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

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.

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First Report of Bacterial Leaf Spot Caused by Xanthomonas campestris pv. olitorii on Jute Grown for Seed in India

Plant Disease ◽

10.1094/pdis-12-12-1196-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1109-1109

Author(s):

C. Biswas ◽

P. Dey ◽

A. Bera ◽

S. Satpathy ◽

B. S. Mahapatra

Keyword(s):

Bacterial Strain ◽

Leaf Spot ◽

Xanthomonas Campestris ◽

Disease Incidence ◽

Negative Control ◽

Nutrient Agar ◽

Distilled Water ◽

Bacterial Leaf Spot ◽

First Report ◽

Pure Cultures

Jute (Corchorus olitorius L.) is the second most important fiber crop after cotton in terms of global production (3). In November 2011, symptoms suggestive of bacterial infection were observed on a seed crop of jute at the CRIJAF research farm, Barrackpore, West Bengal, India. The disease appeared as small, brown, circular spots, usually less than 5 mm in diameter on the leaves and some of the spots were surrounded by a yellow halo. The lesions on the stems were elongated and in some cases were found to girdle the stem. In the later stages of disease, brown sunken spots were found on the green capsules. Disease incidence varied from about 20% to 90% of the total plants in different affected fields at the CRIJAF research farm. Bacterial leaf spot of jute with similar symptoms was reported in 1957 from Sudan (4). Five symptomatic and three asymptomatic leaf samples were collected from different jute fields. Bacterial colonies isolated on nutrient agar medium from infected young leaves were Xanthomonas-like and pale yellow cream in color. Total DNA was extracted from symptomatic as well as asymptomatic leaf samples by using an improved salt concentration and simple sodium acetate CTAB method (2). Single bacterial colonies were transferred to nutrient agar (NA) medium plates and incubated at 28°C for 48 h. Pure colonies from plates were used directly for DNA extraction using the QIAGEN DNeasy Blood and Tissue kit. PCR was carried out with Xanthomonas campestris specific primers NZ8F3/NZ85R3 (1), which generated an amplicon of 530 bp from all the symptomatic leaf samples as well as pure cultures of the isolated bacteria. No amplification was obtained from asymptomatic leaves. The amplicons from the five symptomatic samples collected from the field were sequenced and showed 100% identity with one another, and one sequence (strain JB-CO-13) was deposited in GenBank (Accession No. KC342185). The BLASTn analysis revealed that bacterial strain JB-CO-13 had 100% identity with X. campestris pv. olitorii (EU285213). Nucleotide span and ORF finder (NCBI) analysis indicated the 530-bp PCR amplicon coded part of a gyrase B gene that had 100% identity with a translated gene product (Protein ID: ABX84334). Three leaves of five 1-month-old jute plants (cv. JRO 204) in pot culture were infiltrated each with a separate bacterial strain using suspensions (1 × 105 CFU/ml) in distilled water. The negative control consisted of leaves infiltrated with sterile distilled water. The plants were kept in a greenhouse with mean maximum and minimum temperatures of 28.96 and 21.8°C, respectively. The plants were covered with plastic bags to maintain high relative humidity (>80%). Typical bacterial lesions were recorded on all the inoculated plants after 1 week. No lesions were seen on the negative control. To the best of our knowledge, this is the first report of bacterial leaf spot on C. olitorius caused by X. campestris pv. olitorii from India. References: (1) J. Adriko et al. Plant Pathol. 61:489, 2012. (2) C. Biswas, et al. Lett. Appl. Microbiol. 56:105, 2013. (3) Food and Agriculture Organization of the United Nations. Agricultural Commodities: Profiles and Relevant WTO Negotiating Issues. Online: http://www.fao.org/docrep/006/Y4343E/y4343e03.htm , 2003. (4) K. A. Sabet. Ann. Appl. Biol. 45:516, 1957.

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First report of Xanthomonas campestris pv. campestris as the causal agent of necrotic leaf spot in Phaseolus vulgaris at Puebla, Mexico

Plant Disease ◽

10.1094/pdis-05-21-1010-pdn ◽

2021 ◽

Author(s):

Conrado Parraguirre-Lezama ◽

Omar Romero Arenas ◽

Maria de los Angeles Valencia de Ita ◽

Antonio Rivera ◽

Nemesio Villa-Ruano ◽

...

Keyword(s):

Leaf Spot ◽

Xanthomonas Campestris ◽

Casamino Acid ◽

Bacterial Suspension ◽

Pathogenicity Test ◽

Distilled Water ◽

Tetrazolium Chloride ◽

First Report ◽

Bean Plants ◽

Xanthomonas Campestris Pv Campestris

Beans are the most cultivated legume in the world. In Mexico, it is the second most important crop after corn (FAO 2020; SIAP 2020). Bean plants “Flor de Mayo M38” variety were affected by a foliar disease during the agricultural cycle 2019 in Puebla-Mexico (19°02'46.6" LN and 98°05'15.6" LO). Necrotic V- shaped lesions were observed on the margins of the leaves surrounded by yellow halos followed by foliar necrosis, affecting 40% of the crop. In Mexico this variety of cultivars is in great demand for local consumption and generates income in foreign currency (Castellanos et al. 1997). Sampling was carried out on 50 plants “Flor de Mayo M38” variety, with necrotic leaf symptoms from ten plots of one hectare. Samples were cut into pieces (5 mm), disinfested with 1% hypochlorite 3 min, and washed with sterile distilled water. Subsequently, samples were dried on sterile paper and placed on Petri plates containing yeast extract calcium carbonate dextrose agar (YDC) medium and kept at 36°C for 3 days. Colonies of ten typical bacteria isolated from all symptomatic plants were Gram (-), small and uniform in size with rounded edges, yellow, convex with entire borders and mucoid appearance on YDC. Bacteria did not grow on 0.1% triphenyl tetrazolium chloride amended casamino acid, peptone, and glucose medium (CPG). Biochemical tests showed that isolates did not reduce nitrate to nitrites, had positive catalase and starch hydrolysis, while the Kovac oxidase test was negative (Schaad and White 1974). Genus identity of the representative isolate Xcf1-APJR, was confirmed by 16S rRNA encoding gene partial sequencing, using universal primers 518F (5'-CCAGCAGCCGCGGTAATACG-3') and 800R (5′-TACCAGGGTATCTAATCC-3′) (Halim et al. 2020). BLASTn alignments against the nucleotide collection were 100% identical to Xanthomonas sequences including Xanthomonas campestris pv. campestris strains NZ_AP019684.1, CP025750.1, and MN108237.1. The 1,418 bp sequence was deposited in the GenBank database under accession number MT645246. The identification of species/pathovar was accomplished by serological methods using a polyclonal antiserum specific for X. campestris pv. campestris (Popovic ́ et al. 2013) with the DAS-ELISA commercial kit (catalog number 07122C/096, LOEWE Biochemica GmbH, Germany). The pathogenicity test was carried out on 50 healthy bean plants from the "Flor de Mayo M38" variety. Bacterial culture incubated at 28°C for 48 h in YDC medium was used to prepare the bacterial suspension (108 CFU mL-1). The first two lower leaves of 30-day-old plants were inoculated by sprinkling. Ten plants sprayed with sterile distilled water were used as negative control. All plants were kept for 20 days in greenhouse at 18-26°C and relative humidity of 60%. After seven days, chlorotic lesions developed on all inoculated plants that became necrotic from 14 days after inoculation (dai). Necrotic leaf spots merged at 14 dai to form necrotic areas of more than 20 mm in diameter, reaching total necrosis of the leaf tissue at 20 dai and were similar to the symptoms observed in the field. Koch's postulates were confirmed by the reisolation of Xcf1-APJR strain, which presented the same colony morphology, partial sequence, and polyclonal specific detection. This is the first report of this pathogen causing necrotic leaf spot in beans from the "Flor de Mayo M38" variety in Puebla-Mexico. The author(s) declare no conflict of interest. References: FAO. 2020. FAOSTAT. Food and Agriculture Data. http://www.fao.org/faostat/en/#home/. SIAP. 2020. Atlas Agroalimentario. https://www.gob.mx/siap/. Castellanos, J. Z., et al. 1997. Arch. Latinoam. Nutr. 47:163. Schaad, N. W., and White, W. C. 1974. Phytopathology. 64:876. https://doi.org/10.1094/Phyto-64-876 Halim, R. A., et al. 2020. HAYATI J. Biosciences. 27:215. https://doi.org/10.4308/hjb.27.3.215 Popovic ́, T., et al. 2013. Plant Dis. 97:418. https://doi.org/10.1094/PDIS-05-12-0506-PDN

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

Plant Disease ◽

10.1094/pdis-04-12-0354-pdn ◽

2012 ◽

Vol 96 (11) ◽

pp. 1690-1690 ◽

Cited By ~ 1

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.

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First report of Rhizopus stolonifer causing Rhizopus bunch rot on grapes in Pakistan

International Journal of Phytopathology ◽

10.33687/phytopath.008.01.2776 ◽

2019 ◽

Vol 8 (1) ◽

pp. 29-30

Author(s):

Salman Ghuffar ◽

Gulshan Irshad ◽

Amjad S. Gondal ◽

Raees Ahmad ◽

Hafizi B. Rosli ◽

...

Keyword(s):

Disease Incidence ◽

Management Strategies ◽

Negative Control ◽

Its2 Region ◽

Distilled Water ◽

Rhizopus Stolonifer ◽

First Report ◽

Grape Berries ◽

Table Grapes ◽

Bunch Rot

During June 2016, a postharvest survey of table grapes was carried out in the main fruit markets of Attock (33°46'07.9"N 72°21'43.0"E) and Jehlum (32°56'22.3"N 73°43'31.4"E) districts of Punjab Province. At the time of sampling, two cultivars (King’s Ruby and Perlette) were sampled at five different locations of both districts. Disease incidence % of bunches averaged 3 to 4 %. Some infected fruit appeared water-soaked, light brown and covered by fluffy mycelium consisting of erect sporangiophores with black sporangia on the top (Figure 1). Symptomatic tissue pieces were surface-sterilized with 0.1% NaOCl for 30 sec, rinsed three times with sterile distilled water, dried on filter paper for 45 sec and incubated on potato dextrose agar (PDA) at 25°C. After 1 day, mycelium on PDA was transferred to a fresh PDA plate and incubated at 25°C with a 12-h photoperiod. Within 3 days, white to yellow colonies with black aerial sporangia were formed (Figure 2). A total of 64 isolates were examined morphologically. Sporangiophores were erect, light brown and 623 to 3800 µm long. One to three rhizoids were observed opposite to each sporangiophore. Sporangia were black, globose to sub-globose, 91 to 124 μm in diameter. Columellae were conical to cylindrical and 86 to 187 μm long × 72 to 205 μm wide. Sporangiospores were hyaline to light dark grey, globose, ellipsoidal in shape and 6.8 to 12.4 × 3.6 to 12.5 μm (Figure 3). These features were identical to the description of Rhizopus stolonifer (Ehrenb.) Vuill (Liou et al., 2007). For molecular identification, the ITS1-5.8S-ITS2 region of two representative isolates (Rizo 05 and Rizo 07) was amplified with primers ITS1/ITS4 (White et al., 1990). Sequence comparison of two isolates Rizo05 and Rizo 07 (Accession no. MH348205 and MH356272) revealed 100% identity with previously reported isolates of Rhizopus stolonifer (Accession no. MG865992, KU729185, HM051076, and MF374842). To complete Koch’s postulates, 10-µl aliquots of spore suspensions (106 spores/ml) of Rizo 05 and Rizo 07 were pipetted onto three non-wounded and four wounded asymptomatic grape berries (seven berries per isolate), Sterile distilled water was applied to asymptomatic berries to serve as a negative control. Berries were incubated at 25 ± 2°C in sterile moisture chambers, and the experiment was conducted twice (Ghuffar et al., 2018). Black to light brown, fluffy mycelium with the original symptoms was observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were recorded on the negative control (Figure 4). The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Previously, Rhizopus stolonifer has been reported as a pathogen on grapes in Chile (Latorre et al., 2002). To our knowledge, this is the first report of Rhizopus stolonifer causing bunch rot of grapes in Pakistan. This finding will help to plan effective disease management strategies against Rhizopus rot of grapes in Pakistan.

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First Report of Black Rot on Arugula Caused by Xanthomonas campestris pv. campestris in Argentina

Plant Disease ◽

10.1094/pdis-92-6-0980c ◽

2008 ◽

Vol 92 (6) ◽

pp. 980-980 ◽

Cited By ~ 1

Author(s):

A. M. Romero ◽

R. Zapata ◽

M. S. Montecchia

Keyword(s):

Xanthomonas Campestris ◽

Pathogenic Bacteria ◽

Biochemical Characterization ◽

Bacterial Suspension ◽

Black Rot ◽

Strictly Aerobic ◽

First Report ◽

Plastic Bags ◽

Water Plants ◽

Xanthomonas Campestris Pv Campestris

During the fall of 2005, arugula (Eruca sativa Mill.) plants grown in experimental field plots in Buenos Aires, Argentina presented V-shaped necrotic lesions on leaf margins and blackened veins with broad yellow halos, followed by leaf necrosis. At flowering, 96% of the plants were affected with 27% of the leaves with symptoms. Yellow, round, mucoid, convex, bacterial colonies were isolated from several leaves on yeast dextrose chalk agar. Two strains were further studied. Xanthomonas campestris pv. campestris Xcc8004 was used as a control. Strains were gram negative, rod shaped, strictly aerobic, catalase-positive, oxidase and urease-negative, hydrolyzed starch, gelatine and aesculin, and did not reduce nitrate (2). Pathogenicity was tested by spraying 10 3-week-old arugula plants with either a bacterial suspension (107 CFU/ml) or sterile water. Plants were placed in plastic bags for 72 h after inoculation. All inoculated plants showed necrotic lesions enlarging from the margin of the leaves 7 days after inoculation. No lesions were observed on control plants. On the basis of biochemical characterization (2) and genomic fingerprints generated by BOX-PCR (1), the pathogen was identified as X. campestris pv. campestris. To our knowledge, this is the first report of X. campestris pv. campestris causing black rot on arugula in Argentina. References: (1) J. L. Rademaker et al. Int. J. Syst. Evol. Microbiol. 50:665, 2000. (2) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001.

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

Plant Disease ◽

10.1094/pdis-11-20-2493-pdn ◽

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.

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First Report of Fire Blight Disease on Blackberry in Turkey

Plant Disease ◽

10.1094/pdis-12-11-1073-pdn ◽

2012 ◽

Vol 96 (12) ◽

pp. 1818-1818

Author(s):

K. K. Bastas ◽

F. Sahin

Keyword(s):

Disease Incidence ◽

Acid Methyl Ester ◽

Negative Control ◽

Culture Collection ◽

Bacterial Suspension ◽

Identification System ◽

Distilled Water ◽

Bacterial Strains ◽

First Report ◽

Microbial Identification

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

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First Report of Bacterial Leaf Spot of Lettuce (Lactuca sativa) Caused by Xanthomonas campestris pv. vitians in Saudi Arabia

Plant Disease ◽

10.1094/pdis-93-1-0107b ◽

2009 ◽

Vol 93 (1) ◽

pp. 107-107 ◽

Cited By ~ 5

Author(s):

M. Al-Saleh ◽

Y. Ibrahim

Keyword(s):

Saudi Arabia ◽

Lactuca Sativa ◽

Leaf Spot ◽

Xanthomonas Campestris ◽

Bacterial Suspension ◽

Strictly Aerobic ◽

Distilled Water ◽

Bacterial Leaf Spot ◽

Biochemical Tests ◽

First Report

In April of 2008, lettuce (Lactuca sativa L. cv. Darkland) plants grown in the Al-Ouunia Region of Saudi Arabia were observed with numerous lesions typical of bacterial leaf spot. Leaf lesions were irregular, small, pale green to black, and 2 to 5 mm in diameter. Bacteria were isolated from diseased leaf tissues by cutting leaves into small pieces (0.5 mm) and soaking them in 2 ml of sterile distilled water. The resulting suspension was streaked onto yeast dextrose calcium carbonate agar (YDC) (1) and plates were incubated at 28°C. Large, round, butyrus, bright yellow colonies typical of Xanthomonas spp. formed after 48 h and five strains were selected for further tests. A yellow, mucoid bacterium was consistently isolated from lettuce samples with typical bacterial leaf spot symptoms. All five strains tested in this study were gram negative, oxidase negative, nitrate reduction negative, catalase and esculin hydrolysis positive, motile, and strictly aerobic. All were slightly pectolytic but not amylolytic. All were identified as Xanthomonas campestris pv. vitians. The bacterium was identified with specific oligonucleotide primers (2). This primer pair directed the amplification of an approximately 700-bp DNA fragment from total genomic DNA of all X. campestris pv. vitians strains tested. Pathogenicity tests were performed by using bacterial cultures grown on YDC for 48 h at 28°C. Each strain was suspended in sterile distilled water and the bacterial concentration was adjusted to 106 CFU/ml. Leaves of 5-week-old lettuce plants (cv. Darkland) were sprayed with the bacterial suspension. The inoculated and sterile-water-sprayed control plants were covered with polyethylene bags for 48 h at 25°C, after which the bags were removed and plants were transferred to a greenhouse at 25 to 28°C (1). All strains were pathogenic on the lettuce cv. Darkland, causing typical bacterial leaf spot symptoms by 2 weeks after inoculation. All inoculated plants showed typical symptoms of bacterial leaf spot and symptoms similar to those observed on the samples collected. No symptoms developed on the control plants. The bacterium was reisolated from inoculated plants and identified as X. campestris pv. vitians by morphological, physiological, and biochemical tests as described above. To our knowledge, this is the first report of bacterial leaf spot of lettuce by X. campestris pv. vitians in Saudi Arabia. References: (1) F. Sahin and A. Miller. Plant Dis.81:1443, 1997. (2) J. D. Barak. Plant Dis.85:169, 2001.

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First Report of Flyspeck Caused by Zygophiala wisconsinensis on Sweet Persimmon Fruit in Korea

Plant Disease ◽

10.1094/pdis-12-10-0917 ◽

2011 ◽

Vol 95 (5) ◽

pp. 616-616 ◽

Cited By ~ 1

Author(s):

J. Kim ◽

O. Choi ◽

J.-H. Kwon

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Its Rdna ◽

Control Treatment ◽

Diospyros Kaki ◽

Distilled Water ◽

First Report ◽

Persimmon Fruit ◽

Fungal Isolates ◽

Agar Disc

Sweet persimmon (Diospyros kaki L.), a fruit tree in the Ebenaceae, is cultivated widely in Korea and Japan, the leading producers worldwide (2). Sweet persimmon fruit with flyspeck symptoms were collected from orchards in the Jinju area of Korea in November 2010. The fruit had fungal clusters of black, round to ovoid, sclerotium-like fungal bodies with no visible evidence of a mycelial mat. Orchard inspections revealed that disease incidence ranged from 10 to 20% in the surveyed area (approximately 10 ha) in 2010. Flyspeck symptoms were observed on immature and mature fruit. Sweet persimmon fruit peels with flyspeck symptoms were removed, dried, and individual speck lesions transferred to potato dextrose agar (PDA) and cultured at 22°C in the dark. Fungal isolates were obtained from flyspeck colonies on 10 sweet persimmon fruit harvested from each of three orchards. Fungal isolates that grew from the lesions were identified based on a previous description (1). To confirm identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA sequence of a representative isolate was amplified and sequenced using primers ITS1 and ITS4 (4). The resulting 552-bp sequence was deposited in GenBank (Accession No. HQ698923). Comparison with ITS rDNA sequences showed 100% similarity with a sequence of Zygophiala wisconsinensis Batzer & Crous (GenBank Accession No. AY598855), which infects apple. To fulfill Koch's postulates, mature, intact sweet persimmon fruit were surface sterilized with 70% ethanol and dried. Three fungal isolates from this study were grown on PDA for 1 month. A colonized agar disc (5 mm in diameter) of each isolate was cut from the advancing margin of a colony with a sterilized cork borer, transferred to a 1.5-ml Eppendorf tube, and ground into a suspension of mycelial fragments and conidia in a blender with 1 ml of sterile, distilled water. The inoculum of each isolate was applied by swabbing a sweet persimmon fruit with the suspension. Three sweet persimmon fruit were inoculated per isolate. Three fruit were inoculated similarly with sterile, distilled water as the control treatment. After 1 month of incubation in a moist chamber at 22°C, the same fungal fruiting symptoms were reproduced as observed in the orchards, and the fungus was reisolated from these symptoms, but not from the control fruit, which were asymptomatic. On the basis of morphological characteristics of the fungal colonies, ITS sequence, and pathogenicity to persimmon fruit, the fungus was identified as Z. wisconsinensis (1). Flyspeck is readily isolated from sweet persimmon fruit in Korea and other sweet persimmon growing regions (3). The exposure of fruit to unusual weather conditions in Korea in recent years, including drought, and low-temperature and low-light situations in late spring, which are favorable for flyspeck, might be associated with an increase in occurrence of flyspeck on sweet persimmon fruit in Korea. To our knowledge, this is the first report of Z. wisconsinensis causing flyspeck on sweet persimmon in Korea. References: (1) J. C. Batzer et al. Mycologia 100:246, 2008. (2) FAOSTAT Database. Retrieved from http://faostat.fao.org/ , 2008. (3) H. Nasu and H. Kunoh. Plant Dis. 71:361, 1987. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.

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First report of Colletotrichum siamense Causing Blossom Blight on Thai basil (Ocimum basilicum L.) in Malaysia

Plant Disease ◽

10.1094/pdis-06-20-1371-pdn ◽

2020 ◽

Author(s):

Siti Izera Ismail ◽

Nur Adlina Rahim ◽

Dzarifah Zulperi

Keyword(s):

Disease Incidence ◽

Its Region ◽

Ocimum Basilicum ◽

Distilled Water ◽

Single Spore ◽

First Report ◽

Plastic Bags ◽

Colletotrichum Siamense ◽

Primer Sets ◽

Acca Sellowiana

Thai basil (Ocimum basilicum L.) is widely cultivated in Malaysia and commonly used for culinary purposes. In March 2019, necrotic lesions were observed on the inflorescences of Thai basil plants with a disease incidence of 60% in Organic Edible Garden Unit, Faculty of Agriculture in the Serdang district (2°59'05.5"N 101°43'59.5"E) of Selangor province, Malaysia. Symptoms appeared as sudden, extensive brown spotting on the inflorescences of Thai basil that coalesced and rapidly expanded to cover the entire inflorescences. Diseased tissues (4×4 mm) were cut from the infected lesions, surface disinfected with 0.5% NaOCl for 1 min, rinsed three times with sterile distilled water, placed onto potato dextrose agar (PDA) plates and incubated at 25°C under 12-h photoperiod for 5 days. A total of 8 single-spore isolates were obtained from all sampled inflorescence tissues. The fungal colonies appeared white, turned grayish black with age and pale yellow on the reverse side. Conidia were one-celled, hyaline, subcylindrical with rounded end and 3 to 4 μm (width) and 13 to 15 μm (length) in size. For fungal identification to species level, genomic DNA of representative isolate (isolate C) was extracted using DNeasy Plant Mini Kit (Qiagen, USA). Internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), and chitin synthase-1 (CHS-1) were amplified using ITS5/ITS4 (White et al. 1990), CL1C/CL2C (Weir et al. 2012), ACT-512F/783R, and CHS-79F/CHS-345R primer sets (Carbone and Kohn 1999), respectively. A BLAST nucleotide search of ITS, CHS-1, CAL and ACT sequences showed 100% similarity to Colletotrichum siamense ex-type cultures strain C1315.2 (GenBank accession nos. ITS: JX010171 and CHS-1: JX009865) and isolate BPDI2 (CAL: FJ917505, ACT: FJ907423). The ITS, CHS-1, CAL and ACT sequences were deposited in GenBank as accession numbers MT571330, MW192791, MW192792 and MW140016. Pathogenicity was confirmed by spraying a spore suspension (1×106 spores/ml) of 7-day-old culture of isolate C onto 10 healthy inflorescences on five healthy Thai basil plants. Ten infloresences from an additional five control plants were only sprayed with sterile distilled water and the inoculated plants were covered with plastic bags for 2 days and maintained in a greenhouse at 28 ± 1°C, 98% relative humidity with a photoperiod of 12-h. Blossom blight symptoms resembling those observed in the field developed after 7 days on all inoculated inflorescences, while inflorescences on control plants remained asymptomatic. The experiment was repeated twice. C. siamense was successfully re-isolated from the infected inflorescences fulfilling Koch’s postulates. C. siamense has been reported causing blossom blight of Uraria in India (Srivastava et al. 2017), anthracnose on dragon fruit in India and fruits of Acca sellowiana in Brazil (Abirami et al. 2019; Fantinel et al. 2017). This pathogen can cause a serious threat to cultivation of Thai basil and there is currently no effective disease management strategy to control this disease. To our knowledge, this is the first report of blossom blight caused by C. siamense on Thai basil in Malaysia.

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