Evaluation of Resistance of Banana Genotypes with AAB Genome to Fusarium Wilt Tropical Race 4 in China

Banana cultivars with the AAB genome group comprise diverse subgroups, such as Plantain, Silk, Iholena, and Pisang Raja, among others, which play an important role in food security in many developing countries. Some of these cultivars are susceptible to Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), the most destructive pathogen threatening banana production worldwide, and some of them are still largely unknown. We evaluated the resistance of 37 banana genotypes, including Plantain, Silk, Iholena, Maia Maoli/Popoulu, Pisang Raja, Pome, and Mysore, to Foc TR4 under both greenhouse and field conditions. Genotypes from the Silk and Iholena subgroups were highly susceptible to Foc TR4. Pome and Mysore showed resistance and intermediate resistance, respectively. However, Pisang Raja ranged from susceptible to intermediate resistance. One cultivar from the Maia Maoli/Popoulu subgroup was highly susceptible, while the other displayed significant resistance. Most Plantain cultivars exhibited high resistance to Foc TR4, except two French types of cultivar, ‘Uganda Plantain’ and ‘Njombe N°2’, which were susceptible. The susceptibility to Foc TR4 of some of the AAB genotypes evaluated, especially Plantain and other cooking bananas, indicates that growers dependent on these varieties need to be included as part of the prevention and integrated Foc TR4 management strategies, as these genotypes play a crucial role in food security and livelihoods.


Introduction
Bananas (Musa spp. L.) are one of the world's most important cash crop grown on large plantations for export, and an essential staple food for many developing countries. Most cultivated bananas are seedless triploid varieties (2n = 3× = 33) derived from intraor inter-specific hybridization between the two species M. acuminata (A genome) and M. balbisiana (B genome), resulting in the genome groups AAA, AAB and ABB [1]. The major cultivars are assigned to clusters of subgroups, which are characterized by genotypes that share similar traits of agronomic and fruit quality, such as Cavendish (AAA), East African Highland bananas (EAHB, AAA), and Plantain (AAB) [2].
Fusarium wilt of banana (FWB), caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), has been considered one of the most devastating diseases in agricultural history [3]. The pathogen can be disseminated by plant material, soil, and water [4]. Once Foc is introduced into a banana field, it cannot be eradicated and survives for many decades (as it forms resistant structures called chlamydospores); moreover, losses of up to 100% Existing AAB cultivars, such as those from the Plantain and Iholena subgroups, are poorly evaluated and underrepresented for their resistance to Foc TR4. Hence, it is of critical importance to screen banana cultivars so that the novel Foc TR4-resistant cultivars can be identified and developed [21]. The screening of banana germplasms for Foc TR4 resistance can be achieved through pot or field trials. Pot trials enable the evaluation of a large number of different banana cultivars in a controlled environment, as well as screening to detect the resistant ones in the short run [22]. Field screening trials offer the opportunity to screen plants in sites affected by Foc TR4, where the resistant cultivars would be planted, to provide data on agronomical and yield performance as well as useful insights into the market's potential receptivity [23]. In the present research, pot and field trials were conducted to assess the Foc TR4 reaction of 37 banana AAB cultivars from the Plantain, Iholena, Maia Maoli/Popoulu, Silk, Pisang Raja, Pome, and Mysore subgroups.

Evaluation under Greenhouse Conditions
The strain Vegetative Compatibility Group (VCG 01213/16, Agriculture Culture Collection China, ACCC 37997) was used for inoculation under greenhouse conditions. It was originally collected from the Cavendish (AAA) cultivar 'Baxi' in Guangdong province, China. Previous work confirmed this strain as representing isolates of Foc TR4 [25,26], and it was used in our former germplasm screening [20]. The strain was initially cultured on potato dextrose agar (PDA) medium for 5 days, and then, the mycelium was transferred to potato dextrose broth (PDB) to grow for another 5 days at 28 • C with a shaking speed of 180 rpm. The final concentration of spore suspension was adjusted to 10 6 spores/mL [27].

Evaluation under Field Conditions
Between November 2019 and January 2022, field tests were conducted to evaluate the genotypes for their resistance to Foc TR4 in naturally infested soils located in Dongguan, Guangdong province (N23 • 02 17.25", E113 • 40 52.87"). This site for field testing was the same place in which we previously carried out germplasm screening of bananas for resistance to Foc TR4 [18]. The Cavendish (AAA) cultivar 'Baxi', used as susceptible control, was previously planted in these plots with >80% of FWB TR4 incidence. The experimental plot was arranged as a randomized complete block design with three replications (blocks), and 10-15 plants within each block for each genotype. Each plant was cultivated according to local commercial growing standards for two cropping cycles, with inter-row spacing of 3.5 m and inter-plant spacing of 2.0 m [29]. No chemicals were applied to control pests and diseases. Disease evaluation was conducted when external symptoms of FWB, such as leaf yellowing on the oldest leaves and occasional pseudostem splitting appeared on the susceptible control 'Baxi'. Once plants were dead or harvested, internal symptom on the rhizome were evaluated. The incidence of disease (ID) was calculated when plants were dead or harvested (2 years after planting) as follows: ID (%) = [diseased plants/total plants] × 100. According to the ID values, the following categories of disease reaction were established: 0% ≤ ID ≤ 20%: highly resistant (HR); 20% < ID ≤ 40%: resistant (R); 40% < ID ≤ 60%: intermediate (I); 60% < ID ≤ 80%: susceptible (S); and 80% ≤ ID: very susceptible (VS) [18].

Molecular Characterization of Foc TR4
To verify the presence or identity of Foc TR4, rhizome tissues (10 cm × 10 cm) were sampled from the discolored parts of the plants if they showed typical Fusarium wilt symptoms, or randomly sampled if no symptoms were observed. The samples were analyzed via PCR using Foc TR4 specific primers [27]. At least one sample per replicate was collected from each cultivar. All the samples were surface-sterilized with 70% ethanol for 5 min, washed with sterile distilled water, and allowed to dry on the sterile filter paper. Thereafter, they were cut into segments of 2 mm 2 and added to tubes containing 200 µL cell lysate; then, they were ground, boiled for 5 min, and centrifuged at 12,000 rpm for 2 min. PCR was performed with 2 µL of the sample as the template for PCR amplification using the Mix MF848 kit (Mei5 Biotechnology Co., Beijing, China) under the following PCR conditions: 3 min at 95 • C, 30 s at 94 • C, 40 s at 56 • C, 1 min at 72 • C for 35 cycles, and 5 min at 72 • C. In the control group, we used genomic DNA (gDNA) extracted from Foc TR4 (VCG 01213/16, ACCC 37997) but not from banana plants. Foc TR4-specific primers were used to detect Foc TR4 [27].

Data Collection and Statistical Analysis
ANOVA was adopted using SPSS 19.0 statistics for comparing different banana cultivars with varying levels of resistance to Fusarium wilt, assessed under greenhouse as well as field conditions, using resistance parameters such as ID and RDI. The Fisher's least significant difference (LSD) test was applied for multiple comparisons of variables at 0.05 (p < 0.05).

Evaluation under Greenhouse Conditions
The susceptible control, the Cavendish (AAA) cultivar 'Baxi', developed internal rhizome symptoms of FWB TR4 at 35 days after inoculation, with an RDI value of 6. Plantain cultivars including 'Uganda Plantain', 'French Sombre', and 'Njombe N     As shown in Table 2, the susceptible Cavendish control ('Baxi') exhibited a resistance rating of susceptible. Likewise, 'Uganda Plantain' was susceptible, while among the other Plantain cultivars, six were classified as resistant, and the other five were assigned as intermediate. All of the four Silk banana cultivars were classified as susceptible. Moreover, 'Poingo' had a rating of resistant, whereas 'Kofi' was classified as intermediate, and the As shown in Table 2

Evaluation under Field Conditions
Internal symptoms typical of FWB were first observed in November 2019 ( Figure 4). Infection caused by Foc TR4 was confirmed using PCR analyses (Figure 4). To facilitate the comparison and further discussions, disease reaction will be presented by subgroups referring to the susceptible control 'Baxi'.  All the Silk-type bananas were susceptible to Foc TR4, showing ID values even higher than 'Baxi' (Figure 6). Three genotypes ('Amrithapani', 'Digjowa', and 'Figue Pomme Géante') presented 100% disease incidence after the second cropping cycle, which also evidenced the high Foc TR4 inoculum pressure in the experimental plots ( Figure 6). Most of genotypes from the Iholena subgroup and one from the Maia Maoli/Popoulu subgroup were highly affected by Foc TR4, with the highest ID values occurring in 'Maritú', 'Uzakan', 'Digjowa', 'Luba', 'Wisu', and 'Pacific Plantain' (Figure 6). 'Kofi' and 'Rukumamb' presented ID values ranging from 6.67% to 48.25%. Interestingly, 'Poingo' from the Maia Maoli/Popoulu subgroup did not develop any internal symptoms of the disease (Figure 6). Genotypes from the Pisang Raja subgroup also presented variation regarding Foc TR4 resistance ( Figure 6). 'Pisang Raja Bulu', 'Pisang Raja No.2', and 'Pisang Rajah' were considered susceptible. 'YN2' presented only a 15% ID and the Pome cultivar 'Pisang Radjah' did not show any FWB symptoms ( Figure 6). These were considered intermediate and highly resistant, respectively. The intermediate genotype 'Pisang Ceylan' from the Mysore subgroup showed an ID value of around 40% (Figure 6).
In the Iholena and Maia Maoli/Popoulu subgroups, 'Poingo' and 'Kofi' were designated as highly resistant, and 'Wisu' and 'Pacific Plantain' were rated as very susceptible during both crop cycles. 'Tigua' was noticed to be susceptible during the plant crop cycle and very susceptible during the initial ratoon crop cycle. Additionally, 'Rukumamb' displayed resistance to Foc TR4 during the plant crop cycle but intermediate levels of resistance in the first ratoon crop cycle. Susceptibility was noticed in 'Luba' during the plant crop cycle; however, during the initial ratoon crop cycle, the cultivar was very susceptible. 'Maritú' showed intermediate-level resistance during the crop cycle but susceptibility during the initial ratoon crop cycle. Furthermore, 'Uzakan' was rated as susceptible during the crop cycle but quite susceptible during another crop cycle ( Table 2). 'Figue Pomme Géante', 'Digjowa', and 'Amrithapani' were designated as very susceptible, and 'Malbhog', which belonged to Silk subgroup, was shown to be susceptible during both crop cycles ( Table 2). For the Pisang Raja subgroup, 'YN2' developed high-level resistance in the crop cycle, whereas, throughout the first ratoon crop cycle, it was resistant. The evaluated 'Pisang Raja Bulu' was found to be susceptible during the crop cycle but very susceptible during the initial ratoon cycle. 'Pisang Rajah' demonstrated intermediate-level resistance and susceptibility during the crop cycle and the initial ratoon crop cycle, respectively. The Pome cultivar 'Pisang Radjah' displayed high resistance, and 'Pisang Ceylan' belonging to Mysore subgroup showed intermediate resistance in the plant crop and ratoon crop cycles, respectively ( Table 2).

Discussion
The use of resistant varieties could be the most efficient measure to manage Foc TR4, which is currently recognized as the most devastating disease in bananas [30][31][32]. In this work, we assessed the resistance level of 37 banana cultivars belonging to the AAB genome against Foc TR4 under both greenhouse and field conditions. The resistance of some cultivars evaluated in the field trial was lower than that obtained in the greenhouse, which might be due to the inoculum concentration and uncontrolled soil characteristics in the field [28].
Plantains, a very important subgroup referred to as cooking bananas (the AAB genome), are of major importance in the diets of numerous populations of Africa, Latin America, and the Caribbean [33]. Our previous results grouped some Plantain cultivars as highly resistant (HR) or resistant (R) [18]. However, in the current results, two and five cultivars were found to be intermediate from field and greenhouse evaluation, respectively. This indicates that some Plantain genotypes, despite displaying a resistant phenotype, are infected by Foc TR4 and, consequently, could disseminate the pathogen through the planting material, for instance. The 'Uganda Plantain' genotype was susceptible, ruling out the hypothesis that all Plantain genotypes display some level of resistance to Foc TR4. Altogether, our results also suggest that French types of Plantain could be more susceptible to Foc TR4, though more studies are still necessary to address this hypothesis.
Banana genotypes belonging to the Iholena subgroup are distinguished by the orange color of their pulp fruits, which is indicative of high pro-vitamin A carotenoid content [34]. Previous result showed that this subgroup is susceptible to Foc R1, but its reaction to Foc TR 4 is almost unknown [7]. In the present results, only one cultivar from this subgroup was ranked as resistant to Foc TR4, and the other six cultivars were susceptible (Table 2). Interestingly, the two cultivars from the Maia Maoli/Popoulu subgroup, 'Pacific Plantain' and 'Poingo', exhibited different resistance levels to Foc TR4, which might be associated with diverse resistance genes within the genome.
The Silk subgroup contains genotypes bearing sweet acidic fruits with an apple-like flavor and are well known for their susceptibility to Foc R1 [35]. Indeed, our results showed that all Silk genotypes evaluated were also susceptible to Foc TR4. Viljoen et al. (2017) [28] also found similar results, indicating that Silk genotypes can be used as susceptible references in phenotyping assays for Foc TR4 resistance. Pisang Raja is one of the most economically important local banana cultivars, in Indonesia, particularly in Java [36]. From our results, only one cultivar from this subgroup was found to be resistant, whereas the others were shown to be intermediate or susceptible (Table 2). Pome banana is popularly consumed in India and Australia, and particularly in Brazil, where this subgroup is known as Prata [2]. Mysore banana is widely used in countries such as India, due to its functional and nutraceutical properties, along with great swelling ability to elaborate jellies and meat-based products [37]. In this paper, the Pome cultivar 'Pisang Radjah' and the Mysore cultivar 'Pisang Ceylan' showed resistance and intermediate resistance to Foc TR4, respectively. However, it was reported that the Pome cultivar 'Lady Finger' was susceptible to Foc race 1 and subtropical race 4 in the field [38], whereas it was severely infected by Foc TR4 in a shade house pot trial [22].
It is interesting that the screening result of a total of 258 genotypes against Foc race 1 (VCG 0124) revealed that different reactions (from immune to susceptible) existed in Pome and Mysore subgroups [39]. These results indicate that more cultivars from these two subgroups should be included for further evaluation on the reaction to Foc TR4.
In the field evaluation, some genotypes (Dwarf Nathan, SH-3436, and FHIA-03) showed increased susceptibility throughout their cropping cycles, which was also reported by Mintoff et al. (2021) [20] and Ndayihanzamaso et al. (2020) [40]. The increased inoculum density of Foc TR4 could be related to this fact, as infected plants were not eradicated. Therefore, the secondary inoculum generated by infected plants, mainly from Baxi (susceptible control), and by susceptible genotypes increased after each cropping cycle.

Conclusions
In summary, in this work we assessed the resistance level of 37 banana cultivars against Foc TR4, identifying sources for disease resistance which might support plant breeding. The fact that Plantains can be infected by Foc TR4 is particularly relevant to food security in Africa, Latin America, and the Caribbean, where these genotypes are staple foods, but also provide a major source of income. Therefore, more efforts are needed to evaluate resistance in global Plantain germplasm banks to Foc TR4. In addition, many other varieties play important roles on food security in many countries around the world where Foc TR4 is not yet present. Therefore, information about the behaviors of these genotypes regarding Foc TR4 resistance might support National Plant Protection Organizations through risk analyses and phytosanitary policies.