Potato Progress

Volume 22 Number 4

2 June 2022 

‍Editor's Note: Potato Progress is meant to document the research work that the Northwest Potato Research Consortium (that is, the potato commissions of Washington, Oregon, and Idaho) supports. Some articles are more practical than others. This is intended since Potato Progress reflects the range of research we fund. Sometimes a problem can be addressed with practical short-term field research aimed at directly developing management practices. In other cases, more fundamental research is needed to address difficult problems or lay the groundwork for practical research. Potato Progress and the Consortium website are meant to convey information about all types of research funded by the Consortium, both practical and fundamental.

Isothermal amplification for potato disease diagnosis: 

what have we learned from detection of the powdery scab pathogen?

Kiwamu Tanaka, Washington State University, kiwamu.tanaka@wsu.edu 

Joseph DeShields, Oregon State University, joseph.deshields@oregonstate.edu 

Natalia Moroz, Washington State University, natalia.moroz@wsu.edu 

James Woodhall, University of Idaho, jwoodhall@uidaho.edu 

‍

What is isothermal amplification and what are its advantages?

Timely detection of plant pathogens enables a suitable, effective control strategy to be implemented. Many potato growers adjust their management strategies based on disease diagnosis. However, in some cases disease diagnosis comes too late to manage within a growing season. Rapid and accurate technologies are required for early pathogen detection.

‍Isothermal amplification is the continuous, exponential amplification of nucleic acid sequences (i.e., DNA or RNA) at a constant temperature using enzymes, usually strand displacing polymerases, rather than the temperature changes (called ‘thermal cycling’) used in PCR. These isothermal amplification technologies can overcome the disadvantages of PCR-based methods because they do not require thermal cycling (Lau and Botella, 2017). Isothermal amplification can be performed with less power consumption, making it compatible with hand-held equipment in the field. Another advantage of isothermal amplification is its speed and sensitivity due to continuous exponential amplification at a constant temperature. Therefore, isothermal amplification methods have gained popularity due to the potential to be used for on-site diagnosis (Mora-Romero et al., 2022), which is also known as point-of-care diagnosis. Among the many isothermal amplification technologies, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) have been developed for pathogen detection for many crops including potatoes (Table 1).  

‍Development of isothermal amplification methods for powdery scab pathogen and PMTV

Powdery scab is a soilborne disease caused by the protist Spongospora subterranea f. sp. subterranea (Sss) (Gau et al., 2013). The disease symptoms are scab, which is cosmetic damage to the tuber skin that significantly affects the potato’s market value, and root gall formation, which restricts root growth and the uptake of nutrients and water (Gilchrist et al., 2011). Symptoms on the tuber skin are sometimes confused with common scab caused by Streptomyces bacteria. Tuber lesions of powdery scab and root galls are filled with clusters of resting spores called cystosori in which primary zoospores are highly persistent and remain viable in soil for many years (Calvert, 1968). Notably, the spores are heat resistant and can survive passage through animal digestive tracts (Morse, 1912). Most importantly, Sss transmits potato mop-top virus (PMTV), which causes a significant reduction of plant growth and internal tuber necrosis (Jones and Harrison, 1969; Tenorio et al., 2006). It can also cause surface blemishs in certain varieties. Unfortunately, no varieties of potato are known to exhibit effective resistance to PMTV (Valkonen, 2015). Recent studies revealed a wide distribution of PMTV in the Nordic countries and the United States (Xu et al., 2004; Budziszewska et al., 2010; Crosslin, 2011). The increasing geographic distribution and prevalence of PMTV, combined with the lack of effective management strategies, has become a major economic concern for potato production in the United States. Therefore, it is critical for a grower to know if powdery scab pathogen is present in their fields so they may implement appropriate management strategies. 

‍The detection and quantification of Sss and PMTV in soil and tissue samples is important to study the epidemiology and control the diseases caused by those pathogens. Realtime or quantitative Polymerase Chain Reaction (qPCR) is a powerful technology that allows the highly sensitive and specific detection of low levels of DNA or RNA from the target pathogen. The PCR-based diagnostics requires relatively expensive thermocyclers and imaging systems. Its typical reaction time is several hours with data analysis. Recently, an on-site real-time qPCR method was developed to detect Sss and PMTV, in which a portable thermocycler was employed (Figure 1; DeShields et al., 2018). However, it still required an expensive device to perform thermocycling and quantitative detection. Therefore, we have been developing isothermal amplification methods for Sss and PMTV detections for potential use in on-site diagnosis.

‍We have developed RPA methods for the detection of Sss and PMTV. Both pathogens were collected originally from local potato farms in the Columbia Basin of Washington State. The result was obtained in less than 60 minutes with a conventional RPA (data not shown; see DeShields et al., 2019 for data in detail), while the clear result was obtained within ~15 minutes at earliest in case of real-time RPA (Figure 2). The data suggested that the RPA method is specific and sensitive for the detection of these pathogens, although its sensitivity was ~10 times lower than the contrastive real-time PCR method. The real-time RPA assay and the real-time one-step RT-RPA assay developed can be exemplary approaches for on-site molecular pathogen detection. 

‍LAMP methods have been developed for the detection of Sss and PMTV (Figure 3). The results were obtained in less than 15 min for both pathogen detections, in which an Alkaline polyethylene glycol reagent was used to expedite the DNA isolation procedure (Chomczynski and Rymaszewski, 2006). Using a portable device, such as the Genie II (Figure 3), up to 16 tests can be performed at a time in the field or laboratory.

‍Advatanges of isothermal amplication

• Speed: the results were obtained within ~15 min by isothermal amplification, whereas it took ~60 min by qPCR.
• Rapid nucleic acid purification: Typically isothermal techniques are more tolerant of substances that can inhibit nucleic acid amplifctaion. This can enable rapid nucleic acid extraction methods to be used.
• Portability: Since thermal cycling is not required, smaller, less expensive equipment can be used making on-site testing more economically viable.

‍Disadvatanges of isothermal amplification

• qPCR is a gold standard method: it is an established method in many diagnostic labs and many technicians are familiar with the method. There is an abundance of validated real-time PCR assays available. In contrast, only few reagents and kits are available for isothermal amplification.
• Primer design can be more complicated: For example, LAMP requires four to six primers rather than the two needed for PCR, which sometimes makes primer design more limiting.
• Lack of sensitivity: qPCR is typically more sensitive than isothermal methods, especially when coupled with a nucleic acid extraction methodology that recovers high levels of DNA or RNA.
• Less robust on soil samples than qPCR: Although less robust with crude samples origanting from plant, qPCR can typically tolerate humic acid inhibtion associated with soil samples greater than an isothermal method.
• Dymamic range: qPCR typically has greater dynamic range than isothermal methods. Dynamic range is essentially the range of detection from low to high. In addition qPCR can allow greater resolution in quantification compared to some isolthermal methods.

‍Summary points – what have we learned during developing isothermal amplification methods?

• Speed: the results were obtained within ~15 min by isothermal amplification, whereas it took ~60 min by PCR.
• Sensitivity: the isothermal amplification was ~10 times less sensitive than qPCR in detecting the powdery scab pathogen in symtomatic tissue and soil samples.
• Nucleotide purification vs crude extract: Reportedly, the isothermal amplification is recommended as an ideal method to amplify DNA/RNA fragments even in crude extracts since it is tolerant to interfering compounds (e.g., PCR inhibitors) present in the samples. Indeed, several successful cases have been reported using crude samples from infected plant materials (Silva et al., 2015; Rojas et al., 2017; Qian et al., 2018; Silva et al., 2018). However, success has not been reported in any test of crude soil extracts. We also attempted using the RPA system with a crude soil extract but failed to amplify any nucleotide molecules (data not shown). For soil samples, a DNA/RNA purification step is required for isothermal amplification reaction. 

‍When is isothermal amplification appropriate?

A balance of the factors listed below is needed to decide if isothermal amplification would be a better approach than PCR and other methods.

• Importance of speed in detecting the pathogen of interest.
• Necessity of an extremely sensitive test.
• Need to perform the test on-site.
• Low sample numbers.
• For demonstration purposes to the grower or consulatant.
• As a confirmation test. For example an isothermal test designed to a different gene can be use as an effective confirmation test since it is a different sequence as well as different assay chemistry.

‍Checklist to decide if isothermal amplification is right for your lab

If answers to the questions below were mostly “yes”, isothermal amplification is a more appropriate technique.

• Do you need results within an hour?
• Do you need to perform on-site testing?
• Are you doing a small scale of testing, and not doing large-batch amplification?
• Are you doing a regular diagnostic test that would use the same primers?
• Are you working with small sample amounts needing high sensitivity?
• Do you have equipment that will conduct isothermal amplification?
• Do you have the budget for the components listed in your intended protocol?

‍Future challenges of molecular detections of phytopathogens

Molecular-based technologies have improved diagnostics by identifying causal pathogens in a more sensitive and quantitative manner and have contributed to an increase in diagnostic confidence in many ways. Isothermal amplification enables a quick and easy test for the presence of pathogens in different tissues and soil samples. However, there are a few remaining challenges. There are times when pathogens are present (e.g., detected by highly sensitive molecular methods) without causing disease. To tackle these challenges, we need to characterize the relationship between the inoculum level in the field and the prevalence of disease outbreaks. In addition, there are times when multiple pathogens are present while only one pathogen is primarily responsible for the observed disease symptoms. Studies about pathobiome and pathogen-pathogen interactions might provide some insight into the disease signatures during different potato growth stages. In this case, a high throughput sequencing approach would be useful for future research. Isothermal methods are therefore just another useful tool in the diagnosticians’ toolbox. They won’t completely replace qPCR or isolation for fungi and bacteria but provide another reliable option for diagnosticians to use in certain situations to ensure a correct and timely diagnosis.

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