2024 Volume 49 Issue 1 Pages 22-30
From the 992 samples of culture extracts of microorganisms isolated from soil in Japan, we found that the extract of Streptomyces sp. no. 226 inhibited Orobanche minor seed germination without significantly affecting the seed germination of Trifolium pratense and the growth of Aspergillus oryzae and Escherichia coli. Using ESI-MS, 1H-NMR, and 13C-NMR, we identified the active compound as cycloheximide. Cycloheximide had half-maximum inhibitory concentrations of 2.6 ng/mL for the inhibition of seed germination of O. minor and 2.5 µg/mL for that of the conidial germination of A. oryzae. Since cycloheximide is known to inhibit translation by interacting with ribosomal protein L28 (RPL28) in yeast, we investigated whether RPL protein of O. minor plays a critical role in the inhibition of O. minor seed germination. Our data suggested that O. minor RPL27A was not sensitive to cycloheximide by comparing it to the strain expressing S. cerevisiae RPL28. These findings suggest the presence of an unidentified mechanism by which cycloheximide hinders O. minor seed germination.
Orobanchaceae includes root parasite weeds, which are noxious parasitic weeds that represent a severe danger to numerous crops across the globe, resulting in considerable output losses and financial strain on farmers. Orobanchaceae includes the broomrape, Orobanche, and Phelipanche species, which are mostly found in the Mediterranean area and parasitize a broad variety of crops and beans.1) The genus Orobanche has over 100 species, four of which are recognized as important weeds in Europe: Orobanche crenata Forsk., Orobanche cumana Waller., Orobanche ramosa L., and Orobanche aegyptiaca Pers.2) Although crop loss has not yet been recorded, Orobanche minor, a parasite of red clovers (Trifolium pratense), is known to inflict economic harm. Red clovers thrive in a symbiotic relationship with nitrogen-fixing Rhizobium bacteria. As a result, they were employed as a form of fertilizer in numerous locations around Japan. Since O. minor seeds were introduced to Japan in the first part of the twentieth century together with red clover seeds (unpublished data), they have grown wild in Japan. As a result, in Japan, we initiated research on suppressing seed germination of broomrape using active chemicals from microorganisms or the microorganisms themselves, using O. minor as a model.
Several parasitic weed management strategies have been presented. Only after a few weeds have sprouted in a limited area may mechanical techniques (hand picking) be used to inhibit the production of weed seeds. However, because Orobanche and Phelipanche produce hundreds of thousands of small seeds that can remain dormant in the soil for more than ten years while still germinating,3,4) removing broomrape by hand pulling is impossible once it has spread. As a result, chemical control of broomrape has been studied since the 1970s. Glyphosate proved effective in controlling broomrape in carrots, parsley, and faba beans at low rates. Sulfosulfuron and rimsulfuron, both sulfonylurea herbicides, were also efficient in controlling broomrape in tomatoes and potatoes.5) However, broomrape’s seed dormancy makes chemical management challenging. Host plant roots lengthen and leak strigolactones in root exudates, which promote broomrape seed development at the place where the roots newly grow throughout the harvest season. Because this occurrence happened in the soil, herbicides must be continually fed into the soil, making chemical management of broomrape problematic.5)
Broomrape may be biologically managed by destroying its seed bank or preventing it from identifying its hosts. Phytomyza orobanchia, an insect herbivore, has been used in the particular biological control of broomrape.6) Filamentous fungi were utilized for biological control as well. In many situations, phenolic chemicals found in the testa of broomrape seeds inhibit seed destruction by microbes. Plant diseases, on the other hand, such as Fusarium sp. and Ulocladium botrytis, may disintegrate the endosperm cell walls, enter within, and metabolize the cytoplasm of their seeds.7) Bacteria such as Pseudomonas aeruginosa and Bacillus subtilis may inhibit broomrape radicle formation.8) Arbuscular mycorrhizal fungi such as Rhizobium leguminosarum and Azospirillum brasilense may lower strigolactones in mycorrhizal plant root exudates, promoting the broomrape seed bank to stay dormant.9) Despite the fact that many weed control techniques have been explored, traditional weed management tactics have proved ineffective in regulating its expansion, emphasizing the urgent need for new and long-term solutions to tackle this deadly plant.
We tested roughly 1,000 microorganisms isolated from soil in Japan for active compounds preventing O. minor seed germination as the first stage in developing chemical or biological control methods for broomrape. We discovered Streptomyces, which generates a chemical that inhibits O. minor seed germination but has no influence on the seed germination of T. pratense and the growth of Aspergillus oryzae and Escherichia coli. Purification and identification of the substance indicated that cycloheximide is an inhibitor of O. minor seed germination. Interestingly, cycloheximide decreased O. minor seed germination at nanomolar concentrations. Cycloheximide’s mode of action on seed germination in O. minor was investigated and addressed.
Minimal medium,10) yeast peptone dextrose broth (Takara), and Luria–Bertani (LB) broth were used to culture A. oryzae RIB40 (NBRC 100959), Saccharomyces cerevisiae Y2HGold (Takara, Shiga, Japan), and E. coli DH5α (Takara), respectively.
2. Isolation of actinomycetes from soil samples and preparation of culture extractsActinomycetes were largely isolated from soil samples obtained in Japan using the previously published modified HVG agar medium.11) To keep isolated microorganisms alive, Maltose-Bennett’s agar media12) were utilized. Inhibitors of O. minor seed germination were identified using extracts generated from cultures cultured for 5 days in liquid medium A at 30°C (Supplemental Table S1) by adding an equivalent amount of acetone and mixing.
3. Screening microorganisms for compounds inhibiting seed germination of O. minorIn May, O. minor seeds were harvested in Hyogo, Japan, and kept at 4°C. Conditioned seeds that had been treated to aid germination were produced as previously reported.13) The organic solvent was air-dried after 14 µL of culture extracts was applied to glass filters (Whatman GF/D; Cytiva, Tokyo, Japan), and then 27 µL of GR24 solution (0.1–1.0 mg/L (w/v)) diluted with distilled water was added to filter paper. A glass filter containing conditioned O. minor seeds was then put on the chemically treated filter. The seeds were examined under a microscope after one week, and the germination rate was obtained by dividing the number of germinated seeds by the total number of seeds. Professor Kuse of Kobe University in Japan generously contributed GR24.
Isolated microorganisms were identified based on 16S rRNA DNA sequence analysis. This involved polymerase chain reaction using primers 5′-AGAGTTTGATCCTGGCTCAG and 5′-AAGGAGGTGATCCAGCCGCA)14) followed by Sanger sequencing.
4. Evaluation of the specific inhibition of the extracts and cycloheximideT. pratense seeds were produced on glass filters (Whatman GF/D), put on another glass filter treated with chemicals, 27 µL of distilled water was added, and an average of 20 T. pratense seeds were checked for germination after one week. The germination rate was obtained by dividing the total number of seeds by the number of germinated seeds. 25 µL of culture extracts and 25 µL of submerged minimal medium containing 1×102 conidia of A. oryzae RIB40 were incubated at 30°C without shaking for 20 hr, and then conidium germination was seen under a microscope. The germination rate was estimated by dividing the total number of conidia by the number of germinated conidia. An overnight culture of E. coli DH5α was diluted 1/100 in LB broth and combined with 25 mL of actinomycete culture extract for the E. coli inhibition experiment. As a control, an E. coli dilution in 50% acetone/LB medium was employed. The mixes were incubated at 37°C for 4 hr before being tested at 600 nm for growth. The E. coli growth rate was estimated by dividing the OD600 of the extract-treated culture by the OD600 of the control.
To study the inhibitory impact of cycloheximide, different quantities were applied to grass filters with GR24 for the inhibition of O. minor seed germination or to minimal medium for the suppression of A. oryzae conidium germination.
5. Purification of seed germination inhibitorStreptomyces sp. no. 226 was cultivated in medium A for 2 days at 30°C. Aliquots were transferred to 200 mL of the medium described in media A to F (Supplemental Table S1) and cultured for 3 days at 30°C. EtOAc extracts were produced and tested for their ability to suppress O. minor seed germination. After adjusting the culture conditions, medium B was chosen to grow Streptomyces sp. no. 226. To purify the chemical, this strain was grown in medium B at 30°C for 5 days. The supernatant (51.8 L) was extracted twice using an equal amount of EtOAc and then evaporated. The crude extract (7.22 g) was loaded onto a Wakogel C-200 column (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and eluted using a stepwise gradient of n-hexane/EtOAc (100 : 0 to 0 : 100), followed by evaporation. The dried material obtained from the fraction of n-hexane/EtOAc (25 : 75) was resuspended in MeCN before being purified using an Inertsil ODS-3 column (250×10 mm, 5 µm, GL Sciences, Tokyo, Japan). The gradient elution was as follows: 30 min of H2O/MeCN (from 90 : 10 to 40/60), 10 min of H2O/MeCN (40 : 60), and 10 min of H2O/MeCN (0 : 100). The eluent was fractionated every 1 min, and the flow rate was adjusted to 4.0 mL/min. In the 11th fraction, the seed germination inhibitor was discovered. The bioactive component was subjected to HPLC with the Inertsil ODS-3 column (250×10 mm, 5 µm) at a flow rate of 4.0 mL/min with 30% MeCN. A significant single peak was identified at the retention time (12.6 min) and processed to obtain 4.6 mg of dry powder.
6. Spectroscopic analysis of the purified compound and cycloheximideThe structures of the purified compound and cycloheximide (Fujifilm, Tokyo, Japan) were determined using 1H- and 13C-NMR spectroscopy and COSY. DMSO-d6 was used as the solvent, and the NMR measurements were conducted on a JNM-ECZ500R/S1 NMR spectrometer (JEOL Ltd., Tokyo, Japan). The chemical shifts were referenced to the solvent peak (δH 2.49, δC 39.7) as an internal standard. Additionally, the molecular mass was determined using the LCMS 2020 system (Shimadzu, Kyoto, Japan).
7. Generation of S. cerevisiae strains that expressed the rpl genesThe ribosomal 60S subunit protein L28 gene (rpl28; gene ID, 852775) of S. cerevisiae is homologous to the rpl27A gene of A. oryzae (rpl27A; gene ID, AO090001000413) and the rpl27A gene of O. minor (gene ID, IADW01093067). The rpl genes were expressed using pGADT7 that had the GAL4 activation domain deleted by PCR using primer sets, pGADT7F and pGADT7R, followed by EcoRI digestion and ligation to generate pGBKT7ΔBD. The S. cerevisiae rpl28 gene was amplified by PCR using the primer pairs ScRPL28F and ScRPL28R, then digested with ClaI and BamHI. pGBKT7ΔBD-rpl28 was created by subcloning this fragment into their sites. S. cerevisiae RPL28 was amplified using primer sets ScRPL28F and ScPRL28Q38MR and ScPRL28Q38MF and ScRPL28R, and then fused by PCR with primer sets ScRPL28F and ScRPL28R. This fragment was subcloned into the ClaI and BamHI sites, resulting in pGBKT7ΔBD-rpl28Q38M. The O. minor rpl27A gene was amplified using the OmRPL27AF and OmRPL27AR primer sets and ligated into the ClaI and BamHI sites to produce pGBKT7ΔBD-rpl28Q38M. The ADH1 promoter, rpl genes, and leu2 marker gene were amplified using primer sets RPL_leuF and RPL_leuR. In S. cerevisiae, the 5′ and 3′ franking regions of rpl28 were amplified using primer sets ScRPLbf500F and ScRPLbf500R and ScRPLaf500F and ScRPLaf500R, respectively. These three fragments were constructed using the primer sets ScRPLbf500F and ScRPLaf500R, which include 500 bp homologous to the flanking region of rpl28. The amplified DNA fragments were utilized to alter the Y2HGold strain of S. cerevisiae (Takara Bio, Shiga, Japan). Nested PCR was used to validate transformants in which the rpl28 locus was replaced with the inserted DNA pieces by homologous recombination. For rpl28-WT and rpl28-Q38M, primer sets F1 and R1 were used, while for O. minor rpl27A, primer sets F1 and R2 were used. The second PCR was amplified using primer sets F2 and R1 and F2 and R2. Supplementary Table S2 summarizes the primers utilized in this work.
8. Susceptibility test of yeast strains expressing rpl28 orthologs cycloheximideOvernight cultures of S. cerevisiae strains, which expressed rpl28 orthologs, were adjusted to an optical density (OD600) of 1.0 using MES buffer (pH 6.0). Subsequently, these cultures were serially diluted and spotted onto SD media lacking leucine, supplemented with varying concentrations of cycloheximide as indicated. Media were kept for 3 days at 30°C. Representative images of three biological replicates are shown.
Since actinomycetes are known as prolific producers of natural products with a wide range of biological activities,15) Actinomycetes were primarily screened from soil samples obtained in different locations in Japan to separate microorganisms providing inhibitors for seed germination of O. minor. A small quantity of dirt was combined with water before being diluted with an SDS-yeast solution. The solution was warmed at 40°C for 20 min to decrease bacteria and increase actinomycetes development. A portion of the solution was applied to the humic acid medium. For 5 days, the isolated strains were grown in a submerged culture with shaking at 30°C. Acetone extracts were made by adding an equivalent quantity of acetone to the culture broth and centrifuging the mixture. The produced acetone extracts, as well as the synthetic strigolactone GR24, were employed to treat Japanese O. minor seeds. Seeds were kept at 25°C for 7 days, and O. minor seed germination was studied under a microscope.
From 992 acetone extracts, 452 samples (45%) preventing O. minor seed germination were found. Thus, extracts were tested for selectivity for inhibition of seed germination of T. pratense, a host of O. minor; conidium germination of A. oryzae, a representative of eukaryotic microorganisms; and growth of E. coli, a representative of prokaryotic microorganisms. Samples that preferentially inhibited O. minor seed germination while having little influence on each other’s development were chosen. Samples with an E. coli growth inhibition rate of less than 52% relative to control conditions were selected, yielding 11 samples that met the criteria (Fig. 1). When 11 samples were extracted with ethyl acetate, the active chemicals from eight of them were found in the aqueous layer and three in the organic layer. The hydrophilic compound that inhibited seed germination at the lowest concentrations was derived from a strain similar to Streptomyces nojiriensis strain BCCO10_878 (identity, 98%) (data not shown), which is known to produce nojirimycin, which inhibited O. minor seed germination.12) That information implied that hydrophilic compounds might include nojirimycin. Therefore, we decided to focus on hydrophobic compounds and made progressive dilutions of three hydrophobic samples, nos. 226, 2336, and 2392, and chose sample no. 226, which affected seed germination the least (Fig. 2). The isolated microorganism’s 16S ribosomal DNA sequence is comparable to that of Streptomyces species (Table 1). As a result, the isolated strain was given the name Streptomyces sp. no. 226.
| Top hits of BLAST analysis | Identity (%) | |
|---|---|---|
| no. 226 | Streptomyces sp. strain KCB16C001 | 99% |
| Streptomyces tsukiyonensis strain: NBRC 14353 | 99% | |
| Streptomyces xanthocidicus strain: NBRC 13469 | 99% |
We optimized the culture conditions and proceeded with the purification of an inhibitor of O. minor seed germination from a 51.8 L culture of strain no. 226. The active compound was extracted using ethyl acetate, resulting in 7.2 g of dry material. We performed chromatography using Wakogel C-200 and Inertsil ODS-3 columns, which yielded 4.6 mg of the purified compound as described in the materials and methods. This compound exhibited a single peak with a retention time of 12.6 min and demonstrated inhibition of O. minor seed germination (Figs. 3a and b). ESI-MS analysis of the purified compound revealed a molecular ion [M+H]+ at m/z 282 (Fig. 3c). Further analysis through 1H-NMR, 13C-NMR, COSY, and distortionless enhancement by polarization transfer experiments indicated the presence of two methyl carbons, three ketones, and five CH2 groups in the purified compound. We searched the Streptomyces natural product database StreptomeDB3.016) and identified cycloheximide as a candidate compound that matched the criteria, with a mass of 281 (Fig. 3d). We compared 1H-NMR data of the purified compound with those of cycloheximide reported previously17) and 13C-NMR data of cycloheximide, which we analyzed in this study (Supplementary Table S3). Their spectra were identical, although there were some extra signals in the spectra data in the purified compound by comparing them to those of cycloheximide probably due to contaminants. Chromatograms of the purified compound alone (Fig. 4a), cycloheximide alone (Fig. 4b), and their mixture (Fig. 4c) displayed a single peak at a retention time of 12.6 min, indicating that the purified compound contained cycloheximide. The half maximal inhibitory concentration (IC50) of the purified compound for O. minor seed germination was determined to be 3.5 ng/mL, equivalent to that of cycloheximide, as demonstrated below.
We explored the reason why cycloheximide, a well-known translational inhibitor for Eukaryote,18,19) was identified despite preferentially selecting molecules that did not suppress fungal growth (Fig. 1). Cycloheximide had an IC50 of 2.6 ng/mL for inhibition of seed germination of O. minor and 2.5 µg/mL for that of conidial germination of A. oryzae (Fig. 5).
Cycloheximide has been known to inhibit seed germination of Triticum aestivum, Arabidopsis thaliana, and Striga hermonthica.20–22) Seed germination of S. hermonthica was inhibited to approximately 30% with 1 µM cycloheximide.21) Interestingly, seed germination of T. pratense was not inhibited by 100 ng/mL cycloheximide, which completely inhibited seed germination of O. minor. The mechanisms by which cycloheximide inhibits the seed germination of O. minor at concentrations as low as one-thousandth of that required to inhibit conidium germination in A. oryzae remains unknown.
S. cerevisiae has an IC50 of 0.3 µg/mL for cycloheximide, which is approximately 100 times greater than that of O. minor.23) We looked into the mechanism of action of cycloheximide in S. cerevisiae since genetic modification in Saccharomyces is easier than that in Aspergillus. Cycloheximide is known to inhibit translation by binding to the yeast ribosomal 60S subunit protein L28 (Rpl28) in S. cerevisiae.24) The changes that gave S. cerevisiae cycloheximide resistance occurred in two areas: Gly37/Gln38/His39 and Gly54/Lys55/Arg59, according to a thorough mutational investigation of RPL28 in S. cerevisiae.25) We found that only the 38th methionine of O. minor RPL27A was not conserved among the six amino acids described above by comparing them to the amino acid sequences of RPL28 of S. cerevisiae, RPL27A of an A. oryzae homolog, and RPL27A of an O. minor homolog (Fig. 6a). We created Saccharomyces strains that express the wild-type RPL28 (WT), RPL27A of O. minor (Om), and S. cerevisiae PRL28 with the 38th amino acid substitution of methionine (Q38M), allowing the expression of proteins at the endogenous rpl28 locus (Fig. 6b), in order to determine whether this amino acid change affects the IC50 value of cycloheximide in O. minor. Through homologous recombination, each expression cassette was inserted into the S. cerevisiae rpl28 locus (Fig. 6c). These three strains were cultivated, the cell numbers were adjusted, the medium was successively diluted, and the cells were spotted on it (Figs. 7a–e). All three strains developed uniformly in the absence of cycloheximide (Fig. 7a). The growth of WT and Q38M on cycloheximide-containing media reduced in a dose-dependent manner (Fig. 7e), while the growth of Om did not slow down even on medium containing 50 µg/mL cycloheximide (Figs. 7a–e). These findings show that O. minor RPL27A is not susceptible to cycloheximide and imply that cycloheximide may prevent O. minor seed germination via an unknown mechanism.
It has been widely accepted for more than 50 years that cycloheximide has the ability to inhibit the growth of fungi,19,26) algae,27) protozoa,28) and higher plants,29) but not of bacteria.25) Notably, cycloheximide prevents the production of proteins in both yeast and human cells.30,31) Our research mainly focused on screening actinomycetes for compounds that can efficiently inhibit the seed germination of O. minor without significantly affecting the seed germination of T. pratense, the conidium germination of A. oryzae, or the growth of E. coli. However, our research showed that the substance that specifically inhibited O. minor seed germination was cycloheximide. It is important to note that cycloheximide suppresses O. minor seed germination at a dose that is 1/1,000 times lower than what is necessary to prevent A. oryzae conidia from germinating. O. minor RPL27A in S. cerevisiae was subjected to functional investigation, which revealed that it seems to be insensitive to cycloheximide. So, in contrast to how it affects S. cerevisiae, our work provides the first evidence for the different processes by which cycloheximide limits the germination of O. minor seeds.
Extremely tiny Orobanche species seeds fall into two categories depending on how they are shaped. O. minor and Orobanche crenata seeds are categorized as type II seeds, which may vary in size from 0.27–0.57×0.14–0.41 mm2) and O. crenata seeds average 4.2 mg in weight per thousand.32) This is much less than the T. pratense seed weight of 2 g in weight per thousand.33) Therefore, in our screening procedure, a particular quantity of culture extracts could be more inhibitive to O. minor seed germination than to T. pratense seed germinations. We did not, however, expect that cycloheximide would be more successful in inhibiting O. minor seed germination than A. oryzae conidium germination (Fig. 5).
According to McGeachy, Meacham, and Ingolia (2019), the binding of cycloheximide to RPL28 is responsible for limiting yeast growth. We carried out studies (Fig. 7) to determine if the S. cerevisiae strains expressing O. minor RPL27A are susceptible to cycloheximide or resistant to it. The findings imply that O. minor RPL27A is not responsible for the lower IC50 for cycloheximide in O. minor compared to A. oryzae since strains expressing RPL27A are resistant to cycloheximide.
In yeast, pleiotropic drug resistance genes, which control the expression of multidrug-resistance transporter genes, transcription factors like PDR3,34) and the 20S proteasomal genes in cycloheximide-resistant mutants have all been linked to gene modifications that result in cycloheximide resistance.35) By increasing the transcription of PDR5, which encodes a potential membrane transporter, overexpression of PDR3 boosted cycloheximide resistance.34) In a different instance, proteasome-dependent proteolysis mutations cause an imbalance between protein synthesis and breakdown, which inhibits growth.36) According to Hanna et al., it is not necessary for yeast growth to be inhibited for all proteins to be synthesized and broken down. According to Hannah et al.,37) cycloheximide inhibits the buildup of certain proteins, notably ubiquitin, which results in a growth arrest mechanism.
Protein synthesis in plants is a labor-intensive metabolic process that consumes a substantial amount of the energy needed for plant development.38) On the other hand, according to Scheurwater et al.,39) 20 to 30% of the ATP created by root respiration is a result of protein breakdown. As a result, the disruption of the energy flow balance between synthesis and degradation caused by the restriction of protein synthesis may have an impact on plant development. Cycloheximide has been studied in Arabidopsis, where it has been discovered to stimulate the expression of many genes related to the abscisic acid (ABA) response, including hormone synthesis and response genes.40) However, the effect of cycloheximide on the growth of Orobanchaceae is still unknown. According to Kermode, ABA is known to inhibit seed germination and encourage seed dormancy.41) The increase of enzymes involved in defense metabolism and cell wall formation is similarly accelerated by cycloheximide in Pisum sativum.42) In Arabidopsis thaliana, it has been shown that the production of cell walls also promotes the defensive response and slows plant development.43) The combination of those factors may clarify the processes by which cycloheximide inhibits seed germination in O. minor, while it is still important to confirm if similar effects are present in Orobanche. In addition, the cycloheximide absorption effectiveness and accumulation rate need attention, while the specifics are not yet known. Clarifying these issues could help us get a more thorough grasp of how cycloheximide works in Orobanchaceae.
Cycloheximide has been identified as an inhibitor for the germination of O. minor seeds, derived from culture extracts of Streptomyces sp. no. 226. It is noteworthy that cycloheximide effectively suppresses the germination of O. minor seeds at a dose that is 1/1,000 times lower than the required amount to hinder the A. oryzae conidial germination. The interaction between the 38th glutamine of RPL28 in S. cerevisiae and cycloheximide is essential for translational inhibition. However, it appears that the O. minor ortholog, RPL27A, does not play a critical role in the inhibition of O. minor seed germination by cycloheximide.
We thank Professor Yukihiro Sugimoto and Professor Masaki Kuse for supporting this study.
This work was supported by the JST/JICA SATREPS (JPMJSA1607 to S.T.).
No potential conflict of interest was supported by the authors.
S.T. and T.K. conceived and designed the project. R.N., M.N., and R.I. conducted the experiments. K.K. conducted the part of chemical analysis. S.T wrote the paper. All authors read and approved the final manuscript.
The online version of this article contains supplementary materials (Figs. S1, S2) which are available at https://www.jstage.jst.go.jp/browse/jpestics/.
Composition of media and primers used in this study were summarized in a supplementary file.
Supplementary material is available at the Journal of Pesticide Science online.
