| As a well-known traditional Chinese medicinal herb, the international and domesticdemand of Salvia miltiorrhiza Bge. is increasing fast. Consequently, replant disease is themajor limiting factors of the development of S. miltiorrhiza artificial cultivation. Findingeffective disease control approaches is the first priority to solve this problem. In order toelucidate reasons for the occurrence of S. miltiorrhiza root diseases, changes of rhizospheresoil and leaves chemical properties and microbial community composition between healthyand diseased S. miltiorrhiza plants root, rhizosphere and rhizoplane soil were determined byroutine soil agro-chemistry analysis and culture-depended method, respectively. Meanwhile,based on the research of isolation and identification of pathogenic fungi from root rot plant,screening and identification of antagonistic actinomycetes,8strains broad spectrumantagonistic actinomycetes were obtained.2-years field experiment was conducted to studythe biocontrol and growth promotion effect of actinomycetes and its microecologicalmechanism. The related results studies provided theoretical basis, experimental evidence andbiocontrol actinomycetes for indication of microecological mechanism and research ofmicrobial remediation of replant soil-borne root disease in S. miltiorrhiza. Related results andconclusions are listed as follows:（1） Isolation and identification of endophytic bacteria from root tissues of S. miltiorrhizaand determination of their bioactivitiesThis study examined the occurrence, distribution, growth-promoting and antifungalactivities of endophytes in the root of S. miltiorrhiza. Six endophytic bacterial strains, whichbelong to genera of Pseudomonas, Rhizobium, Bacillus and Novosphingobium, were isolatedfrom the root of healthy S. miltiorrhiza. Cell suspension (approx.109cell·mL-1) of twoisolates namely R. radiobacter, B. aryabhattai and cell-free fermentation filtrate of fourisolates namely Ps. brassicacearum subsp. neoaurantiaca, R. radiobacter, Ps. thivervalensis,B. aryabhattai substantially promoted the growth of hypocotyl and radicle of muskmelonseeds. The cell-free fermentation filtrate of six isolates had no inhibiting effect on testedpathogenic fungi namely Fusarium solani, F. oxysporum f. sp. vasinfectum and F. oxysporum. This work indicates that endophytic bacteria occur in the root of S. miltiorrhiza, and thatassociated bacterial isolates have growth-promoting effect on muskmelon seeds and areexpected to be a potential source for bioactive metabolites.（2） Microecological mechnism of root diseases occurrence of S. miltiorrhizaThe microecological mechanism of the red-leaf diseased, Fusarium wilt infected and rootrot diseased S. miltiorrhiza were studied by determination and comparison of soil nutritioncontents and microflora in the root zones of healthy and infected plants. Normal method wasused to measure the content of soil and leaf nutrition, the amount of bacteria, actionmycetes,fungi and the ratio of B/F, A/F, B/A in the rhizosphere, rhizoplane and bulk soil by dilution,and semar technique was used to do the molecular biology identification of the dominantmicrobes and to study the microecological mechanism of diseased S. miltiorrhiza in rootingzone soil.Red-leaf diseased S. miltiorrhiza:①The available N, P and K contents in rhizospheresoil of diseased S. miltiorrhiza leaves were all significantly lower than in healthy S.miltiorrhiza leaves, while there was no significant difference of available P content inrhizosphere soil between the diseased and the healthy S. miltiorrhiza. These indicated thedeficiency of P in S. miltiorrhiza leaves was the primary cause of the red-leaf disease.②Thenumber of bacteria in the rhizosphere soil of diseased S. miltiorrhiza decreased sharply, whilethe numbers of fungi and actinomycetes increased enormously; compared to the healthy S.miltiorrhiza, the number of bacteria decreased by41.3%, whilst the numbers of fungi andactinomycetes increased by156.6%,189.5%, respectively. The dynamic of the numbers ofbacteria, fungi and actinomycetes in the rhizoplane soil of diseased S. miltiorrhiza was as thesame as the rhizosphere soil.③The numbers of predominant bacteria （B. aryabhattai,Piscinibacter aquaticus）, the predominant fungi （F. solani, Myrothecium roridum, F.tricinctum, Aspergillus calidoustus, F. oxysporum, Dothideomycetes sp.） and the predominantactinomycetes （Streptomyces lateritius, Lentzea waywayandensis, S. stelliscabiei, S. collinus）in the root zone soil were higher in the diseased S. miltiorrhiza than in the healthy plant,which could resulted in the red-leaf disease of S. miltiorrhiza.Fusarium wilt infected S. miltiorrhiza:①Compared to those in the healthy plant leaves,N, P, K contents significantly decreased by8.4%,35.6%,23.5%and23.4%,57.6%,57.7%inwithered and dead plant leaves （P＜0.05）. Compared to those in the healthy plant rhizosphere,soil organic matter and available N, P, K contents increased in infected plant rhizosphere. Itshowed that the occurrence of Fusarium wilt was independent of soil nutrient deficiency.②In dead plant rhizosphere, the numbers of soil bacteria, fungi and actinomycetes respectivelyincreased by168.0%,852.8%and247.4%compared to those in the healthy plant rhizosphere （P＜0.05）. In dead plant rhizoplane, the numbers of soil bacteria, fungi and actinomycetesrespectively increased by72.6%,2020.0%and424.2%compared to those in the healthy plantrhizosphere （P＜0.05）.③In infected plant rhizosphere and rhizoplane soils, predominantmicrobial species that might be harmful were mainly2fungi isolates （F. solani and F.oxysporum）. These predominant soil microbes likely caused Fusarium wilt by negativelyaffecting the growth of roots and their absorption of nutrient from soil.Root rot diseased S. miltiorrhiza:①The content of available N, P and K interminal-stage infected plant leaves were higher than in healthy plant leaves. Compared tothose in the healthy plant rhizosphere, soil organic matter and available nutrient contents allincreased in infected plant rhizosphere soil. Soil organic matter, available P and K contentswere highest in terminal-stage infected plant rhizosphere soil and were respectively2.9,2.3and1.6times of that in the healthy plant rhizosphere soil, and pH decreased by12.3%compared with the control. It showed that the occurrence of root rot was independent of soilnutrient deficiency.②In rhizosphere soil, compared to those in the healthy plant, thenumbers of soil bacteria in initial-stage, middle-stage and terminal-stage infected plantincreased by36.5%,54.1%and80.2%, respectively; the numbers of fungi in initial-stage,middle-stage and terminal-stage infected plant increased by160.8%,191.0%and310.6%,respectively; the numbers of actinomycetes in middle-stage and terminal-stage infected plantdecreased by52.2%and51.9%, respectively. In rhizoplane soil, compared to those in thehealthy plant, the numbers of soil bacteria decreased by35.0%in initial-stage infected plant,while increased by147.7%and498.0%in middle-stage and terminal-stage infected plant,respectively; the numbers of fungi in initial-stage, middle-stage and terminal-stage infectedplant increased by364.0%,365.8%and1312.9%, respectively; the numbers of actinomycetesin initial-stage and terminal-stage infected plant increased by158.4%and989.1%,respectively. In S. miltiorrhiza roots, the numbers of bacteria in initial-stage, middle-stage andterminal-stage infected plant were1.5,24.3and13.1times of the control, respectively. Fungiwere only detected in middle-stage and terminal-stage infected plant, and the number of fungiin terminal-stage infected plant was4.3times of that in middle-stage infected plant; while thenumber of actinomycetes in terminal-stage infected plant was225.6%higher than the control;B/A in middle-stage infected plant was350.0%higher than the control.③6predominantfungi were isolated from S. miltiorrhiza rhizosphere, rhizoplane soils and root, of which4isolates were suspected harmful organisms. They were identified as F. oxysporum, F. solani,Fusarium sp. and Penicillium brefeldianum. The main pathogenic fungi were F. oxysporumand F. solani. These predominant soil microbes likely caused root rot disease by negativelyaffecting the growth of roots and their absorption of nutrient from soil. In conclusion, the occurence of root diseases in S. miltiorrhiza are mainly caused byabnormal soil microflora in plant rhizosphere and rhizoplane soil. In addition, root diseasescould lead a decline of S. miltiorrhiza root absorption function, which made a substantialincrease in residual available P, K in rhizosphere soil.（3） Isolation and identification of pathogenic fungi from root rot diseased S. miltiorrhizaFrom the infected S. miltiorrhiz plant roots,19fungi isolates were collected by tissueseparation method.4pathogenic fungi were screened by in vitro infection test of S. miltiorrhizroots and further attributed to2genus and2species, which were Fusarium solani1, F. solani2, F. oxysporum f. sp. vasinfectum1and F. oxysporum2, according to morphologicalcharacters and rDNA-ITS sequence analysis.（4） Screening and identification of antagonistic actinomycetes against pathogenic fungifrom root rot diseased S. miltiorrhiza22strains of antagonistic actinomycetes, which had obvious inhibitory effects on F.solani and F. oxysporum were screened from the300tested actinomycetes with agar blockmethod and growth rate method, included8strains of broad spectrum actinomycetes （D62,F54, D3, D38, Act8, L8, Act12, Act1） against the4pathogenic fungi. The inhibitory effectsof actinomycetes D62, F54, D3and Act8were obvious on F. solani and F. oxysporum, whichinhibitory rates were31.0%-55.6%. And the unit mycelium inhibitory rates of L8, D62andAct12on the4pathogenic fungi were114.5-691.7%·g-1; The inhibitory rings of D62and F54were7.0mm and6.8mm on F.oxysporum1, and the inhibitory rates reached53.5%and51.4%. The8broad spectrum actinomycetes isolates were identified as S. rubiginosohelvolus（D62）, S. lincolnensis （D3）, S. olivaceus （D38）, S. pactum （L8, Act12）, S. californicus （Act1）and Streptomyces sp.（F54, Act8）.（5） The growth promoting and adjusted effect in rooting zone of biomicrobialactinomycetes Act12on S. miltiorrhiza①Root dipping of actinomycetes Act12preparation showd favorable growth and yieldpromotion effect on S. miltiorrhiza. In the plot experiment, inoculating with actinomycetes of10and100times dilution, the stem-leaf natural weight increased by29.7%and35.5%respectively compared with the control treatment; the root natural weight increased by44.0%and39.6%respectively, and root dry weight increased by26.3%and33.3%respectively. Inthe field experiment, inoculating with actinomycetes preparation, the yield of root naturalweight increased2022.0kg·hm-2.②Root dipping of actinomycetes Act12preparation canimprove medicine quality of S. miltiorrhiza root. In the plot experiment, under the treatmentof actinomyces diluted100times by wood ash, the contents of TanshinoneⅡA, Salvianolicacid B and Danshensu in S. miltiorrhiza all achieved maximum value which increased by 175.0%,102.6%and110.0%respectively compared with that of control. Similarly, thecontents of this three active constituents per plant increased by348.7%、230.6%and242.6%compared with that of control. In the field experiment, the contents of Salvianolic acid B andDanshensu in S. miltiorrhiza were19.4%and20.8%higher than that in the control treatment,also, the contents of this two active constituents per plant improved by27.1%and28.5%compared with the control treatment.③Inoculating with actinomycetes Act12can adjust soilmicroecological balance, modify microflora and microbial community composition byreducing amount of harmful organisms and increasing amount of beneficial organisms. Inrhizosphere soil, compared to those of control, inoculating with Act12of10times dilution,the numbers of bacteria and fungi decreased by58.8%and34.8%respectively, and thenumber of actinomycetes increased by85.3%.③In rhizosphere and rhizoplane soilinoculating with Act12treatment,7predominant microorganisms might be beneficial, whichincluded2bacteria isolates namely Ochrobactrum pseudogrignonense and Sphingobiumaromaticiconvertens,5actinomycetes isolates namely S. xanthophaeus, S. capillispiralis, S.matensis, S. chartreusis and S. lincolnensis;4predominant fungi are suspected harmfulorganisms, which are Aspergillus flavus, As. niger, M. roridum and Nectria haematococca.④Inoculating with actinomycetes may control the disease of root-knot nematode. The diseaseincidence of root-knot nematode in S. miltiorrhiza decreased by50%by using actinomycetesAct12preparation.（6） The growth promoting and adjusted effect in rooting zone of biomicrobialactinomycetes Act12combined with potassium humate on S. miltiorrhiza in fieldCombined application of Act12preparation with potassium humate enhanced the growthpromotion effect on S. miltiorrhiza.①Under Act12combined with potassium humatetreatment, survival rate increased by8.7%compared with the control treatment, and mortalityrate decreased by39.0%; Stem-leaf natural weight, root natural weight, root natural weightper plant, root dry weight and root dry weight per plant were6.1%、28.6%、11.1%、36.3%and9.0%higher than the control, respectively.②C ombined application of Act12preparation withpotassium humate can adjust soil microecological balance, modify microflora and microbialcommunity composition. In rhizoplane soil, compared with the control treatment, A/Fincreased by95.0%under Act12combined with potassium humate treatment. In S.miltiorrhiza roots, under Act12combined with potassium humate treatment, the number ofbacteria was195.0%higher than the control. While fungi and actinomyctes were not detected.③In rhizosphere and rhizoplane soil with Act12and potassium humate,6predominantmicroorganisms might be beneficial, which included3bacteria isolates namely Arthrobacternitroguajacolicus, R. radiobacter and Ps. frederiksbergensis,3actinomycetes isolates namely S. diastatochromogenes, S. lateritius and S. cavourensis;2predominant microorganisms weresuspected harmful isolates, included1bacteria isolate Brevibacterium frigoritolerans and1actinomycetes isolate S. turgidiscabies.③Combined application of Act12preparation withpotassium humate may control the disease of root-knot nematode. The disease incidence ofroot-knot nematode in S. miltiorrhiza decreased by49.6%by using Act12preparationcombined with potassium humate. |
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