Michael Fürnkranz

Microbial Diversity Inside Pumpkins: Microhabitat-Specific Communities Display a High Antagonistic Potential Against Phytopathogens

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.

Characterization of plant growth promoting bacteria from crops in Bolivia.

The use of plant growth promoting bacteria (PGPB) is an economically and environmental friendly alternative to the application of chemical fertilizers resp. pesticides in agriculture. To obtain novel bacterial strains that could be used for plant growth promotion (PGP) in the agriculture of Bolivia, plant associated bacteria derived from horseradish tree (Moringa oleifera), sorghum (Sorghum vulgare), sunflower (Helianthus annuus), and safflower (Carthamus tinctorius) were screened for direct and indirect in vitro PGP traits. Subsequently, most promising strains were selected for ad planta studies. According to in vitro experiments, out of 59 tested isolates, 19% were declared as diazotrophs, 41% as solubilizers of phosphorus, 10% were able to reduce the ethylene precursor 1-aminocy-clopropane-1-carboxylic acid (ACC), and 17% exhibited phytohormone (IAA) synthesis. Only a small proportion of rhizo-bacterial strains (7% out of 276) showed in vitro antagonism against plant pathogenic Colletotrichum sp., whereas none of isolated stem and leaf endophytes inhibited growth of Verticillium chlamydosporum or Bipolaris maydis in dual culture. Based on results of in vitro screens, the rhizospheric strains Pectobacterium cypripedii M56, Pantoea agglomerans M72, and P. agglomerans M81 were selected for ad planta applications. Results revealed significant increases in number of beans per black bean plant resp. diameters of flower heads of sunflower plants compared to controls by P. agglomerans M72. We conclude that the assessment and selection of plant associated bacteria based on traits conferring theoretically PGP can provide the basis for the development of new microbial inoculants for agricultural purposes.ZusammenfassungDie Anwendung pflanzenwachstumsfördernder Bakterien in der Landwirtschaft kann eine profitable und umweltschonende Alternative zum Einsatz von chemischen Düngemitteln und Pestiziden darstellen. Um neue, bakterielle Pflanzenwachstumsförderer für die bolivianische Landwirtschaft zu gewinnen, wurden pflanzenassoziierte Bakterien von Meerrettichbaum (Moringa oleifera), Sorghumhirse (Sorghum vulgare), Sonnenblume (Helianthus annuus) und Färberdistel (Carthamus tinctorius) isoliert und in vitro auf direkte und indirekte Mechanismen der Pflanzenwachstumsförderung untersucht. Ausgehend von 59 unter Laborbedingungen getesteten Isolaten, waren 19% diazotroph, 41% wurden als Phosphor-Solubilisierer deklariert, 17% zeigten Phytohormonsynthese (IAA) und 10% wurden als Verwerter der Ethy- len-Vorläufer-Substanz 1-Aminocyclopropan-1-Carbonsäure (ACC) erklärt. Nur ein geringer Anteil rhizobakterieller Stämme (7% von 276) zeigte antagonistische Aktivität in vitro gegen das Pflanzenpathogen Colletotrichum sp., während keiner der von Stängeln oder Blättern isolierten Endophyten das Wach stum von Verticillium chlamydosporum oder Bipolaris maydis in Dualkultur inhibieren konnte. Basierend auf den Resultaten der in vitro Tests, wurden die Rhizosphären-Stämme Pectobacterium cypripedii M56, Pantoea agglomerans M72 und P. agglomerans M81 für die Applikation in Freilandexperimenten selektiert, wobei P. agglomerans M72 zu signifikant höheren Bohnenzahlen bei schwarzer Bohne bzw. Blütenkorbdurchmessern bei Sonnenblume im Vergleich zu Kontroll-Behandlungen geführt hat. Die Bewertung und Selektion pflanzenassoziierter Bakterien, basierend auf deren pflanzenwachstumsfördernden Eigenschaften in vitro, stellt die Grundlage für die Entwicklung neuer biologischer Produkte für landwirtschaftliche Zwecke dar.

Emerging multi-pathogen disease caused by Didymella bryoniae and pathogenic bacteria on Styrian oil pumpkin

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.

Genome Sequence of Serratia plymuthica Strain S13, an Endophyte with Germination- and Plant-Growth-Promoting Activity from the Flower of Styrian Oil Pumpkin

ABSTRACT The bacterium Serratia plymuthica strain S13 was demonstrated to colonize various plant-associated microhabitats and to suppress damping-off diseases. The completed genome sequence has a size of 5.5 Mb, containing 4,957 putative protein-encoding regions, and will be used to identify genetic determinants enabling the bacterium to escort a plants entire life cycle.

A new textile-based approach to assess the antimicrobial activity of volatiles:

Textiles with antimicrobial properties are required in many areas but it is difficult to assess the effect with current assays based on the cultivation of microorganisms. Using confocal laser scanning microscopy (CLSM) we observed that the complex matrix of textiles provides an ideal niche for the adhesion of microorganisms because polyester fibres showed a high number of irregularly appearing notches. In another experiment with green fluorescent protein-labelled Staphylococcus epidermidis cells were found in these notches, tightly bound to the fibres. Staphylococcus cells shielded in the textile were not killed by conventional decontamination techniques like UV irradiation. Cultivation-dependent evaluation of bacterial survival after 10 minutes of irradiation indicated a good reduction of more than 99%, whereas fluorescent viability staining of cells, in combination with CLSM, displayed more than 5% survival. This latter sensitive assay can also be used to assess the activity of volatile antimicrobials. It was shown that volatiles produced by plant-associated bacteria (Pseudomonas chlororaphis and Paenibacillus polymyxa) irreversibly inhibited the growth of human-associated pathogens like Staphylococcus aureus and Candida albicans on textiles. This new approach enables the detection and evaluation of new volatile antimicrobials for their use in disinfection of garments.