Apple scab, caused by Venturia inaequalis, is one of the most damaging diseases worldwide on apple and is currently managed mainly by scheduled applications of fungicides. The aim of the present study was to understand the pathogen population structure in Jammu and Kashmir which is important for breeding and deployment of resistant cultivars. Twenty-seven isolates of V. inaequalis were sampled from commercial apple growing areas to estimate differences in pathogen populations using Random Amplified Polymorphic DNA (RAPD) 10 -and 20-mer primers. RAPD data analysis grouped 27 isolates in to three major clusters accommodating 10, 10 and 7 isolates each. The categories did not follow any geographic or source cultivar pattern. Allele frequencies among the three populations varied from 0.00 to 1.00. The average genetic diversity within each population (HS) over all loci studied was 0.21, 0.23 and 0.20 in Ganderbal, Pulwama and Srinagar, respectively thereby showing high genetic diversity within each population. The average genetic differentiation at a single locus among all sampled populations (GST) was 0.12. The overall gene flow (Nm) was 3.54 while the gene flow values at a single locus ranged from 0.70 to 69.32. Pair-wise genetic differentiation values (FST) among all loci were low, thereby indicating high diversity among the three populations. Analysis of molecular variance (AMOVA) obtained after clustering the isolates at district level showed highly significant genetic differentiation among populations with 5.46 and 94.54% genetic variability recorded among and within district populations, respectively. Population genetic analysis of V. inaequalis is the first molecular analysis of this pathogen from the India and especially from Jammu and Kashmir, a north-western Himalayan state of India.
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Apple (Malus x domestica) grows well in the hilly regions of India at altitudes ranging from 1500 to 2700 m asl. The agro-climatic conditions of Jammu and Kashmir state of India are suitable for growing different varieties of apple which constitute about 91% of its total fruit production. However, the crop, as also the ornamental Malus spp., suffer quantitative and qualitative losses on account of frequent epidemics of scab caused by a fungal pathogen Venturia inaequalis (Cooke) G. Wint. which has been associated since 1930 with the crop in Jammu and Kashmir (Nath, 1935) where its epiphytotics have been a regular feature since 1973 (Joshi et al., 1973). Reliance on six to eight fungicide foliar sprays and the use of scab-resistant cultivars are the main strategies used for scab control (Ellis et al., 1998; Koeller, 1994). However, these strategies have encountered difficulties owing to evolution of fungicide-resistant strains and new resistance-breaking phenotypes. As a consequence, scientists determined the genetic basis for resistance to several fungicides (Jones et al., 1987) and characterized new races (Parisi and Lespinasse, 1996), leading to better understanding of the resistance mechanism to develop anti-resistance control strategies. Variability in V. inaequalis has been assessed using a standard differential set of cultivars, allowing consistent comparison of data among different research groups for common race spectrum. Eight pathotypes of V. inaequalis have been identified worldwide (Benaouf and Parisi, 2000; Bus et al., 2005; Heaton et al., 1991; MacHardy et al., 2001; Parisi et al., 1993). However, categorization of the fungus isolates into physiologic races based on reaction on differentials depends on environmental conditions, spore count and the inoculation conditions that may vary from one laboratory to another. In order to overcome this problem, application of molecular markers has proved useful in assessing variability at genotypic level which allows direct comparison between pathogenic phenotype and genotype. These markers are almost unlimited in number and are not affected by the environment (Williams et al., 1990). The distinct pattern obtained for each individual can be taken as the specific fingerprint that describes and identifies them. A high level of genetic diversity has been reported in V. inaequalis (Guerin and Le Cam, 2004; Melounova et al., 2004; Schnabel et al., 1999; Tenzer and Gessler, 1997, 1999). Venturia inaequalis isolates obtained from different apple cultivars planted in the same orchard in the UK differed significantly in their virulence characteristics (Barbara et al., 2008) as well as at the molecular level, based on AFLP analysis, whereas the isolates from different cultivars or regions in China did not (Xu et al., 2008). Analysis of microsatellite profiles of V. inaequalis samples from five continents suggested that the fungus originated in Central Asia and is now well established worldwide displaying high within-population diversity (Gladieux et al., 2008). More information on within-population variability is needed to understand better the forces acting on the host pathogen co-evolution.
In India, no studies have been carried out on the population structure of V. inaequalis. Hence, the present investigations were carried out to determine how variability in the causal fungal pathogen, V. inaequalis, is structured in the Kashmir valley with an objective to determine the population structure of the fungus using RAPD 10-and 20-mer primers.
MATERIALS AND METHODS
Twenty-seven isolates of V. inaequalis were collected during June to August 2009 and 2010 from commercial apple growing areas of seven districts viz., Anantnag, Pulwama, Shopian, Kulgam, Ganderbal, Srinagar and Baramulla of the Jammu and Kashmir state of India and their single spore isolations made on water agar medium. The germinated single spores were transferred on to the Potato Dextrose Agar slants, incubated at 20°C and maintained for further use.
Total genomic DNA of each isolate was extracted using CTAB method (Murray and Thompson, 1980). Initially six isolates of V. inaequalis were screened with forty-six 10-mer and twelve 20-mer RAPD primers (Operon Technology) for polymorphism. The primers (OPA-03, OPA-04, OPA-13, U1, U5 and U9) showing maximum consistency of polymorphism among the 2 samples of DNA preps from isolates were selected for RAPD profiling of all the isolates. PCR was carried out in 0.2 mL PCR tube with 25 μL reaction volume containing 1 X buffer (20 mM Tris-HCl pH 8.0; 50 mM KCl, MBI, Fermentas Life Sciences), 1.5 mM MgCl2, 2.0 μL dNTP mix (0.2 mM, MBI, Fermentas Life Sciences), 1 U of Taq DNA polymerase (MBI, Fermentas Life Sciences, 5 U μL-1), 2 μL of DNA tempelate (20 ng), 5 pmols of primer and 17.3 μL of sterilized distilled water. The reaction mixture was vortexed and centrifuged in a microfuge (Thermo Scientific, Thermo electron Corporation). Amplifications were performed using thermal cycler (Whatman Biometra, T-Gradient, Goettingen, Germany) programmed for initial denaturation at 94°C for 5 min followed by 40 cycles at 94°C for 1 min, 37/55°C (37°C for deca-mer and 55°C for twenty-mer) for 1 min, 72°C for 2 min and a final extension at 72°C for 5 min using fastest ramp time between transitions. The amplified PCR products were resolved by electrophoresis using 1.2% (w/v) agarose gel in 1X Tris borate EDTA buffer (0.5 M Tris, 0.05 M boric acid, 1 mM EDTA, pH 8.0). The gels were stained with ethidium bromide (0.5 μg mL-1). DNA ladders of 100 bp and Lambda DNA/EcoR1+Hind-III Marker (MBI Fermentas Life Sciences) were used as markers. The gels were run at 80 V for one and a half hour using Consort Power Pack system (Consort EV 215) and the gel images captured using a combination of Ultracam Digital Imaging (A 650 Canon Camera) and Electronic UV Transilluminator (Ultra.Lum.Inc, 1480 N, Claremont Boulevard, Claremont, CA).
Bands that could be scored univocally for their presence and absence were included in the analysis. The binary matrices were analyzed by DARwin 5.0 (Perrier et al., 2003) and the dissimilarity coefficients used to construct dendrogram using neighbor joining programme, selecting the unweighted pair-group arithmetic mean method (UPGMA). The dendrogram with best fit to dissimilarity matrix based on cophenetic value (COPH) chosen from Tree Fit Criterion Dialog Box. The alleles frequencies, total genetic diversity (HT) and genetic diversity within (HS) and among (GST) population were estimated and used to calculate standard population genetic statistics using POPGENE (version 1.31, Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton, Canada). Heterogeneity in allele frequencies among samples was tested using the likelihood ratio chi-square statistic G2 (Brown, 1996; Hamelin et al., 1994). Pair-wise genetic differentiation (GST) among sub-populations was estimated using Neis coefficient of differentiation GST (Nei, 1973). The population structure was evaluated using the analysis of molecular variance (AMOVA) model in the ARLEQUIN ver 3.0 software (Excoffier et al., 2005) and fixation indices FST analogous to FST (Hudson et al., 2002) estimated. The analysis used a hierarchical structure in which all the isolates belonging to each district were considered as a population except the isolates from district Baramulla and Budgam which could not be included in the analysis owing to paucity of sufficient number of isolates. The isolates from district Shopian were merged with those from district Pulwama on account of their similar topographical and geographical features. The statistical significance of the total and pair-wise fixation indices was estimated by comparing the observed distribution with the null distribution generated by 10,000 permutations of the data matrix. Multiple tests of the same null hypothesis were subject to table-wide sequential Bonferroni correction to avoid elevated Type I error.
Of the six isolates of V. inaequalis screened with 10-mer and 20-mer primer, three 10-mers (OPA-03, OPA-04, OPA-13) and three 20-mers (U1, U5 and U9) giving consistent banding patterns were selected for RAPD analysis of 27 isolates of V. inaequalis. The number of bands obtained with different primers ranged from 8 to 14, of which 8 to 12 were polymorphic. In all, a total of 25 consistently reproducible RAPD bands were obtained with all the primers of which 22 (88%) were polymorphic. Cluster analysis of the RAPD bands generated dendrograms with a cophenetic correlation coefficient of 0.84 which substantiate the accuracy of dendrogram.
|Fig. 1:||Dendrogram of 27 isolates of V. inaequalis generated by UPGMA. Scale at the bottom depicts the dissimilarity obtained using neighbour joining in DARwin 5.0|
The dendrogram analysis of 27 isolates revealed a high genotypic diversity within V. inaequalis populations. Three major clusters were obtained using neighbor joining method in DARwin5.0. Cluster I and III accommodated 10 isolates each, where as cluster II contained 7 isolates. Perusal of the dendrogram (Fig. 1) further reveals that the isolates could not be categorized either on the basis of their geographical distribution or the cultivar from which isolated.
Three populations of V. inaequalis based on 24 isolates collected from district Ganderbal, Pulwama and Srinagar subjected to population genetic analysis revealed 20 out of 25 loci as significantly different (χ2 test, p = 0.05) among all the populations (Table 1). Allele frequencies among the populations varied from 0.00 to 1.00. The allele frequency in Ganderbal and Pulwama populations ranged between 0.00 and 1.00, whereas it ranged between 0.12 and 1.00 in Srinagar population (Table 1). The average genetic diversity within each population (HS) over all loci studied was 0.21, 0.23 and 0.20 in Ganderbal, Pulwama and Srinagar, respectively, thereby showing high genetic diversity within each population (Table 1).
The average genetic differentiation at a single locus among all sampled populations (GST) was 0.12, even though the value at single locus varied from 0.007 to 0.42 (Table 1). The lower GST values of 0.007, 0.02, 0.03, 0.05 and 0.0.7 were found at L11, L1, L3, L4, L5, L9, L14, L16 and L25 loci, respectively (Table 1). To obtain the information on the loci under immigration, gene flow (Nm) was calculated across loci. The overall gene flow (Nm) was 3.54, while the gene flow values at a single locus ranged from 0.70 to 69.32. The results obtained show an Nm value greater than 1 indicating little differentiation.
In order to obtain information regarding diversity within populations, pair-wise genetic differentiation values (FST) among all the loci were calculated (Table 2). Most of the values obtained between a pair of populations were low thereby indicating high diversity among the populations. Value higher than 0.10 (i.e., 0.12) was found only for populations between Ganderbal and Srinagar. To elucidate the diversity existing within each population, analysis of molecular variance (AMOVA) was performed to ascertain the percent diversity among the populations (Table 3).
|Table 1:||Allele frequencies, population diversity and gene flow estimates of RAPD markers in three populations of V. inaequalis collected from three locations of Kashmir valley|
|*χ2 values were significant at p<0.05 **frequency of RAPD band (Plus allele)|
|Table 2:||Pair wise genetic differentiation (FST) between three V. inaequalis populations|
|Table 3:||Summary of analysis of molecular variance (AMOVA) of three V. inaequalis populations from different regions of Kashmir valley|
The analysis revealed highly significant (FST = 0.054, p<0.001; 50175 permutations) genetic differentiation, with 5.46 and 94.54% of the genetic variability occurring among and within district populations, respectively.
Scab (V. inaequalis) is globally the most important disease in apple Malus X domestica (MacHardy, 1996; Gupta, 1990; Verma and Sharma, 2003) and was recorded in Jammu and Kashmir state of India first in 1930 (Nath, 1935), experiencing sporadic occurrence and affecting indigenous cultivar Ambri. The frequent epiphytotics of the disease since 1973, with devastating effects on apple production in the region, made it a house hold name (Gupta, 1990; Joshi et al., 1973) leading to its declaration as a disease of national importance. Even then, diminutive work on assessing the population structure of the causal fungus has been carried out in India. An attempt to ascertain the population structure of V. inaequalis at molecular level was, therefore, made and can be considered as the first molecular analysis of this pathogen in India.
RAPD (10 and 20 mer primers) profiling of 27 V. inaequalis isolates of Kashmir in India revealed high level of diversity without congruence of the phenogram with either the geographic location or the apple cultivars from which isolates were obtained. The high genetic differentiation between isolates may be due to the history of apple cultivation in the region and the vertical and horizantal expansion of host density increased over years of production. Until 50 years ago, apples were grown in the meadow system where trees were planted throughout the landscape often with wide spaces between trees. Splash dispersal of pathogen conidia is mainly important for dispersal within or between neighboring trees, whereas wind mediated long-distance dispersal of its airborne ascospores or infected leaf litter determine recurrence/re-infection. Higher the tree density, higher the probability of occurrence of the disease. The intensive cultivation with the use of cost intensive inputs have further aggravated the situation. However, low-input apple cultivation in house gardens or on meadows could serve as reservoirs for V. inaequalis, preventing effects of genetic drift. This could also be a reason why re-colonization of newly planted apple trees apparently occurs quite quickly, since there has never been a scab-free orchard in the valley.
The isolates from Red delicious cultivar collected from various commercial apple growing areas during the present study differed significantly among and within populations and were much diverse than expected, suggesting, therefore, that several evolutionary forces might be acting on the pathogen. Firstly the different cultivars viz., Golden Delicious, Red Delicious, Ambri, Cox orange Pippien, Shereen, Firdous etc. present in our orchard ecosystem might have exerted selection pressure on V. inaequalis. Secondly, the sexual recombination may be the major reason for creating high genetic diversity.
The low values of HS, GST and pair-wise genetic differentiation among the three populations of V. inaequalis from the valley indicate that the pathogen maintains high genetic and genotypic diversity which is close to that observed in Central Asia and Europe (Gladieux et al., 2008; Guerin et al., 2007; Guerin and Le Cam, 2004; Melounova et al., 2004; Tenzer et al., 1999; Tenzer and Gessler, 1997, 1999). Maintenance of high genetic diversity by the fungus in this part of the world gives an indication about the probable introduction of fungus from Central Asia, the centre of origin of V. inaequalis (Gladieux et al., 2008) and Europe as most of the growers cultivate Red Delicious cultivar along with the cultivars from European origin. Gladieux et al. (2008) observed lower levels of variation in V. inaequalis from non-Central Asian populations and suggested that these populations have lost alleles in the process of movement, arrival and establishment outside their native range. Contrary to this, the present study clearly shows that the fungus is highly diverse in the Kashmir valley and all three populations were far from being clonal and none displayed extreme reduction as did Phytophthora infestans (Goodwin et al.,1994), P. ramorum (Ivors et al., 2006), Sphaeropsis sapinea (Burgess et al., 2001), Ustilago scitaminea (Raboin et al., 2007), Magnaporthe grisea (Zeigler, 1998), Ceratocystis fimbriata f. platani (Engelbrecht et al., 2004) and Fusarium circinatum (Wikler and Gordon, 2000). Rather, the variability observed in V. inaequalis samples could be compared with that reported for the cereal pathogens Rhynchosporium secalis or Stagonospora nodorum outside their centre of origin (Stukenbrock et al., 2006; Zaffarano et al., 2006).
Loci with GST values higher than 0.1 indicate that the loci might be under selection pressure or else linked to loci under selection in some populations. However, V. inaequalis undergoes an annual sexual stage allowing crossovers and recombine allele combinations generating new haplotypes. Selection can thus act on individual loci without affecting drift or gene flow at unlinked loci. Looking at the differentiation between each pair of populations, high FST values (FST>0.1) observed during the study between populations from Srinagar and Ganderbal only with small geographic distance gives the clue that very frequent short distance gene flow occurs in the Jammu and Kashmir and the dispersal over longer distances occurs frequent enough to counteract genetic differentiation due to genetic drift or local selection.
The high genetic diversity between V. inaequalis isolates of Kashmir with no congruence of the phenogram with the geographic locations or the apple cultivars is suggestive of the occurrence of frequent sexual recombinations in the pathogen and / or exertion of selection pressure or by either cultivation of scab resistant cultivars or the application of foliar fungicidal sprays against the disease. There is immediate need to further characterize and quantify this variability in the pathogen to ultimately base on the breeding programmes for evolving durable resistant apple cultivars. Adoption of holistic approach such as integrated disease management strategy for containing this epidemic disease shall help in easing out selection pressure on the pathogen to avoid further increase in its genetic divergence in the region.
The authors are thankful to Department of Science and Technology, Government of India, New Delhi for financial assistance in the form of ad hoc research project (SR/SO/PS47/08). We also thank Mr. Nazir Ahmad, Mr.Arif and Mudasir Gulzar for assisting in preparing the reagents in the laboratory.
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