elocation-id: e2711
The mechanisms of Fusarium oxysporum related to the degradation of structural components of the root, such as xylan, are very important since the colonization of this organ is a key piece in the establishment of the disease. The present study focused on detecting the gene coding for the xylanase xyl3 enzyme in strains of F. oxysporum f. sp. vanillae and searching for homologues to this gene in sequences of other formae speciales and species of the Fusarium genus, in order to determine the phylogenetic relationships between xylanases within the F. oxysporum species complex, as well as to search for evidence of natural selection. The results indicated that, of the nine strains evaluated, only three had a copy of the xyl3 gene. The phylogeny showed eight clades, where clade 3 was consistent with the classification of xyl3, while the other types of xylanases were grouped in clade 2. The natural selection test showed no evidence of positive selection within the phylogeny, suggesting that the neutral mutation is responsible for the diversity in the xylanase gene among the F. oxysporum species complex, leading to the proposal that the gene does not appear to have changed with colonization of new hosts.
mutations, positive selection, xylanase gene.
The fungus Fusarium oxysporum is a ubiquitous inhabitant of soils in almost all ecosystems. It is currently considered a complex of species, based on phylogenies carried out with different genes (O’Donell et al., 2009). F. oxysporum fulfills various ecological roles, although it is mainly considered a saprophyte (Abdul et al., 2016), it has an important role as a beneficial (Waweru et al., 2014) and pathogenic endophyte (Demers et al., 2015). The latter receive special attention since they cause very destructive diseases in various crops.
The pathogenic endophytes of F. oxysporum are named according to their specificity to the host, this is known as formae speciales (Edel-Hermann and Lecomte, 2019). Being a soil inhabitant, most of the formae speciales of F. oxysporum invade the plant through the root (Olivain et al., 2006; Turrá et al., 2015; Koyyapurath et al., 2016). Therefore, it is essential to understand the mechanisms that the fungus uses for the degradation of the main components of the root cell walls. These components include cellulose, hemicellulose and lignin (Pattathil et al., 2015).
Xylan is one of the structural components of hemicellulose and therefore, it is essential to determine the ability of the pathogen to degrade this polysaccharide (Pattathil et al., 2015). Due to the chemical complexity of xylan, a number of enzymes are needed to degrade it and break the resistance of the cell wall (De Vries and Visser, 2001; Kalluri et al., 2014).
Some pathogenic strains of F. oxysporum contain functional genes coding for different variants of the xylanase enzyme, for example, differential expression of the genes xyl2 and xyl3 has been observed during the colonization of tomato plants by F. oxysporum f. sp. lycopersici, with the xyl3 gene being the one that showed activity in the roots (Ruiz-Roldán et al., 1999). It was also determined that the xyl3 gene is expressed differentially between races and pathotypes of F. oxysporum f. sp. ciceris in chickpea, therefore, it can be used as a marker to differentiate between physiological races of this forma specialis (Jorge et al., 2005; Gurjar et al., 2009).
On the other hand, it was demonstrated that the presence and activity of two genes, xyl3 and xyl4, are not directly related to the pathogenic capacity of F. oxysporum f. sp. lycopersici in tomato (Gómez-Gómez et al., 2002). F. oxysporum f. sp. vanillae is the cause of stem and root rot in vanilla (Vanilla planifolia), an orchid of high commercial value because it is the natural source of vanillin (Pinaria et al., 2010; Adame-García et al., 2015; González-Oviedo et al., 2022). For this pathogen, there is evidence of variation in the activity of lytic enzymes related to pathogenic differences found between different isolates of the fungus (Adame-García et al., 2011; Koyyappurath et al., 2015).
Histological analyses performed in the root zone of vanilla infected with the pathogen have shown that, unlike other formae speciales, F. oxysporum f. sp. vanillae invades the root hair zone, penetrates through cortical cells, but does not colonize the vascular system, indicating that its root damage mechanisms are essential for disease establishment (Koyyappurath et al., 2016). However, there is no information on the enzymes that degrade important components of the root, such as xylan, and to date the mechanisms that this pathogen uses to establish the disease have not been clearly established.
The objective of the present work was to detect the presence of the xyl3 gene in strains of F. oxysporum f. sp. vanillae that have shown different levels of pathogenicity, in order to determine phylogenetic relationships between xylanases within the F. oxysporum species complex, as well as to search for evidence of positive natural selection.
Nine strains of F. oxysporum f. sp. vanillae were used, which were previously reported as pathogenic to vanilla (Adame-García et al., 2015) and which belong to the collection of vanilla pathogens under the protection of the Laboratory of Genetics and Plant-Microorganism Interactions of the Faculty of Agricultural Sciences of the Veracruzan University. Each fungal shelter consisted of PDA agar discs with mycelium immersed in sterile distilled water stored at 4 °C. For their use, the strains were reactivated in PDA medium from the inoculation of 10 μl of the fungal suspension of the strain in shelter, incubated for seven days at 27 °C, with a period of 16 h of light and eight of darkness.
DNA extraction was performed according to the protocol established by Adame-García et al. (2016). The conditions for the amplification of the xyl3 gene were based on the protocol described by Gurjar et al. (2009), using the oligonucleotides XYL3-F (5’- GAC AAY AGC ATG AAG TGG GAT- 3’) and XYL3-R (5’- ACA CCC CAD ACR GTR ATD CC-3’). The reaction mixture consisted of 1X PCR buffer, 2.5 mM of MgCl2, 1 U of Taq DNA polymerase (Promega brand), 0.25 mM of dNTPs, 25 pmol of each oligonucleotide and 50 ng of genomic DNA, in a final volume of 25 μl.
The thermal cycle used for amplification was as follows: an initial denaturation phase at 94 °C for 5 min, 30 denaturation cycles at 94 °C for 1 min, annealing at 50 °C for 30 s and polymerization at 72 °C for 30 s and final extension at 72 °C for 10 min. PCR reactions were performed in a T100 thermal cycler (Bio-Rad®). PCR products were visualized in a 1.8% agarose gel in TAE buffer (80 V, 60 min), stained in 2% ethidium bromide (Promega) under UV light in a Gel Doc EZ Imager photodocumenter (Bio-Rad®), a 100 bp molecular weight marker (Promega®) was used to compare the size of the amplification product.
Subsequently, the amplification products were purified using the protocol of the Wizard® SV Gel and PCR Clean-Up System kit (Promega) and were sequenced using the Sanger sequencing method. The amplifications were repeated in triplicate.
The sequences were analyzed and edited in the Bioedit 7.2.5 software (Hall, 1999) to perform a Blast analysis (parameters offered by default) in the genbank database of the NCBI. For this, the analysis included genomes of different formae speciales of F. oxysporum as well as genomes of other species of the Fusarium genus obtained from different electronic databases of open access.
A group of 81 sequences of genes coding for the xylanase enzyme were used, which were annealed using the ClustalW algorithm (gap open= 15; gap extend= 3). The database consisted of 76 sequences of different formae speciales and five of other species of the Fusarium genus. Annealing was performed in the Bioedit 7.2.5 software (Hall, 1999). Unweighted parsimony analyses were performed with the TNT 1.1 software (Goloboff et al., 2008) using the Winclada interface (1.94.1). The search for the most parsimonious tree was executed with 1 000 replicates for each case, using a combination of algorithms (Ratchet + Drift + Sectorial Fusion + TBR-max). Inferences about clade robustness were derived with Bootstrap resampling (1 000 repetitions with the same search characteristics).
The modified Nei-Gojobori model was applied to determine the parameters dN and dS (Nei and Gojobori, 1986). The calculations were performed using a maximum likelihood method based on the phylogenetic tree previously obtained. The general time-reversible (GTR) model was applied as a nucleotide substitution model and a standard genetic code was selected, this analysis was performed with the MEGA 7 software (Kumar et al., 2016).
Only three of the nine strains of F. oxysporum f. sp. vanillae (JAGH5, JAGH10, JAGH12) were positive for amplification of the xyl3 gene. A single 0.7 kb product with no nonspecific bands was observed. The sequencing process allowed obtaining three sequences with high definition in the electropherogram. The Blast analysis linked all sequences to the xylanase xyl3 gene of F. oxysporum f. sp. lycopersici (accession number AF052582.1) with 99% similarity.
These results allowed a genotyped division of F. oxysporum f. sp. vanillae into two groups, one in which the xyl3 gene is present and one in which it is absent. Since so far there were no reports of any type of xylanase in F. oxysporum f. sp. vanillae, the present study reports for the first time the detection of the xyl3 gene in this forma specialis. In addition, since the amplification reactions of the gene did not generate products in all the strains previously studied by Adame-García et al. (2015), it is stated that there are different genotypes within this forma specialis and that among these differences is the xylanase 3 enzyme (XYL3).
The xyl3 gene has been used to distinguish races of F. oxysporum f. sp. ciceris (Gurjar et al., 2009) and its differential activity has supported the differentiation of pathotypes (Jorge et al., 2005). It is noteworthy that the strains of F. oxysporum f. sp. vanillae that generated amplification products of the xyl3 gene belong to the group of moderate virulence for vanilla (Adame-García et al., 2015), while it has been demonstrated that several structural motifs of xylan have changed during the evolution of plant groups (Peña et al., 2016).
Such information will be valuable in determining how much the diversity of xylanase enzymes of F. oxysporum f. sp. vanillae is related to strains that present a higher degree of pathogenicity, considering the structural composition of xylans of the cell wall of the roots of V. planifolia and in comparison with Vanilla pompona, which has the characteristic of being one of the species of the genus most resistant to pathogens (Soto-Arenas and Solano Gómez, 2007).
The Blast analysis in each genome found in the NCBI database allowed identifying some copies of the xylanase xyl3 gene in different species and formae speciales of F. oxysporum. Table 1 shows the percentages of similarity achieved with the sequences of this study.
Parsimony analysis generated a single most parsimonious tree (Figure 1). In this topology, eight clades with appropriate bootstrap support were retrieved. Clade 3 contains the sequence corresponding to F. oxysporum f. sp. lycopersici xyl3 (AF052582) and the three sequences of the xylanase gene of F. oxysporum f. sp. vanillae, this corroborates that these strains contain a homologous gene of xyl3. It is noteworthy to note that this clade contains most of the strains of F. oxysporum f. sp. lycopersici and only one strain of F. oxysporum f. sp. medicaginis.
Other isoforms of the xylanase gene of F. oxysporum f. sp. lycopersici (xyl1, xyl4, xyl5) and xyl4 of F. oxysporum f. sp. ciceris are grouped in clade 4 and are sisters of a lineage with only the xyl2 isoform of F. oxysporum f. sp. lycopersici. Next to this clade, there are two small clades, the first composed of xylanase of F. graminearum (NC026476) and F. pseudograminearum (NC031953) and the second composed of xylanase of F. verticillioides (NC031678), F. fujikuroi (NC031678) and a strain of F. oxysporum f. sp. cubense RT4 (KB726570).
These two clades are very distinctive because these sequences were used as outer groups along with F. culmorum (LT598661). Clades 1, 2, and 8 have no sequences of F. oxysporum f. sp. lycopersici; those clades are composed of pathogenic strains of cucurbits, bananas and other plants. Some individual lineages were not adequately resolved. Eight well-supported clades were obtained for the phylogeny of the xylanase gene. A previous classification was proposed for the genes of the xylanase enzyme, they were classified as xyl1 (Ruiz et al., 1999), xyl2 and xyl3 (Ruiz et al., 1999), xyl4 and xyl5 (Gómez-Gómez et al., 2002). Nevertheless, in the phylogeny shown in the present study, all xyl genes, except for xyl3, are located in the same clade (Clade 2; Figure 1).
The model by Nei-Gojobori (1986) was used to determine whether some codons of the sequence of the xylanase xyl3 gene are affected by positive selection. For this purpose, 108 codons were analyzed for synonymous and nonsynonymous mutations. The most common amino acid found was glycine, with four different codons, GGC (eight times), GGG (five times), GGA (two times), GGT (one time). This shows that synonymous mutations are frequently present in xylanase xyl3 sequences. No significant results were observed regarding positive selection for other amino acids.
To evaluate the differences between xylanase genes according to clade division, natural selection tests based on codons were performed between the different lineages indicated by the gene tree. Synonymous mutations were found to be more abundant and common than nonsynonymous mutations, which in turn is evidence of neutral mutations (Nei and Gojobori, 1986). This approach has been used in other genes to detect positive selection; that is, evidence that natural selection gives rise to the diversity of some gene (Zhang et al., 2005; Hughes and Friedman, 2008; Metzger and Thomas, 2010). According to an exhaustive search of the scientific literature, this is the first study with an approach of positive selection tests on codons used for the analysis of genes in relation to pathogenicity in F. oxysporum.
Differences in the amplification of the xylanase xyl3 gene in strains of F. oxysporum f. sp. vanillae can be explained on the basis of the polyphyletic distribution of this forma specialis among the F. oxysporum Species Complex (Pinaria et al., 2015; Flores-de la Rosa et al., 2018). Some pathogenicity effectors move horizontally between different lineages of F. oxysporum, giving pathogenic capacity to these lineages. Some of these new pathogenic lineages contain the xyl3 gene in their genomes, while others do not, so there are pathogenic strains with and without activity of the gene (Laurence et al., 2015).
This research showed that the presence of the xyl3 gene is not a characteristic of all strains of F. oxysporum f. sp. vanillae, even the presence of the gene could be associated with moderate virulence. Phylogeny suggests different types of xyl genes; however, no evidence of positive selection was observed in the coding sequences for this gene in F. oxysporum.
Abdul-Karim, N. F.; Mohd, M.; Izham-Mohd, N. M and Zakaria, L. 2016. Saprophytic and potentially pathogenic Fusarium species from peat soil in Perak and Pahang. Trop Life Sci. Res. 27(1):1-20. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807956/.
Adame-García, J.; Trigos, Á.; Iglesias-Andreu, L. G.; Flores-Estévez, N. and Luna-Rodríguez, M. 2011. Isozymic and pathogenic variations of Fusarium spp. associated with vanilla stem and root rotting. Trop subtrop agroecosystems. 13(3):299-306. https://www.revista.ccba.uady.mx/ojs/index.php/TSA/article/view/1330/663.
Adame-García, J.; Rodriguez-Guerra, R.; Iglesias-Andreu, L. G.; Ramos-Prado, J. M. and Luna-Rodríguez, M. 2015. Molecular identification and pathogenic variation of Fusarium species isolated from Vanilla planifolia in Papantla Mexico. Bot. Sci. 93(3):669-678. https://doi.org/10.17129/botsci.142.
Adame-García, J.; Flores-Rosa, F. R.; Ricaño-Rodríguez, J. and Luna-Rodríguez, M. 2016. Adequacy of a protocol for amplification of EF-1α gene of Fusarium oxysporum f. sp. vanillae. ARPN J. Agric. Biol. Sci. 11(6):236-241. http://www.arpnjournals.org/jabs/ research-papers/rp-2016/jabs-0616-804.pdf.
Demers, J. E.; Gugino, B. K. and Jiménez-Gasco, M. M. 2015. Highly diverse endophytic and soil Fusarium oxysporum populations associated with field-grown tomato plants. Appl. Environ. Microbiol. 81(1):81-90. https://doi.org/10.1128/AEM.02590-14.
De-Vries, R. P. and Visser J. 2001. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiol Mol. Biol Rev. 65(4):497-522. https://doi.org/10.1128/MMBR.65.4.497-522.2001.
Edel-Hermann, V. and Lecomte, C. 2019. Current status of Fusarium oxysporum formae speciales and races. Phytopathology. 109(4):512-530. https://doi.org/10.1094/PHYTO-08-18-0320-RVW.
Flores-Rosa, F. R.; Luna, E.; Adame-García, J.; Iglesias-Andreu, L. G. and Luna-Rodríguez, M. 2018. Phylogenetic position and nucleotide diversity of Fusarium oxysporum f. sp. vanillae worldwide based on translation elongation factor 1α sequences. Plant Pathol. 67(6):1278-1285. https://doi.org/10.1111/ppa.12847.
Goloboff, P. A.; Farris, J. S. and Nixon, K. C. 2008. TNT, a free program for phylogenetic analysis. Cladistics. 24(5):774-786. https://doi.org/10.1111/j.1096-0031.2008.00217.x.
González-Oviedo, N.; Iglesias-Andreu, L. G.; Flores-Rosa, F. R.; Rivera-Fernández, A. and Luna-Rodríguez M. 2022. Genetic analysis of the fungicide resistance in Fusarium oxysporum associated to Vanilla planifolia. Mex. J. Phytopathol. 40(3):1-19. https://doi.org/10.18781/R.MEX.FIT.2203-3.
Gómez-Gómez, E.; Ruíz-Roldán, M. C.; Pietro, A.; Roncero, M. I. G. and Hera, C. 2002. Role in pathogenesis of two endo-β-1,4-xylanase genes from the vascular wilt fungus Fusarium oxysporum. Fungal Genet Biol. 35(3):213-222. https://doi.org/10.1006/ fgbi.2001.1318.
Gurjar, G.; Barve, M.; Giri, A. and Gupta, V. 2009. Identification of Indian pathogenic races of Fusarium oxysporum f. sp. ciceris with gene specific, ITS and random markers. Mycologia. 101(4):484-495. https://doi.org/10.3852/08-085.
Hughes, A. L. and Friedman, R. 2008. Codon-based tests of positive selection, branch lengths, and the evolution of mammalian immune system genes. Immunogenetics. 60:495-506. https://doi.org/10.1007/s00251-008-0304-4.
Jorge, I.; Rosa, O.; Navas-Cortés, J. A.; Jiménez-Díaz, R. M and Tena, M. 2005. Extracellular xylanases from two pathogenic races of Fusarium oxysporum f. sp. ciceris: enzyme production in culture and purification and characterization of a major isoform as an alkaline endo beta xylanase of low molecular weight. Antonie van Leeuwenhoek. 88:48-59. https://doi.org/10.1007/s10482-004-7584-y.
Kalluri, U. C.; Yin, H.; Yang, X. and Davison, B. H. 2014. Systems and synthetic biology approaches to alter plant cell walls and reduce biomass recalcitrance. Plant Biotechnol J. 12(9):1207-1216. https://doi.org/10.1111/pbi.12283.
Koyyappurath, S.; Conéjéro, G.; Dijoux, J. B.; Lapeyre-Montès, F.; Jade, K.; Chiroleu, F.; Gatineau, F.; Verdeil, J. L.; Besse, P. and Grisoni, M. 2015. Differential responses of vanilla accessions to root rot and colonization by Fusarium oxysporum f. sp. radicis-vanillae. Front Plant Sci. 6:1-16. https://doi.org/10.3389/fpls.2015.01125.
Koyyappurath, S.; Atuahiva, T.; Le Guen, R.; Batina, H.; Le Squin, S.; Gautheron, N.; Edel-Hermann, V.; Peribe, J.; Jahiel, M.; Steinberg, C.; Liew, E. C. Y.; Alabouvette, C.; Besse, P.; Dron, M.; Sache, I.; Laval, V. and Grisoni, M. 2016. Fusarium oxysporum f. sp. radicis-vanillae is the causal agent of root and stem rot of vanilla. Plant Pathol. 65(4):612-625. https://doi.org/10.1111/ppa.12445.
Kumar, S.; Stecher, G. and Tamura, K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33(7):1870-1874. https://doi.org/10.1093/molbev/msw054.
Laurence, M. H.; Summerell, B. A. and Liew, E. C. Y. 2015. Fusarium oxysporum f. sp. canariensis: evidence for horizontal gene transfer of putative pathogenicity genes. Plant Pathol. 64(5):1068-75. https://doi.org/10.1111/ppa.12350.
Metzger, K. J. and Thomas, M. A. 2010. Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins. BMC Evol. Biol. 10(139):1-9. https://doi.org/10.1186/1471-2148-10-139.
Nei, M. and Gojobori, T. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3(5):418-426. https://doi.org/10.1093/oxfordjournals.molbev.a040410.
O’Donnell, K.; Gueidan, C.; Sink, S.; Johnston, P. R.; Crous, P. W.; Glenn, A.; Riley, R.; Zitomer, N. C.; Colyer, P. and Waalwijk, C. 2009. A two-locus DNA sequence database for typing plant and human pathogens within the Fusarium oxysporum species complex. Fungal Gen. Biol. 46(12):936-948. https://doi.org/10.1016/j.fgb.2009.08.006.
Olivain, C.; Humbert, C.; Nahalkova, J.; Fatehi, J.; L’Haridon, F. and Alabouvette, C. 2006. Colonization of tomato root by pathogenic and nonpathogenic Fusarium oxysporum strains inoculated together and separately into the soil. Appl. Environ. Microbiol . 72(2):1523-1531. https://doi.org/10.1128/AEM.72.2.1523-1531.2006.
Pattathil, S.; Hahn, M. G.; Dale, B. E. and Chundawat, S. P. S. 2015. Insights into plant cell wall structure, architecture, and integrity using glycome profiling of native and AFEXTM-pretreated biomass. J. Exp. Bot. 66(14):4279-4294. https://doi.org/10.1093/jxb/ erv107.
Peña, M. J.; Kulkarni, A. R.; Backe, J. Boyd, M. O.; Neill, M. A. and York, W. S. 2016. Structural diversity of xylans in the cell walls of monocots. Planta. 244:589-606. https://doi.org/10.1007/s00425-016-2527-1.
Pinaria, A. G.; Liew, E. C. Y. and Burgess, L. W. 2010. Fusarium species associated with vanilla stem rot in Indonesia. Australasian Plant. Pathol. 39:176-183. https://doi.org/10.1071/AP09079.
Pinaria, A. G.; Laurence, M. H.; Burgess, L. W. and Liew, E. C. Y. 2015. Phylogeny and origin of Fusarium oxysporum f. sp. vanillae in indonesia. Plant Pathol. 64(6):1358-65. https://doi.org/10.1111/ppa.12365.
Ruiz-Roldan, M. C.; Di-Pietro, A.; Huertas-Gonzalez, M. D. and Roncero, M. I. 1999. Two xylanase genes of the vascular wilt pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants. Mol. Gen. Genet. 261:530-536. https://doi.org/10.1007/s004380050997.
Soto-Arenas, M. A y Solano-Gómez, A. R. 2007. Ficha técnica de Vanilla planifolia. En: información actualizada sobre las especies de orquídeas del PROY-NOM-059-ECOL-2000. Instituto Chinoin A.C. Herbario de la Asociación Mexicana de Orquideología AC. Proyecto No. W029. México. DF. 1-18 pp. http://www.conabio.gob.mx/conocimiento/ise/fichasnom/Vanillaplanifolia00.pdf.
Turrà, D.; Ghalid, M.; Rossi, F. and Pietro, A. 2015. Fungal pathogen uses sex pheromone receptor for chemotropic sensing of host plant signals. Nature. 527:521-524. https://doi.org/10.1038/nature15516.
Waweru, B.; Turoop, L.; Kahangi, E.; Coyne, D. and Dubois, T. 2014. Non-pathogenic Fusarium oxysporum endophytes provide field control of nematodes, improving yield of banana (Musa sp.). Biological Control. 74:82-88. https://doi.org/10.1016/j.biocontrol.2014.04.002.
Zhang, J.; Nielsen, R.; and Yang, Z. 2005. Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol. Biol. Evol. 22(12):2472-2479. https://doi.org/10.1093/molbev/msi237.