Fusarium oxysporum f. sp. cubense: infection mechanism and symptoms of the disease

The genus Fusarium is the broadest and most important in the Tuberculareacea family, and from a taxonomical standpoint, it is one of the most difficult to handle out of all the fungus groups. This fungus covers numerous species and diverse special forms (f. sp.) within each species. On the other hand, it presents great variability and its identification in different species is imprecise (^{Dean et al., 2012}). The perfect phase of F. oxysporum (FOX) is still unknown. However, the survival and proliferation of this species and is special forms depend on its asexual spores. FOC produces three types of asexual spores, including macroconidia, microconidia and chlamydospores in its life cycle (Figure 1 A-C), which help it spread and survive. In addition, it shares a similar infection cycle with Fusarium oxysporum f. sp. lycopercisi (FOL), the causal agent of wilt in tomato (Solanum lycopersicum) (^{Guo et al., 2014}). Chlamydospores have thick walls, formed inside the hypha or the macroconidia, and can survive in the absence of the host for several years in a latent state, or they can germinate and grow as saprophytes in the remains of non-host plants to produce new chlamydospores (^{Nelson et al., 1983}). FOC conidia germinate and form fungal hyphae under diverse nutritional and environmental conditions; the hyphae develop around the roots and colonize their surface. They then penetrate the epidermis, invade and colonize the xylem vessels of the root (Figure 1 D-F). After the process of infection in the banana plant roots, the fungus grows towards the rhizome and the pseudostem and causes the death of the tissue or of the entire plant. Between the initial infection by the fungus and the external symptoms, there is a moment in which the limbo of the leaves turns a bright yellow and it wilts or collapses around the pseudostem (Figure 1 G), whereas the vascular bundles of the pseudostem and of the rhizoma of the diseased plant turn reddish or maroon (Figure 1 H-I) (Guo et al., 2014).

The fungus may survive in the absence of the host for extended periods in a latent state or it may germinate and grow as a saprophyte in the remains of non-host plants to produce new chlamydospores. Estas a su vez le permiten al patógeno adaptarse a condiciones extremas (^{Ploetz et al., 2015}). Consequently, the susceptible genotypes cannot be grown in an infected field for up to 30 years (^{Buddenhagen, 2009}). However, the infection process may end and not progress when the plant is not susceptible or is not predisposed by environmental stress. ^{Dong et al. (2012}) found that the resistant host responds to a signal in the vessels infested with the formation of callose, vascular gels and tylose that immobilize the spores around the storage site. The development of these structures was examined in detail under and electron microscope and it was determined that the synthesis and release of the phenolic substances produced and lignified by these structures are the same which stop the invasion of the pathogen during the host-pathogen interaction.

Figure 1 . Vascular wilt infection cycle in banana plants, caused by Fusarium oxysporum f. sp. cubense. A) macroconidia; B) microconidia; C) chlamydospore caused by the isolation FOC, marked with fluorescent green protein; D) connection of the FOC hypha to the roots of the banana plant; E) colonization of the vascular bundles of the roots of the banana plant via the hypha of the FOC (indicated with the arrow); F) longitudinal section of the roots of the banana plant showing the fungal hyphae growing in the vascular bundles; G) diseased banana plants with the predominant symptoms of yellow leaves; H-I) diseased vascular bundles of the pseudostems and the banana plant rhizomes turning reddish or maroon (indicated by the arrow). Source: et al. (2014). 

Pathogenic complexity of FOC

The variability of FOC populations has been differentiated based on its pathogenicity, in which three races have been identified that affect Musa spp.: race 1, responsible of the epiphytotic disease in plantations of Manzano (AAB) and Gros Michel (AAA) cultivars; race 2, pathogenic to the Bluggoe (ABB) cultivar and some tetraploids (AAAA); and race 4, which attacks the cultivars of the Cavendish subgroup (AAA) and all those susceptible to races 1 and 2 (^{Ploetz, 2006}). The latter was discovered in 1990 in Taiwan, and later divided into the tropical and subtropical strains (Ploetz, 2006). Nowadays, race 4 of FOC is found in twenty out of 135 banana-producing countries (^{Martínez et al., 2020}). However, designating FOC races was cumbersome, and other methods were developed which revealed its genetic diversity. The vegetative compatibility analysis is a faster and more reliable technique than the pathogenicity test. Its use has helped determine the diversity of FOC races within a region. The analyses of vegetative compatibility groups (GCV) divided the FOC isolations into 24 GCVs (GCV0120 to GCV0126 and GCV0128 to GCV01224) (Ploetz, 2006). Later deoxyribonucleic acid (DNA) markers revealed the polyphyletic origin of FOC, since some GCV are taxonomically closer to other special forms of FOX than to other FOC GCVs (^{Fourie et al., 2009}). Additionally, the strains belonging to diverse GCVs infest particular banana cultivars and were therefore grouped in the same race. This suggests that the pathogenicity to a specific cultivar has evolved convergently or is a result of the horizontal transfer of genes between the members of the FOX complex (Ploetz, 2006). In general, FOC lineages show a notable dichotomy referred to as types or clades (^{Groenewald et al., 2006}). High resolution genotypification by sequencing analysis using DArTseq generates readings of short sequences after reducing the complexity of a genome by digestion with restriction enzymes. In this sense, markers of entire genomes using DArTseq divided the 24 FOC strains (which represent all the GCVs known to date) into two groups (^{Cruz et al., 2013}). On the other hand, the analysis of the genome revealed that the genomic structures of the isolations of races 1 and 4 were highly synthetic (occurring in the same chromosome) with those of FOL strain Fol4287. ^{Guo et al. (2014}) managed to identify SIX orthologous genes, primarily described for the genome of FOL isolations via the analysis of the genomes of isolations from race 1 and the RT4 of FOC. In this regard, many protocols have been designed to detect FOC isolation molecules, which have been thoroughly revised by ^{Lin and Lin (2016)} and ^{Ying and Yi (2016}). Thus, FOC is composed of three races, eight lineages and 24 GCVs. Most of the damage is caused by the RT4 in FOC, found only in Asia. However, ^{García-Bastidas et al. (2019}) reported the first case of FOC-related RT4 outside of Southeast Asia in the Colombian Guajira region, classified as Fusarium odoratissimum (Figure 2). This represents a high risk for all the other countries in the Americas. Therefore, strategies must be developed to fight this disease and minimize the hazards of the effects caused by the entry of this new race of the pathogen into exporting Latin American and Caribbean countries.

Controlling wilt by Fusarium in banana

The perennial production of the banana crop and the polycyclic nature of wilt by Fusarium hinder the development of efficient management strategies. In spite of this, using the available knowledge and technologies, it is now easier to implement quarantine policies and therefore avoid pathogens from spreading. In relation to this, in Mexico, the SADER (Secretariat of Agriculture and Rural Development) via the SENASICA (National Agrifood Health, Safety and Quality Service) are taking important measures to prevent the entry and spreading of the RT4 of FOC into the country. These measures include phytosanitary surveillance actions for the timely detection of RT4, training technicians and farmers in order to raise awareness regarding the risk of moving vegetative material, and finally, exploring and monitoring the banana-producing regions (^{Manzo et al., 2014}). Other measures for management also included are planting annually in phases, the use of tissue culture plants, crop rotation, having an adequate drainage system in the plantation, the incorporation of organic matter and applying biocontrol agents via Trichoderma harzianum. However, these control methods only allow for planting these cultivars for short periods, since the fungus destroys the plantations soon after (^{Fu et al., 2017}). Due to this, the only efficient control method has been considered to be planting disease-resistant hosts (^{Dita et al., 2011}). In this sense, since the appearance of wilt by Fusarium in different banana-producing countries, a wide genetic breeding program has been developed for the resistance to the three races of FOC, using conventional and biotechnological techniques, with a recent emphasis on the RT4 of FOC.

Figure 2 Distribution of tropical race 4 of Fusarium osyxporum f. sp. cubense in banana plants. Map produced by International Biodiversity for ProMusa (image courtesy of ProMusa.com). 

Biotechnological techniques to support the genetic breeding of Musa spp. for the resistance to FOC

The greatest possibilities provided by biotechnological techniques are found in the improvement of the resistance to pathogens, which offer several advantages over conventional methods, due to the possibility of selecting individuals from large populations of plants in a short timeframe. This increases the possibility of finding the desired traits. On the other hand, they help control inoculants and the environmental conditions that may interfere with the results obtained. In turn, they help access plants that are free of diseases and offer the efficiency of the genetic breeding programs for crops (^{Ortiz and Swennen, 2014}). For these reasons, the results of the breeding of banana and plantain plants have been introduced faster than with the conventional genetic breeding methods (^{Li et al., 2015}); micropropagation has played an important role, and particularly, it has improved the management of healthy germplasm on a global scale. Somaclonal variation, in vitro mutagenesis, genetic transformation and early selection in the Musa genus have been used successfully in several genetic breeding programs.

Utilization of the somaclonal variation

Somaclonal variation, which comprises genetic or epigenetic changes induced during the callus phase of the plant cells grown in vitro has been used for genetic breeding in several agronomic traits. ^{Molina et al. (2011}) determined the response to susceptibility or resistance to the RT4 in FOC in two somaclonal variants of Cavendish (AAA) from Taiwan, two commercial cultivars of the Cavendish subgroup, three local cultivars, and an improved hybrid from Honduras, in a field previously infested with the pathogen from the Philippines. The investigation observed the highest values of incidence of the disease (100%) in two commercial Cavendish cultivars (Gran Enano and Williams), as well as in the local cultivar ‘Lakatan’. However, no plants infected with the FOC RT4 were found in the somaclonal variants from Taiwan (‘GCTCV119’ and ‘Formosana’). In other studies, ^{Molina et al. (2016)} compared four selections of somaclonal variations (‘GCTCV-105’, ‘GCTCV-119’, ‘GCTCV-218’ and ‘GCTCV-219’) of Cavendish (AAA) from Taiwan and three important local cultivars from the Philippines: ‘Latundan’ (AAB, subgroup Seda), ‘Lakatan’ (AAA, subgroup Lakatan) and ‘Saba’ (ABB, subgroup Saba) with the commercial Cavendish Gran Enano (AAA, subgroup Cavendish) in a soil severely infested with FOC RT4 in southern Philippines. Results displayed susceptibility with an incidence of the disease of 64 and 76% respectively in the commercial cultivars of Gran Enano and ‘Lakatan’ in the first cycle of evaluation of resistance, and 79 and 92% respectively in the second cycle. However, ‘GCTCV’ somaclonal variants expressed an incidence of the disease of 0 to 6% in the first cycle and 0 to 8% in the second cycle of the tests to evaluate the symptoms of the disease. The cultivar ‘Saba’ displayed an incidence of the disease of 0% in both the first and second cycles of evaluation of resistance to the fungus. These results confirmed the stability in the resistance to FOC RT4 in the ‘GCTCV’ somaclonal variants. On the other hand, they show that the selection of somaclonal variants are a feasible tool in genetic breeding for the search for resistance to FOC in banana plants.

Utilization of induced in vitro mutagenesis

Mutagenic techniques that induce inheritable changes in the genetic composition of a cell by altering its deoxyribonucleic acid (DNA) have been used as a very efficient tool in the genetic breeding of plants. In this sense, ^{Chen et al. (2013}) used in vitro mutagenesis, induced by ethyl methanesulfonate to evaluate the obtaining of lines of Brazilian banana (Musa spp., AAA) resistant to FOC RT4. The plants regenerated from the five Brazilian banana lines resistant to the RT4 of the pathogen then underwent an in vitro selection method, in which they displayed a reduction in the incidence of the disease in relation to plants used as a control. Meanwhile, ^{Saraswathi et al. (2016}) obtained banana mutants from the cultivar Rasthali (Silk, AAB), resistant to race 1 of FOC, vegetative compatibility group (GCV) [0124/5]. These results show that it is possible to adopt different biotechnological techniques for the genetic breeding of banana and obtain resistance to FOC.

Utilization of genetic transformation

The genetic transformation methods used to introduce into the cells the exogenous DNA to be expressed in the plant is another biotechnological technique for the development of resistance to fungi in economically important crops such as banana. Several genes have been used to fight this pathogen. In this sense, ^{Paul et al. (2011}) tested genes Bcl-xl, Ced-9 and Bcl-2 3’ UTR in the banana cultivar ‘Lady Finger’ (AA). The results showed the overexpression of gene Bcl-2 3’ UTR in the lines that displayed resistance to FOC. On the other hand, they noticed apoptosis to be characteristic in host plants against necrotrophic pathogens, in which the pathogen caused the death of tissue and increased its growth potential faster. In another study, ^{Ghag et al. (2012}) inserted petunia (Petunia hybrida) genes that codify defensins PhDef1 and PhDef2 in banana plants, using embryogenic suspensions as explants and Agrobacterium tumefaciens as a transformation system, and found high levels of constitutive expressions of these defensins in elite banana plants in the cultivar Rasthali (Musa AAB), which, in turn, displayed resistance to the infection with race 1 of FOC, in both in vitro and in vivo studies.

On the other hand, ^{Mahdavi et al. (2012}) showed the expression of genes of thaumatin in rice (Oriza sativa) in transgenic banana plants resistant to FOC TR4. Later, genes obtained from onions (Allium cepa) which are codifying for an antimicrobial protein (Ace-AMP1) were introduced and expressed in transgenic bananas (^{Mohandas et al., 2013}). Likewise, ^{Ghag et al. (2014}) developed a procedure for transformation in bananas mediated by Agrobacterium for the expression of siRNAs aimed at vital FOC genes. After eight months of evaluations, they obtained five transgenic lines with levels of resistance to FOC. In turn, ^{Zhuang et al. (2016}) demonstrated the antagonistic effect of multifunctional protein B4 from the virus Banana bunchy top virus(BBTV) against F. oxysporum in bananas. This offers new insights for the breeding of transgenic resistance to Fusarium, taking the virus-fungus interaction into consideration. Likewise, ^{Dale et al. (2017}) obtained two transgenic Cavendish lines, the first of which was transformed using gene RGA2, isolated from a diploid banana plant resistant to FOC RT4. The second line was transformed, using gene Ced9 from nematodes. After three years of field tests, they displayed resistance to FOC RT4.

As these examples show, genetic transformation has been, in recent years, the biotechnological technique with the highest expectation for genetic breeding in the crop. However, most studies show no long-term field results to help evaluate the efficiency of the genes in situ (^{Ploetz, 2015}). Hence the comprehension of the host-pathogen interaction in terms of defense, and the paths related to virulence mechanisms help identify the critical steps to develops resistant cultivars using genetic approaches.

Early resistance selection

The early selection of the resistance in plants to different special forms (f. sp.) of F. oxysporum has been an essential goal in conventional and biotechnological genetic breeding. This type of selection has increased due to the advances on studies on plant processes, on the biology of pathogens and on the understanding of the plant-pathogen interaction. The use of fungus culture filtrates (FCH) and toxins, and even dual cultures are promising for this process. The first and most crucial step when evaluating a new banana clone is selecting the resistance to the difference races of wilt by FOC (^{Buddenhagen, 2009}). Therefore, the development of efficient methods to help select susceptible and resistant to this pathogen is a priority in the genetic breeding of this crop. To date, different groupd of researchers have developed two selection systems for the resistance of banana plants to FOC: 1) selection ex vitro and in vivo by planting banana propagules in pots, or in the field with the inoculant of the natural or artificial fungus; 2) selection in vitro, using propagules or FCH and toxins as selection agents. Below are some of the experiences of the selection of the resistance of banana plants to FOC with the use of the selection systems mentioned.

Ex vitro and in vivo evaluations of the resistance to the RT4 of FOC in banana propagules

The ex vitro and in vivo selections to evaluate the resistance to diseases have been the main selection methods for many years. They consist of the natural infection with spores from the fungus and are currently still being used. Below are the results of the evaluation for resistance to FOC RT4 of banana cultivars, plantains and Cavendish somaclonal variants with those selection systems, provided by the Taiwan Banana Research Institute (^{Huang et al., 2005}).

^{Dita et al. (2011}) obtained a quick and reliable bioassay for the evaluation of resistance to FOC RT4 under greenhouse conditions. They used a double pot system, following ^{Mohamed et al. (2000}). The roots of seedlings from the in vitro culture of the cultivar Gran Enano (grupo AAA) were cut at a length of approximately 40 cm. They then carried out the inoculation process individually with three isolations of the RT4 of the fungus (NRRL36114, FOC-115 and BPS3.4) of the GCV [01213] and one isolation of race 1, CNPMFO8-R1. The inoculation was carried out by submerging the root for 30 minutes in a suspension of 10⁵ conidia mL^-1 of the fungus. The inoculated seedlings were planted in 8 L pots containing sterile river sand as a substrate and supplemented with 20 g of maize grains pre-colonized with FOC. The evaluation of the resistance was carried out between days 7 and 40 after the inoculation of the seedlings by evaluating the internal symptoms and the decoloring of the rhizome. The results showed that the bioassay described under controlled greenhouse conditions was reliable to obtain a response of resistance on the reaction of the host to FOC. In addition, the bioassay presented a useful tool to carry out accurate studies in the plant-pathogen interaction. In this sense, ^{Li et al. (2015}) studied the resistance to FOC RT4 in eight genotypes of wild banana plants (Musa acuminata subsp. burmannica, M. balbisiana, M. basjoo, M. itinerans, M. nagensium, M. ruiliensis, M. velutina and M. yunnanensis) under greenhouse and field conditions. For the greenhouse tests, the banana seedlings from the in vitro culture were inoculated by punching holes at the bottom of the pseudostem, along with a suspension of 5 x 10⁶ spores mL^-1 of the fungus. After this process, the resistance was selected every day for 65 days by evaluating the expression of internal and external symptoms of the disease in the seedlings. For external symptoms, it was carried out using a grading scale with four categories: 0 = no symptoms; 1 = initial yellowing, mainly on the lower leaves; 2 = yellowing of all the lower leaves with a certain decoloring of the youngest leaves; 3 = all leaves with intense yellowing or dead plant. Meanwhile, the internal symptoms were evaluated using a scale based on the decoloring of the rhizome: 0 = no symptoms, 1 = 1 - 20%, 2 = 21 - 40% and 3 = > 40% of decolored rhizomes. The field selecion test was carried out on an experimental field naturally infested with FOC RT4. The evaluation of the expression of external symptoms of the disease was carried out every two weeks for 12 months after planting and internal symptoms were evaluated at the end of the experiment (12 months) according to the scale. The results showed that there are different sources of resistance to FOC RT4, which is an important genetic resource for banana genetic breeding programs that intend to obtain cultivars resistant to wilt by FOC RT4. However, studies related to the resistance to FOC RT4 continue, as indicated with examples in Table 1.

In vitro selection using propagules or filtrates of the fungal culture and toxins

^{Wu et al. (2010}) developed an in vitro bioassay methodology for the early detection of resistance or susceptibility to FOC RT4 in six cultivars of Musa (Brazil Xiangjiao subgroup AAA, Guangfen Num.1 subgruoup ABB, Formosana subgroup AAA, Nongke No.1 subgroup AAA, GCTCV-119 subgroup AAA and Dongguan Dajiao subgroup AAA). The rooted in vitro banana seedlings were inoculated in their culture medium via a suspension of 10⁶ conidia mL^-1 of FOC. After 24 days of inoculation, the selection of the resistance to the pathogen was carried out on the banana seedlings using a scale of 0 to 6 degrees of severity, suggested by the same authors. The results showed that the two susceptible cultivars (Brazil Xiangjiao and Guangfen No.1) presented the highest degrees of severity of the disease in comparison with the four resistant cultivars tested (Formosana, Nongke No.1, GCTCV-119 and Dongguan Dajiao). This shows that the promising resistant clones obtained with traditional genetic breeding techniques can be directly analyzed using the in vitro bioassay described. This method is more time-efficient, since it requires no acclimatization stage for the plants. Meanwhile, ^{Saraswathi et al. (2016}) carried out the selection of mutants of banana plants of the Rasthali cultivar (Silk, AAB) with resistance to race 1 of the FOC GCV [0124/5] by in vitro selection, using FCH and the toxin (fusaric acid) as selection agents. The individual buds of the banana mutants obtained after the third or fourth subculture were transferred to the multiplication culture medium and supplemented with different concentrations of the toxin (commercial fusaric acid) (Sigma Aldrich, U.S.A.) (0.0125; 0.025; 0.0375; 0.05 and 0.0625 mM) and with concentrations of 3, 4, 5, 6, 7 and 8% of the FCH; in addition, they added growth regulator. After three weeks, they observed a survival rate of 50% for the explants in the concentration of 0.050 mM of the toxin and 7% with the filtrate of the fungal culture. In higher concentrations, they observed a rapid reduction in the growth of the banana mutant buds. After three months, the selected mutants were transferred in order to carry out the tests in pots under conditions controlled by the inoculation into the substrate with a suspension of spores of 12 × 10⁹ conidia mL^-1 of the fungus. After six months of the process of evaluation of the resistance, three putative mutants were obtained with resistance to FOC race 1, which were massively multiplied in vitro to continue other studies on the interaction.

It is worth pointing out that in order to establish resistance selection systems, determining a correct concentration of the filtrate of the pathogen culture or toxin is necessary for the expression of the differential phytotoxic activity between varieties. This increases the possibilities of obtaining stable lines for disease-resistant plants (^{Švábová and Lebeda, 2005}). As indicated, even when in vitro selections are carried out, allegedly resistant plants must undergo field studies. The disease caused by FOC has relatively long incubation periods under these conditions. In other words, many months can go by between the infection of the roots and the yellowing and collapse of the limbo of the leaves (external symptoms). Therefore, a large number of plants must be followed up for a prolonged time.

The above statements indicate the need to develop alternative, quick and non-destructive methods for the selection of resistance of banana to FOC in the resistance selection test under field conditions. In this sense, ^{Companioni et al. (2003}) developed a procedure for the foliar differentiation of resistance and susceptibility of banana cultivars to FOC race 1, GCV [01210] by means of leaves-boring bioassay (bioassay on banana detached leaves) in and the application of FCH. The evaluation bioassay consisted in collecting middle-aged banana cultivar leaves in the field: Gros Michel (group AAA, susceptible) and FHIA-01 (group AAAB, resistant), on which FCH was applied. The application was made on three different positions of the adaxial side of the leaf limbo: distal, middle and proximal. After 48 hours, the phytotoxic activity of the fungal culture filtrates was evaluated with the symptomatological expression of necrosis formed around the point of application of FCH, expressed as the area of the elliptic lesion (mm2) (Figure 3). Forty-eight hours after the FCH was applied on banana leaves, the greatest statistical differences between cultivars were observed.

Other studies (^{Companioni et al., 2005}; ²⁰¹²) included evaluations of other indicators, such as biochemical components (Figure 4). The discriminant analysis for the differentiation of resistance and susceptibility of banana cultivars, used in the genetic breeding program, is a novel aspect in this investigation.

This estimation was carried out using a data matrix that included the effect of the FCH (area of the lesion and levels of phenols, both free and linked to the cell walls, aldehydes except malondialdehyde, and proteins) on the leaves of 18 cultivars. After the discriminant analysis, two functions were obtained; one for resistant cultivars and the other for susceptible ones. Each plant of the matrix was evaluated according to discriminant functions and classified as resistant or susceptible for 94.4% of all cases (68 plants out of 72). The described method is a useful tool to increase the efficiency of the selection under ex vitro and in vivo conditions, regardless of environmental conditions or the favorable season for the development of the disease. It also helps evaluate an important number of samples in the laboratory and to speed up the genetic breeding programs for this disease. For this reason, ^{García et al. (2020)} proved that the method to differentiate the susceptibility or resistance to Fusarium described earlier can be carried out quickly and in a non-destructive way with the use of treated FCH on the leaves for different populations of the pathogens. This could be applicable to the genetic breeding programs for musaceae, both conventional and biotechnological. On the other hand, they lay the foundations for future projects related to the identification of the specificity of alleged avirulence genes of the causal agent. In this regard, ^{Portal et al. (2018}) studied the toxic components of FCH of race 1 of FOC GCV [01210] using the bioassay described by ^{Companioni et al. (2003}) and they isolated the extracellular microbial metabolites implied in the response of the plant. In the latter, they obtained a culture filtrate of the specific host and they characterized the phytotoxic compounds such as fusaric acid, bovericin and fumonisin B1. These results are the basis for the direct isolation of avirulence genes of the pathogen and genes for the resistance in banana plants via advanced genetics for use in genetic breeding programs for the crop.

The results presented in this revision show the potential of plant biotechnology and other tools in the field of genetic breeding for the crop, in which biotechnological techniques constitute feasible tools in the search for resistance to FOC. In this regard, genetic transformation is the biotechnological technique with the highest expectations in recent years. However, most field studies lack long-term results that contribute to the evaluation of genes in situ. Therefore, understanding the host-pathogen interaction in terms of defense and the paths related to the virulence mechanisms help identify critical steps towards the development of resistant cultivars using genetic approaches. However, the selection systems for resistance to this disease under field conditions are laborious, destructive and time-consuming. For these reasons, the development of efficient methods for the early selection of resistance to FOC are a useful tool for accurately studying the plant-pathogen interaction and increasing the efficiency of genetic breeding for the crop.

Figure 3 Symptomatologic expression of necrosis formed around the point of application of the fungal culture filtrate, expressed as the area of the elliptical lesion (mm²). 

Perspectives

Scientific studies reported globally, related to the host-pathogen interaction of the binome Musa spp. and Fusarium oxysporum f. sp. cubense, indicate that the current and future tendency is the search for materials resistant to the pathogen via diverse biotechnology techniques, such as somaclonal variation, induced in vitro mutagenesis and genetic transformation itself, which are supported by methodologies for the early detection of resistance, with partially successful results thus far. Diverse work groups have been formed to face this phytopathological problem; those in Southeast Asia are particularly important, since it is where FOC RT4 was first detected, in 1990. In June of 2019, plants with FOC symptoms were observed in Colombia, only 19 years later. This was a warning for OIRSA member countries, including Mexico, and by July 25, 2019, the ^{SADER-SENASICA (2019)} claimed to be ready to prevent the entry of the fungus Fusarium oxysporum f. sp. cubense RT4 into the country, and reinforced security and prevention measures to mitigate the risk of its entry. It also made a priority out of training technicians and farmers, in which epidemiological surveillance was crucial. Regarding scientific research to face the problem that draws near, little progress has been made in Mexico. It is therefore crucial for research-focused institutions to address this problem.

Figure 4 Method to differentiate the resistance to Fusarium oxysporum f. sp. cubense race 1 in banana plants at a foliar level, according to Companioni et al. (2012). 

CONCLUSIONS

The results presented in this revision show the potential of plant biotechnology and other tools in the field of genetic breeding in the crop. They help speed up the genetic breeding programs for resistance to this disease. The genetic transformation is the biotechnological technique that has provided the greatest expectations in recent years. However, most field studies lack long-term results that help evaluate the efficiency of the genes in situ. Due to this, the development of efficient methods for the early selection of resistance to FOC is a useful tool to carry out accurate studies in the plant-pathogen interaction and to increase the efficiency of the genetic breeding programs for resistance to FOC in the crop.