The bryophyte genus Sphagnum is a reservoir for powerful and extraordinary antagonists and potentially facultative human pathogens

Sphagnum plants grow in natural, species-poor carpets at low pH but without any known substantial fungal disease. To investigate this phenomenon, we analysed bacterial populations associated with two Sphagnum species with different ecological behaviour, namely S. magellanicum and S. fallax, from three sites in Germany and three in Norway, with a special focus on the functional group of antagonists. The screening of 493 bacterial isolates for antagonistic activity against fungal pathogens resulted in 237 (48%) active isolates. We found a higher proportion of antagonists for S. magellanicum (24%) than we did for S. fallax (19%) in general. The majority of the antagonists belonged to the genera Serratia (15%), Burkholderia (13.5%), Staphylococcus (13.5%), and Pseudomonas (10%). In contrast to the high moss specificity found for antagonistic bacteria, Burkholderia as well as Serratia isolates with highly similar molecular fingerprints as ascertained by BOX-PCR for both Sphagnum species were found. Interestingly, a high proportion of antagonists, for example Staphylococcus, Hafnia, Yersinia, and Pantoea, were identified as strains that are known as facultative pathogens of humans. Sphagnum plants represent an ecological niche not only for diverse and extraordinary microbial populations with a high potential for biological control of plant pathogens but also for opportunistic human pathogens.     

Diversity and antagonistic potential of bacteria associated with bryophytes from nutrient-poor habitats of the Baltic Sea Coast

Very little is known about the interaction of bryophytes with bacteria. Therefore, we analyzed bacteria associated with three bryophyte species, Tortula ruralis, Aulacomnium palustre, and Sphagnum rubellum, which represent typical moss species of three nutrient-poor plant communities at the southern Baltic Sea coast in Germany. By use of two cultivation-independent techniques, denaturing gradient gel electrophoresis and single-strand conformation polymorphism analysis of the 16S ribosomal DNA, a high degree of moss specificity was found for associated bacterial communities. This specificity could be further evidenced by a cultivation-dependent approach for the following parameters: (i) plate counts of bacteria on R2A medium, (ii) proportion of antagonistic isolates, (iii) antagonistic activity as well as spectrum against pathogens, and (iv) diversity and richness of antagonistic isolates. The proportion of isolates with antagonistic activity against the pathogenic model fungus Verticillium dahliae was highest for S. rubellum (31%), followed by A. palustre (17%) and T. ruralis (5%). A high percentage (99%) of moss-associated antagonistic bacteria produced antifungal compounds. The high recovery of antagonistic isolates strongly suggests that bryophytes represent an ecological niche which harbors a diverse and hitherto largely uncharacterized microbial population with yet unknown and untapped potential biotechnological applications, e.g., for biological control of plant pathogens.     

Diversity and Antagonistic Potential of Bacteria Associated with Bryophytes from Nutrient-Poor Habitats of the Baltic Sea Coast

Very little is known about the interaction of bryophytes with bacteria. Therefore, we analyzed bacteria associated with three bryophyte species, Tortula ruralis, Aulacomnium palustre, and Sphagnum rubellum, which represent typical moss species of three nutrient-poor plant communities at the southern Baltic Sea coast in Germany. By use of two cultivation-independent techniques, denaturing gradient gel electrophoresis and single-strand conformation polymorphism analysis of the 16S ribosomal DNA, a high degree of moss specificity was found for associated bacterial communities. This specificity could be further evidenced by a cultivation-dependent approach for the following parameters: (i) plate counts of bacteria on R2A medium, (ii) proportion of antagonistic isolates, (iii) antagonistic activity as well as spectrum against pathogens, and (iv) diversity and richness of antagonistic isolates. The proportion of isolates with antagonistic activity against the pathogenic model fungus Verticillium dahliae was highest for S. rubellum (31%), followed by A. palustre (17%) and T. ruralis (5%). A high percentage (99%) of moss-associated antagonistic bacteria produced antifungal compounds. The high recovery of antagonistic isolates strongly suggests that bryophytes represent an ecological niche which harbors a diverse and hitherto largely uncharacterized microbial population with yet unknown and untapped potential biotechnological applications, e.g., for biological control of plant pathogens.     

Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi

Differences between endophytic and ectophytic bacterial communities with stress on antagonistic bacteria, were studied by comparing the composition of communities isolated from the rhizosphere, phyllosphere, endorhiza and endosphere of field-grown potato plants using a multiphasic approach. Terminal restriction fragment length polymorphism analysis of 16S rDNA of the bacterial communities revealed discrete microenvironment-specific patterns. To measure the antagonistic potential of potato-associated bacteria, a total of 2648 bacteria were screened by dual testing of antagonism to the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. Composition and diversity of bacterial antagonists were mainly specific for each microenvironment. The rhizosphere and endorhiza were the main reservoirs for antagonistic bacteria and showed the highest similarity in their colonisation by antagonists. The most prominent species of all microenvironments was Pseudomonas putida, and rep-PCR with BOX primers showed that these isolates showed microenvironment-specific DNA fingerprints. P. putida isolates from the rhizosphere and endorhiza gave nearly identical fingerprints confirming the high similarity of bacterial populations. The phlD gene, involved in the production of the antibiotic 2,4-diacetyl-phloroglucinol, was found only among Pseudomonas isolates from the rhizosphere and endorhiza. Evaluation of the bacterial isolates for biocontrol potential based on fungal antagonism and physiological characteristics resulted in the selection of five promising isolates from each microenvironment. The most effective isolate was Serratia plymuthica 3Re4-18 isolated from the endorhiza.     

Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematode Meloidogyne incognita (Kofoid & White) Chitwood

To study the effect of microenvironments on potato-associated bacteria, the abundance and diversity of bacteria isolated from the rhizosphere, phyllosphere, endorhiza, and endosphere of field grown potato was analyzed. Culturable bacteria were obtained after plating on R2A medium. The endophytic populations averaged 10(3) and 10(5) CFU/g (fresh wt.) for the endosphere and endorhiza. respectively, which were lower than those for the ectophytic microenvironments, with 10(5) and 10(7) CFU/g (fresh wt.) for the phyllosphere and rhizosphere, respectively. The composition and richness of bacterial species was microenvironment-dependent. The occurrence and diversity of potato-associated bacteria was additionally monitored by a cultivation-independent approach using terminal restriction fragment length polymorphism analysis of 16S rDNA. The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of microenvironment-specific communities. In an approach to measure the antagonistic potential of potato-associated bacteria, a total of 440 bacteria was screened by dual testing for in vitro antagonism towards the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. The proportion of isolates with antagonistic activity was highest for the rhizosphere (10%), followed by the endorhiza (9%), phyllosphere (6%), and endosphere (5%). All 33 fungal antagonists were characterized by testing their in vitro antagonistic mechanisms, including their glucanolytic, chitinolytic, pectinolytic, cellulolytic, and proteolytic activity, and by their BOX-PCR fingerprints. In addition, they were screened for their biocontrol activity against Meloidogyne incognita. Overall, nine isolates belonging to Pseudomonas and Streptomyces species were found to control both fungal pathogens and M. incognita and were therefore considered as promising biological control agents.     

Endophytic bacterial communities of field-grown potato plants and their plant-growth-promoting and antagonistic abilities

To study the effect of plant growth on potato-associated bacteria, the composition and properties of bacteria colonizing the endosphere of field-grown potato were analyzed by a multiphasic approach. The occurrence and diversity of potato-associated bacteria were monitored by a cultivation-independent approach, using terminal restriction fragment length polymorphism analysis of 16S rDNA. The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of plant-specific communities. However, endophytic populations correlated to a certain extent with plant growth performance. Endophytes were also isolated from plants that grew well or grew poorly and were identified by partial sequencing of the 16S rRNA genes. A broad phylogenetic spectrum was found among isolates and differently growing plants hosted different bacterial populations. In an approach to investigate the plant-growth-promoting potential of potato-associated bacteria, a total of 35 bacteria were screened by dual testing for in vitro antagonism towards (i) the fungal pathogens Verticillium dahliae, Rhizoctonia solani, Sclerotinia sclerotiorum, and Phytophthora cactorum and (ii) the bacterial pathogens Erwinia carotovora, Streptomyces scabies, and Xanthomonas campestris. The proportion of isolates with antagonistic activity was highest against Streptomyces sp. (43%) followed by those against Xanthomonas sp. (29%). As all plants showed more or less severe disease symptoms of scab disease caused by Streptomyces scabies, we assume that the presence of the pathogen induced the colonization of antagonists. The antifungal activity of the isolates was generally low. The biotechnological potential of endophytic isolates assessed by their antagonistic activity and by in vitro production of enzymes, antibiotics, siderophores, and the plant growth hormone indole-1,3-acetic acid was generally high. Overall, seven endophytes were found to antagonize fungal as well as bacterial pathogens and showed a high production of active compounds and were therefore considered promising biological control agents.     

不同生境下五唇兰根部可培养内生细菌多样性研究

兰科植物内生细菌与菌根真菌的协作对宿主植物的生长、抗病、抗逆及植物修复环境能力等具有重要意义,揭示其内生细菌多样性及与生境之间的关系有助于阐明兰科植物的适应与进化机制。本研究基于16SrDNA序列分析探讨了不同生境下东南亚特有种五唇兰根部可培养内生细菌多样性及其空间异质性。结果表明:从不同生境下五唇兰根部共分离出内生细菌59株,其中从土生型五唇兰根部分离出内生细菌45株(76.27%),从石生型五唇兰根部分离出内生细菌14株(23.73%);基于内生细菌16SrDNA序列同源性分析及构建的系统发育树显示,五唇兰根部内生细菌分属于7属,即芽孢杆菌属(Bacillus)、伯克氏菌属(Burkholderia)、草酸菌属(Pandoraea)、土壤杆菌属(Agrobacterium)、类芽孢杆菌属(Paenibacillus)、泛菌属(Pantoea)、欧文氏菌属(Erwinia),其中优势属为芽孢杆菌属,次优势属为泛菌属和伯克氏菌属;多样性分析显示,土生型五唇兰根部内生细菌群落的Shannon多样性指数大于石生型五唇兰,不同生境下五唇兰根部内生细菌群落结构差异极显著(P0.01)。土生型五唇兰根部内生细菌群落优势属为芽孢杆菌属和泛菌属,石生型五唇兰根部内生细菌群落优势属为芽孢杆菌属和伯克氏菌属。     

The rhizosphere effect on bacteria antagonistic towards the pathogenic fungus Verticillium differs depending on plant species and site

Rhizobacteria with antagonistic activity towards plant pathogens play an essential role in root growth and plant health and are influenced by plant species in their abundance and composition. To determine the extent of the effect of the plant species and of the site on the abundance and composition of bacteria with antagonistic activity towards Verticillium dahliae, bacteria isolated from the rhizosphere of two Verticillium host plants, oilseed rape and strawberry, and from bulk soil were analysed at three different locations in Germany over two growing seasons. A total of 6732 bacterial isolates screened for in vitro antagonism towards Verticillium resulted in 560 active isolates, among which Pseudomonas (77%) and Serratia (6%) were the most dominant genera. The rhizosphere effect on the antagonistic bacterial community was shown by an enhanced proportion of antagonistic isolates, by enrichment of specific amplified ribosomal DNA restriction analysis types, species and genotypes, and by a reduced diversity in the rhizosphere in comparison to bulk soil. Such an effect was influenced by the plant species and by the site of its cultivation. Altogether, 16S rRNA gene sequencing of 66 isolates resulted in the identification of 22 different species. Antagonists of the genus Serratia were preferentially isolated from oilseed rape rhizosphere, with the exception of one site. For isolates of Pseudomonas and Serratia, plant-specific and site-specific genotypes were found.     

Microbial communities associated with the root system of wild olives (Olea europaea L. subsp. europaea var. sylvestris) are good reservoirs of bacteria with antagonistic potential against Verticillium dahliae

Wild olive trees, namely oleaster, are considered the ancestor of cultivated olive and a unexplored source of genetic variability that might contain important traits of agronomic and biotechnological interest. The longevity and genetic diversity of oleasters may have favoured selection of specific and well adapted rhizosphere microbial populations that can constitute unique reservoirs of microbial antagonists of Verticillium dahliae, the main soilborne fungal pathogen of olive worldwide. The objective of this present study was to determine the structure and diversity of bacterial communities in the rhizosphere and endosphere of oleaster from 11 havens in Cádiz and Córdoba provinces of Andalusia, southern Spain. To carry out the study we used a multiphasic approach. First, the occurrence and diversity of rhizosphere bacteria was monitored by a cultivation-independent-approach, using fluorescent terminal restriction fragment length polymorphism (FT-RFLP) analyses of amplified 16S rDNA sequences. FT-RFLP patterns revealed a high heterogeneity in the composition of the sampled rhizosphere bacterial communities and suggested the existence of plant genotype-site-specific communities, with each oleaster haven being a unique reservoir of bacterial diversity. Secondly, to investigate the antagonistic potential of these root-associated bacterial populations, a total of 675 bacterial isolates obtained from oleaster rhizosphere and endosphere were screened by dual testing for inhibition of in vitro growth of the highly virulent, olive defoliating pathotype of V. dahliae. Out of 675 tested bacterial isolates, 94 (14%) showed a strong antagonistic activity against a defoliating V. dahliae pathotype. Of the antagonistic bacteria, a slightly lower proportion (12.9% of total bacteria) were inhabitant of the oleaster rhizosphere compared to that in the endosphere (16.5%). The biotechnological potential of those isolates was assessed by in vitro production of different hydrolytic enzymes, indole-1.3-acetic acid (IAA), siderophores, and antimicrobial compounds. Overall, most of bacterial antagonists (58.5 to 78.3%) showed proteolytic, lipolytic, and chitinolytic activity, and produced IAA and siderophores. Finally, analysis of the 16S rDNA gene sequence indicated that most of the 94 bacterial antagonists belong to genera Bacillus (56.4%), Pseudomonas (27.7%), and Paenibacillus (7.4%). Overall, the rhizosphere and endosphere of wild olives were proved as a good reservoir of bacteria antagonists against V. dahliae. Several of those bacteria showing high and broad antagonism potential may therefore be considered for further analyses as promising biocontrol agents against V. dahliae in olive.     

Successful strategy for the selection of new strawberry-associated rhizobacteria antagonistic to Verticillium wilt

In order to isolate and characterize new strawberry-associated bacteria antagonistic to the soil-borne pathogenic fungus Verticillium dahliae Kleb., rhizobacterial populations from two different strawberry species, Greenish Strawberry (Fragaria viridis) and Garden Strawberry (F. x ananassa) obtained after plating onto King's B and glycerol-arginine agar, were screened for in vitro antagonism toward V. dahliae. The proportion of isolates with antifungal activity determined in in vitro assay against V. dahliae was higher for the Garden Strawberry than for the Greenish Strawberry. From 300 isolates, 20 isolates with strong antifungal activity were selected characterized by physiological profiling and molecular fingerprinting methods. Diversity among the isolates was characterized with molecular fingerprints using amplified ribosomal DNA restriction analysis (ARDRA) and the more discriminating BOX-PCR fingerprint method. The physiological profiles were well correlated with molecular fingerprinting pattern analysis. Significant reduction of Verticillium wilt by bacterial dipping bath treatment was shown in the greenhouse and in fields naturally infested by V. dahliae. The relative increase of yield ranged from 117% (Streptomyces albidoflavus S1) to 344% (Pseudomonas fluorescens P10) in greenhouse trials, and 113% (Streptomyces albidoflavus S1) to 247% (Pseudomonas fluorescens P6) in field trials. Evaluation resulted in the selection of three effective biocontrol agents (Pseudomonas fluorescens P6, P10, and Streptomyces diastatochromogenes S9) antagonistic to the Verticillium wilt pathogen.     

Endophytic Bacterial Communities in Ginseng and their Antifungal Activity Against Pathogens

Plant roots are associated with diverse communities of endophytic bacteria which do not exert adverse effects. The diversity of bacterial endophytes associated with ginseng roots cultivated in three different areas in Korea was investigated. Sixty-three colonies were isolated from the interior of ginseng roots. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates belonged to three major phylogenetic groups: the high G+C Gram-positive bacteria (HGCGPB), low G+C Gram-positive bacteria (LGCGPB), and the Proteobacteria. The dominant species at the three different ginseng growing areas were: HGCGPB at Ganghwa (55.0%), LGCGPB at Geumsan (45.5%), and Proteobacteria at Jinan (61.9%). Most cellulase-, xylanase-, and pectinase-producing colonies among the isolates belong to the LGCGPB group, except for Pectobacterium carotovora which belonged to the Proteobacteria. The 13 isolates belonging to LGCGPB and Proteobacteria were assessed for their antifungal activity against phytopathogenic fungi such as Rhizoctonia solani. Among them, Paenibacillus polymyxa GS01, Bacillus sp. GS07, and Pseudomonas poae JA01 show potential activity as biocontrol agents against phytopathogenic fungi. Finally, most of the low G+C Gram-positive bacteria with antifungal activity against phytopathogenic microorganisms showed cellulolytic enzyme activity while some Proteobacteria with the antifungal activity and the high G+C Gram-positive bacteria did not show any cellulolytic activity.     

Phylogenetic distribution of symbiotic bacteria from Panamanian amphibians that inhibit growth of the lethal fungal pathogen Batrachochytrium dendrobatidis

The introduction of next‐generation sequencing has allowed for greater understanding of community composition of symbiotic microbial communities. However, determining the function of individual members of these microbial communities still largely relies on culture‐based methods. Here, we present results on the phylogenetic distribution of a defensive functional trait of cultured symbiotic bacteria associated with amphibians. Amphibians are host to a diverse community of cutaneous bacteria and some of these bacteria protect their host from the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd) by secreting antifungal metabolites. We cultured over 450 bacterial isolates from the skins of Panamanian amphibian species and tested their interactions with Bd using an in vitro challenge assay. For a subset of isolates, we also completed coculture experiments and found that culturing isolates with Bd had no effect on inhibitory properties of the bacteria, but it significantly decreased metabolite secretion. In challenge assays, approximately 75% of the bacterial isolates inhibited Bd to some extent and these inhibitory isolates were widely distributed among all bacterial phyla. Although there was no clear phylogenetic signal of inhibition, three genera, Stenotrophomonas, Aeromonas and Pseudomonas, had a high proportion of inhibitory isolates (100%, 77% and 73%, respectively). Overall, our results demonstrate that antifungal properties are phylogenetically widespread in symbiotic microbial communities of Panamanian amphibians and that some functional redundancy for fungal inhibition occurs in these communities. We hope that these findings contribute to the discovery and development of probiotics for amphibians that can mitigate the threat of chytridiomycosis.     

Bacterial diversity associated with subalpine fir (Abies lasiocarpa) ectomycorrhizae following wildfire and salvage-logging in central British Columbia

To assess the effect of fire and salvage logging on the diversity of mycorrhizal-bacterial communities, bacteria associated with Cenococcum, Thelephora, Tomentella, Russulaceae, and E-strain ectomycorrhizae (ECM) of Abies lasiocarpa seedlings were characterized using two approaches. First, bacteria were isolated and characterized by Biolog, gas chromatography fatty acid methyl ester (GC-FAME), and amplified 16S rDNA restriction analysis (ARDRA). The bacterial communities retrieved from ECM from both sites were dominated by Proteobacteria (groups gamma and beta). Pseudomonas was the most common genus isolated, followed by Variovorax, Burkholderia, and Xanthomonas. Gram-positive isolates (mostly high-G+C Gram-positive bacteria) were more frequently retrieved on the burned-salvaged site, many commonly associated with the two ascomycete ECM, Cenococcum and E-strain. Pseudomonas species were retrieved more frequently from Thelephora. Although actinomycetes were isolated from all sites, almost no actinomycetes or other Gram-positive bacteria were isolated from either Thelephora or Tomentella. Second, amplified 16S rRNA gene sequences were amplified directly from root tips and then cloned into the plasmid vector pAMP1, followed by restriction analysis. This technique distinguished more genotypes than isolates retrieved by culturing methods, but generally, results were similar in that the largest proportion of the bacteria were putatively Gram-negative; putative Gram-positive bacteria were fewer and most were from the burned-salvaged site. Direct cloning resulted in many patterns that did not match any identified isolates, suggesting that a large proportion of clones were unique or not culturable by the methods used. Analysis for both protocols showed no significant difference in bacterial diversity between the burned-salvaged and unburned sites.     

Endophytic bacteria of Sphagnum mosses as promising objects of agricultural microbiology

Sphagnum mosses serve as a unique habitat for microorganisms, which play an important role both for the host plants and the peatland ecosystems. The aim of the present study was to isolate endophytic bacteria from the tissues of Sphagnum mosses and to screen them for strains promising for further application in agricultural microbiology. About 50 samples of Sphagnum fallax (H. Klinggr.) H. Klinggr. and Sphagnum magellanicum Brid. were collected in the Austrian Alps and the Lenindgrad Region of Russia in 2009–2010. Endophytic bacteria were detected inside the moss plants using fluorescent in situ hybridization (FISH) followed by confocal laser scanning microscopy (CLSM). Altogether, 283 isolates were obtained by cultivation on the nutrient media. Examination of the isolates for the antagonistic activity revealed that more than 50% of them could suppress the growth of phytopathogenic and toxigenic fungi. More than 30% of isolates showed some antagonistic activity against microbial phytopathogens. The isolated strains could colonize crops and promote their growth. Molecular-genetic identification coupled with physiological/biochemical characterization showed that the dominant endophytic groups belonged to the genera Burkholderia, Pseudomonas, Flavobacterium, Serratia and Collimonas. The isolated endophytes were shown to be promising objects for the development of effective growth-promoting and protective microbiological preparations to be used in agriculture.     

Antagonistic interactions among marine pelagic bacteria.

Recent studies suggest that bacterial abundance and species diversity in the ocean's water column are variable at the millimeter scale, apparently in response to the small-scale heterogeneity in the distribution of organic matter. We hypothesized that bacterium-bacterium antagonistic interactions may contribute to variations in community structure at the microscale. We examined each of the 86 isolates for their inhibition of growth of the remaining 85 isolates by the Burkholder agar diffusion assay. More than one-half of the isolates expressed antagonistic activity, and this trait was more common with particle-associated bacteria than with free-living bacteria. This was exemplified by members of the alpha subclass of the class Proteobacteria (alpha-proteobacteria), in which production of antagonistic molecules was dominated by attached bacteria. We found that gamma-proteobacteria (members of the orders Alteromonadales and Vibrionales) are the most prolific producers of inhibitory materials and also the most resilient to them, while members of the Bacteriodetes were the organisms that were least productive and most sensitive to antagonistic interactions. Widespread interspecies growth inhibition is consistent with the role of this phenomenon in structuring bacterial communities at the microscale. Furthermore, our results suggest that bacteria from pelagic marine particles may be an underutilized source of novel antibiotics.     

Diversity and characterization of antagonistic bacteria from tropical estuarine habitats of Cochin, India for fish health management

Mortalities due to pathogenic bacteria are a major problem in aquaculture, especially in larval rearing systems. Use of antibiotics to overcome this problem is not an option any more due to the increasing antibiotic resistance among pathogens. The present study aims to understand the diversity of bacteria with antagonistic properties in the tropical estuarine habitats of Cochin, located along the southwest coast of India, and to use them as an alternative to antibiotics in aquaculture. Among the 4,870 isolates screened, approximately 1 % showed significant antibacterial activity against six common aquaculture pathogens belonging to the genera Aeromonas and Vibrio. The antagonistic bacteria were identified as Bacillus (81 %) and Pseudomonas (19 %) using biochemical and 16S rRNA gene sequence homology. The isolates showing stable and higher levels of antibacterial activity were subjected to enzymatic expression profile, antibiotic resistance pattern and abiotic stress tolerance assays. As a result, five Pseudomonas spp. and four Bacillus spp., were identified as promising antagonistic isolates that could be exploited as probionts or microbial products (MP's), to control bacterial diseases in aquaculture rearing systems.     

Sugar Beet-Associated Bacterial and Fungal Communities Show a High Indigenous Antagonistic Potential Against Plant Pathogens

The aim of this study was to analyze microbial communities in/on sugar beet with special focus on antagonists toward plant pathogens. For this purpose, the composition of microorganisms isolated from the rhizosphere, phyllosphere, endorhiza, and endosphere of field-grown sugar beet plants was analyzed by a multiphasic approach at three different plant development stages at six locations in Europe. The analysis of microbial communities by Single Strand Conformation Polymorphism (SSCP) of 16S/18S rRNA clearly revealed the existence of discrete microenvironment- and site-specific patterns. A total of 1952 bacterial and 1344 fungal isolates screened by dual testing for antagonism toward the pathogens Aphanomyces cochlioides, Phoma betae, Pythium ultimum, and Rhizoctonia solani resulted in 885 bacterial (=45%) and 437 fungal (=33%) antagonists. In general, the indigenous antagonistic potential was very high and influenced by (a) the location, (b) the plant developmental stage, and (3) the microenvironment. Furthermore, we showed for the first time that the antagonistic potential was highly specific for each target pathogen. The majority of antagonistic microorganisms suppressed only one pathogen (bacteria: 664 = 75%; fungi: 256 = 59%), whereas the minority showed a broad host range (bacteria: 4 = 0.5%; fungi: 7 = 1.6%). The bacterial communities harbored the highest antagonistic potential against P. ultimum, whereas the fungal communities contained more antagonists against A. cochlioides and R. solani. In contrast to their high proportion, only a low diversity of antagonists at genotypic and species level was found. Novel antagonistic species, e.g., Subtercola pratensis or Microbacterium testaceum were found in the internal part of the sugar beet body.     

Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards VERTICILLIUM: The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.     

Molecular analysis of lichen-associated bacterial communities

The bacterial communities associated with 11 different lichen samples (belonging to eight different species) from different habitats were investigated. The culturable aerobic-heterotrophic fraction of the bacterial communities was isolated from nine lichen samples on protein-rich and sugar-rich/N-free media. Thirty-four bacterial isolates were purified and pooled into groups (phylotypes) by analysis of the ribosomal internal transcribed spacer polymorphism. Twenty five phylotypes were identified, each comprising between one and three isolates. One isolate of each phylotype was partially sequenced and the resulting 16S rRNA gene sequences were compared in a phylogenetic analysis. Three genera of Firmicutes, four of Actinobacteria and three of Proteobacteria were identified. Two phylotypes, belonging to the phyla Actinobacteria and Proteobacteria, respectively, were not identified at genus level. Some bacterial taxa were retrieved frequently in different lichen species sampled in the same or different sites. Paenibacillus and Burkholderia phylotypes seem to be common in lichens. Luteibactor rhizovicina was found in three different lichens of two different regions. In a cultivation-independent approach, total DNA was extracted from 11 lichen samples. Molecular fingerprints of the bacterial communities were obtained by PCR-amplification of the internal transcribed spacer region, and sequencing of selected bands indicated the presence of additional bacteria.     

Diversity and Antimicrobial Activity of Culturable Fungi Isolated from Six Species of the South China Sea Gorgonians

Fungi in gorgonians are now known to cause gorgonian diseases, but little attention has been paid to the nature of fungal communities associated with gorgonians. The diversity of culturable fungi associated with six species of healthy South China Sea gorgonians were investigated using a culture-dependent method followed by analysis of fungal internal transcribed spacer sequences. A total of 121 fungal isolates were recovered and identified using the Basic Local Alignment Search Tool search program. These belonged to 41 fungal species from 20 genera. Of these, 30 species and 12 genera are new reports for gorgonians, and the genera Aspergillus and Penicillium were the most diverse and common in the six gorgonian species. Comparison of the fungal communities in the six gorgonian species, together with results from previous relevant studies, indicated that different gorgonian species and the same gorgonian species living in different geographic locations had different fungal communities. The gorgonian Dichotella gemmacea harbored the most fungal species and isolates, while Echinogorgia aurantiaca had the least fungal diversity. Among the six media used for fungal isolation, potato glucose agar yielded the highest isolates (27 isolates), while glucose peptone starch agar had the best recoverability of fungal species (15 species). The antimicrobial activity of the 121 fungal isolates was tested against three marine bacteria and two marine gorgonian pathogenic fungi. A relatively high proportion (38?%) of fungal isolates displayed distinct antibacterial and antifungal activity, suggesting that the gorgonian-associated fungi may aid their hosts in protection against pathogens. This is the first report comparing the diversity of fungal communities among the South China Sea gorgonians. It contributes to our knowledge of gorgonian-associated fungi and further increases the pool of fungi available for natural bioactive product screening.