Priyanka by many fungal species. The food

Priyanka V,Kanchana S, Rajesh Kumar
N, Saiganesh V S, Santhosh M, Usha B and  Iyappan S


Screening of plant growth promoting Rhizobacteria
with antifungal activity for Fusarium

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The plant growth promoting rhizobacteria (PGPR) present in
rhizosphere of many plants species have a beneficial effect on plants either in
a direct (nutrients and hormones) or indirect manner (defence mechanism). The antagonistic
property of plant growth promoting bacteria was used to resist the growth of
various fungal pathogens. The isolates of bacteria from the plant of Solanum lycopersicum and Arachis hypogaea which showed positive
for Indole-3-acetic acid (IAA) production and phosphate solubilisation were
subjected for antifungal activity against Fusarium
oxysporum. The four isolates were found to contain antifungal property
towards the plant pathogen Fusarium oxysporum.
The plant growth promotion assay was done using the five isolates using Vigna radiata.  This
resulted in increase of  root length,
shoot length, wet and dry biomass for five isolates. 

Keywords- Rhizosphere · Plant Growth Promoting Rhizobacteria
· Fungal pathogens · Auxin · Phosphate  solubilisation



Plants are prone to the infection by many fungal species. The
food and agriculture organization states that pests and disease are responsible
for most of the crop loss worldwide1.
It has been stated that plant disease are responsible for 10% of yield loss
every year in more developed area and 20% in less developed area. Among these
most virulent diseases are caused by fungal species. Most of the pathogenic
fungi belong to the class Ascomycetes (e.g. Fusarium wilt disease by Fusarium
sp.). The Fusarium
sp. mostly infects banana,
tomato and rice pants, which are most predominantly used food crops all over
the world2.
Fungi reproduce by both sexually and asexually via spores. Spores are widely
distributed in soil and associated with many plants. Fungal diseases are controlled by use of chemical
fungicides, however the fungi developed resistance to various fungicides as
time prolongs and the usage of fungicide 
leads to environmental pollution3. Therefore bio-control for fungal
pathogens can be developed by antagonistic rhizobacteria which probably does
not cause any environmental pollution. Most of the Plant Growth Promoting Rhizobacteria
(PGPR) were able to control the number of pathogenic bacteria and
fungi through microbial antagonism.  PGPR
helps plant growth and defense by either direct or indirect mechanism. In
direct mechanism rhizhobacteria synthesize Phytohormones for plant growth and
promotes growth further by fixing nitrogen and solubilizing organic phosphates4.Where
in indirect mechanism, Rhizobacteria provide defense by producing various
antibiotics and lytic enzymes that inhibits the growth of other plant pathogens. Besides
antagonism, certain plant-microbe interactions can induce mechanisms in which
the plant can better defend itself against pathogenic bacteria, fungi and other
microorganisms. This kind of resistance is called Induced systemic resistance
where the bacterial components like lipopolysaccharides, homoserine lactone,
acetoin, 2, 3-butanediol stimulates the plant defense mechanism by inducing the
jasmonate and ethylene signaling6.      


and methods

Isolation and identification of fungi

For fungi isolation, leaves were surface
sterilized with 95% ethanol; lesions were cut from the infected leaves of Solanum lycopersicum and placed on
potato dextrose agar. After incubation at 28ºC for 3-5 days7, the fungi were
stained with lacto phenol cotton blue and observed under microscope.   The
molecular identification was done by amplifying ITS region using the primers
………….. and ……………….. The PCR condition for the designed
primers was 95?C for 10 minutes as initial denaturation, followed by 35 cycles
of denaturation at 95?C for 30 seconds, annealing at 38?C for 30 seconds,
extension at 72?C for 20 seconds and at last final extension at 72?C for 7
minutes8. The amplified
PCR product was subjected to purification and then sequencing. 


Isolation and identification of


plants such as Solanum lycopersicum and Arachis hypogaea, was
collected from the agriculture fields.  The
roots of the plants were washed in autoclaved distilled water and the roots of
the plants were cut into small pieces and were allowed to incubate for 1-2
hours in the conical flasks containing distilled water at 37ºC. From the
incubated sample 1ml was serially diluted and spread on LB agar plates and the
plates were incubated at 37ºC overnight. The bacteria with unique morphology were isolated and identified by ribotyping
using 16S universal primers  …………
and ……………..



activity by PGPR


antifungal activity was screened by well diffusion method. The 100µl of fungal culture was spread on Potato dextrose agar plates
and overnight grown exponential phase bacterial cultures were adjusted to 0.4
OD at 580nm and 5µl of culture was added into the wells of PDA plates. The
plates were incubated at 28ºC for 2-3 days.


for IAA production


The isolates were screened for IAA production.
The qualitative and quantitative determination of IAA production was performed
by isolates
showing antifungal activity which were grown in LB broth added with 0.1mg per
ml tryptophan and incubated at 30°C for 3 days. Broth containing bacterial
isolates was centrifuged, 100µl of supernatant
from each sample was transferred to 96-microwell plate and 150µl of Salkowski reagent (1mL of
0.5M FeCl3 and 50mL of 35% HClO4) was added to each well. The samples were
incubated at room temperature for 25minutes in dark and in presence of IAA, colour
of the mixture in plate changes to pink or deep red colour and their absorbance
was measured at 540nm10. IAA quantification
was done using IAA standards11.


Assay for phosphate solubilisation


The qualitative estimation of phosphate solubilisation
was performed by well diffusion method using Pikovskaya agar12.
Overnight grown bacterial culture was adjusted to 0.4 OD at 580nm and 5µl of sample were loaded onto the wells punctured on Pikovskaya
agar plate. The isolates which were able to solubilise the inorganic phosphate shows
halo zone of clearance4.


Plant growth promotion assay


The bacterial isolates B-53, rh-1, rh-2, 7-1, 11-5,
13-1 were grown in LB broth and adjusted to 0.4 OD at 580nm. 10 ml of culture
was taken and centrifuged and the pellet was dissolved in saline water, and
then used for growth promotion assay. In order to estimate the growth promotion
the seeds of Vigna radiata were surface sterilized with sodium
hypochlorite and tween 20 and kept overnight for germination. Germinated seeds in
equal size were selected for planting. The soil used for growing plants was
autoclaved. In a 1500g of soil the bacterial isolates containing 109cfu per ml were mixed and germinated seeds were
planted. Plants were kept in light for 16 hours and 8 hours in dark. Plants were
irrigated with Hoagland solution and sterilized water in the ratio of 1:1 for
every 48 h (300 mL per pot).The  plants
were uprooted  after 12 days and measurements
such as shoot length, root length, fresh and dry weights were determined13.


Results and discussions


characterization of isolated bacteria and fungi


1.5 Kb 16S rDNA gene was
amplified for the isolated bacterial strains (fig).  The PCR products were purified using Quiagen
purification kit and sequenced using 16s primers. Among six bacterial isolates,
five shows 99% similarity to different Pseudomonas aeruginosa strains and one strain
shows 99% similarity to Pseudomonas putida.






Fig.1.Gel picture for 16s  gene for





400 bp of ITS was
amplified (fig) and sequenced.  This
sequence was subjected to blast analysis against NCBI database which showed
that the isolated fungi are Fusarium oxysporum
with 99% similarity.




Antifungal activity


The bacterial strains
are subjected to antifungal activity against Fusarium oxysporum (fig). All six bacterial isolates were able to
inhibit the growth of Fusarium oxyporum
species. Among six 7-1, 11-5, 13-1 strain shows more zone of clearance compared
to the other bacterial strains. These bacteria able to inhibit the growth of
fungi after two days of incubation under normal room temperature.

Fig.3. Inhibition of Fusarium
oxysporum by bacterial isolates. (A)  Inhibition by bacteria Rh-1 and Rh-2. (B)
Inhibition by bacteria 7-1 and 11-5. (C) Inhibition by bacteria 13-1 and








Plant Growth promotion

The growth promotion assays includes, tests for auxin
production, phosphate solublisation, and testing for plant growth promotion. The
plant growth promotion was performed by growing Vigna radiata  in presence and absence of isolated bacterial

Indole acetic acid

IAA was one of the important plant
growth hormone which helps in cell proliferation and elongation. The bacterial
isolates have the ability to produce IAA with different pathways at different
concentration. These bacterial strains produce IAA in presence of tryptophan in
the concentration of 1µg- 5µg per ml. Among six
bacterial isolates 53 and 13-1 shows highest production of IAA compared to
other strains (fig).

Fig.4.IAA produced by rhizobacteria
represented in µg/ml



qualitative estimation of phosphate solubilisation was done in Pikovskaya agar
which is rich in tri calcium phosphate. After three days of incubation, zone of
clearance were observed in plate and was documented (fig).  The isolated rhizobacteria have the ability
to reduce tri calcium phosphate to mono calcium phosphate which is readily
absorbed by the plant. Other than B-53 all shows more zone of clearance. This
shows bacteria reduced phosphate and this will help potential plant growth promoter
in agriculture.


solubilisation by bacteria shows positive result. Rh-1, Rh-2, 7-1, 11-5, 13-1 shows
more halo zone of clearance.

Plant growth promotion

For growth promotion assay plants were inoculated
with bacteria at 109 cfu per ml.  After
twelve days of planting, the plants are uprooted carefully to measure shoot
length, root length and biomass. Plants which are inoculated with rhizobacteria
shows increase in root and shoot length compared to control. In five bacterial
strains B-53 and 7-1 Shows significant increase (26% – 30%) in shoot length. While
comparing root length B53 and rh2 shows significant increase in root length
about 15% – 20%, compared to other bacteria (fig). Bacterial isolates B53 shows
more yield of biomass nearly 27% compared to non-inoculant and other bacteria
shows slight increase in biomass (fig).

Fig.6.Graph representing growth promotion by bacteria increases shoot and
root length













Fig.7.Graph representing rhizobacteria increases the biomass of plant
compared to control













is a basic study of screening rhizobacteria from Solanum lycopersicum and Arachis hypogaea Rhizosphere. We have
concluded that among all the rhizobacteria obtained, B-53, Rh-1, B-7(1),
B-13(1) and B-11(5) were shown more potential to produce plant growth hormone, solubilise
inorganic phosphate to organic form and also inhibit the activity of Fusarium
oxysporum, hence these bacterial species can be used as a potential
bio-fertilizers and fungicide for plants infected by Fusarium.


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