Effects Of Phomopsis Liquidambari B3 On Nodule Endophytes And Rhizosphere Diazotroph Of Continuously Cropped Peanut

Peanut(Arachis hypogaea L.)is an important legume oil and commercial crop worldwide.Because of the increasing scarcity of land and agro-industrialization,peanuts are increasingly grown repetitiously on the same land without any crop rotation,particularly in the hilly red soil region of southern China.The consecutive monoculture leads to a serious decline in nodulation and quality of peanuts.Because of the problems in operation and economic benefit,the existing methods for alleviating consecutive monoculture obstacle are hard in application.We have tried to overcome continuous cropping obstacles by using endophytic fungus Phomopsis liquidambari B3,and the results showed that the endophtic fungi significantly improve the yield of peanut,nodulation and nitrogen fixation.The mechanisms of overcoming continuous cropping obstacles are optimization of soil microbial community,regulation the root exudate of peanut and accelerating the elimination ofphenolic acids in peanut continuous cropping soil.However,whether there are other important mechanisms are still not clear.Nitrogen is impotant for peanut growth and peanut can obtain nitrogen from nodule nitrogen fixation,soil and fertilizer supply.In this study,we try to investigate other mechanisms following two aspects:(1)liquidambari B3 may increase the diversity of nodular endophytic beneficial bacteria of peanut.(2)P.liquidambari B3 may improve the abundance and community structure of diazotroph in rhizosphere soil of peanut.Nodular endophytic bacteria play an important role in nodulation.Therefore,a pot experiment was conducted under natural conditions to evaluate the effect of P.liquidambari on the diversity of endophytic bacteria associated with continuously cropped peanut nodules.Four treatments were performed:non-continuous cropping(NS);continuous cropping(CK);continuous cropping soil with P.liquidambari(CSB3);continuous cropping soil with sterilized P.liquidambari(CSIB3).A total of 120 isolates were obtained belonging to three phyla(Bacteroidetes,Firmicutes,Proteobacteria),16 genera and 33 species.The diversity of bacteria in NS,including three phyla,17 species and 34 isolates,was greater than that in CK,which only contained one phylum,12 species and 27 isolates.The diversity in CSB3,including 3 phyla,18 species and 34 isolates,was higher than that in CK and CSIB3(2 phyla,11 species and 25 isolates).The correlation analysis showed that the 16S rRNA gene was positively correlated with the pH and total N content of the soil.This report is the first to describe the effect of a fungal endophyte on the diversity of endophytic bacteria associated with continuously cropped peanut nodules.The results showed that activated P.liquidambari promote nodulation by increasing the diversity of nodular endophytic bacteria,which are potentially associated with the improvements of soil environment caused by P.liquidambari.Futhermore,we analysied the plant growth-promoting properties including phosphate solubilization,siderophore-producing attributes,IAA production and the pathogens resistance ability of the 33 representative isolates.Only NS-13(Bradyrhizobium cytisi)could induce peanut nodulation when it was reinoculated.Five isolates(both 3 striains were in NS and CSB3,1 strains were in CK and CSIB3 respectively)promoted peanut nodulation and increased root and shoot length and peanut biomass when they co-inoculated with NS-13.Total 13 strains could ehance the root and shoot length and peanut biomass.Both CSB3 and NS had 9 strains,while only 3 and 2 strains were found in CSIB3 and CK respectively.The number of phosphate solubilization,siderophore-producing attributes,IAA production and the pathogens resistance ability bacteria were 16,16,11 and 7 respectively.The number of phosphate solubilization strains in NS were same as CSB3(10),while 5 and 4 isolates were found in CSIB3 and CK respectively.Eleven ironophore production strains were found in CSB3 which is the most in four treatments,followed by NS(10),CSIB3(5)and CK(4).Both CSB3 and NS had 7 the number of IAA production and strains,while CK and CSIB3 had 4 and 1 stranis respectively.Both five antagonism of strains were isolated from CSB3 and NS,while only 3 and 1 strains were isolated from CK and CSIB3 respectively.The above results showed that,compared with uncontinuous cropping treatment,long-term continuous cropping may reduce the diversity of nodular endophytic beneficial bacteria of peanut,and the application of P.liquidambari alleviate this decrease,and then promote the nodulation of peanut under continuous cropping systerm.Although the 40-50%nitrogen is root nodule nitrogen fixation,peanut need obtain nitrogen from soil.Biological nitrogen fixation plays an important role in agriculture ecosystem as it is a main way for organisms getting nitrogen from nature.We therefore investigated the diazotroph abundance and community structure in rhizosphere soil of peanut among four treatments using qPCR and T-RFLP.We found that the diazotroph abundance and diazotroph abundance percent(the nifH copies/16S rDNA copies in per gram dry soil)in NS and CSB3 were significantly higher than other two treatments in the same stage.Clustering analysis showed that diazotroph community structure in NS and CSB3 were also different from CK and CSIB3.We futher analysed the relationship between the diazotroph community structure by RDA analysis.The factors which affects diazotroph community structure in different periods were different.In seedling stage,the factors were organic matter,ammonia nitrogen,nitre nitrogen and total nitrogen of soil.In flowering phase,the factors were pH and ammonia,while available phosphorus,ammonia nitrogen and total nitrogen in havest time.In short,our study showed that diazotroph abundance and diazotroph abundance percent in long-term continuous cropping system were significantly lower than uncontinuous cropping system,and the diazotroph community structure were also changed.The application of endophytic fungi not only increase the diazotroph abundance and diazotroph abundance percent,but also change the diazotroph community structure in rhizosphere soil of peanut under continuous cropping system.This may be related with the change of soil nutrient.This may be another reason for P.ease peanut continuous cropping obstacle.