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

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.

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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 disease caused by Corynespora cassiicola on American sweetgum (Liquidambar styraciflua L.) in China

Plant Disease ◽

10.1094/pdis-08-20-1842-pdn ◽

2021 ◽

Author(s):

Yun-fei Mao ◽

Xiang-rong Zheng ◽

Fengmao Chen

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Liquidambar Styraciflua ◽

Bootstrap Support ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Corynespora Cassiicola ◽

First Report ◽

Leaf Spots

American sweetgum (Liquidambar styraciflua L.) is a forest plant native to North America, which has been introduced into other countries due to its ornamental and medicinal values. In June 2019, symptoms of leaf spots on sweetgum were observed in a field (5 ha) located in Xuzhou, Jiangsu Province, China. On this field, approximately 45% of 1,000 trees showed the same symptoms. Symptoms were observed showing irregular or circular dark brown necrotic lesions approximately 5 to 15 mm in diameter with a yellowish margin on the leaves. To isolate the pathogen, diseased leaf sections (4×4mm) were excised from the margin of the lesion, surface-sterilized with 0.1% NaOCl for 90 s, rinsed 4 times in sterile distilled water, air dried and then transferred on potato dextrose agar (PDA) medium at 25°C in the dark. Pure cultures were obtained by monospore isolation after subculture. Ten purified isolates, named FXI to FXR, were transferred to fresh PDA and incubated as above to allow for morphological and molecular identification. After 7 days, the aerial mycelium was abundant, fluffy and exhibited white to greyish-green coloration. The conidia were dark brown or olive, solitary or produced in chains, obclavate, with 1 to 15 pseudosepta, and measured 45 to 200µm  10 to 18µm. Based on morphological features, these 10 isolates were identified as Corynespora cassiicola (Ellis et al. 1971). Genomic DNA of each isolate was extracted from mycelia using the cetyltrimethylammonium bromide (CTAB) method. The EF-1α gene and ITS region were amplified and sequenced with the primer pairs rDNA ITS primers (ITS4/ITS5) (White et al. 1990) and EF1-728F/EF-986R (Carbone et al.1999) respectively. The sequences were deposited in GenBank. BLAST analysis revealed that the ITS sequence had 99.66% similarity to C. cassiicola MH255527 and that the EF-1α sequence had 100% similarity to C. cassiicola KX429668A. maximum likelihood phylogenetic analysis based on EF-1α and ITS sequences using MEGA 7 revealed that ten isolates were placed in the same clade as C. cassiicola (Isolate: XQ3-1; accession numbers: MH572687 and MH569606, respectively) at 98% bootstrap support. Based on the morphological characteristics and phylogenetic analyses, all isolates were identified as C. cassiicola. For the pathogenicity test, a 10 µl conidial suspension (1×105 spores/ml) of each isolate was dripped onto healthy leaves of 2-year-old sweetgum potted seedlings respectively. Leaves inoculated with sterile water served as controls. Three plants (3 leaves per plant) were conducted for each treatment. The experiment was repeat twice. All seedlings were enclosed in plastic transparent incubators to maintain high relative humidity (90% to 100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. After 10 days, leaves inoculated with conidial suspension of each isolate showed symptoms of leaf spots, similar to those observed in the field. Control plants were remained healthy. In order to reisolate the pathogen, surface-sterilized and monosporic isolation was conducted as described above. The same fungus was reisolated from the lesions of symptomatic leaves, and its identity was confirmed by molecular and morphological approaches, thus fulfilling Koch’s postulates. Chlorothalonil and Boscalid can be used to effectively control Corynespora leaf spot (Chairin T et al.2017). To our knowledge, this is the first report of leaf spot caused by C. cassiicola on L. styraciflua in China.

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First Report of Corynespora Leaf Spot on Beach Vitex Caused by Corynespora cassiicola in Korea

Plant Disease ◽

10.1094/pdis-05-13-0480-pdn ◽

2013 ◽

Vol 97 (11) ◽

pp. 1512-1512 ◽

Cited By ~ 3

Author(s):

J. H. Park ◽

M. J. Park ◽

S. H. Lee ◽

C. K. Lee ◽

H. D. Shin

Keyword(s):

Invasive Species ◽

Leaf Spot ◽

Native Species ◽

Invasive Plant ◽

Sand Dunes ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Corynespora Cassiicola ◽

Single Spore ◽

Leaf Spots

Beach vitex, Vitex rotundifolia L. fil., is a perennial that grows in temperate and tropical areas of the Pacific. In areas where it has been introduced outside of its native range, beach vitex has proven to be an invasive species. This plant dominates dune ecosystems leading to a reduction in the prevalence of native species (1). In October 2010, previously unknown leaf spots were observed on the beach vitex growing on sand dunes in Incheon City of Korea. The same symptoms were repeated in 2011 and 2012. In September 2012, the same leaf spots were found on the beach vitex in Samcheok and Gyeongju in Korea. The symptoms usually started as small, dark brown to purplish leaf spots with more or less concentric rings, eventually causing leaf blights or yellowing with 50% or more defoliation by the end of September. Representative samples (n = 6) 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 occasionally in clusters, 80 to 500 × 5 to 9 μm, and mostly arose on the abaxial surface of symptomatic leaves. Conidia were borne singly or in short chains of 2 to 4, ranging from cylindrical to broadest at the base and tapering apically, straight to slightly curved, pale olivaceous brown, 1 to 12 pseudoseptate, 50 to 250 × 8 to 18 μm, each with a conspicuous thickened hilum. On potato dextrose agar (PDA), 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 (3). Two monoconidial isolates were preserved at the Korean Agricultural Culture Collection (Accession Nos. KACC45712 and KACC46953). Isolate KACC45712 was used for molecular works and pathogenicity test. 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 sequence of 520 bp was deposited in GenBank (Accession No. KC987359). A BLAST search in GenBank revealed that the sequence showed 100% identity with those of C. cassiicola (e.g., JQ801302). To conduct a pathogenicity test, a conidial suspension (ca. 2 × 104 conidia/ml) was prepared by harvesting conidia from 2-week-old cultures, and the suspension was sprayed onto the leaves of three healthy seedlings. Inoculated plants were kept in humid chambers for 48 h in a glasshouse. After 5 days, typical leaf spot symptoms started to develop 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 (2,4). To our knowledge, C. cassiicola has not been reported on Vitex spp. anywhere in the world. According to field observations in Korea, Corynespora leaf spot was most severe in August and September, especially following a prolonged period of moist weather. C. cassiicola may be a potential biocontrol agent for this highly invasive species. References: (1) M. C. Cousins et al. Invasive Plant Sci. Manag. 3:340, 2010. (2) L. J. Dixon et al. Phytopathology 99:1015, 2009. (3) M. B. Ellis. Dematiaceous Hyphomycetes. Commonw. Mycol. Inst.: Kew, UK, 1971. (4) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, Retrieved April 30, 2013.

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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.

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

Plant Disease ◽

10.1094/pdis-08-20-1773-pdn ◽

2020 ◽

Author(s):

Quan Shen ◽

Xixu Peng ◽

Feng He ◽

Shaoqing Li ◽

Zuyin Xiao ◽

...

Keyword(s):

Relative Humidity ◽

Leaf Spot ◽

Plant Pathogens ◽

Hunan Province ◽

Tartary Buckwheat ◽

Tween 20 ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

First Report

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.

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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.

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First Report of Curvularia gladioli Causing a Leaf Spot on Gladiolus grandiflorus in Brazil

Plant Disease ◽

10.1094/pdis-12-12-1118-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 847-847 ◽

Cited By ~ 5

Author(s):

D. P. Torres ◽

M. A. Silva ◽

D. B. Pinho ◽

O. L. Pereira ◽

G. Q. Furtado

Keyword(s):

Leaf Spot ◽

Central Cell ◽

Post Inoculation ◽

Intermediate Cell ◽

Conidial Suspension ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

First Report ◽

The Third ◽

Leaf Spots

Gladiolus (Iridaceae) is a popular bulbous plant grown worldwide as an ornamental garden plant or cut flower due to its attractive color, size, and flower shape. In April 2012, leaf spots were observed on plants of Gladiolus grandiflorus varieties T-704 and Amsterdam growing in a production area of cut flowers located in the city of Viçosa, Minas Gerais. The oval to round leaf spots were brown with a dark border surrounded by a halo of yellow tissue. Infected leaf samples were deposited in the herbarium at the Universidade Federal de Viçosa (VIC31897). A fungus was isolated from the leaf spots and a single-spore pure culture was initiated and grown on corn meal carrot agar (CCA) medium in petri dishes incubated at 25°C under a 12-h photoperiod for 4 weeks. A sporulating single-spore culture was deposited at the Coleção de Culturas de fungos fitopatogênicos “Prof. Maria Menezes” (UFRPE, Brazil) code CMM 4055. On CCA medium, the fungal isolate initially appeared white, becoming dark after 14 days. Thirty conidia and conidiophores were measured for identification to species. The septate, smooth to pale brown conidiophores were present singly or in groups. The simple, straight or flexuous conidiophores were 42.5 to 82.5 × 3.5 to 7.5 μm and some had a geniculate growth pattern. The majority of conidia were curved at the third (central) cell from the base, which was usually enlarged compared to the end cells. The cells at each end of the 3-distoseptate conidia were pale brown, the intermediate cell brown or dark brown, and the third (central) cell was often the darkest. The basal cell had a protuberant hilum. Conidia were smooth and 20.0 to 33.5 × 10 to 17.5 μm. These characteristics matched well with the description of Curvularia gladioli (1). To confirm this identification, DNA was extracted using a Wizard Genomic DNA Purification Kit and the internal transcribed spacer region (ITS) of rDNA was amplified using ITS1 and ITS4 primers and the partial 28S rDNA region using primers LR0R and LR5. The sequences were deposited in GenBank as accession nos. JX995106 and JX995107, respectively. The ITS sequence matched sequence AF071337, C. gladioli, with 100% identity. This pathogen was first identified as C. lunata, but based on the characteristic of the hilum, spore size, and pathogenicity testing, the fungus was renamed C. trifolii f. sp. gladioli (3). Due to the explicit curvature of the conidia at the third cell and molecular data, the fungus was reclassified as C. gladioli (1,2). To confirm Koch's postulates, 1-month-old healthy plants of G. grandiflorus var. T-704 and Amsterdam (five plants each) were inoculated with a conidial suspension (2 × 104 conidia mL–1) by spraying the foliage and then placed on a growth chamber at 25°C. The control plants were sprayed with distilled water. Symptoms were consistent with those initially observed and all plants developed leaf spots by 4 days post-inoculation. C. gladioli was consistently recovered from the symptomatic tissue and control plants remained symptomless. To our knowledge, this is the first report of C. gladioli causing leaf spot on G. grandiflorus in Brazil. Due to a lack of chemical fungicides for management of this pathogen, further studies to evaluate the susceptibility of the main varieties of gladiolus grown in Brazil to C. gladioli may be necessary. References: (1) G. H. Boerema and M. E. C. Hamers. Neth. J. Plant Pathol. 95:1, 1989. (2) D. S. Manamgoda et al. Fungal Divers. 56:131, 2012. (3) J. A. Parmelee. Mycologia 48:558, 1956.

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First Report of Corynespora Leaf Spot on Ailanthus altissima Caused by Corynespora cassiicola in Korea

Plant Disease ◽

10.1094/pdis-11-11-0938 ◽

2012 ◽

Vol 96 (4) ◽

pp. 586-586 ◽

Cited By ~ 1

Author(s):

J. H. Park ◽

M. J. Park ◽

S. H. Lee ◽

H. D. Shin

Keyword(s):

Leaf Spot ◽

Central China ◽

Ailanthus Altissima ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Commonwealth Mycological Institute ◽

Conidial Suspension ◽

Corynespora Cassiicola ◽

Leaf Spots ◽

Adult Trees

Ailanthus altissima (Mill.) Swingle, known as tree-of-heaven, is a deciduous tree belonging to the family Simaroubaceae, which is native to both northeast and central China and Taiwan. The trees often have the ability to replace indigenous plants and disrupt native ecosystems (3). In August 2010, a leaf spot disease was observed on young trees in Yangpyeong, Korea. Field observation in 2010 and 2011 showed that infections are common on 1- or 2-year-old trees. Adult trees were rarely infected. Symptoms usually started at the margin of leaves and expanded into irregular, dark brown leaf spots, eventually causing significant premature defoliation. Representative samples were deposited in the herbarium of Korea University (KUS-F25174 and -F25304). Conidiophores of fungi observed microscopically on the leaf spots were erect, brown to dark brown, single or occasionally in clusters, 80 to 550 × 5 to 8 μm, and mostly arose on the abaxial surface of symptomatic leaves. Conidia were borne singly or in short chains of two to four, ranging from cylindrical to broadest at the base and tapering apically, straight to slightly curved, pale olivaceous brown, 3 to 18 pseudoseptate, 70 to 450 × 8 to 22 μm, each with a conspicuous thickened hilum. On potato dextrose agar, single-spore cultures of five isolates were identified as Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei on the basis of morphological and cultural characteristics (1,4). A monoconidial isolate was preserved at the Korean Agricultural Culture Collection (Accession No. KACC45510). Genomic DNA was extracted with 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 with an ABI Prism 337 automatic DNA sequencer (Applied Biosystems, Foster, CA). The resulting sequence of 548 bp was deposited in GenBank (Accession No. JN974462). The sequence showed >99% similarity (1-bp substitution) with a sequence of C. cassiicola from Ipomoea batatas (GenBank Accession No. FJ852716). To conduct a pathogenicity test, a conidial suspension (~2 × 104 conidia/ml) was prepared by harvesting conidia from 2-week-old cultures of KACC45510 and the suspension sprayed onto the leaves of three healthy seedlings. Three noninoculated seedlings served as control plants. Inoculated and noninoculated plants were kept in humid chambers for 48 h in a glasshouse. After 5 days, typical leaf spot symptoms started to develop on the leaves of all three inoculated plants. C. cassiicola was reisolated from the lesions, confirming Koch's postulates. No symptoms were observed on control plants. C. cassiicola is cosmopolitan with a very wide host range (2). To our knowledge, C. cassiicola has not been reported on A. altissima anywhere in the world. According to field observations in Korea, Corynespora leaf spot was most severe in August and September, especially following a prolonged period of moist weather. C. cassiicola may be a potential biocontrol agent for this highly invasive tree species. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute: Kew, Surrey, England, 1971. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA, Retrieved from http://nt.ars-grin.gov/fungaldatabes/ , October 28, 2011. (3) L. B. Knapp and C. D. Canham. J. Torrey Bot. Soc. 127:307, 2000. (4) J. H. Kwon et al. Plant Pathol. J. 17:180, 2001.

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First Report of Corynespora cassiicola Causing Leaf Spot of Cassava in China

Plant Disease ◽

10.1094/pdis-94-7-0916a ◽

2010 ◽

Vol 94 (7) ◽

pp. 916-916 ◽

Cited By ~ 1

Author(s):

X.-B. Liu ◽

T. Shi ◽

C.-P. Li ◽

J.-M. Cai ◽

G.-X. Huang

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Its Region ◽

Pathogenic Fungi ◽

Commonwealth Mycological Institute ◽

Corynespora Cassiicola ◽

Single Spore ◽

First Report ◽

Leaf Spots ◽

Main Vein

Cassava (Manihot esculenta) is an important economic crop in the tropical area of China. During a survey of diseases in July and September of 2009, leaf spots were observed on cassava plants at three separate plantations in Guangxi (Yunfu and Wuming) and Hainan (Baisha) provinces. Circular or irregular-shaped leaf spots were present on more than one-third of the plants. Spots were dark brown or had white papery centers delimited by dark brown rims and surrounded by a yellow halo. Usually, the main vein or small veinlets adjacent to the spots were dark. Some defoliation of plants was evident at the Wuming location. A fungus was isolated from symptomatic leaves from each of the three locations and designated CCCGX01, CCCGX02, and CCCHN01. Single-spore cultures of these isolates were incubated on potato dextrose agar (PDA) for 7 days with a 12-h light/dark cycle at a temperature of 28 ± 1°C. Conidiophores were straight to slightly curved, unbranched, and pale to light brown. Conidia were formed singly or in chains, obclavate to cylindrical, straight or curved, subhyaline-to-pale olivaceous brown, 19.6 to 150.3 μm long and 5.5 to 10.7 μm wide at the base, with 4 to 13 pseudosepta. Morphological characteristics of the specimen and their conidia were similar to the descriptions for Corynespora cassiicola (2). The isolate CCCGX01 was selected as a representative for molecular identification. Genomic DNA was extracted by the cetyltrimethylammoniumbromide protocol (3) from mycelia and used as a template for amplification of the internal transcribed spacer (ITS) region of rDNA with primer pair ITS1/ITS4. The sequence (GenBank Accession No. GU138988) exactly matched several sequences (e.g., GenBank Accession Nos. FJ852715, EF198117, and AY238606) of C. cassiicola (1). Young, healthy, and fully expanded green leaves of cassava cv. SC205 were surface sterilized. Ten leaves were inoculated with 10-μl drops of 104 ml suspension of conidia and five leaves were inoculated with the same volume of sterile water to serve as controls. After inoculation, leaves were placed in a dew and dark chamber for 36 h at 25°C and subsequently transferred to the light for 5 days. All inoculated leaves with isolates showed symptoms similar to those observed in natural conditions, whereas the controls remained symptom free. The morphological characteristics of reisolated conidia that formed on the diseased parts were identical with the nature isolates. To our knowledge, this is the first report of leaf spot caused by C. cassiicola on cassava in China. References: (1) L. J. Dixon et al. Phytopathology 99:1015, 2009. (2) M. B. Ellis et al. Corynespora cassiicola. No. 303 in: CMI Description of Pathogenic Fungi and Bacteria. Commonwealth Mycological Institute, Kew, UK 1971. (3) J. R. Xu et al. Genetics 143:175, 1996.

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First Report of Corynespora Leaf Spot of Eucalyptus in China

Plant Disease ◽

10.1094/pdis-07-14-0697-pdn ◽

2015 ◽

Vol 99 (3) ◽

pp. 419-419 ◽

Cited By ~ 1

Author(s):

C. K. Phan ◽

J. G. Wei ◽

F. Liu ◽

B. S. Chen ◽

J. T. Luo ◽

...

Keyword(s):

Leaf Spot ◽

Southern China ◽

Tap Water ◽

Pathogenic Fungi ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Commonwealth Mycological Institute ◽

Conidial Suspension ◽

Single Spore ◽

First Report

Eucalyptus is widely planted in the tropics and subtropics, and it has become an important cash crop in Southern China because of its fast-growing nature. In the Guangxi Province of southern China, Eucalyptus is produced on approximately 2 million ha, and two dominant asexual clones, Guanglin No. 9 (E. grandis × E. urophylla) and DH3229 (E. urophylla × E. grandis), are grown. Diseases are an increasing threat to Eucalyptus production in Guangxi since vast areas are monocultured with this plant. In June 2013, a leaf spot disease was observed in eight out of 14 regions in the province on a total of approximately 0.08 million ha of Eucalyptus. Initially, the lesions appeared as water-soaked dots on leaves, which then became circular or irregular shaped with central gray-brown necrotic lesions and dark red-brown margins. The size of leaf spots ranged between 1 and 3 mm in diameter. The main vein or small veins adjacent to the spots were dark. The lesions expanded rapidly during rainy days, producing reproductive structures. In severe cases, the spots coalesced and formed large irregular necrotic areas followed by defoliation. The causal fungus was isolated from diseased leaves. Briefly, the affected leaves were washed with running tap water, sterilized with 75% ethanol (30 s) and 0.1% mercuric dichloride (3 min), and then rinsed three times with sterilized water. Small segments (0.5 to 0.6 cm2) were cut from the leading edge of the lesions and plated on PDA. The plates were incubated at 25°C for 7 to 10 days. When mycelial growth and spores were observed, a single-spore culture was placed on PDA and grown in the dark at 25°C for 10 days. A pathogenicity test was done by spraying a conidial suspension (5 × 105 conidia ml–1) of isolated fungus onto 30 3-month-old leaves of Guanglin No. 9 seedlings. The plants were covered with plain plastic sheets for 7 days to keep the humidity high. Lesions similar to those observed in the forests were observed on the inoculated leaves 7 to 10 days after incubation. The same fungus was re-isolated. Leaves of control plants (sprayed with sterilized water) were disease free. Conidiophores of the fungus were straight to slightly curved, erect, unbranched, septate, and pale to light brown. Conidia were formed in chains or singly with 4 to 15 pseudosepta, which were oblong oval to cylindrical, subhyaline to pale olivaceous brown, straight to curved, 14.5 to 92.3 μm long, and 3.5 to 7.1 μm wide. The fungus was morphologically identified as Corynespora cassiicola (1). DNA of the isolate was extracted, and the internal transcribed spacer (ITS) region (which included ITS 1, 5.8S rDNA gene of rDNA, and ITS 2) was amplified with primers ITS5 and ITS4. 529 base pair (bp) of PCR product was obtained and sequenced. The sequence was compared by BLAST search to the GenBank database and showed 99% similarity to C. cassiicola (Accession No. JX087447). Our sequence was deposited into GenBank (KF669890). The biological characters of the fungus were tested. Its minimum and maximum growth temperatures on PDA were 7 and 37°C with an optimum range of 25 to 30°C. At 25°C in 100% humidity, 90% of conidia germinated after 20 h. The optimum pH for germination was 5 to 8, and the lethal temperature of conidia was 55°C. C. cassiicola has been reported causing leaf blight on Eucalyptus in India and Brazil (2,3) and causing leaf spot on Akebia trifoliate in Guangxi (4). This is the first report of this disease on Eucalyptus in China. References: (1) M. B. Ellis and P. Holliday. CMI Descriptions of Pathogenic Fungi and Bacteria, No. 303. Commonwealth Mycological Institute, Kew, Surrey, UK, 1971. (2) B. P. Reis, et al. New Dis. Rep. 29:7, 2014. (3) K. I. Wilson and L. R. Devi. Ind. Phytopathol. 19:393, 1966. (4) Y. F. Ye et al. Plant Dis. 97:1659, 2013.

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