Early Selection for Resistance to Fusarium Wilt in Banana

Abstract

Early selection for plant resistance to different special forms of Fusarium oxysporum has been a key goal in conventional and biotechnological breeding. We previously developed a straightforward procedure to differentiate resistant and susceptible field-grown banana cultivars for Fusarium wilt at the leaf level. This chapter presents compiled results from several years of work by our group, developing a method to differentiate resistance and susceptibility to Fusarium wilt vegetative compatibility group (VCG) [01210] race 1 at the leaf level in banana cultivars. This is achieved using the leaf pit bioassay and application of fungal culture filtrates.

1. Introduction

Bananas and plantains, belonging to the Musa spp., stand as pivotal crops in tropical and subtropical regions. They serve as a crucial food source for a substantial portion of the global population [1]. However, the production of these crops faces a significant threat from diseases like Fusarium wilt, primarily caused by Fusarium oxysporum f. sp. cubense (FOC) [2]. The introduction of FOC tropical race 4 (RT4) into Cavendish cultivar plantations has marked it as one of the top ten diseases in agricultural history [3, 4]. Controlling this disease with agrochemicals comes at a high cost and inflicts severe environmental damage [1]. As a result, there has been a growing consensus in recent years that the most effective and environmentally friendly approach to combat this disease is genetic enhancement for resistance [5].

In this context, the initial and pivotal step in assessing a new banana clone within a breeding program for this crop is selecting for resistance to different races of FOC wilt [6]. Simultaneously, early selection for plant resistance against various specialized forms (f. sp.) of F. oxysporum has been a primary focus in conventional and biotechnological breeding. Advancements in our understanding of plant processes, pathogen biology, and plant-pathogen interactions have bolstered this effort. Nevertheless, existing methods for screening resistance to this disease under field conditions are labor-intensive, destructive, and time-consuming [7, 8]. Consequently, developing efficient techniques for selecting susceptible and resistant cultivars to combat this pathogen in crop breeding has become a top priority.

Hence, the utilization of fungal culture filtrates (FCH), toxins, and even dual cultures holds promise in this endeavor. This chapter presents the results of several years of research conducted by our working group in developing a method for distinguishing resistance and susceptibility to Fusarium wilt race 1 (VCG) [01210] in bananas at the leaf level, employing FCH and the puncture bioassay in an efficient and non-destructive manner.

2. Early resistance selection

For many years, assessing plant disease resistance has primarily relied on ex vitro and in vivo selection. These approaches involve exposing the plants to natural infection by fungus spores and continue to be widely employed even today. The results of evaluating resistance to FOC’s RT4 strain in banana cultivars, plantains, and Cavendish somaclonal variants using these selection techniques, as provided by the Taiwan Banana Research Institute, are presented in Table 1 [9].

Cultivars	Place	Response to FOC RT4	Reference
‘FHIA 01’	FAO, Malasia	Resistant	[9]
‘FHIA 02’	International Musa; Testing; Program, phase III, China (IMTP III, China)	Resistant	[9]
‘FHIA 03’	Journal of Tropical Crops China	Resistant	[10]
‘FHIA 17’	Papua New Guinea	Resistant	[11]
‘FHIA 18’	Journal of Tropical Crops China	Highly resistant	[10]
‘FHIA 21’	International Musa; Testing; Program, phase III, China (IMTP III, China)	Resistant	[11]
‘FHIA 23’	FHIA: Bananas Database	Susceptible	[12]
‘FHIA 25’	Papua New Guinea	Resistant	[11]
‘FORMOSANA’	Taiwan	Resistant	[13]

Table 1.

Results in the evaluation for resistance to RT4 of FOC in banana, plantains, and somaclonal variants of Cavendish cultivars.

Li et al. [14] assessed the resistance of eight wild banana genotypes (Musa acuminata subsp. burmannica, M. balbisiana, M. basjoo, M. itinerans, M. nagensium, M. ruiliensis, M. velutina, and M. yunnanensis) to FOC’s RT4 strain under both greenhouse and field conditions. In the greenhouse experiments, banana seedlings from in vitro cultures were infected by puncturing holes at the base of the pseudo stem with a fungus spore suspension of 5×10⁶ spores mL⁻¹. They then monitored resistance every 2 days for 65 days by assessing the seedlings’ internal and external disease symptoms. External symptoms were evaluated using a four-class rating scale: 0 = no symptoms; 1 = initial yellowing, mainly on lower leaves; 2 = yellowing of all lower leaves with some discoloration of younger leaves; 3 = all leaves with intense yellowing or dead plant. Internal symptoms were assessed based on rhizome discoloration: 0 = no symptoms, 1 = 1–20%, 2 = 21–40%, and 3 = > 40% discolored rhizome. They conducted experiments in a plot naturally infested with FOC RT4 for the field screening test. They evaluated external disease symptom expression every 2 weeks for 12 months after planting, and assessed internal symptoms at the end of the 12-month experiment using the same scale. The results demonstrated the presence of various sources of resistance to FOC’s RT4, representing valuable genetic resources for banana breeding programs aiming to develop wilt-resistant cultivars against this strain. However, research on resistance to RT4 of FOC continues to be an ongoing endeavor.

In a similar approach [5] screened banana mutants from the Rasthali cultivar (Silk, AAB) were resistant to GCV race 1 [0124/5] of FOC through in vitro selection. They employed FCH (fungal culture filtrates) and the toxin fusaric acid as selection agents. They initiated the process by transferring individual banana mutant shoots, obtained after the third or fourth subculture, to a multiplication culture medium. This medium was enriched with varying concentrations of the toxin (commercial fusaric acid from Sigma Aldrich, USA) ranging from 0.0125 to 0.0625 mM, along with FCH concentrations spanning from 3–8%. Additionally, a growth regulator was introduced into the medium. After 3 weeks, they observed that 50% of the explants survived at the 0.050 mM toxin concentration and 7% with FCH. Higher concentrations resulted in a noticeable decline in the shoot growth of the banana mutants. The selected mutants were transferred to pot tests under controlled conditions 3 months later. The substrate was inoculated with a spore suspension containing 12 × 109 conidia mL⁻¹ of the fungus to initiate these tests. After 6 months of evaluating resistance, they successfully identified three potential mutants that exhibited resistance to race 1 of FOC. These promising mutants were subsequently propagated extensively in vitro to enable further studies on their interaction with the fungus.

It’s important to note that the selection agent chosen plays a crucial role in establishing resistance selection systems. To achieve this, determining the correct inoculum concentration of the FCH toxin is essential to highlight the differential phytotoxic effects among different varieties. This step significantly enhances the likelihood of obtaining stable plant lines with disease resistance [15]. Moreover, even when in vitro selections are conducted, subjecting the supposedly resistant plants to field studies is imperative. It’s worth mentioning that the disease caused by FOC exhibits relatively long incubation periods under these conditions. In simpler terms, it may take several months from root infection to the appearance of external symptoms like yellowing and leaf blade collapse. Consequently, monitoring a large number of plants over an extended period becomes necessary.

These approaches underscore the urgency of developing alternative, swift, and non-destructive methods for screening banana resistance to FOC in resistance screening tests conducted under field and ex vitro conditions.

2.1 Use of fungal culture filtrates for leaf-level differentiation of resistance to Fusarium wilt race 1 (VCG) [01210]

Considering the earlier approaches, [16] devised a method to distinguish between resistance and susceptibility of banana cultivars to FOC race 1, GCV [01210]. They achieved this using a leaf spotting bioassay combined with applying FCH. The assessment involved gathering middle-aged leaves from two banana cultivars: Gros Michel (group AAA, susceptible) and FHIA-01 (group AAAB, resistant). These leaves were then subjected to FCH application at three distinct positions on the upper side of the leaf blade: distal, middle, and proximal. After 48 hours, they evaluated the phytotoxic effects of the FCH by examining the symptomatological manifestation of necrosis around the area where FCH was applied, quantified as the size of the elliptical lesion (mm²) (Table 2) [17]. The most significant differences between cultivars were observed 48 hours after applying FCH to banana leaves.

Table 2.

Symptom expression of necrosis formed around the point of application of the fungal culture filtrate, expressed as the area of the elliptical lesion (mm²) [17].

In previous studies [18, 19] additional evaluations, including the analysis of biochemical components were conducted. The steps involved in this analysis are presented straightforwardly. Notably, a novel aspect of this research is the analysis to differentiate between resistance and susceptibility of banana cultivars within the genetic improvement program. This estimation was derived from a data matrix encompassing the impact of FCH, including factors such as lesion area, levels of free and cell wall-bound phenols, aldehydes (excluding malondialdehyde), and proteins in leaves from eighteen cultivars. Following the discriminant analysis, two functions were derived—one for resistant cultivars and another for susceptible cultivars. Each plant in the matrix underwent assessment based on these discriminant functions, resulting in a classification as resistant or susceptible in 94.4% of the cases (68 out of 72 plants). This described method proves to be a valuable tool for enhancing selection efficiency under both ex vitro and in vivo conditions, irrespective of environmental factors and the time of year conducive to disease development. Moreover, it facilitates evaluating a substantial number of samples in a laboratory setting, thus expediting genetic improvement programs for combatting this disease.

The described method introduces the possibility of screening for resistance to FOC in leaf fragments of plants aged between 8 to 9 months. In this approach, FCH is applied to the leaves of individuals under study for resistance or susceptibility, while a control group receives a non-inoculated culture medium. Comparing this selection method with existing techniques for evaluating banana resistance to Fusarium oxysporum f. sp. cubense (as previously developed by [20, 21], it becomes evident that working with FCH is notably more straightforward. Furthermore, this method offers advantages such as reducing the space requirements for the selection process and shortening the time needed to determine whether a plant is resistant or susceptible. Additionally, it addresses a drawback of resistance evaluations conducted under ex vitro conditions, which often prove ineffective due to the brief infection period and challenges associated with controlling inoculum concentration distribution. It also mitigates the issues with resistance selection under natural field conditions, which tend to be time-consuming, costly, and influenced by fluctuations in inoculum abundance and climatic factors impacting disease dissemination, infection, development, and symptom expression [22].

As previously mentioned, [16, 19] developed a method for distinguishing the resistance of banana cultivars to FOC race 1 using FCH. However, the broad application of these results in genetic improvement programs for this crop necessitates validation against different races of the fungus. Specifically, validation was required for two isolates belonging to different vegetative compatibility groups (GCV): GCV [01210] race 1 and GCV [0124/125] race 2. To address this, [23] undertook the validation of this method for the rapid differentiation of Panama disease using FCH from both FOC race 1 and race 2. For this validation, they utilized foliar samples from banana plants aged 8 to 9 months, cultivated at the Banana Germplasm Bank of the National Institute for Tropical Viandas Research in Santo Domingo, Villa Clara, Cuba. These banana cultivars had previously been classified as either resistant or susceptible to FOC races 1 and 2 by [24]. The fungal strains employed were FOC race 1 GCV [01210] and race 2 GCV [0124/125], sourced from the strain collection of the Instituto de Investigaciones de Sanidad Vegetal (INISAV), Havana, Cuba. In the initial part of the experiment, [23] focused on obtaining FCH during the in vitro growth of both FOC race 1 GCV [01210] and race 2 GCV [0124/125]. To achieve this, they determined the optimal harvesting time for FCH for both races on a daily basis for up to 30 days. The phytotoxic activity of the FCH was assessed using the puncture bioassay method outlined by [16] at each evaluated time point (ranging from 1 to 30 days). During this research stage, they compared the responses of 15 banana cultivars with varying resistance levels to the pathogen in vivo, as previously categorized by [24]. FCH for FOC race 1 was harvested at 15 days, while that for race 2 was collected at 29 days after inoculation in Caldo Czapek culture medium. Subsequently, they evaluated how cultivars of Musa spp., responded to the application of fungal filtrates from both races.

The FCH obtained at 15 and 29 days from strains of races 1 and 2 successfully differentiated over 93% of individuals with known responses to the pathogen in vivo (Table 3). Among the cultivars most susceptible to FCH from race 1 were Manzano Criollo, Gros Michel, Pisang Lilin, and Yangambi km 5, all showing no statistical differences in their responses. Paka followed as the next most susceptible cultivar. Conversely, all FHIA cultivars assessed (01, 02, 03, 04, 18, and 21) displayed resistance to the phytotoxic effects of FCH from race 1 GCV [01210], with no statistical variations in their respective responses. Furthermore, the cultivars ‘Burro Criollo,’ ‘Pelipita,’ ‘Pisang jari guaya,’ and ‘Bluggoe’ also exhibited resistance, with responses similar to FHIA. Researchers in various plant-pathogen interactions have successfully obtained culture filtrates with phytotoxic properties for early resistance screening. Moreover, methods have been developed for the isolation and purification of metabolites that play a role in the selective response of plants [25].

Cultivar	Elliptical lesion area (mm^2)
	FOC race 1 GCV [01210]; (Day 15)	FOC race 2 GCV [0124/125]; (Day 29)
Manzano Criollo (AAB)	***53.6a	*2.3b
Gros Michel (AAA)	***55.4a	*2.8b
Yangambi Km 5 (AAA)	***55.3a	*4.1b
Paka (AA)	***52.4a	*3.5b
Pisang Lilin (AA)	**54.4a	*3.4b
Pisang jari guaya (AA)	**4.1b	**52.8a
Burro Criollo (ABB)	*3.2b	***54.0a
Bluggoe (ABB)	*2.1b	***53.7a
Pelipita (ABB)	*3.4a	*2.6b
FHIA-01 (AAAB)	*3.6a	*2.3b
FHIA-02 (AAAB)	*2.5a	*2.6a
FHIA-03 (AABB)	**2.8b	***53.5a
FHIA-04 (AAAB)	*3.4a	*3.2a
FHIA-18 (AAAB)	**3.2b	*4.1a
FHIA-21 (AAAB)	*4.0b	**4.8a

Table 3.

Responses of the Musa spp., cultivars to the application of filtrates of the fungus culture of races 1 and 2, evaluated with the bioassay on banana detached leaves and in earlier field evaluations faced with the microorganism in vivo [9].

Means with equal letters indicate that there are no statistically significant differences (univariate analysis of variance, one-factor ANOVA, ANOVA, and Tukey’s HSD, p ≤ 0.05). Response of cultivars in field evaluations according to [24]: *Resistant, **Tolerant, ***Susceptible/Averages with same letters indicate no statistically significant differences (One-factor univariate analysis of variance, ANOVA, Tukey ANOVA, and HSD, p ≤ 0.05). Responses of the cultivars in field evaluations according to [24]: *Resistant, **Tolerant, ***Susceptible.

The results indicate that banana plants’ resistance or susceptibility to Fusarium oxysporum f. sp. cubense can be distinguished at the leaf level using fungal culture filtrates, applicable in conventional and biotechnological Musaceae genetic improvement programs. In this context, one challenge in testing resistance under in vivo conditions is ensuring a uniform distribution of the pathogen inoculum among all plants to be evaluated. Some plants may receive excessive inoculum, while others might not get enough to develop the disease [26]. Additionally, environmental effects on inoculated plants studied in the field raise concerns [22]. Furthermore, it’s not always feasible to characterize FOC populations through pathogenicity testing due to the influence of plant-environment interactions. There’s clear evidence of differential plant responses under varying environmental conditions, even within established differential groups [27]. It’s worth noting that to establish a disease resistance evaluation method, it must outperform the traditional approach in terms of labor, space, and time requirements. These considerations have been taken into account in the method tested for both pathogen races.

The results from [23] highlight the feasibility of a rapid and non-destructive method for distinguishing susceptibility from resistance to Fusarium blight. This method utilizes FCH applied to leaves and can be employed for different pathogen populations, offering potential benefits for conventional and biotechnological Musaceae breeding programs. Moreover, these findings represent a pioneering step in validating a method that disrupts the natural biological cycle of the disease.

They also lay the groundwork for future research to identify phytotoxic metabolites and examine the specificity of putative avirulence genes in the pathogen. This, in turn, will facilitate an understanding of the role of these metabolites in the response of Musa spp., to Fusarium wilt. In this context, [28] conducted a study using the bioassay described by Companioni et al. [16] to investigate the toxic components of FCHs from FOC GCV race 1 [01210]. They successfully isolated extracellular microbial metabolites involved in the plant’s response, generating a host-specific culture filtrate. This research led to the characterization of phytotoxic compounds such as fusaric acid, bovericin, and fumonisin B1. These results lay the foundation for the direct isolation of avirulence genes in the pathogen and resistance genes in bananas, employing advanced genetics to enhance genetic improvement programs for the crop.

The results presented here underscore the viability of biotechnological techniques as valuable tools in the quest for FOC resistance. Nonetheless, many field studies lack the long-term data necessary to assess genes’ effectiveness in real-world conditions. To address this, comprehending the host-pathogen interaction, particularly in terms of defense mechanisms and virulence pathways, becomes pivotal. Such understanding helps to pinpoint critical stages for developing resistant cultivars through genetic approaches. Nevertheless, selecting for resistance to this disease in field conditions remains a labor-intensive, destructive, and time-consuming process. This highlights the significance of developing efficient methods for early resistance selection. Such methods enable not only precise investigations into plant-pathogen interactions but also enhance the overall efficiency of breeding programs for this crop.

2.2 Evaluation of resistance to Fusarium wilt race 1 in banana mutants

The application of biotechnological techniques to bolster resistance breeding for Fusarium wilt in bananas has accelerated the introduction of breeding outcomes when contrasted with conventional methods [29]. Additionally, these techniques have enhanced the overall efficiency of breeding programs for this crop [5].

Ventura et al. [30] utilized a combination of tissue culture and in vitro mutagenesis in meristematic apices of the ‘Zanzibar’ cultivar (group AAB). This approach yielded a wide range of genetic variability in the ‘Zanzibar’ clone, leading to the selection of 15 mutants. Among these mutants, ‘Z 13,’ ‘Z 30,’ and ‘Z 30 A’ emerged as the most promising candidates for crop genetic improvement due to their impressive traits, including high yield, elimination of superficial corms, reduced height, and organized tillering. Given these promising findings, [31] assessed resistance to Fusarium wilt race 1 GCV [01210] in these selected banana clones. These clones were derived from the genetic improvement program for Musa spp., conducted at the Instituto Nacional de Investigaciones en Viandas Tropicales de Santo Domingo, Villa Clara, Cuba (INIVIT). These 15 mutants were initially obtained from the ‘Zanzibar’ clone through ionizing radiation (gamma rays) applied to meristematic apices in an in vitro culture. Following this, the most advanced materials for crop genetic improvement, specifically ‘Z 13,’ ‘Z 30,’ and ‘Z 30 A’ were chosen after three vegetative cycles under field conditions. These lines are being considered for registration as new genotypes in Musa spp., by the International Atomic Energy Agency (IAEA) [30]. To evaluate resistance to FOC race 1 GCV [01210], the methodology described by [18, 19] was employed. Foliar samples were collected from the ‘Z 13,’ ‘Z 30,’ and ‘Z 30 A’ lines 8 to 9 months after planting in the Banana Germplasm Bank of INIVIT. The evaluation of resistance to Fusarium wilt race 1 GCV [01210] in each line and at each assessment time, based on discriminant functions, is detailed in Table 4. After 48 hours following the application of treatments and subsequent biochemical analyses on the leaves of the ‘Zanzibar’ clone lines, it was observed that the discriminant functions classified 93.3% of ‘Z 30’ plants as resistant (56 out of 60 plants). Similarly, 91.6% of ‘Z 13’ plants were classified as resistant (55 out of 60 plants), and ‘Z 30 A’ exhibited a classification of 96.6% of plants as resistant (58 out of 60 plants). These results demonstrate that the selected lines derived from the irradiated ‘Zanzibar’ clone, namely ‘Z 13,’ ‘Z 30,’ and ‘Z 30 A’ hold substantial promise for genetic improvement in the crop, particularly for resistance to FOC race 1 GCV [01210], with resistance levels ranging from 91.6 to 96.6%. However, the necessity of continuing additional field studies to confirm resistance in the evaluated lines cannot be discounted. Even when in vitro resistance evaluations are conducted, it remains essential to subject putatively resistant plants to field studies [32].

Evaluation of moments	Evaluated lines	Total number of plants evaluated	Total number of potentially resistant plants according to discriminant functions
1	‘Z-30’	10	10 (100%)
2	‘Z-30’	10	8 (80%)
3	‘Z-30’	10	9 (90%)
4	‘Z-30’	10	9 (90%)
5	‘Z-30’	10	10 (100%)
6	‘Z-30’	10	10 (100%)
1	‘Z-13’	10	8 (80%)
2	‘Z-13’	10	9 (90%)
3	‘Z-13’	10	9 (90%)
4	‘Z-13’	10	10 (100%)
5	‘Z-13’	10	10 (100%)
6	‘Z-13’	10	9 (90%)
1	‘Z-30A’	10	10 (100%)
2	‘Z-30A’	10	9 (90%)
3	‘Z-30A’	10	9 (90%)
4	‘Z-30A’	10	10 (100%)
5	‘Z-30A’	10	10 (100%)
6	‘Z-30A’	10	10 (100%)

Table 4.

Classification into susceptible or resistant lines made by discriminant functions on selected lines of the irradiated ‘Zanzíbar’ clone [17].

The results obtained in this work showed that the selected lines of the irradiated ‘Zanzibar’ clone (‘Z 13’, ‘Z 30,’ and ‘Z 30 A’) evaluated at different times of the collection showed resistance to FOC race 1 GCV [01210] according to the method for the differentiation of susceptibility or resistance to Fusarium wilt described by [18, 19]. On the other hand, it is easy to perform the work of resistance evaluation with FC of the fungus and reduce the response time necessary to know if a plant is resistant or susceptible (48 hours). Finally, it allows the evaluation of a significant number of samples under laboratory conditions, thus accelerating genetic improvement programs for this disease. This shows the potential of biotechnology in the field of crop genetic improvement.

3. Conclusions

The creation of effective methods for the early selection of resistance to Fusarium oxysporum f. sp. cubense serves as a valuable tool for conducting precise studies on the plant-pathogen interaction and enhancing the efficiency of genetic improvement programs in this crop. Moreover, the method for distinguishing susceptibility from resistance to Fusarium wilt, outlined in this chapter, has the potential for application across various pathogen populations. It can prove beneficial in both conventional and biotechnological breeding programs for Musaceae.

Acknowledgments

This research was supported by the International Foundation for Science (Stockholm, Sweden) and by the Cuban Ministry for Science, Technology and the Environment.