Journal of Agricultural Science and Technology A 3 (2013) 955-959 Earlier title: Journal of Agricultural Science and Technology, ISSN

1939-1250

DAVID

PUBLISHING

Tunisian Population of the Wheat Pathogen Mycosphaerella graminicola is Still Fully Sensitive to Strobilurin Fungicides
Mouna Naouari1, Ali Siah2, Mohamed Elgazzah1, Philippe Reignault3 and Patrice Halama2
1. Laboratory of Population Genetics and Biological Resources, El Manar University, Tunis 2092, Tunisia 2. Laboratory of Biotechnology and Management of Pathogens in Agriculture, GIS PhyNoPi, Higher Institute of Agriculture, Catholic University of Lille, Lille cedex F-59046, France 3. Plant-Fungus Interactions and Remediation Team, UCEIV Laboratory, GIS PhyNoPi, Littoral Cte dOpale University, Calais cedex F-62228, France Received: October 7, 2013 / Published: December 20, 2013. Abstract: Mycosphaerella graminicola (anamorph: Zymoseptoria tritici) is the causal agent of Septoria tritici blotch, the most frequently occurring disease on wheat crops worldwide. A set of 163 monoconidial isolates of this fungus were sampled in 2012 from five geographical locations of Tunisia (Bizerte, Bja, Kef, Jendouba and Siliana) in order to examine the status of strobilurin resistance of M. graminicola in this country. The resistance was assessed by using PCR-based mismatch mutation assay that determined the cytochrome b substitution G143A responsible for strobilurin resistance. All isolates were found sensitive since they possessed the wild-type allele G143 conferring sensitivity. This study confirms previous reports on the fungus in Tunisia and reveals that the Tunisian population of M. graminicola remains fully sensitive to strobilurin fungicides. An appropriate management of strobilurin applications in Tunisia is thereby recommended to prevent local development and widespread of resistance, as in Europe, where pathogen populations are fully resistant to strobilurins today. Key words: Mycosphaerella graminicola, G143A substitution, strobilurin resistance.

1. Introduction
Septoria tritici blotch caused by the fungal pathogen Mycosphaerella graminicola (anamorph: Zymoseptoria tritici) is one of the most frequently occurring diseases on wheat crops in Tunisia and worldwide. Severe disease epidemics can reduce yields by 35% to 50% [1]. Since host resistance against M. graminicola is not completely effective in most wheat cultivars, disease control relies mainly on the use of fungicides. Benzimidazoles and sterol-demethylation inhibitors have been the most widely used chemical classes to control M.
Corresponding author: Patrice Halama, professor, research fields: plant pathology, crop protection, fungal genetics and biology. E-mail: patrice.halama@isa-lille.fr.

graminicola for a long time since early and late 1970s, respectively. Since their introduction in 1996, strobilurin fungicides were intensively applied for the management of most agricultural fungal pathogens, including M. graminicola [2]. Strobilurins belong to the QoI (Quinone outside Inhibitor) fungicide class. They inhibit mitochondrial respiration in fungi by binding to the Quinone outside (Qo) binding site of the cytochrome bc1 enzyme complex, thereby blocking electron transfer and halting ATP synthesis [3]. The success of strobilurins is due to their higher efficacy and additional favorable effects on the plant physiology [4]. The intensive use of strobilurins in the field and a lack in the management of their applications led to a

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Tunisian Population of the Wheat Pathogen Mycosphaerella graminicola is Still Fully Sensitive to Strobilurin Fungicides Table 1 Location Bizerte Bja Jendouba Kef Siliana Total Origin and number of isolates used in this study. Number of isolates 50 64 18 14 17 163

rapid development of resistant isolates of M. graminicola in Europe. The first resistant isolate was detected in the United Kingdom in 2001 [4]. Then, their frequency rapidly increased in different regions over Europe [5-9]. As for most other fungi, the resistance is conferred by a point mutation in the mitochondrial cytochrome b gene, giving rise to a substitution from glycine to alanine at position 143 (G143A) of the cytochrome b enzyme [4]. This mutation prevents the binding of strobilurins to the Qo site, hence restoring the ability of the fungus to carry out mitochondrial respiration. Monitoring of fungicide resistance is of particular importance in pest management strategies to ensure effective disease control and is crucial for understanding the distribution, evolution and the impact of resistance in the field. Although strobilurin resistance of M. graminicola is currently widespread throughout Europe, recent investigations performed with 2008 and 2010 isolates revealed an absence of such a resistance in Tunisia [10, 11]. However, no information is available on resistance within the current fungal population in this country. The aim of the present work was to determine whether strobilurin resistance occurs in the Tunisian population of M. graminicola during the 2012 growing season.

2.2 DNA Extraction and Mismatch PCR Assay DNA extraction was carried out from two-week PDA-growing cultures according to Siah et al. [12]. Sensitivity or resistance to strobilurins among the isolates was determined mutation using on a part mismatch of the amplification assay

cytochrome b gene [8]. Primers were designed with a mismatch on the penultimate nucleotide of the 3 end of the forward primer, in which the ultimate nucleotide was at the point mutation position of the codon 143 of the cytochrome b gene. The primer set used to amplify a 639 bp DNA fragment in sensitive isolates was a sense primer StrobSNP2fwd NT (5-CTTATGGTCAAATGTCTTTATGATG-3,

404-428 of the cytochrome b gene) with a mismatch of T instead of G at nucleotide position 427 and an antisense primer StrobSNP1rvs NT (5-GGTGACTCAACGTGATAGC-3,

1024-1043). The primer set used to amplify a 302 bp DNA fragment in resistant isolates was an antisense primer StrobSNPrcF7 (5-CAATAAGTTAGTTATAACTGTTGCGG-3, NT 428-453 of the cytochrome b gene) with a mismatch of T instead of G at nucleotide 429 and a sense primer StrobSNPrcR1 (5-CTATGCATTATAACCCTAGCGT-3, NT 152173). This resulted in a single nucleotide mismatch on the sequence of the sensitive isolates and a double nucleotide mismatch on the sequence of the resistant isolates and vice versa for each primer set. PCR reactions as well as PCR thermal cycling conditions were performed according to Siah et al. [8]. PCR products were separated by electrophoresis in 1.5%

2. Materials and Methods

2.1 Fungal Sampling and Isolation A set of 163 monoconidial isolates of M. graminicola was collected in 2012 from infected plants of durum wheat (Triticum durum) in five distinct geographical locations of Tunisia (Bizerte, Bja, Jendouba, Kef and Siliana) (Table 1). The isolates were collected according to the protocol described by Siah et al. [12] from un-treated and naturally infected wheat plants. All isolates were collected from different plants. Once obtained, the isolates were grown on potatoes dextrose agar (PDA) medium and stored at -80 C for further analyses.

Tunisian Population of the Wheat Pathogen Mycosphaerella graminicola is Still Fully Sensitive to Strobilurin Fungicides

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agarose gels running at 100 V for 45 min. After ethidium bromide staining and visualizing under UV light, images were captured with a digital camera (Clara Vision, France) and scored manually.

3. Results and Discussion

A set of 163 M. graminicola isolates from five distinct geographical locations of Tunisia were assessed using a mismatch PCR assay for the presence/absence of the mitochondrial cytochrome b substitution G143A associated with strobilurin resistance. All isolates generated a single amplicon of 639 bp corresponding to the sensitive wild-type allele G143 (Fig. 1). No 302 bp fragment corresponding to the resistant allele A143 was found among isolates from all sampled locations (Figs. 1 and 2). The results indicate an absence or an occurrence at undetectable frequencies of strobilurin-resistant isolates in the current Tunisian population of M. graminicola, thereby agreeing with previous reports. Indeed, Boukef et al. [10] showed an absence of the resistant allele A143 in a sample of 357 M. graminicola isolates collected in Tunisia during the 2007/2008 growing season. Likewise, Stammler et al.

[11] analyzed phenotypically 52 Tunisian isolates sampled in 2010 and found that all isolates were highly sensitive to pyraclostrobin. The status of strobilurin resistance in Tunisia strongly differs from the present situation in Western Europe, where M. graminicola populations are currently fully resistant to strobilurins (A. Siah, unpublished data). This difference is likely due to lower fungicide exposure in Tunisia compared to Europe, where fungicide applications are more frequent. Recent investigations revealed that strobilurin resistance of M. graminicola is also widespread in Eastern Europe, especially in Czech Republic, where a rapid increase in resistance was observed during the period from 2005 to 2011 coinciding with the widespread application of strobilurins in this country [9]. Regarding the other parts of the word, Saidi et al. [13] assessed the G143A substitution in 89 M. graminicola isolates sampled in five provinces of Iran and concluded that all isolates were sensitive to strobilurins. But more recently, Estep et al. [14] analyzed two fungal collections from two locations of Western Oregon in USA and found frequencies of 9.5% (16/169) and 83.4% (146/175) of resistant isolates in the two collections, respectively,

Fig. 1 Mismatch PCR assay on a part of the cytochrome b gene to determine G143 or A143 allele in 18 M. gramincola isolates from Tunisia. Sensitive isolates carrying the G143 allele generated an amplicon of 639 bp. No resistant isolate generated an amplicon of 302 bp corresponding to the A143 allele. Lane M, molecular size standard XIV (100-bp ladder).

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Tunisian Population of the Wheat Pathogen Mycosphaerella graminicola is Still Fully Sensitive to Strobilurin Fungicides

Fig. 2 Map of Tunisia showing the frequency of strobilurin-sensitive and strobilurin-resistant M. graminicola isolates in the five locations sampled in 2012. Numbers in the circles indicate the number of isolates assessed. (A: Bizerte, B: Bja, C: Jendouba, D: Kef, E: Manouba).

hence reporting for the first time the occurrence of strobilurin-resistant genotypes of M. graminicola in America.

of the resistance, such as in Europe, where pathogen populations are nowadays fully resistant to strobilurins.

4. Conclusions
This study reveals that the Tunisian population of M. graminicola remains fully sensitive to strobilurin fungicides. This finding indicates that the Mediterranean separating Tunisia from Europe constitutes a geographical barrier that prevents the migration of resistance from Europe into Tunisia throughout wind-born ascospores. However, an appropriate management of strobilurin applications, for example their use in mixtures or alternation with other classes of fungicides, is recommended in Tunisia to prevent local development and widespread

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