2014 — 2015 RESEARCH REPORT California Strawberry Commission California Strawberry ANNUAL PRODUCTION PRODUCTION ANNUAL

CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 2014 — 2015 P.O. Box 269 P.O. 831-724-5973 fax 831-724-1301 phone Watsonville, CA 95077 CA Watsonville, www.calstrawberry.com [email protected] © 2016 California Strawberry Commission CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

2014 - 2015 Table of Contents

Introduction – Message from the Research Committee Chairman ...... 5

Pathology Fungicide Trials for Fruit and Foliar Pathogens of Strawberry 2013-2014...... 9 Mark Bolda Steven Koike

Management of Diseases Caused by Fusarium oxysporum, Verticillium dahliae and Macrophomina phaseolina...... 17 Dr. Thomas R. Gordon

Operating a State-wide Strawberry Disease Diagnostic Services Center ...... 31 Steven T. Koike

Evaluation of the Population Structure of Macrophomina phaseolina and Optimization of Quantification Assays...... 37 Dr. Frank N. Martin

Entomology

Strawberry Insect and Mite Control...... 47 Dr. Frank G. Zalom

Collection, Release and Establishment of Peristenus digoneutis, a European Lygus Bug Parasitoid, on the California Central Coast...... 65 Dr. Kim A. Hoelmer

2014 Lygus and Mite Management Program...... 71 Dr. Hillary Q. Thomas

2015 California Strawberry Commission Lygus Management Program...... 81 Dr. Hillary Q. Thomas

Weed Science Weed Management in Strawberry...... 93 Dr. Steven Fennimore

2 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Farming Without Fumigants Non-fumigant Strategies for Soilborne Disease Control in California Strawberry Production Systems ...... 101 Dr. Carol Shennan Dr. Joji Muramoto

A Review of the Final Three Seasons (2012, 2013 and 2014) of Research and Grower Demonstrations on the Raised Bed Trough (RaBeT) Substrate Production Systems...... 117 Dr. Hillary Q. Thomas Dr. Dan E. Legard

Innovation Automated Strawberry Calyx Removal Technology for Increasing Profit and Minimizing Field Labors...... 143 Dr. Yang Tao, P.E.

Automated Orientating and Capping of Strawberries for Processing...... 153 Dr. Stavros G. Vougioukas

Appendices Commission Members and Alternates for 2014-2015 ...... 170 Research Committee Members 2014-2015 ...... 172 2016 Grower Resource and Contact Information ...... 173

3 2014 - 2015 RESEARCH PROJECTS 4 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Introduction A Message from the Research Committee Chairman

The 2014-2015 Annual Production Research Report summarizes the results of 16 research and extension projects funded by the California Strawberry Commission (CSC). These reports are progress updates for the research and extension projects funded by the CSC during the 2013-2014 fiscal year. These projects include the development of a de-capping machine, management of soilborne diseases, Lygus bug-monitoring, and the use of Anaerobic Soil Disinfestation (ASD) to provide clean soil for producing strawberries.

The CSC is the leading funding source for world class strawberry production research in the United States and we hope that these reports will help strawberry growers, Pest Control Advisors and other industry members address production problems. These reports are also intended to document research that may not be readily available in other publications. We hope our research is helpful to researchers in California and around the world.

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 efforts and dedication to the California strawberry industry. California strawberry growers continue to face increasing production and regulatory challenges and the research efforts of these researchers are vital for the continued success of the California strawberry industry.

I also want to thank the members of the Science Advisory Committee for their help in reviewing these projects for the Research Committee. I would like to especially thank the many growers, Pest Control Advisors 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, the University of California Cooperative Extension and the United States Department of Agriculture for their continuing support of the Strawberry Commission’s research programs.

Sincerely,

Tom AmRhein

Research Committee Chairman

5 2014 - 2015 RESEARCH PROJECTS 6 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT PATHOLOGY

7 2014 - 2015 RESEARCH PROJECTS 8 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Fungicide Trials for Fruit and Foliar Pathogens of Strawberry 2013-2014

Principle Investigators Mark Bolda UCCE Santa Cruz County 1432 Freedom Boulevard Watsonville, CA 95076 (831) 763-8040 [email protected]

Steve Koike UCCE Monterey County 1432 Abbott Street Salinas, CA 93901 (831) 759-7350 [email protected]

Summary In 2014, we evaluated fungicide products against a slate of several registered fungicides and an untreated control. Fungicides were tested for efficacy against gray mold caused by Botrytis cinerea and powdery mildew caused by Podosphaera aphanis.

Introduction The authors have been engaged in studying fruit and foliar pathogens in strawberry since 2007. While this group of pathogens includes anthracnose caused by species of Colletotrichum, leather rot caused by Phytophthora cactorum and angular leaf spot caused by Xanthomonas fragariae, the fungicide screening of 2014 concentrated on the two most common problems: gray mold caused by Botrytis cinerea, and powdery mildew caused by Podosphaera aphanis. Year after year these diseases are the most important foliar/fruit concerns for growers, and subsequently garner the most attention from fungicide distributors and manufacturers.

9 2014 - 2015 RESEARCH PROJECTS Materials and Methods Powdery Mildew The trial consisted of treatments (Table 1) arranged in a randomized complete block design with each treatment replicated four times, on the Holly Ranch managed by Dole on the variety Monterey. Applications of all materials were made in the equivalent of 150 gallons per acre with a motorized backpack sprayer and hand held boom configured with ten 8001 flat fan nozzles operating at 120 psi, which is intended to closely mimic the conditions of commercial application. Applications of all materials were made two weeks apart: May 16, May 28, June 16, and June 24, 2014.

Powdery mildew evaluations were done on June 5, June 19 and July 7 and consisted of taking ten strawberry leaflets (per plot) of young to mid-age and assigning a percentage on a scale of 0 to100% (in 10% increments) to the level of powdery mildew severity on the underside.

Table 1. 2014 Powdery Mildew Treatments

Test Material Rate/acre 1 Thiram 24/7 2.6 qt 2 Thiram 24/7 2.6 qt+ Procure 480 SC 8 fl oz 3 Thiram Granu-Flo 4.4 lb 4 Pyriofenone 4 fl oz 5 Pyriofenone 5 fl oz 6 Pyriofenone 4 fl oz + Quintec 6 fl oz 7 Quintec 6 fl oz 8 Rally 5 oz 9 Rally 5 oz w/ Quintec 5 fl oz 10 Merivon + Nufilm P 8 fl oz + 0.01% v/v 11 Merivon + Nufilm P 10 fl oz + 0.01% v/v 12 Pristine 23 oz 13 Torino 3.4 fl oz w/ Mettle 5 fl oz 14 Isofetamid 17 fl oz 15 Isofetamid + IB8111 10.3 fl oz + 5.57 fl oz 16 Isofetamid + 18121 10.3 fl oz + 16.5 fl oz 17 Isofetamid + IB18220 10.3 fl oz + 7.6 fl oz 18 Untreated check -

10 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Results for 2014 Trial

Table 2. Powdery Mildew Evaluation- Percent Leaf Disease by Treatment

Character Rated % Infest % Infest % Infest Rating Date Jun-5-2014 Jun-19-2014 Jul-7-2014 Number of Decimals 2 2 2 Trt Treatment 1 2 3 No. Name 1 Thiram 24/7 2.6 qt 23.44 a 17.25 bcd 23.08 abc 2 Thiram 24/7 2.6 qt + Procure 480 SC 8 fl oz 11.88 cde 7.25 gh 14.64 def 3 Thiram Granu-Flo 4.4 lb 16.25 bc 18.00 bc 26.35 a 4 Pyriofenone 4 fl oz 13.44 cde 13.00 def 15.85 cde 5 Pyriofenone 5 fl oz 13.13 cde 8.25 fgh 8.90 fgh 6 Pyriofenone 4 fl oz + Quintec 6 fl oz 15.31 bcd 11.25 efg 13.60 d-g 7 Quintec 6 fl oz 15.00 bcd 15.50 b-e 23.55 ab 8 Rally 5 oz 16.25 bc 19.25 b 29.40 a 9 Rally 5 oz w/ Quintec 5 fl oz 10.31 c-f 14.00 cde 17.26 bcd 10 Merivon 8 fl oz + Nufilm P 0.01% 6.88 ef 4.25 h 6.76 hi 11 Merivon 10 fl oz + Nufilm P 0.01% 3.75 f 3.75 h 3.49 i 12 Pristine 23 oz 21.25 ab 13.00 def 22.66 abc 13 Torino 3.4 fl oz w/ Mettle 5 fl oz 7.19 ef 7.00 gh 8.67 gh 14 Isofetamid 17 fl oz 11.88 cde 8.75 gh 11.47 d-h 15 Isofetamid 10.3 fl oz + IB8111 5.57 fl oz 10.63 cde 11.75 efg 15.71 cde 16 Isofetamid 10.3 fl oz + 18121 16.5 fl oz 11.94 cde 4.75 h 10.84 e-h 17 Isofetamid 10.3 fl oz + IB18220 7.6 fl oz 9.06 def 7.25 gh 10.98 d-h 18 Untreated check 20.94 ab 24.75 a 30.04 a LSD (P=0.05) 6.768 4.920 0.838t Standard Deviation 4.786 3.479 0.593t CV 36.12 29.53 14.89

Means followed by same letter do not significantly differ (P=0.05, LSD)

Botrytis Gray Mold The trial consisted of treatments (Table 3) arranged in a randomized complete block design with each treatment replicated four times, on the Holly Ranch managed by Dole on the variety Monterey. Applications of all materials were made in the equivalent of 150 gallons per acre with a motorized backpack sprayer and hand held boom configured with ten 8001 flat fan nozzles operating at 120 psi, which is intended to closely mimic the conditions of commercial application. Applications of all materials were made two weeks apart May 16, May 28, June 16, and June 24, 2014.

11 2014 - 2015 RESEARCH PROJECTS Evaluations for Botrytis infected fruit were made during the weekly fruit harvest by a professional crew of research plot harvesters beginning May 21 and continuing until July 16. Culls were sorted and examined for symptoms and signs of gray mold disease, and a percentage of gray mold infected fruit was calculated from the total fruit harvested from that plot (Table 4).

Table 3. Test materials for Botrytis study

Test Material Rate/acre 1 Thiram 24/7 2.6 qt 2 Thiram 24/7 2.6 qt+ Procure 480 SC 8 fl oz 3 Thiram Granu-Flo 4.4 lb 10 Merivon + Nufilm P 8 fl oz + 0.01% v/v 11 Merivon + Nufilm P 10 fl oz + 0.01% v/v 12 Pristine 23 oz 14 Isofetamid 17 fl oz 15 Isofetamid + IB8111 10.3 fl oz + 5.57 fl oz 16 Isofetamid + 18121 10.3 fl oz + 16.5 fl oz 17 Isofetamid + IB18220 10.3 fl oz + 7.6 fl oz 18 Untreated check -

12 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Results for 2014 Trial

Table 4. Results for 2014 Botrytis Evaluation – Percentage Fruit Infected Fruit

Character Rated % Bot % Bot % Bot % Bot % Bot % Bot % Bot % Bot % Bot Rating Date May-21- May-28- Jun-4- Jun-11- Jun-18- Jun-25- Jul-3- Jul-9- Jul-16- 2014 2014 2014 2014 2014 2014 2014 2014 2014 No. Treatment Name 1 2 3 4 5 6 7 8 9 1 Thiram 24/7 2.6 qt 18.70 a 9.25 a 9 a 1.68 a 3.15 ab 8.75 a 18.01 a 15.91 a 11.62 a 2 Thiram Granuflo 4.4 lbs 26.74 a 7.52 a 7.22 a 1.07 a 1.95 abc 15.17 a 18.39 a 12.05 a 6.91 a 3 Thiram 24/7 + Procure 480 SC 27.47 a 8.40 a 9.27 a 2.49 a 1.64 abc 14.36 a 16.198 a 20.18 a 9.00 a 4 Merivon @ 8 fl oz 30.91 a 8.14 a 5.52 a 1.49 a 0.74 c 15.56 a 16.268 a 12.67 a 6.51 a 5 Merivon @ 10 fl oz 20.96 a 6.62 a 5.96 a 1.25 a 0.69 c 15.73 a 17.233 a 15.86 a 7.10 a 6 Pristine @ 23 oz 20.34 a 10.04 a 7.46 a 1.44 a 3.49 a 12.32 a 14.708 a 18.94 a 10.70 a 7 Isofetamid @ 17 fl oz 16.99 a 4.32 a 6.54 a 1.07 a 1.67 abc 12.70 a 17.975 a 11.04 a 9.21 a 8 Isofetamid @ 10.3 fl oz 24.39 a 9.63 a 5.67 a 1.58 a 1.35 bc 10.86 a 20.643 a 13.64 a 10.40 a + IB8111 @ 5.57 fl oz 9 Isofetamid @ 10.3 fl oz 18.64 a 5.95 a 4.69 a 2.11 a 2.63 ab 8.60 a 12.815 a 12.84 a 8.28 a + IB18121 @ 16.5 fl oz 10 Isofetamid @ 10.3 fl oz 30.17 a 10.98 a 5.59 a 1.85 a 1.87 abc 9.22 a 19.803 a 11.83 a 6.64 a + IB18220 7.6 fl oz 11 UTC 20.26 a 15.82 a 13.52 a 1.68 a 3.14 ab 7.66 a 14.720 a 10.82 a 9.26 a LSD P=0.05 0.323t 1.655t 0.344t 0.276t 0.265t 0.282t 8.9782 6.8736 0.2239 Standard Deviation 0.224t 1.146t 0.238t 0.191t 0.183t 0.196t 6.2180 4.7604 0.1551 CV 16.26t 38.05t 27.11t 46.46t 39.85t 17.79t 36.62 33.61 15.82

Means followed by same letter do not significantly differ (P=0.05, LSD)

13 2014 - 2015 RESEARCH PROJECTS Discussion of Results for 2014 Trial Powdery Mildew Control of powdery mildew by both rates of Merivon and a rotation of Torino and Mettle was exceptional, with percentages of infection significantly lower than many treatments in each of the three evaluation dates.

Looking at the last rating date on July 7, neither formulation of Thiram controlled powdery mildew, but the inclusion of Procure together with Thiram 24/7 did result in disease percentages below the untreated control. Test compounds isofetamid and pyriofenone had lower percentages of mildew than the untreated control. Rally 40W did not have significantly lower percentages of mildew than the untreated control. Botrytis With the exception of the June 18 evaluation date, no significant differences were found between any of treatments. On June 18, both treatments of Merivon demonstrated lower levels of Botrytis infected fruit than the untreated control, Thiram 24/7, isofetamid at10.3 fl oz + IB8111 and Pristine.

Acknowledgments We thank Patty Ayala, Kat Kammeijer and Monise Sheehan for their assistance with this trial. We acknowledge the California Strawberry Commission and cooperating companies for supporting this work.

14 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

15 2014 - 2015 RESEARCH PROJECTS 16 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Management of Diseases Caused by Fusarium oxysporum, Verticillium dahliae and Macrophomina phaseolina

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 During 2014, our primary focus was on Fusarium wilt, caused by Fusarium oxysporum f. sp. fragariae. This included work related to development and characterization of genetic resistance, which will make an increasingly important contribution toward management of this disease in the future. Based on our 2014 evaluations, UC cultivars, ‘San Andreas’, ‘Portola’ and ‘Ventana’ continue to appear resistant to Fusarium wilt. For resistance to be effective, it is necessary to test breeding lines with pathogen isolates that are representative of the population that plants will encounter under field conditions. To ensure that this is the case, we have characterized the California population of the Fusarium wilt pathogen and determined that it is comprised of three genetically distinct strains. All these strains will be tested to determine if they differ in virulence on strawberry. Previous work with F. oxysporum from blackberry showed that this fungus may also be damaging to strawberry. We conducted further tests that confirmed this possibility, at least under controlled conditions. We have also evaluated a number of factors that can influence the ability ofF. o. fragariae (F,o,f,) to cause disease. This included further testing of soil pH effects on disease. Our findings showed no evidence that soil acidification increases the likelihood of infection or enhances the severity of Fusarium wilt. An end of season Vapam application significantly reduced the survival rate of the pathogen in soil but was not effective in eliminating the pathogen from infected crowns. Anaerobic

17 2014 - 2015 RESEARCH PROJECTS soil disinfestation appeared to be more effective when rice bran was used as a carbon source and was more effective deeper in the soil than above a depth of 6 inches. However, in no cases was the pathogen completely eliminated. We tested three crops that might be grown in rotation with strawberries (lettuce, broccoli and spinach) and found that roots and stems of all three could be colonized by the Fusarium wilt pathogen. Further work will be required to determine if the extent of colonization will allow these crops to support a significant increase in pathogen inoculum. In our third year of testing strawberry genotypes for resistance to crown rot, caused by M. phaseolina, we recorded a wide range of reactions. Some cultivars displayed no symptoms and others became severely diseased. Overall, the ranking of cultivars was very similar to what we have recorded in previous years. Results of host range tests showed Macrophomina to have little or no capacity to colonize the following cover crops: fava bean, mustard, oat, and common vetch. The only crop confirmed to be infected was mustard, from which M. phaseolina was recovered from a single root piece. Based on these results, none of the crops tested appear to pose a risk of increasing soil inoculum of M. phaseolina.

Introduction Fusarium wilt, caused by Fusarium oxysporum f. sp. fragariae, and crown rot caused by Macrophomina phaseolina, have continued to become more widespread, and now occur in all major strawberry production areas in California (Koike et al., 2012). Diminished availability of methyl bromide and less complete treatment of soil (application to beds rather than flat fumigation of an entire field) are key factors contributing to the emergence of these diseases in California. The origin of the pathogens is not known, but M. phaseolina is of long residence in California and is known to cause disease on numerous crops. Consequently, this pathogen was likely to have been widely distributed prior to its recognition as a cause of disease on strawberries. On the other hand, F. oxysporum f. sp. fragariae is not known to affect other crops and so was likely introduced to California from another strawberry growing area (Koike and Gordon, 2015). Fusarium wilt has been a problem primarily in Ventura County, but over the past two years, this disease has been confirmed at many new locations in Santa Cruz and Monterey counties. Both pathogens can be moved with soil and so movement of equipment between fields will likely result in both diseases becoming even more widespread.

Key elements in management of diseases caused by soilborne pathogens is maintaining soil inoculum below damaging levels, and growing varieties that are resistant to disease. As fumigation becomes a progressively less reliable means of suppressing pathogen populations, genetic resistance will become correspondingly more important. It was in anticipation of this need that the UC breeding program initiated screening for resistance to Verticillium wilt. This on-going effort has succeeded in significantly increasing the level of resistance in the UC breeding population. As a result, breeding lines advanced to cultivar status are typically more resistant than cultivars released in earlier years. In 2009, we initiated a similar program to increase resistance to Fusarium wilt. Although we have found that some cultivars adapted to California growing conditions are highly resistant to Fusarium wilt (e.g., ‘San Andreas’ and ‘Ventana’), others are quite susceptible (‘Albion’ and ‘Monterey’).

The success of a breeding program in developing genetically resistant cultivars is contingent on screening germplasm against representative strains of the pathogen. To date we have used a mix of isolates of F.o.f. from diverse locations within the state, but we now know that the population in California includes three genetically distinct strains, only one of which is represented in the mix we have been using. The basis for the recognition of these strains is described in this report. In the coming year we will be testing all three strains to determine if they differ in virulence on strawberry genotypes that we regard as resistant to Fusarium wilt.

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As our capacity to suppress pathogen populations in soil using conventional flat fumigation diminishes over time, alternative practices will become more important. Growers have expressed interest in two procedures that may contribute to this effort: anaerobic soil disinfestation (ASD) and end-of-season Vapam application, both of which have been subject of tests that are described herein. Crop rotation is another practice that can help to maintain pathogen populations below damaging levels. In this report, we describe the potential for the Fusarium wilt pathogen to increase on crops other than strawberry. We also assessed the ability of the blackberry strain of F. oxysporum to cause disease on strawberries.

Presently no strawberry varieties are considered highly resistant to crown rot caused by M. phaseolina. In principle, a resistant variety could be developed by transferring a major gene from a known resistant genotype, but as yet there is no known source of major gene resistance to Macrophomina. Future efforts will be devoted to searching for such a gene but presently our focus is on elevating resistance within the UC breeding population. To this end, we have developed a test that reveals variation in susceptibility within the UC breeding population. This test has now been used in three successive years, and the results are described herein. We also report on studies of the host range of M. phaseolina, to better understand the risk that may be posed by various cover crops. These studies were motivated by questions from growers about whether or not cover crops, planted in rotation with strawberry, could become infected and thereby enhance disease on strawberries planted subsequent to the cover crop.

Materials and Methods Screening for Resistance to Fusarium wilt Fourteen cultivars were tested for susceptibility to Fusarium wilt by immersing roots of runner plants in an aqueous suspension of pathogen spores. A spore suspension was prepared from plates of potato dextrose agar (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 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 29.4 million spores/fluid ounce by the addition of 0.1% water agar as needed. Spore suspensions of five isolates were combined so each of the five was represented by approximately 5.9 million spores per ounce. After inoculation, plants were transplanted into plots that had been fumigated with methyl bromide and chloropicrin (2:1 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 inoculated in fall of 2013, and rated during the spring of 2014. Each plant was 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. The Potential for Isolates of Fusarium oxysporum from Blackberry to Damage Strawberry Dormant crowns of the cultivars ‘Albion’ and ‘Chandler’ were root dip inoculated, as described above, and maintained in a greenhouse for eight weeks. For each experiment, ten plants of each cultivar were inoculated with a suspension of 29.4 million spores/fluid ounce and an additional ten of each cultivar were inoculated with a suspension of 58.8 million spores per ounce. Ten plants of each cultivar were dipped in water instead of a spore suspension to serve as negative controls. At eight weeks after inoculation, plants were rated for symptoms of Fusarium wilt. In addition, petioles were assayed for the presence of F.o.f. by culturing them on plates of Komada’s selective medium (KM), which were incubated at room temperature under fluorescent lights for up to two weeks. Infected petioles were identified by the emergence of colonies with the distinctive appearance of the pathogen. The experiment was conducted twice.

19 2014 - 2015 RESEARCH PROJECTS The Population Structure of F.o.f. in California Isolates of F.o.f. were obtained from symptomatic strawberry plants in commercial fruit production fields throughout California between 2008 and 2014. Collection sites included fields in all major strawberry growing regions in the state. Tissue samples were surface-sterilized by submergence in 70% ethanol for 30 seconds, followed by 1 minute in 1% sodium hypochlorite. Surface-treated tissue was placed on plates of KM, which were incubated at room temperature under fluorescent lights for up to two weeks. Colonies morphologically similar toF. oxysporum were sub-cultured as single hyphal tips, and stored on dried filter paper at 4 C. To confirm pathogenicity, roots of dormant strawberry crowns were dipped in a spore suspension that was prepared as described above. Inoculated plants were placed in 0.25 gallon pots filled with sterilized potting mix (one plant per pot). Plants were maintained in a growth chamber, as described above, for six to eight weeks, after which plants were rated for symptoms of Fusarium wilt, using the scale described above. Assigning isolates to vegetative compatibility groups requires development of mutant strains with complementary deficiencies in utilization of an essential nutrient. For this purpose, we use mutants that affect the nitrate utilization pathway. To obtain these mutants, isolates of F.o.f. were cultured on PDA amended with 4% potassium chlorate. Growth on this medium only develops from mutants that are resistant to chlorate. Such mutants are usually also unable to utilize nitrate. Chlorate resistant growth was transferred to Czapek’s medium, which has no reduced source of nitrogen. Nitrate non- utilizing (nit) colonies were identified by their flat growth and lack of aerial mycelium. Complementary nit mutants (those with mutations affecting different parts of the nitrate utilization pathway) served as tester strains and were paired with nits from other isolates (meaning placed in proximity to each other on the same plate) on Czapek’s medium. If two isolates are compatible, robust growth with abundant aerial mycelium will develop where they come into contact. If isolates are not compatible, growth remains flat. DNA was extracted from mycelium of each isolate growing on PDA, using the OmniGen-X Purespin gDNA Purification Kit as per manufacturer’s instructions. The polymerase chain reaction (PCR) was used to amplify the translation elongation factor and intergenic spacer regions. PCR products were sequenced at the UC Davis DNA Sequencing Facility. Phylogenetic trees were constructed in PAUP v4.0a145 using Maximum Likelihood and Parsimony analyses with 10,000 bootstrap replicates. Trees were created for a dataset that included sequences of both regions.

The Effect of Soil pH on Root Infection and Disease Caused by Fusarium oxysporum f. sp. fragariae Soil was collected from a field in Ventura County where Fusarium wilt was a cause of extensive mortality in the previous strawberry crop. Sand that had been sterilized by autoclaving was blended with soil to achieve a 60:40 mix of sand:soil. The pH of the blended soil was adjusted either to pH 5.1, with sulfuric acid, or to pH 7.2, with sodium hydroxide. Following pH adjustment, inoculum density of the Fusarium wilt pathogen was assayed by soil dilution plating. This was accomplished by suspending soil in a sterilized solution of 0.1% water agar and spreading subsamples of this suspension on plates of KM. Inoculated plates were maintained for seven days at room temperature under fluorescent lights, after which colonies corresponding to the pathogen were identified based on their distinctive appearance. Counts of pathogen colonies on soil dilution plates confirmed there was not a significant difference in inoculum density between acidified and non-acidified soils. Two dormant strawberry crowns (cv ‘San Andreas’ or ‘Albion’) were positioned in the center of a 0.5 gallon pot, which was then filled with pH adjusted soil. Plants were maintained in controlled environment chamber with a 12 hour photoperiod and temperatures of 25 C (77 F) and 18 C (64 F) for light and dark periods, respectively. Beginning between 15 and 18 days after the start of the experiment, plants from one pot of each pH were gently separated from the soil. Newly emerged root tips were removed and placed in a flask containing sterile 0.1% sodium hexametaphosphate and agitated on rotary shaker at approximately 75 rpm for 20 minutes. This process was repeated three times, followed by 20 minutes in sterile water. Washed roots were blotted on paper towels to remove excess moisture and placed on plates of KM. 15 root tips (0.5 – 1.5 cm in length; = 0.2 – 0.6 inches) were placed on each plate. After five to seven days under

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fluorescent lights at room temperature, root tips were scored as infected by the Fusarium wilt pathogen or not. Infected root tips were identified by the emergence of a colony with the distinctive appearance of F.o.f.. To confirm the identity of the pathogen, colonies emerging from all root tips scored as positive were transferred to fresh plates of KM, along with a known isolate of F.o.f. for comparison. Plates were incubated as described above and colonies identified as the pathogen were transferred to potato dextrose agar (PDA). After a minimum of one week on PDA, DNA was extracted and used as a template for amplification of a diagnostic DNA sequence using PCR. Positive amplification was taken as confirmation that the colony in question corresponded to the pathogen. If the diagnostic fragment was not amplified, the isolate in question was tested for somatic compatibility with known isolates of the pathogen. Each pot (including roots from two plants) was scored for the percentage of root tips confirmed to be infected byF.o.f.. An experiment included five pots at each pH. This experiment was conducted twice using the cultivar ‘San Andreas’ and twice using the cultivar ‘Albion’. To test for an effect of soil pH on development of Fusarium wilt, ‘Albion’ was grown in potting mix that had been combined with sand containing inoculum of F.o.f.. Inoculum was prepared by blending fully colonized plates of PDA with water and adding the slurry to sterilized sand. Inoculated sand was allowed to dry at room temperature and combined with potting soil to obtain an inoculum density of 250 colony forming units (CFUs) per gram [= 7143 per ounce of soil]. Soil was adjusted to either pH 5.1 with sulfuric acid or to pH 7.1 with sodium hydroxide. Four plants of each cultivar were transplanted into 0.5 gallon pots filled with pH adjusted, infested potting mix (one plant per pot) and maintained for six weeks in a growth chamber with conditions as described above. Plants were rated at weekly intervals on a 1 to 5 scale, with 1 corresponding to a healthy plant and 5 for a plant killed by the disease. The experiment was conducted twice.

Colonization of Rotation Crops by F.o.f. Three crops were tested to determine the frequency with which they were colonized by F.o.f.: broccoli (Brassica oleracea), lettuce (Lactuca sativa) and spinach (Spinacia oleracea). For each crop, seed was sown in potting soil and following emergence, young seedlings were dipped into a spore suspension of the Fusarium wilt pathogen, which was prepared as described above. Strawberry (cv ‘Albion’) plants, included for comparison with rotation crops, were inoculated as described above. All inoculated plants were transplanted into potting soil and maintained in a controlled environment chamber under the conditions described above. After six weeks, plants were removed, roots and stems were separated, washed to remove soil, immersed briefly in 70% ethanol, followed by one minute in 1% sodium hypochlorite, and placed on plates of KM. Colonies corresponding to F.o.f. were identified based on morphological criteria, with follow-up tests for the presence of the diagnostic DNA sequence using PCR, as described above. The experiment was conducted three times. Effect of Anaerobic Soil Disinfestation on Survival of the Fusarium wilt Pathogen in Soil The experiment from which soil was assayed was located at the Monterey Bay Academy (MBA) in Santa Cruz County. Soil samples were taken from each of four replications of treatments that included ASD applied to flat ground with incorporation of different carbon sources (rice bran or molasses) prior to irrigation. Soil samples were taken after application of treatments and stored at 40 F until assayed. To estimate the abundance of inoculum in each sample, various quantities of soil, initially 10 grams (= 0.35 ounces), were suspended in 0.1% water agar, and spread over the surface of plates of KM. Plates inoculated in this manner were incubated at room temperature under fluorescent lights for five to seven days, and colonies of F.o.f. were identified as described above. Based on the number of pathogen colonies and the weight of soil assayed, the inoculum density was calculated and expressed as the number of CFUs per unit weight of soil. Effect of End-of-season Vapam Treatment on Survival of the Fusarium wilt Pathogen in Soil 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

21 2014 - 2015 RESEARCH PROJECTS until it was dry. Thereafter, the infested sand was packaged in nylon pouches, which were buried in various locations in beds at the end of the season in a strawberry field in Ventura County. Pouches were placed either directly under a drip line or midway between two drip lines, at both 6 and 12 inches below the surface. In addition, infected strawberry crowns were buried in similar locations, relative to drip tapes. Following treatment, pouches and crowns were deployed in this manner in four beds that were treated with metam sodium, which was applied through drip lines at a rate of 30 gallons per acre. The experiment included four replicate beds that were untreated. Pouches were recovered and infested sand was assayed by soil dilution plating to quantify the viability of F.o.f., as described above. Crown pieces were assayed by placement on KM and scored as positive or negative if the Fusarium wilt pathogen emerged, or not, respectively. Susceptibility of Cover Crops to Macrophomina phaseolina The pathogen was grown in a sand-cornmeal medium for approximately two months, after which the colonized medium was allowed to dry at room temperature. The following cover crops were used in this study: fava bean (Vicia faba), mustard (Sinapis alba cv ‘Ida Gold’), oat (Avena sativa cv ‘Cayuse’), rye (Secale cereale cv ‘Merced’), and common vetch (Vicia sativa). Strawberry (cv ‘Albion’) was used as a positive control. All cover crop species were grown by placing seeds into transplant trays and growing them in a greenhouse for one month. Dormant strawberry plants were planted into shallow trays containing potting mix in order to allow plants to break dormancy and grow roots and foliage. All plants were inoculated by sprinkling the sand-cornmeal inoculum onto the root balls of each plant and then misting the root balls with distilled water to secure the material to the roots. Inoculated plants were potted into either 4-inch (cover crops) or 6-inch (strawberry) pots. Control plants of strawberry and all cover crops were inoculated with sterile sand-cornmeal medium and planted in similar fashion. All plants were maintained in a greenhouse, and evaluated for symptoms after six weeks. After disease evaluation, roots of all Macrophomina-inoculated plants were sampled and tested for the pathogen. Cover crop and strawberry root pieces were selected if any discoloration or disease symptom was evident; if no roots showed any symptom or discoloration, randomly collected healthy roots were tested. For strawberry, 20 root pieces were tested and for cover crops 30 root sections were tested. Evaluating Susceptibility of Strawberry Genotypes to Crown rot Caused by Macrophomina phaseolina A known pathogenic isolate of M. phaseolina was grown on PDA at room temperature. Twelve fully colonized plates were blended in sterile water, and the resulting suspension was diluted with 0.35% water agar to obtain a final volume of 2.5 liters. Plants were inoculated by dipping their roots into the aqueous suspension of propagules. Inoculated plants were immediately transplanted into plots at the UC field station in Watsonville, CA that had been fumigated with methyl bromide and chloropicrin (2:1 at 350 pounds per acre). Eleven cultivars were tested, with two replicate plots of 20 plants for each cultivar. 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 Screening for Resistance to Fusarium wilt Of the cultivars tested, ‘Grenada’ and ‘Albion’ were the most severely affected by Fusarium wilt, with resistance scores of 2.4 and 2.7, respectively. As has been observed in previous tests, ‘San Andreas’ and ‘Ventana’ were highly resistant, with scores ≥ 4.9, which means that inoculated plants remained symptom-free or nearly so throughout the season. Overall, scores recorded in the 2014 test were consistent with the five-year average for cultivars that have been tested in multiple years (Table 1).

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Table 1. Fusarium wilt resistance scores for UC cultivars tested in multiple years.

Cultivar Resistance score1 2014 2013 2012 2011 2010 Average2 ‘Monterey’ 3.6 2.5 2.5 3 4 3.1 ± 0.29 ‘Albion’ 2.7 2.3 4.3 2.5 4.1 3.2 ± 0.42 ‘Camarosa’ 3.6 2.4 3.4 3.5 3.9 3.4 ± 0.26 ‘Benicia’ 4.2 4.0 2.9 2.5 ---3 3.4 ± 0.37 ‘Palomar’ 3.7 4.1 4.4 4.0 4.2 4.1 ± 0.11 ‘Camino Real’ 4.7 4.3 4.3 3.5 4.9 4.3 ± 0.24 ‘Ventana’ 5.0 4.8 3.6 5.0 4.4 4.6 ± 0.26 ‘Portola’ 4.6 4.7 5.0 5.0 5 4.9 ± 0.08 ‘San Andreas’ 4.9 4.8 5.0 5.0 4.9 4.9 ± 0.04

1 Resistance score is on a 1 to 5 scale, with 1 given to a plant that is severely diseased or Killed by Fusarium wilt and 5 corresponding to a plant with no symptoms of disease. 2 The average for tests conducted in 2010, 2011, 2012, 2013 and 2014 followed by the standard error. 3 Not tested. The Potential for Isolates of Fusarium oxysporum from Blackberry to Damage Strawberry In the first experiment, no symptoms were observed on either ‘Chandler’ or ‘Albion’, when plants were inoculated at the lower level (29.4 million spores per ounce), and the pathogen was not recovered from petioles on any plants. At the higher inoculum level (58.8 million spores per ounce) two ‘Albion’ plants died and most of the others were symptomatic. The average rating for Albion was 2.4 ± 0.5. The Fusarium wilt pathogen was recovered from petioles on three of the most severely affected plants, and not from any that were symptomless or mildly stunted (rating < 2.0). Nearly all Chandler plants inoculated at the higher level remained symptomless, with one plant showing mild stunting. The pathogen was not isolated from petioles on any of the plants. When the experiment was repeated, a number of ‘Albion’ plants were symptomatic at both inoculum levels; average ratings were 2.6 ± 0.5 and 1.9 ± 0.4 for the low and high inoculum levels, respectively. Based on a t-test, there was not a significant difference between ratings at the two different inoculum levels for ‘Albion’ (P = 0.2835). The Fusarium wilt pathogen was isolated from all plants with a rating ≥ 3.0. ‘Chandler’ was also more severely affected in the second experiment than in the first. Average ratings at the low and high inoculum levels were 1.8 ± 0.4 and 1.6 ± 0.2, respectively. This difference was not significant (P =0.4328). The pathogen was isolated from petioles on only two of seven symptomatic plants of cv ‘Chandler’. The Population Structure of F. o. fragariae in California Fifty-nine isolates of F. oxysporum obtained from symptomatic strawberry plants from throughout California were found to be pathogenic to strawberry based on root-dip inoculations. Fifty-five of these isolates were associated with a single somatic compatibility group (SCG 1). The remaining isolates were associated with two other groups: SCG 2 (2 isolates) and SCG 3 (3 isolates). Isolates associated with SCG 1 were recovered from all major strawberry production areas, whereas SCG 2 and SCG 3 were not found north of San Luis Obispo County. Based on a comparison of DNA sequences of the translation elongation factor and the intergenic spacer, the three compatibility groups corresponded to three well-separated clusters. Isolates of F.o.f. from Japan, included for comparison, were affiliated with two of the three clusters. Non-pathogenic isolates of F. oxysporum recovered from soil could not be separated from pathogenic isolates, based on the DNA sequences that were compared.

23 2014 - 2015 RESEARCH PROJECTS The Effect of Soil pH on Root Infection and Disease Caused by Fusarium oxysporum f. sp. fragariae During the short interval of growth in pH adjusted soil (< three weeks), all plants appeared healthy and grew vigorously. Thirty or more root tips were collected from each pair of plants in the same pot and determined to be infected by F.o.f. or not. Across all four experiments (two each for ‘Albion’ and ‘San Andreas’), 96% of the colonies identified as the pathogen based on colony morphology on KM were confirmed by the PCR test. Most of the colonies that lacked the diagnostic DNA sequence were found to be somatically compatible with tester strains for known pathogenic isolates, indicating a low rate of false negatives with the PCR test. The percentage of root tips infected was computed for plants in each of five pots in each treatment (soil acidified or not) of each experiment. Percentage data were transformed (arcsin square root) prior to analysis. Across treatments in both experiments, infection rates ranged from 22.4 ± 3.6% to 43.8 ± 2.1% for ‘San Andreas’ and from 20.0 ± 5.8% to 45.0 ± 4.4% for ‘Albion’ (Table 2). Averaged across both experiments, roots of ‘Albion’ became infected more frequently in soil near neutrality (36.5 ± 4.1%) than in the acidified soil 24.2 ± 4.1%. Analysis of variance showed this difference to be significant (P = 0.0105). For ‘San Andreas’, roots became infected at a similar rate in both acidified (31.0 ± 6.0%) and non-acidified soils (34.0 ± 3.6%). Analysis of variance indicated this difference was not significant (P = 0.0741).

Table 2. Colonization of strawberry root tips by Fusarium oxysporum f. sp. fragariae

Treatment1 Cultivar Block2 Soil pH3 Root tips colonized (%) Initial Final Acidified ‘San Andreas’ 1 5.02 ± 0.06 5.60 ± 0.04 26.1 ± 3.2 Neutral ‘San Andreas’ 1 7.30 ± 0.01 7.44 ± 0.06 22.4 ± 3.6 Acidified ‘San Andreas’ 2 4.97 ± 0.03 5.26 ± 0.02 43.8 ± 2.1 Neutral ‘San Andreas’ 2 7.23 ± 0.02 7.05 ± 0.02 41.7 ± 6.6 Acidified ‘Albion’ 1 4.94 ± 0.03 5.11 ± 0.16 20.0 ± 5.8 Neutral ‘Albion’ 1 7.19 ± 0.02 7.05 ± 0.02 27.9 ± 4.6 Acidified ‘Albion’ 2 4.89 ± 0.03 5.08 ± 0.03 45.0 ± 4.4 Neutral ‘Albion’ 2 7.20 ± 0.03 6.95 ± 0.04 28.5 ± 5.7

1 Soil was acidified or not. 2 Replications of the experiment 3 pH of the soil prior to planting (initial) and at the end of the experiment (final)

To determine if there were significant effects of soil pH on damage caused by Fusarium wilt, plants of the cultivar ‘Albion’ were maintained in pH-adjusted soil for six weeks. Averaged across both experiments, pH was 5.15 ± 0.01 and 7.2 ± 0.02 for the acidified and non-acidified soils, respectively. Each time the experiment was conducted, symptoms were evident in both treatments by four weeks after planting, and by six weeks all plants were either dead or severely diseased. Pooling data across both experiments, average disease severity scores (on a 1 to 5 scale) were 4.2 ± 0.2 and 3.5 ± 0.3, for acidified and neutral soils, respectively. A t-test showed the effect of pH on disease severity not to be significant (P = 0.236).

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Colonization of Rotation Crops by F. o. fragariae No symptoms developed on any plants during the course of these experiments. However, for all crops, root infections were confirmed on a number of plants (Figure 1). For both strawberry and broccoli, 94% of plants had infected roots, whereas lettuce had the lowest frequency of infection (56%). These differences bordered on significance (P = 0.054). Colonization of the stem was confirmed for 44% of both lettuce and strawberry plants, and 22 and 27% of spinach and broccoli plants, respectively. These differences were also not significant (P = 0.4557).

Figure 1. The percentage of plants from which the Fusarium wilt pathogen was recovered for three crops that could be grown in rotation with strawberries.

25 2014 - 2015 RESEARCH PROJECTS Effect of Anaerobic Soil Disinfestation on Survival of the Fusarium wilt Pathogen in Soil Based on post-fumigation samples that were taken from 0 to 6 inches in the soil profile, the inoculum density of F.o.f. was highest in the ASD – molasses treatment (460 ± 251 CFUs per gram of soil) [= 13031 ± 7139 CFUs per ounce] and lowest in the ASD – rice bran treatment (180 ± 89 CFUs per gram of soil) [= 5059 ± 2521 CFUs per ounce]. The untreated plots were intermediate. Based on analysis of variance, the effect of treatment on inoculum density of the Fusarium wilt pathogen at this level in the soil was not significant (P = 0.3610). For soil at 6 – 12 inches below the soil surface, inoculum density was highest in the untreated plots (375 ± 217 CFUs per gram) [= 10623 ± 6147 CFUs per ounce] and lowest in the ASD – rice bran treatment (60 ± 20 CFUs per gram of soil) [=1700 ± 567 CFUs per ounce]. The ASD – molasses treatment was intermediate. Although the differences were substantial, they were not statistically significant (P = 0.2702). Effect of End-of-season Vapam Treatment on Survival of the Fusarium wilt Pathogen in Soil The Fusarium wilt pathogen was isolated from 100% of the crown segments recovered from soil in beds that were untreated. In treated beds, at a depth of 6 inches, the pathogen was isolated from 50% of the crowns both directly beneath a tape and between tapes. At 12 inches, the pathogen grew from 92% of the crowns placed between tapes but from none of the crowns directly under a tape. A high rate of survival in buried sand was apparent in untreated beds, regardless of location. Inoculum densities were greater than 13,000 CFUs per gram of sand [= 368,272 CFUs per ounce] in all cases. Survival was dramatically reduced at all locations in treated beds but the pathogen was not completely eliminated. Inoculum densities ranged from 1.3 to 6.0 CFUs per gram of sand [= 39 to 170 CFUs per ounce] recovered from treated beds. Susceptibility of Cover Crops to Macrophomina phaseolina After six weeks, the majority of the Macrophomina-inoculated strawberry plants exhibited foliar dieback and wilting (8 symptomatic plants out of 10 for isolate 08-12). None of the Macrophomina-inoculated or sterile sand-cornmeal inoculated cover crop species showed any foliar symptoms of disease. Roots of mustard, oat, rye, and vetch showed no discolored, necrotic, or symptomatic roots. For fava bean, however, eight of nine plants inoculated with isolate 08-12 exhibited externally dark, discolored roots. In contrast, control fava bean plants, along with all other control cover crops, had white, asymptomatic roots. For Macrophomina-inoculated mustard, oat, rye, and vetch, all roots appeared symptomless, so 30 pieces of white, roots were randomly selected and tested; all such root pieces were negative for M. phaseolina with the exception of one root piece from mustard. For the darkened roots of inoculated fava bean, all 30 pieces were negative for M. phaseolina. Evaluating Susceptibility of Strawberry Genotypes to Crown rot Caused by Macrophomina phaseolina Resistance scores (1 to 5 scale, with 1 being severely diseased and 5 being healthy) for the cultivars evaluated in 2014 ranged from a low of 1.95, for ‘Camarosa’, to a high of 4.78 for ‘Grenada’. High resistance scores (> 4.0) were also recorded for ‘Monterey’, ‘Fronteras’ and ‘Petaluma’, whereas all other cultivars received intermediate ratings. A number of cultivars have been tested in three successive years, and the rankings have been reasonably consistent (Figure 2).

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Figure 2. Disease resistance score on a 1 to 5 scale (with 1 corresponding to a plant killed by Macrophomina and 5 corresponding to a plant with no symptoms of disease) for seven UC cultivars

Discussion The results of our tests of UC cultivars for resistance to Fusarium wilt are consistent with results from previous years. This is apparent from the five-year averages and low standard errors shown in Table 1. ‘San Andreas’ and ‘Portola’ both appear highly resistant, which is confirmed by their performance in trials conducted in heavily infested fields. ‘Ventana’ has also performed well in field trials but our tests show a slightly lower resistance score and more variation over time. Whereas, ‘Ventana’ scored as low as 3.6 in one year (2012), ‘San Andreas’ has never had a score below 4.8 (Table 1). This differential may indicate that resistance in ‘Ventana’ is more sensitive to differences in environmental conditions, such as may occur over time. It is a goal of our program to understand the inheritance of resistance to Fusarium wilt so that all breeding lines can be elevated to the level now seen in ‘San Andreas’.

In 2014, Fusarium wilt of blackberry was recognized in California for the first time. Host range tests indicated that strawberries may show some susceptibility to this pathogen. To follow up on these findings, we conducted additional tests with two cultivars, and with a higher inoculum level than used in the original tests, which was 29.4 million spores per ounce. The results reinforce the impression that the blackberry Fusarium is capable of damaging strawberries. However, given the high inoculum level that was required to see an effect, this fungus seems likely to pose a limited risk to strawberries at the levels of exposure that would occur in most situations. To complete the picture, we are testing a broader range of UC germplasm, with an emphasis on cultivars that are resistant to F.o.f..

27 2014 - 2015 RESEARCH PROJECTS The occurrence of three genetically distinct strains of F.o.f. in California suggests that this pathogen has been introduced to the state more than once. As yet we do not know if these strains differ in virulence to strawberry. They have all been identified as F.o.f. based on their ability to cause disease on ‘Albion’ in controlled inoculations. This fall we will initiate a test designed to determine if these three strains interact differentially with strawberry genotypes that have thus far proven to be resistant to Fusarium wilt.

Wilt diseases result from systemic infections, in which the pathogen colonizes the water conducting tissue (xylem) and thereby restricts the flow of water to the shoot. This cannot occur until the pathogen has established residence within the root. A number of factors in the soil environment may influence the ability to infect strawberry roots, including pH. Grower and pest control advisor (PCA) observations in Ventura County suggested that Fusarium wilt was more severe in fields where irrigation water was acidified with sulfuric acid. To address this question, we conducted controlled environment studies that did not show any relationship between soil pH and disease severity. Since that time, a report from Australia indicated that Fusarium wilt of strawberry was less severe in soils near neutrality (pH 7.0) than in more acidic soils. Consequently, we conducted additional experiments to determine if soil pH was a factor that California growers could manipulate to reduce severity of disease caused by F.o.f.. For the resistant cultivar, ‘San Andreas’, our findings showed that soil pH does not significantly affect the frequency of root infections. For ‘Albion’, the frequency of infection was significantly higher at neutrality than in acidified soil, the reverse of what would be expected if acidification increased severity of disease. It should be noted that reports from Australia are based on very sandy soils and that improvement resulted from the addition of lime. In contrast, our work was with heavy clay soils, and contrasting pH values were obtained by lowering pH of the native soil with sulfuric acid. So, it may be that liming a sandy soil can provide a benefit to disease control, whereas acidifying a clay soil does not increase the risk.

That F.o.f. can colonize the roots of lettuce, broccoli and spinach is not surprising. Most Fusarium wilt pathogens colonize the roots of most crops. However, these colonies are usually limited to the root cortex and the amount of inoculum produced is not sufficient to substantially degrade the value of crop rotation. More unexpected is the frequency with which F.o.f. was recovered from the stems of all three crops. Our next step will be to quantify pathogen biomass within each crop to determine the extent to which each may support development of the pathogen and thereby compromise the benefits of crop rotation.

Anaerobic soil disinfestation did not eliminate F.o.f. in soil. In the upper soil profile, the ASD treatment that included molasses as a carbon source actually had higher levels of the pathogen than the untreated soil. This difference was not shown to be significant, due to the large variances in our estimates of pathogen population size in each treatment. Larges variances presumably reflect the non-uniform distribution of inoculum in the field. ASD appeared to be more effective deeper in the soil (below 6 inches), probably because conditions remained anaerobic longer than closer to the surface. One limitation on the efficacy of ASD is temperature. Control of Fusarium wilt may be difficult to achieve under cool conditions that prevail near the coast.

Application of Vapam at the end of the season was effective in reducing, though not completely eliminating inoculum present in sand. Pathogen survival in crowns was high, regardless of location in the bed. This finding underscores the importance of residue decomposition prior to the application of a fumigant. More complete break-down of infected tissue will enhance the efficacy of fumigation.

A third year of encouraging results in screening UC cultivars for resistance to Macrophomina crown rot augurs well for progress in elevating resistance to this disease. An important next step will be to evaluate the performance of cultivars that vary in susceptibility, under field conditions. In our test of cover crops,M. phaseolina failed to infect and cause disease on fava bean, mustard, oat, rye, and vetch.

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Other researchers have reported M. phaseolina as causing disease on fava bean, mustard, oat, and various Vicia species but not V. sativa (vetch). Although roots of inoculated fava bean developed dark, external discoloration, Macrophomina was not recovered from any symptomatic fava bean roots. These cover crops, therefore, do not appear to be hosts of M. phaseolina that would increase soil inoculum levels if grown in rotation with strawberry in California.

Selected references • Koike, S. T. and T. R. Gordon. 2015. Management of Fusarium wilt of strawberry. Crop Protection 73: 67-72.

• Koike S. T. and T. R. Gordonet al., 2012. Recent developments on strawberry plant collapse problems in California caused by Fusarium and Macrophomina. International J. Fruit Science.

29 2014 - 2015 RESEARCH PROJECTS 30 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Operating a State-wide Strawberry Disease Diagnostic Services Center

Principle Investigator Steven T. Koike Plant Pathology Farm Advisor University of California Cooperative Extension Monterey and Santa Cruz Counties 1432 Abbott Street Salinas, CA 93901 (831) 759-7350 [email protected]

Cooperating Investigators Mark Bolda UC Cooperative Extension, Santa Cruz County

Oleg Daugovish UC Cooperative Extension, Ventura County

Surendra Dura UC Cooperative Extension, Santa Barbara County

Tom Gordon Department of Plant Pathology, UC Davis

Frank Martin USDA-ARS, Salinas

Bill Wintermantel USDA-ARS, Salinas

Summary This strawberry diagnostic program is a service-oriented extension activity. With the support of the California Strawberry Commission and the strawberry industry, we operate a county-based diagnostic lab in Salinas that focuses on diagnosing problems for strawberry growers throughout the state of California. This diagnostic service is intended to confirm the presence of common, well known strawberry pathogens as well as to detect new diseases and problems as they might arise. We also continue to offer soil tests for Verticillium inoculum levels. Diagnostic services are available to all segments of the strawberry industry, ranging from large operations to limited resource growers, and includes transplant growers, production growers, pest control advisors, and other field professionals. Lab findings are communicated to growers and also to other strawberry researchers, increasing the opportunity for collaborative research. Our local presence on the coast also facilitates timely analyses for those who submit samples.

31 2014 - 2015 RESEARCH PROJECTS In 2014 through 2015, our lab was particularly occupied with confirming cases of Verticillium wilt, Macrophomina crown rot, and Fusarium wilt, three key soilborne diseases, for growers and pest control advisors throughout the state. All three pathogens are significant concerns for growers in all the coastal strawberry production regions from Ventura in the south through Santa Cruz in the north.

In collaboration with other extension advisors and field personnel, we also assisted in diagnosing the numerous cases of abiotic problems caused by high salts and other field conditions that resulted in poor establishment of transplants and damage to established plants.

Introduction The general goals of the project are the following: 1) General strawberry diagnostics Appropriate strawberry pathogen assays were standardized and implemented at the UC Cooperative Extension lab in Salinas, CA. Standard protocols were used for testing plants for Macrophomina, Fusarium, Phytophthora, Verticillium, Rhizoctonia, Colletotrichum, Cylindrocarpon, and Xanthomonas. Soil samples were tested for Verticillium propagules using a dry sieve method and NP-10 semi-selective medium. Nematode extractions are conducted if requested by the grower, though it is our current understanding that nematode-related problems do not yet occur on strawberry in California (one request was received in 2014-2015; results were negative for plant parasitic nematodes). Our diagnostic services also identified non-pathogen problems such as plant establishment problems due to stress and production issues, and fruit bronzing due to environmental factors (fruit bronzing complaints were few in 2014-2015).

2) Statewide diagnostic services To increase services to growers in various strawberry production areas in the state, we enlisted the involvement of Cooperative Extension personnel in various counties and researchers at UC Davis and the USDA-ARS in Salinas. Services were provided to strawberry growers, transplant producers, and pest control advisors from throughout the state. 3) Coordinate with other researchers working with strawberries To integrate this extension effort with the overall research program for strawberries, we communicated with various UC and USDA researchers so as to inform them about pertinent lab findings. We are actively collaborating with Tom Gordon (UC Davis) and Frank Martin (USDA) on the Macrophomina and Fusarium soilborne disease concerns. We consulted with Bill Wintermantel (USDA) regarding virus disease cases.

4) Focus on current soilborne issues Because of the development of Macrophomina and Fusarium soilborne problems, we particularly focused on strawberry dieback cases and advised growers on this issue. This aspect of the diagnostic program, along with confirmation of Verticillium wilt, comprised the majority of the samples received in 2014-2015

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Methods Submitted samples are always tested for general pathogens using non-selective, non-specialized media (acidified corn meal agar and other agars for fungi; sucrose peptone agar for foliar bacteria). As deemed appropriate, other selective, specialized media are added to the isolation protocols (PARP for Phytophthora and Pythium, NP-10 for Verticillium, FS for Fusarium, modified Mather’s for Colletotrichum). The dry sieve soil assay and NP-10 medium are used for Verticillium soil tests. We particularly focused on the developing Macrophomina and Fusarium problems on strawberry. Collapsed strawberry plants were always examined specifically for both of these soilborne pathogens as well as for Verticillium.

Results and Discussion We regularly tested for and detected both of the recently reported soilborne diseases caused by Macrophomina and Fusarium. Both pathogens continue to be significant concerns for growers in all of the coastal counties. Additional fields having Verticillium wilt cases occurred in 2014-2015, demonstrating that growers need to remain aware of this damaging pathogen. Because the dieback symptoms caused by Verticillium, Macrophomina, and Fusarium are very similar, laboratory testing will be required to identify which pathogen is involved.

Extension Diagnostic findings were communicated with growers and PCAs who submitted the samples. Disease developments were highlighted in our UC Cooperative Extension newsletter (Crop Notes) and also the extension blog sponsored by Farm Advisor Mark Bolda http://ucanr.org/blogs/strawberries_caneberries. Important findings from our diagnostic lab were also communicated to Spanish-only speakers by translating and posting the articles on the Spanish language blog: http://ucanr.org/blogs/fresamora/.

Disease management: Both Macrophomina crown rot and Fusarium wilt diseases cause significant plant decline, particularly when plants are bearing heavy fruit loads and when stress factors (elevated temperatures, especially) are present. Both pathogens are soilborne, so growers should beware of spreading the fungi to uninfested fields via mud and dirt on implements and tractors. Growers should minimize plant stress by irrigating appropriately and managing factors such as mites; however, infected plants likely will develop disease even in the absence of stress. Some strawberry cultivars are tolerant of one or the other of these pathogens and do not become as severely diseased compared to very susceptible cultivars.

Selected References

• Bolda, M. and S. T. Koike. 2011. Verticillium wilt showing up in Watsonville-Salinas strawberry fields. June 9. ANR blog: http://ucanr.org/blogs/strawberries_caneberries/. (Also in Spanish: Bolda, M. and S. T. Koike. Aparece marchitez de Verticillium en campos de fresa en Watsonville-Salinas. July 8)

• Bolda, M. and S. T. Koike. 2015. Powdery mildew of strawberry. Production Guideline Issue 12. September. California Strawberry Commission. Also published in Spanish: Mildiu polvoroso en la fresa (Publicacion 12. Septiembre. CSC).

• Koike, S. T. 2012. Soilborne diseases of strawberry: a review. Monterey County Crop Notes. March/April.

33 2014 - 2015 RESEARCH PROJECTS • Koike, S. T., and Ajwa, H. 2012. Soilborne pathogens a concern for strawberry growers. Vegetables West 16:12-13. June issue.

• Koike, S. T. and T. R. Gordon. 2015. Management of Fusarium wilt of strawberry. Special issue: Management of Fusarium diseases. Crop Protection 73:67-72.

• Koike, S. T., Gordon, T., R. and G. Browne. 2013. UC IPM Pest Management Guidelines: Strawberry. Disease section. UC ANR Publication 3468. Website: http://www.ipm.ucdavis.edu/PMG/selectnewpest.strawberry.html.

• Koike, S. T., Gordon, T. R., Daugovish, O. and H. Ajwa. 2013. Fusarium wilt of strawberry. Production Guideline Issue 11. December. California Strawberry Commission. Also published in Spanish: Marchites por Fusarium de la fresa (Publicacion 11. Diciembre. CSC).

• Koike, S. T., Gordon, T. R., Daugovish, O., Ajwa, H., Bolda, M. and K. Subbarao. 2012. Recent developments on strawberry plant collapse problems in California caused by Fusarium and Macrophomina. International Journal of Fruit Science 13:76-83.

• Koike, S. T., Gordon, T. R., Daugovish, O., Ajwa, H. and F. Martin, F. 2013. Charcoal rot of strawberry. Production Guideline Issue 10. November. California Strawberry Commission. Also published in Spanish: Pudricion carbonosa en la fresa (Publicacion 10. Noviembre. CSC).

34 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Acknowledgments The California Strawberry Commission and the strawberry industry in California have been consistently supportive of this program. We acknowledge the California Strawberry Commission for making this program possible. We thank the growers and pest control advisors who have participated in this service and submitted samples. We thank the following persons for their assistance: Patty Ayala, Mark Bolda, Oleg Daugovish, Tom Gordon, Keita Goto, Sofia Hernandez, Joy Jacobs, Kat Kammeijer, Dan Legard, Frank Martin, Krishna Subbarao, Bill Wintermantel, Alexandra Wollenman. We thank Eric Lauritzen (Monterey County Agricultural Commissioner) for his assistance with the greenhouse and laboratory facilities at UC Cooperative Extension—Monterey County.

35 2014 - 2015 RESEARCH PROJECTS 36 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Evaluation of the Population Structure of Macrophomina phaseolina and Optimization of Quantification Assays

Principle Investigator Dr. Frank N. Martin Research Plant Pathologist USDA-ARS 1636 E. Alisal Street Salinas, CA 93905 (831) 755-2873 [email protected]

Collaborators Steve Koike UC Cooperative Extension 1432 Abbott Street Salinas, CA 93901

Dr. Marina Ramon USDA-ARS 1636 E. Alisal Street Salinas, CA 93905

Dr. Renee Arias USDA-ARS National Peanut Research Laboratory 1011 Forrester Drive, SE P.O. Box 509 Dawson, GA 39842-0509

Summary With the transition from broadcast preplant fumigation to individual bed treatment using methyl bromide + chloropicrin alternative fumigants, Macrophomina phaseolina has become an emerging disease problem in California strawberry production, causing serious losses in all production districts. Population analysis using simple sequence repeats (SSR) markers of 266 isolates recovered from different hosts in California combined with results previously reported on east coast isolates recovered from a variety of hosts identified a single clade containing 98% of the strawberry isolates. Four strawberry isolates didn’t group with the rest of the isolates from this host, three of these represent new genotypes recovered from Ventura County in the 2014 season. Five isolates from cantaloupe, almond and pistachio from California and sunflower from Tennessee also grouped within the main strawberry clade. Genotypic analysis of 62 isolates recovered from adjacent fields in Santa Maria revealed 60 of the isolates were clonal. A similar analysis of 28 isolates from the same field in Salinas revealed more diversity, with two main groupings and 3 singletons that were similar to one of the groupings. Pathogenicity tests with a subset of isolates recovered from strawberry were virulent on this host but did not cause disease on the other plant species tested; however, the strawberry isolate collected in 2011 that did not group with the rest of the isolates from this host exhibited low virulence on strawberry.

37 2014 - 2015 RESEARCH PROJECTS Additional pathogenicity tests are in progress to better evaluate if there is a correlation between genotype and host range. In an effort to identify sequences unique to the main genotype of isolates that attack strawberry that will be used in developing a specific real time PCR assay the genomes of several California isolates ofM. phaseolina were sequenced and assembled contigs. Comparative analyses are in progress to identify the best regions to use.

Introduction Beginning at least as early as 2005 and continuing through 2014, the industry has seen the significant increase of charcoal rot caused by M. phaseolina. The problem was initially confined to two counties, Orange and Ventura. However, by 2014 the pathogen had been confirmed by researchers in all the major strawberry producing regions as well as in other parts of the state that have smaller acreage. Confirmed counties are the following: San Diego, Orange, Ventura, Santa Barbara, San Luis Obispo, Monterey, Santa Cruz, San Benito, Santa Clara, Alameda, Sacramento, and Fresno. In addition to the increasing list of counties reported to have diseased strawberries, for each county the number of infected fields likewise increases each season.

Symptoms consist of wilting foliage, plant stunting, and drying and death of older leaves, though the central youngest leaves often remain green and alive. Plants can eventually collapse and die. When plant crowns are cut open, internal vascular and cortex tissues are dark to orange brown. In locations where the disease has occurred for more than one season, the patches can be quite large and appear to have spread from the initial problem area. In Ventura, Santa Barbara and Monterey counties M. phaseolina and Fusarium oxysporum together cause significant plant decline. It is noteworthy that in these cases we have never isolated other important, well known pathogens such as Colletotrichum, Phytophthora, or Verticillium.

In California, there is a strong correlation between fields most seriously affected by these plant collapse problems and the use of pre-plant, bed-applied alternatives to standard methyl bromide + chloropicrin applications. Because of the political/regulatory trend away from available, effective fumigants that are used by the strawberry industry, this disease situation is a critical threat to the long term health of the strawberry industry. The nine-year spread of Macrophomina to previously uninfested parts of California indicates that charcoal rot is currently the number one threat to the industry which at present does not have satisfactory plant resistance with which to combat the pathogen.

Macrophomina phaseolina has a broad host range encompassing over 100 plant families and 500 species on a worldwide basis. The pathogen forms resting structures called microsclerotia are capable of surviving in the soil for long periods of time, hence the need for effective management strategies for preventing pathogen establishment and disease outbreaks. There are reports in the literature of some level of host specificity of field isolates and of grouping of isolates from particular hosts or geographic regions based on molecular criteria (reviewed below). With this in mind, it would be important to know more about the population structure of the isolates in California causing disease problems on strawberry. This would clarify if specific genotypes of the pathogen are responsible for the current disease problem. This information in turn would be useful for developing soil assays for the pathogen that would focus only on the genotypes virulent on strawberry as well as provide important background information on strains of the pathogen that should be used in strawberry resistance screening programs.

38 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Backround on the Pathogen Host specificity and population structure of M. phaseolina

There have been a number of reports in the literature suggesting host specialization of isolates of the pathogen. For example, Pearson et al., (1987) observed that isolates recovered from soil that corn was growing in exhibited different growth morphologies when grown on medium amended with chlorate, yet isolates recovered from corn roots recovered from the same soil exhibited a single morphology type; there was no differential colonization of isolates on soybean roots in the same soil. When looking at fields that were monocropped to corn, cotton, sorghum and soybean for 15 years, Su et al., (2001) reported some level of host specialization for isolates recovered from corn and that all isolates grouped according to host when DNA fingerprint analysis was done using random amplified polymorphic DNA (RAPD) analysis. Interestingly, isolates exhibited variation in their growth morphology on chlorate medium that reflected differential grouping of isolates in RAPD analysis. Zveibil et al., (2012) recently reported that isolates of M. phaseolina from other hosts in Israel were equally virulent on strawberry.

A number of other studies examining population structures of isolates using various techniques have been reported. Similar groupings of isolates from different hosts, and in some cases geographic origin, have also been reported in other studies using RAPD primers (reviewed in Das et al., 2008), amplified fragment length polymorphism analysis (AFLP; reviewed in Reyes-Franco et al., 2006), simple sequence repeat analysis (SSR; Jana et al., 2005b) and primers developed from repetitive sequences of rice (Jana et al., 2005a). However, other studies did not find a consistent correlation between molecular grouping and location of isolate recovery or virulence on a particular host (Reyes-Franco et al., 2006). Two additional SSR studies reporting on new primer sets have recently been published and in both cases the grouping of genotypes was correlated with the host of recovery. Baird et al., (2010) used 12 SSR loci to evaluate 109 isolates of the pathogen recovered from different geographic regions and hosts and observed that some genotype clustering by host and geographic region was observed. However, strawberry isolates that were included in the analysis (1 from CA and 3 from FL) were identical and were located on a branch separate from most isolates from other hosts. Arias et al., (2011) reported on new primer pairs for 182 SSR loci and examined 24 isolates recovered from a range of hosts and observed grouping of genotypes for isolates recovered from the same host (isolates from snap bean, pumpkin, sunflower and corn each had their own clade while isolates from soybean, cotton, and sorghum grouped together in a larger clade).

Objectives The specific objectives of this project were:

• Develop an agar plate detection method by evaluating differential media for M. phaseolina in soil.

• Evaluate the population structure of M. phaseolina in strawberry production areas of California to see if there is any genetic grouping of isolates that attack or are more virulent on strawberry.

• Evaluate the real time PCR molecular diagnostic technique reported by Babu et al., (2011) for detection of M. phaseolina on strawberry. The technique will be modified or redesigned as necessary. The focus will be on detection from plants as well as soil quantification.

39 2014 - 2015 RESEARCH PROJECTS Materials and Methods Soil plating assays

Given the importance of M. phaseolina to the strawberry industry, research is needed to develop soil assays designed to detect and quantify this fungus. Research will first be conducted to compare several agar plate methods (Mihal, 1992) for relative sensitivity and recoverability of M. phaseolina. This approach will mirror the current situation that exists for detection of Verticillium in soil; we have an agar plate method (NP-10 medium) that is routinely used by UC Cooperative Extension to quantify V. dahliae from field soils, as well as a molecular technique (Bilodeau et al., 2012) that has been developed in the Martin lab and could be used commercially for soil tests.

Culture collection

Through the CSC-supported diagnostic lab program (operated by UC Cooperative Extension in Monterey County), growers from Ventura, Santa Barbara, Monterey, Santa Clara, and Santa Cruz regularly submitted diseased strawberry samples for analysis. These cultures are forwarded to the Martin lab for storage and genotyping. The Koike lab is also conducting pathogenicity tests on strawberry and other reported hosts of M. phaseolina with these isolates.

Isolate genotyping

Simple Sequence Repeats (SSRs or Microsatellites) analysis as described by Arias et al., (2011) will be used to genotype isolates in this project. A collaboration has been established with Renee Arias to use the same 182 SSR loci she used in her previous analysis of the pathogen. Also, the SSR data for strawberry isolates that have been collected thus far were generated on the same genotyping unit that she used to collect her data (Arias et al., 2011), so the datasets will be able to be combined for a larger scale analysis comparing the populations from strawberry to isolates collected from other hosts.

Molecular detection of Macrophomina phaseolina

The real time PCR molecular diagnostic technique reported by Babu et al., (2011) for detection of M. phaseolina will be evaluated for pathogen detection on strawberry. The technique will be modified or redesigned as necessary. The focus will be on detection from plants as well as soil quantification.

Results Develop an agar plate detection method by evaluating differential media for M. phaseolina in soil.

Experiments were conducted in the attempt to further develop soil assays for detecting M. phaseolina. Using the published techniques of Mihal et al., (1992); after repeated attempts with variation in the procedures, none of them worked with the strawberry isolates. Additional trials were conducted with modification of a technique that is used in the Midwest for quantification of M. phaseolina in soybean production areas; this assay did not work when plating soil from infested strawberry fields. Colonies of M. phaseolina were occasionally observed when soil was processed for V. dahliae soil assays (NP-10 medium), but the numbers were low and inconsistent.

Establish a culture collection of M. phaseolina representing production areas in California

In 2012 through 2015, purified isolates from these samples were sent to the Martin lab for storage and possible inclusion in the genetic grouping analysis. Isolates from other hosts have also been collected and added to the culture collection to facilitate evaluation for host specificity. Based on collections through the 2014 production season there were

40 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

over 247 isolates recovered from California in the culture collection (188 from strawberry). Two fields, one in Salinas and the other in Santa Maria, were extensively sampled to evaluate if there was variation in pathogen genotype within the field (28 and 62 isolates each, respectively).

Simple Sequence Repeats (SSRs or Microsatellites)

To identify the best SSR markers to genotype the isolates recovered from strawberry, in collaboration with the Arias lab, several isolates were genotyped with all 182 markers previously described (Arias et al., 2011). A total of 65 possible markers were selected and used to examine 15 isolates from California, 24 subsequent markers were selected to use to genotype the culture collection (some of these markers were included to allow the addition of data that our collaborator had collected on isolates from a broader selection of hosts in the analysis).

Simple Sequence Repeats (SSRs or Microsatellites)

SSR analysis has been completed on 247 isolates with 24 loci (Figure 1) with the results indicating:

• 98% of the isolates recovered from strawberry are placed on the main strawberry clade. For the four isolates from this host that were not in this clade:

• One isolate was from Sacramento (collected in 2011) and is less aggressive in pathogenicity tests on strawberry than isolates from the main strawberry clade.

• Three isolates recovered from Ventura in 2014, pathogenicity tests with these isolates are in progress.

• Multiple isolates from a single field were examined: • Salinas – 28 isolates represented five closely related genotypes (three were represented by single isolates). • Santa Maria - two adjacent field separated by a road were sampled and 60 out of 62 isolates had an identi- cal genotype.

New observations for 2014 isolates

• Three isolates from Ventura were not placed in the main strawberry clade • One isolate was on a clade basal to the main strawberry clade grouping with isolates recovered from a range of hosts. • Two isolates had a nearly identical genotype and were placed on the same primary clade as the 2011 isolate from Sacramento. • Almond and pistachio isolates from California and sunflower fromTennessee were placed in the main strawberry clade: • An isolate collected from cantaloupe in Los Baños in 2011 previously was the only non-strawberry isolate to group within the main strawberry clade. • Pathogenicity tests with these isolates on strawberry are in progress.

Pathogenicity tests

Steve Koike has been conducting pathogenicity tests (funded from CSC (Gordon) and other sources) using two different techniques. For the rapid screen, plants are inoculated by inserting a toothpick colonized by the pathogen into the crown and incubating for several weeks. Using this approach strawberry isolates from the main clade were highly aggressive on strawberry, whereas the strawberry isolate from Sacramento that grouped with isolates from other hosts caused little disease on strawberry. When tested on other hosts (snap bean, corn, soybean, sunflower and cantaloupe)

41 2014 - 2015 RESEARCH PROJECTS by a toothpick/stem inoculation, the strawberry isolates caused limited disease on cantaloupe but none of the other plants. When tests were done with plants grown in infested soil, no disease was observed except for strawberry plants. Additional tests evaluating the pathogenicity of non-strawberry isolates on strawberry are in progress, but preliminary results suggest most isolates from other hosts are either nonpathogenic or are much less aggressive than isolates in the main strawberry clade.

Molecular detection of Macrophomina phaseolina

It is important to make sure we know what pathogen genotypes are important for causing disease in California production fields so any soil quantification assays that are developed accurately reflect the risk of disease. Additional pathogenicity tests evaluating isolates recovered from other hosts on strawberry are in progress but results obtained thus far suggest the focus should be on detection of the clade that contains 98% of the strawberry isolates genotyped to date. In an effort to identify unique sequences in these isolates that can be used for designing clade specific markers, genomic DNA from several strawberry isolates and one alfalfa isolate were sequenced by Illumina and the nuclear genomes assembled (the strawberry isolate is assembled into 2,452 contigs with 50x depth of coverage and totaling 50 MB in size; additional sequencing is in progress to improve the assembly by reducing the final number of contigs), comparative genomics has been used to identify unique regions in the strawberry isolates that are highly conserved. Once the pathogenicity tests have been completed and if the results confirm only isolates from the primary strawberry clade are important, clade specific markers will be designed from these regions.

Discussion Soil dilution plating on selective media using published techniques has not been successful for enumeration of M. phaseolina from California strawberry soils. Kelly Ivors (Cal Poly, San Luis Obispo) recently forwarded a technique her lab was using that we are also finding to work well for recovery of M. phaseolina from strawberry fields.

The results of the genotype analysis indicate a high level of grouping of isolates recovered from strawberry with limited variation observed. Work is also in progress with the Koike lab to determine if there is a correlation between genotype and host range. The results obtained thus far indicate that strawberry isolates have a host preference for strawberry but additional tests evaluating the pathogenicity of isolates recovered from other hosts on strawberry are needed before firm conclusions can be drawn. Trials are also in progress to evaluate virulence differences among isolates (some isolates have been recovered from strawberry that are not in the main strawberry clade and appear to be less aggressive on strawberry, this difference in aggressiveness on the host needs to be quantified). Isolates collected in 2015 from California and DNA provided by collaborators in Florida and Israel from isolates recovered there are in the process of being genotyped by SSR analysis and will be added to the current analysis (an additional 96 isolates).

42 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Pathology

Figure 1. SSR analysis of broader collection of California isolates of M. phaseolina

Selected References

• Arias, R.S. Ray, J.D., Mengistu, A. and B. E. Scheffler. (2011) Discriminating microsatellites from Macrophomina phaseolina and their potential association to biological functions. Plant Pathology 60:709-718.

• Babu, B.K., Mesapogu, S., Sharma, A., Somasani, S.R. and D. K. Arora. (2011) Quantitative real-time PCR assay for rapid detection of plant and human pathogenic Macrophomina phaseolina from field and environmental sam- ples. Mycologia 103:466-473.

• Babu, B.K., Saxena, A.K., Srivastava, A.K. and D. K. Arora. (2007) Identification and detection of Macrophomina phaseolina by using species-specific oligonucleotide primers and probe. Mycologia 99:797-803.

• Baird, R.E. Wadl, P.A., Allen, T., McNeill, D., Wang, X. Moulton, J.K., Rinehart, T.A., Abbas, H.K., Shier, T. and R. N. Trigiano. (2010) Variability of United States Isolates of Macrophomina phaseolina based on simple sequence repeats and cross genus transferability to related genera within Botryosphaeriaceae. Mycopathologia 170:169- 180. Bilodeau, G.J., Koike, S.T., Uribe, P. and F. N. Martin. (2012). Development of an assay for rapid detection and quantification of Verticillium dahliae in soil. Phytopathology 102:331-343

• Das, I.K., Fakrudin, B. and D. K. Arora. (2008) RAPD cluster analysis and chlorate sensitivity of some Indian isolates of Macrophomina phaseolina from sorghum and their relationship with pathogenicity. Microbiol. Res. 163:215-224.

• Jana, T.K., Singh, N.K., Koundal, K.R. and T. R. Sharma. (2005a). Genetic differentiation of charcoal rot pathogen, Macrophomina phaseolina, into specific groups using URP-PCR. Can. J. Microbiol. 51:159-164

43 2014 - 2015 RESEARCH PROJECTS • Jana, T.K., Sharma, T.R. and N. K. Singh. (2005b). SSR-based detection of genetic variability in the charcoal rot pathogen Macrophomina phaseolina. Mycol. Res. 109:81-86.

• Koike, S. T., Gordon, T. R., Daugovish, O., Ajwa, H., Bolda, M. and K. Subbarao. (2012) Recent developments on strawberry plant collapse problems in California caused by Fusarium and Macrophomina. International Journal of Fruit Science 13:76-83.

• Mihal, J (1992) Macrophomina. Pages 134-136. In: Methods for Research on Soil Borne Phytopathogenic Fungi. L. Singleton et al., eds. American Phytopathological Society St. Paul, MN. 265 pp.

• Pearson, C.A.S., Leslie, J.F. and F. W. Schwenk. (1987) Host preference correlated with chlorate resistance in Macrophomina phaseolina. Plant Dis. 71:828-831.

• Purkayastha, S., Kaur, B., Dilbaghi, N. and A. Chaudhury. (2006) Characterization of Macrophomina phaseolina, the charcoal rot pathogen of cluster bean, using conventional techniques and PCR based molecular markers. Plant Pathology 55:106-116

• Reyes-Franco, M.C., Hernandez-Delgado, S., Beas-Fernandez, R., Medina-Fernandez, M. Simpson, J. and N. Mayek-Perez. (2006) Pathogenic and genetic variability within Macrophomina phaseolina from Mexico and other countries. J. Phytopathology 154:447-453.

• Su, G., Suh, S.-O., Schneider, R.W. and J. S. Russin. (2001) Host specialization in the charcoal rot fungus, Mac- rophomina phaseolina. Phytopathology 91:120-126.

• Suga, H., Hirayama, Y., Morishima, M., Suzuki, T., Kageyama, K. and M. Hyakumachi. (2013). Development of PCR primers to identify Fusarium oxysporum f. sp. fragariae. Plant Dis. 97:619-625.

• Zveibil, A., Mor, N., Gnayem, N. and S. Freeman. 2012. Survival, host–pathogen interaction, and management of Macrophomina phaseolina on strawberry in Israel. Plant Dis. 96:265-272.

44 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT ENTOMOLOGY

45 2014 - 2015 RESEARCH PROJECTS 46 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

Strawberry Insect and Mite Control

Principle Investigator Dr. Frank G. Zalom Dept. of Entomology One Shields Avenue UC Davis Davis, CA 95616 (530) 752-3687 [email protected]

Summary Arthropod pests cause significant problems for California strawberry growers, and I have worked with the strawberry industry for over 25 years on a variety of different pest problems. This is my final report for the California Strawberry Commission on my continuing extension/general support project, so I am taking this opportunity to thank the growers and commission for their support over the years and providing a complete bibliography and brief summary of some of the more useful outcomes of the applied research and extension activities conducted by my lab related to those documents. I am also preparing a special webpage on my laboratory website that will include selected presentations that I have made on various strawberry-related topics over the years that present useful information on various insect and mite pests and results of our studies in addressing their management.

Introduction I have been conducting a comprehensive applied research and extension program to address insect and mite problems of California’s strawberry growers for over 25 years. The results of these studies have been published in over 40 journal articles and book chapters and almost 100 extension-type publications to date, most of which were produced and distributed by the California Strawberry Commission and UC Cooperative Extension. Results were also presented directly to growers and others involved in the strawberry industry at almost 150 meetings held by UC or the commission in every production area. During this time, my lab has almost continuously addressed what I consider to be the two key arthropod pests of California strawberries, Lygus bug (primarily Lygus hesperus) and two-spotted spider mite (TSSM), Tetranychus urticae, which are responsible for the greatest crop losses in most years. My first published work on Lygus was in1990, with former UC Cooperative Extension Farm Advisors Carolyn Pickel and Norm Welch (1), and my first publication on TSSM

47 2014 - 2015 RESEARCH PROJECTS was in 1993 with Doug Walsh, Norm Welch and Doug Shaw (E2). A number of other problems arose over the years that I addressed as reqested by strawberry growers including thrips (primarily western flower thrips,Frankliniella occidentalis), whiteflies (primarily greenhouse whitefly, Trialeurodes vaporariorum), several Lepidoptera (moth) pests including corn earworm (Helicopverpa zea), beet armyworm (Spodoptera exigua) and light brown apple moth (Epiphyas postvittana), vinegar flies (Drosophila species including spotted wing drosophila, Drosophila suzukii), and other Tetranychid spider mites including carmine mite (Tetranychus cinnabarinus), Lewis mite (Eotetranychus lewisi), persea mite (Oligonychus perseae) as well as a Tarsonemid, the cyclamen mite (Phytonemus pallidus). Interestingly, while some of these proved to be ‘one time’ problems, several recur periodically and often for the same reasons that they became outbreak pests originally. Because this will be my final report to the Commission, I intend to summarize the work conducted by my lab with reference to documents that can be accessed for further information and by making presentations on these pest problems available on my lab’s website. I hope that this report, the bibliography of publications that have come from this project, and the presentations will serve as useful sources of information on these pests for growers as well as a baseline of knowledge for future California strawberry research.

Objectives The specific objectives of my proposal were:

1. Summarize and extend information on Lygus, Lepidoptera larvae, mites, drosophila and whiteflies.

2. Develop dose response data for populations of two-spotted spider mites from different strawberry growing areas for their susceptibility to Agri-Mek and Acramite, and determine mechanisms of resistance development.

3. Determine efficacy of new products for control of two-spotted spider mite.

Methods This report focuses and expands on the first objective by summarizing information on strawberry pest problems that I have worked on, and referencing the summary to a comprehensive rpublication list and a new website of presentations of many presentations that have made that provide more detailed results. Results of the other two objectives involving TSSM are incorporated into the summary.

Lygus. That Lygus cause economic damage to strawberries at very low densities was first established by Dr. Bill Allen. Field cage studies we conducted during the late 1980s using different Lygus densities affirmed his observations (1). Sampling methods including beating and vacuuming were compared to actual numbers of Lygus per plant enabling action thresholds and monitoring guidelines to be developed (1, 2). Lygus resistance to organophosphate insecticides was becoming a problem in the mid-1980s, and finding new controls became an ongoing goal for my lab over the next 25 years. Seasonal surveys of alternate Lygus hosts was one approach we pursued at that time to determine external sources of Lygus invasion (E8), and how to use that knowledge for more effective management. Other non-chemical approaches we took were to evaluate effectiveness of insect vacuum machines that some growers had developed at that time were compared to malathion and unregistered insecticides (4, 5, E!), and somewhat later we evaluated the potential for introducing parasitoids as Lygus biocontrol agents (E54). Registering new pesticides on strawberries is a challenge. My lab conducted Lygus insecticide efficacy field studies annually until 2012. All of these studies produced replicated data that included damage estimates for products both with and in the absence of treatment that were provided to registrants and the Commission, and through them to regulators to help encourage and facilitate emergency and full registrations (see 35 and Commission annual reports for methods).

48 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

We also measured resistance to organophosphate, carbamate and pyrethroid insecticides in Lygus populations from strawberries using plastic baggie (E27) and tube bioassays (35) both to alert growers of susceptibility in different regions as well as to assist in justifying emergency registrations.

Two-spotted spider mite and other mites. TSSM is a key pest of strawberries in all production regions, and its management requires some type of control intervention virtually every year (E65). My lab has conducted biology and control studies for TSSM in every year to present. We worked collaboratively with Drs. Doug Shaw and Kirk Larson for over 20 years to screen UC advanced selections measuring mite densities and yield in treated and untreated plots on the UC Davis field station at Watsonville (see methods in 16). We conducted field studies to measure factors such as seasonal effects of feeding by TSSM by manipulating mite densities at different periods of the growing season (8, E12), influence of plant vigor on mite densities and yield as influenced by pre-transplant nursery chilling (7) and fumigation (E16), and other stressors such as temperature (E24). We also studied effects of miticides on predatory mites to determine their compatibility with biocontrol releases in both field and detailed laboratory studies. When I began to work on strawberries, the primary miticides available were Omite, Plictran, Vendex and Kelthane, but it was obvious that harvest restrictions on all of these would eventually preclude their use on strawberries. At that point I began to evaluate miticides for potential registration in replicated field plot studies and with lab bioassays, and made those data available to registrants. Efficacy tests using methods similar to those for TSSM were also conducted against carmine mite, Lewis mite and cyclamen mite when these occasional mite pests became problematic and sites were available for conducting replicated and controlled studies. My lab also completed laboratory research on development of Lewis mite and TSSM under different temperature regimes and differential effects of predation on both species.

Thrips. Thrips have primarily been of concern to strawberry growers because of fruit bronzing that has been attributed to their feeding. Starting in the late 1990s, I conducted several collaborative studies with Drs. Steve Koike and Kirk Larson that identified three types of bronzing, only one of which is caused by thrips feeding. The studies involving comparisons of plots grown with different mulches, overhead sprinkling and deficit irrigation to alter temperature, and applications of lignins as a sunscreen was used to identify potential causes of type II bronzing which was of most concern to growers and seemed to be associated with hot days with high UV penetration. Methods for these studies are described in papers 18 and 33. During the same period, my lab compared different approaches to sampling thrips in strawberries and we sampled fields with thrips bronzing to establish damage thresholds. We also conducted replicated insecticide field trials to facilitate registration of an effective product for thrips control (E47), and determined ability of the predatory mite Amblyseius swirskii to control thrips by making releases in plots within commercial fields and applying acaricdes between the plots to prevent movement of the predators and monitoring thrips abundance (E72).

Whiteflies. Strawberry was not a recorded host for greenhouse whitefly before their occurrence was noted in the late 1990s first in the Oxnard area and shortly later in the Watsonville/ Salinas area. High populations were observed causing extreme damage in both areas, and whitefly-borne viruses were identified in the Watsonville area. Dr. Nick Toscano and I began sampling whitefly populations in both areas to determine seasonal hosts of the whiteflies. We also did insecticide efficacy studies to identify products that could be registered for their control. Since the whiteflies were occurring across fields and on multiple crops, an areawide approach was necessary that involved considerable grower education and collaboration (E32, E39, E41, E50, E61).

Lepidoptera. Native Lepidoptera larvae including corn earworm, beet armyworm and several cutworm species can damage strawberry fruit and young transplants. Because they are often controlled by insecticides applied for other pests in most growing areas, they are not considered significant problems by most growers. However, southern California growers who do not need to treat annually for key pests such as Lygus do experience losses from these insects, so most of my work has been limited to studies of newer, less toxic pesticides for their control in southern California field studies

49 2014 - 2015 RESEARCH PROJECTS and the timing of those treatments. In most cases, I have done these studies using artificial infestations of strawberry plots with eggs or newly hatched larvae to insure data uniformity. The light brown apple moth (LBAM) was introduced into the Monterey Bay area in 2007, and has become a regulatory issue because of export restrictions on fruit coming from quarantine zones. I identified this insect to the commission as a potential pest before it was first detected in Santa Cruz County, and most of my work on LBAM in strawberries has been of an educational nature. I am proud to have worked with the commission to produce the first publication on identification of LBAM that was published soon after the first moth was detected in the Watsonville area (E64, E66).

Vinegar flies. Most vinegar flies (Drosophila species) are primarily a problem in processed fruit because they infest over-ripe and decaying fruit. An outbreak resulting in economic losses occurred in the Oxnard area in 1995 (E9) that Dr. Toscano and I responded to by identifying species present, comparing potential sampling methods, and evaluating efficacy of cultural controls and registered insecticides. In 2008,Drosophila suzukii, the spotted wing drosophila, was first found infesting commercially viable ripe strawberries in Watsonville by Mark Bolda (37). Since then, my lab, particularly Dr. Kelly Hamby, has worked with Mr. Bolda and others to study its seasonal biology, compare sampling methods (41,43), and identify insecticides for its control. My lab has been collaborating with UC Davis colleague Dr. Joanna Chiu to study the genomics of D. suzukii (44) that presents a tremendous opportunity to apply extensive molecular research already conducted on the laboratory model species Drosophila melanogaster to this pest species and other pest vinegar flies as well.

Relevant Powerpoint presentations that I have made on many of these insect and mite pests are accessible on my lab website at: http://ucanr.edu/sites/zalomlab/Strawberries/.

Results and Discussion

Lygus. Although several Lygus species may be found on California strawberries, molecular analysis of field collections confirmed that the dominant species is Lygus hesperus (39). Field cage studies we conducted during the late 1980s determined damage relationships for both Lygus nymphs and adults per plant (1). Monitoring guidelines and action thresholds based on these per plant damage relationships and data we obtained on per plant estimates of Lygus densities using beating hoops or hand-held vacuums (1, 2) provided the sampling recommendations still used in the UCIPM Guidelines (E99). Our survey of seasonal hosts for Lygus conducted during the early 1990s (E8) resulted in the UC recommendation of monitoring Lygus hatch on weed hosts and destroying them before adults emerge and migrate to strawberries to help reduce catfacing (E5, E69). We updated our understanding of Lygus movement in the landscape surrounding new strawberry plantings on several occasions over the years as production practices changed. This was particularly useful in establishing the role of second year strawberries (E91) and planted trap crops such as alfalfa as Lygus sources for new plantings if not appropriately managed (E80). Insect vacuum machines developed as an alternative control method by growers who were experiencing losses due to organophosphate-resistant Lygus resistance in the late1980s were documented to provide similar or better control than did malathion, but were much less effective than the pyrethroid insecticides that were not yet registered. Lygus parasitoids provided up to 30 percent control of Lygus populations in cage studies and became established in the field, but they did not provide economic control(E54) . We also found that full bed reflective mulch reduced Lygus damage to strawberries, but these data are only available in my oral presentations. An effective insecticide will provide the least damage, most consistent results, and most cost-effective option for Lygus control on strawberries, however registering a new insecticide on strawberries takes years and requires considerable supporting data. For example, it took eight years from when we began working with pyrethroid insecticides to when Danitol (fenpropathrin) became registered on strawberries in 1996 (E17). At that time, use of pyrethroid insecticides reduced damage by organophosphate resistant Lygus by over 90%, and growers came to reply on them. We were concerned by potential

50 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

development of resistance and began to monitor resistance levels (E26, E27, E30, E42). Resistance to pyrethroids was not detected until the mid-2000s. By that time several newer insecticide classes had become available that had at least some effect on Lygus when used alone or in combination with an insecticide from another class. None however was as effective against all life stages as were the pyrethroids in our studies (see commission annual reports since 2005-06). Rimon (novaluron) (E45), the first of these newer products was finally registered on strawberries in 2011 (E92) and has been followed by a few others including Beleaf (E95). It will be important moving forward to time what insecticides are available against the most susceptible life stages and combine these treatments with other controls such as vacuums and timely removal of alternate hosts to reduce overall Lygus damage. It will also be critical to work with agrochemical companies to be aware of new products for Lygus control and help them to generate data from strawberries to facilitate their registration, a process that can take eight or more years.

Two-spotted spider mite. Results of my collaborative work on varietal susceptibility to TSSM indicated that there is little difference in genotypes to different mite levels in the UC breeding lines (16), but that there is a distinct difference between day-neutral and short-day varieties (13). Over the 20 years that we conducted this work, yield results were summarized annually and used by UC breeders as one criterion for advanced selections. Some of these results are presented in annual commission reports and presentations on my lab website. It became apparent that tolerance for TSSM feeding had improved as newer varieties replaced ‘Selva’ which was the standard when our studies began when began the work, and this was confirmed when on occasion ‘Selva’ was included in our annual varietal evaluations. Results from these 20+ years were never compiled in their entirety and are ripe for meta-analysis. Many of our studies of TSSM and strawberry yield have led to now standard recommendations in UC management guidelines (E66, E99). For example, we documented that increased plant vigor helps strawberries tolerate greater mite densities, although there is a balance between vegetative growth and fruiting that must be considered. We showed that ‘Selva’ (8) and ‘Chandler’ (E29) were most susceptible to TSSM feeding early season as indicated by yield reduction during that time and could tolerate much greater TSSM populations later in the season with 5% yield loss recorded at densities of only 5 TSSM per midtier leaflet. A similar seasonal yield loss was confirmed in newer varieties such as ‘Diamante’. My lab clearly showed that pre-transplant nursery chilling of transplants increases tolerance of mite feeding of day-neutral varieties (7, E10). We also showed that lack of fumigation increased susceptibility to TSSM feeding, resulting in reduced yield (E16). It is likely that stressors, even those such as temperature that are beyond grower control will increase damage by spider mite feeding and perhaps even the number of mites present (E24). The first miticide registered that replaced Vendex, Plictran and Dicofol was Agri-Mek (abamectin) (E2, E3), and it became available through annual emergency registrations for several years before finally receiving a full registration. My lab did considerable work to detail its translaminar activity (6, E4) and its optimal use in the field (E6). We also began to evaluate any new miticides that could have a fit in strawberries on an ongoing basis and in field trials in different production regions (9, 10, 11, 17, 19, 20, 21, 26, E18, E20, E22, E23, E31, E33, E34, E36, E38, E43, E44, E48, E51, E53 and several Commission annual reports). A number of new products became registered starting with Acramite (bifenezate) in 2002 (E48) which is fortunate since we have confirmed that resistance to Agri-Mek and generic products containing abamectin is becoming fairly widespread as I have reported in my recent Commission annual reports. We continue to evaluate new products as they arise and have been particularly focused on Nealta which was first registered nationally this year and is the only product available from its chemical class so it should not be cross- resistant to another pesticide. However, through a national survey we conducted at the request of its registrant BASF, my lab has identified greenhouse populations in other states where Nealta is resistant and we have identified a few strawberry fields in California where only the highest label rate is effective even though it has not been used to date. As part of this commission project my lab has been studying the genetic sites for Nealta resistance to help identify what the source of the tolerance might be, but this work is still in progress. We are maintaining a Nealta-resistant TSSM colony in my lab for further work. Because predatory mites are commonly released by strawberry growers, it is important to consider non-target effects of other chemicals, particularly miticides, that are used in strawberries. Through our research,

51 2014 - 2015 RESEARCH PROJECTS we have been able to identify and rank toxicity of most acaricides to Phytoseiulus persimilis as well as the period after application during which it remains toxic to the predators (9, 10, 27 36, E72). We also documented the sublethal effects of the mite growth regulator Zeal (etoxazole) and its transmission through feeding on TSSM killed by the chemical that makes it virtually incompatible with maintenance of predatory mites in a treated field (40). Using Omite before flowering to reduce overwintering TSSM populations was an effective practice to cleanse fields that was compatible with predatory mite releases. With the loss of Omite in 1995, my lab showed that horticultural mineral oils could be applied before bloom to achieve similar results without damaging plants (14, 15, E21, E28). This is a little used but highly effective practice that should be considered by growers. Miticide efficacy was also determined for carmine mite (25,E11, E90) and cyclamen mite (34, E7, E14) in field studies similar to those for TSSM. The presence of similar non-pest Tarsonemid mites that could be confused with cyclamen mite was observed and noted (34). Our studies on development of Lewis mite and TSSM under different temperature regimes and on different hosts indicated TSSM to be more successful than Lewis mite at higher temperatures on strawberries (2012 Commission Annual Production Research Report), and that Lewis mite was more successful on a reported alternate host than on strawberry which may help explain why it is so rarely a problem. My lab is maintaining a Lewis mite colony should future research become necessary.

Thrips. Our studies showed that there was no association between thrips abundance and type II bronzingwhich is characterized by one surface of a fruit showing symptoms (18). Instead, our studies indicate that type II bronzing is likely caused by environmental stress that occurs on clear and hot days. Type II bronzing (not thrips) was significantly reduced when either lignins or overhead irrigation was applied to reduce temperature (22, 33, E45, E46, E77, E79). Both treatments reduce temperature and UV light exposure as well as transpiration which might be the actual cause of this type of bronzing on hot days. My insecticide trials showed that the spinosyn insecticides which include Success, Entrust and Radiant were most effective for thrips control and were also compatible with the use of predatory mites in strawberries (24, E84). Company imposed restrictions on the number of applications that can be made with these products to prevent resistance from developing prompted my lab to conduct whole field studies to determine if resistance could be managed with insecticide rotations or by other means (E86, E92, E95), but we were not successful in reversing the label restrictions. Sampling for thrips and action thresholds to prevent bronzing caused by thrips through our research are the basis for recommendations in UC guidelines (29, E99). When a new predatory mite Amblyseius swirskii became commercially available, we determined their ability to control thrips as well as whiteflies by making releases in plots within commercial fields and monitoring thrips abundance (E71). Indeed A. swirskii had a broader host range than is typical of other predatory mites released in strawberries and they reduced thrips populations relative to no release control plots, but the cost of their use and effectiveness are not competitive with the spinosyns including the OMRI approved Entrust for organic producers. In Europe, minute pirate bugs are released in protected crops for thrips control. My lab developed an artificial diet to mass-rear one species,Orius insidiosus, that could be used by insectaries to improve their productions and availability (3), however we never attempted field releases in strawberries to determine feasibility of their use.

Whiteflies. Although it remains unknown why or how greenhouse whitefly adapted to strawberries as a host, the outbreaks that occurred could be attributed to the presence of overlapping hosts in both areas, primarily the presence of overlapping summer and fall plantings in the Oxnard area, and second year and new fall plantings in the Watsonville area. In the Oxnard area, the problem was largely overcome by instituting a brief strawberry-free period between the termination of the fall planting and the transplanting of the summer planting together with the use of the synthetic neonicotinoid Admire (E55) on the new planting to extend the infestation-free period. In the Watsonville area, most growers stopped the practice of maintaining second year fields and most growers in affected areas began using Admire (imidacloprid) (E55) . Although our insecticide trials identified that registered organophosphates reduced whitelfy populations, environmental and resistance concerns for other insects such as Lygus, made registration of effective new products essential to control the outbreak. Admire and the insect growth regulator Esteem (pyriproxyfen) received emergency registrations in 2003 supported by our efficacy studies and my labs research on yield losses due to whiteflies (28). An economic assessment of

52 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

returns provided by these products that I conducted with UC Davis agricultural economists Drs. Greg McKee and Rachael Goodhue (31) also helped justify a full registration. Optimum timing of these treatments was predicted using a model of whitefly development that we developed and included these products (32). Recently, a greenhouse whitefly outbreak occurred in the Santa Maria area, seemingly caused by similar factors to those responsible for the previous outbreaks. An areawide educational program facilitated by the commission that included lessons learned from the previous outbreaks seems to have limited the potential damage, and I was ready and able to contribute to the effort in an advisory capacity.

Lepidoptera. I have presented information on monitoring the spring flight of corn earworm and beet armyworm in southern California using pheromone traps and degree-days at a number of meetings, particularly at the annual UC South Coast Strawberry Day at Irvine, CA. Field trials that my lab have done (24, E47) indicated that insect growth regulators such as Intrepid (methoxyfenozide), the spinosyns Radiant and Entrust, Rimon, and even some Bts applied more than once timed to peak egg hatch were acceptable alternatives to organophosphate and carbamate insecticides that had been used previously. I worked closely with the commission and local agricultural commissioners even before the first LBAM moth was captured in Santa Cruz County in 2007 to develop a grower education and inspection/certification plan to help cope with quarantines that would be imposed following LBAM detection. I worked with the commission to produce the first publication on identification and biology of LBAM, and it was later distributed as an educational tool for other growers in the Monterey Bay area (E64, E66). It was updated in 2010 (E84, E85. I was also an author of a UC publication on LBAM (E67). By the end of the 2007 growing season, it became apparent to me that LBAM was not an important pest of strawberries and that the greatest loss to growers was due to the regulation on shipments should a larva be detected. I made a number of presentations on LBAM at grower and public meetings during the period of the eradication program (see presentations on my lab website). My lab, in particular Dr. Hillary Thomas together with Mark Bolda, conducted field research on LBAM biology, sampling and focused on caneberries where LBAM was a greater issue.

Vinegar flies. There are several native Drosophila species that are always present in strawberry growing areas that are capable of infesting over-ripe fruit and that can rapidly build to high populations (E9). Our observations during the 1995 vinegar fly outbreak provided the basis for UC guidelines on prevention of damage to the processing crop (29,E99 ) that included cultural approaches such as removing culled and overripe unharvested fruit from fields and shortening harvest intervals (E9, E75, E76) as well as use of registered insecticides (E35). Occasional outbreaks that have occurred since then are attributable to poor sanitation during harvest of the processing crop. The most recent outbreak (in 2012 and 2013) was largely caused by insufficient availability of labor for performing sanitation. Although the invasive spotted wing drosophila (SWD) was first found on Watsonville area strawberries and it has been reported as a pest of ripe strawberries elsewhere (37), its economic damage in California has been almost entirely to the processing crop (38, E74) where its management is similar to that of the native vinegar flies. Monitoring methods developed for SWD including use of traps containing liquid baits such as apple cider vinegar and yeast, sugar and water are not selective (41, 43) and can be used to measure presence and population trends of all vinegar flies that might be present in strawberries. Effective insecticides for SWD control include spinosyns including the OMRI-approved Entrust, malathion, and some pyrethroids including Brigade (bifenthrin), and these are also effective against native vinegar flies. I collaborated with Dr. Joanna Chiu to sequence the SWD genome (44). This work provides an opportunity for the extensive basic molecular research that has been conducted on Drosophila melanogaster and a few other species related to insecticide resistance, olfaction, and other biological processes to be applied to a pest species. We have already started to apply this to insecticide detoxification as it relates to periods of the day when SWD are more susceptible to insecticides (42) and to chemicals that specifically attract SWD versus other Drosophila species (journal article in press). We have also developed a molecular diagnostic tool that scan be used to distinguish SWD larvae in fruit from other common Drosophila species.

53 2014 - 2015 RESEARCH PROJECTS As I mentioned earlier, I am presenting this summary of work conducted by my lab as my final report to the commission, In it, I have referenced documents representing journal articles, book chapters and extension-type publication (some of which might be considered by some to be ‘gray literature’) that can be accessed for further information. I am also making presentations on these pest problems available on my lab’s website at: http://ucanr.edu/sites/zalomlab/Strawberries/ since considerable data generated by my lab has not been provided in written documents. My intent is to provide references to information on strawberry pests that I hope will be useful for growers as well as to inform future California strawberry research.

Acknowledgements I want to thank the strawberry growers that have supported our work over the years and especially those who let us work on their farms, the California Strawberry Commission for sustained financial support, my UC collaborators over the years especially Carolyn Pickel, Norm Welch, Mark Bolda, Doug Shaw, Kirk Larson, Nick Toscano, Jianlong Bi, and Oleg Daugovish, and current and former members of my lab including Pat Thompson, Nikki Nicola, Doug Walsh, Robert Yu Yi and David Limburg. Bibliographic references associated with this project: Journal articles and book chapters:

1. Zalom, F.G., Pickel, C. and N. Welch. 1990. Recent trends in strawberry arthropod management for coastal areas of the western United States, pp 239-259. In L.T. Wilson, N.J. Bostanian and T.J. Dennehy, [eds.], Monitoring and integrated management of arthropod pests of small fruit crops. Intercept Press, LTD. Winborne, England.

2. Zalom, F.G., Pickel, C., Walsh, D.B. and N.C. Welch. 1993. Sampling for Lygus hesperus Knight (Hemiptera: Miridae) in strawberries. J. Econ. Entomol. 86(4): 1191-1195.

3. Castane, C. and F.G. Zalom. 1994. Artificial oviposition substrate for the mass-rearing of Orius insidiosus (Hem. Anthocoridae). Biol. Control. 4: 88-91.

4. Pickel, C., Zalom, F.G., Walsh, D.B. and N.C. Welch. 1994. Efficacy of vacuum machines forLygus hesperus Knight (Hemiptera: Miridae) control in coastal California strawberries. J. Econ. Entomol. 87(6): 1636-1640.

5. Pickel, C., Zalom, F.G., Walsh, D.B. and N.C. Welch. 1995. Vacuums provide limited Lygus control in strawberries. Calif. Agric. 49(2):19-22.

6. Walsh, D.B., Zalom, F.G., Shaw, D.V. and N.C. Welch. 1996. Effect of strawberry plant physiological status on the translaminar activity of avermectin B1 and its efficacy against the twospotted spider mite (Acari: Tetranychidae). J. Econ. Entomol. 89(5): 1250-1253.

7. Walsh, D.B., Zalom, F.G. , Welch, N.C., Pickel, C. and D.V. Shaw. 1997. Pretransplant cold storage of strawberries: effects on plant vigor, yield and spider mite (Acari: Tetranychidae) abundance. J. Econ. Entomol. 90(3): 818-823.

8. Walsh, D.B., Zalom, F.G. and D.V. Shaw. 1998. Interaction of the two spotted spider mite (Acari: Tetranychidae) with yield of day-neutral strawberries in California. J. Econ. Entomol. 91(3): 678-685.

9. Zalom, F., Walsh, D., Tobia, C. and C. Ingels. 1999. Spider mite control on central valley strawberries with acaricides and predatory mites, 1998. Arthropod Managem. Tests. 24: C27.

10. Walsh, D., Zalom, F., Tobia, C. and D. Shaw. 1999. Acaricide efficacy and effects on predatory mites in central coast strawberries, 1998. Arthropod Managem. Tests. 24: C24.

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11. Zalom, F., Tobia, C., Larson, K., Toscano, N. and G. Ballmer. 2000. Acaricide efficacy against spider mites on south coast strawberries, 1998-99. Arthropod Managem. Tests. 25: 75-76.

12. Walsh, D. B. and F. G. Zalom. 2000. Programa de manejo integral de plagas de artropodos de la fresa en las regiones productoras de California. Pp. 109-122, In J. Z. Castellanos and F. G. O’Hart, eds. Simposio Internacional de Fresa. Zamora, Mexico.

13. Walsh, D. B., Zalom, F. G., Shaw, D. V. and K. D. Larson. 2002. Yield reduction caused by twospotted spider mite feeding in an advanced-cycle strawberry breeding population. J. Amer. Soc. Hort. Sci. 127(2):230-237.

14. Walsh, D. and F. Zalom. 2002. Winter horticultural and agricultural mineral oil treatments for control of two-spotted spider mite on California strawberries. pp. 481-491, In A. Beattie, D, Watson, M. Stevens, D. Rae and R. Spooner-Hart (eds.). Spray Oils Beyond 2000: Sustainable Pest and Disease Management. Veritage Press. Lisarow, New South Wales, Australia.

15. Zalom, F. and D. Walsh. 2002. Petroleum-derived spray oils: a useful tactic in California IPM systems. pp. 379-386, In A. Beattie, D, Watson, M. Stevens, D. Rae and R. Spooner-Hart (eds.). Spray Oils Beyond 2000: Sustainable Pest and Disease Management. Veritage Press. Lisarow, New South Wales, Australia.

16. Shaw, D. V., Zalom, F. G. and K. D. Larson. 2003. Relative differences in yield for strawberry (Fragariaxananassa Duch.) genotypes are stable over differing levels of infestation by twospotted spidermites (Tetranychus urticae Koch.). J. Amer. Soc. Hort. Sci. 128(5): 678-681.

17. Zalom, F.G., Glik, T., Limburg, D. and D. Shaw. 2004. Two-spotted spider mite control, 2001. Arthropod Management Tests. 29: C21.

18. Larson, K. D., Koike, S. T. and F. G. Zalom. 2005. Bed mulch treatment affects strawberry fruit bronzing and yield performance. HortScience. 40(1) 72-75.

19. Zalom, F., Thompson, P., Nicola, N. and D. Shaw. 2005. Central coast acaricide evaluation, 2002. Arthropod Managem. Tests. 30: C29.

20. Zalom, F., Thompson, P., Nicola, N., and D. Shaw. 2005. Central coast acaricide evaluation, 2003. Arthropod Managem. Tests. 30: C30.

21. Zalom, F., Thompson, P., Nicola, N. and D. Shaw. 2005. Central coast acaricide evaluation, 2004. Arthropod Managem. Tests. 30: C31.

22. Larson, K. D., Koike, S. T. and F. G. Zalom. 2006. Polyethylene mulch, deficit irrigation, overhead sprinkling and strawberry fruit bronzing. Acta Horticulturae. 708:51-57.

23. Zalom, F G., Shaw, D. V. and K. D. Larson. 2006. Strawberry insects and mites in California, ecology and control. In: Encyclopedia of Pest Management. Marcel Dekker, Inc. New York. http://www.dekker.com/sdek/abstract~db=enc~content =a713627116. [DOI: 10.1081/E-EPM-120041243].

24. Zalom, F. G., Thompson, P., Larson, K., Smith, C. and J. Palacios. 2006. Central of beet armyworm and western flower thrips with reduced-risk insecticides, 2004. Arthropod Managem. Tests. 31: C19.

25. Zalom, F. G., Thompson, P., Nicola, N. and K. Larson. 2006. Evaluation of miticides against the carmine spider mite, 2005. Arthropod Managem. Tests. 31: C20.

55 2014 - 2015 RESEARCH PROJECTS 26. Zalom, F. G., Thompson, P., Nicola, N. and K. Larson. 2006. Evaluation of miticides against the two-spotted spider mite, 2005. Arthropod Managem. Tests. 31: C21.

27. Saenz de Cabazon Irigaray, F. J., Zalom, F. G. and P. B. Thompson. 2007. Residual toxicity of acaricides to Galendromus occidentalis and Phytoseiulus persimilis reproductive potential. Biol. Contr. 40:153-159.

28. McKee, G.J., Zalom, F.G. and R.E. Goodhue. 2007. Management and yield impact of the greenhouse whitefly (Trialeurodes vaporariorum) on California strawberries. HortScience. 42(2): 280-287.

29. Bolda, M.A., Daugovish, O., Fennimore, S.A., Koike, S.T., Larson, K.D., Marcum, D.B. and F.G. Zalom. (technical coordinators) 2008. Integrated pest management for strawberries, 2nd ed. University of California, Division of Agriculture and Natural Resources Publication 3351.

30. Zalom, F. G. 2008. Challenges of managing Lygus in strawberry plantations on the central coast of California. J. Insect Sci. 8 (49): 26-27.

31. McKee, G.J., Goodhue, R.E., Zalom, F.G., Carter, C.A. and J.A. Chalfant. 2009. Population dynamics and the economics of invasive species management: the greenhouse whitefly in California-grown strawberries. J. Environ. Managem. 90: 561-570.

32. McKee, G. and F.G. Zalom. 2009. A model of greenhouse whiteflyTrialeurodes vaporariorum (Westwood) population development and management on Camarosa variety strawberry plants. J. Asia-Pacific Entomol. 12: 117-122.

33. Koike, S.T., Zalom, F.G. and K.D. Larson. 2009. Bronzing of strawberry fruit as affected by production practices, environmental factors, and thrips. HortScience. 44(6): 1-6.

34. Zalom, F.G., Thompson, P.B. and N. Nicola. 2009. Cyclamen mite, Phytonemus pallidus (Banks), and other tarsonemid mites in strawberries. Acta Hortuculturae 842: 243-246.

35. Thompson, P.B., Zalom, F.G. and M.P. Bolda. 2009. Evaluating insecticides for control of adult Lygus bugs (Miridae: Hemiptera) in California strawberries. Acta Horticulturae 842: 219-223.

36. Zalom, F.G. and F.J. Saenz de Cabezon Irigaray. 2009. Integrating pesticides and biocontrol of mites in agricultural systems. pp. 471-476, In Trends in Acarology, M.W. Sabelis and J. Bruin, eds. Springer, Dordrecht, The Netherlands.

37. Walsh, D.B., Bolda, M.P., Goodhue, R.E., Dreves, A.J., Lee, J., Bruck, D.J., Walton, V.M., O’Neal, S.D. and F.G. Zalom. 2011. Drosophila suzukii (Diptera: Drosophilidae): Invasive pest of ripening soft fruit expanding its geographic range and damage potential. J. Integr. Pest Managem. 2(1): G1-G7.

38. Goodhue, R.E, Bolda, M., Farnsworth, D., Williams, J.C. and F.G. Zalom. 2011. Spotted wing drosophila infestation of California strawberries and raspberries: economic analysis of potential revenue losses and control costs. Pest Managem. Sci. 67: 1396-1402.

39. Zhou, C., Kandemir, I., Walsh, D.B., Zalom, F.G. and Lavine L.C. 2012. Identification of Lygus hesperus by DNA barcoding reveals insignificant levels of genetic structure among geographically distant and habitat diverse populations: Implications for integrated pest management. PLoS One. 7(3): e34528. doi:10.1371/journal.pone.0034528.

40. Saenz de Cabezon Irigaray, F.J. and F.G. Zalom. 2012. Transovarial biotransference of etoxazole through a terrestrial trophic web. Pest Managem. Sci. 68: 1467-1470.

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41. Lee, J.C., Burrack, H.J., Barrantes, L.D., Beers, E.H., Dreves, A.J., Hamby, K., Haviland, D.R., Isaacs, R., Richardson, T., Shearer, P., Stanley, C.A., Walsh, D.B., Walton, V.M., Zalom, F.G. and D.J. Bruck. 2012. Evaluation of monitoring traps for Drosophila suzukii (Diptera: Drosophilidae) in North America. J Econ. Entomol. 105: 1350-1357.

42. Hamby, K.A., Kwok, R.S., Zalom, F.G. and J.C. Chiu. 2013. Integrating circadian activity and gene expression profiles to predict chronotoxicity of Drosophila suzukii response to insecticides. PLOS One. 8(7): e68472

43. Lee, J.C., Shearer, P.W., Barrantes, L.D., Beers, E.H., Burrack, H.J., Dalton, D.T., Dreeves, A.J., Gut., L.J., Hamby, K.A., Haviland, D.R., Isaacs, R., Nielsen, A.L., Richardson, T., Rodriguez-Saona, C.R., Stanley, C.A., Walsh, D.B., Walton, V.M., Yee, W.L., Zalom, F.G. and D.J. Bruck. 2013. Trap designs for monitoring Drosophila suzukii (Diptera: Drosophilidae). Environ. Entomol. 42: 1348-1355.

44. Chiu, J.C., Jiang, X., Zhao, L., Hamm, C.A., Cridland, J.M., Saelao, P., Hamby, K.A., Lee, E.K., Kwok, R.S., Zhang, G., Zalom, F.G., Walton, V.M. and D.J. Begun. 2013. Genome of Drosophila suzukii, the spotted wing drosophila. G3: Genes, Genomes, Genetics. 3: 2257-2271.

45. Murphy, K.A., Unruh, T.R., Zhou, L.M., Zalom, F.G., Shearer, P.W., Beers, E.H., Walton, V.M., Miller, B. and J.C. Chiu. 2015. Using comparative genomics to develop a molecular diagnostic for the identification of an emerging pestDrosophila suzukii. Bull. Entomol. Res. 105(3): 364-72.

Extension-type publications:

E1 Pickel, C., Walsh, D.B., Welch, N.C. and F. Zalom. 1991. Efficacy of suction machines for lygus control. The Pink Sheet, Strawberry News Bulletin, 91-3.

E2 Walsh, D., Zalom, F., Welch, N. and D.V. Shaw. 1993. I. Avid 0.15 EC field studies, Winter 1992. The Pink Sheet, Strawberry News Bulletin 93-21.

E3 Walsh, D., Zalom, F., Welch, N. and D.V. Shaw. 1993. II. Avid laboratory studies, Winter 1992. Susceptibility of Tetranychus urticae populations. The Pink Sheet, Strawberry News Bulletin 93-22.

E4 Walsh, D., Zalom, F., Welch, N. and D.V. Shaw. 1993 III. Strawberry plant physiological status: Effects on translaminar activity of Avid. The Pink Sheet, Strawberry News Bulletin 93-23.

E5 Gubler, W.D., Phillips, P.A., Pickel, C., Shaw, D.V., Welch N.C. and F.G. Zalom. 1994. Integrated pest management for strawberries. Univ. Calif. Div. Agric. Nat. Res. Publ. 3351. 142 pp.

E6 Walsh, D., Welch, N.and F. Zalom. 1995. Experimental anti-metabolites, field trials on spider mites, 1994. The Pink Sheet, California Strawberry Commission. 95-4.

E7 Walsh, D., Welch, N. and F. Zalom. 1995. Experimental anti-metabolites. 1995. Field trials on cyclamen mite, 1994. The Pink Sheet, California Strawberry Commission. 95-5.

E8 Walsh, D., Zalom, F., Toscano N. and E. Borden. 1995. Lygus bug hatch on winter weeds in Watsonville and Santa Maria. The Pink Sheet, California Strawberry Commission. (special issue).

E9 Zalom, F.G., Walsh, D., Toscano N.C. and L. Beehler. 1995. Vinegar flies, Drosophila spp. and strawberries. The Pink Sheet, California Strawberry Commission, 95-8.

E10 Walsh, D., Zalom, F., Shaw, D.V. and N. Welch. 1995. Selva-supplemental cold storage & twospotted spider mite control influence on fruit production, size and number. The Pink Sheet, California Strawberry Commission. 95-15.

57 2014 - 2015 RESEARCH PROJECTS E11 Walsh, D., Welch, N., Larson, K., Zalom, F. and N. Toscano. 1995. Cyclamen mite update, April 1995. The Pink Sheet, 95-17, California Strawberry Commission. 95-17.

E12 Walsh, D., Larson, K., Zalom, F., Shaw, D. and N. Toscano. 1995. Chandler-timing of two-spotted mite control influence on fruit production, size and number on fall transplanted strawberries in southern California. The Pink Sheet, California Strawberry Commission. 95-20.

E13 Pickel, C., Phillips, P., Trumble, J., Welch, N., Zalom, F., Gubler, D., Westerdahl B.B. and H. Agamalian. 1995. Strawberry pest management guidelines. UCPMG Publication 22, 40 pp. (first year of UCIPM Guidelines for Strawberries, issued biennially thereafter and I have been an author on all to date).

E14 Walsh, D., Welch, N., Larson, K., Zalom, F. and N. Toscano. 1996. Cyclamen mite update. The Pink Sheet, California Strawberry Commission. 96-2.

E15 Walsh, D.B., Zalom, F., Shaw, D.V. and N. Welch. 1996. Strawberry cultivar, soil fumigation, spider mite interrelationship field trials - mite abundance and effect on fruit yield. The Pink Sheet, California Strawberry Commission. 96-4.

E16 Walsh, D., Zalom, F., Shaw, D.V. and N. Welch. 1996. Selva - Interaction of soil fumigation, supplemental pre-transplant cold storage and timing of two-spotted spider mite control. Influences on fruit production, size and number. The Pink Sheet, California Strawberry Commission. 96-5.

E17 Zalom, F., Welch, N., Walsh, D. and N. Toscano. 1996. Danitol use on strawberries. The Pink Sheet, California Strawberry Commission. 96-10.

E18 Walsh, D. and F. Zalom. 1996. Chemical control of the two-spotted spider mite. The Pink Sheet, California Strawberry Commission. 96-11.

E19 Pickel, C., Phillips, P., Trumble, J., Welch, N., Zalom, F., Gubler, D., Westerdahl B.B. and H. Agamalian. Strawberry pest management guidelines. UCPMG Publication 22, 41 pp.

E20 Walsh, D., Larson, K., Zalom F. and N. Toscano. Early season treatments for spider mites in southern California. The Pink Sheet, California Strawberry Commission. 96-20.

E21 Walsh, D., Zalom, F., Welch N. and D. Shaw. 1996. Alternative treatments to propargite for winter mite control on central coast strawberries. The Pink Sheet, California Strawberry Commission. 96-21.

E22 Walsh, D., Larson, K. and F. Zalom. 1997. Mite timing trials on southern California strawberries. The Pink Sheet, California Strawberry Commission. 96-22.

E23 Toscano, N., Walsh, D., Zalom, F. and L. Beehler. 1997. Acaricide tests in southern California, spring, 1996. The Pink Sheet, California Strawberry Commission. 97-1.

E24 Walsh, D., Zalom, F., Shaw D. and N. Welch. 1997. Warm fall temperatures may lead to mite outbreaks in spring. The Pink Sheet, California Strawberry Commission. 97-4.

E25 Zalom, F.G. 1997. IPM for strawberry insects in California. Proceedings North American Strawberry Growers Assoc. 13:62-67.

E26 Walsh, D., Zalom, F., Toscano, N. and W. Brindley. 1997. A comparison of Lygus controls on central coast strawberries. The Pink Sheet, California Strawberry Commission. 97-12.

58 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

E27 Walsh, D., Zalom, F., Welch, N. and N. Toscano. 1997. Brigade and danitol use on strawberries, a pest resistance management approach. The Pink Sheet, California Strawberry Commission. 97-13.

E28 Walsh, D., Zalom, F. and N. Toscano. 1998. Omni supreme spray - a narrow range horticultural oil which may be useful for cool season spider-mite control. The Pink Sheet, Strawberry News Bulletin, 1 p.

E29 Walsh, D., Shaw, D., Larson, K. and F. Zalom. 1998. El Niño update – nursery chilling and fall temperatures: Importance to plantation establishment and mite susceptibility. The Pink Sheet, Strawberry News Bulletin, 2 pp.

E30 Walsh, D., Brindley, W., Toscano, N. and F. Zalom. 1998. Lygus bug insecticide susceptibility studies 1997. The Pink Sheet, Strawberry News Bulletin, 2 pp.

E31 Walsh, D., Toscano, N., Balmer, G., Zalom, F. and K. Larson. 1998. Acaricide tests on southern California strawberries, Spring 1997. The Pink Sheet, Strawberry News Bulletin, 2 pp.

E32 Toscano, N.C. and F. Zalom. 1998. Greenhouse whitefly Trialeuordes vaporiorum. Pest Alert, Strawberry News Bulletin, 2 pp.

E33 Walsh, D., Ingels, C. and F. Zalom. 1998. Spider mite control tests on Central Valley strawberries. The Pink Sheet, Strawberry News Bulletin, 2 pp.

E34 Zalom, F., Walsh, D. and D. Shaw. 1999. Acaricide efficacy and effects on predator mites in Central Coast strawberries, 1998. The Pink Sheet, Strawberry News Bulletin, 2 pp.

E35 Toscano, N.C., Ballmer, G. and F. Zalom. 1999. Vinegar flies, Drosophila spp. and strawberries. The Pink Sheet, 2 pp.

E36 Ingels, C., Zalom, F. and P. Thompson. 1999. Spider mite control tests on Central Valley strawberries. The Pink Sheet, 2 pp.

E37 Zalom, F., Limburg, D. and D. Shaw. 1999. Acaricide trial for Central Coast strawberries. The Pink Sheet, California Strawberry Commission. 2 pp.

E38 Zalom, F., Larson, K., Tobia, C., Ballmer, G. and N. Toscano. 1999. Acaricide efficacy on spider mites in South Coast strawberries, winter 1998-99. The Pink Sheet, California Strawberry Commission. 2 pp.

E39 Toscano, N. and F. Zalom. 1999. Whitefly identification and management for California strawberries. California Strawberry Commission leaflet. 2 pp.

E40 Zalom, F.G., Phillips, P.A. and N.C. Toscano. 2000. Insects and mites. UC IPM Pest Management Guidelines: Strawberry. University of California, Div. Agric. Nat. Res., Publ. 3468, 29 pp.

E41 Udayagiri, S., Zalom, F. and N. Toscano. 2000. The greenhouse whitefly, an emerging pest on Central Coast strawberries. The Pink Sheet, California Strawberry Commission. 2 pp.

E42 Zalom, F., Toscano, N., Walsh, D., Brindley, W. and D. Limburg. 2000. Lygus bug insecticide susceptibility studies 1996-1999. The Pink Sheet, California Strawberry Commission. 2 pp.

E43 Limburg, D., Zalom, F., Larson, K., Ballmer, G. and N. Toscano. 2000. Acaricide evaluations for twospotted spider mites in southern California strawberries. The Pink Sheet, California Strawberry Commission. 00-10. 2 pp.

E44 Limburg, D., Zalom, F. and D. Shaw. 2001. Watsonville and bay area mite control on strawberries. The Pink Sheet, California Strawberry Commission. 01-01. 2pp.

59 2014 - 2015 RESEARCH PROJECTS E45 Larsen, K. D., Koike, S., Zalom, F. and T. Sjulin. 2001. Strawberry fruit bronzing research summary, 1999-2000. The Pink Sheet, California Strawberry Commission. 01-08. 2pp.

E46 Limburg, D., Zalom, F., Larson, K., Koike, S. and D. Shaw. 2001. Thrips control treatments and fruit bronzing. The Pink Sheet, California Strawberry Commission. 01-10. 2pp.

E47 Limburg, D. and F. Zalom. 2002. Success for insect control on strawberries, a new insecticide registration. The Pink Sheet, California Strawberry Commission. 02-01. 2pp.

E48 Zalom, F. and D. Limburg. 2002. Acramite for spider mite control on strawberries. The Pink Sheet, California Strawberry Commission. 02-10. 2pp.

E49 Larson, K. D., Koike, S., Zalom, F., Polito, V. and E. Show. 2002. Strawberry fruit bronzing research summary. The Pink Sheet, California Strawberry Commission. 02-11. 2pp.

E50 Toscano, N., Bi, J., Ballmer, J.G. and F. Zalom. 2002. Greenhouse whitefly update. The Pink Sheet, California Strawberry Commission Leaflet, 02-16, 2pp.

E51 Zalom, F., Limburg, D. and D. Shaw. 2002. Acaricide trial for Watsonville strawberries. The Pink Sheet, California Strawberry Commission. 02-15. 2pp.

E52 Zalom, F.G. Strawberries, 2002. Appendix A UC Specialist’s Reports. pp. 187-199, In M. Metcalfe, B. McWilliams, B. Hueth, R. Van Steenwyk, D. Sunding, A. Swoboda and D. Zilberman, The Economic Impacts of Organophosphates in California Agriculture. California Department of Food and Agriculture Report. Sacramento, CA.

E53 Zalom, F., Thompson, P., Limburg, D. and D. Shaw. 2003. Strawberry miticide evaluation, summer 2002. The Pink Sheet, California Strawberry Commission. 03-04. 2pp.

E54 Waters, T., Walsh, D., Ferguson, H. and F. Zalom. 2003. Lygus parasites show promise, but no silver bullet; surveys for Peristenus wasps for 2002 and 2003. Washington State Univ. Agricultural and Environmental News. 209. 7 pp.

E55 Zalom, F.G. and N. Toscano. 2003. Management of greenhouse whiteflies: Admire and Esteem emergency registrations. Pesticide Alert: Strawberry News Bulletin, California Strawberry Commission. Sept. 24, 2003, 2pp.

E56 Zalom, F., Thompson, P. and M. Bolda. 2003. Central coast whitefly studies, 2002-03. The Pink Sheet, California Strawberry Commission. 03-10. 2pp.

E57 Toscano, N. and F. Zalom. 2003. Greenhouse whitefly identification and management (revised). California Strawberry Commission Leaflet.

E58 Zalom, F.G. 2004. Statewide strawberry entomology research program. pp. 30-60, In: 2003-04 California Strawberry Commission Annual Production Research Report.

E59 Zalom, F.G. 2005. Statewide strawberry entomology research program. pp. 31-52, In: 2004-05 California Strawberry Commission Annual Production Research Report.

E60 Zalom, F.G. 2006. Statewide strawberry entomology research program. pp. 29-42, In: 2005-06 California Strawberry Commission Annual Production Research Report.

E61 Toscano, N., Zalom, F.G. and J. Bi. 2007. Greenhouse whitefly management. Calif. Strawberry Commission Production Guidelines, Issue 2. 4 pp.

60 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

E62 Zalom, F.G., Toscano, N., Smith, H. and P. Thompson. 2007. Managing Lygus Bugs in Strawberries. Calif. Strawberry Commission Production Guidelines, Issue 3. 4 pp.

E63 Zalom, F.G. 2007. Challenges of managing Lygus in strawberry plantations on the central coast of California. In 2nd International Lygus Bug Symposium, Pacific Grove, CA. 2 pp.

E64 Zalom, F.G. 2007. Identifying leafrollers including the light brown apple moth. Calif. Strawberry Commission Production Guidelines, Issue 5. 4 pp

E65 Zalom, F.G., Shaw, D., Larson, K. and P. Thompson. 2007. Managing Spider Mites in Strawberries. Calif. Strawberry Commission Production Guidelines, Issue 7. 6 pp.

E66 Zalom, F.G. 2007. Identificando los insectos que causan enrollamiento de la hoya incluyendo la palomilla café claro de la manzana, Publicacion 5. 4 pp.

E67 Johnson, M.W., Pickel, C., Strand, L.L., Varela, L.G., Wilen, C.A., Bolda, M.P., Flint, M.L., Lam, W.K. and F.G. Zalom. 2007. Light brown apple moth in California: quarantine, management and potential impacts. http://www.ipm.ucdavis.edu/ EXOTIC/lightbrownapple moth.html. 21 pp.

E68 Zalom, F.G. 2007. Statewide strawberry entomology research program. pp. 214-226, In: 2006-07 California Strawberry Commission Annual Production Research Report.

E69 Bolda, M.P., Daugovish, O., Fennimore, S.A., Koike, S.T., Larson, K.D., Marcum, D.B. and F.G. Zalom (technical coordinators). 2008. Integrated pest management for strawberries, 2nd ed. University of California, Division of Agriculture and Natural Resources Publication 3351.

E70 Zalom, F.G. 2008. Strawberry insect and mite control. pp. 131-144, In: 2007-08 California Strawberry Commission Annual Production Research Report.

E71 Zalom, F.G. 2008. Biology of Amblyseius swirskii, a potential biocontrol agent for whiteflies, thrips and mites in California strawberries. pp. 145-156, In: 2007-08 California Strawberry Commission Annual Production Research Report.

E72 Saenz-de-Cabezon Irigaray, F.J., Zalom, F.G., Moreno-Grijalba, F., Marco, V., Perez-Moreno, I. and L. Carvajal Montoya. 2008. Terrestrial bioaccumulation: experimental approaches on an arthropod prey-predatory mite system. O.59, In ENDURE International Conf., Diversifying Crop Protection, 12-15 Oct., 2008. LaGrande-Motte, France.

E73 Zalom, F.G. 2009. Statewide strawberry entomology research program. pp. 9-21, In: 2008-09 California Strawberry Commission Annual Production Research Report.

E74 Bolda, M.P., Goodhue, R.E. and F.G. Zalom. 2010. Spotted wing drosophila: potential economic impact of a newly established pest. Agric. Resource Econ. Update. 13(3): 5-8.

E75 Zalom, F.G. and M.P. Bolda. 2010. Manejo de la mosca del vinegre (Drosophila). The Pink Sheet, Commission de la Fresa de California, 7 Abril, 2010, 2 pp.

E76 Zalom, F.G. and M.P. Bolda. 2010. Vinegar fly (Drosophila) management. The Pink Sheet, California Strawberry Commission, April 7, 2010, 2 pp.

E77 Koike, S., Zalom, F. and K. Larson. 2010. Strawberry bronzing; research indicates that Type II bronzing is associated with stressful environmental conditions. American Vegetable Grower. May, 2010, pp. 24-26.

61 2014 - 2015 RESEARCH PROJECTS E78 Bi, J., Dara, S., Bolda, M. and F. Zalom. 2010. Development of an area-wide Lygus bug monitoring program for the central coast and Santa Maria Valley. Central Coast Agriculture Highlights (UCCE Santa Barbara Co.). June, 2010, 2 pp.

E79 Koike, S., Zalom, F. and K. Larson. 2010. Strawberry fruit bronzing caused by stress and not thrips. Crop Notes (UCCE Monterey Co.). May/June, 2010, pp. 1-3.

E80 Bi, J., Dara, S., Bolda, M. and F. Zalom. 2010. Population dynamics and developmental biology of Lygus bugs in strawberries and the trap crop alfalfa. Crop Notes (UCCE Monterey Co.). May/June, 2010, pp. 5-7.

E81 Bi, J., Dara, S., Bolda, M. and F. Zalom. 2010. Development of an area-wide Lygus bug monitoring program for the central coast and Santa Maria Valley. Crop Notes (UCCE Monterey Co.). May/June, 2010, pp. 10-11.

E82 Zalom, F.G., Bolda, M.P. and P.A. Phillips. Insects and mites. 2010. pp. 17-61, In UC IPM Pest Management Guidelines: Strawberry. Univ. Calif Div. Agric. Natur. Res. Publ. 3468.

E83 Bi, J., Yi, Y., Zalom, F. and M. Bolda. 2010. Susceptibility of western flower thrips to spinetoram (Radiant) on strawberries in early production season. Crop Notes (UCCE Monterey Co.). July, 2010, pp. 11-13.

E84 Zalom, F.G. 2010. Identifying leafrollers including the light brown apple moth. Calif. Strawberry Commission Production Guidelines, Issue 5.1. June, 2010, 6 pp.

E85 Zalom, F.G. 2010. Como identificar los insectos que causan enrollamiento de la hoja incluyendo la palomilla marron de la manzana. Comision de la Fresa de California Publicacion 5.1. Junio, 2010. 6 pp.

E86 Bi, J., Yi, Y., Zalom, F. and M. Bolda.2010. Impact of a management strategy on susceptibility of western flower thrips in a susceptible site to Radiant insecticide on strawberries. Crop Notes (UCCE Monterey Co.). November/December, 2010, pp. 1-3.

E87 Bi, J., Toscano, N. and F. Zalom. 2010. Susceptibility of the two-spotted spider mite to selected acaricides. Crop Notes (UCCE Monterey Co.). November/December, 2010, pp. 11-12.

E88 Zalom, F.G. 2010. Statewide strawberry entomology research program. pp. 105-119, In: 2009-10 California Strawberry Commission Annual Production Research Reports.

E89 Bi, J., Dara, S., Bolda, M. and F. Zalom. 2010. Statewide strawberry entomology research program. pp. 71-87, In: 2009-10 California Strawberry Commission Annual Production Research Reports.

E90 Bi, J., Toscano, N. and F. Zalom. 2011. Susceptibility of the carmine spider mite to selected acaricides. Crop Notes (UCCE Monterey Co.). January/February, 2011, pp. 1-2.

E91 Bi, J., Greene, I., Kramer, A., Bolda, M. and F. Zalom. 2011. First generation of Lygus nymphs is hatching in second year strawberries in the 2011 production season. Salinas Valley Agriculture. February 23, 2011. http://ucanr.org/blogs/ SalinasValleyAgriculture

E92 Bolda, M. and F. Zalom. 2011. The use of Rimon (novaluron) for early season management of Lygus bugs in strawberries. ANR Strawberry and Caneberry Blog. March 23, 2011. http://ucanr.org/blogs/strawberries_caneberries/

E93 Yu, Y., Bi, J., Zalom, F. and M. Bolda. 2011. Ecological impact on susceptibility of western flower thrips to the insecticide Radiant in strawberry production. Crop Notes (UCCE Monterey Co.). March/April, 2011, pp. 1-3.

E94 Bi, J., Zalom, F., Greene, I. and M. Bolda. 2011. Susceptibility of Lygus bugs to commonly used insecticides in strawberries. Crop Notes (UCCE Monterey Co.). May/June, 2011, pp. 4-6.

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E95 Bi, J., Zalom, F. and M. Bolda. 2011. Susceptibility of western flower thrips to Radiant insecticide on strawberries in an intensive production area. Crop Notes (UCCE Monterey Co.). July/August, 2011, pp. 19-20.

E96 Zalom, F.G. 2011. Statewide strawberry entomology research program. pp. 67-79, In: 2010-11 California Strawberry Commission Annual Production Research Reports.

E97 Bi, J. and F. Zalom. 2011. Development of an area-wide Lygus bug monitoring program for strawberry production in California. pp. 53-65, In: 2010-11 California Strawberry Commission Annual Production Research Reports.

E98 Zalom, F.G. 2012. Statewide strawberry entomology research program. pp. 85-97, In: 2011-12 California Strawberry Commission Annual Production Research Reports.

E99 Zalom, F.G., Bolda, M.P., Phillips, P.A. and S. Joseph. 2014. Insects and mites. pp. 17-60, In UC IPM Pest Management Guidelines: Strawberry. Univ. Calif Div. Agric. Natur. Res. Publ. 3468.

63 2014 - 2015 RESEARCH PROJECTS 64 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

Collection, Release and Establishment of Peristenus digoneutis, a European Lygus Bug Parasitoid, on the California Central Coast

Principle Investigators Dr. Kim A. Hoelmer Research Entomologist, USDA ARS, Beneficial Insects Introduction Research Unit and Acting Director, ARS Overseas Biological Control Laboratories 501 S. Chapel St. Newark, DE 19713 (302) 731-7330 ext. 242 [email protected]

Co-Investigators Dr. Carol Shennan, Professor Department of Environmental Studies University of California Santa Cruz, CA 95064

Dr. Charles H. Pickett Senior Environmental Scientist California Department Food & Agriculture Sacramento, CA

Summary Western tarnished plant bugs (lygus bugs, Lygus hesperus) are a key pest of strawberries on the California central coast. Two European lygus bug natural enemies, the parasitic wasps Peristenus relictus and Peristenus digoneutis, were previously released in Santa Cruz and Monterey Counties as part of an earlier California Strawberry Commission-funded project to reduce lygus bug populations. One of these wasps, P. relictus, has since become established in California and has contributed to lygus bug reductions in strawberries. The second, P. digoneutis, did not become established; this was likely attributable to a poor climatic match between where it was collected and released. This project was intended to improve lygus bug control through the collection, release and establishment of a new P. digoneutis population collected in the region of Brittany, France, which was identified by climate matching as the area in the wasps’ native range with a climate that best-matches the strawberry-growing regions of coastal central

65 2014 - 2015 RESEARCH PROJECTS California. Based on this wasp’s performance in Europe and the northeastern United States where it has already established, we anticipate that its establishment will benefit California strawberry growers by further diminishing lygus bug populations. Initial surveys began in late 2012 in Brittany to locate sites for collection of P. digoneutis, and collections were made during summer and fall months of 2013 and 2014 for shipment to the U.S. for mass rearing and field release in California. The first releases were made in fall 2013 and were continued in 2014 and 2015, although the project did not receive the planned third year of funding. Surveys are being conducted in California to monitor for establishment. Plans are for the NJDA cooperator to continue rearing P. digoneutis for additional releases as rearing resources allow.

Introduction In North America tarnished plant bugs (mirid plant bugs of the genus Lygus) are serious pests of many crop cultures. Lygus bugs are a key pest of strawberries on the California central coast. A European natural enemy of lygus bugs, the parasitoid wasp Peristenus digoneutis, was released by USDA researchers several decades ago and became established in the northeastern US states (Day 1996), and has been expanding into the north central states and northwards into Canada (Day et al., 2000, 2008; Tilmon and Hoffman, 2003). Because lygus bugs lack an effective native North American nymphal parasitoid that can reduce its numbers in California strawberries, efforts have been made to identify and release European lygus bug parasitoids to reduce lygus populations. As part of a California Strawberry Commission-funded project, two European parasitoids, P. relictus and P. digoneutis, were released in Santa Cruz and Monterey counties from 2002 to 2006 (Pickett et al,. 2007, 2009). Peristenus relictus established in central and northern California and has contributed to significant lygus bug reductions in strawberries. It has dispersed into surrounding regions and can be expected to gradually decrease lygus bug populations along the central coast (Pickett et al., 2013). Peristenus digoneutis was also released on the central coast, but did not become established. Because P. relictus does not typically maintain lygus bug populations below economic thresholds we anticipate that by releasing a population of P. digoneutis on the central coast with greater climatic compatibility, the biological control of lygus will be improved. We believe the population of P. digoneutis that was initially released was not climatically well-matched to Monterey Bay strawberry growing regions and thus failed to establish.

Additional parasitism by P. digoneutis can be expected to augment the existing biological control provided by P. relictus. In Europe P. digoneutis tends to dominate in cooler regions, with P. relictus dominating in warmer areas. P. digoneutis should also attack lygus in the spring when P. relictus is less active. Increasing parasitism in the spring would be especially beneficial for second-year strawberries, which often face nymphal lygus populations very early in their second growing season.

Thus, the goal of this project has been to permanently establish a population of P. digoneutis that is well-suited to the climate of strawberry-growing regions of coastal central California.

Materials and Methods Using CLIMEX software as described in last year’s project report, French Brittany was determined to be the closest climate match for the Monterey Bay region within the known geographic range of P. digoneutis in Europe. Explorations and a series of field collections were made in 2012, 2013 and 2014 in the French Brittany region in the general vicinity of Matignon (department/county of Côtes d’Armor) (Figure 1) by Mr. Dominique Coutinot (now retired senior support scientist and quarantine officer, USDA ARS European Biological Control Laboratory in Montpellier, France).

66 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

Figure 1. France with the region of French Brittany (Bretagne) indicated by the ellipse

During repeat visits to the area during the spring, summer and early fall of 2014, European tarnished plant bug nymphs (Lygus rugulipennis) were again collected by sweep-netting in alfalfa (Medicago sativa) in the area of Henanbihen where collections were also made during 2013. Nymphs were placed in containers with fresh green beans as food and the collections were returned to EBCL and held in the laboratory until parasitoid larvae emerged from parasitized lygus nymphs and spun cocoons for pupation. The cocoons of P. digoneutis were placed in dishes with moistened vermiculite and were sent in two shipments of several hundred cocoons in August and October 2014 to the USDA ARS Beneficial Insect Introduction Research Unit (BIIR) in Newark, DE via airfreight (SDV LI Montpellier, France) under a current APHIS foreign importation permit.

After arrival at the ARS BIIR laboratory, the material was held in the quarantine facility for emergence of adult parasitoids from the cocoons. Emerged adults were screened for hyperparasitoids and were identified to species. Live, emerged adult P. digoneutis wasps were sent to Mr. Thomas Dorsey (Director, Philip Alampi Beneficial Insect Laboratory - New Jersey Department of Agriculture, Division of Plant Industry, Bureau of Biological Pest Control), for mass rearing at the Alampi Laboratory in Trenton, NJ. A colony of lygus bugs maintained on the Cohen artificial diet provided the nymphs for parasitism by female P. digoneutis. The parasitized nymphs were maintained at the Alampi lamp until emergence of F1 generation adult wasps, which were then shipped to California for field release by UC Santa Cruz research associate Diego Nieto and colleagues in alfalfa trap crops adjacent to strawberry fields in Prunedale, CA. In the fall of 2014, two additional sites in Aromas and Marina were selected for releases in 2015. At the Marina site, a 0.5 acre plot of un- managed alfalfa was planted to facilitate parasitoid establishment. Alfalfa at all sites was continuously maintained (e.g., weeded, trimmed, fertilized and irrigated), to help facilitate colonization resulting from 2014 releases and to encourage high host densities in preparation for releases in 2015.

1,000 P. digoneutis adults were also shipped from NJDA to UCSC on July 28th, 2015 for field release in California.

67 2014 - 2015 RESEARCH PROJECTS Results and Discussion In May and June of 2014, approximately two dozen Closterotomus norvegicus nymphs were collected from mustard, radish and yarrow at the Prunedale release site. Collected nymphs were reared to allow adult parasitoids to emerge and be identified (i.e., P. relictus versus P. digoneutis). In July, parasitism data were collected at the Prunedale release site using three different techniques: first, hand-held vacuums were used to collect adult parasitoids. Thirty-two such 50-suction samples were collected and analyzed. Second, approximately 185 Lygus spp. nymphs were collected and reared. Third, 25 Peristenus spp. larvae, which were collected from dissected nymphs, were shipped and identified to species via polymerase chain reaction (PCR) by Dr. Martin Erlandson at the Insect Pathology Laboratory, Agriculture and Agri-Food Canada (Figure 2).

Figure 2. Gel electrophoresis image distinguishing Peristenus species from larvae specimens. Base pair results in lanes 4, 5, 6 and 11 all indicate larval species as P. relictus. Image provided by M. Erlandson. Parasitoid releases in 2014 were conducted from 6 August to 11 September. In Prunedale and Salinas, 650 and 550 P. digoneutis adults were released at each site, respectively. Autumn sampling was conducted on 8 October 2014, as 214 Lygus spp. nymphs were collected and reared. While P. relictus adults were collected using these methods in 2014, P. digoneutis adults were not recovered.

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Overwintering pupae from October 2014 collections were reared, with 12 parasitoids emerging in March. Spring collections were made in March and April from Prunedale and Salinas release sites; 53 mirid (Lygus spp. and Closterotomus norvegicus) nymphs were collected from alfalfa, mustard and radish, and reared. In July, approximately 200 lygus bug nymphs were also collected and are currently being reared. Also in July, 80 100-suction samples were taken to recover adult parasitoids at the Prunedale site. One hundred sixty additional such samples are scheduled in August and September.

The first releases of 2015 occurred in Marina and Salinas, where 625 and 375 parasitoids were released, respectively (Figure 3). Additional releases are scheduled in August and September for sites located in Marina, Salinas, Prunedale and Aromas. Another robust round of nymphal collections at these locations is also scheduled for September. Thus far, several P. relictus adults have been collected using these methods, but P. digoneutis adults have yet to be recovered. Monitoring for establishment will continue beyond the conclusion of the funded project as resources permit. If establishment occurs as anticipated, the buildup of the population, with attendant impact on lygus populations, is likely to take time and may not be immediately evident.

Figure 3. Release of Peristenus digoneutis in a 0.5 acre alfalfa plot in Marina, CA on 29 July 2015.

69 2014 - 2015 RESEARCH PROJECTS Selected References

• Day, WH. 1996. An evaluation of biological control of the tarnished plant bug, Lygus lineolaris, in alfalfa by the introduced parasitoid Peristenus digoneutis (Braconidae). Envir. Entomol. 25: 512-518.

• Day, WH., Tilmon, KJ., Romig, RF., Eaton, AT. and KD Murray. 2000. Recent range expansions of Peristenus digoneutis (Hymenoptera: Braconidae), a parasite of the tarnished plant bug (Hemiptera: Miridae), and high temperatures limiting its geographic distribution in North America. Journal of the New York Entomological Society 108: 326-331.

• Day, WH., Romig, RF., Faubert, HH. and KM Tatman. 2008. The continuing dispersion of Peristenus digonuetis Loan (Hymenoptera: Braconidae), an introduced parasite of the tarnished plant bug Lygus lineolaris (Palisot) (Hemiptera: Miridae) in Northeastern U.S.A. and Southeastern Canada. Entomological News. 119: 77-80.

• Pickett, CH., Rodriguez, R., Brown, J., Coutinot, D., Hoelmer, KA., Kuhlmann, U., Goulet, H., Schwartz, MD. and PD Goodell. 2007. Establishment of Peristenus digoneutis and P. relictus (Hymenoptera: Braconidae) in California for the control of Lygus spp. (Heteroptera: Miridae). Biocontrol Science and Technology 17, 261–272.

• Pickett, CH., Swezey, SL., Nieto, DJ., Bryer, JA., Erlandson, M., Goulet, H. and MD Schwartz. 2009. Colonization and establishment of Peristenus relictus (Hymenoptera: Braconidae) for control of Lygus spp. (Hemiptera: Miridae) in strawberries on the California Central Coast. Biological Control. 49: 27-37.

• Pickett, CH., Nieto, DJ., Bryer, JA., Swezey, SL., Stadtherr, M., Wisheropp, D., Erlandson, M. and M. Pitcairn. 2013. Post-release dispersal of the introduced lygus bug parasitoid Peristenus relictus in California. Biocontrol Science and Technology, 23: 861-871.

• Tilmon, KJ and MP Hoffmann. 2003. Biological control of Lygus lineolaris by Peristenus spp. in strawberry. Biological Control. 26: 287-292.

70 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

2014 Lygus and Mite Management Program

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

Dr. Dan Legard Mark Edsall Kyle Blauer (intern) Martin Morones (intern)

Cooperators DG Specialty Farms, Sundance Berry Farms and additional anonymous strawberry industry growers.

Summary The California Strawberry Commission’s (CSC) in-house entomology program has worked to address priority pest issues in the strawberry industry by conducting area-wide monitoring programs and on-farm management trials for lygus bugs and spider mites. The 2014 lygus bug management program focus was to improve the performance and efficiency of commercial bug vacuums. For spider mites, we conducted laboratory bioassays of miticide efficacy for three key products to estimate the impact that miticide resistance might have on outbreaks of spider mites in the Oxnard and Santa Maria production districts. Results of the lygus bug vacuum program demonstrate that modifications to the angle of louvers and the introduction of holes into the louvers of commercial bug vacuums can double vacuum efficiency. Miticide bioassays showed low average efficacy for materials tested to control two-spotted spider mites that were field-collected from seven sites in each district. Best management practices resulting from this year’s bug vacuum work are being developed into a training program. Future mite work will focus on the integration of chemical and biological control, and evaluation of spray program efficacy.

71 2014 - 2015 RESEARCH PROJECTS Introduction The research department of the California Strawberry Commission (CSC) has in an-house entomology program that works directly with strawberry growers to identify and help address pest issues. Our primary role is to evaluate and improve current integrated pest management practices (IPM), identify remaining gaps in IPM tools and opportunities for research programs, help in the development of training and programs, and to identify and coordinate management planning for season-specific pest issues. Lygus bug and two-spotted spider mites have been identified by the strawberry industry as priority pests and our research efforts have focused on them due to the significant economic losses they cause.

Lygus hesperus (Hemiptera: Miridae) is a generalist feeder and a native pest of numerous agricultural crops in the western United States. In strawberry production, lygus feeds on the early developing achenes which causes direct deformation of fruit, known as “cat-facing” and renders the fruit unmarketable. Adult lygus are able to locate stable food sources at long distances and the prevalence of this pest in the landscape contributes to its economic importance, in combination with the lack of effective management tools. For lygus management, strawberry growers rely primarily on insecticides and the use of commercial bug vacuums in addition to good cultural practices. However, a low economic threshold for this pest means that it is common for growers to lose control of lygus bug in strawberry production fields.

The CSC has been conducting pest monitoring in collaboration with approximately 10% of acreage in Oxnard, Santa Maria, and Watsonville production regions. The area-wide monitoring program over the past four seasons has found that lygus is seldom controlled at or below the established economic threshold (1 lygus bug per 20 plants) at most of the fields we monitored, despite well implemented insecticide management programs. Economic loss from fruit deformation caused by lygus bug feeding ranged between 10 to 70% of marketable fruit in those fields. Lygus resistance to key insecticides has been observed since the early 1990’s and new registrations of effective products are rare. Our monitoring data also suggested that growers who used commercial bug vacuums had fewer problems with lygus (Thomas and Legard, 2014). For these reasons, the CSC program has focused on evaluating and improving the performance of bug vacuums used by growers to control lygus.

Commercial bug vacuums are tractor-mounted mechanical control tools that work by sucking insects off of the plants. They are widely used by strawberry growers due to the absence of other management tools. In strawberry production, 3-to-4 vacuum hoods with individual fans are mounted on the front end of a tractor, each tied into a central hydraulic system that is powered by the tractor’s power take off (PTO). The tractor is usually solely used for vacuuming because growers use them daily for most of the season and they are not easily removed.

Prior trial work during 2013 demonstrated that vacuums reduce lygus damage to strawberry fruit (Thomas and Legard, 2014). However, we also identified that most vacuums we tested had very low efficiency, there was high variability in performance between vacuums, and the strawberry industry was lacking standard operating procedures for the vacuums. During the 2014 season, our main objective for the lygus research program was to increase vacuum efficiency and develop best management practices for using bug vacuums.

Similar to the situation with lygus bug, spider mites have been an issue in commercial cultivation since the California strawberry industry started expanding in the late 1950’s. However, in the last four to five seasons, serious losses have been caused by spider mite feeding in the Oxnard-Ventura production area, where entire fields were damaged severely enough that they were abandoned. More typically in northern production strawberry fields, there is a background level of mite infestation that causes some level of yield loss due to their feeding on strawberry leaves. The most common and important pest mite in strawberry is the two-spotted spider mite, Tetranychus urticae Koch. Our objectives for the 2014 season for the spider mite program was to obtain further information about the cause for the increased difficulty of managing spider mites in strawberry.

72 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

Materials and Methods During the 2014 season, the California Strawberry Commission conducted field trials in cooperation with growers in each of California’s major strawberry growing regions, in coordination with our annual area-wide pest monitoring program. Trials were conducted between April and October, 2014 as described below.

Bug Vacuum Program

Building on data that was collected during the 2013 season demonstrated that there were potential issues that were lowering the performance of many vacuums, we evaluated if there were quick and cost effective solutions that could improve vacuum performance. Vacuum Frequency Trial We wanted to know if increasing the frequency of vacuuming might improve their overall performance. The industry standard for vacuuming frequency is two passes per week, which conveniently matches their picking schedule. Our objective was to evaluate if this was the optimal frequency. This study was conducted over seven weeks between July and August, 2014 on four ranches in the Santa Maria district with a single one-acre replicate of the following vacuuming frequency treatments at each ranch (for a total of four reps): (1) one time per week (2) two times per week, 3) four times per week and 4) an unvacuumed grower’s standard. We purchased a new industry standard C & N Tractor, Watsonville, CA, manufactured tractor with three fume hoods and fans sized for 64 inch strawberry bed center to center spacing, typical of Santa Maria production. We assessed the efficiency of the vacuum for picking up lygus bugs prior to conducting the vacuum frequency trial. We determined lygus populations in the study using a standard beat sampling method for lygus and sampled each treatment plot on each of the four ranches. Treatment plots were sampled the day after vacuuming occurred, and every week for the duration of the experiment (seven weeks). Six beat-box samples were taking from the center beds of each plot in a grid design and pooled to produce an average count for each treatment plot. Data was analyzed by repeated measures ANOVA and in post-hoc individual treatment comparisons by t-test. Field Vacuum Assessments We performed a series of step-wise assessments to compare performance characteristics of commercial bug vacuums with modifications intended to improve their performance and overall efficiency. Our goal was to identify specific modifications that would (1) increase wind speed at the intake to pull more insects off of the plant, and 2) ensure 100% kill of lygus bugs that went through the vacuum apparatus, since survival of viable lygus passing through fans had been documented during the previous season. Vacuum parameters were field-tested on 64inch beds in Santa Maria using an industry standard C & N Tractor vacuum manufactured with intake dimensions approximately 43 inches wide and 9 inches deep. We tested the performance of 1) increasing the speed (RPM) of the vacuum fan with the goal of increasing wind speed at the fan intake in the hood, 2) lowering the angle of the louver from 45 degrees to 20 degrees to increase the percentage of lygus killed by impacting them onto the louver and 3) modifying the baffles using perforated sheet metal on the louvers by using steel punch plate perforated with 1/8 inch holes to maintain the wind speed at the intake while decreasing the baffle angle from 45 to 20 degrees. Vacuum Wind Speed Comparisons Wind speed measurements were taken for the vacuums with the different configurations using a Windscribe Ultrasonic aenometer (Davis Instruments Corp, CA) by taking the highest reading recorded in the same locations of the left, center, and right, as well as front, center and back of the mouth of each vacuum intake on the fume hood (Figure X).

73 2014 - 2015 RESEARCH PROJECTS These measurements were repeated three times. Wind speed comparisons were conducted on adjacent hoods of the CSC research vacuum. We pre-tested each hood to ensure that there were no major discrepancies in their wind speed performance before modifications were applied. Baffles on the back of commercial bug vacuums typically have 5 to 7 angled louvers, i.e., angled slats (Figure X). For the comparison tests, we fitted the hoods with louvers set to (1) 45 degrees, (2) 20 degrees and (3) 20 degrees perforated with 1/8 inch holes throughout, at two different hydraulic pressure levels – 2000 and 2500 psi. Both pressure settings are within the operating tolerances of current industry standard motors.

Figure X. Pictures of a commercial lygus vacuum. A. Front view of a tractor mounted commercial lygus vacuum with three hoods, each with a hydraulic powered fan. Each hood was designed to fit over a standard 64 inch wide strawberry bed with 4 rows of plants. B. The intake opening for the fan on a lygus hood. The two outside hoods can angle up on a hinge to ease the movement of the tractor mounted hoods between fields. C. Solid baffles at a 205 angle mounted on the output opening on each of the hoods. D. View from above of a punch-plate 20% baffle with 1/8 inch holes mounted on a vacuum output opening.

74 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

Vacuum Efficiency Estimates

• Pre-sampling: A minimum of five 20-plant beat style pre-samples were taken from each trial field the day before, or just prior to conducting each assessment to estimate background populations. Each sample was taken at least five rows away from the other samples, and the plants within each sample were 10 to 20 feet apart.

• Vacuum Uptake Estimates (Pre-Vacuum): To quantify the number of lygus and other insects being pulled off the plants, we fashioned a device using window screen frames and sewn mesh bags fitted within the frame. The frame dimensions were cut and fitted to the opening on the front of the vacuum.The bags were approxi- mately 8 inches deep, and shallow enough that they would not get drawn into the fan blade. To collect lygus, the sampling device was affixed to the front end of the vacuum using bungee cords and the operator drove the vacuum a set distance depending on the background pressure of lygus in the field and the length of the rows, typically this distance was 250 to 300 feet. Where low population pressure was observed, the vacuums would cover up to 600 feet. Plastic bins were placed under each fume hood at the end of the run and the vacuum was turned off to allow the samples to drop into the bin. The bins were closed with lids to prevent insects from escaping and the insects were counted later that day in the laboratory. The number of small nymphs, large nymphs and adult lygus were counted and recorded by viewing the collected material under a dissecting scope. The numbers were then compared with the beatbox sample numbers by conversion to the average number of lygus per plant and the subsequent percent collected by the vacuum. At least three replicates of the pre-vacu- um samples were collected at each site for each trial.

• Survival after Vacuuming (Post-Vacuum): Sewn mesh bags approximately 4 feet long and were placed over the baffles after the fan blades and secured in place using bungee cords. The vacuum operator drove the same set distance for vacuum samples as they did for the pre-vacuum samples, and the two types of samples were paired and repeated the same number of times in each field in a paired design. The samples were collected in the same region of the field as the pre-vacuum samples but there were at least five rows between each replicate sample. Lygus bugs were transferred from the bags after the vacuum had been shut off and the bags removed, to 2.5 quart containers for transport to a cooler, and counted as soon as possible to estimate the percent alive and dead of each lygus life stage. We collected lygus post-vacuum samples from at least two vacuum hoods (sub-samples) on each pass. A minimum of three replicates of the post-vacuum samples were collected at each site.

• Vacuum Efficiency Estimates: Vacuum efficiency was estimated as the percent of the per-plant absolute lygus population that was picked up by the vacuums, using the population estimated by the beat-box sample. Zalom and Pickels estimated that the beat sample picks up approximately 52% of the absolute population (Zalom and Pickels, 1993). Miticide Bioassays

We conducted a series of bioassays to evaluate the efficacy of the four most commonly used miticides on populations of two-spotted spider mite collected from grower’s fields.

• Field Populations of mites.: Fields were selected based on high mite pressure. Strawberry leaflets that were naturally infested with two-spotted spider mite were collected in commercial strawberry fields and placed in brown paper bags for transport to the laboratory. Adult female mites were identified under a stereoscope and 15 were used per treatment-replicate in each bioassay, with at least three replicates. Seven field populations were tested from each of the three major districts: Watsonville-Salinas, Santa Maria-Guadalupe and Oxnard-Ventura.

75 2014 - 2015 RESEARCH PROJECTS • Bioassay Treatments.: Miticide treatments included Dyne-amic adjuvant added at 0.25% volume to volume. A standard adjuvant-distilled water solution was used to make the dose solutions. Solutions were tested using a pH meter to ensure that the pH of the test solution was within the range recommended by the miticides label. All solutions were at maximum allowable label rate per 200 gallons of water. The treatments we evaluated were: (1) Control (Dyne-amic at .25% volume:volume), (2) Acramite 1lb/acre , (3) Agri-Mek SC at 3.5 oz, and (4) Kanemite at 31 oz.

• Test Procedure: The procedure was modified from a protocol from Frank Zalom’s lab at UC Davis (N. Nicola, pers. com.). Treatments were prepared following all personal protective equipment (PPE) requirements, label instructions and restrictions. Cowpea leaves were dipped in the previously listed bioassay treatment solutions and dried, then placed onto moist filter paper in a Petri dish (underside/abaxial side up). Mites were transferred to the leaves using a paintbrush. We also put a lid on the dish with air holes in it to reduce desiccation of the leaf. Fifteen adult female mites were transferred per replicate (leaflet) with three replicates per treatment.

• Scoring: Mite mortality was determined 72 hours after the mites were placed on the leaves. Percent mortality was calculated and corrected using Schneider Orelli’s formula where corrected mortality (%) = ((Mortality % in treated plot - Mortality % in control plot)/(100 - Mortality % in control plot)) * 100.

Results Vacuum Frequency Trial

The standard lygus vacuum used in the frequency trial was estimated to be 2.5% efficient; i.e., it killed 2.5% of the estimated in-field lygus population per pass based on sampling conducted prior to the start of this trial. We did not observe significant differences between any of the vacuuming frequency treatments after seven weeks of vacuuming, but all vacuumed treatments were significantly different from the growers (un-vacuumed) standard treatment (Figure 1). It took four weeks before the 2x/week and 4x/week treatments were significantly different from the growers standard and by week 5 the 1x/week treatment was also significantly different from the growers standard (t=1.98, p=0.05). By week 7 the growers used insecticide sprays to drop the lygus population back down to similar levels for all treatments.

Using a post-vacuum bagging method, where we catch all the material that passes through the vacuum, we compared the survival of lygus after exiting a vacuum with a 45 degree louver-angle baffle and a 20 degree louver-angle baffle (N=6 samples each), and found that an average of 11% of lygus survived with the 45 degree baffle and an average of only 6% of lygus survived if the baffle angle was decreased to 20 degrees.

76 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

Figure 1. Average total lygus by date during a vacuum frequency trial with treatments of 1x, 2x and 4x per week compared against a grower standard on four ranches (N=4). Lygus were sampled using a beat method (N=4 weekly samples per treatment-plot) explain error bars.

Vacuum Design Assessments

When fitted with a 45 degree angle baffles the vacuum had wind speeds averaging 42 mph at 2000 psi (Table 1) in the hydraulic pump used to power the vacuum. When the psi was increased to 2500, wind speed increased to an average of 47 mph. When fitted with a 20 degree solid louver, the wind speed decreased to 27 mph at 2500 psi. However, there was no difference in the wind speed of baffles fitted with 20 degree louvers with the 1/8 inch holes (pinch plate) when compared to the industry standard 45 degree solid louvers. Perforated louvers at 20 degree angles also performed as well an industry standard baffles with 45 degree angle solid louvers at 2500 psi in pulling lygus off of strawberry plants, averaging over 4% efficiency (Table 2). Operating the hydraulic pump at at 2500 p.s.i. combined with the perforated 20 degree angle baffles resulted in a 100% kill of lygus that passed through the vacuum.

Table 1. Average wind speed measurements on an industry standard vacuum with the hoods fitted with different baffle styles and operation parameters. Six measurements were averaged for each fume hood-treatment combination. All wind speed measurements were taking using a handheld ultrasonic wind speed meter at the left, middle and right sides of each hood intake.

Baffle Louver Style Operating Avg. Windspeed SEM Notes Angle PSI (N=6) 45 degrees Solid louvers 2000 42.43 1.17 Industry Standard 45 degrees Solid louvers 2500 46.92 1.29 20 degrees Solid louvers 2500 26.90 3.04 Industry Standard 20 degrees Louvers perforated 2500 46.94 1.08 with 1/8 inch holes

77 2014 - 2015 RESEARCH PROJECTS Table 2. Average vacuum efficiency (percentage of total lygus removed by the vacuum) when fitted with different baffle styles during one-day assessments. Vacuum efficiency was calculated as the average percent of the field population that was removed from the field during a single pass with the vacuum. Beat sampling was used to estimate the field population and calculate the per plant populations.

Baffle Treatment Date Beat Sample Estimate of Avgerage Lygus Vacuum Average Lygus per Plant Vacuumed per Plant Efficiency Grower Standard, 45 degree baffles* 8/17/14 0.75 0.043 3.04% Standard Baffle, 45 degrees* 10/2/14 0.81 0.029 3.58% Standard Baffle, 45 degrees* 10/20/14 0.265 0.016 3.17% Perforated Louvers, 20 degrees* 10/20/14 0.265 0.021 4.18%

Miticide Bioassays

The results from the miticide bioassay were as follows: Bifenazate (Acramite) averaged 43% mortality, Abamectin (Agri-mek) averaged 48% mortality and Adecquinocyl (Kanemite) averaged 65% mortality (Figure 2). These results suggest that there is a fairly high level of resistance to these miticides since they were tested by spraying detached leaves with the full labeled rate.

Discussion Lygus Bug

The newly purchased vacuum used during this trial had an efficiency comparable to the industry average from work we conducted during the 2013 season. These vacuums had an average efficiency of about 2.5% when their fans were operated at 2000 p.s.i. Although all vacuuming treatments lowered lygus numbers compared to the grower’s standard during the vacuum frequency trial, no significant effects were observed from increasing the number of times a field is vacuumed per week from one to four passes per week.

78 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

The relatively low efficiency (4% of the lygus picked up) of the vacuum used in this study, which also allowed 11% of lygus to survive after passing through the vacuum, probably accounted for our inability to produce significant differences in lygus populations even when increasing the frequency from one to four times per week.

By evaluating the effect of higher p.s.i/wind speeds and changes in the angle and type of baffles on the efficiency of the vacuum, we were able to improve the performance of the vacuum in removing and killing lygus in strawberry fields. Decreasing the baffle angle from 45 to 20 degrees increased the percentage of lygus killed, presumably because more lygus strike, and are killed, by the lower angled baffle. But this baffle angle also decreased wind speed at the intake by up to 15 mph, which reduced the number of lygus removed from the plants. Replacing the solid plate with 1/8 inch punch plate at a 20 degree angle maintained the higher wind speed and the higher lygus mortality rate.

We recommend that growers modify their vacuums by replacing solid louver baffles with perforated louvers, and reducing the angle of the louvers on the baffles to a 20 degree angle. Work remains to identify the ideal louver angle and hole size for the punch plate used on the louvers. Small punch-plate holes may become clogged by plant material in the field and will require additional maintenance procedures. We currently recommend that the holes in the punch plate be at least 1/8 inch. Growers should also take wind speed measurements and evaluate the current performance of their vacuums. The operating pressure of the hydraulic pump should be increased from 2000 psi to 2500 psi on newer vacuums to increase wind speed at the intake. Care should be taken to monitor the fan motors when operating at the higher psi to ensure there is not excessive wear, and that this pressure setting is within the manufacturer recommended specifications for their vacuum motors. We have developed a training class to share the latest best operating practices for vacuums and relevant maintenance procedures necessary to maximize the performance of lygus vacuums.

Two-spotted Mite

Miticide bioassays indicated that the efficacy of the key miticides was low on mites collected from all the strawberry districts and are likely contributing to the poor mite control observed in the industry. We plan to expand our program to evaluate spray application and miticide use in combination with biological control agents for mite control, to identify additional opportunities for improving mite management practices.

Acknowledgments This work was conducted in collaboration with growers of the California strawberry industry, notably DG Specialty Farms and Sundance Berry Farms.

Selected References • Abbott, W. S. 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265-267.

• Miller A.L., Tindall K. and B. R. Leonard. (2010). Bioassays for Monitoring Insecticide Resistance. JoVE. 46. http:// www.jove.com/details.php?id=2129, doi: 10.3791/21.

• Thomas, H.Q. and D. E. Legard. 2014. 2013 Management of Lygus with bug vacuums in strawberry. Annual Production Research Report: 2013-2014, pp 61-70. California Strawberry Commission.Zalom, F. G., C. Pickel, Walsh, D. B and N. C. Welch. 1993. Sampling for Lygus hesperus Knight (Hemiptera: Miridae) in Strawberries. J. Econ. Entomol. 86(4): 1191-1195.

79 2014 - 2015 RESEARCH PROJECTS 80 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

2015 California Strawberry Commission Lygus Management Program

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

Dr. Dan Legard, VP Research and Education Mark Edsall, Field Research Supervisor Daniel Olivier, Field Research Specialist Myles Shoemaker (intern) Jose Valdez (intern) Jimmy Wells (intern) Vanessa Castillo (intern) Joseph Ugalde (intern)

Summary Since 2013, the California Strawberry Commission’s research department has led a program focused on improving and standardizing the performance of commercial bug vacuums for management of lygus bug in strawberry production. During the 2015 season, we conducted a series of stepwise field tests to increase vacuum efficiency by testing several physical modifications to the vacuum and testing changes to standard operating procedures. By replacing the motor with one that is rated to run at higher pressure (increase from 2000 to 3000 psi) and increase the wind speed of the vacuum, we were able to increase its efficiency from around 4% to an 8% average. We further increased the vacuums efficiency to 18% by modifying the front end of the intake by adding a plate to narrow the opening and increase suction through the plant canopy. For standard operating procedures, we determined that at least two consecutive passes can be made on the same area of the field without a drop off in the number of lygus removed, which suggests that there is further room to increase vacuum efficiency. We tested two different tractor ground speeds and found that slowing down to 1.1 mph from 1.9 mph did not improve vacuum performance, therefore 2 mph remains the recommended operating ground speed.

81 2014 - 2015 RESEARCH PROJECTS We also evaluated the effect of vacuuming at different times of day, morning, afternoon and evening and found that more lygus were picked up in the afternoon than in the morning or evening. We also documented that modified vacuums capture a significant proportion of small nymphs, in addition to large nymphs and adults. We have incorporated these findings and our best management practice recommendations into a training program that was held in each of the three major strawberry production districts in the spring. While we significantly improved the performance of the standard vacuum from 4% efficiency to 18%, our target goal is 25% efficiency (the vacuums should pick up 25% of the lygus in a field with each pass). We plan to continue modifying the vacuum and optimizing its operating procedures next season with the goal of further improving efficiency.

Introduction The research department of the California Strawberry Commission (CSC) has a program that works directly with strawberry growers to identify and address pest issues. Since 2013, our research has focused on improving the performance of commercial bug vacuums in strawberry production for management of lygus bug. Lygus is a key economic pest in California strawberry production, costing the industry an estimated $100 to $200 million annually in fruit damage due to feeding of the lygus on the fruit.

Commercial bug vacuums are tractor-mounted mechanical control tools that work by sucking the insects off of the plants and killing them. They are widely used by strawberry growers for lygus control in combination with pesticide applications. In strawberry production, 3-to-4 vacuum hoods (one hood per bed), each mounted with a motorized fan blade to create the vacuum’s suction are mounted on the front end of a tractor, and powered by a central hydraulic system connected to the tractor’s power take off (PTO).

Prior research conducted by the CSC during the 2014 season resulted in a recommendation to modify the baffle component of the vacuum fume hoods to 1) lower the angle of the louvers, or slats, to ensure that lygus were killed as they were sucked through the hood, and 2) to make the louvers out of perforated sheet metal, otherwise known as punch plate, rather than solid metal, to allow air to move through the plates and increase the overall wind speed (Thomas and Legard, 2014). During the 2014 season, we were able to increase the efficiency of the vacuums from 2.5% to 4.2% on average (i.e., the modified vacuums collected 4.2% of the lygus population in the field). This season we set a goal of increasing the average vacuum efficiency to 25%. To reach this target, we continued stepwise testing of additional modifications to the vacuums and conducted field tests to evaluate operational practices that could improve the performance of the bug vacuum.

Methods We performed a series of step-wise assessments to test modifications to the bug vacuum intended to improve its performance and efficiency. We also evaluated different operational approaches to see if they would improve operating procedures.

Test Procedures For each of the field evaluations, we used the same methods to measure the wind speed of the vacuum (i.e., suction) and to estimate vacuum efficiency (percentage of lygus on vacuumed plants that are removed and killed by the vacuum).

82 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

Wind Speed Measurement: Wind speed measurements were taken for the vacuums with the different configurations using a Windscribe Ultrasonic anemometer (Davis Instruments Corp, CA). The reported speed was determined by taking the highest reading recorded in different locations (left, center, and right, as well as front, center and back) of the mouth of each vacuum intake on the fume hood. These measurements were repeated three times for each evaluation. Wind speed comparisons were conducted on adjacent hoods of the test vacuum. We tested the wind speed on each hood to ensure that there were no major differences in their original wind speed before we made the modifications in their performance. Baffles on the outlet of commercial bug vacuums typically have five to seven angled louvers (Figure 1A). For the comparison tests, we fitted the hoods with louvers set to 20 degree perforated with 1/8 inch holes throughout. We operated the vacuum at 3000 psi using a new motor (Danfoss SNM2) for all of the tests.

Vacuum Efficiency Estimates

• Pre-sampling: A minimum of five 20-plant beat-style pre-samples were taken from each trial field the day before, or just prior to conducting each assessment to estimate background populations. Each sample was taken at least five rows away from the other samples, and the plants within each sample were 10 to 20 feet apart. We ran t-tests to ensure that there were no differences in background pressure in areas of the field that were assigned to different treatments.

• Vacuum Uptake Estimates (Pre-Vacuum): To quantify the number of lygus and other insects being pulled off the plants, we fashioned a device using window screen frames and sewn mesh bags fitted within the frame. The frame dimensions were cut and fitted to the opening on the intake of the vacuum (bottom of the fan opening). The bags were approximately 8 inches deep, and shallow enough that they would not get drawn into the fan blade. To collect lygus, the sampling device was affixed to the intake end of the vacuum open- ing using bungee cords and the operator drove the vacuum a set distance depending on the background pressure of lygus in the field and the length of the rows, typically this distance was 250 to 300 feet. Where low lygus populations were observed, the vacuums would run for up to 600 feet. Plastic bins were placed under each fume hood at the end of the run and the vacuum was turned off to allow the samples to drop into the bin. The bins were closed with lids to prevent insects from escaping and the insects were counted later that day in the laboratory. The number of small nymphs, large nymphs and adult lygus were counted and recorded by viewing the collected material under a dissecting scope. The numbers were then compared with beat-box samples that had been converted to the average number of lygus per plant and the subsequent percent collected by the vacuum. At least three replicates of these pre-vacuum samples were collected at each site for each trial.

• Survival after Vacuuming (Post-Vacuum): Sewn mesh bags, approximately 4 feet long, were placed over the baffles after the fan blades and secured in place with bungee cords. The vacuum operator drove the same set distance for vacuum samples as they did for the pre-vacuum samples, and the two types of samples were paired and repeated the same number of times in each field in a paired design. The post-vacuum samples were collected in the same region of the field as the pre-vacuum samples but with at least five rows between each sample. Lygus bugs were counted as soon as possible after collection. A minimum of three replicates of the post-vacuum samples were collected at each site.

83 2014 - 2015 RESEARCH PROJECTS Vacuum Efficiency Estimates: Vacuum efficiency was estimated as the percent of the per-plant absolute lygus population that was picked up by the vacuums, using the population estimated by the beat-box sample adjusted to reflect that the this type of sampling collects approximately 52% of the actual population (Zalom and Pickels, 1993).

Modifications Tested

New High Pressure Motor

In early 2015, we contracted a private engineering company, Radius West (Modesto, CA), to model and eval- uate air flow in the grower standard vacuum configuration. At their direction we replaced the standard motors with new model that would run at higher psi, and require less maintenance (Danfoss SNM2). The previous motors ran at 2000 psi, whereas the specifications for the new motor allows safe operation at 3000 psi eW reconfigured our vacuum with the new motor and replaced the pressure lines to ensure they could handle the pressures we were running the motors at. We field tested the performance of the modified bug vacuum’s efficiency using our standard procedures on August 26, 2015 (N=4).

Front Opening Modification on the Front of the Hood

The hood of current commercial vacuums have a taller opening at the front end (Figure 1A) then the rear, which apparently reduced the potential for mechanical damage to fruit as the hood passed over the plant and pre- vented the hood from “digging” into the plants as the tractor dipped when entering the field at end of the rows. eW hypothesized that reducing the height of this opening would improve vacuum performance by increasing the amount of airflow through the plant canopy. We tested a modification of the hood where we attached a sheet metal plate, with a flexible strip of rubber attached to its lower edge to the front of the hood to reduce the height of the front opening by approximately 9 inches (Figure 1B). Three tests of the standard and modified hood were conducted on different dates (October 8, 21 and 28, 2015) in fields with different background lygus populations as estimated with pre-samples using the beat box method. All tests were conducted running the vacuum at 3000 psi Counts were analyzed globally (for all three dates) by matched pair analysis for each life stage and for total lygus counts.

Figure 1A. Picture of the front opening on the hoods of a standard 4-row bed commercial lygus vacuum used to evaluate the effect of installing a plate to increase airflow through the plant canopy. The first hood on the far left has a plate installed to reduce the height of the front opening. There are six steel baffles on the outlet opening (top) of each fan hood.

84 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

Figure 1B. The metal plate used to reduce the height of the front opening of the hood. There is a rubber fringe added to the lower edge to reduce the amount of damage the barrier might cause to plants it brushes across.

Operating Procedures Tested

Time of Day and Plant Moisture

Vacuuming during the night when normal production practices are not occurring (i.e., harvesting, spraying, etc.) could extend the period when a vacuum could be used and avoid disrupting other farming operations. We tested whether vacuuming at night could be as effective as at during the day. For field scouting we had previously observed higher variance and lower lygus estimates when we counted lygus using the beat box method when the plants were wet. So we evaluated the effect of leaf wetness on the efficiency of the vacuum on two dates (May 31 and 21, 2015) by noting if the foliage was wet during early morning or evening tests and comparing the efficiency to tests in the same field after the foliage had dried during the day.

Tractor Ground Speed Comparison

We wanted to see if a slower tractor ground speed (1.2 mph), would improve performance of the vacuum compared to the recommended speed of ~2.0 mph that we identified in 2014. One test was conducted on May 22, 2015, to compare the two speeds. The treatments (1.2 vs 2.0 mph) were replicated three times.

Multiple Sequential Vacuum Pass Trial

The grower standard vacuuming frequency is two passes per field per week. eW tested whether you could intensively vacuum down the lygus population in a field by completing repeated sequential passes on one day, and we counted the number of lygus that were picked up on each sequential pass. We conducted separate trials on two dates (August 11 and 18, 2015) and evaluated the effect of four consecutive sequential pass conducted on each date. The treatment was replicated four times on each date. Lygus counts in the treatment blocks were made before the first pass and immediately after each pass using a beat-box.

85 2014 - 2015 RESEARCH PROJECTS Results Modifications New High-pressure Motor

A side-by-side comparison of the research vacuum equipped with the high pressure motor with an industry standard vacuum showed that the lygus collection efficiency of the grower standard vacuum running at 33 mph wind speed (2000 psi) was 2.51% (i.e., they sucked up 2.51% of the estimated lygus in the field) whereas the modified vacuum fitted with high-pressure motors (Danfoss SNM2) running at 3000 psi had an average wind speed of 47 mph and was 8.75% (F(1,6)=6.2, p=0.047) efficient at picking up lygus from the field. This demonstrated that the newly modified vacuum picked up more than double the amount of lygus than the grower standard vacuum running when it was running at 2000 psi.

Front Opening Modification on the Front of the Hood

The installation of the 9-inch wide plate to reduce the height of the front opening of the vacuum hood, increased the number of all lygus life stages that were picked up by the vacuum compared to the hood without the plate (Figure 2). On average, the hood (run at 3000 psi) with the front plate picked up 2.57±0.35 times more total lygus than without the plate, this significantly increased the efficiency of the vacuum to an average of 16% to 18% efficiency compared to a 5% to 8% efficiency without the plate. The increased efficiency of the vacuums with the front plate installed basically doubled the number of each of the life stages of lygus picked up (Table 1) compared to the vacuum without the plate.

Figure 2. Average number of lygus of each life stage sucked up by the research bug vacuum when fitted with and without, a front plate designed to reduce the height of the opening at the front of the vacuum. The vacuums were run at 3000 psi Trial conducted on October 28, 2015. Each sample is the number of lygus sucked up by the vacuum over 300 feet of bed, there were four replicates of each treatment (with and without front plate).

86 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

Table 1. Mean lygus picked up by the research bug vacuum run at 3000 psi when fitted with a front plate modification to the front of the hood compared to the standard vacuum without the modification. Trials were conducted with four replicates of each treatment on October 8, 21 and 28, 2015, and analyzed globally as matched pairs (N=12).

Operating Procedures Time of Day and Moisture

There was a lower background lygus population during the May 13, 2016 trial (3.8±0.73 avg total lygus per beat-box sample) than during the trial on May 21, 2015 (8.45±0.77 avg total lygus per beat-box sample). There were significantly

more lygus of all life stages picked up during the afternoon than morning or night during the test on May 13, 2015 (F(2,9)=9.55,

p=0.006 for adults; F(2,9)=11.32, p=0.004 for large nymphs, F(2,9)=4.18, p=0.05 for small nymphs). However, there was no significant difference on May 21, 2015. During both trials it was dry during the afternoon samples, whereas the plants were wet during the morning both days, and at night during the May 21, 2015 sample. It was not possible to determine whether time of day or moisture contributed to the results because it was never wet during the afternoon.

Tractor Ground Speed Comparison

There was no significant improvement in the number of lygus picked when the vacuum was slowed from 1.9 to 1.1 mph during our trial conducted (Figure 3) on May 22, 2015.

Figure 3. Average number of lygus picked up by the research vacuum when operating at 1.1 mph and 1.9 mph. Trial conducted on May 22, 2015; there were four replicates of each treatment and each pass was 350 feet.

87 2014 - 2015 RESEARCH PROJECTS Multiple Sequential Vacuum Pass Trial

It is possible to continue to pick up lygus on sequential passes of the vacuum over the same beds, and there was not a significant drop off in the number of lygus picked up in subsequent passes (Figure 4) until the third pass in our trial in a field with a low lygus population density, and until the fourth pass in a field with a high lygus population density (Figure 5). One interesting result was that during the multiple passes, the average percent of each sample that was comprised of small nymphs was relatively high (44.6±1.5%). Beat box sampling of the vacuumed beds in the field after the end of the trial suggested that overall lygus populations were 42% lower than before the trial in the field with the high population density, a larger reduction than would be predicted from the lygus in the vacuum samplings.

Figure 4. Average number of lygus picked up during repeated passes of bug vacuums over the same beds for a field with a low population density. Trial was conducted on August 11, 2015; there were three replicates.

Figure 5. Average number of lygus picked up during repeated passes of bug vacuums over the same beds for a field with a high population density. Trial was conducted on August 18, 2015; there were three replicates.

88 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Entomology

Discussion Our recommendations from the 2014 season were to modify the baffles on the exit side of the hood’s fan opening. This modification, using punch plate with at least ¼ in holes in it and using a 20% deflector angle), raised the efficiency of bug vacuums from 2.5% to above 4% (Thomas & Legard, 2014). Testing of new high pressure motors (Danfoss SNM2) this season further increased the efficiency of our test vacuum to approximately 8%. The new motors are able to run at a higher pressure (3000 psi) which enables the fans to generate a higher wind speed. It will likely require more fuel to run the motors at the higher psi, but we feel that the benefits from the increased performance outweigh the greater costs. The addition of a front plate to reduce the height of the intake further increased the efficiency of the bug vacuum efficiency to 15 to 18%. If growers modify their bug vacuums by: 1) installing the modifications previously outlined in the 2014 report (tractor speed, wind speed, punch plate baffle at 20% angle), 2) install the larger motors and run the vacuum fans at 3000 psi, and 3) install a plate to reduce the height of the front opening of the vacuum hoods, we expect that they will dramatically improve the performance of vacuums in managing lygus in their fields. More research is needed on scheduling the use of the modified vacuum to optimize its efficiency in removing lygus from strawberry fields as well as on further modifications to the vacuum that could further improve its efficiency.

The number of lygus collected by the vacuum on consecutive passes over the same beds suggests that there may be an opportunity to improve on the grower standard operating procedure of vacuuming twice a week. Our test suggests that under some situations it may be better to intensively vacuum areas of the field with high numbers of lygus rather than using the standard twice-a-week schedule. The high percent of small nymphs picked up with the modified vacuum suggest that this tool may be effective at controlling new hatches of lygus. However, a rapid rebound of lygus populations in regions of the field where we intensively vacuumed to reduce the population suggest that if there are significant egg hatches within a field, then a long term vacuum scheduling strategy would be needed to effectively manage lygus.

Selected References • Thomas, H.Q. and Legard, D.E. 2014. 2014 Lygus and Mite Management Program. Annual Production Research Report. California Strawberry Commission.

89 2014 - 2015 RESEARCH PROJECTS 90 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Weed Science

WEED SCIENCE

91 2014 - 2015 RESEARCH PROJECTS 92 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Weed Science

Weed Management in Strawberry

Principal Investigator Dr. Steven Fennimore Cooperative Extension Weed Specialist Dept. of Plant Sciences University of California, Davis USDA Agricultural Research Station 1636 East Alisal Street Salinas, CA 93905 (831) 755-2896 [email protected]

Cooperating Investigators Dr. Oleg Daugovish UCCE Ventura Ventura, CA

Cooperating Personnel & Collaborators Dr. Thomas C. Miller & John S. Rachuy Dept. of Plant Sciences U.C. Davis Salinas, CA

Jose Garcia Salinas, CA

Summary Weed management in strawberry is costly due to need for hand weeding labor. Alternative fumigants are not as effective on weeds as methyl bromide and chloropicrin (MB/Pic). Therefore, we continue to evaluate potential new herbicides and fumigants to contain or reduce weed management costs in California strawberry. In the 2013/14 season we evaluated the herbicide clopyralid (Stinger ®) and the fumigant dazomet (Temozad ®). Our objectives were: 1) to investigate efficacy and timing of clopyralid application by drip chemigation and 2) to investigate efficacy of dazomet incorporated by two methods: a) sprinkler irrigation; b) mechanical incorporation with a rototiller. Clorpyralid was found to provide good weed control when applied mid-winter through drip tape and was safe to strawberry. With the exception of nutsedge, dazomet provided good weed control either applied to the surface or mechanically incorporated and was found to have similar weed control efficacy as standards K-Pam and Pic-Clor. Mechanically incorporated dazomet placed the fumigant 6 to 8 inches below the surface which controlled nutsedge better than surface applied dazomet.

93 2014 - 2015 RESEARCH PROJECTS Introduction Due to the transition from methyl bromide (MB) to alternative fumigants and increasing regulatory actions, weed management times in California strawberry are increasing. MB alternative fumigants provide less weed control than MB provides (e.g., of yellow nutsedge, Malva, clover, etc.) and fumigation options are limited in buffer zones. Shortages of labor for fruit harvest mean even less labor is available for hand weeding. In combination with increasing labor costs, weed management significantly contributes to increasing strawberry production costs in California. Herbicides can significantly reduce production costs in strawberries by reducing need for hand weeding. Herbicides can provide residual weed control during the production season. In previous projects, we have determined safe and effective rates of novel herbicides (Clorpyralid) and MB alternatives. The purpose of this project was to assess the weed control efficacy and the timing of Clopyralid by drip irrigation and the efficacy of the fumigant dazomet. Field studies were conducted to address the difficulties with management of nutsedge, clovers and other weeds in strawberry. The main aim of this research was to contain or lower weed control costs for strawberry producers through development of herbicide treatments that complement existing fumigants.

Objectives (1) To evaluate the efficacy and timing of clopyralid applied by drip chemigation.

(2) To investigate efficacy of dazomet incorporated by sprinkler irrigation versus mechanical incorporation.

Objective 1: Efficacy and timing of Clorpyralid (Stinger ®) applied by drip chemigation

Materials, Procedures Clopyralid was applied at 1/6 and 1/3 pint/acre on September 19, 2013 to 26 inch wide finished bed tops with a CO2 powered backpack sprayer (R&D Systems) at 40 GPA (374 l/ha), 30 psi (207 kPa), 61 d prior to transplanting. For comparison, standards Chateau 51 WDG (flumioxazin) 3 oz/acre and GoalTender 1 pint/acre were also similarly applied. Beds were covered with plastic mulch within one week after application. On October 24, 2013, Pic-Clor 60 was applied through the drip tape at 29 GPA (271 l/ha) to the entire trial, including the controls. Beds were 48 inches (= 1.22 m) wide on center, 2 rows per bed, planted at 12.5 inch in-row spacing to ‘Albion’ strawberry (November 19, 2013). Plots, replicated four times per treatment, were arranged in a randomized complete block design. Plots were 50 ft. (= 15.2 m) long with approximately 95 plants per plot at a plant population of 21,125 plants/A. Clopyralid was applied to the respective plots 30 days following transplanting, again at 1/6 and 1/3 pint/A, both by spray application over the top of the plants and by drip chemigation on December 19, 2013. These four clopyralid treatments were applied to an additional four plots mid- winter (February 12, 2014). Phytotoxicity, canopy diameters, plant stand, weed density, weed biomass and fruit yield were monitored.

Results While Chateau and GoalTender provide good weed control, the only chance to apply them to the bedtop under the plastic is 30 days before transplanting. An application that could be applied under the plastic during the season would be valuable for providing supplemental weed control in-season. However, the clopyralid 30 post transplant drip application caused slight to moderate injury to strawberry, higher than was seen for the clopyralid spray application on the same

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date (Table 1). The clopyralid 30 day pre-transplant, and midwinter application were safe to strawberry. In the 15 other states which have approved Stinger for strawberry, some have pre-harvest intervals as short as seven days, allowing the possibility of later season control than afforded by current options. Future studies might focus upon season-long control opportunities afforded by product combinations such as Chateau followed by a midwinter clopyralid application. Clopyralid has 18-month plant back restrictions for vegetable crops such as carrot, celery and lettuce. Clopyralid would be most useful in fields where strawberry is grown continuously and in those areas where the field can be rotated to more tolerant crops such as beans, onion or spinach following strawberry.

Conclusion Clopyralid has potential for use in California strawberry and should be evaluated further.

Table 1. Weed control, stand, phytotoxicity, plant diameters and fruit yield of drip and spray applied Stinger at different rates and timings in Strawberry.

z accumulation of counts on January 15, March 5, April 8 and May 27, 2014. y scale of 0 = no phytotoxicity to 10 = death; values < 2.0 are commercially insignificant. x mean separations by Duncan’s multiple range test. Means not sharing common letters are significantly different at P = 0.05. w separation and probability on transformed [square root (x + 0.5)] data to homogenize variance.

Objective 2: Efficacy of broadcast application of Dazomet (Temozad ®) incorporated by two methods sprinkler versus rototiller.

Materials, Procedures Dazomet was applied at 220, 350, and 421 lb/acre to the surface of the bed tops with a Gandy applicator (Owatonna, MN) on September 23, 2013, 57 d prior to transplanting (November 19, 2013). Half of dazomet plots were then rototilled and shaped to incorporate the material 6 to 8 inches into the bed. The other half of the dazomet plots were incorporated by sprinkler irrigation over a five day period as per label instructions.

95 2014 - 2015 RESEARCH PROJECTS Beds were then covered with plastic mulch. For comparison, the fumigants K-Pam 50 GPA and Pic-Clor 60 29 GPA were applied by drip chemigation on October 10, 2013 and October 24, 2013, respectively. Beds were 48inches (= 1.22 m) wide on center, 2 rows per bed, planted at 12.5 inches within row spacing to ‘Albion’ strawberry 11.19.13. Plant population was 21,125 plants/A. Treatments were replicated four times and arranged in a randomized complete block design, with single bed plots 50 ft. long (= 15.2 m) long containing approximately 95 plants per plot. Phytotoxicity, canopy diameters, weed densities, weed biomass and fruit yield data were collected.

Results Mechanical or sprinkler incorporation of dazomet at the medium and high rates controlled weeds similar to K-pam or Pic-Clor 60 drip with no negative impact on yield (Table 2). The higher yields in dazomet treated beds than in non-treated beds likely are associated with the pathogen suppression afforded by dazomet, as the low rate of dazomet, while not sufficient to control weeds, still showed yields greater than those in non-treated plots. The dazomet 220 lb/A treatment was too low at this site, and growers should consider their field histories in choosing a sufficient rate. Controlled tests of seed of shepherd’s purse, burning nettle, ryegrass, and tubers of nutsedge were all controlled at even the lowest rates, when dazomet was incorporated, but nutsedge control was not as effective when dazomet was sprinkled in, likely due to failure of the fumigant to reach sufficient concentration at depths where tubers were placed (Table 3). Also, the dazomet 421 lb/A rate when sprinkled in reduced plant stand, requiring more replants. Aside from the nutsedge, there was no difference in weed control results observed between the two dazomet incorporation methods, and the high rate replant issue associated with sprinkling in tips the balance toward rototill as the safer method. The sandy soil at this site made the sprinkler incorporation difficult because the beds tended to collapse.

Conclusions Dazomet has potential for use in California strawberry. The best way to use dazomet is at low rates applied before or after chloropicrin is applied.

Table 2. Strawberry stand (%), phytotoxicity estimates, plant diameters and fruit yield in plots treated with dazomet.

z scale of 0 = no phytotoxicity to 10 = death; values < 2.0 are commercially insignificant. y mean separations by Duncan’s multiple range test. Means not sharing common letters are significantly different at P = 0.05.

96 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Weed Science

Table 3. Weed seed survival and field weed densities resulting from several rates of dazomet incorporated by rototiller and sprinkler.

z accumulation of counts on January 14, February 19, April 9 and May 16, 2014. y mean separations by Duncan’s multiple range test. Means not sharing common letters are significantly different at P = 0.05. x gallons per acre (gpa) wmean separations and P value from data transformed to arcsine square root percent to address heterogeneity of variance. vbinomial data with no variance

97 2014 - 2015 RESEARCH PROJECTS 98 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT FARMING WITHOUT FUMIGANTS

99 2014 - 2015 RESEARCH PROJECTS 100 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Farming Without Fumigants

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

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]

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

Dr. Dan Legard Vice President, Research & Education California Strawberry Commission P O Box 269 Watsonville, CA 95077 (831) 724-1301 [email protected]

101 2014 - 2015 RESEARCH PROJECTS Summary Project objectives were to: 1) test anaerobic soil disinfestation (ASD) and mustard seed meal (MSM) at a large scale field demonstration site infested withMacrophomina phaseolina in the Ventura district; 2) better understand interactions between temperature, disease control, fruit yield and N dynamics under summer flat ASD treatment at the Monterey Bay Academy (MBA) site, and 3) examine whether changes in soil microbial community composition are responsible for soilborne disease suppression by ASD and MSM in above mentioned field trial.

For objectives 1 and 3, a demonstration trial was established at M. phaseolina-infested organic site used for the last year’s trial in Oxnard, CA in August 2014. One acre treatments of ASD rice bran 9 t/ac (ASD-RB9), ASD rice bran 6t/ ac (ASD-RB6), MSM 2.5 t/ac and GS practices were established. One t/ac of pre-plant organic fertilizer (10-10-2.5) was applied only to the GS plot. ASD-RB9 treatment greatly improved crops yields relative to the grower’s standard practice without pre-plant fertilizer and reduced disease severity. Soil fungal community analysis showed that only ASD-RB9 treatment maintained a distinctive community from the 2013-14 season to the 2014-15 season, suggesting that a certain rate of C-source may be necessary to maintain specific soil microbial communities and associated disease suppression function. Pelleted MSM did not increase yield nor decrease plant mortality in this trial though powdered MSM warrants further study.

For objectives 2 and 3, a replicated trial included ASD-flat with rice bran 9 t/ac (ASD-flat RB9), ASD-flat with molasses 6 t/ac (ASD-flat-ML6), chloropicrin 300 lbs/ac (Pic 300) and untreated check (UTC) as main plots, and with and without pre-plant fertilizer (PPF) as sub plots was established on Fusarium oxysporum f. sp. fragariae (F.o.f.)-infested MBA site in Watsonville, CA. Unlike the 2013-14 season, due to the late start of the treatment, soil temperatures in ASD-flat-RB9 barely reached the cumulative temperature threshold and did not reach the threshold in ASD-flat-ML6. As a result, ASD treatments did not reduce F.o.f. in the soil compared to UTC regardless of type of C-source whereas Pic 300 reduced the populations significantly. Reflecting the soil F.o.f level, Pic 300 possessed the highest yield, while yields for ASD-flat- RB9 was ~50%, and ASD-ML6 and UTC was ~30% of Pic 300. Changes in soil inorganic N distribution across the soil profile before and after the flat ASD treatment indicated mineralized N accumulation in the subsoil rather than top soil suggesting N from rice bran in flat-ASD treatment may not be used efficiently by strawberries. T-RFLP analysis indicated that a distinctive soil bacterial community was formed by each main treatment immediately post-treatment though it did not affect the soil F.o.f. population. To reduce Fusarium wilt by ASD, summer flat ASD must start by mid-August at latest in the central coast of CA.

Introduction Anaerobic soil disinfestation (ASD) has been shown to be effective in reducing Verticillium dahliae microsclerotia in soil by 80 to 100% and to be able to provide marketable fruit yields compatible to fumigation in California strawberry systems (Shennan et al., 2014a). In response, California berry growers started to adopt ASD at a commercial scale; ASD acreage has rapidly increased from 5 acres to 1,017 acres in the last four years (Farm Fuel Inc. personal communication). Although this represents roughly 20% of California organic strawberry acreage, it is only 2.5% of total strawberry acreage.

Mustard seed meal (MSM) has been successfully used for controlling the replant disease complex of apples, and providing similar or superior yield compared to fumigant controls (Mazzola and Brown, 2010; Mazzola et al., 2015). It has also been evaluated in strawberry systems and showed neutral to positive effects on fruit production and disease suppression when used alone or in combination with other non-fumigant approaches such as ASD and steam (Fennimore et al., 2014).

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To further expand adoption of non-fumigant approaches and to evaluate the practicality of these methods, large scale demonstrations in fields with different soil types, production regions, and disease pressure are necessary.

In the 2013-14 seasons, ASD and MSM were tested for controlling two emerging lethal diseases of California strawberries; Charcoal rot caused by M. phaseolina and Fusarium wilt caused by F. oxysporum f. sp. fragariae (F.o.f.) (Shennan et al., 2014b). In the demonstration trial in Oxnard, a doubling of yields and a 50% reduction of mortality relative to the GS was achieved by using ASD with rice bran 9 t/ac at this M. phaseolina-infested organic site. ASD lowered the soil pH to more optimal levels for strawberry growth relative to other treatments. In contrast MSM applied at 2 t/ac failed to control M. phaseolina and resulted in yields that were more than 20% below the ASD-RB9 yields, although still substantially higher than the GS. A combination of phytotoxicity following transplanting, and subsequent salinity damage due to high nitrate levels over the winter may have reduced yields in the MSM treatment. As previously observed for other locations, application of ASD with RB or use of MSM led to distinctly different microbial community profiles which may be important for disease control.

Previously we had shown that fall applied ASD did not control F.o.f. at the MBA location in Watsonville, and hypothesized that this was due to soil temperatures that were too low, since ASD successfully controlled Fusarium elsewhere (Rosskopf et al., 2014; Shennan et al., 2014b). Data for the 2013-14 season at MBA generally support this notion, with the greatest reduction in Fusarium in the soil, and lowest incidence of wilt for the susceptible cultivar ‘Albion’, observed following summer applied ASD (flat treatment). Soil temperatures during summer ASD exceeded the cumulative soil temperature threshold for Fusarium wilt control based on research in Japan (Yonemoto et al., 2006). Nonetheless, fruit yield was low due probably to unexpectedly low nitrogen availability in this treatment.

In the 2014-15 season, two trials mentioned above are being repeated with refinements at the same locations to see if greater pathogen control can be achieved.

The project objectives were to:

1. Test ASD and MSM in a large scale field demonstration (1 acre) infested with M. phaseolina in Ventura district.

2. Better understand interactions between temperature, disease control, fruit yield and N dynamics under summer flat ASD treatment at Fusarium-infested MBA site.

3. Examine whether changes in soil microbial community composition are responsible for soilborne disease suppression by ASD and MSM in the above mentioned field trials.

Here we report results from the 2014-2015 experiments thus far.

Materials and Methods Objectives 1 and 3 In August 2014, a non-replicated strawberry demonstration trial was established at the same organic site with the last year’s trial in Oxnard, CA. The soil type is Pico sandy loam and the site had seven to eight years of organic management history. Treatments were ASD rice bran (RB) 9 t/ac (ASD-RB9), ASD RB 6 t/ac (ASD-RB6), MSM 2.5 t/ac (MSM pelleted) and grower standard practices (GS). Plot size was 1 acre for each treatment. A 2,000 lbs/ac of pre-plant organic fertilizer (PPF) was applied only to the GS plot. The same treatments, except for the RB 6 t/ac plot, had been applied to the same plots for the previous strawberry crop which was followed by a wheat cover crop incorporated in early August 2014. Instead of RB 6 t/ac, the previous treatment had been ASD using RB 3 t/ac plus MSM 2 t/ac as C-sources. Rice bran, MSM, and

103 2014 - 2015 RESEARCH PROJECTS PPF were applied to assigned plots on August 25 by broadcaster then incorporated with a spring hallow, beds shaped, rotor-tilled with a bad shaper-attached rotor-tiller, drip lines added and TIF plastic tarp laid down. Approximately 9 a/in of water was applied to ASD and MSM plots from August 25 to September 26 (treatment period: 32 days). Soil temperature and soil Eh, an indicator of anaerobisis, at 6 inch depth, were measured daily at ASD plots using soil temperature probes and ORP sensors (Sensorex, Garden Grove, CA) during the treatment.

A ‘Propriatary’ strawberry variety known to be susceptible to Fusarium wilt and charcoal rot, was planted on October 14, 2014 ensuring a six week plant back time for MSM treatment to avoid the phytotoxicity. Strawberries were harvested and marketable yield at each plot was recorded by the grower from January to May 2015. Plant mortality was measured by counting dead or nearly dead plants in each plot. Each plot was divided into four sections and soil samples taken from 0 to 6 inch depth pre-treatment, post-treatment, and then monthly throughout the growth period from each section for inorganic N and EC (1:1) and pH (1:1) analysis. Pre- and post-treatment soils were also subject to microbial analysis by USDA-ARS, WA using real-time quantitative PCR (RT-PCR) and terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate the treatment effect on nutrient status of strawberry plants, 20 young-matured leaf blades were randomly sampled from each section of the plot on April 23, 2015. After drying and milling to pass through 0.5 mm sieves, tissue samples were shipped to UC Davis Analytical Laboratory for N, P, K, S, B, Ca, Mg, Zn, Mn, Fe, and Cu analysis. Data were expressed as averages and standard errors when data were collected from pseudo-replicates.

Objectives 2 and 3 A randomized block split plot experiment with four replicates was designed with ASD with RB 9 t/ac (ASD-RB), ASD with molasses 6 t/ac (ASD-Mol), chloropicrin 300 lbs/ac (Pic 300), and untreated check (UTC) as main plots, and with or without preplant fertilizer (1,000 lbs/ac of 18-8-13, six to seven month slow release fertilizer) as sub plots. Plot size was a 50 foot long bed with 52 inch wide. Rice bran was broadcasted to the assigned plots and rotor tilled on September 3, 2014. On the same day, drip tapes and then clear TIF were laid to all ASD plots. For ASD-RB plots, 2.3 a/in of water was drip irrigated on September 3 and 4. For the ASD-Mol plots, molasses was injected via drip tapes on September 4 and 5, with 5.9 ac-in of water. In both ASD plots, no additional water was added during the treatment period that lasted for 21 days. Soil temperature at 6 inch and 8 inch depths and soil Eh, an indicator of anaerobisis, at 6 inch depth, were monitored at ASD and UTC plots using 5TE sensors (Decagon Inc., Pullman, WA) and ORP sensors (Sensorex, Garden Grove, CA) connected to data loggers during the treatment.

TIF and the monitoring system were removed on September 26. The Pic 300 plots were bed fumigated on October 2. Strawberry ‘Albion’ was planted on November 18. To examine N dynamics, a composite soil samples of 10 to 20 subsamples from 0 to 6 inches, 6 to 12 inches, 12 to 18 inches, and 18 to 24 inches depth was collected from each plot at pre-treatment (September 2), post-treatment (September 26), and immediately after bed listing (October 23). After planting, 0 to 6 inch soil depth samples have been collected monthly for inorganic N analysis. Post-treatment soil samples (0 to 6 inch and 6 to 12 inch depth) were also sent to Tree Fruit Research Lab, USDA-ARS, WA for pathogen analysis. Wilt score using scale of 1 (no dead leaves) to 5 (76 – 100% dead leaves) for all plants per plot has been measured biweekly and the maximum plant canopy diameters of 20 random samples per plot has been measured monthly both from March 25 to August 12, 2015. Marketable and cull fruit yield of each plot has been monitored for all plants per plot biweekly since April 2, 2015 till August 6, 2015. For statistical analysis, data were subject to ANOVA and mean separation using Tukey’s HSD test (P<0.05).

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Results Oxnard Site Objectives 1 and 3 Strawberry plants grew well from November 2014 to February 2015. Unlike previous season, MSM plot did not show any die-out soon after planting due to the six week plant-back time and the irrigation during the treatment period in MSM plot. Plant mortality was observed initially in late March (Figure 1) and the major pathogen was identified as M. phaseolina, though F. oxysporum was also identified at less frequency. Overall crop mortality in late season (as of May 4, 2015) was about a half of the previous year (Figure 1). ASD-RB9 treatment reduced plant mortality by 50% compared to GS plot, but ASD-RB6 and MSM treatments did not. Plant size was largest in ASD-RB9 plot, followed by ASD-RB6 plot and MSM plot, and the smallest in GS plot (Figure 2).

Figure 1. Mortality of strawberry crop in the Oxnard demonstration trial in the 2013-14 and the 2014-15 seasons.

105 2014 - 2015 RESEARCH PROJECTS Figure 2. Plant growth at Macrophomina-infested demonstration trial at an organic site in Oxnard, CA (April 23, 2015). From left to right, mustard seed meal 2.5 t/ac, ASD rice bran 6 t/ac, ASD rice bran 9 t/ac, and the growers standard treatments. Only the GS plot received pre-plant fertilizer (10-10-2.5, 2,000 lbs/ac).

Marketable yield of the 2014-15 trial was much lower than the previous year; approximately 50% of the 2013-14 season (Figure 3). The lack of market in spring 2015, caused by the early production of the northern districts as a result of the warm spring weather, forced many growers in the southern district to leave ripened fruits unharvested and these fruits were not included in marketable yield data. For the same reason, yield data collection ended on April 23, a month earlier than the previous year. This also contributed to the relatively low and probably somewhat skewed yield data of this season. Regardless, as seen in previous season, ASD-RB9 and ASD-RB6 treatments doubled marketable fruit yield relative to GS plot even without pre-plant fertilizer application. Mustard seed meal treatment did not increase fruit yield compared to GS plot.

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Figure 3. Relative cumulative marketable fruit yield in the Oxnard demonstration trial in the 2013-14 and the 2014-15 seasons. MSM 2 t/ac (2014) or 2.5 t/ac (2015), ASD RB6; ASD with rice bran 3 t/ ac + MSM 2 t/ac (2014) or rice bran 6 t/ac (2015), ASD RB9; ASD with rice bran 9 t/ac (2014 and 2015), GS; growers’ standard practice (2014 and 2015).

The ASD-RB9 treatment lowered the soil pH further to more optimal levels for strawberry growth relative to other treatments (Figure 4A). Unlike the 2013-14 season, a high soil EC (1:1) immediately post-treatment in MSM plot was not observed in the 2014-15 season (Figures 4A & B). Soil nitrate concentration had a positive linear correlation with soil EC (1:1) (R2 = 0.49***. n=240) and a negative linear correlation with soil pH (1:1) (R2 = 0.11**. n=240) indicating the great influence of this ion on both soil pH and EC (Figures 5A & B).

107 2014 - 2015 RESEARCH PROJECTS A) B)

Figure 4A & 4B. Changes in a) soil pH (1:1) and b) soil EC (1:1) during the two seasons of the Oxnard demonstration trial. See Figure 3 for legend.

A) B)

Figure 5A & 5B. Correlations between nitrate concentration and a) pH (1:1) or b) EC (1:1) in topsoil (0 to 6 inch depth) during the two seasons of the Oxnard demonstration trials.

There was a significant shift in fungal community composition in soils post ASD, relative to the MSM and the GS (Figure 6A). However, prior to treatment the following year and after an intervening wheat cover crop, fungal communities from ASD-RB9 treated plots still clustered together and were distinct from the other plots (Figure 6B) again indicating longer term biological changes in soil following ASD. Following the second year with ASD treatment soil fungal communities again showed very distinct clustering based on treatment (Figure 6C). This pattern has been observed in other field trials and we are in the process of identifying which species become more prevalent following ASD and if this is related to type of carbon source.

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A) B) C)

Figure 6A, B & C Fungal community similarity based on ITS T-RFLP analysis A) post-treatment October 2013, B) pre-treatment August 2014 and C) post-treatment September 2014 from the Oxnard demonstration trial.

Nutrient status of strawberry plants in April was evaluated by contrasting nutrient content in leaf blades to the sufficient range established for strawberries during the main harvest season in coastal California (Bottoms et al., 2013). Assuming the sufficiency ranges developed based on data from the Santa Maria and Salinas/Watsonville area are applicable to these samples, all nutrients except Ca (in all plots) and Mg (in ASD-RB6 and 9 plots) were in or slightly above the sufficient range (Table 1).

Table 1. Mineral nutrient content in strawberry leaf blades from the Oxnard demonstration trial. ’Proprietary’ cultivar sampled on April 23, 2015.

109 2014 - 2015 RESEARCH PROJECTS MBA Site Objectives 2 and 3 Unlike the 2013-14 season, due to the late start of the treatment, soil temperatures in ASD-flat-RB9 barely reached the cumulative temperature threshold (> 300 cumulative hours above 86 F at 8 inch soil depth (Yonemoto et al., 2006)) and did not reach the threshold in ASD-flat-ML6 where additional water was used to inject molasses (Table 2).

Table 2. ASD conditions of the summer flat ASD trials (the 2013-14 and 2014-15 trials) at the MBA site, Watsonville.

As a result, unlike the 2013-14 season (Figures 7A & B), ASD treatments did not reduce F.o.f. in soil compared to UTC regardless of C-source type whereas Pic 300 reduced populations significantly (Figure 7b). Reflecting the soil F.o.f. level, plants ASD and UTC plots started to show disease symptom in late March. Throughout the growth season, wilt score was lowest for Pic 300 and no difference was observed between ASD and UTC though use of PPF slowed disease progression until May (Figure 8A). Pic 300 possessed the highest yield, while yields for ASD- flat-RB9 was ~50%, and ASD-ML6 and UTC was ~30% of Pic 300 (Figure 8B). Soil inorganic N dynamics in 0 to 6 inch soil in ASD-RB9 plots during the growth season showed a similar low level with the previous season (Figures 9A & B). Changes in soil inorganic N distribution across the soil profile (0 to 24 inch) before and after the flat ASD treatment indicated mineralized N accumulation in the subsoil (6 to 12 inch depth) rather than top soil (0 to 6 inch depth) (Figures 10A, B & C). T-RFLP analysis indicated that a distinctive soil bacterial community was formed by each main treatment immediately post-treatment though it did not affect the soil F.o.f. population (Figure 11).

A) B)

Figure 7A & 7B. Fusarium oxysporum population in soil post-treatment at A) the 2013-14 trial and B) the 2014-15 trial at the MBA site, Watsonville.

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A) B)

Figure 8A & 8B. Wilt score development, and B): cumulative marketable fruit yield in ASD trial in MBA, Watsonville. Wilt score 1 = healthy plant, 2 = 1-25% dead leaves, 3 = 26-50% dead leave, 4 = 51-75% dead leaves, 5 = 76-100% dead leaves.

A) B)

Figure 9A & 9B. Soil inorganic N (NH4-N + NO3-N) dynamics in selected plots of the 2013-14 (A) and the 2014-15 (B) trials in MBA site, Watsonville. 0 to 6 inch depth. PPF: pre-plant fertilizer. UTC: untreated check. In the 2013-14 trial, PPF (640 lbs/ac of 18-6-12, 12 to 14 months slow release) was applied only to UTC. In the 2014-15 trial, PPF (1,000 lbs/ac 18-8-13, seven to nine months slow release) was applied to PPF sub plots. There was 0.76 inch of precipitation on April 7, 2015 lowering inorganic N content in the soil sampled on April 14, 2015.

111 2014 - 2015 RESEARCH PROJECTS A) B) C)

Figure 10. Soil inorganic N dynamics across the soil profile in summer flat ASD without pre-plant fertilizer treatment from A): pre-ASD flat treatment (September 2, 2014), B) post-ASD flat treatment (September 26, 2014), and C) immediately after bed listing (October 23, 2014) in the MBA trial, Watsonville.

Figure 11. Similarity of soil bacterial community composition among treatment plots immediately post- treatment application at the MBA trial Watsonville as determined by T-RFLP analysis. UTC: un-treated check, ASD-RB: ASD with rice bran 9 t/ac, ASD-ML: ASD with molasses 6 t/ac, PIC 300: chloropicrin 300 lbs/ac.

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Discussion Trials were repeated at the same locations as the previous season to determine whether greater control of charcoal rot by M. phaseolina and Fusarium wilt by F. oxysporum f. sp. fragariae could be achieved.

At the Oxnard demonstration trial where the field was infested byM. phaseolina, ASD-RB9 treatment again doubled fruit yield relative to the GS treatment as observed during the last season (Figure 3). In particular, this year’s trial demonstrated that pre-plant fertilizer is not necessary in ASD-RB9 treated plots. ASD-RB9 was also the only treatment that reduced strawberry mortality (by ~50%) compared to the GS plot (Figure 1). Although relative yield of ASD-RB6 plot was similar to ASD-RB9 plot (Figure 3), given the fact that the plant size was much larger in ASD-RB9 plot (Figure 2), it is likely that the yield data were skewed by the lack of yield data collection in April to May (see results for more details). Further, soil fungal community analysis showed that only ASD-RB9 treatment maintained a distinctive community that persisted from the 2013-14 season to the 2014-15 season (Figure 6) suggesting a certain rate of C-source may be necessary to maintain specific soil microbial communities and associated disease suppression function for an entire season.

Soil nitrate appeared to play a key role in pH and EC dynamics of this low-buffered sandy-loam soil (Figure 5). To better understand overall ion dynamics in this field, analysis of other soluble ions are required. Level of nitrate leaching should also be monitored in ASD plots in future experiments.

Mustard seed meal did not increase yield nor decrease plant mortality in this trial. Recent studies indicated that the release of AITC from pelleted MSM was ~1/10 of that from powdered MSM (Mazzola, unpublished). Thus use of powdered MSM for disease control warrants further study.

In the MBA trial in Watsonville, the site highly infested with F.o.f., summer flat ASD failed to control Fusarium wilt this year due most likely to insufficient cumulative soil temperature (Table 1). To reduce Fusarium wilt by ASD, summer flat ASD must start by mid-August at latest in the central coast of CA. In controlled environment experiments (88/76 F, two weeks), ASD with grass effectively suppressed populations of an introduced F.o.f. isolate and suppressed wilt (Figures 12A & B). Effectiveness of summer flat ASD with varying C-source on F.o.f. will be examined in the next field trial.

N in C-source for summer flat ASD may not be efficiently used by strawberries. Thus, a low-N C-source would be more appropriate for the summer flat ASD. A shift in bacterial community induced by ASD was not related to immediate disease suppression in this case though other studies indicate that it may be related to the development of disease suppressiveness. At this point, use of a resistant cultivar, crop rotation, good sanitation and a combination of all three are the most effective non-fumigant Fusarium wilt management strategies for strawberries in the central coastal CA.

113 2014 - 2015 RESEARCH PROJECTS A) B)

Figure 12A & 12B. Populations of F. oxysporum f. sp. fragariae (A) and resulting crown infection of strawberry (B) as affected by ASD conducted using 9 t/ac of dry grass as the carbon input. Error bars indicate one standard deviation of the mean.

Acknowledgements This project was co-funded by the California Strawberry Commission and USDA Areawide Project (2094-21220-001- 15S). We thank our collaborators and cooperators Will Doyle of WD Farms, Dole Berry Company, Inc., Mark Edsall, Alex Orozco, Daniel Olivier, Myles Shoemaker, and Hillary Thomas of the California Strawberry Commission, and staff, students and volunteers of the Shennan lab, UC Santa Cruz; and Shashika S. Hewavitharana, Department of Plant Pathology, Washington State University.

Selected References

• Fennimore, S.A., Martin, F.N., Miller, T.C., Broome, J.C., Dorn, N. and I. Greene. 2014. Evaluation of a Mobile Steam Applicator for Soil Disinfestation in California Strawberry. Hortscience 49:1542-1549.

• 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.

• Mazzola, M., Hewavitharana, S. S. and S. L. Strauss. 2015. Brassica seed meal soil amendments transform the rhizosphere microbiome and improve apple production though resistance to pathogen re-infestation. Phytopathology 105:460-469.

• Rosskopf, E.N., Burelle, N., Hong, J., Butler, D.M., Noling, J.W., He, Z., Booker, B. and F. Sances. 2014. Comparison of anaerobic soil disinfestation and drip-applied organic acids for raised-bed specialty crop production in Florida. Acta Horticulturae, 1044:221-228.

• Shennan, C., Muramoto, J., Bolda, M.P., Koike, S.T. and O. Daugovish. 2009. Optimizing anaerobic soil disinfestation for non-fumigated strawberry production in California. 2009 Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions:101-1- 101-3

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• Shennan, C., Muramoto, J., Lamers, J., Mazzola, M., Rosskopf, E.N., Kokalis-Burelle, Momma, N., Butler, D.M. and Y. Kobara. 2014a. Anaerobic Soil Disinfestation for Soil Borne Disease Control in Strawberry and Vegetable Systems: Current Knowledge and Future Directions. Acta Horticulturae, 1044:165-175.

• Shennan, C., Muramoto, J., Zavatta, M. and M. Mazzola. 2014. Non-fumigant approaches for controlling fusarium wilt and charcoal rot of strawberry. 2014b. Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions:11-1- 11-4.

• Yonemoto, K., Hirota, K., Mizuguchi, S. and K. Sakaguchi. 2006. Utilization of the sterilization by sooil reduction in an open air field and its efficacy against Fusarium wilt of strawberry.Proc. Assoc. Pl. Protec. Shikoku (41):15-24 (In Japanese with English summary).

115 2014 - 2015 RESEARCH PROJECTS 116 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Farming Without Fumigants

A Review of the Final Three Seasons (2012, 2013 and 2014) of Research and Grower Demonstrations on the Raised Bed Trough (RaBeT) Substrate Production System

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

Dr. Dan E. Legard Mark Edsall Alex Orozco California Strawberry Commission

Dwight Rowe, SunGro Horticulture Tom Sjulin, Horticultural Consultant Cliff Low, Perry Laboratory

Summary This report is a review of the final three years (2012, 2013 and 2104) of our research project on the raised bed trough (RaBeT) substrate based production system. During this period, our research focused on reducing the setup and operating costs of the RaBeT system while maintain high marketable yield. We conducted a series of small plot experiments at Monterey Bay Academy (MBA) focused on identifying the best treatments and enlarged those treatments on 1-acre grower demonstrations in each of the main production regions. During the three years of these trials, we primarily focused our research on lowering the relatively high cost of the substrate mix we used (coconut coir and peat) in the RaBeT system by amending them with clean field soil. We also evaluated the effect of reducing the volumes of substrate per plant and if we could reuse substrate from the previous season(s). Compost (2012), reduced fertility (2013), and plant spacing (2014) were also investigated during these studies. We learned that it was essential to use computerize fertigation controllers to properly manage the irrigations and fertilizations needed in a substrate based system. We found that substrates amended with soil were significantly less productive than the substrate treatments not amended with soil. Recycled substrates did not consistently produce equivalent marketable yields as virgin substrates, and in one grower demonstration, severe plant collapse occurred due to infestation of the reused substrate with a pathogen that had been introduced on transplants the previous season. In the grower demonstration plots the substrate treatments were often able to match or exceed the marketable yields of the grower standard treatment. The cost of the RaBeT system was significantly greater than that of the current soil based production system with just the annual costs of substrate estimated to be $3,300 to $7,700 more

117 2014 - 2015 RESEARCH PROJECTS per season. The main take away from the three years of research reported here is that while there is potential to increase marketable yields compared to conventional production systems, the significantly higher cost of substrate based production systems keeps the cost benefit analysis heavily weighted towards the current soil based production system in California.

Introduction One of the most important limiting factors for the production of strawberries in California are soilborne diseases caused by Verticillium dahliae, Macrophomina phaseolina, and Fusarium oxysporum. Because of these diseases, almost all conventional strawberry growers fumigate their ground before planting every season. The risk of serious losses to soilborne diseases and subsequent infestation of the soil with these pathogens is currently too great for conventional growers to attempt in soil not pretreated with fumigants. Historically, methyl bromide has been the most effective and widely used fumigant to prevent field loss to soilborne pathogens in strawberry production. However, under the Montreal Protocol, methyl bromide use will be phased out completely after 2016, due to its impact as an ozone-depleting substance. Fumigant alternatives to methyl bromide are less effective than methyl bromide and are also under increasing regulatory constraints in California. In other countries where the use of fumigants has been restricted or banned, a portion of the strawberry fruit production has been transitioned to substrate-based production systems to minimize the risk of soilborne diseases (Leitin and Baetes, 1991; Leitin, 2001, 2004).

The California Strawberry Commission (CSC) began evaluating strawberry substrate production systems in 2008, as part of the Farming without Fumigants Initiative. European substrate production typically employs a table-top system with either bags, pots or troughs of substrate elevated at a height intended to facilitate fruit harvesting. The raised bed trough (RaBeT) system is an adaptation of the table-top soilless production system where strawberries are grown on top of soil beds instead of on a raised platform. Our previous research (Wang et al., 2010, 2011) focused on modifying substrate production to make it compatible with the California open-field production system and evaluated media alternatives to peat and coir, which are the standard substrates used in strawberry production (Lao and Jimenez, 2004; Leitin, 2005; Paranjpe et al., 2003, 2008; Urrestarazu et al., 2004).

Our objectives for the three-year period from 2012 to 2014 were 1) to further develop the RaBeT production system with a focus on cost reduction, 2) to adapt and optimize the system for use in commercial strawberry production settings in California, and 3) to provide the strawberry industry with sufficient information to enable them to install and produce strawberries in substrate using the RaBeT system. The CSC conducted replicated experiments at the Monterey Bay Academy (MBA) research site in Watsonville, CA. To optimize the RaBeT system in commercial production settings, 1-acre demonstration trials were setup annually with grower collaborators on strawberry farms in each of the major production districts: Watsonville-Salinas, Santa Maria-Guadalupe and Oxnard-Ventura. Results from each season’s replicated experiments were used to improve the design of the following year’s grower trials.

Methods General Methods Raised Bed Trough Production System. Standard commercial strawberry production in the Watsonville/Salinas area begins by working soil into raised beds that are on 48 to 56 inch centers, with two rows of strawberry plants per bed. In the southern production regions of Santa Maria/Guadalupe and Ventura/Oxnard, beds are spaced 64 to 68 inches apart and there are four strawberry rows per bed. In each system, drip irrigation is used to provide water and fertilizer and the bed top is covered in plastic mulch either at planting or just following establishment.

The RaBeT substrate system is a soilless production system that mimics the physical structure of a traditional soil

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production system. It is a modified version of the Dutch table-top soilless production system wherein strawberries are grown in substrates, typically peat moss or coconut coir. The primary difference is that the RaBeT system is structurally more similar to a traditional soil-based raised bed plasticulture system (Figure 1). In the RaBeT system, soil beds are constructed as a structural element, but are modified with custom troughs cut into the bed top to accommodate the media or substrate. The bed formation process currently requires a custom bed shaper to be fabricated to specification. The full-sized trough standard at the beginning of this project was 11 to 12 inches wide by 5 to 6 inches deep, with each trough accommodating two rows of plants (approximately 6 to 7 L of media per plant). A single trough was cut into the 2-row beds used in the northern production district (Watsonville/Salinas), whereas two troughs were cut into the 4-row beds used in the two southern production districts (Santa Maria/Guadalupe and Oxnard/Ventura).

Landscaping fabric was then attached and molded to the bed top using mulch pins and into the trough bottom to form a water-permeable root barrier. This barrier allows water and nutrients to drain from the substrate into the soil bed below (leach), and also provided a barrier to restrict the growth of roots into the underlying soil and minimize the contact of growth media with soil. For this project, Sunbelt 3.2 (Dewitt Company, MO) polypropylene groundcover fabric was utilized, based on the results of previous work comparing the performance of various landscape fabrics (Cabrera et al., 2011).

Substrate (primarily composed of either peat or coconut coir), was then mounded into each trough manually. A single line of pressure compensating drip tape was layed into each trough on top of the substrate. The entire system was then covered in plastic mulch, and planting holes were cut prior to planting. The final setup is comparable to traditional soil bed culture, in shape and appearance and the finished bed is indistinguishable from the appearance of commercial soil production bed. This allows growers to utilize the same farm equipment and harvesting practices as for soil based production, reducing the infrastructure costs in comparison to a table-top system (Figure 1).

Figure 1 Diagram of the RaBeT substrate system setup (left) with a picture (right) of a 2-row, landscape fabric covered RaBeT bed with a single coconut coir filled trough, a single drip line and two rows of strawberry plants, mulch was laid over the bed and holes were cut for the plants to grow through.

119 2014 - 2015 RESEARCH PROJECTS Irrigation and Nutrition Management. In traditional strawberry production, a combination of slow-release and liquid fertilizers are used, and plants are watered every three to four days using longer irrigation sets. In order to maintain adequate moisture and fertility in substrate, a pulse-irrigation management program was used in the RaBeT system. This program consisted of short, frequent irrigations with liquid fertilizer applied daily during irrigation. Monitoring of leachate amounts and electrical conductivity (EC) is required in this system to aid with irrigation and nutrition management decisions (volume, frequency and fertility program). Custom pressure compensating drip tubing (Netafim Uniram, 18 mm, .26 GPH, 8 inch spacing; Netafim USA, inc) was employed rather than traditional drip tape to improve distribution of water and nutrients when irrigating with intervals typically shorter than 10 minutes.

Optimum values for liquid fertilizer programs were modeled after European substrate production values. Irrigation programs for the substrate plots were determined empirically by maintaining a leachate volume percentage between 10 to 15% of volume applied at MBA (Watsonville, CA) where water quality was good (base electrical conductivity was 0.5 dS/m) and a leachate volume of 25 to 30% at grower demonstration sites where irrigation water quality was poor and base electrical conductivity was 1.7 dS/m or higher. To monitor the leachate, a five foot PVC trough was placed in one bed of each treatment to collect leachate into a container placed into a hole below the PVC trough. The leachate was retrieved from the container each day and its volume, and electrical conductivity were measured and recorded. When salt levels in the substrate were detected above target values of 2.0 dS/m during the season, irrigations with only water were added to flush salts until substrate ECs decreased to reasonable levels (<2.0 dS/m). More sophisticated monitoring methods were added to the system in years 2 and 3 of the project as described in more detail below.

Yield. Strawberries in California are grown in an annual cycle, with new plants established each year from October to November, and fruit harvesting beginning in January (Oxnard-Ventura), February-March (Santa Maria-Guadalupe) or March-April (Watsonville-Salinas) for the fall-plant production system. Strawberries are harvested twice a week for the duration of the season. Harvest continues through July (Oxnard-Ventura) or as late as November or December (Santa Maria-Guadalupe and Watsonville-Salinas regions). Yield data for each harvest was collected in units of kilograms (kgs) at the MBA experiments and at Ramos Farms (2013), subplot yields were recorded in kgs as well. At the remaining grower demonstration plots, marketable yields were recorded in trays per acre (eight 1 pound containers per tray). For comparison, marketable trays per acre were also reported for an adjacent grower standard commercial soil plot at each demonstration site.

Statistics. For the replicated experiments at MBA, analyses of variance was conducted on the season total marketable yield and percent culls for treatments in each experiment using a mixed model with treatments as fixed effects, and replicates as random effects (NCSS8 statistical software, NCSS, Kaysville, UT). Each trial was further analyzed with a set of planned contrasts to compare specific treatment combinations. No statistical analysis were conducted on the un-replicated grower demonstration marketable yield data.

Methods for the 2012 Season The primary objective of this season was to evaluate the performance of three cost reduction measures in the substrate system. The first cost reduction method was to amend clean soil into the substrate, providing the benefits of reducing the overall volume of substrate used, since substrate was the most expensive recurring cost in the system. The tradeoff is that use of amended soil has the potential to be infested with pathogens. The second cost reduction method we evaluated was to reduce the volume of the trough and thus the amount of the substrate used per plant. The third cost reduction method was to reuse substrate for a second season. We also evaluated the use of a compost in place of higher priced substrates.

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2012 MBA Experiments. Two cost reduction replicated experiments were conducted at the MBA research plots in Watsonville, CA. An amended soil experiment compared five treatments against a substrate standard and an untreated soil standard (Table 1). An anaerobic soil disinfestation (ASD) and a steam treatment were included as possible in-bed mechanisms to disinfest the soil. Treatments were compared against an untreated soil control. With the exception of the 50% coir treatment, all amended treatments were a mix of 50% soil, with 25% rice hull and 25% coir. A second experiment compared the performance of reduced trough size, reused substrate and compost treatments against three substrate standards, 100% coir, 100% peat and a 60:40 blend of peat:coir (Table 1). Both experiments were planted with cultivar ‘Albion’ on November 21, 2011, at a 14 inch between plant spacing. There were four replicates of each treatment in a randomized complete block design. A 38 plant subplot was harvested in each plot, twice a week from April 30 through August 30, 2012 (36 harvests). All pest management practices were managed by Dole Berry Company and matched their commercial production practices.

2012 Grower Demonstration Plots. Grower demonstration RaBeT plots were setup in each of the main production regions. At each site, approximately 1 acre of RaBeT beds were constructed and ½ acre was filled with 60% peat: 40% coir blend, and an adjacent ½ acre was filled with 100% coconut coir. There was one site each in Oxnard, Santa Maria, and Guadalupe, and two sites in Watsonville (five sites in total). At the Oxnard site, a ‘Proprietary’ variety was planted on October 10, 2011, in four rows per bed top on 68 inch center-to-center beds. Sites in Santa Maria and Guadalupe were planted with cultivar ‘Albion’ in four rows per bed top on October 28, 2011 and November 4, 2011, respectively, on 64 inch center beds. The two Watsonville sites were planted on November 6, 2011 and November 18, 2011, with cultivar ‘Albion’ in two rows per bed top on 56 inch or 48 inch center beds. At all sites, a 14 inch plant spacing was used. Whole plot yield was reported in trays per acre for the substrate plots and compared to an adjacent half acre of commercial grower standard produced in fumigated soil. In 2012, the substrate plots were fertigated with two D8R, 40 gallon per minute (gpm) fertilizer injectors (Dosatron USA, FL) hooked up to a two-tank (A & B) fertilizer system.

Methods for the 2013 Season During the 2013 season, we continued to evaluate the performance of a reduced trough volume, reused substrate and amended soil treatments in replicated experiments at MBA. We also evaluated the effect of reducing early season fertility. At the grower demonstration trials, we evaluated the large plot performance of standard substrate blends to see how they would perform on commercial farms and installed computerized fertigation controllers at all the sites to improve the monitoring, reliability and precision of the fertigation.

2013 MBA Experiments. At MBA, two replicated experiments were conducted to evaluate different methods for reducing the cost of producing strawberry fruit in the RaBeT system (Table 1). In the first experiment, the effect of reducing the volume of substrate used in the trough was evaluated by comparing the standard trough volume (5.74 liters) with treatments using 20% (4.6 liters), or 40% (3.4 liters) less of the 60:40 peat: coir substrate blend and of the 100% coir mix. To reduce the trough volume by 20 and 40% without reforming the soil beds, a flexible plastic corrugated drain pipe (2.5 inch or a 4.5 inch, respectively) was placed in the center of the substrate trough for the entire length of the bed, prior to adding substrate, to reduce the amount used per plant to 80% (4.6 liters), or 60% (3.4 liters) of the original 5.74 liter volume of substrate per plant. The beds were then covered with green plastic mulch. Each treatment consisted of a 50 foot long bed and were replicated four times in a randomized complete block design.

In the second experiment, we evaluated a number of alternative methods to reduce cost including: reusing substrate from the previous season; amending soil to a substrate mix; using ASD to treat the amended soil and reused substrate (Shennan et al., 2011); and the effect of reducing the rate of nitrogen early in the season on productivity in substrate. These treatments were compared to standard substrate treatments using virgin 100% coir and 60:40 peat:coir. The amended soil treatments evaluated the productivity of a 50:50 soil:coir mixture.

121 2014 - 2015 RESEARCH PROJECTS For the second year treatments, substrate and amended soil used in the previous season (2012) was collected, separated from plant material and reused for a second season. Anaerobic soil disinfestation was applied to a number of different treatments to see if it would improve the productivity of reused and amended substrates as well as a virgin 100% coir treatment. Four weeks prior to planting, we worked 89.5 lbs of rice bran into the substrate in each 50 foot bed (9 tons per acre) and covered them with plastic mulch. Water was applied through the drip irrigation system to saturate the substrate with the goal of creating anaerobic conditions for three weeks. Unfortunately, due to the well-drained nature of our RaBeT system at MBA, we were unable to maintain anaerobic conditions in these treatments. However, it was hoped that rice bran additions would still create a disease suppressive and growth promoting microbial community similar to what happens when rice bran and other biologically active amendments are added to soil (Shennan et al., 2013).

For the nitrogen rate treatments, a rate of 80 ppm of nitrogen in the irrigation solution was set as the standard, since that was the amount of nitrogen we had used in our irrigations over the past two seasons. We evaluated the effect of both a lower and a higher nitrogen fertility program during the early stages of plant growth. We compared three different rates of nitrogen (40 ppm, 80 ppm and 120 ppm) in the irrigation water during the first four months after planting (November 9, 2012 to March 16, 2013), then switched to the standard rate (80 ppm) for all treatments for the remainder of the season. Due to the previously found challenges of fertilizer and water management, a more sophisticated Fertidan fert-irrigation controller (Famidan, Spain) was used at MBA to manage the fertigations. The soil bed troughs and landscaping fabric were reused from the 2012 season experiments. Compaction and weathering resulted in a slightly smaller trough size measuring 11 x 5 inches rather than the original 12 x 6 inch dimensions. The variety ‘Albion’ was planted on November 9, 2012, at a 14 inch between plant spacing with two plant rows per bed/trough. The experiments were harvested twice a week and graded into marketable and unmarketable fruit from April 5, 2013 to August 15, 2013 (39 harvests).

2013 Grower Demonstration Plots. Grower demonstrations were conducted at five sites during the 2013 season, but the two sites in Ventura County suffered from severe disease problems that prevented the collection of meaningful yield data. For the substrate treatments, soil beds were formed using a modified bed-shaper to cut a 5 to 6 inch deep by 11 to 12 inch wide trough in the bed top, covered with ground fabric and 100% coconut coir or 60:40 peat:coconut coir blend were mounded into the troughs manually. Treatments at each site were installed in approximately 0.25 acre blocks, but were not replicated. The planted area prepared with substrate for each grower demonstration were approximately 1 acre in size. The substrate plots were covered with plastic mulch. A 0.25 acre block of standard soil beds prepared with fumigated soil adjacent to the substrate treatments was prepared according to grower standard practices at each ranch and was used as the grower standard treatment. There were two grower demonstration sites in the Watsonville area. The first was at the Jensen Ranch, planted with ‘Monterey’ in two rows at 14 inch plant spacing on a 52 inch center to center beds on November 17, 2012. To see if a second drip-line would improve the productivity of strawberry plants grown in 100% coir or 60:40 peat:coir, we installed 0.25 acre plots of each substrate with either one or two lines of pressure compensating drip tape (Custom Netafim USA Uniram; 18 mm, .26 GPH, 8 inch spacing) per trough. Irrigation and fertility application were controlled by a Netafim Fertikit 3G (Netafim).

The second demonstration at Ramos Farms, was planted in two rows on 56 inch bed spacing and 14 inches between plant spacing with variety ‘Monterey’ on November 24, 2013. At Ramos Farms, the beds (troughs) were reused from the previous season along with the Sunbelt 3.2 ground mulch and the single line of Netafim drip tape (Custom Netafim Uniram; 18 mm, .26 GPH, 8 inch spacing). Irrigation and fertility was controlled by a Priva Nurtijet 100 (Priva North America).

The third grower demonstration was conducted at Mar Vista Berry Ranch 5 in the Santa Maria-Guadalupe area. This demo was planted in two troughs per bed (two rows per trough) with the variety ‘Monterey’ at 14 inches between plant spacing on November 7, 2013, on a 64 inch bed spacing. To compare the effect of reduced substrate volume on productivity, we installed a 4.5 inch drain pipe in the center of each trough in the reduced area experiments. This trough

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reduced the volume of the substrates (100% coir and 60:40 peat:coir) to 60% of the standard substrate treatment. A single line of pressure compensating Netafim (Custom Netafim Uniram; 18 mm, .26 GPH, 8 inch drip emitter spacing) was laid in each trough. Irrigation and fertility were controlled by a Priva Nurtijet 100.

The grower demonstration trials were harvested twice a week, although occasionally they were only harvested once a week. Commercial crews harvested and recorded the number of trays harvested per treatment for the whole demonstration planting at both the Jensen and Mar Vista farm sites. A 20 plant subplot was harvested in each treatment at the Ramos Farms site and the weight of the marketable yield was recorded. There were 43 harvests (March 26, 2013 to September 10, 2013) at Mar Vista, 47 harvests (April 16, 2013 to October 17, 2013) at Jensen Ranch and 36 harvests (April 15, 2013 to September 11, 2013) at Ramos Farms. Foliar pesticide treatments were applied as needed to both the grower standard and substrate treatments by tractor as per standard commercial practice by the grower.

Methods for the 2014 Season 2014 MBA Experiments. At MBA, we continued experiments looking at lowering the cost of the RaBeT system by reducing and reusing the substrate (Table 1). The trough area was reduced by modifying the original bed press to produce two troughs per bed (one for each plant row) with the approximate dimensions of 5 to 6 inches wide by 2.5 to 3 inches deep. We also compared the reuse of substrate for a both a second and a third year with virgin 100% peat, 100% coir and 60:40 peat:coir treatments. The effect of plant spacing on strawberry cultivar performance in substrate production was evaluated in a separate experiment by comparing the normal 14 inches between plant spacing to a tighter 10 inches between plant spacing treatment for the three public varieties most commonly used by the California strawberry industry in the northern district: ‘Albion’, ‘Monterey’ and ‘San Andreas’.

123 2014 - 2015 RESEARCH PROJECTS Table 1. Substrate experiments and treatments applied at MBA research trial site during the 2012, 2013 and 2014 seasons.

2014 Grower Demonstration Plots. Grower demonstrations were conducted at four sites during the 2014 season: at conventional sites in Oxnard and Guadalupe, and two sites in Watsonville. One Watsonville site was the first attempt at using the RaBeT system for organic production and on a site with heavy clay soil. At the second Watsonville site, which had been used for RaBeT demonstrations the previous two seasons, the grower opted to reuse both the existing RaBeT beds and the substrate from the previous two seasons giving us yield data for substrate reused for a third season. A computer controlled Netafim fertigation unit was used at the organic east Watsonville site, and Priva fertigation units were used at the Guadalupe, Oxnard and west Watsonville sites. Each substrate demonstration plot was approximately 1 acre in size. The substrate mixture at all sites was 60:40 peat:coir, which was established as the standard after the 2013 season, because it was consistently the highest performing and least variable substrate mixture in previous experiments. Each substrate demonstration plot was conducted on commercial strawberry farms with the goal of comparing substrate production to conventional strawberry production in soil. Marketable yield data from the substrate block and an adjacent 1-acre soil production block of the grower’s field were collected for comparison.

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The standard size of the troughs was reduced to approximately 5 to 6 inches wide by 2.5 to 3 inches deep based on results from the 2013 season, which resulted in troughs of approximately 3.4 to 4.3 cubic liters of substrate per plant. Troughs were custom cut using newly fabricated bed shapers. Each trough was planted with a single line of plants. In the southern districts the new system resulted in four troughs and four rows of plants; at the northern site in Watsonville there were two troughs and two rows of plants. The Oxnard site was planted with a ‘Proprietary’ variety and harvested 20 times between January 13 and May 15, 2014. The Guadalupe site was planted with variety ‘Albion’ and harvested 39 times between April 14 and September 9, 2014. The organic Watsonville site was planted with a ‘Propriety’ variety; yield data collection at this site was terminated early and not reported here due to the significant challenges during the setup and production at this site. The third year reused substrate site in west Watsonville planted variety ‘Monterey; and only seven harvests were recorded between April 7 and May 6, 2014.

Results Results for the 2012 Season MBA Experiments. In the amended soil experiment at MBA, the untreated amended soil produced significantly lower marketable yields than other treatments (Figure 2; post-hoc analysis at P<0.05; Figure 3). Low-fertility, 50% coir and 50% silt were the highest yielding treatments but they were not significantly higher than the steam, ASD and clay amended soil treatments. Un-amended coconut coir remained the standard in the amended soil experiment and had the highest marketable yields. In the substrate experiment, marketable yields were significant different (F8,27 = 8.73, P <0.0001) between treatments. The compost blend produced the lowest marketable yield, followed by recycled coir, and the compost-coir blend (Figure 2; Figure 4). Reduced volume performed as well as 100% coir and 100% peat.

Figure 2 Average total marketable yield (kg) per plot (n=4) in (1) an amended soil experiment comparing soil-substrate mixtures as well as non-fumigant soil treatments and (2) a cost reduction experiment comparing reduced volume, reused substrate and compost treatments to substrate media standards. Cultivar ‘Albion’ was planted on November 19, 2011, in RaBeT system beds spaced 52 in apart with 14 inch plant spacing. There were 36 plants per plot, planted in two rows per bed. Experiments were and harvested from April 30 to August 30, 2012 (36 picking dates). Error bars represent standard error of the mean (SEM).

125 2014 - 2015 RESEARCH PROJECTS Figure 3 Average marketable yield (kg) on each harvest date in 2012 (36 harvests), in the amended soil experiment (N=4) at MBA (Watsonville, CA) in 2012. Cultivar ‘Albion’ was planted on November 19, 2011, in RaBeT system beds spaced 52 inches apart with14 inch between plant spacing. There were 36 plants of cultivar ‘Albion’ per plot, planted in 2-rows per bed.

Figure 4 Average marketable yield (kg) on each harvest date (36 harvests) in the cost reduction experiment (N=4) at MBA (Watsonville, CA) comparing reused, reduced area and compost treatments to 100% coir, 100% peat and the 60:40 blend of peat and coir in 2012. Cultivar ‘Albion’ was planted on November 19, 2011 in RaBeT system beds spaced 52 in apart with a 14 in between plants. There were 36 plants of cultivar ‘Albion’ per plot, planted in 2-rows per bed.

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Grower Demonstration Plots. Early season transplant establishment challenges occurred at the Oxnard, Santa Maria and both Watsonville grower demonstration trial sites, due to a dry winter and several issues with the fertilizer injection system. At the worst impacted site in Watsonville, the grower had difficulty allocating labor towards installation of the system infrastructure, so there was a two month delay before any fertilizer was applied through the drip system.

The Guadalupe site was well-managed owing to existing infrastructure from a prior trial. Salt issues were detected early in the season at the majority of sites, due to insufficient leaching volumes and irrigation schedules were adjusted to correct this issue. Early season establishment issues were addressed and high salt issues were corrected by the addition of daily water-only irrigations. Properly implementing these corrections was difficult due to the type of fertilizer injection systems we were using (Dosatrons). We addressed the challenges with monitoring the application of fertilizer and water at the grower trials by installing in-field Decagon sensors (Decagon Devices, Inc., WA) to remotely track EC, temperature and water content at each site. With access to the data from the decagon data loggers by cellular connection we were able to quickly respond to fertility and water management issues. At all but the site where early season fertilizer had not been applied, yield in both the peat-coir and coir treatments improved by June (Figures 5, 6 and 8) but season-long yield was reduced at the affected sites in Watsonville, Santa Maria and Guadalupe for both treatments.

At the Santa Maria and Guadalupe sites, the totals for marketable yield would have continued to improve during the late-season, but the field was taken out of production relatively early (Figures 7 and 8). At the Oxnard site, the marketable yield trend was similar to the grower standard, but overall yield was lower (Figure 9). Notably, marketable yields at all sites were lower in the coconut coir treatment than the peat:coir treatment.

Figure 5 Average marketable yield (trays per acre) on each harvest date in half-acre grower demonstration plots and a half-acre grower standard planted in fumigated soil adjacent to the RaBeT plots on a Watsonville farm in 2012. Cultivar ‘Albion’ was planted on November 6, 2011 in RaBeT system beds spaced 56 inches apart with 14 inches between plants. A tray contains eight 1 lb clamshells of strawberries.

127 2014 - 2015 RESEARCH PROJECTS Figure 6 Average marketable yield (trays per acre) on each harvest date in half-acre grower demonstration plots and a half-acre grower standard planted in fumigated soil adjacent to the RaBeT plots on a Watsonville farm in 2012. Cultivar ‘Albion’ was planted on November 18, 2011, in RaBeT system beds spaced 48 inches apart with 14 inch between plant spacing. A tray contains eight 1 lb clamshells of strawberries.

Figure 7 Average marketable yield (trays per acre) on each harvest date in half-acre grower demonstration plots and a half-acre grower standard planted in fumigated soil adjacent to the RaBeT plots on a Santa Maria farm in 2012. Cultivar ‘Albion’ was planted on November 6, 2011, in RaBeT system beds spaced 64 in apart with 14-inch plant spacing and four rows of plants per bed. A tray contains eight 1 lb clamshells of strawberries.

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Figure 8 Average marketable yield (trays per acre) on each harvest date in half-acre grower demonstration plots and a half-acre grower standard planted in fumigated soil adjacent to the RaBeT plots on a Guadalupe farm in 2012. Cultivar ‘Albion’ was planted on October 28, 2011, in RaBeT system beds spaced 64 inches apart with 14 inch between plant spacing and four rows of plants per bed. A tray contains eight 1 lb clamshells of strawberries.

Figure 9 Average marketable yield (trays per acre) on each harvest date in half-acre grower demonstration plots and a half-acre grower standard planted in fumigated soil adjacent to the RaBeT plots on an Oxnard farm in 2012. A proprietary cultivar was planted on October 10, 2011, in RaBeT system beds spaced 64 inches apart with 14 inch between plant spacing and four rows of plants per bed. Plants were harvested 31 times between January 20, 2012 and June 4, 2012. A tray contains eight 1 lb clamshells of strawberries.

129 2014 - 2015 RESEARCH PROJECTS Results for the 2013 Season Experiments. For the replicated studies conducted at MBA, total marketable yields for the season (average of four replicates) are presented in Figure 10 for the two replicated experiments. There were significant treatment differences in both experiments. In the reduced substrate volume experiment, treatment effects were significant (P<0.05) for both marketable yield and percent culls. Based on the planned contrasts, marketable yields were not significantly different between the 100% coir and peat:coir substrates. Interestingly, marketable yields for the combined 100% coir and peat:coir substrates increased as substrate volumes decreased in a strong linear response (P<0.01). Percent culls in this trial were significantly higher in the 100% coir treatments compared to the peat:coir treatments (P<0.05, data not shown).

In the second experiment, treatment effects were highly significant (P<0.01) for both marketable yield and percent culls. Based on the planned contrasts, the 100% coir and peat:coir substrate treatments, as a group, were higher yielding (P<0.01) than the amended soil treatments. The peat:coir substrate treatments were also higher yielding (P<0.01) than the 100% coir substrate treatments. Differences in yields were not significant between comparable ASD and non-ASD treatments, or between comparable freshly-prepared and reused substrates. Percent culls were higher in amended soil treatments compared to the combined 100% coir and peat:coir treatments (P<0.01). Percent culls were not significantly different in the comparison between 100% coir treatments and peat:coir treatments. Percent culls were higher (P<0.05) in reused substrates compared to new substrates (data not shown). Also, ASD-treated substrates had significantly higher percent culls (P<0.05) than non-ASD treatments (data not shown).

Figure 10 Average marketable yield (lbs) during the 2013 season, per plot (n=4) in two replicated experiments conducted at MBA in a reduced substrate volume experiment and a reused substrate, nitrogen fertility and amended soil experiment. The experiments were planted with ‘Albion’ on November 9, 2012 in RaBeT system beds spaced 52 in apart with 14 inch plant spacing and harvested from April 5, 2013 to August 15, 2013 for a total of 39 harvests.

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Grower Demonstration Plots. For the grower demonstration trials, the accumulative marketable yields are shown in Figures 11 (Santa Maria), 12 (Jensen Ranch, Watsonville), and 13 (Ramos Farms, Watsonville). At the Santa Maria location, all the substrate treatments, including the two 60% substrate volume treatments, produced markedly higher yields than the grower standard. At Jensen Ranch, the substrate treatments produced similar marketable yields as the grower standard and there did not appear to be any large differences between the different substrates or the use of 1 vs 2 drip lines. At Ramos Farms, the grower standard produced higher marketable yields than the substrate treatments. Marketable yields of the new substrates did not differ greatly from the substrates that were reused from the previous season.

Figure 11 Weekly accumulative marketable yield (trays per acre) during the 2013 season, at the Santa Maria grower demonstration site. The 60% peat, 40% coir and 100% coir treatments were grown either using the full volume of substrate in a 12 inch x 5.5 inch trough or only 60% of the volume of the trough. These treatment plots were approximately 0.25 acre of cultivar ‘Monterey’ planted in four rows (two rows per trough, two troughs per bed) in a 64 inch bed spacing with a 14 inch between plant spacing on November 7, 2013. The grower standard was the same variety, bed size and plant spacing in fumigated soil with water and fertilizer provided by the grower. A tray contains eight 1 lb clamshells of strawberries.

131 2014 - 2015 RESEARCH PROJECTS Figure 12 Weekly accumulative marketable yield (trays per acre) during the 2013 season, at the Jensen Farm (Watsonville, CA) grower demonstrate site. Treatments compared a grower standard in fumigated soil with 100% coir and 60% coir: 40% peat substrate treatments where either one or two pressure compensating drip lines were used. There were 47 harvests (April 16, 2013 to October 17, 2013) by a commercial crew during the season. The plots were approximately 0.25 acre of cultivar ‘Monterey’ planted in a two row bed (52 in apart) with a 14 inch between plant spacing. A tray contains eight 1 lb clamshells of strawberries.

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Figure 13 Weekly accumulative marketable yield (lbs) during the 2013 season, at the Ramos Farm, Watsonville, CA, grower demonstrate site. Treatments compared a grower standard in fumigated soil with 100% coir and 60% coir: 40% peat substrate treatments where either new substrate was used or the substrate from the previous season was reused. A 20 plant subplot was harvested for each treatment and the marketable yield is reported in pounds. There were 36 harvests (April 15, 2013 to September 11, 2013) during the season. The plots were approximately .25 acre of cultivar ‘Monterey’ planted in two rows in a 56 inch bed spacing with 14 inch between plant spacing.

Results for the 2014 Season MBA Experiments. In the plant density experiment, the tighter 10 inch spacing between plants in the RaBeT system produced greater marketable yield on average than the standard 14 inch plant spacing for varieties ‘Monterey’ and ‘San Andreas’ (F1,15 =43.4, P <0.001), and both ‘Monterey’ and ‘San Andreas’ produced significantly greater marketable yield than ‘Albion’ (Figure 14). There was no significant treatment effect in the substrate experiment comparing either the reduced substrate volume or the reuse of substrate for a second or third season. However, post hoc analysis comparing specific treatments revealed that the second-year coir treatment produced significantly lower marketable yield (Figure 15).

133 2014 - 2015 RESEARCH PROJECTS Figure 14 Comparison of the effect of 10 inch versus 14 inch between plant spacing on the average marketable yield (kg) for three varieties (n=4), ‘Albion’, ‘Monterey’ and ‘San Andreas’. Strawberry transplants were grown in 60:40 peat:coir mixture in replicated experiments at MBA in 2014. There were 36 harvests between April 10, 2014 and July 31, 2014. Error bars are standard error of the mean (SEM).

Figure 15 Average marketable yield (kg) for substrate treatments in 2014 at the MBA RaBeT production system cost reduction experiments. Only the reused second-year peat:coir treatment produced significantly lower marketable yield by post-hoc analysis (P=0.001). There were 36 harvests between April 10, 2014 and July 31, 2014. Error bars are standard error of the mean (SEM).

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Grower Demonstration Plots. At the Guadalupe and Oxnard trial sites, total marketable yield was not apparently different from the grower standard, but the harvests were off cycle with the grower standard at the Guadalupe site (Figure 16) and on-cycle at the Oxnard site (Figure 17). Also, there was also no apparent differences in marketable yield between the substrate reused for a third season at the west Watsonville site (Figure 18), however, harvest data was only reported at that site for a one-month period. Practical challenges in the setup and the use of organic fertilizers that plugged the drip lines at the organic east Watsonville site prevented useful data from being collected at that site.

Figure 16 Marketable yield per harvest (trays per acre) on a 1-acre RaBeT demonstration plot and adjacent 1-acre grower standard (fumigated soil bed) plot in Guadalupe, CA, during the 2014 season. The Guadalupe site was planted with variety ‘Albion’ and harvested 39 times between April 14, 2014 and September 9, 2014. A tray contains eight 1 lb clamshells of strawberries.

Figure 17 Weekly accumulative marketable yield (trays per acre) on a 1-acre RaBeT demonstration plot and adjacent 1-acre grower standard (fumigated soil) in Oxnard, CA during the 2014 season. This site was planted with a ‘Proprietary’ variety and harvested 40 times between January 13 and May 15, 2014. A tray contains eight 1 lb clamshells of strawberries.

135 2014 - 2015 RESEARCH PROJECTS Figure 18 Accumulative marketable yield in trays per acre on a 1-acre RaBeT demonstration plot and adjacent grower standard (soil bed) acre production of production in Watsonville, CA, during the 2014 season. The third-year reused substrate site in west Watsonville was planted with variety ‘Monterey’. There were seven harvests between April 7 and May 6, 2014. A tray contains eight 1 lb clamshells of strawberries.

Cost of the RaBeT system An important consideration in evaluating the practicality for growers in adopting the RaBeT system is its relative cost compared to conventional production in fumigated soil. While many of the costs are similar between the two systems (plants, water, land rent, fertilizer, pesticides, harvest labor, etc.), there are several significant differences. The cost of the substrate is a key difference since it is the largest single expense in the RaBeT system. The cost of the peat, coir or the peat:coir mixture are similar so we reported an average cost for the substrates.

In 2014, the substrate cost per acre ranged from $5,581 (in a 2-row bed system) to $9,949 (in a 4-row bed system) in the smallest troughs that we evaluated in growers’ fields. This is significantly higher than the cost of drip fumigating beds with a chloropicrin based fumigant at approximately $2,300 per acre (200 lbs per acre with totally impermeable (TIF) film). While some components in the RaBeT system can be reused for up to 10 years, they are also relatively expensive: landscape fabric, $2,173 (2-row) to $3,059 (4-row) per acre; pressure compensating drip lines, $6,212 to $9,318 per acre. The cost of these components alone raised the relative cost of the RaBeT system to $8,000 to $12,000 more per acre for initial setup than a conventional fumigated field, and $3,300 to $7,700 more for just the substrate than fumigation.

The overall setup costs for the RaBeT system are $11,000 (in 2-row beds) to almost $20,000 (in 4-row beds) more expensive per acre than a conventional fumigated soil production system. It would be very difficult for a grower to recover these additional expenses unless the marketplace would pay a significant premium for conventional strawberries produced without the use of fumigants.

136 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Farming Without Fumigants

Conclusions This research and demonstration project has shown that strawberry fruit of sufficient marketable quality can be produced for the US market in a RaBeT production system. Grower demonstration trials of approximately 1 acre each were conducted in each of the main strawberry production regions from 2012 to 2104, and replicated small plot experiments were conducted at MBA each year. The replicated trials at MBA successfully identified ways to reduce the overall cost of the RaBeT production system while maintaining commercially acceptable yields of marketable fruit.

The grower demonstration plots were, in many cases, able to match or exceed the commercial yield of adjacent conventional production in fumigated soil, and served as excellent sites for demonstration of the RaBeT production system to the industry. The CSC also extended the results from this work to the industry through annual field days in each region and at presentations at extension research meetings. Despite this, there has been no adoption of the RaBeT system by commercial growers and there are only a few small trials being conducted by the industry using other substrate based production systems. The lack of adoption by the industry was likely due to a number of significant barriers that will prevent the California strawberry industry from the widespread adoption of substrate based production systems in the near future.

The main challenges we identified during this project included: 1) extremely high per acre costs for the substrate and other materials needed to set up the system; 2) problems with managing soilborne diseases in the RaBeT system, and 3) the need for training on the technical skills necessary to manage substrate based production systems. Overall, potential increases in marketable yield using the RaBeT system were not sufficient to entice growers to transition away from the fumigated-soil based production systems they used for generations. With the cost of substrate averaging from $3,330 (2-row bed) to $7,700 (4-row bed) an acre more than drip fumigation, and with overall setup costs being from $11,000 to almost $20,000 more per acre than the current conventional fumigated soil production system, it is difficult to envision a significant portion of the California strawberry industry adopting substrate production of strawberry fruit in the near future. While it is likely that some California strawberry growers would experiment with substrate-based production systems if effective fumigants were not available, most growers are likely to continue to produce strawberries in soil due to their familiarity with that system and the lower costs.

The overarching goal of this project was to develop a substrate based production system that eliminated the need to use fumigants to produce strawberry fruit in California. Fumigation is primarily used to manage soilborne diseases in strawberry production in California in conventional production systems. We were able to avoid soilborne diseases initially in the RaBeT system, but after we setup 1-acre grower demonstration plots, we learned that it was susceptible to the same soilborne diseases as soil based production. This had not been observed during three seasons of small plot trial work. Strawberry plants at two of our grower demonstration sites developed severe infestations of Fusarium wilt, resulting in widespread plant collapse and contamination of the substrate and beds with the pathogen. It appeared that the plants in RaBeT system at these sites developed disease due to either infested soil or infected strawberry transplants.

Once a soilborne disease develops in a substrate production system, then all the components of the system that come in contact with the substrate and the plants need to be treated to control the pathogen(s). Currently, the only effective tool for accomplishing this in the RaBeT system are fumigants, leaving the grower with a more expensive production system that still requires the use of fumigants to manage soilborne diseases. Replacing the substrate yearly would reduce the risk of crop loss from soilborne diseases, but due to the high cost of substrates, that approach is not economically practical.

One of the goals of the grower demonstration plots was to allow growers and their employees to learn how to manage strawberry production in a substrate based system and adapt the RaBeT system for use in commercial production. What became apparent throughout the project, was that most of the growers and their employees lacked the technical expertise to properly manage a substrate based production system. Several of these trials suffered severe setbacks in production due to the lack of understanding of the different water and nutrient needs of strawberry grown in substrate.

137 2014 - 2015 RESEARCH PROJECTS These challenges were so severe, that remote monitoring equipment was needed at all the demonstration sites to enable us to determine if fertilizer and water were being properly applied. We also had to provide daily on-site support for the maintenance and upkeep of the fertigation systems at each farm. Even growers who participated in the grower demonstrations for several years, were not prepared to install or manage the RaBeT system without regular technical support from our team. Training programs on the strawberry production in substrate and professional technical support are needed before strawberry growers can successfully transition to substrate production of strawberry fruit.

This project has demonstrated that although the use of substrate in a RaBeT production system was possible for conventional production of strawberry fruit, it is not currently economically viable, sufficiently free of risk of soilborne diseases problems, nor easily adopted by California strawberry growers due the technical expertise need to successfully operate substrate based production systems. RaBeT or other substrate based production systems as an alternative to production in soil with the use of fumigants. However, we recommend that further research in substrate production of strawberries in California focus on evaluating production in climate controlled greenhouses to determine if these might provide sufficient increases in production during high value periods to offset the high cost of these systems.

Selected References • Cabrera, J. A., Wang, D., Gerik, J.S. and J. Gan. 2011. Effects of Landscape Fabrics on Pest Control In A Raised-Bed Trough System For Strawberry Production Without Fumigation. Paper presented at: Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions. Print and online: www.mbao.org.

• Kempler, C. 2002. ‘Out-of-Season’ greenhouse production of raspberry and strawberry. Proc. XXVI IHC – Protected Cultivation. Acta0 Hort. 633:459-465.

• 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 Ramieson (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.

• Shennan, C., Muramoto, J., Koike, S.T. and O. Daugovish. 2011. Optimizing anaerobic soil disinfestation for non- fumigated strawberry production in California. California Strawberry Commission Annual Production Research Report, 2010-2011: 111-123.

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• 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 Strawberry Commission Annual Production Research Report: 2009-2010.

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141 2014 - 2015 RESEARCH PROJECTS 142 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Innovation

Automated Strawberry Calyx Removal Technology for Increasing Profit and Minimizing Field Labors

Principal Investigator Dr. Yang Tao, P.E. Professor Dept. of Bioengineering Clark School of Engineering Univerity of Maryland College Park, MD 20742 [email protected] http://www.bioe.umd.edu/research/laboratories.html#tao

Summary Strawberries harvested for processing as frozen fruits are currently de-calyxed manually in the field. This process requires the removal of the stem cap with green leaves (i.e., the calyx) and incurs many disadvantages when performed by hand. Not only does it necessitate the need to maintain cutting tool sanitation, but it also increases labor time and exposure of the de-capped strawberries before in-plant processing. This leads to labor inefficiency and decreased harvest yield. By moving the calyx removal process from the fields to the processing plants, this new practice would reduce field labor and improve management and logistics, while increasing annual yield. The strawberry industry has shown great interest in the development and implementation of an automated calyx removal system. The goal of this project is to develop an automated de-calyxing machine consisting of three main components: a strawberry loading and orientation conveyor, a machine vision system for calyx identification, and a synchronized multi-waterjet knife calyx removal system.

A stainless steel wash-down capable automated strawberry de-clayxing machine has been built and is currently being optimized for field integration. The operating machine has been demonstrated to California Strawberry Commission (CSC) representatives and has received their enthusiastic support. Shortly, we will be meeting with processing plant managers and their affiliates to begin preparing the necessary utilities and protocols for machine installation. Before the machine is shipped, safety guards and interlocks will be installed to meet occupational safety and health administration standards. In addition, a user-friendly machine interface will be developed to facilitate ease of operation.

Our ongoing tasks include the design and fabrication of the machine safety features, mechanical and electronic optimization, and increasing throughput yield. In the next few months, we will continue to run small-scaled tests of 100lb strawberry batches for further optimization. Furthermore, we will collaborate with California plant managers to organize the logistics of machine field installation. We expect to have our machine ready to ship by the end of 2015.

143 2014 - 2015 RESEARCH PROJECTS Introduction Strawberries consist of two markets: the fresh and the processed. The growth of both markets is dependent on each other’s success. The delicate and perishable nature of fresh strawberries requires that when markets are saturated, any excess berries be quickly frozen. This allows frozen strawberries to gradually be introduced into the market similar to grain from silos. The processed strawberry market provides economic stability and diversity to the industry.

Trailing only grapes and apples, fresh strawberries are the 3nd largest non-citrus fruit crop in the U.S., with annual production valued at $2,865,432,000 (Noncitrus Fruits and Nuts 2015 Summary, 2015). Strawberry production involves repeated fruit-bearing and multiple harvests per season, making strawberries the highest tonnage per acre (25 tons/acre) crop among all fruits (Noncitrus Fruits and Nuts 2015 Summary, 2015). The labor costs of this fruit can be up to 60 percent of production cost (Chase, 2012).

In 2014, processed strawberries in the U.S. reached annual valuation of over $241 million, growing roughly 30 percent over the previous year (Noncitrus Fruits and Nuts 2015 Summary, 2015). This represents over 560 million pounds of strawberries harvested for processing. These processed strawberries end up in foods such as ice cream, yogurt, juices, jams and jellies, and baked goods. However, before these strawberries reach the consumer, the calyx must be removed before each strawberry is individually quick-frozen to preserve taste and quality.

The current method of calyx removal involves infield use of cutting blades. The strawberry is picked from the vine, de-calyxed on the spot, and then tossed in a fruit bin (see Figure 1). This bin is then transported to a nearby processing facility, where berries are stored in refrigerated areas until enough quantity is accumulated for the instant quick freezing (IQF) process. De-calyxed berries can at times be stored for up to two days in refrigerated areas before they are sent to IQF. This can result in yield loss, decreased quality, and increased nutrient decline (Daming Dong, 2013) (Youngjae Shina, 2007) (Mohammad Ali Sahari, 2004).

Figure 1. The system used to de-calyx strawberries is depicted. Cutting blades are used to remove the strawberry calyxes in the field. The berries are then transported and stored at a local processing plant to await IQF (Tao)

In addition, according to the CSC, this especially labor-intensive process can double the amount of time required for fruit picking. Furthermore, strawberry growers face significant challenges in finding field workers willing to use the sharp hazardous instruments for harvesting.

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Today, we have built a prototype that has proved the concept and feasibility of our system. This model will be occupationally safe and field-ready for beta test runs by the end of 2015. Materials and Methods An Overview The calyx removal machine consists of three main components: 1) a strawberry loading and orientation conveyor, 2) a machine vision system for calyx identification, and 3) a synchronized multi-waterjet knife removal actuation system. Please see our previous year report for further details (Tao, California Strawberry Commission Annual Report 2012-2013, 2013).

Model II Beta System A functional beta-version of the Automated Vision-guided Intelligent De-calyxing (AVID) machine has been completed (Figures 2 & 3). This stainless steel system is wash-down capable and has been designed to meet the USDA/FDA requirements for food safety and sanitation of food processing equipment. The preliminary results that are discussed below were obtained from this setup.

Figure 2. Exposed view of the Model II Beta AVID. The final design will contain guards and safety features to prevent misuse and injury. This version is wash-down capable with a rating of IP66 / NEMA4X (Tao).

145 2014 - 2015 RESEARCH PROJECTS Figure 3. A functioning construction of the Model II Beta AVID machine (Tao, Lin, Chen, & Seibel, 2014). Patent pending.

Handling System A rotational energy-driven strawberry orientation method is developed to order the initial randomly oriented strawberry input. A rotational force is applied to the strawberry through the use of a custom-designed roller. This causes the typical strawberry to uniformly orient itself into one of two positions due to its conical-like shape. Given a distribution of sizes and geometries that were sampled over a 24 month period, the roller rods were developed to orient over 90% of strawberries. Both empirical and dynamic simulations were used to optimize design. The resultant roller rod geometry resembles an hourglass figure. This creates a pseudo-cup in between the valley of adjacent roller rods. The contact surface of the roller rod material consists entirely of 316 stainless steel. Attention was placed on ease of cleaning and maintenance. The roller design is optimized based on strawberry size, shape, slope, and roller material to produce the highest percentage of strawberry orientation and singulation. Many variations of the roller rod were investigated. Further details can be found in our previous report (Tao, California Strawberry Commission Annual Report 2013-2014, 2014).

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Preliminary Results and Discussion The optimal strawberry orienting roller design is determined using both empirical and dynamic simulation data (Tao, Lin, Chen, & Seibel, 2014). The resultant design oriented a majority of strawberries over a 24-month period. The berries that the handling system did not orient well were due to characteristics that significantly deviated from the typical conical strawberry shape. These characteristics were categorized as spherical, fanned wedge, and other. The other category was observed less than 1% of the time and consisted of damaged berries or berries that had mutant-like features.

Based on batch runs of 100 pounds, currently the beta material handling system can perfectly orient 83% of strawberries (See Figure 4). Of the 17% not oriented, 13% are oriented enough for the current system to remove the calyx, but with higher than optimal fruit loss. The other 4% are oriented too poorly and cannot be cut at all. These berries will be flagged, and a later sorting system will remove them from the product stream. It was also observed that by initially sizing the berries to a small, medium, and large group, the overall orientation improved.

Figure 4. Orientation results of the material handling system are shown. Perfectly-Oriented strawberries will lead to the least amount of fruit loss during calyx removal. Un-Oriented strawberries will be flagged and sorted away from the product line (Tao).

147 2014 - 2015 RESEARCH PROJECTS Ongoing Improvements Additional attachments to the current material handling system are being investigated to increase overall perfect strawberry orientation. Early results indicate that perfectly oriented berries could be improved by 5to 10% with minimal mechanical adjustment. Machine Vision System The machine vision system consists of two industrial CCD cameras; each views half of the conveyor area. These cameras provide a pixel resolution of about 0.25mm2. The system detects each strawberry calyx through distinguishing features, including its color, relative location to fruit shape, and other combinations of spectral imaging and geometry. Once the calyx location is identified, parameters including size, position, orientation, and tilt angle are used to guide the waterjet knife to the appropriate cut location. Preliminary Results and Discussion The imaging system is capable of identifying the precise calyx location in real-time (Figure 5). The average cutline error was roughly 1 mm. Non-uniform lighting conditions, as well as water mist, were simulated to replicate potential factory environments. It was observed that under low lighting or high mist conditions, significant noise in the image would appear.

Figure 5. AVID vision system has identified the calyx location and calculated the appropriate cutline (shown in white) for their on-line removal. Non-uniformed lighting conditions were used to stress test the system (Tao, 2014).

Ongoing Improvements More advanced algorithms are being investigated to improve cutline accuracy and to better determine optimal calyx removal coordinates under larger ranges of environmental conditions. We are exploring many image processing techniques to identify both strawberry fruit and calyx features to assist in algorithm development. Higher intensity lights are being installed to decrease potential low-lighting noise. A fan will be mounted inside the vision chamber to avoid any significant water mist accumulation. Sorting System A sorting system was developed to separate poorly oriented strawberries from the main product stream (Figure 6). The system is attached to the end of the conveyor. The vision system first identifies the poorly oriented strawberry and then commands the waterjet knife to not cut this particular berry. This uncut berry is then pushed into a rejection stream by a burst of air from a solenoid valve.

148 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Innovation

Figure 6. AVID vision system has identified poorly oriented strawberries for removal from the product stream. The system moves the cutting line away from the strawberry to avoid cutting. This is the case for the two berries on the right. The left berry scenario is currently being corrected.

The uncut rejected berries could then be diverted to a return flow for additional processing. Preliminary Results and Discussion The vision system algorithms necessary to identify un-oriented strawberries have been developed. A Sensitivity vs. Specificity test was performed on a small batch of roughly 300 strawberries (Table 1). The analysis indicates that un-oriented identification sensitivity is high at 99.6%; however, at 57.2%, the specificity can be improved. This indicates that the algorithm can correctly identify a majority of un-oriented strawberries. Given the high sensitivity, it is possible to increase the specificity by increasing the calyx identification threshold.

Table 1. Sorting Algorithm Sensitivity and specificity data (preliminary test run).

149 2014 - 2015 RESEARCH PROJECTS Ongoing Improvements The un-oriented identification algorithm is iteratively being improved. Based on early results, a significant improvement can be achieved by enhancing the current feature resolution of the strawberry calyx. Synchronized Multi-Waterjet Knife Removal Actuation System A high pressure waterjet stream is used to cut the calyx off the strawberry with knife-like cut quality. The high pressure decreases water consumption and increases cut precision. The pressure was optimized to increase cut quality and extend the pump maintenance cycle. Each water nozzle is stationed over a lane of strawberries formed by the material handling section. Given the precise coordinates of the calyx from the vision system, actuators will position the knife accordingly. Preliminary Results and Discussion Based on testing hundreds of strawberries, a live run of the AVID machine produces fairly precise knife-like cut quality when removing the calyx (Figure 7). The current system is configured to cut off white shoulders along with the calyx.

The system produced three groups of outputs: strawberry meet, calyx, and strawberry with residual. The majority of strawberries with residual resulted from un-oriented strawberries. These berries will be flagged and sorted from the product line. At the time of this report, this flagged sorting mechanism has been configured and mounted to the machine. The strawberry fruit and calyx will be separated using a common bar shaker, where calyxes fall through and fruit tumbles along top. A slightly larger than expected fruit loss was observed during tests. This was due to strawberries being slightly tilted down or up. This can be corrected in a later version of the machine by increasing nozzle actuation degrees of freedom.

Figure 7. Cut quality results from live run of AVID machine. The system allows the user to adjust the closeness of the calyx cut through the user-friendly touch screen. Currently it is configured to cut off white shoulders along with the calyx (Tao, 2014).

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Ongoing Improvements The precision and sensitivity of the waterjet cutting line is being improved. Strawberries that have un-even cutting lines are a result of the berry rocking while under the waterjet cutting stream. A number of additional components to the system are being developed to minimize this effect. Early results indicate that the rocking problem has been solved. Summary and Discussion Based on our results and on-going progress, we believe an automated calyx removal system is nearing the field integration stage, thanks to the collaborative support of the CSC. Our material handling system has shown that strawberries can be oriented and positioned reliably for efficient processing. The vision system currently works; and with on-going improvements, the overall robustness of the system will increase. We have designed a reliable and easy means to synchronize the position of the waterjet knifes with the calyx location coordinates provided by the vision system. The results of our high-pressure waterjet knife experiments allowed us to design an appropriate pressure system to produce excellent calyx removal while decreasing the overall cost of the machine. Manufacturers, distributors, and fabricators of the machine components have been found and relationships developed. This year’s results have significantly advanced us to the goal of bringing calyx removal out of the fields and into an autonomous, controlled, reliable environment. Our ongoing tasks include the installation of machine safety features and iterative improvements to main subsystems. We are expecting to have our machine ready to ship out by the end of this year.

Selected References • Chase, C. (2012, Nov). Whole Farm Financial Analysis. Retrieved 9 14, 2015, from Iowa StateUniversity Extension and Outreach: https://www.extension.iastate.edu/agdm/wholefarm/html/c3-65.html

• Daming Dong, C. Z. (2013). Analyzing Strawberry Spoilage via its Volatile Compounds Using Longpath Fourier Transform Infrared Spectroscopy. Nature, 2585 .

• FAOSTAT. (2013). Production Crops. Retrieved from Food And Agriculture Organization Of The United Nations: http:// faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor

• Hartman, K. R. and A. W. Gerrans. (1963). Patent No. 3092160. USA.

• Leban, E. (1976). Patent No. 3952646. USA.

• Ledebuhr, R. L., Hansen, C. M. and R. J. Patterson. (1978). Patent No. 4122766. USA.

• Mohammad Ali Sahari, F. M. (2004). Effect of low temperature on the ascorbic acid content and quality characteristics of frozen strawberry. Food Chemistry, 357–363.

• (2015). Noncitrus Fruits and Nuts 2015 Summary. Washington DC: USDA, National Agricultural Statistics Service.

• Tao, Y. (2013). California Strawberry Commission Annual Report 2012-2013. Watsonville: California Strawberry Commission.

• Tao, Y. (2014). California Strawberry Commission Annual Report 2013-2014. Watsonville: California Strawberry Commission

• Tao, Y., Lin, J., Chen, X. and G. Seibel. (2014). Patent No. 028602. USA.

• Youngjae Shina, b. R. (2007). Temperature and relative humidity effects on quality, total ascorbic acid, phenolics and flavonoid concentrations, and antioxidant activity of strawberry.Postharvest Biology and Technology, 349–357.

151 2014 - 2015 RESEARCH PROJECTS 152 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Innovation

Automated Orientating and Capping of Strawberries for Processing

Principal Investigator Dr. Stavros G. Vougioukas The Regents of the University of California University of California, Davis Department of Biological and Agricultural Engineering One Shields Avenue Bainer Hall 3054 Davis, CA 95616 (530) 752-1890 [email protected]

Co-investigator Dr. David Slaughter University of California, Davis. Department of Biological and Agricultural Engineering. One Shields Ave. Davis, CA 95616

Summary Strawberries that are to be processed and frozen must be free of their calyx and stem. Manual capping in the field results in higher labor costs, and potential labor shortages pose severe threats to the future of the frozen strawberries industry and its supplying growers. Capping at the processing plant would be cleaner, safer, and provide greater yields if a high speed capping machine is used. However, despite numerous attempts in the past hundred years, a reliable and economically justifiable machine has yet to be developed. We are addressing this challenging problem by exploiting recent advances in computer-aided engineering (CAE), mechatronics and real-time image processing. Our envisioned system has a high-throughput stage that singulates and orients a large percentage of the strawberries into a consistent cutting pose as it transports them to an array of high-speed cutters. Misaligned strawberries are visually detected and fed back into the system probabilistically, in order to increase the overall alignment rate without having to track each individual berry. A customized image processing program determines the ideal cutting plane which will remove the calyx with minimal fruit loss. When the berry enters the cutting array, an electronic signal activates a precisely controlled pneumatically operated high speed blade closest to the ideal cutting plane to cap the berry.

The overall goal for this project period was the design and construction of a single line orienting and capping machine. Small physical prototypes were developed and strengths and weaknesses of different systems were evaluated. The use of a waterjet to cap berries was extensively explored, but was found to be impractical due to the large cutting forces exerted upon the berries and the high cost and structural constraints associated with a moving waterjet cutting head.

153 2014 - 2015 RESEARCH PROJECTS The prevailing design (patent pending) consists of a conveyor of slotted concave rollers, a high speed imaging system, and computer-controlled high speed pneumatic blades. A lab-scale prototype has been built and experimentation and fine-tuning has begun. Initial tests show this prototype can orient a large percentage of properly sized strawberries in a single pass and deliver them in a good resting pose to the cutter array. A camera and imaging software can quickly determine if a berry is in a good position to be cut. If so, then the software chooses the ideal cutting plane to remove the calyx while minimizing fruit loss and sends a signal to a PLC to activate the appropriate blade as the berry passes beneath it. Berries which are not in a good cutting pose pass through the blade array unscathed. A mechanism ejects them from the line so they may be re-introduced into the orientation section for another attempt. Berries which have been successfully capped are delivered to a grating mechanism which separates the cut caps from the berries. Caps may be sent to a juicing line and capped berries move on for further processing.

This research report covers the 12 month period from August 1, 2014 until July 31, 2015.

Introduction Strawberries that are to be processed and frozen must be free of their calyx and stem. Manual capping in the field results in higher labor costs and potential labor shortages pose severe threats to the future of the frozen strawberries industry and its supplying growers.

At the processing plant strawberries are unloaded on conveyor belts where they end up in random positions and orientations. Automatic capping requires that each strawberry enter the cutting mechanism with consistent position and orientation (referred to as cutting pose), and that capping is performed at high - economically justifiable - rates. Bringing strawberries from a random pose to the desired cutting pose cannot be done by handling each berry independently (e.g., with a robotic actuator) due to the high-throughput requirements. Also, agitating all berries with a common mechanism (e.g., vibration) does not bring all berries to the desired cutting pose because of large variability in berry size and shape. Limitations in sensing and actuation technologies have prevented the purely mechanical prototypes developed during the last hundred years from capping strawberries automatically at acceptable success and throughput rates.

We are addressing this challenging problem by exploiting recent advances in computer-aided engineering (CAE), mechatronics and real-time image processing. Our envisioned system comprises of two main stages: Stage 1) A high-throughput stage sizes and singulates the strawberries, and provides a means that causes them to come to rest at a ‘minimum energy’ pose; this pose coincides with the cutting pose for a large percentage of the fruits. Misaligned strawberries are visually detected and fed back into the system probabilistically, in order to increase the overall alignment rate without having to track each individual berry.

Stage 2) A lower-throughput stage aligns individual strawberries and feeds them to the cutting system using mechatronic actuation coupled with real-time computer vision for strawberry pose estimation.

The strawberries from the outputs of both stages enter the cutting system that consists of an array of precisely controlled pneumatically operated high speed blades. After the berries have been cut, they are delivered to a grating which separates the cut cap from the fruit. The caps may be diverted to a juicing line and the capped berries proceed on for further processing.

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The specific goals for this project period were the following:

1. Continued Investigation of the Waterjet Cutting Process

Early tests showed that unless a strawberry is restrained, the force of the waterjet upon initial contact would cause the berry to move, resulting in a misdirected or poor quality cut. Measurement of the forces exerted by the waterjet upon the berry during the capping operation proved that substantial pressure would be needed to hold the berry in place if a good cut is to be obtained.

In our original concept, the head of the waterjet would be moved rapidly along an optimal cutting path to remove the calyx and minimize fruit loss. However, we learned that due to the weight of the nozzle head and acceleration required, the reaction forces would be enormous, necessitating a massive structure to handle the loads and maintain precision cuts. In addition, the linear motors and controllers required to precisely move the head are very expensive, adding to the already high cost of the waterjet. Therefore, though it initially looked promising, the idea of using a waterjet to cap berries was deemed impractical.

2. Examination of Alternative Methods to Orient Strawberries

Because of their unique shape and fragility, it is difficult to coax strawberries into a resting pose that is suitable for high speed capping. Making extensive use of computer modeling and 3D printing, we examined a number of different methods to orient strawberries and hold them in a good cutting pose. One method used small movable arms and linkages in a carrier to push and hold berries into the proper position. A second used shaped slotted troughs which vibrated as they lifted berries which were floating in water. A third used concave slotted rollers which spun the berries as they travelled along a conveyor. Details of this work are presented in the Materials and Methods section of this report.

3. Design and Construction of a Single Line Orienting and Capping Prototype

A benchtop prototype orienting and capping machine was designed and constructed. This machine uses partially slotted concave rollers mounted on drive chains to form a conveyor which both orients the berries and carries them to a cutting assembly. A feeding mechanism singulates and deposits berries onto the moving rollers just as they enter the orientation section of the machine. Upon leaving the orientation section, computer vision is used to determine if the berry is in a good cutting pose, and if so, locates the ideal cutting plane to remove the calyx with minimal loss of flesh. The cutting assembly uses computer controlled high speed pneumatically actuated blades to cut the cap from the berry. Capped berries drop onto a grating where the caps are separated from the berries. Berries in a poor cutting pose pass through the cutting array unscathed and a mechanism at the end of the conveyor sorts them from the capped berries and redirects them for further attempts at orientation. UC Davis has a patent pending on this machine and its associated software.

4. Development of Image Processing and System Control Software

The brains of the orientation and capping machine is the image processing software and algorithms which control the system. High speed color image processing is needed to detect the calyx and determine if the berry is in a good cutting pose. Algorithms calculate the best cutting plane and control the actuators for the pneumatic blades so that the correct blade descends just as the berry passes beneath it.

155 2014 - 2015 RESEARCH PROJECTS Materials and Methods Objective 1: Continued Investigation of Waterjet Cutting Process Previously we had shown that the cutting forces exerted upon a strawberry by a waterjet were very significant— about two to three times the weight of the berry in the sideward direction and 10 to 15 times the weight of the berry in the downward direction. Thus, unless securely restrained, a strawberry will jump upon initial contact with a waterjet, resulting in a poor or incomplete cut. Several different restraining methods were tested, including suction cups, shaped troughs, and mechanical fingers shown in Figures 1, 2, and 3, respectively. The outcome of tests with these methods are given in the Results section of this report. In addition to examining ways to hold the strawberry during cutting, we also looked at potential damage that may be caused by the waterjet impacting the carriers. As the jet can very quickly score the surface of plastic rollers and troughs, we looked at its effect upon stainless steel. A one-sixteenth inch thick plate of hardened stainless steel was subjected to the impact of a waterjet located four inches above the plate operating at 43,000 PSI. This was done in three different locations for durations of 15 seconds, 5 minutes, and 10 minutes, respectively. The stream was pure water with no added abrasive material.

While efforts were being made to orient and restrain berries for waterjet cutting, we were also exploring the use of linear motors to move the waterjet head along an idealized cutting path as determined by our algorithms. Waterjets operate at extremely high pressures (40,000 to 100,000 PSI), so the nozzles, tubing, and fittings are quite substantial and not lightweight. Discussions with industry experts in linear motors indicate that moving the nozzle head with speed and precision requires a rather large and expensive linear motor. Also, to withstand the resulting reaction forces, a very heavy framework will be needed on which to mount the motor.

Figure 1. Commercial and Custom-made Suction Cups

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Figure 2. Various 3D Printed Troughs

Figure 3. Mechanical Strawberry Positioner

157 2014 - 2015 RESEARCH PROJECTS Objective 2. Examination of Alternative Methods to Orient Strawberries We have already discussed some methods of orienting strawberries (troughs, suction cups, and mechanical fingers) in the previous section and now turn to one method that looks very promising—the use of specially shaped rollers on a moving conveyor. Concave rollers have been used for decades in the inspection and packing of fruits such as apples, oranges, and pears. Strawberries are somewhat more challenging, but we have found that if sized properly, concave rollers can be used to orient most shapes of strawberries. For example, conic (including long-conic and globose-conic) tend to orient nicely on concave rollers whereas wedge and pure globose-shaped berries do not. Fortunately, our study completed last year of strawberries destined for processing showed that from a sample of over 1,300 strawberries representing 14 different cultivars, only about 11% were either wedge or pure globose-shaped. For greater efficiency in automated capping, processors can choose to purchase strawberries from cultivars which are most likely to produce conic-shaped fruit.

An apparatus was constructed which allowed the testing of rollers having different sizes, shapes, materials, and rotational speeds (Figure 4). Strawberries can be placed upon the rollers in random orientations and the rollers spin for a predetermined amount of time, after which they quickly stop. A photograph is taken of the final resting position of the strawberry and a determination is made as to whether or not the berry is in a good cutting pose.

Figure 4. Roller Testing Apparatus

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Objective 3: Design and Construction of a Single Line Orienting and Capping Prototype A major advantage of using shaped rollers on a conveyor is that they can transport the strawberries to a cutting station at the same time that they orient the berries. By adding a series of slots perpendicular to the axis of the roller, any pair of adjacent rollers may be used to cradle a berry while a knife blade descends into one of the slots, cutting off the calyx as it does so. This is the principle behind the design of the UC Davis orienting and capping machine.

Blades have long been used to cap strawberries in the field and to slice and dice them in the processing plant. When a berry is resting in a slotted trough or between a pair of slotted rollers, a fast moving blade can cut cleanly through the berry without having it jump as it would when struck by a waterjet. Pneumatic cylinders are inexpensive and can extend and retract blades at very high speeds. An array of blades, one corresponding to each slot in the roller, can be used to cap a strawberry by activating the blade closest to the calyx as the berry passes beneath the array.

A benchtop prototype orienting and capping machine approximately six feet long was designed and constructed (Figure 5). This machine uses 88 partially slotted concave rollers mounted on drive chains to form a conveyor which both orients the berries and carries them to a cutting assembly. A feeding mechanism singulates and deposits berries onto the moving rollers just as they enter the orientation section of the machine. Upon leaving the orientation section, the rollers stop spinning and the berry comes to a resting pose on the rollers with the slotted side up. A camera captures an image of each successive pair of rollers as they pass by and computer vision is used to determine if the berry is in a good cutting pose, and if so, locates the ideal cutting plane to remove the calyx with minimal loss of flesh. The cutting assembly uses computer controlled high speed pneumatically actuated blades to cut the cap from the berry. Algorithms determine which blade in the array is closest to the ideal cutting plane and activates that blade as the berry passes beneath it. Capped berries drop onto a grating where the caps are separated from the berries. Berries in a poor cutting pose pass through the cutting array unscathed and a mechanism at the end of the conveyor sorts them from the capped berries and redirects them for further attempts at orientation.

Figure 5. Prototype Strawberry Orienting and Capping Machine

159 2014 - 2015 RESEARCH PROJECTS As this is an experimental machine, a good deal of flexibility has been designed into it. Conveyor speed can be precisely controlled using a variable frequency drive. Spacing of rollers can be altered along the length of the drive chains. Components such as the camera, lights, and cutting array can be moved within the frame to optimize the use of space. These features will be used to improve the efficiency and performance of the machine without having to completely rebuild it.

Objective 4: Development of Image Processing and System Control Software Previously we had obtained thousands of images of strawberries in known orientations using a Lynxmotion robotic arm with 4 degrees of freedom coupled with a Basler AVA 1900-50c camera. To control the robot and camera, we designed C++ based software that positioned berries in a series of preset orientations while taking photos. Using this information we designed an algorithm which estimates the orientation of an imaged berry and evaluated its accuracy by comparing its results with our ground truth photographs. Image processing is rapidly completed using the CUDA parallel processing architecture from NVIDIA. Figure 6 outlines the image processing algorithm flowchart. To optimize processing speed, the image is first resized to eliminate non-essential portions of the image. The green calyx and the red strawberry flesh are digitally extracted by first creating Red minus Green layers and Green minus Blue layers. To actually extract the flesh and calyx images, a binarisation based on two thresholds is performed. Finally to remove the noise, an opening operation is realized allowing one to obtain separated images of the calyx and the strawberry flesh. This information is used to determine if the calyx can be cut from that berry.

Another decision processing algorithm controls the actions of the cutting head. First, it’s possible that a berry was not on the rollers due perhaps to a misfeed, in which case the cutting head does nothing. Next, if there is a berry present, then a particular blade is assigned to cut the calyx depending upon the orientation and location of the calyx. A determination is made as to whether cutting the calyx will remove too much flesh, in which case the berry would be ejected. The threshold for this decision to eject can be set by the machine operator and adjusted to the needs of the processor. If the berry is in a good cutting pose and an acceptable cut can be made, then the proper pneumatic cylinder is activated to move the correct blade when the roller pair reaches that blade. Figure 7 shows the flowchart for this algorithm.

Tests of the imaging system and software were conducted without activating the blades. Strawberries of various sizes and shapes were randomly dropped onto the roller/conveyor which was moving at 4 inches/second (10 cm/sec). A photograph was taken as each pair of rollers passed beneath the camera. The resulting image was processed and the output was saved to a file. Included in the output were codes indicating if a berry was detected, and if so, was it in a good cutting pose. If it was in a good pose, then the blade number best suited to cut the berry was listed. The estimated percentage of flesh which would be cut and lost with the cap was also calculated. Each data line in the file could be linked to the photograph from which that data was derived. By comparing computed data with the actual photograph, we could determine the accuracy of the imaging system and decision algorithms and make adjustments to fine tune the processing code.

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Figure 6. Image Processing Flowchart

161 2014 - 2015 RESEARCH PROJECTS Figure 7. Cutting Decision Flowchart

Results 1. Continued Investigation of the Waterjet Cutting Process Commercially available suction cups did not work well at all—they lacked sufficient gripping power and were very sensitive to the size and shape of the berry. We designed and molded our own suction cups which worked much better, but it proved to be very difficult to pick up a berry in the correct cutting pose at high speeds. It also took a significant amount of energy to operate a number of suction cups simultaneously.

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Shaped troughs in which a berry could nest were studied extensively because it was believed that such troughs could be used to singulate, orient, and transport the berry to the waterjet. Complex trough shapes could also be 3D printed in a reasonable time. Troughs were used to lift individual strawberries floating in a water bath (singula- tion) and then vibrated to try to cause the berry to settle into a low energy resting pose in the trough (orientation). The success rate in achieving a good orientation varied from 40 to 70 percent and was highly dependent upon the size and shape of the berries. Troughs were slotted in an attempt to keep the waterjet from cutting into the trough and introducing foreign material into the strawberry stream. However, no matter how well the berry seemed to be nestled in the trough, it still jumped upon contact with the waterjet. Therefore, we attempted to use a mechanical pushing and restraining mechanism in each trough, as was previously shown in Figure 3. After a berry has settled into the trough, the cam-activated mechanism would push the berry against a restraining plate and side arms would move together to position and hold the berry in a good cutting pose. Though it looked feasible, this somewhat complicated mechanism did not work very well in practice, again due to the high variability in the size and shape of strawberries.

The results of the waterjet impacting a stainless steel plate were disappointing. After 15 seconds exposure there was a visible divot in the plate, indicating some material had been dislodged by the jet. After five minutes expo- sure there was clear evidence of erosion. When the plate was examined after 10 minutes exposure, a small hole had been cut through it. So, even if the carriers, whether troughs or rollers, were made of stainless steel, there is a good chance they will be damaged by the waterjet and eject small particles into the product.

Given the already high cost of the waterjet pumps, the added expense of the linear motor and supporting framework ($70,000 to $100,000 per nozzle) makes the machine bulky and economically unjustifiable.

Though initially promising, it has been shown that the use of a waterjet to cap strawberries is both very expensive and impractical. The forces exerted on the berry upon initial contact with the waterjet cause it to jump from its holder, resulting in poor or incomplete cuts. Restraining the berry during cutting is difficult and may bruise the fruit. A waterjet striking the strawberry carrier or other components can rapidly erode and deposit foreign materials into the product. The cost of waterjet pumps is very high, and linear motors to control the path of the cutting head add signifi- cantly to the price of the system. It is for these reasons that we are no longer actively looking at waterjets for use in capping strawberries.

2. Examination of Alternative Methods to Orient Strawberries We tested suction cups, shaped troughs, mechanical fingers, and concave rollers for use in orienting strawberries prior to capping. Each has its own advantages and disadvantages, but ultimately we found that slotted concave rollers work better than the other methods. Not only do they orient berries quite well, but the same rollers also transport the berries and hold them during the capping procedure.

In one test, 150 strawberries of random size and shape were divided into five groups representing five possible orientations in which a berry may land upon a pair of spinning rollers: aligned with roller axis, perpendicular to roller axis, rotated 45 degrees to roller axis, calyx vertically down, and calyx vertically up. Each berry was dropped onto the rollers in the same orientation five times to test repeatability. The rollers were made of Delrin and were spinning at 200 RPM. After landing upon the rollers, they spun for 2.67 seconds (about nine revolutions) before coming to a quick stop. The results from the 750 drops showed that 81% came to rest in a good cutting pose. Ninety-three berries were either conic or long conic and weighed less than 25 grams. Of the 465 drops for those berries, 92% came to rest in a good cutting pose.

163 2014 - 2015 RESEARCH PROJECTS Tests at different rotational speeds showed similar results. From observations of spinning berries, it appears that most berries will reach some stable orientation after about six to eight complete rotations, regardless of their original starting position or speed of rotation. This information is important in sizing the orientation section of a conveyor made of rollers. In such a conveyor, the rollers can be made to spin if they are in contact with a friction pad as the conveyor moves forward. The length of the friction pad and the diameter of the portion of the roller in contact with the pad will determine the number of rotations made by the roller, regardless of the speed of the conveyor.

3. Design and Construction of a Single Line Orienting and Capping Prototype We have designed, built, and filed a patent for a strawberry orienting and capping machine. As it is currently undergoing testing and optimization, we do not yet know its overall throughput rate, but we estimate it will handle five to eight berries per second. However, the machine is modular in nature and can be readily expanded or ganged with other machines to increase the capping rate. Furthermore, some of the components of this machine, such as the drivetrain motor and gearbox, could be shared by multiple lines, thus lowering the system cost. We have demonstrated that berries moving on a conveyor line can be cleanly cut by fast acting pneumatically powered blades. Our imaging system can determine if berries are in a good orientation to be cut and berries not in a good pose can be automatically removed from the line and, if desired, fed back into the system for another orientation attempt.

4. Development of Image Processing and System Control Software We have developed high speed imaging software and algorithms to control decision making processes and cutting blade movement. The accuracy of the imaging algorithms has been validated by comparing their output against thousands of images of berries in known orientations. The system control software can determine which blade is closest to the ideal cutting plane to remove the calyx while preserving the maximum amount of flesh and actuate that blade just as the berry passes beneath it.

Real time testing with strawberries on a moving conveyor showed that images could be reliably captured and processed in less than 15 milliseconds. Figures 8A and 8B show two such images captured during testing. The red, green, and blue lines represent cutting blades and correspond to slots in the rollers. The green line is the blade which will completely separate the flesh from the calyx. In Figure 8A, the berry is not well aligned with the axis of the roller, so if a cut were attempted, only the small sliver of flesh below the green line would remain, and the portion above the green line would be discarded as the cap. The algorithm calculates the amount of flesh which would be lost with the cap, and if that amount exceeds a predetermined threshold, then the cut will not be made and the berry will be ejected from the conveyor. The algorithm correctly rejected all berries in poses like this. In Figure 8B, the berry is in a good cutting pose. The algorithm calculated the amount of flesh which would be lost if any of the blades above the calyx were used and determined that the one represented by the green line would completely remove the calyx while minimizing the amount of lost flesh. Thresholds for acceptable limits of percentage of flesh lost can be adjusted by the operator in order to fine tune the machine as experience is gained.

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Figure 8A. Poor Cutting Pose

Figure 8B. Good Cutting Pose

165 2014 - 2015 RESEARCH PROJECTS Discussion Over a half billion pounds of capped strawberries are used by the strawberry processing industry each year in the United States. Numerous attempts have been made to develop mechanical strawberry capping machines during the past 100 years, and no completely automated machine has been sufficiently successful as to be commercially viable. The current state of the art is that strawberries still are capped in the field by pickers with blades, just as was done a hundred years ago. Not only is this extremely labor intensive, but it is also dangerous and unsanitary. Workers risk cutting their fingers, fruit is wasted due to inconsistent cuts, and strawberries begin to degrade immediately upon being cut, so by the time the capped berries reach the processing plant a significant portion are not suitable for use. Machinery developed to handle other types of fruit do not work well with strawberries because of their fragility and unique shape. In addition, because there are fewer workers willing to pick and cap strawberries in the field, or to work in processing plants because of the seasonal nature of the industry, there is great demand from the processors for automation to replace this labor- intensive and costly procedure. While several prior designs have been developed for portions of the berry capping task, none of the designs have been successful in integrating all of the required tasks to complete the capping process. A key to the novelty and success of our design is the feature that berry orientation and capping are accomplished in the same carrier, without a device-to-device transfer step. Loss of orientation, at the device-to-device transfer step has been one of the main roadblocks to commercial success of several alternative techniques. Many of the other failed alternatives were too mechanically aggressive in handling the berries, resulting in damage to the fruit. Our design, in contrast, does not grasp the berry during cutting, resulting in less damage to the berry during capping.

In the project period from August 1, 2014 until July 31, 2015 we have extensively tested, and ultimately rejected, the use of waterjets to cap strawberries. We have designed and built a bench scale prototype machine which can orient and cap strawberries by making use of modern advances in computer vision, image processing, and high speed mechatronics. We have demonstrated that the imaging system can capture and process images of berries on a moving conveyor and the algorithms can correctly determine if the berries are in a good cutting pose. Additionally, we have shown that the decision algorithms can reliably choose the blade which will remove the calyx while minimizing the amount of flesh lost with the cap. Our machine may be used as a standalone device or ganged with similar machines to increase throughput. We have met the primary objectives set for this period and are on schedule to complete the testing, refinement, and commercialization of our automated strawberry orienting and capping machine.

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167 2014 - 2015 RESEARCH PROJECTS 168 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT APPENDICES

169 2014 - 2015 RESEARCH PROJECTS California Strawberry Commission Commission Members and Alternates for 2014-2015

Producer Members and Alternates

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

District 2: Ventura County Member: Will Doyle Alternate: Rick Meck Sean Stevens Michael Cleugh Dave Murray Mike Ferro Hector Gutierrez Edgar Terry

District 3: Santa Barbara and San Luis Obispo Counties Member: Lorena Chavez Alternate: Don Bowden 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: Ed Kelly Alternate: Esteban Arreguin Peter Navarro Tony Marci Rogelio Ponce Jr. Walt Maitoza Tom AmRhein Victor Ramirez Richard Uyematsu

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

District 6: All Counties (Producer Member-at-Large) Mark Murai

170 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT Appendices

Processor Members and Alternates Member: Ed Haft Alternate: Terry Sebastian Bob Barnhouse Mike Collins

Shipper Members and Alternates Member: Rich Amirsehhi Alternate: John McPike Michael Hollister Allen Davis Bill Moncovich Vince Lopes George Schaaf

Public Members Member: Gloria Sakata Alternate: Vacant

171 2014 - 2015 RESEARCH PROJECTS 2014-2015 Research Committee

Dave Murray, Chairman Thomas AmRhein Don Bowden Allen Davis Will Doyle Greg France Jack Fujishige Daren Gee Bryan Gresser Hector Gutierrez Vince Lopes Tony Marci Neil Nagata Victor Ramirez Sean Stevens Richard Uyematsu Ron Vargas

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2016 Grower Resource and Contact Information

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

Dan Legard Mark Edsall Director of Research & Education Field Research Supervisor [email protected] [email protected] Ariel Zajdband Sofia Hernandez Production Research & Education Manager Special Projects / Meeting Coordinator [email protected] [email protected] Daniel Olivier Field Research Specialist [email protected]

UCCE County Farm Advisors

Fresno/Tulare Ruth Dahlquist-Willard (559) 241-7513 [email protected] Merced N/A (209) 385-7403 Orange N/A (949) 733-3970 Sacramento Chuck Ingels (916) 875-6527 [email protected] San Diego Ramiro Lobo (858) 246-1860 [email protected] Santa Barbara, San Luis Obispo & Ventura /Statewide IPM Program Surendra Dara (805) 781-5940 [email protected] Santa Cruz, Monterey & San Benito Michael Cahn (831) 759-7377 [email protected] Santa Cruz/Monterey Steve Koike (831) 759-7356 [email protected] Santa Cruz, Monterey & San Benito Mark Bolda (831) 763-8040 [email protected] Santa Cruz, Monterey & San Benito Shimat Joseph (831) 759-7359 [email protected] Shasta, Lassen & Trinity Larry Forero (530) 224-4900 [email protected] Stanislaus N/A (209) 525-6800 Ventura Oleg Daugovish (805) 645-1454 [email protected] Ventura/San Bernardino/Los Angeles Andre Biscaro (805) 645-1465 [email protected] Yolo County Margaret Lloyd (530) 564-8642 [email protected]

173 2014 - 2015 RESEARCH PROJECTS County Agricultural Commissioners

Fresno Les Wright (559) 600-7510 [email protected] Merced David Robinson (209) 385-7431 [email protected] Monterey Eric Lauritzen (831) 759-7325 [email protected] Orange Jeff Croy (714) 955-0100 [email protected] Riverside John Snyder (951) 955-3000 [email protected] Sacramento Juli Jensen (916) 875-6603 [email protected] San Benito Karen Overstreet (831) 637-5344 [email protected] San Bernardino Roberta Y. Willhite (909) 387-2105 [email protected] San Diego Ha Dang (858) 694-2739 [email protected] San Luis Obispo Martin Settevendemie (805) 781-5910 [email protected] Santa Barbara Cathy Fisher (805) 681-5600 [email protected] Santa Cruz Juan Hidalgo (831) 763-8080 [email protected]

Ventura Henry Gonzales (805) 388-4343 [email protected]

Strawberry Researchers

Dr. Husein Ajwa, UC Davis (831) 970-8621 [email protected] Dr. Steven Fennimore, UC Davis (831) 755-2896 [email protected] Dr. Tom Gordon, UC Davis (530) 754-9893 [email protected] Dr. Kim Hoelmer, USDA/ARS +33-499-62-3020 [email protected] Dr. Gerald Holmes, CalPoly (805) 756-2120 [email protected] Dr. Kelly Ivors, CalPoly (805) 756-6157 [email protected] Dr. Frank Martin, USDA-ARS (831) 755-2873 [email protected] Dr. Mark Mazzola, USDA-ARS (509) 664-2280 [email protected] Dr. Joji Muramoto, UC Santa Cruz (831) 459-4181 [email protected] Dr. Carol Shennan, UC Santa Cruz (831) 459-4181 [email protected] Dr. Yang Tao, University of Maryland (301) 405-1189 [email protected] Dr. Stavros Vougioukas, UC Davis (530) 752-1890 [email protected] Dr. Frank G. Zalom, UC Davis (530) 752-3687 [email protected]

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175 2014 - 2015 RESEARCH PROJECTS 176 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 2014 — 2015 RESEARCH REPORT California Strawberry Commission California Strawberry ANNUAL PRODUCTION PRODUCTION ANNUAL

CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT 2014 — 2015 P.O. Box 269 P.O. 831-724-5973 fax 831-724-1301 phone Watsonville, CA 95077 CA Watsonville, www.calstrawberry.com [email protected] © 2016 California Strawberry Commission