Intermittent induction of LEA peptide by lactose enhances the expression of insecticidal proteins in Bacillus thuringiensis.

Cry toxins from Bacillus thuringiensis (Bt) have been extensively applied in agriculture to substitute the use of chemical insecticides. We have previously reported the use of a coexpression system in which late embryogenesis abundant (LEA) peptides under the control of the lac promoter increase the expression of insecticidal proteins in Bt. The use of lactose to induce the expression of LEA peptides may be a desirable alternative to isopropyl ß-D-thiogalactopyranoside, the most frequently used inducer for recombinant protein expression. In this study we investigated the use of lactose as an inducer for optimal protein expression. We observed enhanced insecticidal Cry protein expression by applying a simple technique based on intermittent induction, and then optimized concentration and the point of induction time from the 11th h to the 15th h. Our data suggest that intermittent induction of lactose might be a new technique for the enhancement of bacterial protein expression.

Enhanced insecticidal activity of Bacillus thuringiensis using a late embryogenesis abundant peptide co-expression system.

Bacillus thuringiensis (Bt) is a ubiquitous, gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing crystal protein, some of which are toxic against a wide range of insect orders like caterpillars, beetles, and flies, including mosquitoes. Regarding the biological control of insects, Bt is the mostly used microorganism worldwide and also alternatives to chemical insecticides for environmental conservation. Some strains of Bt are showing a promising activity against a wide variety of mosquito like Aedes, Culex, and Anopheles and so on with extremely damages in the larval midgut and ultimate death. Here, we introduced a late embryogenesis abundant (LEA) peptide co-expression system based on the expression vector pHT01 with a strong σA-dependent promoter to enhance the expression of insecticidal crystal proteins in native Bt. Two types of LEA peptide (LEA-II and LEA-K) were designed based on the sequence of group-3 LEA protein, which consists of a repetitive sequence of 11 amino acids. The LEA-II mediated co-expression system enhanced the production of crystal protein 3-fold after 12 h of induction of the peptide with 0.5 mM IPTG. Enhanced expression of crystal protein was confirmed by bioassay using 4th instar Aedes albopictus larvae. This unique approach has great potential to produce bio-pesticides by enhanced crystal protein expression not only for mosquitoes but also for other insects.

Component features, odor-active volatiles, and acute oral toxicity of novel white-colored truffle Tuber japonicum native to Japan.

Component analysis of a novel white-colored truffle native to Japan, Tuber japonicum, was performed to determine its characteristic features. The analysis of odor-active volatile compound showed a high contribution of 1-octen-3-ol and 3-methyl-2,4-dithiapentane to the odor of T. japonicum. Although 2,4-dithiapentane is a key odorant of well-known white truffle T. magnatum, 3-methyl-2,4-dithiapentane was detected from the ripe T. japonicum. The chemical components of T. japonicum showed no clear difference with those of edible truffles T. magnatum and T. melanosporum. It was rich in crude protein, crude fiber, and minerals (especially potassium), and low in crude fat. Glutamine and glutamic acid were detected in T. japonicum as free amino acids, while T. magnatum contained a large amount of alanine. Acute oral toxicity tests showed no abnormality, with an LD50 value of over 2000 mg/kg under the test conditions. The present study may support future market distribution of T. japonicum as a high-class foodstuff.

Inside Cover Image, Volume 8, Issue 1.

The cover image is based on the ORIGINAL RESEARCH Component features, odor-active volatiles, and acute oral toxicity of novel white-colored truffle Tuber japonicum native to Japan by Tomoko Shimokawa et al., https://doi.org/10.1002/fsn3.1325. Cover Credit: Cover image ©Tomoko Shimokawa Images.

Heavy-ion beam mutagenesis of the ectomycorrhizal agaricomycete Tricholoma matsutake that produces the prized mushroom "matsutake" in conifer forests.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized mushroom "matsutake" in Pinaceae forests. Currently, there are no available cultivars or cultivation methods that produce fruiting bodies. Heavy-ion beams, which induce mutations through double-stranded DNA breaks, have been used widely for plant breeding. In the present study, we examined whether heavy-ion beams could be useful in isolating T. matsutake mutants. An argon-ion beam gave a suitable lethality curve in relation to irradiation doses, accelerating killing at 100-150 Gy. Argon-ion beam irradiation of the agar plate cultures yielded several transient mutants whose colony morphologies differed from that of the wild-type strain at the first screening, but which did not persist following culture transfer. It also generated a mutant whose phenotype remained stable after repeated culture transfers. The stable pleiotropic mutant not only exhibited a different colony morphology to the wild type, but also showed increased degradation of dye-linked water-insoluble amylose and cellulose substrates. Thus, heavy-ion beams may be useful for isolating mutants of T. matsutake, although precautions may be required to maintain the mutants, without phenotypic reversion, during repetitive culture of their mycelia.

Atomic and nanoscale imaging of a cellulose nanofiber and Pd nanoparticles composite using lower-voltage high-resolution TEM.

We have examined the advanced application of transmission electron microscopy (TEM) for the structural characterization of a composite of cellulose nanofiber (CNF) and palladium (Pd) nanoparticles. In the present study, we focused on electron-irradiation damage and optimization of high-resolution TEM imaging of the composite. The investigation indicates that the CNF breaks even under low-electron-dose conditions at an acceleration voltage of 200 kV. We then applied lower-voltage TEM at 60 kV using a spherical aberration corrector and a monochromator, in order to reduce electron-irradiation damage and improve the spatial resolution. The TEM observation achieved high-resolution imaging and revealed the existence of small Pd nanoparticles, around 2 nm in diameter, supported on the CNF. It is considered that the use of a monochromator in combination with spherical aberration correction contributed to the atomic and nanoscale imaging of the composite, owing to the improvement of the information limit under a lower-acceleration voltage.

A qPCR assay that specifically quantifies Tricholoma matsutake biomass in natural soil.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, "matsutake" mushrooms along densely developed mycelia, called "shiro," in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A0 horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.

Synthesis of Polyaniline (PANI) in Nano-Reaction Field of Cellulose Nanofiber (CNF), and Carbonization.

Polymerization of aniline in the presence of cellulose nano-fiber (CNF) is carried out. We used dried CNF, CNF suspension, and CNF treated by enzyme and ultra-sonification to obtain polyaniline (PANI)/CNF as a synthetic polymer/natural nano-polymer composite. The polymerization proceeds on the surface of CNF as a nano-reaction field. Resultant composites show extended effective π-conjugation length because CNF as a reaction field in molecular level produced polymer with expanded coil structure with an aid of orientation effect of CNF. Possibility of PANI ß-pleats structure in molecular level of PANI on the CNF is also discussed. SEM observation showed that fine structure is easily obtained by combining PANI with CNF. Carbonization of PANI/CNF allows production of nano-fine form with shape preserved carbonization (SPC).

Root endophyte interaction between ectomycorrhizal basidiomycete Tricholoma matsutake and arbuscular mycorrhizal tree Cedrela odorata, allowing in vitro synthesis of rhizospheric "shiro".

The ectomycorrhizal basidiomycete Tricholoma matsutake associates with members of the Pinaceae such as Pinus densiflora (red pine), forming a rhizospheric colony or "shiro," which produces the prized "matsutake" mushroom. We investigated whether the host specificity of T. matsutake to conifers is innately determined using somatic plants of Cedrela odorata, a tropical broad-leaved tree (Meliaceae) that naturally harbors arbuscular mycorrhizal fungi. We found that T. matsutake could form in vitro shiro with C. odorata 140 days after inoculation, as with P. densiflora. The shiro was typically aromatic like that of P. densiflora. However, this was a root endophytic interaction unlike the mycorrhizal association with P. densiflora. Infected plants had epidermal tissues and thick exodermal tissues outside the inner cortex. The mycelial sheath surrounded the outside of the epidermis, and the hyphae penetrated into intra- and intercellular spaces, often forming hyphal bundles or a pseudoparenchymatous organization. However, the hyphae grew only in the direction of vascular bundles and did not form Hartig nets. Tricholoma fulvocastaneum or "false matsutake" naturally associates with Fagaceae and was also able to associate with C. odorata as a root endophyte. With T. matsutake, C. odorata generated a number of roots and showed greatly enhanced vigor, while with T. fulvocastaneum, it generated a smaller number of roots and showed somewhat lesser vigor. We argue that the host-plant specificity of ectomycorrhizal matsutake is not innately determined, and that somatic arbuscular mycorrhizal plants have a great potential to form mutualistic relationships with ectomycorrhizal fungi.

Effects of growth stage on enzymatic saccharification and simultaneous saccharification and fermentation of bamboo shoots for bioethanol production.

Bamboo is a fast-growing renewable biomass that is widely distributed in Asia. Although bamboo is recognised as a useful resource, its utilization is limited and further development is required. Immature bamboo shoots harvested before branch spread were found to be a good biomass resource to achieve a high saccharification yield. The saccharification yield of the shoots increased (up to 98% for immature Phyllostachys bambusoides) when xylanase was used in addition to cellulase. Simultaneous saccharification and fermentation (SSF) processing converted immature shoots of P. bambusoides and Phyllostachys pubescens to ethanol with an ethanol yield of 169 and 139 g kg(-1), respectively (98% and 81%, respectively, of the theoretical yields based on hexose conversion) when 12 FPU g(-1) enzyme and the yeast Saccharomyces cerevisiae were used.

Purification, molecular cloning, and enzymatic properties of a family 12 endoglucanase (EG-II) from fomitopsis palustris: role of EG-II in larch holocellulose hydrolysis.

A family 12 endoglucanase with a molecular mass of 23,926 Da (EG-II) from the brown-rot basidiomycete Fomitopsis palustris was purified and characterized. One of the roles of EG-II in wood degradation is thought to be to loosen the polysaccharide network in cell walls by disentangling hemicelluloses that are associated with cellulose.