First report of strains within the Pythium spinosum species complex causing carrot cavity spot in California

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). References: Hiltunen, L.H., and White, J. G. 2002. Ann. Appl. Biol. 141:201. Lévesque, C. A., and De Cock, W. A. M. 2004. Mycol. Res. 108:1363. Robideau, G.P., et al. 2011. Mol. Ecol. Resour. 11:1002. Schrandt K. K., et al. 1994. Plant Dis. 78:335. Schroeder, K.L., et al. 2006. Phytopathology 96:637. Supplementary material: Supplementary figure S1 Supplementary figure S2

Incidence and Severity of Cavity Spot of Carrot as Affected by Pigmentation, Temperature, and Rainfall

Field trials to determine the effect of carrot pigmentation and weather parameters on cavity spot (CS) of carrot were conducted in the Holland/ Bradford Marsh region of Ontario between 2002 and 2009. In all, 23 colored carrot cultivars from the United States Department of Agriculture (USDA) Agricultural Research Service breeding program at the University of Wisconsin (n = 5) and commercial seed companies (n = 18) were seeded in organic soil (pH 6 to 7, 45 to 75% organic matter) in late May to early June and harvested in late October or early November. Carrot roots were assessed for CS severity midseason and postharvest. Evaluations postharvest indicated that the purple pigmented carrot from breeding line ‘USDA 106-3’ and cultivars ‘Purple Rain’ and ‘Purple Haze’ consistently had low CS severity. The orange-pigmented ‘USDA 101-23’, ‘Cellobunch’, ‘YaYa’, and ‘Envy’ had moderate CS; and the red-pigmented carrot breeding line ‘USDA 104-3’ and cultivars ‘Atomic Red’, ‘Proline Red’, ‘Dragon’, and an unnamed line from India had high CS. Differences in CS severity in carrot cultivars between evaluations at midseason and postharvest suggest that some carrot cultivars are more susceptible to Pythium spp. inoculum in soil (alloinfection) and others to secondary infection (autoinfection) that can be attributed to the Pythium sp. involved in CS. CS severity was positively correlated with total rainfall 2 and 3 months after seeding, and was negatively correlated with number of days with air temperature ≥30°C 3 and 4 months after seeding. Soil temperature and total rainfall were the best predictors of CS incidence and severity. These results could allow a forecast of disease incidence and severity at harvest.

First Report of Pythium sulcatum Causing Cavity Spot in Processing Carrot Crops in the Columbia Basin of Washington State

In October 2012, symptoms of cavity spot (1) were observed on roots of two 50 ha, Red Core Chantenay processing carrot (Daucus carota L. subsp. sativus (Hoffm.)) crops in the Columbia Basin of central Washington. Symptoms consisted of sunken, elliptical lesions (3 to 15 mm long) on the root surface. Approximately 6% of the roots in each crop were affected, which was sufficient to present sorting problems for the processor. Symptomatic roots were washed thoroughly in tap water, and then small sections of tissue from the lesion margins were removed aseptically and plated onto water agar (WA) without surface-sterilization. Isolates with morphological characteristics typical of Pythium sulcatum Pratt & Mitchell (2) were obtained consistently from the symptomatic tissue. The genus and species identity of seven isolates was confirmed by sequence analysis of the internal transcribed spacer (ITS) 1-5.8S-ITS2 region of ribosomal DNA (rDNA) using universal eukaryotic primers UN-UP18S42 and UN-LO28S576B with the PCR protocol described by Schroeder et al. (3). The ITS consensus sequences of the seven isolates (Accession Nos. KF509939 to KF509945) were 98 to 99% homologous to ITS sequences of P. sulcatum in GenBank. Pathogenicity of all seven isolates was confirmed by inoculating mature carrot roots of cv. Bolero. Each root was washed with tap water, sprayed to runoff with 70% isopropanol, and dried in a laminar flow hood on sterilized paper toweling. The roots were then placed in plastic bins lined with paper toweling moistened with sterilized, deionized water. Each root was inoculated by placing two 5 mm-diameter agar plugs, taken from the edge of an actively growing WA culture of the appropriate isolate, on the root surface approximately 3 cm apart. Non-colonized agar plugs were used for a non-inoculated control treatment. Four replicate roots were inoculated for each isolate and the control treatment. After inoculation, the roots were misted with sterilized, deionized water, a lid was placed on each bin, and the roots were incubated in the dark at 22°C. Roots were misted daily to maintain high relative humidity. Dark, sunken lesions were first observed 3 days post-inoculation on roots inoculated with the P. sulcatum isolates, and all inoculated roots displayed cavity spot lesions by 7 days. No symptoms were observed on the non-inoculated control roots. Colonies with morphology typical of P. sulcatum were re-isolated from the symptomatic tissue of roots inoculated with the P. sulcatum isolates, and the species identity of the re-isolates was confirmed by ITS rDNA sequence analysis, as described above. Although P. sulcatum is one of several Pythium species that can cause cavity spot of carrot (1), to our knowledge, this is the first report of P. sulcatum causing cavity spot in Washington State, which has the largest acreage of processing carrot crops in the United States (4). References: (1) R. M. Davis and R. N. Raid. Compendium of Umbelliferous Crop Diseases. The American Phytopathological Society, St. Paul, MN, 2002. (2) A. J. van der Plaats-Niterink. Monograph of the Genus Pythium. Stud. Mycol. No. 21. CBS, Baarn, The Netherlands, 1981. (3) K. L. Schroeder et al. Phytopathology 96:637, 2006. (4) E. J. Sorensen. Crop Profile for Carrots in Washington State. U.S. Dept. Agric. National Pest Manage. Centers, 2000.

First Report of Pythium recalcitrans Causing Cavity Spot of Carrot in Michigan

During a survey of carrot (Daucus carota L.) cavity spot in Michigan in September 2010, carrot roots with typical cavity spot symptoms were collected from production fields in Fremont Co. The lesions were excised from infected roots, surface-disinfested with 0.62% NaClO for 3 min, rinsed in sterilized, distilled water three times, cut into 0.5 cm long pieces, and then plated on water agar (WA) amended with carbendazim (10 μg/ml), ampicillin (50 μg/ml), rifampicin (50 μg/ml), and pentachloronitrobenzene (10 μg/ml) (cumulatively referred to as CARP). Plates were incubated at 22 ± 1°C in the dark for 3 days. Pure cultures of the isolates were obtained by transferring a single hyphal tip of each colony to potato dextrose agar (PDA) amended with CARP. Among the 33 isolates obtained, M2-05 was identified as a Pythium sp. that differed from the known cavity spot pathogens of carrot. The isolate has spherical hyphal swellings but no other distinguishing morphological characteristics. M2-05 was further classified by analyzing the partial sequences of four genes: the internal transcribed spacer (ITS) region of ribosomal DNA, beta-tubulin (β-tub), cytochrome c oxidase subunit 2 (cox 2), and NADH dehydrogenase subunit 1 (nadh 1) (1,3). A BLAST search of these sequences for M2-05 was conducted using the nucleotide database of GenBank, resulting in 100% similarity to all four sequenced genes of P. recalcitrans (2). The DNA sequences of M2-05 were deposited in GenBank (JQ734349, JQ734229, JQ734289, and JQ734409 for ITS, β-tub, cox 2, and nadh 1, respectively). Koch's postulates were conducted by inoculating mature carrot roots (cv. Nantindo) with mycelial plugs (4 mm in diameter) cut from the margin of actively growing colonies of M2-05 on PDA plates. Two mycelial plugs were placed on each carrot root at 3-cm intervals, with the mycelial side facing the root; and two non-colonized agar plugs were placed similarly for the non-inoculated control treatment. In comparison, carrot roots also were inoculated with an isolate of each of P. sulcatum and P. violae using the same method. There were four replicate carrot roots inoculated for each isolate and each of the control treatments. The inoculated roots were placed on a plastic grid (7 mm in height) in a closed plastic container, with moist paper towels underneath the grids. The container was incubated in the dark at 22 ± 1°C, and the roots were sprayed gently daily with sterilized, distilled water to maintain high humidity. Brown lesions were observed on all inoculated carrot roots 5 days after inoculation. The lesions measured 0.68 ± 0.48, 1.20 ± 0.71, and 0.56 ± 0.31 mm2 averaged over all eight lesions for the isolates of P. recalcitrans, P. sulcatum, and P. violae, respectively. Symptomatic tissues from the inoculated roots were excised and incubated on WA-CARP plates, and the culture from each lesion confirmed as the isolates inoculated using the same molecular methods described above. The carrot tissue under the control agar plugs remained symptomless, and no Pythium was recovered from the control roots. P. recalcitrans was described in 2008 as infecting roots of Beta vulgaris and Vitis vinifera (2). To our knowledge, this is the first published report of P. recalcitrans naturally infecting carrot roots, not only in Michigan, but anywhere in the world. References: (1) L. Kroon et al. Fungal. Genet. Biol. 41:766, 2004. (2) E. Moralejo et al. Mycologia 100:310, 2008. (3) N. O. Villa et al. Mycologia 98:410, 2006.

Fungicide Sensitivity of Pythium spp. Associated with Cavity Spot of Carrot in California and Michigan

The identity of 172 isolates of Pythium spp. from cavity spot lesions on carrot produced in California and Michigan was determined, and their sensitivity to three fungicides was examined. Pythium violae accounted for 85% of California isolates, with P. irregulare, P. dissotocum (the first report as a carrot pathogen in the United States), P. ultimum, and P. sulcatum making the balance. P. sulcatum, P. sylvaticum, and P. intermedium were the most commonly recovered (85%) species in Michigan; others from Michigan included P. intermedium, P. irregulare, and an unclassified strain, M2-05. On fungicide-amended media, 93% of isolates were sensitive to mefenoxam (inhibition of mycelial growth was >60% at 10 μg active ingredient [a.i.]/ml); however, two of five isolates of P. irregulare from California were highly resistant (≤60% inhibition at 100 μg a.i./ml); about half of the isolates of P. intermedium and P. sylvaticum and a single isolate of P. violae were highly or intermediately resistant to mefenoxam (>60% inhibition at 100 μg a.i./ml, or ≤60% inhibition at 10 μg a.i./ml). P. dissotocum, P. irregulare, P. sulcatum, M2-05, and three of seven isolates of P. intermedium were insensitive to fluopicolide (effective concentrations for 50% growth inhibition [EC50] were >50 μg a.i./ml), while P. sylvaticum, P. ultimum, P. violae, and some isolates in P. intermedium were sensitive (EC50 < 1 μg a.i./ml). All isolates were sensitive to zoxamide (EC50 < 1 μg a.i./ml). Sensitivity baselines of P. violae to zoxamide and fluopicolide were established.

Antimicrobial efficacy of cinnamon, ginger, horseradish and nutmeg extracts against spoilage pathogens

In the search for alternatives to the use of synthetic fungicides, aqueous spice extracts were evaluated for their effects on the mycelial growth of various spoilage pathogens and their ability to control potato dry rot and carrot cavity spot in vivo. Results showed that cinnamon, ginger and nutmeg significantly inhibited the mycelial growth of Aspergillus niger (Ascomycota), Fusarium sambucinum (Ascomycota), Pythium sulcatum (Oomycota) or Rhizopus stolonifer (Zygomycota), whereas horseradish extract did not lead to the inhibition of any microorganism at the tested concentration. Among the most effective extracts, 0.05 g mL‑1 of cinnamon extract completely inhibited A. niger and P. sulcatum, and 0.10 g mL‑1 of cinnamon extract completely inhibited F. sambucinum. A concentration of 0.05 g mL‑1 of ginger extract also caused 100% inhibition of P. sulcatum. In vivo, cinnamon extract significantly reduced lesions of potato dry rot and carrot cavity spot, and ginger extract reduced lesions of carrot cavity spot. These results indicate that aqueous cinnamon and ginger extracts could provide an alternative to the use of synthetic fungicides to control these pathogens.