First report of Curvularia plantarum causing leaf spot on sweet potato (Ipomoea batatas) in China

Sweet potato [Ipomoea batatas (L.) Lam], is an extremely versatile vegetable that possesses high nutritional values. It is also a valuable medicinal plant having anti-cancer, antidiabetic, and anti-inflammatory activities. In July 2020, leaf spot was observed on leaves of sweet potato in Nanchang, China (28°45'51"N, 115°50'52"E), which affected the growth and development of the crop and caused tuberous roots yield losses of 25%. The disease incidence (total number of diseased plants / total number of surveyed plants × 100%) was 57% from a sampled population of 100 plants in the field. Symptomatic plants initially exhibited small, light brown, irregular-shaped spots on the leaves, subsequently coalescing to form large irregular brown lesions and some lesions finally fell off. Fifteen small pieces (each 5 mm2) of symptomatic leaves were excised from the junction of diseased and healthy tissue, surface sterilized in 75% ethanol solution for 30 sec and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water and incubated on potato dextrose agar (PDA) plates at 28°C in darkness. A total of seven fungal isolates with similar morphological characteristics were obtained as pure cultures by single-spore isolation. After 5 days of cultivation at 28°C, dark brown or blackish green colonies were observed, which developed brown, thick-walled, simple, or branched, and septate conidiophores. Conidia were 18.28 to 24.91 × 7.46 to 11.69 µm (average 21.27 × 9.48 µm, n = 100) in size, straight or slightly curved, middle cell unequally enlarged, brown to dark brown, apical, and basal cells slightly paler than the middle cells, with three septa. Based on morphological characteristics, the fungal isolates were suspected to be Curvularia plantarum (Raza et al. 2019). To further confirm the identification, three isolates (LGZ1, LGZ4 and LGZ5) were selected for molecular identification. The internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and translation elongation factor 1-alpha (EF1-α) genes were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004), gpd1/gpd2 (Berbee et al. 1999), EF-983F/EF-2218R (Rehner and Buckley 2005), respectively. The sequences of ITS region of the three isolates (accession nos. MW581905, MZ209268, and MZ227555) shared 100% identity with those of C. plantarum (accession nos. MT410571-72, MN044754-55). Their GAPDH gene sequences were identical (accession nos. MZ224017-19) and shared 100% identity with C. plantarum (accession nos. MN264120, MT432926, and MN053037-38). Similarly, EF1-α gene sequences were identical (accession nos. MZ224020-22) and had 100% identity with C. plantarum (accession nos. MT628901, MN263982-83). A maximum likelihood phylogenetic tree was built based on concatenated data from the sequences of ITS, GAPDH, and EF-1α by using MEGA 5. The three isolates LGZ1, LGZ4, and LGZ5 clustered with C. plantarum. The fungus was identified as C. plantarum by combining morphological and molecular characteristics. Pathogenicity tests were conducted by inoculating a conidial suspension (106 conidia/ml) on three healthy potted I. batatas plants (five leaves wounded with sterile needle of each potted plant were inoculated). In addition, fifteen wounded leaves of three potted plants were sprayed with sterile distilled water as a control. All plants were maintained in a climate box (12 h light/dark) at 25°C with 80% relative humidity. All the inoculated leaves started showing light brown flecks after 7 days, whereas the control leaves showed no symptoms. The pathogenicity test was conducted three times. The fungus was reisolated from all infected leaves of potted plants and confirmed as C. plantarum by morphological and molecular identification, fulfilling Koch’s postulates. To our knowledge, this is the first report of C. plantarum causing leaf spot on sweet potato in China. The discovery of this new disease and the identification of the pathogen will contribute to the disease management, provide useful information for reducing economic losses caused by C. plantarum, and lay a foundation for the further research of resistance breeding.

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First Report of a Chaetomella sp. Causing a Leaf Spot on Sansevieria trifasciata in China

Plant Disease ◽

10.1094/pdis-10-12-0993-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 992-992 ◽

Cited By ~ 4

Author(s):

Y. L. Li ◽

Z. Zhou ◽

W. Lu ◽

J. R. Ye

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Leaf Tissue ◽

Aerial Mycelium ◽

Control Treatment ◽

Distilled Water ◽

First Report ◽

Potted Plants ◽

Fungal Isolates

Sansevieria trifasciata originates from tropical West Africa. It is widely planted as a potted ornamental in China for improving indoor air quality (1). In February 2011, leaves of S. trifasciata plants in an ornamental market of Anle, Luoyang City, China, were observed with sunken brown lesions up to 20 mm in diameter, and with black pycnidia present in the lesions. One hundred potted plants were examined, with disease incidence at 20%. The symptomatic leaves affected the ornamental value of the plants. A section of leaf tissue from the periphery of two lesions from a plant was cut into 1 cm2 pieces, soaked in 70% ethanol for 30 s, sterilized with 0.1% HgCl2 for 2 min, then washed five times in sterilized distilled water. The pieces were incubated at 28°C on potato dextrose agar (PDA). Colonies of two isolates were brown with submerged hyphae, and aerial mycelium was rare. Abundant and scattered pycnidia were reniform, dark brown, and 200 to 350 × 100 to 250 μm. There were two types of setae on the pycnidia: 1) dark brown setae with inward curved tops, and 2) straight, brown setae. Conidia were hyaline, unicellular, cylindrical, and 3.75 to 6.25 × 1.25 to 2.50 μm. Morphological characteristics suggested the two fungal isolates were a Chaetomella sp. To confirm pathogenicity, six mature leaves of a potted S. trifasciata plant were wounded with a sterile pin after wiping each leaf surface with 70% ethanol and washing each leaf with sterilized distilled water three times. A 0.5 cm mycelial disk cut from the margin of a 5-day-old colony on a PDA plate was placed on each pin-wounded leaf, ensuring that the mycelium was in contact with the wound. Non-colonized PDA discs were placed on pin-wounded leaves as the control treatment. Each of two fungal isolates was inoculated on two leaves, and the control treatment was done similarly on two leaves. The inoculated plant was placed in a growth chamber at 28°C with 80% relative humidity. After 7 days, inoculated leaves produced brown lesions with black pycnidia, but no symptoms developed on the control leaves. A Chaetomella sp. was reisolated from the lesions of inoculated leaves, but not from the control leaves. An additional two potted plants were inoculated using the same methods as replications of the experiment, with identical results. To confirm the fungal identification, the internal transcribed spacer (ITS) region of rDNA of the two isolates was amplified using primers ITS1 and ITS4 (2) and sequenced. The sequences were identical (GenBank Accession No. KC515097) and exhibited 99% nucleotide identity to the ITS sequence of an isolate of Chaetomella sp. in GenBank (AJ301961). To our knowledge, this is the first report of a leaf spot of S. trifasciata caused by Chaetomella sp. in China as well as anywhere in the world. References: (1) X. Z. Guo et al. Subtropical Crops Commun. Zhejiang 27:9, 2005. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

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First report of Coniella castaneicola causing leaf blight on blueberry (Vaccinium virgatum) in China

Plant Disease ◽

10.1094/pdis-05-21-1122-pdn ◽

2021 ◽

Author(s):

Jiahao Lai ◽

Guihong Xiong ◽

Bing Liu ◽

Weigang Kuang ◽

Shuilin Song

Keyword(s):

Molecular Identification ◽

Morphological Characteristics ◽

Leaf Blight ◽

Yield Potential ◽

Effective Control ◽

Economic Losses ◽

Distilled Water ◽

First Report ◽

Fungal Isolates ◽

Blight Disease

Blueberry (Vaccinium virgatum), an economically important small fruit crop, is characterized by its highly nutritive compounds and high content and wide diversity of bioactive compounds (Miller et al. 2019). In September 2020, an unknown leaf blight disease was observed on Rabbiteye blueberry at the Agricultural Science and Technology Park of Jiangxi Agricultural University in Nanchang, China (28°45'51"N, 115°50'52"E). Disease surveys were conducted at that time, the results showed that disease incidence was 90% from a sampled population of 100 plants in the field, and this disease had not been found at other cultivation fields in Nanchang. Leaf blight disease on blueberry caused the leaves to shrivel and curl, or even fall off, which hindered floral bud development and subsequent yield potential. Symptoms of the disease initially appeared as irregular brown spots (1 to 7 mm in diameter) on the leaves, subsequently coalescing to form large irregular taupe lesions (4 to 15 mm in diameter) which became curly. As the disease progressed, irregular grey-brown and blighted lesion ran throughout the leaf lamina from leaf tip to entire leaf sheath and finally caused dieback and even shoot blight. To identify the causal agent, 15 small pieces (5 mm2) of symptomatic leaves were excised from the junction of diseased and healthy tissue, surface-sterilized in 75% ethanol solution for 30 sec and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water, and then incubated on potato dextrose agar (PDA) at 28°C for 5-7 days in darkness. Five fungal isolates showing similar morphological characteristics were obtained as pure cultures by single-spore isolation. All fungal colonies on PDA were white with sparse creeping hyphae. Pycnidia were spherical, light brown, and produced numerous conidia. Conidia were 10.60 to 20.12 × 1.98 to 3.11 µm (average 15.27 × 2.52 µm, n = 100), fusiform, sickle-shaped, light brown, without septa. Based on morphological characteristics, the fungal isolates were suspected to be Coniella castaneicola (Cui 2015). To further confirm the identity of this putative pathogen, two representative isolates LGZ2 and LGZ3 were selected for molecular identification. The internal transcribed spacer region (ITS) and large subunit (LSU) were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004) and LROR/LR7 (Castlebury and Rossman 2002). The sequences of ITS region (GenBank accession nos. MW672530 and MW856809) showed 100% identity with accessions numbers KF564280 (576/576 bp), MW208111 (544/544 bp), MW208112 (544/544 bp) of C. castaneicola. LSU gene sequences (GenBank accession nos. MW856810 to 11) was 99.85% (1324/1326 bp, 1329/1331 bp) identical to the sequences of C. castaneicola (KY473971, KR232683 to 84). Pathogenicity was tested on three blueberry varieties (‘Rabbiteye’, ‘Double Peak’ and ‘Pink Lemonade’), and four healthy young leaves of a potted blueberry of each variety with and without injury were inoculated with 20 μl suspension of prepared spores (106 conidia/mL) derived from 7-day-old cultures of LGZ2, respectively. In addition, four leaves of each variety with and without injury were sprayed with sterile distilled water as a control, respectively. The experiment was repeated three times, and all plants were incubated in a growth chamber (a 12h light and 12h dark period, 25°C, RH greater than 80%). After 4 days, all the inoculated leaves started showing disease symptoms (large irregular grey-brown lesions) as those observed in the field and there was no difference in severity recorded between the blueberry varieties, whereas the control leaves showed no symptoms. The fungus was reisolated from the inoculated leaves and confirmed as C. castaneicola by morphological and molecular identification, fulfilling Koch’s postulates. To our knowledge, this is the first report of C. castaneicola causing leaf blight on blueberries in China. The discovery of this new disease and the identification of the pathogen will provide useful information for developing effective control strategies, reducing economic losses in blueberry production, and promoting the development of the blueberry industry.

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First Report of Flyspeck Caused by Zygophiala wisconsinensis on Sweet Persimmon Fruit in Korea

Plant Disease ◽

10.1094/pdis-12-10-0917 ◽

2011 ◽

Vol 95 (5) ◽

pp. 616-616 ◽

Cited By ~ 1

Author(s):

J. Kim ◽

O. Choi ◽

J.-H. Kwon

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Its Rdna ◽

Control Treatment ◽

Diospyros Kaki ◽

Distilled Water ◽

First Report ◽

Persimmon Fruit ◽

Fungal Isolates ◽

Agar Disc

Sweet persimmon (Diospyros kaki L.), a fruit tree in the Ebenaceae, is cultivated widely in Korea and Japan, the leading producers worldwide (2). Sweet persimmon fruit with flyspeck symptoms were collected from orchards in the Jinju area of Korea in November 2010. The fruit had fungal clusters of black, round to ovoid, sclerotium-like fungal bodies with no visible evidence of a mycelial mat. Orchard inspections revealed that disease incidence ranged from 10 to 20% in the surveyed area (approximately 10 ha) in 2010. Flyspeck symptoms were observed on immature and mature fruit. Sweet persimmon fruit peels with flyspeck symptoms were removed, dried, and individual speck lesions transferred to potato dextrose agar (PDA) and cultured at 22°C in the dark. Fungal isolates were obtained from flyspeck colonies on 10 sweet persimmon fruit harvested from each of three orchards. Fungal isolates that grew from the lesions were identified based on a previous description (1). To confirm identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA sequence of a representative isolate was amplified and sequenced using primers ITS1 and ITS4 (4). The resulting 552-bp sequence was deposited in GenBank (Accession No. HQ698923). Comparison with ITS rDNA sequences showed 100% similarity with a sequence of Zygophiala wisconsinensis Batzer & Crous (GenBank Accession No. AY598855), which infects apple. To fulfill Koch's postulates, mature, intact sweet persimmon fruit were surface sterilized with 70% ethanol and dried. Three fungal isolates from this study were grown on PDA for 1 month. A colonized agar disc (5 mm in diameter) of each isolate was cut from the advancing margin of a colony with a sterilized cork borer, transferred to a 1.5-ml Eppendorf tube, and ground into a suspension of mycelial fragments and conidia in a blender with 1 ml of sterile, distilled water. The inoculum of each isolate was applied by swabbing a sweet persimmon fruit with the suspension. Three sweet persimmon fruit were inoculated per isolate. Three fruit were inoculated similarly with sterile, distilled water as the control treatment. After 1 month of incubation in a moist chamber at 22°C, the same fungal fruiting symptoms were reproduced as observed in the orchards, and the fungus was reisolated from these symptoms, but not from the control fruit, which were asymptomatic. On the basis of morphological characteristics of the fungal colonies, ITS sequence, and pathogenicity to persimmon fruit, the fungus was identified as Z. wisconsinensis (1). Flyspeck is readily isolated from sweet persimmon fruit in Korea and other sweet persimmon growing regions (3). The exposure of fruit to unusual weather conditions in Korea in recent years, including drought, and low-temperature and low-light situations in late spring, which are favorable for flyspeck, might be associated with an increase in occurrence of flyspeck on sweet persimmon fruit in Korea. To our knowledge, this is the first report of Z. wisconsinensis causing flyspeck on sweet persimmon in Korea. References: (1) J. C. Batzer et al. Mycologia 100:246, 2008. (2) FAOSTAT Database. Retrieved from http://faostat.fao.org/ , 2008. (3) H. Nasu and H. Kunoh. Plant Dis. 71:361, 1987. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.

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First Report of Leaf Spot of Sweet Basil Caused by Cercospora guatemalensis in Korea

Plant Disease ◽

10.1094/pdis-05-12-0436-pdn ◽

2012 ◽

Vol 96 (10) ◽

pp. 1580-1580

Author(s):

J. H. Park ◽

K. S. Han ◽

J. Y. Kim ◽

H. D. Shin

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Its Region ◽

Culture Collection ◽

Distilled Water ◽

First Report ◽

Sweet Basil ◽

Organic Farm ◽

Leaf Spots ◽

Close Phylogenetic Relationship

Sweet basil, Ocimum basilicum L., is a fragrant herb belonging to the family Lamiaceae. Originated in India 5,000 years ago, sweet basil plays a significant role in diverse cuisines across the world, especially in Asian and Italian cooking. In October 2008, hundreds of plants showing symptoms of leaf spot with nearly 100% incidence were found in polyethylene tunnels at an organic farm in Icheon, Korea. Leaf spots were circular to subcircular, water-soaked, dark brown with grayish center, and reached 10 mm or more in diameter. Diseased leaves defoliated prematurely. The damage purportedly due to this disease has reappeared every year with confirmation of the causal agent made again in 2011. A cercosporoid fungus was consistently associated with disease symptoms. Stromata were brown, consisting of brown cells, and 10 to 40 μm in width. Conidiophores were fasciculate (n = 2 to 10), olivaceous brown, paler upwards, straight to mildly curved, not geniculate in shorter ones or one to two times geniculate in longer ones, 40 to 200 μm long, occasionally reaching up to 350 μm long, 3.5 to 6 μm wide, and two- to six-septate. Conidia were hyaline, acicular to cylindric, straight in shorter ones, flexuous to curved in longer ones, truncate to obconically truncate at the base, three- to 16-septate, and 50 to 300 × 3.5 to 4.5 μm. Morphological characteristics of the fungus were consistent with the previous reports of Cercospora guatemalensis A.S. Mull. & Chupp (1,3). Voucher specimens were housed at Korea University herbarium (KUS). An isolate from KUS-F23757 was deposited in the Korean Agricultural Culture Collection (Accession No. KACC43980). Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The resulting sequence of 548 bp was deposited in GenBank (Accession No. JQ995781). This showed >99% similarity with sequences of many Cercospora species, indicating their close phylogenetic relationship. Isolate of KACC43980 was used in the pathogenicity tests. Hyphal suspensions were prepared by grinding 3-week-old colonies grown on PDA with distilled water using a mortar and pestle. Five plants were inoculated with hyphal suspensions and five plants were sprayed with sterile distilled water. The plants were covered with plastic bags to maintain a relative humidity of 100% for 24 h and then transferred to a 25 ± 2°C greenhouse with a 12-h photoperiod. Typical symptoms of necrotic spots appeared on the inoculated leaves 6 days after inoculation, and were identical to the ones observed in the field. C. guatemalensis was reisolated from symptomatic leaf tissues, confirming Koch's postulates. No symptoms were observed on control plants. Previously, the disease was reported in Malawi, India, China, and Japan (2,3), but not in Korea. To our knowledge, this is the first report of C. guatemalensis on sweet basil in Korea. Since farming of sweet basil has recently started on a commercial scale in Korea, the disease poses a serious threat to safe production of this herb, especially in organic farming. References: (1) C. Chupp. A Monograph of the Fungus Genus Cercospora. Ithaca, NY, 1953. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology & Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , May 5, 2012. (3) J. Nishikawa et al. J. Gen. Plant Pathol. 68:46, 2002.

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First Report of Fusarium Wilt on Eremophila spp. Caused by Fusarium oxysporum in Italy

Plant Disease ◽

10.1094/pdis-08-10-0597 ◽

2010 ◽

Vol 94 (12) ◽

pp. 1509-1509 ◽

Cited By ~ 1

Author(s):

G. Polizzi ◽

D. Aiello ◽

V. Guarnaccia ◽

A. Vitale ◽

G. Perrone ◽

...

Keyword(s):

Fusarium Oxysporum ◽

Morphological Characteristics ◽

Gene Sequences ◽

Single Spore ◽

First Report ◽

Potted Plants ◽

Plastic Bags ◽

Gene Coding ◽

Affected Plant ◽

Tubulin Protein

Eremophila spp. (Myoporaceae family), endemic to Australia, are evergreen shrubs or small trees occurring in arid, semi-arid, tropical, or temperate regions. In Europe, Eremophila spp. are grown for their horticultural appeal. During 2009 and 2010, extensive wilting was observed on 2-month to 1-year-old potted plants of Eremophila laanii F. Muell., E. glabra subsp. carnosa Chinnock, and E. maculata (Ker Gawl.) F. Muell. grown in a commercial nursery near Catania (southern Italy). Internally, symptomatic plants had conspicuous vascular discoloration from the crown to the canopy. Diseased crown and stem tissues were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissues. Colonies with purple mycelia and violet reverse colors developed after 9 days. On carnation leaf agar, single-spore isolates produced microconidia on short monophialides, macroconidia that were three to five septate with a pedicellate base, and solitary and double-celled or aggregated chlamydospores. A PCR assay was conducted on two representative isolates (ITEM 12591 and ITEM 12592) by analyzing sequences of the partial CaM gene (coding calmodulin protein) and benA (coding beta-tubulin protein) using the primers as reported by O'Donnell et al. (1). Calmodulin sequences of ITEM 12951 and ITEM 12952 isolates (GenBank Nos. FR671157 and FR671158) exhibited 99.8 and 99.5% identity with Fusarium oxysporum strain ITEM 2367 (GenBank No. AJ560774), respectively, and had 99.5% homology between them. BenA gene sequences of ITEM 12951 (GenBank No. FR671426) exhibited an identity of 100% to F. oxysporum f. sp. vasinfectum strain CC-612-3 (GenBank No. AY714092.1), and benA gene sequences of ITEM 12952 (GenBank No. FR671427) exhibited an identity of 100% to F. oxysporum f. sp. vasinfectum strain LA 140 (GenBank No. FJ466740.1), whereas the homology between the two strains is 99.5%. Morphological characteristics, as well as CaM and benA sequences, identified the isolates as F. oxysporum Schlechtend:Fr. Pathogenicity tests were performed by placing 1-cm2 plugs of PDA from 9-day-old mycelial cultures near the crown on potted, healthy, 3-month-old cuttings of E. laanii, E. glabra subsp. carnosa, and E. maculata. Twenty plants for each species were inoculated with each isolate. The same number of plants served as noninoculated controls. All plants were enclosed for 4 days in plastic bags and placed in a growth chamber at 24 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 23 to 27°C. Symptoms identical to those observed in the nursery developed 20 days after inoculation with both strains. Crown and stem discoloration was detected in all inoculated plants after 45 days. Wilting was detected on 15% of plants. Control plants remained symptomless. F. oxysporum was consistently reisolated from symptomatic tissues and identified as previously above. To our knowledge, this is the first report of F. oxysporum causing disease of Eremophila spp. worldwide. Reference: (1) K. O'Donnell et al. Mycoscience 41:61, 2000.

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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 Bacterial Leaf Spot of Coriander Caused by Pseudomonas syringae pv. coriandricola in China

Plant Disease ◽

10.1094/pdis-09-21-1901-pdn ◽

2021 ◽

Author(s):

Lei Li ◽

Yishuo Huang ◽

Yanxia Shi ◽

A LI CHAI ◽

Xuewen Xie ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Leaf Spot ◽

Citrate Synthase ◽

Morphological Characteristics ◽

Likelihood Method ◽

Bacterial Suspension ◽

Leaf Spot Disease ◽

Distilled Water ◽

Bacterial Leaf Spot ◽

First Report

Coriander (Coriandrum sativum L.) or Chinese parsley is a culinary herb with multiple medicinal effects that are widely used in cooking and traditional medicine. From September to November 2019, symptoms were observed in 2-month-old coriander plants from coriander ﬁelds in Lanzhou and Wenzhou, China. The disease developed rapidly under cold and wet climatic conditions, and the infection rate was almost 80% in open coriander fields. Typical symptoms on leaves included small, water-soaked blotches and irregular brown spots surrounding haloes; as the disease progressed, the spots coalesced into necrotic areas. Symptomatic leaf tissue was surface sterilized, macerated in sterile distilled water, and cultured on nutrient agar plates at 28 °C for 48 h (Koike and Bull, 2006). After incubation, six bacterial colonies, which were individually isolated from collected samples from two different areas, were selected for further study. Colonies on NA plate were small, round, raised, white to cream-colored, and had smooth margins. All bacterial isolates were gram-negative, rod-shaped and nonfluorescent on King's B medium. The bacteria were positive for levan production, Tween 80 hydrolysis, and tobacco hypersensitivity but negative for oxidase, potato slice rot test, arginine dihydrolase, ice nucleation activity, indole production and H2S production. The suspension of representative isolate for inoculating of plants was obtained from single colony on King's B medium for 2-3 days at 28 °C. DNA was extracted from bacterial suspensions of YS2003200102 cultured in 20 ml of King’s B medium broth at 28 °C for 1 day. Extraction was performed with a TIANamp Bacterial DNA Kit (TIANGEN, China) according to the manufacturer’s recommendations. The pathogen was confirmed by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase A (gapA) gene, the citrate synthase (gltA) gene, the DNA gyrase B (gyrB) gene and the RNA polymerase sigma factor 70 (rpoD) gene using gapA-For/gapA-Rev, gltA-For/gltA-Rev, gyrB-For/gryB-Rev, rpoD-For/rpoD-Rev primers, respectively (Popović et al., 2019). The sequences of the PCR products were deposited in GenBank with accession numbers MZ681931 (gapA), MZ681932 (gltA), MZ681933 (gyrB), and MZ681934 (rpoD). Phylogenetic analysis of multiple genes (Xu and Miller, 2013) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolates and ten published sequences of P. syringae pv. coriandricola were clustered into one clade with a 100% confidence level. To confirm the pathogenicity of isolate YS2003200102, 2-month-old healthy coriander plants were inoculated by spraying the leaves with a bacterial suspension (108 CFU ml−1) at 28 °C incubation temperature and 70% relative humidity condition, and sterile distilled water was applied as a negative control treatment (Cazorla et al. 2005). Three replicates were conducted for every isolate, and each replicate included 6 coriander plants. After twelve days, only the inoculated leaves with bacterial suspension showed bacterial leaf spot resembling those observed on naturally infected coriander leaves. Cultures re-isolated from symptomatic leaves showed the same morphological characteristics and molecular traits as those initially isolated from infected leaves in the field. This bacterium was previously reported causing leaf spot of coriander in India and Spain (Gupta et al. 2013; Cazorla et al. 2005). To our knowledge, this is the first report of P. syringae pv. coriandricola causing leaf spot disease on coriander in China. Studies are needed on strategies to manage P. syringae pv. coriandricola in crops, because its prevalence may cause yield loss on coriander in China.

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First Report of Colletotrichum tropicale Causing Anthracnose on Common Fig in Taiwan

Plant Disease ◽

10.1094/pdis-06-21-1329-pdn ◽

2021 ◽

Author(s):

Chung-hang Duan ◽

Guan-ying Chen

Keyword(s):

Manganese Superoxide Dismutase ◽

Morphological Characteristics ◽

Width Ratio ◽

Conidial Suspension ◽

Gene Sequences ◽

First Report ◽

Fungal Isolates ◽

Common Fig ◽

Colletotrichum Tropicale ◽

Central Taiwan

Ficus carica L. known as common fig is one of the most profitable fruit crops in Taiwan. Their fruit are harvested for high-priced market. Common fig can be eaten fresh or dried and processed to make different food products. In September 2015, an anthracnose-like disease was widely observed on common fig fruit planted in an orchard in Lukang township (24°04'36" N, 120°27'15" E) in Changhua County, central Taiwan. Symptoms were sunken, water-soaked lesions covered with salmon-colored spore masses and were observed on all stages of fruit, especially when fruit was ripe. Four fungal isolates were collected from four diseased fruit of different plants in the same orchard. Conidia were spread on 2% water agar, and a single conidium was separated by a handmade glass needle. Fungal isolates were grown on potato dextrose agar (PDA) at 24 to 28°C with diffused light. All four strains produced white, aerial, and cottony mycelia covered with abundant salmon-colored conidial masses on PDA. The conidia were hyaline, single celled, round cylindrical on both ends, thin walled, and the contents guttulate. The sizes of conidia were 15.4 (18.5 to 13.1) × 4.73 (5.8 to 3.6) μm [average (max. to min.); length/width ratio = 3.25, n = 40]. DNA was isolated from the representative isolate FC1 and used for amplification of partial sequences of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin 2 (TUB2), manganese-superoxide dismutase (SOD2), calmodulin (CAL), chitin synthase 1 (CHS-1) (Weir et al. 2012) and the intergenic region of apn2 and MAT1-2-1 gene (ApMat) genes (Sharma et al. 2013). A BLAST search against the NCBI database revealed that FC1 gene sequences [GenBank accession nos. MT192648 (ITS), MT155819 (GAPDH), MT199873 (ACT), MT199874 (TUB2), MT815916 (SOD2), MT815917 (CAL), MW684717 (CHS-1) and MT221652 (ApMat)] displayed 99.1, 98.2, 99.3, 99.6, 99.5, 100.0, 92.8 and 100.0% nucleotide identity to the respective gene sequences of Colletotrichum tropicale CBS 124949 (ICMP18653) (JX010264, JX010007, JX009489, JX010407, JX010329, JX009719, JX009870 and KC790728). Multilocus phylogenetic analysis performed with reference sequences showed that the isolate FC1 clustered with C. tropicale in accordance with BLAST results. A conidial suspension (1 × 106 conidia/mL) prepared from FC1 isolate was inoculated by spraying onto detached, ripe, healthy, non-wounded and surface-disinfected common fig fruit (cv. China, n = 4). Fruit sprayed with sterile water were used as control. Fruit were kept in a moist chamber (greater than 90% relative humidity, 24 to 28°C) for 24 h and then maintained in the lab for additional 5 days. The inoculated fruit developed lesions similar to the disease symptoms in the orchard. No symptom was observed on fruit treated with water. C. tropicale was re-isolated from symptomatic fruits and had similar morphological characteristics to FC1 isolate, thus fulfilling Koch’s postulates. The experiment was repeated once showing similar results. The FC1 isolate of C. tropicale with the identification number BCRC FU31436 has been deposited at Taiwan Bioresource Collection and Research Center. This fungus had previously been found on lotus and mango in Taiwan (Chen and Kirschner 2018; Wu et al. 2020), while the pathogenicity among the isolates from different origins is not yet known. To our knowledge, this is the first report of C. tropicale causing anthracnose on common fig fruit in Taiwan.

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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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Brown leaf spot of Cycas debaoensis Caused by Colletotrichum siamense in Sichuan, China

Plant Disease ◽

10.1094/pdis-10-20-2149-pdn ◽

2021 ◽

Author(s):

Shan Han ◽

Jimin Ma ◽

Yanyue Li ◽

Shujiang Li ◽

yinggao Liu ◽

...

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Negative Control ◽

Wild Plants ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

Potted Plants ◽

Brown Leaf Spot ◽

Brown Leaf

Cycas debaoensis Y. C. Zhong et C. J. Chen is an endemic species in China that is listed among China’s national key preserved wild plants (Class I) (Xie et al. 2005). It is mainly distributed in south China (Guangxi, Guizhou, and other regions). In April 2017, a new leaf disease of C. debaoensis was found in Chengdu (30°35′32″ N; 104°05′11″E) in China with an incidence over 40%. Symptoms on C. debaoensis initially appeared as brown necrotic lesions on the margin or in the center of leaves. The lesions then enlarged gradually and developed into brown spots, necrotic lesions with dark brown margins. Many small and black dots were observed on necrotic lesions. Eventually, the diseased leaves withered and died. Ten samples were collected and surface-sterilized by 3% NaClO and 75% ehanol respectively for 60s and 90s, rinsed with autoclaved distilled water and then blot-dried with autoclaved paper towels. Five isolates from diseased leaves with similar morphology were isolated from single spores. Morphological characteristics were recorded from pure cultures grown on potato dextrose agar (PDA) incubated at 25°C for 3-9 days. Initially, the colonies grown on PDA were white, then, became pale gray with concentric zones and greenish black beneath. Conidia were single-celled, smooth-walled, straight, colorless, cylindrical with both ends bluntly rounded,13.0-16.5 × 4.7-5.8 μm in size (n = 100 spores). For molecular identification, the genomic DNA of the isolates was extracted using a DNeasyTM Plant Mini Kit (Qiagen). The internal transcribed spacer (ITS) (ITS1/ITS4 White et al., 1990), β-tubulin (TUB2) (BT2A/BT2B (O’Donnell et al., 1997)), actin (ACT) (ACT512F/ACT (Carbone & Kohn, 1999)), calmodulin (CAL) (CL1C/CL2C (Weir et al., 2012)), mating type protein and chitin synthase (CHS-1) (CHS-1) (CHS-9 79F/CHS-345R (Carbone & Kohn, 1999)) were amplified. BLAST results indicated that the ITS, TUB2, ACT, CAL, CHS-1 sequences (GenBank MN305712, MN605072, MT478663, MT465591 and MT478664) showed 99-100% identity with C. siamense sequences at NCBI (GenBank JF710564, MK341542, MK855094, MH351155 and MK471373). The Phylogenetic tree inferred from the combined dataesets (TEF, TUB and ACT) show that the isolate belongs to C. siamense clade with a credibility value of 99%. Two-year-old potted plants of C. debaoensis (10 plants) were used for pathogenicity test. On each plant, 5 leaves were sprayed with a conidial suspension (1 × 106 conidia/ml) on both sides of the leaves. Autoclaved distilled water was used as negative control (10 plants). Plants were kept in the greenhouse at 25 °C under 16h/8h photoperiod and 70-75% relative humidity (RH). The symptoms observed on the inoculated plants were similar to those observed in the field, while the controls remained asymptomatic. C. siamense was re-isolated from all diseased inoculated plants, and the culture and fungus characteristics were the same as the original isolate. The morphological characteristics and molecular analyses of the isolate matched the description of C. siamense (Prihastuti et al., 2009). C. siamense was previously reported infecting Citrus reticulata (Cheng et al. 2013), but this is the first report of brown leaf spot on C. debaoensis caused by C. siamense in China. This finding provides important basis for further research on the control of the disease.

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