INTRODUCTION

Dieback of pistachio (Pistacia vera L.) is one of the most important, destructive and threatening diseases of Iran pistachio orchards. The disease affects different parts of the tree such as branch and trunk. Dieback of pistachios was first reported in 1987 in Iran (Aminaei 1987). Beside its plant pathogenic activity, it is also associated with many types of human infections (Abbas et al. 2009). Hyphomycosis disease in human caused by two species of Paecilomyces lilacinus and P. variotii (Houbraken et al. 2008).

At this time, the majority of studies on phylogeny of Paecilomyces species using molecular markers have been performed on entomopathogenic species (Dalleau–Clouet et al. 2005; Luangsa–ard et al. 2004). Recently, researchers have attempted to find out more information about the relationships between the different species of Paecilomyces, especiallyinsect–pathogen species. Genetic similarities in unidentified isolates of P. fumosoroseus and some selected strains were observed using ITS and RAPD markers (Azevedo et al. 2000). Arbitrarily primed PCR and PCR with tRNA consensus primers have been used to analyse genetic variability among P. fumosoroseus isolates (Tigano–Milani et al. 1995).

The conserved sequence of rDNA–ITS regions has been used for molecular phylogenetic analysis of fungi (Kiss 1997; Nilsson et al. 2008). Sequence variation within the ribosomal DNA region has been used extensively for the phylogenetic analysis of both closely related and distantly related organisms (White et al. 1990). This can also provide an alternative approach to RAPD–PCR and tRNA–PCR for both the estimation of genetic diversity and the determination of phylogenetic relationships. Furthermore, the fast–evolving ITS region has been found to be a powerful tool for characterization of most fungal bio–control agents (Avis et al. 2001).

Ribosomal genes evolve cohesively within a single species and exhibit only limited sequence divergence between rDNA copies. In contrast, comparison between species showed normal levels of sequence divergence (Arnheim et al. 1980). There is not enough information about the genetic variability of this species in the literatures.

The aim of the present study was to investigate the genetic diversity among P. variotii isolates of different geo–climatic regions from Kerman province, using ITS and RFLP analysis.

MATERIALS AND METHODS

Sampling

Samples were collected from different pistachio farms of Kerman province in Iran during 2011–2012 (Fig. 1). Sampling area were divided to seven geographical zone based on GPS information (Table1). 

The infected branches showing necrosis symptom were cut, kept in nylon pockets and transferred immediately to the refrigerator at 20 ^°C.

Fig. 1. The location of sampling regions on map of Karman province, Iran. Sampling regions are indicated by black filled circle.

Isolation and purification of isolates

The small pieces from the central core of infected barks of pistachio branches were surface–sterilized with 3% chloramine T (Sigma Co., Germany) and were placed on PDA (potato dextrose agar; Merck, Germany) culture medium for fungal growth at 22–25 ^°C for one week (Ebrahimi et al. 2015). Purification of fungal isolates was conducted by the hyphal–tip method and fungal identification at the genus/species level was carried out by morphological criteria (Brown & Smith 1957; Hoog et al. 2000; Samson 1974). Out of 116 P. variotii isolates, 28 selected isolates were recovered from all sampling region (four isolates from each region) which showed that typical species characters were selected to assess genetic diversity for further analysis.

DNA extraction

A piece of ten–day–old fungal colony on PDA medium was transferred to 100 mL Erlenmeyer flasks containing 200 mL of PDB liquid medium (Merck, Germany). The flasks were placed on a rotary shaker (120 rpm min^–1) for eight days at 25 ^°C and then the mycelia were harvested by filtering. Total genomic DNA was extracted from dried mycelium using the CTAB method (Nicholson et al. 1997). Total DNA was quantified using a Scanodrop 200 (Analytik Jena, Germany) spectrophotometer and the concentration of DNA was adjusted to 25 ng.µL^–1 for use in PCR assay. DNA quality was assessed by 1% agarose gel electrophoresis stained by ethidium bromide.

Table 1. Code number, Location and geographic position calculated by GPS of Paecilomyces variotii isolates used in this study.

DNA amplification

Primers TW81 (5′–GTTCCGTAGGTGAACCTG C–3′) and AB28 (5′–TATGCTTAAGTTCAGCGG GT–3′) were used to amplify the ITS–rDNA region (White et al., 1990). Amplification were carried out in volumes of 25 μL containing: 1 μL of genomic DNA (25 ng), 1.5 μL of 10×buffer PCR (100 mM Tris–HCl, 15 mM MgCl₂, 500 mM KCl, pH 8), 1 μL of MgCl₂ (50 mM), 0.25 μl of dNTPs (100 mM),
5U Master Taq DNA polymerase (Genall, Sout Korea), and 25 μL of each primer (20 mM). The PCR reaction was performed with the following steps: an initial denaturation step at 95 ^°C for 5 min, 35 cycles at 95 ^°C (30 s)/56 ^°C (60 s)/72 ^°C (60 s), and a final extension step at 72 ^°C for 10 min. A negative control deleting DNA template was used in every set of reactions. PCR products were separated by electrophoresis on 1.2% agarose gels stained with ethidium bromide (0.5 µg.mL^–1) and photographed under UV light.

PCR–RFLP analysis

The PCR products were purified using the PCR purification kit (Genall, South Korea,) for the PCR–RFLP analysis. Thirteen restriction enzymes including EcoR I, Hpyf 3I, Hinf I, Msp I, Apa I, Mbo I, Pst I, Not I, Rsa I, Dra I, BamH I, Hind III, and Mse I (SinaClon, Iran) were used to digest ITS–rDNA PCR products. Ten units of each enzyme, with a total volume of 15 μL were used in the reaction. The reaction was incubated for 18 h at 37 ^°C.

Genetic diversity

ITS–RFLP patterns were used to estimate similarities among the isolates. Restriction–enzyme digests were used to generate ITS–RFLPs. For this purpose, each DNA band formed by the digestion in RFLP analysis was considered to be a character, and only the presence or absence of RFLPs fragments was recorded. A dendrogram was constructed from the resulting distance matrix using the Unweighted Pair Group Method with Arithmetic Mean Algorithms (UPGMA) and genetics similarity determined using Jaccard's similarity coefficient (Sneath & Sokal, 1973). The software PopGene 32 was used to perform the distance analysis (Kumar et al. 2008).ThePAUP version 0.4.0 beta program was used for phylogenetic analysis of the various data sets (Swofford, 2003). Genetic variations within and between populations was estimated by analysis of molecular variance (AMOVA) performed with GenALEX version 6.1.

Sequencing

Both strands of each PCR products were sequenced by PishgamBiotech Company (Tehran, Iran). DNA sequences were queried using the NCBI stand–alone BlastAll program (Altschul 1990) against the NCBI non–redundant (nr) protein reference library, Swissprot version 6, UniProt and UniRef100. Sequence similarities above 90% with an E value less than 1E^–10 were considered as statistically significant positive matches. Deposited sequences were retrieved from GenBank. The obtained sequences were aligned with a rDNA–ITS sequence of P. variotti isolates in gene bank using the Clustal W program, version 1.81 (Thompson et al. 2002).

RESULTS

Identification of fungal isolates

All recovered fungal isolates from infected twigs were identified by morphological criteria using valid mycology keys. One hundred sixteen isolates out of 180 were identified as Paecilomyces variotii. After two weeks growth, the isolates showed a brown or yellow– brownish colour on the surface of solid medium. A powdery yellow–brownish colony with a high growth rate at 25 ^°C and 37 ^°C was observed on PDA medium. Single–celled and hyaline conidia were born in chains with the youngest cell at the base of conidiophores. The phialides were swollen at the base and gradually taper to a sharp point at the tip. To confirm morphological diagnosis, the sequences of five represented isolates from different geographic regions were queried against data base. Analysis of alignment showed a high similarity of our sequences (96–99%) with deposited sequences of P.variotii in geneBank (Table 2, Fig. 2).

Polymorphism of ITS–RFLP patterns

Amplification of the region from the 3´ end of the 18S rDNA to the 5´ end of the 28S of rDNA resulted in an approximately 600–800 base pair (bp) fragment (Fig. 2). The ITS1–ITS2 amplicons were subjected to digestion with thirteen different restriction enzymes. Seven out of the 13 restriction endonuclease (EcoR І, Hpyf 3І, Apa І, Hinf І, Mbo І, Msp І, showed restriction pattern. No restriction sites were found when DNA was treated with Rsa І, Not І, Pst І, BamH І and Hind ІІІ. The banding patterns obtained with restriction endonuclease digestion, the number and the size of the fragments from 28 P. variotii isolates are characterized in Table 3. Based on resulted patterns of digested PCR products, all isolates were divided into three distinct groups. The sixteen isolates from various graphic regions (Ravar, Sirjan, Rafsanjan and Kerman) were clustered in group 1 based on ITS–RFLP patterns. Group 2 consisted of 8 isolates originated from diverse geographic locations representing four isolates from Zarand, two isolates from Bardsir and  two isolates from Tahroud origins and group 3 contain three isolates from two different geographical regions including Bardsir (2 isolates) and Tahroud (one isolates) isolates ( Table 3).

The enzyme BamH1 digested the fragment, but showed no polymorphisms among isolates. The highest number of restricted fragment was obtained for the Aps I enzyme, whereas the EcoR I and Msp І showed the lowest digestion. The Mbo І enzyme revealed a higher variety and the Msp І enzyme showed a low diversity among isolates. The maximum number of nucleic acid band ranged from 45 – 325 was obtained for Aps I pattern (Table 3).

The Mbo I and Msp I enzymes revealed the highest and lowest values for H^c (0.453 and 0.347 respectively. The highest (0.644) and lowest (0.525) I^d values were obtained for Mbo I and Msp 1 (Table 4). Cluster analysis using NTSYSpc software (version 2.2) based on the Jaccard’s coefficient showed that all isolates were divided to nine separate groups with a high similarity value of 70%. Isolates were grouped into nine clusters designated from A to I. Isolates of group A–B and group D–E contain isolates from Zarand and Rafsanjan regions with similarity value of 66% and 50% respectively. Other isolates were placed in a distinct group (C, D, E, F, I) (Fig. 4).

Table 2. Similarity percentage of studied isolates of Paecilomyces variotii with deposited sequences in GeneBank

Analysis of molecular variance showed a high proportion of total variation is supported by variability (85%) among isolates and less proportionately (15%) within isolates (Table 5).

SUMS0303        TGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAA-GGATCATTACCGA 59

KUC5015         ------------TCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAA-GGATCATTACCGA 47

BCC14365        -------------------------------------------GAA-GGATCATTACCGA 16

Z2              -----------------------GTTCCGTAGGTGAACCTGCGGAA-GGATCATTGCAGC 36

B3              -----------------------GTTCCGTAGGTGAACCTGCGGAA-GGATCGTAAACCT 36

K2              -----------------------GTTCCGTAGGTGAACCTGCGGAA-GGATCATTACCAC 36

X3              -----------------------GTTCCGTAGGTGAACCTGCGGAAAGGATCATTACCGA 37

R1              -----------------------GTTCCGTAGGTGAACCTGCGGAA-GGATCATTACCGA 36

Isolate15       --------------------------TCCGTAGGTGAACCTGCGGAAGGATCATTACCGA 34

SCSGAF0038      ------------------------------------------------------TACCGG 6

SUMS0303        GTGAGGGTCC-CACGAGGCCCAACCTCCCATCCGTGTTG-AACTACACCTGTTGCTTCGG 117

KUC5015         GTGAGGGTCC-CACGAGGCCCAACCTCCCATCCGTGTTG-AACTACACCTGTTGCTTCGG 105

BCC14365        GTGAGGGTCC--ACGAGGCCCAACCTCCCATCCGTGTTG-AACTACACCTGTTGCTTCGG 73

Z2              GTGCGGGACC-CACGCAGATACACCCTCCACCCGTGTTATAACTACACCTGTTGCTTCGG 95

B3              GCGTGGGTCT-CATGAGTGACAATGCTGCATCCGTGTTG-AACTACACCTGTTGCTTCGG 94

K2              GGGCTGGTCCACGCAGAGAAGAACCTCCCATCCGTGTTG-AACTACACCTGTTGCTTCGG 95

X3              GTGAGGGTCA-CGCATATACCAACCTCCCATCCGTGTTG-AACTACACCTGTTGCTTCGG 95

R1              GTGAGGGTCC-CACGAGGCCCAACCTCCCATCCGTGTTGGAACTACACCTGTTGCTTCGG 95

Isolate15       GTGAGGGTCC-CTCGAGGCCCAACCTCCCATCCGTGTTG-AACTACACCTGTTGCTTCGG 92

SCSGAF0038      ATTAGA--TC-CACGAG--CTAACCTCC-ATCCGTGTTG-AACTACACCTGTTGCTTCGG 59

SUMS0303        CGGGCCCGCCGTGGTTCACGCCCGGCCGCCGGGGGGCCTTGTGCTCCCGGGCCCGCGCCC 177

KUC5015         CGGGCCCGCCGTGGTTCACGCCCGGCCGCCGGGGGGCCTTGTGCTCCCGGGTCCGCGCCC 165

BCC14365        CGGGCCCGCCGTGGTTCACGCCCGGCCGCCGGGGGGCCTTGTGCCCCCGGGCCCGCGCTC 133

Z2              CGGGCCCGTCGAGGTTCACGCCCGGCCGCCGGGGGGCCTTGTGCCCCCGGGCCCGCGCCC 155

B3              CGGGCCCGCCGTGGTTCACGCCCGGCCGCCGGGGGGCCTTGTGCCCCCGGGCCCGCGCCC 154

K2              CGGGCCCGCCGTGGTTCACGCCCGGCCGCCGGGGGGCCTTGTGCCCCCGGGCCCGCGCCC 155

X3              CGGGCCCGCCGTGGTTCACGCCCGGCCGCCGGGGGGCCTTGTGCCCCCGGGCCCGCGCCC 155

R1              CGGGCCCGCCGTGGTTCACGCCCGGCCGCCGGGGGGCCTTGTGCCCCCGGGCCCGCGCCC 155

Isolate15       CGGGCCCGCCGTGGTTCACGCCCGGCCGCCGGGGGGCCTTGTGCTCCCGGGCCCGCGCCC 152

SCSGAF0038      CGGGCCCGCCGTGGTTCACGCCCGGCCGCCGGGGGGCCTTGTGCCCCCGGGCCCGCGCCC 119

SUMS0303        GCCGAAGACCCCTCGAACGCTGCCCTGAAGGTTGCCGTCTGAGTATAAAATCAATCATTA 237

KUC5015         GCCGAAGACCCCTCGAACGCTGCCCTGAAGGTTGCCGTCTGAGTATAAAATCAATCATTA 225

BCC14365        GCCGAAGACCCCTCGAACGCTGCCCTGAAGGTTGCCGTCTGAGTATAAAATCAATCATTA 193

Z2              GCCGAAGACCCCTCGAACGCTGCCCTGAAGGTTGCCGTCTGAGTATAAAATCAATCATTA 215

B3              GCCGAAGACCCCTCGAACGCTGCCCTGAAGGTTGCCGTCTGAGTATAAAATCAATCATTA 214

K2              GCCGAAGACCCCTCGAACGCTGCCCTGAAGGTTGCCGTCTGAGTATAAAATCAATCATTA 215

X3              GCCGAAGACCCCTCGAACGCTGCCCTGAAGGTTGCCGTCTGAGTATAAAATCAATCATTA 215

R1              GCCGAAGACCCCTCGAACGCTGCCCTGAAGGTTGCCGTCTGAGTATAAAATCAATCATTA 215

Isolate15       GCCGAAGACCCCTCGAACGCTGCCCTGAAGGTTGCCGTCTGAGTATAAAATCAATCATTA 212

SCSGAF0038      GCCGAAGACCCCTCGAACGCTGCCCTGAAGGTTGCCGTCTGAGTATAAAATCAATCGTTA 179

SUMS0303        AAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGAT 297

KUC5015         AAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGAT 285

BCC14365        AAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGAT 253

Z2              AAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGAT 275

B3              AAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGAT 274

K2              AAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGAT 275

X3              AAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGAT 275

R1              AAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGAT 275

Isolate15       AAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGAT 272

SCSGAF0038      AAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGAT 239

SUMS0303        AAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCC 357

KUC5015         AAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCC 345

BCC14365        AAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCC 313

Z2              AAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCC 335

B3              AAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCC 334

K2              AAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCC 335

X3              AAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCC 335

R1              AAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCC 335

Isolate15       AAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCC 332

SCSGAF0038      AAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCC 299

Fig. 2. A part of sequence alignment showing high similarity between the studied sequences of Paecilomyces variotii isolates and deposited sequences of this specie in GeneBank.

Table 3. Patterns within the Paecilomyces variotii rDNA–ITS–rDNA region after digestion with EcoR І, Hpyf 3 І, Apa І, Hinf І, Mbo І, Msp І, Mse І restriction endonucleases.

Table 4. Genetic diversity indices of Paecilomyces variotii isolates. 

Na: Number of different alleles; Ne: Number of effective alleles; He: Nei's Unbiased Expected Heterozygosity. Id: Shannon Index

Fig. 4. Dendrogram constructed from analysis of DNA fragments 28 Paecilomyces variotii isolates amplified by PCR–RFLPP. The matrix was created with the Jacard similarity coefficient, and clustering was performed with UPGMA algorithm.

Table 5. Analysis of molecular variance of P. variotii isolates.