First Report of Paramyrothecium breviseta Causing Leaf Spot Disease of Coffea canephora in China.

Coffee is a tropical plant with two widely cultivated species, namely Coffea arabica and Coffea canephora. A leaf spot disease causing brownish and necrotic lesions was broken out on the C. canephora coffee seedlings in a nursery in Ruili County, Yunnan Province, China, during 2018 to 2019. The incidence of the disease was 15% ~ 20%. Ten diseased leaf samples from five diseased plants were collected for pathogen isolation by tissue separation method. Leaf pieces were cut from the margin of the necrotic lesions (4 × 6 mm), surface-sterilized for 30 s in 75% ethanol, followed by 0.1% arsenic mercury solution for 15 s, then washed 3~4 times with sterilized distilled water and transferred onto potato dextrose agar (PDA) medium in petri plates. Four morphologically similar isolates were obtained from lesions and cultivated on PDA at 25°C. Initial colonies of isolates were round, neat edge, white, floccose mycelium and developed dark green-to-black concentric rings that were sporodochia bearing viscid spore masses after 5~7 days. Conidia were acetates, hyaline and cylindrical with both rounded ends and 4.8 to 6.4 µm long × 1.6 to 2.6 µm wide. Koch's test were conducted on three healthy plants leaves of original source variety C. canephora No.2 and C.arabica Catimor CIFC7963 (control plants) with spore suspension (1 × 106/mL), respectively. Meanwhile, equal numbers of healthy plants were inoculated with water as controls. After inoculation, the plants were transferred into an incubator at 25℃ with saturated humidity. After 10 days of inoculation, all the tested plants presented similar typical symptoms with the diseased leaves under natural conditions; whereas the controls remained healthy. Koch's postulates were performed by re-isolating the fungus from the inoculated leaves and verifying its colony and morphological characters. Two single spore isolates cultured on PDA medium were selected for DNA extraction. The ribosomal internal transcribed spacer (ITS) was PCR amplified by using primers ITS1 and ITS4 (White et al., 1990), β-tubulin gene by Bt2a and Bt2b (Glass and Donaldson, 1995), the RNA polymerase II second largest subunit (rpb2) by RPB2-5F2 and RPB2-7cR (O'Donnell et al, 2007), calmodulin (cmda) gene by CAL-228F and CAL2Rd (Groenewald et al., 2013). The sequences of ITS (MT853067 ~ MT853068), β-tubulin (MT897899 ~ MT897900), rpb2 (MW256264~ MW286265) and cmda (MT897897~ MT897898) were deposited in GenBank databases. BLAST analysis revealed that the representative isolates sequences shared 99.31%~99.65% similarities to the ITS sequence of Paramyrothecium breviseta (Accession Nos. NR_155670.1), 99.43% similarities to the β-tubulin sequence of P. breviseta (Accession Nos. KU846406.1), 98.98% similarities to the rpb2 sequence of P. breviseta (Accession Nos. KU846351.1), and 98.54%~98.71% similarities to the cmda sequence of P. breviseta (Accession Nos. KU846262.1). As it shown in the phylogenetic tree derived from combined ITS, β-tubulin, rpb2, and cmda gene sequences, the two representative isolates were clustered together with P. breviseta CBS 544.75 with 98% strong bootstrap support, which confirmed that P. breviseta is the causal agent of leaf spot of Coffea canephora. To our knowledge, this is the first report of a leaf spot disease caused by P. breviseta on C. canephora in China, which raised the caution that P. breviseta is also pathogenic to Coffea Arabica.