Sreening And Identification Of Endophytic Actinomycetes From Soybean Roots And Study Of Their Metabolites With Bioactivity

Phytophthora root rot, which is caused by Phytophthora sojae, results in considerable loss ofsoybean crop yield worldwide. However, in some soybean-growing fields with Phytophthora rootrot symptoms, a small population of asymptomatic soybean plants (called escape plants) that areadjacent to affected plants have been consistently observed. That is the possibility that any healingproperties of the plant used by indigenous peoples may in fact be related to the presence of one ormore endophytic actinomycetes within the tissues of the plant. Therefore, the overall aims of thiswork were to isolate and identify the strains present in asymptomatic soybean plants, and toevaluate the antifungal activity of these actinomycetes against P. sojae. A bioactivity-guidedapproach was then employed to isolate and determine the chemical identity of bioactiveconstituents with antifungal activity against P. sojae.(1) A total of29endophytic actinomycete strains were successfully isolated. The dominantgenus was Streptomyces (19), while Micromonospora (1), Nonomuraea (1), Streptosporangium (1)and Wangella (1) were also recovered.(2) Strain NEAU-J3Tcharacterized using a polyphasic approach. Phylogenetic analysis basedon16S rRNA gene sequences suggested that strain NEAU-J3Tfell within the familyMicromonosporaceae. The strain was observed to form an extensively branched substratemycelium, which carried non-motile oval spores with a smooth surface. The cell walls of strainNEAU-J3Twere determined to contain meso-diaminopimelic acid and galactose, ribose and glucosewere detected as whole-cell sugars. The major menaquinones were determined to be MK-9(H4) andMK-9(H6). The phospholipids detected were phosphatidylcholine and phosphatidylethanolamine.The major cellular fatty acids were determined to be C16:0, C18:1ω9c, C18:0, C17:0, C17:1ω7c,anteiso-C17:0, C16:1ω7c and C15:0. The DNA G+C content was62.5mol%. On the basis of themorphological, chemotaxonomic characteristics, phylogenetic analysis and characteristic patterns of16S rRNA gene signature nucleotides, strain NEAU-J3Tis considered to represent a novel speciesof a new genus within the family Micromonosporaceae, for which the name Wangella harbinensisgen. nov., sp. nov. is proposed. The type strain of Wangella harbinensis is strain NEAU-J3T(=CGMCC4.7039T=DSM45747T).(3) Strain NEAU-Da3Twas characterized using a polyphasic approach.16S rRNA genesequence similarity studies showed that strain NEAU-Da3Tbelonged to the genus Streptomyces,being most closely related to Streptomyces carpaticus NRRL B-16359T(99.5%), Streptomyces cheonanensis VC-A46T(99.3%) and Streptomyces xiamenensis MCCC1A01550T(97.2%),similarities to other type strains of the genus Streptomyces were lower than97.1%. Themaximum-likelihood phylogenetic tree based on16S rRNA gene sequences showed that the isolateformed a distinct phyletic line with S. carpaticus NRRL B-16359T, S. cheonanensis VC-A46Tand S.xiamenensis MCCC1A01550T. This branching pattern was also supported by the tree constructedwith the neighbour-joining method. A comparative study between strain NEAU-Da3Tand theclosest related Streptomyces type strains revealed that it differed from them in morphological,physiological and biochemical characteristics. Therefore, it is proposed that strain NEAU-Da3Trepresents a novel Streptomyces species. Streptomyces harbinensis sp. nov. The type strain ofStreptomyces harbinensis is NEAU-Da3T(=CGMCC4.7047T=DSM42076T).(4) Strain NEAU-W2Twas characterized using a polyphasic approach. The organism wasfound to have morphological and chemotaxonomic characteristics typical of streptomycetes. TheG+C content of the DNA was66.12mol%. Analysis of the16S rRNA gene sequence of strainNEAU-W2Trevealed that the strain formed a distinct clade within the16S rRNA gene sequencephylogenetic tree and the highest similarity (99.61%) was to Streptomyces neyagawaensis strainATCC27449T. However, the DNA-DNA relatedness between strain NEAU-W2Tand S.neyagawaensis ATCC27449Twas found to be58.51%. Strain NEAU-W2Tcould also bedifferentiated from S. neyagawaensis ATCC27449Tand other Streptomyces species showing high16S rRNA gene sequence similarity (98–99%), as well as other borrelidin-producing strains bymorphological and physiological characteristics. On the basis of its physiological and molecularproperties, it is proposed that strain NEAU-W2Trepresents a novel Streptomyces species.Streptomyces heilongjiangensis sp. nov. The type strain of Streptomyces heilongjiangensis isNEAU-W2T(=CGMCC4.7004T=DSM42073T=ATCC BAA-2424T).(5) Streptomyces heilongjiangensis NEAU-W2with strong antifungal activity against P. sojaewas isolated from healthy soybean root, using an in vitro screening technique. A bioactivity-guidedapproach was then employed to isolate and determine the chemical identity of bioactiveconstituents with antifungal activity from strain NEAU-W2. The structure of the antifungalmetabolite was elucidated as borrelidin on the basis of spectral analysis. To our knowledge, this isthe first report that borrelidin has strong antifungal activity against P. sojae with EC50and EC95of0.0056and0.026mg/L against the dominant race1. The values were62.5-and262.3-fold lowerthan those of the commercial fungicide metalaxyl, which was used primarily to treat soybean seedfor the control of P. sojae, respectively. The in situ antifungal activity demonstrated that borrelidinreduced the symptoms of soybean seedlings infected with P. sojae race1by94.72%at10mg/L.Thus, borrelidin might be a promising candidate for new antifungal agents against P. sojae.