微生物学

Ralstonia solanacearum is a bacterial phytopathogen able to cause bacterial wilt disease in more than 200 plant species. Plant disease biocontrol strategies are used for controlling this disease and tomato is used as a model plant to conduct R. solanacearum associated studies. Conventional screening methods such as seed bacterization, soil drenching and root bacterization (in grown plants) to assess the ability of biocontrol bacteria to antagonize R. solanacearum under in planta conditions in different hosts are time-consuming and costly. A fast, cost effective method is a key requirement to advance the research on R. solanacearum biocontrol. In this protocol, we have inoculated the roots of tomato seedlings with bacterial isolates showing antagonistic activity against R. solanacearum under in vitro conditions. After 16 h of treatment with the antagonizing bacteria, seedlings were inoculated with R. solanacearum by a well-established root-dip method. Then the seedlings were maintained at controlled conditions and the number of wilted/dead seedlings were recorded up to 10^th day post R. solanacearum inoculation. Biocontrol efficacy was calculated from the records for each tested isolate. This protocol is advantageous than already available protocols in the sense that it can be completed within a very short duration (~18 days for tomato) and there is no requirement of culture media to maintain the seedlings. This method can be used for quickly screening large number of bacterial isolates and different host genotypes within a short period of time and at a minimum cost.

Soil organisms are diverse taxonomically and functionally. This ecosystem experiences highly complex networks of interactions, but may also present functionally independent entities. Plant roots, a metabolically active hotspot in the soil, take an essential part in shaping the rhizosphere. Tracking the dynamics of root-microbe interactions at high spatial resolution is currently limited due to methodological intricacy. In this study, we developed a novel microfluidics-based device enabling direct imaging of root-bacteria interactions in real time.

Besides analyzing the composition and dynamics of microbial communities, plant microbiome research aims to understanding the mechanism of plant microbiota assembly and their biological functions. Here, we describe procedures to investigate the role of bacterial interspecies interactions in root microbiome assembly and the beneficial effects of the root microbiota on hosts by using a maize root-associated simplified seven-species (Stenotrophomonas maltophilia, Ochrobactrum pituitosum, Curtobacterium pusillum, Enterobacter cloacae, Chryseobacterium indologenes, Herbaspirillum frisingense and Pseudomonas putida) synthetic bacterial community described in our previous work. Surface-sterilized maize seeds were grown in a gnotobiotic system based on double-tube growth chambers after being soaked in suspensions containing multiple species of bacteria. The dynamics of the composition of the bacterial communities colonized on maize roots were tracked by a culture-dependent method with a selective medium for each of the seven strains. The impact of bacterial interactions on the community assembly was evaluated by monitoring the changes of community structure. The plant-protection effects of the simplified seven-species community were assessed by quantifying (1) the growth of a fungal phytopathogen, Fusarium verticillioides on the surfaces of the seeds and (2) the severity of seedling blight disease the fungus causes, in the presence and absence of the bacterial community. Our protocol will serve as useful guidance for studying plant-microbial community interactions under the laboratory conditions.

The present protocol to visualize living bacteria at the pore level of cauliflower hydathodes is simple and trained users in confocal microscopy can execute it successfully. It can be easily adapted to capture images with other plant-microorganism interactions at the leaf surface and should be useful to obtain important information on pore and stomatal biology. A critical limitation to methods used to observe plant-microorganism interactions in the pore is the application of too much pressure to the sample during observations and z-stack acquisitions. To solve this issue, we recommend the use of a long working-distance water immersion objective lens that allows observations even with thick samples.

To prevent yield losses in plant cultivation due to plant pathogens, it is an important task to find new disease resistance mechanisms. Recently, Weidenbach et al. (2016) reported about the capacity of the rice gene OsJAC1 to enhance resistance in rice and barley against a broad spectrum of different pathogens. Here, we describe the respective protocols used by Weidenbach and colleagues for inoculation of rice with the basidiomycete Rhizoctonia solani, the oomycete Pythium graminicola and the ascomycete Blumeria graminis f. sp. hordei (Bgh).

We performed a growth inhibition assay to test antibacterial compounds in leaf extracts from transgenic rice plants. The assay is based on over-night co-incubation of a defined concentration of colony forming units (cfu) of the respective bacteria together with aqueous extracts of ground leaf tissue.

Fusarium head blight (FHB) caused by Fusarium pathogens is a globally important cereal disease. To study Fusarium pathogenicity and host disease resistance, robust methods for disease assessment and quantification are needed. Here we describe the procedure of a detached leaves assay emphasizing the image analysis. The protocol provides the different steps of a rapid, automatic and quantitative image analysis to evaluate leaf area infected by Fusarium graminearum.

Grapevine (Vitis vinifera L.) is susceptible to an array of diseases among them the grey mold caused by the necrotrophic fungus Botrytis cinerea that decreases grape productivity and quality. To ensure a satisfactory yield and harvest quality numerous chemical fungicides are required, but they have serious drawbacks. One alternative is the use of beneficial bacteria to improve plant health. Pseudomonas fluorescens has been shown to trigger a plant-mediated resistance response in aboveground plant tissues against fungal, oomycete, bacterial, and viral pathogens. Triggered plant resistance exploits mechanisms of the plant immune system through a priming state that provides plants with enhanced capacity for rapid and strong activation of defense responses after pathogen infection, resulting in a lower fitness-cost. The primed responses by beneficial bacteria include induced expression of defense-related genes, cell wall reinforcement, and the production of secondary metabolites after pathogen infection. In this protocol, we describe the experimental design to evaluate the priming state of grapevine plants by the beneficial bacterium Pseudomonas fluorescens PTA-CT2 and their resistance level to Botrytis cinerea according to Verhagen et al. (2011) and Gruau et al. (2015).

The basidiomycetous smut fungus Ustilago maydis (U. maydis) infects all aerial parts of its host plant maize (Zea mays L.). Infection symptoms are seen in the form of prominent tumors on all aerial parts of maize, after the establishment of a biotrophic interaction with the host usually around 5-6 days post infection (dpi). The fungus colonizes the various developmentally distinct aerial organs at different stages of development to form these prominent symptoms. Although being a biotrophic plant pathogen, U. maydis can easily be cultivated under axenic conditions to produce a standardized inoculum. The infections can be carried out under laboratory conditions by syringe inoculation on all the aerial organs of maize. This protocol has been successfully utilized to infect all the aerial organs of maize and formulate the virulence assays in U. maydis making it an excellent model system to study phyto-pathological investigations (Schilling et al., 2014; Redkar et al., 2015).

The basidiomycetous smut fungus Ustilago maydis (U. maydis) infects all aerial parts of its host plant maize (Zea mays L.). Infection is seen in the form of prominent tumorous symptoms after the establishment of a biotrophic interaction with the host, usually around 5-6 days after infection. The fungus colonizes the various developmentally distinct aerial organs at different stages of development. Formation of tumors is coupled with the induction of host cell division. Activation of cell division can be understood as a measure of DNA synthesis which is triggered to induce rapid divisions in host cell. This developed protocol helps in tracking tumor induction in U. maydis by monitoring of DNA synthesis in planta. Infected leaves were treated with 5-ethynyl-2-deoxyuridine (EdU) at several stages of infection in the seedling leaves and labeled. EdU incorporation in the S phase cells, was visualized by attaching a fluorescent tag and non-dividing maize nuclei were stained with propidium iodide (PI). This protocol helped to understand the tumor development in U. maydis by confocal laser scanning microscopy (Kelliher and Walbot, 2011; Redkar et al., 2015)