First report of Colletotrichum chlorophyti causing peanut anthracnose in China.

Peanut (Arachis hypogaea L.) is an important oil crop - mainly in Shandong and Henan Provinces, in China. In 2018-2019, the occurrence of a black spot disease on the leaves and stems were found in two fields (a total of 10 ha) in Niulingguanzhuang village of Yishui county in Linyi, Shandong Province, China, and 20% to 40% of plants were infected, thereby reducing the amount of marketable product. Natural symptoms were circular, dark brown-to-black lesions (2-6 mm in diameter) and coalescent necrosis on leaves and black necrosis in stems. Six symptomatic leaves and stems collected from six plants of two fields, were used for isolation. Portions of infected tissue were surfaced-sterilized with 0.5% NaClO for two minutes, 70% alcohol for 30 seconds and washed twice with sterile water. The tissues were placed on potato dextrose agar (PDA) at 28℃ for 5 days. Felt or fleece forming colonies about 4 cm in diameter that were circular, flat, and with dark center and white narrow margins were formed. Three isolates (LSJF-4, HSGF, HSJF) were purified and one culture (LSJF-4) was deposited in the China General Microbiological Culture Collection Center (CGMCC, NO. 3.19617). The chlamydospore were dark brown, verruculose, organized in chains and clusters, yellow-brown to black-brown color, and mostly spherical, 10.3 - 18.4 μm × 8.3-11.4 μm (n = 50, av. 12.6 ± 2.1× 10.1 ± 1.1 µm). The conidia were colorless, crescent or sickle-shaped, with one acute end and one blunt end. They were 16.7 - 24.3 μm × 3.6 - 5.5 μm (n= 50, av. 21.2 ± 1.7× 4.5 ± 0.36 µm), with dark brown, straight, septate setae, 40.3 - 86.9 μm × 3.6-5.5 μm (n=50, av. 70.0 ± 19.1 × 4.5 ± 0.7 µm) in size. Setae were straight, dark brown. These morphological characteristics of our isolates were identical to Colletotrichum chlorophyti (Damm et al. 2009). The internal transcribed spacer (ITS, ITS1/ITS4) rDNA of three fungus (LSJF-4, HSGF, HSJF) were amplified using PCR and sequenced, as described by Damm et al (2009). The sequencing results (GenBank Accession No. MK796409, MN756650, MN756651) were submitted to the GenBankand blast analysis showed that it had 99.0% identity with those of Colletotrichum chlorophyti (No. GU227894). Actin (ACT, ACT-512F/ACT-783R), beta-tubulin (Tub2, T1/T2 ), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GDF1/GDR1), chitin synthase (CHS1, CHS-79f/CHS-345R), histone (H3, Cy1H3F/ Cy1H3R) of LSJF-4 (Nos. MN688800, MN688797, MN097811, MN688799 and MN688798, respectively) (Damm et al. 2009) were also sequenced and BLAST. The results showed high identity of all the five sequences to the CGMCC 3.19617 (Nos. GU228286/GU228287 in GAPDH = 99.6%, GU228384/GU22385 in CHS1 = 96.8%, GU228090/GU228091 in H3 = 99.0%, GU227992/GU227993 in ACT = 98.7%, and GU228188/GU228189 in Tub2 = 98.8%). Therefore, isolate CGMCC 3.19617 was identified as C. chlorophyti based on morphological and molecular characteristics. To determine pathogenicity, a conidial suspension containing 106 conidia/mL was sprayed on leaves and stems of six 40-day old peanut seedlings. Three control plants were similarly sprayed with sterile water. All treated plants were kept moist (>85% relative humidity) at 30℃ for 48 h in darkness, and then kept in 60% relative humidity and 28℃ conditions. Two weeks post-inoculation, small, dark, near elliptical lesions appeared on inoculated leaves and stems, which were similar to those naturally infected plants. While controls remained symptomless. C. chlorophyti was reisolated from infected tissues and identified based on the previous methods, fulfilling Koch's postulates. The test was repeated twice. C. chlorophyti has been reported as a pathogen associated with soybean anthracnose isolated from the Netherlands (Damm et al. 2009). C. chlorophyti was first detected on soybean plants imported from Uruguay (Li et al. 2017) in China. To our knowledge, this is the first report of C. chlorophyti causing peanut anthracnose in China. C. chlorophyti may be a new threat to Leguminous plant species, especially peanuts plants. We proposed paying close attention to taking necessary control meatures.