First report of root rot caused by Fusarium armeniacum on soybean in Korea

Fusarium wilt samples were collected in 2017 and 2019 from two soybean (Glycine max) fields, Yesan (36°73′N, 126°81′E) and Gimje (35°76′N, 126°80′E), in Korea. The disease incidence rate in each field was approximately 1%. For fungal isolation, root lesion fragments were surface-sterilized in 1% sodium hypochlorite for 2 min, rinsed thrice with sterile distilled water, and then incubated on water agar (WA) plates at 28 °C in an incubator for 5 days. Two isolates (YS37231 and GJ3050) were obtained using the hyphal tip method. Colonies of the isolates on potato dextrose agar (PDA) produced white aerial mycelia, which later turned pinkish yellow. The isolates on PDA formed abundant chlamydospores and macroconidia, but microconidia were absent. Macroconidia were 3–5 septate and prominently curved, measuring 12.4 to 41.2 × 3.3 to 4.3 µm (Leslie and Summerell, 2006). For the identification of the isolates, translation elongation factor 1 alpha (EF-1α) and RNA polymerase second largest subunit (RPB2) regions were amplified and sequenced using EF1, EF2, RPB2-5f2, and RPB2-7cr primers, respectively (O’Donnell et al. 2010). EF-1α sequences of YS37231 (MT445439) and GJ3050 (MT445440) showed 99.01 and 99.67% identity with F. armeniacum (FD_01843 and FD_01305; FUSARIUM-ID database), respectively. The RPB sequences of YS37231 (MT445442) and GJ3050 (MT445441) showed 100 and 98.48% identity with that of F. armeniacum (FD_01869; FUSARIUM-ID database), respectively. The sequences MT445439, MT445440, MT445441, and MT445442 were deposited in NCBI GenBank. The pathogenicity of the two isolates on the soybean cultivar Daewonkong was determined using two inoculation methods. In the first method, a pathogenicity assay was performed on seedlings using WA plates (Cruz Jimenez et al. 2018). Eight surface-sterilized seeds were transferred to WA plates, with or without actively growing cultures, for 3 days; and then incubated at 25 °C in a growth chamber (12 h photoperiod) for 7 days. After 7 days, brown lesions were observed on the roots in inoculated plates; however, no symptoms were observed in the control. -In the second method, 10-day old soybean seedlings were inoculated by cutting and soaking the roots in the spore suspension (1 × 106 conidia/mL) for 2 h. The inoculum was prepared by incubating isolates on PDA for 10 days, then adding sterile distilled water, scraping the growth medium, and filtering the suspension. The seedlings were then transplanted into 18 cm plastic pots (20 cm height) and grown under greenhouse conditions (26 °C ± 3 °C, 13 h photoperiod) for 2 weeks. After 7 days, the inoculated plants showed wilting symptoms, developed brown lesions in the roots, and eventually died within 2 weeks after inoculation. No such symptoms were observed in the control (inoculated with sterile distilled water). The isolates were re-isolated from the inoculated seedlings for confirmation. Although the fungus and associated mycotoxins have been reported in the rice produced in Korea (Hong et al. 2015), to the best of our knowledge, this is the first report of F. armeniacum causing Fusarium wilt on soybean in Korea. In the US, it was first reported by Ellis et al. (2012). Fusarium wilt is a soilborne disease of growing concern in soybean cultivation worldwide. Our findings will help increase awareness about the global spread of this disease.

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First Report of Phomopsis longicolla Causing Stem Canker of Eggplant in Guangdong, China

Plant Disease ◽

10.1094/pdis-07-13-0790-pdn ◽

2014 ◽

Vol 98 (3) ◽

pp. 426-426 ◽

Cited By ~ 4

Author(s):

C. Shu ◽

J. Chen ◽

H. Huang ◽

Y. He ◽

E. Zhou

Keyword(s):

Disease Incidence ◽

Guangdong Province ◽

Stem Canker ◽

Universal Primers ◽

Distilled Water ◽

Conidial Suspension ◽

Phomopsis Longicolla ◽

Fungal Isolation ◽

First Report ◽

Absorbent Cotton

Eggplant (Solanum melongena L.) is an economically important vegetable crop worldwide. In August 2012, severe stem cankers were observed on eggplant at the early stage of maturation in several fields in Guangdong Province, China. Diseased plants raised cankers on the stems and branches, which resulted in wilting and stunting. No symptoms developed on eggplant fruit. Disease incidence was as high as 40% within affected fields. By using routine fungal-isolation methods and single-spore purification technique, five single-conidial isolates were obtained from each diseased stem. Colonies were grayish-white, circular, and got yellow pigmentation when placed in acidified potato dextrose agar (PDA) in an incubator at pH 4.5 and 25°C with a 12-h photoperiod. Stromata were black, large, and spreading in a concentric pattern. Conidiomata were pycnidial, and the pycnidia were round, oblate, triangular or irregular, and unilocular. Conidiophores were colorless, separated, dichotomous, and 10.0 to 18.0 × 1.5 to 2.0 μm. Alpha conidia were single-celled, ellipsoidal to fusiform, guttulate, and 6.0 to 8.0 × 2.0 to 2.5 μm. Beta conidia, produced on oat meal agar in 2 weeks at 25°C in the dark, were filiform, hamate, and 16.0 to 28.0 × 0.7 to 1.0 μm. Based on these morphological characters, the fungus was identified as Phomopsis longicolla Hobbs (1). The ITS-rDNA sequence (GenBank Accession No. KC886605) of the isolate EPPL1 of this fungus (P. longicolla EPPL1) was obtained by using universal primers ITS5/ITS4 (1). BLAST searches showed a 98% homology with the sequence of the ITS region of rDNA of P. longicolla. Phylogenetic analysis showed that P. longicolla EPPL1 clustered with P. longicolla SYJM15 and formed a distinct clade distantly related to P. vexans PV3 (GU373630), a well-known pathogen of eggplant. Digestion of PCR-amplified DNA with Alu I yielded two restriction fragments of sizes consistent with those reported for P. longicolla (2). Pathogenicity tests were performed on 30-day-old plants of cv. Yuefengzihongqie grown in a plastic pot (1 liter) in a greenhouse by using mycelial plugs and conidial suspensions of isolate EPPL1 as inocula. A mycelial plug (4 mm in diameter) from a 7-day-old PDA culture was placed on stems of both wounded and non-wounded plants and covered with sterile absorbent cotton moistened with sterile distilled water. Both wounded and non-wounded plants were inoculated with 0.5 ml of conidial suspension (1 × 106 conidia ml–1) dropped onto sterile absorbent cotton covering the stems. Control assays were performed with agar plugs and sterile distilled water only. Inoculated plants were placed in a greenhouse with a 12-h photoperiod at 28°C. Each treatment was replicated on five plants, and the test was repeated. Twenty-five days after inoculation, both wounded and non-wounded plants inoculated with either method showed raised cankers at the points of inoculation and canker lesions similar to those observed in the field expanded up and down the stems to reach lengths of 15 to 30 mm. Later, sparse, small, black pycnidia formed on the surface of the lesions. The inoculated plants exhibited stunting and premature senescence compared to controls. P. longicolla was re-isolated from the infected stems of inoculated plants. Control plants were asymptomatic. To our knowledge, this is the first report of P. longicolla causing stem canker in eggplant in Guangdong, China. Considering the economic importance of eggplant in Guangdong Province and throughout the world, further study of phomopsis stem canker of eggplant is warranted. References: (1) T. W. Hobbs et al. Mycologia 77:535, 1985. (2) A. W. Zhang et al. Plant Dis. 81:1143, 1997.

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First Report of Fusarium wilt disease on Watermelon Caused by Fusarium oxysporum f. sp. niveum (FON) in Malaysia

Plant Disease ◽

10.1094/pdis-04-21-0780-pdn ◽

2021 ◽

Author(s):

Muhammad Ziaur Rahman ◽

Khairulmazmi Ahmad ◽

Yasmeen Siddiqui ◽

Norsazilawati Saad ◽

Tan Geok Hun ◽

...

Keyword(s):

Fusarium Oxysporum ◽

Fusarium Wilt ◽

Cover Crops ◽

Fungal Pathogen ◽

Disease Incidence ◽

Wilt Disease ◽

Likelihood Method ◽

Distilled Water ◽

First Report ◽

Fusarium Wilt Disease

Fusarium wilt disease incited by Fusarium oxysporum f. sp. niveum (FON) is the utmost devastating soil-inhabiting fungal pathogen limiting watermelon (Citrullus lanatus) production in Malaysia and globally. The field disease survey of fusarium wilt was carried out during December 2019 and November 2020, in three major production areas (3 farmer fields per location) in Peninsular Malaysia namely, Mersing, Serdang and Kuantan and disease incidence of 30 and 45%, was recorded for each year, respectively. Infected watermelon plants showed symptoms such as vascular discoloration, brown necrotic lesions to the soil line or the crown, one-sided wilt of a plant, or a runner or the whole plant. Infected root and stem tissues, 1-2 cm pieces were surface sterilized with 0.6% NaOCl for 1 minute followed by double washing with sterile water. The disinfected tissues were air-dried and transferred onto semi-selective Komada’s medium (Komada 1975) and incubated for 5 days. The fungal colonies produced were placed on potato dextrose agar (PDA) to attain a pure culture and incubated at 25±2℃ for 15 days. The pure fungal colony was flat, round and light purple in color. Macroconidia were straight to slightly curved, 18.56-42.22 µm in length, 2.69-4.08 µm width, predominantly 3 septate and formed in sporodochia. Microconidia measured 6.16-10.86 µm in length and 2.49-3.83 µm in width, kidney-shaped, aseptate and were formed on short monophialides in false-heads. Chlamydospores were single or in pairs with smooth or rough walls, found both terminally or intercalary. To confirm their pathogenicity, two-week-old watermelon seedlings (cv. NEW BEAUTY) were dipped into spore suspension (1 ˟ 106 spores/ml) of representative isolates of JO20 (Mersing), UPM4 (Serdang) and KU41 (Kuantan) for 30 second and then moved into 10 cm diameter plastic pots containing 300 g sterilized soil mix. Disease symptoms were assessed weekly for one month. Control seedlings were immersed in sterile distilled water before transplanting. The inoculated seedlings showed typical Fusarium wilt symptoms like yellowing, stunted growth, and wilting, which is similar to the farmer field infected plants. However, the seedlings inoculated by sterile distilled water remained asymptomatic. The pathogen was successfully re-isolated from the infected seedlings onto Komada’s medium, fulfilling the Koch’s postulate. For the PCR amplification, primers EF-1 and EF-2 were used to amplify the tef1-α region. A Blastn analysis of the tef1-α sequences of the isolates JO20 (accession nos. MW315902), UPM4 (MW839560) and KU41 (MW839562) showed 100% similarity; with e-value of zero, to the reference sequences of F. oxysporum isolate FJAT-31690 (MN507110) and F. oxysporum f. sp. niveum isolate FON2 790-2 (MN057702). In Fusarium MLST database, isolates JO20, UPM4 and KU41 revealed 100% identity with the reference isolate of NRRL 22518 (accession no. FJ985265). Though isolate FJ985265 belongs to the f. sp. melonis, earlier findings had revealed Fusarium oxysporum f. sp. are naturally polyphyletic and making clusters with diverse groups of the Fusarium oxysporum species complex (O’Donnell et al. 2015). The isolates JO20, UPM4 and KU41 were identified as F. oxysporum f. sp. niveum based on the aligned sequences of tef1-α and molecular phylogenetic exploration by the maximum likelihood method. To the best of our knowledge, this is the first report of F. oxysporum f. sp. niveum as a causative pathogen of Fusarium wilt disease of watermelon in Malaysia. Malaysia enables to export watermelon all-year-round in different countries like Singapore, Hong-Kong, The United Arab Emirates (UAE), and Netherlands. The outburst of this destructive soil-borne fungal pathogen could cause hindrance to watermelon cultivation in Malaysia. Thus, growers need to choice multiple management tactics such as resistant varieties, cultural practices (soil amendments and solarization), grafting, cover crops and fungicide application to control this new pathogen.

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First Report of Fusarium Wilt of Paper Flower (Bougainvillea glabra) Caused by Fusarium oxysporum in Italy

Plant Disease ◽

10.1094/pdis-94-4-0483a ◽

2010 ◽

Vol 94 (4) ◽

pp. 483-483 ◽

Cited By ~ 3

Author(s):

G. Polizzi ◽

D. Aiello ◽

V. Guarnaccia ◽

A. Vitale ◽

G. Perrone ◽

...

Keyword(s):

Fusarium Oxysporum ◽

Fusarium Wilt ◽

Disease Incidence ◽

Elongation Factor ◽

Gene Sequences ◽

Single Spore ◽

Potential Threat ◽

First Report ◽

Potted Plants ◽

Bougainvillea Glabra

Paper flower (Bougainvillea glabra Choisy), native to Brazil, is the most widely and intensively cultivated species of bougainvillea as a potted plant in Sicily (Italy). During 2008 and 2009, a wilting of vegetatively produced B. glabra cv. Sanderiana was observed in several nurseries in eastern Sicily (Catania and Messina provinces). Disease incidence was higher (~10 to 30%) in the tree-shaped potted plants (standards). Occasionally, wilting was detected on plants that were not tree shaped. Internally, symptomatic plants showed conspicuous vascular orange discoloration from the crown to the canopy. Diseased crown and stem tissues were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissue. Colonies with light purple or purple mycelia and violet reverse colony colors developed after 10 days. On carnation leaf agar, single-spore isolates produced microconidia in false heads on short monophialides, macroconidia that were 3-septate with a pedicellate base, and solitary and double-celled or aggregate chlamydospores. A PCR assay was conducted on two representative strains (DISTEF-BGS1 and DISTEF-BGS2) by analyzing sequences of the parzial translation elongation factor alpha gene (TEF-1α) and CaM gene (coding calmodulin protein). The primers used are previously used by O'Donnell et al. (1,2). Calmodulin sequences of BGS1 and BGS2 strains (GenBank Nos. FN645740 and FN645741, respectively) exhibited 99% homology with Fusarium oxysporum strain ITEM 2367 (GenBank No. AJ560774), and have homology of 99.6% between them. TEF-1 gene sequences of BGS1 (GenBank No. FN645739) exhibited an identity of 100% to F. oxysporum f. sp. lycopersici MUCL 22544 GenBank No. EF056785.1) and TEF-1α gene sequences of BGS2 (GenBank No. FN655742) exhibited an identity of 100% to F. oxysporum strain NRRL 45954 (GenBank No. FJ985431.1), whereas the homology between the two strains is 98.5%. Both PCR approaches established the identity of the isolates to the F. oxysporum Schlechtend:Fr (1,2). Pathogenicity tests were performed by placing 1-cm2 plugs of PDA from 10-day-old mycelial cultures near the crown on 40 potted, healthy, 6-month-old cuttings of paper flower. Twenty plants for each isolate were used. The same number of plants served as noninoculated controls. All plants were enclosed for 5 days in plastic bags and placed in a growth chamber at 24 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 24 to 26°C. Symptoms identical to those observed in nurseries developed 1 month after inoculation with both strains. Crown and stem orange discoloration was detected in all inoculated plants after 2 months. Control plants remained symptomless. F. oxysporum was consistently reisolated from symptomatic tissues and identified as previously described. To our knowledge, F. oxysporum was previously reported on paper flower in Ghana (3). However, this is the first demonstration of the pathogenicity of F. oxysporum on paper flower and it is the first report in Europe of the disease. The presence of Fusarium wilt in Sicily is a potential threat to paper flower production in nurseries. References: (1) K. O'Donnell et al. Proc. Natl. Acad. Sci. USA 95:2044, 1998. (2) K. O'Donnell et al. Mycoscience 41:61, 2000. (3) P. Spaulding. USDA Agric. Handb. 197:1, 1961.

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First Report of Cercospora apiicola Causing Leaf Spots and Stem Lesions on Celery in China

Plant Disease ◽

10.1094/pdis-09-20-1879-pdn ◽

2020 ◽

Author(s):

Qian Zhao ◽

A LI CHAI ◽

Yanxia Shi ◽

Xuewen Xie ◽

Baoju Li

Keyword(s):

Disease Incidence ◽

Elongation Factor ◽

Morphological Characteristics ◽

Internal Transcribed Spacers ◽

Apium Graveolens ◽

Malt Extract ◽

Translation Elongation ◽

First Report ◽

Leaf Spots ◽

Stem Lesions

Celery (Apium graveolens L.) is a vegetable crop cultivated widely in the Mediterranean, Europe and parts of Asia. From March to May in 2014, leaf spots and stem lesions were observed on celery plants in Yanqing (116°03′E, 40°32′N), Beijing and Chengdu (104°06′E, 30°67′N), Sichuan Province. Plants developed 0.3-1.8 cm diameter subcircular leaf spots with brown centers surrounded by pale yellow halos. Spots on leaves were amphigenous. Necrotic areas on stems were subcircular to elongated, pale brown to brown. Plants in five greenhouses were surveyed with 30 to 60% disease incidence. Necrotic tissue from 8 stems and 12 leaves were cut from the margins of lesions and divided into two parts. One part was treated with lactophenol and used for microscopic examination. The other part was surface sterilized with 4% sodium hypochlorite for 2 min, rinsed three times in sterile water, placed onto 2% malt extract agar (MEA), and incubated at 26°C for seven days with natural daylight. Stromata on leaves and stems were not well developed. Four-to-ten conidiophores (15.3-56.5 × 2.8-5.5 μm) formed in fascicles, emerged through stomata or erupted through the cuticle. Conidia (n=50) were 60-135 × 2.5-4.5 μm, solitary, septate, cylindrical to obclavate-cylindrical, hila thickened and darkened. Colonies were white to smoke-gray, and aerial mycelia were sparse to moderate. Morphological characteristics of the pathogen were similar to Cercospora apiicola (Groenewald et al. 2006; Groenewald et al. 2013). The gDNA of 20 isolates was extracted from mycelium using the Plant Genomic DNA Kit (Tiangen, China). The internal transcribed spacers (ITS), actin (ACT), translation elongation factor 1-α (TEF1) and histone H3 (HIS3) regions were amplified with primer pairs ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone and Kohn 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006). Phylogenetic analysis of multiple genes (Bakhshi et al. 2018) was conducted with the neighbor-joining method using MEGA7. The sequences of our isolate (QC14030702) and five published sequences of C. apiicola were clustered into one clade with a 99% confidence level. The sequences of QC14030702 have been deposited in GenBank with accessions KU870468 for ITS, KU870469 for ACT, KU870470 for TEF1, and KU870471 for HIS3. Pathogenicity of the isolates was tested on plants (cv. Jia Yuan Xi Yang Qin). Because the pathogen sporulated poorly on various media, mycelial fragments were sprayed on leaves in a suspension of 1×106 mL-1 in a greenhouse (temperature 26±0.5°C; RH 98%; photoperiod 12 h). Healthy plants were sprayed with sterilized water as controls. Three replicates of every isolate were conducted, and each replicate included 5 celery plants. After 7 days, leaf spots appeared on all inoculated plants, which were similar to those on celery in the field. All control plants remained asymptomatic. Re-isolation of the fungus from infected tissues showed same morphological and cultural characteristics of C. apiicola as the original isolates. C. apiicola has been reported in Greece, Korea, South Korea and Venezuela on celery, but never been reported in China (Farr and Rossman 2020). C. apiicola potential threatens celery production, and this the first report of the disease in China.

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First Report of Flyspeck Caused by Zygophiala wisconsinensis on Sweet Persimmon Fruit in Korea

Plant Disease ◽

10.1094/pdis-12-10-0917 ◽

2011 ◽

Vol 95 (5) ◽

pp. 616-616 ◽

Cited By ~ 1

Author(s):

J. Kim ◽

O. Choi ◽

J.-H. Kwon

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Its Rdna ◽

Control Treatment ◽

Diospyros Kaki ◽

Distilled Water ◽

First Report ◽

Persimmon Fruit ◽

Fungal Isolates ◽

Agar Disc

Sweet persimmon (Diospyros kaki L.), a fruit tree in the Ebenaceae, is cultivated widely in Korea and Japan, the leading producers worldwide (2). Sweet persimmon fruit with flyspeck symptoms were collected from orchards in the Jinju area of Korea in November 2010. The fruit had fungal clusters of black, round to ovoid, sclerotium-like fungal bodies with no visible evidence of a mycelial mat. Orchard inspections revealed that disease incidence ranged from 10 to 20% in the surveyed area (approximately 10 ha) in 2010. Flyspeck symptoms were observed on immature and mature fruit. Sweet persimmon fruit peels with flyspeck symptoms were removed, dried, and individual speck lesions transferred to potato dextrose agar (PDA) and cultured at 22°C in the dark. Fungal isolates were obtained from flyspeck colonies on 10 sweet persimmon fruit harvested from each of three orchards. Fungal isolates that grew from the lesions were identified based on a previous description (1). To confirm identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA sequence of a representative isolate was amplified and sequenced using primers ITS1 and ITS4 (4). The resulting 552-bp sequence was deposited in GenBank (Accession No. HQ698923). Comparison with ITS rDNA sequences showed 100% similarity with a sequence of Zygophiala wisconsinensis Batzer & Crous (GenBank Accession No. AY598855), which infects apple. To fulfill Koch's postulates, mature, intact sweet persimmon fruit were surface sterilized with 70% ethanol and dried. Three fungal isolates from this study were grown on PDA for 1 month. A colonized agar disc (5 mm in diameter) of each isolate was cut from the advancing margin of a colony with a sterilized cork borer, transferred to a 1.5-ml Eppendorf tube, and ground into a suspension of mycelial fragments and conidia in a blender with 1 ml of sterile, distilled water. The inoculum of each isolate was applied by swabbing a sweet persimmon fruit with the suspension. Three sweet persimmon fruit were inoculated per isolate. Three fruit were inoculated similarly with sterile, distilled water as the control treatment. After 1 month of incubation in a moist chamber at 22°C, the same fungal fruiting symptoms were reproduced as observed in the orchards, and the fungus was reisolated from these symptoms, but not from the control fruit, which were asymptomatic. On the basis of morphological characteristics of the fungal colonies, ITS sequence, and pathogenicity to persimmon fruit, the fungus was identified as Z. wisconsinensis (1). Flyspeck is readily isolated from sweet persimmon fruit in Korea and other sweet persimmon growing regions (3). The exposure of fruit to unusual weather conditions in Korea in recent years, including drought, and low-temperature and low-light situations in late spring, which are favorable for flyspeck, might be associated with an increase in occurrence of flyspeck on sweet persimmon fruit in Korea. To our knowledge, this is the first report of Z. wisconsinensis causing flyspeck on sweet persimmon in Korea. References: (1) J. C. Batzer et al. Mycologia 100:246, 2008. (2) FAOSTAT Database. Retrieved from http://faostat.fao.org/ , 2008. (3) H. Nasu and H. Kunoh. Plant Dis. 71:361, 1987. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.

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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 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 Gibellulopsis nigrescens on peppermint in Germany

Journal of Plant Diseases and Protection ◽

10.1007/s41348-021-00540-0 ◽

2021 ◽

Author(s):

Elias Alisaac ◽

Monika Götz

Keyword(s):

Verticillium Wilt ◽

Elongation Factor ◽

Economic Losses ◽

Fungal Culture ◽

Translation Elongation ◽

First Report ◽

Regular Control ◽

Fungal Dna ◽

Elongation Factor 1 ◽

Important Medicinal Plant

AbstractPeppermint is an important medicinal plant, and it is known for its essential oils and phenolic acids. Verticillium wilt is a vascular disease resulted from several Verticillium spp. causing significant economic losses in peppermint cultivation. In this study, the fungus Gibellulopsis nigrescens (syn. Verticillium nigrescens) was isolated from symptomless peppermint plants during the regular control of Verticillium wilt on peppermint in Germany. A pure fungal culture was prepared, and fungal DNA was extracted. Ribosomal internal transcribed spacer (ITS), beta-tubulin (TUB), and translation elongation factor 1-α (TEF1-α) were amplified, sequenced, and deposited in the GenBank. These sequences are located within the Gibellulopsis nigrescens cluster. Koch’s postulate was fulfilled, and the fungus was re-isolated from the inoculated plants. Up to our knowledge, this is the first report of Gibellulopsis nigrescens on peppermint in Germany.

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First Report of Phytophthora drechsleri Associated with Stem and Foliar Blight of Gynura bicolor in Taiwan

Plant Disease ◽

10.1094/pdis-12-10-0931 ◽

2011 ◽

Vol 95 (7) ◽

pp. 874-874 ◽

Cited By ~ 1

Author(s):

Y. M. Shen ◽

C. H. Chao ◽

H. L. Liu

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Hyphal Growth ◽

Pathogenicity Test ◽

Distilled Water ◽

First Report ◽

Foliar Blight ◽

Dual Cultures ◽

Phytophthora Drechsleri ◽

Gynura Bicolor

Gynura bicolor (Roxb. ex Willd.) DC., known as Okinawa spinach or hong-feng-cai, is a commonly consumed vegetable in Asian countries. In May 2010, plants with blight and wilt symptoms were observed in commercial vegetable farms in Changhua, Taiwan. Light brown-to-black blight lesions developed from the top of the stems to the petioles and extended to the base of the leaves. Severely infected plants declined and eventually died. Disease incidence was approximately 20%. Samples of symptomatic tissues were surface sterilized in 0.6% NaOCl and plated on water agar. A Phytophthora sp. was consistently isolated and further plated on 10% unclarified V8 juice agar, with daily radial growths of 7.6, 8.6, 5.7, and 2.4 mm at 25, 30, 35, and 37°C, respectively. Four replicates were measured for each temperature. No hyphal growth was observed at 39°C. Intercalary hyphal swellings and proliferating sporangia were produced in culture plates flooded with sterile distilled water. Sporangia were nonpapillate, obpyriform to ellipsoid, base tapered or rounded, and 43.3 (27.5 to 59.3) × 27.6 (18.5 to 36.3) μm. Clamydospores and oospores were not observed. Oospores were present in dual cultures with an isolate of P. nicotianae (p731) (1) A2 mating type, indicating that the isolate was heterothallic. A portion of the internal transcribed spacer sequence was deposited in GenBank (Accession No. HQ717146). The sequence was 99% identical to that of P. drechsleri SCRP232 (ATCC46724) (3), a type isolate of the species. The pathogen was identified as P. drechsleri Tucker based on temperature growth, morphological characteristics, and ITS sequence homology (3). To evaluate pathogenicity, the isolated P. drechsleri was inoculated on greenhouse-potted G. bicolor plants. Inoculum was obtained by grinding two dishes of the pathogen cultured on potato dextrose agar (PDA) with sterile distilled water in a blender. After filtering through a gauze layer, the filtrate was aliquoted to 240 ml. The inoculum (approximately 180 sporangia/ml) was sprayed on 24 plants of G. bicolor. An equal number of plants treated with sterile PDA processed in the same way served as controls. After 1 week, incubation at an average temperature of 29°C, blight and wilt symptoms similar to those observed in the fields appeared on 12 inoculated plants. The pathogen was reisolated from the lesions of diseased stems and leaves, fulfilling Koch's postulates. The controls remained symptomless. The pathogenicity test was repeated once with similar results. G. bicolor in Taiwan has been recorded to be infected by P. cryptogea (1,2), a species that resembles P. drechsleri. The recorded isolates of P. cryptogea did not have a maximal growth temperature at or above 35°C (1,2), a distinctive characteristic to discriminate between the two species (3). To our knowledge, this is the first report of P. drechsleri being associated with stem and foliar blight of G. bicolor. References: (1) P. J. Ann. Plant Pathol. Bull. 5:146, 1996. (2) H. H. Ho et al. The Genus Phytophthora in Taiwan. Institute of Botany, Academia Sinica, Taipei, 1995. (3) R. Mostowfizadeh-Ghalamfarsa et al. Fungal Biol. 114:325, 2010.

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First Report of Curvularia aeria on Etlingera linguiformis from Nagaland, India

Plant Disease ◽

10.1094/pdis-01-14-0060-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1580-1580 ◽

Cited By ~ 2

Author(s):

C. Kithan ◽

L. Daiho

Keyword(s):

Leaf Surface ◽

Agricultural Research ◽

Disease Incidence ◽

Indian Agricultural Research Institute ◽

Mountain Range ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Initial Symptoms

Etlingera linguiformis (Roxb.) R.M.Sm. of Zingiberaceae family is an important indigenous medicinal and aromatic plant of Nagaland, India, that grows well in warm climates with loamy soil rich in humus (1). The plant rhizome has medicinal benefits in treating sore throats, stomachache, rheumatism, and respiratory complaints, while its essential oil is used in perfumery. A severe disease incidence of leaf blight was observed on the foliar portion of E. linguiformis at the Patkai mountain range of northeast India in September 2012. Initial symptoms of the disease are small brown water soaked flecks appearing on the upper leaf surface with diameter ranging from 0.5 to 3 cm, which later coalesced to form dark brown lesions with a well-defined border. Lesions often merged to form large necrotic areas, covering more than 90% of the leaf surface, which contributed to plant death. The disease significantly reduces the number of functional leaves. As disease progresses, stems and rhizomes were also affected, reducing quality and yield. The diseased leaf tissues were surface sterilized with 0.2% sodium hypochlorite for 2 min followed by rinsing in sterile distilled water and transferred into potato dextrose agar (PDA) medium. After 3 days, the growing tips of the mycelium were transferred to PDA slants and incubated at 25 ± 2°C until conidia formation. Fungal colonies on PDA were dark gray to dark brown, usually zonate; stromata regularly and abundantly formed in culture. Conidia were straight to curved, ellipsoidal, 3-septate, rarely 4-septate, middle cells broad and darker than other two end cells, middle septum not median, smooth, 18 to 32 × 8 to 16 μm (mean 25.15 × 12.10 μm). Conidiophores were terminal and lateral on hyphae and stromata, simple or branched, straight or flexuous, often geniculate, septate, pale brown to brown, smooth, and up to 800 μm thick (2,3). Pathogen identification was performed by the Indian Type Culture Collection, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi (ITCC Accession No. 7895.10). Further molecular identity of the pathogen was confirmed as Curvularia aeria by PCR amplification and sequencing of the internal transcribed spacer (ITS) regions of the ribosomal DNA by using primers ITS4 and ITS5 (4). The sequence was submitted to GenBank (Accession No. MTCC11875). BLAST analysis of the fungal sequence showed 100% nucleotide similarity with Cochliobolus lunatus and Curvularia aeria. Pathogenicity tests were performed by spraying with an aqueous conidial suspension (1 × 106 conidia /ml) on leaves of three healthy Etlingera plants. Three plants sprayed with sterile distilled water served as controls. The first foliar lesions developed on leaves 7 days after inoculation and after 10 to 12 days, 80% of the leaves were severely infected. Control plants remained healthy. The inoculated leaves developed similar blight symptoms to those observed on naturally infected leaves. C. aeria was re-isolated from the inoculated leaves, thus fulfilling Koch's postulates. The pathogenicity test was repeated twice. To our knowledge, this is the first report of the presence of C. aeria on E. linguiformis. References: (1) M. H. Arafat et al. Pharm. J. 16:33, 2013. (2) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (3) K. J. Martin and P. T. Rygiewicz. BMC Microbiol. 5:28, 2005. (4) C. V. Suberamanian. Proc. Indian Acad. Sci. 38:27, 1955.

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