Skip to main content
Download PDF

Contrasting regulation of live Bacillus cereus No.1 and its volatiles on Shiraia perylenequinone production

Microbial Cell Factories volume 21, Article number: 172 (2022) Cite this article

Abstract

Background

Fungal perylenequinones (PQs) are a class of photoactivated polyketide mycotoxins produced by plant-associated fungi. Hypocrellins, the effective anticancer photodynamic therapy (PDT) agents are main bioactive PQs isolated from a bambusicolous Shiraia fruiting bodies. We found previously that bacterial communities inhabiting fungal fruiting bodies are diverse, but with unknown functions. Bacillus is the most dominant genus inside Shiraia fruiting body. To understand the regulation role of the dominant Bacillus isolates on host fungus, we continued our work on co-culture of the dominant bacterium B. cereus No.1 with host fungus Shiraia sp. S9 to elucidate bacterial regulation on fungal hypocrellin production.

Results

Results from "donut" plate tests indicated that the bacterial culture could promote significantly fungal PQ production including hypocrellin A (HA), HC and elsinochrome A-C through bacterial volatiles. After analysis by gas chromatograph/mass spectrometer and confirmation with commercial pure compounds, the volatiles produced by the bacterium were characterized. The eliciting roles of bacterial volatile organic compounds (VOCs) on HA production via transcriptional regulation of host Shiraia fungus were confirmed. In the established submerged bacterial volatile co-culture, bacterial volatiles could not only promote HA production in the mycelium culture, but also facilitate the release of HA into the medium. The total production of HA was reached to 225.9 mg/L, about 1.87 times that of the fungal mono-culture. In contrast, the live bacterium suppressed markedly fungal PQ production in both confrontation plates and mycelium cultures by direct contact. The live bacterium not only down-regulated the transcript levels of HA biosynthetic genes, but also degraded extracellular HA quickly to its reductive product.

Conclusion

Our results indicated that bacterial volatile release could be a long-distance signal to elicit fungal PQ production. Biodegradation and inhibition by direct contact on fungal PQs were induced by the dominate Bacillus to protect themselves in the fruiting bodies. This is the first report on the regulation of Bacillus volatiles on fungal PQ production. These findings could be helpful for both understanding the intimate fungal–bacterial interactions in a fruiting body and establishing novel cultures for the enhanced production of bioactive PQs.

Graphical Abstract

Background

Shiraia bambusicola is a medicinally important fungus that parasitizes small bamboo branches and is widely distributed in Southern China and Japan [1]. Shiraia fruiting body has been traditionally used in Chinese medicine as “Zhu Huang” for the treatment of rheumatoid arthritis, tracheitis, stomachache and psoriasis [2]. Perylenequinones (PQs) are the main bioactive components from the fruiting bodies, including hypocrellins (HA-HD) and elsinochrome A (EA) [3]. HA and HB have been developing as new non-porphyrin photosensitizers for the photodynamic therapy on cancers [4], virus [5] and multidrug-resistant bacteria [6]. In the natural habitats, diverse bacteria live together with host fungus from the surface and internal tissues of fungal fruiting bodies. Fifty bacteria strains were identified from fruiting bodies of wild Himalayan Cantharellus spp. [7]. Fifty-five strains of bacteria including Bacillus, Lysinibacillus, Paenibacillus, Pandorea, Streptomyces, Alcaligenes and Pseudomonas existed in the fruiting body of Agaricus bisporus [8]. Pseudomonas fluorescens was found as the dominant bacterial species in the fruiting body of Cantharellus cibarius [9]. In our previous study, a total of 31 strains of bacteria were isolated from Shiraia fruiting bodies, whereas Bacillus was one of the dominant genera [10].

Most Bacillus species are isolated from bulk and rhizosphere soil, and some have been investigated as promising biocontrol agents of plant pathogens. Their efficacy for the plant protection is due to their biosynthesis of various bioactive secondary metabolites [11]. Some Bacillus strains released the antifungal cyclic lipopeptides to induce the generation of reactive oxygen species (ROS) and plant systemic resistance against phytopathogenic fungi [12, 13]. B. amyloliquefaciens SQR9 could exhibit different antifungal activities by the production of lipopeptides and siderophore bacillibactin [14]. Except such soluble bioactive secondary metabolites, a wide range of volatile organic compounds (VOCs) are also formed in bacilli. Kai (2020) reviewed the diversity and distribution of VOCs from B. subtilis [15]. There were totally 231 VOCs produced by 26 strains of B. subtilis including hydrocarbons, ketones, alcohols, aldehydes, ester, acids, aromatics and sulfur- and nitrogen-containing compounds. The released volatiles were found to inhibit fungal growth, promote plant growth and induce plant systemic resistance [16, 17]. The VOC benzaldehyde and 1,3-butadiene inhibited significantly the bacterial growth and motility of Ralstonia solanacearum and triggered salicylic acid pathway to induce the systemic resistance of tobaccos [18]. The mixture of VOCs produced by endophytic B. tomentosa VM11 have biocontrol activity against necrotrophic fungus Sclerotinia sclerotiorum via inhibiting mycelial growth and decreasing sclerotia production [19]. Although the high abundance of Bacillus was found to be one of the most frequent bacterial communities in fungal fruiting bodies [8, 20], the bioactivity and the physiological roles of Bacillus in fruiting bodies were scarcely reported. The spore-forming Bacillaceae were found during the maturation process of Tuber borchii fruiting bodies and their bacterial cellulolytic, chitinolytic activities were more likely to be involved in the ascus opening [21]. Xiang et al. (2017) reported that Bacillus inside the fruiting body of A. bisporus had broad-spectrum antimicrobial activities and potential mushroom growth-promoting abilities [8]. In our previous studies on Shiraia fruiting body [10], 31 culturable bacteria were isolated from the fruiting bodies, among which 14 Bacillus isolates were found. Illumina high-throughput sequencing results showed that Bacillus was the most dominant genus in the fruiting body. All Bacillus isolates exhibited various degrees of suppression on fungal red PQ pigments from the mycelium in the confrontation test [10]. Out of 14 isolates, B. cereus (No.1, No.15), B. safensis (No.18) and B. anthracis (No.19) were able completely to inhibit the fungal HA accumulation. However, there has been so far neither report concerning effects of Bacillus strains on the growth of host fungus Shiraia, nor regarding its regulation on biosynthesis of fungal PQs. To understand the regulation role of the fruiting body associated Bacillus on host fungus, we continued our work to investigate the relationship between B. cereus No.1 and host fungus Shiraia sp. S9 previously isolated from the fruiting body [10, 22]. This work has been undertaken to compare the effects of live bacterium No.1 and its volatiles on Shiraia PQ production. The eliciting role and characterization of the bacterial volatiles were investigated. Based on the impact of VOCs, a novel submerged volatile co-culture of Shiraia with B. cereus No.1 was established for the enhanced HA production. Simultaneously, the inhibitory mechanism of the live bacterium on HA was also revealed preliminarily. This is the first report on the elicitation role of Bacillus VOCs on fungal PQ production.

Results

Effects of live Bacillus and its volatiles in solid culture

An in vitro confrontation bioassay between the host fungus Shiraia sp. S9 and bacterium B. cereus No.1 were conducted (Fig. 1A). After 8 days of the co-culture, the accumulation of PQ pigments was significantly suppressed by the direct contact (Fig. 1B). The hyphal growth diameter showed an obvious suppression from day 4–8 compared with the control group (Fig. 2A, B). PQs including HA, HC, EA and EB were inhibited under the direct contact of live No.1, about 27.8%, 40.1%, 54.4%, 67.5% decrease of that in the control group (fungal mono-culture) (Fig. 2C, D). Among the PQs, the content of HA was suppressed most significantly (Fig. 2D). The HA content in hyphae was inhibited to the lowest content (0.56 mg/cm2) on day 8 (Fig. 2E).

Fig. 1
figure 1

The mode diagrams and plate cultures for the confrontation tests of the live bacterium Bacillus cereus No.1 and its host fungus Shiraia sp. S9 (A, B). The mode diagrams and the "donut" plate for the bacterial volatiles and the fungus S9 (C,D). The cultures were maintained on PDA plate at 28℃ for 8 days

Full size image
Fig. 2
figure 2

Effects of live B. cereus No.1 on the fungal growth (A), colony diameter (B), individual PQ in HPLC chromatogram (C), PQ contents (D) and HA content (E) of Shiraia sp. S9. The co-culture was maintained on PDA plate at 28 °C for 8 days. Values are mean ± SD from three independent experiments. (*p < 0.05, **p < 0.01 versus fungal monoculture as the control group)

Full size image

A "donut" plate assay (Fig. 1C) was conducted for the investigation of the bacterial volatile-mediated effects on fungus S9. It was an interesting finding that the bacterial volatiles could promote the secretion of red PQ pigments in the plates (Fig. 1D). The contents of PQs including HA, HC, EA, EB and EC were enhanced significantly, about 4.74-, 4.67-, 5.93-, 2.81-, 7.00-fold higher than that of the control group, respectively (Fig. 3A, B). However, the bacterial volatiles shortened the distance of hyphal branches (Fig. 3C–E) and suppressed fungal conidiation (Fig. 3F).

Fig. 3
figure 3

The effects of bacterial volatiles on the fungal growth and PQ production of Shiraia sp. S9. The fungus was kept on PDA treated with bacterial volatiles for 7 days. The chromatogram of individual PQs in mycelium (A), and the PQ contents in mycelium (B) were measured. The mycelial morphology was observed (C and D, × 400). The length between mycelial branches (E) and conidium concentration of S9 were measured on day 7 (F). The bacterial suspension and fungal mycelial plugs were inoculated simultaneously. Values are mean ± SD from three independent experiments. (**p < 0.01 versus fungal monoculture as the control group)

Full size image

Identification of bacterial VOCs and their effects on fungal HA production

A total of 34 VOCs were detected from B. cereus No.1 by GC–MS (Fig. 4A) and listed in Table 1 for 13 compounds (a relative area of more than 1.0% and more than 70% identity), and Additional file 1: Table S1 for other compounds (a relative area of less than 1.0% or less than 70% identity). The most abundant volatile metabolites were identified as ketones, alkanes, aldehydes, esters, alcohols and sulfides. The tested commercial VOCs at 5 mg/mL exhibited no significant influence on the fungal growth (Fig. 4B), but fungal HA production was promoted markedly by most of them (phenylacetaldehyde, dimethyl disulfide, phenylethyl alcohol, hexadecane and benzaldehyde) (Fig. 4C). The content of HA in hyphae reached 1.28 mg/cm2 on day 5, about 4.06 times that of the control group under the treatment of dimethyl disulfide.

Fig. 4
figure 4

The VOC identification and their effects on the fungus S9. The total ion current diagram of VOCs from No.1 after 24-h culture (A). Main peaks: 1, dimethyl disulfide (tR 4.03 min); 2, styrene (tR 7.88 min); 3, benzaldehyde (tR 10.09 min); 4, phenylacetaldehyde (tR 12.71 min); 5, phenylethyl alcohol (tR 14.84 min); 6, hexadecane (tR 28.09 min). Effects of main VOCs on fungal growth diameter (B) and HA content (C). Different VOC standards were added at 5 mg/mL on day 3 of the plate cultures of fungus S9 at 28 ℃ and the measurement was token after 2 days of the treatment. VOCs such as phenylacetaldehyde (PAA), hexadecane (HD), benzaldehyde (BD), phenylethyl alcohol (PEA), styrene (SN) and dimethyl disulfide (DMDS) were selected. Values are mean ± SD from three independent experiments. (*p < 0.05, **p < 0.01 versus fungal monoculture as the control group)

Full size image
Table 1 The analysis of VOCs from strain No.1 by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS–SPME–GC–MS)
Full size table

Effects of the bacterium on Shiraia growth and HA production in liquid culture

We continued to investigate the effects of the live bacterium on HA production in mycelium culture. After the application of bacterium No.1 at 50–500 cells/mL to 3-day-old fungal cultures, the fungal biomass was not altered during the culture (Fig. 5A). The content of HA was suppressed markedly by live bacterium at a density of 50–500 cells/mL (Fig. 5B). When No.1 at 500 cells/mL was added, the fungal biomass was suppressed only under the addition on day 0 and 1 (Fig. 5C). The addition of the bacterium in early stages (day 0–3) caused the full suppression of HA production (Fig. 5D). We chose the addition at 500 cells/mL on day 4 of initial culture for the time-course investigation. We found the addition on day 4 had no effects on the fungal biomass (Fig. 5E), whereas the accumulation of HA dropped rapidly to 0.02 mg/g dry weight (DW) after 24 h, but rose to the control level gradually (Fig. 5F).

Fig. 5
figure 5

Effects of live bacterium No.1 at 50–500 cells/mL on fungal biomass (A) and HA content (B) in liquid cultures. Live No.1 was added on day 3 of the mycelium culture (8 days). The effects of different adding time (day 0–7) on fungal biomass (C) and HA content (D) were also measured. Time profiles of fungal biomass (E) and HA content in mycelium (F) under the bacterial treatment. Live No.1 at 500 cells/mL was added on day 4 of the mycelium culture (8 days). The arrow represents the addition time of live No.1. Values are mean ± SD from three independent experiments (*p < 0.05, **p < 0.01 versus fungal monoculture as the control group). Different letters above the bars mean significant differences (p < 0.05)

Full size image

To analyze the influence of the bacterial VOCs on HA production in liquid culture, a submerged volatile co-culture was established (Fig. 6A). In this culture system, the flask containing bacterial suspension at 500 cells/mL was connected with fungal culture on day 0, 3, 6. The fungal biomass of the culture was not altered (Fig. 6B). However, the treatment could not only promote HA production in the mycelium culture (Fig. 6C), but also facilitated the release of HA into the medium after the connection on day 3 (Fig. 6D). The total production of HA reached the maximum 225.9 mg/L on day 8, about 1.87 times that of the control group (Fig. 6E).

Fig. 6
figure 6

Effects of bacterial volatiles on fungal growth and HA production in the submerged volatile co-cultures (A). The fungal dry biomass (B), HA contents in mycelium (C), the released HA in cultural broth (D) and total HA production (E) were measured in the culture. Two culture flasks were connected through sealed glass tube. The culture was maintained in 250-mL flask containing 100 mL of the liquid medium at 150 rpm and 28 ℃ for 8 days. An equal volume of sterile LB broth instead of bacterial suspension added to flask was used as control group. Values are mean ± SD from three independent experiments. Different letters above the bars mean significant differences (p < 0.05)

Full size image

Degradation of HA by live bacterium

In order to find out the reason why fungal HA was decreased in the presence of live bacterium, we applied live No.1 to culture medium containing standard HA (Fig. 7A). Live bacterium at 500 cells/mL could rapidly reduce HA from the initial content 62.3 mg/L to 16.3 mg/L in the medium within 24 h, and the percentage of HA degradation was approximately 74.8%. HA degradation by live No.1 was also confirmed in the HPLC chromatogram (Fig. 7B), where a new peak for the degradation product was found at the retention time of 10.2 min. The possible degradation product in the medium was identified by UPLC-Q-TOF–MS/MS. Compared with the standard HA with a molecular formula of C30H26O10 with [M + H] + ion peaks at m/z 547.1621 (Fig. 7C), the suggested molecular formula of degraded product was C30H28O10 with [M + H] + ion peaks at m/z 549.1767 (Fig. 7D). The results suggested that the HA could be hydrogenated and reduced by live No.1 into HAH2 (Fig. 7E).

Fig. 7
figure 7

The degradation of HA by B. cereus No.1. The degradation kinetic (A) and HPLC chromatogram (B) of standard HA by the bacterium. MS and MS2 spectra scan relative to HA standardat 40 mg/L (C) and its degradation products treated with live bacterium No.1 after 48 h (D). Structures of HA and a possible degradation product HAH2 (E). The live bacterial No.1 was added to the culture medium containing HA (40 μg/mL) and kept at 28 ℃ in the dark at 150 r/min for 2 days

Full size image

Effects of bacterial components on Shiraia growth and HA

Four different bacterial components including bacterial ethyl acetate extract (EAE), crude bacterial polysaccharide (BPS), bacterium No.1 in dialysis tubes and live bacterium were applied to Shiraia mycelium culture on day 3. The different concentrations of EAE and BPS were compared (Fig. 8A–D). The results revealed that these extracts had no effect on the fungal biomass (Fig. 8A, C), but EAE ≥ 50 mg/L and BPS at 100 mg/L could suppress significantly the mycelial content of HA (Fig. 8B, D). Although the fungal biomass was not altered in the cultures under the various treatments (Fig. 8E), the live No.1 and bacterial extracts (EAE and BPS) in the cultures suppressed the HA biosynthesis (Fig. 8F). The inhibitory effect of live No.1 on HA was the most significant and the content of HA was reduced by 56.2% to 0.13 mg/g DW.

Fig. 8
figure 8

Effects of live bacterium of B. cereus No.1 and the bacterial components on fungal growth and HA production of Shiraia sp. S9. The effects of different concentration of crude polysaccharides (BPS) and ethyl acetate extract (EAE) on fungal biomass (A, C) and HA contents in the mycelium (B, D). Effects of live bacterium at 500 cells/mL, BPS at 100 mg/L, EAE at 200 mg/L and bacterium in the dialysis tubing on fungal biomass (E) and HA contents (F) in mycelium culture. The live No.1, EAE, BPS and bacterium in the dialysis tubing were added on day 3 of the 8-day-culture of the fungus S9. Fungal monoculture was used as the control group. Values are mean ± SD from three independent experiments (*p < 0.05, **p < 0.01 versus control groups). Different letters above the bars mean significant differences (p < 0.05)

Full size image

Effects of No.1 on key gene expression for HA biosynthesis

After 8 days of the treatments including live bacterium and bacterial volatiles in the plates, seven HA biosynthesis-related genes including multicopper oxidase (MCO), polyketide synthase (PKS), monooxygenase (Mono), FAD/FMN-dependent oxidoreductase (FAD), O-methyltransferase (Omef), zinc finger transcription factor (ZFTF) and ATP-binding cassette (ABC) were quantified by using qRT-PCR (Fig. 9). The expression of all genes related to HA biosynthesis were down-regulated by live bacterium No.1 in direct contact, about 6.6-, 2.0-, 3.7-, 1.5-, 2.2-, 5.3- and 3.7-fold of the control group, respectively. On the other hand, the bacterial volatiles showed an opposite way on the expression of HA biosynthetic genes, up-regulating the expression of most tested genes including Mono, Omef, MCO, FAD and PKS.

Fig. 9
figure 9

Effects of live B. cereus No.1 and its volatiles on the expression of HA biosynthetic genes of Shiraia sp. S9 in solid culture. The confrontation tests between live bacterium or bacterial volatiles with the fungus S9 were the same as specified in Fig. 1. The cultures were maintained on PDA plate at 28℃ for 8 days. Values are mean ± SD from three independent experiments. (*p < 0.05, **p < 0.01 versus fungal monoculture as the control group)

Full size image

Discussion

Shiraia fruiting bodies are main resources for Chinese folk medicine (Zhu Huang) and the important photosensitizer hypocrellins for photodynamic therapy [23]. Shiraia-associated microbial communities are rich and diverse in the fruiting bodies or inside bamboo tissues. The associated fungi were isolated from the fruiting bodies, shoots and leaves of fresh bamboos (Phyllostachys sp., Sasa sp. and Brachystachyum densiflorum) [24,25,26,27]. Yan et al. (2021) isolated 17 endophytic fungi from the bamboo branches with fruiting bodies of S. bambusicola and the endophytic Arthrinium sp. AF-5 was found to stimulate mycelial growth and hypocrellin production of S. bambusicola. [28]. Phoma sp. BZJ6, an endophytic fungus could promote laccase from S. bambusicola [27]. In the fruiting bodies, the host Shiraia and bacteria were found living together [10] and one of fruiting body-associated bacteria P. fulva SB1 stimulated fungal PQ production [22]. These results indicated that Shiraia-associated microbes may develop intimate interactions with Shiraia on regulation of the fungal metabolites. In this study, the live dominant bacterium B. cereus No.1 and its volatiles exerted the contrasting effects on Shiraia PQ production. The contents of PQs including HA, HC and EA-EC were increased by about 2.81–7.00-fold under the treatment of bacterial volatiles (Fig. 3A, B), whereas the live bacterium in direct contact had inhibitory effects on fungal PQ production in both the confronting test (Fig. 2) and the mycelium co-cultures (Fig. 5). To date, there is scarce report on volatile-mediated promotion and the contacting suppression by the same bacterium on fungus. P. aeruginosa, the most frequently colonizing bacterium in chronic lung infection was associated with the fungal pathogens Aspergillus fumigatus. The bacterium was found to inhibit conidial germination and biofilm formation of A. fumigatus by direct contact [29]. However, bacterial VOCs released by P. aeruginosa had the stimulatory effects on the fungal growth [30]. We reported for the first time that the volatiles released by Bacillus had stimulatory effects on fungal PQ production. Since the dominant bacterium B. cereus No.1 and the host fungus Shiraia sp. S9 shared the same habitat in the fruiting bodies, these results suggested the existence of competitive or even more complex interactions between the bacteria and fungi in fruiting bodies.

HA, the main hypocrellin from Shiraia fruiting bodies has a photodynamic antibacterial activity against both Gram-positive Staphylococcus aureus, B. subtilis and Gram-negative Escherichia coli, Salmonella typhimurium [31, 32]. The antibacterial activities are contributed by HA-induced ROS targeting to bacterial cell wall and membranous enzymes. HA was synthesized in Shiraia cells, but could be released to extracellular space [22], leading to the bacterial inability to grow around the host fungi. Usually, bacteria release VOCs as defensive weapons to antagonize fungi at a long distance [33]. Inhibition on mycelium growth or spore germination was reported most frequently by bacterial VOCs [19, 34]. In our study, Shiraia conidiation was suppressed and the distance of hyphal branches was decreased by volatiles of B. cereus No.1 (Fig. 3), indicating a slight stress applied to the fungus. The volatiles triggered the upregulation of various genes of PQ biosynthesis gene cluster (Fig. 9), including FAD, MCO, Mono, Omef and PKS [35, 36]. The enhanced transcript levels were responsible for the induced PQs (HA, HC and EA-EC) by the volatiles. The main VOCs from the bacterium are aldehydes, ketones, alkanes, sulfides, esters and alcohols (Table 1 and Additional file 1: Table S1). Some VOCs such as phenylacetaldehyde, dimethyl disulfide, phenylethyl alcohol, hexadecane and benzaldehyde showed the significant promotion on the fungal HA accumulation (Fig. 4C). The VOCs were proved to be responsible for the unexpected stimulatory effects on Shiraia hypocrellins. These results also demonstrated that the fungus may boost up its antibacterial activity via the accumulation of PQs in response to the bacterial volatile-mediated antagonism. On the other hand, this bacterial-fungal interaction could be harnessed to improve biotechnological production of important fungal metabolites. Based on the stimulatory effects of VOCs by edible mushroom Pleurotus ostreatus on exopolysaccharide production of the medicinal fungus Ganoderma lucidum, Asadi et al. (2021) established a novel submerged volatile co-culture system in which the specific yield of exopolysaccharides was increased about 2.2-fold by 6-day treatment of P. ostreatus VOCs [37]. Currently, versatile systems for microbial mono- and co‑cultures have been constructed for production of value‑added compounds [38, 39]. In this study, we also made an effort to establish the submerged volatile co-culture system (Fig. 6). This is the first report on the use of bacterial volatiles as a novel elicitor to simulate PQ production in mycelium cultures. With the optimization on medium components, culture condition and other bioprocess parameters, the production of fungal metabolites in the submerged volatile co-culture system could be enhanced greatly.

In contrast to the enhanced Shiraia PQ production by the elicitation of the bacterial VOCs, significant inhibition on PQ production was observed in situations of direct contact of live bacterium No.1 and Shiraia sp. S9 in both solid plates (Fig. 2) and mycelium cultures (Fig. 5). The suppression on the fungal growth was obvious by direct contact with the live bacterium after day 4 on the cultural plates (Fig. 2B). The bacterial extracts (BPS and EAE) were also proved to have the inhibitory effects on fungal HA production in the mycelium culture (Fig. 8), suggesting that not only direct contact but some bacterial cell components could contribute to the suppression. In our study, the expressions of all tested genes for PQ biosynthesis were down-regulated by live bacterium in solid culture (Fig. 9). This is in agreement with the finding that B. megaterium inhibited fungal aflatoxin and cyclopiazonic acid production of A. flavus through down-regulating the pathway gene expression [40]. On the other hand, live bacterium No.1 could quickly degrade HA in the culture medium by 74.8% within 24 h (Fig. 7A) and conversed it to a possible HAH2 by the transfer hydrogenation reduction (Fig. 7C–E). Therefore, it can be suggested that the bacterial degradation capacity was linked to the antagonistic action of the bactericidal HA produced by Shiraia. The bacterial degradation mechanism and degraded metabolites need further investigation. As photoactivated PQ toxins are involved in the fungal pathogenicity of Cercospora, Mycosphaerella, and Alternaria in plant diseases [41], our results also indicated that the B. cereus No.1 hold a promise as potential biocontrol agent due to its inhibition of PQ biosynthesis and its biodegradation capacity.

Conclusion

In summary, this study presented the first assessment of dual effects of a fruiting body-associated bacterium (B. cereus No.1) on metabolite production (PQs) of host fungus: promotion by bacterial VOCs and inhibition by direct contact. Shiraia fruiting bodies host a wide diversity and high abundance of bacteria and produce pharmaceutically and agriculturally important PQs such as hypocrellins and elsinochromes. It is an interesting finding that VOCs from one of the predominant Bacillus strains (No.1) stimulated PQ biosynthesis via transcriptional regulation. The study showed unexpectedly that host fungus could interact with the associated bacteria by PQ production at a distance via sensing bacterial VOCs. Based on this impact of VOCs, a novel submerged bacterial volatile co-culture was constructed for biotechnological production of the important photosensitizer HA. In contrast, the live bacterium not only down-regulated the transcript levels of HA biosynthetic genes, leading to suppression on HA accumulation by direct contact, but also degraded extracellular HA quickly. Although bacterial signal compounds and VOCs perception mechanisms in fungus need to be further elucidated, our study provided a new understanding of specific cues of bacterial VOCs in bacteria-fungi interactions. These findings could be harnessed for the potential biotechnological application of VOCs in submerged co-culture for PQ production and agricultural application for the live bacterium as a biocontrol agent for plant diseases.

Material and methods

Strains, media and culture conditions

The PQ-producing strain Shiraia sp. S9 and its associated bacterium B. cereus No.1 used in this study were isolated in our Lab [10] and deposited at the China General Microbiological Culture Collection Center (CGMCC) with registered number CGMCC16369 and 21176, respectively. The strain of Bacillus (B. cereus No.1) selected for this study was based on our previous report [10] for the inhibition of hypocrellin production by bacteria associated with the fruiting body. The physiological, biochemical characteristics and 16S rDNA gene sequence analysis were used for the identification of the stain [10]. The phylogenetic tree of B. cereus No.1 was presented in Additional file 1: Fig. S1. The fungus strain S9 was stored at 4 °C on a potato dextrose agar (PDA) plate and bacterial strain No.1 was conserved on Luria–Bertani (LB) agar plate at 4 °C. To initiate the liquid culture, S9 was cultured on a PDA plate in the dark for 8 days at 28 °C. The seed culture preparation, the medium component and conditions for the liquid cultures were in the same as described in our previous report [42]. Then, 5 mL of the seed broth was transferred into a 150-mL flask with 50 mL liquid medium and was incubated at 28 °C and 150 rpm on a rotary shaker (ZD-8802, Hualida, Suzhou, China).

Co-cultures of live bacterium and its volatiles with Shiraia sp. S9

The confrontation bioassay in vitro between the fungus S9 and the bacterial strain No.1 was conducted according to our previously reported method [22]. Mycelial plug (5-mm-diameter) was taken from the margin of 8-day-old culture and placed on the center of a 90 mm PDA Petri dish (Fig. 1A). After 24 h of incubation, 10 μL of bacterial suspension (105 cells/mL) was streaked in parallelly on both sides of the PDA, approximately 7 cm apart from each other. The equivalent LB broth without the bacterium was used as the control group. The “donut” plate assay was used to measure the effects of bacterial VOCs on the fungus with a slight modification [30]. A smaller 60-mm petri dish lid was placed upside down in a 90-mm petri dish to separate physically the bacterial media in the outside ring and fungal media in the inside (Fig. 1C). A volume of 12 mL LB media was pipetted into the outer ring and 10 mL of PDA media was added to the inside. The bacterial suspension (105 cells/mL) of 15 μL after 24 h of cultivation was inoculated on LB medium and the fungal mycelial plugs of 5-mm-diameter cultured for 8 days were inoculated on the PDA medium simultaneously. The diameter of fungal growth and HA content were measured when the co-culture was kept for 2–7 days at 28°C in a constant temperature incubator (GNP-9080BS-III, CIMO, Shanghai, China).

The submerged volatile co-culture (Fig. 6A) was established to determine the effects of bacterial VOCs on the fungus in liquid culture according to the report by Asadi et al. [37]. This co-culture system was composed of a 250 mL culture flask for fungus S9 and one 250-mL culture flask for bacterium containing 100 mL LB broth. Two flasks were connected through a glass tube. The S9 seed culture (10 mL) was added into a 250-mL flask with 100 mL liquid medium. After 24 h of cultivation at 37°C, bacterial suspension at a final density of 500 cells/mL was added to the flask for bacterial volatile producer on day 0, 3, 6 of the fungal culture and connected to the flask for mycelial culture. Then, the co-cultures were kept on a rotary shaker at 150 rpm at 28 °C for 8 days. The equivalent volume of fresh LB broth without inoculating bacterium was added to the flask as a control.

Elicitation of bacterial components on fungal PQ production

To compare effects of bacterial extracts and live bacterium on fungal PQ production of Shiraia sp. S9, ethyl acetate extract (EAE) from the bacterial fermentation broth, bacterial polysaccharides (BPS) and live bacterium in dialysis tubes were prepared according to our previous study [22]. The mycelium cultures were added with live bacteria No.1 (500 cells/mL), EAE (10–200 mg/L), BPS (10–100 mg/L), and 2 mL of live bacteria in dialysis tubes (5 × 103 cells/mL), respectively on day 3 and kept on a rotary shaker at 150 rpm at 28 °C for another 5 days. The mono-culture of S9 treated with the equal volumes of sterile LB broth was used as the control group.

Analysis of bacterial VOCs

VOCs from B. cereus No.1 after 24 h of incubation at 28℃ were collected and analyzed as described by Li et al. (2010) [43]. Briefly, the VOCs were determined by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC–MS, Agilent 6890N-5975B, American) equipped with an HP-5MS column (Agilent, CA, USA). Mass spectra were obtained using the scan modus (total ion count, 50–500 m/z). Compounds were identified by comparison of mass spectra and retention times with those of available standards in the library of the National Institute of Standards and Technology (NIST).

PQ extraction and quantification

The fungal PQs in PDA and mycelium culture of Shiraia sp. S9 were extracted according to our previous study [44]. The PQ contents were measured by the reverse-phase Agilent 1260 HPLC system (Agilent Co., Wilmington, DE, USA) equipped with the Agilent HC-C18 column (Santa Clara, USA) with a mobile phase of acetonitrile: water (65:35, v/v).

Bacterial degradation on fungal HA

For the degradation activity of B. cereus No.1 on fungal HA, the standard HA (purity > 98%, Chinese National Compound Library, Shanghai, China) was added to LB medium (50 mL) in flask at 40 μg/mL and bacterial suspension (500 cells/mL) of live No.1 after 24 h-incubation was inoculated into culture medium. Then, this mixture was subsequently incubated at 28℃ in the dark at 150 r/min for 2 days and the culture medium without bacterial addition was used as a control group. The remnant HA and degradation products in the liquid medium were analyzed by UPLC-Q-TOF–MS/MS. The samples were extracted by using equal volume ethyl acetate, subsequently dried and re-dissolved in absolute ethanol and filtered with a 0.45 μm organic phase filter. HPLC was performed on a system equipped with an Agilent HC- C18 column (250 × 4.6 mm, 5 μm) with a mobile phase (acetonitrile: water at 65: 35) at 1 mL/min. High-resolution mass spectral analysis was executed on Agilent 6538A Q-TOF mass spectrometer equipped with an electrospray ion (ESI) source in positive mode. The capillary voltage and fragmentor voltage were maintained at 3.5 kV and 135 V respectively. The flow rate of drying gas was running at 350℃ at 10 L/min. The constituents of samples were detected by using full-scan MS analysis from m/z 50–1700. The collision energy of HA and degradation products was 30 eV.

Quantitative real-time PCR analysis

RNA samples from the fungal mycelia were obtained using the RNAprep pure Plant Kit (Tiangen, Beijing, China). Specific primers for each gene were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA) and listed in Additional file 1: Table S2. The method of real-time quantitative PCR (RT-qPCR) was based on previous research report [45] and performed using CFX96-C1000 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative transcript levels were determined according to the 2−ΔΔCt method described by Zhang et al. (2016) [46].

Statistical analysis

Student's t-test was used as a significance test to compare the means between two groups. One-way analysis of variance (ANOVA) was performed using statistical analysis system (SAS 9.2, Cary, USA) to assess the significance of differences of the means among multiple groups. All results are expressed as Mean ± Standard Deviation (SD). The level of significance was set at p < 0.05.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its Additional files.

References

  1. Cheng TF, Jia XM, Ma XH, Lin HP, Zhao YH. Phylogenetic study on Shiraia bambusicola by rDNA sequence analyses. J Basic Microbiol. 2004;44:339–50.

    Article  CAS  PubMed  Google Scholar 

  2. Zhong JJ, Xiao JH. Secondary metabolites from higher fungi: discovery, bioactivity, and bioproduction. Adv Biochem Eng Biotechnol. 2009;113:79–150.

    CAS  PubMed  Google Scholar 

  3. Khiralla A, Mohammed AO, Yagi S. Fungal perylenequinones. Mycol Prog. 2022;21:38.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Zhang J, Cao EH, Li JF, Zhang TC, Ma WJ. Photodynamic effects of hypocrellin A on three human malignant cell lines by inducing apoptotic cell death. J Photochem Photobiol B. 1998;43:106–11.

    Article  CAS  PubMed  Google Scholar 

  5. Hirayama J, Ikebuchi K, Abe H, Kwon KW, Ohnishi Y, Horiuchi M, Shinagawa M, Ikuta K, Kamo N, Sekiguchi S. Photoinactivation of virus infectivity by hypocrellin A. Photochem Photobiol. 1997;66:697–700.

    Article  CAS  PubMed  Google Scholar 

  6. Guo L-Y, Yan S-Z, Tao X, Yang Q, Li Q, Wang T-S, Yu S-Q, Chen S-L. Evaluation of hypocrellin A-loaded lipase sensitive polymer micelles for intervening methicillin-resistant Staphylococcus aureus antibiotic-resistant bacterial infection. Mat Sci Eng C. 2020;106: 110230.

    Article  CAS  Google Scholar 

  7. Kumari D, Reddy MS, Upadhyay RC. Diversity of cultivable bacteria associated with fruiting bodies of wild Himalayan Cantharellus spp. Ann Microbiol. 2013;63:845–53.

    Article  CAS  Google Scholar 

  8. Xiang Q, Luo L, Liang Y, Chen Q, Zhang X, Gu Y. The diversity, growth promoting abilities and anti-microbial activities of bacteria isolated from the fruiting body of Agaricus bisporus. Pol J Microbiol. 2017;66:201–7.

    Article  PubMed  Google Scholar 

  9. Danell E, Alström S, Ternström A. Pseudomonas fluorescens in association with fruit bodies of the ectomycorrhizal mushroom Cantharellus cibarius. Mycol Res. 1993;97:1148–52.

    Article  Google Scholar 

  10. Ma YJ, Zheng LP, Wang JW. Bacteria associated with Shiraia fruiting bodies influence fungal production of hypocrellin A. Front Microbiol. 2019;10:2023.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Andrić S, Meyer T, Ongena M. Bacillus responses to plant-associated fungal and bacterial communities. Front Microbiol. 2020;11:1350.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Cawoy H, Mariutto M, Henry G, Fisher C, Vasilyeva N, Thonart P, Dommes J, Ongena M. Plant defense stimulation by natural isolates of Bacillus depends on efficient surfactin production. Mol Plant Microbe Interact. 2014;27:87–100.

    Article  CAS  PubMed  Google Scholar 

  13. Cho SJ, Lee SK, Cha BJ, Kim YH, Shin KS. Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03. FEMS Microbiol Lett. 2003;223:47–51.

    Article  CAS  PubMed  Google Scholar 

  14. Li B, Li Q, Xu Z, Zhang N, Shen Q, Zhang R. Responses of beneficial Bacillus amyloliquefaciens SQR9 to different soilborne fungal pathogens through the alteration of antifungal compounds production. Front Microbiol. 2014;5:636.

    PubMed  PubMed Central  Google Scholar 

  15. Kai M. Diversity and distribution of volatile secondary metabolites throughout Bacillus subtilis isolates. Front Microbiol. 2020;11:559.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Fiddaman PJ, Rossall S. The production of antifungal volatiles by Bacillus subtilis. J Appl Bacteriol. 1993;74:119–26.

    Article  CAS  PubMed  Google Scholar 

  17. Ryu C-M, Farag MA, Hu C-H, Reddy MS, Kloepper JW, Paré PW. Bacterial volatiles induce systemic resistance in Arabidopsis. Plant Physiol. 2004;134:1017–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tahir HA, Gu Q, Wu H, Niu Y, Huo R, Gao X. Bacillus volatiles adversely affect the physiology and ultra-structure of Ralstonia solanacearum and induce systemic resistance in tobacco against bacterial wilt. Sci Rep. 2017;7:40481.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Massawe VC, Hanif A, Farzand A, Mburu DK, Ochola SO, Wu L, Tahir HAS, Gu Q, Wu H, Gao X. Volatile compounds of endophytic Bacillus spp. have biocontrol activity against Sclerotinia sclerotiorum. Phytopathology. 2018;108:1373–85.

    Article  CAS  PubMed  Google Scholar 

  20. Li Q, Li X, Chen C, Li S, Huang W, Xiong C, Jin X, Zheng L. Analysis of bacterial diversity and communities associated with Tricholoma matsutake fruiting bodies by barcoded pyrosequencing in Sichuan province, southwest China. J Microbiol Biotechnol. 2016;26:89–98.

    Article  CAS  PubMed  Google Scholar 

  21. Citterio B, Malatesta M, Battistelli S, Marcheggiani F, Baffone W, Saltarelli R, Stocchi V, Gazzanelli G. Possible involvement of Pseudomonas fluorescens and Bacillaceae in structural modifications of Tuber borchii fruit bodies. Can J Microbiol. 2001;47:264–8.

    Article  CAS  PubMed  Google Scholar 

  22. Ma YJ, Zheng LP, Wang JW. Inducing perylenequinone production from a bambusicolous fungus Shiraia sp. S9 through co-culture with a fruiting body-associated bacterium Pseudomonas fulva SB1. Microb Cell Fact. 2019;18:121.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Zhang C, Wu J, Liu W, Zheng X, Wang P. Natural-origin hypocrellin-HSA assembly for highly efficient NIR light-responsive phototheranostics against hypoxic tumors. ACS Appl Mater Interfaces. 2019;11:44989–98.

    Article  CAS  PubMed  Google Scholar 

  24. Zhang L, Gao J, Hou CL. Molecular identification and diversity analyses of fungi associated with ascomata of Shiraia bambusicola from southern Anhui Province, Eastern China. J Beijing Forest Univ. 2009;31:19–26 (in Chinese).

    CAS  Google Scholar 

  25. Liu YX, Hyde KD, Ariyawansa HA, Li WJ, Zhou DQ, Yang YL, Chen YM, Liu ZY. Shiraiaceae, new family of Pleosporales (Dothideomycetes, Ascomycota). Phytotaxa. 2013;103:51–60.

    Article  Google Scholar 

  26. Morakotkarn D, Kawasaki H, Seki T. Molecular diversity of bamboo-associated fungi isolated from Japan. FEMS Microbiol Lett. 2007;266:10–9.

    Article  CAS  PubMed  Google Scholar 

  27. Du W, Sun CL, Wang J, Xie WJ, Wang BQ, Liu XH, Zhang YM, Fan YH. Conditions and regulation of mixed culture to promote Shiraia bambusicola and Phoma sp. BZJ6 for laccase production. Sci Rep. 2017;7:17801.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Yan XX, Wen YD, Hu MH, Wu ZQ, Tian XF. Promotion of the hypocrellin yield by a co-culture of Shiraia bambusicola (GDMCC 60438) with Arthrinium sp. AF-5 fungus. Fermentation. 2021;7:316.

    Article  CAS  Google Scholar 

  29. Mowat E, Rajendran R, Williams C, McCulloch E, Jones B, Lang S, Ramage G. Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation. FEMS Microbiol Lett. 2010;313:96–102.

    Article  CAS  PubMed  Google Scholar 

  30. Briard B, Heddergott C, Latgé JP. Volatile compounds emitted by Pseudomonas aeruginosa stimulate growth of the fungal pathogen Aspergillus fumigatus. MBio. 2016;7:e00219.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Su YJ, Sun J, Rao SQ, Cai YJ, Yang YJ. Photodynamic antimicrobial activity of hypocrellin A. J Photochem Photobiol B. 2011;103:29–34.

    Article  CAS  PubMed  Google Scholar 

  32. Du W, Sun CL, Liang ZQ, Han YF, Yu JP. Antibacterial activity of hypocrellin A against Staphylococcus aureus. World J Microbiol Biotechnol. 2012;28:3151–7.

    Article  CAS  PubMed  Google Scholar 

  33. Effmert U, Kalderás J, Warnke R, Piechulla B. Volatile mediated interactions between bacteria and fungi in the soil. J Chem Ecol. 2012;38:665–703.

    Article  CAS  PubMed  Google Scholar 

  34. Chaurasia B, Pandey A, Palni LM, Trivedi P, Kumar B, Colvin N. Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro. Microbiol Res. 2005;160:75–81.

    Article  CAS  PubMed  Google Scholar 

  35. Yang H, Wang Y, Zhang Z, Yan R, Zhu D. Whole-genome shotgun assembly and analysis of the genome of Shiraia sp. Strain Slf14, a novel endophytic fungus producing huperzine A and hypocrellin A. Genome Announc. 2014;2:e00011-14.

    PubMed  PubMed Central  Google Scholar 

  36. Ren XY, Liu YX, Tan YM, Huang YH, Liu ZY, Jiang XL. Sequencing and functional annotation of the whole genome of Shiraia bambusicola. Genes Genom Genet. 2020;10:23–35.

    CAS  Google Scholar 

  37. Asadi F, Barshan-Tashnizi M, Hatamian-Zarmi A, Davoodi-Dehaghani F, Ebrahimi-Hosseinzadeh B. Enhancement of exopolysaccharide production from Ganoderma lucidum using a novel submerged volatile co-culture system. Fungal Biol. 2021;125:25–31.

    Article  CAS  PubMed  Google Scholar 

  38. Ávila N, Tarragó-Castellanos MR, Barrios-González J. Environmental cues that induce the physiology of solid medium: a study on lovastatin production by Aspergillus terreus. J Appl Microbiol. 2017;122:1029–38.

    Article  PubMed  CAS  Google Scholar 

  39. Thuan NH, Tatipamula VB, Canh NX, Giang NV. Recent advances in microbial co-culture for production of value-added compounds. 3Biotech. 2022;12:115.

    Google Scholar 

  40. Kong Q, Chi C, Yu JJ, Shan SH, Li QY, Li QT, Guan B, Nierman WC, Bennett JW. The inhibitory effect of Bacillus megaterium on aflatoxin and cyclopiazonic acid biosynthetic pathway gene expression in Aspergillus flavus. Appl Microbiol Biotechnol. 2014;98:5161–72.

    Article  CAS  PubMed  Google Scholar 

  41. Daub ME, Herrero S, Chung KR. Photoactivated perylenequinone toxins in fungal pathogenesis of plants. FEMS Microbiol Lett. 2005;252:197–206.

    Article  CAS  PubMed  Google Scholar 

  42. Sun CX, Ma YJ, Wang JW. Enhanced production of hypocrellin A by ultrasound stimulation in submerged cultures of Shiraia bambusicola. Ultrason Sonochem. 2017;38:214–24.

    Article  CAS  PubMed  Google Scholar 

  43. Li Q, Ning P, Zheng L, Zheng L, Huang JB, Li GQ, Hsiang T. Fumigant activity of volatiles of Streptomyces globisporus JK-1 against Penicillium italicum on Citrus microcarpa. Postharvest Biol Tec. 2010;58:157–65.

    Article  CAS  Google Scholar 

  44. Lei XY, Zhang MY, Ma YJ, Wang JW. Transcriptomic responses involved in enhanced production of hypocrellin A by addition of Triton X-100 in submerged cultures of Shiraia bambusicola. J Ind Microbiol Biotechnol. 2017;44:1415–29.

    Article  CAS  PubMed  Google Scholar 

  45. Ma YJ, Lu CS, Wang JW. Effects of 5-Azacytidine on growth and hypocrellin production of Shiraia bambusicola. Front Microbiol. 2018;9:2508.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Zhang J, Zhu L, Chen H, Li M, Zhu X, Gao Q, Wang D, Zhang Y. A polyketide synthase encoded by the gene An15g07920 is involved in the biosynthesis of ochratoxin A in Aspergillus niger. J Agric Food Chem. 2016;64:9680–8.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82073955 and 81773696) and the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD).

Author information

Authors and Affiliations

  1. College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, China

    Rui Xu, Xin Ping Li, Xiang Zhang, Wen Hao Shen & Jian Wen Wang

  2. Suzhou Institute for Food and Drug Control, Suzhou, 215104, China

    Chun Yan Min

Authors
  1. Rui Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xin Ping Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wen Hao Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chun Yan Min
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jian Wen Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JWW and RX conceived the study and participated in its design. RX, XPL, XZ, WHS and CYM undertook experiments and data analysis. JWW and RX drafted the manuscript. JWW supervised the research and revised the paper. All authors discussed the results, commented and approved the final manuscript.

Corresponding author

Correspondence to Jian Wen Wang.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Table S1.

The analysis of VOCs from strain No.1 by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS–SPME–GC–MS). Table S2. Primers and relevant information of reference and target genes. F: forward primer, R: reverse primer. Fig. S1. (A) Macroscopic colony appearance of No.1 strain on LB agar plate for 24 h. (B) Phylogenic tree of Bacillus cereus No.1 was built by using Clustal W in MEGA software (Molecular Evolutionary Genetics Analysis, version 7.0), with distance options according to the Kimura two-parameter model and clustering with the neighbor-joining (NJ) method. The type strain of the type species, B. subtilis ATCC 6051T (CP003329.1), was used as an outgroup. Type strains are indicated by the superscriptT. Bootstrap values higher than 50% (of 1000 cycles) were indicated. Scale bar indicated substitutions per nucleotide position

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, R., Li, X.P., Zhang, X. et al. Contrasting regulation of live Bacillus cereus No.1 and its volatiles on Shiraia perylenequinone production. Microb Cell Fact 21, 172 (2022). https://doi.org/10.1186/s12934-022-01897-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12934-022-01897-z

Keywords

Microbial Cell Factories

ISSN: 1475-2859

Contact us