The Fungus Metarhizium sp. BCC 4849 Is an Effective and Safe Mycoinsecticide for the Management of Spider Mites and Other Insect Pests.

Five isolates of Metarhizium sp. were evaluated for their pathogenicity against the spider mite (Tetranychus truncatus Ehara) (Acari: Tetranychidae) and Metarhizium sp. BCC 4849 resulted in the highest mortality (82%) on the 5th day post-inoculation (DPI). Subsequent insect bioassay data indicated similar high virulence against five other insects: African red mites (Eutetranychus africanus Tucker) (Acari: Tetranychidae), bean aphid (Aphis craccivora Koch) (Hemiptera: Aphididae), cassava mealybug (Phenacoccus manihoti Matile-Ferrero) (Hemiptera: Pseudococcidae), sweet potato weevil (Cylas formicarius Fabricius) (Coleoptera: Brentidae), and oriental fruit fly (Bactrocera dorsalis Hendel) (Diptera: Tephritidae), at mortalities of 92-99%, on 3rd-6th DPI, and in laboratory conditions. The pathogenicity assay against E. africanus in hemp plants under greenhouse conditions indicated 85-100% insect mortality on 10th DPI using the fungus alone or in combination with synthetic acaricide. Genome sequencing of Metarhizium sp. BCC 4849 revealed the high abundance of proteins associated with zinc-, heme-, and iron-binding; oxidation-reduction; and transmembrane transport, implicating its versatile mode of interaction with the environment and adaptation to various ion homeostasis. The light and scanning electron microscopy indicated that at 24 h post inoculation (PI), adhesion and appressorial formation occurred, notably near the setae. Most infected mites had stopped moving and started dying by 48-72 h PI. Elongated hyphal bodies and oval blastospores were detected in the legs. At 96-120 h PI or longer, dense mycelia and conidial mass had colonized the interior and exterior of dead mites, primarily at the bottom than the upper part. The shelf-life study also indicated that conidial formulation combined with an oxygen-moisture absorber markedly enhanced the viability and germination after storage at 35 °C for four months. The fungus was tested as safe for humans and animals, according to our toxicological assays.

Understanding carbon utilization routes between high and low starch-producing cultivars of cassava through Flux Balance Analysis.

Analysis of metabolic flux was used for system level assessment of carbon partitioning in Kasetsart 50 (KU50) and Hanatee (HN) cassava cultivars to understand the metabolic routes for their distinct phenotypes. First, the constraint-based metabolic model of cassava storage roots, rMeCBM, was developed based on the carbon assimilation pathway of cassava. Following the subcellular compartmentalization and curation to ensure full network connectivity and reflect the complexity of eukaryotic cells, cultivar specific data on sucrose uptake and biomass synthesis were input, and rMeCBM model was used to simulate storage root growth in KU50 and HN. Results showed that rMeCBM-KU50 and rMeCBM-HN models well imitated the storage root growth. The flux-sum analysis revealed that both cultivars utilized different metabolic precursors to produce energy in plastid. More carbon flux was invested in the syntheses of carbohydrates and amino acids in KU50 than in HN. Also, KU50 utilized less flux for respiration and less energy to synthesize one gram of dry storage root. These results may disclose metabolic potential of KU50 underlying its higher storage root and starch yield over HN. Moreover, sensitivity analysis indicated the robustness of rMeCBM model. The knowledge gained might be useful for identifying engineering targets for cassava yield improvement.

Unlocking conserved and diverged metabolic characteristics in cassava carbon assimilation via comparative genomics approach.

Globally, cassava is an important source of starch, which is synthesized through carbon assimilation in cellular metabolism whereby harvested atmospheric carbon is assimilated into macromolecules. Although the carbon assimilation pathway is highly conserved across species, metabolic phenotypes could differ in composition, type, and quantity. To unravel the metabolic complexity and advantage of cassava over other starch crops, in terms of starch production, we investigated the carbon assimilation mechanisms in cassava through genome-based pathway reconstruction and comparative network analysis. First, MeRecon - the carbon assimilation pathway of cassava was reconstructed based upon six plant templates: Arabidopsis, rice, maize, castor bean, potato, and turnip. MeRecon, available at http://bml.sbi.kmutt.ac.th/MeRecon, comprises 259 reactions (199 EC numbers), 1,052 proteins (870 genes) and 259 metabolites in eight sub-metabolisms. Analysis of MeRecon and the carbon assimilation pathways of the plant templates revealed the overall topology is highly conserved, but variations at sub metabolism level were found in relation to complexity underlying each biochemical reaction, such as numbers of responsible enzymatic proteins and their evolved functions, which likely explain the distinct metabolic phenotype. Thus, this study provides insights into the network characteristics and mechanisms that regulate the synthesis of metabolic phenotypes of cassava.

Rice ORMDL controls sphingolipid homeostasis affecting fertility resulting from abnormal pollen development.

The orosomucoids (ORM) are ER-resisdent polypeptides encoded by ORM and ORMDL (ORM-like) genes. In humans, ORMDL3 was reported as genetic risk factor associated to asthma. In yeast, ORM proteins act as negative regulators of sphingolipid synthesis. Sphingolipids are important molecules regulating several processes including stress responses and apoptosis. However, the function of ORM/ORMDL genes in plants has not yet been reported. Previously, we found that temperature sensitive genetic male sterility (TGMS) rice lines controlled by tms2 contain a deletion of about 70 kb in chromosome 7. We identified four genes expressed in panicles, including an ORMDL ortholog, as candidates for tms2. In this report, we quantified expression of the only two candidate genes normally expressed in anthers of wild type plants grown in controlled growth rooms for fertile and sterile conditions. We found that only the ORMDL gene (LOC_Os07g26940) showed differential expression under these conditions. To better understand the function of rice ORMDL genes, we generated RNAi transgenic rice plants suppressing either LOC_Os07g26940, or all three ORMDL genes present in rice. We found that the RNAi transgenic plants with low expression of either LOC_Os07g26940 alone or all three ORMDL genes were sterile, having abnormal pollen morphology and staining. In addition, we found that both sphingolipid metabolism and expression of genes involved in sphingolipid synthesis were perturbed in the tms2 mutant, analogous to the role of ORMs in yeast. Our results indicated that plant ORMDL proteins influence sphingolipid homeostasis, and deletion of this gene affected fertility resulting from abnormal pollen development.

Starch biosynthesis in cassava: a genome-based pathway reconstruction and its exploitation in data integration.

BACKGROUND: Cassava is a well-known starchy root crop utilized for food, feed and biofuel production. However, the comprehension underlying the process of starch production in cassava is not yet available. RESULTS: In this work, we exploited the recently released genome information and utilized the post-genomic approaches to reconstruct the metabolic pathway of starch biosynthesis in cassava using multiple plant templates. The quality of pathway reconstruction was assured by the employed parsimonious reconstruction framework and the collective validation steps. Our reconstructed pathway is presented in the form of an informative map, which describes all important information of the pathway, and an interactive map, which facilitates the integration of omics data into the metabolic pathway. Additionally, to demonstrate the advantage of the reconstructed pathways beyond just the schematic presentation, the pathway could be used for incorporating the gene expression data obtained from various developmental stages of cassava roots. Our results exhibited the distinct activities of the starch biosynthesis pathway in different stages of root development at the transcriptional level whereby the activity of the pathway is higher toward the development of mature storage roots. CONCLUSIONS: To expand its applications, the interactive map of the reconstructed starch biosynthesis pathway is available for download at the SBI group's website (http://sbi.pdti.kmutt.ac.th/?page_id=33). This work is considered a big step in the quantitative modeling pipeline aiming to investigate the dynamic regulation of starch biosynthesis in cassava roots.

Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants.

The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar "KU50" was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19-21% was observed. Among the total number of ESP explants, 32-38% regained normal extended shoot phenotype and 88-96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries.