First Report of Strains Within the Pythium spinosum Species Complex Causing Carrot Cavity Spot in California.

Abstract

Carrots (Daucus carota L. subsp. sativus (Hoffm.)) with typical symptoms of cavity spot, i.e., sunken, round to elliptical lesions of 2-5 mm long (Hiltunen and White 2002), were collected from two locations in California, Bakersfield and Riverside, in January and July 2019, respectively. Carrots were rinsed in tap water, 4-mm2 lesion fragments were pressed into selective corn meal agar (CMA-PARP; Schrandt et al. 1994) and incubated at 23ºC in the dark for four days. Identification of pure cultures was performed via amplification and sequence analysis of two genomic regions, the Internal Transcribed Spacer 1-5.8S-ITS2 (ITS) region and the cytochrome C oxidase subunit 1 (COI) gene, using the universal primers UN-UP18S42/UN-LO28S576B (Schroeder et al. 2006) and OomCOXI-Levup/OomCOXI-Levlo (Robideau et al. 2011), respectively. Via BLAST, two isolates from organically grown carrots in Bakersfield (MCIF19) and Riverside (JSCS19), with identical ITS sequences (GenBank Acc. Nos. MZ799354 and MZ799355, respectively), showed 99.61% similarity (1021/1025 bp) to that of Pythium spinosum (AY598701.2). Yet, the COI of MCIF19 (MZ803207) showed 98.72% similarity (692/701 bp) to that of Pythium paroecandrum (GU071818.1), while the COI of JSCS19 (MZ803208) was identical (701/701 bp) to that of Pythium kunmingense (GU071820.1), a rarely isolated species considered within the species complex of P. spinosum (Robideau et al. 2011). According to these results, the isolates were identified as belonging to the P. spinosum species complex, part of Pythium Clade F (Lévesque and De Cock 2004; Robideau et al. 2011). Further research is needed to clarify the exact taxonomic status of both isolates. Koch's postulates were completed using two different assays. Each assay was done twice and with carrots of the cultivar Maverick. Surface-sterilized, freshly harvested, mature carrots, in a plastic box lined with moistened sterile paper towels, were inoculated each with four CMA plugs (5-mm diameter) with actively growing mycelium of each isolate. CMA plugs, non-inoculated or colonized by a known pathogenic P. violae strain, were used as the negative and positive control, respectively. Boxes were closed to maintain humidity and incubated at 23ºC in the dark. Lesions similar to the ones caused by P. violae were observed at day 3 for all plugs of both isolates. No symptoms were observed for the negative control, even after extending the incubation to 7 days. In a more natural assay, four non-treated carrot seeds were planted in tree seedling pots (25 x 6.5 cm) containing sterilized 50/50 peat moss/sand combined with 15-ml V8 broth (Schrandt et al. 1994) with densely grown mycelium. The same inoculation treatments were used as for the carrot disk assay. Plants (one plant/pot, four plants/treatment) were maintained at 23ºC under a 16 h photoperiod with daily watering (20 ml). At 14 weeks, the carrots inoculated with P. violae and the two test isolates showed cavity spot lesions while no symptoms were observed on carrots growing in non-inoculated medium. For both assays, pathogens were re-isolated from rinsed symptomatic tissue and their identity was confirmed using the molecular analysis described above. No oomycetes were recovered from the non-inoculated carrots. Although several Pythium species have been associated with cavity spot before, this is, to our knowledge, the first report of strains within the P. spinosum species complex causing carrot cavity spot in California and elsewhere. Funding: This research was made possible by the California Fresh Carrot Advisory Board (FRA-21).