Sustainable root rot and soil management in raspberry
The Pacific Northwest of the US encompasses 90% of processed raspberry acreage nationwide. The duration of harvestable plantings has declined from >10 to approximately 5 yrs. Root rot damage by Phytophthora rubi 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 soilborne 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.
1. Develop a real-time PCR assay and complementary 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)
Jessica Gigot traveled to Univ. of Kentucky and participated in the Real-Time Workshop for Plant Pathologists (January 2009). For the quantitative real-time PCR assay, primers were designed to target the ITS region and to provide adequate specificity for P. rubi and produce an appropriately sized amplicon for quantitative PCR (~100-300 bp). Primers for the SYBR green and Taq-man assay and the Taq-man probe were adapted from a previous publication (Table 1). All PCR reactions were 25 ul and all PCR protocols were developed using Quantifast protocols (Qiagen, Inc.; Valencia, CA). All DNA samples from culture, soil and roots were extracted with the Power Soil kit (MoBio Laboratories, Inc., Carlsbad, CA). The reactions were run on a Rotor-gene 2000 (Qiagen, Inc., Valencia, CA).
Both a Taq-man and SYBR green real-time PCR assay are functional and can be used to identify and quantify P. rubi. The amplicon produced using these primers was sequenced, is appropriate for our target organism and is consistent with published results on Genbank. A standard curve was created from our original isolate (ATCC 16184) using both the SYBR green assay (Graph 1) and the Taq-man assay (Graph 2). Both assays require optimization. However the correlation coefficients of the standard curves r-squared values are appropriate indicating that our assays can detect a range of P.rubi DNA levels. The efficiency of the Taq-man assay (.92) is better than that of the SYBR green assay (.79). Additional isolates of P. rubi have been obtained from the Grünwald lab (USDA-ARS Corvallis) and Weber lab (Cornell University) and will be tested with both real-time assays. Preliminary results show that the primers are compatible with multiple isolates of P. rubi (Graph 3). Additionally, more work will be done to verify the specificity of primers and probes against related oomycetes (Pythium spp, Phytopthora spp.) from using isolates obtained from the Inglis lab (Washington State University-NWREC) and Forge lab (Agri-Canda). Initial soil and root samples from infested field soils and raspberry roots have been tested with the real time assays and minimal inhibition has been detected which indicates that extraction methods are adequate for our samples.
To assess the effect of various densities of P. rubi on root rot, a greenhouse bioassay was developed. Tissue culture plants (cv. Meeker) were inoculated with oopsores (0, 10, 100 or 1000/g soil) and grown in containers for ~6 weeks. Following plants were analyzed for root rot disease (Graph 4). Disease was assessed using a rating scale (0=healthy to 9=diseased) and plants that had been inoculated with 1000 oospores/ g soil have significantly more disease than the control.
In order to relate DNA quantity in the standard curve to oospore density, DNA will be extracted from solutions with known densities of P. rubi oospores. Additionally, known densities of P.rubi will be added to various soil types. Following, DNA will be extracted and analyzed using both of the real-time assay protocols. Raspberry roots from infested fields that were confirmed with P.rubi using conventional PCR will now be examined using this quantitative molecular technique.
Field trials were established at WSU-Mount Vernon in July 08 and July 09 to investigate the effect of solarization plus organic amendment on P. rubi and P. penetrans. Plant pathogen control was assessed for all of the treatments (Table 2). 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. Soil solarization did not significantly affect the severity of disease caused by P. rubi but the plots with Inline alone, solarization + InLine, solarization + S. alba, and solarization + linseed meal had significantly greater disease ratings than the control plots. The InLine treatment significantly increased the P. rubi root rot rating (6.2) and the cfu/g of A. tumefaciens (4.3) compared to the control plots (4.9 and 2.2, respectively). Aerial biomass was lowest in the InLine treatment and there was no significant difference among treatments for root biomass. The combination of solarization + S. alba also significantly increased the cfu/g of A. tumefaciens (4.9) compared to the control plots (2.2). In 2008, accumulated hours above 29oC only reached 34 hrs at a soil depth of 15 cm in the solarization plots. The lack of heat accumulation most likely explains the failure of solarization treatments to control P. rubi or A. tumefaciens.
P. penetrans counts were generally low in this field and the observed reductions in nematode counts from Jul 08 to Jan and Apr 09 may have been a result of natural population fluxes. However, in Jan 09 the solarization plots had significantly greater nematode populations (24.4/50 g soil) compared to counts in all of the non-solarization plots (0 to 7.2/50 g soil) except those treated with solarization + linseed meal (14.6/50 g soil). By the final sampling date, there was no significant difference in P. penetrans counts among treatments although no nematodes of this species were detected in plots with either of the InLine treatments.
Weed biomass, number of weeds per plot and percentage of weed coverage were all significantly lower in the S.alba amended plots versus the control (Table 3). For soil properties (Table 4) sulfur was significantly higher in the solarization+S.alba treatment. Values for nitrate and pH ranged from 4- 186 ppm and 5.4-6.4, respectively.
Results from the 08-09 trial have been used to create a Plant Disease Management Report (submitted, Dec 7 09) and will be published in Hort Technology (in progress).
Another field trial examining solarization and brassicaceous seed meal has been established at WSU-NWREC and will be assessed in Spring 2010. Results from this trial will be incorporated into the final report. Additional greenhouse trials using field soil and the brassicaceous seed meal will be performed in Spring 2010 for further analysis of pathogen control.
Impacts and Contributions/Outcomes
As fumigants become less available, growers in this region will need to transition to more sustainable, alternative technologies that will require a new set of assessment tools. Implementation of EPA fumigation restrictions is underway and new regulations will make whole-field fumigation economically and socially prohibitive by 2011. 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 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.
On December 8th 2009, Jessica Gigot presented some of her first-year results to over 200 growers and professionals in the small fruit industry in western Washington at the Small Fruit Workshop in Lynden, WA. Her presentation was entitled, “Dynamics of soilborne pathogens and alternative to fumigation.” A condensed version of this talk was also given at the Agriculture and Northwest Ecosystems Conference at WSU-NWREC (Nov. 10, poster attached).
Washington State University-NWREC
16650 State Route 536
Mount Vernon, WA 98273
Office Phone: 3608486129
Washington State University-NWREC
16650 State Route 536
Mount Vernon, WA 98273
Office Phone: 3608486124