Bactericidal metabolites from Phellinus noxius HN-1 against Microcystis aeruginosa

Harmful algal blooms cause serious problems worldwide due to large quantities of cyanotoxins produced by cyanobacteria in eutrophic water. In this study, a new compound named 2-(3, 4-dihydroxy-2-methoxyphenyl)-1, 3-benzodioxole-5-carbaldehyde (Compound 1), together with one known compound, 3, 4-dihydroxybenzalacetone (DBL), was purified from Phellinus noxius HN-1 (CCTCC M 2016242). Compound 1 and DBL displayed activity against the cyanobacteria Microcystis aeruginosa with a half maximal effective concentration of 21 and 5 μg/mL, respectively. Scanning electron and transmission electron microscopic observations showed that the compounds caused serious damage and significant lysis to M. aeruginosa cells. qRT-PCR assay indicated that compound 1 and DBL exposure up-regulated the expression of gene mcyB and down-regulated the expression of genes ftsZ, psbA1, and glmS in M. aeruginosa. This study provides the first evidence of bactericidal activity of a new compound and DBL. In summary, our results suggest that compound 1 and DBL might be developed as naturally-based biocontrol agents.

Compound 2 was elucidated as 3, 4-dihydroxybenzalacetone (DBL) (   35 reported. Bactericidal activities of compound 1 and DBL. The experimental aim was to determine the inhibitory potency against the growth of M. aeruginosa by measuring the cell density after exposed to compound 1 and DBL for 72 h, (Fig. 2A). The two compounds exhibited bactericidal activities against an M. aeruginosa culture as the cell densities significantly decreased in comparison to that of the control. As the data shown in Fig. 2, DBL has more efficient anti-cyanobacterial activity against M. aeruginosa. The EC 50,72h values of compound 1 and DBL were 20.6 and 5.1 μg/mL, respectively. The algicidal assay indicated that the anti-M. aeruginosa activities of two compounds increased with the dosage. As shown in the Fig. 2, with the increase of concentration of DBL, the content of chlorophyll a was decreased gradually from 0.28 to 0.04 μg/mL at 72 h, which was 90.45% lower than that of the control. Compound 1 has little inhibitory effect on algae at low concentration, which was 0.37 μg/mL at 1 μg/mL, while the content of chlorophyll a was decreased to 0.07 μg/mL with the increasing concentration of compound 1 (200 μg/mL). According to the OD value, the EC 50 values of DBL and compound 1 were 5.86 and 18.24 μg/mL, respectively, which were close to the cell density test results. Based on the above, we conclude that compound 1 and DBL can inhibit the growth of M. aeruginosa in a dose-dependant manner.  and the content of O 2 •− increased from 0. 36 ± 0.001 μg/g 3 , which was higher than that of compound 1 with peak ratio of 0.36 ± 0.002 μg/g 3 . The content of O 2 •− in cells exposed to DBL and compound 1 were maximum value of 0.40 ± 0.001 and 0.39 ± 0.001 μg/g 3 at 72 h (Fig. 3A). Figure 3B shows effects of two compounds on the electric conductivity (EC) ratio. The EC ratio of DBL was 622 μS/cm initially and increased to 880 ± 6.03 μS/cm on 72 h, which was higher than that of compound 1 (745 ± 5.25 μS/cm). Compared with the control, DBL and compound 1 significantly effected on the EC ratios of M. aeruginosa.
Micro and ultrastructure changes of M. aeruginosa exposed to compound 1 and DBL. Our results demonstrated that compound 1 and DBL significantly affected the morphology of M. aeruginosa cells. Compared to the control cells (Fig. 4A), the morphological changes of the cells after exposure to 4 μg/mL of compound 1 and DBL were observed under SEM and TEM to evaluate the bactericidal mechanism of tested compounds on morphological micro and ultrastructures ( Fig. 4B and C). The M. aeruginosa cells appeared to be normal shaped as plump, and round with smooth exteriors in the control (Fig. 4D). After exposure to compound 1 or DBL, majority of M. aeruginosa cells exhibited obvious changed in morphology and lost their integrity. Figure 4E and F show that the cytoplasm became notably condensed and plasmolysis occurred in the cells. The untreated cell had complete cell wall and a basic structure, including a nuclear area, vesicle, and other cell organelles (Fig. 4G), whereas the exposed cells were disrupted and lysed. The compounds severely damaged the cell-walls and caused cell disruption, collapsed, perforation and content lysis ( Fig. 4H and I). DBL damage was more severe as loss of nuclear area and gas vesicle and is integration of cell architecture.

Effects on transcription level of M. aeruginosa genes. Based on the experiments of microscopic observation and determinations of chlorophyll a, electrical conductivity and superoxide anion O 2
•− , to further clarify the bactericidal mechanism on gene expression, we tested the key synthesis gene of chlorophyll a and related genes of cell membrane. The four targeted genes, including microcystin in several cyanobacterial generasynthesis genes mcyB 23 , cell division gene ftsZ, photosynthesis gene psbA1, and peptidoglycan synthesis gene glmS, were chosen to analyze the effects of the compound 1 and DBL on gene transcription. We detected the transcriptional expression changes of these genes of M. aeruginosa exposed to the two compounds (Fig. 5). Compared to the control, ftsZ, glmS and psbA1 genes were slightly down-regulated after 24 h, while expression was reduced significantly after 48 h exposures to compound 1. The mcyB was up-regulated and then reduced. The qRT-PCR analysis demonstrated that DBL increased the transcriptional expressions of mcyB then decrease it. Consequently, a decrease in ftsZ gene, psbA1 gene, and glmS gene, were observed. The results suggested that DBL seriously influenced the transcription of genes in M. aeruginosa.
In this study, we isolated two compounds from P. noxius HN-1. Based on the 1 H and 13 C NMR spectra the structure of compound 1 is similar to that of the known compound 2-(3′,4′-dihydroxyphenyl)-1,3-benzod ioxole-5-aldehyde isolated from Melissa officinalis 32 , differing in a methoxy group is replaced by H at C-11 (δ C 49.8). The known compound is 10-fold more active than ascorbic acid and is easily degraded into two molecules of protocatechualdehyde 32 . Accordingly a hypothesis is suggested that they probably have homogeneous activities.
DBL is a polyphenol derived from the medicinal fungus Chaga (Inonotus obliquus) in Japan, and is used as a folk medicine to treat cancers in Russia 33,34 . DBL has growth-inhibitory effects 35 and shows strong antioxidant activity in terms of both superoxide and hydroxyl radical scavenging activities 34 , suggesting the therapeutic effects of DBL. However, to our best knowledge, there is no report available on the bactericidal activity of DBL as a natural metabolite produced by P. noxius.
The present study is for the first time to show that compound 1 and DBL exhibit anti-cyanobacterial activities against M. aeruginosa with EC 50 values of 20.6 and 5.1 μg/mL. The differential effects of the two compounds may be due to their structural differences. It is similar to other previously reported compounds. It was shown that the EC 50 values of salcolin A and B isolated from Hordeum vulgare, were 6.0 and 9.6 μg/mL against M. aeruginosa 36 . The antialgal allelochemical ethyl 2-methylacetoacetate was isolated from Phragmites communis and with the The cell membrane is the target for many antimicrobial agents 37,38 and some electrolytes tend to leach out first, then large molecules such as DNA, RNA, and other materials leak out 39 . The release of intracellular components is a good indicator of membrane integrity 38,39 . In recent studies, it was suggested that some compounds, which act as an environmental stress, can increase the production of O 2 •− in cells 40,41 . O 2 •− is the precursor of active free radicals that have the potential for reacting with biological macromolecules inducing cell damage.
Exposure to compound 1 and DBL lead to increase of O 2 •− contents in M. aeruginosa cells, which may induce lipid peroxidation, indicated the leakage and release of electrolytes, nucleic acids, and proteins from the cyanobacteria and contribute to the increase of EC. Compound 1 and DBL belong to phenolic compounds which are similar to phenolic acid compounds, therefore we infer that target of these two compounds might be the cell membrane. Other report indicated that antioxidant enzyme (superoxide dismutase) activities and specific activities of A. flos-aquae were enhanced at the beginning of ρ-hydroxybenzoic acid and ferulic acid oxidative stress conditions 42 .
Although some substances have been reported to control M. aeruginosa, their inhibition mechanism remain unknown. Previous studies suggested that those compounds destroy cell structure, cause oxidative damage, and affect algal photosynthesis and enzymatic activities 6, 17, 43 . Zhang et al. 44 demonstrated that 2′-deoxyadenosine produced by Streptomyces jiujiangensis strain JXJ 0074 T led to severe crumpling, collapse, and perforation of M. aeruginosa, and a reduction in chlorophyll content. Bacilysin, isolated from B. amyloliquefaciens FZB42, acts against cell walls and also has significant anti-cyanobacterial effects 5 . In the present study, the morphometric analysis at the microstructural and ultrastructural levels by SEM and TEM indicate that compound 1 and DBL primarily affected the cell wall and increase cell permeability, leading to the efflux of intracellular components and eventually cell lysis. Based on the O 2 •− and EC contents assay, M. aeruginosa cell membrane was irreversibly damaged under the conditions of two compounds deoxidize stress.
To define the molecular bactericidal mechanism, the expression of microcystin peptide synthesis gene mcyB, cell division gene ftsZ, photosynthesis gene psbA1, and peptidoglycan synthesis gene glmS were analyzed by qRT-PCR. The expression abundance of these genes was reduced by compound 1 and DBL and the growth of M. aeruginosa was significantly suppressed. Our results are similar to the previous studies that also suggest that the transcript abundance of regulated genes were obviously reduced when M. aeruginosa under pyrogallol stress or algicidal bacterium stress 45,46 . The ftsZ gene encodes cell division protein FtsZ, which is essential to the cyanobacterium Synechocystis sp. PCC 6803 survival 47 . Combined with cell wall breakage, the decrease in the expression of genes ftsZ and glmS, indicates that membrane damage may be the bactericidal mechanism for DBL in M. aeruginosa cells. Compound 1 had less effect on the cell membrane than DBL.
M. aeruginosa, a toxic cyanobacterium, can produce microcystins. Microcystin formation is catalyzed by a complex multifunctional enzyme containing peptide synthetase (mcyABC) and hybrid polyketide-peptide synthetase (mcyDE) 48 . After M. aeruginosa cells were stimulated by compound 1 and DBL, the mcyB expression increased, which might be related to increase of the microcystin content caused by release of microcystin from dead M. aeruginosa cells. Dziga et al. concluded that the expression of mcyB is up-regulated under exposure to pyrogallol because of the release of hepatotoxin from dead Microcystis cells 49 , which increase microcystin content. Zhang et al. have also proved that the transcription expression of the microcystin synthetase gene is affected by ginkgolic acid 6 .
The photosynthetic gene expression is possibly regulated at the transcriptional level 50,51 . Some studies have indicated that the interruption of the electron transfer chain which affects photosynthetic processes, and oxidant damage may be the inhibitory mechanisms 6,45,49,52 . It has been known that PS II was sensitive to the environment 53 . The reduced abundances of psbA1 in PS II implies that the repair rate does not keep up with the damage rate and that compound 1 and DBL stress would interfere with electron transport. The psbA1 gene, the possible target for compound 1, was significant and rapid downregulated than that of DBL. It may be another factor in the effect on M. aeruginosa growth. This result is similar to other compound such as amoxicillin and levofloxacin hydrochloride that decrease PS II activity in Synechocystis sp 54,55 . Based on the qRT-PCR analysis, we suggest that the psbA1 gene is the potential binding site of compound 1 affecting algal photosynthesis. DBL multisite action, including releasing of microcystin, the cells membrane and cell structure damage, and reduction photosynthesis cause M. aeruginosa death. The morphological and molecular analysis results indicated that compound 1 and DBL might have different mechanisms against M. aeruginosa and we will study the protein expression changes in the future to clarify the bactericidal mechanism. In conclusion, compound 1 and DBL, isolated from P. noxius HN-1, show potent bactericidal activity and may be useful to mitigate harmful algal blooms in a synergistic manner.
Microorganisms. P. noxius strain HN-1 was isolated from brown root pathogens collected in Changjiang city, Hainan Province, China 21 , was cultured in potato dextrose agar (PDA) medium at 28 °C and stored in our lab (see Supplementary Fig. S1). The strain HN-1 was deposited in China Center for Type Culture Collection (CCTCC) (CCTCC M 2016242) (GenBank accession number KX592167).
Isolation and identification of the compounds. P. noxius strain HN-1 was cultured on PDA at 28 °C for 7 days. Two pieces of mycelial agar plugs (0.5 cm × 0.5 cm) were inoculated into 1 L Erlenmeyer flasks containing 400 mL potato dextrose broth (PDB). The cultivation was shaken at 120 r/min at 28 °C for 7 days, and then kept in still at 28 °C for 45 days. The culture broth (60 L) was filtered to give the filtrate and mycelia. The crude extract was reduced in vacuo to approximately 1 L and partitioned in succession between H 2 O and petroleum ether, ethyl acetate (EtOAc) and n-butyl alcohol 56 (20 mL) was centrifuged at 4000 × g for 20 min and then was homogenized with ice-cold phosphate buffered saline (PBS) (6 mL, 65 mM, pH 7.8), filtered with filter paper, and centrifuged at 5000 × g for 10 min at 4 °C. 2 mL supernatant was added to 1.5 mL PBS (65 mM, pH 7.8) and 0.5 mL hydroxylamine hydrochloride (10 mM), followed by incubation at 25 °C for 20 min. After that, 2 mL of the mixture was added to 2 mL sulfanilic acid (17 mM) and 2 mL α-naphthylamine (17 mM), incubated for 20 min at 25 °C. The samples were settled for 10 min at room temperature and was measured at 530 nm. O 2 •− was determined by using the following equation: M (μg/ g 3 ) = 2 × V t × n/(F W × V S ), n is concentration of NO 2 − (μg/mL), V t is total volume, F W is weight of sample, V S is the crude enzyme extract volume 59 . Cells microstructure and ultrastructural analysis. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis were used to test the influence of compound 1 and DBL. M. aeruginosa was cultivated at 25 °C under 60 μmol photons/(m 2 s) and a 12 h:12 h (light: dark) cycle. The cells were exposed to compound 1 and DBL with the final concentration of 4 μg/mL for 72 h, respectively. The fixed cells were collected by centrifugation, prefixed in 2.5% glutaraldehyde and washed three times in 0.1 M phosphate buffer for 10 min. Dehydration was done with a gradient series of ethanol. For SEM analysis samples, the cells were coated with gold, and examined with a Hitachi S-3000N SEM (Hitachi, Japan). For TEM analysis samples, the cells were postfixed in 1% osmium tetroxide for 1 h and dehydrated with a gradient series of ethanol. After dehydration, the samples were embedded in Epon 812 and sectioned with an ultramicrotome (LKB-V, Sweden). The sections were examined under a Hitachi H-600 TEM (Hitachi, Japan). Micrographs were taken at 10.0 kV 5 .
qRT-PCR Analysis. M. aeruginosa was exposed to 4 μg/mL compound 1, DBL or water as the control for 24 , 48 , 72 h. After incubation, the cells were collected by centrifuging at 10,000 rpm for 10 min at 4 °C. Total RNA was extracted with TRIzol reagent (Invitrogen, USA). cDNA was synthesized with the reverse transcriptase kit (TaKaRa Bio Inc, Dalian, China). qRT-PCR was performed with SYBR Premix Ex Taq (TaKaRa Bio) and an ABI 7500 Fast Real-Time PCR Detection System in a 20 μL volume. The conditions consisted of one cycle of 3 min at 95 °C followed by 40 cycles of 95 °C for 15 s, 56 °C for 30 s. Primers of target genes were listed in Table 2 Table 2. Primers designed for qRT-PCR analysis. Reference Wu et al. 5 reported.