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: Targeted microbial control| 13.1: Controlling Microbial Growth | As such, it has been used in either liquid form or as a vapor for the sterilization of medical instruments and tissue grafts, and it is a common component of vaccines, used to maintain their sterility. Chemically, fluoride can become incorporated into the hydroxyapatite of tooth enamel, making it more resistant to corrosive acids produced by the fermentation of oral microbes. e Target-selective bactericidal effect of PIAS. Since an antimicrobial action of a single metabolite is not relevant in many cases, the existing EU regulations may require such a rethinking in the registration of MBCAs as long as antimicrobial metabolites are not present in the formulated MBCA at relevant concentrations. This leads to an overestimation of the importance of this mode of action in comparison to other mechanisms which cannot be detected in such in vitro assays. Sections Sections. |
| Using Chemicals to Control Microorganisms | Microbiology | The data requirements for plant protection products preparations are set out in Commission Regulation EU No. Microbial interactions and biocontrol in the rhizosphere. Let us help you optimize your membrane system. Dysbiosis of the salivary microbiota in pediatric-onset primary sclerosing cholangitis and its potential as a biomarker. The objective of any treatment program should be to expose the attached microbial population to an antimicrobial dosage sufficient to penetrate and disrupt the biofilm. |
| Implementing microbial control interventions on beef and veal in provincially licensed plants | For example, invasive applications that require insertion into the human body require a much higher level of cleanliness than applications that do not. The second factor is the level of resistance to antimicrobial treatment by potential pathogens. For example, foods preserved by canning often become contaminated with the bacterium Clostridium botulinum , which produces the neurotoxin that causes botulism. Because C. botulinum can produce endospores that can survive harsh conditions, extreme temperatures and pressures must be used to eliminate the endospores. Other organisms may not require such extreme measures and can be controlled by a procedure such as washing clothes in a laundry machine. For researchers or laboratory personnel working with pathogens, the risks associated with specific pathogens determine the levels of cleanliness and control required. Various organizations around the world, including the World Health Organization WHO and the European Union EU , use a similar classification scheme. Each BSL requires a different level of biocontainment to prevent contamination and spread of infectious agents to laboratory personnel and, ultimately, the community. For example, the lowest BSL, BSL-1, requires the fewest precautions because it applies to situations with the lowest risk for microbial infection. BSL-1 agents are those that generally do not cause infection in healthy human adults. These include noninfectious bacteria, such as nonpathogenic strains of Escherichia coli and Bacillus subtilis , and viruses known to infect animals other than humans, such as baculoviruses insect viruses. Because working with BSL-1 agents poses very little risk, few precautions are necessary. Laboratory workers use standard aseptic technique and may work with these agents at an open laboratory bench or table, wearing personal protective equipment PPE such as a laboratory coat, goggles, and gloves, as needed. Other than a sink for handwashing and doors to separate the laboratory from the rest of the building, no additional modifications are needed. These include bacteria such as Staphylococcus aureus and Salmonella spp. BSL-2 laboratories are equipped with self-closing doors, an eyewash station, and an autoclave, which is a specialized device for sterilizing materials with pressurized steam before use or disposal. BSL-1 laboratories may also have an autoclave. BSL-3 agents have the potential to cause lethal infections by inhalation. Because of the serious nature of the infections caused by BSL-3 agents, laboratories working with them require restricted access. Laboratory workers are under medical surveillance, possibly receiving vaccinations for the microbes with which they work. In addition to the standard PPE already mentioned, laboratory personnel in BSL-3 laboratories must also wear a respirator and work with microbes and infectious agents in a biological safety cabinet at all times. BSL-3 laboratories require a hands-free sink, an eyewash station near the exit, and two sets of self-closing and locking doors at the entrance. These laboratories are equipped with directional airflow, meaning that clean air is pulled through the laboratory from clean areas to potentially contaminated areas. This air cannot be recirculated, so a constant supply of clean air is required. BSL-4 agents are the most dangerous and often fatal. These microbes are typically exotic, are easily transmitted by inhalation, and cause infections for which there are no treatments or vaccinations. Examples include Ebola virus and Marburg virus, both of which cause hemorrhagic fevers, and smallpox virus. There are only a small number of laboratories in the United States and around the world appropriately equipped to work with these agents. In addition to BSL-3 precautions, laboratory workers in BSL-4 facilities must also change their clothing on entering the laboratory, shower on exiting, and decontaminate all material on exiting. While working in the laboratory, they must either wear a full-body protective suit with a designated air supply or conduct all work within a biological safety cabinet with a high-efficiency particulate air HEPA -filtered air supply and a doubly HEPA-filtered exhaust. The laboratory itself must be located either in a separate building or in an isolated portion of a building and have its own air supply and exhaust system, as well as its own decontamination system. What are some factors used to determine the BSL necessary for working with a specific pathogen? The most extreme protocols for microbial control aim to achieve sterilization: the complete removal or killing of all vegetative cells, endospores, and viruses from the targeted item or environment. Sterilization protocols are generally reserved for laboratory, medical, manufacturing, and food industry settings, where it may be imperative for certain items to be completely free of potentially infectious agents. Sterilization can be accomplished through either physical means, such as exposure to high heat, pressure, or filtration through an appropriate filter, or by chemical means. Chemicals that can be used to achieve sterilization are called sterilant s. Sterilants effectively kill all microbes and viruses, and, with appropriate exposure time, can also kill endospores. For many clinical purposes, aseptic technique is necessary to prevent contamination of sterile surfaces. Aseptic technique involves a combination of protocols that collectively maintain sterility, or asepsis, thus preventing contamination of the patient with microbes and infectious agents. Medical procedures that carry risk of contamination must be performed in a sterile field, a designated area that is kept free of all vegetative microbes, endospores, and viruses. Sterile fields are created according to protocols requiring the use of sterilized materials, such as packaging and drapings, and strict procedures for washing and application of sterilants. Other protocols are followed to maintain the sterile field while the medical procedure is being performed. One food sterilization protocol, commercial sterilization, uses heat at a temperature low enough to preserve food quality but high enough to destroy common pathogens responsible for food poisoning, such as C. botulinum and its endospores are commonly found in soil, they may easily contaminate crops during harvesting, and these endospores can later germinate within the anaerobic environment once foods are canned. Metal cans of food contaminated with C. To eliminate the risk for C. botulinum contamination, commercial food-canning protocols are designed with a large margin of error. They assume an impossibly large population of endospores 10 12 per can and aim to reduce this population to 1 endospore per can to ensure the safety of canned foods. For example, low- and medium-acid foods are heated to °C for a minimum of 2. Even so, commercial sterilization does not eliminate the presence of all microbes; rather, it targets those pathogens that cause spoilage and foodborne diseases, while allowing many nonpathogenic organisms to survive. The Association of Surgical Technologists publishes standards for aseptic technique, including creating and maintaining a sterile field. Sterilization protocols require procedures that are not practical, or necessary, in many settings. Various other methods are used in clinical and nonclinical settings to reduce the microbial load on items. The process of disinfection inactivates most microbes on the surface of a fomite by using antimicrobial chemicals or heat. Because some microbes remain, the disinfected item is not considered sterile. Ideally, disinfectants should be fast acting, stable, easy to prepare, inexpensive, and easy to use. An example of a natural disinfectant is vinegar; its acidity kills most microbes. Chemical disinfectants, such as chlorine bleach or products containing chlorine, are used to clean nonliving surfaces such as laboratory benches, clinical surfaces, and bathroom sinks. Typical disinfection does not lead to sterilization because endospores tend to survive even when all vegetative cells have been killed. Unlike disinfectants, antiseptics are antimicrobial chemicals safe for use on living skin or tissues. Examples of antiseptics include hydrogen peroxide and isopropyl alcohol. The process of applying an antiseptic is called antisepsis. In addition to the characteristics of a good disinfectant, antiseptics must also be selectively effective against microorganisms and able to penetrate tissue deeply without causing tissue damage. The type of protocol required to achieve the desired level of cleanliness depends on the particular item to be cleaned. For example, those used clinically are categorized as critical, semicritical, and noncritical. Critical items must be sterile because they will be used inside the body, often penetrating sterile tissues or the bloodstream; examples of critical items include surgical instruments, catheters, and intravenous fluids. Gastrointestinal endoscopes and various types of equipment for respiratory therapies are examples of semicritical items; they may contact mucous membranes or nonintact skin but do not penetrate tissues. CURRENTLY ON BACKORDER - ARRIVING EARLY FEB TUDCA is a bile acid naturally formed in the body that supports the digestion and utilization of fats and oils Search for " {{ result }} ". For New Zealand: We offer free tracked shipping on all orders over 50NZD. For Australia: We offer free tracked DHL EXPRESS shipping over NZD. For United States of America: We offer free tracked DHL EXPRESS shipping over NZD. For remaining countries we are working to achieve better rates but for the moment shipping is DHL EXPRESS shipping for 99NZD. Click and Collect is also available to those local shoppers. Please allow some time to prepare your order. You will receive a pick up notification email once your order is ready for collection. If you wish to pop into our physical store we operate out of the gorgeous Matakana Village nestled 20 minutes north of Auckland. Find our address and hours on our contact us page. You may return any unopened item that is still in its original packaging to Matakana Online Pharmacy within SEVEN days of receipt of your order as recorded by the courier for an exchange for another product or, under special circumstances, a full refund see below. We can process returns and refunds only for items purchased from Matakana Pharmacy. Please be aware that return courier charges are the responsibility of the consumer. You should expect to receive your refund within four weeks of giving your package to the return shipper, however, in many cases you will receive a refund more quickly. This time period includes the transit time for us to receive your return from the shipper 5 to 10 business days , the time it takes us to process your return once we receive it 3 to 5 business days , and the time it takes your bank to process our refund request 5 to 10 business days. We will meet all our obligations under the Consumer Guarantees Act. We cannot accept items for exchange that are returned after SEVEN days. Please note that we are not obligated to refund items unless they are defective or damaged. A full refund will be given in this instance. If you are returning because item is no longer required, or ordered in error you may be asked to pay the re-shipping fee for exchanges alongside any other additional costs. Home RN Labs RNLabs Micro Clear 60caps. A - Z, Practitioner Only Products, R-T, RN Labs. RNLabs Micro Clear 60caps. Default variant. Add To Cart Added Sold Out Add to Cart. Add To Wishlist Added To Wishlist. Add To Compare Added To Compare. 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Targeted microbial control -
In these interactions, different enzymes are secreted in subsequent events, regulated by signaling by different secondary metabolites Karlsson et al. Furthermore, isolates selected for high constitutive enzyme production may not be strong competitors in competitive environments because they continuously invest into formation of metabolites which are needed only for their function in the particular situations of antagonism in close contact with the host.
Due to this high complexity of a hyperparasitism, which often is a cascade of events, all depending on each other and leading to ultimate cell death only after activating the whole cascade, screening assays should not focus in a simplified way on single events, such as formation of a single enzyme, but should measure the final results of the entire cascade of events.
Enzymes such as CWDEs are complex proteins consisting of several or amino acids with the function to catalyze the conversion of specific substrates into specific products. Functioning of enzymes depends not only on amino acid sequences but also on their complex tertiary structures Iyer and Ananthanarayan, Unfolding of these structure or disordered polypeptides lead to enzyme denaturation and irreversible loss of the enzymatic activity.
Enzymes are sensitive to physical denaturation, e. The generally high sensitivity of enzymes to denaturation is a main obstacle in technological processes so that enzyme stabilization during production and application is common in technological applications.
Proteases, cellulases, lipases, amylases, and other enzymes are produced at industrial scales by microorganisms and are commonly used in paper processing, food manufacture, medical device cleaning, ethanol manufacture, as well as many common household cleaning processes such as laundry and dishwashing Anonymous, Enzymes used for such technical applications have been tested through many years and it has been proven that enzymes have a very safe toxicological profile with a good record of occupational health and safety for the consumer.
Studies revealed that enzymes seem unlikely to be dangerous to the aquatic environment due to their ready biodegradability and the low effects on aquatic organisms observed Anonymous, Cell wall-degrading enzymes are commonly produced in the environment by microorganisms during decomposition of organic matter originating from dead plant tissues and dead microorganisms including dead fungal hyphae, and continuously play an essential role in nutrient cycling in all ecosystems.
Given this background activity of enzymatic CWDEs in natural ecosystems, application of hyperparasites in biological control will not significantly increase cell wall degrading activities in the environment.
Hyperparasites produce low amounts of fungal CWDEs during short time periods locally in micro-niches when they interact with their hosts. The produced low amounts of chitinases, β-1,3-glucanases and proteases present in the environment very locally during short time periods are substrate-specific and highly sensitive to denaturation in the environment with its usually high microbial activity combined with chemical and physical factors enhancing enzyme denaturation.
In conclusions relevant toxicological and ecotoxicological risks of hyperparasite applications can be considered as very low because activities are highly specific, production is restricted in time and space and rapid denaturation is common. The development of resistance by a plant pathogen against hyperparasitism by a biological control agents has not yet been reported.
Pathogens can develop resting structures such as endospores, chlamydospores, and melanised sclerotia with high resistance against hyperparasitism by naturally occurring antagonistic microorganisms Bardin et al. Pathogens can also repress synthesis of enzymes needed by the antagonist for hyperparasitic interactions.
A considerable variation in susceptibility of S. sclerotiorum to the commercially applied hyperparasite Coniothyrium minitans has been observed in different regions in France Nicot et al. Sclerotia produced by the various strains of S.
sclerotiorum differed in average thickness and thickness of their melanised cortical tissue. However, both morphological traits did not correlate with susceptibility to hyperparasitism by C. minitans Nicot et al.
With the background of continuous selection pressure by hyperparasites present in the natural microbiome it is not likely that a temporal increase of this pressure by an antagonist application will enhance resistance of the pathogen.
In conclusion, antagonists with hyperparasitism as mode of action can be selected using adequate bioassays Table 1. They generally have a narrow host range and their activity depends on environmental conditions because their antagonistic activity depends on active growth.
The risk for development of resistance against hyperparasites by pathogens can be considered as low. Hyperparasitism acts through CWDEs which production is highly regulated by signaling from the potential host pathogen.
Since these enzymes, ubiquitously produced in all ecosystems, are highly substrate specific and highly susceptible to rapid degradation, toxicological and eco-toxicological risks can be considered as very low Table 2 and do not warrant a risk assessment. Antimicrobial metabolites are secondary metabolites belonging to heterogeneous groups of organic, low-molecular weight compounds produced by microorganisms that are deleterious to the growth or metabolic activities of other microorganisms Thomashow et al.
They are produced and released to the environment in small quantities by many microorganisms. Huge numbers of known antibiotics are produced by actinomycetes different antibiotics , bacteria and fungi Bérdy, Approximately one-third of the bacterial divisions have no cultured representatives and are known only through rRNA sequences Clardy et al.
It thus can be assumed that the majority of antibiotics produced in situ in the environment is still unknown Raaijmakers and Mazzola, Microbial genome analysis revealed huge numbers of cryptic antibiotic gene clusters encoding still unknown antibiotics.
Antimicrobial metabolites are often considered as the most potent mode of action of microorganisms against competitors allowing antibiotic producing microorganisms competitive advantages in resource-limited environments Raaijmakers and Mazzola, Production of antimicrobial metabolites, mostly with broad-spectrum activity, has been reported for biocontrol bacteria belonging to Agrobacterium , Bacillus , Pantoea , Pseudomonas , Serratia , Stenotrophomonas , Streptomyces , and many other genera.
In Bacillus , especially lipopeptides such as iturin, surfactin, and fengycin have been investigated Ongena and Jacques, , in Pseudomonas many antibiotic metabolites such as DAPG, pyrrolnitrin and phenazine have been studied Raaijmakers and Mazzola, Many antibiotics are produced only when a microbial population reaches certain thresholds.
This quorum-sensing phenomenon is well described for phenazine-producing Pseudomonas. Genomic information reveals that also these genera have the potential to produce many still unknown secondary metabolites with possible antimicrobial activity. Also fungal antagonists can produce antimicrobial compounds.
For Trichoderma and closely related Clonostachys former Gliocladium , 6-PAP, gliovirin, gliotoxin, viridin and many more compounds with antimicrobial activity have been investigated Ghorbanpour et al.
Microorganisms producing antimicrobial metabolites with the potential to interfere with antibiotics in human and veterinary medicine must be excluded from use as MBCAs Anonymous, a. The inhibitory effect of secondary metabolites on spore germination or hyphal growth of pathogens can be quantified in vitro on nutrient media testing the effects of the antagonistic microorganisms cultured in dual cultures, their metabolites as present in supernatants of cultures of these microorganisms or the purified concentrations of the metabolite.
In vitro assays are used since the early beginning of scientific research on microbial antagonists, e. Studying inhibitory effects of potential antagonists on agar or in liquid media in dual cultures has several advantages.
Assays are fast, resource efficient, highly reproducible and effects are easily to be quantified by measuring colony sizes or percentages of germinated spores.
The resulting inhibition zones visualize clearly biocontrol effects and are often used to explain the principles of biocontrol. These advantages may also have led to a bias in biocontrol research. Screening of new antagonists often starts with using in vitro assays which are very suitable to detect in vitro antagonists which act via antimicrobial metabolites in the artificial environment.
This leads to an overestimation of the importance of this mode of action in comparison to other mechanisms which cannot be detected in such in vitro assays. As a biased result, in self-fulfilling prophecy, in vitro assays may confirm the importance of in vitro antibiosis in biocontrol by systematically excluding other modes of action.
The main disadvantage of in vitro dual cultures is that production of secondary metabolites depends on nutrient concentration and composition of the chosen medium.
Common nutrient media are approximately times richer in nutrients compared to the rhizosphere, and bulk soils are even much less rich in nutrients, supporting even 10— times less bacteria than the rhizosphere Lugtenberg et al.
Consequently, amounts of secondary metabolites in in vitro systems are much higher than reached in natural habitats. Furthermore, agar media or liquid media are ideal for diffusion of the antibiotic compounds which is not the case in habitats such as soil or leaf surfaces.
Several studies demonstrated that in vitro antagonism does not predict antagonism in complex assays including host plants which simulate the natural habitat situation under controlled or even in field situations Knudsen et al.
An example is the screening of Trichoderma isolates for their potential to control R. Köhl tested isolates belonging to T. viride , T. hamatum , T. harzianum , or T. koningii in dual cultures with R. solani and in pot experiments with lambs lettuce seeds planted in R. solani infested soil.
Dual cultures on yeast dextrose agar revealed antagonistic isolates. For these isolates, the average efficacy in reduction of damping off in the pot experiments was For the remaining 64 isolates, showing no in vitro antagonism, the average efficacy in pot experiments was similar with This example demonstrates that in vitro antagonism depends on the chosen conditions and by far does not explain the antagonistic potential of isolates.
Also recent transcriptomic studies confirm that in vitro produced metabolites may not be expressed or play a minor role in situ Koch et al. Antibiosis observed on agar plates historically resulted in the development of pharmaceutical antibiotics. With similar expectations, results of agar plates often are translated to the control of plant pathogens in the field situation with antimicrobial metabolites seen as sole mode of action against competitors.
There is very limited information on measured antimicrobial effects of antagonists in situ compared to the large number of publications of in vitro effects. Transcriptome analyses of microbial activities in soil confirms that antimicrobial metabolites are produced in soil.
Raaijmakers and Mazzola listed results of various authors who quantified different antibiotics produced in situ in soils by bacterial strains introduced at high densities.
Production of 5 ng to 5 μg per gram of soil or plant tissue were reported depending on experimental conditions, strains used and type of produced antibiotic with exceptional higher values up to μg per gram for a Bacillus subtilis isolate.
Antibiotic concentration may be higher in certain microniches, but an important fraction of the antibiotics may be bound to the producing cells and may not diffuse in the habitat Raaijmakers and Mazzola, Antibiotics are not stable in the soil environment.
Arseneault and Filion report on half-life of antibiotics produced by biocontrol strains in soil ranging between 0. Such short life spans can be due to microbial decomposition but also to chemical and physical inactivation. Information on in situ concentration of antimicrobial metabolites produced by MBCAs against plant disease and their life span is hardly to be quantified and therefore often missing and not included in risk assessments on non-target effects Mudgal et al.
Despite the low concentrations, the inhomogeneous distribution and short lifespan of antimicrobial compounds produced by biocontrol strains in situ , studies with mutants of biocontrol strains disrupted in specific antibiotic synthesis demonstrated that antibiotic metabolites play an important role in microbial interactions in soil and plant surfaces Handelsman and Stabb, ; Raaijmakers and Mazzola, There is increasing evidence that antimicrobial metabolites have important functions for the producing microorganisms at subinhibitory concentrations.
In other words: such compounds are characterized as being antibiotic because of their effect on microorganisms at high concentration under in vitro conditions although their function in the natural habitat is very different at the prevailing lower concentrations.
Arseneault and Filion discuss modulation of gene expression by low antibiotic concentrations instead of inciting of cell death at high concentrations. Antibiotics at low concentrations can be involved in signaling and microbial community interactions, communication with plants, and regulation of biofilm formation.
Raaijmakers and Mazzola discussed a range of functions of antimicrobial metabolites at low concentrations: there is evidence that antimicrobials including lipopetides protect bacteria from grazing by bacteriovorus nematodes such as Caenorhabditis elegans.
Also volatile antibiotic compounds may play a role in long-distance interactions amongst soil organisms including bacterial predators. Lipopeptides of Bacillus and Pseudomonas are involved in the surface attachment of bacterial cells and biofilm formation by activating signaling cascades finally resulting in the formation of extracellular matrices which protect microorganisms from adverse environmental stresses.
Some antibiotics, especially lipopeptides support the mobility of bacteria, most likely via changing the viscosity of the colonized surfaces. Surface-active antibiotics allow bacteria to move to nutrient rich locations and also change the water dynamics on leaf surfaces which indirectly affects pathogen development.
Other groups of antibiotics influence the nutritional status of plants. For example, DAPG-producing Pseudomonas upregulates the nitrogen fixation by plant growth-promoting Azospirillum brasilense , and redox-active antibiotics support mobilization of limiting nutrients such as manganese and iron.
Screening of new antagonists acting through antimicrobial metabolites needs to address the insights in ecological functioning of such compounds.
Efficient antagonists produce antimicrobial metabolites in situ in microniches at sufficiently high concentrations to gain advantage over competitors or at low concentration to fulfill various functions like signaling or nutrient mobilizations, thus functions different from antibiosis.
As for most other modes of action, the design of adequate bioassays is essential which combine interactions between potential antagonist, pathogen, plant and are conducted under representative environmental conditions regarding soil environment and microclimate.
The often applied in vitro screening by far does not mimic the real conditions under which antagonists should be active. However, screening under in vitro conditions for strong producers of potential antimicrobial compounds is the first method if the exploitation of the metabolites is envisaged.
Antimicrobial metabolites can be produced by selected isolates of antagonistic bacteria or fungi in bioreactors in fermentation processes optimized for high yield of the preferred metabolite. Commercial biological control products may contain microbial metabolites as active ingredient together with the producing microbial antagonist so that after application the direct effect of the metabolite is combined with the potential production of additional metabolite in situ.
Other products may contain only the produced metabolites, possibly in combination with remains of dead cells of the producing antagonist. Such a use of microbial metabolites is strictly speaking outside the scientific definition of biological control which is defined as the use of living beneficial organisms to suppress populations of plant pathogens Heimpel and Mills, , but in a broader definition, use of metabolites is also considered as biological control Glare et al.
Several reports demonstrate variability within pathogen populations in their sensitivity to antimicrobial secondary metabolites. Selected isolates of Pseudomonas spp. produce DAPG with antimicrobial activity against several plant pathogens.
A high diversity in sensitivity to DAPG between isolates for Gaeumannomyces graminis var. tritici has been reported by Mazzola et al.
cinerea by Schouten et al. Isolates of B. cinerea also differ in sensitivity to pyrrolnitrin Ajouz et al. These examples indicate that selection pressure by broad use of biological control agents with a single antimicrobial secondary metabolite as mode of action may result in the selection of less sensitive pathogen strains so that the efficacy of the MBCA will not be durable.
For B. cinerea , a pathogen with high potential to develop resistance against chemical fungicides through adaptation, adaptation to antimicrobial compounds produced by MBCAs has been found Li and Leifert, A similar adaptation to pyrrolnitrin, produced by P.
chlororaphis , was developed by strains of B. cinerea in in vitro assays with increasing concentrations of the antimicrobial compound in agar growth media Ajouz et al. Interestingly, the build-up of resistance resulted in reduced fitness of the strains so that such strains will not persist in absence of selection pressure by pyrrolnitrin.
Pathogen strains with higher resistance against antimicrobial compound produced by MBCAs are able to excrete such compounds, e. Since selection pressure depends on dose and exposure duration, the risk for building up resistance is lower if the antimicrobial compounds are produced by the antagonist in situ only during direct interaction with the pathogen, often even at subinhibitory concentrations, compared to situations were formulated antimicrobial compounds produced by antagonists already during fermentation are applied at higher dose to the entire crop.
Risk assessments are required for registration of MBCAs as plant protection products for antimicrobial metabolites which are considered as relevant Anonymous, Plant pathogenic microorganisms potentially producing mycotoxins and human and animal pathogens potentially producing neurotoxins are excluded from use in biological control.
Other secondary metabolites with proven antimicrobial activity which are produced by MBCAs in bioreactors and applied as formulated bioactive compounds included in the end product in amounts effective in disease control Glare et al.
If such metabolites potentially are produced in vitro , but not present in the MBCA or only at low concentration, they are not relevant for risk assessment Sachana, However, for the majority of MBCAs, antimicrobial metabolites are produced at low concentrations in situ in microniches with low nutrient availability.
Concentrations are subinhibitory if modes of action different from antibiosis are exploited Raaijmakers and Mazzola, In other situations, metabolite production may be locally and temporally above a minimal inhibitory concentration resulting in inhibition or killing of the targeted pathogen.
Such an antibiosis will be restricted in time because of the short life span of antimicrobial metabolites in the environment. Furthermore, the producing antagonist populations will drop after application Scheepmaker and van de Kassteele, There is a continuum of microbial activity including production of a great variety of secondary metabolites in the natural environment.
Unlimited growth of applied saprophytic microorganisms, often a fear of regulators, will not occur in the environment where saprophytic microbial populations are regulated by competitive exploitation of limited resources.
Thus, applications of MBCAs with potential in situ production of antimicrobial metabolites will not add relevant toxicological or eco-toxicological risks to the cropping system. In conclusion, antagonists with antimicrobial metabolites as mode of action can be selected using adequate bioassays if in situ production by living antagonists is envisaged or in vitro if the application of the formulated metabolites is envisaged Table 1.
They generally have a broad host range and their activity depends on environmental conditions if their antagonistic activity depends on in situ production, thus on active growth. The risk for development of resistance against antimicrobial metabolites by pathogens can be considered as low in cases where metabolites are produced in situ.
In cases where a single formulated microbial metabolite is applied on crops, the risk of development of resistance will be, depending on the genetics of the targeted pathogen and the stability of the metabolite in the environment, comparable to risk for chemical active substances. Because of the low concentrations of in situ produced antimicrobial metabolites in microniches with low nutrient availability in combination with the typically short lifespans of the metabolites in the environment and the presence of antimicrobial metabolites produced by indigenous microorganisms, toxicological and eco-toxicological risks can be considered as low.
If formulated metabolites are applied, their toxicological and eco-toxicological risks are determined by their toxicological profile, the applied concentration and their stability in the environment Table 2.
The research on mode of action of MBCAs usually focuses on induced resistance and priming, competition, hyperparasitism, and antibiosis, but more modes of action are known. For example, fungal viruses in the family Hypoviridae are used to induce hypovirulence in Cryphonectria parasitica , the causing agent of chestnut blight Milgroom and Cortesi, ; Double et al.
Other antagonists act via the inactivation of enzymes involved in pathogen infections Elad, , see below or the enzymatic degradation of pathogen structures such as a lectin needed by the rice blast pathogen Magnaporthe oryzae for spore attachment on the host leaf surface which can be degraded by a specifically selected isolate of Chryseobacterium sp.
Ikeda et al. It can be expected that employing multi-omics will identify many still undetected ways of interactions between microorganisms. It is also known that secondary metabolites and other compounds produced by MBCAs can act through different modes of action.
For example, DAPG can have a direct effect as antimicrobial metabolite against the pathogen but also acts as MAMP Pieterse et al. Thus, both antibiosis and induced resistance act simultaneously and an artificial separation between the in situ effect of DAPG on a single mode of action is hardly possible.
Another example is the production of iron-binding siderophores for nutrient competition with the pathogen that are also recognized by the plants as MAMPs inducing resistance Höfte and Bakker, The systematic discrimination of the modes of action of MBCAs is a scientific exercise to unravel how MBCAs act.
This information is important for optimizing the use of MBCAs but also asked for registration where the mode of action has to be indicated Anonymous, a. However, nature of microbial interactions is more complex and does not fit into such pragmatic categories of scientists, regulators, and risk managers.
In many cases where the mode of action intensively has been studied for a single biocontrol strain, results confirm that antagonistic interactions are driven by more than one mode of action. Separation into different modes of action is also not always clear and seems to be artificial.
For example, Trichoderma spp. produce hydrolytic enzymes that permeabilize and degrade the fungal cell wall as one of the key steps in the successful attack of the fungal hosts Karlsson et al. The increased permeability of the cell wall is facilitating the subsequent entry of secondary antimicrobial metabolites.
Isolate T39 of Trichoderma harzianum , originally selected for the control of B. cinerea , also controls the foliar pathogens Pseudoperonospora cubensis , S. sclerotiorum , and Sphaerotheca fusca Elad, Isolates of antagonistic Trichoderma spp. are generally known to produce antimicrobial metabolites and to act via hyperparasitism Harman et al.
Detailed studies on T. harzianum T39 revealed that no antimicrobial metabolites are interfering with the targeted pathogens. The isolate is able to produce chitinases but Elad found no correlation between the ability of this strain or other, non-antagonistic strains of T.
harzianum with their biocontrol activity. harzianum T39 produces several proteases in situ on bean leaves which restrain enzymes of B.
The proteases reduced the activities of the pathogen enzymes exo- and endopolygalacturonase, pectin methyl esterase, pectate lyase, chitinase, β-1,3-glucanase, and cutinase, that are essential for the pathogen during host infection. In experiments with protease inhibitors the biocontrol effect was fully or partially nullified.
The biocontrol effect of T. harzianum T39 can thus partly be explained by the production of enzymes which suppress pathogen enzymes. The other proven modes of action of T. harzianum T39 were nutrient competition, ISR and locally induced resistance.
Elad concluded that various modes of action are responsible for the control of biotrophic and necrotrophic foliar pathogens by T. harzianum T39 and he assumed that multiple mechanisms are also involved in other biocontrol systems, but in most cases only part of the possible mechanisms have been elucidated.
Pseudozyma flocculosa is an efficient antagonist of Erysiphales Bélanger et al. flocculosa can produce 6-methylheptadecanoic acid and the glycolipid flocculosin. Since there was no evidence for induced resistance in treated plants and nutrient competition seemed to be unlikely in antagonism against a biotrophic pathogen, it was concluded that antibiosis is the sole mode of action.
However, gene expression studies revealed that there was no significant increase in expression of the relevant genes at any time during the antagonistic process so that other modes of action must be responsible Bélanger et al.
There is now increasing evidence that competition for the micronutrients Zn and Mn plays a role during the dedicated tritrophic interaction: powdery mildew takes up these elements from the host plant and P. flocculosa draws these elements then from the pathogen.
Both examples of in depth investigations of the mode of action of MBCAs illustrate that tritrophic interactions between host, pathogen and MBCA are complex and often different from what is initially expected Elad, ; Bélanger et al. New, rather unexpected combinations of mechanisms may be revealed by future analysis of the increasing genomic and transcriptomic information.
Current examples are studies on gene expression of Clonostachys rosea Nygren et al. Such a highly regulated in situ production of various ubiquitous mechanisms commonly used in the microbial interplay in the environment makes the use of MBCAs a particular safe and sustainable technology.
Because of the ubiquitous character of in situ modes of action specific risk assessments are not relevant. Because of the complexity of the cascades of physiological events the indication of the principal single mode of action as data requirement of Commission Regulation Anonymous, a ; see Box 1 is impossible.
Box 1. What are the data requirements and the uniform principles concerning the mode of action of the microorganism against plant diseases in the EU? The most important data requirements related to the mode of action of active substances are set out in Commission Regulation EU No.
The data requirements for plant protection products preparations are set out in Commission Regulation EU No.
must be stated. It must also be stated whether or not the product is translocated in plants and, where relevant, if such translocation is apoplastic, symplastic or both. The uniform principles for evaluation and authorisation of plant protection products are set out in Commission Regulation EU No.
The micro-organism in the plant protection product should ideally function as a cell factory working directly on the spot where the target organism is harmful.
The characterization and identification of relevant metabolites must be assessed and the toxicity of these metabolites must be addressed. The mode of action of the micro-organism shall be evaluated in as much detail as appropriate. a antibiosis;. b induction of plant resistance;. c interference with the virulence of a pathogenic target organism;.
d endophytic growth;. e root colonization;. f competition of ecological niche e. g parasitization;. h invertebrate pathogenicity. Mode of action is taken into account at evaluation of the degree of adverse effects on the treated crop, operator exposure, viable residues, fate, and behavior in the environment and at risk assessment of birds, mammals, aquatic organisms, bees, arthropods other than bees and earthworms and nitrogen and carbon mineralization in the soil.
Microbial biological control agents interact with the plant, the targeted pathogen and the resident microflora.
Studies on the interactions with the resident microflora have been hampered in the past because of limitations of available methods.
This changed drastically with arrival of Next Generation Sequencing NGS methods such as metagenomics and metatranscriptomics allowing to identify the composition and functions of the microbiome Massart et al.
Further steps by adding information of metametabolomics and signalomics Mhlongo et al. As a result, a holistic in depth understanding on MBCA-microbiota interactions will support better timing, formulation and application of MBCAs and prevent failures.
It is expected that three new developments will have significant impact on biological control of plant diseases. Application of specific compounds or complex substrates will modulate indigenous microbiota compositions with the aim to enhance microbial suppression of plant pathogens Mazzola and Freilich, Such a manipulation of resident microbiota toward disease suppression may be comparable to conservation biological control applied in insect pest control, e.
Simple or complex substrates applied for such a prebiotic approach may not be considered as plant protection products. A third expectation is that core microbiomes will be designed Gopal et al. Gopal et al. The ecological considerations supporting the idea of assembled consortia are sound Table 1.
However, practical considerations may hamper their introduction. Validation and optimization of in silico -designed consortia under ranges of relevant environmental conditions will be complex and will need substantial resources.
In a commercial setting, development of mass production, down streaming and storage procedures separately for each individual consortium member will need substantially more resources and investments compared to production of single strain MBCAs Table 1.
Registration of assembled consortia as plant protection products will add further difficulties. Regulations in the EU demand the risk assessment of each active ingredient before the product can be registered.
In case of assembled consortia, costs will thus increase substantially. In this context, strategies to develop helper strains or to shape the indigenous microbiota may clearly have advantages above the use of assembled core consortia Table 2.
On the other hand, an adapted legislation for novel disease control systems would benefit society as a whole as well as the environment. Microbial biological control agents use a broad arsenal of modes of action which are used wherever microorganisms interact, communicate, and regulate their co-existence between microbial cells and between microorganisms and plants.
The exploitation of different modes of action has different advantages and disadvantages in relation to the development of commercial MBCAs by industries and their practical use by growers Table 1 , but also regarding the perception of possible toxicological and ecotoxicological risks for producers, users, consumers, and the environment Table 2.
Studies on mode of action of well-documented antagonists show that antagonism generally is not based on a single action of a certain mode of action, but on a sequence of events with the use of different modes of action over time.
During such cascades of physiological events signals often are the result of the earlier used modes of action, e. For the development of specific biocontrol products, certain modes of action may be preferred. In such cases, screening of new MBCAs can be very focused, e.
This screening strategy may be powerful if new strains are being selected superior to an existing, well characterized antagonist or for further strain improvement within an existing antagonist strain. However, in most other cases, selection procedures should be preferred that allow the selection of new combinations of known and still unknown modes of action which are produced directly at the site of interaction.
The key challenge for screening projects is thus the development of suitable robust bioassays which combine the interactions between pathogen, host, and antagonist under controlled conditions.
Attractive alternative routes via in vitro tests should not be used to avoid biased selection with emphasis on one mode of action, thus excluding many other powerful modes of action or combination thereof, which may be even ineffective at all if evaluated on their own.
The efficacy of biological control agents against plant diseases may not be durable because pathogen populations may develop resistance comparable to the frequently observed build-up of resistance against chemical fungicides with a single mode of action. Important factors for an erosion of effectiveness are variation in susceptibility to the mode of action within the pathogen population, selection pressure resulting in shifts within pathogen populations toward less susceptible strains and the fitness of the selected strains in the environment under conditions without selection pressure Bardin et al.
Variation in susceptibility of pathogens has been found for some pathogens such as S. sclerotiorum and G. graminis var. tritici Mazzola et al.
However, development of resistance has not reported yet for commercially used biological control products for control of plant diseases Nicot et al.
The risk of resistance development in MBCAs used in sustainable IPM systems is also low because IPM combines a variety of measures to prevent damage by diseases without relying on a single control method. The build-up of resistance is a serious problem in single molecule-single mode of action chemical fungicides which shorten their economic life span.
For MBCAs the principle modes of action exhibit much less selection pressure on pathogens additional to the always present selection pressure during natural competitive interactions of organisms.
Furthermore, it is common that a combination of different modes of action are active and each mode of action is based on multiple actors, e. For MBCAs it thus can be concluded that build-up of resistance is much less likely compared to the build-up of resistance against chemical plant protection products.
Only exceptional uses of MBCAs such as the use of in vitro produced highly concentrated and purified secondary metabolites or the use of genetically modified MBCAs with extraordinarily high expression of a single antimicrobial metabolite may result in selection pressures comparable to single site fungicides.
Knowledge of the mode of action of the microorganisms is required and has also to be considered in the context of other potential risks before a MBCA can be approved for use as plant protection product. Risk assessments of MBCAs are regulated in the EU by Regulation EC No.
The regulations focus strongly on the possible risks of secondary metabolites and toxins potentially produced by microorganisms. Several groups of fungi are known to produce mycotoxins, several groups of bacteria are known to produce toxins including the botulinum-neurotoxin BoNT.
Microorganisms producing such mycotoxins or toxins in relevant amounts are excluded from the use in biological control because of the potential risks for humans and animals. MBCAs may produce other secondary metabolites as sole mode of action, or — in the majority of cases — as component of a cascade of different secondary metabolites in combination or alternation with other metabolites such as CWDEs or MAMPS.
The function of the produced metabolites often is not antibiosis but to fulfill other functions including signaling at subinhibitory concentrations. Secondary metabolite production is highly regulated and restricted to micro niches and in time.
Such metabolites are rapidly degraded and thus have short life spans in the environment. Only for MBCAs which produce potential antimicrobial metabolites in vitro or during the mass production fermentation process and contain such metabolites in the formulated end product at effective concentration, thorough risk assessment is indicated and the minimal effective concentration against the target and representative non-target organisms can be established.
However, in all other cases, such metabolites are not relevant for a risk assessment. Furthermore, reliable quantification of temporal metabolite concentrations in microniches in the in situ situation can hardly to be achieved. The perception of risks caused by antimicrobial metabolites in biological control may be more a result of the broad use of in vitro studies on antibiosis in biocontrol research rather than the result of studies on on-site production of such metabolites in the environment.
In vitro antagonism can easily be visualized through inhibition zones on culture media. The similar method is used for the screening of pharmaceutical antibiotics that aims at the development of products containing single molecules for medical treatments.
Communication on biocontrol research based on in vitro assays, showing inhibition zones, may create a wrong view on the nature of biocontrol control resulting in the fear of the use of antibiotics in crop protection. Since results of in vitro assays generally do not correlate with results obtained in bioassays or with crops Koch et al.
Biocontrol research unraveling the mechanisms in the much more complex in situ situations may reduce the unjustified fears for microbial metabolites produced by MBCAs. In conclusion, MBCAs are functioning directly on the spot where the targeted organism is harmful Anonymous, , generally combining different modes of action to highly regulated cascades of events.
Current thinking on how to consider the mode of action during the risk assessment and registration procedure of MBCAs focuses on a single mode of action and potential risks of in vitro produced metabolites, very similar to the risk assessment of synthetic fungicides with a single compound as active ingredient.
A rethinking is needed considering that the effectiveness of MBCAs in most cases is based on natural, complex, highly regulated interactions between microbial cells and plants on site but are not the results of a single action of a single metabolite.
Toxicological and ecotoxicological risks of such complex processes of interaction can be considered as very low. Moreover, humans and other organisms have been and still are exposed to such processes in evolutionary terms and adverse effects are not known.
Since an antimicrobial action of a single metabolite is not relevant in many cases, the existing EU regulations may require such a rethinking in the registration of MBCAs as long as antimicrobial metabolites are not present in the formulated MBCA at relevant concentrations. Microbial biological control agents use a great variety of mechanisms to protect plants from pathogens.
Important modes of action strengthen the resistance of the plant, e. Hyperparasitism and secondary metabolites are directly affecting the targeted pathogen via highly regulated cascades of physiological events but not by a single constitutively produced metabolite.
Secondary metabolites produced in vitro may have antimicrobial activity at high concentration but low amounts are produced in situ very locally during interaction and metabolites have short life spans, often with functions such as signaling, very different from antibiosis.
During the cascades of events a range of different compounds with different modes of action are used to outcompete the pathogen. Such events of signaling and interaction are common wherever microorganisms interact.
The highly regulated in situ production of ubiquitous mechanisms commonly used in the microbial interplay makes the use of MBCAs a safe and sustainable technology.
In situ produced compounds such as MAMPs, enzymes or secondary metabolites are not relevant for risk assessments so that detailed toxicology and ecotoxicological studies of these compounds are not relevant, and should not be required.
The fear of antimicrobial metabolites produced by MBCAs after their release is not based on real risks but fed by the wrong perception on how biocontrol acts if studied under in vitro conditions. If antimicrobial metabolites are the active ingredient in the formulation of the biocontrol product, risk assessment of such metabolites is relevant.
Better screening assays for finding the next generation of MBCAs are needed to measure the overall effect of the interplay of different modes of action. Multi-omics will help to further understand the complex events during microbial interactions in the environment. Current EU regulations on registration of MBCAs should allow a science-based differentiation between the majority of compounds involved in modes of action to be considered as safe and not relevant for detailed risk assessment and the limited number of cases relevant for risk assessments where secondary metabolites are present as active ingredients in MBCAs formulations at high concentrations.
JK conceived and designed the research. JK, RK, and WR contributed to the manuscript and revised it critically for important intellectual content. All authors approved the final version of the manuscript. RK was employed by company Linge Agroconsultancy b.
and WR was employed by company Koppert Biological Systems. JK declares no competing interests. We thank the members of the greenTEAM of the Board for the Authorisation of Plant Protection Products and Biocides CTGB , Ede, Netherlands, for their valuable comments on earlier versions of the manuscript.
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Variability of Botrytis cinerea sensitivity to pyrrolnitrin, an antibiotic produced by biological control agents. Biocontrol 56, — Amann, R. Phylogenetic identification and in situ detection of individual microbial cells without cultivation.
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Bakker, R. Barton and B. Hemming San Diego: Academic Press , — Bardin, M. Is the efficacy of biological control against plant diseases likely to be more durable than that of chemical pesticides?
Plant Sci. PubMed Abstract CrossRef Full Text Google Scholar. Bélanger, R. Mode of action of biocontrol agents: all that glitters is not gold. Bérdy, J. Bioactive microbial metabolites. Boller, T. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors.
Plant Biol. Calvo-Garrido, C. Suppression of Botrytis cinerea on necrotic grapevine tissues by early-season applications of natural products and biological control agents.
Carisse, O. Effect of fall application of fungal antagonist on spring ascospore production of the apple scab pathogen. Venturia inaequalis. Phytopathology 90, 31— Chou, M. The effect of pollen grains on infections caused by Botrytis cinerea. Clardy, J. New antibiotics from bacterial natural products.
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Phytopathology 96, — Trichoderma species — opportunistic, avirulent plant symbionts. Heimpel, G. Biological Control - Ecology and Applications. Cambridge: Cambridge University Press. Hijmegen, T. Isolation and identification of hyperparasitic fungi associated with Erysiphaceae. Höfte, M.
Varma and S. Chincholkar Berlin: Springer-Verlag , — Ikeda, K. Attachment of airborne pathogens to their host: a potential target for disease control. Acta Phytopathol. Please leave us a note to let us know if you have been prescribed this product before by a Practitioner. RN Labs Micro Clear is a potent combination of botanicals to provide a targeted yet comprehensive anti-microbial effect.
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Figure 2. Triclosan is a common ingredient in antibacterial soaps despite evidence that it poses environmental and health risks and offers no significant health benefit compared to conventional soaps. credit b, c: modification of work by FDA.
Some of the first chemical disinfectants and antiseptics to be used were heavy metals. Heavy metals kill microbes by binding to proteins, thus inhibiting enzymatic activity. Heavy metals are oligodynamic, meaning that very small concentrations show significant antimicrobial activity.
Ions of heavy metals bind to sulfur-containing amino acids strongly and bioaccumulate within cells, allowing these metals to reach high localized concentrations. This causes proteins to denature. Heavy metals are not selectively toxic to microbial cells.
They may bioaccumulate in human or animal cells, as well, and excessive concentrations can have toxic effects on humans. If too much silver accumulates in the body, for example, it can result in a condition called argyria , in which the skin turns irreversibly blue-gray.
One way to reduce the potential toxicity of heavy metals is by carefully controlling the duration of exposure and concentration of the heavy metal.
Figure 3. Heavy metals denature proteins, impairing cell function and, thus, giving them strong antimicrobial properties. a Copper in fixtures like this door handle kills microbes that otherwise might accumulate on frequently touched surfaces.
b Eating utensils contain small amounts of silver to inhibit microbial growth. c Copper commonly lines incubators to minimize contamination of cell cultures stored inside. d Antiseptic mouthwashes commonly contain zinc chloride. e This patient is suffering from argyria, an irreversible condition caused by bioaccumulation of silver in the body.
Fred and Hendrik A. van Dijk. Mercury is an example of a heavy metal that has been used for many years to control microbial growth. It was used for many centuries to treat syphilis. Mercury compounds like mercuric chloride are mainly bacteriostatic and have a very broad spectrum of activity.
Various forms of mercury bind to sulfur-containing amino acids within proteins, inhibiting their functions. It is toxic to the central nervous, digestive, and renal systems at high concentrations, and has negative environmental effects, including bioaccumulation in fish. Topical antiseptics such as mercurochrome , which contains mercury in low concentrations, and merthiolate , a tincture a solution of mercury dissolved in alcohol were once commonly used.
However, because of concerns about using mercury compounds, these antiseptics are no longer sold in the United States. Silver has long been used as an antiseptic. In ancient times, drinking water was stored in silver jugs. Silver nitrate drops were once routinely applied to the eyes of newborns to protect against ophthalmia neonatorum , eye infections that can occur due to exposure to pathogens in the birth canal, but antibiotic creams are more now commonly used.
Silver is often combined with antibiotics, making the antibiotics thousands of times more effective. Several other heavy metals also exhibit antimicrobial activity. Copper sulfate is a common algicide used to control algal growth in swimming pools and fish tanks. The use of metallic copper to minimize microbial growth is also becoming more widespread.
Copper linings in incubators help reduce contamination of cell cultures. The use of copper pots for water storage in underdeveloped countries is being investigated as a way to combat diarrheal diseases. Copper coatings are also becoming popular for frequently handled objects such as doorknobs, cabinet hardware, and other fixtures in health-care facilities in an attempt to reduce the spread of microbes.
Nickel and zinc coatings are now being used in a similar way. Other forms of zinc, including zinc chloride and zinc oxide , are also used commercially.
Zinc chloride is quite safe for humans and is commonly found in mouthwashes, substantially increasing their length of effectiveness.
Zinc oxide is found in a variety of products, including topical antiseptic creams such as calamine lotion, diaper ointments, baby powder, and dandruff shampoos.
Other chemicals commonly used for disinfection are the halogens iodine , chlorine , and fluorine. Iodine works by oxidizing cellular components, including sulfur-containing amino acids, nucleotides, and fatty acids, and destabilizing the macromolecules that contain these molecules.
It is often used as a topical tincture, but it may cause staining or skin irritation. One common iodophor is povidone-iodine , which includes a wetting agent that releases iodine relatively slowly.
Figure 4. a Betadine is a solution of the iodophor povidone-iodine. credit b: modification of work by Andrew Ratto. Chlorine is another halogen commonly used for disinfection.
When chlorine gas is mixed with water, it produces a strong oxidant called hypochlorous acid, which is uncharged and enters cells easily.
Chlorine gas is commonly used in municipal drinking water and wastewater treatment plants, with the resulting hypochlorous acid producing the actual antimicrobial effect.
Those working at water treatment facilities need to take great care to minimize personal exposure to chlorine gas. Sodium hypochlorite is the chemical component of common household bleach , and it is also used for a wide variety of disinfecting purposes.
Hypochlorite salts, including sodium and calcium hypochlorites, are used to disinfect swimming pools. Chlorine gas, sodium hypochlorite, and calcium hypochlorite are also commonly used disinfectants in the food processing and restaurant industries to reduce the spread of foodborne diseases.
Workers in these industries also need to take care to use these products correctly to ensure their own safety as well as the safety of consumers. A recent joint statement published by the Food and Agriculture Organization FAO of the United Nations and WHO indicated that none of the many beneficial uses of chlorine products in food processing to reduce the spread of foodborne illness posed risks to consumers.
Another class of chlorinated compounds called chloramines are widely used as disinfectants. Chloramines are relatively stable, releasing chlorine over long periods time. Chloramines are derivatives of ammonia by substitution of one, two, or all three hydrogen atoms with chlorine atoms.
Figure 5. Monochloroamine, one of the chloramines, is derived from ammonia by the replacement of one hydrogen atom with a chlorine atom.
Chloramines and other cholorine compounds may be used for disinfection of drinking water, and chloramine tablets are frequently used by the military for this purpose. After a natural disaster or other event that compromises the public water supply, the CDC recommends disinfecting tap water by adding small amounts of regular household bleach.
Recent research suggests that sodium dichloroisocyanurate NaDCC may also be a good alternative for drinking water disinfection. Currently, NaDCC tablets are available for general use and for use by the military, campers, or those with emergency needs; for these uses, NaDCC is preferable to chloramine tablets.
Chlorine dioxide, a gaseous agent used for fumigation and sterilization of enclosed areas, is also commonly used for the disinfection of water. Although chlorinated compounds are relatively effective disinfectants, they have their disadvantages.
Some may irritate the skin, nose, or eyes of some individuals, and they may not completely eliminate certain hardy organisms from contaminated drinking water. The fungus Cryptosporidium , for example, has a protective outer shell that makes it resistant to chlorinated disinfectants.
Thus, boiling of drinking water in emergency situations is recommended when possible. The halogen fluorine is also known to have antimicrobial properties that contribute to the prevention of dental caries cavities.
Chemically, fluoride can become incorporated into the hydroxyapatite of tooth enamel, making it more resistant to corrosive acids produced by the fermentation of oral microbes.
Fluoride also enhances the uptake of calcium and phosphate ions in tooth enamel, promoting remineralization. In addition to strengthening enamel, fluoride also seems to be bacteriostatic.
It accumulates in plaque-forming bacteria, interfering with their metabolism and reducing their production of the acids that contribute to tooth decay. Alcohols make up another group of chemicals commonly used as disinfectants and antiseptics.
They work by rapidly denaturing proteins, which inhibits cell metabolism, and by disrupting membranes, which leads to cell lysis.
Once denatured, the proteins may potentially refold if enough water is present in the solution. This is because alcohols coagulate proteins. In higher alcohol concentrations, rapid coagulation of surface proteins prevents effective penetration of cells.
The most commonly used alcohols for disinfection are ethyl alcohol ethanol and isopropyl alcohol isopropanol, rubbing alcohol.
Alcohols tend to be bactericidal and fungicidal, but may also be viricidal for enveloped viruses only. Although alcohols are not sporicidal, they do inhibit the processes of sporulation and germination.
Alcohols are volatile and dry quickly, but they may also cause skin irritation because they dehydrate the skin at the site of application. One common clinical use of alcohols is swabbing the skin for degerming before needle injection. Alcohols also are the active ingredients in instant hand sanitizers , which have gained popularity in recent years.
The alcohol in these hand sanitizers works both by denaturing proteins and by disrupting the microbial cell membrane, but will not work effectively in the presence of visible dirt.
Last, alcohols are used to make tinctures with other antiseptics, such as the iodine tinctures discussed previously in this chapter. All in all, alcohols are inexpensive and quite effective for the disinfection of a broad range of vegetative microbes. However, one disadvantage of alcohols is their high volatility, limiting their effectiveness to immediately after application.
Figure 6. a Ethyl alcohol, the intoxicating ingredient found in alcoholic drinks, is also used commonly as a disinfectant. b Isopropyl alcohol, also called rubbing alcohol, has a related molecular structure and is another commonly used disinfectant.
credit a photo: modification of work by D Coetzee; credit b photo: modification of work by Craig Spurrier. Surface-active agents, or surfactants , are a group of chemical compounds that lower the surface tension of water.
Surfactants are the major ingredients in soaps and detergents. Soaps are salts of long-chain fatty acids and have both polar and nonpolar regions, allowing them to interact with polar and nonpolar regions in other molecules. They can interact with nonpolar oils and grease to create emulsions in water, loosening and lifting away dirt and microbes from surfaces and skin.
Soaps do not kill or inhibit microbial growth and so are not considered antiseptics or disinfectants. However, proper use of soaps mechanically carries away microorganisms, effectively degerming a surface. Some soaps contain added bacteriostatic agents such as triclocarban or cloflucarban , compounds structurally related to triclosan, that introduce antiseptic or disinfectant properties to the soaps.
Figure 7. Soaps are the salts sodium salt in the illustration of fatty acids and have the ability to emulsify lipids, fats, and oils by interacting with water through their hydrophilic heads and with the lipid at their hydrophobic tails.
Soaps, however, often form films that are difficult to rinse away, especially in hard water, which contains high concentrations of calcium and magnesium mineral salts. Detergents contain synthetic surfactant molecules with both polar and nonpolar regions that have strong cleansing activity but are more soluble, even in hard water, and, therefore, leave behind no soapy deposits.
Anionic detergents , such as those used for laundry, have a negatively charged anion at one end attached to a long hydrophobic chain, whereas cationic detergents have a positively charged cation instead.
Cationic detergents include an important class of disinfectants and antiseptics called the quaternary ammonium salts quats , named for the characteristic quaternary nitrogen atom that confers the positive charge.
Overall, quats have properties similar to phospholipids, having hydrophilic and hydrophobic ends. As such, quats have the ability to insert into the bacterial phospholipid bilayer and disrupt membrane integrity.
The cationic charge of quats appears to confer their antimicrobial properties, which are diminished when neutralized. Quats have several useful properties. They are stable, nontoxic, inexpensive, colorless, odorless, and tasteless.
They tend to be bactericidal by disrupting membranes. They are also active against fungi, protozoans, and enveloped viruses, but endospores are unaffected. In clinical settings, they may be used as antiseptics or to disinfect surfaces. Mixtures of quats are also commonly found in household cleaners and disinfectants, including many current formulations of Lysol brand products, which contain benzalkonium chlorides as the active ingredients.
Benzalkonium chlorides, along with the quat cetylpyrimidine chloride , are also found in products such as skin antiseptics, oral rinses, and mouthwashes. Figure 8. a Two common quats are benzylalkonium chloride and cetylpyrimidine chloride. Note the hydrophobic nonpolar carbon chain at one end and the nitrogen-containing cationic component at the other end.
b Quats are able to infiltrate the phospholipid plasma membranes of bacterial cells and disrupt their integrity, leading to death of the cell.
Handwashing is critical for public health and should be emphasized in a clinical setting. For the general public, the CDC recommends handwashing before, during, and after food handling; before eating; before and after interacting with someone who is ill; before and after treating a wound; after using the toilet or changing diapers; after coughing, sneezing, or blowing the nose; after handling garbage; and after interacting with an animal, its feed, or its waste.
Figure 9 illustrates the five steps of proper handwashing recommended by the CDC. Handwashing is even more important for health-care workers, who should wash their hands thoroughly between every patient contact, after the removal of gloves, after contact with bodily fluids and potentially infectious fomites, and before and after assisting a surgeon with invasive procedures.
Even with the use of proper surgical attire, including gloves, scrubbing for surgery is more involved than routine handwashing. There is no single widely accepted protocol for surgical scrubbing.
According to the Association of Surgical Technologists AST , surgical scrubs may be performed with or without the use of brushes. Figure 9. a The CDC recommends five steps as part of typical handwashing for the general public. b Surgical scrubbing is more extensive, requiring scrubbing starting from the fingertips, extending to the hands and forearms, and then up beyond the elbows, as shown here.
credit a: modification of work by World Health Organization. Bisbiguanides were first synthesized in the 20th century and are cationic positively charged molecules known for their antiseptic properties.
One important bisbiguanide antiseptic is chlorhexidine. It has broad-spectrum activity against yeasts, gram-positive bacteria, and gram-negative bacteria, with the exception of Pseudomonas aeruginosa , which may develop resistance on repeated exposure.
It also has activity against enveloped viruses. However, chlorhexidine is poorly effective against Mycobacterium tuberculosis and nonenveloped viruses, and it is not sporicidal. Chlorhexidine is typically used in the clinical setting as a surgical scrub and for other handwashing needs for medical personnel, as well as for topical antisepsis for patients before surgery or needle injection.
It is more persistent than iodophors, providing long-lasting antimicrobial activity. Chlorhexidine solutions may also be used as oral rinses after oral procedures or to treat gingivitis. Another bisbiguanide, alexidine , is gaining popularity as a surgical scrub and an oral rinse because it acts faster than chlorhexidine.
Figure The bisbiguanides chlorhexadine and alexidine are cationic antiseptic compounds commonly used as surgical scrubs. It is a strong, broad-spectrum disinfectant and biocide that has the ability to kill bacteria, viruses, fungi, and endospores, leading to sterilization at low temperatures, which is sometimes a convenient alternative to the more labor-intensive heat sterilization methods.
It also cross-links proteins and has been widely used as a chemical fixative. Because of this, it is used for the storage of tissue specimens and as an embalming fluid. It also has been used to inactivate infectious agents in vaccine preparation.
Formaldehyde is very irritating to living tissues and is also carcinogenic; therefore, it is not used as an antiseptic. Glutaraldehyde is structurally similar to formaldehyde but has two reactive aldehyde groups, allowing it to act more quickly than formaldehyde.
It is used to disinfect a variety of surfaces and surgical and medical equipment. However, similar to formaldehyde, glutaraldehyde irritates the skin and is not used as an antiseptic. A new type of disinfectant gaining popularity for the disinfection of medical equipment is o-phthalaldehyde OPA , which is found in some newer formulations of Cidex and similar products, replacing glutaraldehyde.
o-Phthalaldehyde also has two reactive aldehyde groups, but they are linked by an aromatic bridge. o-Phthalaldehyde is thought to work similarly to glutaraldehyde and formaldehyde, but is much less irritating to skin and nasal passages, produces a minimal odor, does not require processing before use, and is more effective against mycobacteria.
Ethylene oxide is a type of alkylating agent that is used for gaseous sterilization. It is highly penetrating and can sterilize items within plastic bags such as catheters, disposable items in laboratories and clinical settings like packaged Petri dishes , and other pieces of equipment.
Ethylene oxide exposure is a form of cold sterilization, making it useful for the sterilization of heat-sensitive items. Great care needs to be taken with the use of ethylene oxide, however; it is carcinogenic, like the other alkylating agents, and is also highly explosive. With careful use and proper aeration of the products after treatment, ethylene oxide is highly effective, and ethylene oxide sterilizers are commonly found in medical settings for sterilizing packaged materials.
β-Propionolactone is an alkylating agent with a different chemical structure than the others already discussed. Like other alkylating agents, β-propionolactone binds to DNA, thereby inactivating it. It is a clear liquid with a strong odor and has the ability to kill endospores.
As such, it has been used in either liquid form or as a vapor for the sterilization of medical instruments and tissue grafts, and it is a common component of vaccines, used to maintain their sterility. It has also been used for the sterilization of nutrient broth, as well as blood plasma, milk, and water.
It is quickly metabolized by animals and humans to lactic acid. It is also an irritant, however, and may lead to permanent damage of the eyes, kidneys, or liver. Additionally, it has been shown to be carcinogenic in animals; thus, precautions are necessary to minimize human exposure to β-propionolactone.
a Alkylating agents replace hydrogen atoms with alkyl groups. Here, guanine is alkylated, resulting in its hydrogen bonding with thymine, instead of cytosine. b The chemical structures of several alkylating agents.
Prions, the acellular, misfolded proteins responsible for incurable and fatal diseases such as kuru and Creutzfeldt-Jakob disease see Viroids, Virusoids, and Prions , are notoriously difficult to destroy.
Prions are extremely resistant to heat, chemicals, and radiation. They are also extremely infectious and deadly; thus, handling and disposing of prion-infected items requires extensive training and extreme caution.
Typical methods of disinfection can reduce but not eliminate the infectivity of prions. Autoclaving is not completely effective, nor are chemicals such as phenol, alcohols, formalin, and β-propiolactone. Even when fixed in formalin, affected brain and spinal cord tissues remain infectious.
Personnel who handle contaminated specimens or equipment or work with infected patients must wear a protective coat, face protection, and cut-resistant gloves.
Any contact with skin must be immediately washed with detergent and warm water without scrubbing. The skin should then be washed with 1 N NaOH or a dilution of bleach for 1 minute.
Contaminated waste must be incinerated or autoclaved in a strong basic solution, and instruments must be cleaned and soaked in a strong basic solution. Peroxygens are strong oxidizing agents that can be used as disinfectants or antiseptics.
The most widely used peroxygen is hydrogen peroxide H 2 O 2 , which is often used in solution to disinfect surfaces and may also be used as a gaseous agent. Hydrogen peroxide solutions are inexpensive skin antiseptics that break down into water and oxygen gas, both of which are environmentally safe.
carotovora , Pseudomonas syringae pv. glycinea , P. syringae pv. tomato , P. marginalis , and Erwinia herbicola was confirmed in similar tests McNeely et al. In biotrophic mycoparasitism, the hyperparasite depends on the living host fungus and gains nutrients from the host cells via haustoria without killing the host.
Host and mycoparasitic fungus interact in a stable and balanced way Jeffries, These often species—specific interactions may be important components in disease suppressiveness in ecosystems but are hardly to be exploited for commercial augmentative biocontrol because mass production of the hyperparasite depends on living host mycelium as substrate.
Hyperparasites with a necrotrophic life style gain nutrients from dead host cells but also from other commonly available organic matter which allow mass production on artificial media making this group of hyperparasites much more favorable for commercial use as MBCA compared to biotrophic hyperparasites.
Necrotrophic hyperparasites invade host spores or hyphal cells after killing such cells. Main mechanisms of parasitism is the excretion of CWDEs combined in some cases with excretion of secondary metabolites in close contact with the host cell leading to openings in the cell wall and subsequent disorganization of the cytoplasm.
Cell wall degradation is typically caused by a range of chitinases, β-1,3-glucanases and proteases or, in case of hyperparasites of oomycota, cellulases. Such a necrotrophic hyperparasitism with invasion of killed host cells is frequently observed by microscopy and electron microscopy.
The assumed nutrient transfer from the dead host cell to the invading fungus often has not been proven because it is technically difficult to investigate such processes, especially in the in situ situation in the field Jeffries, For some pathogen groups, researchers thoroughly investigated the phenomenon of hyperparasitism and found many antagonistic fungal species.
For example, 30 hyperparasitic species against Rhizoctonia solani belonging to 16 genera have been reported by Jeffries Obligate biotrophic pathogens have been of particular interest for biocontrol using hyperparasites.
Hijmegen and Buchenauer report on eight hyperparasitic species of powdery mildews. Zheng et al. tritici Zheng et al. In bioassays on rust-inoculated wheat seedlings, A.
alternata germ tubes contacted with and penetrated into urediniospores of the pathogen at 24 hpi, and caused complete urediniospore collapse at 36—48 hpi. The most studied mycoparasites are belonging to the genera Trichoderma and Clonostachys. Antagonistic isolates of these genera vary in host range and individual strains mostly have a range of plant pathogenic hosts.
They produce structures for attachment and infection, and kill their hosts by CWDEs, often in combination with antimicrobial secondary metabolites Harman et al. These lytic enzymes are not constitutive but their production is triggered by complex signaling after recognition of the host.
Surface compounds such as lectins from the host cell wall, surface properties and diffusible host-released secondary metabolites play important roles in the recognition and signaling pathways such as MAPK cascades, cAMP pathway and G-protein signaling Karlsson et al.
Mycoparasitism-related gene families in Trichoderma such as ech42 and prb1 are upregulated during mycoparasitism. As result of the initial activities of CWDEs, oligosaccharides and oligopeptides are released by the host that are then recognized by Trichoderma receptors and thereby act as inducers Karlsson et al.
This attack by a necrotrophic mycoparasite results in further increase of permeability and degradation of host cell walls and death of the host.
A synergistic transcription of various genes involved in cell wall degradation was also reported for Trichoderma atroviride in interaction with B. cinerea and Phytophthora capsici Reithner et al. Screening for hyperparasitic strains often is done by using host structures as baits, especially if such structures are large for easy handling and observations.
Sclerotia, e. solani , individual urediniospores or pustules of rust pathogens, and individual conidia or pustules of powdery mildew have been exposed to potential antagonist candidates and macroscopical and microscopical observations were made to find strains which invade the host structures, often accompanied with discoloration of these structures.
Such studies generally are completed by assessments of the viability of invaded host structures, e. Alternative screening of candidate antagonists for their activity of fungal CWDEs under in vitro conditions seems to be less adequate because activity levels of single enzymes in situations without interaction with the hosts will not be representative for the highly regulated interplay between antagonist and pathogen.
In these interactions, different enzymes are secreted in subsequent events, regulated by signaling by different secondary metabolites Karlsson et al. Furthermore, isolates selected for high constitutive enzyme production may not be strong competitors in competitive environments because they continuously invest into formation of metabolites which are needed only for their function in the particular situations of antagonism in close contact with the host.
Due to this high complexity of a hyperparasitism, which often is a cascade of events, all depending on each other and leading to ultimate cell death only after activating the whole cascade, screening assays should not focus in a simplified way on single events, such as formation of a single enzyme, but should measure the final results of the entire cascade of events.
Enzymes such as CWDEs are complex proteins consisting of several or amino acids with the function to catalyze the conversion of specific substrates into specific products. Functioning of enzymes depends not only on amino acid sequences but also on their complex tertiary structures Iyer and Ananthanarayan, Unfolding of these structure or disordered polypeptides lead to enzyme denaturation and irreversible loss of the enzymatic activity.
Enzymes are sensitive to physical denaturation, e. The generally high sensitivity of enzymes to denaturation is a main obstacle in technological processes so that enzyme stabilization during production and application is common in technological applications.
Proteases, cellulases, lipases, amylases, and other enzymes are produced at industrial scales by microorganisms and are commonly used in paper processing, food manufacture, medical device cleaning, ethanol manufacture, as well as many common household cleaning processes such as laundry and dishwashing Anonymous, Enzymes used for such technical applications have been tested through many years and it has been proven that enzymes have a very safe toxicological profile with a good record of occupational health and safety for the consumer.
Studies revealed that enzymes seem unlikely to be dangerous to the aquatic environment due to their ready biodegradability and the low effects on aquatic organisms observed Anonymous, Cell wall-degrading enzymes are commonly produced in the environment by microorganisms during decomposition of organic matter originating from dead plant tissues and dead microorganisms including dead fungal hyphae, and continuously play an essential role in nutrient cycling in all ecosystems.
Given this background activity of enzymatic CWDEs in natural ecosystems, application of hyperparasites in biological control will not significantly increase cell wall degrading activities in the environment.
Hyperparasites produce low amounts of fungal CWDEs during short time periods locally in micro-niches when they interact with their hosts. The produced low amounts of chitinases, β-1,3-glucanases and proteases present in the environment very locally during short time periods are substrate-specific and highly sensitive to denaturation in the environment with its usually high microbial activity combined with chemical and physical factors enhancing enzyme denaturation.
In conclusions relevant toxicological and ecotoxicological risks of hyperparasite applications can be considered as very low because activities are highly specific, production is restricted in time and space and rapid denaturation is common.
The development of resistance by a plant pathogen against hyperparasitism by a biological control agents has not yet been reported.
Pathogens can develop resting structures such as endospores, chlamydospores, and melanised sclerotia with high resistance against hyperparasitism by naturally occurring antagonistic microorganisms Bardin et al.
Pathogens can also repress synthesis of enzymes needed by the antagonist for hyperparasitic interactions. A considerable variation in susceptibility of S. sclerotiorum to the commercially applied hyperparasite Coniothyrium minitans has been observed in different regions in France Nicot et al.
Sclerotia produced by the various strains of S. sclerotiorum differed in average thickness and thickness of their melanised cortical tissue. However, both morphological traits did not correlate with susceptibility to hyperparasitism by C. minitans Nicot et al. With the background of continuous selection pressure by hyperparasites present in the natural microbiome it is not likely that a temporal increase of this pressure by an antagonist application will enhance resistance of the pathogen.
In conclusion, antagonists with hyperparasitism as mode of action can be selected using adequate bioassays Table 1. They generally have a narrow host range and their activity depends on environmental conditions because their antagonistic activity depends on active growth.
The risk for development of resistance against hyperparasites by pathogens can be considered as low. Hyperparasitism acts through CWDEs which production is highly regulated by signaling from the potential host pathogen.
Since these enzymes, ubiquitously produced in all ecosystems, are highly substrate specific and highly susceptible to rapid degradation, toxicological and eco-toxicological risks can be considered as very low Table 2 and do not warrant a risk assessment.
Antimicrobial metabolites are secondary metabolites belonging to heterogeneous groups of organic, low-molecular weight compounds produced by microorganisms that are deleterious to the growth or metabolic activities of other microorganisms Thomashow et al.
They are produced and released to the environment in small quantities by many microorganisms. Huge numbers of known antibiotics are produced by actinomycetes different antibiotics , bacteria and fungi Bérdy, Approximately one-third of the bacterial divisions have no cultured representatives and are known only through rRNA sequences Clardy et al.
It thus can be assumed that the majority of antibiotics produced in situ in the environment is still unknown Raaijmakers and Mazzola, Microbial genome analysis revealed huge numbers of cryptic antibiotic gene clusters encoding still unknown antibiotics.
Antimicrobial metabolites are often considered as the most potent mode of action of microorganisms against competitors allowing antibiotic producing microorganisms competitive advantages in resource-limited environments Raaijmakers and Mazzola, Production of antimicrobial metabolites, mostly with broad-spectrum activity, has been reported for biocontrol bacteria belonging to Agrobacterium , Bacillus , Pantoea , Pseudomonas , Serratia , Stenotrophomonas , Streptomyces , and many other genera.
In Bacillus , especially lipopeptides such as iturin, surfactin, and fengycin have been investigated Ongena and Jacques, , in Pseudomonas many antibiotic metabolites such as DAPG, pyrrolnitrin and phenazine have been studied Raaijmakers and Mazzola, Many antibiotics are produced only when a microbial population reaches certain thresholds.
This quorum-sensing phenomenon is well described for phenazine-producing Pseudomonas. Genomic information reveals that also these genera have the potential to produce many still unknown secondary metabolites with possible antimicrobial activity.
Also fungal antagonists can produce antimicrobial compounds. For Trichoderma and closely related Clonostachys former Gliocladium , 6-PAP, gliovirin, gliotoxin, viridin and many more compounds with antimicrobial activity have been investigated Ghorbanpour et al.
Microorganisms producing antimicrobial metabolites with the potential to interfere with antibiotics in human and veterinary medicine must be excluded from use as MBCAs Anonymous, a. The inhibitory effect of secondary metabolites on spore germination or hyphal growth of pathogens can be quantified in vitro on nutrient media testing the effects of the antagonistic microorganisms cultured in dual cultures, their metabolites as present in supernatants of cultures of these microorganisms or the purified concentrations of the metabolite.
In vitro assays are used since the early beginning of scientific research on microbial antagonists, e. Studying inhibitory effects of potential antagonists on agar or in liquid media in dual cultures has several advantages. Assays are fast, resource efficient, highly reproducible and effects are easily to be quantified by measuring colony sizes or percentages of germinated spores.
The resulting inhibition zones visualize clearly biocontrol effects and are often used to explain the principles of biocontrol. These advantages may also have led to a bias in biocontrol research.
Screening of new antagonists often starts with using in vitro assays which are very suitable to detect in vitro antagonists which act via antimicrobial metabolites in the artificial environment. This leads to an overestimation of the importance of this mode of action in comparison to other mechanisms which cannot be detected in such in vitro assays.
As a biased result, in self-fulfilling prophecy, in vitro assays may confirm the importance of in vitro antibiosis in biocontrol by systematically excluding other modes of action.
The main disadvantage of in vitro dual cultures is that production of secondary metabolites depends on nutrient concentration and composition of the chosen medium. Common nutrient media are approximately times richer in nutrients compared to the rhizosphere, and bulk soils are even much less rich in nutrients, supporting even 10— times less bacteria than the rhizosphere Lugtenberg et al.
Consequently, amounts of secondary metabolites in in vitro systems are much higher than reached in natural habitats. Furthermore, agar media or liquid media are ideal for diffusion of the antibiotic compounds which is not the case in habitats such as soil or leaf surfaces.
Several studies demonstrated that in vitro antagonism does not predict antagonism in complex assays including host plants which simulate the natural habitat situation under controlled or even in field situations Knudsen et al.
An example is the screening of Trichoderma isolates for their potential to control R. Köhl tested isolates belonging to T. viride , T. hamatum , T. harzianum , or T. koningii in dual cultures with R. solani and in pot experiments with lambs lettuce seeds planted in R. solani infested soil.
Dual cultures on yeast dextrose agar revealed antagonistic isolates. For these isolates, the average efficacy in reduction of damping off in the pot experiments was For the remaining 64 isolates, showing no in vitro antagonism, the average efficacy in pot experiments was similar with This example demonstrates that in vitro antagonism depends on the chosen conditions and by far does not explain the antagonistic potential of isolates.
Also recent transcriptomic studies confirm that in vitro produced metabolites may not be expressed or play a minor role in situ Koch et al.
Antibiosis observed on agar plates historically resulted in the development of pharmaceutical antibiotics.
With similar expectations, results of agar plates often are translated to the control of plant pathogens in the field situation with antimicrobial metabolites seen as sole mode of action against competitors.
There is very limited information on measured antimicrobial effects of antagonists in situ compared to the large number of publications of in vitro effects. Transcriptome analyses of microbial activities in soil confirms that antimicrobial metabolites are produced in soil.
Raaijmakers and Mazzola listed results of various authors who quantified different antibiotics produced in situ in soils by bacterial strains introduced at high densities.
Production of 5 ng to 5 μg per gram of soil or plant tissue were reported depending on experimental conditions, strains used and type of produced antibiotic with exceptional higher values up to μg per gram for a Bacillus subtilis isolate. Antibiotic concentration may be higher in certain microniches, but an important fraction of the antibiotics may be bound to the producing cells and may not diffuse in the habitat Raaijmakers and Mazzola, Antibiotics are not stable in the soil environment.
Arseneault and Filion report on half-life of antibiotics produced by biocontrol strains in soil ranging between 0. Such short life spans can be due to microbial decomposition but also to chemical and physical inactivation.
Information on in situ concentration of antimicrobial metabolites produced by MBCAs against plant disease and their life span is hardly to be quantified and therefore often missing and not included in risk assessments on non-target effects Mudgal et al. Despite the low concentrations, the inhomogeneous distribution and short lifespan of antimicrobial compounds produced by biocontrol strains in situ , studies with mutants of biocontrol strains disrupted in specific antibiotic synthesis demonstrated that antibiotic metabolites play an important role in microbial interactions in soil and plant surfaces Handelsman and Stabb, ; Raaijmakers and Mazzola, There is increasing evidence that antimicrobial metabolites have important functions for the producing microorganisms at subinhibitory concentrations.
In other words: such compounds are characterized as being antibiotic because of their effect on microorganisms at high concentration under in vitro conditions although their function in the natural habitat is very different at the prevailing lower concentrations. Arseneault and Filion discuss modulation of gene expression by low antibiotic concentrations instead of inciting of cell death at high concentrations.
Antibiotics at low concentrations can be involved in signaling and microbial community interactions, communication with plants, and regulation of biofilm formation. Raaijmakers and Mazzola discussed a range of functions of antimicrobial metabolites at low concentrations: there is evidence that antimicrobials including lipopetides protect bacteria from grazing by bacteriovorus nematodes such as Caenorhabditis elegans.
Also volatile antibiotic compounds may play a role in long-distance interactions amongst soil organisms including bacterial predators. Lipopeptides of Bacillus and Pseudomonas are involved in the surface attachment of bacterial cells and biofilm formation by activating signaling cascades finally resulting in the formation of extracellular matrices which protect microorganisms from adverse environmental stresses.
Some antibiotics, especially lipopeptides support the mobility of bacteria, most likely via changing the viscosity of the colonized surfaces.
Surface-active antibiotics allow bacteria to move to nutrient rich locations and also change the water dynamics on leaf surfaces which indirectly affects pathogen development. Other groups of antibiotics influence the nutritional status of plants. For example, DAPG-producing Pseudomonas upregulates the nitrogen fixation by plant growth-promoting Azospirillum brasilense , and redox-active antibiotics support mobilization of limiting nutrients such as manganese and iron.
Screening of new antagonists acting through antimicrobial metabolites needs to address the insights in ecological functioning of such compounds.
Efficient antagonists produce antimicrobial metabolites in situ in microniches at sufficiently high concentrations to gain advantage over competitors or at low concentration to fulfill various functions like signaling or nutrient mobilizations, thus functions different from antibiosis.
As for most other modes of action, the design of adequate bioassays is essential which combine interactions between potential antagonist, pathogen, plant and are conducted under representative environmental conditions regarding soil environment and microclimate.
The often applied in vitro screening by far does not mimic the real conditions under which antagonists should be active. However, screening under in vitro conditions for strong producers of potential antimicrobial compounds is the first method if the exploitation of the metabolites is envisaged.
Antimicrobial metabolites can be produced by selected isolates of antagonistic bacteria or fungi in bioreactors in fermentation processes optimized for high yield of the preferred metabolite. Commercial biological control products may contain microbial metabolites as active ingredient together with the producing microbial antagonist so that after application the direct effect of the metabolite is combined with the potential production of additional metabolite in situ.
Other products may contain only the produced metabolites, possibly in combination with remains of dead cells of the producing antagonist. Such a use of microbial metabolites is strictly speaking outside the scientific definition of biological control which is defined as the use of living beneficial organisms to suppress populations of plant pathogens Heimpel and Mills, , but in a broader definition, use of metabolites is also considered as biological control Glare et al.
Several reports demonstrate variability within pathogen populations in their sensitivity to antimicrobial secondary metabolites. Selected isolates of Pseudomonas spp. produce DAPG with antimicrobial activity against several plant pathogens.
A high diversity in sensitivity to DAPG between isolates for Gaeumannomyces graminis var. tritici has been reported by Mazzola et al. cinerea by Schouten et al. Isolates of B. cinerea also differ in sensitivity to pyrrolnitrin Ajouz et al.
These examples indicate that selection pressure by broad use of biological control agents with a single antimicrobial secondary metabolite as mode of action may result in the selection of less sensitive pathogen strains so that the efficacy of the MBCA will not be durable.
For B. cinerea , a pathogen with high potential to develop resistance against chemical fungicides through adaptation, adaptation to antimicrobial compounds produced by MBCAs has been found Li and Leifert, A similar adaptation to pyrrolnitrin, produced by P.
chlororaphis , was developed by strains of B. cinerea in in vitro assays with increasing concentrations of the antimicrobial compound in agar growth media Ajouz et al.
Interestingly, the build-up of resistance resulted in reduced fitness of the strains so that such strains will not persist in absence of selection pressure by pyrrolnitrin. Pathogen strains with higher resistance against antimicrobial compound produced by MBCAs are able to excrete such compounds, e.
Since selection pressure depends on dose and exposure duration, the risk for building up resistance is lower if the antimicrobial compounds are produced by the antagonist in situ only during direct interaction with the pathogen, often even at subinhibitory concentrations, compared to situations were formulated antimicrobial compounds produced by antagonists already during fermentation are applied at higher dose to the entire crop.
Risk assessments are required for registration of MBCAs as plant protection products for antimicrobial metabolites which are considered as relevant Anonymous, Plant pathogenic microorganisms potentially producing mycotoxins and human and animal pathogens potentially producing neurotoxins are excluded from use in biological control.
Other secondary metabolites with proven antimicrobial activity which are produced by MBCAs in bioreactors and applied as formulated bioactive compounds included in the end product in amounts effective in disease control Glare et al. If such metabolites potentially are produced in vitro , but not present in the MBCA or only at low concentration, they are not relevant for risk assessment Sachana, However, for the majority of MBCAs, antimicrobial metabolites are produced at low concentrations in situ in microniches with low nutrient availability.
Concentrations are subinhibitory if modes of action different from antibiosis are exploited Raaijmakers and Mazzola, In other situations, metabolite production may be locally and temporally above a minimal inhibitory concentration resulting in inhibition or killing of the targeted pathogen.
Such an antibiosis will be restricted in time because of the short life span of antimicrobial metabolites in the environment. Furthermore, the producing antagonist populations will drop after application Scheepmaker and van de Kassteele, There is a continuum of microbial activity including production of a great variety of secondary metabolites in the natural environment.
Unlimited growth of applied saprophytic microorganisms, often a fear of regulators, will not occur in the environment where saprophytic microbial populations are regulated by competitive exploitation of limited resources.
Thus, applications of MBCAs with potential in situ production of antimicrobial metabolites will not add relevant toxicological or eco-toxicological risks to the cropping system. In conclusion, antagonists with antimicrobial metabolites as mode of action can be selected using adequate bioassays if in situ production by living antagonists is envisaged or in vitro if the application of the formulated metabolites is envisaged Table 1.
They generally have a broad host range and their activity depends on environmental conditions if their antagonistic activity depends on in situ production, thus on active growth. The risk for development of resistance against antimicrobial metabolites by pathogens can be considered as low in cases where metabolites are produced in situ.
In cases where a single formulated microbial metabolite is applied on crops, the risk of development of resistance will be, depending on the genetics of the targeted pathogen and the stability of the metabolite in the environment, comparable to risk for chemical active substances.
Because of the low concentrations of in situ produced antimicrobial metabolites in microniches with low nutrient availability in combination with the typically short lifespans of the metabolites in the environment and the presence of antimicrobial metabolites produced by indigenous microorganisms, toxicological and eco-toxicological risks can be considered as low.
If formulated metabolites are applied, their toxicological and eco-toxicological risks are determined by their toxicological profile, the applied concentration and their stability in the environment Table 2. The research on mode of action of MBCAs usually focuses on induced resistance and priming, competition, hyperparasitism, and antibiosis, but more modes of action are known.
For example, fungal viruses in the family Hypoviridae are used to induce hypovirulence in Cryphonectria parasitica , the causing agent of chestnut blight Milgroom and Cortesi, ; Double et al. Other antagonists act via the inactivation of enzymes involved in pathogen infections Elad, , see below or the enzymatic degradation of pathogen structures such as a lectin needed by the rice blast pathogen Magnaporthe oryzae for spore attachment on the host leaf surface which can be degraded by a specifically selected isolate of Chryseobacterium sp.
Ikeda et al. It can be expected that employing multi-omics will identify many still undetected ways of interactions between microorganisms. It is also known that secondary metabolites and other compounds produced by MBCAs can act through different modes of action.
For example, DAPG can have a direct effect as antimicrobial metabolite against the pathogen but also acts as MAMP Pieterse et al. Thus, both antibiosis and induced resistance act simultaneously and an artificial separation between the in situ effect of DAPG on a single mode of action is hardly possible.
Another example is the production of iron-binding siderophores for nutrient competition with the pathogen that are also recognized by the plants as MAMPs inducing resistance Höfte and Bakker, The systematic discrimination of the modes of action of MBCAs is a scientific exercise to unravel how MBCAs act.
This information is important for optimizing the use of MBCAs but also asked for registration where the mode of action has to be indicated Anonymous, a. However, nature of microbial interactions is more complex and does not fit into such pragmatic categories of scientists, regulators, and risk managers.
In many cases where the mode of action intensively has been studied for a single biocontrol strain, results confirm that antagonistic interactions are driven by more than one mode of action. Separation into different modes of action is also not always clear and seems to be artificial.
For example, Trichoderma spp. produce hydrolytic enzymes that permeabilize and degrade the fungal cell wall as one of the key steps in the successful attack of the fungal hosts Karlsson et al. The increased permeability of the cell wall is facilitating the subsequent entry of secondary antimicrobial metabolites.
Isolate T39 of Trichoderma harzianum , originally selected for the control of B. cinerea , also controls the foliar pathogens Pseudoperonospora cubensis , S. sclerotiorum , and Sphaerotheca fusca Elad, Isolates of antagonistic Trichoderma spp. are generally known to produce antimicrobial metabolites and to act via hyperparasitism Harman et al.
Detailed studies on T. harzianum T39 revealed that no antimicrobial metabolites are interfering with the targeted pathogens. The isolate is able to produce chitinases but Elad found no correlation between the ability of this strain or other, non-antagonistic strains of T.
harzianum with their biocontrol activity. harzianum T39 produces several proteases in situ on bean leaves which restrain enzymes of B. The proteases reduced the activities of the pathogen enzymes exo- and endopolygalacturonase, pectin methyl esterase, pectate lyase, chitinase, β-1,3-glucanase, and cutinase, that are essential for the pathogen during host infection.
In experiments with protease inhibitors the biocontrol effect was fully or partially nullified. The biocontrol effect of T.
harzianum T39 can thus partly be explained by the production of enzymes which suppress pathogen enzymes. The other proven modes of action of T. harzianum T39 were nutrient competition, ISR and locally induced resistance.
Elad concluded that various modes of action are responsible for the control of biotrophic and necrotrophic foliar pathogens by T. harzianum T39 and he assumed that multiple mechanisms are also involved in other biocontrol systems, but in most cases only part of the possible mechanisms have been elucidated.
Pseudozyma flocculosa is an efficient antagonist of Erysiphales Bélanger et al. flocculosa can produce 6-methylheptadecanoic acid and the glycolipid flocculosin. Since there was no evidence for induced resistance in treated plants and nutrient competition seemed to be unlikely in antagonism against a biotrophic pathogen, it was concluded that antibiosis is the sole mode of action.
However, gene expression studies revealed that there was no significant increase in expression of the relevant genes at any time during the antagonistic process so that other modes of action must be responsible Bélanger et al. There is now increasing evidence that competition for the micronutrients Zn and Mn plays a role during the dedicated tritrophic interaction: powdery mildew takes up these elements from the host plant and P.
flocculosa draws these elements then from the pathogen. Both examples of in depth investigations of the mode of action of MBCAs illustrate that tritrophic interactions between host, pathogen and MBCA are complex and often different from what is initially expected Elad, ; Bélanger et al.
New, rather unexpected combinations of mechanisms may be revealed by future analysis of the increasing genomic and transcriptomic information. Current examples are studies on gene expression of Clonostachys rosea Nygren et al.
Such a highly regulated in situ production of various ubiquitous mechanisms commonly used in the microbial interplay in the environment makes the use of MBCAs a particular safe and sustainable technology.
Because of the ubiquitous character of in situ modes of action specific risk assessments are not relevant. Because of the complexity of the cascades of physiological events the indication of the principal single mode of action as data requirement of Commission Regulation Anonymous, a ; see Box 1 is impossible.
Box 1. What are the data requirements and the uniform principles concerning the mode of action of the microorganism against plant diseases in the EU? The most important data requirements related to the mode of action of active substances are set out in Commission Regulation EU No.
The data requirements for plant protection products preparations are set out in Commission Regulation EU No. must be stated. It must also be stated whether or not the product is translocated in plants and, where relevant, if such translocation is apoplastic, symplastic or both.
The uniform principles for evaluation and authorisation of plant protection products are set out in Commission Regulation EU No. The micro-organism in the plant protection product should ideally function as a cell factory working directly on the spot where the target organism is harmful.
The characterization and identification of relevant metabolites must be assessed and the toxicity of these metabolites must be addressed.
The mode of action of the micro-organism shall be evaluated in as much detail as appropriate. a antibiosis;. b induction of plant resistance;. c interference with the virulence of a pathogenic target organism;. d endophytic growth;. e root colonization;. f competition of ecological niche e.
g parasitization;. h invertebrate pathogenicity. Mode of action is taken into account at evaluation of the degree of adverse effects on the treated crop, operator exposure, viable residues, fate, and behavior in the environment and at risk assessment of birds, mammals, aquatic organisms, bees, arthropods other than bees and earthworms and nitrogen and carbon mineralization in the soil.
Microbial biological control agents interact with the plant, the targeted pathogen and the resident microflora. Studies on the interactions with the resident microflora have been hampered in the past because of limitations of available methods.
This changed drastically with arrival of Next Generation Sequencing NGS methods such as metagenomics and metatranscriptomics allowing to identify the composition and functions of the microbiome Massart et al.
Further steps by adding information of metametabolomics and signalomics Mhlongo et al. As a result, a holistic in depth understanding on MBCA-microbiota interactions will support better timing, formulation and application of MBCAs and prevent failures.
It is expected that three new developments will have significant impact on biological control of plant diseases. Application of specific compounds or complex substrates will modulate indigenous microbiota compositions with the aim to enhance microbial suppression of plant pathogens Mazzola and Freilich, Such a manipulation of resident microbiota toward disease suppression may be comparable to conservation biological control applied in insect pest control, e.
Simple or complex substrates applied for such a prebiotic approach may not be considered as plant protection products.
A third expectation is that core microbiomes will be designed Gopal et al. Gopal et al. The ecological considerations supporting the idea of assembled consortia are sound Table 1.
However, practical considerations may hamper their introduction. Validation and optimization of in silico -designed consortia under ranges of relevant environmental conditions will be complex and will need substantial resources.
In a commercial setting, development of mass production, down streaming and storage procedures separately for each individual consortium member will need substantially more resources and investments compared to production of single strain MBCAs Table 1.
Registration of assembled consortia as plant protection products will add further difficulties. Regulations in the EU demand the risk assessment of each active ingredient before the product can be registered.
In case of assembled consortia, costs will thus increase substantially. In this context, strategies to develop helper strains or to shape the indigenous microbiota may clearly have advantages above the use of assembled core consortia Table 2.
On the other hand, an adapted legislation for novel disease control systems would benefit society as a whole as well as the environment. Microbial biological control agents use a broad arsenal of modes of action which are used wherever microorganisms interact, communicate, and regulate their co-existence between microbial cells and between microorganisms and plants.
The exploitation of different modes of action has different advantages and disadvantages in relation to the development of commercial MBCAs by industries and their practical use by growers Table 1 , but also regarding the perception of possible toxicological and ecotoxicological risks for producers, users, consumers, and the environment Table 2.
Studies on mode of action of well-documented antagonists show that antagonism generally is not based on a single action of a certain mode of action, but on a sequence of events with the use of different modes of action over time.
During such cascades of physiological events signals often are the result of the earlier used modes of action, e. For the development of specific biocontrol products, certain modes of action may be preferred.
In such cases, screening of new MBCAs can be very focused, e. This screening strategy may be powerful if new strains are being selected superior to an existing, well characterized antagonist or for further strain improvement within an existing antagonist strain. However, in most other cases, selection procedures should be preferred that allow the selection of new combinations of known and still unknown modes of action which are produced directly at the site of interaction.
The key challenge for screening projects is thus the development of suitable robust bioassays which combine the interactions between pathogen, host, and antagonist under controlled conditions. Attractive alternative routes via in vitro tests should not be used to avoid biased selection with emphasis on one mode of action, thus excluding many other powerful modes of action or combination thereof, which may be even ineffective at all if evaluated on their own.
The efficacy of biological control agents against plant diseases may not be durable because pathogen populations may develop resistance comparable to the frequently observed build-up of resistance against chemical fungicides with a single mode of action.
Important factors for an erosion of effectiveness are variation in susceptibility to the mode of action within the pathogen population, selection pressure resulting in shifts within pathogen populations toward less susceptible strains and the fitness of the selected strains in the environment under conditions without selection pressure Bardin et al.
Variation in susceptibility of pathogens has been found for some pathogens such as S. sclerotiorum and G. graminis var. tritici Mazzola et al. However, development of resistance has not reported yet for commercially used biological control products for control of plant diseases Nicot et al.
The risk of resistance development in MBCAs used in sustainable IPM systems is also low because IPM combines a variety of measures to prevent damage by diseases without relying on a single control method.
The build-up of resistance is a serious problem in single molecule-single mode of action chemical fungicides which shorten their economic life span. For MBCAs the principle modes of action exhibit much less selection pressure on pathogens additional to the always present selection pressure during natural competitive interactions of organisms.
Furthermore, it is common that a combination of different modes of action are active and each mode of action is based on multiple actors, e. For MBCAs it thus can be concluded that build-up of resistance is much less likely compared to the build-up of resistance against chemical plant protection products.
Only exceptional uses of MBCAs such as the use of in vitro produced highly concentrated and purified secondary metabolites or the use of genetically modified MBCAs with extraordinarily high expression of a single antimicrobial metabolite may result in selection pressures comparable to single site fungicides.
Knowledge of the mode of action of the microorganisms is required and has also to be considered in the context of other potential risks before a MBCA can be approved for use as plant protection product. Risk assessments of MBCAs are regulated in the EU by Regulation EC No.
The regulations focus strongly on the possible risks of secondary metabolites and toxins potentially produced by microorganisms. Several groups of fungi are known to produce mycotoxins, several groups of bacteria are known to produce toxins including the botulinum-neurotoxin BoNT.
Microorganisms producing such mycotoxins or toxins in relevant amounts are excluded from the use in biological control because of the potential risks for humans and animals.
MBCAs may produce other secondary metabolites as sole mode of action, or — in the majority of cases — as component of a cascade of different secondary metabolites in combination or alternation with other metabolites such as CWDEs or MAMPS.
The function of the produced metabolites often is not antibiosis but to fulfill other functions including signaling at subinhibitory concentrations. Secondary metabolite production is highly regulated and restricted to micro niches and in time.
Such metabolites are rapidly degraded and thus have short life spans in the environment. Only for MBCAs which produce potential antimicrobial metabolites in vitro or during the mass production fermentation process and contain such metabolites in the formulated end product at effective concentration, thorough risk assessment is indicated and the minimal effective concentration against the target and representative non-target organisms can be established.
However, in all other cases, such metabolites are not relevant for a risk assessment. Furthermore, reliable quantification of temporal metabolite concentrations in microniches in the in situ situation can hardly to be achieved. The perception of risks caused by antimicrobial metabolites in biological control may be more a result of the broad use of in vitro studies on antibiosis in biocontrol research rather than the result of studies on on-site production of such metabolites in the environment.
In vitro antagonism can easily be visualized through inhibition zones on culture media. The similar method is used for the screening of pharmaceutical antibiotics that aims at the development of products containing single molecules for medical treatments.
Communication on biocontrol research based on in vitro assays, showing inhibition zones, may create a wrong view on the nature of biocontrol control resulting in the fear of the use of antibiotics in crop protection.
Since results of in vitro assays generally do not correlate with results obtained in bioassays or with crops Koch et al.
Biocontrol research unraveling the mechanisms in the much more complex in situ situations may reduce the unjustified fears for microbial metabolites produced by MBCAs. In conclusion, MBCAs are functioning directly on the spot where the targeted organism is harmful Anonymous, , generally combining different modes of action to highly regulated cascades of events.
Current thinking on how to consider the mode of action during the risk assessment and registration procedure of MBCAs focuses on a single mode of action and potential risks of in vitro produced metabolites, very similar to the risk assessment of synthetic fungicides with a single compound as active ingredient.
A rethinking is needed considering that the effectiveness of MBCAs in most cases is based on natural, complex, highly regulated interactions between microbial cells and plants on site but are not the results of a single action of a single metabolite.
Toxicological and ecotoxicological risks of such complex processes of interaction can be considered as very low. Moreover, humans and other organisms have been and still are exposed to such processes in evolutionary terms and adverse effects are not known.
Since an antimicrobial action of a single metabolite is not relevant in many cases, the existing EU regulations may require such a rethinking in the registration of MBCAs as long as antimicrobial metabolites are not present in the formulated MBCA at relevant concentrations.
Microbial biological control agents use a great variety of mechanisms to protect plants from pathogens. Important modes of action strengthen the resistance of the plant, e. Hyperparasitism and secondary metabolites are directly affecting the targeted pathogen via highly regulated cascades of physiological events but not by a single constitutively produced metabolite.
Secondary metabolites produced in vitro may have antimicrobial activity at high concentration but low amounts are produced in situ very locally during interaction and metabolites have short life spans, often with functions such as signaling, very different from antibiosis.
During the cascades of events a range of different compounds with different modes of action are used to outcompete the pathogen. Such events of signaling and interaction are common wherever microorganisms interact. The highly regulated in situ production of ubiquitous mechanisms commonly used in the microbial interplay makes the use of MBCAs a safe and sustainable technology.
In situ produced compounds such as MAMPs, enzymes or secondary metabolites are not relevant for risk assessments so that detailed toxicology and ecotoxicological studies of these compounds are not relevant, and should not be required.
The fear of antimicrobial metabolites produced by MBCAs after their release is not based on real risks but fed by the wrong perception on how biocontrol acts if studied under in vitro conditions. If antimicrobial metabolites are the active ingredient in the formulation of the biocontrol product, risk assessment of such metabolites is relevant.
Better screening assays for finding the next generation of MBCAs are needed to measure the overall effect of the interplay of different modes of action. Multi-omics will help to further understand the complex events during microbial interactions in the environment.
Current EU regulations on registration of MBCAs should allow a science-based differentiation between the majority of compounds involved in modes of action to be considered as safe and not relevant for detailed risk assessment and the limited number of cases relevant for risk assessments where secondary metabolites are present as active ingredients in MBCAs formulations at high concentrations.
JK conceived and designed the research. JK, RK, and WR contributed to the manuscript and revised it critically for important intellectual content.
All authors approved the final version of the manuscript. RK was employed by company Linge Agroconsultancy b.
and WR was employed by company Koppert Biological Systems. JK declares no competing interests. We thank the members of the greenTEAM of the Board for the Authorisation of Plant Protection Products and Biocides CTGB , Ede, Netherlands, for their valuable comments on earlier versions of the manuscript.
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In addition to physical methods Targetef microbial control, chemicals are also used to control microbial growth. Mucrobial wide Targeted microbial control of chemicals can Targeted microbial control used as disinfectants or antiseptics. This section Coenzyme Q and fatigue the variety Targeted microbial control chemicals used as Tzrgeted and antiseptics, including their mechanisms of mmicrobial and common uses. In the s, scientists began experimenting with a variety of chemicals for disinfection. In the s, British surgeon Joseph Lister — began using carbolic acid, known as phenolas a disinfectant for the treatment of surgical wounds see Foundations of Modern Cell Theory. Today, carbolic acid is no longer used as a surgical disinfectant because it is a skin irritant, but the chemical compounds found in antiseptic mouthwashes and throat lozenges are called phenolics. Chemically, phenol consists of a benzene ring with an —OH group, and phenolics are compounds that have this group as part of their chemical structure.
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