Identification of potential marker genes for <i>Trichoderma harzianum</i> strains with high antagonistic potential against <i>Rhizoctonia solani</i> by a rapid subtraction hybridization approach

A rapid subtraction hybridization approach was used to isolate genes differentially expressed during mycelial contact between Trichoderma harzianum (Hypocrea lixii) and Rhizoctonia solani, and could serve as marker genes for selection of superior biocontrol strains. Putatively positive clones were evaluated by transcription analysis during mycelial contact with R. solani versus growth on glucose, and for their differential transcription between two strains with either strong or poor biocontrol capability before, at, and after contact with R. solani. Besides four clones, which had similarity to putative but as yet uncharacterized proteins, they comprised ribosomal proteins, proteins involved in transcriptional switch and regulation, amino acid and energy catabolism, multidrug resistance, and degradation of proteins and glucans. Transcription of three clones was evaluated in five T. harzianum strains under confrontation conditions with R. solani. Two clones&#8212;acetyl-xylane esterase AXE1 and endoglucanase Cel61b&#8212;showed significant upregulation during in vivo confrontation of a T. harzianum strain that successively demonstrated a very high antagonistic capability towards R. solani, while expression was progressively lower in a series of T. harzianum strains with intermediate to poor antagonistic activity. These clones are promising candidates for use as markers in the screening of improved T. harzianum biocontrol strains

Fine-Scale Genetic Structure of \u3ci\u3eMonilinia fructicola\u3c/i\u3e During Brown Rot Epidemics Within Individual Peach Tree Canopies

The purpose of this study was to determine the fine-scale genetic structure of populations of the brown rot pathogen Monilinia fructicola within individual peach tree canopies to better understand within-tree plant pathogen diversity and to complement previous work on spatiotemporal development of brown rot disease at the canopy level. Across 3 years in a total of six trees, we monitored disease development, collected isolates from every M. fructicola symptom during the course of the season, and created high-resolution three-dimensional maps of all symptom and isolate locations within individual canopies using an electromagnetic digitizer. Each canopy population (65 to 173 isolates per tree) was characterized using a set of 13 microsatellite markers and analyzed for evidence of spatial genetic autocorrelation among isolates during the epidemic phase of the disease. Results showed high genetic diversity (average uh = 0.529) and high genotypic diversity (average D = 0.928) within canopies. The percentage of unique multilocus genotypes within trees was greater for blossom blight isolates (78.2%) than for fruit rot isolates (51.3%), indicating a greater contribution of clonal reproduction during the preharvest epidemic. For fruit rot isolates, between 54.2 and 81.7% of isolates were contained in one to four dominant clonal genotypes per tree having at least 10 members. All six fruit rot populations showed positive and significant spatial genetic autocorrelation for distance classes between 0.37 and 1.48 m. Despite high levels of within-tree pathogen diversity, the contribution of locally available inoculum combined with short-distance dispersal is likely the main factor generating clonal population foci and associated spatial genetic clustering within trees

Die Ablehnung der Softwarepatentrichtlinie: Ein zweifelhafter Erfolg

Anfang Juli 2005 hat das Europäische Parlament die Softwarepatentrichtlinie zurückgewiesen. Nach welchen Regeln werden computerimplementierte Erfindungen derzeit behandelt? Welche Folgen hat die Ablehnung der Richtlinie für die zukünftige Behandlung von Software-Entwicklungen? --

Genetic Diversity of Fusarium oxysporum f. sp. dianthi in Southern Spain

The diversity of races and prevalence of pathogenic populations of Fusarium oxysporum f. sp. dianthi (Fod) were surveyed in an area in southern Spain. From 54 farms, 132 isolates were collected from wilted carnation plants. Isolates were characterized by RAPD-PCR, DNA sequence analysis of the TEF1-α gene, and race-specific molecular markers. Selected isolates from RAPD groups were phenotypically evaluated by pathogenicity tests. Data analysis showed that Fod race 2 was the most frequent and prevalent race in the study area, followed by race 1/8. Moreover, phylogenetic analyses showed similar results, which were different to those of the race-specific PCR assays. It was concluded that (i) seven isolates were not classified in groups where Fod testers were clustered; even they showed different results when race-specific markers were used, (ii) ten isolates with retarded race 1 or race 8 specific band were characterized as F. proliferatum by TEF1-α gene sequencing and clustered into an outgroup, and (iii) six isolates failed to generate an amplification signal using race-specific markers. Furthermore, three of them were grouped close to race 2 tester according to the phylogenetic analyses, showing the same differential pathogenicity as race 2. This may indicate a Fod race 2 subgroup in this region.Ministerio de Educación y Ciencia AGL2005-08137-C03-02Junta de Andalucía P06-AGR-02313Ministerio de Ciencia e Innovación AGL2008-05414-C03-0

Relationships Between Blueberry Flower Age, Pollination, and Conidial Infection by Monilinia vaccinii-corymbosi

Monilinia vaccinii-corymbosi infects open blueberry flowers via the gynoecial pathway, leading to mummification of the developing fruit. To determine the effect of flower age on infection, stigmata were inoculated with conidia of M. vaccinii-corymbosi between 0 and 5 days after anthesis, fungal growth rates through the stylar canal were measured in detached flowers in the laboratory, and fruit disease incidence was determined in plants grown in the greenhouse. Hyphal growth rates were greatest in flowers inoculated on the day of anthesis, declined linearly with increasing flower age at inoculation (r = 0.921; P \u3c 0.0001; n = 12), and were unaffected by the presence or absence of pollen applied at the time of inoculation. In greenhouse-grown plants, the percentage of infected fruit decreased exponentially with increasing flower age at inoculation (R = 0.878; P = 0.0057; n = 10), with disease incidence ranging from 76.4% for flowers inoculated on the day of anthesis to 15.5% for those inoculated 4 days later. Fruit disease incidence in the greenhouse was linearly correlated with hyphal growth rates in detached flowers (r = 0.985; P \u3c 0.0001; n = 9), justifying the use of detached flowers when investigating gynoecial infection by M. vaccinii-corymbosi. In separate experiments, the effects of timing and sequence of pollination and inoculation on hyphal growth rates through the stylar canal and on disease incidence were investigated. Application of pollen to detached flowers 1 or 2 days before inoculation reduced hyphal growth rates by between 14.0 and 42.9% compared with flowers that received pollen and conidia simultaneously. Similarly, reductions in fruit disease incidence by between 9.5 and 18.3% were observed on greenhouse-grown plants for pollination-to-inoculation intervals ranging from 1 to 4 days. These results document that newly opened flowers are most susceptible to infection by M. vaccinii-corymbosi and that fruit disease incidence is reduced if pollination occurs at least 1 day before inoculation. Strategies that lead to early pollination of newly opened flowers may be useful for managing mummy berry disease in the field

Analyse Hitzeschockprotein-kodierender Gene von Mykoplasmen

[no abstract

Biodiversity of the genus Trichoderma and identification of marker genes involved in the antagonism between Trichoderma spp. and plant pathogenic fungi

As Mediterranean islands are known for their high diversity of vascular plants and a high level of endemism, they can provide basic insights into the relationship between geographical patterns and ecological processes. Studying the soil fungus Trichoderma/Hypocrea is of special interest to our working group due to its world-wide distribution, the continuos ongoing identification of new isolates, and its high percentage of strains being effective as biocontrol agents. We isolated 482 strains of Hypocrea/Trichoderma from 15 soils comprising undisturbed and disturbed soil environments. Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea/Trichoderma, sequence similarity analysis and phylogenetic inferences. The majority of the isolates were positively identified as pan- European and/or pan-global Hypocrea/Trichoderma species from Sections Trichoderma and Pachybasium. T. harzianum (H. lixii) is well known as a biocontrol agent against a range of economically important plant pathogens. In the present study we used a Rapid Subtraction Hybridisation approach to isolate genes which are differentially expressed during mycelial contact between T. harzianum and R. solani, and could serve as marker genes for selection of superior biocontrol strains. The 50 characterised clones comprised proteins involved (i) in protein synthesis and turn-over, (ii) in metabolism, and (iii) in the hydrolysis of macromolecules. An acetyl xylane esterase and an endoglucanase showed significant upregulation during in vivo confrontation of a T. harzianum strain with high antagonistic capability towards R. solani, while the expression was low or absent in a T. harzianum strain with poor antagonistic activity

Spatio-temporal Patterns of Pre-harvest Brown Rot Epidemics within Individual Peach Tree Canopies

Tree canopies are architecturally complex and pose several challenges for measuring and character-izing spatial patterns of disease. Recently developed methods for fine-scale canopy mapping and three-dimensional spatial pattern analysis were applied in a 3-year study to characterize spatio-temporal development of pre-harvest brown rot of peach, caused by Monilinia fructicola, in 13 trees of different maturity classes. We observed a negative correlation between an index of disease aggregation and disease incidence in the same tree (r = −0.653, P \u3c 0.0001), showing that trees with higher brown rot incidence had lower aggregation of affected fruit in their canopies. Significant (P ≤ 0.05) within-canopy aggregation among symptomatic fruit was most pronounced for early-maturing cultivars and/or early in the epidemic. This is consistent with the notion of a greater importance of localized, within-tree sources of inoculum at the beginning of the epidemic. Four of five trees having \u3e10 blossom blight symptoms per tree showed a significant positive spatial association of pre-harvest fruit rot to blossom blight within the same canopy. Spatial association analyses further revealed one of two out-comes for the association of new fruit rot symptoms with previous fruit rot symptoms in the same tree, whereby the relationship was either not significant or exhibited a significant negative associa-tion. In the latter scenario, the newly diseased fruit were farther apart from previously symptomatic fruit than expected by random chance. This unexpected result could have been due to uneven fruit ripening in different sectors of the canopy, which could have affected the timing of symptom devel-opment and thus led to negative spatial associations among symptoms developing over time in a tree