First Report of Leaf Spot Caused by Alternaria alternata on Yucca gloriosa in China

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

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First Report of 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 Alternaria spp. Causing Leaf Spot on Sweet Viburnum in China

Plant Disease ◽

10.1094/pdis-06-20-1407-pdn ◽

2021 ◽

Author(s):

Chaodong Qiu ◽

Yingying Zhang ◽

Zhenyu Liu

Keyword(s):

Leaf Spot ◽

Genomic Dna ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Pathogenicity Test ◽

Distilled Water ◽

Single Spore ◽

First Report ◽

Sweet Viburnum

Sweet viburnum [Viburnum odoratissimum (L.) Ker Gawl] is an evergreen shrub mainly cultivated along roadsides in urban landscapes and also in parks and residential areas. A foliar disease occurred on about 40% of sweet viburnum plants near Anhui Grand Theatre, Anhui Province of China in June 2019. In early stages of sweet viburnum infection, the symptoms appeared as small brown spots ranged in length from 2 to 3 millimeters on the leaves. The spots developed on the upper, middle, and lower leaves of the plant, however, the upper leaves got more severely affected. As the disease develops, the spots enlarged and became rectangular or oval, brown to dark-brown, and their centers became ashen gray. In later stages of infection, the diseased leaves became wilting. Diseased leaves were surface disinfested and three small sections (2-3 mm2) were cut from the margin of the lesions. Sections were placed in 1.5% NaClO for 2 min, submerged in three changes of sterilized distilled water for 1 min each, placed onto potato dextrose agar (PDA) medium amended with 50 μg/ml of ampicillin and kanamycin, and incubated at 25℃ for 3 days. The mycelium from the leading edge of colonies growing from the tissue was sub-cultured onto a PDA plate for 3 days, followed by spore induction (Simmons 2007) and single spore isolation to obtain a pure culture of the putative pathogen. Colonies of one single spore isolate HF0719 were rounded, grayish white with dense aerial mycelium viewed from above and dark brown viewed from below. On potato carrot agar (PCA) medium, conidiophores were branched or occasionally unbranched. On branched conidiophores, conidia were in dwarf tree-like branched chains of 2-5 conidia. On unbranched conidiophores, conidia were simple or in chains of 2-8 conidia. Conidia were light brown or dark brown, ovoid, ellipsoidal to fusiform, and ranged in size from 7 to 26.5 × 4.5 to 11 μm with an average size of 16 × 7 µm based on 500 spore observations, with one beak and 1-7 transverse, 0-3 longitudinal, and 0-3 oblique septa. Beaks were ranged in (1.5-)2-10(-16) μm long. Based on cultural and morphological characteristics, isolate HF0719 was identified as Alternaria spp. (Simmons 2007). For molecular identification, total genomic DNA was isolated from mycelia collected from 7 day-old colonies of isolate HF0719 using the fungal genomic DNA extraction kit (Solarbio, Beijing, China). Fragments of five genes, including those encoding glyceraldehyde-3-phosphate dehydrogenase (gpd), plasma membrane ATPase, actin, calmodulin, and the Alternaria major allergen (Alt a1) regions of isolate HF0719 were amplified and sequenced using primer pairs gpd1/gpd2 (Berbee et al. 1999), ATPDF1/ATPDR1, ACTDF1/ACTDR1, CALDF1/CALDR1 (Lawrence et al. 2013), and Alt-for/Alt-rev (Hong et al. 2005), respectively. The obtained nucleotide sequences were deposited into GenBank as accession numbers: gpd, MT614365; ATPase, MT614364; actin, MT614363; calmodulin, MN706159; and Alt a1, MN304720. Phylogenetic tree using a maximum likelihood bootstrapping method based on the five-gene combined dataset in the following order: gpd, ATPase, actin, calmodulin, Alt a1 of HF0719 and standard strains representing 120 Alternaria species (Lawrence et al. 2013) was constructed. Isolate HF0719 formed a separate branch. On the basis of morphological characteristics and phylogenetic pattern, isolate HF0719 was identified as Alternaria spp.. A pathogenicity test was performed by rubbing 32 healthy leaves of six 5-year-old sweet viburnum plants with a cotton swab dipped in spore suspension containing 2.6 × 106 spores/ml, following leaf surface disinfection with 70% ethanol in the open field. Sterilized distilled water was used as control. The average air temperature was about 28℃ during the period of pathogenicity test. Eleven days after inoculation, 100% of inoculated leaves showed the leaf spot symptom identical to symptoms observed in the field. Control leaves were symptomless. The experiment was done three times. The re-isolated pathogen from the leaf lesion had the same morphological and molecular characteristics as isolate HF0719, thus satisfying Koch’s postulates. The genus Alternaria has been reported to cause leaf spot on sweet viburnum in Florida, USA (Alfieri et al. 1984). To our knowledge, this is the first report of Alternaria spp. causing leaf spot on sweet viburnum in China, a highly valued ornamental plant. Our findings will contribute to monitoring and adopting strategies for manage leaf spot disease on sweet viburnum.

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First Report of Cylindrocarpon sp. Associated with Root Rot Disease of Strawberry in North Carolina

Plant Disease ◽

10.1094/pdis-01-13-0116-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1251-1251 ◽

Cited By ~ 1

Author(s):

T. B. Adhikari ◽

C. S. Hodges ◽

F. J. Louws

Keyword(s):

North Carolina ◽

Root Rot ◽

Management Strategies ◽

Morphological Characteristics ◽

Internal Transcribed Spacers ◽

Universal Primers ◽

Sodium Hypochlorite Solution ◽

Tissue Samples ◽

First Report ◽

Root Necrosis

Strawberry (Fragaria × ananassa Duchesne) is an economically important fruit crop in North Carolina for domestic consumption and export. In April 2012, outbreaks of a destructive root disease were observed in strawberry cv. Chandler in Buncombe, New Hanover, and Roman counties, North Carolina. Samples from Rowan (ID 13175) and Buncombe (ID 13193) counties submitted to the Plant Disease and Insect Clinic of the Department of Plant Pathology, North Carolina State University, exhibited yellowing and wilting of leaves and extensive root necrosis, and disease severity based on field symptoms ranged from 20 to 30%. To identify the pathogen, five small pieces of necrotic crown and root tissues were taken from each sample, surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, and plated onto potato dextrose agar (PDA) with 0.5 g liter–1 of streptomycin sulfate. Colonies developing from the tissue samples were transferred to PDA. Colonies from both samples were identical, grew relatively slowly, and gradually turned yellowish to partially brownish. After about 7 days, abundant conidia were formed. These were hyaline, mostly straight with both ends rounded, predominantly three septate, and 40 to 50 × 5 to 10 μm. Based on morphological characteristics, these isolates were identified as a species of Cylindrocarpon (1) To confirm the original identification of the fungus as a species of Cylindrocarpon, genomic DNA of both isolates was extracted from mycelia using DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA) and analyzed using PCR (2). The internal transcribed spacers (ITS)1 and (ITS)2 flanking the 5.8S rRNA regions were amplified and sequenced using universal primers ITS1 (forward) and ITS4 (reverse). The sequences of the 421 bp (GenBank KC847090 and KC847091) of both isolates were identical. Furthermore, a BLASTn search of these sequences showed homology of 99% with the sequences of Cylidrocarpon species (AB369421.1, AM419069.1, AM419074.1, AY295332.1, JN031017.1, JN253505.1, and JQ886422.1), To fulfill Koch's postulates, inoculum of each isolate was prepared and adjusted to 1.5 × 107 conidia/ml using a hemacytometer. ‘Chandler’ strawberry plants were grown in 25-cm diameter plastic pots (one seedling per pot) in the greenhouse and five 6-week-old plants were injected with conidia of each isolate into the base of crown using a 5-ml syringe. The plants were covered with clear plastic for 24 h and left on the greenhouse bench with a 16-h photoperiod and 25/20°C day/night temperatures and assessed for disease development 14 days after inoculation. The inoculated plants exhibited wilting and root necrosis, consistent with the symptoms observed on strawberry plants in the field. Control plants treated with distilled water remained healthy. Isolations were made from the inoculated plants and the fungus used for inoculation was recovered from all plants. The morphology of these isolates was in agreement with published descriptions of Cylindrocarpon (1). To our knowledge, this is the first report of a Cylindrocarpon sp. causing crown and root rot on strawberry in North Carolina and effective disease management strategies need to be explored. References: (1) C. D. Booth. Mycol. Pap. (CMI) 104:1, 1996. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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Leaf Spot Caused by Boeremia linicola on Siberian ginseng in China

Plant Disease ◽

10.1094/pdis-09-20-2076-pdn ◽

2020 ◽

Author(s):

Yi Ming Guan ◽

Shu Na Zhang ◽

Ying Ying Ma ◽

Yue Zhang

Keyword(s):

Phylogenetic Analysis ◽

Rna Polymerase Ii ◽

Leaf Spot ◽

Management Strategies ◽

Elongation Factor ◽

Morphological Characteristics ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

Siberian Ginseng ◽

Pathogenicity Testing

Siberian ginseng (Eleutherococcus sessiliflorus (Rupr. & Maxim.) S. Y. Hu, Araliaceae), is a perennial medicinal plant that is widely cultivated in China. Leaf spot was observed in 2- and 3-year-old Siberian ginseng in Zuojia County (126°05′23.2″E, 44°03′09.5″N), northeast China, in August 2019. Polygonal or irregular black spots ranging from 2 to 9 mm in diameter were found on infected leaves, and each leaf had dozens of spots. The green color around the lesions gradually faded. As the disease progressed, the spots withered and multiple lesions merged into large disease spots, causing leaf wilting (Fig. 1). More than 38% of plants in one 25-ha field were infected in 2019. Fifteen diseased leaves were collected from those plants and cut into 5-mm pieces. The pieces were surface-disinfected by immersion in 1% NaOCl for 2 min and then rinsing twice with sterile distilled water. The leaf pieces were placed on acidified potato dextrose agar (PDA, pH 4.7) in Petri plates, and incubated in the dark at 25°C. Nineteen isolates were obtained and all were purified from a single spore in water agar. Isolate CWJ7 was randomly selected for identification and pathogenicity testing. The colonies on PDA were olivaceous gray to olivaceous black, velvet, with dense hyphae and a scalloped or irregular margin. The reverse side was gray-black and surrounded by tawny halos. The conidia were aseptate and variable in shape and dimension: piriform, columnar, drop-shaped, dumbbell-shaped or oval, measuring 4.90 (7.03) 9.50 × 2.10 (2.78) 3.40 µm (n=100), and chlamydospores were absent. Black pycnidia (132.2–241.5 µm in diameter) appeared after 7 days. The pathogen was initially identified as Phoma or Phoma-like (Boerema et al. 2004). Further confirmation was also determined by sequencing the nuclear ribosomal internal transcribed spacer region (GenBank accession no. MT912950), 28S ribosomal RNA gene (MT912968), and genes encoding β-tubulin (MT920618), the second largest subunit of RNA polymerase II (MT920619) and translation elongation factor (MT946526) (de Hoog and Gerrits van den Ende 1998; Rehner & Samuels 1994; Liu et al. 1999; Vilgalys & Hester 1990), and Blast searches showed 90%–100% homology with GU237754, GU237938, KT389780, KT389575, and KY484705, respectively. In a phylogenetic analysis combining all loci, CWJ7 and the type strains of Boeremia linicola clustered in one group (Fig. 2). Based on its morphological characteristics and phylogenetic analysis, isolate CWJ7 was identified as B. linicola as revised in 2019 (Jayawardena et al. 2019). Healthy 2-year-old plants were used for pathogenicity testing. The leaves of nine potted plants (one plant per pot, three plants per replicate) were spray-inoculated with a suspension of conidia (1×105 spores/ml) from colonies on PDA for 7 days and cultured for 48 h under continuous black light. Nine plants were sprayed with sterile water as the control. This experiment was repeated twice. All plants were cultured in a greenhouse (25°C, 12-h photoperiod, 78% relative humidity). Clear plastic bags were used to maintain high humidity. After 7 days, the inoculated plants showed lesions on the leaves, similar to those observed in the field. The control plants remained symptomless. The pathogen was reisolated and identified by sequencing. This is the first report of B.linicola causing Siberian ginseng leaf spot, and a new record of this species in China. This disease poses a threat to production and management strategies should be developed.

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First Report of Diaporthe longicolla Causing Leaf Spot on Kalanchoe pinnata in China

Plant Disease ◽

10.1094/pdis-12-20-2681-pdn ◽

2021 ◽

Author(s):

Xiaodong Sun ◽

Xinglai Cai ◽

Qiangqiang Pang ◽

Man Zhou ◽

Wen Zhang ◽

...

Keyword(s):

Leaf Spot ◽

Southern China ◽

Elongation Factor ◽

Morphological Characteristics ◽

Negative Control ◽

Hainan Province ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Kalanchoe Pinnata

Kalanchoe pinnata (Lam.) Pers. [syn.: Bryophyllum pinnatum (Lam.) Oken] is an important medicinal agent in southern China. The succulent leaves of this plant are used in the treatment of cholera, bruises, urinary diseases and whitlow. In Oct. 2019, leaf spots were detected on K. pinnata plants in Chengmai County, Hainan Province, China. Lesions with brown to black margins were irregularly shaped and associated with leaf margins. Spots coalesced to form larger lesions (Fig. S1-A), with black pycnidia present in more mature lesions. Symptomatic K. pinnata were found with 10-20% incidence during the humid winters of Hainan Province. Leaf tissues of 10 symptomatic plants were collected and surface sterilized in 70% ETOH for 30s, 0.1% HgCl2 for 30 s, rinsed 3x with sterile distilled water for 30s, placed on potato dextrose agar (PDA) amended with 30mg/L of kanamycin sulfate, and incubated at 25°C in the dark for 3-5 days. Four fungal isolates were obtained using a single-spore isolation method. The colonies were floccose, dense, and white with forming on older colonies grown on PDA (Fig. S1-B-1&2). Alpha conidia exuded from ostiole, rostrate, long-beaked pycnidia in creamy-to-yellowish drops. Alpha conidia were hyaline, ellipsoidal, separated and averaged 6.3μm (SD ± 1.13) long × 1.9μm (SD ± 0.33) wide (n=50). Beta conidia were not seen. The morphological characteristics matched the previous description of Diaporthe longicolla (syn. Phomopsis longicolla) (Hobbs et al. 1985). Mycelial genomic DNA of the representative isolate LDSG3-2 was extracted as template. The internal transcribed spacer (ITS) , translation elongation factor 1α gene (TEF) and β-tubulin (TUB2) regions were amplified. These loci were amplified using primer pairs ITS4/ITS5 (White, et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. A BLAST search of GenBank showed ITS (MN960195), TEF (MN974483) and TUB2 (MN974482) sequences of the isolate were 99%, 100%, and 99% homologous with D. longicolla strains DL11 (MF125048, 557/563 bp), D55 (MN584792, 347/347 bp) and DPC-HOH-32 (MK161506, 502/504 bp). Maximum likelihood trees based on concatenated nucleotide sequences of the three genes were constructed using MEGA 7.0, and bootstrap values indicated the isolate was D. longicolla (Fig. S1-D). Pathogenicity testing was performed using isolate LDSG3-2 by depositing 5µl droplets of a conidial suspension (1 × 106 ml-1) into 5 artificially wounded leaves (using a sterile needle) of 10 healthy 3-month-old K. pinnata plants. An equal number of artificially wounded control leaves were inoculated with sterile water to serve as a negative control. The test was conducted three times. Plants were kept at 25°C in 80% relative humidity and observed for symptoms. Two weeks after inoculation, no symptoms were observed on control plants (Fig. S1-C-1) and all inoculated plants showed symptoms (Fig. S1-C-2) similar to those observed in the field. The fungus was re-isolated from the infected tissues and showed the same cultural and morphological characteristics of the strain inoculated and could not be isolated from the controls fulfilling Koch’s postulates. To our knowledge, this is the first report of leaf spot on K. pinnata caused by D. longicolla in China. This disease is of concern since Phomopsis diseases are common in K. pinnata fields and can cause significant reduction in yield. References: White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. DOI: 10.1016/0167-7799(90)90215-J Carbone, I., and Kohn, L. M. 1999. Mycologia. 91:553. DOI: 10.2307/3761358 Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. DOI: 10.1002/bit.260460112 Hobbs, T. W. et al. 1985. Mycologia. 77: 535. DOI: 10.2307/3793352

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First report of Diaporthe ueckerae causing stem canker on soybean (Glycine max L.) in Colombia

Plant Disease ◽

10.1094/pdis-04-21-0718-pdn ◽

2021 ◽

Author(s):

Nathali López-Cardona ◽

YUDY ALEJANDRA GUEVARA ◽

Lederson Gañán-Betancur ◽

Carol Viviana Amaya Gomez

Keyword(s):

Management Strategies ◽

Elongation Factor ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Stem Canker ◽

Growth Stages ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Glycine Max L ◽

Soybean Plants

In October 2018, soybean plants displaying elongated black to reddish-brown lesions on stems were observed in a field planted to the cv. BRS Serena in the locality of Puerto López (Meta, Colombia), with 20% incidence of diseased plants. Symptomatic stems were collected from five plants, and small pieces (∼5 mm2) were surface sterilized, plated on potato dextrose agar (PDA) and incubated for 2 weeks at 25°C in darkness. Three fungal isolates with similar morphology were obtained, i.e., by subculturing single hyphal tips, and their colonies on PDA were grayish-white, fluffy, with aerial mycelium, dark colored substrate mycelium, and produced circular black stroma. Pycnidia were globose, black, occurred as clusters, embedded in tissue, erumpent at maturity, with an elongated neck, and often had yellowish conidial cirrus extruding from the ostiole. Alpha conidia were observed for all isolates after 30 days growth on sterile soybean stem pieces (5 cm) on water agar, under 25ºC and 12 h light/12h darkness photoperiod. Alpha conidia (n = 50) measured 6.0 – 7.0 µm (6.4 ± 0.4 µm) × 2.0 – 3.0 µm (2.5± 0.4 µm), were aseptate, hyaline, smooth, ellipsoidal, often biguttulate, with subtruncate base. Beta conidia were not observed. Observed morphological characteristics of these isolates were similar to those reported in Diaporthe spp. by Udayanga et al. (2015). DNA from each fungal isolate was used to sequence the internal transcribed spacer region (ITS), and the translation elongation factor 1-α (TEF1) gene, using the primer pairs ITS5/ITS4 (White et al. 1990) and EF1-728F/EF1- 986R (Carbone & Kohn, 1999), respectively. Results from an NCBI-BLASTn, revealed that the ITS sequences of the three isolates (GenBank accessions MW566593 to MW566595) had 98% (581/584 bp) identity with D. miriciae strain BRIP 54736j (NR_147535.1), whereas the TEF1 sequences (GenBank accessions MW597410 to MW597412) had 97 to 100% (330-339/339 bp) identity with D. ueckerae strain FAU656 (KJ590747). The species Diaporthe miriciae R.G. Shivas, S.M. Thomps. & Y.P. Tan, and Diaporthe ueckerae Udayanga & Castl. are synonymous, with the latter taking the nomenclature priority (Gao et al. 2016). According to a multilocus phylogenetic analysis, by maximum likelihood, the three isolates clustered together in a clade with reference type strains of D. ueckerae (Udayanga et al. 2015). Soybean plants cv. BRS Serena (growth stages V3 to V4) were used to verify the pathogenicity of each isolate using a toothpick inoculation method (Mena et al. 2020). A single toothpick colonized by D. ueckerae was inserted directly into the stem of each plant (10 plants per isolate) approximately 1 cm below the first trifoliate node. Noncolonized sterile toothpicks, inserted in 10 soybean plants served as the non-inoculated control. Plants were arbitrarily distributed inside a glasshouse, and incubated at high relative humidity (>90% HR). After 15 days, inoculated plants showed elongated reddish-brown necrosis at the inoculated sites, that were similar to symptoms observed in the field. Non-inoculated control plants were asymptomatic. Fungal cultures recovered from symptomatic stems were morphologically identical to the original isolates. This is the first report of soybean stem canker caused by D. ueckerae in Colombia. Due to the economic importance of this disease elsewhere (Backman et al. 1985; Mena et al. 2020), further research on disease management strategies to mitigate potential crop losses is warranted.

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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 Blueberry Leaf Spot Caused by Cylindrocladium colhounii in China

Plant Disease ◽

10.1094/pd-90-1553a ◽

2006 ◽

Vol 90 (12) ◽

pp. 1553-1553 ◽

Cited By ~ 2

Author(s):

Y. S. Luan ◽

L. Feng ◽

L. J. An

Keyword(s):

Leaf Spot ◽

Morphological Traits ◽

Internal Transcribed Spacers ◽

Fluorescent Light ◽

Adaxial Side ◽

Single Spore ◽

Tubulin Gene ◽

First Report ◽

Plastic Bags ◽

Β Tubulin

During late July and early August of 2005, leaf spot symptoms were observed in a blueberry nursery at a plantation in Dalian, which to our knowledge, lies within the largest blueberry-production area in China. Symptoms were observed primarily on lowbush species, for example Blomidon, as well as half-highbush cultivars. A slow-growing, white mycelium from the margin of necrotic leaf spots was recovered on potato dextrose agar (PDA). The following morphological traits were observed: erect conidiophores that branch twice and were terminated in a stiped, clavate phialide; hyaline, cylindrical, four-celled conidia; and globose, reddish brown, aggregated chlamydospores. Conidiophores (including stipes and terminal phialides) were 305 to 420 × 5 to 9 μm; primary branches were 9 to 45 × 5 to 6.3 μm; secondary branches were 9 to 17.3 × 3.1 to 4.5 μm; phialides were 7.8 to 17.5 × 2.5 to 6 μm; stipes (from the highest branch area to vesicle) were 150 to 270 μm long; and vesicles were 13 to 30 × 2 to 4.5 μm. Conidia were 50 to 72 × 4 to 5.5 μm. Chlamydospores were 15 to 20 μm in diameter. Koch's postulates were fulfilled by spray inoculating two healthy cultivars with conidiophores homogenized in axenic water. As a control, two healthy plants were sprayed with axenic water. Plants were placed inside plastic bags to maintain humidity and incubated in a growth chamber at 26°C under fluorescent light for 14 h and 20°C in darkness for 10 h. After 2 days, the plastic bags were removed and plants were maintained under the same conditions. After 4 days, small-to-medium brown spots with purplish margins were observed on the adaxial side of leaves from inoculated plants, but not from control plants. Fungi isolated from these lesions had the same morphological traits as the ones isolated previously from field plants. The morphological descriptions and measurements were similar to Cylindorocladium colhounii (2). The 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA and the β-tubulin gene were amplified from DNA extracted from single-spore cultures using the ITS1/ITS4 primers and T1/Bt2b primers, respectively, and sequenced (1). The ITS and β-tubulin gene sequences were similar to C. colhounii STE-U 1237 (99%; GenBank Accession No. AF231953) and C. colhounii STE-U 705 (99%; GenBank Accession No. AF231954), respectively. The morphology, secondary conidiation, and sequences of ITS and β-tubulin gene identify the causal fungus as C. colhounii. To our knowledge, this is the first report of C. colhounii on blueberry in China or in the world. References: (1) P. W. Crous et al. Can. J. Bot. 77:1813, 1999. (2) T. Watanabe. Page 222 in: Dictorial Atlas of Soil and Seed Fungi. CRC Press, Inc., Boca Raton, Fl, 1994.

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First Report of Leaf Spot Caused by Alternaria alternata on Kiwifruit in Italy

Plant Disease ◽

10.1094/pdis.1999.83.5.487d ◽

1999 ◽

Vol 83 (5) ◽

pp. 487-487 ◽

Cited By ~ 3

Author(s):

L. Corazza ◽

L. Luongo ◽

M. Parisi

Keyword(s):

New Zealand ◽

Alternaria Alternata ◽

Leaf Spot ◽

Southern Italy ◽

Morphological Characteristics ◽

Potato Dextrose Agar ◽

First Report ◽

Leaf Spots ◽

Detached Leaves ◽

Pathogenicity Tests

A leaf spot of kiwifruit (Actinidia deliciosa (A. Chev.) C. F. Liang & A. R. Ferg.) leaves was recently observed on plants of the cultivar Hayward in an orchard near Salerno, in southern Italy. The affected plants showed early severe defoliation. The fungus isolated from the infected leaves was identified as Alternaria alternata (Fr.:Fr.) Keissl., based on conidial morphological characteristics. Pathogenicity tests were made by inoculating detached leaves of male pollinator cultivar Tomuri and the female cultivars Hayward and Bruno with a 7-mm disk taken from actively growing cultures of the fungus on potato dextrose agar (PDA). After 14 days, necrotic leaf spots developed and A. alternata was consistently isolated from the inoculated leaves. A. alternata has been observed as a pathogen on leaves and fruits in New Zealand. In the Mediterranean, it has been reported in Israel (2) and in the island of Crete (1). This is the first report of Alternaria leaf spot on kiwifruit in Italy. References: (1) V. A. Bourbos and M. T. Skoudridakis. Petria 7:111, 1997. (2) A. Sive and D. Resnizky. Alon Hanotea 41:409, 1987.

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First Report of Sea buckthorn Stem Wilt Caused by Fusarium sporotrichioides in Gansu, China

Plant Disease ◽

10.1094/pdis-03-21-0627-pdn ◽

2021 ◽

Author(s):

Bo Xia ◽

Yue Liang ◽

Jianzhong Hu ◽

Xiaoling Yan ◽

Liqiang Yin ◽

...

Keyword(s):

Elongation Factor ◽

Morphological Characteristics ◽

Gansu Province ◽

Sea Buckthorn ◽

Molecular Characteristics ◽

Ear Rot ◽

Fusarium Sporotrichioides ◽

Tree Roots ◽

Internal Transcribed Spacer Region ◽

First Report

Sea buckthorn (Hippophae rhamnoides) is an important deciduous shrub for fruit and ecological restoration in arid and semi-arid regions of China. Twelve Chinese and Russian cultivars (cv. Shenqiuhong, eshi01, ... eshi11) were planted about 1.6 acre area in a seedling nursery, located in Qingyang City of Gansu province in northwest China, where high mortality (more than 70%) of sea buckthorn was observed in late July 2019. Symptoms consisted of massive chlorosis, drooping leaves and dried-up stems on 5-year-old trees. Pieces of tree roots and stems with irregular light-brown discoloration in the xylem vessels were selected. Small pieces of discolored tissue were surface disinfested (1 min in 1% sodium hypochlorite, followed by three rinses with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues. Colonies on PDA grew rapidly. Dense mycelia were pinky-white initially, and became carmine red color with age on the undersurface of the plate. Macroconidia were moderately curved, 3 to 5 marked septa, hyaline, thick walled, and measuring 27.8± 3.6 µm × 4.8 ± 0.5 µm (n = 30). Microconidia were abundant, pear-shaped, ellipsoid to fusoid, often with a papilla at the base, and 8.4 ± 2.2 µm ×3.1 ± 0.3 µm (n = 30). Genomic DNA was extracted for amplification and sequencing of the internal transcribed spacer region (ITS1 and ITS4 primers) (White et al. 1990) of the ribosomal DNA (Accession Nos. MN160235 to MN160238) and translation elongation factor-1 alpha (EF1 and EF2 primers, accession Nos. MN429075 to MN429078) (O’Donnell et al. 1998). The sequences revealed 99% similarity to the sequences of the ITS (AY188917), and 100% identity with EF1-α (JF740808) regions of Fusarium sporotrichioides. Based on morphological and molecular characteristics, the fungus was identified as F. sporotrichioides (Leslie and Summerell 2006). Koch’s postulates were fulfilled on healthy, potted 1-year-old sea buckthorn seedings using two isolates in a greenhouse at 25 °C, 90% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedings were inoculated with PDA plugs as controls. Seven to ten days after inoculation, typical symptoms of dark-brown necrotic lesions on chlorotic leaf margins were observed. About 2 weeks after inoculation, the inoculated stems were gradually dry up, accompanied by withering and fallen leaves. Control plants remained asymptomatic. Pathogens were successfully isolated from the inoculated stems again, exhibiting morphological characteristics identical to those of F. sporotrichioides. Previous papers reported F. sporotrichioides as a common pathogen caused lavender wilt (Cosic et al. 2012), foliar spots on forage corn (Moya-Elizondo et al. 2013) and maize ear rot (Wang et al. 2019). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. sporotrichioides on several Chinese and Russian cultivars in Gansu province of China. In Heilongjiang province, the same disease was reported in 2010 (Song et al. 2010), nearly 30 longitudes away from Gansu province. Therefore, this disease appears to be a serious risk for future sea buckthorn production.

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