Leaf surfaces (phyllospheres) have been shown to provide appropriate conditions for colonization by microorganisms including diazotrophic bacteria that are able to fix atmospheric nitrogen (N 2 ). In this study, we determined leaf-associated N 2 fixation of a range of rainforest plants in Costa Rica, under different environmental conditions, by tracing biomass N incorporation from 15 N 2 . N 2 -fixing bacterial communities of the plant species Carludovica drudei, Grias cauliflora and Costus laevis were investigated in more detail by analysis of the nifH gene and leaf-associated bacteria were identified by 16S rRNA gene analysis. N 2 fixation rates varied among plant species, their growth sites (different microclimatic conditions) and light exposure. Leaf-associated diazotrophic bacterial communities detected on C. drudei and C. laevis were mainly composed of cyanobacteria (Nostoc spp.), whereas on the leaves of G. cauliflora c-proteobacteria were dominant in addition to cyanobacteria. The complexity of diazotrophic communities on leaves was not correlated with N 2 fixation activity. 16S rRNA gene sequence analysis suggested the presence of complex microbial communities in association with leaves, however, cyanobacteria showed only low abundance. Our findings suggest that cyanobacteria as well as c-proteobacteria associated with leaf-colonizing epiphytes may provide significant nitrogen input into this rainforest ecosystem. The ISME Journal (2008) 2, 561-570; doi:10.1038/ismej.2008.14; published online 14 February 2008Subject Category: microbial ecology and functional diversity of natural habitats

Recent and substantial yield losses of Styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) are primarily caused by the ascomycetous fungus Didymella bryoniae but bacterial pathogens are frequently involved as well. The diversity of endophytic microbial communities from seeds (spermosphere), roots (endorhiza), flowers (anthosphere), and fruits (carposphere) of three different pumpkin cultivars was studied to develop a biocontrol strategy. A multiphasic approach combining molecular, microscopic, and cultivation techniques was applied to select a consortium of endophytes for biocontrol. Specific community structures for Pseudomonas and Bacillus, two important plant-associated genera, were found for each microenvironment by fingerprinting of 16S ribosomal RNA genes. All microenvironments were dominated by bacteria; fungi were less abundant. Of the 2,320 microbial isolates analyzed in dual culture assays, 165 (7%) were tested positively for in vitro antagonism against D. bryoniae. Out of these, 43 isolates inhibited the growth of bacterial pumpkin pathogens (Pectobacterium carotovorum, Pseudomonas viridiflava, Xanthomonas cucurbitae); here only bacteria were selected. Microenvironment-specific antagonists were found, and the spermosphere and anthosphere were revealed as underexplored reservoirs for antagonists. In the latter, a potential role of pollen grains as bacterial vectors between flowers was recognized. Six broad spectrum antagonists selected according to their activity, genotypic diversity, and occurrence were evaluated under greenhouse conditions. Disease severity on pumpkins of D. bryoniae was significantly reduced by Pseudomonas chlororaphis treatment and by a combined treatment of strains (Lysobacter gummosus, P. chlororaphis, Paenibacillus polymyxa, and Serratia plymuthica). This result provides a promising prospect to biologically control pumpkin diseases.

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The Styrian oil pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca Greb. is a crop of cultural, commercial, and medical importance. In the last decade, yield losses of pumpkins increased dramatically. The ascomycetous fungus Didymella bryoniae (Fuckel) Rehm was identified as main causal agent provoking gummy stem blight as well as black rot of pumpkins. We observed a remarkable phenotypic diversity of the fungal pathogen, which contrasted with a high genotypic similarity. Evidence of pathogenictiy of D. bryoniae on Styrian oil pumpkin was demonstrated in a newly developed greenhouse assay. Isolates representing the five observed phenotypic groups fulfilled the Koch's postulates. In the field, the fungal disease was often associated with bacterial colonization by Pectobacterium carotovorum, Pseudomonas viridiflava, Pseudomonas syringae and Xanthomonas cucurbitae. The pathogenic behaviour of bacterial isolates on pumpkin was confirmed in the greenhouse assay. The high co-incidence of fungal and bacterial disease suggests mutualistic effects in pathogenesis. With a new assay, we found that bacteria can use the mycelium of D. bryoniae for translocation. We argue that the rapid rise of the multi-pathogen disease of pumpkins results from combined action of versatile pathogenic bacteria and the rapid translocation on a structurally versatile mycelium of the fungal pathogen.