Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens

ABSTRACT The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.     

Genetic and functional leaf traits variability of Quercus laurina along an oak diversity gradient in Mexico

The ecological literature has documented the effects of plant hybridization on phenotypic variation, and dominant, intermediate, or novel morphological, chemical and physiological traits in hybrids. It is important to understand the ecological consequences of hybridization by evaluating their impact on phenotypic expression of functional traits. We evaluated the relationship between genetic diversity of Quercus laurina and functional foliar traits along an oak diversity gradient. We selected five study sites that represent an oak diversity gradient where Q. laurina is present. Using chloroplast and nuclear microsatellites, we evaluated genetic diversity, measured functional foliar traits of Q. laurina in each site and assessed the effects of local climate variables on the oak community and functional traits. We found a greater abundance of Q. laurina in all study sites. We did not find a relationship between the number of accompanying red oak species and the population genetic diversity in Q. laurina, but higher genetic diversity was found in all study sites in comparison with European oak species. Sites with more oak species had more variation of foliar functional traits. Our results do not support the hypothesis that predicts higher levels of genetic diversity of Q. laurina in communities with greater oak diversity from the same section, but we demonstrated an increase in the foliar functional traits of Q. laurina associated with oak richness and climate variables. We highlight the need to consider environmental and ecological variables linkages as regulatory mechanisms of the phenotypic plasticity expressed in changes of some functional attributes of oaks.

     

An investigation of the effects of ketoprofen following rumen fistulation surgery in lactating dairy cows

Post-operative pain management following rumen surgery is not common practice. We examined the effect of providing the pain medication ketoprofen to dairy cattle following the first stage of a rumen cannulation surgery, which involves an incision in the body wall and exteriorizing and clamping the rumen. The results of this study provide clear evidence that the first stage of the surgery was painful and ketoprofen at the time of and 24 h following surgery, alleviated some, but not all, of the post-surgical pain. Pain mitigation should be included when performing flank surgery in cattle.     

Herd diagnosis of low pathogen diarrhoea in growing pigs – a pilot study

Background

The major indication for antibiotic use in Danish pigs is treatment of intestinal diseases post weaning. Clinical decisions on antibiotic batch medication are often based on inspection of diarrhoeic pools on the pen floor. In some of these treated diarrhoea outbreaks, intestinal pathogens can only be demonstrated in a small number of pigs within the treated group (low pathogen diarrhoea). Termination of antibiotic batch medication in herds suffering from such diarrhoea could potentially reduce the consumption of antibiotics in the pig industry. The objective of the present pilot study was to suggest criteria for herd diagnosis of low pathogen diarrhoea in growing pigs.Data previously collected from 20 Danish herds were used to create a case series of clinical diarrhoea outbreaks normally subjected to antibiotic treatment. In the present study, these diarrhoea outbreaks were classified as low pathogen (<15% of the pigs having bacterial intestinal disease) (n =5 outbreaks) or high pathogen (≥15% of the pigs having bacterial intestinal disease) (n =15 outbreaks). Based on the case series, different diagnostic procedures were explored, and criteria for herd diagnosis of low pathogen diarrhoea were suggested. The effect of sampling variation was explored by simulation.

Results

The diagnostic procedure with the highest combined herd-level sensitivity and specificity was qPCR testing of a pooled sample containing 20 randomly selected faecal samples. The criteria for a positive test result (high pathogen diarrhoea outbreak) were an average of 1.5 diarrhoeic faecal pools on the floor of each pen in the room under investigation and a pathogenic bacterial load ≥35,000 per gram in the faecal pool tested by qPCR. The bacterial load was the sum of Lawsonia intracellularis, Brachyspira pilosicoli and Escherichia coli F4 and F18 bacteria per gram faeces. The herd-diagnostic performance was (herd-level) diagnostic sensitivity =0.99, diagnostic specificity =0.80, positive predictive value =0.94 and negative predictive value =0.96.

Conclusions

The pilot study suggests criteria for herd diagnosis of low pathogen diarrhoea in growing pigs. The suggested criteria should now be evaluated, and the effect of terminating antibiotic batch medication in herds identified as suffering from low pathogen diarrhoea should be explored.

Electronic supplementary material

The online version of this article (doi:10.1186/2046-0481-67-24) contains supplementary material, which is available to authorized users.     

不同产区宁夏枸杞果实的主成分分析与综合评价

以宁夏、青海、新疆3个产区的宁夏枸杞品种为试验材料,分析其外在品质(百粒质量、果形指数)和内在品质(多糖、甜菜碱、胡萝卜素)的差异,利用主成分分析建立综合评价模型进行品质综合评价。结果表明,不同产区的宁夏枸杞品质存在差异。主成分分析提取了前3个主成分,累计方差贡献率为91.71%。主成分综合评价结果表明,青海产区宁杞5号、1号和宁夏产区宁杞7号果实品质较好。     

Suppression of fish meat softening by strict control of storage temperature

ABSTRACT:   Changes in meat firmness and structure of cultured yellowtail at several storage temperatures, from −1.5°C to 10°C, were compared. Firmness decreased less at −1.5°C and 10°C than at the other temperatures. During storage at temperatures from −0.5 to 4.0°C, expansion of intercellular space, which indicates weakening of the connecting force between muscle cells, began to increase after 8 h storage, and the ratio of space to the whole area increased by 3.4–4.9 times after 24 h of storage. However, at −1.5°C and 10°C, the increase of intercellular space was suppressed, and the ratio was 2.4–2.6 times, even after 24 h storage. Collagen fibrils connecting muscle cells disintegrated after 24 h storage at all storage temperatures. Conversely, in 10°C storage, the fine structure of muscle, except for collagen fibrils, was maintained better than at other storage temperatures. These findings indicate that −1.5 and 10°C storage could maintain fish-meat quality for longer periods than storage at −0.5, 1.0, or 4.0°C, the normal refrigeration temperature.     

Isolation of chicken taste buds for real‐time Ca2+ imaging

We isolated chicken taste buds and used a real‐time Ca²⁺ imaging technique to investigate the functions of the taste cells. With RT‐PCR, we found that isolated chicken taste bud‐like cell subsets express chicken gustducin messenger RNA. Immunocytochemical techniques revealed that the cell subsets were also immunopositive for chicken gustducin. These results provided strong evidence that the isolated cell subsets contain chicken taste buds. The isolated cell subsets were spindle‐shaped and approximately 61–75 μm wide and 88–98 μm long, and these characteristics are similar to those of sectional chicken taste buds. Using Ca²⁺ imaging, we observed the buds' response to 2 mmol/L quinine hydrochloride (a bitter substance) and their response to a mixture of 25 mmol/L L‐glutamic acid monopotassium salt monohydrate and 1 mmol/L inosine 5′‐monophosphate disodium salt, umami substances. The present study is the first morphological demonstration of isolated chicken taste buds, and our results indicate that the isolated taste buds were intact and functional approaches for examining the taste senses of the chicken using Ca²⁺ imaging can be informative.