Quantitative detection of pear-pathogenic Stemphylium vesicarium in orchards.

ABSTRACT Isolates of Stemphylium vesicarium causing brown spot of pear can be distinguished from nonpathogenic isolates of S. vesicarium from pear or from other hosts on the basis of distinctive amplified fragment length polymorphism fingerprinting profiles. DNA fragments specific for isolates pathogenic to pear were identified and a quantitative polymerase chain reaction (PCR) was developed on the sequence from one of these specific DNA loci. This TaqMan PCR has a high sensitivity with a dynamic range for reliable quantification between 1 ng and 100 fg of DNA. The method detected pear-pathogenic isolates of S. vesicarium originating from four different European countries and various regions within those countries. No cross-reaction was found with either the nonpathogenic isolates of S. vesicarium tested or isolates belonging to other Stemphylium spp. or related fungi. The pathogen was detected on leaves with brown-spot symptoms originating from six different locations in The Netherlands, Italy, and Spain. Pear-pathogenic S. vesicarium populations were monitored on crop residues in two Dutch orchards between October 2007 and October 2008. Brown spot had been observed at both orchards at the end of the growing season of 2007. In one location, pear-pathogenic S. vesicarium was detected only sporadically on crop residues and no brown-spot symptoms were observed on fruit in 2008. At the other location, a pathogenic population was found on fallen pear leaves and on other crop residues but this population decreased during winter. From the beginning of the growing season in 2008 onward, the pathogen population could not be detected and the disease incidence was only 0.6%. The TaqMan PCR will allow more detailed studies on epidemiology of brown spot and on the effect of disease control measures.

Risks for infection of strawberry plants with an aerosolized inoculum of Xanthomonas fragariae.

Xanthomonas fragariae is the causative agent of angular leaf spot of strawberry, a quarantine organism in plant propagation material in the European Union. Field experiments were conducted to assess the risks for infection of strawberry plants through dispersal of an aerosolized inoculum. In practice, pathogen aerosols can be formed during mowing of an infected crop or by water splashing on symptomatic plants during overhead irrigation or rain. In our experiments, aerosols were generated by spraying suspensions of X. fragariae with a density of 108 cfu ml-1 or water under pressure vertically up into the air. In strawberry plants (cv Elsanta) placed at 1.3, 5 and 10 m distance downwind from the spray boom, infections were found, as evidenced with a combination of dilution-plating and molecular techniques, but more frequently in plants wetted prior to inoculation than in plants kept dry. A logarithmic decrease in infection incidence was found with the distance to the inoculum source. Symptomatic plants were found up to 5 m distance from the inoculum source. No infected plants were found in plants placed 4 m upwind or treated with water. In glasshouse studies, it was shown that under conditions favorable for disease development, spray-inoculation of strawberry plants with estimated densities of X. fragariae as low as 2000 cfu per plant were able to cause symptoms both in cv Elsanta and cv Sonata. Results indicate that there is a considerable risk on infections of strawberry plants exposed to aerosolized inoculum.

Population Dynamics of Fusarium spp. and Microdochium nivale in Crops and Crop Residues of Winter Wheat.

ABSTRACT Naturally occurring populations of Fusarium avenaceum, F. culmorum, F. graminearum, F. poae, and Microdochium nivale were studied in two field experiments from anthesis in June 2003 until harvest in crops of winter wheat, and subsequently during 10 months after harvest until June 2004 on their residues exposed on the soil surface under field conditions. The dynamics of the different pathogens were estimated by quantifying the amount of DNA present in wheat tissues using TaqMan-polymerase chain reaction. While colonization of grain by Fusarium spp. and M. nivale was low, high amounts of DNA of F. avenaceum, F. graminearum, and F. culmorum were found in ear residues, internodes, and nodes of the mature crop. Amounts of DNA of pathogens decreased significantly during the following 10 months in residues of internodes and nodes, but not in residues of stem bases. Knowledge on population dynamics of pathogens will help to develop preventive measures aimed at reduction of inoculum sources of head blight pathogens.

Survival of Ralstonia solanacearum Biovar 2, the Causative Agent of Potato Brown Rot, in Field and Microcosm Soils in Temperate Climates.

ABSTRACT After outbreaks of potato brown rot in three different fields in the Netherlands, the fate of the brown rot pathogen, Ralstonia solanacearum biovar 2, was monitored in soil by immunofluorescence colony staining (IFC) supported by R. solanacearum division-2 specific polymerase chain reaction. In selected areas of all fields, the R. solanacearum population densities were initially on the order 10(4) to 10(6) per g of topsoil. These population densities then declined progressively over time. In two fields, however, the pathogen persisted for periods of 10 to 12 months. The survival of a selected R. solanacearum biovar 2 isolate, strain 1609, in three soils, a loamy sand and two different silt loam soils, was further studied in soil microcosm experiments. The effects of temperature and soil moisture content were assessed. At 12 or 15 and 20 degrees C, a gradual decline of the population densities was observed in all three soils, from the established 10(5) to 10(6) CFU g(-1) of dry soil to significantly reduced levels, occasionally bordering the limit of detection (10(2) CFU g(-1)of dry soil), in periods of approximately 90 to 210 days. Soil type affected the rate of population decline at 20 degrees C, with the greatest decline occurring in loamy sand soil. In all three soils, the survival of IFC-detectable R. solanacearum 1609 cells at 4 degrees C was severely impaired, reflected in an accelerated decline of CFU counts, to undetectable numbers. Moreover, indications were found for the occurrence of viable but nonculturable strain 1609 cells in the loamy sand as well as in one silt loam soil under these conditions. In addition, a single freezing-thawing cycle caused a significant additional reduction of the culturable R. solanacearum 1609 populations in the three soils, though detectable populations remained. Moderate soil moisture fluctuations of approximately pF 2 did not affect the survival of R. solanacearum 1609 in soil. Severe drought, however, drastically reduced the populations of strain 1609 CFU in all three soils.

A Dutch field survey on fungal infection and mycotoxin concentrations in maize.

Mycotoxins are secondary metabolites produced by fungi that can cause adverse health effects. Due to climate change, temperatures are expected to rise and changes in rainfall patterns are foreseen. These developments may increase fungal occurrence and mycotoxin concentrations in maize. It is therefore useful to monitor mycotoxin levels in maize and record the accompanying agronomic factors and weather parameters. This paper describes a field survey in the Netherlands in which information on soil, cultivar, green manure, tillage as well as sowing, emergence, flowering and harvest dates of silage maize were collected from 148 growers. A small number of these growers (42 in total) were visited to collect maize samples revealing that 50% of the samples were contaminated with Fusarium species and mycotoxins were detected in 25% of the samples. The Fusarium species that was most commonly found was F. crookwellense followed by F. graminearum, F. culmorum, F. sporotrichiodes and F. equiseti. In total 31 mycotoxins were analysed. The predominant mycotoxins present were (sum of 3 and 15)-acetyl-DON and nivalenol; other mycotoxins found were alternariol, beauvericin, deoxynivalenol, diacetoxyscirpenol, moniliformin and zearalenone. Nivalenol was present in concentrations up to 1670 µg kg⁻¹ and acetylated DON was usually present at higher concentrations than DON. Statistical analysis of the current data showed no correlation between mycotoxins present and agronomic factors recorded. Field studies as described in this paper are useful and need to be continued in the future in order to observe trends in mycotoxin occurrence.

Trypanosomatid protozoa in fruit of Solanaceae in southeastern Brazil.

Fruits of cultivated and indigenous Solanaceae from Southeastern Brazil have been examined for the presence of trypanosomatid flagellates. The 14 species found infected were: Capsicum annuum, C. praetermissum, Lycopersicon esculentum, Nicandra physaloides, Physalis angulata, Solanum sp., S. americanum, S. concinnum, S. diflorum, S. erianthum, S. gilo, S. robustum, S. variable and S. viarum. The pentatomid hemipteran Arvelius albopunctatus experimentally transmitted flagellates to fruits of some species. Cultures of flagellates were obtained from fruits of eight species of Solanaceae and from A. albopunctatus.

Trypanosomatid parasites of plants (phytomonas).

Trypanosomatids of the genus Phytomonas have been known as parasites of lactiferous plants since the beginning of the century and have been the subject of renewed attention in the past decade, as they are now recognized to be pathogenic in plants of economic interest. Nevertheless, information about these flagellates is still scanty. Until recently they had not been cultured, or studied biochemically or ultrastructurally. Phytophagous insects are their putative vectors but exactly which species are involved remains to be established. There are many unanswered questions about the taxonomic identification, pathogenecity and transmission of Phytomonas spp as well as about their natural hosts and reservoirs; this article by Erney Camargo, Pieter Kastelein and Isaac Roitman highlights some of them.

Effects of ecological factors on the survival and physiology of Ralstonia solanacearum bv. 2 in irrigation water.

The fate of Ralstonia solanacearum bv. 2, the causative agent of brown rot in potato, in aquatic habitats of temperate climate regions is still poorly understood. In this study, the population dynamics and the physiological response of R. solanacearum bv. 2 were tested in sterile pure water and in agricultural drainage water obtained from waterways near potato cropping fields in The Netherlands. The behaviour of five different biovar 2 isolates in drainage water at 20 degrees C was very similar among strains. One typical isolate with consistent virulence (strain 1609) was selected for further studies. The effects of temperature, light, canal sediment, seawater salts, and the presence of competing microorganisms on the survival of strain 1609 were assessed. Moreover, the impacts of the physiological state of the inoculum and the inoculum density were analyzed. The population dynamics of strain 1609 in sterile pure water were also characterized. In sterile pure water, the fate of R. solanacearum 1609 cells depended strongly on temperature, irrespective of inoculum density or physiological state. At 4 degrees C and 44 degrees C, strain 1609 CFU numbers showed declines, whereas the strain was able to undergo several cell divisions at 12 degrees C, 20 degrees C, and 28 degrees C. At 20 degrees C and 28 degrees C, repeated growth took place when the organism was serially transferred, at low inoculum density, from grown water cultures into fresh water devoid of nutrients. Both at low and high cell densities and regardless of physiological state, R. solanacearum 1609 cells persisted as culturable cells for limited periods of time in drainage water. A major effect of temperature was found, with survival being maximal at 12 degrees C, 20 degrees C, and 28 degrees C. Temperatures of 4 degrees C, 36 degrees C, or 44 degrees C induced accelerated declines of the culturable cell numbers. The drainage water biota had a strong effect on survival at 12 degrees C, 20 degrees C, and 28 degrees C, as the persistence of strain 1609 was significantly enhanced in sterile drainage water systems. Furthermore, there was a negative effect of incident light, in a light:dark regime, on the survival of R. solanacearum 1609 in natural drainage water. Also, levels of seawater salts realistic for drainage water in coastal areas were detrimental to strain survival. Ralstonia solanacearum 1609 showed considerable persistence in canal sediment saturated with drainage water, but died out quickly when this sediment was subjected to drying. Evidence was obtained for the conversion of R. solanacearum 1609 cells to nonculturable cells in water microcosms kept at 4 degrees C, but not in those kept at 20 degrees C. A substantial fraction of the cells found to be nonculturable were still viable, as evidenced by the direct viable count and by staining with the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride. The potential occurrence of viable-but-nonculturable cells in natural waters poses a problem for the detection of R. solanacearum by cultivation-based methods.

Inoculum sources of the tan spot fungus Pyrenophora tritici-repentis in The Netherlands.

Since 1994 the importance of tan spot of wheat has increased in the wheat growing areas of the Netherlands. The purpose of the present study was to determine inoculum sources of this disease caused by Pyrenophora tritici-repentis. Both in 1999 and 2000, the incidence of tan spot was assessed in 40 commercial fields of winter wheat scattered over the main wheat growing areas of the Netherlands. Adjoining fields were checked for presence of stubble or crops with straw covers and the surrounding vegetation was searched for grasses with leaf spots. Straw and affected leaves of wheat and grasses were examined for P. tritici-repentis. In greenhouse experiments the pathogenicity of isolates from alternative hosts was compared with that of isolates from wheat. The possible development of P. tritici-repentis perithecia on straw of crops other than wheat was explored for barley, oat, rye grass and rape grown in fields nearby a tan spot affected wheat field. Furthermore, dispersal of tan spot was studied in a field trial in which winter wheat was sown leeward to stubble of above-mentioned severely tan spot affected wheat crop. During the surveys three cases were found of wheat crops adjoining fields with P. tritici-repentis infested stubble or straw covers. It was only after flowering that the first symptoms of tan spot appeared in the three commercial wheat crops. Couch grass (Elymus repens) was often found as host of P. tritici-repentis. In the surroundings of more then half of the wheat crops affected by tan spot this weed was also infected. Pyrenophora tritici-repentis isolates from couch grass were found to be as pathogenic to wheat as isolates from wheat to both wheat and couch grass. The observations on straw of barley, oat, rye grass, rape and wheat revealed P. tritici-repentis perithecia only on wheat straw. In the field trial with wheat sown leeward to P. tritici-repentis infested stubble, first symptoms of tan spot appeared on wheat during April and May when the release of ascospores was at a maximum. Disease severity gradually decreased with increasing distance from the side adjoining the stubble. The results of this study indicate that straw covers and stubble from tan spot diseased wheat crops and cough grass are inoculum sources of P. tritici-repentis.

An experimentally induced phlegmonous abscess by a strain of Bacteroides gingivalis in guinea pigs and mice.

The virulence of B. gingivalis strain W83 was studied in an experimental animal model. Cells grown overnight, washed and resuspended in broth, were injected intradermally or subcutaneously in the back of guinea pigs, rats and mice. This strain proved to be very virulent, causing a severe phlegmonous abscess in guinea pigs. Also in mice, which are thought to be resistant to infections with black-pigmented Bacteroides strains, the same type of infection could be induced. Rats proved to be rather insensitive. The model presented can be used as a simple virulence test for these anaerobic bacteria.