scholarly journals First Report of Colletotrichum godetiae Causing Anthracnose and Twig Blight on Persian Walnut in Hungary

Plant Disease ◽  
2020 ◽  
Author(s):  
Virág Varjas ◽  
Tamás Lakatos ◽  
Tímea Tóth ◽  
Csilla Kovács

Persian walnut (Juglans regia L.) fruit with preharvest anthracnose symptoms, necrotic fruit stalks, and twigs with necrotic buds, and peaks were collected in a Hungarian orchard next to Nágocs, in September 2018. Disease incidence was approximately 15% on a Hungarian bred walnut cultivar ‘Milotai 10’. Similar symptoms were found on Persian walnut in other locations (eg. Milota, Érd, Sarród, and Kocs). Acervuli were observed on necrotic lesions on fruit, and twigs with pale orange conidial masses. Conidia were hyaline, unicellular, and fusiform. Morphometric measurements of conidia showed mean length ± SD × width ± SD = 15.9 ± 1.7 × 4.5 ± 0.4 μm, length/width ratio 1:0.3 (n=100). The fungus was isolated from conidial masses on potato dextrose agar (PDA) medium amended with Chlorampenicol (25 mg/L). A total of 12 isolates were obtained as pure cultures by single-spore isolations and incubated at 23°C in dark for 10 days. The colonies were white to gray or grayish-orange on the upper side and with black spots on the reverse side. The isolates showed morphological characteristics of Colletotrichum acutatum in sensu lato (Jayawardena et al. 2016). Molecular analyses were conducted to identify the exact species. Internal transcribed spacer (ITS) region, actin (ACT), and calmodulin (CAL) partial genes were amplified by ITS1F/ITS4R, ACT512F/ACT783R and CAL1/CAL2 primers (White at al. 1990, Carbone and Kohn 1999, O’Donnell et al. 2000). The sequences of ITS region (GenBank Accession Nos: MK367398-99, MK367401-02) showed 100% identity with C. godetiae sequence. Based on ACT gene (GenBank Accession Nos: MK415991-92, MK415994-95) were 100% identity with the deposited C. godetiae type strains from walnut. The obtained sequences of CAL gene (GenBank Accession Nos: MK415998-99, MK416001-02) were same and showed 100% with other C. godetiae sequences from other host plants. The fungus was identified as Colletotrichum godetiae Neerg. Pathogenicity tests were accomplished in the field and under laboratory conditions (25°C on thermostat) on 10 green ‘Milotai 10’ walnut fruit, and 10 walnut twigs each. Tests were conducted on living trees, collected fruit, and two-year-old twigs by inserting mycelial agar plugs (5 mm in diameter) onto wounded pericarp tissues, which were then wrapped with wet cotton and parafilm. Wounded tissues on 5 fruit and 5 two-year-old twigs were treated with non-colonized PDA plugs as noninoculated controls. After 14 d necrotic lesions 9 to 17 mm in diameter developed on fruit on living trees. Lengths of 12 to 17 mm and width of 7 to 12 mm necrosis was measured on phloem of walnut twigs, and almost two times larger in cambium. No necrosis developed around control wounds. Koch's postulates were fulfilled with the reisolation of the pathogen from symptomatic tissues, isolates were identical morphologically and by sequence analysis of ITS region, ACT, and CAL partial genes to the original isolates. Damm et al. (2012) described two C. godetiae strains associated with walnut, one isolated in Austria and another one of unknown origin. An epidemic event of walnut anthracnose caused by Colletotrichum species mainly C. godetiae was reported in France (Da Lio et al. 2018). The pathogen was isolated from nuts, buds, insects, and stems. To our knowledge, this is the first report of anthracnose of walnut fruit caused by C. godetiae in Hungary. Anthracnose caused by C. godetiae, and previously reported C. fioriniae (Varjas et al. 2019) is becoming an increasing preharvest problem on Persian walnut in Hungary.

Plant Disease ◽  
2014 ◽  
Vol 98 (7) ◽  
pp. 1007-1007 ◽  
Author(s):  
B. J. Li ◽  
J. X. Chuan ◽  
M. Yang ◽  
G. F. Du

Gynura (Gynura bicolor DC.) is a perennial herbaceous plant in the family Compositae. It is an important Chinese vegetable, and is commonly used as a Chinese herbal medicine. In 2010, a severe leaf spot disease was observed on gynura grown in the main production areas in Tong Nan County, Chongqing City, China. Some farms experienced 60% disease incidence. Symptoms usually began on the lower leaves, as circular to elliptical or irregular spots with concentric rings. Individual spots were dark brown with grayish centers, sometimes coalescing and leading to extensive necrosis. The fungus associated with lesions was characterized as follows: Conidiophores were single or in clusters, straight or flexuous, unbranched, percurrent, cylindrical, pale to dark brown, 87.5 to 375.0 μm long and 5.0 to 10.5 μm wide. Conidia were solitary or catenate, straight to slightly curved, obclavate to cylindrical, 3 to 14 pseudoseptate, 82.8 to 237.5 μm long and 7.0 to 7.8 μm wide, and pale brown. The morphological characteristics of the conidia and conidiophores agreed with the descriptions for Corynespora cassiicola (1). To isolate the causal pathogen, surface-sterilized tissue at the margin of lesions was immersed in 75% ethanol for 30 s, rinsed in sterile water, dried in a laminar flow bench, transferred to PDA, and incubated at 28°C. Four single-spore cultures of the isolates were obtained and named from ZBTK10110637 to ZBTK10110640. All strains were identified as C. cassiicola. The isolate ZBTK10110637 was selected as representative for molecular identification. Genomic DNA was extracted by CTAB (2). The internal transcribed spacer (ITS) region of the rDNA was amplified using primers with ITS1 (5′-TCCGATGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). Amplicons were 433 bp (GenBank Accession No. JX867272) and shared 100% similarity with that of C. cassiicola (NRC2-1 No. AB539285.1). To confirm pathogenicity, four isolates were used to inoculate 12 gynura plants (6 weeks old) by mist spray-inoculation with 108 spores/ml suspension in sterile distilled water on the leaves. Control plants were misted with sterile distilled water. After inoculation, all plants were incubated in a greenhouse maintained at 20 to 28°C with relative humidity of 80 to 85%. Five days after inoculation, dark brown spots with a grayish center typical of field symptoms were observed on all inoculated plants. No symptoms were seen on water-treated control plants. The fungus was re-isolated from inoculated plants. The morphological characteristics of isolates were identical with the pathogen recovered originally. This is the first report of C. cassiicola on gynura. References: (1) M. B. Ellis. CMI Mycological Papers 65(9):1-15, 1957. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.


Plant Disease ◽  
2015 ◽  
Vol 99 (1) ◽  
pp. 161-161 ◽  
Author(s):  
I. Y. Choi ◽  
S. H. Hong ◽  
S. E. Cho ◽  
J. H. Park ◽  
H. D. Shin

Peucedanum japonicum Thunb., belonging to the family Apiaceae, is distributed in many Asian countries, including Korea. This plant was recently developed as an edible green and is cultivated under organic farming in Korea. In June 2013, plants showing typical symptoms of powdery mildew were found with approximately 50% disease incidence in polyethylene-film-covered greenhouses in Iksan City, Korea. Symptoms first appeared as circular white colonies, which subsequently showed abundant mycelial growth on the leaves, often covering the whole surface. Infected plants were unmarketable mainly due to signs of white fungal growths and reddish discoloration on the leaves. The same symptoms were found on P. japonicum in poly-tunnels in Iksan City and Jinan County of Korea in 2014. Voucher specimens (n = 3) were deposited in the Korea University Herbarium (KUS). Appressoria were lobed, and solitary or in opposite pairs. Conidiophores were cylindrical, 80 to 145 × 8 to 10 μm, and composed of three to four cells. Foot-cells of conidiophores were straight to substraight, cylindrical, and 25 to 63 μm long. Singly produced conidia were oblong-elliptical to oblong, occasionally ovate, 35 to 50 × 13 to 16 μm with a length/width ratio of 2.3:3.1, with angular/rectangular wrinkling of outer walls, and lacked distinct fibrosin bodies. Germ tubes were produced on the perihilar position of conidia. Primary conidia were apically conical, basally truncate, and generally smaller than the secondary conidia. No chasmothecia were found. These structures are typical of the powdery mildew Pseudoidium anamorph of the genus Erysiphe. The specific measurements and morphological characteristics were consistent with those of E. heraclei DC. (2). To confirm the identification, the complete internal transcribed spacer (ITS) region of rDNA from KUS-F27872 was amplified with primers ITS1/ITS4 and sequenced. The resulting 560-bp sequence was deposited in GenBank (Accession No. KM491178). The obtained ITS sequence shared >99% similarity with those of E. heraclei from apiaceous hosts, e.g., Daucus carota (KC480605), Pimpinella affinis (AB104513), and Petroselinum crispum (KF931139). Pathogenicity was confirmed through inoculation by gently dusting conidia onto leaves of five healthy potted plants. Five non-inoculated plants served as controls. Inoculated plants developed symptoms after 6 days, whereas the control plants remained symptomless. The fungus present on the inoculated plants was identical in morphology to those observed in the field. Powdery mildew of P. japonicum caused by E. heraclei has been reported in Japan (4), and numerous reports of E. heraclei on various species of Peucedanum plants have been made in most part of Europe and East Asia (Japan and far eastern Russia) (1,3). However, this is the first report of powdery mildew caused by E. heraclei on P. japonicum in Korea. Occurrence of powdery mildews is a threat to the quality and marketability of this plant, especially in organic farming. References: (1) K. Amano. Host Range and Geographical Distribution of the Powdery Mildew Fungi. Japan Scientific Societies Press, Tokyo, 1986. (2) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No.11. CBS, Utrecht, 2012. (3) D. F. Farr and A. Y. Rossman. Fungal Databases, Syst. Mycol. Microbiol. Lab., online publication. ARS, USDA. Retrieved August 18, 2014. (4) S. Tanda and C. Nakashima. J. Agric. Sci., Tokyo Univ. Agric. 47:54, 2002.


Plant Disease ◽  
2020 ◽  
Author(s):  
Siti Izera Ismail ◽  
Nur Adlina Rahim ◽  
Dzarifah Zulperi

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.


Plant Disease ◽  
2020 ◽  
Author(s):  
Fangmin Hao ◽  
Quanyu Zang ◽  
Weihong Ding ◽  
Erlei Ma ◽  
Yunping Huang ◽  
...  

Melon (Cucumis melo L.) is a member of the Cucurbitaceae family, an important economical and horticultural crop, which is widely grown in China. In May 2020, fruit rot disease with water-soaked lesions and pink molds on cantaloupe melons was observed in several greenhouses with 50% disease incidence in Ningbo, Zhejiang Province in China. In order to know the causal agent, diseased fruits were cut into pieces, surface sterilized for 1 min with 1% sodium hypochlorite (NaClO), 2 min with 75% ethyl alcohol, rinsed in sterile distilled water three times (Zhou et al. 2018), and then placed on potato dextrose agar (PDA) medium amended with streptomycin sulfate (100 μg/ml) plates at 25°C for 4 days. The growing hyphae were transferred to new PDA plates using the hyphal tip method, putative Fusarium colonies were purified by single-sporing. Twenty-five fungal isolates were obtained and formed red colonies with white aerial mycelia at 25°C for 7 days, which were identified as Fusarium isolates based on the morphological characteristics and microscopic examination. The average radial mycelial growth rate of Fusarium isolate Fa-25 was 11.44 mm/day at 25°C in the dark on PDA. Macroconidia were stout with curved apical and basal cells, usually with 4 to 6 septa, and 29.5 to 44.2 × 3.7 to 5.2 μm on Spezieller Nährstoffarmer agar (SNA) medium at 25°C for 10 days (Leslie and Summerell 2006). To identify the species, the internal transcribed spacer (ITS) region and translational elongation factor 1-alpha (TEF1-α) gene of the isolates were amplified and cloned. ITS and TEF1-α was amplified using primers ITS1/ITS4 and EF1/EF2 (O’Donnell et al. 1998), respectively. Sequences of ITS (545 bp, GenBank Accession No. MT811812) and TEF1-α (707 bp, GenBank Acc. No. MT856659) for isolate Fa-25 were 100% and 99.72% identical to those of F. asiaticum strains MSBL-4 (ITS, GenBank Acc. MT322117.1) and Daya350-3 (TEF1-α, GenBank Acc. KT380124.1) in GenBank, respectively. A phylogenetic tree was established based on the TEF1-α sequences of Fa-25 and other Fusarium spp., and Fa-25 was clustered with F. asiaticum. Thus, both morphological and molecular characterizations supported the isolate as F. asiaticum. To confirm the pathogenicity, mycelium agar plugs (6 mm in diameter) removed from the colony margin of a 2-day-old culture of strain Fa-25 were used to inoculate melon fruits. Before inoculation, healthy melon fruits were selected, soaked in 2% NaClO solution for 2 min, and washed in sterile water. After wounding the melon fruits with a sterile needle, the fruits were inoculated by placing mycelium agar plugs on the wounds, and mock inoculation with mycelium-free PDA plugs was used as control. Five fruits were used in each treatment. The inoculated and mock-inoculated fruits were incubated at 25°C with high relative humidity. Symptoms were observed on all inoculated melon fruits 10 days post inoculation, which were similar to those naturally infected fruits, whereas the mock-inoculated fruits remained symptomless. The fungus re-isolated from the diseased fruits resembled colony morphology of the original isolate. The experiment was conducted three times and produced the same results. To our knowledge, this is the first report of fruit rot of melon caused by F. asiaticum in China.


Plant Disease ◽  
2020 ◽  
Author(s):  
Yue Lian Liu ◽  
Jian Rong Tang ◽  
Yu Han Zhou

Monstera deliciosa Liebm is an ornamental foliage plant (Zhen et al. 2020De Lojo and De Benedetto 2014). In July of 2019, anthracnose lesions were observed on leaves of M. deliciosa cv. Duokong with 20% disease incidence of 100 plants at Guangdong Ocean University campus (21.17N,110.18E), Guangdong Province, China. Initially affected leaves showed chlorotic spots, which coalesced into larger irregular or circular lesions. The centers of spots were gray with a brown border surrounded by a yellow halo (Supplementary figure 1). Twenty diseased leaves were collected for pathogen isolation. Margins of diseased tissue was cut into 2 × 2 mm pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 60 s, rinsed three times with sterile water before isolation. Potato dextrose agar (PDA) was used to culture pathogens at 28℃ in dark. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. Fourteen isolates were obtained from 20 leaves. Three single-spore isolates (PSC-1, PSC-2, and PSC-3) were obtained ,obtained, which were identical in morphology and molecular analysis (ITS). Therefore, the representative isolate PSC-1 was used for further study. The culture of isolate PSC-1 on PDA was initially white and later became cottony, light gray in 4 days, at 28 °C. Conidia were single celled, hyaline, cylindrical, clavate, and measured 13.2 to 18.3 µm × 3.3 to 6.5 µm (n = 30). Appressoria were elliptical or subglobose, dark brown, and ranged from 6.3 to 9.5 µm × 5.7 to 6.5 µm (n = 30). Morphological characteristics of isolate PSC-1 were consistent with the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). DNA of the isolate PSC-1 was extracted for PCR sequencing using primers for the rDNA ITS (ITS1/ITS4), GAPDH (GDF1/GDR1), ACT (ACT-512F/ACT-783R), CAL (CL1C/CL2C), and TUB2 (βT2a/βT2b) (Weir et al. 2012). Analysis of the ITS (accession no. MN243535), GAPDH (MN243538), ACT (MN512640), CAL (MT163731), and TUB2 (MN512643) sequences revealed a 97-100% identity with the corresponding ITS (JX010161), GAPDH (JX010002), ACT (FJ907423), CAL (JX009714) and TUB2 (KP703502) sequences of C. siamense in GenBank. A phylogenetic tree was generated based on the concatenated sequences of ITS, GAPDH, ACT, CAL, and TUB2 which clustered the isolate PSC-1 with C. siamense the type strain ICMP 18578 (Supplementary figure 2). Based on morphological characteristics and phylogenetic analysis, the isolate PSC-1 associated with anthracnose of M. deliciosa was identified as C. siamense. Pathogenicity test was performed in a greenhouse at 24 to 30oC with 80% relative humidity. Ten healthy plants of cv. Duokong (3-month-old) were grown in pots with one plant in each pot. Five plants were inoculated by spraying a spore suspension (105 spores ml-1) of the isolate PSC-1 onto leaves until runoff, and five plants were sprayed with sterile water as controls. The test was conducted three times. Anthracnose lesions as earlier were observed on the leaves after two weeks, whereas control plants remained symptomless. The pathogen re-isolated from all inoculated leaves was identical to the isolate PSC-1 by morphology and ITS analysis, but not from control plants. C. gloeosporioides has been reported to cause anthracnose of M. deliciosa (Katakam, et al. 2017). To the best of our knowledge, this is the first report of C. siamense causing anthracnose on M. deliciosa in ChinaC. siamense causes anthracnose on a variety of plant hosts, but not including M. deliciosa (Yanan, et al. 2019). To the best of our knowledge, this is the first report of C. siamense causing anthracnose on M. deliciosa, which provides a basis for focusing on the management of the disease in future.


Plant Disease ◽  
2013 ◽  
Vol 97 (1) ◽  
pp. 147-147
Author(s):  
J. H. Park ◽  
S. E. Cho ◽  
K. S. Han ◽  
H. D. Shin

Garlic chives, Allium tuberosum Roth., are widely cultivated in Asia and are the fourth most important Allium crop in Korea. In June 2011, a leaf blight of garlic chives associated with a Septoria spp. was observed on an organic farm in Hongcheon County, Korea. Similar symptoms were also found in fields within Samcheok City and Yangku County of Korea during the 2011 and 2012 seasons. Disease incidence (percentage of plants affected) was 5 to 10% in organic farms surveyed. Diseased voucher specimens (n = 5) were deposited at the Korea University Herbarium (KUS). The disease first appeared as yellowish specks on leaves, expanding to cause a leaf tip dieback. Half of the leaves may be diseased within a week, especially during wet weather. Pycnidia were directly observed in leaf lesions. Pycnidia were amphigenous, but mostly epigenous, scattered, dark brown to rusty brown, globose, embedded in host tissue or partly erumpent, separate, unilocular, 50 to 150 μm in diameter, with ostioles of 20 to 40 μm in diameter. Conidia were acicular, straight to sub-straight, truncate at the base, obtuse at the apex, hyaline, aguttulate, 22 to 44 × 1.8 to 3 μm, mostly 3-septate, occasionally 1- or 2-septate. These morphological characteristics matched those of Septoria allii Moesz, which is differentiated from S. alliacea on conidial dimensions (50 to 60 μm long) (1,2). A monoconidial isolate was cultured on potato dextrose agar (PDA). Two isolates have been deposited in the Korean Agricultural Culture Collection (Accession Nos. KACC46119 and 46688). Genomic DNA was extracted using the DNeasy Plant Mini DNA Extraction Kit (Qiagen Inc., Valencia, CA). The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1/ITS4 primers and sequenced. The resulting sequence of 482-bp was deposited in GenBank (JX531648 and JX531649). ITS sequence information was at least 99% similar to those of many Septoria species, however no information was available for S. allii. Pathogenicity was tested by spraying leaves of three potted young plants with a conidial suspension (2 × 105 conidia/ml), which was harvested from a 4-week-old culture on PDA. Control leaves were sprayed with sterile water. The plants were placed in humid chambers (relative humidity 100%) for the first 48 h. After 7 days, typical leaf blight symptoms started to develop on the leaves of inoculated plants. S. allii was reisolated from the lesions of inoculated plants, confirming Koch's postulates. No symptoms were observed on control plants. The host-parasite association of A. tuberosum and S. allii has been known only from China (1). S. alliacea has been recorded on several species of Allium, e.g. A. cepa, A. chinense, A. fistulosum, and A. tuberosum from Japan (4) and A. cepa from Korea (3). To the best of our knowledge, this is the first report of S. allii on garlic chives. No diseased plants were observed in commercial fields of garlic chives which involved regular application of fungicides. The disease therefore seems to be limited to organic garlic chive production. References: (1) P. K. Chi et al. Fungous Diseases on Cultivated Plants of Jilin Province, Science Press, Beijing, China, 1966. (2) P. A. Saccardo. Sylloge Fungorum Omnium Hucusque Congnitorum. XXV. Berlin, 1931. (3) The Korean Society of Plant Pathology. List of Plant Diseases in Korea, Suwon, Korea, 2009. (4) The Phytopathological Society of Japan. Common Names of Plant Diseases in Japan, Tokyo, Japan, 2000.


Plant Disease ◽  
2013 ◽  
Vol 97 (1) ◽  
pp. 138-138 ◽  
Author(s):  
X. Y. Chen ◽  
J. D. Feng ◽  
Z. Su ◽  
C. Sui ◽  
X. Huang

Curcuma wenyujin Y.H. Chen & C. Ling is a traditional Chinese medicinal herb in the Zingiberaceae family. Commonly known as Wen yujin, the root is widely used for alleviating pain and protecting the liver. A severe leaf blight disease was observed in three C. wenyujin farms in Hainan Province of China in October 2010. The obvious symptoms of leaf blight, yellow to brown irregular lesions (1 to 20 cm) on C. wenyujin, usually began at the tips of leaves and the main veins. This disease, especially severe from August to October, caused heavy damage and 100% of mature plants (10 months old) in farms were infected. The disease was most severe when continuous cropping was performed and showed slight improvement when rotation was adopted. Farmers usually sprayed carbendazim (50% WP) and thiophanate-methyl (70% WP) to control this disease, but these treatments were not effective. To isolate the causal pathogen, diseased plants were collected in October 2010 from a field of the Hainan Branch Institute of Medicinal Plant Development in Hainan Province. Lesion tissue was removed from the border between symptomatic and healthy tissue, surface sterilized in 75% ethanol for 1 min, washed in three changes of sterile distilled water, transferred to potato dextrose agar (PDA) plates, and incubated at 28°C for 7 days. Single spore cultures of five isolates were obtained and identified as Curvularia clavata based on morphological characteristics (1). Conidia measured 20 to 29 × 7.5 to 10.5 μm (n = 100), were curved, 3-septate, and the third cell from the base was larger and darker than the others. Mycelia of single spore cultures growing on PDA for 5 days were used for DNA extraction using a plant genomic DNA kit (TIANGEN, Beijing). The internal transcribed spacer (ITS) region of the rDNA was amplified using primers ITS1 and ITS4. The amplicons were 562 bp in length (GenBank Accession No. JQ730852) and had 99% nucleotide identity with the GenBank Accession No. JN021115 and AF071336 of C. clavata. Pathogenicity tests were conducted using fresh and healthy detached Curcuma wenyujin leaves. Mycelial discs (10 mm) removed from a 5-day-old colony on PDA were used for inoculation. Each isolate was inoculated on three distinct leaves (two distinct inoculations per leaf). Three additional leaves inoculated with sterile PDA discs were used as control. Inoculated leaves were covered with a polythene film to maintain high humidity. Leaves in trays were kept in a growth chamber at 28°C and observed for symptom appearance every day. Five days after inoculation, inoculated leaves developed blight symptoms similar to those observed on naturally infected leaves. No symptoms were observed on non-inoculated leaves. C. clavata was reisolated from the inoculated leaves, thus fulfilling Koch's postulates. C. clavata has been previously reported to be economically important on a number of other hosts (2). To our knowledge, this is the first report of Curvularia leaf blight on Curcuma wenyujin caused by C. clavata in China. References: (1) A. M Mandokhot et al. Eur. J. Plant Pathol.78:65, 1972. (2) T. Y. Zhang et al. Flora fungorum sinicorum: Beijing, China, 2010.


Plant Disease ◽  
2021 ◽  
Author(s):  
Francisco Bruno da Silva Café ◽  
Rhannaldy Benício Rebouças ◽  
Juvenil H. Cares ◽  
Cristiano Souza Lima ◽  
Francisco de Assis Câmara Rabelo Filho ◽  
...  

During a survey in 2018 for plant nematodes associated with roots and soil in cactus cultivation areas in Ceará State (3°44'48"S, 38°34'29"W), cysts were found on roots of mandacaru, Cereus jamacaru DC. This cactus is native to Brazil, can grow to 6-10 meters in height, and is widely distributed in the Northeast region (Romeiro-Brito et al. 2016) where it is used in construction, in disease remedies, as forage, and as an ornamental (Sales et al. 2014). Several cysts, second-stage juveniles (J2) and eggs extracted from the soil and roots, using sucrose centrifugation, were examined by scanning electron microscopy (SEM) and light microscopy (LM) to determine morphological and morphometric characteristics. Molecular characteristics were determined by DNA extraction from J2 and embryonated eggs using a protocol specific for Heteroderidae (Subbotin et al., 2018). The internal transcribed spacer sequence (ITS) region of the rDNA and D2-D3 regions of the 28S rDNA were amplified using the universal primers TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′), D2A(5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B(5′-TCGGAAGGAACCAGCTACTA-3′), respectively. To confirm that mandacaru is a host for C. cacti, six plantlets of mandacaru were inoculated with 1,800 eggs of the nematode, and kept in a greenhouse at 31 ± 3 ºC and irrigated daily. Six non inoculated mandacaru plantlets served as control treatment. Morphometric characteristics of cysts (n=35) were body length, excluding neck, 555.8 ± 87.8 (354,9 - 727,6) μm, body width 392.1 ± 63.4 (297.9 - 553.7) μm, neck length 63.5 ± 25.8 (49.8-105.0) μm, length to width ratio 1.4 ± 0.2 (1.0-1.8) μm and vulval cone length 48.4 ± 15.2 (40.7 –53.6) μm. Cysts had a rough surface, were lemon-shaped to rounded and had a zigzag cuticular pattern with a protruding vulval cone. They were circumfenestrate without underbridge and bullae, but with the presence of vulval denticles. Measurements of second-stage juveniles (n = 13) included the body length 511.2 ± 33.7 (452.7 - 551.5) μm, stylet length 28.0 ± 2.8 (25.4 - 34.0) μm, tail length 50.7 ± 5.1 (40.6 - 57.4) μm, tail hyaline region 22.7 ± 2.2 (18.9 – 27.1), with a = 20.9 ± 2.2 (17.7-24.3) μm, b = 5.4 ± 0.4 (5.1-5.8) μm, b'= 3.4 ± 0.4 (3.1-3.9) μm, c = 10.2 ± 1.3 (8.9-13.3) μm and c' = 3.8 ± 0.4 (3.0-4.5) μm. The observations of essential morphological characteristics for identification indicated that the species found on C. jamacaru was Cactodera cacti (Filipjev & Schuurmans-Stekhoven, 1941) Krall & Krall, 1978. The sequences of the studied rDNA regions were submitted to GenBank (ITS: MW562829 and D2–D3 regions of 28S: MW562830). The samples used for molecular analysis showed a high degree of sequence identity (99.59%) with C. cacti, from China, Iran and USA for the ITS region. The identity of the D2-D3 regions of 28S sequence was 99.54% with C. cacti isolates from Germany and 99.41% with isolates from USA. Phylogenetic analyses were performed using Maximum likelihood (ML) method for both individual loci, confirming the species as Cactodera cacti. All inoculated mandacaru plantlets showed C. cacti cysts on the roots after 60 days, confirming that mandacaru is a host for C. cacti. This species was reported in São Paulo State, in 2001, associated with ornamental cactus cultivated in pots, but plant species were not identified (Santos et al., 2001). The second report in Brazil was to Schlumbergera sp., an ornamental plant (Oliveira et al. 2007). In both studies, the nematode was not morphologically nor molecularly characterized. Cactodera cacti has been commonly associated with cactus worldwide (Esser, 1992). It has been reported in association with C. jamacaru was first reported in 2011 in China (Duan et al. 2012). This is the first report of the occurrence of C. cacti on C. jamacaru in field conditions in Brazil, and its presence in cactus cultivation areas with agricultural importance represents a threat to cactus production in the country.


Plant Disease ◽  
2011 ◽  
Vol 95 (6) ◽  
pp. 775-775 ◽  
Author(s):  
V. Ayala-Escobar ◽  
V. Santiago-Santiago ◽  
A. Madariaga-Navarrete ◽  
A. Castañeda-Vildozola ◽  
C. Nava-Diaz

Bougainvillea (Bougainvillea spectabilis Willd) growing in 28 gardens during 2009 showed 100% disease incidence and 3 to 7% disease severity. Bougainvilleas with white flowers were the most affected. Symptoms consisted of light brown spots with dark brown margins visible on adaxial and abaxial sides of the leaves. Spots were circular, 2 to 7 mm in diameter, often surrounded by a chlorotic halo, and delimited by major leaf veins. Single-spore cultures were incubated at 24°C under near UV light for 7 days to obtain conidia. Pathogenicity was confirmed by spraying a conidial suspension (1 × 104 spores/ml) on leaves of potted bougainvillea plants (white, red, yellow, and purple flowers), incubating the plants in a dew chamber for 48 h and maintaining them in a greenhouse (20 to 24°C). Identical symptoms to those observed at the residential gardens appeared on inoculated plants after 45 to 60 days. The fungus was reisolated from inoculated plants that showed typical symptoms. No symptoms developed on control plants treated with sterile distilled water. The fungus produced distinct stromata that were dark brown, spherical to irregular, and 20 to 24 μm in diameter. Conidiophores were simple, born from the stromata, loose to dense fascicles, brown, straight to curved, not branched, zero to two septate, 14 × 2 μm, with two to four conspicuous and darkened scars. The conidia formed singly, were brown, broad, ellipsoid, obclavate, straight to curved with three to four septa, 40 × 4 μm, and finely verrucous with thick hilum at the end. Fungal DNA from the single-spore cultures was obtained using a commercial DNA Extraction Kit (Qiagen, Valencia, CA); ribosomal DNA was amplified with ITS5 and ITS4 primers and sequenced. The sequence was deposited at the National Center for Biotechnology Information Database (GenBank Accession Nos. HQ231216 and HQ231217). The symptoms (4), morphological characteristics (1,2,4), and pathogenicity test confirm the identity of the fungus as Passalora bougainvilleae (Muntañola) Castañeda & Braun (= Cercosporidium bougainvilleae Muntañola). This pathogen has been reported from Argentina, Brazil, Brunei, China, Cuba, El Salvador, India, Indonesia, Jamaica, Japan, Thailand, the United States, and Venezuela (3). To our knowledge, this is the first report of this disease on B. spectabilis Willd in Mexico. P. bougainvilleae may become an important disease of bougainvillea plants in tropical and subtropical areas of Mexico. References: (1) U. Braun and R. R. Castañeda. Cryptogam. Bot. 2/3:289, 1991. (2) M. B. Ellis. More Dematiaceous Hypomycetes. Commonwealth Mycological Institute, Kew, Surrey, UK, 1976. (3) C. Nakashima et al. Fungal Divers. 26:257, 2007. (4) K. L. Nechet and B. A. Halfeld-Vieira. Acta Amazonica 38:585, 2008.


Plant Disease ◽  
2011 ◽  
Vol 95 (12) ◽  
pp. 1588-1588 ◽  
Author(s):  
G. Polizzi ◽  
D. Aiello ◽  
V. Guarnaccia ◽  
A. Vitale ◽  
G. Perrone ◽  
...  

In June 2010, a widespread damping-off was noticed in a commercial nursery in eastern Sicily on ~20,000 potted 2-month-old strawberry tree (Arbutus unedo L.) seedlings. More than 40% of the seedlings showed disease symptoms including brown lesions at the seedling crown above and below the soil line that expanded rapidly to girdle the stem. Stem lesions were followed by death of the entire seedling in a few days. Diseased stem and crown tissues of 20 seedlings were surface disinfested for 2 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C in the dark. Fungal isolates with mycelial and morphological characteristics of Colletotrichum spp. were isolated from all seedlings. Fungal colonies were pale orange or gray without carmine pigments. On carnation leaf agar (CLA), single-spore isolates produced many orange masses of hyaline, aseptate conidia with a cylindrical to ellipsoidal shape, rounded apex, and 11 to 15 μm long and 3 to 4.5 μm wide (average 13.2 × 3.7 μm). The pointed conidia of 10 isolates were morphologically similar. DNA isolation was performed with the Wizard Magnetic DNA Purification Kit (Promega, Madison, WI) following the manufacturer's instructions with some modifications. A PCR assay was conducted on two representative isolates (ITEM 13492 and ITEM 13493) by analyzing sequences of gene benA (coding β-tubulin protein) using the primers T1 and T10 reported by O'Donnell and Cigelnik (1). BenA gene sequence of ITEM 13492 exhibited an identity of 99.8% to C. simmondsii strain BRIP 4704 (GenBank No. GU183277), while BenA gene sequence of ITEM 13493 exhibited an identity of 100% to C. acutatum strain BRIP52695 (GenBank No. GU183314). The identification of these two species was made by comparing the internal transcribed spacer region and BenA sequences of these two strains with that deposited by Shivas and Tan (2). Morphological characteristics, as well as the PCR assay, identified the isolates as Colletotrichum acutatum J.H. Simmonds and C. simmondsii R.G. Shivas & Y. P. Tan (2,3). Pathogenicity tests were carried out on 2-month-old seedlings of strawberry tree grown on alveolar trays. Conidial suspensions of two isolates (ITEM 13492 and ITEM 13493) were obtained from 14-day-old single-spore colonies on CLA, then adjusted to 105 conidia per ml and sprayed on seedlings. Fifty seedlings for each isolate were used. The same number of seedlings was mock inoculated with sterile distilled water. All seedlings were enclosed for 4 days in plastic bags and placed in a growth chamber at 24 ± 1°C for 45 days. Identical symptoms to those observed in the nurseries appeared 30 days after inoculation, and after 45 days, 80% of the plants were dead. No difference in virulence between the two isolates was observed and no symptoms were detected on the control plants. C. acutatum and C. simmondsii were successfully reisolated from all symptomatic tissues and identified as previously described, completing Koch's postulates. To our knowledge, this is the first report in the world of C. acutatum and C. simmondsii on strawberry tree. This suggests that Colletotrichum spp. may be important pathogens of young seedlings of strawberry tree in nurseries. References: (1) K. O'Donnell and E. Cigelnik. Mol. Phylo. Evol. 7:103, 1997. (2) R. G. Shivas and Y. P. Tan. Fungal Divers. 39:111, 2009. (3) B. C. Sutton. Page 523 in: The Coelomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 1980.


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