Effects Of Endophytic Fungi On The Selective Degradation Of Host Volatile Oil And Host Seedling Growth

Atractylodes lancea（Thunb.）DC is a kind of perennial herb,which belongs to compositae,and with the function of drying dampness and strengthening the spleen,expelling wind and clearing away cold,improving eyesight.The rhizome is the native medicinal materials and volatile oil is the main active component of atractylodes.Due to the exhaustion of resources of A.lancea and the non-geoherbs ingredients can change curative effect of traditional Chinese medicine.Currently,most herbs traded on the market are artificially cultivated,which with continous cropping obstacle.A key question is that how the quality of the herbs can be ensured or even improved when they are cultivated away from their native environment.Preliminary studies showed that A.lancea contains various of endophytic fungi which contribute to host growth and accumulation of active ingrdients.However,the endophytic fungi exist in host,whether they are related to genuineness formation or tolerance has no research.We try to attempt from the microorganisms,microorganism and plants point,to research the symbiotic relationship between A.lancea and their endophytic fungi.The main contents are as follows:First,in order to study endophytic fungi are involved in the formation of plant genuineness,non-genuine A.lcncea was chose as experiment materials endophytic fungi were screened from the root of A.lancea.Transformation method was used in vitro.The changes of volatile oil were traced by gas chromatography.One endophytic fungus（strain ALG-13）which could utilize volatile oil selectively was screened.Single foctor experiment were conducted for exploring the effects of various factors that including kinds of carbon source,speed,liquid volume,pH and concentration of plant tissue on degradation by this strain.Subsequently,the main affecting factors carbon source,speed,pH and liquid volume were optimized using orthogonal arrary design.Results showed that endophytic fungus ALG-13 could selectively use the volatile oil,change the relative percentage of the main components of volatile oil,Atractylon and atractydin were increased,β-eudesmol and atractylol decreased.After selectively degradation by fungus,volatile oil components percentage was closer to the geo-herbs.Strain ALG-13 was identified as Bionectria ochroleuca according to its morphological characteristics and systematic analysis of ITS sequence.The optimal conditions were as follows:sucrose used as carbon source,rotating speed was 200 r·min-1,initial pH for medium was 4.5,50ml liquid was added in 250ml flask.The endophytic fungus ALG-13 could degrade the volatile oil selectively,which was benefit for forming geo-herbs A.lancea volatile oil composition.Since endophytic fungi can degrade volatile oil to ease restrictive growth by host plant,what mechanism did plants limit endophyte overgrowth?whether such restriction mechanism was distinguished from pathogens?A.lancea and its endophytic fungi were chosen,water distillation was used to extract volatile oil and separated each component.Plate growth method,paper disc assary,drop method and SEM were applied to study the volatile oil antimicrobial activity against endophytic fungi and pathogen.Results show that volatile oil has the strongest antimicrobial activity against Alternaria solani;Fusarium solani and Fusarium oxysporum were not obvious;for endophytic fungi,it has smaller inhibition.β-eudesmol and atractylol both had no inhibitory effect on the five species of fungi,while atractydin has stronger inhibition activity than Atractylon against pathogen.Under the stress of atractydin and atractylon,the sporulation of pathogen were suppressed,the hyphal branches of tested endophytic fungi increased,the distance between the branches became shorter.Mycelium of Alternaria solani breaks down,fold,warty surface and only caused blister of endophytic fungi when under atractydin and atractylon stress.This indicated that volatile oil and its component can inhibit growth of endophytic fungi,but such inhibition activity weaker than pathogen infection.Atractydin and atractylon were the key substance to balance endophytic fungi and host plant,it elaborate in theory that antagonistic balance mechanism between A.lancea and endophytic fungi.Allelopathic effect is an ever-increasing worldwide issue due to its toxic effects on plant metabolism and public health.Volatile oil has been documented that their constituents are potent seed germination inhibitors and retard plant growth.In the study,we design experiment whether application of exogenous endophytic fungus ALG-13 enhances the resistance of A.lancea to volatile oil stress.To address this issue,the effects of exogenous endophytic fungus on the chlorophyll content,soluble protein,root activity and osmotic regulation substances as well as growth index in plantlets under volatile oil stress were analyzed.The results show that:12.5-100μg·ml^-1 volatile oil has no significantly difference on the growth of seedlings,200μg·ml^-1 volatile oil significantly inhibited the growth,and 200-600 μg·ml^-1 changed with no difference,therefore 200 μg·ml^-1 choose to research allelopathic effect;Volatile oil treatment severely reduced the chlorophyll concentration,chlorophyll a/b ratio,and soluble protein content.Exogenous application of endophytic fungus ALG-13 markedly retarded the degradation of photosynthetic pigments and promoted the synthesis of soluble protein in volatile oil-treated Atractylodes lancea;In comparison with the control,volatile oil treatment induced a massive accumulation of MDA significantly in A.lancea,when applied with exogenous endophytic fungus,the content of MDA declined.However the effect of endophytic fungus on proline was contrary to that on MDA in A.lancea;volatile oil treatment significantly reduced root activity of A.lancea,it increased in exogenous endophytic fungus treatment.In conclusion,our results have shown that the supplement of exogenous endophytic fungus effectively relieved the phytotoxicity of volatile oil to A.lancea by degradation of photosynthetic pigments,promoting the synthesis of soluble protein,increasing the root activity and maintaining the balance of osmotic regulation substances.