Experimental evidence of a pathogenic change caused by homologous recombination between endogenous and introduced dysfunctional Avr-Pita genes in Pyricularia oryzae

To provide experimental evidence that somatic homologous recombination (HR) is involved in the instability and diversification of the avirulence gene Avr-Pita in Pyricularia oryzae, we generated a dysfunctional Avr-Pita homolog and integrated it into strain Hoku-1 containing Avr-Pita. In the transformants, the occurrence of somatic HR events between Avr-Pita and the dysfunctional homolog was confirmed by PCR–RFLP. Germlings from conidia from the HR-positive transformants had lost the avirulence function, which enabled it to infect rice cultivar Yashiromochi containing the corresponding resistant gene Pi-ta. These results suggested that genetic mutation caused by somatic HR is one of the mechanisms responsible for virulence diversity.     

Characterization of plant-derived water extractable organic matter by multiple spectroscopic techniques

Water extractable organic matter (WEOM) derived from fresh- or early-stage decomposing soil amendment materials may play an important role in the process of organic matter accumulation. In this study, eight WEOM samples extracted with a 40:1 (v/w) water to sample ratio from alfalfa (Medicago sativa L.), corn (Zea mays L.), crimson clover (Trifolium incarnatum L.), hairy vetch (Vicia villosa L.), lupin (Lupinus albus L.), soybean (Glycine max L. Merr.), wheat (Triticum aestivum L.), and dairy manure were investigated using ultraviolet (UV)–visible, Fourier transform infrared (FT-IR), solution ³¹P nuclear magnetic resonance (NMR), and solid state ¹³C NMR spectroscopies. UV–visible and FT-IR spectra of the plant-derived WEOM samples were typical for natural organic matter, but possessed less humic-like characteristics than dairy manure-derived WEOM. Solution ³¹P NMR spectra indicated that WEOM samples extracted from alfalfa, corn, and soybean shoots contained both orthophosphate and monoester P. Of the monoester P in WEOM from soybean shoot, 70% was phytate P. WEOM from crimson clover, hairy vetch, lupin, and wheat shoots contained orthophosphate only. The solid-state ¹³C NMR spectra of the seven plant-derived WEOM samples indicated that they all were primarily composed of sugars, amino acids or peptides, and low molecular mass carboxylic acids. Carbohydrates were dominant components with very few aromatics present in these samples. In addition, WEOM from crimson clover and lupin, but not other three leguminous plant WEOM samples, contained significant asparagine. On the other hand, WEOM from corn and wheat contained less amino acids or peptides. The spectra of WEOM of dairy manure revealed the presence of significant amounts of nonprotonated carbons and lignin residues, suggesting humification of the manure-derived WEOM. Significant carbohydrates as well as aromatics were present in this WEOM. The P and C bonding information for these WEOM samples may be useful for understanding the effects of WEOM on soil nutrient availability to plants. Trade or manufacturers' names mentioned in the paper are for information only and do not constitute endorsement, recommendation, or exclusion by the USDA-ARS.     

Methodology for the study of the hypothalamic-pituitary hormone secretion in cattle

Studies on the neuroregulatory mechanisms on the secretion of anterior pituitary (AP) hormones in domestic animals are important because nearly all complex physiological and metabolic processes are regulated by the AP hormones. To examine them, this article considers in vivo approaches such as the techniques of intrahypothalamic injection, intracerebroventricular injection, push-pull perfusion, and microdialysis, which have been employed in our own research group for the study in cattle. Also, in vitro approaches such as bovine AP cell culture and the AP explants superfusion system are described. This article clarifies the potential of neuroendocrine study techniques in cattle.     

Effects of sika deer (Cervus nippon) and dwarf bamboo (Sasamorpha borealis) on seedling emergence and survival in cool-temperate mixed forests in the Kyushu Mountains

Effects of sika deer (Cervus nippon) and dwarf bamboo (“sasa;” Sasamorpha borealis) on seedling emergence and survival were investigated in cool-temperate mixed forests in the Kyushu mountain range, Japan. We compared the effects of sika deer between two sites with different sika deer densities. One site (no-sasa forest) has sparse cover of S. borealis and a high density of sika deer, and the other site (sasa forest) has dense cover of S. borealis with a low density of sika deer. In the no-sasa forest, more seedlings emerged and the survival rate of the seedlings was higher compared with the sasa forest. Compared with the sasa forest, the canopy in the no-sasa forest was more open, the organic layer was shallower and drier, the mean daily soil temperature was higher, and soil temperature fluctuated more widely. Those environmental parameters did not differ between unfenced and fenced plots. The comparison between the sasa and no-sasa forests suggested that removal of S. borealis by sika deer had an indirect positive effect on seedling emergence and survival. However, the fenced-plot experiments indicated that sika deer inhibited seedling emergence and survival. These contradictory conclusions could result from the much larger negative effects of S. borealis on seedling emergence and survival compared with the effects of sika deer browsing.     

Comparative study of plasma leptin concentration between solid ruminal and liquid abomasal feeding in weaned adult sheep

This experiment was conducted to investigate the difference between ruminal (solid feed, SF) and abomasal (liquid feed, LF) feeding on the plasma leptin concentration in sheep. The experiment consisted of 2 weeks to adapt the animals to SF, 4 weeks of feeding on SF, 2 weeks adaptation to LF, 8 weeks of feeding on LF, 2 weeks of adaptation to SF, and 4 weeks of feeding on SF. The LF directory flowed into the abomasums of sheep by bottle feeding. Plasma leptin concentration before morning feeding was almost constant in the SF periods, whereas it showed between‐day variations when measured during the LF periods. Mean plasma leptin levels were higher for LF (7.77 ± 0.76 ng/mL; mean ± SE) than for SF periods (3.95 ± 0.16 ng/mL; mean ± SE). Although plasma leptin concentration did not show any change after feeding in the SF and LF periods, plasma insulin and glucose levels increased within 15 min after liquid abomasal feeding, but not after solid ruminal feeding. The high plasma leptin concentration during the LF periods in weaned sheep could be due to change of digestible energy intake and changes in plasma insulin and glucose levels accompanying the changes in digestive processes and nutrient supply.     

A possible role of central serotonin in L‐tryptophan‐induced GH secretion in cattle

To clarify the role of serotonin (5‐HT) in the regulatory mechanism of L‐tryptophan (TRP)‐induced growth hormone (GH) secretion in cattle, changes in 5‐HT concentrations in the cerebrospinal fluid (CSF) in the third ventricle (3V) and GH in plasma before and after the peripheral infusion of TRP were determined simultaneously. The direct effect of TRP on GH release from the dispersed anterior pituitary cells was also assessed. A chronic cannula was placed in 3V by stereotaxic surgery, then CSF and blood were withdrawn under physiological conditions. TRP (38.5 mg/kg BW) was infused through an intravenous catheter from 12.00 to 14.00 hours and CSF and blood sampling were performed from 11.00 to 18.00 hours at 1‐h intervals. The concentration of 5‐HT in CSF was determined by high‐performance liquid chromatography with electrochemical detection. GH, melatonin (MEL), and cortisol (CORT) concentrations were measured by radio‐immunoassay and enzyme‐immunoassay. Concentrations of 5‐HT were increased by TRP infusion. The TRP infusion significantly increased GH release. On the other hand, TRP did not stimulate GH release from the bovine pituitary cells. MEL and CORT concentrations were not altered by TRP infusion. These results suggest that TRP induced GH release via the activation of serotonergic neurons in cattle.     

Development of Novel Water-extraction System with Thermoelectric Module Using Solar and Wind Power in Arid Land

This study aimed to develop a water-extraction system which could produce the fresh water from the air in arid regions and which used renewable energies as the electric power source. In this paper, the experiments for water extraction from the air were carried out by using the novel multi-stage water-extraction device with Peltier deices for two cases of temperature and related humidity of the air. One was the case where the temperature and the related humidity of the air were constant, and the other was the case where they were simulated the variation of the temperature and related humidity of the air in a day of summer and spring in Loess Plateau, China. The effects of the temperature and related humidity of the atmospheric air and supply the electric power to Peltier devices on performance of water production of the device were investigated and reported.     

Comparing the carbon sequestration capacity of temperate deciduous forests between urban and rural landscapes in central Japan

Urbanisation is increasing tremendously in some parts of the world. Consequently, many rural forests may become depleted, although many opportunities exist for urban forests to increase. However, few studies have quantified the carbon (C) sequestration capacities of urban and rural forests in specific climatic zones. The present study compared carbon sequestration in two temperate deciduous forests located in Nagoya and Toyota, central Japan. The Nagoya University forest represented an urban forest, and a site in Toyota represented a rural forest. The urban forest at Nagoya University had comparatively smaller areas of green space and larger areas of buildings and roads. Land uses for building and road, which are typical of urban areas, result in smaller diurnal temperature ranges but higher air temperature, vapour pressure deficit, and atmospheric carbon dioxide (CO₂) concentration. The urban forest in this study exhibited higher gross ecosystem exchange (GEE), especially in the active growing season from May to September, suggesting the possible effect of CO₂ fertilisation. However, higher air temperatures caused comparatively smaller net ecosystem exchange (NEE) because of higher ecosystem respiration (RE). Although both forests functioned as CO₂ sinks at annual time scales, the rural Toyota forest site (5.43 t C ha^–1 yr^–1) had 36% higher net ecosystem production (NEP=–NEE; the negative sign indicates uptake by the forest ecosystem from the atmosphere) than that at the urban forest. The higher normalised respiration (i.e., RE/GPP ratio; GPP=–GEE where GPP represents gross primary production) at the Nagoya University forest might be attributable to factors associated with the degree of urbanisation. Thus, in temperate forests, factors associated with urbanisation may reduce the atmospheric carbon sink function by accelerating respiration. This is an issue of global interest, as many countries are experiencing rapid urbanisation.     

An avirulence gene homologue in the tomato wilt fungus Fusarium oxysporum f. sp. lycopersici race 1 functions as a virulence gene in the cabbage yellows fungus F. oxysporum f. sp. conglutinans

Six4, a small protein secreted by Fusarium oxysporum f. sp. lycopersici (Fol) in tomato xylem sap during infection, triggers Fol race 1-specific resistance (I) in tomato. SIX4 is regarded as an avirulence gene. Although SIX4 is considered unique to Fol race 1, we detected this gene in the cabbage yellows fungus F. oxysporum f. sp. conglutinans (Foc) by PCR. Because the genes from Foc and SIX4 in Fol were >99 % identical at the nucleotide level, the Foc gene was designated FocSIX4. The expression of FocSIX4 was detected by RT-PCR in stems and roots of cabbage 8 days after infection with Foc. In contrast with Fol, disruption of FocSIX4 in Foc did not increase virulence to Foc-resistant cabbage cvs. Shutoku-SP and Koikaze. On the contrary, the disruptants had reduced virulence not only on Foc-resistant cultivars but also on Foc-susceptible cv. Shikidori. These results suggested that FocSIX4 is involved in virulence, but not in avirulence, in the cabbage yellows fungus.     

Construction of a system for exploring mitotic homologous recombination in the genome of Pyricularia oryzae

To investigate mitotic homologous recombination (HR) in Pyricularia oryzae, we created an HR detection system. The system consists of two non-functional enhanced yellow fluorescent protein (YFP) and blasticidin S deaminase (BSD) fusion genes (YFP::BSD). If mitotic HR occurs between the two non-functional genes in the genome, restoration of the functional YFP::BSD gene can be expected. The expression of the functional YFP::BSD gene can be detected by both YFP fluorescence and resistance against BS. When the P. oryzae genome was transformed simultaneously with two non-functional genes, all six lines of transformants with both genes had some portion of their hyphae exhibiting YFP fluorescence and BS resistance during growth. Up to ca. 10 % of conidia harvested from the mycelium of each of the six lines had YFP fluorescence, suggesting that HR consistently occurs during mycelium growth. To determine whether and how the HR-mediated phenotypic changes occurred at the DNA level, we analyzed the genomic DNA of BS-resistant mycelia by PCR-RFLP and sequencing and were able to confirm the existence of a restored functional YFP::BSD gene and a non-functional recombinant gene. Collectively, these findings demonstrate that we established a successful HR detection system for P. oryzae, which can be used for other plant pathogens, and that mitotic HR actually occurs in P. oryzae and constitute the first experimental evidence for mitotic HR in a fungus.