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.

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Artificial symbiosis was established between diazotrophic Azomonas insignia and strawbeH'y (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 btM N"-benzyladenine (BA), 0.3 gM gibberellic acid (GA3) , 2.2 gM indole-3-butyric acid (IBA), and 3% sucrose] and plant regeneration [R3 = MS mineral salts + 555 gM myo-inositol, 1.2 I.tM thiamine HC1, 4.4 gM BA, 0.5 gM IBA, 0.3 gM c~-naphthaleneacetic acid (NAA), 0.5 gM 2,4-dichlorophenoxyacetic acid (2,4-D)] media were developed for the test cultivar Fert(;di 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.

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.

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Symbiotic associations were established between nitrogen‐fixing Azotobacter zettuovii (CRS‐H6) cells and carrot (Daucus carota L. cv. ‘Rother’ Half Long) tissues based on the induced carbon and energy dependency of diazotrophs on plant metabolic activity. Symbiotic associations were grown on nitrogen‐free media for four years. Plant regeneration was achieved from callus‐bacterium associations on nitrogen‐containing media. Light and electron micrographs are used to show the localization of bacteria in intercellular spaces of callus and regenerated plant tissues. The nitrogen‐fixing ability of the partnership was proved on nitrogen‐free media.

Biological nitrogen fixation is the most important process in which some prokaryotic organisms fix N 2 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.