Occurrence of boscalid resistance in cucumber powdery mildew in Japan and molecular characterization of the iron–sulfur protein of succinate dehydrogenase of the causal fungus

A total of 74 mass isolates of cucumber powdery mildew fungus (Podosphaera xanthii) were collected from commercial greenhouses with a history of boscalid use in Ibaraki Prefecture, Japan, and tested in a leaf disk assay for their sensitivity to boscalid. The mildew development of 40 of 74 isolates and five sensitive reference isolates on the disks was completely suppressed at 5 μg boscalid/ml. The minimum inhibitory concentrations (MIC) for the remaining 34 isolates were 50 μg/ml or higher, and 21 of these isolates also grew well at 500 μg/ml. Six single-spore isolates were resistant to boscalid with MIC values higher than 500 μg/ml; four of these were moderately resistant (MR), and two were very highly resistant (VHR) isolates. The growth of MR isolates was almost completely suppressed at 500 μg/ml, whereas two VHR isolates grew vigorously at 500 μg/ml. In foliar inoculation tests of potted cucumber plants, the efficacy of boscalid (500 μg/ml) against both MR and VHR isolates was very low. Partial DNA fragment of the iron–sulphur protein subunit (SdhB) gene of succinate dehydrogenase was PCR-amplified from five sensitive and five resistant isolates and directly sequenced, revealing that VHR isolates possess a substitution from a highly conserved histidine (CAT) to tyrosine (TAT) in a third cysteine-rich center of a putative SdhB, whereas MR isolates so far have not been found to have such substitution in SdhB.     

Molecular characteristics of horse phospholipase C zeta (PLCζ)

A sperm‐specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca²⁺]_i) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT‐PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long‐lasting [Ca²⁺]_i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca²⁺]_i oscillation‐inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.     

Regulation of the intestinal barrier by nutrients: The role of tight junctions

Tight junctions (TJs) play an important role in intestinal barrier function. TJs in intestinal epithelial cells are composed of different junctional molecules, such as claudin and occludin, and regulate the paracellular permeability of water, ions, and macromolecules in adjacent cells. One of the most important roles of the TJ structure is to provide a physical barrier to luminal inflammatory molecules. Impaired integrity and structure of the TJ barrier result in a forcible activation of immune cells and chronic inflammation in different tissues. According to recent studies, the intestinal TJ barrier could be regulated, as a potential target, by dietary factors to prevent and reduce different inflammatory disorders, although the precise mechanisms underlying the dietary regulation remain unclear. This review summarizes currently available information on the regulation of the intestinal TJ barrier by food components.     

Successful measures to prevent the spread of bovine papular stomatitis in a dairy farm

Nasal papules and oral ulcers were observed in calves that were group-housed at a dairy farm. The calves were diagnosed with bovine papular stomatitis (BPS) due to parapoxvirus (PPV) infection based on virologic examinations using polymerase chain reaction to detect PPV. To prevent the spread of BPS, we isolated the affected calves, made procedural changes so that the affected herd was managed after the healthy herd, disinfected the bedding with slaked lime, disinfected the stalls and fences with invert soap, and changed the animals’ feed to soft grass which does not damage the oral cavity. As a result, we succeeded in control the infection quickly.     

Morphological analyses of nephrin expression in progressive glomerulonephropathy of common marmosets

In this study, we focused on nephrin, one of the key molecules within the slit diaphragm of podocytes, as although there have been reports on its expression in humans and rats, their presence in common marmosets has not been reported. We investigated nephrin expression and changes in glomeruli, depending on the development of spontaneous progressive glomerulonephropathy in common marmosets. Nineteen common marmosets at two to ten years of age were evaluated. The kidney was examined by microscopy with hematoxylin and eosin and immunohistochemical staining for nephrin. The lesions were classified into three grades according to a renal lesion grading system reported previously. The nephrin-positive area was measured by morphometric analysis, and the nephrin-positive ratio was calculated. Nephrin expression was observed along the glomerular capillary loop in a continuous linear pattern in renal lesion grades 0 to 2 and either discontinuous linear or coarse granular pattern in grade 3. Nephrin expression tended to decrease significantly depending on the grade of renal lesions. Alteration in nephrin expression has been suggested to play an important role in the progression of renal lesions.     

Histological Characteristics of the Regression of Corpora Lutea in Wistar Hannover Rats: the Comparisons with Sprague-Dawley Rats

We examined the ovaries of 44 Wistar Hannover (RccHan^TM:WIST) (WH) and 30 Sprague-Dawley (SD) rats at 32-weeks of age to determine whether the ovarian structure and formation/regression of the corpora lutea (CLs) differ between the two strains. The average ovary weight was higher in WH rats. The average number of all CLs, including currently formed and previously formed CLs, was higher in WH rats in all cycles; however, no appreciable difference was detected in the number of newly or currently formed CLs between the two strains. CLs regression characterized by degeneration and necrosis of luteal cells began to appear in diestrus in both strains; however, the distribution of degenerated/necrotic cells in CLs differed. Necrotic cells were scattered in SD rats but were focally observed in the center of the CL in WH rats. The reduction in size of previously formed CLs accompanied by regression started about 2 or more stages later in WH rats than in those of SD rats. In conclusion, the higher number of CLs in WH rats is considered to be due to slow CL regression compared with in SD rats.     

Effect of food amount and temperature on growth rate and aerobic scope of juvenile chum salmon

Offshore migration of Pacific salmon Oncorhynchus spp. is partly triggered by increasing body size and high motility in the early stages of life. The survival of juvenile salmon may depend on their growth rate during the first few months in the sea, and this factor partly regulates the dynamics of adult populations. Here, we assessed the effects of water temperature and food availability on the growth of juvenile chum salmon O. keta. In addition, by combining the measurements of metabolic performance for growth and activity (Absolute Aerobic Scope: AAS) with a bioenergetics model, we estimated the energy allocation for different activities in the juveniles. Under high temperatures (14 °C), juveniles reared at low food levels (1% body weight) allocated less than half their energy for growth than those reared at high food levels (4% body weight). These findings suggest that high temperature and low food level constrain the growth of juveniles, providing an insight into the effect of the recent increase in warm and low-nutrient water masses on survival of juveniles and catches of adult chum salmon on the Pacific side of Honshu Island, Japan.

     

Construction of a linkage map based on retrotransposon insertion polymorphisms in sweetpotato via high-throughput sequencing

Sweetpotato (Ipomoea batatas L.) is an outcrossing hexaploid species with a large number of chromosomes (2n = 6x = 90). Although sweetpotato is one of the world’s most important crops, genetic analysis of the species has been hindered by its genetic complexity combined with the lack of a whole genome sequence. In the present study, we constructed a genetic linkage map based on retrotransposon insertion polymorphisms using a mapping population derived from a cross between ‘Purple Sweet Lord’ (PSL) and ‘90IDN-47’ cultivars. High-throughput sequencing and subsequent data analyses identified many Rtsp-1 retrotransposon insertion sites, and their allele dosages (simplex, duplex, triplex, or double-simplex) were determined based on segregation ratios in the mapping population. Using a pseudo-testcross strategy, 43 and 47 linkage groups were generated for PSL and 90IDN-47, respectively. Interestingly, most of these insertions (~90%) were present in a simplex manner, indicating their utility for linkage map construction in polyploid species. Additionally, our approach led to savings of time and labor for genotyping. Although the number of markers herein was insufficient for map-based cloning, our trial analysis exhibited the utility of retrotransposon-based markers for linkage map construction in sweetpotato.     

A RAPD-derived STS marker is linked to a bacterial wilt (Burkholderia caryophylli) resistance gene in carnation

Bacterial wilt caused by Burkholderia caryophylli is one of the most important and damaging diseases of carnations (Dianthus caryophyllus) in Japan. We aimed to identify random amplified polymorphic DNA (RAPD) markers associated with the genes controlling bacterial wilt resistance in a resistance-segregating population of 134 progeny plants derived from a cross between Carnation Nou No. 1 (a carnation breeding line resistant to bacterial wilt) and Pretty Favvare (a susceptible cultivar). We screened a total of 505 primers to obtain RAPD markers useful for selecting resistant carnation lines: 8 RAPD markers identified by bulked segregant analysis were linked to a major resistance gene; of these, WG44-1050 had the greatest effect on resistance to bacterial wilt. A locus with large effect on bacterial resistance was mapped around WG44-1050 through QTL analysis. The RAPD marker WG44-1050 was successfully converted to a sequence-tagged site (STS) marker suitable for marker-assisted selection (MAS). Five combinations of primers were designed for specific amplification of WG44-1050. In addition, the STS marker we developed was useful and reliable as a selection marker for breeding for resistance to bacterial wilt, using a highly resistant wild species, D. capitatus ssp. andrzejowskianus and a resistant line, Carnation Nou No. 1, as breeding materials.