Nigrospora oryzae Causing Black Leaf Spot Disease of Hibiscus mutabilis in China

Cotton rose (Hibiscus mutabilis Linn.) is a deciduous shrub native to China. It has been widely cultivated in many provinces in China for its ornamental and ecological value (Shang et al., 2020). In May 2017, an unknown leaf spot symptom was first observed on H. mutabilis at the Chengdu Campus of Sichuan Agricultural University (30°42′31″ N, 103°51′28″ E). The disease occurred from May to September with approximately 81% incidence by field sample survey of 300 plants in Chengdu Greenway. The symptoms at first appeared as irregular black spots on the leaves. Then the lesions grew and coalesced into large, black necrotic areas, which later produced leaf chlorosis and abscission (Fig. 1-A). This disease seriously reduced the ornamental value of H. mutabilis. Forty diseased lesions (4 × 5 mm) were surface sterilized with 75% alcohol for 60 s and 3% NaClO for 45 s, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and then incubated in a dark at 25°C. From the 7 obtained isolates, 4 isolates exhibited the morphology described as Nigrospora oryzae (Hao et al., 2020). The fungus produced initially circular white colonies, and then the centers turned dark gray or black with age on the PDA. Hyphae were smooth, branched, septate, hyaline, or pale brown. Conidia (N = 100 spores) were abundant, and were solitary, dark-brown to black, smooth, aseptate, and measured 11 to 15 μm in diameter (Fig. 1). DNA was extracted from the fungal colonies using a DNeasyTM Plant Mini Kit (Qiagen). The internally transcribed spacer (ITS), β-tubulin gene (TUB), and translation elongation factor 1-alpha (TEF1) were amplified with primers ITS1/ITS4 (White et al., 1990), BT2A/BT2B (Glass and Donaldson 1995), and EF1-728F/EF1-986R (O'Donnell et al., 1998; Carbone and Kohn 1999), respectively. BLAST results indicated that the ITS, TUB, and TEF1 sequences (GenBank accession Nos. MN515070, MN733956, and MN635723, respectively) had 99% identity with N. oryzae sequences (GenBank accession Nos. KX986031, KY019553, and KY019358). The result was confirmed by multilocus phylogenetic analysis (Fig. 2). The morphological characteristics and molecular analyses of the isolate matched the description of N. oryzae. To confirm pathogenicity, Koch’s postulates were fulfilled under controlle conditions. The seedlings of 20 two-year-old potted H. mutabilis plants were inoculated by spraying conidial suspension at the concentration of 1 × 106 conidia/ml on both sides of leaves. Sterilized distilled water (20 seedlings) were used as negative controls. The experiment was performed three times. All plants were incubated at 25°C ± 2°C under a 16 h/8 h photoperiod and 70%–75% relative humidity (RH) after inoculation, and observed daily for disease development. Two weeks later, the inoculated plants showed the same symptoms as the original diseased plants and the controls remained asymptomatic. The pathogen N. oryzae was re-isolated from all ioculated plants, and the culture and fungus characteristics were the same as those of the original isolate. But N. oryzae was not isolated from the control plants. The results indicated that N. oryzae is a causal agent of the disease. N. oryzae was reported as a leaf pathogen on cotton (Zhang et al., 2012), but this is the first report of N. oryzae causing leaf black spot on H. mutabilis in the world. The identification could provide relevant information for adopting appropriate management strategies to control the disease.

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First Report of Leaf Spot on Kidney Bean Caused by Nigrospora oryzae in China

Plant Disease ◽

10.1094/pdis-06-21-1171-pdn ◽

2021 ◽

Author(s):

Mingyan Ming Luo ◽

Yulan Yu Jiang

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Kidney Bean ◽

Leaf Spot Disease ◽

Conidial Suspension ◽

First Report ◽

Spot Disease ◽

Bean Cultivar ◽

Leaf Spots ◽

Nigrospora Oryzae

Kidney bean (Phaseolus vulgaris L.) is a legume with high nutritional and economic value. This vegetable crop is widely cultivated in China, providing a year-round supply of young edible pods. In July 2020, a leaf spot disease on kidney bean cultivar ‘Dabailong’ was observed on a two-hectare field in Longli County (26°16′15.66″ N, 106°48′12″ E), Guizhou Province, China. Disease incidence was estimated to be nearly 50%. Foliar symptoms manifested as black circular spots, surrounded by a yellow halo and accompanied by white mycelium. To identify the pathogen, small portions of tissue (5×5 mm) from margins of leaf spots were cut from 20 symptomatic leaves, surface disinfected with 75% ethanol for 30 s, rinsed two times with sterile distilled water, dried on a sterile filter paper, and incubated on potato dextrose agar (PDA) at 28°C for 3 days. A total of 39 single-spore isolates were obtained. The colonies on PDA were fluffy, changing from white to gray or black with age, and reaching 7-cm diameter in 5 days at 28°C. Conidia were black, globose to subglobose, smooth, solitary, measuring 13.0 to 16.0 × 10.5 to 16.0 µm (n=30). Morphological characteristics were consistent with Nigrospora oryzae. In addition, the rDNA internal transcribed spacer (ITS), large subunit (LSU), β-tubulin (TUB) and translation elongation factor 1-alpha (TEF1) loci were amplified by PCR and sequenced (White et al. 1990, Glass and Donaldson 1995, O'Donnell et al. 1998; Carbone and Kohn 1999). The ITS, LSU, TUB and TEF1 sequences of two isolates, GUCC19-5105 and GUCC19-5192, were submitted to GenBank. BLASTn analysis of these sequences showed >98% homology with those of N. oryzae strain LC 7293 in GenBank (ITS, 99.80% (MZ145361 vs KX985931 – 498/499 bp) and 99.62% (MZ148445 vs KX985931 – 525/527 bp); LSU, 100% (MZ146317 vs KY806236 – 837/837 bp) and 99.76% (MZ148446 vs KY806236 – 847/849 bp); TUB, 98.72% (MZ329335 vs KY019601 – 386/391 bp) and 98.67% (MZ329337 vs KY019601 – 373/378 bp) and TEF1, 98.91% (MZ329336 vs KY019396 – 452/457 bp) and 98.89% (MZ329334 vs KY019396 – 444/449 bp) respectively). The phylogenetic tree of the combined 4 sequences showed that both isolates clustered with N. oryzae. Based on morphological characteristics and the multigene phylogenetic analysis, GUCC19-5105 and GUCC19-5192 isolates were identified as N. oryzae. Pathogenicity tests were performed twice by spraying conidial suspension (1×105 conidia/mL) of the two isolates (GUCC19-5105 and GUCC19-5192) on leaves of ten (five per isolate) healthy 5-week-old kidney bean cultivar ‘Dabailong’ plants. Two plants sprayed with sterile water served as controls. After inoculation, all the plants were kept moist in plastic bags for 24 hours and incubated in a greenhouse at 25°C for 20 days. Leaf spots similar to those observed in the field were observed 20 days post inoculation, but no lesions were observed on control plants. N. oryzae was reisolated from the infected tissues of inoculated kidney bean plants and the identity of the reisolated pathogen was confirmed as N. oryzae through morphology and sequencing ITS, LSU, TUB and TEF1 loci. In recent years, N. oryzae has been reported to infect a variety of plants such as Aloe vera, Citrullus lanatus and Costus speciosus (Begum et al. 2018; Chen et al. 2019; Sun et al. 2020). To our knowledge, this is the first report of leaf spot disease on kidney bean caused by N. oryzae in the world and provides a basis for diagnosticians and researchers to identify the disease and develop disease management strategies.

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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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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 Leaf Spot Caused by Alternaria alternata on Yucca gloriosa in China

Plant Disease ◽

10.1094/pdis-04-21-0857-pdn ◽

2021 ◽

Author(s):

Qiang Zhang ◽

Yanru Zhang ◽

Hongli Shi ◽

Yunfeng Huo

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Management Strategies ◽

Elongation Factor ◽

Morphological Characteristics ◽

Internal Transcribed Spacers ◽

Molecular Characteristics ◽

Single Spore ◽

Tissue Samples ◽

First Report

Yucca gloriosa L. is introduced to China as a garden plant because of its attractive tubular flowers (Ding et al. 2020). In 2020 and 2021, a foliar disease occurred on approximately 10% of the Y. gloriosa plants in the campus of Henan Institute of Science and Technology, Xinxiang (35°18′N, 113°54′E), Henan Province, China. At the early stages, symptoms appeared as small brown spots on the tip of the leaves. As the disease developed, the spots gradually expanded and turned into necrotic tissue with a clear brown border. The length of lesions ranged from 1 to 3 cm. Infected tissue samples were cut into small pieces, surface sterilized with 75% ethanol for 30 s followed by 0.5% NaClO for 2 min, rinsed thrice with sterile water and plated on potato dextrose agar (PDA). After incubation at 25℃ for 3 days, five fungal isolates were collected and purified using single spore culturing. Morphological observations were made on the 7-day-old cultures. Colonies on PDA were white at first and then turned to dark olive or black along with profuse sporulation. Conidia were borne on branched conidiophores, light brown to dark brown, ellipsoidal to obpyriform, and 20.5 to 43.6 ×7.5 to 15.4 μm in size, with 2-6 transverse septa and 0-3 longitudinal septa (n = 50). The morphological characteristics of the five isolates were consistent with the description for Alternaria alternata (Simmons 2007). One representative isolate (ZQ20) was selected for molecular identification. The internal transcribed spacers (ITS)-rDNA, translation elongation factor-1 alpha (TEF-1α), Alternaria major allergen (Alt a1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene regions were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EFl-728F/ EFI-986R (Carbone and Kohn, 1999), Alt-for/Alt-rev (Hong et al. 2005), and gpd1/gpd2 (Berbee et al. 1999), respectively. Their sequences were submitted to GenBank (ITS, MW832377; TEF-1α, MW848791; Alt a1, MW848792; GAPDH, MW848793). BLAST searches showed ≥99% nucleotide identity to the sequences of A. alternata (ITS, 100% to KF465761; TEF-1α, 100% to MT133312; Alt a1, 100% to KY923227; and GAPDH, 99% to MK683863). Thus, the fungus was identified as A. alternata based on its morphological and molecular characteristics. To confirm its pathogenicity, 25 healthy leaves of five 2-year-old Y. gloriosa plants were used. Leaves were wounded with one sterile needle and inoculated with 5-mm-diameter fungal agar disks obtained from 5-day-old cultures. Sterile PDA disks of the same size were used as the controls. Treated plants were covered with a plastic bag at 12 to 25℃ for 48 h to ensure a high level of moisture. After 15 days, the inoculated plants developed the symptoms similar to those observed in naturally infected plants, whereas the control plants were symptomless. The fungus was reisolated from the symptomatic leaves with the same morphological and molecular characteristics as the original isolates, fulfilling the Koch's postulates. Leaf spot caused by A. alternata in the Yucca plants has been reported in India (Pandey 2019). To our knowledge, this is the first report of A. alternata causing leaf spot on Y. gloriosa in China. Identification of the cause of the disease is important to developing effective disease management strategies.

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First Report of Colletotrichum cliviicola Causing Leaf Spot on Tobacco (Nicotiana tabacum) in Hunan Province of China

Plant Disease ◽

10.1094/pdis-02-21-0409-pdn ◽

2021 ◽

Author(s):

Ya Rong Wang ◽

Zhao Hu ◽

Jie Zhong ◽

Yi Chen ◽

Jun Zi Zhu

Keyword(s):

Nicotiana Tabacum ◽

Leaf Spot ◽

Morphological Characteristics ◽

Disease Diagnosis ◽

Hunan Province ◽

Leaf Spot Disease ◽

Post Inoculation ◽

Conidial Suspension ◽

First Report ◽

Colletotrichum Species

Tobacco (Nicotiana tabacum L.) is an annual, leafy, herb of the genus Nicotiana in the family Solanaceae. It is an important commercial crop in China. In 2020, a leaf spot disease was observed on tobacco leaves in commercial fields in the Hunan Province of China. Symptoms appeared as water-soaked, yellow-green spots, then turned dark brown, and coalesced into larger necrotic lesions, often leading to leaf wilt. Approximately 20% of the plants in a 50-ha area were infected, exhibiting symptomatic spots on 60% of these leaves. Symptomatic leaf samples were collected and cut into small pieces, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40s, rinsed with sterile distilled water for three times, plated on potato dextrose agar (PDA) and incubated at 26°C in the dark. Isolates with similar morphology were developed from ten samples. Fungal isolates produced densely, white to dark green, aerial mycelium. Conidia were straight, hyaline, aseptate, cylindrical, contained oil globules, and 15 to 25 µm × 3.0 to 4.0 µm (n=50). Appressoria were dark brown, irregularly shaped, 5.5 to 10.0 μm × 4.5 to 6.5 μm (n=50). These morphological characteristics were typical of Colletotrichum cliviicola (Yang et al. 2009). For molecular identification, the internal transcribed spacer (ITS) region of rDNA, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS-1) genes of a representative isolate CS16-2 were amplified and sequenced using the primer pairs as described previously (Weir et al. 2012). These sequences were deposited in GenBank (GenBank Accession Nos. MW649137 for ITS, MW656181 for ACT, MW656182 for GAPDH and MW656183 for CHS-1). BLAST analysis showed that they had 99.46% to 100% identity to the corresponding sequences of C. cliviicola strains. A concatenated phylogenetic tree was generated, using the ACT, GAPDH and CHS-1 sequences of the isolate CS16-2 and other closely matching Colletotrichum species obtained from the GenBank. We found that the CS16-2 was grouped with the C. cliviicola clade with 97% bootstrap support, including the C. cliviicola strain AH1B6 (Wang et al. 2016). Pathogenicity was tested spraying 2-month-old potted tobacco plants until runoff with a conidial suspension (105 spores/ml). Leaves were mock inoculated with sterilized water. The pathogenicity tests were performed twice, with three replicate plants each. Plants were kept in humid chambers at 26°C with a 12-h photoperiod. Five days post-inoculation, the inoculated plants developed symptoms of consisting of the yellow-brown necrotic lesion resembling the symptoms that were observed in fields, while the control plants remained symptomless. C. cliviicola was re-isolated and identified by morphological and molecular methods as described above. Currently, C. cliviicola has been reported to be the causal agent of anthracnose in some plants, such as soybean (Zhou et al. 2017) and Zamioculcas zamiifolia (Barbieri et al. 2017). However, to our knowledge, this is the first report of C. cliviicola causing leaf spot on tobacco in China and even in the word. Given that the may greatly affect the yield and quality of tobacco production, growers should be prepared to manage this new disease. This work might provide further insight for disease diagnosis on tobacco as some other Colletotrichum species, such as C. fructicola (Wang et al. 2016) and C. karsti (Zhao et al. 2020), have also been responsible for anthracnose.

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First report of leaf spot disease caused by Stemphylium eturmiunum on American sweetgum (Liquidambar styraciflua L.) in China

Plant Disease ◽

10.1094/pdis-09-20-1877-pdn ◽

2021 ◽

Author(s):

Yun-fei Mao ◽

Li Jin ◽

Huiyue Chen ◽

Xiang-rong Zheng ◽

Minjia Wang ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Liquidambar Styraciflua ◽

Leaf Spot Disease ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Wood Processing ◽

Light Yellow

American sweetgum (Liquidambar styraciflua L.) is an important tree for landscaping and wood processing. In recent years, leaf spots on American sweetgum with disease incidence of about 53% were observed in about 1200 full grown plants in a field (about 8 ha) located in Pizhou, Jiangsu Province, China. Initially, dense reddish-brown spots appeared on both old and new leaves. Later, the spots expanded into dark brown lesions with yellow halos. Symptomatic leaf samples from different trees were collected and processed in the laboratory. For pathogen isolation, leaf sections (4×4mm) removed from the lesion margin were surface sterilized with 75% ethanol for 20s and then sterilized in 2% NaOCl for 30s, rinsed three times in sterile distilled water, incubated on potato dextrose agar (PDA) at 25 °C in the darkness. After 5 days of cultivation, the pure culture was obtained by single spore separation. 6 isolate samples from different leaves named FXA1 to FXA6 shared nearly identical morphological features. The isolate FXA1 (codes CFCC 54675) was deposited in the China Center for Type Culture Collection. On the PDA, the colonies were light yellow with dense mycelium, rough margin, and reverse brownish yellow. Conidiophores (23–35 × 6–10 µm) (n=60) were solitary, straight to flexuous. Conidia (19–34 × 10–21 µm) (n=60) were single, muriform, oblong, mid to deep brown, with 1 to 6 transverse septa. These morphological characteristics resemble Stemphylium eturmiunum (Simmons 2001). Genomic DNA was extracted from mycelium following the CTAB method. The ITS region, gapdh, and cmdA genes were amplified and sequenced with the primers ITS5/ITS4 (Woudenberg et al. 2017), gpd1/gpd2 (Berbee et al. 1999), and CALDF1/CALDR2 (Lawrence et al. 2013), respectively. A maximum likelihood phylogenetic analysis based on ITS, gapdh and cmdA (accession nos. MT898502-MT898507, MT902342-MT902347, MT902336-MT902341) sequences using MEGA 7.0 revealed that the isolates were placed in the same clade as S. eturmiunum with 98% bootstrap support. All seedlings for pathogenicity tests were enclosed in plastic transparent incubators to maintain high relative humidity (90%-100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. For pathogenicity, the conidial suspension (105 spores/ml) of each isolate was sprayed respectively onto healthy leaves of L. styraciflua potted seedlings (2-year-old, 3 replicate plants per isolate). As a control, 3 seedlings were sprayed with sterile distilled water. After 7 days, dense reddish-brown spots were observed on all inoculated leaves. In another set of tests, healthy plants (3 leaves per plant, 3 replicate plants per isolate) were wound-inoculated with mycelial plugs (4×4mm) and inoculated with sterile PDA plugs as a control. After 7 days, brown lesions with light yellow halo were observed on all inoculation sites with the mycelial plugs. Controls remained asymptomatic in the entire experiment. The pathogen was reisolated from symptomatic tissues and identified as S. eturmiunum but was not recovered from the control. The experiment was repeated twice with the similar results, fulfilling Koch’s postulates. S. eturmiunum had been reported on tomato (Andersen et al. 2004), wheat (Poursafar et al. 2016), garlic (L. Fu et al. 2019) but not on woody plant leaves. To our knowledge, this is the first report of S. eturmiunum causing leaf spot on L. styraciflua in the world. This disease poses a potential threat to American sweetgum and wheat in Pizhou.

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First Report of Anthracnose on Bletilla striata Caused by Colletotrichum fructicola in China

Plant Disease ◽

10.1094/pdis-05-21-1008-pdn ◽

2021 ◽

Author(s):

Shao-Mei Wang ◽

Juan Huang ◽

Miao-Hua Zheng ◽

Ying-Na Wang ◽

Qing Yuan ◽

...

Keyword(s):

Leaf Spot ◽

Manganese Superoxide Dismutase ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Infected Leaf ◽

Post Inoculation ◽

Conidial Suspension ◽

Sterile Water ◽

Transparent Plastic ◽

Bletilla Striata

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is a traditional Chinese medicinal plant. In April 2018 and 2019, a leaf spot disease was observed on ∼20% of B. striata plants in two fields (∼1.4 h) in Guilin, Guangxi Province, China (Fig.1 A). Small, circular, brown spots were initially observed on the leaf surfaces, which progressively expanded into large, sunken, dark brown, necrotic areas. As the disease progressed, lesions merged into large, irregular spots, ultimately resulting in abscission. To determine the causal agent, small pieces (5 mm x 5 mm) were collected from the infected leaf tissues (n = 18), surface sterilized in 1% NaOCl for 2 min, and rinsed three times with sterile water. Then, the tissues were placed on potato dextrose agar (PDA) with chloramphenicol (0.1 g/L) and incubated under 12 h photoperiod at 26°C for 3 days. Seventeen isolates were obtained, of which twelve isolates with similar morphological characteristics were obtained from the germinated spores on PDA. Seven-day-old colonies on PDA appeared cottony, pale white to pale gray from above, and grayish-green from below. Conidia of strain BJ-101.3 were hyaline, aseptate, straight, and cylindrical, with rounded ends (Fig.1 E-G), measuring 11.3 to 15.9 μm × 4.0 to 6.4 μm (n = 50). Appressoria were brown to dark brown, with different shapes and a smooth edge (Fig.1 H-I), measuring 6.3 to 10.0 μm × 4.1 to 8.0 μm (n = 50). Morphological features were similar to C. gloeosporioides species complex (Weir et al. 2012, Fuentes-Aragón et al. 2018). For molecular identification, DNA was extracted from two isolates BJ-101.3 and BJ-101.13, following the CTAB method (Guo et al. 2000). The internal transcribed spacer (ITS) region, partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), manganese superoxide dismutase (SOD2), beta-tubulin (TUB2), glutamine synthetase (GS), and Apn2-Mat1-2 intergenic spacer and partial mating-type (ApMat) genes were amplified by PCR and sequenced (Weir et al. 2012, Silva et al. 2012, Vieira et al. 2017). The obtained sequences were deposited in GenBank (MW386818, MW386819, MW403508 to MW403519, and MW888410 to MW888413). BLASTN analysis of the obtained sequences showed 99% identity with those of C. fructicola (JX010165，JX010033, FJ917508, FJ907426, JX009866, JX010095, JX010327, JX010405, JQ807838) (Weir et al. 2012, Liu et al. 2015). A phylogenetic tree based on the concatenated sequences confirmed the isolates as C. fructicola (Fig.2). Furthermore, pathogenicity tests were conducted on six 1.5-year-old B. striata plants. Healthy leaves on the plants were inoculated with the conidial suspensions (106 conidia/mL; 10 μL) of the strains BJ-101.3 and BJ101.13. The conidial suspension of each isolate was inoculated onto at least three leaves. Another three plants inoculated with sterile water served as the control. All plants were covered with transparent plastic bags and incubated in a greenhouse at 26°C for 14 days with a 12 h photoperiod. Nine days post-inoculation, the inoculated leaves showed leaf spot symptoms, while the control plants remained symptomless (Fig.1 B-C). The experiments repeated three times showed similar results. Finally, C. fructicola was consistently reisolated from the infected leaves and confirmed by morphology and sequencing, fulfilling Koch’s postulates. The outcome of this study will help in developing effective management measures against anthracnose of B. striata.

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First Report of Leaf Spot Caused by Cladosporium tenuissimum on Panicle Hydrangea (Hydrangea paniculate) in China

Plant Disease ◽

10.1094/pdis-12-20-2640-pdn ◽

2021 ◽

Author(s):

Chenxu Li ◽

Peng Cao ◽

Chuanjiao Du ◽

Xi Xu ◽

Wensheng Xiang ◽

...

Keyword(s):

Dna Sequences ◽

Leaf Spot ◽

Disease Incidence ◽

Elongation Factor ◽

Flowering Plant ◽

Leaf Spot Disease ◽

Infected Leaf ◽

Conidial Suspension ◽

First Report ◽

Dark Green

Panicle Hydrangea (Hydrangea paniculate) is an ornamental flowering plant native to China and Japan. In August 2019, leaf spot symptoms with about 30% disease incidence were observed on panicle hydrangea in two grower fields (about 0.1 ha in total) of Northeast Agriculture University, China (126.72°E, 45.74°N). Symptoms initially appeared on the lower and older leaves and showed small subcircular brown spots with dark-brown edges on both sides. As the disease progressed, the necrotic spots enlarged, became irregular, coalesced, and the infected leaf blighted in approximately 2 weeks. Panicle hydrangea leaf samples (n=15) from different plants that showed spot symptoms were collected and surface sterilized with 70% ethanol for 10 s, followed by 0.5% NaClO treatment for 4 min, and rinsed in sterile water 3 times. Thereafter, leaf samples were placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. Fifteen hyphal-tipped pure cultures were obtained. Colonies growing on PDA for 7 days were olive green to dark green, exhibited a velvet-like texture and sometimes were radially furrowed and wrinkled. Margins varied from white gray to dark green without prominent exudates. The back of the plate showed dark green to black. Conidiophores were up to 180 to 600 µm long, 2.8 to 4.5 µm wide (n=50), subcylindrical-filiform, straight, septate, and unbranched or rarely branched. Ramoconidia were 0 to 1 septate, cylindrical to clavate, smooth-walled, 8 to 22 μm long (n=50). Conidia were single-celled, lemon-shaped, smooth-walled and 2.0 to 5.0 µm (diameter) (n=50). To confirm the identity, three genomic DNA regions, internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (EF), and actin (ACT) of the representative isolate BAI-1 were amplified with primer pairs ITS1/4, EF1-728F/986R, and ACT-512F/783R, respectively (Bensch et al. 2012; Jo et al. 2018). DNA sequences of the isolate from ITS, EF, and ACT showed 99.81% (514/515 bp), 99.10% (219/221 bp), and 99.54% (216/217 bp) nucleotide identity with those of C. tenuissimum CBS 125995, respectively (GenBank accession nos. HM148197, HM148442, and HM148687). The sequences of isolate BAI-1 were deposited in GenBank (accession nos. MW045455, MW052465, and MW052466). To fulfill Koch’s postulates, five healthy 2-year-old panicle hydrangea plants grown in pots were surface sterilized with 70% ethanol, washed twice with sterile distilled water, and sprayed with a conidial suspension of strain BAI-1 (adjusted to 1×106 conidia/ml using a hemocytometer), maintained in a greenhouse at 25°C and 85% relative humidity. Five plants sprayed with sterilized water served as controls. The inoculated plants showed leaf spot symptoms that were similar to those previously observed in the fields after 7 days, whereas control leaves remained healthy. The fungus was reisolated from symptomatic leaves and its identity was confirmed by morphological and molecular method. These experiments were repeated twice. So far, C. tenuissimum was reported to cause leaf spot of alfalfa (Han et al. 2019) and castor (Liu et al. 2019). To our knowledge, this is the first report of leaf spot disease in panicle hydrangea caused by C. tenuissimum in China. Leaf spot has a negative effect on the aesthetic value of panicle hydrangea, and this report will assist with monitoring distribution of the disease as well as developing management recommendations.

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