Final Report for GNE11-027
The purpose of this project was to quantify differences between cultivated and wild blueberry plants (Vaccinium spp.) with respect to entomopathogenic nematodes (EPN). The primary objective was to explore the difference in diversity in entomopathogenic nematode communities in wild and cultivated blueberry. This proved to be difficult as the color of EPN-killed wax moth larvae used for baiting EPN from soil samples did not reliably correlate with EPN species and turnaround time with our collaborating lab using molecular methods was slow. Although this objective could not be completed to date, we are currently working towards it at Rutgers University using molecular methods not included in the proposal. Nonetheless, EPN activity could still be assessed over three sampling points during the course of two blueberry seasons and was compared between the two habitats. This information is necessary in the assessment of these communities, especially in the agricultural setting, because various species may be affected differently by predictable changes over the course of the season (e.g., water management, fertilizer application, etc.).
We found EPN activity and total number of plant parasitic nematodes (PPN) to vary significantly during the season. There were no significant differences in EPN activity by treatment but apparent trend was for more infections in the cultivated soil. The 2011 season had significantly more EPN activity and PPNs than 2012. Activity and PPN totals in 2012 appeared to be consistently lower than the 2011 data points. These results suggest that nematode populations are variable over time in both cultivated and wild blueberry fields. A different analysis and more data are needed to address what factors are most important in this variation.
Highbush blueberries (Vaccinium corymbosum) are one of the few commercial plant commodities native to the United States. Unlike many other crops, wild plants grow nearby commercially grown plants and present an opportunity to optimize biocontrol through study of wild plants and their associated community. Biocontrol, the use of natural enemies to control pest populations, is particularly important for commercial blueberries in New Jersey which are grown historically in the Pinelands National Reserve (PNR) and subject to its regulations. The oriental beetle (Coleoptera: Scarabaeidae: Anomala orientalis) is among the hardest for blueberry growers to manage because the larval stage is the most damaging and feeds belowground. Larval feeding can cause the blueberry bush to weaken and in extreme cases, even cause plant death.
Regulations in the PNR heavily restrict the use of some pesticides that would be usable outside of the protected woodlands. Managing soil pests generally requires the use of noxious agrochemicals (e.g. methyl bromide, thionazin, aldicarb) but due to the protected nature of the PNR and the high water table in the coastal plain, growers are limited to and dependant on imidcloprid (1-[(6-chloropyridin-3-yl) methyl]-N-nitro-4, 5-dihydroimidazol-2-amine). This systemic chemical, among other potential environmental issues, is likely harmful to commercial bees (Cresswell et al. 2011) but its overuse could cause resistance issues and thus, other options are needed. Because the larvae of the Oriental Beetle feed belowground, biological control using entomopathogenic nematodes (EPN) is a viable option.
EPN (Rhabditida: Steinernematidae and Heterorhabditidae) are found in soils throughout the world (Kaya 1990). They are important to soil communities in their connection with members of other trophic levels such as plants and insects. They have been shown to regulate insect populations (Strong et al. 1996) and to respond to herbivore induced signals as host finding cues (e.g. Van Tol et al. 2001, Ali et al. 2010). In the soil, EPN are present as “infective juveniles”(IJ), a dauer stage that is sensitive to host-finding cues, which often can be inactive for long periods of time. When searching for a host, their behavior can be classified along a continuum from cruiser, a highly mobile searcher, to ambusher. Adult and other stages of EPNs occur inside the insect host where they go through multiple generations and result in many IJs exiting a depleted host.
Biocontrol requires a foundation of working knowledge. To begin the process of developing a biocontrol program for the use of EPN in blueberry within the PNR, it first must be determined what EPNs are already present as well as their activity levels over the course of the season. This is important because the PNR is a protected environment and thus, it would be ideal to use already active, endemic species of EPN rather than introducing new species.
Identifying EPN to species proved to be harder than proposed. Cadaver coloration was not dependably linked to species identity and turn around time for having them processed molecularly at the University of Arizona did not have as timely a turn around as we needed. Therefore, we decided in 2013 to process the samples molecularly at Rutgers with the help of Dr. Dina Fonseca. Thus, the differences in EPN diversity have not yet been quantified. However, we were able to analyze the results of EPN activity over the 2 years from the various sites to see differences in EPN activity over the course of the blueberry producing season in both settings.
From the same soil samples, we also looked at PPN and soil characteristics from each sampling site. We planned to have PPN extractions from all three samples per year but in the second year, the second sample was lost by our collaborator. Therefore, we used PPN only from the first and third samples from both years. With all of these data, we hope to be able to coalesce all of the data we collected to indicate which of the factors are most important to the system and can be used in manipulative lab studies.
To approach the objectives of this project, 5-10 grower fields and associated wild blueberry stands in Atlantic County, New Jersey were utilized for sampling. During the 2011 season, 5 grower fields were used and in 2012, 10 grower fields were used. These fields were selected based on two criteria: 1. High oriental beetle trap captures (collected by Dean Polk, Rutgers Fruit IPM Coordinator, Rutgers Cooperative Extension); 2. Presence of wild blueberry in adjacent wooded areas. In each grower field, a 50’x50’ plot containing 100-150 blueberry bushes was denoted by flags and 10 random plants plants within this plot were sampled. At each selected plant, eight soil cores were taken with an Oakfield sampler (2.5 cm diameter x 30 cm depth, approximately 100 mL) within the area below the plant’s canopy and transferred to and combined in a plastic bag. An analogous 50’x50’ plot was selected in the natural habitat. Wild plants were also chosen at random from this plot and handled the same way as samples from the managed habitat. Sampling was conducted three times: at blueberry bloom (early May), harvest (early July), and post-harvest (early September).
Soil samples were brought to the Koppenhöfer lab in New Brunswick, NJ. Sub-samples (100g sample per plant sampled) of each sample were baited with 5 waxworm larvae each (Galleria mellonella). Petri dishes used to contain the 100 g sample were checked after 5 days and 10 days and any dead or infected larvae found after 5 days replaced with new live larvae. Larvae with typical signs of EPN infection were placed individually on emergence traps (White traps) and any emerging nematode progeny were collected and kept in tap water in tissue culture flasks at 8 °C until further processing (Kaya and Stock 1999). In emergence traps, infective juveniles migrate from the infection dish with the cadavers into the water in the bottom of the larger petri dish that contains it. These IJs were used to confirm the infection in additional G. mellonella. Infections per plant was calculated from the number of infections occurring over the course of the baiting rounds which occurred until there were no infections for two weeks.
As mentioned above, identifying EPN to species was harder than we planned in the original proposal. While there were certain types of colorations (e.g., orange-red, tan, chestnut, brown-grey), there was a lot of variability within each type which led us to worry that more that several very similar, possibly yet to be described species may be mixed up without the use of molecular methods.. In 2013, we decided to begin processing the samples with molecular methods at Rutgers with the help of Dr. Dina Fonseca. To do this, individual nematodes from an additional round of soil collection and baiting were used for DNA extraction according to methods from Floyd et al. 2002. PCR products are then sent off for sequencing and Basic Local Alignment Search Tool (BLAST) in GenBank is being used to locate species identity.
Preliminary results suggest diversity of EPN species is higher in wild settings. Additionally, Steinernema glaseri is dominant in cultivated fields. Other species that have been identified so far: Heterohabditis georgiana and Steinernema feltiae.
While no significant differences were found between cultivated or wild settings (Figure 1a), which will be referred to as treatment from here, significant differences in interactive effects were found in year by sample by treatment (Figure 1b). Overall, there was a trend for more infections in cultivated soil. Although activity does not significantly differ between wild and cultivated fields, it is still likely that diversity will be different between the two areas because nematode communities tend to vary with plant identity (Bezemer et al. 2011). Furthermore, high densities of nematodes can also induce the proliferation of nematode predators such as nematode trapping fungi (Jaffee and Strong 2005) which could potentially contribute to differences in more diverse, stable soil environments where such natural enemies are able to persist.
Plant Parasitic nematodes (PPNs) are important to agroecosystems. Because PPNs were found to be also attracted to herbivore induced signals (Ali et al. 2011), we chose to include them as a part of this preliminary study of blueberries in the PNR. The PPNs extracted were identified to genera which did not allow us to use true diversity indices. However, a plant parasitic nematode index (PPI) developed by Tom Bongers in 1990 distinguishes PPN genera along a continuum from colonizer to persister in order to describe the maturity index of a soil. We used this index but found no differences between cultivated and wild settings (F(1/21.2)=0.15, P=0.7019). The total number of PPNs per sample was also analyzed and we found that multiple interactive effects were significant. Year by sample (Figure 2a) was significant at alpha equals 0.05 (F(1/36.1)=4.69, P=0.0370) as well as year by treatment (Figure 2b) (F(1/46.7)=4.53, P=0.0386). These results suggest that while the overall state of the fields may not be different by maturity, how they function over time can potentially be very different.
While EPN activity did not differ significantly by treatment, some soil characteristics did (Table 1). Because infective juvenile nematodes must seek their hosts in the soil, these characteristics may affect the success of EPNs and may affect different species differently. The pH, notoriously acidic in blueberry soil, did not differ between cultivated and wild (F(1/18)=4.32, P= 0.05221) but there was a trend for a slightly higher pH in the cultivated setting. Phosphorus (F(1/18)=12.63, P=0.002268) and potassium (F(1/18)=6.9951, P=0.01647)) differed significantly with higher concentrations in the cultivated setting in both instances. Nutrient differences by treatment were expected due to the supplementation in the cultivated treatment.
Both EPN and PPN data were analyzed using a mixed model in SAS 9.2.3. A mixed model was necessary because site is considered a random variable nested in treatment. Soil data was analyzed using analysis of variance using RStudio version 0.97.551. Data was considered significant if alpha was less than or equal to 0.05.
Education & Outreach Activities and Participation Summary
During the course of this project, I attended the annual meeting of the Entomological Society of America (ESA) meeting in 2012 where I presented this work and interacted with many people working on similar or related projects. In 2013, I will be presenting at the Society for Invertebrate Pathology meeting in Pittsburgh, PA in August and at the ESA meeting in Austin, TX in November. Before presenting at grower meetings, I want to have a more complete story associated with the data. By this, I mean I would like to be able to tell them more about correlations between PPN, soil properties and EPN activity and identity. This requires a more complex multivariate analysis than presented here. I am taking multivariate statistics in Fall 2013 and I will likely start presenting at extension meetings during this time.
In general, farmers seemed very enthusiastic about the idea of harnessing endemic populations of EPN. However, because the project is not resulting in a finalized methodology for using them, they are not as intensely interested. Preserving natural enemies is already a common theme in IPM training and this is an extension of that idea. Before farmers could truly adopt this as a treatment for oriental beetle populations, much more information on the efficacy of using EPN will have to be developed.
Areas needing additional study
A number of studies would need to be done in order for this to be a usable system by farmers. This includes: 1. efficacy of various species on oriental beetle, 2. developing an EPN attractant, 3. developing a way of easily monitoring EPN populations.