Botrytis cinerea Infection in Grape Flowers: Defense Reaction, Latency, and Disease Expression

ABSTRACT Inflorescences of field-grown grapevines (Vitis vinifera L. cv. Gamay) were inoculated with a Botrytis cinerea conidia suspension or dried conidia at different stages during bloom in moist weather. Approximately 10% of the conidia germinated within 72 h, resulting in two to three times more latent infections than uninoculated controls in pea-size (7 mm in diameter) berries. In surface-sterilized pea-size berries, latent B. cinerea was present predominantly in the receptacle area. After veraison, latent B. cinerea also was found in the style and, in mature berries, latent colonies were distributed throughout the pulp. Inoculation at full bloom led to the highest disease severity (66%) at harvest, compared with 38% in controls. Stilbene stress metabolites in the flowers were measured by high-performance liquid chromatography. Resveratrol accumulated mainly after pre-bloom and full-bloom inoculation, but did not prevent infection. Piceid levels did not change following inoculation, while epsilon-viniferin was found in necrotic tissues only, and pterostilbene and alphaviniferin were not detected at all. B. cinerea conidia suspensions also were applied to various locations on flowers of pot-grown cvs. Pinot Noir and Chardonnay. Inoculation of the receptacle area, but not that of the stigma and ovary, resulted in latent infections. Stilbene synthesis was similar to the field results, with resveratrol accumulating mainly in the calyptra and receptacle area. Constitutive soluble phenolic compounds (mainly derivatives of quercetin and hydroxy-cinnamic acid) were present at high concentrations in the calyptra but at low levels in the receptacle area. These experiments confirmed bloom as a critical time for B. cinerea infection in grapes and suggest that the most likely site of infection is the receptacle area or cap scar exposed at anthesis. Stilbenes may have a limited role in inhibition of flower infection and latency in susceptible grape cultivars, and epsilon-viniferin may be a by-product rather than a deterrent of infection.     

Intraspecific Relationship of Plasmopara halstedii Isolates Differing in Pathogenicity and Geographic Origin Based on ITS Sequence Data

Sequence parts of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA were analysed to screen for the intraspecific variability of a non-coding genomic region in 15 Plasmopara halstedii populations of different pathotype and geographic origin. Samples revealed uniformity in a ca. 790 Bp fragment comprising of the ITS-1, 5.8S and front parts of the ITS-2. In contrast, clear differences were found in a ca. 810 Bp fragment of the ITS-2 thus allowing differentiation between populations of pathotype 100, 310 and 330 and a group of populations representing pathotypes 700, 701, 703, 710 and 730. Samples of pathotypes 700 to730 originated from Slovakia, France, and Germany, but were uniform in both ITS sequence parts, thus indicating very recent origin of these highly aggressive physiological races. The potential use of ITS sequences for pathotype differentiation and phylogenetic studies in P. halstedii is discussed.     

A novel fluorescent pH probe for expression in plants

Background  

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants.     

Artenspektrum und Befallshäufigkeit von Fusarium spp. in Bt- und konventionellem Mais im Maiszünsler-Befallsgebiet Oderbruch

The European corn borer (Ostrinia nubilalis) is one of the most important pests of maize (Zea mays). Injuries to the plants caused by the larvae of the European corn borer may represent entrance gates for fungal-spores. The cultivation of Bacillus thuringiensis maize (Bt-maize) is one possibility to reduce infestation by the European corn borer. The aim of the present project was to determine and to compare the number of species and the frequency of Fusarium spp. infestation in Bt-maize (cry1Ab) and conventional maize. In 2003, we analysed the Fusarium spp. infestation of samples of chaffed Bt-maize and its isogenic variety on two experimental fields in the Oderbruch region (Germany), an European corn borer infested area. The conventional variety on the first of the experimental fields (previous crop wheat and forking cultivation) showed a small infestation (16%) of Ostrinia nubilalis while in the conventional variety on the second field (previous crop maize and not forking cultivation) the infestation of the European corn borer was almost three times higher (47%). In the conventional variety on both of the experimental fields we found a high Fusarium spp. infestation (70%). Especially species of the section Liseola dominates, among them: F. subglutinans, F. proliferatum und F. verticillioides. The Fusarium infestation in the samples of Bt-maize from the field with previous crop wheat and forking cultivation was just as high as in the conventional variety (70%). The infestation of Fusarium spp. in the samples of Bt-maize from the field with previous crop maize and forking cultivation was more than 20?% lower.     

Assemblages of wood-inhabiting fungi related to silvicultural management intensity in beech forests in southern Germany

Current silvicultural treatments in beech forests are aimed at achieving thick logs without discoloured hardwood. Therefore intensive thinning is applied already in younger stands with the objective of large-sized trunks at an age of 100 years. However, this approach bears the risk that dead wood structures and broken trees are completely removed from the forest. The impact of three different silvicultural management intensity levels on wood-inhabiting fungi over decades was investigated in a large beech forest (>10,000 ha) in southern Germany in 69 sampling plots: A Intensive Thinning and Logging with high-value trees, B Conservation-Oriented Logging with integration of special structures such as dead wood and broken trees and C Strict Forest Reserves with no logging for 30 years. The analysis of community showed marked differences in the fungus species composition of the three treatments, independent of stand age. The relative frequencies of species between treatments were statistically different. Indicator species for naturalness were more abundant at sites with low silvicultural management intensity. Fomes fomentarius, the most common fungus in virgin forests and strict forest reserves, is almost missing in forests with high-management intensity. The species richness seemed to be lower where intensive thinning was applied (P = 0.051). Species characteristic for coarse woody debris were associated to low management intensity, whereas species with a significant preference for stumps became more frequent with increasing management intensity. A total amount of dead wood higher than 60 m³/ha was found to enable significantly higher numbers of species indicators of naturalness (P = 0.013). In conclusion, when applying intensive silvicultural treatment, the role of dead wood needs to be actively considered in order to maintain the natural biocoenosis of beech forests.     

Influence of soil acidity on depth gradients of microbial biomass in beech forest soils

The objectives of the study were to investigate mineral soil profiles as a living space for microbial decomposers and the relation of microbial properties to soil acidity. We estimated microbial biomass C on concentration (g g^–1 DW) as well as on volume basis (g m^–2) and the microbial biomass C to soil organic C ratio along a vertical gradient from L horizon to 20 cm in the mineral soil and along a gradient of increasing acidity at five beech forest stands in Germany. Microbial biomass C concentration ranged from 17,000–34,000 g C_mic g^–1 DW in the litter layer and decreased dramatically down the profile to 29–264 g C_mic g^–1 DW at 15–20 cm depth in the mineral soil. This represents depth gradients of microbial biomass C concentrations ranging from a factor of 65 in slightly acidic and up to 875 in acidic soils. In contrast, microbial biomass C calculated on a volume basis (g C_mic m^–2) showed a different pattern since a considerable part of the microbial biomass C was located in the mineral soils. In the soil profile 22–34% of the microbial biomass C was found in the mineral soil at strictly acidic sites and as much as 64–88% in slightly acidic soils. The microbial biomass C to soil organic carbon ratios decreased in general down from the L horizon in the forest floor to 0–5 cm depth in the mineral soils. In strongly acidic mineral soils however, the C to soil organic carbon ratio increased with depth, suggesting a positive relation to increasing pH. We conclude from depth gradients of soil pH and microbial biomass C to soil organic carbon ratio that pH affects this ratio at acidic sites. The inter-site comparison indicates that acidity restricts microbial biomass C in the mineral soils.     

Short-term competition between crop plants and soil microbes for inorganic N fertilizer

Agricultural systems that receive high amounts of inorganic nitrogen (N) fertilizer in the form of either ammonium (NH₄⁺), nitrate (NO₃⁻) or a combination thereof are expected to differ in soil N transformation rates and fates of NH₄⁺ and NO₃⁻. Using ¹⁵N tracer techniques this study examines how crop plants and soil microbes vary in their ability to take up and compete for fertilizer N on a short time scale (hours to days). Single plants of barley (Hordeum vulgare L. cv. Morex) were grown on two agricultural soils in microcosms which received either NH₄⁺, NO₃⁻ or NH₄NO₃. Within each fertilizer treatment traces of ¹⁵NH₄⁺ and ¹⁵NO₃⁻ were added separately. During 8 days of fertilization the fate of fertilizer ¹⁵N into plants, microbial biomass and inorganic soil N pools as well as changes in gross N transformation rates were investigated. One week after fertilization 45-80% of initially applied ¹⁵N was recovered in crop plants compared to only 1-10% in soil microbes, proving that plants were the strongest competitors for fertilizer N. In terms of N uptake soil microbes out-competed plants only during the first 4 h of N application independent of soil and fertilizer N form. Within one day microbial N uptake declined substantially, probably due to carbon limitation. In both soils, plants and soil microbes took up more NO₃⁻ than NH₄⁺ independent of initially applied N form. Surprisingly, no inhibitory effect of NH₄⁺ on the uptake and assimilation of nitrate in both, plants and microbes, was observed, probably because fast nitrification rates led to a swift depletion of the ammonium pool. Compared to plant and microbial NH₄⁺ uptake rates, gross nitrification rates were 3-75-fold higher, indicating that nitrifiers were the strongest competitors for NH₄⁺ in both soils. The rapid conversion of NH₄⁺ to NO₃⁻ and preferential use of NO₃⁻ by soil microbes suggest that in agricultural systems with high inorganic N fertilizer inputs the soil microbial community could adapt to high concentrations of NO₃⁻ and shift towards enhanced reliance on NO₃⁻ for their N supply.