First Report of Botrytis pseudocinerea Causing Gray Mold on Blueberry in North America

Botrytis cinerea has previously been shown to consist of two sibling species, referred to as Group I and Group II, that can be differentiated by PCR-RFLP analysis of the Bc-hch gene, a vegetative incompatibility locus (1). Group I has recently been described as a new cryptic species, B. pseudocinerea (4). Gray mold caused by B. cinerea is a major postharvest disease of blueberries in the Central Valley of California. In 2012 and 2013, blueberry fruit were sampled at harvest from various locations in the region and stored at 0 to 1°C for 5 weeks, and fungi were isolated from decayed fruit. In total, 526 isolates of Botrytis spp. were obtained. Genomic DNA was extracted and PCR-RFLP of a fragment of the Bc-hch gene was performed. Four isolates showed the distinctive restriction band pattern associated with Group I (1). The identity of these four isolates was further investigated by sequencing portions of four genes: internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPBII), using the primers described previously (3,4). Sequences were deposited in GenBank (Accession Nos. KJ796643 to 58). BLAST analysis showed that sequences of all four genes for the four isolates were 99.8 to 100% similar to those of B. pseudocinerea. Morphological characteristics of the four blueberry isolates were examined as described previously (4). On potato dextrose agar, colonies were gray; the mycelial growth rate was 26 mm/day at 19°C in the dark. Conidiophores were simple and erect, and conidia were borne in grapelike clusters, one celled, hyaline, elliptical to ovoid, 6.5 to 15.7 × 5.6 to 9.8 μm (average of 7.4 × 10.1 μm). As reported previously, none of the morphological characters was able to differentiate between B. cinerea and B. pseudocinerea (4). To test pathogenicity, freshly harvested organic blueberry fruits were treated with 0.5% sodium hypochlorite for 2 min, rinsed with sterile water, wounded using a sterile needle, and inoculated by placing 1 μl of a conidial suspension (1.0 × 105 spores/ml) from each isolate into the wound with a pipette. Inoculated fruit (10 for each isolate) were incubated at 20°C for 5 days in the dark. Experiments were performed twice. All inoculated fruit developed rot, and no decay was observed on the noninoculated controls. All four isolates of B. pseudocinerea were pathogenic, and the fungus was re-isolated from decayed fruit. B. pseudocinerea isolates are known to be naturally insensitive to fenhexamid (1,4). Sensitivity of the four isolates to fenhexamid was examined in vitro as previously described (4). The EC50 values for fenhexamid for the four isolates ranged from 7.7 to 9.9 μg/ml and isolates were considered resistant to fenhexamid (1,4). Based on the morphological, physiological, and genetic characteristics, the four blueberry isolates were identified as B. pseudocinerea. It appeared that this species was present at very low frequency (0.76%) in blueberry fields in California. Previously, B. pseudocinerea has been reported from French, German, and New Zealand vineyards (1,2,4). To our knowledge, this is the first report of B. pseudocinerea causing gray mold in blueberry in California and in North America. References: (1) E. Fournier et al. Mycologia 95:251, 2003. (2) P. R. Johnston et al. Plant Pathol. 63:888, 2014. (3) M. Staats et al. Mol. Biol. Evol. 22: 333, 2005. (4) A.-S. Walker et al. Phytopathology 101:1433, 2011.

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First Report of Leaf Spot Caused by Botrytis cinerea on Cardamine hupingshanensis in China

Plant Disease ◽

10.1094/pdis-04-21-0698-pdn ◽

2021 ◽

Author(s):

Jun Guo ◽

Jin Chen ◽

Zhao Hu ◽

Jie Zhong ◽

Jun Zi Zhu

Keyword(s):

Botrytis Cinerea ◽

Morphological Characteristics ◽

Gray Mold ◽

Hunan Province ◽

Conidial Suspension ◽

Heat Shock Protein 60 ◽

Basal Cells ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

First Report

Cardamine hupingshanensis is a selenium (Se) and cadmium (Cd) hyperaccumulator plant distributed in wetlands along the Wuling Mountains of China (Zhou et al. 2018). In March of 2020, a disease with symptoms similar to gray mold was observed on leaves of C. hupingshanensis in a nursery located in Changsha, Hunan Province, China. Almost 40% of the C. hupingshanensis (200 plants) were infected. Initially, small spots were scattered across the leaf surface or margin. As disease progressed, small spots enlarged to dark brown lesions, with green-gray, conidia containing mold layer under humid conditions. Small leaf pieces were cut from the lesion margins and were sterilized with 70% ethanol for 10 s, 2% NaOCl for 2 min, rinsed with sterilized distilled water for three times, and then placed on potato dextrose agar (PDA) medium at 22°C in the dark. Seven similar colonies were consistently isolated from seven samples and further purified by single-spore isolation. Strains cultured on PDA were initially white, forming gray-white aerial mycelia, then turned gray and produced sclerotia after incubation for 2 weeks, which were brown to blackish, irregular, 0.8 to 3.0 × 1.2 to 3.5 mm (n=50). Conidia were unicellular, globose or oval, colourless, 7.5 to 12.0 × 5.5 to 8.3 μm (n=50). Conidiophores arose singly or in group, straight or flexuous, septate, brownish to light brown, with enlarged basal cells, 12.5 to 22.1 × 120.7 to 310.3 μm. Based on their morphological characteristics in culture, the isolates were putatively identified as Botrytis cinerea (Ellis 1971). Genomic DNA of four representative isolates, HNSMJ-1 to HNSMJ-4, were extracted by CTAB method. The internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH), heat-shock protein 60 gene (HSP60), ATP-dependent RNA helicaseDBP7 gene (MS547) and DNA-dependent RNA polymerase subunit II gene (RPB2) were amplified and sequenced using the primers described previously (Aktaruzzaman et al. 2018) (MW820311, MW831620, MW831628, MW831623 and MW831629 for HNSMJ-1; MW314722, MW316616, MW316617, MW316618 and MW316619 for HNSMJ-2; MW820519, MW831621, MW831627, MW831624 and MW831631 for HNSMJ-3; MW820601, MW831622, MW831626, MW831625 and MW831630 for HNSMJ-4). BLAST searches showed 99.43 to 99.90% identity to the corresponding sequences of B. cinerea strains, such as HJ-5 (MF426032.1, MN448500.1, MK791187.1, MH727700.1 and KX867998.1). A combined phylogenetic tree using the ITS, G3PDH, HSP60 and RPB2 sequences was constructed by neighbor-joining method in MEGA 6. It revealed that HNSMJ-1 to HNSMJ-4 clustered in the B. cinerea clade. Pathogenicity tests were performed on healthy pot-grown C. hupingshanensis plants. Leaves were surface-sterilized and sprayed with conidial suspension (106 conidia/ mL), with sterile water served as controls. All plants were kept in growth chamber with 85% humidity at 25℃ following a 16 h day-8 h night cycle. The experiment was repeated twice, with each three replications. After 4 to 7 days, symptoms similar to those observed in the field developed on the inoculated leaves, whereas controls remained healthy. The pathogen was reisolated from symptomatic tissues and identified using molecular methods, confirming Koch’s postulates. B. cinerea has already been reported from China on C. lyrate (Zhang 2006), a different species of C. hupingshanensis. To the best of our knowledge, this is the first report of B. cinerea causing gray mold on C. hupingshanensis in China and worldwide. Based on the widespread damage in the nursery, appropriate control strategies should be adopted. This study provides a basis for studying the epidemic and management of the disease.

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First Report of Fusarium ipomoeae Causing Peanut leaf Spot in China

Plant Disease ◽

10.1094/pdis-01-21-0226-pdn ◽

2021 ◽

Author(s):

Manlin Xu ◽

Xia Zhang ◽

Jing Yu ◽

zhiqing Guo ◽

Ying Li ◽

...

Keyword(s):

Leaf Spot ◽

Genomic Dna ◽

Block Design ◽

Management Strategies ◽

Morphological Characteristics ◽

Ethanol Solution ◽

Distilled Water ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report

Peanut (Arachis hypogaea L.) is one of the most economically important crops as an important source of edible oil and protein. In August 2020, circular to oval-shaped brown leaf spots (2-6 mm in diameter) with well-defined borders surrounded by a yellow margin were observed on peanut plant leaves in Laixi City, Shandong Province, China. Symptomatic plants randomly distributed in the field, the incidence was approximately 5%. Leave samples were collected consisted of diseased tissue and the adjacent healthy tissue. The samples were dipped in a 70% (v/v) ethanol solution for 30 s and then soaked in a 0.1% (w/v) mercuric chloride solution for 60 s. The surface-sterilized tissues were then rinsed three times with sterile distilled water, dried and placed on Czapek Dox agar supplemented with 100 μg/ml of chloramphenicol. The cultures were incubated in darkness at 25 °C for 3–5 days. Fungal colonies were initially white and radial, turning to orange-brown in color, with abundant aerial mycelia. Macroconidia were abundant, 4 to 7 septate, with a dorsiventral curvature, and were 3.3–4.5 × 18.5–38.1 μm (n=100) in size; microconidia were absent; chlamydospores were produced in chains or clumps, ellipsoidal to subglobose, and thick walled. The morphological characteristics of the conidia were consistent with those of Fusarium spp. To identify the fungus, an EasyPure Genomic DNA Kit (TransGEN, Beijing, China) was used to extract the total genomic DNA from mycelia. The internal transcribed spacer region (ITS rDNA) and the translation elongation factor 1-α gene (TEF1) were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1/EF2 (O’Donnell et al. 1998), respectively. Based on BLAST analysis, sequences of ITS (MT928727) and TEF1 (MT952337) showed 99.64% and 100% similarity to the ITS (MT939248.1), TEF1 (GQ505636.1) of F. ipomoeae isolates. Sequence analysis confirmed that the fungus isolated from the infected peanut was F. ipomoeae (Xia et al. 2019). The pathogenicity of the fungus was tested in the greenhouse. Twenty two-week-old peanut seedlings (cv. Huayu20) grown in 20-cm pots (containing autoclaved soil) were sprayed with a conidial suspension (105 ml−1) from a 15-day-old culture. Control plants were sprayed with distilled water. The experiment was conducted as a randomized complete block design, and placed at 25 °C under a 12-h photoperiod with 90% humidity. Symptoms similar to those in the field were observed on leaves treated with the conidial suspension ten days after inoculation, but not on control plants. F. ipomoeae was re-isolated from symptomatic leaves but not from the control plants. Reisolation of F. ipomoeae from inoculated plants fulfilled Koch's postulates. To our knowledge, this is the first report of F. ipomoeae causing peanut leaf spot in China. Our report indicates the potential spread of this pathogen in China and a systematic survey is required to develop effective disease management strategies.

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First report of Fusarium incarnatum causing fruit rot of litchi in China

Plant Disease ◽

10.1094/pdis-11-20-2393-pdn ◽

2021 ◽

Author(s):

Zhaoyin Gao ◽

Jiaobao Wang ◽

Zhengke Zhang ◽

Min Li ◽

Deqiang Gong ◽

...

Keyword(s):

Southern China ◽

Fusarium Species ◽

Morphological Characteristics ◽

Fruit Rot ◽

Conidial Suspension ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Litchi Fruit ◽

His3 Gene

Litchi (Litchi chinensis Sonn.) is an indigenous tropical and subtropical fruit in Southern China with an attractive appearance, delicious taste, and good nutritional value (Jiang et al. 2003). In March 2020, brown rots were observed on nearly ripe litchi fruits (cv. Guihuaxiang) in an orchard of Lingshui county, Hainan province of China (18.615877° N, 109.948871° E). About 5% fruits were symptomatic in the field, and the disease caused postharvest losses during storage. The initial infected fruits had no obvious symptoms on the outer pericarp surfaces, but appeared irregular, brown to black-brown lesions in the inner pericarps around the pedicels. Then lesions expanded and became brown rots. Small tissues (4 mm × 4 mm) of fruit pericarps were cut from symptomatic fruits, surface-sterilized in 1% sodium hypochlorite for 3 min, rinsed in sterilized water three times, plated on potato dextrose agar (PDA) and incubated at 28℃ in the darkness. Morphologically similar colonies were isolated from 85% of 20 samples after 4 days of incubation. Ten isolates were purified using a single-spore isolation method. The isolates grown on PDA had abundant, fluffy, whitish to yellowish aerial mycelia, and the reverse side of the Petri dish was pale brown. Morphological characteristics of conidia were further determined on carnation leaf-piece agar (CLA) (Leslie et al. 2006). Macroconidia were straight to slightly curved, 3- to 5-septates with a foot-shaped basal cell, tapered at the apex, 2.70 to 4.43 µm × 18.63 to 37.58 µm (3.56 ± 0.36 × 28.68 ± 4.34 µm) (n = 100). Microconidia were fusoid to ovoid, 0- to 1-septate, 2.10 to 3.57 µm × 8.18 to 18.20 µm (2.88 ± 0.34 × 11.71 ± 1.97 µm) (n = 100). Chlamydospores on hyphae singly or in chains were globose, subglobose, or ellipsoidal. Based on cultural features and morphological characteristics, the fungus was identified as a Fusarium species (Leslie et al. 2006). To further confirm the pathogen, DNA was extracted from the 7-day-old aerial mycelia of three isolates (LZ-1, LZ-3, and LZ-5) following Chohan et al. (2019). The sequences of the internal transcribed spacer region of rDNA (ITS), translation elongation factor-1 alpha (tef1) gene, and histone H3 (his3) gene were partially amplified using primers ITS1/ITS4, EF1-728F/EF1-986R, and CYLH3F/CYLH3R, respectively (Funnell-Harris et al. 2017). The nucleotide sequences were deposited in GenBank (ITS: 515 bp, MW029882, 533 bp, MW092186, and 465 bp, MW092187; tef1: 292 bp, MW034437, 262 bp, MW159143, and 292 bp, MW159141; his3: 489 bp, MW034438, 477 bp, MW159142, and 474 bp, MW159140). The ITS, tef1, and his3 genes showed 99-100% similarity with the ITS (MH979697), tef1 (MH979698), and his3 (MH979696) genes, respectively of Fusarium incarnatum (TG0520) from muskmelon fruit. The phylogenetic analysis of the tef1 and his3 gene sequences showed that the three isolates clustered with F. incarnatum. Pathogenicity tests were conducted by spraying conidial suspension (1×106 conidia/ml) on wounded young fruits in the orchid. Negative controls were sprayed with sterilized water. Fruits were bagged with polythene bags for 24 hours and then unbagged for 10 days. Each treatment had 30 fruits. The inoculated fruits developed symptoms similar to those observed in the orchard and showed light brown lesions on the outer pericarp surfaces and irregular, brown to black-brown lesions in the inner pericarps, while the fruits of negative control remained symptomless. The same fungus was successfully recovered from symptomatic fruits, and thus, the test for the Koch’s postulates was completed. F. semitectum (synonym: F. incarnatum) (Saha et al. 2005), F. oxysporum (Bashar et al. 2012), and F. moniliforme (Rashid et al. 2015) have been previously reported as pathogens causing litchi fruit rots in India and Bangladesh. To our knowledge, this is the first report of Fusarium incarnatum causing litchi fruit rot in China.

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First Report of Oak Anthracnose Caused by Apiognomonia errabunda on Oriental White Oak in Korea

Plant Disease ◽

10.1094/pdis-02-13-0181-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1121-1121

Author(s):

C. K. Lee ◽

S. H. Lee ◽

J. H. Park ◽

S. E. Cho ◽

H. D. Shin

Keyword(s):

North America ◽

Morphological Characteristics ◽

Its Region ◽

Culture Collection ◽

Deciduous Tree ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Natural Forests ◽

White Oak ◽

First Report

Oriental white oak, Quercus aliena Blume, is native to East Asia including Korea. It is one of the major deciduous tree species in natural forests in Korea. In May 2012, several hundred trees were found to be heavily damaged by a previously unknown leaf disease in a forest near Songjiho Lake in Goseong County of central Korea. Leaf symptoms began as small, water-soaked, pale greenish to grayish lesions, which enlarged to follow the veins or midribs and to be bounded by them, often killing part of the leaf. Leaf distortion and blight resulted in the later stage of disease development. A number of grayish brown to nearly black acervuli were formed on the lesions, especially on the midribs and veins. Acervuli were mostly hypophyllous, intraepidermal, erumpent, circular to ellipsoid in outline, cushion-like, and 70 to 220 μm in diameter. Conidia (n = 30) were elliptical to fusiform-elliptical, occasionally obclavate, aguttulate or guttulate, hyaline, aseptate, and 7.5 to 20 × 5 to 7.5 μm (mean 14.6 × 6.1 μm). These morphological characteristics of the fungus were consistent with the description of conidial state of Apiognomonia errabunda (Roberge ex Desm.) Höhn. (3,4). Voucher specimens were deposited in the Korea University Herbarium (KUS). An isolate obtained from KUS-F26690 was deposited in the Korean Agricultural Culture Collection (Accession No. KACC46842). 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 549-bp sequence was deposited in GenBank (KC426947). This showed >99% similarity with sequences of A. errabunda (AJ888475 to 888477). For pathogenicity test, inoculum was prepared by harvesting conidia from 4-week-old cultures on potato dextrose agar. A conidial suspension (1 × 106 conidia/ml) was sprayed onto young leaves of three potted seedlings. Three seedlings treated with sterile distilled water served as controls. Plants were covered with plastic bags to maintain 100% relative humidity for 24 h and then kept in a greenhouse (20 to 26°C and 60 to 80% RH). After 26 days, typical leaf spot symptoms, identical to the ones observed in the field, developed on the inoculated leaves. No symptoms were observed on controls. A. errabunda was reisolated from the lesions of inoculated plants, fulfilling Koch's postulates. Oak anthracnose associated with A. errabunda (including A. quercina) has been recorded in Europe and North America (1,4). Oak anthracnose of evergreen Quercus glauca Thunb. (syn. Cyclobalanopsis glauca (Thunb.) Oerst.) associated with A. supraseptata in Japan is not related to this disease (2). To our knowledge, this is the first report of oak anthracnose of Q. aliena globally and also the first finding of A. errabunda in Asia as well as in Korea. This pathogen is known as one of the major forest pathogens in oak stand in Europe and North America (3). Pending further studies, including a risk assessment, A. errabunda may be considered as a potentially new and serious threat in native and planted ranges of Q. aliena in Korea. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, retrieved February 18, 2013. (2) S. Kaneko and T. Kobayashi. Trans. Mycol. Soc. Japan 25:11, 1984. (3) A. Ragazzi et al. Phytopathol. Mediterr. 46:295, 2007. (4) M. V. Sogonov et al. Mycol. Res. 111:693, 2007.

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First Report of Gray Mold of Strawberry Caused by Botrytis cinerea in South Carolina

Plant Disease ◽

10.1094/pdis-06-11-0529 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1482-1482 ◽

Cited By ~ 4

Author(s):

D. Fernández-Ortuño ◽

X. Li ◽

W. Chai ◽

G. Schnabel

Keyword(s):

South Carolina ◽

Botrytis Cinerea ◽

Species Identification ◽

Morphological Characteristics ◽

Pcr Amplification ◽

Gray Mold ◽

Tween 20 ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

Control Fruit

Gray mold caused by Botrytis spp. is one of the most economically important diseases of cultivated strawberry (Fragaria × ananassa) worldwide. From April to June 2011, strawberries with symptoms resembling gray mold disease were collected from different locations (Chesnee, Florence, Lexington, McBee, Monetta, and North Augusta) in South Carolina. Fruit infections began as small, firm, light brown lesions that enlarged quickly, becoming covered with a gray, fuzzy mass of spores followed by a soft rot. To isolate the causal agent, spores from symptomatic fruit were suspended in 1% Tween 20, streaked onto the surface of potato dextrose agar plates, and incubated at 22°C. Fungal colonies from single spores were at first colorless and later became gray to brown when the conidiphores and conidia developed. Conidia were identified by their morphological characteristics: an average size of 14 × 9 μm, ellipsoid to rounded without internal structure, and with a scar on the point of union to the conidiophore (1). Sclerotia produced in culture were hard, dark, irregular shaped, and formed after 2 weeks. The pathogen was identified as Botrytis cinerea Pers.: on the basis of morphology and confirmed by a restriction digest with ApoI of the 413-kb PCR amplification product obtained with BA2f/BA1r primers (2). Koch's postulates were conducted by inoculating 10 surface-sterilized strawberries with a conidial suspension (105 spores/ml) of a randomly chosen B. cinerea isolate previously characterized; 10 control fruit received sterile water without conidia. The inoculated fruit were incubated for 3 days at room temperature in air-tight plastic bags. Inoculated fruit developed typical gray mold symptoms with gray sporulating lesions. The developing spores on inoculated fruit were confirmed to be B. cinerea. All control fruit remained healthy. For many Botrytis spp., the internal transcribed spacer region does not reveal nucleotide variations and thus is useless for species identification. We used additional, more appropriate genetic markers for molecular-based species identification and verified that strawberries in South Carolina are affected by gray mold disease caused by B. cinerea. To our knowledge, this is the first scientific report of B. cinerea causing gray mold of strawberry in South Carolina. References: (1) W. R. Jarvis. Botryotinia and Botrytis Species: Taxonomy, Physiology and Pathogenicity. A Guide to the Literature. Monograph no. 15. Canada Department of Agriculture, Research Branch, Ottawa, 1977. (2) K. Nielsen et al. Plant Dis. 86:682, 2002.

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First Report of Brown Rot of fruit on Vitis amurensis Caused by Monilinia polystroma in China

Plant Disease ◽

10.1094/pdis-12-20-2636-pdn ◽

2021 ◽

Author(s):

Yue Wang ◽

Yiming Yang ◽

Shutian Fan ◽

Wenpeng Lu

Keyword(s):

Russian Far East ◽

Morphological Characteristics ◽

Brown Rot ◽

Effective Control ◽

Vitis Amurensis ◽

Conidial Suspension ◽

Sterile Water ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Germ Tubes

Vitis amurensis Rupr. (Amur grape) is a wild grape genetic resource widely distributed in Heilongjiang, Jilin, Liaoning, and Inner Mongolia, among other places in China (Song et al. 2009) and the Russian Far East and Korean Peninsula. In September 2018, brown rot symptoms were observed at ripening stage on the fruits of a 5-year-old Amur grapevine germplasm resources nursery of the cultivar ‘Beibinghong’ and a few Russian resources in Zuojia Town, Jilin City, Jilin Province, China. The diseased fruit surface became brown with soft rot and produced buff to brownish-grey sporodochia with conidia. Around 180 plants of ‘Beibinghong’ were examined which had 8 % incidence. Forty five samples were collected from symptomatic fruits of 15 randomly sampled ‘Beibinghong’ grape clusters, cut into 5-mm2 pieces of diseased tissue, surface sterilized with 1% NaOCl for 2 min, rinsed three times with sterile water, dried on sterilized filter paper, and plated on potato dextrose agar (PDA). Thirteen monosporic isolates were obtained using the single-spore isolation method with incubation at 25°C and a 12-h light/12-h dark cycle. The average colony diameter was 46–49 mm after 4 days of culture on PDA. Colonies were white to grayish with even margins. Irregular black stromatal crusts were observed on the reverse side of dishes 10 days after inoculation. Conidial spores were produced when cultured on cherry agar at 25°C under near-ultravolet light. Spores were single-celled and hyaline, limoniform or ellipsoid, and were produced in branched monilioid chains, 12–22 × 8–13 µm (mean: 15.4 ± 1.03 × 9.01 ± 0.72 µm, n = 50). When conidia were cultured on water agar at 25°C for 18 h, the germ tubes were straight, 700–1,000 µm long, and often with two germ tubes per conidium. Morphological characteristics were consistent with those of Monilinia polystroma (van Leeuwen et al. 2002). To confirm the species identification, two DNA regions of the selected isolate ‘VAMPWYZSH8’ were amplified by polymerase chain reaction (PCR) and sequenced: the internal transcribed spacer region (ITS) was generated using primers ITS1/ ITS4 (Munda 2015) and β-tubulin (TUB2) was amplified using primers Bt2a/Bt2b (Zhu et al. 2016). A BLAST analysis of the nucleotide sequence of the PCR products revealed 100% identity with two M. polystroma sequences in the NCBI GenBank (KJ814976 for ITS, KR778970 for TUB2). Our sequences were deposited in GenBank with accession nos. MT038413 for ITS and MT038414 for TUB2. On the basis of these results, the isolate was identified as M. polystroma. To confirm pathogenicity, 78 fresh and healthy bunches of ‘Beibinghong’ grapes at ripening were collected, surface disinfected by immersion in 1% NaOCl for 1 min, rinsed three times with sterile water, then allowed to air dry. Under dry aseptic conditions, the fruits were inoculated using the pin prick method. Each wound was inoculated with 10 μl conidial suspension (106 spore ml−1) and incubated at 25°C with about 90% relative humidity and natural light. Inoculation with water was used as control and the experiment was repeated three times. After a 10-day incubation, typical symptoms of brown rot developed on inoculated fruits, while control fruits were symptomless. The fungus was consistently re-isolated only from diseased fruits and showed the same morphological characteristics as the original isolates, thus fulfilling Koch’s postulates. This is the first report of M. polystroma on V. amurensis in China. The resulting disease decreases fruit quality and yield, necessitating the development of effective control measures.

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First Report of Stemphylium solani as the Causal Agent of a Leaf Spot on Greenhouse Cucumber

Plant Disease ◽

10.1094/pdis-08-12-0776-pdn ◽

2013 ◽

Vol 97 (2) ◽

pp. 287-287 ◽

Cited By ~ 5

Author(s):

D. J. Vakalounakis ◽

E. A. Markakis

Keyword(s):

Leaf Spot ◽

Natural Infection ◽

Apical Cell ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

First Report

During the 2011 to 2012 crop season, a severe leaf spot disease of cucumber (Cucumis sativus) cv. Cadiz was noticed on crops in some greenhouses in the Goudouras area, Lasithi, Crete, Greece. Symptoms appeared in late winter, mainly on the leaves of the middle and upper part of the plants. Initially, small necrotic pinpoint lesions with white centers, surrounded by chlorotic halos, 1 to 3 mm in diameter, appeared on the upper leaf surfaces, and these progressively enlarged to spots that could coalesce to form nearly circular lesions up to 2 cm or more in diameter. Stemphylium-like fructifications appeared on necrotic tissue of older lesions. Severely affected leaves became chlorotic and died. No other part of the plant was affected. Small tissue pieces from the edges of lesions were surface disinfected in 0.5% NaClO for 5 min, rinsed in sterile distilled water, plated on acidified potato dextrose agar and incubated at 22 ± 0.5°C with a 12-h photoperiod. Stemphylium sp. was consistently isolated from diseased samples. Colonies showed a typical septate mycelium with the young hyphae subhyaline and gradually became greyish green to dark brown with age. Conidiophores were subhyaline to light brown, 3- to 10-septate, up to 200 μm in length, and 4 to 7 μm in width, with apical cell slightly to distinctly swollen, bearing a single spore at the apex. Conidia were muriform, mostly oblong to ovoid, but occasionally nearly globose, subhyline to variant shades of brown, mostly constricted at the median septum, 22.6 ± 6.22 (11.9 to 36.9) μm in length, and 15.1 ± 2.85 (8.3 to 22.6) μm in width, with 1 to 8 transverse and 0 to 5 longitudinal septa. DNA from a representative single-spore isolate was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was amplified using the universal primers ITS5 and ITS4. The PCR product was sequenced and deposited in GenBank (Accession No. JX481911). On the basis of morphological characteristics (3) and a BLAST search with 100% identity to the published ITS sequence of a S. solani isolate in GenBank (EF0767501), the fungus was identified as S. solani. Pathogenicity tests were performed by spraying a conidial suspension (105 conidia ml–1) on healthy cucumber (cv. Knossos), melon (C. melo, cv. Galia), watermelon (Citrullus lanatus cv. Crimson sweet), pumpkin (Cucurbita pepo, cv. Rigas), and sponge gourd (Luffa aegyptiaca, local variety) plants, at the 5-true-leaf stage. Disease symptoms appeared on cucumber and melon only, which were similar to those observed under natural infection conditions on cucumber. S. solani was consistently reisolated from artificially infected cucumber and melon tissues, thus confirming Koch's postulates. The pathogenicity test was repeated with similar results. In 1918, a report of a Stemphylium leaf spot of cucumber in Indiana and Ohio was attributed to Stemphylium cucurbitacearum Osner (4), but that pathogen has since been reclassified as Leandria momordicae Rangel (2). That disease was later reported from Florida (1) and net spot was suggested as a common name for that disease. For the disease reported here, we suggest the name Stemphylium leaf spot. This is the first report of a disease of cucumber caused by a species of Stemphylium. References: (1) C. H. Blazquez. Plant Dis. 67:534, 1983. (2) P. Holliday. Page 243 in: A Dictionary of Plant Pathology. Cambridge University Press, Cambridge, UK, 1998. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) G. A. Osner. J. Agric. Res. 13:295, 1918.

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First Report of Wilt Caused by Verticillium dahliae on Cosmos (Cosmos bipinnatus) in Italy

Plant Disease ◽

10.1094/pdis-93-8-0846b ◽

2009 ◽

Vol 93 (8) ◽

pp. 846-846 ◽

Cited By ~ 1

Author(s):

A. Carlucci ◽

F. Lops ◽

S. Frisullo

Keyword(s):

Verticillium Wilt ◽

Verticillium Dahliae ◽

Vascular Tissue ◽

Sequence Similarity ◽

Morphological Characteristics ◽

Herbaceous Plant ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Cosmos Bipinnatus

Cosmos (Cosmos bipinnatus Cav., Asteraceae) is an herbaceous plant that is grown for landscape use. During August and September of 2008 in five public and three private gardens located in Monopoli (Apulia, southern Italy), 3 to 8% of the plants showed severe symptoms of vine decline, stunting, gradual yellowing and wilting of the leaves, and final collapse of the whole plant. External symptoms were associated with brown or black streaking of the vascular tissue of roots, collar, and stem. Dead plants had numerous microsclerotia embedded in the xylem of plant tissues. Stem, collar, and root sections (0.5 cm long) from symptomatic plants collected in five gardens were surface disinfested in 5% NaOCl for 1 min and transferred to petri dishes containing potato dextrose agar (PDA) amended with 100 μg ml–1 of streptomycin sulfate and 10 μg ml–1 of neomycin. After 10 days of incubation, at 25°C in the dark, hyaline hyphae with dark microsclerotia (37 to 112 μm) and verticillate conidiophores were produced. Conidia were single celled and hyaline with dimensions of 3.3 to 7.8 × 1.8 to 3.3 μm (mean dimensions 4.2 × 2.5 μm). According to morphological characteristics, the fungus was identified as Verticillium dahliae Kleb. (1) (isolates no. Vd1818, Vd1819, and Vd1820 stored in a collection at the Department DiSACD, University of Foggia). Molecular analyses were performed on the basis of nucleotide sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2) of ribosomal DNA. ITS sequences of this fungus, compared with sequences found in GenBank and attributed to V. dahliae (no. GQ130129, GQ130130, GQ130131), showed 98 to 99% sequence similarity. Healthy 40-day-old plants of C. bipinnatus (garden cosmos) cv. Sonata Pink Blusk and C. sulphurous (yellow cosmos) cv. Bilbo, obtained from seeds previously disinfested for 1 min in 3% NaOCl and ascertained to be healthy by isolation on PDA medium, were used for pathogenicity tests. Plants were grown in 3-liter pots in a steam-disinfested peat, sand, and soil mixture (2:1:1) in the greenhouse at 23 to 26°C. Ten plants of each cultivar were inoculated by root dipping into a conidial suspension of each fungal isolate (1.5 × 106 CFU ml–1). Six noninoculated cosmos plants of each cultivar served as controls. The experiment was repeated three times. First symptoms of wilting were observed on all inoculated plants of each cultivar 20 days after the inoculation; at 40 days, symptom severity ratings on plants were taken, in which 1 = asymptomatic, 2 = stunted, 3 = wilting, and 4 = dead. All three isolates caused vascular discoloration, stunting, wilting, and plant death. The mean disease rating was 3.2 and did not differ significantly among isolates. The pathogen was consistently reisolated from infected plants, fulfilling Koch's postulates. Noninoculated plants remained healthy. To our knowledge, this is the first report of Verticillium wilt on cosmos in Italy. The finding is important since other ornamental plants that are susceptible to Verticillium wilt are also grown in landscapes in the region. The disease was previously reported in Turkey (2). References: (1) G. F. Pegg and B. L. Brandy. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002. (2) E. Sezgin et al. Turk. Phytopathol. 14:43, 1985.

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First Report of Botrytis Leaf Blight and Fruit Rot on Schisandra chinensis Caused by Botrytis cinerea in China

Plant Disease ◽

10.1094/pdis-02-11-0086 ◽

2011 ◽

Vol 95 (6) ◽

pp. 769-769

Author(s):

R. H. Yu ◽

J. Gao ◽

J. Wang ◽

X. Wang

Keyword(s):

Botrytis Cinerea ◽

Morphological Characteristics ◽

Gray Mold ◽

Leaf Blight ◽

Jilin Province ◽

Fruit Rot ◽

Conidial Suspension ◽

Schisandra Chinensis ◽

Mycelium Growth ◽

First Report

Schisandra (Schisandra chinensis (Turcz.) Baill) is a perennial plant belonging to Magnoliaceae. It is a very important medicinal herb in China and is mainly used for treatment of insomnia and memory decay. From July to September 2008, an unknown leaf blight and fruit rot on schisandra were first observed at Jingyu County, Jilin Province. The same symptoms were detected in other areas of Jilin Province, such as Ji'an City, Baishan City, and Hunchun City. Initially, some small, brown spots appeared on the tip or margin of the leaves. Light brown or brown necrotic lesions developed and eventually covered entire leaves. Seriously affected leaves were rolled or distorted and eventually became completely dry and brittle. Small spots appeared on the surface of mature fruits, coalesced, and the fruits finally dropped. Gray mycelia and conidiophores developed on the diseased leaves and fruits. To isolate the causal agent, conidia and conidiophores were scraped aseptically from the internal tissues, suspended in sterile water, and streaked onto the surface of potato dextrose agar (PDA). Single-hyphal tips were transferred on PDA and the isolated fungus was identified as Botrytis cinerea Pers.: Fr. on the basis of its morphological characteristics and internal transcribed spacer (ITS) sequence. Colonies of B. cinerea on PDA were colorless at first and became gray to brown 20 days later with the mycelium growth and conidia producing in cultures. Conidia are single celled, lemon shaped, colorless to a light color, and 4.4 to 15.0 × 7.0 to 10.0 μm. Sclerotia formed about 1 week later, were black-brown and varied in size (2.0 to 5.0 × 2.0 to 4.0 mm) and shape. The ITS region of rDNA was amplified from DNA extracted from single-spore isolate BC12 of B. cinerea using primers ITS4/ITS5 and sequenced (GenBank Accession No. GU724512), BLAST analysis (1) of the 535-bp segment showed 99% similarity with the sequence of Botryotinia fuckeliana (perfect stage of B. cinerea). Pathogenicity tests were carried out on healthy schisandra plants that were 4 years old. After the surface of the leaves and fruits was disinfected with 5% sodium hypochlorite, a conidial suspension of 105 conidia/ml was sprayed on 10 schisandra leaves, and plugs of the fungus obtained from the colony margins were transferred onto a 3- × 3-mm wound on the surface of disinfected fruit. Ten control schisandra leaves and 10 fruits were inoculated at the same time. Plants were covered with polyethylene bags and incubated at 25°C in a greenhouse with relative humidity of 85% for 3 days. Similar symptoms to those observed on diseased leaves and fruits in the field were observed on inoculated schisandra leaves and fruits 7 days after inoculation, whereas control leaves and fruits showed no symptoms. The pathogen was successfully reisolated. The gray mold disease caused by B. cinerea was reported in many plants, such as Lavandula stoechas and Chamelaucium uncinatum in Italy (2,3). However, to our knowledge, this is the first report of gray mold disease of schisandra caused by B. cinerea in China. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) A. Garibaldi et al. Plant Dis. 94:968, 2009. (3) A. Garibaldi et al. Plant Dis. 94:380, 2010.

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First Report of Colletotrichum gloeosporioides Causing Anthracnose of Banana (Musa spp.) in Malaysia

Plant Disease ◽

10.1094/pdis-10-12-0985-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 991-991 ◽

Cited By ~ 5

Author(s):

M. A. Intan Sakinah ◽

I. V. Suzianti ◽

Z. Latiffah

Keyword(s):

Morphological Characteristics ◽

Pathogenic Fungi ◽

Culture Collection ◽

Postharvest Disease ◽

Conidial Suspension ◽

First Report ◽

Musa Spp ◽

Initial Symptoms ◽

Dark Green ◽

Average Size

Banana is the second largest cultivated fruit crop in Malaysia, and is cultivated for both the domestic market and also for export. Anthranose is a well-known postharvest disease of banana and with high potential for damaging market value, as infection commonly occurs during storage. Anthracnose symptoms were observed on several varieties of banana such as mas, berangan, awak, nangka, and rastali in the states of Perak and Penang between August and October 2011. Approximately 80% of the fruits became infected with initial symptoms characterized as brown to black spots that later became sunken lesions with orange or salmon-colored conidial masses. Infected tissues (5 × 5 mm) were surface sterilized by dipping in 1% sodium hypochlorite (NaOCl) for 3 to 5 min, rinsed with sterile distilled water, and plated onto potato dextrose agar (PDA). Direct isolation was done by transferring the conidia from conidial masses using an inoculation loop and plating onto PDA. For both methods, the PDA plates were incubated at 27 ± 1°C with cycles of 12 h light and 12 h darkness. Visible growth of mycelium was observed after 4 to 5 days of incubation. Twenty isolates with conidial masses were recovered after 7 days of incubation. The isolates produced grayish white to grayish green and grey to moss dark green colony on PDA, pale orange conidial masses, and fusiform to cylindrical and hyaline conidia with an average size of 15 to 19 × 5 to 6 μm. Appresoria were ovate to obovate, dark brown, and 9 to 15 × 7 to 12 μm and setae were present, slightly swollen at the base, with a tapered apex, and brown. The cultural and morphological characteristics of the isolates were similar to those described for C. gleosporioides (1,2,3). All the C. gloeosporioides isolates were deposited in culture collection at Plant Pathology Lab, University Sains Malaysia. For confirmation of the identity of the isolates, ITS regions were sequenced using ITS4 and ITS5 primers. The isolates were deposited in GenBank with accessions JX163228, JX163231, JX163201, JX163230, JX163215, JX163223, JX163219, JX163202, JX163225, JX163222, JX163206, JX163218, JX163208, JX163209, JX163210, JX431560, JX163212, JX163213, JX431540, and JX431562. The resulting sequences showed 99% to 100% similarity with multiple C. gloeosporioides isolates in GenBank. Pathogenicity tests were conducted using mas, berangan, awak, nangka, and rastali bananas. Fruit surfaces were sterilized with 70% ethanol and wounded using a sterile scalpel. Two inoculation techniques were performed separately: mycelia plug and conidial suspension. Mycelial disc (5 mm) and a drop of 20 μl spore suspension (106 conidia/ml) were prepared from 7-day-old culture and placed on the fruit surface. The inoculated fruits were incubated at 27 ± 1°C for 10 days at 96.1% humidity. After 3 to 4 days of inoculation, brown to black spotted lesions were observed and coalesced to become black sunken lesions. Similar anthracnose symptoms were observed on all banana varieties tested. C. gloeosporioides was reisolated from the anthracnose lesions of all the inoculated fruit in which the cultural and morphological characteristics were the same as the original isolates. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose of Musa spp. in Malaysia. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) J. E. M. Mordue. Glomerella cingulata. CMI Description of Pathogenic Fungi and Bacteria, No. 315. CAB International,1971. (3) H. Prihastuti et al. Fungal Diversity 39:89, 2009.

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