Sustainable root rot and soil management in raspberry

Brassicaceous seed meal field trials

Inoculum preparation

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,
2) solarization,
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,
2) solarization,
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).

Inoculum evaluation

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).

Nematode samples

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 Analysis

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.