Cassava Bacterial Blight Research Articles

Early Detection of Cassava Vascular Bacteriosis (Xanthomonas Axonopodis pv. Manihotis) by Visible Near-infrared Spectroscopy Before the Appearance of Critical Symptoms

Cassava is a crucial crop for food and nutritional security. It is an important source of carbohydrates in African countries. Cassava crops can sometimes be infected by leaf diseases, affecting overall production and reducing farmers' incomes. Early detection is therefore essential to reduce the impact of these diseases. Light reflections from leaves are known to convey information about plant health. In this study, healthy and inoculated cassava were detected from the reflections obtained from the leaves using a USB 4000 spectrometer with a spectral range of (350-1050) nm. Artificial neural networks (ANNs) and nearest neighbours (KNNs) were used with principal components and vegetation indices as input for classification. Various performance metrics, i.e. Precision, Recall, Accuracy and F1_score for KNN, Accuracy, RMSE and R2 for ANN were calculated to evaluate the different established models. Using the principal components (PC1, PC2) we obtained the best models with the ANNs with the following metrics (R2=0.9678; RMSE=0.0146; Accuracy =98.30%) for the variety IM 84 and (R2=0.9291; RMSE=0.0354 Accuracy=91.30%) for the Yacé variety. With vegetation indices, the best performing models were obtained with KNN whose input parameters are RatiodRE_703 ((Accuracy =92.50% and F1-score=0.9449) and fWBI ((Accuracy =90% and F1-score=0.9326) for Yacé and vog 3 (Accuracy=100% and F1-score=1), DD (Accuracy=100% and F1_score=1) and dNIRmin920_980 (Accuracy=95% and F1_score=0.9373) for the IM 84. Overall, the results showed that leaf spectral reflectance can be used successfully for the early detection of cassava bacterial blight.

Editing of the MeSWEET10a promoter yields bacterial blight resistance in cassava cultivar SC8.

Cassava starch is a widely used raw material for industrial production and food source for people. However, cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) results in severe yield losses and is the most destructive bacterial disease in all worldwide cassava-growing regions. Xam11 is a highly pathogenic subspecies from China that infects the Chinese local cassava South China No. 8 (SC8) cultivar with marked symptoms. This study showed that the transcription activator-like effector TALE20Xam11 of Xam11 strain regulates the expression of disease-susceptibility gene MeSWEET10a by binding to the EBETALE20 region of the MeSWEET10a promoter in cassava cultivar SC8. CRISPR/Cas9-generated mutations of the EBETALE20 region resulted in a significant reduction in MeSWEET10a expression after infection by Xam11, correlating with reduced disease symptoms, smaller lesion sizes and decreased bacterial proliferation compared with the wild type. Importantly, the edited plants maintained normal growth, development and yield characteristics under greenhouse conditions. The results lay a research foundation for breeding resistant cassava cultivar SC8 to bacterial blight.

Expression Pattern and Functional Analysis of MebHLH149 Gene in Response to Cassava Bacterial Blight.

The significant reduction in cassava (Manihot esculenta Crantz) yields attributed to cassava bacterial blight (CBB) constitutes an urgent matter demanding prompt attention. The current study centered on the MebHLH149 transcription factor, which is acknowledged to be reactive to CBB and exhibits augmented expression levels, as indicated by laboratory transcriptome data. Our exploration, encompassing Xanthomonas phaseoli pv. manihotis strain CHN01 (Xpm CHN01) and hormone stress, disclosed that the MebHLH149 gene interacts with the pathogen at the early stage of infection. Furthermore, the MebHLH149 gene has been discovered to be responsive to the plant hormones abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA), intimating a potential role in the signaling pathways mediated by these hormones. An analysis of the protein's subcellular localization suggested that MebHLH149 is predominantly located within the nucleus. Through virus-induced gene silencing (VIGS) in cassava, we discovered that MebHLH149-silenced plants manifested higher disease susceptibility, less ROS accumulation, and significantly larger leaf spot areas compared to control plants. The proteins MePRE5 and MePRE6, which are predicted to interact with MebHLH149, demonstrated complementary downregulation and upregulation patterns in response to silencing and overexpression of the MebHLH149 gene. This implies a potential interaction between MebHLH149 and these proteins. Both MePRE5 and MePRE6 genes are involved in the initial immune response to CBB. Notably, MebHLH149 was identified as a protein that physically interacts with MePRE5 and MePRE6. Based on these findings, it is hypothesized that the MebHLH149 gene likely functions as a positive regulator in the defense mechanisms of cassava against CBB.

Analysis of inbreeding depression in five S1 Cassava families of elite varieties at the seedling nursery and clonal evaluation trial stages.

Background Cassava is an outcrossing, highly heterozygous plant that is reported to suffer from inbreeding depression. However, unraveling recessive traits and exploring additive genes would require a limited level of inbreeding for the genetic improvement of cassava. Method The impact of inbreeding depression (ID) on agronomic and biotic tolerance of cassava was evaluated using the S1 progenies of five African cassava varieties (TMS 30572, TME 419, TMS 98/0505, TMS 01/1371, and TMS 98/0002) at the seedling and clonal evaluation stages. Results At both trial stages, the effects of ID were severe on average performance of fresh root yield and fresh foliage yield; moderate on harvest index, dry matter content, vigor, and tolerance to cassava mosaic disease, cassava bacterial blight, and cassava anthracnose diseases; and less severe on plant height. Further examination of S1 families using molecular markers showed varying levels of heterozygosity at several loci across the genome. In addition, the degree of heterozygosity and ID varied by the S1 family. The TMS 01/1371 family showed the lowest degree of heterozygosity and ID on the average performance of different agronomic attributes, indicating that inbreeding may be strategically explored in this family to increase genetic gain and identify useful recessive traits. Lastly, the observed depression from inbreeding was higher in seedling evaluation than in clonal evaluation trials for fresh root yield, fresh foliage yield, harvest index, and dry matter content. Conclusion S1 individuals showing relatively low heterozygosity based on SNP data were selected as parents to advance genetic gain in cassava. Generally, reduced heterozygosity was prominent in traits with severe ID impacts on average phenotypic performance. Our results highlight the relative importance of exploring non-additive genetic effects and transgressive segregations for favorable allele combinations in cassava improvement.

Molecular characterization of cassava zinc finger-homeodomain (ZF-HD) transcription factors reveals their role in disease resistance

The production of cassava (Manihot esculenta Crantz) is constantly threatened by cassava bacterial blight (CBB), caused by Xanthomonas phaseoli pv. manihotis (Xpm). Zinc finger-homeodomain (ZF-HD) belongs to a family of homozygous heterotypic cassette genes widely implicated in various developmental and physiological processes in plants. Despite their importance, a comprehensive analysis of ZF-HD genes, particularly those involved in disease resistance, has not been performed for cassava. In the present study, we utilized bioinformatics methods to identify 21 ZF-HD genes distributed across 11 chromosomes of cassava genome, with the majority exhibiting gene structure without introns. Phylogenetic analysis categorized these genes into two major groups (MIF and ZHD) with five subgroups. We observed fourteen pairs of duplicated genes, suggesting that segmental duplication has likely facilitated the expansion of the cassava ZF-HD gene family. Comparative orthologous analyses between cassava and other plant species shed light on the evolutionary trajectory of this gene family. Promoter analyses revealed multiple hormone- and stress-related elements, indicative of a functional role in stress responses. Expression profiling through RNA-seq and quantitative real-time PCR (qRT-PCR) demonstrated that certain cassava ZF-HD genes are up-regulated in response to Xpm infection, suggesting their involvement in defense mechanisms. Notably, MeZHD7 gene was identified via virus induced gene silencing (VIGS) as potentially crucial in conferring resistance against CBB. Results from subcellular localization experiments indicated that MeZHD7 was localized in the nucleus. The Luciferase reporter assay demonstrated an interaction between MeZHD7 and MeMIF5. These findings may lay the foundation for further cloning and functional analyses of cassava ZF-HD genes, particularly those associated with pathogen resistance.

KIN10-mediated HB16 protein phosphorylation and self-association improve cassava disease resistance by transcriptional activation of lignin biosynthesis genes.

Cassava bacterial blight significantly affects cassava yield worldwide, while major cassava cultivars are susceptible to this disease. Therefore, it is crucial to identify cassava disease resistance gene networks and defence molecules for the genetic improvement of cassava cultivars. In this study, we found that MeHB16 transcription factor as a differentially expressed gene in cassava cultivars with contrasting disease resistance, positively modulated disease resistance by modulating defence molecule lignin accumulation. Further investigation showed that MeHB16 physically interacted with itself via the leucine-Zippe domain (L-Zip), which was necessary for the transcriptional activation of downstream lignin biosynthesis genes. In addition, protein kinase MeKIN10 directly interacted with MeHB16 to promote its phosphorylation at Ser6, which in turn enhanced MeHB16 self-association and downstream lignin biosynthesis. In summary, this study revealed the molecular network of MeKIN10-mediated MeHB16 protein phosphorylation improved cassava bacterial blight resistance by fine-tuning lignin biosynthesis and provides candidate genes and the defence molecule for improving cassava disease resistance.

First report of Cassava Bacterial Blight caused by Xanthomonas phaseoli pv. manihotis in the Amazonian forest of Ecuador.

Xanthomonas phaseoli pv. manihotis (Xpm) is a plant pathogenic bacterium known as the causal agent of cassava bacterial blight (CBB). CBB is the most limiting bacterial disease affecting cassava (Manihot esculenta Crantz), characterized by diverse symptoms including angular water-soaked leaf lesions, blight, wilting, stem exudates, stem cankers and dieback. CBB has been reported in most cassava-growing regions around the world, and, under conducive conditions, crop yield losses can reach up to 100% (Zárate-Chaves et al. 2021). While Xpm genetic diversity is remarkably high in South America (Bart et al. 2012) and cassava originates and was domesticated in the Amazon basin (Allem 2002), reports of CBB in the Amazonian region are missing. To fill this gap, in October 2018 we surveyed for CBB symptoms in cassava fields of the Orellana Province, located in the Amazon forest of the Republic of Ecuador. Adult cassava plants exhibiting typical angular, water-soaked leaf lesions were found in polyculture plots, i.e. intercrops of cassava with other species such as plantains and fruit trees (a.k.a. chakras). After surface disinfection with 5% sodium hypochlorite followed by 70% ethanol, white Xpm-like colonies were isolated from diseased leaf tissues of four plants on YPGA medium (yeast extract, 5 g/l; peptone, 5 g/l; glucose, 5 g/l; agar-agar, 15 g/l) supplemented with cephalexin (40 mg/l) and cycloheximide (50 mg/l). Pathogenicity tests were performed on peat-potted, 2-month-old cassava plants of the cultivar 60444. Bacterial suspensions were adjusted to an OD600 of 0.2 (2 × 108 CFU/ml) in sterile 10-mM MgCl2 and syringe infiltrated in fully-expanded leaves. In parallel, 20 µl of each bacterial suspension adjusted to an OD600 of 0.02 (2 × 107 CFU/ml) were inoculated on stems inside a hole previously punched with a sterile needle in the junction of the third-top petiole. Sterile 10-mM MgCl2 was used for mock inoculations in both leaves and stems, and experiments were replicated in three plants. Plants were incubated in a greenhouse at 28 ± 1°C with a 12-h photoperiod. Infiltrated leaves developed watersoaking 3 days post inoculation, while wilted leaves, stem exudates, and dieback were observed 21 days after stem inoculation. Control plants remained symptomless. White Xpm-like colonies were re-isolated from symptomatic leaves (Fig S1). One colony of each of the four Xpm isolates (before and after re-isolation) was assessed using diagnostic PCRs (Bernal-Galeano et al. 2018; Flores et al. 2019), using strain Xam668 as positive control. All four candidates were positive for both diagnostic tools. The sequences of the housekeeping genes atpD, dnaK, efp, glnA, gyrB and rpoD of our isolates were extracted from full genome sequences obtained through Oxford Nanopore Technologies (ONT) (GenBank OR288194 to OR288217) and compared to their homologs in four close Xanthomonas species and a reference Xpm strain (Table S1). The sequences of the tested strains aligned with that of Xpm CIO151 (GCA_004025275.1) (Arrieta-Ortiz et al. 2013) with nucleotide identity above 99.92% (Fig S2). The four strains were named CIX4169, CIX4170, CIX4171 and CIX4172, stored in the IRD Collection of Xanthomonas, where they are available upon request. To our knowledge, this is the first report of CBB in the Amazonian region and in Ecuador, where cassava is a central element for local culture and economy. Further surveys will be necessary to evaluate the distribution and prevalence of CBB in other ecozones of Ecuador where cassava is cultivated.

CRISPR/Cas9-generated mutations in a sugar transporter gene reduce cassava susceptibility to bacterial blight.

Bacteria from the genus Xanthomonas are prolific phytopathogens that elicit disease in over 400 plant species. Xanthomonads carry a repertoire of specialized proteins called transcription activator-like (TAL) effectors that promote disease and pathogen virulence by inducing the expression of host susceptibility (S) genes. Xanthomonas phaseoli pv. manihotis (Xpm) causes bacterial blight on the staple food crop cassava (Manihot esculenta Crantz). The Xpm effector TAL20 induces ectopic expression of the S gene Manihot esculenta Sugars Will Eventually be Exported Transporter 10a (MeSWEET10a), which encodes a sugar transporter that contributes to cassava bacterial blight (CBB) susceptibility. We used CRISPR/Cas9 to generate multiple cassava lines with edits to the MeSWEET10a TAL20 effector binding site and/or coding sequence. In several of the regenerated lines, MeSWEET10a expression was no longer induced by Xpm, and in these cases, we observed reduced CBB disease symptoms post Xpm infection. Because MeSWEET10a is expressed in cassava flowers, we further characterized the reproductive capability of the MeSWEET10a promoter and coding sequence mutants. Lines were crossed to themselves and to wild-type plants. The results indicated that expression of MeSWEET10a in female, but not male, flowers is critical to produce viable F1 seed. In the case of promoter mutations that left the coding sequence intact, viable F1 progeny were recovered. Taken together, these results demonstrate that blocking MeSWEET10a induction is a viable strategy for decreasing cassava susceptibility to CBB and that ideal lines will contain promoter mutations that block TAL effector binding while leaving endogenous expression of MeSWEET10a unaltered.

Interactions between the nitrate reductase 2 and catalase 1 fine-tune disease resistance in cassava.

Cassava is one of the most important tuber crops that is used for food, starch and bio-energy. However, cassava is susceptible to a number of diseases, especially cassava bacterial blight (CBB). Nitric oxide (NO) and hydrogen peroxide (H2O2) regulate plant growth and development, as well as stress responses. However, no direct relationships between the enzymes involved in the metabolic enzymes that produce and process these key signaling molecules has been demonstrated. Here, we provide evidence for the interaction between the nitrate reductase 2 (MeNR2) and catalase 1 (MeCAT1) proteins in vitro and in vivo, using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays, respectively. MeNR2 is a positive regulator and MeCAT1 is a negative regulator of CBB resistance. MeNR2 was localized in the nucleus, cell membrane and peroxisome, while MeCAT1 was localized in the peroxisomes. The interactions between MeNR2 and MeCAT1 also had effects of their respective enzyme activities. Taken together, the data presented here suggested that there is coordination between H2O2 and NO signaling in cassava disease resistance, through the interactions between MeCAT1 and MeNR2.

NF-YC15 transcription factor activates ethylene biosynthesis and improves cassava disease resistance.

The nuclear factor Y (NF-Y) transcription factors play important roles in plant development and physiological responses. However, the relationship between NF-Y, plant hormone and plant stress resistance in tropical crops remains unclear. In this study, we identified MeNF-YC15 gene in the NF-Y family that significantly responded to Xanthomonas axonopodis pv. manihotis (Xam) treatment. Using MeNF-YC15-silenced and -overexpressed cassava plants, we elucidated that MeNF-YC15 positively regulated disease resistance to cassava bacterial blight (CBB). Notably, we illustrated MeNF-YC15 downstream genes and revealed the direct genetic relationship between MeNF-YC15 and 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (MeACO1)-ethylene module in disease resistance, as evidenced by the rescued disease susceptibility of MeNF-YC15 silenced cassava plants with ethylene treatment or overexpressing MeACO1. In addition, the physical interaction between 2C-type protein phosphatase 1 (MePP2C1) and MeNF-YC15 inhibited the transcriptional activation of MeACO1 by MeNF-YC15. In summary, MePP2C1-MeNF-YC15 interaction modulates ethylene biosynthesis and cassava disease resistance, providing gene network for cassava genetic improvement.

Effect of farmers’ knowledge and production practices on the intensity of cassava bacterial blight in Western Kenya

The study was conducted to determine the association between farmers’ knowledge and production practices on the intensity of cassava bacterial blight (CBB) in Western Kenya. Multistage sampling was used to select 193 farms in Nambale and Teso south sub-counties in Western Kenya. A semi-structured questionnaire was administered to obtain information on farmer’s knowledge and cassava production practices. Within each farm, bacterial blight incidence was determined and 30 cassava plants were evaluated for disease severity. Symptomatic leaf samples were collected for isolation and confirmation of the CBB pathogens. GPS coordinates of each farm were taken for the development of disease distribution maps. Data analysis included descriptive statistics and the Chi-square test which was used to determine the association between sociodemographic traits and disease incidence. CBB was prevalent in both sub-counties and Xanthomonas phaseoli pv manihotis was the more widespread compared to Xanthomonas axonopodis pv cassavae. However, there was no association between CBB incidence, training, seed source, and intercropping suggesting that other factors contributed to the high prevalence of the disease. About 85% of the farmers interviewed were unaware of the disease suggesting that the farmers could be spreading the disease unknowingly through the use of self-recycled and neighbor-obtained cassava cuttings. The findings of this study will contribute toward measures aimed at curbing the disease and its spread.

Superoxide dismutase gene family in cassava revealed their involvement in environmental stress via genome-wide analysis

Superoxide dismutase (SOD) is a crucial metal-containing enzyme that plays a vital role in catalyzing the dismutation of superoxide anions, converting them into molecular oxygen and hydrogen peroxide, essential for enhancing plant stress tolerance. We identified 8 SOD genes (4 CSODs, 2 FSODs, and 2 MSODs) in cassava. Bioinformatics analyses provided insights into chromosomal location, phylogenetic relationships, gene structure, conserved motifs, and gene ontology annotations. MeSOD genes were classified into two groups through phylogenetic analysis, revealing evolutionary connections. Promoters of these genes harbored stress-related cis-elements. Duplication analysis indicated the functional significance of MeCSOD2/MeCSOD4 and MeMSOD1/MeMSOD2. Through qRT-PCR, MeCSOD2 responded to salt stress, MeMSOD2 to drought, and cassava bacterial blight. Silencing MeMSOD2 increased XpmCHN11 virulence, indicating MeMSOD2 is essential for cassava's defense against XpmCHN11 infection. These findings enhance our understanding of the SOD gene family's role in cassava and contribute to strategies for stress tolerance improvement.

Isolation and Identification of Endophytic Bacteria Bacillus sp. ME9 That Exhibits Biocontrol Activity against Xanthomonas phaseoli pv. manihotis.

In recent years, the bacterial blight of cassava has caused substantial economic losses to the Chinese cassava industry. Chemical control methods have become the primary approach to control this disease; however, their widespread usage and harmful residues have raised concerns about environmental pollution. In order to avoid this, it is urgent to seek a green ecological method to prevent and control it. Biological control through the utilization of microorganisms not only effectively inhibits the disease, but also gives consideration to environmental friendliness. Therefore, investigating an endophytic biological control method for cassava bacterial blight is of great importance. In this study, cassava leaf tissues were used as test specimens in order to isolate endophytic bacteria by using dilution and separation methods. Bacillus ME9, derived from cassava endophytic bacteria, exhibits good antagonism against a diverse range of pathogens, including Xpm11. Its genome consists of a series of genes encoding antibacterial lipopeptides, which may be directly related to its antibacterial capabilities. Furthermore, inoculation resulted in a substantial change in the diversity of the endophytic bacterial community, characterized by improved diversity, and displayed an obvious inhibition of pathogenic bacterial growth, demonstrating successful colonization within plants. The results laid a foundation and provided theoretical support for the development and utilization of cassava endophytic bacterial diversity and endogenous disease control strategies.

Evaluation of Weeds as Alternative Hosts of Xanthomonas phaseoli pv. manihotis, the Causal Agent of Cassava Bacterial Blight in Burkina Faso

Cassava bacterial blight (CBB), caused by Xanthomonas phaseoli pv . manihotis ( Xpm), is one of the most important diseases threatening cassava and has recently been reported in Burkina Faso. Better management of CBB calls for the combined use of genetically resistant varieties and appropriate cultural practices. The objectives of this study were to evaluate the potential role of weed plants present in cassava fields as inoculum reservoirs for CBB. An inventory of weeds growing in eight cassava fields at four locations in Burkina Faso, and the cultivation of soil samples from the same eight fields, indicated the existence of 27 different species, reaching 28.32 plants per square meter. Field characterization led to the identification of seven species of weeds displaying symptoms reminiscent of Xpm infection, but no Xpm could be isolated from or detected in those plants. Artificial inoculations with Xpm resulted in 21 species producing symptoms of necrosis around the inoculation points. The presence of Xpm in the leaves of all these weed species was confirmed by re-isolation of the pathogen 2 weeks after inoculation. Thus, weeds that can harbor Xpm were prevalent in the cassava fields we studied, but their role as potential sources of Xpm inoculum for CBB epidemics remains to be demonstrated. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Multilocus Sequence Analysis and Detection of Copper Ion Resistance of Xanthomonas phaseoli pv. manihotis Causing Bacterial Blight in Cassava.

Cassava (Manihot esculenta Crantz) is an important tropical tuber crop around the world. Cassava bacterial blight, caused by Xanthomonas phaseoli pv. manihotis, is a key disease that influences cassava production worldwide. Between 2008 and 2020, 50 X. phaseoli pv. manihotis strains were isolated from diseased plant samples or acquired from China, Uganda, Cambodia, Colombia, Malaysia, and Micronesia. Using multilocus sequence analysis, the genetic diversity of X. phaseoli pv. manihotis strains was evaluated. A neighbor-joining phylogenetic dendrogram was constructed based on partial sequences of five housekeeping genes (atpD-dnaK-gyrB-efp-rpoD). The strains clustered into three groups whose clusters were consistent with atpD and RpoD gene sequences. Group I contained 46 strains from China, Uganda, Cambodia, and Micronesia, and the other two groups were comprised of strains from Colombia and Malaysia, respectively. The resistance of all these strains to copper ion (Cu2+) was determined, the minimal inhibitory concentration was between 1.3 and 1.7 mM, and there was no significant difference between strains from different geographic region. During genome annotation of the X. phaseoli pv. manihotis strain CHN01, homologous gene clusters of copLAB and xmeRSA were identified. The predicted amino acid sequences of two gene clusters were highly homologous with the copper-resistant protein from Xanthomonas strains. CopLAB and xmeRSA were amplified from all these strains, suggesting that the regulation of copper resistance is associated with two distinct metabolic pathways. CopLAB and xmeRSA were highly conserved among strains from different geographic regions, possibly associated with other conserved function.

The self-association of cytoplasmic malate dehydrogenase 1 promotes malate biosynthesis and confers disease resistance in cassava

Malate dehydrogenase (MDH) as an essential metabolic enzyme is widely involved in plant developmental processes. However, the direct relationship between its structural basis and in vivo roles especially in plant immunity remains elusive. In this study, we found that cytoplasmic cassava (Manihot esculenta, Me) MDH1 was essential for plant disease resistance against cassava bacterial blight (CBB). Further investigation revealed that MeMDH1 positively modulated cassava disease resistance, accompanying the regulation of salicylic acid (SA) accumulation and pathogensis-related protein 1 (MePR1) expression. Notably, the metabolic product of MeMDH1 (malate) also improved disease resistance in cassava, and its application rescued the disease susceptibility and decreased immune responses of MeMDH1-silenced plants, indicating that malate was responsible for MeMDH1-mediated disease resistance. Interestingly, MeMDH1 relied on Cys330 residues to form homodimer, which was directly related with MeMDH1 enzyme activity and the corresponding malate biosynthesis. The crucial role of Cys330 residue in MeMDH1 was further confirmed by in vivo functional comparison between overexpression of MeMDH1 and MeMDH1C330A in cassava disease resistance. Taken together, this study highlights that MeMDH1 confers improved plant disease resistance through protein self-association to promote malate biosynthesis, extending the knowledge of the relationship between its structure and cassava disease resistance.

A minisatellite-based MLVA for deciphering the global epidemiology of the bacterial cassava pathogen Xanthomonas phaseoli pv. manihotis.

Cassava Bacterial Blight (CBB) is a destructive disease widely distributed in the different areas where this crop is grown. Populations studies have been performed at local and national scales revealing a geographical genetic structure with temporal variations. A global epidemiology analysis of its causal agent Xanthomonas phaseoli pv. manihotis (Xpm) is needed to better understand the expansion of the disease for improving the monitoring of CBB. We targeted new tandem repeat (TR) loci with large repeat units, i.e. minisatellites, that we multiplexed in a scheme of Multi-Locus Variable number of TR Analysis (MLVA-8). This genotyping scheme separated 31 multilocus haplotypes in three clusters of single-locus variants and a singleton within a worldwide collection of 93 Xpm strains isolated over a period of fifty years. The major MLVA-8 cluster 1 grouped strains originating from all countries, except the unique Chinese strain. On the contrary, all the Xpm strains genotyped using the previously developed MLVA-14 microsatellite scheme were separated as unique haplotypes. We further propose an MLVA-12 scheme which takes advantage of combining TR loci with different mutation rates: the eight minisatellites and four faster evolving microsatellite markers, for global epidemiological surveillance. This MLVA-12 scheme identified 78 haplotypes and separated most of the strains in groups of double-locus variants (DLV) supporting some phylogenetic relationships. DLV groups were subdivided into closely related clusters of strains most often sharing the same geographical origin and isolated over a short period, supporting epidemiological relationships. The main MLVA-12 DLV group#1 was composed by strains from South America and all the African strains. The MLVA-12 scheme combining both minisatellite and microsatellite loci with different discriminatory power is expected to increase the accuracy of the phylogenetic signal and to minimize the homoplasy effects. Further investigation of the global epidemiology of Xpm will be helpful for a better control of CBB worldwide.

Genome-wide analyses of LATERAL ORGAN BOUNDARIES in cassava reveal the role of LBD47 in defence against bacterial blight.

The Arabidopsis thaliana ASYMMETRIC LEAVES2 (AS2) gene is responsible for the development of flat, symmetric, and extended leaf laminae and their veins. The AS2 gene belongs to the plant-specific AS2-LIKE/LATERAL ORGAN BOUNDARIES (LOB)-domain (ASL/LBD), which consists of 42 proteins in Arabidopsis with a conserved amino-terminal domain known as the AS2/LOB domain, and a variable carboxyl-terminal region. AS2/LOB domain consists of an amino-terminal (N-terminal) that contains a cysteine repeat (the C-motif), a conserved glycine residue, and a leucine-zipper-like. AS2/LOB domain has been characterised in plants such as A. thaliana, Zea mays, and Oryza sativum. Nevertheless, it remains uncharacterised in cassava (Manihot esculenta). Characterisation and identification of cassava ASL/LBD genes using the computational algorithms, hidden Markov model profiles (PF03195), determined 55 ASL/LBD genes (MeASLBD1 to MeASLBD55). The gene structure and motif composition were conserved in MeASLBDs, while the expression profiles of these genes were highly diverse, implying that they are associated with diverse functions. Weighted gene co-expression network analysis (WGCNA) of target genes and promoter analysis suggest that these MeASLBDs may be involved in hormone and stress responses. Furthermore, the analysis of cis-regulatory elements in promoter regions suggested that MeASLBDs may be involved in the plant phytohormone signal response. The transcriptome data of cassava under biotic and abiotic stresses revealed that MeASLBD46 and MeASLBD47 greatly respond to disease and drought. The MeASLBD47 gene was selected for functional analysis. The result indicated that MeASLBD47 significantly mitigated the virulence of cassava bacterial blight (XamCHN11) through Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and Virus-induced gene silencing (VIGS). These findings provided a comprehensive analysis of ASL/LBD genes and laid the groundwork for future research to understand ASL/LBD genes.

Histone deacetylase 9 regulates disease resistance through fine-tuning histone deacetylation of melatonin biosynthetic genes and melatonin accumulation in cassava.

Melatonin participates in plant growth and developmentand bioticand abiotic stress responses. Histone acetylation regulates many plant biological processes via transcriptional reprogramming. However, the direct relationship between melatonin and histone acetylation in plant disease resistance remains unclear. In this study, we identified cassava bacterial blight (CBB) responsive histone deacetylase 9 (HDA9), which negatively regulated disease resistance to CBB by reducing melatonin content. In addition, exogenous melatonin alleviated disease sensitivity of MeHDA9 overexpressed plants to CBB. Importantly, MeHDA9 inhibited the expression of melatonin biosynthetic genes through decreasing lysine 5 of histone 4 (H4K5) acetylation at the promoter regions of melatonin biosynthetic genes, thereby modulating melatonin accumulation in cassava. Furthermore, protein phosphatase 2C 12 (MePP2C12) interacted with MeHDA9 in vivo and in vitro, and it was involved in MeHDA9-mediated disease resistance via melatonin biosynthetic pathway. In summary, this study highlights the direct interaction between histone deacetylation and melatonin biosynthetic genes in cassava disease resistance via histone deacetylation, providing new insights into the genetic improvement of disease resistance via epigenetic regulation of melatonin level in tropical crops.

Improving cassava bacterial blight resistance by editing the epigenome

Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas phaseoli pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. In this work, we direct methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display decreased CBB symptoms while maintaining normal growth and development. This work therefore presents an epigenome editing approach useful for crop improvement.