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.

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.

Quantification of Mycelium of Botrytis spp. and the Antagonist Ulocladium atrum in Necrotic Leaf Tissue of Cyclamen and Lily by Fluorescence Microscopy and Image Analysis.

ABSTRACT A technique was developed to localize and quantify the internal mycelial colonization of necrotic leaf tissue of cyclamen (Cyclamen persicum) or lily (Lilium) by pathogenic Botrytis spp. and the antagonist Ulocladium atrum. This technique allows investigation of competitive substrate colonization by both fungi, which is a key process for biological control of Botrytis spp. by U. atrum. A combination of differential fluorescent labeling and image analysis was applied on cryostat sections of necrotic leaf tissue. Botrytis mycelium was labeled specifically by indirect immunofluorescence using a monoclonal antibody specific for Botrytis spp. And an antimouse fluorescein conjugate. Wheat germ agglutinin conjugated to the fluorochrome TRITC was used to label mycelium of both fungi. Image analysis was used to measure the relative surface area of the cryostat section covered by fluorescing hyphae of Botrytis spp. and by fluorescing hyphae of both fungi. A mathematical conversion was derived and used to calculate the relative mycelial volume of each fungal species in the necrotic tissue based on the measured relative surface areas. Temporal aspects of substrate colonization were studied in a short time series. An analysis of components of variance provided insight into spatial colonization patterns for the fungal species involved and allowed the design of efficient sampling strategies for future experiments.

Biological Control of Botrytis cinerea in Cyclamen with Ulocladium atrum and Gliocladium roseum Under Commercial Growing Conditions.

ABSTRACT The effect of treatments with conidial suspensions of Ulocladium atrum and Gliocladium roseum on leaf rot of cyclamen caused by Botrytis cinerea was investigated under commercial greenhouse conditions. Spraying U. atrum (1 x 10(6) conidia per ml) or G. roseum (2 x 10(6) conidia per ml and 1 x 10(7) conidia per ml) at intervals of 2 to 3 weeks during the production period and spraying U. atrum (1 x 10(6) conidia per ml) at intervals of 4 to 6 weeks resulted in a significant reduction of natural infections of petioles by B. cinerea. U. atrum or G. roseum (1 x 10(7)conidia per ml) was as effective as the standard fungicide program. B. cinerea colonized senesced leaves within the plant canopy and infected adjacent petioles and leaves later. The antagonists colonized senesced leaves and reduced B. cinerea development on these leaves. Thus, the inoculum potential on petioles adjacent to necrotic leaf tissues was reduced. The fate of U. atrum conidia on surfaces of green cyclamen leaves during a 70-day period after application was studied. The number of conidia per square centimeter of leaf surface remained relatively constant during the entire experiment. Sixty percent of the conidia sampled during the experiments retained the ability to germinate. When green leaves were removed from the plants to induce senescence and subsequently were incubated in a moist chamber, U. atrum colonized the dead leaves. Senesced leaves also were colonized by other naturally occurring fungi including B. cinerea. On leaves treated with U. atrum from all sampling dates, sporulation of B. cinerea was significantly less as compared with the untreated control. Our results indicate that early applications of U. atrum before canopy closure may be sufficient to achieve commercially satisfactory control of Botrytis leaf rot in cyclamen.