Effects Of Endophytic Fungi On The Soil Allelochemicals And Peanut Root Exudate

Peanut is one of most important economic oil crops in China.Due to the limited area of arable land and the economic benefits of peanut,the phenomenon of continuous cropping is very common.The yield of peanut decrease sharply and the occurrence of diseases are frequent in peanut continuous cropping.The suggestions from scholars are hard in application because of the problems in operation and economic benefit.We try to overcome the continuous cropping obstacles by using endophytic fungi,which significantly improve the yield of peanut,nodulation and nitrogen fixation.The mechanisms of overcoming continuous cropping obstacles that we have invested are optimization of soil microbial community and accelerating the elimination of phenolic acids in peanut continuous cropping soil.However,whether there are other important mechanisms are still not clear.In this study,we hypothesized that the mechanisms of overcoming peanut continuous cropping obstacle by endophytic fungi also included:(1)endophytic fungi may regulate peanut root exudates which may promote nodulation;(2)laccase,an important extracellular enzyme secreted endophytic fungi,may play an important role in restoring peanut continuous cropping soil;(3)accelerating the degradation of allelopathic compounds such as flavonoid luteolin and stilbenoid resveratrol released from peanut residue;.To test these hypotheses,we designed a series of experiments which could provide more theories for the application of endophytic fungi to overcome peanut continuous cropping obstacles.Peanut root exudates were collected in the incubator conditions.Peanut root exudates were collected at 0 to 35 d,and the content of phenolic acids,flavonoids,organic acids and amino acids in root exudate were analysised.The effects of peanut root exudates and single substances in root exudates on rhizobium nodulation characteristics were examined.The results showed that endophyte infection did not change the species of phenolic acids,flavonoids,organic acids and amino acids in root exudate,but their content(benzoic acid,p-hydroxy benzoic acid,ferulic acid,cinnamon acid,quercetin,biochanin A,isoliquiritigenin,citric acid,oxalic acid,tartaric acid,formic acid,glutamate,glycine and glutamine)were significantly increased.Compared with non-infected treatment,the root exudates in endophyte infected treatment could significantly increase the growth biofilm formation,expression of nodC gene,nodulation of rhizobia.Finally,the effects of a single substance on rhizobia nodulation characteristics were studied,and the results showed that four phenolic acids in root exudates increased rhizobial chemotaxis,three flavonoids increased the gene experession of nodC gene expression and chemotaxis of rhizobium,and citric acid,oxalic acid and amino acid improved bacteria growth,biofilm formation and chemotaxis.These results showed that endophyte infection increased the content of phenolic acids flavonoids,organic acids and amino acids in root exudates,and the nodulation abilities of rhizobium were also enhanced.In order to study whether laccase secreted by endophytic P.liquidambari playes an important role in overcoming peanut continuous cropping obstacle,the laccase gene of endophytic fungi was cloned and heterologous expression,and then was add into the continuous cropping soil to investigate its reparation effects..TAIL-PCR technology was used to obtained the full-length 1867-bp laccase gene,and the 1731-bp full-length cDNA sequence was also obtained by RT-PCR.The laccase protein was successfully heterologous expressed in Schizosaccharomyces pombe,and its catalytic properties were studied.When pure laccase was added into continuous cropping soil,peanut seedling height,total dry weight,root dry weight and nodule numbers increased by 10.4%?11.2%;soil bacteria,actinomyces,nitrogen fixing bacteria and SMBN enhanced by 15.5%?27.7%;and toxic vanillic acid,coumaric acid and 4-hydroxy benzoic acid were reduced by 21%,27%and 40%.These results showed that the endophytic fungus laccase could increase soil microbial activity,promote the growth of peanut and reduce harmful phenolic acids contents,which were helpful to overcome peanut continuous cropping obstacle.Laboratory residue degradation experiments and pot experiments were used to study the allelopathic autotoxicity of luteolin on peanut growth,and degradation ability of endophytic fungi on luteolin and its metabolic processes were also examined.The results showed that luteolin released from peanut residue persisted in soil,and reached the highest concentration of 0.42 ?g g-1 soil.Peanut growth,plant dry weight and the number of nodues were significantly reduced when luteolin was added in pots.Similarly,bacterial content,soil microbial biomass carbon,soil bacterial and fungal community diversity and soil dehydrogenase in peanut rhizosphere soil were significantly decreased.It was also found that luteolin could inhibit the growth of peanut nodule bacteria,biofilm formation and chemotaxis.The optimum concentration of luteolin as sole carbon source supplied in culture was 200 mg L-1,which allowed 97%and 99%degradation of luteolin by P.liquidambari in liquid culture and soil conditions,respectively.The investigation of the fungal metabolic pathway showed that luteolin was first decomposed to caffeic acid and phloroglucinol.These intermediate products were degraded to protocatechuic acid and hydroxyquinol,respectively,and then rings were opened by ring-cleavage dioxygenases.Two novel genes encoding the protocatechuate 3,4-dioxygenase and hydroxyquinol 1,2-dioxygenase were successfully cloned.Reverse-transcription quantitative polymerase chain reaction demonstrated that expression levels of mRNA of these two genes increased significantly after P.liquidambari was induced by the intermediate products caffeic acid and phloroglucinol,respectively.These results revealed that P.liquidambari can biodegrade luteolin efficiently and could potentially be used to bioremediate phytoestrogen contamination.The release of resveratrol in peanut residue and its allelopathy on peanut seedling were also conducted in laboratory residue degradation experiments and pot experiments.The results showed that resveratrol from the peanut residue could be released into the soil,and it reached the highest concentration of 0.18 ?g g-1 soil.Resveratrol significantly inhibited the growth of peanuts in pot experiments.The soil microbial activity,soil microbial biomass carbon,bacteria content and diversities of microbial community in rhizosphere soil were also redueced by the releasing of resveratrol.Rhizobium nodulation ablities such as growth,biofilm formation,chemotaxis and nodule number were inhibited by resveratrol.The optimum concentration of resveratrol as sole carbon source supplied in culture was 400 mg L-1,and after 168 hours P.liquidambari could completely degrade resveratrol in soil conditions.The liquid chromatography mass spectrometry(HPLC-MS),the enzyme activities and qRT-PCR were used to analyze the metabolic pathways of resveratrol by endophyte.The results showed that the double bonds between the two benzene rings in the resveratrol were first broken with the products of ?-hydroxy benzaldehyde and 3,5-dihydroxy benzaldehyde.Then they were transformed to ?-hydroxybenzoic acid and 3,5-dihydroxy benzoic acid.These results showed resveratrol from peanut residue could be released into the soil,was toxic toward peanut seedling,and could be biodegraded efficiently by P.liquidambari.