| Astragalus membranaceus is a widely cultivated herbaceous perennial medicinal plant whose roots are highly valued for clinical applications.The yield and quality of artificially cultivated A.membranaceus are always reduced by drought stress,which seriously limits its growth,medicinal quality and industrial development.Dark septate endophyte(DSE)is a group of fungi that are designated to colonize the roots of plants and do not cause host diseases,with dark septate hyphae and microscopic nuclei.Trichoderma spp.originates from Moniliaceae which is a widely distributed biological control agent and new growth promoter in nature.They have been proven to perform a variety of ecological functions,such as promoting plant growth,improving soil conditions,and activating plant resistance.In this study,three strains of DSE fungi(Acrocalymma.aquatica,Macrophomina pseudophaseolina,Paraphoma radicina)isolated from the root system of cultivated A.membranaceus in Anguo Chinese herbal medicine plantation in Hebei were selected as the research object and simulated drought stress by PEG-6000.The tolerance and adaptability of DSE fungi to in vitro drought stress were investigated;in combination with Trichoderma afroharzianum,Trichoderma longibrachiatum isolated from the soil of the root zone of medicinal plants,plate co-culture tests as well as pot tests were conducted to investigate the effects of dual inoculation in sterilized and non-sterilized soil on the growth and resistance of A.membranaceus seedings.We investigated the effects of double inoculation in sterilized and non-sterilized soil on the growth and drought resistance of A.membranaceus plants.Combined with the determination of growth,physiological indexes,accumulation of medicinal components in the root system and soil physicochemical properties of A.membranaceus,we aimed to reveal the mechanism of the pro-growth effect of endophytic fungal double inoculation on A.membranaceus under drought stress.It provides a basis for the research and development of efficient mixed microbial inocula.The main experimental results are as follows:1.In vitro drought tolerance assays of DSE fungi revealed that M.pseudophaseolina and P.radicina showed stronger drought tolerance,which was significantly associated with increased growth rate,biomass,melanin,soluble protein and superoxide dismutase under drought stress conditions.Principal component analysis(PCA)demonstrated that soluble protein and biomass were the main indicators of the response of DSE fungi to drought stress.DSE strains may alleviate the damage of drought stress by regulating their own growth,melanin accumulation,osmoregulatory substances and antioxidant defense system.2.The plate tests proved that the co-culture operation had significant effects on the growth and metabolism of DSE and Trichoderma strains.Compared with single culture DSE strain,Trichoderma caused a significant reduction in biomass and a significant increase in mycelial diameter of co-cultured DSE strains,and the changes in spacing and melanin content varied with DSE species.There were significant differences in the effects of different DSE strains on the sporulation of Trichoderma.P.radicina significantly increased the sporulation of T.afroharzianum by 37.92 % compared to monoculture.Principal component analysis showed that mycelial diameter,colony radius,biomass and inhibition rate were main index reflecting the characteristics of co culture.3.DSE strains developed a stable symbiotic relationship with A.membranaceus.Dual inoculation with M.pseudophaseolina and T.longibrachiatum were significantly increased by23.33 %,6.67 % and 13.34 % in terms of mycelial,micromycorrhizal and total colonization rates of DSE fungi compared to M.pseudophaseolina alone,respectively.4.Soil water deficit significantly promoted the growth of A.membranaceus seedlings in a sterile culture pot experiment,and the inoculation effect differed significantly among single inoculation treatments.Inoculation of M.pseudophaseolina alone significantly increased root biomass,total root length,root surface area,root diameter and root volume of A.membranaceus by 155.56 %,55.54 %,82.62 %,16.67 % and 115.56 %,respectively,compared to noninoculated.Single inoculation of T.afroharzianum significantly increased leaf number and aboveground biomass by 28.68 % and 31.88 %,respectively,compared to the uninoculated group.5.Dual inoculation of DSE fungi with Trichoderma significantly improved growth and drought tolerance of A.membranaceus by exerting the complementary ecological functions of the strains during soil water deficit.Double inoculation with P.radicina and T.longibrachiatum significantly increased plant height,soluble protein,glutathione and soil fast-acting phosphorus content,while root MDA,soil organic carbon and ammonium nitrogen accumulation were significantly reduced.Double inoculation with M.pseudophaseolina and T.longibrachiatum significantly increased proline,root glutathione and nitrate reductase of A.membranaceus.The results of the variation partitioning analysis showed that the effect of Trichoderma species on drought resistance indexes was more dependent on DSE species.After combining growth and stress tolerance indicators,it was found that the double inoculation of P.radicina and T.longibrachiatum had the optimal drought-promoting performance.6.Conventional culture pot experiment found that single inoculation could promote the root growth of A.membranaceus.M.pseudophaseolina significantly increased root diameter;T.afroharzianum increased root biomass,root surface area,total root volume,soil available phosphorus content and urease activity by 46.84 %,102.33 %,94.44 %,34.48 % and 24.63 %respectively.Variance decomposition showed that soil nutrients and Trichoderma species had great effects on the growth of A.membranaceus.As a typical representative of beneficial rhizosphere microbial groups,Trichoderma can stimulate root cell division and differentiation,improve soil productivity,and regulate plant root development.7.DSE and Trichoderma strains double inoculation showed neutral or positive effects on the accumulation of pharmacodynamic components and drought resistance of A.membranaceus,which was closely related to soil nutrients and Trichoderma species.Dual inoculation by P.radicina and T.longibrachiatum significantly increased the plant height,root ascorbic acid and glutathione by 2.18 %,54.32 % and 405.67 %.Double inoculation with M.pseudophaseolina and T.afroharzianum significantly increased the root length,mancoxanthin and soil nitrate nitrogen of A.membranaceus.Aboveground biomass and Calycosin 7-O-β-D-Glucopyranoside were highest and soil alkaline phosphatase was lowest in P.radicina and T.afroharzianum double inoculation of A.membranaceus.Redundancy analysis showed that ammonium nitrogen,nitrate nitrogen,available phosphorus and urease were the main soil factors affecting the root physiological indexes of A.membranaceus.8.DSE fungi and Trichoderma can play a synergistic role in improving plant growth,promoting nutrient absorption,increasing the accumulation of effective components and improving the stress resistance of roots.Combined with the practical needs of root medicine and water-saving agriculture,three groups of strain combinations with good inoculation effect were selected,which provided a theoretical basis for developing mixed bacterial agents,improving the cultivation ability and the accumulation of bioactive substances of artificially cultivated medicinal plants. |
PDF Full Text Request