Baldani Et Al, 1996 - Recent Advances in Bnf With Non-legume Plants

Pergmon Soit Bidt. Biochem. Vol. 29, No. 516, pp. 911-922, 1997

c 1997 Elsevier Science Ltd. Al1 rights reserved Printed in Great Britain

PII: S0038-0717(%)0021&0 0038-0717/97 $17.00 + 0.00

RECENT ADVANCES IN BNF WITH NON-LEGUME PLANTS

JOS 1. BALDANI,* LEONARDO CARUSO, VERA L. D. BALDANI, SILVIA R. GOE and JOHANNA DBEREINER

EMBRAPA-Centro Nacional de Pesquisa de Agrobiologia (CNPAB), 23851-970, Seropdica, RJ, Brazil. and UFRRJ, IF, DCA, 23850-970, Seropdica, RJ, Brazil.

( Accepted 5 July 1996 )

Summary-It is now wel1 accepted that nitrogen-fxing bacteria colonising graminaceous plants can be grouped into three categories: 1, rhizosphere organisms; 2, facultative endophytes and 3, obligate endophytes. In the first category are included al1 species that colonise the root surface such as Arotobacter pasputi, Beijerinckia spp. Facultative endophytes are those nitrogen-fixing bacteria that can colonise the surface and interior of the roots principally the four species of Azospirihum, except Azospirillum hato- praeferuns. The third category is constituted mainly by diazotrophs isolated more recently such as Acet- obacter diazotrophicus, Herbaspirillum spp and Azoarcus spp which are able to colonise the root interior and aerial tissues of the plan& Although most of the studies related to nitrogen fixing bacteria have been concentrated on Azospirillum spp, it is the obligate endophytes, isolated more recently, that have attracted the attention of scientists working in this field. The ability to colonise the entire plant interior and locate themselves within niches protected from oxygen or other factors make them the most prom- ising group of diazotrophs associated with graminaceous and other non-leguminous plants. In this review we compare these three groups of nitrogen-fixing bacteria, their interaction with the host plants and discuss the potential of their use in agricu1ture.O 1997 Elsevier Science Ltd

INTRODIJCTION

It has now been almost 40 years since Dbereiner and Ruschel (1958) isolated the nitrogen-fixing bacterium named Beijerinckiu flurninensis from the rhizosphere of sugarcane plants grown in tropical soils and demonstrated the potential of diazotrophs to associate with graminaceous plants. However, it was only after the rediscovery of the genus Azospirillum by Dbereiner and Day (1975) that scientists in the world became interested in diazotrophic bacteria associated with graminaceous plants. During the last two decades many new nitrogen-fixing bacteria have been isolated and identified, including species of the genera Azospirillum, Herbaspirillum, Acetobacter and Azonrcus. The greater part of these diazotrophs have been isolated from tropical regions, especially in Brazil, and they have been the main source for groups in the world working with associative diazotrophs. Other plant-associated nitrogen-fixing bacteria have been identified, but probably because of their low number, or restricted occurrence, they are not wel1 explored. The interest in the association of diazotrophs with graminaceous plants reinforces the importante of the biological nitrogen fixation process to sustainable agriculture systems in Brazil where low amounts of nitrogen fertilisers have always been applied (Dbereiner, 1995).

Author for correspondence: Fax: 55 021 682-1230.

An analysis of the studies carried out on the interactions of diazotrophic bacteria with graminaceous plants over the last 40 years has shown a clear change in the way scientists have focused on these interactions. Initially, most of the studies were concentrated on bacteria isolated from soil or rhizosphere soil. The best example is Azotobacter puspali, a bacterium that colonises specifically the rhizosphere of Puspalum notatum (Dbereiner, 1966). Later, came the era of Azospirillum, nitrogen-fixing bacteria able to colonise either the rhizosphere or the root interior (here called facultative endophytes) of several forage grasses and cereals (Dbereiner and Baldani, 1982). Five species have been identified so far: A. Iipoferum, A. brasilense, A. amazonense, A. halopraeferans and A. irakense (Tarrand et Magalhaes et al., 1983; Reinhold et al., 1987; Khammas et al., 1989). In the last decade, new nitrogen-fixing genera were identified and because of their occurrence mainly within plant tissues, they have been called endophytes (obligate) instead of endorhizosphere-associated bacteria, a term used until recently for the root interior (Dbereiner, 1992a). Among the obligate endophytic diazotrophs are Acetobacter diuzotrophicus (Cavalcante and Dbereiner, 1988), Azoarcus spp (Reinhold-Hurek et al., 1993), Herbaspirillum seropedicae (Baldani et al., 1986a), the recently described species Herbaspirillum rubrisubalbicans (Gillis et al., 1991;

911

912 Jos 1. Baldani ef al.

Baldani et al., 1996) and the partially characterised Burkholderia sp. (Hartmann et al., 1995b; Baldani et al., unpublished). Very recently many of these diazotrophs (facultative and obligate) were isolated from palm oil trees (Ferreira et al., 1995), fruit (Weber et al., 1995) and toffee plants (Jimnez- Salgado et al., 1995) and therefore extending the endophytic colonisation of diazotrophic bacteria to plants other than graminaceous.

RHIZOSPHERE DIAZOTROPHS

Rhizosphere diazotrophs were mainly isolated in the early sixties and their biological nitrogen fixation (BNF) contribution to the sustainable agriculture system is stil1 unknown. There is a debate about the actual role of these bacteria in the association with graminaceous plants. Much evidente has pointed towards the role of phytohormones (Zimmer et al., 1988), and because of their ability to interfere with the root morphology and growth of the plant they have also been considered plant- growth promoting rhizobacteria (PGPR) (Bashan et al., 1993).

At least in one case, however, there is strong evi- dence of a rhizosphere nitrogen-fixing bacterium contributing to the nitrogen accumulated in the plant (Boddey et al., 1983). Using the N isotope dilution technique the authors demonstrated that about 20 kg ha-y- of the N accumulated in Paspalum notatum CV. batatais was derived from BNF. An important aspect of this association is the strict specificity of this plant with the rhizosphere diazotroph named Azotobacter paspali (Dbereiner, 1966). A survey on the occurrence of this diazotroph among several ecotypes or varieties of Paspalum and other grasses showed the presence of A. puspali mainly in the rhizosphere of the ecotype P. notatum CV. batatais (93% of 247 samples tested), rarely associated with other Paspalum spp and none in other plants. The specificity makes this association unique and could be compared with the endophytic association of Acetobacter diazotrophicus with sugar cane (Dbereiner, 1992b). The compounds involved in the plant-bacteria interaction are stil1 unknown, although one could speculate about the role of molecular signals similar to those observed in the rhizobia-legume interaction. A complete understanding of this association could open new avenues to manipulate other associations, such as the facultative endophytic association, so that their maximum potential of nitrogen fixation could be explored.

Another rhizosphere diazotroph that seems to behave quite differently from the common rhizosphere diazotrophs is Beijerinckia, principally B. indica and B. fluminensis found mainly in association with sugarcane plants grown in tropical climates (Dbereiner, 1959). Although no biological

nitrogen fixation contribution by these diazotrophs has been demonstrated, the release of compounds by the sugar cane plants, regulating the colonisation of the root as we11 as establishing the bacteria limiting area around the plant, led US to speculate about the importante of Beijerinckia spp in association with sugar cane. It has been shown that these bacteria preferentially colonise the rhizoplane and rhizosphere of sugar cane plants as do other rhizosphere diazotrophs, and that the exudates, mainly sugar compounds, are involved in the association because the bacteria are found principally in areas where these compounds are released or run off from the leaves (Dbereiner and Alvahydo, 1959). Nothing is known about the interaction of rhizosphere diazotrophs like Beijerinckia spp with obligate endophytes like Acetobacter diazotrophicus that appear to colonise sugar cane plants exclu- sively. It is possible that competition for these compounds, or the amount released by the plant, may account for some of the variability in biological nitrogen fixation observed among sugar cane varieties.

More recently, a very specific rhizosphere association was also demonstrated between Azospirillum halopraeferans and kallar grass plants grown in saline-sodic soil in Pakistan (Reinhold et al., 1987). Attempts to isolate this bacterium from grasses grown in saline regions of the Brazilian toast were unsuccessful (Reinhold et al., 1989). However, no survey on the occurrence of this rhizosphere diazotroph has yet been carried out in other countries.

ENDOPHYTIC DIAZOTROPHS

The term endophyte was coined some years ago to refer to root interior colonisation of plants by micro-organisms (bacteria and fungi) that usually do not cause damage to the host and live most of their life inside of the plant tissue without eliciting any pathogenic symptoms (Pereira, 1995). Later, the definition was extended to bacteria and fungi that colonise the root interior and promote benefits to the plant (Kloepper and Beauchamp, 1992). Due to the ability of some diazotrophs to colonise primar- ily the root interior of graminaceous plants, to survive very poorly in soil and to fix nitrogen in association with these plants, the term endophyte was also introduced into this area by Dbereiner (1992a, 1992b) and became widespread among scientists interested in nitrogen-fixing bacteria. Splitting the term endophyte into facultative and obligate was suggested to distinguish, respectively, strains that are able to colonise both the surface and root interior and to survive we11 in soil from those that do not survive we11 in soil but colonise the root interior and aerial parts. The ecological aspects used to distinguish these two groups as we11

BNF with non-legume plants 913

as the rhizosphere diazotrophs Table 1.

are presented in Table 2. Occurrence of faculatative and obligate endophytic diazotrophs in non-legurne vlants

Facultative endophytic diazotrophs

This group of diazotrophs, composed mostly of Azospirillum species, can be considered the starting point of most ongoing biological nitrogen fixation programs with non-legume plants worldwide. Although Azospirillum hpoferum (Tarrand et al., 1978) was the first species of the genus to be isolated, it is A. brasilense among al1 of the five known species that is the best characterised diazotroph at the physiological and molecular level. Azospirillum spp have been found associated with several cereals and forage grasses grown in temperate and tropical climates (Dbereiner and Baldani, 1982; Baldani, 1984; Germida, 1986). However, not al1 species have been demonstrated to colonise graminaceous plants grown al1 over the world (Table 2). The species A. amazonense (Magalhes et al., 1983), for example, has been isolated only from cereals, forage grasses and palm trees collected in Brazil (Baldani, 1984; Magalhes and Dbereiner, 1984). The species A. irakense, described more recently, has been isolated from roots of rite plants grown in the subtropical region in Iraq (Khammas et al., 1989). NO other report on the occurrence of this diazotroph has been published as yet.

Among the several physiological characteristics used to distinguish the Azospirillum species, there are a few that may play a role in the association with the plants. Both A. lipoferum and A. brasilense utilise malate as a carbon source, but only the for- mer is able to use glucose. Besides the ability to fix nitrogen both species are able to produce phytohormones like IAA as demonstrated by the presence of the IAA biosynthesis genes (vande Broek and Vanderleyden, 1995). The authors also showed the presence of these genes in A. amazonense but not in A. irakense. The ability to utilise sucrose is the main characteristic of A. amazonense and A. irakense species, although only the first is able to grow in a

Table 1. Ecological aspezts to differentiate rhizosphere and endophytic diazotrophs associated with non-legume plants

Aspects Rhizosphere Endophytes

Facultative Obligate

Survival in soil Natura1 Good Good Vely poor Sterile Good Good POOI

Colonization Root surface Yes Yes NO Root interior NO Yes Yes Aerial parts NO Yesjno Yes

More Host plant Various/restricted Various restrictedb

Arotobacter paspoli/Paspolum notatum CV. batatais; A. halopraeferans/Kallar grass.

b Acetobacter diazotrophicus/sugarane, weet potato and Pennisetum; Azoarcus/Kallar grass.

Diazotroph Plant Plant parts

A. brasilense

A. lipoferum

A. amazoneme

A. irakense

Forage grasses Sugar cane

Tuber plants Palm trees

Cereals Sugar cane Palm trees

Rite Obligates

H. seropedicae Cereals

H. rubrisubalbicans

A. diazotrophicus

Aroarcus spp Burkholderia spp

Faculatatives Cereals

Forage grasses Sugar cane Palm trees

Cereals

Sugar cane Forage grasses

Palm trees Sugar cane

Rite Sugar cane Pennisetum

Sweet potato Kallar grass

Cereals Sugar cane

Tuber plants Palm trees

Roots, sterns, seeds Roots, sterns

Roots, sterns, leaves Roots, sterns, fruits Roots, sterns, seeds,

xylem sap Roots, leaves

Roots, sterns, leaves Tubers, roots

Roots, sterns, fruits Roots, sterns, seeds

Roots, sterns Roots, sterns, fruits

Roots

Roots, sterns, leaves, seeds

Roots, sterns Roots, sterns, leaves

Roots, sterns Roots, sterns, leaves Roots, sterns, leaves Roots, sterns, leaves

Roots, sterns Roots, sterns, tubers Roots, base of sterns Roots, sterns, leaves Roots, sterns, leaves

Roots, tubers Roots, sterns

After Dbereiner et al. (1994).

wide range of pH. In addition, A. irakense hydroly- ses pectin and tolerates high concentrations of salts (3%). The knowledge that many Azospirillum strains dissimilate nitrite to nitrous oxide and dinitrogen based on physiological tests, was recently confrmed by the detection of the N20 reductase gene involved in the denitrification with a probe constructed from the enzymes present in Pseudomonas stutzeri (Bothe et al., 1994). A signal of about 20 kb was observed in A. lipoferum and A. brasilense but not in A. amazonense and A. irakense.

Due to their ability to colonise the root surface and interior of many cereals and forage grasses, the first report showing the presence of Azospirillum in cells of the cortex, in intercellular spaces between the cortex and endodermis and in the xylem cells of maize roots by applying the tetrazolium reduction staining technique (Patriquin and Dbereiner, 1978) was accepted with some skepticism, because the for- masan crystals formed could be produced by any type of reducing agent. Later on, using a very specific method of root sterilisation, Baldani (1984) and Baldani et al. (1986b) showed that certain strains of Azospirillum spp in fact colonise the root interior and that there are sites of colonisation by the bacteria along the root. Afterwards, the same group working on the colonisation process of wheat plants by Azospirillum spp demonstrated that a homologous strain A. brasilense Sp245, isolated

914 Jos 1. Baldani et al.

from surface sterilised roots of wheat, colonised and established itself inside the roots, whereas strains considered heterologous like A. brasilense Sp7 were unable to colonise the root interior as evaluated by the Most Probable Number counting of these strains in N-free semisolid NFb medium after surface sterilisation of the roots (Baldani et al., 1987). In addition, it was shown that homologous strains contributed much more to the nitrogen incorporated in the plant. Recently, studies with strain-specific monoclonal antibodies confirmed the ability of strain Sp245 but not Sp7 to colonise the root interior of wheat plants (Schloter et al., 1994). Identical results were obtained using a more sophis- ticated approach, TRITC-labelled species-specific oligonucleotide probe (BRA 18bl6b) for A. brasilense coupled with confocal laser scanning microscopy (Hartmann et al., 1995a). The methodology clearly visualised an in-situ interior colonisation of wheat roots by strain Sp 245 whereas strains Sp7 or strain Wa3, an isolate from wheat rhizosphere, only colonised the root surface. Also quite recently, vande Broek et al. (1993) study- ing the colonisation of wheat roots under axenic conditions by strain Sp245 expressing the gusA gene (GUS reporter system) showed that the bacteria initially concentrate in the root hair zones and at sites of lateral root emergence and that the proliferation to other parts of the root is dependent on the status of the nitrogen and carbon source present in the solution.

Although many ecological and physiological properties of Azospirillum are already known, there is stil1 a lack of information on the mechanisms involved in the plant-bacteria interaction. Studies of this interaction at the molecular leve1 have not advanced rapidly, despite the availability of tech- niques, probably because of the difficulties in isolation of the genes directly involved in the processes, and because of the absente of an easily detectable plant phenotype after inoculation with the mutants strains (see review of vande Broek and Vanderleyden, 1995). Nevertheless, many genes homologous to rhizobia genes already known to be involved in the legume symbiosis have been detected in Azospirillum using indirect approaches. Homologies to the Rhizobium meliloti genes essen- tial for nodulation in alfalfa such as nod DABCIJ and nodQPGEFG with A. brasilense and A. lipoferum (Fogher et al., 1985) as wel1 as to the chvA and chvB genes involved in the root attachment of A. tumefaciens (Waelkens et al., 1987) have been demonstrated. More recently, Raina et al. (1995) confirmed the presence of the chvB homologous gene in A. brasilense Sp7 and demonstrated that this lotus has a good homology to the ndvB gene of Rhizobium meliloti. Loei exoB and exoC that comp- lement R. meliloti exoB and exoC mutants have also been identified in A. brasilense (Michiels et al.,

1988), but a mutation in the exoC lotus in strain Sp7, affecting the EPS biosynthesis, suggests that, at least in this case, this lotus does not play a role in the root attachment capacity of this strain (Michiels et al., 1991). In contrast, recent results with a mutant of strain Sp7 impaired in exopolysaccharide production and hence the flocculation process, suggest that exopolysaccharide is involved in the root colonisation of wheat (Katupitiya et al., 1995). The mutant showed a different pattern of colonisation (concentrated on crevices surrounding the site of lateral root emergence) and a superior ability to fix nitrogen in association with the plant. Similar to that observed for rhizobia species, some of these homologous genes observed in A. brasilense are located in the plasmid p90 (Croes et al., 1991). The authors also demonstrated the presence of three loei involved in motility of A. brasilense in solid (Mot1 and Mot2) and liquid (Mot3) medium in the plasmid ~90. In addition, the same group working with a mutant affected in the lotus Mot3, which lost both polar and lateral flagella, demonstrated that the polar flagellum could be involved in the first step of the root attachment (Croes et al., 1993; more detail in vande Broek and Vanderleyden, 1995). Recently, signal molecules were isolated from the gramineous plants and partially identified that might provide new insights on the plant-bacteria associations (van Bastelaere et al., 1995). Nevertheless, we need to keep in mind that other species of Azospirillum and other facultative diazotrophs also colonise graminaceous plants and therefore a much more complex interaction should be expected when seeds are sown in the field.

Although many inoculation experiments with Azospirillum spp have been carried out al1 over the world showing a positive effect of the bacteria on yield (Boddey and Dbereiner, 1995) there is stil1 a debate about the main mode of action by which facultative endophytic diazotrophs contribute to the nitrogen accumulated in the plants. Effects of plant- growth promoting substances (Zimmer et al., 1988) nitrogen fixation per se (I.E. Garcia de Salamone, unpubl. M.Sc. thesis, University of Buenos Aires, 1993) or the ability of the bacterial nitrate reductase to help in the incorporation of the nitrogen assimi- lated from soil by the plant (Boddey et al., 1986; Ferreira et al., 1987) have been demonstrated. Despite these different mechanisms exerted by facultative endophytic diazotrophs in association with graminaceous plants, increases in the range of 5 to 30% in yield have been observed in several inoculation experiments with Azospirillum (Baldani et al., 1983; Okon and Labandera-Gonzalez, 1994).

Since Azospirillum spp do not excrete ammonium during the process of nitrogen fixation in culture, several approaches have been developed to provide this characteristic to Azospirillum envisaging improvement of the association. Mutants able to


excrete ammonium have been obtained from A. brasilense Sp7 using EDA mutagenesis (Machado et al., 1991) but no effect on the plant was determined. Almost at the same time, Christiansen-Weniger and van Veen (1991) showed that an ammonium excreting mutant of A. brasilense (Wa3) promoted better growth of the wheat plant as compared with the wild-type. The same authors, using another ammonium excreting mutant (strain C3) tested with the 15Nz gas i o p s to e dilution technique, concluded that the strain was able to transfer the nitrogen fixed directly to the maize plant and that this amount transferred was increased in para-nodulat- ing plants obtained by treatment with 2,4D (Christiansen-Weniger, 1994). Responses of graminaceous plants to inoculation with facultative endophytic diazotrophs like Azospirillum should always include the interaction with other diazotrophs as wel1 as additional characteristics such as excretion of ammonium or IAA production.

Obligate endophytic diazotrophs

This group includes Acetobacter diazotrophicus, a nitrogen-fixing bacterium clustered in the alpha subclass of the Proteobacteria and Azoarcus spp, Herbaspirillum seropedicae, H. rubrisubalbicans and a partially identified Burkholderia sp.; these are clustered in the beta subclass of the Proteobacteria.

Acetobacter diazotrophicus

Studies on the occurrence of this diazotroph have shown that it has restricted host range (Dbereiner, 1992b). A. diazotrophicus, has been found mainly associated with sugar-rich plants such as sugarcane, sweet potato and Cameroon grass, al1 of which pro- pagate vegetatively (Table 2). It colonises roots, sterns and leaves of sugar cane in numbers up to 106 cells g- fresh weight or more if an improved testing medium is used (Reis et al., 1994); in sweet potato it has been found in numbers up to 105 (Paula et al., 1993). Similar values als0 were observed in sugar cane grown in Australia by Li and MacRae (1992) using the ELISA method. In our laboratory with the ELISA technique it was possible to detect differences in the colonisation of various nitrogen-fixing varieties of sugar cane by A. diazotrophicus. However, no correlation was observed in the number of the bacteria and the capacity of the sugarcane varieties to obtain N from BNF (Silva et al., 1995). The restricted occurrence of A. diazotrophicus was confirmed by its complete absente in soil and in tissue of weed plants grown between rows in a sugar cane field (Dbereiner et al., 1988). These results also were confirmed using a species-specific primer designed to discriminate A. diazotrophicus from Acetobacter species and other diazotrophs (Reis et al., 1995). The best confirmation that this bacterium is an obligate endophyte comes from the results obtained with 10 month old

plants grown in a sugarcane field which had been planted by the micropropagation process normally used to produce disease-free plants. This bacterium could not be detected in plants at ages varying from 3 to 10 months (Reis Jr, unpubl. results). Other sup- port for this endophytic characteristic comes from recent results from our laboratory showing that A. diazotrophicus does not survive in the soil (Fig. 1). Using a lacZ-fusion it was observed that this bacterium cannot be detected 2 days after inoculation in natura1 soil, although it can survive longer (up to 10 days) in sterile soil (Caruso and Baldani, 1995).

The infection and colonisation processes of sugar cane by Acetobacter diazotrophicus have been exam- ined by James et al. (1994) and Reis Jr et al. (1995). On the root surface the bacteria were concentrated at the root tips and around lateral root junctions, and the bacteria infected the plants at these sites. Inside the roots, the bacteria were observed in apparently intact, enlarged epidermal cells, and also at the base of the stem within xylem vessels through which the bacteria appeared to migrate to the shoot tissues. The confirmation that A. diazotrophicus colonises the intercellular spaces of sugar cane stem parenchyma comes from the work of Dong et al. (1994) who showed the presence of the bacteria in numbers of 104 cells ml- of fluid inside the apo- plast. Despite the restricted host plant occurrence, a few isolates with many characteristics similar to Acetobacter were isolated recently from toffee plants in Mexico (Jimnez-Salgado et al., 1995).

The most common physiological characteristics of A. diazotrophicus are the high sucrose tolerante (10%) growth and nitrogen fixation at low pH (5.0 or less), chocolate colonies on potato agar medium with 10% sucrose, absente of nitrate reductase, nitrogen fixation not affected by high concentrations of NO; (25 mM) and partial inhibition by NH,+ especially at high sucrose concentrations (Teixeira et al., 1987; Boddey et al., 1991; Stephan et al., 1991). Another unique characteristic of this bacterium is the ability to excrete part of the fixed nitrogen into the medium as demonstrated by Cojho et al. (1993) using an amylolytic yeast to mimic the plant. More recently, Cruz et al. (1995) determined that ammonium was the product excreted by A. diazotrophicus under nitrogen fixation conditions.

Not much is known about the genetics of A. diuzotrophicus, as the bacterium was isolated quite recently. Nevertheless, it is known that some strains carry plasmids of sizes varying from 50 to 110 Mda and that the nif genes are located in the chromo- somes (Teixeira et al., 1994). It was also shown that this obligate diazotroph has a narrow genetic diver- sity (Caballero-Mellado and Martinez-Romero, 1994); n$HDK and nifA have already been sequenced (Sevilla et al., 1995; Teixeira, unpubl. results). Other genes like ntrBC, n$B, (Meletzus,

916 Jos 1. Baldam et ul

10

Q Sterile soil 8 0 Natura1 soil

z % OO 6 2

0 4 !!

2

0 0 2 7 15

Days after inoculation

Fig. 1. Survival of Acetobucter diuzotrophicus strain PAL5 in natura1 and sterile soil with moisture tension at -0.03 MPa and temperature of 32C. Asterisks indicate that the population had declined to less than the leve1 detected by the Most Probable Number (MPN) method

unpubl. results) and nifV and n!fE have also been detected in A. diuzotrophicus by complementation (Sevilla et ~1.. 1995).

Herbuspirillum seropedicue

The second obligate endophyte is Herbaspirillum seropedicae that has a wider host range specificity than A. diazotrophicus since it has been isolated from many graminaceous plants such as maize, sor- ghum, rite, sugar cane and forage grasses grown in Brazil (Table 2). Quite recently, it was isolated from sugar cane plants grown in South Africa (J. Thomson. pers commun.). It has also been isolated from other non-legume plants including oil palm trees (Ferreira et al., 1995). It has been found inside roots, sterns and leaves of these graminaceous plants (Dbereiner ef al., 1994) but not within leaves of sugar cane plants harvested from the field (Olivares et al.. 1996). Artificial inoculation of sugar cane leaves with H. seropedicue showed that the bacteria do not spread into the xylem vessels of the leaves but remain localised at the point of inoculation. The pattern of sugar cane root colonisation by H. seropedicae is quite similar to that observed for A. diazotrophicus, except that the attachment of the cells occurs al1 along the roots and that invasion of the roots occurs mostly at junctions of secondary roots where through the intercellular space it colonises the xylem. (F.L. Olivares, pers commun.). On the other hand, the colonisation of rite plants by a selected strain of H. seropedicue showed that the bacteria first colonise the epidermal cells ofthe root surface and then enter through the intercellular spaces derived from cells or secondary root emergence (Baldani et al., 1995).

The natura1 dissemination of H. seropedicue is not yet clear, although it seems to be transferred mainly through the seeds, as it can occasionally be isolated from seeds of cereals (Baldani et al., 1992).

In plants which are micropropagated vegetatively like sugarcane, it can be transmitted in the same way as observed for A. diazotrophicus. This was confirmed by the presence of the bacteria in sugar cane plants originating from the micropropagation process where the innermost apical meristems have not been extracted carefully (Olivares et UI., 1996). Like other endophytes, H. seropedicae does not survive wel1 in natura1 soil; it is less affected in sterile soil indicating that biotic factors interfere with the survival of this bacterium in natura1 soil. Nevertheless, the survival of H. seropedicue in both sterile and natura1 soils was better than that observed for A. diazotrophicus (Caruso and Baldani, 1995).

Herhaspirillum rubrisubulbicans

This obligate endophytic diazotroph was found recently among several strains of Pseudomonus rubrisubulbicans. a species considered a mild phyto- pathogenic agent causing mottle stripe disease in some susceptible varieties of sugarcane grown in countries other than Brazil. (Pimentel et al.. 1991). Based on its DNA:rRNA homology, and some physiological characteristics as wel1 as 15Nz gas incorporation, these strains were included in the genus Herbuspirillum (Gillis ef al., 1991; Baldani et al., 1996). Inoculation of H. rubrisubulbicuns by injection into leaves of the sugarcane variety B- 4362, susceptible to mottled stripe disease, produced the typical symptoms of the disease (Olivares et al., 1993). NO symptoms, however, were observed in the commercial Brazilian sugar cane varieties CB45-3, NA 56-79 or SP 70-1143 inoculated with this bacterium. Similarly, inoculation of these commercial varieties as wel1 as the susceptible variety B-4362, with a strain of H. seropedicae did not produce any red and white stripes symptomatic of the mottled stripe disease (Olivares et al., 1993). Studies on the colonisation of sugar cane by this endophyte, have concentrated mainly on the aerial tissue parts and have shown the presence of the bacterium within the meta- and protoxylem (Olivares et al., 1993). In the case of the variety B-4362, H. rubrisubulbicuns was found to completely black some of the xylem vessels and to colonise the intercellular spaces of the mesophyll cells. On the other hand, in the variety SP 70-1143, resistant to mottled stripe disease, the bacteria also colonise the xylem but form clusters of 10 to 20 cells encapsulated by membranes, probably of plant origin (Boddey, 1995). More recently, activity of nitrogenase was detected in the xylem vessels of sugar cane leaves infected with this obligate endophyte; nitrogenase was demonstrated with an antiserum raised against the FeMoCo subunit of nitrogenase (F.L. Olivares et al., unpubl. data). The pattern of colonisation of roots of sugarcane varieties showed an infection and colonisation process similar to that of the H. seropedicue. Preliminary


results on the colonisation of rite roots by H. rubrisubalbicans showed that the bacteria concentrate at the point of secondary root emergence and first colonise cells from the epidermis and hypodermis where they begin to multiply and colonise the upper part of the plant, probably through the xylem (Fig. 2).

Although it was initially thought that H. rubrisubalbicans would be restricted to sugarcane (Table 2), it was recently isolated from rite plants (V.L.D. Baldani, unpubl. results) and palm tree plants (Ferreira et al, 1995). Occurrence of H. rubrisubalbicans in rite plants may not be a surprise because inoculation of rite seedlings grown axenically with strain M4 increased nitrogen accumulated in the plant about 30% (Baldani et al., 1995).

Azoarcus spp

This obligate endophytic diazotroph, including the species A. indigens, A. communis and a few isolates that do not show high homology to these two species but are called Azoarcus sp. (Reinhold-Hurek et al., 1993), has been isolated from Kallar grass grown in saline-sodic soils in Pakistan. In contrast to Azospirillum halopraeferans, that has been found only on the surface of the roots of this plant (Reinhold et al., 1987), Azoarcus was repeatedly found inside Kallar grass roots or stem bases (Table 2). More recently, the authors developed genus-specific primers for Azoarcus which could differentiate strains of this genus from other nitrogen-fixing bacteria and suggested PCR amplification using the genus-specific primers to determine the presence of Azoarcus in plant tissues of Kallar grass (Hurek et al., 1993a). Like other endophytes already discussed, Azoarcus does not cause pathogenic reac- tions when inoculated into Kallar grass grown axenically (Hurek et al., 1994). The authors showed that strain BH72 is capable of invading roots of the original host as wel1 as rite plants, infecting the cortex region and penetrating the stele of the roots. Spreading of the bacteria into the plant shoot is probably by the colonisation of the xylem vessels where it could be detected by the use of specific antibodies or by its nitrogenase activity. Enzymes like cellulases, an exoglucanase and an endogluca- nase, already detected in Azoarcus, might contribute to the process of infection of this obligate endophyte into these plants (Hurek and Reinhold- Hurek, 1994; Reinhold-Hurek et al., 1994). A close interaction of this bacterium with a rhizosphere fun- gus was also demonstrated by these authors. They observed the induction of tubular arrays of internal membrane stacks in Azoarcus which they demonstrated to be functional membranes related to nitrogen fixation (confirmed by antiserum raised against nitrogenase), and they named these structures diazo- somes (Hurek er al., 1993b).

Burkholderiu spp

This genus was recently created to include strains belonging to the (Pseudomonas) rRNA complex including (P.) solanacearum and (P.) cepuciu (Yabuuchi et al., 1992). Quite recently, a new nitrogen-fixing bacterium was isolated from the rhizosphere of a rite plant grown in Vietnam, and because.of its high DNA:DNA, DNA:rDNA homology and phenotypic characteristics close to the genus Burkholderia, the name B. vietnamiensis was proposed (Tran Van et al., 1994). It cannot be concluded yet that this species is an endophyte, because it has been isolated only from rhizosphere macerates (roots plus adhering soil).

In a survey to determine the occurrence of nitrogen-fxing bacteria in rite plants grown in Brazil, Oliveira (unpubl. MSc thesis, Federal Rural University of Rio de Janeiro 1992) isolated a new group of bacteria on a N-free semi-solid medium containing glucose, oxalate and citrate. The bacteria were different from the other nitrogen-fixing bacteria known in our Centre and were designated E. This bacterium was also isolated at the same time from cassava plants (roots, tubers and sterns) in numbers quite similar to Azospirillum (Balota et UI., 1994). Using an improved N-free semi-solid medium called JMV (mannitol as a carbon source and pH 4.5 to 5.0), several isolates were obtained from roots, sterns and leaves of rite plants (Table 2) (V.L.D. Baldani, pers commun.). Morphological and physiological characteristics as wel1 as a partial sequencing of the 23s rRNA indicate that these strains as wel1 as isolates from cassava and sweet potato belong to the Burkholderia genus (Hartmann et al., 1995b) but do not share similarities that per- mit them to be included in the species B. vietnamiensis. It is interesting that a specific oligonucleotide probe generated to identify these rite isolates per- mitted the identification of another group of strains that had been isolated from sugar cane. This new group was also confirmed to belong to the genus Burkholderia by partial sequencing of the 16s rRNA of a strain Ppe8; its phenotypical and morphological characteristics suggest it may represent a new species or subspecies (Hartmann et al., 1995b). A oligonucleotide probe generated from the hyper- variable region of the 23s rRNA of the strain PPe8 confirmed these results and distinguished them from the other group (Hartmann et al., 1995b). More recently, strains of these two groups were also isolated from fruit plants (Weber et al., 1995). The role of these new endophytic nitrogen-fixing bacteria to an association of a plant and bacteria is not yet known, although they may exercise the same func- tions as the other endophytes.

Preliminary results from the infection and colonisation process of rite by Burkholderia sp. strains showed that the bacteria first colonise the root sur- faces and them enter the cells through the intercellu-

Fig. 2. Rite root Lissues inoculated with ffe~hu.cfJi,-i//rlr,l ~~h~rsd~o/l~i~ UI.\ straln M4. (21) Entrance of the bacteria through a wound on the root surface. Bar = 10 pm. (b) Detail of hacteria growing under the

loose epidermis (B). Bar ~ 10 pm.


Fig. 3. Detail of the root surface colonisation of rite by the Burkhoideriu sp. strain M209. Bar = 10 Pm

lar spaces caused by broken membranes (Fig. 3). needed to identify the limiting factors of the associ- The bacteria also can penetrate through crdcks of ation. Nif minus or other regulatory nif mutants as the epidermal cells caused by loss of surface cells we11 as mutants affected by molecular signal inter- and also at the points of secondary root emergence actions wil1 be very helpful in providing a better (Baldani et al., 1995). A massive number of bacteria understanding of associative endophytic bacteria. have been found inside rite roots without causing cel1 damage (V.L.D. Baldani and S.R. Goi, unpubl. results).

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Baldani Et Al, 1996 - Recent Advances in Bnf With Non-legume Plants