Heat resistance,biology and prevention of Diehliomyces microsporus in crops of Agaricus species

Soon after its introduction the mushroom speciesAgaricus bitorquis, which is immune to virus disease and prefers a warm climate, was threatened by the competitorDiehliomyces microsporus, false truffle. This fungus also likes warmth, and used to occur in crops ofA. bisporus.Mycelium and ascocarps were grown on several nutrient media. Optimum temperatures for mycelial growth were 26°C and 32°C, with a slight depression at 30°C. In trials in isolated growingrooms strain Somycel 2.017 ofA. bitorquis was generally used since it appeared to be highly sensitive to the competition of false truffle. Inoculation with mycelium, ascocarps or ascospores ofD. microsporus nearly always resulted in the presence of the competitor and in decreased mushroom yields. Even ten spores per m² causedD. microsporus. The time of inoculation was most important: irrespective of the kind of inoculum, inoculation only resulted in both false truffle and yeild loss, if applied from spawning until a few days after casing. Inoculation at a later date could result in false truffle, but yield was not decreased.As germination in vitro of ascospores failed, even after addition of various triggers, ascospore suspensions were treated at various temperatures for several periods. Then mushroom growing trays spawned with Somycel 2.017 were inoculated with the treated suspensions giving 7–11×10⁷ spores/m². The ascospores could not withstand 85°C for 0.5 h, 80°C for 1 h and 70°C for 3 h. Spontaneous incidence of false truffle, however, could not always be prevented and interfered with the results of these trials. It is possible that the thermal death-point of the ascospores is below 85°C. Fruiting bodies and ascospores did not survive peak-heating at the beginning and cooking out (compost temperature 12 h at 70°C) at the end of a crop. After cooking out, however,D. microsporus could still be present in the wood of trays and contaminate a following crop if no wood preservative was applied.Yield of Somycel 2.017 was reduced by the competition ofD. microsporus much more than yeilds of other strains ofA. bitorquis. The least sensitive were the highly productive strains Horst K26 and Horst K32.The effects of fungicides onD. microsporus in vitro and in growing trials did not correspond. The fungicides tested so far could not prevent or controlD. microsporus. Growing of less sensitive strains ofA. bitorquis together with sanitary measures early in the crop and at the end of the crop, however, can prevent the competitor. failure to turn up of false truffle. To understand the discrepancy between the in vitro effects of several fungicides and their effect in inoculated mushroom trays, the rate of adsorption of benomyl in the substrate and probably the interrelationships between antagonists andD. microsporus require further research. Other strains ofA. bitorquis than Somycel 2.017 appeared to be less sensitive to the competition. Among these, highly productive strains Horst K26 and Horst K32 will not be hindered byD. microsporus if the following precautions are exercised: cooking out at the end of a crop (compost temperature 70°C for 12 hours), followed by treatment of the wood with SPCP; protection by hygiene early in the crop, i.e. covering of the compost by a thin plastic sheet during mycelial growth followed by a quick execution of casing.Samenvatting De teelt van de warmteminnende champignonsoortAgaricus bitorquis, die immuun is voor virusziekte, werd al spoedig na introductie bedreigd door de eveneens warmteminnende concurrentDiehliomyces microsporus, valse truffel. Deze schimmel kwam vroeger voor in teelten vanA. bisporus; de sporen zouden een temperatuur van 82°C gedurende 5 uur kunnen overleven (Lambert, 1932). Tabel 1 geeft de myceliumgroei op verschillende voedingsbodems en de vorming van vruchtlichamen (Fig. 1A, B) weer. De optimale temperaturen voor myceliumgroei waren 26°C en 32°C, met een licht depressie bij 30°C (Fig. 2). Proeven in geïsoleerde teeltruimten werden voornamelijk uitgevoerd met Somycel 2.017, een ras vanA. bitorquis. Inoculatie met mycelium, vruchtlichamen en/of ascosporen vanD. microsporus, al of niet in reincultuur gekweekt, leidde vrijwel steeds tot de aanwezigheid van de concurrent in de geïnoculeerde teeltkisten (Fig. 1C, D), waarbij vruchtlichamen met ascosporen (Fig. 1E) gevormd werden en tot een reductie van het aantal champignons. Tien sporen per m² waren al voldoende omD. microsporus te doen aanslaan (Fig. 3). Het tijdstip van inoculatie bleek van groot belang te zijn: onafhankelijk van de aard van het inoculum leverde dit slechts zowel valse truffel als oogstreductie op, indien het werd aangebracht in de periode vanaf enten tot enkele dagen na het afdekken (Tabel 2 en Fig. 4). Inoculatie op latere tijdstippen kon wel tot valse truffel leiden, maar niet tot oogstreductie.Aangezien de kieming van ascosporen in vitro slechte resultaten opleverede, ook na toevoeging van diverse stimulantia, werden ascosporensuspensies in vitro gedurende verschillende tijden bij verschillende temperaturen behandeld; vervolgens werden teeltkisten met de behandelde suspensies geïnoculeerd (7 tot 11×10⁷ sporen/m²). De kisten waren tevoren geënt met Somycel 2.017. Een aantal proeven wees uit, dat de ascosporen 1/2 uur 85°C, 1 uur 80°C en 3 uur 70°C, niet overleefden (Tabel 3). Het spontaan optreden van valse truffel kon echter niet altijd worden voorkomen en beïnvloedde de uitkomsten van deze proeven. Daarom is het mogelijk, dat de sporen al bij een lagere temperatuur worden gedood Vruchtlichamen en ascosporen werden gedood door het uitzweten aan het begin van een teelt en door het doodstomen aan het einde van een teelt (composttemperatuur 12 uur 70°C) maar de schimmel bleek in het laatste geval wel over te kunnen blijven in het hout van teeltkisten als er vervolgens geen houtontsmettingsmiddel werd toegepast.Somycel 2.017 leed verhoudingsgewijs meer schade door concurrentie vanD. microsporus dan enkele andere rassen (Tabel 4 en. 5). Inoculatie met ascosporen bleek bij de minst gevoelige en meest produktieve rassen Horst K26 en Horst K32 slechts te gelukken in extreem droge compost; bij Somycel 2.017 daarentegen zowel in compost met een laag als met een hoog vochtgehalte. Inoculatie met mycelium veroorzaakte meer valse truffel en meer schade naarmate de compost natter was (Tabel 5).De werking van een aantal fungiciden in vitro (Tabel 6) en in teeltkisten (Tabel 7) stemde niet overeen. Aangezien de tot nu toe getoetste fungicidenD. microsporus niet kunnen voorkomen of bestrijden, moet preventie van deze concurrent worden gezocht in het telen van weinig gevoelige rassen vanA. bitorquis in combinatie met hygiënische maatregelen vroeg in en aan het eind van de teelt.     

CONTRAST-ENHANCED ULTRASONOGRAPHY IN NORMAL CANINE LIVER. EVALUATION OF IMAGING AND SAFETY PARAMETERS

Contrast-enhanced ultrasonography, a new imaging modality in veterinary medicine, can provide data on tissue perfusion. The objective of this study was to use the ultrasonographic contrast agent SonoVue to evaluate various transit time indices in the normal canine liver, to examine the effect of anesthesia on these parameters, and to evaluate the safety of this agent in dogs. The liver of 11 healthy dogs was studied by ultrasound during an intravenous bolus injection of SonoVue. Each dog underwent the examination twice, first with and later without the use of anesthesia. A time-intensity curve was generated from a selected region of interest within the liver from each scanning session. Ratios derived from peak enhancement, time to peak enhancement, up-slope and full-width half-maximum (FWHM) of the curve were calculated from the time-intensity curves, and are reported. There were no statistically significant differences (P > 0.05) in peak enhancement, up-slope and FWHM between dogs that were anesthetized and dogs that were not. Time to peak enhancement, however, was significantly shorter when the dogs were anesthetized than when they were nonanesthetized (P < 0.05). There were no biologically significant changes in clinical laboratory findings. This study indicates that contrast-enhanced ultrasound using SonoVue gives reproducible liver perfusion data, and appears to be a safe and well-tolerated agent for use in dogs. When considering normal values, the use of anesthetic drugs has to be considered.     

Decreased platelet function in Cavalier King Charles Spaniels with mitral valve regurgitation

With aggregometry, increased platelet activity has been reported in Cavalier King Charles Spaniels (CKCS) without mitral regurgitation (MR). In contrast, dogs with MR have been found to have decreased platelet activity. The purpose of this study was to test an easy bedside test of platelet function (the Platelet Function Analyzer [PFA-100]) to see if it could detect an increase in platelet activity in CKCS without MR and a decrease in platelet activity in CKCS with MR. This study included 101 clinically healthy dogs > 1 year of age: 15 control dogs of different breeds and 86 CKCS. None of the dogs received medication or had a history of bleeding. The PFA-100 evaluates platelet function in anticoagulated whole blood under high shear stress. Results are given as closure times (CT): the time it takes before a platelet plug occludes a hole in a membrane coated by agonists. The CT with collagen and adenosine-diphosphate as agonists was similar in control dogs (median 62 seconds; interquartile interval 55-66 seconds) and CKCS with no or minimal MR (55; 52-64 seconds). The CT was higher in CKCS with mild MR (regurgitant jet occupying 15-50% of the left atrial area) (75; 60-84 seconds; P = .0007) and in CKCS with moderate to severe MR (jet > 50%) (87: 66-102 seconds; P < .0001). CKCS with mild, moderate, and severe, clinically inapparent MR have decreased platelet function. The previous finding of increased platelet reactivity in nonthrombocytopenic CKCS without MR could not be reproduced with the PFA-100 device.     

Brucellosis in the European Union and Norway at the turn of the twenty-first century

Control and eradication programs of brucellosis in cattle, sheep, goats and pigs have been more or less successfully implemented within the Member States (MS) of the European Union (EU) and Norway after Word War II. As a result, the epidemiological situation of animal brucellosis is extremely diverse among different MS or regions within a MS and among the different animal species. Some MS, mainly North European countries, and Norway are declared “officially bovine brucellosis free” and/or “officially ovine and caprine (Brucella melitensis) free”. The situation is less favorable in Southern European countries, particularly as far as sheep and goat brucellosis are concerned. This situation has important zoonotic consequences as reflected in the number of human brucellosis cases due to B. melitensis that are still encountered in those countries. Brucellosis in swine has re-emerged as a result of spillover from the wild boar brucellosis (Brucella suis biovar 2) reservoir, particularly in outdoor reared pigs. Besides the actual challenge to eradicate brucellosis, further issues have to be addressed: (1) the management of false positive serological results that occur in the course of brucellosis testing, particularly in cattle; (2) the impact of wildlife brucellosis, particularly wild boar brucellosis in domestic animals; and (3) the importance of B. melitensis infection in cattle that are in contact with infected sheep.     

Selection of timothy (Phleum pratense L.) under different conditions of culture

Summary Recurrent selection for herbage weight under different conditions of competition and cutting frequency between hexaploid timothy cultivars and within their open-pollinated progenies was inefficient in producing in two generations either highly improved material as compared to the control (Climax) or any pronounced cultural ecotypes as shown by genotype x treatment interaction analyses, but some Climax and Hungarian selections were promising.Parent-offspring correlations indicated high heritability of heading and morphological characters in the first generation but not in vigor and yield which were the only characters measured in the second generation. The correlations may have been enhanced by the technique of open-pollination. A few selfings included showed very high inbreeding depression.Very early characters were useful in indicating growth types. Plant x season interactions were obvious but not tested. There were grass x legume interactions in the F₁ showing the different effects of alfalfa and white clover. A leaf marking was useful in differentiating white clover cultivars. In the F₂, spacing x cutting system interactions were significant. Light interception was significantly correlated to yields in the grass rows. Discriminant functions indicated the importance of tiller thickness for a selection index.The results are discussed in relation to experimental errors and to the possible polysomic segregation in hexaploid timothy.Published with the approval of the Director of the Ottawa Research Station, Central Experimental Farm, Ottawa. Contribution No 264.     

Modelling and mapping the suitability of European forest formations at 1-km resolution

Proactive forest conservation planning requires spatially accurate information about the potential distribution of tree species. The most cost-efficient way to obtain this information is habitat suitability modelling i.e. predicting the potential distribution of biota as a function of environmental factors. Here, we used the bootstrap-aggregating machine-learning ensemble classifier Random Forest (RF) to derive a 1-km resolution European forest formation suitability map. The statistical model use as inputs more than 6,000 field data forest inventory plots and a large set of environmental variables. The field data plots were classified into different forest formations using the forest category classification scheme of the European Environmental Agency. The ten most dominant forest categories excluding plantations were chosen for the analysis. Model results have an overall accuracy of 76%. Between categories scores were unbalanced and Mesophitic deciduous forests were found to be the least correctly classified forest category. The model’s variable ranking scores are used to discuss relationship between forest category/environmental factors and to gain insight into the model’s limits and strengths for map applicability. The European forest suitability map is now available for further applications in forest conservation and climate change issues.     

Filamentous and polyhedral virus-like particles in Boletus edulis

In the north-east and east of the Netherlands a disease ofBoletus edulis was noticed. Symptoms are described. The name ‘little-cap disease’ is proposed. In all cell-free preparations made from diseased, but also from healthy carpophores polyhedral virus-like particles about 33 nm and 42 nm in diameter were observed, the 33 nm particles prevailing. Occasionally a few polyheral particles of about 28 nm were isolated from healthy and diseased carpophores. Some healthy and diseased mushroom samples yielded filamentous virus-like particles about 500 nm long and 13 nm wide, with an axial canal. To our knowledge no other filamentous virus-like particles have been isolated from fungi so far. The role of the virus-like particles inB. edulis and the cause of ‘little-cap disease’ are still unknown.     

Comparison of the sensitivity of in vitro and in vivo tests for detection of the presence of a bovine viral diarrhoea virus type 1 strain

Veterinary vaccines are usually tested for the absence of contaminants. However, the quality control does not always imply that vaccines are not contaminated as, for example, illustrated by the bovine herpes virus 1 (BHV1) vaccine used in The Netherlands in 1999 that contained a small amount of bovine viral diarrhoea virus (BVDV1). Thousands of cows were vaccinated with BHV1 vaccine batches, and the question arose as to whether these small amounts of BVDV1, most likely not detected with in vitro tests, could have infected cattle. More in general, the question was whether the outcome of the in vitro tests, i.e. the in vitro infectivity, was indicative for the infectivity for cattle, i.e. the in vivo infectivity. We therefore carried out in vitro experiments to determine the sensitivity of a BVDV1 isolation assay. In addition, we performed two animal experiments, in which we estimated the lowest dose needed to infect calves with BVDV1. We extrapolated the experimental in vitro and in vivo results from a tissue culture infectious dose (TCID50) to a cattle infectious dose (CID50). We observed a partial response in the calves inoculated with this dose: four out of six calves turned out to be infected. In the tissue culture test, all 20 samples tested negative. The response in vivo, however, was not significantly higher than the in vitro response, which implies that no difference in susceptibility was observed between the animal test and the tissue culture test. Based on the results in our experiments, some cattle may have been infected with BVDV1 after the application of the contaminated BHV1 vaccine during the vaccination campaign. The question remains that how many cattle received contaminated vaccine, and became infected with BVDV1.