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

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

Summary

The Northern root-knot nematode Meloidogyne hapla is an important soilborne pathogen of potatoes and other vegetables in New York. This pathogen reduces tuber yield and quality. Management decisions for nematodes need to be made prior to planting. Information regarding nematode population densities is critical in determining the implementation of management tactics, but is not readily available to most growers. Decisions made therefore are frequently risk averse, and assumptions of the ubiquitous presence of the nematode frequently leads to purblind application of chemical treatments. These chemistries may be environmentally and economically costly, raising the cost of production and have negative impacts on the environment.

The holistic objective of my project is to reduce unnecessary applications by increasing informed decision making through the development of a new quantitative risk algorithm for Meloidogyne hapla that will estimate tuber damage at harvest based upon initial pathogen population densities. This tool will aid in decision making by incorporating information on nematode population densities prior to planting, host plant susceptibility, and other edaphic factors to determine a risk category for the field, from which management decisions may be directed.  

Objectives/Performance Targets

A specific objective of this project is to assess the utility of the molecular quantification techniques to quantify inoculum of M. hapla across a range of soil types. Traditionally, nematode population densities are determined through identifying and counting nematodes through a microscope. Nematode identification on the basis of morphological characteristics is technically difficult, laborious, and may only provide genus level identification. I look to provide a step-change by quantifying populations through measuring DNA of the target organism rather than through morphological examination. This may offer a test that is more sensitive and specific to the pathogen species of interest. Whole genomic DNA from nematodes and other soilborne microbes will be extracted directly from soil and measured using qPCR with a unique the primer and probe set developed within this project.

To achieve this objective, it was necessary to determine a suitable method for isolating nematode DNA of sufficient quantity and quality needs from the soil prior to performing the qPCR tests. Soil provides a unique challenge 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 of limited utility for this study as they only process a maximum of 1 gram of soil. This volume of soil if far too small to draw 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 identified three unique methods for isolating bulk genomic DNA from soil that have been successfully employed by other researchers. The protocols selected were more economical than commercially available kits and were selected on the basis of their potential to scale up the protocol to process larger soil samples (~ 10 g). I designed and conducted an experiment to determine which of these protocols would be most appropriate for my project. These methods include a phenol extraction/skim milk purification¹ a phenol extraction/hydroxyapatide purification², and a proteinase K extraction/ polyvinylpolypyrrolidone purification³.

I tested each method by performing the steps as described in each of the original publications. Data collection and analysis consisted of measuring the quality (ng/mL) and quality (260/280 ratio; values of 1.8 or greater are considered high quality) of the resultant DNA. Each method was performed with six biological replicates using soil with a known infestation of M. hapla. A commercial kit (MoBio PowerSoil PowerLyser, 0.25 g) was included in the experiment as the “gold standard” of DNA quantity and quality produced for comparative purposes.

Each method was analyzed using the statistical least significant difference (LSD) test. The LSD is the value a protocol must differ from another in order to be deemed significantly different, in this case at the 5% level. Protocols were grouped into significance groups based upon the results of the LSD test, and different groups were denoted by letter. Further, the coefficient of variation (CV) was also calculated to measure the dispersion of results between the six replicates used for each method. The CV was reported as a percentage, with lower values indicating a lower dispersion and a greater level of repeatability in measurements within a protocol. Finally, the concordance correlation coefficient (CCC) was also reported. This statistic measures the agreement between results produced by each experimental protocol to the baseline results obtained with the commercial kit. Large CCC values of a protocol indicate results that are more similar to those obtained by the commercial kit.

A second objective of my project was to develop and test a quantitative PCR procedure for quantifying M. hapla populations in soil across a wide range of soil types. To accomplish this objective, this year I developed numerous sets of qPCR primer and probe sequences with the aim of developing a set that was specific to M. hapla. The primer and probe sets were designed to preferentially bind to two effector genes within M. hapla: chorismate mutase and 16D10. I tested the primer and probe sets against 13 different species of common plant parasitic nematodes using qualitative PCR to determine each set’s specificity.  A primer and probe set were considered specific if amplification was only observed in M. hapla DNA samples.

References

¹Kheyrodin, H. and Ghazvinian, K. 2015. Soil DNA isolation to use in polymerase chain reaction (PCR) amplification. Afric. J. of Ag. Res. 10(11):1158-1163.

²Purdy, K.J., Embley, T.M., Takii, S., and Nedwell, D.B. 1996. Rapid extraction of DNA and rRNA from sediments by novel hydroxyapatite spin-column method. Appl. And Envrio. Microbio. 62(10):3905-3907.  

³Porteous, L.A. and Armstrong, J.L. 1991. Recovery of bulk DNA from soil by a rapid, small scale extraction method. Current Microbio. 22:345-348.

Accomplishments/Milestones

The phenol extraction/skim milk purification method produced quantity and quality results most similar to those obtained by the MoBio PowerSoil PowerLyser commercial kit, which was used as the “gold standard” in comparing methods. However, the phenol extraction/hydroxyapatide purification method produced the optimal combination of DNA quantity (Table 1) and quality (Table 2). I am exploring further how to scale up the phenol extraction/hydroxyapatide purification protocol to process larger volumes of soil which will provide more biologically relevant conclusions. It is anticipated that this method will be used for processing field samples.

Table 1. The DNA isolation from soil methods tested produced a range of DNA concentrations, from 9.3 to 75.96 ng/uL. The Proteinase K extraction/polyvinylpolypyrrolidone purification methods produced the highest quantity of DNA, however, the phenol extraction/skim milk purification method produced results most similar to the positive control. The MoBio PowerSoil PowerLyser 0.25 g commercial kit was used as positive control and “gold standard” to which each method was compared. ^aCV, coefficient of variation. ^bCCC, concordance correlation coefficient.

Table 2. The DNA isolation from soil methods tested produced a range of DNA quality, from 1.39 to 1.88 260/280 ratios. The phenol extraction/skim milk purification method produced quality results most similar to the positive control (MoBio PowerSoil PowerLyser commercial kit). However, the phenol extraction/hydroxyapatide purification produced the highest quality DNA, above the quality produced with the positive control

 
In the development of a primer and probe for use in qPCR tests, I identified one set targeting the 16D10 effector gene that was highly sensitive and specific to M. hapla (Fig. 1), producing amplification only in M. hapla DNA samples. Further verification of amplification of the correct target was conducted by cloning and sequencing the amplicon. I have selected this set to use in further qPCR tests of field samples within this project.

 Figure 1. Results of primer and probe development revealed an optimal primer set that produced amplification only in M. hapla DNA samples. This set (designated Mha17) is show in the lower gel. Bands are visible only in lanes with M. hapla samples. The internal transcribed spacer (ITS, upper gel) was used as a positive PCR control.

• Figure 1

Impacts and Contributions/Outcomes

Management decisions regarding pesticide application for the northern root-knot nematode M. hapla often need to be made prior to planting. In the absence of data on inoculum densities, decisions are often risk averse, requiring pesticide application in the form of fumigant or non-fumigant nematicides. The frequency of unnecessary pesticide applications may be reduced by the development and adoption of molecular based quantification tools. Work conducted since receiving the Northeast SARE grant has consisted of selecting optimal whole DNA isolation methods and development of qPCR primer and probe sets for use in molecular quantification. This work is the foundation for the development of a molecular based quantification test for quantification of nematode populations to be used by growers in conjunction with extension agents and scientists. This molecular test will provide growers timely indication of nematode population densities, supporting informed management decisions.  Immediate future work consists of using selected qPCR primer and probe set and soil DNA isolation method in a greenhouse experiment to develop risk categories for crop loss or damage due to M. hapla.

Collaborators: