First report of Alternaria alternata causing Alternaria leaf spot of Fig in Pakistan

Fig (Ficus carica) is a species of flowering plants within the mulberry family. During June 2020, leaf spots were observed on several fig plants (31°26'15.0"N 73°04'25.6"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms were small, oval to circular, light brown, sunken spots that were uniformly distributed on the leaves. Spots gradually enlarged and coalesced into circular to irregular dark brown to black spots that could be up to 3cm diam. with no or small sized fruit. Disease incidence was approximately 25%. To identify the causal agent of the disease, 15 symptomatic leaves were collected. Small pieces from all diseased samples were removed from the margin between healthy and diseased tissues were surface disinfested in 70% ethanol for 2 min, rinsed three times with sterile distilled water, plated on Potato dextrose agar and incubated at 25 ± 2°C with a 12-h photoperiod. Fungal isolation on PDA medium frequency was 95% from diseases leaves. Morphological observations were made on 7- day- old single-spore cultures. The colonies initially appeared light grayish which turned sooty black in color. All fungal isolates were characterized by small, short-beaked, multicellular conidia. The conidia were ellipsoidal or ovoid and measured 9 to 25 μm × 5 to 10 μm (n = 40) with longitudinal and transverse septa. The morphological characters matched those of Alternaria alternata (Simmons et al. 2007). Genomic DNA of a representative isolate (FG01-FG03) was extracted using DNAzol reagent (Thermo Fisher Scientific MA, USA) and PCR amplification of the internal transcribed spacer (ITS) rDNA region, was performed with primers ITS1/ITS4 (White et al. 1990), partial RNA polymerase II largest subunit (RPB2) with RPB2-5F/RPB2-7cR (Liu et al. 1999) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene regions was performed with gpd1/gpd2 (Berbee et al. 1999). The obtained sequences were deposited in GenBank with accession numbers MW692903.1 to MW692905.1 for ITS-rDNA gene, MZ066731.1 to MZ066733.1 for RPB2 and MZ066728.1 to MZ066730.1 for GAPDH. BLASTn analysis showed 100% identity with the submitted sequences of A. alternata for ITS rDNA, RPB2, and GAPDH. To confirm pathogenicity, 2-month-old 15 healthy potted F. carica plants were sprayed at true leaf stage with conidial suspension by using an atomizer in a greenhouse. Each representative A. alternata isolate (FG01-FG03) was inoculated on every three plants with conidial suspensions (106 conidia/ml; obtained from 1-week-old cultures) amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant) whereas, three control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. All plants were incubated at 25 ± 2°C in a greenhouse, and the experiment was conducted twice. After 10 days of inoculation, each isolate induced leaf spots similar to typical spots observed in the field, whereas the control plants remained symptomless. The fungus was re-isolated from symptomatic tissues and reisolation frequency was 100%. Re-isolated fungal cultures were again morphologically and molecularly identical to A. alternata, thus fulfilling Koch’s postulates. Previously, A. alternata has been reported cause fruit disease of fig in Pakistan and California, USA (Alam et al. 2021; Latinović et al. 2014). To our knowledge, this is the first report of A. alternata causing leaf spot on common fig in Pakistan. In Pakistan, fig is widely grown for drying, and this disease may represent a threat to fig cultivation.

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Alternaria alternata causing Alternaria Leaf Spot of Cucumis melo (Muskmelon) in Pakistan

Plant Disease ◽

10.1094/pdis-05-20-0973-pdn ◽

2021 ◽

Author(s):

Muhammad Zeshan Ahmed ◽

Saba Saeed ◽

Ahmad Hassan ◽

Salman Ghuffar ◽

Ahsan Abdullah ◽

...

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Cucumis Melo ◽

Causal Agent ◽

The Body ◽

Its Rdna ◽

Tween 20 ◽

Distilled Water ◽

Conidial Suspension ◽

Cucumis Melo L

In July 2019, leaf spot symptoms were observed on muskmelon (Cucumis melo L.) cv. Jackball-1 plants in an experimental field of 2.02 ha with a disease incidence of 30% (31°26'05.4"N 73°04'30.3"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms consisted of small, circular, brown, necrotic spots 1 to 2 mm in size covering 10 to 30% of the leaf blade, which gradually enlarged and developed concentric rings. To identify the causal agent of the disease, a total of 20 symptomatic leaves were collected. Small pieces removed from the margin between healthy and diseased tissues were surface disinfected in 70% ethanol for 2 min, rinsed three times with sterile distilled water, plated on Potato dextrose agar and incubated at 25 ± 2°C with a 12-h photoperiod. Morphological observations were made on 7-day-old single-spore cultures. The colonies initially appeared white and then turned olive-green. All 20 fungal isolates were characterized by small, short-beaked, multicellular conidia. The conidia were ellipsoidal or ovoid and measured 11.5 to 30 μm × 7.5 to 15 μm (n = 50) with longitudinal and transverse septa. Conidia were produced on short conidiophores in chains. The beaks were short (often less than one-third the body length) and conical or cylindrical. These morphological features concur with the description of Alternaria alternata (Fr.) Keissler (Woudenberg et al. 2013). For molecular identification, genomic DNA of four representative isolates (HMSMZA 07, 08, 09, 10) were extracted and PCR amplification of the internal transcribed spacer (ITS)-rDNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and translation elongation factor-1 alpha (TEF-1α) gene regions were performed (White et al. 1990, Berbee et al. 1999, Carbone & Kohn, 1999) respectively. The obtained sequences were deposited in GenBank with accession numbers MT253643.1-MT253646.1 (ITS-rDNA), MT318260.1-MT318263.1 (GAPDH), and MT318280.1-MT318283.1 (TEF-1α). BLASTn analysis of HMSMZA 07 sequences showed 100% identity with ITS rDNA (MN615420.1), GAPDH (MK637438.1) and TEF-1α (MN807795.1) sequences of A. alternata. To confirm pathogenicity, 5-6 weeks-old Muskmelon (Cucumis melo L.) cv. Jackball-1 plants (true leaf stage) were sprayed until runoff (1.5 to 2 ml per plant) with A. alternata conidial suspension (106 conidia/ml; obtained from 1 week-old cultures) amended with 0.1% (vol/vol) of Tween 20 using an atomizer in the green house. The experiment included four A. alternata isolates inoculated onto three muskmelon plants per each isolate, whereas control plants (n = 3) were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse and the experiment was conducted twice. After 5 to 7 days post inoculation, necrotic leaf spots were observed on the inoculated plants and A. alternata was reisolated and confirmed by morphological and molecular (ITS) features. No disease was observed on control plants. Previously, A. alternata on muskmelon has been reported in Pakistan (Ahmad et al. 1997), however this study provides a detailed description of disease symptoms, morphological and molecular identity of the causal agent including completion of Koch’s postulates. The disease could represent a threat for muskmelon crop in Pakistan due to its increasing cultivation and therefore warrants the need to develop disease management strategies.

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First Report of Alternaria alternata f. sp. cucurbitae Causing Alternaria Leaf Spot of Melon in the Mid-Atlantic Region of the United States

Plant Disease ◽

10.1094/pdis-92-4-0652b ◽

2008 ◽

Vol 92 (4) ◽

pp. 652-652 ◽

Cited By ~ 2

Author(s):

X. G. Zhou ◽

K. L. Everts

Keyword(s):

United States ◽

Disease Severity ◽

Body Length ◽

Alternaria Alternata ◽

Leaf Spot ◽

The United States ◽

Tween 20 ◽

Conidial Suspension ◽

First Report ◽

Alternaria Leaf Spot

Alternaria alternata f. sp. cucurbitae, the casual agent of Alternaria leaf spot, was first described in Greece where it caused severe losses to greenhouse-grown cucumbers (Cucumis sativus) (3,4). The fungus also attacks melon (C. melo) and watermelon (Citrullus lanatus) (1–3). In late June of 2006, following a period of windy and rainy days, numerous dark brown, circular lesions, 0.5 to 1 mm in diameter, were observed on leaves of melons in a field in Wicomico County, Maryland. The lesions gradually enlarged and coalesced into large, nearly circular, or irregularly shaped lesions that could be as long as 3 cm. The center of the lesions was light tan, surrounded by a dark brown ring and a chlorotic halo, and tended to split in the later development stages. Most of the lesions appeared on the edge of the leaves and no lesions developed on the stems and fruit. Lesions first started on old leaves and then developed on leaves in the middle part of the canopy. Leaf lesions were observed on melon cvs. Ananas, Honeydew Greenflesh, and Israeli. Disease severity ranged from 3 to 20% of the leaf area affected. Small pieces (3 × 3 mm) of tissue removed from the margin between healthy and diseased tissue were surface disinfected in 0.5% NaOCl for 2 min and plated on acidified, ¼-strength potato dextrose agar. Isolations made from diseased tissue frequently (61%) yielded fungal colonies with morphological features and spore dimensions that were consistent with the description of A. alternata f. sp. cucurbitae (1,3). Fungal isolates were characterized by small, short-beaked, multicellular conidia. Conidia were ovoid, obclavate, and sometimes ellipsoidal with the average overall body length of 39 μm (range, 17 to 80 μm) and width of 14 μm (range, 7 to 20 μm). Conidia were produced on short conidiophores in chains. The beaks were short (often less than one-third the body length) and conical or cylindrical. Pathogenicity of six single-spore isolates was determined on four melon cultivars (Honeydew Greenflesh, Israeli, Tam Dew, and Topmark) and one watermelon cultivar (Sugar Baby) in a greenhouse. Twenty plants of each cultivar at the one-true-leaf stage were sprayed with a conidial suspension (106 conidia/ml) of each isolate amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant). Inoculation with sterile distilled water amended with 0.1% Tween 20 served as controls. The plants were placed in a dew growth chamber for 48 h at 24°C and subsequently maintained in a greenhouse at 21 to 29°C. At 4 to 5 days after inoculation, each isolate induced leaf lesions on each inoculated cultivar similar to typical lesions observed in the field. There was no significant difference in disease severity among the cultivars tested or between melon and watermelon. Control plants remained symptomless. The fungus was readily reisolated from symptomatic tissues. To our knowledge, this is the first report of A. alternata f. sp. cucurbitae causing Alternaria leaf spot of melon in the Mid-Atlantic United States and the only report outside Georgia in the southern region of the United States (D. B. Langston, personal communication) and Greece. References: (1) D. L. Vakalounakis. Plant Dis. 74:227, 1990. (2) D. L. Vakalounakis. Ann. Appl. Biol. 117:507, 1990. (3) D. L. Vakalounakis. Alternaria leaf spot. Page 24 in: Compendium of Cucurbit Diseases. T. A. Zitter et al., eds. The American Phytopathological Society, St. Paul, MN, 1996. (4) D. L. Vakalounakis and N. E. Malathrakis. J. Phytopathol. 121:325, 1988.

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First Report of Leaf Spot in Farfugium japonicum Caused by Alternaria alternata in China

Plant Disease ◽

10.1094/pdis-02-21-0395-pdn ◽

2021 ◽

Author(s):

Hui Wang ◽

Hong Liu ◽

Xun Lu ◽

Qian Zhou

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Economic Losses ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

Isolation Technique ◽

First Report ◽

Farfugium Japonicum

Farfugium japonicum (L.) Kitam (with the common name leopard plant) is known as a garden and medical herb, and belongs to the family Asteraceae. In May 2019, a leaf spot disease was observed on the upper leaf surface of F. japonicum in Changsha city, Hunan province, China. More than 98% of the F. japonicum plants were infected in a garden of Donghu district (28°13′ N; 112°56′ E). Leaf symptoms included small (1 to 10 mm in diameter), brown spots that were circular, tan to gray in the center and distinct brownish-yellow margins. Severely affected leaves were blighted and plants were dying. For isolation, symptomatic leaf tissue was surface sterilized, rinsed in sterile distilled water, and plated on potato dextrose agar (PDA) amended with a 50 μg/ml streptomycin sulfate followed by incubation at 25°C in darkness. By a single-spore isolation technique, pure fungal cultures were obtained and displayed gray-brown and gray-white aerial mycelia after five days of incubation. One representative isolate (HnAa-1) was selected for further studies. Conidia of HnAa-1 were olive brown, obpyriform, either branched or unbranched with a short beak, 1 to 5 transverse septa, and 0 to 3 longitudinal or oblique septa. The conidia were 10 to 35 μm long and 2 to 12 μm wide. HnAa-1 was identified as an Alternaria sp. on the basis on morphological characterization by Simmons (1). Further identification to species level was made by molecular analyses. DNA of HnAa-1 was extracted from the regions internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and partial Alt a 1 major allergen (ALT) gene. Amplification and sequencing was carried out with the method described by Woudenberg et al.(2) . BLASTn searches showed that the ITS, GAPDH and ALT sequences had the highest similarity with A. alternata strains, with 100% (548/548) identities for ITS (GQ169728), 100% (567/567) identities for GAPDH (MK903028) and 99.36% (466/469) identities for ALT (MN184998). Moreover, the ITS, GAPDH and ALT sequences had more than 99% identities with the epitype CBS 916.96 of A. alternata (ITS: AF347031; GAPDH: AY278808; ALT: AY563301). The ITS, GAPDH and ALT sequences of HnAa-1 were submitted to GenBank (Accession No. MT767170, No. MW115639 and No. MW316727). Pathogenicity tests were conducted by spraying a 10 ml conidial suspension (1.0 ×105 conidia /mL) on surfaces of leaves of three healthy plants (8-week-old). Leaves of three healthy plants were sprayed with sterile distilled water as a control treatment. All inoculated plants were maintained in growth chamber at 25°C with a 12-h photoperiod. The pathogenicity test was repeated twice. After five days inoculation, typical brown spots and necrotic lesions similar to those observed in the field, had developed on all inoculated plants but not on water-treated control plants. Alternaria alternata was re-isolated from the symptomatic tissue of inoculated plants but not from the control plants, and re-identified with morphological and molecular methods, which fulfilled Koch's postulates. This host-pathogen association has been reported in Korea (3), but it is the first report of A. alternata causing leaf spots on F. japonicum in China. Since A. alternata is a ubiquitous and very important plant pathogen causing leaf spot diseases in over 100 species plant, the occurrence of this disease is a serious threat to F.japonicum and might lead to economic losses. Therefore, appropriate prevention strategies to F.japonicum should be adopted.

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The First Report of Leaf Spot Caused by Alternaria alternata on Italian Ryegrass (Lolium multiflorum) in China

Plant Disease ◽

10.1094/pdis-08-20-1737-pdn ◽

2020 ◽

Author(s):

Xuekai Wei ◽

Longhai Xue ◽

Chunjie Li

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Lolium Multiflorum ◽

Italian Ryegrass ◽

Post Inoculation ◽

Distilled Water ◽

Conidial Suspension ◽

Forage Crops ◽

Isolation Technique ◽

First Report

Italian ryegrass (Lolium multiflorum Lam.) is one of the most important forage crops in southwestern China. In 2018, a leaf spot was observed in a field of Italian ryegrass in Mengyang, Sichuan province, China (30.96925°N, 104.10223°E). From January to early March, this leaf spot developed sporadically and appeared as brown to dark brown lesions. In late May, this disease reached a peak with incidence up to 80% and appeared as reddish-brown necrotic spots with a grayish white to brown center. To isolate the pathogen, sections (0.5 × 1 cm2) of 30 diseased leaves collected from 10 plants were surface-disinfested in 70% ethanol solution for 30 s, 5% NaOCl solution for 5 min, rinsed thrice in sterilized distilled water, air dried, plated on potato dextrose agar (PDA), and incubated at 25°C in the dark for 4 days. To obtain pure isolates, the single-spore isolation technique (Cai et al. 2009) was used. The conidial suspensions were diluted to a reasonable concentration, spread onto PDA, and incubated at 25°C in dark for 24 to 48 h, and then single germinated conidia were transferred onto new PDA plates (Cai et al. 2009). Nine pure isolates showing similar morphology were obtained for further study. Colonies on PDA were dark gray in the center surrounded by white to gray, with gossypine mycelia on the upper side, and red to dark red on the reverse side. Conidia were obclavate or pyriform, olivaceous to dark brown, with 0 to 6 transverse septa and 0 to 4 longitudinal septa, 13.2 to 55.0 (27.9) × 6.3 to 12.5 (9.8) µm. Conidiophores were septate, hyaline to olivaceous brown, either branched or unbranched, geniculate at the tip, 2.5 to 5.9 μm wide and up to 70 μm long. These morphological and cultural characteristics were consistent with the descriptions of Alternaria alternata (Fr.) Keissl. isolated from Apple (Elfar et al. 2018). To confirm the pathogenicity on Italian ryegrass, healthy plants (8-week-old) of cultivar Splendor grown in five pots filled with potting soil were spray-inoculated with conidial suspension (1 × 106 conidia/ml). Plants in another, five pots were sprayed with sterilized distilled water as controls. All pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 18 to 25°C. At 14 days post inoculation, symptoms typical of brown to dark brown leaf spots developed on the plants inoculated with conidial suspension, whereas no symptoms on the control plants. The pathogenicity tests were carried out three times. The same pathogen was consistently re-isolated from inoculated leaves and confirmed by morphological characterization as described above. To further identify this pathogen, isolate HMCH-9 (=CGMCC 3.19924) was selected as a representative for molecular characterization. Following Woudenberg et al. (2015), the internal transcribed spacer regions 1 and 2 and intervening 5.8S rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPD), translation elongation factor 1-alpha (TEF), RNA polymerase second largest subunit (RPB2), and Alternaria major allergen (Alt) genes were partially amplified and sequenced. Sequences were deposited in GenBank (accession nos. MH567106 for ITS, MH567107 for GPD, MH567109 for TEF, MH567110 for RPB2, and MH567108 for Alt). BLAST analysis of all these five segments showed >99.8% identity with those sequences of ex-type isolate CBS 916.96 of A. alternata (Woudenberg et al. 2015). To our knowledge, this is the first report of A. alternata causing leaf spot on Italian ryegrass in China. The accurate identification of this pathogen would be useful for the prevention and control of leaf spot on Italian ryegrass in the future.

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

Plant Disease ◽

10.1094/pdis-12-20-2706-pdn ◽

2021 ◽

Author(s):

Luqman Amrao ◽

Muhammad Zeshan Ahmed ◽

Saba Saeed ◽

Rashida Atiq ◽

Muhammad Subhan Shafique ◽

...

Keyword(s):

Capsicum Annuum ◽

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Its Rdna ◽

Tween 20 ◽

Conidial Suspension ◽

First Report ◽

Capsicum Annuum L ◽

Gapdh Gene

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

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First Report of Alternaria Leaf Spot Caused by Alternaria sp. on Highbush Blueberry (Vaccinium corymbosum) in South Korea

Plant Disease ◽

10.1094/pdis-04-14-0344-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1434-1434

Author(s):

J.-H. Kwon ◽

D.-W. Kang ◽

M.-G. Cheon ◽

J. Kim

Keyword(s):

South Korea ◽

Leaf Spot ◽

Disease Incidence ◽

Vaccinium Corymbosum ◽

Its Rdna ◽

Universal Primers ◽

Conidial Suspension ◽

First Report ◽

Representative Isolate ◽

Alternaria Sp

In South Korea, the culture, production, and consumption of blueberry (Vaccinium corymbosum) have increased rapidly over the past 10 years. In June and July 2012, blueberry plants with leaf spots (~10% of disease incidence) were sampled from a blueberry orchard in Jinju, South Korea. Leaf symptoms included small (1 to 5 mm in diameter) brown spots that were circular to irregular in shape. The spots expanded and fused into irregularly shaped, large lesions with distinct dark, brownish-red borders. The leaves with severe infection dropped early. A fungus was recovered consistently from sections of surface-disinfested (1% NaOCl) symptomatic leaf tissue after transfer onto water agar and sub-culture on PDA at 25°C. Fungal colonies were dark olive and produced loose, aerial hyphae on the culture surfaces. Conidia, which had 3 to 6 transverse septa, 1 to 2 longitudinal septa, and sometimes also a few oblique septa, were pale brown to golden brown, ellipsoid to ovoid, obclavate to obpyriform, and 16 to 42 × 7 to 16 μm (n = 50). Conidiophores were pale to mid-brown, solitary or fasciculate, and 28 to 116 × 3 to 5 μm (n = 50). The species was placed in the Alternaria alternata group (1). To confirm the identity of the fungus, the complete internal transcribed spacer (ITS) rDNA region of a representative isolate, AAVC-01, was amplified using ITS1 and ITS4 primers (2). The DNA products were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and the resulting pOR13 plasmid was sequenced using universal primers. The resulting 570-bp sequence was deposited in GenBank (Accession No. KJ636460). Comparison of ITS rDNA sequences with other Alternaria spp. using ClustalX showed ≥99% similarity with the sequences of A. alternata causing blight on Jatropha curcas (JQ660842) from Mexico and Cajannus cajan (JQ074093) from India, citrus black rot (AF404664) from South Africa, and other Alternaria species, including A. tenuissima (WAC13639) (3), A. lini (Y17071), and A. longipes (AF267137). Two base substitutions, C to T at positions 345 and 426, were found in the 570-bp amplicon. Phylogenetic analysis revealed that the present Alternaria sp. infecting blueberry grouped separately from A. tenuissima and A. alternata reported from other hosts. A representative isolate of the pathogen was used to inoculate V. corymbosum Northland leaves for pathogenicity testing. A conidial suspension (2 × 104 conidia/ml) from a single spore culture and 0.025% Tween was spot inoculated onto 30 leaves, ranging from recently emerged to oldest, of 2-year-old V. corymbosum Northland plants. Ten leaves were treated with sterilized distilled water and 0.025% Tween as a control. The plants were kept in a moist chamber with >90% relative humidity at 25°C for 48 h and then moved to a greenhouse. After 15 days, leaf spot symptoms similar to those observed in the field developed on the inoculated leaves, whereas the control plants remained asymptomatic. The causal fungus was re-isolated from the lesions of the inoculated plants to fulfill Koch's postulates. To our knowledge, this is the first report of Alternaria sp. on V. corymbosum in South Korea. References: (1) E. G. Simmons. Page 1797 in: Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands, 2007. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (3) M. P. You et al. Plant Dis. 98:423, 2014.

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First Report of Alternaria Leaf Spot of Banana Caused by Alternaria alternata in the United States

Plant Disease ◽

10.1094/pdis-01-13-0007-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1116-1116 ◽

Cited By ~ 3

Author(s):

V. Parkunan ◽

S. Li ◽

E. G. Fonsah ◽

P. Ji

Keyword(s):

United States ◽

Alternaria Alternata ◽

Leaf Spot ◽

Morphological Characteristics ◽

The United States ◽

Its Sequencing ◽

Single Spore ◽

First Report ◽

Alternaria Leaf Spot ◽

Leaf Spots

Research efforts were initiated in 2003 to identify and introduce banana (Musa spp.) cultivars suitable for production in Georgia (1). Selected cultivars have been evaluated since 2009 in Tifton Banana Garden, Tifton, GA, comprising of cold hardy, short cycle, and ornamental types. In spring and summer of 2012, 7 out of 13 cultivars (African Red, Blue Torres Island, Cacambou, Chinese Cavendish, Novaria, Raja Puri, and Veinte Cohol) showed tiny, oval (0.5 to 1.0 mm long and 0.3 to 0.9 mm wide), light to dark brown spots on the adaxial surface of the leaves. Spots were more concentrated along the midrib than the rest of the leaf and occurred on all except the newly emerged leaves. Leaf spots did not expand much in size, but the numbers approximately doubled during the season. Disease incidences on the seven cultivars ranged from 10 to 63% (10% on Blue Torres Island and 63% on Novaria), with an average of 35% when a total of 52 plants were evaluated. Six cultivars including Belle, Ice Cream, Dwarf Namwah, Kandarian, Praying Hands, and Saba did not show any spots. Tissue from infected leaves of the seven cultivars were surface sterilized with 0.5% NaOCl, plated onto potato dextrose agar (PDA) media and incubated at 25°C in the dark for 5 days. The plates were then incubated at room temperature (23 ± 2°C) under a 12-hour photoperiod for 3 days. Grayish black colonies developed from all the samples, which were further identified as Alternaria spp. based on the dark, brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (2). Conidia were 23 to 73 μm long and 15 to 35 μm wide, with a beak length of 5 to 10 μm, and had 3 to 6 transverse and 0 to 5 longitudinal septa. Single spore cultures of four isolates from four different cultivars were obtained and genomic DNA was extracted and the internal transcribed spacer (ITS1-5.8S-ITS2) regions of rDNA (562 bp) were amplified and sequenced with primers ITS1 and ITS4. MegaBLAST analysis of the four sequences showed that they were 100% identical to two Alternaria alternata isolates (GQ916545 and GQ169766). ITS sequence of a representative isolate VCT1FT1 from cv. Veinte Cohol was submitted to GenBank (JX985742). Pathogenicity assay was conducted using 1-month-old banana plants (cv. Veinte Cohol) grown in pots under greenhouse conditions (25 to 27°C). Three plants were spray inoculated with the isolate VCT1FT1 (100 ml suspension per plant containing 105 spores per ml) and incubated under 100% humidity for 2 days and then kept in the greenhouse. Three plants sprayed with water were used as a control. Leaf spots identical to those observed in the field were developed in a week on the inoculated plants but not on the non-inoculated control. The fungus was reisolated from the inoculated plants and the identity was confirmed by morphological characteristics and ITS sequencing. To our knowledge, this is the first report of Alternaria leaf spot caused by A. alternata on banana in the United States. Occurrence of the disease on some banana cultivars in Georgia provides useful information to potential producers, and the cultivars that were observed to be resistant to the disease may be more suitable for production. References: (1) E. G. Fonsah et al. J. Food Distrib. Res. 37:2, 2006. (2) E. G. Simmons. Alternaria: An identification manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands, 2007.

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

Plant Disease ◽

10.1094/pdis.2003.87.2.203a ◽

2003 ◽

Vol 87 (2) ◽

pp. 203-203

Author(s):

S. T. Koike ◽

S. A. Tjosvold ◽

J. Z. Groenewald ◽

P. W. Crous

Keyword(s):

Leaf Spot ◽

Sequence Data ◽

Disease Incidence ◽

Leaf Spot Disease ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Spot Disease ◽

Leaf Spots ◽

Initial Symptoms

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

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First Report of Nigrospora osmanthi Causing Leaf Spot on Tartary Buckwheat in China

Plant Disease ◽

10.1094/pdis-08-20-1773-pdn ◽

2020 ◽

Author(s):

Quan Shen ◽

Xixu Peng ◽

Feng He ◽

Shaoqing Li ◽

Zuyin Xiao ◽

...

Keyword(s):

Relative Humidity ◽

Leaf Spot ◽

Plant Pathogens ◽

Hunan Province ◽

Tartary Buckwheat ◽

Tween 20 ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

First Report

Buckwheat (Fagopyrum tataricum) is a traditional short-season pseudocereal crop originating in southwest China and is cultivated around the world. Antioxidative substances in buckwheat have been shown to provide many potential cardiovascular health benefits. Between August and November in 2019, a leaf spot was found in several Tartary buckwheat cv. Pinku1 fields in Xiangxiang County, Hunan Province, China. The disease occurred throughout the growth cycle of buckwheat after leaves emerged, and disease incidence was approximately 50 to 60%. Initially infected leaves developed a few round lesions, light yellow to light brown spots. Several days later, lesions began to enlarge with reddish brown borders, and eventually withered and fell off. Thirty lesions (2×2 mm) collected from three locations with ten leaves in each location were sterilized in 70% ethanol for 10 sec, in 2% sodium hypochlorite for 30 sec, rinsed in sterile water for three times, dried on sterilized filter paper, and placed on a potato dextrose PDA with lactic acid (3 ml/L), and incubated at 28°C in the dark for 3 to 5 days. Fungal colonies were initially white and later turned black with the onset ofsporulation. Conidia were single-celled, black, smooth, spherical to subspherical, and measured 9.2 to 15.6 µm long, and 7.1 to 11.6 µm wide (n=30). Each conidium was terminal and borne on a hyaline vesicle at the tip of conidiophores. Morphologically, the fungus was identified as Nigrospora osmanthi (Wang et al. 2017). Identification was confirmed by amplifying and sequencing the ITS region, and translation elongation factor 1-alpha (TEF1-α) and partial beta-tublin (TUB2) genes using primers ITS1/ITS4 (Mills et al. 1992), EF1-728F/EF-2 (Carbone and Kohn 1999; O’Donnell et al. 1998) and Bt-2a/Bt-2b (Glass et al. 1995), respectively. BLAST searches in GenBank indicated the ITS (MT860338), TUB2 (MT882054) and TEF1-α (MT882055) sequences had 99.80%, 99% and 100% similarity to sequences KX986010.1, KY019461.1 and KY019421.1 of Nigrospora osmanthi ex-type strain CGMCC 3.18126, respectively. A neighbor-joining phylogenetic tree constructed using MEGA7.0 with 1,000 bootstraps based on the concatenated nucleotide sequences of the three genes indicated that our isolate was closely related to N. osmanthi. Pathogenicity test was performed using leaves of healthy F. tataricum plants. The conidial suspension (1 × 106 conidia/ml) collected from PDA cultures with 0.05% Tween 20 buffer was used for inoculation by spraying leaves of potted 20-day-old Tartary buckwheat cv. Pinku1. Five leaves of each plant were inoculated with spore suspensions (1 ml per leaf). An equal number of control leaves were sprayed with sterile water to serve as a control. The treated plants were kept in a greenhouse at 28°C and 80% relative humidity for 24 h, and then transferred to natural conditions with temperature ranging from 22 to 30°C and relative humidity ranging from 50 to 60%. Five days later, all N. osmanthi-inoculated leaves developed leaf spot symptoms similar to those observed in the field, whereas control leaves remained healthy. N. osmanthi was re-isolated from twelve infected leaves with frequency of 100%, fulfilling Koch’s postulates. The genus Nigrospora has been regarded by many scholars as plant pathogens (Fukushima et al. 1998) and N. osmanthi is a known leaf blight pathogen for Stenotaphrum secundatum (Mei et al. 2019) and Ficus pandurata (Liu et al. 2019) but has not been reported on F. tataricum. Nigrospora sphaerica was also detected in vegetative buds of healthy Fagopyrum esculentum Moench (Jain et al. 2012). To our knowledge, this is the first report of N. osmanthi causing leaf spot on F. tataricum in China and worldwide. Appropriate strategies should be developed to manage this disease.

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