Real time PCR assays to detect and quantify the nematodes Pratylenchus vulnus and Mesocriconema xenoplax

Highlights

• We developed and tested two novel qPCR assays to quantify lesion and ring nematode.

• Primers did not cross react with tested species of other plant-parasitic nematodes.

• Both assays predicted nematode counts well in laboratory experiments.

• Both assays predicted nematode counts well in solutions extracted from field soil.

• Molecular diagnostics could efficiently quantify and identify pest nematodes.

Abstract

Economically damaging populations of lesion nematode and ring nematode are managed in tree crops largely through pre-plant chemical fumigation, the use of which is increasingly restricted due to human health and environmental concerns. Reducing the use of fumigants requires precise knowledge of pest nematodes’ density and distribution, however; extensive sampling is costly due to the time intensive process of nematode counting and identification. In this study, species specific primers were designed and real time PCR (qPCR) assays developed separately for both species of nematodes. The assays successfully detected each species and did not show significant amplification of non-target nematode groups. Both assays related well with microscopic counts of prepared solutions of nematodes, as well as solutions extracted from field samples. Such high-throughput molecular quantification could reduce diagnostic costs, allowing a more accurate picture of nematode populations in the field.

Introduction

Close to 80% of the world's almonds and 75% of walnuts are produced in the Central Valley of California (Beede 1998; California Walnut Board, 2019; Almond Board of California, 2017). Over 400,000 ha of almonds are produced in California with an estimated annual economic impact of 21.5 billion dollars (California Department of Food and Agriculture, 2018, Sumner et al., 2014). Although smaller by comparison, the walnut industry recently generated 1.4 billion dollars in annual revenue from 161,874 ha (California Department of Food and Agriculture, 2019). Two of the main nematodes of concern in these production systems are ring nematode, Mesocriconema xenoplax, and root lesion nematode, Pratylenchus vulnus (Micke, 1996; Beede 1998). While other species of Pratylenchus and Mesocriconema are also occasionally present in California almond and walnut orchards (Siddiqui et al., 1973), they are considered less of an economic concern. P. vulnus are migratory plant endoparasites, and produce black necrotic lesions throughout the cortex of infected roots (Jones et al., 2013). In contrast, M. xenoplax are migratory ectoparasites, living in the soil and feeding on root tips, reducing root mass by up to 85% (Micke, 1996). To controls these nematodes, growers use resistant rootstocks and chemical fumigation (UCIPM 2017), although annual fumigant applications have recently been restricted due to public health concerns (Marks, 2016).

To minimize fumigant use, growers need to know precisely the density and distribution of nematodes present. However, currently, it is difficult to estimate the extent of nematode problems because accurately representing their often-patchy distributions requires extensive soil sampling (Goodell and Ferris 1980). This leads directly into problems with nematode diagnostics. Decisions must be made about how many soil samples to take for a given area, with some recommendations citing extensive soil sampling in as small as 0.1 ha blocks to be sure that pest nematodes are not present and that fumigation treatments are unnecessary (Schneider and Hanson 2009). Since increasing the number of soil samples submitted to a laboratory for testing increases diagnostic fees, soil samples are often composited from as large an area as is practical. The benefits of compositing samples from a large area must be weighed against the variation potentially introduced by incorporating additional samples, which could affect reliability (Ferris et al., 1981).

The most labor and time intensive part of nematode quantification involves their extraction from soil, as well as identification and counting under the microscope (Ferris et al., 1981). Compounding the problem, the extraction efficiency of nematodes from soil is often low and counts can vary greatly between laboratories (Duncan and Phillips 2009). Once isolated, specialized training is additionally needed to correctly identify nematodes, making quantification a lengthy and expensive process, with costs ranging from $30-$125 per sample. Molecular methods of identification using real time PCR (qPCR) can overcome some of these drawbacks, and predict damage more accurately and consistently than traditional methods of quantification (Yan et al., 2013; Berry et al., 2007; Atkins et al., 2005), potentially at a lower cost. For example, while it can take up to 30 min to identify pest nematodes from a single sample under the microscope, 26–43 samples can be analyzed in 2 h using a qPCR approach, depending on the number of technical replicates. The reduced time required for identification (6–10 times less) could potentially make up for increased reagent costs (approximately $6 per sample).

Building on polymerase chain reaction (PCR), qPCR simultaneously quantifies and identifies nematode populations by comparing the intensity of the amplified signal to a standard curve calculated from known densities (Berry et al., 2007). Although few qPCR assays exist to quantify and detect M. xenoplax, several have been developed for Pratylenchus spp., but none for P. vulnus specifically. Yan et al. (2013) designed a qPCR assay that detected and quantified Pratylenchus neglectus from DNA extracts of 0.5 g soil, while Berry et al. (2008) developed qPCR methods for Pratylenchus zeae extracted DNA from nematode solutions which were enriched from larger 200 cm³ samples. For other nematodes groups, such as M. incognita, there has also been work on direct quantification of nematodes from dried, pulverized soil samples (Min et al., 2012), although it remains to be examined how quantification from small soil samples (<1g) interacts with field heterogeneity and the sample size limitations discussed above. qPCR methods can be quite sensitive. For example Sato et al. (2007) designed PCR primers for P. penetrans which were able to detect a single target nematode in over 800 non-target nematodes. Such methods have also occasionally been tested in the field with good relationships between microscopic counts and qPCR predictions for P. thornei (Yan et al., 2012). Despite these advances, nematodes are largely still quantified commercially using traditional microscopic counting by analytical labs, although notable exceptions have existed (Ophel-Keller et al., 2008).

Creating a qPCR diagnostic tool for P. vulnus and M. xenoplax could lead to new knowledge of their precise densities and distributions in orchards, allowing growers to make more informed management decisions. As a step towards this goal, the objectives of this study were: 1) to develop qPCR assays for identifying and quantifying P. vulnus and X. xenoplax and 2) to compare the method's performance to standard microscopic methods on field samples with native nematode populations.