The Interaction Between Endophytes Associated With Schisandraceae Plants And Schitriterpenoids

Triterpenes are one of the largest and most diverse families of plant natural products,which have many important functions such as antibacterial,plant defense and signal transmission,and play an important role in regulating the interaction between plants and microorganisms.Compared with triterpenoids from other plants,schitriterpenoids and schinortriterpenoids are unique components of Schisandraceae plants,which are characterized by complex polycyclic lactones paired with highly oxygenated multiple chiral,and have a variety of pharmacological activities.However,the contents of most schitriterpenoids and schinortriterpenoids in plants are very low.Moreover,the chemical synthesis of these specialized metabolites is quite difficult and commercially unfeasible due to the complicated stereo-chemical rings with multiple chiral centers.This has led to a lack of research on their physiological and pharmacological activities.Studies have shown that endophytic microorganisms are closely related to the active ingredients and growth of traditional Chinese medicine.To tap the potential of endophytes and continuously improving the yield and quality of medicinal plants,it is important to further study the structure and function of endophytic microorganisms in medicinal plants and the interaction between endophytes with secondary metabolites of host plant.In our previous studies,we have confirmed that certain endophytic fungi isolated from Kadsura angustifolia can specifically transform and modify the main components of the host plant,and promote the host growth,these indicating that the correlation between Schisandraceae plants and endophytes is worthy of further exploration in order to improve the yield of schitriterpenoids and schinortriterpenoids in Schisandraceae plants.Additionally,few reports on the composition and assembly of endophytic and rhizospheric microbiome in Schisandraceae plants,and the interaction mechanism between endophytes with schitriterpenoids has not been deeply studied.Accordingly,in this study,Kadsura angustifolia and Schisandra sphenanthera collected from the same geographical environment in Xichou county of Yunnan province were used to investigate the interaction between schitriterpenoids and endophytes of Schisandraceae plants.The main results are as follows:(1)The composition of the endophytic and rhizospheric microbiome of K.angustifolia and S.sphenanthera was analyzed by high-throughput 16 Sr RNA and ITS amplicon sequencing.The results of OTUs analysis among different samples showed that the number of OTUs shared by rhizosphere microorganisms was significantly higher than that shared by rhizosphere microorganisms and endophytes,indicating a significant difference in the composition of rhizosphere microorganisms and endophytes of two Schisandraceae plants.The number of OTUs of endophytes shared by two Schisandraceae plants in the same niche was significantly higher than that of endophytes shared by K.angustifolia/S.sphenanthera in different niche,which indicated that the effect of niche on the population distribution of endophytes was greater than that of host species.Additionally,OTUs annotation and relative abundance analysis showed that the rhizosphere bacteria of K.angustifolia and S.sphenanthera are mainly composed of Proteobacteria,Acidobacteria and Actinomycetes,and the dominant genera are mainly composed of Pseudomonas,Acidothermus,Acidibacter,Pantoea,Candidatus＿Solibacter,Bradyhizobium,and unidentified＿Burkholderiaceae.The rhizosphere fungi of K.angustifolia and S.sphenanthera are mainly composed of Ascomycota and Basidiomycota,and the dominant genera are unidentified-Trimorphomycetaceae,Archaeorhizomyces and unidentified＿Archaeorhizomycetes.Compared with rhizosphere microorganisms,the endophytic bacteria in two Schisandraceae plants are mainly composed of Cyanobacteria,Proteobacteria,Firmicutes and Bacteroidetes bacteria,and the dominant genera are Pseudomonas,Bacteroides,Sphingomonas,Pantoea,Bacillus,unidentified＿Cyanobacteria,unidentified＿Burkholderiaceae,and unidentified＿Rickettsiales.Similarly,endophytic fungi of K.angustifolia and S.sphenanthera are mainly composed of Ascomycota and Basidiomycota.However,at the genus level,the dominant genera of endophytic fungi in roots,stems,leaves and fruits of K.angustifolia and S.sphenanthera are significantly different.The common dominant genera of K.angustifolia and S.sphenanthera are Trichoderma and Pestalotiopsis.Therefore,compared with rhizosphere microorganisms,endophytic microorganisms of K.angustifolia and S.sphenanthera have unique composition groups.The results of alpha diversity analysis showed that the diversity of bacteria and fungi gradually decreased from rhizosphere soil to plant tissue,indicating that the two Schisandraceae plants selectively enriched bacteria and fungi in rhizosphere soil.The results of PCo A analysis of beta diversity showed that the rhizosphere and endophytic microorganisms of K.angustifolia and S.sphenanthera were highly clustered,which indicated that the rhizosphere and endophytic microorganisms of the two plants were highly similar,respectively.Based on the results of high-throughput sequencing,endophytic bacteria and fungi from different tissues of K.angustifolia and S.sphenanthera were isolated by various culture strategies.A total of 119 and 127 strains of endophytic bacteria and 98 and 99 strains of endophytic fungi were isolated from K.angustifolia and S.sphenanthera,which were identified to belong to different taxa.Mainly,these endophytes isolated from K.angustifolia and S.sphenanthera were all detected in the above high-throughput sequencing results,which provided strain materials for further research.(2)To explore whether schitriterpenoids play a role in the regulation of endophytes in Schisandraceae plants,the chemical constituents of K.angustifolia and S.sphenanthera were studied.A major triterpenoid,nigranoic acid(K-1),was isolated from K.angustifolia and four major triterpenoids,iso-anwuweizicacid(S-1),nigranoic acid(S-2),mirandilactone C(S-3)and lancifodilactone D(S-4)were isolated from S.sphenanthera.The effects of different concentrations of nigranoic acid and Mix(mix of compounds S-1,S-2,S-3 and S-4)on the growth of 27 representative endophytic bacteria(belonging to 27 genera and 4 phyla)and 26 representative endophytic fungi(belonging to 26 genera and 2 phyla)of two Schisandraceae plants were determined in vitro.The results showed that both nigranoic acid and Mix could significantly promote the growth of endophytes in the dominant genera of different phyla,including Firmicutes,Bacteroides,Proteobacteria,Ascomycetes,Basidiomycetes and Zygomycota.However,nigranoic acid and Mix have inhibitory effects on the growth of non-dominant endophytes in these different phyla.Specially,both nigranoic acid and mix significantly inhibited the growth of all the endophytic bacteria belonging to Actinomycetes.Therefore,the main triterpenoids of K.angustifolia and S.sphenanthera have similar selective regulation on the growth of endophytic bacteria and endophytic fungi.(3)Different microbial groups have inter-species competition or cooperation during the growth process.It is an important strategy to analyze the composition of plant microorganisms and construct a reasonable microbial community to study the interaction mode between microorganisms.By studying the interaction between 27 representative endophytic bacteria and 26 endophytic fungi in vitro,it was found that the interaction between most endophytic bacteria and most endophytic fungi promoted each other’s growth,respectively.In contrast,most endophytic bacteria and endophytic fungi inhibited each other’s growth.Besides,combined with the regulatory analysis results of triterpenoids on endophytes,it was found that all tested endophytic bacteria had a pronounced growth-promoting effect on bacteria whose growth was positively regulated by schitriterpenoids(TPB),and this promoting effect was significantly higher than that of the bacteria whose growth was negatively regulated by schitriterpenoids(TNB).It shows that bacteria whose growth is positively regulated by schitriterpenoids have a more obvious growth advantage in the interaction of the whole endophytic bacteria group.Unlike the interaction between bacteria,only the growth of Trichoderma hamatum,Lasiodiplodia theobromae,Clitopilus prunulus and Ceratobasidium sp.was positively regulated by schitriterpenes(TPF)significantly inhibited the growth of other endophytic fungi and occupied the absolute growth advantage in the entire interaction of endophytic fungi.However,compared with those endophytic fungi whose growth was negatively regulated by triterpenes(TNF),other endophytic fungi whose growth was positively regulated by triterpenes did not occupy a more obvious growth advantage in fungal interactions,such as Pestalotiopsis microspore,Fusarium decemcellulare,Phlebiopsis sp.and Bipolaris setariae.In addition,although endophytic bacteria and endophytic fungi have an obvious antagonistic effect,the inhibition effect of all tested endophytic bacteria on TPF is significantly lower than TNF.These results indicate a significant correlation between the endophyte’s growth regulating by schtriterpenoids and the interaction between the endophytes.In other words,schitriterpenoids may affect the growth of different endophytes,and then regulate the interaction intensity of them to maintain the stability and balance of the endophytic community.(4)Secondary metabolites not only regulate the composition of the plant microbiome,but also affect the ecological functions of microorganisms.In order to further explore the ecological functions of endophytes selectively regulated by schitriterpenoids,we evaluated the ability of 27 strains of endophytic bacteria and 26 strains of endophytic fungi to promote the growth(including phosphate solubilization,IAA production,iron carrier secretion,DPPH scavenging and antioxidant)and the ability of synthesis of terpenoids in vitro.It was found that all tested endophytes have terpenoid synthesis ability and at least one growth-promoting ability.Interestingly,the growth-promoting and triterpenoid synthesis ability of endophytes whose growth was positively regulated by schitriterpenes(TPB/TPF)were significantly higher than those whose growth was negatively regulated by schitriterpenes(TNB/TNF).Furthermore,we constructed a co-culture system of five different synthetic communities(including TPB&TNB,TPF&TNF,TNB&TNF,TPB&TPF,and TNB&TNF&TPB&TPF)with sterile S.sphenanthera plants,and then studied the effects of five synthetic communities on the growth and metabolism of S.sphenanthera.The results showed that,compared with the control group without microbial treatment,the treatments of five synthetic communities could promote the elongation of fibrous root and plant height and the accumulation of various metabolic components(including total sugar,total terpenoids,flavonoids and polyphenols)of S.sphenanthera to varying degrees.Among them,the TPB&TPF treatment group showed most potent promotion effect on the growth and metabolic accumulation of S.sphenanthera,such as the fibrous root,plant height,soluble sugar,flavonoids,polyphenols and total triterpenoids content of S.sphenanthera increased 318.98%,104.53%,103.96%,102.21%,56.66% and 240.54% respectively compared with the control.Additionally,compared with the TPB&TNB and TPF&TNF treatment groups,the TNB&TNF&TPB&TPF,TNB&TNF and TPB&TPF treatment groups all showed a more significant promotion effect on the host plant,indicating that endophytic bacteria and endophytic fungi have a synergistic effect on growth-promoting of host plant.In particular,among the five synthetic communities,only TPF&TNF treatment group showed wilt symptom of leaves in the late growth period,which indicated that a large number of endophytic fungi existed have a certain toxic effect on the host plants.The co-existence of endophytic bacteria and fungi weakend the virulence of fungi and maintained the microecological balance of the endophytic community and plant health.In addition,the effect of TPB&TPF treatment group on the growth and metabolic accumulation of S.sphenanthera was significantly stronger than that of the other four synthetic communities.Therefore,endophytic bacteria and fungi whose growth is positively regulated by schitriterpenoids have the ecological function of significantly promoting the growth of host plants and the accumulation of secondary metabolism,indicating that schitriterpenoids may be selective regulators for the assembly and optimization of beneficial endophytic communities in Schisandraceae plants.(5)Plant secondary metabolites can regulate the assembly of the microbiome,and in return,microorganisms can also affect plant secondary metabolism.We investigated the fermented products of an endophytic fungi Penicillium ochrochloron SWUKD4.1850 grown on host-plant K.angustifolia and wheat bran,respectively,to assess the impact of SWUKD4.1850 on the secondary metabolites of K.angustifolia.The results showed that the presence of the host plant can significantly change the secondary metabolism of P.ochrochloron.Twenty-four rare schitriterpenoids and schinortriterpenoids were isolated from co-fermentation cultures,which were characterized by complex polycyclic lactones paired with highly oxygenated multiple chiral,and they have not been found in previous chemical study of P.ochrochloron and K.angustifolia.According to the biogenetic pathway of these compounds and the correlation analysis between the production of endophytic fungi and the characteristic active components of the host plant,it is revealed that these metabolites are most likely to be derived from nigranoic acid,the main chemical active component of the K.angustifolia.Obtained results indicated that endophytes associated with Schisandraceae plants have a strong ability to specifically transform and modify schitriterpenoids of host plant.In this study,by analyzing the structure of P.ochrochloron and K.angustifolia associated microbiome,the dominant species and assembly rules of the two plantsassociated microbiome were deciphered,and the mechanism of interaction between endophytes associated with Schisandraceae plants and schitriterpenoids was preliminarily revealed.Thus,this study will lay a solid foundation for the use of endophytes to regulate the growth of Schisandraceae plants and improve their quality.