| In the future,the demand for rice will be higher because of high consumption of rice by the increasing world population.The reduction in agricultural lands and severe diseases of rice crop further aggravate the situation.Among these diseases,rice sheath blight is the second most prevalent disease after rice blast known to be a globally important disease of rice crop.Rice sheath blight is caused by Rhizoctonia solani which is one of the most highly destructive fungi on rice crop.The only reliable method to control this disease was the application of fungicides however fungicides have created environmental and organism’s health issues.Despite ongoing management practices,rice sheath blight is continuously threatening rice crop in the world.This necessitates screening of Biocontrol agents（BCAs）which should be cost-effective and ecofriendly as an alternative to fungicides.Keeping in view the importance of BCAs,the following are abstracts of the results achieved.In the present study,fungal BCAs candidates were screened from the fallow and cultivated soil of rice fields.The rice field soil samples were divided into two sets,one set of soil were subjected to soil microbiome analysis to determine the relative abundance of BCAs candidates,rice pathogens i.e.R.solani and microbial diversity.The other set of soil samples were subjected to traditional BCAs candidate’s isolation method by burying sclerotia of R.solani in the soil as a bait to trap BCAs candidates.The BCAs candidates which infected（colonized and/or rotted）the sclerotia were further evaluated against the sclerotia and mycelia of R.solani.Soil microbiome analysis revealed that phyla Ascomycota was predominantly found in both soil types.The relative abundance of BCAs such as Talaromyces,Coniothyrium minitans,rice pathogens such as Rhizoctonia and microbial diversity was higher in the cultivated soil than in the fallow soil.Using the traditional method of isolation,a total of 17 fungal BCAs candidates were isolated using Almost all the BCAs candidates significantly caused sclerotial infection.Further,these BCAs candidates were subjected to dual culture assays to evaluate their overgrowth potential against R.solani.All BCAs candidates varied in their overgrowth on R.solani against the mycelia and no considerable zone of inhibition was found between the BCAs candidates and R.solani.The most effective BCAs candidates were identified as Trichoderma isolates and Talaromyces isolates.Majority of BCAs candidates related to Talaromyces isolates were retrieved from the cultivated soil.Hence,both soil microbiome analysis and traditional method indicated the importance of these novel Talaromyces isolates and therefore,the biological potential of these Talaromyces isolates were further evaluated.Five Talaromyces isolates（namely,TF-04,TF-03,TF-02,TF-01 and TA-02）from the above experiments were further evaluated against the sclerotia and hyphae of R.solani under different in-vitro conditions.These isolates were grown in different culture media（PDA,PDB,MEA,and CYA）,p H（3,4,5,6,7 and 8）,osmotic potentials（KCL（g/L）:0,1,2,3,4,and 5）and temperatures（20,24 and 28oC）.The most remarkable growth of all the isolates was achieved at 28°C,p H 4-5.0,culture media（PDA）and Osmotic potentials（0-0.3 g/L）.The colony growth（cm）,no of spores and biomass of all the isolates were recorded higher in such conditions.Among the isolates,TF-04 gave better results followed by TF-01,TF-02,TF-03 and TA-02.About morphology,all Talaromyces isolates produced ellipsoid smooth-walled conidia and spiny type ascospores.The TA-02 conidiophore branching was monoverticillate to biverticillate while TF-04,TF-01 and TF-02 were monoverticillate and TF-03 was monoverticillate with subterminal or side branches.Basing on the DNA sequences of ITS,β-tubulin（Ben A）,Calmadolin（Ca M）and RNA polymerase II second largest subunit（RPB2）,isolates TF-01,TF-02,TF-03 and TF-04 were close to Ta.flavus,while TA-02 were close to Ta.assiutensis.After setting optimum temperature,culture media and p H,the isolates were subjected to evaluate their parasitic abilities against the hypha and sclerotia of R.solani.In dual culture assays,among the isolates,TF-04 significantly debilitated the mycelia of all the R.solani.Isolate TA-02 was not highly effective in suppression of hyphal growth of the pathogen as compared to other isolates.Moreover,light microscopy revealed coiling of hyphae of R.solani by TF-04,TF-03,TF-02 and TF-01,however no coiling around the hyphae of R.solani were noted in case of isolate TA-02.Further antifungal compounds（Cultural filtrates,Volatiles and Crude extracts）of all the isolates significantly reduced the hyphae of R.solani.Afterwards,sclerotia of R.solani were treated with the spore’s suspension of the isolates and then buried on autoclaved sand,in soil types（autoclaved and non-autoclaved）with different temperatures（20-32°C）and in different soil water potentials（1-11 MPa）.In sand,among the isolates,TF-04 and TF-03significantly rot the sclerotia with values 85%and 83%respectively,followed by TF-01and TF-02 with values 81.66%and 80%.Meanwhile,the isolate TA-02 caused only38.33%sclerotia rotted.Similarly,in soil types and different soil water potentials,the percentage of sclerotia viability fluctuated with the majority of the isolates characterized by high anti-sclerotial activity.In conclusion,isolates TF-04 followed by TF-01,TF-02and TF-03 in the majority of trials significantly inhibited the mycelial growth and sclerotia of R.solani;meanwhile,isolate TA-02 was declared weak antagonist and were omitted from other experiments.The most effective isolate TF-04 from the above experiment was optimized by treating the rice seeds and seedlings with the mycelia and spores at different period in growth chamber to determine an optimum spore suspension and time which would be used in the pot-based greenhouse and outdoor experiments.The optimum spore suspension for treating rice seeds and seedlings was found to be 107spores m L-1for 6 h which significantly enhanced vegetative parameters（shoot height,root height,fresh shoot weight,fresh root weight,dry shoot weight and dry root weight）in the treated rice seedlings as compared to untreated control rice seedlings in series of growth chamber trials.Afterwards,rice seeds and seedlings were treated with the optimum spore suspensions of isolates TF-04,TF-03,TF-02 and TF-01 to study various physiological,biochemical,pathological and yield parameters in a pot-based greenhouse and outdoor conditions by inoculating R.solani.In both pot-based greenhouse and outdoor conditions,application of Talaromyces isolates through seed treatment was found to be more effective in suppression of rice sheath blight disease.All isolates significantly reduce disease index of rice sheath blight in the treated plants as compared to untreated control plants.In pot-based greenhouse experiment in seed treated plants with four isolates suggested that among the isolates,TF-04 had significantly reduced rice sheath blight index（1.93 fold,37.7%）in comparison to untreated control plants（72.75%）on 20 days post-inoculation with R.solani on rice sheaths.Similarly,in the seedling treated plants,TF-04 reduced rice sheath blight index（1.45 fold,48.40%）in comparison to untreated control plants（79.36%）.Moreover,in the TF-04 treated plants higher plant height（cm）,biomass（g）,no of tillers,grain yield per/pot and 1000 filled grain wt.were recorded as compared to other isolates and untreated control plants.Furthermore,in pot-based outdoor conditions,on 20 days post-inoculation with R.solani highest disease index was recorded in untreated control plants as compared to Talaromyces isolates treated plants.Among the isolates tested,TF-04 treated plants were found to have increased total plant height（1.16 fold）,fresh biomass（1.74 fold）,dry biomass（1.55 fold）,number of tillers per hills（1.89 fold）,productive tillers per hill（2.24 fold）,panicle length（1.06 fold）,1000g yield wt.（1.16 fold）and grain yield（1.41fold）folds as compared to untreated control plants.In another pot-based outdoor trial,the rice seeds were treated with spore suspensions(107,106and 105spores m L-1)of the most effective isolate TF-04 and control rice seeds were treated with sterilized distilled water.Results suggested that all the spore’s suspensions have significantly increased the vegetative parameters as compared to untreated control plants but the most effective suspension was considered 107spores m L-1.Also,the scanning electron microscopy and GFP-labelled TF-04 isolate could colonize on the rice roots and defense enzyme activities,total chlorophyll contents and relative expression of defense genes were also significantly higher in the treated rice plants as compared to untreated control plants.In conclusion,Ta.flavus isolates which could significantly antagonize rice sheath blight and promote rice yield,was screened out from rice soil by combining traditional isolation method and soil microbiome analysis.As far as we know,this is the first time that we show Ta.flavus has a considerable potential to control rice diseases. |
