0.5 g of phloem tissue from leaf midribs and stems of each sample. DNA samples were analyzed with a nested PCR assay using phytoplasma 16S rDNA universal primers R16mF2/R16mR1 followed by specific primers R16F2n/R16R2 (1), which amplified a 1,452- and 1,246-bp product, respectively. We tested all 30 lilac samples, 20 of which had symptoms of leafroll and yellowing. These produced the expected 1,452- and 1,246-bp PCR products In contrast, the remaining 10 samples from symptomless trees yielded no PCR products. We also surveyed another lilac variety (Syringa reticulata), which is widely grown on the campus, and tested 50 samples with the above method. Again, 1.4- and 1.2-kb PCR products were amplified from all 30 trees displaying leafroll and yellowing symptoms, but not from the other 20 samples from symptomless trees. A comparative analysis of sequences derived from the two hosts showed that the phytoplasmas infecting them were most similar (>99%) to paulownia witches'-broom (PaWB) phytoplasma (GenBank Accession No. EF199937). Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with endonucleases AluI and MseI indicated that all symptomatic plants were infected by the phytoplasmas belonging to aster yellow group (16SrI) subgroup D (16SrI-D) PaWB phytoplasma (2). 16S rDNA sequence comparisons and RFLP analysis of the cloned 16S rDNA from S. oblata (GenBank Accession No. FJ445224) and S. reticulate (GenBank Accession No. FJ445225) indicated that the phytoplasmas infecting them were nearly identical (99.8% identity). To our knowledge, this is the first report of the presence of the phytoplasma associated with a leafroll disease of S. oblata and S. reticulata in China. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

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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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First Report of Pectobacterium polaris Causing Aerial Stem Rot of Potato in China

Plant Disease ◽

10.1094/pdis-06-21-1269-pdn ◽

2021 ◽

Author(s):

Jinhui Wang ◽

Wanxin Han ◽

Zheng Li ◽

Jianing Cheng ◽

Yang Pan ◽

...

Keyword(s):

16S Rdna ◽

Disease Incidence ◽

Housekeeping Genes ◽

Luria Bertani ◽

Stem Rot ◽

Universal Primers ◽

Management Options ◽

First Report ◽

Potato Plants ◽

Aerial Stem

In July 2020, potato plants (cv. Xisen 6) showing characteristic symptoms of aerial stem rot were observed in a field in Fengning Manchu Autonomous County, Chengde, Hebei Province (northern China). The disease incidence in that field (5 ha in size) was more than 50%. Aerial stem rot of potato has increased in prevalence over recent years in Chengde, it can cause significant yield loss on susceptible cultivars such as Xisen 6 and Huangxin 226. Affected stem (light brown and water-soaked stem sections) pieces ca. 0.5 cm in length were surface-sterilized by dipping them in 75% ethanol for one min and then three successive rinses with sterile distilled water. Then, the tissues were soaked in 200 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the crystal violet pectate (CVP) medium. The CVP plates were incubated at 28°C for 48 h. Colonies producing pits were restreaked and purified on Luria-Bertani (LB) agar plates. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primers 27F/1492R (Weisburg et al. 1991) and sequenced. Results of the Blastn analysis of the 16S rDNA amplicons (MZ348607, MZ348608) suggested that the isolates FN20211 and FN20222 belonged to the genus Pectobacterium. Housekeeping genes including acnA, gapA, icdA, mdh, proA and rpoS were also amplified using a set of primers (Ma et al. 2007; Waleron et al. 2008) followed by sequencing (MZ356250-MZ356261). To determine the species of the stem rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes, and phylogenetic tree was reconstructed using RAxML (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between FN20211 and FN20222. The result of phylogenetic analysis showed that the isolates clustered with P. polaris type strain NIBIO1006T, which was isolated from potato (Dees et al. 2017). And the concatenated sequence of the six loci of isolate FN20211/FN20222 is 100% identical to those of the strains PZ1 (CP046377.1) and WBC1 (GCF_011378945.1), which were isolated from potato in South Korea and from Chinese cabbage in China, respectively. Potato seedlings (cv. Xisen 6 and Favorita) were inoculated with the isolates FN20211 and FN20222 by injecting 100 µl of bacterial suspensions (108 CFU·mL-1) into the upper parts of the stems of potato plants, or injected with 100 µl of 0.9% saline as control. The seedlings were grown at 25°C and 50% relative humidity. Three days after inoculation, only the bacteria-inoculated seedlings showed disease symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected stems and were identified using the same PCR primers as described above. Therefore, P. polaris isolates FN20211 and FN20222 fulfill Koch’s postulates for aerial stem rot of potato. P. polaris causing blackleg and soft rot on potato plants has been reported in European countries including Netherlands, Norway (Dees et al. 2017) and Poland (Waleron et al. 2019), and also in Pakistan (Sarfraz et al. 2019) and Russia (Voronina et al. 2021). To our knowledge, this is the first report of P. polaris causing aerial stem rot of potato in China. The stem rot poses a significant threat to the local potato industry, and further research on epidemiology and disease management options is needed.

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