First Report of Leptosphaerulina Leaf Spot Caused by <i>Leptosphaerulina trifolii</i> on Alfalfa in Heilongjiang Province, China

HomePlant DiseaseVol. 103, No. 10First Report of Leptosphaerulina Leaf Spot Caused by Leptosphaerulina trifolii on Alfalfa in Heilongjiang Province, China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Leptosphaerulina Leaf Spot Caused by Leptosphaerulina trifolii on Alfalfa in Heilongjiang Province, ChinaX. P. Liu, X. M. Jing, H. X. Yan, G. L. Li, and Y. H. LuoX. P. Liu†Corresponding author: X. P. Liu; E-mail Address: [email protected]http://orcid.org/0000-0002-9977-7295College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, China, X. M. JingCollege of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, China, H. X. YanCollege of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, China, G. L. LiCollege of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, China, and Y. H. LuoCollege of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, ChinaAffiliationsAuthors and Affiliations X. P. Liu † X. M. Jing H. X. Yan G. L. Li Y. H. Luo College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, China Published Online:7 Aug 2019https://doi.org/10.1094/PDIS-04-19-0693-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Foliar pathogens significantly reduce alfalfa (Medicago sativa L.) yield and quality (Nutter et al. 2002). In July 2015, a major foliar disease was found in most alfalfa fields, especially on regrown plant foliage after the first harvest, in Heilongjiang province of China. Infected plants had small black pepper spots on young leaves or larger "eye spots" (diameter, 1 to 3 mm). The lesion center was light to yellowish brown with a dark brown edge. A chlorotic area usually surrounded each lesion. Lesions merged in some cases, causing leaf scorch and leaflet death. Infected leaflet tissue fragments (∼5 mm) were surface sterilized with 75% ethanol for 30 s and 0.1% HgCl2 for 3 min, rinsed three times with sterile water, dried, and then incubated for 10 days on 20% V8 juice agar (Miller 1955) plates at 22°C for 14 h of light per day. Several strains were isolated from different leaflet fragments. Colonies on V8 plates were round and appressed, had neat edges and radial growth, and had a few white mycelia with many ascocarps distributed in black globules on the entire surface of, or within, the medium. After 10 days, dispersed ascospores formed many small colonies around the main colony. Ascocarps were moved to a clean microscope slide to release asci and ascospores. Ascocarps measured 53 to 98 × 31 to 48 µm (length × width) and contained several asci, each containing eight ascospores. Ascospores (25 to 48 × 11 to 19 µm) were oval or obovate-elliptic, transparent, and mostly muriform (two to four cross walls, zero to two longitudinal walls). Morphological characters were consistent with Leptosphaerulina trifolii (Olanya et al. 1990). Five representative isolates with similar morphological characters were chosen for internal transcribed spacer (ITS) sequencing. Total DNA of each isolate was extracted from a 20-day-old colony grown on V8 juice agar. PCR analysis was conducted using the primer pairs ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). BLAST analysis of ITS sequences showed that all five isolates had identical ITS sequences (accession no. MK379997) that were 99 to 100% identical to L. trifolii (acc. nos. AY131203 and MH860866). Isolate pathogenicity was tested on 20 alfalfa plants at the eight-leaf stage by inverting sporulating cultures of each isolate over plants for 2 days. Another 20 plants served as controls without inoculation. All plants were maintained at room temperature (22 to 24°C) and 65% relative humidity in a growth chamber. Typical pepper spot lesions, similar to symptoms observed on samples collected in the field, appeared on infected plants within 10 days after inoculation. Control plants showed no symptoms. The pathogen reisolated from the infected leaves was identical to the original culture. Following Koch's postulates, the strains were capable of causing Leptosphaerulina leaf spot on alfalfa. Based on disease symptoms and pathogen identification, the strains isolated caused Leptosphaerulina leaf spot on alfalfa. This disease is known from Australia, Canada, and North America (Barbetti 1991; Hwang et al. 2006; Rizvi et al. 1993); it causes pepper spot disease in most annual Medicago species (Barbetti 2007). To our knowledge, this is the first report of Leptosphaerulina leaf spot caused by L. trifolii on alfalfa in China. This disease may reduce alfalfa yields, with important economic consequences (Nutter et al. 2002).The author(s) declare no conflict of interest.References: Barbetti, M. J. 1991. Plant Pathol. 40:296. https://doi.org/10.1111/j.1365-3059.1991.tb02379.x Crossref, ISI, Google ScholarBarbetti, M. J. 2007. Plant Dis. 91:239. https://doi.org/10.1094/PDIS-91-3-0239 Link, ISI, Google ScholarHwang, S.-F., et al. 2006. Eur. J. Plant Pathol. 115:389. https://doi.org/10.1007/s10658-006-9027-2 Crossref, ISI, Google ScholarMiller, P. M. 1955. Phytopathology 45:461. ISI, Google ScholarNutter, F. W., et al. 2002. Plant Dis. 86:269. https://doi.org/10.1094/PDIS.2002.86.3.269 Link, ISI, Google ScholarOlanya, O. M., et al. 1990. Phytopathology 80:1278. https://doi.org/10.1094/Phyto-80-1278 Crossref, ISI, Google ScholarRizvi, S. S. A., et al. 1993. Plant Dis. 77:1126. https://doi.org/10.1094/PD-77-1126 Crossref, ISI, Google ScholarThe author(s) declare no conflict of interest.Funding: This work was supported by the program for the National Natural Science Foundation of China (project no. 31702169) and Young Scholars with Creative Talents in Heilongjiang Bayi Agricultural University (project no. CXRC2017006).DetailsFiguresLiterature CitedRelated Vol. 103, No. 10 October 2019SubscribeISSN:0191-2917e-ISSN:1943-7692 DownloadCaptionBell pepper cv. Bomby infected with zucchini yellow mosaic virus along with cucumber mosaic virus, pepper mild mottle virus, and tobacco mosaic virus (Verma et al.). Photo credit: S. Tripathi. 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Metrics Article History Issue Date: 3 Oct 2019Published: 7 Aug 2019First Look: 11 Jun 2019Accepted: 5 Jun 2019 Pages: 2673-2673 Information© 2019 The American Phytopathological SocietyFundingNational Natural Science Foundation of ChinaGrant/Award Number: 31702169Young Scholars with Creative Talents in Heilongjiang Bayi Agricultural UniversityGrant/Award Number: CXRC2017006KeywordsalfalfaMedicago sativa L.Leptosphaerulina leaf spotfungiLeptosphaerulina trifoliiThe author(s) declare no conflict of interest.Cited byOccurrence and Nutrition Indicators of Alfalfa withLeptosphaerulina in Chifeng, Inner Mongolia14 September 2022 | Agriculture, Vol. 12, No. 9First Report of Alfalfa Leaf Spot Caused by Leptosphaerulina australis in ChinaLi Li Zhang and Yan Zhong Li22 September 2021 | Plant Disease, Vol. 105, No. 8First Report of Leptosphaerulina Leaf Spot Caused by Leptosphaerulina trifolii on Trifolium repens in BrazilA. A. D. Victoria, B. E. Furtado, M. T. Holz, O. Romero-Arenas, and L. J. 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