Yoh Sakuma

Changes in the water status and osmotic solute contents in response to drought and salicylic acid treatments in four different cultivars of wheat (Triticum aestivum)

Salicylic acid (SA) controls growth and stress responses in plants. It also induces drought tolerance in plants. In this paper, four wheat (Triticum aestivum L.) cultivars with different drought responses were treated with SA in three levels of drain (90, 60, 30% of maximum field capacity) to examine its interactive effects on drought responses and contents of osmotic solutes that may be involved in growth and osmotic adjustment. Under drought condition, the cultivars Geza 164 and Sakha 69 had the plant biomass and leaf relative water content (LRWC) greater than the cultivars Gemaza 1 and Gemaza 3. In all cultivars, drought stress decreased the biomass, LRWC, and the contents of inorganic solutes (Ca, K, Mg) and largely increased the contents of organic solutes (soluble sugars and proline). By contrast, SA increased the biomass, LRWC and the inorganic and organic solute contents, except proline. Correlation analysis revealed that the LRWC correlated positively with the inorganic solute contents but negatively with proline in all cultivars. SA caused maximum accumulations of soluble sugars in roots under drought. These results indicated that SA-enhanced tolerance might involve solute accumulations but independently of proline biosynthesis. Drought-sensitive cultivars had a trait lowering Ca and K levels especially in shoots. Possible functions of the ions and different traits of cultivars were discussed.

Qualitative and quantitative PCR methods using species-specific primer for detection and identification of wood rot fungi

Species-specific oligonucleotide primers for detecting wood rot fungi, Gloeophyllum trabeum, Trametes versicolor, Coniophora puteana, and Serpula lacrymans, and the primer detecting a group of related fungi to G. sepiarium were developed. These primer sequences were picked up from the internal transcribed spacer region between small-subunit rDNA and large-subunit rDNA. The species selectivities of the developed primers were checked. Real-time polymerase chain reaction (PCR) was carried out using these highly specific primers to quantitatively detect at least of 0.01 ng genome DNA of the target species. This quantitative PCR was also used to differentiate the target species DNA from mixed species DNA. A PCR-based technique using the species-specific primers would be applicable to multiple-sample assay in diagnosis of wood decay and to investigation of environmental fungal populations.

Effect of moisture content of a wood matrix on a small-scale biodegradation system for organic solid waste

The optimum working moisture content of a wood matrix for the garbage automatic decomposer-extinguisher (GADE) machine was investigated using a small-scale degradation reactor. A formula feed for rabbits was used as the model waste. The degradation experiment was conducted under controlled conditions such as moisture content, environmental temperature, and airflow rate. The degradation rate was estimated precisely from weight loss and the CO2 evolution rate. The degradation rate were nearly constant at a moisture content of 30%–80% on a-wet-weight basis. Microorganisms from the environment propagated in the reactor with no inoculums added. The number of microorganisms showed a trend similar to that of the degradation rate. The microorganism community changed according to the moisture content of the matrix and were considered to attain a constant degradation rate at a wide range of moisture content of a matrix.

Direct ethanol production from cellulosic materials by consolidated biological processing using the wood rot fungus Schizophyllum commune.

In the present study, ethanol production from polysaccharides or wood chips was conducted in a single reactor under anaerobic conditions using the white rot fungus Schizophyllum commune NBRC 4928, which produces enzymes that degrade lignin, cellulose and hemicellulose. The ethanol yields produced from glucose and xylose were 80.5%, and 52.5%, respectively. The absolute yields of ethanol per microcrystalline cellulose (MCC), xylan and arabinogalactan were 0.26g/g-MCC, 0.0419g/g-xylan and 0.0508g/g-arabinogalactan, respectively. By comparing the actual ethanol yields from polysaccharides with monosaccharide fermentation, it was shown that the rate of saccharification was slower than that in fermentation. S. commune NBRC 4928 is concluded to be suitable for CBP because it can produce ethanol from various types of sugar. From the autoclaved cedar chip, only little ethanol was produced by S. commune NBRC 4928 alone but ethanol production was enhanced by combined use of ethanol fermenting and lignin degrading fungi.

Detection of intra- and interspecific variation of the dry rot fungus Serpula lacrymans by PCR-RFLP and RAPD analysis

We investigated a genotype-based assay to discriminate the dry rot fungi Serpula lacrymans. DNAs were extracted from 74 isolates from the northern half of Japan, and internal transcribed spacers (ITS) were amplified by polymerase chain reaction. Genotypes of isolates were checked by restriction fragment length polymorphism (RFLP) using two enzymes, Taq I and Hha I. Among the 74 isolates identified as S. lacrymans in terms of morphologic features, 5 isolates were shown to have been misidentified. Random amplified polymorphic DNA (RAPD) analysis was conducted in order to detect the intraspecific diversity of S. lacrymans isolated in Japan. Because no relation between geographical origin and genetic distances was observed, the intraspecific diversity of S. lacrymans is suggested to be small.

Effect of environmental temperature on a small-scale biodegradation system for organic solid waste

The optimum environmental temperature for a biodegrading machine using wood particles as a matrix was investigated using a small-scale degradation reactor and model waste. The biodegradation rate was evaluated by weight loss of waste and CO2 evolution. The degradation reaction was restricted only by adjusting the environmental temperature while sufficient oxygen and substrates were supplied. Results suggested that the optimum temperature for degradation was 30°–40°C for exploiting biological activity effectively with the lowest use of energy. Bacteria from the environment propagated in the reactor with no inoculum added. The microbial flora changed during the operation time but had no effect on the biodegradation rate.

Analysis of the electrophoretic karyotype of Flammulina velutipes

The karyotype ofFlammulina velutipes (Curt.: Fr.) Sing. was analyzed electrophoretically using contourclamped homogeneous electric field gel electrophoresis and hybridization with DNA probes. The chromosomal DNA from the monokaryon (Fv-4K) and the dikaryon (Fv-4) were resolved into six and eight bands, respectively. The sizes of the chromosomes ranged from 1.9 to 6.0 megabase (Mb) pairs. Each of the separated bands of chromosomal DNA was identified by use of five cloned probes. The number of these chromosomes was estimated to be 6 and 12, respectively; and the size of the entire genome was estimated to be about 20.1 and 38.6Mb, respectively. From a comparison of the hybridization patterns, the existence of allelic chromosomes of different sizes was deduced in the Fv-4 strain.

General Roles of Phytochelatins and Other Peptides in Plant Defense Mechanisms Against Oxidative Stress/Primary and Secondary Damages Induced by Heavy Metals

Phytochelatins (PCs) are nonprotein cysteine-rich oligopeptides having the general structure of (γ-glutamyl-cysteinyl)n-glycine (n = 2–11). They are synthesized from the precursor glutathione (a reduced form, GSH) by the activity of phytochelatin synthase (PCS). The biosynthesis is stimulated by several heavy metals (HMs), especially Cd and metalloid As. PCs can bind to various HMs like Cd, As, Cu, Pb, Zn, and Ag, via their sulfhydryl (–SH) and carboxyl (–COOH) groups. The complexations become more stable and massive in vacuole where acid-labile sulfides (S2−) are incorporated to make the PCs–S–HMs conjugates. Both the thiols and S2− are originated from sulfate through a partially common energy-dependent metabolism (sulfur assimilation), which is again enhanced by Cd, besides essential metals (Co, Mg). To date, fundamental roles of PCs and also related iso-peptides such as hPCs in intracellular detoxification and/or transport of HMs are well demonstrated in various plants, especially in experiments targeting genes and enzymes for PC and GSH biosynthesis. However, how they function as a defense molecule in the oxidative stresses or other biological processes are still unknown or conceiving subtle problems. Some of the possible functions are highlighted in this chapter as tentative examples for further discussion: (1) PCs–S–HMs complex as a potent pool/stock of thiols or reducing powers to be reusable for further robustious responses by the tolerant plants against various abiotic and biotic stresses including oxidative stress and (2) PCs as a possible mediator for metal translocation or redistribution via phloem rather than xylem, regardless of a trait of “hyperaccumulator” for HMs in land plants. Apart from the positive roles of PCs in HM-tolerant plants, arguments still hot arise an issue (3) the roles of PCs, GSH, and other thiols as delicate barometer or indicators in the mineral and redox balance and/or homeostasis, in addition to their well-known functions being substrates and antidotes. In the absence of HMs, the levels of PCs are too minute to account for their sufficient bindings to the essential metals. Although GSH is ubiquitous and abundant, it is a multifunctional peptide that rapidly consumed or oxidized for numerous enzymic or nonenzymic antioxidants/redox systems as well as direct substrate for PCS. Eventually, importance of preservation of thiols and sulfide (S2−) as resource for reducing powers in sensitive sessile plants against various oxidative stresses is again emphasized in return for PCs in the HM-tolerant plants in metalliferous habitats.

Identification and species-typing of wood rotting fungi using melting curve analysis

A method for identification and typing of wood rotting fungi using the melting temperature [T(m)] of DNA fragments coding rRNA (rDNA) was examined. The T(m)s of four DNA fragments, inter transcribed spacer (ITS) I, ITS II, and two partial fragments of 28S rDNA from each of 20 species of wood rotting fungi, were measured by melting curve analysis. The T(m) variation was large enough between species to enable identification based on the T(m) values. A pair of T(m)s of the ITS I region (between the primers ITS1 and ITS2) and the ITS II region (between the primers ITS3 and ITS4) had the highest resolution for identifying wood rotting fungi. To assess about the diversity of the T(m), intraspecific diversity of these DNA fragment sequences was evaluated using test strain sequences and data from the GenBank/EMBL/DDBJ biological database. The intraspecific diversity of T(m) was considered to be small because the nucleotide diversity of each fragment was small within the species.

Effects of wood species on degradation rates and bacterial communities in a small-scale biodegradation system for garbage using wood matrices

Simulated organic waste was biodegraded in a laboratory-scale machine using matrices prepared from four wood species to investigate the effects of wood species on the degradation rate and the bacterial community. The degradation rate, estimated by measuring weight loss and CO2 evolution, was found to be equal among the four wood species. Changes in viable cell counts and microbial communities over time were examined. Viable cell counts were also similar among the wood species, but initial bacterial communities differed owing to differences in wood species, although these communities became similar with time. The sensitivity of isolates to wood extractives was examined using paper discs. The extractive-insensitive bacteria species were dominant at the initial stage of biodegradation. However, occupancy of sensitive bacteria increased with time. It was thought that antibacterial extractives were degraded or inactivated after some time.