Introduction

Pythium oligandrum Dreschler is a cosmopolitan oomycete and is one of the five mycoparasite species assigned to clade D of the Pythium genus (Levesque and De Cock 2004). It is considered as non-pathogenic (Kilpatrick 1968; Al-Hamdani and Cooke 1983; Martin and Hancock 1986) and has received considerable attention as a potential biocontrol agent. It is effective in control of damping-off and root diseases caused by several soil-borne fungal phytopathogens (McQuilken et al. 1990; Al-Rawahi and Hancock 1998; Hase et al. 2008; Horner et al. 2012; Yacoub et al. 2016). In protection of plants it acts both directly - through mycoparasitism, antibiosis, competition for nutrients and space, and indirectly - by inducing plant resistance (Gerbore et al. 2014).

Pythium oligandrum was isolated from the soil and rhizosphere of various plants in Iran, including amaryllis (Tehran Province), aptenia (Fars Province), barley (Fars Province), beetroot (Hamadan, Khorasan, Khuzestan, West Azerbaijan Provinces), cucumber, eggplant (Razavi Khorasan Province), lantana (Tehran Province), pine (Mazandaran Province), safflower (Kermanshah Province) and turfgrass (Fars and Tehran Provinces) (Bolboli and Mostowfizadeh-Ghalamfarsa 2015; Mostowfizadeh-Ghalamfarsa 2016). Such a wide geographical distribution would suggest the high variation. However, no information is available on genetic relationship among P. oligandrum isolates from different crops.

DNA molecular markers provide valuable tools to study fungal genetic diversity. Inter simple sequence repeat (ISSR)-PCR has been developed, tested and used in genetic studies of various organisms (Poyraz 2016), including fungal intraspecific genetic variation (Lindblom and Ekman 2005). This technique includes an amplifying DNA sequences between simple sequence repeats (SSRs) with anchored or non-anchored SSR homologous primers (Zietkiewicz et al. 1994). This method does not require genome sequences and generates specific and reproducible patterns due to the highly stringent conditions of the reaction (Bornet and Branchard 2001).

Additionally, the ISSR analysis has a low cost. This method may reveal higher polymorphism compared to the random amplified polymorphic DNA (RAPD) analysis due to the repeated regions in the genome (Es-selman et al. 1999). ISSR has been used to identify repeated motifs in the genome of Pythium group F isolates by Vasseur et al. (2005).

The objective of the present research is to find out the genetic diversity among P. oligandrum isolates from different crops cultivated in various regions of Iran. Pythium oligandrum genetic variation, the relationship among populations, geographical and nutritional preferences may determine the host preferences which can be useful in potential practical biological control.

Materials and methods

Sampling and Pythium isolation

Soil samples were collected around roots of alfalfa (1 location in Kerman Province - southern Iran), barley (1 location in Fars Province - south-western Iran), chickpea (10 locations in Kermanshah, western Iran), eggplant (2 locations in Bushehr Province - south-western Iran), grapevine (1 location in Fars Province), lemon (1 location in Fars Province), lentil (8 locations Kermanshah Province - western Iran), maize (1 location), pine (1 location in Fars Province), pistachio (2 locations in Yazd Province - central Iran), potato (2 locations in Fars Province), rice (1 location in Golestan - north-eastern Iran), soybean (1 location in Golestan), tomato (8 locations in Bushehr, Fars, Ilam - western Iran), turfgrass (2 locations in West Azerbaijan - north-western Iran), walnut (1 location in Chaharmahal and Bakhtiari - central Iran), wheat (2 locations in Chaharmahal and Bakhtiari, and Golestan north-eastern Iran) in December 2013–August 2017 (Fig. 1, Table 1). Pythium spp. were isolated by baiting with 5-mm sour orange (Citrus aurantium L.) leaf disks at 20 °C for 24 h (Banihashemi et al. 1992) and plating on PARP-CMA (PARP: pimaricin 0.01 g, ampicillin 0.25 g, rifampin 0.01 g, pentachloronitrobenzene 0.1 g, CMA: cornmeal 20 g, peptone 20 g, glucose 20 g, agar 15 g, distilled water 1 L) (Jeffers and Martin 1986). After 36 h the single colonies were prepared with hyphal tip method on 1.5% water agar. Cultures were subcultured regularly on CMA and incubated at 25 °C in the dark.

Fig. 1
figure 1

Geographical locations of Pythium oligandrum isolates studied (Original map is from https://d-maps.com/carte.php?num_car=5494&lang=fr)

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Table 1 Pythium oligandrum isolates recovered from soils of various crops in Iran. Isolates used in this study, origin, hosts, and coordinates
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Identification of P. oligandrum isolates

Pythium oligandrum was identified on morphological characters (oogonia, sporangia, antheridia) and molecular data of the ITS rDNA (with primers P.OLIG.F1 (5’-CTGTGCTTCGTCGCAAGACT-3′ and P.OLIG.R.04 (5′- CTTTAAAAAGACAGCGCGAGA-3′) and specific genes encoding elicitin-like proteins (with primers OilgandrinF (5’-ATGTTCACCAAGACCTTGG-3′ and OligandrinR (5′- TTAAGCGGAGCCAACCACGG-3′) (Godfrey et al. 2003; Masunaka et al. 2010). Amplifications were performed in a 20-μL reaction volume (Vasseur et al. 2005), with an initial denaturation for 2 min at 95 °C, followed by 30 cycles of 40 s denaturation at 94 °C, 50 s of primer annealing at 60 °C for P.OLIG.F1/P.OLIG.R.04 and at 58 °C for OligandrinF/OligandrinR, 60 s extension at 72 °C, and a final extension at 72 °C for 10 min. The presence of PCR products was confirmed by gel electrophoresis.

Genetic diversity analysis with inter simple sequence repeat (ISSR)-PCR

The ISSR markers were amplified using seven primers (Table 2). The PCR amplification was carried out in 20-μL reaction mixture (Weiland et al. 2015) with an initial denaturation for 2 min at 95 °C, followed by 35 cycles of 1 min denaturation at 94 °C, 50 s of primer annealing at the specific temperature, 60 s extension at 72 °C, and a final extension at 72 °C for 10 min. Effectiveness of amplification was checked by electrophoresis on a 1.5% agarose gel.

Table 2 Inter simple sequence repeat (ISSR) primers used for analysis of Pythium oligandrum genetic variation
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Amplification products were scored for the presence (1) or absence (0) of bands, and a binary matrix was used to estimate the genetic similarities between pairs. The unweighted pair group method with arithmetic mean (UPGMA) clustering method was used to generate a dendrogram for P. oligandrum by computing the GS values with Jaccard coefficient in NTSYSpc 2.2 program (Rohlf 2000). The genetic diversity was analyzed in populations of 5,10 and 18 isolates, from Bushehr, Fars and Kermanshah Provinces, respectively. Genetic variation for each locus and population was analyzed with Nei’s gene diversity index (H), Shannon index (I), observed (Na) and effective (Ne) number of alleles, number of polymorphic loci (PL), percentage of polymorphic loci (PPL) and also Nei’s genetic distance (Yeh et al. 1999). Total genetic diversity (Ht), mean diversity within each population (Hs), Nei’s coefficient of genetic diversity (Gst) and gene flow (Nm) were calculated according to Nei (1986). Principal coordinate analysis (PCA) and analysis of molecular variance (AMOVA) was performed using GenAlEx 6.41 (Peakall and Smouse 2006).

Results

Collection of P. oligandrum

Forty-five P. oligandrum isolates were collected from soil around roots of alfalfa, barley, chickpea, eggplant, grapevine, lemon, lentil, maize, pine, pistachio, potato, rice, soybean, tomato, turfgrass, walnut and wheat in eight provinces in Iran (Fig. 1, Table 1). The isolates were characterized by ornamented oogonia with acute spines, contiguous sporangia with (sub-) globose elements connected by hypha and often absence of antheridia (Van der Plaäts-Niterink 1981). Identification was confirmed by PCR with specific primers.

Cluster analysis

A total of 102 DNA fragments were amplified using ISSR primers. Three clusters were obtained with UPGMA (Fig. 2). The cluster 1 consisted of 11 isolates from lemon, lentil, pistachio, rice, soybean, tomato and wheat soils in Chaharmahal & Bakhtiari, Fars, Golestan and Kermanshah. The major cluster 2 consisted of 32 isolates from alfalfa, chickpea, grapevine, grass, lentil, maize, pine, potato, tomato and walnut soils in Chaharmahal & Bakhtiari, Fars, Kerman, Kermanshah, Uromia, Yazd. The cluster 3 included only two isolates from barley and tomato soils in Fars Province. All eighteen isolates from Kermanshah Province were in clusters 1 and 2 and all 10 isolates from Fars Province were in clusters 2 and 3. Genetic similarity between isolates from soils of chickpea and lentil was 100% and 96% respectively in Kermanshah Province. Genetic similarity between isolates from soil of eggplant in Bushehr Province, and soils of soybean and rice in Golestan Province was 90%.

Fig. 2
figure 2

Dendrogram of Pythium oligandrum isolates from Iran based on inter simple sequence repeat (ISSR) markers

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Genetic diversity of populations

Nei’s gene diversity index (H), Shannon’s index (I), observed (Na) and effective (Ne) number of alleles, number of polymorphic loci (PL) and percentage of polymorphism (PPL) indicate the highest and the lowest polymorphs of P. oligandrum from Fars and Bushehr, respectively (Table 3). Nei’s pairwise genetic distances between the populations ranged from 0.037 to 0.077 (Table 4). The highest genetic distance was obtained between isolates from Bushehr and Fars, and the lowest genetic distance was obtained between isolates from Bushehr and Kermanshah. Nei’s gene diversity statistics, including total genetic diversity (Ht) and mean diversity within each population (Hs) indices were 0.31 and 0.27, respectively. Nei’s coefficient of genetic diversity (Gst) was 0.11, indicating that genetic variance among the populations was 11%. Gene flow (Nm) showing the average number of migrants among the population was 3.96. The PCA scatter plot based on the ISSR dataset showed vague borders between the three populations (Fig. 3). No clear separation between the different populations was highlighted. The first and the second principal coordinates accounted for 21.81% and 33.21% of variation, respectively. Variation among and within populations from three different provinces was 7% and 93%, respectively (Table 5).

Table 3 Genetic variation statistics for Pythium oligandrum populations from three different provinces in Iran
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Table 4 Nei’s genetic distance between pairs of Pythium oligandrum populations from three different provinces in Iran
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Fig. 3
figure 3

Scatter plots of the first and second principal coordinates analysis (PCA) based on inter simple sequence repeat (ISSR) data of three populations of Pythium oligandrum in Iran. Individuals within the same population are represented with the same icon

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Table 5 Data from analysis of molecular variance (AMOVA) of Pythium oligandrum populations
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Discussion

The genetic structure and diversity of P. oligandrum population from Iran was evaluated using ISSR markers. A moderate level of genetic diversity was found within three P. oligandrum populations. The level of genetic diversity of our P. oligandrum was: (i) similar to diversity in Pythium aphanidermatum (Edson) Fitzp. in Pennsylvania (USA) (Lee et al. 2010) and (ii) higher than diversity of P. aphanidermatum in Oman (Al-Sa'di et al. 2012).

Pythium oligandrum is a homothallic organism, and self-fertilization in sexual reproduction (Huzar-Novakowiski and Dorrance 2018) should reduce the genetic variation. It happened in homothallic Globisporangium irregulare (Buisman) Uzuhashi, Tojo & Kakish. (syn. Pythium irregulare Buisman) and Globisporangium ultimum (Trow) Uzuhashi, Tojo & Kakish. (syn. Pythium ultimum Trow) from forest nursery soils (Weiland et al. 2015). However, different oomycetes behave differently. Genetic diversity among populations of various oomycetes seems to be variable, depending on the level of inbreeding and out-crossing, location and climate. The dominating out-crossing in sexual reproduction of Globisporangium sylvaticum (W.A. Campb. & F.F. Hendrix) Uzuhashi, Tojo & Kakish. (syn. Pythium sylvaticum W.A. Campb. & F.F. Hendrix) and P. aphanidermatum contributed to the high diversity of the former (Weiland et al. 2015) but not of the latter (Al-Sa'di et al. 2012).

High and low genetic variability respectively within and among populations emphasize that the most of the variation exists within populations. Similar results were reported for another oomycete, e.g. Phytophthora capsici Leonian (Li et al. 2012). Many individuals within such populations are likely to be genetically different.

Grouping of the isolates in three major clusters was not associated with geographical or nutritional preferences. It partly agrees with Zhao et al. (2007), Al-Sa'di et al. (2008), Amini et al. (2014) and Bentes et al. (2018) who reported that there is not significant positive correlation between genetic variation and geographical or nutritional preferences of species, including P. aphanidermatum. We showed that the external factors, such as host, location and climate do not affect the genetic structure of P. oligandrum.

Gene flow is one of the evolutionary forces that may have a significant impact on the genetic diversity of a population (Nourollahi et al. 2011). It indicates the frequent migration and high genetic exchange (Weiland et al. 2015). The high level of gene flow among our populations indicates the lack of clear-cut boundaries. This is shown also by principal coordinate analysis (PCA), which showed no definite separation between individuals from different populations. The high level of gene flow has also been observed in G. irregulare and G. sylvaticum in forest nursery soils (Weiland et al. 2015). In the absence of gene flow, genetic drift causes different allele frequencies at neutral loci, leading to differentiation in isolated populations (Keller et al. 1997). Genetic diversity of population can be caused by migration or transfer (with soil or plants) of microorganisms’ genotypes. It is limited, but possible (Lee et al. 2010; Weiland et al. 2015; Huzar-Novakowiski and Dorrance 2018).

Conclusion

Study show the diversity and evolutionary potential of antagonistic P. oligandrum. The collection of P. oligandrum isolates representing the range of its genetic diversity in Iran could help to screen isolates for biocontrol agent.