First Report of Pantoea ananatis causing leaf spot disease of maize in Ecuador

Maize (Zea mays) is the second most cultivated grain crop in Ecuador, with growing significance as a source of fodder and food. During the rainy season (November and December) of 2018 and 2019, a disease of maize that was not previously observed in Ecuador was found at commercial fields of Misqui Sara variety, at four parishes of canton Quito (Tumbaco, Pifo, Puembo, and Checa), province of Pichincha. Infected plants, at tassel initiation, displayed symptoms of localized chlorotic streaks on leaves that expanded with time, and around a month later turned necrotic. Severely affected plants wilted and died. Symptoms appeared in lower leaves first and were later observed in upper leaves as the disease progressed. Disease incidence was between 20 and 30% in the affected plantations, with around 30% of infected plants wilting and dying, resulting in 20-25% of yield losses. Upper leaves from ten symptomatic plants, five from Puembo and five from Checa, were collected randomly. Two 0.5 cm2 pieces of leaf from each plant were excised from the margins of the necrotic lesions, surface sterilized and macerated in 9 mL of sterile peptone water. The 10-3 dilutions were plated onto nutrient agar and incubated at 28°C for 24 hours. Yellow, mucoid colonies were isolated on nutrient agar. Three isolates from Puembo and two from Checa were selected for testing Koch´s postulates and further biochemical and molecular characterization. Isolates were Gram-negative rods, oxidase negative, catalase, indol and citrate positive. Fragments of the 16S, gyrB, and rpoB loci were amplified and sequenced using the 27F/1492R (Lane, D. J., 1991), UP-1/UP-2r (Yamamoto & Harayama, 1995), and rpoBCM81-F/rpoBCM32b-R (Brady, C., et al., 2008) primer pairs, respectively. All isolates presented identical sequences for the different loci, therefore only sequences from isolate FP191505 were deposited in GenBank (GenBank accession no. MW528428-MW528430). A search of homologous sequences using BLAST resulted in identities of 99.3, 99.7, and 100 % for 16S, gyrB, and rpoB, respectively, with sequences from Pantoea ananatis type specimen LGM 2665 (Brady, C., et al., 2008; Hauben, L., et al., 1998; GenBank accession nos NR_119362.1, EF988824.1 EF988996.1), indicating that our isolates belong to this species. Pathogenicity tests were performed by syringe infiltration of bacterial suspensions. Each one of the five characterized P. ananatis isolates was inoculated in four leaves (500 ul of 1 x 108 CFU mL-1 per leave) of three healthy maize plants. Negative control plants were infiltrated with sterile distilled water. Plants were incubated at 28-30°C and 60% relative humidity for 24 hours. Later, plants were maintained in a greenhouse with 27°C/21°C day/night temperatures and observed daily. After six weeks all bacteria-inoculated plants developed symptoms of chlorosis and necrosis while the control was symptomless. Bacteria were re-isolated from symptomatic leaves and identified as P. ananatis following the same methodologies used for the initial identification. To our knowledge, this is the first report of P. ananatis causing leaf spot of maize in Ecuador.

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First Report of Bacterial Leaf Spot Caused by Xanthomonas campestris pv. olitorii on Jute Grown for Seed in India

Plant Disease ◽

10.1094/pdis-12-12-1196-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1109-1109

Author(s):

C. Biswas ◽

P. Dey ◽

A. Bera ◽

S. Satpathy ◽

B. S. Mahapatra

Keyword(s):

Bacterial Strain ◽

Leaf Spot ◽

Xanthomonas Campestris ◽

Disease Incidence ◽

Negative Control ◽

Nutrient Agar ◽

Distilled Water ◽

Bacterial Leaf Spot ◽

First Report ◽

Pure Cultures

Jute (Corchorus olitorius L.) is the second most important fiber crop after cotton in terms of global production (3). In November 2011, symptoms suggestive of bacterial infection were observed on a seed crop of jute at the CRIJAF research farm, Barrackpore, West Bengal, India. The disease appeared as small, brown, circular spots, usually less than 5 mm in diameter on the leaves and some of the spots were surrounded by a yellow halo. The lesions on the stems were elongated and in some cases were found to girdle the stem. In the later stages of disease, brown sunken spots were found on the green capsules. Disease incidence varied from about 20% to 90% of the total plants in different affected fields at the CRIJAF research farm. Bacterial leaf spot of jute with similar symptoms was reported in 1957 from Sudan (4). Five symptomatic and three asymptomatic leaf samples were collected from different jute fields. Bacterial colonies isolated on nutrient agar medium from infected young leaves were Xanthomonas-like and pale yellow cream in color. Total DNA was extracted from symptomatic as well as asymptomatic leaf samples by using an improved salt concentration and simple sodium acetate CTAB method (2). Single bacterial colonies were transferred to nutrient agar (NA) medium plates and incubated at 28°C for 48 h. Pure colonies from plates were used directly for DNA extraction using the QIAGEN DNeasy Blood and Tissue kit. PCR was carried out with Xanthomonas campestris specific primers NZ8F3/NZ85R3 (1), which generated an amplicon of 530 bp from all the symptomatic leaf samples as well as pure cultures of the isolated bacteria. No amplification was obtained from asymptomatic leaves. The amplicons from the five symptomatic samples collected from the field were sequenced and showed 100% identity with one another, and one sequence (strain JB-CO-13) was deposited in GenBank (Accession No. KC342185). The BLASTn analysis revealed that bacterial strain JB-CO-13 had 100% identity with X. campestris pv. olitorii (EU285213). Nucleotide span and ORF finder (NCBI) analysis indicated the 530-bp PCR amplicon coded part of a gyrase B gene that had 100% identity with a translated gene product (Protein ID: ABX84334). Three leaves of five 1-month-old jute plants (cv. JRO 204) in pot culture were infiltrated each with a separate bacterial strain using suspensions (1 × 105 CFU/ml) in distilled water. The negative control consisted of leaves infiltrated with sterile distilled water. The plants were kept in a greenhouse with mean maximum and minimum temperatures of 28.96 and 21.8°C, respectively. The plants were covered with plastic bags to maintain high relative humidity (>80%). Typical bacterial lesions were recorded on all the inoculated plants after 1 week. No lesions were seen on the negative control. To the best of our knowledge, this is the first report of bacterial leaf spot on C. olitorius caused by X. campestris pv. olitorii from India. References: (1) J. Adriko et al. Plant Pathol. 61:489, 2012. (2) C. Biswas, et al. Lett. Appl. Microbiol. 56:105, 2013. (3) Food and Agriculture Organization of the United Nations. Agricultural Commodities: Profiles and Relevant WTO Negotiating Issues. Online: http://www.fao.org/docrep/006/Y4343E/y4343e03.htm , 2003. (4) K. A. Sabet. Ann. Appl. Biol. 45:516, 1957.

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First report of banana (Musa acuminate L. AAA Cavendish, cv. Formosana) leaf spot disease caused by Curvularia geniculata in China

Plant Disease ◽

10.1094/pdis-09-21-2001-pdn ◽

2021 ◽

Author(s):

Yanxiang Qi ◽

Yanping Fu ◽

Jun Peng ◽

Fanyun Zeng ◽

Yanwei Wang ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Universal Primers ◽

Leaf Spot Disease ◽

Leaf Margin ◽

Conidial Suspension ◽

Tropical Fruit ◽

First Report ◽

Spot Disease

Banana (Musa acuminate L.) is an important tropical fruit in China. During 2019-2020, a new leaf spot disease was observed on banana (M. acuminate L. AAA Cavendish, cv. Formosana) at two orchards of Chengmai county (19°48ʹ41.79″ N, 109°58ʹ44.95″ E), Hainan province, China. In total, the disease incidence was about 5% of banana trees (6 000 trees). The leaf spots occurred sporadically and were mostly confined to the leaf margin, and the percentage of the leaf area covered by lesions was less than 1%. Symptoms on the leaves were initially reddish brown spots that gradually expanded to ovoid-shaped lesions and eventually become necrotic, dry, and gray with a yellow halo. The conidia obtained from leaf lesions were brown, erect or curved, fusiform or elliptical, 3 to 4 septa with dimensions of 13.75 to 31.39 µm × 5.91 to 13.35 µm (avg. 22.39 × 8.83 µm). The cells of both ends were small and hyaline while the middle cells were larger and darker (Zhang et al. 2010). Morphological characteristics of the conidia matched the description of Curvularia geniculata (Tracy & Earle) Boedijn. To acquire the pathogen, tissue pieces (15 mm2) of symptomatic leaves were surface disinfected in 70% ethanol (10 s) and 0.8% NaClO (2 min), rinsed in sterile water three times, and transferred to potato dextrose agar (PDA) for three days at 28°C. Grayish green fungal colonies appeared, and then turned fluffy with grey and white aerial mycelium with age. Two representative isolates (CATAS-CG01 and CATAS-CG92) of single-spore cultures were selected for molecular identification. Genomic DNA was extracted from the two isolates, the internal transcribed spacer (ITS), large subunit ribosomal DNA (LSU rDNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1-α) and RNA polymerase II second largest subunit (RPB2) were amplified and sequenced with universal primers ITS1/ITS4, LROR/LR5, GPD1/GPD2, EF1-983F/EF1-2218R and 5F2/7cR, respectively (Huang et al. 2017; Raza et al. 2019). The sequences were deposited in GenBank (MW186196, MW186197, OK091651, OK721009 and OK491081 for CATAS-CG01; MZ734453, MZ734465, OK091652, OK721100 and OK642748 for CATAS-CG92, respectively). For phylogenetic analysis, MEGA7.0 (Kumar et al. 2016) was used to construct a Maximum Likelihood (ML) tree with 1 000 bootstrap replicates, based on a concatenation alignment of five gene sequences of the two isolates in this study as well as sequences of other Curvularia species obtained from GenBank. The cluster analysis revealed that isolates CATAS-CG01 and CATAS-CG92 were C. geniculata. Pathogenicity assays were conducted on 7-leaf-old banana seedlings. Two leaves from potted plants were stab inoculated by puncturing into 1-mm using a sterilized needle and placing 10 μl conidial suspension (2×106 conidia/ml) on the surface of wounded leaves and equal number of leaves were inoculated with sterile distilled water serving as control (three replicates). Inoculated plants were grown in the greenhouse (12 h/12 h light/dark, 28°C, 90% relative humidity). Necrotic lesions on inoculated leaves appeared seven days after inoculation, whereas control leaves remained healthy. The fungus was recovered from inoculated leaves, and its taxonomy was confirmed morphologically and molecularly, fulfilling Koch’s postulates. C. geniculata has been reported to cause leaf spot on banana in Jamaica (Meredith, 1963). To our knowledge, this is the first report of C. geniculata on banana in China.

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First Report of a Leaf Spot on Snow Lotus Caused by Alternaria carthami in China

Plant Disease ◽

10.1094/pdis-92-2-0318b ◽

2008 ◽

Vol 92 (2) ◽

pp. 318-318

Author(s):

S. Zhao ◽

G. Xie ◽

H. Zhao ◽

H. Li ◽

C. Li

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Leaf Spot Disease ◽

Tianshan Mountain ◽

Causal Organism ◽

Saussurea Involucrata ◽

First Report ◽

Spot Disease ◽

Initial Symptoms ◽

Snow Lotus

Snow lotus (Saussurea involucrata Karel. & Kir. ex Sch. Bip.) is an economically important medicinal herb increasingly grown in China in recent years. In June of 2005, a leaf spot disease on commercially grown plants was found in the QiTai Region, south of the Tianshan Mountain area of Xinjiang, China at 2,100 m above sea level. Disease incidence was approximately 60 to 70% of the plants during the 2006 and 2007 growing seasons. Initial symptoms appeared on older leaves as irregularly shaped, minute, dark brown-to-black spots, with yellow borders on the edge of the leaflet blade by July. As the disease progressed, the lesions expanded, causing the leaflets to turn brown, shrivel, and die. A fungus was consistently isolated from the margins of these lesions on potato dextrose agar. Fifty-eight isolates were obtained that produced abundant conidia in the dark. Conidia were usually solitary, rarely in chains of two, ellipsoid to obclavate, with 6 to 11 transverse and one longitudinal or oblique septum. Conidia measured 60 to 80 × 20 to 30 μm, including a filamentous beak (13 to 47 × 3.5 to 6 μm). According to the morphology, and when compared with the standard reference strains, the causal organism of leaf spot of snow lotus was identified as Alternaria carthami (1,4). Pathogenicity of the strains was tested on snow lotus seedlings at the six-leaf stage. The lower leaves of 20 plants were sprayed until runoff with conidial suspensions of 1 × 104 spores mL–1, and five plants sprayed with sterile distilled water served as controls. All plants were covered with a polyethylene bag, incubated at 25°C for 2 days, and subsequently transferred to a growth chamber at 25°C with a 16-h photoperiod. Light brown lesions developed within 10 days on leaflet margins in all inoculated plants. The pathogen was reisolated from inoculated leaves, and isolates were deposited at the Key Oasis Eco-agriculture Laboratory of Xinjiang Production and Construction Group, Xinjiang and the Institute of Biotechnology, Zhejiang University. No reports of a spot disease caused by A. carthami on snow lotus leaves have been found, although this pathogen has been reported on safflower in western Canada (3), Australia (2), India (1), and China (4). To our knowledge, this is the first report of a leaf spot caused by A. carthami on snow lotus in China. References: (1) S. Chowdhury. J. Indian Bot. Soc. 23:59, 1944. (2) J. A. G. Irwin. Aust. J. Exp. Agric. Anim. Husb. 16:921, 1976. (3) G. A. Petrie. Can. Plant Dis. Surv. 54:155, 1974. (4) T. Y. Zhang. J. Yunnan Agric. Univ.17:320, 2002.

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

Plant Disease ◽

10.1094/pdis.2003.87.2.203a ◽

2003 ◽

Vol 87 (2) ◽

pp. 203-203

Author(s):

S. T. Koike ◽

S. A. Tjosvold ◽

J. Z. Groenewald ◽

P. W. Crous

Keyword(s):

Leaf Spot ◽

Sequence Data ◽

Disease Incidence ◽

Leaf Spot Disease ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Spot Disease ◽

Leaf Spots ◽

Initial Symptoms

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

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First Report of Leaf Spot Disease Caused by Colletotrichum gloeosporioides on Chinese Bean Tree in China

Plant Disease ◽

10.1094/pdis-03-12-0261-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 138-138 ◽

Cited By ~ 1

Author(s):

B. Z. Fu ◽

M. Yang ◽

G. Y. Li ◽

J. R. Wu ◽

J. Z. Zhang ◽

...

Keyword(s):

Leaf Spot ◽

Colletotrichum Gloeosporioides ◽

Disease Incidence ◽

Phylogenetic Analyses ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

First Report ◽

Spot Disease ◽

Rdna Its ◽

Its Sequence Analysis

Chinese bean tree, Catalpa fargesii f. duciouxii (Dode) Gilmour, is an ornamental arbor plant. Its roots, leaves, and flowers have long been used for medicinal purposes in China. During July 2010, severe outbreaks of leaf spot disease on this plant occurred in Kunming, Yunnan Province. The disease incidence was greater than 90%. The symptoms on leaves began as dark brown lesions surrounded by chlorotic halos, and later became larger, round or irregular spots with gray to off-white centers surrounded by dark brown margins. Leaf tissues (3 × 3 mm), cut from the margins of lesions, were surface disinfected in 0.1% HgCl2 solution for 3 min, rinsed three times in sterile water, plated on potato dextrose agar (PDA), and incubated at 28°C. The same fungus was consistently isolated from the diseased leaves. Colonies of white-to-dark gray mycelia formed on PDA, and were slightly brown on the underside of the colony. The hyphae were achromatic, branching, septate, and 4.59 (±1.38) μm in diameter on average. Perithecia were brown to black, globose in shape, and 275.9 to 379.3 × 245.3 to 344.8 μm. Asci that formed after 3 to 4 weeks in culture were eight-spored, clavate to cylindrical. The ascospores were fusiform, slightly curved, unicellular and hyaline, and 13.05 to 24.03 × 10.68 to 16.02 μm. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5 (2). Sequencing of the PCR products of DQ1 (GenBank Accession No. JN165746) revealed 99% similarity (100% coverage) with Colletotrichum gloeosporioides isolates (GenBank Accession No. FJ456938.1, No. EU326190.1, No. DQ682572.1, and No. AY423474.1). Phylogenetic analyses (MEGA 4.1) using the neighbor-joining (NJ) algorithm placed the isolate in a well-supported cluster (>90% bootstrap value based on 1,000 replicates) with other C. gloeosporioides isolates. The pathogen was identified as C. gloeosporioides (Penz.) Penz. & Sacc. (teleomorph Glomerella cingulata (Stoneman) Spauld & H. Schrenk) based on the morphological characteristics and rDNA-ITS sequence analysis (1). To confirm pathogenicity, Koch's postulates were performed on detached leaves of C. fargesii f. duciouxii, inoculated with a solution of 1.0 × 106 conidia per ml. Symptoms similar to the original ones started to appear after 10 days, while untreated leaves remained healthy. The inoculation assay used three leaves for untreated and six leaves for treated. The experiments were repeated once. C. gloeosporioides was consistently reisolated from the diseased tissue. C. gloeosporioides is distributed worldwide causing anthracnose on a wide variety of plants (3). To the best of our knowledge, this is the first report of C. gloeosporioides causing leaf spots on C. fargesii f. duciouxii in China. References: (1) B. C. Sutton. Page 1 in: Colletotrichum: Biology, Pathology and Control. CAB International. Wallingford, UK, 1992. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (3) J. Yan et al. Plant Dis. 95:880, 2011.

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First Report of Leaf Spot Disease Caused by Epicoccum nigrum on Lablab purpureus in India

Plant Disease ◽

10.1094/pdis-07-13-0798-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 284-284 ◽

Cited By ~ 12

Author(s):

S. Mahadevakumar ◽

K. M. Jayaramaiah ◽

G. R. Janardhana

Keyword(s):

Leaf Spot ◽

Leaf Surface ◽

Disease Incidence ◽

Leaf Spot Disease ◽

Post Inoculation ◽

Conidial Suspension ◽

Lablab Purpureus ◽

First Report ◽

Spot Disease ◽

Epicoccum Nigrum

Lablab purpureus (L.) Sweet (Indian bean) is an important pulse crop grown in arid and semi-arid regions of India. It is one of the most widely cultivated legume species and has multiple uses. During a September 2010 survey, we recorded a new leaf spot disease on L. purpureus in and around Mysore district (Karnataka state) with 40 to 80% disease incidence in 130 ha of field crop studied, which accounted for 20 to 35% estimated yield loss. The symptoms appeared as small necrotic spots on the upper leaf surface. The leaf spots were persistent under mild infection throughout the season with production of conidia in clusters on abaxial leaf surface. A Dueteromyceteous fungus was isolated from affected leaf tissues that were surface sterilized with 2% NaOCl2 solution then washed thrice, dried, inoculated on potato dextrose agar (PDA) medium, and incubated at 28 ± 2°C at 12 h alternate light and dark period for 7 days. The fungal colony with aerial mycelia interspersed with dark cushion-shaped sporodochia consists of short, compact conidiophores bearing large isodiametric, solitary, muricate, brown, globular to pear shaped conidia (29.43 to 23.92 μm). Fungal isolate was identified as Epicoccum sp. based on micro-morphological and cultural features (1). Further authenticity of the fungus was confirmed by PCR amplification of the internal transcribed spacer (ITS) region using ITS1/ITS4 universal primer. The amplified PCR product was purified, sequenced directly, and BLASTn search revealed 100% homology to Epicoccum nigrum Link. (DQ093668.1 and JX914480.1). A representative sequence of E. nigrum was deposited in GenBank (Accession No. KC568289.1). The isolated fungus was further tested for its pathogenicity on 30-day-old healthy L. purpureus plants under greenhouse conditions. A conidial suspension (106 conidia/ml) was applied as foliar spray (three replicates of 15 plants each) along with suitable controls. The plants were kept under high humidity (80%) for 5 days and at ambient temperature (28 ± 2°C). The appearance of leaf spot symptoms were observed after 25 days post inoculation. Further, the pathogen was re-isolated and confirmed by micro-morphological characteristics. E. nigrum has been reported to cause post-harvest decay of cantaloupe in Oklahoma (2). It has also been reported as an endophyte (3). Occurrence as a pathogen on lablab bean has not been previously reported. To our knowledge, this is the first report of the occurrence of E. nigrum on L. purpureus in India causing leaf spot disease. References: (1) H. L. Barnet and B. B. Hunter. Page 150 in: Illustrated Genera of Imperfect Fungi, 1972. (2) B. D. Bruten et al. Plant Dis. 77:1060, 1993. (3) L. C. Fávaro et al. PLoS One 7(6):e36826, 2012.

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First Report of Tomato Gray Leaf Spot Disease Caused by Stemphylium solani in Malaysia

Plant Disease ◽

10.1094/pdis-03-12-0223-pdn ◽

2012 ◽

Vol 96 (8) ◽

pp. 1226-1226

Author(s):

A. Nasehi ◽

J. B. Kadir ◽

M. A. Zainal Abidin ◽

M. Y. Wong ◽

F. Mahmodi

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Gray Leaf Spot ◽

Leaf Spot Disease ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Spot Disease ◽

Pcr Product ◽

Morphological Criteria

In June 2011, tomatoes (Solanum lycopersicum) in major growing areas of the Cameron Highlands and the Johor state in Malaysia were affected by a leaf spot disease. Disease incidence exceeded 80% in some severely infected regions. Symptoms on 50 observed plants initially appeared on leaves as small, brownish black specks, which later became grayish brown, angular lesions surrounded by a yellow border. As the lesions matured, the affected leaves dried up and became brittle and later developed cracks in the center of the lesions. A survey was performed in these growing areas and 27 isolates of the pathogen were isolated from the tomato leaves on potato carrot agar (PCA). The isolates were purified by the single spore technique and were transferred onto PCA and V8 agar media for conidiophore and conidia production under alternating light (8 hours per day) and darkness (16 hours per day) (4). Colonies on PCA and V8 agar exhibited grey mycelium and numerous conidia were formed at the terminal end of conidiophores. The conidiophores were up to 240 μm long. Conidia were oblong with 2 to 11 transverse and 1 to 6 longitudinal septa and were 24 to 69.6 μm long × 9.6 to 14.4 μm wide. The pathogen was identified as Stemphylium solani on the basis of morphological criteria (2). In addition, DNA was extracted and the internal transcribed spacer region (ITS) was amplified by universal primers ITS5 and ITS4 (1). The PCR product was purified by the commercial PCR purification kit and the purified PCR product sequenced. The resulting sequences were 100% identical to published S. solani sequences (GenBank Accestion Nos. AF203451 and HQ840713). The amplified ITS region was deposited with NCBI GenBank under Accession No. JQ657726. A representative isolate of the pathogen was inoculated on detached 45-day-old tomato leaves of Malaysian cultivar 152177-A for pathogenicity testing. One wounded and two nonwounded leaflets per leaf were used in this experiment. The leaves were wounded by applying pressure to leaf blades with the serrated edge of a forceps. A 20-μl drop of conidial suspension containing 105 conidia/ml was used to inoculate these leaves (3). The inoculated leaves were placed on moist filter paper in petri dishes and incubated for 48 h at 25°C. Control leaves were inoculated with sterilized distilled water. After 7 days, typical symptoms for S. solani similar to those observed in the farmers' fields developed on both wounded and nonwounded inoculated leaves, but not on noninoculated controls, and S. solani was consistently reisolated. To our knowledge, this is the first report of S. solani causing gray leaf spot of tomato in Malaysia. References: (1) M. P. S. Camara et al. Mycologia 94:660, 2002. (2) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (3) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (4) E. G. Simmons. CBS Biodiversity Series 6:775, 2007.

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First Report of White Leaf Spot of Dry Bean Caused by Pseudocercosporella albida in North America

Plant Disease ◽

10.1094/pdis.2003.87.12.1537c ◽

2003 ◽

Vol 87 (12) ◽

pp. 1537-1537

Author(s):

L. E. del Río ◽

C. A. Bradley ◽

R. S. Lamppa

Keyword(s):

North America ◽

Leaf Spot ◽

Disease Incidence ◽

Infected Plant ◽

Leaf Spot Disease ◽

Dry Bean ◽

First Report ◽

White Leaf ◽

Electron Microscopes ◽

The Impact

In August 2002, white leaf spot disease was observed on red kidney bean (Phaseolus vulgaris L.) at three fields near Staples, MN. Disease incidence in these fields, calculated as the proportion of plants showing visible symptoms, ranged from 20 to 100%. Symptoms and signs consisted of abundant, white sporulation on the abaxial side of leaves that was limited by secondary veins, resulting in angular-shaped lesions. Corresponding with fungal growth, yellow, angular spots were observed on the upper leaf surface. In older leaves, lesions coalesced, covering most of their surface, while younger leaves were relatively free of symptoms. Heavily diseased plants senesced faster and defoliated earlier than nonsymptomatic plants. Identification of the pathogen was conducted by direct observation of infected plant tissues with light and electron microscopes. Conidia were hyaline, filiform, rounded at their apex, and with no visible scar at their point of attachment to the conidiophore. Conidia had one to four septa, were 57 to 68 μm long, and approximately 2.4 μm in diameter. Conidia were produced at the tip of colorless, short conidiophores that emerged through stomata in groups of five or more. These traits are in agreement with the description of Pseudocercosporella albida (Matta & Belliard) Deighton (1,2). A sample of infected tissues has been deposited at the U.S. National Fungus Collection (BPI 842303) at Beltsville, MD. The impact of this disease on bean yields in Minnesota was not estimated. However, in the cool highlands of Colombia, where white leaf spot is endemic, yields can be reduced by as much as 47% (3). To our knowledge, this is the first report of the presence of this pathogen on dry bean in North America. References: (1) F. C. Deighton. Trans. Br. Mycol. Soc. 66:547, 1976. (2) A. Matta and L. Belliard. Phytopathol. Mediterr. 12:102, 1973. (3) H. F. Schwartz et al. Plant Dis. 65:494, 1981.

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First report of leaf spot disease caused by Stemphylium eturmiunum on American sweetgum (Liquidambar styraciflua L.) in China

Plant Disease ◽

10.1094/pdis-09-20-1877-pdn ◽

2021 ◽

Author(s):

Yun-fei Mao ◽

Li Jin ◽

Huiyue Chen ◽

Xiang-rong Zheng ◽

Minjia Wang ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Liquidambar Styraciflua ◽

Leaf Spot Disease ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Wood Processing ◽

Light Yellow

American sweetgum (Liquidambar styraciflua L.) is an important tree for landscaping and wood processing. In recent years, leaf spots on American sweetgum with disease incidence of about 53% were observed in about 1200 full grown plants in a field (about 8 ha) located in Pizhou, Jiangsu Province, China. Initially, dense reddish-brown spots appeared on both old and new leaves. Later, the spots expanded into dark brown lesions with yellow halos. Symptomatic leaf samples from different trees were collected and processed in the laboratory. For pathogen isolation, leaf sections (4×4mm) removed from the lesion margin were surface sterilized with 75% ethanol for 20s and then sterilized in 2% NaOCl for 30s, rinsed three times in sterile distilled water, incubated on potato dextrose agar (PDA) at 25 °C in the darkness. After 5 days of cultivation, the pure culture was obtained by single spore separation. 6 isolate samples from different leaves named FXA1 to FXA6 shared nearly identical morphological features. The isolate FXA1 (codes CFCC 54675) was deposited in the China Center for Type Culture Collection. On the PDA, the colonies were light yellow with dense mycelium, rough margin, and reverse brownish yellow. Conidiophores (23–35 × 6–10 µm) (n=60) were solitary, straight to flexuous. Conidia (19–34 × 10–21 µm) (n=60) were single, muriform, oblong, mid to deep brown, with 1 to 6 transverse septa. These morphological characteristics resemble Stemphylium eturmiunum (Simmons 2001). Genomic DNA was extracted from mycelium following the CTAB method. The ITS region, gapdh, and cmdA genes were amplified and sequenced with the primers ITS5/ITS4 (Woudenberg et al. 2017), gpd1/gpd2 (Berbee et al. 1999), and CALDF1/CALDR2 (Lawrence et al. 2013), respectively. A maximum likelihood phylogenetic analysis based on ITS, gapdh and cmdA (accession nos. MT898502-MT898507, MT902342-MT902347, MT902336-MT902341) sequences using MEGA 7.0 revealed that the isolates were placed in the same clade as S. eturmiunum with 98% bootstrap support. All seedlings for pathogenicity tests were enclosed in plastic transparent incubators to maintain high relative humidity (90%-100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. For pathogenicity, the conidial suspension (105 spores/ml) of each isolate was sprayed respectively onto healthy leaves of L. styraciflua potted seedlings (2-year-old, 3 replicate plants per isolate). As a control, 3 seedlings were sprayed with sterile distilled water. After 7 days, dense reddish-brown spots were observed on all inoculated leaves. In another set of tests, healthy plants (3 leaves per plant, 3 replicate plants per isolate) were wound-inoculated with mycelial plugs (4×4mm) and inoculated with sterile PDA plugs as a control. After 7 days, brown lesions with light yellow halo were observed on all inoculation sites with the mycelial plugs. Controls remained asymptomatic in the entire experiment. The pathogen was reisolated from symptomatic tissues and identified as S. eturmiunum but was not recovered from the control. The experiment was repeated twice with the similar results, fulfilling Koch’s postulates. S. eturmiunum had been reported on tomato (Andersen et al. 2004), wheat (Poursafar et al. 2016), garlic (L. Fu et al. 2019) but not on woody plant leaves. To our knowledge, this is the first report of S. eturmiunum causing leaf spot on L. styraciflua in the world. This disease poses a potential threat to American sweetgum and wheat in Pizhou.

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First report of leaf spot caused by Colletotrichum fructicola on Dalbergia hupeana in China

Plant Disease ◽

10.1094/pdis-08-21-1842-pdn ◽

2021 ◽

Author(s):

Yang Zhou ◽

Rou Ye ◽

Qin Ying ◽

Yang Zhang ◽

Linping Zhang

Keyword(s):

Phylogenetic Tree ◽

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Jiangxi Province ◽

Leaf Spot Disease ◽

Sterile Water ◽

First Report ◽

Spot Disease ◽

Colletotrichum Fructicola

Dalbergia hupeana is a kind of wood and medicinal tree widely distributed in southern China. Since 2019, a leaf spot disease was observed on the leaves of D. hupeana in Gangxia village, Luoting town in Jiangxi Province, China (28°52′53″N, 115°44′58″E). The disease incidence was estimated to be above 50%. The symptoms began as small spots that gradually expanded, developing a brown central and dark brown to black margin. The spots ranged from 4 to 6 mm in diameter. Leaf pieces (5 × 5 mm) from lesion margins were surface sterilized in 70% ethanol for 30 s followed by 2% NaOCl for 1 min and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Fifteen strains with similar morphological characterizations were isolated, and three representative isolates (JHT-1, JHT-2, and JHT-3) were chosen and used for further study. Colonies on PDA of three isolates were grayish-green with white edges and dark green on the reverse side. Conidia were transparent, cylindrical with rounded ends, and measured 3.6-5.3 µm × 9.5-15.2 µm (3.7 ± 0.2 × 13.6 ± 1.1 µm, n = 100). Appressoria were dark brown, globose or subcylindrical, and ranged from 6.2-9.2 µm× 5.1-6.8 µm (7.9 ± 0.4 × 5.9 ± 0.3 µm, n=100). The morphological characteristics of the three strains were consistent with the description of species in the Colletotrichum gloeosporioides complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin 2 (TUB2) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, CHS-79F/CHS-345R, GDF/GDR and T1/Bt2b (Weir et al. 2012), respectively. The sequences were deposited in GenBank (Accession Nos. MZ482016 - MZ482018 for ITS; MZ463636 - MZ463638 for ACT; MZ463648- MZ463650 for CAL; MZ463639 - MZ463641 for CHS-1; MZ463642 - MZ463644 for GAPDH; MZ463645 - MZ463647 for TUB2). A neighbor-joining phylogenetic tree was constructed with MEGA 7.0 using the concatenation of multiple sequences (ITS, ACT, GAPDH, TUB2, CHS-1, CAL) (Kumar et al. 2016). According to the phylogenetic tree, three isolates fall within the Colletotrichum fructicola clade (boot support 99%). Based on morphological characteristics and phylogenetic analysis, three isolates were identified as C. fructicola. The pathogenicity of three isolates was conducted on two-yr-old seedlings (30 cm tall) of D. hupeana. Healthy leaves were wounded with a sterile needle and then inoculated with 10 μL spore suspension (106 conidia per mL). Controls were treated with sterile water. All plants were covered with transparent plastic bags and incubated in a greenhouse at 28°C with a 12 h photoperiod (relative humidity > 80%). Within five days, the inoculated leaves developed lesions similar to those observed in the field, whereas controls were asymptomatic. The experiments repeated three times showed similar results. The infection rate was 100%. C. fructicola was re-isolated from the lesions, whereas no fungus was isolated from control leaves. C. fructicola can cause leaf diseases in a variety of hosts, including Aesculus chinensis (Sun et al. 2020), Peucedanum praeruptorum (Ma et al. 2020), and Mandevilla × amabilis (Sun et al. 2020). C. brevisporum and C. gigasporum were also reported to infect Dalbergia odorifera (Chen et al. 2021; Wan et al. 2018). However, This is the first report of C. fructicola associated with leaf spot disease on D. hupeana in China. These results will help to develop effective strategies for appropriately managing this newly emerging disease.

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