First Report of Leaf Spot Caused by Paramyrothecium foliicola on Tomato (Solanum lycopersicum) in China

Tomato (Solanum lycopersicum) is a staple vegetable across the world. In October 2019, leaf spots were observed on tomato (cv. Tianmi) in a greenhouse in JiZhou District Tianjin, China(117°10 'E; 39°55 'N). Symptoms initially appeared as small brown spots, which gradually expanded and turned into circular, oval or irregular spots (some spots with distinct concentric zones). In severe cases, some spots coalesced and eventually covered the whole leaf. Disease incidence ranged between 12 and 18%. Twenty symptomatic leaves from five plants were collected and cut into small pieces, surface disinfested in 2% NaClO for 60 s, rinsed three times in sterile water, and subsequently plated on potato dextrose agar (PDA). Plates were incubated at 25°C in the dark for 7 days. A total of 102 isolates were obtained and 92 isolates had the same morphology. Colonies were initially white with abundant aerial mycelia and formed sporodochia with conidial masses in olivaceous green concentric rings. All isolates formed single-celled, hyaline, and rod-shaped conidia were 4.91 to 7.43 (avg. 6.53±0.72) × 1.41 to 2.45 （avg. 2.11±0.30）μm with rounded ends (n=50). Conidiophores were highly branched. These characteristics resembled a Paramyrothecium-like fungus (Lombard et al. 2016). The genomic DNA of three representative single-spored isolates TJJXPF1-3 were extracted and the internal transcribed spacer (ITS) region, β-tubulin (tub2), large subunit ribosomal RNA (LSU), calmodulin (cmdA) and translation elongation factor 1-alpha (tef1) genes were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), LR0R/LR5 (Rehner and Samuels 1995; Vilgalys and Hester 1990), CAL-228F/CAL2Rd (Carbone and Kohn 1999; Groenewald et al. 2013) and EF1-728F/EF2 (O'Donnell et al. 1998), respectively. All sequences were deposited in GenBank (ITS: MW463444, OM368178, OM368179; tub2: MW269542，OM714930，OM714931; LSU: OM349050, OM397398, OM390582; cmdA: MW280443, OM350474, OM350476; tef1: MW560083, OM350475, OM350477). BLASTN analysis showed 99.3-100% similarity with reference isolate QB1 of P. foliicola (MK335967, MT415353, MT415362, MT415356 and MT415359). Multilocus phylogenetic analysis showed that TJJXPF1-3 best grouped with the P. foliicola clade, which was identified by morphological characteristics and phylogenetic analysis. To fulfill Koch's postulates, pathogenicity tests were conducted by spray-inoculation with a conidial suspension of isolate TJJXPF1 prepared with distilled water (1×105 conidia/mL) on five 45-day old tomato plants. Three healthy plants were sprayed with sterile water as control. All treatments were incubated in an artificial climate chamber (25°C, 80% RH, 12h light/12h dark ). After two weeks, leaf spots were observed on all inoculated plants, which were similar to those in the greenhouse of JiZhou District, while control plants remained asymptomatic. Additionally, the pathogens were reisolated from symptomatic leaves and three representative isolates TJJXPF4-6 were identified as P. foliicola. The pathogenicity tests were repeated thrice. To our knowledge, this is the first report of leaf spot caused by P. foliicola on tomato in China. This disease could be a serious threat to tomato production in the future. Our findings will help to differentiate this disease from other leaf spot-like diseases and develop disease control strategies.