scholarly journals First Report of Potato Blackleg Disease Caused by Pectobacterium atrosepticum in Guangdong China

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1652-1652
Author(s):  
X. M. She ◽  
Z. F. He ◽  
Y. F. Tang ◽  
Z. G. Du ◽  
G. B. Lan
Keyword(s):  
16S Rdna ◽  
Vascular Tissue ◽  
Guangdong Province ◽  
Bacterial Suspension ◽  
Post Inoculation ◽  
Blackleg Disease ◽  
Potato Blackleg ◽  
Pectobacterium Atrosepticum ◽  
First Report ◽  
Potato Plants

Potato (Solanum tuberosum L.) is an important crop in China. In 2013, diseased potatoes exhibiting blackleg and soft rot symptoms were found in the winter potato growing areas of Huizhou city, Guangdong Province, China, with an incidence of approximately 20%. Initially, the stem bases of infected plants blackened and this symptom spread upward. Later, foliage of the diseased plants became yellow and the stem rotted with vascular discoloration. Twenty diseased plants with typical black leg symptoms were collected from a 10-ha potato field with approximately 60,000 potato plants per hectare. A bacterium with small, irregular, round, fluidal, white colonies was isolated from the vascular tissue of all diseased plants on nutrient agar at 26°C for 2 days. Ten strains were randomly selected for pathogenicity assays. Potato plants (cv. Favorita) at the five- to six-leaf stage were inoculated by injecting their stems with 1 ml of each strain in a bacterial suspension (3 × 108 CFU/ml). The inoculated potato plants were incubated at 16 to 21°C and 65 to 85% humidity, and exhibited the same symptoms as the diseased potato plants in the field by 3 to 5 days post inoculation (dpi). The bacterium was reisolated from the diseased tissue (stem) of the inoculated potato plants and produced characteristic pits on crystal violet pectate medium (1). The bacterium utilized a-methyl glucoside, glucose, lactose, maltose, cellobiose, raffinose, melibiose, and citrate, but not d-arabitol, sorbitol, or malonate. The bacteria also gave a positive reaction for catalase and production of reducing substances from sucrose, but gave a negative reaction for oxidase, production of phosphatase, and indole. Using the universal bacterial 16S rDNA primer set, 27f/1541R (4), 1,400-bp fragments were amplified from the 10 strains. The sequences of the 10 fragments (GenBank Accessions KC695819 to KC695828) were identical and had 100% sequence identity with 16S rDNA of Pectobacterium atrosepticum CFBP 1526 (JN600332). Further, the 438-bp and 690-bp fragments were respectively amplified from all 10 strains with the P. atrosepticum-specific primers Y45/Y46 (3) and ECA1f/ECA2r (2). To our knowledge, this is the first report of potato blackleg disease caused by P. atrosepticum (formerly named as Erwinia carotovora subsp. atroseptica) in Guangdong Province, China. References: (1) D. Cupples et al. Phytopathology 64:468, 1974. (2) S. H. De Boer et al. Phytopathology 85:854, 1995. (3) D. Frenchon et al. Potato Research 41:63, 1995. (4) M. Horita et al. J. Gen. Plant Pathol. 70:278, 2004.

scholarly journals First Report of Pectobacterium versatile Causing Aerial Stem Rot of Potato in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Wanxin Han ◽  
Jinhui Wang ◽  
Zheng Li ◽  
Yang Pan ◽  
Dai Zhang ◽  
...  
Keyword(s):  
16S Rdna ◽  
Disease Incidence ◽  
New York State ◽  
Housekeeping Genes ◽  
Soft Rot ◽  
Stem Rot ◽  
Post Inoculation ◽  
First Report ◽  
Potato Plants ◽  
Aerial Stem

Pectobacterium species cause blackleg, soft rot and stem rot in potato and many other vegetable crops (Charkowski 2015). In July 2020, potato plants showing characteristic symptoms of aerial stem rot were observed in a field (cv. Xisen 6) in Fengning Manchu Autonomous County, Chengde, Hebei Province (North China). The disease incidence in that field (5 ha in size) was more than 50%. Putative pectolytic bacteria were obtained from symptomatic stem tissues (light brown and water-soaked stem sections) by culturing on the crystal violet pectate (CVP) medium. Bacterial colonies producing pits, were restreaked and purified on Luria-Bertani (LB) agar. The isolates causing stem rot were gram negative and rod shaped, negative for oxidase, urease, indole production, gelatin liquefaction and acid production from maltose and D-sorbitol. All isolates were catalase positive, produced acid from lactose, rhamnose, saccharose, raffinose and D-arabinose, and were tolerant to 5% NaCl, and able to utilize citrate. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech). The 16S rDNA region was amplified by PCR using the universal primer pair 27F/1492R and sequenced. Result of the Blastn analysis of the 16S rDNA amplicons (MZ379788, MZ379789) suggested that the isolates FN20111 and FN20121 belonged to the genus Pectobacterium. To determine the species of the stem rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes acnA, gapA, icdA, mdh, proA and rpoS (MZ403781-MZ403792), and phylogenetic tree was reconstructed using RAxML v8.2.12 (https://github.com/stamatak/standard-RAxML). The result of phylogenetic analysis showed that the stem rot Pectobacterium isolates FN20111 and FN20121 clustered with P. versatile (syn. ‘Candidatus Pectobacterium maceratum’) strains CFBP6051T (Portier et al. 2019), SCC1 (Niemi et al. 2017) and F131 (Shirshikov et al. 2018). And the isolates FN20111 and FN20121 were more closely related to the type strain CFBP6051T than to strains SCC1 and F131. Potato seedlings (cv. Xisen 6 and Favorita) were inoculated with the isolates FN20111 and FN20121 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 solution as control. The seedlings were grown at 28°C and 50% relative humidity. Three days post-inoculation, only the bacteria-inoculated seedlings showed diseased symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected stems and were identified using the same PCR primers of housekeeping genes as described above, fulfill Koch’s postulates. P. versatile causing soft rot and blackleg on potato plants has been reported in Finland (Niemi et al. 2017), Russia (Shirshikov et al. 2018), Netherlands (Portier et al. 2019), Poland (Waleron et al. 2019) and in New York State (Ma et al. 2021). To our knowledge, this is the first report of P. versatile causing aerial stem rot of potato in China.

scholarly journals First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Mesona chinensis in China

Plant Disease ◽  
2011 ◽  
Vol 95 (2) ◽  
pp. 222-222
Author(s):  
Q. Liu ◽  
Y. Li ◽  
J. Chen
Keyword(s):  
16S Rdna ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Vascular Tissue ◽  
Negative Control ◽  
Bacterial Suspension ◽  
Herbaceous Plant ◽  
Disease Etiology ◽  
Tetrazolium Chloride ◽  
First Report

Jellywort (Mesona chinensis Benth) is an herbaceous plant in the Lamiaceae (mint) family. The plant is referred to as ‘Xiancao’ (weed from angels) in Chinese and primarily used to make grass jelly, a popular refreshing drink. Currently, Xiancao cultivation is a fast-growing industry with a high profit margin in southern China. An estimated 7,000 ha is grown with a value of more than $50 million USD. In June, 2009, a wilting disease of Xiancao was observed in Guangdong and the neighboring Guangxi and Fujian provinces with incidence up to 50% in the severest case. Affected plants initially show withering symptoms on apical leaves during the daytime with recovery at night. As the disease develops, withering leaves spread downward, eventually encompassing the whole plant. Leaves lose vigor but remain green. After 3 to 4 days, wilting becomes irreversible. Roots and basal stem tissues blacken and rot, leading to plant death. Longitudinal sectioning of the basal stem shows browning of vascular tissues with whitish ooze visible when compressed. To investigate the disease etiology, 12 Xiancao plants from three cultivars showing typical wilting symptoms were collected from a production field in Zengcheng City of Guangdong Province in June 2010. A total of 27 bacterial isolates showing large, elevated, and fluidal colonies with a pale red center were isolated from vascular tissue on tripheny tetrazolium chloride medium (3) after incubation at 30° for 2 days. Fifteen 45-day-old Xiancao plants (cv. Zhengcheng 1) were inoculated by injection of 20 μl of bacterial suspension (1 × 108 CFU/ml) into the middle stem. Sterile water was used as a negative control. After 4 to 6 days of incubation in a greenhouse (28 to 30°), all (15 of 15) inoculated plants developed wilting symptoms as described above. The same bacterium was reisolated from inoculated plants. The five negative control plants did not show any wilting symptoms. With the same artificial inoculation procedure, this bacterium also caused similar wilting disease in tobacco, potato, tomato, pepper, and eggplant. An inoculation test with a tomato strain of Ralstonia solanacearum resulted in similar symptoms. On the basis of symptomatology and bacterial culture characteristics, R. solanacearum (formerly Pseudomonas solanacearum) was suspected as the causal agent. For confirmation, the universal bacterial 16S rDNA primer set E8F/E1115R (1) was used to amplify DNA from pure culture. A 1,027-bp DNA sequence was obtained and deposited in GenBank with Accession No. HQ159392. BLAST search against the current version of GenBank database showed 100% similarity with the 16S rDNA sequences of 26 R. solanacearum strains. Furthermore, primer set 759/760 (4) amplified a specific 280-bp fragment. Along with the result from multiplex PCR (2), the bacterium was identified as R. solanacearum Phylotype I. To our knowledge, this is the first report of a disease caused by R. solanacearum on M. chinensis. References: (1) G. Baker et al. J. Microbiol. Methods 55:541, 2003. (2) M. Fegan and P. Prior. Page 449 in Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. C. Allen et al., eds. The American Phytopathological Society. St. Paul, MN, 2005. (3) A. Kelman, Phytopathology 44:693, 1954. (4) N. Opina et al. Asia Pac. J. Mol. Biol. Biotechnol. 5:19, 1997.

scholarly journals First Report of Bacterial Foot Rot of Rice Caused by a Dickeya zeae in China

Plant Disease ◽  
2012 ◽  
Vol 96 (12) ◽  
pp. 1818-1818 ◽  
Author(s):  
X. M. Pu ◽  
J. N. Zhou ◽  
B. R. Lin ◽  
H. F. Shen
Keyword(s):  
16S Rdna ◽  
Guangdong Province ◽  
Gyrb Gene ◽  
Bacterial Suspension ◽  
Bacterial Disease ◽  
Sterile Water ◽  
Foot Rot ◽  
Rice Seedlings ◽  
First Report ◽  
Negative Controls

A bacterial disease of rice, bacterial foot rot, was found in Guangdong Province, China in September 2011, with an incidence about 10%. The typical symptom was a dark brown decay of the tillers. In the early stages of the disease, a brown sheath rot seemed to spread from the ligulae regions. The lesions quickly extended down to the nodes, culms, and finally to the crowns. Neighboring tillers of the same crown were invaded systemically, causing foot rot symptoms. A soft rot with an unpleasant odor developed in young tissues of infected tillers. In the advanced stage, many tillers decayed, so that entire diseased plants could easily be pulled from the soil. Six diseased samples were collected and bacteria were isolated from the edge of symptomatic tissues, after samples were sterilized in 0.3% NaOCl for 10 min, rinsed in sterile water three times, and placed on nutrient agar (beef extract 3 g, yeast extract 1 g, peptone 5 g, glucose 10 g, agar 16 g, distilled water 1 L, pH 6.8 to 7.0). For identification, a total of 12 representative isolates were selected. 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) identified isolate EC1 as Pectobacterium chrysanthemi (SIM 0.827), which has since been transferred to genus Dickeya. PCR was used to amplify the 16S rDNA gene with primers 27f and 1492r, the dnaX gene with primers dnaXf and dnaXr (2), and the gyrB gene with primers gyrBf1 (5′-ATGTCGAATTCTTATGACTCCTC-3′) and gyrB-r1 (5′-TCARATATCRATATTCGCYGCTTTC-3′), which were designed based on published gyrB gene sequences of genus Dickeya. A BLASTn search of all three loci [16S rDNA (JQ284040), dnaX (JQ284041), and gyrB (JQ284042)] revealed that EC1 had 100% sequence identify to Dickeya zeae [16S rDNA (AB713560), dnaX (AB713593), gyrB (AB713635)]. Pathogenicity tests were conducted by injecting 10 rice seedlings with 100 μl of the bacterial suspension (1 × 108 CFU/ml) in the stem base, and an additional 10 rice seedlings were injected with 100 μl of sterile water as negative controls. Inoculations were carried out in a greenhouse at 28 to 32°C and 90% relative humidity. Foot rot symptoms identical to those described above were observed after 7 days on inoculated plants, but not on the negative controls. The bacterium was reisolated from the lesions and had 100% sequence identity for all three loci to EC1. Previously, similar symptoms were reported on rice in Guangdong province of China, and the causal agent was identified as Erwinia chrysanthemi (1). To our knowledge, this is the first report of D. zeae causing foot rot disease on rice in China. References: (1) Q. G. Liu et al. J. South China Agric. Univ. 18:128, 1997. (2) M. Sławiak et al. Eur. J. Plant Pathol. 125:245, 2009.

scholarly journals First Report of Corn Bacterial Leaf Stripe Caused by Acidovorax avenae in Guangdong Province, China

Plant Disease ◽  
2014 ◽  
Vol 98 (10) ◽  
pp. 1424-1424 ◽  
Author(s):  
C. Ji ◽  
J. Ou ◽  
D. Xu ◽  
R. Pan
Keyword(s):  
16S Rdna ◽  
Coming Out ◽  
Pathogenic Bacteria ◽  
Reference Strain ◽  
Guangdong Province ◽  
Metabolic Phenotype ◽  
Post Inoculation ◽  
First Report ◽  
Leaf Stripe ◽  
Initial Symptoms

Each autumn of 2011 to 2013, a disease was observed on corn (cv. Huameitian 8) in the Experimental Farm of South China Agricultural University, Guangzhou, Guangdong Province, with an incidence ranging from 20 to 65%. Initial symptoms appeared as small, water-soaked lesions on the leaves. Under warm and humid conditions, lesions expanded along the veins producing necrotic stripes. Stripes later became dry and brown, often with shredding of the infected tissue. Bacterial ooze was observed coming out of sections of leaves showing stripe symptoms. Diseased leaf tissues were surface disinfected in 75% ethanol for 1 min followed by three rinses with sterile distilled water. Bacteria were isolated from symptomatic tissues. Colonies were creamy white, circular, convex, smooth with entire margins on nutrient agar, and non-fluorescent and slow-growing on King's medium B. The strains were gram-negative, rod-shaped with a unidirectional flagellum, aerobic, positive for gelatin liquefaction and catalase, amylase activity, and negative for nitrate reduction. The strains utilized mannose, fructose, and citrate, but not lactose and maltose. 16S rDNA genes of two strains, GDHN01 (Accession No. JQ904301) and GDHN02 (JQ904302), were amplified using universal primers (3). A 1,433-bp amplification product was obtained. Using BLAST analysis of NCBI GenBank, these sequences showed 99 to 100% sequence identity with the type strain Acidovorax avenae subsp. avenae ICMP 3183 (NR041757). These two strains were identified as A. avenae by Biolog metabolic phenotype analysis (Biolog, Hayward, CA) with similarity indices ranging from 0.550 to 0.823. The strains induced hypersensitive response on leaves of 2-month-old tobacco plants within 24 h. The pathogenicity of GDHN01 and GDHN02 was tested three times by needle puncture with bacterial suspensions containing of 6 × 108 CFU/ml on five-leaf-stage healthy corn seedlings (cv. Huameitian 8) (n = 5 seedlings/isolate/experiment). Positive controls were inoculated with A. avenae reference strain Yu13 (obtained from Nanjing Agricultural University, China) (1) and negative controls were inoculated with sterile water. Plants were kept in the greenhouse at 28°C and 80% RH. Yellow to brown stripes were observed on all inoculated leaves at 4 to 6 days post-inoculation; similar symptoms were caused by the reference strain, but no symptoms developed on the negative control plants. Re-isolated bacteria from symptomatic tissues were confirmed to be A. anevae based on morphology, physiological assays, and 16S rDNA sequence analysis described above. No target bacteria were isolated from the control plants. A. avenae was previously identified as the causal agent of bacterial leaf stripe on corn in Jiangsu Province (1) and rice in Zhejiang Province (2). To our knowledge, this is the first report of A. avenae naturally infecting corn in Guangdong Province. Guangdong is the largest sweet corn-producing and consuming province in China. This seed-borne pathogen may pose a threat to corn production in Guangdong Province. References: (1) Y. Gao et al. Jiangsu J. Agric. Sci. 23:22, 2007. (2) L. H. Xu et al. Chinese J. Rice Sci. 22:302, 2008. (3) N. W. Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria, 3rd edition. APS Press. St. Paul, MN, 2001.

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

Plant Disease ◽  
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.

scholarly journals First Report of Bleeding Canker of Pear Tree Trunks Caused by Dickeya fangzhongdai in Korea

Plant Disease ◽  
2021 ◽  
Author(s):  
Eu Ddeum Choi ◽  
Youngmin Kim ◽  
Yerim Lee ◽  
Min-Hye Jeong ◽  
Gyoung Hee Kim ◽  
...  
Keyword(s):  
16S Rrna ◽  
Intergenic Spacer ◽  
Fine Powder ◽  
Luria Bertani ◽  
Bacterial Suspension ◽  
Post Inoculation ◽  
Pathogenicity Test ◽  
Flower Bud ◽  
First Report ◽  
Pear Tree

Pears (Pyrus pylifolia L.) are cultivated nationwide as one of the most economically important fruit trees in Korea. At the end of October 2019, bleeding canker was observed in a pear orchard located in Naju, Jeonnam Province (34°53′50.54″ N, 126°39′00.32″ E). The canker was observed on trunks and branches of two 25-year-old trees, and the diseased trunks and branches displayed partial die-back or complete death. When the bark was peeled off from the diseased trunks or branches, brown spots or red streaks were found in the trees. Bacterial ooze showed a rusty color and the lesion was sap-filled with a yeasty smell. Trunks displaying bleeding symptoms were collected from two trees. Infected bark tissues (3 × 3 mm) from the samples were immersed in 70% ethanol for 1 minute, rinsed three times in sterilized water, ground to fine powder using a mortar and pestle, and suspended in sterilized water. After streaking each suspension on Luria-Bertani (LB) agar, the plates were incubated at 25°C without light for 2 days. Small yellow-white bacterial colonies with irregular margins were predominantly obtained from all the samples. Three representative isolates (ECM-1, ECM-2 and ECM-3) were subjected to further characterization. These isolates were cultivated at 39 C, and utilized (-)-D-arabinose, (+) melibiose, (+)raffinose, mannitol and myo-inositol but not 5-keto-D-gluconate, -gentiobiose, or casein. These isolates were identified as Dickeya sp. based on the sequence of 16S rRNA (MT820458-820460) gene amplified using primers 27f and 1492r (Heuer et al. 2000). The 16S rRNA sequences matched with D. fangzhongdai strain ND14b (99.93%; CP009460.1) and D. fangzhongdai strain PA1(99.86%; CP020872.1). The recA, fusA, gapA, purA, rplB, and dnaX genes and the intergenic spacer (IGS) regions were also sequenced as described in Van der wolf et al. (2014). The recA (MT820437-820439), fusA (MT820440-820442), gapA (MT820443-820445), purA (MT820446-820448), rplB (MT820449-820451), dnaX (MT820452-820454) and IGS (MT820455-820457) sequences matched with D. fangzhongdai strains JS5, LN1 and QZH3 (KT992693-992695, KT992697-992699, KT992701-992703, KT992705-992707, KT992709-992711, KT992713-992715, and KT992717-992719, respectively). A neighbor-joining phylogenetic analysis based on the concatenated recA, fusA, gapA, purA, rplB, dnaX and IGS sequences placed the representative isolates within a clade comprising D. fangzhongdai. ECM-1 to 3 were grouped into a clade with one strain isolated from waterfall, D. fangzhongdai ND14b from Malaysia. Pathogenicity test was performed using isolate ECM-1. Three two-year-old branches and flower buds on 10-year-old pear tree (cv. Nittaka), grown at the National Institute of Horticultural and Herbal Science Pear Research Institute (Naju, Jeonnam Province in Korea), were inoculated with 10 μl and 2 μl of a bacterial suspension (108 cfu/ml), respectively, after wounding inoculation site with a sterile scalpel (for branch) or injecting with syringe (for flower bud). Control plants were inoculated with water. Inoculated branches and buds in a plastic bag were placed in a 30℃ incubator without light for 2 days (Chen et al. 2020). Both colorless and transparent bacterial ooze and typical bleeding canker were observed on both branches and buds at 3 and 2 weeks post inoculation, respectively. No symptoms were observed on control branches and buds. This pathogenicity assay was conducted three times. We reisolated three colonies from samples displaying the typical symptoms and checked the identity of one by sequencing the dnaX locus. Dickeya fangzhongdai has been reported to cause bleeding canker on pears in China (Tian et al. 2016; Chen et al. 2020). This study will contribute to facilitate identification and control strategies of this disease in Korea. This is the first report of D. fangzhongdai causing bleeding canker on pears in Korea.

scholarly journals First Report of Candidatus Phytoplasma solani Associated with Potato Plants in Greece

Plant Disease ◽  
2014 ◽  
Vol 98 (12) ◽  
pp. 1739-1739 ◽  
Author(s):  
M. C. Holeva ◽  
P. E. Glynos ◽  
C. D. Karafla ◽  
E. M. Koutsioumari ◽  
K. B. Simoglou ◽  
...  
Keyword(s):  
16S Rdna ◽  
Nested Pcr ◽  
Pathogenic Bacteria ◽  
Related Strain ◽  
Northern Greece ◽  
First Report ◽  
Potato Plants ◽  
Pcr Product ◽  
16S Rdna Pcr ◽  
Stolbur Phytoplasma

In August 2013, potato plants (Solanum tuberosum) cv. Banba displaying symptoms resembling those caused by Candidatus Phytoplasma solani (potato stolbur phytoplasma) were observed in a 2-ha field in the area of the Peripheral Unit of Drama (northern Greece). The plants were 10 weeks old and their symptoms included reddening and upward rolling of leaflets, reduced size of leaves, shortened internodes, and aerial tuber formation. Incidence of affected plants was estimated to be 40% in the field. Four symptomatic potato plants were collected for laboratory testing of possible phytoplasma infection. From each of these four plants, total DNA was extracted from mid veins of reddish leaflets from apical shoot parts and of leaflets emerging from aerial tubers, using a phytoplasma enrichment procedure (1). A nested PCR using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (3) amplified the expected ~1.2-kb 16S rDNA fragment in all four symptomatic potato plants. No amplification was observed with DNA similarly extracted from leaflets of asymptomatic potato plants of the same variety collected from an apparently healthy crop. One of the four 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced by Beckman Coulter Genomics (United Kingdom). At least twofold coverage per base position of the cloned PCR product was achieved. BLAST analysis showed that the obtained sequence of the PCR 16S rDNA product was: i) 100% identical to several GenBank sequences of Ca. P. solani strains, including strains detected previously in Greece infecting tomato (GenBank Accession No. JX311953) and Datura stramonium (HE598778 and HE598779), and ii) 99.7% similar to that of the Ca. P. solani reference strain STOL11 (AF248959). Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup (AF248959). The sequence of this PCR product was deposited in NCBI GenBank database under the accession no. KJ810575. The presence of the stolbur phytoplasma in all four symptomatic potato plants examined was further confirmed by nested PCR using the stolbur-specific STOL11 primers (3) targeting non-ribosomal DNA. Based on the observed symptoms in the field and laboratory molecular examinations, we concluded that the potato plants were infected by a Ca. P. solani related strain. The stolbur disease has been previously reported in Greece affecting tomato (2,5) and varieties of D. stramonium (4). To our knowledge, this is the first report of a Ca. P. solani related strain infecting a potato crop in Greece. As northern Greece is a center of potato production, the source of this pathogen is to be investigated. References: (1) U. Ahrens and E. Seemuller. Phytopathology 82:828, 1992. (2) A. S. Alivizatos. Pages 945-950 in: Proceedings of the 7th International Conference of Plant Pathogenic Bacteria. Academiai Kiado, Budapest, Hungary, 1989. (3) J. Jović et al. Bull. Insectol. 64:S83, 2011. (4) L. Lotos et al. J. Plant Pathol. 95:447, 2013. (5) E. Vellios and F. Lioliopoulou. Bull. Insectol. 60:157, 2007.

scholarly journals First Report of Petiole (Rachis) Blight of Washingtonia filifera Caused by Phoma glomerata in Greece

Plant Disease ◽  
2013 ◽  
Vol 97 (11) ◽  
pp. 1509-1509 ◽  
Author(s):  
E. K. Ligoxigakis ◽  
E. A. Markakis ◽  
I. A. Papaioannou ◽  
M. A. Typas
Keyword(s):  
Vascular Tissue ◽  
Economic Loss ◽  
Rrna Genes ◽  
Its2 Region ◽  
Post Inoculation ◽  
28S Rrna ◽  
First Report ◽  
Palm Species ◽  
Dark Green ◽  
Washingtonia Filifera

In July 2007, a severe petiole (rachis) blight disease was observed on several California fan palms (Washingtonia filifera) in the vicinity of Heraklion (Crete), Greece. Typical symptoms included discolored (brown to reddish-brown), reversed V-shaped lesions on the petiole bases of the oldest (lowest) leaves, and elongated yellow to dark-brown stripes along the petiole. The lesions progressively expanded and penetrated the petioles, resulting in gradual discoloration (from tan to brown-black) of the internal petiole tissues, including the vascular tissue. The bases of infected petioles occasionally became fragile and burst open, while the corresponding leaf blades were characterized initially by yellowing and one-sided or uneven wilt and, later, desiccation and death with the entire leaves curving downwards. The disease gradually moved upward to younger leaves, severely debilitating but rarely killing the infected trees. A filamentous fungus was consistently isolated onto potato dextrose agar (PDA) plates from sections of diseased petioles, forming dense, dark green colonies with abundant light to dark brown, subglobose pycnidia (diameter ranging between 36.4 to 177.4 μm, and averaging 99.4 μm, n = 50) on the agar surface or immersed in the medium. Chlamydospores and numerous dictyochlamydospores were also observed, with the latter being initially light to dark brown and later becoming black. The numerous conidia were hyaline, ovoid to ellipsoid, and single-celled. Their dimensions were 5.3 to 7.3 × 2.4 to 4.9 μm, averaging 6.5 × 3.2 μm (n = 100). The ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (3), were amplified with PCR from total DNA extracted from two representative isolates, and sequenced (GenBank Accession Nos. KC802086 to KC802087). Using BLASTn, both sequences were 100% identical to Phoma glomerata ITS sequences (FJ427018, FJ427011, AF126816). Based on morphological and molecular analyses, the pathogen was identified as Phoma glomerata (Corda) Wollenw. & Hochapfel, also known as Peyronellaea glomerata (Corda) Goid. ex Togliani or Coniothyrium glomeratum Corda (1,2). To prove pathogenicity and fulfill Koch's postulates, petioles of the older leaves of eight W. filifera 2-year-old seedlings were wounded with a sterile scalpel (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface), inoculated with agar discs from a 2-week-old PDA culture of the fungus, and sealed with Parafilm. For controls, sterile PDA plugs were placed on the artificial wounds of five more seedlings. All plants were maintained in the greenhouse at 15 ± 5°C, with 90% humidity. Petiole blight and leaf necrosis symptoms—identical to those observed in the infected plants—were evident 5 weeks post-inoculation, and P. glomerata was consistently reisolated from all inoculated plants. No symptoms were observed on control plants. This is the first report of petiole blight of a palm species caused by P. glomerata in Greece. Due to the extensive use of palms as ornamentals in Greece, the occurrence of P. glomerata can potentially cause economic loss to the local ornamental industry. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) R. M. Hosford, Jr. Phytopathology 65:1236, 1975. (3) M. P. Pantou et al. Mycol. Res. 109:889, 2005.

scholarly journals First Report of Pectobacterium aroidearum Causing Soft Rot in Olecranon Honey Peach (Prunus persica L.) in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Zhibin Liang ◽  
Huidi Liu ◽  
Zeling Xu ◽  
Lian-hui Zhang
Keyword(s):  
16S Rdna ◽  
Dna Sequences ◽  
Prunus Persica ◽  
Bacterial Species ◽  
Guangdong Province ◽  
Housekeeping Genes ◽  
Soft Rot ◽  
Accession Number ◽  
First Report ◽  
Conserved Region

Olecranon honey peach (Prunus persica L.) is a popular fruit tree cultivated in Guangdong Province of China. Due to its excellent economic values and popularity, it has recently been widely adopted and planted in several other southern Provinces and Autonomous Region in China, including Yunnan, Hunan, Jiangxi, Guizhou, and Guangxi. In Lianping County of Guangdong Province alone, the annual peach fruit production was about 78,800 tonnes (Xie et al. 2017). In July 2021, peach fruits showing soft rot symptoms were collected from an olecranon honey peach plantation in Lechang, Guangdong, China. Symptoms included tissue disintegration with bacterial oozes and rotting smells. To isolate the causal agent of soft rot in the peach fruits, the bacterial oozes from various rotted fruits were streaked on the modified YEB agar plate (Huang et al. 2021), and 21 bacterial colonies were selected for PCR amplification using the primers targeting the conserved region of 16S rDNA gene (Wei et al. 2020). A blastN analysis of the DNA sequences of the obtained PCR fragments in NCBI website indicated that 17 isolates named as ZL strains were potential bacterial species of Pectobacterium with about 99% similarity (Genbank accession number of ZL1: OK189602) to Pectobacterium aroidearum SCRI 109T (Genbank accession number: NR_159926). Three of them (ZL1, ZL2 and ZL3) were selected for assay of pathogenicity. The bacterial suspensions (10 μl, 1×106 CFU/ml) of strains ZL1, ZL2 and ZL3 were injected into olecranon honey peach fruits by using a syringe. A portion of peach fruits were similarly injected with sterile distilled water as the negative control. After 18 h incubation at 25 °C, the typical symptom of soft rot, i.e., tissue decay, became visible on the peach fruits inoculated with the bacterial suspensions. After inoculation for 42 h, bacterial oozes were exuded from rotting tissues. Peach fruits without injuries were also sprayed with the bacterial suspensions under the same conditions, but decay symptoms were not observed, suggesting that the bacterial infection needs the wounding or injuries. To fulfill the Koch’s postulates, bacterial colonies were re-isolated from bacterial oozes, and their conserved region of 16S rDNA fragments were amplified and sequenced. Bioinformatics analysis of the DNA sequence data confirmed that all the isolated colonies were Pectobacterium strains. Using the Biolog Gen III system, the representative strain ZL1 was identified as Pectobacterium (SIM 0.56). Transmission electron microscopy analysis showed that the bacterial cells of strain ZL1 were rod-shaped with peripheral flagella. To further determine the species of ZL strains, eight housekeeping genes (acnA, gapA, icd, mdh, mtlD, pgi, proA and rpoS) were analyzed by the methods described previously (Nabhan et al. 2013). The amplified DNA sequences analyzed by the blastN program in NCBI showed that the sequences of eight housekeeping genes from strains ZL1, ZL2 and ZL3 were identical to each other (Genbank accession number: OK274248 to OK274255), and most of the gene sequences shared over 99% similarity to their counterparts in P. aroidearum L6 (Genbank accession number: NZ_CP065044) (Xu et al. 2021), except that the acnA and proA genes showed about 98% and 96% similarity respectively to the corresponding genes of P. aroidearum L6. In addition, the multi-locus sequence analysis (MLSA) using DNA sequences of above eight housekeeping genes showed that ZL strains were grouped with other P. aroidearum strains. Taken together, the results of molecular and biochemical assays confirmed that ZL strains isolated from olecranon honey peach fruits were P. aroidearum. To our knowledge, this is the first report of P. aroidearum causing soft rot disease in olecranon honey peach in China. P. aroidearum is a relatively newly described soft rot pathogen (Nabhan et al. 2013). More recently, the pathogen was found causing soft rot infections in lettuce, Chinese cabbage, pepper (Capsicum annuum) fruits, konjac, carrot and Syngonium podophyllum (Barroso et al. 2019; Moraes et al. 2020; Sun et al. 2019; Tang et al. 2020; Xu et al. 2021). The results of this study add a new plant species to the host range of P. aroidearum.

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

Plant Disease ◽  
2012 ◽  
Vol 96 (5) ◽  
pp. 760-760 ◽  
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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