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Specific association of diazotrophic acetobacters with sugarcane
00384717~91$3.00+0.00
.Soi/ Brol. fhochem.Vol.23.No. 10.pp. 999-1002.1991 Printedin Great Bntain. All rightsreserved
copyright 5 1991 PorgamonPressplc
SPECIFIC ASSOCIATION OF DIAZOTROPHIC ACETOBACTERS WITH SUGARCANE R. P. Lr and I. C. MACRAE* Department of Microbiology, The University of Queensland. Queensland 4072, Australia (Accepted I5 March 1991) Summary-By repeated s&culturing in N-free semisolid sucrose medium, diazotrophic acetobacters were isolated from sugarcane roots or soil collected from four regions of Queensland, Australia. No such organisms were isolated from other grasses from the same locations. It was suggested that these microorganisms are specifically associated with sugarcane hecause they preferred high concentrations of sucrose for growth and N2 fixation and did not utilize common amino acids or organic acids for growth.
INTRODtiCTION Lima ct al. (I 987) employing “N techniques have yielded promising results indicating that associative NZ fixation may contribute to the N economy of sugarcane. Although thcrc have been reports of the sclcctive stimulation of Beijerinckiu in the sugarcane rhizosphcrc (Dobcrcincr, 1961) and the isolation of diazotrophic bacteria belonging to the gcncru A.-OWhuc~cr. A ~c~spirilluttr, Bucilhrs. Klehsiellu. Enrerobucrer and Etwiniu (Rcnnic el cd., 1982) from sugarcanc roots, thcrc arc no data available as to which of thcsc microorganisms is gcncraly associated with sugarcanc from difrcrcnt regions around the world. Also, it is not known which of thcsc microorganisms was rcsponsible for the obscrvcd associative NZ fixation (Lima CI (II., 1987). A new group of acid-tolerant diazotrophs isolated from sugarcane in Brazil has been characterized and named Succharohucfer niwocupruns (Cavalcante and Doberciner. 1988). and later rcnamcd Acetobacrer diuxtrruphicus (Gillis er ul.. 1989). Although sugarcane is a major crop in Australia very little is known about the diazotrophs associated with this plant. Our objectives were (a) to examine sugarcane and other tropical grass roots from different regions and soil types of Australia for the presence of diazotrophic acetobactcrs and to isolate these bacteria when detected, and (b) to examine some of the phenotypic characters of these microorganisms which may be important in their association with sugarcane. .\lATERIAlS
AND
METHODS
Isolurion of ucelobucrers Sugarcane roots from individual plants of I2 varieties of sugarcane or soil between sugarcane rows were collected from several sites near Brisbane (Alluvial soil, 28 ‘S and I53”E). Nambour (Clay soil, 26.6”s and 153’E). Mackay (Pioneer soil, 21.5’S and 148-E and Childcrs (Red earth, 25‘S and 152’E). Queensland, Australia from March 1988 to Dccem*Author for correspondence. 999
ber 1989. Eight different unidentified grasses collected around The University of Queensland campus, one sea grass Zosreru sp. from Morcton Bay, and grasses including Buffcl grass (Cenchrus ciliuris), Couch grass (Cynodon duc~ylon). Molasses grass (Melinis minuriflr,ra), Green panic (funicum maximum). Rhodes grass (Chloris gup~o), Sabi grass (Urochloa mosambicensis), Sudan grass (Sorghum sudanense) and Ruzi grass (Bruchiariu ruziziensis) collcctcd from Childcrs. Queensland. Australia were also examined as sources of diazotrophic acctobactcrs. Diazotrophic acctobactcrs wcrc enriched using 5 ml semisolid medium containing basal N-free salts (Rcnnic et ul., IY82). 1.5% sucrose as carbon source, 0.002% yeast extract and 0.2% agar in I3 ml serum bottles, The pH of semisolid medium was 6.5 initially but was changed to 4.5 later for better selection of diazotrophic acetobactcrs. Appropriate dilutions of rhizosphere soil, rhizoplane or cndorhizosphcrc sections of sugarcane roots, or small pieces of sugarcane and grass roots were inoculated into the bottom of the semisolid enrichment medium and kept at 28°C for 3-4 days. Enrichment cultures were subculturcd for up to IO times every 2 or 3 days and the C2H, reduction test performed between each subculturing. The CZ Hz reduction test was performed using 20% (v/v) C2H, in the headspace above the semisolid medium and an exposure period of 1-3 h. CZH, production was measured using a Shimadzu Gas Chromatograph 8A having a 1 m stainless steel column of Porapak Type N (mesh 50-80) and a flame ionization detector connected to a Delta Chromatography Data Computer System. Pure cultures were finally isolated using a medium having the same composition as the semisolid medium but with 0.01% yeast extract and 1.5% agar. Upon isolation, each strain was checked for purity by streaking onto purification medium which was similar to the isolation medium but contained 0.2% NH,CI and 0.2% yeast extract. The pure cultures were checked for CIH, reduction activity in semisolid sucrose enrichment medium. Stock cultures were maintained on agar slants of purification medium in
R. P. Lt and I. C. MACRM
loo0
5 ml bijou bottles at 4‘C and transferred monthly. Growth or characterization tests were carried out at 28C. Phenot_spic properties
All C2 H,;reduction positive isolates were subjected to a few pnmary diagnostic characterization tests to confirm identity. Two-day old cultures grown on purification medium were used as inocula or used directly for these diagnostic characterization studies. Gram reaction and motility were tested according to Skerman (1967). Oxidation or fermentation of glucose was determined according to Hayward (1964). Oxidase reaction was determined using Oxidase Detection Strips (Disposable Products Pty Ltd. Adelaide. South Australia). Ethanol medium (De Ley et pi., 1984) modified by the addition of 8 ml 1% aqueous bromothymol blue was used to test the ability of isolates to oxidize ethanol first to acetate and further to CO:. GYC agar (De Ley er al., 1984) and potato agar which contained an extract of 200 g potato and 10% sucrose, were included for pigmentation determination. Growth in the presence of 30% glucose or 10% ethanol was tested in liquid medium containing I% yeast extract as the basal medium. Semisolid enrichment medium modified by addition of various amounts of KNO, was inoculated and kept at 28‘C for 3 days to test the ability of each isolate to synthesize nitrogcnasc and cxprcss its activity in the prcscnce of high concentrations of nitrate, and to test for nitrate reduction (Skcrman, 1967). The ability of the isolates to utilize various C substrates as sole C sources was tcstcd on agar plates using the isolation medium in which the yeast extract was rcplaccd with 0.2% NH,CI. and sucrose with rclcvant C substrates. Agar plates of the snmc composition but without the addition of C source were used as controls. Each C substrate was mcmbranc-sterilizcd (0.2 pm port size) and added to the sterile basal medium to give a final concentration of I% for sugars or 0.5% for ncutralizcd organic acids. RESULTS
Isolution
o$ diozotrophic
acetobucrers
After incubation for 4 days, surface pellicles developed in semisolid sucrose medium inoculated with sugarcane root samples and C2Hz reduction was observed with these cultures. Success in isolating diazotrophic acetobacters by streaking onto agar plates at this stage was poor as diazotrophic acetobatters grew much slower on agar plates than dizotrophs belonging to the family Enlerobacreriuceue and the genus Pseudomonas which were also prcscnt (data not shown). After repeated subculturing and finally streak inoculation of plates onto isolation medium, most enrichments yielded only one type of growth after 48 h. Bacterial growth along the initial inoculum lines was very slimy and separated colonies were < I mm dia. Colonies became bigger (3-4 mm) only after 5-6 days. When these colonies were transferred into semisolid sucrose enrichment medium, obvious growth was observed, a surface pellicle was formed and the pH of the semisolid medium dropped below 3 after 2 days. Most isolates obtained by using the above procedures were later confirmed as
diazotrophic acetobacters although a few proved to be Eeijerinckia spp. Beijerinckia spp isolates could be easily separated from acetobacters by their extremely slimy growth both on purification medium and in semisolid sucrose medium. and by their much slower growth (> 5 days) on purification medium. Isolates which had been designated as acetobacters by their characteristic bacterial growth were all Gram-negative, strictly aerobic, oxidase-negative rods able to oxidize ethanol first to acetate and further to CO2 and were therefore confirmed as belonging to the genus Acetobacter (De Ley et al., 1984; Gillis et al., 1989). A total of 114 diazotrophic acetobacters were obtained from roots of 12 sugarcane varieties grown in the various soil types and two were obtained from soil collected between sugarcane rows. Diazotrophic acetobacters were not isolated from any of the grass samples examined by the same procedure. Enrichment cultures of these grass samples lost C?H, reduction activity after the second or third subculture. Earlier streaking onto isolation medium after one subculturing did not recover any diazotrophic acetobatters. Numbers of isolates of diazotrophic acetobatters isolated from the sugarcane root and soil samples are summarized in Table I. Similar media with mannitol, malate or glucose at pH 7 were also included for isolation studies for the Brisbane samples, but diazotrophic isolates from these media mostly belonged to the family Enferubacteriaceae and the genus Pseudonu~nas. No acctobactcrs were isolatcd. Phenotypic
properties
Typical characteristics of the isolates are presented in Table 2. Water-soluble brown pigments were produccd on GYC plate. Non-diffusible brown pigments were produced on Potato agar supplemented with 10% sucrose and on isolation and purification media after prolonged incubation. Surface pelliclc was formed on the surface of semisolid medium after growth for l-2 days. Nitrate reduction was negative for all the isolates and nitrogcnasc was synthesized and its activity was expressed in the presence of 80 mM nitrate although partial inhibition of growth Table I. Number of isolates of diarorrophic acclobackn obtained from rugarcane roots and soil Region and dare Brisbane. 4 March 1988
Brisbane. X Augusl 1988
Number of isolltlm 5 6 I4 23
5
7 Mackay. July 1989
8
Nambour. 25 September 1989
5 6 2 7 5 6 I7
Childcrs. 6 December 1989
Suglrrcanc varietlcs and soil First raoon of Q I37 Second ra~oon of QI 37 Planr cane oTQI37 Fourth ri11oonof CP44lOl without N-fcrtdizcr Fourth ra~oon of CP44lOl Hith N-fcrtdizcr Second ra~oon of QI IO with N-fertilizer 4138. 46X. Qla. 4124. QSO. 987 Plan1 cane of CP44101 First ratoon of CP44101 Soil bccweenrows First ratoon of Q137 Second ratoon of 4137 Plant cane of 0137 QI47.4146. Qb7
Diazotrophic acetobacters in sugarcane was noted in the presence of SO-80 mu nitrate. At nitrate concentrations below 30 mu. there was no difference in nitrogenase activity between nitrateamended cultures and nitrogen-free control cultures. Growth proceeded in the presence of 30% glucose or 10% ethanol. Results for C substrates utilized as sole C source are presented in Table 3. Only very few of the tested carbon substrates supported growth. These included sucrose, fructose, glucose, galactose, gluconate. mannitol, sorbitol and glycerol. Xylose. ribose, mannose, arabitol, ethanol and xylitol only supported limited growth while i-inositol and D-arabinose gave varying results. The isolates preferred high concentrations of sucrose such as 10% for both growth and NZ fixation. None of the tested common amino acids and organic acids (except gluconate) supported growth.
Table
3. Substrate
diazotrophic
1001
utdization
acctobacter
as sole carbon
isolates
from
and energy
rugarcane
Substrate
roots
source
by
and soil
Utilizatton
Sucrose.
glucose.
rafinosc.
fructose.
gluconatc.
galactose.
glycerol.
turanose,
mannitol.
+
sorbitol, Ethanol.
xylitol,
o-arabitol,
ribose,
mannose.
xylosc. i-lnositol, Rhamnose.
1~x0s~. maltose,
dcxtrin.
starch,
dulcitol. Lactate.
inulin,
methanol, acetate.
lactose.
salicin.
maleate. acid.
adipic
acid.
L-aspartic
acid.
formic
acid.
hippuric alaninc.
acid.
L-lcucinc.
glutamic Lcystcinc
results
oxalate.
malonic
r-prolinc.
thiamine. good
growth; among
-
citrate.
fumarate.
acids,
ctllobiose,
adonitol.
crythritol,
malatc.
hcptanoic
Valine,
+:
+Y
o-arabinose
benzoatc. n-vale&
acid.
hydroxybutyric acid
tx-omithine.
acid.
+ -:
succinatc.
glycinc.
t.-tyrosine. -
slight
growth;
-:
no growth;
v: varying
isolates.
DISCUSSION
Diazotrophic acetobacters were isolated from most of the sugarcane root samples collected from sugarcane fields in Queensland, Australia from March 1988 to December 1989. Diazotrophic acetobacters were also isolated from sugarcanc roots in Canberra, Australia (A. H. Gibson 1989 pcrs. commun.) and from sugarcane roots and stems grown in four regions of Brazil (Cavalcantc and Dobcreincr. 1988). Extcnsivc taxonomic studies with ELISA, gel immunodiffusion, G + C% content of DNA and DNADNA hybridization (data not shown) rcvcalcd that our diazotrophic Acefobactcr isolalcs and the diazotrophic Acetobtrcter strains from A. H. Gibson, Canberra also belong to A. diu~otrophicus (Gillis rf ol., 1989). The Fact that diazotrophic acelobactcrs were only isolated from sugarcanc and not from the various grasses and sea grass tcstcd indicates that diazotrophic acctobactcrs are generally associated with sugarcanc grown in diverse geographic regions around the world but not with grasses. The inability of these organisms to utilize common amino acids and organic acids which were believed to be major components of plant exudates and to be the major C and energy sources for rhizosphcre, rhizoplanc and endorhizosphere microorganisms may be the reason of the absence of these organisms from the grasses. These organisms preferred high concentrations of
Table
2.
Diagnostic
characteristics associated
of
with
diazotrophic
acetobactcrs
sugarcane Result
Character Gram-reaction OtF
Ondative
of glucose
Oxldaw Motlhty
+
Oxidwm
of ethanol
first to acetate
and further
to +
Dark
brown
10% Brown
pigments
produced
on potato
pigments
on GYC
agar with
sucrose water
+
soluble
agnr
+
Growth
at the presence
of 30%
sucrose
t
Grouch
at the prcxncc
of
ethanol
+
Nitrate
10%
reduction
Nitrogcnax
synthesis
and
activity
at
prcxncc
of
80 rnti nitrate Surface
pclliclc
semwAid days
t formatmn
sucrose
medium
and after
pH
below
incubation
3 for
in ? +
sucrose for growth and N: fixation. In terms of available C substrate, it was not surprising that the sugarcane root surroundings (rhizosphere, rhizoplane. endorhizosphcre) or stems and leaves where a high concentration of sucrose exists might provide a favourable environment for these organisms. Therefort our data also indicate a specific association of diazotrophic acctobacters with sugarcanc. Grasses or ccrcals which do not produce and cxudc sucrose may not harbour thcsc organisms. It is cvidcnt lhat the selection of C substrates and pH of the isolation medium play a very important role in the isolation of new diazotrophs such as acctobactcrs. Besides the rcquircment for sucrose as a C source. the repeated subculturing procedure is also csscntial for the succcss of rccovcring diazotrophic acetobactcrs. Our attempts to isolate diazotrophs by directly plating root preparations on N-free agar medium with sucrose as carbon source incubated under various partial oxygen pressures did not recover any acetobactcrs at all but yielded Enrerobucreriuceue and Pseudomonus spp exclusively. Similarly, streaking growth from semisolid sucrose enrichment cultures after the first or second subculturing did not yield acetobacters either. Actually, the acetobacter isolates grew much slower on agar plates even with sucrose as C source than the Enterobocteriuceae and Pseudomonas isolates and it was very likely that organisms of Enrerobacreriocene and Pseudomonas spp would overgrow acetobacters if repeated subculturing in semisolid medium was not employed. These organisms had some unusual and important properties compared with other diazotrophs. They were tolerant of stress conditions such as low pH and extremely high concentrations of sucrose and glucose. Most noteworthy was the fact that these microorganisms could synthcsizc nitrogenase and express its activity in the presence of 80 tTtM nitrate. The A. diozotrophicus isolated from Brazil could fix N2 in the presence of 10 tnM nitrate and no data on higher concentrations of nitrate was reported by Cavalcante and Dobereiner (1988). It is not known if the Brazil A, diozofrophicus is also tolerant of nitrate concentrations as high as 80m~.
R. P. LI and 1. C. MACRAE
1002
Ackno~ledgemeno-Ruiping Li is in receipt of a University of Queensland Postgraduate Research Scholarship. We acknowledge the cooperation of the Bureau of Sugar Experimentation Stations in facilitating the sampling of field sugarcane.
Gilhs M.. Kersters K., Hoste 8.. Janssens D.. Kroppenstedt R. M. and Stephen M. P. (1989) Acetobacrer d&orrophicus sp. nov., a nitrogen fixing acetic acid bacterium associated with sugarcane. International Journal of Sysremafic
Bacteriology
39, 36 I-364.
Hayward A. C. (1964) Characteristics REFERENCES
Cavalcante V. A. and Dobereiner J. (1988) A new acidtolerant nitrogen-fixing bacterium associated with sugarcane. Plum and Soil 108. 23-31. De Ley J.. Swings J. and Gassele F. (1984) Genus 1. Acetobacrer Beijerink 1898. 215. In Bergey’s Manual of SyJremaric Bacreriology (N. R. Kreig and J. Golt, Eds), Vol. I, pp. 270-274. Williams & Wilkins, Baltimore. Dobereiner J. (1961) Nitrogen fixing bacteria of the genus Beijerinckia Derx in the rhizosphere of sugarcane. Planr and Soil 15, 21 l-216.
solanacearwn. 265-277.
The Journal of Applied
of Pseudomonas Bacteriology
27,
Lima E.. Boddey R. M. and Dobereiner J. (1987) Quantification of biological nitrogen tixation associated with sugarcane using a 15-N aided nitrogen balance. Soil Biology & Biochemtifry
19. 165-170.
Rennie R. J., de. Freitas J.. Ruschel A. P. and Vose P. B. (1982) Isolation and identification of N,-fixing bacteria associated with sugarcane (Succhurum sp.). Cunadiun Journal of Microbiology
28. 462-467.
Skerman V. B. D. (I 967) A Guide to rhe Identi/ication of the Genera of Bacteria, 2nd edn. pp. 219-220 and 276-280. Williams & Wilkins, Baltimore.
.Soi/ Brol. fhochem.Vol.23.No. 10.pp. 999-1002.1991 Printedin Great Bntain. All rightsreserved
copyright 5 1991 PorgamonPressplc
SPECIFIC ASSOCIATION OF DIAZOTROPHIC ACETOBACTERS WITH SUGARCANE R. P. Lr and I. C. MACRAE* Department of Microbiology, The University of Queensland. Queensland 4072, Australia (Accepted I5 March 1991) Summary-By repeated s&culturing in N-free semisolid sucrose medium, diazotrophic acetobacters were isolated from sugarcane roots or soil collected from four regions of Queensland, Australia. No such organisms were isolated from other grasses from the same locations. It was suggested that these microorganisms are specifically associated with sugarcane hecause they preferred high concentrations of sucrose for growth and N2 fixation and did not utilize common amino acids or organic acids for growth.
INTRODtiCTION Lima ct al. (I 987) employing “N techniques have yielded promising results indicating that associative NZ fixation may contribute to the N economy of sugarcane. Although thcrc have been reports of the sclcctive stimulation of Beijerinckiu in the sugarcane rhizosphcrc (Dobcrcincr, 1961) and the isolation of diazotrophic bacteria belonging to the gcncru A.-OWhuc~cr. A ~c~spirilluttr, Bucilhrs. Klehsiellu. Enrerobucrer and Etwiniu (Rcnnic el cd., 1982) from sugarcanc roots, thcrc arc no data available as to which of thcsc microorganisms is gcncraly associated with sugarcanc from difrcrcnt regions around the world. Also, it is not known which of thcsc microorganisms was rcsponsible for the obscrvcd associative NZ fixation (Lima CI (II., 1987). A new group of acid-tolerant diazotrophs isolated from sugarcane in Brazil has been characterized and named Succharohucfer niwocupruns (Cavalcante and Doberciner. 1988). and later rcnamcd Acetobacrer diuxtrruphicus (Gillis er ul.. 1989). Although sugarcane is a major crop in Australia very little is known about the diazotrophs associated with this plant. Our objectives were (a) to examine sugarcane and other tropical grass roots from different regions and soil types of Australia for the presence of diazotrophic acetobactcrs and to isolate these bacteria when detected, and (b) to examine some of the phenotypic characters of these microorganisms which may be important in their association with sugarcane. .\lATERIAlS
AND
METHODS
Isolurion of ucelobucrers Sugarcane roots from individual plants of I2 varieties of sugarcane or soil between sugarcane rows were collected from several sites near Brisbane (Alluvial soil, 28 ‘S and I53”E). Nambour (Clay soil, 26.6”s and 153’E). Mackay (Pioneer soil, 21.5’S and 148-E and Childcrs (Red earth, 25‘S and 152’E). Queensland, Australia from March 1988 to Dccem*Author for correspondence. 999
ber 1989. Eight different unidentified grasses collected around The University of Queensland campus, one sea grass Zosreru sp. from Morcton Bay, and grasses including Buffcl grass (Cenchrus ciliuris), Couch grass (Cynodon duc~ylon). Molasses grass (Melinis minuriflr,ra), Green panic (funicum maximum). Rhodes grass (Chloris gup~o), Sabi grass (Urochloa mosambicensis), Sudan grass (Sorghum sudanense) and Ruzi grass (Bruchiariu ruziziensis) collcctcd from Childcrs. Queensland. Australia were also examined as sources of diazotrophic acctobactcrs. Diazotrophic acctobactcrs wcrc enriched using 5 ml semisolid medium containing basal N-free salts (Rcnnic et ul., IY82). 1.5% sucrose as carbon source, 0.002% yeast extract and 0.2% agar in I3 ml serum bottles, The pH of semisolid medium was 6.5 initially but was changed to 4.5 later for better selection of diazotrophic acetobactcrs. Appropriate dilutions of rhizosphere soil, rhizoplane or cndorhizosphcrc sections of sugarcane roots, or small pieces of sugarcane and grass roots were inoculated into the bottom of the semisolid enrichment medium and kept at 28°C for 3-4 days. Enrichment cultures were subculturcd for up to IO times every 2 or 3 days and the C2H, reduction test performed between each subculturing. The CZ Hz reduction test was performed using 20% (v/v) C2H, in the headspace above the semisolid medium and an exposure period of 1-3 h. CZH, production was measured using a Shimadzu Gas Chromatograph 8A having a 1 m stainless steel column of Porapak Type N (mesh 50-80) and a flame ionization detector connected to a Delta Chromatography Data Computer System. Pure cultures were finally isolated using a medium having the same composition as the semisolid medium but with 0.01% yeast extract and 1.5% agar. Upon isolation, each strain was checked for purity by streaking onto purification medium which was similar to the isolation medium but contained 0.2% NH,CI and 0.2% yeast extract. The pure cultures were checked for CIH, reduction activity in semisolid sucrose enrichment medium. Stock cultures were maintained on agar slants of purification medium in
R. P. Lt and I. C. MACRM
loo0
5 ml bijou bottles at 4‘C and transferred monthly. Growth or characterization tests were carried out at 28C. Phenot_spic properties
All C2 H,;reduction positive isolates were subjected to a few pnmary diagnostic characterization tests to confirm identity. Two-day old cultures grown on purification medium were used as inocula or used directly for these diagnostic characterization studies. Gram reaction and motility were tested according to Skerman (1967). Oxidation or fermentation of glucose was determined according to Hayward (1964). Oxidase reaction was determined using Oxidase Detection Strips (Disposable Products Pty Ltd. Adelaide. South Australia). Ethanol medium (De Ley et pi., 1984) modified by the addition of 8 ml 1% aqueous bromothymol blue was used to test the ability of isolates to oxidize ethanol first to acetate and further to CO:. GYC agar (De Ley er al., 1984) and potato agar which contained an extract of 200 g potato and 10% sucrose, were included for pigmentation determination. Growth in the presence of 30% glucose or 10% ethanol was tested in liquid medium containing I% yeast extract as the basal medium. Semisolid enrichment medium modified by addition of various amounts of KNO, was inoculated and kept at 28‘C for 3 days to test the ability of each isolate to synthesize nitrogcnasc and cxprcss its activity in the prcscnce of high concentrations of nitrate, and to test for nitrate reduction (Skcrman, 1967). The ability of the isolates to utilize various C substrates as sole C sources was tcstcd on agar plates using the isolation medium in which the yeast extract was rcplaccd with 0.2% NH,CI. and sucrose with rclcvant C substrates. Agar plates of the snmc composition but without the addition of C source were used as controls. Each C substrate was mcmbranc-sterilizcd (0.2 pm port size) and added to the sterile basal medium to give a final concentration of I% for sugars or 0.5% for ncutralizcd organic acids. RESULTS
Isolution
o$ diozotrophic
acetobucrers
After incubation for 4 days, surface pellicles developed in semisolid sucrose medium inoculated with sugarcane root samples and C2Hz reduction was observed with these cultures. Success in isolating diazotrophic acetobacters by streaking onto agar plates at this stage was poor as diazotrophic acetobatters grew much slower on agar plates than dizotrophs belonging to the family Enlerobacreriuceue and the genus Pseudomonas which were also prcscnt (data not shown). After repeated subculturing and finally streak inoculation of plates onto isolation medium, most enrichments yielded only one type of growth after 48 h. Bacterial growth along the initial inoculum lines was very slimy and separated colonies were < I mm dia. Colonies became bigger (3-4 mm) only after 5-6 days. When these colonies were transferred into semisolid sucrose enrichment medium, obvious growth was observed, a surface pellicle was formed and the pH of the semisolid medium dropped below 3 after 2 days. Most isolates obtained by using the above procedures were later confirmed as
diazotrophic acetobacters although a few proved to be Eeijerinckia spp. Beijerinckia spp isolates could be easily separated from acetobacters by their extremely slimy growth both on purification medium and in semisolid sucrose medium. and by their much slower growth (> 5 days) on purification medium. Isolates which had been designated as acetobacters by their characteristic bacterial growth were all Gram-negative, strictly aerobic, oxidase-negative rods able to oxidize ethanol first to acetate and further to CO2 and were therefore confirmed as belonging to the genus Acetobacter (De Ley et al., 1984; Gillis et al., 1989). A total of 114 diazotrophic acetobacters were obtained from roots of 12 sugarcane varieties grown in the various soil types and two were obtained from soil collected between sugarcane rows. Diazotrophic acetobacters were not isolated from any of the grass samples examined by the same procedure. Enrichment cultures of these grass samples lost C?H, reduction activity after the second or third subculture. Earlier streaking onto isolation medium after one subculturing did not recover any diazotrophic acetobatters. Numbers of isolates of diazotrophic acetobatters isolated from the sugarcane root and soil samples are summarized in Table I. Similar media with mannitol, malate or glucose at pH 7 were also included for isolation studies for the Brisbane samples, but diazotrophic isolates from these media mostly belonged to the family Enferubacteriaceae and the genus Pseudonu~nas. No acctobactcrs were isolatcd. Phenotypic
properties
Typical characteristics of the isolates are presented in Table 2. Water-soluble brown pigments were produccd on GYC plate. Non-diffusible brown pigments were produced on Potato agar supplemented with 10% sucrose and on isolation and purification media after prolonged incubation. Surface pelliclc was formed on the surface of semisolid medium after growth for l-2 days. Nitrate reduction was negative for all the isolates and nitrogcnasc was synthesized and its activity was expressed in the presence of 80 mM nitrate although partial inhibition of growth Table I. Number of isolates of diarorrophic acclobackn obtained from rugarcane roots and soil Region and dare Brisbane. 4 March 1988
Brisbane. X Augusl 1988
Number of isolltlm 5 6 I4 23
5
7 Mackay. July 1989
8
Nambour. 25 September 1989
5 6 2 7 5 6 I7
Childcrs. 6 December 1989
Suglrrcanc varietlcs and soil First raoon of Q I37 Second ra~oon of QI 37 Planr cane oTQI37 Fourth ri11oonof CP44lOl without N-fcrtdizcr Fourth ra~oon of CP44lOl Hith N-fcrtdizcr Second ra~oon of QI IO with N-fertilizer 4138. 46X. Qla. 4124. QSO. 987 Plan1 cane of CP44101 First ratoon of CP44101 Soil bccweenrows First ratoon of Q137 Second ratoon of 4137 Plant cane of 0137 QI47.4146. Qb7
Diazotrophic acetobacters in sugarcane was noted in the presence of SO-80 mu nitrate. At nitrate concentrations below 30 mu. there was no difference in nitrogenase activity between nitrateamended cultures and nitrogen-free control cultures. Growth proceeded in the presence of 30% glucose or 10% ethanol. Results for C substrates utilized as sole C source are presented in Table 3. Only very few of the tested carbon substrates supported growth. These included sucrose, fructose, glucose, galactose, gluconate. mannitol, sorbitol and glycerol. Xylose. ribose, mannose, arabitol, ethanol and xylitol only supported limited growth while i-inositol and D-arabinose gave varying results. The isolates preferred high concentrations of sucrose such as 10% for both growth and NZ fixation. None of the tested common amino acids and organic acids (except gluconate) supported growth.
Table
3. Substrate
diazotrophic
1001
utdization
acctobacter
as sole carbon
isolates
from
and energy
rugarcane
Substrate
roots
source
by
and soil
Utilizatton
Sucrose.
glucose.
rafinosc.
fructose.
gluconatc.
galactose.
glycerol.
turanose,
mannitol.
+
sorbitol, Ethanol.
xylitol,
o-arabitol,
ribose,
mannose.
xylosc. i-lnositol, Rhamnose.
1~x0s~. maltose,
dcxtrin.
starch,
dulcitol. Lactate.
inulin,
methanol, acetate.
lactose.
salicin.
maleate. acid.
adipic
acid.
L-aspartic
acid.
formic
acid.
hippuric alaninc.
acid.
L-lcucinc.
glutamic Lcystcinc
results
oxalate.
malonic
r-prolinc.
thiamine. good
growth; among
-
citrate.
fumarate.
acids,
ctllobiose,
adonitol.
crythritol,
malatc.
hcptanoic
Valine,
+:
+Y
o-arabinose
benzoatc. n-vale&
acid.
hydroxybutyric acid
tx-omithine.
acid.
+ -:
succinatc.
glycinc.
t.-tyrosine. -
slight
growth;
-:
no growth;
v: varying
isolates.
DISCUSSION
Diazotrophic acetobacters were isolated from most of the sugarcane root samples collected from sugarcane fields in Queensland, Australia from March 1988 to December 1989. Diazotrophic acetobacters were also isolated from sugarcanc roots in Canberra, Australia (A. H. Gibson 1989 pcrs. commun.) and from sugarcane roots and stems grown in four regions of Brazil (Cavalcantc and Dobcreincr. 1988). Extcnsivc taxonomic studies with ELISA, gel immunodiffusion, G + C% content of DNA and DNADNA hybridization (data not shown) rcvcalcd that our diazotrophic Acefobactcr isolalcs and the diazotrophic Acetobtrcter strains from A. H. Gibson, Canberra also belong to A. diu~otrophicus (Gillis rf ol., 1989). The Fact that diazotrophic acelobactcrs were only isolated from sugarcanc and not from the various grasses and sea grass tcstcd indicates that diazotrophic acctobactcrs are generally associated with sugarcanc grown in diverse geographic regions around the world but not with grasses. The inability of these organisms to utilize common amino acids and organic acids which were believed to be major components of plant exudates and to be the major C and energy sources for rhizosphcre, rhizoplanc and endorhizosphere microorganisms may be the reason of the absence of these organisms from the grasses. These organisms preferred high concentrations of
Table
2.
Diagnostic
characteristics associated
of
with
diazotrophic
acetobactcrs
sugarcane Result
Character Gram-reaction OtF
Ondative
of glucose
Oxldaw Motlhty
+
Oxidwm
of ethanol
first to acetate
and further
to +
Dark
brown
10% Brown
pigments
produced
on potato
pigments
on GYC
agar with
sucrose water
+
soluble
agnr
+
Growth
at the presence
of 30%
sucrose
t
Grouch
at the prcxncc
of
ethanol
+
Nitrate
10%
reduction
Nitrogcnax
synthesis
and
activity
at
prcxncc
of
80 rnti nitrate Surface
pclliclc
semwAid days
t formatmn
sucrose
medium
and after
pH
below
incubation
3 for
in ? +
sucrose for growth and N: fixation. In terms of available C substrate, it was not surprising that the sugarcane root surroundings (rhizosphere, rhizoplane. endorhizosphcre) or stems and leaves where a high concentration of sucrose exists might provide a favourable environment for these organisms. Therefort our data also indicate a specific association of diazotrophic acctobacters with sugarcanc. Grasses or ccrcals which do not produce and cxudc sucrose may not harbour thcsc organisms. It is cvidcnt lhat the selection of C substrates and pH of the isolation medium play a very important role in the isolation of new diazotrophs such as acctobactcrs. Besides the rcquircment for sucrose as a C source. the repeated subculturing procedure is also csscntial for the succcss of rccovcring diazotrophic acetobactcrs. Our attempts to isolate diazotrophs by directly plating root preparations on N-free agar medium with sucrose as carbon source incubated under various partial oxygen pressures did not recover any acetobactcrs at all but yielded Enrerobucreriuceue and Pseudomonus spp exclusively. Similarly, streaking growth from semisolid sucrose enrichment cultures after the first or second subculturing did not yield acetobacters either. Actually, the acetobacter isolates grew much slower on agar plates even with sucrose as C source than the Enterobocteriuceae and Pseudomonas isolates and it was very likely that organisms of Enrerobacreriocene and Pseudomonas spp would overgrow acetobacters if repeated subculturing in semisolid medium was not employed. These organisms had some unusual and important properties compared with other diazotrophs. They were tolerant of stress conditions such as low pH and extremely high concentrations of sucrose and glucose. Most noteworthy was the fact that these microorganisms could synthcsizc nitrogenase and express its activity in the presence of 80 tTtM nitrate. The A. diozotrophicus isolated from Brazil could fix N2 in the presence of 10 tnM nitrate and no data on higher concentrations of nitrate was reported by Cavalcante and Dobereiner (1988). It is not known if the Brazil A, diozofrophicus is also tolerant of nitrate concentrations as high as 80m~.
R. P. LI and 1. C. MACRAE
1002
Ackno~ledgemeno-Ruiping Li is in receipt of a University of Queensland Postgraduate Research Scholarship. We acknowledge the cooperation of the Bureau of Sugar Experimentation Stations in facilitating the sampling of field sugarcane.
Gilhs M.. Kersters K., Hoste 8.. Janssens D.. Kroppenstedt R. M. and Stephen M. P. (1989) Acetobacrer d&orrophicus sp. nov., a nitrogen fixing acetic acid bacterium associated with sugarcane. International Journal of Sysremafic
Bacteriology
39, 36 I-364.
Hayward A. C. (1964) Characteristics REFERENCES
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