Final Report for GW09-021
The Pacific Northwest of the U.S. encompasses 90% of processed raspberry acreage nationwide. The duration of harvestable plantings has declined from >10 years to approximately 5 years. Root rot damage by Phytophthora rubi (Pr) and Pratylenchus penetrans has been associated with this decline, but soil characteristics that promote these pathogens are not well understood. Currently, broadcast pre-plant fumigation is used to manage soil-borne pathogens; a practice that is expensive and chemically intensive. This research aims to develop a quantitative molecular assay for Pr in raspberry soil and roots and investigate alternatives to fumigation for pre-plant management of these pathogens.
In the Pacific Northwest, commercial raspberry growers have several pathogens and plant-parasitic nematodes that can cause yield decline over time (McElroy, 1992). New restrictions on whole-field fumigation and on many common fumigant products (USEPA, 2009) will require growers to implement novel approaches to manage soil-borne pathogens and plant-parasitic nematodes that are less reliant on chemical treatments. Biologically-based approaches for managing pathogens have been used in other perennial crop production systems (Mazzola et al., 2010; Bailey and Lazarovits, 2003; Zasada et al., 2003), but these require further investigation to be effectively implemented into raspberry production systems.
Integrated control of pests in raspberry production systems has been investigated (Pinkerton et al., 2009; Pinkerton et al., 2002; Maloney et al., 2005; Wilcox et al., 1999). Integrated management requires an input of organic matter into the soil, addition of a biocontrol agent or bio-pesticide, or manipulation of the soil ecosystem to encourage beneficial organisms and minimize pest activity and survival. One such component of this type of system is biofumigation.
Biofumigation has been defined as the suppressive effect of brassicaceous materials on soil-borne pests in part due to the release of toxic isothiocyanates (ITC) and other compounds, byproducts of glucosinolate hydrolysis (Motisi et al., 2010). Release of these compounds is mediated by several factors, including particle size, soil temperature and water availability. The composition of ITCs will vary by species (Fahey et al., 2001), thus influencing how this material influences plant health and affects soil biota. Historically, brassicaceous plant materials have been incorporated into soil as green manures for pathogen control and nitrogen supplementation, however the use of brassicaceous seed meals is also of interest (Mazzola and Brown, 2010; Snyder et al., 2009; Zasada et al., 2009). Seed meals are a byproduct of the biofuel industry oil extraction process and may increase in volume and availability in the Pacific Northwest in the future (Higgins, S, personal communication).
Solarization is another fumigant alternative method that has been used in annual and perennial cropping systems (Stapleton, 2000). This approach uses passive solar heating of moist soil mulched with plastic sheeting. In Washington, the use of solarization did control common raspberry pathogens in the field (Pinkerton et al., 2009; Pinkerton et al., 2002), but results were not consistent across years due to the variability of the mild, marine climate in this region. Exposure time required to kill P. rubi was estimated to be 222 hours at 29o C, and all locations in this study exceeded the desired heat units (Pinkerton et al. 2009). Solarization in combination with organic amendments has also been investigated (Gigot et al., 2010 (APPENDIX A); Klein et al., 2007; Gelsomino et al., 2006) and may be a viable choice for raspberry growers as opposed to solarization alone, but further research is needed. Tarping of brassicaceous plants increased the efficacy of this material alone in coastal areas of California (Zasada et al. 2003)
Application of biofumigants will require more information on how the biofumigant affects target and non-target organisms, as well as how these products influence crop performance and the long-term suppression of pathogens. This study investigates the effects of brassicaceous seed meals in combination with soil solarization on two raspberry pathogens, Phytophthora rubi and Pratylenchus penetrans, in field and greenhouse trials. In addition, we address some of the issues associated with application of this material, including application rate, host plant toxicity and impact on non-target organisms, such as beneficial nematode communities.
1. Develop a real-time PCR assay and complimentary bait assay for quantifying P. rubi inoculum in raspberry roots and field soil
2. Measure P. rubi and plant pathogenic nematode survival and infectivity in alternative bed management treatments
3. Assess common soil quality indicators in alternative bed management treatments (nematode community, particulate organic matter, water stable aggregates, water infiltration, pH, nutrient status)
Brassicaceous seed meal field trials
Inoculum of Phytophtora rubi (strain ATCC 16184) was prepared by modifying the procedure of (Dissanayake et al., 1997). Vermiculite, V-8 broth and oat seed (450 cm3:350 ml: 20 cm3) were added to 900 ml glass jars and covered with foil. Inoculum mixtures were autoclaved twice over a 24 hour time period. After cooling, five agar plugs (0.7-cm-diam.) from 14- to 21-day old V-8 plates containing P.rubi were added to each jar, and jars were incubated at room temperature (18 to 20 °C) for four to five weeks. Inoculum was rinsed, drained and thoroughly mixed with 20.4 kg autoclaved (2 x) field soil (Skagit silt loam; Fine-silty, mixed, superactive, nonacid, mesic Fluvaquentic Endoaquepts). Inoculated soil (150 g) was placed in nylon mesh bags (L’eggs; Hanesbrand, Inc. Winston-Salem, NC) in a processing laboratory in preparation of burial.
Field trial establishment
Field plots were established at the Washington State University-Northwestern Washington Research and Extension Center (WSU-NWREC) in Mount Vernon, WA July 2008 and July 2009. Each plot was a single raised bed (5 m x 20 cm x 94 cm). Beds were shaped, and drip tape and solarization film (Robert Marvel Plastic Mulch; Annville, PA) were applied with a Rain-Flo model 2600 bed shaper (Rain-Flo Irrigation, East Earl, PA). In 2008, the following treatments were evaluated:
1) a non-treated control,
3) Inline™ (1,3-dichloropropene: chloropicrin, 61:33, 400 L/ha) drip fumigant
4) Sinapis alba ‘IdaGold’ seed meal at a rate of 1% v/v,
5) soil solarization plus Inline™ dripline fumigant,
6) solarization plus S. alba at 1% v/v, and
7) solarization and linseed meal at 1% v/v (non-ITC control).
In 2009, the following treatment were evaluated:
1) a non-treated control,
3) B. juncea ‘Pacific Gold’ seed meal at a rate of 1% v/v,
4) S. alba ‘IdaGold’ seed meal at a rate of 1% v/v,
5) soil solarization plus B. juncea seed meal at 1.0% v/v,
6) solarization plus S. alba at 1% v/v, and
7) solarization and linseed meal at 1% v/v (non-ITC control).
In both experiments, plots were arranged in randomized complete blocks, and treatments were replicated five times.
Both brassicaceious seed meals were obtained from within Washington State University (Dept. of Crop and Soil Science/Steward Higgins) and linseed meal was obtained from Skagit Farmer’s Supply (Mount Vernon, WA) and was included in the trials as a non-glucosinolate containing meal with approximately the same N-content as the brassicaceous seed meals. Brassicaceous seed meals were obtained in a pelletized form and ground prior to use. Seed meals were ground in a grain grinder (Kitchen Aid, St. Joseph, MI) to a consistent particle size (< 20 mm). Prior to solarization film, seed meals were sprinkled on the surface of pre-formed beds and incorporated to a depth of approximately 20 cm with a shovel. Nylon mesh bags containing P. rubi (~10^3 oospores per gram of soil) were placed at 15, 30, and 40 cm in the center of these beds spaced 45 cm apart. To bury, holes were hand dug to 45 cm, and the soil removed was piled adjacent to the plot. Three bags, representing each depth, were tied together with plastic wire, and each bag was buried in sequential order. Re-fill soil was patted down to ensure depth placement of the bags. Bags were placed within 3 m of the south end of the plot. Bags were retrieved from each plot February 12, 2009 and January 7, 2010 for each trial. All plots were saturated (0 kPa) following seed meal incorporation to a depth of 45 cm.
Temperature sensors (HOBO, Onset Corp., Bourne, MA) were buried in solarization and control plots in each of two replications at 15, 30 and 45 cm. Soils samples (composite of 10, 2.5-cm diam. cores) were collected from all treatments. In 2010, samples were collected from three replications of each treatment in March and analyzed for nitrogen content (NH4+, N03-), carbon, percentage organic matter, pH and sulfur (A&L Labs West, Portland, OR).
Survival of P. rubi inoculum from the field trial was evaluated with a greenhouse bioassay using susceptible raspberry ‘Tulameen’ plants grown in D40H Deepots (Stuewe and Sons, Inc, Tangent OR) with 20 ml inoculum from each plot being mixed with an autoclaved field soil (Skagit silt loam) and vermiculite mixture (1:2/v:v). Three plants were inoculated per replication and treatment (20 ml inoculum per plant) for each field trial assay. Non-inoculated checks were also included in the greenhouse assay. The plants were watered and fertigated with 20-20-20 NPK (Plant Marvel Laboratories, Chicago Heights, IL) as needed, up to three times a week, by immersion in 473 ml (12 cm high) cups for six to eight hours. To encourage infection by P.rubi, plants were flooded by immersion in 946 ml (17-cm high) cups for 48 hours every two weeks (Walters et al., 2008). Greenhouse conditions were set at 16o C 12 hour day/night light cycle.
Tissue culture plants were harvested after noticeable above ground root rot symptoms (leaf necrosis and wilting) occurred. Plants were harvested from containers and roots were washed in cold water to remove excess soil. Roots were set in trays by replication and treatment. Root rot and proportion of root disease were evaluated using a standardized, visual rating scale (Walters et al., 2008). Root rot was measure on a 0 to 9 (0=healthy, 9=dead) scale and proportion of root disease was measured on a 0 to 7 (0=0-10%, 7=100%) scale. Root dry weight was recorded after 24 hours at 72o C. Aerial biomass was also recorded after 24 hour at 72o C. A sub-sample of symptomatic root material was taken from one replication and viewed under the microscope for presence of P.rubi oospores. Sterilized root fragments were plated onto selective media (PARP) for verification of P. rubi germination (Walters et al., 2008).
Soil samples were collected at three dates: pre-bed formation, late season and early spring. Ten soil cores (2.5-cm diam. by 15 cm deep) were collected from within each plot. Soil cores were pooled and P. penetrans were extracted from soil by placing 50 g of soil on a Baermann funnel for five days (Ingham, 1994). Extracted nematodes were collected and the number of nematodes was determined using a dissecting microscope at 40x magnification.
Seed meal rate greenhouse experiment
A greenhouse trial was established to determine rate of B. juncea and S. alba seed meals needed to suppress P. rubi and P. penetrans. Phytophthora rubi inoculum was produced using a method adapted from Dissanayake et al. (1997). Phytophthora rubi (strain ATCC 16184) was produced as described above. Oospore density of the inoculum was evaluated by taking 6 g of inoculum and homogenizing (Ultra-turrax T25; Janke& Kunkel Labortechnik IKA, Wilmington, NC) the material in 60 ml H20. Four 10 ul aliquots of this mixture were distributed onto microspore slides and the number of oospores was determined and averaged across the four samples (~24,000 oopsores/g).
Field soil (Skagit silt loam) was collected, sieved (2-mm), and autoclaved twice. Sterilized soil was then mixed with fin vermiculite (Steubers, Snomomish, OR) in a 1:2 ratio. Appropriate quantities of P.rubi inoculum were added to the soil:vermiculute mixture to obtain an oospore density of 200 oospores per g/soil. Seed meals were added to soil at 0.5, 1.0 and 2.0% weight to volume (w/v) rates. A non-treated control was included in the experimental design. Seed meals were incorporated into soil and placed in a 18.8 L plastic buckets, and covered loosely with plastic and incubated at 20-23o C for 6 weeks.
Following this treatment period, tissue culture plants ‘Meeker’ (3 months) were planted into the P. rubi infested mixture (400 ml/150 g) in D40H Deepots and set-up in a greenhouse bioassay. Plants were watered to maintain the soil potting mixture at field capacity. Plants were flooded every two weeks by placing Deepots in 16 oz cups filled with water. Plants were fertilized by cup fertilization (Walters et al., 2008). Overhead watering was done as needed to maintain a uniform moisture level throughout the container. Greenhouse conditions were 16o C with a 12 hour day night light cycle.
Tissue culture plants were harvested after noticeable above ground root rot symptoms (leaf necrosis and wilting) occurred. Plants were harvested from containers and roots were washed in cold water to remove excess soil. Roots were set in trays by replication and treatment. Root rot and proportion of root disease were evaluated using a standardized, visual rating scale as described above. Root dry weight was recorded after 24 hour at 72o C. Aerial biomass was also recorded after 24 hours at 72o C.
For the experiment in which P. penetrans was evaluated, similar treatments and soil preparation occurred as described above. The P. penetrans population used in all studies was obtained from a red raspberry field in Lynden, WA. Soil, a Kickerville silt loam (Isotic, Mesic Typic Haplorthods), was collected from the root zone (15-30 cm depth) of established plants. The soil was passed through a 4-mm sieve with material retained on the sieve being discarded. Mustard seed meals were applied to soil to achieve rates of 0.5, 1.0 and 2.0% w/w dry soil for both B. juncea and S. alba. Treated soils were placed in plastic bags and the bags were sealed at the top using a plastic 250 ml cup in which holes had been made. Bags containing soil were incubated in the dark at 23o C for six weeks. Weekly, approximately 5 ml of water was added to the bags to account for moisture loss. After the incubation period, treated soils were placed in D40H Deepots and a tissue cultured red raspberry ‘Meeker’ planted in each Deepot. Plants were allowed to grow for six weeks in a greenhouse with 24/18o C day/night temperatures under ambient light. The plants were fertigated weekly with 20-20-20 NPK (20N-8.8P-16.6K) (Scotts, location). At termination, the aboveground portion of the plant was removed and placed in a 70o C oven for one week before determining dry weight. The contents of the pot were emptied onto a tray and the roots were shaken free of soil and a 50 g soil sample was collected. The 50-g soil sample was placed on a Baermann funnel and nematodes were extracted for five days. Roots were washed free of soil and P. penetrans was extracted by intermittent mist for one week (Ingham, 1994). The roots were then placed in a 70 °C oven for one week before determining dry weight. Nematodes were enumerated using a dissecting microscope at 40x magnification and are expressed as number of P. penetrans per gram dry root or number per 50 g soil.
All experiments were conducted twice and the seed meal/rate combinations, as well as the non-treated controls, were replicated six times. Deepots were arranged in a randomized block design on greenhouse benches in all experiments.
Seed meal and nematode community composition experiment
A micro-plot experiment was established in a field at WSU-NWREC June 2010. Two field soils (Soil A and Soil B) were amended with B. juncea, S. alba, or left non-treated. Field soil was collected from two raspberry field survey sites [Soil A, Skagit County (Briscot fine sandy loam) and Soil B, Whatcom County (Kickerville silt loam)] located in Burlington and Lynden, WA, respectively. Soils were collected on June 15, 2010 from both locations. The soils were passed through a 4-mm-sieve and mixed by hand with either B. juncea (0.5 % w/v), S.alba (1% v/v) on no meal (non-treated control). Rates were determined based on effectiveness from greenhouse trials mentioned above. Soil for each treatment and replication (16 L) was placed in a clean 18.8 L plastic buckets with five holes (2-cm diam.) in the bottom. A field plot was prepared by rototilling two times and pulling four beds. Buckets were placed in holes (45-cm deep) that were dug by hand within the formed beds. One, tissue cultured raspberry ‘Meeker’ plant was planted into each bucket and watered regularly to maintain field capacity. The experiment was arranged in a randomized, complete block design and treatments were replicated four times for each soil type.
Composite soil sample consisting of eight cores (2.5-cm diam. to a depth of 15 cm) were collected from each bucket at one week and five weeks after planting. A soil chemical analysis (A&L Labs Soil Labs, Portland, OR) was performed on a subsample of soil after one week measuring total N, P, K, % organic matter and pH. Nematodes were extracted from 250 g of soil by decanting/wet sieving through a series of sieves with nematodes being caught on a 400-mesh sieve. The 400-mesh sieve was backwashed into a container and the contents transferred to a Baermann funnel; nematodes were collected after five days. Nematodes were immediately fixed in 5% formalin solution for future enumeration. A dissecting microscope was used to count the total number of nematodes per sample. The sample was centrifuged, the supernatant removed, and the pellet and remaining solution were spread on a microscope slide and covered with a cover slip (Ferris and Manute, 2003). Nematodes for each slide were identified to family when possible using a compound microscope; in those samples with few nematodes all encountered nematodes were identified. The actual abundance of each taxon was adjusted according to the total number of nematode in the sample.
Plant back experiments
Three rates of each B. juncea and S. alba seed meals (0.5, 1.0, and 2.0% w/v) were incorporated into soil to determine phytotoxicity to raspberry in greenhouse experiments. A non-treated control was included, and all treatments were replicated three times and arranged in a randomized block design. The experiment was conducted twice. Ground meals of both species obtained from WSU were applied to sterilized field soil (Skagit silt loam), the mixture was transferred to 10-cm pots and tissue culture raspberry ‘Meeker’ (Sakuma Brothers, Burlington, WA) plants were planted into the mixtures. Plants were watered daily to maintain field capacity. Greenhouse conditions were 16o C with a 12 hour day/night light cycle. Plant toxicity measurements, assayed as a percentage of total plant aerial material damaged on a 0 to 10 scale (0=dead, 10=healthy), was collected one, four, and six weeks after seed meal application.
Additionally, 100 g of seed meal amended soil (mentioned above) was placed into small plastic bags and brought to field capacity. For each time period (one, four and six weeks), twenty seeds of lettuce (‘Black Seeded Simpson’ Burbee, Lot 8) and radish (‘Cherry Belle’, Burbee Lot 3) were placed in the top 1 cm of the bag and the bags were partially sealed and observed for percentage of germinated seed after one week. Bags were held at 18 °C in a plastic container in the dark. This experiment had three replications and was repeated.
Data from all experiments were analyzed using PROC GLM (SAS Institute, Cary, NC). In the field trial bioassay, there was a depth by treatment interaction for root rot, proportion of diseased roots, aerial weight and root weight, therefore all treatments were analyzed by depth. For the P.rubi seed meal rate greenhouse trial there was no interaction between tests for any treatment parament so data were combined. For the P.penetrans seed meal rate greenhouse trial there was an interaction between test and treatments so data were analyzed and presented separately. Means were separated by Fisher’s protected least significant difference (LSD) (P < 0.05).
Nematodes from the community study were assigned to trophic groups based on Yeates et al., (1993) and faunal indices were calculated as described by Ferris et al. (2001) including the Basal Index (BI), Structure Index (SI), Enrichment Index (EI) and Channel Index (CI). The enrichment and structure index are both indicators of the food web conditions with a focus on functional nematode guilds (Ferris and Bongers, 2001). Proportions (bacterial feeders, fungal feeders, omnivores and predators and plant feeders) was based on the sum of these groups divided by the adjusted total. All data (total nematodes, P.penetrans, BI, SI, EI and CI, and proportions were analyzed using PROC GLM and means were separated as above.
Quantitative real-time PCR assay
Inoculum of P.rubi was also evaluated using a quantitative real-time PCR assay. Selected treatments (Non-fumigated VIF control, Telone C-35 with VIF, MIDAS 50:50, 250 HDPE, and MB:chloropicrin) were evaluated with this method. The assay utilized a Taq-man probe and primers (Invitrogen/Life Technology, Carlsbad, CA) developed by Bonants et al. (2004). DNA was extracted from soil samples using Soil Mo Bio soil extraction kit (Mo Bio, Inc., Carlsbad, CA). The primers and probe targeted the ITS region and amplified a 130 base pair amplicon (Bonants et al., 2004). All assays were run on a Rotorgene 6000 (Qiagen, Inc., Valencia, CA) and used Fast Probe reagents (Qiagen, Inc.). Each run was 25 µl (1 µl sample DNA) and included internal and external controls (Bonants et al., 2004). Samples of known Phytophthora spp. and Pythium spp. isolates, as well as known oospore concentrations per gram of soil, were used to validate the assay (Gigot et al., 2009). Inoculum samples were run in triplicate and concentrations were determined based on an established standard curve. Assay results are presented in ng/µl.
Brassicaceous seed meal trial 2008-2009
For all the plant pathogens evaluated, depth of sampling in the soil was not a significant factor in the analyses of variance, so data were combined over the sampling depths for each pathogen. In 2008, accumulated hours above 29o C reached 34 hours at a soil depth of 15 cm only in the solarization plots. Soil solarization did not significantly affect the severity of disease caused by P. rubi, but the plots with Inline alone, solarization plus InLine, solarization plus S. alba, and solarization plus linseed meal had significantly greater disease ratings than the non-treated control (Table 11). The InLine treatment significantly increased the P. rubi disease rating (6.2) compared to the non-treated control (4.9). There was no difference between root weight, however the Inline treatment alone significantly increased aerial weight in the greenhouse bioassay. At the first sampling (January 2009), there were significantly more nematodes in the solarized plots than in the control, Inline, S.alba, solarization and Inline and solarization and S. alba plots. By the final sampling date (April 2009), there was no significant difference in P. penetrans population densities among treatments, however there were no P. penetrans detected in plots with either of the InLine treatments. Soil pH was significantly higher in the control (6.4) as compared to solarization (6.0), S.alba (5.8), solarization and Inline (5.6) and solarization and S. alba (5.3) (P=0.0025). Sulfur was significantly higher in the solarization and S. alba treatment than any other treatment (P=0.01). Nitrogen (N03 and NH4) was not affected by any treatment in this field trial.
Brassicaceous seed meal field trial 2009-2010
For root rot, differences between treatments were only detected at 15 cm (Figure 11A), with solarization plus both brassicacous seed meals having the lowest root rot rating (P < 0.05) compared to the non-treated control and non-solarized-amended plots. The level of root rot in solarized plots that received brassicaceous seed meals was not different from root rot levels in solarized, linseed meal-amended plots. Solarization alone and seed meals alone did not reduce the level of root rot caused by P. rubi with root rot ratings similar to the non-treated control at 15 cm.
Pratylenchus penetrans population densities at this site were generally low (< 10/50 g soil). At the final sampling date there was no significant difference in P. penetrans population densities between treatments (data not shown). Root weight was significant at the 45 cm depth only with solarization plus B. juncea, S. alba, and linseed seed meal having significantly higher root mass than the non-treated control (Figure 11 B). At 15 cm, aerial weight was significantly higher for these treatments as well.
The accumulated number of hours above 29o C were 104 hours (15 cm), 8 hours (30 cm) and 0 hours (45 cm) and were higher than in the 2008-2009 trial. Nitrogen (N03-) levels were significantly higher in all solarization plus seed meal treatments than the non-treated control and seed meal alone treatments (Table 12). Ammonium (NH4+) was significantly higher in the solarization plus B. juncea and S. alba treatments (P < 0.05). Seed meals and solarization, alone or combined, did not consistently alter percentage organic matter or soil pH. Sulfur was significantly higher in the solarization plus S. alba treatment (50 ppm) followed by the solarization plus B. juncea and S. alba alone treatments.
Seed meal rate greenhouse trial
There was no significant difference between experiments for P. rubi, therefore data for this organism was combined for analysis. All treatment resulted in significantly lower levels of root rot than the control (Figure 12). In general, all rates of B. juncea had lower root rot levels than S. alba, with 2.0% having the lowest level of root rot of any of the tested treatments. All rates of B. juncea resulted in greater reductions in root rot levels compared to S. alba at 0.5%, while the higher rates of S. alba resulted in comparable root rot levels as B. juncea at 0.5% and 1.0% w/v. There was a non-significant dose response between S. alba rates and root rot levels, with higher rates resulting in less root rot. Aerial and root biomass were lower for all plants treated with S. alba, but this was not significant (Figure 12).
There was a significant interaction between P. penetrans experiments, therefore experiments were analyzed separately (Figure 13A). All rates of B. juncea reduced P. penetrans population densities to near zero in both experiments. Rate of 1.0% or 2.0% of S. alba was needed to reduce P. penetrans population densities to the same extent as B. juncea at any rate. Interestingly, S. alba at 0.5% actually increased P. penetrans population densities compared to the non-treated control in both experiments, however this increase was not significant. In the P. penetrans experiment, root weight was significantly higher in the control and S. alba (0.5%) treatments and lowest for the 2.0% application rate of B .juncea and S. alba in Test 1 (Figure 13B). Aerial weight was lowest for the 2.0% application rate of B. juncea and S. alba and highest for the 0.5% rate of B. juncea and S. alba as well as the control.
Seed meal and nematode community composition experiment
Total nematode abundance before treatment was 1,254 (+ 143.7) nematodes/250 g soil for Soil A and 315 (+ 51.6) nematodes/ 250 g soil for Soil B (Table 13). Total P.penetrans populations was significantly higher in Soil A (277.2 per 250 g soil) than Soil B (32.2 per 250 g soil). There were no community composition differences between soils pre-treatment. Total nematode numbers were reduced in Soil A between pretreatment and one week, but values were similar for Soil B.
One week after application, both meals significantly reduced total nematode abundance and P.penetrans populations compared to their respective controls (Table 13). Brassica juncea seed meal application resulted in the greatest reduction in total nematode abundance, although this was not significant in either soil in comparison to S. alba. The community composition of Soil B was not significantly affected at one week, but the CI and proportion of bacterial feeders and fungal feeders was reduced in the seed meal amended soils.
Five weeks after application, total nematode abundance had increased in all treatments compared to non-amended soils. For both soils, P.penetrans populations were not significantly different between amended and non-amended soils, although levels were lower in B.juncea amended soils. At five weeks, the application of seed meals significantly increased the enrichment index (EI) for Soil B. However, for both soils, B. juncea applications significantly reduced the structure index (SI), while S. alba had a significantly higher SI, similar to the control, in comparison to B.junceae treatment. The proportion of omnivores and predators as well as plant feeders was significantly reduced in the B.junceae treatments for both soils at five weeks. However, B.junceae has the highest (P>0.05) proportion of bacterial feeders in Soil B.
Plant back test
There was no interaction between experiments; therefore data was combined for analysis (Table 14). Brassica juncea (0.5% v/v) was not phytotoxic to raspberry at any time period. At one week, phytotoxicity was detected in the S. alba (1.0% v/v) application (> 40% plant damage). However by four and six weeks no phytotoxicity was detected on raspberry plants as a result of S. alba seed meal. Across and within time there was no effect of seed meal on the germination of lettuce and radish seeds (Table 15). Neither of these germination assays detected the phytotoxicity to raspberry from S. alba seed meal that was observed at one week.
Quantitative real-time molecular assay
In the quantitative real-time PCR assay, the three treatments evaluated by this method (Telone C-35 with VIF, MIDAS 50:50, 250 HDPE, and MB:chloropicrin) had significantly lower DNA concentrations (ng/µl) than the non-fumigated VIF control (0.32 ng/µl) (Figure 10). There were no significant difference among the fumigant treatments, although the MB treatment had the lowest DNA concentration (0.00035 ng/µl) as compared to Telone C-35 with VIF (0.01 ng/µl) and MIDAS 50:50 250 HDPE (0.003 ng/µl). The standard curve for this assay was based on samples ranging from 1 ng to 100 femtogram. These results are similar to root rot and proportion of diseased root results from the greenhouse bioassay.
While the quantitative real-time assay was able to detect differences in DNA content across treatments, many of these differences were not significant. The only significant difference was between the control and all fumigant treatments. These results were similar to the bioassay results, but an increased level of specificity is still needed and in the absence of known thresholds for P.rubi, it is hard to maintain that enough control was reached with these chemical treatment options. Precision of inoculum evaluation and assay sensitivity require more investigation and a more specific primer/probe combination may be valuable for future research with artificial P. rubi inoculum and naturally occurring soil-borne inoculum. Also, it is hard to know from the molecular assay alone if the inoculum would be pathogenic on the raspberry roots, so it is not recommended as a replacement to the bioassay approach at this time. However, it can potentially lead to a better understanding of inoculum levels in soil and how they are affecting this perennial crop over time.
Educational & Outreach Activities
Gigot, J., Zasada, I. and Walters, T. Integration of brassciaceous seed meals into raspberry production systems. Soil Biology and Biochem. (draft); Gigot, J. WSU Dissertation, Chapter 3.
Walters, T., Gigot, J and Zasaba, I. 2011. Effective assessment and implementation of methyl bromide alternatives for raspberry nursery growers. HortScience (draft); Gigot, J. WSU Dissertation, Chapter 2.
Gigot, J. Walters, T., Zasada, I. 2011. Pre-plant management alternatives for raspberry growers. WSU Extension Fact Sheet (revisions)
Gigot, J., and Walters, T. and Particka, M. Pre-plant management alternatives for raspberry growers, 2008-2009. Plant Disease Management Reports: 4:SMF006.
Gigot, J and Walters, T. Development of a quantitative real-time PCR assay for assessment of Phytophthora rubi in soil. American Phytopathological Society/Canadian Phytopathological Society Annual Joint Mtg. June 20-23, 2010. Vancouver, BC.
Gigot, J, Zasada, I, Forge T and Walters, T. Occurrence of Phytophthora rubi and Pratylenchus penetrans in northwestern Washington red raspberry fields. Phytopathology. 99:S43. American
Phytopathological Society Annual Meeting. August 1-5, 2009. Portland, OR.
Gigot, J, Zasada, I, and Walters, T. Effectiveness of soil solarization on management of Phytophthora rubi and Pratylenchus penetrans in northwestern Washington. Soil Ecology Society/Society of Nematologists Annual Joint Mtg. July 12-15, 2010. Burlington, VT.
Is it easy to go organic? December 2010. Cultivating Regional Food Security Conference: UW/WSU Center for Urban Horticulture, UW.
Integration of biofumigants in root health management in raspberry. December 2010. Small FruitsWorkshop. Lynden, WA. Whatcom County Extension. Annual grower meeting with 100+ growers/ag professionals in attendance.
Dynamics of soil borne pathogens and alternatives to fumigation in raspberry. December 2009. Small Fruits Workshop. Lynden, WA. Whatcom County Extension. Annual grower meeting with 100+ growers/ag professionals in attendance.
Root Lesion nematode survey in raspberry. April 2009. Integrated Pest Management Scouting Tools For Blueberry and Raspberry. Session 1: Pre-Bloom. Whatcom County Extension. Boxx Berry Farm. Ferndale, WA.
Lead organizer, “Agriculture and Northwest Ecosystems: Graduate Student Symposium,”November 10, 2009. WSU-Mount Vernon NWREC. Presented solarization and seed meal work.
Through a combination of field and greenhouse experiments, it was demonstrated that brassicaceous seed meals have the potential to effectively reduce populations of P. rubi and P. penetrans, two important pathogens of raspberry. However, this pest management strategy will require a deeper understanding of the effects of B. juncea and S. alba seed meals on pathogen populations, raspberry health and soil nematode communities. Factors that will require consideration include seed meal rates, application timing, combination with other pest management practice and non-target effects.
Both seed meals were effective against both pathogens. For B. juncea, a rate of 0.5% was effective against both pathogens. However, for S. alba, a slightly higher rate of 1.0% was needed to reduce populations of both P. rubi and P. penetrans. The limitations of a greenhouse study are that there is no way to assess how populations of these pathogens will respond over time to organic matter additions and these results may be short lived or non-existent in actual soil ecosystems or with various native isolates of P.rubi. Artificial inoculum was used in both the field and greenhouse trials. However, it did seem that P. rubi propagules were either destroyed or germination and colonization suppressed by B. juncea, although no specific mechanism was explored.
Another study reported the inability of seed meals to affect Phytopthora cactorum, while the seed meal used (B. napus) did control other root pathogens such as Rhizoctonia spp, and Pythium spp. found in apple production soils in the same study (Mazzola and Brown, 2010). Also, B. napus was found to suppress pathogens regardless of glucosinolate content (Mazzola et al., 2001). Mazzola et al. (2007) proposed alternative suppressive mechanisms for seed meals in apple systems. Beyond the fungicidal impacts, they reported that seed meals enhanced native populations of Streptomyces spp. that may have had a biocontrol effect on target pathogens. Previous work found Streptomyces spp. can help hydrolyze cell walls and limit growth of P. rubi (Toussaint et al., 1997). Since it is known that seed meals can have both a biological as well as a chemical effect on pathogens, more research is needed on the mechanism involved in the control of P. rubi.
Brassicaceae crops and seed meal applications have both been found to control other plant-parasitic nematodes, including Heterodera schachtii, Tylenchulus semipenetrans and Meloidogyne incognita (; Lazzeri et al., 2009; Zasada, et al., 2009; Zasada et al., 2003; Lazzeri et al., 1993). In our studies, P. penetrans was controlled by both species (B. juncea and S. alba) of seed meal tested. It was found that the lowest rate of B. juncea effectively controlled P. penetrans, whereas 1.0% or more of S. alba was needed for control of this nematode. This is in accordance with previous work by Zasada et al. (2009) who determined lower application rates of B. juncea and higher (> 1.0%) application rates of S. alba were needed to suppress P. penetrans.
The amount of glucosinolate released from the seed meals will be dependent on particle size, as well as moisture and temperature (Mora and Borek, 2010), and these factors need to be taken into account when determining applications for both pathogens, as well as depth of effectiveness. Particle size was an important consideration and a necessary particle size of < 20 mm was identified for control of M. incognita and P. penetrans with S. alba seed meal (Zasada et al., 2009). In apple replant research, both fine (< 1.0 mm) and course (2.0 to 4.0 mm) ground B. juncea seed meal controlled P. penetrans (Mazzola and Zhao, 2010).
Solarization has been examined in previous work by Pinkerton et al. (2005 and 2009) and Gigot et al. (2010) in the Pacific Northwest as an alternative soil-borne pathogen and weed control option for raspberry. In this study, effectiveness of the seed meals with and without solarization was found only at 15 cm depth, and this will limit the long-term pathogen control needed for a perennial crops developing a root systems capable of growing below this depth. Temperatures in the 2009 summer season were variable, but target heat unit hours > 29o C were achieved at both 15 and 30 cm. Despite the success of the greenhouse rate trials, work is still needed towards the application of brassica seed meals in field soil systems. The ability of solarization to enhance the effectiveness of brassicaceous seed meals was not conclusively demonstrated. In the 2009-2010 field trial, solarization enhanced the P. rubi suppressive effects of B. juncea and S. alba seed meals compared to either seed meal alone. However, in the 2008-2009 field trial where S. alba was combined with solarization, there was no effect of this treatment.
The combination of solarization, or potentially tarping, with these amendments is promising for the raspberry industry. However, both incorporation depth as well as solarization depth could be a limitation of these approached. In the case of nematodes, vertical migration or distribution (Pudisaini et al., 2006; Forge et al., 1998) may allow these pests to avoid these management options, making their success void further along in the growing season. A better understanding of how heat units as well as organic matter incorporation affect the soil profile to relevant roots depths, depending on host crop, will be important.
In general, nematode populations in the field trial were low so it is hard to assess the effectiveness of the meals on P. penetrans at this location. However, P. penetrans population densities did return to similar levels measured at the spring sampling date, which may indicate that the meals will not offer nematode control over time and be suitable for planting requirements in the spring. While temperatures in this region are more variable and less consistently as high as other regions where solarization has been used effectively (Stapleton, 2000; Katan, 1981), it may be a viable control option used in conjunction with seed meal applications depending on the season.
Following, additional amendment types used with solarization or brassica seed meals may be an interesting area of exploration in the future for raspberry systems. In a controlled laboratory study, herb plant matter (thyme (Thymus vulgaris) and wild rocket (Sisymbrium officinale)) added to solarization helped to control Fusarium oxysporum f. sp. radices-lycopersici and improve soil quality factors, like microbial biomass (ref). Manure amendment plus solarization helped to control M. incognita and also improved soil enzyme production, which can be an indirect measure of soil quality (Ros et al., 2008). The addition of a biocontrol agent (Trichoderma spp.) with solarization was effective at controlling P. cactorum on strawberry (Porras et al., 2007).
The seed meals, in general, also affected general soil properties. All seed meals increased available nitrogen and S. alba increased sulfur levels. The extra addition of organic N to the soil may be another benefit of the application of brassicaceous seed meals (Gruver et al., 2007). Brassica juncea and S. alba increased microbial biomass in carrot systems as well as soil N mineralization at four and eight days after application (Synder et al., 2009). Gelsomino et al. (2006) found that solarization helped to promote mineralization of soil organic matter into N and P forms. Fertility enhancement, which is another aspect or, potential benefit, of biological inputs should be integrated into further disease focused research in raspberry systems. Consequently, the impact of nitrogenous compounds on nematode survival is another aspect of brassicaceous seed meals that requires further attention (Zasada et al., 2009; Cohen et al., 2005).
In the nematode community study, both seed meals reduced total nematode numbers and P.penetrans populations at one week, but levels were not significantly different than the control treatments at five weeks. However, community composition was different between the two soils and between seed meal application at five weeks, indicating a longer-term effect of this type of organic amendment on non-target organisms. The seed meals used in this study had difference effects on soil community composition. Gruver et al., (2010) determined that cover crop tissue quality and quantity will largely affect nematode community response after incorporation.
At five weeks, the proportion of bacterial feeders was significantly higher in Soil B for the B.juncea amended soils as compared to S.alba. Bulluck et al. (2002) found that populations of bacterial-feeding nematodes mainly in the Rhabditidae and Cephalobidae and fungal-feeding nematodes were greater after planting in soils amended with swine manure, composted cotton-gin trash or rye-vetch, than in soils amended with synthetic fertilizer. Bierdman et al. (2008) found that surface application of untreated urban wood waste significantly increased bacterivorous, plant parasitic, omnivorous and predator nematode densities. The application of seed meals increased the EI for both meals in Soil B after five weeks. However, for both soils at five weeks, B. juncea reduced the structure index (SI), while S. alba had a higher SI, similar to the control, in comparison.
The two soils used in this study had different responses to seed meal treatments. In transfer from the field to the buckets used in the microplot, total nematode numbers were reduced in Soil A and remained similar in Soil B, although this was not analyzed statistically. In a transition study genus composition was greatly associated with the level of disturbance connected to management style (conventional to organic and low-input) and nematode populations stabilized and were more diverse in the less intensive systems (Berkelsmans et al., 2003). At 1 week the CI and proportion of bacterial and fungal feeders was altered in Soil A but not Soil B. At five weeks, the SI, EI and proportion of bacterial feeds, omnivores/predators and plant feeders was affected in Soil B but only SI, omnivores/predators and plant feeders was affected in Soil A.
This is a narrow window to view application rates, but seed meal applications seemed to have some initial beneficial effects on reducing plant-parasitic nematodes and enhancing beneficial nutrient cycling N releasing nematodes. However, soil type should be taken into account when using seems meals. As shown here, indigenous nematode populations will respond differently to seed meals, thus accentuating the varying effect of seed meal type on non-target organisms.
There was no phytotoxicity effect to raspberry at any date for B. juncea, however, at one week S. alba seed meal was highly phytotoxic to raspberry. However, this phytotoxic effect was not observable four or six weeks after S. alba seed meal application. Sinapis alba has been used as an herbicide, and it isnot surprising that some phytoxicity was seen in this experiment (reference). Growers interested in using seed meal applications will need to determine planting time following meal application. Vegetable seed germination assays are a common way to screen soils for planting suitability following fumigation (Walters, personal communication). In this study, this technique was not effective in assessing seed meal applications for phytotoxicity. Further work is needed in this area to help growers optimize planting time post-seed meal application and avoid plant injury. Brassicaceous seed meals may be a useful tool for raspberry growers to manage soil-borne pathogens in the future. However, these byproducts cannot currently be used alone as a management tool. Seed meal species, particle size (Mazzola and Brown, 2010; Zasada et al., 2009) and application rates will all influence efficacy. The use of seed meals in concert with other biologically-related management strategies, such as solarization, as well as selection of resistant germplasm, may also help to improve consistency and reliability of this material in the field. More work is needed examining the effect of these types of management combinations on root health in raspberry as well as the related fertility impacts of these materials on soil biological activity and root growth and development.
The real-time assay will be useful for this endeavor because it could be a more rapid, sensitive and quantitative measure of P. rubi in soils than the traditional bioassay. While the protocol has been established, a more refined primer probe combination is needed. This is being developed in partnership with Frank Martin and Nik Grunwald (USDA) and wil be used in future fumigant alternative work using seed meals and cover crops in raspberry systens. While the real-time assay may help to make better assessments of the pathogen in soil, it will still be used in conjunction with the traditional bioassay in order to incorporate a measure of overall plant response.
Growers in both Skagit and Whatcom county are interested in both solarization and the use of seed meals in rows and for alley management as well. Several farms are participating in on-farm research trials addressing these technologies with the Small Fruit Horticulture program at WSU-Mount Vernon. Until brassicaceous seed meals become a readily available product, their full adoption will be delayed. This is reliant on the biofuels industry and future development in this state. Solarization with other types of organic materials will be investigated in the future.
Areas needing additional study
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