First Report of Nigrospora osmanthi Causing Leaf Spot on Tartary Buckwheat in China

Buckwheat (Fagopyrum tataricum) is a traditional short-season pseudocereal crop originating in southwest China and is cultivated around the world. Antioxidative substances in buckwheat have been shown to provide many potential cardiovascular health benefits. Between August and November in 2019, a leaf spot was found in several Tartary buckwheat cv. Pinku1 fields in Xiangxiang County, Hunan Province, China. The disease occurred throughout the growth cycle of buckwheat after leaves emerged, and disease incidence was approximately 50 to 60%. Initially infected leaves developed a few round lesions, light yellow to light brown spots. Several days later, lesions began to enlarge with reddish brown borders, and eventually withered and fell off. Thirty lesions (2×2 mm) collected from three locations with ten leaves in each location were sterilized in 70% ethanol for 10 sec, in 2% sodium hypochlorite for 30 sec, rinsed in sterile water for three times, dried on sterilized filter paper, and placed on a potato dextrose PDA with lactic acid (3 ml/L), and incubated at 28°C in the dark for 3 to 5 days. Fungal colonies were initially white and later turned black with the onset ofsporulation. Conidia were single-celled, black, smooth, spherical to subspherical, and measured 9.2 to 15.6 µm long, and 7.1 to 11.6 µm wide (n=30). Each conidium was terminal and borne on a hyaline vesicle at the tip of conidiophores. Morphologically, the fungus was identified as Nigrospora osmanthi (Wang et al. 2017). Identification was confirmed by amplifying and sequencing the ITS region, and translation elongation factor 1-alpha (TEF1-α) and partial beta-tublin (TUB2) genes using primers ITS1/ITS4 (Mills et al. 1992), EF1-728F/EF-2 (Carbone and Kohn 1999; O’Donnell et al. 1998) and Bt-2a/Bt-2b (Glass et al. 1995), respectively. BLAST searches in GenBank indicated the ITS (MT860338), TUB2 (MT882054) and TEF1-α (MT882055) sequences had 99.80%, 99% and 100% similarity to sequences KX986010.1, KY019461.1 and KY019421.1 of Nigrospora osmanthi ex-type strain CGMCC 3.18126, respectively. A neighbor-joining phylogenetic tree constructed using MEGA7.0 with 1,000 bootstraps based on the concatenated nucleotide sequences of the three genes indicated that our isolate was closely related to N. osmanthi. Pathogenicity test was performed using leaves of healthy F. tataricum plants. The conidial suspension (1 × 106 conidia/ml) collected from PDA cultures with 0.05% Tween 20 buffer was used for inoculation by spraying leaves of potted 20-day-old Tartary buckwheat cv. Pinku1. Five leaves of each plant were inoculated with spore suspensions (1 ml per leaf). An equal number of control leaves were sprayed with sterile water to serve as a control. The treated plants were kept in a greenhouse at 28°C and 80% relative humidity for 24 h, and then transferred to natural conditions with temperature ranging from 22 to 30°C and relative humidity ranging from 50 to 60%. Five days later, all N. osmanthi-inoculated leaves developed leaf spot symptoms similar to those observed in the field, whereas control leaves remained healthy. N. osmanthi was re-isolated from twelve infected leaves with frequency of 100%, fulfilling Koch’s postulates. The genus Nigrospora has been regarded by many scholars as plant pathogens (Fukushima et al. 1998) and N. osmanthi is a known leaf blight pathogen for Stenotaphrum secundatum (Mei et al. 2019) and Ficus pandurata (Liu et al. 2019) but has not been reported on F. tataricum. Nigrospora sphaerica was also detected in vegetative buds of healthy Fagopyrum esculentum Moench (Jain et al. 2012). To our knowledge, this is the first report of N. osmanthi causing leaf spot on F. tataricum in China and worldwide. Appropriate strategies should be developed to manage this disease.

Download Full-text

First Report of Alternaria alternata Causing Leaf Spot of Tartary Buckwheat (Fagopyrum tataricum) in China

Plant Disease ◽

10.1094/pdis-01-21-0191-pdn ◽

2021 ◽

Author(s):

Shaoqing Li ◽

quan shen ◽

Haihua Wang ◽

Feng He ◽

Zuyin Xiao ◽

...

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Tartary Buckwheat ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

Fagopyrum Tataricum ◽

First Report ◽

Fungal Isolates ◽

Initial Symptoms

Buckwheat (Fagopyrum tataricum) is recognized as a healthy food with abundant nutrients and high levels of rutin. In April and May of 2020, an unknown tartary buckwheat leaf spot distinct from Nigrospora leaf spot (Shen et al. 2020) was observed in Xiangxiang, Hunan, China (27°49′54″N, 112°span style="font-family:'Times New Roman'; color:#0000ff">18′48″E.). Disease incidence was 60-70% within three fields (totally 7, 000 m2). The disease occurred after plants emerged. Initial symptoms began as circular, or ellipsoid, chlorotic, water-soaked spots, mostly on leaf apexes or leaf margins. The small spots gradually enlarged and often coalesced to form large circular or irregular, pale to light brown lesions, and the infected leaves eventually withered and fell off. Thirty 2 × 2 mm infected tissue pieces collected from five locations were sterilized in 70% ethanol for 10 S, in 2% NaClO for 30 S, rinsed in sterile water for three times, dried, and placed on PDA with lactic acid (3 ml/L). After 3-5 days at 28°C in the dark, 17 fungal isolates were purified using single-spore isolation method. Almost all fungal isolates had similar morphology. Colonies were initially olive green with white margin and later turned dark olive or black with profuse sporulation. Conidia were borne in long chains, tawny to brownish green, with 1-3 longitudinal and 1-7 transverse septa, pyriform, and measured 9.5-39.6 µm long, and 5.1-12.6 µm wide (n=50). Based on morphological characteristics, the fungus was identified as Alternaria alternata (Simmons 2007). Partial internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α(TEF) and Alternaria major allergen (Alt a1) genes of isolate BLS-1 were amplified using ITS1/ITS4 (Mills et al. 1992), EF1-728F/EF1-986R (Carbone and Kohn 1999), Gpd1/Gpd2 and Alt-4for/Alt-4rev (Lawrence et al. 2013), respectively. Sequences were deposited into GenBank with acc. nos MW453091 (ITS), MW480219 (GAPDH), MW480218 (TEF), and MW480220 (Alt a1). BLASTn analysis showed 99.8% (ITS, MH854758.1), 100% (GAPDH, KP124155.1), 99.8% (TEF, KP125073.1) and 100% (Alt a1, KP123847.1) identity with reference strain CBS 106.24 of A. alternata, confirming isolate BSL-1 to be A. alternata. A neighbor-joining phylogenetic tree constructed by MEGA7.0 based on concatenated sequences of the four genes indicated that BSL-1 formed a distinct clade with A. alternata CBS 106.24 with 100% bootstrap values. Pathogenicity test was triplicately performed on healthy leaves. Twenty leaves of five 20-day-old plants (cv. Pinku1) were sprayed with conidial suspension (1×106 conidia/ml) collected from PDA cultures with 0.05% Tween 20. An equal number of control leaves were sprayed with sterile water to serve as the controls. Treated plants were kept in a greenhouse at 28±3 °C with relative humidity of 80±5% for 24 h and transferred to natural conditions (22-30°C, RH 50-60%). After 4 to 6 days, all inoculated leaves developed symptoms similar to those observed in the fields, while the control leaves remained healthy. A. alternata was re-isolated from all infected leaves. Occasionally-isolated Diaporthe isolates were not pathogenic. A. alternata causes leaf spot of oat (Zhao et al. 2020) and leaf blight of F. esculentum (Lu et al. 2019). To our knowledge, this is the first report of A. alternata causing leaf spot on F. tataricum in China and the world. Effective strategies should be developed to manage the disease.

Download Full-text

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.

Download Full-text

First Report of Leaf Spot Caused by Pseudocercospora pruni-persicicola on Sweet Cherry in Korea

Plant Disease ◽

10.1094/pdis-09-13-0968-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 693-693

Author(s):

I. Y. Choi ◽

U. Braun ◽

J. H. Park ◽

H. D. Shin

Keyword(s):

Leaf Spot ◽

Sweet Cherry ◽

Prunus Persica ◽

Morphological Characteristics ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

Voucher Specimen ◽

First Report ◽

Close Phylogenetic Relationship

Sweet cherry, Prunus avium (L.) L., is not much cultivated in Korea, with only 150 ha planted for domestic consumption. In September 2012, a previously unknown leaf spot was observed with nearly 100% incidence on trees (cv. Seneca) planted in a plastic greenhouse in Iksan City of Korea. Interestingly, the same cultivar as well as other cultivars planted outdoors did not show these symptoms. Leaf spots were irregular to subcircular, dark brown with or without a yellow halo, and becoming coalesced to cause leaf blight and premature defoliation. A cercosporoid fungus was consistently associated with disease symptoms. Fungal structures within the lesion developed on both leaf sides but mostly on the upper side. Stromata were well-developed, globular, dark brown, composed of textura angularis-globosa, and 30 to 80 μm in diameter. Conidiophores were densely fasciculate, pale olivaceous to pale brown, subcylindrical, geniculate-sinuous, 8 to 24 × 3 to 4 μm, and aseptate to 2-septate. Conidiogenous loci were inconspicuous, neither thickened nor darkened. Conidia were olivaceous, generally darker than conidiophores, cylindrical to obclavate, almost straight to mildly curved, short obconically truncate at the base, obtuse at the apex, 1- to 10-septate, constricted at the septa, 12 to 86 × 3.5 to 5 μm, guttulate, and had unthickened, not darkened hila. Morphological characteristics of the fungus were consistent with previous descriptions of Pseudocercospora pruni-persicicola (J.M. Yen) J.M. Yen (1,3). A voucher specimen was deposited in the Korea University herbarium (Accession No. KUS-F27264) and a monoconidial isolate was deposited in the Korean Agricultural Culture Collection (Accession No. KACC47019). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 (4) and sequenced. The resulting 505-bp sequence was deposited in GenBank (Accession No. KF670713). A BLAST search in GenBank revealed that the sequence showed >99% similarity with sequences of many Pseudocercospora species, indicating the close phylogenetic relationship of species in this genus. To conduct a pathogenicity test, a conidial suspension (~1 × 104 conidia/ml) was prepared in sterile water by harvesting conidia from 2-week-old cultures on V8 juice agar, and the suspension was sprayed until runoff onto the leaves of five healthy seedlings. Control plants were sprayed with sterile water. The plants were covered with plastic bags to maintain a relative humidity of 100% for 48 h and then transferred to a greenhouse. Necrotic spots appeared on the inoculated leaves 20 days after inoculation, and were identical to the ones observed in the field. P. pruni-persicicola was re-isolated from symptomatic leaf tissues, fulfilling Koch's postulates. Control plants remained symptomless. The fungus has previously been recorded on Prunus persica (L.) Stokes in Taiwan (2,3). To our knowledge, this is the first report of this fungus on P. avium globally as well as in Korea. The disease poses a new threat to the sweet cherry industry in Korea. References: (1) U. Braun and V. A. Melnik. Cercosporoid Fungi from Russia and Adjacent Countries. Rus. Acad. Sci., St.-Petersburg, 1997. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, Retrieved August 24, 2013. (3) J. M. Yen. Rev. Mycol. 42:57, 1978. (4) T. J. White et al. PCR Protocols. Academic Press, San Diego, CA, 1990.

Download Full-text

First report of Curvularia lunata causing Curvularia leaf spot of corn in Delaware

Plant Disease ◽

10.1094/pdis-04-21-0742-pdn ◽

2021 ◽

Author(s):

Madeline Grace Henrickson ◽

Alyssa M Koehler

Keyword(s):

Leaf Spot ◽

The United States ◽

Reference Sequence ◽

Penicillin G ◽

Tween 20 ◽

Curvularia Lunata ◽

Conidial Suspension ◽

Sterile Water ◽

Moist Chamber ◽

First Report

In mid-August 2020, small circular tan-colored lesions were detected in leaves of corn, Zea mays, in Sussex county Delaware. Symptoms were observed in many fields, affecting multiple hybrids that were at approximately the dough growth stage (R4). Lesions were present on most plants and individual ear leaf severity ratings ranged from 3 to 14 %. Symptomatic corn leaves were selected from the field and kept in a moist chamber for 24 hrs at 25 °C. In lesions, melanized spores were observed ranging from 21.94 µm to 30.51 µm in length and 7.83µm to 11.70µm in width (n=20). One cm2 leaf sections were extracted and sterilized in a 0.85% sodium hypochlorite solution for 30s followed by a sterile water rinse for 30s. Leaf pieces were then plated onto potato dextrose agar amended with 50µg/ml penicillin-G-sodium salt and streptomycin sulfate. Petri dishes were incubated at 25 °C with a 12-hr photoperiod for 14 days. Colonies were brown-black to lighter gray in color. Media was stained from orange, to dark brown, or not at all. Conidia were melanized and curved with three transverse septa matching the size above. Colony and spore morphology were consistent with the description of Curvularia lunata (Garcia-Aroca et al. 2018). Pure cultures were obtained, and a representative isolate was sequenced to confirm fungal identity by amplifying the internal transcribed spacer (ITS) region with primers ITS4 and ITS5. BLAST search results confirmed 100% similarity (483/531 bp) to the C. lunata reference sequence (MK623264). The sequence was deposited in GenBank as accession number MW794323. To complete Koch’s postulates, corn (hybrid: Hubner H6187RCSS) was planted into 10 cm pots and maintained at 25°C and a 12 hr photoperiod. A conidial suspension was made by soaking a Petri dish of a 3-week-old fungal colony with 20 ml of sterile water and a drop of Tween-20 solution then, scraping mycelia to dislodge spores. The conidial concentration was calibrated to 5 x 105 spores/ml and 15 ml were applied to each whorl of four three-collar stage plants using a Preval sprayer (Nakoma Products, Bridgeview, IL). Four plants were inoculated with 15 ml sterile DI water as a control. This experiment was repeated twice. Each plant was moved to an incubator and covered with a plastic bag for 24 hr to maintain humidity. Conditions in the incubator were maintained at 25 °C with a 12-hr photoperiod. Inoculated plants displayed small, oval-shaped lesions within four days. No symptoms were observed on the control plants. Symptomatic leaves were harvested and placed into a moist chamber for 24 hrs to sporulate and lesions were plated onto PDA as described above. Culture morphology was consistent with the original isolate, with spores slightly larger ranging from 22.35 µm to 39.29 µm in length, and 8.36 µm to 11.69 µm in width (n=20). Isolates obtained from inoculated plants were sequenced and maintained 100% identity with the reference sequence described above. C. lunata was first reported in Louisiana in 2017 (Garcia-Aroca et al. 2018) and in Kentucky in 2018 (Anderson et al. 2019). This is the first report of Curvularia leaf spot on corn in Delaware. Symptoms developed late in the season, so it is unlikely that yield was affected in 2020. However, the economic impact of this disease in the United States is still unclear, it will be important to monitor potential impacts of this disease in Delaware corn production. References 1.Anderson NR, et al., 2019, Plant Disease, 103, 2692, doi: 0.1094/PDIS-03-19-0629-PDN. 2. Garcia-Aroca T, et al., 2018, Plant health progress, 19, 140, doi: 10.1094/PHP-02-18-0008-BR.

Download Full-text

First Report of Leaf Spot Caused by Corynespora cassiicola on Rose of Sharon in Korea

Plant Disease ◽

10.1094/pdis-11-12-1099-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 847-847 ◽

Cited By ~ 1

Author(s):

S. T. Seo ◽

J. H. Park ◽

S. E. Cho ◽

H. D. Shin

Keyword(s):

Leaf Spot ◽

Brown Spot ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Corynespora Cassiicola ◽

Sterile Water ◽

Single Spore ◽

First Report ◽

Leaf Spots ◽

Leaf Yellowing

Rose of Sharon, Hibiscus syriacus L., is a flowering shrub in the family Malvaceae planted as the national flower of South Korea. In September 2012, previously unknown leaf spots with premature defoliation were observed on dozens of Rose of Sharon plants growing in the shaded area in a park of Dongducheon, Korea. The same symptoms were found on Rose of Sharon in several localities of Korea in 2012. The symptoms usually started as small, dark brown to grayish leaf spots, eventually causing leaf yellowing with significant premature defoliation. The diseased leaves retained for a while green color at the margin of the spots. Representative samples (n = 5) were deposited in the Korea University Herbarium (KUS). Conidiophores of the fungus observed microscopically on the leaf spots were erect, brown to dark brown, single or in clusters, amphigenous but mostly hypophyllous, and measured 80 to 400 × 5 to 10 μm. Conidia were borne singly or in short chains, ranging from cylindrical to broadest at the base and tapering apically, straight to slightly curved, pale olivaceous brown, 2 to 16 pseudoseptate, 50 to 260 × 9 to 20 μm, each with a conspicuous thickened hilum. On potato dextrose agar, single-spore cultures of two isolates were identified as Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei on the basis of morphological and cultural characteristics (1,2). Two monoconidial isolates were preserved at the Korean Agricultural Culture Collection (KACC46956 and KACC46957). Genomic DNA was extracted using the DNeasy Plant Mini DNA Extraction Kit (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The resulting sequences of 520 bp were deposited in GenBank (Accession Nos. KC193256, KC193257). A BLAST search in GenBank revealed that the sequences showed 100% identity with those of numerous C. cassiicola isolates from diverse substrates. To conduct a pathogenicity test, a conidial suspension (ca. 2 × 104 conidia/ml) was prepared in sterile water by harvesting conidia from 2-week-old cultures of KACC46956, and the suspension was sprayed onto the leaves of three healthy 2-year-old plants. Inoculated plants were kept in humid chambers for the first 48 h and thereafter placed in the glasshouse. After 10 days, typical leaf spot symptoms developed on the leaves of all three inoculated plants. C. cassiicola was reisolated from the lesions, confirming Koch's postulates. Control plants treated with sterile water remained symptomless. C. cassiicola is cosmopolitan with a very wide host range (1,2). Though Corynespora hibisci Goto was recorded to be associated with brown spot disease of H. syriacus in Japan (4), there is no previous record of C. cassiicola on H. syriacus (3). To our knowledge, this is the first report of Corynespora leaf spot on Rose of Sharon in Korea. According to our field observations in Korea, this disease was found in August and September, following a prolonged period of moist weather. Severe infection resulted in leaf yellowing and premature defoliation, reducing tree vigor and detracting the beauty of green leaves. References: (1) L. J. Dixon et al. Phytopathology 99:1015, 2009. (2) M. B. Ellis. Dematiaceous Hyphomycetes. Commonw. Mycol. Inst., Kew, UK, 1971. (3) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, Retrieved November 22, 2012. (4) K. Goto. Ann. Phytopathol. Soc. Japan 12:14, 1942.

Download Full-text

First Report of Alternaria tenuissima Causing Leaf Spot on Eggplant in Malaysia

Plant Disease ◽

10.1094/pdis-03-12-0237-pdn ◽

2012 ◽

Vol 96 (8) ◽

pp. 1226-1226 ◽

Cited By ~ 1

Author(s):

A. Nasehi ◽

J. B. Kadir ◽

M. A. Zainal Abidin ◽

M. Y. Wong ◽

F. Mahmodi

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Plant Diseases ◽

Fruit Rot ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

Dark Light ◽

Alternaria Tenuissima ◽

First Report

A leaf spot on eggplant (Solanum melongena) was observed in major eggplant growing regions in Malaysia, including the Cameron Highlands and Johor State, during 2011. Disease incidence averaged approximately 30% in severely infected regions in about 150 ha of eggplant fields and greenhouses examined. Early symptoms consisted of small, circular, brown, necrotic spots uniformly distributed on leaves. The spots gradually enlarged and developed concentric rings. Eventually, the spots coalesced and caused extensive leaf senescence. A fungus was recovered consistently by plating surface-sterilized (1% NaOCl) sections of symptomatic leaf tissue onto potato dextrose agar (PDA). For conidial production, the fungus was grown on potato carrot agar (PCA) and V8 agar media under a 16-h/8-h dark/light photoperiod at 25°C (4). Fungal colonies were a dark olive color with loose, cottony mycelium. Simple conidiophores were ≤120 μm long and produced numerous conidia in long chains. Conidia averaged 20.0 × 7.5 μm and contained two to five transverse septa and the occasional longitudinal septum. Twelve isolates of the fungus were identified as Alternaria tenuissima on the basis of morphological characterization (4). Confirmation of the species identification was obtained by molecular characterization of the internal transcribed spacer (ITS) region of rDNA amplified from DNA extracted from a representative isolate using universal primers ITS4 and ITS5 (2). The 558 bp DNA band amplified was sent for direct sequencing. The sequence (GenBank Accession No. JQ736021) was subjected to BLAST analysis (1) and was 99% identical to published ITS rDNA sequences of isolates of A. tenuissima (GenBank Accession Nos. DQ323692 and AY154712). Pathogenicity tests were performed by inoculating four detached leaves from 45-day-old plants of the eggplant cv. 125066x with 20 μl drops (three drops/leaf) of a conidial suspension containing 105 conidia/ml in sterile distilled water. Four control leaves were inoculated with sterile water. Leaves inoculated with the fungus and those treated with sterile water were incubated in chambers at 25°C and 95% RH with a 12-h photoperiod/day (2). Leaf spot symptoms typical of those caused by A. tenuissima developed on leaves inoculated with the fungus 7 days after inoculation, and the fungus was consistently reisolated from these leaves. The control leaves remained asymptomatic and the pathogen was not reisolated from the leaves. The pathogenicity test was repeated with similar results. To our knowledge, this is the first report of A. tenuissima causing a leaf spot on eggplant in Malaysia. A. tenuissima has been reported to cause leaf spot and fruit rot on eggplant in India (3). References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (3) P. Raja et al. New Disease Rep. 12:31, 2005. (4) E. G. Simmons. Page 1 in: Alternaria Biology, Plant Diseases and Metabolites. J. Chelchowski and A. Visconti, eds. Elsevier, Amsterdam, 1992.

Download Full-text

First Report of Ramularia coleosporii causing Leaf Spot on Perilla frutescens in Korea

Plant Disease ◽

10.1094/pdis-01-21-0223-pdn ◽

2021 ◽

Author(s):

Md Aktaruzzaman ◽

Tania Afroz ◽

Hyo-Won Choi ◽

Byung Sup Kim

Keyword(s):

Leaf Spot ◽

Ipomoea Batatas ◽

Rust Fungus ◽

Morphological Characteristics ◽

Perilla Frutescens ◽

Herbaceous Plant ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

First Report

Perilla (Perilla frutescens var. japonica), a member of the family Labiatae, is an annual herbaceous plant native to Asia. Its fresh leaves are directly consumed and its seeds are used for cooking oil. In July 2018, leaf spots symptoms were observed in an experimental field at Gangneung-Wonju National University, Gangneung, Gangwon province, Korea. Approximately 30% of the perilla plants growing in an area of about 0.1 ha were affected. Small, circular to oval, necrotic spots with yellow borders were scattered across upper leaves. Masses of white spores were observed on the leaf underside. Ten small pieces of tissue were removed from the lesion margins of the lesions, surface disinfected with NaOCl (1% v/v) for 30 s, and then rinsed three times with distilled water for 60 s. The tissue pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. Five single spore isolates were obtained and cultured on PDA. The fungus was slow-growing and produced 30-50 mm diameter, whitish colonies on PDA when incubated at 25ºC for 15 days. Conidia (n= 50) ranged from 5.5 to 21.3 × 3.5 to 5.8 μm, were catenate, in simple or branched chains, ellipsoid-ovoid, fusiform, and old conidia sometimes had 1 to 3 conspicuous hila. Conidiophores (n= 10) were 21.3 to 125.8 × 1.3 to 3.6 μm in size, unbranched, straight or flexuous, and hyaline. The morphological characteristics of five isolates were similar. Morphological characteristics were consistent with those described for Ramularia coleosporii (Braun, 1998). Two representative isolates (PLS 001 & PLS003) were deposited in the Korean Agricultural Culture Collection (KACC48670 & KACC 48671). For molecular identification, a multi-locus sequence analysis was conducted. The internal transcribed spacer (ITS) regions of the rDNA, partial actin (ACT) gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified using primer sets ITS1/4, ACT-512F/ACT-783R and gpd1/gpd2, respectively (Videira et al. 2016). Sequences obtained from each of the three loci for isolate PLS001 and PLS003 were deposited in GenBank with accession numbers MH974744, MW470869 (ITS); MW470867, MW470870 (ACT); and MW470868, MW470871 (GAPDH), respectively. Sequences for all three genes exhibited 100% identity with R. coleosporii, GenBank accession nos. GU214692 (ITS), KX287643 (ACT), and 288200 (GAPDH) for both isolates. A multi-locus phylogenetic tree, constructed by the neighbor-joining method with closely related reference sequences downloaded from the GenBank database and these two isolates demonstrated alignment with R. coleosporii. To confirm pathogenicity, 150 mL of a conidial suspension (2 × 105 spores per mL) was sprayed on five, 45 days old perilla plants. An additional five plants, to serve as controls, were sprayed with sterile water. All plants were placed in a humidity chamber (>90% relative humidity) at 25°C for 48 h after inoculation and then placed in a greenhouse at 22/28°C (night/day). After 15 days leaf spot symptoms, similar to the original symptoms, developed on the leaves of the inoculated plants, whereas the control plants remained symptomless. The pathogenicity test was repeated twice with similar results. A fungus was re-isolated from the leaf lesions on the inoculated plants which exhibited the same morphological characteristics as the original isolates, fulfilling Koch’s postulates. R. coleosporii has been reported as a hyperparasite on the rust fungus Coleosporium plumeriae in India & Thailand and also as a pathogen infecting leaves of Campanula rapunculoides in Armenia, Clematis gouriana in Taiwan, Ipomoea batatas in Puerto Rico, and Perilla frutescens var. acuta in China (Baiswar et al. 2015; Farr and Rossman 2021). To the best of our knowledge, this is the first report of R. coleosporii causing leaf spot on P. frutescens var. japonica in Korea. This disease poses a threat to production and management strategies to minimize leaf spot should be developed.

Download Full-text

First Report of Epicoccum layuense Causing Leaf Brown Spot on Camellia oleifera in Hefei, China

Plant Disease ◽

10.1094/pdis-08-21-1726-pdn ◽

2022 ◽

Author(s):

Xiang Xie ◽

Shiqiang Zhang ◽

Qingjie Yu ◽

Xinye Li ◽

Yongsheng Liu ◽

...

Keyword(s):

Leaf Spot ◽

Brown Spot ◽

Likelihood Method ◽

Leaf Spot Disease ◽

Original Strain ◽

Conidial Suspension ◽

Camellia Oleifera ◽

Sterile Water ◽

Beta Tubulin ◽

First Report

Camellia oleifera, a major tree species for producing edible oil, is originated in China. Its oil is also called ‘‘eastern olive oil’’ with high economic value due to richness in a variety of healthy fatty acids (Lin et al. 218). However, leaves are susceptible to leaf spot disease (Zhu et al. 2014). In May 2021, we found circular to irregular reddish-brown lesions, 4-11 mm in diameter, near the leaf veins or leaf edges on 30%-50% leaves of 1/3 oil tea trees in a garden of Hefei City, Anhui Province, China (East longitude 117.27, North latitude 31.86) (Figure S1 A). To isolate the causal agents, symptomatic leaves were cut from the junction of diseased and healthy tissues (5X5 mm) and treated with 70 % alcohol for 30 secs and 1 % NaClO for 5 min, and subsequently inoculated onto PDA medium for culture. After 3 days, hyphal tips were transferred to PDA. Eventually, five isolates were obtained. Then the isolates were cultured on PDA at 25°C for 7 days and the mycelia appeared yellow with a white edge and secreted a large amount of orange-red material to the PDA (Figure S1 B and C). Twenty days later, the mycelium appeared reddish-brown, and sub-circular (3-10 mm) raised white or yellow mycelium was commonly seen on the Petri dish, and black particles were occasionally seen. Meanwhile, the colonies on the PDA produced abundant conidia. Microscopy revealed that conidia were globular to pyriform, dark, verrucose, and multicellular with 14.2 to 25.3 μm (=19.34 μm, n = 30) diameter (Figure S1 D). The morphological characteristics of mycelial and conidia from these isolates are similar to that of Epicoccum layuense (Chen et al.2020). To further determine the species classification of the isolates, DNA was extracted from 7-day-old mycelia cultures and the PCR-amplified fragments were sequenced for internal transcribed spacer (ITS), beta-tubulin and 28S large subunit ribosomal RNA (LSU) gene regions ITS1/ITS4, Bt2a/Bt2b and LR0R/LR5, followed by sequencing and molecular phylogenetic analysis of the sequences analysis (White et al. 1990; Glass and Donaldson 1995; Vilgalys and Hester 1990). Sequence analysis revealed that ITS, beta-tubulin, and LSU divided these isolates into two groups. The isolates AAU-NCY1 and AAU-NCY2, representing the first group (AAU-NCY1 and AAU-NCY5) and the second group (AAU-NCY2, AAU-NCY3 and AAU-NCY4), respectively, were used for further studies. Based on BLASTn analysis, the ITS sequences of AAU-NCY1 (MZ477250) and AAU-NCY2 (MZ477251) showed 100 and 99.6% identity with E. layuense accessions MN396393 and KY742108, respectively. And, the beta-tubulin sequences (MZ552310; MZ552311) showed 99.03 and 99.35% identity with E. layuense accessions MN397247 and MN397248, respectively. Consistently, their LSU (MZ477254; MZ477255) showed 99.88 and 99.77% identity with E. layuense accessions MN328724 and MN396395, respectively. Phylogenetic trees were built by maximum likelihood method (1,000 replicates) using MEGA v.6.0 based on the concatenated sequences of ITS, beta-tubulin and LSU (Figure S2). Phylogenetic tree analysis confirmed that AAU-NCY1 and AAU-NCY2 are closely clustered with E. layuense stains (Figure S2). To test the pathogenicity, conidial suspension of AAU-NCY2 (106 spores/mL) was prepared and sterile water was used as the control. Twelve healthy leaves (six for each treatment) on C. oleifera tree were punched with sterile needle (0.8-1mm), the sterile water or spore suspension was added dropwise at the pinhole respectively (Figure S1 E and F). The experiment was repeated three times. By ten-day post inoculation, the leaves infected by the conidia gradually developed reddish-brown necrotic spots that were similar to those observed in the garden, while the control leaves remained asymptomatic (Figure S1 G and H). DNA sequences derived from the strain re-isolated from the infected leaves was identical to that of the original strain. E. layuense has been reported to cause leaf spot on C. sinensis (Chen et al. 2020), and similar pathogenic phenotypes were reported on Weigela florida (Tian et al. 2021) and Prunus x yedoensis Matsumura in Korea ( Han et al. 2021). To our knowledge, this is the first report of E. layuense causing leaf spot on C. oleifera in Hefei, China.

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 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