First Report of Pythium Root Rot Caused by Pythium mastophorum on Parsley in the United States

Abstract

In California, parsley (Petroselinum crispum [Mill.] Fuss) is a versatile leafy green commodity that is grown for fresh, frozen, dried, and processed products. In May 2017, parsley grown in a commercial field in Santa Barbara County, CA, showed symptoms of an unknown disease. Plants were stunted, wilted, and had chlorotic foliage. The large taproots were discolored, and small feeder roots were sparse and necrotic. Affected plants appeared in clusters, and disease incidence was approximately 5%. To recover possible pathogens, roots were surface sterilized in 0.01% bleach for 30 s and plated on corn meal agar amended with pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP) (Kannwischer et al. 1978). After 3 days, rapidly growing, coenocytic mycelia typical of a Pythium species grew out from all roots. Older cultures showed globose sporangia and ornamented oogonia. Oogonia had an average diameter of 23 µm, and projections were conical, measuring an average length of 3 µm. Five isolates were tested using a recombinase polymerase amplification genus-specific Phytophthora assay (targeting the trnM-trnP-trnM region in the mitochondria) and were found to be negative (Miles et al. 2015). To determine the species, DNA was extracted from mycelial plugs using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and the internal transcribed spacer (ITS) regions 1 and 2 were amplified and sequenced using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 primers (5′-TCCTCCGCTTATTGATATGC-3′) (GenBank accession no. MG593942). The sequences were 99% homologous to Pythium mastophorum strain CBS 375.72 (GenBank accession no. AY598661). To test for pathogenicity, 4-week-old plants of flat leaf (cv. Italian Dark Green Flat) and curly leaf (cv. Moss Curled) parsley were inoculated using a root-dip method for five isolates. Inoculum was made by blending five fully colonized V8 plates with 250 ml of sterile water. Parsley root balls were soaked for 10 to 15 min in the inoculum and then repotted in a 1:2 sand/soil mixture. In 2 to 3 weeks, inoculated plants became stunted and chlorotic and had symptoms that matched those seen in the field. Roots were severely decayed. Roots plated onto PARP resulted in colonies that morphologically and molecularly were identical to the original isolates. Control plants of both cultivars were inoculated with a mixture of sterile V8 media and water using the above protocol. These plants developed no foliar or root symptoms. This test was completed three times with consistent results. We believe this is the first report of P. mastophorum infecting parsley in California. P. mastophorum has been reported on parsley only in southeastern Australia (Petkowski et al. 2013). In California, this species has been reported on celery (Vazquez et al. 1996). Because affected parsley plants were completely unharvestable, this disease could be a significant concern for growers if inoculum spreads throughout this and other parsley growing regions in the state.