First Report of Diaporthe Fruit Rot and Stem Canker of Hydroponic Cucumber Caused by Diaporthe sp. in the United States

Abstract

Multiple Diaporthe spp. cause root and fruit rots or stem lesions on Cucumis spp.: D. cucurbitae, D. melonis, D. longicolla (syn. D. eres), D. pterocarpi, D. sclerotioides, D. sojae, and D. ueckerae (Broge et al. 2020; Fukada et al. 2018; Udayanga et al. 2012, 2015). From May-August 2021, cucumbers (Cucumis sativus) ‘Katrina’ and ‘Alcazar’ were grown in a 24-plant, commercial Bato bucket system with rockwool blocks on a perlite substrate in a research greenhouse in Wooster, Ohio. At maturity, plants collapsed rapidly from stem lesions without foliar chlorosis (25% of ‘Katrina’ and 17% of ‘Alcazar’). Lesions were 7.5 to 15 cm in length, tan to golden-brown with black pycnidia and located 5 to 15 cm above the crown. Stems shredded easily with vascular discoloration around the lesion. Two identical fungal strains were isolated on ½ acidified potato dextrose agar (APDA) following surface disinfestation with 0.6% sodium hypochlorite for 30 s and sterile water rinse. Fungal cultures were floccose, white to tan mycelia with pycnidia. Oblong, elliptical, biguttulate, aseptate alpha conidia were observed with mean dimensions: 8.0 µm (5.2-9.8 µm) by 3.1 µm (2.5-3.8 µm) on ½ APDA and 9.8 µm (6.6-12.4 µm) by 3.0 µm (1.9-5.3 µm) on petioles. On prune extract agar, beta conidia mean dimensions were: 19.7 µm (12.0-27.7 µm) by 1.2 µm (0.8-1.8 µm). Fungal DNA was amplified and sequenced bidirectionally with ITS (ITS4/ITS5), CAL (CAL228F/737R), HIS (CYLH3F/H3-1B), TEF1 (EF1-728f/EF1-986R), and TUB2 (Bt1a/Bt1b) primers (Carbone and Kohn 1999; Glass and Donaldson 1995) (GenBank: OP265712-13, OP288460-65, OQ418506-07). Based on a maximum likelihood phylogenetic tree of concatenated genes, this novel Diaporthe sp., most closely related to D. stewartii, has not been reported on Cucumis spp. Strains were deposited in the USDA-ARS Culture Collection (NRRL# 64461-62). Koch’s postulates were conducted in a greenhouse with mean day temperature of 25°C and 12 hr supplemental lighting. One-month old cucumbers ‘Katrina,’ grown in rockwool cubes (5 plants per isolate) and potting mix (6 plants per isolate), were inoculated with a one-week-old culture of either strain. The second true leaf was cut and a pipette tip containing an inoculated plug of ½ APDA was placed on the remaining petiole (Mathew et al. 2018). Non-inoculated ½ APDA was used for controls. Plants were tarped for 24 hours to increase humidity and pipette tips removed after one week. After two weeks, petioles were shrunken, tan to golden brown with pycnidia. After 3-4 weeks, stem lesions matching those above were observed on inoculated plants, and plants collapsed. For fruit rot, three Beit Alpha cucumbers were rinsed with tap water, dried, a 5 mm plug was removed from the fruit and replaced with a 5 mm plug of one-week-old fungus on ½ APDA. After 3 days, fruits were water soaked and soft. For root rot, two plates of one-week-old cultures were macerated in 500 mL of sterile water and mixed with 1500 mL of vermiculite. Two seeds of cucumber ‘Katrina’ were planted into three reps of each isolate and control. All control seeds germinated, but all inoculated seeds experienced pre- or post-emergence damping off. No symptoms were ever observed on any controls. Fungi were isolated from all inoculated tissues as described above. Based on morphology, Diaporthe sp. was isolated from all inoculated plants but never from controls. This Diaporthe sp. may be a new constraint to hydroponic cucumber production, but incidence needs to be determined globally.