Elaeagnusspecies (Torrey and Racette, 1989). In some cases strains that show typical Frankiamorphology have been isolated from nodules, but have not been induced to reinfect any host plant in controlled culture (Hahnet al., 1989a; Mirzaet al., 1991).
While such observations may initially seem to cast doubt on the identification of an isolate as aFrankiastrain, suggesting that they could beFrankia-like contaminants, there are other possible explanations. For example, infection by aFrankiastrain may require the action of some helper microorganism such as a fungal or bacterial species that is present in soil but not in axenic culture where the test is carried out.
Another observation that complicates the concept of cross-inoculation groups is that, no matter what taxonomic criteria are used, it is clear that a remarkable diversity ofFrankiastrains can be isolated from a single nodule (Rouvieret al., 1996). On the other hand, these taxonomic approaches, while generally failing to distinguish clear species, indicate that strains tend to cluster together according to the host plant of origin, lending some weight to a loose cross-inoculation concept. An observation that is of relevance here is that nodulation ofCasuarinaandAllocasuarinaspecies tends to be relatively specific, as they are only infected by strains that have been isolated from these hosts (Reddell and Bowen, 1986; Torrey and Racette, 1989). Thus strains and sources of inoculum for use withCasuarinaspecies in the field should be selected with considerable care.
Rippka’s classification divided 178 living strains into 22 genera, based on mor- phological and developmental features that could readily be determined in cultured material and that were, as far as possible, constant for a given strain (because the pre- vious classification had been botanical in nature, it had been based on descriptions of dead type-specimens). These 22 genera were placed in five main sections, as described in Table 2.3. As indicated, one of the primary morphological criteria used in this classification is whether the strains are unicellular or filamentous in nature.
Most cyanobacteria are obligate photoautotrophs; that is, they must fix their own carbon by photosynthesis and cannot grow by heterotrophic metabolism of existing sources of organic carbon. This obligate photoautotrophy is probably due to an inability of cyanobacteria to take organic carbon sources into the cell, rather than to a lack of enzymes for the metabolism of these compounds. Facultative
Cell
arrangement Group Reproduction Heterocysts Division Genera Unicellular
forms
Filamentous forms (cells form a trichome)
I
II
III
IV
V
Multiple fission, possibly also with binary fission
Intercalary cell division and trichome breakage
As above, plus may form homogonia
As section IV No
Yes
Yes
One plane
One plane
More than one plane
Gloeothece Gloeobacter Gloeocapsa Synechococcus Synechocystis Chamaesiphon Dermocarpa Xenococcus Dermocarpella Myxosarcina Chroococcidopsis Pleurocapsagroup Spirulina
Oscillatoria Pseudanabaena Lyngbya Phormidium Plectonema Anabaena Nodularia Cylindrospermum Nostoc
Scytonema Calothrix Chlorogloeopsis Fischerella Table 2.3. The major taxonomic groups of cyanobacteria (after Rippkaet al., 1979; see also Castenholz and Waterbury, 1989).
heterotrophy in certain strains is one of the taxonomic criteria used by Rippkaet al.
(1979), who presented data on individual strains.
N2-fixing species are found among members of all five taxonomic sections of the cyanobacteria. This widespread distribution of N2-fixation ability is initially sur- prising because, as discussed above, cyanobacteria carry out oxygenic photosynthesis.
This creates a problem for N2-fixation, as O2is invariably damaging to nitrogenase activity, and mechanisms adopted by cyanobacteria for protecting nitrogenase from O2are discussed further in Chapter 3. The ability to carry out N2-fixation in anaerobic conditions was analysed and is reported for all strains described by Rippka et al. (1979). A number of free-living species of cyanobacteria are of possible importance in tropical cropping systems and these are discussed further in Chapter 6.
Symbiotic species are found in group IV and are primarily, if not exclusively,Nostoc species. A key characteristic of group IV strains is the ability to differentiate special- ized cells for N2-fixation, known as heterocysts (described further in Chapter 3). The key cyanobacterial symbiosis of importance in tropical cropping systems is that with Azolla.
TheAzolla–cyanobacterial symbiosis
Azolla Lam. is a genus of aquatic ferns, members of which occur throughout the world. The ferns are usually found free-floating on the surface of the water. The seven recognized species of Azolla are divided into two subgenera (Stergianou and Fowler, 1990; Saunders and Fowler, 1993). Subgenus Azolla contains two sections: sectionAzollacontainingAzolla caroliniana,A. filiculoides,A. mexicana,A.
microphyllaandA. rubra; and sectionRhizosperma, which contains onlyA. pinnata, within which two susbspecies are recognized, A. pinnata subsp. pinnata and A. pinnatasubsp.imbricata. The second subgenus,Tetrasporocarpia, contains only A. nilotica. A combination of morphological and molecular data indicates that A. caroliniana,A. mexicanaandA. microphyllaare very close taxonomically, as areA.
filiculoidesand A. rubra, although there has been no formal reclassification (Van Coppenolleet al., 1995a,b). All species ofAzollacontain a heterocystous N2-fixing cyanobacterial symbiont, referred to asAnabaena azollaeStrasburger and placed in the orderNostocales, familyNostocaceae.
TheAzollacyanobacterial symbiont is still formally referred to as anAnabaena species,Anabaena azollae. However, there is now considerable evidence supporting the contention that theAzollasymbiont is in fact aNostocspecies proper, the genus to which all other symbiotic cyanobacteria are assigned. The primary taxonomic criterion used to assign cyanobacteria to this genus is the formation of homogonia filaments (Rippkaet al., 1979), and theAzollasymbiont does form such structures at specific developmental stages of the symbiosis (Peters and Meeks, 1989). In addition, DNA probe techniques were used to analyse cyanobacterial symbionts isolated from Azollaspecies and it was found that the symbionts were indeed more closely related to a free-livingNostocspecies than to a free-livingAnabaenaspecies by these criteria (Plazinskiet al., 1990). However, the fact that the symbionts ofAzollaappear to be
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obligate, i.e. it has never been possible to culture them and use the cultures to reinfect Azolla, makes taxonomic classification very difficult. Thus, there has been no formal taxonomic reclassification of theAzolla symbiont and the nameAnabaena azollae continues to be used.
Molecular techniques have been used to look at genetic variation in the symbi- onts associated with the differentAzollaspecies. These experiments are conducted by extracting microbial symbionts/endophytes from theAzollafern and analysing these directly without any culture. Hence there is no guarantee that a single microbial species is being examined – in fact this is highly unlikely. Nevertheless, DNA and fatty acid composition analysis methods have given remarkably concordant results, indicating that there are characteristic differences in the symbionts associated with each species. In particular, symbionts from A. nilotica, A. pinnata (section Rhizosperma) andA. rubracluster together and those fromA. filiculoides,A. micro- phylla,A. mexicana andA. carolinianacluster together, symbionts from the latter three forming a very tight cluster consistent with the idea that they may in fact constitute a single species. These data have led to the suggestion that there may have been coevolution of host and symbiont (Caudales et al., 1995; Rasmussen and Svenning, 2000).
The symbiont remains associated withAzollathroughout the life cycle of the fern, being located in specialized cavities in the upper surface of the leaves. TheAzolla leaves themselves are only 1–3 cm in diameter and occur in tight clusters, this dense packing enabling formation of the typical dense mats ofAzolla seen on the still surfaces of ponds, drainage ditches and paddy fields (Fig. 2.9). These mats may be reddish or purple in colour due to the presence of anthocyanins.Azollais not able to colonize turbulent waters because the mats become fragmented and cannot grow vigorously enough.
The structure of the fern is based around the floating stem or rhizome. On the surface of the water are borne alternately arranged leaves, and adventitious roots arise from the rhizome beneath the water surface. During growth of new leaves the Anabaenacolonization is maintained from a colony of undifferentiated Anabaena filaments that is located at the tip of each stem. As a new leaf differentiates, some of this apical colony becomes transferred into the cavity of the developing leaf. This process is facilitated by a structure called the primary branched hair, a multicellular epidermal trichome that extends from the new leaf primordium into the apical Anabaenacolony.
The mature leaf cavity contains two such branched hairs and about 25 simple hairs, all with ultrastructure as seen in transfer cells, and these may function both in provision of carbon to the symbiont and in assimilation of ammonia by the host (Calvertet al., 1985). TheAnabaenafilaments within the mature leaf cavity show differentiation of 20–30% of cells into heterocysts – a higher proportion than observed in the free-living strains – and have high nitrogenase activity.
While most reproduction ofAzolla is by vegetative means, and thus poses no problem for maintenance of the cyanobacterial symbiont,Azolladoes sporulate, if unpredictably. When this occurs, filaments of the A. azollae microsymbiont are incorporated into the developing micro- and megasporocarps, and their presence
N2-fixing Organisms49
Fig. 2.9. TheAzolla–Anabaenasymbiosis. (a) A dense mat ofAzolla pinnatafronds in Rwanda. (b) A singleA. pinnatafrond. (c) Trans- verse section through anA. pinnatafrond showing symbioticAnabaenain the leaf cavity. (d) Scanning electron micrograph ofAnabaena azollaefilaments with heterocysts inAzollaleaf cavity. (Photographs b, c, d: J.H. Becking.)
67
in the megasporocarps on fertilization and subsequent germination of a fresh sporophyte ensures continuity of the symbiosis. This life cycle makes reinfection by free-living cyanobacteria unnecessary, and, as discussed above, it is not certain whether the trueAzollasymbiont is capable of free-living growth.