First Report of Crown Rot of Banana Caused by Fusarium porliferatum in Georgia, USA

Crown rot is one of the most damaging disease of banana fruit characterized by rot and necrosis of crown tissues. In severe cases, the disease can spread to the pedicel and banana pulp. Crown rot can be infected by several common fungi, including Lasiodiplodia theobromae, Musicillium theobromae, Colletotrichum musae, and a complex of Fusarium spp. and lead to softening and blackening of tissues (Lassois et al., 2010; Kamel et al., 2016; Triest et al., 2016; Snowdon, 1990). In November 2020, typical crown rot of banana fruits (cv. Pisang Awak, belonging to the tetraploid AABB genome) were observed from UGA Banana Research 12 Plots, Tifton, GA, with incidence rates of 15%. Initial symptoms appeared in the infected crown of green banana fruits. As the infection progressed, the crown tissues became blackened and softened, followed by an internal development of infection affecting the peduncle and the fruit, triggered early ripening of bananas. At last, the development of necrosis on the pedicels and fruits appeared and caused the fingers to fall off. To identify the pathogen, tissue pieces (~0.25 cm2) from the infected crown and pedicles were surface-sterilized in a 10% bleach solution for 1 min, followed by 30 s in 70% EtOH. The disinfected tissues were rinsed in sterile water 3 times and cultured on potato dextrose agar (PDA) amended with 50 µg/ml streptomycin at 25°C in the dark for 5–10 days. Isolates of the pathogen were purified using the single-spore isolation method (Leslie and Summerell 2006). Colonies on PDA produced fluffy aerial mycelium and developed an intense purple pigment when viewed from the underside. A range of colony pigmentation and growth rates were observed among the isolates. The microconidia were ovoid, hyaline, or ellipse in shape. The morphological features of the isolates were identified as Fusarium proliferatum (Leslie and Summerell, 2006). To further identify the isolates, genomic DNA was extracted from a representative isolate. And the internal transcribed spacer (ITS) region, the partial elongation factor (TEF1-α) gene and the β-tubulin gene (TUB2)were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012), EF-1 /EF-2 (O’Donnell et al. 1998) and B-tub1 /B-tub2 (O’Donnell and Cigelnik, 1997), respectively. The amplicons were sequenced and deposited in NCBI (accessions no. MZ292989, MZ293071 for ITS: MZ346602, MZ346603 for TEF1-α and MZ346600 and MZ346601 for B-tub). The ITS, TEF1-α, and B-tub sequences of the isolates showed 100% sequence similarity with Fusarium proliferatum isolates (accessions no. MT560212, LS42312, and LT575130, respectively) using BLASTn in Genbank. For pathogenicity testing, three whole bunched bananas sterilized with 10% bleach solutions and washed by sterilized water, were cut into 5 bananas per brunch. The cut surface of the banana crown was inoculated with conidial suspension (1.0 × 107 cfu/ml) of the pathogen with pipette tips. Equal number of bananas were treated with sterilized water in the same volume as a control. All bananas were sealed in a plastic bag and incubated at 25°C. After 7 days post inoculation, all inoculated bananas showed initial crown rot symptoms while no symptoms were observed on the control bananas. The fungus was re-isolated from the symptomatic tissues of infected bananas and confirmed to be genetically identical to F. proliferatum of the original inoculated strains according to morphological characteristics and molecular identification, fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of F. proliferatum causing crown rot on bananas in Georgia, USA.

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First Report of Paraphoma chrysanthemicola Causing Crown and Leaf Rot of Atractylodes lancea in China

Plant Disease ◽

10.1094/pdis-11-21-2408-pdn ◽

2022 ◽

Author(s):

Hongyang Wang ◽

Chuanzhi Kang ◽

Wang Yue-Feng ◽

Sheng Wang ◽

Zhang Yan ◽

...

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Crown Rot ◽

Post Inoculation ◽

Conidial Suspension ◽

Sterile Water ◽

First Report ◽

Atractylodes Lancea ◽

Detached Leaves ◽

Leaf Rot

Atractylodes lancea is an important traditional Chinese medicinal plant whose rhizome is used for treating complaints such as rheumatic diseases, digestive disorders, night blindness and influenza. Jiangsu Province is the optimal cultivation location for high-quality A. lancea rhizome. Since June 2019, symptoms of crown rot and leaf rot were observed in about 10-20% of the A. lancea in a plantation (31° 36' 1" N, 119° 6' 40" W) in Lishui, Jiangsu, China. Lesions occurred on the stem near the soil line and on the leaves (Fig. 1A). Disease incidence reached approximately 80-90% by September, 2021 (Fig. 1B) and resulted in severe loss of rhizome and seed yields. For pathogen isolation, ten samples of symptomatic stem segments and ten diseased leaves were collected, surface-sterilized using 5% NaClO solution, rinsed with sterile water, cut into 0.5-2 cm segments, and plated to potato dextrose agar (PDA), and then incubated at 30°C in darkness. Pure cultures of four isolates showing morphological characteristics of Paraphoma spp. were obtained, identified as a single P. chrysanthemicola strain, and named LSL3f2. Newly formed colonies initially consisted of white mycelia; the five-day-old colonies developed a layer of whitish grey mycelia with a grey underside. 20-day-old colonies had white mycelium along the margin and with a faint yellow inner circular part with irregular radial furrows, and the reverse side looking caramel and russet (Fig. 1C). Pycnidia were subglobose (diameter: 5 to 15 μm; Fig. 1D). Unicellular, bicellular or strings of globose or subglobose chlamydospores developed from hyphal cells (Fig. 1E and 1F). The internal transcribed spacer (ITS) region and large subulin-28S of LSL3f2 were cloned using primers ITS1/ITS4 and LR0R/LR7 (Aveskamp et al. 2010, Li et al. 2013), and deposited in GenBank (OK559658 and OK598973, respectively). BLASTn search and phylogenetic analysis showed the highest identity between LSL3f2 and P. chrysanthemicola sequences (Fig. 1G) and confirmed LSL3f2 as P. chrysanthemicola. Koch’s postulates were completed using one-month-old vegetatively propagated A. lancea plantlets growing on autoclaved vermiculite/peat mixture at 26°C with a light/dark cycle of 12/12 hours. Each plantlet was inoculated with 5 ml of conidial suspension in water (1 × 108 cfu/ml) by applying to soil close to the plantlet, with sterile water used as a mock control (n = 10). By 20 days post-inoculation, inoculated plantlets showed a range of disease symptoms consistent to those observed in infested fields (Fig. 1H). Pathogenicity was additionally confirmed using detached leaves inoculated with a colonized agar plug of LSL3f2 or an uninoculated control comparison (diameter = 5 mm) and incubated at 26℃ in the dark. Five to seven days post-inoculation, detached leaves showed leaf rot symptoms including lesions, yellowing and withering consistent with those in infested fields, while control leaves remained healthy (n = 10, Fig. 1I). The pathogen was reisolated from the diseased plantlets and detached leaves, in both cases demonstrating the micromorphological characteristics of LSL3f2. P. chrysanthemicola has been reported to cause leaf and crown rot on other plants such as Tanacetum cinerariifolium (Moslemi et al. 2018), and leaf spot on A. japonicain (Ge et al. 2016). However, this is the first report of P. chrysanthemicola causing crown and leaf rot on A. lancea in China.

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First Report of Colletotrichum cereale Causing Anthracnose on Avena nuda in China

Plant Disease ◽

10.1094/pdis-10-20-2109-pdn ◽

2020 ◽

Author(s):

Na Zhao ◽

Junyu Yang ◽

Xiaoli Fang ◽

lingrui Li ◽

Hongfei Yan ◽

...

Keyword(s):

Morphological Characteristics ◽

Causal Agent ◽

Post Inoculation ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Fungal Isolates ◽

Colletotrichum Cereale ◽

Avena Nuda

Naked oats (Avena nuda L.) is rich in protein, fat, vitamin, mineral elements and so on, and is one of the world's recognized cereal crops with the highest nutritional and healthcare value. In July 2019, leaf spot was detected on A. nuda in Zhangbei experimental station of Hebei Agricultural University. The incidence of disease is 10% to 20%. The symptoms were similar to anthracnose disease, the infected leaves had fusiform or nearly fusiform yellowish-brown spots, yellow halo around the spots. Numerous acervuli with black setae diagnostic of fungi in the genus Colletotrichum were present on necrotic lesions. To identify the pathogen, ten symptomatic leaves were collected, and only one disease spot was isolated from each leaf. Small square leaf pieces (3 to 5 mm) were excised from the junction of diseased and healthy tissues with a sterile scalpel and surface disinfested with 75% alcohol for 30s, 0.1% corrosive sublimate for 1 min, rinsed three times in sterile water. Plant tissues were then transferred on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Two fungal isolates were obtained and purified by single-spore isolation method. All fungi have the same morphology and no other fungi were isolated. The aerial mycelium was gray black. The conidia were colorless and transparent, falcate, slightly curved, tapered toward the tips, and produced in acervuli with brown setae. The length and width of 100 conidia were measured and size ranged from 1.86 to 3.84 × 8.62 to 29.81 μm. These morphological characteristics were consistent with the description of Colletotrichum cereale (Crouch et al. 2006). To further assess the identity of the species, the genomic DNA of two fungal isolates (LYM19-4 and LYM19-10) was extracted by a CTAB protocol. The ribosomal DNA internal transcribed spacer (ITS) region as well as, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and the beta-tubulin 2 (Tub2) partial genes were amplified and sequenced with primers ITS4/5, GDF/GDR, ACT-512F/ACT-783R, and T1/Bt2b, respectively (Carbone et al. 1999; Templeton et al. 1992; O'Donnell et al. 1997; Glass et al. 1995). The sequences of the ITS-rDNA region (MW040121, MW040122), the GAPDH sequences (MW052554, MW052555), the ACT sequences (MW052556, MW052551) and the Tub2 sequences (MW052552, MW052553) of the two single-spore isolates were more than 99% identical to C. cereale isolate CGMCC3.15110 (JX625159, KC843517, KC843534 and JX625186). Maximum likelihood tree based on concatenated sequences of the four genes were constructed using MEGA7. The results showed the strains isolated from A. nuda were closely related to C. cereale, as supported by high bootstrap values. A pathogenicity test of the C. cereale isolates was performed on first unfolding leaves of A. nuda. Koch's postulates were carried out with isolates by spraying a conidial suspension of 106 conidia/mL on leaves of healthy A. nuda. Four replicated pots were inoculated at a time, 10 leaves each pot, while sterile distilled water was used as the control. All treated plants were placed in a moist chamber (25°C, 16-h light and 8-h dark period). Anthracnose symptoms developed on the inoculated plants 7 days post inoculation while all control plants remained healthy. Microscopic examination showed the surface of infected leaves had the same acervuli, setae, and conidia as the original isolate. The pathogenicity test was repeated three times. C. cereale was previously reported as the causal agent of anthracnose on feather reed grass in US (Crouch et al. 2009). To our knowledge, this is the first report of C. cereale as the causal agent of A. nuda anthracnose in China.

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First Report of Ergot (Claviceps purpurea) on Drunken Horse Grass (Achnatherum inebrians) in China

Plant Disease ◽

10.1094/pdis-12-20-2649-pdn ◽

2021 ◽

Author(s):

Haijuan Zhang ◽

Taixiang Chen ◽

Yali He ◽

Chunjie Li

Keyword(s):

Molecular Characterization ◽

Morphological Characteristics ◽

Post Inoculation ◽

Conidial Suspension ◽

Claviceps Purpurea ◽

Large Area ◽

Single Spore ◽

First Report ◽

Achnatherum Inebrians ◽

Natural Grasslands

Drunken horse grass (Achnatherum inebrians) belongs to the family Poaceae: it is mainly distributed in the natural grasslands of northern and northwestern in China. Ergot is a disease that can not only affect the growth of the grass, but also cause livestock poisoning (Coufal-Majewski et al. 2016). In September 2018, ergot was observed in a large area (about 15 ha) in Xinghai county, Qinghai province, China (35° 47′ N, 99° 53′ E, Altitude 3559 m). Around 65% of the plants of Achnatherum inebrians were affected. Symptoms initially showed drop-like honeydew on the ears of drunken horse grass, and later brown to dark brown sclerotia were observed. These were straight to slightly curved, measured 6.7 to 13.5 × 1.5 to 2.1 mm, which was approximately 1 to 4 times the size of healthy seeds. Sixteen spikes with typical symptoms were collected from eight different fields. Sclerotia were disinfested by immersion in 75% ethanol for 30 s and 1% NaClO for 90 s, rinsed three times in sterilized water, plated on potato dextrose agar (PDA) medium, incubated at 24°C in the dark and isolates purified by culturing from single spores. Finally, 16 single-spore cultures with similar phenotypes were obtained from these sclerotia. Colonies produced on PDA for 15 days at 24 ℃ were grayish white with fluffy aerial mycelium, about 60 mm in diameter. Conidia were hyaline, ovoid to cylindrical, 5.42 to 7.69 × 2.85 to 3.75 μm (avg. 5.67 × 3.2; n = 50). These morphological characteristics were consistent with the descriptions of Claviceps species in general (PíchovÁ et al. 2018). To further identify the Claviceps spp., isolate NSZJ (=MHLZU-AI20201012) was selected as a representative for molecular characterization. Two nuclear protein-coding genes TUB2 and MCM7 were amplified by T2/T12 (O’Donnell and Cigelnik 1997) and CARCA-F/M456-5R (Rehner and Buckley 2005), respectively, and sequenced. Sequences were deposited in GenBank (accession nos. MW115640 for TUB2 and MW115641 for MCM7). A BLAST analysis of these two segments showed >99% identity with those sequences of isolate W3 of C. purpurea (Pazoutová et al. 2014). To confirm the pathogenicity on drunken horse grass, 20 healthy plants (2-year-old) grown in an experimental field at the College of Pasture Agriculture Science and Technology, Yuzhong Campus of Lanzhou University in China (104° 39′ E, 35° 89′ N, altitude1653m) were spray-inoculated with conidial suspension (1 × 106 conidia/ml) during the flowering period. Another 20 plants in the field were sprayed with sterilized distilled water as controls. All plants were individually covered with transparent polyethylene bags for 24 h to maintain high relative humidity. After 7 to 10 days, small yellowish-white drops of honeydew were observed in some florets. At 22 days post inoculation, all the inoculated panicles developed three to six sclerotia per head, ranging in size from 6.7 to 13.5 mm, while control plants remained healthy. The same pathogen was consistently re-isolated from inoculated spikes and confirmed by morphological and molecular characterization as described above. Claviceps purpurea was reported to be associated with ergot in A. lemmonii in Idaho and A. robustum in Montana (Alderman et al. 2004). To our knowledge, this is the first report of C. purpurea causing ergot in A. inebrians in China.

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First Report of Anthracnose of Alocasia macrorrhiza Caused by Colletotrichum karstii in Guangdong, China

Plant Disease ◽

10.1094/pdis-10-13-1046-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 696-696 ◽

Cited By ~ 1

Author(s):

Y. He ◽

C. Shu ◽

J. Chen ◽

E. Zhou

Keyword(s):

Morphological Characteristics ◽

Pcr Amplification ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Leaf Spots ◽

Purification Technique ◽

Initial Symptoms

Alocasia macrorrhiza (L.) Schott. (Araceae), native to South America, is a common, herbaceous perennial ornamental plant in tropical and subtropical areas (1). A severe leaf spot disease was observed on this plant in several places on the campus of authors' university in Guangzhou, Guangdong Province, China, in April 2013. Initial symptoms were water-soaked, dark green leaf spots. These small spots gradually expanded to 6- to 11-mm circular lesions. They were grayish-white in color with a yellow halo and many small, black, concentric dots were observed on them. Microscopic examination revealed that these small dots were acervuli, which were 100 to 300 μm in diameter, developing beneath the epidermis and becoming erumpent with age. By using routine tissue-isolation method and single-spore purification technique, four single-conidial isolates were obtained from each of four diseased leaves. These isolates formed a grayish-white colony with numerous pink spore masses on PDA at 28°C. Their mycelial radial growth rate was about 4.5 mm per day. Conidia were single-celled, hyaline, and cylindrical with an obtuse apex and protruding base; they were 12.7 to 14.2 × 4.8 to 5.9 μm in size. Conidial appressoria were irregular in shape, sepia to dark brown, solitary, and 6.9 to 8.5 × 4.6 to 5.9 μm. These morphological characteristics were consistent with the description of Colletotrichum karstii (2). The sequences of beta-tubulin gene (TUB2) and partial actin gene (ACT) of a representative isolate CAM1 were obtained by PCR amplification with primers BT2a/BT2b and ACT512F/ACT783R, respectively. These sequences were deposited in GenBank under the accession numbers of KF444947 and KF460435. BLAST searches showed a 99% homology with the TUB2 and ACT sequences of C. karstii (JX625209, KC843559). Therefore, the fungus isolated from A. macrorrhiza was identified as C. karstii by morphological and molecular characteristics. Pathogenicity tests were performed on 30-day-old plants of A. macrorrhiza grown in plastic pots (0.8 L) by spraying 15 ml conidial suspension (1 × 106 conidia ml–1) of this fungus onto each plant. The control plants were sprayed only with sterile distilled water. These plants then were placed in an intelligent artificial climate incubator with 12-h photoperiod and 100% relative humidity at 24 ± 1°C. Three replicates, each with five plants, were included in a test, and the test was repeated twice. Seven days after inoculation, the inoculated plants showed necrotic lesions on leaves similar to those observed on the campus, but no symptoms were observed on any non-inoculated controls. The same fungus C. karstii was re-isolated from the infected leaves. Although C. karstii is a well-known anthracnose pathogen on some plants belonging to family Orchidaceae (2), this is the first report of the same pathogen causing anthracnose on A. macrorrhiza in Guangdong, China. References: (1) S. Li et al. PLoS ONE 8(6):e66016, 2013. (2) Y. Yang et al. Cryptogr. Mycol. 32:229, 2011.

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First Report of Anthracnose Caused by Colletotrichum fructicola on Hybrid Pear Fruit in Korea

Plant Disease ◽

10.1094/pdis-09-20-1866-pdn ◽

2021 ◽

Author(s):

Eu Ddeum Choi ◽

Sook-Young Park

Keyword(s):

Control Strategies ◽

Morphological Characteristics ◽

Likelihood Method ◽

Pyrus Pyrifolia ◽

Post Inoculation ◽

Conidial Suspension ◽

Pear Fruit ◽

Single Spore ◽

First Report ◽

Other Information

In August 2020, anthracnose-like symptoms was observed on pear fruit (Pyrus pyrifolia  P. communis) cultivated at 0.2 ha by the National Institute of Horticultural and Herbal Science Pear Research Institute at the Rural Development Administration (Naju, Jeonnam Province in Korea). Symptoms were observed only on fruit (112 days after full bloom (DAFB)), and disease incidences was at least 90%. Initial black specks developed into larger brown or black lesions on fruit after 3 days. Later, sunken lesions with orange conidial masses were observed. Finally, infected fruit dropped prematurely. To isolate and identify the pathogen, small pieces (5  5 mm) from the margin of lesions on fruit were surface sterilized by immersing in 70% ethanol for 1 minute, washed three times with sterile water, dried, and placed on water agar amended with 100 ppm streptomycin, then incubated in the dark at 25°C. Hyphae emerging from the three independent tissues were subcultured on Potato Dextrose Agar (PDA), resulting in three independent isolates (CP-1, CP-2, CP-3) after single spore isolation. Colonies were pale gray on PDA, but the colony edges were white. Conidia were transparent, cylindrical with rounded ends, and 13.8 to 20.1 μm  4.8 to 6.2 μm (avg. 18.3 μm  5.4 μm, n = 100) in size. Appressoria were dark brown, globose or subcylindrical, and 6.3 to 9.5 μm  5.2 to 6.9 μm in size (8.1  6.1 μm, n = 100). The morphological characteristics were similar to the descriptions of C. gloeosporioides species complex (Weir et al. 2012). Sequences of ITS (MT921589-91), GAPDH (MT921987-89), CAL (MT921990-92), ACT (MT921993-95), CHS-1 (MT921996-98), TUB2 (921999-01), and ApMAT (MT922002-04) sequences from CP-1, CP-2, and CP-3 matched with C. fruiticola strain BRIP 62871 (100%; MK298285), HXQT-2 (100%; MN52588), HXQT-2 (100%; MN52839), HXQT-2 (99.65; MN525801), ICKP18B4 (99.34%; LC494275), HB5 (100%; MH985245), and GQHZJ23 (100%; MN338294), respectively. Concatenated gene sequences were used for a phylogenetic analysis based on the maximum likelihood method. The reference gene accessions and other information are presented in Weir et al. (2012). The analysis placed the isolates within a clade comprising C. fructicola. Pathogenicity of CP-1 was tested using 120 healthy pear fruits. The fruit surfaces were sterilized with 70% ethyl alcohol for 2 min and washed twice with sterilized water. Three 120 DAFB fruits were inoculated with 10 l of a conidial suspension (1×106 conidia/ml) with and without wounding. Another three control fruits were inoculated with sterile distilled with and without wounding. The inoculated fruit were placed in a plastic box to maintain high humidity and incubated in the dark at 25°C. Symptoms were observed on both wounded fruits after 3 days post inoculation (dpi) and 5 dpi on the unwounded fruits. No symptoms were observed in the control on both the wounded fruits. Pathogenicity tests was performed in duplicate. The pathogen was re-isolated from symptomatic tissues (100%) on treatments on both the wounded and unwounded fruits, but not control. The identity of the both re-isolated pathogen from the wounded and unwounded fruits was confirmed via analysis of seven genes and morphological characteristics, thus fulfilling Koch’s postulates. Although C. fructicola has been reported on apples and peaches in Korea (Kim et al. 2018; Lee et al. 2020), this is the first report of anthracnose caused by C. fructicola on pear fruit in Korea, highlighting the need for systematically investigating the diversity and incidence of pear anthracnose in Korea. This study will contribute to the development of control strategies for anthracnose disease on pear fruit in Korea.

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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 Leaf Spot Caused by Botrytis cinerea on Cardamine hupingshanensis in China

Plant Disease ◽

10.1094/pdis-04-21-0698-pdn ◽

2021 ◽

Author(s):

Jun Guo ◽

Jin Chen ◽

Zhao Hu ◽

Jie Zhong ◽

Jun Zi Zhu

Keyword(s):

Botrytis Cinerea ◽

Morphological Characteristics ◽

Gray Mold ◽

Hunan Province ◽

Conidial Suspension ◽

Heat Shock Protein 60 ◽

Basal Cells ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

First Report

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

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First Report of Leaf Spot Caused by Ascochyta marginata on Aralia elata in Korea

Plant Disease ◽

10.1094/pdis-08-11-0658 ◽

2012 ◽

Vol 96 (1) ◽

pp. 147-147

Author(s):

S. H. Lee ◽

C. K. Lee ◽

M. J. Park ◽

H. D. Shin

Keyword(s):

East Asia ◽

Leaf Spot ◽

Morphological Characteristics ◽

Culture Collection ◽

Research Report ◽

Conidial Suspension ◽

First Report ◽

Commercial Cultivation ◽

Aralia Elata ◽

Initial Symptoms

Aralia elata (Miq.) Seem., known as Japanese angelica tree, is a deciduous shrub belonging to the Araliaceae, which is native to East Asia. The young shoots have long been used in various dishes in East Asia. Commercial cultivation of this shrub, especially in polytunnels, is expanding in Korea. Several diseases including Sclerotinia rot have been known to be present on this plant (1,2). In early September 2007, leaf spot symptoms were first observed on several trees in Hongcheon, Korea. Microscopic observations revealed that the leaf spots were associated with an Ascochyta sp. Further surveys of the Ascochyta leaf spot showed the occurrence of the disease in approximately 5 to 10% of the trees in the 3 ha of commercial fields surveyed in Chuncheon, Gapyeong, Inje, and Jinju, Korea. Initial symptoms on leaves were circular to irregular, brown to dark brown, becoming zonate, and finally fading to grayish brown in the center with a yellow halo. Representative samples were deposited in the herbarium of Korea University. Conidiomata on leaf lesions were pycnidial, amphigenous, but mostly epiphyllous, immersed or semi-immersed in host tissue, light brown to olive brown, and 60 to 200 μm in diameter. Ostioles were papillate, 20 to 35 μm wide, and surrounded by a ring of darker cells. Conidia were hyaline, smooth, cylindrical to clavate, straight to mildly curved, slightly constricted at the septa, medianly one-septate, sometimes aseptate, 8 to 16 × 2.5 to 3.5 μm, and contained small oil drops. These morphological characteristics were consistent with the previous reports of Ascochyta marginata J.J. Davis (3,4). A monoconidial isolate was cultured on potato dextrose agar (PDA) plates and accessioned in the Korea Agricultural Culture Collection (Accession KACC43082). The conidia were readily formed on PDA. Inoculum for the pathogenicity tests was prepared by harvesting conidia from 30-day-old cultures of KACC43082 and a conidial suspension (approximately 2 × 106 conidia/ml) was sprayed onto leaves of three healthy seedlings. Three noninoculated seedlings served as controls. Inoculated and noninoculated plants were covered with plastic bags for 48 h in a glasshouse. After 7 days, typical leaf spot symptoms started to develop on the leaves of the inoculated plants. The fungus, A. marginata, was reisolated from those lesions, confirming Koch's postulates. No symptoms were observed on control plants. Previously, the disease was reported in Japan (4) and China (3). To our knowledge, this is the first report of A. marginata on Japanese angelica trees in Korea. According to our field observations in Korea, the Ascochyta leaf spot mostly occurred on plants growing in a humid environment, especially during the rainy season. The seedlings as well as the trees growing in sunny, well-ventilated plots were nearly free from this disease. Therefore, the growing conditions seemed to be the most important factor for the development and severity of the disease. References: (1) C. K. Lee et al. Plant Pathol. J. 26:426, 2010. (2) S. H. Lee et al. Diseases of Japanese Angelica Tree and Their Control. Research Report 08-10. Korea Forest Research Institute. Seoul, Korea, 2008. (3) J. Sun et al. Acta Mycol. Sin. 14:107, 1995. (4) M. Yoshikawa and T. Yokoyama. Mycoscience 36:67, 1995.

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First Report of Nigrospora Leaf Blight on Sesame Caused by Nigrospora sphaerica in China

Plant Disease ◽

10.1094/pdis-07-13-0684-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 842-842 ◽

Cited By ~ 3

Author(s):

H. Zhao ◽

H. Y. Liu ◽

X. S. Yang ◽

Y. X. Liu ◽

Y. X. Ni ◽

...

Keyword(s):

Morphological Characteristics ◽

Leaf Blight ◽

Morphological Data ◽

Its2 Region ◽

Oilseed Crop ◽

Conidial Suspension ◽

First Report ◽

Initial Symptoms ◽

Central Regions ◽

Leaf Pathogen

Sesame (Sesamum indicum L.) is an important oilseed crop widely grown in the central regions of China. A new leaf blight has increasingly been observed in sesame fields in Anhui, Hubei, and Henan provinces since 2010. Approximately 30 to 40% of the plants were symptomatic in the affected fields. Initial symptoms were yellow to brown, irregularly shaped lesions. Lesions later expanded and the affected leaves tuned grayish to dark brown and wilted, with a layer of whitish mycelial growth on the underside. Severe blighting caused the center of lesions to fall out, leaving holes in the leaves. Sections of symptomatic leaf tissues were surface-sterilized in 75% ethanol for 30 s, then in 1% HgCl2 for 30 s, rinsed three times in sterile distilled water, and plated onto potato dextrose agar (PDA). The resulting fungal colonies were initially white, and then became grayish-brown with sporulation. Conidia were single-celled, black, smooth, spherical, 14.2 to 19.8 μm (average 17.1 μm) in diameter, and borne on a hyaline vesicle at the tip of each conidiophore. Morphological characteristics of the isolates were similar to those of Nigrospora sphaerica (1). To verify the identification based on morphological features, the ITS1-5.8S-ITS2 region of the ribosomal RNA was amplified using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers (3), and then sequenced and compared to the GenBank database through a BLAST search. Comparison of the sequence revealed 100% similarity to N. sphaerica (GenBank Accession No. JF817271.1). On the basis of morphological data and the ITS rDNA sequence, the isolate was determined to be N. sphaerica. Pathogenicity tests were conducted using fresh and healthy sesame leaves of 10 plants. A conidial suspension (106 conidia/ml) collected from a 7-day-old culture on PDA was used for inoculation. Leaves of 10 plants were spray-inoculated with the spore suspension at the 6-week-old growth stage, and an additional 10 plants were sprayed with sterile water. Inoculated plants were covered with polyethylene bags to maintain high humidity. Plants were kept at 28°C and observed for symptom every day. Ten to 15 days after inoculation, inoculated leaves developed blight symptoms similar to those observed on naturally infected leaves. No symptoms were observed on the control leaves. N. sphaerica was re-isolated from the inoculated leaves, thus fulfilling Koch's postulates. N. sphaerica has been reported as a leaf pathogen on several hosts worldwide (2). To our knowledge, this is the first report of Nigrospora leaf blight on sesame caused by N. sphaerica in China. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ . July 01, 2013. (3) M. A. Innis et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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First Report of Nigrospora Leaf Blight on Tea Caused by Nigrospora sphaerica in India

Plant Disease ◽

10.1094/pdis-05-14-0545-pdn ◽

2015 ◽

Vol 99 (3) ◽

pp. 417-417 ◽

Cited By ~ 11

Author(s):

J. Dutta ◽

S. Gupta ◽

D. Thakur ◽

P. J. Handique

Keyword(s):

Camellia Sinensis ◽

West Bengal ◽

Morphological Characteristics ◽

Its Region ◽

Causative Organism ◽

Distilled Water ◽

Conidial Suspension ◽

Tea Leaves ◽

First Report ◽

Initial Symptoms

Tea [Camellia sinensis (L.) O. Kuntze] is an economically important non-alcoholic caffeine-containing beverage crop widely cultivated for leaves in India, especially in the Darjeeling district of West Bengal. In May 2012, distinct blight symptoms were observed on leaves of popular tea cultivars AV-2, Tukdah 78, Rungli Rungliot 17/144, and Bannockburn 157 in commercial tea estates of the Darjeeling district. This disease reduces yield and quality of the leaves. The initial symptoms were frequently observed on the young leaf margins and apices. Foliar symptoms are characterized by grayish to brown, semicircular or irregular shaped lesions, often surrounded by pale yellow zones up to 9 mm in diameter. The lesions later expand and the affected leaves turn grayish to dark brown and eventually the dried tissue falls, leading to complete defoliation of the plant. The disease causes damage to leaves of all ages and is severe in young leaves. A portion of the symptomatic leaf tissues were surface sterilized in 70% ethanol for 30 s, then in 2% NaClO for 3 min, rinsed three times in sterile distilled water, and plated onto potato dextrose agar (PDA). The fungal colonies were initially white and then became grayish to brown with sporulation. Conidia were spherical to sub spherical, single-celled, black, 19 to 21 μm in diameter, and were borne on a hyaline vesicle at the tip of each conidiophore. Morphological characteristics of the isolates were concurring to those of Nigrospora sphaerica (1). Moreover, the internal transcribed spacer (ITS) region of the ribosomal RNA was amplified by using primers ITS1 and ITS4 and sequenced (GenBank Accession No. KJ767520). The sequence was compared to the GenBank database through nucleotide BLAST search and the isolate showed 100% similarity to N. sphaerica (KC519729.1). On the basis of morphological characteristics and nucleotide homology, the isolate was identified as N. sphaerica. Koch's postulates were fulfilled in the laboratory on tea leaves inoculated with N. sphaerica conidial suspension (106 conidia ml−1) collected from a 7-day-old culture on PDA. Six inoculated 8-month-old seedlings of tea cultivars AV-2 and S.3/3 were incubated in a controlled environment chamber at 25°C and 80 to 85% humidity with a 12-h photoperiod. In addition, three plants of each cultivar were sprayed with sterile distilled water to serve as controls. Twelve to 14 days after inoculation, inoculated leaves developed blight symptoms similar to those observed on naturally infected tea leaves in the field. No symptoms were observed on the control leaves. The pathogen was re-isolated from lesions and its identity was confirmed by morphological characteristics. It was reported that N. sphaerica is frequently encountered as a secondary invader or as a saprophyte on many plant species and also as a causative organism of foliar disease on several hosts worldwide (2,3). To our knowledge, this is first report of N. sphaerica as a foliar pathogen of Camellia sinensis in Darjeeling, West Bengal, India, or worldwide. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, Syst. Mycol. Microbiol. Lab., ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ July 01, 2013. (3) E. R. Wright et al. Plant Dis. 92:171, 2008.

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