International Journal of Fruit Science, 13:357–372, 2013Copyright © Taylor & Francis Group, LLCISSN: 1553-8362 print/1553-8621 onlineDOI: 10.1080/15538362.2013.748373

Impact and Occurrence of Phytophthora rubiand Pratylenchus penetrans in Commercial

Red Raspberry (Rubus ideaus) Fieldsin Northwestern Washington

JESSICA GIGOT1, THOMAS W. WALTERS2, and INGA A. ZASADA3

1Science Department, Northwest Indian College, LaConner, Washington, USA2Washington State University-Mount Vernon NWREC, Mount Vernon, Washington, USA

3USDA, Agricultural Research Service, Horticultural Crops Research Laboratory,Corvallis, Oregon, USA

Red raspberry (Rubus idaeus) production is a vital componentof northwestern Washington’s agriculture. The main objectives ofthis study were to document the occurrence of soilborne pathogensPhytophthora rubi and Pratylenchus penetrans in early stage pro-duction fields, relate this information to soil properties, and betterunderstand the individual and combined effect of P. rubi andP. penetrans on raspberry root health. P. rubi was found at eachfield and P. penetrans population densities were variable (0 to∼8000 nematodes/g dry root) across locations. In controlled green-house studies, P. rubi was very pathogenic to red raspberry ‘Meeker’at densities >10 oospore/gram soil and there was no interactionbetween P. rubi and P. penetrans. P. rubi is endemic to raspberryproduction in this region, and is an aggressive pathogen on rasp-berry. However, the chronic damage to roots caused by P. penetransshould not be ignored.

KEYWORDS Phytophthora, raspberry, nematode, root, Rubus

INTRODUCTION

The Pacific Northwest region of the United States comprises 92% of theprocessed red raspberry (Rubus idaeus) acreage nationwide (USDA, 2009).

Address correspondence to Jessica Gigot, Northwest Indian College, SwinomishCampus, Science Department, 17113 Tallawhalt Lane, Box C-11, LaConner, WA 98257, USA.E-mail: jgigot@nwic.edu

357

358 J. Gigot et al.

In northwestern Washington, the raspberry industry generates $59 millionin revenue annually and encompasses 3926 hectares of production (USDA,2009). Over the past few decades the productive lifetime of plantings inthis region has decreased from >10 years to ∼5 years due to apparent roothealth decline. As a consequence, commercial raspberry growers have madeunderstanding the ecology of soilborne pathogens and their effect on planthealth and crop yield a top research priority (www.nwsmallfruit.org).

In red raspberry, root damage caused by Phytophthora rubi Wilcoxand Duncan and Pratylenchus penetrans (Cobb) Filipjev and SchurmansStekhoven have been implicated with this decline (McElroy, 1992; Wilcoxet al., 1993). Phytophthora rubi is a homothallic oomycete that is consid-ered to be most active from November to March (Erwin and Ribeiro, 1996).In cool and saturated conditions, this pathogen causes Phytophthora root rotin most varieties of red raspberry. Root rot symptoms include a blackeningdiscoloration of the roots and crowns and a wilting of leaves and canes inlate summer (Erwin and Ribiero, 1996). Although raspberry germplasm withresistance to Phytophthora root rot is available, fruit from these plants arenot considered commercially suitable because they are not of individuallyquick frozen (IQF) and other industry processing standards (Bristow et al.,1988; Pattison et al., 2004).

Pratylenchus penetrans, the root lesion nematode, is a migratoryendoparasite with over 400 host plant species, including commercial crops,cover crops, and weeds (Davis and MacGuidwin, 2005). Pratylenchus pene-trans can complete several generations in a single growing season dependingupon soil temperature. All stages of the nematode move between soil androots and feed on and migrate in root cortical cells. Damage caused byP. penetrans generally includes a reduction in fine root abundance andthe wounding of root tissue, which appear as necrotic lesions on the roots(McElroy, 1992). Raspberry germplasm resistant to P. penetrans is yet to befound and newly developed cultivars would be subject to the same con-straints over IQF standards and yield parameters that exist for those resistantto Phytophthora root rot (Vrain and Daubeny, 1986).

While soil fumigation reduces soilborne pathogen and plant-parasiticnematode populations initially, replanted acreage in western Washington isconsistently less productive than ground that does not have a history ofraspberry production (R. Honcoop, personal communication). Many growersattest that the effect is most noticeable on marginal soil with poor drainage.While there has not been a typical replant disease characterized on raspberry,current research in British Columbia (S. Sabaratnam, personal communica-tion) and Michigan (A.M. Schilder, personal communication) suggests thatseveral soilborne pathogens may be involved. This research focused onP. rubi and P. penetrans, both well-known pathogens in the region.

Vrain and Pepin (1989) found that over time the presence of bothP. rubi and P. penetrans caused severe stunting of canes, poor emergence

Impact and Occurrence of Red Raspberry Pathogens 359

of primocanes, and death of floricanes and primocanes. However, furtherinformation on the combined effect of P. rubi and P. penetrans on rasp-berry establishment and productivity is needed in order to develop effectivemanagement strategies. In other cropping systems, the presence of both anematode and fungal pathogen has been found to intensify disease severitycompared to when either is present alone. Examples include sudden deathsyndrome on soybean (Glycine max) where Heterodera glycines increasesthe severity of root rot caused by Phytophthora sojae (Xing and Wesphal,2006).

The main objective of this study was to perform a survey in order todocument the occurrence of P. rubi and P. penetrans in early production agefields (3–5 years) in northwestern Washington raspberry growing regions andto relate this information to soil properties. In addition, controlled experi-ments were conducted to better understand the interaction of P. rubi and P.penetrans and their effect on root growth and development. Understandingsoil properties or management conditions that support or promote either orboth of these organisms will help to address the long-term chronic problemof root health that is endemic to this production system.

MATERIALS AND METHODS

Commercial Red Raspberry Field Survey

Ten raspberry fields, representative of production fields in Skagit (5 fields)and Whatcom (5 fields) counties in northwestern Washington, were sam-pled. These fields had histories of root rot or were experiencing eitherabove-ground root rot or replant disease symptoms and were identified withguidance from the Skagit County Extension and Whatcom County RaspberryIPM programs. Field history, variety, age of planting, and soil type wererecorded. Root and soil samples were collected from 10 random sites (4.6 mlong × 0.9 m wide) within each field that had varying above-ground symp-toms of root rot (yellowing leaves, poor growth, and stand establishment).At each site, 10 soil cores (15 cm deep × 5 cm diameter) were collected inthe root zone of established plants in October 2008. The soil cores from a sitewere combined and then the soil was passed through a 4-mm-diameter Sieve,and root fragments retained on the sieve were collected and reserved for iso-lation of Phytophthora spp. and extraction of P. penetrans (see below). Thesieved soil samples were subdivided for subsequent nematode extraction andsoil chemical and physical analyses. Pratylenchus penetrans were extractedfrom soil by placing 50 g of soil on a Baermann funnel for 5 days (Ingham,1994). Extracted nematodes were collected and the number of nematodeswas determined using a dissecting microscope at 40× magnification; soilnematode populations are expressed as P. penetrans/100 g soil. Soil chemicaland physical analyses were conducted by A&L Laboratories (Portland, OR)

360 J. Gigot et al.

and included % organic matter (OM), nitrate-N (NO3−), sulfur, potassium,

phosphorus, calcium, cation exchange capacity (CEC), pH, and soil texture(% sand, silt, and clay).

Root sections (1 cm long) that were symptomatic (discolored with obvi-ous lesions) were surface-sterilized (Sabaratnum, personal communication)for 3 min with 0.02% Tween 20, rinsed with sterile distilled water (repeatedthree times), and cultured on PARP medium to recover Phytophthora spp.(Duncan and Kennedy, 1989). A 0.3 g subsample of root tissue from eachsite/field location was evaluated for Phytophthora spp. by enzyme-linkedimmunosorbent assay (ELISA) using the Phytophthora Complete Kit (AgDia,Elkhart, IN). Polymerase chain reaction (PCR) testing was also performedbased on a protocol by Bonants et al. (1997) through the Whatcom CountyPhytophthora spp. survey lab (WSU Puyallup Research and Extension Center,Puyallup, WA), and was used to detect the presence of P. rubi in rootsamples (3 sites per field). The remaining roots were washed free of soil,and P. penetrans was extracted by intermittent mist for 1 week (Ingham,1994). Roots from which nematodes were extracted were dried at 72◦C for24 h and then dry weights determined. Extracted nematodes were collected,population densities were determined using a dissecting microscope at 40×magnification, and the density expressed as number of P. penetrans/g dryroot.

Infested Field Soil Bioassay

In order to assess disease potential of field soil with varying frequency ofPhytophthora spp. inoculums and P. penetrans population densities, bioas-says using tissue culture ‘Meeker’ red raspberry plants were performed. Sixfields (1, 2, 3, 6, 7, and 8) from the fall 2008 survey were revisited in spring2009 and 2010 (Table 1). In 2010, field 8 was not included (grower hadremoved raspberry plants) in the bioassay. Soil and roots were collected witha shovel from one site per field (4.6 m long × 0.9 m wide), placed in four19-liter buckets, passed through a 2-mm-diameter sieve, and root fragmentsretained on the sieve were collected. Prior to bioassay set-up, root and soilsamples were analyzed for P. penetrans in both years, as described above.Randomly selected root samples were also tested to confirm the presenceof P. rubi using conventional PCR at WSU-Puyallup, as described above, in2009 only. The same soil chemical and physical analyses as described above,except textural analysis, were performed by A&L Laboratories during bothyears on collected soil.

Aliquots of sieved soil were loaded into 15-cm-wide Deepots (D40H;Stuewe and Sons Inc., Tangent, OR) and one tissue cultured red raspberry‘Meeker’ plant (Sakuma Bros Farm, Burlington, WA) was planted into the soil.Tissue culture plants were 6 months and 3 months old prior to transplanting

TAB

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361

362 J. Gigot et al.

in 2009 and 2010, respectively. Plants were grown in a greenhouse main-tained at 16◦C with a 12 h day/night light cycle. Plants were watered regularlyusing a combination of below (immersion of Deepots in 473 ml (12-cm-high)cups for 6 to 8 h) and overhead watering to maintain field capacity and fer-tigated with 20-20-20 NPK (Plant Marvel Laboratories, Chicago Heights, IL)as needed. Each trial was arranged in a randomized, complete block designwith four replications of four plants per field soil.

Plants were harvested approximately 8 weeks after planting. The aerialportions of the plants was removed and dried at 72◦C for 24 h and dryweights were then determined. Adhering soil was gently removed from rootsthat were then washed with tap water to remove excess soil and roots wereset in trays by replication and survey field. Root rot was evaluated using astandardized, 0 to 9 (0 = healthy, 9 = dead), continuous, visual rating scale(Walters and Pinkerton, 2008). A subsample of symptomatic root material wascollected from one plant per survey location and viewed under the micro-scope for presence of Phytophthora spp. oospores by cutting roots with asterilized scalpel and observing the roots with a squash mount technique.Surface sterilized (see above) root fragments were plated onto selectivemedia (PARP) for verification of Phytophthora spp. Pratylenchus penetranswas extracted and quantified from a handful of the root as described above.The remaining roots were dried at 72◦C for 24 h and dry weights thendetermined.

CO-INOCULATION OF RASPBERRY WITH P. RUBI AND P. PENETRANS

P. rubi (strain ATCC 16184) inoculum was produced using a method adaptedfrom Dissanayake et al. (1997). P. rubi was grown on a mixture of vermi-culite, V-8 broth, and oats in a 900-ml glass jar. The mixture was autoclavedtwice over a 24-h period and then inoculated with five 5-mm plugs from themargin of a 3-week-old P. rubi culture. After 4 weeks, jars were inspectedfor contamination after which the inoculum mixture was removed fromthe jar and dried in a fume hood for 1 week. The inoculum mixture wasthen ground into a powder using a grain grinder (Kitchen Aid, St. Joseph,MI). Oospore density of the inoculum was evaluated by taking 6 g ofinoculum and homogenizing the material in 60 ml of water in a Janke &Kunkel Labortechnik Ultra-turrax T25 (IKA, Wilmington, NC). Four 10-µlaliquots of this mixture were distributed onto glass slides and the total num-ber of oospores was determined. The inoculum contained approximately17,000 oopspores/ml. Pratylenchus penetrans, originally collected from araspberry field in Lynden, WA and maintained on peppermint (Menthapiperita), was used in this experiment. Nematodes were extracted fromroots by intermittent mist and suspensions of nematodes were adjusted toapproximately 100 P. penetrans/ml.

Impact and Occurrence of Red Raspberry Pathogens 363

Prior to planting, field soil (Skagit silt loam) was collected at WSU-Mount Vernon, passed through a 2-mm-diameter sieve, and autoclavedtwice (121◦C, 15 min). Soil was then mixed with fine vermiculite (Steubers,Snohomish, WA) at a 1:2 ratio. P. rubi inoculum was added to thesoil:vermiculite mixture to attain oospore densities of 0, 10, 100, and1,000 oospores/g soil. Raspberry ‘Meeker’ tissue culture plants were plantedinto the P. rubi infested mixture (400 ml P. rubi inoculum/150 g soil mix-ture per container) in 15-cm-diameter Deepots. Phytophthora rubi infestedsoil was either inoculated or not inoculated with P. penetrans at all oosporedensities. To inoculate with P. penetrans, small holes (2.5 cm deep) werecreated on two sides of the tissue culture plant and the nematode solution(1.5 ml) was pipetted into each hole to obtain 1 P. penetrans/g soil andthen covered. This experiment was a 4 × 2 factorial design (4 oospore levels× 2 nematode densities) and was arranged as a randomized block designwith each treatment combination replicated six times; the experiment wasconducted twice.

The plants were watered and fertigated as needed similar to above.In addition, Deepots were flooded to encourage P. rubi infection by placingDeepots in 946 ml (17 cm high) cups for 48 h every 2 weeks (Walters andPinkerton, 2008). Greenhouse conditions were set at 16◦C, 12-h day/nightlight cycle. At experiment termination (∼6 weeks), root rot ratings, P. pen-etrans population densities in roots, and aerial and root biomass wereevaluated using methods described above.

Data Analysis

Pearson correlation coefficients were calculated to examine the relation-ship between fall soil properties (n = 100) and number of P. penetrans/groot (PROC CORR) and frequency of Phytophthora spp. detection fromELISA (KENDAL) in the field survey. All parameters in the infested field soilbioassay and co-inoculation experiment were analyzed using a general linearmodel (PROC GLM; SAS Institute, Cary, NC). Trials from the co-inoculationexperiment were analyzed separately because there was a significant interac-tion between trials (p < 0.001). Means were separated by Fisher’s protectedleast significant difference test (p < 0.05).

RESULTS

Commercial Red Raspberry Field Survey

In the fall of 2008, P. rubi and Phytophthora spp. were detected in roots at alllocations using PCR- and ELISA-based methods, respectively (Table 1). Thefrequency of P. rubi detection by PCR in each location ranged from 33% to

364 J. Gigot et al.

100% while the frequency of Phytophthora spp. detection by ELISA rangedfrom 30% to 100%. P. penetrans was recovered from soil and root samples atall ten survey locations (Table 1). Average P. penetrans population densitiesvaried greatly within and between survey locations from 0 to approximately8,000 nematodes/g root tissue (Table 1). Average soil population densitiesof P. penetrans were much lower than densities detected in correspondingroots and ranged from 0 to 242 P. penetrans/100 g soil.

Dominant soil types for each location are listed in Table 2. In fall 2008,NO3

− levels in surveyed fields were variable, but were very high, >80 PPM,in locations 1, 6, and 7 (data not shown). Across locations, OM ranged from3.3% to 8.7% and pH values ranged from 4.2 to 6.8. Percentage organic matterwas positively, but only moderately correlated (r = 0.34; p < 0.01) with P.penetrans population densities in roots while percentage clay was negatively,but only moderately correlated (r = −0.33; p < 0.01) with P. penetranspopulation densities in roots. The frequency of Phytophthora spp. identifiedin grower fields as determined by ELISA was negatively, but only moderatelycorrelated (r = −0.25; p < 0.01) with the textural analysis for percentagesilt. No other soil properties showed correlations with Phytophthora spp.frequency or P. penentrans population densities in roots.

Infested Field Soil Bioassay

In spring 2009 and 2010, P. rubi was detected by PCR at all six surveylocations sampled. P. penetrans population densities in soil were lower inspring 2009 and 2010 (data not shown) compared to samples collected inthe fall of 2008 (Table 1). In 2009, P. penetrans were not detected in fields1, 3, or 6 and there were only 2 P. penetrans/100 g soil detected at field8. Similar to 2008, fields 2 and 7 had the highest P. penetrans populationdensities with 88 and 40 P. penetrans/100 g soil, respectively. In 2010, there

TABLE 2 Predominant Soil Types within Each Survey Field in Skagit and Whatcom Counties,Washingtonz

Survey field Predominant soil type

1 Sumas silt loam, Field silt loam, Mt. Vernon very fine sandy loam2 Mt. Vernon very fine sandy loam, Pilchuck variant fine sandy loam3 Briscot fine sandy loam, Skagit silt loam4 Field silt loam, Skagit Silt loam5 Mt. Vernon very fine sandy loam, Skagit silt loam6 Hale silt loam, Laxton silt loam7 Kickerville silt loam8 Hale silt loam, Edmonds-Woodlyn loam, Laxton silt loam9 Mount Vernon fine sandy loam, Oridia silt loam

10 Laxton loam, Everett very gravelly sandy loam

zSource: NRCS Web Soil Survey (websoilsurvey.nrcs.usda.gov/).

Impact and Occurrence of Red Raspberry Pathogens 365

FIGURE 1 Root rot rating (0–9 scale; 0 = healthy, 9 = dead) of raspberry (Rubus ideaus)‘Meeker’ plants assayed field soil containing Phytophthora spp. and Pratylenchus spp. col-lected from northwestern Washington raspberry survey sites during 2009 and 2010. Columnswith the same letter are not significantly (P < 0.05) different according to Fisher’s protectedleast significant difference. N = 24.

were still no nematodes detected in soil samples from fields 1, 3, or 6, while198 and 226 P. penetrans/100 g soil were found in soils from survey locations2 and 7, respectively.

In the 2009 bioassay, raspberry plants planted into soil from field 6 hadthe lowest root rot ratings, and these ratings were lower than those observedon plants from fields 2, 4, and 5 (Fig. 1). Root rot ratings of plants planted intosoil from the other survey locations were similar. In contrast to 2009 bioassayresults, raspberry plants planted in soil from location 5 had significantlygreater root rot ratings compared to plants planted into soils from other sur-vey fields. Fields 2 and 4 had significantly higher root rot than fields 3 and6. Oospores of P. rubi in bioassay plant roots were observed in both years(data not shown), although no clear pattern of colonization across fields orreplications was discernable. P. penetrans population densities were signifi-cantly higher in plant roots grown in soil from survey fields 2 and 5 duringboth years (Fig. 2). In 2010, P. penetrans population densities in bioassayplant roots were higher than in 2009 (Fig. 2). In 2009, aerial biomass of redraspberry grown in soil from field 4 was lower than the other survey fieldsexcept location 5 (Fig. 3). There was no significant difference in root biomassacross survey fields in 2009. In 2010, plants grown in soil from field 5 hadthe lowest aerial and root biomass among the survey locations.

CO-INOCULATION OF RASPBERRY WITH P. RUBI AND P. PENETRANS

As stated above, results from trials were different (p < 0.01), therefore datawere analyzed separately; however, similar trends were observed in both

366 J. Gigot et al.

FIGURE 2 Number of Pratylenchus penetrans/g recovered from raspberry (Rubus ideaus)‘Meeker’ assayed with field soil containing Phytophthora spp. and Pratylenchus spp. collectedfrom northwestern Washington raspberry survey sites during 2009 and 2010. Columns withthe same letter are not significantly (P < 0.05) different according to Fisher’s protected leastsignificant difference. N = 24.

FIGURE 3 Aerial biomass (A) and root biomass (B) of raspberry (Rubus ideaus) ‘Meeker’assayed with field soil containing Phytophthora spp. and Pratylenchus spp. collected fromnorthwestern Washington raspberry survey sites during 2009 and 2010. Columns with thesame letter are not significantly (P < 0.05) different according to Fisher’s protected leastsignificant difference. N = 24.

trials. There was no interaction between P. rubi oospore density and thepresence or absence of P. penetrans in both trials (p = 0.40 and 0.85); there-fore, P. rubi data across nematode treatments were combined for analysis.In both trials, the level of root rot was significantly affected by oosporedensity, with increasing root rot with increasing levels of oospore inoculum(Fig. 4). The same trend was observed for proportion diseased root (data notshown), which ranged from 5.5 to 6.1 at 1,000 oospores/g soil and <0.8 at0 oospores/g soil across trials. P. penetrans was recovered from inoculated

Impact and Occurrence of Red Raspberry Pathogens 367

FIGURE 4 Root rot rating (0–9 scale; 0 = healthy, 9 = dead) of raspberry (Rubusideaus) ‘Meeker’ inoculated with different densities of Phytophthora rubi (0, 10, 100, and1,000 oospores/g soil) and Pratylenchus penetrans (0 or 200 nematodes/g soil). There wasno interaction between P. rubi and P. penetrans so data were combined. Columns identi-fied by the same letter are not significantly (P < 0.05) different according to least significantdifference. NSD = No significant difference.

plants in trial 1 (181.6 + 16.1 nematodes/g dry root) and trial 2 (136 +14.7 nematodes/g dry root). On average, the highest densities of P. pene-trans in root tissues (223.24 + 16.2 P. penetrans/g dry root) were found inplants that had been inoculated with 1,000 P. rubi oospores/g soil, but thisdifference was not significant. Root biomass was significantly lower for plantsinoculated with 1,000 P. rubi oospores/g soil than for plants inoculated withlower densities of P. rubi in both trials (Table 3). There was no impact ofP. rubi oospore density on aerial biomass in either trial. P. penetrans at aninoculation density of 1 nematode/g soil did not affect root or aerial biomassin either trial (data not shown).

TABLE 3 Aerial and Root Biomass (g) of Red Raspberry (Rubus idaeus) ‘Meeker’ Co-inoculated with Phytophthora rubi and Pratylenchus penetransz

Trial 1 Trial 2

P. rubi oospores/g soil Root Aerial Root Aerial

0 1.1ay 1.0 1.0a 0.910 1.0a 1.0 1.0a 0.8100 0.9a 0.9 0.9ab 0.61,000 0.6b 0.7 0.7b 0.5

zThere was no significant difference between P. penetrans inoculated and non-inoculated plants(p = 0.40 and 0.85 in trials 1 and 2, respectively); therefore, data was combined for analysis (n = 48).yMeans followed by the same letter within a column are not significantly different as determined byFisher’s protected least significant difference (p < 0.05).

368 J. Gigot et al.

DISCUSSION

Phytophthora spp. and P. penetrans are prominent soilborne pathogens ofred raspberry in northwestern Washington, posing a threat to the long-termviability of this industry. Our survey is the first to demonstrate the widespreadoccurrence, frequency, and population densities of these pathogens in redraspberry. In our production field survey, P. rubi, Phytophthora spp., andP. penetrans were all commonly encountered. While we did not quantify P.rubi propagules in soil, PCR-based tests showed that P. rubi was detectablein >50% of samples/site from 8 out of 10 fields. All surveyed fields wereELISA-positive for Phytophthora spp., while the frequency of detection withina field was >50% in 7 out of 10 fields. Other species of Phytophthoramay also be affecting root rot and root health in raspberry (Wilcox et al.,1993). The identification and impact of other Phytophthora spp. on raspberryproductivity are unknown.

For most Phytophthora diseases, propagule recovery and quantificationis problematic with dilution plating methods and oospore germination fromplant roots and in culture being unpredictable (Erwin and Ribeiro, 1996).It was reported that P .rubi was most active from November to March, whichmay also affect how easily P. rubi is detected during other times of the yearand how inoculum levels are assessed in soil. Examples of the difficultyin propagule recovery and quantification can be drawn from P. fragariae,which is closely related to P. rubi (formerly P. fragariae var. rubi; Man andWillem, 2007). In strawberry, the amount of P. fragariae sporulation variedby host genotype with susceptible plants supporting a faster rate and greateramount of sporulation by the pathogen compared to the traditionally resistantgermplasm (Milholland and Daykon, 1993). An inspection of strawberry rootsover 2 years found that oospores of P. fragariae formed in roots during themonths when temperatures were over 16◦C, with the maximum recoveryoccurring in March of both years (Forge et al., 1998). Similar to P. fragariae,the activity and ability to recover P. rubi may be related to sporulation levelsand germination rates of dormant spore structures. More work is needed onisolate recovery techniques and monitoring to determine the activity periodof P. rubi in northwestern Washington.

The majority of the fields sampled in fall 2008 had P. penetrans rootpopulation densities >500 nematodes/g root and population densities in soil<100 nematodes/g soil. McElroy (1992) suggested that P. penetrans popula-tion densities exceeding 1 nematode/cm3 soil affected stand establishmentand 2 to 8 nematodes/cm3 soil decreased plant growth in established plant-ings. We rarely detected P. penetrans soil population densities greater than1 nematode/g soil; therefore, according to McElroy (1992) treatment wouldnot be warranted in the majority of the survey locations. However, popu-lation densities of P. penetrans detected in root samples were higher, andin three locations exceeded 1,000 nematodes/g dry root. The relationship

Impact and Occurrence of Red Raspberry Pathogens 369

between P. penetrans population densities in roots on raspberry productiv-ity has not been investigated. This survey demonstrated that raspberries innorthwestern Washington are grown on a wide range of soil types. SkagitCounty soils generally were fine to sandy loams, while the predominantKickerville loam of Whatcom County is characterized by high organic mat-ter content and a low sand content. Phytophthora root rots have historicallybeen associated with high water content in soil (Duncan and Cowan, 1980;Duncan and Kennedy, 1989). Following this reasoning, it was assumed thatsoils in our survey with higher clay contents would be associated withhigher moisture levels, which could also exacerbate Phytophthora root rot.However, it was found that Phytophthora spp. frequency in raspberry soilswas negatively correlated with silt content and there was no correlation withany other soil property. Klironomos et al. (1999) documented spatial vari-ability in soil properties and suggested that in-field heterogeneity makesit difficult to detect relationships between soil properties and pathogens.While host genotype and relative resistance of roots to P. rubi can miti-gate pathogen sporulation duration and number of oospores formed in roots(Laun and Zinkernagel, 1997), increasing soil moisture content can encour-age persistence and infectivity of Phytophthora spp. over time (Duncanand Cowan, 1980). Interestingly, P. cryptogea zoospore movement in soilcolumns was measured to be 8–12 mm in a clay loam soil and 40 mm in acoarse soil mix (Duniway, 1992). It was concluded that pore space betweenprimary particles of finer textured soils are too small to permit significantzoospore movement. Other, still unknown, factors in the soil biosphere maybe affecting P. rubi persistence in northwestern Washington soils.

Pratylenchus penetrans population densities did increase with decreas-ing clay content in our survey. Population densities of this nematode alsoincreased as percentage organic matter increased. A strong relationshipbetween sand content and high nematode population densities has beenreported (Townshend, 1972), however, other researchers found varyingresults when investigating other soil properties on plant-parasitic nema-tode distributions, such as pH and organic matter content (Noe and Barker,1985; Wyse-Pester et al., 2002). Relationships between nematodes and soilproperties vary by species, and in a study of soil properties and nematodediversity, less than 30% of the variability could be explained by edaphic fac-tors (Robertson and Freckman, 1995). The addition of organic matter to thesoil will also influence nematode population densities, especially if a fieldis under management that employs cultural practices like cover croppingand manure additions that can cause temporal augmentation of differentnematode populations (Widmer et al., 2002).

Greenhouse experiments were conducted to evaluate the diseaseresponse of young raspberry plants to infested soil from grower’s fieldswhere mature raspberry plants where demonstrating yield decline and

370 J. Gigot et al.

above-ground plant symptoms (i.e., wilt, yellowing, poor growth and estab-lishment, etc.). Due to the constraint of working in production fields whereroot rot ratings are not possible without using destructive methods, field soilwas imported into a greenhouse setting. In these experiments, only fieldsoils for which P. rubi was detected with the PCR assay were used (Table 1).Pratylenchus spp. population densities varied between fields, with fields2 and 5 having the highest densities in both years. These fields also had highroot rot ratings in the bioassay. We observed minimal P. rubi colonizationof roots due to unsaturated conditions in the assay (i.e., no flooding as inco-inoculation trial). However, despite the potential inoculum in the soil andoospore formation in plant roots, the amount of root damage caused by P.penetrans alone was interesting and may be an indication of pre-plant issuesfor raspberry growers. In 2010, the degree of root rot was greater than in2009. In this year, tissue culture raspberry plants were 3 months youngerand this may have affected their susceptibility to soilborne pathogens.

A controlled greenhouse study was performed to further assess the effectand interaction of P. rubi and P. penetrans. In the co-inoculation study, whichincluded flooding to encourage P. rubi infection, P. rubi inoculation lev-els above 10 oospores/g soil caused substantial disease on raspberry roots,regardless of the presence of P. penetrans. These results are in accordancewith previous work by Vrain and Pepin (1989). Although P. penetrans didnot significantly affect the dry weight of raspberry roots, the amount of fineroots was observed to be less on plants that had been inoculated with P.penetrans. On the roots with the most root rot that had been inoculatedwith 1,000 oospores/g soil, high population densities of P. penetrans werestill detected. As an aggressive soilborne pathogen, at higher inoculum lev-els, P. rubi can destroy raspberry roots. While P. penetrans do not necessarilyexacerbate Phytophthora root rot symptoms, P. penetrans presence in all dis-eased roots is interesting and suggests that P. penetrans might contribute tolong-term root health decline especially at the lower P. rubi inoculum levels.

CONCLUSIONS

While P. rubi, Phytophthora spp., and P. penetrans are present in rasp-berry systems in western Washington, it seems that population densitiesof both pathogens vary and are affected differently by various confound-ing factors like management and soil type. In this study, the dynamicsof two main pathogens on raspberry in northwestern Washington havebeen explored. While it is obvious that P. rubi is common in raspberryproduction in this region, and is an aggressive pathogen on raspberry,the chronic damage to roots caused by P. penetrans should not beignored. Furthermore, investigation into additional indigenous soil pathogencommunities is warranted.

Impact and Occurrence of Red Raspberry Pathogens 371

ACKNOWLEDGMENTS

This research was partially funded by the WSU-BIOAg program. We wouldlike to thank Jerry Weiland for his critical review of this manuscript and MikePartika for his support in all greenhouse and field work.

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