First report of Cercospora cf. citrulina causing leaf spot of Ipomoea pes-caprae in China

Ipomoea pes-caprae plays an important role in protecting the tropical and subtropical coastal beach of the world. In 2018, a leaf spot was observed on I. pes-caprae in Xisha islands of China, 13.2–25.8% of leaves were infected. The initial symptoms were small (1–3 mm diameter), single, circular, dark gray spots with a light-yellow center on the leaves. The lesions enlarged and were scattered or confluent, distinct and circular, subcircular or irregular, occasionally vein-limited, pale to dark gray-brown, with a narrow dark brown border surrounded by a diffuse yellow margin. Microscopic observations of the spots revealed that caespituli were dark brown and amphigenous, but abundant on the underside of the leaves. Mycelia were internal. Conidiophores were fasciculate, occasionally solitary, pale olivaceous-brown throughout, 0- to 3-septate, 27.9–115.8 (63.4±22.5) µm × 3.2–5.3 (4.3±0.87) µm (n=100). Conidial scars were conspicuously thickened. Conidia were solitary, hyaline, filiform, acicular to obclavate, straight to slightly curved, subacute to obtuse at the apex, truncate at the base, multi-septate, 21.0–125.5 (60.2±20.1) µm × 2.0–5.0 (3.8±0.83) µm (n=100). Single-conidium isolates were obtained from representative colonies grown on potato dextrose agar (PDA) incubated at 25℃ in the dark. The colonies grew slowly and were dense, white to gray and flat with aerial mycelium. Mycelia were initially white, and then became gray. Conidia were borne on the conidiophores directly. The pure isolate HTW-1 was selected for molecular identification and pathogenicity test, which were deposited in Microbiological Culture Collection Center of Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (tef1) and histone H3 (his3) genes were amplified with ITS1/ITS4, EF-1 / EF-2, and CYLH3F / CYLH3R primers, respectively (Groenewald et al. 2013). The obtained sequences of HTW-1 were all deposited in GenBank with accession numbers MT410467 for ITS, MT418903 for tef1 and MT418904 for his3. The ITS, tef1 and his3 genes all showed 100% similarity for ITS (JX143582), tef1 (JX143340) and his3 (JX142602) with C. cf. citrulina (MUCC 588; MAFF 239409) from I. pes-caprae in Japan. Based on the morphological characteristics and molecular identification, the pathogen was identified as Cercospora cf. citrulina (Groenewald et al. 2013). The pathogenicity test was conducted by spraying conidial suspension (1×104 conidia/mL) on wounded and unwounded leaves for seedling of I. pes-caprae in greenhouse and in sterile vitro condition. The conidial suspension was prepared using conidia from 30-day-old culture grown on PDA at 25℃ in the dark. Leaf surfaces of seedling in greenhouse were wounded by lightly rubbing with a steel sponge and detached leaf surfaces were wounded by sterile needles. the treatments were sprayed with conidial suspensions on wounded and unwounded leaf surfaces. The control was sprayed with sterile water. After eight days, the typical symptoms of spots which were small, single, circular and dark gray appeared on the inoculated wounded leaves, while the inoculated unwounded leaves and the control leaves were symptomless. The pathogen was only re-isolated from the inoculated wounded leaves. The pathogen may be infected by wound. A total of 20 Cercospora and related species was found on Ipomoea spp. (García et al. 1996). Cercospora cf. citrulina has been reported on I. pes-caprae in Japan, although it was unclear if it was a pathogen or saprophyte (Groenewald et al. 2013). To our knowledge, this is the first report of C. cf. citrulina causing leaf spot of I. pes-caprae in China. This disease could threat the cultivation of I. pes-caprae in China.

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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 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 Septoria Leaf Spot of Lavandin Caused by Septoria lavandulae in Croatia

Plant Disease ◽

10.1094/pdis-07-13-0735-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 282-282

Author(s):

K. Vrandečić ◽

J. Ćosić ◽

D. Jurković ◽

I. Stanković ◽

A. Vučurović ◽

...

Keyword(s):

Leaf Spot ◽

Antibacterial Properties ◽

Morphological Characteristics ◽

Its Region ◽

Morphological Identification ◽

Conidial Suspension ◽

Necrotic Tissue ◽

First Report ◽

Initial Symptoms ◽

Septoria Leaf Spot

Lavandula × intermedia Emeric ex Loiseleur, commonly known as lavandin, is an aromatic and medicinal perennial shrub widely and traditionally grown in Croatia. The lavandin essential oil is primarily used in perfumery and cosmetic industries, but also possesses anti-inflammatory, sedative, and antibacterial properties. In June 2012, severe foliar and stem symptoms were observed on approximately 40% of plants growing in a commercial lavandin crop in the locality of Banovo Brdo, Republic of Croatia. Initial symptoms on lower leaves included numerous, small, oval to irregular, grayish brown lesions with a slightly darker brown margin of necrotic tissue. Further development of the disease resulted in yellowing and necrosis of the infected leaves followed by premature defoliation. Similar necrotic oval-shaped lesions were observed on stems as well. The lesions contained numerous, dark, sub-globose pycnidia that were immersed in the necrotic tissue or partly erumpent. Small pieces of infected internal tissues were superficially disinfected with 50% commercial bleach (4% NaOCl) and placed on potato dextrose agar (PDA). A total of 10 isolates from leaves and five from stems of lavandin formed a slow-growing, dark, circular colonies with raised center that produced pycnidia at 23°C, under 12 h of fluorescent light per day. All 15 recovered isolates formed uniform hyaline, elongate, straight or slightly curved conidia with 3 to 4 septa, with average dimensions of 17.5 to 35 × 1.5 to 2.5 μm. Based on the morphological characteristics, the pathogen was identified as Septoria lavandulae Desm., the causal agent of lavender leaf spot (1,2). Pathogenicity of one selected isolate (428-12) was tested by spraying 10 lavandin seedlings (8 weeks old) with a conidial suspension (106 conidia/ml) harvested from a 4-week-old monoconidial culture on PDA. Five lavandin seedlings, sprayed with sterile distilled water, were used as negative control. After 5 to 7 days, leaf spot symptoms identical to those observed on the source plants developed on all inoculated seedlings and the pathogen was successfully re-isolated. No symptoms were observed on any of the control plants. Morphological identification was confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA (3). Total DNA was extracted directly from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and PCR amplification performed with primers ITS1F/ITS4. Sequence analysis of ITS region revealed at least 99% identity between the isolate 428-12 (GenBank Accession No. KF373078) and isolates of many Septoria species; however, no information was available for S. lavandulae. To our knowledge, this is the first report of Septoria leaf spot of lavandin caused by S. lavandulae in Croatia. Since the cultivation area of lavandin plants has been increasing in many continental parts of Croatia, especially in Slavonia and Baranja counties, the presence of a new and potentially harmful disease may represent a serious constraint for lavandin production and further monitoring is needed. References: (1) T. V. Andrianova and D. W. Minter. IMI Descriptions of Fungi and Bacteria, 142, Sheet 1416, 1999. (2) R. Bounaurio et al. Petria 6:183, 1996. (3) G. J. M. Verkley et al. Mycologia 96:558, 2004.

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First report of leaf spot disease caused by Corynespora cassiicola on American sweetgum (Liquidambar styraciflua L.) in China

Plant Disease ◽

10.1094/pdis-08-20-1842-pdn ◽

2021 ◽

Author(s):

Yun-fei Mao ◽

Xiang-rong Zheng ◽

Fengmao Chen

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Liquidambar Styraciflua ◽

Bootstrap Support ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Corynespora Cassiicola ◽

First Report ◽

Leaf Spots

American sweetgum (Liquidambar styraciflua L.) is a forest plant native to North America, which has been introduced into other countries due to its ornamental and medicinal values. In June 2019, symptoms of leaf spots on sweetgum were observed in a field (5 ha) located in Xuzhou, Jiangsu Province, China. On this field, approximately 45% of 1,000 trees showed the same symptoms. Symptoms were observed showing irregular or circular dark brown necrotic lesions approximately 5 to 15 mm in diameter with a yellowish margin on the leaves. To isolate the pathogen, diseased leaf sections (4×4mm) were excised from the margin of the lesion, surface-sterilized with 0.1% NaOCl for 90 s, rinsed 4 times in sterile distilled water, air dried and then transferred on potato dextrose agar (PDA) medium at 25°C in the dark. Pure cultures were obtained by monospore isolation after subculture. Ten purified isolates, named FXI to FXR, were transferred to fresh PDA and incubated as above to allow for morphological and molecular identification. After 7 days, the aerial mycelium was abundant, fluffy and exhibited white to greyish-green coloration. The conidia were dark brown or olive, solitary or produced in chains, obclavate, with 1 to 15 pseudosepta, and measured 45 to 200µm  10 to 18µm. Based on morphological features, these 10 isolates were identified as Corynespora cassiicola (Ellis et al. 1971). Genomic DNA of each isolate was extracted from mycelia using the cetyltrimethylammonium bromide (CTAB) method. The EF-1α gene and ITS region were amplified and sequenced with the primer pairs rDNA ITS primers (ITS4/ITS5) (White et al. 1990) and EF1-728F/EF-986R (Carbone et al.1999) respectively. The sequences were deposited in GenBank. BLAST analysis revealed that the ITS sequence had 99.66% similarity to C. cassiicola MH255527 and that the EF-1α sequence had 100% similarity to C. cassiicola KX429668A. maximum likelihood phylogenetic analysis based on EF-1α and ITS sequences using MEGA 7 revealed that ten isolates were placed in the same clade as C. cassiicola (Isolate: XQ3-1; accession numbers: MH572687 and MH569606, respectively) at 98% bootstrap support. Based on the morphological characteristics and phylogenetic analyses, all isolates were identified as C. cassiicola. For the pathogenicity test, a 10 µl conidial suspension (1×105 spores/ml) of each isolate was dripped onto healthy leaves of 2-year-old sweetgum potted seedlings respectively. Leaves inoculated with sterile water served as controls. Three plants (3 leaves per plant) were conducted for each treatment. The experiment was repeat twice. All seedlings were enclosed in plastic transparent incubators to maintain high relative humidity (90% to 100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. After 10 days, leaves inoculated with conidial suspension of each isolate showed symptoms of leaf spots, similar to those observed in the field. Control plants were remained healthy. In order to reisolate the pathogen, surface-sterilized and monosporic isolation was conducted as described above. The same fungus was reisolated from the lesions of symptomatic leaves, and its identity was confirmed by molecular and morphological approaches, thus fulfilling Koch’s postulates. Chlorothalonil and Boscalid can be used to effectively control Corynespora leaf spot (Chairin T et al.2017). To our knowledge, this is the first report of leaf spot caused by C. cassiicola on L. styraciflua in China.

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First Report of Stemphylium solani as the Causal Agent of a Leaf Spot on Greenhouse Cucumber

Plant Disease ◽

10.1094/pdis-08-12-0776-pdn ◽

2013 ◽

Vol 97 (2) ◽

pp. 287-287 ◽

Cited By ~ 5

Author(s):

D. J. Vakalounakis ◽

E. A. Markakis

Keyword(s):

Leaf Spot ◽

Natural Infection ◽

Apical Cell ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

First Report

During the 2011 to 2012 crop season, a severe leaf spot disease of cucumber (Cucumis sativus) cv. Cadiz was noticed on crops in some greenhouses in the Goudouras area, Lasithi, Crete, Greece. Symptoms appeared in late winter, mainly on the leaves of the middle and upper part of the plants. Initially, small necrotic pinpoint lesions with white centers, surrounded by chlorotic halos, 1 to 3 mm in diameter, appeared on the upper leaf surfaces, and these progressively enlarged to spots that could coalesce to form nearly circular lesions up to 2 cm or more in diameter. Stemphylium-like fructifications appeared on necrotic tissue of older lesions. Severely affected leaves became chlorotic and died. No other part of the plant was affected. Small tissue pieces from the edges of lesions were surface disinfected in 0.5% NaClO for 5 min, rinsed in sterile distilled water, plated on acidified potato dextrose agar and incubated at 22 ± 0.5°C with a 12-h photoperiod. Stemphylium sp. was consistently isolated from diseased samples. Colonies showed a typical septate mycelium with the young hyphae subhyaline and gradually became greyish green to dark brown with age. Conidiophores were subhyaline to light brown, 3- to 10-septate, up to 200 μm in length, and 4 to 7 μm in width, with apical cell slightly to distinctly swollen, bearing a single spore at the apex. Conidia were muriform, mostly oblong to ovoid, but occasionally nearly globose, subhyline to variant shades of brown, mostly constricted at the median septum, 22.6 ± 6.22 (11.9 to 36.9) μm in length, and 15.1 ± 2.85 (8.3 to 22.6) μm in width, with 1 to 8 transverse and 0 to 5 longitudinal septa. DNA from a representative single-spore isolate was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was amplified using the universal primers ITS5 and ITS4. The PCR product was sequenced and deposited in GenBank (Accession No. JX481911). On the basis of morphological characteristics (3) and a BLAST search with 100% identity to the published ITS sequence of a S. solani isolate in GenBank (EF0767501), the fungus was identified as S. solani. Pathogenicity tests were performed by spraying a conidial suspension (105 conidia ml–1) on healthy cucumber (cv. Knossos), melon (C. melo, cv. Galia), watermelon (Citrullus lanatus cv. Crimson sweet), pumpkin (Cucurbita pepo, cv. Rigas), and sponge gourd (Luffa aegyptiaca, local variety) plants, at the 5-true-leaf stage. Disease symptoms appeared on cucumber and melon only, which were similar to those observed under natural infection conditions on cucumber. S. solani was consistently reisolated from artificially infected cucumber and melon tissues, thus confirming Koch's postulates. The pathogenicity test was repeated with similar results. In 1918, a report of a Stemphylium leaf spot of cucumber in Indiana and Ohio was attributed to Stemphylium cucurbitacearum Osner (4), but that pathogen has since been reclassified as Leandria momordicae Rangel (2). That disease was later reported from Florida (1) and net spot was suggested as a common name for that disease. For the disease reported here, we suggest the name Stemphylium leaf spot. This is the first report of a disease of cucumber caused by a species of Stemphylium. References: (1) C. H. Blazquez. Plant Dis. 67:534, 1983. (2) P. Holliday. Page 243 in: A Dictionary of Plant Pathology. Cambridge University Press, Cambridge, UK, 1998. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) G. A. Osner. J. Agric. Res. 13:295, 1918.

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First report of Colletotrichum fioriniae causing anthracnose on mu oil tree in China

Plant Disease ◽

10.1094/pdis-03-21-0502-pdn ◽

2021 ◽

Author(s):

Yang Zhang ◽

Guangqiang Li ◽

Dou Yang ◽

Ruoling Zhang ◽

Songze Wan

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Juglans Regia ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

First Report ◽

Spot Disease ◽

Initial Symptoms ◽

Oil Plant

Mu oil tree (Vernicia montana) is an economically important woody oil plant, which is widely distributed in southern China. In mid-May 2020, a leaf spot disease was observed on the leaves of mu oil tree in Taihe County in Jiangxi Province, China (26°55′25.55″N, 114°49′5.85″E). The disease incidence was estimated to be above 40%. Initial symptoms were circular red-brown spots which were 1-2 mm in diameter, then enlarged with red-brown center. In later stages, the spots coalesced and formed large patches, and subsequently red-brown centers of lesions gradually dried and fell out, forming a “shot hole” appearance. To identify the pathogen, diseased leaves were collected from Taihe County. Leaf tissues (5 × 5 mm) were cut from the margins of typical symptomatic lesions, surface- sterilized in 75% ethanol for 30 seconds and 3% sodium hypochlorite for 60 seconds, then rinsed with sterile distilled water three times. Leaf pieces were placed on potato dextrose agar (PDA; 1.5%, Difco-BD Diagnostics) and incubated at 25 °C in the dark. Pure cultures were obtained from individual conidia by recovering single spores. On PDA, colonies were initially white and cottony. The mycelia then became pinkish to deep-pink with time at the center on the front side and pink on the reverse side. Colonies produced pale orange conidial masses after 9 days. Conidia were fusiform with acute ends, smooth-walled, hyaline, and measured 3.6–5.5 × 8.1–14.5 µm (4.5 ± 0.5 × 10.6 ± 1.0 µm, n = 100). The morphological characteristics of the isolate matched the descriptions of Colletotrichum acutatum complex (Damm et al. 2012). For molecular identification, the internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), beta-tubulin 2 (TUB2), and actin (ACT) were sequenced using the primers ITS1/ITS4, GDF/GDR, CHS-79F/CHS-345R, T1/Bt2b, ACT-512F/ACT-783R, respectively (Weir et al. 2012). The obtained sequences were deposited into the GenBank [accession nos. MW584317 (ITS); MW656269 (GAPDH); MW656270 (TUB2); MW656268 (CHS-1); MW656267 (ACT)]. All the sequences showed 94 to 100% similarity with those of C. fioriniae. A neighbor-joining phylogenetic tree was generated by combining all the sequenced loci using MEGA7.0 (Kumar et al. 2016). The isolate TH-M4 clustered with C. fioriniae, having 99% bootstrap support. Base on the morphology and multi-gene phylogeny, isolate TH-M4 was identified as C. fioriniae (Damm et al. 2012). To confirm pathogenicity, 20 healthy leaves of 10 mu oil trees (3-year-old) grown outdoors were inoculated with a drop of spore suspension (106 conidia per mL) of the isolate TH-M4 in September 2020. Another 10 plants were inoculated with sterile water as the control. The leaves were wounded with a sterile toothpick. All the inoculated leaves were covered with black plastic bags to maintain humidity for 2 days. The pathogenicity test was repeated twice. The resulting symptoms were similar to those on the original infected plants, whereas the control leaves remained asymptomatic. The same fungus was re-isolated from the lesions on the inoculated plant, fulfilling Koch’s postulates. C. fioriniae has been recorded as anthracnose pathogen on Mahonia aquifolium (Garibaldi et al. 2020), Paeonia lactiflora (Park et al. 2020), Solanum melongena (Xu et al. 2020), and Juglans regia (Varjas et al. 2020). To our knowledge, this is the first report of C. fioriniae associated with leaf spot disease on mu oil tree in China. This study provided crucial information for epidemiologic studies and appropriate control strategies for this oil plant disease.

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First Report of Leaf Spot Caused by Pseudocercospora pruni-persicicola on Sweet Cherry in Korea

Plant Disease ◽

10.1094/pdis-09-13-0968-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 693-693

Author(s):

I. Y. Choi ◽

U. Braun ◽

J. H. Park ◽

H. D. Shin

Keyword(s):

Leaf Spot ◽

Sweet Cherry ◽

Prunus Persica ◽

Morphological Characteristics ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

Voucher Specimen ◽

First Report ◽

Close Phylogenetic Relationship

Sweet cherry, Prunus avium (L.) L., is not much cultivated in Korea, with only 150 ha planted for domestic consumption. In September 2012, a previously unknown leaf spot was observed with nearly 100% incidence on trees (cv. Seneca) planted in a plastic greenhouse in Iksan City of Korea. Interestingly, the same cultivar as well as other cultivars planted outdoors did not show these symptoms. Leaf spots were irregular to subcircular, dark brown with or without a yellow halo, and becoming coalesced to cause leaf blight and premature defoliation. A cercosporoid fungus was consistently associated with disease symptoms. Fungal structures within the lesion developed on both leaf sides but mostly on the upper side. Stromata were well-developed, globular, dark brown, composed of textura angularis-globosa, and 30 to 80 μm in diameter. Conidiophores were densely fasciculate, pale olivaceous to pale brown, subcylindrical, geniculate-sinuous, 8 to 24 × 3 to 4 μm, and aseptate to 2-septate. Conidiogenous loci were inconspicuous, neither thickened nor darkened. Conidia were olivaceous, generally darker than conidiophores, cylindrical to obclavate, almost straight to mildly curved, short obconically truncate at the base, obtuse at the apex, 1- to 10-septate, constricted at the septa, 12 to 86 × 3.5 to 5 μm, guttulate, and had unthickened, not darkened hila. Morphological characteristics of the fungus were consistent with previous descriptions of Pseudocercospora pruni-persicicola (J.M. Yen) J.M. Yen (1,3). A voucher specimen was deposited in the Korea University herbarium (Accession No. KUS-F27264) and a monoconidial isolate was deposited in the Korean Agricultural Culture Collection (Accession No. KACC47019). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 (4) and sequenced. The resulting 505-bp sequence was deposited in GenBank (Accession No. KF670713). A BLAST search in GenBank revealed that the sequence showed >99% similarity with sequences of many Pseudocercospora species, indicating the close phylogenetic relationship of species in this genus. To conduct a pathogenicity test, a conidial suspension (~1 × 104 conidia/ml) was prepared in sterile water by harvesting conidia from 2-week-old cultures on V8 juice agar, and the suspension was sprayed until runoff onto the leaves of five healthy seedlings. Control plants were sprayed with sterile water. The plants were covered with plastic bags to maintain a relative humidity of 100% for 48 h and then transferred to a greenhouse. Necrotic spots appeared on the inoculated leaves 20 days after inoculation, and were identical to the ones observed in the field. P. pruni-persicicola was re-isolated from symptomatic leaf tissues, fulfilling Koch's postulates. Control plants remained symptomless. The fungus has previously been recorded on Prunus persica (L.) Stokes in Taiwan (2,3). To our knowledge, this is the first report of this fungus on P. avium globally as well as in Korea. The disease poses a new threat to the sweet cherry industry in Korea. References: (1) U. Braun and V. A. Melnik. Cercosporoid Fungi from Russia and Adjacent Countries. Rus. Acad. Sci., St.-Petersburg, 1997. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, Retrieved August 24, 2013. (3) J. M. Yen. Rev. Mycol. 42:57, 1978. (4) T. J. White et al. PCR Protocols. Academic Press, San Diego, CA, 1990.

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First Report of Leaf Spot Caused by Didymella americana on Cassia nomame in China

Plant Disease ◽

10.1094/pdis-08-21-1799-pdn ◽

2021 ◽

Author(s):

Weiming Sun ◽

Lina Feng ◽

Xiaolei Wen ◽

Bojia Han ◽

Danrun Xing ◽

...

Keyword(s):

Sequence Analysis ◽

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Rrna Gene ◽

Pathogenicity Test ◽

Koch’S Postulates ◽

First Report ◽

Initial Symptoms ◽

Koch's Postulates

Cassia nomame (Sieb.) Kitagawa is an annual plant in the Leguminousae family. The aerial parts of C. nomame have been used as tonic and diuretic in Korea and Japan (Syed et al. 2019). A leaf spot was observed on the leaves of a 1-year-old C. nomame landrace in Changli County (39.42°N, 119.10°E), Qinhuangdao City, Hebei Province during August to October in 2018. In many fields (n≥3), the disease incidence over 80% in the middle and late stage of plant growth. Symptoms on leaves in one field began with many small, dark necrotic spot lesions. Later, the lesions spread to round-to-oval, slightly sunken in the center, and large necrotic patches with indefinite margins. Finally, lesions coalesced and resulted in defoliation. Lesions were occasionally observed on the pods. Symptoms on the pods were initially small, dark spots and then expanded to large necrotic patches with irregular edges. Symptomatic tissues (n=32) from pods and leaves were cut into 3 to 8 mm2 squares, surface disinfested with 75% ethanol for 10 s, rinsed with sterile distilled water, then placed on potato dextrose agar (PDA) at 28℃. After 3 days, ten isolates with consistent characteristics were obtained with a frequency 52.6%. The isolates on PDA were round, initially pale and had little aerial mycelium, gradually turned olive green and had dense wool-like dense aerial mycelia after 3 days. Conidia were hyaline, smooth, solitary, and elliptical. The conidia measured 5.4 to 8.2 μm × 2.5 to 3.8 μm (n=50), and has two oil bodies positioning at opposite poles. Pigmented chlamydospores were spherical or nearly pear-shaped, and solitary. Black fructifications (pycnidia) were produced profusely on PDA after subculture for 3 days. All the isolates were similar to Didymella sp. in morphology (Aveskamp et al. 2009). Choice three isolates YSGUO8 YSGGUO8-a and YSGGUO8-b to be further characterized by sequencing of the internal transcribed spacer (ITS), actin gene, and 28S large subunit of the nuclear rRNA gene (LSU) (Zhang et al. 2017). The sequences of three strains (MK836417 MZ484072 and MZ484073 for ITS, MK837604 MZ593675 and MZ593676 for actin, MK843781 MZ836208 and MZ836207 for LSU, respectively) showed 99% to 100% similarity with Didymella americana K-004 (KY070279 for ITS,KY070285 for LSU), Phoma americana CBS 256.65 (FJ426973 for ITS, FJ426871 for actin, MH870196 for LSU) and P. americana CBS 185.85 (FJ426972 for ITS, FJ426870 for actin, GU237990 for LSU) in GenBank. The fungi were identified as D. americana (formerly P. americana or Peyronellaea americana) on the basis of morphological characteristics and sequence analysis. A pathogenicity test was conducted with three times on 1-year-old C. nomame strain at the 4 to 6 compound leaf stage. Conidia were obtained from 7-day-old PDA cultures grown at 28℃ with a 12-h photoperiod. Koch’s postulates were fulfilled by spray inoculating ten healthy young plants with 106 conidia per milliliter of D. americana strain YSGUO8, and sterile water as the control. After inoculation, the plants were managed at 28℃, 60% relative humidity and a 12-h photoperiod. After 5 to 8 days, the inoculated leaves developed small and dark spots lesions similar to those observed on the leaves with initial symptoms in the field. The control leaves remained symptomless. The same fungi were re-isolated from infected leaves by morphology observation and sequence analysis, confirming Koch's postulates. D. americana has caused leaves spot on Table Beet in New York (Vaghefi et al. 2016). To our knowledge, this is the first report of D. americana causing leaf spot of C. nomame in China.

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First Report of Alternaria alternata Causing Leaf Spot of Tartary Buckwheat (Fagopyrum tataricum) in China

Plant Disease ◽

10.1094/pdis-01-21-0191-pdn ◽

2021 ◽

Author(s):

Shaoqing Li ◽

quan shen ◽

Haihua Wang ◽

Feng He ◽

Zuyin Xiao ◽

...

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Tartary Buckwheat ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

Fagopyrum Tataricum ◽

First Report ◽

Fungal Isolates ◽

Initial Symptoms

Buckwheat (Fagopyrum tataricum) is recognized as a healthy food with abundant nutrients and high levels of rutin. In April and May of 2020, an unknown tartary buckwheat leaf spot distinct from Nigrospora leaf spot (Shen et al. 2020) was observed in Xiangxiang, Hunan, China (27°49′54″N, 112°span style="font-family:'Times New Roman'; color:#0000ff">18′48″E.). Disease incidence was 60-70% within three fields (totally 7, 000 m2). The disease occurred after plants emerged. Initial symptoms began as circular, or ellipsoid, chlorotic, water-soaked spots, mostly on leaf apexes or leaf margins. The small spots gradually enlarged and often coalesced to form large circular or irregular, pale to light brown lesions, and the infected leaves eventually withered and fell off. Thirty 2 × 2 mm infected tissue pieces collected from five locations were sterilized in 70% ethanol for 10 S, in 2% NaClO for 30 S, rinsed in sterile water for three times, dried, and placed on PDA with lactic acid (3 ml/L). After 3-5 days at 28°C in the dark, 17 fungal isolates were purified using single-spore isolation method. Almost all fungal isolates had similar morphology. Colonies were initially olive green with white margin and later turned dark olive or black with profuse sporulation. Conidia were borne in long chains, tawny to brownish green, with 1-3 longitudinal and 1-7 transverse septa, pyriform, and measured 9.5-39.6 µm long, and 5.1-12.6 µm wide (n=50). Based on morphological characteristics, the fungus was identified as Alternaria alternata (Simmons 2007). Partial internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α(TEF) and Alternaria major allergen (Alt a1) genes of isolate BLS-1 were amplified using ITS1/ITS4 (Mills et al. 1992), EF1-728F/EF1-986R (Carbone and Kohn 1999), Gpd1/Gpd2 and Alt-4for/Alt-4rev (Lawrence et al. 2013), respectively. Sequences were deposited into GenBank with acc. nos MW453091 (ITS), MW480219 (GAPDH), MW480218 (TEF), and MW480220 (Alt a1). BLASTn analysis showed 99.8% (ITS, MH854758.1), 100% (GAPDH, KP124155.1), 99.8% (TEF, KP125073.1) and 100% (Alt a1, KP123847.1) identity with reference strain CBS 106.24 of A. alternata, confirming isolate BSL-1 to be A. alternata. A neighbor-joining phylogenetic tree constructed by MEGA7.0 based on concatenated sequences of the four genes indicated that BSL-1 formed a distinct clade with A. alternata CBS 106.24 with 100% bootstrap values. Pathogenicity test was triplicately performed on healthy leaves. Twenty leaves of five 20-day-old plants (cv. Pinku1) were sprayed with conidial suspension (1×106 conidia/ml) collected from PDA cultures with 0.05% Tween 20. An equal number of control leaves were sprayed with sterile water to serve as the controls. Treated plants were kept in a greenhouse at 28±3 °C with relative humidity of 80±5% for 24 h and transferred to natural conditions (22-30°C, RH 50-60%). After 4 to 6 days, all inoculated leaves developed symptoms similar to those observed in the fields, while the control leaves remained healthy. A. alternata was re-isolated from all infected leaves. Occasionally-isolated Diaporthe isolates were not pathogenic. A. alternata causes leaf spot of oat (Zhao et al. 2020) and leaf blight of F. esculentum (Lu et al. 2019). To our knowledge, this is the first report of A. alternata causing leaf spot on F. tataricum in China and the world. Effective strategies should be developed to manage the disease.

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First report of banana (Musa acuminate L. AAA Cavendish, cv. Formosana) leaf spot disease caused by Curvularia geniculata in China

Plant Disease ◽

10.1094/pdis-09-21-2001-pdn ◽

2021 ◽

Author(s):

Yanxiang Qi ◽

Yanping Fu ◽

Jun Peng ◽

Fanyun Zeng ◽

Yanwei Wang ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Universal Primers ◽

Leaf Spot Disease ◽

Leaf Margin ◽

Conidial Suspension ◽

Tropical Fruit ◽

First Report ◽

Spot Disease

Banana (Musa acuminate L.) is an important tropical fruit in China. During 2019-2020, a new leaf spot disease was observed on banana (M. acuminate L. AAA Cavendish, cv. Formosana) at two orchards of Chengmai county (19°48ʹ41.79″ N, 109°58ʹ44.95″ E), Hainan province, China. In total, the disease incidence was about 5% of banana trees (6 000 trees). The leaf spots occurred sporadically and were mostly confined to the leaf margin, and the percentage of the leaf area covered by lesions was less than 1%. Symptoms on the leaves were initially reddish brown spots that gradually expanded to ovoid-shaped lesions and eventually become necrotic, dry, and gray with a yellow halo. The conidia obtained from leaf lesions were brown, erect or curved, fusiform or elliptical, 3 to 4 septa with dimensions of 13.75 to 31.39 µm × 5.91 to 13.35 µm (avg. 22.39 × 8.83 µm). The cells of both ends were small and hyaline while the middle cells were larger and darker (Zhang et al. 2010). Morphological characteristics of the conidia matched the description of Curvularia geniculata (Tracy & Earle) Boedijn. To acquire the pathogen, tissue pieces (15 mm2) of symptomatic leaves were surface disinfected in 70% ethanol (10 s) and 0.8% NaClO (2 min), rinsed in sterile water three times, and transferred to potato dextrose agar (PDA) for three days at 28°C. Grayish green fungal colonies appeared, and then turned fluffy with grey and white aerial mycelium with age. Two representative isolates (CATAS-CG01 and CATAS-CG92) of single-spore cultures were selected for molecular identification. Genomic DNA was extracted from the two isolates, the internal transcribed spacer (ITS), large subunit ribosomal DNA (LSU rDNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1-α) and RNA polymerase II second largest subunit (RPB2) were amplified and sequenced with universal primers ITS1/ITS4, LROR/LR5, GPD1/GPD2, EF1-983F/EF1-2218R and 5F2/7cR, respectively (Huang et al. 2017; Raza et al. 2019). The sequences were deposited in GenBank (MW186196, MW186197, OK091651, OK721009 and OK491081 for CATAS-CG01; MZ734453, MZ734465, OK091652, OK721100 and OK642748 for CATAS-CG92, respectively). For phylogenetic analysis, MEGA7.0 (Kumar et al. 2016) was used to construct a Maximum Likelihood (ML) tree with 1 000 bootstrap replicates, based on a concatenation alignment of five gene sequences of the two isolates in this study as well as sequences of other Curvularia species obtained from GenBank. The cluster analysis revealed that isolates CATAS-CG01 and CATAS-CG92 were C. geniculata. Pathogenicity assays were conducted on 7-leaf-old banana seedlings. Two leaves from potted plants were stab inoculated by puncturing into 1-mm using a sterilized needle and placing 10 μl conidial suspension (2×106 conidia/ml) on the surface of wounded leaves and equal number of leaves were inoculated with sterile distilled water serving as control (three replicates). Inoculated plants were grown in the greenhouse (12 h/12 h light/dark, 28°C, 90% relative humidity). Necrotic lesions on inoculated leaves appeared seven days after inoculation, whereas control leaves remained healthy. The fungus was recovered from inoculated leaves, and its taxonomy was confirmed morphologically and molecularly, fulfilling Koch’s postulates. C. geniculata has been reported to cause leaf spot on banana in Jamaica (Meredith, 1963). To our knowledge, this is the first report of C. geniculata on banana in China.

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