Occurrence and Microbiological Characteristics of Azospirillum Strains Associated With Wheat Rhizoplane and Endorhizosphere
Occurrence and Microbiological Characteristics of Azospirillum Strains Associated With Wheat Rhizoplane and Endorhizosphere
Occurrence and Microbiological Characteristics of Azospirillum Strains Associated With Wheat Rhizoplane and Endorhizosphere
ABSTRACT : Azosprillum spp. were isolated from the endorhizosphere of 27 wheat genotypes and 32 Azospirillum strains
were obtained. Among these six were Azospirillum lipoferum, 13 were Azosprillum brasilense and 13 isolates could not be
grouped into either of the two categories. And all the isolates were subjected to various biochemical characterization. All the
isolates were tested for siderophore production by growing them on M-9 medium. Out of 32 isolates, 18 strains showed good
growth on the medium, where as five others showed moderate growth and rest others totally failed to grow on the medium. The
variations in the isolates might be due to the genetic variations in the plant genotypes and their properties.
with respective 1 per cent carbon sources. The different WAS-28 however, showed weak growth. Only WAS-10
carbon sources used were citrate, aconitate, isocitrate, strain could utilize glutamate as sole carbon source. Eleven
á-ketoglutarate, succinate, fumarate, L- malate, isolates could grow in the presence of 1 per cent bile
oxaloacetate, pyruvate, L-lactate, malonate, acetate, â- concentration and 13 strains showed good response in
hydroxybutyrate, n-propanol, L-histidine, L-tyrosine, L- triple sugar iron slants. Out of 32 isolates, 15 isolates
phenylalanine, L-alanine, L-glutamate, L-aspartate, L- required biotin for its growth. Growth was observed at
asparagine, L-proline, L-hydroxyproline, L-serine, glycine, pH 6.0, in only 5 isolates. Azospirillum is a general root
putrescine, D-gluconate, D-mannitol, D-fructose, glycerol. colonizer and is not a plant-specific bacterium (Bashan
The growth from the Master plate Luria agar was and Holguin, 1997; Hartmann and Baldani, 2006). In the
replicated on different plates containing medium with present investigation 32 spirilla were isolated from the
different carbon sources and plates were incubated at endorhizosphere of different wheat genotypes. After
29ºC and readings were observed after 2 h of incubation. several transfers, 15 strains were identified as bacteria
RESULTS AND DISCUSSION belonging to the genus Azospirillum based on to their
common cultural and cell morphological characteristics.
Investigations were carried out to isolate native
The characteristics were the formation of a veil like pellicle
isolates of Azospirillum from wheat rhizoplane and
or ballon often 10mm below the surface of semi solid N-
endorhizosphere. The isolates were further identified
free media. The formation of this pellicle is due to an
based on their physiological characteristics. All the isolates
aerotactic response of the motile bacteria towards low
formed subsurface pellicle in the semi solid medium. The
levels of PO4 that permit N2 fixation (Okon, 1985). The
isolates were urease positive, could grow in anaerobic
dissolved O2 concentration in the media was just enough
condition in presence of nitrate phosphatase positive, could
for optimal respiration rates without inhibiting N2 fixation
grow in anaerobic condition in presence of nitrate
(Day and Dobereiner, 1976), as a result of nitrogenase
phosphatase positive and most of them were catalase
inhibition.
positive, however but some of the isolates showed weak
catalase activity (WAS-3, WAS-5, WAS-19, WAS-27, Utilization of carbon compounds
WAS-29 and WAS-31). None of the isolates could The ability of isolates to use several carbonaceous
produce H2S from cystine or could utilize gelatine. All the compounds as sole carbon source were checked using
isolates did not grow on EMB agar, one per cent bile, 31 different carbon sources. The observations were taken
MacConkey agar, MRVP broth and sellers slants. None with respect to growth in comparison with malate as
of the strains could hydrolyse the starch. Very few strains control. The results obtained are presented in Table 2. All
WAS-6, WAS-11, WAS-29, WAS-30 could utilize the isolated strains could utilize citrate, L- malate,
succinate as sole carbon source. WAS-31 could utilize oxaloacetate, pyruvate, L-lactate, â- hydroxybutyrate, D-
succinate as sole carbon source and WAS-20, WAS-26, gluconate, D-mannitol and glycerol. Screening of different
Table 2 : Utilization of different compounds as the sole carbon sources by Azospirillum isolates.
Isolate Citrate Aconitate Isocitrate α –ketoglu Succinate Fumarate L-malate Oxaloac- Pyruvate L-lactate Malonate Acetate β–Hydroxy- n-
tarate etate butyrate propanol
WAS-1 + - - - - + + + + + - - + -
WAS-2 + - - - - + + + + + - - + -
WAS-3 + - - - + + + + + + - - + -
WAS-4 + - - - - + + + + + - - + -
WAS-5 + - - - - + + + + + - - + -
WAS-6 + - - - +/- + + + + + - + + -
WAS-7 + - - - - + + + + + + - + +
WAS-8 + - - + - +/- + + + + + - + -
WAS-9 + - - - - +/- + + + + + - + -
WAS-10 + - - - - + + + + + - - + -
WAS-11 + + - + +/- + + + + + - - + -
WAS-12 + - - - - + + + + + + - + -
WAS-13 + - - - - + + + + + - - + -
WAS-14 + - - - - + + + + + - - + -
S. Shubha et al
WAS-15 + - - - - +/- + + + + - - + -
WAS-16 + - - - - + + + + + - - + -
WAS-17 + - - - - + + + + + - - + -
WAS-18 + - - - - +/- + + + +/- + - + -
WAS-19 + - - - - + + + + + - - + -
WAS-20 + + - + +/- + + + + + - - + -
WAS-21 + - - - - + + + + + - - + -
WAS-22 + - - - - + + + + + - - + -
WAS-23 + - - - - + + + + + - - + -
WAS-24 + - - - - + + + + + - - + -
WAS-25 + - - - - + + + + + - - + -
WAS-26 + - - + + + + + + + - - + -
WAS-27 + - - - - + + + + + + - + -
WAS-28 + - - + + + + + + + - - + -
WAS-29 + - - + + +/- + + + + - - + -
WAS-30 + - - + + + + + + + - - + -
WAS-31 + - - - + + + + + + - - + -
WAS-32 + - - - - + + + + + - - + -
A.brasilence - - - - + + + + + + - - + -
A.lipoferum + - - - + + + + + + - - + -
Table 2 contd...
Table 2 : Utilization of different compounds as the sole carbon sources by Azospirillum isolates (Contd....).
Isolate L- L- L-Phenyl- L- L-Glu- L-Aspar- L- L-Hydroxl L- Glycine Putres- D-Gl- D-Glu- D- D- Glyce- L-aspp-
histidine Tyrosine alanine Alanine tomate agine Proline proline serine cine ucose conate Mannitol fructose rol artate
WAS-1 + - - + - + + + - - - - + + + + -
WAS-2 - - - - + - - - - - - + + + +
WAS-3 - - - - + - - - - - ++ + + + -
WAS-4 - - - - - - - - - - - + + + +
WAS-5 + - - - - - + + - - - +/- + + + + +
WAS-6 - - + - - - - - - - +/- + + + -
WAS-7 - - - - + - - - - - +/- + + + -
WAS-8 - - - - - - - - - - - + + + -
strains for carbohydrate utilization differed markedly with ketoglutarate as sole carbon source. None of the isolates
respect to the Azospirillum species and to the carbon could utilize isocitrate. Six strains could utilize succinate
source. However, all the 32 strains effectively oxidized and three of them however showed only weak growth.
the tested organic acids (Succinate, Malate and Pyruvate) All the isolates except 5 strains WAS-8, WAS-9, WAS-
when used as a sole carbon source auzanotrophically [in 15, WAS-18 and WAS-29 showed good growth on
presence of (NH2SO4)]. The preference of the organic fumarate medium. On L-lactate as sole carbon source,
acids by different Azospirillum species was reported all strains except WAS-18 showed good growth. On
earlier by (Reinhold et al, 1985). This can be explained malonate as sole carbon source, growth was observed in
on the basis that organic acids were the major source of only 6 strains. In acetate as carbon source, only WAS-6
nutrients for the microflora in the rhizosphere (Curl and strain could show good growth. Only WAS-7 isolate could
Truelove, 1986). utilize n-propanol as sole carbon source. None of the
On aconitate, growth was observed in only WAS -11 isolates could utilize L-histidine, L-tyrosine, L-
and WAS-20 strains. Only seven isolates could utilize á- phenylalanine, L-serine, glycine and putrescine. Only 7
among the 32 isolates could utilize L-alanine as a sole
Microbiological characteristics of Azospirillum strains
carbon source. On L- aspargaine as a sole carbon source approach and the Lon gene was found to be involved in
8 isolates could grow well. On L-proline, only few isolates the iron uptake of A. brasilense (Mori et al, 1992). Our
could grow well. 4 isolates grew well on L-hydroxy Azospirillum strains were examined for siderophore
proline. In D-glucose as a sole carbon source, only 6 production by testing their capability to grow in iron limiting
isolates could grew very well. All others showed weak (M-9) medium (Hugh and Leifson, 1953). Table 3 indicates
growth. Only 7 strains could utilize L- aspirate as sole that seven strains are able to grow well in the later medium,
carbon source. The additions of certain carbon source whereas three other strains exhibited moderate growth
encourage the growth of certain group of bacteria. For and the remaining strains cannot grow in that special
instance, addition of manitol or glucose in N2- free medium medium. More studies are needed to examine the
leads to the frequent isolation of Azotobacteraceae efficiency of such indigenous Azospirillum strains as it is
(Thompson et al, 1979), whereas malate leads to the well proved that native strains perform better in all the
isolation of Azospirillum (Okon et al, 1977) In the present plant nutrient management rather than new strain isolated
study, Azospirillum was isolated and enriched from the from elsewhere in plant microbial interactions studies
rhizosphere, rhizoplane or bulk soil using the nitrogen-free which could be better utilized for plant nutrient
biotin medium (NFb) in which L-malic was the sole C- management aspects.
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