Molecular fingerprinting of Fusarium oxysporum f . sp . passiflorae isolates using AFLP

Fusarium oxysporum f. sp. passiflorae W.L. Gordon (FOP) is one of the most important fungal pathogens of passion fruits. Understanding molecular variation of isolates from different areas is of utmost importance. Molecular fingerprinting on 14 isolates of FOP were conducted using AFLP molecular markers (Amplified Fragment Length Polymorphism), and their genetic variability were estimated. Twenty-five AFLP primer combinations were selected for amplification of FOP isolates and one for Fusarium oxysporum f. sp. cubense W.C. Snyder & H.N. Hansen (FOC), resulting in 99% polymorphic fragments, with an average of 40 fragments per primer combination. Specific fingerprints could be generated for most of the isolates evaluated; we observed a high power of discrimination of the AFLP primer combinations, with the presence/ absence of up to 26 specific fragments per isolate. Thus, specific fingerprinting was obtained for 10 of the 15 isolates analyzed. The values of the polymorphic information content, the index and the resolving power of the markers showed wide variation and re� ected the high informative contents of the primers used in the characterization of the FOP isolates. The FOP isolates were divided into four groups, irrespective of their geographic origins, with the allocation of 5, 7, 1 and 1 FOP isolates into Groups II, III, IV and V, respectively. A wide genetic diversity was observed in FOP isolates, which should be taken into consideration when implementing strategies for the improvement of passion fruit in the search for cultivars with multiple resistance to


Introduction
Brazil is the world's largest producer and consumer of passion fruit (Passiflora edulis Sims f. flavicarpa Deg.), with approximately 62,000 hectares cultivated and an annual production of 920,000 tons.The states of Bahia, Espírito Santo, and Ceará are responsible for approximately 73% of the national production.However, recently yield has decreased due to disease problems, especially with the occurrence of fusarium wilt caused by Fusarium oxysporum f. sp.passiflorae W.L. Gordon (FOP) (McKnight, 1951).
Knowledge of the genetic diversity of FOP has contributed to the development of disease control strategies (Kistler et al., 2001), as some of these pathogens have a high capacity of variation, and their apparent absence of a sexual stage is compensated by other processes that ensure permutation and maintenance of genetic diversity (Kistler et al., 1992;Leslie, 1993).Fungi with a high level of genetic diversity can be difficult to control, as they tend to adapt more quickly to any control measure, such as the introduction of a resistant cultivar.
FOP isolates obtained from infected plants in various production regions show some morphological differences in colony growth, although the degree of pathogen aggressiveness in relation to the appearance of disease symptoms in different varieties of passion fruit is not effectively known.Morphological and genetic differences between FOP isolates may be associated with their pathogenicity, and these observations must be taken into consideration when the passion fruit germplasm is screened for sources of resistance and, later, in the development of resistant cultivars.
In order to do so, it is vital to better understand the genetic variability of the FOP isolates, which are still unknown.Recently, molecular techniques have been increasingly used in studies of genetic diversity and species phylogeny, or for the characterization of populations of F. oxysporum within and between formae speciales (Baayen et al., 2000;O'Donnell, 2000;Abd-Elsalam et al., 2004;Bogale et al., 2006;Groenewald et al., 2006;Stewart et al., 2006).Among these techniques, AFLP (Amplified Fragment Length Polymorphism) is a class of marker frequently used (Zeller et al., 2003;Belabid et al., 2004).
Amplified Fragment Length Polymorphism (AFLP) markers are extremely powerful because of their high multiplex power, i.e., enabling the generation of a large number of bands per gel (amplification).Therefore, it has high potential for the development of specific primers in the identification of isolates.Given these needs and the molecular tools available, this study was conducted to assess the possibility of developing specific fingerprinting within the formae speciales of FOP to be able to trace the presence of these isolates in the main passion fruit-producing regions and to find the different ways of genetic structuring of FOP isolates, one of the principal infectious agents of passion fruit.

Biological material
Fourteen monosporic cultures of FOP from the Embrapa Cassava and Fruits fungus collection were used.The isolates were taken from passion fruit plants displaying typical symptoms of wilt disease and obtained from farms in the states of Bahia and Espirito Santo.After isolation, these were subjected to morphological studies to confirm the identity of the pathogen.In addition to this, one isolate of Fusarium oxysporum f. sp.cubense W.C. Snyder & H.N. Hansen (FOC) was used as an outgroup (Table 1).

Growth of the fungus in liquid medium, and extraction of DNA
To obtain the mycelial mass, the isolates were chopped up and incubated at 25 °C in potato-dextrose (PD) liquid medium (200 g of potatoes peeled and cut into small pieces, cooked in distilled water for 20 min, filtered through a muslin cloth and mixed with 5 g of unhydrated dextrose (Impex).The isolates were shaken continuously for a period depending on each isolate.The mycelial mass of each isolate was filtered and frozen in liquid nitrogen for the extraction of DNA.
The extraction of DNA from the isolates was performed using the CTAB method ((C 16 H 33 )N(CH 3 ) 3 Br, cetyltrimethylammonium bromide hexadecyltrimethylammonium bromide) described by Zolan and Pukilla (1986).DNA quantification was performed using visual comparison to the intensities of fragments of a series of known concentrations of Lambda DNA (Invitrogen), after electrophoresis (3 v cm -1 ) in 1.0 % agarose gels containing ethidium bromide (1.0 µg mL -1 ).

Evaluation of primer combinations
A preliminary study was performed with 64 prim-er combinations and two FOP isolates (013 and 071) to identify the more polymorphic combinations for definitive analyses with the marker.The best combinations were used for characterization of the 14 FOP isolates and the one FOC isolate (Table 2).

Amplification with AFLP markers
Analyses were performed using the Vos et al. (1995) protocol with minor modifications, using the commercial AFLP ® Core Reagent Kit (Invitrogen).Two combinations of endonucleases were employed.In the digestion reaction, 250 ng of genomic DNA were digested using EcoRI and MseI for 2 hours at 37 o C. Reactions were then incubated at 70 °C for 15 min to deactivate the enzymes.For connection of the adapters, fragments of digested DNA were added to 5X buffer of T4 DNA ligase (Invitrogen), 0.1 µM of each enzyme's adapter (EcoRI and MseI), 1 U of T4 DNA ligase (1 U µL -1 , Invitrogen) and 5.0 µL of ultra-pure sterilized water, and the reactions were incubated at 20 °C for 3 h.The mixture was diluted 1:5 in a 1:10 mixture of TE and ultra-pure water.
The pre-amplification was performed in a final volume of 20 µL, consisting of 3.0 µL of the solution containing the digested DNA linked to the adapter, 0.15  mM dNTPs (Invitrogen), 1X PCR buffer without MgCl 2 (Fermentas), 1.5 mM MgCl 2 (Invitrogen), 1U of Taq DNA polymerase (Fermentas), 0.5 µM of the primer for the rare-cutting enzyme with a selective base (EcoRI + C or EcoRI + A) and 0.5 µM of the primer for the frequent-cutting enzyme (MseI + A or MseI + C).The PCR program consisted of 95 °C for 2 min, followed by 30 cycles of 94 °C for 30 s, 56 °C for 1 min and 72 °C for 2 min.This was followed by a final extension step for five minutes at 72 °C.The pre-amplified material was diluted 1:60 in a 1:10 mixture of TE and ultra-pure water.
In the selective amplification stage, 2.5 µL of the pre-amplified products were used, with 0.15 µM dNTPs (Invitrogen), 1X PCR buffer (Fermentas), 2 mM MgCl 2 (Invitrogen), 8 µg mL -1 BSA (New England Biolabs), 1.5 U of Taq DNA polymerase (Fermentas), 0.5 µM of the primer for the rare-cutting enzyme (EcoRI + CN or EcoRI + CN), where N can be any nucleotide, and 0.5 µM of the primer for the frequent-cutting enzyme (MseI + CNN or MseI + ANN), with a final volume of 20 µL.The amplification program consisted of 95 °C for 2 min, followed by 12 cycles of 94 °C for 30 s, 65 °C for 30 s with a reduction of 0.7 °C per cycle, and 72 °C for 2 min; followed by 24 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 2 min; with a final incubation at 72 °C for 2 min.
The amplified fragments were separated by electrophoresis in a 6% polyacrylamide denaturing gel at 70 W for 3 h.The amplification products were visualized by silver staining according to Creste et al. (2001).The molecular masses of the fragments were determined by comparison with a 50-bp ladder (New England Biolabs).

Molecular analysis of the data
For each primer pair, the number of polymorphic fragments (NPF), polymorphic information content (PIC), marker index (MI) and primer resolving power (Rp) was calculated.The polymorphic information content (PIC) was calculated using PIC i =2f i (1-f i ), where i is the information of marker I, f i is the frequency of the amplified allele (presence of fragments) and (1 -f i ) is the frequency of the null alleles (Roldan-Ruiz et al., 2000).The PIC was taken as the average of each primer combination.
The marker index (MI) was calculated by MI = PIC x EMR, where EMR is the "effective multiplex relationship" given by the product of the total number of fragments per primer (n) and the fraction of polymorphic fragments (b) (Varshney et al., 2007).The resolving power (Rp) was calculated using the formula Rp = ∑ I b , where I b =1-[2x|0.5-p|]and p is the proportion of accessions with fragments (Prevost and Wilkison, 1999).
AFLP polymorphic markers were computed as the absence (0) and presence (1) of fragments.The dissimilarities between the genotypes were calculated by the complement of simple matching, using the Genes program (Cruz, 2006).From the dissimilarity matrix, the dendrogram was generated with the UPGMA method (Unweighted Pair Group Method with Arithmetic Mean), using Statistica 7.1 software (Statsoft, 2005).Fusion point criteria were used as cutting point to define clusters (Mingoti, 2005), where a graph with the number of groups versus the distance at each stage (fusion) is made, and jumping points indicate the optimal number of clusters.The consistency between the dissimilarities of the clusters and of the matrices was verified using cophenetic correlations with the Genes 1.0 program (Cruz, 2006).The presence of private alleles was determined using the GenAlEx program (Peakall and Smouse, 2006).

Informative content of the AFLP marker
Consistent molecular profiles were obtained for all of the isolates evaluated.The 25 AFLP primer pairs produced 1,050 fragments (ranging from 30 to 916 bp).Of this total, 989 (94.0 %) fragments were polymorphic (Table 2).On average, each primer pair produced 40 polymorphic fragments.The lowest number of fragments was detected for the EcoRI + CC / MseI + ATC pair and the highest for EcoRI + CT / MseI + AAA, with 20 and 58 fragments, respectively.Figure 1 illustrates three primer combinations that were evaluated (E+CT / M+AAC, E+CT / M+AAG and E+CT / M+ATA).
The polymorphic information content for dominant markers such as the AFLP ranged from 0 to 0.50 (Roldan-Ruiz et al., 2000).Thus, on average for the primers pairs evaluated, the polymorphic information content (PIC) varied from 0.15 (primers E+CA / M+ATC and E+AA / M+CTG) to 0.28 (primers E+CT / M+ATA), with an average of 0.20, which reflects the informative content of the primers used (Table 2).
The marker index (MI), which incorporates the informative content of the marker (PIC), the number of fragments per primer pair and the fraction of polymorphic fragments, varied from 3.60 (EcoRI + CC / M + ATC) to 12.72 (EcoRI + CA / MseI + AAT), with an average of 7.92.In the isolates evaluated, Rp varied from 4.62 to 17.90 for the combinations EcoRI + CC / MseI + ATC and EcoRI + CA / MseI+AAT respectively, following the same tendency as the MI.This fact is shown by the high correlation between these two indices (r = 0.97, p < 0.01).

Discriminatory capacity of AFLP markers
The evaluation of the discriminatory capacity of the AFLP primers was also evaluated on the basis of the presence of unique fragments (+) and on the absence of specific fragments (-) in certain isolates.High discriminatory powers of the combinations of AFLP primers were observed, with a range of 2 to 12 presences of unique fragments (+) for the combinations EcoRI + CG / MseI  3).Considering presence and absence of unique fragments, 575 differences were observed among the isolates evaluated.Only isolates FOP003, FOP004, FOP022, FOP057 and FOP069 had no unique fingerprinting.However, a large number of exclusive FOC fragments were observed, highly differentiated from the passiflorae form.

Molecular characterization of the FOP
The genetic dissimilarity varied from 4% to 48%, with an average of 25%.Although the isolates had different geographical origins, the most similar isolates were FOP004 and FOP022, with only 4% dissimilarity.The cophenetic correlation was 0.95, indicating a high level of accuracy between the original data of the genetic distance matrix and the data represented in the dendrogram (Figure 2).

Discussion
AFLP markers have been used to access the genetic variation of intimately related breeds or species, which would be impossible to discover using morphological traits or other molecular mechanisms (Abdel-Satar et al., 2003).In this study, these markers served to analyze the genetic variation of 14 isolates of FOP, representative of different passion fruit-producing fields.In general, the haplotypes generated by the AFLP markers showed high informative capacity and indicated high genetic variability present in FOP isolates.
For the selection of combinations of more informative markers, the polymorphic information content (PIC), marker index (MI) and resolving power (Rp) have traditionally been used.The Rp takes into consideration the capacity of a given primer combination to discrimi-primer combination to discrimi-to discriminate the genotype.In the isolates evaluated, the Rp varied in the same manner as the marker index (MI), which is shown by the high correlation between these two indices (r = 0.97 p < 0.01).This reinforces the fact that  2).
The highly informative nature of these markers contributes to the observation of polymorphisms between the FOP isolates, as well as contributes to identify specific marks for each isolate.The 395 differences of fragments among the 14 FOP isolates and one FOC isolate allow the identification of specific fingerprinting for 10 isolates.Of these isolates, FOP002 and FOP028 have particularly high numbers of polymorphic and specific fragments.As a consequence of the higher number of unique fragments; these isolates were grouped separately (Figure 3).
The five isolates that had no unique fragments were placed in Groups II and III, as these probably had only slight differences in relation to the others in the cluster, using AFLP markers.However, isolate FOC143 had completely different standard fingerprinting from the FOP isolates, separate from the others in the group, Sci.Agric.v.70, n.2, p.108-115, March/April 2013 which emphasizes the highly diverse natures of these two pathogens of the same species.
Studies of the diversity among pathogenic isolates of Fusarium have been done using mitochondrial DNA, ITS (Internal Transcribed Spacer) regions, PCR and realtime PCR techniques to determine genetic relationships between formae speciales (Kim et al., 1993;Oliveira and Costa, 2002) and to differentiate them into pathogenic races (Hirano and Airie, 2006;Inami et al., 2010).However, the success in the differentiation of pathogenic isolates between different formae speciales has given rise to contradictions, for example, in not distinguishing isolates of F. solani that are pathogenic to either soybeans or beans from those that are pathogenic to both crops with the use of the ARDRA technique (Amplified Ribosomal DNA Restriction Analysis) (Oliveira and Costa, 2002).On the other hand, Werner and Irzykowska (2007) demonstrated the applicability of RAPD in differentiating between pathogenic and non-pathogenic isolates of F. oxysporum, and Hirano and Arie (2006) used molecular differences in the endo-polygalacturonase (pg1) and exo-polygalacturonase (pgx4) genes of F. oxysporum f. sp.lycopersici and radicis-lycopersici isolates to design a set of primers able to differentiate between the pathogenic types of F. oxysporum in tomato.
Although the number of FOP isolates is reduced, the 14 analyzed in this study are representative of the principal passion fruit-producing State in Brazil, and therefore, the molecular results demonstrate that the AFLP technique has high potential for the identification of specific marks among the forma specialis passiflorae.The specific fragments could be cloned and transformed into SCAR (Sequence-Characterized Amplified Region) markers, enabling molecular analyses to be conducted more rapidly, as SCAR technique has fewer stages.The use of this information may help in the rapid identifi ca-identification of FOP, even within this forma specialis.
This is one of the first studies conducted to characterize FOP isolates from producing regions with a high rate of infestation of the disease.The results show that there are no differences in the gene pool in relation to geographic locations, as there is no grouping according to these criteria.Thus, the drawing up of a genetic improvement program for the selection of genotypes resistant to wilt disease should focus on the genetic diversity of the pathogen, verifying whether this standard does or does not reflect the aggressiveness and pathogenicity of the FOP isolates.
The isolates from the same geographic origin were found not to have identical fingerprinting.This observation corroborates the work of other authors showing that molecular information does not detect a strong relationship between genetic variation and geographic distribution or in the groupings generated (Abdel-Satar et al., 2003).
Group III included half of the isolates analyzed (Figure 2), showing genetic similarities between isolates from quite geographically distant areas in the state of Bahia, although significant molecular diversity was found.These considerations can also apply to Group II, including isolates with even higher genetic divergence, even from different states such as Bahia and Espirito Santo.
The information on the specificity of fragments is directly or indirectly related to the dissimilarity matrix between the FOP isolates, which is also shown by the dendrogram formed (Figure 2).Isolates FOP003, FOP004 and FOP022 had no specific fragments belonging to the same group.Furthermore, isolates FOP004 and FOP022 showed the lowest dissimilarity.Similarly, FOP069 from Linhares (ES), which also showed no specific fingerprinting, was allocated to Group II, along with isolates from different geographic areas of the Bahia.
As F. oxysporum has no known sexual stage, one hypothesis that has been suggested is that the genetic variation observed is in part generated by parasexuality (Kistler, 1997), amplifying the variability created by high mutations rates.Parasexuality occurs within heterokaryon fungi by the occasional fusion of two nuclei and the resulting formation of a diploid nucleus.During multiplication, there must be crossing-over in some mitotic divisions, resulting in the appearance of genetic recombinants with diploid nuclei, which quickly and progressively loose individual chromosomes to revert to their haploid state (Agrios, 2005).Earlier research suggested genetic exchanges between and within individual lineages.Fourier et al. (2009), studying the relationships between VCG of FOC, demonstrated recombination of genetic material within some lineages of this pathogen, which must be due to parasexuality or heterokaryosis between individuals with very similar genomes.
The results found here are relevant to the study of passion fruit-FOP interactions, as the separation of the FOP isolates into different groups will help in screening for sources of resistance to this pathogen.The selection of the genotypes must be performed by inoculation of the plants with isolates from different groups.Thus, the genotypes with resistance to multiple isolates from different groups will have a greater chance of having longerlasting resistance under field conditions.Understanding the diversity of this pathogen adds value to research on resistance to wilt disease, allowing the identification of future races and helping in the identification of sources of genetic resistance that are consistent in the various regions of the country.

Figure 2 -
Figure 2 -Dendogram of the genetic relationships among 15 isolates (14 FOP and one FOC) obtained with the use of the dissimilarity matrix with simple matching coefficient.The grouping method was UPGMA.The vertical bar represents fusion point.Bootstrap values are shown in the branches nodes, and 0.95 of cophenetic value.

Figure 3 -
Figure 3 -Distribution of unique fragments (presence) for nine isolates of FOP and one of FOC using AFLP markers.A: graphic of the number of unique fragments in the isolates analyzed.B: fingerprinting of the isolates on the basis of the unique fragments.

Table 1 -
Isolates of Fusarium oxysporum f. sp.passiflorae and Fusarium oxysporum f. sp.cubense, used on the molecular analysis with Amplified Fragment Length Polymorphism (AFLP) markers.

Table 2 -
Characterization of the AFLP primers by the total number of fragments (NB), number of polymorphic fragments (NBP), polymorphic information content (PIC), marker index (MI) and resolving power of the marker (Rp).
1+= presence of fragment and -= absence of fragment, numbers between brackets indicate the number of marks absent or present.