Plant viruses and viroids in Japan

Journal of General Plant Pathology - An increasing number of plant viruses and viroids have been reported from all over the world due largely to metavirogenomics approaches with technological...     

Attenuated plant viruses: preventing virus diseases and understanding the molecular mechanism

Attenuated viruses have been isolated and studied not only as a practical means of controlling virus diseases but also to gain a molecular understanding of viral virulence and cross protection. They have been isolated from crop fields and generated through high/low temperature treatment or by mutagens such as nitrous acid and ultraviolet irradiation. Some viruses have been beneficially used in fields and evaluated for one or more decades. Molecular genetic studies on attenuated viruses have revealed that amino acid substitutions are located in replicase and the movement protein in tobamovirus, protein 2b for cucumovirus, and P1 and HC-Pro for potyvirus. In most cases, with a few exceptions, symptom attenuation is positively correlated with a reduced level of RNA silencing suppression. Molecular mechanisms underlying virus attenuation and cross protection and the rationale for practical use of attenuated viruses for effective virus disease control are discussed.     

Low-temperature preservation (at 4 °C) of marine rotifer Brachionus

Mass production of rotifers is essential as live food during the larval rearing season, but a major problem of rotifer culture is unpredictable culture collapse. If mass‐produced rotifers could be kept alive at low temperature for an extended period of time, they could be supplied as live food to cultured marine fish larvae without interruption. Four experiments were performed to test this possibility in six strains of Brachionus plicatilis O. F. Müller and eight strains of Brachionus rotundiformis Tschugunoff. The results showed that: (1) B. rotundiformis strains were less tolerant to 4 °C than B. plicatilis strains. Among the B. rotundiformis strains, the strains known as SS type were the most susceptible and showed the lowest survival. (2) Exchange of culture media during the incubation at 4 °C in B. plicatilis and B. rotundiformis resulted in higher survival than not changing the culture media, but there were no differences in the regression slope with or without changing the culture media. (3) Acclimation at 15 °C for 96 h for B. plicatilis and B. rotundiformis before transfer to 4 °C resulted in higher survival rates than acclimation at 10 °C. (4) The combination of frequent exchange of culture media and acclimation significantly improved the survival of B. plicatilis and B. rotundiformis compared with controls that were maintained at 4 °C without exchange of the culture media. Large‐scale trials using B. plicatilis (Kamiura strain) cultured in 30‐L tanks were conducted in a hatchery at a density of 2000–20 000 individuals mL^?1. Rotifers were transferred directly from 25 °C to 4 °C. About 50% of the rotifers at 20 000 individuals mL^?1 survived after 14 days at 4 °C. These preserved rotifers could be cultured at 20 °C, recovering within 4 days.     

Epidermal structure created by canine hair follicle keratinocytes enriched with bulge cells in a three‐dimensional skin equivalent model in vitro: implications for regenerative therapy of canine epidermis

Background – Keratinocytes in the hair follicle bulge region have a high proliferative capacity, with characteristics of epithelial stem cells. This cell population might thus be an ideal source for generating the interfollicular epidermis in a canine skin equivalent. Hypothesis/Objectives – This study was designed to determine the ability of canine hair follicle bulge cell‐enriched keratinocytes to construct canine living skin equivalents with interfollicular epidermis in vitro. Animals – Four healthy beagle dogs from a research colony. Methods – Bulge cell‐enriched keratinocytes showing keratin 15 immunoreactivity were isolated from canine hair follicles and cultured on dermal equivalent containing canine fibroblasts. Skin equivalents were subjected to histological, immunohistochemical, western blot and RT‐PCR analyses after 10–14 days of culture at the air–liquid interface. Results – The keratinocyte sheets showed an interfollicular epidermal structure comprising four to five living cell layers covered with a horny layer. Immunoreactivities for keratin 14 and desmoglein 3 were detected in the basal and immediate suprabasilar layers of the epidermis, while keratin 10 and desmoglein 1 occurred in more superficial layers. Claudin 1 immunoreactivity was seen in the suprabasalar layer of the constructed epidermis, and filaggrin monomers and loricrin were detected in the uppermost layer. Basal keratinocytes in the skin equivalent demonstrated immunoreactivity to antibodies against basement membrane zone molecules. Conclusions and clinical importance – A bulge stem cell‐enriched population from canine hair follicles formed interfollicular epidermis within 2 weeks in vitro, and thus represents a promising model for regenerative therapy of canine skin.     

Usefulness of cefovecin disk‐diffusion test for predicting mecA gene‐containing strains of Staphylococcus pseudintermedius and clinical efficacy of cefovecin in dogs with superficial pyoderma

Background –  Cefovecin has been widely used to treat skin infections in dogs. The relationship of the cefovecin disk‐diffusion test results to the presence of the mecA gene and the clinical efficacy of cefovecin have not been fully evaluated. Hypothesis/Objectives –  To determine the usefulness of an in vitro cefovecin disk‐diffusion test in predicting the presence of the mecA gene in Staphylococcus pseudintermedius, as well as the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma. Methods –  Twenty‐six S. pseudintermedius strains isolated from 22 dogs with pyoderma were used. In vitro disk‐diffusion test results of cefovecin were compared with agar‐dilution test results, the presence of the mecA gene, and the improvement in clinical scores of dogs with superficial pyoderma at 14 days post treatment. Results –  There was a significant linear correlation (r = ?0.83) between the diameter of the obvious zone of inhibition by disk diffusion and the minimal inhibitory concentration for cefovecin (P < 0.0001). Receiver operating characteristic analysis revealed that zone diameters between 25 and 27 mm exhibited better sensitivity (92.9%) and specificity (100.0%) for detection of strains carrying the mecA gene. The mean improvement in clinical scores in dogs carrying cefovecin‐resistant strains was significantly lower than in dogs carrying cefovecin‐susceptible strains (P < 0.01). Conclusions and clinical importance –  The cefovecin disk‐diffusion test with a cut‐off value estimated in this study was valuable for predicting mecA gene carriage in S. pseudintermedius, as well as the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma caused by S. pseudintermedius.     

Proposal of screening method for intestinal mucus adhesive lactobacilli using the enzymatic activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)

Adhesion tests are complex, time‐consuming and expensive, while the most important criterion for a probiotic lactobacilli is the ability to adhere to the human intestine. Thirty lactobacilli isolates from human intestinal tissues were measured for cell surface glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) activity using a microtiter plate screening method. GAPDH activities were detected in 21 out of 30 samples from 12 h cultures and in all samples from 18 h cultures. This suggests GAPDH is universally expressed on the bacterial cell surfaces from many lactobacilli. A statistically significant positive correlation was shown between GAPDH activity and adhesion using the BIACORE adhesion assay (P < 0.01). The new screening method using GAPDH enzymatic activity without an adhesion test may be possible due to the significant positive correlation of GAPDH activity with adhesion of lactobacilli derived from the human intestine.     

Dataset of CarboEastAsia and uncertainties in the CO2 budget evaluation caused by different data processing

The datasets of net ecosystem CO₂ exchange (NEE) were acquired from 21 forests, 3 grasslands, and 3 croplands in the eastern part of Asia based on the eddy covariance measurements of the international joint program, CarboEastAsia. The program was conducted by three networks in Asia, ChinaFLUX, JapanFlux, and KoFlux, to quantify, synthesize, and understand the carbon budget of the eastern part of Asia. An intercomparison was conducted for NEE estimated by three gap-filling procedures adopted by ChinaFLUX, JapanFlux, and KoFlux to test the range of uncertainty in the estimation of NEE. The overall comparison indicated good agreement among the procedures in the seasonal patterns of NEE, although a bias was observed in dormant seasons depending on the different criteria of data screening. Based on the gap-filled datasets, the magnitude and seasonality of the carbon budget were compared among various biome types, phenology, and stress conditions throughout Asia. The annual values of gross primary production and ecosystem respiration were almost proportional to the annual air temperature. Forest management, including clear-cutting, plantation, and artificial drainage, was significant and obviously affected the annual carbon uptake within the forests. Agricultural management resulted in notable seasonal patterns in the crop sites. The dataset obtained from a variety of biome types would be an essential source of knowledge for ecosystem science as well as a valuable validation dataset for modeling and remote sensing to upscale the carbon budget estimations in Asia.     

Characterization of glycoconjugates and sialic acid modification in the olfactory bulb of the Chinese fire-bellied newt (Cynops orientalis)

Diverse glycoconjugates are expressed in the vertebrate olfactory bulb and serve as guidance cues for axons of nasal receptor neurons. Although the involvement of glycoconjugates in the segregation of the olfactory pathway has been suggested, it is poorly understood in salamanders. In this study, lectin histochemistry was used to determine glycoconjugate distribution in the olfactory bulb of the Chinese fire-bellied newt (Cynops orientalis). Succinylated wheat germ agglutinin (sWGA), Ricinus communis agglutinin-I and Lens culinaris agglutinin showed different bindings in the nerve fibre layer or glomerular layer, or both, between the main and accessory olfactory bulbs. We then investigated the lectin-binding pattern after the removal of terminal sialic acids using neuraminidase. Desialylation resulted in a change in the binding reactivities with seven lectins. Wheat germ agglutinin, sWGA, soybean agglutinin (SBA) and peanut agglutinin showed different degrees of binding between the main and accessory olfactory bulbs. In addition, SBA showed a heterogeneous labelling of glomeruli in the rostral region of the main olfactory bulb. Our results suggest that terminal sialic acids mask the heterogeneity of glycoconjugates in the olfactory bulb of C. orientalis.     

Improved method for in situ biolistic transformation to analyze barley–powdery mildew interactions

Although the use of stable transformants is indispensable to elucidate mechanisms underlying molecular plant–pathogen interactions, this approach remains difficult to apply to crops. Alternatively, biolistic transformation has often been used as a transient expression method in various plants. In this study, we developed a method for in situ biolistic transformation without separating leaves from barley seedlings by using a hand-held particle bombardment system because unwounded leaves are preferable for analyzing interactions between barley and Blumeria graminis f. sp. hordei which requires healthy living cells. As a result, we found that the infection rate in intact leaves was higher than in separated leaves and that the transformation efficiencies in leaves were higher when plants were grown in vermiculite rather than in culture soil. Furthermore, we determined the appropriate inoculation time after bombardment to analyze the incompatible interaction and successfully monitored the gradual occurrence of cell death over time. Our system was suitable for relatively long-term follow-up analysis of the fate of each single cell during plant–pathogen interactions.