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.

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.

Although synthetic pesticides play a major role in plant protection, their application needs to be reduced because of their negative impact on the environment. This applies also to copper preparations, which are used in organic farming. For this reason, alternatives with less impact on the environment are urgently needed. In this context, we evaluated eight isolates of the genus Lysobacter (mainly Lysobacter enzymogenes) for their activity against plant pathogens. In vitro, the investigated Lysobacter isolates showed broad antagonistic activity against several phytopathogenic fungi, oomycetes and bacteria. Enzyme assays revealed diverse activities for the tested isolates. The most promising L. enzymogenes isolate (LEC) was used for further detailed analyses of its efficacy and effective working concentrations. The experiments included in vitro spore and sporangia germination tests and leaf disc assays as well as ad planta growth chamber trials against Alternaria solani and Phytophthora infestans on tomato plants, Pseudoperonospora cubensis on cucumbers and Venturia inaequalis on young potted apple trees. When applied on leaves, dilutions of a culture suspension of LEC had a concentration-dependent, protective effect against the tested pathogens. In all pathosystems tested, the effective concentrations were in the range of 2.5–5% and similarly efficacious to common plant protection agents containing copper hydroxide, wettable sulphur or fenhexamid. Thus, the isolate of L. enzymogenes identified in this study exhibits a broad activity against common plant pathogens and is therefore a promising candidate for the development of a microbial biocontrol agent.

In previous experiments, Trichoderma sp. BI 7376 and Pseudomonas chlororaphis subsp. aurantiaca BI 7439, applied as seed treatments, controlled soilborne Fusarium culmorum on maize. The current paper is focused on a deeper characterization of the effects of both strains. The experiments were conducted as pot tests with artificial inoculation of the substrate with F. culmorum, or with maize seed lots infected with Fusarium spp. When seeds were treated with Trichoderma strain BI 7376, Pseudomonas strain BI 7439 or with the chemical active ingredient thiram and stored before they were sown in substrate inoculated with F. culmorum, the protection by all agents declined. During the storage period of 211 days, the activity of thiram and Trichoderma strain BI 7376 dropped by about 38% and 57%, respectively. After 36 days of storage, Pseudomonas strain BI 7439 failed to provide any protection, which was obviously related to the observed total loss of viable cells of this agent. Moreover, we observed that both strains protected against soilborne F. subglutinans and F. verticillioides, showing that their activity was not limited to F. culmorum which was used in the previous experiments. Further, experiments with seed lots suspected or known to be infected with Fusarium species indicated that Trichoderma strain BI 7376 also controlled seedborne inoculum. When electron seed treatment was followed by application of Trichoderma strain BI 7376, both seed- and soilborne infections were controlled, showing that the concept of using a combination of a physical seed treatment and microbial antagonists appears feasible.

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.

The aim of this study was to determine the impact of the location of the pathogen inoculum on damage caused by Globisporangium (syn. Pythium) ultimum, Fusarium culmorum and Rhizoctonia solani in pot tests with maize. For this purpose, pathogen inoculum was added to potting substrate, and the resulting mix was used to fill the whole pot volume, the upper half, or the lower half of pots. The remaining volume was filled with non-inoculated substrate. In a second experimental approach, maize seeds were germinated in non-inoculated potting substrate and the seedlings were transferred to inoculated substrate. The seeds were untreated, treated with the chemical thiram, or treated with a bacterial or a fungal biocontrol agent. With each of the pathogens, the damage to the developing maize seedlings was the strongest when the seeds germinated in the inoculated potting substrate. When only the roots were in contact with the inoculum, there was limited damage by R. solani and F. culmorum, and no damage by G. ultimum. This implies that in experiments with artificial inoculation, the seeds should always be in immediate contact with the inoculum if a strong pathogenic effect is desired. Conversely, seed treatments must, in the first place, be able to protect the spermosphere, while the requirement to protect the roots at a distance from the seed seems to depend on the pathogen.

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.

Plant-based screening experiments were conducted with the aim of identifying biocontrol bacteria and fungi for seed treatment of maize. Candidate microorganisms were evaluated for their protective effects against soilborne infections by species of Fusarium , Globisporangium (syn. Pythium ) and Rhizoctonia. The microorganisms tested were bacteria and fungi from maize roots or other sources, including some active microbial components of commercial biocontrol products. Due to the method of isolation chosen, the majority of bacteria from maize roots were spore formers, most of them species of the genera Bacillus, Brevibacillus and Paenibacillus . In pot tests with the potting substrate inoculated with F. culmorum, the level of control provided by seed treatment with the most efficacious bacterial and fungal isolates was comparable or close to the chemical reference seed treatment thiram. The most effective bacteria were species of Pseudomonas , Burkholderia and Streptomyces . Among a subset of approx. 100 bacteria studied, the in vivo and in vitro activities against F. culmorum were only weakly correlated, although some strains deviated from this pattern. The most effective fungi were two strains of Clonostachys rosea and isolates of Trichoderma . The latter and a strain of Gliocladium virens provided also protection against R. solani . Activity against Globisporangium ultimum was recorded for one isolate of Trichoderma and the two strains of C. rosea. A reduction in the impact of seedborne F. culmorum was also observed after seed treatment with two strains of F. oxysporum f. sp. strigae. The results are discussed in relation to previous reports on rhizosphere bacteria of maize and their use in biocontrol of plant pathogens or for plant growth promotion.

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 Chaetomium 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. 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 the Study We provide the first systematic study comparing six different Chaetomium species with regard to their use as biocontrol agents.

The study describes the development and employment of plant tests based on artificial inoculation of seeds or the potting substrate for evaluating the potential of microorganisms to control seedling blight of maize caused by seed- and soil-borne fusaria. Nine strains ofFusariumwere isolated from maize kernels and identified morphologically and by molecular methods as belonging to the speciesFusarium verticillioides, F. subglutinans, F. cerealis, F. poaeandF. proliferatum. In order to determine pathogenicity, maize kernels were inoculated by immersion in suspensions of conidia of these isolates and sown in a pasteurized substrate in seed trays. Based on plant dry weight, the isolates ofF. verticillioidesandF. subglutinanswere more pathogenic than the other isolates. Using an isolate ofF. subglutinans, the efficacy of a set of 25 potential fungal and bacterial antagonists was assessed using inoculation of maize kernels by placement in mixtures of the pathogen and the antagonists. The results obtained with this methodology indicate the potential of a number of different microorganisms applied as seed treatments, including some reported previously as biocontrol agents, to control seed-borne seedling blight of maize. In order to develop a method for the testing of biocontrol agents against soil-borne attack, isolates ofF. subglutinans,F. cerealisandF poaefrom maize kernels together with isolates ofF. avenaceum,F. culmorumandF. graminearumoriginating from maize silage and wheat were used to artificially inoculate the potting substrate. The results showed large differences in pathogenicity, with the most aggressive isolates belonging toF. culmorumandF. graminearum.

Common bean root microbiome was used to research for potential biocontrol agents of phytopathogenic fungi as Fusarium sp., Macrophomina sp., and Alternaria sp. causal agents of root rot disease on host plants. Therefore, a bacterial collection of 90 endophytic and rhizospheric isolates was established from field-grown common bean plants in Tunisia and screened for their antifungal activity against pathogenic fungal strains. Antifungal activity was checked at biochemical and genetic levels. Twelve bacterial strains exhibited up to 71% of inhibition of the three pathogenic strains of Fusarium sp., Macrophomina sp., and Alternaria sp. Biocontrol assays conducted under controlled conditions demonstrated that Bacillus amyloliquefaciens , Bacillus halotolerans , Bacillus velezensis , Agrobacterium fabrum , and Pseudomonas lini displayed the highest protective effect on common bean cv. Coco blanc. These bacterial strains were associated with significant plant growth promotion up to 217%, in comparison with control plants. Biochemical analysis of the antagonistic and plant growth promoting activity revealed the production of xylanases, chitinases, siderophore, hydrogen cyanide, phosphate-solubilizing activity, and the production of indole-3-acetic acid, particularly in Bacillus spp. strains. Polymerase chain reaction revealed the presence of lipopeptide biosynthetic genes encoding surfactin, iturin, bacillomycin, and fengycin. The study unveiled that common bean root microbiome contains potential bacterial strains that exhibit efficient biocontrol activity of Fusarium sp., Macrophomina sp., and Alternaria sp., and act as plant growth promoters. Considering various plant growth promoting and biocontrol traits, the study showed the superiority of the Bacillus spp. strains among different common bean root microbiomes.

Pythium isolates from diseased and dead bait plants of maize and cress grown in compost or various soils (maize fields, parkland under deciduous trees, grassland) were characterised and tested for pathogenicity to maize ( Zea mays L . ). In pot tests performed under controlled conditions, pathogenicity of the isolates to maize was apparent by reduction of root and shoot growth, whereas damping-off of maize seedlings was less frequent. Contrarily, pea seedlings were killed by pathogenic Pythium isolates. Pythium isolates from diseased maize seedlings and pathogenic strains from other gramineous plants ( P. phragmitis , P. aff. phragmitis, P. catenulatum ) were not necessarily more virulent to maize compared to isolates originating from dicotyledonous plants (cress). The most virulent isolates originated from compost and caused a reduction of maize shoot growth of up to 60%. Phylogenetic analysis revealed that they were very closely related to P. ultimum var. ultimum and P. arrhenomanes , respectively. Isolates originating from maize fields, grassland and parkland under deciduous trees, a reference culture of P. arrhenomanes and strains of P. phragmitis , P. aff . phragmitis and P. catenulatum with known pathogenicity on reed were non-pathogenic on maize. Isolates from compost, and from maize fields generally had a higher temperature optimum for mycelial growth (30 °C) and a faster growth rate (1.5–2.0 mm h −1 ) compared to the isolates from parkland under deciduous trees and grassland soil (20–25 °C, ~1.0 mm h −1 ), respectively. This study indicates a potential impact of pathogenic Pythium on maize plants even in the absence of visible symptoms.

For biological control of plant pests, e.g. cockchafer grubs, in the rhizosphere of oak, apple or pine trees, ento­mopathogenic Beauveria spp. are increasingly applied. For successful use, it is important to monitor the spread and persistence of the inoculated fungi, both qualitati­vely and quantitatively. The determination of both para­meters by plating on selective nutrient media or by mole­cular methods such as PCR of soil samples are quite laborious and often do not yield satisfactory results. Therefore, the aim of the present study was to develop a spe­cific in situ method using immunofluorescence labelling of Beauveria spp. growing on young fine roots of three-year old oak saplings. All fine roots investigated were covered with a dense net of soil rhizosphere fungi, as visua­lized by staining with the nonspecific dye blankophor. On non-inoculated roots, polyclonal Beauveria anti­bodies did not label any of these naturally growing fungi. Only samples of roots inoculated with Beauveria brongniartii displayed specific labelling up to ten months after inocu­lation. Whereas the natural rhizosphere fungi were detected growing in the intercellular space of the root cortex in an ectomycorrhiza-like manner up to the endodermis, hyphae of the inoculated B. brongniartii were never seen within the root tissue but only growing on the surface of the rhizodermis. These observations indicate that B. brongniartii does not grow endophytically, and that the method used allows to discriminate B. brongniartii from the resident fungal flora in the oak tree rhizosphere. Detection by immunofluorescence labelling employed in the current study may be a useful tool to follow B. brongniartii in experiments aimed at establishing the entomopathogen in the rhizosphere and to monitor its fate in long-term control of entomopathogens.

The study involved six test fungi previously recorded in the literature as being endophytes (Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea, Trichoderma harzianum, Fusarium proliferatum, Chaetomium globosum), two plant pathogenic fungi (Ascochyta fabae, Plenodomus lingam) and four host plants (Vicia faba, Bras­sica napus, Phaseolus vulgaris, Zea mays). Aerial conidia, blastospores, or ascospores, respectively were applied to leaf surfaces by spraying or by infiltrating spore suspensions through stomata directly into the leaves. Obser­vations using light microscopy showed that the test fungi germinated on the leaf surface but did not enter actively into the leaves. Within the leaves, germination of spores and growth of hyphae appeared to depend on the presence of damaged plant tissue. Various host reactions such as browning of epidermal cells and formation of papillae were observed. Colonization of healthy leaves by the test fungi in a manner similar to the pathogens A. fabae (on Faba bean) and P. lingam (on oilseed rape) was not observed. Spore germination and hyphal growth commenced when inoculated leaves were placed on agar medium. The results indicate that the test fungi possessed a saprotrophic rather than an endo­phytic life style when associated with leaf tissue of the studied hosts.