Title: Quantification of soilborne pathogens of potato using real-time PCR

Transcription

1 1 Title: Quantification of soilborne pathogens of potato using real-time PCR Principle Investigator: Neil C. Gudmestad, Department of Plant Pathology, North Dakota State University, Fargo, ND (O); (F) Submitted to Minnesota Area II Potato Growers and NPPGA Executive Summary: Soilborne diseases of potato are generally regarded as the one of the most serious economic constraints facing the potato industry when disease losses are coupled with the cost of control. The principle pathogens involved in early dying are Verticillium dahliae and Colletotrichum coccodes. C. coccodes also causes black dot and is a major tuber blemish disease of potato in this region. Another important soilborne disease emerging in this region is powdery scab, caused by Spongospora subterranea. This pathogen is also the vector of potato mop top virus, an important tuber necrosis virus recently detected in North Dakota. Methods to determine the presence and concentration of these important plant pathogens are costly, time-consuming, and in the case of powdery scab, nonexistent. The development of a multiplex real-time PCR method capable of detecting and quantifying soil inocula of these three soilborne pathogens of potato would assist growers in making management decisions. Rationale: A number of important soilborne pathogens affect potato development and tuber quality. These diseases include powdery scab, caused by Spongospora subterranea, Verticillium wilt, caused by Verticillium dahliae, and black dot, caused by Colletotrichum coccodes. Powdery scab can affect crop development by infecting roots and negatively impacting nutrient and water uptake but this disease also affects tuber quality. The powdery scab pathogen is also the vector of the potato mop top virus. V. dahliae and C. coccodes affect crop development and are components of the early dying complex, although the former is generally acknowledged as the primary pathogen (Davis and Johnson, 2001; Johnson, 1994; Tsror et al., 1999). Vegetative group 4 is the most important group of V. dahliae affecting potato. C. coccodes also affects tuber quality, particularly as it affects the fresh market and the chip processing sectors of the potato industry (Read and Hide, 1988). Disease management of these diseases usually involved the implementation of various agrochemicals, which are expensive and in some cases, such as with metam sodium used to control Verticillium wilt, can potentially harm the environment. Diseases such as powdery scab cannot be controlled chemically leaving growers with the option of avoiding the disease by not planting a field or using less susceptible cultivars. Soil population levels of a pathogen usually impacts disease development and severity. In the Midwestern US, soil populations of V. dahliae >8 microsclerotia/g of soil are generally regarded as economically damaging. Preliminary studies on C. coccodes suggest soil populations >70 microsclerotia are yield limiting (Gudmestad et al., 2005). Soil population studies on these two pathogens were performed using classical soil dilution and culture plating in the laboratory. These types of studies are very difficult or

2 2 impossible to do with pathogens such as S. subterranea, a pathogen that cannot be cultured. Additional studies on the relationships between populations of soilborne pathogens and yield and quality losses would be aided by methods of detection and quantification that are precise, rapid and relatively inexpensive. Quantification of soilborne pathogen inocula has been recently facilitated by the implementation of polymerase chain reaction (PCR) technology, either in classical or in real-time format (Cullen, et al., 2002; Gudmestad, et al., 2007; Qu, et al., 2006). Realtime PCR has proven to be useful in quantifying C. coccodes resulting in a relationship being detected between soil population of the pathogen and the number of potato crops in a given field (Figure 1). This work was funded by MN Area II in Similar studies have been performed using S. subterranea (Qu et al., 2006). The goal of the research proposed here is to develop a multiplex real-time PCR method that will allow quantification of several soilborne pathogens simultaneously within the same sample. The target pathogens include V. dahliae, C. coccodes, and S. subterranea. The development of the real-time PCR assay for C. coccodes, and S. subterranea is complete. The research proposed here will concentrate on improving the PCR assay for V. dahliae and the development of the multiplex so that one soil sample can be tested for all three pathogens simultaneously. Studies on soil sampling and soil processing will also be performed to optimize DNA extraction for the PCR detection protocol. Research Objectives: 1) Develop sensitive and genomic primers for V. dahliae that can be used in realtime PCR to detect this pathogen in soil. 2) Develop a multiplex real-time PCR method for the detection and quantification of V. dahliae, C. coccodes, and S. subterranea in a single reaction. Research Plan: The real-time PCR method for the black dot pathogen, C. coccodes, has been successfully developed by our laboratory with funding from MN Area II in 2006 and 2007 and was used on a trial basis during the last two growing seasons (see MN Area II research report submitted in November, 2006 & 2007). A primer for the detection of S. subterranea in real-time PCR is now available and had been used to quantify this pathogen in 186 commercial and seed production fields. The only remaining pathogen remaining for the multiplex real-time PCR system is V. dahliae. The entire genome for V. dahliae has now been successfully sequenced which provides this project with an excellent opportunity to screen the genome and to evaluate a very large number of primer candidates.

3 3 A real-time PCR assay for V. dahliae will be developed by analyzing DNA sequences of V. dahliae with Primer Express Software. The species specificity of each real-time assay will be confirmed by amplifying target DNA and non-target DNAs including the three other fungi and purified potato DNA. Real-time PCR reactions and thermocycling conditions will be as described by Vandemark et al. (2000). In order to develop multiplex real-time PCR assays that can detect all possible combinations of the four pathogens (V. dahliae x C. coccodes, V. dahliae x S. subterranea, C. coccodes x S. subterranea), two Taqman probes will be synthesized for each pathogen, with one being labeled at the 5' terminus with the fluorochrome 6- carboxyfluorescein (FAM), and the other labeled at the 5' terminus with the fluorochrome VIC (Applied Biosystems). Initial real-time PCR assays will include purified DNA (25 ng) of each pathogen in all six pair-wise combinations. Primer and probe limiting experiments will be performed according to manufacturer s recommendations (Applied Biosystems) to determine the minimum, most cost effective amount of primers and Taqman probe that can be used in multiplex reactions. After verifying the sensitivity and specificity of the multiplex real-time PCR assays with purified pathogen DNAs, soil will be collected from potato fields. Mycelia of V. dahliae and C. coccodes, along with spore balls of S. subterannea, will be added in known quantities to the soil samples. DNA will be extracted from 5 g soil samples with the MegaPrep DNA extraction kit (Mo Bio Inc.) and multiplex real-time PCR will be performed using 10 μl of the soil DNA extract in a 50 μl reaction volume. The amount of each respective pathogen detected in the soil sample will be determined based on standard curves using purified pathogen DNA as template. Serial dilutions of the DNA extracts from pathogen infested soil will be made and examined by real-time PCR to determine threshold levels for reliable detection of each pathogen. DNA extracted from the original soil sample prior to infestation by the four respective pathogens will also be amplified by real-time PCR to examine baseline levels of detection from non-infested soil. Results: The three PCR primers for V. dahliae, C. coccodes, and S. subterannea have been successfully multiplexed so that the relative quantity of each pathogen can be determined with one reaction (Figure 1). However, we still have some optimization to do to make this test consistent from soil type to soil type. Nonetheless, the progress to date has been positive. We have performed a number of beta tests of using the soil PCR to detect and quantify the pathogens that cause Verticillium wilt, black dot and powdery scab (Table 2). Using these data, a number of potato growers have been able to avoid planting red-skinned cultivars, which are extremely susceptible to powdery scab, into fields with high levels of powdery scab. We have optimized the relationship between PCR and the plate counts of microsclerotia of V. dahliae which is now very high (Figure 2). We feel this will dramatically improve the results we will obtain in determining soil populations of this pathogen.

4 4 Literature Cited: Cullen DW, Lees AK, Toth IK, and Duncan JM Plant Pathol. 51: Davis, JR and Johnson, DA Black dot. In Compendium of Potato Diseases, 2 nd edition. American Phytopathology Press, St. Paul, MN. 106pp. Dobinson KF, Harrington MA, Omer M, and Rowe RC Plant Dis. 84: Gudmestad NC, Pasche JS, and Taylor RJ Infection frequency of Colletotrichum coccodes during the growing season. Proceedings of the 16 th Triennial European Association forpotato Research Conference p Gudmestad, NC, Taylor, RJ, and Pasche, JS Austral. Plant Pathol. 36: Johnson, DA Plant Dis. 78: Lees, AK, Cullen, DW, Sullivan, L, and Nicolson, MJ Plant Pathol. 51: Qu, X, Kavanagh, JA, Egan, D, and Christ, B Am. J. Potato Res. 83: Read, P.J. and Hide, G.A Potato Res. 31: Tsror (Lahkim) L., Erlich, O. and Hazanovsky, M Plant Dis. 83: Vandemark, G. J., B. M. Barker and M. A. Gritsenko Phytopathology 92:

5 5 Table 1. Frequency of real-time PCR detection of Spongospora subterranea, the causal agent of Powdery Scab, in soil samples collected in North Dakota and Minnesota in Farm # Samples # Positive Samples % Positive Samples ND Farm A % ND Farm B % ND Farm C % ND Farm E % MN Farm A % Table 2. Frequency of real-time PCR detection of Colletotrichum coccodes, the causal agent of Black Dot, in soil samples collected in North Dakota and Wisconsin in Farm # Samples # Positive Samples % Positive Samples WI Farm % ND Farm E % Table 3. Frequency of real-time PCR detection of Verticillium dahliae, the causal agent of Verticillium Wilt, in soil samples collected in North Dakota, Minnesota and Wisconsin in Farm # Samples # Positive Samples % Positive Samples WI Farm % MN Farm A % MN Farm B % MN Farm C % ND Farm D %

6 Black Dot Verticillium Powdery Scab Fluorescence (dr) Cycles Figure 1. Flourescence emission (dr) in a real-time PCR assay for the quantification of Colletotrichum coccodes (Black Dot), Verticillium dahlliae (Verticillium Wilt) and Spongospora subterranea (Powdery Scab) from field soil R² = Threshold cycle (Ct) Verticillium ppg Figure 2. Relationship between real-time PCR crossing threshold (Ct) and number of Verticillium dahliae propagules per gram (ppg) in sterile soil infested with microscerotia.