A TaqMan real-time PCR assay for Rhizoctonia cerealis and its use in wheat and soil

Rhizoctonia cerealis causes sharp eyespot in cereals and the pathogen survives as mycelia or sclerotia in soil. Real-time Polymerase Chain Reaction (qPCR) assays based on TaqMan chemistry are highly suitable for use on DNA extracted from soil. We report here the first qPCR assay for R. cerealis using TaqMan primers and a probe based on a unique Sequence Characterised Amplified Region (SCAR). The assay is highly specific and did not amplify DNA from a range of other binucleate Rhizoctonia species or isolates of anastomosis groups of Rhizoctonia solani. The high sensitivity of the assay was demonstrated in soils using a bulk DNA extraction method where 200 μg sclerotia in 50 g of soil were detected. DNA of the pathogen could also be amplified from asymptomatic wheat plants. Using the assay on soil samples from fields under different crop rotations, R. cerealis was most frequently detected in soils where wheat was grown or soil under pasture. It was detected least frequently in fields where potatoes were grown. This study demonstrates that assays derived from SCAR sequences can produce specific and sensitive qPCR assays.


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PCR-based methods for identification of R. cerealis include conventional PCR assays designed from RAPD 4 fragments (Nicholson and Parry, 1996) and ITS sequences (Chen et al., 2005). Real-time PCR approaches until 5 now have been based on SYBR green chemistry with primers designed to either b-tubulin sequences (Guo et al., 6 2012) or unique sequences amplified with the microsatellite primer M13 (Hamada et al., 2012). However, 7 previous studies suggest SYBR green assays are strongly influenced by the presence of humic acids which are 8 present in DNA samples extracted from soil (Alaeddini, 2012). Since R. cerealis is a soil-borne pathogen, 9 alternative real-time PCR chemistry may be desirable for studies which require detection of the fungus in soil.
Real-time PCR assays based on TaqMan chemistry have been shown to be highly suitable with DNA isolated 11 from soil (Ophel-Keller et al., 2008). Therefore we report the development and validation of a sensitive and 12 specific real-time PCR assay based on TaqMan chemistry for R. cerealis, its use in plant material and on DNA 13 extracted from a range of UK field soils under different crops. By determining the relative levels of the fungus 14 in soil under different crops, the effectiveness of crop rotations in managing the levels of the pathogen in the soil 15 and providing inoculum in subsequent wheat crops can be determined.

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Isolates 20 All isolates used in the study are shown in Table 1. Isolates were obtained from existing culture collections at 21 Fera and the University of Nottingham. Additional isolates were kindly provided by Dr Marc Cubeta, University of North Carolina. Isolates were routinely maintained on potato dextrose agar (PDA) at 20 °C in the dark with 23 longer term storage on frozen barley grains (Sneh et al. 1991). DNA was extracted from 2-3 week old cultures 24 using a Wizard Magnetic DNA Purification System for Food (Promega UK, Southampton) in conjunction with a 25 Kingfisher ML magnetic particle processor (Thermo Scientific UK Ltd) following the manufacturers 26 recommended protocols.

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Assay design and real-time PCR initial denaturation at 95°C for 5 min, followed by 35 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 60 s, 5 and a final extension step at 72°C for 5 min. Template DNA used was from isolates RC1, RC2, RC3 and RC4. 6 PCR products were visualised by agarose gel electrophoresis using 2% gels containing 0·5 µg ml −1 ethidium 7 bromide in TAE buffer (40 mmol I -1 tris-acetate, 1 mmol l -1 EDTA, pH 8·0). A QIAquick PCR Purification Kit 8 (Qiagen) was used to purify samples for DNA sequencing which was undertaken by MWG biotech. Resulting 9 DNA sequences were aligned by using ClustalW in Mega5 (Tamara et al., 2011) and Primer Express 3.0 10 software was used (Life Technologies) to design specific TaqMan ® primers and a probe for R. cerealis.

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Real-time PCR (TaqMan®) was carried out in 96 well plates using the ABI Prism7900HT Sequence Detector  Determining assay sensitivity 27 A ten-fold dilution series of pure culture DNA from isolate RC1 was used to determine the technical sensitivity 28 of the assay. Sensitivity of the assay in soil was determined by spiking 50 g of sandy-loam soil (determined to isolate RC5), a series of 12 spiked samples were prepared. Sclerotia were prepared by growing the isolate on 1 PDA for four weeks in 90-mm petri-dishes and removing them with a scalpel. Sclerotia were then air-dried 2 overnight then macerated using a scalpel, the appropriate weight was added to each soil sample and then left 3 overnight prior to DNA extraction.

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Plant material displaying a range of symptoms from healthy to severe were prepared by inoculating 7 day old 6 seedlings of wheat (cv. Gladiator) with sand-maize meal inoculum of R. cerealis (isolate RC1) prepared as   (Table 2).

Results
Primers RC2F and RC2R amplified a 780 bp fragment for isolates RC1 to RC4. The sequence of each was 1 identical. A forward and reverse primer with probe were designed between nucleotides 186 and 260. Primer and 2 probe sequences for the assay are given in  The relationship between Ct and pure DNA concentration is shown in Figure 1. An R 2 value of over 0.99 was 12 observed and the reaction efficiency was determined to be 101%. The limit of detection was determined to be 5 13 fg although the detection was inconsistent at this level. A strong linear relationship (R 2 =0.92) was also observed  (Table 3). The highest incidence of R. cerealis was in soil where wheat was most recently 25 grown (72.2% in spring of 78.9% in Autumn) or under pasture in spring (71.4%). In addition to incidence, the 26 highest levels of R. cerealis were also detected in soil from wheat in spring (4782 pg DNA/g soil) and pasture 27 (1186.5 pg DNA/g soil). Incidence and mean levels of R. cerealis was lowest in soil samples from potato fields 28 (12% incidence and 9 pg/g level). It was possible to sample nine fields, of potatoes following wheat, both in

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In this study we describe a new qPCR assay based on TaqMan chemistry for the detection of the BNR fungus R. 6 cerealis, the causal agent of sharp eyespot in cereals. This assay, designed to a unique SCAR sequence, was 7 highly specific for R. cerealis. This was shown by testing the assay with DNA against a wide range of

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In addition to specificity, the developed assay was also highly sensitive. We were able to detect 200 µg of 16 sclerotia in 50 g of soil. This is similar to the previous study for the detection of R. solani AG3-PT in soil where 17 a single 200 µg sclerotia was detected in a 250 g bulk soil sample. The assay was also able to detect R. cerealis 18 in plants prior to symptom development (asymptomatic). Combined with a robust sampling methodology and an 19 accurate risk framework, the assay could be used to screen soils for the presence of the pathogen to inform 20 planting and crop rotation decisions.

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The sensitivity and specificity demonstrated with this assay demonstrates the versatility of using unique SCAR 23 fragments for developing real-time PCR assays. In contrast to assays designed to conserved genes such as rDNA 24 ITS and β-tubulin, assays based on unique sequences such as SCAR fragments ensure the assay designed is not 25 constrained to the location of sequence polymorphisms, likely resulting in more optimised primer design and 26 assays with enhanced performance. Here we demonstrate that a SCAR based R. cerealis assay had similar 27 sensitivity to a previously designed assay for R. solani AG3-PT (Woodhall et al., 2013) designed to ITS 28 sequences which are present in multiple copies in the fungal genome.
Using the assay to test 92 soil samples taken from fields grown under five different crops showed that R.
1 cerealis was detected in over half the fields tested. R. cerealis was found present in the majority of the wheat 2 and pasture fields tested. This can be expected since cereals are the main host of the pathogen and the role of a 3 previous cereal crop in building up R. cerealis inoculum levels in soil has been reported previously (Colbach et 4 al., 1997;Lemańczyk, 2012). The level of R. cerealis detected in soil was higher than described in previous 5 studies. Guo et al. (2012) determined the average levels in R. cerealis in four wheat fields ranged from 20.3 to 6 133.0 pg DNA per g of soil, whilst here we found the average level in wheat fields in the UK was higher with 7 levels of 629.6 pg DNA/g soil observed in spring and 135.82 pg DNA/g soil observed in autumn. Soils 8 originating from fields where potato was grown had the lowest levels of R. cerealis detected and the lowest 9 incidence of detection. Growing potatoes in the crop rotation may reduce the abundance of the pathogen in soil.

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In this study, six fields that tested positive for R. cerealis in spring were also sampled again, following potatoes,

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in Autumn. In five of the fields following potatoes no R. cerealis was detected in autumn, and in one instance where it was the levels were reduced considerably (by a factor of 33). R. cerealis has been shown to be capable 13 of causing infection on potatoes (Hollins et al., 1983) so this reduction in R. cerealis abundance may be due to        Disease rating