A Study On Mechanism Of Synergistic Symbiosis Of Endophytic Fungus Acremonium Strictum And Bacterium Acinetobacter Sp.in Their Shared Host Atractylodes Lancea

Atractylodes lancea is a traditional Chinese medicinal plant and its rhizomes contain many bioactive components,including sesquiterpenoids(atractylone,P-eudesmol,hinesol),polyacetylenes(atractylodin).This medicinal herb has been widely used for treatment of rheumatic disease,gastrointestinal disorders and influenza.Many studies have examined pair-wise interactions between plants and endophytes,while overlooking the interplays among multiple endosymbionts and their combined impacts on hosts.In order to further investigate the interplays between multiple symbionts in natural environment,we employed a three-way symbiosis system,containing endophytic fungus,endophytic bacterium and the host plant.Our aim was to figure out that,1)the impacts of the three-way symbiosis on the host growth,defense-related responses;2)the possible interplays between multiple symbionts;3)the impacts of bacterial endophyte on plant secondary metabolism and signaling pathways involved.Firstly,Atractylodes lancea plantlets were inoculated with endophytic fungus Acremonium strictum AL16,or endophytic bacterium Acinetobacter sp.,or both,to investigate the impacts of the three-way symbiosis on the host.Our results showed that defense-related responses of the co-inoculated plantlets were delayed and weakened relative to plantlets with single inoculants,but no detrimental effects on phyto-physiology(growth,photosynthesis)were observed after combined inoculations.Quantitative PCR analysis verified a decrease(200%)in AL16 colonization density within plants after co-inoculation with the endobacteria.An in vitro assay was then performed to elucidate the suppressed plant defense responses and reduced fungal colonization by dual inoculation.The data showed that the presence of Acinetobacter sp.reduced AL16 colony diameter by 32.62%and decreased spore germination rate by 31.02%without negatively affecting fungal morphology.Additionally,direct hyphal attachment of the bacterium to AL16 in vitro was visualized by scanning electronic microscopy.Therefore,we propose that a balanced and compatible symbiosis might require constraints conferred by the antagonistic endophyte Acinetobacter sp.on the fungus AL16 in the tripartite endophytic bacterium-fungus-plant system.In the second place,we found that,in addition to inhibitory metabolites,endophytic bacterium ALEB16 also emited volatile organic compounds(VOCs)able to inhibit hyphal growth and viability of fungus AL16.The results showed that among all the three treatments,VOCs produced by bacterium Acinetobacter sp.ALEB16 exhibited the greatest inhibitory rate(18.09%)by comparison with that in treatment of bacterial thalli(13.88%)and fermentation broth(10.09%).Then the VOCs produced by ALEB16 were analyzed by GC-MS.Overall,17 compounds were detected and the identified compounds were mainly categorized into four classes including 7 acids,two naphthyls,one benzene,and one sulfide.Naphthalene,1,6,7-trimethyl-and benzeneacetic acid,methyl ester(BAME)were two other main volatile compounds produced by bacterium ALEB16.Since the inhibitory effect of naphthyl compounds on fungi has been studied widely in other reports,BAME was selected for further individual testing of antifungal activity against the endophytic fungus AL16.The inhibition test showed that mycelial growth and sporulation of fungus AL16 were significantly inhibited at the presence of BAME of tested concentrations and bacterial VOCs blend.Further,the cytochemical approach in situ demonstrated that fungal viability(mitochondrial NADH dehydrogenase and mitotic ability)were inhibited in both the BAME-and bacterial VOCs-treated hyphae.This paper showed that volatile secondary metabolites produced by an endophytic bacterium Acinetobacter sp.exterted an antagonistic impact on endophytic fungus Acremonium strictum and might play a role in the multiple symbioses among host plant and its interspecific endophytes.Finally,we investigated the impacts of the bacterial endophyte ALEB16 on plant secondary metabolism and signaling pathways involved.This work showed that the endophytic Acinetobacter sp.ALEB16,capable of activating volatile oil accumulation of Atractylodes lancea(increased by 30.37%),also induced abscisic acid(ABA)and salicylic acid(SA)generation of plants.Pretreatment of plantlets with the ABA biosynthetic inhibitor or SA inhibitor blocked the bacterium-induced volatile production.Further,relationships between ABA and SA in the bacterium-induced volatile production were elucidated.ABA inhibitors suppressed not only the bacterium-induced volatile accumulation but also the induced ABA and SA generations;nevertheless,SA inhibitors showed no significant inhibition on the bacterium-induced ABA generation,implying that SA acted downstream of ABA production.These results were confirmed by observations that exogenous ABA and SA not only promoted volatile oil production but also reversed the inhibition of bacterium-induced volatile accumulation by biosynthetic inhibitors.Transcriptional activities of genes involved in sesquiterpenoid biosynthesis also increased significantly with bacterium,ABA and SA treatments.Mevalonic acid pathway was proved to be the main contributor to the bacterium-induced production of volatile sesquiterpenoids by using specific inhibitors for terpenoids.In conclusion,the present work demonstrates that Acinetobacter sp.acts as an endophytic elicitor to stimulate volatile biosynthesis of host A.lancea via an ABA/SA-dependent pathway,thereby yielding additional insight into interconnection between ABA and SA in biosynthesis-related signaling pathways.