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Journal of (2012), 94 (1), 99-108 Edizioni ETS Pisa, 2012 99

POPULATION STRUCTURE AND MATING TYPE DISTRIBUTION OF THE BLIGHT PATHOGEN RABIEI FROM PAKISTAN AND THE UNITED STATES

H. Ali1, S.S. Alam1, R.N. Attanayake2, M. Rahman3 and W. Chen2

1 Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan 2 USDA-ARS, Washington State University, Pullman, WA, USA 3 National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan

SUMMARY INTRODUCTION

Ascochyta blight caused by the Ascochyta ra- Chickpea ( arietinum), a cool season grain biei (AR) depresses chickpea production in Pakistan grown on a surface of 11 million ha with world- and worldwide. Thirty two AR isolates representing six wide production of about 9 million tons (FAOSTAT, geographical regions of Pakistan were compared with a 2009), is cultivated in more than 45 countries through- US-AR population for mating type frequency and genet- out the world including Pakistan where it is grown ic variation. Mating type results showed that the Pak- mostly in rainfed areas and on marginal lands in the istani AR (PAR) population had an apparent skewed (3 province of Punjab. Its yield in Pakistan is about 583 kg Mat1-2: 1 Mat1-1) distribution, although Chi-square ha-1 (GOP, 2009), much lower than its potential yield as tests showed non-significant deviation from equal distri- its production is under the constant threat of diseases bution due to small sample sizes. The US population and pests (Akhtar et al., 2008, 2009, 2011). Among fun- showed a 1:1 distribution of the two mating types. The gal diseases, wilt and blight are the two most important uneven distribution of mating types indicates that sexu- limiting factors to chickpea productivity. al reproduction among the PAR is rare due to either un- Chickpea blight is caused by Ascochyta rabiei (Pass.) availability of both mating types or lack of conducive Lab. (AR), whose perfect stage [telemorph: Didymella ra- environment, but statistical analysis showed that pan- biei (Kovachevski) von Arx] was discovered by Ko- mixia is there reflecting past recombinational events. vachevski in 1936 on overwintering chickpea debris in Genetic variation at six microsatellite loci was assessed southern Bulgaria (Kaiser, 1997). AR has been reported and each isolate was assigned to a microsatellite haplo- from 29 countries including the Middle East and the type. Population structure of the isolates was inferred Mediterranean and is usually associated with severe re- using Bayesian analyses implemented in the structure duction in chickpea yield and quality, especially under software which differentiated isolates into three distinct cool and wet weather conditions (Kaiser, 1997; Singh and clusters, two clusters of PAR and one of the US isolates. Reddy, 1996). It attacks all of the above-ground parts of However, few isolates from the US shared the same ge- the plant including stems, leaflets, pods and seeds, caus- netic background with one cluster of the PAR isolates, ing necrotic lesions that are circular on leaves and pods providing a link of inter-continental migration of the and elongate on petioles and stems. The disease may pathogen. Additionally, the two clusters of PAR-isolates cause a total yield loss if the environmental conditions are are not strictly associated with geographic locations in favourable to both the crop and the pathogen (>350 mm Pakistan, suggesting frequent gene flow of AR among annual rainfall, 20-22°C) (Nene and Reddy, 1987; different locations. Future studies should extend the Jimenez-Diaz et al., 1993; Acikgoz et al., 1994). sampling of representative populations to overcome the AR survives in plant debris, soil and infected seeds limitations of the small sample size for more accurate as- and it reproduces asexually, through the production of sessment of population structure. conidia in flask-shaped pycnidia, and sexually, through the production of from pseudothecia. As- Key words: mating type, chickpea, Ascochyta rabiei, cospores and conidia spread through wind and rain simple sequence repeats, genetic diversity. splashes, travelling distances up to hundreds of meters (Rhaiem et al., 2007). AR is heterothallic with two mat- ing types encoded by alternate alleles at a single mating type (MAT) locus. The MAT specific locus is governed by a single gene and its idiomorphs exist in the forms MAT1-1 and MAT1-2 (Turgeon and Yoder, 2000). The Corresponding author: H. Ali advantage of sexual reproduction is the adaptation to Fax: +92.419201776 E-mail: [email protected] new environment through recombination of genetic ma- 010_JPP834RP_Ali_99 19-03-2012 16:51 Pagina 100

100 Ascochyta rabies population structure in Pakistan and the US Journal of Plant Pathology (2012), 94 (1), 99-108

terial between the two mating strains. It also helps over- of the fact that less virulent pathotypes (pathotype I and coming the selection pressure imposed by the introduc- II) prevail there (Chen et al., 2004) as compared to Pak- tion of resistant host genotypes and fungicides. Deter- istan (Jamil et al., 2000; H. Ali, personal communication). mining the frequency of mating types in a fungal popu- The present study was undertaken to: (i) generate lation may help to infer the frequency and the relative knowledge about the genetic diversity of PAR isolates in importance of the sexual stage in the production region comparison with the US isolates; (ii) assess the mating (Cherif et al., 2006). Both AR mating types have been type distribution in Pakistan using MAT specific primers; reported in at least 15 countries including Tunisia, (iii) determine the AR reproductive mode in Pakistan. Turkey, USA, Canada and Spain (Rhaiem et al., 2007). Initially, mating types were identified by crossing iso- lates of unknown mating type with known tester strains MATERIAL AND METHODS (Wilson and Kaiser, 1995; Kaiser and Kusmenoglu, 1997), a time-consuming and laborious method. A spe- Fungal isolates. A total of 71 AR isolates were used cific-PCR assay has more recently been developed by in this study to analyze mating types and genetic vari- Barve et al. (2003), using multiplex PCR specific for ability. Thirtyfour isolates were collected during 2000- each idiomorph at the MAT locus. 2006 from six different geographical locations of Pak- Knowledge of the genetic structure of the fungal istan, namely Chakwal, Islamabad (northern Punjab), population is useful for predicting the genetic variation, Thal (central Punjab), Swabi, Karak and Attock (Khy- as the level of variability reflects the genetic evolution in ber Pakhtunkhwa), whereas 37 isolates were collected the fungal population. Genetic diversity of AR in differ- from one field in North Dakota, USA (Table 1). ent regions of the world has been studied using differ- ent DNA based approaches like restriction fragment DNA extraction. The isolates were purified using length polymorphism (RFLP), random amplified poly- single spore technique. Conidial suspensions of each morphic DNA (RAPD) and simple sequence repeats isolate were spread on potato dextrose agar with lactic (SSRs) markers (Geistlinger et al., 1997; Udupa and acid (APDA). After two or three days, single germinat- Weigand, 1998; Geistlinger et al., 2000; Santra et al., ing conidia were picked up, transferred and cultivated 2001; Vail and Banniza, 2009; Varshney et al., 2009; on PDA. DNA was extracted from fungal mycelium Nourollahi et al., 2010). Only a single study has been and isolated using FastDNA Kits (MP Biomedicals, conducted on the diversity and mating types of Pak- USA) according to manufacturer’s instructions. DNA istani AR (PAR) isolates using RAPD marker (Jamil et concentration was checked on Nano drop (USA). DNA al., 2000, 2010). Since RAPDs are dominant markers was diluted in sterile deionized distilled water to 10 known to be poorly reproducible (Jones et al., 1997), ng/µl before use in PCR. we have used the more robust SSR markers to detect ge- netic variation in PAR isolates collected from different Mating type assay. Mating types of all the 71 AR iso- geographical locations of Pakistan. lates (34 from Pakistan and 37 from USA) were deter- Pakistan has faced AR epidemics from 1928 to 1982 mined using the multiplex MAT-specific PCR assay which resulted in very heavy economic losses to farmers (Barve et al., 2003). Primer combinations of SP21, Tail5 (Kauser, 1965). Therefore, Ascochyta blight is a constant and Com1 were used in a single PCR carried out in 25 threat for Pakistani agriculture for the cold and humid µl vol containing 10 ng of genomic DNA, 1X PCR

climate together provide congenial conditions to its at- buffer (containing 1.5 mM MgCl2), 0.2 mM dNTPs, 1 tacks. In the US, the disease causes severe losses in spite unit Taq DNA polymerase (Promega, USA) and 10-20

Table 1. Isolates of Ascochyta rabiei collected from different areas of Pakistan and the US and their mating types as determined by PCR.

Province /State Geographic region Isolates (No.) Mat 1-1 Mat1-2 X2 P value

Northern Punjab Islamabad 4 1 3 1.000 0.317 Chakwal 18 5 13 3.556 0.059 Khyber Pakhtunkhwa Swabi 2 0 2 - Karak 1 0 1 - Attock 2 0 2 - Central Punjab Thal 7 1 6 3.571 0.059 North Dakota, USA Minot 37 22 15 1.324 0.250 010_JPP834RP_Ali_99 19-03-2012 16:51 Pagina 101

Journal of Plant Pathology (2012), 94 (1), 99-108 Ali et al. 101

pMol each of the primers. Amplification was performed SSR amplification and analyses. PCR was performed in BioRad Cycler thermal cycler (Bio-Rad Laboratories, in a 12 µl reaction mixture containing 0.2 mM dNTP,

USA) and cycling conditions consisted of an initial de- 1X PCR buffer, 1.5 mM MgCl2, and 1 unit Taq DNA naturation at 95°C for 3 min followed by 35 cycles of polymerase (Promega, USA), with the addition of 1 ng 94°C for 20 sec, 58°C for 20 sec, 72°C for 40 sec, and a genomic DNA, 0.24 µl (10 nM) of the forward primer final extension at 72°C for 10 min. DNA amplicons labeled at the 5’ end with M13 sequence, 0.3 µl (10 nM) were separated in 1.5% ethidium bromide-stained of the reverse primer and 0.06 µl (10 nM), one of the agarose gels and visualized under UV light on gel docu- four fluorescent dyes PET, NED, FAM and VIC (Ap- mentation system ChemiDOCTM XRS (Bio-Rad, USA). plied Biosystems, USA). Following an initial denatura- Amplicon size was estimated using a DNA ladder (Hy- tion step of 2 min at 94°C, PCR was performed for a to- per ladder II, USA). tal of 50 cycles, 20 sec at 94°C, 25 sec at 58°C, 23 sec at 67°C, with a final extension for 10 min at 72°C. PCR SSR assay using M13-tailed primers. Six SSR loci products from up to four SSR loci labeled with different and their primer sequences (Table 2) were obtained fluorescent dyes were pooled for multiplex genotyping from a published report on AR (Geistlinger et al., (fragment analysis) with the DNA analyser (ABI ZETO 2000). We used the M13-tailed primer to detect genetic 3730, Applied Biosystems, USA). Because of the differ- diversity of 67 isolates (31 from Pakistan and 36 from ences in the relative fluorescence among the four fluo- the USA). The M13 sequence (CACGACGTTG- rophores, different dilutions were employed for the TAAAACGAC) was added to the 5’ end of the forward PCR products labeled with the different fluorophores. primers which were synthesized by MWG Biotech Inc. The dilution factor for PCR products labeled with Fam (USA). M13 primer strategy consists of three primers, a and Vic was 0.12 (3 µl in final 25 µl), and dilution fac- SSR targeting forward primer with a 5’ M13 tail, a SSR tors for PCR products labeled with Ned and Pet were targeting reverse primer and a fluorescently labeled 0.16 and 0.24 (4 µl and 6 µl in final 25 µl), respectively. M13 primer. During PCR, the SSR product is fluores- For analysis on the genetic analyzer, 3 µl of the 25 µl di- cently labeled following participation of the M13 luted and pooled PCR products were mixed with 9.9 µl primer after the first few cycles of amplification. There- of formamide and 0.1 µl of LIZ-445 size standard (Ap- fore, instead of synthesizing one specific fluorescently plied Biosystem, USA) in a 96 well PCR plate (Gene- labeled primer for each SSR marker locus, only a dye la- Mate, USA). The mixture was heated for 5 min at 95°C beled M13 primer is needed. This allows the forward then chilled on ice and analyzed with the DNA analyser. M13-tailed primer and reverse primer to initiate the re- action and when the limited forward primer is depleted, Data analysis. SSR allelic sizes were determined us- the labeled primer takes its place in the remaining PCR ing Gene Marker v 1.5 software. To group the different cycles (Hayden et al., 2008). genotypes based on SSR allelic size dissimilarity, cluster analysis was performed on the Euclidean distance ma-

Table 2. Sequences of SSR primers used in the study and private and common alleles detected in both Pakistani and US populations.

Private allelesPrivate alleles Locusa Repeating motif(s) a PrimerPrimer sequence sequence (5’ to 3(5’)’ ato 3’) a Common allelesCommon Totalalleles allelesT PakistanPakistan US US CTCGAAACACATTCCTGTGCCTCGAAACACATTCCTGTGC 0 0 ArA06T (CAACAC)7(N)9(CAC)3 1313 4 4 GGTAGAAACGACGAATAGGGGGTAGAAACGACGAATAGGG CTGTATAGCGTTACTGTGTGCTGTATAGCGTTACTGTGTG ArH02T (GAA)58(GTA)6 5 5636 3 14 14 TCCATCCGTCTTGACATCCGTCCATCCGTCTTGACATCCG CATTGTGGCATCTGACATCACCATTGTGGCATCTGACATCAC ArH05T (CTT)18 1 1737 3 11 1 TGGATGGGAGGTTTTTGGTATGGATGGGAGGTTTTTGGTA CTGTCACAGTAACGACAACGCTGTCACAGTAACGACAACG ArH06T (CAA)9(CAG)7(CAA)21 5 5424 2 11 1 ATTCCAGAGAGCCTTGATTGATTCCAGAGAGCCTTGATTG CAGAGGGGAGTCACAAGTATCCAGAGGGGAGTCACAAGTATC ArR01D (GTGTGTGG)6 102102 3 3 GAGTTACAGCTGCAAGACATTCGAGTTACAGCTGCAAGACATTC ATACACCCAAACCGGGTATCATACACCCAAACCGGGTATC ArR12D (CA)15 513513 9 9 GTATGGAATGTGCGATAGGAGTATGGAATGTGCGATAGGA

aLocus designation, repeating motifs and primer sequences are from Geistlinger et al. (2000). 010_JPP834RP_Ali_99 19-03-2012 16:51 Pagina 102

102 Ascochyta rabies population structure in Pakistan and the US Journal of Plant Pathology (2012), 94 (1), 99-108

trix with the unweighted pair group method based on Cortes et al. (1998) reported only one mating type arithmetic means (UPGMA) using the clustering pro- (MAT1-1) in 11 PAR isolates studied. Our results are gramme XLSTAT-2011 software (Addinsoft’s http:// consistent with those of Kaiser (1997) and Navas-Cortes www.xlstat.com). For population structure analysis et al. (1998). All evidence showed the dominance of mat- Structure software 2.23 was used (Pritchard et al., ing type Mat 1-2 in Pakistan, although the sampling loca- 2000). Structure program gives the estimation of log tions were not mentioned in the early studies (Kaiser, probability of the data for each value of number of pop- 1997; Navas-Cortes et al., 1998). In this study it appeared ulations (K). We performed a series of independent that the two mating types are in skewed distribution (3:1) runs for K = 1-5 populations, a burn in period of 50,000 for all the isolates from different localities of Pakistan. Markov chain Monte Carlo (MCMC) iterations, and a However, Chi-square tests showed that the mating type data collection period of 1 million MCMC iterations. distribution for the samples from Chakwal, Islamabad Each simulation of K was repeated 5 times. The hypoth- and Thal were not significantly different from 1:1 distri- esis of random mating was tested by calculating the In- bution (Table 1), whereas the samples from other loca-

dex of association (IA) using statistical programme tions were too small to be tested. A similar situation was MULTILOCUS v1.32 (Agapow and Burt, 2001) using also reported from Syria, where about 64% of a total 133 5000 randomizations. Due to the number of missing isolates were MAT1-1, and the remaining isolates were values it was necessary to choose the option of fixing MAT1-2. The overall distribution showed significant de- missing values in place so that each randomization had viation from 1:1 ratio. However, Chi-square tests of the the same data structure as suggested by the MultiLocus subsamples from six provinces showed they were not sig- software manual. Evolutionary relationship among PAR nificantly different from 1:1 distribution (Atik et al., and US-AR population was determined through mini- 2011). MAT1-2 has been reported as dominant in Turkey mum spanning network (MSN) using Arlequin v3.1 and (MAT1-1) in Tunisia (Taylor and Ford, 2007; Rhaiem (Excoffier et al., 2005) and MSN was visualized using et al., 2007). In some countries including India and Aus- the graphic software Inskscape (http://inkscape.org/). tralia, only one mating type (MAT1-2) has been found; however it should be noted that in Australia the teleo- morph stage was found in the field (Taylor and Ford, RESULTS AND DISCUSSION 2007), suggesting the the other mating type may be rare, but present. Mating type distribution: The multiplex PCR easily Both AR mating types and sexual stage were detected differentiated the two mating types as shown by the pres- in Pakistan by Kaiser (1997) indicating that the intro- ence of 490 bp and 700 bp amplicons for MAT1-2 and duction of MAT1-1 in this region dates to many years MAT1-1, respectively (Fig. 1A). Isolates could be unam- ago. The reason of its unequal distribution is not biguously assigned to one of the two mating types. known. It could be due to insufficient sampling. It was Among the 34 Pakistani isolates, MAT1-2 was more com- also known that mating types of some fungi can differ in mon in Pakistan than MAT1-1 in a ratio of 3:1 (Table 1). pathogenicity and fitness (Zhan et al., 2002). However, It should be noted that the mating type designation based whether there is difference in pathogenicity and fitness on DNA sequences of the mating type locus reversed the between the two AR mating types is not known. previous mating type designation based on pairing with Among the 37 isolates from North Dakota, the two mating type testers. Mating type 2 had been found domi- mating types showed a 1:1 ratio (Table 1). This finding is nant in Pakistan also in early studies. Kaiser (1997) found in line with the study of Peever et al. (2004) in which 41 MAT1-1 and 4 MAT1-2 types out of 45 PAR isolates both mating types showed equal ratio in the US Pacific through conventional laboratory procedure, and Navas- northwest region whereas only one mating type (MAT1-

Fig. 1. Mating type profiles as detected through MAT specific primers. A) PAR isolates. B) US isolates. 010_JPP834RP_Ali_99 19-03-2012 16:51 Pagina 103

Journal of Plant Pathology (2012), 94 (1), 99-108 Ali et al. 103

Fig. 2. Clusters of Pak and US AR populations with STRUCTURE software using six SSR loci. Pak-isolates (Group I and Group II), US isolates (Group III).

1) was found in California. Both mating types have been monomorphic in Varshney et al. (2009) study, but was detected in many countries including Italy, Portugal, polymorphic in our study (3 alleles in PAR and 2 alleles Egypt, Morocco, Algeria, Greece, Turkey, Tunisia, Spain, in US isolates). More recently Nourollahi et al. (2010) USA and Canada (Navas-Cortes et al., 1998; Peever et al., detected large number of alleles at three SSR loci 2004; Barve et al., 2004; Rhaiem et al., 2007). (ArH06T, ArA03T and ArH05T) in Iranian AR isolates, demonstrating high levels of polymorphisms. These da- Genetic diversity and recombination. All six mi- ta suggest that AR populations may be genetically di- crosatellite loci were found to be polymorphic among verse in countries where the host and the pathogen have the 67 isolates from Pakistan and the US, with each lo- long been sympatric (Taylor and Ford, 2007). cus generating 3 to 14 alleles for a total of 52 alleles In Pakistan only limited research has previously (Table 2). The number of alleles detected in this study been carried out on the genetic diversity of AR isolates. was more diverse than that reported earlier (Geistlinger Jamil et al. (2000) studied 46 PAR isolates using et al., 2000). High levels of GD were detected between oligonucleotide fingerprinting and grouped them into PAR and US isolates as populations from both countries six distinct clusters based on their geographical distri- shared very few common alleles at four SSR loci bution. Most of the isolates were area-specific. More (ArH02T, ArH05T, ArH06T and ArR12D) out of the recently, Jamil et al. (2010) using RAPD primers on six loci tested as reported in Table 2. Narrow GD was twelve PAR isolates from Peshawar, Kaghan, Islam- found within each of the two regions, reflecting abun- abad, Attock and Chakwal showed that isolates from dant intraregional gene flow and recombination as fur- the same region were genetically distinct. We tried to

ther supported by the IA test. Peever et al. (2004) re- use more robust co-dominant SSR markers and includ- ported significant GD among AR collected from Pacific ed two isolates, C50 (PAR13) and C51 (PAR14), from northwest of the US. By contrast, Varshney et al. (2009) Chakwal analyzed earlier by Jamil et al. (2010) obtain- detected lower levels of genetic variation among 64 In- ing different results. In fact, according to Jamil et al. dian AR isolates for only 12 out of 20 SSR loci were (2010) these two isolates exhibited significant differ- polymorphic with fewer alleles (2-4 alleles). This may be ences, with genetic distance of 0.94 based on RAPD due to clonal reproduction, as only one mating type markers, whereas in our study they appeared to be ge- (MAT1-2) has been detected in India and closer geo- netically very similar, differing only in one microsatel- graphic locations. In particular, locus ArR01D was lite locus (Fig. 2 and 3).

Fig. 3. UPGMA dendrogram of 67 AR isolates from Pakistan and US based on Euclidean distance. 010_JPP834RP_Ali_99 19-03-2012 16:51 Pagina 104

104 Ascochyta rabies population structure in Pakistan and the US Journal of Plant Pathology (2012), 94 (1), 99-108

Table 3. Ascochyta rabiei isolates with their mating type and their geographical origin in Pakistan and the USA.

Origin Isolate ID Locus MAT type

* ** ArA06T ArH02T ArH05T ArH06T ArR01D ArR12D Attock 1 PAR1 180 336 227 198 220 178 2 Islamabad 2 PAR3 180 333 227 201 220 178 2 3 PAR4 156 336 230 192 220 178 2 Chakwal 4 PAR5 156 351 209 189 212 178 1 5 PAR6 156 351 227 198 220 178 2 6 PAR7 180 333 227 198 220 178 2 7 PAR8 180 333 227 198 220 178 2 8 PAR9 180 333 227 198 220 178 2 9 PAR10 180 330 227 198 -9 180 2 10 PAR11 180 330 227 -9 220 182 2 11 PAR12 180 354 227 198 220 178 2 12 PAR13 156 348 209 198 220 178 2 13 PAR14 156 351 209 198 220 178 2 14 PAR15 180 333 230 -9 212 178 1 15 PAR16 180 -9 227 189 -9 178 1 16 PAR17 156 333 227 198 220 178 2 17 PAR18 156 348 209 198 220 178 2 18 PAR19 180 348 209 -9 220 178 2 19 PAR20 162 333 227 189 212 200 1 20 PAR21 162 360 227 189 212 202 2 Attock 21 PAR22 162 360 215 198 220 202 2 Swabi 22 PAR24 180 363 215 225 188 178 2 23 PAR25 -9 360 215 225 188 -9 2 Karak 24 PAR27 180 336 215 243 188 176 2 Thal 25 PAR28 180 360 -9 222 188 178 2 26 PAR29 -9 351 209 198 220 -9 2 27 PAR30 162 330 227 189 220 198 1 28 PAR31 156 333 227 198 220 178 2 29 PAR32 -9 360 215 198 220 190 2 30 PAR33 180 354 209 198 -9 178 2 31 PAR34 180 330 227 189 212 178 2 US, ND 32 AR1 180 360 269 210 212 178 1 33 AR2 180 324 209 189 212 178 1 34 AR3 156 -9 227 -9 212 190 1 35 AR4 156 360 227 210 212 190 1 36 AR5 180 360 203 210 212 200 2 37 AR6 180 366 209 210 212 178 1 38 AR7 180 366 209 210 212 178 1 39 AR8 180 -9 209 210 212 178 1 010_JPP834RP_Ali_99 19-03-2012 16:51 Pagina 105

Journal of Plant Pathology (2012), 94 (1), 99-108 Ali et al. 105

40 AR9 180 288 -9 192 -9 178 1 41 AR10 180 288 218 192 220 178 1 42 AR11 180 288 218 192 220 178 1 43 AR12 156 360 218 192 220 190 1 44 AR13 180 312 233 210 212 178 1 45 AR14 180 351 230 207 212 190 1 46 AR15 156 288 206 210 220 190 2 47 AR16 156 288 218 192 220 178 1 48 AR17 180 357 227 210 212 190 2 49 AR18 180 357 233 210 212 178 2 50 AR19 156 288 221 192 220 190 2 51 AR20 180 288 203 210 212 178 1 52 AR21 156 360 221 210 212 190 2 53 AR22 180 360 233 192 212 178 2 54 AR23 156 288 221 192 220 190 1 55 AR24 156 360 -9 210 -9 178 1 56 AR25 156 360 224 210 212 190 1 57 AR26 180 360 203 189 212 178 2 58 AR27 180 -9 218 210 220 178 2 59 AR28 180 360 218 210 220 178 2 60 AR29 180 360 218 210 220 178 1 61 AR30 180 360 227 210 212 178 2 62 AR31 -9 360 227 210 212 -9 2 63 AR32 180 360 227 210 212 190 2 64 AR33 168 279 -9 165 -9 188 2 65 AR34 168 -9 -9 189 212 178 2 66 AR36 180 333 206 195 212 178 1 67 AR37 180 366 203 210 212 178 1 * Isolates ID for population structure.; ** Isolate ID with PAR and AR for dendrogram.

Bayesian analysis of population structure using Struc- cospores from one region to another or movement of ture software grouped the isolates from Pakistan and AR-infected seeds from one area to another through the US into three major clusters, i.e. two clusters of PAR seed exchange among farmers. Group III comprised AR isolates (Group I and Group II) and one of US isolates population from US with 17 isolates having admixture (Group III) as shown in Fig. 2. Group I comprised PAR of alleles from group I. Another overlapping of clusters isolates collected from the Swabi and Karak regions between group I (PAR population) and group III (US (Khyber Pakhtunkhwa), one from Chakwal (PAR21) AR population) as reported in Fig. 2, also show that and one US isolate (AR33) and also included some ad- there might be intercontinental flow of genetic material. mixed genotypes from Chakwal, Thal, Attock and from This grouping of isolates is further supported by UP- the US. Group II included most of the isolates from GMA analysis based on the size of microsatellite alleles Chakwal, three isolates from Thal, and two from Islam- using XLSTAT software. UPGMA-based dendrogram abad. No strong correlation was seen between PAR iso- separated both US AR and PAR populations into four lates and their geographical distribution except for the different groups (Fig. 3). Group 1 comprised nine US isolates from Karak and Swabi, suggesting frequent isolates whereas the second group included three iso- gene flow among these geographical locations. Such lates from Khyber Pakhtunkhwa (Swabi and Karak ar- overlapping of clusters and sharing of genetic structure eas) and one isolate from Thal (PAR28). Eight isolates of PAR could be explained either by dispersal of as- from Chakwal, with one isolate both from Islamabad 010_JPP834RP_Ali_99 19-03-2012 16:51 Pagina 106

106 Ascochyta rabies population structure in Pakistan and the US Journal of Plant Pathology (2012), 94 (1), 99-108

Fig. 4. Minimum spanning network (MSN) for haplotypes in PAR (grey) and US- AR (black). The size of the circles reflects the frequency of haplotype and length of line indicates the distance due to allelic difference among them.

and Attock and two isolates from Thal made up the ferent sources. The existence of private alleles shown in third group. Group 4 could be further subdivided into Table 2 indicates population differentiation and lack of two subgroups, one representing mixed populations significant gene flow between these two populations. both of US AR and PAR showing relatedness among However, intercontinental migration may be possible as them, and the second subgroup mainly composed of some isolates from the two continents grouped together PAR isolates with three isolates from chakwal and Thal. not only in the clustering analyses mentioned above but UPGMA clustering indicated that clustering was not also in the MSN (Fig. 4). The MSN differentiated hap- strictly region-specific since each cluster included iso- lotypes from both populations into two distinct clades lates from different regions. Genetic similarity among (A and B) with clade A constituted by both Pakistani isolates from different regions of Pakistan could be due and US haplotypes while clade B was constituted mostly to movement of isolates from one region to another in by US haplotypes with two Pakistani haplotypes. This the form of infected seeds, infected debris through agri- intercontinental genetic flow might be due to the trade cultural vehicle or exchange of seeds between farmers. of infected seeds as discussed by Kaiser (1997). This can be controlled through the sale of certified The presence of both mating types is essential for seeds that may restrain the spread of pathogen. The dis- sexual reproduction to occur and sexual reproduction

tinct clustering of US and PAR isolates obtained both was confirmed by the Index of association (IA) in both by Bayesian and UPGMA clustering revealed that the AR and PAR populations (Table 4). It is interesting to US isolates from North Dakota may originate from dif- observe that random mating seems to occur even in 010_JPP834RP_Ali_99 19-03-2012 16:51 Pagina 107

Journal of Plant Pathology (2012), 94 (1), 99-108 Ali et al. 107

Table 4. Test for random mating among Pakistani and USA population of Ascochyta rabiei from SSR data.

Without clone correction With clone correction

Population NIA rd P value N IA rd P value PAR 31 0.367 0.074 0.005 25 0.165 0.034 0.073 USAR 36 0.392 0.078 0.004 26 0.223 0.045 0.216

Analyses were performed with 5000 randomizations. Note that IA and rd have the same P value.

Pakistan where the ratio between mating types may be Agapow P. M., Burt A., 2001. Indices of multilocus linkage skewed to differ from 1:1. It is also possible that the disequilibrium. Molecular Ecology Notes 1: 101-102. lack of linkage disequilibrium may be due to historical Akhtar K.P., Shah T.M., Atta B.M., Dickinson M., Jamil F.F., sexual recombination. It has in fact been reported that Haq M.A., Hameed S., Iqbal M.J., 2008. Natural occur- for the AR-chickpea pathosystem, the signature of past rence of phytoplasma associated with chickpea phyllody recombination cannot be eroded in field population disease in Pakistan - a new record. Plant Pathology 57: 771. even if asexual reproduction was enforced for 40 gener- Akhtar K.P., Shah T.M., Atta B.M., Dickinson M., Hodgetts ations. It is not clear whether sexual reproduction and J., Khan R.A., Haq M.A., Hameed S., 2009. Symptomatol- ogy, etiology and transmission of chickpea phyllody disease the existence of virulent pathotypes may be correlated in Pakistan. Journal of Plant Pathology 91: 649-653. or not (Udupa et al., 1998; Imtiaz et al., 2011; Jamil et Akhtar K.P., Ahmad M., Shah T.M, Atta B.M., 2011. Trans- al., 2000; Ali et al., unpublished information). mission of Chlorotic dwarf virus in chickpea by the Although the sample size used in this study was limit- leafhopper Orosius albicinctus (Matsumura) in Pakistan. ed for a comprehensive analysis of mating type distribu- Plant Protection Science 47: 1-4. tion and population structure in Pakistan, this is the Atik O., Baum M., El Ahmed A., Ahmed S., Abang M.M., first attempt applying powerful microsatellite markers Yabrak M.M., Murad S., Kabbabeh S., Hamwieh A., 2011. to PAR populations. Microsatellite data may be subject- Chickpea ascochyta blight: disease status and pathogen ed to conventional analysis of Wright’s Fst or Slatkin’s mating type distribution in Syria. Journal of Phytopathology Rst, an Fst-analogue assuming stepwise mutation model. 159: 443-449. Both of these statistics have their drawbacks and there Barve M.P., Arie T., Salimath S.S., Muehlbauer F.J., Peever is no clear consensus over their relative accuracy (Bal- T.L., 2003. Cloning and characterization of the mating type lous and Lugan-Moulin, 2002). (MAT) locus from Ascochyta rabiei (teleomorph: Didymella The current study and analyses have shown interest- rabiei) and a MAT phylogeny of legume-associated Ascochy- ing features of the PAR populations and pointed out the ta spp. Fungal Genetics and Biology 39: 151-167. needs for future investigations. These should employ Barve M.P., Santra D.K., Ranjekar P.K., Gupta V.S., 2004. Ge- well-planed sampling strategies and detailed analysis netic diversity analysis of a world-wide collection of As- and confirmation of microsatellite repeat units in the al- cochyta rabiei isolates using sequence tagged microsatellite markers. World Journal of Microbiology & Biotechnology leles to provide a more complete picture of the popula- 20: 735-741. tion structure and differentiation as well as the AR mi- Balloux F., Lugon-Moulin N., 2002. The estimation of popu- gration rates. lation differentiation with microsatellite markers. Molecu- lar Ecology 11:155-165. Butler E.J., 1918. Fungi and Diseases in Plants. Thatcher, ACKNOWLEDGEMENTS Spink and Co., Calcutta, India. Chen W., Coyne C.J., Peever T.L., Muehlbauer F.J., 2004. H. Ali is thankful to the Higher Education Commis- Characterization of chickpea differentials for pathogenicity sion of Pakistan for providing funds to conduct this re- assay of ascochyta blight and identification of chickpea ac- search work at WSU, Pullman, WA, USA, and thanks cessions resistant to Didymella rabiei. Plant Pathology 53: also Mr. Tony Chen for his help with some of the exper- 759-769. iments. Chérif M., Chilvers M.I., Akamatsu H., Peever T.L., Kaiser W.J., 2006. Cloning of the mating type locus from Ascochy- ta lentis (teleomorph: Didymella lentis) and development REFERENCES of a multiplex PCR mating assay for Ascochyta specie. Cur- rent Genetics 50: 203-215. Acikgoz N., Karaca M., Er C., Meyveci K., 1994. Chickpea Excoffier L., Laval G., Schneider S., 2005. ARLEQUIN, ver- and lentil production in Turkey. In: Muehlbauer F.J., sion 3.0: an integrated software package for population ge- Kaiser W.J. (eds). Expanding the Production and Use of netics data analysis. Evolutionary Bioinformatics 1: 47-50. Cool Season Food , pp.388-398. Kluwer Academ- FAOSTAT, 2009. FAO Statistical Database. http://faostat. ic Publishers, Dordrecht, The Netherlands. fao.org/ 010_JPP834RP_Ali_99 19-03-2012 16:51 Pagina 108

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Received June 18, 2011 Accepted December 12, 2011