First Report of Colletotrichum siamense Causing Leaf Spot on Alocasia macrorrhiza in China

Alocasia macrorrhiza (L.) Schott, known as Alocasia is found in the Araceae, and is widely planted in southern China for its ornamental and medicinal value. This plant has a wide range of pharmacological effects, and has potential anti-tumor activity (Lei et al. 2013). In July of 2019, leaf spots were observed on A. macrorrhiza in the Xixiangtang Area, Nanning, Guangxi, China. Disease symptoms began with water-soaked yellow-green spots and progressed to form brown, round or oval lesions with yellow halos. Under severe conditions, spots merged into larger irregular lesions. More than 60% of the plants in a 0.5 ha field showed disease symptoms. Symptomatic leaves were collected and cut into small pieces (3×3 mm). Leaf pieces from the margin of the necrotic tissue were surface sterilized in 75% alcohol for 10 s, followed by 2% sodium hypochlorite solution for 2 min, then rinsed three times in sterile distilled water. Tissues were plated on potato dextrose agar (PDA) and incubated at 28°C for 5 days in the dark. Among over 30 isolates, most shared a similar morphology, the isolation rate of these was 86.7% and three of these (GY1-1A, GY1-1B, and GY1-1C) were chosen for single-spore purification and used for fungal morphological characterization and identification. White feathery aerial mycelia with olivaceous gray mycelia below were observed in 7-day cultures. After 14 days, orange conidia were observed. Conidia were hyaline, guttulate, smooth, one-celled, and cylindrical, averaged 13.79 μm × 5.26 μm, 13.89 μm × 5.33 μm and 13.92 μm × 5.42 μm for GY1-1A, GY1-1B and GY1-1C, respectively. Appressoria were mostly irregular in outline, deeply lobed or lightly lobed, gray brown to dark brown, conidial appressoria were 7.93 to 8.74 μm × 5.26 to 5.42 μm, mycelial appressoria were 7.15 to 10.11 μm × 5.60 to 7.44 μm. These morphological characteristics were similar to the C. siamense as previously described (Weir et al. 2012). The partial internal transcribed spacer (ITS) regions, actin (ACT), chitin synthase (CHS-1), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), β-tubulin (TUB2), and the intergenic region of apn2 and MAT1-2-1 (ApMAT) were amplified from genomic DNA for the three isolates using primers ITS4/ITS1 (White et al. 1990), ACT-512F/ACT-783R, CHS-79F/CHS-354R, GDF1/GDR1, CL1C/CL2C, Bt2a/Bt2b (Weir et al. 2012), and AM-F/AM-R (Silva et al. 2012) and sequenced. All sequences showed over 99% identity with C. siamense and were deposited in GenBank (ITS, MW040179-MW040181; ACT, MW049220-MW049222; CHS-1, MW049229-MW049231; GAPDH, MW049232-MW049234; CAL, MW049226-MW049228; TUB, MW049235-MW049237; ApMAT, MW049223-MW049225). Maximum Likelihood (ML) phylogenetic tree was constructed with MEGA 5 using the concatenation of multiple sequences (ACT, CHS-1, GAPDH, ITS, TUB2, CAL). According to the phylogenetic tree, all three isolates were found with C. siamense with 95% bootstrap support. To confirm pathogenicity, three sets (three plants per set) of healthy leaves were slightly scratched with autoclaved toothpicks at each of eight locations. Each inoculation location was a cross (2 mm length) and inoculation location was at least 3 cm apart. Ten μl of conidial suspension (106 conidia /ml in 0.1% sterile Tween 20) was applied to the inoculation areas. A control group was mock inoculated with 0.1% sterile Tween 20. Plants were covered with plastic bags to maintain a high humidity environment and placed in a 28°C growth chamber with constant light for 7 days. Inoculated leaves showed yellowish brown spots (0.4 × 0.65 cm), but no symptoms were observed in the control group. The fungus was reisolated from inoculated leaves, and these isolates matched the molecular and morphological characteristics of the original isolates confirming Koch’s postulates. Reported hosts of this pathogen include Coffea arabica, Carica papaya, Melilotus indicus and Litchi chinensis (Weir et al. 2012; Qin et al. 2017; Ling et al. 2019) and so on. To our knowledge, this is the first report of C. siamense causing leaf spot on A. macrorrhiza in China. The identification of this pathogen provides a foundation for the management of leaf spot on this medicinal plant.

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First Report of Alternaria alternata Causing Leaf Spot on Chili (Capsicum annuum L.) in Pakistan

Plant Disease ◽

10.1094/pdis-12-20-2706-pdn ◽

2021 ◽

Author(s):

Luqman Amrao ◽

Muhammad Zeshan Ahmed ◽

Saba Saeed ◽

Rashida Atiq ◽

Muhammad Subhan Shafique ◽

...

Keyword(s):

Capsicum Annuum ◽

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Its Rdna ◽

Tween 20 ◽

Conidial Suspension ◽

First Report ◽

Capsicum Annuum L ◽

Gapdh Gene

Chili (Capsicum annuum L.) is an important vegetable crop in Pakistan. During summer of 2019, chili leaf spot symptoms were observed on 3-month-old plants in the fields, with 30 to 40% of disease incidence, in District Faisalabad, Punjab, Pakistan. Diseased leaves were characterized by numerous tiny round spots (0.5 to 2.0 mm in diameter, average 1 mm) that were white to grey with a sunken center, surrounded with dark brown edge and chlorotic halo. The lesions gradually enlarged and coalesced into large, nearly circular, or irregularly shaped lesions that could be as long as 3 cm. Small pieces of symptomatic leaf tissues were surface sterilized in 1% sodium hypochlorite for 1 min, rinsed in sterile water, and plated on potato dextrose agar (PDA) amended with streptomycin (100 ppm). After 5 days at 25°C with a 12-hour photoperiod, same fungal colonies developed. The colonies initially appeared white and then turned olive-green. The conidiophores were brown septate and generally branched. Conidia borne singly or in short chains were multicellular, obclavate to obpyriform, and 16.2 to 38.5 µm (average 27.35 ± 2.1 µm) in length and 8 to 16.5 µm (average 12.25 ± 1.6 µm) in width, with zero to three longitudinal and two to five transverse septa (n=35). The fungus was identified as Alternaria sp. (Fr.) Keisel based on its morphological characteristics (Simmons et al. 2007). For molecular identification, genomic DNA of two representative isolates (SSUAF1 and SSUAF2) was extracted using DNAzol reagent and PCR amplification of the internal transcribed spacer (ITS)-rDNA region, Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) gene and RNA polymerase II second largest subunit (rpb2) were performed with primers ITS1/ITS4 (White et al. 1990), gpd1 and gpd2 (Berbee et al. 1999), RPB2-5F/RPB2-7cR (Liu, et al. 1999), respectively. The obtained sequences were deposited in GenBank with acc. nos. MT249008.1 and MT249009.1 for ITS-rDNA; MT318220.1 and MT318221.1 for the GAPDH; and MT318236.1, and MT318237.1 for RPB2 gene. A BLAST search in GenBank showed 100% identity with A. alternata for both ITS region (MT279999.1), GAPDH gene (MK637438.1) and RBP2 gene (MK605900.1). To confirm pathogenicity, 2-month-old healthy potted C. annuum plants were inoculated using an atomizer in a greenhouse. A total of 12 plants at the true leaf stage in each experiment were sprayed with a conidial suspension (106 conidia/ml) of both isolates amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant). Four plants were inoculated with each of the two isolates, whereas four control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse. After 10 days of inoculation, each isolate induced leaf lesions that were similar to typical lesions observed in the field. The experiment was conducted twice with similar results. The fungus was readily reisolated from symptomatic tissues whereas the control plants remained symptomless. Re-isolated fungal cultures were morphologically and molecularly identical to A. alternata, thus fulfilling the Koch’s postulates. Previously, A. alternata has been reported in Italy and India (Devappa et al. 2016; Garibaldi et al. 2019). To our knowledge, this is the first report of A. alternata causing leaf spot of C. annuum in Pakistan. This report will help the identification of leaf spot of chili and the development of management strategies for control of this disease in Pakistan.

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First Report of Anthracnose on Rubus rosaefolius Smith Caused by Colletotrichum boninense in China

Plant Disease ◽

10.1094/pdis-06-20-1309-pdn ◽

2021 ◽

Author(s):

Xiao Zheng ◽

Li-tao Tan ◽

Sheng Cheng ◽

Pengyu Liang ◽

Lan Fang ◽

...

Keyword(s):

Leaf Spot ◽

Management Strategies ◽

Morphological Characteristics ◽

Its Region ◽

Essential Amino Acids ◽

Leaf Spot Disease ◽

Control Group ◽

Conidial Suspension ◽

First Report ◽

Vegetable Juice

Raspberry (Rubus rosaefolius Smith), also called march bubble or milk bubble, is widely distributed and economically important in China. Raspberries are rich in nutrients such as essential amino acids, vitamin C, dietary fiber, superoxide dismutase (SOD) and minerals (Yang et al. 2019). In May 2019, a leaf spot disease was observed on raspberry in Enshi (N29°07'10', E108°23'12'), Hubei province of China. The symptoms were small dark-brown spots (Fig.1) on over 90% of observed plants. To isolate the pathogen, leaf sections (5 mm × 3 mm) from the border of the symptomatic tissue were cut and sterilized with 75% ethanol for 30 s, followed by 2% sodium hypochlorite (NaClO) for 2 min, and then rinsed three times with sterile water. Leaf sections were placed on potato dextrose agar (PDA) medium amended with 25 μg / ml ampicillin and incubated at 25 °C in the dark for 3 days. Isolated colonies were sub-cultured on PDA by hyphal tip transfer. Eight fungal isolates with similar morphology, abundant white aerial hyphae, were collected. Colonies on PDA grew up to 80 mm in diameter by 7 days at 25 °C. The center of each colony became black (Fig.2). Conidia were unicellular, oval and hyaline. Conidia ranged in size from 14.5 to 19.75 µm × 5.80 to 10.20 µm (n=50) in 20% (v/v) V8 vegetable juice medium. No appressoria were observed. Morphological characteristics are similar to those of Colletotrichum spp. (Moriwaki et al. 2003). Total genomic DNA of a representative isolate S1 was extracted with a CTAB method (Stenglein et al. 2006). Internal transcribed spacer (ITS) region of rDNA, actin (ACT) , beta-tubulin (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced with the primer pairs of ITS4 / ITS5, ACT512F / ACT783R, Bt-2a / Bt-2b and GDF1 / GDR1, respectively (Weir et al. 2012). BLAST results showed that ITS, ACT, TUB2 and GAPDH gene sequences (GenBank accession nos. MN498030, MT780498, MT780496 and MT780497, respectively) were 99% identical to those of Colletotrichum boninense Moriwaki, Sato & Tsukiboshi (GenBank accession nos. MF076598, JX009583, JQ005588 and JX009905, respectively). Concatenated sequences of the four genes were used to conduct a phylogenetic analysis using neighbor-joining method in MEGA7 (Toussaint et al. 2016). The isolate S1 clustered with above C. boninense strains retrieved from NCBI database. Therefore, the present isolate S1 was identified as C. boninense. Pathogenicity tests were performed using one-month-old raspberry plants, 24 controls and 30 inoculated. The plants were sprayed with conidial suspension ( 106 conidia / mL) cultured on 20% (v/v) V8 vegetable juice medium for 15 days. The control plants were sprayed with sterile distilled water. All plants were covered with plastic bags 24h to maintain the relative humidity in the field. Fifteen days after inoculation, typical symptoms of brown spots were observed on leaves similar to the disease on field plants, while the leaves from the control group remained asymptomatic. C. boninense was reisolated and identified from inoculated symptomatic leaves. Anthracnose on raspberry caused by Colletotrichum gloeosporioides (Dai et al. 2013) and C. fioriniae (Schoeneberg et al. 2020) has previously been reported. However, to the best of our knowledge, this is the first report of Colletotrichum boninense causing leaf spot on Raspberry in China. If more reports of this pathogen are found on raspberries, then it may be necessary to develop effective management strategies for controlling this disease.

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First report of banana (Musa acuminate L. AAA Cavendish, cv. Formosana) leaf spot disease caused by Curvularia geniculata in China

Plant Disease ◽

10.1094/pdis-09-21-2001-pdn ◽

2021 ◽

Author(s):

Yanxiang Qi ◽

Yanping Fu ◽

Jun Peng ◽

Fanyun Zeng ◽

Yanwei Wang ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Universal Primers ◽

Leaf Spot Disease ◽

Leaf Margin ◽

Conidial Suspension ◽

Tropical Fruit ◽

First Report ◽

Spot Disease

Banana (Musa acuminate L.) is an important tropical fruit in China. During 2019-2020, a new leaf spot disease was observed on banana (M. acuminate L. AAA Cavendish, cv. Formosana) at two orchards of Chengmai county (19°48ʹ41.79″ N, 109°58ʹ44.95″ E), Hainan province, China. In total, the disease incidence was about 5% of banana trees (6 000 trees). The leaf spots occurred sporadically and were mostly confined to the leaf margin, and the percentage of the leaf area covered by lesions was less than 1%. Symptoms on the leaves were initially reddish brown spots that gradually expanded to ovoid-shaped lesions and eventually become necrotic, dry, and gray with a yellow halo. The conidia obtained from leaf lesions were brown, erect or curved, fusiform or elliptical, 3 to 4 septa with dimensions of 13.75 to 31.39 µm × 5.91 to 13.35 µm (avg. 22.39 × 8.83 µm). The cells of both ends were small and hyaline while the middle cells were larger and darker (Zhang et al. 2010). Morphological characteristics of the conidia matched the description of Curvularia geniculata (Tracy & Earle) Boedijn. To acquire the pathogen, tissue pieces (15 mm2) of symptomatic leaves were surface disinfected in 70% ethanol (10 s) and 0.8% NaClO (2 min), rinsed in sterile water three times, and transferred to potato dextrose agar (PDA) for three days at 28°C. Grayish green fungal colonies appeared, and then turned fluffy with grey and white aerial mycelium with age. Two representative isolates (CATAS-CG01 and CATAS-CG92) of single-spore cultures were selected for molecular identification. Genomic DNA was extracted from the two isolates, the internal transcribed spacer (ITS), large subunit ribosomal DNA (LSU rDNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1-α) and RNA polymerase II second largest subunit (RPB2) were amplified and sequenced with universal primers ITS1/ITS4, LROR/LR5, GPD1/GPD2, EF1-983F/EF1-2218R and 5F2/7cR, respectively (Huang et al. 2017; Raza et al. 2019). The sequences were deposited in GenBank (MW186196, MW186197, OK091651, OK721009 and OK491081 for CATAS-CG01; MZ734453, MZ734465, OK091652, OK721100 and OK642748 for CATAS-CG92, respectively). For phylogenetic analysis, MEGA7.0 (Kumar et al. 2016) was used to construct a Maximum Likelihood (ML) tree with 1 000 bootstrap replicates, based on a concatenation alignment of five gene sequences of the two isolates in this study as well as sequences of other Curvularia species obtained from GenBank. The cluster analysis revealed that isolates CATAS-CG01 and CATAS-CG92 were C. geniculata. Pathogenicity assays were conducted on 7-leaf-old banana seedlings. Two leaves from potted plants were stab inoculated by puncturing into 1-mm using a sterilized needle and placing 10 μl conidial suspension (2×106 conidia/ml) on the surface of wounded leaves and equal number of leaves were inoculated with sterile distilled water serving as control (three replicates). Inoculated plants were grown in the greenhouse (12 h/12 h light/dark, 28°C, 90% relative humidity). Necrotic lesions on inoculated leaves appeared seven days after inoculation, whereas control leaves remained healthy. The fungus was recovered from inoculated leaves, and its taxonomy was confirmed morphologically and molecularly, fulfilling Koch’s postulates. C. geniculata has been reported to cause leaf spot on banana in Jamaica (Meredith, 1963). To our knowledge, this is the first report of C. geniculata on banana in China.

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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 Leaf Spot Caused by Ascochyta marginata on Aralia elata in Korea

Plant Disease ◽

10.1094/pdis-08-11-0658 ◽

2012 ◽

Vol 96 (1) ◽

pp. 147-147

Author(s):

S. H. Lee ◽

C. K. Lee ◽

M. J. Park ◽

H. D. Shin

Keyword(s):

East Asia ◽

Leaf Spot ◽

Morphological Characteristics ◽

Culture Collection ◽

Research Report ◽

Conidial Suspension ◽

First Report ◽

Commercial Cultivation ◽

Aralia Elata ◽

Initial Symptoms

Aralia elata (Miq.) Seem., known as Japanese angelica tree, is a deciduous shrub belonging to the Araliaceae, which is native to East Asia. The young shoots have long been used in various dishes in East Asia. Commercial cultivation of this shrub, especially in polytunnels, is expanding in Korea. Several diseases including Sclerotinia rot have been known to be present on this plant (1,2). In early September 2007, leaf spot symptoms were first observed on several trees in Hongcheon, Korea. Microscopic observations revealed that the leaf spots were associated with an Ascochyta sp. Further surveys of the Ascochyta leaf spot showed the occurrence of the disease in approximately 5 to 10% of the trees in the 3 ha of commercial fields surveyed in Chuncheon, Gapyeong, Inje, and Jinju, Korea. Initial symptoms on leaves were circular to irregular, brown to dark brown, becoming zonate, and finally fading to grayish brown in the center with a yellow halo. Representative samples were deposited in the herbarium of Korea University. Conidiomata on leaf lesions were pycnidial, amphigenous, but mostly epiphyllous, immersed or semi-immersed in host tissue, light brown to olive brown, and 60 to 200 μm in diameter. Ostioles were papillate, 20 to 35 μm wide, and surrounded by a ring of darker cells. Conidia were hyaline, smooth, cylindrical to clavate, straight to mildly curved, slightly constricted at the septa, medianly one-septate, sometimes aseptate, 8 to 16 × 2.5 to 3.5 μm, and contained small oil drops. These morphological characteristics were consistent with the previous reports of Ascochyta marginata J.J. Davis (3,4). A monoconidial isolate was cultured on potato dextrose agar (PDA) plates and accessioned in the Korea Agricultural Culture Collection (Accession KACC43082). The conidia were readily formed on PDA. Inoculum for the pathogenicity tests was prepared by harvesting conidia from 30-day-old cultures of KACC43082 and a conidial suspension (approximately 2 × 106 conidia/ml) was sprayed onto leaves of three healthy seedlings. Three noninoculated seedlings served as controls. Inoculated and noninoculated plants were covered with plastic bags for 48 h in a glasshouse. After 7 days, typical leaf spot symptoms started to develop on the leaves of the inoculated plants. The fungus, A. marginata, was reisolated from those lesions, confirming Koch's postulates. No symptoms were observed on control plants. Previously, the disease was reported in Japan (4) and China (3). To our knowledge, this is the first report of A. marginata on Japanese angelica trees in Korea. According to our field observations in Korea, the Ascochyta leaf spot mostly occurred on plants growing in a humid environment, especially during the rainy season. The seedlings as well as the trees growing in sunny, well-ventilated plots were nearly free from this disease. Therefore, the growing conditions seemed to be the most important factor for the development and severity of the disease. References: (1) C. K. Lee et al. Plant Pathol. J. 26:426, 2010. (2) S. H. Lee et al. Diseases of Japanese Angelica Tree and Their Control. Research Report 08-10. Korea Forest Research Institute. Seoul, Korea, 2008. (3) J. Sun et al. Acta Mycol. Sin. 14:107, 1995. (4) M. Yoshikawa and T. Yokoyama. Mycoscience 36:67, 1995.

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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 Leaf Blight of Rice Caused by Cochliobolus lunatus in China

Plant Disease ◽

10.1094/pdis-03-13-0303-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 686-686 ◽

Cited By ~ 4

Author(s):

L. M. Liu ◽

S. W. Huang ◽

L. Wang ◽

E. Q. Hou ◽

D. F. Xiao

Keyword(s):

Leaf Blight ◽

Green Water ◽

Tween 20 ◽

Curvularia Lunata ◽

Conidial Suspension ◽

First Report ◽

Rice Plants ◽

Rdna Its ◽

Cochliobolus Lunatus ◽

Wide Range

Leaf-streak symptoms were observed on rice (Oryza sativa L.) starting at the booting stage through harvest in Zhejiang Province, China, in 2012. Based on Fuyang County, only 15% of the rice fields were estimated to show these symptoms. However, incidence could be 40 to 80% when the rice got infected. Typical symptoms started as green water-soaked streaks from the tip or edge of leaf blades, similar to bacterial leaf blight caused by Xanthomonas oryzae. Infected leaves turned yellow, then eventually became wilted and dry. No bacterial streaming was observed and no bacteria were isolated. Pieces of infected leaf tissue were surface sterilized using 0.1% (v/v) mercuric chloride, rinsed with sterilized water, then placed on water agar (WA). After 2 or 3 days on WA at 28°C, only fungal growth was observed from surface sterilized tissues. Fungi were isolated, purified by single spore separation process, and subcultured to potato dextrose agar (PDA) plates. Growing on PDA, the surface of the colony was circular, fluffy, and shiny velvety-black, whereas the under surface was dark Prussian blue. Conidiophores were single or fascicled, brown to dark brown, rarely branched, multiseptate, and straight or often geniculate near the apex. Conidia were brown, smooth, fusiform, geniculate or hook-shaped, 17.5 to 28.5 × 8.5 to 14.0 μm, and 3-septate, with the third cell from the base larger and darker than the others. Molecular identification was performed by analysis of the rDNA internal transcribed spacer region (ITS1-5.8S-ITS2). The rDNA-ITS region was amplified with primer pair ITS1 and ITS4 (5), sequenced, and deposited in GenBank (Accession No. KC462186). The sequence of rDNA-ITS (KC462186) showed 100% identity with Cochliobolus lunatus R.R. Nelson & Haasis (JN943422) after BLAST. Based on the results of morphological and molecular analyses, the fungus isolated from infected leaves was identified as C. lunatus (anamorph: Curvularia lunata (Wakk.) Boedijn) (3). Pathogenicity tests were conducted three times by spraying a conidial suspension (1 × 105 spores/ml) with 0.1% (v/v) Tween 20 on 12 healthy rice plants at late tillering stage. The same number of the healthy rice plants sprayed with sterilized water with 0.1% (v/v) Tween 20 were used as control. All plants were kept at 30°C and 75 to 85% relative humidity (RH) under a 12-h light/dark rotation. About 5 to 7 days after inoculation, green water-soaked streaks began to appear on inoculated plants. From 7 to 14 days after inoculation, the lesions developed quickly and the leaves began to wilt. After 14 days, inoculated plants showed symptoms similar to those originally observed in the field, while control plants (sprayed with sterilized water) remained healthy. C. lunatus was re-isolated from all inoculated plants, and re-identified by the same methods (morphological and molecular methods) as described above, thereby satisfying Koch's postulates, and confirming C. lunatus as the cause of the disease. C. lunatus is a pathogen of a wide range of plants and is common in paddy environments. It was reported as one of the causal agents of black kernel of rice (4) and rice spikelet rot disease (SRD) (1,2). The level of incidence observed in the affected fields suggest that this disease could potentially cause major losses under favorable weather conditions if susceptible cultivars are grown. To our knowledge, this is the first report of C. lunatus causing leaf blight of rice in China. References: (1) S. W. Huang et al. Crop Prot. 30:1, 2011. (2) S. W. Huang et al. Crop Prot. 30:10, 2011. (3) D. S. Manamgoda et al. Fungal Divers. 51:3. (4) S. H. Ou. Rice diseases [M]. CABI, 1985. (5) T. J. White et al. PCR Protocols: a Guide to Methods and Application. Academic Press, San Diego, CA, 1990.

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Leaf spot disease of Orychophragmus violaceus caused by Alternaria tenuissima in China

Plant Disease ◽

10.1094/pdis-10-20-2287-pdn ◽

2021 ◽

Author(s):

Dongli Liu ◽

Jing Li ◽

Saisai Zhang ◽

Xiangjing Wang ◽

Wensheng Xiang ◽

...

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Its Sequence ◽

Conidial Suspension ◽

Orychophragmus Violaceus ◽

Disease Symptoms ◽

Alternaria Tenuissima ◽

Spot Disease ◽

Pathogenicity Tests

Orychophragmus violaceus (L.) O. E. Schulz, also called February orchid or Chinese violet cress, belongs to the Brassicaceae family and is widely cultivated as a green manure and garden plant in China. During the prolonged rainy period in August 2020, leaf spot disease of O. violaceus was observed in the garden of Northeast Agricultural University, Harbin, Heilongjiang province. One week after the rainy days, the disease became more serious and the disease incidence ultimately reached approximately 80%. The disease symptoms began as small brown spots on the leaves, and gradually expanded to irregular or circular spots. As the disease progressed, spots became withered with grayish-white centers and surrounded by dark brown margins. Later on, the centers collapsed into holes. For severely affected plants, the spots coalesced into large necrotic areas and resulted in premature defoliation. No conidiophores or hyphae were present, and disease symptoms were not observed on other tissues of O. violaceus. To isolate the pathogen, ten leaves with typical symptoms were collected from different individual plants. Small square leaf pieces (5×5 mm) were excised from the junction of diseased and healthy tissues, disinfected in 75% ethanol solution for 1 min, rinsed in sterile distilled water, and then transferred to Petri dishes (9 cm in diameter) containing potato dextrose agar (PDA). After 3 days of incubation at 25 oC in darkness, newly grown-out mycelia were transferred onto fresh PDA and purified by single-spore isolation. Nine fungal isolates (NEAU-1 ~ NEAU-9) showing similar morphological characteristics were obtained and no other fungi were isolated. The isolation frequency from the leaves was almost 90%. On PDA plates, all colonies were grey-white with loose and cottony aerial hyphae, and then turned olive-green and eventually brown with grey-white margins. The fungus formed pale brown conidiophores with sparsely branched chains on potato carrot agar (PCA) plates after incubation at 25 oC in darkness for 7 days. Conidia were ellipsoidal or ovoid, light brown, and ranged from 18.4 to 59.1 × 9.2 to 22.3 µm in size, with zero to two longitudinal septa and one to five transverse septa and with a cylindrical light brown beak (n = 50). Based on the cultural and morphological characteristics, the fungus was tentatively identified as Alternaria tenuissima (Simmons 2007). Genomic DNA was extracted from the mycelia of five selected isolates (NEAU-1 ~ NEAU-5). The internal transcribed spacer region (ITS) was amplified and sequenced using primers ITS1/ITS4 (White et al., 1990). Blast analysis demonstrated that these five isolates had the same ITS sequence, and the ITS sequence of representative strain NEAU-5 (GenBank accession No. MW139354) showed 100% identity with the type strains of Alternaria alternata CBS916.96 and Alternaria tenuissima CBS918.96. Furthermore, the translation elongation factor 1-α gene (TEF), RNA polymerase II second largest subunit (RPB2), and glyceraldehyde 3-phosphate dehydrogenase (GPD) of representative strain NEAU-5 were amplified and sequenced using primers EF1-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-5R (Sung et al., 2007), and Gpd1/Gpd2 (Berbee et al., 1999), respectively. The sequences of RPB2, GPD, and TEF of strain NEAU-5 were submitted to GenBank with accession numbers of MW401634, MW165223, and MW165221, respectively. Phylogenetic trees based on ITS, RPB2, GPD, and TEF were constructed with the neighbour-joining and maximum-likelihood algorithms using MEGA software version 7.0. The results demonstrated that strain NEAU-5 formed a robust clade with A. tenuissima CBS918.96 (supported by 99% and 96% bootstrap values) in the neighbour-joining and maximum-likelihood trees. As mentioned above, strain NEAU-5 produced seldomly branched conidial chains on PCA plates. The pattern is consistent with that of A. tenuissima (Kunze) Wiltshire, but distinct from that of A. alternata which could produce abundant secondary ramification (Simmons 2007). Thus, strain NEAU-5 was identified as A. tenuissima based on its morphology and phylogeny. Pathogenicity tests were carried out by inoculating five unwounded leaves with a conidial suspension of strain NEAU-5 (approximately 106 conidia/ml) on five different healthy plants cultivated in garden, and an equal number of leaves on the same plants inoculated with sterilized ddH2O served as negative controls. Inoculated and control leaves were covered with clear plastic bags for 3 days. After 6 days, small brown and irregular or circular spots were observed on all leaves inoculated with conidial suspension, while no such symptoms were observed in the control. The tests were repeated three times. Furthermore, the pathogenicity tests were also performed using 2-month-old potted plants in a growth chamber (28 oC, 90% relative humidity, 12 h/12 h light/dark) with two repetitions. Five healthy plants were inoculated by spraying 20 ml of a conidial suspension of strain NEAU-5 (approximately 106 conidia/ml) onto unwounded leaves. Five other healthy plants were inoculated with sterilized ddH2O as controls. After 7 days, similar symptoms were observed on leaves inoculated with strain NEAU-5, whereas no symptoms were observed in the control. The pathogen was reisolated from the inoculated leaves and identified as A. tenuissima by morphological and molecular methods. In all pathogenicity tests, A. tenuissima could successfully infect unwounded leaves of O. violaceus, indicating a direct interaction between leaves and A. tenuissima. It is known that high humidity and fairly high temperatures can favor the epidemics of Alternaria leaf spot (Yang et al., 2018), and this may explain why severe leaf spot disease of O. violaceus was observed after prolonged rain. Previously, it has been reported that Alternaria brassicicola and Alternaria japonica could cause leaf blight and spot disease on O. violaceus in Hebei and Jiangsu Provinces, China, respectively (Guo et al., 2019; Sein et al., 2020). Although these pathogens could lead to similar disease symptoms on the leaves of O. violaceus, it is easy to distinguish them by the morphological characteristics of conidiophores and ITS gene sequences. To our knowledge, this is the first report of A. tenuissima causing leaf spot disease of O. violaceus in China.

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First Report of Fusarium ipomoeae Causing Peanut leaf Spot in China

Plant Disease ◽

10.1094/pdis-01-21-0226-pdn ◽

2021 ◽

Author(s):

Manlin Xu ◽

Xia Zhang ◽

Jing Yu ◽

zhiqing Guo ◽

Ying Li ◽

...

Keyword(s):

Leaf Spot ◽

Genomic Dna ◽

Block Design ◽

Management Strategies ◽

Morphological Characteristics ◽

Ethanol Solution ◽

Distilled Water ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report

Peanut (Arachis hypogaea L.) is one of the most economically important crops as an important source of edible oil and protein. In August 2020, circular to oval-shaped brown leaf spots (2-6 mm in diameter) with well-defined borders surrounded by a yellow margin were observed on peanut plant leaves in Laixi City, Shandong Province, China. Symptomatic plants randomly distributed in the field, the incidence was approximately 5%. Leave samples were collected consisted of diseased tissue and the adjacent healthy tissue. The samples were dipped in a 70% (v/v) ethanol solution for 30 s and then soaked in a 0.1% (w/v) mercuric chloride solution for 60 s. The surface-sterilized tissues were then rinsed three times with sterile distilled water, dried and placed on Czapek Dox agar supplemented with 100 μg/ml of chloramphenicol. The cultures were incubated in darkness at 25 °C for 3–5 days. Fungal colonies were initially white and radial, turning to orange-brown in color, with abundant aerial mycelia. Macroconidia were abundant, 4 to 7 septate, with a dorsiventral curvature, and were 3.3–4.5 × 18.5–38.1 μm (n=100) in size; microconidia were absent; chlamydospores were produced in chains or clumps, ellipsoidal to subglobose, and thick walled. The morphological characteristics of the conidia were consistent with those of Fusarium spp. To identify the fungus, an EasyPure Genomic DNA Kit (TransGEN, Beijing, China) was used to extract the total genomic DNA from mycelia. The internal transcribed spacer region (ITS rDNA) and the translation elongation factor 1-α gene (TEF1) were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1/EF2 (O’Donnell et al. 1998), respectively. Based on BLAST analysis, sequences of ITS (MT928727) and TEF1 (MT952337) showed 99.64% and 100% similarity to the ITS (MT939248.1), TEF1 (GQ505636.1) of F. ipomoeae isolates. Sequence analysis confirmed that the fungus isolated from the infected peanut was F. ipomoeae (Xia et al. 2019). The pathogenicity of the fungus was tested in the greenhouse. Twenty two-week-old peanut seedlings (cv. Huayu20) grown in 20-cm pots (containing autoclaved soil) were sprayed with a conidial suspension (105 ml−1) from a 15-day-old culture. Control plants were sprayed with distilled water. The experiment was conducted as a randomized complete block design, and placed at 25 °C under a 12-h photoperiod with 90% humidity. Symptoms similar to those in the field were observed on leaves treated with the conidial suspension ten days after inoculation, but not on control plants. F. ipomoeae was re-isolated from symptomatic leaves but not from the control plants. Reisolation of F. ipomoeae from inoculated plants fulfilled Koch's postulates. To our knowledge, this is the first report of F. ipomoeae causing peanut leaf spot in China. Our report indicates the potential spread of this pathogen in China and a systematic survey is required to develop effective disease management strategies.

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First report of Cercospora cf. citrulina causing leaf spot of Ipomoea pes-caprae in China

Plant Disease ◽

10.1094/pdis-05-20-1081-pdn ◽

2020 ◽

Author(s):

Min Li ◽

Meijiao Hu ◽

Zhaoyin Gao ◽

Xiaoyu Hong ◽

Chao Zhao ◽

...

Keyword(s):

Molecular Identification ◽

Leaf Spot ◽

Plant Protection ◽

Morphological Characteristics ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Xisha Islands ◽

First Report ◽

Initial Symptoms ◽

Leaf Surfaces

Ipomoea pes-caprae plays an important role in protecting the tropical and subtropical coastal beach of the world. In 2018, a leaf spot was observed on I. pes-caprae in Xisha islands of China, 13.2–25.8% of leaves were infected. The initial symptoms were small (1–3 mm diameter), single, circular, dark gray spots with a light-yellow center on the leaves. The lesions enlarged and were scattered or confluent, distinct and circular, subcircular or irregular, occasionally vein-limited, pale to dark gray-brown, with a narrow dark brown border surrounded by a diffuse yellow margin. Microscopic observations of the spots revealed that caespituli were dark brown and amphigenous, but abundant on the underside of the leaves. Mycelia were internal. Conidiophores were fasciculate, occasionally solitary, pale olivaceous-brown throughout, 0- to 3-septate, 27.9–115.8 (63.4±22.5) µm × 3.2–5.3 (4.3±0.87) µm (n=100). Conidial scars were conspicuously thickened. Conidia were solitary, hyaline, filiform, acicular to obclavate, straight to slightly curved, subacute to obtuse at the apex, truncate at the base, multi-septate, 21.0–125.5 (60.2±20.1) µm × 2.0–5.0 (3.8±0.83) µm (n=100). Single-conidium isolates were obtained from representative colonies grown on potato dextrose agar (PDA) incubated at 25℃ in the dark. The colonies grew slowly and were dense, white to gray and flat with aerial mycelium. Mycelia were initially white, and then became gray. Conidia were borne on the conidiophores directly. The pure isolate HTW-1 was selected for molecular identification and pathogenicity test, which were deposited in Microbiological Culture Collection Center of Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (tef1) and histone H3 (his3) genes were amplified with ITS1/ITS4, EF-1 / EF-2, and CYLH3F / CYLH3R primers, respectively (Groenewald et al. 2013). The obtained sequences of HTW-1 were all deposited in GenBank with accession numbers MT410467 for ITS, MT418903 for tef1 and MT418904 for his3. The ITS, tef1 and his3 genes all showed 100% similarity for ITS (JX143582), tef1 (JX143340) and his3 (JX142602) with C. cf. citrulina (MUCC 588; MAFF 239409) from I. pes-caprae in Japan. Based on the morphological characteristics and molecular identification, the pathogen was identified as Cercospora cf. citrulina (Groenewald et al. 2013). The pathogenicity test was conducted by spraying conidial suspension (1×104 conidia/mL) on wounded and unwounded leaves for seedling of I. pes-caprae in greenhouse and in sterile vitro condition. The conidial suspension was prepared using conidia from 30-day-old culture grown on PDA at 25℃ in the dark. Leaf surfaces of seedling in greenhouse were wounded by lightly rubbing with a steel sponge and detached leaf surfaces were wounded by sterile needles. the treatments were sprayed with conidial suspensions on wounded and unwounded leaf surfaces. The control was sprayed with sterile water. After eight days, the typical symptoms of spots which were small, single, circular and dark gray appeared on the inoculated wounded leaves, while the inoculated unwounded leaves and the control leaves were symptomless. The pathogen was only re-isolated from the inoculated wounded leaves. The pathogen may be infected by wound. A total of 20 Cercospora and related species was found on Ipomoea spp. (García et al. 1996). Cercospora cf. citrulina has been reported on I. pes-caprae in Japan, although it was unclear if it was a pathogen or saprophyte (Groenewald et al. 2013). To our knowledge, this is the first report of C. cf. citrulina causing leaf spot of I. pes-caprae in China. This disease could threat the cultivation of I. pes-caprae in China.

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