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

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 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 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 Agrobacterium tumefaciens causing crown gall disease of roselle (Hibiscus sabdariffa L.) in Taiwan

Plant Disease ◽

10.1094/pdis-05-21-1007-pdn ◽

2021 ◽

Author(s):

Huan-Yu Chen ◽

Chun-Chi Lin ◽

Chih-Wei Wang ◽

NAI-CHUN LIN

Keyword(s):

Agrobacterium Tumefaciens ◽

Sequence Analysis ◽

Crown Gall ◽

Causal Agent ◽

Reca Gene ◽

Bacterial Suspension ◽

Hibiscus Sabdariffa ◽

Agrobacterium Vitis ◽

First Report ◽

Crown Gall Disease

Roselle (Hibiscus sabdariffa L.) plants, whose calyces are used for production of beverages or jams, are mainly cultivated in Taitung County of eastern Taiwan. Since 2016, large crown galls were observed on the roselle plants in the commercial plantations at Taimali and Jinfong Townships of Taitung County. A follow-up survey in July and August of 2017 revealed spreading of this disease to the neighboring areas including Beinan and Dawu Townships. Disease incidence was estimated to be 0.6-10%. Galls of varying sizes (2-15 cm in diameter) were usually found on the roots and crowns of the roselle plants, starting with small swellings at the infection sites. Galls were light-colored, and smooth and tender in texture at the early stage, but later turned dark-colored, and appeared rough and woody. In some cases, adventitious roots extruding from the larger crown galls could be seen. Isolation of the causal agent was performed by quadrantally streaking bacterial suspension made from surface-sterilized, macerated galls on trypticase soy agar (TSA). After incubating at 28°C for 5 days, single colonies were transferred onto new TSA plates for further cultivation at 28°C. Finally, circular, convex, viscous and milky white colonies with smooth surface similar to colony morphology of Agrobacterium tumefaciens C58 were obtained for further identification. First, all six candidate isolates (TZ-1, TL1-2, TL2-1, TD1-1, TD1-24 and TD2-1) were identified as Agrobacterium spp. using the partial sequences of the 16S rRNA gene (accession numbers MW205820 to MW205825 in the GenBank database). The selected isolates also showed some biochemical and physiological characteristics similar to A. tumefaciens, including oxidase positive, growth at 35°C and in 2% NaCl, and alkalinity from litmus milk. Moreover, they were tested negative for utilization of citrate and acid production on potato dextrose agar (PDA) supplemented with calcium carbonate. Under a transmission electron microscope, the bacterium was rod-shaped and possessed peritrichous flagella. By means of multiplex PCR using primers designed for differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2, a 184 bp product was detected in all six isolates, indicating that they all belong to Agrobacterium biovar 1. Furthermore, the recA allele of each isolate was PCR amplified using primers F2898/F2899, and recA sequence analysis assigned all six isolates to A. tumefaciens genomospecies G7 (GenBank accession numbers MZ570905-MZ570910). Pathogenicity assay was carried out by inoculating the stems of 2-week-old roselle seedlings through wounds made with a sterile needle with bacteria on it. The inoculated seedlings were kept in plastic bags to maintain high humidity. Symptoms similar to those observed in the field developed at the inoculation sites after 7 days, and Koch’s postulates were fulfilled when the bacteria re-isolated from the galls were also identified as A. tumefaciens genomospecies G7 using recA gene sequence analysis. To our knowledge, this is the first report of crown gall disease caused by A. tumefaciens on Hibiscus sabdariffa in Taiwan. This disease may potentially damage the roselle industry if no action is taken to stop its spreading. Identification of the causal agent of roselle crown gall disease could help us further investigate its ecology and develop integrated pest management strategies for prevention of this disease in the future.

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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 Fire Blight Disease on Blackberry in Turkey

Plant Disease ◽

10.1094/pdis-12-11-1073-pdn ◽

2012 ◽

Vol 96 (12) ◽

pp. 1818-1818

Author(s):

K. K. Bastas ◽

F. Sahin

Keyword(s):

Disease Incidence ◽

Acid Methyl Ester ◽

Negative Control ◽

Culture Collection ◽

Bacterial Suspension ◽

Identification System ◽

Distilled Water ◽

Bacterial Strains ◽

First Report ◽

Microbial Identification

During 2008 and 2009, a new disease on blackberry (Rubus fruticosus cv. Chester) causing leaf and shoot blight and cankers with brown discoloration of necrotic tissues on mature branches was observed in Isparta and Konya provinces of Turkey. Disease incidence was estimated to be 4% for the two years. Isolations were made from lesions on leaves and shoots on nutrient sucrose agar (NSA) medium. Bacteria consistently isolated from the diseased tissues were identified on the basis of biochemical, physiological (2), and molecular tests (1). Eleven representative bacterial strains were gram-negative, rod-shaped, mucoid, fermentative, yellow-orange on Miller and Scroth (MS) medium, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, acid production from lactose, and inositol. Two reference strains of Erwinia amylovora (EaP28 and NCPPB 2791) obtained from the culture collection unit of Selcuk University were used as positive controls. All strains induced a hypersensitive response in tobacco (Nicotiana tobaccum cv White Burley) 24 h after inoculation with a 108 CFU/ml bacterial suspension in water. All strains were identified as E. amylovora using the species-specific primers set A/B (1), which amplified a 1-kb DNA fragment in PCR, and fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (TSBA 6 v. 6.00; Microbial ID, Newark, DE) with similarity indices ranging from of 79 to 99%. Pathogenicity was confirmed by injecting bacterial suspensions (108 CFU/ml–1) in sterile distilled water into the shoot tips of 2-year-old R. fruticosus cv. Chester and the first blighting symptoms were observed on leaves within 3 days and also 10 days later after inoculation on shoots. Sterile distilled water was used as a negative control. No symptoms were observed on control plants. All tests were repeated three times. The bacterium was reisolated from inoculated plants and identified as. E. amylovora. To our knowledge, this is the first report of E. amylovora on blackberry in Turkey. Phytosanitary measures are needed to prevent any further spread of the bacterium to new blackberry areas. References: (1) S. Bereswill et al. App. Environ. Microbiol. 58:3522, 1992. (2) A. L. Jones and K. Geider. Lab. Guide for Identification of Plant Pathological Bacteria, 40, 2001.

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First Report of Acidovorax avenae subsp. citrulli as the Causal Agent of Bacterial Leaf Blight of Betelvine in Taiwan

Plant Disease ◽

10.1094/pdis-94-8-1065a ◽

2010 ◽

Vol 94 (8) ◽

pp. 1065-1065 ◽

Cited By ~ 1

Author(s):

W.-L. Deng ◽

T.-C. Huang ◽

Y.-C. Tsai

Keyword(s):

Leaf Blight ◽

Causal Agent ◽

Bacterial Leaf Blight ◽

Bacterial Suspension ◽

Identification System ◽

Distilled Water ◽

First Report ◽

Microbial Identification ◽

Sequence Identity ◽

Disease Free

In November 2008, betelvines (Piper betle L., Piperaceae) exhibiting leaf blight symptoms were observed in central Taiwan. Infections resulted in a 30 to 70% loss of leaf yield in the investigated betel leaf-producing facilities. Symptoms began with small, necrotic, water-soaked spots that progressed to circular to irregularly shaped brown lesions, 5 to 10 mm in diameter, with chlorotic halos on leaves; some lesions started from the edge of leaves and later fused to form dried, necrotic margins. Bacteria-like streaming fluid was visible from the edges of freshly cut lesions at the junctions of chlorotic and necrotic leaf tissues when observed with a light microscope at ×100. When the streaming fluid was streaked onto King's medium B (3), a slow-growing, gram-negative, nonfluorescent bacterium was identified from the whitish colonies that consistently developed on the medium. Five bacterial isolates from three lesions were characterized with fatty acid methyl ester analysis (Agilent Technologies, Santa Clara, CA) and Sherlock Microbial Identification System (Microbial IDentification Inc., Newark, DE), and for each isolate, the bacterium was confirmed as Acidovorax avenae subsp. citrulli with a similarity index >0.70. In addition, the Biolog system (Biolog, Hayward, CA) and 16S ribosomal RNA sequence identity comparison were performed to confirm that the five betelvine-isolated bacteria were A. avenae subsp. citrulli based on a similarity of 0.54 with Biolog and 99% sequence identity for 16S rRNA gene. Koch's postulates were fulfilled by infiltrating a bacterial suspension of 3 × 105 CFU/ml into 40 leaves of four greenhouse-grown, disease-free, mature betelvine plants. After inoculation, plants were kept in a humidified greenhouse at 28°C to favor symptom development and symptoms similar to those observed in the greenhouse were evident at 7 days post inoculation (dpi) on all bacterium-infiltrated leaves. Control leaves infiltrated with distilled water remained symptomless. Bacteria showing morphological and biochemical similarities (2) to the ones used for inoculation were isolated from all of the inoculated betelvine leaves. In addition, a bacterial suspension at 3 × 108 CFU/ml was sprayed at the amount of 5 ml per plant onto 6 to 10 plants each of 4-week-old disease-free seedlings of watermelon (Citrullus lanatus (Thunb.) Matsum & Nakai, cv. Empire No. 2), oriental sweet melon (Cucumis melo L. var. saccharinus Naudin, cv. Silver Beam), and waxgourd (Benincasa hispida (Thunb.) Cogn., cv. Cheerer) for bioassays, and the inoculated seedlings were enclosed in plastic bags for 36 h at 28°C. Water-soaked lesions were observed on leaves of watermelon and waxgourd at 2 dpi and on sweet melon at 4 dpi on all inoculated plants but not on distilled water-sprayed control plants, indicating that A. avenae subsp. citrulli strains from betelvine could also infect melon plants. A. avenae subsp. citrulli was previously identified as the causal agent of bacterial fruit blotch on melon and bitter gourd in Taiwan (1). To our knowledge, this is the first report that A. avenae subsp. citrulli can naturally infect betelvine, a noncucurbit crop, to elicit bacterial leaf blight disease. References: (1) A.-H. Cheng and T.-C. Huang. Plant Pathol. Bull. 7:216, 1998. (2) J. B. Jones et al. Page 121 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (3) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954.

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First Report of Pectobacterium punjabense causing potato soft rot and blackleg in Serbia

Plant Disease ◽

10.1094/pdis-06-21-1199-pdn ◽

2021 ◽

Author(s):

Marta Loc ◽

Dragana Milošević ◽

Maja Ignjatov ◽

Žarko Ivanović ◽

Dragana Budakov ◽

...

Keyword(s):

16S Rdna ◽

Potato Plant ◽

Soft Rot ◽

Economic Losses ◽

Bacterial Suspension ◽

Distilled Water ◽

Potato Tubers ◽

First Report ◽

Potato Plants ◽

16S Rdna Pcr

Soft rot and blackleg are common diseases affecting potato (Solanum tuberosum) production in Serbia. Pectinolytic plant pathogens belonging to the genera Pectobacterium cause soft rot and wilt diseases by plant cell wall degradation. These opportunistic phytopathogens lead to considerable economic losses in many potato-growing regions worldwide and are listed among top 10 plant pathogenic bacteria (Mansfield et al. 2012). Potato plants (cv. VR808) with symptoms of wilting, slow growth, stem blackening and tubers softening, were collected from a commercial potato field in Zobnatica (Serbia) in July 2019 and subjected to analysis. All symptoms occurred in the same field and the incidence of symptomatic plants was approximately 5%. Isolation was performed from 10 randomly chosen potato plant and tuber samples, expressing wilting and soft rot symptoms. Plant tissue was surface-disinfected and 1 cm length sections from the margins of lesions were macerated in sterile distilled water for 25 min and streaked on nutrient-agar medium. After 48 h of incubation at 26°C, predominant shiny, cream-colored, round colonies were obtained from all samples. Three representative isolates (MMZKVR1, MMZCVR2, and MMZKVR3) from independent samples were selected randomly and subjected to biochemical and pathogenicity tests. Isolates were gram-negative, nonfluorescent facultative anaerobes, exhibiting pectinolytic activity on potato tuber slices and hypersensitive response on tobacco leaves. They expressed catalase activity but did not express oxidase or acid phosphatase activity or produce indole. All strains grew at 37°C, in 5% NaCl, and reduced nitrate. Pathogenicity of the obtained isolates was tested on 3-week-old healthy potato plants (cv. VR808 and cv. Kiebitz) grown in commercial Baltic Tray Substrate (Hawita) in the greenhouse, as well as on potato tubers of the same varieties. Three potato plant stems per isolate were inoculated by the toothpick piercing method (Duarte et al. 2004) using bacterial suspension (approx. 1 × 108 CFU/ml). Inoculated plants were incubated under plastic bags in a greenhouse at 25 ± 2°C. Blackleg symptoms and stem wilting developed 48 hours after inoculation. No symptoms were observed on plants inoculated with sterile toothpicks dipped in sterile distilled water. The pathogen was re-isolated from symptomatic plants, fulfilling Koch's postulates and sequencing of 16S rDNA confirmed the originally isolated pathogen. Three potato tubers per isolate were inoculated by toothpicks dipped in bacterial suspension (approx. 1 × 108 CFU/ml). Inoculated tubers were placed in a sealed plastic container at 25 ± 2°C. Treatment with sterile distilled water was used as a negative control. Softening of the tissue around the inoculation point developed within 48 h from inoculation, and no symptoms developed on the control tubers. For molecular analyses, total DNA of the isolates was extracted using the DNeasy Plant Mini Kit (Qiagen). The isolates were not detected in diagnostic PCR assays using specific primers Br1F/L1R for the detection of P. brasiliense (Duarte et al. 2004) and primers EXPCCF/EXPCCR for P. catotovorum subsp. carotovorum (Kang et al. 2003). The 16S rDNA PCR amplification was performed using the universal PCR primer pair 27F/1492R (Fredriksson et al. 2013) and followed by Sanger sequencing (Macrogen Europe BV). The BLASTn analysis of sequences (GenBank Accession Numbers MZ048661, MZ048662, and MZ157274) revealed 100% query coverage and 100% identity to the sequences of Pectobacterium punjabense in NCBI (MT242589 and CP038498) isolated from potato in China and Pakistan (Sarfraz et al. 2018), respectively. All three obtained isolates were proposed to belong to Pectobacterium punjabense sp. nov. To further validate the identification, isolate MMZCVR2 of P. punjabense was selected for multilocus sequence analyses of 5 housekeeping genes (gyrA, recA, recN, rpoA and rpoS). The gyrA (MZ161817), recA (MZ161818), recN (MZ161819), rpoA (MZ161820) and rpoS (MZ161821) sequence analysis showed the highest nucleotide identity (99.44 to 100%) with P. punjabense strain SS95 (Sarfraz et al. 2018) previously deposited in NCBI GenBank database. To our knowledge, this is the first report of blackleg and soft rot caused by P. punjabense on potato in Serbia. Pectobacterium punjabense is a newly described species causing soft rot and blackleg disease in potato plants (Sarfraz et al. 2018). Its current geographic distribution is not well-described but important to know since soft rot bacteria are easily transported long distances in latently infected seed tubers and can cause significant economic losses in potato production worldwide.

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First Report of Crown Gall, Caused by Agrobacterium tumefaciens on Geraldton Wax (Chamelaucium uncinatum) in South Africa

Plant Disease ◽

10.1094/pdis.1999.83.8.783a ◽

1999 ◽

Vol 83 (8) ◽

pp. 783-783

Author(s):

E. Carstens ◽

Z. Dawood ◽

E. L. Mansvelt ◽

S. Serfontein ◽

D. G. Malan

Keyword(s):

South Africa ◽

Pathogenic Bacteria ◽

Export Market ◽

Bacterial Suspension ◽

Identification System ◽

Poor Growth ◽

First Report ◽

Plant Pathogenic Bacteria ◽

Pure Cultures ◽

Chamelaucium Uncinatum

Geraldton wax (Chamelaucium uncinatum, family Myrtaceae) plants are grown as cutflowers for the export market in South Africa. In July 1998, gall-like structures were observed on collars and roots of Geraldton wax plants in commercial fields in Wellington. The galls were observed after plants exhibited poor growth. The galls varied in size and in texture from soft and spongy to hard. Secondary symptoms involved poor root development and browning of stem tissues near galls. Isolations from the galls yielded nearly pure cultures of a Gram negative, rod-shaped bacterium on Roy Sauer medium (2), typical of an Agrobacterium sp. Carbon source utilization testing with the Biolog GN Bacterial Identification System (version 3.50) confirmed the bacterium as a biovar of A. tumefaciens with a similarity of 0.88. Pathogenicity was confirmed by injecting 4- to 6-week old tomato and tobacco plants and 1-year-old Geraldton wax plants with approximately 5 μl of the bacterial suspension (108 CFU/ml) in sterile, distilled water. Inoculated plants were then transferred to a greenhouse at 25°C. Galls developed 1 month after inoculation. The bacterium was readily reisolated from the inoculated plants. A. tumefaciens is endemic to South Africa and has a very wide host range that includes several ornamentals (1). This is the first report of A. tumefaciens on Geraldton wax plants in South Africa. References: (1) J. F. Bradbury. 1986. Guide to Plant Pathogenic Bacteria. CAB Int., Slough, U.K. (2) N. W. Schaad. 1988. Laboratory Guide for Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, MN.

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First Report of Leaf Spot of Dracaena reflexa Caused by Burkholderia gladioli Worldwide

Plant Disease ◽

10.1094/pdis-94-6-0781c ◽

2010 ◽

Vol 94 (6) ◽

pp. 781-781

Author(s):

M. K. Romberg ◽

R. L. Griffin ◽

S. Murugan ◽

B. D. Quinn ◽

B. J. R. Alexander

Keyword(s):

16S Rdna ◽

Leaf Spot ◽

Natural Infection ◽

Similarity Index ◽

Leading Edge ◽

Bacterial Suspension ◽

Distilled Water ◽

Carbon Utilization ◽

First Report ◽

Burkholderia Gladioli

In December 2008 (austral summer), a new disease of Dracaena reflexa Lam. cv. Anita was observed in a postentry quarantine greenhouse near Auckland, New Zealand on plants imported from Costa Rica. Symptoms included rust-colored, water-soaked lesions with chlorotic margins approximately 5 by 10 mm. When the disease was first noticed, incidence approached 80%, but subsequent reduction in greenhouse temperature dramatically reduced symptom expression and lesions were only visible on some leaf tips. Bacteria consistently isolated from the lesions on King's medium B (KB) were cream-colored, shiny, and produced a yellow, diffusible, nonfluorescent pigment. All isolates were able to rot onion slices. On the basis of BIOLOG (Hayward, CA) carbon utilization profiles, isolates were initially identified as Burkholderia gladioli (Severini 1913) Yabuuchi et al. 1993 with a probability index of 100% and a similarity index of 0.85. For molecular identification, a near full-length sequence of the 16S rDNA gene was amplified from all isolates using primers fD2 and rP1 (1), obtaining a PCR product of approximately 1,500 bp. The nucleotide sequences were 100% identical to a number of B. gladioli GenBank entries, including Accession Nos. EF193645 and EF088209. To confirm pathogenicity, three isolates (two isolated prior to greenhouse temperature reduction and one after) were used. Three D. reflexa plants were inoculated per bacterial isolate by wounding three young fully expanded leaves on each plant (four wounds per leaf) and spraying the leaves with a bacterial suspension in sterile distilled water at 108 CFU/ml. At the same time, Gladiolus nanus plants were inoculated in a similar manner. Control plants (D. reflexa and G. nanus) were wounded and sprayed with sterile distilled water. All inoculated plants were covered with plastic bags to maintain humidity and placed in a growth chamber at 25°C. At 3 days, all inoculated plants began to show water soaking and reddish coloration around the inoculation sites, and by 7 days, the lesions had expanded to resemble natural infection. Bacteria isolated on KB from the leading edge of each lesion were morphologically identical to the initial isolates. No bacteria were recovered from the wound sites on the control plants. The 16S rDNA sequences of selected isolates from inoculated plants showed 100% identity to the sequences of the initial isolates, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of B. gladioli causing leaf spot of D. reflexa in the world. Reference: (1) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

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First Report of Bacterial Leaf Spot of Coriander Caused by Pseudomonas syringae pv. coriandricola in China

Plant Disease ◽

10.1094/pdis-09-21-1901-pdn ◽

2021 ◽

Author(s):

Lei Li ◽

Yishuo Huang ◽

Yanxia Shi ◽

A LI CHAI ◽

Xuewen Xie ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Leaf Spot ◽

Citrate Synthase ◽

Morphological Characteristics ◽

Likelihood Method ◽

Bacterial Suspension ◽

Leaf Spot Disease ◽

Distilled Water ◽

Bacterial Leaf Spot ◽

First Report

Coriander (Coriandrum sativum L.) or Chinese parsley is a culinary herb with multiple medicinal effects that are widely used in cooking and traditional medicine. From September to November 2019, symptoms were observed in 2-month-old coriander plants from coriander ﬁelds in Lanzhou and Wenzhou, China. The disease developed rapidly under cold and wet climatic conditions, and the infection rate was almost 80% in open coriander fields. Typical symptoms on leaves included small, water-soaked blotches and irregular brown spots surrounding haloes; as the disease progressed, the spots coalesced into necrotic areas. Symptomatic leaf tissue was surface sterilized, macerated in sterile distilled water, and cultured on nutrient agar plates at 28 °C for 48 h (Koike and Bull, 2006). After incubation, six bacterial colonies, which were individually isolated from collected samples from two different areas, were selected for further study. Colonies on NA plate were small, round, raised, white to cream-colored, and had smooth margins. All bacterial isolates were gram-negative, rod-shaped and nonfluorescent on King's B medium. The bacteria were positive for levan production, Tween 80 hydrolysis, and tobacco hypersensitivity but negative for oxidase, potato slice rot test, arginine dihydrolase, ice nucleation activity, indole production and H2S production. The suspension of representative isolate for inoculating of plants was obtained from single colony on King's B medium for 2-3 days at 28 °C. DNA was extracted from bacterial suspensions of YS2003200102 cultured in 20 ml of King’s B medium broth at 28 °C for 1 day. Extraction was performed with a TIANamp Bacterial DNA Kit (TIANGEN, China) according to the manufacturer’s recommendations. The pathogen was confirmed by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase A (gapA) gene, the citrate synthase (gltA) gene, the DNA gyrase B (gyrB) gene and the RNA polymerase sigma factor 70 (rpoD) gene using gapA-For/gapA-Rev, gltA-For/gltA-Rev, gyrB-For/gryB-Rev, rpoD-For/rpoD-Rev primers, respectively (Popović et al., 2019). The sequences of the PCR products were deposited in GenBank with accession numbers MZ681931 (gapA), MZ681932 (gltA), MZ681933 (gyrB), and MZ681934 (rpoD). Phylogenetic analysis of multiple genes (Xu and Miller, 2013) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolates and ten published sequences of P. syringae pv. coriandricola were clustered into one clade with a 100% confidence level. To confirm the pathogenicity of isolate YS2003200102, 2-month-old healthy coriander plants were inoculated by spraying the leaves with a bacterial suspension (108 CFU ml−1) at 28 °C incubation temperature and 70% relative humidity condition, and sterile distilled water was applied as a negative control treatment (Cazorla et al. 2005). Three replicates were conducted for every isolate, and each replicate included 6 coriander plants. After twelve days, only the inoculated leaves with bacterial suspension showed bacterial leaf spot resembling those observed on naturally infected coriander leaves. Cultures re-isolated from symptomatic leaves showed the same morphological characteristics and molecular traits as those initially isolated from infected leaves in the field. This bacterium was previously reported causing leaf spot of coriander in India and Spain (Gupta et al. 2013; Cazorla et al. 2005). To our knowledge, this is the first report of P. syringae pv. coriandricola causing leaf spot disease on coriander in China. Studies are needed on strategies to manage P. syringae pv. coriandricola in crops, because its prevalence may cause yield loss on coriander in China.

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