Transgenic East African Highland Banana Plants Are Protected against Radopholus similis through Host-Delivered RNAi.

The burrowing nematode Radopholus similis is considered a major problem of intensive banana cultivation. It can cause extensive root damage resulting in the toppling disease of banana, which means that plants fall to the ground. Soaking R. similis in double-stranded (ds) RNA of the nematode genes Rps13, chitin synthase (Chs-2), Unc-87, Pat-10 or beta-1,4-endoglucanase (Eng1a) suppressed reproduction on carrot discs, from 2.8-fold (Chs-2) to 7-fold (Rps13). The East African Highland Banana cultivar Nakitembe was then transformed with constructs for expression of dsRNA against the same genes, and for each construct, 30 independent transformants were tested with nematode infection. Four months after transfer from in vitro culture to the greenhouse, the banana plants were transferred to a screenhouse and inoculated with 2000 nematodes per plant, and thirteen weeks later, they were analyzed for several parameters including plant growth, root necrosis and final nematode population. Plants with dsRNA constructs against the nematode genes were on average showing lower nematode multiplication and root damage than the nontransformed controls or the banana plants expressing dsRNA against the nonendogenous gene. In conclusion, RNAi seems to efficiently protect banana against damage caused by R. similis, opening perspectives to control this pest.

Evaluating combined effects of pesticide and crop nutrition (with N, P, K and Si) on weevil damage in East African Highland Bananas.

Banana weevil (Cosmopolites sordidus, Germar) is a major pest in East African Highland Banana. The influence of crop nutritional status on weevil damage is poorly understood. Nutrient availability affects the nutritional quality of plants for weevils and may affect weevil damage. Here, we evaluate the effect of insecticides alone and in combination with fertilisers (N, P, K and Si) on weevil damage using data from two experiments in central and southwest Uganda. In the first experiment, we varied chlorpyrifos and application rates of N, P and K. In the second experiment, we varied the application rates of K and Si. Treatment effects were analysed using generalised linear mixed models with a negative binomial distribution. In the first experiment, chlorpyrifos reduced and N increased weevil damage, while P and K had no significant effect. In the K or Si application rates reduced weevil damage compared with the control. We conclude that the combined application of chlorpyrifos with K and Si fertilisers can contribute to weevil damage control on sites with low nutrient availability and should form part of integrated weevil management in bananas. Future studies should assess how much reduction in insecticide use is possible in EAHB with judicious input rates.

Internal Quality Assessment of East African Highland Cooking Banana (Musa spp.) Flour: Significance for Breeding and Industrial Applications.

This study assessed the internal quality traits of East African Highland cooking banana flours, exploring their significance for breeding and potential industrial applications. Twenty cultivars (nine hybrids and eleven landraces) were used. Swelling power capacity, water solubility, water absorption capacity, water absorption index, freeze-thawing stability, and pasting characteristics of banana flour were assessed using standard methods. The results showed that cultivars with high swelling power also exhibited a high water absorption capacity and water absorption index, thus making them suitable for bakery industries. The water absorption capacity ranged between 5.66% (N2) and 11.68% (N11). Landraces KBZ (9.01) and NKYK (8.05), and hybrids N11 (11.68) and N9 (8.48) are suitable as thickeners due to high WAC. Hybrids (N7, 27.83%, and N9, 22.59%) and landraces (NMZ, 32.69%, and NFK, 34.24%) had low freeze-thawing stability, hence it is applicable as a food stabilizer. Landrace NKT (19.14%) and hybrid N9 (16.95%) had the highest solubility, and landrace KBZ (6.93%) and hybrid N3 (6.66%) had the lowest solubility. Landraces MSK (6265), NKY (3980), and NFK (3957), and hybrids N6 (3608), N7 (3505), and N9 (3281 RVU) had high peak viscosity. The trough viscosity, final viscosity, and breakdown viscosity of cultivars varied from 422.5 to 5004 RVU. The landraces MSK (5021 RVU) and NFK (4111 RVU) had the highest final viscosity, making them suitable for application in the food industry for thick and stable sauces. Landrace TRZ had the lowest pasting temperature (62.7 °C), making it advantageous for use where fast gelatinization is required, hence saving energy costs and cooking time. These findings suggest that the genetic attributes inherent in cultivars can be incorporated into breeding programs targeting required traits for industrial application.

Mapping spatial distribution and geographic shifts of East African highland banana (Musa spp.) in Uganda.

East African highland banana (Musa acuminata genome group AAA-EA; hereafter referred to as banana) is critical for Uganda's food supply, hence our aim to map current distribution and to understand changes in banana production areas over the past five decades. We collected banana presence/absence data through an online survey based on high-resolution satellite images and coupled this data with independent covariates as inputs for ensemble machine learning prediction of current banana distribution. We assessed geographic shifts of production areas using spatially explicit differences between the 1958 and 2016 banana distribution maps. The biophysical factors associated with banana spatial distribution and geographic shift were determined using a logistic regression model and classification and regression tree, respectively. Ensemble models were superior (AUC = 0.895; 0.907) compared to their constituent algorithms trained with 12 and 17 covariates, respectively: random forests (AUC = 0.883; 0.901), gradient boosting machines (AUC = 0.878; 0.903), and neural networks (AUC = 0.870; 0.890). The logistic regression model (AUC = 0.879) performance was similar to that for the ensemble model and its constituent algorithms. In 2016, banana cultivation was concentrated in the western (44%) and central (36%) regions, while only a small proportion was in the eastern (18%) and northern (2%) regions. About 60% of increased cultivation since 1958 was in the western region; 50% of decreased cultivation in the eastern region; and 44% of continued cultivation in the central region. Soil organic carbon, soil pH, annual precipitation, slope gradient, bulk density and blue reflectance were associated with increased banana cultivation while precipitation seasonality and mean annual temperature were associated with decreased banana cultivation over the past 50 years. The maps of spatial distribution and geographic shift of banana can support targeting of context-specific intensification options and policy advocacy to avert agriculture driven environmental degradation.

Analysis of Genetic Diversity of Banana Weevils (Cosmopolites sordidus) (Coleoptera: Curculionidae) Using Transcriptome-Derived Simple Sequence Repeat Markers.

The banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) is an economically important insect pest of bananas. It causes up to 100% yield losses and substantial lifespan reduction in bananas. Advances in genomics, proteomics, and sequencing technologies have provided powerful pathways to genotyping disastrous pests such as C. sordidus. However, such technologies are often not available to the majority of rural subtropical African banana growers and pest control managers. This study was therefore motivated by the need to create cheap and easily accessible C. sordidus genotyping methods that could be deployed by banana pest control managers to the benefit of C. sordidus control programs in the tropics where such advanced technologies are not readily accessible. We used an in-house C. sordidus transcriptome from the an-ongoing study from which we mined an array of simple sequence repeat (SSR) markers. Of these, six highly polymorphic transcriptome-derived SSR markers were used to successfully genotype within and among banana weevil population genetic diversity of 12 C. sordidus populations collected from four banana-growing agro-ecological zones (AEZs) in Uganda. The developed transcriptome-derived SSR markers can be used by researchers in population genetics for characterization of the C. sordidus and identification of new genes that are linked to traits of particular interest. The significant genetic diversity revealed in C. sordidus provides pertinent information for integrated pest management strategies.

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.

First Evidence of Feeding-Induced RNAi in Banana Weevil via Exogenous Application of dsRNA.

Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound, and no resistant farmer-preferred cultivars are known to date. In this paper, we examined the ability to induce RNA interference (RNAi) in the banana weevil via feeding. We first developed an agar- and banana corm (rhizome) flour-based artificial diet in a multi-well plate setup that allowed the banana weevils to complete their life cycle from egg through the larval instars to the pupal stage in an average period of 53 days. Adults emerged about 20 days later. The artificial diet allowed the tunneling and burrowing habits of the larvae and successful metamorphosis up to adult eclosion. Adding dsRNA for laccase2 to the artificial diet resulted in albino phenotypes, confirming gene-silencing. Finally, C. sordidus was fed with dsRNA against a selection of essential target genes: snf7, rps13, mad1, vha-a, vha-d, and lgl for a period of 45 days. 100% mortality within 9-16 days was realized with dssnf7, dsrps13, and dsmad1 at 200 ng/mL artificial diet, and this corresponded to a strong reduction in gene expression. Feeding the dsRNA targeting the two vha genes resulted in 100% mortality after about 3-4 weeks, while treatment with dslgl resulted in no mortality above the dsgfp-control and the water-control. Our results have implications for the development of RNAi approaches for managing important crop pests, in that banana weevils can be controlled based on the silencing of essential target genes as snf7, rps13, and mad1. They also highlight the need for research into the development of RNAi for banana protection, eventually the engineering of host-induced gene-silencing (HIGS) cultivars, given the high RNAi efficacy and its species-specific mode of action, adding the RNAi approach to the armory of integrated pest management (IPM).

Evaluation of banana germplasm and genetic analysis of an F1 population for resistance to Fusarium oxysporum f. sp. cubense race 1.

Fusarium wilt of bananas (Musa spp.), caused by Fusarium oxysporum f. sp. cubense (Foc) causes up to 100% yield loss in bananas. Foc race 1 in particular is very devastating to dessert bananas in Uganda. One of the effective control strategies for the disease is the development of resistant cultivars through breeding. The objectives of this study were to identify suitable banana germplasm for generating a segregating population for resistance to Foc race 1 and understand the mode of inheritance of resistance to Foc race 1. Twenty-two banana accessions sourced from the National Agricultural Research Organisation in Uganda were challenged with Foc race 1 in a screen house experiment. Monyet, resistant to Foc race 1 and Kokopo, susceptible, were selected and crossed to generate 142 F1 genotypes. These F1 genotypes were also challenged with Foc race 1 in a screen house experiment. Data were collected on rhizome discoloration index (RDI), leaf symptom index (LSI) and pseudo-stem splitting (PSS), and analysed for variability. The banana accessions evaluated showed varying degrees of resistance to Foc race 1. Segregation ratios for resistant versus susceptible progenies fitted 13:3 (χ2 = 0.12, P = 0.73) for RDI and 11:5 (χ2 = 3.04, P = 0.08) for PSS. Estimated broad sense heritability was 27.8% for RDI, 13.9% for LSI and 14.7% for PSS. The results suggest that resistance to Foc race 1 in banana is controlled by at least two dominant genes with epistatic interaction and that heritability of resistance to Foc race 1 is low in Musa spp.

Silencing of the Mitogen-Activated Protein Kinases (MAPK) Fus3 and Slt2 in Pseudocercospora fijiensis Reduces Growth and Virulence on Host Plants.

Pseudocercospora fijiensis, causal agent of the black Sigatoka disease (BSD) of Musa spp., has spread globally since its discovery in Fiji 1963 to all the banana and plantain growing areas across the globe. It is becoming the most damaging and economically important disease of this crop. The identification and characterization of genes that regulate infection processes and pathogenicity in P. fijiensis will provide important knowledge for the development of disease-resistant cultivars. In many fungal plant pathogens, the Fus3 and Slt2 are reported to be essential for pathogenicity. Fus3 regulates filamentous-invasion pathways including the formation of infection structures, sporulation, virulence, and invasive and filamentous growth, whereas Slt2 is involved in the cell-wall integrity pathway, virulence, invasive growth, and colonization in host tissues. Here, we used RNAi-mediated gene silencing to investigate the role of the Slt2 and Fus3 homologs in P. fijiensis in pathogen invasiveness, growth and pathogenicity. The PfSlt2 and PfFus3 silenced P. fijiensis transformants showed significantly lower gene expression and reduced virulence, invasive growth, and lower biomass in infected leaf tissues of East African Highland Banana (EAHB). This study suggests that Slt2 and Fus3 MAPK signaling pathways play important roles in plant infection and pathogenic growth of fungal pathogens. The silencing of these vital fungal genes through host-induced gene silencing (HIG) could be an alternative strategy for developing transgenic banana and plantain resistant to BSD.

Genetically engineered bananas resistant to Xanthomonas wilt disease and nematodes.

Banana is an important staple food crop feeding more than 100 million Africans, but is subject to severe productivity constraints due to a range of pests and diseases. Banana Xanthomonas wilt caused by Xanthomonas campestris pv. musacearum is capable of entirely destroying a plantation while nematodes can cause losses up to 50% and increase susceptibility to other pests and diseases. Development of improved varieties of banana is fundamental in order to tackle these challenges. However, the sterile nature of the crop and the lack of resistance in Musa germplasm make improvement by traditional breeding techniques either impossible or extremely slow. Recent developments using genetic engineering have begun to address these problems. Transgenic banana expressing sweet pepper Hrap and Pflp genes have demonstrated complete resistance against X. campestris pv. musacearum in the field. Transgenic plantains expressing a cysteine proteinase inhibitors and/or synthetic peptide showed enhanced resistance to a mixed species population of nematodes in the field. Here, we review the genetic engineering technologies which have potential to improve agriculture and food security in Africa.

Agrobacterium tumefaciens-Mediated Transformation of Pseudocercospora fijiensis to Determine the Role of PfHog1 in Osmotic Stress Regulation and Virulence Modulation.

Black Sigatoka disease, caused by Pseudocercospora fijiensis is a serious constraint to banana production worldwide. The disease continues to spread in new ecological niches and there is an urgent need to develop strategies for its control. The high osmolarity glycerol (HOG) pathway in Saccharomyces cerevisiae is well known to respond to changes in external osmolarity. HOG pathway activation leads to phosphorylation, activation and nuclear transduction of the HOG1 mitogen-activated protein kinases (MAPKs). The activated HOG1 triggers several responses to osmotic stress, including up or down regulation of different genes, regulation of protein translation, adjustments to cell cycle progression and synthesis of osmolyte glycerol. This study investigated the role of the MAPK-encoding PfHog1 gene on osmotic stress adaptation and virulence of P. fijiensis. RNA interference-mediated gene silencing of PfHog1 significantly suppressed growth of P. fijiensis on potato dextrose agar media supplemented with 1 M NaCl, indicating that PfHog1 regulates osmotic stress. In addition, virulence of the PfHog1-silenced mutants of P. fijiensis on banana was significantly reduced, as observed from the low rates of necrosis and disease development on the infected leaves. Staining with lacto phenol cotton blue further confirmed the impaired mycelial growth of the PfHog1 in the infected leaf tissues, which was further confirmed with quantification of the fungal biomass using absolute- quantitative PCR. Collectively, these findings demonstrate that PfHog1 plays a critical role in osmotic stress regulation and virulence of P. fijiensis on its host banana. Thus, PfHog1 could be an interesting target for the control of black Sigatoka disease in banana.

Transgenic banana plants expressing Xanthomonas wilt resistance genes revealed a stable non-target bacterial colonization structure.

Africa is among the continents where the battle over genetically modified crops is currently being played out. The impact of GM in Africa could potentially be very positive. In Uganda, researchers have developed transgenic banana lines resistant to banana Xanthomonas wilt. The transgenic lines expressing hrap and pflp can provide a timely solution to the pandemic. However, the impact of the transgenes expression on non-target microorganisms has not yet been investigated. To study this effect, transgenic and control lines were grown under field conditions and their associated microbiome was investigated by 16S rRNA gene profiling combining amplicon sequencing and molecular fingerprinting. Three years after sucker planting, no statistically significant differences between transgenic lines and their non-modified predecessors were detected for their associated bacterial communities. The overall gammaproteobacterial rhizosphere microbiome was highly dominated by Xanthomonadales, while Pseudomonadales and Enterobacteriales were accumulated in the pseudostem. Shannon indices revealed much higher diversity in the rhizosphere than in the pseudostem endosphere. However, the expression of the transgenes did not result in changes in the diversity of Gammaproteobacteria, the closest relatives of the target pathogen. In this field experiment, the expression of the resistance genes appears to have no consequences for non-target rhizobacteria and endophytes.

Genome Sequencing of Xanthomonas vasicola Pathovar vasculorum Reveals Variation in Plasmids and Genes Encoding Lipopolysaccharide Synthesis, Type-IV Pilus and Type-III Secretion Effectors.

Xanthomonas vasicola pathovar vasculorum (Xvv) is the bacterial agent causing gumming disease in sugarcane. Here, we compare complete genome sequences for five isolates of Xvv originating from sugarcane and one from maize. This identified two distinct types of lipopolysaccharide synthesis gene clusters among Xvv isolates: one is similar to that of Xanthomonas axonopodis pathovar citri (Xac) and is probably the ancestral type, while the other is similar to those of the sugarcane-inhabiting species, Xanthomonas sacchari. Four of six Xvv isolates harboured sequences similar to the Xac plasmid, pXAC47, and showed a distinct Type-IV pilus (T4P) sequence type, whereas the T4P locus of the other two isolates resembled that of the closely related banana pathogen, Xanthomonas campestris pathovar musacearum (Xcm). The Xvv isolate from maize has lost a gene encoding a homologue of the virulence effector, xopAF, which was present in all five of the sugarcane isolates, while xopL contained a premature stop codon in four out of six isolates. These findings shed new light on evolutionary events since the divergence of Xvv and Xcm, as well as further elucidating the relationships between the two closely related pathogens.

Genome-wide sequencing reveals two major sub-lineages in the genetically monomorphic pathogen xanthomonas campestris pathovar musacearum.

The bacterium Xanthomonas campestris pathovar musacearum (Xcm) is the causal agent of banana Xanthomonas wilt (BXW). This disease has devastated economies based on banana and plantain crops (Musa species) in East Africa. Here we use genome-wide sequencing to discover a set of single-nucleotide polymorphisms (SNPs) among East African isolates of Xcm. These SNPs have potential as molecular markers for phylogeographic studies of the epidemiology and spread of the pathogen. Our analysis reveals two major sub-lineages of the pathogen, suggesting that the current outbreaks of BXW on Musa species in the region may have more than one introductory event, perhaps from Ethiopia. Also, based on comparisons of genome-wide sequence data from multiple isolates of Xcm and multiple strains of X. vasicola pathovar vasculorum, we identify genes specific to Xcm that could be used to specifically detect Xcm by PCR-based methods.