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

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 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.

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First Report of a Leaf Spot Disease of Bells-of-Ireland (Moluccella laevis) Caused by Cercospora apii in California

Plant Disease ◽

10.1094/pdis.2003.87.2.203a ◽

2003 ◽

Vol 87 (2) ◽

pp. 203-203

Author(s):

S. T. Koike ◽

S. A. Tjosvold ◽

J. Z. Groenewald ◽

P. W. Crous

Keyword(s):

Leaf Spot ◽

Sequence Data ◽

Disease Incidence ◽

Leaf Spot Disease ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Spot Disease ◽

Leaf Spots ◽

Initial Symptoms

Bells-of-Ireland (Moluccella laevis) (Lamiaceae) is an annual plant that is field planted in coastal California (Santa Cruz County) for commercial cutflower production. In 2001, a new leaf spot disease was found in these commercially grown cutflowers. The disease was most serious in the winter-grown crops in 2001 and 2002, with a few plantings having as much as 100% disease incidence. All other plantings that were surveyed during this time had at least 50% disease. Initial symptoms consisted of gray-green leaf spots. Spots were generally oval in shape, often delimited by the major leaf veins, and later turned tan. Lesions were apparent on both adaxial and abaxial sides of the leaves. A cercosporoid fungus having fasciculate conidiophores, which formed primarily on the abaxial leaf surface, was consistently associated with the spots. Based on morphology and its host, this fungus was initially considered to be Cercospora molucellae Bremer & Petr., which was previously reported on leaves of M. laevis in Turkey (1). However, sequence data obtained from the internal transcribed spacer region (ITS1, ITS2) and the 5.8S gene (STE-U 5110, 5111; GenBank Accession Nos. AY156918 and AY156919) indicated there were no base pair differences between the bells-of-Ireland isolates from California, our own reference isolates of C. apii, as well as GenBank sequences deposited as C. apii. Based on these data, the fungus was subsequently identified as C. apii sensu lato. Pathogenicity was confirmed by spraying a conidial suspension (1.0 × 105 conidia/ml) on leaves of potted bells-of-Ireland plants, incubating the plants in a dew chamber for 24 h, and maintaining them in a greenhouse (23 to 25°C). After 2 weeks, all inoculated plants developed leaf spots that were identical to those observed in the field. C. apii was again associated with all leaf spots. Control plants, which were treated with water, did not develop any symptoms. The test was repeated and the results were similar. To our knowledge this is the first report of C. apii as a pathogen of bells-of-Ireland in California. Reference: (1) C. Chupp. A Monograph of the Fungus Genus Cercospora. Cornell University Press, Ithaca, New York, 1954.

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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 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.

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First Report of Leaf Spot Caused by Alternaria alternata on Marigold (Tagetes erecta) in Beijing, China

Plant Disease ◽

10.1094/pdis-09-13-1005-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1153-1153 ◽

Cited By ~ 1

Author(s):

Y. Li ◽

J. Shen ◽

B. H. Pan ◽

M. X. Guo ◽

Q. X. Wang ◽

...

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Leaf Spot Disease ◽

Tagetes Erecta ◽

Pathogenicity Test ◽

First Report ◽

Leaf Spots ◽

Commercial Crop ◽

Necrotic Spots ◽

Beijing China

Marigold (Tagetes erecta) is an important commercial crop and 200 ha are planted every year in the Beijing district of China. A leaf spot disease of T. erecta was observed during 2012 and 2013 in the Beijing district. The disease was widespread, with 60 to 75% of the fields affected. Leaves of the affected plants had small, brown, necrotic spots on most of the foliage. Yield losses of flowers of up to 20 to 30% were reported. The spots gradually enlarged, becoming irregular in shape, or remained circular, and with concentric rings or zones. In the later stages of infection, the spots coalesced, and the leaves withered, dried, and fell from the plants (4). A fungus was consistently isolated on potato dextrose agar (PDA) from the infected leaves of T. erecta. After 6 days of incubation at 26°C and a 12-h photoperiod, the fungus produced colonies that were flat, with a rough upper surface (2). The conidiophores were short. Conidia varied from 18 × 6 to 47 × 15 μm and were medium to dark brown or olive-brown in color, short beaked, borne in long chains, oval and bean shaped, with 1 to 5 transverse septa and 0 to 2 longitudinal septa. The rDNA of the internal transcribed spacer regions 1 and 2 and the 5.8S gene in seven isolates were amplified using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The nucleotide sequence was the same as isolate No. 7, which was deposited in GenBank (Accession No. KF307207). A BLAST search showed 97% identity with the strain Alternaria alternata GNU-F10 (KC752593). Seven isolates were also confirmed as A. alternata by PCR identification performed by specific primers (C_for/C_rev) of A. alternata (1). Seven isolates were grown on PDA for 2 weeks and the conidia harvested. A 5-μl drop of spore suspension (1 × 105 spores/ml) was placed on each leaflet of 140 detached, surface-sterilized T. erecta leaves. Twenty leaves were inoculated with sterile distilled water as a control. The leaves were incubated in a growth chamber at 80 to 90% relative humidity, 50 to 60 klx/m2 light intensity, and a 12-h photoperiod. After 6 days, leaf spots similar to the original developed at inoculation sites for all isolates and A. alternata was consistently re-isolated. The control leaves remained symptomless. The pathogenicity test was performed three times. Leaf spot of T. erecta caused by Alternaria spp. is well known in Asian countries such as Japan (3). To our knowledge, this is the first report of A. alternata on T. erecta in the Beijing district of China. References: (1) T. Gat. Plant Dis. 96:1513, 2012. (2) E. Mirkova. J. Phytopathol. 151:323, 2003. (3) K. Tomioka. J. Gen. Plant Pathol. 66:294, 2000. (4) T. Y. Zhang. Page 284 in: Flora Fungorum Sinicorum, Volume 16: Alternaria. Science Press, Beijing, 2003.

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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 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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First report of Bipolaris yamadae leaf spot disease on Guinea grass (Panicum maximum) in Florida.

Plant Disease ◽

10.1094/pdis-07-20-1486-pdn ◽

2020 ◽

Author(s):

Ashish Adhikari ◽

Xuechun Wang ◽

Brett Lane ◽

Philip F Harmon ◽

Erica Goss

Keyword(s):

Leaf Spot ◽

Phylogenetic Trees ◽

Leaf Spot Disease ◽

Panicum Maximum ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Leaf Spots ◽

Guinea Grass ◽

Symptomatic Leaf

Guinea grass is an invasive perennial C4 grass and is a common weed around agricultural crops in Louisiana, Texas, and Hawaii, USA (Overholt and Franck 2019). In November 2018, leaf spots were observed on Guinea grass occurring in an organic garden located in Gainesville, Florida, USA. Lesions were oblong to irregular, dark grey to brownish center with pale-yellow to brownish black margin. Lesions had coalesced, forming necrotic margins that spread from the leaf tip, resulting in leaf blight and collapse of the canopy. Pieces of symptomatic leaf blades (5 sq cm) were surface sterilized (1 min), washed with sterile distilled water and plated onto water agar media plates. Plates were incubated at 27°C under 12-h light/dark for 3 to 5 days. Grey to black cottony mycelium was consistent on all plates and produced conidia characteristic of Bipolaris spp. Conidia were transferred to potato dextrose agar (PDA) plates with a 0.5 mm diameter sterile needle. Three isolates GG1, GG2 and GG3 were successfully grown on PDA. Conidia were black to brown colored, distoseptate with 3 to 8 septa and measured from (60.6- )70-105(-139.8) × (16.0-)17-23(-25.9) μm (avg: 93.3 μm, n=35, SD = 20.6; avg = 21.3 μm, n = 35, SD = 2.89). Conidiophores were in groups or single, brown, smooth and straight, septate and swollen at upper tip. Sigma Extract-N-Amp was used for genomic DNA extraction. Primers ITS1/ITS4 and GPD1/GPD2 (Berbee et al. 1999) were used to amplify and sequence the internal transcribed spacer region (ITS) and partial glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene, respectively. Sequences were aligned using MUSCLE and alignment was trimmed for length. Maximum likelihood phylogenetic trees were constructed with 1,000 bootstrap samples based on the K2+G substitution model, selected by BIC for these two loci using Mega X (Kumar et al. 2018). The ITS and GPDH sequences of GG1, GG2 and GG3 (Genbank accessions MT514518-20, MT576654-56), grouped with B. yamadae isolates CPC_28807 and CBS_202.29 in phylogenetic trees (Marin-Felix et al. 2017). All three isolates from Guinea grass were inoculated on Sach’s agar (Luttrell 1958) at 27°C under 12-h light/dark for a week, but no sexual morph was observed, and consistent for two repeated inoculations. To fulfill Koch’s postulates, one isolate, GG1, was used. Conidia were harvested from a one-week-old colony grown on PDA incubated at 27°C and 12-h light/dark cycle. The concentration of the conidial suspension was adjusted to 105 conidia/ml using a hemocytometer. Using a Passche H-202S airbrush sprayer, five-week-old seedlings of Guinea grass were sprayed until runoff with the conidia suspension or 0.5% tween water only. Each treatment included four replicates and the experiment was repeated. Leaf spot symptoms were observed on the seedlings inoculated with conidia, whereas seedlings sprayed with water were asymptomatic. Cultures with the expected morphology were isolated from symptomatic leaf blades and absent from control plants. To our knowledge, this is the first report of leaf spot on Guinea grass caused by B. yamadae in Florida, USA. B. yamadae was previously reported from Guinea grass in India, and from other Panicum species in the northern USA (Farr and Rossman 2019). B. yamadae was also isolated from sugarcane in Cuba and China, and corn in Japan (Manamgoda et al. 2014, Raza et al. 2019), which suggests that it has the potential to impact agronomic crops in Florida, such as sugarcane and corn.

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