Flavonoids Enhancement And Alleviation Of Stress In Brassica Campestris Ssp Chinensis By The Exogenous Inoculation Of Piriformospora Indica

Chinese cabbage(Brassica Campestris spp.chinensis),which belongs to the Brassicaceae family is a predominantly consumed green leafy vegetable in China.It has the noteworthy health-promoting properties due to its high contents of fibers and phytochemicals.The quality of fresh vegetables could be assessed based on their nutritional value,growing conditions and usage of fertilizer.The major Phytochemicals in plants are secondary metabolites that play important role in plant survival as well in human health.Flavonoids are widely-distributed polyphenolic secondary metabolites which are biologically active molecules with a wide range of biological activities particularly in relation to plant physiology,growth,and development.Flavonoid not only provides protection against harmful factors but also facilitates to interact with other plants and microorganisms due to their physical and biochemical properties.One of the most important functions of flavonoids is to protect plants against abiotic(salt,drought,UV radiation and heat)and biotic stresses(pathogen attack and herbivore).Scavenging of ROS is very important to maintain the redox state balance inside the plant cells.Functional groups in the rings and the presence of conjugated double bonds in the structure of these molecules enrich them with notable antioxidant property.Largely,flavonoids quench the ROS by reducing the singlet oxygen's,hindering of enzymes involved in ROS generation(lipoxygenase,cyclooxygenase xanthine oxidase,monooxygenase),by chelating transition metal ions which trigger the ROS production,and quenching lipid peroxidation by number of free radical reactions,and help in the recycling of other antioxidants.In plants,the majority of their functions are attributed to its strong anti-oxidative properties.They participate in plants'responses to harsh environmental conditions and effectively regulate the cell differentiation and growth.Flavonoids also reduce various biotic stresses in plants,including pathogenic fungi and bacteria.More specifically,the exudation of flavonoids to the rhizosphere has been shown as signal molecules in response to symbiont or pathogenic microorganism.Some flavonoids may be involved directly or indirectly to inhibit pathogen's enzymes,particularly,the plant cell wall digesting enzymes by chelating metals required for their activities.Generally,the most common compounds acting as antimicrobials include flavanones,flavans,and isoflavonoids which trigger after an attack of the pathogen(phytoalexins)or be pre-synthesized(phytoanticipins),while some are found in the form of root exudates.Despite the fact that the genetic modification and agronomic manipulation methods are widely used to improve the biomass and beneficial phytochemicals in plants,the inadequate public acceptance and soil specificity of genetically modified food are still the challenges.To date,the use of beneficial microorganisms has become the sole alternative solution to ensure nutrient use efficiency and future food security because of the environmental concerns regarding excess utilization of chemical fertilizer.In general,the beneficial microorganisms exert positive effects on the growth characteristics by developing a holistic and functional relationship with plants.Applying beneficial microbes in agriculture has a long history started from 60 years ago and becomes more supported as they were proven to reduce the biotic and abiotic stresses in plants.An endosymbiont Piriformospora indica is the member of the order Sebacinales which exhibit mycorrhiza like properties.Interestingly,P.indica has the ability to build a mutualistic association with a variety of plants by direct manipulation in its hormonal signaling processes and production of primary and secondary metabolites which are correlated with the stronger immune responses.Symbiotically associated P.indica is crucially useful in the plant by providing miscellaneous services such as growth-promotion,nutrient uptake,disease resistance and stress tolerance.P.indica-root colonization by entering to the rhizodermal cells through root hairs and elevated flavonoids was evidenced in barley plants which is used as a second model plant.It can conclude from the available literature that P.indica can be utilized as bio priming agent for the better plant yield.In series of experiments we investigated the impact of exogenous inoculation of beneficial fungal strain P.indica on phytochemical modification and the putative genes expression of Chinese cabbage(Brassica campestris ssp.chinensis L.)under normal and stress conditions.Initially,the impact of exogenous inoculation of beneficial fungal strain P.indica was investigated in the greenhouse experiment,the following 9 treatments were applied:(1)CK(un-inoculated sterile soil),(2)CF(chemical fertilizer),(3)OF(organic fertilizer),(4)B(biochar),(5)OB(organic fertilizer and Biochar),(6)BP(biochar and fungi),(7)OP(organic fertilizer and fungi),(8)OBP(organic fertilizer,biochar and fungi),(9)(fungi).The natural soil collected from an organic farm which located in Shanghai,China was used in pot experiments.Growth attributes such as leaf number,leaf area,fresh and dry biomass were determined after harvesting the experimental samples under different treatments as well as root colonization of experimental plants by P.indica was also assayed in inoculated plants.The expression levels of flavonoid biosynthetic pathway genes from Pakchoi were assessed by quantitative real-time PCR(qRT-PCR)and the results were also confirmed through High performance liquid chromatography and spectrophotometric analysis.Results showed that the exogenous application of P.indica significantly(P<0.05)enhanced plant growth,chlorophyll and carotenoids content as well as other antioxidant related pathways.The RT-qPCR analysis of 5 key flavonoids pathway genes including CHS,CHI,F3H,ANS and FLS,indicated that FLS,triggering the synthesis of kaemferol was up-regulated by the inoculation of P.indica.High performance liquid chromatography(HPLC)affirmed that among the different combinations of fungal and organic fertilizer treatments,the phenolic acids and flavonoids were considerably enriched in organic fertilizer and fungi(OP)followed by fungi alone(P)and organic fertilizer,biochar,fungi(OBP)treated plants.The antiradical activity was higher in OP(61.29%)followed by P(60%)and organic fertilizer(OF)(53.84%)inoculated plants which positively correlated with chlorophyll,carotenoids and flavonoids level(P<0.05).In conclusion,the results revealed that P.indica alone or in combination with organic fertilizer can improve phytochemical and antiradical properties in Pakchoi.Furthermore,the data provide a new contribution by unraveling the effect of different fertilizers and a beneficial endosymbiont P.indica on the secondary metabolites of Pakchoi and as well as for the first time report the usefulness of Pakchoi-P.indica association.As the elevated level of flavonoids were found in P.indica co-cultivated plants and due to the significant and broad function of flavonoids and antioxidants in several plants defense mechanism against biotic and abiotic stresses,P.indica symbiotic colonization with pakchoi was studied under salinity and clubroot disease condition.The morphological attributes and physiological markers such as antioxidant enzyme,malondialdehyde(MDA),abscisic acid(ABA),salicylic acid(SA),ion analysis and electrolyte leakage were assessed in pakchoi under two NaCl concentrations(100 and 200 mM).In addition,the expression level of some well-known genes involved in Na~+/K~+homeostasis was also elucidated in different plant tissues.Under abiotic stress(salinity)P.indica co-cultivation significantly increased biomass of Pakchoi as well as antioxidant enzymes such as superoxide dismutase(SOD),peroxidase(POD),catalase(CAT)and augmented the level of salicylic acid(SA)and gibberellic acid(GA),assisting the plants to cope salinity stress.The salt tolerance mechanism of the experimental plant was studied at the cellular level by quantifying the gene expression level of salt overly sensitive(SOS)signaling pathway including,SOS1 and SOS2 as well as NHX-type Na~+/H~+antiporter(NHX1)from the shoot and root samples.The gene expression analysis showed the higher expression level of the candidate genes in inoculated plants under the given salt concentration(100 and 200mM).The findings suggest that the symbiotic association of endosymbiont P.indica can help the plants to overcome the salinity stress.The goal of the last part of the study is to contribute to basic understanding of P.indica-B.campestris ssp.chinensis interactions and to assess its impact on clubroot infection as well as the expression level of known P.brassicae genes in root of infected B.campestris ssp.chinensis.The morphological attributes including fresh weight of root,shoot,leaf number and area as well as shoot diameter were evaluated after 57 days of plant's growth.Like,that microscopy and pathological analysis was also carried out.Major flavonoids were investigated in samples from all treatments with high performance liquid chromatography(HPLC)using six kinds of flavonoid as standards,Chlorogenic acid,coffeic acid,kaempferol,quercitin(Shanghai Yuanye Bio-Technology Co.,Ltd.)rutin(BBI life sciences,life sciences research products and services),and luteolin(Antibodies,Proteins,Kits and Reagents for Life Science).From a molecular perspective,it was studied that whether the endophyte plays the role of antagonist directly to the pathogenic attack or induces systemic resistance.Furthermore,to identify changes at molecular level induced by the P.indica which provide multifaceted amenities to the host plant and induce systemic resistance,we compared the root transcriptome of P.indica colonized and non-inoculated B.campestris ssp.chinensis plants after challenge with biotrophic clubroot disease pathogen P.brassicae.Triplicate root samples were collected and used for transcriptome analysis at harvest.Analysis of biotic stress reducing capability of P.indica showed the reduction in formation of clubroot in Brassica campestris spp.chinensis which is extremely susceptible to the root pathogen Plasmodiophora brassicae.Roots of the P.brassicae-infected plant were colonized with P.indica,as demonstrated by staining technique and microscopy.Like that,enhanced biomass was observed in P.indica co-inoculated pakchoi plants by comparing with Plants infected with the pathogen only.Co-inoculation of Pakchoi plant with P.indica and P.brassicae reduced gall formation up to 61.60%by comparing plants infected with only P.brassicae as shown by the"disease Index"(DI).In addition,higher level of flavonoids was observed in P.indica inoculated plants which has been excessively reported to contribute in enhancing the plant immune system.Thus,a hypothesis was proposed that P.brassicae development in B.campestris ssp.chinensis plants could be delayed with the inoculation of P.indica.Expression of P.brassicae genes which were expressed differentially during disease development were monitored using quantitative RT-PCR and was corroborated with delayed pathogen development.These results suggest that P.indica and perhaps other beneficial endosymbiont could be an effective tool for the management of clubroot infection.This study also addresses for the first time the transcriptome level changes in B.campestris ssp.chinensis after inoculation with P.indica alone(P),P.brassicae alone(Pb),both organisms such that P.indica and P.brassicae together(PP),or neither organism(c)in comparison.DEG's were identified in comparison such that 8272 in control(C)vs P.indica co-inoculated plants,7173 in control(C)vs P.brassicae(Pb)co-inoculated plants,12915 in control(C)vs P.indica and P.brassicae(PP)co-inoculated plants,9416 in P.indica(P)vs P.indica and P.brassicae(PP)co-inoculated plants and 705 in P.brassicae(Pb)vs P.indica(P)and P.brassicae(PP)co-inoculated plants.Flavonoids synthesis pathways were induced,terpenoids,glucosinolates and proanthocyanidins accumulated and plant hormone signal transduction related genes were expressed in response to P.indica or P.brassicae co-inoculation in different combination.KEGG enrichment analysis of DEG's also showed that genes related to Starch,sucrose and galactose metabolism as well as alkaloids were significantly enriched under both P.indica and P.brassicae treatment.Thus priming of these genes might be involved in P.indica-induced systemic disease resistance and the results also provide a better information and a contribution for understanding of interaction between P.indica,P.brassicae and host plant,as well as new insights in to crop improvement/protection for sustainable agriculture production.Conclusion:In summary,we conclude that P.indica helps plants to overcome the biotic and abiotic stresses by mediating the biosynthesis of essential metabolites and antioxidant enzyme defense system.Our data suggest that,bio-protective action of P.indica might be mediated via systemic induction of antioxidant defense in the host plants.The study highlights that symbiotically associated P.indica can provide miscellaneous services such as growth-promotion,nutrient uptake,disease resistance and stress tolerance in Pakchoi plants.