First Report of Leaf Blight Caused by Curvularia aeria and C. senegalensis on Tomato (Solanum lycopersicum L.) in Florida, USA.

Abstract

Tomato (Solanum lycopersicum L.) is an important economic crop in Florida and worldwide. In November 2021, a leaf blight was reported on tomato plants (hybrid cherry and artisan tomatoes) from a small farm in Miami-Dade County, Florida. About 100 plants showed symptoms with disease severity of 15% and disease incidence of 80%. Symptoms on the leaves started as small dark spots and coalesced to form larger necrotic lesions over time. Symptomatic leaf tissues were cut into 5-mm pieces, surface disinfected with 70% ethanol for 30 s and 1% NaClO for 5 min, then cultured on PDA for 3 to 5 days at 25°C. Isolations were conducted in three rounds, with 15 samples in each round. Except for the saprophytes, fungal isolates of Curvularia were consistently recovered from tissues in each round. Single spore isolates grouped in two morphotypes (CT1 and CT3, CT2 and CT4) were examined for morphological and molecular identification. Colonies on PDA were dark yellow-green, with a fluffy surface, then both morphotypes turned black, although CT2 and CT4 were light yellow at the edges. CT1 and CT3 produced light-brown, straight to curved conidia with smooth walls and 1 to 3 septa, 18 to 28 ✕ 9 to 12 µm (n=60), and dark-brown stromatic synnemata (> 200 µm in length) in the center of the colony after ~30 days of incubation in PDA. CT2 and CT4 produced brown, mostly curved conidia, 14 to 23 ✕ 8 to 9 µm (n=60), with slightly rugose walls and 3-4 septa, without synnemata. A dehydrated culture of each isolate was deposited in the Plant Industry Gainesville Herbarium [(PIGH, accession numbers 17443 (CT1), 17444 (CT2), 17445 (CT3), 17446 (CT4)]. Total DNA was extracted using DNeasy Plant Pro Kit (Qiagen, Germantown, MD) followed by PCR amplification and Sanger sequencing of the internal transcribed spacers (ITS) and the Large Subunit (LSU) of the rRNA gene, together with the protein coding gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1- (TEF1) and -tubulin (TUB) (Marin-Felix et al. 2020; Himashi et al. 2021; Manamgoda et al. 2012; Myllys et al. 2002) (GenBank accession numbers ITS-LSU: OQ657944-OQ657947, GAPDH: OQ689438 to OQ689441, TEF1: OQ689442 to OQ689445, TUB: OQ689446 to OQ689449). Curvularia clavata as the molecular marker (96% identity) was used for identification. Sequence similarity of 100% in GAPDH, ITS and LSU was obtained in megaBLAST searches for both groups of morphotypes, CT1 and CT3 to Curvularia aeria (Bat., J.A. Lima & C.T. Vasconc.) Tsuda type culture CBS 294.61, and CT2 and CT4 to Curvularia senegalensis (Speg.) Subram. culture CBS 149.71. Pathogenicity tests were conducted with each isolate on six tomato plants that were 6- weeks-old. The seeds used in the tests were provided by the farm, and the variety 'Red Bounty' was also used. Inoculation was accomplished by spraying a spore suspension (1 x 106 spores/ml) of each of the four isolates (CT1 to CT4) and by placing 6-mm PDA plugs of the isolates on the leaves. Six tomato plants were used as the control. All plants were covered by plastic bags and placed in a greenhouse at 23-27°C. The inoculated plants developed small dark spots on leaves 2 weeks after inoculation, and the leaves inoculated by plugs of the fungal isolates had large necrotic lesions, which were similar to those observed on tomato plants from the field. The pathogenicity tests were repeated three times, Curvularia was consistently isolated from inoculated leaves after the symptoms developed, and they were confirmed morphologically in each test. No symptoms were observed from the control plants. Curvularia aeria and C. senegalensis are known foliar pathogens on several important crops, but not tomatoes. To our knowledge, this is the first report of C. aeria and C. senegalensis causing leaf blight in tomatoes worldwide. This finding is important because it will extend the host range of C. aeria and C. senegalensis to tomato, it also implied the essentiality of crop rotation in disease management.