Azolla anabaena

• Fujian Academy of Agricultural Sciences, China

The nitrogen-fixing symbiotic association between the equatic fern Azolla and the cyanobacterium Anabaena azollae is already widely used to supplement nitrogen supplies for rice crops. It also has uses in production of animal foodstuff supplement, fish farming and biomass production, and these have much unrealised potential. This project seeks to develop a reliable system for classifying Anabaena azollae varieties, and thence to develop and exploit methods for making new Azolla-Anabaena combinations.

Although the Azolla genus contains only seven known species, the large collections held in the National Azolla Research Centre in Fuzhou and at IRRI comprise hundreds of strains. Few of these strains have been characterised, but some have potentially valuable properties. However, it is not known whether the wide variation that occurs is a property of the host plant, of the symbiotic alga or of both. More importantly, almost nothing is known about the taxonomy of Anabaena azollae.

In 1984, scientists at ANU conducted pilot studies on A. azollae, using techniques of recombinant DNA research, which showed that it is possible to devise a molecular taxonomy based on DNA sequences, and established that strain differences do exist. The DNA fingerprinting involves extracting DNA from Anabaena cells obtained from axenic cultures of Azolla-Anabaena. The DNA is 'cut' by a range of restriction enzymes and the molecular size of the fragments containing nitrogenase genes is determined. This can be used as a diagnostic feature.

Fundamental research on Anabaena azollae will continue in Canberra. Initially; researchers aim to develop a classification that can be used in all future research on the system, to elucidate host specificities and natural affinities among Anabaena varieties found in Azolla species and to investigate the stability and rate of mutation of Anabaena genotypes. It is also planned to raise monoclonal antibodies to Anabaena antigens to see whether serological methods can be used alongside the DNA fingerprinting procedure.

This research will use sterile cultures of Azolla species carrying Anabaena symbionts, making use of the culture collection in Fuzhou and as many new isolates as can be obtained. Australian material is very under-represented in the strain collections, and a special effort will be made to obtain samples from different regions.

Work on the recombination of Azolla plants with characterised Anabaena azollae strains - already in progress in Fuzhou - can then expand. The methods involve preparation of alga-free host plants and viable samples of Anabaena. Two categories of recombination are under study. One depends on cross inoculations, introducing algal cells into vegetative host plants: micro-injection into the region of the stem apex is the best prospect. The other category exploits the sexual reproductioon cycle and uses hybridisation of Anabaena-free microspores (male) with megaspores (which carry parental Anabaena) from other strains or species. Unequivocal
Characterisation of Anabaena strains is essential in this work, in order to prove successful recombination.

Both laboratory and field trials, which will take place in China, will then evaluate new recombinants for nitrogen fixation, disease resistance and environmental responses. Such trials are already under way in Fuzhou, using existing strains, but again a reliable taxonomic system will be beneficial.

As well as providing new technology to the Chinese research team, the project will increase knowledge of the basic biology of an economically important nitrogen-fixing association. In addition, improved strains of the symbiotic association may arise from the research. This would benefit rice, foodstuff and biomass production methods involving Azolla in all areas where it is grown, including China, the Philippines, Vietnam, India, Africa and South America.