Azospirillum brasilense ATCC 29145t - Journal of Bacteriology

Vol. 169, No. 4

JOURNAL OF BACTERIOLOGY, Apr. 1987, p. 1670-1677

0021-9193/87/041670-08$02.00/0 Copyright © 1987, American Society for Microbiology

Cyst Production and Brown Pigment Formation in Aging Cultures of Azospirillum brasilense ATCC 29145t LAKSHMI SADASIVAN* AND CARLOS A. NEYRA Department of Biochemistry and Microbiology, New Jersey Agricultural Experiment Station, Cook College, Rutgers

University, New Brunswick, New Jersey 08903 Received 2 October 1986/Accepted 22 December 1986

Encystation in Azospinillum brasilense ATCC 29145 was observed by using routine laboratory staining and phase-contrast and electron microscopy. Encystment occurred in liquid and in solid or semisolid media containing fructose (8 mM) and KNO3 (0.5 mM). The encysted forms consisted of a central body filled with poly-p-hydroxybutyric acid granules, an electron-transparent intinelike region, and a thick outer layer. Enlarged giant encysted forms with multiple central bodies were also observed during the germination of a desiccated brown colony. Morphogenetically different forms in an aging culture could be resolved by sucrose density gradient centrifugation. The dense encysted forms along with numerous granules in a fibrillar network pelleted at 70% sucrose, while empty saclike envelopes along with vegetative cells and coccoid bodies peileted at 55% sucrose. Different media induced various degrees of pigmentation in A. brasilense ATCC 29145 after aging. The pigment possessed several of the properties reported for microbial melanins, including insolubility in water and organic solvents, solubility in cold and hot alkali, and bleaching in hydrogen peroxide. The UV absorption maxima of the alkali extract were at 280 and 310 nm. Electron micrographs of the brown pigment showed that it occurred as aggregated granules surrounding the encysting celis as well as being excreted into the medium in an aging culture. It is concluded that A. brasilense ATCC 29145 produces compounds that form a brown pigment similar to melanin and are expressed under the influence of certain cultural conditions conducive for encystment.

Azospirillum brasilense is known to fix nitrogen in culture media and in association with the roots of several grasses (3, 23, 28, 33, 50). This microorganism has been used in field inoculation studies over the last decade with relative success by many workers (19, 20, 31, 42, 45), and crop yield increases as a result of inoculation are also well documented (3, 10, 40, 42, 48). Nonetheless, the response to inoculation by treated plants is known to be highly unpredictable (1, 50). It is generally assumed that bacteria added to the rhizosphere will survive, multiply, and remain metabolically active to support nitrogen fixation. Survival of the bacteria is rarely monitored after inoculation in the field. These bacteria are known to be highly pleomorphic and to change their metabolic activities swiftly in the face of changing environmental conditions (5, 15, 37). In the soil these may include the availability of carbon and nitrogen sources, moisture content, and oxygen tension. Resistance to desiccation in these bacteria is achieved by encystation, a phenomenon in which the vegetative cells undergo certain morphological and biochemical changes to form resistant resting cysts, which enables them to survive deleterious physical conditions, particularly desiccation (38, 39). Cyst formation in Azobacter spp. has been well documented by morphogenetic, ultrastructural, and biochemical studies (17, 35, 43, 44, 51, 52). In Azospirillum spp., cysts are cells which have undergone morphological changes as observed under phase-contrast and scanning electron microscopy and found to be resistant to desiccation from a few hours to 1 month (24, 32). We have presented some morphological and ultrastructural details of

the encysting cells from a desiccated floc which was induced in a liquid culture medium containing low levels of fructose (8 mM) as the carbon source and KNO3 (0.5 mM) as the nitrogen source (37). Under these conditions, the vegetative cells lost motility, assumed an enlarged spherical form, and accumulated abundant poly-,-hydroxybutyric acid (PHB) granules and developed an outer undifferentiated layer (coat) of polysaccharides. We proposed that the phenomenon was related to encystation. In an attempt to recognize the cultural conditions for complete encystment as defined for Azobacter spp., we grew Azospirillum brasilense ATCC 29145 cells in the same medium containing fructose and nitrate but on solid agar. Growth under these cultural conditions produced brown-black pigmentation in a 1-week period, which intensified as the culture aged. Pigmentation thus far reported in azospirilla has been primarily related to the synthesis of carotenoids, which have been shown to protect the nitrogenase enzyme of A. brasilense and also other strains from oxidative damages (15, 29). The objectives of this paper are to define the culture conditions under which A. brasilense ATCC 29145 differentiates into cysts, to compare and present evidence that the PHB-rich encysted cells confer a survival advantage to A. brasilense ATCC 29145, and to report pigment (brown pigment) formation in azospirilla as a result of aging and partially characterize the pigment. (A preliminary report of this work has been presented [L. Sadasivan and C. A. Neyra, in W. Klingmuller, ed., Proc. Third Bayreuth Azospirillum Workshop, p. 230, 1985].) MATERIALS AND METHODS

Corresponding author. t New Jersey Agricultural Experiment Station publication D-01204-02-86. *

Bacterial strain and culture conditions. A. brasilense Sp7 was used for the encystation and pigment

no.

(ATCC 29145) 1670

VOL. 169, 1987

CYST PRODUCTION AND PIGMENT FORMATION IN A. BRASILENSE

formation studies. The bacterium was routinely subcultured biweekly on nutrient agar (Difco Laboratories, Detroit, Mich.) slants and maintained at 30°C. Minimal salts medium (MSM) as described previously (37) was used as the basic medium to induce encystation, with modifications to incorporate different carbon sources. Malate, fructose (at a concentration of 8 mM), and n-butanol (at 0.3% concentration) were added individually as carbon sources. Three different conditions were used: liquid, solid, and semisolid media (0.175% agar [Difco]). In liquid and solid media, the nitrogen source was 0.5 mM KNO3. A log-phase culture from nutrient broth (NB; Difco) was harvested by centrifugation at 5,000 rpm at 4°C and washed three times in 100 mM KPO4 buffer (pH 6.8) to give an OD60 of 0.8 when read in a spectrophotometer (model 240; Gilford). From this suspension, a loopful of bacteria was streaked on solid agar plates, and 0.1 ml was inoculated into 100 ml of semisolid nitrogen-free basal (NFb) medium (37) and 10 ml into 100 ml of liquid medium. The solid plates and the flasks with semisolid medium were kept stationary, while the liquid medium cultures were continuously shaken at 200 rpm in a gyrotory shaker (New Brunswick Scientific Co., Inc., New Brunswick, N.J.). All the cultures were incubated at 37°C. The cultures inoculated in NFb semisolid medium were incubated in two batches, one under light and the other in dark to see any effect of light on pigment formation. Phase-contrast and electron microscopy of encysted cells. A. brasilense cells grown in three conditions (solid, semisolid, and liquid) were desiccated at room temperature (28 ± 2°C) and observed for more than 6 months. The desiccating colonies from solid agar plates and pellicular growth from semisolid medium were periodically observed under a phasecontrast microscope either unstained or stained as described by Vela and Wyss (52). After desiccation, they were scraped and inoculated into a fresh NFb semisolid medium containing fructose (8 mM) as the carbon source, incubated at 35°C for 24 to 48 h, and allowed to germinate. The newly developing growth from the inoculum which was observed to contain various encysted forms with single and multiple cental bodies was detached and photographed under a Nikon phase-contrast microscope. Ultrastructural studies. The desiccated growth from all three conditions was fixed in 1% glutaraldehyde-0.2 M cacodylate buffer, pH 7.4, followed by 1% osmium tetroxide fixation for 10 h, and dehydrated in a series of graded ethyl alcohol solutions after staining in uranyl acetate or ruthenium red by the procedures of Cole and Popkin (11) and Cagle and Vela (9). The stained preparations were finally embedded in Spurr plastic as described by Cole and Popkin (11). Ultrathin sections were cut on an ultramicrotome and observed under a transmission electron microscope (Siemens model 1A Elmscope). Separation of various encysted forms by sucrose density gradients. Gradients of sucrose in water (55, 60, 65, and 70%, wt/wt) were prepared, and each concentration was layered carefully on top of the others with 2 ml of 70% sucrose at the bottom of a polypropylene tube. The desiccated pellicular growth from semisolid medium was removed, suspended in 100 mM KPO4 buffer (pH 6.8), and layered on top of the 55% sucrose gradient. The tubes were then centrifuged at 20°C for 4 h at 40,000 rpm in an ultracentrifuge (Beckman model L5-75B). The three bands resolved were individually withdrawn in a 1-ml hypodermic syringe from outside the polypropylene tube and then observed under the transmission

1671

electron microscope after negative staining with uranyl acetate.

Thermal resistance experiments. One milliliter of a suspension of cells from (i) 24-h-old NB-grown, highly motile vegetative cells lacking PHB granules, (ii) 24-h-old MSM liquid medium (supplemented with 8 mM malate and 5 mM NH4Cl)-grown cells containing mixed populations of highly motile vibrioid and nonmotile round forms with PHB granules, and (iii) 1-month-old MSM semisolid nitrogen-free medium-grown cells having developed a brown pigment in the pellicle were serially diluted in sterile distilled water. Dilutions of 105 and 106 of each of the above cultures were immersed in a series of water baths adjusted to temperatures ranging from 30 to 60°C. After 20 min, the tubes were removed and rapidly cooled before plating 100-pul samples on tryptic soy agar (TSA; Difco). Desiccation experiments. One milliliter of a suspension of the three different types of cells described above was serially diluted in sterile distilled water. One hundred microliters from dilutions of 104 and 105 were placed in sterile 1.5-ml Eppendorf microfuge tubes, and the tubes were in turn kept open in a sterile petri dish. The petri dishes with the tubes were then placed in an incubator at 37°C. At selected intervals, the dried cells from the microfuge tubes were washed with 100 ptl of sterile distilled water with vigorous agitation to remove them quantitatively, and their viability was determined by plating on TSA. Pigment induction studies and characterization of brown pigment. Cells from a 24-h-old NB-grown culture were washed twice in sterile saline, and then a loopful was inoculated into various solid and semisolid agar media such as nutrient agar and MSM containing malate or fructose as the carbon source. The media were also supplemented with various amino acids, such as 0.1% tyrosine, 0.1% phenylalanine, 0.1% cysteine, and 0.1% tyrosine plus cysteine, which are known to be the precursors of brown pigment (melanin) formation. Induction of pigment formation on TSA plates was achieved as follows. One loopful of cells was inoculated into 100 ml of NB medium in 250-ml flasks and incubated at 37°C for 1 to 4 weeks under constant shaking at 200 rpm on a gyrotory shaker (New Brunswick Scientific). Every week, 100-pu samples were withdrawn from the aging cultures and plated on TSA. The plates were then incubated at 30°C for 7 days. Colonies forming brown pigments were counted and compared with non-pigment-forming colonies. The solubility of the pigment formed in an NFb semisolid medium was tested in water (cold and hot), alcohol, acetone, benzene, ether, 1 N HCI, and 1 N NaOH or KOH by the method of Lingappa et al. (25). The absorption spectrum of the brown pigment soluble in cold and hot alkali was done by the method of Ivins and Holmes (18) with a Beckman DU 40 recording spectrophotometer on a range of wavelengths from 200 to 400 nm. The spectrum obtained was compared with spectra obtained from other brown pigments formed as a result of autooxidation of phenolic compounds (3,4-dihydroxyphenyl-

alanine, catechol, and melanin).

RESULTS A. brasilense ATCC 29145 cells grown in semisolid nitrogen-free malate medium were found to produce brown pigment in the aging pellicles. In nutrient agar plates, pigmentation was rarely seen, but when the aging cultures from NB were plated on TSA and incubated for 1 week or more,

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SADASIVAN AND NEYRA

J. BACTERIOL. TABLE 2. Desiccation resistance of A. brasilense ATCC 29145 Disiccation at 37'Ca (days)

NB

No. of viable cellsb NFb

N-free

0 2 4 6 8 10 12

4 x 1010 2.5 x 105 0 0 0 0 0

1.6 x 107 6 x 104 0 0 0 0 0

8 x 105 9 x 104 2.3x 104 2x104 7x104 8 x 104 5 x 104

a

Cells from 100 ,ul of suspension were first air dried at 30'C and then kept

at 37°C for specified periods.

b See Table 1, footnote b.

FIG. 1. Pigmented colonies of A. brasilense formed on NFb solid agar medium supplemented with 8 mM fructose plus 0.5 mM KNO3.

approximately 60 to 70% of the colonies in the plate developed pigmentation. Growth in media containing low levels of a carbon source such as 8 mM fructose or malate supplemented with a minimal quantity of nitrogen in the form of KNO3 (0.5 mM) also induced pigment production (Fig. 1). In a liquid medium with 0.5 mM KNO3 as the nitrogen source, no diffusible pigment was observed in the medium. However, when cultures were kept undisturbed for more than 1 month in liquid medium, a flocculant brown deposit of cells appeared adhering to the walls of the flasks at the surface of the medium. Under anaerobic conditions, no pigment formation was observed. In general, pigment production was more intense under microaerobic conditions than in aerobic conditions. Incubating the flasks in either light or dark had no effect on pigmentation. Thermal resistance of the actively motile vegetative cells from NB and NFb-fructose medium was compared with that of the largely nonmotile encysted forms from a 1-month-old browning culture in nitrogen-free semisolid medium (Table 1). Suspensions of nonmotile encysted forms exhibited greater tolerance to heat, with only 50% reduction in viable counts per 10°C rise in temperature, than the actively motile 24-h-old vegetative cells from NB- and NFb-fructose-grown cells, which had a 90% population decrease.

The viability after desiccation of the vegetative and encysted A. brasilense is presented in Table 2. Cells from NB and liquid minimal medium cultures could not survive desiccation for more than 2 days, while the PHB-rich browning cultures exhibited resistance up to 10 to 12 days of desiccation at 37°C. The PHB-rich cells with no pigmentation did not show resistance. Phase-contrast microscopy. The cells in the brown colonies or brown pellicle formed on a semisolid nitrogen-free medium were round cystlike cells highly refractile in phasecontrast microscopy (Fig. 2). The brown colony, after complete dessiccation for 6 months, was also found to germinate in a nitrogen-free semisolid malate agar medium (Fig. 3). The horseshoe-shaped residual coats appeared similar to those of Azotobacter vinelandii cyst germination (43). Encysted forms with multiple central bodies. Figure 4 illustrates the phase-contrast microscopic observation of the germination of desiccated encysted forms. At 48 h after transfer into a fructose-containing NFb semisolid medium, the encysted form enlarged and the central body began to multiply. Figure 4A shows a normal enlarged encysted form with a single central body and another encysted form in which the central body is undergoing division within a single outer coat. In Fig. 4B, C, and D, the increasing number of multiple central bodies is clear and ranges from 2 to 4 per giant form. A similar phenomenon of the formation of giant cysts or cysts with multiple central bodies has been reported for Azotobacter vinelandii (4, 9). Ultrastructural studies of the encysting cells. Figure 5 shows ultrathin sections of A. brasilense cells from a desiccating brown colony. Different morphogenetic forms of encystation were observed and photographed. Figure 5A

TABLE 1. Resistance of A. brasilense ATCC 29145 to thermal destructiona No. of viable cellsb NFb

Temp

(OC) 30 40 50 60

NB

6 6

x x

0 0

109 108

1 x 108 3 x 107 1.7 x 106 0

N-free

7 x 10 3 x 105 1.6 x 105 0

a Cells were subjected to the indicated temperatures for 20 min and then plated on nutrient agar plates to determine the number of viable cells. b NB, 24-h-old NB-grown, highly motile, vibrioid cells devoid of PHB granules; NFb, 24-h-old cells from NFb liquid medium containing 8 mM fructose and 0.5 mM NH4Cl (the cells were motile with many PHB granules, and many were also encapsulated); N-free, cyst forms from a 1-month-old culture in nitrogen-free semisolid medium containing 8 mM fructose (the pellicular growth was dark brown).

FIG. 2. Phase-contrast micrograph of encysted cells of A. brasilense from a brown desiccated colony. Bar, 10 ,um.


VOL. 169, 1987

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FIG. 3. Phase-contrast micrograph of germinating encysted cells of A. brasilense from a brown colony. Note the vegetative cells emerging out of a horseshoe-shaped saclike exinic layer. Bar, 10 Lm.

shows a vibrioid vegetative cell undergoing shrinking of its protoplasm, which is a primary step in cyst formation in bacteria (17, 38). Figure SB shows the complete change in cell morphology with the development of a distinct central body filled with PHB granules. The central body is also seen surrounded by differentiating capsular layers and numerous tiny granular blebs arising out of the central body membrane. In Fig. SC, three completely encysted cells are shown which possess a thick outer coat (exinelike layer). These forms were also seen surrounded by brown-black granular particles which appeared to be responsible for the observed pigmentation associated with the colonies and pellicles. Figure SD is a higher magnification of a single fully matured encysted form of A. brasilense in which a thick outer coat similar to exine is clear and is separated from the central body by a highly refractile, less electron-dense intinelike region. These encysted forms, when stained by the procedure of Vela and Wyss (52), showed the central body to be green and the outer coat red or brown, while the intermediate intinelike region remained unstained. Characterization of the brown pigment. A. brasilense ATCC 29145 was tested for its ability to produce pigment on several solid and semisolid media during growth under aerobic conditions at 30°C (Table 3). It was evident that pigment formation by this strain was dependent on the nutritional content of the medium and the growth condition. Strain Sp7 did not produce pigment in liquid culture but readily produced it in a nitrogen-free semisolid medium and more slowly in certain solid media (Table 3). The dark brown to black color of the pigment suggested that it might be a melanin. Incorporation of amino acids in the medium induces pigment formation in certain media. These pigments did not dissolve in water or organic solvents but were soluble in cold or hot alkali (1 N NaOH or KOH). The pigment also showed bleaching properties similar to melanins, as reported by other workers. The spectrum of crude alkali extracts of the pigment was similar to that of other types of melanins (Fig. 6). The pigment had an absorption maximum at 280 nm and a peak in the range between 310 and 320 nm, which has also been reported for other kinds of microbial melanins (18). Sucrose density gradient separation. Separation of the

1673

different cell forms by sucrose density gradient yielded three forms different in their density, as revealed by three bands obtained at 55, 65, and 70% sucrose. The large encysted forms along with the brown aggregated granular material were pelleted at the bottom of the 70% sucrose gradient. Mixtures of vegetative cells and a few isolated granular particles and small nonmotile encysted forms were observed at 65%, while the band at 55% sucrose consisted mostly of cell forms which were coccoidlike bodies and empty saclike structures.

DISCUSSION A. brasilense ATCC 29145 cells differentiate into forms that are more resistant to desiccation than vegetative cells and hence are considered cysts. Under the conditions tested, the incidence of the various morphogenetic stages of encystation and germination can be seen among the mixed population of vegetative cells, coccoid bodies, and other pleomorphic forms. In the case of Azotobacter vinelandii, however, the induction of cysts has been achieved in a butanol-containing medium up to 90 to 95% recovery within a specified period (39, 43). Even though fructose-containing medium induced the occurrence of A. brasilense cyst forms to a greater extent (visual observation) than other carbon sources, there may be either other factors influencing complete encystation or only a specific pleomorphic form of A. brasilense which responds to induction by fructose. We have noted various morphogenetic forms of A. brasilense never reported before and which, in some respects, appear to resemble Azotobacter vinelandii cysts. Production of the reserve material PHB and its role in survival of various stress and deleterious conditions in a related strain, A. brasilense Cd (ATCC 29729), have been reported (47). From our data with A. brasilense ATCC

I

FIG. 4. Phase-contrast micrographs taken during germination of encysted forms of A. brasilense cells from the desiccated brown pellicular growth, 48 h after transfer into a semisolid nitrogen-free medium containing 8 mM fructose. (A) Encysted form with a single and dividing central body; (B) encysted form with three central bodies; (C and D) giant form with four central bodies. Bar, 10 ,um.

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SADASIVAN AND NEYRA

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D FIG. 5. Ultrastructures of the encysted forms of A. brasilense. (A) Vegetative cell undergoing protoplasmic shrinkage. (B) An encysting form with a complete central body containing PHB granules and numerous tiny granular blebs on the outside wall. The polysaccharidelike capsular layer (P) is evident. (C) A completely encysted form surrounded by numerous melaninlike granules (Mg). (D) Higher magnification of the encysted form showing the central body (cb) with PHB granules and an electron-dense body, an intinelike layer (in) separating a thick outer coat (oc). Bar, 0.5 F±m.

29145, it is also apparent now that the PHB-rich cells confer more resistance to desiccation than the NB-grown PHBdeficient cells (Table 2). A unique observation during this study was the formation of granular brown-black pigmentation by A. brasilense ATCC 29145 in aging cultures. This pigment appears to be melaninlike according to preliminary tests performed as suggested for any melanin pigment (25, 27, 46). Production

of melanin pigments by bacteria has been reported for species within several genera, including Rhizobium (6, 7), Pseudomonas (13, 30), Micrococcus (27), Gluconobacter (34), Mycobacterium (36), and Azotobacter (41). The brownish black pigment exhibited in most bacteria has been partially characterized and suggested to be melanins (18). The decomposition of PHB granules has also been shown to induce the production of brown melaninlike pigments (13).

VOL. 169, 1987


TABLE 3. Influence of various media on pigment formation by A. brasilense ATCC 29145 Amount of pigment formedb Mediuma

1 day

NB NA Tryptic soy broth TSA NA + L-tyrosine NA + L-tyrosine + L-cysteine NA + L-phenylalanine

-

MSMFKA

-

MSMFKA + L-tyrosine MSMFKA + L-tyrosine + L-cysteine MSMMKA MSMMKA + L-tyrosine MSMMKA +L-tyrosine + L-Cysteine NFb + malate (semisolid) NFb + fructose (semisolid)

-

7 days

+

++ + +++

+ ++

-

+++ ++ ++

+ + + ++++ ++++

a NA, Nutrient agar; MSMFKA, MSM agar with fructose and KNO3; MSMMKA, MSM agar plus malate and KNO3. Fructose and malate were used at 8 mM, KNO3 at 0.5 mM, and amino acids at 0.1%. b Pigment production was observed visually as well as microscopically and was scored semiquantitatively from - (no visible pigment) to + + + + (maximum pigment observed). Plates and tubes were observed after incubation for 1 or 7 days at 30°C.

We have shown that the brown pigmentation observed in A. brasilense is triggered in aging cultures under carbon and nitrogen limitation, and under these conditions the cells also tend to form abundant PHB granules (37). Because this pigment shows certain preliminary characteristics of melanins, including resistance to organic solvents, bleaching in oxidants such as sodium hypochlorite and hydrogen peroxide, and solubility in hot alkali, we consider them to be some

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ACKNOWLEDGMENTS Collaboration in electron microscopic studies with R. Triemer and Laura Wood at Nelson Biological Laboratories, suggestions by B. A. Zilinskas, Rutgers University, and technical assistance by Mary Lou Tobin are gratefully acknowledged. This work was supported by the New Jersey Agricultural Experiment Station, financed by state and federal Hatch funds. Partial support was also provided by NJAES Project No. 04400 on rhizosphere research. L.S. is a postdoctoral fellow supported by funds partially provided by the NJAES, Department of Biochemistry and Microbiology, and the International Agriculture and Food Programs at Cook College, Rutgers University.

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type of melanin (25). Furthermore, the spectroscopic properties of the alkali-soluble pigment, an absorption maximum at 280 nm, and a second shoulder peak at 315 nm are indicative of an indole type of melanin (12). The ultrastructure of the developing granules also provides evidence that they are similar to melanin. The granules here were observed to be clustered in a network of fibrillar material encapsulating the aging cells, particularly around encysting cells. A similar observation has been reported previously for a fungus, Verticillium dahliae (53, 54). However, the origin of these granules in A. brasilense is not clearly evident from the micrographs, and therefore we are not commenting on whether they are intracellular or extracellular granules formed as a result of auto-oxidation of the compounds which may have been excreted by the aging cultures. The function of these melaninlike granules in A. brasilense is unknown. In fungi and actinomycetes melanins are known to exist in structures that are extremely resistant to deleterious environmental conditions, such as spores and conidial and sclerotial walls (2, 8, 14, 16, 21, 26, 46). The melanized dark-pigmented fungi are reported to be particularly resistant to lysis by other microorganisms (2, 8, 14, 21). Granular materials have also been reported to be formed when A. brasilense Sp7 cells were inoculated with grass roots (33, 49). Umali-Garcia et al. observed granule formation around the encasement envelope which surrounds the 4-day-old inoculum cells in a nitrogen-free medium with pearl millet (49). These granules have been proposed to originate from grass root exudates and not from the bacterium, because when the bacterium was inoculated into tryptic soy broth, it did not produce these granules in 24-h-old cultures. From this study it appears that granule formation in strain Sp7 is triggered easily under nitrogenlimiting conditions and after aging in a complete medium such as TSA. The granular material reported here withstood various manipulations of specimen preparation for transmission electron microscopy and was of bacterial origin. There are several reports for other systems that show correlation between melanin formation and virulence or pathogenesis (7, 18, 22). Whether the granules formed by A. brasilense ATCC 29145 have any specific role in recognition and adsorption to grass roots in addition to their role in survival is yet to be

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LITERATURE CITED 1. Albrecht, S. L., M. H. Gaskin, J. R. Milam, S. C. Schank, and R. L. Smith. 1983. Ecological factors affecting survival and activity of Azospirillum in the rhizosphere. Experientia (Suppl.) 48:138-148. 2. BalHesta, J. P. G., and M. Alexander. 1971. Resistance of Zygorhynchus species to lysis. J. Bacteriol. 106:938-946. 3. Barber, L. E., J. D. Tjepkema, S. A. Russell, and H. J. Evans. 1976. Acetylene reduction (nitrogen fixation) associated with

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