Host status and damage potential of Paratrichodorus renifer and Pratylenchus penetrans (Nematoda) to blueberry (Vaccinium spp.)

Abstract

Stubby root nematodes (Paratrichodorus species) and root lesion nematodes (Pratylenchus species) have been found in root zone soil of blueberry plants in most blueberry-growing regions of North America. Relatively little is known, however, of the reproductive potential and damage caused by these nematodes to blueberry. We performed controlled inoculation studies in a greenhouse in order to assess population growth and damage caused by Paratrichodorus renifer and Pratylenchus penetrans on a range of blueberry cultivars, including representatives of Vaccinium corymbosum (four cultivars), V. angustifolium (one cultivar) and V. ashei (one cultivar). All tested blueberry cultivars were considered good hosts to P. renifer (reproduction factor (RF) values > 1.0) except V. ashei ‘Powderblue’ which was considered a poor host (RF = 0.2 and 0.1 for trials 1 and 2, respectively). Paratrichodorus renifer reduced root biomass of susceptible blueberry cultivars; however, this effect was not consistent across trials. Pratylenchus penetrans did not reproduce on any blueberry cultivars under greenhouse conditions. Population growth and damage caused by P. renifer on V. corymbosum × angustifolium ‘Chippewa’ was assessed using field microplots. Paratrichodorus renifer reduced berry yield, canopy volume and top dry weights two years after inoculation by 40, 25 and 25%, respectively. Our results clearly indicate that P. renifer population densities increase on V. corymbosum and V. angustifolium and the nematode is potentially damaging to blueberry.

Résumé

Dans la majorité des régions nord-américaines où pousse le bleuet, on a trouvé, dans la rhizosphère, des nématodes du genre Trichodorus (Paratrichodorus sp.) et des nématodes des lésions des racines (Pratylenchus sp.). Toutefois, nous en savons peu sur le potentiel de reproduction de ces vers parasites et des dommages qu'ils peuvent infliger aux bleuetiers. Nous avons effectué des études d'inoculation contrôlée en serre afin d'évaluer la croissance des populations et les dommages causés par Paratrichodorus renifer et Pratylenchus penetrans sur une variété de cultivars de bleuets, y compris des cultivars de Vaccinium corymbosum (4), de V. angustifolium (1) et de V. ashei (1). Tous les cultivars testés se sont révélés être de bons hôtes pour P. renifer (valeurs du facteur de reproduction [FR] > 1.0), sauf V. ashei ‘Powderblue’ qui a été considéré comme une mauvaise plante hôte (FR = 0.2 et 0.1 pour les tests 1 et 2, respectivement). P. renifer a réduit la biomasse racinaire des cultivars réceptifs; toutefois, les tests n'affichaient pas tous les mêmes résultats. En serre, P. penetrans ne s'est reproduit sur aucun des cultivars. La croissance de la population de P. renifer et les dommages qu'il a infligés à V. corymbosum × angustifolium ‘Chippewa’ ont été évalués dans des microparcelles en plein champ. Deux années après inoculation, P. renifer a diminué le rendement, le volume du couvert et le poids de matière sèche de 40, 25 et 25 %, respectivement. Nos résultats indiquent clairement que, sur V. corymbosum et sur V. angustifolium, les densités de population de P. renifer augmentent et que le nématode est potentiellement dommageable pour le bleuet.

Keywords:

• Ericaceae
• nematode
• plant-parasitic nematode
• root disease
• root lesion nematode
• stubby root nematode
• Vaccinium

Mots clés:

• Ericaceae
• maladie racinaire
• nématode
• nématode des lésions des racines
• nématode du genre Trichodorus
• nématode phytoparasite
• Vaccinium

Introduction

Blueberry (Vaccinium spp.) has become an economically important crop in the Pacific Northwest (PNW) region of the USA and coastal British Columbia (BC). The blueberry industry was established fairly recently, with only a small percentage of fields being over 20 years old (Strik & Yarborough, 2005). Most perennial fruit crops develop root disease complexes, and plant-parasitic nematodes often contribute to these complexes. Relatively little is known, however, of the impacts of plant-parasitic nematodes on blueberry plants.

Stubby root nematodes (Trichodorus and Paratrichodorus species) have been found in root zone soil of blueberry plants in most other major blueberry-growing areas of North America, particularly in the northeastern and southern Midwest regions of the USA (Goheen & Braun, 1955; Zuckerman, 1962; Childress & Ramsdell, 1986; Clark et al., 1987; Clark & Robbins, 1994). Converse & Ramsdell (1982) reported finding Paratrichodorus spp. in three out of 10 fields in Oregon. More recently, Zasada et al. (2010) surveyed blueberry fields in the PNW and BC for plant-parasitic nematodes and found stubby root nematodes more frequently than any other nematode group, with a frequency of detection ranging from 14% in Oregon to 62% in BC. Using morphological and molecular characteristics (sequence analysis of the 18S and ITS1 regions of ribosomal DNA), Forge et al. (2009) identified some of the stubby root nematode populations reported by Zasada et al. (2010) from BC and the PNW as Paratrichodorus renifer Siddiqi (GenBank accessions GQ489248-GQ489244). Despite the common association of stubby root nematodes with blueberry plants, their impact on this host is not well understood. Zuckerman (1962) found that P. minor (Colbran) Siddiqi (syn. Trichodorus christei Allen) reduced root growth of young highbush blueberry cuttings by 67% over 6 months under greenhouse conditions, indicating that stubby root nematodes have the potential to damage blueberry plants. No previous studies have assessed the host–parasite relationship of P. renifer with any Vaccinium species.

Root lesion nematodes (Pratylenchus species) have also frequently been recovered from the root zone of blueberry plants in the PNW and BC (Zasada et al., 2010) and other blueberry-growing areas (Mai et al., 1960; Converse & Ramsdell, 1982; Clark et al., 1987; Clark & Robbins, 1994). Most surveys did not identify Pratylenchus populations to species, whereas Clark & Robbins (1994) identified Pr. scribneri and Pr. zeae. In the PNW and BC, Pr. penetrans (Cobb) Schuurmans-Stekhoven is well recognized as a pathogen of tree-fruit crops and other small-fruit crops (raspberry, strawberry) (Potter & Noling, 1984; McElroy, 1991). In some parts of the PNW and BC, highbush blueberry is being planted on Pr. penetrans-infested sites previously used for raspberry, and there is concern that Pr. penetrans could be damaging to blueberry plants in these situations. Previous studies using an unspecified blueberry genotype (Race & Hutchinson, 1959) or using sterile roots of an unspecified genotype of lowbush blueberry (McCrum & Hilborn, 1962), however, suggest that Pr. penetrans may not be pathogenic to blueberry plants.

The objectives of this research were to: (i) assess the potential for P. renifer and Pr. penetrans to reproduce on, and affect the growth of, several cultivars of blueberry under greenhouse conditions; and (ii) measure the effects of P. renifer on growth and yield of ‘Chippewa’ blueberry plants under field conditions.

Materials and methods

Greenhouse experiments

Two greenhouse trials were conducted at the USDA Horticultural Crops Research Laboratory in Corvallis, Oregon, to evaluate the host response of several cultivars of blueberry to P. renifer and Pr. penetrans. Vaccinium corymbosum L. ‘Bluecrop’, ‘O'Neal’, ‘Misty’, and ‘Duke’, V. angustifolium Aiton ‘Brunswick’, and V. ashei Reade ‘Powderblue’ (provided by Fall Creek Farm & Nursery, Inc., Lowell, OR) were transplanted into 3.8 L pots containing a steam pasteurized soil mix (1:2 washed sand : Willamette loam, v/v). In the second greenhouse trial, peppermint (Mentha x piperita L.) and sudangrass (Sorghum bicolor spp. drummondii (L.) Moench) were also included in the experimental design as they are known hosts to Pr. penetrans and P. renifer, respectively (Merrifield & Ingham, 1996; Crow et al., 2001). Plants were grown in a greenhouse under long-day conditions (16-h photoperiod) with 26/18 °C day/night temperatures and fertilized weekly with 21-7-7 (N-P-K acid) (J.R. Peters Inc., Allentown, PA) for approximately one month before inoculation with the nematodes.

The Pr. penetrans population used in this experiment was obtained from a red raspberry (Rubus idaeus L.) field in Lynden, WA and maintained on peppermint plants in the greenhouse. Inoculum was obtained by extracting nematodes from peppermint roots under intermittent mist for 48 hr (Ingham, 1994). The P. renifer population was obtained from a blueberry field in Lynden, WA and was maintained on sudangrass plants in the greenhouse. Inoculum was obtained by extracting P. renifer from soil by wet sieving–centrifugation (Ingham, 1994). At inoculation, approximately 2500 mixed-stage individuals of Pr. penetrans and 3200 mixed-stage individuals of P. renifer were inoculated into the root zone in 1-mL aliquots placed in five 2.5-cm deep holes made around the base of the plant. The holes were filled after inoculation. Non-infested controls included adding 1 mL water to five holes. Each nematode/plant combination was replicated 8 times in each of two trials and plants were arranged in a randomized block design on separate benches according to plant-parasitic nematode. The plants were allowed to mature for approximately 5 months. Trial 1 was conducted during June to November 2008 and trial 2 during June to November 2010.

At harvest, the foliage and stems were removed, air-dried, and then placed in a 70 °C oven overnight before determining dry weight. The remaining contents of each pot were placed in an 18.9 L bucket containing 10 L of water. The soil was dislodged from the root mass and the root mass was removed from the slurry, which was then mixed and allowed to settle for 15 s. Two 250 mL aliquots were removed from the supernatant and poured over an 18-mesh (1 mm opening) sieve. This step was repeated again to provide a total of 1 L of supernatant removed from the slurry. The supernatant was mixed, allowed to settle for 1 min and then poured over a 400-mesh (38 μm opening) sieve. Debris and nematodes captured on the sieve were washed into a container and allowed to settle overnight. Nematodes were further extracted using sugar centrifugation (Ingham, 1994).

The roots from each plant were washed free of soil and any remaining debris. The original root ball was dissected from the emerging roots and discarded. The roots were air-dried and then placed in a 70 °C oven overnight before determining dry weight. Prior to drying the roots of plants inoculated with Pr. penetrans, a subsample of the root systems was removed and nematodes extracted from the subsample in a mist chamber (Ingham, 1994) for 7 days. All nematodes collected from soil and root samples were enumerated using a dissecting microscope at 40× magnification.

Nematode population densities were expressed as number per pot. Reproduction factors (RF = final population/initial population) were determined for each pot. Nematode data were log transformed (log₁₀ (x+1)) prior to analysis to meet the assumptions of normality and homogeneity of variance; non-transformed data are presented. ANOVA was used to determine if there was a significant effect of cultivar within nematode-inoculated or non-inoculated groups, with P ≤ 0.05 used as the cut-off for significance. Means were compared with Tukey's adjustment for multiple comparisons (calculated with P ≤ 0.05). Plant biomass was expressed on a dry weight basis; transformation of these data was not necessary prior to analysis. The effects of P. renifer inoculation on shoot and root growth within trials and cultivars were tested using t-test for comparison of means. All data were analyzed using the computer software JMP (SAS Institute, Cary, NC, USA).

Microplot experiment

A BC population of P. renifer, which was isolated from blueberry plants on the grounds of the Pacific Agri-Food Research Centre (PARC) in Agassiz, British Columbia and designated ‘PARC’, was used in the field microplot experiment. The PARC population was isolated by hand-picking approximately 100 individual nematodes and transferring into each of six individual 30 L pots of ‘Stevens’ cranberry (Vaccinium macrocarpon Aiton) planted in pasteurized peat : sand (1:1) mixture. The nematodes were left to reproduce for about 6 months before the infested soil was used as inoculum.

Rubbermaid^(tm) rubbish bins (80 L, 60 cm high × 46 cm inside diameter) were used to construct the microplots at the PARC location. Three separate 5-cm diameter holes were drilled in the bottom of each bin for drainage. A backhoe was used to dig four 100-cm wide trenches with a 3-m space between the trenches. Twenty bins were placed into each of the four trenches, with 1 m spacing between the centres of adjacent bins within a row. Approximately 10-cm depth of pea-sized gravel was placed in the bottom of each bin, and then native soil (Monroe series sandy loam, eluviated eutric brunisol) (Luttmerding, 1981) was backfilled in and around each bin.

In May 2007, the microplots were fumigated by mixing 16 g of Basamid® granular fumigant (Mitsui & Co., Toronto) into the top 30 cm of soil with a spade, and then all microplots received about 3 cm of water. One month later, 10-cm depth of Douglas-fir sawdust was incorporated into the top 20 cm of soil in each microplot and one blueberry ‘Chippewa’ plant was placed in the centre. The plants were previously propagated by a commercial nursery (Sidhu & Sons Nursery, Mission, BC), from cuttings rooted in pasteurized media and grown for one year in 2 L pots.

On 27 July 2007, 40 of the microplots were inoculated with 300 mL P. renifer-infested soil from the cranberry pots. The other 40 microplots were inoculated with 300 mL soil mix from non-inoculated cranberry. The inoculation treatment was allocated in a completely randomized design. The inoculation procedure involved using a trowel to remove approximately 500 mL soil to a depth of 20 cm midway between the blueberry plant and the edge of the microplot, filling the hole with the 300 mL infested or non-infested soil from greenhouse-grown cranberry, and then covering the area with the removed soil. The infested soil had an inoculum density of 5 P. renifer mL⁻¹ soil (based on Baermann pan extraction from five subsamples) (Forge & Kimpinski, 2007), resulting in an initial inoculum of 1500 P. renifer per microplot. Given a microplot radius of 23 cm and assuming an effective root zone depth of 30 cm, we estimated a root zone soil volume of 50 L and an initial inoculum density of 0.03 P. renifer mL⁻¹ microplot soil.

The microplots were fitted with drip irrigation emitters and jet fill tensiometers (Soil Moisture Corp., Santa Barbara, CA) were installed in five randomly chosen microplots to monitor soil moistures. The pots were irrigated as needed during the growing season to maintain water potentials between −20 and −100 kPa.

The microplots were sampled in October 2008, 2009 and 2010. Each time, three soil cores (1.9-cm diameter) were removed from each microplot to a depth of 30 cm. The three cores were combined, mixed, and nematodes were extracted from 60 mL subsamples using Baermann pans; the three sample holes in each microplot were partially filled and covered with aged sawdust mulch. All berries were harvested from each plant on 9 July and again on 22 July in 2009. Berries were not harvested in 2010 due to severe bird predation that occurred before the planned harvest. Canopy volume of each plant was estimated in August 2009 by measuring canopy height and diameter in east–west and north–south directions. Canopy volume was estimated using the formula for a sphere, 4/3πr³. In December 2010, after leaf drop, the tops of all plants were removed, dried and weighed. The effects of nematode inoculation on plant growth parameters were tested using t-tests for comparison of means in Excel (Microsoft Corp., Bellevue, WA).

Results and discussion

Greenhouse experiments

There was a significant difference in nematode population densities, RF values and plant biomass between trials; therefore, data from the trials are presented separately (P ≤ 0.001). In general, P. renifer population density increase was at least two times greater on blueberries in trial 1 when compared with trial 2. However, despite this difference in population density increase, similar trends were observed in both trials. ‘Bluecrop’ and ‘Brunswick’ supported the highest P. renifer population densities among the blueberry cultivars evaluated in both trials (Table 1). In trial 1, these population densities were significantly different (P ≤ 0.001) from the other blueberry cultivars, while in trial 2, ‘O'Neal’ had similar P. renifer population densities (P = 0.05). Also in trial 2, where sudangrass was included in the experimental design as a known host, ‘Bluecrop’, ‘Brunswick’ and ‘O'Neal’ supported similar P. renifer populations densities as sudangrass (P = 0.05). In both trials, ‘Powderblue’ supported the lowest P. renifer population densities, which were different from those on all other tested blueberry cultivars, and sudangrass in trial 2 (P ≤ 0.001). The remaining blueberry cultivars were intermediate in host status to P. renifer. The same trends were observed for RF values. All blueberry cultivars except ‘Powderblue’ were considered good hosts for P. renifer with RF values > 1.0 (Table 1).

Table 1.  Population densities and reproduction factors (RF)^a of Paratrichodorus renifer on blueberry (Vaccinium spp.)

	Trial 1		Trial 2
Species and cultivar	P. renifer/pot	RF	P. renifer/pot	RF
V. corymbosum ‘Bluecrop’	29.731 (±2.592) a ^b	9.3 (±0.8) a	13.071 (±1.396) a	4.1 (±0.4) a
V. angustifolium ‘Brunswick’	38.785 (±2.682) a	12.1 (±0.8) a	10.816 (±1.888) ab	3.4 (±0.6) a
V. corymbosum ‘Duke’	11.687 (±997.8) b	3.7 (±2.9) b	3.096 (±1.176) c	1.0 (±0.4) c
V. corymbosum ‘Misty’	12.813 (±1.202) b	4.0 (±0.4) b	4.547 (±949) bc	1.4 (±0.3) c
V. corymbosum ‘O'Neal’	10.888 (±716) b	3.4 (±0.2) b	5.170 (±764) abc	1.6 (±0.2) bc
V. ashei ‘Powderblue’	600 (±113) c	0.2 (±0.1) c	509 (±175) d	0.2 (±0.1) d
Sudangrass	na^c	na	9.238 (±1.162) ab	2. 9 (±0.4) ab
^aRF = final nematode population/initial nematode population.
^bData were log(10) + 1 transformed prior to analysis. Means (± standard errors) followed by the same letter are not different according to Tukey's adjustment for multiple comparisons (P < 0.05).
^cNA = not included in trial.

In trial 1, only root biomass of ‘Brunswick’ was reduced by P. renifer (P ≤ 0.001) with root weights of 4.79 (±0.25) g and 3.11 (±0.27) g in P. renifer non-inoculated and inoculated plants, respectively. In trial 1, there was no effect of P. renifer on shoot weight of any blueberry cultivar compared with corresponding non-inoculated cultivars (P = 0.21). In trial 2, P. renifer did not reduce root weight of ‘Brunswick’ (P = 0.21) compared with the non-inoculated ‘Brunswick’ as in trial 1. However, in trial 2, P. renifer did reduce root weights of ‘Bluecrop’, ‘Duke’, ‘Misty’ and ‘O'Neal’ when compared with corresponding non-inoculated cultivars (P ≤ 0.007). Similar to trial 1, there was no effect of P. renifer on blueberry shoot weights compared with corresponding non-inoculated cultivars (P = 0.08). Variable reduction in root biomass by P. renifer in this study may be explained by the extent to which roots were established at inoculation. The effect of P. minor (syn. Trichodorus christiei) feeding on blueberry roots was more pronounced on cuttings inoculated at the time of setting compared with cuttings inoculated after rooting (Zuckerman, 1962).

Pratylenchus penetrans did not reproduce on any of the blueberry cultivars evaluated but did reproduce on mint, the known susceptible host (data not shown). The total number of Pr. penetrans recovered from inoculated blueberry plants across trials ranged from 4 to 48 nematodes per pot. RF values for Pr. penetrans were never greater than 0.02 on the blueberry cultivars. In trial 2, mint was a good host for Pr. penetrans with a total population density of 2692 (±147) nematodes/pot and a RF > 1.0 (±0.06). Pratylenchus penetrans is widespread in raspberry fields in the region and is well recognized as a significant pathogen of raspberry (McElroy, 1991). There is concern that Pr. penetrans could damage blueberry if planted on sites previously grown to raspberry. Our data, along with the inability to extract root lesion nematodes from blueberry roots in sites with Pr. penetrans in soil (Forge, unpub. data), indicate that Pr. penetrans is not able to parasitize blueberry plants and should not present a concern to growers replacing a raspberry crop with blueberry. These results are also in accordance with previous reports where unidentified highbush and lowbush blueberry plants were not hosts for P. penetrans (Race & Hutchinson, 1959; McCrum & Hilborn, 1962). Nonetheless, longer-term studies to determine if Pr. penetrans populations can eventually adapt to blueberry are worth pursuing.

Microplot experiment

The mean P. renifer population density increased 71 times over the first 14 months (two growing seasons) after inoculation (Table 2), indicating that ‘Chippewa’ blueberry is an extremely good host for P. renifer. Berry yields of plants grown in P. renifer-inoculated soil in the second growing season were approximately 40% lower than yields of plants growing in non-inoculated soil (Table 3). Similarly, canopy volume in the second growing season was reduced about 25% by P. renifer, and at the end of the third growing season, shoot dry weights were approximately 25% lower in P. renifer-inoculated soil than in non-inoculated soil.

Table 2.  Population densities (nematodes mL⁻¹ soil) of Paratrichodorus renifer in October of 2008, 2009 and 2010. The microplots were inoculated with 0.03 P. renifer mL⁻¹ root zone soil in July 2007. Values in parentheses are the mean population density expressed as a fraction of the initial population density

	2008	2009	2010
Mean	2.133 (71)	3.617 (121)	1.433 (48)
Median	1.450	1.767	0.817
S.D.	2.417	5.133	1.533

Table 3.  Effects of Paratrichodorus renifer on berry yield and canopy volume of 3-year-old, and top dry weights of 4-year-old, ‘Chippewa’ blueberry plants

	Berry yield (g plant^-1)	Canopy volume (L plant^-1)	Top dry wt (g)
With P. renifer	113	143	339
Without P. renifer	197	191	444
P (T ≤ t)	<0.001	0.001	<0.001

By 2010, 29 microplots had become contaminated with a species of Rotylenchus. For these 29 microplots, mean population densities were 0.2, 0.3 and 0.05 Rotylenchus mL⁻¹ soil in 2008, 2009 and 2010, respectively. Fifteen of the 29 Rotylenchus-contaminated microplots were inoculated with P. renifer. T-tests were used to compare growth parameters in Rotylenchus-contaminated microplots to non-contaminated microplots over the entire set of microplots, as well as in P. renifer-inoculated and non-inoculated microplots. None of these analyses indicated an effect of the Rotylenchus contamination.

Our microplot results clearly indicate that P. renifer is potentially damaging to blueberry plants. However, it is important to note that, on average, P. renifer population densities that developed in the microplots were much larger than Paratrichodorus population densities usually observed under field conditions (Zasada et al., 2010). The BC data of the most recent survey (Zasada et al., 2010) involved sampling primarily in June, whereas the microplots were sampled in October of each year. Other factors that could have contributed to the differential population build-up include: elimination of antagonists (e.g. nematode-trapping fungi, predaceous nematodes) from the fumigated microplots, different abiotic conditions (e.g. more preferable or uniform soil moisture regime) and differential suitability of blueberry cultivars as hosts for P. renifer. The cultivar used in this microplot experiment, ‘Chippewa’, is a hybrid genotype of highbush (V. corymbosum) and lowbush (V. angustifolium) blueberry species, and is not a commercially grown cultivar in the PNW or BC. Future research will use a similar microplot approach to assess population development and effects on more widely grown cultivars of highbush blueberry such as ‘Duke’ and ‘Bluecrop’.

Most Paratrichodorus spp. are vectors of Tobraviruses (Tobacco rattle virus, Pea early browning virus, Pepper ringspot virus) (Brown et al., 1989). While none of these viruses are reported to be pathogens of blueberry, currently there is no information on whether P. renifer can vector these or any other Tobraviruses. The possibility that P. renifer could transmit viruses to blueberry deserves additional research.

Acknowledgements

The authors wish to thank Shaobing Yu and James Hall for assistance with microplot establishment, Amy Peetz for assistance with greenhouse experiments, and AAFC's SAGES program for funding the microplot experiments.