Leaf spot caused by Colletotrichum fructicola on star anise (Illicium verum) in China

Star anise (Illicium verum) has been cultivated for centuries in southern China, and its fruit is an important seasoning spice, and can be used as a medicine (Wang et al. 2011). It is grown mainly in Guangxi, Guangdong, Guizhou, and Yunnan provinces, in China. Anthracnose is one of the important diseases of star anise, which seriously affects the yield and quality by infecting twigs, pedicels, fruit stalks and fruits (Liao et al. 2017). When leaf spots first appear, they are round, water-stained, small, dark brown spots, which expands into round separated spots, then the spots become yellowish brown with small black acervuli arranged in a circular pattern. On 22 August 2019, four leaf spot samples of star anise were collected, with two each from Shanglin County and Jinxiu County in Guangxi Province. The plantations in this area of around 8 ha had more than 80% leaf spot incidence. Small pieces of tissues (5 mm × 5 mm) were taken from the zone between symptomatic and healthy plant tissues, surface-disinfected in 75% ethanol for 10 s and 1% NaClO (sodium hypochlorite) for 1 min, and washed three times in sterilized distilled water. The sterilized leaf tissues were placed on potato dextrose agar (PDA) and incubated at 28°C in darkness for a week. Hyphae growing from tissue pieces were subcultured onto fresh PDA. Three of the four leaves yielded cultures resembling Colletotrichum spp. Four fungal isolates were obtained by a single-spore isolation method. The isolates JX1-2 and JX1-5 were collected from Jinxiu County while SL1-2 and SL2-1 were collected from Shanglin County. Genomic DNA was extracted from these four fungal isolates, followed by PCR amplification and sequencing of the rDNA internal transcribed spacer (ITS), actin (ACT), Apn2-Mat1-2 intergenic spacer, partial mating type (Mat1-2) (ApMat), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Weir et al. 2012). The sequences have been deposited in GenBank (ITS: MW301215 to MW301218; ACT: MW348965 to MW348968; ApMat: MW348973 to MW348976; CAL: MW348957 to MW348960; CHS-1: MW348969 to MW348972; GAPDH: MW348961 to MW348964). For phylogenetic analysis, MEGAX (Kumar et al. 2018) was used to produce a Maximum Likelihood (ML) tree with 1000 bootstrap replicates, based on a concatenation of the sequenced genomic regions for each of the four isolates from this study as well as sequences of other Colletotrichum species obtained from GenBank. The results revealed that isolates JX1-2, JX1-5, and SL1-2 were C. horii, and SL2-1 was C. fructicola (Weir et al.2012). The resulting colonies were initially white with abundant aerial hyphae, and white-gray after three days at 28°C on PDA. Isolate SL2-1 eventually turned greenish-grey after 14 days, while the center of C. horii isolates turned iron-gray with white-gray marginal. Both species of Colletotrichum had hyaline conidia that were terete, smooth, apex obtuse, base truncate, and there were no significant differences (P>0.05) in conidial size between C. horii (10.5 to 33.6 × 3.6 to 9.3 μm) (n=300) and C. fructicola (13.1 to 16.2 × 4.7 to 7.1 μm) (n=100). Pathogenicity tests were conducted in the greenhouse on 1-year-old star anise seedlings, and performed with a conidial suspension (10 µL of 106 conidia/mL) containing 0.1% Tween 20 placed onto lightly wounded sites on healthy leaves. Light cross-shaped wounds were made with sterilized toothpicks, gently scratching the surface without piercing the leaf. Each isolate was inoculated onto three seedlings, with at least eight leaves per seedling inoculated in two spots after light wounding. Control seedlings were inoculated with water containing 0.1% Tween 20. All inoculated seedlings were maintained in the greenhouse (12 h/12 h light/dark, 25±2°C), and covered with plastic bags to maintain high humidity throughout. The wounded sites inoculated with C. horii darkened to greenish-brown after 24 h, and C. fructicola gave similar symptoms after 36 h. Then the wounds turned to light brown round spots, and after 5 days, the spots expanded to water-stained spots with dots of acervuli arranged in a circular pattern. No symptoms were observed for the non-inoculated control. Each fungal isolate was consistently re-isolated from inoculated leaves, thus fulfilling Koch's postulates. There were significant differences (P<0.05) in aggressiveness between the two species, with C. horii showing larger diameter lesions (averaging 10.2 mm) than C. fructicola (averaging 8.4 mm). Anthracnose of star anise caused by C. horii (Liao et al. 2017) and C. coccdes (Wu et al. 2003) has been previously reported in China; however, to our knowledge, this is the first report of C. fructicola infecting star anise in China. This study may provide reference for further epidemiological study and prevention of anthracnose on star anise.

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First Report of Corynespora Leaf Spot of Eucalyptus in China

Plant Disease ◽

10.1094/pdis-07-14-0697-pdn ◽

2015 ◽

Vol 99 (3) ◽

pp. 419-419 ◽

Cited By ~ 1

Author(s):

C. K. Phan ◽

J. G. Wei ◽

F. Liu ◽

B. S. Chen ◽

J. T. Luo ◽

...

Keyword(s):

Leaf Spot ◽

Southern China ◽

Tap Water ◽

Pathogenic Fungi ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Commonwealth Mycological Institute ◽

Conidial Suspension ◽

Single Spore ◽

First Report

Eucalyptus is widely planted in the tropics and subtropics, and it has become an important cash crop in Southern China because of its fast-growing nature. In the Guangxi Province of southern China, Eucalyptus is produced on approximately 2 million ha, and two dominant asexual clones, Guanglin No. 9 (E. grandis × E. urophylla) and DH3229 (E. urophylla × E. grandis), are grown. Diseases are an increasing threat to Eucalyptus production in Guangxi since vast areas are monocultured with this plant. In June 2013, a leaf spot disease was observed in eight out of 14 regions in the province on a total of approximately 0.08 million ha of Eucalyptus. Initially, the lesions appeared as water-soaked dots on leaves, which then became circular or irregular shaped with central gray-brown necrotic lesions and dark red-brown margins. The size of leaf spots ranged between 1 and 3 mm in diameter. The main vein or small veins adjacent to the spots were dark. The lesions expanded rapidly during rainy days, producing reproductive structures. In severe cases, the spots coalesced and formed large irregular necrotic areas followed by defoliation. The causal fungus was isolated from diseased leaves. Briefly, the affected leaves were washed with running tap water, sterilized with 75% ethanol (30 s) and 0.1% mercuric dichloride (3 min), and then rinsed three times with sterilized water. Small segments (0.5 to 0.6 cm2) were cut from the leading edge of the lesions and plated on PDA. The plates were incubated at 25°C for 7 to 10 days. When mycelial growth and spores were observed, a single-spore culture was placed on PDA and grown in the dark at 25°C for 10 days. A pathogenicity test was done by spraying a conidial suspension (5 × 105 conidia ml–1) of isolated fungus onto 30 3-month-old leaves of Guanglin No. 9 seedlings. The plants were covered with plain plastic sheets for 7 days to keep the humidity high. Lesions similar to those observed in the forests were observed on the inoculated leaves 7 to 10 days after incubation. The same fungus was re-isolated. Leaves of control plants (sprayed with sterilized water) were disease free. Conidiophores of the fungus were straight to slightly curved, erect, unbranched, septate, and pale to light brown. Conidia were formed in chains or singly with 4 to 15 pseudosepta, which were oblong oval to cylindrical, subhyaline to pale olivaceous brown, straight to curved, 14.5 to 92.3 μm long, and 3.5 to 7.1 μm wide. The fungus was morphologically identified as Corynespora cassiicola (1). DNA of the isolate was extracted, and the internal transcribed spacer (ITS) region (which included ITS 1, 5.8S rDNA gene of rDNA, and ITS 2) was amplified with primers ITS5 and ITS4. 529 base pair (bp) of PCR product was obtained and sequenced. The sequence was compared by BLAST search to the GenBank database and showed 99% similarity to C. cassiicola (Accession No. JX087447). Our sequence was deposited into GenBank (KF669890). The biological characters of the fungus were tested. Its minimum and maximum growth temperatures on PDA were 7 and 37°C with an optimum range of 25 to 30°C. At 25°C in 100% humidity, 90% of conidia germinated after 20 h. The optimum pH for germination was 5 to 8, and the lethal temperature of conidia was 55°C. C. cassiicola has been reported causing leaf blight on Eucalyptus in India and Brazil (2,3) and causing leaf spot on Akebia trifoliate in Guangxi (4). This is the first report of this disease on Eucalyptus in China. References: (1) M. B. Ellis and P. Holliday. CMI Descriptions of Pathogenic Fungi and Bacteria, No. 303. Commonwealth Mycological Institute, Kew, Surrey, UK, 1971. (2) B. P. Reis, et al. New Dis. Rep. 29:7, 2014. (3) K. I. Wilson and L. R. Devi. Ind. Phytopathol. 19:393, 1966. (4) Y. F. Ye et al. Plant Dis. 97:1659, 2013.

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

Plant Disease ◽

10.1094/pdis-12-20-2681-pdn ◽

2021 ◽

Author(s):

Xiaodong Sun ◽

Xinglai Cai ◽

Qiangqiang Pang ◽

Man Zhou ◽

Wen Zhang ◽

...

Keyword(s):

Leaf Spot ◽

Southern China ◽

Elongation Factor ◽

Morphological Characteristics ◽

Negative Control ◽

Hainan Province ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Kalanchoe Pinnata

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

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

Plant Disease ◽

10.1094/pdis-08-20-1773-pdn ◽

2020 ◽

Author(s):

Quan Shen ◽

Xixu Peng ◽

Feng He ◽

Shaoqing Li ◽

Zuyin Xiao ◽

...

Keyword(s):

Relative Humidity ◽

Leaf Spot ◽

Plant Pathogens ◽

Hunan Province ◽

Tartary Buckwheat ◽

Tween 20 ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

First Report

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

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First Report of Leaf Spot Caused by Corynespora cassiicola on Baphicacanthus cusia in China

Plant Disease ◽

10.1094/pdis-10-12-0921-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 690-690

Author(s):

Q.-L. Li ◽

S.-P. Huang ◽

T.-X. Guo ◽

Z.-B. Pan ◽

J.-Y. Mo ◽

...

Keyword(s):

Leaf Spot ◽

Ipomoea Batatas ◽

Its Region ◽

Tween 20 ◽

Herbaceous Plant ◽

Commonwealth Mycological Institute ◽

Corynespora Cassiicola ◽

Single Spore ◽

First Report ◽

Perennial Herbaceous Plant

Baphicacanthus cusia is a perennial herbaceous plant in the family Acanthaceae that is native to China, where it grows in warm temperate mountainous or hilly regions. It is commonly used as a Chinese herbal medicine. In March 2012, symptoms of leaf spot were observed on leaves of B. cusia in Long'an County, Guangxi, China, where this plant is extensively cultivated. Symptoms were initially small brown dots which developed into irregular to circular leaf spots. These spots enlarged and overlapped, extending until the 7- to 9-cm-long and 3- to 4-cm-wide leaves withered entirely, mostly within 2 months. On potato dextrose agar (PDA), the same fungus was cultured from 92% of 75 symptomatic leaf samples that had been surface sterilized in a 45-second dip in 0.1% mercuric chloride. Fungal structures were observed on diseased leaves: conidiophores (85 to 460 × 4 to 8 μm) were erect, brown, single or in clusters, and conidia (36 to 90 × 5 to 16 μm) were single or in chains of two to four, brown, cylindrical or obclavate, straight or slightly curved, with 3 to 18 pseudosepta and a conspicuous hilum. Three single-spore isolates were identified as Corynespora cassiicola (Berk & Curt.) Wei based on morphological and cultural characteristics (1). The rDNA internal transcribed spacer (ITS) region of one isolate, ZY-1, was sequenced (GenBank Accession No. JX908713), and it showed 100% identity to C. cassiicola, GenBank FJ852716, an isolate from Micronesia cultured from Ipomoea batatas (2). Pathogenicity tests were performed with each of the three isolates by spraying conidial suspensions (5 × 104 conidia/ml) containing 0.1% Tween 20 onto the surfaces of leaves of 60-day-old, 20-cm tall plants. For each isolate, 30 leaves from five replicate plants were treated. Control plants were treated with sterilized water containing 0.1% Tween 20. All plants were incubated for 36 h at 25°C and 90% relative humidity in an artificial climate chamber, and then moved into a greenhouse. Seven days after inoculation, dark brown spots typical of field symptoms were observed on all inoculated leaves, but no symptoms were seen on water-treated control plants. Koch's postulates were fulfilled by reisolation of C. cassiicola from diseased leaves. To our knowledge, this is the first report of C. cassiicola infecting B. cusia worldwide. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute: Kew, Surrey, England, 1971. (2) L. J. Dixon et al. Phytopathology 99:1015, 2009.

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Brown Culm Rot of Dendrocalamus latiflorus Munro Caused by Diaporthe guangxiensis in Sichuan Province, China

Plant Disease ◽

10.1094/pdis-04-21-0837-pdn ◽

2021 ◽

Author(s):

Wenjian Wei ◽

Han Zhang ◽

Liling Xie ◽

Han Liu ◽

Fengying Luo ◽

...

Keyword(s):

Genomic Dna ◽

Southern China ◽

Disease Incidence ◽

Its Region ◽

Sichuan Province ◽

Control Measures ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

Dendrocalamus Latiflorus

Dendrocalamus latiflorus Munro, the most widely cultivated bamboo species in southern China, has high ornamental value used in gardens, while culms are also used for buildings and as fibers and edibles (Gao et al. 2011). In June 2020, brown culm rot of bamboo was observed in Yibin city, Sichuan Province, in an area of approximately 1000 hectares. Disease incidence was approximately 60%, of which 30% of the plants had died. At the end of June, the lesions expanded but did not surround the base of the culm. From the end of June to the beginning of September, the lesions expanded upward and formed a streak, of which the color gradually deepened to purple-brown and black-brown. At the same time, the disease spots at the base of the culm also expanded horizontally. After the spots surrounded the base of the culm, the diseased bamboo died. Ten culms showing typical symptoms were collected and cut into 5×5 mm pieces at the junction of infected and healthy tissues. The tissues were sterilized for 1 to 2 min in 3% sodium hypochlorite, decontaminated in 75% alcohol for 3 to 5 min, placed on modified potato glucose agar (PDA) with streptomycin sulfate (50 μg/ml), and incubated at 26°C. Two isolates were obtained by the single-spore method (Sivan et al. 1992). The isolates both produced white round colonies similar to Diaporthe guangxiensis and two types of conidia: one was α type (5.5 to 8.2×1.0 to 2.8 µm, n=30), colourless, single-celled, undivided, and oval, containing two oil droplets; and β type (21.1 to 30.2×0.8 to 1.4 µm, n=30), colourless, single celled and hook shaped. Genomic DNA was extracted from the two isolates by using a fungal genomic DNA extraction kit (Solarbio, Beijing). The products were amplified by polymerase chain reaction (PCR) with primers for the internal transcribed spacer 1 (ITS) region (White et al. 1990), calmodulin (CAL) gene (Carbone and Kohn 1999), translation elongation factor 1-alpha (TEF) gene (Glass and Donaldson 1995) and beta-tubulin (TUB) gene (Soares et al. 2018). The amplified products were sequenced and blasted in GenBank (accession numbers MW380383, MW431318, MW431317 and MW431316 for ITS, CAL, TEF, and TUB, respectively). The ITS, CAL, TEF, and TUB sequences showed 100%, 99.33%, 100%, and 99.80% identity to D. guangxiensis JZB320094 (accession numbers MK335772.1, MK736727.1, MK523566.1, MK500168.1 in GenBank), respectively. To evaluate the pathogenicity of the isolates, five plants were each inoculated with two isolates. The cortex of potted bamboo were injured locally with sterilized needle, and the bamboo culms were inoculated with 100 μl of conidial suspension (105 cfu/ml). The surface of the inoculation wound was covered with gauze soaked with sterilized water. Five plants inoculated with sterile water were used as controls. The treated plants were maintained in a greenhouse at a temperature of 22 to 29°C and relative humidity of 70 to 80%. One month later, of all inoculated plants showed similar symptoms as those observed in the field. D. guangxiensis was re-isolated from all inoculated plants. The pathogenicity test was repeated three times with similar results. This is the first report of D. guangxiensis causing brown culm rot of D. latiflorus in China. These results will facilitate an enhanced understanding of factors affecting bamboo and the design of effective management strategies of the pathogenic species on bamboo and thus to develop corresponding control measures.

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First Report of Leaf Spot of Houttuynia cordata Caused by Alternaria alternata in China

Plant Disease ◽

10.1094/pdis-10-10-0724 ◽

2011 ◽

Vol 95 (3) ◽

pp. 359-359

Author(s):

L. Zheng ◽

R. Lv ◽

Q. Li ◽

J. Huang ◽

Y. Wang ◽

...

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Southern China ◽

Sclerotium Rolfsii ◽

Tween 20 ◽

Herbaceous Plant ◽

Houttuynia Cordata ◽

First Report ◽

Perennial Herbaceous Plant ◽

Pathogenicity Tests

Houttuynia cordata is a perennial herbaceous plant (family Saururaceae) that is native to southern China, Japan, Korea, and Southeast Asia where it grows well in moist to wet soils. It is commonly used as a Chinese herbal medicine and as a vegetable. In North America and Europe it is also used as an ornamental. From September 2007 to November 2009, symptoms of leaf spot were found on H. cordata leaves in Dangyang County, Hubei, China, with the crop area affected estimated to be over 600 ha per year. Rhizome yield was reduced by 20% on average, with up to 70% yield losses in some fields during the autumn growing season. Lesions were initially small, brown, and oval or circular that developed into dark spots and sometimes formed target spots with white centers. These spots enlarged and overlapped, extending until the leaves withered entirely usually within 2 months. A fungus was consistently recovered from symptomatic leaf samples collected in October 2008 or 2009 with an average 90% isolation rate from ~60 leaf pieces that were surface sterilized with 0.1% mercuric chloride solution. Three isolates, HCDY-2, HCDY-3, and HCDY-4, were used to further evaluate characteristics of the pathogen. On potato dextrose agar, all cultures initially developed white colonies and the centers turned gray or brown after 4 days of incubation. Conidiophores were single or fasciculate, straight or knee curved, gray-brown with regular septa, and 42 to 61 × 4 to 5 μm. Conidia were obclavate or ovate, brown, and 26 to 38 × 12 to 20 μm with three to five transverse and one to three longitudinal or oblique septa. The tops of some conidia developed into secondary conidiophores, which were cylindrical, beige, and 5 to 17 × 3 to 5 μm. The pathogen was identified as Alternaria alternata based on descriptions in Simmons (3). Genomic DNA of HCDY-2 was extracted, and the rDNA-internal transcribed spacer sequence showed 99.6% identity to A. alternata (GenBank No. AY513941). Pathogenicity tests were performed with the three isolates by spraying conidial suspensions (1 × 106 conidia/ml) containing 0.1% Tween 20 onto upper and lower surfaces of leaves of 40-day-old 15-cm high plants. There were 20 leaves from five replicate plants for each isolate. Control plants were treated with sterilized water containing 0.1% Tween 20 only. All plants were incubated with a 16-h photoperiod at 25°C and 90% relative humidity in an artificial climate chamber. Five days after inoculation, typical brown spots were observed on all inoculated leaves but no symptoms were seen on water-treated control plants. Koch's postulates were fulfilled by reisolation of A. alternata from diseased leaves. The pathogenicity tests were carried out twice. A survey of the literature revealed only a few fungal diseases associated with H. cordata (1,2,4), including Phyllosticta houttuyniae, Pseudocercospora houttuyniae, Rhizoctonia solani, and Sclerotium rolfsii. Although A. alternata is a cosmopolitan plant pathogen, it has not been reported on any species in the four genera in Saururaceae (Anemopsis, Gymnotheca, Houttuynia, and Saururus) (3). To our knowledge, this is the first report of A. alternata infecting H. cordata worldwide. References: (1) Y. L. Guo and W. X. Zhao. Acta Mycol. Sin. 8:118, 1989. (2) K. Sawada. Spec. Publ. Taiwan Univ. 8:138, 1959. (3) E. G. Simmons. Alternaria: An Identification Manual. The American Phytopathological Society, St. Paul, MN, 2007. (4) Y. Wu et al. J. Changjiang Vegetables (In Chinese) 2:19, 2007.

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First Report of Curvularia gladioli Causing a Leaf Spot on Gladiolus grandiflorus in Brazil

Plant Disease ◽

10.1094/pdis-12-12-1118-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 847-847 ◽

Cited By ~ 5

Author(s):

D. P. Torres ◽

M. A. Silva ◽

D. B. Pinho ◽

O. L. Pereira ◽

G. Q. Furtado

Keyword(s):

Leaf Spot ◽

Central Cell ◽

Post Inoculation ◽

Intermediate Cell ◽

Conidial Suspension ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

First Report ◽

The Third ◽

Leaf Spots

Gladiolus (Iridaceae) is a popular bulbous plant grown worldwide as an ornamental garden plant or cut flower due to its attractive color, size, and flower shape. In April 2012, leaf spots were observed on plants of Gladiolus grandiflorus varieties T-704 and Amsterdam growing in a production area of cut flowers located in the city of Viçosa, Minas Gerais. The oval to round leaf spots were brown with a dark border surrounded by a halo of yellow tissue. Infected leaf samples were deposited in the herbarium at the Universidade Federal de Viçosa (VIC31897). A fungus was isolated from the leaf spots and a single-spore pure culture was initiated and grown on corn meal carrot agar (CCA) medium in petri dishes incubated at 25°C under a 12-h photoperiod for 4 weeks. A sporulating single-spore culture was deposited at the Coleção de Culturas de fungos fitopatogênicos “Prof. Maria Menezes” (UFRPE, Brazil) code CMM 4055. On CCA medium, the fungal isolate initially appeared white, becoming dark after 14 days. Thirty conidia and conidiophores were measured for identification to species. The septate, smooth to pale brown conidiophores were present singly or in groups. The simple, straight or flexuous conidiophores were 42.5 to 82.5 × 3.5 to 7.5 μm and some had a geniculate growth pattern. The majority of conidia were curved at the third (central) cell from the base, which was usually enlarged compared to the end cells. The cells at each end of the 3-distoseptate conidia were pale brown, the intermediate cell brown or dark brown, and the third (central) cell was often the darkest. The basal cell had a protuberant hilum. Conidia were smooth and 20.0 to 33.5 × 10 to 17.5 μm. These characteristics matched well with the description of Curvularia gladioli (1). To confirm this identification, DNA was extracted using a Wizard Genomic DNA Purification Kit and the internal transcribed spacer region (ITS) of rDNA was amplified using ITS1 and ITS4 primers and the partial 28S rDNA region using primers LR0R and LR5. The sequences were deposited in GenBank as accession nos. JX995106 and JX995107, respectively. The ITS sequence matched sequence AF071337, C. gladioli, with 100% identity. This pathogen was first identified as C. lunata, but based on the characteristic of the hilum, spore size, and pathogenicity testing, the fungus was renamed C. trifolii f. sp. gladioli (3). Due to the explicit curvature of the conidia at the third cell and molecular data, the fungus was reclassified as C. gladioli (1,2). To confirm Koch's postulates, 1-month-old healthy plants of G. grandiflorus var. T-704 and Amsterdam (five plants each) were inoculated with a conidial suspension (2 × 104 conidia mL–1) by spraying the foliage and then placed on a growth chamber at 25°C. The control plants were sprayed with distilled water. Symptoms were consistent with those initially observed and all plants developed leaf spots by 4 days post-inoculation. C. gladioli was consistently recovered from the symptomatic tissue and control plants remained symptomless. To our knowledge, this is the first report of C. gladioli causing leaf spot on G. grandiflorus in Brazil. Due to a lack of chemical fungicides for management of this pathogen, further studies to evaluate the susceptibility of the main varieties of gladiolus grown in Brazil to C. gladioli may be necessary. References: (1) G. H. Boerema and M. E. C. Hamers. Neth. J. Plant Pathol. 95:1, 1989. (2) D. S. Manamgoda et al. Fungal Divers. 56:131, 2012. (3) J. A. Parmelee. Mycologia 48:558, 1956.

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

Plant Disease ◽

10.1094/pdis-92-4-0652b ◽

2008 ◽

Vol 92 (4) ◽

pp. 652-652 ◽

Cited By ~ 2

Author(s):

X. G. Zhou ◽

K. L. Everts

Keyword(s):

United States ◽

Disease Severity ◽

Body Length ◽

Alternaria Alternata ◽

Leaf Spot ◽

The United States ◽

Tween 20 ◽

Conidial Suspension ◽

First Report ◽

Alternaria Leaf Spot

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

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