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.

Tuber pathogenicity of Macrophomina phaseolina strain 3 a isolated from rotten cassava tuber from farm lands in Osogbo, Osun State, Nigeria, its virulence genes and ADMET properties.

BACKGROUND: Macrophomina phaseolina is a pathogen that causes an opportunistic disease that spreads by soil and seeds and affects more than 500 different plant species, like fruits, trees, and row crops. Mycotoxins, such as phaseolinic acid, and phaseolinone, are produced by M. phaseolina isolates in previous investigations; however, the production of these mycotoxins seems to vary depending on the host and the region. METHODS AND RESULTS: In this study, Macrophomina phaseolina strain 3 A was isolated from rotten cassava tuber and identified using the analysis of the sequences of the internal transcribed spacer region. The isolate was inoculated on a fresh healthy cassava tuber at 25 °C and tuber-rotting potential was monitored for 4 weeks. Virulence genes MPH_06603, MPH_06955, and MPH_01521 were determined with designed primers, and secondary metabolites were characterized by FTIR and GCMS. The rotten tuber effect was observed from the 2nd week of the experiment with severe tuber rot and weight reduction. The PCR showed the presence of MPH_06603 virulence gene. The GCMS showed N-Methylpivalamide (115.0 m/z), Butane, 1,4-dimethoxy- (119.0 m/z), and 5-Hydroxymethylfurfural (126.0 m/z) were the predominant metabolites produced by the pathogen. The compounds in the metabolites inhibit CYP3A4 enzymes, cause eye irritation, and Human Ether-a-go-go-related gene inhibition. CONCLUSION: This study revealed that M. phaseolina was responsible for the cassava tuber rot which leads to a lower yield of farm produce. The metabolites produced are toxic and unsafe for human consumption. It is suggested that farmers should destroy any cassava affected by this pathogen to prevent its toxic effects on humans and animals.

Isolation, genome analysis and tissue localization of Ceratobasidium theobromae, a new encounter pathogen of cassava in Southeast Asia.

In Southeast Asia (SEA) fastidious fungi of the Ceratobasidium genus are associated with proliferation of sprouts and vascular necrosis in cacao and cassava, crops that were introduced from the tropical Americas to this region. Here, we report the isolation and in vitro culture of a Ceratobasidium sp. isolated from cassava with symptoms of witches' broom disease (CWBD), a devastating disease of this crop in SEA. The genome characterization using a hybrid assembly strategy identifies the fungus as an isolate of the species C. theobromae, the causal agent of vascular streak dieback of cacao in SEA. Both fungi have a genome size > 31 Mb (G+C content 49%), share > 98% nucleotide identity of the Internal Transcribed Spacer (ITS) and > 94% in genes used for species-level identification. Using RNAscope® we traced the pathogen and confirmed its irregular distribution in the xylem and epidermis along the cassava stem, which explains the obtention of healthy planting material from symptom-free parts of a diseased plant. These results are essential for understanding the epidemiology of CWBD, as a basis for disease management including measures to prevent further spread and minimize the risk of introducing C. theobromae via long-distance movement of cassava materials to Africa and the Americas.

Influence of Drought Stress on the Rhizosphere Bacterial Community Structure of Cassava (Manihot esculenta Crantz).

Drought presents a significant abiotic stress that threatens crop productivity worldwide. Rhizosphere bacteria play pivotal roles in modulating plant growth and resilience to environmental stresses. Despite this, the extent to which rhizosphere bacteria are instrumental in plant responses to drought, and whether distinct cassava (Manihot esculenta Crantz) varieties harbor specific rhizosphere bacterial assemblages, remains unclear. In this study, we measured the growth and physiological characteristics, as well as the physical and chemical properties of the rhizosphere soil of drought-tolerant (SC124) and drought-sensitive (SC8) cassava varieties under conditions of both well-watered and drought stress. Employing 16S rDNA high-throughput sequencing, we analyzed the composition and dynamics of the rhizosphere bacterial community. Under drought stress, biomass, plant height, stem diameter, quantum efficiency of photosystem II (Fv/Fm), and soluble sugar of cassava decreased for both SC8 and SC124. The two varieties' rhizosphere bacterial communities' overall taxonomic structure was highly similar, but there were slight differences in relative abundance. SC124 mainly relied on Gamma-proteobacteria and Acidobacteriae in response to drought stress, and the abundance of this class was positively correlated with soil acid phosphatase. SC8 mainly relied on Actinobacteria in response to drought stress, and the abundance of this class was positively correlated with soil urease and soil saccharase. Overall, this study confirmed the key role of drought-induced rhizosphere bacteria in improving the adaptation of cassava to drought stress and clarified that this process is significantly related to variety.

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.

Bio-synthesis and characterization of silver nanoparticles from Trichoderma species against cassava root rot disease.

Cassava root rot disease caused by the fungal pathogens Fusarium solani and Lasiodiplodia theobromae produces severe damages on cassava production. This research was conducted to produce and assess silver nanoparticles (AgNPs) synthesized by Trichoderma harzianum for reducing root rot disease. The results revealed that using the supernatants of T. harzianum on a silver nitrate solution changed it to reddish color at 48 h, indicating the formation of AgNPs. Further characterization was identified using dynamic light scattering (DLS) and scanning electron microscope (SEM). DLS supported that the Z-average size is at 39.79 nm and the mean zeta potential is at - 36.5 mV. SEM revealed the formation of monodispersed spherical shape with a diameter between 60-75 nm. The antibacterial action of AgNPs as an antifungal agent was demonstrated by an observed decrease in the size of the fungal colonies using an increasing concentration of AgNPs until the complete inhibition growth of L. theobromae and F. solani at > 58 µg mL-1 and at ≥ 50 µg mL-1, respectively. At in vitro conditions, the applied AgNPs caused a decrease in the percentage of healthy aerial hyphae of L. theobromae (32.5%) and of F. solani (70.0%) compared to control (100%). The SR-FTIR spectra showed the highest peaks in the first region (3000-2800 cm-1) associated with lipids and fatty acids located at 2962, 2927, and 2854 cm-1 in the AgNPs treated samples. The second region (1700-1450 cm-1) consisting of proteins and peptides revealed the highest peaks at 1658, 1641, and 1548 cm-1 in the AgNPs treated samples. The third region (1300-900 cm-1), which involves nucleic acid, phospholipids, polysaccharides, and carbohydrates, revealed the highest peaks at 1155, 1079, and 1027 cm-1 in the readings from the untreated samples. Finally, the observed root rot severity on cassava roots treated with AgNPs (1.75 ± 0.50) was significantly lower than the control samples (5.00 ± 0.00).

Comparative analysis of infected cassava root transcriptomics reveals candidate genes for root rot disease resistance.

Cassava root-rot incited by soil-borne pathogens is one of the major diseases that reduces root yield. Although the use of resistant cultivars is the most effective method of management, the genetic basis for root-rot resistance remains poorly understood. Therefore, our work analyzed the transcriptome of two contrasting genotypes (BRS Kiriris/resistant and BGM-1345/susceptible) using RNA-Seq to understand the molecular response and identify candidate genes for resistance. Cassava seedlings (resistant and susceptible to root-rot) were both planted in infested and sterilized soil and samples from Initial-time and Final-time periods, pooled. Two controls were used: (i) seedlings collected before planting in infested soil (absolute control) and, (ii) plants grown in sterilized soil (mock treatments). For the differentially expressed genes (DEGs) analysis 23.912 were expressed in the resistant genotype, where 10.307 were differentially expressed in the control treatment, 15 DEGs in the Initial Time-period and 366 DEGs in the Final Time-period. Eighteen candidate genes from the resistant genotype were related to plant defense, such as the MLP-like protein 31 and the peroxidase A2-like gene. This is the first model of resistance at the transcriptional level proposed for the cassava × root-rot pathosystem. Gene validation will contribute to screening for resistance of germplasm, segregating populations and/or use in gene editing in the pursuit to develop most promising cassava clones with resistance to root-rot.

CPK1-HSP90 phosphorylation and effector XopC2-HSP90 interaction underpin the antagonism during cassava defense-pathogen infection.

Cassava is one of the most important tropical crops, but it is seriously affected by cassava bacteria blight (CBB) caused by the bacterial pathogen Xanthomonas phaseoli pv manihotis (Xam). So far, how pathogen Xam infects and how host cassava defends during pathogen-host interaction remains elusive, restricting the prevention and control of CBB. Here, the illustration of HEAT SHOCK PROTEIN 90 kDa (MeHSP90.9) interacting proteins in both cassava and bacterial pathogen revealed the dual roles of MeHSP90.9 in cassava-Xam interaction. On the one hand, calmodulin-domain protein kinase 1 (MeCPK1) directly interacted with MeHSP90.9 to promote its protein phosphorylation at serine 175 residue. The protein phosphorylation of MeHSP90.9 improved the transcriptional activation of MeHSP90.9 clients (SHI-RELATED SEQUENCE 1 (MeSRS1) and MeWRKY20) to the downstream target genes (avrPphB Susceptible 3 (MePBS3) and N-aceylserotonin O-methyltransferase 2 (MeASMT2)) and immune responses. On the other hand, Xanthomonas outer protein C2 (XopC2) physically associated with MeHSP90.9 to inhibit its interaction with MeCPK1 and the corresponding protein phosphorylation by MeCPK1, so as to repress host immune responses and promote bacterial pathogen infection. In summary, these results provide new insights into genetic improvement of cassava disease resistance and extend our understanding of cassava-bacterial pathogen interaction.

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.

Multiple taxa inoculants of arbuscular mycorrhizal fungi enhanced colonization frequency, biomass production, and water use efficiency of cassava (Manihot esculenta).

Increasing water use efficiency (WUE) in crops is critical to maintaining agricultural production under climate change-exacerbated drought. One of these approaches may consist of leveraging on the beneficial interactions between crops and arbuscular mycorrhizal fungi (AMF). In this study, we investigated how inoculation with AMF from three different taxa (Claroideoglomus etunicatum (T1), Gigaspora margarita (T2), and Rhizophagus irregularis (T3)) and their combination (T123) and a non-inoculated "control" treatment in a greenhouse could achieve increased biomass production and water use efficiency in cassava under three levels of water availability (100% PC, 60%-moderate stress, and 30%-severe stress). Whereas T1 and T2 resulted in a lower growth rate for the plants than the control, T123 enhanced cassava height and the number of petioles and leaves. T123 and T3 increased the total plant dry biomass in comparison with uninoculated plants by 30% and 26%, respectively. The T123 and plants inoculated with T3 significantly increased cassava above-ground biomass by 19% as compared to T1 (8.68 ± 2.44 g) and T2 (8.68 ± 2.44 g) inoculated plants. T123 resulted in higher WUE, which was validated by the leaf carbon (δ13C) isotopic signature, significantly outperforming cassava with T1 and T2, yet there was no difference between the control and T3. Overall, this study demonstrated that the use of multiple AMF from different taxa can increase cassava growth and WUE under greenhouse conditions.

LESION SIMULATING DISEASE 3 regulates disease resistance via fine-tuning histone acetylation in cassava.

Bacterial blight seriously affects the growth and production of cassava (Manihot esculenta Crantz), but disease resistance genes and the underlying molecular mechanism remain unknown. In this study, we found that LESION SIMULATING DISEASE 3 (MeLSD3) is essential for disease resistance in cassava. MeLSD3 physically interacts with SIRTUIN 1 (MeSRT1), inhibiting MeSRT1-mediated deacetylation modification at the acetylation of histone 3 at K9 (H3K9Ac). This leads to increased H3K9Ac levels and transcriptional activation of SUPPRESSOR OF BIR1 (SOBIR1) and FLAGELLIN-SENSITIVE2 (FLS2) in pattern-triggered immunity, resulting in immune responses in cassava. When MeLSD3 was silenced, the release of MeSRT1 directly decreased H3K9Ac levels and inhibited the transcription of SOBIR1 and FLS2, leading to decreased disease resistance. Notably, DELLA protein GIBBERELLIC ACID INSENSITIVE 1 (MeGAI1) also interacted with MeLSD3, which enhanced the interaction between MeLSD3 and MeSRT1 and further strengthened the inhibition of MeSRT1-mediated deacetylation modification at H3K9Ac of defense genes. In summary, this study illustrates the mechanism by which MeLSD3 interacts with MeSRT1 and MeGAI1, thereby mediating the level of H3K9Ac and the transcription of defense genes and immune responses in cassava.

Identification of two cassava receptor-like cytoplasmic kinase genes related to disease resistance via genome-wide and functional analysis.

Receptor-like cytoplasmic kinases (RLCKs) play important roles in various developmental processes and stress responses in plants. Whereas, the detailed information of this family in cassava has not clear yet. In this study, A total of 322 MeRLCK genes were identified in the cassava genome, and they could be divided into twelve clades (Clades I-XII) according to their phylogenetic relationships. Most RLCK members in the same clade have similar characteristics and motif compositions. Over half of the RLCKs possess cis-elements in their promoters that respond to ABA, MeJA, defense reactions, and stress. Under Xpm11 infection, the expression levels of four genes show significant changes, suggesting their involvement in Xpm11 resistance. Two RLCK (MeRLCK11 and MeRLCK84) genes potentially involved in resistance to cassava bacterial blight were identified through VIGS experiments. This work laid the foundation for studying the function of the cassava RLCK genes, especially the genes related to pathogen resistance.

Aspergillus section Flavi and aflatoxins in Brazilian cassava (Manihot esculenta Crantz) and products.

Aflatoxins are carcinogenic compounds produced by some species of Aspergillus, especially those belonging to Aspergillus section Flavi. Their occurrence in food may start in the field, in the post-harvest, or during storage due to inadequate handling and storage. Because cassava is a staple food for a high percentage of the Brazilian population, we evaluated the presence of aflatoxin-producing species in cassava tubers, cassava products (cassava flour, cassava starch, sour starch, and tapioca flour), and in soil samples collected from cassava fields. In addition, the levels of aflatoxin contamination in cassava products were quantified. A total of 101 samples were analyzed, and 45 strains of Aspergillus section Flavi were isolated. Among the identified species, Aspergillus flavus, Aspergillus arachidicola, Aspergillus novoparasiticus, and Aspergillus parasiticus were found. The majority of strains (73.3%) tested for their aflatoxin-producing ability in synthetic media was positive. Despite that, cassava and cassava products were essentially free of aflatoxins, and only one sample of cassava flour contained traces of AFB1 (0.35 µg/kg).

Autophagy-related genes serve as heat shock protein 90 co-chaperones in disease resistance against cassava bacterial blight.

Heat shock protein 90 (HSP90) is involved in plant growth and various stress responses via regulating protein homeostasis. Autophagy keeps cellular homeostasis by recycling the components of cellular cytoplasmic constituents. Although they have similar effects on cellular protein homeostasis, the direct association between HSP90 and autophagy signaling remains unclear in plants, especially in tropical crops. In this study, the correlation between HSP90 and autophagy signaling was systematically analyzed by protein-protein interaction in cassava, one of the most important economy fruit in tropic. In addition, their effects on plant disease response and underlying mechanisms in cassava were investigated by functional genomics and genetic phenotype assay. The potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex interacts with MeATGs and subsequently triggers autophagy signaling, conferring improved disease resistance to cassava bacterial blight (CBB). On the contrary, HSP90 inhibitor and autophagy inhibitor decreased disease resistance against CBB in cassava, and autophagy may be involved in the potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex-mediated multiple immune responses. This study highlights the precise modulation of autophagy signaling by potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex in autophagy-mediated disease resistance to CBB.

Enterotoxin Gene Distribution and Genotypes of Bacillus cereussensu lato Isolated from Cassava Starch.

Bacillus cereus is a human pathogenic bacterium found in foods with the potential to cause emesis and diarrhea. This study estimated the presence, toxigenic and genomic diversity of B. cereus s.l. obtained from cassava starch samples collected in bakeries and powdered food companies in Medellín (Colombia). Bacillus cereuss.l. was found in 43 of 75 (57%) cassava starch samples and 98 isolates were obtained. The nheABC, hblCDAB, cytK2, entFM and cesB toxin genes were detected by multiplex PCR and the most frequent operon was nheABC, whereas cesB gene was not found. Twelve toxigenic profiles were determined by the detection of toxin genes, and the most frequent profiles harbored all enterotoxin genes. A broad genomic diversity was detected according to GTG5-PCR fingerprinting results with 76 B. cereus s.l. grouped in sixteen clusters and the 22 isolates clustering separately. No relationship was observed between genomic background and toxigenic profiles. In general, the results showed a high genomic and enterotoxigenic diversity in B. cereus s.l. found in cassava starch. These results should incentive future studies to understand the distribution of B. cereus s.l. isolated on raw materials in comparison with finished products.

Efficient biorefinery of whole cassava for citrate production using Aspergillus niger mutated by atmospheric and room temperature plasma and enhanced co-saccharification strategy.

BACKGROUND: The non-grain crop cassava has attracted intense attention in the biorefinery process. However, efficient biorefinery of whole cassava is faced with some challenges due to the existence of strain inhibition and refractory cellulose during the citrate production process. RESULTS: Here, a novel breeding method - atmospheric and room temperature plasma (ARTP) - was applied for strain improvement of citrate-producing strain Aspergillus niger from whole cassava. The citrate yield of the mutant obtained using ARTP mutagenesis increased by 36.5% in comparison with the original strain. Moreover, citric acid fermentation was further improved on the basis of an enhanced co-saccharification strategy by supplementing glucoamylase and cellulase. The fermentation efficiency increased by 35.8% with a 17.0 g L-1 reduction in residual sugar on a pilot scale. CONCLUSIONS: All these results confirmed that a combination of the novel breeding method and enhanced co-saccharification strategy could be used to efficiently refine whole cassava. The results also provide inspiration for the production of value-added products and waste disposal in agro-based industries. © 2021 Society of Chemical Industry.

Brewing rich 2-phenylethanol beer from cassava and its producing metabolisms in yeast.

BACKGROUND: Cassava is rich in nutrition and has high edible value, but the development of the cassava industry is limited by the traditional low added value processing and utilization mode. In this study, cassava tuber was used as beer adjunct to develop a complete set of fermentation technology for manufacturing cassava beer. RESULTS: The activities of transaminase, phenylpyruvate decarboxylase and dehydrogenase in 2-phenylethanol Ehrlich biosynthesis pathway of Saccharomyces cerevisiae were higher in cassava beer than that of malt beer. Aminotransferase ARO9 gene and phenylpyruvate decarboxylase ARO10 gene were up-regulated in the late stage of fermentation, which indicated that they were the main regulated genes of 2-phenylethanol Ehrlich pathway with phenylalanine as substrate in cassava beer preparation. CONCLUSIONS: Compared with traditional wheat beer, cassava beer was similar in the content of nutrition elements, diacetyl, total acid, alcohol and carbon dioxide, but has the characteristics of fresh fragrance and better taste. The hydrocyanic acid contained in cassava root tubes was catabolized during fermentation and compliant with the safety standard of beverage. Further study found that the content of 2-phenylethanol in cassava beer increased significantly, which gave cassava beer a unique elegant and delicate rose flavor. © 2020 Society of Chemical Industry.

A phylogeny for the plant pathogen Piptoporellus baudonii using a multigene data set.

Piptoporellus baudonii is proposed as a new combination for Laetiporus baudonii in the Polyporales (Basidiomycota) based on morphological and molecular features. This parasitic macrofungus attacks cashew trees, Eucalyptus, cassava, Tectona, and some indigenous trees in southern regions of Tanzania and poses a serious threat to agroforestry and livelihood conditions in the area. Phylogenetic trees were produced from partial sequences of three rDNA gene regions and a portion of translation elongation factor 1-alpha (TEF1) gene of Laetiporus baudonii for comparisons with samples from the antrodia clade. Our results reveal a strongly supported group of L. baudonii with Piptoporellus in Fomitopsidaceae. Piptoporellus baudonii shares many morphological features with other members of Piptoporellus but differs from them in having broadly ellipsoid or rarely ovoid basidiospores. Both morphological and phylogenetic evidence justify the placement of L. baudonii in Piptoporellus together with the three other known species in the genus.

In vitro propagation of three mosaic disease resistant cassava cultivars.

BACKGROUND: Cassava is a staple food for over 800 million people globally providing a cheap source of carbohydrate. However, the cultivation of cassava in the country is facing to viral diseases, particularly cassava mosaic disease (CMD) which can cause up to 95% yield losses. With aim to supply farmers demand for clean planting materials, there is need to accelerate the production of the elite cultivars by use of tissue culture in order to cope with the demand. METHODS: Nodal explants harvested from the greenhouse grown plants were sterilised using different concentrations of a commercial bleach JIK (3.85% NaOCl) and varying time intervals. Microshoots induction was evaluated using thidiazuron (TDZ), benzyl amino purine (BAP), and kinetin. Rooting was evaluated using different auxins (Naphthalene acetic acid NAA and Indole-3-butyricacid IBA). PCR-based SSR and SCAR markers were used to verify the presence of CMD2 gene in the regenerated plantlets. RESULTS: The highest level of sterility in explants (90%) was obtained when 20% Jik was used for 15 min. The best cytokinin for microshoots regeneration was found to be kinetin with optimum concentrations of 5, 10 and 20 µM for Agric-rouge, Atinwewe, and Agblehoundo respectively. Medium without growth regulators was the best for rooting the three cultivars. A survival rate of 100, 98, and 98% was recorded in the greenhouse for Agric-rouge, Atinwewe, and Agblehoundo respectively and the plantlets appeared to be morphologically normal. The SSR and SCAR analysis of micropropagated plants showed a profile similar to that of the mother plants indicating that the regenerated plantlets retained the CMD2 gene after passing through in vitro culture, as expected with micropropagation. CONCLUSION: The nodal explants was established to be 20% of Jik (3.85% NaOCl) with an exposure time of 15 min. Kinetin was proved to be the best cytokinins for microshoot formation with the optimum concentration of 5, 10 and 20 µM for Agric-rouge, Atinwewe, and Agblehoundo respectively. The protocol developed during this study will be useful for mass propagation of the elite cassava cultivars.

Enhancing of anthracnose disease resistance indicates a potential role of antimicrobial peptide genes in cassava.

Cassava (Manihot esculenta Crantz.) is an important economic crop in tropical countries. Demands for using cassava in food, feed and biofuel industries have been increasing worldwide. Cassava anthracnose disease, caused by Colletotrichum gloeosporioides f.sp. manihotis (CAD), is considered a major problem in cassava production. To minimize the effects of such disease, this study investigated the response of cassava to attack by CAD and how the plants defend themselves against this threat. Genome-wide identification of antimicrobial peptide genes (AMPs) and their expression in response to fungal infection was performed in the resistant cassava cultivar (Huay Bong 60; HB60) in comparison with the highly susceptible cultivar (Hanatee; HN). A total of 114 gene members of AMP were identified in the cassava genome database. Fifty-six gene members were selected for phylogenetic tree construction and analysis of putative cis-acting elements in their promoter regions. Differential expression profiles of six candidate genes were observed in response to CAD infection of both cassava cultivars. Upregulation of snakins, MeSN1 and MeSN2 was found in HB60, whereas MeHEL, Me-AMP-D2 and MeLTP2 were highly induced in HN. The MeLTP1 gene was not expressed in either cultivar. HB60 showed a reduced severity rating in comparison to HN after CAD infection. The biomembrane permeability test of fungal CAD was strongly affected after treatment with protein extract derived from CAD-infected HB60. Altogether, these findings suggest that snakins have a potential function in the CAD defense response in cassava. These results could be useful for cassava improvement programs to fight fungal pathogen.