Éva Preininger

Artificial Tripartite Symbiosis Involving a Green Alga (Chlamydomonas), a Bacterium (Azotobacter) and a Fungus (Alternaria): Morphological and Physiological Characterization

A long-living artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria) was established on carbon- and nitrogen-free medium. The basis of the interdependence is the complementation of photosynthetic CO2 assimilation and atmospheric nitrogen fixation. Green color of the colonies indicated that the algal cells had enough nitrogen to synthesize chlorophylls. The chlorophyll content was nearly 40 % of the control cells. The relatively high rate of photosynthetic oxygen evolution proved that nitrogen was effectively used for building up a well functioning photosynthetic apparatus. This was supported by the analysis of photosystems and ultrastructural investigations. In comparison with degreened algae cultured on nitrogen-free medium, the chloroplasts in the symbiont algal cells contained a well-developed, stacked thylakoid membrane system without extreme starch or lipid accumulation. The occurrence of the fungus in the association greatly increased the chlorophyll content. Far fewer types of amino acids were excreted by the tripartite cultures than by pure cultures. Cystathionine, which is a common intermediate in the sulfur-containing amino acid metabolism, was produced in high quantities by the tripartite symbiosis. This can mostly be attributed to the activity of the fungus.

In vitro establishment of nitrogen-fixing strawberry (Fragaria × ananassa) via artificial symbiosis with Azomonas insignis

SummaryArtificial symbiosis was established between diazotrophic Azomonas insignis and strawberry (Fragaria × ananassa). The partnership was created by in vitro techniques through callus induction and organogenesis. Suitable micropropagation [M3=Murashige and Skoog (1962) (MS) basal medium supplemented with 2.5 µM N6-benzyladenine (BA), 0.3 µM gibberellic acid (GA3), 2.2 µM indole-3-butyric acid (IBA), and 3% sucrose] and plant regeneration [R3=MS mineral salts+555 µM myo-inositol, 1.2 µM thiamine HCl, 4.4 µM BA, 0.5 µM IBA, 0.3 µM α-naphthaleneacetic acid (NAA), 0.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D)] media were developed for the test cultivar Fertödi F5. New shoots containing bacteria were rooted, acclimatized, and planted outdoors. The basis of the partnership during the in vitro phase is the bacterial dependence on the plant metabolic activity, using maltose in the medium as carbon and energy source that can be utilized by the plant cells only. The presence of bacteria in the intercellular spaces of the callus tissues and regenerated plants was proved by re-isolation and microscopic techniques. Nitrogenase activity was also detected in the plant tissues.

Minibeet initiation from derooted sugarbeet (Beta vulgaris L.) seedlings in vitro

Abstract An in vitro method for the initiation of minibeet development from derooted sugarbeet seedlings was developed. The surface sterilized seeds were germinated for 25 days on sugar-free Murashige and Skoog medium containing half-strength nutritive elements, 10 mg l−1 thiamine, 0.1 mg l−1 gibberellic acid and 7.0 g l−1 agar. After germination, seedlings of six different genotypes were derooted at the root neck and incubated for 90–120 days on minibeet initiation medium containing double-strength Murashige and Skoog elements, B5 vitamins, 100 mg l−1 activated charcoal, 30 g l−1 d -glucose, 6-benzylaminopurine (0.2 mg l−1) and abscisic acid (0.2 mg l−1). Without subculturing, minibeets developed on 20–40% of five different genotypes of seedlings. The developed minibeets were characterized structurally and functionally by means of a histological study and relative sucrose accumulation. Minibeets were compared to tuber-like formations which showed neither polycambial structure nor relative sucrose accumulation. Minibeets gave rise to plants after potting in the greenhouse.

Structural and functional changes in the photosynthetic apparatus of Chlamydomonas reinhardtii during nitrogen deprivation and replenishment

Nitrogen is an essential factor for normal plant and algal development. As a component of nucleic acids, proteins, and chlorophyll (Chl) molecules, it has a crucial role in the organization of a functioning photosynthetic apparatus. Our aim was to study the effects of nitrogen starvation in cultures of the unicellular green alga, Chlamydomonas reinhardtii, maintained on nitrogen-free, and then on nitrogen-containing medium. During the three-week-long degreening process, considerable changes were observed in the Chl content, the ratio of Chl-protein complexes, and photosynthetic activity of the cultures as well as in the ultrastructure of single chloroplasts. The regreening process was much faster then the degradation; total greening of the cells occurred within four days. The rate of regeneration depended on the nitrogen content. At least 50% of the normal nitrogen content of Tris-Acetate-Phosphate (TAP) medium was required in the medium for the complete regreening of the cells and regeneration of chloroplasts.

A new approach for the biolistic method: Bombardment of living nitrogen-fixing bacteria into plant tissues

SummaryA new utilization of the biolistic gun was developed for the direct introduction of nitrogen-fixing bacteria (Azotobacter vinelandii) into strawberry (Fragaria x ananassa) tissues. This was the first case of using living bacteria as microprojectiles for the bombardment of plant tissues. Bacterial cells, adhered to tungsten particles, were accelerated by a nitrogen-powered device, and delivered into the target leaves and regenerating shoot meristems. The presence of bacteria in the developing strawberry callus tissues and regenerating plants was detected by microscopy, acetylene reduction assay, and selective polymerase chain reaction. Practically, the elaborated method proved to be suitable for the establishment of artificial intereellular, associations between nitrogen-fixing bacteria and higher plants.

Trials to create artificial nitrogen-fixing symbioses and associations using in vitro methods: An outlook

SummaryBiological nitrogen fixation is the most important process in which some prokaryotic organisms fix N2 into ammonium. From an agricultural standpoint, biological nitrogen fixation (BNF) is critical because industrial production of nitrogen fertilizers seldom meets agricultural demands. To increase the BNF is one of the main challenges for the future. There are different possibilities for extending biological nitrogen fixation to the economically important plants. One of the possibilities is to create new artificial systems between diazotrophic bacteria and different higher plants. This is the main topic of the present review article which discusses the establishment of new associative and/or symbiotic systems, via introduction of diazotrophic bacteria into the roots by different methods; and incorporation of nitrogen-fixing bacteria in the entire plant by in vitro methods, through the establishment of intracellular endosymbioses via induced uptake of bacteria by plant protoplasts (endocytobiosis), and establishment of intercellular associations by forced introduction of bacteria into the plant tissues (exocytobiosis). The common characteristic of the methods to create artificial plant-microbe systems for atmospheric nitrogen fixation is the use of in vitro plant systems: cells, tissues and organ cultures. The review pays particular attention to new bacterial inoculation procedures for introduction of the diazotrophic bacteria inside the plant tissues.

Artificial Plant-Azotobacter Symbiosis for Atmospheric Nitrogen Fixation

The main aim of this work is the direct introduction of diazotrophic prokaryotes into algal cells and into the intercellular spaces of plants and the prolonged cultivation of these artificial systems. They help the better understanding of natural symbioses and the production of plants capable to fix atmospheric nitrogen. The realization of such type of symbiosis may result considerable reduction of production and utilization of nitrogen fertilizers. This would have an essential impact for the developing countries not to speak about the elimination of nitrate contamination from the soils. Our artificial alga-bacterium endosymbiosis (endocytobiosis) as well as plant-bacterium symbiosis (exocytobiosis) prove the possibility to produce stable and functioning nitrogen fixing symbiosis widely among plants. The importance of this can’t be assessed yet.

Creation of Artificial Symbiosis Between Azotobacter and Higher Plants

An artificial symbiosis was established between diazotrophic Azomonas insignis and strawberry (Fragaria x ananassa). The partnership was created by in vitro techniques through callus induction and organogenesis. The basis of this partnership is the bacterial dependence on the plant’s metabolic activity, using maltose in the medium as a carbon and energy source which can be utilized by the plant cells only.

Small RNA NGS Revealed the Presence of Cherry Virus A and Little Cherry Virus 1 on Apricots in Hungary

Fruit trees, such as apricot trees, are constantly exposed to the attack of viruses. As they are propagated in a vegetative way, this risk is present not only in the field, where they remain for decades, but also during their propagation. Metagenomic diagnostic methods, based on next generation sequencing (NGS), offer unique possibilities to reveal all the present pathogens in the investigated sample. Using NGS of small RNAs, a special field of these techniques, we tested leaf samples of different varieties of apricot originating from an isolator house or open field stock nursery. As a result, we identified Cherry virus A (CVA) and little cherry virus 1 (LChV-1) for the first time in Hungary. The NGS results were validated by RT-PCR and also by Northern blot in the case of CVA. Cloned and Sanger sequenced viral-specific PCR products enabled us to investigate their phylogenetic relationships. However, since these pathogens have not been described in our country before, their role in symptom development and modification during co-infection with other viruses requires further investigation.

Experimental System for Creating New Type of N2-Fixing Plants

Artificial associations were established between nitrogen-fixing Azomonas or Azotobacter cells and plant tissues based on the induced carbon and energy dependency of diazotrophs on plant metabolic activity. Plant regeneration was achieved from mixed callus-bacterium associations. Light and electron microscopy was used to show the location of bacteria in intercellular spaces of callus and regenerated plant tissues. The nitrogen-fixing ability of the partnership was proved by acetylene reduction assay.