Reprinted | Plant nutrition and soil improvement
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Abstract: Brevibacillus laterosporus has a variety of biological activities such as insecticide, antibacterial, anti-tumor, biodegradation and transformation, and has shown great application value and potential in biological control, environmental protection, medicine, microbial agent development and many other aspects. In this paper, the main research results and corresponding research methods of Bacillus flasporaii since its discovery are reviewed, focusing on the progress of the specific application of Bacillus flanksporum brevis in production, and some of the contents are mentioned for the first time in China. At the same time, the problems in the application of Bacillus flanksporum Breve are also pointed out, and its future research and development and application are prospected.
In taxonomy, Brevibacillus laterosporus belongs to Cellular organisms, bacterial domains, Firmicutes, Bacilli, Bacillus, Bacillales, Baenibacillaceae, Baenibacillaceae, Bacillus breve (Brevibacillus). It is widely distributed in nature and has been isolated in soil, volcanic mudslides, fresh water, seawater, insects, leaf surfaces, locust beans, compost, milk, cheese, honey, starchy foods, rubber products factory wastewater, animal fur and quail. In the early 20th century, when White studied the pathogenic bacteria of European larval rotting disease, it isolated Bacillus flankspora from its secondary source of infection, and people began to pay attention to its relationship with insects. In 1916 a new species was isolated from the laubach freshwater, named Bacillus laterosporus, and its morphological and physiological and biochemical properties were described in detail. In 1996, Shida et al. analyzed and constructed a phylogenetic tree based on the sequence of 16S rRNA, and identified Bacillus flankspringii as a new genus including Bacillus breve, including Bacillus brevis, according to modern taxonomy. Ruiu reviewed the research history of Bacillus breve diureus laterally in terms of morphological characteristics, lethal effect on invertebrates, antibacterial effect, and other properties. In this paper, the main research and breakthrough progress of Bacillus flasspora brespora are comprehensively reviewed at home and abroad, and the application profile of Bacillus flasspora brespora from early detection to the current production is systematically summarized.
1. Morphological characteristics of Bacillus flanksporum Breve
Bacillus brevis flankspora is a rod-shaped, endospore-producing facultative anaerobic bacterium capable of producing its characteristic canoe-shaped companion spores (CSPB) that cling tightly to one side of the spores, allowing them to be laterally born and colored with toluidine blue or crystal purple dyes. The cell size of Bacillus breve is about (0.5~0.8)μm× (2.5~5.0)μm, the growth temperature range is 15~45 °C, and the suitable pH range is 6~9. For a long time, the special morphological characteristics of Bacillus flanksporum Bresbosporum have attracted the attention of many researchers. In the mid-20th century, Hannay used optical and electron microscopy to observe fixed, in-sporangia cells and spores and came to the conclusion that companion spores formed before spore formation and remained attached to one side of the spore after spore formation, which is part of the spore. Various hypotheses have been proposed about the function of the companion spore, which may contribute to the germination of spores, which are either attached to the walls or deformed by the sporangia walls. However, so far it has not been possible to determine its special features. In 1958, Fitz-James et al. invented a method of isolating canoe-shaped spores from the protoplasts of the spores and analyzed the chemical composition of this structure. In addition to the canoe-like companion spores, Montaldi et al. also found angular spheroid companion spores and intersecting parallel stripes in the sporangia in 1990. The function of these structures is unclear, but it was later discovered that the different shapes and sizes of concomitant spore bodies of Bacillus flanksporum breve were associated with the activity of killing dipteran insects.
2. Biological activity of Bacillus flanksporum Breve
Bacillus flanksporidium breve is a microbial resource that can both be antibacterial and insecticidal and have medical value. Early research mainly focused on insecticidal activity, foreign research is more, and finished biological insecticides have been listed. In recent years, there has been growing concern about the properties of Bacillus breve lateral spores being able to produce antibacterial active substances.
Bacillus flankspora has a variety of biological activities and exhibits broad-spectrum antimicrobial activity, particularly against bacteria and fungi. Recent whole genome sequencing results suggest that Bacillus flaskspora also has the potential to produce substances such as polyketone compounds, non-ribosomal peptides and toxins. It is also able to produce active substances such as aminopeptidase inhibitors, glutaryl 7-ACA acyltransferases, thrombin inhibitors, and lysine, showing good application prospects. In addition, Bacillus flankspora is also a probiotic for mammals and birds, and the antibiotics it produces have been used in the field of medicine.
2.1 Insecticidal activity of Bacillus breve
There are many studies on the insecticidal activity of Bacillus breve. Studies have shown that a variety of Bacillus flaspora are pathogenic to different invertebrates such as insects, nematodes and molluscs. Its insecticidal activity is mainly due to the toxic effect of the protein contained in the companion spore on a variety of insects, and after contact with or invasion of the target, different substances are produced at different stages of the cell growth cycle, thus playing a toxic role. Researchers have explored some specific toxin proteins and how they work, but there are still many questions that need to be further analyzed and elucidated.
2.1.1 Early studies on insecticidal effects found that some strains of Bacillus flaspora had toxic effects on potato beetles and tobacco beetle larvae. Du Rand et al. found that a strain of Bacillus flambossporus breve was more toxic to mealworm larvae than Bacillus thuringiensis at the same biomass, and determined that its semi-lethal concentration was 3.388×106 spores/mL. Boets et al. found that the toxin secreted by Bacillus bresbosporum laterals had protein characteristics, named the new insecticidal secretion protein ISP1A and ISP2A, and determined its DNA coding sequence. Toxicity is only manifested when the main insecticidal secretion protein and the complementary insecticidal secretion protein are ingested by insects at the same time, while there is no toxicity when one insecticidal protein is ingested alone. Differences between different strains of Bacillus breve lateral spores have led to controversy over their resistance to Lepidoptera. Strains of Bacillus breve flanks exhibited different levels of toxicity to nocturnal soy moths, while the same strains showed no insecticidal activity against nocturnal moths in meadows.
The earliest reports of the potential insecticidal effects of Bacillus flanksporidium breve stemmed from studies of mosquitoes. Pathogenicity to mosquitoes is not related to culture supernatant, but is affected by somatic cells. Studies in recent years have found that the spore and canoe-shaped companion spore structure of Bacillus breve lateral spores play a major role in killing mosquitoes. Separation from Russia to obtain B. Laterosporus 615 and 921, which produce cytoplasmic crystal inclusions of different shapes and sizes, have reported their insecticidal activity.
Recent studies have found that a crystal-producing type of lateral Bacillus flanksporum breve has an insecticidal effect on tetramembranous worms when both spores and crystals are present. However, spore-crystal precursors have no toxic effect on protozoan cells.
Ruiu et al. first discovered in 2006 that Bacillus flankspore, which has formed spore, is infectious to houseflies. The toxic effect on houseflies is mainly derived from spores. Bacteria such as B. laterosporus produce insecticidal effects by attaching toxins tightly to live spores. In subsequent histopathological studies, the causative factors and modes of action of Bacillus flasksporum Breve were studied in depth. The value of Bacillus breve as a biological agent for the control of flies is also reflected in another aspect: the safety evaluation results show that it is not toxic to non-target organisms, but only acts on the parasitic bees and bees of the houseflies of the order Hymenoptera.
2.1.2 Nematode effect Some strains of Bacillus flambossinum contain nematode compounds in the spores, which can inhibit the egg hatching and larval development of nematodes, therefore, Bacillus flambossii has become a biological control agent for parasitic nematodes. The insecticidal toxin obtained by HPLC separation is a high-temperature, low molecular weight (approximately 2.9 kD) protein with UV absorption wavelengths of 205, 220, and 268 nm. It has insecticidal activity for both animal parasitic nematodes and plant parasitic nematodes.
The extracellular protein produced by Bacillus bresbosporum lateralis G4 has been determined to be toxic to nematodes, and the alkaline protease BRG4 isolated and purified from the fermentation supernatant of the bacterium has insecticidal activity against independently surviving nematodes and plant parasitic nematodes Bursaphelenchus xylophilus Nickle. Observed under a scanning electron microscope, these proteins severely damage the stratum corneum of nematodes, causing their outer epidermis to shed and form many fissures. This mode of action is similar to that of nematode fungi, which invade the nematode body and are achieved by extracellular proteases degrading the protein cornea of the epidermis and the matrix that connects the chitin fibers of the nematode. Using pure cultures of recombinants, the insecticidal activity of BPG4 was verified in vitro. The BLG4 coding gene cloned and expressed in Bacillus subtilis is highly homologous to the coding genes of the Bacillus subtilis protease family in the serine protease class and other stratum corneal degrading proteases. The lack of mutants of the BRG4 gene is significantly less pathogenic when detected. During the degradation of the nematodes stratum corneum, the fluorescent immunoposition method was used to prove that proteases were involved in bacterial infection, and the research results have been applied.
2.1.3 Mollusc effects have been reported to have been reported to be bacillus shortening (B. f. 2005). Brevis) similar species have insecticidal activities against aquatic snails, smooth double navel snails and zebra mussels. The entire composition of a bacterial culture is related to its pathogenicity to mollusks, in which bacterial antibiotics, such as short bacitracin S and D, play a major role. Bacillus breve is more infectious to animals with shorter life cycles, and zebra mussels in the larval stage of the disc are as pathogenic as Bacillus thuringiensis.
The above study suggests that the mechanism of action of Bacillus flaspora may differ between different strains and between different strains of bacillus flasks and between different targets.
2.2 Antibacterial activity of Bacillus breve
Bacillus flaspora produces a wide range of antibacterial substances and exhibits a broad spectrum of bacteriostatic activity against various types of fungi and bacteria. Many species in the genus Bacillus breve that can produce proteases and antibiotics have this biological property. In this regard, there are more foreign reports, and the antibacterial substances that have been identified are non-peptide bacillusamines, peptide antibiotics, polyketone compounds and chitinase.
2.2.1 B. laterosporus ZQ2, which inhibits plant pathogenic fungi isolated from the rhizosphere soil of apple in China, has broad-spectrum resistance to apple tree pathogenic bacteria, such as Rhizoctonia solani and Fusarium aculis (F. oxysporum), Fusarium solanum (F. solani), Aspergillus fumigatus, Alternaria alternata, Valsa sordida, Apple Anthracnum gloeosporioides, Botrytis cinerea, and Physalospora.) piricola) and so on. Under the condition of 120 °C high temperature treatment for 30 min, ultraviolet radiation and pH value 1 to 11, the antifungal activity in the culture supernatant of the bacterium can still achieve 80% of the bacteriostatic rate. Isolated from Bacillus flanksporum breve BPM3 in mud in India, it was able to inhibit plant pathogenic fungi such as F. Growth of oxysporum, F. semitectum, Magnaporthe grisear, Rhizoctonia oryzae, and the gram-positive bacteria Staphylococcus aureus. The compounds that play an inhibitory role are isolated and identified, and through spectral analysis, the chemical composition of the highest active sites is initially found to include C-H, carbonyl, dimethyl, -CH2 and methyl.
Some strains of Bacillus breve laterals inhibit fungal growth by producing chitinase to degrade the fungal cell wall. Zhao Qiumin et al. screened out 1 strain of Bacillus chitinae breve 1~864 from 524 strains of Bacillus chitinase, and the study showed that the bacterium had obvious inhibitory effect on 6 pathogens such as Gibberella wheat. From the mangrove wetland soil in India, a strain of Bacillus breve lak210 was isolated, and on a colloidal chitin-containing medium, it can produce chitinase and release it into the culture medium. These antimicrobial proteins have an inhibitory effect on pathogenic fungi of the Fusarium species, as opposed to those produced by B. The laterosporus LMG15441 gene encodes a quad-domain chitinase of 89.4 kD and a di-domain chitinase (ChiA1) of 69.4 kD are highly homologous.
2.2.2 Inhibiting the antibacterial properties of plant pathogenic bacteria Bacillus flanksprine breve is related to the antimicrobial peptides it produces, and a method of quantitatively detecting its biological activity using agar diffusion has been studied. Li Weijie et al. identified tomato blight as bacillus breve, and prepared a solid bacterium X10, and the determination of its field application effect showed that the control effect of the agent on tomato blight was better, and at the same time it could promote the growth and yield of tomato plants. In addition, the protein crude extract in X10 fermentation broth has the effect of inhibiting the growth of Solanaceae. Recently, from the fermentation broth of Bacillus flanksporum Breve A60, a novel short-sequence linear antimicrobial peptide with a molecular mass of about 1.6 kD was isolated and identified, named BL-A60, and proved to have inhibitory effects on a variety of plant pathogenic bacteria. The antibacterial active substance has good thermal stability, remains active after 15 minutes of treatment at 100 °C, the antibacterial activity is not affected in the pH range of 3 to 11, and the proteinase K, trypsin and pepsin treatment do not affect its activity. The inhibitory effect of antimicrobial peptides on microorganisms shows that it causes cell disruption and lysis by destroying cell membranes. These antimicrobial peptides enter the cell through the cell membrane and transfer with the flow of the cytoplasm, and they eventually disrupt the synthesis of proteins by interfering with DNA and RNA. BL-A60 may also work in this way, but these mechanisms have not yet been clearly studied. When Li Dingjun et al. studied the 2-Q-9 of Bacillus breve, they found that the bacteriostatic substance it secreted could strongly inhibit the normal growth of the physiological subspecies of 3 green blight bacteria, and further research on the properties of the bacteriostatic substance showed that the bacteriostatic substance may be protein or polypeptide. Chen Wu et al. isolated and purified the exosome 2-Q-9 exotracheic antibacterial peptide BL2Q9. Ren Zhaozhen et al. obtained the antibacterial active substance R21 of Bacillus breve Lh-1, and the bacteriostatic test proved that R21 has bacteriostatic effect on pathogenic gram-negative and positive bacteria, food spoilage bacteria and a few fungi. Zhao Xiuxiang et al. isolated from the tobacco rhizosphere soil to a strain of biocontrobacterium flanks spore Bacillus breve B8, its fermentation liquid on tobacco nigra nigra bacteria bacteriostatic circle diameter of 63 mm, the test inferred that the bacteriostatic substance may be protein or polypeptide.
2.3 Phosphorus dissolving function of Bacillus breve lateral spore
Bacillus breve lateral spore has the functions of phosphorus and potassium solution. Lei Shi studied the phosphorus-dissolving capacity of Bacillus flanks breve strain BL-21, indicating that it was suitable for the development of bacterial fertilizers for dryland crops, and could increase the available phosphorus content in soil and crop yield to a certain extent. It has been found that Bacillus flanksporum breve is able to degrade hydroamine thion. In the process of studying the phosphorus solution of Bacillus flaspora, Du Chunmei et al. found that it had the effect of degrading organophosphorus pesticides such as oxidizing Leguo and water amine thion, analyzed the conditions and pathways for the degradation of water amine thion by Bacillus flanksii strain BL-2l, and obtained the best medium formulation and degradation conditions for the degradation of water amine thion.
2.4 Degradation of organic pollutants by Bacillus breve
The use of microorganisms for bioremediation has become a new research hotspot. The use of biological methods to treat contaminated sites for degradation and removal of sources of contamination or contaminating compounds. It has been found that more and more substances can be degraded by Bacillus breve, the most typical example is the degradation of polyvinyl alcohol to acetate, but also to produce a variety of enzymes such as lignin peroxidase, laccase, aminopyrine-N-demethylase, NADH-DCIP reductase and peacock green reductase. To achieve decolorization of textile azo dyes, Kurade et al. studied the preferential degradation of different structure dyes by Bacillus flaspora Bresbosporum MTCC 2298, and optimized the degradation law and physicochemical parameters of decolored substances using HPTLC. Subsequently, a new bacterial-yeast microbiota BL-GG was found to effectively decolor the textile industry wastewater and the disperse dye Scarlet RR, and it was believed that the combined enzyme catalytic mechanism produced by the combination of bacteria-fungi or yeast showed a synergistic effect and reduced the production of toxic metabolites. Waghmode et al. evaluated Galactomyces geotrichum MTCC 1360 (GG) and B. The effects of laterosporus MTCC 2298 (BL) and the microbial flora GG-BL on the disperse dye Brown 3 REL were further verified.
Studies have demonstrated that Bacillus flankspring are able to degrade plant tannins in tannery wastewater, biodegrade phenol and toluene, bioabsorb toxic metals in aqueous solutions, and detoxify heavy metals in sewage systems.
2.5 Medical function of Bacillus breve
The medical function of Bacillus breve has long been well known, especially in foreign countries in this regard, the technology is more mature, and the related products are rich in variety, which has brought good news to human health. The United States and the United Kingdom have developed probiotic health care drugs such as BOD using Bacillus flanksporum breve for oral prevention or treatment of gastrointestinal disorders. In the European market, the biotin precursors produced by this bacterium have developed many products.
The antifungal and antibacterial properties of certain strains of Bacillus flasks have medicinal value because of the therapeutic effects of the antibiotics they produce. For example, the antibiotic lateral sporeamine has shown broad-spectrum resistance to gram-negative and gram-positive bacteria in both in vivo and in vitro tests. Isolated from tropical water off the coast of Papua New Guinea, a strain of Bacillus breve disporum was obtained, which can produce acyldipeptides tupuseleiamides and antifungal polyketone compound basiliskamides, which can effectively inhibit E. Coli and Candida albicans. The same strain PBG-276 isolated from the ocean can also produce a variety of peptide compounds including loloatins, bogorols and lipopeptide tauramamide. Bacillus breve fermentation brome also contains other antibiotics, such as cyclodecapeptide laterocidin, thrombin inhibitors bacithrocins A, B and C, leuhistin, leuhistin, and the anti-cancer substance Sigerin. Krachkovskii et al. isolated a cyclic decapeptide antibiotic from Bacillus breve diuresis lateralis IGM and identified as loloatinA by nuclear magnetic resonance spectrophotometry. Some lateral Bacillus flasks isolated from soil can produce an important industrial enzyme, cephalosporin acyltransferase, for the industrial production of cephalosporin derivatives.
Bacillus breve flankspora can produce a variety of antitumor active substances, such as spermidon, immunotoxins, 15-deoxygenguanidin, etc. 15-Deoxyspergualin (DSG) is a metabolite produced by Bacillus flaspora breve and has immunosuppressive activity against canines, rodents, and primates. It has great potential for anti-cancer immunosuppression, which can inhibit the proliferation of monocytes, lymphocytes and macrophages, inhibit the release of peroxidase and lysosomal enzymes by monocytes, and significantly reduce cell infiltration after kidney transplantation. Shi et al. to B. Laterosporus A7 mutagenesis, a high-yielding anticancer and genetically stable strain A-94-7 was screened out, and its fermentation medium was optimized, and the anticancer production was 4 times that of the original strain.
2.6 Other functions of Bacillus breve
Crawford et al. studied the degradation of Bacillus to 4-hydroxybenzoic acid and found that strain B. Laterosporus PHB-7a in a medium containing 4-hydroxybenzoic acid is capable of producing high levels of gentian oxygenase and maleic acid pyruvate hydrolase, which converts 4-hydroxybenzoic acid into gentianate, which is further converted to gentian acid by the action of glutathione. Aramori et al. obtained a novel glutaryl-producing 7-ACA acyltransferase lateral Bacillus brevis J1 from soil. A strain of Bacillus breve, lateral spore isolated from soil, Yoshimoto et al., can produce an endo-α-N-acetamidogalosidase. Umerie et al. produce L-lysine by batch fermentation of Bacillus flankspora isolated from soil. The biologically active substances produced by Bacillus flankspora have an algaecidal effect on cyanobacteria in many genera, including harmful algae such as fibrillation, fishy algae, microcystic algae, and candida. It hinders the photosynthesis of microalgae and leads to stagnation of cell division, destruction of cyanobacterial chains, and cell lysis, which can effectively control the pollution of water sources by microalgae. Studies of all these active substances have shown great application potential for Bacillus flaspora.
3. Application status and existing problems of Bacillus flanksporum breve
Studies have demonstrated the potential of Bacillus breve diureus flanks to develop as a biocontrol agent for the control of insects, nematodes, mollusks and plant pathogens. Due to the inhibitory effect of Bacillus breve lateral spores on many harmful microorganisms, it has been applied as a feed additive to the intestinal regulation of mammals. Given its prebiotic effects, oral administration of spore preparations of Bacillus breve to poultry can improve feed utilization and increase body weight. In addition, because it has antibacterial properties and can produce unique antibiotic substances, it has been gradually applied to the pharmaceutical industry. Recent related studies have leaned more towards its biodegradation and decontamination properties and highlighted its potential for bioremediation applications.
Genome sequencing of three Bacillus flanks of Bacillus flankspora has been completed in India. In the United States, Bacillus flaspora is successfully used as a biological insecticide to treat animal nematodes. The results of Bacillus flanksopsis bressporidium of Yunnan University in China have also been applied in production. The NovoTM microbial agent produced by the world's enzyme giant - Novozymes in Denmark also contains Bacillus flavorum Breve. Since 2001, there have been more than 40 patents on Bacillus flankspora, most of which involve the preparation and application of compound microbial fertilizers of Bacillus breve, as well as the specific application of Bacillus flanksprung for the treatment of household garbage, fermentation of antibacterial proteins, production of antimicrobial peptides, and as foliar synergists, water quality and soil amendments, algae suppressants, etc. For example, Sun Jianxin and others invented a microbial fertilizer to promote plant growth and its cultivation method, which contains a complex microorganism composed of Bacillus subtilis, Bacillus licheniformis and Bacillus diapsporus; Cao Fengming et al. provide a rapid detection and identification of microbial fertilizer in The technology of Bacillus flanksprungii, using specific primers, PCR amplification according to the sample genomic DNA, detection and identification of Bacillus flanksfolis in microbial fertilizers.
There are still some problems in the practical application of Bacillus flankspora that need to be solved urgently. Mainly including: (1) how to solve the problem of low fermentation yield of Bacillus brevis lateral spore, in order to improve its fermentation yield in actual production and increase production efficiency; (2) the biosecurity problem of Bacillus flambossii organisms into the environment, which requires a large number of field tests to prove the ecological niche occupied in the surrounding environment after use and the impact on ecological balance, and to examine whether its number can be reduced or disappeared in a short period of time after playing a role, so as to maintain the original ecological balance and ensure ecological safety ;(3) How to establish a rapid, accurate and effective method for detecting the content and distribution status of Bacillus breve in the environment, so as to facilitate timely tracing, and further explore the colonization and survival of strains in soil and even plants, the interrelationship with indigenous microorganisms, and monitor the effect of field application in real time. (4) Further research and development of bacillus flanks breve related biodegradable bacteriotic agents and enzyme preparations.
The multiple biological functions of Bacillus breve have a positive impact on production practices and have great application potential. Based on its insecticidal, bacteriostatic and other characteristics, it can continuously develop corresponding microbial agents, health care drugs, biological prevention and control products and environmental protection chemical products. With the completion of the whole genome sequencing of Bacillus flanksopsis Breve, the isolation, cloning and efficient expression of related genes can be gradually carried out, and Bacillus flanksii will play an increasingly important role in human production and life. With the application of transgenic technology and the in-depth study of the nematode-killing effect of Bacillus brevis lateral spore, the corresponding transgenic anti-nematode crops can be further developed to benefit society.
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