Diversity Of Streptomyces Species From Diseased Potato Scab Tubers And Their Phytotoxic And Antibacterial Activity

Weeds have caused significant losses for crop production in agricultural production.Chemical herbicides have played an important role in ensuring crop production.However,the high reliance on chemical herbicides has led to environmental and herbicide resistance issues,which pose a serious threat to world agricultural production.Thus,there is an urgent need to develop efficient and safe bioherbicides.Plant pathogens and their secondary metabolites are abundant and stable sources,making them important for the development of bioherbicides.In this thesis,the diseased potato common scab tubers were taken as the research object.The culture-dependent method was used to isolate the strains of streptomycetes genera associated with the potato common scab diseased tubers,and identified by molecular biological methods.The isolated strains were further screened by herbicidal and antibacterial activities,and three strains were selected according to the herbicidal activity effect and abundant metabolites.Various chromatographic methods were used to isolate and purify the active metabolites,and the structure of the active substances was determined using spectroscopic analysis.The results are as follows:1.A total of 50 strains of actinomycetes were isolated from the potato common scab tubers and purified by culture-dependent method,and the majority of isolates were recovered from Gauze’s No.1（46%）.They were then identified by molecular biological methods and divided into the streptomycetes genera,belonging to 12 different species.The strains with a homology greater than 99%of S.pratensis ch24^T had the highest number（32%）.2.The screening of the herbicidal activity of the isolated strains’fermented liquid showed that 45 of the 50 isolates（92%）exhibited herbicidal activities against at least one of the growth of weeds roots.Among them,NKY-12,SCA2,and SCA10 have strong inhibitory effects on the growth of Echinochloa crusgalli’root,with an inhibition rate of100.0%;L7-12,NKY-12,NKY-17,GS-49,SCA4,SCA5,SCA7,and AMCC400023 have strong inhibitory effects on the growth of Digitaria sanguinalis’root,with a inhibition rate of over 90%;NKY-12,NKY-29,NKY-30,B4-38,and GS-66 showed good inhibitory effects on the growth of Abutilon theophrastis,with a inhibition rate of more than 70%.The results of antibacterial activities indicate that only 6 isolates showed antibacterial activities against the Staphylococcus aureus at the concentration of 60μg/filter paper.NKY-4 showed excellent antibacterial activities against S.aureus with the disc diameter of inhibition zone diameter of 11.3 mm,which was slightly weaker than the positive gentamicin sulfate with an IZD of 16.0 mm.3.The pathogenicity of obtained strains was verified by the method of radish seedlings,the results showed that 4 isolates of Streptomyces L7-2,L7-7,NKY-4,and GS-67 had a good inhibitory effect on radish seedlings,with inhibition rates of 64.0%,66.2%,68.5%,and 68.8%,respectively.Pathogenicity was further tested through the small potato slice method and pathogenic gene detection.Among them,NKY-4 colonized on tuber slices and produced dark brown necrotic spots.The PCR detection results confirmed that strain NKY-4 contained the biosynthetic gene txt AB of thaxtomins,preliminarily confirming the possibility that NKY-4 had a new pathogenic species of potato common scab.4.Various chromatographic methods were used to isolate and purify the active metabolites.By using spectroscopic data analyses and comparison of their data in the literature,four compounds were identified from AMCC400023,including one new compound N-（2,5-dihydroxyphenyl）-3-acetamide-4-hydroxybenzamide（1）and three known compounds Thaxtomin A（2）,Nicotinic acid（3）and pyrrole-2-carboxylic acid（4）;two compounds were purified from SCA10 and their structures were determined to be N-（2-Hydroxyphenyl）acetamide（5）and 4-Hydroxybenzoic acid（6）;one compound was purified from NKY-4 and its structure was determined to be 2,3-dihydrobenzoic acid（7）.The herbicidal bioassay showed that compound 2 had a strong inhibitory effect on the weeds roots of E.crusgalli,D.sanguinalis,and A.theophrasti with an inhibition rate of100.0%at the concentration of 100μg/m L.Its IC₅₀ values were 0.99,0.78,and 1.95μg/m L,respectively.Compared with the positive control 2,4-Dichlorophenoxyacetic acid（IC50 values of 0.88,<0.1,<0.1μg/m L）,its inhibitory effect on the roots of E.crusgalli was comparable to the positive control,while its inhibitory effect on the roots of D.sanguinalis,and A.theophrasti were weaker than the positive control.The new compound1 has only weak herbicidal activity against the weeds roots of E.crusgalli,D.sanguinalis,and A.theophrasti,with inhibition rates of 9.6%,19.3%,and 20.8%,respectively,and compounds 3-7 showed weak herbicide activity on the roots of some of the tested weeds.The results of antibacterial activities indicated that compounds 1-7 showed no inhibitory effect on three tested bacterial strains:E.coli,M.tetragenus,and S.aureus at the concentration of 60μg/filter paper.The above results show that Streptomyces and its secondary metabolites related to potato scab have good herbicidal activity,which has important research value for the development of new bioherbicides.