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

Plant Disease ◽  
2021 ◽  
Author(s):  
José Luis Palomo Gómez ◽  
Maria Shima ◽  
Adela Monterde ◽  
Inmaculada Navarro ◽  
Silvia Barbé ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Xanthomonas Campestris ◽  
Negative Control ◽  
Leaf Blight ◽  
Bacterial Leaf Blight ◽  
Pcr Analysis ◽  
Bacterial Suspension ◽  
Rrna Gene ◽  
Distilled Water ◽  
First Report

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

scholarly journals First Report of Bacterial Leaf Blight of Carrot Caused by Xanthomonas hortorum pv. carotae in Korea

Plant Disease ◽  
2014 ◽  
Vol 98 (2) ◽  
pp. 275-275 ◽  
Author(s):  
I.-S. Myung ◽  
M.-J. Yoon ◽  
J.-Y. Lee ◽  
G.-D. Kim ◽  
M.-H. Lee ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Negative Control ◽  
Leaf Blight ◽  
Gyrb Gene ◽  
Bacterial Leaf Blight ◽  
Distilled Water ◽  
First Report ◽  
Rpod Gene ◽  
Phenotypic Tests ◽  
Hydrolysis Of

In December 2012, symptoms of typical bacterial leaf blight were observed on carrot plants (Daucus carota L. subsp. sativus) cultivated in commercial fields in Kujwa, Jeju, Korea. The disease was detected in 40% of 50 fields surveyed with an incidence of 10% on average. The bacterial leaf blight lesions on leaf blades were elongated, dark brown to black with water-soaked edges and chlorotic halos. Lesions were also crescent-shaped to V-shaped on leaflets. Four bacterial isolates were recovered on trypticase soy agar from leaf lesions that were surface-sterilized in 70% ethyl alcohol for 20 s. Identity of the isolates was confirmed by PCR product (1,266-bp) using a specific primer set for Xanthomonas hortorum pv. carotae (Kendrick 1934) Vauterin et al. 1995, XhcPP03 (1). All isolates were gram-negative, aerobic rods with a single polar flagellum. Isolates were positive for catalase and negative for oxidase. In phenotypic tests for differentiation of Xanthomonas (2), the isolates positive for mucoid growth on yeast extract-dextrose-calcium carbonate agar, growth at 35°C, hydrolysis of esculin, protein digestion, alkaline in litmus milk, acid production from arabitol, and utilization of glycerol and melibiose. The isolates were negative for growth on SX medium, hydrolysis of starch, and ice nucleation. The gyrB gene (863 bp) and the rpoD gene (870 bp) were sequenced to aid identification of the original isolates using published PCR primer sets, Xgyr1BF/Xgyr1BR and XrpoD1F/XrpoD1R (4), respectively. Sequences of the gyrB gene (GenBank accessions KC920729 to KC920732) from the carrot isolates shared 100% sequence identity with that of the X. hortorum pv. carotae strain NCPPB 425 (EU285243). In phylogenetic analyses based on the partial sequences of the gyrB and the rpoD genes for Xanthomonas spp. available at NCBI (4), and sequences of the carrot isolates (KC920734 to KC920737 for rpoD gene) using the Neighbor-joining method in MEGA Version 5.1 (3), the isolates were clustered in the X. hortorum-cynarae-garnderi group. Pathogenicity of the isolates was tested by spray inoculation with a bacterial suspension (106 CFU/ml) prepared in sterile distilled water at 6 to 7 true-leaf stage (three plants per isolate). Sterile distilled water was used as negative control. The inoculated plants were incubated in a growth chamber (25°C and 95% relative humidity [RH]) for 15 hr, and then transferred to a greenhouse at 24 to 28°C and 65% RH. Characteristic leaf blight symptoms developed on inoculated carrot plants, while no symptoms were observed on the negative control plants 14 days after inoculation. The bacterium was re-isolated from symptomatic tissue and the identity confirmed through gyrB gene sequence analysis (4). Based on PCR, morphological and phenotypic tests, sequence analysis, and pathogenicity assays, the isolates were identified as X. hortorum pv. carotae. To our knowledge, this is the first report of bacterial leaf blight of carrot caused by X. hortorum pv. carotae in Korea. The detection of this pathogen could have a significant economic impact due to yield losses from disease development. Consolidation of quarantine inspection on imported carrot seeds needs to control an outbreak of the disease. Crop rotation and plowing are recommended to reduce incidence of the disease in the infested fields. References: (1) J. A. Kimbrel et al. Mol. Plant Pathol. 12:580, 2011. (2) N. W. Schaad et al. Page 189 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. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

scholarly journals First Report of New Bacterial Leaf Blight of Rice Caused by Pantoea ananatis in Southeast China

Plant Disease ◽  
2021 ◽  
Author(s):  
Lin Yu ◽  
Changdeng Yang ◽  
Zhijuan Ji ◽  
Yuxiang Zeng ◽  
Yan Liang ◽  
...  
Keyword(s):  
Oryza Sativa L ◽  
Sequence Similarity ◽  
Disease Incidence ◽  
Leaf Blight ◽  
Bacterial Leaf Blight ◽  
Bacterial Suspension ◽  
Rrna Gene ◽  
Pantoea Ananatis ◽  
Southeast China ◽  
First Report

In autumn 2020, leaf blight was observed on rice (Oryza sativa L., variety Zhongzao39, Yongyou9, Yongyou12, Yongyou15, Yongyou18, Yongyou1540, Zhongzheyou8, Jiafengyou2, Xiangliangyou900 and Jiyou351) in the fields of 17 towns in Zhejiang and Jiangxi Provinces, China. The disease incidence was 45%-60%. Initially, water-soaked, linear, light brown lesions emerged in the upper blades of the leaves, and then spread down to leaf margins, which ultimately caused leaf curling and blight during the booting-harvest stage (Fig. S1). The disease symptoms were assumed to be caused by Xanthomonas oryzae pv. oryzae (Xoo), the pathogen of rice bacterial blight. 63 isolates were obtained from the collected diseased leaves as previously described (Hou et al. 2020). All isolates showed circular, smooth-margined, yellow colonies when cultured on peptone sugar agar (PSA) medium for 24h at 28℃. The cells were all gram-negative and rod-shaped with three to six peritrichous flagella; positive for catalase, indole, glucose fermentation and citrate utilization, while negative for oxidase, alkaline, phenylalanine deaminase, urease, and nitrate reductase reactions. 16S rRNA gene sequence analysis from the 6 isolates (FY43, JH31, JH99, TZ20, TZ39 and TZ68) revealed that the amplified fragments shared 98% similarity with Pantoea ananatis type strain LMG 2665T (GenBank JFZU01) (Table S3). To further verify P. ananatis identity of these isolates, fragments of three housekeeping genes including gyrB, leuS and rpoB from the 6 isolates were amplified and sequenced, which showed highest homology to LMG 2665T with a sequence similarity of 95%-100% (Table S3). Primers (Brady et al. 2008) and GenBank accession numbers of gene sequences from the 6 isolates are listed in Table S1 and Table S2. Phylogenetic analysis of gyrB, leuS and rpoB concatenated sequences indicated that the 6 isolates were clustered in a stable branch with P. ananatis (Fig. S2). Based on the above morphological, physiological, biochemical and molecular data, the isolates are identified as P. ananatis. For pathogenicity tests, bacterial suspension at 108 CFU/mL was inoculated into flag leaves of rice (cv. Zhongzao39) at the late booting stage using clipping method. Water was used as a negative control. The clipped leaves displayed water-soaked lesions at 3 to 5 days after inoculation (DAI); then the lesion spread downward and turned light brown. At about 14 DAI, blight was shown with similar symptoms to those samples collected from the rice field of Zhejiang and Jiangxi provinces (Fig. S1). In contrast, the control plants remained healthy and symptomless. The same P. ananatis was re-isolated in the inoculated rice plants, fulfilling Koch’s postulates. In the past decade, P. ananatis has been reported to cause grain discoloration in Hangzhou, China (Yan et al. 2010) and induce leaf blight as a companion of Enterobacter asburiae in Sichuan province, China (Xue et al. 2020). Nevertheless, to the best of our knowledge, this is the first report of P. ananatis as the causative agent of rice leaf blight in southeast China. This study raises the alarm that the emerging rice bacterial leaf blight in southeast China might be caused by a new pathogen P. ananatis, instead of Xoo as traditionally assumed. Further, the differences of occurrence, spread and control between two rice bacterial leaf blight diseases caused by P. ananatis and Xoo, respectively need to be determined in the future.

scholarly journals First Report of Bacterial Spot Caused by Xanthomonas campestris pv. vesicatoria on Sweet Pepper (Capsicum annuum L.) in Saudi Arabia

Plant Disease ◽  
2012 ◽  
Vol 96 (11) ◽  
pp. 1690-1690 ◽  
Author(s):  
Y. Ibrahim ◽  
M. Al-Saleh
Keyword(s):  
Saudi Arabia ◽  
Xanthomonas Campestris ◽  
Sweet Pepper ◽  
Negative Control ◽  
Bacterial Suspension ◽  
Distilled Water ◽  
Bacterial Spot ◽  
Biochemical Tests ◽  
First Report ◽  
Pepper Fruit

In the summer of 2009 and 2010, 18 sweet pepper fruit with blister-like, raised, rough lesions were collected from four greenhouses (total of 0.1 ha) in the Al-Kharj region of Saudi Arabia. All samples were collected from commercial crops of the sweet pepper cv. California Wonder. Disease incidence was ≤5%. Isolations were made from all diseased fruits. A small piece (3 mm2) of symptomatic tissue from pepper fruit was placed in a sterile mortar and macerated in sterile distilled water with a pestle. A loopful of bacterial suspension from each sample was streaked onto Tween B agar medium (3). Plates were incubated at 28°C for 48 h. Single yellow, circular, butyrous, shiny colonies were picked from the plates and transferred to nutrient agar plates containing 5% D+ glucose agar (NGA). Gram-negative, rod-shaped bacteria were consistently isolated from the fruit and 10 of the isolates were identified as Xanthomonas campestris pv. vesicatoria on the basis of morphological, physiological, and biochemical tests (1,2). The isolates were oxidase positive and levan negative, arginine-dihydrolase positive, and did not macerate potato discs. The isolates were also non-fluorescent, grew at 37 and 4°C but not at 40°C, did not liquefy gelatine or starch, but did produce H2S. The identity of the 10 bacterial strains was confirmed by PCR assay using primers RST65 and RST69 (4). Four-week old pepper plants (cv. California Wonder) were inoculated by spraying five potted plants with each isolate using a bacterial suspension (108 CFU/ml). Sterile distilled water was sprayed on an additional five plants as a negative control treatment. The bacterial isolates caused necrotic lesions, each with a yellow halo, on leaves of inoculated plants. Bacteria reisolated from the necrotic lesions using the technique previously described were identical to the original strains according to the morphological, cultural, and biochemical tests described above. Negative control plants inoculated with sterile distilled water did not show symptoms and no bacterial colonies were recovered from them. To our knowledge, this is the first report of bacterial spot on pepper fruits in Saudi Arabia. References: (2) R. F. Bradbury. Genus II Xanthomonas Dowson 1939. In: Bergey's Manual of Systematic Bacteriology, Vol. 1, Krieg, R., Holt, J. G. (Eds.), Williams & Wilkins Co., Baltimore, MD, 1987. (3) R. A. Lelliott and D. E. Stead. Methods for the Diagnosis of Bacterial Diseases of Plants. Blackwell Scientific Publications, Oxford, UK. (1) R. G. McGuire et al. Plant Dis 70:887, 1986. (4) A. Obradovic et al. Eur. J. Plant Pathol. 110:285, 2004.

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

Plant Disease ◽  
2010 ◽  
Vol 94 (8) ◽  
pp. 1065-1065 ◽  
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.

scholarly journals First report of stalk bacterial soft rot of sugarcane caused by Dickeya zeae in China

Plant Disease ◽  
2020 ◽  
Author(s):  
yanchang Yang ◽  
Ziting Yao ◽  
Mu-Qing Zhang ◽  
Chengwu Zou ◽  
Baoshan Chen
Keyword(s):  
Pure Water ◽  
Bacterial Species ◽  
Evolutionary Relationship ◽  
Soft Rot ◽  
Bacterial Suspension ◽  
Rrna Gene ◽  
Post Inoculation ◽  
Distilled Water ◽  
Sugarcane Cultivar ◽  
First Report

In late September 2019, seven stalks of about 1400 stalks of sugarcane cultivar Zhongzhe 1 exhibited soft rot symptoms in a trial plot in Beihai city, Guangxi province of China. Symptoms included scorched and collapsed leaves, maceration of stalks, and sour smelling exudates from the stalks (Supplementary Fig. S1). Severely diseased stalks had collapsed and were dead. Internal stalk fragments of 5 × 5 mm were collected at the junction of healthy and diseased tissue after surface-sterilization of stalks with 70% ethanol for one minute, and three times rinsing with sterile distilled water. Stalk fragments were placed on Luria–Bertani agar medium (1 % w/v tryptone, 0.5 % w/v yeast extract, 1 % w/v NaCl, 1 % w/v agar, pH7.0) and plates were put in an incubator at 30°C for 48h. Four types of bacterial colonies were obtained, and small and white colonies with irregular margins were the most dominant. A single colony of each type was diluted in sterile distilled water and aliquots of each suspension were streaked on fresh medium plates to obtain pure cultures. Ten eight-week-old stalks (11 th leaf stage) of sugarcane plants, which derived from cuttings of symptomless cultivar Zhongzhe 1, were inoculated by injection of 300 μl of bacterial suspension (3.5x108 CFU/ml) into the stalks. Another 10 stalks were injected with pure water and served as control. The inoculated plants were kept in a greenhouse at 25-37℃.Among the four types of bacteria, only strain BH9 induced symptoms that were identical to those of diseased canes observed in the field (Supplementary Fig. S1). Elongated water-soaked lesions were observed around the inoculation sites three days post inoculation. Five of the 10 BH9-inoculated plants had collapsed two days later. Water-soaked stalks had a sour smell similar to the filed diseased plants. Eight days post inoculation, all BH9-inoculated plants exhibited symptoms but control plants remained symptomless up to 30 days after inoculation. Uniform white colonies with irregular margins were isolated from the inoculated stalks that developed soft rot symptom, and these bacteria caused again stalk soft rot symptoms when inoculated to a new batch of 10 healthy plants. The 16S rRNA gene of strain BH9 was amplified by PCR with primer pair fD2/rP1 and the PCR amplicons from three independent colonies were sequenced. The sequences of the three amplicons were identical (Accession No. MT723897). BLAST alignments of the 16S rDNA sequence from BH9 strain with the GenBank database revealed that BH9 belonged to the genus Dickeya (98.5% identity between D. zeae BH9 and D. zeae EC1). Further PCR assays and sequencing of three genes, DNA polymerase III gamma subunit gene dnaX with primers dnaXf/dnaXr, DNA gyrase gene gyrB with primers gyrBf1/gyrBr1, and recombinase A gene recA with primers recAf/recAr, were performed to identify the species within the genus Dickeya (Zhang et al., 2014). BH9 sequences of these genes (Accession No. MT723898 to MT723900) had highest identity (97.5%, 97.6%, and 97.7%, respectively) with those from D. zeae EC1 (GenBank accession No. CP006929.1). To determine the evolutionary relationship of BH9 to other Dickeya species and strains, a phylogenetic analysis was performed using dnaX, gyrB, and recA sequences. As shown in Supplementary Fig. S2, BH9 clustered with D. zeae strains and formed a lineage distinguishable from other Dickeya species. Among the closest strains, D. zeae NCPPB3531 (Accession No. CM001980.1) was isolated from potato and D. zeae CSL RW192 (Accession No. CM001972.1) from river water (Pritchard et al., 2013). Consequently, strain BH9 was identified as D. zeae. This bacterial species has been reported to cause soft rot in rice (Pu et al., 2012), banana (Zhang et al., 2014), maize (Martinez-Cisneros et al., 2014), and clivia (Hu et al., 2018). To the best of our knowledge, this is the first report of a bacterial stalk rot caused by D. Zeae in sugarcane. In fact, low incidence of D. zeae-caused stalk soft rot was recently found in sugarcane fields in Fusui County, about 150 km north to Beihai. Given the potential threat of this disease to the local sugarcane industry, the mode of transmission, cultivar resistance, and measures to control the disease should be investigated.

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

Plant Disease ◽  
2015 ◽  
Vol 99 (2) ◽  
pp. 281-281 ◽  
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.

scholarly journals First Report of Xanthomonas campestris pv. campestris Causing Marginal Leaf Necrosis of Arugula (Eruca vesicaria subsp. sativa) in Serbia

Plant Disease ◽  
2021 ◽  
Author(s):  
Andjelka Prokić ◽  
Tamara Marković ◽  
Jelena Menković ◽  
Milan Ivanovic ◽  
Aleksa Obradoviċ
Keyword(s):  
Xanthomonas Campestris ◽  
Disease Incidence ◽  
Positive Control ◽  
Negative Control ◽  
Accurate Information ◽  
Strictly Aerobic ◽  
Distilled Water ◽  
Tetrazolium Chloride ◽  
First Report ◽  
Xanthomonas Campestris Pv Campestris

Arugula (Eruca vesicaria subsp. sativa (Miller) Thell., syn. Brassica eruca L.), is an annual cruciferous crop that is increasingly grown for fresh consumption in Serbia. In November 2018, a few detached leaves of cultivated arugula originating from a local producer, showing necrotic lesions, were observed in a fresh vegetable market in Belgrade, Serbia. Information about the disease incidence and severity was not available. Intensity of the observed symptoms was low, but it could be a consequence of the produce quality selection for the market. The leaves developed irregular chlorotic lesions starting from the leaf edge, and tissue within some of them turned dark brown and necrotic (Fig. 1a). From the lesions on different leaves, smooth, bright yellow pigmented, round and opalescent bacterial colonies were isolated on nutrient agar (NA) medium after 72 h of incubation at 26°C. Six bacterial isolates, obtained from three leaf subsamples which induced hypersensitive reaction in tobacco leaves (Nicotiana tabacum L. cv. Samsun), were selected for further studies. On yeast - dextrose – CaCO3 medium, the strains formed characteristic creamy yellow, mucoid, opaque and convex colonies. All isolates were Gram-negative, strictly aerobic, non-fluorescent and catalase positive, did not produce oxidase nor arginine dehydrolase, and did not show pectynolitic activity on potato tuber slices. They hydrolyzed starch, gelatine and esculin, used glucose and sucrose, but not arabinose as a carbon source, and did not reduce nitrates. They grew at 36°C, and tolerated 5% NaCl and 0.02% triphenyl-tetrazolium chloride (Lelliott and Stead, 1987). These growth characteristics were similar as for the reference Xanthomonas campestris pv. campestris (Xcc) strain KFB 105, used in all tests as a positive control (Obradović et al., 2000). The isolates were further characterized by polymerase chain reaction (PCR) using primers DLH120/DLH125, specific for the hrpF gene region of X. campestris according to Berg et al. (2005). Specific DNA fragment of 619 bp was amplified for all tested isolates. Amplification and partial sequencing of the gyrB gene of four isolates was performed using set of primers described by Parkinson et al. (2007). All obtained partial gyrB sequences were identical to each other. According to BLAST analysis (GenBank Acc. Nos. MW508894 - MW508897) they shared 100% of sequence identity with different Xcc strains and 99.5 % with the X.c. pv. raphani pathotype strain, deposited in the NCBI GenBank database. Pathogenicity of the isolates was tested by spraying leaves of 3-week old E. sativa seedlings grown in a commercial potting mix in a greenhouse, with a 24 h-old bacterial culture suspended in sterile distilled water (107 CFU/ml). Xcc strain KFB 105 was used as positive and sterile distilled water as negative control. Inoculated plants were incubated under plastic bags for 48 h and further maintained in a greenhouse at approx. 28°C. On inoculated plants, chlorotic lesions, spreading from the leaf margins, further coalescing into irregular, V-shaped tissue necrosis associated with blackening of veins, developed up to two weeks after inoculation (Fig. 1b, c). The colonies reisolated from symptomatic leaves were identified using PCR, as described above. Based on studied characteristics, all six isolates associated with arugula leaf lesions in Serbia belong to a clonal population. They were identified as X. campestris pv. campestris, the causal agent of black rot, a major disease affecting crucifers, including arugula worldwide (Romero et al., 2008; Rosenthal, et al., 2018). So far, it has been described on Brassica oleracea and B. napus in Serbia (Obradović et al., 2001; Popović et al., 2019). This is the first report of Xcc infecting arugula in this country. The severity of the symptoms developed on artificially inoculated plants indicated significant potential of the pathogen to affect arugula crop in conditions favoring infection. Being a minor crop, accurate information about severity of arugula diseases in Serbia is not available. Lack of crop rotation and close proximity of other Xcc host species on a farm could contribute to further spreading of this problem. Follow up of this arugula disease should reveal the distribution, population structure and genetic diversity of Xcc strains affecting this crop in Serbia.

scholarly journals First Report of Pseudomonas oryzihabitans Causing Stem and Leaf Rot on Muskmelon in China

Plant Disease ◽  
2021 ◽  
Author(s):  
JuFen Li ◽  
Ganghan Zhou ◽  
Tan Wang ◽  
Tao Lin ◽  
yiwen wang ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Suspension ◽  
Rrna Gene ◽  
Distilled Water ◽  
Phylogenetic Tree Analysis ◽  
First Report ◽  
Rot Disease ◽  
The 16S Rrna Gene ◽  
Leaf Rot

Muskmelon (Cucumis melo L.) is an important economic crop in China, which is planted on more than 376, 000 hectares with over 13 million tons of annual fruit production. In February 2020, a serious bacterial stem and leaf rot disease on muskmelon plants was observed in greenhouses in Liguo Town, Ledong County, Hainan Province, China (18.54° N, 108.87° E), with disease incidences being approximately 10 to 12%. Initially, soft rot symptoms appeared on petioles and stems, showing yellow bacterial ooze signs, which was different from the milky white ooze produced by Erwinia tracheiphila infection. The infected tissues of petioles, stems, and leaves eventually developed into browning and withering symptoms. To isolate and identify the causal agent, the lesion tissues were sterilized by immersion in 75% ethanol for 30 s, washed three times with sterile water, and then cut and soaked in 1 ml of distilled water for 10 min. The suspension was serially diluted and spread on Luria-Bertani agar (LB) medium. After incubation at 28°C for 24 to 36 h, the resulted bacterial colonies were tiny and were streaked on LB plate for further culture. After purification, the colonies were yellow, circular, smooth-margined, and two independent representative isolates CM-11 and CM-12 were used for further validation experiments. The electron microscope analysis showed that the pathogen was rod-shaped, with a length of 1.34 ± 0.22 μm and a width of 0.54 ± 0.06 μm (N=50), and had a single terminal flagellum. The gram staining of the two isolates was negative. Moreover, the tested strains were positive for catalase but negative for oxidase and were able to utilize D-glucose, L-arabinose, and D-mannitol. Morphological, physiological, and biochemical characteristics of both isolates were consistent with those of Pseudomonas spp. To verify the species identity of the bacterial pathogens, genomic DNA of isolates CM-11 and CM-12 was extracted and several conserved genes were amplified and sequenced, including the 16S rRNA gene with primers 27F/1492R (GenBank MW187499 and MW187500), rpoB gene with primers V4/LAPS27 (MW201910 and MW446819), and gyrB gene with primers gyrBBAUP2/APrU (MW187501 and MW187502) (Mulet et al. 2010). In the BLAST analysis, the 16S rRNA sequences showed a 99% similarity to that of Pseudomonas oryzihabitans strains TH19 (LC026009), AA21 (MG571765). The rpoB and gyrB sequences showed high similarity (> 98%) to P. oryzihabitans strains FDAARGOS_657. The phylogenetic tree analysis of rpoB and gyrB genes further verified that the two isolates CM-11 and CM-12 were most closely related to P. oryzihabitans species. Consequently, the two pathogenic isolates CM-11 and CM-12 were identified as P. oryzihabitans. Both strains of CM-11 and CM-12 were tested to accomplish Koch's postulates. Young branches of muskmelons (cultivar Yugu, 10 days after pollination) were chosen as the material for inoculation. Ten healthy detached branches were placed in 15 ml tubes by submerging the cutting wound in 5 ml of the bacterial suspension (108 CFU/ml). Ten additional branches were implemented with sterilized distilled water as a negative control. The inoculated branches were placed in a plastic box containing moistened paper at 28°C. Rotting symptoms appeared within 5 days after infection, while the control samples remained healthy. Bacteria were re-isolated from diseased tissues, and the 16S rRNA gene sequences of the isolates showed the same as those from the original pathogen. Panicle blight and grain discoloration disease caused by P. oryzihabitans on rice has been described in China (Hou et al. 2020). It’s also recently found that P. oryzihabitans caused center blackening disease on muskmelon fruit in Korea (Choi et al. 2019). This study indicated that it was a causative agent of stem and leaf rot disease during the field growth period. To the best of our knowledge, this is the first report of P. oryzihabitans causing muskmelon stem rot in China.

scholarly journals First Report of Xanthomonas Leaf Blight of Onion in California

Plant Disease ◽  
2002 ◽  
Vol 86 (3) ◽  
pp. 330-330 ◽  
Author(s):  
J. J. Nunez ◽  
R. L. Gilbertson ◽  
X. Meng ◽  
R. M. Davis
Keyword(s):  
Dna Sequences ◽  
Xanthomonas Campestris ◽  
Similarity Index ◽  
The United States ◽  
Leaf Blight ◽  
Yield Reduction ◽  
Bacterial Suspension ◽  
Fresh Market ◽  
First Report ◽  
Profile Similarity

In 2000 and 2001, severe leaf blight of fresh market onions occurred in several fields in the Antelope Valley of California, a high desert area located in northern Los Angeles County. In at least two fields, 70% of the canopy was affected, which resulted in an estimated yield reduction of over 50%. Both organically and conventionally grown onions were affected. Symptoms included numerous small chlorotic lesions that appeared first on older leaves. Lesions were often surrounded by water-soaked margins. As the season progressed, the lesions became elongated and necrotic. Entire leaf blades were often killed. The disease never progressed into the bulbs, but bulbs of infected plants never grew to full size. Yellow mucoid bacterial colonies were recovered on yeast extractdextrose-CaCO3 agar from symptomatic tissue. All isolates were gram-negative rods with single polar flagella. Two representative isolates were identified as Xanthomonas campestris based on their carbon utilization profile (similarity index of 0.784 and 0.850; Biolog, Hayward, CA), fatty acid profile (similarity index of 0.588; MIS-TSBA, version 4.10, MIDI Inc., Newark, DE), and 16S-23S intergenic spacer DNA sequences (98% sequence identify with strains of X. campestris). In greenhouse pathogenicity tests, eight white globe onion plants were inoculated with a bacterial suspension (106 CFU/ml) of each of the two isolates. Plants were inoculated by spraying the suspension on leaves lightly injured by rubbing with Carborundum or puncturing with needles dipped in the suspension. Controls were inoculated with water. All plants inoculated with the bacteria developed symptoms in 6 days. The bacterium was reisolated from all inoculated plants and confirmed as Xanthomonas. The trial was conducted twice. To our knowledge, this is the first report of Xanthomonas leaf blight of onion in California. In the United States, the disease has been reported in Texas and Colorado (1,2). References: 1. T. Isakeit et al. Plant Dis. 84:201, 2000. 2. H. F. Schwartz and K. Otto. Plant Dis. 84:922, 2000.

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