Page 1 of 3 1 First Report of Leaf Blight Caused byAlternaria brassicicolaon 2 Orychophragmus violaceus in China 3 C. Guo, C. M. Wang, T. W. Zhou and S. L. Jin, InstituteofPlantProtection,GansuAcademyofAgriculturalSciences,Lanzhou 730070,China; andC. X. Duan, Institute of Crop Science,ChineseAcademyof Agricultural Sciences, Beijing100081, China. Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-08-18-1458-PDN • posted 12/14/2018 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. 4 5 6 7 Orychophragmus violaceus(February orchid), is an annual or 8 perennial cruciferous herbaceous plant thatis widely cultivated and usedas 9 an ornamental plantor as a green manure in China. In September 2017, a 10 new leaf blight disease was observed on O.violaceusp lants with 80% to 11 100% disease incidence in a field located in Huanghua County, Cangzhou 12 City, Hebei Province in northern China. Symptoms on affected leaves 13 began as pinpoint spots that enlarged to form irregular to circular black 14 spots with an obvious wheel striatum, approximately 12 mm in 15 diameter,after fourteen days. After seven days lesions became necrotic 16 and occupied 30% to 70% of the leaf surface.Approximately 5 × 5 mm 17 pieces from the margin of necroticleaves weresurface disinfested with 75% 18 ethanol and 10% sodium hypochlorite, rinsed three times with sterile 19 distilled water, and placed onto potato dextrose agar (PDA). After 2 days 20 of incubation at 25°C in the dark, newly grown-out mycelia were 21 transferred onto fresh PDA and purified by the singlesporemethod. On 22 PDA, olive-green mycelium developed after 2 days of incubationat 25°C, 23 turned dark green, and covered the petri dish 10 days later. Primary 1 Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-08-18-1458-PDN • posted 12/14/2018 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 2 of 3 24 conidiophores were mostly simple with a single conidiogenous site. 25 Conidia formed long branched chains 12 to 125 μm length. The youngest 26 conidia in a chain were ovoid, 8.7 to 26.1 × 4.4 to 6.2 μm. Older conidia 27 were larger, ellipsoid to ovoid with dimensions 25.8 to 35.2 × 10.9 to 28 11.8 μm. Conidiahad 0 to 8 transverse septa, usually without longitudinal 29 septa.A very small conidial beak or no beak was observed on each 30 conidium. On the basis of these morphological characteristicsthe fungus 31 was identified as Alternaria brassicicola(Wiltshire, 1947).For molecular 32 identification, the translation elongation factor-1α (TEF-1α) and internal 33 transcribed spacer (ITS) region of ribosomal RNA were amplified and 34 sequenced from fourisolates (GS1-1, GS1-2, HH2-1, HH3-1) (Carbone& 35 Kohn, 1999, White et al, 1990). Partial translation elongation factor 36 (TEF-1α) gene sequences and ITS(ITS1/ITS2) of isolate GS1-1, GS1-2, 37 HH2-1, and HH3-1weregenerated(GenBank Accession No.MH424919, 38 MK249721, 39 MK257127, MK257128). BLASTn analysis revealed 100% sequence 40 identity to A.brassicicola(KT895946, KF889266, JX213350, KF889266; 41 MH102059,MF462311, MF462313, JF439458). MK249722, MK249723; MH424918, MK249724, 42 Pathogenicity testing with four single conidium isolates (GS1-1, 43 GS1-2, HH2-1, HH3-1) were conducted by inoculating leaves with a 44 conidial suspensionof the individual isolates (diluted with distilled water, 2 Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-08-18-1458-PDN • posted 12/14/2018 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 3 of 3 45 approximately 1×106 conidia/ml) ontwenty plants at the three-leaf stage 46 and placing them in a humidity chamber at 25°C for 24 h.The twenty 47 plants at the three-leaf stage were mock inoculated with distilled water as 48 a control. Initial symptoms developed after 3 days and the typical 49 symptoms 5 days after inoculation withA.brassicicolaand were similar to 50 those observed in the field. The pathogen was reisolatedand confirmed to 51 be A.brassicicola, completing Koch’s postulates. No symptoms were 52 observed on control leaves. To our knowledge, this is the first report of 53 A.brassicicola causing leaf blight on O.violaceus in northern China. The 54 summer in Hebei province is hot and very wet with an average 55 temperature and precipitation in July being 26°Cand 627 mm, 56 respectively. Because of the suitable climatic conditions for leaf blight, 57 strategies for managing A. brassicicola on O. violaceus in this region 58 should be developed. 59 References: 60 Carbone, I., and Kohn, L. M. 1999. Mycologia. 91: 553. 61 White, T. J., et al. 1990. Page 315 in: PCR protocols: a guide to methods 62 and applications. Academic Press, San Diego. 63 Wiltshire, S. P. 1947. Mycological Papers. 20: 1. 3