First Report of Trichoderma crassum Causing Leaf Spot on Tomato (Solanum lycopersicum cv. M82) in Ohio.

Trichoderma is a genus of wood-decaying fungi generally found in soil (Druzhinina and Kubicek 2005). Trichoderma crassum was confirmed to be a sister species to T. virens according to the molecular sequencing results (Chaverri et al. 2003). A foliar disease with ~70% incidence on Solanum lycopersicum was observed in a greenhouse at The Ohio State University (40°0'8'' north latitude, 83°1'36'' west longitude), Columbus, United States, in December 2021. On average up to 60% of the leaves per two-month-old tomato plant were infected. Initially, the dark-grey color and irregular spots appeared at the leaf tips. As the disease progressed, the yellow necrotic lesions were observed surrounding the preformed disease spots. Finally, the infected leaves appeared curled and wilted as a whole. The leaf fragments from three tomato plants 40 inches apart were cut from the diseased lesions and surface sterilized with 75% ethanol (30 seconds) and 1% NaOCl (60 seconds), subsequently rinsed with sterilized deionized water three times. Nine pieces of the sterilized leaf tissues were then placed on the PDA plates at 28℃ in the dark and incubated in one incubator for 4 days. The pure cultures of five isolates were acquired and examined with a light microscope. The fungus from all the isolates changed from white to dark green with the radial pattern and profuse sporulation on the PDA. The produced round conidia were observed under a light microscope (Fig S1). The DNA was extracted from two representative isolates which showed the same morphology. The internal transcribed spacer (ITS) region and a conserved fungal rRNA region were amplified using the primers ITS1/ITS4 (5'-TCCGTAGGTGAACCTGCGG-3' and 5'-TCCTCCGCTTATTGATATGC-3') (White et al. 1990) and SR6f/SR7r (5'-TGTTACGACTTTTACTT-3' and 5'-AGTTAAAAAGCTCGTAGTTG-3') (Hirose et al. 2012), respectively. The PCR products were further sequenced by Sanger sequencing (Table S1). Based on the BLAST results through NCBI website, the ITS sequences of the two isolates were 99% (566/572) and 98% (558/572) identical to Trichoderma crassum DAOM 164916 (EU280067). Their SR sequences both showed 99% (290/293; 289/293) identity to the same strain. The phylogenetic tree was also created with the sequences of ITS region by MEGA software (version 11) (Fig S2). Therefore, the fungus was identified as Trichoderma crassum based on its morphological characteristics (green conidia), Sanger sequencing results, and phylogenetic tree. To complete Koch's postulates, the 5-mm-diameter fungal agar discs of 7-day-old pure cultures were used for the inoculation on 18 healthy leaves of six tomato cv. M82 plants with two-month-old. The sterile pure PDA discs of the equal size were used for the mock inoculation as a comparison. Fungal plug method was chosen in this study because it had been widely applied to characterization of the fungal pathogens causing leaf spot disease (Pornsuriya et al. 2020; Yang et al. 2021). Five days later, the same symptom as those that occurred on the previously naturally infected tomato plants were observed on all the inoculated leaves (Fig S3A). However, there were no symptoms on the leaves with the mock inoculation. The fungus re-isolated from the symptomatic leaves showed the consistent morphology (dark-green color with radial sporulation) with the original isolates (Fig S3B). Thus, Trichoderma crassum was verified as the causal agent of the foliar disease on Solanum lycopersicum cv. M82 in our greenhouse. To our knowledge, it is the first report of Trichoderma crassum leading to the leaf spot and wilt on tomato in Ohio. The identification of the causal agent lays the groundwork for the development of necessary disease management techniques. We acknowledge the funding support from CFAES Internal Grants Program 2021009.