MOESM2 of Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis

Additional file 2: Figure S2. Co-transcriptional analysis of the biosynthetic genes in mil cluster. A. Intergenic regions numbered 1-4 were subjected to RT-PCR analysis and the occurrence of translational coupling between milA1 and milD were marked by “▲”, this location was not subjected to RT-PCT analysis. B. RT-PCR analysis of co-transcribed genes. Primers were designed to amplify 4 intergenic regions (numbered 1-4, Table S1). cDNA was synthesized from RNA samples isolated from S. bingchenggensis BC04 cultures (3 d cultivation in fermentation medium). Positive controls: PCR reactions using genomic DNA of BC04 as template which gave products of the same molecular sizes as those amplified from cDNA template obtained by reverse transcription. Negative controls: PCR amplification of hrdB using “cDNA” template synthesized without reverse transcriptase, which did not give products, thus excluding the possible contamination of genomic DNA. c: cDNA template; g: genomic DNA template; NRT: “cDNA” template synthesized in the absence of reverse transcriptase. These results show that milA2 and milC, milA4 and milE, milR and milA3, milA1 and milD form an operon respectively, whereas the other two genes milF and orf1 are transcribed individually

Image_4_Antifungal, Plant Growth-Promoting, and Genomic Properties of an Endophytic Actinobacterium Streptomyces sp. NEAU-S7GS2.TIF

Diseases caused by Sclerotinia sclerotiorum have caused severe losses of many economically important crops worldwide. Due to the long-term persistence of sclerotia in soil and the production of air-borne ascospores, synthetic fungicides play limited roles in controlling the diseases. The application of antagonistic microorganisms can effectively reduce the number of sclerotia and eventually eradicate S. sclerotiorum from soil, and therefore considerable interest has been focused on biological control. Streptomyces sp. NEAU-S7GS2 was isolated from the root of Glycine max and its rhizosphere soil. It showed significant inhibitory activity against the mycelial growth of S. sclerotiorum (99.1%) and completely inhibited sclerotia germination. Compared to the control, in the pot experiment the application of NEAU-S7GS2 not only demonstrated excellent potential to control sclerotinia stem rot of soybean with 77 and 38% decrease in disease incidence and disease index, respectively, but could promote the growth of soybean. The light microscopy and scanning electron microscopy showed that co-culture of NEAU-S7GS2 with S. sclerotiorum on potato dextrose agar could lead to contorted and fragmented mycelia of S. sclerotiorum, which was associated with the secretion of hydrolytic glucanase and cellulase and the production of active secondary metabolites by NEAU-S7GS2. The plant growth promoting activity of NEAU-S7GS2 was related to the solubilization of inorganic phosphate, and production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase and indole acetic acid (IAA). To further explore the plant growth promoting and antifungal mechanisms, the complete genome of strain NEAU-S7GS2 was sequenced. Several genes associated with ammonia assimilation, phosphate solubilization and IAA synthesis, together with genes encoding ACC deaminase, glucanase and α-amylase, were identified. AntiSMASH analysis led to the identification of four gene clusters responsible for the biosynthesis of siderophores including desferrioxamine B and enterobactin. Moreover, the biosynthetic gene clusters of lydicamycins, phenazines, and a glycosylated polyol macrolide showing 88% gene similarity to PM100117/PM100118 were identified. These results suggested that strain NEAU-S7GS2 may be a potential biocontrol agent and biofertilizer used in agriculture.</p

Image_3_Antifungal, Plant Growth-Promoting, and Genomic Properties of an Endophytic Actinobacterium Streptomyces sp. NEAU-S7GS2.TIF

Diseases caused by Sclerotinia sclerotiorum have caused severe losses of many economically important crops worldwide. Due to the long-term persistence of sclerotia in soil and the production of air-borne ascospores, synthetic fungicides play limited roles in controlling the diseases. The application of antagonistic microorganisms can effectively reduce the number of sclerotia and eventually eradicate S. sclerotiorum from soil, and therefore considerable interest has been focused on biological control. Streptomyces sp. NEAU-S7GS2 was isolated from the root of Glycine max and its rhizosphere soil. It showed significant inhibitory activity against the mycelial growth of S. sclerotiorum (99.1%) and completely inhibited sclerotia germination. Compared to the control, in the pot experiment the application of NEAU-S7GS2 not only demonstrated excellent potential to control sclerotinia stem rot of soybean with 77 and 38% decrease in disease incidence and disease index, respectively, but could promote the growth of soybean. The light microscopy and scanning electron microscopy showed that co-culture of NEAU-S7GS2 with S. sclerotiorum on potato dextrose agar could lead to contorted and fragmented mycelia of S. sclerotiorum, which was associated with the secretion of hydrolytic glucanase and cellulase and the production of active secondary metabolites by NEAU-S7GS2. The plant growth promoting activity of NEAU-S7GS2 was related to the solubilization of inorganic phosphate, and production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase and indole acetic acid (IAA). To further explore the plant growth promoting and antifungal mechanisms, the complete genome of strain NEAU-S7GS2 was sequenced. Several genes associated with ammonia assimilation, phosphate solubilization and IAA synthesis, together with genes encoding ACC deaminase, glucanase and α-amylase, were identified. AntiSMASH analysis led to the identification of four gene clusters responsible for the biosynthesis of siderophores including desferrioxamine B and enterobactin. Moreover, the biosynthetic gene clusters of lydicamycins, phenazines, and a glycosylated polyol macrolide showing 88% gene similarity to PM100117/PM100118 were identified. These results suggested that strain NEAU-S7GS2 may be a potential biocontrol agent and biofertilizer used in agriculture.</p

Table_1_Antifungal, Plant Growth-Promoting, and Genomic Properties of an Endophytic Actinobacterium Streptomyces sp. NEAU-S7GS2.DOCX

Diseases caused by Sclerotinia sclerotiorum have caused severe losses of many economically important crops worldwide. Due to the long-term persistence of sclerotia in soil and the production of air-borne ascospores, synthetic fungicides play limited roles in controlling the diseases. The application of antagonistic microorganisms can effectively reduce the number of sclerotia and eventually eradicate S. sclerotiorum from soil, and therefore considerable interest has been focused on biological control. Streptomyces sp. NEAU-S7GS2 was isolated from the root of Glycine max and its rhizosphere soil. It showed significant inhibitory activity against the mycelial growth of S. sclerotiorum (99.1%) and completely inhibited sclerotia germination. Compared to the control, in the pot experiment the application of NEAU-S7GS2 not only demonstrated excellent potential to control sclerotinia stem rot of soybean with 77 and 38% decrease in disease incidence and disease index, respectively, but could promote the growth of soybean. The light microscopy and scanning electron microscopy showed that co-culture of NEAU-S7GS2 with S. sclerotiorum on potato dextrose agar could lead to contorted and fragmented mycelia of S. sclerotiorum, which was associated with the secretion of hydrolytic glucanase and cellulase and the production of active secondary metabolites by NEAU-S7GS2. The plant growth promoting activity of NEAU-S7GS2 was related to the solubilization of inorganic phosphate, and production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase and indole acetic acid (IAA). To further explore the plant growth promoting and antifungal mechanisms, the complete genome of strain NEAU-S7GS2 was sequenced. Several genes associated with ammonia assimilation, phosphate solubilization and IAA synthesis, together with genes encoding ACC deaminase, glucanase and α-amylase, were identified. AntiSMASH analysis led to the identification of four gene clusters responsible for the biosynthesis of siderophores including desferrioxamine B and enterobactin. Moreover, the biosynthetic gene clusters of lydicamycins, phenazines, and a glycosylated polyol macrolide showing 88% gene similarity to PM100117/PM100118 were identified. These results suggested that strain NEAU-S7GS2 may be a potential biocontrol agent and biofertilizer used in agriculture.</p

Table_2_Antifungal, Plant Growth-Promoting, and Genomic Properties of an Endophytic Actinobacterium Streptomyces sp. NEAU-S7GS2.docx

Diseases caused by Sclerotinia sclerotiorum have caused severe losses of many economically important crops worldwide. Due to the long-term persistence of sclerotia in soil and the production of air-borne ascospores, synthetic fungicides play limited roles in controlling the diseases. The application of antagonistic microorganisms can effectively reduce the number of sclerotia and eventually eradicate S. sclerotiorum from soil, and therefore considerable interest has been focused on biological control. Streptomyces sp. NEAU-S7GS2 was isolated from the root of Glycine max and its rhizosphere soil. It showed significant inhibitory activity against the mycelial growth of S. sclerotiorum (99.1%) and completely inhibited sclerotia germination. Compared to the control, in the pot experiment the application of NEAU-S7GS2 not only demonstrated excellent potential to control sclerotinia stem rot of soybean with 77 and 38% decrease in disease incidence and disease index, respectively, but could promote the growth of soybean. The light microscopy and scanning electron microscopy showed that co-culture of NEAU-S7GS2 with S. sclerotiorum on potato dextrose agar could lead to contorted and fragmented mycelia of S. sclerotiorum, which was associated with the secretion of hydrolytic glucanase and cellulase and the production of active secondary metabolites by NEAU-S7GS2. The plant growth promoting activity of NEAU-S7GS2 was related to the solubilization of inorganic phosphate, and production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase and indole acetic acid (IAA). To further explore the plant growth promoting and antifungal mechanisms, the complete genome of strain NEAU-S7GS2 was sequenced. Several genes associated with ammonia assimilation, phosphate solubilization and IAA synthesis, together with genes encoding ACC deaminase, glucanase and α-amylase, were identified. AntiSMASH analysis led to the identification of four gene clusters responsible for the biosynthesis of siderophores including desferrioxamine B and enterobactin. Moreover, the biosynthetic gene clusters of lydicamycins, phenazines, and a glycosylated polyol macrolide showing 88% gene similarity to PM100117/PM100118 were identified. These results suggested that strain NEAU-S7GS2 may be a potential biocontrol agent and biofertilizer used in agriculture.</p

Image_2_Antifungal, Plant Growth-Promoting, and Genomic Properties of an Endophytic Actinobacterium Streptomyces sp. NEAU-S7GS2.TIF

Diseases caused by Sclerotinia sclerotiorum have caused severe losses of many economically important crops worldwide. Due to the long-term persistence of sclerotia in soil and the production of air-borne ascospores, synthetic fungicides play limited roles in controlling the diseases. The application of antagonistic microorganisms can effectively reduce the number of sclerotia and eventually eradicate S. sclerotiorum from soil, and therefore considerable interest has been focused on biological control. Streptomyces sp. NEAU-S7GS2 was isolated from the root of Glycine max and its rhizosphere soil. It showed significant inhibitory activity against the mycelial growth of S. sclerotiorum (99.1%) and completely inhibited sclerotia germination. Compared to the control, in the pot experiment the application of NEAU-S7GS2 not only demonstrated excellent potential to control sclerotinia stem rot of soybean with 77 and 38% decrease in disease incidence and disease index, respectively, but could promote the growth of soybean. The light microscopy and scanning electron microscopy showed that co-culture of NEAU-S7GS2 with S. sclerotiorum on potato dextrose agar could lead to contorted and fragmented mycelia of S. sclerotiorum, which was associated with the secretion of hydrolytic glucanase and cellulase and the production of active secondary metabolites by NEAU-S7GS2. The plant growth promoting activity of NEAU-S7GS2 was related to the solubilization of inorganic phosphate, and production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase and indole acetic acid (IAA). To further explore the plant growth promoting and antifungal mechanisms, the complete genome of strain NEAU-S7GS2 was sequenced. Several genes associated with ammonia assimilation, phosphate solubilization and IAA synthesis, together with genes encoding ACC deaminase, glucanase and α-amylase, were identified. AntiSMASH analysis led to the identification of four gene clusters responsible for the biosynthesis of siderophores including desferrioxamine B and enterobactin. Moreover, the biosynthetic gene clusters of lydicamycins, phenazines, and a glycosylated polyol macrolide showing 88% gene similarity to PM100117/PM100118 were identified. These results suggested that strain NEAU-S7GS2 may be a potential biocontrol agent and biofertilizer used in agriculture.</p

Image_1_Antifungal, Plant Growth-Promoting, and Genomic Properties of an Endophytic Actinobacterium Streptomyces sp. NEAU-S7GS2.TIF

Diseases caused by Sclerotinia sclerotiorum have caused severe losses of many economically important crops worldwide. Due to the long-term persistence of sclerotia in soil and the production of air-borne ascospores, synthetic fungicides play limited roles in controlling the diseases. The application of antagonistic microorganisms can effectively reduce the number of sclerotia and eventually eradicate S. sclerotiorum from soil, and therefore considerable interest has been focused on biological control. Streptomyces sp. NEAU-S7GS2 was isolated from the root of Glycine max and its rhizosphere soil. It showed significant inhibitory activity against the mycelial growth of S. sclerotiorum (99.1%) and completely inhibited sclerotia germination. Compared to the control, in the pot experiment the application of NEAU-S7GS2 not only demonstrated excellent potential to control sclerotinia stem rot of soybean with 77 and 38% decrease in disease incidence and disease index, respectively, but could promote the growth of soybean. The light microscopy and scanning electron microscopy showed that co-culture of NEAU-S7GS2 with S. sclerotiorum on potato dextrose agar could lead to contorted and fragmented mycelia of S. sclerotiorum, which was associated with the secretion of hydrolytic glucanase and cellulase and the production of active secondary metabolites by NEAU-S7GS2. The plant growth promoting activity of NEAU-S7GS2 was related to the solubilization of inorganic phosphate, and production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase and indole acetic acid (IAA). To further explore the plant growth promoting and antifungal mechanisms, the complete genome of strain NEAU-S7GS2 was sequenced. Several genes associated with ammonia assimilation, phosphate solubilization and IAA synthesis, together with genes encoding ACC deaminase, glucanase and α-amylase, were identified. AntiSMASH analysis led to the identification of four gene clusters responsible for the biosynthesis of siderophores including desferrioxamine B and enterobactin. Moreover, the biosynthetic gene clusters of lydicamycins, phenazines, and a glycosylated polyol macrolide showing 88% gene similarity to PM100117/PM100118 were identified. These results suggested that strain NEAU-S7GS2 may be a potential biocontrol agent and biofertilizer used in agriculture.</p

MOESM6 of Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis

Additional file 6: Table S1. Primers used in this study

Characterization of a Novel Insecticidal Protein Cry9Cb1 from <i>Bacillus thuringiensis</i>

In recent decades, there have been increasing reports of insect resistance in Bacillus thuringiensis (Bt) crops. Alternative use of Cry toxins, with high insecticidal activity and different mechanisms of action, may be an important strategy to manage this resistance. Cry9 protein, with high toxicity to the lepidopteran pests and no cross-resistance with commercial Cry1 proteins, is a valuable relevant resource. A novel insecticidal protein, MP1489, subsequently named as Cry9Cb1, with 88% amino acid sequence identity with Cry9Ca1, was identified from Bt strain SP663; it exhibited high insecticidal activity against Plutella xylostella, Ostrinia furnacalis, and Chilo suppressalis and no cross-resistance with Cry1Fa in Ostrinia furnacalis. Its minimal active fragments against Plutella xylostella and Ostrinia furnacalis were identified to be 72T–657V and 68D–655A, respectively; food-safety assessment showed no sequence homology with any known allergen and rapid degradation and inactivation by both heat and the gastrointestinal environment. Therefore, Cry9Cb1 is proposed to have a brilliant prospect as an insecticidal protein in agriculture

Uptake and Biotransformation of Guvermectin in Three Crops after In Vivo and In Vitro Exposure

Guvermectin, as a novel nucleoside-like biopesticide, could increase the rice yield excellently, but the potential environmental behaviors remain unclear, which pose potential health risks. Therefore, the uptake and biotransformation of guvermectin in three types of crops (rice, lettuce, and carrot) were first evaluated with a hydroponic system. Guvermectin could be rapidly absorbed and reached equilibrium in roots (12–36 h) and shoots (24–60 h) in three plants, and guvermectin was also vulnerable to dissipation in roots (t1/2 1.02–3.65 h) and shoots (t1/2 9.30–17.91 h). In addition, 8 phase I and 2 phase II metabolites, transformed from guvermectin degradation in vivo and in vitro exposure, were identified, and one was confirmed as psicofuranine, which had antibacterial and antitumor properties; other metabolites were nucleoside-like chemicals. Molecular simulation and quantitative polymerase chain reaction further demonstrated that guvermectin was metabolized by the catabolism pathway of an endogenous nucleotide. Guvermectin had similar metabolites in three plants, but the biotransformation ability had a strong species dependence. In addition, all the metabolites exhibit neglectable toxicities (bioconcentration factor 50,rat > 5000 mg/kg b.w.) by prediction. The study provided valuable evidence for the application of guvermectin and a better understanding of the biological behavior of nucleoside-like pesticides