A New Approach: Determining cyt b G143A Allele Frequency in Zymoseptoria tritici by Digital Droplet PCR.

Greta Battistini,Marina Collina,Katia Gazzetti

doi:10.3390/biology11020240

Abstract

Simple SummaryDroplet digital polymerase chain reaction (ddPCR) is an innovative technique for quantifying a target DNA in a diluted target sample, based on the partition of PCR reaction in a large number of sub-reactions (droplets). In recent years there has been an increase in digital PCR (dPCR) utilization in several different fields, and the application of ddPCR in fungicide resistance studies is very recent. Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB), one of the most devasting foliar diseases of wheat grown in temperate climates. STB control relies mostly on fungicide applications and mutations conferring fungicides resistances are concerning phenomena. G143A substitution in fungal cytochrome bc1 confers resistance toward Quinone outside Inhibitor (QoIs) fungicides. In Italy, QoIs are currently sprayed in STB control programs. To the best of our knowledge, we have developed the first ddPCR assay for G143 and A143 alleles detection in samples of gDNA from Z. tritici monocondial cultures. We have also investigated G143 and A143 alleles frequency in Italian Z. tritici populations representative of different fungicide management strategies. The detection of very low G143A substitution percentages in Z. tritici populations is essential for monitoring the emergence of QoIs resistance in field and effectively control STB.Z. tritici first appeared in Italy later than in northern-central European countries. QoIs fungicides currently play a role in STB control, used in combination with Demethylation Inhibitors (DMIs) or Succinate dehydrogenase Inhibitors (SDHIs). In this study, we set up a fast, sensitive, and accurate ddPCR protocol in order to investigate the presence and frequency of G143A substitution, causing a reduction in strobilurins’ efficacy in Z. tritici. The best PCR conditions for the clear separation of positive and negative droplets were identified. The lowest wild-type and resistant alleles frequencies were accurately determined on samples consisting of mixed DNAs from monoconidial cultures of Z. tritici and were expressed as fractional abundance. The protocol was tested by determining the copy number and frequency of alleles on gDNA purified in three Italian Z. tritici field populations representative of different fungicide management strategies. For the first time, the determination of allele concentration and the frequency of a mutation involved in Z. tritici fungicide resistance was carried out by employing digital PCR. This new approach provides a diagnostic tool that is rapid and able to detect very low G143A substitution percentages, which is very useful for fungicide resistance detection at early stages, thus, informing field management strategies for contrasting STB disease.

Highlights

Wheat (Triticum genus) is an important crop in northern‐central Europe and in Mediterranean countries, such as Italy, especially Triticum durum cultivars
To the best of our knowledge, in this study we present the development of the first ddPCR protocol for detecting and quantifying single nucleotide polymorphism, which confer fungicide resistance on Z. tritici
We investigated the presence and frequency of G143A substitution, which causes a reduction in Z. tritici sensitivity to Quinone outside Inhibitors (QoIs), in cultures collected from fields of durum wheat that were treated with different fungicide spray programs

Summary

Introduction

Wheat (Triticum genus) is an important crop in northern‐central Europe and in Mediterranean countries, such as Italy, especially Triticum durum cultivars. Tritici Blotch (STB), caused by Zymoseptoria tritici, can produce heavy yield losses every year when not properly controlled. In Italy the severity of STB infection, on durum wheat varieties, showed an exponential growth trend in the years from 2007 to 2010, reaching a peak in 2013 and a further increase in 2016 [1]. In the plains area of Bologna (Emi‐ lia‐Romagna, a geo‐political region in northern Italy), the infection caused yield losses of up to 30% in years with high inoculum pressure [2]. Genetic resistance against STB is absent or only partial and, disease control relies mostly on fungicide ap‐ plications. Demethylation Inhibitors (DMIs), Quinone outside Inhibitors (QoIs), and Succinate dehydrogenase Inhibitors (SDHIs) have been introduced and multisite fungi‐ cides are routinely used to control Z. tritici [3]

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Discussion

Conclusion