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

Transcription

1 Non-fumigant Strategies for Soilborne Disease Control in California Strawberry Production Systems Principle Investigator Dr. Carol Shennan Professor Department of Environmental Studies University of California, Santa Cruz 1156 High Street Santa Cruz, CA (831) Co-Investigators Dr. Joji Muramoto Associate Researcher Department of Environmental Studies University of California, Santa Cruz 1156 High Street Santa Cruz, CA (831) Dr. Steven Fennimore Department of Plant Sciences University of California, Davis 1636 East Alisal Street Salinas, CA (831) Dr. Mark Mazzola USDA-ARS 1104 N. Western Ave. Wenatchee, WA (509) Dr. George Lazarovits A&L Biologicals Agroecology Research Services Centre 2136 Jetstream Road London, ON, Canada, N5V 3P5 (519) ext 246 Collaborators and Affiliations Dr. Frank Martin USDA-ARS Salinas, CA Dr. Krishna Subbarao University of California, Davis Dr. Surendra Dara University of California Cooperative Extension San Luis Obispo County Santa Barbara County 153

2 Summary Soilborne disease management without chemical fumigants is a major challenge for strawberry production in California. Current re-registrations and regulations are likely to intensify this obstacle by severely limiting availability of fumigants on a large percentage of strawberry acreage. We compared the effect of non-fumigant methodologies such as anaerobic soil disinfestation (ASD), steam, mustard seed meal amendments (MM), and organic acids (OA), alone and in combination, on fruit yield, survival of a range of soilborne pathogens, weed density, soil chemical and biological characteristics, and economics in California strawberry production. Overall, results from the Monterey Bay Academy trials (MBA and ) and the Santa Maria trial ( ) showed ASD and ASD+MM to be the most consistently effective and economically feasible non-fumigant alternatives. However, applying 9 tons/acre of rice bran for ASD, which provides ~360 lbs-n/acre, may release excess nitrogen to the environment. At an on-going trial in Watsonville, cumulative fruit yield by mid-july from ASD plots that utilized 6 tons/acre of rice bran or rice bran + molasses (4.5 tons/acre each) were comparable with 9 tons/acre rice bran or Pic-Clor 60 plots and exceeded methyl bromide plots. Distinct changes in soil microbial community composition were observed immediately after application of soil treatments at the MBA and Santa Maria field trials during the growing season. These differences in soil microbial community composition were maintained over the course of the season and were still apparent at the end of the production season in September Similar to last year s results, pot experiments indicate that strawberry plant growth was significantly improved in response to ASD, MM or MM following ASD treatments relative to the control. However, when MM was applied in conjunction with ASD or two or three weeks prior to ASD treatment, strawberry growth was not improved relative to the control and was diminished relative to the ASD/MM application sequence. Future experiments should examine 1) effects of different ASD carbon sources and input rates on fruit yield, survival of a range of soilborne pathogens (especially of Fusarium spp. and Macrophomina spp.), greenhouse gas emission, N dynamics, and economics, 2) performance of ASD in large scale field experiments, and 3) whether changes in soil microbial community composition are responsible for soilborne disease suppression by ASD. Introduction California s strawberry production system for the last 40 years has relied upon preplant fumigation with methyl bromide (MeBr) (Wilhelm et al., 1961); however MeBr is being phased out through the Montreal Protocol and considerable resources have been invested into replacing it with other fumigants, yet even the most promising option, chloropicrin, has a questionable future as it may not gain re-registration (US-EPA, 2006) due to potential negative health effects. This underscores the critical need for developing a wider range of alternative practices (Carpenter et al., 2001). A number of non-fumigant pre-plant strategies for the control of soilborne diseases in strawberry production systems have been studied. Mustard seed meal amendments (MM) were shown to have the capacity to create disease suppressive soils. Although such materials were commonly viewed as yielding disease control through the processes of biofumigation (release of toxic products during residue decomposition), specific elements of the soil biological community have also been shown to contribute to disease or weed control (Cohen and Mazzola, 2005; Mazzola et al., 2007; Hoagland et al, 2008). It is likely that the modes of action may vary from pathogen to pathogen (Mazzola et al., 2007) and that the source of the product can also modulate disease control efficacy (Mazzola et al., 2009). Anaerobic soil disinfestation (ASD) was developed in Japan (Shinmura, 2000; Momma, 2008) and the Netherlands (Blok et al., 2000; Messiha et al., 2007) as an alternative to MeBr. It has been shown to control soilborne pathogens and nematodes in strawberries. ASD integrates principles behind solarization and flooding to control soilborne pests in situations where neither is effective or feasible. 154 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

3 Studies conducted over the past six years were aimed at optimizing ASD for use in California strawberry systems. Overall, ASD was shown to be consistently effective at suppressing Verticillium dahliae in coastal California when 9 tons ac-1 of rice bran was pre-plant incorporated and 3 to 4 acre-inches of irrigation was applied in sandy-loam to clay-loam soils (Shennan et al., 2011). In trials at Watsonville and Castroville, marketable yields from ASD plots were equal or higher than Pic-Clor 60 plots, and significantly greater than untreated controls. However, applying 9 tons/acre of rice bran for ASD, which provides 360 lbs-n/acre, may release excess nitrogen to the environment. Steam has been used for over 100 years to kill soilborne pathogens and weeds in potting soil (Baker and Roistacher, 1957). It is universally accepted that raising the soil temperature to 158 F for 20 minutes kills all pathogens and weeds. Preliminary data derived from the new bed steamer indicates that it rapidly heats soil to a 14 inch depth at a cost of $5,472 per acre broadcast compared to $3,200 to $3,600 per acre for MeBr applied broadcast (Fennimore, 2011). Soil ph and volatile fatty acids (VFAs) including organic acids can play an important role in soil suppressiveness (Chet & Baker, 1980). The use of fish emulsion containing a large concentration of VFAs reduced the viability of V. dahliae microsclerotia up to 99% (Abbassi et al, 2009). The project objectives were to: 1) Examine effects of anaerobic soil disinfestation (ASD), mustard seed meal (MM), organic acid material (OA), alone and in combination, on fruit yield, a range of soilborne pathogens, weed seed viability, soil chemical and biological characteristics, and economics in strawberry production systems in California. 2) Determine whether changes in soil microbial composition are responsible for soilborne disease suppression detected in response to ASD, MM, and OA. 3) Test whether we can reduce N input from C-sources for ASD without losing the effectiveness by using a lower rate of rice bran, or a low-n material such as molasses. Here we report results from the and trials as well as the progress made in the experiments thus far. Materials and Methods Objective 1: To compare several non-fumigant options, replicated completely randomized field trials were conducted at Watsonville (MBA) in the and seasons; and at Santa Maria (Manzanita Berry Farm) in the season. Please note that to provide the overall picture of the project; some data used in the CSC report are reused in this report. Watsonville Site ( ) A field trial was established on an Elder sandy-loam field with non-detectable levels of V. dahliae at the MBA site, Watsonville, Santa Cruz County. In , treatments were ASD (rice bran 9 tons/acre), mustard seed meal (MM. MPT Mustard Products & Technologies Inc. Saskatchewan, Canada. 1.5 tons/acre), steam, MM+ASD (MM 1.5 tons/acre + rice bran 7.5 tons/acre ), MM+steam (1.5 tons/acre), Pic-Clor 60 fumigation, and untreated control (UTC) arranged in completely randomized block design with four replicates. Each plot was a 4.3 feet wide (center-to-center) x 40 foot long bed. MM was shank applied at 6 inch depth of beds (two rows per bed) on October 7, On the same day rice bran was applied on the bed surface of assigned plots and incorporated into 0 to 6 inch depth by a hand-push rototiller. After reshaping beds and applying drip tapes and standard green plastic mulch, 2.5 acre-inches of water was drip irrigated intermittently to ASD and MM+ASD plots from October 8, 2010 to Novermber 3,

4 Eh, a measure of anaerobiosis, and soil temperature at 6 inch depth were automatically monitored in ASD, MM+ASD, and UTC plots from October 8, 2010 to November 6, 2010 using ORP sensors and soil temperature sensors, respectively. Steam was applied by spike injection from a stationary steam generator for sufficient time to raise the soil temperature to 158 F for 20 min on October 13, 2010 and October 14, Pic-Clor 60 was applied on October 15, Holes were cut through plastic mulch on November 18, 2010 and strawberry plants cv. Albion were planted on November 22, 2010 at a plant density of 20,000 per acre. Marketable fruit yield was assessed twice weekly (28 plants) from April 28, 2011 to November 15, Net return above harvest and treatment costs were calculated for each treatment based on the marketable fruit yield from the trial as well as harvest cost and treatment cost including material, application, incorporation and additional irrigation needed for ASD. Watsonville Site ( ) A trial of the same experimental design and size as for the previous season was repeated in the season at the MBA site. For MM treatments, we used Strawberry Mix from Farm Fuel Inc. (Watsonville) at 1.5 tons/acre. Rice bran and MM were applied as described above and incorporated into 6 inch depth by a hand-push rototiller on October 12, After reshaping beds and applying drip tapes and standard green plastic mulch, 0.8 acre-inches of water was drip irrigated into all plots on October 14, An additional 1 acre-inch of water was intermittently applied via drip tapes to ASD and MM+ASD plots until October 20, 211. Eh and soil temperature were monitored from October 16, 2011 to November 16, Steam was applied during October 18, 2011 and October 20, Pic-Clor 60 fumigation was conducted on November 13, Planting holes were cut on November 17, 2011 and strawberry cv Albion was transplanted at 20,000 plants/acre on November 21, Marketable fruit yield from 35 plants/plot were monitored twice weekly from April 24, 2012 to September 12, Soil samples were taken from 0 to 6 inch depth in all plots pre-treatment (October 12, 2011), post-treatment (November 17, 2011), and the end of harvest (September 17, 2012) for microbial analysis by USDA-ARS WA using real-time quantitative PCR (RT-PCR) and terminal restriction fragment length polymorphism (T-RFLP) analysis. The final soil samples were further tested for V. dahliae microsclerotia population by the Subbarao lab, UC Davis, Salinas, and Pythium spp. population by the Martin lab, USDA-ARS, Salinas. Santa Maria Site ( ) A randomized block design with four replicates and treatments UTC, ASD (rice bran 9 tons/acre), fish emulsion (Fish. True Organic 402 acidified by sulfuric acid (2% v/v) used as a high OA material. ph 4.8), MM+ASD (rice bran 7.5 tons/ acre + MM Strawberry Mix from Farm Fuel Inc. 1.5 tons/acre), ASD+Fish, and Pic-Clor 60 fumigation was established in a Sorrento sandy-loam soil at Manzanita Berry Farm, Santa Maria. Each plot was a 5.3 feet wide x 35 feet long bed. Rice bran and MM were applied on top of the beds and incorporated to 6 inch depth by a bed shaper-attached rototiller on September 22, After applying drip tapes and standard black plastic mulch, ASD and MM+ASD plots were intermittently drip irrigated for a total of 3.0 acre-inches from September 26, 2011 to October 15, acre-inches of 2% (=1:50 diluted) acidified fish emulsion was applied to Fish and ASD+Fish plots on September 30, 2011 and October 15, 2011 to moisten the potential strawberry root zone (0 to 12 inch depth). ASD+Fish plots were also intermittently drip irrigated with 2.4 acre-inches of water to attain a total irrigation amount of 3 acre-inches, and equivalent amounts were applied to the other ASD plots. Eh and soil temperature were monitored in ASD, MM+ASD, ASD+Fish and UTC plots from September 23, 2011 to October 24, 2011 as described above. Pic-Clor 60 fumigation was conducted on October 7, Holes were cut through the plastic mulch on November 11, 2011 and strawberry cv PS-4634 was transplanted at 30,400 plants/acre on November 15, Fifteen gallons/acre of 2% acidified fish emulsion was applied at every other irrigation event to Fish and ASD+Fish plots since January 31, Marketable fruit yield (40 plants/plot) was monitored twice weekly from March 23, 2012 to August 8, The final soil samples were further tested for V. dahliae and Macrophomina species populations by the Subbarao lab, UC Davis, Salinas. 156 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

5 Objective 2: Experiments were conducted to assess the effect of application sequence on integration of ASD and MM applications for disease control in an organic field soil from UC Santa Cruz, CA. Brassica juncea seed meal was applied at 0.3% (wt/wt basis) and rice bran for ASD treatments was applied at a rate of 117 oz./square yard. Treatment sequences consisted of ASD/MM and MM/ASD, with a two or three week interval between treatment applications. Additional treatments consisted of control, MM or ASD alone, and a co-application of MM+ASD at the same time. Soils were dispensed into 1 gallon pots. MM only treatments were sealed in air tight Bitran bags for 24 hours to simulate tarping and concentrate the activity of the active volatile allyl isothiocyanate. All pots containing ASD as a component of the treatment were irrigated with 10.1 oz. water at the time of rice bran application, pots were sealed in a double layer bitran bag, and were incubated for one week at 75 F prior to subsequent application of MM or planting. Soil ph was determined immediately prior to planting. Soils were planted to strawberry (cv Camarosa ) one week after application of the final treatment and controls were fertilized with Hoagland s solution. Plants were grown using a 16-hour photoperiod and a day/night temperature regime of 75/60 F. Plants were harvested after 15 weeks and biomass determined. Objective 3: Watsonville Site ( ) A field trial was established at an Elder sandy-loam field with a non-detectable V. dahliae population at the Plant Sciences Inc (PSI) site, Watsonville, Santa Cruz County. Main treatments were ASD rice bran 9 tons/acre (ASD-RB9), ASD rice bran 6 tons/acre (ASD-RB6), ASD molasses (N 0.5%) 9 tons/acre (ASD-ML9), ASD molasses 6 tons/acre (ASD- ML6), ASD rice bran and molasses 4.5 tons/acre each (ASD-Mix), Pic-Clor 60 fumigation, Methyl bromide/chloropicrin fumigation (MB/Pic) and untreated control (UTC). Further, to examine whether pre-plant fertilizer can be eliminated under ASD treatments, sub plots of with and without pre-plant fertilizer (650 lbs/acre of 6-month slow release fertilizer ) were established within each main plot arranged in completely randomized block split-plot design with four replicates. Due to proximity to a sensitive site and the buffer zone requirement, fumigation plots were established adjacent to ASD plots without randomization. Each plot was a 4 feet wide (center-to-center) x 150 foot long bed. On September 14, 2012 rice bran was applied on the bed surface of assigned plots and incorporated into 0 to 6 inch depth by a rototiller attached with a bed shaper. After applying drip tapes and standard black plastic mulch, ~1:2 water-diluted sugarcane molasses was applied to assigned plots via drip tapes on September 18, Three acre-inches of water was drip irrigated to all ASD plots from September 18, 2012 to October 9, MB/Pic (400 lbs/acre flat fume) and Pic-Clor 60 (38.5 gal/acre drip fume) was applied on September 4, 2012 and September 29, 2012, respectively. Holes were cut through plastic mulch on October 17, 2012 and strawberry plants cv. Albion were planted on November 16, 2012 at a plant density of 21,780 per acre. Marketable fruit yield has been assessed twice weekly (40 plants) since April 15, Weed fresh biomass and weeding time was measured at main plots on January 31, March 11, April 6, May 9, June 13 and July 18, Soil samples (0 to 6 inches) have been taken monthly from September 2012 for inorganic N analysis at UCSC. Post-treatment soil samples taken on October 17, 2012 were tested for microbial analysis by USDA-ARS WA using RT-PCR and T-RFLP analysis and soilborne pathogens using nested PCR with species specific primers by A&L Laboratory, Canada. 157

6 Results Objective 1 Cumulative Eh and Soil Temperature During the ASD Treatment In the sandy loam soil at the MBA Watsonville site, MM+ASD plots developed stronger anaerobic conditions in both seasons than ASD plots during the ASD treatment; at 21 days from the first irrigation, cumulative Eh below 200 mv was 79,000 ( ) to 120,000 mv hrs ( ) in MM+ASD plots, whereas it was 26,000 ( ) to 50,000 mv hrs ( ) in ASD plots. The weak anaerobic development in ASD plots in may be due to insufficient irrigation rate during ASD treatment (total 1.8 acre inches). Soil temperature at 6 inch depth during ASD treatment averaged 66 F in the MBA trial for both years and 73 F in the Santa Maria trial where strong anaerobic conditions (54,000 to 71,000 mv hrs) developed in ASD, ASD+Fish, and MM+ASD plots at 21 days. Marketable Fruit Yield and Economics At MBA, marketable fruit yields in Steam, Steam+MM, ASD, and MM+ASD treatments were similar to Pic-Clor application in (Figure 1A). The cost of MM and the ASD treatment with rice bran were similar, $1,693 and $1,632 per acre respectively (Figure 1B) whereas steam added $10,440 per acre compared to $800 per acre for Pic- Clor. Therefore, while yields and gross revenues were comparable across treatments, net returns above treatment and harvest costs were highest for Pic-Clor followed by ASD and MM+ASD and lowest for Steam+MM. In at MBA, marketable fruit yields only in Steam+MM treatment were comparable to Pic-Clor, followed by MM+ASD, and were significantly greater than ASD, MM, steam, and UTC (Figure 1C). The low yield of ASD may be due to the weak anaerobic development mentioned above. Similar to , net returns above treatment and harvest costs were highest for Pic-Clor, followed by ASD and MM+ASD and lowest for steam (Figure 1D). In Santa Maria, ASD, ASD+Fish, and MM+ASD plots had similar marketable fruit yields as Pic-Clor, followed by Fish, and had significantly greater yields than UTC (Figure 2A). Due to the treatment cost difference, net returns above treatment and harvest costs were highest for Pic-Clor, followed by ASD. Relatively small difference in yields gave a slight advantage to UTC in net returns above treatment and harvest costs over Fish, ASD+Fish, and ASD+MM (Figure 2B). Soil Microbial Community Composition, Root Infection by Specific Pathogens, and Soil Pathogen Populations At the end of the growing season at the MBA site, treatment differences were observed in relative root infection by Cylindrocarpon, Fusarium, Pythium and Rhizoctonia spp. The treatments representing the highest/lowest incidence of recovery from strawberry roots were as follows: Cylindrocarpon, Control (23%)/MM+ASD (3%); Fusarium, MM+ASD (23%)/Pic-Clor, steam, ASD all 4-6%; Pythium, Control (12%)/Pic-Clor, ASD, steam, MM+steam, MM+ASD, all 0-2%; Rhizoctonia, Control (20%)/mustard, ASD and MM+ASD all 1-3%. In addition, Meloidogyne hapla was detected in strawberry roots at the MBA site but were primarily restricted to the steam and MM+steam treatments. At the Santa Maria site, ASD was superior to soil fumigation in suppressing recovery of Fusarium and Rhizoctonia spp. from strawberry roots, but recovery of Pythium spp. from roots was elevated in response to ASD. Pythium isolates recovered from roots of strawberry at this site were identified as P. megacarpum, P. spinosum and P. violae, none of which are known to be significant pathogens of strawberry. 158 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

7 As reported previously, distinct changes in soil microbial community composition were observed immediately after application of soil treatments at the MBA and Santa Maria field trials during the growing season. Somewhat surprisingly, these differences in soil microbial community composition were maintained over the course of the season and were still apparent at the end of the production season in September ASD appeared to induce the most persistent effects on fungal community composition as all except one replicate from ASD or MM+ASD treated plots, clustered in a single branch of the dendrogram (Figure 3). Figure 1. Cumulative marketable fruit yield (A, C), and partial costs and net return (B, D) at the MBA trial in the season (A, B) and the season (C, D). UTC: untreated control, ASD: anaerobic soil disinfestation, and MM: mustard seed meal. Means marked with the same letter have no significant difference according to protected LSD test (P=0.05). 159

8 Figure 2. Cumulative marketable fruit yield (A), and partial costs and net return (B) at the Santa Maria trial in the season. UTC: untreated control, ASD: anaerobic soil disinfestation, and MM: mustard seed meal. Means marked with the same letter have no significant difference according to protected LSD test (P=0.05). 160 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

9 Figure 3. Effect of soil treatments on fungal community composition prior to application (A. September 2011), post application (B. October 2011), and at the end of the growing season (C. September 2012) at the MBA site determined by T-RFLP analysis. X-axis denotes similarity among fungal communities across plots (range 0 to 1). The tree was constructed from the Jaccard similarity coefficient of composite profiles from triplicate digestions of amplified fungal DNA using primers specific for the ITS region of rdna. Bootstrap values are indicated at branch nodes of the tree (10,000 bootstrap replicates). Treatments: Ck=control; ASD=anaerobic soil disinfestation; ASDMus=ASD+mustard seed meal; Mus=mustard seed meal; Pic Clor; Steam; SteamMus=Steam+mustard seed meal. Plot numbers are given at the end of each line. 161

10 At the MBA site at the end of the season, the number of V. dahliae microsclerotia g-1 dry soil varied between 0 and 2.5 in the different treatments with the soil from untreated control having 1.5 microsclerotia g-1 dry soil. Because of the low numbers of microsclerotia, treatment differences were not significant. Pythium spp. population of UTC and ASD were statistically similar and numerically greater than Pic-Clor, steam, and steam+mm treatments although, as mentioned above, root infection by the pathogen in ASD treatments were much lower than UTC. Results from Santa Maria trial were also similar and reductions in microsclerotia from treatments could not be confirmed. Even though the mean numbers of Macrophomina species CFUs varied widely between the different treatments, the variability between replications was high and thus, no significant differences could be ascertained between treatments or between treatments and untreated control (data not shown). The Santa Maria trial had very low populations of Pythium spp. with no meaningful differences among all the treatments at the end of the season. Objective 2 As observed in pot trials conducted in a Santa Maria field soil during , sequence of application significantly influenced the effect of MM+ASD treatments on growth of strawberry in UC Santa Cruz field soil; however unlike the previous trial conducted in an alkaline soil, ph was not significantly altered. Initial soil ph was 6.3 in the non-treated control and ranged from 6.0 to 6.65 in soils that received MM and/or ASD treatment. Plant growth was significantly improved in response to ASD, MM or MM following ASD treatments relative to the control. However, when MM was applied in conjunction with ASD or at two or three weeks prior to ASD treatment, strawberry growth was not improved relative to the control and was diminished relative to the ASD/MM application sequence (Figure 4). This observation is similar to that observed in the Santa Maria soil and although the process causing depressed growth in response to this sequence is not known, the data imply that an extended period between MM and ASD application may alleviate the effect. In terms of pathology, Rhizoctonia sp. AG-G appeared to be a significant growth limiting factor in this soil, being isolated from 43% of root segments from the control soil. Although MM (17.5%) alone ASD alone (25%) or MM following ASD ( %) reduced Rhizoctonia AG-G root infection, this value ranged from 35 to 43% when ASD followed MM in sequence (Figure 4). 162 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

11 Figure 4. Effect of ASD, mustard seed meal (MM), sequence of application and period between treatment applications on strawberry biomass after 10 weeks in UC Santa Cruz field soil 163

12 Objective 3 At the PSI site with pre-plant fertilizers, ASD-RB9, ASD-RB6, ASD-Mix, and Pic-Clor treatments had statistically similar high cumulative marketable fruit yield as of mid-july; and these were greater than ASD-ML6, ASD-ML9, MB/Pic, and UTC (Figure 5). Level of cumulative yield reduction by eliminating pre-plant fertilizer ranged widely; the effect was minimal in MB/Pic (8%), relatively small in ASD-RB9 (22%), ASD-RB6 (20%), ASD-Mix (21%), and greater in ASD-ML6 (37%) and ML9 (31%), Pic-Clor (40%), and UTC (49%). Figure 5. Cumulative marketable fruit yield at the PSI trial in the season (as of August 15, 2013) cv. Albion. UTC: untreated control, ASD-ML6: ASD with molasses 6 tons/acre, ASD- ML9: ASD with molasses 9 tons/acre, ASD Mix: ASD with rice bran 4.5 tons/acre + molasses 4.5 tons/acre, ASD-RB6: ASD with rice bran 6 tons/acre, ASD-RB9: ASD with rice bran 9 tons/acre, Pic-Clor: Pic-Clor 60, and MB/Pic: Methyl bromide/chloropicrin mixture. w/o PPF: without pre-plant fertilizer. w/ppf: with preplant fertilizer (650 lbs of six month slow release fertilizer ). Means marked with the same letter have no significant difference according to protected LSD test (P=0.05). 164 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

13 Soil inorganic N (0 to 6 inch depth) in with pre-plant fertilizer plots were generally much greater and variable compared to without pre-plant fertilizer plots; soil inorganic N in without pre-plant fertilizer plots did not exceed 50 ppm during the period (Figure 6A), whereas it reached 100 to 230 ppm in October to December 2012 in all plots except fumigated plots, then decreased to 10 to 50 ppm in late April 2013 in pre-plant fertilized plots (Figure 6B). A pre-plant fertilizer was band applied to beds from which a part of soil samples were taken. This appeared to have caused greater soil inorganic N concentrations in pre-plant applied plots that were temporally highly variable in some plots (e.g. ASD-Mix, ASD-ML9). ASD-RB9 without pre-plant fertilizer plots maintained 20 to 40 ppm of inorganic N during the period. Figure 6. Changes in soil inorganic N (NO3-N + NH4-N, 0-6 depth) in without pre-plant fertilizer plots (A) and with pre-plant fertilizer plots (B) at the PSI trial in the season (9/14/12 (pre -treatment) - 4/24/13 (early harvest season)). During the PSI trial, soil treatments induced similar general effects on the overall densities of soil microorganisms. ASD conducted using rice bran consistently resulted in higher soil densities of total bacteria, total fungi, fluorescent Pseudomonas spp., Streptomyces spp., Fusarium spp. and Pythium spp. indicating a generalized response to this carbon input. In contrast, ASD conducted using molasses as the C input had no consistent effect on any microbial group monitored across the three field study sites. In particular, soil fumigation with either Pic-Clor or methyl bromide/ chloropicrin reduced densities of all microbial groups, but the effects in general were less evident on bacterial than fungal populations. 165

14 Similar to the season at the MBA site (Figure 3), treatment-specific effects on fungal and bacterial community composition were consistently detected at the PSI site based on examination of the similarity in microbial communities from individual field plots using terminal restriction fragment length polymorphism (T-RFLP) data. Although there was an absence of clustering prior to treatment application, post-application fungal and bacterial communities within a given treatment exhibited a greater degree of similarity than between soil treatments. This treatment separation was typically more robust for the fungal than bacterial community, though for the fumigation treatments there were definitive distinctions regardless of microbial community (Figure 7). In the post-treatment soil, there were detectable amounts of Phytopthora fragariae in one of the untreated soil samples. There were detectable amounts of Pythium irregulare in ¾ UTC samples, ¾ Molasses 9 ton samples, ¾ RB + Mol samples, 1/4 of Molasses 6 ton samples, and 2/4 Pichlor-60 samples, but not in MeBr samples. There were detectable amounts of Pythium ultimum in all soil samples except one of the Molasses 6 ton samples and 3 of the 4 MB/Pic treated soil samples. Figure 7. Effect of soil treatments on fungal (left panel) and bacterial (right panel) community composition post-treatment application at the PSI site determined by T-RFLP analysis. X-axis denotes similarity among fungal communities across plots (range 0 to 1). The tree was constructed from the Jaccard similarity coefficient of composite profiles from triplicate digestions of amplified fungal DNA using primers specific for the ITS region of rdna. Bootstrap values are indicated at branch nodes of the tree (10,000 bootstrap replicates). Treatments: UTC=control; RB= Rice bran-based anaerobic soil disinfestation at either the 6 ton (RB 6) or 9 ton (RB 9) input rate; Mol= molasses-based ASD disinfestation at either the 6 ton (Mol 6) or 9 ton (Mol 9) input rate ; RB/Mol= Rice bran/molasses-based ASD; Pic-Clor and MB/Pic= pre-plant soil fumigation with Pic-Clor or methylbromide/chloropicrin, respectively. 166 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

15 Total fresh weed biomass as of July 18, 2013 at the PSI trial in ASD treatment was statistically similar to or slightly greater than UTC and significantly greater than fumigant treatments (Table 1). Total weeding time (hrs/acre) of ASD-Mix, -RB6, and RB9 were shorter than UTC, but much longer than fumigants. Table 1. Treatment effect on total weed fresh biomass and total weeding time at the PSI trial, Watsonville in the season (as of July 18, 2013). Means marked with the same letter have no significant difference according to protected LSD test (P=0.05). Treatment UTC ASD-ML6 ASD-ML9 ASD-Mix ASD-RB6 ASD-RB9 Pic-Clor MB/Pic P value Total Weed fresh biomass (lbs/500 sq. ft.) 10.7 bc 11.1 abc 9.5 c 10.4 bc 15.9 a 13.7 ab 1.3 e 2.3 d < Total weeding time (hrs/acre) 108 a 100 ab 89.4 abc 78.8 c 84.0 bc 75.3 c 30.6 e 41.1 d <

16 Discussion During the past two years, we compared the effect of non-fumigant methodologies, including ASD, steam, MM, and OA, alone and in combination, on fruit yield, survival of a range of soilborne pathogens, weed density, soil chemical and biological characteristics, and economics in strawberry production systems at the MBA site in Watsonville and the Santa Maria site Although both sites are virtually free from V. dahliae, the MBA site had disease pressure by Pythium and Cylindrocarpon. Overall, results from the MBA trial ( and ) and the Santa Maria trial ( ) indicated that ASD and MM+ASD to be the most consistently effective and economically feasible non-fumigant alternatives at this point. However, there are some issues to be addressed with current ASD technology. First, it did not provide fumigation levels of weed suppression (Table 1) and may need to be combined with herbicide use in severely weed infested sites (see Table 1 of the CSC report for weed densities at the MBA site). Second, although ASD has proved as effective as fumigation in suppressing V. dahliae and improving strawberry yields when using 9 tons/acre rice bran, a reduction in application rate or use of alternative carbon sources should be explored for a variety of reasons including: potential efficacy against other pathogens, reducing N input and cost and ease of application. For example, although rice bran ASD yields certain anti-fungal volatiles, ASD conducted with alternative carbon inputs has generated volatiles that have superior activity toward Pythium and Fusarium spp. (Mazzola, unpublished). Nine tons/acre of rice bran (N 2%, P2O5 5%, K2O 2%) provides N 360 lbs/acre, P2O5 900 lbs/acre, and K2O 360 lbs/acre. Data from both sites in reported in the CSC report showed that N in rice bran can be mineralized quickly resulting in high soil inorganic N post-treatment through to early fruit stage. However, data from the PSI trial in showed that ASD-RB6 (N 240 lbs/acre) and ASD-Mix plots (N 225 lbs/acre) provided statistically comparable cumulative marketable fruit yield by mid-july with ASD-RB9 and Pic-Clor (Figure 5). Further, soil inorganic N data (Figure 6) in ASD-RB6 plot suggest that considerable reduction of soil inorganic N concentration with relatively small yield reduction can be achieved by eliminating or reducing pre-plant N fertilizer application in ASD. Third, the modes of action contributing to disease control in ASD needs to be further explored. Preliminary studies demonstrate that ASD alters soil biology and results in development of a soil community that is suppressive to disease incited by Pythium spp. Reports also suggest that different organic substrates applied in ASD results in production of different volatile profiles which differ in their capacity to suppress fungal pathogens including F. oxysporum and P. ultimum (Mazzola, unpublished). Yet these volatiles would not have had a role in the observed soil suppressiveness towards reintroduced P. ultimum, suggesting that a multiplicity of mechanisms may contribute to ASD-induced disease control (Mazzola et al., 2012). Effective use of ASD or its integration with other methods such as organic acids or MM requires an understanding of mechanisms of action to avoid the diminution or elimination of disease control activity, as was observed in pot trials when MM application preceded ASD (Figure 4). In the MBA trials, MM alone at 1.5 tons/acre was not effective in the season (Figure 1A), and modestly effective in the season (Figure 1C). This variability may be attributed to the different sources of MM material between two seasons. In apple systems, MM applied at 3 tons/acre effectively suppressed soil borne pathogens (Mazzola and Brown, 2010). However, since MM contains 6% N and considerably elevated soil nitrate levels, higher application rates in strawberry may be prohibitive. Further studies should focus on how best to integrate MM and other practices such as ASD in strawberry systems. Fish emulsion significantly increased marketable fruit yield above the control in Santa Maria where disease pressure was very low and warrants further study in fields with greater disease pressure. This study confirms earlier data that steam is as effective as chemical fumigation. However, current bed steamer costs $5,472 per acre and a more efficient steam injection system is critical for adoption in a commercial setting. Recent advances may reduce the cost of steam treatment to less than $5,500 per acre with the potential of further cost reductions. 168 CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

17 In the season, some California berry growers started to implement ASD at commercial scales; ~20 growers used ASD in 32 fields (mostly organic) ranging from 0.5 to 20 acres in size, totaling 123 acres (Farm Fuel Inc, personal communication). As of early June 2013, most growers who implemented rice bran-based ASD appeared to be satisfied with plant performance and yield level especially those possessing fields with high disease pressure, planning expansion of ASD treated fields. Future studies should examine 1) effects of different ASD carbon sources and input rates on fruit yield, survival of a range of soilborne pathogens (especially of Fusarium spp. and Macrophomina spp.), greenhouse gas emission, N dynamics, and economics, 2) performance of ASD in large scale field experiments, and 3) clarification of the role and identity of soil microorganisms in disease suppression and identification of optimal application treatment sequence strategies for MM and ASD systems. 169

18 Acknowledgements This project was partially funded by the California Strawberry Commission and USDA-NIFA Methyl Bromide Transition Program. We thank our collaborators Dole Berry Company, Inc. Dave Peck at Manzanita Berry Farm, and Mike Nelson, Luis Rodriguez, and Patti Wallace of Plant Sciences Inc. Karen Klonsky of UC Davis conducted the economic analysis. Selected References Blok, W.J., Lamers, J.G., Termorshuizen, A.J. and G. J. Bollen Control of soilborne plant pathogens by incorporating fresh organic amendments followed by tarping. Phytopathology. 90: Braun, P. G The combination of Cylindrocarpon lucidum and Pythium irregulare as a possible cause of apple replant disease in Nova Scotia. Can. J. Plant Pathol. 13: Butler, D.M., Kokalis-Burelle, N., Muramoto, J., Shennan, C., McCollum, T.G. and E. N. Rosskopf Impact of anaerobic soil disinfestation combined with soil solarization on plant-parasitic nematodes and introduced inoculum of soilborne plant pathogens in raised-bed vegetable production. Crop Protection 39: Cohen, M.F., Yamasaki, H. and M. Mazzola Brassica napus seed meal soil amendment modifies microbial community structure, nitric oxide production and incidence of Rhizoctonia root rot. Soil Biol. Biochem. 37: Hoagland, L., Carpenter-Boggs, L., Reganold, J.P. and M. Mazzola Role of native soil biology in Brassicaceous seed meal-induced weed suppression. Soil Biol. Biochem. 40: Mazzola, M., Brown, J., Izzo, A.D. and M. F. Cohen Mechanism of action and efficacy of seed meal-induced suppression of pathogens inciting apple replant disease differ in a Brassicaceae species and time-dependent manner. Phytopathology 97: Mazzola, M. and J. Brown Efficacy of brassicaceous seed meal formulations for the control of apple replant disease in organic and conventional orchard production systems. Plant Dis. 94: Mazzola, M., Shennan, C. and J. Muramoto Application sequence and soil biology influence anaerobic soil disinfestation induced disease suppression. Proceedings for the Annual International Conference on Methyl Bromide Alternatives and Emission Reductions, , Nov. 6-8, Orlando, FL. Momma, N., Momma, M. and Y. Kobara Biological soil disinfestation using ethanol: effect on Fusarium oxysporum f. sp lycopersici and soil microorganisms. Journal of General Plant Pathology 76: CALIFORNIA STRAWBERRY COMMISSION ANNUAL PRODUCTION RESEARCH REPORT

19 Shennan, C., Muramoto, J., Baird, G., Fennimore, S., Koike, S.T., Bolda, M.P., Daugovish, O., Dara, S., Mazzola, M. and G. Lazarovits Non-fumigant strategies for soilborne disease control in California strawberry production systems. Proceedings for the Annual International Conference on Methyl Bromide Alternatives and Emission Reductions, , Nov. 6-8, Orlando, FL. Shinmura, A Causal agent and control of root rot of welsh onion. PSJ Soilborne Disease Workshop Report 20: (in Japanese with English Summary). Tewoldemedhin, Y. T., Mazzola, M., Labuschagne, I. and A. McLeod A multi-phasic approach reveals that apple replant disease is caused by multiple biological agents with some agents acting synergistically. Soil Biol. Biochem. 43: