Molecular and Aggressiveness Characterization of Isolates of Fusarium solani and Fusarium oxysporum f . sp . passiflorae Associated to Passion Fruit Wilting

This work was carried out with the objective of performing a molecular and aggressiveness characterization of F. solani and F. oxysporum f.sp. passiflorae collected in the Pantanal, Cerrado and Amazon biomes. We selected the most aggressive isolates for use in breeding programs aiming resistance to Collar Rot and Fusariosis. For inoculation of the isolates of F. oxysporum f.sp. passiflorae the washed root method was used. The molecular characterization of the isolates was carried out by partial sequencing of the Transcribed Internal Spacer of the rDNA region. The isolates of F. solani formed two distinct groups in relation to aggressiveness. Among all isolates, FSUNEMAT 40 and FSUNEMAT 46 were the most aggressive. The model with K=2 was taken as the best model to explain the genetic structure of the F. solani populations, with clear combinations of genes from both gene pools. There were three groups with respect to the aggressiveness of the isolates of F. oxysporum f.sp. passiflorae, with the isolated FOUNEMAT 22 being the most aggressive. In view of these results, the isolates of F. solani collected in P. edulis in the state of Mato Grosso presented a high molecular variability independent from the biome of origin, and this was also observed in the tests of aggressiveness. The results indicate the need to consider the molecular variability and the aggressiveness of the pathogens in the evaluation of genotypes of sour passion fruit in programs of selection of resistant cultivars.


Introduction
The cultivation of passion fruit, Passiflora edulis, has been growing every year in Brazil, occupying a prominent position in tropical fruit growing (MAPA, 2016).The state of Mato Grosso is the largest producer in the Midwest region, totaling 5,275 tons in a cultivated area of 346 hectares.This production is still very low when compared to the state of Bahia, which produces an average of 342 thousand tons/year (IBGE, 2016).
One of the main problems that contributes to the reduction in productivity of the passion fruit crop is the occurrence of diseases, such as collar rot and fusariosis, caused respectively by the fungi Fusarium solani Martius and Fusarium oxysporum f.sp.passiflorae (Fischer & Rezende, 2008).Plants infected by these fungi initially present wilting of the nodes and, with the progress of the disease, there is early defoliation, wilting and death of the plant in a few days.These symptoms are the result of the damage caused by these fungi to the vascular system of plants (Fischer & Rezende, 2016).The presence of F. solani and F. oxysporum f.sp.passiflorae in the soil may even prevent the cultivation of passion fruit for years since these pathogens have a resistance structure (chlamydospore) that guarantees fungus survival (Bueno et al., 2006).population is indispensable for breeding programs aiming the selection of a resistant passion flower cultivar or a hybrid in view of the complex pathosystem, where the pathogen involved presents a genetic variability, as reported by previous studies (Carvalho et al., 2015;Dariva et al., 2015;Silva et al., 2017).
In this sense, this work was carried out with the objective of performing a molecular and aggressiveness characterization of F. solani and F. oxysporum f.sp.passiflorae collected in the Pantanal, Cerrado and Amazon biomes.We selected the most aggressive isolates for use in breeding programs aiming resistance to Collar Rot and Fusariosis.

Characterization of Aggressiveness
Sixteen isolates of F. solani and eight isolates of F. oxysporum f.sp.passiflorae belonging to the collection of the State University of Mato Grosso (UNEMAT) were selected for characterization of aggressiveness in P. edulis (Tables 1 and 2).All isolates were collected from plants presenting symptoms of wilting in passion fruit producing areas, including the three biomes of the state of Mato Grosso: Pantanal, Cerrado and Amazon (Carvalho et al., 2015).All isolates are preserved in the UNEMAT fungi collection in filter paper strips stored in a refrigerator at ± 5 °C.In order to characterize the aggressiveness, clones (cuttings) were produced from a P. edulis plant, accession UFV50, belonging to the GAB (Germplasm Active Bank) of UNEMAT.The cuttings contained three internodes.Then, they were planted in trays of 72 cells containing Plantmax ® substrate (Eucatex Mineral Ltda.) and kept in a greenhouse under controlled irrigation and two weekly applications of leaf fertilizer in order to stimulate plant rooting.The whole period lasted approximately three months until the stakes reached ±20 cm in height.After rooting, for the experiment on F. solani, the cuttings were transplanted into pots containing ½ of substrate, ¼ of sand and ¼ of autoclaved soil.For inoculation, filter paper strips of each isolate of F. solani and of F. oxysporum f.sp.passiflorae were placed in Petri dishes containing PDA (potato-dextrose-agar) culture medium, and then incubated in BOD at 25 °C and a photoperiod of 12 hr.for seven days.After this time, a blade of each plate was made to ensure that all the isolates sporulated.

F. solani
The procedure of inoculation of the isolates of F. Solani was that described by Fischer et al. (2005b), in which a disk of culture medium containing the pathogen (5 mm diameter) was fixed with PVC plastic on a small wound in the plant stem at a height of 2 cm from the soil.The PVC plastic was removed five days after inoculation (DAI), and the same procedure was performed with the controls, except for the application of the fungus.
The experimental design consisted of randomized blocks with 17 treatments (16 isolates + 1 control), with four replications and three plants per plot.
The clones of the inoculated UFV50 genotype were analyzed based on 13 characteristics, as described by Preisigke et al. (2015), which are: The scores in each plot were used to calculate the Disease Index (DI) according to McKinney (1923).The evaluations of all treatments began on the 5 days post inoculation (DPI).They were carried out every two days until the 55 th DAI or until the death of the plant.At the end of the experiment, the isolates were re-isolated in order to complete the Koch Postulates.
Data evaluated were submitted to analysis of variance, and means were compared by Scott-Knott test at 5% probability.
The genetic distance matrix estimation was performed based on Gower's algorithm (1971).Afterwards, the dendrogram was constructed using the UPGMA grouping method.The adjustment between the distances matrix and the dendrogram was estimated by the Cophenetic Correlation Coefficient (CCC) (Sokal and Rohlf, 1962).To establish a cut-off point in the dendrogram and to define the number of groups, the Mojena (1977) procedure was used based on the relative size of the merging (distances) levels in the dendrogram.
All analyses were performed using the GENES software (Cruz, 2016) with interaction of the "ape" package of the R software (R Development Core Team, 2014).
2.1.2F. oxysporum f.sp.passiflorae For the inoculation of the isolates, the washed root method was used.The seedlings were removed from the trays and the roots were washed in distilled water.Then, the roots were cut using sterile scissors and immersed in 100 mL conidia suspension in 400 mL plastic pots (the root was left in the suspension for 24 hours) (Preisigke et al., 2017).For the control, the procedure was the same, replacing only the suspension of conidia for distilled water.
After the 24-hour period, the suspension was withdrawn, and 100 mL of nutrient solution was added as proposed by Clark (1975).This solution was changed every three days.
The experimental design was randomized blocks with 9 treatments (8 isolates + 1 control), with four replications and three plants per plot.
We evaluated the survival period (period in days from inoculation to plant death) and the number of live plants.
Evaluations were carried out periodically up to 50 DAI.During this period, symptomatic seedlings had parts of their stem and roots disinfested and transferred to PDA medium to confirm the etiological agent.After data collection, the data were submitted to graphic dispersion using the means of the characteristics.The statistical analysis was performed using the Genes software (Cruz, 2016).

Molecular Characterization and Population Structure
For the extraction of genomic DNA, the monosporic isolates of F. solani and F. oxysporum f. sp.passiflorae were grown in liquid potato dextrose agar (PDA) medium for two weeks.Subsequently, the mycelium was macerated in liquid nitrogen.DNA extraction was performed using extraction kit (Axygen Biosciences USA) according to the manufacturer.
The molecular characterization of the isolates was carried out by partial sequencing of the internal transcribed spacer (ITS) of the rDNA region.The primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (GGAAGTAAAAGTCGTAACAAGG 5'-3') were used for amplification of the rDNA's ITS region (White et al., 1990).For 25 μl of reaction, approximately 60 ng of total DNA, 2.5 μl of 10x PCR buffer, 2.5 mM of each dNTP, 10 pMol of each primer oligonucleotide, 1 U of Taq polymerase and ultrapure H 2 O s.q.f. for 25 ml were used.The amplification program consisted of an initial stage of denaturation at 94 °C for 2 min, followed by 35 cycles at 94 °C for 45 s, 50 °C for 45 sec and 72 °C for 1 min, and a final step of stretching at 72 °C for 10 min in an Amplitherm thermocycler.The sequencing was performed by the Sanger method using the Big Dye Kit on the Applied Biosystem ABI3100 sequencer (Dunn & Blattner, 1987).
The sequences obtained were edited and aligned using the BioEdit software (Hall, 1999), and then compared to sequences in the GenBank database using the NCBI BLAST software (www.ncbi.nlm.nih.gov).The phylogenetic tree was constructed using the MEGA software, version 5.0 (Tamura et al., 2011), and the Neighbor-Joining method with a bootstrap of 1,000 replications.
For the structuring of the populations, the Structure v2.3.4 software (Pritchard;Stephens;Donnelly, 2000) was used through clustering based on the Bayesian model.The structure of populations was classified into "K" clusters according to their genetic similarities.The number of clusters (K) tested ranged from 1 to 10, with 5 interactions each.The number of steps of the burnin length was 10,000 and the MCMC (Markov chain Monte Carlo) repetitions was 100,000.To determine the number of genetic groups (most likely K), the criterion proposed by Evanno, Regnaut, and Goudet (2005) was used through the Structure Harvester v0.6.94 software (Earl & Vonholdt, 2012).

Characterization of Aggressiveness F. solani
By analyzing the data contained in the analysis of variance, it was possible to observe a significant difference between the means of the isolates at 1% probability by F test for almost all characteristics analyzed, except for LL, which was significant at 5%.

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The results value 3 wa value for t single pop Figure 5 As describ the ITS1 a eight isola alignment 6).
The phylo of the isola the Cerrad isolates FO the third g (number of dead plants and length of injuries in the collar of the plants), and a variability in the resistance between the materials for each isolate.
By analogy between the biome of origin of the isolates studied here and the grouping formed, there was no relation between the degree of aggressiveness and the origin of the pathogen, that is, the isolates from the three biomes of the state of Mato Grosso (Pantanal, Cerrado and Amazon) were distributed indistinctly in the groups formed.
These data corroborate those of Nakatina et al. ( 2009) upon studying the variability of five pathogenic isolates of Xanthomonas axonopodis pv.passiflorae collected at four different locations in the state of São Paulo.In that study, the authors verified the existence of variability in the aggressiveness of the isolates when inoculated in sour passion fruit.However, there was no evident relation between the collection site and the groups' composition.This is different from what was found by Munhoz et al. (2011) by working with isolates of X. axonopodis pv.passiflorae.The authors noticed a relation between the collection region and the genetic diversity of the isolates.
In addition, Dariva et al. (2015) found a high intraspecific genetic variability for isolates of F. solani using the molecular markers ISSR and RAPD, but there was also no clustering of isolates according to the geographic region of origin.
a technical point of view, the genetic variability observed for the pathogen's aggressiveness (F.solani) to P. edulis may be related to the genetic variability of the genus Passiflora since Brazil is an important center of diversity of this genus, and generally the centers of diversity of the hosts are also centers of diversity of pathogens (Leppik. 1970).It is interesting to emphasize that there is a co-evolution process between pathogen and host, generating variability in both plant and fungal populations, which are usually associated with evolutionary forces, such as mutation, migration and selection (Barbieri & Carvalho, 2001).In addition, another explanation would be the existence of the sexed phase of this fungus, which belongs to the genus Haematonectria, where there is genetic recombination and the possibility of crossing between different individuals (Fischer et al., 2005a;Nayaka et al., 2011;Dufresne et al., 2011).

Molecular Characterization and Population Structure-F. solani
According to the results, a great genetic diversity was verified among the isolates of F. Solani studied.This high variability was also observed by Bahar and Shahab (2012) upon investigating 33 isolates of F. solani collected in different regions of Iran and characterized by marked SSR.The DNA analysis showed that the isolates had a high genetic variability and formed 10 distinct groups in the phylogenetic tree.
In studies on passion fruit, Henao-Henao et al. (2018) analyzed populations of Fusarium spp.associated with P. edulis in five municipalities of Valle del Cauca.Colombia.They analyzed 35 isolates by amplifying the region of the TEF 1 alpha gene, and identified three species of Fusarium, among them F. solani.
In this same context, using the ITS-5.8Sregion sequencing and the elongation factor 1α (EF-1α).Bueno et al. (2014) verified an interspecific variability of eight F. solani isolates from P. edulis.The authors identified that the isolates had host specificity and that the phylogenetic trees of the ITS and EF-1a region showed that the isolates formed a distinct group within the F. solani group when compared to other GenBank sequences.
In view of these results, the isolates of F. solani collected in P. edulis in the state of Mato Grosso presented a high molecular variability independent from the biome of origin, and this was also observed in the tests of aggressiveness.

Characterization of Aggressiveness-Fusarium oxysporum f.sp. passiflorae
All isolates of F. oxysporum f.sp.passiflorae were pathogenic.However, the analysis of aggressiveness showed a variation among isolates classified as aggressive and moderately aggressive.Similar results were found by Cunha et al. (2015) working with different isolates of Fusarium oxysporum f. sp.cubense collected in the north and south of the state of Santa Catarina.Brazil.The authors identified that the isolates from Bananeiras of the Pome subgroup were only classified as aggressive (88.5%) and moderately aggressive (11.5%).None was classified as little aggressive.
The data indicate that there is no relationship between the aggressiveness of the isolates analyzed in this study and their biome of origin.This also occurred with the isolates of F. solani.This is probably due to a horizontal transfer of genes between lineages in the species under study.Baayen et al. (2000) found that formae speciales in the complex of F. oxysporum might have independent origins.Pathogenicity and virulence evolve through mutations, transposition or dissemination to distant related organisms through parasexuality or horizontal transfer of genes.
Faced with these facts, Hua- Van et al. (2001) reported that the transposition of DNA sequences into the genome might result in genetic, intra-and inter-specific variability.They also point out that, from an evolutionary point of view, there is the possibility of a horizontal transfer of genes between different species, as observed in an experiment on isolates of F. oxysporum.

Molecular Characterization and Population
Structure-F.oxysporum f.sp.passiflorae According to results obtained by molecular analysis, the groups formed demonstrate a high degree of diversity among the isolates of F. oxysporum f.sp.passiflorae.It should be noted that the isolate FOUNEMAT 5 from the Pantanal biome was the most divergent because it was allocated alone in a group.Note that the clusters formed in the population structure analysis coincide with the groups generated by the phylognetic tree using the Neighbor-Joining method.
Other authors also found a high molecular genetic diversity among isolates of F. oxysporum f.sp.passiflorae.Silva et al. (2013), using AFLP markers, observed a high variability among 14 isolates of F. oxysporum f.sp.passiflorae collected in Bahia.Dariva et al. (2014) determined the genetic variability of 13 isolates of F. oxysporum f.sp.passiflorae pathogenic to passion fruit by using ISSR and RAPD markers.The analyses revealed a population with a high intra-population genetic variability.Similar results were found by Walker et al. ( 2016) using the ITS1 and ITS4 markers on Fusarium acuminatum and Fusarium verticillioides.The authors were able to genetically dissociate these isolates from others of the genus Fusarium.
Contrary to what was found in this study in Iran, Nourollahi and Madahjalali (2017) determined the population structure and the genetic diversity of 101 isolates of Fusarium oxysporum f. sp.lentis by SSR markers.The results showed a high genetic similarity between the isolates probably due to a low gene flow in the collection region.
By making an analogy between the results obtained with the molecular and aggressive tests.a high genetic and pathogenic variability of the isolates can be observed regardless of the biome of origin.

Conclusion
The results indicate a need to consider the molecular variability and the aggressiveness of the pathogens F. solani and F. oxysporum f.sp.passiflorae in the evaluation of genotypes of sour passion fruit in programs of selection of resistant cultivars.The genetic structure of pathogen populations may provide insights into the pathogen's evolutionary potential, as well as lead to the development of integrated disease management strategies and breeding programs.The use of the most aggressive pathogens in this study is recommended for the selection of resistant genotypes of Passiflora spp.Among all isolates of the F. solani, FSUNEMAT 40 and FSUNEMAT 46 were the most aggressive and of the F. oxysporum f.sp.passiflorae the most aggressive was FOUNEMAT 22. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011) a) lesion expansion, length and width (CE and LW); b) number of plants in which the lesion reached less than 50% of the circumference (NPL-50%); c) period of inoculation until the lesion reached more than 50% of the circumference of the damaged plant stem (PIL+50%); d) period of inoculation until the lesion reached 100% of the circumference of the damaged plant stem (PIL100%); e) number of plants in which the lesion reached the medulla (NPLRM); f) normalized area below the expansion of lesion area curve (ABELAC); g) normalized area below the expansion of lesion width curve (ABELWC); f) normalized area below the expansion of lesion length curve (ABELLC); i) period of inoculation until the plant wilts (IPPW); j) number of dead plants (NDP); k) survival period (SP); and l) range of scores (RS).

Figure
Figure 2. G rogram

Figure
Figure 6.D

Table 1 .
Relation of Fusarium solani isolates used in the aggression test

Table 2 .
Relation of Fusarium oxysporum f.sp.passiflorae isolates used in the aggression test

Table 3 .
Aggressiveness averages of the 12 characteristics of 16 isolates of F. solani inoculated in P. edulis Note.Averages followed by the same letter in the columns do not differ from each other by the Scott Knott test at the 5% probability level.Characteristics of LE and LW are expressed in millimeters.Averages followed by the same letter in the columns do not differ from each other by the Scott Knott test at the 5% probability level.Characteristics of ABELAC, ABELWC and ABELLC are expressed in millimeters.
In this sense, the Gower's algorithm was used for the joint analysis of the 13 variables evaluated in this experiment in order to estimate the divergence of the aggressiveness of isolates of F. solani.The isolates FSUNEMAT 21 and FSUNEMAT 40 were the most distant in relation to aggressiveness, presenting a magnitude of 0.62, while the isolates FSUNEMAT 12 and FSUNEMAT 17 were the most similar, with a value of 0.03.It is noteworthy that the jas.ccsenet.