Phytopathologia Mediterranea Firenze University Press The international journal of the www.fupress.com/pm Mediterranean Phytopathological Union Research Papers Population structure of the faba bean blight pathogen Ascochyta fabae (teleomorph, Citation: Omri Ben Youssef N., Kerdraon L., Mieuzet L., Halila I., Didymella fabae) in Tunisia Jammezi N., Mbazia A., Kharrat M., Le May C. (2019) Population struc- ture of the faba bean blight pathogen Ascochyta fabae (teleomorph, Didy- Noura OMRI BEN YOUSSEF1,*, Lydie KERDRAON2, Lucie MIEUZET3, mella fabae) in Tunisia. Phytopatho- Imen HALILA1, Noura JAMMEZI1, Ahlem MBAZIA1, Mohamed KHAR- logia Mediterranea 58(1): 81-94. doi: RAT1, Christophe LE MAY3,4,* 10.14601/Phytopathol_Mediterr-23562 1 Laboratoire des Grandes Cultures, Institut National de la Recherche Agronomique de Accepted: October 25, 2018 Tunisie, Université de Carthage, Rue Hédi Karray, EL Manzah, 1004, Tunis, Tunisie 2 Published: May 15, 2019 INRA, UMR, UMR BIOGER, INRA-AgroParisTech, Avenue Lucien Brétignières BP 01 78850 Thiverval-Grignon, France Copyright: © 2019 Omri Ben Youssef 3 INRA, UMR 1349 IGEPP, Domaine de la Motte, 35653 Le Rheu, France N., Kerdraon L., Mieuzet L., Halila I., 4 Agrocampus Ouest, UP ESP, 65 rue de Saint Brieuc, 35043 Rennes, France Jammezi N., Mbazia A., Kharrat M., Le *Corresponding authors: [email protected]; [email protected] May C. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. Abstract. Ascochyta blight, caused by Ascochyta fabae (teleomorph: Didymella fabae) com/pm) and distributed under the has decreased faba bean production in Tunisia and worldwide. The teleomorph has terms of the Creative Commons Attri- recently been observed in Tunisia, raising new questions of how to control this major bution License, which permits unre- stricted use, distribution, and reproduc- disease. Isolates (317) of the pathogen were obtained between 2011 and 2013, from tion in any medium, provided the origi- four geographical regions in Tunisia (Beja, Bizerte, Jendouba and Tunis). The 240 iso- nal author and source are credited. lates obtained in 2012 were compared for mating type frequency and genetic variation by using ten polymorphic SSR markers. Of these isolates, MAT1-2 was more common Data Availability Statement: All rel- (2:1) in Tunisia than MAT1-1, but this ratio can change according to population. Low evant data are within the paper and its to high genetic variation was detected between locations and among pathogen popula- Supporting Information files. tions. Beja showed the greatest genotypic richness (R = 0.42), followed by Tunis (R = Competing Interests: The Author(s) 0.25), Bizerte (R = 0.13), and Jendouba (R = 0.11). Indices of association (IA) and Rd declare(s) no conflict of interest. were significantly different from 0 in all the populations, suggesting high multilocus linkage disequilibrium and confirming clonal populations. Population structure of the Editor: Jenny Davidson, South Austral- isolates was inferred using Bayesian analyses, Principal Component Analysis (PCA), ian Research and Development Insti- and Minimum Spanning Networks, which all revealed that the populations from each tute (SARDI), Adelaide, Australia. location were not distinct. Evaluating changes in seasonal genetic diversity showed low to high variances of FST values between the two cropping seasons in all regions. How- ever, the PCA analysis failed to separate the A. fabae isolates sampled during the two successive seasons into two groups, indicating that these populations did not consti- tute distinct genetic groups. These results suggest that gene flow was limited among populations, even those separated by short geographic distances. Future studies should enlarge the number of samples of representative populations, to overcome the limi- tations of a small sample size and to provide a more accurate assessment of A. fabae population structure. Keywords. Ascochyta blight, Ascochyta fabae, faba bean, genetic differentiation, popu- lation genetics. Phytopathologia Mediterranea 58(1): 81-94, 2019 ISSN 0031-9465 (print) | ISSN 1593-2095 (online) | DOI: 10.14601/Phytopathol_Mediterr-23562 82 N. Omri-Ben Youssef et alii INTRODUCTION populations, and to assess their ability to evolve (Mil- groom and Peever, 2003; de Meeûs et al., 2007). Sev- The faba bean disease, Ascochyta blight, caused by eral studies have evaluated population diversity of some Ascochyta fabae Speg. (anamorph) and its teleomorph Ascochyta spp. using different molecular tools, in par- Didymella fabae Jellis & Punith, is a widespread dis- ticular for A. rabiei (Bayraktar et al., 2007; Ali et al., ease, particularly in temperate regions, including Can- 2012; Atik et al., 2013) and A. pinodes (Le May et al., ada, Argentina, Europe, the Mediterranean region, the 2012; Padder et al., 2012; Laloi et al., 2016; Le May et Middle East, Australia, New Zealand, Japan, Korea and al., 2017). Studies conducted on populations of A. rabiei China (Maurin et al., 1990). This is also one of the most collected in several countries showed high diversity lev- important foliar diseases affecting faba beans in Tunisia els that were attributed to genetic flow provided by the (Kharrat et al., 2006). The fungus attacks all aerial parts exchange of seeds between farmers (Nourollahi et al., of host plant (leaves, stems, pods and seeds) causing 2011). For A. fabae populations, very few studies evalu- necrotic lesions, and leads to faba bean yield and quality ated the genetic diversity of this fungus. A study using damage. In Western Europe, depending on weather con- Random Amplified Polymorphic DNA (RAPD) markers, ditions during the cropping seasons, Ascochyta blight which included 36 isolates taken from different countries causes averages of 10 to 30% yield losses (Hanounik and (Tunisia, Morocco, Italy, France, Spain, Australia and Robertson, 1989). In Tunisia, yield losses are estimated Algeria), showed high genotypic diversity, with 26 dif- to 15%, and may reach 95% in disease favourable condi- ferent genotypes observed within these isolates (Rouaissi tions (Kharrat et al., 2006). et al., 2001). More recently, Ozkilink et al. (2011, 2015), Ascochyta fabae is an haploid heterothallic patho- using a set of 18 Simple Sequence Repeat (SSR) markers, gen that can survive during intercropping seasons on showed that A. fabae populations from Syria displayed infested seeds and plant debris. Seeds constitute the a high level of polymorphism. They showed high diver- major source of primary inoculum, and seed exchanges sity but no differentiation, and rejected a null hypothesis between and within countries are considered the main of random mating in the studied population, which is sources of introduction and mechanism of disease indicative of predominating asexual reproduction. expansion or dispersal (Kaiser et al. 1997). Ascochyta Ascochyta blight has decreased faba bean produc- fabae is maintained on crop stubble as asexual and tion in Tunisia, a country characterized by variable sexual forms for at least one season (Rubiales and Trap- weather conditions, diverse crop areas, various seed ori- ero-Casas, 2002; Omri Ben Youssef et al., 2012). Due to gins (produced by farmers or otherwise), and cultural the stage of maturation of pseudothecia, the release of practices. Since D. fabae has been observed recently in Tunisia (Omri Ben Youssef et al., 2012), it has become ascospores may overlap with the vegetative development especially important to study the genetic diversity of of faba bean (Rubiales and Trapero-Casas, 2002). the pathogen populations in the main faba bean grow- Although applying fungicides to control A. fabae ing areas affected by Ascochyta blight. The present study on seeds and faba bean plants gives satisfactory results, was undertaken to: (i) collect information about the plant resistance remains the best way to control this genetic diversity of A. fabae populations in the various disease. Considerable efforts have been made to iden- areas of faba bean production in Tunisia; (ii) assess the tify sources of resistance in faba bean germplasm, par- mating type distribution in this country using mating- ticularly in Syria, Egypt, Canada, Morocco, France and type (MAT) specific primers; and (iii) determine wheth- Tunisia (Hanounik and Robertson, 1989; Rashid et al. er the diversity of A. fabae populations evolves from one 1991; Maurin and Tivoli, 1992; Kharrat et al., 2006). Par- cropping season to another. tial host resistance has contributed to reducing chemi- cal intervention to control the disease (Avila et al., 2004; Kharrat et al., 2006). MATERIALS AND METHODS Pathogen variability represents a major challenge for the development and use of resistant cultivars. In gen- Fungal isolates eral, new virulent isolates of pathogens emerge through selection imposed by their hosts. These shifts in viru- A total of 317 A. fabae isolates were used in this lence necessitate the adoption of disease control strate- study to analyze genetic variability. All isolates were col- gies that target pathogen populations rather than indi- lected between 2011 and 2013, from several faba bean viduals (McDonald and Linde, 2002). Studying plant fields in four different geographical locations of Tunisia, pathogen diversity principally aims to identify which including Beja (three to nine fields, 122 isolates), Bizerte forces will heavily influence the evolution of pathogen (two to seven fields, 87 isolates), Jendouba (three to eight