Isolation,long-term culture,and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells

Summary A long-term cell culture system for adult cardiomyopathic hamster cardiac muscle cells has been established. The diseased and control hearts were dissociated into single cell suspension with the modifications of our previous technique using collagenase and hyaluronidase as applied to the dissociation of the adult rat heart. The postperfusion of the diseased heart with Krebs-Ringer phosphate buffer and bovine serum albumin was very helpful in obtaining greater yield of viable diseased muscle cells; the cells were cultured for 4 wk. Approximately 60% of the myocytes from the diseased heart and 85% of the myocytes from the normal heart attached to the substrates and survived throughout the culture period. Approximately 60 to 70% of the cardiac myocytes from the diseased and control hearts were bi- or multinucleated; 30% of the diseased and 80% of the normal myocytes showed rhythmic contractility. Electron microscopy revealed the presence of two kinds of cardiac muscle cells in the diseased cell culture on the basis of their myofibril content: one with scanty myofibrils and another with abundant myofibrils. Myocytes with sparse myofibrils showed certain characteristic features that included autophagic vacuoles, amorphous matrix of fine filamentous texture, scattered strips of myofibrils, and abnormal organization of the Z-line. Cardiac muscle cells with abundant myofibrillar content contained unorganized myofibrils in certain sarcomeres. These studies demonstrate the feasibility of maintaining diseased cardiac muscle cells from adult cardiomyopathic hamsters for at least 4 wk in monolayer culture. This study was supported by a grant from the American Heart Association of Michigan, National Institutes of Health grant HL-25482, and by an Oakland University Biomedical Research Support Grant.     

A useful pathological condition of wood

A fungus known asCytosphaera mangiferae Died, was isolated for the first time from the diseased tissues of standing trees ofAquilaria agallocha Roxb. Irregular patches of diseased wood, a result of natural infection, are found in the trunks of standingA. agallocha trees, which are of great economic importance. A fragrant perfume locally known as ‘attar’ is obtained from the diseased wood by steam distillation. Incomplete or partially diseased wood is employed in the preparation of a joss-stick locally known as ‘agarbatee,’ which gives out fragrant fumes on burning. The fungus colonised wood blocks of A agallocha when these were inoculated artificially. There is a possibility of commercial exploitation for the production of diseased wood by artificial infection.     

The haematological assessment of bacterial infection in rainbow trout, Salmo gairdneri Richardson

The pH, pCO2 and pO2 values for diseased fish did not differ significantly from those values for healthy fish, but fish treated with terramycin showed a highly significant rise in pCO2 with a corresponding fall in pO2. Haematocrit values, as well as haemoglobin and total plasma protein concentrations were significantly lower in diseased fish while treated fish showed a significant rise in total plasma protein. Glucose concentrations fell very significantly in diseased fish and showed a significant increase in treated fish.
The plasma electrolytes, sodium, potassium and chloride, were significantly reduced in diseased fish and potassium levels also fell in treated fish. Diseased fish showed very significantly reduced erythrocyte counts. Of the three parameters MCV, MCH and MCHC, only the values for MCH in diseased fish differed significantly from those for healthy fish.     

Two-dimensional proton magnetic resonance of human muscle extracts

In continuation of our previous work on one-dimensional (1D) proton nuclear magnetic resonance (1H-NMR) of normal and diseased human muscle extracts we recorded the two-dimensional (2D) J-correlated proton magnetic resonance spectra of these extracts. Significant differences between normal and diseased muscle extracts, not observed in the 1D 1H-NMR spectra, were seen from their 2D connectivity contour patterns. Taurine was not present in cerebral palsy muscle extract while both normal and scoliosis muscles contained this metabolite. Only the normal muscle had carnitine. Carnosine was present in all muscles. alpha-Ketoglutarate was found only in the diseased muscle extracts. While the amino acids lysine, cysteine and glutamine were common to normal and diseased muscles, threonine was seen only in the diseased muscles. Additional small differences were detected in the 2D patterns of human muscle extracts.     

Diseases affect cold-water corals too: Eunicella verrucosa (Cnidaria: Gorgonacea) necrosis in SW England

The first recorded incidence of cold-water coral disease was noted in Eunicella verrucosa, a coral on the international 'red list' of threatened species, at a marine protected area in SW England in 2002. Video surveys of 634 separate colonies at 13 sites revealed that disease outbreaks were widespread in SW England from 2003 to 2006. Coenchyme became necrotic in diseased specimens, leading to tissue sloughing and exposing skeletal gorgonin to settlement by fouling organisms. Sites where necrosis was found had significantly higher incidences of fouling. No fungi were isolated from diseased or healthy tissue, but significantly higher concentrations of bacteria occurred in diseased specimens. Of 21 distinct bacteria isolated from diseased tissues, 19 were Vibrionaceae, 15 were strains of Vibrio splendidus and 2 others closely matched Vibrio tasmaniensis. Vibrios isolated from E. verrucosa did not induce disease at 15 degrees C, but, at 20 degrees C, controls remained healthy and test gorgonians became diseased, regardless of whether vibrios were isolated from diseased or healthy colonies. Bacteria associated with diseased tissue produced proteolytic and cytolytic enzymes that damaged E. verrucosa tissue and may be responsible for the necrosis observed. Monitoring at the site where the disease was first noted showed new gorgonian recruitment from 2003 to 2006; some individuals had died and become completely overgrown, whereas others had continued to grow around a dead central area.     

Pollen dispersal is driven by pollinator response to plant disease and plant spatial aggregation

Most plant species are pollinated by animals, mainly insects, who adjust their foraging behaviour to the spatial distribution of rewards. Any changes in rewards of individual plants could then affect pollen dispersal at the level of plant patches or populations. Such change in floral rewards often results from infection by plant pathogens, for example by anther smuts (i.e. no pollen and reduced nectar in diseased flowers). Here, we tested the hypothesis that the infection of plant populations by anther smuts affects the pattern of pollen dispersal.We investigated the patterns of pollen dispersal in experimental arrays of potted plants differing in the presence of diseased plants and the degree of plant spatial aggregation. We tracked pollen dispersal using a fluorescent dye powder as a pollen analogue, while we simultaneously observed pollinator foraging behaviour.We found that the dispersal of the pollen analogue increased in the presence of diseased plants in experimental arrays, but this effect was strongly dependant on plant spatial aggregation. The parallel observations of pollinator behaviour suggest that this pattern resulted from pollinator discrimination against diseased plants and increased movement in arrays with intermingled diseased plants, provided that plant clusters were close to each other.Our study indicates that pollinators respond to diseased plants in a similar way as to healthy plants with low rewards. Consequently, diseased plants should be treated not only as a potential source of infection but also as a factor influencing pollen dispersal in plant populations.     

健康与凋萎病杨梅树体及根围菌群的差异

凋萎病是制约杨梅产业发展的严重病害。为了有效防控凋萎病,本研究分析了杨梅健康和感染凋萎病树体各部位及根表土和根围土中细菌和真菌群落的丰富度与多样性的差异。结果表明: 与健康树相比,病树根围土、根表土、根、枝干、枝皮和叶片的细菌和真菌丰富度均发生了显著变化,其中,根表土细菌和枝皮内真菌的丰富度和多样性均显著降低,而枝皮内细菌和根表土的真菌丰富度和多样性均显著升高。病树各部位及根表、根围土细菌和真菌的优势菌相对丰度在门、纲和属水平上发生了明显的变化,在病树枝干、根和根表土中的假单胞菌属及根表土、根围土中的镰刀菌属的相对丰度明显降低,病树根表土及根围土中青霉菌属的相对丰度明显增加。与凋萎病菌同属的拟盘多毛孢菌在病树根内显著减少,而在其他位置均大量增殖,其相对丰度与多数相对丰度较高的真菌呈正相关。本研究结果将为开发杨梅凋萎病的生态改良、培育健康树体和生物防治技术提供有效的理论依据。     

Occurrence of Peroxidases in Sweet Potato Infected by the Black Rot Fungus

Changes in the peroxidase activity and the patterns in sweet potato tissue infected by Ceratocystis fimbriata were investigated, by the method of starch-gel electrophoresis, DEAE-cellulose column chromatography and immunochemical analysis, compared with that in cut and healthy tissue. Time-course analysis of the increment of the total peroxidase activity also showed noticeable differences between diseased and cut tissues. The peroxidases in diseased and cut tissues were composed of four major and several minor components. Though electrophoretic analysis did not show so striking differences in the patterns of peroxidases between diseased and cut tissues, there were distinct differences in the ratio of activities of major peroxidase components between both tissues. Immunochemical works indicated that peroxidase A which showed the most prominent increase in diseased tissue was found to be formed in cut tissue though the amounts were appreciably small. The activity of peroxidase C in diseased tissue was not so high as seen in cut tissue.     

Fungi in healthy and diseased sea fans (<Emphasis Type="Italic">Gorgonia ventalina)</Emphasis>: is <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> always the pathogen?

Caribbean corals, including sea fans (Gorgonia spp.), are being affected by severe and apparently new diseases. In the case of sea fans, the pathogen is reported to be the fungus Aspergillus sydowii, and the disease is named aspergillosis. In order to understand coral diseases and pathogens, knowledge of the microbes associated with healthy corals is also necessary. In this study the fungal community of healthy Gorgonia ventalina colonies was contrasted with that of diseased colonies. In addition, the fungal community of healthy and diseased tissue within colonies with aspergillosis was contrasted. Fungi were isolated from healthy and diseased fans from 15 reefs around Puerto Rico, and identified by sequencing the nuclear ribosomal ITS region and by morphology. Thirty fungal species belonging to 15 genera were isolated from 203 G. ventalina colonies. Penicillum and Aspergillus were the most common genera isolated from both healthy and diseased fans. However, the fungal community of healthy fans was distinct and more diverse than that of diseased ones. Within diseased fans, fungal communities from diseased tissues were distinct and more diverse than from healthy tissue. The reduction of fungi in diseased colonies may occur prior to infection due to environmental changes affecting the host, or after infection due to increase in dominance of the pathogen, or because of host responses to infection. Data also indicate that the fungal community of an entire sea fan colony is affected even when only a small portion of the colony suffers from aspergillosis. An unexpected result was that A. sydowii was found in healthy sea fans but never in diseased ones. This result suggests that A. sydowii is not the pathogen causing aspergillosis in the studied colonies, and suggests several fungi common to healthy and diseased colonies as opportunistic pathogens. Given that it is not clear that Aspergillus is the sole pathogen, calling this disease aspergillosis is an oversimplification at best. Communicated by Biology Editor Dr Michael Lesser     

Anther-smut infection of Silene alba caused by Ustilago violacea: factors determining fungal reproduction

Summary The anther-smut fungus Ustilago violacea sporulates in flowers of the dioecious host plant Silene alba. Growth chamber comparisons of healthy and diseased plants, with the genetic background of host and pathogen controlled, revealed that fungal infection increases the number of flowers produced per plant and decreases the size of individual flowers. There were few consistent effects of plant genotype or fungal isolate on diseased flower traits, but differences between the plant sexes were apparent. Stimulation of flower production is proportionally greater in females than males: thus, although healthy male plants produce many more flowers than healthy females, sexual differences in diseased flower number are reduced. Sexual differences in diseased flower size also exist, with male flowers smaller than females. A field inoculation study confirmed dimorphism in diseased flower size and demonstrated that spore production per flower was greater for males than females for all flower size classes.     

Rhizosphere mycoflora of healthy and yellow vein mosaic virus infected okra (Abelmoschus esculentus) plants.

Investigations on the rhizosphere mycoflora of healthy and virus (YVMV) infected okra plants showed a higher fungal population in the rhizosphere of healthy plants at preflowering and post-flowering stages than in that of diseased ones. Maximum population was observed during flowering both in healthy and diseased plant rhizosphere as well as in non-rhizosphere soil. However, virus infected plants showed a higher population at the flowering stage than healthy ones. The quantitative differences in the rhizosphere of healthy and diseased plants during flowering seem to be due to a change in C/N ratio and amino acids. The drastic reduction in diseased plant rhizospheres during the post-flowering stage may be due to either change in C/N ratio unfavourable to mycoflora or production of some toxic substances inhibiting multiplication of the mycoflora.     

Composition and activity of rhizosphere microbial communities associated with healthy and diseased greenhouse tomatoes

Aims

The goal of this study was to investigate the structure and functional potential of microbial communities associated with healthy and diseased tomato rhizospheres.

Methods

Composition changes in the bacterial communities inhabiting the rhizospheric soil and roots of tomato plants were detected using 454 pyrosequencing. Microbial functional diversity was investigated with BIOLOG technology.

Results

There were significant shifts in the microbial composition of diseased samples compared with healthy samples, which had the highest bacterial diversity. The predominant phylum in both diseased and healthy samples was Proteobacteria, which accounted for 35.7–97.4 % of species. The class Gammaproteobacteria was more abundant in healthy than in diseased samples, while the Alphaproteobacteria and Betaproteobacteria were more abundant in diseased samples. The proportions of pathogenic Ralstonia solanacearum and Actinobacteria species were also elevated in diseased samples. The proportions of the various bacterial populations showed a similar trend both in rhizosphere soil and plant roots in diseased versus disease-free samples, indicating that pathogen infection altered the composition of bacterial communities in both plant and soil samples. In terms of microbial activity, functional diversity was suppressed in diseased soil samples. Soil enzyme activity, including urease, alkaline phosphatase and catalase activity, also declined.

Conclusions

This is the first report that provides evidence that R. solanacearum infection elicits shifts in the composition and functional potential of microbial communities in a continuous-cropping tomato operation.     

Cations in Hemolymph and Alimentary Tract Tissues of Healthy and Milky Diseased European Chafer (Amphimallon majalis) Larvae

A study was made of certain cations present in hemolymph and alimentary tract tissues of healthy and diseased European chafer larvae and the spores of Bacillus popilliae collected from diseased hemolymph. The major ions found in the hemolymph, in order of decreasing abundance, were potassium, magnesium, sodium, and calcium. Hemolymph of diseased larvae contained relatively higher concentrations of sodium, potassium, magnesium, iron, and zinc than hemolymph of healthy larvae. Concomitantly, the concentrations of ions were lower in the mid-gut and anterior intestinal tissues of diseased larvae. Only sodium decreased slightly in the diseased tissues of the rectum and rectal sac; other ions remained unchanged or increased. Little or no manganese or copper was detected in the hemolymph or tissues. The major cations of spores of B. popilliae were sodium, calcium, and magnesium. Small amounts of potassium, manganese, copper, iron, and zinc were detected in the spores. Based on calcium and dipicolinic acid determinations of the spores, sufficient calcium was found to allow for the formation of calcium dipicolinate in the expected concentrations.     

Changes in the composition of volatile compounds of Spartium junceum induced by the phytoplasmal disease,Spartium witches’‐broom

Abstract

In southern Italy, Spartium junceum (Spanish broom) is severely affected by a phytoplasmal disease, Spartium witches’‐broom (SpaWB). The volatile fractions extracted from flowers of healthy and diseased plants, examined by gas chromatography and gas chromatography–mass spectrometry, appeared to be quantitatively and qualitatively different. In both the healthy and the diseased plants, the main components were n‐alkanes, which occurred at a rate of 55.2% and 38.8%, respectively. The level of aliphatic acids was considerably lower in flowers of the diseased plants than in those of the healthy plants (4.5% vs. 18.7%). Sesquiterpenes were detected only in the diseased plants. It is possible that the changes in the composition of secondary metabolites of diseased plants can be related to plant defense responses.

Abbreviations: AP, apple proliferation; EY, elm yellows; SpaWB, Spartium witches’‐broom     

Effect of epidermal papillomatosis on survival of the freshwater fish Rutilus rutilus

Epidermal papillomatosis occurs in several marine and freshwater fish species. Previously, papillomatosis has been shown to induce mortality in juvenile carp. We studied the effect of epidermal papillomatosis on the survival of adult male roach Rutilus rutilus by caging naturally diseased, marked (by us) fish in the field. Within the constraints of the experimental design, there was no difference in survival between healthy, slightly diseased and heavily diseased fish. Therefore, we conclude that the possible effect of epidermal papillomatosis on the mortality of wild roach is relatively minor.     

Drug Resistance in Strains of Escherichia Coli Isolated from the Intestinal Tract of Pigs in Norway

Faecal samples from 95 healthy pigs and samples of jejunal content from 85 piglets suffering from colienterotoxaemia were tested for the presence of drug resistant E. coli strains. Practically all pigs in both groups harboured E. coli strains resistant to one or more of the 6 antibiotics/chemotherapeutic agents tested (Oxytetracycline, streptomycin, sulphaisodimidin, neomycin, ampicillin, chloramphenicol). Almost 100% of healthy and approx. 90% of diseased pigs harboured strains resistant to Oxytetracycline, streptomycin and sulphaisodimidin. Pigs with strains resistant to neomycin, ampicillin and chloramphenicol were less frequently found. The predominant coliform flora consisted of E. coli strains” resistant to Oxytetracycline, streptomycin and sulphaisodimidin in 71% to 81% of diseased pigs and in 47% to 69% of the healthy pigs. In diseased pigs ¾ of the animals had a coliform flora dominated by neomycinresistant E. coli strains. Of the 721 resistant E. coli strains isolated from healthy pigs, 11% were single resistant while the corresponding figure for the 518 resistant strains isolated from diseased pigs was 6%. Thus 89% and 94% of strains showed simultaneous resistance to 2 or more antibiotics. E. coli strains resistant to 3 or more drugs were found in approx. 60% and 70% of the isolates from healthy and diseased animals, respectively. Oxytetracycline/streptomycin/sulphaisodimidin resistance was most commonly found, approx. 22% and 38% of the strains from healthy and diseased pigs, respectively, showing this resistance pattern. Transmission of drug resistance which was examined in E. coli strains originating from the diseased pigs was demonstrated in approx. 76% of the isolates. The incidence of drug resistance transfer in single, double, triple and quadruple resistant strains was 11%, 68%, 97% and 98%, respectively.     

Characterization of the Constituent Fatty Acids in the Phosphoglycerides of Chemically Induced Polycystic Disease and Gastric Tumor in Rat

The positional distribution of fatty acids in choline- and ethanolamine phosphoglycerides from the livers of polycystic disease-bearing rats and stomachs of gastric tumor-bearing rats was analyzed and compared to that in normal rat liver and stomach. Polycystic disease and gastric tumors were induced with N-Methyl-N'-Nitro-N-Nitrosoguanidine. There was no significant difference between diseased and normal organs with respect to the composition of fatty acids at the 1-position, except that diseased livers exhibited an increase of approximately 22% in 16: 0 fatty acid and a decrease of approximately 12% in 18: 1 fatty acid. However, in diseased organs, there were marked quantitative changes in the positional distribution of fatty acids at the 2-position, their composition exhibiting greater saturation. Furthermore, in diseased organs, there was an increase in 15: 0 fatty acid at the 2-position of ethanolamine phosphoglycerides.     

A laboratory study of the effects of cytoplasmic polyhedrosis virus on Heliothis virescens (Lepidoptera: Noctuidae)

Larvae of Heliothis virescens reared from eggs contaminated with cytoplasmic polyhedrosis virus weighed 50% less than those reared from virus-free eggs. The larval stage in a virus-infected group was 4 days longer than that of a healthy group, and diseased pupae weighed an average of 60 mg less than healthy pupae. Furthermore, adult emergence was reduced 10% in virus-infected pupae, diseased moths lived 9 days less than healthy moths, and production of eggs was reduced 68% in diseased females.     

Changes in double-stranded RNA profiles in Agaricus bisporus during subculture

Abstract A complex double-stranded RNA (dsRNA) pattern found in mycelial cultures of diseased basidiocarps comprised nine major segments between 0.78–3.6 kbp and four minor segments between 8.8–15 kbp. With extensive subculturing, a new pattern of five segments between 1.5–8.8 kbp was detected in cultures expressing the diseased phenotype. Loss of the 1.5 and 3.6 kbp segments following heat therapy at 30°C was associated with remission of symptoms. Some of the dsRNAs co-purified with mitochondria in extracts from diseased cultures and basidiocarps.     

长春花内生细菌多样性与柑橘黄龙病菌的相关性

【目的】分析感柑橘黄龙病长春花植株与健康长春花植株不同部位内生细菌菌群结构变化,为柑橘黄龙病菌与长春花内生细菌的相关性研究提供理论基础。【方法】本研究利用兼性厌氧可培养技术、16S rDNA限制性片段长度多态性分析(Restriction fragment length polymorphism,RFLP)以及16S rDNA序列分析相结合的方法。【结果】分别从感病和健康长春花叶、茎、根的组织中分离获得67株内生细菌,与GenBank中29种细菌的相似性达到97%-100%。其中短小杆菌属(Curtobacterium sp.)、欧文氏菌属(Erwinia sp.)、蜡样芽胞杆菌(Bacillus cereus)为感病长春花内生细菌的优势菌群,鞘胺醇单胞菌属(Brevundimonas sp.)、芽胞杆菌属(Bacillus sp.)为健康长春花内生细菌的优势菌群;马胃葡萄球菌(Staphylococcus equorum)为两者的共同优势菌群。通过RFLP方法分析,感病株得到16个、健株得到23个操作分类单元(Operational TaxonomicUnits,OTUs),感病植株中除柑橘黄龙病菌Candidatus Liberibacter asiaticus外,还有丰富的CandidatusLiberibacter sp.存在。【结论】感病与健康长春花植株中均含有丰富的内生细菌,黄龙病菌的存在改变了长春花原有内生细菌的菌群结构,且菌群多样性下降。可见长春花内生细菌在一定程度上受到柑橘黄龙病菌的抑制。