Nuclear magnetic resonance: a tool for imaging belowground damage caused by Heterodera schachtii and Rhizoctonia solani on sugar beet.

Belowground symptoms of sugar beet caused by the beet cyst nematode (BCN) Heterodera schachtii include the development of compensatory secondary roots and beet deformity, which, thus far, could only be assessed by destructively removing the entire root systems from the soil. Similarly, the symptoms of Rhizoctonia crown and root rot (RCRR) caused by infections of the soil-borne basidiomycete Rhizoctonia solani require the same invasive approach for identification. Here nuclear magnetic resonance imaging (MRI) was used for the non-invasive detection of belowground symptoms caused by BCN and/or RCRR on sugar beet. Excessive lateral root development and beet deformation of plants infected by BCN was obvious 28 days after inoculation (dai) on MRI images when compared with non-infected plants. Three-dimensional images recorded at 56 dai showed BCN cysts attached to the roots in the soil. RCRR was visualized by a lower intensity of the MRI signal at sites where rotting occurred. The disease complex of both organisms together resulted in RCRR development at the site of nematode penetration. Damage analysis of sugar beet plants inoculated with both pathogens indicated a synergistic relationship, which may result from direct and indirect interactions. Nuclear MRI of plants may provide valuable, new insight into the development of pathogens infecting plants below- and aboveground because of its non-destructive nature and the sufficiently high spatial resolution of the method.

Genotyping and phenotyping of Fusarium graminearum isolates from Germany related to their mycotoxin biosynthesis.

Fusarium graminearum is the most important pathogen causing Fusarium head blight (FHB) of small cereal grains worldwide responsible for quantitative and qualitative yield losses. The presence in crops is often associated with mycotoxin contamination of foodstuff limiting its use for human and animal consumption. A collection of isolates of F. graminearum from Germany was characterized genetically and chemically for their potential to produce the B trichothecenes deoxynivalenol (DON) and nivalenol (NIV). Molecular methods with eight PCR assays were implemented based on functional Tri7 and Tri13 genes and on the tri5-tri6 intergenic region to differentiate between chemotaxonomic groups DON and NIV, resulting in a marked majority (61/63) of DON chemotypes. Mycotoxins produced on rice kernels were quantified by means of LC-MSMS including DON, NIV, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON), DON-3-glucoside, fusarenon X, as well as zearalenone; all of them proving to be present in high concentration among the isolates. All DON-chemotype isolates also produced lower amounts of NIV with the amount being positively correlated (R²=0.89) to the DON amount. 15-ADON and 3-ADON are reported to be produced simultaneously by the isolates, the former dominating over the latter in all but one isolate. Fungal biomass, was quantified via ergosterol amount on rice. It was used to calculate specific mycotoxin production per biomass of isolates, ranging from 0.104 to 1.815mg DON mg-1 ergosterol, presenting a Gaussian distribution. Genotype and phenotype characterization revealed discrepancies with respect to mycotoxin production potential of the fungi, i.e. isolates from one chemotype were able to produce mycotoxins from other chemotypes in considerable amounts.

Microbiological and SYBR green real-time PCR detection of major Fusarium head blight pathogens on wheat ears.

Fusarium head blight (FHB) caused by several Fusarium species is one of the most serious diseases affecting wheat throughout the world. The efficiency of microbiological assays and real-time PCRto quantify major FHB pathogens in wheat ears after inoculation with F. graminearum, F. culmorum, F. avenaceum and F. poae undergreenhouse and field conditions were evaluated. The frequency of infected kernel, content of fungal biomass, disease severity and kernel weight were determined. To measure the fungal biomass an improved DNA extraction method and a SYBR Green real-time PCR were developed. The SYBR Green real-time PCR proved to be highly specific for individual detection of the species in a matrix including fungal and plant DNA. The effect of Fusarium infection on visible FHB severity, frequency of infected kernels and thousand-kernel mass (TKM) significantly depended on the Fusarium species/isolate. F. graminearum resulted in highest disease level, frequency of infected kernels, content of fungal biomass, and TKM reduction followed by F. culmorum, EF avenaceum and F. poae, respectively. The comparison of frequency and intensity of kernel colonization proved differences in aggressiveness and development of the fungi in the kernels. Only for F. graminearum, the most aggressive isolate, application of microbiological and real-time PCR assays gave similar results. For the other species, the intensity of kernel colonization was lower than expected from the frequency of infection.

Localized adhesion of nongerminated Venturia inaequalis conidia to leaves and artificial surfaces.

Adhesion to the host surface is the first step for successful plant pathogen development and has been reported to be associated with both passive and active processes. For conidia of Venturia inaequalis, which depend on leaf wetness for germination, this process has not yet been described. Conidia of V. inaequalis adhered to wet hydrophobic surfaces immediately after contact to the surface, hours before initiation of germination. Attachment of nongerminated conidia was much better on hydrophobic surfaces, such as apple leaves and polystyrene, than on hydrophilic glass. Conidia released adhesive material localized in a droplet named spore tip glue (STG) at the spore apex which interacted with a contact surface only when water was present. Histochemical investigations indicated the presence of proteins and carbohydrates in STG, lectin labeling the presence of beta-galactose and N-acetylglucosaminyl residues. Transmission electron microscopy revealed two phases in the STG at the tip of dry mature conidia; as STG was present on the outer side of the intact fungal cell wall its formation should be associated with the secretion of glue through pores of the conidial wall. Surface-active substances affected the adhesion of conidia to hydrophobic surfaces stressing the importance of hydrophobic interactions. The use of protein biosynthesis inhibitors did not affect adhesion of conidia indicating that the adhesive material was preformed. It is concluded that the coincidence of STG, contact to a hydrophobic surface, and free water are essential for the adhesion of V. inaequalis conidia.

Thermal imaging of cucumber leaves affected by downy mildew and environmental conditions.

Pathogenesis of Pseudoperonospora cubensis causing downy mildew of cucumber resulted in changes in the metabolic processes within cucumber leaves including the transpiration rate. Due to the negative correlation between transpiration rate and leaf temperature, digital infrared thermography permitted a non-invasive monitoring and an indirect visualization of downy mildew development. Depending on the stage of pathogenesis and the topology of chloroses and necroses, infection resulted in a typical temperature pattern. Spatial heterogeneity of the leaf temperature could be quantified by the maximum temperature difference (MTD) within a leaf. The MTD increased during pathogenesis with the formation of necrotic tissue and was related to disease severity as described by linear and quadratic regression curves. Under controlled conditions, changes in temperature of infected leaves allowed the discrimination between healthy and infected areas in thermograms, even before visible symptoms of downy mildew appeared. Environmental conditions during thermographic measurement, in particular air temperature and humidity, as well as water content and age of the leaf influenced the temperature of its surface. Conditions enhancing the transpiration rate facilitated the detection of changes in leaf temperature of infected leaves at early stages of infection. As modified by environmental conditions, MTD alone is not suitable for the quantification of downy mildew severity in the field.

Effect of downy mildew development on transpiration of cucumber leaves visualized by digital infrared thermography.

ABSTRACT Disease progress of downy mildew on cucumber leaves, caused by the obligate biotrophic pathogen Pseudoperonospora cubensis, was shown to be associated with various changes in transpiration depending on the stage of pathogenesis. Spatial and temporal changes in the transpiration rate of infected and noninfected cucumber leaves were visualized by digital infrared thermography in combination with measurements of gas exchange as well as microscopic observations of pathogen growth within plant tissue and stomatal aperture during pathogenesis. Transpiration of cucumber leaf tissue was correlated to leaf temperature in a negative linear manner (r = -0.762, P < 0.001, n = 18). Leaf areas colonized by Pseudoperonospora cubensis exhibited a presymptomatic decrease in leaf tem perature up to 0.8 degrees C lower than noninfected tissue due to abnormal stomata opening. The appearance of chlorosis was associated with a cooling effect caused by the loss of integrity of cell membranes leading to a larger amount of apoplastic water in infected tissue. Increased water loss from damaged cells and the inability of infected plant tissue to regulate stomatal opening promoted cell death and desiccation of dying tissue. Ultimately, the lack of natural cooling from necrotic tissue was associated with an increase in leaf temperature. These changes in leaf temperature during downy mildew development resulted in a considerable heterogeneity in temperature distribution of infected leaves. The maximum temperature difference within a thermogram of cucumber leaves allowed the discrimination between healthy and infected leaves before visible symptoms appeared.

Effects of arbuscular mycorrhizal fungi and a non-pathogenic Fusarium oxysporum on Meloidogyne incognita infestation of tomato.

Arbuscular mycorrhizal (AM) fungi and non-pathogenic strains of soil-borne pathogens have been shown to control plant parasitic nematodes. As AM fungi and non-pathogenic fungi improve plant health by different mechanisms, combination of two such partners with complementary mechanisms might increase overall control efficacy and, therefore, provide an environmentally safe alternative to nematicide application. Experiments were conducted to study possible interactions between the AM fungus Glomus coronatum and the non-pathogenic Fusarium oxysporum strain Fo162 in the control of Meloidogyne incognita on tomato. Pre-inoculation of tomato plants with G. coronatum or Fo162 stimulated plant growth and reduced M. incognita infestation. Combined application of the AM fungus and Fo162 enhanced mycorrhization of tomato roots but did not increase overall nematode control or plant growth. A higher number of nematodes per gall was found for mycorrhizal than non-mycorrhizal plants. In synergisms between biocontrol agents, differences in their antagonistic mechanisms seem to be less important than their effects on different growth stages of the pathogen.

The IPM Wheat Model--results of a three-year study in North Rhine-Westphalia, Lower Saxony and Schleswig-Holstein.

Under the primary utilisation of phytosanitary production factors such as selection of variety, crop rotation and N fertilisation according to plant requirements, the IPM Wheat Model comprises the elements diagnosis (qualitative = type of pathogen, quantitative = disease severity), scientifically grounded treatment thresholds which, as critical values in pathogen development, can be applied to define the optimum time of fungicide application, and pathogen-specific effective fungicides and application amounts. This leads to the location and year-specific optimised control of the pathogen and of the associated yield performance. After several years of development in Bavaria (from 1985 on) and Schleswig-Holstein (1993-1999), the model was tested as part of a project involving the Universities of Bonn and Kiel and the plant protection services of the German states of Lower Saxony, North Rhine-Westphalia and Schleswig-Holstein in a three-year study (1999-2001) in interregional locations (usually nine per state) with the winter wheat variety Ritmo (interregional indicator variety) and a further variety of regional importance in different variations (untreated control, three to four times growth stage-oriented variants for the determination of the absolute damage potential, IPM-variant). In exact records (approx. 12 dates per vegetation period), the disease epidemics were recorded weekly. With the genetically uniform indicator variety Ritmo, the results documented substantially differing year- and location-specific disease and yield patterns. Interregionally, a broad wheat pathogen spectrum (Puccinia striiformis, P. recondita, Septoria tritici, Stagonospora (syn. Septoria) nodorum, Blumeria (syn. Erysiphe) graminis, Pseudocercosporella herpotrichoides, Drechslera tritici-repentis) in differing composition, disease severity and damage effect was demonstrated. The heterogeneity of the infection and damage patterns was increased in the case of the second variety, in association with the genetic variability. The epidemiologically-orientated indications (average two, reduced application amounts) according to the IPM Wheat Model in association with time-diverging progressions led, on an interregional basis, with minimum input and in association with the diverging dynamics, to a biologically and economically optimised fungicide application. In the context of economic and ecological performance, the comprehensive results of the project demonstrated the valuable functionality of the IPM Wheat Model.

Impact of strobilurins on physiology and yield formation of wheat.

Strobilurin fungicides have a broad spectrum activity against all major foliar pathogens of wheat. In addition to this extraordinary fungicidal activity side-effects have been reported which result in higher yields of cereals, e.g. the reduction of respiration, delayed leaf senescence, activation of nitrogen metabolism as well as increased tolerance against abiotic stress factors. In the vegetation period 2000/2001 field trials were carried out at three sites in North Rhine-Westphalia to study the effects of three strobilurin fungicides on the yield formation of six winter wheat varieties. The strobilurins were applied two times as the commercial products Stratego (trifloxystrobin + propiconazole), Amistar/Pronto Plus (azoxystrobin/spiroxamine + tebuconazole) and Juwel Top (kresoxim-methyl + epoxiconazole + fenpropimorph. Fungicide-treated plants were kept disease-free by an initial azole-application in GS 31 in order to exclude disease effects on physiological parameters relevant to yield formation. Photosynthetic electron transport of strobilurin-treated wheat, was improved as early as at GS 65 compared to azole-treated plants. Differences often increased with growth stage and were closely related to a delay in leaf senescence. A higher photosynthetic activity of strobilurin-treated plants was confirmed by gas exchange and chlorophyll fluorescence measurements under field conditions. The yield benefit of wheat from strobilurin treatments varied from 2% to 9% depending on an improved photosynthetic capacity due to a higher and/or prolonged activity. Neither yield potential nor disease susceptibility of the cultivar had an effect on the height of the extra yield which, in contrast was modified by location and wheat genotype.

Evaluation of the IPM Wheat Model in the Rhineland 2001.

The results of field trials at nine locations with two wheat cultivars in each case demonstrated the practicability of the action threshold-based fungicide strategy of the IPM Wheat Model under the intensive wheat production conditions in the Rhineland. Action thresholds used and dosage of fungicides applied are outlined. Despite using reduced rates of fungicides the epidemic development of relevant pathogens--mainly Septoria tritici and Puccinia recondita--was controlled in early stages of disease development. In plots treated according to the IPM Wheat Model disease incidence and disease severity were reduced effectively compared to untreated plots with low and high disease incidence, respectively. The effective control of pathogens using pathogen-specific action thresholds resulted in yield levels very similar to those of disease-free plots simultaneously minimising the total amount of fungicides applied. Disease control according to the IPM Wheat Model raised yields by up to 25% with monetary benefits averaging more than [symbol: see text] 100 ha-1 in 2001.

Biological control of grey mould (Botrytis cinerea) with the antagonist Ulocladium atrum.

The competitive saprophytic fungus Ulocladium atrum was selected on the basis of its antagonistic potential for the control of grey mould caused by the necrotrophic pathogen Botrytis cinerea. Field trials were carried out to asses the efficacy of U. atrum as a biocontrol agent against B. cinerea in grapevine. The results demonstrated that under moderate disease pressure U. atrum had the potential to control grey mould, whereas under high disease pressure the efficacy was not sufficient to substitute the use of fungicides completely.

Variation in the spectrum of Fusarium species on winter wheat.

In 1998-2000 a monitoring of the spectrum of Fusarium species on winter wheat was carried out in the Rhineland. The epidemic spread ofFusarium spp. on wheat plants during growing season was investigated as well as the grain contamination after harvest.F avenaceum was the Fusarium species isolated most frequently followed byF culmorum, F poae andF graminearum. Microdochium nivale occurred considerably only in 1998. Both, susceptibility and plant height of the cultivars were correlated to the incidence of Fusarium species /M nivale on harvested kernels; interactions with cropping intensities were detected. The incidence ofF poae seemed to be independent of the cultivar-specific Fusarium susceptibility. Despite the lack of disease symptoms, between growth stages 45-85 mycelium ofFusarium spp. was detectable in the leaves as well as conidia on the leaf surfaces.

Significance of different inoculum sources for the Fusarium infection of wheat ears.

From 1997 to 1999 the occurrence ofFusarium spp. on wheat grain and its contamination with the mycotoxins deoxynivalenol and invalenol were investigated under organic farming conditions in the Rhineland, Germany. For comparison, some trials were also run under integrated farming conditions. The importance of the seed contamination withFusarium spp. as well as the impact of farming system, previous crop and soil preparation on the inoculum sources ofFusarium spp. in the soil were investigated. The data on the inoculum sources was compared to the Fusarium infection of grains and their content of DON and NIV. The crop residues in the soil were the most important inoculum source for the Fusarium species infecting wheat ears and grains. The amount of potential inoculum in the soil largely depended on the previous crop and the system of soil preparation.

Impact of growth conditions on the occurrence ofFusarium spp. and the mycotoxin content of wheat.

In 1997-99 the occurrence ofFusarium spp. on winter wheat and the contamination with mycotoxins was investigated at three locations in the Rhineland, Germany. All cultivation methods investigated had an effect on the level ofFusarium infection, however, rainfall during flowering was the most important factor. The choice of cultivar and soil cultivation proved to be the most promising tools to reduce head scab severity and mycotoxin contamination.

Yield reduction and mycotoxin contamination of wheat due toFusarium culmorum.

The inoculation of wheat ears with 27 isolates ofFusarium culmorum in growth stage 65 reduced 1000-grain weights by 14 to 61%. For the phytopathological characterisation of isolates the virulence on primary wheat leaves and the growth rate an potato-dextrose-agar were assessed. Deoxynivalenol-producing isolates ofF. culmorum reduced the 1000-grain weight more than nivalenol-producing isolates.

Characterisation offusarium graminearum andF. culmorum isolates by mycotoxin production and aggressiveness to wheat.

A total of 27Fusarium culmorum isolates from Germany and 41F. graminearum isolates from Kenya were investigated for aggressiveness and mycotoxin production on wheat ears. In addition, ergosterol content of the kernels from ears inoculated withF. graminearum was determined and theF. culmorum isolates were tested for mycotoxin productionin vitro. For both pathogens, isolates markedly differed in aggressiveness. 59% and 37% of theF. culmorum isolates produced NIV and DON, respectively,in vivo andin vitro. The DON-producing isolates also produced 3-acDONin vitro. The more aggressive isolates produced mainly DON while the less aggressive isolates produced mainly NIV. 12% and 85% of theF. graminearum isolates produced NIV and DON, respectively. The highly aggressive isolates produced higher amounts of DON, aggressiveness being highly correlated to DON content in the kernels. NIV-producing isolates were less aggressive. Ergosterol content of kernels was moderately correlated to aggressiveness but highly correlated to DON content. Disease severity was associated with kernel weight reduction.