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.
N-free medium they show growth of a characteristic ‘pellicle’ – a balloon-like pattern of growth at the area below the surface where the concentration of O2is sufficiently low to allow N2-fixation.
Strains from the first three species ofAzospirillumhave been isolated from the rhizosphere of roots of all the major cereal crops in Africa and Latin America. These include maize, sorghum, wheat, rice and millet (reviewed in Döbereiner and Pedrosa, 1987).A. halopraeferanswas found associated with the roots of Kallar grass, a salt- tolerant grass grown in saline soils in Pakistan (Reinholdet al., 1987). Although azospirilla are primarily discussed in the context of cereal crops, there is also evidence concerning the colonization of non-cereal plants (Bashanet al., 1991).
MostAzospirillumstrains colonize the plant exterior but there are some that are endophytic in root tissue. The location of specific strains within plant tissues has been confirmed with the use of monoclonal antibodies (Schloteret al., 2000) and specific fluorescent rRNA probes (Assmuset al., 1995). This phenomenon appears to be strain- rather than species-specific and has been observed with a variety of Gramineae, including sugarcane.
Herbaspirillum
H. seropedicaewas the first diazotrophic endosymbiont identified. It could be isolated from rhizosphere soil and surface sterilized roots of maize, sorghum and rice, but not from uncropped soil (Baldaniet al., 1986a). It shares several characteristics in common withAzospirillum, including formation of pellicles in semi-solid media, but is differentiated from Azospirillum by being smaller in size with more than one flagellum, and DNA homology experiments have confirmed that it should be placed in a separate genus (Falket al., 1986). It appears to be primarily a root-inhabiting endophyte, having been isolated from the roots of 13 different graminaceous species, and has also been isolated from the stems and leaves of maize and rice and from the stems of sugarcane (James and Olivares, 1998). Curiously, it is able to fix N2in the presence of high concentrations of sucrose (up to 10%) but is unable to use sucrose as a carbon source, hence the benefit thatHerbaspirillumobtains by inhabiting plant tissues remains unclear. Indeed one theory suggested thatHerbaspirillumgrows on organic acids produced by other endosymbionts, such asAcetobacter diazotrophicus (James and Olivares, 1998).
Two further species ofHerbaspirillumhave been identified:H. rubrisubalbicans, also an N2 fixer; and a third, non-fixing species referred to only asHerbaspirillum
‘species 3’ (Baldaniet al., 1996). The latter is found primarily in clinical isolates and only very rarely in plants. H. rubrisubalbicans, formerly known as Pseudomonas rubrisubalbicans, is a mild plant pathogen of certain cultivars of sugarcane and sorghum. Both endophytic Herbaspirillum species are found in the xylem and H. rubrisubalbicanscan also be found in the adjacent apoplast (intercellular spaces) of host plants.H. seropedicae can produce mottle stripe disease on sorghum and Pennisetum(Pimentelet al., 1991). The pathways of infection are not certain, but it appears thatHerbaspirillumspecies infect the roots of host plants either from the rhizosphere or they are carried within the seed coat, entering the roots via cracks in the epidermis at lateral root junctions (Chapter 6). Entry into the xylem can also
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occur at these lateral root junctions where the endodermis surrounding the stele (the pericycle) is disrupted, allowing passage from the root cortex into the vascular tissue.
The bacteria can then be carried to the aerial parts of the plant in the transpiration stream, and it is possible that they may become incorporated into the seed, a common means of transmission of plant-associated bacteria (McInroy and Kloepper, 1995; James and Olivares, 1998).
Acetobacter
Acetobacteris a genus of Gram-negative microaerobic bacteria characterized by an ability to grow at low pH and to form acetic acid from ethanol. There is only one N2-fixing member of this genus known, which is hence calledA. diazotrophicus.A.
diazotrophicuswas isolated from sugarcane and was first thought to constitute a new bacterial genus, ‘Saccharobacter nitrocaptans’ (Cavalcante and Döbereiner, 1988), but further molecular analysis showed that it was anAcetobactersp. (Gilliset al., 1989).
Its host range is much more limited thanHerbaspirillum, being thus far isolated only from sugarcane, the grassPennisetum purpureumand sweet potato, all of which are very rich in either sugar or starch. Like Azospirillum and Herbaspirillum, A.
diazotrophicusis best grown in semi-solid medium, where it forms a pellicle. It can tolerate sucrose concentrations up to 30% and is capable of using sucrose as a carbon substrate, although it turns out that this is due to secretion of an enzyme that hydro- lyses the sucrose to glucose and fructose extracellularly (Alvares and Martinez-Drets, 1995).A. diazotrophicus does not contain nitrate reductase, and is able to fix N2
in the presence of levels of nitrate as high as 25 mM. This may mean that it can continue to fix N2in the host plant, even while the host is assimilating nitrate directly from the soil, thus potentially providing the host plant with two sources of N.
Within the plant,A. diazotrophicushas been observed both in the xylem and in the adjoining apoplast. Its presence in the xylem may seem unexpected, given the low sucrose levels available there, but it is thought that the low pO2in the xylem may favour this tissue as a location for diazotrophic bacteria (James and Olivares, 1998).
The main method of transmission of the bacteria from generation to generation of host plant is within the sets of sugarcane, due to the fact that vegetative propagation is the norm for cane. However, evidence also suggests thatA. diazotrophicus can infect plants by much the same means asHerbaspirillum(see above), including the possibility of being seed borne. An additional possible route of transmission forA.
diazotrophicusis apparently via sap-feeding insects, including mealy bugs and leaf hoppers, as the bacteria have been isolated from a number of members of both groups of insects.
Other heterotrophic N2-fixing bacteria that associate with plants
Several other groups of N2-fixing bacteria have been described in association with the roots of different crop plant species. Members of the genusAzotobacterare free-living bacteria with the remarkable ability to fix N2aerobically (Chapter 3). The two best- known species,A. chroococcumandA. vinelandii, have a rather strict requirement for neutral pH conditions and thus are not abundant in tropical soils except in a few near-neutral soils in the humid tropics (Döbereiner and Pedrosa, 1987). One species,
A. paspali, has been found in association with the roots of one specific ecotype of Paspalum notatum, a subtropical invasion grass (Döbereineret al., 1972), where the requirements for high pH appear to be satisfied specifically in the rhizosphere. A further species has been described:Azotobacter salinestris, which was isolated from saline soils in Canada and Egypt (Page and Shivprasad, 1991).
Studies of bacteria associated with the roots of Kallar grass (Leptochloa fusca) led to the discovery ofAzoarcus, of which two species have been described:A. indigensand A. communis(Reinhold-Hureket al., 1993).Azoarcusis found deep within the grass tissues, where it colonizes the vascular system and aerenchyma (Hureket al., 1993, 1994), and produces nitrogenase (Egeneret al., 1998; Reinhold-Hurek and Hurek, 1998).Azoarcusspecies also occur as endophytes in rice (Engelhardet al., 2000).
Members of the aerobic N2-fixing genusBeijerinckiaare more tolerant of low pH and are therefore more common in tropical soils, where acid conditions often prevail. They occur much less abundantly in temperate regions and this may be due either to the pH conditions or to other aspects of mineral nutrition provided by tropical soils (Becking, 1961a,b). They show characteristic slow growth on N-free agar media, with colonies appearing only after 8–10 days incubation. Four species are recognized:B. indicaandB. mobilis(Derx, 1950),B. derxii(Tchan, 1957) and B. fluminensis(Döbereiner and Ruschel, 1958). Both B. indicaandB. fluminensis have been found associated with the rhizosphere of sugarcane and other plants (Döbereiner and Pedrosa, 1987).
A further group of aerobic N2-fixing bacteria of possible significance in tropical soils is theDerxiaspecies, such asD. gummosa. These were first isolated from a soil in western India (Jensenet al., 1960) and have been found in soils throughout the tropics, but there is no evidence suggesting that they form specific associations with any plant roots. Moreover, although they are described as aerobic N2-fixing bacteria, in culture they do not fix N2until very large (20 mm diameter and 10 mm height) gelatinous colonies form, inside which the environment is presumably microaerobic.
Several species of truly microaerobic N2-fixing bacteria have also been described as associating with plant roots. These include other diazotrophic members of the Spirillaceae (the family that containsHerbaspirillum) which have been found associ- ated with roots, such asAquaspirillum fasciculus,A. perigrenumandCampylobacter nitrofigilis. The latter was isolated from roots ofSpartina alterniflorain a salt marsh in Canada, and is tolerant of 7% NaCl (McClunget al., 1983).
Strains that are classified asPseudomonasspecies are known to be very common rhizosphere organisms, and some strains assigned to this genus possess the ability to fix N2 (e.g. Ballyet al., 1983; Barraquioet al., 1983; Watanabeet al., 1987b;
Jenniet al., 1989). On the other hand, it is widely acknowledged that the genus Pseudomonasis a ‘dumping ground’ for aerobic, polarly flagellated, Gram-negative, rodlike bacteria of uncertain affinity (de Voset al., 1989; Young, 1992; Chanet al., 1994). Anyone who has tried to identify a new bacterial strain they have isolated may well have shared the experience of identifying it as a ‘Pseudomonas’ species. Clearly extreme caution has to be exercised before assigning an unknown bacterium to this genus. The reclassification of the plant pathogenPseudomonas rubrisubalbicans as Herbaspirillumpresents one example, as already discussed. Other N2-fixing bacteria
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isolated from cereal rhizospheres were identified as Pseudomonas cepaciabut have since been transferred to the genusBurkholderia, which contains a number of species not known to fix N2. Isolates from rice rhizospheres in Vietnam were named B.
vietnamiensis(Gilliset al., 1995), and a further group of isolates from Vertisols in Martinique have been named asB. carribensis(Achouaket al., 1999b). Strains ofB.
cepaciahave been used successfully in biocontrol of fungal ‘damping off’ with maize (Hebbaret al., 1998), but others are better known as causal agents of the debilitating human disease, cystic fibrosis. In fact some of the most highly transmissible patho- genic clones ofB. cepaciaare grouped by RFLP analysis of their 16S rRNA genes with B. vietnamiensis(Segonds et al., 1999). Given the severity of cystic fibrosis, use of Burkholderiaas crop inoculants seems unlikely to be worth the potential risks.
Some members of the Enterobacteriaceae also possess the ability to fix N2, the most notable example in the scientific literature beingKlebsiella pneumoniae.
N2-fixing bacteria reported as being Klebsiella or Enterobacter species have been isolated from the rhizosphere of rice (Ladhaet al., 1983), and an N2-fixing enteric bacterium, Rahnella aquatilis, has been isolated from the rhizosphere of rice and maize (Bergeet al., 1991), but, in general, members of theEnterobacteriaceaedo not appear to be of major significance in agriculture. Of 18 strains ofErwinia herbicola and 16 strains ofEnterobacter agglomeranstested, only four of the former and two of the latter were found to possess nitrogenase activity in an acetylene reduction assay (Papen and Werner, 1979). No species ofEscherichiaorSalmonellahave been shown to possess endogenous nitrogenase activity, althoughE. colistrains with the ability to fix N2 have been constructed by the transfer of the nif gene cluster from K.
pneumoniae(Dixon and Postgate, 1972).
There are very few reports of free-living Gram-positive N2-fixing bacteria found associated with plants. A new species ofBacillus,B. azotofixans, was isolated from the rhizosphere of several different grasses and was also found as a free-living species in the soil in Brazil (Seldinet al., 1984). Previously, N2-fixingBacillusstrains had only been isolated from soil, and were assigned to one of two species:B. polymyxa orB.
macerans(Witzet al., 1967). These three species were transferred to a new genus, Paenibacillus(Ash, 1993), together with other species in which N2-fixation has yet to be assessed (Achouaket al., 1999a).
Aside from these bacteria that have been isolated from the rhizosphere of plants, N2-fixing bacteria can also be found in the phyllosphere of many plants. In a review by Ruinen (1974), a compendium of N2-fixing phyllosphere bacteria included representatives of most of the genera listed above. As discussed earlier, even rhizobia can be found epiphytically. Large populations of stem- and root-nodulating bacteria were found on the leaves of host (Aeschynomenespecies orS. rostrata) and non-host plants (Adebayoet al., 1989; Robertsonet al., 1995).
This brings us to conclude this section with an important point. It might seem from the above, and from the accompanying literature, that Brazil is a country particularly favoured with rhizospheric diazotrophic bacteria, and that the only other major location for such bacteria is in rice paddies in the Philippines, in the same way as most rhizobial species seem to arise in China, Mexico or Senegal. It should be remembered that in fact these two locations are favoured with scientists who
have shown remarkable interest in and dedication to the detection of such micro- organisms, and that, yet again, it seems that we learn as much about the ecology of scientists as about the ecology of bacteria from perusing the literature.
Conclusions
The genera, species and strains of bacteria that are capable of N2-fixation are extraordinarily diverse. The primary groups are the symbiotic bacteria (rhizobia, Frankiaand symbiotic cyanobacteria), free-living cyanobacteria, and other free-living diazotrophs that are found in soil and in the rhizospheres of certain plants.
Advances in molecular taxonomy have provided a more rigorous means of classifying bacteria. However, this may say little about the practical competence of a bacterial strain to, say, fix N2or nodulate a specific host plant. Thus, while advances in bacterial taxonomy are of great importance in providing a true systematic description of bacterial species, variation on a fine scale within bacterial ‘species’
is such that no conclusions can ever be drawn about phenotypic properties without rigorous testing.
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