Bryan J. Cassone Ph.D., Brandon University
SOYBEAN AND ITS ROOT/STEM DISEASES
Like all crops, soybean plays host to a variety of disease-causing microorganisms (called pathogens), which can decrease harvesting yields and impact seed quality. Although Manitoba has not yet experienced the same reductions in yields due to these pathogens as other North American soybean growing regions, it is imperative that we continue our efforts to enhance crop protection to prevent these diseases from becoming a serious issue in the near future. Timely and accurate detection of pathogens is one of the important parts of disease control and management. Of particular concern are soybean diseases caused by root/stem pathogens. Notable in the province are the fungal or oomycete diseases root/ stem rot (Phytophthora sojae), white mold (Sclerotinia sclerotiorum), pod/stem blight (Diaporthe phaseolorum) and fusarium root/stem rot (Fusarium oxysporum). These four pathogens are the focus of our diagnostic tool.
WHAT ARE THE ISSUES WITH CURRENT DISEASE DIAGNOSTICS?
Annual disease surveys are carried out in Manitoba, which mostly rely on visual inspection of symptom development. While quick, easy and cheap, this often results in incorrect diagnosis or inaccuracies among surveyors. For instance, stem rot caused by Fusarium oxysporum and Phytophthora sojae can be very challenging to distinguish visually, even by experienced surveyors (Figure 1). Our recent BU pilot study indicated that less than 25% of these visual identifications were accurate. Further, it is impossible to identify co-infections (more than one pathogen infecting a plant), which we have found to frequently occur in the field. In some cases, traditional microbiology techniques (e.g., morphology, motility, sporulation and staining) are incorporated into surveillance activities, though its application is limited by the inability to culture some pathogens and the time between tissue collection and diagnosis.
DEVELOPMENT OF THE DIAGNOSTIC TOOL
Field collection, pure culture isolations
Soybean fields throughout Manitoba were surveyed in 2020 and 2021 to collect symptomatic plant tissue samples for this study. The four targeted pathogenic microorganisms were isolated from stem and roots in the laboratory and grown in pure culture. Basically we grow them in petri dishes supplemented with nutrients that the microorganisms need to survive outside of their natural habitat of soybean.
Diagnostic tool development
A series of molecular biology steps and relevant troubleshooting was required for development of the disease diagnostic tool:
1. RNA extractions from each laboratory isolate and first-strand cDNA synthesis by reverse transcription. The cDNA is a reduced part of the pathogen genome – the “molecular fingerprint” – that allows us to much more accurately identify the pathogen than by just visualizing it.
2. Probe/primer set design and optimization. The cDNA is still many thousands of nucleotides (A, T, C and G’s), making it impractical to work with. Therefore, the next step is to find a smaller region of the cDNA of just hundred or so nucleotides that can 1) accurately detect only the targeted pathogen and no other organisms and 2) uniquely identify the targeted pathogen in a reaction consisting of several other soybean pathogens. Using computers and specialized software, we examine many different cDNA regions for specific properties and the exceedingly small number that pass this filter must then be empirically verified to (A) detect the pathogen in the pure cultures (this is done by conventional PCR) and (B) detect the pathogen but not the other pathogens in the same reaction (this is done by singleplex RT-PCR).
3. Standard curves for quantification of pathogen cDNA. The chosen cDNA region of each pathogen undergoes ten 2-fold serial dilutions to construct standard curves. These dilutions give us a frame of reference of how much pathogen is in each sample, which is needed in situations where we want to know how infected a given sample is with the pathogen.
4. Multiplex assays. Molecular methods are already available for some soybean pathogens that can successfully identify it in an infected sample. However, these are currently limited to testing only one pathogen at a time. If your visual diagnosis is incorrect, the test will not work. Further, if the sample is infected by more than one pathogen you will not be able to resolve this. Multiplex assays allow us to test for multiple pathogens in a given sample. This is like a paternity test for plants where you can test four potential fathers at the same time – with the unusual caveat that all four tested could be the father. We developed these assays to detect (and quantify) up to four stem/root pathogens simultaneously using an approach called RT-qPCR.
Validation with infected soybean
Most farmers are not concerned with identifying pathogens growing in petri dishes. The purpose of the diagnostic tool is to be able to determine which pathogen(s) is/are causing disease(s) in soybean fields. We therefore needed to ensure that the developed tool can also detect the targeted pathogens in infected soybean tissues. We first validated the diagnostic tool by artificially inoculating soybean with known stem/root pathogens in the laboratory and testing whether it could identify single and co-infections in the developing plants. Once confirmed, we validated our tool using symptomatic and asymptomatic soybean stems and roots collected directly from Manitoba soybean fields at various growth stages.
WHAT ARE THE BENEFITS FOR FARMERS?
Fungicide seed treatments are being used by farmers without knowledge of whether the targeted pathogens are present in their field. Not only is this a waste of money on an unnecessary fungicide, the process may create regulatory concerns that jeopardize the availability of the product for times when they are actually needed. Our diagnostic tool can simultaneously detect up to four of the most common and economically important fungal and oomycete pathogens in infected soybean stem/root tissues. Not only is this approach sensitive, accurate and cost-effective (estimated at less than $10 per sample), the time between sample collection and diagnoses can be as little as a few hours. The molecular infrastructure and expertise needed to run these assays are widely available in government and academia laboratories across Canada. Importantly, the tool can accurately detect the pathogens throughout the growing season, even prior to the development of disease symptoms. Overall, this diagnostic tool represents a substantial improvement over current methods to identify soybean diseases in Manitoba.
We thank the farmers that provided us access to their fields for sample collection. We are grateful to Manitoba Pulse & Soybean Growers, Western Grains Research Foundation and the Governments of Manitoba and Canada through the Canadian Agricultural Partnership for funding and supporting this project.