High-throughput phenotyping reveals differential transpiration behaviour within the banana wild relatives highlighting diversity in drought tolerance.

Crop wild relatives, the closely related species of crops, may harbour potentially important sources of new allelic diversity for (a)biotic tolerance or resistance. However, to date, wild diversity is only poorly characterized and evaluated. Banana has a large wild diversity but only a narrow proportion is currently used in breeding programmes. The main objective of this study was to evaluate genotype-dependent transpiration responses in relation to the environment. By applying continuous high-throughput phenotyping, we were able to construct genotype-specific transpiration response models in relation to light, VPD and soil water potential. We characterized and evaluated six (sub)species and discerned four phenotypic clusters. Significant differences were observed in leaf area, cumulative transpiration and transpiration efficiency. We confirmed a general stomatal-driven 'isohydric' drought avoidance behaviour, but discovered genotypic differences in the onset and intensity of stomatal closure. We pinpointed crucial genotype-specific soil water potentials when drought avoidance mechanisms were initiated and when stress kicked in. Differences between (sub)species were dependent on environmental conditions, illustrating the need for high-throughput dynamic phenotyping, modelling and validation. We conclude that the banana wild relatives contain useful drought tolerance traits, emphasising the importance of their conservation and potential for use in breeding programmes.

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.

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.

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.

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.

Maximizing genetic representation in seed collections from populations of self and cross-pollinated banana wild relatives.

BACKGROUND: Conservation of plant genetic resources, including the wild relatives of crops, plays an important and well recognised role in addressing some of the key challenges faced by humanity and the planet including ending hunger and biodiversity loss. However, the genetic diversity and representativeness of ex situ collections, especially that contained in seed collections, is often unknown. This limits meaningful assessments against conservation targets, impairs targeting of future collecting and limits their use. We assessed genetic representation of seed collections compared to source populations for three wild relatives of bananas and plantains. Focal species and sampling regions were M. acuminata subsp. banksii (Papua New Guinea), M. balbisiana (Viet Nam) and M. maclayi s.l. (Bougainville, Papua New Guinea). We sequenced 445 samples using suites of 16-20 existing and newly developed taxon-specific polymorphic microsatellite markers. Samples of each species were from five populations in a region; 15 leaf samples from different individuals and 16 seed samples from one infructescence ('bunch') were analysed for each population. RESULTS: Allelic richness of seeds compared to populations was 51, 81 and 93% (M. acuminata, M. balbisiana and M. maclayi respectively). Seed samples represented all common alleles in populations but omitted some rarer alleles. The number of collections required to achieve the 70% target of the Global Strategy for Plant Conservation was species dependent, relating to mating systems. Musa acuminata populations had low heterozygosity and diversity, indicating self-fertilization; many bunches were needed (> 15) to represent regional alleles to 70%; over 90% of the alleles from a bunch are included in only two seeds. Musa maclayi was characteristically cross-fertilizing; only three bunches were needed to represent regional alleles; within a bunch, 16 seeds represent alleles. Musa balbisiana, considered cross-fertilized, had low genetic diversity; seeds of four bunches are needed to represent regional alleles; only two seeds represent alleles in a bunch. CONCLUSIONS: We demonstrate empirical measurement of representation of genetic material in seeds collections in ex situ conservation towards conservation targets. Species mating systems profoundly affected genetic representation in seed collections and therefore should be a primary consideration to maximize genetic representation. Results are applicable to sampling strategies for other wild species.

Unravelling the complex story of intergenomic recombination in ABB allotriploid bananas.

BACKGROUND AND AIMS: Bananas (Musa spp.) are a major staple food for hundreds of millions of people in developing countries. The cultivated varieties are seedless and parthenocarpic clones of which the ancestral origin remains to be clarified. The most important cultivars are triploids with an AAA, AAB or ABB genome constitution, with A and B genomes provided by M. acuminata and M. balbisiana, respectively. Previous studies suggested that inter-genome recombinations were relatively common in banana cultivars and that triploids were more likely to have passed through an intermediate hybrid. In this study, we investigated the chromosome structure within the ABB group, composed of starchy cooking bananas that play an important role in food security. METHODS: Using SNP markers called from RADSeq data, we studied the chromosome structure of 36 ABB genotypes spanning defined taxonomic subgroups. To complement our understanding, we searched for similar events within nine AB hybrid genotypes. KEY RESULTS: Recurrent homologous exchanges (HEs), i.e. chromatin exchanges between A and B subgenomes, were unravelled with at least nine founding events (HE patterns) at the origin of ABB bananas prior to clonal diversification. Two independent founding events were found for Pisang Awak genotypes. Two HE patterns, corresponding to genotypes Pelipita and Klue Teparod, show an over-representation of B genome contribution. Three HE patterns mainly found in Indian accessions shared some recombined regions and two additional patterns did not correspond to any known subgroups. CONCLUSIONS: The discovery of the nine founding events allowed an investigation of the possible routes that led to the creation of the different subgroups, which resulted in new hypotheses. Based on our observations, we suggest different routes that gave rise to the current diversity in the ABB cultivars, routes involving primary AB hybrids, routes leading to shared HEs and routes leading to a B excess ratio. Genetic fluxes took place between M. acuminata and M. balbisiana, particularly in India, where these unbalanced AB hybrids and ABB allotriploids originated, and where cultivated M. balbisiana are abundant. The result of this study clarifies the classification of ABB cultivars, possibly leading to the revision of the classification of this subgroup.

Comparative Transcriptome and Expression Profiling of Resistant and Susceptible Banana Cultivars during Infection by Fusarium oxysporum.

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive diseases of banana. Methods to control the disease are still inadequate. The present investigation targeted expression of defense-related genes in tissue cultured banana plantlets of Fusarium resistant and susceptible cultivars after infection with biological control agents (BCAs) and Fusarium (Foc race 1). In total 3034 differentially expressed genes were identified which annotated to 58 transcriptional families (TF). TF families such as MYB, bHLH and NAC TFs were mostly up-regulated in response to pathogen stress, whereas AP2/EREBP were mostly down-regulated. Most genes were associated with plant-pathogen response, plant hormone signal transduction, starch and sucrose metabolism, cysteine and methionine metabolism, flavonoid biosynthesis, selenocompound metabolism, phenylpropanoid biosynthesis, mRNA surveillance pathway, mannose type O-glycan biosynthesis, amino acid and nucleotide sugar metabolism, cyanoamino acid metabolism, and hormone signal transduction. Our results showed that the defense mechanisms of resistant and susceptible banana cultivars treated with BCAs, were regulated by differentially expressed genes in various categories of defense pathways. Furthermore, the association with different resistant levels might serve as a strong foundation for the control of Fusarium wilt of banana.

Significant progressive heterobeltiosis in banana crossbreeding.

BACKGROUND: Heterobeltiosis is the phenomenon when the hybrid's performance is superior to its best performing parent. Banana (Musa spp. AAA) breeding is a tedious, time-consuming process, taking up to two decades to develop a consumer acceptable hybrid. Exploiting heterobeltiosis in banana breeding will help to select breeding material with high complementarity, thus increasing banana breeding efficiency. The aim of this study was therefore to determine and document the level of heterobeltiosis of bunch weight and plant stature in the East African highland bananas, in order to identify potential parents that can be used to produce offspring with desired bunch weight and stature after a few crosses. RESULTS: This research found significant progressive heterobeltiosis in cross-bred 'Matooke' (highland cooking) banana hybrids, also known as NARITAs, when grown together across years with their parents and grandparents in Uganda. Most (all except 4) NARITAs exhibited positive heterobeltiosis for bunch weight, whereas slightly more than half of them had negative heterobeltiosis for stature. The secondary triploid NARITA 17 had the highest heterobeltiosis for bunch weight: 249% versus its 'Matooke' grandparent and 136% against its primary tetraploid parent. Broad sense heritability (across three cropping cycles) for yield potential and bunch weight were high (0.84 and 0.76 respectively), while that of plant stature was very low (0.0035). There was a positive significant correlation (P < 0.05) between grandparent heterobeltiosis for bunch weight and genetic distance between parents (r = 0.39, P = 0.036), bunch weight (r = 0.7, P < 0.001), plant stature (r = 0.38, P = 0.033) and yield potential (r = 0.59, P < 0.001). Grandparent heterobeltiosis for plant stature was significantly, but negatively, correlated to the genetic distance between parents (r = - 0.6, P < 0.001). CONCLUSIONS: Such significant heterobeltiosis exhibited for bunch weight is to our knowledge the largest among main food crops. Since bananas are vegetatively propagated, the effect of heterobeltiosis is easily fixed in the hybrids and will not be lost over time after the release and further commercialization of these hybrids.

Chromosome Painting in Cultivated Bananas and Their Wild Relatives (Musa spp.) Reveals Differences in Chromosome Structure.

Edible banana cultivars are diploid, triploid, or tetraploid hybrids, which originated by natural cross hybridization between subspecies of diploid Musa acuminata, or between M. acuminata and diploid Musa balbisiana. The participation of two other wild diploid species Musa schizocarpa and Musa textilis was also indicated by molecular studies. The fusion of gametes with structurally different chromosome sets may give rise to progenies with structural chromosome heterozygosity and reduced fertility due to aberrant chromosome pairing and unbalanced chromosome segregation. Only a few translocations have been classified on the genomic level so far, and a comprehensive molecular cytogenetic characterization of cultivars and species of the family Musaceae is still lacking. Fluorescence in situ hybridization (FISH) with chromosome-arm-specific oligo painting probes was used for comparative karyotype analysis in a set of wild Musa species and edible banana clones. The results revealed large differences in chromosome structure, discriminating individual accessions. These results permitted the identification of putative progenitors of cultivated clones and clarified the genomic constitution and evolution of aneuploid banana clones, which seem to be common among the polyploid banana accessions. New insights into the chromosome organization and structural chromosome changes will be a valuable asset in breeding programs, particularly in the selection of appropriate parents for cross hybridization.

Effect of paleopolyploidy and allopolyploidy on gene expression in banana.

BACKGROUND: Bananas (Musa spp.) are an important crop worldwide. Most modern cultivars resulted from a complex polyploidization history that comprised three whole genome duplications (WGDs) shaping the haploid Musa genome, followed by inter- and intra-specific crosses between Musa acuminata and M. balbisiana (A and B genome, respectively). Unresolved hybridizations finally led to banana diversification into several autotriploid (AAA) and allotriploid cultivars (AAB and ABB). Using transcriptomic data, we investigated the impact of the genome structure on gene expression patterns in roots of 12 different triploid genotypes covering AAA, AAB and ABB subgenome constitutions. RESULTS: We demonstrate that (i) there are different genome structures, (ii) expression patterns go beyond the predicted genomic groups, and (iii) the proportion of the B genome influences the gene expression. The presence of the B genome is associated with a higher expression of genes involved in flavonoid biosynthesis, fatty acid metabolism, amino sugar and nucleotide sugar metabolism and oxidative phosphorylation. There are cultivar-specific chromosome regions with biased B:A gene expression ratios that demonstrate homoeologous exchanges (HE) between A and B sub-genomes. In two cultivars, aneuploidy was detected. We identified 3674 genes with a different expression level between allotriploid and autotriploid with ~ 57% having recently duplicated copies (paralogous). We propose a Paralog Inclusive Expression (PIE) analysis that appears to be suitable for genomes still in a downsizing and fractionation process following whole genome duplications. Our approach allows highlighting the genes with a maximum likelihood to affect the plant phenotype. CONCLUSIONS: This study on banana is a good case to investigate the effects of alloploidy in crops. We conclude that allopolyploidy triggered changes in the genome structure of a crop and it clearly influences the gene.

Association genetics of bunch weight and its component traits in East African highland banana (Musa spp. AAA group).

KEY MESSAGE: The major quantitative trait loci associated with bunch weight and its component traits in the East African highland banana-breeding population are located on chromosome 3. Bunch weight increase is one of the major objectives of banana improvement programs, but little is known about the loci controlling bunch weight and its component traits. Here we report for the first time some genomic loci associated with bunch weight and its component traits in banana as revealed through a genome-wide association study. A banana-breeding population of 307 genotypes varying in ploidy was phenotyped in three locations under different environmental conditions, and data were collected on bunch weight, number of hands and fruits; fruit length and circumference; and diameter of both fruit and pulp for three crop cycles. The population was genotyped with genotyping by sequencing and 27,178 single nucleotide polymorphisms (SNPs) were generated. The association between SNPs and the best linear unbiased predictors of traits was performed with TASSEL v5 using a mixed linear model accounting for population structure and kinship. Using Bonferroni correction, false discovery rate, and long-range linkage disequilibrium (LD), 25 genomic loci were identified with significant SNPs and most were localized on chromosome 3. Most SNPs were located in genes encoding uncharacterized and hypothetical proteins, but some mapped to transcription factors and genes involved in cell cycle regulation. Inter-chromosomal LD of SNPs was present in the population, but none of the SNPs were significantly associated with the traits. The clustering of significant SNPs on chromosome 3 supported our hypothesis that fruit filling in this population was under control of a few quantitative trait loci with major effects.

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.

The Plantain Proteome, a Focus on Allele Specific Proteins Obtained from Plantain Fruits.

Proteomics has been applied with great potential to elucidate molecular mechanisms in plants. This is especially valid in the case of non-model crops of which their genome has not been sequenced yet, or is not well annotated. Plantains are a kind of cooking bananas that are economically very important in Africa, India, and Latin America. The aim of this work was to characterize the fruit proteome of common dessert bananas and plantains and to identify proteins that are only encoded by the plantain genome. We present the first plantain fruit proteome. All data are available via ProteomeXchange with identifier PXD005589. Using our in-house workflow, we found 37 alleles to be unique for plantain covered by 59 peptides. Although we do not have access (yet) to whole-genome sequencing data from triploid banana cultivars, we show that proteomics is an easily accessible complementary alternative to detect different allele specific SNPs/SAAPs. These unique alleles might contribute toward the differences in the metabolism between dessert bananas and plantains. This dataset will stimulate further analysis by the scientific community, boost plantain research, and facilitate plantain breeding.

Phenalenone-type phytoalexins mediate resistance of banana plants (Musa spp.) to the burrowing nematode Radopholus similis.

The global yield of bananas-one of the most important food crops-is severely hampered by parasites, such as nematodes, which cause yield losses up to 75%. Plant-nematode interactions of two banana cultivars differing in susceptibility to Radopholus similis were investigated by combining the conventional and spatially resolved analytical techniques (1)H NMR spectroscopy, matrix-free UV-laser desorption/ionization mass spectrometric imaging, and Raman microspectroscopy. This innovative combination of analytical techniques was applied to isolate, identify, and locate the banana-specific type of phytoalexins, phenylphenalenones, in the R. similis-caused lesions of the plants. The striking antinematode activity of the phenylphenalenone anigorufone, its ingestion by the nematode, and its subsequent localization in lipid droplets within the nematode is reported. The importance of varying local concentrations of these specialized metabolites in infected plant tissues, their involvement in the plant's defense system, and derived strategies for improving banana resistance are highlighted.

Evolutionary dynamics and biogeography of Musaceae reveal a correlation between the diversification of the banana family and the geological and climatic history of Southeast Asia.

Tropical Southeast Asia, which harbors most of the Musaceae biodiversity, is one of the most species-rich regions in the world. Its high degree of endemism is shaped by the region's tectonic and climatic history, with large differences between northern Indo-Burma and the Malayan Archipelago. Here, we aim to find a link between the diversification and biogeography of Musaceae and geological history of the Southeast Asian subcontinent. The Musaceae family (including five Ensete, 45 Musa and one Musella species) was dated using a large phylogenetic framework encompassing 163 species from all Zingiberales families. Evolutionary patterns within Musaceae were inferred using ancestral area reconstruction and diversification rate analyses. All three Musaceae genera - Ensete, Musa and Musella - originated in northern Indo-Burma during the early Eocene. Musa species dispersed from 'northwest to southeast' into Southeast Asia with only few back-dispersals towards northern Indo-Burma. Musaceae colonization events of the Malayan Archipelago subcontinent are clearly linked to the geological and climatic history of the region. Musa species were only able to colonize the region east of Wallace's line after the availability of emergent land from the late Miocene onwards.

Cryopreservation of Bituminaria bituminosa varieties and hybrids.

Bituminaria bituminosa (L.) C.H. Stirton is a drought tolerant, perennial legume pasture species and a source of pharmaceutical compounds. Bituminaria breeding programs aim to develop and conserve hybrids with desirable traits such as high forage quality, tolerance to biotic or abiotic stresses, and high contents of furanocoumarins. In this work we present a cryopreservation study of different B. bituminosa accessions: two varieties and eight intervarietal hybrids resulting from crosses between the three botanical varieties: var. bituminosa, var. crassiuscula, and var. albomarginata. No previous work on cryopreservation of Bituminaria species has been reported. We applied the ultra-fast cooling method, using droplet vitrification on aluminum foil strips. First, we investigated the PVS2 toxicity and cryopreservation damage in two genotypes, comparing three PVS2 treatments and two culture media. An incubation of 30 min in PVS2 resulted in regeneration rates after cryopreservation higher than 80%. The MS medium was selected for optimal meristem outgrowth, in order to avoid the prominent callus formation that was observed in the presence of BAP. These conditions were subsequently used to cryopreserve eight other genotypes. The results were highly variable; 45 days after cryopreservation, survival ranged between 22% and 98% while regeneration ranged between 0% and 96%, depending on the accession. A significant and positive correlation was observed between survival and regeneration. At 90 days post culture plantlets could be recovered from cryopreserved explants of all genotypes. This study shows that the droplet vitrification method is promising for the cryopreservation of eight of the 10 genotypes assayed and the method can thus be applied to develop a cryobank of B. bituminosa.

Unravelling the effect of sucrose and cold pretreatment on cryopreservation of potato through sugar analysis and proteomics.

Apical shoot tips were dissected from donor plants (cultured in several conditions) and cryopreserved using the droplet-vitrification technique. The effect of two preculture treatments (sucrose pretreatment medium or cold-culturing during two weeks) on donor plants of four potato species (Solanum commersonii, S. juzepcukii, S. ajanhuiri, and Solanum tuberosum) was studied. Post-cryopreservation meristem growth and plant recovery were influenced by the treatments, but the effect on the regeneration was strongly genotype-dependent. The highest post-rewarming plant recovery percentage was obtained using meristems dissected from donor plants of S. commersonii cultured on sucrose pretreatment medium or cold-cultured. Both preculture conditions also enhanced plant recovery in S. juzepcukii compared to control cultures. Cold preculture, however, proved to be undesirable for S. tuberosum whereas sucrose pretreatment had a positive impact on the plant regeneration of this species. The determination of changes in the concentration of soluble sugars revealed sugar accumulation, especially of sucrose and the raffinose family of oligosaccharides (RFOs), which can be linked to tolerance towards the cryopreservation. Additionally, a study of the proteome of the donor plantlets after the pretreatments by 2D-fluorescence difference gel electrophoresis (DIGE) was carried out to identify differentially abundant proteins. Carbon metabolism-related proteins, together with stress-response and oxidative-homeostasis related proteins were the main class of proteins that changed in abundance after the pretreatments. Our results suggest that oxidative homeostasis-related proteins and sugars may be associated with the improved tolerance to cryopreservation and the ability to cold acclimate by S. commersonii in contrast to the other genotypes. The increased accumulation of sucrose and RFOs play a fundamental role in the response to stress in potato and may help to acquire tolerance to cryopreservation.

Datasets from fertilized improved and local varieties of cassava grown in the highlands of South Kivu, Democratic Republic of Congo.

The use of mineral fertilizer and organic inputs with an improved and local variety of cassava allows (i) to identify nutrient limitations to cassava production, (ii) to investigate the effects of variety and combined application of mineral and organic inputs on cassava growth and yield and (iii) to evaluate the profitability of the improved variety and fertilizer use in cassava production. Data on growth, yield and yield components of an improved and local variety of cassava, economic analysis, soil and weather, collected during two growing cycles of cassava in farmer's fields in the highlands of the Democratic Republic of Congo (DR Congo) are presented. The data complement the recently published paper "Increased cassava growth and yields through improved variety use and fertilizer application in the highlands of South Kivu, Democratic Republic of Congo" (Munyahali et al., 2023) [1]. Data on plant height and diameter were collected throughout the growing period of the crop while the data on the storage root, stem, tradable storage root, non-tradable storage root and harvest index were determined at 12 months after planting (MAP). An economic analysis was performed using a simplified financial analysis whereby additional benefits were calculated relative to the respective control treatments; the total costs included the purchasing price of fertilizers and the additional net benefits represented the revenue from the increased storage root yield due to fertilizer application. The value cost ratio (VCR) was calculated as the additional net benefits over the cost of fertilizer purchase.

Expression of a rice chitinase gene in transgenic banana ('Gros Michel', AAA genome group) confers resistance to black leaf streak disease.

Transgenic banana (Musa acuminata 'Gros Michel') integrating either of two rice chitinase genes was generated and its resistance to Black Leaf Streak disease caused by the fungus Mycosphaerella fijiensis was tested using a leaf disk bioassay. PCR screening indicated the presence of the hpt selectable marker gene in more than 90 % of the lines tested, whereas more than three quarters of the lines contained the linked rice chitinase gene resulting in a co-transformation frequency of at least 71.4 %. Further, a unique stable integration of the transgenes in each line revealed some false negative PCR results and the expected co-transformation frequency of 100 %. The transgene insert number per line ranged from 1 to 5 and single transgene insert lines (25 % of all) were identified. Considerable delay in disease development (up to 63 days post-incoculation) over a monitoring period of 108 days occurred in nine lines with extracellularly targeted chitinase out of 17 transgenic lines tested and their necrotic leaf area decreased by 73-94 % compared to the untransformed susceptible control line. Finally, correlation between symptom development and rice chitinase expression was confirmed in two lines by Western analysis. The potential of rice chitinase genes to enhance resistance against M. fijiensis in banana was demonstrated as well as the usefulness of the leaf disk bioassay for early disease screening in transgenic banana lines.