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 oxysporum
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 (ISR)5 where the bacterial components like lipopolysaccharides, homoserine lactone, acetoin, 2, 3-butanediol stimulates the plant defense 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; 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 PGPR
The 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 ……………..
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 added into the wells of PDA plates. The plates were incubated at 28ºC for 2-3 days.
Assay 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
Molecular 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 bacteria
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.
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 53.
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 strains.
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
The 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.
Fig.5.Phosphate 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
This 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.