First report of Stenotrophomonas maltophilia causing root soft rot of Sanqi (Panax notoginseng) in China

Sanqi (Panax notoginseng (Burk.) F. H. Chen) is a traditional Chinese medicinal plant with a long planting cycle of 2-3 years that makes it vulnerable to root diseases caused by several pathogens, including Fusarium solani, Alternaria panax, Phytophthoracactorum, and Pseudomonas sp. In April 2019, root soft rot samples of Sanqi were collected from a plantation site in Songming, southwest of China. Typical symptoms included root softening and necrosis, yellow leaf, and stem wilting. Ten diseased roots samples were collected and sterilized with 0.1% HgCl2 for 1 min, 75% ethanol for 2min, and then rinsed thrice with sterile water. Sterilized roots were cut into small pieces of 5 × 5 mm and cultured on the nutrient agar (NA) medium for 48 h at 28°C. From the root cultures, a total of thirteen bacterial strains were obtained. Three strains, SM 2-5, SM 2-13, and SM 2-14 were selected for further study. These three strains were gram-negative, short rod-shaped (1～2×0.5～1μm), non-spore-forming and had polar tufted flagella as observed under a transmission electron microscope (TEM). Also, the strains were positive for oxidase, beta-galactosidase, arginine dihydrolase, and lysine decarboxylase while negative for amylase and urease tested by biochemical methods (Wang 2017). To further determine the pathogenic species, genomic DNA of these three strains was extracted using a Genomic DNA Kit (Tsing Ke, Beijing, China), to PCR amplify 16S rDNA using universal primers 27F/1492R (Wang et al. 2017). Also, S. maltophilia 23S rDNA specific primers SM1/SM4 (Whitby et al. 2000) were used for PCR amplification to confirm the species. 16S rDNA sequence analysis showed that SM 2-5 (GenBank Accession No. MW555227), SM 2-13 (GenBank Accession No. MW555228), and SM 2-14 (GenBank Accession No. MW555229) shared the highest identity (>99.9%) with the S. maltophilia strains (GenBank Accession No. MT323142, MH669295, MN826555). Furthermore, 23S rDNA sequence analysis of SM 2-5 (GenBank Accession No. MZ707732), SM 2-13 (GenBank Accession No. MZ645941) and SM 2-14 (GenBank Accession No. MZ707733) revealed their high identity (>99.8%) with the S. maltophilia species. 16S and 23S rDNA phylogenetic analysis (Mega6.06) using the neighbor-joining (NJ) method with 1,000 bootstrap replicates revealed the three strains clustering with the other S. maltophilia strains. Therefore, based on morphology, metabolic profile, and sequence analysis, the three strains were identified as Stenotrophomonas maltophilia. To test pathogenicity, the strains were grown in the nutrient broth (NB) medium for 48h at 28°C until bacterial suspension reached to OD600≈1.0 (2.0×109CFU/mL). Then, healthy roots of one-year-old Sanqi plants, pre-washed with sterilized water and -poked with a sterilized needle, were soaked in bacterial suspension (2.0×109CFU/mL) of the three strains separately for inoculation 10min. Sterilized water treatment was used as a control. Subsequently, bacteria-inoculated plants were planted in sterile soil pots and cultured in a greenhouse at 28°C with shading rate of 70%. Each treatment group included 3 plants with 3 replicates. Ten days post inoculation, symptoms similar to the ones in natural conditions were observed in the bacteria-inoculated plants. Based on the disease index (Li et al. 2020), we found that among the three strains, SM 2-13 displayed the highest virulence, while no symptoms were observed in the control plants. The same bacterial strains were re-isolated from these inoculated roots and identified by the methods described above. Previous studies showed that some Stenotrophomonas species cause plant diseases such as rice white stripe (Singh et al. 2001), strawberry leaf black spot (Wang et al. 2017), Cyclobalanopsis patelliformis leaf spot (Bian et al. 2020), and Jatropha curcas L. seed borne and stem necrosis (Wang et al. 2018). To our knowledge, this is the first report confirming Stenotrophomonas maltophilia causing root soft rot of Panax notoginseng in China.

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First Report of a Soft Rot of Phalaenopsis aphrodita Caused by Dickeya dieffenbachiae in China

Plant Disease ◽

10.1094/pdis-11-11-0942 ◽

2012 ◽

Vol 96 (5) ◽

pp. 760-760 ◽

Cited By ~ 8

Author(s):

J. N. Zhou ◽

B. R. Lin ◽

H. F. Shen ◽

X. M. Pu ◽

Z. N. Chen ◽

...

Keyword(s):

16S Rdna ◽

Large Scale ◽

Soft Rot ◽

The Philippines ◽

Gyrb Gene ◽

Bacterial Suspension ◽

Sterile Water ◽

Gene Sequences ◽

First Report ◽

Tropical Asia

Phalaenopsis orchids, originally from tropical Asia, are mainly planted in Thailand, Singapore, Malaysia, the Philippines, and Taiwan and have gained popularity from consumers all over the world. The cultivation area of Phalaenopsis orchids has been rising and large-scale bases have been established in mainland China, especially South China because of suitable environmental conditions. In September 2011, a soft rot of Phalaenopsis aphrodita was found in a Phalaenopsis planting base in Guangzhou with an incidence of ~15%. Infected plants initially showed water-soaked, pale-to-dark brown pinpoint spots on leaves that were sometimes surrounded by a yellow halo. Spots expanded rapidly with rising humidity and temperatures, and in a few days, severely extended over the blade with a light tan color and darker brown border. Lesions decayed with odorous fumes and tissues collapsed with inclusions exuding. The bacterium advanced to the stem and pedicle. Finally, leaves became papery dry and the pedicles lodged. Six diseased samples were collected, and bacteria were isolated from the edge of symptomatic tissues after sterilization in 0.3% NaOCl for 10 min, rinsing in sterile water three times, and placing on nutrient agar for culture. Twelve representative isolates were selected for further characterization. All strains were gram negative, grew at 37°C, were positive for indole production, and utilized malonate, glucose, and sucrose but not glucopyranoside, trehalose, or palatinose. Biolog identification (version 4.20.05, Hayward, CA) was performed and Pectobacterium chrysanthemi (SIM 0.868) was confirmed for the tested isolates (transfer to genus Dickeya). PCR was used to amplify the 16S rDNAgene with primers 27f and 1492r, dnaX gene with primers dnaXf and dnaXr (3), and gyrB gene with primers gyrBf (5′-GAAGGYAAAVTKCATCGTCAGG-3′) and gyrB-r1 (5′-TCARATATCRATATTCGCYGCTTTC-3′) designed on the basis of the published gyrB gene sequences of genus Dickeya. BLASTn was performed online, and phylogeny trees (100% bootstrap values) were created by means of MEGA 5.05 for these gene sequences, respectively. Results commonly showed that the representative tested strain, PA1, was most homologous to Dickeya dieffenbachiae with 98% identity for 16S rDNA(JN940859), 97% for dnaX (JN989971), and 96% for gyrB (JN971031). Thus, we recommend calling this isolate D. dieffenbachiae PA1. Pathogenicity tests were conducted by injecting 10 P. aphrodita seedlings with 100 μl of the bacterial suspension (1 × 108 CFU/ml) and another 10 were injected with 100 μl of sterile water as controls. Plants were inoculated in a greenhouse at 28 to 32°C and 90% relative humidity. Soft rot symptoms were observed after 2 days on the inoculated plants, but not on the control ones. The bacterium was isolated from the lesions and demonstrated identity to the inoculated plant by the 16S rDNA sequence comparison. Previously, similar diseases of P. amabilis were reported in Tangshan, Jiangsu, Zhejiang, and Wuhan and causal agents were identified as Erwinia spp. (2), Pseudomonas grimontii (1), E. chrysanthemi, and E. carotovora subsp. carovora (4). To our knowledge, this is the first report of D. dieffenbachiae causing soft rot disease on P. aphrodita in China. References: (1) X. L. Chu and B. Yang. Acta Phytopathol. Sin. 40:90, 2010. (2) Y. M. Li et al. J. Beijing Agric. Coll. 19:41, 2004. (3) M. Sławiak et al. Eur. J. Plant Pathol. 125:245, 2009. (4) Z. Y. Wu et al. J. Zhejiang For. Coll. 27:635, 2010.

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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 Dickeya fangzhongdai Causing Peduncle Soft Rot of Banana in China

Plant Disease ◽

10.1094/pdis-11-21-2513-pdn ◽

2021 ◽

Author(s):

Di Yang ◽

Chan Juan Du ◽

Yunfeng Ye ◽

Lian Fu Pan ◽

Jin Zhang ◽

...

Keyword(s):

16S Rdna ◽

Dna Sequences ◽

Southern China ◽

Intergenic Spacer ◽

Soft Rot ◽

Bacterial Suspension ◽

Sterile Water ◽

Internal Tissue ◽

Flower Bud ◽

First Report

Banana (Musa spp.) is a popular fruit all over the world, and it’s also an important cash crop with a planting area of 358,924 ha in southern China. In July 2020, a peduncle soft rot disease occurred on dwarf banana (Musa sp. cv. Guangfen) in Guigang city (N22°50'29″, E109° 43'34″), Guangxi province, China. More than 20% plants were infected in the banana plantation. The first external sign of the disease appeared on the incisional wound after the flower bud was cut off from the peduncle. The symptom initially appeared as a black lesion on the wound, then extended into the internal tissue of the whole peduncle. In the later stages, the internal tissue became soft and rot, occasionally formed a necrotic cavity, and eventually led to the black rot of the whole peduncle with a foul smell. To isolate the pathogen, the internal lesion tissues of 5 mm × 5 mm were collected between the border of symptomatic and healthy tissue, treated with 75% ethanol for 10 s, and 0.1% HgCl2 for 3 min, then rinsed with sterile water for three times. Sterilized tissue fragments were cut to pieces with sterilized surgical shears and soaked in 5 mL sterile water, then shaken for 10 min in a vortex oscillator. The suspension was diluted 1000 times with sterilized water，then plated on nutrient-agar medium and incubated at 28℃ in darkness for 24 h. Among the 32 isolates, 23 pure bacterial cultures with similar morphology were predominantly obtained from the samples. These bacteria were gram-negative, and their colonies were initially yellowish white with irregular edges and smooth surfaces, then turned to grayish blue after 72 h incubated at 28℃. The representative isolates GZF2-2 and GZF1-8 were selected for further identification. Genomic DNA was isolated from the bacteria and the 16S rDNA was amplified with primers 27F/1492R (Weisburg et al. 1991) and sequenced. The obtained sequences (GenBank Accession No. MZ768922 and OK668082) showed >99% identities to several records of Dickeya fangzhongdai deposited in NCBI GenBank (1400/1404 bps for GZF2-2 to KT992690, 1409/1417 bps for GZF1-8 to MT613398) based on BLAST analysis. In addition, the recA, fusA, gapA, purA, rplB, dnaX genes and the 16S-23S intergenic spacer (IGS) regions of the two isolates were also amplified and sequenced (GenBank Accession Nos. OK634381-OK634382, OK634369- OK634370, OK634373-OK634374, OK634377-OK634378, OK634385-OK634386, OK634365- OK634366 and OK631722-OK631723) as described by Tian et al. (2016). All the DNA sequences matched that of D. fangzhongdai strains JS5T (percent identities>99.06%), PA1 and ECM-1 in GenBank. Neighbor-joining phylogenetic analysis by software MegaX (Kumar et al. 2018) based on the 16S rDNA sequences revealed that the two isolates were in the same clade with reported D. fangzhongdai strains. Multilocus sequence analysis of the other seven regions also showed the two representative isolates were belong to D. fangzhongdai. Therefore, the isolates were identified as D. fangzhongdai. Pathogenicity of isolate GZF2-2 was investigated to demonstrate Koch’s postulate. The end of the banana peduncles of 6 healthy plants were cut off, and 10 mL bacterial suspension (108 CFU/mL) was inoculated to the fresh wound on the plants using sterile brushes. Six control plants were inoculated with sterilized water. All the inoculated peduncles were covered with plastic bags to maintain high humidity. After 28 days, all the peduncles inoculated with strain GZF2-2 showed soft rot symptoms similar to those observed in the field, while the controls remained symptomless. The same bacteria were re-isolated from the symptomatic peduncles and confirmed by sequencing the 16S rDNA. D. fangzhongdai has been reported to cause soft rot on onion (Ma et al. 2020) and bleeding cankers on pear trees (Chen et al. 2020). To the best of our knowledge, this is the first report of D. fangzhongdai causing peduncle soft rot on banana in China.

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First report of soft rot of cabbage caused by Pectobacterium wasabiae in Japan

Plant Disease ◽

10.1094/pdis-02-21-0238-pdn ◽

2021 ◽

Author(s):

Taketo Fujimoto ◽

Takato Nakayama ◽

Takehiro Ohki ◽

Tetsuo MAOKA

Keyword(s):

Sequence Analysis ◽

Dna Sequences ◽

Acid Production ◽

Field Experiments ◽

Soft Rot ◽

Likelihood Method ◽

Bacterial Suspension ◽

Aconitate Hydratase ◽

First Report ◽

Pectolytic Activity

Cabbage (Brassica oleracea var. capitata) is one of the important vegetables in Japan. In the summer of 2019, some cabbages with soft rot were found in commercial fields in Hokkaido, the northern island in Japan. All diseased plants showed grey to brown discoloration and expanding water-soaked lesions on leaves. We obtained two independent strains (NACAB191 and NACAB192) from diseased leaves. DNA from these strains yielded an expected single size amplicon with the primer set of PhF/PhR for P. wasabiae (De Boer et al. 2012) by PCR, but did not yield the expected amplicon with the primer set of BR1f/L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004) and Eca1f/Eca2r for P. atrosepticum (De Boer et al., 1995) by PCR. These two strains grew at 37°C, and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative and rod-shaped. The bacterium was positive for O-nitrophenyl-beta-D-galactopyranoside, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. All strains exhibited pectolytic activity on potato slices. The sequence analysis of 16S rDNA (LC597897 and LC597898) showed more than 98% identities to P. wasabiae strain (e.g. HAFL01 in Switzerland) by BLAST analysis. In addition, Multi-locus sequence analysis (Ma et al. 2007) was performed by MEGA10 (Kumer et al. 2018) using concatenated DNA sequences of seven housekeeping genes (aconitate hydratase(acnA, LC597923 and LC597924), glyceraldehyde-3-phosphate dehydrogenase A(gapA, LC597970 and LC597971), isocitrate dehydrogenase (icdA, LC597996 and LC597997), malate dehydrogenase(mdh, LC598022 and LC598023), mannitol-1-phosphate dehydrogenase (mtlD, LC598048 and LC598049), glucose-6-phosphate isomerase (pgi, LC598074 and LC598075) and gamma-glutamyl phospate reductase (proA, LC598079 and LC598080)), and all clustered NACAB191 and NACAB192 into a clade containing other confirmed strains of P. wasabiae. As a result, these two strains shared high identity with each other (>98%, E-Values showed 0). The clade containing these two strains was consistently placed in a larger clade with the other P. wasabiae and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank when the consensus tree constructed using Maximum Likelihood method. Pathogenicity of these strains against cabbage (cv. ‘Rakuen’) was confirmed by the field experiments with five weeks growth plants sprayed with bacterial suspension (1×107cfu/ml). Thirty cabbages per strain were used in this study, 12 plants treated the suspension of NACAB191 and 16 plants treated the suspension of NACAB192 which died with the same soft rot symptoms about four weeks after inoculation. Whereas water-inoculated plants remained symptomless. Strains re-isolated from the artificially diseased stems were confirmed as P. wasabiae using the methods as biochemical characterization and multiple genetic analyses. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the soft rot symptoms of cabbage in Hokkaido in 2019 were caused by P. wasabiae. To our knowledge, this is the first report of P. wasabiae as the soft rot disease agent of cabbage in Japan.

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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 Bacterial Blight of Guar Caused by Xanthomonas axonopodis pv. cyamopsidis in China

Plant Disease ◽

10.1094/pdis-09-13-0983-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 840-840 ◽

Cited By ~ 2

Author(s):

Y. Z. Ren ◽

Y. L. Yue ◽

G. X. Jin ◽

Q. Du

Keyword(s):

16S Rdna ◽

Bacterial Blight ◽

Negative Control ◽

Bacterial Suspension ◽

Cyamopsis Tetragonoloba ◽

Sterile Water ◽

Bacterial Strains ◽

Xanthomonas Axonopodis ◽

First Report ◽

Pathogenicity Tests

Bacterial blight was observed on field-grown guar (Cyamopsis tetragonoloba L.) for the first time in China. The disease outbreak occurred in the Xinjiang Uyghur Autonomous Region after several weeks of unusually heavy rains during late summer 2013. The disease incidence was generally 40 to 50%, although values as high as 80% were observed in several fields. Initial field symptoms included water-soaked spots on leaves, pods, petioles, and stems. During later stages of infection, the color of the spots became dark. We also observed large, angular, necrotic lesions at leaf tips, black streaks on petioles and stems, split stems, defoliation, wilting or top withering, vascular necrosis, and dieback. Samples of diseased leaves, stems, petioles, pods, and seeds were surface sterilized, ground, and then plated onto King's B medium. Plates were incubated at 28°C for 72 h. Fifteen bacterial strains with yellow-pigmented, opaque, and round colonies were isolated. These strains were aerobic, gram-negative rods with a single, polar flagellum. They were positive for H2S, esculin, oxidase, tobacco hypersensitivity, indole production from tryptophan, nitrate reduction to nitrite, and the utilization of glucose, mannose, trehalose, galactose, and starch. The maximum salt tolerance of the strains was 2 to 3%. Pathogenicity tests using eight strains were conducted in July 2013. A bacterial culture was suspended in sterile water with a final concentration of 108 CFU/ml. Eight 4-week-old guar plants were inoculated by (i) spraying the bacterial suspension on the leaves until runoff, or (ii) puncturing the stems with a needle that had been dipped into the bacterial suspension. Sterile water was used as a negative control. Plants were kept in a mist room with 100% relative humidity for 24 h. Stem and leaf symptoms similar to those of the original plants were observed on the inoculated guar plants within 10 days of inoculation. No symptoms developed on the negative control plants. Yellow bacterial colonies re-isolated from inoculated plant tissues were morphologically identical to the original. 16S rDNA was amplified using universal primers (Pa 5′-AGTTTGATCCTGGCTCAG-3′ and Ph 5′-TACCTTGTTACGACTTCGTCCCA-3′) and sequenced. A BLAST search of the NCBI GenBank database indicated that the 16S rDNA sequences of three strains (accession nos. KF563926, KF563927, and KF563928) had 99.9% identity to Xanthomonas axonopodis strain XV938 (AF123091). Under greenhouse conditions, bacterial strains wilted asparagus bean and pea but rarely infected bean, kidney bean, faba bean, mung bean, soybean, red bean, pea, garbanzo bean, and peanut. Based on morphology, pathogenicity tests, 16S rDNA sequencing, and host plant specificity, the pathogen was confirmed as X. axonopodis pv. cyamopsidis (synonym: X. campestris pv. cyamopsidis [Patel et al., 1953]). To our knowledge, this is the first report of bacterial blight of guar caused by X. axonopodis pv. cyamopsidis in China. Guar has recently been introduced in Xinjiang Province. Our findings indicate that bacterial blight may pose a threat to the economic sustainability of guar production in the region. References: (1) I. A. Milyutina et a1. FEMS Microbiol. Lett. 239:17, 2004. (2) I. M. G. Almeida et al. Summa Phytopathol. 18:255, 1992. (3) J. D. Mihail et al. Plant Dis. 69:811, 1985.

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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 Pectobacterium aroidearum causing bacterial soft rot of carrot in Taiwan

Plant Disease ◽

10.1094/pdis-08-20-1824-pdn ◽

2020 ◽

Author(s):

Wen-Qian Tang ◽

Ching-Yu Chang ◽

Yi-Jin Lee ◽

Chia-Ching Chu

Keyword(s):

16S Rdna ◽

Soft Rot ◽

Control Group ◽

Bacterial Strains ◽

Sequence Analyses ◽

Cloning And Sequencing ◽

Plastic Container ◽

First Report ◽

Maximum Likelihood Tree ◽

Foul Odor

Carrot (Daucus carota) is an important root vegetable planted and consumed worldwide (Stein and Nothnagel 1995). In June 2020, carrots (cv. New Kuroda) showing soft rot symptoms were observed in a 600 sqft plot located in Pitou, Changhua, Taiwan (23°54'00.9"N, 120°28'37.3"E; with around 400 plants). About 10% of the plants on site had similar symptoms; infected taproot tissues were macerated (Figure S1) and emitted a foul odor. In most cases, the peels above the rotten tissues remain intact. Two infected plants were brought to the lab. Macerated tissues were suspended in water and examined under a microscope at 600X (without staining). Rod, motile bacteria were observed in all of the samples and the bacteria were isolated onto nutrient agar. Three bacterial strains were obtained from two taproots; strain Car1 was isolated from one plant, and strains Car2 and Car3 were isolated from the other. Their colonies were translucent, round and convex. All isolates could ferment glucose and induce soft rot symptoms on potato tuber slices (Schaad et al. 2001). They were not able to produce indigoidine on yeast dextrose calcium carbonate agar and were tested negative for phosphatase activity (Schaad et al. 2001). The 16S rDNA of Car1 to Car3 were amplified using primers 27F/1492R (Lane 1991). Cloning and sequencing of their 16S rDNA (GenBank accession no. MT889640) revealed that their sequences shared 99.9% identity (1,463/1,464 bp) with that of Pectobacterium aroidearum CFBP 8168T (SCRI 109T; GenBank accession no. NR_159926.1). Multilocus sequence analyses targeting the three isolates’ dnaX, leuS and recA genes were conducted. The concatenated sequences (1,596 bp) of Car1 to Car3 and those included in a previous work (Portier et al. 2019) were subjected to phylogenetic analysis. The sequences of Car1 to Car3 were identical (GenBank accession nos. MT892671-MT892673). A maximum-likelihood tree showed that the three isolates belonged to the same clade as P. aroidearum CFBP 8168T (GenBank accession nos. MK516971, MK517115 and MK517259; Figure S2). For the concatenated sequences analyzed, the identity between P. aroidearum CFBP 8168T and our three isolates was 99.4% (1,587/1,596 bp). The pathogenicity of these isolates was determined by inoculating the bacteria into carrot (cv. Xiangyang No.2) taproots. Strains Car1 to Car3 were grown on NA for 48 h (28 °C) and cell suspensions with OD600 values of 0.3 (2.4 x 108 CFU/ml; in water) were prepared. The suspensions of each strain (100 μl) were loaded into 200 μl pipette tips. The tips were then pierced into intact carrot taproots (2.4 cm deep), ejected and left on the plants (one tip per plant). Three taproots were tested for each strain. Tips loaded with 100 μl of water were used for the controls (three replicates). The plants were incubated in a sealed plastic container kept in a growth chamber set at 28°C. After 48 h, all of the inoculated taproots produced soft rot symptoms resembling those observed in the field and plants in the control group did not. Bacteria were re-isolated from macerated tissues of the artificially infected plants and found to share the same leuS sequence with Car1 to Car3. Occurrences of carrot soft rot in Taiwan have only been attributed to Dickeya spp. (Erwinia chrysanthemi) in previous studies (Hsu and Tzeng 1981). The present study is the first report of P. aroidearum infecting carrots in Taiwan. The findings may add to our understanding of the diversity of soft rot pathogens affecting carrot production in Taiwan.

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First Report of Bacterial Soft Rot Caused by Pantoea agglomerans on Chinese yam (Dioscorea opposita Thunb.) in China

Plant Disease ◽

10.1094/pdis-10-21-2151-pdn ◽

2022 ◽

Author(s):

Susu Fan ◽

Fangyuan Zhou ◽

Xueying Xie ◽

Xinjian Zhang ◽

Tielin Wang

Keyword(s):

16S Rdna ◽

Soft Rot ◽

Henan Province ◽

Pantoea Agglomerans ◽

Bacterial Suspension ◽

Pathogenicity Test ◽

Bacterial Soft Rot ◽

First Report ◽

Chinese Yam ◽

Dioscorea Opposita

Chinese yam (Dioscorea opposita Thunb.), which belongs to the family of Dioscorea, is widely naturalized throughout China, due to its high economic and medicinal value. Since 2019, water-soaked lesions were frequently observed in the underground tubers of Chinese yam located in Xinyang City, Henan Province. To identify the causal agent, ten pieces of tissue from the underground tubers with disease symptoms were collected. Those infected tissues (5×5 mm) were crushed in 500 μL sterilized water after surface sterilization and streaked onto Luria-Bertani agar plates. Pale-yellowish, rod-shaped, slimy single bacterial colonies with smooth margin were observed after 24 hours of incubation, and three bacterial colonies (named CY-1, CY-2 and CY-3) were randomly selected for further biochemical and molecular characterization. These bacteria were gram-negative with the cell length of 1.0 to 3.0 μm, width of 0.5 to 1.0 μm, and with peritrichous flagella. Subsequently, the bacteria were biochemically analyzed through BIOLOG (Hayward, CA) and identified as Pantoea agglomerans with 99% probability. Furthermore, the phylogenetic analysis results based on 16S rDNA, DNA gyrase subunit B (gyrB), and RNA polymerase sigma factor (rpoD) showed these three isolates were most closely related to P. agglomerans. The sequence of 16S rDNA, gyrB and rpoD of each strain was submitted to GenBank with the accession numbers MZ541065 MZ541066 and MZ541067 for 16S rDNA; MZ669846, MZ669847 and MZ669848 for gyrB; MZ669849, MZ669850 and MZ669851 for ropD. Pathogenicity test was performed to complete Koch’s postulates. Tubers of Chinese yam were wounded by sterile needle and inoculated with 500 μL 108 CFU/mL bacterial suspension. Sterilized water was used as a control. Five pots were inoculated for each isolate. Water-soaked lesions appeared after five days incubation at 25°C in a biochemical incubator and no lesions were observed on the control. Bacteria re-isolated from the lesions were similar in phenotypic and molecular characteristics to the original isolates. In brief, based on colony morphology, biochemical tests, characteristic sequence analysis, and pathogenicity verification, the pathogen responsible for the soft rot of Chinese yam in Henan Province was identified as P. agglomerans. In China, P. agglomerans has been reported to associate with bacterial soft rot on Chinese cabbage (Guo et al., 2020). To our knowledge, this work is the first report of bacterial rot caused by P. agglomerans on Chinese yam.

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