INTRODUCTION

Date palm (Phoenix dactylifera L.) provides an excellent source of food with high nutritional values, called a mine (Zaid & Wet 2002). In addition to providing a wide range of essential nutrients, various parts of the date plant can be used in different industries. To eradicate food insecurity, malnutrition, and food crises, FAO (The Food and Agriculture Organization) has a unique look for the date palm and implement different projects and programs in various fields (Arias et al. 2016). Khuzestan province, located in the southwest of Iran, is considered as one of the most critical palm cultivation areas in Iran, which its date fruits are exported to different countries. Khuzestan province seems to be one of the places of origin and distribution of date in the world (Zaid & Wet 2002). The significant date palm cultivation areas in Khuzestan include the southern, western, and central parts of the province. One of the most important diseases of palm in Khuzestan province is khamedj disease (inflorescence rot). Based on our field observations, due to the favorable environmental conditions for the pathogen, Khuzestan province has a higher incidence of disease than other Iran provinces.

Climate change and heavy rainfall during the last two years in this province led to a conducive environment and pathogen outbreak. Khamedj is the most severe disease in the hot and humid regions, and its outbreaks could occur after a prolonged cold and humid winter (Djebri 1998), the same condition that exists in Khuzestan. Severe outbreaks of the disease occurred in Basrah, Iraq, in 1948–1949 and 1977–1987, causing 80% loss of the annual harvest (Al-Hassan & Waleed 1977). The significant causal fungus of khamedj disease is Mauginiella scaettae (Ascomycota) (Abdollah et al. 2005).

Simple sequence repeats (SSRs) or microsatellites are short motifs (1–6 base pairs) repeated DNA sequences which their length variation occurs primarily due to slipped-strand mispairing during replication, repair, and recombinant genetic mutations (Rossetto 2001, Karaoglu et al. 2005). It has been demonstrated that SSR markers can be a valuable and efficient source for genetic characterization of species and other aspects such as detecting genetic variation in a pathogen population (Adhikari et al. 2008, Kumar et al. 2013). Karaoglu et al. (2005) have shown that the abundance of SSRs in fungi is low in comparison with other organism's genomes. The cross-transferability of SSRs have been widely recognized. It has been proven that SSR primers developed for one species can be used to study polymorphism at homologous loci in related species (Rossetto 2001). Thus, using such SSRs primers that have been previously developed in related species has become fast, efficient, and cheaper. Hence, because
of the lack of sufficient information on the M. scaettae genome, seven SSR primers previously reported in Phaeosphaeria nodorum (Stukenbrock et al. 2005, Adhikari et al. 2008) were evaluated for the cross-transferability to M. scaettae. Microsatellites present in expressed regions of the genome (Expressed Sequence Tags or EST-SSR) have a crucial advantage compared with SSRs from non-coding sequences which is that they are often more transferable across species (Kumar et al. 2013, Singh et al. 2014). EST-SSRs have been used in various genetic studies in different organisms, and a high rate of their transferability has already been reported for animals, plants, and fungi (Cristancho & Escobar 2008).

The present study aimed to evaluate EST-SSRs transfer between P. nodorum and M. scaettae isolates and used this powerful marker to investigate the genetic diversity of M. scaettae isolates collected from eight regions of the most critical locations of date palm cultivation in Khuzestan province in Iran.

MATERIALS AND METHODS

Sampling and isolation of Mauginiella scaettae

Sample collection was made in eight regions of Khuzestan province in the southwest of Iran from March to May during 2018-2019 (Fig. 1; Table 1). Samples were collected from date palms showing symptoms of blackish rust spots on the spathe's outer

Fig. 1. Sampling locations of Mauginiella scaettae in palm date cultivation regions of Khuzestan province. The sampling locations have shown by red stars (https://en.wikipedia.org). 

Table 1. Characterization of the isolates of Mauginiella scaettae used in the present study. All isolates were obtained in different regions of Khuzestan province, Iran.

Fig. 2. Symptoms and signs of the khamedj disease of date palm. a. opened and unopened rotten male inflorescence which deformed slightly and showing blackish rust spots on the outer surface and covered by white Mauginiella scaettae arthrospores, b. Rotten female inflorescence, c. Opened rotten female inflorescence that severely destructed, d. Symptoms and signs on the different part of the inflorescence (spathe, spikes (flowers and strands).

DNA extraction

The mycelium was scratched from the surface of a 20-day-old PDA plate and powdered in liquid nitrogen. The DNA was extracted using phenol-chloroform protocol, according to Zhong & Stephenson (2001). The quality of DNA was determined using 2% agarose gel, and its quantity was adjusted to 50 ng by NanoDrop.

Morphological and molecular characterization

The identification of the M. scaettae was made based on the morphological criteria, colony morphology, mycelium, and arthroconidia according to Abdollah et al. (2005), Sigler & Carmichael (1976), and von Arx et al. (1982). Internal transcribed spacer ITS-nrDNA region of four isolates, including abad-sa1, ahva-ma1, behb-ma1, and hove-gh, was sequenced to confirm the morphological identification. The ITS1-5.8S-ITS2 region was amplified using the ITS1 (5′-CTTGGTC ATTTAGAGGAAGTAA) and ITS4 (5′-TCCTCCGCT TATTGATATGC) primer pair (White et al. 1990) for 8 min at 95°C for the initial denaturation step followed by 35 cycles of 95°C for 30 s, 58°C for 20 s, 72°C for 60 s and a final extension of 72°C for 5 min.

SSR analysis

Seven primer pairs used in the current study are listed in Table 2. All EST-derived microsatellite loci which analyzed in this study were previously isolated and characterized from the wheat pathogen fungus, Phaeosphaeria nodorum (Stukenbrock et al., 2005). PCR reaction was carried out in a total volume of 20 μL containing 3 μl genomic DNA (50 ng), 0.5 ml of each primer (10 μM primers), 6 μL water, and 10 μL

Table 2. Characterization of seven microsatellite loci of Phaeosphaeria nodorum which their transferability to Mauginiella scaettae was investigated.

^a PIC: Polymorphic information content

master mix (1.5X). PCR reaction was programmed as initial denaturation for 2.5 min at 96°C followed by 36 cycles of denaturation for 30 s at 96°C; annealing for 1 min (modifications of annealing temperatures were necessary and different depending on used markers (Table 2)), extension for 1 min at 72°C followed by 5 min at 72°C. The amplified products were separated on 4% polyacrylamide gel. The gels were run in 1X TBE buffer in electrophoresis apparatus at 250 V for two h and 30 min, stained with silver nitrate (Hung et al. 2018).

Statistical analysis

All band lines were visually scored as present (1) or absent (0). Data of SSR markers were entered into Microsoft Excel version 2010. The data were analyzed using NTSYS-pc software version 2.2.

The average of alleles per locus and the polymorphic information content (PIC) were estimated and compared between EST-SSRs. The data were analyzed using a qualitative data route to generate Jaccard's similarity coefficient using NTSYS-pc, software version 2.2. The corresponding dendrograms were constructed by the unweighted paired group method with the arithmetic mean (UPGMA) and SAHN clustering.

RESULTS

Morphological and molecular identification

Thirteen isolates of M. scaettae were recovered from seven cultivars at eight locations of Khuzestan province during 2018-2019 (Table 1.). The mono-
conidial isolates were identified and verified based on morphology (Abdollah et al. (2005), Sigler & Carmichael (1976), and von Arx et al. (1982)). The fungus colonies on PDA were relatively slow-growing, reaching 9 cm in diameter after ten days at 25°C, white; the reverse, creamy, and in some isolates becoming brown to black in old cultures PDA. The hyphae were regularly septate and repeatedly branched. Many abundant arthrospores were prod-uced by segmentation of the hyphae. The arthrospores were unicellular or multicellular (1-6 cellular and rarely up to 13 cellular), hyaline, cylindrical, or ellipsoidal and rarely round. The identification was confirmed by sequencing the ITS-nrDNA region. The size of the amplified region was found to be varying from 564 to 606 bp in the four monosporic isolates. The sequence data alongside with BLAST search showed more than 99 % homology with M. scaettae and proved the identity of M. scaettae isolates which confirmed the morphological identifycation.

Transferability of SSR loci

Seven primer pairs (Table 2) were used on thirteen isolates of M. scaettae (Table 1), and Trichoderma sp. were tested as a control isolate. Five of the primer sets (῀71.4%) successfully amplified and generated DNA bands with expected size range and two EST-SSR markers (῀28.57) showed no amplification (Table 2). The lowest number of alleles (one allele) was detected at the SNOD21 locus, and the loci SNOD1, SNOD26, and SNOD22 generated more allele numbers (four alleles). The three alleles were detected at SNOD17 locus. The highest amplification percentage was in SNOD26 primer, which showed amplification for 92.3% of the tested isolates (Fig. 3). The low amplification rate was in SNOD21 primer, which generated the expected band in only one isolate (7.7% of the tested isolates). The EST- SSR markers, PCR annealing temperature, fragment length, percentage of polymorphism, and PIC are described in Table 2. Eventually, the five EST- SSR loci revealed a total of sixteen alleles with an average of 3.2 alleles per locus.

Fig. 3. SSR banding patterns detected by SNOD26 microsatellite in Mauginiella scattae isolates. Lane 1: hov-gh; Lane 4: ahva-med1; Lane 8: abad-ma1; Lane 12: behb-ma1; Lane 13: ramh-sa1; Lane 14: behb-kh2; Lane 17: behb-kh1; Lane 19: ahva-ma1; Lane 20: shad-sa1; Lane 21: karo-sa1; Lane 23: hend-br1; Lane 26: abad-sa1; Lane 29: karo-khad1. M: DNA size marker.

Fig. 4. Dendrogram showing a genetic relationship among thirteen Mauginiella scaettae isolates based on five microsatellite markers. Scale indicates Jaccard’s coefficient of similarity. Group I, II and III represent three groups of isolates clustered.