First Report of Didymella rhei causing leaf spot on rhubarb in New York.

Abstract

First Report of Didymella rhei causing leaf spot on rhubarb in New York E. J. Indermaur1, C. T. C. Day1, and C. D. Smart1† 1School of Integrative Plant Science, Section of Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva NY 14456 †Corresponding author: C. D. Smart; Email: cds14@cornell.edu Rhubarb (Rheum spp.) is a perennial grown across the northern United States for petiole production (Foust & Marshall 1991). In August 2021, leaf spots were observed on rhubarb growing in a two-acre field in Erie Co., NY (Fig. S1). Approximately 30% of the plants in the field had leaf spot with disease severity of 5%. Initial symptoms on leaves were light brown, circular lesions with red margins that later coalesced into irregular spots. Lesion centers were dry with concentric rings, often perforating as they enlarged. Lesions on petioles were light brown, fusiform, and sunken with red margins. To identify the causal agent(s), symptomatic leaves and petioles from 50 plants (cultivar unknown) were collected with a W-shape sampling scheme. Lesion margins were surface sterilized with 70% ethanol for 60 s, 10% bleach for 60 s, rinsed in sterile water, plated on acidified potato dextrose agar (PDA), and incubated for two to four days at 20˚C. Hyphal tips from colony edges were transferred to new PDA plates. After 20 days, colonies (n=53) were olivaceous buff to grey olivaceous, producing white to grey, sparse aerial mycelium. Brown to black pycnidia were produced within five days in concentric rings around plate centers. Pycnidia were globose to subglobose, with one to two non-papillate or slightly papillate ostioles, and with mean diameter 75.8 (30.8 to 113.5) µm (n=20). Conidia were hyaline, ellipsoid or allantoid, and aseptate with mean ± SD dimensions of 6.2 ± 0.4 (4.9 to 8.1) x 2.2 ± 0.4 (1.3 to 3.3) µm (n=30) (Fig. S2). Based on these morphological characteristics, the isolates were initially identified as Didymella rhei [Ellis & Everh] (Qian Chen & L. Cai) (Boerema 2004). To confirm the identity, mycelia were scraped from PDA plates and homogenized using a TissueLyser II (Qiagen Inc.). Genomic DNA was extracted with a DNeasy Plant Mini Kit following manufacturer's instructions (Qiagen Inc.). PCR assays with primers ITS 4 and ITS 5 and fRPB2-7cR and RPB2-5F2 (Liu et al. 1999; Sung et al. 2007) were used to amplify the internal transcribed spacer (ITS) and the rpb2 gene regions of one representative isolate (strain RHU21204). Products were sequenced using Sanger chemistry. The sequences were deposited in GenBank with accession numbers OM903952 (ITS) and OM925897 (rpb2). The ITS and rpb2 sequences exhibited 99% (492/494 bp) and 100% (846/846 bp) identity with D. rhei accessions KF531831.1 and KP330428.1, respectively. Based on morphological and molecular characteristics, the pathogen was identified as D. rhei. To fulfill Koch's postulates, healthy leaves and petioles of four rhubarb seedlings (cultivars unknown) were spray-inoculated with a conidial suspension (1 × 107 conidia/ml) containing 0.2% Tween-20 from strain RHU21204. A tween suspension with no conidia was used as a control. Each treatment had three replicates. After inoculation, plants were placed in a 19˚C growth chamber with a 12-h photoperiod and misted for 30 min twice daily to maintain humidity above 80%. Initial symptoms were observed five days post inoculation (dpi), while control plants were asymptomatic. The pathogen was isolated 21 dpi from inoculated leaves and petioles with symptoms as described above (Fig. S1) and identified morphologically and molecularly as D. rhei. A representative isolate was deposited in the Cornell Plant Pathology Herbarium as CUP-070923. To our knowledge, this is the first report of D. rhei causing rhubarb leaf spot in New York and reducing the health and marketability of its host. Funding Source This project was funded by the College of Agriculture and Life Sciences, Cornell University. Literature Cited Boerema, G. H. et al. 2004. CABI Publishing. 288. Foust, C. M. and Marshall, D. E. 1991. HortScience 26:1360. DOI: 10.21273/HORTSCI.26.11.1360 Liu, Y. J. et al. 1999. Mol. Biol. Evol. 16:1799. Sung, G. H. et al. 2007. Mol. Phylogenet. Evol. 44:1204. DOI: 10.1016/j.ympev.2007.03.011.