First Report of Xanthomonas Leaf Blight of Onion in California

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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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 Leaf Blight of Japanese Yew Caused by Pestalotiopsis microspora in Korea

Plant Disease ◽

10.1094/pdis-08-13-0821-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 691-691 ◽

Cited By ~ 2

Author(s):

Y. H. Jeon ◽

W. Cheon

Keyword(s):

Dna Sequences ◽

Apical Cell ◽

Morphological Characteristics ◽

Leaf Blight ◽

Economic Damage ◽

Leaf Margin ◽

Conidial Suspension ◽

First Report ◽

Pestalotiopsis Microspora ◽

Large Trees

Worldwide, Japanese yew (Taxus cuspidata Sieb. & Zucc.) is a popular garden tree, with large trees also being used for timber. In July 2012, leaf blight was observed on 10% of Japanese yew seedling leaves planted in a 500-m2 field in Andong, Gyeongsangbuk-do Province, South Korea. Typical symptoms included small, brown lesions that were first visible on the leaf margin, which enlarged and coalesced into the leaf becoming brown and blighted. To isolate potential pathogens from infected leaves, small sections of leaf tissue (5 to 10 mm2) were excised from lesion margins. Eight fungi were isolated from eight symptomatic trees, respectively. These fungi were hyphal tipped twice and transferred to potato dextrose agar (PDA) plates for incubation at 25°C. After 7 days, the fungi produced circular mats of white aerial mycelia. After 12 days, black acervuli containing slimy spore masses formed over the mycelial mats. Two representative isolates were further characterized. Their conidia were straight or slightly curved, fusiform to clavate, five-celled with constrictions at the septa, and 17.4 to 28.5 × 5.8 to 7.1 μm. Two to four 19.8- to 30.7-μm-long hyaline filamentous appendages (mostly three appendages) were attached to each apical cell, whereas one 3.7- to 7.1-μm-long hyaline appendage was attached to each basal cell, matching the description for Pestalotiopsis microspora (2). The pathogenicity of the two isolates was tested using 2-year-old plants (T. cuspidata var. nana Rehder; three plants per isolate) in 30-cm-diameter pots filled with soil under greenhouse conditions. The plants were inoculated by spraying the leaves with an atomizer with a conidial suspension (105 conidia/ml; ~50 ml on each plant) cultured for 10 days on PDA. As a control, three plants were inoculated with sterilized water. The plants were covered with plastic bags for 72 h to maintain high relative humidity (24 to 28°C). At 20 days after inoculation, small dark lesions enlarged into brown blight similar to that observed on naturally infected leaves. P. microspora was isolated from all inoculated plants, but not the controls. The fungus was confirmed by molecular analysis of the 5.8S subunit and flanking internal transcribed spaces (ITS1 and ITS2) of rDNA amplified from DNA extracted from single-spore cultures, and amplified with the ITS1/ITS4 primers and sequenced as previously described (4). Sequences were compared with other DNA sequences in GenBank using a BLASTN search. The P. microspora isolates were 99% homologous to other P. microspora (DQ456865, EU279435, FJ459951, and FJ459950). The morphological characteristics, pathogenicity, and molecular data assimilated in this study corresponded with the fungus P. microspora (2). This fungus has been previously reported as the causal agent of scab disease of Psidium guajava in Hawaii, the decline of Torreya taxifolia in Florida, and the leaf blight of Reineckea carnea in China (1,3). Therefore, this study presents the first report of P. microspora as a pathogen on T. cuspidata in Korea. The degree of pathogenicity of P. microspora to the Korean garden evergreen T. cuspidata requires quantification to determine its potential economic damage and to establish effective management practices. References: (1) D. F. Farr and A. Y. Rossman, Fungal Databases, Syst. Mycol. Microbiol. Lab. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ (2) L. M. Keith et al. Plant Dis. 90:16, 2006. (3) S. S. N. Maharachchikumbura. Fungal Diversity 50:167, 2011. (4) T. J. White et al. PCR Protocols. Academic Press, San Diego, CA, 1990.

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First Report of Xylella fastidiosa Associated with Leaf Scorch in Black Oak in Washington, D.C.

Plant Disease ◽

10.1094/pdis.2004.88.2.224c ◽

2004 ◽

Vol 88 (2) ◽

pp. 224-224 ◽

Cited By ~ 2

Author(s):

Q. Huang

Keyword(s):

Xylella Fastidiosa ◽

The United States ◽

Enzyme Linked Immunosorbent Assay ◽

Bacterial Suspension ◽

White Oak ◽

First Report ◽

Leaf Petiole ◽

Leaf Scorch ◽

Pcr Product ◽

Black Oak

Bacterial leaf scorch caused by Xylella fastidiosa has been reported in 17 species of oak including bur, pin, red, scarlet, shingle, and white oaks (3). In September 2002, a leaf scorch symptom characterized by marginal necrosis of leaves bordered by a darker brown band was observed in a mature black oak (Quercus velutina Lam.) at the U.S. National Arboretum in Washington, D.C. The leaf petiole of the black oak was processed in general extraction buffer (Agdia, Inc., Elkhart, IN) contained in a FastDNA lysing matrix tube using the FastPrep FP120 instrument (Qbiogene, Inc., Carlsbad, CA) (1). The leaf petiole extract reacted with an antiserum specific for X. fastidiosa (Agadia, Inc.) in an enzyme-linked immunosorbent assay (ELISA). A slow-growing bacterium was cultured from leaf petioles of the affected black oak tree by soaking the surface-sterilized, finely cut leaf petioles in sterile water for 30 min, followed by spreading the bacterial suspension on periwinkle wilt plates (1). When the cultured bacterium was subjected to polymerase chain reaction (PCR) with primers specific for X. fastidiosa (2), a 472-bp PCR product was detected. The PCR product was confirmed to be the predicted X. fastidiosa product by sequencing and sequence comparison with the reported genomic sequence of X. fastidiosa. ELISA and bacterial isolation from leaf petioles of a nearby symptomless white oak (Q. alba L.) tree were negative. To our knowledge, this is the first report of X. fastidiosa associated with leaf scorch in black oak in the United States, expanding the host range of the bacterium in economically important landscape tree species. References: (1) Q. Huang and J. L. Sherald. Curr. Microbiol. 48:73, 2004. (2) M. R. Pooler and J. S. Hartung. Curr. Microbiol. 31:377, 1995. (3) J. L. Sherald. Xylella fastidiosa, A bacterial pathogen of landscape trees. Page 191 in: Shade Tree Wilt Diseases, C. L. Ash, ed. The American Phytopathological Society, 2001.

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First Report of Leaf Blight and Stem Canker of Pachysandra terminalis Caused by Pseudonectria pachysandricola in Korea

Plant Disease ◽

10.1094/pdis-09-11-0813 ◽

2012 ◽

Vol 96 (2) ◽

pp. 287-287

Author(s):

K. S. Han ◽

J. H. Park ◽

S. E. Cho ◽

H. D. Shin

Keyword(s):

United States ◽

Its Region ◽

Stem Canker ◽

The United States ◽

Culture Collection ◽

Leaf Blight ◽

First Report ◽

Cornell University ◽

Plastic Bags ◽

Young Leaves

Pachysandra terminalis Siebold & Zucc., known as Japanese pachysandra, is a creeping evergreen perennial belonging to the family Buxaceae. In April 2011, hundreds of plants showing symptoms of leaf blight and stem canker with nearly 100% incidence were found in a private garden in Suwon, Korea. Plants with the same symptoms were found in Seoul in May and Hongcheon in August. Affected leaves contained tan-to-yellow brown blotches. Stem and stolon cankers first appeared as water soaked and developed into necrotic lesions. Sporodochia were solitary, erumpent, circular, 50 to 150 μm in diameter, salmon-colored, pink-orange when wet, and with or without setae. Setae were hyaline, acicular, 60 to 100 μm long, and had a base that was 4 to 6 μm wide. Conidiophores were in a dense fascicle, not branched, hyaline, aseptate or uniseptate, and 8 to 20 × 2 to 3.5 μm. Conidia were long, ellipsoid to cylindric, fusiform, rounded at the apex, subtruncate at the base, straight to slightly bent, guttulate, hyaline, aseptate, 11 to 26 × 2.5 to 4.0 μm. A single-conidial isolate formed cream-colored colonies that turned into salmon-colored colonies on potato dextrose agar (PDA). Morphological and cultural characteristics of the fungus were consistent with previous reports of Pseudonectria pachysandricola B.O. Dodge (1,3,4). Voucher specimens were housed at Korea University (KUS). Two isolates, KACC46110 (ex KUS-F25663) and KACC46111 (ex KUS-F25683), were accessioned in the Korean Agricultural Culture Collection. Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced using ABI Prism 337 automatic DNA sequencer (Applied Biosystems, Foster, CA). The resulting sequence of 487 bp was deposited in GenBank (Accession No. JN797821). This showed 100% similarity with a sequence of P. pachysandricola from the United States (HQ897807). Isolate KACC46110 was used in pathogenicity tests. Inoculum was prepared by harvesting conidia from 2-week-old cultures on PDA. Ten young leaves wounded with needles were sprayed with conidial suspensions (~1 × 106 conidia/ml). Ten young leaves that served as the control were treated with sterile distilled water. Plants were covered with plastic bags to maintain a relative humidity of 100% at 25 ± 2°C for 24 h. Typical symptoms of brown spots appeared on the inoculated leaves 4 days after inoculation and were identical to the ones observed in the field. P. pachysandricola was reisolated from 10 symptomatic leaf tissues, confirming Koch's postulates. No symptoms were observed on control plants. Previously, the disease was reported in the United States, Britain, Japan, and the Czech Republic (2,3), but not in Korea. To our knowledge, this is the first report of P. pachysandricola on Pachysandra terminalis in Korea. Since this plant is popular and widely planted in Korea, this disease could cause significant damage to nurseries and the landscape. References: (1) B. O. Dodge. Mycologia 36:532, 1944. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , September 24, 2011. (3) I. Safrankova. Plant Prot. Sci. 43:10, 2007. (4) W. A. Sinclair and H. H. Lyon. Disease of Trees and Shrubs. 2nd ed. Cornell University Press, Ithaca, NY, 2005.

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First Report of Guignardia citricarpa Associated with Citrus Black Spot on Sweet Orange (Citrus sinensis) in North America

Plant Disease ◽

10.1094/pdis-01-12-0101-pdn ◽

2012 ◽

Vol 96 (8) ◽

pp. 1225-1225 ◽

Cited By ~ 31

Author(s):

T. S. Schubert ◽

M. M. Dewdney ◽

N. A. Peres ◽

M. E. Palm ◽

A. Jeyaprakash ◽

...

Keyword(s):

North America ◽

Sweet Orange ◽

Black Spot ◽

The United States ◽

Yield Reduction ◽

Rrna Gene ◽

Specific Primers ◽

Oatmeal Agar ◽

First Report ◽

Citrus Black Spot

In March 2010, citrus black spot symptoms were observed on sweet orange trees in a grove near Immokalee, FL. Symptoms observed on fruit included hard spot, cracked spot, and early virulent spot. Hard spot lesions were up to 5 mm, depressed with a chocolate margin and a necrotic, tan center, often with black pycnidia (140 to 200 μm) present. Cracked spot lesions were large (15 mm), dark brown, with diffuse margins and raised cracks. In some cases, hard spots formed in the center of lesions. Early virulent spot lesions were small (up to 7 mm long), bright red, irregular, indented, and often with many pycnidia. In addition, small (2 to 3 mm), elliptical, reddish brown leaf lesions with depressed tan centers were observed on some trees with symptomatic fruit. Chlorotic halos appeared as they aged. Most leaves had single lesions, occasionally up to four per leaf. Tissue pieces from hard spots and early virulent spots were placed aseptically on potato dextrose agar (PDA), oatmeal agar, or carrot agar and incubated with 12 h of light and dark at 24°C. Cultures that grew colonies within a week were discarded. Fourteen single-spore cultures were obtained from the isolates that grew slower than the Guignardia mangiferae reference cultures, although pycnidia formed more rapidly in the G. mangiferae cultures (1). No sexual structures were observed. Cultures on half-PDA were black and cordlike with irregular margins with numerous pycnidia, often bearing white cirrhi after 14 days. Conidia (7.1 to 7.8 × 10.3 to 11.8 μm) were hyaline, aseptate, multiguttulate, ovoid with a flattened base surrounded by a hyaline matrix (0.4 to 0.6 μm) and a hyaline appendage on the rounded apex, corresponding to published descriptions of G. citricarpa (anomorph Phyllosticta citricarpa) (1). A yellow pigment was seen in oatmeal agar surrounding G. citricarpa, but not G. mangiferae colonies as previously reported (1,2). DNA was extracted from lesions and cultures and amplified with species-specific primers (2). DNA was also extracted from G. mangiferae and healthy citrus fruit. The G. citricarpa-specific primers produced a 300-bp band from fruit lesions and pure cultures. G. mangiferae-specific primers produced 290-bp bands with DNA from G. mangiferae cultures. The internally transcribed spacer (ITS) of the rRNA gene, translation-elongation factor (TEF), and actin gene regions were sequenced from G. citricarpa isolates and deposited in GenBank. These sequences had 100% homology with G. citricarpa ITS sequences from South Africa and Brazil, 100% homology with TEF, and 99% homology with actin of a Brazilian isolate. Pathogenicity tests with G. citricarpa were not done because the organism infects immature fruit and has an incubation period of at least 6 months (3). In addition, quarantine restrictions limit work with the organism outside a contained facility. To our knowledge, this is the first report of black spot in North America. The initial infested area was ~57 km2. The disease is of great importance to the Florida citrus industry because it causes serious blemishes and significant yield reduction, especially on the most commonly grown ‘Valencia’ sweet orange. Also, the presence of the disease in Florida may affect market access because G. citricarpa is considered a quarantine pathogen by the United States and internationally. References: (1) R. P. Baayen et al. Phytopathology 92:464, 2002. (2) N. A. Peres et al. Plant Dis. 91:525, 2007 (3) R. F. Reis et al. Fitopath Bras. 31:29, 2006.

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Report of Bacterial Leaf Spot on Collards and Turnip Leaves in Ohio

Plant Disease ◽

10.1094/pdis.2002.86.2.186a ◽

2002 ◽

Vol 86 (2) ◽

pp. 186-186 ◽

Cited By ~ 2

Author(s):

M. L. Lewis Ivey ◽

S. Wright ◽

S. A. Miller

Keyword(s):

Pseudomonas Syringae ◽

Leaf Spot ◽

Xanthomonas Campestris ◽

Similarity Index ◽

Acid Methyl Ester ◽

Bacterial Suspension ◽

Water Control ◽

Bacterial Leaf Spot ◽

Turnip Leaves ◽

Negative Controls

In 2000, circular water-soaked lesions typical of bacterial leaf spot were observed on leaves of collards (Brassica oleracea L. var. viridis) throughout commercial fields in northwest Ohio. Light brown, rectangular, water-soaked lesions were observed on turnip leaves (Brassica rapa L.). Bacterial streaming from lesions on both crops was observed microscopically. Cream colored, fluorescent colonies were isolated from diseased tissues on Pseudomonas F medium, and eight representative colonies (four from collards and four from turnip) were selected and purified. Fatty acid methyl ester analysis was performed on all of the isolates. Two from collards and two from turnip were identified as Pseudomonas syringae pv. maculicola (mean similarity index = 0.82 [MIDI Inc., Newark, DE]). DNA extracts from pure cultures of the P. syringae pv. maculicola strains were used as template in a polymerase chain reaction (PCR) assay with primers derived from the region of the coronatine gene cluster controlling synthesis of the coronafacic acid moiety found in P. syringae pv. tomato and P. syringae pv. maculicola (CorR and CorF2) (D. Cuppels, personal communication). DNA from P. syringae pv. tomato strain DC3000 and P. syringae pv. maculicola strain 88–10 (2) served as positive controls, while water and DNA from Xanthomonas campestris pv. vesicatoria strain Xcv 767 were used as negative controls. The expected 0.65-kb PCR product was amplified from three of four strains (two from turnip and one from collards) and the positive control DNA, but not from the negative controls. Pathogenicity tests were performed twice on 6-week-old turnip (‘Forage Star’, ‘Turnip Topper’, ‘Turnip Alamo’, ‘Turnip 7’), collard (‘Champion’) and mustard (Brassica juncea L. ‘Southern Giant Curl’) seedlings using the three PCR-positive strains. Premisted seedlings were spray-inoculated separately with each of the three strains (2 × 108 CFU/ml, 5 ml per plant) and a water control. Greenhouse temperatures were maintained at 20 ± 1°C. For both tests, all strains caused characteristic lesions on all of the crucifer cultivars within 5 days after inoculation; the control plants did not develop symptoms. To satisfy Koch's postulates, one of the turnip strains was reisolated from ‘Turnip Topper’ plants, and the collard strain was reisolated from ‘Champion’ plants. The three original and two reisolated strains induced a hypersensitive response in Mirabilis jalapa L. and Nicotiana tabacum L. var. xanthia plants 24 h after inoculation with a bacterial suspension (1 × 108 CFU/ml). The original and reisolated strains were compared using rep-PCR with the primer BOXA1R (1). The DNA fingerprints of the reisolated strains were identical to those of the original strains. To our knowledge, this is the first report of bacterial leaf spot on commercially grown collards and turnip greens in Ohio. References: (1) B. Martin et al. Nucleic Acids Res. 20:3479, 1992. (2) R. A. Moore et al. Can. J. Microbiol. 35:910, 1989.

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First Report of Leaf Blight on Fan Columbine (Aquilegia flabellata) Caused by Rhizoctonia solani AG 4 in Italy

Plant Disease ◽

10.1094/pdis-93-4-0433a ◽

2009 ◽

Vol 93 (4) ◽

pp. 433-433 ◽

Cited By ~ 2

Author(s):

A. Garibaldi ◽

G. Gilardi ◽

D. Bertetti ◽

M. L. Gullino

Keyword(s):

United States ◽

Rhizoctonia Solani ◽

Aqueous Suspension ◽

Morphological Characteristics ◽

The United States ◽

Leaf Blight ◽

Herbaceous Plant ◽

Pathogenicity Test ◽

First Report ◽

Leaf Petiole

Aquilegia flabellata (Ranunculaceae), fan columbine, is a perennial herbaceous plant with brilliant blue-purple flowers with white petal tips. It can also be grown for cut flower production. In April of 2008, in several nurseries located near Biella (northern Italy), a leaf blight was observed on 10 to 15% of potted 30-day-old plants grown on a sphagnum peat substrate at 15 to 20°C and relative humidity of 80 to 90%. Semicircular, water-soaked lesions developed on leaves just above the soil line at the leaf-petiole junction and later along the leaf margins. Lesions expanded over several days along the midvein until the entire leaf was destroyed. Blighted leaves turned brown, withered, and abscised. Severely infected plants died. Diseased tissue was disinfested for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA) amended with 25 mg/liter streptomycin sulfate. A fungus with the morphological characteristics of Rhizoctonia solani was consistently recovered, then transferred and maintained in pure culture. Ten-day-old mycelium grown on PDA at 22 ± 1°C appeared light brown, rather compact, and had radial growth. Sclerotia were not present. Isolates obtained from affected plants successfully anastomosed with tester isolate AG 4 (AG 4 RT 31, obtained from tobacco plants). Results were consistent with other reports on anastomosis reactions (2). Pairings were also made with tester isolates of AG 1, 2.1, 2.2, 3, 6, 7, 11, and BI with no anastomoses observed between the recovered and tester isolates. The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLASTn analysis (1) of the 648-bp fragment showed a 100% homology with the sequence of R. solani AG-4 AB000018. The nucleotide sequence has been assigned GenBank Accession No. FJ 534555. For pathogenicity tests, the inoculum of R. solani was prepared by growing the pathogen on PDA for 10 days. Five plants of 30-day-old A. flabellata were grown in 3-liter pots. Inoculum consisting of an aqueous suspension of PDA and mycelium disks (5 g of mycelium + agar per plant) was placed at the collar of plants. Five plants inoculated with water and PDA fragments alone served as control treatments. Plants were maintained in a greenhouse at temperatures between 20 and 24°C. The first symptoms, similar to those observed in the nursery, developed 7 days after the artificial inoculation. R. solani was consistently reisolated from infected leaves and stems. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. The presence of R. solani AG1-IB on A. flabellata has been reported in Japan (4), while in the United States, Rhizoctonia sp. is described on Aquilegia sp. (3). This is, to our knowledge, the first report of leaf blight of A. flabellata caused by R. solani in Italy as well as in Europe. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. E. Carling. Grouping in Rhizoctonia solani by hyphal anastomosis reactions. In: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control. Kluwer Academic Publishers, The Netherlands, 1996. (3) D. F. Farr et al. Fungi on Plants and Products in the United States. The American Phytopathological Society, St Paul, MN, 1989. (4) E. Imaizumi et al. J. Gen. Plant Pathol. 66:210, 2000.

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

Plant Disease ◽

10.1094/pdis-05-21-0988-pdn ◽

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.

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First Report of a New Curtovirus Species, Spinach severe curly top virus, in Commercial Spinach Plants (Spinacia oleracea) from South-Central Arizona

Plant Disease ◽

10.1094/pdis-94-7-0917b ◽

2010 ◽

Vol 94 (7) ◽

pp. 917-917 ◽

Cited By ~ 9

Author(s):

C. Hernandez ◽

J. K. Brown

Keyword(s):

Dna Sequencing ◽

Spinacia Oleracea ◽

The United States ◽

Full Length ◽

Yield Reduction ◽

Insect Vector ◽

First Report ◽

South Central ◽

Total Dna ◽

Central Arizona

During April 2009, a commercial spinach field (1 km2 [250 acres]) in south-central Arizona developed geminivirus-like disease symptoms (4). Approximately 40 to 50% of the spinach plants exhibited extreme leaf distortion, foliar interveinal chlorosis, shortened internodes, and ~80% yield reduction. The beet leafhopper, Circulifer tenellus, the only known insect vector of curtoviruses in the United States, was observed on spinach plants. Total DNA was isolated (1) from three plant samples exhibiting the same symptom phenotype and used to PCR-amplify a 446-bp fragment of a suspected curtovirus, using primers F 5′-CTACCATCAGTAATGATGGG-3′and R 5′ CATATTTGCCACCTCCAGTGTC-3′ designed around the coat protein gene (Cp) for several known curtoviruses. DNA sequencing and BLAST analysis of the cloned fragments (n = 3 with 100% identity) revealed BLAST matches at 81 to 83% with the Cp for three isolates of Beet curly top Iran virus (BCTIV) (EU273816–18). To amplify the full-length curtovirus genome, total DNA from one of the three positive samples was used as the template in rolling circle amplification (RCA) employing the non-sequence specific TempliPhi 100 Amplification System (GE Healthcare) that amplifies circular DNA templates. The RCA products were linearized with PstI, yielding a ~3-Kbp fragment that was cloned into pGEM3zf+ (Promega, Madison, WI). To obtain the complete sequence, one plasmid (09-10-8) containing a full-length insert was selected and prepared for sequencing with the Template Generation System II Kit (Finnzymes, Espoo, Finland). The resultant 28 sequences were assembled into a contig using SeqMan software (DNASTAR, Madison, WI). Also, RCA clones (09.10-2, -3, and -4) from the same sample were subjected to DNA sequencing with universal M13F and M13R primers followed by primer walking (>300 bp overlap). The four 3,066-bp genomes shared 99 to 100% nt identity. An alignment (ClustalV; MegAlign, DNASTAR) with sequences of all curtovirus species available in GenBank indicated that the Arizona spinach isolates shared the highest nt sequence identity (59%) with Horseradish curly top virus (HrCTV). The next closest relatives were Beet mild curly top virus, Beet severe curly top virus, and Spinach curly top virus, at 50%. The genome consists of six open reading frames and lacks the AC3 gene, an arrangement most similar to HrCTV (3). The ICTV approved working cut-off for Curtovirus species demarcation at <89% nt identity (2) supports recognition of this isolate from spinach (GU734126) as a new, previously undescribed curtovirus species, for which we propose the name Spinach severe curly top virus (SSCTV-[Arizona:2009]). The curtovirus-like symptoms, presence of the curtovirus leafhopper vector, and isolation of a curtovirus-like genome from symptomatic spinach plants are highly suggestive of curtovirus etiology. To our knowledge, this is the first report of SSCTV worldwide and its association with diseased spinach in Arizona. Although a different curtovirus species was reported from the same infected spinach field (4), this study provides evidence that at least two curtoviruses were present in this spinach field in Arizona. References: (1) J. J. Doyle and J. L. Doyle. Focus 12:13, 1990. (2) C. M. Fauquet et al. Arch. Virol. 153:783, 2008. (3) K. A. Klute et al. J. Gen. Virol. 77:1369, 1996. (4) C. Nischwitz and M. W. Olsen. Online publication. doi:10.1094/PHP-2010-0216-01-BR. Plant Health Progress. 2010.

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First Report of the Root-Knot Nematode Meloidogyne floridensis on Tomato (Lycopersicon esculentum) in Florida

Plant Disease ◽

10.1094/pd-89-0527b ◽

2005 ◽

Vol 89 (5) ◽

pp. 527-527

Author(s):

G. T. Church

Keyword(s):

Lycopersicon Esculentum ◽

Field Experiments ◽

The United States ◽

The State ◽

Bromophenol Blue ◽

Root Knot Nematode ◽

Fresh Market ◽

Root Knot Nematodes ◽

First Report ◽

Meloidogyne Spp

The state of Florida is the largest producer of fresh market tomato (Lycopersicon esculentum L.) in the United States with 2003 yields of 634 million kg on 17,700 ha valued at 516 million dollars. Effective crop management is essential for production of vegetables in Florida because of the presence of intense pest pressure. The identification of the pests present is the first step in the development of a successful IPM (integrated pest management) program. Root-knot nematodes (Meloidogyne spp.) are common nematodes that parasitize vegetables in Florida and cause significant yield reductions when not properly managed. In 2003 field experiments, soil was collected from two research farms in Saint Lucie and Seminole counties in Florida. Galling caused by root-knot nematode was observed on tomato at both locations. Since females suitable for identification are difficult to obtain from field-grown roots, field soil was placed in pots in the greenhouse and planted with Lycopersicon esculentum cv. Rutgers. Standard morphological techniques, differential host tests, and isozyme phenotypes were used in nematode identification. Female root-knot nematodes were extracted from tomato roots and placed in extraction buffer (10% wt/vol sucrose, 2% vol/vol Triton X-100, 0.01% wt/vol bromophenol blue). The females were crushed, loaded on a polyacrylamide gel, and separated by electrophoresis using the PhastSystem (Amersham Biosciences, Piscataway, NJ). The activities of malate dehydrogenase and esterase enzymes were detected using standard techniques. Isozyme phenotypes consistent with Meloidogyne incognita (Kofoid and White) Chitwood and M. javanica (Treub) Chitwood as well as with the newly described M. floridensis Handoo (1) were observed at both locations. To our knowledge, this is the first report of M. floridensis naturally occurring on tomato in Florida. The identification and distribution of M. floridensis in vegetable production fields is important for disease management throughout the state since the host range is likely different from other Meloidogyne spp. Reference: (1) Z. A. Handoo et al. J. Nematol. 36:20, 2004.

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