First Report of Gray Leaf Spot of Maize Caused by Cercospora zeina in China

Gray leaf spot of maize (Zea mays L.) is an important foliar disease in many parts of China. The causal organism of gray leaf spot in China is generally regarded as Cercospora zeae-maydis (3). In October 2011, symptoms similar to gray leaf spot were observed on 77% of maize plants in 25 locations (about 3,000 ha.) of Yunnan Province, China, and the disease could cause yield losses of 35 to 50%. The symptoms of leaf spot were different from those caused by C. zeae-maydis. The lesions on leaves were oblong, pale gray to pale brown, 2 to 3 × 5 to 40 mm, and confined by leaf veins that eventually coalesced. To identify the pathogen, 75 leaf samples were collected from 25 fields (three leaf samples for each field) at the kernel maturity stage. Single, well-separated lesions were excised and surface-sterilized by placing them in 75% ethanol for 5 s, then disinfested in 2% sodium hypochlorite for 5 min and rinsed with sterilized water. The lesions were incubated on water agar (WA) at 24°C for 48 to 72 h to allow sporulation. Seventy-five single-conidial isolates were obtained and cultured as described in Crous (1). Morphology of the isolates was determined on plates containing maize leaf powder agar (MLPA). After 5 days, isolates produced pale brown mycelia that consisted of 3- to 4-μm-wide, septate, branched hyphae. Conidiophores were 5 to 7 × 55 to 100 μm, straight to slightly flexuous, and usually 1- to 5-septate. Conidia were average 7.5 × 68 μm, fusiform, apex subobtuse, base subtruncate, and 3- to 6-septate. These characteristics are similar to C. zeina (2). The internal transcribed spacer (ITS) region of rDNA was amplified from each of the 75 isolates using primers ITS1/ITS4 and sequenced. The same sequences were obtained and the sequence of isolate YNGLS was submitted to GenBank (Accession No. KC878692). BLAST analysis of the sequence showed 100% confirmation to C. zeina (DQ185081). Additionally, a PCR-based diagnostic test using species-specific primers (2) confirmed the identification of the 75 isolates as C. zeina. The pathogenicity of the isolates was tested on greenhouse grown maize variety Huidan 4. The test was performed on 40 plants and replicated three times. The plants were inoculated at the 10 leaf stage by injecting 2 ml of conidial suspensions (2,500 conidia ml–1) into leaf whorl using a hypodermic syringe, and control plants were injected with sterile water. Conidia were collected from 5-day-old cultures grown on MLPA and suspended in sterile water. Forty days after inoculation, all inoculated plants showed characteristic lesions on leaves, but control plants remained asymptomatic. C. zeina was reisolated from the lesions, and the identity of the reisolates was confirmed by the morphological and molecular characteristics as stated above. C. zeina was previously reported as the causal agent of maize gray leaf spot (2). To our knowledge, this is the first report of C. zeina causing gray leaf spot of maize in China. References: (1) P. W. Crous. Mycologia Memoir. 21:1, 1998. (2) P. W. Crous et al. Stud. Mycol. 55:189, 2006. (3) C. H. Lu et al. J. Southwest China Normal Univ. 37:51, 2012.

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First Report of Gray Leaf Spot Caused by Pyricularia oryzae (synonym: Magnaporthe oryzae) in Oat (Avena sativa) in Georgia, USA.

Plant Disease ◽

10.1094/pdis-06-21-1182-pdn ◽

2021 ◽

Author(s):

Willis Turner Spratling ◽

Suraj Sapkota ◽

Brian Christopher Vermeer ◽

Jason Mallard ◽

Emran Ali ◽

...

Keyword(s):

Avena Sativa ◽

Leaf Spot ◽

Pcr Amplification ◽

Its Region ◽

Gray Leaf Spot ◽

Pyricularia Oryzae ◽

Spore Suspension ◽

Sterile Water ◽

Economic Implications ◽

First Report

In southeastern U.S., oat (Avena sativa L.) is predominantly grown as a grain or forage crop due to its exceptional palatability (Buntin et al. 2009). In November 2020, leaf spot symptoms were observed in an oat field (cv. Horizon 720) in Screven County, Georgia (GPS: 32°38'57.6"N 81°31'32.178"W). Lesions were oblong, whitish to gray in color, and surrounded by dark brown borders. Symptomatic oat leaves were sampled from the field and cut into 1 cm2 sections that were surface sterilized, plated onto Potato Dextrose Agar (PDA) media and incubated in the dark at 23°C. To obtain pure cultures, fungal hyphal tips were transferred onto fresh PDA plates 3 times. The pathogen was identified as Pyricularia (Magnaporthe) based on typical conidial morphology (Ellis 1971). Conidia were hyaline, pyriform, 2-septate, and displayed a basal hilum. Conidia measured 5.32 to 10.64 μm (average 8.24 μm) wide by 15.96 to 29.26 μm (average 25.40 μm) long. The identification of Pyricularia was further confirmed genetically via PCR amplification followed by sequencing. Genomic DNA was extracted from a 14-day old pure culture using a CTAB method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CaM) gene, and -tubulin (TUB) gene were amplified using ITS5-ITS4 (White et al. 1990), CMD5-CMD6 (Hong et al. 2005), and Bt2a- Bt2b (Glass and Donaldson 1995) primer sets, respectively. Amplicons were Sanger sequenced and blasted against the NCBI database. Results exhibited 100% (ITS), 100% (CaM), and 99.61% (TUB) homology with Pyricularia oryzae Cavara (GenBank accession no. LC554423.1, CP050920.1, and CP050924.1, respectively). The ITS, CaM, and TUB sequences of the isolate were deposited in GenBank as MZ295207, MZ342893, and MZ342894, respectively. In a greenhouse (23°C, 80% RH), Koch’s postulates were carried out by using oat seedlings cv. Horizon 270 grown in Kord sheet pots filled with Sun Gro professional growing mix, and a P. oryzae spore suspension containing 104 conidia ml−1. The spore suspension (10 ml) was sprayed with an air sprayer onto 7 pots of oat seedlings at the two-leaf stage. Seven supplementary pots of oat seedlings of the same cultivar were sprayed with sterile water to act as controls. After inoculation, plants were covered with black plastic bags that had been sprayed with sterile water to maintain high humidity and incubated overnight in the greenhouse. The bags were removed the next day, and plants were evaluated for symptoms in the following days. Seven days after inoculation, plants displayed symptoms similar to those found in the original field sample. Control plants showed no symptoms. Pyricularia oryzae was consistently re-isolated from inoculated symptomatic oat tissues. To our knowledge, this is the first report of gray leaf spot caused by P. oryzae on oat in the state of Georgia and in the continental United States. Pyricularia oryzae can infect several graminaceous plants, including agronomically important crops such as rice (Oryza sativa) and wheat (Triticum spp.) (Chung et al. 2020). Phylogenetic analysis on the ITS region using 6 different host lineages was performed and revealed that this oat isolate was most closely related to the Lolium lineage. This outbreak could have economic implications in oat production.

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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.

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First Report of Leaf Spot on Industrial Hemp (Cannabis sativa L.) Caused by Alternaria alternata in China

Plant Disease ◽

10.1094/pdis-01-21-0130-pdn ◽

2021 ◽

Author(s):

Lili Tang ◽

Xixia Song ◽

Liguo Zhang ◽

Jing Wang ◽

Shuquan Zhang

Keyword(s):

Leaf Spot ◽

Cannabis Sativa ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

First Report ◽

Leaf Spots ◽

Industrial Hemp

Industrial hemp is an economically important plant with traditional uses for textiles, paper, building materials, food and medicine (Li 1974; Russo et al. 2008; Zlas et al. 1993). In August 2020, an estimated 80% of the industrial hemp plants with leaf spots were observed in greenhouse in Minzhu town, Harbin City, Heilongjiang Province, China (45.8554°N, 126.8167°E), resulting in yield losses of 20%. Leaf symptoms began as small spots on the upper surface of leaves and gradually developed into brown spots with light yellow halos. These irregular spots expanded gradually and eventually covered the entire leaf; the center of the spots was easily perforated. To identify the pathogen, 20 diseased leaves were collected, and small sections of (3 to 5 mm) were taken from the margins of lesions of infected leaves. The pieces were sterilized with 75% alcohol for 30 s, a 0.1% mercuric chloride solution for 1 min, and then rinsed three times with sterile water. Samples were then cultured on potato dextrose agar at 28℃ in darkness for 4 days. A single-spore culture was obtained by monosporic isolation. Conidiophores were simple or branched, straight or flexuous, brown, and measured 22 to 61 μm long × 4 to 5 μm wide (n = 50). Conidia were solitary or in chains, brown or dark brown, obclavate, obpyriform or ellipsoid. Conidia ranged from 23 to 55 μm long × 10 to 15 μm wide (n = 50) with one to eight transverse and several longitudinal septa. For molecular identification (Jayawardena et al. 2019), genomic DNA of pathogenic isolate (MZ1287) was extracted by a cetyltrimethylammonium bromide protocol. Four gene regions including the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosplate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1) and RNA polymerase II beta subunit (RPB2) were amplified with primers ITS1/ITS4, GDF1/GDR1, EF1-728F/EF1-986R and RPB2-5F/RPB2-7cR, respectively (White et al. 1990). Resulting sequences were deposited in GenBank with accession numbers of MW272539.1, MW303956.1, MW415414.1 and MW415413.1, respectively. A BLASTn analysis showed 100% homology with A. alternata (GenBank accession nos. MN615420.1, MH926018.1, MN615423.1 and KP124770.1), respectively. A neighbor-joining phylogenetic tree was constructed by combining all sequenced loci in MEGA7. The isolate MZ1287 clustered in the A. alternata clade with 100% bootstrap support. Thus, based on morphological (Simmons 2007) and molecular characteristics, the pathogen was identified as A. alternata. To test pathogenicity, leaves of ten healthy, 2-month-old potted industrial hemp plants were sprayed using a conidial suspension (1×106 spores/ml). Control plants were sprayed with sterile water. All plants were incubated in a greenhouse at 25℃ for a 16 h light and 8 h dark period at 90% relative humidity. The experiment was repeated three times. After two weeks, leaf spots of industrial hemp developed on the inoculated leaves while the control plants remained asymptomatic. The A. alternata pathogen was re-isolated from the diseased leaves on inoculated plants, fulfilling Koch's postulates. Based on morphology, sequencing, and pathogenicity test, the pathogen was identified as A. alternata. To our knowledge, this is the first report of A. alternata causing leaf spot disease of industrial hemp (Cannabis sativa L.) in China and is worthy of our attention for the harm it may cause to industrial hemp production.

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First Report of Leaf Spot caused by Curvularia lunata on Wild Rice in China

Plant Disease ◽

10.1094/pdis-01-21-0147-pdn ◽

2021 ◽

Author(s):

Hong Kai Zhou ◽

Yue Lian Liu ◽

Jian Rong Tang ◽

Fei Teng Zhong ◽

Ya Li

Keyword(s):

Leaf Spot ◽

Wild Rice ◽

Disease Incidence ◽

Its Region ◽

Oryza Rufipogon ◽

Likelihood Method ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Curvularia Lunata ◽

Sterile Water

Wild rice (Oryza rufipogon), a species only recently cultivated in China, is an invaluable resource for rice breeding and basic research. In June 2019, a leaf spot disease on wild rice (O. rufipogon cv. ‘Haihong-12’) was observed in a 3.3 ha field in Zhanjiang (20.93 N, 109.79 E), China. The early symptoms were the presence of small, brown, and circular to oval spots that eventually turned reddish brown. The size of the spots varied from 1.0–5.0 mm × 1.0–3.0 mm. Disease incidence was higher than 20%. High temperature and high humidity climate were favorable for the disease occurrence. Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, then rinsed three times with sterile water before isolation. The tissues were plated onto 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. Three isolates, namely, Cls-1, Cls-2, and Cls-3, were subjected to further morphological and molecular studies. The colonies of the three isolates on PDA were initially light gray later becoming dark green. Conidiophores were erect, dark brown, geniculate, and unbranched. Conidia were fusiform, geniculate or hook-shaped, smooth-walled, dark-brown, 3-septate, with the second curved cell about 13.4–18.2 μm × 6.5–8.6 μm in size (n = 30). These morphological features agreed with previous descriptions of Curvularia lunata (Wakker) Boed (Macri and Lenna 1974). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor (EF-1α) were amplified using primers ITS1/ITS4, gpp1/gdp2 (Berbee et al. 1999), and EF-1/EF-2 (O’Donnell 1997), respectively. Amplicons of the three isolates were sequenced and submitted to GenBank (accession nos. MW042182, MW042183, and MW042184; MW091453, MW091454, and MW091455; MW090049, MW090050, and MW090051). The sequences of the two isolates were 100% identical to those of C. lunata (accession nos. MG971304, MG979801, MG979800) according to the results of BLAST analysis. A phylogenetic tree was built on the basis of concatenated data from the sequences of ITS, GAPDH, and EF-1α via the maximum likelihood method. The tree clustered Cls-1, Cls-2, and Cls-3 with C. lunata. The three isolates were determined as C. lunata by combining morphological and molecular characteristics. Pathogenicity tests were performed on Cls-1 in a greenhouse at 24 °C–30 °C with 80% relative humidity. Individual rice plants (cv. ‘Haihong-12’) with three leaves were 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 another five pots were sprayed with sterile water and used as controls. The test was conducted three times. Disease symptoms were observed on the leaves after 10 days, but the controls remained healthy. C. lunata occurs on O. sativa (rice) (Liu et al. 2014; Majeed et al. 2016), but it has not been reported on O. rufipogon until now. To the best of our knowledge, this study is the first to report that C. lunata causes leaf spots on O. rufipogon in China. Thus, vigilance is required for breeding O. rufipogon.

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First Report of Leaf Spot on Tibouchina semidecandra Caused by Beltrania rhombica in China

Plant Disease ◽

10.1094/pdis-04-12-0363-pdn ◽

2012 ◽

Vol 96 (9) ◽

pp. 1380-1380 ◽

Cited By ~ 1

Author(s):

Z. R. Shi ◽

M. M. Xiang ◽

Y. X. Zhang ◽

J. H. Huang

Keyword(s):

Leaf Spot ◽

Its Region ◽

Control Measures ◽

Control Treatment ◽

Sterile Water ◽

Single Spore ◽

First Report ◽

Potted Plants ◽

Detached Leaves

Tibouchina semidecandra Cogn. is a popular ornamental plant in tropical and subtropical areas (1). In August 2011, a leaf spot was observed on approximately 70% of 5,000 potted plants of T. semidecandra in a nursery in Zhongshan, Guangdong Province, China. Each leaf spot was round with a brown center surrounded by a reddish brown border, and ranged from 8 to 10 mm in diameter. A fungus was isolated consistently from the lesions by surface-sterilizing symptomatic leaf sections (each 3 cm2) with 75% alcohol for 8 s, washing the sections with sterile water, soaking the sections in 3% NaOCl for 15 s, rinsing the sections with sterile water three times, and then placing the sections on potato dextrose agar (PDA) at 28°C. Each of three single-spore isolates on PDA produced gray, floccose colonies that reached 70 mm in diameter after 5 days at 28°C. Setae were dark brown, straight, erect, distantly and inconspicuously septate, and 125 to 193 × 3.0 to 4.5 μm. Conidiophores were light brown, cylindrical, simple or sometimes branched at the base, and 105 to 202 × 3 to 5 μm. Separating cells were hyaline, oval, and 12 to 13 × 4 to 5 μm. Conidia were unequally biconic, unicellular, dark brown with a pale brown or subhyaline band just above the widest part, and 26 to 31 × 8.5 to 12 μm (mean 27.3 × 10.6 μm) with a conspicuous appendage at the apex that was 6 to 14 × 1 to 1.8 μm. These characteristics were consistent with the description of Beltrania rhombica Penz. (3). The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) of one isolate (GenBank Accession No. JN853777) was amplified using primers ITS4 and ITS5 (4) and sequenced. A BLAST search in GenBank revealed 97% similarity to the ITS sequence of an isolate of B. rhombica (GU797390.1). To confirm pathogenicity of the isolate, ten detached leaves from 3-month-old plants of T. semidecandra ‘Purple Glorybush’ were inoculated in vitro with 5-mm diameter, colonized mycelial plugs from the periphery of 5-day-old cultures of the isolated fungus. The agar plugs were put on the leaf surface and secured with sterile, moist cotton. Sterile PDA plugs were similarly used as the control treatment on ten detached leaves. Leaves were placed in petri dishes and incubated in a growth chamber with 12 h of light/day at 28°C. Necrotic lesions appeared on leaves after 2 to 3 days of incubation, whereas control leaves inoculated with sterile PDA plugs remained asymptomatic. B. rhombica was consistently reisolated from the lesions using the same method described above, but was not reisolated from the control leaves. Although there are approximately 77 reported hosts of B. rhombica (2), to our knowledge, this is the first report of B. rhombica causing a leaf spot on T. semidecandra. Because the disease caused foliar damage and reduced the ornamental value of the nursery plants, control measures may need to be implemented for this species in nurseries. References: (1) M. Faravani and B. H. Bakar. J. Food Agric. Env. Pap. 5:234, 2007. (4) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 30 Mar. 2012. (2) K. A. Pirozyski and S. D. Patil. Can. J. Bot. Pap. 48:567, 1970. (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, CA, 1990.

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First Report of Target Leaf Spot Caused by Corynespora cassiicola on Balsam Pear in China

Plant Disease ◽

10.1094/pdis-94-1-0127c ◽

2010 ◽

Vol 94 (1) ◽

pp. 127-127 ◽

Cited By ~ 1

Author(s):

B.-J. Li ◽

Y.-J. Zhao ◽

W. Gao ◽

Y.-X. Shi ◽

X.-W. Xie

Keyword(s):

Leaf Spot ◽

Its Region ◽

Potato Dextrose Agar ◽

Corynespora Cassiicola ◽

Sterile Water ◽

Exact Match ◽

First Report ◽

Humidity Chamber ◽

Plant Pathol ◽

Dark Green

Balsam pear (Momordica charantia L.) is an economically important vegetable in China with increasing interest as a medicinal plant. In December of 2006, a new foliar disease caused by Corynespora cassiicola was observed on balsam pear growing in greenhouses in Shouguang City, Shandong Province, China. The disease occurred on 35% or less of the plants. Leaves of affected plants developed off-white halos surrounding circular lesions that were 1 to 5 mm broad. The lesions became dark brown, necrotic with concentric rings, and up to 15 mm in diameter. Severely affected plants eventually wilted and defoliated. Pieces of tissue from the leading edges of lesions were disinfected in 1% NaOCl for 1 min, rinsed in sterile water, and plated on potato dextrose agar. Colonies of the fungus were gray to dark green. Conidiophores were erect and simple, pale brown to brown, and 100 to 450 μm long and 3 to 8 μm wide. Conidia were obclavate to cylindrical, pale olivaceous brown to dark brown, smooth, 35 to 100 × 8 to 12 μm, and were produced in chains. On the basis of these characteristics, the fungus was identified as Corynespora cassiicola (1). The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and deposited in GenBank (Accession No. GQ381292). It was an exact match for a sequence of C. cassiicola previously deposited (Accession No. EU364555). To confirm pathogenicity, 30 1-month-old healthy seedlings of balsam pear were inoculated by spraying a suspension of conidia (1 × 105 conidia per ml) of one isolate of C. cassiicola until runoff. Ten seedlings were sprayed with sterile water as controls. Plants were kept in a humidity chamber at 27°C overnight and then placed in a growth chamber at 27°C. After 7 days, symptoms identical to those described above were observed, while no symptoms developed on the control plants. The pathogen was reisolated from inoculated leaves. C. cassiicola causes foliar diseases on many plants, including tomato, eggplant, soybean, and cucumber (2). There is one report on balsam pear in Korea (3). To our knowledge, this is the first report of target leaf spot caused by C. cassiicola on balsam pear in China. References: (1) M. B. Ellis. CMI Mycol. Pap. No. 65, 1957. (2) M. B. Ellis et al. CMI Mycol. Pap. No. 303, 1971. (3) J. H. Kwon et al. Plant Pathol. J. 21:164, 2005.

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First Report of Anthracnose on Cucurbitaceous Crops Caused by Glomerella magna in Taiwan

Plant Disease ◽

10.1094/pdis-94-6-0787a ◽

2010 ◽

Vol 94 (6) ◽

pp. 787-787 ◽

Cited By ~ 3

Author(s):

J.-G. Tsay ◽

R.-S. Chen ◽

W.-L. Wang ◽

B.-C. Weng

Keyword(s):

Pcr Amplification ◽

Leaf Tissue ◽

Its Region ◽

Spore Suspension ◽

Rrna Gene ◽

Sodium Hypochlorite Solution ◽

Sterile Water ◽

Cucumis Sativus L ◽

First Report ◽

Species Specific

During the summer and fall of 2006, leaf anthracnose samples were collected from fields of cucumber (Cucumis sativus L.), calabash gourd (Lagenaria siceraria (Molina) Standley), and luffa (Luffa cylindrica (L.) M. Roem.) in southern Taiwan. On cucumber leaves, spots start as water-soaked areas and expand into brown spots. Leaf lesions on calabash gourd and luffa begin as water soaked and then become light brown-to-reddish spots. Centers of lesions sometimes fall out; giving infected leaves a shot-hole appearance. Small pieces (approximately 2 × 2 mm) of diseased leaf tissue from margins of individual lesions were surface disinfected in 1% sodium hypochlorite solution for 1 min, rinsed in sterile water, plated on water agar, and incubated at 25°C. After 4 days, mycelium was isolated, transferred to potato dextrose agar (PDA), and then incubated at 25°C in a 12-h light/darkness regimen. Fast-growing colonies on PDA were white to orange or pink with abundant acervuli but no perithecium. One-celled conidia were ovoid to oblong and 12 to 20 × 4 to 6 (15.9 × 5.0) μm. The morphological traits were identical to those of Colletotrichum magna (teleomorph Glomerella magna Jenkins & Winstead) and clearly distinct from those of C. orbiculare (Berk. & Mont.) Arx (synonym C. lagenarium (Pass.) Ellis & Halst. (conidia were mostly oblong, measuring 7 to 11 × 2 to 6 [9.3 × 4.2] μm, with slow-growing gray colonies) (2,3). Koch's postulates were performed to verify that the isolates were capable of causing anthracnose on cucurbitaceous crops. Pathogenicity tests were conducted in the greenhouse at 25°C under natural daylight conditions. Isolate C0604 was grown on PDA for 14 days and a spore suspension was made (106 spores/ml). Three 14-day-old seedlings at the two- to three-leaf stage of muskmelon (Cucumis melo L. var. reticulatus Naud., cv. Sapphire), squash (Cucurbita moschata Duch., cv. Achen), calabash gourd (cv. Huapu), and luffa (cv. 623) were sprayed with the spore suspension and then covered with plastic bags. Control treatments were sprayed with sterile water. After 2 days, the bags were removed. Typical anthracnose symptoms developed on all inoculated seedlings 7 days after inoculation. G. magna was reisolated from inoculated leaves following the protocol used for the original isolation. Control seedlings developed no symptoms. To confirm the identity of the fungus, PCR amplification and DNA sequencing of the internal transcribed spacer 1 (ITS1)-5.8S-ITS2 of rRNA gene of the isolate C0604 was performed by using ITS1/ITS4 as the PCR and sequencing primers. Sequence analysis of the 558-bp PCR product (GenBank Accession No. GU358453) showed 100% identity to the rRNA sequence of G. magna (GenBank Accession No. DQ003103) (1). PCR amplification of the ITS region was also carried out using species-specific primer GmF (5′- GTG AAC ATA CCT CAA ACG TTG CC -3′)/GmR (5′- GGA GGG TCC GCC ACT GTA TTT CG -3′) designed in this study. A DNA fragment of approximately 378 bp was amplified from nine isolates of G. magna, whereas no amplification products were obtained from reference cultures of C. gloeosporioides (Penz.) Penz. & Sacc. and C. orbiculare. To our knowledge, this is the first report of G. magna causing anthracnose on cucurbitaceous crops in Taiwan. References: (1) M. Du et al. Mycologia 97:641, 2005. (2) S. F. Jenkins, Jr. and N. N. Winstead. Phytopathology 54:452, 1964. (3) T. A. Zitter et al., eds. Compendium of Cucurbit Diseases. The American Phytopathological Society, St. Paul, MN, 1996.

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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.

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First Report of Target Leaf Spot Caused by Corynespora cassiicola on Cowpea in China

Plant Disease ◽

10.1094/pdis-11-13-1113-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1427-1427

Author(s):

J.-T. Li ◽

S.-X. Mo ◽

H.-B. Fu

Keyword(s):

Leaf Spot ◽

Its Region ◽

American Samoa ◽

Control Measures ◽

Corynespora Cassiicola ◽

Sterile Water ◽

First Report ◽

Humidity Chamber ◽

Causal Pathogen ◽

Its1 And Its2

Cowpea (Vigna unguiculata L.) is an important economic vegetable and is widely planted in China. During a survey of diseases in May 2009, a new leaf disease incited by the fungus Corynespora cassiicola was observed on cowpea growing in greenhouses in Shouguang city, Shandong Province, China. Circular lesions of different sizes were present on approximately 40% of the plants. Lesions were round with grayish brown centers surrounded by brownish concentric rings and ranged from 1 to 13 mm in diameter. Leaves with many lesions resulted in chlorosis, wilt, and defoliation. Yellow disk was observed on lesion edges of partly infected leaves. Abundant conidia and conidiophores appeared on the abaxial surface of leaves. To identify the causal pathogen, pieces of tissue from the leading border of lesions were sterilized in 75% ethanol for 1 min, rinsed in sterile water, transferred to potato dextrose agar (PDA), and then incubated at 28°C in an incubator. Colonies grew to 60 mm and were gray in color after 7 days. Conidiophores were straight and unbranched, pale or dark brown, and 63 to 211 × 4 to 8 μm. Conidia were born singly or in chains, obclavate or cylindrical, brown or olivaceous, 33 to 97 × 5 to 11 μm. Based on the above characteristics, the fungus was similar to C. cassiicola (Berk. & M.A. Curtis.) C.T. Wei (2). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS1 and ITS2 and deposited in GenBank (Accession No. KC894915). A BLAST search in GenBank indicated precise match for a sequence of C. cassiicola from cowpea in American Samoa (1). To satisfy Koch's postulates, 20 one-month-old seedlings of cowpea were sprayed with a spore suspension (1 × 105 spores/ml) of one isolate of C. cassiicola until runoff. Another 20 seedlings, sprayed with sterile water, served as non-inoculated controls. Plants were placed in a humidity chamber at 28°C for 12 h and then transferred to a growth chamber at 28°C. Symptoms similar to those described above appeared after 7 days on inoculated plants; however, no symptoms were observed on non-inoculated controls. C. cassiicola was re-isolated from inoculated plants. The pathogen can cause diseases on a number of plants and lead to losses. In China, this pathogen has previously been recorded on about 20 genera of plants. It also included V. sinensis (3), a close plant with V. unguiculata. However, to our knowledge, this is the first report of target leaf spot caused by C. cassiicola on cowpea (V. unguiculata) in China. Control measures may be needed to manage the disease. References: (1) L. J. Dixon et al. Phytopathology 99:1015, 2009. (2) M. B. Ellis. CMI Mycol. Pap. No. 65, 1957. (3) F. L. Tai. Sylloge Fungorum Sinicorum. Science Press, Beijing, 1979.

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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.

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