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

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

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First Report of Pilidium concavum Causing Tan-Brown Leaf Spot on Strawberry in China

Plant Disease ◽

10.1094/pdis-03-12-0310-pdn ◽

2012 ◽

Vol 96 (9) ◽

pp. 1377-1377 ◽

Cited By ~ 4

Author(s):

W. L. Geng ◽

P. Hu ◽

Z. Ma ◽

X. Y. Zhao ◽

Y. M. Wei

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Leaf Tissue ◽

Brown Rot ◽

Pathogenicity Test ◽

Causal Organism ◽

First Report ◽

Molecular Features ◽

Brown Leaf Spot ◽

Brown Leaf

In April 2011, a survey of diseases was conducted on strawberry plants grown in greenhouses in the town of Xingshou, Changping District, Beijing, China. A tan-brown leaf spot with the presence of pink spore masses was observed on older leaves of strawberry plants. In general, the leaf spots began as small, round, water-soaked lesions in the middle or on the margin of leaves, which enlarged gradually up to 1 to 3 cm in diameter and were circular or irregular and brown to dark brown. Occasionally, the center of some spots cracked in the middle lesion under dry conditions. Eventually, black sporodochia were produced on the upper surface of spots and exuded pink conidial masses under humid conditions. Fungal structures were taken directly from the diseased leaves and examined microscopically for morphological characteristics. Sporodochia, 172 to 451 × 138 to 343 μm, were dark and suborbicular. Conidiophores, 8.4 to 48.3 × 0.8 to 2.1 μm, were hyaline, unicellular, and cylindrical. Conidia, 3.1 to 10.2 × 1.5 to 3.0 μm, were hyaline, aseptate, and canoe-shaped to allantoid, forming singly. For further study, the fungus was isolated in pure culture on potato dextrose agar (PDA) medium from symptomatic leaf tissue. In culture, the mycelium was white at first and then changed to brownish. The sporodochia were light colored at first and turned brownish or almost black in older cultures. To identify the fungus, the ITS1-5.8S-ITS2 rDNA region of the isolate was amplified by PCR with primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and sequenced. The 462-nt sequence (GenBank Accession No. JQ995228) was identical to that of Pilidium concavum (1). To validate Koch's postulates, pathogenicity was tested by inoculating 20 leaves on 10 healthy strawberry plants with a mycelial plug from a 15-day-old colony (0.5 cm in diameter). Controls were treated with plugs of PDA medium. The inoculated and control plants were then maintained in growth chambers at 25°C (12 h of light per day, 80% humidity). After 12 days, 100% of the inoculated leaves showed symptoms identical to those observed on leaves in the field while the control leaves remained healthy. The original fungus was reisolated from inoculated leaves showing the symptoms. Thus, it was concluded that the fungus was the causal organism of the leaf spot. On the basis of morphological characteristics, molecular features, and pathogenicity tests, the pathogen of tan-brown leaf spot on strawberry was identified as P. concavum. To our knowledge, this is the first report of P. concavum causing tan-brown leaf spot on strawberry in China. This fungus was reported to cause leaf spot on Paeonia suffruticosa in China (2). It also caused tan-brown rot on strawberry fruit in our pathogenicity test. References: (1) L. Cardin et al. Plant Dis. 93:548, 2009. (2) Y. B. Duan et al. Plant Dis. 94:271, 2010.

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

Plant Disease ◽

10.1094/pdis-08-21-1665-pdn ◽

2021 ◽

Author(s):

Jiahao Lai ◽

Tongke Liu ◽

Bing Liu ◽

Weigang Kuang ◽

Shuilin Song

Keyword(s):

Sweet Potato ◽

Molecular Identification ◽

Leaf Spot ◽

Ipomoea Batatas ◽

Morphological Characteristics ◽

Distilled Water ◽

Gene Sequences ◽

First Report ◽

Potted Plants ◽

Fungal Isolates

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

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Characterization of Pyrenophora Species Causing Brown Leaf Spot on Italian Ryegrass (Lolium multiflorum) in Southwestern China

Plant Disease ◽

10.1094/pdis-07-19-1457-re ◽

2020 ◽

Vol 104 (7) ◽

pp. 1900-1907

Author(s):

Longhai Xue ◽

Yong Liu ◽

Su Zhou ◽

James F. White ◽

Chunjie Li

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Lolium Multiflorum ◽

Morphological Characteristics ◽

Italian Ryegrass ◽

Large Area ◽

Internal Transcribed Spacer Region ◽

Brown Leaf Spot ◽

Brown Leaf ◽

Pathogenicity Tests

Drechslera leaf spot (DLS) caused by Pyrenophora (Drechslera) species is one of the most serious diseases affecting Italian ryegrass (Lolium multiflorum) in China. Between 2015 and 2018, this disease was observed in three Italian ryegrass fields in the province of Sichuan, China. Average leaf disease incidence was approximately 1 to 12% but could range up to 100%. Symptoms appeared as brown or tan spots surrounded by a yellow halo, or brown to dark brown net blotch; subsequently, spots increased in number and size, and they later covered a large area of leaf, eventually causing leaf death. In this study, 86 strains of Pyrenophora fungi were isolated from leaf lesions of Italian ryegrass. Coupled with phylogenetic analysis of the internal transcribed spacer region, partial 28S ribosomal RNA gene, and glyceraldehyde-3-phosphate dehydrogenase gene, morphological characteristics showed that Pyrenophora dictyoides and P. nobleae are associated with Italian ryegrass in southwest China. Pathogenicity tests confirmed that both species can infect Italian ryegrass, causing leaf spot, whereas the virulence of the two species differed; P. nobleae showed lower pathogenicity to Italian ryegrass. This is the first time that these two Pyrenophora species were formally reported on Italian ryegrass based on both morphological and molecular characters. Overall, this study improves knowledge of the Pyrenophora species associated with Italian ryegrass and provides a foundation for control of this disease in the future.

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Morphology and Molecular Characteristics of Alternaria sonchi Causing Brown Leaf Spot on Sonchus asper in Korea

Research in Plant Disease ◽

10.5423/rpd.2021.27.3.107 ◽

2021 ◽

Vol 27 (3) ◽

pp. 107-114

Author(s):

Huan Luo ◽

Myung Soo Park ◽

Jun Myoung Yu

Keyword(s):

Rna Polymerase Ii ◽

Leaf Spot ◽

Phylogenetic Analyses ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Internal Transcribed Spacer Region ◽

Sonchus Asper ◽

Brown Leaf Spot ◽

Brown Leaf

During a disease survey on weeds and minor cultivated crops in Korea, a brown leaf spot disease was observed on Sonchus asper. Leaf lesions were round or irregular in shape, and grayish brown to brown with a purple margin. In severe infection, lesions enlarged and coalesced, resulting in blighting of the leaves. The isolates from these leaf lesions were identified as Alternaira sonchi based on morphological characteristics and phylogenetic analyses of Internal transcribed spacer region, Alternaria allergen a1, glyceraldehyde 3-phosphate dehydrogenase, RNA polymerase II, and translation elongation factor genes. This study provides a comprehensive description of the morphological characteristics and phylogenetical traits of A. sonchi causing brown leaf spot on S. asper in Korea.

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First Report of Fusarium proliferatum Causing Fruit Rot of Grapes (Vitis vinifera) in Pakistan

International Journal of Phytopathology ◽

10.33687/phytopath.007.02.2589 ◽

2018 ◽

Vol 7 (2) ◽

pp. 85-88 ◽

Cited By ~ 1

Author(s):

Salman Ghuffar ◽

Gulshan Irshad ◽

Fengyan Zhai ◽

Asif Aziz ◽

Hafiz M. Asadullah M. Asadullah ◽

...

Keyword(s):

Vitis Vinifera ◽

Negative Control ◽

Fusarium Proliferatum ◽

Fruit Rot ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

Fruit Crop ◽

First Report ◽

Thin Walled

Grapes (Vitis vinifera) are the important fruit crop in Pakistan, mostly cultivated for edible purpose. In September 2016, unusual fruit rot symptoms were observed 3-5 days after harvesting on grapes cv. Kishmishi in post-harvest packing houses in Jehlum district (32°56'22.3"N 73°43'31.4"E) of Punjab province. To determine the disease incidence, a total of 10 boxes of grapes from 5 different locations were selected randomly. Each box contained average 12 bunches and 30 bunches out of 120 inspected bunches displayed typical symptoms of the disease. The initial Symptoms were small, round, water-soaked lesions that rapidly developed into soft, white to light pink mycelium near the centre of infected fruits (Figure 1). A total of 186 symptomatic berries were surface sterilized with 1% sodium hypochlorite, rinsed three times with sterile distilled water and dried by placing on filter paper for 45 sec. Sterilized tissues (approximately 4 mm3) were excised and incubated on potato dextrose agar (PDA) medium at 25 ± 4°C. One week after incubation, colonies with abundant aerial mycelium were initially white, cottony and turned to violet and dark purple with age (Figure 2). A total of 25 isolates were examined morphologically. Macroconidia were slender, thin-walled, 3 to 5 septate, curved apical cell, with 20.9 to 45.2 × 3.2 to 7.1 μm and Microconidia were thin-walled, aseptate, club-shaped with 4.5 to 11.2 × 2.3 to 4.1 μm (Figure 3). These characteristics best fit for the description of Fusarium proliferatum (Leslie and Summerell, 2006). Portions of the internal transcribed spacer (ITS) region were sequenced (White et al., 1990). Sequences of two isolates Fus 07 and Fus 09 (GenBank Accessions; MH444366 and MH464139) showed 100% identity to the corresponding gene sequences of Fusarium proliferatum (GenBank Accessions; MH368119, MF033172 and KU939071) (Figure 4). Pathogenicity test was performed by inoculation with 50-μl conidial suspension (1 × 106conidia/ml) of two isolates onto three non-wounded and four wounded asymptomatic grapes berries. Sterile distilled water was used for a negative control (Figure 5). The experiment was conducted twice and berries were incubated at 25 ± 2°C in sterile moisture chambers (Ghuffar et al., 2018). White to light pink mycelium in appearance with the original symptoms were observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were observed on the negative control. The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Fusarium proliferatum, one of the destructive species, causes diseases like foot-rot of corn (Farr et al., 1990), root rot of soybean (Díaz Arias et al., 2011), bakanae of rice (Zainudin et al., 2008), wilt of date palm (Khudhair et al., 2014), tomato wilt (Chehri, 2016) and tomato fruit rot (Murad et al., 2016). To our knowledge, this is the first report of Fusarium proliferatum causing fruit rot of grapes in Pakistan, where the disease poses a significant threat to the sustainability of this major fruit crop.

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

Plant Disease ◽

10.1094/pdis-10-12-0993-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 992-992 ◽

Cited By ~ 4

Author(s):

Y. L. Li ◽

Z. Zhou ◽

W. Lu ◽

J. R. Ye

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Leaf Tissue ◽

Aerial Mycelium ◽

Control Treatment ◽

Distilled Water ◽

First Report ◽

Potted Plants ◽

Fungal Isolates

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

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Leaf Spot and Stem Blight Caused by Botrytis cinerea on Poinsettia in Argentina

Plant Disease ◽

10.1094/pd-89-1359c ◽

2005 ◽

Vol 89 (12) ◽

pp. 1359-1359

Author(s):

H. E. Palmucci ◽

P. E. Grijalba

Keyword(s):

Botrytis Cinerea ◽

Leaf Spot ◽

Buenos Aires ◽

Pathogenic Fungi ◽

Pathogenicity Test ◽

Euphorbia Pulcherrima ◽

Conidial Suspension ◽

Stem Blight ◽

Potted Plants ◽

Leaf Spots

Poinsettia (Euphorbia pulcherrima Will. ex. Klotzsch) is a worldwide potted or landscape ornamental plant that belongs to the Euphorbiaceae family. During 2003 and 2004, several symptoms were observed on poinsettia potted plants in nurseries and crops near Buenos Aires. Symptoms included irregular, brown, water-soaked spots on adult plants and leaf spots that extended causing stem blight in seedlings. Small pieces of diseased tissues were surface disinfected for 2 min in 2% sodium hypochlorite, plated on 2% potato dextrose agar (PDA), and incubated at 22°C for 48 h. Dense, whitish mycelia developed on PDA and then turned gray when asexual structures were formed. The fungus conidia were ellipsoid, hyaline, nonseptate, and were formed on botryose heads. The pathogenicity test was carried out on 10 plants using a conidial suspension (2 × 106 spores per ml) that was sprayed on leaves with and without injuries. All plants were incubated in a moist chamber at 22 ± 2°C for 48 h and then maintained in a greenhouse. After 3 days, symptoms similar to the original were observed on the inoculated plants. Control plants sprayed with distilled water remained symptomless. Koch's postulates were confirmed by reisolating the same fungus from diseased plants. In accordance with conidial and cultural characteristics, the pathogen was identified as Botrytis cinerea Pers: Fr. (1). To our knowledge, this is the first report of B. cinerea causing leaf spot and stem rot on poinsettia in Buenos Aires, Argentina. Reference: (1) M. V. Ellis and J. M. Waller. No. 431 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1974.

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First report of Cercospora cf. citrulina causing leaf spot of Ipomoea pes-caprae in China

Plant Disease ◽

10.1094/pdis-05-20-1081-pdn ◽

2020 ◽

Author(s):

Min Li ◽

Meijiao Hu ◽

Zhaoyin Gao ◽

Xiaoyu Hong ◽

Chao Zhao ◽

...

Keyword(s):

Molecular Identification ◽

Leaf Spot ◽

Plant Protection ◽

Morphological Characteristics ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Xisha Islands ◽

First Report ◽

Initial Symptoms ◽

Leaf Surfaces

Ipomoea pes-caprae plays an important role in protecting the tropical and subtropical coastal beach of the world. In 2018, a leaf spot was observed on I. pes-caprae in Xisha islands of China, 13.2–25.8% of leaves were infected. The initial symptoms were small (1–3 mm diameter), single, circular, dark gray spots with a light-yellow center on the leaves. The lesions enlarged and were scattered or confluent, distinct and circular, subcircular or irregular, occasionally vein-limited, pale to dark gray-brown, with a narrow dark brown border surrounded by a diffuse yellow margin. Microscopic observations of the spots revealed that caespituli were dark brown and amphigenous, but abundant on the underside of the leaves. Mycelia were internal. Conidiophores were fasciculate, occasionally solitary, pale olivaceous-brown throughout, 0- to 3-septate, 27.9–115.8 (63.4±22.5) µm × 3.2–5.3 (4.3±0.87) µm (n=100). Conidial scars were conspicuously thickened. Conidia were solitary, hyaline, filiform, acicular to obclavate, straight to slightly curved, subacute to obtuse at the apex, truncate at the base, multi-septate, 21.0–125.5 (60.2±20.1) µm × 2.0–5.0 (3.8±0.83) µm (n=100). Single-conidium isolates were obtained from representative colonies grown on potato dextrose agar (PDA) incubated at 25℃ in the dark. The colonies grew slowly and were dense, white to gray and flat with aerial mycelium. Mycelia were initially white, and then became gray. Conidia were borne on the conidiophores directly. The pure isolate HTW-1 was selected for molecular identification and pathogenicity test, which were deposited in Microbiological Culture Collection Center of Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (tef1) and histone H3 (his3) genes were amplified with ITS1/ITS4, EF-1 / EF-2, and CYLH3F / CYLH3R primers, respectively (Groenewald et al. 2013). The obtained sequences of HTW-1 were all deposited in GenBank with accession numbers MT410467 for ITS, MT418903 for tef1 and MT418904 for his3. The ITS, tef1 and his3 genes all showed 100% similarity for ITS (JX143582), tef1 (JX143340) and his3 (JX142602) with C. cf. citrulina (MUCC 588; MAFF 239409) from I. pes-caprae in Japan. Based on the morphological characteristics and molecular identification, the pathogen was identified as Cercospora cf. citrulina (Groenewald et al. 2013). The pathogenicity test was conducted by spraying conidial suspension (1×104 conidia/mL) on wounded and unwounded leaves for seedling of I. pes-caprae in greenhouse and in sterile vitro condition. The conidial suspension was prepared using conidia from 30-day-old culture grown on PDA at 25℃ in the dark. Leaf surfaces of seedling in greenhouse were wounded by lightly rubbing with a steel sponge and detached leaf surfaces were wounded by sterile needles. the treatments were sprayed with conidial suspensions on wounded and unwounded leaf surfaces. The control was sprayed with sterile water. After eight days, the typical symptoms of spots which were small, single, circular and dark gray appeared on the inoculated wounded leaves, while the inoculated unwounded leaves and the control leaves were symptomless. The pathogen was only re-isolated from the inoculated wounded leaves. The pathogen may be infected by wound. A total of 20 Cercospora and related species was found on Ipomoea spp. (García et al. 1996). Cercospora cf. citrulina has been reported on I. pes-caprae in Japan, although it was unclear if it was a pathogen or saprophyte (Groenewald et al. 2013). To our knowledge, this is the first report of C. cf. citrulina causing leaf spot of I. pes-caprae in China. This disease could threat the cultivation of I. pes-caprae in China.

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