Ann Microbiol (2014) 64:185–197 DOI 10.1007/s13213-013-0651-8

ORIGINAL ARTICLE

Analysis of genetic diversity in colocasiae causing leaf blight of ( esculenta) using AFLP and RAPD markers

Vishnu Sukumari Nath & Muthukrishnan Senthil Alias Sankar & Vinayaka Mahabaleswar Hegde & Muthulekshmi Lajapathy Jeeva & Raj Shekar Misra & Syamala Swayamvaran Veena & Mithun Raj

Received: 19 September 2012 /Accepted: 9 April 2013 /Published online: 2 May 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Abstract The oomycetous fungus Phytophthora colocasiae between dendrogram and original similarity matrix were sig- that causes taro leaf blight is one of the most devastating nificant for RAPD (r=0.904) and AFLP (r=0.825). The re- diseases of taro and is widely distributed in . Molecular sults of this study displayed a high level of genetic variation and cultural techniques were employed for assessing and among the isolates irrespective of the geographical origin. The exploiting the genetic variability among isolates of P. possible mechanisms and implications of this genetic varia- colocasiae obtained from different geographical regions of tion are discussed. India. Analysis of the 5.8-ITS region revealed detectable intraspecific variation among isolates. Ten random ampli- Keywords Phytophthora colocasiae . AFLP . RAPD . fied polymorphic DNA (RAPD) and eight amplified frag- Genetic diversity . Taro . Disease management ment length polymorphism (AFLP) primers produced 198 and 510 reproducible fragments, respectively. AFLP pro- duced 100 % polymorphism, whereas RAPD showed Introduction 93.5 % polymorphism. The average value of the number of observed alleles, the number of effective alleles, mean Taro, a monocotyledonous root crop, is an important staple Nei’s genetic diversity, and Shannon’s information index food crop grown throughout many Pacific Island countries, were 2.00–1.94, 1.53–1.36, 0.31–0.24, and 0.47–0.40, re- parts of Africa, Asia, and the Caribbean for its fleshy spectively, for two DNA markers used. Analysis of molec- and nutritious leaves (Lebot and Aradhya 1991; Sharma et ular variance (AMOVA) for both markers produced similar al. 2008). The corms, leaves, and petioles are used as veg- results with the majority (85 %, AFLP; 89 %, RAPD) of the etables. The taro plant is a rich source of carbohydrates, diversity present within population of P. colocasiae. proteins, minerals, and vitamins, and has medicinal proper- Dendrograms based on two molecular data using the unweighted ties to reduce tuberculosis, ulcers, pulmonary congestion, pair group method with arithmetic mean (UPGMA) was in- and fungal infection (Misra and Sriram 2002). Taro corms congruent and classified the P.colocasiaeisolates into one and are utilized in various industries for the preparation of high two major clusters. Cophenetic correlation coefficient fructose syrup and alcohols (Misra et al. 2008). Besides contributing to sustained food security in the domestic mar- ket, it also brings in export earnings (Jianchu et al. 2001; : : * : : V. S. Nath M. S. A. Sankar V. M. Hegde ( ) M. L. Jeeva Revill et al. 2005). These prospects make taro as one of the S. S. Veena : M. Raj Division of Crop Protection, Central Tuber Crops Research most important tuber crops. Institute, Thiruvananthapuram 695017 Kerala, India Leaf blight caused by Phytophthora colocasiae Raciborski e-mail: [email protected] is the most destructive disease of taro, and is a major constraint for taro cultivation worldwide, including India (Jackson et al. R. S. Misra Regional Centre of CTCRI, Dumduma HBC P.O, 1980;Misra1999; Sahoo et al. 2007).Thepathogenaffects Bhubaneswar 751 019 Orissa, India leaves, stems, and stored corms in the plant. The disease is 186 Ann Microbiol (2014) 64:185–197 characterized by the formation of brownish water-soaked cir- P. colocasiae isolates from and the Pacific cular spots on young and mature leaves (Fig. 1). As the infec- region has been previously described using isozyme and tion progresses, the spots enlarge to form patches, and, as the RAPD markers (Lebot et al. 2003). Little attention has been disease spreads, the whole leaf rots (Lebot et al. 2003). paid to genetic diversity analysis of P.colocasiaefrom India. Zoosporangia, which are produced abundantly on the surfaces Development of effective management strategies for disease of infected leaves, are considered the most important survival caused by P. colocasiae requires an understanding of the structures of this pathogen (Trujillo 1964). During favorable phenotypic and genotypic diversities in the pathogen. conditions (intermittent rainy weather), the entire field is dev- The objectives of the research reported here was to astated within a few weeks of the onset of infection. Leaf blight evaluate P. c o l o c a s i a e diversity in different parts of has become a limiting factor for taro production in all taro- India, using phenotypic (morphological variation, mating growing countries including India, causing yield reductions of type distribution, sensitivity to metalaxyl), and molecu- the magnitude of 50 % (Jackson et al. 1980; Thankappan 1985; lar methods: amplified fragment length polymorphism Misra and Chowdhury 1997). Cultural practices and metalaxyl- (AFLP), and random amplified polymorphic DNA based fungicides have been unsuccessful in protecting the crop (RAPD). It was anticipated that the knowledge of the or have proved too expensive (Misra 1999). Also, development population genetics of P. c o l o c a s i a e may eventually of resistance to fungicide is another major threat (Cohen and contribute to the development of more durable disease Coffey 1986). Although host resistance is the most economi- management strategies. cally viable and environmentally sound practice to manage this disease, popularization of resistant cultivars is limited due to the lack of other desirable economic and market value traits. Materials and methods The limited success in disease management in many situ- ations is due to knowledge gaps in understanding the genetic Plant materials structure of pathogen populations (Martin and English 1997). As compared to other species of Phytophthora, very little Leaves of taro and stem tissue showing typical symptoms of attention has been paid on P.colocasiaeglobally or at the blight, including susceptible and tolerant cultivars, were regional level. Despite the huge economic loss associated with collected from various geographical regions of India leaf blight disease, our understanding of the biology of the (Table 1). The regions showing a high degree of disease pathogen is sparse. The pathogen has a limited host range incidence were given preference for sample collection. (Lebot et al. 2003). P. colocasiae is known to be heterothallic and requires both A1 and A2 mating types for sexual repro- Isolation of pathogen duction, although evidence of homothallic behavior has been reported (Lin and Ko 2008). Variation among P. colocasiae For isolation, leaf tissue segments of 2–3cmfromleaf isolates in phenotypic characters, such as growth rate, colony blight-infected areas were excised. The segments were ster- morphology, metalaxyl resistance, and virulence, have been ilized in 70 % ethanol for 1 min, followed by 1 % sodium recognized (Misra et al. 2011). Significant genetic diversity in hypochlorite for 2 min. The fragments were then rinsed

Fig. 1 Taro leaf blight disease. a Field view of taro leaf blight, b early phase of taro leaf blight showing development of small circular lesions, c the lesions enlarge and coalesce, d at a later stage, the disease spread across the entire leaf, e finally, the entire leaf is destroyed, f first author inspecting and collecting diseased leaf samples Ann Microbiol (2014) 64:185–197 187

Table 1 Details of Phytophthora colocasiae iso- Isolate Location District/sampling Year of Colony morphology Mating lates used in the present study code site collection type along with their colony mor- phology and mating types P1 Kerala Block 2, CTCRI 2010 Uniform without pattern A1 field P3 Kerala Block 1, CTCRI 2010 Uniform without pattern A1 field P21a Kerala Farm, CTCRI 2011 Uniform without pattern A1 P9 Kerala Thiruvananthapuram 2008 Irregular pattern A1 P4 Kerala Aleppy 2011 Plain A1 P7 Kerala Pathanamthitta 2011 Plain A1 P6a Kerala Kottayam 2011 Plain A1 P23a Kerala Kollam 2010 Irregular pattern A1 P15 Kerala Haripad 2012 Plain A1 P28a Kerala Idukki 2010 Flat with concentric rings A1 P11 Kerala Calicut 2007 Stellate A2 P22a Kerala Calicut 2008 Stellate A1 P16a Andhra Veerwada 2010 Plain with irregular A1 Pradesh concentric rings P26 Andhra East Godawari 2011 Plain with irregular A1 Pradesh concentric rings P29 Andhra Parudin pallam 2010 Plain with irregular A1 Pradesh concentric rings P27 Andhra Veerwada 2011 Plain with irregular A1 Pradesh concentric rings P5 Odisha Nayagarh 2007 Cottony A1 P12 Odisha Khandapara 2007 Cottony A1 P2a Odisha RC, CTCRI 2008 Cottony with concentric A1 rings P13 Odisha Salepur 2008 Cottony A1 P24a Odisha Puri 2007 Cottony A1 P25 Odisha Puri 2007 Cottony A1 P14 Uttar Malikpur 2007 Cottony A1 Pradesh P17 Delhi New Delhi 2010 Uniform with concentric A1 rings P19 Assam Nellie Road 2007 Stellate A1 P30 Assam Nellie Road 2010 Stellate A2 P8 Meghalaya Ribhoi 2009 Cottony with concentric A1 rings P20a Meghalaya Nongpoh 2010 Uniform with concentric A1 rings P10 West Nadia 2009 Cottony A1 Bengal P31 West Kalyani 2007 Cottony A1 a Isolates selected for pathoge- Bengal nicity testing and ITS amplifica- P18 Tripura West Tripura 2010 Cottony A1 tion for species confirmation twice with sterile distilled water. Leaf segments were dried 20 g/L agar). Each isolate was stored at −20 °C in 50 % on sterile Whatman filter paper in a laminar flow hood, and glycerol (long-term storage) and at 15 °C on potato dextrose placed onto Phytophthora-selective media, i.e. rye agar agar (PDA) slants in the dark (short-term storage). amended with 20 mg/L rifamycin, 200 mg/L vancomycin, 200 mg/L ampicillin, 68 mg/L pentachloronitrobenzene, and Colony morphology 50 mg/L 50 % benlate (Erwin and Ribeiro 1996). Segments were incubated in Petri dishes for 4–5 days at 28 °C, and Colony morphology was studied on PDA medium. A 5-mm mycelia were then transferred onto potato dextrose agar disc obtained from the periphery of the colony, in areas of medium (PDA; 250 g/L potato, 20 g/L dextrose and active growth, was placed at the center of the Petri dishes 188 Ann Microbiol (2014) 64:185–197 containing PDA, and the plates were incubated at 28 °C in instructions. The nucleic acid obtained was dissolved in TE the dark for 2 weeks. Following incubation, morphology of buffer (100 μl; pH 8.0). The quality and integrity of DNA were P. colocasiae was characterized based on the colony texture. assessed by agarose gel electrophoresis and stored at −20 °C until Three replicates were used for each isolate to confirm the further use. characteristics at similar incubation conditions mentioned above. In order to draw a definite conclusion of colony ITS amplification morphology only one medium was used, as colony mor- phology can be highly influenced by the different media The internal transcribed spacer regions, including the 5.8S composition used. rDNA, were sequenced and analyzed for nine selected P. colocasiae isolates from each morphology group, to repre- Determination of mating type sent the whole set of isolates for validation of species identification using universal ITS1 and ITS4 primers The mating type of isolates was determined by pairing each (White et al. 1990). Each 25 μL of PCR reaction consisted unknown with the isolate of a known A1 (98–111) and A2 of 50 ng of template DNA, 100 μM each deoxynucleotide

(98-35a) mating type on carrot agar (CA) medium (250 g/L triphosphate, 20 ng of each primer, 1.5 mM MgCl2,1×Ta q carrot juice and 20 g/L agar) at 3 cm apart. After incubation buffer (10 mM Tris–HClpH9.0,50mMKCl,0.01% at 28 °C in the dark for 4 weeks, the agar blocks were gelatin), 1 U of Taq DNA polymerase (Merck GeNei, examined microscopically. An isolate was designated to be India). Amplifications were performed in an Agilent sure mating type A1 if oospores were present when paired with a cycler 8800 (Agilent Technologies, USA). The thermal cy- known A2 tester and vice versa. The solo culture of each cler was programmed as follows: 2 min at 94 °C, 35 cycles isolate was examined for oospore formation as a control. of 30 s at 94 °C, 1 min at 57.1 °C, and 1 min 30 s at 72 °C, The positive control was a cross between two tester isolates and finally, 8 min at 72 °C. Amplified products were re- of opposite mating types. The test was replicated three solved on a 1.5 % agarose gel containing 0.5 μgml−1 times. ethidium bromide and photograph was scanned through the Gel Doc System (Alpha Imager; Alpha Innotech, CA, Pathogenicity tests USA). The amplification products were purified to remove excess primers and nucleotides using a QIA-quick PCR Pathogenicity tests were performed with a representative set purification kit (Qiagen) and sequenced with the same of isolates, from all morphological groups using a modified primers as for the PCR amplifications. Sequencing was floating leaf disc method. Taro leaves (cv. Sree Kiran, leaf carried out in the DNA fingerprinting wing of Rajiv blight-susceptible) were disinfected by immersing in 1 % Gandhi Centre for Biotechnology, Thiruvananthapuram. sodium hypochlorite solution for 1 min, rinsed twice with The nucleotide sequences obtained were processed to re- sterile double-distilled water, and blotted dry on sterile move primer sequences and low quality reads, transformed paper towels. Five leaf disks (5×5 cm) were floated on into consensus sequences with Geneious Pro software v.5.6. sterile distilled water in 200-mm glass Petri plates and The resulted high quality sequences were analyzed with inoculated with a mycelial disc (5 mm) excised from the BLASTn (NCBI) to confirm the authenticity of isolates. The margins of actively growing cultures of P. colocasiae. Leaf sequences were aligned using the computer package ClustalW pieces with a noncolonized agar plug served as control (Thompson et al. 1994) and a phylogenetic study was treatments. Plates were covered with a lid containing moist- performed using MEGA v.5 (Tamura et al. 2011). Sequences ened filter paper to maintain high humidity and incubated at were analyzed to determine the relationships between isolates 25 °C in the dark. The leaf discs were daily observed for by the neighbor-joining method (Saitou and Nei 1987). disease symptoms. Lesion length was recorded 5 days after Bootstrap values were generated using 2,000 replicates. The inoculation (d.a.i.). Re-isolation, according to Koch’s pos- ITS-5.8S sequence of Phytophthora palmivora (Accession tulates, was made from all resulting lesions. no. GU111646) was obtained from the GenBank database andusedasanoutgroup. Extraction of genomic DNA AFLP analysis P. colocasiae isolates were grown in potato dextrose broth medium (PDB; 250 g/L potato, 20 g/L dextrose) at 28 °C AFLP analysis was performed as described by Vos et al. with 50 rpm. After 5–7 days, depending on the growth of (1995) with modifications. Genomic DNA (200 ng) was the isolate, mycelium was harvested and dried on sterile double-digested with 0.5 μL EcoRI (10 U/μl) restriction paper towels. DNA was extracted using a Genomic DNA enzyme at 65 °C for 3 h followed by Ta q I for another 3 h purification kit (Fermentas, EU) according to the manufacturer’s in a primary 15 μl reaction volume. To the digested DNA Ann Microbiol (2014) 64:185–197 189

was added 10× T4 Buffer (10 mM MgCl2, 50 M Tris–HCl, random sample of 5 isolates representing different geo- pH7.5, 10 mM DTT, 1 mM ATP, 25 μg/mL BSA, EcoRI graphical origins (data not shown). Based on the initial ligation adapter (30 ng) 0.5 μl, Ta q I ligation adapter screening, a set of 10 random decamer oligonucleotides (150 ng) 0.5 μl, T4 DNA ligase (5 U/μl) 0.5 μl, and 3 μl primers was used for the final study (Table 2). Primers were double-deionized water, to a final volume of 20 μl. The selected based on their ability to detect and resolve poly- mixture was incubated at 25 °C overnight. Pre-amplification morphic amplified products. To ensure reproducibility, the PCR was performed after diluting the ligated DNA 10-fold primers generating weak or complex patterns were not se- with double-deionized water. A total volume of 25 μlreaction lected for this study. Each 25 μl of PCR reaction consisted mixture containing 3 μl of the digestion/ligation mixture, of 50 ng of template DNA, 100 μM each deoxynucleotide 1.0 μl Ta q I primer (200 ng), 1.0 μl EcoRI primer (200 ng), triphosphate, 20 ng of decanucleotide primers (Integrated

2.5 μl 10× PCR Buffer (500mMKCl, 100 mM Tris–HCl, DNA Technologies), 1.5 mM MgCl2,2.5μl Ta q buffer pH 8.3, 15 mM MgCl2), 0.5 μl dNTPs (2.5 mM), 0.5 μl Ta q (10 mM Tris–HCl pH 9.0, 50 mM KCl, 0.01 % gelatin), DNA polymerase (1 U/μl), and 17.5 μl double-deionized 1UofTa q DNA polymerase (Bangalore Genei, Bangalore, water was prepared. PCR reactions were performed with the India). Amplifications were performed in a Biorad C1000 following cycling parameters: 5 min at 95 °C; 30 cycles of thermal cycler (Biorad). The PCR reaction mixtures were 30 s denaturing at 94 °C, 60 s annealing at 56 °C, and 60 s heated at an initial step of 95 °C for 2 min and then elongation at 72 °C, ending with 4 °C pause. After checking subjected to 35 cycles of the following program: 95 °C for for the presence of a smear of fragment by 1.5 % agarose 30 s, 35 °C for 1 min, 72 °C for 1 min 45 s. After the last electrophoresis, the amplification product was diluted 20 cycle, the temperature was maintained at 72 °C for 10 min. times with double-deionized water. After pre-screening of 36 Amplified products were resolved on a 1.8 % agarose gel primer pairs, 8 selective primer pairs were chosen for this containing 0.5 μgml−1 ethidium bromide and visualized study (Table 2). Each selective AFLP reaction was carried under UV light. Gel photographs were scanned by Gel out in a total volume of 25 μl, containing 0.5 μl Ta q Iselective Doc System (Alpha Imager; Alpha Innotech). The size of primer (200 ng), 0.4 μl EcoRI selective primer (200 ng), the amplification products was estimated by comparison 2.5 μl 10× PCR Buffer, 0.8 μL dNTPs (2.5 mM), 0.8 μl Ta q with 1Kb plus DNA ladder (Fermentas). At least two repli- DNApolymerase(1U/μl), 2.5 μl pre-amplification products, cates of the amplification assay were performed with tem- and 17.5 μl double-deionized water. The PCR reactions were plate DNA from two different DNA extractions to ensure performed with the following profile: 2 min at 95 °C; 30 s the consistency of each band. denaturing at 94 °C, 30 s annealing at 65 °C, and 2 min elongation at 72 °C, followed by reduction of the annealing Data analysis temperature in each cycle by 0.7 °C for 12 cycles. The annealing temperature was maintained at 56 °C for the All clearly detectable RAPD and AFLP bands were scored remaining 23 cycles. Amplifications were performed in for their presence (1) or absence (0) by vizual observation; Biorad C1000 thermal cycler (Biorad, Singapore). At least only reproducible and well-defined bands were scored. A two replicates of the amplification assay were performed with dendrogram was constructed using genetic similarity matri- template DNA from two different DNA extractions to ensure ces to display relationships between isolates using the Nei the consistency of each band. To the amplification products, and Li distance (1979) according to the unweighted pair an equal volume of formamide loading buffer was added. The group mean algorithm using the TREECON software pack- amplification product were denatured at 95 °C for 5 min and age v.1.3 (Vandepeer and Dewachter 1994). The relative then electrophoresed on a 6 % denaturing polyacrylamide gel support for the different groups and stability of the dendro- at a constant power of 90 W for approximately 90 min until gram was assessed by bootstrap analysis (2,000 replicates). the forward-running dye reached the end of the gel. AFLP gels The cophenetic correlation coefficient was calculated to were silver-stained according to standardized protocol and provide statistical support for the dendrogram obtained, photographed. Sizes of amplification products were estimated and Mantel’s test (Mantel 1967) was performed to check using a 100-bp DNA ladder. the goodness-of-fit of the cluster analysis of the matrix on which it was based (1,000 permutations). When the value of RAPD analysis a cophenetic correlation coefficient was ≥0.8, this value means that the data within a cluster are most likely to be Random decamer oligonucleotides primers corresponding to highly reliable (Rohlf 1993). RAPD primer kit (T, A, and G) from Integrated DNA The similarity matrix was also used to perform a hierar- Technologies (Coralville, USA) were used for RAPD anal- chical analysis of molecular variance (AMOVA) (Excoffier ysis. To optimize the method, different reagent concentra- et al. 1992) by using FAMD Software v.1.25 (Schluter and tions and PCR reaction conditions were assayed on a Harris 2006). This analysis enables partitioning of the total 190 Ann Microbiol (2014) 64:185–197

Table 2 Summary statistics for Phytophthora colocasiae isolates from different taro-growing regions of India based on AFLP and RAPD amplification

Marker Primer Sequence (5′-3′) No. of bands No. of Mean no. of Polymorphism scored polymorphic bands (%) bands

AFLP E+AT/ CTC GTA GAC TGC GTA CC AT/ 102 102 32.3 100 T+AA TACTCAGGACTGGCAA E+AC/ CTC GTA GAC TGC GTA CC AC/ 56 56 16.1 100 T+AC TACTCAGGACTGGC AC E+GA/ CTC GTA GAC TGC GTA CC GA/ 51 51 12.5 100 T+GT TACTCAGGACTGGC GT E+GT/ CTC GTA GAC TGC GTA CC GT/ 81 81 23.1 100 T+TC TACTCAGGACTGGC TC E+AG/ CTC GTA GAC TGC GTA CC AG/ 60 60 13.6 100 T+AT TACTCAGGACTGGC AT E+AC/ CTC GTA GAC TGC GTA CC AC/ 40 40 6.9 100 T+AT TACTCAGGACTGGC AT E+AT/ CTC GTA GAC TGC GTA CC AT/ 52 52 16.9 100 T+AC TACTCAGGACTGGC AC E+TG// CTC GTA GAC TGC GTA CC TG/ 68 68 22.2 100 T+TC TACTCAGGACTGGC TC Total 510 510 143.6 800 Average 63.7 63.7 17.95 100 RAPD OPG 10 AGGGCCGTCT 24 24 6.8 100 OPG 12 CAGCTCACGA 24 23 8.2 95.8 OPG 16 AGCGTCCTCC 21 19 8.7 90.4 OPG17 ACGACCGACA 14 14 3.9 100 OPG18 GGCTCATGTG 21 21 8.1 100 OPG 19 GTCAGGGCAA 16 14 7.0 87.5 OPG 9 CTGACGTCAC 21 21 7.4 100 OPT 6 CAAGGGCAGA 15 13 6.8 86.6 OPT7 GGCAGGCTGT 22 22 5.4 100 OPT 13 AGGACTGCCA 20 15 5.4 75 Total 198 164 67.7 935.3 Average 19.8 16.4 6.7 93.5

AFLP and RAPD variation into within and among geo- hypothesis (rBarD=0) can be rejected when the ob- graphical region variation components, and provides a mea- served rBarD<0.001, and it can be assumed that the sure of inter-region genetic distances as the proportion of the sampled isolates probably originated from a population total AFLP and RAPD variation residing between P. with a clonal mode of reproduction. colocasiae of any two regions (called Phi statistics). Allelic frequencies of RAPD and AFLP marker were used separately to estimate the percentage of polymor- Results phic loci (P), observed number of alleles (NA), effective number of alleles (NE), Nei’s gene diversity (H), and Isolation of pathogen Shannon index (I) with respect to Hardy–Weinberg equilibrium (Hedrick 2000) using the computer program A total of 31 isolates were obtained from several leaf POPGENE 32 (Yeh and Yang 1999). Loci were consid- blight-infected samples collected from different geo- ered polymorphic if more than one allele was detected. graphical regions of India (Table 1). Isolation was not We evaluated the evidence for recombination by successful from decayed or rotten samples. All isolates performing linkage disequilibrium tests. The standard- were positively identified as P. colocasiae by comparing ized index of association (rBarD) statistic (Agapow their morphology and sporangial characteristics with and Burt 2001) was used to estimate linkage disequilib- authentic cultures maintained by the Central Tuber rium (LD) in each population using the software Crops Research Institute, CTCRI, Thiruvananthapuram, MULTILOCUS v.1.3 (Agapow and Burt 2001). The null India. Ann Microbiol (2014) 64:185–197 191

Colony morphology beginning which turned brown upon the progression of the disease. The results of the colony characteristics of PDA medium indicated considerable morphological differences between ITS amplification isolates of P. colocasiae. Based on the morphological vari- ation, isolates of P. colocasiae were classified into nine The ITS region, including the 5.8S gene of all isolates, was groups (Table 1; Fig. 2). Isolates from the same field/region successfully amplified and sequenced. Amplification had similar growth patterns. yielded a ∼750-bp product in all isolates studied. All isolates were positively identified as P. colocasiae by ITS amplifi- Determination of mating type cation and sequencing. The ITS sequence analysis revealed 97–99 % nucleotide sequence homology with each other The majority of the isolates (29) of P. colocasiae tested were and 95–99 % similarity among the isolates of P. colocasiae of A1 mating type. Only 2 isolates were found to be of A2 available in the GenBank database (data not shown). mating type (Table 1). Alignment of sequences using Clustal W revealed consider- able variation in all the isolates examined. Variations ranged Pathogenicity tests from single base pair changes to multiple changes representing deletions and insertions. More sequence varia- The results of the pathogenicity test are shown in tion was evident in the ITS1 region with only a few short Fig. 3. In general, only the recently obtained isolates regions showing complete homology across all isolates ex- were able to cause serious infection on taro leaf discs. amined (data not shown). The obtained sequences were The P. colocasiae isolates that were isolated earlier were deposited in NCBI and the accession numbers assigned are not or weakly pathogenic. All isolates were able to KC505326–KC505334. reproduce typical leaf blight symptoms. The isolates Phylogenetic analysis of the sequences clearly portrayed initiated lesion development 3 d.a.i. which progressed the variation among the isolates. Phylogram distributed the in a circular fashion from the inoculation point. The isolates into different groups with high bootstrap values inoculated sites showed water-soaked lesions at the indicating the robustness of the clustering (Fig. 4).

Fig. 2 The 31 isolates of P. colocasiae were classified into nine groups based on colony texture when cultures were grown for 14 days on PDA medium. A Cottony. B Stellate. C Cottony with concentric rings. D Plain with irregular concentric rings. E Irregular pattern. F Plain. G Uniform with concentric rings. H Uniform without pattern. I Flat with concentric rings 192 Ann Microbiol (2014) 64:185–197

RAPD analysis

The 10 primer combinations amplified 198 reproducible fragments ranging in size from 200 to 1,800 bp, of which 164 (93.5 %) were polymorphic (Table 2). When finger- prints of these isolates were compared, some bands common to the majority of isolates were observed, while others were unique to one or few isolates. The highest number of am- plification products (24) was obtained with the primer OPG10 and OPG12, while the lowest was with OPG17 (14); the average number of bands among all total 10 Fig. 3 Mean lesion length measured on taro leaf disc (cv Sree Kiran) 5 d.a.i. with P. colocasiae isolates belonging to different morphological groups primers was 19.8. The number of polymorphic fragments detected by each primer varied from 13 to 24, with an average of 16.4. The highest number of polymorphic bands AFLP analysis (24) was produced by the primers OPG10, whereas the primer OPT6 generated the lowest number of polymorphic Eight EcoRI+2/Ta q I+2 primer pair combinations re- bands (13). Based on an UPGMA clustering algorithm, the solved 510 markers that could be scored reliably. genotypes were grouped into two major clusters (Fig. 6) 100 % polymorphism was observed across all 31 iso- with high bootstrap values. Cluster I formed the major group lates evaluated. The highest number of amplification in 19 isolates while cluster II had 12 isolates. The clustering products (102) was obtained with the primer pair E+ of isolates in the dendrogram was not correlated with geo- AT/T+AA, while the lowest (51) with E+GA/T+GT graphical origin. The cophenetic correlation coefficient be- pair; the average number of bands among total 8 primer tween dendrogram and the original similarity matrix were pairs was 63.7. The number of polymorphic fragments significant for RAPD (r=0.904). detected by each primer varied from 51 to 102, with an average of 63.7. The highest number of polymorphic Analysis of genetic diversity bands (102) was produced by the primer pair E+ AT/T+AA, whereas the primer E+GA/T+GT generated Population genetic analysis was performed with the assis- the lowest number of polymorphic bands (51) (Table 2). tance of POPGENE software. The observed number of UPGMA cluster analysis indicated that all isolates were alleles (NA), effective number of alleles (NE) and Nei’s gene grouped under a single major cluster with the exception diversity varied among populations. Isolates were divided of isolate P31 which formed the out-group (Fig. 5). The into subpopulations based on their geographic origin to pattern of clustering of isolates was not associated with project their diversity at the regional level. Both AFLP and geographical origin. RAPD markers produced almost similar population param- The cophenetic correlation coefficient between dendro- eters (Table 3). gram and the original similarity matrix were significant for Analysis of molecular variance (AMOVA) based on AFLP (r=0.825). AFLP and RAPD data shows that a high percentage of the

Fig. 4 A neighbor-joining ITS phylogenetic tree of P. colocasiae isolates belonging to different morphological groups. Numbers at nodes indicate bootstrap values (2,000 replicates). Phytophthora palmivora was used as an out- group Ann Microbiol (2014) 64:185–197 193

Fig. 5 Dendrogram (UPGMA) of 31 isolates of P. colocasiae based on AFLP data. Numbers at nodes indicate bootstrap values (2,000 replicates)

total genetic diversity of P. colocasiae populations in this morphological groups in P. colocasiae. The present study study were distributed on a small spatial scale with an revealed more morphological groups than a previous study average of 87 % of the genetic diversity distributed within by Misra et al. (2011), probably due to the number of iso- populations and only 23 % among populations (Table 4). lates used which provides a broader coverage of different The pairwise Φ statistics ranged from 0.108 to 0.149 indi- geographical regions of India. Mating type analysis showed cating populations are considerably differentiated. a predominance of A1 mating type with only two isolates The observed rBarD for 31 isolates of P.colocasiaewas being the opposite (A2) mating type. Similar results have 0.065 (P<0.040) for AFLP and 0.030 (P<0.020) for RAPD been reported by Misra et al. 2011, who also observed the data. These results indicate that the P. colocasiae populations lack of abundance of compatible mating types (A1 and A2) have a recombination/clonal mode of reproduction. in India. Recently, a study by Tyson and Fullerton (2007) found only one mating type of P. colocasiae (A2) through- out the Pacific region, including Guam, , , Discussion , , and . Oospores and of P. c o l o c a s i a e have not often been The current study represents the first attempt to explore the detected in naturally infested soil or in association with genetic variation existing within P.colocasiaeisolates from naturally infected host tissues (Ko 1979). From the results India using RAPD and AFLP markers. The purpose of this obtained, it can be assumed that may study was to evaluate P. colocasiae diversity in different re- not be playing a direct role in disease epidemics of taro in gions of India using cultural and molecular methods. Our India. Pathogenicity tests showed that the recently obtained results confirm that P.colocasiaeare highly diverse across isolates were more aggressive and were able to cause serious the Indian subcontinent. A high proportion of polymorphic infection on taro leaf discs. There was a considerable differ- loci revealed profound genetic variability. The results demon- ence in the mean lesion diameter of all the isolates studied. strated the utility of AFLP and RAPD markers to assess Variation in lesion length as observed in this study has also genetic diversity among isolates of P. colocasiae. Molecular been reported in other Phytophthora spp. (Granke et al. markers such as RAPD and AFLP are usually used to assess 2011; Costamilan et al. 2012) and in other plant pathogens total genetic variation present among organisms. DNA poly- (Baskarathevan et al. 2012; Mahto et al. 2012). The variable morphism in these markers arises from differences in the lesion lengths produced by the P. colocasiae isolates reflects DNA sequences caused by nucleotide pair substitutions, de- the high degree of genetic diversity present among them. As letions, inversions, and translocations (Waugh et al. 1997). pathogenicity was found to be variable between isolates, We employed a combination of cultural and molecular further studies involving a number of isolates representing techniques to gain insights into the nature of P. colocasiae diverse geographic origins have to be performed for a de- from India. Analysis of colony morphology revealed diverse finitive conclusion. No relationships were observed between 194 Ann Microbiol (2014) 64:185–197

Fig. 6 Dendrogram (UPGMA) of 31 isolates of P. colocasiae based on RAPD data. Numbers at nodes indicate bootstrap values (2,000 replicates)

colony morphology, mating type distribution, pathogenicity single cluster for the latter. The lack of association between tests, and geographical origin of the isolates tested. groups based on AFLP and RAPD markers in this study was The ITS amplification and sequencing confirmed the not surprising, as the two methods used for analysis reveal isolates as P. colocasiae. Alignment of the ITS sequences genetic variation in different regions of the genome. AFLP showed considerable variation in the ITS1 region of all markers usually search for polymorphism in regions of the isolates studied. A similar observation was reported by genome containing restriction sites for the restriction en- Cooke and Duncan (1997), who also observed more poly- zymes used in the analysis (EcoRI and Ta q I sites). On the morphism in the ITS1 region of Phytophthora species in a other hand, RAPD marker loci are distributed throughout phylogenetic study using ITS1 and ITS 2 regions. The the genome. The random decamer primers can bind to any presence of high levels of DNA polymorphism in ITS region in the genome that contain complementary sequence region provides the indication that these pathogens are con- and generate polymorphism between two individuals. The tinuously evolving in nature. Mechanisms such as translo- lack of association between different DNA markers was also cations, chromosome deletions, and duplications are observed by Purvis et al. (2001), who fingerprinted P. common in Phytophthora species (Goodwin 1997), and this infestans isolates with AFLP and RFLP markers. An addi- may also be the case with P. colocasiae. However, a large tional factor affecting genetic diversity assayed by different area has not been sampled, and sequencing of more number marker techniques is the number of markers or probes used of isolates representing diverse geographical regions is de- in an analysis (Messmer et al. 1991). Generally, precision sirable to confirm the overall population structure of P. improves as more probes or marker loci are detected in the colocasiae with respect to ITS region. analysis (Moser and Lee 1994). UPGMA analysis documented greater amount of varia- Several reasons could be attributed to the high intra-zonal tion among P. colocasiae isolates. Nei’s gene diversity ex- diversity detected in the present investigation. Other than amination revealed that the P. colocasiae were highly sexual recombination, the genetic variation seen within the different within close population, which was further con- population may have arisen via asexual mechanisms, such firmed by AMOVA analysis. The clustering of isolates was as mitotic recombination. P. colocasiae is known to rapidly not found to be associated with their geographical origin or reproduce asexually through the formation of large numbers phenotypic characters which were in agreement with previ- of sporangia, which either germinate directly or differentiate ous reports on genetic diversity analysis in P. colocasiae into motile zoospores. Based on the observed rBarD values, (Lebot et al. 2003; Mishra et al. 2010). A reconstituted we speculate that population of P. colocasiae have a recom- dendrogram based on RAPD and AFLP data was incongru- bination mode of reproduction. Evidence of mitotic recom- ent with isolates forming two clusters for the former and the bination in Phytophthora species has been previously Ann Microbiol (2014) 64:185–197 195

Table 3 Population genetic parameters for Phytophthora colocasiae isolates from taro based on molecular markers and geographical regions

a b c d e Marker Population Code Polymorphic bands PPB (%) NA NE H I

AFLP Kerala 495 97.06 1.970±0.169 1.414±0.249 0.271±0.126 0.429±0.164 Andhra Pradesh 315 61.76 1.617±0.486 1.368±0.348 0.222±0.188 0.334±0.274 Meghalaya 209 40.98 1.409±0.492 1.289±0.348 0.169±0.203 0.247±0.297 Assam 175 34.31 1.343±0.475 1.242±0.336 0.142±0.196 0.207±0.287 Odisha 429 84.12 1.841±0.365 1.367±0.280 0.239±0.148 0.378±0.207 West Bengal 305 59.80 1.391±0.365 1.256±0.456 0.145±0.189 0.227±0.275 Total 510 100 2.000±0.000 1.363±0.213 0.249±0.109 0.406±0.138 RAPD Kerala 179 90.40 1.904±0.295 1.445±0.321 0.273±0.161 0.421±0.215 Andhra Pradesh 112 56.57 1.565±0.496 1.374±0.383 0.217±0.203 0.321±0.292 Meghalaya 82 41.41 1.414±0.493 1.292±0.349 0.171±0.204 0.250±0.298 Assam 87 43.94 1.439±0.497 1.310±0.351 0.182±0.206 0.265±0.300 Odisha 153 77.27 1.772±0.420 1.386±0.324 0.239±0.169 0.370±0.239 West Bengal 116 58.59 1.585±0.493 1.327±0.326 0.203±0.182 0.309±0.269 Total 187 94.44 1.944±0.229 1.539±0.324 0.317±0.156 0.478±0.204 a Percentage of polymorphic bands (PPB) b Observed number of alleles (NA) c Effective number of alleles (NE) d Nei’s gene diversity (H) e Shannon’s information index (I) reported (Goodwin 1997;FryandGoodwin1995;Abu-El In conclusion, AFLP and RAPD fingerprints proved to be a Samen et al. 2003). According to Goodwin (1997), mutation powerful tool for characterizing individual isolates of P. is thought to be the primary source of genetic variation in colocasiae. Fundamental factors to disease management and . These mutations in most cases can be neutral and control are (1) how much genetic variability is in a pathogen may not cause any observable changes in phenotype, but it is population, (2) how and when that variability flows through not impossible that at least a part of the genotype variation the population, and (3) how it affects the pathogen’scapacity might have been the result of spontaneous mutation (Silvar et to cause disease, survive, and reproduce. Linking the utility of al. 2006). Alternatively, other mechanisms such as transloca- marker studies to functional level will carry out the research to tions, chromosome deletions, and duplications may occur in the next level with useful results. More survey and population this asexually reproducing fungus that could be the possible studies are required before the origins of this inter- and intra- means for such variation. Variability in the pathogen could specific diversity in P. colocasiae can be determined. The also be elucidated by the fact that the isolates were collected present study is part of our continuing effort to understand from different climatic classifications, although the limited the population structure of P. colocasiae in India. Knowledge number of isolates used in the study would not allow for a of the pathogen population structure and dynamics is useful in robust inference to be made about the influence of climate in determining the most appropriate integrated management the variability in the pathogen population. strategies. With a better understanding of the genetic diversity

Table 4 Analysis of molecular variance (AMOVA) of 31 isolates of Phytophthora colocasiae using AFLP and RAPD markers

Source df SSD Φ statistics Variance Proportion of variation components components (%)

AFLP Among populations 5 2.66 0.149 0.054 14.93 Within populations 25 7.71 0.308 85.06 Total 30 10.37 0.362 RAPD Among populations 1.70 0.108 0.027 10.83 Within populations 5.67 0.226 89.16 Total 7.37 0.254 df degrees of freedom; SSD sums of squared deviations 196 Ann Microbiol (2014) 64:185–197 and population biology of P.colocasiae, regulatory solutions colocasiae isolates from South East Asia and the Pacific. Plant – can be developed for the efficient management of the leaf Pathol 52:303 313 Lin JM, Ko HW (2008) Occurrence of isolates of Phytophthora blight disease globally, particularly in the Indian subcontinent. colocasiae in Taiwan with homothallic behavior and its signifi- cance. Mycologia 100(5):727–734 Acknowledgments The funding provided by the Indian Council of Mahto NB, Gurung S, Nepal A, Adhikari TB (2012) Morphological, Agricultural Research, New Delhi, for conducting the research work, is pathological and genetic variations among isolates of gratefully acknowledged. 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