Anaerobic Soil Disinfestation for Soil Borne Disease Control in Strawberry and Vegetable Systems: Current Knowledge and Future Directions

Transcription

1 Anaerobic Soil Disinfestation for Soil Borne Disease Control in Strawberry and Vegetable Systems: Current Knowledge and Future Directions C. Shennan a and J. Muramoto Department of Environmental Studies University of California, Santa Cruz Santa Cruz, CA USA M. Mazzola USDA-ARS Tree Fruit Research Laboratory Wenatchee, WA USA N. Momma Institute for Horticultural Plant Breeding Kamishiki, Matsudo Chiba Japan J. Lamers PPO-AGV, Applied Plant Research Wageningen UR Lelystad The Netherlands E.N. Rosskopf and N. Kokalis-Burelle USDA-ARS United States Horticultural Research Laboratory Fort Pierce, FL USA D.M. Butler Department of Plant Sciences University of Tennessee Knoxville, TN USA Y. Kobara National Institute for Agro-Environmental Sciences Kannondai, Tsukuba Ibaraki Japan Keywords: fumigant alternatives, microbial communities, soil-borne disease control, nematodes Abstract Anaerobic soil disinfestation (ASD), a biological alternative to soil fumigation, has been shown to control a wide range of soil-borne pathogens and nematodes in numerous crop production systems across Japan, The Netherlands and the US. A brief review of the status to the science behind ASD and its application for commercial settings is discussed for each country. Future work needs to focus on how to optimize the technique (in terms of carbon source used, temperature and degree of anaerobiosis attained) to control specific sets of pathogens, and to better which mechanism(s) are responsible for disease control in different situations. The role of observed microbial community shifts as a result of ASD in immediate disease control and long term disease suppression needs to be more fully explored. Further reductions in the costs of ASD compared to fumigant use will help increase adoption of the technique which is currently limited by cost and uncertainty about its effectiveness at controlling different pathogens across a range of environments. INTRODUCTION In the search for viable alternatives to the use of methyl bromide and other soil fumigants for control of soil borne pathogens, nematodes and weeds, methods using anaerobic decomposition of organic matter have emerged as showing broad spectrum efficacy across a range of environments and production systems. Initially two methods, a cshennan@ucsc.edu Proc. VIII th IS on Chemical and Non-Chemical Soil and Substrate Disinfestation Eds.: M.L. Gullino et al. Acta Hort. 1044, ISHS

2 biological soil disinfestation (BSD) for open fields in The Netherlands (Blok et al., 2000; Goud et al., 2003) and soil reductive sterilization (SRS) for greenhouses in Japan (Shinmura, 2004), were developed independently. Since that time considerable research has led to refinement of the methods and expansion into new areas including strawberry production in California, a major user of soil fumigants, and vegetable and cut flower production in Florida (Shennan et al., 2010, 2013; Butler et al., 2012a,b). Here the term ASD is used to encompass all these similar approaches for simplicity, and because it emphasizes the central role of creating anaerobic soil conditions and aptly identifies the process as a disinfestation rather than a sterilization. The method involves addition of a labile carbon source (to stimulate microbial growth and respiration), tarping with plastic to limit gas exchange, and irrigation to fill soil pore space with water, which allows for diffusion of decomposition by-products through the soil solution and reduces soil oxygen levels. Anaerobic conditions are created as the rapid growth of aerobic microorganisms depletes remaining soil oxygen and the microbial community shifts to facultative and obligate anaerobes. Anaerobic conditions are maintained for a period that varies with soil temperature and C-sources used (Table 1), before the tarp is either removed or planting holes punched through the tarp to allow oxygen back into the soil and stimulate the degradation of remaining by-products of anaerobic decomposition. The exact mechanisms that lead to pest suppression with ASD are not clearly understood. Different mechanisms may prove to be critical for suppressing specific organisms, but production of organic acids via anaerobic decomposition of the added C, release of volatile compounds, and biocontrol by microorganisms that flourish during the process are all potentially important. Fusarium oxysporum f. sp. lycopersici was completely suppressed in membrane-filtered soil solution collected from soil treated by ethanol-based ASD (EtOH-ASD) which is consistent with water-soluble compounds such as acetic acid and butyric acid and metal ions such as Fe 2+ and Mn 2+ contributing to the ASD effect (Momma et al., 2013). Changes in soil microbial communities are also observed following ASD and are discussed later in this paper. In this paper we describe the current state of knowledge and practice of ASD in the US, Japan, and The Netherlands and highlight key questions that remain for optimization of the approach for different production systems. Challenges facing wider adoption of the method by commercial growers are also discussed. CURRENT STATE OF DEVELOPMENT OF ASD Japan ASD is used nationwide for suppressing parasitic nematodes and soil borne pathogens especially in areas where residential and agricultural fields coexist. Wheat bran, rice bran, molasses, or diluted ethanol (<1.0%, v/v) are used as the carbon source. Some limited trials have been done with locally available organic materials such as byproducts from shochu distilleries and silage effluents. Solid organic materials such as wheat bran and rice bran are incorporated easily with a rototiller, but the effect of ASD is then limited to around 20 cm depth. Liquid materials such as molasses and ethanol are applied with irrigation water and penetrate deeper into the soil. With ASD using ethanol (EtOH), nematodes were eliminated down to 60 cm (Uekusa et al., 2013). The amount of irrigation used depends on soil conditions, but is typically 100 up to 200 mm when ethanol is used and applied with drip irrigation. Conventional polyethylene films are used for tarping for a period of 2 to 3 weeks in both open field and greenhouses. The tomato farmers association in Biratoricho, Hokkaido, the northernmost part of Japan, prohibits the use of chemical soil fumigants and recommends ASD using wheat bran. ASD is commonly done in early spring in this region when soil temperatures can rise to C inside plastic houses and ASD works well to suppress Pyrenochaeta lycopersici. Good pest suppression with ASD has been shown in andisol, alluvial, and sandy soils, and several artificial substrates such as coconut husk, cedar bark, and rock wool used in raised platform strawberry systems. ASD with EtOH is preferred over 166

3 chloropicrin fumigation to disinfest these artificial media, using an irrigation system equipped with a nutrient injector for EtOH dilution and application. Results on the spectrum of efficacy of EtOH ASD are summarized in Table 2. ASD is less effective at controlling bacterial pathogens than fungal pathogens and nematodes. However, bacterial wilt of tomato was strongly suppressed by combining rice bran-based ASD and grafting with a resistant cultivar, although each measure alone was not effective (Nakako, 2013). An implementation manual for ASD is now is available in Japanese from Commercial adoption of EtOH ASD by growers has been facilitated by the availability of an ethanol product Ecological exclusively for use in ASD, which does not fall under the category of a pesticide, but is listed under Biological ASD materials. While there are no data on the amount of land where ASD with wheat bran, rice bran, and molasses is being used, EtOH ASD was carried out in 28 out of 47 provinces as trials, demonstrations and commercial uses in The Netherlands Growers utilize residues of a green manure as the carbon source for ASD. It is harvested in summer and incorporated with a rotary spading cultivator in conjunction with a pressure roller into the top 30 cm of the soil at a rate of at least 40 t ha -1 (fresh weight). The lightly compacted soil is irrigated with mm of water and covered the following day with VIF tarp. After 4-6 weeks the plastic is removed (Blok et al., 2000). This method is currently used in the production of strawberry runners and in tree nurseries to control Verticillium dahliae, in asparagus to control Fusarium oxysporum and in greenhouses with soil substrate for organic farming to control Meloidogyne spp., and Verticillium. For control of replant disease in asparagus the method is modified to use a higher rate of 80 t ha -1 of green manure rotary tilled to a depth of 80 cm before plastic mulching. Some practical considerations are key to the success of the ASD: ensuring the plastic does not come loose, avoiding perforation by smoothing the soil surface before laying plastic and incorporating green manures at an early growth stage before stems start to lignify, as well as scaring off birds. Temperature is an important variable, and maximum air temperatures above 20 C are needed at a minimum, restricting ASD application to the summer in this region. ASD has been successfully used on sandy and clay loam soils and since 2004 has been used commercially on about 80 ha. ASD has been shown experimentally to control a wide range of pathogens (Table 3), and a number of studies suggest that the benefits from ASD can last more than a single season. For example, when asparagus is planted in a control soil with F. oxysporum f. sp. asparagi or F. redolens f. sp. asparagi and in a soil treated with ASD no differences are observed for the first two years, but thereafter the ASD treated plots show yield increases of 17-53% and remain in economic production for longer than the control plots (Lamers and Wilms, 2008). Similarly a long term effect on V. dahliae suppression was seen by Goud et al. (2003) in a study of tree nurseries. The extent and reliability of longer term disease suppression is a critical area for future research, since it will greatly affect the comparative economics of ASD versus soil fumigants. United States: Florida Research conducted in Florida on ASD has principally used composted broiler litter (CBL) and blackstrap molasses as carbon sources. The process involves listing a false bed, applying the CBL (sufficient to provide 224 kg N/ha) and molasses (8.2 Mg dry matter/ha), rotovating to approximately 15 cm, reforming the bed, and covering the bed with clear solarization plastic. Two drip lines (20 cm emitter spacing) are added per bed at the time of plastic application and approximately 50 mm of water applied. Based on Eh data, it is necessary to complete this process within 24 hours to accomplish the target Eh level (Rosskopf, unpublished). This process has been tested for use in raised-bed vegetable crop production including pepper, tomato, cucumbers, and strawberries. A similar approach has been tested for the production of cut flowers on flat ground. Soils 167

4 types in the production areas in which ASD has been tested are various types of sands. During the period of ASD, solarization also occurs as temperatures range from C, consistently 5-10 C higher than temperatures found in bare ground or under standard lowor high-density polyethylene tarps. A major limitation to the use of ASD in Florida is thus the need to remove the solarization plastic and replace it with a dark plastic, or to paint the plastic with an opaque water-based paint to lower soil temperatures for crop growth. Early research results have shown that each component of the system serves to control different pests. For example, in factorial experiments evaluating the impact of three levels of initial irrigation, +/- amendment with CBL, and +/- amendment with molasses; molasses amendment was the most significant factor in control of introduced inoculum of Fusarium oxysporum f. sp. lycopersici. In the same trial, initial irrigation was most significant for limiting nematode galling and soil populations (Butler et al., 2012). Similarly, strawberry plant mortality due to Macrophomina phaseolina, was significantly reduced when compared to the untreated check, but ASD did not entirely eliminate the pathogen (Fig. 1). A second limitation to the adoption of ASD by commercial vegetable producers is the use of CBL due to food safety concerns, although the material has been composted and shown to be free of detectable levels of Salmonella. Yet there is evidence that movement of this organism in tomato can occur discouraging conventional vegetable producers from using ASD. Current research is focused on input substitution for the CBL and ways to avoid the need for applying a second type of plastic. Utilizing a summer cover crop as a carbon input for ASD may provide growers more incentive to try the approach. In greenhouse studies, different cover crop inputs were compared to ASD utilizing molasses. Interestingly, pearl millet increased the viable colony forming units of Fusarium oxysporum f. sp. lycopersici, regardless of CBL amendment, but there was a significant interaction between C source and CPL amendment on reducing viability of Sclerotium rolfsii (Butler et al., 2012b). While molasses ASD resulted in a decrease in yellow nutsedge germination compared to no carbon input, few cover crops equaled this reduction. USA: Tennessee In 2010, an effort began to optimize ASD for production systems in Tennessee and regions with similar environments and production systems in southeastern USA. Initially, efforts focused on screening carbon sources including dried molasses (a sugarcane by-product used as livestock feed), wheat bran, and residues of various seasonally-adapted cover crops; for use in warm-season vegetable production. Major soil borne pest issues include Sclerotium rolfsii, Fusarium oxysporum, Ralstonia solanacearum, Meloidogyne incognita and weeds, especially yellow and purple nutsedges. These crops are typically produced in raised-bed, plasticulture systems following mid-spring soil fumigation. Soil temperature at 15 cm depth at this time of the year ranges from 15 to 25 C. Black polyethylene mulch to mm thick is used and the length of ASD treatment in research studies and demonstrations is typically three weeks. Irrigation to implement ASD has proved effective at 50 mm (on the bed area), which assuming that 25% of the soil volume is comprised of air-filled pore space, could effectively flush soil pore space to 20 cm depth. However, ASD can be difficult for Tennessee growers to implement in the early spring given their desire to plant their crop to capture higher early season market prices. Moving ASD treatment to earlier time periods may also not be as feasible because of low soil temperatures and potentially wet conditions delaying equipment use in fields. Initial research results suggested that high C source rates are needed (4 mg C g -1 soil) for effective and consistent ASD due to the relatively low soil temperatures (15 to 25 C) present during treatment (Butler et al., 2012). Cover crop biomass at sufficient rates is not difficult to achieve in field settings, however. In field studies, initial trials have suggested few differences in yields from ASD treatments compared to untreated controls (McCarty et al., 2012) and fumigated or untreated controls (Shrestha et al., 2013), due to relatively low endemic pest pressure. However, the effectiveness of ASD in 168

5 suppressing introduced pathogen inoculum has been promising; colonization of introduced sclerotia of Sclerotium rolfsii by Trichoderma sp. following ASD has been observed in several trials and may lead to the identification of direct parasitism as a potential mechanism for suppression of fungi of this type (Butler et al., 2012; Shrestha et al., 2013). Several growers have trialed ASD in Tennessee on a limited basis, but the method is not yet consistent enough to recommend large-scale grower adoption. Work continues to examine the interaction of C source and temperature on key pathogens, the effect of soil properties on ASD efficacy, the role of biocontrol fungi during ASD, and crop fertility management post ASD. USA: California and Washington State The main focus for ASD in CA strawberries initially was on suppression of Verticillium dahlia. Multiple field trials showed that ASD needs to be completed by the end of October before soil temperatures decline below 17 C at 15 cm depth. When 20 t ha -1 of rice bran (RB) was pre-plant incorporated and at least 75 mm of irrigation applied to sandy-loam to clay-loam soils, ASD consistently reduced the number of V. dahlia microsclerotia in the soil by 85 to 100%. Moreover, marketable yields from ASD plots were generally equal to or higher than Pic-Clor 60 plots, and significantly greater than untreated controls (Shennan et al., 2013). Following this success, a number of berry growers in California started to implement ASD in commercial fields (Table 1) (Farm Fuel Inc., pers. commun.). However, due to economic factors (cost ~$ 6,000 per ha as compared with ~$ 4,400 for PicChlor 60) and high nitrogen application issues (~400 kg total N ha -1 ) associated with use of 20 t ha -1 RB, there is interest in examining alternative carbon sources or reducing the amount of RB used, for example by applying a mix of RB and molasses or incorporating summer cover crops supplemented with RB. The type of carbon source used may however modulate the efficacy of ASD in control of soil borne diseases through modifying multiple aspects of the overall soil environment. Rice bran, but not molasses, used as the ASD carbon input induced significant increases in total fungal (Fig. 2) and bacterial densities in treated strawberry field soils. Similarly, ASD with rice bran resulted in significant and sustained shifts in microbial community composition, but ASD conducted with molasses did not alter community structure relative to the non-treated control soil system (Fig. 3). Correspondingly, ASD conducted with rice bran typically resulted in higher strawberry yields than with ASD using molasses (Shennan et al., 2013). The differential level of plant disease control in response to ASD with contrasting carbon inputs may be determined, in part, by the spectrum of volatiles produced during the anaerobic phase. In apple, three ASD carbon inputs (ethanol, grass residues and Brassica juncea seed meal) that provided the greatest control of P. penetrans (Fig. 3; Hewavitharana and Mazzola, unpublished data) also yielded the most active spectrum of nematicidal volatiles, including allyl isothiocyanate, dimethyl trisulfide and 2-ethyl-1- hexanol. In contrast, ASD with composted cow manure failed to produce any nematicidal volatiles and did not significantly lower lesion nematode numbers recovered from apple roots. Similarly, ASD conducted with ethanol, grass residues, rice bran or SM as the carbon source produced volatiles that were active against Fusarium oxysporum, Pythium ultimum and Rhizoctonia solani AG-5, but no active volatiles were produced when composted steer manure was used as the carbon input (Table 1; Hewavitharana and Mazzola, unpublished data). ASD-derived chemistries alone are not necessarily sufficient for attaining effective disease control, however. Although volatiles emitted during ASD with rice bran suppressed growth of P. ultimum, apple root infection by Pythium spp. in these same soils was exacerbated relative to the no-treatment control. In addition to carbon source affecting disease control efficacy, soil temperature is also key. For strawberries soil temperatures needed to be above 17 C for at least a week to get V. dahliae control, which may be related to a smaller reduction in ph due to lower organic acid production at cooler temperatures even when good anaerobic conditions are 169

6 achieved (Shennan et al., unpublished data). However, it is likely that the relationships between extent of anaerobiosis, temperature and disease suppression differ with the pathogen in question. For example fall applied ASD in CA did not control Fusarium oxysporum, whereas this pathogen is controlled with ASD in Japan and FL where soil temperatures are higher. CONCLUSION In summary ASD as practiced currently in Japan, The Netherlands and the US can provide effective control of a wide range of pathogens and nematodes even though specific techniques vary in terms of carbon source used, timing, and soil conditions. Future work needs to focus on understanding how to optimize the technique (in terms of carbon source used, temperature and degree of anaerobiosis) to control specific sets of pathogens, and to better understand which mechanism(s) are responsible for disease control in different situations. The role of microbial community shifts as a result of ASD and its relationship to both immediate disease control and long term disease suppression needs to be more fully explored. Further reductions in the costs of ASD compared to fumigant use should be explored to help increase adoption of the technique which is currently limited by cost and uncertainty about its effectiveness at controlling different pathogens across a range of environments. Literature Cited Blok, W.J., Lamers, J.G., Termorshuizen, A.J. and Bollen, G.J Control of soilborne plant pathogens by incorporating fresh organic amendments followed by tarping. Phytopathology 90: Butler, D.M., Kokalis-Burelle, N., Muramoto, J., Shennan, C., McCollum, T.G. and Rosskopf. E.N. 2012a. 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: Butler, D., Rosskopf, E., Kokalis-Burelle, N., Albano, J., Muramoto, J. and Shennan, C. 2012b. Exploring warm-season cover crops as carbon sources for anaerobic soil disinfestation (ASD). Plant and Soil, p Goud, J.C., Termorshuizen, A.J., Blok, W.J. and van Bruggen, A.H.C Long-term effect of biological soil disinfestation on Verticillium wilt. Plant Disease 88: Lamers, J. and Wilms, J De lange termijn werking van biologische grondontsmetting. Biologische grondontsmetting doet de opbrengst ieder jaar stijgen tot wel 100% na herinplant van asperges. PPO-AGV report , Lelystad, The Netherlands, p.27. Lamers, J.G., Runia, W.T., Molendijk, L.P.G. and Bleeker, P.O Perspectives of Anaerobic soil disinfestation. Acta Hort. 883: Momma, N., Kobara, Y., Uematsu, S., Kita, N. and Shinmura, A Development of biological soil disinfestations in Japan. Appl. Microbiol. Biotech. doi: /s Nakaho, K Integrated protection of tomato wilt disease by the high grafting method. Agro-technological system: Soil Science and Plant Nutrition 5(1): to (in Japanese). Shennan, C., Muramoto, J., Koike, S.T. and Daugovish, O Optimizing anaerobic soil disinfestation for non-fumigated strawberry production in California. California Strawberry Commission Annual Production Research Report : Shennan, C., Muramoto, J., Fennimore, S., Mazzola, M. and Lazarovits, G Nonfumigant strategies for soilborne disease control in California strawberry production systems. California Strawberry Commission Annual Production Research Report : Shinmura, A Principle and effect of soil sterilization methods by reducing the redox potential of soil. PSJ Soilborne Disease Workshop Report 22:2-12 (in Japanese with English summary). 170

7 Shrestha, U., Ownley, B.H., Rosskopf, E.N., Dee, M.E. and Butler, D.M Optimization of amendment C:N ratio in anaerobic soil disinfestation for control of Sclerotium rolfsii. Proceedings of the Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, p.14-1 to Uekusa et al Application of low concentration ethanol that enhances the efficacy of anaerobic soil disinfestation. Plant Protection 67(4):7-12 (in Japanese). 171

8 172 Tables Table 1. Comparison of ASD methods in different regions. Region USA CA Fl TN Japan Netherlands C-sources 1 and rates (t ha -1 ) RB 13.5 RB 20, RB 10+ Mol 10 Mol 8.2 CBL varied rates Plant residue Mol 6 WB 6 EtOH 2-7, Mol 4-12, RB 10-20, WB Plant residue (fresh wt.) Water appl. (mm) ~75 Depth treated (cm) bed 20 flat Tarp period (weeks) 3 Soil temp. 15 cm ( C) fall summer Crops System Pathogen control Strawberry, raspberry, herbs Vegetables, cut flowers, strawberry Vegetables ~100 to liquid, 20 solid or air temps above 20 o C Strawberry vegetables, cut flowers Maple, catalpa, asparagus, strawberry nurseries Bed, open field; flat, open field Bed, flat, open field Bed, open field flat - plastic houses and open field, substrate in greenhouse Flat open field, substrate in greenhouse 1 EtOH = ethanol; Mol = molasses; CBL = composted broiler litter; RB = rice bran; WB = wheat bran. Verticillium dahliae, Rhizoctonia solani Pythium myriotylum, Macrophomina phaseolarum, Fusarium oxysporum Nematode control? Meloidogyne arenaria, M. incognita Sclerotium rolfsii? See Table 2, V. dahliae, Fusarium oxysporum, F. redolens, Ralstonia solanacearum See Table 2, Meloidogyne incognita Pyrenochaeta lycopersici See Table 3 See Table 3 Weed control Little effect Grasses Limited effect? Some effect (see Table 3) Commercial use 49 ha ha 2013 None None 28 out of 47 regions 70 ha 172

9 Table 2. Pests controlled by ethanol based ASD in Japan. Pest Crop Application rate of ethanol Phomopsis sclerotioides Cucurbits 1-2% (v/v), L/m 2 Burkholderia caryophylli Carnation 2% (v/v), L/m 2 Fusarium oxysporum Spinach, strawberry % (v/v), L/m 2 Glomerella cingulata Strawberry 0.5% (v/v), 100 L/m 2 Pyrenochaeta lycopersici Tomato 0.75% (v/v), 200 L/m 2 Plasmodiophora brassicae Broccoli 1.0% (v/v), 100 L/m 2 Meloidogyne spp. Cucurbits, tomato % (v/v), L/m 2 Pratylenchus spp. Radish 1.0% (v/v), L/m 2 Phytophthora palmivora Southern star 0.7% (v/v), 180 L/m 2 Table 3. Efficacy of ASD on pathogens in The Netherlands (Lamers et al., 2011). Pathogen Efficacy 1 Efficacy Fungi Fusarium oxysporum Phytophthora fragariae Rhizoctonia solani AG3 Rhizoctonia tuliparum Sclerotinia sclerotiorum Synchytrium endobioticum Verticillium dahliae Nematodes Ditylenchus dipsaci Globodera pallida Meloidogyne fallax M. chitwoodi Pratylenchus penetrans P. fallax Trichodoridae ++ + Bacteria Ralstonia solanacearum 1 Efficacy: + some effect, ++ distinct effect, highly effective. Weeds Weeds from rootstock, bulb, etc. Weeds from seed ++ + Table 4. Effect of carbon amendment on activity of volatiles generated during ASD on colony growth of Rhizoctonia solani AG-5, Pythium ultimum and Fusarium oxysporum. Mean colony diameter 2 Carbon amendment 1 F. oxysporum P. ultimum R. solani AG-5 C 14.4a b 24.0a 24.0a ET 4.0cd 11.4b 2.4c EXC 10.6b 24.0a 23.0b GR 2.4d 0.2d 0.6d SM 0.0e 0.0d 0.0e PC 14.8a 24.0a 24.0a RB 5.4c 3.6c 1.0d CM 14.8a 24.0a 24.0a 1 ET = ethanol, 8.9 kl ha -1 ; GR = grass residues, 20.0 t ha -1 ; SM = B. juncea seed meal, 4.9 t ha -1 ; PC = pasteurized control; RB = rice bran, 4.9 t ha -1 ; CM = composed steer manure, 11.1 t ha Values represent mean colony radius (mm) across all replicates, with n=5. Data were analyzed using SAS PROC GLM method. Means within a column designated with the same letter are not significantly different (P>0.05). 173

10 Figures Fig. 1. Percentage of strawberry plants impacted by stunting and mortality caused by a disease complex of Fusarium oxysporum and Macrophomina phaseolina. Bars with the same letter are not significantly different based on Fisher s protected LSD (0.05). cfu g -1 soil 9.00E E E E E E E E E E+00 Fig. 2. Effect of soil treatments on total fungal community density in strawberry field soils assessed using culture-based methods. Values are mean fungal density (n=4) with standard deviations. Treatments: no-treatment control (UTC); PicChlor 60 fumigation (PicChlor); methyl bromide chloropicrin fumigation (MBPic); and ASD using molasses 6 (Mol 6) and 9 t ha -1 (Mol 9), rice bran 6 (RB 6) and 9 t ha -1 (RB 9), or rice bran 4.5 t ha -1 + molasses 4.5 t ha -1 (RBMol). 174

11 Component Component 1 Fig. 3. Effect of soil treatment on fungal community composition in strawberry field soils using principal coordinate analysis of T-RFLP data. Treatments: non-amended control (Δ), PicChlor ( ), MeBr/Pic ( ), and ASD using carbon inputs of rice bran 9 t ha -1 ( ), rice bran 6 t ha -1 ( ), rice bran 4.5 t ha -1 + molasses 4.5 t ha -1 (*), molasses 9 t ha -1 ( ), or molasses 6 t ha -1 ( ). Fig. 4. Effect of carbon amendments used in ASD on Pratylenchus penetrans recovered from roots of Gala apple seedlings grown in orchard soil. Treatments: ASD with 10% ethanol (Et; 8.9 kl ha -1 ), grass residues (GR; 20.0 t ha -1 ), Brassica juncea seed meal (SM; 4.9 t ha -1 ), rice bran (RB; 4.9 t ha -1 ) or composted steer manure (CM; 11.1 t ha -1 ), pasteurized control (PC) and no amendment control (C). Columns with the same letter are not significantly different (P=0.05) based on Friedman s non-parametric test. 175

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