INTRODUCTION

Gibberella intermedia (Kuhlman)Samuels, Nirenberg and Seifertis a member of G. fujikuroi species complex (Leslie 1991). This mating population has been assigned to anamorphic F. proliferatum (Matsushima) Nirenberg known as G. fujikuroi mating population D and is a pathogen of some economically important plants worldwide including rice, maize, citrus fruit, banana, orchids, sorghum (Leslie & Summerell 2006), onion (Galván et al. 2008)and sugarcane (Alizadeh 2010). Particularly in Iran, it has been  known as the causal agent of stalk and ear rot of maize (Bujari et al. 1993, Ghiasian et al. 2004), foot rot disease of rice (Padasht-dehkayi 1993), basal and root rot disease of onion (Rabiee motlagh 2009) and knife cut disease of sugarcane (Alizadeh 2010). The species produce wide variety of mycotoxins often at high levels in seeds and crops through toxic agents including beauvericin, fumonisins, fusaproliferin, fusaric acid, fusarins and moniliformin (Leslie & Summerell 2006). Recently, the incidence and severity of these diseases have increased drastically in Iran. However, to the best of our knowledge, no comprehensive study on the Iranian F. proliferatum populations has been conducted. As well, little is known about genetic variability of its host in Iran. Intraspecific differences in the pathogenicity of individual isolates from different hosts have not been extensively studied either.

In the majority of plant pathogen systems much of the breakdown of plant resistance genes is due to pathogen population evolving virulence, rather than the nature of the employed resistance gene (Garcia-Arenal & McDonald 2003). Thus, to breed for durable disease resistance, a study of the population genetic and evolutionary potential of pathogen populations is in demand. The pathogens with the highest evolutionary potential pose the highest ‘risk’ of defeating resistance genes or counteracting other control methods such as applications of pesticides or antibiotics (McDonald & Linde 2002). Two important factors for pathogen evolution are the reproduction/ mating system and gene/genotypic flow.

Sexual fertility is an important practical parameter to understand the structure of fungal populations as an evidence of sexual cross-fertility. This is usually required when two strains are assigned to a common species (Leslie &Klein 1996). Assessing the potential for mating by toxigenic strains of Fusarium would increase our understanding of the genetic mechanisms that maintain intraspecific diversity as well as biological and evolutionary integrity of the species. The reproduction/mating system will affect the way that alleles are distributed in a population. Also, sexual reproduction combines favorable alleles into the same genetic background, resulting in genotypes with higher fitness or higher levels of virulence/fungicide resistance. Besides the occurrence of sexual reproduction, its frequency is also a prominent parameter to design strategies of plant pathogens. These strategies are often different for clonally and sexually reproducing organisms (McDonald & McDermott1993).

In the previous study, vegetative compatibility grouping of the same Iranian populations of F. proliferatum from maize, rice, sugarcane and onion showed that natural populations of the speciesin Iran are genetically highly divergent and include isolates representing a potential risk for disease development (Alizadeh 2010). A correlation between VCGs grouping and host preferences also were founded. Assessing of genetic diversity based on RAPD molecular marker (Mohamadian et al. 2011) showed that maize and rice isolates of F. proliferatum in Iran are clearly divergent. Therefore, in order to better examination of genetic diversity, we conducted AFLP molecular marker to identify distinct F. proliferatum isolates from different hosts.

The objective of this research was to investigate the genetic structure of F. proliferatum populations from various hosts including maize, rice, sugarcane and onion in different areas of Iran using AFLP molecular markers. Specifically, we investigated the evolutionary potential of pathogen populations by determining their genetic diversity, reproduction system and the degree of gene flow among host populations. We aimed to determine whether observed patterns of genetic diversity are consistent with strict clonality, random mating, or a mixture of asexual and sexual reproduction. Furthermore, this research was conducted to determine whether host populations of the fungus are distinct populations in Iran that has evolved separately, or whether they belong to the same larger panmictic population. Thus, we studied the extent of genetic differentiation within and among populations. Also, we conducted pathogenicity test to determine whether there are differences in the ability of each of these four host populations to infect maize ears.

MATERIALS AND METHODS

Fungal isolates

A total of 142 F. proliferatum single-spore isolates which formerly have been collected from diseased maize, rice, sugarcane and onion from various areas of Iran were selected for population genetic analysis (Fig. 1). Among them, 79 isolates have been recovered from maize stalks and seeds obtained from nineteen locations in 10 maize producing provinces throughout of Iran (i.e. Ardabil, Isfahan, Tehran, Fars, Qazvin, Kermanshah, Golestan, Mazandaran, Hamadan and Kerman provinces) during the 2004-2005 growing season. Others were include 23 isolates from comer- cial rice fields in Guilan and Mazandaran provinces in north of Iran, 20 isolates from onion in Khorasan province and 20 isolates from sugarcane grown in commercial sugarcane farms in Khuzestan. These isolates were identified using the morphological characteristics of the species as described by Leslie & Summerell (2006) and Nelson et al. (1983). Isolates were stored on filter papers at -20 °C in fungal culture collection, at mycology laboratory, University College of agriculture and natural resources, university of Tehran, Karaj, Iran.

Fig. 1. Map of Iran showing sampling regions and data related to F. proliferatum isolated from maize ears in 10 Iranian provinces (i.e. Ardabil, Isfahan, Tehran, Fars, Qazvin, Kermanshah, Golestan, Mazandaran, Hamadan and Kerman provinces) during 2004 and 2005. Rice isolates obtained from commercial rice fields in Guilan and Mazandaran provinces in north of Iran. Onion isolates recovered from in Khorasan province and sugarcane isolates recovered from commercial sugarcane farms in Khuzestan.

The isolates are summarized (including their host plant species, year of isolation, geographical origin, mating type and VCG s groups) in Table 1. For the Maize and onion F. proliferatum isolates used in this study, morphological identification was confirmed by PCR assay using specie-specific primers, conducted by Rahjoo et al. (2008) and Rabiee Motlagh (2009), previously.

Amplified fragment length polymorphism (AFLP) Analysis

For AFLP analysis, fungal isolates were cultured in 100 ml flasks containing 50 ml of potato dextrose broth (PDB), and grown for seven to 10 days in dark, at 25°C. Fungal mycelium was harvested, dried by vacuum filtration through a filter paper, and lyophilized overnight.

Total fungal DNA was extracted from 20-40 mg of lyophilized mycelia grounded in 2 ml tubes using a Core-one^tm Plant Genomic DNA isolation Kit (Corebio, Korea) according to the manufacturer’s instructions.

Four hundred nano-grams of DNA suspension was used for AFLP reactions. AFLP fingerprinting was done as described by Vos et al. (1995) using combination of two restriction enzymes: EcoRI–MseI. After digestion and ligation, pre-amplification was performed in a volume of 20 μl using EcoRI0: 5'-CTCGTAGACTGCGTACCAATTC-3' and MseI0: 5'-CACGATGAGTCCTGAGTAA-3' primers without extra-nucleotides. Subsequently, six primer pair combinations were used for selective amplification (Table 2). Reactions were performed in 25 μl containing 5 μl aliquot of the pre-amplification template (v/v 1/15), 50 ng primer, 0.2 mM of all four dNTPs, and 0.2 U Taq polymerase (Smart Taq, Fermentase, Germany) in PCR buffer (PCR buffer, Sinagen co., Iran). The amplification was done in a termocycler CG1-96 (Corbett Research, Australia), and consisted of an initial denaturation for 2 min at 94 °C followed by 10 cycles of denaturation at 95 °C for 30 s; annealing at 63 °C for 30 s and extension at 72 °C for 2 min, followed by 25 cycles of denaturation at 95 °C for 30 s; annealing at 54 °C for 30 s and extension at 72 °C for 2 min, with a final extension for 5 min at 72 °C. From each sample, 5 μl of AFLP products was loaded on a 6% denaturing polyacrylamide gel (Merk, Germany), and gel electrophoresis was performed in a Biorad gel electrophoresis system (Biorad, USA).

Table 2. Six primer pair combinations were used for selective amplification in AFLP analysis.

Polyacrylamide gels were stained by silver nitrate and then photographed. Images were scored manually for the presence or absence of bands and bands between 200 and 700 bp in length were scored manually. Analysis was performed based on 248 polymorphic AFLP bands. The AFLP fingerprint patterns obtained were converted into binary data matrices containing arrays of 0 and 1.

The presence of a band was scored as 1 and its absence as 0. Only bands that amplified conspicuously over several DNA extractions and PCR experiments with the isolates were considered as reproducible and used for analysis.

The binary matrices was analyzed with NTSYS-pc ver. 2.20 (Rohlf  2000) software using the band-based DICE similarity coefficient and the clustering of fingerprints was performed with the unweighted pair group (UPGMA) method by using average linkages (Nei & Li 1979). DNA samples from three isolates were submitted to the AFLP procedure repeated in triplicate. In order to compare isolates from different hosts, polymorphic bands were analyzed by POPGENE 32 ver. 1.31, GenALEX version 6.1 and Nei coefficient. Nei’s unbiased measure of gene diversity, H (Nei 1978) and Shannon's Information index, I (Lewontin 1972) for all four host populations were estimated in POPGENE 32 ver. 1.31 (Yeh et al. 1999). To estimate the distribution of genetic variation among and within host populations, a hierarchical analysis of molecular variance (AMOVA) and the significance levels of genetic variations were calculated using a permutation test with 1000 permutations using GENALEX ver. 6.1 (Peakall & Smouse 2006). Nei’s unbiased genetic distance was estimated for all four host populations in POPGENE 32 ver. 1.31 (Yeh et al. 1999). The extent of gene flow between populations was estimated from GST (Slatkin & Barton 1989) in POPGENE 32 ver. 1.31 (Yeh et al. 1999).

Sexual crossing and female fertility

Crosses to confirm mating population, investing- ation of sexual fertility status and identifying of mating types were made in carrot agar using the protocol developed by Klittich and Leslie(1988a). Standard tester strains 8549 (MATD-1) and 8550 (MATD-2) as the female parent and the uncharacterized field isolates as male parent were used for crosses. In all crosses, a positive and negative control was included. The female parent was inoculated by transferring a plug of mycelia onto a 60-mm carrot agar plate, and simultaneously the male parent was inoculated onto a PDA slant (8 ml medium in a 25 × 150 mm culture tube). After 7 days, the male conidia were suspended in 3-5 ml of a 2.5% tween 60 solution, and 1-1.5 ml of the suspension were gently spread over the surface of the female parent colony with a glass rod by thoroughly wetting the mycelium. Plates were incubated in the dark at 25°C for 2-3 days in order to provide mycelia contact. Finally, cultures were transferred to an incubator where temperature was maintained at 22-23°C with high humidity for about a month. A mixture of cool white fluorescence and near-UV was used for providing the desired light intensity to cultures. After the incubation period, plates were examined daily for the growth of fertile perithecia under stereomicroscope and crosses recorded positive when ascospore-oozing perithecia were observed. For each cross, fertility was confirmed by the observation of cirrhus on top of the perithecia. Female fertility examination for 40 randomly selected field isolates including 10 isolates of each host populations was conducted in crosses using the protocol described above (Klittich & Leslie1988a) in which the field isolates used as female parents and the standard testers were the male parents. After 4-6 week, plates were examined daily for the growth of the fertile perithecia.

Multiplex PCR for MAT-1 and MAT-2 idiomorphs

Fusarium isolates were grown on PDA plates for 7 days and mycelia were harvested and grounded in liquid nitrogen. Total DNA was extracted from grounded mycelia of each isolate (~200 mg wet weight) using a Core-one^tm Plant Genomic DNA isolation Kit (Corebio, Korea) according to the manufacturer’s instructions.

Multiplex PCR was conducted using the two pairs of degenerate PCR primers as introduced by Ker^'enyi et al. (2004) to amplify highly conserved alpha box and HMG box domains found in the MAT1 and MAT2 portions, respectively. The sequences of the primers used were as follow: FusALPHA forward: 5′-CGCCC TCT(GT)AA(CT)G(GC)CTTCATG-3′, FusALPHA reverse: 5′-GGA(AG)TA(AG)AC (CT)TTAGCAAT (CT)AGGGC-3′, FusHMG  forward: 5′-CGACCTC CCAA(CT)GC(CT) TACAT-3′ and FusHMG reverse: 5′-TGGGCGGTA CTGGTA(AG)TC (AG) GG-3′. The oligonucleotides were synthesized by Eurofins MWG operon (Germany).  Each PCR mixture (20 μl) contained 12.5 μl of sterilized distilled water, 2 μl of 10 × PCR buffer, 0.8 μl (2 mM) MgCl₂, 1 μl (0.5 mM) of dNTPs, 1.2 μl (6 μM) primer, 0.5 μl Smar Taq DNA Polymerase (2.5 U/ μl; CinnaGen Co., Iran) and 2 μl (~10 ng) DNA template. The PCR amplification was performed using the following programs: an initial denaturation at  94 °C for 2 min followed by 35 cycles of 30 s at  94 °C, 45 s at 55 °C and 60 s at 72 °C with a final extension of 10 min at 72 °C in a CG1-96 thermocycler (Corbett Research, Australia). Amplification products were separated by electrophoresis on a 1% (wt/vol) agarose gel for 90 min at 100 V in 1X TBE buffer (0.09 M Tris, 0.09 M boric acid and 0.002 M EDTA, pH 8.0). To visualize the amplified DNA, gels were stained with 1% ethidium bromide and then photographed by trans illumination using a Gel-Documentation (IMAGO, B & L System, the Netherland).

Pathogenicity

A total of 32 isolates (8 isolates from each host: maize, rice, sugarcane and onion) were selected for pathogenicity test on maize ear. A susceptible maize genotype K74/1 line would be a likely good candidate for Fusarium colonization and was selected as the experimental genotype for pathogenicity test. Experiments were conducted during the growing season of 2012 in irrigated sites at the Seed and Plant Improvement Institute (SPII), Karaj, Iran. All soils were fertilized according to regional recommendation. Field inoculations were performed according to Yates and Sparks (2008) at post-pollination during the brown silk stage (Fig. 2a). Each of ears was inoculated with a 5 ml of a suspension of 1×10⁶ F. proliferatum conidia with a 400-gauge needle. The needle was inserted into the ear until resistance from the cob was felt and then was withdrawn gently while the conidial suspension was slowly dispensed. Pathogenicity test performed in a randomized complete block design (RCBD), 3 blocks and 33 lines in each block assigned for the pathogenicity test. Five maize ears in each line of each block inoculated by one F. proliferatum isolate. Also, one line in each block specified as control randomly and inoculated with 5 ml double deionized sterile water. Thus, the total ears analyzed were 2400 based on 32 (isolates) × 5 (replicate) × 3 (plot). Ears were harvested at kernel maturity during mid-October of year. Disease severity was scaled according to scale proposed by Reid & Zhu (2002) (Fig. 2b).

Furthermore, morphological identification of the isolates as F. proliferatum was confirmed by sexual crosses (Fig. 3). Only, eight isolates did not produce perithecium in the crosses. All of the 40 isolates were used for determination of male-female fertility in current study were female-sterile or male-fertile.

Identification of mating type idiomorphs

The applicability of the diagnostic PCR method for mating type identification was tested on 142 F. proliferatum isolates obtained from maize, rice, sugarcane and onion plants. Both MAT-1 and MAT-2 individuals were identified among these isolates (Fig. 4). The MAT-1- and MAT-2-specific fragments of 200 and 260 bp, respectively, were amplified in different isolates of Fusarium. Based on PCR amplification, among 142 isolates, 72 isolates (50.7%) including 40 isolates (50.6%) from maize, 14 isolates (60.8%) from rice, 5 isolates (25%) from onion and 13 isolates (65%) from sugarcane were identified as MAT-1 and 70 isolates (49.2%) including 39 isolates (49.3%) from maize, 9 isolates (39.1%) from rice, 15 isolates (75%)  from onion and 7 isolates (35%) from sugarcane belonged to MAT-2.

AFLP analysis of F. proliferatum isolates

The AFLP analysis of the fungal collection using six primer combinations yielded a total of 301 bands. Table 2 shows the number and percentage of polymorphic bands reproduced by each primer combinations. Finally, analysis was performed based on 248 polymorphic markers. Figure 5shows the phenetic relationships among F.proliferatum isolates from four different hosts including: maize, rice, sugarcane and onion for the combined data set, including all six primer combinations. Based on denderogram, the isolates of F. proliferatumwere separated according to the host plants. In general, F. proliferatumisolates were separated into four distinct groups. The isolates from sugarcane were grouped into two distinct groups.

The isolates from rice were grouped in a unique distinct group. However, maize and onion isolates were grouped in a unit fingerprinting group. Genetic similarity between four mentioned host populations was more than 70%.

Fig. 5. Dendrogram generated from UPGMA cluster analysis of 80 Fusarium proliferatum isolates using DICE similarity coefficient, showing four groups according to the hosts.

Population genetics analysis

Nei’s unbiased gene diversity (H) for four populations ranged from 0.1518 to 0.3928. Largest gene diversity values were for sugarcane and maize populations and smallest values were for rice and onion populations, respectively (Table 3).

Also, Shanon`s information index (I) ranged from 0.2307 to 0.3295. Sugarcane, maize, onion and rice host populations have largest to smallest account of I (Table 3).

In total, from 248 polymorphic AFLP bands, the number of polymorphic bands in each of the populations was from 48% for onion populations to 75% for sugarcane populations (Table 3).

Likewise, gene flow (Nm) values among populations comparisons was 0.86.

Nei’s unbiased genetic distance (Nei 1978) between pairwise comparisons of host populations ranged from 0.0346 to 0.3048. The smallest genetic distance in pairwise comparisons was seen between maize and onion populations and the largest were for rice and onion populations (Fig. 6).

The results of AMOVA showed that 60% of the total genetic variation was attributable to the differences among populations, whereas 40% was due to the variation within populations.  

Pathogenicity

Pathogenicity tests compared the effects of the maize, rice, onion and sugarcane isolates on maize ears. Typical rotting symptoms developed and the fungal mycelia grew over the ears (Fig. 7b). Evaluation of virulence of isolates was done on the basis of disease severity (%DS) index at physiological stage. The results of the analysis using of variance of data by Duncan’s test showed that difference between treatments were statistically significant (α = 1%). The data, summarized in Table 4, showed that all studied F. proliferatum isolates originated from maize, rice, onion and sugarcane were pathogenic to maize ears and there were a considerable differences among the isolates for their virulence ability. So that different levels of pathogenicity were observed among the isolates. No disease symptoms were found in the sterile water control treatments (Fig. 7a). Among all of the isolates the maize isolates had greatest (35%) and the rice isolates showed smallest (6%) degree of pathogenicity in maize ears. Also, sugarcane and onion isolates showed 32% and 24% of pathogenicity, respectively. Totally, MTF4-3 isolate originating from maize showed the highest level of virulence (64%) and isolates MRP29 recovered from rice showed the lowest levels of virulence (2.2%) among all isolates.

Table 3. Accounts of H, I, number of polymorphic bands and percentage of polymorphic bands in the populations of Fusarium proloferatum.

H = Nei gene diversity

I  = Shanon` s information index [Lewontin (1972)]

Fig. 6. Nei’s unbiased genetic distance dendrogram between pairwise comparisons of host populations of Fusarium proliferatum.

Fig. 7. Pathogenicity test of the maize, rice, onion and sugarcane isolates on maize ears. a. Control treatments. b. Typical rotting symptoms developed over the ears by Fusarium proliferatum isolates.