DOI: https://doi.org/10.21203/rs.3.rs-1796940/v1
Blue mold caused by Penicillium sp. is one of the most serious postharvest diseases of citrus fruit. The aim of this study was to isolate and identify native Bacillus with inhibition phenotypes of citrus plants fruits. We investigated the antifungal effect of Bacillus velezensis wr8 on the postharvest pathogens Penicillium sp. inoculated on fruits, as well as on the growth of these fungi on Petri dishes with defined media. MALDI-TOF MS was conducted to illuminate the underlying mechanism. Results showed that Bacillus velezensis wr8 significantly inhibited Penicillium sp. growth in vitro. Moreover, secondary metabolites suppressed the disease development of citrus fruits artificially inoculated with Penicillium sp. in 25℃. Furthermore, MALDI-TOF MS indicated that lipoprotein with the molecular mass of 30.2 kDa was a key component about against Penicillium sp.. In addition, the secondary metabolites with antibacterial activity against Escherichia coli showed antimicrobial peptide with the molecular weight of 9.8 kDa. These results demonstrated that Bacillus velezensis wr8 could produce lipoprotein and antimicrobial peptide to inhibit Penicillium sp. and Escherichia coli which has broad application prospect in the future development. Finally, Bacillus velezensis wr8 is to provide data support for the development and utilization of high activity bacteriocin at room temperature and its application in the field of food safety.
Citrus, due to their high content nutrients, decay is caused by plant fungal pathogens during harvesting and transportation [1–3]. Thus, economic losses caused by postharvest diseases currently represent one of the main dilemmas of the citrus industry worldwide. It has been reported that Penicillium sp. is the main postharvest diseases of citrus, could cause postharvest blue mold of citrus fruits, accounting for 90% of the total losses [4]. The main sources of Penicillium sp. contamination are conidia, which made wound pathogenic and caused a soft and wet rot of citrus. The application of synthetic chemical germicides is a primary method to control postharvest decay [5], However, the frequency of resistant strains increases significantly, due to the increasing demands for food safety, their use is becoming more restricted. There is a growing need to develop alternative treatments of postharvest disease that is more endure and safe. The controlled use of microorganisms which antagonize pathogenic microorganisms also shows immense potential as an alternative method for controlling postharvest diseases [6]. There are several mechanisms of biological control using microorganisms are involved, including competing for nutrients [7]; inhibition microorganism [8]; inducing resistance [9]; regulating the population density at specific sites [10].
Decay caused by food-borne bacterial pathogens also a major concern due to the increasing demands for health safety issues [6]. For instance, Staphylococcus aureus and Escherichia coli in contaminated food are leading causes of gastroenteritis [11]. It has been reported that Bacillus can secrete protein toxins that are lethal to filamentous fungi and bacteria [12–14]. Surfactin, iturin and fengycin lipopeptides antimicrobial substances were showed a broad spectrum of antifungal activity against Fusarium oxysporum, Pythium aphanidermatum and Botrytis cinerea [15]. In addition, Bacillus has shown high efficiency in the control of Penicillium sp. [12–13, 16]. Bacillus velezensis GSBZ09 presented broad spectrum antifungal activity and remarkably inhibited the mycelial growth and spore germination of C. vitis [17]. The iturin family of lipopeptides was identified, that they show strong pathogen inhibition ability of Penicillium crustosum [18]. Interestingly, most antagonistic microorganisms are isolated from local organs of the plants, having local adaptive advantages, and thus may be better antagonists than those from other sources [19–20]. So far there have been a few studies on isolation of indigenous inhibition Bacillus for the biocontrol agent against postharvest pathogens.
This work was to isolate and identify native Bacillus with inhibition Penicillium sp. phenotypes of citrus plants fruits retain freshness. We investigated the antifungal effect of wr8 on the postharvest pathogens Penicillium sp. inoculated on fruits, as well as on the growth of these fungi on Petri dishes with defined media. A range of generally used germicides are under review in many countries due to health safety issues. We also examined the effect of wr8 on Listeria monocytogenes, Escherichia coli. MALDI-TOF MS was used to analyze different kinds of antimicrobial substances in the metabolites of Bacillus velezensis wr8 and establish alternative treatments of postharvest disease that is more endure and safe. Bacillus velezensis wr8 as the biocontrol agent against postharvest pathogens, has certain advantages: i) It can grow rapidly using inexpensive substrates and has simple nutritional requirements being able to colonize dry surfaces for long periods of time. ii) The antibacterial activity can be maintained at room temperature.
The pathogen used in this study was obtained from Guangxi Academy of Sciences (Fusarium oxysporum f. sp. cubense (FOC) 1, Fusarium oxysporum f. sp. cubense (FOC) TR4, Fysarium fujikuroi, Fusarium proliferatum, Penicillium crustosum, Cladosporium sphaeroporum, Escherichia coli, Listeria monocytogenes, Ralstonia solanacearum moderately, Staphylococcus aureus, Pseudomonas fluorescens). Citrus fruits were collected during January 2021 in the town of Nanning. Bacillus was isolated from decaying Citrus and stored in the microbial collection of the Laboratory preservation Center at Guangzhou. Penicillium sp. strain was grown at 25℃ in Potato Dextrose Agar (Potato Dextrose Agar, PDA: bought from Coolaber). Bacillus strain was grown at 37℃ in LB medium (0.5% yeast extract, 1% peptone, 1% sodium chloride).
The Bacillus biological control agents isolation and identification
The Bacillus biological control agents (ACBL) were isolated from the citrus peel during January 2021. They were obtained from 3 citrus producing regions in Nanning, China. The Bacillus isolation was carried out according to Babadoost et al. [21]. The epidermis of fruit disinfected by 75% ethanol was cut into pieces and placed in LB medium to enrich the bacteria. Using the decimal dilution technique and a sterile water solution sterile distilled water. Plating was performed in triplicate using LB culture media (Yeast extract peptone dextrose: 0.5% yeast extract, 1.0% peptone, 1.0% sodium chloride, 1.5% agar dissolved in 1.0 liter of distilled water). The bacteria with inhibitory effect on Penicillium sp. were screened by plate confrontation tests [22]. The strain about the most obvious inhibition zone was sequenced for verification. Briefly, the web-based tool from the National Center for Biotechnology Information (https://www.ncbi.nlm.ni h.gov/) was used to analyze strain types. The strain utilized the universal primers 5′-AGAGT TTGATCCTGGCTCAG-3′ and 5′-GGTTACCTTACGACTT − 3′. Using the wr8 sequence as the target, 27 strains were selected, which were different from the wr8 type. Multiple sequence alignments of wr8 and other strain sequences were generated using ClustalW version 1.83. A phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA 6.0 with 1000 bootstrap replications.
Antifungal efficacy of Bacillus velezensis wr8 against Penicillium sp.
PDA medium was used to grow fungal colonies. After 5 days of culture, agar discs (1 mm in diameter) with mycelia were excised and then transplanted to the center of PDA dishes (90 mm in diameter) under sterile conditions. The growth radius of pathogenic bacteria is 1 cm, Bacillus velezensis wr8 was inoculated. The diameters of the colonies were measured to analyze the growth of Penicillium sp..
Screening of the Bacillus strains for antifungal activity in vitro against Penicillium sp.
Antifungal activity of secondary metabolite against Penicillium sp. was evaluated by agar well diffusion method [23]. Penicillium sp. spore concentration was adjusted to 1 × 106 conidia mL− 1, Then mix 1ml spores into PDA soft agar and we poured evenly on the plate. Agar well diffusion (9 mm) containing 100µL secondary metabolite were gently the agar plates. The plates were incubated at 25°C for 3 days, and the diameter of the inhibition zone was observed. Bacillus strain was grown at 37℃ in LB medium (0.5% yeast extract, 1% peptone, 1% sodium chloride) and Obtaining secondary metabolites by 1 × 106 conidia mL− 1, 0.22um membrane filtration.
Inhibitory activity of Bacillus velezensis wr8 against citrus pathogenic Penicillium sp.
According to the methodology of Ferraz et al. [24], the safe 64 oranges were washed, superficially disinfected with 0.2% (v/v) sodium hypochlorite for 3 min and rinsed in sterile water to eliminate the sodium hypochlorite. Then, the fruits were wounded at two equidistant points, and 20 µL of Penicillium sp. conidial suspension (1 × 106 conidia mL− 1) was inoculated in the wounded area, which was treated 24 h later with 20 µL wr8 secondary metabolites (1 × 107 mL− 1). The control group was treated with the equal amount of sterile water instead of wr8 suspension. Disease severity was assessed on the 15th days after inoculation by observation blue mold lesions region size phenotype.
Inhibition Spectrum of Bacillus velezensis wr8
Antibacterial spectrum of Bacillus velezensis wr8 was examined using agar well diffusion assay [25]. In brief, LB agar plates were overlaid with 9 mL of LB soft agar (0.8% agar) inoculated with 1% of each indicator strain, which was previously grown until the suspensions reached 1 × 106 conidia mL− 1. Wells were made on the seeded plates using a sterile Pasteur pipette tip (9 mm in diameter) and culture 100 µl sterile supernatan t of Bacillus velezensis wr8 was added into each well, by 0.22um filter membrane filter. The plates were incubated overnight at 37℃ and then inhibition zones were examined. Antifungal spectrum of Bacillus velezensis wr8 was detected by plate confrontation method.
Purification of Bacillus velezensis wr8 secondary metabolites
For Bacillus velezensis wr8, incubated 1 L bacterial suspension at 220 r·min− 1 for 24 h and centrifuged to remove cells. After the sterile supernatant was lyophilized and dissolved in methanol at the rate of 1:50 (w/v) and stirred for 6 h. Methanol was evaporated by rotary evaporator. Each Bacillus culture was filtered and a 0.22 µm Millipore membrane after incubation to remove Bacillus cells according to a protocol adapted from Valarini et al. [26]. The active dialyzed fraction was then further purified by AKTA prime plus system (Amersham-Bioscience, Uppsala, Sweden) calibrated with 20 mmol·L− 1 Tris-HCl buffer, at a flow rate of 0.8 mL·min− 1. The active CEC fraction was introduced to preequilibrated (ddH2O with 2% acetonitrile and 0.1% TFA) RP-HPLC purifier and elution was conducted using a linear gradient from 95% solvent A (ddH2O with 2% acetonitrile and 0.1% TFA) and 5% solvent B (100% acetonitrile with 0.1% TFA) for 10 min to 100% solvent B for 50 min. All fractions were procured at 280 nm absorbance and lyophilized. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed by using a MALDI-TOF spectrometer (Bruker Daltonics, Bremen, Germany) in positive ion mode, to determine the molecular mass of pure bacteriocin.
Data was presented as the mean ± standard deviation. The chosen significance level was 0.05 for all statistical tests in this study. Antagonistic activity data were compared using Dunnett's test between control and study groups. Antifungal activity data were compared using Tukey’s HSD test. All treatments were repeated at least two times, and the statistical analysis difference was indicated by asterisks.
Isolation and identification of Bacillus
Retain freshness dominant strain wr8 was obtained (Fig. 1a), which was from the rotten fruits of citrus. The isolated strain was identified according to its 16S rRNA gene, belonging to Bacillus sp.. After which phylogenetic tree was established (Fig. 1b), results indicated that the strain could be classified into Bacillus velezensis, and named Bacillus velezensis wr8.
Screening of the Bacillus strains for antifungal activity against Penicillium sp.
To better understand the impact of Bacillus for inhibition of Penicillium sp. Bacillus strains were isolated from citrus fruits. The antagonistic potential of Bacillus were evaluated, based on the assessment of phenotype in dual culture tests, with the examined fungi grown on PDA plates. Result showed Bacillus velezensis wr8 strain inhibition of Penicillium sp. (Fig. 2a). The inhibition effect of Bacillus velezensis wr8 appeared, When the distance from Penicillium sp. mycelium was 1cm (Fig. 2b).
Detection of “inhibition” activity by Bacillus isolates
We used the pour plate methodology, adapted from Dong et al. [27]. With the aim at evaluating the antagonistic capacity of Bacillus strains secondary metabolites against Penicillium sp. 100 µL aliquots of a suspension containing 1 × 106 cells mL− 1 of the Bacillus strains were filter with 0.22 membrane and added into the wells. The cell-free culture filtrate was then used to check the antifungal Penicillium sp. activity of the secreted extracellular metabolites. After 3 days after incubation, observation of these cultures showed a inhibition (Fig. 3).
Inhibitory activity of Bacillus velezensis wr8 against citrus pathogenic Penicillium sp.
Although the type of biocontrol strain and its inhibitory effect on the pathogenic fungus Penicillium sp. in vitro has demonstrated, the effect on the fruits of citrus remains unclear. Therefore, the experiment of infection and biological control by Penicillium sp. and Bacillus velezensis wr8 on fruits was carried out. It can be seen that the control group inoculated with Penicillium sp. showed evident rot, while the development of Bacillus velezensis wr8 reduced the region lesions of the fruits after treatment (Fig. 4). The result revealed that Bacillus velezensis wr8 strain inhibited the pathogenic fungus Penicillium sp..
Inhibition spectrum of Bacillus velezensis wr8
Bacillus velezensis wr8 showed very strong inhibition against all the indicator fungi in this study. It could not only effectively inhibit Foc 1 and Foc-TR4, the pathogen causing banana Fusarium wilt, Fysarium fujikuroi of Oryza sativa, Fusarium proliferatum of maize ear rot, but also inhibit Penicillium crustosum and Cladosporium sphaeroporum of citrus. In addition, Aspergillus versicolor could be effectively inhibit by wr8, which had a certain broad-spectrum antibacterial effect (Fig. 5).
For bacteria, based on the diameter of the inhibition zone observed. Bacillus velezensis wr8 showed very strong inhibition against Escherichia coli and Listeria monocytogenes. Bacillus velezensis wr8 inhibited Ralstonia solanacearum moderately but did not show any activity against Staphylococcus aureus and Pseudomonas fluorescens (Table 1).
|
Indicator strains |
Bacillus velezensis wr8 |
|---|---|
|
Escherichia coli |
+++ |
|
Listeria monocytogenes |
+++ |
|
Staphylococcus aureus |
- |
|
Pseudomonas fluorescens |
- |
|
Ralstonia solanacearum |
+ |
| -, 0 mm (no inhibitory activity); +, 0 to 5 mm (moderate inhibitory activity); ++, 5 to 10 mm (strong inhibitory activity); +++, more than 10 mm (very strong inhibitory activity) | |
Purification of bacteriocin about against Penicillium sp.
To further examine the effect of key components of secondary metabolites, purification of secondary metabolites were initiated by AKTA purifier [28–29]. Crude peptide mixture was subjected to AKTA purifier and RP-HPLC purifier, introduced to the MALDI-TOF MS, based on the revealed mass spectra of purified secondary metabolites obtained one high intense peak lipoprotein with the molecular mass of 30.2 kDa (Table 2).
|
Description |
Mw (kD) |
Sequence |
|---|---|---|
|
Antimicrobial peptide |
9.873 |
GYWVGIYESVDK |
|
Lipoprotein |
30.199 |
DAIQVESTK/ NKLNPLK |
Antibacterial activity against Escherichia coli
The methanol extract purified products were subjected for AKTA purifier analysis to determine the molecular size of the bacteriocin. We chose the one the highest peak collected purified. The band with antibacterial activity against Escherichia coli showed antimicrobial peptide with molecular weight of 9.8 kDa (Table 2). The result is to provide data support for the development and utilization of new broad-spectrum bacteriocin and its application in the field of food safety.
The results reported in this work, showed the possibility of an alternative strategy for fruit storage, based on the postharvest control of Penicillium sp. through an application of Bacillus during pre-harvest may allow better protection of fruits to the pathogens that occur in the post-harvest. The previous work found that some Bacillus velezensis have been widely used as biological fungicides to control powdery mildew, Botrytis cinerea, sheath blight, ergot durum, late blight, cotton Fusarium wilt and apple putrefying disease [30]. Bacillus is a kind of gram-positive bacteria that can produce stress resistant spores. It has good bacteriostasis and has been recognized as a probiotic. There are several data on an antifungal role of Bacillus velezensis against citrus postharvest pathogens. Usall et al. reported that most Penicillium sp. infections occurred on fruits during harvest or soon after in the packinghouses [31].Thus, the preventive control of Penicillium sp. through an application of Bacillus during pre-harvest may allow better protection of fruits to the pathogens that occur in the post-harvest. We reported that Bacillus velezensis wr8 plays as a role as a fungicide to the pear Penicillium sp. postharvest storage of fresh-cut pears. It is effective for the biocontrol of postharvest diseases on citrus fruits. We found that the secondary metabolites of Bacillus velezensis wr8, could produce antimicrobial peptide to inhibit Penicillium sp.. The data suggested the potential use of this strain for biocontrol of blue mold in harvest citrus.
Because of their toxicity and carcinogenicity, chemical residues pose hazards to both the environment and the health of humans. The microorganism of non-toxic safer alternatives for controlling postharvest diseases becomes more important. Riteshri et al. showed that Bacillus velezensis and Bacillus subtilis encoded multiple secondary metabolite gene clusters correlating, which can effectively inhibit the growth of Gram-negative bacteria such as Escherichia coli, Salmonella paratyphoid B, Shigella flexneri and Salmonella enterica activity [32–33]. Riteshri et al. showed that Bacillus velezensis CGS1.1 displayed antimicrobial activity against Escherichia coli and Salmonella enterica [34]. Consistent with these observations, we found that the secondary metabolites of Bacillus velezensis wr8, could produce lipoprotein to inhibit Escherichia coli which has broad application prospect in the future development.
The purpose of the study on purification and antibacterial mechanism of Bacillus velezensis wr8 is to provide data support for the development and utilization of high activity bacteriocin at room temperature and its application in the field of food safety. Our study also proved that secondary metabolites can maintain their activities for a long time at room temperature. Although the studies presented in this article have helped to understand better the purification and antibacterial mechanism of Bacillus velezensis wr8, further investigations are still essential, since there are many aspects to be explored, especially, the lack of studies to prove which of secondary metabolites is directly linked to in pathogen.
Conflict of interest the authors declare that they have no conflict of interest.
Author contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by XHW and RW. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the Science and Technology Project of Guangxi (2020ZYZX3027, AB2107601), and a Bagui Scholar Program Fund (2016A25) of Guangxi Zhuang Autonomous Region awarded to LQ.Z. We appreciate all scholars enrolled in this study, including the Bagui Scholarship of Guangxi Zhuang Autonomous Region awarded to LQ.Z.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Compliance with Ethics Requirements The authors confirm that this manuscript has not already been published nor is it under consideration for publication elsewhere. This article does not contain any studies with human or animal subjects.