Vegetative compatibility and rep-PCR DNA fingerprinting groups of Fusarium solani isolates obtained from different hosts and their pathogenicity

DNA extraction

Three to four mycelia plugs (each 4 mm in diameter) from PDA cultures were transferred to flasks containing 60 ml of potato dextrose broth, and incubated at room temperature for 6–8 days. Mycelial mass was filtered through a filter paper, washed three times with sterile water, air-dried and then frozen at -80^°C and lyophilized prior to use. Lyophilized mycelia were ground in liquid nitrogen into a fine powder with a mortar and pestle. Fungal genomic DNA was extracted using Core-one ^tm Plant Genomic DNA isolation Kit (Core Bio, Korea) following the manufacturers’ instructions.

Rep-PCR analysis

Rep-PCR reaction was performed using BOX primer (1A-1R 5'-CTACGGCAAGGCGACGCTG ACG-3') (McDonald et al. 2000). PCR was carried out using PCR Master Mix kit (CinnaGen PCR Master Kit). Each reaction consisted of the following components: 10 μL of PCR Master Mix (containing 1 U of Taq DNA polymerase, 2 μL PCR buffer, 3 mM MgCl₂ and 0.25 mM of each dNTPs), 10 pM primer, 3 μL (30 ng/μl) of fungal genomic DNA and 5.5 μL distilled water in a final reaction volume of 20 μL. PCR was performed in a palm Thermal Cycler (CG1-96, Corbett Research, Australia) with the following PCR program. An initial denaturation step of 5 minutes at 95^°C followed by 35 cycles of denaturation at 94^°C for 3s and 92^°C for 30s, 1 min of annealing at 49^°C, extension for 7 min at 72^°C and a final extension for 10 min at 72^°C.

Data analysis

To determine the genetic relationships among the isolates, the presence or absence of DNA bands was converted into binary data (1 for presence and 0 for absence of each band). Similarity matrix was calculated with Dice’s coefficient and the SIMQUAL program of NTSYSpc, ver. 2.1. Cluster analysis was carried out within the SAHN program by using the UPGMA (Unweighted Pair Group Method with Arithmetic Mean).

Virulence of VCGs representatives on potato plants in greenhouse

For greenhouse pathogenicity tests, spore suspensions were created following Romberg & Davis (2007). Macroconidia and microconidia were harvested from 2-week-old cultures grown on PDA at 25^ºC by adding three ml of sterile water to the plates and scraping the surface of the agar plate with a sterile glass slide. The resulting conidial or mycelial suspension was filtered through eight layers of cheesecloth to remove the mycelial fragments. Conidial concentrations were calculated using a hem cytometer and diluted in water to a concentration of 10⁶ macro- and microconidia/ml for pathogenicity tests. The pathogenicity of twenty three F. solani isolates obtained from potato, common bean, chickpea, cucumber, watermelon and melon as VCGs representatives was determined on healthy 8-10 cm-tall potato seedlings of Agria cv., the most frequently potato cultivar in all potato growing regions of Khorasan-Razavi province, Iran. After trimming the roots, the entire root system was immersed in a 10⁶ macro- and microconidia/ml suspension of each isolate. Each seedling was transplanted into three separate 15-cm-diameter pots containing sterile soil (5:3:2:1 mixture of farm soil, sand, animal manure and compost). Each treatment was replicated three times. The inoculated plants were maintained in greenhouse at 25^ºC to 28^ºC for 45- 50 days, and then removed from the pots. Then, they were scored for disease severity based on a 0–3 scale (0 = No disease, 1 = mild root rot, 2 = vascular discoloration of the root and crown, 3 = vascular discoloration of the root, crown and stem) (Moradzadeh Eskandari 2010).

Virulence of VCGs representatives on potato tubers under lab conditions

Tubers of Agria cv. were used to determine the virulence of the same VCGs representatives as those used in the pathogenicity tests on plants. Initially, the tubers appearing healthy and uniform in size (100-120 g) were selected and washed to remove excess soil, surface sterilized in 0.5% sodium hypochlorite solution for 10 min, rinsed with three changes of sterile distilled water and then air dried. Afterwards, the tubers were wounded with a four mm-diameter cork borer to a depth of four mm (Theron & Holz 1989) and inoculated with all of the fresh F. solani mycelia (from 1-week-fresh cultures grown on PDA at 25^°C) by putting 7 mm PDA blocks. All the wounded potato tubers were wrapped in paper bags (Manici & Cerato 1994) and incubated at 20^°C under dark conditions for three weeks. Blocks of PDA medium were used as control. Each treatment was replicated three times. At the end of incubation, tubers were cut through the inoculation points, and the degree of rot was estimated based on a zero to five (or A-F) scale, basically according to Theron and Holz (1989): 0. (A) - Limited discoloration, no exten-
ded dry rot in inoculated areas; 1. (B) - Limited discoloration with the development of dry rot in inoculated areas; 2. (C) - Extensive discoloration with increased dry rot in inoculated areas; 3. (D) - Extensive discoloration with extensive dry rot in inoculated areas; 4. (E) - discoloration and very extensive dry rot, tubers not disappearing completely; 5. (F) - discolora-
tion and very extensive dry rot, tubers disappearing completely.

RESULTS

Determination of VCGs

Production of chlorate-resistant sectors was very low on MMC, PDC and CDAC containing 15 g/l KC10₃, but after increasing the concentration of KClO₃ in the three media to 50-70 g/l, most of the isolates readily formed chlorate-resistant sectors (Fig. 1a). Totally, fifty two isolates produced chlorate-resistant sectors. These sectors were then transferred to MM containing NaNO₃ as the sole nitrogen source. The sectors with thin expansive growth on MM were considered as nit mutants. A few sectors resistant to chlorate were recovered, but they had wild type colony morphology on MM. Such mutants are known as Chlorate resistant isolates utilizing nitrate (Crn) (Bowden & Leslie 1992).

Four different nitrogen sources were used in order to identify phenotypic classes. Three classes of nit mutants were recovered representing mutations at a nitrate reductase structural locus (nit l, unable to utilize nitrate), a nitrate-assimilation pathway-specific regulatory locus (nit 3, unable to utilize nitrate or nitrite) and loci affecting the assembly of a molybdenum-containing cofactor necessary for nitrate reductase activity (Nit M, unable to utilize nitrate, hypoxanthine or uric acid). The frequency of nit 1, nit 3 and Nit M phenotype was 44.74%, 36.84% and 18.42%, respectively. Nit l mutants were recovered at a higher frequency than nit 3 and Nit M mutants.

Complementation between nit mutants was indicated by the development of a dense aerial growth where the mycelia of the colonies grew together and anastomosed. When Nit M mutants were paired-or were involved in the pairing, complementation occurred more rapidly than those of other nit mutant pairs. When nit 1 and nit 3 mutants were paired, weak vegetative compatibility reactions were obtained. Those isolates that had two mutants available for complementation tests, and had positive intra-strain complementation tests were classified as heterokaryon self-compatible (HSC) and those with negative intra strain complementation tests were considered as heterokaryon self-incompatible (HSI). One of the 52 isolates was HSI. Six multi-strain VCGs were identified among the HSC strains (Fig. 1b). These groups were designated as VCG A to VCG W. VCG A as the largest group had 18 members and VCGs B, C and D comprised of 8, 6 and 3 members, respectively. Each of the VCGs E and F consisted of two members. Seventeen isolates out of 51 HSC isolates were incompatible with all of the other isolates and therefore were considered as single-strain VCGs with designation as VCG G to VCG W. Generally, isolates of each of the VCGs originated from different hosts and usually from different regions (Table 2). VCG A included fourteen isolates of potato, three isolates of chickpea and one isolate of common bean, which belonged to several regions (Chenaran, Sabzevar, Fariman, Torbat-e Heydarieh, Quchan, Nishapur, Mashhad and Birjand). VCG B consisted of seven isolates of potato and one isolate of common bean, obtained from four regions (Chenaran, Fariman, Torbat-e Heydarieh and Mashhad). Three isolates of common bean, one isolate of potato, one isolate of chickpea and one isolate of melon were grouped in VCG C. The members of this VCG were obtained from six regions (Chenaran, Torbat-e Heydarieh, Mashhad, Faruj, Kashmar and Ardabil). VCG D contained 3 isolates of three host plants (potato, common bean and chickpea) and three different regions (Kashmar, Qaen and Torbat-e Heydarieh). Two isolates of chickpea from two different regions (Faruj and Mashhad) were grouped in VCG E. VCG F contained 2 isolate of potato, obtained from Birjand. Therefore, there was no correlation between VCGs and hosts and geographical origin of the isolates (Fig. 1c).

Rep-PCR DNA fingerprinting

Totally, 37 fragments were amplified using BOX primer, ranging in size from 2000 to 3500 bp, all of which were polymorphic (Fig. 2). Cluster analysis revealed that all of the isolates examined were placed in two major fingerprinting groups (designated as Fo₁ and Fo₂) with at least 30% genetic similarity (Fig. 3). Table 3 exhibits diversity of VCGs according to rep-PCR analysis using BOX primer.

Pathogenicity test of VCG representatives on potato tubers revealed that among 23 VCGs representatives tested, 21 isolates showed various virulence and only isolates CH-25 (VCG E) and FW-52 (VCG T) caused no symptoms on tubers of Agria cv. Isolates Fpo-5 (VCG B), Fpo-70 (VCG I) and Fpo-69 (VCG A) from potato possessed the highest virulence (Fig. 5). Details of these tests and isolates grouping are given in table 4.

Rep-PCR DNA fingerprinting

Totally, 37 fragments were amplified using BOX primer, ranging in size from 2000 to 3500 bp, all of which were polymorphic (Fig. 2). Cluster analysis revealed that all of the isolates examined were placed in two major fingerprinting groups (designated as Fo₁ and Fo₂) with at least 30% genetic similarity (Fig. 3). Table 3 exhibits diversity of VCGs according to rep-PCR analysis using BOX primer.

Fig. 2. Electrophoretic patterns on 1.4 % agarose gel of amplified fragments generated from the 21 isolates of Fusarium solani by rep-PCR, using BOX-primer. Lane M is the 1 kb DNA ladder; Lanes1-20; F. solani isolates (16, Fpo-5, Fpo-69, Fpo-9, 
Fpo-74, CH-25, LM-40, 12, Fpo-67, Fpo-87, C-102, 14, FW-52, Fpo-76, LM-23, Fpo-44, Fpo-16, LM-25, C-67, CH-2 and Fpo-8).

Fig. 3. Dendrogram constructed by Unweighted Pair Group Method with Arithmetic Mean (UPGMA) based on the data of rep-PCR using BOX primer, indicating relationships among Fusarium solani isolates obtained from different host plants. Similarity matrix was produced by Dice`s coefficient. Defined main groups, designated as Fo₁ and Fo₂ and subgroups of Fo_1, designated as Fo_1-1 to Fo_1-12 are indicated on the right.

Table 3. Diversity of Fusarium solani VCG groups according to rep-PCR DNA fingerprinting analysis, using BOX primer.

Fig. 4. Pathogenicity of Fusarium solani isolates on potato plants in greenhouse at 25°C. (a) Isolate Fpo-74 obtained from potato (b) Isolate CH-25 obtained from chickpea. In each Fig., the infected plant is on the right side and the healthy plant is on the left side. Photographs were taken 30 days after inoculation.

DISCUSSION

Vegetative compatibility groups (VCGs) have been used to examine diversity and population structure in many fungi. Initially, Puhalla (1985) found that there was a correlation between VCG and forma specialis. Isolates in the same VCGs belonged to the same forma specialis and strains in different formae speciales were grouped in different VCGs (Correll 1991).

Vegetative compatibility has been tested in many fungi using nitrate non-utilizing (nit) mutants to demonstrate heterokaryosis (Leslie 1993). Various factors such as temperature, nutrition and the kind of fungus influence generation of KClO₃ resistant sectors in the fungi (Klittich & Leslie 1988). Fusarium solani strains are more tolerant to chlorate than most strains of F. oxysporum and F. moniliform (Correllet al. 1987, Klittich & Leslie 1988). In the present study, most nit mutants were recovered on PDA containing 50-70 g/l KClO₃.

Identification of phenotypes of nit mutants showed that nit 1 and Nit M have highest and lowest frequencies, respectively. Frequency of nit mutations in fungi can be influenced by the nitrogen source in medium containing potassium chlorate. Hawthorne et al. (1996) showed that by adding thereonine to the medium containing chlorate, Nit M of the species F. solani increases.

In this study, Nit mutants were obtained for 52 out of 54 isolates of F. solani. One out of 52 isolates was heterokaryon self- incompatible (HIS). Hawthorne et al. (1996) reported that there are many self-incompatible isolates among different isolates of F. solani, but the importance of self-incompatibility in nature is uncertain. Correll et al.(1989) concluded that HSI in Gibberella fujikuroi (F. monilifome)was under control of a single gene, and Jacobson and Gordon (1990) suggested that it could be a mutant artifact that arises in fungi maintaining for long periods in an artificial culture.

Fifty one heterokaryon self-compatible (HSC) isolates were grouped in 23 VCGs. Seventeen of 51 HSC isolates were incompatible with all the other isolates, and therefore were regarded as single-member VCGs. Presence of high single-member VCGs indicates the high level of genetic diversity among the isolates. Distribution of F. solani isolates obtained from various areas and host plants into different VCGs was observed in the present study. For example, the isolates belonging to VCG A were obtained from potato, common bean and chickpea, and collected from eight regions. VCG D included the isolates from potato, common bean, cucumber and melon, collected from three regions. Besides, only VCG E and VCG F contained the isolates from the same host. VCG E consisted of two isolates from chickpea, and VCG F contained two isolates from potato. Comparison between the isolates from different VCGs and their host origins showed that the isolates from the same host were classified in different VCGs, and had a considerable amount of genetic diversity. Therefore, except for some cases, VCGs grouping in the present study could not separate isolates, especially the potato isolates that had the most frequency in this study based on the host and geographical origin. But in some other studies, VCG grouping could separate isolates of F. solani based on the host origin. For instance, Hawthorn et al. (1996) placed 57 F. solani isolates in 35 VCGs, and reported a direct relationship between VCG and host origin of the isolates. Also, there was no correlation between VCG grouping and geographical origins of the isolates in the present study, except for VCG F with two members that were collected from the same region. On the other hand, the isolates of different geographical areas were placed in the same VCG. This suggests the possibility of distribution of the fungus through infected tubers and agricultural equipment, wind and rain in different regions. According to Elmer (1991), existence of the isolates from different regions in the same VCG, or in the other words, existence of a specific VCG in several areas indicates the selective survival of vegetative compatibility groups. This result was similar to the results of Raouffiet al. (2004) and Mohammadi & Banihashemi (2005). On the other hand, some studies have demonstrated the correlation between vegetative compatibility groups and geographical origins. Rahkhodaei (2000) reported that isolates of F. solani from potato in each VCG were collected from the same region.

Dendrogram generated using data of BOX primers showed that 54 isolates collected from different regions of Khorasan-Razavi province and from different hosts showed >30% similarity. This result suggests a high level of genetic diversity among the isolates. There was no correlation between the identified fingerprinting groups and host and geographical origins of the fungal isolates. These results are in agreement with results from the previous studies on F. solani (Moradzadeh Eskandari 2010, Romberg & Davis 2007, Baghai Raveri et al. 2007). Missing relationships between DNA fingerprinting groups and VCGs observed in this study showed that it could be impossible to separate the isolates based on VCGs using BOX primer. Using VCG grouping and rep-PCR in the present study showed the genetic diversity within F. solani isolates, but both methods were unable to classify the isolates based on the host origin. Therefore, using other methods such as investigation of mating populations will generate further information about the host population of F. solani. Pathogenicity test of twenty three F. solani isolates obtained from different hosts as VCGs representatives on potato plants showed that there was no relation between the host origin and virulence of the isolates, so that two out of four isolates in pathogenicity zero group belonged to potato host. In contrast, non-potato isolates in some cases had more virulence than potato isolates, and were comparable with potato isolates in severity of symptoms. Also, the pathogenicity of twenty three isolates as VCGs representatives on potato tubers showed that except VCG T and VCG E representtatives (from non-potato hosts), the rest of VCGs representatives were able to cause dry rot on potato tubers with diverse virulence.  This result confirmed the results of their pathogenicity on potato plants. In a way, the isolates tested in both pathogenicity tests, regardless of the host origin were grouped in different pathogenicity groups. These results indicated that the pathogenicity of F. solani isolates was not host specific and the isolates obtained from non-potato hosts were able to cause disease on potato plants and tubers. On the other hand, the isolates obtained from potato showed no symptom on potato plants. This result confirmed the results of previous studies (Moradzadeh Eskandari 2010, Romberg & Davis 2007).

ACKNOWLEDGMENTS

We gratefully acknowledge the High Council for Research of University of Tehran for financially support and providing materials under grant number 7110024/6/24.