The interaction between Rhizoglomus irregulare and hyphae attached phosphate solubilizing bacteria increases plant biomass of Solanum lycopersicum

The synergistic interaction between arbuscular mycorrhizal fungi (AMF) and phosphate solubilizing bacteria (PSB) can enhance growth and phosphorous uptake in plants. Since PSBs are well known hyphal colonizers we sought to understand this physical interaction and exploit it in order to design strategies for the application of a combined microbial inoculum. Phosphate-solubilizing bacteria strongly attached to the hyphae of Rhizoglomus irregulare were isolated using a two compartment system (root and hyphal compartments), which were separated by a nylon mesh through which AMF hyphae could pass but not plant roots. Allium ampeloprasum (Leek) was used as the host plant inoculated with R. irregulare. A total of 128 bacteria were isolated, of which 12 showed stable phosphate solubilizing activity. Finally, three bacteria belonging to the genus Pseudomonas showed the potential for inorganic and organic phosphate mobilization along with other plant growth promoting traits. These PSBs were further evaluated for their functional characteristics and their interaction with AMF. The impact of single or co-inoculations of the selected bacteria and AMF on Solanum lycopersicum was tested and we found that plants inoculated with the combination of fungus and bacteria had significantly higher plant biomass compared to single inoculations, indicating synergistic activities of the bacterial-fungal consortium

The Combined Applications of Microbial Inoculants and Organic Fertilizer Improve Plant Growth under Unfavorable Soil Conditions

The performance of two bio-inoculants either in single or in combined applications with organic fertilizer was tested to determine their effect on plant growth and yield under normal and unfavorable field conditions such as low pH value and low content of P. Arbuscular Mycorrhiza Fungi (AMF; three species of Glomus) and the plant-growth-promoting bacterial strain Kosakonia radicincitans DSM16656 were applied to barley in a two-year field experiment with different soil pH levels and available nutrients. Grain yield; contents of P, N, K, and Mg; and soil microbial parameters were measured. Grain yield and the content of nutrients were significantly increased by the applications of mineral fertilizer, organic fertilizer, AMF, and K. radicincitans, and the combined application of organic fertilizer with AMF and with K. radicincitans over the control under normal growth conditions. Under low-pH and low-P conditions, only the combined application of the organic fertilizer with K. radicincitans and organic fertilizer with AMF could increase the grain yield and content of nutrients of barley over the control.European Union’s Horizon 2020 Research and Innovation ProgramProject EXCALIBUR under grant agreement No. 817946EU project SUSTAINABLE, EU grant agreement no. 10100770

Contamination pathways and detection of human pathogenic Escherichia coli from plant foods

Seit dem Ausbruch mit enterohämorrhagischen Escherichia coli (EHEC) O104:H4 im Sommer 2011 in Deutschland sind pflanzliche Lebensmittel als mögliche Infek­tionsquelle für humanpathogene E. coli in das Licht der Öffentlichkeit gerückt. Die Suche nach der Infektionsquelle des EHEC O104:H4 Erregers, der für mehr als 4000 Erkrankte, für über 800 Fälle von Nierenversagen und für 53 Todesfälle verantwortlich war, erwies sich als schwierig, obwohl die für Diagnostik relevanten Eigenschaften anhand der Isolate aus den Patienten früh bekannt waren. Epidemiologische Untersuchungen deuteten auf pflanzliche Lebensmittel als mögliche Infek­tionsquelle und nach den zu Unrecht verdächtigten Gurken konnten schließlich aus Bockshornkleesamen hergestellte Keimsprossen eines bestimmten Herstellers als primäre Infektionsquelle bestimmt werden. Die Diagnostik pathogener E. coli aus pflanzlichen Lebensmitteln ist ungleich schwieriger als aus tierischen Quellen, für die letzteren gibt es seit Jahren amtliche Untersuchungs­verfahren und eine Vielzahl von publizierten Methoden. Untersuchungsmethoden, die für Milch und Fleisch­produkte konzipiert wurden, lassen sich jedoch auf rohe pflanzliche Lebensmittel schlecht übertragen. Die Gründe dafür liegen zum einen an der Menge und der Zusammensetzung der Begleitflora von Mikroorganismen, die bei Pflanzen völlig anders ist, als bei tierischen Produkten. Dazu kommt, dass die natürliche mikrobielle Kontamination bei zum Rohverzehr bestimmten Pflanzen sich je nach Kategorie (Obst, Gemüse, Mischsalat und Sprossen) hinsichtlich ihrer Menge, Eigenschaften und Zusammensetzung stark unterscheidet. E. coli kommen als Kontaminanten bei pflanzlichen Lebensmitteln ebenso vor, häufig jedoch in so geringen Mengen, dass spezifische Anreicherungsverfahren notwendig sind, um mögliche pathogene E. coli wie EHEC überhaupt zu erkennen. Die weitere Hürde liegt in der Isolierung verdächtiger E. coli aus einer Fülle von natürlichen Begleitorganismen. Verfahren der quantitativen PCR (qPCR, Real-Time PCR) haben sich bewährt, um auch geringe Konzentrationen von E. coli in pflanzlichem Untersuchungsmaterial nachzuweisen und gleichzeitig mögliche Pathogenitätseigenschaften bei diesen Keimen zu erkennen. Auch für die Isolierung der nachgewiesenen pathogenen E. coli müssen an die Lebensmittelmatrix Pflanze angepasste Methoden gewählt werden, die hier ebenfalls vorgestellt werden. DOI: 10.5073/JfK.2016.03.01, https://doi.org/10.5073/JfK.2016.03.01With the massive EHEC O104:H4 outbreak in summer 2011, vegetables became interesting to the German public because of their possible role as a source of infections with enterohemorrhagic Escherichia coli (EHEC). The search for the source of infection with EHEC O104:H4, which was responsible for more than 4000 diseases, more than 800 cases of renal failure and 53 deaths, proved difficult, although the relevant diagnostic properties based on the isolates from the patients were early known. Epidemiological studies pointed to plant foods as possible source and after the wrongly suspected cucumbers, fenugreek sprouts from a particular manufacturer were identified as the primary source of infection. The diagnosis of pathogenic E. coli from plant foods is more difficult than from animal sources, for the latter official control procedures and a variety of methods are published since many years. Investigation methods that were designed for dairy and meat products are not well suitable for testing raw foods of plant origin. The reasons are partly due to the quantity and composition of the accompanying flora of microorganisms, which is completely different in plants than in animal products. Depending on the raw food category (fruits, vegetables, mixed salad and sprouts) the natural microbial contamination of certain plants differs largely in its amount, characteristics and composition. E. coli is often present as contaminant in plant foods, but often in such small quantities that specific enrichment methods are needed to identify potential pathogenic E. coli types such as EHEC. The other hurdle lies in the isolation of suspected E. coli from an abundance of natural accompanying organisms. Quantitative PCR (qPCR, real-time PCR) methods have been proven to detect even low levels of E. coli in plant tissue and simultaneously to identify possible human pathogenic bacteria. Finally, for the isolation of pathogenic E. coli from plants specific methods adapted to this particular food matrix must be chosen. These are also presented here. DOI: 10.5073/JfK.2016.03.01, https://doi.org/10.5073/JfK.2016.03.0

Selected Rhizosphere Bacteria Help Tomato Plants Cope with Combined Phosphorus and Salt Stresses

Plants are often challenged by multiple abiotic stresses simultaneously. The inoculation of beneficial bacteria is known to enhance plant growth under these stresses, such as phosphorus starvation or salt stress. Here, for the first time, we assessed the efficiency of selected beneficial bacterial strains in improving tomato plant growth to better cope with double stresses in salty and P-deficient soil conditions. Six strains of Arthrobacter and Bacillus with different reservoirs of plant growth-promoting traits were tested in vitro for their abilities to tolerate 2–16% (w/v) NaCl concentrations, and shown to retain their motility and phosphate-solubilizing capacity under salt stress conditions. Whether these selected bacteria promote tomato plant growth under combined P and salt stresses was investigated in greenhouse experiments. Bacterial isolates from Cameroonian soils mobilized P from different phosphate sources in shaking culture under both non-saline and saline conditions. They also enhanced plant growth in P-deficient and salt-affected soils by 47–115%, and their PGP effect was even increased in higher salt stress conditions. The results provide valuable information for prospective production of effective bio-fertilizers based on the combined application of local rock phosphate and halotolerant phosphate-solubilizing bacteria. This constitutes a promising strategy to improve plant growth in P-deficient and salt-affected soils.Peer Reviewe

Antagonistic Potential of Fluorescent Pseudomonads Colonizing Wheat Heads Against Mycotoxin Producing Alternaria and Fusaria

Natural control of phytopathogenic microorganisms is assumed as a priority function of the commensal plant microbiota. In this study, the suitability of fluorescent pseudomonads in the phyllosphere of crop plants as natural control agents was evaluated. Under field conditions, ears of winter wheat were found to be colonized with high consistency and at a high density by pseudomonads at the late milk dough stage. Isolates of these bacteria were evaluated for their potential to protect the plants from phytopathogenic Alternaria and Fusarium fungi. More Pseudomonas isolates were antagonistically active against alternaria than against fusaria in the dual culture test. The alternaria responded species-specifically and more sensitively to bacterial antagonism than the strain-specific reacting fusaria. A total of 110 randomly selected Pseudomonas isolates were screened for genes involved in the biosynthesis of the antibiotics 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid, pyoluteorin, and pyrrolnitrin. The key gene for production of the phloroglucinol was found in none of these isolates. At least one of the genes, encoding the biosynthesis of the other antibiotics was detected in 81% of the isolates tested. However, the antagonistic effect found in the dual culture assay was not necessarily associated with the presence of these antibiotic genes. Wheat grains as natural substrate were inoculated with selected antagonistic Pseudomonas isolates and Alternaria and Fusarium strains, respectively. The fungal growth was only slightly delayed, but the mycotoxin production was significantly reduced in most of these approaches. In conclusion, the distribution of phytopathogenic fungi of the genera Alternaria and Fusarium in the field is unlikely to be inhibited by naturally occurring pseudomonads, also because the bacterial antagonists were not evenly distributed in the field. However, pseudomonads can reduce the production of Alternaria and Fusarium mycotoxins in wheat grains and thus have the potential to improve the crop quality

Comparative Genomics Reveal a Flagellar System, a Type VI Secretion System and Plant Growth-Promoting Gene Clusters Unique to the Endophytic Bacterium Kosakonia radicincitans

The recent worldwide discovery of plant growth-promoting (PGP) Kosakonia radicincitans in a large variety of crop plants suggests that this species confers significant influence on plants, both in terms of yield increase and product quality improvement. We provide a comparative genome analysis which helps to unravel the genetic basis for K. radicincitans' motility, competitiveness and plant growth-promoting capacities. We discovered that K. radicincitans carries multiple copies of complex gene clusters, among them two flagellar systems and three type VI secretion systems (T6SSs). We speculate that host invasion may be facilitated by different flagella, and bacterial competitor suppression by effector proteins ejected via T6SSs. We found a large plasmid in K. radicincitans DSM 16656T, the species type strain, that confers the potential to exploit plant-derived carbon sources. We propose that multiple copies of complex gene clusters in K. radicincitans are metabolically expensive but provide competitive advantage over other bacterial strains in nutrient-rich environments. The comparison of the DSM 16656T genome to genomes of other genera of enteric plant growth-promoting bacteria (PGPB) exhibits traits unique to DSM 16656T and K. radicincitans, respectively, and traits shared between genera. We used the output of the in silico analysis for predicting the purpose of genomic features unique to K. radicincitans and performed microarray, PhyloChip, and microscopical analyses to gain deeper insight into the interaction of DSM 16656T, plants and associated microbiota. The comparative genome analysis will facilitate the future search for promising candidates of PGPB for sustainable crop production

Progress in cultivation-independent phyllosphere microbiology

Most microorganisms of the phyllosphere are nonculturable in commonly used media and culture conditions, as are those in other natural environments. This review queries the reasons for their ‘noncultivability’ and assesses developments in phyllospere microbiology that have been achieved cultivation independently over the last 4 years. Analyses of total microbial communities have revealed a comprehensive microbial diversity. 16S rRNA gene amplicon sequencing and metagenomic sequencing were applied to investigate plant species, location and season as variables affecting the composition of these communities. In continuation to culture-based enzymatic and metabolic studies with individual isolates, metaproteogenomic approaches reveal a great potential to study the physiology of microbial communities in situ. Culture-independent microbiological technologies as well advances in plant genetics and biochemistry provide methodological preconditions for exploring the interactions between plants and their microbiome in the phyllosphere. Improving and combining cultivation and culture-independent techniques can contribute to a better understanding of the phyllosphere ecology. This is essential, for example, to avoid human–pathogenic bacteria in plant food

It Takes Two to Tango: A Bacterial Biofilm Provides Protection against a Fungus-Feeding Bacterial Predator

Fungus-bacterium interactions are widespread, encompass multiple interaction types from mutualism to parasitism, and have been frequent targets for microbial inoculant development. In this study, using in vitro systems combined with confocal laser scanning microscopy and real-time quantitative PCR, we test whether the nitrogen-fixing bacterium Kosakonia radicincitans can provide protection to the plant-beneficial fungus Serendipita indica, which inhabits the rhizosphere and colonizes plants as an endophyte, from the fungus-feeding bacterium Collimonas fungivorans. We show that K. radicincitans can protect fungal hyphae from bacterial feeding on solid agar medium, with probable mechanisms being quick hyphal colonization and biofilm formation. We furthermore find evidence for different feeding modes of K. radicincitans and C. fungivorans, namely “metabolite” and “hyphal feeding”, respectively. Overall, we demonstrate, to our knowledge, the first evidence for a bacterial, biofilm-based protection of fungal hyphae against attack by a fungus-feeding, bacterial predator on solid agar medium. Besides highlighting the importance of tripartite microbial interactions, we discuss implications of our results for the development and application of microbial consortium-based bioprotectants and biostimulants

A Proteomic Approach Suggests Unbalanced Proteasome Functioning Induced by the Growth-Promoting Bacterium Kosakonia radicincitans in Arabidopsis

Endophytic plant growth-promoting bacteria have significant impact on the plant physiology and understanding this interaction at the molecular level is of particular interest to support crop productivity and sustainable production systems. We used a proteomics approach to investigate the molecular mechanisms underlying plant growth promotion in the interaction of Kosakonia radicincitans DSM 16656 with Arabidopsis thaliana. Four weeks after the inoculation, the proteome of roots from inoculated and control plants was compared using two-dimensional gel electrophoresis and differentially abundant protein spots were identified by liquid chromatography tandem mass spectrometry. Twelve protein spots were responsive to the inoculation, with the majority of them being related to cellular stress reactions. The protein expression of 20S proteasome alpha-3 subunit was increased by the presence of K. radicincitans. Determination of proteasome activity and immuno blotting analysis for ubiquitinated proteins revealed that endophytic colonization interferes with ubiquitin-dependent protein degradation. Inoculation of rpn12a, defective in a 26S proteasome regulatory particle, enhanced the growth-promoting effect. This indicates that the plant proteasome, besides being a known target for plant pathogenic bacteria, is involved in the establishment of beneficial interactions of microorganisms with plants