S, Priyanka. V, Rajesh Kumar. N, Saiganesh.V. S, Santhosh. M, Iyappan. S Screening of plant growth promoting rhizobacteriawith antifungal activity for Fusariumoxysporum AbstractThe plant growth promoting bacteria present in rhizosphere (PGPR)of many plants species have a beneficialeffect on plants either in a direct (nutrients and hormones) or indirect manner(defence mechanism).
The antagonistic propertyof plant growth promoting bacteria was used to resist the growth of variousfungal pathogens. The isolates of bacteria from the plant of solanum lycopersicum and Arachi shypogaea which showed positivefor IAA production and phosphate solubilisation were subjected for antifungalactivity against for Fusarium oxysporum. The four isolates were found to contain antifungalproperty towards the plant pathogen Fusarium oxysporum. The plant growthpromotion assay was done using the four isolates and control for which theseeds of Vigna radiata were used.This resulted in the increase of rootlength, shoot length, wet weight and dry weight for the four isolates whencompared to control.Keywords- Rhizosphere · Plant Growth PromotingBacteria · Antagonist · Fungal pathogens .
Auxin · Phosphate solubilisation· IntroductionPlants are prone to the infectionby many fungal species. The food and agriculture organization states that pestsand disease are responsible for most of the crop loss worldwide1.It has been stated that plant disease are responsible for 10% of yield lossevery year in more developed area and 20% in less developed area. Among thesemost virulent diseases are caused by fungal species. Most of the pathogenicfungi belong to the class Ascomycetes (e.g.
Fusarium wilt disease by Fusariumsp). 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 otherstructures. Spores are widely distributed in soil and associated with manyplants. Fungal diseases arecontrolled by the use of chemical fungicides, however the fungi developedresistance to various fungicides as time prolongs and the fungicide practisecauses environmental pollution3. Therefore bio-control for fungalpathogens can be developed by antagonistic rhizobacteria which probably doesnot cause any environmental pollution. Most of the rhizobacteria are PlantGrowth Promoting Bacteria (PGPR).
The PGPR wereable to control the number of pathogenic bacteria and fungi through microbialantagonism, which is achieved by competing with the pathogens. PGPR helps plantgrowth and defense by either direct or indirect mechanism. In direct mechanismrhizhobacteria synthesize phytohormones for plant growth and promotes growthfurther by fixing nitrogen and solubilizing organic phosphates4.
Where in indirect mechanism rhizobacteria provide defense by producing variousantibiotics and lytic enzymes that inhibits the growth of other plantpathogenic microorganisms. Besides antagonism, certainplant-microbe interactions can induce mechanisms in which the plant can betterdefend itself against pathogenic bacteria, fungi and other micro organisms.This kind of resistance is called Induced systemic resistance (ISR)5where the bacterial components like lipopolysaccharides, homoserine lactone,acetoin, 2, 3-butanediol stimulates the plant defence mechanism by inducing thejasmonate and ethylene signaling6. Materialand methodsIsolation and identification of fungiFor fungi isolation, leaves were surfacesterilized with 95% ethanol, and lesions were cut from the infected leaves of Solanum lycopersicum.
The infectedleaves were cut and placed on petriplate containing potato dextrose agar. Theplates were incubated at 28ºC for 3-5 days7. After thegrowth of fungi the microscopic observation was done with lactophenol cottonblue staining method. The molecular identification was done by amplifying ITSregion using PCR. Specific primers for ITS region were designed and then PCRamplification was done for fungal DNA sample. The PCR condition for thedesigned primers was 95?C for 10 minutes as initial denaturation, followed by35 cycles of denaturation at 95?C for 30 seconds, annealing at 38?C for 30seconds, extension at 72?C for 20 seconds and at last final extension at 72?Cfor 7 minutes8.
The amplifiedPCR product was subjected to purification and then sequenced. Isolation and identification ofPGPR Theplants such as Solanum lycopersicum and Arachis hypogaea, wascollected from the fields located in kancheepuram. The roots of the plants werewashed in autoclaved distilled water and the roots of the plants were cut intosmall pieces and were allowed to incubate for 1-2 hrs in the conical flaskscontaining distilled water at 37ºC. From the incubated sample 1ml wasserially diluted and spread on LB agar plates and the plates were incubated at37ºC overnight. The bacteria withunique morphology were pure cultured and were subjected to DNA isolation9.For molecular identication the DNA samples were amplified for 16S rRNA usinguniversal 16S ribotyping primers. The PCR condition for the primers was 95?Cfor 10 minutes as initial denaturation followed by 35 cycles for denaturationat 95?C for 30 seconds, annealing at 55?C for 30 seconds, extension at 72?C for1.
5 minutes with a final elongation step at 72?C for 5 minutes. Antifungalactivity by PGPR Theantifungal activity was screened by well diffusion method. The 100µl of fungal culture was spread on Potato dextrose agar platesand overnight grown exponential phase bacterial cultures were adjusted to 0.4OD at 580nm and 5µl of culture was diffused into the wells of PDA plates. Theplates were incubated at 28ºC for 2-3 days. The bacterium with antifungalactivity shows the zone of inhibition. Assayfor IAA production The isolates were screened for indole aceticacid production.
The qualitative and quantitative determination of IAAproduction was performed by isolates showing antifungal activity which weregrown in LB broth added with 0.1mg per ml tryptophan and incubated at 32°C for3 days. Broth containing bacterial isolates was centrifuged, 100µl of supernatant from each samplewas transferred to 96-microwell plate and 150µlof Salkowski reagent (1mL of 0.5M FeCl3 and 50mL of 35% HClO4) was added toeach well. The samples were incubated at room temperature for 25 minutes indark and in presence of auxin color of the mixture in plate changes to pink ordeep red color and their absorbance was measured at 540nm10. Auxinquantification value was done by extrapolating calibration curve made using IAAas standard11. Assay for phosphate solubilisation The qualitative estimation of phosphate solubilisationwas 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 Pikovskayaagar plate. The isolates which were able to solubilise the inorganic phosphate showshalo zone of clearance4. Plant growth promotion assay The bacterial isolates B-53,rh-1,11-5,13-1 weregrown in LB broth and adjusted to 0.4 OD at 580nm. 10 ml of culture was takenand centrifuged and the pellet was dissolved in saline water, then used forgrowth promotion assay. In order to estimate the growth promotion the seeds of Vignaradiata were surface sterilized with sodium hypochlorite and tween 20 andkept overnight for germination. The seeds germinated in equal size wereselected for planting. The soil used for plant growth was autoclaved to avoidcontamination.
In a 1500g of soil the bacterial isolates containing 109 cfu per ml were mixedand germinated seeds were planted. Plants were kept in light for 16 hours and 8hours in dark. Plantswere irrigated with Hoagland solution and sterilized water in the ratio of 1:1for every 48 h (300 mL per pot).The plants were uprooted after 12days and measurements such as shoot length, root length, fresh and dry weightswere determined13. Results and discussions Molecularcharacterization of isolated bacteria and fungi Colony PCR wasperformed for the bacterial isolated using 16s FP and RP.
100µl of PCR mixture was purified by using Quiagen purification kit. Thepurified DNA template are sequenced using 16s primers. Among five bacterialisolates four bacteria shows 99% similarity to different pseudomonasaeruginosa strains and one bacterial strain shows 99% similarity to pseudomonas putida.. The fungal DNA wasextracted using glass bead method.
And the extracted DNA was amplified using ITS FP and RP with the genesize of 400 bp. From the sequencing result the isolated fungi is identified asfusarium oxysporum, which is a plant pathogen. Antifungal activity The bacterial strainsare subjected to antifungal activity for fusarium oxysporum. All six bacterialisolates were able to inhibit the growth of fusarium oxyporum species. Amongsix two pseudomonas aeruginosa strain shows more zone of clearance compared tothe other bacterial strains. This bacteria able to inhibit the growth of fungiafter two days of incubation under normal room temperature. Plant Growth promotionThe growth promotion assays include the tests for auxinproduction, phosphate solublisation, and testing for improved growth of plants(Vigna radiata) by mixing bacteria in soil. This test includes shootlength, root length, dry and wet biomass for determining the increase in growthby the rhizobacteria.
Indole acetic acidIAAwas one of the important plant growth hormones. Its function in plants was cellproliferation and cell elongation. The bacterial isolates has able to produceIAA with different pathways at different concentration. This bacterial trainsproduce IAA in presence of tryptophan in the concentration of 1µg-5µgper ml. Among six bacterial isolates 53 and rh-1 shows highest production ofIAA compared to other strain. Other bacterial isolates produce IAA but inminimal level. This result is based on the standard curve made by differentconcentration of IAA. PhosphatesolubilizationThequalitative estimation of phosphate solubilization was done in pikovskaya agarwhich is rich in tri calcium phosphate.
Rhizobacteria have ability to reducetri calcium phosphate to mono calcium phosphate which is readily absorbed bythe plant. This bacterial isolates have ability to degrade tri calciumphosphate in two days. After one days of incubation halo zone was formed aroundthe well. Pseudomonas putida shows more zone of clearance then other bacteria. Thisshows bacteria reduced phosphate and this will help potential plant growth promoterin agriculture.
Plantgrowth promotionFor growth promotion assay plants were inoculatedwith bacteria at CFU of log 9 after twelve days of planting the plants areuprooted carefully to measure shoot length, root length and biomass.Plantswhich are inoculated with rhizobacteria shows increase in root and shoot lengthcompared to control. In five bacterial strains pseudomonas putida and one ofpseudomonas aeruginosa shows significant increase 26% – 30% increase in shootlength.
While comparing root length 53 and rh2 shows significant increase inroot length about 15% – 20%,compared to other bacteria. Bacterial isolates b53shows more yield of biomass nearly 27% compared to non-inoculant and otherbacteria shows slight increase in biomass. ConclusionThisis a basic study of screening rhizobacteria from groundnut rhizosphere. We haveconcluded 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 organicform and also inhibit the activity of Fusarium oxysporum, hence thesebacterial species can be used as a potential bio-fertilizers and fungicide forplants infected by Fusarium.