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

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 Bipolaris oryzae Causing Leaf Spot of Switchgrass in Mississippi

Plant Disease ◽

10.1094/pdis-94-5-0643a ◽

2010 ◽

Vol 94 (5) ◽

pp. 643-643 ◽

Cited By ~ 5

Author(s):

M. Tomaso-Peterson ◽

C. J. Balbalian

Keyword(s):

Leaf Spot ◽

Panicum Virgatum ◽

Morphological Characteristics ◽

Seed Transmission ◽

Forest Products ◽

Its Region ◽

Infested Soil ◽

Bipolaris Oryzae ◽

Diagnostic Laboratory ◽

First Report

‘Alamo’ switchgrass (Panicum virgatum L.) seedlings growing in a soilless mix exhibiting dark brown, irregular-shaped foliar lesions with black borders were submitted to the Mississippi State University Plant Disease Diagnostic Laboratory in the summer of 2009 from a local forest products company. Symptomatic tissues were plated onto water agar (WA) and incubated for 21 days on a laboratory bench top with a 12-h photoperiod at 22°C. An asexual, sporulating, dematiaceous hyphomycete identified as Bipolaris oryzae (Breda de Haan) Shoemaker was observed. Conidiophores were single, mostly straight, multiseptate, brown, and ranging from 138 to 306 × 7.7 to 15.3 μm and averaged 221.6 × 10.7 μm. Conidia were golden brown, multiseptate, ranging from 3 to 10 septa, straight to slightly curved to fusoid, wider midway, and tapering toward the terminal cells. Conidia measured 40.8 to 109.7 × 10.2 to 20.4 μm and averaged 75.8 × 13.8 μm. Morphological characteristics of B. oryzae were similar to those described by Drechsler (1) and Sivanesan (3). The internal transcribed spacer (ITS) region of ribosomal DNA from four pure culture colonies derived from single conidia was amplified by PCR using ITS1 and ITS4 primers. The resultant 572 bp was sequenced for isolates 86 through 89 (GenBank Accession Nos. GU222690–93). The sequences were 99% similar to the sequence of B. oryzae strain ATCC-MYA 3330 (GenBank No. FJ746665) isolated from P. virgatum. Pathogenicity of isolates 86 and 88 was confirmed by inoculating sterile potting mix with a fungal slurry. Sterile Alamo switchgrass seeds were sown into the infested soil in Magenta boxes and incubated for 6 weeks in a growth chamber with a 14-h photoperiod at 30°C. Leaf lesions and leaf blight were observed in seedling stands growing in B. oryzae-infested soil. Lesions were excised and plated onto WA. Sporulation of B. oryzae was observed on symptomatic tissue. In the interim, 300 nonsterilized Alamo switchgrass seeds of the same seed lot as the original symptomatic seedlings and originating from Oklahoma were plated onto WA (10 seed per plate). The seeds were incubated on the bench top as previously described. The experiment was repeated and B. oryzae colonized 1.4% of the total switchgrass seeds evaluated, indicating seed transmission and subsequent seedling infection as previously observed in the original seedlings. Leaf spot, caused by B. oryzae, was first reported as a new disease of switchgrass in North Dakota (2). In the summer of 2009, the authors observed leaf spot in four cultivars of switchgrass, including Alamo, growing in research plots in Webster County, MS. Twenty-two isolates of B. oryzae were recovered from diseased leaves of these switchgrass cultivars. To our knowledge, this is the first report of B. oryzae causing leaf spot of switchgrass in Mississippi, which broadens the natural distribution of this disease. References: (1) C. Drechlser. J. Agric. Res. 24:641, 1923. (2) J. M. Krupinsky et al. Can. J. Plant Pathol. 26:371, 2004. (3) A. Sivanesan. Mycol. Pap. 158:201, 1987.

Download Full-text

First Report of Bipolaris oryzae Causing Leaf Spot on Cultivated Wild Rice (Oryza rufipogon) in China

Plant Disease ◽

10.1094/pdis-11-20-2529-pdn ◽

2021 ◽

Author(s):

Yue Lian Liu ◽

Jian Rong Tang ◽

Ya Li ◽

Hong Kai Zhou

Keyword(s):

Leaf Spot ◽

Wild Rice ◽

Morphological Characteristics ◽

Oryza Rufipogon ◽

Likelihood Method ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Sterile Water ◽

Bipolaris Oryzae ◽

First Report

Wild rice (Oryza rufipogon) has been widely studied and cultivated in China in recent years due to its antioxidant activities and health-promoting effects. In December 2018, leaf spot disease on wild rice (O. rufipogon cv. Haihong-12) was observed in Zhanjiang (20.93 N, 109.79 E), China. The early symptom was small purple-brown lesions on the leaves. Then, the once-localized lesions coalesced into a larger lesion with a tan to brown necrotic center surrounded by a chlorotic halo. The diseased leaves eventually died. Disease incidence was higher than 30%. Twenty diseased leaves were collected from the fields. The margin of diseased tissues was cut into 2 × 2 mm2 pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, and then rinsed three times with sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28 °C in the dark for 4 days. Pure cultures were produced by transferring hyphal tips to new PDA plates. Fifteen isolates were obtained. Two isolates (OrL-1 and OrL-2) were subjected to further morphological and molecular studies. The colonies of OrL-1 and OrL-1 on PDA were initially light gray, but it became dark gray with age. Conidiophores were single, straight to flexuous, multiseptate, and brown. Conidia were oblong, slightly curved, and light brown with four to nine septa, and measured 35.2–120.3 µm × 10.3–22.5 µm (n = 30). The morphological characteristics of OrL-1 and OrL-2 were consistent with the description on Bipolaris oryzae (Breda de Haan) Shoemaker (Manamgoda et al. 2014). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor (EF-1α) were amplified using primers ITS1/ITS4, GDF1gpp1/GDR1 gdp2 (Berbee et al. 1999), and EF-1α-F/EF-1α-R EF-1/EF-2 (O’Donnell 2000), respectively. Amplicons of OrL-1 and OrL-2 were sequenced and submitted to GenBank (accession nos. MN880261 and MN880262, MT027091 and MT027092, and MT027093 and MT027094). The sequences of the two isolates were 99.83%–100% identical to that of B. oryzae (accession nos. MF490854，MF490831，MF490810) in accordance with BLAST analysis. A phylogenetic tree was generated on the basis of concatenated data from the sequences of ITS, GAPDH, and EF-1α via Maximum Likelihood method, which clustered OrL-1 and OrL-2 with B. oryzae. The two isolates were determined as B. oryzae by combining morphological and molecular characteristics. Pathogenicity test was performed on OrL-1 in a greenhouse at 24 °C to 30 °C with 80% relative humidity. Rice (cv. Haihong-12) with 3 leaves was grown in 10 pots, with approximately 50 plants per pot. Five pots were inoculated by spraying a spore suspension (105 spores/mL) onto leaves until runoff occurred, and five pots were sprayed with sterile water and used as controls. The test was conducted three times. Disease symptoms were observed on leaves after 10 days, but the controls remained healthy. The morphological characteristics and ITS sequences of the fungal isolates re-isolated from the diseased leaves were identical to those of B. oryzae. B. oryzae has been confirmed to cause leaf spot on Oryza sativa (Barnwal et al. 2013), but as an endophyte has been reported in O. rufipogon (Wang et al. 2015).. Thus, this study is the first report of B. oryzae causing leaf spot in O. rufipogon in China. This disease has become a risk for cultivated wild rice with the expansion of cultivation areas. Thus, vigilance is required.

Download Full-text

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

Plant Disease ◽

10.1094/pdis-01-13-0117-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1655-1655 ◽

Cited By ~ 2

Author(s):

A. L. Vu ◽

K. D. Gwinn ◽

B. H. Ownley

Keyword(s):

Leaf Spot ◽

Panicum Virgatum ◽

Leaf Tissue ◽

Its Region ◽

The United States ◽

Growth Chamber ◽

Post Inoculation ◽

Commonwealth Mycological Institute ◽

Sterile Water ◽

Plastic Bag

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 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 Leaf Spot of Sweet Basil Caused by Cercospora guatemalensis in Korea

Plant Disease ◽

10.1094/pdis-05-12-0436-pdn ◽

2012 ◽

Vol 96 (10) ◽

pp. 1580-1580

Author(s):

J. H. Park ◽

K. S. Han ◽

J. Y. Kim ◽

H. D. Shin

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Its Region ◽

Culture Collection ◽

Distilled Water ◽

First Report ◽

Sweet Basil ◽

Organic Farm ◽

Leaf Spots ◽

Close Phylogenetic Relationship

Sweet basil, Ocimum basilicum L., is a fragrant herb belonging to the family Lamiaceae. Originated in India 5,000 years ago, sweet basil plays a significant role in diverse cuisines across the world, especially in Asian and Italian cooking. In October 2008, hundreds of plants showing symptoms of leaf spot with nearly 100% incidence were found in polyethylene tunnels at an organic farm in Icheon, Korea. Leaf spots were circular to subcircular, water-soaked, dark brown with grayish center, and reached 10 mm or more in diameter. Diseased leaves defoliated prematurely. The damage purportedly due to this disease has reappeared every year with confirmation of the causal agent made again in 2011. A cercosporoid fungus was consistently associated with disease symptoms. Stromata were brown, consisting of brown cells, and 10 to 40 μm in width. Conidiophores were fasciculate (n = 2 to 10), olivaceous brown, paler upwards, straight to mildly curved, not geniculate in shorter ones or one to two times geniculate in longer ones, 40 to 200 μm long, occasionally reaching up to 350 μm long, 3.5 to 6 μm wide, and two- to six-septate. Conidia were hyaline, acicular to cylindric, straight in shorter ones, flexuous to curved in longer ones, truncate to obconically truncate at the base, three- to 16-septate, and 50 to 300 × 3.5 to 4.5 μm. Morphological characteristics of the fungus were consistent with the previous reports of Cercospora guatemalensis A.S. Mull. & Chupp (1,3). Voucher specimens were housed at Korea University herbarium (KUS). An isolate from KUS-F23757 was deposited in the Korean Agricultural Culture Collection (Accession No. KACC43980). 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. The resulting sequence of 548 bp was deposited in GenBank (Accession No. JQ995781). This showed >99% similarity with sequences of many Cercospora species, indicating their close phylogenetic relationship. Isolate of KACC43980 was used in the pathogenicity tests. Hyphal suspensions were prepared by grinding 3-week-old colonies grown on PDA with distilled water using a mortar and pestle. Five plants were inoculated with hyphal suspensions and five plants were sprayed with sterile distilled water. The plants were covered with plastic bags to maintain a relative humidity of 100% for 24 h and then transferred to a 25 ± 2°C greenhouse with a 12-h photoperiod. Typical symptoms of necrotic spots appeared on the inoculated leaves 6 days after inoculation, and were identical to the ones observed in the field. C. guatemalensis was reisolated from symptomatic leaf tissues, confirming Koch's postulates. No symptoms were observed on control plants. Previously, the disease was reported in Malawi, India, China, and Japan (2,3), but not in Korea. To our knowledge, this is the first report of C. guatemalensis on sweet basil in Korea. Since farming of sweet basil has recently started on a commercial scale in Korea, the disease poses a serious threat to safe production of this herb, especially in organic farming. References: (1) C. Chupp. A Monograph of the Fungus Genus Cercospora. Ithaca, NY, 1953. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology & Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , May 5, 2012. (3) J. Nishikawa et al. J. Gen. Plant Pathol. 68:46, 2002.

Download Full-text

First Report of Fruit Rot of Melon Caused by Fusarium asiaticum in China

Plant Disease ◽

10.1094/pdis-08-20-1857-pdn ◽

2020 ◽

Author(s):

Fangmin Hao ◽

Quanyu Zang ◽

Weihong Ding ◽

Erlei Ma ◽

Yunping Huang ◽

...

Keyword(s):

Disease Incidence ◽

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Fruit Rot ◽

Post Inoculation ◽

Basal Cells ◽

First Report ◽

Fusarium Asiaticum ◽

Sterile Needle

Melon (Cucumis melo L.) is a member of the Cucurbitaceae family, an important economical and horticultural crop, which is widely grown in China. In May 2020, fruit rot disease with water-soaked lesions and pink molds on cantaloupe melons was observed in several greenhouses with 50% disease incidence in Ningbo, Zhejiang Province in China. In order to know the causal agent, diseased fruits were cut into pieces, surface sterilized for 1 min with 1% sodium hypochlorite (NaClO), 2 min with 75% ethyl alcohol, rinsed in sterile distilled water three times (Zhou et al. 2018), and then placed on potato dextrose agar (PDA) medium amended with streptomycin sulfate (100 μg/ml) plates at 25°C for 4 days. The growing hyphae were transferred to new PDA plates using the hyphal tip method, putative Fusarium colonies were purified by single-sporing. Twenty-five fungal isolates were obtained and formed red colonies with white aerial mycelia at 25°C for 7 days, which were identified as Fusarium isolates based on the morphological characteristics and microscopic examination. The average radial mycelial growth rate of Fusarium isolate Fa-25 was 11.44 mm/day at 25°C in the dark on PDA. Macroconidia were stout with curved apical and basal cells, usually with 4 to 6 septa, and 29.5 to 44.2 × 3.7 to 5.2 μm on Spezieller Nährstoffarmer agar (SNA) medium at 25°C for 10 days (Leslie and Summerell 2006). To identify the species, the internal transcribed spacer (ITS) region and translational elongation factor 1-alpha (TEF1-α) gene of the isolates were amplified and cloned. ITS and TEF1-α was amplified using primers ITS1/ITS4 and EF1/EF2 (O’Donnell et al. 1998), respectively. Sequences of ITS (545 bp, GenBank Accession No. MT811812) and TEF1-α (707 bp, GenBank Acc. No. MT856659) for isolate Fa-25 were 100% and 99.72% identical to those of F. asiaticum strains MSBL-4 (ITS, GenBank Acc. MT322117.1) and Daya350-3 (TEF1-α, GenBank Acc. KT380124.1) in GenBank, respectively. A phylogenetic tree was established based on the TEF1-α sequences of Fa-25 and other Fusarium spp., and Fa-25 was clustered with F. asiaticum. Thus, both morphological and molecular characterizations supported the isolate as F. asiaticum. To confirm the pathogenicity, mycelium agar plugs (6 mm in diameter) removed from the colony margin of a 2-day-old culture of strain Fa-25 were used to inoculate melon fruits. Before inoculation, healthy melon fruits were selected, soaked in 2% NaClO solution for 2 min, and washed in sterile water. After wounding the melon fruits with a sterile needle, the fruits were inoculated by placing mycelium agar plugs on the wounds, and mock inoculation with mycelium-free PDA plugs was used as control. Five fruits were used in each treatment. The inoculated and mock-inoculated fruits were incubated at 25°C with high relative humidity. Symptoms were observed on all inoculated melon fruits 10 days post inoculation, which were similar to those naturally infected fruits, whereas the mock-inoculated fruits remained symptomless. The fungus re-isolated from the diseased fruits resembled colony morphology of the original isolate. The experiment was conducted three times and produced the same results. To our knowledge, this is the first report of fruit rot of melon caused by F. asiaticum in China.

Download Full-text

First Report of Alternaria heveae Causing Black Leaf Spot of Rubber Tree in China

Plant Disease ◽

10.1094/pdis-01-14-0065-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 1011-1011 ◽

Cited By ~ 1

Author(s):

Z. Y. Cai ◽

Y. X. Liu ◽

G. X. Huang ◽

M. Zhou ◽

G. Z. Jiang ◽

...

Keyword(s):

Rubber Tree ◽

Morphological Characteristics ◽

Black Spot ◽

Its Region ◽

Post Inoculation ◽

First Report ◽

Leaf Spots ◽

Plastic Bags ◽

Manual Pressure ◽

Tree Leaf

Rubber tree (Hevea brasiliensis Muell. Arg.) is an important industrial crop of tropical areas for natural rubber production. In October 2013, foliar spots (0.1 to 0.4 mm in diameter), black surrounded by a yellow halo, and with lesions slightly sunken were observed on the rubber tree leaf in a growing area in Heikou County of Yunnan Province. Lesion tissues removed from the border between symptomatic and healthy tissue were surface sterilized in 75% ethanol and air-dried, plated on PDA plates, and incubated at 28°C with alternating day/night cycles of light. The pathogen was observed growing out of many of the leaf pieces, and produced abundant conidia. Colonies 6.1 cm in diameter developed on potato carrot agar (PCA) after 7 days, with well-defined concentric rings of growth. Colonies on PCA were composed of fine, dark, radiating, surface and subsurface hyphae. Conidia produced in PCA culture were mostly solitary or in short chains of 2 to 5 spores, long ovoid to clavate, and light brown, 40 to 81.25 × 8 to 20 μm (200 colonies were measured), with 3 to 6 transverse septa and 0 to 2 longitudinal or oblique septa. Morphological characteristics were similar to those described for Alternaria heveae (3,4). A disease of rubber tree caused by Alternaria sp. had been reported in Mexico in 1947 (2). DNA of Ah01HK13 isolate was extracted for PCR and sequencing of the ITS region with ITS1 and ITS4 primers was completed. From the BLAST analysis, the sequence of Ah01HK13 (GenBank Accession No. KF953884), had 97% similarity to A. dauci, 96% identical to A. macrospora (AY154701.1 and DQ156342.1, respectively), indicating the pathogen belonged to Alternaria genus. According to morphological characteristics, this pathogen was identified as A. heveae. Pathogenicity of representative isolate, Ah01HK13 was confirmed using a field rubber tree inoculation method. Three rubber plants (the clone of rubber tree Yunyan77-4) were grown to the copper-colored leaf stage and inoculated by spraying spore suspension (concentration = 104 conidia/ml) to the copper-colored leaves until drops were equally distributed on it using manual pressure sprayer. Three rubber plants sprayed with sterile distilled water were used as controls. After inoculation, the plants were covered with plastic bags. The plastic bags were removed after 2 days post-inoculation (dpi) and monitored daily for symptom development (1). The experiment was repeated three times. The typical 0.1 to 0.4 mm black leaf spots were observed 7 dpi. No symptoms were observed on control plants. A fungus with the same colony and conidial morphology as A. heveae were re-isolated from leaf lesions on inoculated rubber plants, but not from asymptomatic leaves of control plants, fulfilling Koch's postulates. Based on these results, the disease was identified as black spot of rubber tree caused by A. heveae. To our knowledge, this is the first report of A. heveae on rubber tree in China. References: (1) Z. Y. Cai et al. Microbiol Res. 168:340, 2013. (2) W. J. Martin. Plant Dis. Rep. 31:155, 1947. (3) E. G. Simmons. Mycotaxon 50:262, 1994. (4) T. Y. Zhang. Page 111 in: Flora Fungorum Sinicorum: Alternaria, Science Press, Beijing, 2003.

Download Full-text

First Report of Leaf Spot Caused by Colletotrichum fructicola on Myrica rubra in China

Plant Disease ◽

10.1094/pdis-10-21-2138-pdn ◽

2021 ◽

Author(s):

Shucheng Li ◽

Liuhua Xiao ◽

Fan Wu ◽

Yinbao Wang ◽

Mingshu Jia ◽

...

Keyword(s):

Leaf Spot ◽

Juglans Regia ◽

Morphological Characteristics ◽

Effective Control ◽

Bootstrap Support ◽

Sterile Water ◽

First Report ◽

Myrica Rubra ◽

Initial Symptoms ◽

Colletotrichum Fructicola

Myrica rubra is an important fruit tree with high nutritional and economic value, which is widely cultivated in multiple regions of China. In January 2021, an unknown disease which caused leaf spot with approximately 20% (n=100 investigated plants) of incidence was discovered on the leaves of M.rubra in Jiujiang City of Jiangxi Province (29.71° N, 115.97° E). The initial symptoms were small pale brown spots (1 to 2 mm diameter) on the leaves, which gradually expanded into round or irregular dark brown spots with the occurrence of the disease, and the lesion developed necrotic tissues in the center at later stages, eventually leading the leaves to chlorotic and wilted. Ten diseased leaves with typical symptoms were collected and the leaf tissue (5 × 5 mm) at junction of diseased and healthy portion were cut. The surfaces were disinfected with 75% ethanol for 45 s, 1% sodium hypochlorite for 1 min, and rinsed in sterile water for 3 times then transferred to potato dextrose agar (PDA) at 28 ± 1 ℃ for 3 days. Five fungal single isolates with similar morphology were purified from single spores. On PDA medium, the colonies initially appeared white with numerous aerial hyphae, and the center of the colony turned gray at later stages, less sporulation. While on modified czapek-dox medium (Peptone 3g, K2HPO4 1g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4 0.01g, Maltose 30g, Agar 15g, Distilled water 1000 mL, pH=7.0), the mycelia of the colony were sparse and produced a large number of small bright orange particles (conidial masses). Conidia were single-celled, transparent, smooth-walled, 1-2 oil globule, cylindrical with slightly blunt rounded ends, 14.45-18.44 × 5.54-6.98 μm (av=16.27 μm × 6.19 μm, n=50) in size. These morphological characteristics of the pathogen were similar to the descriptions of Colletotrichum fructicola (Ruan et al, 2017; Yang et al, 2021). To further confirm the identity of the pathogen, genomic DNA from a representative isolate was extracted with DNA Extraction Kit (Yeasen, Shanghai, China), and the internal transcribed spacer (ITS), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), calmodulin gene (CAL), actin (ACT) and chitin synthase 1 (CHS 1) were amplified by using the primers ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Weir et al, 2012), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999), respectively. The PCR amplified sequences were submitted to GenBank (GenBank Accession No. ITS, MW740334; GAPDH, MW759805; CAL, MW759804; ACT, MW812384; CHS-1, MW759803) and aligned with GenBank showed 100% identity with C. fructicola (GenBank Accession No. ITS, MT355821.1 (546/546 bp); GAPDH, MT374664.1 (255/255 bp); CAL, MK681354.1 (741/741 bp); ACT, MT364655.1 (262/262 bp); CHS, MT374618.1 (271/271 bp)). Phylogenetic tree using the maximum likelihood methods with Kimura 2-parameter model and combined ITS-ACT-GAPDH-CHS-CAL concatenated sequences, bootstrap nodal support for 1000 replicates in MEGA7.0, revealed that the isolate was assigned to C. fructicola strain (ICMP 18581 and CBS 125397) (Yang et al. 2021) with 98% bootstrap support. Pathogenicities of were tested on fifteen healthy M. rubra plants (five for wounded inoculation, five for nonwounded inoculation, and five for controls) in the orchard. Twenty leaves were marked from each plant, and disinfected the surface with 75% ethanol. Ten μL spore suspension (1.0 × 106 conidia/ml) of each isolate from 7-day-old culture were inoculated on the surface of 20 needle-wounded and 20 nonwounded leaves, respectively. Healthy leaves were inoculated with sterile water as controls by the same method. All inoculated leaves were sprayed with sterile water and covered with plastic film to remained humidification. After 5 days, all the wounded leaves which were inoculated with C. fructicola showed similar symptoms to those observed on the original leaves. Symptoms of nonwounded leaves were milder than the wounded inoculated leaves, while control leaves remained healthy. Finally, the C. fructicola was re-isolated from the inoculated leaves. C. fructicola has been reported on Juglans regia, Peucedanum praeruptorum, Paris polyphylla var. Chinensis in China (Wang et al, 2017; Ma et al, 2020; Zhou et al, 2020). As far as we know, this is the first report of C. fructicola causing leaf spot on M.rubra in China. This result contributes to better understand the pathogens causing diseases of M.rubra in this region of China and develop effective control strategies.

Download Full-text

First Report of Septoria Leaf Spot of Lavandin Caused by Septoria lavandulae in Croatia

Plant Disease ◽

10.1094/pdis-07-13-0735-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 282-282

Author(s):

K. Vrandečić ◽

J. Ćosić ◽

D. Jurković ◽

I. Stanković ◽

A. Vučurović ◽

...

Keyword(s):

Leaf Spot ◽

Antibacterial Properties ◽

Morphological Characteristics ◽

Its Region ◽

Morphological Identification ◽

Conidial Suspension ◽

Necrotic Tissue ◽

First Report ◽

Initial Symptoms ◽

Septoria Leaf Spot

Lavandula × intermedia Emeric ex Loiseleur, commonly known as lavandin, is an aromatic and medicinal perennial shrub widely and traditionally grown in Croatia. The lavandin essential oil is primarily used in perfumery and cosmetic industries, but also possesses anti-inflammatory, sedative, and antibacterial properties. In June 2012, severe foliar and stem symptoms were observed on approximately 40% of plants growing in a commercial lavandin crop in the locality of Banovo Brdo, Republic of Croatia. Initial symptoms on lower leaves included numerous, small, oval to irregular, grayish brown lesions with a slightly darker brown margin of necrotic tissue. Further development of the disease resulted in yellowing and necrosis of the infected leaves followed by premature defoliation. Similar necrotic oval-shaped lesions were observed on stems as well. The lesions contained numerous, dark, sub-globose pycnidia that were immersed in the necrotic tissue or partly erumpent. Small pieces of infected internal tissues were superficially disinfected with 50% commercial bleach (4% NaOCl) and placed on potato dextrose agar (PDA). A total of 10 isolates from leaves and five from stems of lavandin formed a slow-growing, dark, circular colonies with raised center that produced pycnidia at 23°C, under 12 h of fluorescent light per day. All 15 recovered isolates formed uniform hyaline, elongate, straight or slightly curved conidia with 3 to 4 septa, with average dimensions of 17.5 to 35 × 1.5 to 2.5 μm. Based on the morphological characteristics, the pathogen was identified as Septoria lavandulae Desm., the causal agent of lavender leaf spot (1,2). Pathogenicity of one selected isolate (428-12) was tested by spraying 10 lavandin seedlings (8 weeks old) with a conidial suspension (106 conidia/ml) harvested from a 4-week-old monoconidial culture on PDA. Five lavandin seedlings, sprayed with sterile distilled water, were used as negative control. After 5 to 7 days, leaf spot symptoms identical to those observed on the source plants developed on all inoculated seedlings and the pathogen was successfully re-isolated. No symptoms were observed on any of the control plants. Morphological identification was confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA (3). Total DNA was extracted directly from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and PCR amplification performed with primers ITS1F/ITS4. Sequence analysis of ITS region revealed at least 99% identity between the isolate 428-12 (GenBank Accession No. KF373078) and isolates of many Septoria species; however, no information was available for S. lavandulae. To our knowledge, this is the first report of Septoria leaf spot of lavandin caused by S. lavandulae in Croatia. Since the cultivation area of lavandin plants has been increasing in many continental parts of Croatia, especially in Slavonia and Baranja counties, the presence of a new and potentially harmful disease may represent a serious constraint for lavandin production and further monitoring is needed. References: (1) T. V. Andrianova and D. W. Minter. IMI Descriptions of Fungi and Bacteria, 142, Sheet 1416, 1999. (2) R. Bounaurio et al. Petria 6:183, 1996. (3) G. J. M. Verkley et al. Mycologia 96:558, 2004.

Download Full-text

First Report of a Leaf Spot Caused by Bipolaris oryzae on Switchgrass in New York

Plant Disease ◽

10.1094/pdis-03-11-0231 ◽

2011 ◽

Vol 95 (9) ◽

pp. 1192-1192 ◽

Cited By ~ 8

Author(s):

K. D. Waxman ◽

G. C. Bergstrom

Keyword(s):

New York ◽

North Dakota ◽

Leaf Spot ◽

Panicum Virgatum ◽

Perennial Grass ◽

Leaf Tissue ◽

Nucleotide Identity ◽

Bipolaris Oryzae ◽

First Report ◽

Mist Chamber

Switchgrass (Panicum virgatum L.) is a perennial grass with biofuel potential. From 2007 to 2010, foliar lesions were observed on first year and mature stands of switchgrass in various locations in New York. Foliar lesions were purple, elliptical (up to 1 cm) with either distinct or diffuse margins, and occasionally with yellow halos and/or white necrotic centers. After 2 to 5 days of moist chamber incubation, surface-sterilized, symptomatic leaf tissue produced conidia that when streaked onto potato dextrose agar containing 0.3 g of streptomycin per liter gave rise to cultures with gray-to-black mycelium that developed brown conidia. The fungus was identified as Bipolaris oryzae (Breda de Haan) Shoemaker on the basis of conidial morphology (1,2). Conidiophores were brown, straight, cylindrical, and multiseptate. Conidia were brown, curved, ellipsoidal tapering to rounded ends, with 3 to 14 septa. Conidia averaged 105 μm (54 to 160 μm) long and 16 μm (12 to 20 μm) wide. Sequences of the glyceraldehyde-3-phosphate dehydrogenase (GDP) gene of three isolates from Tompkins County (Cornell Accession and corresponding GenBank Nos.: Bo005NY07 [cv. Cave-in-Rock], JF521648; Bo006NY07 [cv. Kanlow], JF521649; and Bo038NY07 [cv. Shawnee], JF521650) exhibited 100% nucleotide identity to B. oryzae isolates (GenBank Nos. AY277282–AY277285) collected from switchgrass in North Dakota (1). Sequences of the rDNA internal transcribed spacer (ITS) regions of the isolates (Cornell Accession and corresponding GenBank Nos.: Bo005NY07, JF693908; Bo006NY07, JF693909; and Bo038NY07, JF693910) exhibited 100% nucleotide identity to B. oryzae isolates (GenBank Nos. GU222690–GU222693) collected from switchgrass in Mississippi (3). Pathogenicity of two of the sequenced isolates (Bo006NY07 and Bo038NY07) along with one other isolate (Bo116NY09 from ‘Cave-in-Rock’ in Cayuga County) was evaluated in the greenhouse. Six- to eight-week-old switchgrass plants were inoculated with conidial suspensions (40,000 conidia/ml) of B. oryzae. Inoculum or sterilized water was applied until runoff. There were three plants per treatment of each of ‘Blackwell’, ‘Carthage’, ‘Cave-in-Rock’, ‘Kanlow’, ‘Shawnee’, ‘Shelter’, and ‘Sunburst’. After inoculum had dried, plants were placed in a mist chamber for 24 h and then returned to the greenhouse. Symptoms developed 2 to 4 days after inoculation for all cultivars. No symptoms developed on the control plants. Foliar lesions closely resembled those observed in the field. B. oryzae was consistently reisolated from symptomatic tissue collected from greenhouse experiments. B. oryzae was first reported as a pathogen of switchgrass in North Dakota (1) and more recently in Mississippi (3). To our knowledge, this is the first report of B. oryzae causing a leaf spot on switchgrass in New York. Observation of severe leaf spot in several field plots suggests that switchgrass populations should be screened for their reaction to regional isolates of B. oryzae prior to expanded production of switchgrass as a biofuel crop. References: (1) J. M. Krupinsky et al. Can. J. Plant Pathol. 26:371 2004. (2) R. A. Shoemaker. Can. J. Bot. 37:883, 1959. (3) M. Tomaso-Peterson and C. J. Balbalian. Plant Dis. 94:643 2010.

Download Full-text