M Meenakshisundaram & K Santhaguru / Int J Cur Sci Res. 2011; 1(2): 23 - 30.
24
Gluconacetobacter diazotrophicus is an endophytic bacterium
first isolated from the sugarcane growing regions of Brazil [5]. It
was widely studied and used as a model system to assess the
bacterial endophyte- plant interactions. After its first discovery, it
was reported from variety of crops viz, coffee [6, 7, 8] and a latest
report states Gluconactobacter sp as a natural colonizer of the wild
rice (Porteresia coarctata Tateoka, formerly Oryza coarctata
Roxb.) and a salt tolerant pokali rice variety [9] . These reports
clearly indicated the wide occurrence of G.diazotrophicus in
different plants than initially expected. G.diazotrophicus may fix
atmospheric nitrogen in the presence of nitrate [10]. Such
incomplete inhibition of nitrogen fixation is of ecological and
agronomic relevance, since it may allow the complementation of
biological nitrogen fixation in the presence of other N sources. This
aspect may favour the re-cultivation of agronomic lands, following
a prolonged period of chemical fertilization, alleviating
immobilization of N by soil microorganisms.
In recent times, attention has been focused on dual inoculation
involving AM fungi and nitrogen fixing bacteria on the growth of
several crop plants [11]. Positive effects of dual colonization of
non-leguminous plant roots by AM fungi and diazotrophic bacteria
were investigated on sugarcane [12] and rice [13]. Dual
inoculation could be especially advantageous in the case of
G.diazotrophicus, since this bacterium has not been isolated from
soil and the bacteria are mainly transmitted from plant to plant
through vegetative propogation by stem pieces [5]. Investigations
have shown that G.diazotrophicus may also be introduced in to
sterile micropropgated sugarcane, sweet potato and sweet
sorghum seedlings via arbuscular mycorrhizae [14, 15] .
Inoculation of G.diazotrophicus with or inside AM spores allowed
this bacterium to penetrate and to colonize the roots of plants and
then passing to the aerial tissue. Inoculation of AM fungi seems
therefore to be an essential condition for colonization of whole
plants by nitrogen fixing bacteria.
Selection of AM fungal strains for the improvement of crop
yields and diazotrophs efficiency should therefore consider inter
symbiotic compatibility besides host-plant compatibility, in order
to avoid unsuccessful field inoculations. In this paper an attempt is
made to identify AM fungi compatible to G.diazotrophicus by an
interaction study involving ten species of AM fungi and
G.diazotrophicus on Sorghum bicolor, a sugar rich crop widely
cultivated in India.
2. Materials and methods
2.1.Location of the sampling sites
Soil samples were collected from rhizosphere of Sorghum
bicolor grown in different localities of south Tamilnadu viz
Dindigul (Dindigul district), Kannivadi and Kovilpatti
(Virudhunagar district), Paravai and Samanatham (Madurai
district) for isolation of AM fungi and Saccharum officinarum from
Madurai district for isolation of G.diazotrophicus.
2.2.Determination of soil characters
Soil pH was determined in the soil: water 1:1 ratio soon after
bringing the soil samples to the laboratory. The total nitrogen (N),
Electrical conductivity (EC) and total phosphorus (P),
exchangeable potassium (K) were determined according to [16].
2.3.Media and cultural conditions
N-free semisolid LG1 medium supplemented with 0.5%
sugarcane juice at pH 4.5 and cycloheximide (150 mgl-1) was used
for enrichment culturing of N2-fixing G.diazotrophicus. For
isolation and culturing, acetic acid LG1 agar plates supplemented
with yeast extract (50mgl-1) and cyclohexamide (150mgl-1) and
potato agar plates with 10% cane sugar were used [5].
2.4.Isolation of G.diazotrophicus
Stem of the selected plants were washed with tap water and the
bud roots were exposed by removing the oldest leaves. The stems
were washed with sterile distilled water and surface sterilized for
5 min with 5% calcium hypochlorite, and then washed five times
with sterile distilled water. Thereafter, the samples were weighed
and homogenized in a sterile sucrose solution using a sterile using
pestle and mortar. Aliquots (500μl) were inoculated in to
semisolid LG1 and incubated at 30°C for 4-6 days. Yellowish
bacterial growth from the tubes was streaked on to LG1 plates [5]
and incubated at 30°C for 5-6 days. The colony morphology was
compared with G.diazotrophicus type strain PAL5.
2.5.Isolation of AM fungi
AM fungal spores were isolated from rhizosphere soils of
S.bicolor by a modification of the wet sieving and decanting
technique [17]. Aliquots (100g) of soil sample were dispersed in
1:1 water and the suspension left undisturbed for 15 min to allow
soil particles to settle. The suspension was then decanted through
710-and 38μm sieves. The sievates were dispersed in water and
filtered through gridded filter papers. Each filter paper was then
spread on a petri dish and scanned under a dissection microscope
at 40 X magnification and all intact spores (non-collapsed spores
with cytoplasmic contents and free from parasitic attack) were
counted. Sporocarps and spores clusters were considered as one
unit. Intact AM fungal spores were transferred using a wet needle
to polyvinyl alcohol-lactoglycerol with or without Melzers reagent
on a glass slide for identification. Spores were identified from
spore morphology and subcellular characters and compared to
original descriptions [18]. Spore morphology was also compared
to the culture database established by INVAM
(http//invam.cag.wvu.edu).
2.6.Estimates of AM fungal colonization
The AM infected roots were cut into 1-cm fragments, cleared in
2.5% KOH [19] acidified with 5N HCL and stained with trypan
blue (0.05% in lactophenol). Roots that remained dark after
clearing were bleached in alkaline H2O2 prior to acidification. The
stained roots were examined with a compound microscope (200-
400X) for AM fungal structures and the percentage of root
colonization was estimated according to a magnified intersection
method [20].
2.7.Experimental design
Sweet sorghum (Sorghum bicolor) seeds obtained from var
Co27 from Tamil Nadu seed testing centre, Madurai were
sterilized with H2O2 for one hour, and germinated under aseptic
conditions in steam-sterilized sand and soil mixture (twice at
120°C for 30 min, with an interval of 24h between both
autoclaving). Ten seeds after germination, were transplanted in 4: