First Report of Leaf Blight on Parthenium hysterophorus Caused by Nigrospora sphaerica in Malaysia

Weeds may act as inoculum reservoirs for fungal pathogens that could affect other economically important crops (Karimi et al. 2019). In February 2019, leaves of the ubiquitous invasive weed, Parthenium hysterophorus L. (parthenium weed) exhibiting symptom of blight were observed at Ladang Infoternak Sg. Siput (U), a state–owned livestock center in Perak, Malaysia. Symptoms appeared as irregularly shaped, brown–to–black necrotic lesions across the entire leaf visible from both surfaces, and frequently on the older leaves. The disease incidence was approximately 30% of 1,000 plants. Twenty symptomatic parthenium weed leaves were collected from several infested livestock feeding plots for pathogen isolation. The infected tissues were sectioned and surface–sterilized with 70% ethyl alcohol for 1 min, rinsed three times with sterile distilled water, transferred onto potato dextrose agar, and incubated at 25°C under continuous dark for 7 days. Microscopic observation revealed fungal colonies with similar characteristics. Mycelium was initially white and gradually changed to pale orange on the back of the plate but later turned black as sporulation began. Conidia were spherical or sub–spherical, single–celled, smooth–walled, 12 to 21 μm diameter (mean = 15.56 ± 0.42 μm, n= 30) and were borne on a hyaline vesicle. Based on morphological features, the fungus was preliminarily identified as Nigrospora sphaerica (Sacc) E. W. Mason (Wang et al. 2017). To confirm identity, molecular identification was conducted using isolate 1SS which was selected as a representative isolate from the 20 isolates obtained. Genomic DNA was extracted from mycelia using a SDS–based extraction method (Xia et al. 2019). Amplification of the rDNA internal transcribed spacer (ITS) region was conducted with universal primer ITS1/ITS4 (White et al. 1990; Úrbez–Torres et al. 2008). The amplicon served as a template for Sanger sequencing conducted at a commercial service provider (Apical Scientific, Malaysia). The generated sequence trace data was analyzed with BioEdit v7.2. From BLASTn analysis, the ITS sequence (GenBank accession number. MN339998) had at least 99% nucleotide identity to that of N. sphaerica (GenBank accession number. MK108917). Pathogenicity was confirmed by spraying the leaf surfaces of 12 healthy parthenium weed plants (2–months–old) with a conidial suspension (106 conidia per ml) collected from a 7 day–old culture. Another 12 plants served as a control treatment and received only sterile distilled water. Inoculation was done 2 h before sunset and the inoculated plants were covered with plastic bags for 24 h to promote conidial germination. All plants were maintained in a glasshouse (24 to 35°C) for the development of the disease. After 7 days, typical leaf blight symptoms developed on the inoculated plants consistent with the symptoms observed in the field. The pathogen was re–isolated from the diseased leaves and morphological identification revealed the same characteristics as the original isolate with 100% re–isolation frequency, thus, fulfilling Koch’s postulates. All leaves of the control plants remained symptomless and the experiment was repeated twice. In Malaysia, the incidence of N. sphaerica as a plant pathogen has been recorded on several important crops such as watermelon and dragon fruit (Kee et al. 2019; Ismail and Abd Razak 2021). To our knowledge, this is the first report of leaf blight on P. hysterophorus caused by N. sphaerica from this country. This report justifies the significant potential of P. hysterophorus as an alternative weed host for the distribution of N. sphaerica. Acknowledgement This research was funded by Universiti Putra Malaysia (UPM/GP–IPB/2017/9523402). References Ismail, S. I., and Abd Razak, N. F. 2021. Plant Dis. 105:488. Karimi, K., et al. 2019. Front Microbiol. 10:19. Kee, Y. J., et al. 2019. Crop Prot. 122:165. Úrbez–Torres, J. R., et al. 2008. Plant Dis. 92:519. Wang, M., et al. 2017. Persoonia 39:118. White, T. J. et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Xia, Y., et al. 2019. Biosci Rep. 39:BSR20182271.

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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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First report of Alternaria alternata causing leaf blight on little millet (Panicum sumatrense) in India.

Plant Disease ◽

10.1094/pdis-06-20-1373-pdn ◽

2020 ◽

Author(s):

Boda Praveen ◽

A. Nagaraja ◽

M. K. Prasanna Kumar ◽

Devanna Pramesh ◽

K. B. Palanna ◽

...

Keyword(s):

Crop Yields ◽

Disease Incidence ◽

Leaf Blight ◽

Post Inoculation ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Pcr Assay ◽

Causal Organism ◽

First Report ◽

Little Millet

Little millet (LM) is a minor cereal crop grown in the Indian sub-continent. During October 2018, dark brown, circular to oval necrotic spots surrounded by concentric rings were observed on the upper leaf surface of the LM (cv. VS-13) grown in the fields of the University of Agricultural Sciences, Bengaluru, India (13.0784oN, 77.5793oE). As the disease progressed, infected leaves became blighted. Disease incidence up to 53% was recorded in 3 fields of 0.4-hectare area each. Thirty symptomatic leaves were collected to isolate the associated causal organism. The margins of diseased tissue were cut into 5 × 5-mm pieces, surface-sterilized in 75% ethanol for 45 seconds followed by 1% sodium hypochlorite for 1 min, finally rinsed in sterile distilled water five times and placed on PDA. After 7 days of incubation at 25°C, greyish fungal colonies appeared on PDA. Single-spore isolations were performed to obtain ten isolates. Pure cultures of the fungus initially produced light gray aerial mycelia that later turned to dark grey. All isolates formed obclavate to pyriform conidia measured 22.66-48.97μm long and 6.55-13.79µm wide with 1-3 longitudinal and 2-7 transverse septa with a short beak (2.55-13.26µm) (n=50). Based on the conidial morphology, the fungus was identified as Alternaria sp. Further, the taxonomic identity of all ten isolates was confirmed as A. alternata using species-specific primers (AAF2/AAR3, Konstantinova et al. 2002) in a PCR assay. Later, one of the isolate UASB1 was selected, and its internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (gapdh), major allergen Alt a 1 (Alt a 1), major endo-polygalacturonase (endoPG), OPA10-2, and KOG1058 genes were amplified in PCR (White et al. 1990; Berbee et al. 1999; Woudenberg et al. 2015), and the resultant products were sequenced and deposited in the NCBI GenBank (ITS, MN919390; gapdh, MT637185; Alt a 1, MT882339; endoPG, MT882340; OPA10-2, MT882341; KOG1058, MT882342). Blastn analysis of ITS, gapdh, Alt a 1, endoPG, OPA10-2, KOG1058 gene sequences showed 99.62% (with AF347031), 97.36% (with AY278808), 99.58% (with AY563301), 99.10% (with JQ811978), 99.05% (with KP124632) and 99.23% (with KP125233) respectively, identity with reference strain CBS916.96 of A. alternata, confirming UASB1 isolate to be A. alternata. For pathogenicity assay, conidial suspension of UASB1 isolate was spray inoculated to ten healthy LM (cv. VS-13) plants (45 days old) maintained under protected conditions. The spore suspension was sprayed until runoff on healthy leaves, and ten healthy plants sprayed with sterile water served as controls. Later, all inoculated and control plants were covered with transparent polyethylene bags and were maintained in a greenhouse at 28±2 ◦C and 90% RH. The pathogenicity test was repeated three times. After 8 days post-inoculation, inoculated plants showed leaf blight symptoms as observed in the field, whereas no disease symptoms were observed on non-inoculated plants. Re-isolations were performed from inoculated plants, and the re-isolated pathogen was confirmed as A. alternata based on morphological and PCR assay (Konstantinova et al. 2002). No pathogens were isolated from control plants. There is an increasing acreage of LM crop in India, and this first report indicates the need for further studies on leaf blight management and the disease impacts on crop yields.

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First Report of Leaf Blight Caused by Septoria allii on Garlic Chives in Korea

Plant Disease ◽

10.1094/pdis-08-12-0809-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 147-147

Author(s):

J. H. Park ◽

S. E. Cho ◽

K. S. Han ◽

H. D. Shin

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Its Region ◽

Leaf Blight ◽

Plant Diseases ◽

Cultivated Plants ◽

Sequence Information ◽

Conidial Suspension ◽

Korean Society ◽

First Report

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.

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First report of Fusarium incarnatum causing spikelet rot on rice in China

Plant Disease ◽

10.1094/pdis-12-20-2660-pdn ◽

2021 ◽

Author(s):

Ling Wang ◽

S. L. Ge ◽

Kehan Zhao ◽

huang Shiwen

Keyword(s):

Natural Science ◽

Disease Incidence ◽

Science And Technology ◽

Agricultural Science ◽

Zhejiang Province ◽

Maturity Stage ◽

Post Inoculation ◽

Distilled Water ◽

Conidial Suspension ◽

First Report

Rice (Oryza sativa L.) is the most important and widely grown crop, covering about 29.9 million ha of total cultivation area in China. In the last decade, spikelet rot disease on rice became much more frequent in the middle and lower reaches of the Yangtze River, China. Fusarium proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg was reported to be a causal agent of spikelet rot on rice in Hangzhou, Zhejiang province (Huang et al. 2012). In September 2019, a survey was conducted to understand the etiology of the disease in the main rice growing regions of Jinshan District of Shanghai. Symptomatic panicles exhibiting reddish or brown discoloration on the glumes were collected from different rice fields, where disease incidence was estimated to be between 20 to 80%. Diseased glumes were cut into small sections (5 × 5 mm) from the boundary of necrotic and healthy tissues, surface-sterilized with 75% ethanol for 30 s and 3% sodium hypochlorite for 90 s, rinsed twice with sterile distilled water, then placed onto 1/5 strength potato dextrose agar (PDA). After 3 to 5 days of incubation at 28°C in the dark, fungal growth with Fusarium-like colonies were transferred to PDA and purified by the single-spore isolation method. A total of 12 isolates were obtained and colonies showed loosely floccose, white mycelium and pale-yellow pigmentation on PDA. Microconidia were ovoid mostly with 0 to 1 septum, and measured 4.2 to 16.6 × 2.5 to 4.1 μm (n = 50). After 5-7 days of inoculation on carnation leaf agar (CLA), macroconidia produced usually had 3 to 5 septa, slightly curved at the apex, ranging from 15.7 to 39.1 × 3.3 to 5.0 μm (n = 50). Chlamydospores were produced in hyphae, most often solitary in short chains or in clumps, ellipsoidal or subglobose with thick and roughened walls. Molecular identification was performed on the representative isolates (JS3, JS9, and JS21). The rDNA internal transcribed spacer (ITS), translation elongation factor (TEF-1α) and β-tubulin (β-TUB) genes were amplified and sequenced using the paired primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (O’Donnell et al. 1998) and T1/T22 (O’Donnell and Cigelnik 1997), respectively. The obtained sequences were deposited in GenBank under accession numbers MT889972 to MT889974 (ITS), MT895844 to MT895846 (TEF-1α), and MT895841 to MT895843 (β-TUB), respectively. BLASTn search of the sequences revealed 99 to 100% identity with ITS (MF356578), TEF-1α (HM770725) and β-TUB (GQ915444) of Fusarium incarnatum isolates. FUSARIUM-ID (Geiser et al. 2004) analysis showed 99 to 100% similarity with sequences of the F. incarnatum-equiseti species complex (FIESC) (FD_01651 and FD_01628). In addition, a phylogenetic analysis based on the concatenated nucleotide sequences placed the isolates in the F. incarnatum clade at 100% bootstrap support. Thus, both morphological observations and molecular criteria supported identification of the isolates as F. incarnatum (Desm.) Sacc (synonym: Fusarium semitectum) (Leslie and Summerell 2006, Nirenberg 1990). Pathogenicity tests were performed on susceptible rice cultivar ‘Xiushui134’. At pollen cell maturity stage, a 2-ml conidial suspension (5 × 105 macroconidia/ml) of each isolate was injected into 10 rice panicles. Control plants were inoculated with sterile distilled water. Then, the pots were kept in a growth chamber at 28°C, 80% relative humidity, and 12 h/12 h light (10,000 lux)/dark. The experiment was repeated two times for each isolate. Two weeks post-inoculation, all inoculated panicles showed similar symptoms with the original samples, whereas no symptoms were observed on the control. The pathogen was re-isolated from inoculated panicles and identified by the method described above to fulfill Koch's postulates. Previous studies reported that F. incarnatum reproduced perithecia to overwinter on rice stubble as the inoculum of Fusarium head blight of wheat in southern China (Yang et al. 2018). To our knowledge, this is the first report of spikelet rot on rice caused by F. incarnatum in China. Further investigation is needed to gain a better understanding its potential geographic distribution of this new pathogen on rice crop. References: (1) Huang, S. W., et al. 2011. Crop Prot. 30: 10. (2) White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. (3) O’Donnell, K., et al. 1998. Proc. Natl. Acad. Sci. U.S.A. 95: 2044. (4) O'Donnell, K., Cigelnik, E. 1997. Mol. Phylogenet. Evol. 7: 103. (5) Geiser, D. M., et al. 2004. Eur. J. Plant Pathol. 110: 473. (6) Leslie, J. F., and Summerell, B. A. 2006. The Fusarium Laboratory Manual. Blackwell, Ames, IA. (7) Nirenberg, H. I. 1990. Stud. Mycol. 32: 91. (8) Yang, M. X., et al. 2018. Toxins. 10: 115. The author(s) declare no conflict of interest. Funding: Funding was provided by National Natural Science Foundation of China (grant no. 31800133), Zhejiang Provincial Natural Science Foundation of China (grant no. LQ18C140005), Key Research and Development Program of Zhejiang Province (grant no. 2019C02018), Shanghai Science and Technology for Agriculture Promotion Project (2019-02-08-00-08-F01127), and the Agricultural Science and Technology Innovation Program of China Academy of Agricultural Science (CAAS-ASTIP-2013- CNRRI).

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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 Leaf Blight of Cyperus iria Caused by Fusarium equiseti in India

Plant Disease ◽

10.1094/pdis-07-12-0690-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 838-838 ◽

Cited By ~ 1

Author(s):

V. Gupta ◽

V. K. Razdan ◽

D. John ◽

B. C. Sharma

Keyword(s):

Leaf Blight ◽

Spore Suspension ◽

Commonwealth Mycological Institute ◽

Fungal Culture ◽

Distilled Water ◽

Total Production ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

Fusarium Equiseti ◽

First Report

In India, rice (Oryza sativa L.) plays a major role in national food security, with total production of 102.75 million t, harvested from 44 million ha during 2011 (1). Weeds are one of the major causes of losses in rice. Cyperus iria, locally known as chatriwala dela (rice flat sedge), is an annual weed in the Cyperaceae that can reach 50 to 60 cm tall. A leaf blight of C. iria was observed during August 2010 in a 20-ha rice field (cv. Basmati 370) at the University Research Farm, Chatha, Jammu (32° 43′ N, 74° 54′ E). Symptomatic plants were scattered randomly in the field and had water-soaked spots on the upper leaf surfaces initially, which turned brown after 4 days and developed a yellow halo, resulting in a blighted appearance. The diseased leaves shriveled and infected plants died. Infected C. iria leaf pieces with adjacent healthy tissue were collected, surface-sterilized in 0.1% mercuric chloride for 20 s, then rinsed three times in sterilized distilled water. The pieces were plated onto potato dextrose agar (PDA) and incubated at 27 ± 1°C for 4 days. A pure fungal culture was obtained by single-spore technique on 2% water agar and maintained on PDA at 10°C. The fungus initially produced white mycelium that became brown with age. Dark brown spots or flecks of pigment formed in the agar. Macroconidia were long and slender, with tapered apical cells that were elongated or even whip-like. Basal cells of macroconidia were prominent, foot shaped, and elongated. Macroconidia were 39.55 to 56.74 × 3.75 to 4.5 μm with 3 to 5 septa. Conidiophores were compact, penicillately branched, and arose from lateral branches which initially were one-celled and bore 2 to 4 phialides at the apex. Chlamydospores were intercalary, solitary, in chains or in knots, globose, and 7 to 9 μm in diameter. On the basis of morphological characteristics (2), the fungus was identified as Fusarium equiseti (Corda) Sacc. and deposited in the Indian Type Culture Collection, New Delhi (8424.11). The ITS (internal transcribed spacer) region of rDNA was amplified by PCR with primers ITS1/ITS2 and sequenced. BLASTn analysis of the sequence showed 100% homology with the ITS sequence of F. equiseti in the NCBI database (JN596252.1), and the sequence was deposited in GenBank (KC434458). To confirm pathogenicity of the F. equiseti isolate, 10 seeds of C. iria were planted in five clay pots (each 38 cm in diameter) filled with sterilized soil. Three seedlings were used for the experiment and the remaining seedlings removed from each pot. A total of 15 seedlings (5 pots × 3 seedlings per pot) at the two-leaf stage were spray-inoculated with a 50-ml conidial suspension of the isolate (105 cfu/ml) using a hand atomizer. The control treatment included three seedlings treated similarly with sterile distilled water. The spore suspension was prepared in potato dextrose broth using a culture of the fungus incubated for 10 days and then homogenized at 140 rpm. Tween 20 (1%) was added to the spore suspension. Small spots developed 4 days after inoculation, and the lesions then coalesced into large necrotic areas, resulting in leaf blight 10 days after inoculation. F. equiseti was reisolated from inoculated leaves using the method described above, whereas no fungus was reisolated from control plants, fulfilling Koch's postulates. The isolated fungus displayed the same morphological and cultural features as the original isolate. F. equiseti has been reported to infect Echinochloa spp. in Iran (3), but to our knowledge, this is the first report of F. equiseti infecting C. iria in India. Thus, F. equiseti represents a potential biocontrol agent for managing C. iria in rice fields. References: (1) Anonymous. Direct. Rice Res. Newslett. 10:2, 2012. (2) C. Booth. The Genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, England, p. 157, 1971. (3) M. R. S. Motlagh. Austral. J. Crop Sci. 4:457, 2010.

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

Plant Disease ◽

10.1094/pdis-09-20-1877-pdn ◽

2021 ◽

Author(s):

Yun-fei Mao ◽

Li Jin ◽

Huiyue Chen ◽

Xiang-rong Zheng ◽

Minjia Wang ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Liquidambar Styraciflua ◽

Leaf Spot Disease ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Wood Processing ◽

Light Yellow

American sweetgum (Liquidambar styraciflua L.) is an important tree for landscaping and wood processing. In recent years, leaf spots on American sweetgum with disease incidence of about 53% were observed in about 1200 full grown plants in a field (about 8 ha) located in Pizhou, Jiangsu Province, China. Initially, dense reddish-brown spots appeared on both old and new leaves. Later, the spots expanded into dark brown lesions with yellow halos. Symptomatic leaf samples from different trees were collected and processed in the laboratory. For pathogen isolation, leaf sections (4×4mm) removed from the lesion margin were surface sterilized with 75% ethanol for 20s and then sterilized in 2% NaOCl for 30s, rinsed three times in sterile distilled water, incubated on potato dextrose agar (PDA) at 25 °C in the darkness. After 5 days of cultivation, the pure culture was obtained by single spore separation. 6 isolate samples from different leaves named FXA1 to FXA6 shared nearly identical morphological features. The isolate FXA1 (codes CFCC 54675) was deposited in the China Center for Type Culture Collection. On the PDA, the colonies were light yellow with dense mycelium, rough margin, and reverse brownish yellow. Conidiophores (23–35 × 6–10 µm) (n=60) were solitary, straight to flexuous. Conidia (19–34 × 10–21 µm) (n=60) were single, muriform, oblong, mid to deep brown, with 1 to 6 transverse septa. These morphological characteristics resemble Stemphylium eturmiunum (Simmons 2001). Genomic DNA was extracted from mycelium following the CTAB method. The ITS region, gapdh, and cmdA genes were amplified and sequenced with the primers ITS5/ITS4 (Woudenberg et al. 2017), gpd1/gpd2 (Berbee et al. 1999), and CALDF1/CALDR2 (Lawrence et al. 2013), respectively. A maximum likelihood phylogenetic analysis based on ITS, gapdh and cmdA (accession nos. MT898502-MT898507, MT902342-MT902347, MT902336-MT902341) sequences using MEGA 7.0 revealed that the isolates were placed in the same clade as S. eturmiunum with 98% bootstrap support. All seedlings for pathogenicity tests were enclosed in plastic transparent incubators to maintain high relative humidity (90%-100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. For pathogenicity, the conidial suspension (105 spores/ml) of each isolate was sprayed respectively onto healthy leaves of L. styraciflua potted seedlings (2-year-old, 3 replicate plants per isolate). As a control, 3 seedlings were sprayed with sterile distilled water. After 7 days, dense reddish-brown spots were observed on all inoculated leaves. In another set of tests, healthy plants (3 leaves per plant, 3 replicate plants per isolate) were wound-inoculated with mycelial plugs (4×4mm) and inoculated with sterile PDA plugs as a control. After 7 days, brown lesions with light yellow halo were observed on all inoculation sites with the mycelial plugs. Controls remained asymptomatic in the entire experiment. The pathogen was reisolated from symptomatic tissues and identified as S. eturmiunum but was not recovered from the control. The experiment was repeated twice with the similar results, fulfilling Koch’s postulates. S. eturmiunum had been reported on tomato (Andersen et al. 2004), wheat (Poursafar et al. 2016), garlic (L. Fu et al. 2019) but not on woody plant leaves. To our knowledge, this is the first report of S. eturmiunum causing leaf spot on L. styraciflua in the world. This disease poses a potential threat to American sweetgum and wheat in Pizhou.

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First Report of Alternaria petroselini Causing Leaf Blight of Fennel in Spain

Plant Disease ◽

10.1094/pdis-01-12-0059-pdn ◽

2012 ◽

Vol 96 (6) ◽

pp. 907-907 ◽

Cited By ~ 2

Author(s):

D. D. M. Bassimba ◽

J. L. Mira ◽

C. Baixauli ◽

A. Vicent

Keyword(s):

Disease Incidence ◽

Leaf Blight ◽

Conidial Suspension ◽

Morphological Examination ◽

Foeniculum Vulgare ◽

Medicinal Uses ◽

First Report ◽

Plastic Bags ◽

Water Plants ◽

Production Areas

Fennel (Foeniculum vulgare Mill.) is an aromatic herb widely cultivated in Mediterranean areas for culinary and medicinal uses. In 2010, symptoms consisting of leaf blight and necrosis were observed in commercial organic fennel production areas in Valencia Province in east-central Spain. Disease incidence in affected fields was approximately 20%. Symptomatic leaves from four fields were surface disinfected with 0.5% NaOCl for 2 min, and small fragments from necrotic lesions were then plated on potato dextrose agar (PDA) amended with 0.5 g of streptomycin sulfate/liter. After 7 days at 25°C, isolates of the genus Alternaria were consistently isolated. Single conidium cultures were grown on PDA and V8 agar for morphological examination. On both agar media, colonies were dark olive brown without production of pigments. On V8 agar, conidia were solitary, darkly pigmented, and predominantly ovoid-subsphaeroid. Mature conidia were 25 to 59 × 12 to 23 μm with up to six to seven transepta and one to three longisepta. The 5.8S, ITS2, and 28S ribosomal RNA (rRNA) regions were amplified with the primers ITS3 and ITS4 (3) from DNA extracted from the isolate IVIA-A029, and sequenced (GenBank Accession No. JQ240204). The sequence had 100% identity (total score 399, 97% coverage) with that of Alternaria petroselini (Neergard) Simmons strain EGS 09-159 (GenBank Accession No. AF229454.1) (1). Pathogenicity tests were conducted on four 3-month-old fennel plants (cv. Giotto) by spraying a conidial suspension of the fungus (10 ml/plant, 103 conidia/ml of water). Four control plants were sprayed with sterile, distilled water. Plants were covered with plastic bags and incubated in a growth chamber for 72 h at 25°C. Leaf necrosis was visible on inoculated plants after 4 days, but symptoms were not observed on control plants. The fungus was reisolated from leaf lesions on inoculated plants, but not from leaves of control plants, confirming Koch's postulates. On the basis of the morphological (2), molecular, and pathogenicity data, the disease was identified as Alternaria leaf blight of fennel caused by A. petroselini. To our knowledge, this is the first report of A. petroselini in Spain. References: (1) B. M. Pryor and R. L. Gilbertson. Mycol. Res. 104:1312, 2000. (2) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands, 2007. (3) T. J. White et al. Pages 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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First report of Diplodia mutila causing leaf blight on Magnolia grandiflora in China

Plant Disease ◽

10.1094/pdis-08-21-1787-pdn ◽

2022 ◽

Author(s):

Xiaosheng Zhao ◽

Chaorong Meng ◽

Xiang-Yu Zeng ◽

Zaifu Yang ◽

Xue-Jun Pan

Keyword(s):

Molecular Characterization ◽

Leaf Blight ◽

Distilled Water ◽

Conidial Suspension ◽

Plastic Film ◽

First Report ◽

Magnolia Grandiflora ◽

Symptomatic Tissue ◽

Grey Center ◽

Β Tubulin

Magnolia grandiflora is a widely cultivated ornamental tree in China. In June 2020, a leaf blight disease was observed on M. grandiflora in Guizhou University (26° 44' 57'' N, 106° 65' 94'' E) in Guiyang, China. The initial symptoms on leaves were expanding round necrotic lesions with a grey center and dark brown edge, and twigs were withered when the disease was serious. Of the 100 plants surveyed 65% had symptoms. To isolate the potential causal pathogen, diseased leaves were collected from an M. grandiflora tree at Guizhou University. Isolations from made form the junction between healthy and symptomatic tissue and disinfested by immersing in 75% ethanol for 30 seconds, 3% NaOCl for 2 minutes, and then washed 3 times in sterile distilled water. Symptomatic tissue was then plated on potato dextrose agar (PDA) and incubated at 25ºC with 12-hour light for 3–5 days. Three isolates (GUCC 21235.1, GUCC 21235.2 and GUCC 21235.3) were obtained. Colonies on PDA after 7 d were dark brown, pycnidia embedded in the mydelium were dark brown to black, single and separated. Conidiophores were transparent measuring 7–12.5 × 2.5–4.5 µm (mean = 9.5 × 3.6 µm, n = 30) in length. Conidia were transparent becoming brown when mature with a diaphragm, with round ends measuring, 21–27 × 10–15 µm (mean = 23.6 × 12.6 µm, n = 30). To confirm the pathogen by molecular characterization, four genes or DNA fragments, ITS, LSU, tef1 and β-tubulin, were amplified using the following primer pairs: ITS4-F/ ITS5-R (White et al., 1990), LR0R/ LR5 (Rehner & Samuels, 1994), EF1-688F/ EF1-986R (Carbone & Kohn, 1999) and Bt2a/ Bt2b (O'Donnell & Cigelnik, 1997). The sequences of four PCR fragments of GUCC 21235.1 were deposited in GenBank, and the accession numbers were MZ519778 (ITS), MZ520367 (LSU), MZ508428 (tef1) and MZ542354 (β-tubulin). Bayesian inference was performed based on a concatenated dataset of ITS, LSU, tef1 and β-tubulin gene using MrBayes 3.2.10, and the isolates GUCC 21235.1 formed a single clade with the reference isolates of Diplodia mutila (Diplodia mutila strain CBS 112553). BLASTn analysis indicated that the sequences of ITS, LSU, tef1 and β-tubulin revealed 100% (546/546 nucleotides), 99.82% (568/569 nucleotides), 100% (302/302 nucleotides), and 100% (437/437 nucleotides) similarity with that of D. mutila in GenBank (AY259093, AY928049, AY573219 and DQ458850), respectively. For confirmation of the pathogenicity of this fungus, a conidial suspension (1×105 conidia mL-1) was prepared from GUCC 21235.1, and healthy leaves of M. grandiflora trees were surface-disinfested by 75% ethanol, rinsed with sterilized distilled water and dried by absorbent paper. Small pieces of filter paper (5 mm ×5 mm), dipped with 20 µL conidial suspension (1×105 conidia mL-1) or sterilized distilled water (as control), were placed on the bottom-left of the leaves for inoculation. Then the leaves were sprayed with sterile distilled water, wrapped with a plastic film and tin foil successively to maintain high humidity in the dark dark. After 36 h, the plastic film and tin foil on the leaves was removed, and the leaves were sprayed with distilled water three times each day at natural condition (average temperature was about 25 °C, 14 h light/10 h dark). After 10 days of inoculation, the same leaf blight began to appear on the leaves inoculated with conidial suspension. No lesion was appeared on the control leaves. The fungus was re-isolated from the symptomatic tissue. Based on the morphological information and molecular characterization, the isolate GUCC 21235.1 is D. mutila. Previous reports indicated that D. mutila infects a broad host range and gives rise to a canker disease of olive, apple and jujube (Úrbez-Torres et al., 2013; Úrbez-Torres et al., 2016; Feng et al., 2019). This is the first report of leaf blight on M. grandiflora caused by D. mutila in China.

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

Plant Disease ◽

10.1094/pdis-93-5-0546b ◽

2009 ◽

Vol 93 (5) ◽

pp. 546-546 ◽

Cited By ~ 2

Author(s):

B. G. Lou ◽

Y. D. Xu ◽

C. Sun ◽

X. M. Lou

Keyword(s):

Southern China ◽

Disease Incidence ◽

Its Region ◽

Culture Collection ◽

Leaf Blight ◽

Its Sequences ◽

Dead Leaf ◽

Conidial Suspension ◽

First Report ◽

Branch Dieback

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

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