First Report of Colletotrichum siamense Causing Daylily (Hemerocallis citrina) Anthracnose in China

Daylily (Hemerocallis citrina Baroni) is a perennial herb whose flowers are commonly used in traditional Chinese cuisine. It is commercially cultivated in the Loess plateau of Gansu province, China. From July to October 2020, necrotic lesions were observed on the foliage of daylily plants in Huan County, Gansu, China, with an average disease incidence of 90%, and 52 to 86 disease index across four fields (approximate 6 hectares). Lesions were fusiform or nearly fusiform yellowish-brown spots of different sizes and a yellow irregular border. Older lesions were almost dark brown that often coalesced and expanded to cover the entire leaves. Thirty-four samples were collected from plants with typical foliar symptoms. Symptomatic tissues were excised from the margins of the lesions and sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, rinsed with sterilized water four times, dried on sterile paper towels, and cultured on Potato Dextrose Agar medium at 25°C for 7 days. A total of 34 fungal isolates with 100% isolation frequency were obtained and characterized. Colonies were white, becoming pale brown with age, reverse turned grayish black with age and irregular pale yellowish borders on the reverse side. Conidia (n=50) were hyaline, one-celled, subcylindrical with obtuse to slightly rounded ends, of 12-18.5×3.5-6 µm in size, (avg. 15.5×4.8 µm). The isolates were designated as K2010301 (51-54) and deposited in the Microbiological Culture Collection Center at College of Pastoral Agriculture Science and Technology, Lanzhou University (China). For fungal identification to species level, genomic DNA of a representative isolate (isolate MG) was extracted. Internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase-1 (CHS-1) and beta-tubulin (TUB2) were amplified using V9G/ITS4, GDF1/GDR1, CHS-354R/CHS-79F, and T1/Bt-2b primer sets (Damm et al., 2012), respectively, and deposited in GenBank under accession numbers MW811458, MW836582, MW836581, and MW836584. BLASTn showed higher than 99% identity with Colletotrichum siamense (GenBank: KP703350 (ITS), MN884050 (GAPDH), MN894598 (CHS-1), and KX578815 (TUB2)). A Bayesian inference analysis of the four concatenated loci showed that isolate MG grouped in the C. siamense clade. Pathogenicity tests were performed by spraying a spore suspension (1×105 conidia/mL) of a 10-day-old culture of isolate “MG” onto 3 healthy and asymptomatic daylily plants. Three control plants were only sprayed with the same volume of sterile distilled water. The inoculated plants were covered with black plastic bags for 2 days to maintain high relative humidity. Anthracnose symptoms resembling those observed in the field developed after 7 days on all inoculated plants, while no symptoms were observed on the control plants. The fungus was reisolated and identified as C. siamense based on morphological features and DNA sequence analysis, fulfilling Koch’s postulates. It has been demonstrated that C. liliacearum (Zhuang, 2005), C. gloeosporioides, and C. spaethianum (Yang et al., 2012) are anthracnose pathogens of H. citrina. To our knowledge, this is the first report of C. siamense causing daylily anthracnose worldwide. This fungal pathogen represents a severe threat and has the potential to cause yield losses of daylily, so further studies should focus on epidemiology and effective management strategies of this disease.

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First report of Colletotrichum siamense Causing Blossom Blight on Thai basil (Ocimum basilicum L.) in Malaysia

Plant Disease ◽

10.1094/pdis-06-20-1371-pdn ◽

2020 ◽

Author(s):

Siti Izera Ismail ◽

Nur Adlina Rahim ◽

Dzarifah Zulperi

Keyword(s):

Disease Incidence ◽

Its Region ◽

Ocimum Basilicum ◽

Distilled Water ◽

Single Spore ◽

First Report ◽

Plastic Bags ◽

Colletotrichum Siamense ◽

Primer Sets ◽

Acca Sellowiana

Thai basil (Ocimum basilicum L.) is widely cultivated in Malaysia and commonly used for culinary purposes. In March 2019, necrotic lesions were observed on the inflorescences of Thai basil plants with a disease incidence of 60% in Organic Edible Garden Unit, Faculty of Agriculture in the Serdang district (2°59'05.5"N 101°43'59.5"E) of Selangor province, Malaysia. Symptoms appeared as sudden, extensive brown spotting on the inflorescences of Thai basil that coalesced and rapidly expanded to cover the entire inflorescences. Diseased tissues (4×4 mm) were cut from the infected lesions, surface disinfected with 0.5% NaOCl for 1 min, rinsed three times with sterile distilled water, placed onto potato dextrose agar (PDA) plates and incubated at 25°C under 12-h photoperiod for 5 days. A total of 8 single-spore isolates were obtained from all sampled inflorescence tissues. The fungal colonies appeared white, turned grayish black with age and pale yellow on the reverse side. Conidia were one-celled, hyaline, subcylindrical with rounded end and 3 to 4 μm (width) and 13 to 15 μm (length) in size. For fungal identification to species level, genomic DNA of representative isolate (isolate C) was extracted using DNeasy Plant Mini Kit (Qiagen, USA). Internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), and chitin synthase-1 (CHS-1) were amplified using ITS5/ITS4 (White et al. 1990), CL1C/CL2C (Weir et al. 2012), ACT-512F/783R, and CHS-79F/CHS-345R primer sets (Carbone and Kohn 1999), respectively. A BLAST nucleotide search of ITS, CHS-1, CAL and ACT sequences showed 100% similarity to Colletotrichum siamense ex-type cultures strain C1315.2 (GenBank accession nos. ITS: JX010171 and CHS-1: JX009865) and isolate BPDI2 (CAL: FJ917505, ACT: FJ907423). The ITS, CHS-1, CAL and ACT sequences were deposited in GenBank as accession numbers MT571330, MW192791, MW192792 and MW140016. Pathogenicity was confirmed by spraying a spore suspension (1×106 spores/ml) of 7-day-old culture of isolate C onto 10 healthy inflorescences on five healthy Thai basil plants. Ten infloresences from an additional five control plants were only sprayed with sterile distilled water and the inoculated plants were covered with plastic bags for 2 days and maintained in a greenhouse at 28 ± 1°C, 98% relative humidity with a photoperiod of 12-h. Blossom blight symptoms resembling those observed in the field developed after 7 days on all inoculated inflorescences, while inflorescences on control plants remained asymptomatic. The experiment was repeated twice. C. siamense was successfully re-isolated from the infected inflorescences fulfilling Koch’s postulates. C. siamense has been reported causing blossom blight of Uraria in India (Srivastava et al. 2017), anthracnose on dragon fruit in India and fruits of Acca sellowiana in Brazil (Abirami et al. 2019; Fantinel et al. 2017). This pathogen can cause a serious threat to cultivation of Thai basil and there is currently no effective disease management strategy to control this disease. To our knowledge, this is the first report of blossom blight caused by C. siamense on Thai basil in Malaysia.

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First Report of Anthracnose of Tricyrtis macropoda Caused by Colletotrichum gloeosporioides in Korea

Plant Disease ◽

10.1094/pdis-03-12-0277-pdn ◽

2012 ◽

Vol 96 (7) ◽

pp. 1070-1070 ◽

Cited By ~ 1

Author(s):

J. H. Park ◽

K. S. Han ◽

Y. D. Kwon ◽

H. D. Shin

Keyword(s):

Colletotrichum Gloeosporioides ◽

Morphological Characteristics ◽

Its Region ◽

Culture Collection ◽

Perennial Herb ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Natural Habitats ◽

First Report ◽

Small Water

Tricyrtis macropoda Miq. (syn. T. dilatata Nakai), known as speckled toadlily, is a perennial herb native to China, Japan, and Korea. The plant has been highly praised for its beautiful flowers and rare populations in natural habitats. In September 2006, several dozen plants were heavily damaged by leaf spots and blight in cultivated plantings in the city of Pocheon, Korea. The infections with the same symptoms were repeated every year. In July 2011, the same symptoms were found on T. macropoda in the cities of Gapyeong and Osan, Korea. The leaf lesions began as small, water-soaked, pale greenish to grayish spots, which enlarged to form concentric rings and ultimately coalesced. A number of blackish acervuli were formed in the lesions. Acervuli were mostly epiphyllous, circular to ellipsoid, and 40 to 200 μm in diameter. Setae were two- to three-septate, dark brown at the base, paler upwards, acicular, and up to 100 μm long. Conidia (n = 30) were long obclavate to oblong-elliptical, sometimes fusiform-elliptical, guttulate, hyaline, and 12 to 20 × 4 to 6.5 μm (mean 15.4 × 5.2 μm). These morphological characteristics of the fungus were consistent with the description of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (2). Voucher specimens (n = 7) were deposited in the Korea University herbarium (KUS). Two isolates, KACC46374 (ex KUS-F25916) and KACC46405 (ex KUS-F26063), were deposited in the Korean Agricultural Culture Collection. Fungal DNA was extracted and the complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The resulting sequences of 549 bp were deposited in Genbank (Accession Nos. JQ619480 and JQ619481). They showed 100% similarity with a sequence of C. gloeosporioides (EU32619). Isolate KACC46374 was used in a pathogenicity test. Inoculum was prepared by harvesting conidia from 3-week-old cultures on potato dextrose agar. A conidial suspension (2 × 106 conidia/ml) was sprayed onto 15 leaves of three plants. Three noninoculated plants served as controls. Plants were covered with plastic bags to maintain 100% relative humidity for 24 h and then kept in a greenhouse (22 to 28°C and 70 to 80% RH). After 5 days, typical leaf spot symptoms, identical to the ones observed in the field, started to develop on the leaves of inoculated plants. No symptoms were observed on control plants. C. gloeosporioides was reisolated from the lesions of inoculated plants, thus fulfilling Koch's postulates. An anthracnose associated with C. tricyrtii (Teng) Teng was recorded on T. formosana and T. latifolia in China (3) and on T. formosana in Taiwan (1), respectively, without etiological studies. The morphological features of C. tricyrtii are within the variation of C. gloeosporioides (2). To our knowledge, this is the first report of anthracnose of T. macropoda. This report has significance to indigenous plant resource conservation managers and scientists because T. macropoda has been listed as one of the 126 “Rare and Endangered Plants” by the Korea Forest Service since 1991. References: (1) K. Sawada. Rep. Dept. Agric. Gov. Res. Inst. Formosa 87: 1, 1944. (2) B. C. Sutton. Pages 1–27 in: Colletotrichum Biology, Pathology and Control. J. A. Bailey and M. J. Jeger, eds. CAB International, Wallingford, U.K. 1992. (3) S. C. Teng. Contrib. Biol. Lab. Sci. Soc. China 8:36, 1932.

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First Report of Colletotrichum siamense Causing Anthracnose of Guava (Psidium guajava) in Mexico

Plant Disease ◽

10.1094/pdis-03-21-0530-pdn ◽

2021 ◽

Author(s):

Edgar Edel Rodríguez-Palafox ◽

Alfonso Vásquez-López ◽

Guillermo Márquez-Licona ◽

Nelson Bernardi Lima ◽

Erika Lagunes-Fortiz ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Species Complex ◽

Aerial Mycelium ◽

Its Region ◽

Psidium Guajava ◽

Culture Collection ◽

First Report ◽

Colletotrichum Species ◽

Guava Fruit ◽

Colletotrichum Siamense

Guava (Psidium guajava L.) is a small tree belonging to the Myrtaceae family and it is distributed worldwide in the tropical and subtropical areas. During the summer of 2019, symptoms of fruit anthracnose were observed on approx. 90% of 250 guava trees located in backyards in Juan Jose Rios, Sinaloa, Mexico. Lesions on guava fruit were irregular, necrotic, and sunken. On advanced infections, acervuli containing salmon-pink masses of spores were observed on the lesions. Twenty fruits were collected from 10 trees (2 fruits per tree). Colletotrichum-like colonies were consistently isolated on PDA medium and 20 monoconidial isolates were obtained. Four isolates were selected as representatives for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of El Fuerte Valley at the Sinaloa Autonomous University (Accession nos. FAVF205–FAVF208). Colonies on PDA medium were flat with an entire margin, with abundant felty and white aerial mycelium, with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with ends rounded, and measuring 14.8 to 18.1 × 4.4 to 5.3 μm. Based on morphological features, the isolates were tentatively allocated in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), as well as partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB2), chitin synthase (CHS-1) and glutamine synthetase (GS) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference and including published ITS, GAPDH, TUB2, ACT, CHS-1, and GS data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the four isolates FAVF205–FAVF208 as C. siamense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accessions nos. ITS: MW598512–MW598515; GAPDH: MW595216–MW595219; TUB2: MW618012–MW618015; ACT: MW595208–MW595211; CHS-1: MW595212–MW595215; and GS: MW618008–MW618011). Pathogenicity of the four isolates was verified on 40 healthy guava fruits. Twenty fruits were wounded with a sterile toothpick (2 mm in depth) and a mycelial plug (6 mm of diameter) was placed on each wound. Ten fruits inoculated with a PDA plug without mycelial growth served as controls. The fruit was kept in a moist plastic chamber at 25°C for 7 days. Pathogenicity of each isolate was tested with both non-wound and wound inoculation methods. The experiments were repeated twice with similar results. All inoculated fruits developed sunken necrotic lesions 4 days after inoculation, whereas no symptoms were observed on the control fruits. The fungi were consistently re-isolated only from the diseased fruits, fulfilling Koch´s postulates. Colletotrichum siamense has been previously reported on guava fruit in India (Sharma et al. 2015). However, to our best knowledge, this is the first report of C. siamense causing fruit anthracnose on guava in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species on guava in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.

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First Report of Anthracnose on Pecan (Carya illinoiensis) Caused by Colletotrichum siamense in Korea

Plant Disease ◽

10.1094/pdis-12-20-2709-pdn ◽

2021 ◽

Author(s):

Ji Yeon Oh ◽

Jeong-In Heo ◽

Dong-Hyeon Lee

Keyword(s):

Its Region ◽

Culture Collection ◽

Likelihood Method ◽

Malt Extract ◽

Distilled Water ◽

Sterile Water ◽

First Report ◽

Colletotrichum Siamense ◽

Plastic Bag ◽

Water Drops

In 2020, severely infected fruit of pecan, Carya illinoiensis, showing distinct anthracnose symptoms were observed from pecan orchards in Uiseong (36°21'31.5"N 128°27'15.9"E) and Miryang (35°22'54.9"N 128°48'06.5"E) in South Korea. Visible symptoms occurred on fruit of the tree between June and July, which included dark, depressed and covered with irregularly shaped lesions. As the disease progressed, the lesions expanded and merged over time, leading to abscission of the fruit, which resulted in severe yield loss of pecan fruit. Of pecan varieties including Caddo, Giles and Peruque that were cultivated in each pecan orchard, Caddo appeared to be most susceptible to the disease. Estimated losses were approximately 30% and 70% for the Uiseong and Miryang pecan orchard, respectively. For pathogen isolation, ten symptomatic fruits of pecan were randomly collected and brought to the laboratory. The fruits were surface disinfested for 30 s with 70% ethanol and 1% sodium hypochlorite. These were then rinsed with sterile distilled water twice, placed in a humid chamber, and incubated at 25 ± 1°C with a photoperiod of 12 h. Acervuli filled with salmon-colored conidial masses were produced abundantly on the fruit a day after the incubation. Conidia were single celled, hyaline, cylindrical having rounded ends, smooth walls, guttulate, 15.5 to 17.7 µm long, and 3.4 to 4.8 µm wide (n = 20). Monoconidial isolates were made on 2% malt extract agar and incubated at 25°Ϲ for two weeks in the dark condition. Of those that were successfully retained, two representative isolates from each orchard were deposited in the culture collection (CDH) of the National Institute of Forest Science, Korea (Accession No. CDH2020-17–18). To ensure the identity of the pathogen, molecular identification was made based on three gene regions, the internal transcribed spacer (ITS) region of rDNA, beta-tubulin (TUB2) gene and a partial sequence of the actin (ACT), which were amplified with ITS1F/ITS4, T1/Bt2b and ACT-512F/ACT-783R, respectively (Weir et al. 2012). These were then submitted to GenBank with accession numbers of MW380423–24 for ITS, MW387129–30 for TUB2 and MW387127–28 for ACT. A BLAST search in GenBank revealed that the sequences showed high similarity to those of Colletotrichum siamense, which were identical to MT434615 and MT274214 for ITS and ACT, respectively, and 99.7% to MK967337 for TUB2. Phylogenetic analysis based on the maximum likelihood method further showed that the isolates recovered in this study clustered with C. siamense, confirming its identity. Pathogenicity was confirmed by inoculating living pecan trees. Healthy fruits from five trees were surface cleaned with cotton soaked in sterile water and air-dried. To inoculate the pathogen, three to five fruit per tree were wounded with a sterilized needle, and an aliquot of 10 μl of spore suspension (1.0 × 105 conidia/ml) of C. siamense (CDH2020-18) was dropped on each wound. To keep moisture, each treated fruit was enveloped by a plastic bag where the cotton soaked in sterile water had been placed. Controls were treated with sterile distilled water drops. The symptoms with dark, depressed and irregularly shaped lesions developed from all inoculated treatments six weeks after the inoculations, which were identical to those observed in the field. However, no symptom was observed on the control. Colletotrichum siamense was successfully re-isolated, fulfilling Koch’s postulates. Taken together, it was confirmed that C. siamense is the causal agent of anthracnose on pecan. In Korea, C. siamense was reported causing anthracnose on apple, persimmon and plum (Farr and Rossman 2020). However, to our knowledge, this is the first report of anthracnose on pecan caused by C. siamense in Korea. To control the disease effectively, more attention should be paid to other regions of the country where the disease caused by the pathogen might occur.

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First Report of Ceratocystis changhui Causing Postharvest Fruit Rot of Peach in Kunming, China

Plant Disease ◽

10.1094/pdis-07-20-1492-pdn ◽

2020 ◽

Author(s):

Xue Li ◽

Ruiqi Zhang ◽

Kecheng Xu ◽

Jie Li ◽

Yu Zhang ◽

...

Keyword(s):

Phylogenetic Trees ◽

Prunus Persica ◽

Management Strategies ◽

Its Region ◽

Its Sequences ◽

Fruit Rot ◽

Single Spore ◽

Early Maturity ◽

Potato Dextrose Agar Medium ◽

First Report

The peach (Prunus persica (L) Batsch) is a predominant commercially grown stone fruit in China (Lee et al. 1990). Ceratocystis changhui is an aggressive pathogen causing typical black rot symptoms on corms of taro (Colocasia esculenta) (Liu et al. 2018), it has not been reported on other hosts. During the summer and autumn of 2013, a postharvest fruit rot disease was observed on several peaches at a farmer's market (N 25°02′; E 102°42′) in Kunming City, Yunnan Province, China. The incidence of the disease varied from 5 to 20%. Necrotic spots were first observed on the infected peach fruit (Prunus persica cv. shuimitao). The spots enlarged gradually and developed into a brown, water-soaked and rotted lesion. Eventually, the whole fruit became soft, rotted and covered with a gray-brown mycelium (Fig. 1 A, B). The isolates were obtained from the symptomatic tissues incubated on slices of fresh carrot root (Moller et al. 1968). After 5 to 10 days of incubation, perithecia and mycelium were observed growing on carrot slices. Spore masses were removed from the apices of perithecia, transferred to potato dextrose agar medium (PDA) and incubated at 25°C for 5 to 10 days, followed by single-spore isolation. All eight single-spore isolates from peach fruits obtained in this study were deposited in the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, China. In culture, mycelium was initially white, gradually turned to greyish-green or brown (Fig. 1E, F). Measurements were made 7 days after the formation of perithecia. Perithecia (Fig. 1G) were black, globose, 185.71 to 305.56 μm × 142.86 to 264.29 µm and showed a long black neck, 600 to 957.14 µm (Fig. 1H). Ascospores (Fig. 1I) were helmet-hat shaped and 2.86 to 6.67 µm ×3.81 to 4.76 µm. Cylindrical conidia (Fig. 1J) 6.67 to 38. 95 µm × 2.86 to7.62 µm were observed. Chlamydospore (Fig. 1K), 8.57 to 13.33 μm × 5.71 to 9.52 μm, were ovoid or obpyriform, smooth. To further verify pathogen identity the internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS1F and ITS4 (Thorpe et al. 2005), and the total genomic DNA from the mycelia of ﬁve isolates was extracted using a CTAB method (Lee &Taylor 1990). The nucleotide sequences have been blasted and deposited in the GenBank database. Analysis of the ITS sequences from the isolates T1-1yp, T1-2yp, T2-1yp (GenBank accession no. KY580895-KY580897) showed 99% to 100% similarity with isolates C. changhui CMW43272 (KY643886), CMW43281 (KY643884), CMW46112 (KY643891) and CMW46113 (KY643892) from taro in China. Phylogenetic trees based on the maximum-likelihood (ML) method were constructed using MEGA 7. ITS sequences of other Ceratocystis spp. were attained from NCBI for comparative analysis (Liu et al. 2018), and Davidsoniella virescens (CMW11164) served as outgroup. The robustness of ML tree was evaluated with 1,000 bootstrap (BS) values. The pathogen was identified as C. changhui based on the phylogenetic analysis (Fig. 2). Three isolates (T1-1yp, T1-2yp, T2-1yp) were used for pathogenicity. Nine Prunus persica cv. yingzuitao fruits at early maturity (8 points out of 10) were wound inoculated with 200μL conidia suspension of the fungus (approximately 2.0 × 106 conidia / mL). Degreasing cotton dipped in sterile water was used to raise the humidity in preservation boxes. Boxes were incubated for 10 days at 25°C. Three peaches as controls were treated only with sterile distilled water in the same way. Symptoms of sunken lesions and fruit rot were observed two days after inoculation, and measured at 1.8 to 3.2 cm from the inoculation point within 5 days (Fig. 1C: right, D). The same pathogen was re-isolated from them confirming Koch’s postulates. Control peaches remained symptomless. This fungus was morphologically and phylogenetically identified as C. changhui. To our knowledge, this is the first report of C. changhui on postharvest peach in Yunnan, China. The disease will affect quality and taste of peach, so it is critical to deploy appropriate management strategies to limit the fungus spread.

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First Report of Leaf Blight on Duying Caused by Phyllosticta anacardiacearum in China

Plant Disease ◽

10.1094/pdis-93-5-0546b ◽

2009 ◽

Vol 93 (5) ◽

pp. 546-546 ◽

Cited By ~ 2

Author(s):

B. G. Lou ◽

Y. D. Xu ◽

C. Sun ◽

X. M. Lou

Keyword(s):

Southern China ◽

Disease Incidence ◽

Its Region ◽

Culture Collection ◽

Leaf Blight ◽

Its Sequences ◽

Dead Leaf ◽

Conidial Suspension ◽

First Report ◽

Branch Dieback

Duying (Elaeocarpus glabripetalus Merr.; Elaeocarpaceae) is widely cultivated as an ornamental tree of commercial importance in southern China. From 2003 to 2008, severe outbreaks of Duying leaf blight occurred in the Hangzhou area, Zhejiang Province. Disease incidence was greater than 20% and mainly infected young leaves and shoots in the spring and autumn. Severely infected leaves and shoots died and eventually led to branch dieback. The overall growth decline of affected trees occurs over 4 to 6 years before tree death. Infection symptoms are characterized by grayish, round, semicircular- or irregular-shaped spots (5 mm to 5 cm long) with dark brown borders and the appearance of black, granular pycnidia within the dead leaf tissues. The primary infection zones are commonly observed on the leaf margins and apices, are brown, up to 2 mm in diameter, and often surrounded by a yellow zone. Pycnidia were globose and 122 to 127 μm (average 123.5 μm) in diameter. A fungus was consistently isolated from symptomatic tissues on potato dextrose agar (PDA). Ash-black pycnidia appeared on PDA after 10 days. Ascospores developed on modified PDA (1 liter of PDA + 20 g of Duying leaves) after 18 days. Conidiogenous cells were cylindrical to obpyriform. The hyaline conidia were obovoid and guttulate, 10 to 13 × 6 to 8 μm (average 11.5 × 7.5 μm), and usually surrounded by a mucilaginous sheath with a hyaline apical appendage that was 5 to 8 μm long. Pseudothecia were solitary and subglobose with long necks. Asci were 45 to 70 × 7.5 to 12 μm (average 62.5 × 10.8 μm). Ascospores were 12 to 13 × 4 to 5 μm with rounded apices and hyaline, mucilaginous, apical caps. The fungus was morphologically identified as Phyllosticta anacardiacearum van der Aa (teleomorph Guignardia mangiferae A. J. Roy). This identification was also confirmed by the China General Microbiological Culture Collection Center (CGMCC). Six representative fungal isolates were identified by sequencing the internal transcribed spacer (ITS) region of the rDNA and comparing the sequences with those in GenBank using BLAST searches. The ITS sequences of six cultures (GenBank Accession Nos. EU821356–EU821361) showed 100% identity with the ITS sequences of an isolate of a Phyllosticta sp. (GenBank Accession No. AF532314) (2) and G. mangiferae (GenBank Accession No. AY277717) (1). To fulfill Koch's postulates, a conidial suspension (106 conidia per ml) collected from PDA cultures (isolate phy01) was used to spray inoculate leaves of potted 3-year-old Duying trees. Inoculated trees were kept for 48 h under a polyethylene sheet cover and grown at 10 to 15°C in a greenhouse. A total of 30 leaves of five healthy trees were inoculated with the pathogen. In addition, five 3-year-old trees were sprayed with sterile water to serve as uninoculated controls. After 10 to 14 days, inoculated leaves showed infection symptoms resembling those observed on Duying trees naturally infected with P. anacardiacearum. The pathogen was reisolated from the margins of necrotic tissues, but not from controls. To our knowledge, this is the first report of leaf blight on E. glabripetalus caused by P. anacardiacearum in China. Reference: (1) F. R. Katia et al. Mycol. Res. 108:45, 2004. (2) A. K. Pandey et al. Mycol. Res. 107:439, 2003.

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First Report of Leaf Anthracnose Caused by Colletotrichum karstii of Piper nigrum in Yunnan Province, China

Plant Disease ◽

10.1094/pdis-08-21-1798-pdn ◽

2021 ◽

Author(s):

Chunhua Lin ◽

Lixia Fu ◽

Zhenzhen Fan ◽

Chun Fang Duan ◽

Tailing Jiang ◽

...

Keyword(s):

Management Strategies ◽

Rubber Tree ◽

Aerial Mycelium ◽

Its Region ◽

Piper Nigrum ◽

Single Spore ◽

Potato Dextrose Agar Medium ◽

First Report ◽

Wide Range ◽

Pepper Leaves

Pepper (Piper nigrum L.) is one of the economically important spice crops of China and mainly grown in the Hainan and Yunnan provinces. In January 2021, the classic anthracnose lesions were observed on pepper leaves at a plantation (24°57’50"N, 98°53’00"E) in Baoshan city, Yunnan, China. Most of the diseased spots occurred at the tips and margins of the old pepper leaves. Lesions were grayish brown or pale white with a slight yellow halo, concentric whorl black dots or scattered black dots were observed on the leaves spots sometimes (Fig. 1). Five symptomatic leaves from different parts of the field were sampled for pathogen isolation. Lesion tissues removed from the border between symptomatic and healthy tissue were surface sterilized in 75% ethanol, then air-dried, plated on potato dextrose agar medium plates (PDA), and incubated in a 12-h photoperiod at 28℃. Similar fungal colonies developed from all plated tissues after 5 days. And five isolates from different leaves (one isolate per leaf) were sub-cultured using the single-spore method. The colonies appeared white, cottony, aerial mycelium dense and slow-growing (mean 1.01 mm day–1) on PDA plates in 6 days. Conidia were short-cylindric, straight, sometimes slightly constricted near the center, ends broadly rounded, measuring 11.05 to 14.43 × 3.78 to 6.08 µm (average = 12.03 × 5.48 µm, n=200). Appressoria were single, subglobose to elliptic, light brown to dark black. Among them, genomic DNA of two isolates (21HJ0301-1 and 21HJ0301-2) were extracted from mycelium and used as a template for molecular identification. The internal transcribed spacer (ITS) region of ribosomal DNA, and partial sequence of chitin synthase (CHS-1), actin (ACT) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene regions were amplified with primer pairs ITS1/ITS4, CHS-79F/CHS-354R, ACT-512F/ACT-783R, GDF/GDR, respectively (Weir et al. 2012). These four gene sequences were deposited in GenBank (Accession No. MZ725047 and MZ725048 for ITS, MZ733415 and MZ733416 for GAPDH, MZ733408 and MZ733409 for ACT, MZ733422 and MZ733423 for CHS-1). A multilocus phylogenetic analysis performed with the reference sequences revealed that both 21HJ0301-1 and 21HJ0301-2 isolates clustered with C. karstii (Fig.2). Based on morphology and molecular results, isolates were confirmed to be C. karstii. Pathogenicity tests were carried out on potted seedlings in the greenhouse, six healthy leaves per isolate were inoculated with six-day-old cultures of C. karstii mycelial discs of 5 mm in diameter after being wounded with a needle or non-wounded. Control leaves were inoculated with PDA agar. Inoculated plants were incubated under high relative humidity at room temperature. Anthracnose symptoms appeared within 5 days using non-wounded or wounded inoculation methods. All control leaves remained asymptomatic. The fungus was re-isolated from inoculated leaves fulfilling Koch’s postulates, but not on controls. C. karstii has a wide range of hosts, such as rubber tree, tea-oil tree, chili, and some other plants belonging to the family Orchidaceae in China (Cai et al. 2016; Jiang and Li 2018; Diao et al. 2017; Yang et al. 2011). To the best of our knowledge, this is the first report of C. karstii on Piper nigrum in China. This report will help us to recognize the anthracnose disease of Piper nigrum and establish a foundation for future studies on C.karstii to address effective management strategies.

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First Report of Sclerotinia Rot of Chinese Atractylodes Caused by Sclerotinia nivalis in China

Plant Disease ◽

10.1094/pdis-06-12-0516-pdn ◽

2012 ◽

Vol 96 (12) ◽

pp. 1823-1823 ◽

Cited By ~ 2

Author(s):

R. J. Zhou ◽

H. J. Xu ◽

J. F. Fu ◽

F. Y. Yang ◽

Z. Liu

Keyword(s):

Dna Sequences ◽

Disease Incidence ◽

Aerial Mycelium ◽

Signs And Symptoms ◽

Its Region ◽

Liaoning Province ◽

Perennial Herb ◽

First Report ◽

Sclerotinia Rot ◽

Its Sequence Analysis

Chinese atractylodes (Atractylodes japonica Koidz.ez Kitam.) is a perennial herb in the Compositae family, and is widely distributed in China. The dried rhizomes of the plant are used in traditional Chinese medicine. During the summer of 2011, typical signs and symptoms of Sclerotinia rot were observed on Chinese atractylodes in a production field of Liaoning Province of China. Symptoms were observed in plants at the flowering stage, distributed in patches throughout the rows, and with a disease incidence of approximately 10 to 15%. The lower mature leaves of infected plants first became yellow and wilted, basal stem areas showed a black-brown rot at the same time under conditions of high humidity, and white cottony mycelium formed along the basal stem and soil surfaces. Ultimately, the basal stem and roots rotted and the plants wilted and died quickly. Black, irregular sclerotia (average 0.8 to 6.9 mm in diameter) were also observed within the pith cavity of split stems and rotted roots. The pathogen was isolated from symptomatic tissues and sclerotia, surface disinfested with 2% sodium hypochlorite, and cultured on potato dextrose agar (PDA) (1). The fungus was mesophilic, with an optimum temperature for mycelial growth in culture of about 20°C. Colonies on PDA produced masses of white aerial mycelium, with small white flocci distributed among sclerotia. After 2 weeks, sclerotia 0.5 to 4.5 mm in diameter were produced near the margin in a uniform distribution. Sclerotia were spherical, elongated, or fused to form irregular shapes and tightly attached to the agar surface by their under surface, which could be seen through the bottom of the petri dishes. DNA sequences of five replicates were obtained using the TianGen DNA secure plant kit. The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and sequenced. BLAST analysis of the 513-bp segment showed high similarity (99%) with a sequence of Sclerotinia nivalis (GenBank Accession No. AB516670). A representative sequence was deposited in GenBank (Accession No. JX294862). The fungus isolated from symptomatic tissues was identified as S. nivalis Saito on the basis of morphological and cultural characteristics (2,3) and ITS sequence analysis. Symptoms were reproduced in the greenhouse by inoculating the basal stem and roots of 15 atractylodes plants at the 7- to 10-leaf stage. Inoculum was prepared by macerating 14-day-old PDA cultures of the fungus in a blender and placing the mixture (approximately 20 g) into the potting medium of each plant. Sterile PDA was used to inoculate the five control plants. Plants were maintained in a greenhouse at 22 to 25°C and about 75% relative humidity. After 7 to 10 days, symptoms were similar to those in the fields. Lower leaves of inoculated plants became yellow and wilted, and infected plants died 2 weeks after inoculation, whereas control plants remained healthy. The pathogen was successfully recovered from symptomatic tissues, completing Koch's postulates. To our knowledge, this is the first report of Sclerotinia rot of Chinese atractylodes. Given its wide host range, S. nivalis has great potential to become an economically important plant pathogen. References: (1) W. G. Kim and W. D. Cho. Mycobiology 30:41, 2002. (2) G. Q. Li et al. Mycol. Res. 104:232, 2000. (3) I. Saito. Mycoscience 38:227, 1997.

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First Report of Anthracnose of Papaya (Carica papaya L.) Caused by Colletotrichum siamense in China

Plant Disease ◽

10.1094/pdis-10-20-2154-pdn ◽

2021 ◽

Author(s):

Yujie Zhang ◽

Wenxiu Sun ◽

Ping Ning ◽

Tangxun Guo ◽

SuiPing Huang ◽

...

Keyword(s):

Carica Papaya ◽

Disease Incidence ◽

Aerial Mycelium ◽

Postharvest Disease ◽

Distilled Water ◽

First Report ◽

Surface Samples ◽

Initial Symptoms ◽

Colletotrichum Siamense ◽

Pathogenicity Tests

Papaya (Carica papaya L.) is a rosaceous plant widely grown in China, which is economically important. Anthracnose caused by Colletotrichum sp. is an important postharvest disease, which severely affects the quality of papaya fruits (Liu et al., 2019). During April 2020, some mature papaya fruits with typical anthracnose symptoms were observed in Fusui, Nanning, Guangxi, China with an average of 30% disease incidence (DI) and over 60% DI in some orchards. Initial symptoms of these papayas appeared as watery lesions, which turned dark brown, sunken, with a conidial mass appearing on the lesions under humid and warm conditions. The disease severity varied among fruits, with some showing tiny light brown spots, and some ripe fruits presenting brownish, rounded, necrotic and depressed lesions over part of their surface. Samples from two papaya plantations (107.54°E, 22.38°N) were collected, and brought to the laboratory. Symptomatic diseased tissues were cut into 5 × 5 mm pieces, surface sterilized with 2% (v/v) sodium hypochlorite for 1 minute, and rinsed three times with sterilized water. The pieces were then placed on potato dextrose agar (PDA). After incubation at 25°C in the dark for one week, colonies with uniform morphology were obtained. The aerial mycelium on PDA was white on top side, and concentric rings of salmon acervuli on the underside. A gelatinous layer of spores was observed on part of PDA plates after 7 days at 28°C. The conidia were elliptical, aseptate and hyaline (Zhang et al., 2020). The length and width of 60 conidia were measured for each of the two representative isolates, MG2-1 and MG3-1, and these averaged 13.10 × 5.11 μm and 14.45 × 5.95 μm. DNA was extracted from mycelia of these two isolates with the DNA secure Plant Kit (TIANGEN, Biotech, China). The internal transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS), β-tubulin 2 (TUB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) regions were amplified by PCR and sequenced. The sequences were deposited into GenBank with accessions MT904003, MT904004, and MT898650 to MT898659. BLASTN analyses against the GenBank database showed that they all had over 99% identity to the type strain of Colletotrichum siamense isolate ICMP 18642 (GenBank accession numbers JX010278, GQ856775, JX009709, GQ856730, JX010410, JX010019) (Weir et al., 2012). A phylogenetic tree based on the combined ITS, ACT, CAL, CHS, TUB2 and GAPDH sequences using the Neighbor-joining algorithm also showed that the isolates were C. siamense. Pathogenicity tests were conducted on 24 mature, healthy and surface-sterilized papaya fruits. On 12 papaya fruits, three well separated wounded sites were made for inoculation, and for each wounded site, six adjacent pinhole wounds were made in a 5-mm-diameter circular area using a sterilized needle. A 10 µl aliquot of 1 × 106 conidia/ml suspension of each of the isolates (MG2-1 and MG3-1) was inoculated into each wound. For each isolate, there were six replicate fruits. The control fruits were inoculated with sterile distilled water. The same inoculation was applied to 12 non-wound papaya fruits. Fruits were then placed in boxes which were first washed with 75% alcohol and lined with autoclaved filter paper moistened with sterilized distilled water to maintain high humidity. The boxes were then sealed and incubated at 28°C. After 10 days, all the inoculated fruits showed symptoms, while the fruits that were mock inoculated were without symptoms. Koch's postulates were fulfilled by re-isolation of C. siamense from diseased fruits. To our knowledge, this is the first report of C. siamense causing anthracnose of papaya in China. This finding will enable better control of anthracnose disease caused by C. siamense on papaya.

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First report of Anthracnose on Peach Fruit Caused by Colletotrichum siamense in Uruguay

Plant Disease ◽

10.1094/pdis-05-21-0950-pdn ◽

2021 ◽

Author(s):

María Julia Carbone ◽

Victoria Moreira ◽

Pedro Mondino ◽

Sandra Alaniz

Keyword(s):

South Carolina ◽

Prunus Persica ◽

Disease Incidence ◽

Management Strategies ◽

Control Treatment ◽

Fluorescent Light ◽

Peach Fruit ◽

First Report ◽

Anthracnose Disease ◽

Equidistant Points

Peach (Prunus persica L.) is an economically important deciduous fruit crop in Uruguay. Anthracnose caused by species of the genus Colletotrichum is one of the major diseases in peach production, originating significant yield losses in United States (Hu et al. 2015), China (Du et al. 2017), Korea (Lee et al. 2018) and Brazil (Moreira et al. 2020). In February 2017, mature peach fruits cv. Pavia Canario with symptoms resembling anthracnose disease were collected from a commercial orchard located in Rincon del Colorado, Canelones, in the Southern region of Uruguay. Symptoms on peach fruit surface were characterized as circular, sunken, brown to dark-brown lesions ranging from 1 to 5 cm in diameter. Lesions were firm to touch with wrinkled concentric rings. All lesions progressed to the fruit core in a V-shaped pattern. The centers of the lesions were covered by orange conidial masses. Monosporic isolates obtained from the advancing margin of anthracnose lesions were grown on PDA at 25ºC and 12h photoperiod under fluorescent light. The representative isolates DzC1, DzC2 and DzC6 were morphologically and molecularly characterized. Upper surface of colonies varied from white or pale-gray to gray and on the reverse dark-gray with white to pale-gray margins. Conidia were cylindrical, with both ends predominantly rounded or one slightly acute, hyaline and aseptate. The length and width of conidia ranged from 9.5 to 18.9 µm (x ̅=14.1) and from 3.8 to 5.8 µm (x ̅=4.6), respectively. The ACT, βTUB2, GAPDH, APN2, APN2/MAT-IGS, and GAP2-IGS gene regions were amplified and sequenced with primers ACT-512F/ACT-783R (Carbone and Kohn, 1999), BT2Fd/BT4R (Woudenberg et al. 2009), GDF1/GDR1 (Guerber et al. 2003), CgDLR1/ColDLF3, CgDLF6/CgMAT1F2 (Rojas et al. 2010) and GAP1041/GAP-IGS2044 (Vieira et al. 2017) respectively and deposited in the GenBank database (MZ097888 to MZ097905). Multilocus phylogenetic analysis revealed that Uruguayan isolates clustered in a separate and well supported clade with sequences of the ex-type (isolate ICMP 18578) and other C. siamense strains (isolates Coll6, 1092, LF139 and CMM 4248). To confirm pathogenicity, mature and apparently healthy peach fruit cv. Pavia Canario were inoculated with the three representative isolates of C. siamense (six fruit per isolate). Fruit were surface disinfested with 70% ethanol and wounded with a sterile needle at two equidistant points (1 mm diameter x 1 mm deep). Then, fruit were inoculated with 5 µl of a spore suspension (1×106 conidia mL-1) in four inoculation points per fruit (two wounded and two unwounded). Six fruit mock-inoculated with 5 µl sterile water were used as controls. Inoculated fruit were placed in moist chamber and incubated at 25°C during 10 days. Anthracnose lesions appeared at 2 and 4 days after inoculation in wounded and unwounded points, respectively. After 7 days, disease incidence was 100% and 67% for wounded and unwounded fruit, respectively. The control treatment remained symptomless. The pathogens were re-isolated from all lesions and re-identified as C. siamense. C. siamense was previously reported in South Carolina causing anthracnose on peach (Hu et al. 2015). To our knowledge, this is the first report of anthracnose disease on peach caused by C. siamense in Uruguay. Effective management strategies should be implemented to control anthracnose and prevent the spread of this disease to other commercial peach orchards.

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