Genetic Mapping, Candidate Gene Identification and Marker Validation for Host Plant Resistance to the Race 4 of Fusarium oxysporum f. sp. cubense Using Musa acuminata ssp. malaccensis.

Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to Fusarium oxysporum f. sp. Cubense (Foc), the causal agent of this disease, is genetically dissected in this study using two Musa acuminata ssp. Malaccensis segregating populations, segregating for Foc Tropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on chromosome 3 of 'DH-Pahang' reference assembly v4. Within this region, there was a cluster of pattern recognition receptors, namely leucine-rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell-wall-associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins, positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant progenies but not in the susceptible F2 progenies at the onset of infection. This suggests that one or several of these genes may control resistance at this locus. To confirm the segregation of single-gene resistance, we generated an inter-cross between the resistant parent 'Ma850' and a susceptible line 'Ma848', to show that the STR4 resistance co-segregated with marker '28820' at this locus. Finally, an informative SNP marker 29730 allowed the locus-specific resistance to be assessed in a collection of diploid and polyploid banana plants. Of the 60 lines screened, 22 lines were predicted to carry resistance at this locus, including lines known to be TR4-resistant, such as 'Pahang', 'SH-3362', 'SH-3217', 'Ma-ITC0250', and 'DH-Pahang/CIRAD 930'. Additional screening in the International Institute for Tropical Agriculture's collection suggests that the dominant allele is common among the elite 'Matooke' NARITA hybrids, as well as in other triploid or tetraploid hybrids derived from East African highland bananas. Fine mapping and candidate gene identification will allow characterization of molecular mechanisms underlying the TR4 resistance. The markers developed in this study can now aid the marker-assisted selection of TR4 resistance in breeding programs around the world.

First Report of the Root-Knot Nematode Meloidogyne enterolobii Parasitizing Plantain (Musa spp., AAB) in Nigeria.

Plantain (Musa spp., AAB), an important staple food in Africa with West Africa accounting for 32% of global production, is prone to numerous pests and diseases of which plant-parasitic nematodes are a key concern. This includes root-knot nematodes (RKN; Meloidogyne spp.), which infect the roots, causing them to become galled, deformed and swollen. The nematode Meloidogyne enterolobii is considered a global threat to production of many important agricultural crops due to its extremely virulent and aggressive nature (Philbrick et al. 2020). In 2019, during a survey to identify the diversity of nematodes associated with Musa spp. in Nigeria, RKN females (n = 13) were isolated from a heavily galled root (50-75% galling) from a single plantain cv. Agbagba (Musa spp., AAB) plant in Onne, Rivers State, Nigeria (4°43'08.8"N 7°10'37.5"E). Genomic DNA was extracted from three females and processed individually using worm lysis buffer and proteinase K (Bert et al. 2008). The females were identified as M. enterolobii based on Nad5 mtDNA (Janssen et al. 2016), with GenBank accession no. ON010028, ON010027, ON010026, and were 100% homologous to the M. enterolobii sequences MW965454, KU372358 and KU372359 (Supplementary Figure S1). The sampled plant did not show any specific above-ground symptoms but swellings were apparent on the secondary and tertiary roots, which were associated with RKN females that were embedded in the root tissue. All the life stages were found clustered together in the root cortex, where they created necrotic brown-black lesions. A mean value of 2,604 ± 820 (mean ± standard deviation) males, eggs and second-stage juveniles (J2) were extracted from 5 g of root sub-samples (n = 6) using the Hussey and Barker (1973) NaOCl method. On average 39 females were hand-picked (n = 6) from 5 g fresh root. Pure cultures were established from single egg masses and maintained on RKN-susceptible tomato plants (Solanum lycopersicum cv. Marmande). To conduct Koch's postulates, two-month old plantlets of plantain cv. Agbagba (n = 5) were inoculated with 8000 J2s and eggs (initial population) of M. enterolobii pure cultures in 8 L pots in a screenhouse in Nigeria. Non-inoculated plantlets were included as negative controls. The nematode reproduction factor (RF = final density / initial population) and root damage symptoms were assessed 90 days post-inoculation. All the inoculated plantlets had similar galling symptoms and extensive necrosis as was observed in the field (Supplementary Figure S2), with an average RF = 25.9. No symptoms were observed on control plants. Adult females (n = 2) removed from the roots were identified as M. enterolobii based on Nad5 mtDNA (ON532789, ON532790) confirming that plantain cv. Agbagba is a host of M. enterolobii. In Nigeria, M. enterolobii has been reported to be associated with four plant species belonging to four plant families: Euphorbiaceae (Oyetunde et al. 2022), Cucurbitaceae (Bello et al. 2020), Dioscoreaceae (Kolombia et al. 2016), and Solanaceae (dos Santos et al. 2019). To our knowledge, this is the first report of M. enterolobii on a member of the Musaceae in Nigeria and globally the first report on plantain (Musa spp., AAB). The impact of M. enterolobii on plantain productivity has yet to be determined but given the RF value obtained in the pathogenicity test, plantain is a suitable host. This calls for a comprehensive RKN diversity study to evaluate the geographic spread of M. enterolobii on this important staple food crop in West Africa.

First Report of Lasiodiplodia theobromae Causing Dieback Symptoms on Plantain (Musa AAB subgroup) in Nigeria.

Bananas (banana and plantains) rank sixth among staple food crops (FAO 2018), with production challenged by biotic factors, mainly fungal diseases that may cause a total loss in some orchards (Jones 2018). In April 2017, dieback symptoms (progressive blackening and necrotic aerial plant parts, leaves, fruits and peduncles) were observed on plantain (Musa AAB subgroup), in Onne, Rivers State, Nigeria (4°42'55.4012″N, 7°10'35.92128″E). Diseased plants (n=112) were either wilted with blackened necrotic areas, or dead (Fig. S1). Nearly 10% of the plants had blackened pseudostems and fruits with slate gray to black internal tissues when sliced (Fig. S1) and black, erumpent pycnidia were observed on diseased fruits. A fungal species was consistently isolated when surface disinfected pieces of diseased samples were cultured on PDA plates. Plates were incubated at 25±2°C for 4 to 15 d to observe conidia. Isolates had colonies and conidia consistent with members of the Botryosphaeriaceae family (Phillips et al. 2013). Immature conidia were single-celled, ellipsoidal and hyaline while mature conidia were two-celled, had a thick wall, a central septum, longitudinal striations, and a dark brown, cinnamon-like color. Size of mature conidia (n = 20) ranged 22.9 to 30.0 × 14.2 to 18.4 µm ( = 27.0 × 15.6 µm; Fig. S1). DNA templates of three isolates (23688-2_R16; 19144-18_R15 and PITA_22-1) were amplified using primers ITS1 and ITS4 for the ITS locus, EF1-688F and EF1-1251R for the translation elongation factor 1-α (TEF-1α) locus (Phillips et al. 2013) and sequenced (GenBank accession Nos. MZ413346, MZ413347, and MZ413348 for ITS; and MZ420177, MZ420178, and MZ420179 for TEF-1α). BLASTn query showed 100% identity with reference sequences of various isolates of Lasiodiplodia theobromae. Based on morphological characters and nucleotide homology, the isolates were identified as L. theobromae (Fig. S1 & S2). To fulfil Koch's postulates, 4-month-old plants of plantain hybrid PITA 24, and mature fruits from three genotypes (PITA 24, plantain cultivar Obino L'ewai) were inoculated with mycelial plugs from the margins of 5-d-old cultures of the three L. theobromae isolates. Pseudostems were drilled with a sterile 5 -mm cork borer, a mycelial plug placed down into the wound, covered with sterilized cotton, and sealed with parafilm. Sterile water was injected every third day to maintain moisture at the inoculated area. Toothpicks containing mycelia were used to inoculate fruits, placed in plastic Crisper boxes. Sterile PDA plugs or toothpicks were used for the controls. Inoculated plants and fruits were kept in a screenhouse at room temperature (~26°C) for 14 d. All inoculated materials developed symptoms similar to the diseased plants in the field. Control plants and fruits remained asymptomatic. L. theobromae was re-isolated from the artificially inoculated plant parts and its identity was confirmed. The fungus L. theobromae is distributed in tropical and subtropical regions and has a wide host range (Phillips et al. 2013; Mehl et al. 2017). This fungus was previously reported in grey literature as the causal agent of Musa spp. basal rot at Onne, Nigeria (Mwangi et al. 2005) but its molecular identification was not conducted; it was unknown whether the isolates were indeed L. theobromae or other cryptic species (L. pseudotheobromae or L. parva) (Alves et al. 2008). Over 15 years later, the present study confirms L. theobromae as the causal agent of basal rot of bananas based on nucleotide homology, and to our knowledge, this is the first report of L. theobromae causing dieback disease on plantain in Nigeria and in Africa. There is need to conduct a more comprehensive distribution surveys and develop appropriate control strategies in Nigeria.

From fruit growth to ripening in plantain: a careful balance between carbohydrate synthesis and breakdown.

In this study, we aimed to investigate for the first time different fruit development stages in plantain banana in order gain insights into the order of appearance and dominance of specific enzymes and fluxes. We examined fruit development in two plantain banana cultivars during the period between 2-12 weeks after bunch emergence using high-throughput proteomics, quantification of major metabolites, and analyses of metabolic fluxes. Starch synthesis and breakdown are processes that take place simultaneously. During the first 10 weeks fruits accumulated up to 48% of their dry weight as starch, and glucose 6-phosphate and fructose were important precursors. We found a unique amyloplast transporter and hypothesize that it facilitates the import of fructose. We identified an invertase originating from the Musa balbisiana genome that would enable carbon flow back to growth and starch synthesis and maintain a high starch content even during ripening. Enzymes associated with the initiation of ripening were involved in ethylene and auxin metabolism, starch breakdown, pulp softening, and ascorbate biosynthesis. The initiation of ripening was cultivar specific, with faster initiation being particularly linked to the 1-aminocyclopropane-1-carboxylate oxidase and 4-alpha glucanotransferase disproportionating enzymes. Information of this kind is fundamental to determining the optimal time for picking the fruit in order to reduce post-harvest losses, and has potential applications for breeding to improve fruit quality.

Datasets from harmonised metabolic phenotyping of root, tuber and banana crop.

Biochemical characterisation of germplasm collections and crop wild relatives (CWRs) facilitates the assessment of biological potential and the selection of breeding lines for crop improvement. Data from the biochemical characterisation of staple root, tuber and banana (RTB) crops, i.e. banana (Musa spp.), cassava (Manihot esculenta), potato (Solanum tuberosum), sweet potato (Ipomoea batatas) and yam (Dioscorea spp.), using a metabolomics approach is presented. The data support the previously published research article "Metabolite database for root, tuber, and banana crops to facilitate modern breeding in understudied crops" (Price et al., 2020) [1]. Diversity panels for each crop, which included a variety of species, accessions, landraces and CWRs, were characterised. The biochemical profile for potato was based on five elite lines under abiotic stress. Metabolites were extracted from the tissue of foliage and storage organs (tuber, root and banana pulp) via solvent partition. Extracts were analysed via a combination of liquid chromatography - mass spectrometry (LC-MS), gas chromatography (GC)-MS, high pressure liquid chromatography with photodiode array detector (HPLC-PDA) and ultra performance liquid chromatography (UPLC)-PDA. Metabolites were identified by mass spectral matching to in-house libraries comprised from authentic standards and comparison to databases or previously published literature.

Subterranean Microbiome Affiliations of Plantain (Musa spp.) Under Diverse Agroecologies of Western and Central Africa.

Plantain (Musa spp.) is a staple food crop and an important source of income for millions of smallholder farmers in sub-Saharan Africa (SSA). However, there is a paucity of knowledge on soil microbial diversity in agroecologies where plantains are grown. Microbial diversity that increases plant performance with multi-trophic interactions involving resiliency to environmental constraints is greatly needed. For this purpose, the bacterial and fungal communities of plantain fields in high rainfall forests (HR) and derived savannas (SV) were studied using Illumina MiSeq for 16S rDNA and ITS amplicon deep sequencing. Microbial richness (α- and ß-diversity), operational taxonomic units, and Simpson and Shannon-Wiener indexes (observed species (Sobs), Chao, ACE; P < 0.05) suggested that there were significant differences between HR and SV agroecologies among the most abundant bacterial communities, and some specific dynamic response observed from fungal communities. Proteobacteria formed the predominant bacterial phylum (43.7%) succeeded by Firmicutes (24.7%), and Bacteroidetes (17.6%). Ascomycota, Basidiomycota, and Zygomycota were the three most dominant fungal phyla in both agroecologies. The results also revealed an immense array of beneficial microbes in the roots and rhizosphere of plantain, including Acinetobacter, Bacillus, and Pseudomonas spp. COG and KEGG Orthology database depicted significant variations in the functional attributes of microbes found in the rhizosphere to roots. This result indicates that the different agroecologies and host habitats differentially support the dynamic microbial profile and that helps in altering the structure in the rhizosphere zone for the sake of promoting synergistic host-microbe interactions particularly under resource-poor conditions of SSA.

Use of timelapse photography to determine flower opening time and pattern in banana (Musa spp.) for efficient hand pollination.

Sterility and low seed set in bananas is the main challenge to their conventional genetic improvement. The first step to seed set in a banana breeding program depends on pollination at the right time to ensure effective fertilization. This study aimed at determining bract opening time (BOT) to enhance efficient pollination and seed set in bananas. A Nikon D810 digital camera was set-up to take pictures of growing banana inflorescences at five-minute intervals and time-lapse movies were developed at a speed of 30 frames per second to allow real-time monitoring of BOT. Genotypes studied included wild banana (1), Mchare (2), Matooke (4), Matooke hybrid (1), and plantain (1). Events of bract opening initiated by bract lift for female flowers (P < 0.01) started at 16:32 h and at 18:54 h for male flowers. Start of bract rolling was at 18:51 h among female flowers (P < 0.001) and 20:48 h for male flowers. Bracts ended rolling at 02:33 h and 01:16 h for female and flowers respectively (P < 0.05). Total time of bract opening (from lift to end of rolling) for female flowers was significantly longer than that of male flowers (P < 0.001). On average, the number of bracts subtending female flowers opening increased from one on the first day, to between one and four on the fourth day. The number regressed to one bract on day eight before start of opening of bracts subtending male flowers. There was a longer opening interval between bracts subtending female and male flowers constituting spatial and temporal separation. Bract rolling increased from partial to complete rolling from proximal to the distal end of the inflorescence among female flower. On the other hand, bracts subtending male flowers completely rolled. Differences in BOT of genotypes with the same reference time of assessment may be partly responsible for variable fertility. Hand pollination time between 07:00 and 10:00 h is slightly late thus an early feasible time should be tried.

Assessment of metabolic variability and diversity present in leaf, peel and pulp tissue of diploid and triploid Musa spp.

Banana (Musa spp.) plants produce many health promoting compounds in leaf, peel and pulp. For a robust metabolic analysis of these tissues, leaf at five developmental stages were compared to assess suitable sampling practices. Results confirmed that the common sampling practise of leaf 3 is applicable for metabolic comparisons. The developed work flow was applied to analyse the metabolite diversity present in 18 different Musa varieties, providing baseline levels of metabolites in leaf, peel and pulp tissue. Correlation analysis was then used to ascertain whether similar trends can be detected in the three plant tissues of the diversity panel. The genome group displayed a dominant role in the composition of the metabolome in all three tissues. This led to the conclusion that a correlation between tissues was only possible within a genome group as the different parental backgrounds caused too great a variation in the metabolomes. It also suggests the metabolome could be used to monitor the interaction/hybridisation of genomes during breeding programmes.

Metabolite database for root, tuber, and banana crops to facilitate modern breeding in understudied crops.

Roots, tubers, and bananas (RTB) are vital staples for food security in the world's poorest nations. A major constraint to current RTB breeding programmes is limited knowledge on the available diversity due to lack of efficient germplasm characterization and structure. In recent years large-scale efforts have begun to elucidate the genetic and phenotypic diversity of germplasm collections and populations and, yet, biochemical measurements have often been overlooked despite metabolite composition being directly associated with agronomic and consumer traits. Here we present a compound database and concentration range for metabolites detected in the major RTB crops: banana (Musa spp.), cassava (Manihot esculenta), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), and yam (Dioscorea spp.), following metabolomics-based diversity screening of global collections held within the CGIAR institutes. The dataset including 711 chemical features provides a valuable resource regarding the comparative biochemical composition of each RTB crop and highlights the potential diversity available for incorporation into crop improvement programmes. Particularly, the tropical crops cassava, sweet potato and banana displayed more complex compositional metabolite profiles with representations of up to 22 chemical classes (unknowns excluded) than that of potato, for which only metabolites from 10 chemical classes were detected. Additionally, over 20% of biochemical signatures remained unidentified for every crop analyzed. Integration of metabolomics with the on-going genomic and phenotypic studies will enhance 'omics-wide associations of molecular signatures with agronomic and consumer traits via easily quantifiable biochemical markers to aid gene discovery and functional characterization.

Effects of In Vitro Polyploidization on Agronomic Characteristics and Fruit Carotenoid Content; Implications for Banana Genetic Improvement.

Bananas (Musa spp.), native to South East Asia, have spread worldwide and are integrated into the diets of millions of people in tropical regions. Carotenoid content varies dramatically between different banana genotypes, providing an opportunity for vitamin A biofortification. Polyploidization is a useful tool for crop improvement with potential to generate new diversity, especially in a polyploid crop like bananas. Ten induced tetraploids generated from six diploid banana genotypes were evaluated for their agronomic attributes and fruit carotenoid content in comparison to their diploid progenitors. Tetraploids had distinct plant morphology, but generally displayed inferior vegetative and yield characteristics with 20% lower bunch weights than their original diploids. Similarly, a 50% decrease in fruit provitamin A carotenoids (α-carotene, 13-cis ß-carotene, 9-cis ß-carotene, trans-ß-carotene) accompanied by a corresponding increase in lutein was recorded in induced tetraploids in comparison to their original diploids. Additionally, all lines were subjected to pollen viability tests to assess their fertility. Pollen viability tests indicated over 70% viability for induced tetraploids and diploid controls, suggesting their possible use in crosses. These findings provide a basis for the application of induced polyploidization in bananas to generate useful genetic material for integration in hybridization programmes aiming to produce vitamin A enriched triploids valuable to malnourished populations.

Variability of carotenoids in a Musa germplasm collection and implications for provitamin A biofortification.

Bananas are important staples in tropical and sub-tropical regions and their potential as a source of provitamin A has recently attracted attention for biofortification. A collection of 189 banana genotypes (AAB-plantains, M. acuminata cultivars and bred hybrids) was screened to determine variability in fruit pulp provitamin A carotenoid (pVAC) content using high performance liquid chromatography. Total carotenoid content in tested genotypes varied from 1.45 µg/g for hybrid 25447-S7 R2P8 to 36.21 µg/g for M. acuminata cultivar ITC.0601 Hung Tu with a mean of 8.00 µg/g fresh weight. Predominant carotenoids identified were α-carotene (38.67%), trans-ß-carotene (22.08%), lutein (22.08%), 13-cis-ß-carotene (14.45%) and 9-cis-ß-carotene (2.92%), indicating that about 78% of the carotenoids in bananas are pVAC. High pVAC genotypes were identified for integration into biofortification strategies to combat vitamin A deficiency in developing countries.

Metabolite profiling characterises chemotypes of Musa diploids and triploids at juvenile and pre-flowering growth stages.

Bananas (Musa spp.) are consumed worldwide as dessert and cooking types. Edible banana varieties are for the most part seedless and sterile and therefore vegetatively propagated. This confers difficulties for breeding approaches against pressing biotic and abiotic threats and for the nutritional enhancement of banana pulp. A panel of banana accessions, representative of the diversity of wild and cultivated bananas, was analysed to assess the range of chemotypes available globally. The focus of this assessment was banana leaves at two growth stages (juvenile and pre-flowering), to see when during the plant growth metabolic differences can be established. The metabolic data corresponded to genomic trends reported in previous studies and demonstrated a link between metabolites/pathways and the genomes of M. acuminata and M. balbisiana. Furthermore, the vigour and resistance traits of M. balbisiana was connected to the phenolic composition and showed differences with the number of B genes in the hybrid accessions. Differences in the juvenile and pre-flowering data led to low correlation between the growth stages for prediction purposes.

Recent advances in banana (musa spp.) biofortification to alleviate vitamin A deficiency.

Vitamin A deficiency (VAD) is one of the most prevalent micronutrient deficiencies that disproportionately affects low income populations in developing countries. Traditional breeding and modern biotechnology have significant potential to enhance micronutrient bioavailability in crops through biofortification. Bananas (Musa spp.) are economically important fruit crops grown throughout tropical and sub-tropical regions of the world where VAD is most prevalent. Some banana genotypes are rich in provitamin A carotenoids (pVACs), providing an opportunity to use bananas as a readily available vehicle for provitamin A delivery. This review summarizes the progress made in carotenoid research in bananas relative to banana diversity and the use of conventional breeding and transgenic approaches aimed at banana biofortification to address vitamin A deficiency. Existing reports on sampling strategies, pVAC retention and bioavailability are also evaluated as essential components for a successful banana biofortification effort. The wide variability of pVACs reported in banana cultivars coupled with recent advances in unraveling the diversity and genetic improvement of this globally important but often-neglected staple fruit crop underscores their importance in biofortification schemes.