An improved method for bright-field imaging of arbuscular mycorrhizal fungi in plant roots

Easy observation methods to assess the colonization levels of arbuscular mycorrhizal (AM) fungi in plant roots are crucial for studying the biology of AM symbiosis and for considering agricultural use. Many AM studies employ Trypan Blue (TB) coupled with lactic acid to stain AM fungal structures as bright-field images; however, TB staining can be difficult to use owing to its noxiousness and high viscosity. Here, we report the development of an easy method for visualizing AM fungal structures as bright-field images using 3,3′-diaminobenzidine (DAB). Wheat germ agglutinin (WGA)-conjugated horseradish peroxidase (HRP), which specifically targets N-acetylglucosamine polymers and detects AM fungal cell walls, penetrated the cortical layers of 10% potassium hydroxide (KOH)-treated soybean roots and stained AM fungal mycelia in the presence of DAB and hydrogen peroxide (H₂O₂). Comparison between DAB and TB staining of soybean (Glycine max L.) roots suggested that the intactness of root systems and image contrast using DAB staining were superior. Background signals in stele observed by DAB staining were negligible as compared with those observed by WGA-fluorescein isothiocyanate staining. DAB staining, which combines the advantages of TB (easy bright-field imaging) and WGA-fluorophore (specific and high-quality) staining, provides a robust imaging method for macro- and micro-level analyses of AM roots and is applicable to at least six crops: soybean, onion (Alium cepa L.), potato (Solanum tuberosum L.), maize (Zea mays L.), rice (Oryza sativa L.), and sunflower (Helianthus annuus L.).     

Allometric equations for accurate estimation of above-ground biomass in logged-over tropical rainforests in Sarawak, Malaysia

Although allometric equations can be used to accurately estimate biomass and/or carbon stock in forest ecosystems, few have been developed for logged-over tropical rainforests in Southeast Asia. We developed allometric relationships between tree size variables (stem diameter at breast height (dbh) and tree height) and leaf, branch, stem and total above-ground biomass in two logged-over tropical rainforests with different soil conditions in Sarawak, Malaysia. The study sites were originally classified as mainly lowland dipterocarp forest and have been selectively logged in the past 20 years. In total, 30 individuals from 27 species were harvested to measure above-ground parts. The correlation coefficients for the allometric relationships obtained for total above-ground biomass as a function of dbh had high values (0.99), although the relationships for leaf biomass had a relatively low coefficient (0.83). We also found relatively high coefficients for allometric relationships between tree height and plant-part biomass, ranging from 0.82 to 0.97. Moreover, there were no differences for allometric equations of total above-ground biomass between study sites. A comparison of equations of above-ground biomass in various previously reported tropical rainforests and pan-tropic general equations imply that our allometric equations differ largely from the equations for tropical primary forests, early successional secondary forest, and even for the general models. Therefore, choosing the biomass estimation models for above-ground biomass in the logged-over forests of Southeast Asia requires careful consideration of their suitability.     

Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus

The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse.     

Possibilities of direct introgression from Brassica napus to B. juncea and indirect introgression from B. napus to related Brassicaceae through B. juncea

The impact of genetically modified canola (Brassica napus) on biodiversity has been examined since its initial stage of commercialization. Various research groups have extensively investigated crossability and introgression among species of Brassicaceae. B. rapa and B. juncea are ranked first and second as the recipients of cross-pollination and introgression from B. napus, respectively. Crossability between B. napus and B. rapa has been examined, specifically in terms of introgression from B. napus to B. rapa, which is mainly considered a weed in America and European countries. On the other hand, knowledge on introgression from B. napus to B. juncea is insufficient, although B. juncea is recognized as the main Brassicaceae weed species in Asia. It is therefore essential to gather information regarding the direct introgression of B. napus into B. juncea and indirect introgression of B. napus into other species of Brassicaceae through B. juncea to evaluate the influence of genetically modified canola on biodiversity. We review information on crossability and introgression between B. juncea and other related Brassicaseae in this report.     

Hepatic AA amyloidosis in a cat: cytologic and histologic identification of AA amyloid in macrophages

A 3‐year‐old, spayed female, Domestic Shorthair cat presented with anorexia, lethargy, vomiting, probable hemoabdomen, and multiple masses on the right lateral liver lobe. Clinicopathologic and imaging abnormalities included anemia, azotemia, icterus, and hepatomegaly with hypoechoic masses. On cytologic evaluation of a fine‐needle aspiration of a liver mass there was abundant extracellular pink‐ to purple‐colored material between hepatocytes. The amorphous material was stained with direct fast scarlet (DFS), and green birefringent areas were observed under polarized light, confirming the presence of amyloid. A unique finding on the cytologic smear were macrophages containing amorphous and fibrillar amyloid‐like protein. Histopathologic examination using H&E and Congo red staining confirmed amyloid deposits within the space of Disse, along the sinusoids, portal tracts, blood vessel walls, and within the cytoplasm of macrophages. Immunohistochemical staining with anti‐AA amyloid antibodies further confirmed the presence of AA amyloid. To the author's knowledge, this is the first report of the cytologic finding of AA amyloid protein within macrophages and DFS stain detection of amyloid on a cytologic smear.     

Polymorphisms in growth hormone gene and their associations with calf weight in Japanese Black cattle

The objectives of this study were to detect effective genetic polymorphisms of bovine growth hormone (bGH) gene associated with calf weight in Japanese Black cattle. Fifty‐eight sires and 47 breeding cows were used to detect the polymorphisms in exons by single‐strand conformation polymorphism (SSCP). Four homozygous and six heterozygous SSCP genotypes were identified in exon 5. Although each single nucleotide polymorphism (SNP) had been reported, these genotypes were caused by three SNPs at the nucleotide positions 2141, 2277 and 2291. Four haplotypes C‐C‐A, G‐C‐A, C‐C‐C and G‐T‐A were newly identified. It was suggested that other haplotypes not detected in this study may not exist, considering the allele frequencies reported in Bos taurus and Bos indicus, and the migrating process of native Japanese cattle. Thereafter, we examined associations between the detected polymorphic sites in exon 5 by PCR – restriction fragment length polymorphism and calf weight using 53 breeding dams and 135 calves. The birth weights of calves with haplotype G‐C‐A are significantly lighter and calves' weights produced by cows with such haplotype are also lighter at 30 days old, using regression analysis. Although further research is necessary, these results may serve as a useful criterion to select breeding stocks, especially in maternal abilities.     

In vitro development and postvitrification survival of cloned feline embryos derived from preadipocytes

The aim of the present study was to optimize the conditions for in vitro development and postvitrification survival of somatic cell cloned feline embryos. To determine the effects of cell cycle synchronization of the nuclear donor cells, we cultured preadipocytes under serum starvation or conventional conditions. After two days in serum starvation culture, the proportion of synchronized donor cells at the G0/G1 phase was 91.6%. This was significantly higher than the proportion of non-synchronized cells in the proliferative phase (72.6%, P<0.05). The in vitro development of somatic cell nuclear transfer (SCNT) embryos reconstructed using donor cells treated under serum starvation conditions (normal cleavage rate of 65.7%, 46/70, and blastocyst formation rate of 20.0%, 14/70) was comparable to that of the serum supplemented group (52.5%, 31/59, and 20.3%, 12/59). Use of in vitro or in vivo matured oocytes as recipient cytoplasts equally supported development of the SCNT embryos to the blastocyst stage (11.9%, 5/42, vs. 9.5%, 2/21). SCNT-derived blastocysts were vitrified using the original minimum volume cooling (MVC) or the modified (stepwise) MVC method. Although none (n=10) of the SCNT blastocysts survived following vitrification by the original MVC method, the stepwise MVC method resulted in 100% survival after rewarming (n=11). In conclusion, we demonstrated that feline somatic cell cloned embryos with a high developmental ability can be produced irrespective of cell cycle synchronization of donor cells using either in vivo or in vitro matured oocytes. Furthermore, by utilizing a stepwise vitrification method, we showed that it is possible to cryopreserve cloned feline blastocysts.     

Amaranthus genetic resources in Indonesia: morphological and protein content assessment in comparison with worldwide amaranths

The morphological variations (growth habit, leaf shape, leaf color, height, stem diameter, number of branches and internodes, leaf area, blade ratio, leaf thickness, number of leaves, and total leaf area) and protein content of 53 Indonesian amaranths (Amaranthus spp.), consisting of weedy-, vegetable-, and ornamental-types, were assessed. The extent of variation in the Indonesian Amaranthus accessions were compared with the worldwide collection (26 accessions from United States Department of Agriculture (USDA) and 5 ornamentals from Nepal) in the experimental field of the University of Tsukuba, Japan. The variation in average values of most morphological traits and protein content in the Indonesian accessions were similar to those of the worldwide germplasm, but the important parameters that influence vegetable yield (e.g., number of leaves and stem diameter) were superior in the Indonesian accessions. Protein content showed a positive correlation with the number of leaves, whereas a negative correlation was observed with leaf thickness. The Indonesian accession of A. viridis and A. dubius showed a great potential to be further selected as parental lines for high protein content and number of leaves. The relatively high protein content of the leaves of Celosia may also potentially serve as an alternative protein source in the tropics.     

Squamous cell carcinoma in a digit of the hind limb with systemic metastasis in a 17-year-old female koala

We encountered a case of cutaneous squamous cell carcinoma (SCC) in a 17-year-old female koala at a zoo. A fragile, papillary, elevated mass was found on the third digit of the right hind limb. SCC was identified histopathologically: squamous cell-like polygonal tumor cells showed a nest-like growth pattern with epidermal down growth, central keratinization and necrotic foci, and invaded dermal connective tissues. Metastatic lesions were observed in various organs, including the lung and axillary lymph node: in the lung, multiple metastatic foci similar to the primary lesion, and in the axillary lymph node, individual polygonal tumor cells infiltrated the sinusoids. Immunohistochemistry revealed that the tumor cells were positive for proliferating cell nuclear antigen, which exhibited 32–33% of labeling indices in the tumor cells. To our knowledge, this is the first report of a case of SCC in a digit of a koala.     

Induction of Soil Suppressiveness Against Rhizoctonia solani by Incorporation of Dried Plant Residues into Soil

ABSTRACT Suppressive effects of soil amendment with residues of 12 cultivars of Brassica rapa on damping-off of sugar beet were evaluated in soils infested with Rhizoctonia solani. Residues of clover and peanut were tested as noncruciferous controls. The incidence of damping-off was significantly and consistently suppressed in the soils amended with residues of clover, peanut, and B. rapa subsp. rapifera 'Saori', but only the volatile substance produced from water-imbibed residue of cv. Saori exhibited a distinct inhibitory effect on mycelial growth of R. solani. Nonetheless, disease suppression in such residue-amended soils was diminished or nullified when antibacterial antibiotics were applied to the soils, suggesting that proliferation of antagonistic bacteria resident to the soils were responsible for disease suppression. When the seed (pericarps) colonized by R. solani in the infested soil without residues were replanted into the soils amended with such residues, damping-off was suppressed in all cases. In contrast, when seed that had been colonized by microorganisms in the soils containing the residues were replanted into the infested soil, damping-off was not suppressed. The evidence indicates that the laimosphere, but not the spermosphere, is the site for the antagonistic microbial interaction, which is the chief principle of soil suppressiveness against Rhizoctonia damping-off.