Whole-genome sequencing and analysis of Streptomyces strains producing multiple antinematode drugs
BMC Genomics volume 23, Article number: 610 (2022)
Nematodes are parasitic animals that cause over 100 billion US dollars loss in agricultural business. The whole-genomes of two Streptomyces strains, Streptomyces spectabilis KCTC9218T and Streptomyces sp. AN091965, were sequenced. Both strains produce spectinabilin, an antinematode drug. Its secondary metabolism was examined to aid the development of an efficient nematicidal drug-producing host strain.
The whole-genome sequences of S. spectabilis KCTC9218T and Streptomyces sp. AN091965 were analyzed using PacBio and Illumina sequencing platforms, and assembled using hybrid methodology. The total contig lengths for KCTC9218T and AN091965 were 9.97 Mb and 9.84 Mb, respectively. A total of 8,374 and 8,054 protein-coding genes, as well as 39 and 45 secondary metabolite biosynthetic gene clusters were identified in KCTC9218T and AN091965, respectively. 18.4 ± 6.45 mg/L and 213.89 ± 21.30 mg/L of spectinabilin were produced by S. spectabilis KCTC9218T and Streptomyces sp. AN091965, respectively. Pine wilt disease caused by nematode was successfully prevented by lower concentration of spectinabilin injection than that of abamectin recommended by its manufacturer. Production of multiple antinematode drugs, including spectinabilin, streptorubin B, and undecylprodigiosin was observed in both strains using high-resolution liquid chromatography mass spectrometry (LC–MS) analysis.
Whole-genome sequencing of spectinabilin-producing strains, coupled with bioinformatics and mass spectrometry analyses, revealed the production of multiple nematicidal drugs in the KCTC9218T and AN091965 strains. Especially, Streptomyces sp. AN091965 showed high production level of spectinabilin, and this study provides crucial information for the development of potential nematicidal drug producers.
Nematodes are parasites that cause harm to humans, other animals, and plants . Nematode infections in humans result in gastrointestinal diseases such as inflammatory bowel disease, and skin allergic reactions [2,3,4]. In addition, they cause various types of damages including stunting and yellow leaves in plants [5, 6]. The abundance of nematodes ranges from a dozen to over a thousand individual per 100 g of soil, depending on the geographic region. They are mostly found in the tundra, with a median of 2,695 nematodes per 100 g of dry soil . This makes nematodes the most abundant animals on earth . Nematode infections resulted in a reported loss of over 100 billion US dollars in 2018,  making them a major cause of financial strain in the agricultural sector. As a result, there have been more efforts to eliminate nematodes from the soil, with Streptomyces species as a promising source for the development of potent and environmentally-friendly nematicidal agents .
Streptomyces species are well known to produce various pharmaceutical products, including antibiotics and anticancer drugs [11, 12], as well as valuable agricultural chemicals . Nematicides are no exception, and many have been discovered from members of the Streptomyces genus. Avermectin, isolated from Streptomyces avermitilis, has been one of the most widely used nematicides since its discovery in 1978 in Japan. In 2020, the annual global sales of avermectin reached $850 million . The avermectin class of molecules includes ivermectin, moxidectin, selamectin, abamectin, and nemadectin . This nematicide interferes with glutamate-gated chloride channels and GABA receptors, resulting in the restriction of movement and reproduction, which leads to nematode death . Spectinabilin (also known as neoaureothin), another potent nematicide, was discovered in Streptomyces spectabilis in 1976 [17, 18] and is produced from chorismate, five molecules of methylmalonyl-CoA, and one malonyl-CoA by polyketide synthase (PKS) . The detailed mode of action of spectinabilin against nematodes is yet to be determined, but it is one of the only two drugs that cause vacuolar death. Treatment of nematode-infected pine trees with spectinabilin increases the survival rate of the trees from 10 to 80% [20, 21]. Prodigiosin, a member of the prodiginine group of compounds produced by several Streptomyces species, is also one of the well-studied nematicide . Biosynthesis of prodigiosin or its close derivative, undecylprodigiosin, (also known as RED due to its color) begins with proline and undergoes condensation with malonyl-CoA and serine, followed by hybridization with fatty acids [23, 24]. However, the mechanism of action of prodigiosin against nematodes also remains unknown.
Here, we present the genome sequences of two Streptomyces strains, Streptomyces spectabilis KCTC9218T and Streptomyces sp. AN091965, which can produce antinematode agents. They were sequenced using both short-read Illumina and long-read PacBio technologies to obtain high-quality genomic sequences. Comparative genome analysis confirmed the presence of spectinabilin and the RED biosynthetic gene cluster (BGC). 18.4 ± 6.45 mg/L and 213.89 ± 21.30 mg/L of spectinabilin were produced by S. spectabilis KCTC9218T and Streptomyces sp. AN091965, respectively. Especially for Streptomyces sp. AN091965, spectinabilin production level was tenfold higher than that from previous reports . In addition, spectinabilin was more effective than abamectin in the prevention of pine wilt disease (PWD) in pine wood, Pinus densiflora, caused by nematode, Bursaphelenchus xylophilus. Further analysis of bacterial culture extracts revealed that the two Streptomyces strains actively produced multiple nematicidal drugs: spectinabilin, RED, and streptorubin B, another member of the prodiginine produced by RED BGC. Our study highlights the possibility of developing a highly potent nematicide-producing microbial strain for agricultural use.
Results and discussion
Strain isolation and characterization of Streptomyces sp. AN091965
In our previous study, we identified 29 Streptomyces strains that have nematicidal activities from 5000 actinomycete isolates. The mortality rate of these strains was above 80% against B. xylophilus . They were isolated from forest soil samples collected from Chollipo Arboretum, Taean-gun, Chungcheongnam-do, Korea (36°47′56.6"N 126°08′58.3"E) in July 2009 (Fig. S1). Among the strains, Streptomyces sp. AN091965 was selected for further study because of its superior nematicidal properties . In this study, the mortality rate of Streptomyces sp. AN091965 crude extract against B. xylophilus was quantitatively determined to be 98.1 ± 2.9%. Only the morphology of Streptomyces sp. AN091965 colonies was briefly reported in a previous study ; thus, more detailed morphological analyses, including physiological analysis, were carried out.
The AN091965 strain was found to grow on Bennett’s agar, Reasoner’s 2A (R2A) agar, and other International Streptomyces Project (ISP) media. The phenotypes of the strain are listed in Table 1. Strain AN091965 grew slowly on ISP 5 medium but grew moderately to rapidly on the other test media. Diffusible pigments were not produced in any of the media tested, except for the G.S.S. liquid culture media. Aerial mycelia were observed only on the Bennett’s agar medium. The substrate mycelia in all the tested media, except ISP 2, were red in color (Fig. 1A), and the development of spore-like bodies was not observed in the aerial mycelium in any of the media tested (Fig. 1B). Physiology of the S. spectabilis KCTC9218T was very similar to that of AN091965 strain (Table S1; Fig. S2).
Genomic feature and phylogenetic study
S. spectabilis KCTC9218T is one of the best-known spectinabilin producers. It shared 99.86% 16S rRNA sequence similarity with Streptomyces sp. AN091965 in our previous study . The high homology of 16S rRNA gene sequences between these two strains suggests that Streptomyces sp. AN091965 may also be a spectinabilin producer. Production of spectinabilin by S. spectabilis KCTC9218T and a new Streptomyces sp. AN091965 was confirmed using high-resolution liquid chromatography mass spectrometry (LC–MS) analysis (Fig. S3).
Spectinabilin was extracted from the cell pellet and culture broth separately, and a major amount of the compound (approximately 86% and 89% from KCTC9218T and AN091965, respectively) was extracted from the cell pellets, indicating that it was not secreted efficiently from either strain (Fig. 2). 16.08 ± 5.06 mg/L and 185.57 ± 13.20 mg/L of spectinabilin were extracted from cell pellets, while 2.32 ± 1.39 mg/L and 28.32 ± 8.10 mg/L were extracted from culture broth of S. spectabilis KCTC9218T and Streptomyces sp. AN091965, respectively. Production of spectinabilin from AN091965 was over tenfold higher than that from KCTC9218T and Streptomyces orinoci ATCC23202T , one of the strains identified earlier as a spectinabilin producer. In our previous study, AN091965 was identified to have the highest level of nematicidal activity among 29 nematicide producing strains we isolated . Such significant differences in nematicidal activity of AN091965 may be explained by the extraordinary levels of spectinabilin production. Further experiments such as RNA-seq and metabolite profiling would be required to explain the differences between secondary metabolism of S. spectabilis KCTC9218T and Streptomyces sp. AN091965, but AN091965 bears a potential for industrial scale production of spectinabilin by further engineering.
To better understand the genomic features of the two strains, high-quality draft genome sequences from S. spectabilis KCTC9218T and Streptomyces sp. AN091965, consisting of six and four contigs, respectively, were obtained using PacBio and Illumina sequencing technologies, with a hybrid assembly of the two (Fig. 3). The total contig length was 9,967,045 bp for KCTC9218T, with an average G + C content of 72.4%, and 9,837,143 bp for AN091965, with an average G + C content of 72.7%. In total, 8,374 protein-coding genes (CDS), 18 rRNA coding genes, and 64 tRNA coding genes were present in KCTC9218T. A total of 8,054 CDS, 18 rRNA, and 67 tRNA genes were identified in AN091965. Detailed genomic data on KCTC9218T and AN091965 is provided in Table 2.
Phylogenetic analysis of S. spectabilis KCTC9218T and Streptomyces sp. AN091965 was first conducted by MEGAX with 16S rRNA sequences (Fig. 4A). Besides S. spectabilis ATCC27465T strain, close relative of the two strains were Streptomyces coeruleorubidus ISP-5145 T (97.73% and 97.73% 16S rRNA gene sequence similarities for AN091965 and KCTC9218T, respectively) and Streptomyces albogriseolus NRRL B-1305 T (98.29% and 98.22% for AN091965 and KCTC9218T, respectively). Next, the taxonomic positions of the two strains were analyzed using autoMLST software. A total of 43 strains, including two spectinabilin-producing Streptomyces species, 40 type Streptomyces strains, and one out-group strain, were analyzed (Fig. 4B). The autoMLST software provides high-quality genome sequence datasets from archives and determines the phylogeny of tested strains by Multi-Locus Sequence Analysis, also known as MLSA, comparing single-copy homologous genes shared among the type and out-group strains . More detailed phylogeny of the analyzed strains can be constructed in this way. All the selected genes for MLST analysis are listed in Table S2. KCTC9218T and AN091965 were identified as close relatives of Streptomyces aureocirculatus NRRL ISP-5386 T and Streptomyces alboflavus NRRL B-2373 T.
Analysis of genomic features
CDSs from KCTC9218T and AN091965 were analyzed and assigned to Clusters of Orthologous Groups (COG) by EggNOG  (Fig. 5). A total of 7902 CDSs from KCTC9218T and 7667 CDSs from AN091965 were assigned to COG. The largest proportion of all genes belonged to the S category. (Genes with unknown function): 2747 genes from KCTC9218T and 2737 genes from AN091965 were assigned to category S, which accounted for 37.92% and 38.13% of the entire CDSs, respectively. Genes related to transcription were the second largest COG, with 10.67% for KCTC9218T and 10.80% for AN091965. Genes coupled with amino acid transport and metabolism accounted for 6.52% for KCTC9218T and 6.43% for AN091965, whereas carbohydrate transport and metabolism accounted for 5.83% in KCTC9218T and 5.67% in AN091965. The distribution of the annotated genes was similar to that of various other Streptomyces strains , as transcription and primary metabolism, including carbohydrate and amino acid metabolism, are among the most essential features in living organisms. The distribution of all other genes among the COG categories was also similar between the two spectinabilin-producing Streptomyces strains.
Phylogenetic analysis revealed that KCTC9218T and AN091965 were closely related (Fig. 4B). The two strains also shared high similarities in genetic features and orthologous protein groups. The 16S rRNA sequences of KCTC9218T and AN091965 acquired from whole genome sequencing data shared 100% and 99.74% identity with Streptomyces spectabilis ATCC27465T (a well-known spectinabilin producer), respectively . To examine the differences between the three spectinabilin-producing strains, especially for KCTC9218T and ATCC27465T, having 100% identical 16S rRNA genes, the compositions of orthologous gene clusters were analyzed using OrthoVenn2 . Streptomyces spectabilis NRRL2792 , another spectinabilin producing Streptomyces strain was included for the analysis (Fig. 6). A total of 8029 and 7105 orthologous gene clusters were found in KCTC9218T and AN091965, respectively. In total, 6504 clusters were common among the four Streptomyces strains. A total of 41 clusters were unique in AN091965 and KCTC9218T strains. 15 clusters from KCTC9218T and 31 clusters from AN091965 were unique to themselves. One cluster for ATCC27465T, and 8 clusters from NRRL2792 were unique to themselves. Although the four spectinabilin-producing strains shared high genomic similarities, such as COG, there were distinctive differences in the distribution of predicted protein function of the orthologous clusters. Especially for KCTC9218T with 100% identical 16S rRNA gene sequences compared to those of ATCC27465T, such differences in organization of orthologous gene clusters were remarkable. Previously, all eleven Brevundimonas alba strains isolated from freshwater community were verified to have 100% identical 16S rRNA gene sequences to each other, but determined to have great differences in their genomic contexts . The KCTC9218T and ATCC27465T strains showed similar results. Therefore, whole genome sequencing of KCTC9218T strain demonstrated the diversity of S. spectabilis community.
Identification of secondary metabolite gene clusters using antiSMASH
The antiSMASH  analysis predicted 45 and 39 secondary metabolite BGCs in KCTC9218T and AN091965, respectively, and we manually examined the predicted BGCs if they really contain the core biosynthetic genes (Fig. 7). This included the BGCs of nine PKSs, eleven nonribosomal peptide synthetases (NRPSs), and three PKS/NRPS hybrids from KCTC9218T. The BGCs of eight PKSs, ten NRPSs, and four PKS/NRPS hybrids were observed from AN091965. In both strains, NRPS was the most abundant secondary metabolite BGC. Type I PKS accounted for the most, seven in each strain, among the PKS BGCs. Three terpene BGCs, namely hopene, albaflavenone, and geosmin, were present in both KCTC9218T and AN091965 (Table 3). Despite differences in orthologous protein clusters, antiSMASH predictions of secondary metabolite BGCs were very similar between KCTC9218T and AN091965. There were 10 similar BGCs, which were expected to produce known compounds with identical predicted functions at high levels of similarity (> 80%) between the two strains. A notable difference was the position of lagmycin-like BGC on the largest contig. While the lagmycin BGC was located upstream of the ectoine BGC in contig 1 of KCTC9218T, it was located between actinospectacin and coelichelin BGCs in contig 1 of AN091965.
Comparison of spectinabilin BGCs
Two different types of spectinabilin BGCs have been reported in S. spectabilis ATCC27465T and Streptomyces orinoci ATCC23202T. Overall, spectinabilin is produced from seven modules of PKS, which are composed of four genes: spnA, spnA′, spnB, and spnC . Spectinabilin biosynthesis is predicted to begin with a chorismate as a starting unit . This is followed by polyketide chain elongation, with six methylmalonyl-CoA and one malonyl-CoA molecules as extender units (Fig. 8A). The major difference between the two spectinabilin BGCs is the function of a regulatory protein present in the cluster. SpnD (AfsR family transcriptional regulator) in S. spectabilis is a positive regulator of the BGC, whereas NorD (ArsR family transcriptional regulator) is a repressor in S. orinoci ATCC23202T .
The spectinabilin BGCs from five producers and aureothin (a structurally and biosynthetically similar compound to spectinabilin) BGC from S. thioluteus were compared (Fig. 8B) to investigate the genetic structure of spectinabilin BGC and propose its biosynthetic mechanism in KCTC9218T and AN091965 strains. The domain and modular organization of PKSs are identical in all spectinabilin-producers, which indicates that spectinabilin biosynthesis follows the same series of PKS-catalyzed reactions in all producers (Fig. 8A). The SpnD of KCTC9218T and AN091965 shared 96.06% identity. When compared to SpnD of ATCC27465T, SpnD of KCTC9218T and AN091965 shared 99.62% and 96.23% identity, respectively. However, no similarity with NorD of S. orinoci ATCC23202T was observed. This suggests that SpnD of KCTC9218T and AN091965 are positive regulators of spectinabilin BGC. In addition, the spnK gene was found in S. spectabilis ATCC27465T, KCTC9218T, and NRRL2792, Streptomyces sp. AN091965 , whereas it was absent in S. orinoci ATCC23202T and S. thioluteus ATCC12310T. Although the specific function of spnK is not known, it is nonessential for spectinabilin production . Finally, a putative spectinabilin membrane transporter, spnM, was identified in all strains.
Although there are high similarities in the spectinabilin BGC between KCTC9218T and AN091965, it would require further studies to identify the cause of differences in spectinabilin production level of the two strains. Secondary metabolism is affected by many factors such as carbon and nitrogen metabolism, gene regulations, physical stress, and availability of nutrients in surroundings . Gene regulation is a complex series of regulatory networks. Although a pathway-specific regulator governing spectinabilin production, SpnD, is identified, the complex upstream regulatory network affecting SpnD cannot be understood solely based on genome sequences. On the other hand, polyketides draw their precursors from carbon sources such as glucose and fatty acids, and the differences in carbon metabolism of KCTC9218T and AN091965 should also be understood . Further studies including metabolite profiling and RNA-seq in combination with genomic information may explain such differences in spectinabilin producing behavior.
Nematicidal activity and toxicity test on nematode infected pine woods
B. xylophilus infection in trunks and branches of pine woods, causing PWD, is possibly due to phytotoxins released by the nematode . To evaluate the nematicidal activities of spectinabilin, spectinabilin purified from Streptomyces sp. AN091965 (Fig. S4) was treated to pine wood, Pinus densiflora, before B. xylophilus infection (Fig. 9). Successful nematode infection in pine was visualized as the presence of needle discoloration, and these visual symptoms allowed us to evaluate the effect of spectinabilin on survival of the trees. 100% of nematicide untreated pine woods died after 60 days of B. xylophilus infection.
Spectinabilin at the concentration of 0.9 mg, 1.8 mg, and 3.6 mg per cm of pine wood trunk diameter was injected into trunks of pine woods before the B. xylophilus infection. The test was performed at quintuplicate, and commercially available nematicide, abamectin, was treated at 1.8 mg per cm of pine wood trunk diameter, a recommended concentration by its manufacturer, as a positive control. Supplement of spectinabilin or abamectin at the same concentration resulted in the prevention of nematode infection for three out of the five tested pine woods. Toxicity of spectinabilin to the pine woods was observed with increasing concentration, up to 3.6 mg/cmTrunk diameter. Spectinabilin treatment at 0.9 mg/cmTrunk diameter successfully inhibited the development of PWD in all pine trees that are artificially infected with B. xylophilus from the quintuplicate test, implying that lower PWD prevention rate at 1.8 mg/cmTrunk diameter spectinabilin treatment may have been due to the toxicity to the tree.
Alhough the rates of PWD outbreak was suppressed in pine woods by abamectin and spectinabilin treatments, some portion of B. xylophilus still survived from the nematicides. Thus, density of the nematode was measured. Number of B. xylophilus in the nematicide untreated pine wood increased from approximately 2000 to 3572 ± 322 per gram of trunk tissue. Abamectin and spectinabilin injection at the concentration of 1.8 mg/cmTree diameter decreased B. xylophilus density from approximately 2000 to 453 ± 51 and 211 ± 53 per gram of trunk tissue, respectively. This result indicated that spectinabilin was more effective than abamectin in the treatment of B. xylophilus infection. At the concentration of 0.9 mg/cmTrunk diameter of spectinabilin injection, number of nematodes decreased from approximately 2000 to 298 ± 93 per gram of trunk tissue.
Analysis of secondary metabolite production
The presence of secondary metabolite BGCs does not indicate that the gene clusters would actively produce the corresponding compounds. Secondary metabolism is affected by many factors, including environmental conditions and the type of media used for bacterial cultures [40, 41]. Cryptic BGCs are functional only under defined conditions . Therefore, KCTC9218T and AN091965 strains were cultured under standard laboratory conditions  using G.S.S. media to investigate whether they produce other nematicidal compounds other than spectinabilin. We reasoned that the red colors of KCTC9218T and AN091965 cells, and antiSMASH prediction of the presence of undecylprodigiosin BGC suggested the two strains are possible producers of prodigiosin-type secondary metabolites in these strains. Thus, LC–MS analysis of crude extracts from the two strains focused on a search for prodigiosin or its derivatives.
LC–MS analysis of extracts from the cell pellets and supernatants of KCTC9218T and AN091965 cultures showed three major peaks. The major product was spectinabilin and the other two were streptorubin B and RED. Despite having complete BGCs of several known NRPS and PKS metabolites (Table 3), only three were detected under the culture conditions used in this study. Generally, RED is not secreted from cells and is often extracted from cell pellets for quantification . Similarly, it was not detected in the supernatants of KCTC9218T and AN091965 cells. However, secretion of streptorubin B was detected in both strains (Fig. S5). Streptorubin B and RED are known for their nematicidal activities . All three bioactive compounds produced by KCTC9218T and AN091965 exhibited antinematode activity, suggesting that these two strains have great potential as sources of antinematode drugs.
In this study, the genomes of two antinematode agent-producing strains, S. spectabilis KCTC9218T and Streptomyces sp. AN091965, were sequenced and annotated. These strains were found to possess an additional secondary metabolite (RED) BGC, in addition to spectinabilin BGC, with antinematicide potential. Liquid cultures of the two strains and identification of bioactive molecules confirmed the active production of multiple nematicidal compounds: spectinabilin, streptorubin B, and RED. Efficacy test against nematode revealed that spectinabilin, at only half of the recommended concentration of abamectin, was sufficient for prevention of PWD. Especially, Streptomyces sp. AN091965 strain has a great potential as an inexpensive industrial nematicidal drug producer due to its ability to produce 200 mg/L of spectinabilin, which is tenfold higher than other spectinabilin producers reported.
Strains and culture conditions
S. spectabilis KCTC9218T and Streptomyces sp. AN091965 strains were cultured in Bennett’s agar media at 28 ℃ before they were transferred to G.S.S. media (10 g of soluble starch, 20 g of glucose, 25 g of soybean meal, 1 g of beef extract, 4 g of yeast extract, 2 g of NaCl, 0.25 g of K2HPO4, and 2 g of CaCO3 in 1 L of distilled water) for antibiotic production. Cultures in ISP2 medium (Becton, Dickinson and Company, New Jersey, USA) were used to extract high molecular weight DNA for whole-genome sequencing. All the 50 mL liquid cultures were carried out at 28 ℃. They were shaken at 180 rpm in 50 mL volume using 250 mL baffled glass flasks. Bacterial cultures were harvested on day 2 for extraction of high molecular weight DNA for whole genome sequencing, and on day 7 for extraction and quantification of secondary metabolites.
Extraction of secondary metabolites
Spectinabilin, RED, and streptorubin B were extracted separately from the mycelium and media broth. Mycelia were isolated through centrifugation. The pellets were suspended in a 10 × vol. of 80% methanol and sonicated for 20 min. The supernatant was collected through centrifugation, 7 min at 5000 × g and room temperature. The cell debris were further removed using paper filtration. The three compounds were extracted from the media broth using an equal volume of EtOAc. The EtOAc fraction was harvested, dried, and resolved in 15 mL methanol. The extracts from both mycelium and media broth were run through Sep-Pak Vac 3 cc silica solid-phase extraction column (Waters, Massachusetts, USA). The silica column was wet with 3 mL methanol, and pre-conditioned with 3 mL of 75% n-hexane in EtOAc. 20 μL of mycelia extract or 100 μL of media broth extract was loaded to the silica column. And then, the three compounds were eluted from the silica column using 3 mL of 75% n-hexane in EtOAc. The eluents were dried using SpeedVac, and reconstituted with 1 mL of acetone for nematicidal activity screening or MeCN for MS analysis.
Nematode culture conditions
B. xylophilus was obtained from the National Institute of Forest Science (Seoul, Korea). It was separated from the xylem of PWD-infected trees using the Baermann funnel technique. The nematodes were maintained on B. cinerea-cultured Potato Dextrose Agar (PDA; Becton, Dickinson and Company, New Jersey, USA) at 25 ℃ for seven days. Cultured nematodes were harvested using sterilized water and subsequently counted.
Determination of nematicidal activity of Streptomyces sp. AN091965 crude extract
The nematicidal activity of Streptomyces sp. AN091965 was determined, with purified spectinabilin from S. spectabilis KCTC9218T as a control. 5 μL of 0.5 mg/mL spectinabilin or Streptomyces sp. AN091965 crude extract in acetone was added to 50 nematodes in 95 μL suspension in a 96-well plate. After 24 h, the nematodes were observed under a microscope to determine mortality. If the nematode body was motionless and straight, it was considered dead. Mortality was calculated as follows:
Morphological characterization of Streptomyces sp. AN091965
To investigate its morphological characteristics, the AN091965 strain was streaked on Bennett’s agar medium. This was cultured at 28 ℃ for 10 days, after which spore form, size, and color of the mycelia of the metal-bearing strain were observed using a scanning electron microscope (FEI Quanta 250 FEG; FEI).
The culture characteristics of strain AN091965 were determined on ISP2, ISP3, ISP4, ISP5, Bennett’s agar, and R2A agar (Becton, Dickinson and Company, New Jersey, USA) media. After incubation at 28 °C for seven days, the morphological characteristics of the strain were observed under an optical microscope (Nikon Labophot-2).
High molecular weight DNA extraction
High-molecular-weight genomic DNA was extracted from S. spectabilis KCTC9218T and Streptomyces sp. AN091965 using the phenol/chloroform extraction method. Cell pellets were collected and washed twice with 10 mL of distilled water in 50 mL conical tubes. A 10 mL lysis buffer containing 3 mg/mL lysozyme (Sigma-Aldrich, Missouri, USA) and 100 μg/mL RNase (Sigma-Aldrich, Missouri, USA) in TE buffer (25 mM Tris–HCl, pH 8.0, 25 mM EDTA, and 0.3 M sucrose) was added. Cells were lysed in the conical tubes at 37 ℃ for 1 h. Proteinase K in distilled water was added to the lysate at a final concentration of 100 μg/mL, and the mixture was incubated at 55 ℃ for 2 h. 8 mL of phenol/chloroform (1:1) were added to the lysate and mixed by inversion for 6 min. The mixture was centrifuged for 15 min at 5000 × g and 4 ℃. The aqueous phase was transferred to a fresh tube, and 6 volume of ethanol was added. The mixture was stored at –20 ℃ for 12 h to enable DNA precipitation. DNA pellets were washed with 5 mL of ice-cold 70% ethanol twice, and the high molecular weight DNA was dissolved in 3 mL of distilled water.
Whole-genome sequencing and assembly
The quality of the extracted DNA was measured using a NanoDrop ND-1000 (Thermo Scientific, Massachusetts, USA) and a Qubit fluorometer (Thermo Scientific) following the manufacturers’ protocol. The 16S rRNA gene was also sequenced to identify foreign gene contamination in the DNA samples. Whole-genome sequencing was performed by DNAlink (Seoul, Republic of Korea) using the Sequel (Pacific Biosciences, California, USA) and Novaseq6000 (Illumina, California, USA) systems. Whole-genome assembly was conducted by ChunLab (Seoul, Republic of Korea) using their Illumina-PacBio hybrid assembly method. Protein-coding sequences from the assembled strains were analyzed by ChunLab. Assembled genome sequences of Streptomyces sp. AN091965 and S. spectabilis KCTC9218T have been submitted to NCBI GenBank, under BioProject ID PRJNA817371, and BioSample accessions SAMN26764618 and SAMN26764619, respectively. The genome sequences are available from the NCBI Nucleotide database by searching accessions JALDMY000000000 and JALDMZ000000000 for Streptomyces sp. AN091965 and S. spectabilis KCTC9218T, respectively.
A phylogenetic tree of the two spectinabilin-producing strains was produced through MEGAX (https://www.megasoftware.net/)  and autoMLST . Phylogenetic tree was constructed from Clustal Omega aligned DNA sequences using the Maximum Likelihood method based on the general time reversible model. It was evaluated with bootstrap analysis of 1000. autoMLST analysis was performed using the fast alignment mode (MAFFT FFT-NS-2) under advanced settings with bootstrap analysis of 1000 replicates (http://automlst.ziemertlab.com/analyze). 50 type strains, reference genomes, and 3 outgroup strains were provided by the software. We reduced the number of reference genomes to 37 by removing repetitive strains. Concatenated alignment option was used to build the phylogenetic tree.
The coding sequences of S. spectabilis KCTC9218T and Streptomyces sp. AN091965 were annotated using EggNOG v5.0 (http://eggnog5.embl.de/#/app/home) . A minimum hit 2-value of 0.001, minimum hit bit score of 60, minimum percent identity of 40, minimum percent of query coverage of 20, minimum percent of subject coverage of 20, auto tax scope, and non-electronic GO evidence settings were used for the analysis. Orthologous gene clusters were analyzed through OrthoVenn2 (https://orthovenn2.bioinfotoolkits.net/home) , using default settings, 1e-2 E-value, and a 1.5 inflation value. Genomic data of S. avermitilis and S. coelicolor were provided by OrthoVenn2, whereas that of S. spectabilis ATCC27465T was acquired from NCBI. Further, antiSMASH analysis was used to identify putative secondary metabolite BGCs (https://antismash.secondarymetabolites.org/#!/start) .
LC–MS analysis of nematicidal metabolite extracts
Spectinabilin, RED, and streptorubin B were identified and quantified using an XEVO® G2S Q-ToF mass spectrometer coupled with an Acquity I-Class system (Waters, Massachusetts, USA). 2 μL of sample were injected, and UPLC was operated at a flow rate of 0.4 mL/min with solvent A, 0.1% formic acid (FA), and solvent B, 80% MeCN with 0.1% FA. Solvent gradient was applied, starting from 40% A to reach 5% A by 10 min, a 2 min hold at 5% A, and returning to 40% A in 0.1 min with an additional 0.9 min hold. An Xselect™ CSH™ C18 (2.1 × 100 mm, 2.5 μm) column (Waters, Massachusetts, USA) was maintained at 40 ℃. The MS system was operated in the ESI mode with positive ionization. The analyzer was operated with an extended dynamic range at a 60,000 resolution. Mass correction was performed using leucine enkephalin (400 pg/μL, 50% MeCN with 0.1% FA) as a lockspray infused at a rate of 5 μL/min. Authentic spectinabilin was provided by Korea Research Institute of Bioscience and Biotechnology for quantification.
Purification of spectinabilin
Spectinabilin was purified from the crude extract before the suppression test. Silica open column was made by suspending 400 mL volume of silica with 500 mL 50% n-hexane in ethyl acetate, and the silica emulsion was tightly packed in a vertically setup, cylinder type separation funnel. The silica open column was washed with 1.5 L of n-hexane–EtOAc (10:1 volume by volume). Compounds were eluted from the silica open column by n-hexane–EtOAc gradient system (10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1, each 1 L), and finally by 2 L of 100% MeOH. Each fraction was dried under rotary vacuum evaporator, and dissolved in 15 mL of MeCN for LC–MS analysis. Conditions for LC–MS were the same as that for the screening of spectinabilin production in the crude extract. A majority of spectinabilin, at high purity, was detected in 100% MeOH fraction (Fig. S4), and spectinabilin was collected from HPLC fractions.
Suppression of PWD under greenhouse conditions
Pot experiments were performed following the procedure from our previous study  with some modifications. One month before the experiment, five-year-old P. densiflora trees were transplanted and stored in a greenhouse with temperature maintained around 22 ℃ to 25 ℃ at the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea). Pine wood growing pots were watered every other day. The height and basal diameter of each tree used in the pot experiment were recorded on the day of nematicide injection. Chemical solutions of spectinabilin were prepared using DPPT as a solvent (20% DMSO, 20% propylene glycol monomethyl ether, 50% propylene glycol, and 10% Tween 20) at the final concentration of 0.9 and 1.8 mg per 100 μL of DPPT. 18 mg/mL of abamectin was prepared with DPPT solution as a positive control. DPPT solution without nematicides was treated as a negative control. A hole (5 mm depth × 3 mm diameter) was drilled on trunks of pine trees about 5 cm above the soil at an angle of 45°, and prepared nematicide solutions were injected into the holes. The holes were covered with parafilm to prevent dryness.
Another holes were drilled on the trunk of pine trees approximately 20 cm above the soil at an angle of 45°. A 0.1 mL aliquot of the nematode suspension (containing about 2,000 nematodes, mixture of adults and juveniles) was pipetted into the holes, and the holes were also covered with parafilm. Average size of the subject pine trees was 120 cm in height, and 1 cm in basal trunk diameter. The pot experiments for spectinabilin efficacy tests were conducted in April 2022. Five replicates of triplicate experiments were conducted for each concentration of 0.9, 1.8, and 3.6 mg per cm of trunk diameter.
Nematode extraction from pine woods and counting for survival test
After the pot experiment, pine tissue samples were collected to quantify nematodes that survived from abamectin and spectinabilin treatments. 2 g of trunk tissue was weighed and transferred onto a gauze in a 50 mL conical tube, filled with 30 mL of distilled water. After soaking the tissue in water for 24 h, it was carefully removed with the gauze from the tube. Nematodes released from trunk tissues were allowed to settle down onto the bottom of the conical tube for 12 h. Water was removed carefully from the top, reducing down to the final volume of 10 mL. 100 μL suspension of nematodes from the 10 mL were spotted onto glass slide, and the nematodes were counted under a microscope. The nematode population was calculated as the number of nematodes per gram of tree trunk.
Availability of data and materials
Assembled genome sequences of Streptomyces sp. AN091965 and S. spectabilis KCTC9218T have been submitted to NCBI GenBank, under BioProject ID PRJNA817371, and BioSample accessions SAMN26764618 and SAMN26764619 respectively. The genome sequences are available from the NCBI Nucleotide database by searching accessions JALDMY000000000 and JALDMZ000000000 for Streptomyces sp. AN091965 and S. spectabilis KCTC9218T respectively.
Clusters of orthologous groups
Biosynthetic gene cluster
Nonribosomal peptide synthetase
Pine wilt disease
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The authors would like to thank the members of ChunLab for technical support and a hybrid assembly from PacBio and Illumina sequencing.
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through “Crop Viruses and Pests Response Industry Technology Development” Program (No. 321110–4) funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA), National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No.2022R1A2C3004621) and the Ministry of Education (2022R1I1A1A01068507), and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. 2020M3H1A1073304).
Ethics approval and consent to participate
The authors report that experimental works on the plant, pine wood Pinus densiflora, comply with institutional, national, and international guidelines. Permissions are only required for field tests of insecticides on plants by the government of Korea, Republic of. No permissions were necessary to perform experiments and collect the plant samples under the greenhouse conditions. All the experiments on pine woods were performed in the greenhouse set up at the private property of Korea Research Institute of Bioscience and Biotechnology (Daejeon, Republic of Korea).
Consent for publication
The authors declare that they have no competing interests.
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Table S1. Cultural characteristics of Streptomyces spectabilis KCTC9218T on different growth media. Table S2. List of genes used for autoMLST analysis. Fig. S1. Location of the sampling sites in South Korea. Red marks indicate areas where soil samples were obtained. Fig. S2. Morphological characteristics of Streptomyces spectabilis KCTC9218T grown on Bennett’s agar media. Fig. S3. LC-MS analysis of (A) spectinabilin extracted from the cell pellets and media broth of S. spectabilis KCTC9218T and Streptomyces sp. AN091965 cultures and (B) their MS/MS fragmentation analysis. Fig. S4. LC-MS chromatogram of metabolite fractions collected during purification of spectinabilin from the crude extract of Streptomyces sp. AN091965 culture broth and cell pellets. Spectinabilin was eluted from silica column at 100 % methanol. Fig. S5. LC-MS spectrum of spectinabilin (yellow), undecylprodigiosin (orange), and streptorubin B (green) from S. spectabilis KCTC9218T and Streptomyces sp. AN091965 cultures.
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Yi, J.S., Kim, J.M., Kang, MK. et al. Whole-genome sequencing and analysis of Streptomyces strains producing multiple antinematode drugs. BMC Genomics 23, 610 (2022). https://doi.org/10.1186/s12864-022-08847-4
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