**6. Genetic diversity and evolution of Fusarium wilt of bananas**

*Fusarium oxysporum* Species Complex (FOSC) are widely distributed and it is mostly non-pathogenic and it is commonly found in roots and soil associated fungus in asymptomatic crop plants. It has been found to be associated with plants as endophyte, saprophyte or just latent in agro-ecosystems [30]. Both, studies on FOSC isolated from non-cultivated species and form cultivated crops have reported a considerable variability based on the morphology of the asexual reproductive structures [31] and latter at the DNA sequence [32, 33]. Understanding its genetic variability is relevant to implement an earlier detection system and implement a proper disease surveillance program.

Recent studies on molecular genetics of *Fusarium* from cultivated plants have shown a high diversity and this variation relays on environmental conditions and are classified in groups and vegetative compatible groups (VCG) as described latter in this chapter. *Fusarium* has evolved heavily depending on its interaction with plant genotypes, such is the case for both 'tropical' and 'subtropical' race 4, which attacks different cultivars, depending of the geographical region [34] as well as for those FOSC from non-cultivated species [30].

Knowledge of the genetic diversity of populations of phytopathogenic fungi and their mode of reproduction are important for the application of management strategies, this with the aim of reducing the impact of the disease [35]. In the case of Foc, this pathogen shows a relatively diverse population genetic structure for a fungus apparently of asexual reproduction and is composed of different evolutionary lineages [33], which has 24 groups of vegetative compatibility (VCGs, VCG0120 to VCG0126 and VCG0128 to VCG01224) distributed worldwide [34, 36–40].

However, in recent samplings in Latin America it was possible to identify 20 new VCGs (new VCG 1 to new VCG 20), these were distributed over the three main clades (clade 1, clade 2 and clade 3), these results show that the majority of the new VCG are grouped in clade 3 and these originate from Latin America [41], this supports the hypothesis on the evolution of Foc, in which it is mentioned that the local populations of *F. oxysporum* evolved and they became pathogenic in the introduced bananas [36, 42–44].

Studying VCGs has been a useful means of subdividing Foc into genetically isolated groups, but it does not, however, measure the genetic relationship between

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*Genetic Diversity of Fusarium Wilt Disease of Banana DOI: http://dx.doi.org/10.5772/intechopen.94158*

the pathogen can potentially reproduce by sexual means.

other molecular tools.

the appearance of Foc-TR4.

considered to be part of Foc [5].

genome B.

the isolates. Furthermore, VCG are phenotypic markers that can undergo a selection process. Direct identification of VCG is a relatively objective, but time-consuming test, and the results indicate genetic similarity rather than genetic difference [31]. Therefore, VCGs represent good phenotypic traits for assessing diversity within populations, but the genetic relationships between VCGs must be assessed using

Fourie and collaborators [39], classified Foc into two clades, clade 1 and clade 2, these based mainly on their evolutionary origins. In the case of clade A, the Foc groups that co-evolved with bananas of genome A belong, while those that belong to clade B evolved with their hosts having genome B or both genome A and

The teleomorph for Foc has never been reported and the pathogen is likely to manifest mutations or parasexualism, as the main basis for its genetic diversity. Although PCR analysis has shown the presence of both MAT idiomorphs, therefore

The race concept has been widely used in the *F. oxysporum* classification system

Three races of Foc are known; but nevertheless, the term race is used in a less formal way in relation to this pathosystem (*Musa*-Foc), since the genetic bases of susceptibility and resistance have not yet been characterized. The Foc races currently described refer to strains of the pathogen, which have been found to be pathogenic to specific cultivars in the field [9, 38]. For example, race (R1) is pathogenic to cultivars of 'Silk', 'Manzano' (AAB) and 'Gros Michel' (AAA). While race 2 (R2) is pathogenic to cooking bananas such as 'Bluggoe' and 'Pear' (ABB) and race 4 (R4) affects all cultivars of the Cavendish subgroup (AAA) and those susceptible to R1 and R2 [4, 5, 45]. Previously, a population of *Fusarium oxysporum* in Central America was considered as race 3 causing wilt in *Heliconia* spp., but is no longer

A Foc race 4 variant was reported in Taiwan affecting Cavendish cultivars in the tropics in 1967 [4, 38]. Therefore, it was necessary to separate the populations that only affected Cavendish cultivars in the subtropics from those populations that affected in the tropics, so two divisions of Foc R4 were generated: race 4 sutropical (STR4) and race 4 tropical (TR4) [38], however, TR4 was pathogenic under tropical and subtropical conditions affecting Cavendish cultivars [25, 39]. In the case of VCGs, they have been associated with STR4 (0120, 01201, 01202, 01209, 01210, 01211, 01215, 0120/15; 0129/11), while only one VCG to TR4 (01213/16) [25, 40]. Visser and collaborators [46], carried out a study on the characterization of tropical Foc race 4 populations affecting 'Cavendish' plantations in South Africa. Only VCG 0120 and idiomorph MAT-2 could be identified, while phylogenetic analysis of the TEF sequence revealed that the isolates from South Africa were pooled with other isolates belonging to VCG 0120 from Australia and Asia. Suggesting, the

introduction and dispersal mainly by infected material within the country.

In Latin America and the Caribbean, the composition of the populations has been limitedly studied. For example, the Cuban populations belong to VCG 01210 (mostly race 1), 0124, 0124/0125 and 0128 (mostly race 2); the isolates did not produce lacinias in K2 medium and the production of volatiles was independent of the race, while in Venezuela VCG 01215 and race 1 are reported. A study using AFLP markers grouped VCG 01210 into a subgroup and showed the presence of common

by plant pathologists. Based on the published data, it can be inferred that the Foc-TR4 isolates recently evolved from the predecessors in Foc-R1. Foc-R1 showed greater phylogenetic diversity than Foc-TR4. Once established, both races apparently co-evolved in the same region, which means that possible horizontal gene transfer could be involved in the high level of diversity seen in Foc-R1, as well as in *Genetic Variation*

Foc has the ability to spread in the soil, which indirectly contaminates in and around plantations, but unfortunately it is also used in nurseries for the propagation of seedlings used for field establishment [25]. Surface waters are easily polluted and use for irrigation of polluted river or pond water is highly risky. In addition, Foc is spread by contaminated tools (shovels, machetes, hoes, etc.), agricultural machinery, clothing and footwear [9, 28]. Any or all of these ways can facilitate the spread of Foc in and around a plantation, and may be possible through other means [6, 28]. Studies carried out in Australia detected TR4 spores in the exoskeletons of the banana weevil (*Cosmopolites sordidus*) and suggested that the insect could be a

The recent transcontinental disseminations of TR4, suggest that something other than vegetative material (suckers) was responsible for these long-distance disseminations. Although these outbreaks may have been the result of something as simple as workers' boots impregnated with soil contaminated by Foc spores from plantations in Southeast Asia, or some other means could be responsible such as the entry of machinery from affected areas. Better knowledge is needed to understand

**6. Genetic diversity and evolution of Fusarium wilt of bananas**

*Fusarium oxysporum* Species Complex (FOSC) are widely distributed and it is mostly non-pathogenic and it is commonly found in roots and soil associated fungus in asymptomatic crop plants. It has been found to be associated with plants as endophyte, saprophyte or just latent in agro-ecosystems [30]. Both, studies on FOSC isolated from non-cultivated species and form cultivated crops have reported a considerable variability based on the morphology of the asexual reproductive structures [31] and latter at the DNA sequence [32, 33]. Understanding its genetic variability is relevant to implement an earlier detection system and implement a

Recent studies on molecular genetics of *Fusarium* from cultivated plants have shown a high diversity and this variation relays on environmental conditions and are classified in groups and vegetative compatible groups (VCG) as described latter in this chapter. *Fusarium* has evolved heavily depending on its interaction with plant genotypes, such is the case for both 'tropical' and 'subtropical' race 4, which attacks different cultivars, depending of the geographical region [34] as well as for those

Knowledge of the genetic diversity of populations of phytopathogenic fungi and their mode of reproduction are important for the application of management strategies, this with the aim of reducing the impact of the disease [35]. In the case of Foc, this pathogen shows a relatively diverse population genetic structure for a fungus apparently of asexual reproduction and is composed of different evolutionary lineages [33], which has 24 groups of vegetative compatibility (VCGs, VCG0120 to

However, in recent samplings in Latin America it was possible to identify 20 new VCGs (new VCG 1 to new VCG 20), these were distributed over the three main clades (clade 1, clade 2 and clade 3), these results show that the majority of the new VCG are grouped in clade 3 and these originate from Latin America [41], this supports the hypothesis on the evolution of Foc, in which it is mentioned that the local populations of *F. oxysporum* evolved and they became pathogenic in the introduced

Studying VCGs has been a useful means of subdividing Foc into genetically isolated groups, but it does not, however, measure the genetic relationship between

VCG0126 and VCG0128 to VCG01224) distributed worldwide [34, 36–40].

predisposing agent as a vector of the disease [29].

the long-distance spread of this pathogen [6].

proper disease surveillance program.

FOSC from non-cultivated species [30].

**134**

bananas [36, 42–44].

the isolates. Furthermore, VCG are phenotypic markers that can undergo a selection process. Direct identification of VCG is a relatively objective, but time-consuming test, and the results indicate genetic similarity rather than genetic difference [31]. Therefore, VCGs represent good phenotypic traits for assessing diversity within populations, but the genetic relationships between VCGs must be assessed using other molecular tools.

Fourie and collaborators [39], classified Foc into two clades, clade 1 and clade 2, these based mainly on their evolutionary origins. In the case of clade A, the Foc groups that co-evolved with bananas of genome A belong, while those that belong to clade B evolved with their hosts having genome B or both genome A and genome B.

The teleomorph for Foc has never been reported and the pathogen is likely to manifest mutations or parasexualism, as the main basis for its genetic diversity. Although PCR analysis has shown the presence of both MAT idiomorphs, therefore the pathogen can potentially reproduce by sexual means.

The race concept has been widely used in the *F. oxysporum* classification system by plant pathologists. Based on the published data, it can be inferred that the Foc-TR4 isolates recently evolved from the predecessors in Foc-R1. Foc-R1 showed greater phylogenetic diversity than Foc-TR4. Once established, both races apparently co-evolved in the same region, which means that possible horizontal gene transfer could be involved in the high level of diversity seen in Foc-R1, as well as in the appearance of Foc-TR4.

Three races of Foc are known; but nevertheless, the term race is used in a less formal way in relation to this pathosystem (*Musa*-Foc), since the genetic bases of susceptibility and resistance have not yet been characterized. The Foc races currently described refer to strains of the pathogen, which have been found to be pathogenic to specific cultivars in the field [9, 38]. For example, race (R1) is pathogenic to cultivars of 'Silk', 'Manzano' (AAB) and 'Gros Michel' (AAA). While race 2 (R2) is pathogenic to cooking bananas such as 'Bluggoe' and 'Pear' (ABB) and race 4 (R4) affects all cultivars of the Cavendish subgroup (AAA) and those susceptible to R1 and R2 [4, 5, 45]. Previously, a population of *Fusarium oxysporum* in Central America was considered as race 3 causing wilt in *Heliconia* spp., but is no longer considered to be part of Foc [5].

A Foc race 4 variant was reported in Taiwan affecting Cavendish cultivars in the tropics in 1967 [4, 38]. Therefore, it was necessary to separate the populations that only affected Cavendish cultivars in the subtropics from those populations that affected in the tropics, so two divisions of Foc R4 were generated: race 4 sutropical (STR4) and race 4 tropical (TR4) [38], however, TR4 was pathogenic under tropical and subtropical conditions affecting Cavendish cultivars [25, 39]. In the case of VCGs, they have been associated with STR4 (0120, 01201, 01202, 01209, 01210, 01211, 01215, 0120/15; 0129/11), while only one VCG to TR4 (01213/16) [25, 40].

Visser and collaborators [46], carried out a study on the characterization of tropical Foc race 4 populations affecting 'Cavendish' plantations in South Africa. Only VCG 0120 and idiomorph MAT-2 could be identified, while phylogenetic analysis of the TEF sequence revealed that the isolates from South Africa were pooled with other isolates belonging to VCG 0120 from Australia and Asia. Suggesting, the introduction and dispersal mainly by infected material within the country.

In Latin America and the Caribbean, the composition of the populations has been limitedly studied. For example, the Cuban populations belong to VCG 01210 (mostly race 1), 0124, 0124/0125 and 0128 (mostly race 2); the isolates did not produce lacinias in K2 medium and the production of volatiles was independent of the race, while in Venezuela VCG 01215 and race 1 are reported. A study using AFLP markers grouped VCG 01210 into a subgroup and showed the presence of common

alleles with VCG 0124 [47]. On the other hand, the pathogenicity studies with representative isolates of each VCG in Cuba, showed a differentiated aggressiveness on different clones between VCG 0124 and 0128, belonging to race 2, indicating lack of genetic sense in the racial classification. It is required to determine in Latin America and the Caribbean the VCG present in the different countries and the pathogenic relationships between them.

In order to better understand how races 1 and 4 are related, genome and transcriptome analysis of *F. oxysporum* f. sp. *cubense* has shown common sequences of single-copy genes from Race 1 and Race 4, showing that there is a close relationship and suggesting that they share a common ancestor. Furthermore, a comparative genomics study among *F. oxysporum* f. sp. *licoperci*, *F. graminearum* and *F. verticillioides* showed that there is transfer of lineage-specific (LS) genomic regions that have pathogenicity related genes with distinct evolutionary profiles, indicative of horizontal acquisition and suggesting that there is transfer of LS chromosomes between genetically isolated *Fusarium* species. This is of high relevance and of particular concern for agricultural systems, because non-pathogenic *F. oxysporum* strains that are already endophytic to crop plants could suddenly become pathogenic [48] and give origin to new pathogenic lineages of *F. oxysporum*. It is clear that in the last decade a large amount of DNA sequence information has been published on *F. oxysporum*, but there is a lack of consistency in the data and a larger study needs to be conducted in which DNA sequences of isolates from non-cultivated species is included and even from *Fusarium* species that are thought not to be related.

Foc genetic diversity studies were initiated using various molecular methods, including random amplified polymorphic DNA markers (RAPDs) [49]; Restriction Fragment Length Polymorphisms (RFLP) [43]; Amplified fragment length polymorphism (AFLP) [50]; DNA sequence analysis [32, 44]; microsatellites or simple repetitive sequences [51]; simple repetitive inter sequence (ISSR) [52]. These studies showed that the population of this fungus in Southeast Asia shows a high degree of variation, suggesting that the Foc lineages evolved together with their hosts in Southeast Asia.

Alternatively, Foc has been suggested to have multiple independent evolutionary origins, both within and outside the *Musa* genetic center [36]. Using the phylogenetic genealogical approach, [32] identified five Foc-independent genetic lineages in a global population. Using a similar approach and additional data, [44] found three additional lineages. However, none of these studies included Indonesian populations, and therefore there is only limited information available on Foc diversity at the center of origin of bananas.

*F. oxysporum* f.sp. *cubense* probably coevolved with its host species within its center of origin [32, 36, 44]. For example, various studies that have used deoxyribonucleic acid (DNA) markers have revealed the polyphyletic origin of Foc and the separation of two main clades and eight to ten lineages, as some VCGs are taxonomically closer to other special forms of *F. oxysporum* than some Foc VCGs [32, 36, 42, 44, 50, 53].

Furthermore, strains belonging to various VCGs infect particular banana cultivars and, therefore, were grouped in the same race, suggesting that the pathogenicity towards a specific cultivar evolved in a convergent way [32, 38, 44] or as a result of horizontal gene transfer between members of the *F. oxysporum* complex [48, 54].

High resolution genotyping sequencing analyzes using (DArTseq) validated and expanded these findings [55]. According to the DArTseq markers of 24 Foc strains (representing all the known VCG so far) they were divided into two groups. These results strongly corroborate the clades mentioned in previous studies, except VCG0123, VCG01210, VCG01212 and VCG01214, which were occasionally grouped into opposing clades, VCG 01221 and 01224, which were never classified before but now clearly belong to clade 2 [55].

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*Genetic Diversity of Fusarium Wilt Disease of Banana DOI: http://dx.doi.org/10.5772/intechopen.94158*

lack of chromosome stability [57].

*F. odoratissimum* II-5 comprises TR4 [58].

two large clades and nine clonal lineages.

**7. Conclusions**

In the advent of high throughput DNA sequencing technology [56] has allowed scientist to better understand the molecular weaponry used by this pathogen. The pathogen molecular tools include genes involved in root attachment, cell degradation, detoxification of toxins produced by the plant's defense mechanism and signal transduction, among others [16]. In Ref. [57], the authors have reported a predicted genome size for several *F. oxysporum* f. sp. *cubense* with a size of 48.56 Mb for Foc Race 1 and 48.81 for Foc Race 4, comprising and estimated of 15,865 and 14,506 genes, respectively. This genome information was compared and aligned to 11 of the 15 chromosomes contained in *F. oxysporum* f. sp. *licopersici*, including those regions reach in transposable elements; which might explain its high genetic variability and

Recently, in a study samples of musaceae with wilt symptoms were collected in the regions of Indonesia, Java, Sumatra, Kalimantan, Sulawesi, Papua and Nusa Tenggara, this demonstrated by phylogenetic analysis that the Foc lineages were genetically different, and it was achieved to identify 11 new species of *Fusarium* affecting musaceae, these were: *Fusarium cugenangense*, *F. duoseptatum*, *F. grosmichelii*, *F. hexaseptatum*, *F. kalimantanense, F. odoratissimum, F. phialophorum, F. purpurascens, F. sangayamense, F. tardichlamydosporum,* and *F. tardicrescens*, placing them in the Banana Fusarium Complex (FOBC), as well as showing that

Fusarium wilt disease of banana caused by soil-born pathogen *Fusarium oxysporum* f.sp. *cubense* (Foc) is considered of the most destructive diseases of bananas and plantains worldwide. Foc produces three types of asexual spores, these are macroconidia, microconidia and chlamydospores, which function as mechanisms of dispersal, reproduction and survival. Foc is a genetically diverse pathogen, although the available evidence so far indicates that it does not use the mechanisms of sexual reproduction, such as recombination, to increase its genetic diversity. Furthermore, the population of this fungus in Southeast Asia shows a high degree of variation, suggesting that Foc lineages evolved together with their hosts in Southeast Asia. Alternatively, it has been suggested that Foc has multiple independent evolutionary origins, both within and outside of the Musaceae origin center. Actually, more than 24 vegetative compatibility groups and three races have been reported. This genetic diversity is accommodated in

*Genetic Diversity of Fusarium Wilt Disease of Banana DOI: http://dx.doi.org/10.5772/intechopen.94158*

In the advent of high throughput DNA sequencing technology [56] has allowed scientist to better understand the molecular weaponry used by this pathogen. The pathogen molecular tools include genes involved in root attachment, cell degradation, detoxification of toxins produced by the plant's defense mechanism and signal transduction, among others [16]. In Ref. [57], the authors have reported a predicted genome size for several *F. oxysporum* f. sp. *cubense* with a size of 48.56 Mb for Foc Race 1 and 48.81 for Foc Race 4, comprising and estimated of 15,865 and 14,506 genes, respectively. This genome information was compared and aligned to 11 of the 15 chromosomes contained in *F. oxysporum* f. sp. *licopersici*, including those regions reach in transposable elements; which might explain its high genetic variability and lack of chromosome stability [57].

Recently, in a study samples of musaceae with wilt symptoms were collected in the regions of Indonesia, Java, Sumatra, Kalimantan, Sulawesi, Papua and Nusa Tenggara, this demonstrated by phylogenetic analysis that the Foc lineages were genetically different, and it was achieved to identify 11 new species of *Fusarium* affecting musaceae, these were: *Fusarium cugenangense*, *F. duoseptatum*, *F. grosmichelii*, *F. hexaseptatum*, *F. kalimantanense, F. odoratissimum, F. phialophorum, F. purpurascens, F. sangayamense, F. tardichlamydosporum,* and *F. tardicrescens*, placing them in the Banana Fusarium Complex (FOBC), as well as showing that *F. odoratissimum* II-5 comprises TR4 [58].
