California Strawberry Commission ANNUAL PRODUCTION RESEARCH REPORT 2011 — 2012

® CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

2011 - 2012 Table of Contents

Introduction – Message From The Research Committee Chairman ...... 5

Pathology A Comprehensive Approach To Management of Wilt Diseases Caused by Fusarium oxysporum and Verticillium dahlia...... 9 Dr. Thomas R. Gordon

Operating A State-wide Strawberry Disease Diagnostic Services Center ...... 21 Steven Koike

Continuing Development of Management Strategies for Charcoal Rot (Macrophomina phaseolina) of Strawberry in California...... 27 Steven Koike

Fungicide Trials For Fruit and Foliar Pathogens of Strawberry...... 35 Mark Bolda Steven Koike

Plant Nutrition Establishing Nutrient Management Practices For High-yield Strawberry Production...... 41 Dr. Timothy Hartz

Irrigation Management Effects of Sprinkler, Partial Sprinkler/Drip and Drip Only Irrigation on Strawberry Transplants...... 59 Dr. Stuart Styles

Entomology California Strawberry Commission Lygus Management Program in Strawberries: Evaluating the Degree-day Model and Insecticide Resistance...... 77 Dr. Hillary Thomas

Strawberry Insect and Mite Control...... 85 Dr. Frank G. Zalom

Weed Science Weed Management In Strawberry...... 101 Dr. Steven Fennimore

2 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Farming Without Fumigants Efficacy of Drip Treatments for Management of Macrophomina and Fusarium Pathogens of Strawberry...... 111 Dr. Husein Ajwa Steven Koikev

Effect of Substrate Air-filled Porosity on Growth of Strawberry...... 121 Dr. Richard Evans

Active Management of Soil Microbial Communities to Limit Soilborne Disease Development in Strawberry Production Systems...... 133 Dr. Mark Mazzola

Non-fumigant Strategies for Soilborne Disease Control in California Strawberry Production Systems...... 145 Dr. Carol Shennan

Evaluation of a Substrate Based Raised Bed Trough (RaBeT) Strawberry Production System in California...... 161 Dr. Hillary Thomas

Regulatory Predicting Harvester Pesticide Exposure From Leaf Residues...... 171 Dr. Robert Krieger

Appendices Commission Members and Alternates for 2011-2012 ...... 184 Research Committee Members 2011-2012 ...... 186 2012 Grower Resource and Contact Information ...... 187

3 2011 - 2012 RESEARCH PROJECTS 4 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Introduction A Message From the Research Committee Chairman

The 2011-2012 Annual Production Research Report summarizes the results of 15 research and extension projects funded by the California Strawberry Commission. These reports are progress updates for the research projects funded by the commission during the 2010-2011 fiscal year. These projects include strawberry nutrition, Lygus bug monitoring, farming without fumigants and fumigant emission reduction, and many others. The commission is a leading funding source for strawberry production research and we hope that these reports help strawberry growers address production problems. These reports are also intended to document research that may not be readily available in other in other publications. It is hoped that these reports will be used by researchers in California and elsewhere to guide their own research.

On the behalf of the California strawberry industry and the California Strawberry Commission, I would like to thank the researchers and their associates for their dedication to the needs of the California strawberry industry. California strawberry growers face increasing production and regulatory challenges and the research efforts of these researchers are critical for the continued viability of the California strawberry industry.

I also want to thank the Research Committee, the Research Committee Leadership Group (Will Doyle, Bryan Gresser, Brian Driscoll and Dan Legard) and the members of the Science Advisory Committee for their help to ensure that we fund projects appropriate for the California strawberry industry. I would like to especially thank the many growers, PCA’s and other members of the strawberry industry who have provided assistance, plants, field plots, labor and materials for this work. I also want to thank the California Strawberry Nurserymen’s Association, The University of California and the USDA for their continuing support of the commission’s research programs.

Sincerely,

Carl Lindgren Research Committee Chairman

5 2011 - 2012 RESEARCH PROJECTS 6 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT PATHOLOGY

7 2011 - 2012 RESEARCH PROJECTS 8 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

A Comprehensive Approach to Management of Wilt Diseases Caused by Fusarium oxysporum and Verticillium dahliae

Principle Investigator Dr. Thomas R. Gordon University of California Department of Plant Pathology One Shields Avenue Davis, CA 95616 (530) 754-9893 [email protected]

Co-Principal Investigators Steven T. Koike, UC Cooperative Extension Oleg Daugovish, UC Cooperative Extension

Cooperators Douglas V. Shaw and Kirk D. Larson Plant Sciences Department U.C. Davis

Summary Our research in 2011 was directed toward the study of vascular wilt diseases caused by the soilborne pathogens, Verticillium dahliae and Fusarium oxysporum f. sp. fragariae. A central focus was on development and characterization of genetic resistance, which will make an increasingly important contribution toward management of wilt diseases in the future. Selecting for resistance to Verticillium wilt over many years has significantly increased levels of resistance to this disease, and in 2011, 63% of 61 breeding lines tested had resistance scores of 4.5 or higher (on a 1 to 5 scale, with 5 being a disease-free plant). We conducted a second year of tests in naturally infested soil to confirm the efficacy of resistance to Verticillium wilt. The results showed that ranking of cultivars based on susceptibility was essentially the same when infection occurred from exposure to inoculum in soil as when plants were root-dip inoculated. This indicates that genotypes identified as resistant by the screening procedure used in the University of California (UC) breeding program should be resistant under field conditions as well. In 2011, we continued development and implementation of a procedure to screen for resistance to Fusarium wilt. This included a comparison of inoculations with a single isolate and a mix of five isolates, and also a comparison of two different inoculum levels. Although disease was somewhat more severe in plants inoculated with the higher dose, the ranking of genotypes was similar. There was not a significant difference in results obtained with the single isolate or the mix.

9 2011 - 2012 RESEARCH PROJECTS As in past years, ‘Ventana’ and ‘San Andreas’ proved to be resistant to Fusarium wilt, whereas ‘Camarosa’ and ‘Albion’ were highly susceptible. Resistance scores for 26 breeding lines ranged from 1.0 to 5.0, with a mean of 3.3. Because grower observations suggested that soil acidification might render plants more prone to Fusarium wilt, we conducted experiments to test for an effect of pH on development of disease. Our results indicate that soil pH has at most only a weak effect on disease development, at least under controlled conditions. We continued to monitor the occurrence of Fusarium wilt and dieback caused by Macrophomina phaseolina, within California. Although Fusarium wilt has thus far been a serious problem only in Ventura County, recent finds indicate it may now also be established in the Santa Maria and Watsonville areas. Macrophomina has been confirmed to occur in all of the major strawberry production regions in the state.

Introduction Historically, Verticillium wilt, caused by the pathogenic soilborne fungus, Verticillium dahliae, has been a major constraint on strawberry production in California. This disease remains a serious problem for organic strawberry growers and is of increasing concern for conventional producers where flat fumigation with methyl bromide and chloropicrin is no longer an option. When a plant suffering from Verticillium wilt dies, the pathogen produces large numbers of melanized, multi-cellular survival structures known as microsclerotia, which are incorporated into soil with crop residue. Microsclerotia can survive for one or more years and infect the roots of a susceptible crop subsequently grown at the same location. Even in fields where pre-plant fumigation has been applied for many years, the fungus can still be detected. Once fumigation is discontinued or ceases to be fully effective, populations of V. dahliae can be expected to increase to damaging levels. The risk is greatest where strawberries are grown in rotation with crops such as potato (high elevation nurseries) and lettuce (fruit production fields), both of which are susceptible to the same strain ofV. dahliae that causes disease in strawberries.

Where pre-plant fumigation cannot eliminate the risk of disease caused by V. dahliae, the extent of damage from Verticillium wilt will likely be determined primarily by the susceptibility of the crop. In other words, it will be necessary to develop strawberry cultivars that are resistant to the disease. In many crops, resistance has been achieved through breeding to combine a single gene for resistance with the characteristics required for a commercial cultivar. However, the efficacy of single gene resistance is often short-lived, as pathogen strains capable of overcoming it can quickly increase in abundance. The alternative is to develop disease resistance based on multiple genes. A longer timeframe is needed to achieve this type of resistance but it is likely to be much more durable. This is the approach being taken by the UC strawberry breeding program.

Since 1994, all strawberry genotypes used as parents have been tested for susceptibility to Verticillium wilt, as part of a multiple trait selection strategy. By using resistance to Verticllium wilt as a selection criterion, resistance scores for the parents used in the UC breeding population have increased by over 60%, and the percentage of genotypes that are at least moderately resistant has increased from 35% in the original germplasm to 78.5% in genotypes used as parents for recent crosses (Shaw et al., 2010a). As a result, breeding lines advanced to cultivar status are typically more resistant than cultivars released in earlier years. This report describes the results of the annual screening conducted in spring of 2011.

One objective of our research has been to verify that strawberry genotypes identified as resistant to Verticillium wilt based on the root-dip inoculation test used in the screening program will manifest resistance when exposed to V. dahliae under field conditions. For this purpose, we have developed naturally infested soils in which to grow plants that differ in susceptibility to Verticillium wilt based on a root-dip assay. The first tests were conducted during the 2009-10 season and the results showed that disease severity resulting from root dip inoculations and from exposure to soilborne inoculum were highly correlated. This report describes results from the second year of the study.

10 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Plant collapse caused by Fusarium oxysporum has become a serious problem in southern California production areas (Koike et al., 2009). It is not known when this pathogenic strain was introduced to California but it seems likely that its recent emergence is due to changing production practices. In particular, the combination of less effective materials (i.e., fumigants not including methyl bromide) and less complete treatment of soil (application to beds rather flat fumigation of an entire field) has allowed the Fusarium wilt pathogen to increase to levels that are damaging. Like V. dahliae, F. oxysporum causes a wilt disease by invading the plant’s water conducting tissue, and as with Verticillium wilt, genetic resistance offers a promising approach to disease management. For this reason, we have developed procedures to be used for screening breeding lines and current cultivars for resistance to Fusarium wilt. We also conducted experiments to test for an effect of pH on development of disease, because grower observations suggested that soil acidification might render plants more prone to this problem.

Materials and Methods Assessment of Susceptibility to Verticillium wilt Eleven cultivars and 61 breeding lines were tested for susceptibility to Verticillium wilt. This was accomplished by immersing roots of runner plants in an aqueous suspension of pathogen spores (Gordon et al., 2006). The spore suspension was prepared from plates of potato dextrose agar (PDA) that were fully colonized by a known virulent isolate of V. dahliae. Spores were washed from the plate using sterile distilled water, and the density of spores was adjusted to 29.4 million spores/fluid ounce (= one million spores/ml). Inoculated plants were immediately transplanted into plots at the Wolfskill experimental orchard (Winters, CA) that had been fumigated with 2 methyl bromide:1 chloropicrin at 350 pounds per acre. Two replicate plots of five seedlings per plot were established for each entry. Plants were given a resistance score based on symptoms of disease using a 1 to 5 scale, with 5 corresponding to a healthy plant and 1 corresponding to a severely diseased or dead plant.

Evaluation of Disease Resistance in Naturally Infested Soil In April of 2010, potato tubers were planted at the UC facility in Watsonville, in ground that had been fumigated with 2 methyl bromide:1 chloropicrin at 350 pounds per acre. Stems were inoculated with a spore suspension of V. dahliae in May, and the disease was allowed to develop for approximately three months. In August, stems were cut and moved into a small area (approximately 100 square feet) within the plot and incorporated into the soil, which was tilled periodically to facilitate decomposition. In October, soil was removed from the incorporation area and placed in a mixer to obtain a more uniform distribution of inoculum. After mixing, soil samples were taken and the inoculum density was estimated to be approximately 451.4 ± 11.4 colony forming units per gram of soil (= 15.8 ± 3.5 per gram gram). This Verticillium- infested soil was used to replace approximately one gallon of fumigated soil at each site where a transplant was placed. In this way, nine cultivars and seven breeding lines were established in two replicate beds, with 20 plants per bed. For comparison, 40 plants of each genotype were also subjected to a root-dip inoculation and 40 plants were not inoculated. Disease severity ratings from all three treatments (infested soil, root-dip and control) were taken throughout the 2011 growing season.

11 2011 - 2012 RESEARCH PROJECTS Evaluation of Soil pH on Severity of Fusarium wilt Dormant strawberry plants of the cultivar Camarosa were grown in one gallon pots filled with Sunshine Mix #1 (Sun Gro Horticulture, Canada) that was adjusted to one of four pH levels (pH 5, 6, 7 or 8), and one of four inoculum densities (0; 14,175; 141,750 or 1,417,500 spores per ounce of potting mix or = 500, 5000, and 50 000 spores per gram), for a total of 16 treatments in a factorial design, with four plants (= replications) per treatment. Inoculum for this experiment was produced by growing cultures of F. oxysporum (isolate GL 1080) on PDA for 14 days under ambient lighting at room temperature (20.2 to 22.8 C). Thirty-eight plates were macerated in a blender with 700 ml sterilized de-ionized water, mixed with 6.5 L of twice autoclaved sand, and dried at room temperature. The inoculum density of the sand was estimated using dilution plating on malachite green agar. Inoculated sand was amended with sterile sand as needed to obtain target inoculum densities. Sand mixed with a slurry of non-inoculated PDA and sterilized de-ionized water was used for the control treatment (0 spores /ounce). To obtain the desired pH, phosphoric acid (H3PO4) or sodium hydroxide (NaOH) was added to 2.3 pounds of moistened potting mix as follows: pH 5: 0.5 ounces of 10% H3PO4; pH 6: 0.2 ounces of 1 M NaOH; pH 7: 1.3 ounces of 1 M NaOH; pH 8: 0.2 ounces of 10 M NaOH. Each batch of pH adjusted potting soil was mixed with 1,200 g of sand of the appropriate inoculum density. The inoculated potting mixture was divided among four one gallon pots and placed in a single plastic tray. This process was repeated as needed to obtain the potting mixtures required for all treatments. Soil pH was measured and re-adjusted over the next three days until it remained at the desired level.

Plants were maintained in a growth chamber at day/night temperatures of 77/ 64 F (25/18 C), with a 12 hour photoperiod and supplied with dilute fertilizer (2:1:2 N-P-K in de-ionized water) as needed. Prior to each watering event, a small sample of potting mix from one pot at each inoculum density of each pH level was collected from around the base of the plant. Each sample was suspended in water and the pH was measured. If the pH was more than 0.5 units away from the desired level, either 10% H3PO4 or 1 M NaOH diluted in de-ionized water was added to the pots needing pH adjustment. Soil pH was monitored and adjusted as needed throughout the experiment in this manner. Approximately nine weeks after planting, all plants were rated on a scale of 1 to 5 (with 1 corresponding to a healthy plant and 5 for a plant that died from the disease). The experiment was conducted twice.

A similar experiment to the one described above was conducted using a combination of potting mix and field soil. This experiment included four soil pH levels (pH 5, 6, 7 and 8), three inoculum densities (0, 283,500 and 708,750 spores/ ounce) and two soil treatments (potting mix alone or potting mix combined with field soil at the rate of approximately 30 grams per pot), for a total of 24 treatments in a factorial design, with four plants per treatment. Field soil was collected from a recently fumigated plot at the Wolfskill experimental orchard that was previously cropped to strawberries. Soil was assayed on Komada’s selective medium to confirm the absence ofF. oxysporum f. sp. fragariae. Inoculum was prepared and pH adjusted as described above. Eight weeks after planting, all plants were rated on a scale of 1 to 5, based on symptom development. The experiment was conducted twice.

Efficacy of Fumigation in Eradication of F. oxysporum f. sp. fragariae Inoculum of F. oxysporum f. sp. fragariae was produced by growing the fungus on PDA, blending fully colonized agar in water and adding the resulting slurry to sand. The sand agar mix was stirred periodically over a period over several days until it was dry. Thereafter, the infested sand was packaged in nylon pouches, which were buried at a depth of approximately 6 inches below the surface both at the center and on the shoulder in four replicate beds for each treatment. The following fumigation treatments were applied: 1) Midas EC Gold (33:62) at 169 pounds per acre through drip lines, 2) Midas EC Gold (33:62) at 225 pounds per acre through drip lines, 3) shank applied Pic60 (62% chloropicrin and 38% 1,3 dichloropropene) at 200 pounds per acre, 4) same as treatment #3 but followed by application of 30 gallons per acre of metam sodium through drip lines, 5) shank applied Pic60 at 300 pounds per acre and 6) same as treatment #5 but

12 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

followed by application of 30 gallons per acre of metam sodium through drip lines. Pouches were recovered and the infested sand was assayed by soil dilution plating. In this procedure, sand was suspended in water, and the suspension was spread over the surface of a selective medium. Colonies of F. oxysporum f. sp. fragariae were enumerated in order to quantify the viability of inoculum recovered from each pouch.

Screening for Resistance To Fusarium wilt Ten cultivars and 26 breeding lines (= 36 genotypes) were tested for susceptibility to Fusarium wilt. This was accomplished by immersing roots of runner plants in an aqueous suspension of pathogen spores. A spore suspension was prepared from plates of PDA that were fully colonized by one of five isolates of F. oxysporum f. sp. fragariae. Spores were washed from each plate using sterile distilled water and adjusted by the addition of sterile water to obtain a density of approximately 29.4 million spores/fluid ounce (= one million spores/ml). A spore suspension of each isolate was used to inoculate 400 mL of potato dextrose broth (PDB) in a 1 L flask, which was maintained on a rotary shaker for approximately seven days at room temperature. Colonized PDB from each flask was filtered through three layers of sterilized cheesecloth. The spore density was adjusted to two different levels: 44.1 and 29.4 million spores/fluid ounce by the addition of 0.1% water agar as needed. At both densities, spore suspensions of five isolates were combined so each of the five was represented by approximately 982,00 and 588,000 spores per ounce at the high and low densities, respectively. The high dose five-isolate mix was used to inoculate all 36 genotypes. Four cultivars (‘Albion,’ ‘Ventana,’ ‘San Andreas’ and ‘Camarosa’) were also inoculated with the low dose of the isolate mix and both the high and low doses of a single isolate (GL 1080). Inoculated plants were immediately transplanted into plots that had been fumigated with 2 methyl bromide:1 chloropicrin at 350 pounds per acre at the Wolfskill experimental orchard. Two replicate plots of five seedlings per plot were established for each entry. Plants were given a resistance score based on symptoms of disease using a 1 to 5 scale, with 5 corresponding to a healthy plant and 1 corresponding to a severely diseased or dead plant.

Results Assessment of Susceptibility To Verticillium wilt The eleven cultivars tested in 2010-11 were included mainly to provide a point of reference with previous years. ‘Albion’ and ‘Camino Real’ both had scores of 5.0, whereas ‘Camarosa’ was rated at 4.0. These scores are higher than seen for the same cultivars in most prior years, which is probably due to a weather pattern during 2010-11 that was less favorable to development of Verticillium wilt. Unlike most recently released cultivars, ‘Benicia’ appears to be relatively susceptible to Verticillium wilt, with a resistance score of 3.5. Of the 61 breeding lines tested, resistance scores ranged from 3.0 to 5.0, with an average of 4.6. Sixty-three percent had scores equal to or greater than 4.5 and 44% had the maximum resistance score of 5.0, meaning they were entirely free of symptoms at the end of the season.

Evaluation of Disease Resistance In Naturally Infested Soil The lowest incidence of disease was in the breeding line 94-256-607. This was true for both root dip inoculated plants (1.3%) and plants grown in infested soil (2.5%). The highest incidence was seen in breeding line 4-39-1, with 70% of root-dip inoculated plants and 46% of plants grown in infested soil showing symptoms of Verticillium wilt. Overall, disease incidence was higher in root-dip inoculated plants, for which the average across all genotypes was 23.4%, than in plants grown in infested soil (16.4%). However, the results of the two inoculations methods were strongly and significantly correlated (R2 = 0.893; P < 0.001) (Figure 1).

13 2011 - 2012 RESEARCH PROJECTS 50

45 R2 = 0.893 40 P < 0.001

35

30

25

20

15 10 5

Percentage of symptomatic plants in infested soil 0 0 10 20 30 40 50 60 70 80

Percentage of root-dip inoculated plants symptomatic

Figure 1. Each point corresponds to a single cultivar or breeding line. The location of the point is defined by the percentage of plants showing symptoms of Verticillium wilt when subjected to a root-dip inoculation (X-axis) or when grown in infested soil (Y-axis).

The Effect of pH on Severity of Fusarium wilt in Infested Potting Mix Plants grown in the absence of inoculum (controls) appeared healthy at all pH levels throughout both experiments, whereas disease severity was clearly influenced by inoculum density. On the other hand, there was not an obvious trend in disease development that correlated with soil pH. The final ratings of all plants from both experiments, excluding negative controls, were evaluated using analysis of variance (ANOVA). Experiment (P < 0.001), replication (P < 0.001) and inoculum density (P < 0.001) were significant factors but pH (P = 0.527) was not a significant factor. The effects of several interaction terms were significant (P < 0.050), and consequently data were analyzed separately by experiment.

For the first experiment, the effect of inoculum density on disease severity was significant (P < 0.001), whereas the effects of soil pH (P = 0.408) and replication (P = 0.130) were not. The pH by inoculum density interaction was significant (P < 0.001), so data were analyzed separately by inoculum density. At the low inoculum level (14,175 spores /ounce), the effect of pH was significant (P < 0.001), whereas replication was not (P = 1.000). Mean disease severity ratings (± standard error) were: 2.0 ± 0.0 at pH 5, 1.3 ± 0.1 at pH 6, 1.5 ± 0.0 at pH 7, and 2.3 ± 0.3 at pH 8. At the intermediate inoculum level (141,750 spores /ounce), replication was significant (P < 0.001) and pH was not (P = 0.127). Mean disease severity ratings were: 2.3 ± 0.3 at pH 5, 2.3 ± 0.2 at pH 6, 3.6 ± 0.4 at pH 7, and 2.3 ± 0.8 at pH 8. At the high inoculum density (1,417,500 spores /ounce), the effects of pH (P = 0.270) and replication (P = 0.987) were both not significant. Mean disease severity ratings for plants grown at this inoculum density were: 4.9 ± 0.1 at pH 5, 5.0 ± 0.0 at pH 6, 4.8 ± 0.3 at pH 7, and 4.5 ± 0.3 at pH 8 (Figure 2).

14 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

6 pH 5

5 pH 6

pH 7 4 pH 8

3

2

Disease severity rating 1

0 Low Intermediate High Inoculum density

Figure 2. Severity of disease on a 1 to 5 scale (with 1 corresponding to a healthy plant and 5 corresponding to a plant killed by Fusarium wilt) for the cultivar ‘Camarosa’ grown in potting mix with three different levels of inoculum (low, intermediate and high) and at four different levels of soil pH (5, 6, 7 and 8). This figure presents the results of the first experiment.

Based on data from the second experiment, the effect of inoculum density was significant (P < 0.001), whereas the effects of soil pH (P = 0.305) and replication (P = 0.136) were not significant. The pH by inoculum density interaction was significant (P = 0.018), and consequently data were analyzed separately by inoculum density. At 14,175 spores / ounce, pH (P = 0.007) and replication (P = 0.028) were significant. Mean disease severity ratings for plants grown at this inoculum density were: 1.1 ± 0.1 at pH 5, 2.8 ± 0.8 at pH 6, 2.8 ± 0.3 at pH 7, and 3.6 ± 0.4 at pH 8. At 141,750 spores /ounce, the effects of pH (P = 0.045) and replication (P < 0.001) were significant. Mean disease severity ratings for plants grown at 141,750 spores /ounce were: 3.9 ± 0.7 at pH 5, 4.4 ± 0.6 at pH 6, 4.5 ± 0.5 at pH 7, and 3.1 ± 0.7 at pH 8. At 1,417,500 spores /ounce, the effect of pH was significant (P < 0.001) but replication was not (P = 0.972). Mean disease severity ratings were: 5.0 ± 0.0 at pH 5, 5.0 ± 0.0 at pH 6, 4.1 ± 0.1 at pH 7, and 4.4 ± 0.2 at pH 8 (Figure 3).

Figure 3. Severity of disease on a 1 to 5 scale (with 1 corresponding to a healthy plant and 5 corresponding to a plant killed by Fusarium wilt) for the cultivar ‘Camarosa’ grown in potting mix with three different levels of inoculum (low, intermediate and high) and at four different levels of soil pH (5, 6, 7 and 8). This figure presents the results of the second experiment.

15 2011 - 2012 RESEARCH PROJECTS Effect of pH on Disease Severity in Field Soil All plants grown in non-inoculated soil remained free of Fusarium wilt symptoms throughout the course of both experiments. At the high inoculum level (708,750 spores/ounce), all plants developed severe symptoms of Fusarium wilt and there was extensive mortality, regardless of pH or soil composition. At the low inoculum level (283,500 spores/ounce), plants grown in potting mix combined with field soil often had slightly higher disease severity ratings than those grown in potting mix alone (Figure 4). However, based on data from both experiments at the lower inoculum level, ANOVA showed pH to be the only significant factor (P = 0.039). Experiment (P = 0.211), inoculum density (P = 0.215), treatment (field soil present or absent, P = 0.554), and replication (P = 0.054) were all not significant. Likewise, all interactions between these factors were not significant (P ≥ 0.198). Mean disease severity ratings averaged across treatment were 2.7, 3.5, 4.0 and 4.0 for plants grown at pH 5, 6, 7 and 8.

Figure 4. Severity of disease on a 1 to 5 scale (with 1 corresponding to a healthy plant and 5 corresponding to a plant killed by Fusarium wilt) for the cultivar ‘Camarosa’ grown in potting mix with and without field soil at four different pH levels (5, 6, 7 and 8).

Effect of Fumigation in Eradication of F. oxysporum f. sp. fragariae Fusarium oxysporum f. sp. fragariae was not detectable in infested sand buried at the center of beds treated with Midas EC Gold at either 169 or 225 pounds per acre, but 44,192 spores per ounce were recovered from pouches buried at the side of the bed, where the low rate was applied. Both rates of shank-applied Pic60 eliminated the pathogen at the sides of the bed but the pathogen remained viable at the center of the bed, with 10,894 and 1,210 spores per ounce surviving at the low (200 pounds per acre) and high rates (300 pounds per acre), respectively. Where the low rate of Pic60 was followed by 30 gallons per acre of metam sodium, surviving inoculum densities were 303 and 9,104 spores per ounce at the center and side of the bed, respectively. The high rate of Pic60 followed by metam sodium eliminated the pathogen at the center of the bed but 605 spores per ounce survived at the side of the bed.

16 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Screening for Resistance to Fusarium wilt Disease developed as expected, with susceptible cultivars such as ‘Camarosa’ and ‘Albion’ displaying severe symptoms by the final rating, whereas the resistant cultivar, ‘Ventana’, showed at most mild stunting, regardless of treatment. The higher inoculation dose (982,000 spores per ounce) tended to result in more severe disease than the lower dose (588,000 spores per ounce). For example, resistance scores for ‘Albion’ inoculated with GL1080 were 3.0 and 1.5 at the low and high doses, respectively. However, analysis of the full data set showed the effect of dose on disease resistance scores not to be significant (P = 0.077). Likewise the effect of isolate (P = 0.141) and all interactions (P ≥ 0.184) were not significant. When non-significant terms were removed from the model, differences among genotypes were significant (P < 0.001). Resistance scores for 26 breeding lines ranged from 1.0 to 5.0, with a mean of 3.3.

Discussion Scores for resistance to Verticillium wilt among UC breeding lines are slightly above what was recorded in 2010. Sixty-three percent had scores at or above 4.5 as compared to 56% last year. However, year over year comparisons of the same genotypes indicate that disease severity was lower overall in 2011. Consequently, the actual differences in susceptibility between breeding lines in the two years are probably less than what is implied by data from 2011. Although most recently released cultivars are significantly more resistant than those developed prior to adoption of the current screening protocol, ‘Benicia’ appears to be an exception, based on a relatively low resistance score in the 2011 test.

Tests conducted in soil naturally infested with V. dahliae were intended to assess the performance of cultivars and breeding lines that have been selected based on their reaction to artificial inoculations. The 2011 experiment was a repeat of a similar experiment conducted in 2010. In 2010, plants were exposed to approximately 300 microsclerotia per ounce of soil, whereas in the 2011 tests inoculum density was estimated to be about 450 microsclerotia per ounce. For comparison, something in the range of 100 microsclerotia per ounce of soil would be expected to cause a significant economic loss in most years. In both 2010 and 2011 there was a strong and highly significant correlation between resistance scores observed for plants subjected to root-dip inoculations and those becoming infected by exposure to soilborne inoculum. This indicates that the screening procedure being used by the UC breeding program has been effective in identifying differences in susceptibility that correspond with how strawberry genotypes respond to the disease under natural conditions.

Previous work has shown that liming to bring soil pH to 6.5 and above can reduce severity of Fusarium wilt affecting watermelon (Everett and Blazquez, 1967) and tomato (Jones and Woltz, 1967). This effect has been attributed to greater availability of micro-nutrients for the pathogen under acidic conditions and more intense competition with bacteria when soil pH is closer to neutral. Grower observations suggested that Fusarium wilt of strawberry in California was more commonly seen in fields where fertigation (the application of nutrient solutions through drip irrigation lines) acidified soil. Whereas growers have found this practice beneficial because lowering soil pH makes micro-nutrients more available to plants, it may also enhance disease severity by releasing the Fusarium wilt pathogen from nutrient limitations on growth and infection.

17 2011 - 2012 RESEARCH PROJECTS The results of our experiments showed that the effect of pH on severity of Fusarium wilt was significant at some inoculum levels when plants were grown in potting mix. At the highest inoculum level disease was severe in all treatments so it was not possible to assess an effect of soil pH. At the lowest inoculum level, disease was relatively mild and only small differences in disease were observed. The most conspicuous effect of pH was apparent at the intermediate inoculum level, with disease being less severe at pH 5.0 than at 7.0 or 8.0. This is the opposite of what would be expected based on published results of pH effects on Fusarium wilt diseases affecting other crops. Of course, potting mix lacks the usual complement of bacteria found in soil and their absence may obscure an effect that would be evident under field conditions. However, even where field soil was included with potting mix, disease severity on average was lowest at pH 5.0 and greatest at pH 7.0 and pH 8.0.

It is not clear why our findings differ from what has been reported for Fusarium wilt of watermelon and tomato. It may reflect differences in the causal pathogens and/or conditions under which tests were conducted. However, it is also important to note that there are conflicting results in the literature regarding the effect of pH on development of Fusarium wilt in watermelon. In a study conducted in a naturally infested field, increased soil pH did not reduce disease severity (Hopkins and Elmstrom, 1976). Notwithstanding these negative results and our own findings under controlled conditions, we cannot exclude the possibility that soil pH affects severity of Fusarium wilt of strawberry under some circumstances. However, even if this is so, it seems unlikely that the effect of pH is strong enough to be a determinative factor in disease development in most fields, which precludes making any general recommendations on adjustment of soil pH for disease management.

Our fumigation experiment documented a differential effect of bed location on efficacy where fumigant is applied through drip lines. The lesser reduction at the side as opposed to the center of the bed is consistent with results of previous experiments and underscores the need to use fumigation practices that will maximize distribution within the bed. This may include using additional drip lines and/or increasing the volume of water used to deliver the fumigant. Where fumigants were applied by shank injection, efficacy tended to be lower at the center of the bed than on the sides. This result indicates that shank injection may also fail to achieve adequate distribution of fumigant within a bed.

As in 2010, the results of screening for resistance to Fusarium wilt showed some cultivars and breeding lines to be highly resistant to Fusarium wilt. ‘Ventana’ and ‘San Andreas’ both fall into the resistant category and this classification is supported by previous field trials where both cultivars were exposed to high inoculum levels. Conditions during our 2011 test were conducive to disease development as indicated by severe disease affecting both ‘Albion’ and ‘Camarosa.’ A significant number of breeding lines appeared to be highly resistant to Fusarium wilt, with 6 of 26 tested lines (23%) receiving the maximum score of 5.0 However, five breeding lines (19%) had resistance scores of 1.5 or less, indicating that considerable variation in susceptibility remains within the UC breeding population.

We have continued to monitor the occurrence of Fusarium wilt and charcoal rot, caused by M. phaseolina, within California. Charcoal rot was originally found in only two counties (Orange and Ventura), but has now been confirmed to occur in all major strawberry production regions in the state, including fields in Alameda, Contra Costa, Los Angeles, Monterey, Orange, Sacramento, San Diego, San Luis Obispo, Santa Barbara, Santa Clara, Santa Cruz, Ventura and Yolo Counties. Fusarium wilt of strawberry was originally limited to Ventura County. However, in early 2012, we isolated what appears to be the Fusarium wilt pathogen from declining strawberry plants in San Luis Obispo and Santa Cruz counties. These isolates cannot be considered pathogenic until further testing (inoculation tests) is completed. The Gordon and Koike labs are currently working to confirm pathogenicity of these isolates. In each succeeding year since their initial discoveries, the number of ranches infested with these two pathogens has continued to increase. In most cases, the disease is being found in fields where standard pre-plant, flat fumigation application of methyl bromide + chloropicrin has not been used.

18 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Selected References

• Everett P.H. and C.H. Blazquez. 1967. Influence of lime on the development of Fusarium wilt of watermelons. Florida Agricultural Experiment Stations Journal Series 2845:143-148.

• Gordon T.R., Kirkpatrick S.C., Hansen J. and D.V. Shaw. 2006. The response of strawberry genotypes to inoculation with isolates of Verticillium dahliae differing in host origin. Plant Pathology 55:766-769.

• Hopkins D.L. and G.W. Elmstrom. 1976. Effect of soil pH and nitrogen source on Fusarium wilt of watermelon on land previously cropped in watermelons. Proceedings of the Florida State Horticultural Society 89:141-143.

• Jones J.P. and S.S. Woltz. 1967. Fusarium wilt (race 2) of tomato: Effect of lime and micronutrient soil amendments on disease development. Plant Disease Report 51:645-648.

• Koike S.T., Kirkpatrick S.C. and T.R. Gordon. 2009. Fusarium wilt of strawberry caused by Fusarium oxysporum in California. Plant Disease 93:1077-1077.

• Shaw D.V., Gordon T.R., Larson K.D., Gubler W.D., Hansen, J. and S.C. Kirkpatrick. 2010a. Genetic progress in breeding for resistance of strawberry (Fragaria x ananassa Duch.) to Verticillium wilt (Verticillium dahliae Kleb.) California Agriculture 64:37-41.

• Shaw D.V., Gordon T.R., Hansen J. and S.C. Kirkpatrick. 2010b. Relationship between the extent of colonization by Verticillium dahliae and symptom expression in strawberry (Fragaria x ananassa) genotypes resistant to Verticillium wilt. Plant Pathology, 59:376-381.

19 2011 - 2012 RESEARCH PROJECTS 20 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 26 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 34 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 38 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT PLANT NUTRITION

39 2011 - 2012 RESEARCH PROJECTS 40 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 56 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT IRRIGATION MANAGEMENT

57 2011 - 2012 RESEARCH PROJECTS 58 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Irrigation Management

73 2011 - 2012 RESEARCH PROJECTS 74 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT ENTOMOLOGY

75 2011 - 2012 RESEARCH PROJECTS 76 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 84 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 98 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT WEED SCIENCE

99 2011 - 2012 RESEARCH PROJECTS 100 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 108 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT FARMING WITHOUT FUMIGANTS

109 2011 - 2012 RESEARCH PROJECTS 110 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 120 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Farming Without Fumigants

131 2011 - 2012 RESEARCH PROJECTS 132 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Farming Without Fumigants

Active Management of Soil Microbial Communities to Limit Soilborne Disease Development in Strawberry Production Systems

Principle Investigator Dr. Mark Mazzola, Research Plant Pathologist USDA/ARS 1104 N. Western Ave. Wenatchee, WA 98801 (509) 664-2280 [email protected]

Collaborator Dr. Michael F. Cohen Department of Biology Sonoma State University Rohnert Park, CA

Summary Studies were conducted to evaluate non-fumigant methods, including Brassicaceae seed meal amendment and wheat cultivation, as a means to suppress strawberry root infection by Macrophomina phaseolina, causal agent of charcoal rot of strawberry. As a follow up to the field trial conducted at Rohnert Park, CA (Sonoma State University) during the 2009-2010 growing season, a field trial was conducted at the same site during 2010-2011to assess seed meal efficacy for the control of charcoal rot. A seed meal formulation of Brassica juncea/Sinapis alba amendment suppressed M. phaseolina soil density and root infection but failed to provide a significant increase in plant growth or yield. The lack of a positive plant growth response was associated with symptoms of phytotoxicity and demonstrated the need for additional knowledge concerning the effect of soil properties on activity and persistence of seed meal derived chemistries, including those possessing herbicidal activity. Such information is required to determine duration of plant back periods that are suitable to avoid plant damage while yielding effective pest control. Similarly this knowledge will be useful in predicting effectiveness of seed meal as a bioherbicide, an activity which demonstrated a high level of efficacy in these trials but which has demonstrated inconsistency across production systems. Wheat cropping of soils proved to be statistically as effective as B. juncea seed meal amendment in controlling strawberry root infection by M. phaseolina when evaluated under controlled environment conditions. Likewise, cropping of a fumigated soil system with wheat prior to artificial infestation with M. phaseolina induced development of a soil microbial population suppressive to charcoal root rot of strawberry. Disease suppression in response to prior wheat cultivation was more complete, though not statistically different, from the seed meal alone or seed meal integrated with wheat treatments. Changes in microbial populations suggest that specific transformations in the fungal community resident to these soils contributed to the observed disease suppression, but the specific functional group(s) responsible for disease control is not currently known. The value of this approach for soil-borne disease management in strawberry requires analysis at the field level as a multiplicity of pathogens are known to function in these systems and may respond differentially to the wheat cropping protocol.

133 2011 - 2012 RESEARCH PROJECTS Introduction Intensive strawberry production in California and worldwide, has relied on pre-plant soil fumigation to control key soilborne pathogens, weeds and pests. Continuing review and regulatory actions will potentially limit access to or use of fumigants on a state-wide or regional basis. Efforts to develop effective non-fumigant alternatives for the control of yield limiting soilborne pests in strawberry are needed, but in general have attained minimal progress for numerous reasons including the often overwhelming complexity of the biological system.

Naturally occurring disease suppressive soils have been documented in numerous crop production systems, and in many instances the biological attributes contributing to suppressiveness have been identified (Mazzola, 2007). Occurrence of such soils suggests that the management of resident biological forces to limit populations or activity of soilborne pathogens can be a valuable component in development of a non-fumigant disease management strategy. However, the most often employed strategy in this regard has been the introduction of single or multiple strain microbial inoculants utilizing an inundative release model, a method that has yielded little success in field level production systems. Rather than inoculating soils or propagative materials with mass produced formulations of non-native biological agents, our strategy has employed cropping practices or amendments to manage the native soil biology with the goal of inducing soil suppressiveness. Such an approach requires an adequate knowledge base to effectively and specifically manipulate this biological resource. For instance, the use of organic amendments or cropping sequences to manage soil suppressiveness cannot be undertaken without basic information about the functional biological entities contributing to disease suppression.

The goal of the proposed program is to effectively manage the biological resources native to strawberry production systems for the management of soilborne diseases, with particular emphasis on control of charcoal rot incited by M. phaseolina. The specific goals for the second year of the project were to assess the capacity of Brassicaceae seed meals to suppress M. phaseolina in small scale field trials and assess the capacity of wheat cropping, with or withoutB. juncea seed meal amendment, to suppress re-infestation of fumigated soils by M. phaseolina.

Materials and Methods During the 2010-2011 growing season a field trial was conducted in raised planter beds (4.1m x 2.0m x 0.23m) at research plots near Sonoma State University, Rohnert Park, CA. Soil (Wright Loam, pH 7.4) treatments were distributed using a randomized split-split plot design with four replicates per treatment. Plots had been artificially infested with M. phaseolina during the 2010 growing season, and inoculum densities were amplified by cultivation of a soybean crop prior to establishment of the strawberry planting. Seed meal treatment consisted of a Brassica juncea/Sinapis alba formulation applied at a rate of 3 tons per acre. The site was planted to strawberry 28 days after seed meal application. Fruit production, runner biomass and plant mortality were monitored throughout the growing season.

Studies were conducted to access the effect of wheat cropping in conjunction with B. juncea seed meal (BjSM) or individually in suppression of disease incited by M. phaseolina. The experiment was carried out in a Santa Maria, CA field soil which exhibited plant collapse in response to a natural M. phaseolina infestation. Soil was amended with BjSM at a rate of 0.3% (wt/wt) and incubated for a period of 10 days, at which time one-half of the seed meal treated soil, and an equivalent volume of non-treated field soil, was planted with a wheat mixture of the ‘Penewawa’ and ‘Lewjain.’ After two months, aerial portions of the wheat plants were harvested and all soils (non-treated control, BjSM alone, BjjSM+wheat and wheat alone) were planted with strawberry (‘Camarosa’) with five replicates per treatment. Hoagland solution (macronutrients only) was applied to the no treatment control to attain equivalent N across soil treatments. Plants were grown in environmental chambers using a day/night temperature profile of 90/65 F with a 12 h photoperiod. Soil and root samples were collected periodically over the course of plant growth and M. phaseolina populations were determined by qPCR.

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Additional studies were conducted to assess whether post-fumigation methods could effectively promote the development of a microbial community suppressive toward M. phaseolina. Soil was fumigated in the field by a commercial applicator using the fumigant 1,3 dichloropropene (Telone)/chloropicrin. Soils were collected six weeks after treatment and stored in a cold room prior to use. The utility of a wheat cropping system, B. juncea seed meal amendment or the integration of these treatments was assessed for the ability to limit re-infestation of fumigated soils by M. phaseolina and subsequent strawberry root infection. Soil treatments included a no treatment control, B. juncea seed meal (0.3% wt/ wt), cultivation of soils with the wheat cultivar mixture (‘Lewjain’ and ‘Penewawa’), and seed meal amendment followed by white cultivation. Seed meal treated soils were incubated for 10 days in polyethylene bags to simulate tarping in the field. Soils were removed from bags and decanted into pots with five replicates per soil treatment and incubated on a greenhouse bench for eight weeks. For wheat treatments, soils were planted and wheat was grown for the same eight week incubation period at which time shoot tissue was harvested and discarded. Each pot was infested with M. phaseolina at a rate of 70 sclerotia per ounce of soil and planted with one strawberry (‘Camerosa’) plant per pot. Plants were harvested after three months and soil, root and crown samples were collected and stored in the cold room until processing. DNA was extracted from all tissue and soil samples and real-time quantitative PCR was conducted to detect and quantify the presence of M. phaseolina.

Data were analyzed using SigmaStat (version 3.1; Systat Software Inc., Point Richmond, CA). Soil population data were transformed to log10 values. Data were subjected to one way analysis of variance and mean separation was performed using the Student-Newman-Keuls test in which P < 0.05 was considered significant. In instance where data failed the Shapiro-Wilk test for normality, data were analyzed using the Kruskal-Wallis test on ranks

135 2011 - 2012 RESEARCH PROJECTS Results As previously observed in a field trial conducted in 2010, the Brassicaceae seed meal amendment significantly reduced weed biomass production irrespective of the presence or absence of M. phaseolina. As a result, weed biomass in seed meal treated plots was near zero at the time of strawberry planting and throughout the duration of the experiment (Figure 1). The seed meal treatment had no significant effect on strawberry plant biomass, total number of fruit produced or total fruit biomass over the course of this experiment when plants were cultivated in M. phaseolina infested soil (data not shown). In fact, when cultivated in M. phaseolina-infested soil strawberry plants exhibited a trend towards reduced productivity in seed meal treated soil relative to non-treated soil though these differences were not statistically (P > 0.05) different. Results obtained in soils that were not infested with the pathogen indicated that phytotoxicity of the seed meal amendment contributed to this plant response. In the absence of M. phaseolina, biomass production in control plots treated with the seed meal was significantly lower than that in control plots that did not receive the seed meal amendment. Likewise, in the absence of the pathogen, plant death in seed meal treated plots was approximately 16% while no plant death was observed in the control plots. In the pathogen infested plots, plant death in seed meal treated plots was 22% and again no plant death was observed in the pathogen-infested control plots. The direct role for seed meal phytotoxicity in the observed plant death, rather than disease incited by M. phaseolina, is also supported by the observation that M. phaseolina was detected in the control-pathogen infested soil but was below the limit of detection in the seed meal treated-pathogen infested (Figure 2). Likewise, M. phaseolina strawberry root infection was significantly higher in the control infested soil than the seed meal amended soil infested with the pathogen (Figure 3).

Figure 1. Effect of B. juncea/S.alba seed meal amendment on weed biomass harvested from strawberry field plots established at Rohnert Park, CA. Weed biomass was harvested at day 28 after seed meal amendment and prior to planting strawberry, and at completion of the experiment (day 142) in all plots. “Mp” indicates plots artificially infested with M. phaseolina. Columns designated with the same letter represent mean values that are not significantly different (P > 0.05).

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Figure 2. Effect of B.juncea/S. alba seed meal amendment on density of M. phaseolina in soils at Rohnert Park, CA, as determined by real-time quantitative PCR. Samples were collected 142 days after planting the site to strawberry (‘Camarosa’). “Mp” indicates plots artificially infested with M. phaseolina Columns designated with the same letter represent mean values that are not significantly different (P > 0.05).

137 2011 - 2012 RESEARCH PROJECTS Figure 3. Effect of B.juncea/S. alba seed meal amendment on strawberry root infection by M. phaseolina at Rohnert Park, CA, as determined by real-time quantitative PCR. Samples were collected 142 days after planting the site to strawberry (‘Camarosa’). “Mp” indicates plots artificially infested with M. phaseolina Columns designated with the same letter represent mean values that are not significantly different (P > 0.05).

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As was observed in experiments conducted in 2010, both B. juncea SM amendment and cultivation of the naturally infested Santa Maria, CA field soil with wheat prior to planting strawberry, significantly reducedM. phaseolina soil densities (Figure 4A). However, there was no additional reduction in density of the pathogen in soil when the treatments were combined in a manner where B. juncea seed meal amendment preceded wheat cultivation of soils. Similarly, prior wheat cultivation and B. juncea SM amendment, or the integration of the two methods, significantly reduced strawberry root infection but the combination treatment did not yield additional disease control benefit relative to wheat cultivation alone (Figure 4B).

Figure 4. Effect of B. juncea seed meal amendment and prior wheat cropping of a naturally infested field soil from Santa Maria, CA, on M. phaseolina soil populations (A) and infection of strawberry (‘Camarosa’) roots (B) at four, eight and 20 weeks after planting as determined by real-time quantitative PCR analysis. Plants were grown using a 90/64 F day/night temperature regime and 12 h photoperiod. For the same sampling period (e.g. 4 weeks), columns designated with the same letter represent mean values that are not significantly different (P > 0.05).

139 2011 - 2012 RESEARCH PROJECTS Wheat cropping and B. juncea SM amendment of a fumigated soil system prior to artificial infestation with M. phaseolina had perhaps functionally though not statistically differential effects on infection of strawberry plants planted in these soils. The pathogen was introduced at a level exceeding that encountered in the naturally infested Santa Maria, CA strawberry field soil by approximately one order of magnitude. M. phaseolina root infection was detected in roots of plants cultivated in the non-treated fumigated soil and soils that were amended with B. juncea SM or were amended with the seed meal and subsequently cultivated with wheat (Figure 5). M. phaseolina was not detected in roots of strawberry grown in soil that only received the wheat cultivation treatment. Although the wheat cultivation treatment alone did not statistically improve suppression of M. phaseolina relative to seed meal alone or seed meal/wheat cultivation treatments, the fact that the pathogen was not detected in plants could indicate a superior value of this treatment as a means to induce a soil system suppressive to charcoal rot of strawberry.

Figure 5. Effect of B. juncea seed meal amendment and prior wheat cropping of a 1,3 dichloropropene- chloropicrin fumigated soil prior to artificial infestation with Macrophomina phaseolina on subsequent strawberry root colonization by the pathogen. Pathogen density in strawberry roots was determined by real-time quantitative PCR analysis. Plants were grown using a 90/64 F day/night temperature regime and 12 h photoperiod. Columns designated with the same letter represent mean values that are not significantly different (P > 0.05).

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Discussion Results from the 2010-2011 field trial conducted at Rohnert Park, CA demonstrate the need for a more complete understanding of how environmental parameters (e.g. soil properties) influence the efficacy of Brassicaceae seed meal amendments for pest suppression in strawberry production systems. There has been little attention given to garnering insight into how management practices or soil characteristics will influence overall productivity of the cropping system, or provide desired outcomes such as weed control, in response to Brassicaceae seed meal amendments. For instance, there are reports of inconsistent weed control in response to mustard seed meal amendments, and in some cases seed meal amendments have failed to provide weed control in strawberry (Fennimore 2011). Similarly, in apple, a B. juncea/S. alba seed meal amendment when applied to two different orchard soils during the same week provided 85% weed suppression at one site but provided no significant weed suppression at the second site (Mazzola, 2011). In the current study, the B. juncea/S. alba seed meal formulation provided excellent weed control during the initial 43 day growth period of the 2009-2010 field study and weed biomass was reduced greater than 90% over the duration of the strawberry growth period (142 days) of the 2010-2011 field trial. Seed meal induced weed suppression can be influenced by numerous parameters including seed meal formulation, composition of the weed seed bank, particle size of the seed meal utilized, soil organic matter content, and even composition of the Pythium spp. community resident to the soil system (Hoagland et al., 2008; Handiseni et al., 2011). Until these materials are utilized in a manner that takes such attributes of the production system into account, it is unlikely that consistently effective weed control will be attained in response to mustard seed meal amendments.

Similarly, much as is the case in using soil fumigants, care must be taken in the identification of appropriate plant back periods between mustard seed meal application and establishment of strawberry. This also will be affected by several attributes of the production system including soil characteristics and seed meal formulation. In the initial field trial conducted in 2009-2010, B. juncea was applied independently and no symptoms of phytotoxicity were observed in strawberry. In the 2010-2011 field trial conducted as part of this program, B. juncea was used in concert with S. alba seed meal and the 28 day plant-back period was not sufficient to avoid damage to strawberry. S. alba seed meal exhibits greater herbicidal activity toward various broad leaf plants and its active chemistry (4-hydroxyl benzyl ionic thiocyanate) demonstrates greater persistence in soil systems than does the most prominent active chemistry of B. juncea seed meal (allyl isothiocyanate) (Handiseni et al., 2011). In apple, a six week plant back period was sufficient to avoid potential B. juncea/S. alba seed meal-induced phytotoxicity in a high organic matter (4.2%) sandy loam orchard soil but use of the same plant back period resulted in 21% tree mortality at an orchard possessing a sandy soil containing 1.7% organic matter (Mazzola, 2011). Attention to such detail is a notable characteristic when utilizing chemical fumigants for soilborne pest control with extensive knowledge concerning the movement and persistence of such chemistries, and the effect of soil properties on these attributes, well documented over many decades. Similar information is required to effectively utilize Brassicaceae seed meals as a pest control measure.

141 2011 - 2012 RESEARCH PROJECTS As was observed in 2010, wheat cultivation of a strawberry field soil naturally infested withM. phaseolina resulted in significant suppression of pathogen soil density and significant reduction in strawberry root infection in a manner similar to that attained in response to Brassicaceae seed meal amendment. In addition, wheat cultivation of a fumigated soil prior to artificial infestation of with M. phaseolina induced effective disease control, most likely through the induction of soil suppressiveness. Integration of the seed meal amendment with wheat cultivation did not provide any additional level of disease control, and in fact, although not statistically different, the density of the pathogen detected in roots of strawberry grown in the seed meal/wheat treatment was higher than the wheat cultivation alone treatment. The basis for this wheat cultivation induced disease suppression is not known. As reported previously, the microbial response showing association with disease suppression was an increase in total culturable fungal densities across soil treatments, with densities being highest in soils cropped to wheat prior to planting strawberry. The utility of this approach in field application for control of soil-borne disease in strawberry is not known. In apple, such a wheat cultivation approach prior to orchard establishment on a replant site effectively controlled Rhizoctonia root rot. However, the method failed to control other fungal root pathogens of apple including Cylindrocarpon spp., and thus was not suitable as a stand-alone treatment in such settings. Therefore, the application of wheat cultivation as an independent treatment for suppression of charcoal rot in strawberry production systems clearly requires investigation at the field level.

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References • Fennimore, S. 2011. Weed management in strawberry. pp 83-88, In, California Strawberry Commission Annual Production Research Report.

• Handiseni, M., Brown, J., Zemetra, R. and Mazzola, M. 2011. Herbicidal activity of Brassicaceae seed meal on wild oat (Avena fatua), Italian ryegrass (Lolium multiflorum), redroot pigweed (Amaranthus retroflexus) and prickly lettuce (Lactuca serriola). Weed Technology 25:127-134.

• Hoagland, L., Carpenter-Boggs, L., Granatstein, D. G., Reganold, J. P. and Mazzola, M. 2008. Role of native soil biology in brassicaceae seed meal-induced weed suppression. Soil Biology & Biochemistry 40:1689-1697.

• Mazzola, M. Advances in Brassica seed meal formulation for apple replant disease control. pp 6.1-6.4, In, Proceedings Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions. MBAO, Fresno, CA. 2011.

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Non-fumigant Strategies for Soilborne Disease Control in California Strawberry Production Systems

Principle Investigator Dr. Carol Shennan, Professor Department of Environmental Studies University of California, Santa Cruz 1156 High Street Santa Cruz, CA 95064 (831) 459-4181 [email protected]

Co-Investigators Dr. Joji Muramoto, Associate Researcher Department of Environmental Studies University of California, Santa Cruz 1156 High Street Santa Cruz, CA 95064 (831) 247-3804 [email protected]

Dr. Steven Fennimore Department of Plant Sciences University of California, Davis 1636 East Alisal Street Salinas, CA 93905 (831) 755-2896 [email protected]

Dr. Mark Mazzola USDA-ARS 1104 N. Western Ave. Wenatchee, WA 98801 (509) 664-2280 [email protected]

Dr. George Lazarovits A&L Biologicals Agroecology Research Services Centre 2136 Jetstream Road London, ON, Canada, N5V 3P5 (519) 457-2575 ext 246 [email protected]

145 2011 - 2012 RESEARCH PROJECTS Summary Soilborne disease management without chemical fumigants is a major challenge for strawberry production in California. Current re-registrations and regulations are likely to intensify this obstacle by severely limiting availability of fumigants on a large percentage of strawberry acreage. Over the past two years, we compared the effect of non-fumigant methodologies such as anaerobic soil disinfestation (ASD), steam, mustard seed meal amendments (MM), and organic acids (OA), alone and in combination, on fruit yield, survival of a range of soilborne pathogens, weed density, soil chemical and biological characteristics, and economics in California strawberry production. Overall, results from the Monterey Bay Academy (MBA) (2010-2011 and 2011-2012) and Santa Maria trials (2011-2012) showed ASD and MM+ASD to be the most consistently effective and economically feasible non-fumigant alternatives. However, applying 9 tons/acre of rice bran for ASD, which provides 360 lbs-N/acre, may release excess nitrogen to the environment. Also pot experiments indicate that MM application after ASD treatment increases plant biomass compared to MM alone, ASD with rice bran, or MM applied as a carbon source for ASD. Future experiments should examine 1) effects of different ASD carbon sources and input rates on fruit yield, survival of a range of soilborne pathogens, weed density, soil characteristics, and economics, 2) performance of ASD in large scale field experiments, and 3) whether changes in soil microbial community composition are responsible for soilborne disease suppression by ASD and MM.

Introduction California’s strawberry production system for the last 40 years has relied upon preplant fumigation with methyl bromide (MeBr) (Wilhelm et al., 1961); however MeBr is being phased out through the Montreal Protocol and considerable resources have been invested into replacing it with other fumigants. Yet, even the most promising option, chloropicrin, has a questionable future as it may not gain re-registration (US-EPA, 2006) due to potential negative health effects. This underscores the critical need for developing a wider range of alternative practices (Carpenter et al., 2001).

A number of non-fumigant pre-plant strategies for the control of soilborne diseases in strawberry production systems have been studied. Mustard seed meal amendments (MM) were shown to have the capacity to create disease suppressive soils. Although such materials were commonly viewed as yielding disease control through the processes of “biofumigation” (release of toxic products during residue decomposition), specific elements of the soil biological community have also been shown to contribute to disease or weed control (Cohen and Mazzola, 2005; Mazzola et al., 2007; Hoagland et al, 2008). It is likely that the modes of action may vary from pathogen to pathogen (Mazzola et al., 2007) and that the source of the product can also modulate disease control efficacy (Mazzola et al., 2009).

Anaerobic soil disinfestation (ASD) was developed in Japan (Shinmura, 2000; Momma, 2008) and the Netherlands (Blok et al., 2000; Messiha et al., 2007) as an alternative to MeBr. It has been shown to control soilborne pathogens and nematodes in strawberries. Anaerobic soil disinfestation integrates principles behind solarization and flooding to control soilborne pests in situations where neither is effective or feasible. Studies conducted over the past five years were aimed at optimizing ASD for use in California strawberry systems. Overall, ASD was shown to be consistently effective at suppressing Verticillium dahliae in coastal California when 9 tons ac-1 of rice bran was pre-plant incorporated and 3 to 4 acre-inches of irrigation was applied in sandy-loam to clay-loam soils (Shennan et al., 2011). In trials at Watsonville and Castroville marketable yields from ASD plots were equal or higher than Pic-Clor 60 plots, and significantly greater than untreated controls.

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Steam has been used for over 100 years to kill soilborne pathogens and weeds in potting soil (Baker and Roistacher, 1957). It is universally accepted that raising the soil temperature to 158 F for 20 min kills all pathogens and weeds. Preliminary data derived from the new bed steamer indicates that it rapidly heats soil to a 14 inch depth at a cost of $5,472 per acre broadcast compared to $3,200 to $3,600 per acre for MeBr applied broadcast (Fennimore, 2011).

Soil pH and volatile fatty acids (VFAs) including organic acids can play an important role in soil suppressiveness (Chet & Baker, 1980). The use of fish emulsion containing a large concentration of VFAs reduced the viability ofV. dahliae microsclerotia up to 99% (Abbassi et al, 2009).

The project objectives were to:

1) Examine effects of anaerobic soil disinfestation (ASD), mustard seed meal (MM), organic acid material (OA), alone and in combination, on fruit yield, a range of soilborne pathogens, weed seed viability, soil chemical and biological characteristics, and economics in strawberry production systems in California.

2) Determine whether changes in soil microbial composition are responsible for soilborne disease suppression detected in response to ASD, MM, and OA.

Here we report results from the 2010-2011 trials as well as the progress made in the 2011-2012 experiments thus far

Materials and Methods

Objective 1: To compare several non-fumigant options, replicated completely randomized field trials were conducted at Watsonville (MBA) in the 2010-11 and 2011-12 seasons; and at Santa Maria (Manzanita Berry Farm) in the 2011-12 season.

Watsonville Site (2010-11) A field trial was established at an Elder sandy-loam field with non-detectable V. dahliae population in the Monterey Bay Academy (MBA) site, Watsonville, Santa Cruz County. In 2010-2011, treatments were ASD (rice bran 9 tons/acre), mustard seed meal (MM. MPT Mustard Products & Technologies Inc. Saskatchewan, Canada. 1.5 tons/acre), steam, MM+ASD (MM 1.5 tons/acre + rice bran 7.5 tons/acre ), MM+steam (1.5 tons/acre), Pic-Clor 60 fumigation, and untreated control (UTC) arranged in completely randomized block design with four replicates. Each plot was a 4.3 feet wide (center- to-center) x 40 foot long bed. MM was shank applied at 6 inch depth of beds (two rows per bed) on October 7, 2010. On the same day rice bran was applied on the bed surface of assigned plots and incorporated into 0 to 6 inch depth by a hand-push rototiller. After reshaping beds and applying drip tapes and standard green plastic mulch, 2.5 acre-inches of water was drip irrigated intermittently to ASD and MM+ASD plots from October 8, 2010 to November 3, 2010. Eh, a measure of anaerobisis, and soil temperature at 6” depth were automatically monitored in ASD, MM+ASD, and UTC plots from October 8, 2010 to November 6, 2010 using ORP sensors and soil temperature sensors, respectively. Steam was applied by spike injection from a stationary steam generator for sufficient time to raise the soil temperature to 158 F for 20 min on October 13, 2010 and October 14, 2010. Pic-Clor 60 was applied on October 15, 2010. Holes were cut through plastic mulch on November 18, 2010 and strawberry plants ‘Albion’ were planted on November 22, 2010 at a plant density of 20,000 per acre. Weed densities were measured in 25 ft2 areas covered with clear tarp on December 15, 2010, January 21, 2011, February 23, 2011 and April 6, 2011. Marketable fruit yield was assessed twice weekly (28 plants) from April 28, 2011 to September 15, 2011. Net return above harvest and treatment costs were calculated for each treatment based on the marketable fruit yield from the trial as well as harvest cost and treatment cost including material, spreading, incorporation and additional irrigation needed for ASD.

147 2011 - 2012 RESEARCH PROJECTS Watsonville Site (2011-2012) A trial of the same experimental design and size as for the previous season was repeated in the 2011-2012 season at the MBA site. For MM treatments, we used “Strawberry Mix” from Farm Fuel Inc. (Watsonville) at 1.5 tons/acre. Rice bran and MM were applied as described above and incorporated into 6 inch depth by a hand-push rototiller on October 12, 2011. After reshaping beds and applying drip tapes and standard green plastic mulch, 0.8 acre-inches of water was drip irrigated into all plots on October 14, 2011. An additional 1 acre-inch of water was intermittently applied via drip tapes to ASD and MM+ASD plots until October 20, 2011. Eh and soil temperature were monitored from October 16, 2011 to November 16, 2011. Steam was applied during October 18, 2011 and October 20, 2011. Pic-Clor 60 fumigation was conducted on November 3, 2011. Planting holes were cut on November 17, 2011 and strawberry ‘Albion’ was transplanted at 20,000 plants/acre on November 21, 2011. Weed densities were measured on January 17, 2012, March 8, 2012, and April 24, 2012 from 20 ft2 sample areas. Marketable fruit yield from 35 plants/plot have been monitored twice weekly since April 24, 2012. Soil samples were taken from 0 to 6 inch depth in all plots pre-treatment (October 12, 2011), post-treatment (November 17, 2011), early growth (January 13, 2012. inorganic N analysis only), and early harvest (May 2, 2012) for chemical analysis by A&L Biologicals and microbial analysis by USDA-ARS Washington using real-time quantitative PCR (RT-PCR) and terminal restriction fragment length polymorphism (T-RFLP) analysis.

Santa Maria Site (2011-2012) A randomized block design with four replicates and treatments UTC, ASD (rice bran 9 tons/acre), fish emulsion (Fish. True Organic 402 acidified by sulfuric acid (2% v/v) used as a high OA material. pH 4.8), MM+ASD (rice bran 7.5 tons/ acre + MM “Strawberry Mix” from Farm Fuel Inc. 1.5 tons/acre), ASD+Fish, and Pic-Clor 60 fumigation was established in a Sorrento sandy-loam soil at Manzanita Berry Farm, Santa Maria. Each plot was a 5.3 feet wide x 35 feet long bed. Rice bran and MM were applied on top of the beds and incorporated to 6 inch depth by a bed shaper-attached rototiller on September 22, 2011. After applying drip tapes and standard black plastic mulch, ASD and MM+ASD plots were intermittently drip irrigated for a total of 3.0 acre-inches from September 26, 2011 to October 15, 2011. 0.3 acre-inches of 2% (=1:50 diluted) acidified fish emulsion was applied to Fish and ASD+Fish plots on September 30, 2011 and October 15, 2011 to moisten the potential strawberry root zone (0’ to 12” depth). ASD+Fish plots were also intermittently drip irrigated with 2.4 acre-inches of water to attain total irrigation amount of 3 acre-inches, and equivalent amounts applied to the other ASD plots. Eh and soil temperature were monitored in ASD, MM+ASD, ASD+Fish and UTC plots from September 23, 2011 to October 24, 2011 as described above. Pic-Clor 60 fumigation was conducted on October 7, 2011. Holes were cut through the plastic mulch on November 11, 2011 and strawberry cv “PS-4634” was transplanted at 30,400 plants/acre on November 15, 2011. Fifteen gallons/acre of 2% acidified fish emulsion was applied at every other irrigation event to Fish and ASD+Fish plots since January 31, 2012. Marketable fruit yield (40 plants/plot) has been monitored twice weekly since March 23, 2012. Soil samples were taken from 0 to 6 inch depth in all plots at pre-treatment (September 22, 2011), post-treatment (October 24, 2011), early growth (January 18, 2012. inorganic N analysis only), and early harvest season (April 2, 2012) for chemical analysis and microbial analysis using methods described previously.

TerraBioGen Rate Trial (2011-2012) To find out the optimum rate of an OA amendment, a randomized block trial was conducted at the MBA site; 0% (water), 0.5%, 1%, and 2% solution of TerraBioGen (TBG), a liquid organic amendment containing high concentration of organic acids, were compared with four replicates. Each plot was a 4.3 feet wide (center-to-center) x 20 feet long bed. Six gallons of each solution was applied to the bed top by watering can on October 21, 2011 and October 28, 2011. Regular management practices at MBA were followed. Pre- and post-treated soil (0 to 6 inches) were sampled for microbiological analysis and fruit yield monitored twice weekly since April 24, 2012.

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Objective 2: Experiments were conducted to assess the effect of application sequence on integration of ASD and MM applications for disease control in a natural field soil from Santa Maria, CA. Brassica juncea seed meal was applied at 0.3% (wt/wt basis) and rice bran for ASD treatments was applied at a rate of 117 oz./square yard. Treatment sequences consisted of ASD/MM and MM/ASD, with a two or three week interval between treatment applications. Additional treatments consisted of control, MM or ASD alone, and a co-application of MM+ASD at the same time. Soils were dispensed into 1 gallon pots. MM only treatments were sealed in air tight Bitran bags for 24 h to simulate tarping and concentrate the activity of the active volatile allyl isothiocyanate. All pots containing ASD as a component of the treatment were irrigated with 10.1 oz. water at the time of rice bran application, pots were sealed in a double layer bitran bag, and were incubated for one week at 75 F prior to subsequent application of MM or planting. Soil pH was determined immediately prior to planting. Soils were planted to strawberry (cv ‘Camarosa’) one week after application of the final treatment and controls were fertilized with Hoagland’s solution. Plants were grown using a 16 hour photoperiod and a day/night temperature regime of 75/60 F. Plants were harvested after 15 weeks and biomass determined.

Results

Objective 1: Cumulative Eh and Soil Temperature During the ASD Treatment In the sandy loam soil at the MBA Watsonville site, ASD and MM+ASD plots developed strong anaerobic condition in both seasons during the ASD treatment; cumulative Eh was 50,000 to 80,000 mV hrs in the 2010-2011 season, and 43,000 to114,700 mV hrs in the 2011-2012 season. In the 2011-2012 season this level of anaerobisis was accomplished with only 1.8 acre-inches of drip irrigated water. Soil temperature at 6 inch depth during ASD treatment averaged 66 F in the MBA trial for both years and 73 F in the Santa Maria trial where very strong anaerobic conditions (88,600 to 115,000 mV hrs) developed in ASD, ASD+Fish, and MM+ASD plots.

Marketable Fruit Yield and Economics At MBA, marketable fruit yields in Steam, Steam+MM, ASD, and MM+ASD were similar to Pic-Clor application in 2010-2011 (Figure 1A). The cost of MM and the ASD treatment with rice bran were similar, $1,693 and $1,632 per acre respectively (Figure 1B) whereas steam added $10,440 per acre compared to $800 per acre for Pic-Clor 60. Therefore, while yields and gross revenues were comparable across treatments, net returns above treatment and harvest costs were highest for Pic-Clor followed by ASD and MM+ASD and lowest for Steam+MM.

149 2011 - 2012 RESEARCH PROJECTS Figure 1A-B. Cumulative marketable fruit yield (A), and costs and net return (B) at the MBA trial in the 2010-2011 season. Means marked with the same letter have no significant difference according to protected-LSD (P=0.05). UTC: untreated control, ASD: anaerobic soil disinfestation, and MM: mustard seed meal.

Mid-season marketable fruit yield in 2011-2012 for ASD, MM+ASD, and Steam+MM plots at MBA were comparable to Pic-Clor and significantly greater than steam, MM, and UTC (as of July 12, 2012. Figure 2A). In Santa Maria ASD, Fish, ASD+Fish, and MM+ASD plots had similar mid-season marketable fruit yields as Pic-Clor and greater than UTC (as of July 15, 2012. Figure 2B).

A. MBA Trial B. Santa Maria Trial

Figure 2A-B. Early to mid-season cumulative marketable fruit yield at A: the MBA trial (as of July 12, 2012. Plant density 20,000/acre), and B: the Santa Maria trial (as of July 19, 2012. Plant density 30,400/acre) in the 2011-2012 season. Means marked with the same letter have no significant difference according to protected-LSD (P=0.05).

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Weed Densities In both seasons at MBA, steam alone or with MM was as effective as Pic-Clor 60 at weed control (Table 1). ASD alone (the 2010-2011 season), or MM+ASD (the 2011-2012 season) suppressed weed densities over UTC, but was less effective than Steam and Pic-Clor 60.

Table 1. Treatment effect on total weed density at the MBA trial, Watsonville in the 2010-2011 and 2011- 2012 seasons.

2010-2011 2011-2012 Treament (weed #/25 ft2) (weed #/20 ft2) Untreated control 702 a 107 a MM 636 ab 101 a ASD 496 b 91 ab Steam 119 c 9 c MM+ASD 569 ab 66 b Steam+MM 94 c 11 c Pic-Clor 60 94 c 2 c

Soil Inorganic N Dynamics and other Chemical Soil Characteristics At the MBA site in 2011-12, MM, ASD, MM+ASD, and Steam+MM treatments greatly increased inorganic N (0 to 6 inches) (November 17, 2011) (Figure 3A). Thereafter, a further increase was observed to 100 to 150 ppm in January, then decreasing to ~25 ppm in May 2012. In contrast, soil inorganic N in Pic-Clor, Steam, and UTC plots remained < 30 ppm during the entire period. The Santa Maria site showed a similar pattern but overall inorganic N was greater than at MBA (Figure 3B); increasing to 30 (UTC) to 70 ppm (MM+ASD) by October 24, 2011 and reaching 100 to 170 ppm in January in all plots. In May, it dropped to 50 ppm in UTC, Fish, and Pic-Clor plots but remained ~100 ppm in ASD, ASD+Fish, and MM+ASD plots. At both sites, ASD plots had lower pH and higher EC compared to other plots (Table 2 for the MBA site. Table 3 for the Santa Maria site). Olsen-P and exchangeable Mg (MBA only) and K were also high in the ASD treated soils.

151 2011 - 2012 RESEARCH PROJECTS A. B.

Figure 3A-B. Changes in soil inorganic N content (0 to 6 inch depth) at MBA (A) and Santa Maria (B). Samples were taken pre-treatment (MBA: October 12, 2011, Santa Maria: September 22, 2011), post-treatment (November 17, 2011 and October 24, 2011), early growth stage (January 13, 2012 and January 18, 2012), and early fruit stage (May 3, 2012 and April 2, 2012). Means with the same letter have no significant difference (protected-LSD (P=0.05).

Table 2. Treatment effect on chemical characteristics of top soil at the MBA trial, Watsonville (0 to 6 inch depth. Sampled on May 2, 2012). Numbers with the same letter do not have significant difference by protected-LSD (P=0.05).

Olsen-

EC 1:2 P2O5 Ex. Ca Ex. Mg Ex. K Ex. Na Treatment pH dS/m ppm ppm ppm ppm

UTC 6.7 0.14 44.8 b 10500 3125 bc 1328 b 305

MM 6.6 0.16 46.5 b 9800 2925 c 1298 b 270

ASD 6.3 0.31 79.8 a 9800 3775 a 2362 a 270

Steam 6.6 0.18 44.0 b 10100 3138 bc 1995 b 310

MM+ASD 6.4 0.29 74.3 a 10275 3863 a 2420 a 295

Steam+MM 6.4 0.27 45.8 b 10725 3325 b 1463 b 323

Pic-Clor 6.7 0.14 43.3 b 10025 3175 bc 1188 b 308

P value 0.07 0.05 0.0001 0.77 0.0001 <0.0001 0.48

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Table 3. Treatment effect on soil characteristics at Santa Maria (0 to 6 inch depth. April 2, 2012). Numbers with the same letter are not significantly different (protected-LSD (P=0.05).

Olsen-

EC 1:2 P2O5 Ex. Ca Ex. Mg Ex. K Ex. Na Treatment pH dS/m ppm ppm ppm ppm

UTC 7.6 a 1.15 70.0 c 33150 3775 2265 bc 873

Fish eml* 7.5 ab 1.30 80.3 bc 30475 3688 2443 bc 890

ASD 7.1 c 1.60 99.8 a 30950 3988 3155 a 715

ASD+Fish eml 7.2 c 1.78 99.3 a 30775 4088 3105 a 920

MM+ASD 7.3 bc 1.48 93.0 ab 30775 3975 2805 ab 800

Pic-Clor 7.6 a 1.15 70.3 c 34000 3825 2125 c 905

P value 0.001 0.21 0.0006 0.38 0.31 0.008 0.11

* Fish emulsion. See text for more details

Soil Microbial Community Composition and Root Infection by Specific Pathogens The effect of soil treatments on soil microbial community composition and incidence of root infection by specific pathogens was determined using molecular and culture-based methods. At the MBA and Santa Maria trials, treatment- specific effects on fungal and bacterial community composition were detected based on examination of the similarity in microbial communities from individual field plots using terminal restriction fragment length polymorphism (T-RFLP) data. At MBA prior to treatment applications, there was a general randomness in the relative similarity of the fungal community among plots, with no clustering of treatments. However, post-treatment application all plots for a particular treatment grouped together. At both sites, all treatments with ASD as a component were clustered and at MBA this was also seen for all treatments with a MM component (Figure 4). Similar treatment effects were observed on bacterial community composition, but were less definitive at Santa Maria than MBA (data not shown).

153 2011 - 2012 RESEARCH PROJECTS Figure 4A-B. Effect of soil treatments on fungal community composition prior to (left) and post (right) application at the MBA site determined by T-RFLP analysis. X-axis denotes similarity among fungal communities across plots (range 0 to 1). The tree was constructed from the Jaccard similarity coefficient of composite profiles from triplicate digestions of amplified fungal DNA using primers specific for the ITS region of rDNA. Bootstrap values are indicated at branch nodes of the tree (10,000 bootstrap replicates). Treatments: Ck=control; ASD=anaerobic soil disinfestation; ASDMus=ASD+mustard seed meal; Mus=mustard seed meal; PicChlor; Steam; SteamMus=Steam+mustard seed meal. Plot numbers given at the end of each line.

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Quantitative changes in soil microbial populations were also observed in response to soil treatments. Total bacterial populations increased in response to all soil treatments except steam and soil fumigation which were similar to the control. However, among bacterial sub-groups soil fumigation did result in a significant increase in fluorescentPseudomonas spp. densities. Pic-Clor soil fumigation consistently suppressed total soil fungal densities.

Colonization of strawberry roots by fungi and oomycetes was assessed by real-time quantitative PCR (qPCR) or by culture-based methods. Neither Macrophomina phaseolina nor V. dahlia were detected by qPCR in roots of plants sampled in April 2012 from either the MBA or Santa Maria sites. At Santa Maria, Fusarium spp. were recovered from strawberry roots at a high frequency for all treatments, however, the highest recovery was from plants cultivated in fumigated soils (78% of root fragments colonized). Isolates were identified by DNA sequence analysis and were identified as Fusarium solani or Fusarium tabacinum (Plectosphaerella cucumerina), with no isolates of Fusarium oxysporum. Isolates of Rhizoctonia spp., representing anastomosis groups A and I were recovered from strawberry roots in control and fumigated soils, but not in soils from ASD treatment. Pythium spp. were not recovered from strawberry roots grown in fumigated Santa Maria field soils but were isolated from 2.5% of root fragments from ASD treated soils. Isolates were identified as Pythium spinosum, Pythium megacarpum and Pythium violae, none of which have been reported as pathogens of strawberry. Pythium and Cylindrocarpon spp. were the dominant fungi recovered from strawberry roots at the MBA site. All Pythium isolates recovered were either P. ultimum or P. sylvaticum, both of which are highly virulent pathogens. The fungal community was dominated by representatives from one of these genera; Cylindrocarpon spp. for steam, MM, ASD, and PicChlor soil fumigation but Pythium spp. for the ASD+MM treatment. Cylindrocarpon spp. exhibit great variation in virulence among isolates, and are often considered unimportant as a plant pathogen. However, studies have shown that this fungus can act in concert with Pythium spp. to cause damage greater than either pathogen alone (Braun, 1991; Tewoldemedhin et al., 2011). Both genera were present in abundance in the control treatment with Cylindrocarpon and Pythium spp. recovered from approximately 30% and 19% of root segments (400 total), respectively. This may account, in part, for the lower yields obtained in control plots. Rhizoctonia spp. were rarely recovered. Fusarium spp. were recovered from strawberry roots for all treatments except PicChlor fumigation, and were identified as F. oxysporum or F. equiseti, the former a significant pathogen of strawberry and the latter known to promote plant growth.

TerraBioGen Rate Trial (2011-2012) Regardless of concentration, mid-season marketable fruit yields (as of July 12, 2012) were similar to UTC, and no shifts in bacterial or fungal populations were observed (data not shown).

Objective 2: To determine if soil biology contributed to disease suppression, a greenhouse bioassay using sterilized and unsterilized soils from the Santa Maria trial was conducted. ASD treated soil was suppressive to root infection by the introduced isolate of P. ultimum, but suppressiveness was abolished when ASD treated soils were pasteurized prior to pathogen infestation. Sequence of application significantly influenced the effect of MM+ASD treatments on soil pH and growth of strawberry in Santa Maria field soil. Initial soil pH was 7.9 in the un-treated control and ranged from 7.6 to 8.06 in soils that received MM alone or MM after ASD treatment. In contrast, pH ranged from 6.2 to 6.4 in soils that received ASD alone or last in the application sequence. Plant growth was significantly improved in response to all ASD or MM treatments relative to the control. However, when MM was applied to the soil two or three weeks after ASD treatment, strawberry growth was significantly improved relative to the individual treatments or when ASD followed MM application. (Figure 5A). Bran alone, with MM, or when applied in the treatment sequence MM/Bran, produced a bacterial community that was distinct from that realized in response to MM alone or the treatment sequence Bran/MM (Figure 5B).

155 2011 - 2012 RESEARCH PROJECTS A. Growth of Strawberries B. Bacterial Communities

Figure 5A-B. Effect of ASD, mustard seed meal (MSM), sequence of application and period between treatment applications on A: strawberry biomass after 15 weeks and B: relative similarity of bacterial communities as determined by T-RFLP analysis, in Santa Maria field soil.

Discussion During the past two years, we compared effect of non-fumigant methodologies: ASD, steam, MM, and organic acids (OA), alone and in combination, on fruit yield, survival of a range of soilborne pathogens, weed density, soil chemical and biological characteristics, and economics in strawberry production systems at the MBA site in Watsonville and the Santa Maria site Although both sites are virtually free from V. dahliae, the MBA site had disease pressure by Pythium and Cylindrocarpon. Overall, results from the MBA trial (2010-2011 and 2011-2012) and the Santa Maria trial (2011-2012) indicated that ASD and MM+ASD to be the most consistently effective and economically feasible non-fumigant alternatives at this point.

However, there are some issues to be addressed on current ASD technology. First, it did not provide fumigation levels of weed suppression and may need to be combined with herbicide use in severely weed infested sites. Second, although ASD has proved as effective as fumigation in suppressing V. dahliae and improving strawberry yields when using 9 tons/acre rice bran, a reduction in application rate or use of alternative carbon sources should be explored. Nine tons/acre of rice bran (N 2%, P2O5 5%, K2O 2%) provides N 360 lbs/acre, P2O5 900 lbs/acre, and K2O 360 lbs/acre. Data from both sites in 2011-2012 showed that N in rice bran can be mineralized quickly resulting in high soil inorganic N post-treatment through to early fruit stage (Figures 3 A & B). In particular,100 ppm or more of inorganic N in the early growth stage raises concerns in wet winters that excess nitrate may be lost into the environment, or in dry winters high soil nitrate may cause salinity damage to the strawberry plants. Olsen-P and exchangeable K also increased in ASD plots (Table 2 and 3). Further research on methods to reduce NPK input from the carbon source for ASD with and without pre-plant fertilizer in ASD are needed. Third, ASD needs to be evaluated for its adoptability and cost effectiveness on a commercial scale in large-scale trials. Funded by other sources, a 0.5 acre ASD demonstration trial is in progress at the USDA Spence site, Salinas. Mid-season marketable fruit yield of the ASD plot was equivalent to nearby fumigated plots and higher than the untreated check (Fennimore et al., unpublished).

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Fourth, the modes of action contributing to disease control in ASD needs to be further explored. Preliminary studies demonstrate that ASD alters soil biology and results in development of a soil community that is suppressive to disease incited by Pythium spp. Reports also suggest that different organic substrates applied in ASD results in production of different volatile profiles which differ in their capacity to suppress fungal pathogens includingF. oxysporum and P.ultimum (Mazzola, unpublished). Yet these volatiles would not have had a role in the observed soil suppressiveness towards reintroduced P. ultimum, suggesting that a multiplicity of mechanisms may contribute to ASD-induced disease control. Effective use of ASD or its integration with other methods such as organic acids or MM requires an understanding of mechanisms of action to avoid the diminution or elimination of disease control activity, as was observed in pot trials when MM application preceded ASD (Figure 5).

In the present study at MBA, MM alone at 1.5 tons/acre was not effective in the 2010-2011 season (Figure 1A), and modestly effective in the 2011-2012 season (Figure 2A). This variability may be attributed to the different sources of MM material between two seasons. In apple systems, MM applied at 3 tons/acre effectively suppressed soil borne pathogens (Mazzola and Brown, 2010). However, since MM contains 6% N and considerably elevated soil nitrate levels (Figure 3A), higher application rates in strawberry may be prohibitive. Further studies should focus on how best to integrate MM and other practices such as ASD in strawberry systems.

Two types of organic acid materials were tested. Application of the Terra BioGen extract did not improve in strawberry plant growth or fruit yield relative to control at MBA, but fish emulsion significantly increased marketable fruit yield above the control in Santa Maria where disease pressure was very low and warrants further study in fields with greater disease pressure.

This study confirms earlier data that steam is as effective as chemical fumigation. However, current steam technology costs $10,440 per acre and a more efficient steam injection system is critical for adoption in a commercial setting. Recent advances may reduce the cost of steam treatment to less than $5,500 per acre with the potential of further cost reductions.

157 2011 - 2012 RESEARCH PROJECTS Acknowledgements This project was partially funded by the California Strawberry Commission. We thank our collabors Dole Food Company, Inc. and Dave Peck at Manzanita Berry Farm. Karen Klonsky of UC Davis conducted the economic analysis, Surendra Dara helped manage the Santa Maria trial.

Selected References

• Blok, W.J., Lamers, J.G., Termorshuizen, A.J. and G. J. Bollen. 2000. Control of soilborne plant pathogens by incorporating fresh organic amendments followed by tarping. Phytopathology. 90:253-259.

• Braun, P. G. 1991. The combination of Cylindrocarpon lucidum and Pythium irregulare as a possible cause of apple replant disease in Nova Scotia. Can. J. Plant Pathol. 13:291-297.

• Butler, D.M., Kokalis-Burelle, N., Muramoto, J., Shennan, C., McCollum, T.G. and E.N. Rosskopf, E.N. 2012. Impact of anaerobic soil disinfestation combined with soil solarization on plant-parasitic nematodes and introduced inoculum of soilborne plant pathogens in raised-bed vegetable production. Crop Protection 39:33-40.

• Cohen, M.F., Yamasaki, H. and M. Mazzola. 2005. Brassica napus seed meal soil amendment modifies microbial community structure, nitric oxide production and incidence of Rhizoctonia root rot. Soil Biol. Biochem. 37:1215-1227.

• Hoagland, L., Carpenter-Boggs, L., Reganold, J.P. and M. Mazzola. 2008. Role of native soil biology in Brassicaceous seed meal-induced weed suppression. Soil Biol. Biochem. 40:1689-1697.

• Mazzola, M., Brown, J., Izzo, A.D. and M.F. Cohen. 2007. Mechanism of action and efficacy of seed meal-induced suppression of pathogens inciting apple replant disease differ in a Brassicaceae species and time-dependent manner. Phytopathology 97:454-460.

• Mazzola, M. and J. Brown. 2010. Efficacy of brassicaceous seed meal formulations for the control of apple replant disease in organic and conventional orchard production systems. Plant Dis. 94:835-842.

• Momma, N., Momma, M., and Y. Kobara. 2010. Biological soil disinfestation using ethanol: effect on Fusarium oxysporum f. sp lycopersici and soil microorganisms. Journal of General Plant Pathology 76:336-344.

• Shennan, C, Muramoto, J., Baird, G., Daugovish, O., Koike, S. and M. Bolda. 2011. Anaerobic soil disinfestation : California. Proc. Annu. Intl. Res. Conf. on Methyl Bromide Alternatives and Emissions Reductions., San Diego, CA. 31 Oct. – 2 Nov. p 44.1 – 44.4.

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• Shinmura, A. 2000. Causal agent and control of root rot of welsh onion. PSJ Soilborne Disease Workshop Report 20:133-143 (in Japanese with English Summary).

• Tewoldemedhin, Y. T., Mazzola, M., Labuschagne, I. and A. McLeod. 2011. A multi-phasic approach reveals that apple replant disease is caused by multiple biological agents with some agents acting synergistically. Soil Biol. Biochem. 43:1917-1927.

159 2011 - 2012 RESEARCH PROJECTS 160 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Farming Without Fumigants

Evaluation of a Substrate Based Raised Bed Trough (RaBeT) Strawberry Production System in California

Co- Investigators Dr. Hillary Q. Thomas California Strawberry Commission P.O. Box 269 Watsonville, CA 95076 (831) 724-1301 [email protected]

Dr. Dan Legard California Strawberry Commission [email protected]

Cooperators Tom Sjulin, Horticultural Consultant Dwight Rowe and Shiv Reddy, SunGro Horticulture Cliff Low, Perry Laboratory Drs. Steve Fennimore and Raquel Serohijos, UC Davis

Summary The California Strawberry Commission began evaluating strawberry substrate production systems in 2008 as part of its Farming without Fumigants initiative. Growers identified a modified raised bed substrate trough (RaBeT) based on a Dutch design as the type of substrate system that would best work in California, due to its low setup costs relative to table-top systems, and its compatibility with current harvest and production practices. In 2010-2011, our primary objective was to improve marketable yields in two preferred substrates and to evaluate an amended soil treatment that combined substrate and steamed soil, which may serve as a reduced cost alternative. The performance of strawberry grown in the RaBeT system was compared to a grower’s standard fumigated treatment and a non-unfumigated treatment at two field sites. The treatments included: (1) 70% peat: 30% perlite blend, (2) 100% coconut coir, (3) 50% silt loam, 25% coir, 25% rice hulls, (4) grower’s standard fumigated soil beds and (5) unfumigated soil beds. Peat-perlite and coir yielded significantly higher than grower’s standard and amended soil treatments at the Monterey Bay Academy (La Selva Beach, CA) trial and all treatments at this site had significantly higher yields than the unfumigated soil control. However, poor fertility management in the soil treatments likely reduced the performance of the grower’s standard at this site. At the Mar Vista (Guadalupe, CA) trial site, there were no significant yield differences between substrate and soil treatments. The 2010-11 season results suggest that strawberries grown in the RaBeT production system can match grower’s standard marketable yields and that a mixture of substrate and soil may be a viable cost reduction measure. The high cost of substrates used in the system are a significant hurdle for implementation of the RaBeT production system.

161 2011 - 2012 RESEARCH PROJECTS Introduction Soil borne diseases caused by Verticillium dahliae, Macrophomina phaseolina, Fusarium oxysporum and other pathogens are the main limiting factor for strawberry production without fumigants (Koike, et. al., 2010). These diseases also cause serious losses in fumigated fields when (1) a portion of the field remains untreated due to regulatory buffer zones and inoculum gets spread throughout the field during bed preparation and other activities, (2) when fumigants are applied at insufficient rates and (3) when the drip applications are used instead of broadcast applications. The phase out of methyl bromide and increasing regulatory constraints on all fumigants threaten the continued viability of the 400 strawberry growers in California. In other countries where fumigant use has been severely restricted, strawberry growers have turned to substrate based production systems to avoid soilborne diseases (Leitin and Baetes, 1991; Leitin, 2001, 2004). Substrate production systems have also been evaluated in the US and Canada as potential alternatives for strawberry production (Kempler, 2002; Paranjpe et. al, 2003, 2008; Takeda, 1999).

The California Strawberry Commission (CSC) began evaluating strawberry substrate production systems in 2008 as part of its Farming without Fumigants initiative. The raised bed trough (RaBeT) substrate system is an adaption of the Dutch table-top soilless production system where strawberries are grown in peat or coconut coir based substrate. The Commission selected the RaBeT system to evaluate, due to its low setup costs relative to other substrate production systems, and its compatibility with the harvest and tractor-based management practices in the current soil-based production system. Prior CSC research (Wang et. al., 2010, 2011) focused on modifying these systems to make them compatible with the California open-field production system and on the evaluation of substrate alternatives to peat and coir media (Lao and Jimenez, 2004; Leitin, 2005; Paranjpe et. al, 2003, 2008; Urrestarazu et. al., 2004). For the 2010-2011 season, the primary objective was to improve production in the best substrates as compared to the growers standard and to evaluate the performance of an amended soil treatment combining substrate and clean soil, which might serve as a cost-saving alternative. The research was conducted on replicated small scale plots at two sites in the Watsonville and Santa Maria production regions.

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Materials and Methods The performance of strawberries grown in the RaBeT substrate system was evaluated against a grower’s standard fumigated treatment at both research sites and an additional standard unfumigated treatment at one site. The northern Watsonville-Salinas district trial was conducted at the Monterey Bay Academy (MBA) research plots in La Selva Beach, CA. A Santa Maria district trial was conducted on-farm at Mar Vista Berry Ranch 5 in Guadalupe, CA. The following treatments were included: (1) 70% peat: 30% perlite blend, (2) 100% coconut coir, (3) 50% silt loam, 25% coir, 25% rice hulls, (4) grower’s standard fumigated soil beds and (5) unfumigated soil beds. At Mar Vista 5, the grower chose not to include an unfumigated control due to the possible risk for his conventional production and for disease pressure in the soil the following season. Albion variety strawberries were planted at both sites using 14 inch spacing on 50 foot long beds. As typical in the Watsonville/Salinas production region, two rows were planted with a 52 inch bed spacing at MBA, while there were four plant rows with a 64 inch bed spacing at Mar Vista 5. For the substrate treatments, soil beds were shaped using a modified bed-shaper to cut a 6 inch deep by 12 inch wide trough in the bedtop. Sunbelt 3.2 (Dewitt Company, MO) polypropylene groundcover fabric was laid over the modified bedtop as a barrier between the soil and substrate, and secured with mulch pins. Each substrate was mounded into the troughs by hand. Both substrate beds and soil beds were covered with green plastic mulch. There were four replicates. At MBA, a havest plot of 25 plants per bed replicate were harvested for yield data; at Mar Vista 5, the harvest plots were 80 plants per bed per replicate. All harvesting and general production practices were managed by Dole Berry Company or their collaborating grower and employed the same methods as the commercial production on each ranch. Marketable and unmarketable yield were measured in kilograms. Monterey Bay Academy was harvested twice a week (41 total harvest dates) from April 28, 2011 to September 15, 2011. Mar Vista 5 was harvested twice a week from April 13, 2011 to October 9, 2011, with gaps in picking during August and September (43 total harvest dates).

Irrigation and fertilizer programs on the grower’s standard and unfumigated plots were managed by the grower. On the substrate plots a pulse-irrigation management program was implemented to maintain adequate moisture and fertility. Custom Netafim Uniram (18 mm, .26 GPH, 8 inch spacing) drip tubing was employed rather than traditional drip tape due to the short irrigation times (typically 3 to 5 minutes). Substrate plots were fertigated using Superdos 20 2.5% injectors (Dosematic/Hydrosystems Co, Ohio) on a two-tank system. Fertilizer programs were prescribed by Perry Laboratories (Watsonville, CA). Optimum values for liquid fertilizer programs were modeled on European substrate production target values and the fertility program was modified accordingly in response to monthly leaf, petiole and substrate sample testing. Irrigation programs for the substrate plots were determined empirically by maintaining a target percentage leachate volume between 10 to 15% at MBA where water quality was higher (base electrical conductivity was 0.5 dS/m) and a target leachate of 25-30% at Mar Vista 5 where irrigation water quality was poor and base electrical conductivity was 1.7 dS/m. A five-foot PVC trough was placed in one bed-replicate of each treatment to collect leachate into a graduated cylinder. Leachate volumes, leachate electrical conductivity (EC), electrical conductivity of the applied fertilizer solution and water usage were recorded by an irrigator on a daily basis. When salt levels were detected to be above target values during the season based on target EC value, clear water irrigations were added to flush salts until line and leachate ECs returned to target values.

163 2011 - 2012 RESEARCH PROJECTS Results and Discussion At Monterey Bay Academy, an analysis of variance of total yield by treatment revealed significant treatment differences (F4,15 = 64, P <0.0001). Peat-perlite and coir yielded significantly higher than grower’s standard and amended soil treatments. All treatments at MBA had significantly higher yields than the unfumigated control (Figure 1A). However, at MBA a lapse in fertilizer management occurred on the grower’s standard in May after most of the pre-plant fertilizer had been utilized, which undoubtedly had a negative impact on the performance of the grower’s standard and nonfumigated soil treatments. At Mar Vista 5, there were no significant yield differences between treatments (Figure 1B).

Figure 1. Average yield per plant in peat-perlite, coconut coir, amended soil, grower’s standard, and unfumigated treatments (N=4) at A) Monterey Bay Academy (La Selva Beach, CA) and B) Mar Vista 5 (Guadalupe, CA) research plots. Treatments labeled with different letters were significantly different at P = 0.05 by Tukey’s Studentized Range (HSD) test.

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There were substantial irrigation and fertility management challenges in the substrate plots at both sites and they likely had some impact on total yield. Watering lapsed at Monterey Bay Academy for 1 to 2 days on several occasions and electrical conductivity in the substrate and soil treatments were above target EC values (2.0 ds/m) at Mar Vista 5 for most of the season. Watering and fertilization challenges did not appear to have shifted yield curve patterns over time at either site. Yield curves followed similar patterns for all treatments at Monterey Bay Academy, but appeared lower in the unfumigated soil treatment (Figure 2A). At Mar Vista 5, we observed that the substrate treatments had higher yields than the grower’s standard on pick dates towards the end of the season (Figure 2B) but the grower terminated the experiment at that ranch before additional data could be collected.

Figure 2. Average yield over time in peat-perlite, coconut coir, amended soil, grower’s standard, and unfumigated treatments (N=4) at A) Monterey Bay Academy (La Selva Beach, CA) and B) Mar Vista 5 (Guadalupe, CA) research plots. There were 25 plants per plot at MBA on a 2-row 52” bed spacing and 80 plants per plot at Mar Vista 5 on a 4-row, 64” bed spacing.

165 2011 - 2012 RESEARCH PROJECTS The properties of the substrates themselves may have also contributed to trends we observed in yield. The amended soil treatment had average pH values of 3.9 and 4.0 for the season at Mar Vista 5 and MBA respectively. At Mar Vista 5, the peat-coir and coir treatments also had lower than target pH value on average during the season (5.9). These values are well below the target pH of 5.7 for substrate production.

The 2010-11 season results suggest that strawberries grown in the RaBeT production system has the potential to match grower’s standard marketable yields. If production yields for strawberry grown in substrate are validated as equivalent to a grower’s standard in subsequent trials, then the remaining hurdle for implementation of substrate production systems in California is the high cost associated with the setup of a raised-bed trough system. The price of substrates like peat and coir are the main barrier to making a RaBeT production system economical. Other equipment and material costs are roughly equivalent to the standard in soil production systems if amortized over 5 to 10 years, and the additional labor required to install the system will become more manageable once equipment is designed for distributing the substrates into field. One way to reduce costs would be to recycle the substrates and reuse them for more than one season. Another means would be to combine the substrate with native soil from the grower’s field. The amended soil treatment performed well and used 50% of the media requirements, reducing those costs by half. The major challenge with using native soil is that it must be disinfested to enable the substrate mixture to produce optimal yields.

Future research will further investigate cost-reducing measures for substrate production by evaluating higher percentage amended soil blends, reuse of substrate for second year production, and the impacts of reducing substrate trough size. As small plots data may not correlate well with commercial yields, we will compare yields in the RaBeT system with standard in soil production in one acre demonstration trials in grower fields.

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References • Kempler, C. 2002. ‘Out-of-Season’ greenhouse production of raspberry and strawberry. Proc. XXVI IHC – Protected Cultivation. Acta Hort. 633:459-465.

• Koike, S. T., T. Gordon, H. Ajwa, O. Daugovish, M. Bolda and D. Legard. 2010. Disease Management studies on strawberry plant collapse problems in California. California Strawberry Commission Annual Production Research Report: 2008-2009, p 41-51.

• Lao, M.T. and S. Jimenez. 2004. Evaluation of almond shell as a culture substrate for ornamental plants. I. Characterization. Phyton. 53:69-78.

• Lieten, F. and W. Baetes. 1991. Greenhouse strawberry culture in peat bags. Adv. Strawberry Production 10:56-57.

• Lieten, F. 2001. Protected cultivation of strawberries in Central Europe, p. 102-107. In S. C. Hokanson and R]amieson (eds.). Strawberry Research to 2001. Proc. 5th North America Strawberry Conf. ASHS Press, Alexandria, VA.

• Lieten, F. 2004. Substrates as an alternative to methyl bromide for strawberry fruit production in northern Europe in both protected and field production. Proc. Int. Conf. Alternatives to Methyl Bromide, Lisbon, Portugal, 27-30 September 2004.

• Lieten, F. 2005. Strawberry production in central Europe. Int. J. Fruit Sci. 5:91-105.

• Paranjpe, A.V., Cantliffe, D.J., Lamb, E.M., Stoffella, P.J. and C.A. Powell. 2003. Winter strawberry production in greenhouses using soilless substrates: an alternative to methyl bromide soil fumigation. Proc. Fla. State Hort. Soc. 116:98-105.

• Paranjpe, A.V., Cantliffe, D.J., Stoffella, P.J., Lamb, E.M. and C.A. Powell. 2008. Relationship of plant density to fruit yield of ‘Sweet Charlie’ strawberry grown in a pine bark soilless medium in a high-roof passively ventilated greenhouse. Scientia Horticulturae 115:117–123.

• Takeda, F. 1999. Out-of-season greenhouse strawberry production in soilless substrate. Adv. Strawberry Res. 18:4–15.

• Urrestarazu, M., G.A. Martınez and M.C. Salas. 2004. Almond shell waste: possible local rockwool substitute in soilless crop culture. Scientia Horticulturae103:453-460.

• Wang, D., J. Gartung, P. Vaughan, J. Ayars, J. Garik, M.Z. Gabriel and M. Gonzales. 2010. Design of a field raised- bed trough system using soiless substrates for strawberry production in California. California Strawberry Commission Annual Production Research Report: 2008-2009, p 123- 133.

• Wang, D., J. Gartung, J. Gerik, A. Cabrera, M. Z. Gabriel and M. Gonzales. 2011. Evaluation of a raised-bed trough (RaBeT) system for strawberry production in California. California.

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169 2011 - 2012 RESEARCH PROJECTS 170 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Regulatory

Predicting Harvester Pesticide Exposures From Leaf Residues

Principal Investigator Dr. Robert Krieger Personal Chemical Exposure Program Dept. of Entomology UC Riverside Riverside, CA 92521 [email protected] (951) 827-3724

Co-Investigators Helen Vega, Zhenshan Chen, Terry Lopez, and Gayatri Sankaran

Co-Investigator Volunteers Li Chen, Ana Krieger, William Krieger, Yu Liu, Loi Tang and Taifeng Zhao

Cooperators Daren Gee and Joe Coehlo Darrensberries Santa Maria, CA Rob Sheehy, Jesus and Rudy Garcia Safari Farms Santa Maria, CA

Gosia Meyer and Julie Penola primuslabs.com Santa Maria, CA

Summary Accurate information about pesticide exposures that happen during strawberry production is important to the industry, to agencies that regulate use, and, increasingly, to the consumer public who enjoy year-around production of wholesome, nutritious strawberries. Previous studies of harvester exposure have concentrated on measurements of harvester exposure at the pre-harvest interval (PHI). This practice protected workers from the acute effects of anticholinesterase agents, but exposure assessments for current risk assessment are inflated when based upon data collected at the PHI.

171 2011 - 2012 RESEARCH PROJECTS Although pesticide residues persist in fields for varying periods of time, knowledge of the availability of leaf residues is required to predict harvester exposure. Three independent measurements of leaf residues have been made during the natural dissipation of surface levels of malathion and Danitol® (fenpropathrin) following their use in crop protection. First, the total surface residue is Dislodgable Foliar Residue (DFR) removed by a detergent rinse of the leaf surface. Second, Transferable Foliar Residue (TFR), the part of the surface deposit removed by touching was measured as residue transferred by mechanical or physical contact. And finally, the available residue transferred to rubber latex gloves, Pesticide Glove Residue, was measured following 2 to 2.5 h work periods. Each of these measurements of available pesticide residue declined very rapidly between the first pick at the PHI and the second pick three days later.

Urine biomonitoring of harvesters after successive picks of sprayed fields in commercial production will be used to determine the long term predictive value of these measurements. Biomonitoring of gloved and barehanded harvesters showed that both groups of workers absorbed less than 0.005 mg malathion/kg body weight per day. This dose likely represents other pesticides with similar chemical and physical properties. Harvesters’ hands contributed about 55% of the absorbed dose. This finding is important to the possible future use of gloves as exposure dosimeters in risk assessment. Available residues and DFR declined more rapidly than urine biomarker excretion resulting in the suggestion that pesticide biomarkers may be formed and absorbed from foliage.

Introduction In spite of plant diseases like Botrytis and powdery mildew, insect pests like lygus bugs, moth and beetle larvae, and thrips, and a variety of weeds competing for nutrients and water, in 2010 the California strawberry industry produced 2.58 billion pounds of berries, amounting to about 91% of U.S. production and valued at $1.8 billion. Pesticides are an extremely important production factor to the California strawberry industry. Industry pesticide use represented 6.4% of the 173 million pounds reported in the 2010 California Pesticide Use Reports. Preplant fumigation accounted for 84% of the total with the remaining 1,755,679 pounds used later in crop protection. Those later uses result in applicator, harvester, and consumer pesticide exposures that are the continuing focus of research of the Personal Chemical Exposure Program (PCEP), Department of Entomology, UC Riverside.

Use of registered materials in crop protection assures safe use and negligible environmental impacts. Harvester and consumer pesticide exposures and risk assessment have historically been the special concerns of UC Riverside’s PCEP. Potential pesticide exposures among strawberry harvesters are minimized by cultural practices related to application schedules, sound work practices, food safety initiatives, field entry times, and worker hygiene and protective clothing. Harvester pesticide exposures come from the application of chemicals to protect the strawberries grown on 25,500 plants per acre. The leaf residues available for contact-transfer as the surface levels naturally dissipate are the primary source of harvester exposures. The record is clear that acute (1-day at the PHI) harvester exposures are below thresholds of health concern, but the nature and extent of later exposures that could be classified as subchronic exposures are uncertain (and will be addressed in pilot studies reported here). Consumer research concerning pesticide residues in strawberries that was performed by PCEP will not be reported here, but current results may be reviewed in published literature (Chen et al. 2012a and b; Krieger et al., 2012).

Predicting Harvester Pesticide Exposures from Leaf Residues requires knowledge of the amount and availability of residue in sprayed fields. Although it is possible to roughly estimate external or dermal harvester exposure potential at the pre-harvest interval based upon Dislodgable Foliar Residues, those estimates are often not reliably converted to absorbed dose. Furthermore, the DFR measurement is not a sound indicator of available surface residue at the PHI and

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later times since only a small part of it is actually transferable to harvester clothing or exposed skin. Here, DFR and two additional measures of available leaf residues were utilized: first, the Transferable Foliar Residue (TFR) resulting from physical or mechanical contact with treated foliage, and second, the Pesticide Glove Residue (PGR) accumulated on latex gloves during harvesting.

Absorbed dose was estimated using post-shift 16 h urine specimens after day 4 and day 7 following the pesticide applications on ranches where harvesters worked either barehanded or wearing rubber latex gloves.

Methods Malathion (2 lbs/A) and Danitol® (1 lb/A; fenpropathrin) were tank mixed with a spreader-sticker adjuvant and applied by a ground rig at 150 gallons/A at DB Specialty Farms and Safari Farms, Santa Maria, CA. The first study period was the fourth day after application and the second was the seventh day. Twenty-six harvesters voluntarily consented to participate at each ranch (plus four other unexposed workers). Signed informed consent forms required by the University of California, Riverside, Human Subjects Institutional Review Board and the California Environmental Protection Agency were obtained from each participant.

Harvesters at DB Specialty Farms wore rubber latex gloves that were changed four times during the day and harvesters at Safari Farms worked barehanded. Each Dislodgable Foliar Residue (DFR) and Transferable Foliar Residue (TFR) sample contained one randomly-selected leaf from each of the 10 sampling sites (Figure 1). Five samples of 10 leaves each were taken after four and seven days for each of the foliar residue determinations (DFR and TFR).

Figure 1. Strawberry Field Study: Leaf Sample Collection Sites. The leaf sampling grid included 10 sampling sites arranged about 100 feet from any field service roads to avoid dust. The arms of the letter “X” will included at least 60 rows. The sampling sites were marked with surveyor flags that remained at the study site during the entire strawberry sield study. At each of the 10 sites a leaf was collected. The 10 leaves comprised one (1) sample for analysis as indicated below. Five samples of 10 leaves each were taken at each time period for determinations of DFR and TFR (Table 1).

173 2011 - 2012 RESEARCH PROJECTS Three independent measurements of surface leaf residues were made and an estimate of absorbed dose was prepared from urine biomonitoring of crews at the PHI and three days later at two ranches where berries were picked by gloved and barehanded harvesters. The specific methods are briefly described below.

1) Dislodgable Foliar Residues, DFR: Total available residues were measured by the Iwata et al. (1977) procedure using diluted detergent to rinse residues from the leaf surface followed by a liquid-liquid extraction. This is Total Residue: the entire residue on the front and back of the leaf surface (some of the DFR is transferred to harvesters and may be absorbed, but most is not transferable by contact).

Whole Leaf samples were placed in 100 ml 0.01% of Surten solution in a plastic bag. The samples were shaken at high frequency for 10 minutes which removed all the surface pesticide. This process was repeated two more times and the final volume extracted with solvent and analyzed for pesticide.

After the leaves were rinsed and dried at room temperature, the leaf areas were measured using a leaf area meter. DFR are reported as ug pesticide/cm2 leaf area (both side of the leaves; Table 1).

Table 1. Dislodgable and Transferable Foliar Residues (DFR and TFR; µg/cm2)

Foliar Residues Mean + Standard deviation (n=5; RSD)1 Malathion Fenpropathrin Dislodgable Foliar Residues, DFR DB Specialty Safari Farms DB Specialty Safari Farms

Study Day 42 40.24 + 0.05 (21%) 0.15 + 0.01 (9%) 0.03 + 0.01 (38%) 0.02 + 0.005 (23%)

Study Day 7 0.03 + 0.01 (42%) 0.02 + 0.005 (30%) 0.02 + 0.01 (71%) 0.01 + 0.008 (61%)

Transferrable Foliar Residues, TFR3 Study Day 4 0.03 + 0.004 (13%) 0.02 + 0.001 (7%) 0.02 + 0.005 (21%) 0.01 + 0.006 (57%)

Study Day 7 0.003 + 0.001 (33%) 0.003 + 0.002 (69%) 0.003 + 0.001 (10%) 0.002 + 0.001 (29%)

1 RSD stands for Relative Standard Deviation which is calculated as [(Standard deviation/Mean) x100]. RSD indicates the variation within the collected samples. 2 On each study day, five sets of 10 leaves each were collected near 10 pre-marked sites at each study site. Samples were stored in gallon sized bags and kept on frozen blue ice. 3 Transferrable foliar residues are available surface residues sampled using the bench top surface roller (BSR). On each study day, five sets of 10 whole leaves each was collected, one from each leaf sample collection site. The leaves were plucked gently without contact of leaf surface and carefully stacked in a rectangular polypropylene box (5.5” x 10” x 3”; 2.24 L) that was kept chilled on frozen blue ice until analysis.

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) 2 Transferable Foliar Residues, TFR: Transferable foliar residue was measured using the Bench-top Surface Roller (BSR) developed in this laboratory (Li, 2009). TFR is the amount removed by mechanical (physical) contact. The sampling device simulates direct worker touching treated foliage.

An aluminum foil was spread out and a cotton cloth of the same size was placed over it. Whole leaves were placed on one-half of the cotton cloth and the other half of the cotton cloth was placed over the leaves along the aluminum foil (like a sandwich). This cotton/foil sandwich was rolled through the sampling device.

The transferred residue was analyzed from an ethyl acetate extract of the cotton cloth. Transferable residue levels representing the part of surface residue removed by contact (rather than a Surten detergent rinse) was a small portion of DFR. Both are measured in ug pesticide/cm2 of leaf surface (Table 1).

3) Pesticide Glove Residue, PGR. Pesticide Glove Residues are a continuous indicator of surface residue accumulated on rubber latex gloves during harvesting. The residue is unique since it directly reflects available residue, but it is not a perfect dosimeter since an uncertain portion of the glove residue is available for dermal absorption.

Gloves were collected from each worker in separate zip lock bags after normal 2 to 2.5 h work periods and stored frozen until analysis. Gloves were thawed, minced with scissors, and extracted with ethyl acetate in double zip-lock bags. The extracts were concentrated using a rotary evaporator for chromatographic analysis. Residues were reported as ug/pair of gloves-h or as ug/cm2 glove area-h. This is the only dosimeter available that can give measurements of potential dose in real work time (Table 2).

175 2011 - 2012 RESEARCH PROJECTS Table 2. Pesticide Glove Residues (PGR; µg/pair) 4,2

Malathion Glove Residue (PGR) Mean + Standard deviation (n=26; RSD)1

Work Period Sum For All Study Day Morning Noon Afternoon Evening Four Work Periods

4 4037+1303 (32%) 3080+1278 (42%) 4613+1921 (42%) 2214+883 (40%) 13944+3319 (24%) 7 1596+ 581 (36%) 966+395 (41%) 481+258 (54%) 392+113 (29%) 3435+1017 (30%) 11 - 216+402 (186%) - - - 14 - 236+113 (48%) - - - 18 - 34+26 (77%) - - - 21 - 41+23 (55%) - - -

Fenpropathrin Glove Residue (PGR) Mean + Standard deviation (n=26; RSD)1

Work Period Sum For All Study Day Morning Noon Afternoon Evening Four Work Periods

4 3826+5226 (137%) 1029+360 (35%) 1813+655 (36%) 1058+303 (29%) 7725+5132 (66%) 7 351+108 (31%) 503+67 (13%) 763+250 (33%) 666+220 (33%) 2282+399.3 (17%) 11 - 326+224 (69%) - - - 14 - 407+222 (54%) - - - 18 - 223+ 81 (36%) - - -

1 Used gloves were collected every work period (2-2.5 hrs) from 26 harvesters on study days 4 and 7. On other study days, gloves were collected only before lunch break. There are 4 work periods on each study day. 2 Gloves supplied by the grower were worn as PPE (this abbreviation is not defined elsewhere in the paper) were collected in separate zip-lock bags and stored frozen on dry ice for transport to UC Riverside.

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4) Urine Metabolite Biomonitoring. Urine pesticide biomarkers were used to evaluate worker exposure. The extent and intensity of harvester contact with treated foliage was used to estimate potential external exposure (clothing and exposed skin). The absorbed dose was measured using the non-toxic, urine biomarkers that are rapidly excreted. These studies used overnight urine specimens from two separate ranches at the PHI and 3 days later. Harvesters worked either bare-handed or wearing rubber latex gloves. Separate studies not reported here have demonstrated that there is little or no breakthrough of either pesticide through the rubber latex during harvesting.

Each worker was used as their own control (check) for possible dietary and environmental pesticide exposure (air, water, dust). The extent of formation of urine biomarkers including malathion specific (MMA, MDA; malathion mono- and diacids), organophosphorus generic (DMP, DMTP, DMDTP; dimethyl-, dimethylthio- and dimethydithiophosphate), and fenpropathrin pyrethroid generic biomarker (3-PBA; 3-phenoxybenzoic acid) were measured. Exposure estimates were adjusted using urinary creatinine to represent a work day and a 24 h urine collection, harvester residue exposure (mg/kg body weight-day). This aspect of PCEP research is under continuing investigation and results will be reported in future peer-reviewed publications.

The harvester residue exposures (mg/kg body weight-day) were substantially less than exposures (Nigg et al. 1984, Zweig et al. 1985) predicted from DFR (Iwata et al. 1977), transfer coefficient (USEPA 2000), and hours worked for times beyond the PHI.

The pesticides selected for study were malathion and fenpropathrin (Danitol®). The fate of both insecticides is well established in harvesters (an importantly, as a food safety issue not discussed here, in fresh market strawberries; see Chen et al. 2012).

Results The available foliar residues of malathion and fenpropathrin on strawberry plants declined rapidly. This was true whether the insecticide residues were measured as DFRs, TFRs or PGRs (Tables 1 and 2). The exposure potential of these residues declines rapidly, but it is measurable and significant for risk assessment during the natural dissipation of the available part of the residue. It is very clear that the transferable residue (TFR; the residue measured by physical contact) is a small part of the residue actually transferred following spraying, and an even smaller part of DFR (the residue removed by a detergent rinse of the leaf surfaces).

Knowledge of the part of the available residue that is actually transferable to harvesters is important for accurately and responsibly estimating worker exposure (Table 1). If a glove residue can be used as a direct dosimeter to estimate worker exposure it would be a valuable research and regulatory tool to assure safe short term, e.g. at the PHI, and longer term harvester exposures that may become concerns in future subchronic aggregate risk assessments (Tables 2 and 3).

177 2011 - 2012 RESEARCH PROJECTS T able 3. Transfer coefficients are not constant during the dissipation of surface residues

PGR Transfer Coefficients1,2 Harvest Following Pesticide Application

Pesticide Residue 1st 2nd 3rd 4th (year) (PHI) (PHI + 3 days) (PHI + 7 days) (PHI + 10 days)

Malathion 4,0005 26,000 27,000 24,000 (2010) (0.25)6 (0.02) (0.004) (0.003)

Malathion 6,100 13,000 NA NA (2011) (0.24) (0.03) NA NA

Fenpropathrin 34,000 39,000 45,000 72,000 (2010) (0.02) (0.01) (0.003) (0.003)

Fenpropathrin 31,000 15,000 NA NA (2011) (0.03) (0.02) NA NA

1 Glove Transfer Coefficient (cm2/hr) in all white cells; NA not available. 2 DFR (µg/cm2) is shown below the Glove Transfer Coefficient (cm2/hr).

Harvester exposures of barehanded or gloved workers were used to show that harvesters’ hands contributed about 55% of absorbed dose. The exposures of both gloved and barehanded harvesters were low (less than 0.005 mg malathion/ kg-day) relative to regulatory levels of concern. This is because foliar residues are low and decline rapidly under field conditions whether measured by DFR or TFR.

Harvester gloves reduce exposure about 45% when work time and foliage residues are adjusted to be equal. This estimate comes from measured dislodgable foliar residue, a strawberry harvester transfer coefficient cm2/h, and work time. In our study the barehanded harvesters worked shorter days and at lower DFRs than the gloved harvesters, resulting in the estimated glove protection factor of 45%, even though the absorbed dose of the two groups was about the same.

Knowledge of the importance of the hands as a route of exposure contributes to the possible use of gloves as direct dosimeters of harvester exposures for a wide variety of pesticides used in crop protection by the strawberry industry.

178 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Regulatory

Discussion To determine the ultimate source of pesticide biomarkers in urine, the measurements of foliar residues must be carefully studied. Modeling harvester exposure based upon environmental measurements such as leaf residues requires detailed understanding of the fate and transport of pesticides on strawberries and validation of key concepts and practices using biomonitoring. This is particularly true with fungicides, insecticides, and miticides used in the strawberry industry that produce prolonged, low level harvester exposures.

Dislodgable foliar residues (DFR) and each of the independent measurements of available surface residues (TFR and PGR) declined 75 to 90% from day 4 to day 7 after malathion application (Table 4). End of shift handwashes during the same period at Safari Farms contained 85% less malathion after the second harvest, seven days after application. Estimates of worker exposure based upon urine biomarkers were more stable. During the same interval the excretion of malathion biomarkers declined only 43% prompting the untested speculation that biomarkers are being dermally absorbed and excreted during harvesting of sprayed fields. This possibility deserves critical evaluation with respect to the use of urine biomonitoring for occupational and residential pesticide exposure and risk assessment (Krieger et al., 2012).

T able 4. Leaf Residues and Harvester Exposure Estimates, DB Specialty Farms, Santa Maria

Factor Used to Estimate 1st Study 2nd Study Monterey Bay Academy Harvester Exposure Period Period

Dislodgable Foliar Residues 0.238 + 0.05 0.027 + 0.01 -90% (ug/cm2)

Transferrable Foliar Residues 0.029 + 0.003 0.0026 + 0.0008 -90% (ug/cm2)

Pesticide Glove Residues 13944 + 3318.7 3435 + 1017.2 -75% (ug/pair)

Handwashes 33.5 + 43.0 5.2 + 1.7 -85% (data not shown @ Safari Farms) (ug/person)

Urine biomonitoring 529.2 + 600.4 300.3 + 444.6 -43% (nmol/g.Cn)

Means followed by a different letter are significantly different at P = 0.05 by Tukey’s Studentized Range (HSD) test.

179 2011 - 2012 RESEARCH PROJECTS Dislodgable foliar residues (DFR) and each of the independent measurements of available surface residues (TFR and PGR) declined 75 to 90% from day 4 to day 7 after malathion application. End of shift handwashes at Safari Farms contained 85% less malathion after the second harvest, 7 days after application. During the same interval the excretion of malathion biomarkers declined only 43% prompting the untested speculation that biomarkers are being dermally absorbed and excreted during harvest.

180 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Regulatory

Selected References

• Chen.L, T. Zhao, C. Pan, J. H. Ross and Krieger, R.I. 2012a. Preformed Biomarkers Including Dialkylphosphates (DAPs) in Produce May Confound Biomonitoring in Pesticide Exposure and Risk Assessment. J. Agric. Food Chem., In Press.

• Iwata, Y., J. B. Knaak, R. C. Spear and Foster, R.J. 1977. Worker Reentry into Pesticide-Treated Crops. I. Procedure for the Determination of Dislodgeable Pesticide Residues on Foliage. Bulletin of Environmental Contamination & Toxicology.18 (6):649-655.

• Krieger R.I. and Dinoff, T.M. 2000. Captan Fungicide Exposures of Strawberry Harvesters Using THPI as a Urinary Biomarker. Arch. Environ. Contam. Toxicol. 38, 398–403.

• Krieger, R. I., L. Chen, M. Ginevan, D. Watkins, R.C. Cochran and Ross, J.H. 2012. Implications of Estimates of Residential Organophosphate Exposure from Dialkylphosphates (DAPs) and Their Relevance to Risk. Regulatory Toxicology and Pharmacology, In Press.

• Li,Y., L. Chen, Z. Chen, J. Coehlo, L. Cui, T. Lopez, G. Sankaran, H. Vega and Krieger, R. 2011. Glove Accumulation of Pesticide Residues for Strawberry Harvester Exposure Assessment. Bulletin of Environmental Contamination and Toxicology 86: 615-620.

• Li, Y. 2009. Occurrence and the Exposure Potential of Selected Pesticide Residues in Strawberries, Particularly Preformed Human Malathion Biomarkers in Leaves and Berries. PhD Dissertation, University of California, Riverside.

• Zhang, X., J.H. Driver, Y. Li, J.H. Ross, and Krieger, R.I. 2008. Dialkylphosphates (DAPs) in Fruits and Vegetables Can Confound Biomonitoring in Organophosphorus Insecticide Exposure and Risk Assessment. J. Agric. Food Chem. 56: 10638-10645.

• Zweig G., J. T. Leffingwell and Popendorf, W. 1985. The relationship between dermal pesticide exposure by fruit harvesters and dislodgeable foliar residues. J. Environ Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 1532-4109, 20(1): 27 – 59.

In Review

Chen, L, T. Zhao, C. Pan, J. Ross, M. Ginevan, H. Vega and Krieger, R. 2012b. Absorption and excretion of organophosphorous insecticide biomarkers of malathion in the rat: Implications for overestimation bias from environmental biomonitoring. Submitted to Reg. Tox. Pharmacol. May 8, 2012. In Revision.

181 2011 - 2012 RESEARCH PROJECTS 182 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT APPENDICES

183 2011 - 2012 RESEARCH PROJECTS California Strawberry Commission Commission Members and Alternates 2011-2012

Producer Members and Alternates

District 1: San Diego, Orange, Imperial, Riverside, Los Angeles & San Bernardino Counties Member: Neil Nagata Alternate: Todd Etchandy Jack Fujishige

District 2: Ventura County Member: Will Doyle Alternate: Hector Gutierrez Sean Stevens Glen Hasegawa Bill Reiman Michael Cleugh Mike Ferro Rob Hiji Edgar Terry

District 3: Santa Barbara and San Luis Obispo Counties Member: Lorena Chavez Alternate: Daniel Chavez Daren Gee George Chavez Greg France Bryan Gresser

District 4: Santa Cruz, Monterey, San Benito, Santa Clara, San Mateo, San Francisco and Alameda Counties Member: Erik Jertberg Alternate: Brian Driscoll Ed Kelly Tom Jones Stuart Yamamoto Walt Maitoza Tom AmRhein Victor Ramirez Mario Aguas

District 5: Sacramento, Madera, Merced, Fresno, & All Other Counties Not Listed Member: Brian Saetern Alternate: Lao Chang

District 6: All Counties (Producer Member - at - Large) Richard Uyematsu

184 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Appendices

Processor Members and Alternates Member: Ed Haft Alternate: Bob Barnhouse P.J. Mecozzi Mike Collins Al Yamamoto

Shipper Members and Alternates Member: Rich Amirsehhi Alternate: Fritz Koontz Michael Hollister Carl Lindgren Matt Kawamura Vince Lopes Bill Moncovich George Schaaf

Public Members Member: Gloria Sakata Alternate: Vacant

185 2011 - 2012 RESEARCH PROJECTS 2011-2012 Research Committee Carl Lindgren, Chairman Thomas AmRhein Daniel Chavez George Chavez Will Doyle Brian Driscoll Todd Etchandy Greg France Jack Fujishige Daren Gee Bryan Gresser Hector Gutierrez Glen Hasegawa Erik Jertberg Tom Jones Neil Nagata Victor Ramirez Sean Stevens Edgar Terry Richard Uyematsu Stuart Yamamoto

186 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Appendices

2012 Grower Resource and Contact Information

California Strawberry Commission Phone: (831) 724-1301, Fax: (831) 724-5973 P.O. Box 269 Watsonville, CA 95077 www.calstrawberry.com

Dan Legard Andrew Kramer Vice President, Research and Education Director of Grower Education [email protected] [email protected] Hillary Thomas Andrew Wiemers Production Research Manager Grower Communications Specialist [email protected] [email protected] Elizabeth Church Laura Gregory Production Research Specialist Grower Communications Specialist [email protected] [email protected] Alex Orozco Jason Sharrett Production Research Specialist Training Development Specialist [email protected]@calstrawberry.org [email protected] Sofia Hernandez Jazmin Lopez Special Projects / Meeting Coordinator Grower Education Program Assistant [email protected] [email protected]

UCCE County Farm Advisors

Santa Cruz, Monterey and San Benito Mark Bolda (831) 763-8040 [email protected] Monterey and Santa Cruz Steve Koike (831) 759-7350 [email protected] Ventura Oleg Daugovish (805) 645-1457 [email protected] Orange John Kabashima (714) 708-1606 [email protected] Fresno Richard Molinar (559) 456-7555 [email protected] Santa Barbara and San Luis Obispo Surendra Dara (805) 934-6240 [email protected] Merced Maxwell Norton (209) 385-7403 [email protected] Shasta / Lassen Dan Marcum (530) 336-5784 [email protected] Sacramento Chuck Ingels (916) 875-6913 [email protected] San Diego Ramiro Lobo (858) 694-2845 [email protected]

187 2011 - 2012 RESEARCH PROJECTS County Agricultural Commissioners Santa Cruz Mary Lou Nicoletti (831) 763-8080 [email protected] Monterey Eric Lauritzen (831) 759-7325 [email protected] Santa Barbara Cathleen Fisher (805) 681-5600 [email protected] San Luis Obispo Robert Lilley (805) 781-5924 [email protected] Ventura Henry Gonzalez (805) 933-2926 [email protected] Orange Rick Lefeuvre (714) 955-0100 [email protected] San Diego Robert Atkins (858) 694-2741 [email protected] Fresno Carol Hafner (559) 456-7510 [email protected] Merced David Robinson (209) 385-7431 [email protected] Riverside John Snyder (951) 955-3045 [email protected] Sacramento Frank Carl (916) 875-6603 [email protected] San Benito Ronald Ross (831) 637-5344 [email protected] San Bernardino John Gardner (909) 387-2115 [email protected]

Strawberry Researchers Dr. Husein Ajwa, UC Davis (831) 755-2823 [email protected] Dr. Richard Evans, UC Davis (530) 752-6617 [email protected] Dr. Steven Fennimore, UC Davis (831) 755-2896 [email protected] Dr. Tom Gordon, UC Davis (530) 754-9893 [email protected] Dr. Robert Krieger, UC Riverside (951) 827-3724 [email protected] Dr. Timothy Hartz, UC Davis (530) 752-1738 [email protected] Dr. Kirk Larson, UC SCREC Irvine (949) 857-0136 [email protected] Dr. Mark Mazzola, USDA/ARS (509) 664-2280 [email protected] Dr. Joji Muramoto, UC Santa Cruz (831) 459-4181 [email protected] Dr. Doug Shaw, UC Davis (530) 752-0905 [email protected] Dr. Carol Shennan, UC Santa Cruz (831) 459-4181 [email protected] Dr. Stuart Styles, Cal Poly State University (805) 756-2429 [email protected] Dr. Frank G. Zalom, UC Davis (530) 752-3687 [email protected]

188 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT NOTES

189 2011 - 2012 RESEARCH PROJECTS 190 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT P.O. Box 269 Watsonville, CA 95077 831-724-1301 phone 831-724-5973 fax [email protected] ® www.calstrawberry.com © 2013 California Strawberry Commission