Repeated soil applications of fungicide reduce activity against cavity spot in

James J. Farrar ■ J. Joseph Nunez ■ R. Michael Davis

In recent years, growers in the San Joaquin Valley have suffered economic losses due to cavity spot, a soilborne disease, despite frequent applications of the fungicide mefenoxam. Although the pathogen remained highly sensitive to mefenoxam in laboratory studies, the effect- ive dosage of the fungicide was apparently compromised in certain fields. Compared to its longevity in soils with no history of mefenoxam use, such as fields using organic production methods, the fungicide degraded rapidly in soil from fields with repeated mefenoxam use. Our research Carrots infected with cavity spot are usually deemed unsuitable for the fresh market. reveals that repeated ap- Caused by the fungus , cavity spot can also infect other crops such as beets, plications of the fungicide to broccoli and . soil can increase the activity of microorganisms that de- avity spot is one of the most dam- Cavity spot is caused by at least grade it, potentially reducing Caging diseases of carrots in the two species of Pythium, including its efficacy against cavity San Joaquin Valley, where more than P. violae and P. ultimum. In California, spot. This is problematic in 50,000 acres are planted annually. Al- cavity spot is most often attributed to California since mefenoxam is though the disease does not reduce P. violae (Vivoda et al. 1991). Little is the only fungicide available to carrot weights, infected carrots are known about the life cycle and popu- carrot growers for cavity spot deemed unsuitable for the fresh mar- lation dynamics of P. violae in soil. It is control. It may be prudent to ket due to the unsightly blemishes. assumed that the fungus survives as practice long crop rotations The economic impact of the disease thick-walled oospores, which germi- and to limit use of mefenox- can be substantial. In extreme cases, nate in response to sugars and other am, where possible. growers have abandoned fields of car- nutrients that exude from roots. Be- rots with high levels of cavity spot. cause the fungus has a wide host The disease is characterized by root range on such diverse plants as , lesions that are elliptical (oriented beets, blackeye beans, broccoli, celery, across the breadth of the root), gener- cucumber and wheat, it probably per- ally less than 0.5 inch in length, and sists in the soil for years (Schrandt et shallow. The lesions are generally al. 1994). None of the carrot cultivars more numerous on the upper third of commonly grown in California pos- the taproot. There are no symptoms on sess useful levels of resistance. Grow- the aboveground parts of the plant. ers have observed that disease

76 CALIFORNIA AGRICULTURE, VOLUME 56, NUMBER 2 incidence in a field increases in pro- meal agar amended with antibiotics. portion to the number of successive When the colonies were 2 to 4 days carrot crops. old, the isolates were transferred to Timely harvests and good water and subsequently maintained on corn- management can help reduce the oc- meal agar plates. Isolates were identi- currence of cavity spot. Unfortunately, fied based on morphological charac- there is no accurate means of predict- teristics of the colonies grown on ster- ing disease risk in an individual field, ile grass blades in water culture. and applications of a fungicide are Pathogenicity of the isolates was sometimes necessary. Today, only one confirmed by placing 4-millimeter- Fig. 1. Relative growth of Pythium violae fungicide, mefenoxam (Ridomil Gold diameter agar plugs from the margin (31 isolates) and P. ultimum (26 isolates) EC), is registered to control cavity spot of 3-day-old cultures on surface- on cornmeal agar amended with various concentrations of the fungicide on carrots. Applied to the soil up to disinfested carrots. After the sites mefenoxam. Colony diameters were three times in a growing season, it of inoculations were covered with expressed as a percentage of the colony usually results in adequate disease sterile moist cotton, the carrots were diameter on unamended cornmeal agar. Bars represent standard errors. Both fungi control (Davis et al. 1991). Recently, incubated for 14 days at 68˚F (20˚C) were highly sensitive to the fungicide. however, the fungicide has failed to in the dark. control the disease in a number of A total of 31 isolates of P. violae and fields. In at least two fields, the entire 26 isolates of P. ultimum pathogenic to carrot crop was abandoned due to a carrot were evaluated for sensitivity to high incidence of cavity spot. The pur- mefenoxam. Four-millimeter-diameter sistance to metalaxyl or mefenoxam pose of this study was to determine plugs from the margin of 3-day-old has been identified in carrot isolates of the cause of the reduced efficacy of cultures of each isolate were placed on P. violae and P. ultimum. mefenoxam on carrots in the San cornmeal agar plates amended with 0, Degradation of mefenoxam Joaquin Valley. 0.1, 1, 10, 50 or 100 parts per million (ppm) mefenoxam. Colony diameters Since we found no evidence that Isolate sensitivity were measured after the plates were isolates of P. violae and P. ultimum We first examined the possibility incubated at room temperature in the were insensitive to mefenoxam, we de- that the loss of cavity spot control was dark for 3 days. Each treatment was signed two experiments to determine due to the development of resistance replicated three times. whether mefenoxam degraded more to mefenoxam within the pathogen Based on the inhibition of the rapidly in soil where it had been used population. To obtain isolates of the growth of the colonies on mefenoxam- repeatedly. An increased rate of pathogen, carrots with cavity spot le- amended agar, all P. violae and P. metalaxyl (and, therefore, mefenoxam) sions were collected from 13 fields ultimum isolates were sensitive to the degradation has developed in avo- from 1998 to 2000. The history of fungicide (fig. 1). The average colony cado, citrus and tobacco soils with a mefenoxam applications ranged from diameter of both species on plates history of metalaxyl use (Droby and no applications in organic fields to amended with 1 ppm mefenoxam was Coffey 1991). many applications in conventional less than 40% of the colony diameter Soil was collected from four con- fields, including those fields where on unamended agar. These results ventionally farmed carrot fields in the mefenoxam failed to control cavity were similar to the results of our un- San Joaquin Valley with a history of spot. After the carrots were washed in published 10-year study on the sensi- repeated mefenoxam applications, and running tap water, cavity spot lesions tivity of Pythium spp. to metalaxyl and from four certified organic fields. Each and a minimal amount of surrounding mefenoxam, which is the active isomer field served as a replication (the trial healthy tissue were removed with a of metalaxyl, and the only compound was repeated once with a different set sterilized scalpel and plated on corn- in use today on carrots. To date, no re- of fields). Soil from the surface to 8

CALIFORNIA AGRICULTURE, MARCH-APRIL 2002 77 Fig. 2. Relative growth of Pythium violae on agar amended with the fungicide mefenoxam and soil extracts from four organic and four conventional fields. Bars represent standard errors. After 8 days, the fungicide in soil extracts from conventional fields was significantly less inhibitory to P. violae than in soil extracts The fungicide mefenoxam initially provides effective control of cavity spot, from organic fields. but after repeated applications over several years its efficacy can be reduced. Carrots are harvested in Kern County. inches deep was collected in a W– agar was expressed as a percentage of Bioassay designed, conducted shaped pattern across each field with a the diameter of colonies grown on soil core that was 1 inch in diameter. In the first experiment on plates amended with nonspiked soil. Twenty cores from each field were mefenoxam degradation, a bioassay Data from the two trials were com- bulked, sifted and placed in a large designed to quantify mefenoxam ac- bined. plastic tub to receive and facilitate the tivity was conducted with an isolate of The colony diameter of P. violae on incorporation of 350 microliters (µl) P. violae (isolate ctl-4a) originally iso- plates prepared from spiked organic mefenoxam in 50 milliliters deionized lated in carrots from Kern County. The soils over the duration of the experi- water. In order to quantify the degra- bioassay method was similar to meth- ment remained at 25% to 30% of the dation of the fungicide, the amount ods used in earlier studies (Bailey and control (fig. 2). The mefenoxam ap- added to the soil was 100 times the la- Coffey 1984; Wicks 1988). An aliquot plied to the organic soil on day zero beled rate. Immediately after the fun- of 50 grams of dried soil from each was not markedly degraded during gicide was thoroughly mixed into the sample was agitated in 100 milliliters the 16 days of the experiment. In con- soil, a 100-cubic-centimeter (cc) sample deionized water for 1 hour on an or- trast, the colony diameter on plates of soil was collected and dried in a bital shaker at 150 rpm. After the soil prepared from conventionally farmed 158˚F (70˚C) oven. Once dried, the soil suspension was filtered through #1 soils increased on days 12 and 16, indi- was ground with a mortar and pestle Whatman paper, cornmeal agar was cating reduced activity of mefenoxam and stored in a plastic bag. This pro- added to the filtrate at a rate of 0.017 in these soils after 8 days. cess was repeated at 4-day intervals grams of cornmeal agar per milliliter for 16 days. Between samplings, the of filtrate. The amended agar was then Soil analysis soil was stored at room temperature in autoclaved and poured into three petri In the second experiment on a plastic pot with a top diameter and plates. A 4-millimeter plug from the mefenoxam degradation, a direct mea- depth of 5 inches. The soil moisture margin of a 3-day-old colony of surement of mefenoxam levels in soil content was adjusted to approximately P. violae was placed in the center of was determined with a gas chromato- field capacity by the addition of deion- each plate. After 4 days at room tem- graph (Farrar and Davis 2000). An ali- ized water each time the soil was perature, the colony diameter on each quot of 5 grams of dried soil from each sampled. A 200-cubic-centimeter ali- plate was measured. The experiment sample was added to 5 milliliters of quot (portion) of soil collected prior to was performed twice and the data methanol and shaken. After the spiking with mefenoxam served as the combined. The inhibition of colony methanol was allowed to evaporate at control for each field. growth on the fungicide-amended room temperature, the contents of the

78 CALIFORNIA AGRICULTURE, VOLUME 56, NUMBER 2 Agricultural Research Service/USDA

Fig. 3. Degradation of mefenoxam in four organic and four conventional field soils spiked with the fungicide. Mefenoxam concentration (ppm) was determined by gas chromatography. Bars represent standard errors. After 8 days, the concentration of mefenoxam in soils from conventional fields was significantly lower than that in organic soils. Soil microflora apparently degrades mefenoxam more rapidly after repeated applications. Carrot growers may need to rely on longer crop rotations and limit use of the fungicide in order to ensure a healthy crop. vials were resuspended in 1 milliliter conventional soils decreased rapidly References of hexane. Standards, controls and after 8 days. These results indicate a Bailey AM, Coffey MD. 1984. A sensitive samples were analyzed in a gas chro- more rapid degradation of mefenoxam bioassay for quantification of metalaxyl in matograph. The concentration of in carrot field soils with a history of soils. Phytopath 74:667–9. mefenoxam for each soil extraction mefenoxam application. Bailey AM, Coffey MD. 1985. Biodegrada- tion of metalaxyl in avocado soils. Phytopath was calculated using a standard curve Apparently, the soil microflora that 75:135–7. prepared for each field. Data from the degrades mefenoxam is stimulated af- Bailey AM, Coffey MD. 1986. Characteriza- two trials were combined. ter repeated applications of the fungi- tion of microorganisms involved in accelerated biodegradation of metalaxyl and metalachlor in In the organic soil, the concentra- cide (Bailey and Coffey 1985, 1986); soils. Can J Microbiol 32:562–9. tion of mefenoxam remained at about they are stimulated simply because the Davis RM, Nunez JJ, Guerard JP, Vivoda 125 ppm from day zero to day 12, and fungicide is a source of nutrition. E. 1991. If registered, fungicide could reduce then decreased to 100 ppm (fig. 3). In This is problematic in California’s cavity spot in carrots. Cal Ag 45(2):29–30. Droby S, Coffey MD. 1991. Biodegradation contrast, the concentration of carrot fields since mefenoxam is the process and the nature of metabolism of mefenoxam in the conventional soil only fungicide available to growers for metalaxyl in soil. Ann Appl Biol 118:543–53. degraded rapidly after 8 days. At 16 cavity spot control. Because of the re- Farrar JJ, Davis RM. 2000. Detection and quantification of metalaxyl using the MIDI Mi- days, the concentration of mefenoxam cent failures to control the disease, it crobial Identification System. Phytopath in soil from conventionally farmed may be prudent to practice long crop 91:S184. fields was less than half the concentra- rotations and to limit the use of the Schrandt JK, Davis RM, Nunez JJ. 1994. tion of mefenoxam in the organic soils. fungicide, where possible. Host range and influence of nutrition, tempera- ture and pH on growth of Pythium violae from Microflora degrades fungicide carrot. Plant Dis 78:335–8. Vivoda E, Davis RM, Nunez JJ, Guerard Both the bioassay and gas chroma- J.J. Farrar is Assistant Professor, Plant JP. 1991. Factors affecting the development of tography methods of quantifying the Sciences Department, California State cavity spot on carrot. Plant Dis 75:519–22. Wicks TJ. 1988. Effect of metalaxyl on the concentration of mefenoxam gave University, Fresno; J.J. Nunez is Farm control of Phytophthora crown rot of almonds. similar results. In the organic soils, Advisor, UC Cooperative Extension Aust J Exp Agric 28:547–52. mefenoxam levels decreased slightly (UCCE), Kern County; and R.M. Davis is over the 16 days of the experiment, UCCE Specialist, Department of Plant whereas levels of mefenoxam in the Pathology, UC Davis.

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