Kanchana. S, Priyanka. V, Rajesh
Kumar. N, Saiganesh.V. S, Santhosh. M, Iyappan. S

 

Screening of plant growth promoting rhizobacteria
with antifungal activity for Fusarium
oxysporum

 

Abstract

The plant growth promoting bacteria present in rhizosphere (PGPR)
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 Arachi shypogaea which showed positive
for IAA production and phosphate solubilisation were subjected for antifungal
activity against  for 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 four isolates and control for which the
seeds of Vigna radiata were used.
This resulted in the increase of  root
length, shoot length, wet weight and dry weight for the four isolates when
compared to control.

Keywords- Rhizosphere · Plant Growth Promoting
Bacteria · Antagonist · Fungal pathogens  .  Auxin  ·  Phosphate  solubilisation

·        
 

Introduction

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 mainly infects banana, tomato and rice pants,
which are most predominantly used food crops all over the world2.
The fungi reproduce by both sexually and asexually via spores and other
structures. Spores are widely distributed in soil and associated with many
plants. Fungal diseases are
controlled by the use of chemical fungicides, however the fungi developed
resistance to various fungicides as time prolongs and the fungicide practise
causes 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 rhizobacteria are Plant
Growth Promoting Bacteria (PGPR). The PGPR were
able to control the number of pathogenic bacteria and fungi through microbial
antagonism, which is achieved by competing with the pathogens. 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
pathogenic microorganisms. Besides antagonism, certain
plant-microbe interactions can induce mechanisms in which the plant can better
defend itself against pathogenic bacteria, fungi and other micro organisms.
This kind of resistance is called Induced systemic resistance (ISR)5
where the bacterial components like lipopolysaccharides, homoserine lactone,
acetoin, 2, 3-butanediol stimulates the plant defence mechanism by inducing the
jasmonate and ethylene signaling6.      

 

Material
and methods

Isolation and identification of fungi

For fungi isolation, leaves were surface
sterilized with 95% ethanol, and lesions were cut from the infected leaves of Solanum lycopersicum. The infected
leaves were cut and placed on petriplate containing potato dextrose agar. The
plates were incubated at 28ºC for 3-5 days7. After the
growth of fungi the microscopic observation was done with lactophenol cotton
blue staining method. The molecular identification was done by amplifying ITS
region using PCR. Specific primers for ITS region were designed and then PCR
amplification was done for fungal DNA sample. 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 sequenced.

 

Isolation and identification of
PGPR

 

The
plants such as Solanum lycopersicum and Arachis hypogaea, was
collected from the fields located in kancheepuram. 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 hrs 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 pure cultured and were subjected to DNA isolation9.
For molecular identication the DNA samples were amplified for 16S rRNA using
universal 16S ribotyping primers. The PCR condition for the primers was 95?C
for 10 minutes as initial denaturation followed by 35 cycles for denaturation
at 95?C for 30 seconds, annealing at 55?C for 30 seconds, extension at 72?C for
1.5 minutes with a final elongation step at 72?C for 5 minutes.      

 

Antifungal
activity by PGPR

 

The
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 diffused into the wells of PDA plates. The
plates were incubated at 28ºC for 2-3 days. The bacterium with antifungal
activity shows the zone of inhibition.

 

Assay
for IAA  production

 

The isolates were screened for indole acetic
acid 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 32°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 25 minutes in
dark and in presence of auxin color of the mixture in plate changes to pink or
deep red color and their absorbance was measured at 540nm10. Auxin
quantification value was done by extrapolating calibration curve made using IAA
as standard11.

 

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-1,11-5,13-1 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, 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. The seeds germinated in equal size were
selected for planting. The soil used for plant growth was autoclaved to avoid
contamination. In a 1500g of soil the bacterial isolates containing 109 cfu 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

 

Molecular
characterization of isolated bacteria and fungi

 

Colony PCR was
performed for the bacterial isolated using 16s FP and RP. 100µl of PCR mixture was purified by using Quiagen purification kit. The
purified DNA template are sequenced using 16s primers. Among five bacterial
isolates four bacteria shows 99% similarity to different pseudomonas
aeruginosa strains and one bacterial strain shows 99% similarity to pseudomonas putida.

.

                             

 

The fungal DNA was
extracted using glass bead method. And the extracted DNA was  amplified using ITS FP and RP with the gene
size of 400 bp. From the sequencing result the isolated fungi is identified as
fusarium oxysporum, which is a plant pathogen.

 

Antifungal activity

 

The bacterial strains
are subjected to antifungal activity for fusarium oxysporum. All six bacterial
isolates were able to inhibit the growth of fusarium oxyporum species. Among
six two pseudomonas aeruginosa strain shows more zone of clearance compared to
the other bacterial strains. This bacteria able to inhibit the growth of fungi
after two days of incubation under normal room temperature.

 

                                                                            

 

 

 

 

 

 

 

 

    
             Plant Growth promotion

The growth promotion assays include the tests for auxin
production, phosphate solublisation, and testing for improved growth of plants
(Vigna radiata) by mixing bacteria in soil. This test includes shoot
length, root length, dry and wet biomass for determining the increase in growth
by the rhizobacteria.

Indole acetic acid

IAA
was one of the important plant growth hormones. Its function in plants was cell
proliferation and cell elongation. The bacterial isolates has able to produce
IAA with different pathways at different concentration. This bacterial trains
produce IAA in presence of tryptophan in the concentration of 1µg-
5µg
per ml. Among six bacterial isolates 53 and rh-1 shows highest production of
IAA compared to other strain. Other bacterial isolates produce IAA but in
minimal level. This result is based on the standard curve made by different
concentration of IAA.

 

Phosphate
solubilization

The
qualitative estimation of phosphate solubilization was done in pikovskaya agar
which is rich in tri calcium phosphate. Rhizobacteria have ability to reduce
tri calcium phosphate to mono calcium phosphate which is readily absorbed by
the plant. This bacterial isolates have ability to degrade tri calcium
phosphate in two days. After one days of incubation halo zone was formed around
the well. Pseudomonas putida shows more zone of clearance then other bacteria. This
shows bacteria reduced phosphate and this will help potential plant growth promoter
in agriculture.

 

Plant
growth promotion

For growth promotion assay plants were inoculated
with bacteria at CFU of log 9 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 pseudomonas putida and one of
pseudomonas aeruginosa shows significant increase 26% – 30% increase in shoot
length. While comparing root length 53 and rh2 shows significant increase in
root length about 15% – 20%,compared to other bacteria. Bacterial isolates b53
shows more yield of biomass nearly 27% compared to non-inoculant and other
bacteria shows slight increase in biomass.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Conclusion

This
is a basic study of screening rhizobacteria from groundnut rhizosphere. We have
concluded that among all the rhizobacteria obtained, B-53, Rh-1, B-7(1),
B-13(1), B-11(5) has shown more potential to produce plant growth hormone,  solublise inorganic phosphates 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.