Isolation, Characterization, and Herbicidal Activity of Streptomyces spp. from Diseased Potato Scab Tubers

Isolation, Characterization, and Herbicidal Activity of Streptomyces spp. from Diseased Potato Scab Tubers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation, Characterization, and Herbicidal Activity of Streptomyces spp. from Diseased Potato Scab Tubers Zhong-di HUANG, Shu-ping SHI, Yi ZHANG, Cai-ping YIN, Shu-xiang ZHANG, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3991115/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background A highly effective and environmentally friendly method of controlling weeds is biological herbicides, which typically constitute of naturally secondary metabolites, such as bioherbicidal metabolites produced by Streptomyces sp. However, the isolation of phytotoxic compounds from pathogenic Streptomyces has not been fully studied. Results Here, a total of 50 strains of Streptomyces genera were isolated from the potato tubers with typical common scab (CS) symptoms using the culture-dependent method. The radish seedling test indicated that 32 fermentation broths of potato common scab-associated Streptomyces could produce phytotoxic metabolites that affect the normal growth of radish seedlings’ radicles. Of note, two potential new pathogens (NKY-4 and L7-2) of potato scab were discovered by combining the methods of radish seedlings, potato tuber slices, and PCR detection of pathogenic genes txtAB . Moreover, the phytotoxic test demonstrated that the fermentation broths of 31 strains exhibited phytotoxic activities against at least one of the tested weeds ( Echinochloa crusgalli , Digitaria sanguinalis , and Abutilon theophrastis ). Furthermore, one novel metabolite and three known compounds, including new N-(2,5-dihydroxyphenyl)-3-acetamide-4-hydroxybenzamide ( 1 ), thaxtomin A ( 2 ), nicotinic acid ( 3 ) and pyrrole-2-carboxylic acid ( 4 ) were isolated from S. bottropensis (AMCC400023). Among them, compound 2 exhibited strong phytotoxic activity against E. crusgalli , D. sanguinalis , and A. theophrast with IC 50 values of 0.99, 0.78, and 1.95 µg/mL, respectively, which was comparable to those of the positive control 2,4-D. Conclusions Based on the results of these findings, phytotoxic metabolites from the potato scab pathogen may be one of the effective ways to develop new biological herbicides. Bioherbicide Potato common scab Streptomyces spp. Herbicidal activity Natural products Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Weeds are one of the most universal and challenging problems faced by agricultural production worldwide, causing detrimental effects on both crop yields and production costs. Generally, the control of weeds mainly includes physical control and the use of chemical or biological herbicides [ 1 ]. Physical weeding is mainly completed by manual and mechanical means, which is labor- and time-intensive and with adverse effects on the soil [ 2 ]. At present, chemical herbicides have high efficiency in controlling weeds, but alter the balance of environmental ecosystems. Problems specifically manifest as herbicide resistance in agrestal weeds, accumulation of toxic substances changes the soil and water environment, and dangerous to human health [ 3 , 4 ]. Therefore, the search for environmental-friendly biological herbicides has drawn much more attention [ 5 ]. Biological herbicides are secondary metabolites or microbial agents that are produced by microorganisms such as fungi, bacteria, and actinomycetes [ 6 ]. The rationale for biological herbicides is to cause plant phytotoxic activities that affect the normal growth of plants through the phytotoxins produced by microorganisms [ 7 ]. Streptomyces is rich in natural products with structurally diverse and phytotoxic activities, which might be used to create as a good candidate for biological weed control [ 8 ]. For instance, the compound rubiginone D2 with strong herbicidal activity was purified and identified from Streptomyces sp. KRA18-249, which was isolated from a natural recreational forest [ 6 ], and herbicidins A and B produced by S. saganonensis also had potent herbicidal activity [ 9 ]. Up to now, more than 900 Streptomyces species have been described, and relatively few species have the capacity to cause plant diseases, such as elicit scab symptoms on potato, radish, beet, and other tap root crops [ 10 , 11 ]. Among them, S. scabies was the first species to be identified as the cause of common scabs on potatoes, which adversely affects its quality [ 12 ]. Thaxtomin A (TA), the main phytotoxin produced by the pathogen S. scabies , not only induced scab symptoms on the surface of healthy potato stems, but also exhibited potent herbicidal activity against weeds [ 13 , 14 ]. In addition, the severity of potato scab outbreaks is rising, causing new pathogens Streptomyces spp. are constantly being discovered, which are due to continuous potato cropping and the increasing resistance of pathogens against bactericides [ 15 ]. Therefore, identifying undiscovered new pathogens from potato scabs and isolating phytotoxins has become a fundamental and urgent issue. In this study, we isolate and purify cultivable actinobacteria from potato tubers with lesions by using culture-dependent, assessed the pathogenicity and herbicidal activity of culturable actinobacteria. Additionally, we described the isolation, and structural characterization of secondary metabolites produced by one pathogenic for pathogen ( S. bottropensis AMCC400023) with herbicidal activity. Materials and methods Sample collection Potato samples with typical CS symptoms were collected from Anhui Academy of Agricultural Sciences, Hefei, China (GPS: 31 ◦ 53ʹ N, 117 ◦ 20ʹ E) and Wuwei, China (GPS: 37 ◦ 94ʹ N, 102 ◦ 61ʹ E), respectively, in December 2020 and September 2021. Isolation of actinobacteria The potato tubers with typical CS symptoms were selected for isolation of actinobacteria. The samples were washed to discard soil residues, then placed on sterile filter paper and air dry naturally in dark conditions. For each isolation, small pieces (1 cm 2 × 0.5 cm) were cut from typical potato scab lesions with a sterile scalpel. Then, the cut lesion tissue was separately placed in 10 mL 75% ethanol for 20-30s [ 16 ], followed by soaking them in sterile water for three times and fully homogenized separately in 10 mL of sterile water. Lastly, an aliquot of 100 µL serial dilution (10 − 1 , 10 − 2 , 10 − 3 ) of homogenates was spread to twelve different actinobacteria-selective media types [ 17 ] (Table S1 ), including actinobacteria isolation agar (AIA), cellulose-casamino acid (CCM), B4 medium, Gause’s No. 1 (G1), glucose-yeast-malt (GYM), luria bertani (LB), ISP medium No.2 (ISP2), ISP medium No.4 (ISP4), oatmeal agar (OMA), Reasoner’s 2A agar (R2A), starch casein agar (SCA), minimal medium (MM). All isolation media were supplemented with a final concentration of 75 mg/L nystatin, 25 mg/L nalidixic acid, 100 mg/L cycloheximide, and 50 mg/L potassium dichromate to suppress Gram-negative bacterial and fungal growth [ 18 ]. Incubation proceeded at 28°C for 1–4 weeks until colonies appeared. Typical Streptomyces colonies were purified by single spore methodology and transferred onto Gause’s No.1 agar. All isolates were preserved on slants at 4 ℃ or stored in 10% glycerol (-80 ℃). Identification and phylogenetic analysis of isolates Isolates were cultivated on G1 medium at 28 ℃ for one week, then initially identified by their phenotypic characteristics including the characteristics of colonies on plates and production of diffusible pigment. All isolates were further identified based on 16S rRNA gene sequencing [ 19 ]. The 16S rRNA gene of isolates was amplified using 2 × Taq Plus Master Max Ⅱ (Day Plus) and the specific primer pair 27F (5′-TCCTCCGCTTATTGATATGC-3′) / 1492R (5′-GGTTACCTTGTTACG ACTT-3′), with the following PCR conditions: 95°C for 3 min, and 33 cycles of (95°C for 20 s, 56°C for 20 s, 72°C for 90 s), followed by 72°C for 70 min. In addition, the trpB and rpoB gene of four strains (NKY-15, NKY-17, L7-2 and NKY-4) was amplified using the specific primer pair trpB-F (5′-GCGCGAGGACCTGAACCACACCGGCTCAC-3′) / trpB-R (5′- TCGATGGCCGGGATGATGCCCTCGG-3′) and rpoB-F (5′-GAGCGCATGACCACCCAGGACGTCGAGGC-3′) / rpoB-R (5′-CCTCGTAGTTGTGACCCTCCCACGGCATGA-3′) [ 20 ]. The PCR products were sent to Tsingke Biotechnology Co., Ltd. (Nanjing, China) for sequencing, then the sequencing results were submitted to EzTaxone server [ 21 ] ( https://www.ezbiocloud.net/ ) for comparison with type material sequences. The 16S rRNA gene sequences obtained in this study were deposited in the GenBank database (accession numbers OR186221-OR186270). The rpoB gene and trpB gene of four strains (NKY-15, NKY-17, L7-2 and NKY-4) sequences obtained in this study were deposited in the GenBank database (accession numbers PP212887- PP212890 and PP212891- PP212894). Pathogenicity tests Virulence assays of isolated strains on radish seedlings were performed according to the reported method [ 16 ]. The radish seeds were washed with sterile water and placed in culture dish moistened with sterile filter paper to induce germination for 24 h. 30 seeds were allowed to grow for 4 days (at 24 ℃) in Petri dishes (9 cm diameter) containing 5 ml of Actinomyces fermentation broth. Then root length of each seedling was recorded. The negative control was sterile water and the positive control was S. bottropensis AMCC400023. The maceration ability of the isolated strains was evaluated by performing the potato tuber slice assay as previously described [ 22 ]. Firstly, the fresh potato tubers (no lesion on the surface) were washed with water, then soaked and disinfected in 75% anhydrous ethanol for 5 minutes, washed in sterilized tap water. The 2 cm thick potato slices with a diameter of 15 mm were placed on moistened filter paper in 90 mm Petri dishes. Five strains (AMCC400023, L7-2, L7-7, NKY-4, and G1-67) with potent inhibitory activity on radish seedlings were selected to grow on G1 medium for one week at 28°C. Following incubation, agar plugs (5 mm diameter) containing sporulating colonies were sampled and placed at the centre of each tuber slice. Potatoes were incubated at 28°C for 7–10 days and observed for severity of browning and necrosis of potato tuber slice. The G1 solid culture medium and AMCC400023 were used as the negative and positive controls, respectively. All experiments were repeated in triplicate. The txtAB gene was involved in the biosynthesis of the main toxin (thaxtomins) of potato scab pathogens [ 23 ]. Some genes including nec1 and tomA were also somehow required for the virulence. But the prevalence of CS pathogens without txtAB genes was either very rare [ 24 ]. We used the most common pathogenic genes txtAB to perform pathogenicity validation on the isolated strains. Polymerase chain reaction was used to amplify txtAB , and the specific primer pair were txtAB-F (5′-CCACCAGGACCTGCTCTTC-3′) / txtAB-R (5′-TCGAGTGGACCTCA CAGATG-3′) [ 23 ]. Finally, the reactions were analyzed by 1.2% agarose gel electrophoresis. Screening for phytotoxic activities 32 strains with inhibitory activity to radish seedlings were selected for small-scale fermentation to screen for further test of herbicide activity. The strains were grown in 150 mL G1 liquid medium in 250 mL Erlenmeyer flask at 28 ℃, 180 rpm for 7 days. The fermentation broth of cultures was obtained by filtering through cotton gauze, and screened for potential herbicidal activity against two monocotyledonous weeds, E. crusgalli and D. sanguinalis , and a dicotyledonous weed, A. theophrasti by the method described in the previous literature [ 25 ]. 30 pregerminated seeds with consistent status were selected to place in Petri dishes (9 cm diameter) with filter paper, and filled with 5 ml of Actinomyces fermentation broth. Distilled water and 2,4-dichlorophenoxyacetic acid (2,4-D, Shanghai Chuangsai Technology Co., Ltd.) were used as negative and positive controls, respectively. After 2–3 days, the length of the radicle of each weed seed was measured. Isolation and characterization of secondary metabolite Based on bioassay-oriented, the strain S. bottropensis AMCC400023 with significant herbicidal activity was selected for identification of compounds in this study. The strain AMCC400023 was inoculated into a 250 mL Erlenmeyer flask containing 150 mL of SCM3 liquid medium and incubated at 28 ℃ (180 rpm) for 3 days. The seed inoculates were then transferred to 400 mL of the same medium in 1000 mL Erlenmeyer flasks, and cultured at 28 ℃ (180 rpm) for 7 days. The fermentation broth (16 L) was filtered through four layers of gauze, and the supernatant extracted three times with EtOAc. The ethyl acetate fractions were concentrated in vacuo with the aid of a rotary evaporator to obtain crude extract (6.0 g). The crude extract was fractionated into seven fractions (100:0, 100:1, 100:2, 100:4, 100:8, 100:16, and 100:32, v/v) by silica gel column (200–300 mesh) using a dichloromethane (CH 2 Cl 2 ) and methanol (MeOH) gradient. Fraction 5 was further purified on a silica gel column, and followed by fractionation on a Sephadex LH-20 column (MeOH) to yield compound 1 (120 mg) and subfraction (R1). The subfraction R1 was recrystallized from methanol, yielding an orange-yellow crystals 2 (148 mg). The fraction (Fr 4) was repeatedly purified on silica gel using CH 2 Cl 2 /MeOH (100:1, 100:2, 100:4) to give compound 3 (10 mg). Fr 2 was fractionated by silica gel column chromatography (CH 2 Cl 2 /MeOH) and recrystallized from methanol to give compound 4 (30 mg). Structural identifications of the compounds were determined via spectroscopic analysis. NMR spectra were measured with Agilent II DD2 spectrometers (Agilent, USA) at 600 MHz for 1 H and 150 MHz for 13 C, and the 2D spectra (COSY, HMQC, HMBC and DETP) were obtained by using standard Agilent software. Phytotoxic assay of compounds Compounds 1 – 4 were determined for phytotoxic activity against E. crusgalli , D. sanguinalis and A. theophrasti by using Petri dish bioassay [ 26 ]. All test compounds were dissolved in acetone and diluted to 100 µg/mL with distilled water. The 2,4-D was used as the positive control. Statistical analysis Statistical analysis was performed using origin 2021 software, considering a significance level of 95%. For the bio-herbicidal assay on tested seeds, each treatment was composed of 30 seeds with three replicates of 10 seeds. Results Isolation of actinomycetes strains from the potato tubers with typical common scab Here, a total of 50 isolates were isolated and purified from the scab lesions on potatoes using 12 different isolation media. As shown in Figure S1 , the number of actinomycetes isolated from different media exhibited appreciably differences. Among them, G1 were the most successful media for isolating actinobacterial strains with the number of 24 strains (about 48%), followed by SCA with 10 strains (20%). The number of strains obtained from the following six culture media were relatively small, including B4 (4 strains), AIA (4 strains), GYM (3 strains), CCM (2 strains), R2A (2 strains) and MM (1 strain). However, no strains were isolated from LB, ISP2, ISP4 and OMA media. Identification and phylogenetic analysis PCR amplification and sequencing analysis of the 16S rRNA gene was performed on 50 isolated actinomycetes, then the results of sequences were analyzed using BLAST and Ezbiocloud database. All isolates were identified as the genus Streptomyces belonging to the family Streptomycetaceae, which belongs to 12 species (Table S2). Among them, S. pratensis (32% of strains) and S. violascens (24%) were the dominant species. Notably, the 16S rRNA gene sequences were analyzed by EzTaxon indicated that some isolates showed relatively low similarities to the type strains of the corresponding genera. Using 16s rRNA, trpB and rpoB to analyse Streptomyces spp. with low similarity (NKY-15 and NKY-17), the results indicate that the strains NKY-15 and NKY-17 were in separate branches (Figure S2, S3), which indicated potential new species. Pathogenicity assay of the Streptomyces isolates The pathogenicity of selected Streptomyces spp. isolates was determined via the following three assays, including radish seed assay, potato tuber slice assay and detection of txtAB gene. A total of 63% (32 strains) of the selected Streptomyces spp. were positive in the radish seed assay (Fig. 1 ). Among them, 5 strains exhibited outstanding inhibitory activity against radish seedlings with an inhibition rate of 60%-70%, which were comparable to that of the positive AMCC400023 with an inhibition rate of 70%. A moderate inhibitory activity was found for 5 strains with an inhibition rate of 40%-60%. In addition, 21 strains showed relatively weak phytotoxic activity against radish seed with the inhibition rate of less than 40%. Furthermore, thaxtomin A (TA) is considered the primary phytotoxin produced by the pathogen Streptomyces scabies , which is the most important pathogenicity factor of potato common scab. Results of PCR analysis with the primer pair txtAB-F/txtAB-R showed that three strains (L7-2, L7-7 and NKY-4) were positive (Fig. 2 A). In the potato tuber slice assay, two isolates (L7-2 and NKY-4) caused CS lesions on potato tubers (Fig. 2 B). Similar to the positive AMCC400023, the strains L7-2 and NKY-4 could successfully colonize on the surface of potato tuber slice with different degree. Specially, the obvious black brown necrotic lesions were observed on potato slice. Therefore, the strains L7-2 and NKY-4 were positive in all three pathogenicity assays, which indicated that they were potential species causing potato scab disease (Figure S2, S3). Phytotoxic assay of fermentation broth of actinomycetes 32 bioactive actinomycetes in the radish seed assay were evaluated for their herbicide activity against the seedlings of E. crusgalli , D. sanguinalis and A. theophrasti using Petri dish bioassays. As shown in Fig. 1 , 31 of the 32 isolates (97%) exhibited herbicidal activities against at least one of the growth of weeds roots. Among them, three strains (NKY-12, SCA2, and SCA10) showed strong phytotoxic activity against E. crusgalli with the inhibition rate of 100%. Ten strains (L7-12, NKY-N, NKY-12, GS-49, GS-67, SCA-2, SCA4, SCA7, SCA10, and AMCC400023) also had strong inhibitory effects on the growth of Digitaria sanguinalis ’ root, with the inhibition rate of over 60%. Besides, 14 strains presented moderate phytotoxic activity against A. theophrasti with the inhibition rate of over 50%. Especially, 18 isolates exhibited inhibitory effects on the growth of both monocotyledonous weeds ( E. crusgalli and D. sanguinalis ) and a dicotyledonous weed ( A. theophrasti ). Structural characterization of secondary metabolites from AMCC400023 Four pure compounds (Fig. 3 ) were purified from SCM3 liquid medium fermentation product of Streptomyces sp. AMCC400023, and their structures were analyzed by NMR and EI-MS techniques, including one new compound N-(2,5-dihydroxyphenyl)-3-acetamide-4-hydroxybenzamide ( 1 ) and three known compounds thaxtomin A ( 2 ) [ 27 ], nicotinic acid ( 3 ) [ 28 ] and pyrrole-2-carboxylic acid ( 4 ) [ 29 ]. N-(2,5-dihydroxyphenyl)-3-acetamide-4-hydroxybenzamide ( 1 ): brown powder; HR-ESI-MS: m/z : 301.0824 [M-H] − , calculated for C 15 H 14 N 2 O 5 302.0903, which was consistent with 1 H and 13 C NMR data (Table 1 and Figures S4-S10). The 1 H NMR spectrum of 1 exhibited three phenolic hydroxyl group signals δ H [8.82 (brs, 1H), 9.04 (brs, 1H) and 10.58 (brs,1H)], two amide group signals δ H [9.93 (s, 1H) and 9.14 (s, 1H)], and a methyl group signal δ H 2.11 (s, 1H). 13 C NMR and DEPT135 spectrum showed two amide carbon signals ( δ C 164.5, and 169.1), twelve olefinic carbons signals ( δ C 109.1, 111.1, 115.3, 116.3, 121.9, 124.1, 124.9, 126.3, 126.8, 140.3, 149.9 and 151.2), and one methyl carbon signal ( δ C 23.6). The HMBC spectrum of 1 exhibited correlations (Fig. 3 ) from H-2 ( δ H 8.36) to C-6( δ C 124.1), and C-4 ( δ C 151.2), from H-3 ( δ H 9.39) to C-2 ( δ C 121.9), C-4, and C-7 ( δ C 169.1), from H-4 ( δ H 10.58) to C-3 ( δ C 126.3) and C-4, from H-5 ( δ H 6.95) to C-1 ( δ C 124.9), C-3, from H-6 ( δ H 7.57) to C-2 and C-4, and from H-8 ( δ H 2.11) to C-7, together with 1 H- 1 H-correlation spectroscopy (COSY) correlations (Fig. 3 ) observed between H-5 and H-6 indicated that compound 1 possessed trisubstituted benzene and acetamide moieties. Further analysis of HMBC data, the correlations from H-1′ ( δ H 9.14) to C-6′( δ C 109.1), C-2′ ( δ C 140.3), and C-7′( δ C 164.5), from H-3′ ( δ H 6.69) to C-1′( δ C 126.8) and C-5′ ( δ C 149.9), from H-4′ ( δ H 6.39) to C-2′and C-6, from H-5′ ( δ H 8.82) to C-4′ ( δ C 111.1), C-5′, and C-6′, and from H-6′ ( δ H 7.33) to C-2′, C-4′, together with 1 H- 1 H-correlation spectroscopy (COSY) correlations (Fig. 3 ) observed between H-3′ and H-4′ revealed the presence of trisubstituted benzene and phenolic hydroxyl groups. Accordingly, the structure of 1 was established as a new compound, and N-(2,5-dihydroxyphenyl)-3-acetamide-4-hydroxybenzamide was proposed as its trivial name. Table 1 1 H and 13 C NMR data for compounds 1 in DMSO- d 6 . Position δ C δ H , mult, J in Hz 1 124.9, C 2 121.9, CH 8.36, s 3 126.3, C 4 151.2, C 5 115.3, CH 6.95, d (8.4) 6 124.1, CH 7.57, d (8.0) 7 169.1, C 8 23.6, CH 3 2.11, s 1' 126.8, C 2' 140.3, C 3' 116.3, CH 6.69, d (8.6) 4' 111.1, CH 6.39, dd (2.8, 5.4) 5' 149.9, C 6' 109.1, CH 7.33, d (2.3) 7' 164.5, C 4-OH 10.58, br s 2'-OH 9.04, br s 5'-OH 8.82, br s 3-NH 9.93, s 1'-NH 9.14, s Thaxtomin A ( 2 ): orange-yellow crystals; HR-ESI-MS: m/z : 461.1401 [M + Na] + , calculated for C 22 H 22 N 4 O 6 438.1539; 1 H NMR (600 MHz, CD 3 OD) δ : 1.64 (1H, dd, J = 14.1, 8.8 Hz, H-10 b ), 2.61 (1H, dd, J = 14.2, 6.3 Hz, H-10 a ), 2.81 (3H, s, N-CH 3 ), 3.03 (3H, s, N-CH 3 ), 3.11 (2H, d, J = 13.6 Hz, H-17), 3.87 (1H, dd, J = 8.4, 6.6 Hz, H-11), 6.71 (3H, m, H-19, H-21, H-23), 6.95 (1H, s, H-2), 7.18 (1H, t, J = 7.9 Hz, H-6), 7.23 (1H, t, J = 8.1 Hz, H-22), 7.69 (1H, d, J = 8.0 Hz, H-7), 7.83 (1H, d, J = 7.8 Hz, H-5). 13 C NMR (150 MHz, CD 3 OD) δ : 168.5 (C, C-16), 167.0 (C, C-13), 159.2 (C, C-20), 143.9 (C, C-4), 141.3 (C, C-9), 137.5 (C, C-18), 132.6 (CH, C-2), 131.3 (CH, C-22), 122.9 (CH, C-23), 121.1 (CH, C-6), 120.0 (C, C-8), 119.3 (CH, C-5), 118.8 (CH, C-7), 118.5 (CH, C-19), 116.0 (CH, C-21), 110.7 (C, C-3), 88.2 (C, C-14), 64.8 (C, C-11), 43.8 (CH 2 , C-17), 34.3 (CH 3 , C-15), 33.7 (CH 2 , C-10), 28.6 (CH 3 , C-12). Nicotinic acid ( 3 ): white crystals; HR-ESI-MS: m/z : 124.0391 [M + H] + , calculated for C 6 H 5 NO 2 123.0320; 1 H NMR (600 MHz, CD 3 OD) δ : 7.55 (1H, dd, J = 5.0, 7.7 Hz, H-5), 8.40 (1H, d, J = 7.9 Hz, H-4), 8.72 (1H, d, J = 3.7 Hz, H-6), 9.12 (1H, s, H-2). 13 C NMR (150 MHz, CD 3 OD) δ : 125.3 (CH, C-5), 129.1 (C, C-3), 139.3 (CH, C-4), 151. 5 (CH, C-6), 153.7 (CH, C-2), 168.2 (CH, C-7). Pyrrole-2-carboxylic acid ( 4 ): brownish crystals; HR-ESI-MS: m/z : 110.0247 [M-H] − , calculated for C 5 H 5 NO 2 111.0320; 1 H NMR (600 MHz, CD 3 OD) δ : 6.16 (1H, s, H-4), 6.83 (1H, s, H-3), 6.92 (1H, s, H-5). 13 C NMR (150 MHz, CD 3 OD) δ : 124.8 (CH, C-5), 124.2 (C, C-2), 116.4 (CH, C-3), 110.6 (CH, C-4). Phytotoxic activities of compounds The phytotoxic activities of the test compounds ( 1 – 4 ) against radicle growth of E. crusgalli , D. sanguinalis and A. theophrasti were shown in Table 2 . The results showed that compound 2 exhibited strong phytotoxic activity against E. crusgalli , D. sanguinalis and A. theophrasti with the inhibition rate of 100%, which was same as that of the positive 2,4-D at a concentration of 100 µg/mL. However, the new compound 1 had weak inhibitory activity against above three weeds with the inhibition rate of less than 30%. Similarly, the compound 4 exhibited weak phytotoxic activity against two monocotyledonous weeds ( E. crusgalli , D. sanguinalis) with the inhibition rate of less than 40%. Further testing was conducted on the herbicidal activity of compound 2 with different concentrations. The results also showed that the metabolite 2 presented strong phytotoxic activity with the IC 50 values of 0.99, 0.78, and 1.95 µg/mL, respectively, which were almost comparable with those of the positive 2,4-D with IC 50 values of 0.88, < 0.1, and < 0.1 µg/mL (Fig. 4 ). Table 2 Inhibitory effects of compounds on the growth of weeds roots (%). Compounds E. crusgalli D. sanguinalis A. theophrasti 1 9.6 ± 6.8 19.3 ± 8.1 20.8 ± 7.9 2 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 3 NI 22.3 ± 8.4 NI 4 20.0 ± 7.2 31.3 ± 6.5 NI 2,4-D 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 Results are presented as the mean ± standard; “NI” means not inhibited; the concentration for the test is 100 µg/mL; 2,4-D was the positive control. Discussion Potato common scab (CS) scab was a soil-borne disease caused by the typical phytopathogenic Streptomyces sp. and was one of the most devastating diseases in potatoes, reducing both crop growth and quality [ 30 ]. In this study, 50 potato-associated Streptomyces , including two potentially new species, were isolated from the potato tubers with typical common scab and identified by morphological and molecular biological methods. As reported previously, a new species was defined by a 16S rRNA gene sequence homology below 98.7% [ 31 , 32 ]. Specifically, based on 16s rRNA and the high-resolution Streptomyces sp. housekeeping genes: trpB (tryptophan synthase subunit beta) and rpoB (RNA polymerase subunit beta) [ 33 ] the phylogenetic trees indicated that Streptomyces spp. with low similarity (NKY-15 and NKY-17) were in separate branches and might potential new species. Here, twelve selective isolation media were employed to isolate as many actinobacteria as feasible. Among them, the G1 and SCA media were the most effective as regards the number of obtained isolates. The G1 and SCA media, which were used to find saccharolytic bacteria, including actinobacteria [ 18 , 34 , 35 ], were mostly made of soluble starch which facilitated the mycelial extension and cell growth of actinomycetes as a sole carbon source [ 19 ]. Till now, approximately 30 species of Streptomyces causing CS have been identified worldwide [ 36 ], of which S. scabies, S. acidiscabies, and S. turgidiscabies were the most characterized [ 36 , 37 ]. Noticeably, the strains NKY-4 and L7-7 were positive in pathogenicity assays, which have never been reported to cause potato scab, indicating that might be newly discovered pathogen of potato scab disease. However, 38% of all the isolates were negative in all pathogenicity assays, indicating that nonpathogenic Streptomyces were also colonizing potato tubers, which was consistent with the previous reports [ 16 , 38 ]. Our results show that about 63% (32 strains) of the tested Streptomyces spp. were positive in the radish seed assay, but most of which were negative in other two pathogenicity assays. This indicates that although thaxtomin family, which is encoded by txtAB gene, is considered as a major player toward plant pathogenicity, other phytotoxic may exist in different species of Streptomyces and can affect the normal growth of radish seedlings [ 39 ]. Other Streptomyces phytotoxic specialized metabolites have been reported, such as two phytotoxic nigericin and geldanamycin were isolated from plant pathogen Streptomyces sp. 11-1-2 [ 40 ], desmethylmensacarcin is a novel phytotoxicity was isolated from pathogen S. niveiscabiei [ 41 ]. The herbicidal activity of 32 strains of Streptomyces spp. exhibiting inhibitory activity on the root length of radish seedlings was further tested against three weeds. The results revealed that 31 Streptomyces strains showed inhibitory activity against at least one of the weeds’ roots. This provides new ideas for the development of novel herbicides of microbial-pathogen origin. Glufosinate was the first microbial-origin herbicide developed and synthesized as a lead compound by the secondary metabolite of bilanafos from Streptomyces [ 42 ]. We investigated the secondary metabolites from the pathogen S. bottropensis AMCC400023, which resulted in the isolation of compounds 1 – 4 . Among them, compound 2 displayed strong inhibitory activity against E. crusgalli , D. sanguinalis , and A. theophrast , with IC 50 values of 0.99, 0.78, and 1.95 µg/mL, respectively. Indeed, compound 2 (TA) was the main phytotoxin synthesized by the potato common scab-causing pathogen Streptomyces spp., which has been reported to be phytotoxic to both broadleaf and the dicotyledonous weed, A. theophrasti [ 43 ]. However, this study found for the first time that compound 2 (TA) showed potential herbicidal activity against two monocotyledonous weeds, E. crusgalli and D. sanguinalis . Morever, it was common to find that the mechanisms of action of phytotoxin was an impact on plant chlorophyll content, lipid peroxidation, and electrolytic leakage [ 6 ]. Therefore, Streptomyces was one of the important sources of lead compounds in natural herbicides. Conclusion In summary, 50 actinomycetes were isolated and identified from potato samples using culture-dependent methods. Sequences analysis demonstrated that all actinomycetes were attached to the Streptomyces genera. 32 strains exhibited a certain degree of virulence as determined by a pathogenicity assay on radish plants. Furthermore, potential new pathogens NKY-4 and L7-2 were positive in three pathogenicity assays. The results of phytotoxic tests showed that 31 extracts (97%) exhibited phytotoxic activities against at least one of the tested weeds. In addition, one novel metabolite and three known compounds were purified from pathogen S. bottropensis AMCC400023. Compound 2 displayed outstanding phytotoxic activity against E. crusgalli , D. sanguinalis , and A. theophrast with IC 50 values of 0.99, 0.78, and 1.95 µg/mL, respectively. Therefore, our results suggest that metabolites produced by S. bottropensis AMCC400023 with herbicidal activity may be a new bioherbicide candidate or leads molecule for a more efficient herbicide. Abbreviations CS Common scab PCR Polymerase chain reaction 2,4-D 2,4-dichlorophenoxyacetic acid TA Thaxtomin A AIA Actinobacteria isolation agar CCM Cellulose-casamino acid G1 Gause’s No. 1 GYM Glucose-yeast-malt LB Luria bertani ISP2 ISP medium No.2 ISP4 ISP medium No.4 OMA Oatmeal agar R2A Reasoner’s 2A agar SCA Starch casein agar MM Minimal medium CH 2 Cl 2 Dichloromethane MeOH Methanol COSY 1 H- 1 H-correlation spectroscopy correlation HMBC 1 H detected heteronuclear multiple bond correlation DEPT Distortionless enhancement by polarization transfer Declarations Acknowledgments We acknowledge Professor Bo Zhou and Agricultural Microbial Resources and Utilization Center, Shandong Agricultural University, China for kindly providing one pathogen strain ( S. bottropensis AMCC400023). Author contribution Ying-lao ZHANG and Shu-xiang ZHANG conceived and designed research. Shu-ping SHI and Yi ZHANG conducted experiments. Zhong-di HUANG and Cai-ping YIN analyzed the data. Zhong-di HUANG wrote the manuscript. All authors read and approved the manuscript. Funding This work was co-financed by the National Natural Science Foundation of China (32270015 and 32102272) and Anhui Outstanding Youth Science Fund Project (2108085J18). Availability of data and materials All data generated or analyzed during this study are included in this manuscript, its supplementary information files and in GenBank database. The 16S rRNA gene sequences obtained in this study were deposited in the GenBank database (accession numbers OR186221-OR186270). The rpoB gene and trpB gene of four strains (NKY-15, NKY-17, L7-2 and NKY-4) sequences obtained in this study were deposited in the GenBank database (accession numbers PP212887- PP212890 and PP212891- PP212894). Ethics approval and consent to participate The research conducted in this study did not involve any animal subjects, therefore obtaining consent to participate was not applicable. Consent for publication Not applicable. Declaration of competing interest The authors declare that they have no conflict of interest. Author information Zhong-di HUANG, Shu-ping SHI, Yi ZHANG, Cai-ping YIN and Ying-lao ZHANG School of Life Sciences, Anhui Agricultural University, Hefei, China References Kim H. J., Bo A. B., Kim J. D., Kim Y. S., Khaitov B., et al. 2020. Herbicidal Characteristics and Structural Identification of the Potential Active Compounds from Streptomyces sp. KRA17-580. J. Agric. Food Chem. 68 , 15373−15380. Bruciene I., Buragiene S., Sarauskis E. 2022. Weeding Effectiveness and Changes in Soil Physical Properties Using Inter-Row Hoeing and a Robot. AGRONOMY-BASEL, 12 , 1514. Dai P., Yan Z., Ma S., Yang Y., Wang Q., et al. 2018. The herbicide glyphosate negatively affects midgut bacterial communities and survival of honey bee during larvae reared in vitro. J. Agric. Food Chem. 66 , 7786–7793. Cho K. M., Shin S. C., Bo A. B., Umurzokov M., Jia W., et al. 2022. 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Characterization of streptomycetes causing potato common scab in Korea. Plant Dis . 87 , 1290–1296. Wu Z., Liu F., Ke S., Zhang Z., Hu H., et al. 2023. Secondary metabolites from Aspergillus sparsus NBERC_28952 and their herbicidal activities. Plants. 12 , 203. Cai Z. F., Zhang W. L., Cao Y. Y., Du X. H., Heterocycl J. 2022. Synthesis and herbicidal activities of 2-phenylpyridine compounds containing alkenyl moieties. J Heterocyclic Chem. 59 , 1247–1252. King R. R., Lawrence C. H., Clark M. C., et al. 1989. Isolation and characterization of phytotoxins associated with Streptomyces scabie . J. Agr. Food Chem. 13 , 849-850. Cheng M. J., Tseng M., Chen I. S., et al. 2009. Secondary metabolites from the culture broth of actinomycete Acrocarpospora sp. firdi 001 and their antimicrobial activity. J. Chil. Chem. Soc. 54 , 198-200. Lewis E. A., Adamek T. L., Vining L. C., et al. 2003. Metabolites of a blocked chloramphenicol produce. J. Nat. Prod. 66 , 62-66. Hao J. J., Ashley K. 2021. Irreplaceable role of amendment-based strategies to enhance soil health and disease suppression in potato production. Microorganisms . 9 , 1660. Rossi-Tamisier M., Benamar S., Raoult D., Fournier P. E. 2015. Cautionary tale of using 16S rRNA gene sequence similarity values in identification of human-associated bacterial species. Int. J. Syst. Evol. Microbiol. 65 , 1929-1934. Molina-Menor E., Gimeno-Valero H., Pascual J., Peretó J., Porcar M. 2021. High culturable bacterial diversity from a European desert: the tabernas desert. Front. Microbiol. 11 , 583120. Yang, Z., Qiao, Y., Konakalla, N.C. et al. 2023. Streptomyces alleviate abiotic stress in plant by producing pteridic acids. Nat Commun 14 , 7398. Weeraphan T., Somphong A., Poengsungnoen V. 2023. Bacterial microbiome in tropical lichens and the effect of the isolation method on culturable lichen-derived actinobacteria. Sci Rep. 13 , 5483. Sapkota A., Thapa A., Budhathoki A., Sainju M., Shrestha P., et al. 2020. Isolation, characterization, and screening of antimicrobial-producing actinomycetes from soil samples. Int. J. Food Microbiol. 2020 , 2716584. Wei Q., Li J., Yang S., Wang W. Z., Min F. X., et al. 2022. Streptomyces rhizophilus causes potato common scab disease. Plant Dis. 106 , 266-274. Biessy A., Filion M. 2022. Biological control of potato common scab by plant-beneficial bacteria. Biol. Control. 165 , 104808. Jordaan E., Van der Waals J. E. 2016. Streptomyces species associated with common scab lesions of potatoes in South Africa. Eur. J. Plant. Pathol. 144 , 631-643. Li Y., Liu J., Díaz-Cruz G., Cheng Z., Bignell D. R. 2019. Virulence mechanisms of plant-pathogenic Streptomyces species: an updated review. Microbiology. 165 , 1025–1040. Díaz-Cruz, G.A., Liu J. Y., Tahlan K., Bignell, DRD. 2022. Nigericin and geldanamycin are phytotoxic specialized metabolites produced by the plant pathogen Streptomyces sp. 11-1-2. Microbiol. Spectr. 10 , e02314-21. Lapaz M. I., López A., Huguet-Tapia J. C., Pérez-Baldassari F. M., Iglesias C., et al. 2018. Isolation and structural characterization of a non-diketopiperazine phytotoxin from a potato pathogenic Streptomyces strain. Nat. Prod. Res. 33 , 2951-2957. Hoerlein G. 1994. Glufosinate (phosphinothricin), a natural amino acid with unexpected herbicidal propertie. Rev. Environ. Contam. T . 138 , 73-145. Wolfe J. C., Neal J. C., Harlow C. D., Gannon T. 2016. Efficacy of the bioherbicide Thaxtomin A on smooth crabgrass and annual bluegrass and safety in cool-season turfgrasses. Weed Technol . 30 , 733-742. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3991115","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289011225,"identity":"d82e4b38-0bd1-43a3-994d-cd17446ae3e3","order_by":0,"name":"Zhong-di HUANG","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhong-di","middleName":"","lastName":"HUANG","suffix":""},{"id":289011226,"identity":"5adccebd-a87f-41c7-999d-1c3bb7fd9ccd","order_by":1,"name":"Shu-ping SHI","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shu-ping","middleName":"","lastName":"SHI","suffix":""},{"id":289011227,"identity":"9b0cc0d2-11c3-415c-bc8a-627c1b616078","order_by":2,"name":"Yi ZHANG","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"ZHANG","suffix":""},{"id":289011228,"identity":"7a270eb4-8b85-4251-866a-7a583c7aa562","order_by":3,"name":"Cai-ping YIN","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Cai-ping","middleName":"","lastName":"YIN","suffix":""},{"id":289011229,"identity":"197dfe2b-c380-4db1-b389-cbba9edcc205","order_by":4,"name":"Shu-xiang ZHANG","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shu-xiang","middleName":"","lastName":"ZHANG","suffix":""},{"id":289011230,"identity":"8bc697cc-ac89-4d40-91af-6ada8d63d28e","order_by":5,"name":"Yinglao Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDACCTBpIQdlMBOtRcKYdC2JDURrkZ/d/Ozh1zaJ9H4gQ4Khwjqxgf3sAbxaGOccMzeWbZPInXHnmJkEw5n0xAaevAS8WpglEsykJYFaGm4kmEkwth0GupDHAK8WNon0byAt6fI30r9JMP4jQguPRI6Z5Mc2iQSDGzlAWxqI0CIhkVMmzXBOwnDjjZxii4Rj6cZtPDn4tcjPSN8m+aPMRl7uRvrGGx9qrGX72c/g1wICzLxsUFYCyHcE1QMB448/xCgbBaNgFIyCEQsA5vc/OldZ918AAAAASUVORK5CYII=","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Yinglao","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-02-26 14:02:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3991115/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3991115/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54339920,"identity":"5ac261d4-0b8b-44fc-9a55-77f269b8f030","added_by":"auto","created_at":"2024-04-09 04:44:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75823,"visible":true,"origin":"","legend":"\u003cp\u003eThe heatmap depicts inhibitory effects of the fermentation broth on the growth of radish seedlings and weeds roots (%).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3991115/v1/184de62123cdf79931423a22.png"},{"id":54339925,"identity":"5f266efe-6b37-49ce-8548-0b0496716042","added_by":"auto","created_at":"2024-04-09 04:44:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":877692,"visible":true,"origin":"","legend":"\u003cp\u003ePathogenicity test of isolate. (A) PCR detection electrophoresis results of pathogenic genes \u003cem\u003eTxtAB\u003c/em\u003e. (B) Pathogenicity verification results of small potato chip. (A) DL2000 Marker (M); Strain AMCC400023 (1); Strain L7-2 (2) Strain L7-7 (3); Strain NKY-4 (4); Strain GS-67 (5). (B) GS media (a); Strain AMCC400023 (b); Strain NKY-4 (c); Strain L7-2 (d).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3991115/v1/6ca8e97d302ccd9d50f72392.png"},{"id":54339917,"identity":"b4ca6bd2-d3e1-4703-8383-979157704e4e","added_by":"auto","created_at":"2024-04-09 04:44:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82391,"visible":true,"origin":"","legend":"\u003cp\u003eThe secondary metabolites of strain \u003cem\u003eS. bottropensis\u003c/em\u003e AMCC400023.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3991115/v1/d474840a81f5e73df4d6655a.png"},{"id":54339926,"identity":"73b3194d-f284-4a1b-88de-bde876b51342","added_by":"auto","created_at":"2024-04-09 04:44:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60263,"visible":true,"origin":"","legend":"\u003cp\u003eIC50 values of compound \u003cstrong\u003e2\u003c/strong\u003e in inhibiting the growth of weeds roots.\u003c/p\u003e\n\u003cp\u003eThe 2,4-D as positive control. (A) \u003cem\u003eE. crusgalli\u003c/em\u003e; (B) \u003cem\u003eD. sanguinalis\u003c/em\u003e; (C) \u003cem\u003eA. theophrasti.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3991115/v1/16d968dafee0e9feae0b3762.png"},{"id":97664525,"identity":"f4aa8146-626b-49d4-9e60-808882dcb0ae","added_by":"auto","created_at":"2025-12-08 09:08:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2108548,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3991115/v1/359fe347-2505-4848-81d4-8e943b5f2159.pdf"},{"id":54339921,"identity":"9d0d7b85-5bfa-48d7-8b00-23b709d02757","added_by":"auto","created_at":"2024-04-09 04:44:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":917663,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3991115/v1/277930fce7b9ec5bc2aeaf16.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation, Characterization, and Herbicidal Activity of Streptomyces spp. from Diseased Potato Scab Tubers","fulltext":[{"header":"Background","content":"\u003cp\u003eWeeds are one of the most universal and challenging problems faced by agricultural production worldwide, causing detrimental effects on both crop yields and production costs. Generally, the control of weeds mainly includes physical control and the use of chemical or biological herbicides [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Physical weeding is mainly completed by manual and mechanical means, which is labor- and time-intensive and with adverse effects on the soil [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At present, chemical herbicides have high efficiency in controlling weeds, but alter the balance of environmental ecosystems. Problems specifically manifest as herbicide resistance in agrestal weeds, accumulation of toxic substances changes the soil and water environment, and dangerous to human health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, the search for environmental-friendly biological herbicides has drawn much more attention [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiological herbicides are secondary metabolites or microbial agents that are produced by microorganisms such as fungi, bacteria, and actinomycetes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The rationale for biological herbicides is to cause plant phytotoxic activities that affect the normal growth of plants through the phytotoxins produced by microorganisms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eStreptomyces\u003c/em\u003e is rich in natural products with structurally diverse and phytotoxic activities, which might be used to create as a good candidate for biological weed control [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For instance, the compound rubiginone D2 with strong herbicidal activity was purified and identified from \u003cem\u003eStreptomyces\u003c/em\u003e sp. KRA18-249, which was isolated from a natural recreational forest [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and herbicidins A and B produced by \u003cem\u003eS. saganonensis\u003c/em\u003e also had potent herbicidal activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUp to now, more than 900 \u003cem\u003eStreptomyces\u003c/em\u003e species have been described, and relatively few species have the capacity to cause plant diseases, such as elicit scab symptoms on potato, radish, beet, and other tap root crops [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among them, \u003cem\u003eS. scabies\u003c/em\u003e was the first species to be identified as the cause of common scabs on potatoes, which adversely affects its quality [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thaxtomin A (TA), the main phytotoxin produced by the pathogen \u003cem\u003eS. scabies\u003c/em\u003e, not only induced scab symptoms on the surface of healthy potato stems, but also exhibited potent herbicidal activity against weeds [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, the severity of potato scab outbreaks is rising, causing new pathogens \u003cem\u003eStreptomyces\u003c/em\u003e spp. are constantly being discovered, which are due to continuous potato cropping and the increasing resistance of pathogens against bactericides [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, identifying undiscovered new pathogens from potato scabs and isolating phytotoxins has become a fundamental and urgent issue.\u003c/p\u003e \u003cp\u003eIn this study, we isolate and purify cultivable actinobacteria from potato tubers with lesions by using culture-dependent, assessed the pathogenicity and herbicidal activity of culturable actinobacteria. Additionally, we described the isolation, and structural characterization of secondary metabolites produced by one pathogenic for pathogen (\u003cem\u003eS. bottropensis\u003c/em\u003e AMCC400023) with herbicidal activity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003ePotato samples with typical CS symptoms were collected from Anhui Academy of Agricultural Sciences, Hefei, China (GPS: 31\u003csup\u003e◦\u003c/sup\u003e53ʹ N, 117\u003csup\u003e◦\u003c/sup\u003e20ʹ E) and Wuwei, China (GPS: 37\u003csup\u003e◦\u003c/sup\u003e94ʹ N, 102\u003csup\u003e◦\u003c/sup\u003e61ʹ E), respectively, in December 2020 and September 2021.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of actinobacteria\u003c/h2\u003e \u003cp\u003eThe potato tubers with typical CS symptoms were selected for isolation of actinobacteria. The samples were washed to discard soil residues, then placed on sterile filter paper and air dry naturally in dark conditions. For each isolation, small pieces (1 cm\u003csup\u003e2\u003c/sup\u003e \u0026times; 0.5 cm) were cut from typical potato scab lesions with a sterile scalpel. Then, the cut lesion tissue was separately placed in 10 mL 75% ethanol for 20-30s [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], followed by soaking them in sterile water for three times and fully homogenized separately in 10 mL of sterile water. Lastly, an aliquot of 100 \u0026micro;L serial dilution (10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) of homogenates was spread to twelve different actinobacteria-selective media types [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), including actinobacteria isolation agar (AIA), cellulose-casamino acid (CCM), B4 medium, Gause\u0026rsquo;s No. 1 (G1), glucose-yeast-malt (GYM), luria bertani (LB), ISP medium No.2 (ISP2), ISP medium No.4 (ISP4), oatmeal agar (OMA), Reasoner\u0026rsquo;s 2A agar (R2A), starch casein agar (SCA), minimal medium (MM). All isolation media were supplemented with a final concentration of 75 mg/L nystatin, 25 mg/L nalidixic acid, 100 mg/L cycloheximide, and 50 mg/L potassium dichromate to suppress Gram-negative bacterial and fungal growth [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Incubation proceeded at 28\u0026deg;C for 1\u0026ndash;4 weeks until colonies appeared. Typical \u003cem\u003eStreptomyces\u003c/em\u003e colonies were purified by single spore methodology and transferred onto Gause\u0026rsquo;s No.1 agar. All isolates were preserved on slants at 4 ℃ or stored in 10% glycerol (-80 ℃).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and phylogenetic analysis of isolates\u003c/h2\u003e \u003cp\u003eIsolates were cultivated on G1 medium at 28 ℃ for one week, then initially identified by their phenotypic characteristics including the characteristics of colonies on plates and production of diffusible pigment. All isolates were further identified based on 16S rRNA gene sequencing [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The 16S rRNA gene of isolates was amplified using 2 \u0026times; Taq Plus Master Max Ⅱ (Day Plus) and the specific primer pair 27F (5\u0026prime;-TCCTCCGCTTATTGATATGC-3\u0026prime;) / 1492R (5\u0026prime;-GGTTACCTTGTTACG ACTT-3\u0026prime;), with the following PCR conditions: 95\u0026deg;C for 3 min, and 33 cycles of (95\u0026deg;C for 20 s, 56\u0026deg;C for 20 s, 72\u0026deg;C for 90 s), followed by 72\u0026deg;C for 70 min. In addition, the \u003cem\u003etrpB\u003c/em\u003e and \u003cem\u003erpoB\u003c/em\u003e gene of four strains (NKY-15, NKY-17, L7-2 and NKY-4) was amplified using the specific primer pair trpB-F (5\u0026prime;-GCGCGAGGACCTGAACCACACCGGCTCAC-3\u0026prime;) / trpB-R (5\u0026prime;- TCGATGGCCGGGATGATGCCCTCGG-3\u0026prime;) and rpoB-F (5\u0026prime;-GAGCGCATGACCACCCAGGACGTCGAGGC-3\u0026prime;) / rpoB-R (5\u0026prime;-CCTCGTAGTTGTGACCCTCCCACGGCATGA-3\u0026prime;) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The PCR products were sent to Tsingke Biotechnology Co., Ltd. (Nanjing, China) for sequencing, then the sequencing results were submitted to EzTaxone server [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/\u003c/span\u003e\u003cspan address=\"https://www.ezbiocloud.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for comparison with type material sequences. The 16S rRNA gene sequences obtained in this study were deposited in the GenBank database (accession numbers OR186221-OR186270). The \u003cem\u003erpoB\u003c/em\u003e gene and \u003cem\u003etrpB\u003c/em\u003e gene of four strains (NKY-15, NKY-17, L7-2 and NKY-4) sequences obtained in this study were deposited in the GenBank database (accession numbers PP212887- PP212890 and PP212891- PP212894).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePathogenicity tests\u003c/h2\u003e \u003cp\u003eVirulence assays of isolated strains on radish seedlings were performed according to the reported method [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The radish seeds were washed with sterile water and placed in culture dish moistened with sterile filter paper to induce germination for 24 h. 30 seeds were allowed to grow for 4 days (at 24 ℃) in Petri dishes (9 cm diameter) containing 5 ml of Actinomyces fermentation broth. Then root length of each seedling was recorded. The negative control was sterile water and the positive control was \u003cem\u003eS. bottropensis\u003c/em\u003e AMCC400023.\u003c/p\u003e \u003cp\u003eThe maceration ability of the isolated strains was evaluated by performing the potato tuber slice assay as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Firstly, the fresh potato tubers (no lesion on the surface) were washed with water, then soaked and disinfected in 75% anhydrous ethanol for 5 minutes, washed in sterilized tap water. The 2 cm thick potato slices with a diameter of 15 mm were placed on moistened filter paper in 90 mm Petri dishes. Five strains (AMCC400023, L7-2, L7-7, NKY-4, and G1-67) with potent inhibitory activity on radish seedlings were selected to grow on G1 medium for one week at 28\u0026deg;C. Following incubation, agar plugs (5 mm diameter) containing sporulating colonies were sampled and placed at the centre of each tuber slice. Potatoes were incubated at 28\u0026deg;C for 7\u0026ndash;10 days and observed for severity of browning and necrosis of potato tuber slice. The G1 solid culture medium and AMCC400023 were used as the negative and positive controls, respectively. All experiments were repeated in triplicate.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003etxtAB\u003c/em\u003e gene was involved in the biosynthesis of the main toxin (thaxtomins) of potato scab pathogens [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Some genes including \u003cem\u003enec1\u003c/em\u003e and \u003cem\u003etomA\u003c/em\u003e were also somehow required for the virulence. But the prevalence of CS pathogens without \u003cem\u003etxtAB\u003c/em\u003e genes was either very rare [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. We used the most common pathogenic genes \u003cem\u003etxtAB\u003c/em\u003e to perform pathogenicity validation on the isolated strains. Polymerase chain reaction was used to amplify \u003cem\u003etxtAB\u003c/em\u003e, and the specific primer pair were txtAB-F (5\u0026prime;-CCACCAGGACCTGCTCTTC-3\u0026prime;) / txtAB-R (5\u0026prime;-TCGAGTGGACCTCA CAGATG-3\u0026prime;) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Finally, the reactions were analyzed by 1.2% agarose gel electrophoresis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eScreening for phytotoxic activities\u003c/h2\u003e \u003cp\u003e32 strains with inhibitory activity to radish seedlings were selected for small-scale fermentation to screen for further test of herbicide activity. The strains were grown in 150 mL G1 liquid medium in 250 mL Erlenmeyer flask at 28 ℃, 180 rpm for 7 days. The fermentation broth of cultures was obtained by filtering through cotton gauze, and screened for potential herbicidal activity against two monocotyledonous weeds, \u003cem\u003eE. crusgalli\u003c/em\u003e and \u003cem\u003eD. sanguinalis\u003c/em\u003e, and a dicotyledonous weed, \u003cem\u003eA. theophrasti\u003c/em\u003e by the method described in the previous literature [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. 30 pregerminated seeds with consistent status were selected to place in Petri dishes (9 cm diameter) with filter paper, and filled with 5 ml of Actinomyces fermentation broth. Distilled water and 2,4-dichlorophenoxyacetic acid (2,4-D, Shanghai Chuangsai Technology Co., Ltd.) were used as negative and positive controls, respectively. After 2\u0026ndash;3 days, the length of the radicle of each weed seed was measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and characterization of secondary metabolite\u003c/h2\u003e \u003cp\u003eBased on bioassay-oriented, the strain \u003cem\u003eS. bottropensis\u003c/em\u003e AMCC400023 with significant herbicidal activity was selected for identification of compounds in this study. The strain AMCC400023 was inoculated into a 250 mL Erlenmeyer flask containing 150 mL of SCM3 liquid medium and incubated at 28 ℃ (180 rpm) for 3 days. The seed inoculates were then transferred to 400 mL of the same medium in 1000 mL Erlenmeyer flasks, and cultured at 28 ℃ (180 rpm) for 7 days. The fermentation broth (16 L) was filtered through four layers of gauze, and the supernatant extracted three times with EtOAc. The ethyl acetate fractions were concentrated in vacuo with the aid of a rotary evaporator to obtain crude extract (6.0 g).\u003c/p\u003e \u003cp\u003eThe crude extract was fractionated into seven fractions (100:0, 100:1, 100:2, 100:4, 100:8, 100:16, and 100:32, v/v) by silica gel column (200\u0026ndash;300 mesh) using a dichloromethane (CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e) and methanol (MeOH) gradient. Fraction 5 was further purified on a silica gel column, and followed by fractionation on a Sephadex LH-20 column (MeOH) to yield compound \u003cb\u003e1\u003c/b\u003e (120 mg) and subfraction (R1). The subfraction R1 was recrystallized from methanol, yielding an orange-yellow crystals \u003cb\u003e2\u003c/b\u003e (148 mg). The fraction (Fr 4) was repeatedly purified on silica gel using CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/MeOH (100:1, 100:2, 100:4) to give compound \u003cb\u003e3\u003c/b\u003e (10 mg). Fr 2 was fractionated by silica gel column chromatography (CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/MeOH) and recrystallized from methanol to give compound \u003cb\u003e4\u003c/b\u003e (30 mg).\u003c/p\u003e \u003cp\u003eStructural identifications of the compounds were determined via spectroscopic analysis. NMR spectra were measured with Agilent II DD2 spectrometers (Agilent, USA) at 600 MHz for \u003csup\u003e1\u003c/sup\u003eH and 150 MHz for \u003csup\u003e13\u003c/sup\u003eC, and the 2D spectra (COSY, HMQC, HMBC and DETP) were obtained by using standard Agilent software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePhytotoxic assay of compounds\u003c/h2\u003e \u003cp\u003eCompounds \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e4\u003c/b\u003e were determined for phytotoxic activity against \u003cem\u003eE. crusgalli\u003c/em\u003e, \u003cem\u003eD. sanguinalis\u003c/em\u003e and \u003cem\u003eA. theophrasti\u003c/em\u003e by using Petri dish bioassay [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. All test compounds were dissolved in acetone and diluted to 100 \u0026micro;g/mL with distilled water. The 2,4-D was used as the positive control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using origin 2021 software, considering a significance level of 95%. For the bio-herbicidal assay on tested seeds, each treatment was composed of 30 seeds with three replicates of 10 seeds.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of actinomycetes strains from the potato tubers with typical common scab\u003c/h2\u003e \u003cp\u003eHere, a total of 50 isolates were isolated and purified from the scab lesions on potatoes using 12 different isolation media. As shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, the number of actinomycetes isolated from different media exhibited appreciably differences. Among them, G1 were the most successful media for isolating actinobacterial strains with the number of 24 strains (about 48%), followed by SCA with 10 strains (20%). The number of strains obtained from the following six culture media were relatively small, including B4 (4 strains), AIA (4 strains), GYM (3 strains), CCM (2 strains), R2A (2 strains) and MM (1 strain). However, no strains were isolated from LB, ISP2, ISP4 and OMA media.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and phylogenetic analysis\u003c/h2\u003e \u003cp\u003ePCR amplification and sequencing analysis of the 16S rRNA gene was performed on 50 isolated actinomycetes, then the results of sequences were analyzed using BLAST and Ezbiocloud database. All isolates were identified as the genus \u003cem\u003eStreptomyces\u003c/em\u003e belonging to the family Streptomycetaceae, which belongs to 12 species (Table S2). Among them, \u003cem\u003eS. pratensis\u003c/em\u003e (32% of strains) and \u003cem\u003eS. violascens\u003c/em\u003e (24%) were the dominant species. Notably, the 16S rRNA gene sequences were analyzed by EzTaxon indicated that some isolates showed relatively low similarities to the type strains of the corresponding genera. Using 16s rRNA, \u003cem\u003etrpB\u003c/em\u003e and \u003cem\u003erpoB\u003c/em\u003e to analyse \u003cem\u003eStreptomyces\u003c/em\u003e spp. with low similarity (NKY-15 and NKY-17), the results indicate that the strains NKY-15 and NKY-17 were in separate branches (Figure S2, S3), which indicated potential new species.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePathogenicity assay of the\u003c/b\u003e \u003cb\u003eStreptomyces\u003c/b\u003e \u003cb\u003eisolates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe pathogenicity of selected \u003cem\u003eStreptomyces\u003c/em\u003e spp. isolates was determined via the following three assays, including radish seed assay, potato tuber slice assay and detection of \u003cem\u003etxtAB\u003c/em\u003e gene. A total of 63% (32 strains) of the selected \u003cem\u003eStreptomyces\u003c/em\u003e spp. were positive in the radish seed assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among them, 5 strains exhibited outstanding inhibitory activity against radish seedlings with an inhibition rate of 60%-70%, which were comparable to that of the positive AMCC400023 with an inhibition rate of 70%. A moderate inhibitory activity was found for 5 strains with an inhibition rate of 40%-60%. In addition, 21 strains showed relatively weak phytotoxic activity against radish seed with the inhibition rate of less than 40%. Furthermore, thaxtomin A (TA) is considered the primary phytotoxin produced by the pathogen \u003cem\u003eStreptomyces scabies\u003c/em\u003e, which is the most important pathogenicity factor of potato common scab. Results of PCR analysis with the primer pair txtAB-F/txtAB-R showed that three strains (L7-2, L7-7 and NKY-4) were positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the potato tuber slice assay, two isolates (L7-2 and NKY-4) caused CS lesions on potato tubers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Similar to the positive AMCC400023, the strains L7-2 and NKY-4 could successfully colonize on the surface of potato tuber slice with different degree. Specially, the obvious black brown necrotic lesions were observed on potato slice. Therefore, the strains L7-2 and NKY-4 were positive in all three pathogenicity assays, which indicated that they were potential species causing potato scab disease (Figure S2, S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhytotoxic assay of fermentation broth of actinomycetes\u003c/h2\u003e \u003cp\u003e32 bioactive actinomycetes in the radish seed assay were evaluated for their herbicide activity against the seedlings of \u003cem\u003eE. crusgalli\u003c/em\u003e, \u003cem\u003eD. sanguinalis\u003c/em\u003e and \u003cem\u003eA. theophrasti\u003c/em\u003e using Petri dish bioassays. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e, 31 of the 32 isolates (97%) exhibited herbicidal activities against at least one of the growth of weeds roots. Among them, three strains (NKY-12, SCA2, and SCA10) showed strong phytotoxic activity against \u003cem\u003eE. crusgalli\u003c/em\u003e with the inhibition rate of 100%. Ten strains (L7-12, NKY-N, NKY-12, GS-49, GS-67, SCA-2, SCA4, SCA7, SCA10, and AMCC400023) also had strong inhibitory effects on the growth of \u003cem\u003eDigitaria sanguinalis\u003c/em\u003e\u0026rsquo; root, with the inhibition rate of over 60%. Besides, 14 strains presented moderate phytotoxic activity against \u003cem\u003eA. theophrasti\u003c/em\u003e with the inhibition rate of over 50%. Especially, 18 isolates exhibited inhibitory effects on the growth of both monocotyledonous weeds (\u003cem\u003eE. crusgalli\u003c/em\u003e and \u003cem\u003eD. sanguinalis\u003c/em\u003e) and a dicotyledonous weed (\u003cem\u003eA. theophrasti\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStructural characterization of secondary metabolites from AMCC400023\u003c/h2\u003e \u003cp\u003eFour pure compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e) were purified from SCM3 liquid medium fermentation product of \u003cem\u003eStreptomyces\u003c/em\u003e sp. AMCC400023, and their structures were analyzed by NMR and EI-MS techniques, including one new compound N-(2,5-dihydroxyphenyl)-3-acetamide-4-hydroxybenzamide (\u003cb\u003e1\u003c/b\u003e) and three known compounds thaxtomin A (\u003cb\u003e2\u003c/b\u003e) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], nicotinic acid (\u003cb\u003e3\u003c/b\u003e) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and pyrrole-2-carboxylic acid (\u003cb\u003e4\u003c/b\u003e) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eN-(2,5-dihydroxyphenyl)-3-acetamide-4-hydroxybenzamide (\u003cb\u003e1\u003c/b\u003e): brown powder; HR-ESI-MS: \u003cem\u003em/z\u003c/em\u003e: 301.0824 [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e, calculated for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e 302.0903, which was consistent with \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR data (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Figures S4-S10). The \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of \u003cb\u003e1\u003c/b\u003e exhibited three phenolic hydroxyl group signals \u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e [8.82 (brs, 1H), 9.04 (brs, 1H) and 10.58 (brs,1H)], two amide group signals \u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e [9.93 (s, 1H) and 9.14 (s, 1H)], and a methyl group signal \u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 2.11 (s, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR and DEPT135 spectrum showed two amide carbon signals (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e164.5, and 169.1), twelve olefinic carbons signals (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 109.1, 111.1, 115.3, 116.3, 121.9, 124.1, 124.9, 126.3, 126.8, 140.3, 149.9 and 151.2), and one methyl carbon signal (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 23.6). The HMBC spectrum of \u003cb\u003e1\u003c/b\u003e exhibited correlations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e) from H-2 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.36) to C-6(\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 124.1), and C-4 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 151.2), from H-3 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 9.39) to C-2 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 121.9), C-4, and C-7 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 169.1), from H-4 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 10.58) to C-3 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 126.3) and C-4, from H-5 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 6.95) to C-1 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 124.9), C-3, from H-6 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 7.57) to C-2 and C-4, and from H-8 (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 2.11) to C-7, together with \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH-correlation spectroscopy (COSY) correlations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e) observed between H-5 and H-6 indicated that compound \u003cb\u003e1\u003c/b\u003e possessed trisubstituted benzene and acetamide moieties. Further analysis of HMBC data, the correlations from H-1\u0026prime; (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 9.14) to C-6\u0026prime;(\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 109.1), C-2\u0026prime; (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 140.3), and C-7\u0026prime;(\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 164.5), from H-3\u0026prime; (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 6.69) to C-1\u0026prime;(\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 126.8) and C-5\u0026prime; (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 149.9), from H-4\u0026prime; (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 6.39) to C-2\u0026prime;and C-6, from H-5\u0026prime; (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.82) to C-4\u0026prime; (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 111.1), C-5\u0026prime;, and C-6\u0026prime;, and from H-6\u0026prime; (\u003cem\u003eδ\u003c/em\u003e\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 7.33) to C-2\u0026prime;, C-4\u0026prime;, together with \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH-correlation spectroscopy (COSY) correlations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e) observed between H-3\u0026prime; and H-4\u0026prime; revealed the presence of trisubstituted benzene and phenolic hydroxyl groups. Accordingly, the structure of 1 was established as a new compound, and N-(2,5-dihydroxyphenyl)-3-acetamide-4-hydroxybenzamide was proposed as its trivial name.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR data for compounds \u003cb\u003e1\u003c/b\u003e in DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePosition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eδ\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eδ\u003csub\u003eH\u003c/sub\u003e, mult, \u003cem\u003eJ\u003c/em\u003e in Hz\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e124.9, C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e121.9, CH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.36, s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e126.3, C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e151.2, C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e115.3, CH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.95, d (8.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e124.1, CH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.57, d (8.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e169.1, C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.6, CH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.11, s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e126.8, C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e140.3, C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e116.3, CH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.69, d (8.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e111.1, CH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.39, dd (2.8, 5.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e149.9, C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109.1, CH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.33, d (2.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e164.5, C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.58, br s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2'-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.04, br s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5'-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.82, br s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3-NH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.93, s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1'-NH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.14, s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThaxtomin A (\u003cb\u003e2\u003c/b\u003e): orange-yellow crystals; HR-ESI-MS: \u003cem\u003em/z\u003c/em\u003e: 461.1401 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e, calculated for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e 438.1539; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CD\u003csub\u003e3\u003c/sub\u003eOD) \u003cem\u003eδ\u003c/em\u003e: 1.64 (1H, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14.1, 8.8 Hz, H-10\u003csub\u003eb\u003c/sub\u003e), 2.61 (1H, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14.2, 6.3 Hz, H-10\u003csub\u003ea\u003c/sub\u003e), 2.81 (3H, s, N-CH\u003csub\u003e3\u003c/sub\u003e), 3.03 (3H, s, N-CH\u003csub\u003e3\u003c/sub\u003e), 3.11 (2H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.6 Hz, H-17), 3.87 (1H, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 6.6 Hz, H-11), 6.71 (3H, m, H-19, H-21, H-23), 6.95 (1H, s, H-2), 7.18 (1H, t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, H-6), 7.23 (1H, t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, H-22), 7.69 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, H-7), 7.83 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, H-5). \u003csup\u003e13\u003c/sup\u003eC NMR (150 MHz, CD\u003csub\u003e3\u003c/sub\u003eOD) \u003cem\u003eδ\u003c/em\u003e: 168.5 (C, C-16), 167.0 (C, C-13), 159.2 (C, C-20), 143.9 (C, C-4), 141.3 (C, C-9), 137.5 (C, C-18), 132.6 (CH, C-2), 131.3 (CH, C-22), 122.9 (CH, C-23), 121.1 (CH, C-6), 120.0 (C, C-8), 119.3 (CH, C-5), 118.8 (CH, C-7), 118.5 (CH, C-19), 116.0 (CH, C-21), 110.7 (C, C-3), 88.2 (C, C-14), 64.8 (C, C-11), 43.8 (CH\u003csub\u003e2\u003c/sub\u003e, C-17), 34.3 (CH\u003csub\u003e3\u003c/sub\u003e, C-15), 33.7 (CH\u003csub\u003e2\u003c/sub\u003e, C-10), 28.6 (CH\u003csub\u003e3\u003c/sub\u003e, C-12).\u003c/p\u003e \u003cp\u003eNicotinic acid (\u003cb\u003e3\u003c/b\u003e): white crystals; HR-ESI-MS: \u003cem\u003em/z\u003c/em\u003e: 124.0391 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, calculated for C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e 123.0320; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CD\u003csub\u003e3\u003c/sub\u003eOD) \u003cem\u003eδ\u003c/em\u003e: 7.55 (1H, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.0, 7.7 Hz, H-5), 8.40 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, H-4), 8.72 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.7 Hz, H-6), 9.12 (1H, s, H-2). \u003csup\u003e13\u003c/sup\u003eC NMR (150 MHz, CD\u003csub\u003e3\u003c/sub\u003eOD) \u003cem\u003eδ\u003c/em\u003e: 125.3 (CH, C-5), 129.1 (C, C-3), 139.3 (CH, C-4), 151. 5 (CH, C-6), 153.7 (CH, C-2), 168.2 (CH, C-7).\u003c/p\u003e \u003cp\u003ePyrrole-2-carboxylic acid (\u003cb\u003e4\u003c/b\u003e): brownish crystals; HR-ESI-MS: \u003cem\u003em/z\u003c/em\u003e: 110.0247 [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e, calculated for C\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e 111.0320; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CD\u003csub\u003e3\u003c/sub\u003eOD) \u003cem\u003eδ\u003c/em\u003e: 6.16 (1H, s, H-4), 6.83 (1H, s, H-3), 6.92 (1H, s, H-5). \u003csup\u003e13\u003c/sup\u003eC NMR (150 MHz, CD\u003csub\u003e3\u003c/sub\u003eOD) \u003cem\u003eδ\u003c/em\u003e: 124.8 (CH, C-5), 124.2 (C, C-2), 116.4 (CH, C-3), 110.6 (CH, C-4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhytotoxic activities of compounds\u003c/h2\u003e \u003cp\u003eThe phytotoxic activities of the test compounds (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e4\u003c/b\u003e) against radicle growth of \u003cem\u003eE. crusgalli\u003c/em\u003e, \u003cem\u003eD. sanguinalis\u003c/em\u003e and \u003cem\u003eA. theophrasti\u003c/em\u003e were shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results showed that compound \u003cb\u003e2\u003c/b\u003e exhibited strong phytotoxic activity against \u003cem\u003eE. crusgalli\u003c/em\u003e, \u003cem\u003eD. sanguinalis\u003c/em\u003e and \u003cem\u003eA. theophrasti\u003c/em\u003e with the inhibition rate of 100%, which was same as that of the positive 2,4-D at a concentration of 100 \u0026micro;g/mL. However, the new compound \u003cb\u003e1\u003c/b\u003e had weak inhibitory activity against above three weeds with the inhibition rate of less than 30%. Similarly, the compound \u003cb\u003e4\u003c/b\u003e exhibited weak phytotoxic activity against two monocotyledonous weeds (\u003cem\u003eE. crusgalli\u003c/em\u003e, \u003cem\u003eD. sanguinalis)\u003c/em\u003e with the inhibition rate of less than 40%. Further testing was conducted on the herbicidal activity of compound \u003cb\u003e2\u003c/b\u003e with different concentrations. The results also showed that the metabolite \u003cb\u003e2\u003c/b\u003e presented strong phytotoxic activity with the IC\u003csub\u003e50\u003c/sub\u003e values of 0.99, 0.78, and 1.95 \u0026micro;g/mL, respectively, which were almost comparable with those of the positive 2,4-D with IC\u003csub\u003e50\u003c/sub\u003e values of 0.88, \u0026lt;\u0026thinsp;0.1, and \u0026lt;\u0026thinsp;0.1 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInhibitory effects of compounds on the growth of weeds roots (%).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. crusgalli\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eD. sanguinalis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eA. theophrasti\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e19.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e31.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2,4-D\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eResults are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard; \u0026ldquo;NI\u0026rdquo; means not inhibited; the concentration for the test is 100 \u0026micro;g/mL;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e2,4-D was the positive control.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePotato common scab (CS) scab was a soil-borne disease caused by the typical phytopathogenic \u003cem\u003eStreptomyces\u003c/em\u003e sp. and was one of the most devastating diseases in potatoes, reducing both crop growth and quality [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In this study, 50 potato-associated \u003cem\u003eStreptomyces\u003c/em\u003e, including two potentially new species, were isolated from the potato tubers with typical common scab and identified by morphological and molecular biological methods. As reported previously, a new species was defined by a 16S rRNA gene sequence homology below 98.7% [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Specifically, based on 16s rRNA and the high-resolution \u003cem\u003eStreptomyces\u003c/em\u003e sp. housekeeping genes: \u003cem\u003etrpB\u003c/em\u003e (tryptophan synthase subunit beta) and \u003cem\u003erpoB\u003c/em\u003e (RNA polymerase subunit beta) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] the phylogenetic trees indicated that \u003cem\u003eStreptomyces\u003c/em\u003e spp. with low similarity (NKY-15 and NKY-17) were in separate branches and might potential new species.\u003c/p\u003e \u003cp\u003eHere, twelve selective isolation media were employed to isolate as many actinobacteria as feasible. Among them, the G1 and SCA media were the most effective as regards the number of obtained isolates. The G1 and SCA media, which were used to find saccharolytic bacteria, including actinobacteria [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], were mostly made of soluble starch which facilitated the mycelial extension and cell growth of actinomycetes as a sole carbon source [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTill now, approximately 30 species of \u003cem\u003eStreptomyces\u003c/em\u003e causing CS have been identified worldwide [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], of which \u003cem\u003eS. scabies, S. acidiscabies, and S. turgidiscabies\u003c/em\u003e were the most characterized [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Noticeably, the strains NKY-4 and L7-7 were positive in pathogenicity assays, which have never been reported to cause potato scab, indicating that might be newly discovered pathogen of potato scab disease. However, 38% of all the isolates were negative in all pathogenicity assays, indicating that nonpathogenic \u003cem\u003eStreptomyces\u003c/em\u003e were also colonizing potato tubers, which was consistent with the previous reports [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our results show that about 63% (32 strains) of the tested \u003cem\u003eStreptomyces\u003c/em\u003e spp. were positive in the radish seed assay, but most of which were negative in other two pathogenicity assays. This indicates that although thaxtomin family, which is encoded by \u003cem\u003etxtAB\u003c/em\u003e gene, is considered as a major player toward plant pathogenicity, other phytotoxic may exist in different species of \u003cem\u003eStreptomyces\u003c/em\u003e and can affect the normal growth of radish seedlings [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Other \u003cem\u003eStreptomyces\u003c/em\u003e phytotoxic specialized metabolites have been reported, such as two phytotoxic nigericin and geldanamycin were isolated from plant pathogen \u003cem\u003eStreptomyces\u003c/em\u003e sp. 11-1-2 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], desmethylmensacarcin is a novel phytotoxicity was isolated from pathogen \u003cem\u003eS. niveiscabiei\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The herbicidal activity of 32 strains of \u003cem\u003eStreptomyces\u003c/em\u003e spp. exhibiting inhibitory activity on the root length of radish seedlings was further tested against three weeds. The results revealed that 31 \u003cem\u003eStreptomyces\u003c/em\u003e strains showed inhibitory activity against at least one of the weeds\u0026rsquo; roots. This provides new ideas for the development of novel herbicides of microbial-pathogen origin.\u003c/p\u003e \u003cp\u003eGlufosinate was the first microbial-origin herbicide developed and synthesized as a lead compound by the secondary metabolite of bilanafos from \u003cem\u003eStreptomyces\u003c/em\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. We investigated the secondary metabolites from the pathogen \u003cem\u003eS. bottropensis\u003c/em\u003e AMCC400023, which resulted in the isolation of compounds \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e4\u003c/b\u003e. Among them, compound \u003cb\u003e2\u003c/b\u003e displayed strong inhibitory activity against \u003cem\u003eE. crusgalli\u003c/em\u003e, \u003cem\u003eD. sanguinalis\u003c/em\u003e, and \u003cem\u003eA. theophrast\u003c/em\u003e, with IC\u003csub\u003e50\u003c/sub\u003e values of 0.99, 0.78, and 1.95 \u0026micro;g/mL, respectively. Indeed, compound 2 (TA) was the main phytotoxin synthesized by the potato common scab-causing pathogen \u003cem\u003eStreptomyces\u003c/em\u003e spp., which has been reported to be phytotoxic to both broadleaf and the dicotyledonous weed, \u003cem\u003eA. theophrasti\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, this study found for the first time that compound 2 (TA) showed potential herbicidal activity against two monocotyledonous weeds, \u003cem\u003eE. crusgalli\u003c/em\u003e and \u003cem\u003eD. sanguinalis\u003c/em\u003e. Morever, it was common to find that the mechanisms of action of phytotoxin was an impact on plant chlorophyll content, lipid peroxidation, and electrolytic leakage [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, \u003cem\u003eStreptomyces\u003c/em\u003e was one of the important sources of lead compounds in natural herbicides.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, 50 actinomycetes were isolated and identified from potato samples using culture-dependent methods. Sequences analysis demonstrated that all actinomycetes were attached to the \u003cem\u003eStreptomyces\u003c/em\u003e genera. 32 strains exhibited a certain degree of virulence as determined by a pathogenicity assay on radish plants. Furthermore, potential new pathogens NKY-4 and L7-2 were positive in three pathogenicity assays. The results of phytotoxic tests showed that 31 extracts (97%) exhibited phytotoxic activities against at least one of the tested weeds. In addition, one novel metabolite and three known compounds were purified from pathogen \u003cem\u003eS. bottropensis\u003c/em\u003e AMCC400023. Compound \u003cb\u003e2\u003c/b\u003e displayed outstanding phytotoxic activity against \u003cem\u003eE. crusgalli\u003c/em\u003e, \u003cem\u003eD. sanguinalis\u003c/em\u003e, and \u003cem\u003eA. theophrast\u003c/em\u003e with IC\u003csub\u003e50\u003c/sub\u003e values of 0.99, 0.78, and 1.95 \u0026micro;g/mL, respectively. Therefore, our results suggest that metabolites produced by \u003cem\u003eS. bottropensis\u003c/em\u003e AMCC400023 with herbicidal activity may be a new bioherbicide candidate or leads molecule for a more efficient herbicide.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Common scab\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Polymerase chain reaction\u003c/p\u003e\n\u003cp\u003e2,4-D \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 2,4-dichlorophenoxyacetic acid\u003c/p\u003e\n\u003cp\u003eTA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Thaxtomin A\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAIA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Actinobacteria isolation agar\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCCM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cellulose-casamino acid\u003c/p\u003e\n\u003cp\u003eG1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Gause\u0026rsquo;s No. 1\u003c/p\u003e\n\u003cp\u003eGYM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Glucose-yeast-malt\u003c/p\u003e\n\u003cp\u003eLB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Luria bertani\u003c/p\u003e\n\u003cp\u003eISP2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;ISP medium No.2\u003c/p\u003e\n\u003cp\u003eISP4 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;ISP medium No.4\u003c/p\u003e\n\u003cp\u003eOMA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Oatmeal agar\u003c/p\u003e\n\u003cp\u003eR2A \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reasoner\u0026rsquo;s 2A agar\u003c/p\u003e\n\u003cp\u003eSCA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Starch casein agar\u003c/p\u003e\n\u003cp\u003eMM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Minimal medium\u003c/p\u003e\n\u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sub\u003eDichloromethane\u003c/p\u003e\n\u003cp\u003eMeOH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Methanol\u003c/p\u003e\n\u003cp\u003eCOSY \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH-correlation spectroscopy correlation\u003c/p\u003e\n\u003cp\u003eHMBC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u003csup\u003e1\u003c/sup\u003eH detected heteronuclear multiple bond correlation\u003c/p\u003e\n\u003cp\u003eDEPT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Distortionless enhancement by polarization transfer\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Professor Bo Zhou and Agricultural Microbial Resources and Utilization Center, Shandong Agricultural University, China for kindly providing one pathogen strain (\u003cem\u003eS. bottropensis\u0026nbsp;\u003c/em\u003eAMCC400023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYing-lao ZHANG and Shu-xiang ZHANG conceived and designed research. Shu-ping SHI and Yi ZHANG conducted experiments. Zhong-di HUANG and Cai-ping YIN analyzed the data. Zhong-di HUANG wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was co-financed by the National Natural Science Foundation of China (32270015 and 32102272) and Anhui Outstanding Youth Science Fund Project\u0026nbsp;(2108085J18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All data generated or analyzed during this study are included in this manuscript, its supplementary information files and in GenBank database. The 16S rRNA gene sequences obtained in this study were deposited in the GenBank database (accession numbers OR186221-OR186270). The \u003cem\u003erpoB\u003c/em\u003e gene and \u003cem\u003etrpB\u003c/em\u003e gene of four strains (NKY-15, NKY-17, L7-2 and NKY-4) \u0026nbsp;sequences obtained in this study were deposited in the GenBank database (accession numbers PP212887- PP212890 and PP212891- PP212894).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The research conducted in this study did not involve any animal subjects, therefore obtaining consent to participate was not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e \u003cstrong\u003einformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhong-di HUANG, Shu-ping SHI, Yi ZHANG, Cai-ping YIN and Ying-lao ZHANG\u003c/p\u003e\n\u003cp\u003eSchool of Life Sciences, Anhui Agricultural University, Hefei, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKim H. 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Efficacy of the bioherbicide Thaxtomin A on smooth crabgrass and annual bluegrass and safety in cool-season turfgrasses. \u003cem\u003eWeed Technol\u003c/em\u003e. \u003cstrong\u003e30\u003c/strong\u003e, 733-742.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bioherbicide, Potato common scab, Streptomyces spp., Herbicidal activity, Natural products","lastPublishedDoi":"10.21203/rs.3.rs-3991115/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3991115/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eA highly effective and environmentally friendly method of controlling weeds is biological herbicides, which typically constitute of naturally secondary metabolites, such as bioherbicidal metabolites produced by \u003cem\u003eStreptomyces\u003c/em\u003e sp. However, the isolation of phytotoxic compounds from pathogenic \u003cem\u003eStreptomyces\u003c/em\u003e has not been fully studied.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere, a total of 50 strains of \u003cem\u003eStreptomyces\u003c/em\u003e genera were isolated from the potato tubers with typical common scab (CS) symptoms using the culture-dependent method. The radish seedling test indicated that 32 fermentation broths of potato common scab-associated \u003cem\u003eStreptomyces\u003c/em\u003e could produce phytotoxic metabolites that affect the normal growth of radish seedlings\u0026rsquo; radicles. Of note, two potential new pathogens (NKY-4 and L7-2) of potato scab were discovered by combining the methods of radish seedlings, potato tuber slices, and PCR detection of pathogenic genes \u003cem\u003etxtAB\u003c/em\u003e. Moreover, the phytotoxic test demonstrated that the fermentation broths of 31 strains exhibited phytotoxic activities against at least one of the tested weeds (\u003cem\u003eEchinochloa crusgalli\u003c/em\u003e, \u003cem\u003eDigitaria sanguinalis\u003c/em\u003e, and \u003cem\u003eAbutilon theophrastis\u003c/em\u003e). Furthermore, one novel metabolite and three known compounds, including new N-(2,5-dihydroxyphenyl)-3-acetamide-4-hydroxybenzamide (\u003cb\u003e1\u003c/b\u003e), thaxtomin A (\u003cb\u003e2\u003c/b\u003e), nicotinic acid (\u003cb\u003e3\u003c/b\u003e) and pyrrole-2-carboxylic acid (\u003cb\u003e4\u003c/b\u003e) were isolated from \u003cem\u003eS. bottropensis\u003c/em\u003e (AMCC400023). Among them, compound \u003cb\u003e2\u003c/b\u003e exhibited strong phytotoxic activity against \u003cem\u003eE. crusgalli\u003c/em\u003e, \u003cem\u003eD. sanguinalis\u003c/em\u003e, and \u003cem\u003eA. theophrast\u003c/em\u003e with IC\u003csub\u003e50\u003c/sub\u003e values of 0.99, 0.78, and 1.95 \u0026micro;g/mL, respectively, which was comparable to those of the positive control 2,4-D.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eBased on the results of these findings, phytotoxic metabolites from the potato scab pathogen may be one of the effective ways to develop new biological herbicides.\u003c/p\u003e","manuscriptTitle":"Isolation, Characterization, and Herbicidal Activity of Streptomyces spp. from Diseased Potato Scab Tubers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-09 04:44:18","doi":"10.21203/rs.3.rs-3991115/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"39ff1504-9be4-45dd-b225-acde93c6a764","owner":[],"postedDate":"April 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-02T17:38:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-09 04:44:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3991115","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3991115","identity":"rs-3991115","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}