Regulation of seed germination in the close Arabidopsis relative Lepidium sativum : a global tissue-specific transcript analysis

The completion of germination in Lepidium sativum and other endospermic seeds (e.g. Arabidopsis [Arabidopsis thaliana]) is regulated by two opposing forces, the growth potential of the radicle (RAD) and the resistance to this growth from the micropylar endosperm cap (CAP) surrounding it. We show by puncture force measurement that the CAP progressively weakens during germination, and we have conducted a time-course transcript analysis of RAD and CAP tissues throughout this process. We have also used specific inhibitors to investigate the importance of transcription, translation, and posttranslation levels of regulation of endosperm weakening in isolated CAPs. Although the impact of inhibiting translation is greater, both transcription and translation are required for the completion of endosperm weakening in the whole seed population. The majority of genes expressed during this process occur in both tissues, but where they are uniquely expressed, or significantly differentially expressed between tissues, this relates to the functions of the RAD as growing tissue and the CAP as a regulator of germination through weakening. More detailed analysis showed that putative orthologs of cell wall-remodeling genes are expressed in a complex manner during CAP weakening, suggesting distinct roles in the RAD and CAP. Expression patterns are also consistent with the CAP being a receptor for environmental signals influencing germination. Inhibitors of the aspartic, serine, and cysteine proteases reduced the number of isolated CAPs in which weakening developed, and inhibition of the 26S proteasome resulted in its complete cessation. This indicates that targeted protein degradation is a major control point for endosperm weakening

Isolation and Characterization of fungal causal agents of black bark disease of apple using the “apple test”

In den letzten, häufig von Trockenheit geprägten Jahren hat sich der Schwarze Rindenbrand an Apfel- und Birnenbäumen in verschiedenen Teilen Deutschlands zunehmend zu einem Problem entwickelt. Ziel der vorliegenden Arbeit war es daher, methodische Grundlagen zur Isolierung der Erreger aus Apfelbäumen und ihrer Charakterisierung im Labor zu erarbeiten. Die Experimente beinhalteten die klassische Isolierung durch Auslegen von erkranktem Gewebe auf Agarmedien sowie ein in der Literatur als „Apfeltest“ beschriebenes Verfahren, bei dem befallene Rinde in Apfelfrüchte gesteckt wird. In der Folge reichern sich Pathogene im Gewebe an und können aus den entstehenden Faulstellen isoliert werden.Aus dem Stammholz eines im Wipfelbereich welkenden Baumes wurden nach Auslegen auf Agarmedium Trametes versicolor, Diaporthe eres sowie Diplodia seriata (bekannt als einer von mehreren Erregern des Schwarzen Rindenbrandes) isoliert. Diese Art wurde auch nach Auslegen befallener Rinde auf Kartoffel-Dextrose-Agar erhalten, ebenso wie Diplodia malorum. Durch Einstecken befallener Rindenstücke in Apfelfrüchte wurden weitere Isolate von D. malorum, drei Isolate von Diplodia bulgarica sowie Lambertella corni-maris, Penicillium sp. und Sclerotinia sclerotiorum gewonnen. Im Biotest mit künstlicher Inokulation mit Reinkulturen riefen die drei letztgenannten Isolate, alle Diplodia-Isolate sowie Diaporthe eres Fruchtfäulen hervor. Die Diplodia-Arten wurden darüber hinaus hinsichtlich der Geschwindigkeit des Hyphenwachstums und der Sporenmorphologie charakterisiert.During the previous years characterized by summer-drought, black canker disease of apple and pear has become a problem in different parts of Germany. The aim of the current work was therefore to develop basic techniques for isolation of the inciting pathogens from apple trees and their characterization in the laboratory. The experiments included the standard isolation of putative pathogens by placing symptomatic tissue on agar media as well as a method described in the literature as „apple test”. For the latter, symptomatic bark pieces are inserted into apple fruits and colonization of the apple tissue is visible as rotting spots. Candidate pathogens can be easily isolated from the colonized apple tissue.After placement of tissue from the trunk of the top of an apple tree expressing wilt symptoms, Trametes versicolor, Diaporthe eres and Diplodia seriata (one of several agents known to cause black canker disease) were isolated. Together with Diplodia malorum, the latter species was also obtained after placement of symptomatic bark pieces on potato dextrose agar. Insertion of diseased bark into apple fruits led to the isolation of further isolates of D. malorum, three isolates of Diplodia bulgarica, Lambertella corni-maris, Penicillium sp. and Sclerotinia sclerotiorum. After inoculation with pure cultures into apple fruits, the latter three, all Diplodia-isolates and Diaporthe eres were pathogenic and caused fruit rots. The Diplodia isolates were further characterised regarding speed of hyphal growth and spore morphology

Characterization of cultural traits and fungicidal activity of strains belonging to the fungal genus Chaetomium

AIMS:Compare and characterize Chaetomium strains with special regard to their potentialities as biocontrol agents. METHODS AND RESULTS:Twelve strains of the fungal genus Chaetomium from diverse ecological niches were identified as belonging to six different species. Large differences were observed between the strains with regard to temperature requirements for mycelial growth and pigmentation of culture filtrates. Culture filtrates and ethyl acetate extracts were assayed for fungicidal effects against important phytopathogens both on agar media and in multiwell plates. The samples from C. globosum were particularly active against Botrytis cinerea, Pyrenophora graminea and Bipolaris sorokiniana, while those from C. cochliodes and C. aureum were inhibitory towards Phytophthora infestans, and P. infestans and Fusarium culmorum, respectively. In order to narrow down the active principle, the most promising extracts were separated by preparative HPLC and the resulting fractions tested in bioassays. Chaetoglobosins were identified as active compounds produced by C. globosum. CONCLUSIONS:The bioassays revealed C. aureum and C. cochliodes as promising candidates for use in biocontrol. Both showed remarkably good activity against the prominent plant pathogen P. infestans. SIGNIFICANCE AND IMPACT OF STUDY:We provide the first systematic study comparing six different Chaetomium species with regard to their use as biocontrol agents

Biocontrol of plant pathogens by Lysobacter enzymogenes isolate LEC: efficacy of shake flask broth, fermentation broth and dried preparations

The aim of our study was to upscale the production of Lysobacter enzymogenes strain LEC from shake flasks to a laboratory bioreactor. The obtained fermentation broth was to be downstream-processed by lyophilisation and fluidised bed-drying, followed by testing of the dried preparations against plant pathogens. Different batches of broth from shake flasks and from individual fermenter runs were characterised and compared. Number of viable cells, in vitro efficacy (ED90 value) against the apple scab pathogen Ventura inaequalis, as well as efficacy against Phytophthora infestans on potato plants were all reproducible and not impaired by upscaling the production volume from shake flask to the bioreactor. Fermentation broth was processed by lyophilisation and fluidised bed-drying. In bioassays, the activity against downy mildew (Pseudoperonospora cubensis) on cucumber were comparable for the unformulated fermentation broth and the fluidised bed-dried product, while the lyophilised product was significantly more effective. Strain LEC showed reproducible pathogen suppressive activity, independent from production scale and batch. Production upscaling and formulation by drying are possible without loss of efficacy

Secondary metabolites from Lysobacter enzymogenes isolate LEC possess suppressive activity against microbial plant pathogens in vitro and in planta

In previous studies Lysobacter enzymogenes isolate LEC exhibited broad biocontrol activity against microbial phytopathogens on different crops. The aim of the present study was to identify metabolites responsible for the antimicrobial and disease-suppressive activities of this isolate. Fermentation broth of LEC was fractionated by preparative HPLC and the resulting single fractions were analyzed by HPLC-ESI-MS. In vitro growth suppression by single fractions was apparent in agar diffusion and microtiter plate assays for Fusarium culmorum, Pythium ultimum, and Bacillus megaterium, but not for Escherichia coli. For F. culmorum and Py. ultimum, suppression was caused by fractions containing precursor ions m/z 511.28 (z = 1) and m/z 513.30 (z = 1), that are characteristic for members of the group of polycyclic tetramate macrolactams (PoTeMs) like alteramides, heat stable antifungal factor (HSAF) and derivatives thereof. The inhibition of B. megaterium was caused by a much broader spectrum of fractions that also contained different precursor ions. In vitro tests with Phytophthora infestans and climate chamber tests with Pseudoperonospora cubensis on cucumber plants were carried out with pooled groups from the single fractions. Significant and dose-dependent activity was exclusively observed for the pooled group containing PoTeMs, which was in agreement with the results of the plate assays. Activity comparable to a copper-containing fungicide was apparent in a concentration that corresponded to 3% or higher of the original fermentation broth. We therefore assume that PoTeMs are responsible for the biocontrol activity of isolate LEC. These findings confirm the capability and suitability of LEC as a biocontrol agent