symbiotic bacteria and fungi

SYMBIOTIC BACTERIA AND FUNGI

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SYMBIOTIC BACTERIA AND FUNGI E. K. James Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK M. Fomina College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK S.M. de Faria EMBRAPA-Agrobiologia, km 47, Seropédica, 23851-970, RJ, Brazil Keywords: Nitrogen fixation, legumes, Actinorhizal plants, nodulation, Rhizobium, Burkholderia, Frankia, Ecto-Mycorrhizas (EcM), Arbuscular Mycorrhizas (AMs), phosphorus, bioremediation Contents

1. Introduction 2. Nitrogen-fixing Legumes and their Symbionts 3. Infection of legumes by rhizobia 4. Other Nitrogen-Fixing Organisms 5. The Role of Nitrogen Fixation in the Nitrogen Cycle 6. Mycorrhizal Fungi and the Phosphorus Cycle 7. Use of Symbiotic Fungi and Bacteria in Bioremediation 8. Conclusion Related Chapters Glossary Bibliography Biographical Sketches Summary Nitrogen (N) and phosphorus (P) are the two most important nutrients for plant growth, but in many environments both are in short supply and/or are unavailable to plants. This is particularly true in tropical forests, where high temperatures and humidity cause mineralization of organic N, and the very high precipitation results in rapid leaching of the remaining soil N. Therefore, the fixation of atmospheric dinitrogen into ammonia and hence into organic compounds is a crucial part of the N- cycle of these forests. Nitrogenase, the enzyme responsible for biological nitrogen fixation (BNF), has a wide taxonomic distribution in the bacteria and archaea, but is confined to a limited number http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (1 of 25)10/2/2010 11:16:14


of species. Among these, soil bacteria called rhizobia, which form symbiotic nodules on the roots of legumes (Fabaceae), are recognized as the main contributors of biologicallyfixed nitrogen to pristine environments, most particularly in tropical forests where they are so abundant and diverse, but there are other symbiotic, endophytic, associative and free-living diazotrophs that make a significant contribution, and this chapter describes these as well as the leguminous symbioses. Similarly, the availability of the second most major essential nutrient for plants, P, is greatly limited by its occurrence and low availability in the soil, most particularly owing to the low solubility of phosphates, and hence plants very early in their evolutionary history developed a symbiotic relationship with fungi called mycorrhizas. There are two types of mycorrhizas: ecto- and endo- (e.g. arbuscular and ericoid) mycorrhizas, and most plant families (> 95 %) are capable of forming a symbiosis with at least one type. In this chapter we review current knowledge about symbiotic mycorrhizal associations within tropical forests and assess their likely contributions to the P-cycle.

1. Introduction Nitrogen and phosphorous are the two main limiting nutrients in both natural and agricultural systems. In agriculture both nutrients are supplied in forms available to plants as fertilizers produced either through chemical processes (eg. the Haber-Bosch process that converts or "fixes" atmospheric nitrogen into ammonia using very high quantities of energy) or from mining mineral deposits (eg. from rock phosphate). Regardless of whether the fertilizers are produced chemically or by mining, the energetic and environmental costs of their production are prodigious. However, in most undisturbed non-agricultural ecosystems (as well as some agricultural ones) soluble nitrogen (ammonium) is made available to plants via a process known as biological nitrogen fixation (BNF), in which bacteria that contain the enzyme complex called nitrogenase (termed "diazotrophs") can fix atmospheric N2 into ammonia using energy from ATP and reducing agent (electrons) supplied by respiration (which can be aerobic or anaerobic): N2 + 8H+ + 8e- + 16ATP ® 2NH3 + H2 + 16ADP + 16Pi This ammonia, which is potentially toxic to the organism, is usually then immediately converted into amino acids or amides for use by the diazotrophic bacterium in the production of proteins and peptides to facilitate its growth. The fixed N incorporated into diazotrophic bacteria is then released into the environment when they die, usually in the form of amino acids that then become mineralized and available for uptake by other bacteria and/or by plants. Interestingly, although nitrogenase, the enzyme responsible for nitrogen fixation, has a wide taxonomic distribution in the bacteria and http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (2 of 25)10/2/2010 11:16:14


archaea, it is actually confined to a relatively limited number of species (J.P.W. Young in PALACIOS & NEWTON, 2005). Nevertheless, many plants, particularly legumes, but also some other higher plants (eg. Gunnera, and "actinorhizal" plants, such as Alnus, Casuarina and Myrica), as well as some cycads and the fern Azolla, form mutualistic symbiotic relationships with nitrogen-fixing bacteria. In these systems, soil bacteria located in specialized organs take nitrogen from the atmosphere and convert it to soluble nitrogen that is available for plant growth. Nitrogen-fixing legumes are one of the main sources of nitrogen in both natural environments and many agricultural systems. However, although pathways controlling nitrogen-fixation and the bacterial symbionts involved are well understood in a handful of model and crop legume species, very little is known about the diversity of symbiotic systems in natural environments in terms of the bacteria involved and their host relationships. This is especially so in the tropics where most legume diversity is found.

2. Nitrogen-fixing Legumes and their Symbionts The family Leguminosae (or Fabaceae) is one of the three largest families of flowering plants, and consists of more than 640 genera with 18,000 plus species (Allen and Allen, 1981; Sprent, 2001). It has been divided taxonomically into three sub-families, the Papilionoideae (468 genera), Mimosoideae (78 genera) and Caesalpinioideae (157 genera), with only the Papilionoideae being represented in temperate regions. The vast majority of legume species of all three sub-families are confined to the tropics and subtropics, where they are particularly abundant and diverse in forests, wetlands and savannahs. Although it is often assumed that all legumes can nodulate and fix N, in fact most genera (95 %) in the Caesalpinioideae do not nodulate, and this is also the case with 7 % and 12 % of genera in the Papilionoideae and Mimosoideae, respectively. However, it should be noted that the vast majority of legumes, particularly in the Caesalpinioideae have so far not actually been examined for their nodulation status, and so these proportions will inevitably change as more legumes species are examined (SPRENT, 2001). Examples of nodules formed on members of all three sub-families are shown in Figures 1 to 4. Briefly, they consist of a central zone that contains the Nfixing bacteria, and this is surrounded by an uninfected "cortex", which may be surmounted by an epidermis; this cortex/epidermis serves to keep out oxygen, to deter pathogens and to prevent desiccation (Figures 1, 2). The enzyme nitrogenase is highly sensitive to oxygen, but the N-fixing bacteria require it for aerobic respiration to supply the ATP necessary for high rates of BNF and, therefore, legume nodules contain a high concentration of an oxygen-carrying protein called leghemoglobin, which surrounds the N-fixing bacteria in the infected cells. This leghemoglobin transfers very rapidly a low concentration of oxygen to the bacteria, and its presence in nodules is essential for their functioning.

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Figure 1. Internal structure of a pea nodule (Papilionoideae) infected by Rhizobium leguminosarum showing the apical meristem (m), blue-stained N-fixing cells within the infected zone (iz), and the peripheral uninfected cortex (c).

Figure 2. Internal structure of a nodule on the tree Erythrophleum ivorense (Caesalpinioideae). Labels are as in Figure 1.

Figure 3. Nodules (arrow) on Piptadenia viridiflora (Mimosoideae) inoculated with Burkholderia phymatum.

Figure 4. Nodules (arrow) on Acacia schaffneri (Mimosoideae) inoculated with Cupriavidus taiwanensis. Nodulation in legumes evolved approximately 55 million years ago, most likely in a semiarid region north of the Tethys seaway, and it may have been driven by peaks in temperature, humidity and atmospheric carbon dioxide concentrations (SPRENT & JAMES, 2007). However, in spite of their semiarid origin, most extant legume diversity is now found in tropical forests and tropical wetlands, and there appears to be high http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (4 of 25)10/2/2010 11:16:14


selection pressure for the symbiotic nodulation trait in these areas (SPRENT, 2001). Until very recently, all the bacteria known to form nitrogen-fixing symbioses with legumes were members of the family Rhizobiaceae and a set of related genera in the order Rhizobiales of the Alpha-proteobacteria. This includes Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium (Ensifer) (Table 1; P. Graham in DILWORTH et al. 2008). The advent of molecular techniques, such as the polymerase chain reaction (PCR) method which can be used for amplifying DNA from bacteria, has allowed for their identification via comparisons of the sequences of certain genes that are known to be highly conserved (so called "housekeeping" genes) with sequences from other bacteria using large databases. This has resulted in an explosion in the number of new rhizobial strains, and new species are being described almost every month. Indeed, in the last few years, using molecular techniques, a number of other α-proteobacteria that are not closely related to Rhizobium have been isolated and identified from legumes, including strains of Methylobacterium, Phyllobacterium, Blastobacter and Devosia. On the other hand, it should be noted that legume nodules provide excellent niches for many opportunistic microorganisms, and so it cannot always be assumed that just because a bacterium comes from a nodule that it is actually a symbiont, and hence it is essential that further evidence is obtained (such as nodulation of its original host under sterile conditions) before deciding that a bacterial strain is really a nodulator (SPRENT, 2001). Nevertheless, and in spite of this caveat, at least some strains in the "new" genera of alpha-proteobacterial rhizobia have been shown to nodulate their hosts. It has long been recognized that the tropics are the main source of new rhizobial isolates (which probably reflects the very high diversity of nodulated legumes in tropical forests), and the vast majority of newly-described rhizobial legume symbionts are also from the tropics, which are also the source of highly promiscuous and very broad host range rhizobia, such as Sinorhizobium strain NGR234, which has the ability to nodulate more than 150 species across all three leguminous sub-families (H. Kobayashi & W. Broughton in DILWORTH et al. 2008). Table 1. However, in the last few years more distantly related Beta-proteobacteria in the genera Burkholderia and Cupriavidus, collectively termed beta-rhizobia, were discovered in nodules of a number of tropical legumes, but most notably from species of Mimosa. This is a large genus in the Mimosoideae that is native to tropical forests and savannahs in the New World, but which also has some Pantropical weed species. The list of genera known to nodulate with Beta-rhizobia is increasing, and almost all of these are from tropical forests, wetlands and savannahs (SPRENT & JAMES, 2007). Phylogenetic analyses of nodulation gene (nodA, nodC) sequences suggest that betarhizobia have acquired their symbiosis genes from "conventional" alpha-rhizobia (eg. Rhizobium) and that there has been more than one independent transfer between the alpha and beta bacteria. However, there is as yet insufficient evidence to suggest exactly where, when, how many times, or indeed in which direction this transfer occurred. Certainly, and whatever the processes by which "Beta-rhizobia" acquired their nodulation ability, the more tropical legumes that are examined then the more potential http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (5 of 25)10/2/2010 11:16:14


there is to isolate even more unusual putative symbionts. However, although it is now relatively easy to identify bacterial isolates via sequences of their core "housekeeping" genes, it is not so easy to show that they are actually symbiotic, and so many of these "strange" bacteria will inevitably remain as curiosities until the hard work is done to confirm (or not) their symbiotic potential.

Table 1: Recognized genera and species of root nodule bacteria from tropical forest legumes (and other relevant legumes). Adapted from P. Graham in DILWORTH et al. (2008) with acknowledgements. The process by which a bacterium that has been isolated from a legume nodule could be shown to be the actual symbiont of that legume and not an opportunistic "contaminant" would be as follows (see Figures 5 to 10): a. Isolation from a surface-sterilized fresh (or rehydrated dried) nodule via streaking of its internal contents onto an agar plate containing an appropriate growth medium (eg. yeast-mannitol agar or YMA), and selection of colonies of bacteria that subsequently grow on the agar (Figures 5, 6). These colonies can grow at different rates, and can take many forms and colors, and now that it is understood that many different types of bacteria can be symbionts, particularly of tropical legumes, it can no longer be assumed that any particular growth rate and/or colony type is either a "contaminant" or a "symbiont", and hence it may be necessary to do further tests on all colony types.

Figure 5. Bacterial colonies (*) growing on agar supplemented with a mixture of nutrients that favor the growth of symbiotic rhizobia from legume nodules. A nodule has been crushed and the contents streaked onto the agar.

Figure 6. Same as Figure 5, except that in this case the bacteria have been put through a purification process, so that all the colonies (*) have been formed by the same strain. http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (6 of 25)10/2/2010 11:16:14


b. After purification of each colony type it is necessary to test each one on the original legume host via inoculation of the putatively symbiotic bacteria onto seedlings growing in sterile conditions. A good symbiont will produce a green and healthy plant with plenty of nodules, whereas a poor symbiont will either produce no nodules at all or else will produce ineffective ones that do not fix N, and hence the plants will be small, yellow and unhealthy (Figures 7 and 8).

Figure 7. Mimosa debilis (Mimosoideae) plants after inoculation with different strains of rhizobia. Both plants are nodulated, but the green plant on the left is nodulated (arrow) by an effective N-fixing strain, whereas the yellow plant on the right is nodulated by an ineffective, non-symbiotic strain.

Figure 8. Comparison of the ability of 2 rhizobial strains to nodulate Mimosa setosissima. Strain JPY164 was isolated from nodules on this plant, and is therefore more compatible with it as a symbiont than strain JPY461 which was isolated from another Mimosa species. This is shown by the green and healthy plant on the left (JPY164) and the small yellow plant on the right (JPY461). Interestingly, when it is inoculated with both strains together, M. setosissima is not as healthy as when it is inoculated only with JPY164. c. Any nodules thus formed should contain the original inoculated strain, but this needs to be confirmed via reisolating that strain from the nodules. The bacteria thus isolated can then be compared to the inoculated strain via markers, such as DNA patterns ("fingerprints") or whole cell protein profiles. d. Another method is to genetically engineer the inoculated bacteria so that they express a fluorescent protein; if the bacteria are genuinely symbiotic then the nodules formed by them should fluoresce under certain defined conditions, and those formed on control plants inoculated with "wild type" bacteria without the green fluorescent protein (GFP) should not (Figures 9 and 10). http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (7 of 25)10/2/2010 11:16:14


Figure 9. Nodules on the roots of Cyclopia genistoides (Papilionoideae) that have been cut open to reveal the pink-colored infected tissue in the centre of the nodules. The pink color (*) is due to the oxygen-carrying protein leghemoglobin which is essential for the functioning of the N-fixing symbiosis.

Figure 10. The same nodules as in Figure 9 after they have been viewed under a fluorescent microscope. The green fluorescent protein (GFP) that has been "tagged" onto the N-fixing bacteria within the nodules has been excited by low wavelength light, and has consequently emitted light of a longer wavelength (i.e. it has fluoresced green light). As the GFP was incorporated into the DNA of the bacteria before they were inoculated on to the roots, this technique shows how GFP can be used to prove that the inoculated strain really is a symbiont.

3. Infection of legumes by rhizobia There has been considerable research into the processes by which nodules form on legumes, but most of this has focused on only a few Papilionoid species, such as the "model" legumes, Lotus japonicus and Medicago truncatula (L. Schauser et al. in DILWORTH et al. 2008), and the important crop/pasture species soybean, alfalfa, pea and common bean. The vast majority of legumes that can nodulate (and these are mostly tropical) have not been examined in anything like such detail, and very little is known about nodulation processes in the Caesalpinioideae and Mimosoideae (SPRENT, 2001). A recent exception to this is Mimosa (see above). In most legumes so far studied, and regardless of the microsymbiont (i.e whether it is alpha or beta-rhizobial), the soilbased rhizobia are attracted to the roots of compatible plants via chemicals released from the roots called flavonoids. These flavonoids can "switch on" the nodulation genes of the rhizobia, which results in the release by the bacteria of chemicals called "Nod Factors" that stimulate the plant to allow the bacteria to enter root hairs and, http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (8 of 25)10/2/2010 11:16:14


simultaneously, stimulate the formation within the root cortex (or epidermis) of a nodule meristem. The bacteria within the root hairs then divide repeatedly and progress into the root cortex via a tube made of by host plant cell wall material called an infection thread (which both directs the bacteria and prevents them from randomly entering the plant cells that the infection thread crosses) until the infection thread containing the bacteria encounters the nodule meristem that is growing outwards to meet it. The infection thread(s) then enters the newly-divided nodule cells and rhizobia are released from the tip of the thread into the cytoplasm. At this point the host cell encloses the newly-released bacteria within a cell membrane so that it is not actually in contact with the plant cytoplasm. These bacteria then differentiate into their symbiotic N-fixing state, and effectively become part of (and dependent upon) the host plant. This process occurs repeatedly and in most legumes the nodule meristem continues to grow outwards from the root, and the resultant mature nodules (Figure 1) are usually elongated structures with a meristem at the tip, a region of cells immediately behind the meristem that are being invaded by infection threads, and behind this (and closest to the root) a large infected zone of N-fixing cells. In older nodules, the region immediately adjacent to the connection to the root contains cells that are senescing, and these will eventually become dominant as the nodule ages and finally degrades, thereby releasing rhizobia into the soil to start the nodulation cycle anew. The aforementioned process seems to be generally true for most legumes, and the elongated nodule shape with a persistent meristem is found in all three sub-families (Figure 1), and these nodules are termed "indeterminate". There are a few Papilionoid legumes, however, that form nodules with a transient meristem, such as those on soybean (Glycine max) and common bean (Phaseolus vulgaris). These "determinate" nodules are usually spherical, as the centrally-located meristem ceases early in the developmental process, and the cells containing the newly-released rhizobia then divide to form the functional N-fixing nodule. As they don’t have a persistent meristem these nodules tend to have a shorter life than indeterminate ones, but this is sufficient to encompass the relatively short period over which an annual plant (such as soybean) requires symbiotic BNF to supply it with N for its vegetative growth before it flowers and sets seed. It is not surprising, therefore, that all the perennial woody legumes that are encountered in tropical forests, have nodules of the persistent indeterminate type, and that determinate nodules are confined to such a small range of species (SPRENT, 2001). It should also be noted at this point that there are variations on the determinate and indeterminate "theme" (eg. "collar" nodules on Lupinus, and "aeschynomenoid" nodules on peanut and Aeschynomene), but these are relatively few in number and all in the Papilionoideae. Although most legumes that can nodulate usually do so only on their roots, there are a few genera (most in the Papilionoideae) that can also nodulate their stems. This nodulation can be either on the roots that arise from the flooded stems (ie. the adventitious roots, such as those arising from the floating stems on Neptunia; Figure



11), or directly on the stems themselves, and these "true" stem nodules are so far reported only on species of Aeschynomene, Discolobium and Sesbania (Figures 12 14). These are all hydrophytic, and grow in tropical wetlands, such as the Pantanal in Brazil (POTT & POTT, 2000; SPRENT, 2001), as well as in other seasonally-flooded regions of the tropics (eg. Amazon and Orinoco basins). Nodulation of their stems in a flooded environment allows these legumes to continue symbiotic N-fixation in conditions that would normally prevent it in non-hydrophytic legumes, as both nodulation and nodule functioning (ie. symbiotic BNF) are very much dependent on a continued supply of oxygen to support the aerobic respiration required by these highly energetically costly processes. Interestingly, the nodules formed on the aerial stems of these plants are slightly unusual in developmental and structural terms and don’t fit easily into the determinate and indeterminate categories (see above). Another unusual feature is that they often contain fully functional photosynthetic chloroplasts, and hence are able to supply the N-fixing bacteroids with both oxygen and possibly also a more direct supply of carbohydrate. The symbiotic bacteria within stem nodules also tend to be different from the norm eg. in the case of Sesbania rostrata, the main symbiont is Azorhizobium (Table 1), a bacterium that is not closely related to Rhizobium, and which has the unusual (for rhizobia) capability of fixing N in a free-living (ie. non-symbiotic) state. Similarly, the symbionts of Aeschynomene spp, although they are usually Bradyrhizobium (Table 1), the strains that form stem nodules contain bacteriochlorophyll and can photosynthesise. The photosynthetic ability of stem nodules by both the plant partner (via chloroplasts) and the microsymbiont (via bacteriochlorophyll) has been linked to the very high rates of BNF recorded by these legumes, and is the reason for their promotion as "green manures" in the cultivation of paddy rice in tropical countries (Figure 13). This use is likely to become more widespread as the costs of mineral N fertilizers rise owing to the inexorable increase in fossil fuel prices that are projected over the next 20 years or so.

Figure 11. Neptunia oleracea (Mimosoideae) plants floating on a pond in the Brazilian Pantanal wetlands. Note the thick, spongy stems that allow the plants to float (arrows); the adventitious roots that arise from these are nodulated.



Figure 12. Nodules (arrows) on the stem of the hydrophytic legume, Discolobium pulchellum (Papilionoideae), which is very abundant in South American wetlands, such as the Pantanal.

Figure 13. A rice paddy field filled with the semi-aquatic legume Sesbania rostrata (Papilionoideae) as a "green manure". This plant, which nodulates profusely both on its roots and stems (see Figure 14), fixes large quantities of N, and it can be ploughed into the paddy field prior to the planting of the rice crop. The fixed N which is released from the green manure can then be taken up by the growing rice and hence is a very good substitute for mineral fertilizer.

Figure 14. Stem nodules on S. rostrata (arrows).

4. Other Nitrogen-Fixing Organisms Although they are globally most likely to the most important in terms of their diversity and their contribution of fixed N, legumes are not the only plants that have symbiotic associations with N-fixing micro-organisms and which make a contribution to the Ncycle (DILWORTH et al. 2008; PAWLOWSKI & NEWTON, 2008; WERNER & NEWTON, 2005). The so-called Actinorhizal plants, which are all woody members of several related families (all in the Rosid clade of plants, which also includes the legumes), such as the Betulaceae, Casuarinaceae, and Rosaceae, have root-nodulating symbioses with Frankia, a diazotrophic filamentous actinomycete, and these symbioses http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (11 of 25)10/2/2010 11:16:14


can be just as effective at fixing N as legume symbioses. The nodules formed are superficially similar to those on legumes, but their anatomy and physiology differ in a number of respects (PAWLOWSKI & NEWTON, 2008). Most Actinorhizal plants (many of which are trees) occur in temperate forest ecosystems, and are not generally associated with tropical forests, but there are exceptions, such as some Alnus, Casuarina, and Allocasuarina species (R.O. Russo in WERNER & NEWTON, 2005). However, at present it is impossible to assess the relative contributions of fixed N made by legume and actinorhizal trees to tropical forests, but it is likely that that made by the legumes is usually much greater, simply because they are more numerous. Generally speaking, the opposite situation is likely to pertain in temperate forests, in which Actinorhizal plants appear to fill the same niche as woody legumes do in tropical forests. There are also higher plant symbioses with N-fixing heterocystous cyanobacteria, such as Anabaena and Nostoc. which are likely to be important to the N-cycle of tropical forests, but as with actinorhizal plants, relatively little is known about them. They include the southern hemisphere native plant, Gunnera, which fixes N via Nostoc heterocysts housed in special vesicles within the host plant cells at the bases of the petioles. Gunnera spp. are very efficient at BNF, as are the N-fixing root nodule symbioses that the Cycads (which are Gymnosperms) form with cyanobacteria. Similarly, the floating fern Azolla, which forms an extracellular symbiosis with cyanobacteria (mainly Anabaena) is common in tropical wetlands, and is so effective at BNF that it is commonly used as a green manure in paddy rice production. Free-living (non-symbiotic) cyanobacteria are also, in themselves, excellent N-fixers (partly because of their photosynthetic ability), and are very common in standing water, such as lakes, ponds and paddy fields. Numerous types of non-photosynthetic diazotrophs, both free-living and those closelyassociated with plants, can also be isolated from tropical soils (eg. Azospirillum). The plant-associated bacteria include those that actually live within the tissues of nonnodulated and/or non-legume plants as endophytes (eg. sugarcane), and it has been suggested by numerous researchers that these could be supplying the plants with fixed N. However, as there are no obvious symbiotic structures analogous to legume or actinorhizal nodules, it is difficult to see how such associations can be efficient at providing the host plants with fixed N, and definitive evidence that they do is still lacking. On the other hand, given that it is estimated that only 1 % of bacteria can, at present, be cultured, it is likely that the amount of diazotrophs in tropical soils has been vastly underestimated.

5. The Role of Nitrogen Fixation in the Nitrogen Cycle The fixation of atmospheric dinitrogen into ammonia and hence into organic compounds is a crucial part of the nitrogen cycle. Globally, BNF accounts for approximately for 50 to 200 megatonnes of N per year contributed to terrestrial http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (12 of 25)10/2/2010 11:16:14


ecosystems, and makes up 80 % of total N fixed, the remainder being via other, physical, processes, such as lightning (R.J.M. van Spenning et al. in WERNER & NEWTON, 2005). Although it is assumed that terrestrial BNF contributions are largely due to the legume-rhizobial symbiosis (both natural and agriculturally-based), in fact it is not easy to make estimates of BNF by legumes in the field. Most methods for estimating BNF in the field are based on the selective discrimination of the heavy stable isotope of nitrogen,15N, and the only method that is practical in a natural forest or savannah is the 15N natural abundance (or delta 15N) method (SPRENT, 2001; R.O. Russo in WERNER & NEWTON, 2005). In spite of complications in using this method, a few delta 15N analyses have succeeded in estimating the amounts of N fixed by nodulated legumes in tropical forests, but the amounts vary greatly from species to species and site to site. A common theme, however, is that N-fixing legume symbioses thrive in tropical wetlands (eg. the Pantanal) and seasonally-flooded forests (Amazon, Orinoco), and this has been linked to the intense leaching of soluble N from the soils caused by flooding during the wet season and mineralization during the dry season (LOUREIRO et al. 1998). Indeed, it has been noted that all legumes in such regions tend to be nodulated (SPRENT, 2001), and they undoubtedly make a major contribution to the N-budget in such systems. Tropical forests in different parts of the World vary greatly in both the number and diversity of legume species present within them (SPRENT 2001). In Asian tropical forests, for example, there are very few nodulated species compared to those in tropical African, and, even more contrastingly, in tropical American forests. However, even in Africa and South America, many of the legumes in forests are non-nodulating. Those that do, tend to nodulate during the wet season, and hence it is possible nodulation ability, and certainly legume BNF, has been underestimated in tropical forests. Interestingly, in spite of the obvious abundance of nodulated legumes in the tropics, it is generally considered that tropical forests are not N-limited, but actually P-limited (see next section), whereas the opposite is true for temperate forests. This is regarded as an ecological mystery, but may be linked to the relatively low efficiency of BNF at temperatures below 26° C.

6. Mycorrhizal Fungi and the Phosphorus Cycle Fungi are a fundamental and vital part of the soil microbiota and have the largest bulk biomass. There are approximately 1.5 million fungal species, of which only 72, 000 have so far been described, and hence the Fungal Kingdom is one of the least well known. However, more than 90 % of terrestrial plants form a symbiotic association with a particular type of fungi called mycorrhizas. This mutualistic association, which dates back more than 400 million years, provides the fungus with relatively constant and direct access to carbohydrates. The latter are mainly glucose and sucrose produced by plant photosynthesis, and they are translocated from the aerial parts of the plant to the http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (13 of 25)10/2/2010 11:16:14


root tissues and then to the fungal partners. In return, the plant uses the fungal mycelium’s very large surface area to absorb water and mineral nutrients from the soil, thus improving the mineral absorption capabilities of the plant roots. Plant roots alone may be incapable of taking up phosphate ions that are immobilized, for example, in soils with a high (alkaline) pH. The mycelium of the mycorrhizal fungus can, however, access these P sources, and make them available to the plants they colonize. The mechanisms of increased absorption are both physical and chemical. Mycorrhizal mycelia are much smaller in diameter than the smallest root, and can explore a greater volume of soil, providing a larger surface area for absorption. Also, the cell membrane chemistry of fungi is different from that of plants and this also assists in the uptake of P and other minerals. Although there are numerous soil microorganisms, both fungal and bacterial, that can assist in the solubilization of unavailable P, mycorrhizas are undoubtedly the most important in most ecosystems, including tropical forests. Indeed, mycorrhizal fungi are a particularly important group of organisms in the cycling of elements in ecosystems, such as forests (M. Fomina et al. in GADD, 2006). Fungi, in general, are very well suited for transformation of metals and minerals, they can acidify their environment and produce chelating agents, and are highly resistant against extreme environmental factors such as metal toxicity, UV, radiation, and desiccation (M. Fomina & G.M. Gadd in ROBSON et al. 2007). One of the main functions of mycorrhizal fungi is their contribution to plant adaptation to extreme environments (SMITH & READ, 1997). Mycorrhizas are especially beneficial for the plant partner in nutrient-poor soils. Mycorrhizal plants are also often more resistant to diseases, such as those caused by microbial soil-borne pathogens, and are more resistant to the effects of drought. These effects are probably due to the improved water and mineral uptake in mycorrhizal plants. Plants grown in sterile soils and growth media often perform poorly without the addition of spores or hyphae of mycorrhizal fungi to colonize the plant roots and aid in the uptake of soil mineral nutrients. The absence of mycorrhizal fungi can also slow plant growth in early succession or on degraded landscapes.

Figure 15. Ectomycorrhizas (EcM) (Rhizopogon rubescens) (arrows) on Pinus sylvestris (Scots pine) roots.



Figure 16. Ectomycorrhizas (EcM) (Paxillus involutus) (arrows) on Pinus sylvestris roots.

Figure 17. Scanning electron micrograph (SEM) of freeze-fractured root of Pinus sylvestris that has a symbiosis with the EcM Paxillus involutus showing the internal structure of the mycorrhizal root. Note the mantle (m) of fungal hyphae covering the root exterior and the Hartig net (arrows) of fungal hyphae growing between the root cortical cells. Depending on the ability of symbiotic fungi to colonize host root cells intra- or extracellularly, the two main types of mycorrhizas are ectomycorrhizas and endomycorrhizas (ericoid and arbuscular mycorrhizas) (SMITH & READ, 1997). Ecto-mycorrhizas (EcM) (Figures 15 – 17) are relatively less abundant than endo-mycorrhizas and are only found in approximately 10 % of plant families. They also form a less intimate association with the (mostly woody) plants with which they are symbiotic, and this generally takes the form of a "sheath" of hyphae covering the root tips (Figures 15, 16), and a Hartig Net of hyphae surrounding the root cortical cells (Figure 17). In some cases, the hyphae may also penetrate the plant cells, in which case the mycorrhiza is called an ectendomycorrhiza. Outside the root, the fungal mycelia form an extensive network within the soil and leaf litter. They are particularly important in temperate coniferous forests, and most EcM belong to the Basidiomycota, Ascomyscota and the Zygomycota. In contrast to EcM fungi that colonize spaces between host plant root cells, endo-mycorrhizal fungi (e.g. ericoid and arbuscular mycorrhiza; Figure 18) colonize root cells intracellularly. Arbuscular mycorrhizas (AM), which were formerly known as vesicular-arbuscular mycorrhizas (VAM), are fungi in the division Glomeromycota, whose hyphae enter into the plant cells, producing structures that are either balloon-like (vesicles) or dichotomously-branching invaginations (arbuscules) (Figure 19). The fungal hyphae do not actually penetrate the cytoplasm of the plant cell, but invaginate the cell membrane in a manner analogous to that of symbiotic bacteroids being surrounded by the symbiosome membrane in legume nodule cells (see earlier).



The structure of the arbuscules greatly increases the contact surface area between the hypha and the cytoplasm of the host cell to facilitate the transfer of nutrients between them. As with EcM the mycelia of the AM extend into the soil and leaf litter, and greatly assist in the uptake of relatively insoluble nutrients from the soil, and they may also allow host plants to interact and exchange nutrients (eg. N and P).

Figure 18. Ericoid mycorrhizal fungus Hymenoscyphus ericae (axenic culture) growing on metallic depleted uranium. The yellow color (arrow) indicates migration of the uranyl ion within the mycelia. Although mycorrhizas can assist in the uptake of a number of plant nutrients, including N and potassium, their role in enhancing P uptake is generally considered to be their most important attribute. Low P availability limits plant growth in many acid soils of the tropics, and P deficiency is mainly caused by strong adsorption of H2PO4 to aluminum (Al) and iron (Fe), and hence most soils are not actually deficient in P, as such. The ability of mycorrhizas to "mine" this unavailable P has been linked to the production by the fungi of the iron-containing protein glomalin, which appears to be capable of releasing P from unavailable Fe – P forms in the NaOH-Pi fraction of the soil (CARDOSO & KUYPER, 2006).

7. Use of Symbiotic Fungi and Bacteria in Bioremediation Many fungi survive and even flourish in environments that are heavily contaminated with toxic metal, and they employ a variety of biochemical and morphological strategies to do this (M. Fomina & G.M. Gadd in ROBSON et al. 2007). Numerous studies have shown that mycorrhizal associations enhance plant growth on metalcontaminated soils. Ectomycorrhizal mycobionts can filter toxic metals in the hyphal sheath or Hartig net by adsorption, by restricting metal mobility in the fungal apoplast via the hydrophobicity of the fungal sheath, by chelating metals via the release of organic acids and other substances, and by the adsorbtion of metal on the external mycelia. Indeed, the geochemical activity of mycorrhizal fungi in soil may significantly alter the physico-chemical environment of the mycorrhizospere (M. Fomina & G.M. Gadd in ROBSON et al. 2007). In terms of the phosphorus acquisition function of mycorrhizas, the solubilization of toxic metal phosphates in the mycorrhizosphere in metal-polluted soils is of special interest. It was shown that many ericoid- and ectomycorrhizal fungi were able to solubilize toxic metal phosphates (eg. copper, http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (16 of 25)10/2/2010 11:16:14


cadmium, and zinc phosphates and lead chlorophosphate) (M. Fomina et al. in GADD, 2006). For the majority of the mycorrhizal fungi tested the main mechanism was acidification (or protonolysis), with a clear correlation between pH and solubilization values. Zinc phosphate was the least toxic of the metals tested, and also the most readily solubilized by mycorrhizal fungi. Studies of ericoid- and ecto-mycorrhizal fungi grown in axenic culture has shown that, in general, the solubilization of toxic metal minerals is related to the metal tolerance of the fungi (M. Fomina et al. in GADD, 2006). The relation of toxic metal mineral solubilization and metal tolerance was confirmed by principal component analysis where copper tolerant isolates of the ericoid mycorrhizal fungus Hymescyphus ericae from the Devon Consol copper mine (UK) demonstrated a much higher ability to solubilize cadmium, copper and zinc phosphates than isolates from non-polluted areas. Similarly, zinc tolerant isolates of ectomycorrhizal fungi (Paxillus involutus, Suillus bovinus, Suillus luteus) from around the zinc smelter at Lommel (Belgium) demonstrated enhanced abilities to dissolve zinc and cadmium phosphates than those from non-polluted soils. Experiments with EcM associations of Scots pine with Paxillus involutus strains demonstrated that zinc phosphate dissolution, and zinc accumulation by roots and whole plants depended on the strain of mycobiont, its zinc tolerance and the phosphorus status of the soil matrix (M. Fomina et al. in GADD, 2006). Under P-rich conditions the EcM association with zinc-tolerant fungus isolated from zinc-polluted soils in Lommel (Belgium) showed the least zinc mobilization from zinc phosphate and the least zinc accumulation within roots and whole plant, whereas the highest zinc mobilization and accumulation was observed for non-mycorrhizal plants. In contrast, under P deficiency, an EcM association with the zinc-tolerant fungus demonstrated the highest zinc mobilization from zinc phosphate and zinc accumulation by the plants. In both Pdepleted and P–replete conditions EcM mycobionts efficiently assisted in P uptake, accumulating significantly more P in EcM roots than in non-mycorrhizal roots. This means that the biogeochemical activity of EcMs in the mycorrhizosphere is conditional and can be altered by additives, e.g. by addition of phosphate fertilizers, and by physicochemical conditions, and the nutritional status of the metal-polluted environment can shift the toxic metal transformation processes mediated by mycorrhizas from metal mobilization to immobilization and vice versa. Mycorrhizal fungi can also be involved in the formation of new phosphate minerals. It has been shown that ericoid and EcM fungi exposed to uranium solids (such as uranium oxides or chemically-unstable metallic depleted uranium derived from ammunition) transform them into thermodynamically stable uranyl phosphate minerals of the metaautunite group (Figure 18). As fungi are perfectly suited for biogeochemical activity, and they comprise the largest bulk biomass in soil, they inevitably comprise the dominant part of the microflora in http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (17 of 25)10/2/2010 11:16:14


toxic metal-polluted soils, and hence play a tremendous role in the establishment and survival of host plants as their partners in mycorrhizal associations. Indeed, fungalbased remediation approaches should always be considered in any attempts at phytoremediation or at any other remediation of metal-polluted soils. There have been many successful examples of revegetation of drastically disturbed mining areas using native trees, shrubs and grasses inoculated with specific EcM and AM species (e.g. Ohio Abandoned Minelands Program, US, 1982-1999) (M. Fomina & G.M. Gadd in ROBSON et al. 2007). Nodulated legumes can also be mycorrhizal, EcM and AM (SPRENT, 2001), and hence may have a "tripartite" symbiosis involving plant, bacterial and fungal partners (J.M. Barea et al. in WERNER and NEWTON, 2005) (Figure 20). There have even been sporadic reports of the colonization of legume nodules by AMs, but closer studies have revealed that this is unlikely in healthy functional nodules. Nevertheless, there is clearly a close association between the two types of symbioses (nodulation and mycorrhizal). Indeed, as AMs evolved much earlier than legumes, it is now assumed that all legumes have the capacity to form symbioses with them. The only known exception to this is the genus Lupinus, which, interestingly, has compensated for the lack of AM-forming ability, by producing cluster roots, which seem to have a Pscavenging function analogous to mycorrhizas. It is now generally accepted that the evolution of symbiotic nodulation in legumes "co-opted" the pre-existing mycorrhizal symbiosis in using many of the same signaling processes and mechanisms (SPRENT & JAMES, 2007). In contrast to AM, the occurrence of EcM in legumes is much more sporadic, being largely (but not exclusively) confined to trees in the Caesalpinioideae, and some of these can also be AM. Indeed, although it was originally thought that EcM and nodulation were mutually incompatible, especially as so many EcM Caesalpinioid legumes are non-nodulating, it is now recognized that this is not always the case, and that some nodulated legumes can also form symbioses with both AM and EcM.

Figure 19. Arbuscules (arrows) formed in a nodulated clover (Papilionoideae) root after inoculation with the arbuscular mycorrhiza (AM), Glomus clarum.



Figure 20. Growth of Mimosa scabrella (Mimosoideae) nodulated with rhizobia and infected with mycorrhizas after inoculation with a mixture consisting of the AM species Gigaspora margarita and Glomus clarum. M + R = AM + rhizobia, R = rhizobia only, TN = no inoculation + mineral N, T = no inoculation. It has long been recognized that tropical legumes, particularly trees, are greatly dependent on mycorrhizas, and that this is partly based on their role in enhancing nodulation and N-fixation. This role could be due to the fact that nodulation is an expensive process in terms of producing symbiotic nodules, and BNF itself is highly costly in energetic terms (ie. in terms of ATP; see earlier), and hence any process that can enhance nutrient uptake (particularly P) in the kind of nutrient-poor soils that legumes thrive in, will improve the legumes ability to devote resources to nodule formation and BNF. In return, nodulated mycorrhizal legumes may make a very substantial contribution to the N and P dynamics of the ecosystems in which they live. For example, nodulated EcM legumes make a very significant contribution to the Pcycle of African rainforests, and nutrient exchange between nodulated and nonnodulated plants via AM mycelial connections are likely to be important in other forests. Tripartite legume symbioses (eg. with Acacia and Mimosa spp.) have been used in bioremediation programs, particularly in Brazil, where extensive mine spoils have been recovered by using nodulated mycorrhizal legumes that can grow well even though the "soils" in such degraded areas contain extremely low levels of plantavailable nutrients (Figures 21 - 26).

Figure 21. Planting of legumes on a slope formed from the tailing of an iron mine in the state of Minas Gerais in Brazil.

Figure 22. Overview of nodulated mycorrhizal legumes growing on an iron mine tailing three months after planting.



Figure 23. Nitrogen-fixing mycorrhizal legume trees growing on a slope formed from a stone quarry waste.

Figure 24. Nitrogen-fixing mycorrhizal legume trees growing on a slope formed from a stone quarry waste.

Figure 25. Nitrogen-fixing mycorrhizal Piptadenia adiantoides (Mimosoideae) growing on a quarry slope in "soil" that is devoid of organic matter.

Figure 26. Young nodules (arrows) on Crotalaria (Papilionoideae) that have been grown on an iron mine tailing.

8. Conclusion Bacterial and fungal symbioses are an essential part of the ecosystem in all tropical forests, and most (indeed, probably all) plants will form at least one type of symbiosis, and many will form more than one. Mycorrhizal symbioses allow plants to exploit http://greenplanet.eolss.net/EolssLogn/mss/C20/E6-142/E6-142-TB/E6-142-TB-16/E6-142-TB-16-TXT.aspx (20 of 25)10/2/2010 11:16:14


scarce reserves of soluble P (and other minerals), whereas N-fixing nodulated plants can tap into the inexhaustible pool of atmospheric gaseous N2 and thereby make themselves independent of soil reserves of this most important plant nutrient. The interconnectedness of plants in tropical forests means that non-nodulated plants can also benefit from N derived from BNF, either directly through mycorrhizal mycelia that they share with nodulated plants, or indirectly via the uptake from the soil of soluble Ncontaining compounds that had originated from mineralized organic matter derived from N-fixing plants. However, although this interconnectedness has clearly benefited tropical forests, it also highlights the fragility of such ecosystems, such that if nodulated legumes, for example, were selectively logged, then an important component of the Ncycle would be removed to the detriment of the whole forest. We are only just beginning to understand the full diversity of bacterial and fungal symbioses, and recently there has been an explosion in information about them from molecular (ie. DNA-based) methods. Tropical forests and wetlands are clearly some of the main centers of diversity of both bacterial and fungal symbioses, and they are likely to be dependent on nodulated legumes to maintain a positive N-balance and on mycorrhizas for a positive P-balance. The roles of these symbioses, therefore, must be considered in any attempts to manage tropical forests/wetlands. In addition, nodulated mycorrhizal plants (legumes as well as actinorhizal plants) should be used increasingly as pioneer plants to help rejuvenate deforested areas, as well as areas that have been degraded through over-intensive agriculture (eg. overgrazed pastures), open cast mining and other industrial activities

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Glossary Actinorhizal plants

ATP Bioremediation

: Dicotyledonous plants that form a nodulating N-fixing symbiosis with a filamentous actinomycete called Frankia. They are in 8 families (Betulaceae, Casuarinaceae, Coriariaceae, Datiscaceae, Elaeagnaceae, Myricaceae, Rhamnaceae, Rosaceae), and most are woody temperate species. : Adenosine Tri-Phosphate. : A process by which polluted and/or degraded lands are returned to their original states by the use of plants, fungi and/or bacteria.



Leghemoglobin

Legume

Mycorrhiza

Nitrogenase

Nodule

: An oxygen-carrying hemo-protein found in legume nodules. It is essential for the functioning of the symbiosis as it rapidly conveys low concentrations of oxygen to support the high respiratory needs of the N-fixing bacteria, but at a concentration sufficiently low not to inactivate the oxygen-sensitive enzyme, nitrogenase. : Organism in the higher plant dicotyledonous family Leguminosae (Fabaceae). This family is now divided into 3 sub-families, the Caesalpinioideae, Mimosoideae, Papilionoideae. One of the main distinguishing features of legumes is that many of them (but not all) can form nodules on their roots (and sometimes stems) that house nitrogen-fixing (N-fixing) symbioses with bacteria called rhizobia. They are very widely distributed and are present in all continents and terrestrial ecosystems, except for Antarctica. They range in growth habit from herbs through to large trees (mainly in the tropics and sub-tropics). : Symbiotic association between a fungus and the roots of a plant.. This symbiosis is formed in the vast majority of terrestrial plants by the fungal partner colonizing their roots either extracellularly (ecto-mycorrhizas or EcM) or intracellularly (arbuscular mycorrhizas or AM). The mycorrhizas function by greatly increasing the area of absorption of the plant roots, and assist greatly in the uptake of essential minerals, particularly phosphorus (P). : Enzyme complex responsible for “fixing” atmospheric nitrogen (dinitrogen) consisting of a molybdenum-iron (MoFe) protein and an iron (Fe) protein. It uses several molecules of ATP (typically 16) to reduce just one molecule of dinitrogen, so it is highly expensive energetically. It is also highly sensitive to oxygen and can be easily denatured unless the organism (free-living or symbiotic) takes steps to protect it (eg. by living inside a root nodule). : A root (and sometimes stem)-borne symbiotic organ on the roots of legumes and Actinorhizal plants that allow N-fixing bacteria to fix atmospheric dinitrogen (N2) into soluble, ammonium-based compounds that can be used by the host plant for all its nutritional N-requirements. The nodule acts as an interface between the 2 symbionts and allows for the supply of sugars from the plant to the bacteria in exchange for fixed N (ammonium). It also provides a low oxygen environment, which is essential for the expression and function of the bacterial enzyme nitrogenase.



Rhizobia

Symbiosis

: Term which used to refer only to bacteria in the genus Rhizobium, but is now a generic term for all soil bacteria that form symbiotic N-fixing nodules on legumes (and the non-legume Parasponia). : Narrowly defined as a mutually beneficial relationship between two different organisms.

Bibliography Allen, O.N., and Allen, E.K. (1981) The Leguminosae. A Source Book of Characteristics, Uses and Nodulation. The University of Wisconsin Press, Madison Wisconsin. 812 p. [Seminal book on nodulation in legumes, and still highly relevant]. Cardoso, I.M., Kuyper, T.W. (2006). Mycorrhizas and tropical soil fertility. Agriculture, Ecosystems and Environment 116: 72-84. [Up to date review on the current state of mycorrhizal research in the tropics]. Dilworth, M.J., James, E.K., Sprent, J.I., and Newton, W.E. (eds). (2008) Nitrogen-fixing legume symbioses. Volume 7 of Nitrogen Fixation: Origins, Applications and Research Progress. Springer, Dordrecht. 402 p. [Arguably the most comprehensive set of text books currently available on nitrogen fixation comprising 7 volumes covering all aspects of the subject in great depth]. Gadd, G.M. (ed.). (2006) Fungi in Biogeochemical Cycles. Cambridge University Press, Cambridge, p. 236-266. [Fundamental text book covering the roles that fungi play in altering their environments and how this impacts upon the larger scale in biogeochemical terms]. Loureiro, M.F., James, E.K., and Franco, A.A. (1998). Nitrogen fixation by legumes in flooded regions. Oecologia Brasiliensis 4: 191-219. [A review that covers most aspects of legume N fixation research in the flooded regions of the tropics]. Palacios, R., and Newton, W.E. (2005). Genomes and Genomics of Nitrogen-fixing Organisms. Volume 3 of Nitrogen Fixation: Origins, Applications and Research Progress. Springer, Dordrecht. 246 p. [Arguably the most comprehensive set of text books currently available on nitrogen fixation comprising 7 volumes covering all aspects of the subject in great depth]. Pawlowski, K., and Newton, W.E. (2008). Nitrogen-fixing Actinorhizal Symbioses. Volume 6 of Nitrogen Fixation: Origins, Applications and Research Progress. Springer, Dordrecht. 310 p. [Arguably the most comprehensive set of text books currently available on nitrogen fixation comprising 7 volumes covering all aspects of the subject in great depth]. Pott, V.J., and Pott, A. (2000). Plantas aquaticas do Pantanal. Embrapa, Brasilia. 400 p. [Beautifully illustrated book describing the aquatic plants that live in the largest wetland in the World]. Robson, G.D., Van West, P. and Gadd, G.M. (eds.). (2007). Exploitation of Fungi. Cambridge



University Press, Cambridge, p. 236-254. [Several review papers covering all current aspects of research into beneficial fungi, including mycorrhizas]. Smith, S.E., and Read, D.J. (1997) Mycorrhizal Symbiosis. Academic Press, London, UK. [Excellent fundamental text book on mycorrhizas]. Sprent, J.I. (2001). Nodulation in legumes. London, UK: Royal Botanic Gardens, Kew. [The most important single volume of information about nodulation in legumes currently available. Comprehensive and easy to read]. Sprent, J.I., and James, E.K. (2007) Legume evolution: Where do nodules and mycorrhizas fit in? Plant Physiology 144, 575-581. [Recent discussion paper examining the roles of the mycorrhizal and rhizobial symbioses in the evolution of legumes]. Werner D., and Newton, W.E. (eds). (2005). Nitrogen fixation in agriculture, forestry, ecology, and the environment. Volume 4 of Nitrogen Fixation: Origins, Applications and Research Progress. Springer, Dordrecht. 347 p. [Arguably the most comprehensive set of text books currently available on nitrogen fixation comprising 7 volumes covering all aspects of the subject in great depth].

Biographical Sketches Euan K. James is a consultant for the Scottish Crop Research Institute. His present research interests are focused on nodulation of legumes, particularly those from seasonally-dry tropical savannahs and forests. Sérgio Miana de Faria is a researcher at Embrapa-Agrobiologia, km47, Seropédica, Rio de Janeiro, Brazil. His present research interests are plant-microbe interactions with an emphasis on the use of symbioses in the rehabilitation of degraded land. Marina Fomina is a researcher at the University of Dundee. Her current research interests are the role of fungal communities and mycorrhizas in geochemical transformations of metals and minerals. To cite this chapter E. K. James, M. Fomina, S. M. de Faria, (2009/Rev.2009), SYMBIOTIC BACTERIA AND FUNGI, in International Commission on Tropical Biology and Natural Resources, [Eds. Kleber Del Claro, Paulo S. Oliveira, Victor Rico-Gray, Ana Angelica Almeida Barbosa, Arturo Bonet, Fabio Rubio Scarano, Francisco Jose Morales Garzon, Gloria Carrion Villarnovo, Lisias Coelho, Marcus Vinicius Sampaio, Mauricio Quesada, Molly R.Morris, Nelson Ramirez, Oswaldo Marcal Junior, Regina Helena Ferraz Macedo, Robert J.Marquis, Rogerio Parentoni Martins, Silvio Carlos Rodrigues, Ulrich Luttge], in Encyclopedia of Life Support Systems (EOLSS), Developed under the Auspices of the UNESCO, Eolss Publishers, Oxford,UK, [http://www.eolss.net] [Retrieved February 10, 2010]



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