First Report of Leaf Spot on Switchgrass Caused by Pithomyces chartarum in the United States

There are few reports on diseases of switchgrass. In November 2009, light brown to white bleached spots (1 to 2 × 3 to 4 μm) were observed on ‘Alamo’ switchgrass (Panicum virgatum L.) grown in a growth chamber in Knoxville, TN, from surface-disinfested seed produced in Colorado. Symptomatic leaf tissue was surface sterilized, air dried, and plated on 2% water agar (WA) amended with 6.9 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). Plates were incubated at 26°C in the dark for 5 days. A sporulating, dematiaceous, mitosporic fungus was observed and transferred to potato dextrose agar. Colonies were white to gray, with brown as conidia increased. Conidia ranged in size from 10 to 22.5 × 20 to 37.5 (average 15.2 × 26.5) μm. Conidia were golden to dark brown, broadly ellipsoidal, some pyriform, with one longitudinal septum and two to three transverse septa, sometimes constricted at the transverse septa. Based on microscopic examination, the fungus was identified as Pithomyces chartarum (Berk. & Curt.) M.B. Ellis (1); observations were consistent with the authority (2). Pathogenicity assays were conducted with 5-week-old ‘Alamo’ switchgrass grown from seed scarified with 60% sulfuric acid and surface-sterilized with 50% bleach. Seed were sown in 9 × 9-cm pots containing 50% (v/v) ProMix Potting and Seeding Mix (Premier Tech Horticulture, Québec, Canada) and 50% Turface ProLeague (Profile Products, Buffalo Grove, IL). Eight replicate pots with ~20 plants each were sprayed with a spore suspension of 5.7 × 105 spores/ml sterile water prepared from 6-day-old cultures grown on V8 juice agar in the dark. Two more pots were sprayed with sterile water to serve as controls. All plants were subjected to high humidity for 72 h by enclosure in a plastic bag. Plants were placed in a growth chamber at 25/20°C with a 12-h photoperiod. Leaf spot symptoms similar to the original disease were evident on plants in each of the eight replicate pots 6 to 10 days post-inoculation. Control plants had no symptoms. Lesions were excised from leaves, surface sterilized, and plated on WA. The resulting cultures were again identified as P. chartarum based on morphology. The internal transcribed spacer (ITS) region of rDNA from the original isolate and the pathogen recovered from plants in the pathogenicity tests were amplified with PCR using primers ITS4 and ITS5. PCR amplicons were obtained from both isolates, sequenced, and found to have 100% identity. A 580-bp sequence was deposited at GenBank (Accession No. JQ406588). The nucleotide sequence had 98 to 100% identity to the ITS sequences of isolates of Leptosphaerulina chartarum (anamorph: P. chartarum), including isolate Mxg-KY09-s4 (GU195649) from leaf spot on Miscanthus × giganteus in Kentucky (1), and isolates from leaf lesions on wheat (EF489400 and JX442978). To our knowledge, leaf spot caused by P. chartarum has not been described on switchgrass (3). Pithomyces chartarum is a seedborne pathogen of switchgrass, and may play a role in stand establishment. References: (1) M. O. Ahonsi et al. Plant Dis. 94:480, 2010. (2) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England. 1971. (3) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA, Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 18 January 2013.

Download Full-text

First Report of Leaf Spot caused by Bipolaris oryzae on Switchgrass in Tennessee

Plant Disease ◽

10.1094/pdis-01-13-0070-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1654-1654 ◽

Cited By ~ 3

Author(s):

A. L. Vu ◽

M. M. Dee ◽

J. Zale ◽

K. D. Gwinn ◽

B. H. Ownley

Keyword(s):

Leaf Spot ◽

Panicum Virgatum ◽

Morphological Characteristics ◽

Its Region ◽

Spore Production ◽

Post Inoculation ◽

Sterile Water ◽

Bipolaris Oryzae ◽

First Report ◽

Symptomatic Leaf

Knowledge of pathogens in switchgrass, a potential biofuels crop, is limited. In December 2007, dark brown to black irregularly shaped foliar spots were observed on ‘Alamo’ switchgrass (Panicum virgatum L.) on the campus of the University of Tennessee. Symptomatic leaf samples were surface-sterilized (95% ethanol, 1 min; 20% commercial bleach, 3 min; 95% ethanol, 1 min), rinsed in sterile water, air-dried, and plated on 2% water agar amended with 3.45 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). A sparsely sporulating, dematiaceous mitosporic fungus was observed. Fungal plugs were transferred to surface-sterilized detached ‘Alamo’ leaves on sterile filter paper in a moist chamber to increase spore production. Conidia were ovate, oblong, mostly straight to slightly curved, and light to olive-brown with 3 to 10 septa. Conidial dimensions were 12.5 to 17 × 27.5 to 95 (average 14.5 × 72) μm. Conidiophores were light brown, single, multiseptate, and geniculate. Conidial production was polytretic. Morphological characteristics and disease symptoms were similar to those described for Bipolaris oryzae (Breda de Haan) Shoemaker (2). Disease assays were done with 6-week-old ‘Alamo’ switchgrass grown from seed scarified with 60% sulfuric acid and surface-sterilized in 50% bleach. Nine 9 × 9-cm square pots with approximately 20 plants per pot were inoculated with a mycelial slurry (due to low spore production) prepared from cultures grown on potato dextrose agar for 7 days. Cultures were flooded with sterile water and rubbed gently to loosen mycelium. Two additional pots were inoculated with sterile water and subjected to the same conditions to serve as controls. Plants were exposed to high humidity by enclosure in a plastic bag for 72 h. Bags were removed, and plants were incubated at 25/20°C with 50 to 60% relative humidity. During the disease assay, plants were kept in a growth chamber with a 12-h photoperiod of fluorescent and incandescent lighting. Foliar leaf spot symptoms appeared 5 to 14 days post-inoculation for eight of nine replicates. Control plants had no symptoms. Symptomatic leaf tissue was processed and plated as described above. The original fungal isolate and the pathogen recovered in the disease assay were identified using internal transcribed spacer (ITS) region sequences. The ITS region of rDNA was amplified with PCR and primer pairs ITS4 and ITS5 (4). PCR amplicons of 553 bp were sequenced, and sequences from the original isolate and the reisolated pathogen were identical (GenBank Accession No. JQ237248). The sequence had 100% nucleotide identity to B. oryzae from switchgrass in Mississippi (GU222690, GU222691, GU222692, and GU222693) and New York (JF693908). Leaf spot caused by B. oryzae on switchgrass has also been described in North Dakota (1) and was seedborne in Mississippi (3). To our knowledge, this is the first report of B. oryzae from switchgrass in Tennessee. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/, 28 June 2012. (2) J. M. Krupinsky et al. Can. J. Plant Pathol. 26:371, 2004. (3) M. Tomaso-Peterson and C. J. Balbalian. Plant Dis. 94:643, 2010. (4) T. J. White et al. Pages 315-322 in: PCR Protocols: a Guide to Methods and Applications. M. A. Innis et al. (eds), Acad. Press, San Diego, 1990.

Download Full-text

First Report of Leaf Spot Caused by Bipolaris spicifera on Switchgrass in the United States

Plant Disease ◽

10.1094/pdis-10-10-0774 ◽

2011 ◽

Vol 95 (9) ◽

pp. 1191-1191 ◽

Cited By ~ 8

Author(s):

A. L. Vu ◽

M. M. Dee ◽

R. J. Gualandi ◽

S. Huff ◽

J. Zale ◽

...

Keyword(s):

United States ◽

Leaf Spot ◽

Panicum Virgatum ◽

Management Strategies ◽

Leaf Tissue ◽

Severe Infection ◽

Its Region ◽

Cost Effective ◽

The United States ◽

Bipolaris Spicifera

Light-to-dark brown leaf spots and general chlorosis were observed on ‘Alamo’ switchgrass (Panicum virgatum L.) grown in ornamental plantings on the campus of the University of Tennessee in Knoxville in December 2007. Disease distribution was patchy, infecting ~10% of plants. Patches had mild to severely infected plants with stunting in areas of severe infection. Symptomatic leaf tissue was surface sterilized, air dried on sterile filter paper, and plated on 2% water agar amended with 10 mg/liter of rifampicin (Sigma-Aldrich, St. Louis, MO) and 10 μl/liter of 2.4 EC Danitol miticide (Valent Chemical, Walnut Creek, CA). Plates were incubated at 26°C in darkness for 5 days. A sporulating, dematiaceous mitosporic fungus was observed and transferred to potato dextrose agar (PDA). Conidiophores were single, light brown, multiseptate, mostly straight, polytretic, geniculate, and sympodial. Conidia were 17.5 × 12 (22) to 30 × 14 (12.5) μm, oval, light brown, and distoseptate, with one to three septa and a flattened hilum on the basal cell. Conidia germinated from both poles. The causal agent was identified as Bipolaris spicifera (Bainier) Subram. Morphological features were as described for B. spicifera (2). Pathogenicity studies were conducted with 5-week-old ‘Alamo’ switchgrass plants grown from surface-sterilized seed in 9 × 9-cm pots containing 50% ProMix Potting and Seeding Mix (Premier Tech Horticulture, Rivière-du-Loup, Québec, Canada) and 50% Turface ProLeague (Profile Products, Buffalo Grove, IL) (vol/vol). Ten replicate pots with ~20 plants each were sprayed with a spore suspension of 4.5 × 106 spores/ml of sterile water prepared from 6-day-old cultures grown on PDA. Plants were subjected to high humidity for 45 h then incubated at 25/20°C with a 12-h photoperiod in a growth chamber. Leaf spot symptoms similar to the original disease appeared on plants in each of the 10 replicate pots 6 days postinoculation. Lesions were excised from leaves, surface sterilized, plated on water agar, and the resulting cultures were again identified as B. spicifera. The internal transcribed spacer (ITS) region of ribosomal DNA from the original isolate used for inoculation and the reisolated culture recovered from plants in the pathogenicity studies were amplified with PCR using primers ITS4 and ITS5 (3). PCR amplicons of ~560 bp were obtained from both isolates and sequenced. Amplicon sequences were identical and the sequence was submitted to GenBank (Accession No. HQ015445). The DNA sequence had 100% homology to the ITS sequence of B. spicifera strain NRRL 47508 (GenBank Accession No. GU183125.1) that had been isolated from sorghum seed. To our knowledge, leaf spot caused by B. spicifera has not been described on switchgrass (1). B. spicifera can be seedborne and has been reported on turfgrass seed exported from the United States to Korea (2). As switchgrass is transitioned from a prairie grass to a biofuels crop planted in large acreages, disease incidences and severities will likely increase, necessitating rapid disease identification and cost effective management strategies. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 4 August 2010. (2) H.-M. Koo et al. Plant Pathol. J. 19:133, 2003. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds, Academic Press, San Diego, 1990.

Download Full-text

First Report of Leaf Spot Caused by Alternaria alternata on Switchgrass in Tennessee

Plant Disease ◽

10.1094/pdis-09-11-0785 ◽

2012 ◽

Vol 96 (5) ◽

pp. 763-763 ◽

Cited By ~ 2

Author(s):

A. L. Vu ◽

M. M. Dee ◽

T. Russell ◽

J. Zale ◽

K. D. Gwinn ◽

...

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Panicum Virgatum ◽

Leaf Tissue ◽

Its Region ◽

Sporulation Medium ◽

Sterile Water ◽

One Pot ◽

First Report ◽

Leaf Spots

Field-grown seedlings of ‘Alamo’ switchgrass (Panicum virgatum L.) from Vonore, TN exhibited light brown-to-dark brown leaf spots and general chlorosis in June 2009. Symptomatic leaf tissue was surface sterilized (95% ethanol for 1 min, 20% commercial bleach for 3 min, and 95% ethanol for 1 min), air dried on sterile filter paper, and plated on 2% water agar amended with 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO) and 5 μl/liter miticide (2.4 EC Danitol, Valent Chemical, Walnut Creek, CA). Plates were incubated at 26°C for 4 days in darkness. An asexual, dematiaceous mitosporic fungus was isolated and transferred to potato dextrose agar. Cultures were transferred to Alternaria sporulation medium (3) to induce conidial production. Club-shaped conidia were produced in chains with branching of chains present. Conidia were 27 to 50 × 10 to 15 μm, with an average of 42.5 × 12.5 μm. Morphological features and growth on dichloran rose bengal yeast extract sucrose agar were consistent with characteristics described previously for Alternaria alternata (1). Pathogenicity studies were conducted with 5-week-old ‘Alamo’ switchgrass plants grown from surface-sterilized seed. Nine pots with approximately 20 plants each were prepared. Plants were wounded by trimming the tops. Eight replicate pots were sprayed with a conidial spore suspension of 5.0 × 106 spores/ml sterile water and subjected to high humidity by enclosure in a plastic bag for 7 days. One pot was sprayed with sterile water and subjected to the same conditions to serve as a control. Plants were maintained in a growth chamber at 25/20°C with a 12-h photoperiod. Foliar leaf spot symptoms appeared 5 to 10 days postinoculation for all replicate pots inoculated with A. alternata. Symptoms of A. alternata infection were not observed on the control. Lesions were excised, surface sterilized, plated on water agar, and identified in the same manner as previously described. The internal transcribed spacer (ITS) region of ribosomal DNA and the mitochondrial small sub-unit region (SSU) from the original isolate and the reisolate recovered from the pathogenicity assay were amplified with PCR, with primer pairs ITS4 and ITS5 and NMS1 and NMS2, respectively. Resultant DNA fragments were sequenced and submitted to GenBank (Accession Nos. HQ130485.1 and HQ130486.1). A BLAST search (BLASTn, NCBI) was run against GenBank isolates. The ITS region sequences were 537 bp and matched 100% max identity with eight A. alternata isolates, including GenBank Accession No. AB470838. The SSU sequences were 551 bp and matched 100% max identity with seven A. alternata isolates, including GenBank Accession No. AF229648. A. alternata has been reported from switchgrass in Iowa and Oklahoma (2); however, this is the first report of A. alternata causing leaf spot on switchgrass in Tennessee. Switchgrass is being studied in several countries as a potentially important biofuel source, but understanding of the scope of its key diseases is limited. References: (1) B. Andersen et al. Mycol. Res. 105:291, 2001. (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 22, 2011. (3) E. A. Shahin and J. F. Shepard. Phytopathology 69:618, 1979.

Download Full-text

First Report of Botrytis cinerea Causing Leaf Spot of Aquilegia vulgaris in Japan

Plant Disease ◽

10.1094/pdis-93-4-0425a ◽

2009 ◽

Vol 93 (4) ◽

pp. 425-425 ◽

Cited By ~ 2

Author(s):

M. Zhang ◽

T. Tsukiboshi ◽

I. Okabe

Keyword(s):

United States ◽

Botrytis Cinerea ◽

Leaf Spot ◽

Leaf Tissue ◽

The United States ◽

Plant Diseases ◽

Its2 Region ◽

Commonwealth Mycological Institute ◽

First Report ◽

Leaf Spots

European columbine, Aquilegia vulgaris L., Ranunculaceae, is an herbaceous flower widely used in gardens, parterres, and courtyards and is a traditional herbal plant. During the summer of 2008, leaf spots were observed on a plant cultivated along a roadside area in Nasushiobara, Tochigi, Japan. In some courtyards, the leaf spot affected more than 60% of the plants. Early symptoms appeared as small, round or elliptic, brown lesions on the leaves. Lesions expanded to 5 to 15 × 4 to 10 mm, irregular spots that were dark brown to black in the middle, with pale yellow-brown or purple-brown margins. In continuously wet or humid conditions, thick, gray mycelium and conidia appeared on the surface of leaf spots. Conidiophores were melanized at the base and hyaline near the apex, branched, and septated (approximately 3 mm × 16 to 18 μm). Conidia were hyaline, aseptate, ellipsoidal to obovoid, with a slightly protuberant hilum, and ranged from 9 to 14.5 × 5.5 to 6.5 μm. The pathogen was identified as Botrytis cinerea Pers.:Fr on the basis of morphology and sequence of ITS1-5.8s-ITS2 region of rDNA. The sequence (GenBank Accession No. FJ424510) exactly matched the sequences of two Botryotinia fuckeliana (anamorph Botrytis cinerea), (e.g., GenBank Accession Nos. AY686865 and FJ169666) (2). The fungus was isolated on potato dextrose agar (PDA) from a single conidium found on the symptomatic leaf tissue. Colonies of B. cinerea were first hyaline and later turned gray to black when the spores differentiated. Koch's postulates were performed with three whole plants of potted aquilegia. Leaves were inoculated with mycelia plugs harvested from the periphery of a 7-day-old colony; an equal number of plants were inoculated with the plugs of PDA medium only and served as controls. All plants were covered with plastic bags for 24 h to maintain high relative humidity and incubated at 25°C. After 8 days, all mycelium-inoculated plants showed symptoms identical to those observed on leaves from A. vulgaris infected in the field, whereas controls remained symptom free. Reisolation of the fungus from lesions on inoculated leaves confirmed that the causal agent was B. cinerea. B. cinerea has been previously reported on A. vulgaris in the United States and China (1,3). To our knowledge, this is the first report of leaf spots caused by B. cinerea on A. vulgaris in Japan. References: (1) Anonymous. Index of Plant Diseases in the United States. USDA Agric. Handb. No 165, 1960. (2) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, England, 1971. (3) Z. Y. Zhang. Flora Fungorum Sinicorum. Vol. 26. Botrytis, Ramularia. Science Press, Beijing, 2006.

Download Full-text

First Report of Anthracnose of Onion Caused by Colletotrichum coccodes in Ohio

Plant Disease ◽

10.1094/pdis-01-14-0032-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1271-1271 ◽

Cited By ~ 2

Author(s):

F. Baysal-Gurel ◽

N. Subedi ◽

D. P. Mamiro ◽

S. A. Miller

Keyword(s):

Leaf Tissue ◽

Its Region ◽

The United States ◽

Tween 20 ◽

Colletotrichum Coccodes ◽

Academic Press ◽

Conidial Suspension ◽

First Report ◽

Allium Cepa L ◽

Total Dna

Dry bulb onion (Allium cepa L. cvs. Pulsar, Bradley, and Livingston) plants with symptoms of anthracnose were observed in three commercial fields totaling 76.5 ha in Huron Co., Ohio, in July 2013. Symptoms were oval leaf lesions and yellowing, curling, twisting, chlorosis, and death of leaves. Nearly half of the plants in a 32.8-ha field of the cv. Pulsar were symptomatic. Concentric rings of acervuli with salmon-colored conidial masses were observed in the lesions. Conidia were straight with tapered ends and 16 to 23 × 3 to 6 μm (2). Colletotrichum coccodes (Wallr.) S. Hughes was regularly isolated from infected plants (2). Culturing diseased leaf tissue on potato dextrose agar (PDA) amended with 30 ppm rifampicin and 100 ppm ampicillin at room temperature yielded white aerial mycelia and salmon-colored conidial masses in acervuli. Numerous spherical, black microsclerotia were produced on the surface of colonies after 10 to 14 days. To confirm pathogen identity, total DNA was extracted directly from a 7-day-old culture of isolate SAM30-13 grown on PDA, using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI) following the manufacturer's instructions. The ribosomal DNA internal transcribed spacer (ITS) region was amplified by PCR using the primer pair ITS1 and ITS4 (2), and sequenced. The sequence, deposited in GenBank (KF894404), was 99% identical to that of a C. coccodes isolate from Michigan (JQ682644) (1). Ten onion seedlings cv. Ebenezer White at the two- to three-leaf stage of growth were spray-inoculated with a conidial suspension (1 × 105 conidia/ml containing 0.01% Tween 20, with 10 ml applied/plant). Plants were maintained in a greenhouse (21 to 23°C) until symptoms appeared. Control plants were sprayed with sterilized water containing 0.01% Tween 20, and maintained in the same environment. After 30 days, sunken, oval lesions each with a salmon-colored center developed on the inoculated plants, and microscopic examination revealed the same pathogen morphology as the original isolates. C. coccodes was re-isolated consistently from leaf lesions. All non-inoculated control plants remained disease-free, and C. coccodes was not re-isolated from leaves of control plants. C. coccodes was reported infecting onions in the United States for the first time in Michigan in 2012 (1). This is the first report of anthracnose of onion caused by C. coccodes in Ohio. Unusually wet, warm conditions in Ohio in 2013 likely contributed to the outbreak of this disease. Timely fungicide applications will be necessary to manage this disease in affected areas. References: (1) A. K. Lees and A. J. Hilton. Plant Pathol. 52:3. 2003. (2) L. M. Rodriguez-Salamanca et al. Plant Dis. 96:769. 2012. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Download Full-text

First Report of Bacterial Spot of Peony Caused by a Xanthomonas sp. in the United States

Plant Disease ◽

10.1094/pdis-11-11-0919 ◽

2012 ◽

Vol 96 (4) ◽

pp. 581-581 ◽

Cited By ~ 5

Author(s):

C. L. Oliver ◽

R. Cai ◽

B. A. Vinatzer ◽

E. A. Bush ◽

M. A. Hansen

Keyword(s):

Leaf Spot ◽

Consensus Sequence ◽

Perfect Match ◽

Leaf Tissue ◽

The United States ◽

Genetic Distances ◽

Biochemical Tests ◽

First Report ◽

Herbaceous Perennials ◽

Phosphate Buffered Saline

In early May 2008 and 2009, peony samples (Paeonia spp.) with symptoms of leaf spot and blight were submitted to the Virginia Tech Plant Disease Clinic. The 2008 peony was an unknown cultivar from a northern Virginia landscape. The three cultivars (Dr. Alexander Fleming, Felix Crousse, and Karl Rosenfield) submitted in 2009 were from a commercial nursery in southwestern Virginia that was reporting leaf spot progressing to severe blight, which rendered plants unsalable, on 75% of a 1,219 m2 block during a 10-day period of heavy rainfall. Bacterial streaming from spots was observed. On the basis of phenotypic and biochemical tests, the isolates were determined to be xanthomonads. Two isolates (one recovered from the 2008 sample and one from the 2009 sample) were used in the following work. Isolates were characterized by multilocus sequencing (MLST) (4). PCR reactions were prepared and cycled using 2X ImmoMix (Bioline, Tauton, MA) according to manufacturer's recommendations with an annealing temperature of 58°C. Template DNA was added by touching a single colony with a 20-μl pipette tip and placing the tip into the reaction mix for 1 min. Four bands of the expected size were visualized on an electrophoresis gel and cleaned products were sequenced in forward and reverse directions at the University of Chicago, Cancer Research Center DNA Sequencing Facility. Corresponding gene fragments of each isolate were identical. A consensus sequence (PAMDB Isolate ID No. 936) for each of the four gene fragments was constructed and compared with sequences in NCBI ( http://www.ncbi.nlm.nih.gov/nuccore/ ) and PAMDB ( http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl ) (1) databases using Blastn (2). No perfect match was found. Genetic distances between the peony isolates and all strains in PAMDB were determined by MegAlign (Lasergene; DNAStar, Madison, WI). The Xanthomonas strain most similar to the isolates recovered from the peony samples was Xanthomonas hortorum pv. hederae ICMP 1661 with a genetic distance of 0.023; this strongly suggests that the peony isolates belong to X. hortorum. For Koch's postulates, six surface-disinfested young leaflets from Paeonia lactiflora ‘Karl Rosenfield’ were inoculated by forcefully spraying a phosphate-buffered saline suspension of each bacterial isolate (~4.3 × 109 CFU/ml) into the underside of the leaf until leaf tissue appeared water soaked. Controls were inoculated similarly with phosphate-buffered saline solution. Moist chambers with inoculated leaves were incubated at ambient temperature under two 48W fluorescent grow lights with 12 h of light and dark. Circular spots were observed on leaves inoculated with the 2009 and 2008 isolates in 18 and 20 days, respectively. No symptoms were observed on controls. Bacterial streaming from leaf spots was observed by phase-contrast microscopy; bacteria were isolated and confirmed to be identical to the original isolates by the methods described above. To our knowledge, this is the first report of a Xanthomonas sp. causing leaf spot and blight on peony. Although bacterial blight of peony has been attributed to a xanthomonad in recent years, the pathogen had not been further characterized (3). References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) D. J. Altschul et al. J. Mol. Biol. 215:403, 1990. (3) M. L. Gleason et al. Diseases of Herbaceous Perennials. The American Phytopathological Society, St. Paul, MN. 2009. (4) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

Download Full-text

First Report of Leaf Spot Caused by Corynespora cassiicola on Baphicacanthus cusia in China

Plant Disease ◽

10.1094/pdis-10-12-0921-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 690-690

Author(s):

Q.-L. Li ◽

S.-P. Huang ◽

T.-X. Guo ◽

Z.-B. Pan ◽

J.-Y. Mo ◽

...

Keyword(s):

Leaf Spot ◽

Ipomoea Batatas ◽

Its Region ◽

Tween 20 ◽

Herbaceous Plant ◽

Commonwealth Mycological Institute ◽

Corynespora Cassiicola ◽

Single Spore ◽

First Report ◽

Perennial Herbaceous Plant

Baphicacanthus cusia is a perennial herbaceous plant in the family Acanthaceae that is native to China, where it grows in warm temperate mountainous or hilly regions. It is commonly used as a Chinese herbal medicine. In March 2012, symptoms of leaf spot were observed on leaves of B. cusia in Long'an County, Guangxi, China, where this plant is extensively cultivated. Symptoms were initially small brown dots which developed into irregular to circular leaf spots. These spots enlarged and overlapped, extending until the 7- to 9-cm-long and 3- to 4-cm-wide leaves withered entirely, mostly within 2 months. On potato dextrose agar (PDA), the same fungus was cultured from 92% of 75 symptomatic leaf samples that had been surface sterilized in a 45-second dip in 0.1% mercuric chloride. Fungal structures were observed on diseased leaves: conidiophores (85 to 460 × 4 to 8 μm) were erect, brown, single or in clusters, and conidia (36 to 90 × 5 to 16 μm) were single or in chains of two to four, brown, cylindrical or obclavate, straight or slightly curved, with 3 to 18 pseudosepta and a conspicuous hilum. Three single-spore isolates were identified as Corynespora cassiicola (Berk & Curt.) Wei based on morphological and cultural characteristics (1). The rDNA internal transcribed spacer (ITS) region of one isolate, ZY-1, was sequenced (GenBank Accession No. JX908713), and it showed 100% identity to C. cassiicola, GenBank FJ852716, an isolate from Micronesia cultured from Ipomoea batatas (2). Pathogenicity tests were performed with each of the three isolates by spraying conidial suspensions (5 × 104 conidia/ml) containing 0.1% Tween 20 onto the surfaces of leaves of 60-day-old, 20-cm tall plants. For each isolate, 30 leaves from five replicate plants were treated. Control plants were treated with sterilized water containing 0.1% Tween 20. All plants were incubated for 36 h at 25°C and 90% relative humidity in an artificial climate chamber, and then moved into a greenhouse. Seven days after inoculation, dark brown spots typical of field symptoms were observed on all inoculated leaves, but no symptoms were seen on water-treated control plants. Koch's postulates were fulfilled by reisolation of C. cassiicola from diseased leaves. To our knowledge, this is the first report of C. cassiicola infecting B. cusia worldwide. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute: Kew, Surrey, England, 1971. (2) L. J. Dixon et al. Phytopathology 99:1015, 2009.

Download Full-text

First Report of Cochliobolus sativus Causing Leaf Spot on Paper Mulberry in China

Plant Disease ◽

10.1094/pdis-04-11-0319 ◽

2011 ◽

Vol 95 (12) ◽

pp. 1586-1586 ◽

Cited By ~ 1

Author(s):

P. S. Wu ◽

K. Chen ◽

H. Z. Du ◽

J. Yan ◽

Q. E. Zhang

Keyword(s):

Leaf Spot ◽

Leaf Tissue ◽

Pathogenic Fungi ◽

Bipolaris Sorokiniana ◽

Causal Agent ◽

Cochliobolus Sativus ◽

Conidial Suspension ◽

Sterile Water ◽

First Report ◽

Forest Park

Paper mulberry, Broussonetia papyrifera (L.) Vent., is a highly adaptable, fast-growing tree that is native to eastern Asia. Its ability to absorb pollutants makes it ideal for ornamental landscapes, especially in industrial and mining areas. During the summer of 2010, brown lesions were observed on leaves of paper mulberry in Baiwangshan Forest Park, Beijing, China. These lesions were ovoid to fusiform and 4 to 9 × 2 to 4 mm with dark brown centers and light brown irregular edges. Spots on severely infected leaves sometimes coalesced to form long stripes with gray centers. To isolate the causal agent of the lesions, 4-mm2 pieces of diseased leaf tissue from 12 leaves were collected at the lesion margins and surface disinfected in 0.5% NaOCl for 3 min, rinsed three times with sterile water, plated on water agar, and incubated at 25°C with a 12-h photoperiod. After 5 days, the cultures, which became dark brown to black, were observed. Conidiophores (120 to 220 × 4 to 7 μm) were solitary or in groups of two to five, straight or flexuous with swollen bases, and light or dark brown. Conidia were dark olive brown, spindle- or oval-shaped with truncated ends (60 to 120 × 15 to 30 μm), slightly curved, and containing 3 to 12 distoseptate (mostly 6 to 10). Pseudothecia, produced after 14 days in culture, were dark brown to black and flask shaped (420 to 530 μm in diameter with 85 to 100 × 75 to 90 μm ostiolar beaks). Asci were cylindrical (100 to 220 × 30 to 40 μm) and contained eight ascospores. Ascospores were filiform, (150 to 360 × 6 to 9 μm), hyaline, with 6 to 11 septations. Isolates were identified as Cochliobolus sativus (Ito & Kurib.) Drechsler & Dastur (anamorph Bipolaris sorokiniana (Sacc. & Sorok.) Shoem.) on the basis of culture color and dimensions and colors of pseudothecia, asci, ascospores, conidiophores, and conidia (2,3). The identity of one isolate was confirmed by ITS1-5.8S-ITS2 rDNA sequence (GenBank Accession No. HQ 654781) analysis that showed 100% homology to C. sativus listed in Berbee et al. (1). Koch's postulates were performed with six potted 3-month-old paper mulberry plants. An isolate was grown on potato dextrose agar for 14 days to obtain conidia for a conidial suspension (3 × 104 conidia/ml). Three of the potted plants were sprayed with the conidial suspension and three were sprayed with sterile water as controls. Each plant was covered with a plastic bag for 24 h to maintain high humidity and incubated at 25°C with a 12-h photoperiod. After 7 days, the inoculated plants showed leaf symptoms identical to those previously observed on paper mulberry trees in the Baiwangshan Forest Park, while control trees remained symptom free. Reisolation of the fungus from the inoculated plants confirmed that the causal agent was C. sativus. C. sativus is widely distributed worldwide causing a variety of cereal diseases. Wheat and barley are the most economically important hosts. To our knowledge, this is the first report of C. sativus as a pathogen causing leaf spot of paper mulberry in China. References: (1) M. L. Berbee et al. Mycologia 91:964, 1999. (2) M. B. Ellis. Dematiaceous Hyphomycetes. CABI, Oxon, UK, 1971. (3) A. Sivanesan et al. No.701 in: Descriptions of Pathogenic Fungi and Bacteria. CAB, Kew, Surrey, U.K., 1981.

Download Full-text

First report of Curvularia leaf spot of field corn, caused by Curvularia lunata, in Mississippi

Plant Disease ◽

10.1094/pdis-10-21-2189-pdn ◽

2022 ◽

Author(s):

Alejandra M. Jimenez Madrid ◽

Tom Allen ◽

Amilcar Vargas ◽

Adam Connor ◽

Tessie Wilkerson

Keyword(s):

Dna Sequences ◽

Leaf Spot ◽

Growth Chamber ◽

Tween 20 ◽

Curvularia Lunata ◽

Pathogenicity Test ◽

Commonwealth Mycological Institute ◽

Field Corn ◽

Foliar Symptoms ◽

Corn Plants

In July 2021, foliar symptoms characterized by small, circular, light brown to tan lesions (0.5 to 3 mm diameter) with reddish-brown margins were observed on field corn (Zea mays L.) in two commercial fields in Hinds and Marion counties, Mississippi. Disease severity ranged from 2 to 15% on observed leaves. Symptomatic leaves were sealed in plastic bags, stored on ice, and transferred to the laboratory. Lesions were cut into small sections (≈4 mm2) and surface-sterilized with 70% ethanol for 30 s then rinsed with sterile water. Sterilized sections were transferred to potato dextrose agar (PDA) amended with chloramphenicol (75 mg/liter) and streptomycin sulfate (125 mg/liter) and incubated at 25°C in the dark for 7 days. Gray to brown-black colonies with orange margins and melanized, curved conidia with three transverse septa were observed microscopically (Fig. 1; ×400). Conidia measurements ranged from 15 to 25 μm in length and 7.5 to 12.5 μm in width (x̄= 20 × 9.8 μm; n= 44). Colony and conidia morphology were consistent with previous descriptions of Curvularia lunata (Wakker) Boedijn (Mabadeje 1969; Ellis 1971). Pure cultures were obtained, and DNA was extracted from 9-day old cultures. Two isolates (TW003-21; TW008-21) were selected for sequencing of the internal transcribed spacer (ITS) region using ITS4 and ITS5 primers. The 530-bp consensus sequences were deposited in GenBank under the accession No. OK095277 and OK095278. BLASTn queries of NCBI GenBank showed that the sequences shared 100% identity with C. lunata isolate DMCC2087 from Louisiana (MG971304) and isolate CX-3 from China (KR633084). A pathogenicity test was performed on V4/V5 stage corn plants (Progeny 9114VT2P) grown in 10.2 cm pots in the greenhouse. Plants were transferred to a growth chamber one-week prior to inoculation. The two isolates were grown on amended PDA for 14 days at 25°C and an inoculum suspension was prepared for each isolate by rinsing culture plates with 2 ml of autoclaved reverse osmosis (RO) water amended with Tween 20 (0.01%) and re-suspended into 40 ml of RO water containing Tween 20. The final concentration was adjusted to 2.6×105 conidia/ml (TW003-21) and 2×105 conidia/ml (TW008-21). Ten corn plants were sprayed with 10 ml of inoculum suspension for each isolate using a Preval sprayer with a CO2 canister, and 10 plants were sprayed with water containing Tween 20 only. Plants were incubated in a growth chamber at ≈79% relative humidity and 25°C. Foliar symptoms including small, circular, and tan lesions, similar to those observed in the field, developed 3 days after inoculation. No symptoms were observed on control plants. Following incubation, symptomatic leaves were collected and C. lunata was re-isolated as described above. Colony, spore morphology and DNA sequences from inoculated plants were consistent with the original isolates as described above. The disease has been recently reported in Louisiana (Garcia-Aroca et al. 2018), Kentucky (Anderson et al. 2019), and Delaware (Henrickson et al. 2021). Although Curvularia leaf spot has been observed sporadically in MS corn fields since 2009 (Allen, personal communication), to our knowledge, this is the first official report of the disease in MS. While this disease has been more frequently encountered in MS, the economic impact associated with C. lunata is currently unknown. References Anderson, N. R., et al. 2019. Plant Dis. 103:2692. Chang, J., et al. 2020. J. Integr. Agr. 19:551-560. Ellis, M. B. 1971. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, England, p. 452-458. Garcia-Aroca T., et al. 2018. Plant Health Prog. 19:140. Henrickson M., et al. 2021. Plant Dis. First Look. Mabadeje, S. A. 1969. Trans. Br. Mycol. Soc. 52:267-271. † Indicates the corresponding author. E-mail: [email protected]

Download Full-text

First Report of Leaf Spot of Sesame Caused by Xanthomonas sp. In the United States

Plant Disease ◽

10.1094/pdis-03-12-0284-pdn ◽

2012 ◽

Vol 96 (8) ◽

pp. 1222-1222 ◽

Cited By ~ 2

Author(s):

T. Isakeit ◽

B. T. Hassett ◽

K. L. Ong

Keyword(s):

United States ◽

Leaf Area ◽

Its Region ◽

The United States ◽

Pcr Analysis ◽

Sterile Water ◽

First Report ◽

Genomic Dna Isolation ◽

Leaf Spots ◽

Mist Chamber

In July 2010 in Texas, extensive leaf spots (10 to 30% leaf area affected) occurred on a commercial planting of sesame (Sesamum indicum L.) in Hidalgo County and to a lesser extent (1 to 5% leaf area) on leaves of several varieties in experimental trials in Colorado and Victoria Counties. The leaf spots were light to dark brown, somewhat circular, and 1 to 3 mm in diameter. A symptomatic leaf from each of three to five plants per county was sampled for isolations. Leaves were sprayed with 70% ethanol and immediately blotted dry with a paper towel. The margins of spots (2 mm2) were excised with a scalpel and placed in a drop of sterile water for 5 min. Drops were streaked on nutrient agar (NA) and incubated at 30°C. The 12 isolations consistently yielded gram-negative, rod-shaped bacteria with yellow, translucent colonies that were visible after 2 days of incubation. The DNA of 11 isolates was extracted with the Norgen (Thorold, ON) Bacterial genomic DNA isolation kit (Cat. #17900) and the ITS region was amplified by 16S uni 1330 and 23S uni 322 anti primers (1). PCR products were treated with the ZymoResearch (Irvine, CA) DNA clean & concentrator kit (Cat. #D4003) and sequenced. With the NCBI database, a BLAST search of the 1,100 bp amplicons showed 93 to 99% identity with pathovars of either Xanthomonas oryzae or X. axonopodis (GenBank Accession Nos. CP003057.1 and CP002914.1, respectively). Amplicon sequences of the sesame isolates were deposited in GenBank as Accession Nos. JQ975037 through JQ975047. The reported species on sesame is X. campestris pv. sesami (2). To fulfill Koch's postulates, potted sesame plants (var. Sesaco 25), 15 to 20 cm tall, were sprayed until runoff with a suspension of bacteria (106 to 107 CFU/ml) from a 2-day-old NA culture. All 12 isolates were evaluated, with five to seven plants per isolate. Plants were maintained in a mist chamber in a greenhouse at 27 to 30°C and 100% relative humidity. The pathogenicity trial was repeated once. Leaf spots were first seen 7 days after inoculation and were prevalent 14 days after inoculation. All 12 isolates were pathogenic. There were no symptoms on leaves sprayed with sterile water. Bacteria that produced colonies consistent with Xanthomonas were reisolated on NA from symptomatic leaves but not from controls. The identities of three isolates were reconfirmed with PCR analysis and sequencing. In 2007, more than 2,000 ha of sesame were grown in the continental United States, with 80% of that in Texas. Currently, acreage of shatter-free varieties of sesame is increasing in arid areas of Texas, Oklahoma, and Kansas. In such areas, the yield impact of this disease is likely to be minimal, except in years with above-average rainfall. To our knowledge, this is the first report of this disease in the United States. References: (1) E. R. Gonçalves and Y. B. Rosato. Int. J. Syst. Evol. Microbiol. 52:355, 2002. (2) J. M. Young et al., New Zealand J. Agric. Res. 21:153, 1978.

Download Full-text