Reducing Pesticide Use in Vegetable Production by Calculating Soil-borne Disease Risk

Final report for GNE15-103

Project Type: Graduate Student
Funds awarded in 2015: $14,633.00
Projected End Date: 12/31/2016
Grant Recipient: Cornell University
Region: Northeast
State: New York
Graduate Student:
Faculty Advisor:
Dr. Sarah Pethybridge
Cornell University
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Project Information

Summary:

The Northern root-knot nematode (Meloidogyne hapla) is an important soilborne pathogen of potatoes in New York, reducing tuber yield and quality.  Information regarding nematode populations within the soil at planting is critical in determining the implementation of management tactics, but is not readily available to most growers due to challenges in delivering timely and accurate population estimates.  Therefore, nematode management decisions made by the grower are often risk averse, frequently resulting in the unnecessary application of chemical treatments.  The holistic objective of my project was to reduce unnecessary applications by increasing informed decision making through the development of a new quantitative risk algorithm for M. hapla that estimates yield loss and tuber damage at harvest based upon quantification of initial (i.e. pre-plant) pathogen DNA in the soil. 

Within this project, I laid key groundwork in the development of a new risk algorithm by (1) isolating high quality DNA from soil, (2) developing primers and probes for quantification of DNA through quantitative PCR, and (3) investigating correlation of pre-plant nematode populations to yield observed at harvest, complementing this work with information on potato cultivar susceptibility to M. hapla.

Firstly, I developed a DNA extraction protocol from 100 g of soil using super paramagnetic iron oxide nanoparticles (SPION) DNA capture. The SPION-based extraction method developed here offers substantial benefits of greater consistency of quantity and quality, reduced processing time and costs, and eliminates the use of hazardous materials used in standard phenol extraction methods.  Additionally, the method had the ability to process soil volumes of 100 g, which can increase the accuracy of disease estimates due to the spatial heterogenetity of nematode populations within the soil.  The sensitivity of the extraction protocol and the binding capacity of the SPION were investigated to assess utility of the method. 

Secondly, I developed a species-specific quantitative PCR primer and probe set for quantification of initial populations.  Primer and probe sequences that targeted the chorismate mutase and 16D10 effector genes from M. hapla were generated and assayed against 13 different species of common plant parasitic nematodes using qualitative PCR to determine the specificity of each set. One set aligning with the 16D10 effector gene was identified that was highly sensitive and specific to M. hapla, and was verified through cloning and sequencing of the target amplicon. 

Thirdly, I investigated the relationship between yield loss and populations of M. hapla and the lesion nematode Pratylenchus spp. under field conditions.  Three commercial potato fields in New York with high populations of M. hapla and Pratylenchus spp. were selected in collaboration with two participating growers.  The fields were intensively sampled at planting and at harvest, nematode populations quantified, and populations correlated with yield data collected at the same sampling locations.  It was found that populations of M. hapla and Pratylenchus spp. exhibited a generally aggregated spatial pattern with high localized variability within each of the three fields sampled, both at planting and at harvest.  No significant linear association was found between total weight (kg) of tubers per plant and M. hapla and Pratylenchus spp. populations at planting (initial population) within any of the three fields.  Furthermore, to support more robust predications of disease risk posed by M. hapla, I quantified the susceptibility of eleven different potato cultivars commonly grown in New York to M. hapla in a greenhouse pot trial. The effects of inoculation and cultivar type on tuber yield, tuber damage, and plant growth characteristics were assessed.  In this greenhouse study, it was found that cultivar type and M. hapla inoculation level had individual significant effects on potato tuber yield. However, the interaction between the two factors on tuber yield was not significant.  Additional effects of inoculation level and cultivar type were also observed. 

High reaching impacts of this project include the development of a DNA extraction method that is easy and rapid to perform, and does not produce hazardous waste, leading to timely results and more sustainable practices in the diagnostic laboratory.  Further, the research conducted during this project has increased our knowledge about disease risk thresholds for M. hapla in potato through population densities and cultivar susceptibility, impacting the agricultural community in New York by supporting more economically and environmentally sustainable nematode management practices in potato production. 

 

Introduction:

Nematodes (and other soilborne pathogens) are associated with substantial reduction in yield and quality in potatoes crops worldwide.  In New York and the northeastern United States, the Northern root-knot nematode (RKN), Meloidogyne hapla, is associated with substantial reductions in tuber yield and quality.  Losses from M. hapla damage occur in all aspects of productivity, such as seed-certification, and in the fresh market and processing industries.  In Idaho, an entire field of fresh market potatoes may be rejected if only 5% of tubers have defects attributed to damage from Colombia root-knot nematode (Meloidogyne chitwoodi) upon visual inspection. This represents an economic loss to the grower of thousands of dollars per acre (King and Taberna, 2013). 

Decisions regarding the management of M. hapla and other plant parasitic nematodes made in intensively managed agricultural systems are integrated functions of risk of crop loss, driven by overriding assumptions of the ubiquitous presence of disease inoculum (i.e. nematode individuals and eggs).  RKN populations are often controlled by pesticide applications in the form of fumigants (e.g methyl bromide) or non-fumigant nematicides (e.g. Vydate®).  The majority of decisions surrounding these pesticide applications are routine and prophylactic in nature, frequently resulting in unnecessary applications.  This purblind approach increases variable costs of production, increases the probability of resistance development or amplified biodegradation of the compound by soil microorganisms, and potentially has off-site deleterious effects on the environment. 

In contrast to foliar diseases, predicting risk of disease due to nematodes and other soilborne pathogens is strongly reliant upon effectively quantifying the presence of inoculum within a field.  Traditionally, risk algorithms used to predict tuber damage from nematodes relate populations within a unit of soil to expected damage.  However, the protocol for extraction, quantification, and identification of nematodes from soil is often a time consuming and difficult process, requiring identification by an experienced nematologist.  It therefore limits the ability of growers to make time-sensitive decisions concerning prophylactic pesticide application. 

Furthermore, management decisions for plant-parasitic nematodes often need to be made prior to planting.  Unfortunately, these decisions are usually made in the absence of data on inoculum densities, and consequently they are often risk averse, necessitating the application of costly preventative treatments before or at planting, with high environmental impact quotients, multiplying the effect of making false positive decisions. Prediction of damage from soilborne diseases may be substantially improved by the provision and adoption of soil tests, the results of which are provided prior to planting and are highly sensitive and specific for pathogens and races according to the planned crop.  To illustrate the achievable benefits, King and Taberna used a variable rate fumigation program in an experimental trial in Idaho for control of M. chitwoodi in potato.  The researchers intensively sampled for M. chitwoodi in the field to generate a map of the spatially variable nematode densities across the field.   From this map, quantities of fumigant applied was dictated by nematode density at that particular location in the field.  The use of variable rate fumigation within the field resulted in an average of a 30% reduction in chemical usage without compromising yield (King and Taberna, 2013).  Rapid detection of the heterogeneity in nematode population densities across a field may thus enable variable application rates of pesticides, promoting a more conservative and efficient use of pesticides.  

The holistic objective of my project was to reduce unnecessary applications by increasing informed decision making through the development of a new quantitative risk algorithm for M. hapla that estimated tuber damage at harvest based upon initial pathogen populations.  I sought to provide a step-change by quantifying populations through measuring DNA of the target organism rather than through morphological examination.  To achieve this goal, I developed a method for extracting whole genomic DNA from nematodes and other soilborne microbes directly from soil and developed unique primer and probe sets for use in qPCR quantification within this project.  Further, I began to establish the relationship between crop loss and initial populations by intensively sampling three commercial potato fields and conducting a greenhouse.

Quantifying the amount of pathogen DNA present in the soil will provide a faster, more accurate estimation of whether M. hapla poses a significant risk to potato prior to planting.  This information may then be used to better inform management decisions for soilborne diseases, including pesticide application, cover crops, and rotation options. 

References:

King B.A. and Taberna J.P. Jr. (2013) Site-specific management of Meloidogyne chitwoodi in Idaho potatoes using 1,3-dichloropropene; approach, experiences and economics.  Journal of Nematology. 45: 202-213.

Project Objectives:

The objective of this multi-year project is to develop new a quantitative risk algorithm for Meloidogyne hapla that will predict tuber damage at harvest based upon initial pathogen population densities prior to planting and other edaphic factors such as soil type.  The specific objectives are to:

(1) Develop and test a quantitative PCR (qPCR) procedure for quantifying inoculum of M. hapla across a range of soil types.  To differentiate between M. hapla and other nematode species within soil samples species-specific primers based upon chorismate mutase, an effector gene present within root knot nematode species, will be utilized.

(2) Assess the utility of the qPCR technique in establishing the relationship between soilborne pathogen DNA levels and tuber damage, supporting the development of new decision theory applications for monocyclic soilborne diseases. 

(3) Test the relationship between M. hapla DNA and tuber damage by intensively sampling three commercial potato fields within New York State.  This will further refine the risk algorithms developed and assess their utility under field conditions.

(4) Present the conceptual framework developed here for reducing pesticide usage through soil testing before planting developed in this study to New York growers though appropriate forums, including Cornell Cooperative Extension meetings, the NY Potato Advisory Group assemblies, and American Phytopathological Society Northeast Division conferences. 

Cooperators

Click linked name(s) to expand
  • Dr. Xiaohong Wang (Researcher)
  • Dr. Frank Hay (Researcher)

Research

Materials and methods:

Isolation of Nematode DNA from Soil

During the course of this study, it was found that optimizing the method for isolating nematode DNA from soil was essential as a precursor to the development of the qPCR assay. Soil provides a unique and challenging media to isolating DNA from soilborne organisms due to humic compounds which may act as PCR inhibitors. Several commercial kits were available for extracting total DNA from soil, however they were suboptimal for this study as they are limited by the ability to extract from only small volumes (less than 10 g) of soil. This volume of soil limits the ability to drawing biologically relevant conclusions for pathogen detection and infer management decisions. Commercials kit are also costly and thus prohibitive for this study and implementation in practice. 

To circumvent this problem, I developed a novel, high-throughput, and economical extraction method using laundry detergent powder lysis and super paramagnetic iron oxide nanoparticles (SPION) DNA capture from 100 g of soil.  The extraction method was compared to a standard phenol-based extraction method and a commercial DNA extraction kit using 0.5 g of pasteurized soil each inoculated with 10 M. hapla second stage juveniles.  Five replicates of each method were performed, and the experiment conducted twice.  The binding capacity of the SPION were assessed within a 100 g of soil extraction by evaluating DNA yield and quantity by performing the protocol with increasing SPION volumes.  Pasteurized soil of 100 g aliquots were each inoculated with 200 M. hapla second stage juveniles, and subject to the extraction procedure using SPION volumes ranging from 1 to 50 mg. Each SPION volume was replicated five times and the experiment was conducted twice. Further, the sensitivity of the extraction method was evaluated by performing the SPION-based extraction method on 100 g pasteurized soil aliquots inoculated with 1, 10, 100, and 1,000 M. hapla second stage juvenile nematodes. Each inoculation level was replicated five times and the experiment was conducted twice. Data were collected by measuring DNA yield with a Qubit fluorometer and DNA quality using a NanoDrop spectrophotometer. Data were recorded and analyzed within the R Studio statistical platform (R Core Team, version 3.1.1). 

Development of qPCR Primers and Probes for Detection of M. hapla

A quantitative PCR (qPCR) procedure for quantifying M. hapla populations in soil across a broad range of soil types was developed. To accomplish this objective, I developed sets of qPCR primer and probe sequences targeting the M. hapla chorismate mutase and 16D10 effector genes using the web-based primer design tool Primer3.  I tested the primer and probe sets against 13 different species of common plant parasitic nematodes using qualitative PCR to determine the specificity of each set. A primer and probe set were considered specific if amplification was only observed from M. hapla DNA. 

Investigation of the Relationship between Initial Nematode populations and Yield at Harvest, 2016 Study

An integral component of the project was to establish the relationship between M. hapla populations and potato tuber yield and damage. To accomplish this, three commercial potato fields in New York with high populations of M. hapla and the lesion nematode Pratylenchus spp. were selected in collaboration with two participating growers. Two fields were located in Springville and established with the fresh market variety ‘Eva’. The third field was located in Wayland and planted with the chipping variety ‘Lamoka’. 

The fields were intensively sampled soon after planting and prior to emergence (May 2016; initial population at planting). Soil samples (~ 1 kg) were collected from the hills at a depth of approximately 6 inches at each point.  Samples were collected on a 10 by 10 point grid, giving 100 total points per field.  Each point was marked with a flag and the location recorded by GPS. Distance between the points varied from 20 to 50 feet, depending on the size and shape of the field. The soil samples were divided into smaller portions for use in three quantification experiments. First, a subsample was subjected to manual counting using a modified Whitehead tray to extract live, motile-stage nematodes. An aliquot of the extraction suspension was then quantified under the microscope to quantify populations by identifying nematode genera.  Secondly, a subsample was used in a tomato “bait plant” experiment. Field soil was placed into 5 inch clay pots, and a tomato seedling was planted into the soil. Plants were maintained for seven weeks, after which they were up-rooted, the roots washed, and root galling severity scored. Bait plants are a common research method for quantifying Meloidogyne spp. populations. A final subsample was prepared for storage and DNA extraction. 

The fields were sampled again after vine kill and prior to harvest (September/October 2016, final population at harvest). Soil samples were collected from the same points as the initial sampling. As before, approximately 1 kg of soil was collected from the hills at a depth of approximately 6 inches. Soil was portioned into two subsamples, the first for manual counting using a modified Whitehead tray, and the remaining for storage and DNA extraction. Additionally, one plant was collected at each sampling point. Data on total yield (kg), number of tubers, diameter of tubers, root mass, root galling severity, and tuber damage were collected. 

Data was analyzed using a paired Student’s t-test to compare the initial and final populations as determined by the manual quantification of motile nematodes from the Whitehead trays.  Correlation analysis and regression models were used to model the relationships between initial populations and the various yield components and crop damage within R Studio. 

Geostatistics was used to assess spatial patterns of nematode populations and determine the degree of spatial dependence between two points in space. Manual count data at each sampling point was interpolated to estimate populations at non-sampled locations, i.e. areas between sampling points.  In the case of spatial dependence, locations closer together are assumed to be more similar in characteristics than those farther apart. To accomplish the interpolation, a semivariogram for each field × species × sampling time combination was calculated. This quantifies the degree of spatial correlation between observations at different sampling locations (semivariance vs. separation distance).  From the semivariogram, ordinary kriging was employed to estimate the populations over the entire sampling area. Ordinary kriging uses a function of weighted averages of known values (the sampling points) to predict the unknown values (locations between the sampling points) (Cressie, 1988). Semivariograms were calculated, fit, and kriging was performed in R Studio using the package ‘gstat’. 

Response of Potato Cultivars to M. hapla

To better inform the level of risk to a crop posed by M. hapla, we quantified the susceptibility of potato cultivars commonly grown in New York to M. hapla in the greenhouse. Similar studies have been conducted (Abawi et al. 2008; Van der Beek et al. 1998).  However these studies only assessed cultivar differences in M. hapla reproduction and provided no quantitative assessment of effects on growth or yield. Additionally, new cultivars of potato have since been released and augmentation of this work was essential to this study.    

Eleven potato cultivars (‘Adirondack Blue’, ‘Altantic’, ‘Eva’, ‘Lamoka’, “Nordana’, ‘Norland’, ‘NY140’, ‘Reba’, ‘Snowden’, ‘Upstate’, and ‘Waneta’) were identified and obtained from Prof. Walter de Jong, Cornell University. Seed pieces were placed into 1.5 gallon plastics pots filled with pasteurized soil (3:1 top-soil to sand). Three weeks after sprouting, plants were inoculated with varying concentrations of M. hapla second stage juveniles, obtained from a hydroponics rearing system.  Plants were inoculated with one of three inoculation treatment groups: 500 nematodes per pot (“Medium” level), 1,500 nematodes per pot (“High” level), and 0 nematodes per pot (“Control” level, inoculated with sterile water). Plants were inoculated by pouring the inoculum solution into three to four holes around the base of the plant.  After inoculation, plants were maintained in the greenhouse for nine weeks.  A highly susceptible tomato variety (‘Rutgers’) was included as a positive control.   The experiment was conducted April to July 2017. 

At harvest, plants were removed from pots and data on foliage fresh weight (g), root mass fresh weight (g), root galling score (assessed on a 0 to 100% scale), number of tubers formed, tuber yield (total tuber weight; g), and tuber diameters (width of long axis; mm) were collected.   Motile, second stage juveniles were also extracted from soil (200 g) obtained from each pot using a pie pan extraction to quantify nematode reproduction factor (number of nematodes at harvest divided by initial population level). 

Data were analyzed in R Studio. Data were visualized by generating box-plots of the response variables and outlying data points with high leverage were removed. Normality in the response variables was assessed using the Shapiro-Wilk test (function ‘shapiro.test’), and transformations (log(x+1)) performed where necessary to achieve normality. Homogeneity of population variances was assessed using Levene’s Test (function ‘LeveneTest’). Differences in responses between cultivars, inoculation levels, and the interaction between cultivar and inoculation level were then assessed using Analysis of Variance (function ‘anova’). Treatment effects that were significant at the 0.05 level were separated means using Tukey’s Method for multiple comparison of means (function ‘HSD.test’). 

Extension of findings and presentation of results

A complementary objective of the project was to present findings and results of this research in multiple forums, including academic conferences and extension events, oral presentations and written publications.  Findings from the 2016 field studies that were relevant and useful to key stakeholder groups, including New York potato farmers, industry leaders, and Cornell Cooperative Extension personnel were organized into a handout and presented at a cooperative extension event.  Further, results from the DNA isolation method and the development of M. hapla specific primers were presented to academic audiences in poster and oral talk format.  Experiments and results detailing the development and testing of the DNA extraction method from soil using SPION were prepared for submission to a scientific journal. 

References

Abawi G.S., Ludwig, J.W., and Gugino B.S. (2008) Diagnosis, biology, and management of root-knot and lesion nematodes on potato.  URL: http://vegetablemdonline.ppath.cornell.edu/NewsArticles/Pot_Nematodes.htm/

Cressie N. (1988) Spatial prediction and ordinary kriging. Mathematical Geology 20: 405-421. 

R Core Team (2014) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL: http://www.R-project.org/

Van der Beek J.G., Vereijken P.F.G., Poleij L.M., and Van Silfhout C.H. (1998) Isolate-by-cultivar interaction in root-knot nematodes Meloidogyne hapla, M. chitwoodi, and M. fallax on potato. Canadian Journal of Botany 76: 75-82. 

Research results and discussion:

Isolation of Nematode DNA from Soil

A SPION-based extraction method for extracting from 100 g of soil that is quick, easy to perform, and economical was developed.  Briefly, air dried soil was suspended in a lysis buffer, then heated in a water bath until the internal temperature of the solution reached 80 degrees C.  The solution was held at 80 degrees C for 5 minutes, then removed and vortexed at maximum speed for 30 seconds.  The extraction mixture was then centrifuged for 5 minutes at 5,000 g.  Next the supernatant was removed and transferred to a fresh container, to which SPION and binding buffer were added. The mixture was incubated for 20 minutes at room temperature. The SPION were then immobilized with a magnetic stand, and the supernatant discarded.  The SPION were then washed three times with ethanol and dried to remove trace ethanol. DNA was then eluted from the particles with Tris-EDTA (TE) buffer. The DNA was then purified using a polyvinylpolypyrrolidone spin column. The method was compared to a standard phenol extraction and commercial extraction kit by performing each method twice, with five replicates.  The SPION method produced DNA that was approximately 100-fold less, but of similar quality to the standard phenol method and the commercial kit (Table 1). Target DNA was amplified from resultant DNA in 70% of samples, indicating consistency of detection. Investigation of the binding capacity of the nanoparticles revealed 10 mg of nanoparticles to be optimal in maximizing DNA yield while minimizing co-capture of contaminants (Fig. 1).  The detection limit of the procedure was determined to be approximately 100 nematodes per 100 g of soil (Table 2), indicating the method was useful for informing nematode management decisions for potato.  The SPION-based extraction method developed here offers substantial benefits of greater consistency of quantity and quality, reduced processing time and costs, and eliminates the use of hazardous materials used in standard phenol extraction methods. 

Development of qPCR Primers and Probes for Detection of M. hapla

In the development of a primer and probe for use in qPCR tests, one set targeting the 16D10 effector gene was identified that was highly sensitive and specific to M. hapla (Fig. 2). Further verification of amplification of the correct target was conducted by cloning and sequencing the amplicon. This set will be used in further qPCR tests of field samples. 

Investigation of the Relationship between Initial Nematode populations and Yield at Harvest, 2016 Study

Counting of M. hapla and Pratylenchus spp., across all sampling locations within each of the fields found high localized variability in populations at planting and harvest (Table 3). Populations of M. hapla at harvest were significantly higher than at planting in two of the three potato fields. Pratylenchus spp. populations were significantly higher at harvest than at planting in one of the three fields (Table 3).  Manual counting of nematodes from individual locations were also used to estimate the nematode populations over the entire sampling area at both pre-plant and pre-harvest sampling times using geostatistical analyses (Fig. 3). This indicated that nematode populations were highly variable within the fields, in a generally aggregated spatial pattern.  At many locations, high populations of M. hapla and Pratylenchus spp. did not co-occur. 

Regression analyses of bait plant root galling severity with M. hapla populations at planting identified a significant positive linear correlation, indicating that root galling severity in the bait plants was strongly associated with the populations of M. hapla in the soil.  This supports the hypothesis that this common technique for quantifying M. hapla populations is accurate, albeit prohibitively laborious for use as a pre-plant risk assessment for growers (Fig. 4). 

No significant association was found between total weight (kg) of tubers per plant and M. hapla and Pratylenchus spp. populations at planting within any of the three fields (initial populations; Table 4).  This finding suggests that ‘Eva’ and ‘Lamoka’ may be good hosts for these nematodes but may also have some tolerance.  A significant positive association was identified between the total weight (kg) of tubers per plant and M. hapla and Pratylenchus spp. populations at harvest (final populations; Table 4).  This finding suggests that a higher root volume may simply be providing support for the multiplication of nematode populations in the absence of a yield penalty.

Response of Potato Cultivars to M. hapla

Cultivar and M. hapla inoculation level had a significant effect on potato tuber yield (Tables 5 and 6). However, the interaction between the two factors on tuber yield was not significant (P > 0.05). Cultivar had a significant effect on shoot weight (P < 0.0001), number of tubers (P < 0.0001), and tuber diameter (P < 0.0001; Table 6). As a main effect, M. hapla inoculation level had no significant effect on shoot weight, root weight, number of tubers formed, or tuber diameter (Table 6). However, the interaction between cultivar and cultivar × M. hapla inoculation level had a significant effect on fresh root weight (P = 0.048). This finding supports the hypothesis that the different potato cultivars exhibited varying root architectures that lead to differences in weight. Additionally, both main factors and the interaction between them had a significant effect on root galling and M. hapla reproduction factor (Table 7). For example galling score was significantly higher in cultivar ‘Lamoka’ than all other potato cultivars tested. Meloidogyne hapla reproduction factor was also significantly higher in ‘Lamoka’ than other cultivars with the exception of ‘Reba’ (Table 5).

One of the dominant effects observed was M. hapla inoculation level on tuber yield, with the ‘Medium’ level being significantly lower than the non-inoculated control group (P = 0.001; Table 6). The plants inoculated at the ‘Medium’ level had lower tuber yields across cultivars, indicating that an initial population level of 500 nematodes per pot (3.4 kg of soil) had a deleterious effect on yield.  However, inoculation level did not have a significant effect on the number of tubers formed or the tuber diameter (two other measures of plant productivity) at the 0.05 level, indicating that these features were not influenced by initial nematode populations, even up to 1,500 nematodes per pot.

Another important result observed was the significant effect of cultivar on reproduction factor, a measure of virulence and host plant suitability.  Several cultivars including ‘Lamoka’, ‘Reba’, ‘Snowden’, and ‘NY140’ had reproduction factors significantly higher than the tomato susceptible controls. Inoculation level also had an effect on reproduction factor, with the ‘Medium’ and ‘High’ levels being significantly greater than the ‘Control’. 

Extension of findings and presentation of results

Research findings from the 2016 soil sampling of three potato fields were summarized into an extension bulletin (attached to this report) that detailed the results and explained the holistic goals of the project including the establishment of a DNA-based pre-plant soil test for detection and quantification of plant parasitic nematodes.  The results and handout were presented at the Fresh Market Potato Variety and Pest Management Meeting on August 25, 2016, a Cornell Cooperative Extension event held in Marion, New York.  Attendees (approximately 50) included growers, crop consultants, and Cornell Cooperative Extension personnel.  The presentation consisted of a 15 minute talk followed by a robust question and answer session.

NE-SARE-Final-Project-Report-Tables-and-Figures

Potato-meeting-25-Aug-2016-v32

Research conclusions:

The SPION-based DNA extraction method from soil was shown to produce high quality, PCR grade DNA from M. hapla present in the soil.  Approximately 10 mg of SPION was identified as the optimal volume of nanopartices to add to the reaction to maximize DNA yield while minimizing co-capture of contaminants.  The SPION-based extraction method developed here offers substantial benefits of greater consistency of quantity and quality, reduced processing time and costs, and eliminates the use of hazardous materials used in standard phenol extraction methods. 

A single qPCR primer and probe set targeting the 16D10 effector gene was identified as highly sensitive and specific to M. hapla (Fig. 2)Verification of the correct target amplification was conducted by cloning and sequencing the amplicon. This set will be used in further qPCR tests of field samples. 

Counting of M. hapla and Pratylenchus spp., across all sampling locations within each of the fields found high localized variability in populations at planting and harvest (Table 3; Fig. 3), indicating that nematode populations exhibit a generally aggregated spatial pattern over the field and certain disease foci are present.  At many locations, high populations of M. hapla and Pratylenchus spp. did not necessarily occur together.  

Regression analyses of bait plant root galling severity with initial populations of M. hapla identified a significant positive linear correlation, indicated that root galling severity in the bait plants was highly associated with the populations of M. hapla in the soil.  The bait plant method is therefore an accurate, but laborious and slow, method for estimating initial populations of M. hapla in the soil. 

No significant association was found between total weight (kg) of tubers per plant and M. hapla and Pratylenchus spp. populations at planting within any of the three fields (Table 4).  This finding suggests that the potato cultivars ‘Eva’ and ‘Lamoka’ may be good hosts for M. hapla and Pratylenchus spp., but may also exhibit some tolerance for these two nematodes.  Future studies are being planned to investigate further this interaction under field conditions. 

In assessing the response of several potato cultivars to M. hapla, the factors of cultivar and M. hapla inoculation level each had a significant effect on potato tuber yield (Tables 5 and 6).  The effect of cultivar reflected a difference in cultivar horticultural characteristics. The effect of inoculation level indicated that an initial population level of 500 nematodes resulted in a significantly lower yield across cultivars compared to the non-inoculated control.  

Participation Summary
2 Farmers participating in research

Education & Outreach Activities and Participation Summary

6 Consultations
1 Curricula, factsheets or educational tools
4 Webinars / talks / presentations

Participation Summary

50 Farmers
2 Number of agricultural educator or service providers reached through education and outreach activities
Education/outreach description:

Stakeholder engagement was a key facet of this project.  Mr. Karl Hofmann of Springville, NY and Mr. Kurt Brehm of Wayland, NY were two potato growers involved in the project for the on-farm sampling.  My faculty mentor and I met with the two participating growers on a bi-monthly basis to inform and present findings from the project.  Participating growers received information about the types and relative populations of plant-parasitic nematodes present in their fields, and potential management tactics discussed. The growers expressed interest and satisfaction with the research taking place on their farms, and conveyed interest in continued collaboration. 

I presented some of the project findings at the Fresh Market Potato Variety and Pest Management Meeting in Marion, NY on August 25, 2016.  The event was hosted by Cornell Cooperative Extension, and the presentation consisted of a 15 minute talk followed by a question and answer session, in which questions were fielded from New York potato growers, crop consultants, and Cornell Cooperative Extension personnel (estimated 50 people in attendance).  A bulletin was prepared for the meeting (attached here and also in Project Results) for attendees to follow along with and take-home. The talk and bulletin detailed results of field sampling for quantifying the relationships between initial nematode populations and yield at harvest, and additionally explained the holistic goals of the project.

Potato-meeting-25-Aug-2016-v31  

Further, I presented results from the project to academic audiences at three scientific conferences, including the 2016 American Phytopathological Society (APS) Annual Meeting in Tampa, FL (July 30 to August 3, 2016); the 2017 APS Annual Meeting in San Antonio, TX (August 5 to 9, 2017); and the APS Northeast Division Meeting in Ithaca, NY (October 19 to 21, 2016).  It is anticipated that the work will also be presented at the 2018 International Congress of Plant Pathology in Boston, MA (July 29 to August 3, 2018). 

I have prepared a journal manuscript summarizing the novel SPION-based DNA extraction method for isolating high quality DNA from soil.  This manuscript has been submitted to the scientific journal Nematology for peer review.  Following publication, other researchers within the areas of plant pathology, microbiology, and soil ecology may use the method for enhanced, environmentally sustainable, study of soil microbiota. 

An extension type factsheet is anticipated to be written, and will include information on nematode biology and summarize key points of new information derived from the experiments conducted during this project award period.  The factsheet will be disseminated to grower stakeholders through cooperative extension channels and will also be available through the lab website (http://evade.pppmb.cals.cornell.edu/).

Project Outcomes

2 Farmers reporting change in knowledge, attitudes, skills and/or awareness
2 Grants applied for that built upon this project
1 Grant received that built upon this project
$10,000.00 Dollar amount of grant received that built upon this project
Project outcomes:

During this project, I developed a novel DNA isolation method from soil, which is quicker, easier, and more economical to perform than DNA isolation through standard phenol methods or commercial extraction kits.  This DNA extraction method does not produce hazardous waste, leading to more sustainable practices in the diagnostic laboratory. 

This work has also enhanced scientific knowledge of the susceptibility of potato cultivars grown in New York and the Northeastern U.S. to M. hapla.  The field trials what were conducted have enhanced the information available for establishing thresholds for pre-plant M. hapla populations for acceptable levels of crop damage or yield losses.

The tools developed and knowledge outcomes reached during this project will lead to more sustainable farming practices by providing information on pathogen populations and making more conserved or targeted applications of nematicides at the field or subfield level possible.  Benefits of this approach include increasing farm profitability by reducing inputs, reducing environmental impacts from nematicide application, and increasing positive relationships between local farmers and their surrounding communities.  Growers involved with the on-farm sampling, as well as those in attendance at the Fresh Market Potato Variety and Pest Management Meeting, expressed support for these benefits.

Important qualitative outcomes of the project included enhancement of my capacity to address the research needs of growers and effectiveness in communicating with growers through on farm research and visits.  These skills are invaluable in my developing career as a plant pathologist with an emphasis on research and cooperative extension. The NE SARE Graduate Student grant has provided financial support to enable me to accomplish key components of my doctoral thesis research and has also provided me a unique opportunity for networking with other researchers and growers involved in the NE SARE program, strengthening my network of professional contacts and collaborators.

Knowledge Gained:

Collaboration with growers for on-farm sampling increased the project director’s awareness that many growers are concerned with maintaining positive relationships with neighbors and communities surrounding their farms.  The growers view the application of nematicides as potentially detrimental to this relationship due to the pervasiveness of odors that accompany application and perceived health risks.  Thus, growers believe reducing or eliminating application where possible will lead to sustainable farming within their communities.     

Through the research activities conducted during this project, I was able to enhance my capabilities as a scientist by planning, conducting, and analyzing data from field sampling studies, a greenhouse pot trial, and laboratory experiments.  These skills have increased my capacity to meet the research needs of growers in my region.  Further, I was able to enhance my effectiveness in communicating the results and outcomes of the research by presenting to diverse audiences.  These qualitative skills are invaluable in my developing career as a plant pathologist, and bolstered my interest in conducting agricultural research and extension upon graduation.  Moreover, involvement with on-farm sampling and presenting at extension events has increased my network of growers.  

 

Assessment of Project Approach and Areas of Further Study:

Key to the success of this project was grower participation for on-farm sampling and discussions.  Three potato fields at two separate farms were intensively sampled at planting and at harvest, and the growers visited for consultations and updates throughout and beyond the project award period.  This collaboration resulted in generation of data under field conditions for establishing the quantitative relationship between initial nematode populations and crop loss at harvest. The collaboration also resulted in increased awareness of nematode issues within the sampled potato fields and both farmers involved expressed interest in continuing the collaboration for future studies. 

An additional grant was secured to repeat on-farm sampling experiments and repeat greenhouse work assessing the response of potato cultivars to M. hapla infestation.  This additional data will provide more robust estimates and refine the algorithm for prediction of damage and yield reductions.   

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.