First report of leaf spot caused by Colletotrichum siamense on Salix matsudana in China

Salix matsudana Koidz. (Chinese willow) is an important landscaping tree species widely grown in China (Zhang et al. 2017). In October 2019, a characteristic leaf spot disease of S. matsudana was found on the campus of Nanjing Forestry University. Most 25-year-old S. matsudana trees (13 out of 21, approximately 62%) on campus showed the leaf spot disease. On average, 70% of the leaves per individual tree were affected by this disease. Foliar symptoms began as dark brown, irregular spots and the centers were gray-white, gradually enlarging with time. Leaf spot symptomatic leaves were collected from three infected S. matsudana trees (10 leaves/tree), and small infected tissues (3–4 mm2) were surface-sterilized in 75% ethanol for 30 s, 1% NaClO for 90 s, rinsed in ddH2O, dried on sterilized filter paper, and plated on potato dextrose agar (PDA), and then incubated at 25°C. Three isolates (NHY1-1, NHY1-2, and NHY1-3) of the same fungus were obtained in 85% of the samples and deposited in China's Forestry Culture Collection Center (NHY1-1: cfcc55354, NHY1-2: cfcc55355, NHY1-3: cfcc55359). The colonies of three isolates were white, but the reverse side was grayish-white. The conidia of NHY1-1 were one-celled, straight, subcylindrical, hyaline, 14.4 ± 0.9 × 5.4 ± 0.4 µm (n = 50), with a rounded end. Conidiophores were hyaline to pale brown, septate, and branched. Appressoria were one-celled, ellipsoidal, brown or dark brown, thick-walled, 8.0 ± 0.9 × 5.9 ± 0.5 µm (n = 50). The conidia and appressoria of the other two isolates weralmost identical to NHY1-1. The morphological characters of the three isolates were matched with those of the Colletotrichum gloeosporioides complex (Weir et al. 2012). For accurate identification, the DNA of the three isolates was extracted. The internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), superoxide dismutase (SOD2), and β-tubulin 2 (TUB2) genes were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF1/GDR1, SODglo2-F/SODglo2-R, and Bt2a/Bt2b, respectively (Weir et al. 2012). The sequences were deposited in GenBank [Accession Nos. MW784679 and MW808959 to MW808964 for NHY1-1; MW784726 and MW808965 to MW808970 for NHY1-2; MW784729 and MW808971 to MW808976 for NHY1-3]. A BLAST search of GenBank showed that ITS, ACT, CAL, GAPDH, SOD2, and TUB2 sequences of the three isolates were identical to Colletotrichum siamense at a high level (>99%), and CHS-1 sequences of three isolates were consistent with Colletotrichum fructicola at a high level (>99%). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, SOD2, and TUB2) placed NHY1-1, NHY1-2, and NHY1-3 in the clade of C. siamense with high bootstrap support values (ML/BI = 93/1). The pathogenicity of three isolates were tested on potted 2-yr-old seedlings (50-cm tall) of S. matsudana, which were grown in a greenhouse. Healthy leaves were wounded with a sterile needle and then inoculated with 10 µL of conidial suspension (106 conidia/mL). Controls were treated with ddH2O (Zhu et al. 2019). In total, 12 seedlings were inoculated including controls. Three seedlings/isolate and 10 leaves/seedling were used for each treatment. The plants were covered with plastic bags after inoculation and sterilized H2O was sprayed into the bags twice/day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2 / 16 ± 2°C. Within 7 days, all the inoculated points showed lesions similar to those observed in field, whereas controls were asymptomatic. The infection rate of each of the three isolates is 100%. C. siamense was re-isolated from the lesions, whereas no fungus was isolated from control leaves. The diseases caused by C. siamense often occur in tropical and subtropical regions of China, with a wide range of hosts, such as Hevea brasiliensis and Coffea arabica, etc. (Cao et al. 2019; Liu et al. 2018). This is the first report of C. siamense causing leaf spot of S. matsudana in China and the world. These data will help to develop effective strategies for managing this newly emerging 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 a Leaf Spot Disease of Bells-of-Ireland (Moluccella laevis) Caused by Cercospora apii in California

Plant Disease ◽

10.1094/pdis.2003.87.2.203a ◽

2003 ◽

Vol 87 (2) ◽

pp. 203-203

Author(s):

S. T. Koike ◽

S. A. Tjosvold ◽

J. Z. Groenewald ◽

P. W. Crous

Keyword(s):

Leaf Spot ◽

Sequence Data ◽

Disease Incidence ◽

Leaf Spot Disease ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Spot Disease ◽

Leaf Spots ◽

Initial Symptoms

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

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First Report of Leaf Spot Disease Caused by Exserohilum rostratum on Pineapple in Hainan Province, China

Plant Disease ◽

10.1094/pdis-11-11-0979 ◽

2012 ◽

Vol 96 (3) ◽

pp. 458-458 ◽

Cited By ~ 4

Author(s):

Z. W. Luo ◽

F. He ◽

H. Y. Fan ◽

X. H. Wang ◽

M. Hua ◽

...

Keyword(s):

Leaf Spot ◽

Leaf Spot Disease ◽

Hainan Province ◽

Conidial Suspension ◽

Potato Dextrose Agar Medium ◽

First Report ◽

Spot Disease ◽

Exserohilum Rostratum ◽

Plant Pathol ◽

Pineapple Leaves

Pineapple (Ananas comosus (L.) Merr.) is an important perennial monocotyledonous plant that serves as an important fruit crop globally and is also produced in the Hainan Province of China where production in 2009 was 296,600 t. In July 2009, atypical symptoms of a leaf spot disease were observed on mature pineapple leaves in Chengmai County; approximately 15% of plants propagated from suckers became symptomatic after 150 to 300 days, eventually causing a 3 to 10% yield loss. In the initial infection stage, grayish white-to-yellowish white spots emerged on the leaf surfaces that ranged from 1.0 to 2.4 × 0.3 to 0.7 cm; black specks were not always present in the spots. Leaf spots also had distinctive light brown-to-reddish brown banding pattern on the edges. Several spots would often merge to form large lesions, 6.5 to 15.4 × 2.5 to 5.6 cm, covering more than 67% of the leaf surface, which can lead to death of the plant. Infected pineapple leaves collected from an orchard of Chengmai County were surface sterilized (75% ethanol for 30 s, 0.1% HgCl2 for 2 min, and rinsed three times in sterile distilled water). Leaf pieces were placed on potato dextrose agar medium and then incubated at 25°C. The emerging fungal colonies were grayish white to brown. Similar strains were obtained from Qionghai City and Wanning City subsequently. Two isolates, ITF0706-1 and ITF0706-2, were used in confirmation of the identity of the pathogen and in pathogenicity tests. Colonies were fast growing (more than 15 mm per day at 25 to 30°C) with dense aerial mycelia. Conidia were fusiform, pyriform to oval or cylindrical, olive brown to dark brown, 3 to 10 septate (typically 5 to 8), 33.2 to 102.5 × 9.0 to 21.3 μm, with a strongly protruding hilum bulged from the basal cell, which were similar to the Type A conidia described by Lin et al. (3). The strains were subjected to PCR amplification of the internal transcribed spacer (ITS)1-5.8S-ITS2 regions with universal primer pair ITS1/ITS4. The ITS sequence comparisons (GenBank Accession Nos. JN711431 and JN711432) shared between 99.60 and 99.83% identity with the isolate CATAS-ER01 (GenBank Accession No. GQ169762). According to morphological and molecular analysis, the two strains were identified as Exserohilum rostratum (Drechs.) Leonard & Suggs. Pathogenicity experiments were conducted five times and carried out by spraying a conidial suspension (105 CFU/ml) on newly matured leaves of healthy pineapple plants; plants sprayed with sterile water served as the negative control. Plants were incubated in the growth chamber at 20 to 25°C. Symptoms of leaf spot developed on test plants 7 days after inoculation while the control plants remained asymptomatic. Koch's postulates were fulfilled with the reisolation of the two fungal strains. Currently, E. rostratum is one of the most common pathogens on Bromeliads in Florida (2) and has been reported on Zea mays (4), Musa paradisiacal (3), and Calathea picturata (1) in China, but to our knowledge, this is the first report of leaf spot disease caused by E. rostratum on pineapple in Hainan Province of P.R. China. References: (1) L. L. Chern et al. Plant Dis. 95:1033, 2011. (2) R. M. Leahy. Plant Pathol. Circ. No. 393. Florida Department of Agriculture and Consumer Services Division of Plant Industry, 1999. (3) S. H. Lin et al. Australas. Plant Pathol. 40:246, 2011. (4) J. N. Tsai et al. Plant Pathol. Bull. 10:181, 2001.

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First Report of Leaf Spot Disease Caused by Epicoccum nigrum on Lablab purpureus in India

Plant Disease ◽

10.1094/pdis-07-13-0798-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 284-284 ◽

Cited By ~ 12

Author(s):

S. Mahadevakumar ◽

K. M. Jayaramaiah ◽

G. R. Janardhana

Keyword(s):

Leaf Spot ◽

Leaf Surface ◽

Disease Incidence ◽

Leaf Spot Disease ◽

Post Inoculation ◽

Conidial Suspension ◽

Lablab Purpureus ◽

First Report ◽

Spot Disease ◽

Epicoccum Nigrum

Lablab purpureus (L.) Sweet (Indian bean) is an important pulse crop grown in arid and semi-arid regions of India. It is one of the most widely cultivated legume species and has multiple uses. During a September 2010 survey, we recorded a new leaf spot disease on L. purpureus in and around Mysore district (Karnataka state) with 40 to 80% disease incidence in 130 ha of field crop studied, which accounted for 20 to 35% estimated yield loss. The symptoms appeared as small necrotic spots on the upper leaf surface. The leaf spots were persistent under mild infection throughout the season with production of conidia in clusters on abaxial leaf surface. A Dueteromyceteous fungus was isolated from affected leaf tissues that were surface sterilized with 2% NaOCl2 solution then washed thrice, dried, inoculated on potato dextrose agar (PDA) medium, and incubated at 28 ± 2°C at 12 h alternate light and dark period for 7 days. The fungal colony with aerial mycelia interspersed with dark cushion-shaped sporodochia consists of short, compact conidiophores bearing large isodiametric, solitary, muricate, brown, globular to pear shaped conidia (29.43 to 23.92 μm). Fungal isolate was identified as Epicoccum sp. based on micro-morphological and cultural features (1). Further authenticity of the fungus was confirmed by PCR amplification of the internal transcribed spacer (ITS) region using ITS1/ITS4 universal primer. The amplified PCR product was purified, sequenced directly, and BLASTn search revealed 100% homology to Epicoccum nigrum Link. (DQ093668.1 and JX914480.1). A representative sequence of E. nigrum was deposited in GenBank (Accession No. KC568289.1). The isolated fungus was further tested for its pathogenicity on 30-day-old healthy L. purpureus plants under greenhouse conditions. A conidial suspension (106 conidia/ml) was applied as foliar spray (three replicates of 15 plants each) along with suitable controls. The plants were kept under high humidity (80%) for 5 days and at ambient temperature (28 ± 2°C). The appearance of leaf spot symptoms were observed after 25 days post inoculation. Further, the pathogen was re-isolated and confirmed by micro-morphological characteristics. E. nigrum has been reported to cause post-harvest decay of cantaloupe in Oklahoma (2). It has also been reported as an endophyte (3). Occurrence as a pathogen on lablab bean has not been previously reported. To our knowledge, this is the first report of the occurrence of E. nigrum on L. purpureus in India causing leaf spot disease. References: (1) H. L. Barnet and B. B. Hunter. Page 150 in: Illustrated Genera of Imperfect Fungi, 1972. (2) B. D. Bruten et al. Plant Dis. 77:1060, 1993. (3) L. C. Fávaro et al. PLoS One 7(6):e36826, 2012.

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First Report of Leaf Spot Disease Caused by Colletotrichum siamense on Vernicia montana in China

Plant Disease ◽

10.1094/pdis-12-20-2609-pdn ◽

2021 ◽

pp. PDIS-12-20-2609

Author(s):

D. Yang ◽

Y. Zhang ◽

Y. T. Zhang ◽

Q. H. Wang ◽

Y. K. Qi ◽

...

Keyword(s):

Leaf Spot ◽

Leaf Spot Disease ◽

First Report ◽

Spot Disease ◽

Colletotrichum Siamense

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First Report of Tomato Gray Leaf Spot Disease Caused by Stemphylium solani in Malaysia

Plant Disease ◽

10.1094/pdis-03-12-0223-pdn ◽

2012 ◽

Vol 96 (8) ◽

pp. 1226-1226

Author(s):

A. Nasehi ◽

J. B. Kadir ◽

M. A. Zainal Abidin ◽

M. Y. Wong ◽

F. Mahmodi

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Gray Leaf Spot ◽

Leaf Spot Disease ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Spot Disease ◽

Pcr Product ◽

Morphological Criteria

In June 2011, tomatoes (Solanum lycopersicum) in major growing areas of the Cameron Highlands and the Johor state in Malaysia were affected by a leaf spot disease. Disease incidence exceeded 80% in some severely infected regions. Symptoms on 50 observed plants initially appeared on leaves as small, brownish black specks, which later became grayish brown, angular lesions surrounded by a yellow border. As the lesions matured, the affected leaves dried up and became brittle and later developed cracks in the center of the lesions. A survey was performed in these growing areas and 27 isolates of the pathogen were isolated from the tomato leaves on potato carrot agar (PCA). The isolates were purified by the single spore technique and were transferred onto PCA and V8 agar media for conidiophore and conidia production under alternating light (8 hours per day) and darkness (16 hours per day) (4). Colonies on PCA and V8 agar exhibited grey mycelium and numerous conidia were formed at the terminal end of conidiophores. The conidiophores were up to 240 μm long. Conidia were oblong with 2 to 11 transverse and 1 to 6 longitudinal septa and were 24 to 69.6 μm long × 9.6 to 14.4 μm wide. The pathogen was identified as Stemphylium solani on the basis of morphological criteria (2). In addition, DNA was extracted and the internal transcribed spacer region (ITS) was amplified by universal primers ITS5 and ITS4 (1). The PCR product was purified by the commercial PCR purification kit and the purified PCR product sequenced. The resulting sequences were 100% identical to published S. solani sequences (GenBank Accestion Nos. AF203451 and HQ840713). The amplified ITS region was deposited with NCBI GenBank under Accession No. JQ657726. A representative isolate of the pathogen was inoculated on detached 45-day-old tomato leaves of Malaysian cultivar 152177-A for pathogenicity testing. One wounded and two nonwounded leaflets per leaf were used in this experiment. The leaves were wounded by applying pressure to leaf blades with the serrated edge of a forceps. A 20-μl drop of conidial suspension containing 105 conidia/ml was used to inoculate these leaves (3). The inoculated leaves were placed on moist filter paper in petri dishes and incubated for 48 h at 25°C. Control leaves were inoculated with sterilized distilled water. After 7 days, typical symptoms for S. solani similar to those observed in the farmers' fields developed on both wounded and nonwounded inoculated leaves, but not on noninoculated controls, and S. solani was consistently reisolated. To our knowledge, this is the first report of S. solani causing gray leaf spot of tomato in Malaysia. References: (1) M. P. S. Camara et al. Mycologia 94:660, 2002. (2) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (3) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (4) E. G. Simmons. CBS Biodiversity Series 6:775, 2007.

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First Report of Leaf Spot Caused by Colletotrichum siamense on Magnolia grandiflora in China

Plant Disease ◽

10.1094/pdis-08-21-1698-pdn ◽

2021 ◽

Author(s):

Yang Zhou ◽

Dou Yang ◽

Lingping Zhang ◽

Yong-Chun Zeng ◽

Yang Zhang

Keyword(s):

Phylogenetic Tree ◽

Leaf Spot ◽

Control Strategies ◽

Epidemiologic Studies ◽

Leaf Spot Disease ◽

Single Spore ◽

Individual Tree ◽

First Report ◽

Magnolia Grandiflora ◽

Colletotrichum Siamense

Magnolia grandiflora (Southern magnolia) is a popular evergreen tree, planted especially as an ornamental for landscaping. In September 2019, leaf spots were observed on M. grandiflora at the campus of Jiangxi Agricultural University (28°45′56″N, 115°50′21″E). Approximately 64% (23 out of 36) M. grandiflora trees (most 24-year-old) occurred leaf spot disease at the campus. On average, 40% of the leaves per individual tree were affected. Foliar symptoms began as small dark brown lesions formed along the leaf margins. As the disease developed, the lesions’ center was sunken with a dark brown border. Symptomatic leaves were collected and cut into 5 × 5 mm pieces. Leaf pieces from the margin of the necrotic tissue were surface sterilized in 70% ethanol for 30 s followed by 2% NaOCl for 1 min and then rinsed in sterile water three times. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Of more than 35 isolates, most shared a similar morphology, with an isolation rate of 85%. Three isolates (JNG-1, JNG-2, and JNG-3) were chosen for single-spore purification and used for morphological characterization and identification. Colonies on PDA of the three isolates were white, cottony, and grayish-white on the undersides of the culture. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 4.4-5.6 × 13.2-17.8 µm (4.7 ± 0.3 × 14.6 ± 1.0 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.5-9.2 × 4.6-6.5 µm (7.3 ± 0.4 × 5.4 ± 0.3 µm, n=100). Morphological features were similar to Colletotrichum siamense as previously described (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), beta-tubulin 2 (TUB2), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-345R and GDF/GDR (Weir et al. 2012), respectively and sequenced. All sequences were deposited into GenBank (ITS, MZ325948-MZ325950; ACT, MZ461477 - MZ461479; GAPDH, MZ461483 - MZ461485; TUB2, MZ461486 - MZ461488; CHS-1, MZ441182 - MZ441184; CAL, MZ461480 - MZ461482). A neighbor-joining phylogenetic tree was constructed with MEGA 7.0 using the concatenation of multiple sequences (Kumar et al. 2016). According to the phylogenetic tree, all three isolates fall within the C. siamense clade (boot support 96%). The pathogenicity of three isolates were tested on M. grandiflora plants, which were grown in the field. Healthy leaves were wounded with a sterile needle and then inoculated with 10 µL of spore suspension (106 conidia/mL). Controls were treated with ddH2O (Zhu et al. 2019). All the inoculated leaves were covered with black plastic bags to keep a high-humidity environment for 2 days. All the inoculated leaves showed similar symptoms to those observed in field, whereas control leaves were asymptomatic for 10 days. The infection rate was 100%. C. siamense was re-isolated from the lesions, whereas no fungus was isolated from control leaves. It was confirmed that C. gloeosporioides is the causal agent of leaf spot on Magnolia virginiana in America (Xiao et al. 2004). However, this is the first report of C. siamense causing leaf spot on M. grandiflora in China. This study provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

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First Report of Gray Leaf Spot of Mango (Mangifera indica) Caused by Pestalotiopsis mangiferae in Taiwan

Plant Disease ◽

10.1094/pdis-91-12-1684b ◽

2007 ◽

Vol 91 (12) ◽

pp. 1684-1684 ◽

Cited By ~ 6

Author(s):

Y. Ko ◽

K. S. Yao ◽

C. Y. Chen ◽

C. H. Lin

Keyword(s):

Leaf Spot ◽

Mangifera Indica ◽

Gray Leaf Spot ◽

Leaf Spot Disease ◽

Continuous Exposure ◽

Conidial Suspension ◽

Tropical Fruit ◽

First Report ◽

Spot Disease ◽

Initial Symptoms

Mango (Mangifera indica L.; family Anacardiaceae) is one of the world's most important fruit crops and is widely grown in tropical and subtropical regions. Since 2001, a leaf spot disease was found in mango orchards of Taiwan. Now, the disease was observed throughout (approximately 21,000 ha) Taiwan in moderate to severe form, thus affecting the general health of mango trees and orchards. Initial symptoms were small, yellow-to-brown spots on leaves. Later, the irregularly shaped spots, ranging from a few millimeters to a few centimeters in diameter, turned white to gray and coalesced to form larger gray patches. Lesions had slightly raised dark margins. On mature lesions, numerous black acervuli, measuring 290 to 328 μm in diameter, developed on the gray necrotic areas. Single conidial isolates of the fungus were identified morphologically as Pestalotiopsis mangiferae (Henn.) Steyaert (2,3) and were consistently isolated from the diseased mango leaves on acidified (0.06% lactic acid) potato dextrose agar (PDA) medium incubated at 25 ± 1°C. Initially, the fungus grew (3 mm per day) on PDA as a white, chalky colony that subsequently turned gray after 2 weeks. Acervuli developed in culture after continuous exposure to light for 9 to 12 days at 20 to 30°C. Abundant conidia oozed from the acervulus as a creamy mass. The conidia (17.6 to 25.4 μm long and 4.8 to 7.1 μm wide) were fusiform and usually straight to slightly curved with four septa. Three median cells were olivaceous and larger than the hyaline apical and basal cells. The apical cells bore three (rarely four) cylindrical appendages. Pathogenicity tests were conducted with either 3-day-old mycelial discs or conidial suspension (105 conidia per ml) obtained from 8- to 10-day-old cultures. Four leaves on each of 10 trees were inoculated. Before inoculation, the leaves were washed with a mild detergent, rinsed with tap water, and then surface sterilized with 70% ethanol. Leaves were wounded with a needle and exposed to either a 5-mm mycelial disc or 0.2 ml of the spore suspension. The inoculated areas were wrapped with cotton pads saturated with sterile water and the leaves were covered with polyethylene bags for 3 days to maintain high relative humidity. Wounded leaves inoculated with PDA discs alone served as controls. The symptoms described above were observed on all inoculated leaves, whereas uninoculated leaves remained completely free from symptoms. Reisolation from the inoculated leaves consistently yielded P. mangiferae, thus fulfilling Koch's postulates. Gray leaf spot is a common disease of mangos in the tropics and is widely distributed in Africa and Asia (1–3); however, to our knowledge, this is the first report of gray leaf spot disease affecting mango in Taiwan. References: (1) T. K. Lim and K. C. Khoo. Diseases and Disorders of Mango in Malaysia. Tropical Press. Malaysia, 1985. (2) J. E. M. Mordue. No. 676 in: CMI Descriptions of Pathogenic Fungi and Bacteria. Surrey, England, 1980. (3) R. C. Ploetz et al. Compendium of Tropical Fruit Diseases. The American Phytopathological Society. St. Paul, MN, 1994.

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First Report of Leaf Spot Disease of Peony Caused by Seimatosporium botan in China

Plant Disease ◽

10.1094/pdis-10-10-0735 ◽

2011 ◽

Vol 95 (2) ◽

pp. 226-226

Author(s):

Y. B. Duan ◽

Z. Z. Yu ◽

Y. B. Kang

Keyword(s):

Basal Cell ◽

Leaf Spot ◽

Apical Cell ◽

Leaf Spot Disease ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

Humid Atmosphere ◽

First Report ◽

Spot Disease ◽

Initial Symptoms

Tree peony (Paeonia suffruticosa Andrews), a perennial ligneous deciduous shrub in the Paeoniaceae family, is known for its beautiful and charming flowers. It is regarded as the flower symbol of China and is cultivated throughout the country. In August 2008, a previously unknown leaf spot was observed on peony cultivated in the Mountain Peony Garden located in the Luoyang area of Henan Province, China. In 2009, the leaf spot disease was observed in some gardens in the city of Luoyang, China. Initial symptoms appeared as small, round or irregular, brown, necrotic lesions in the middle of leaves. These lesions gradually enlarged up to 1 cm in diameter and were circular or irregular, brown to dark brown, and brown on the margins. In a humid atmosphere, black, sessile, discoid acervuli developed on the lesions, and the lesions sometimes became waxy-like, eventually coalesced, and nearly covered the entire leaf. Conidia produced in acervuli had two morphologically different types. One type had a single basal appendage, ellipsoid to fusiform, transversely three septate, 16 to 20 × 5 to 7 μm, smooth, basal cell obconic with a truncate base, subhyaline, 3 to 5 μm long; two central cells subcylindrical to dolioform, brown to dark brown, 8 to 10 μm long, apical cell conical with rounded apex, concolorous with the central cells, 4 to 5 μm long, basal appendage filiform, unbranched, excentric, 4 to 8 μm long. The other type had a single appendage at both ends, fusiform to subcylindrical, transversely three septate, 16 to 20 × 4 to 5 μm, smooth; basal cell obconic with a truncate base, subhyaline, 4 to 5 μm long; two central cells subcylindrical to dolioform, pale brown, 8 to 11 μm long; apical cell conical with an acute apex, hyaline to subhyaline, 4 to 5 μm long; basal appendage filiform, unbranched, excentric, 4 to 8 μm long; apical appendage filiform, unbranched, 4 to 8 μm long. Single conidial isolates of both types of conidia yielded identical colonies, which produced both types of conidia on potato dextrose agar (PDA), thus showing that both types of conidia belonged to the same fungus. Colonies on PDA were slimy in appearance, yellow to villous with an irregular taupe margin; reverse brown to grayish brown. Cultural and conidial characteristics of the isolates were similar to those of Seimatosporium botan (1). The DNA sequence for the fungus showed internal transcribed spacer region (ITS1-5.8S-ITS2) sequences (GenBank Accession No. HM067840) with 93% sequence identity to S. discosioides (Accession Nos. EF600970.1 and EF600969.1). This is the first submission of a S. botan sequence to GenBank. To determine pathogenicity, 20 healthy leaves of P. suffruticosa were inoculated by spraying a conidial suspension of S. botan onto the foliage. Ten leaves were sprayed with sterile water and served as controls. Plants were covered with plastic for 24 h to maintain high relative humidity. After 15 days, the symptoms described above were observed on leaves in all inoculated plants, whereas symptoms did not develop on the control plants. The pathogen was reisolated from inoculated leaves, fulfilling Koch's postulates. On the basis of morphology and ITS region sequences, we conclude that S. botan is the causal agent of leaf spots of P. suffruticosa. There is a report of S. botan on P. suffruticosa stems in Japan (1), but to our knowledge, this is the first report of leaf spot disease of peony caused by S. botan in China. References: (1) S. Hatakeyama et al. Mycoscience 45:106, 2004.

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First report of Alternaria alternata causing leaf spot on Oat (Avena sativa) in India

Plant Disease ◽

10.1094/pdis-02-21-0322-pdn ◽

2021 ◽

Author(s):

Boda Praveen ◽

Prasanna Kumar M.K. ◽

Devanna Pramesh ◽

PALANNA K B ◽

Buela P.P. ◽

...

Keyword(s):

Avena Sativa ◽

Leaf Spot ◽

Leaf Spot Disease ◽

Post Inoculation ◽

Conidial Suspension ◽

Cultural Characteristics ◽

Leaf Quality ◽

First Report ◽

Spot Disease ◽

Pathogenicity Assay

Oat (Avena sativa L.) is an important cereal crop grown worldwide primarily for food and animal feed. In November 2019, a leaf spot disease was observed on the oat plants at Mandya (12.5218° N, 76.8951° E), Karnataka, India. The disease incidence on plants was ranged between 43 to 57 percent. Initially, the symptoms appeared on leaves as small dark-brown spots surrounded by a yellow halo later turned to irregular necrotic spots with a yellow halo. A total of thirty leaves showing typical symptoms were collected from ten plants (three leaves per plant), cut into an area of 4-5 mm pieces at the junction of infected and healthy tissues. Cut tissues were soaked in 75% ethanol for 45 seconds, followed by 1% sodium hypochlorite solution for 1 min, rinsed five times in sterile distilled water, air dried, and placed on PDA and incubated at 25 ± 1 ℃. After 7 days of incubation, greyish fungal colonies appeared on PDA. Single-spore isolation method was employed to recover the pure cultures for five isolates. The colonies initially produced light-greyish aerial mycelia, then turned to dark-greyish upon maturity. Conidia were obclavate to pyriform and measured 17.34 to 46.97 µm long, 5.38 to 14.31 µm wide with 0 to 3 longitudinal, and 1 to 6 transverse septa with short beak (2.73 to 10.17µm) (n = 50.) Based on the morpho-cultural characteristics, the isolates were identified as Alternaria spp., and PCR assay using species-specific primers (AAF2/AAR3; Konstantinova, et al. 2002) confirmed the taxonomic identity of all five isolates as A. alternata. To further confirm the identity, the internal transcribed spacer (ITS), small subunit (SSU), glyceraldehyde-3-phosphate dehydrogenase (gapdh), RNA polymerase second largest subunit (rpb2), Alternaria major allergen (Alt a1), endopolygalacturonase (endoPG), an anonymous gene region OPA10-2, KOG1058 and translation elongation factor 1-alpha (tef1) of two isolates MAAS-1 and MAAS-2 were PCR amplified using the primers described previously (Woudenberg et al. 2015; Praveen et al. 2020) and the resultant PCR products were sequenced and deposited in NCBI GenBank (ITS: MW487388, MW741962, SSU: MW506220, MW752854, gapdh: MW506221, MW752855, rpb2: MW506222, MW752856, Alt a1: MW506223, MW752857, endoPG: MW506224, MW752858, OPA10-2: MW506225, MW752859, KOG1058: MW506226, MW752860, and tef1: MW506227, MW752861) which showed (99.62%, 99.81%), (100%, 100%), (100%, 99.66%), (100%, 100%), (99.58%, 99.15%), (99.55%, 99.32%), (99.53%, 99.68%), (99.23%, 99.56%) and (99.17%, 99.58%) identity with ITS (AF347031), SSU (KC584507), gapdh (AY278808), rpb2 (KC584375), Alt a1 (AY563301), endoPG (JQ811978), OPA10-2 (KP124632), KOG1058 (KP125233) and tef1 (KC584634) genes/genomic regions of type strain CBS916.96 of A. alternata respectively, confirming the identity of MAAS-1 as A. alternata. For pathogenicity assay, the conidial suspension (2 × 106 conidia/ml) of MAAS-1 isolate was artificially sprayed until runoff on ten healthy oat plants (cv. Kent, 35 days old) and ten plants sprayed with sterile water served as control. All plants were covered with polyethylene covers and kept under the greenhouse at 28 ± 1 ℃. The pathogenicity assay was repeated three times. After six days post-inoculation, small dark-brown spots with light-yellow halo appeared on leaves of MAAS-1inoculated plants. In comparison, no symptoms were observed on control plants. The fungal pathogen was re-isolated from the artificially infected plants and confirmed as A. alternata based on morpho-cultural characteristics and PCR assays. The leaf spot disease of Oat caused by A. alternata has already been reported from China (Chen et al. 2020); to our knowledge, it is the first report of A. alternata causing leaf spot on Oat in India. The leaf spot disease is an emerging threat to oat cultivation, and it reduces the grain yield and leaf quality; therefore, its management is essential for increasing productivity.

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