Biological control is a potential nonchemical method to manage plant pathogens by beneficial microorganisms. To improve antagonistic potential of biocontrol agents, mutation by radiations, chemicals, and genetic manipulations has been used. Genetic techniques and ionizing radiation containing direct or indirect emissions play the greatest role for selection of useful microorganisms to enhance the efficiency of biological systems. Indeed, genetic engineering has a main role in increasing antimicrobial metabolites, host colonization ability, and endurance in micro-ecosystem. Genetic improvement can be achieved by protoplast fusion, genetic modification (GM), and chemical (genotoxic agents) and physical mutations. However, ultraviolet light and ionizing radiations can induce modifications in the genome of an organism. Irradiation, particularly gamma rays, is also applied for controlling postharvest diseases. Indeed, irradiation cannot completely eliminate pathogens, but it might result in cell injury and directly damage the chromosomal DNA of a living cell. This technology has been used for many reasons including disinfestation of foods, reducing foodborne pathogens, and extending shelf life many fruits, vegetables, and nuts. In the current review, we discuss advances in the radiation and molecular genetic techniques with the aim to improve antagonistic potential of microorganisms as it is applied to the suppression of plant pathogens.
- biocontrol agents
- genetic engineering
- ionizing radiations
The risks associated with chemical residues on the leaves and fruits have highlighted the need for more useful and safer alternative control treatments. Biological control is a potential nonchemical mean to manage plant pathogens including fungi, bacteria, nematodes, or weeds by beneficial microorganisms such as fungi, yeast, or bacteria . The virulence of pathogens probably changes if genetic mutations occur in the genes related to pathogenicity of microorganisms upon mutagenic treatments . It is quite obvious that the application of ionizing radiations such as gamma rays and X-rays and genetic methods plays the greatest role in the development of techniques for the selection of useful microorganisms . Indeed, irradiation is the process of exposing an amount of energy in the form of speed particles or rays for improving food safety and reducing microorganisms that destroy agricultural crops. For instance, studies on transcriptional changes of
2. Genetic improvement of microorganisms
The biological and molecular characterizations of biocontrol agents and bioactive compound producers are very important for the modern agriculture . Since environmental conditions are subject to change, the biocontrol agent requires genetic improvement for effective performance . To improve the efficiency and productivity of biological systems, genetic engineering has a main role in increasing antifungal and antibacterial metabolites, host colonization ability, and endurance in micro-ecosystem. Genetic improvement can be achieved by chemical and physical mutations, protoplast fusion, and transformation .
2.1 Use of protoplast fusion
Protoplast fusion is an important technique for gene manipulation. It breaks down the barriers to genetic exchange and is a relatively new flexible technique to induce or promote genetic recombination in a variety of prokaryotic and eukaryotic cells . By protoplast fusion ( Figure 1 ), interspecific or even intergeneric hybrids can be produced, and it is feasible to transfer useful genes, for attributes (such as disease resistance, enzyme and phytotoxin production, rapid growth rate, nitrogen fixation, protein quality, and drought resistance), from one species to another .
To breed new strains with improved production of the spore and phytotoxin, protoplast fusion between the
2.2 Genetic transformation
Nowadays, some wild-type fungal and bacterial species are used as biocontrol agents. But, there are limitations that restrict their efficacies. Poor survival of the inoculant in particular soils and low production of required metabolites are the most important limitations . The technology of genetic modification (GM) has the capacity to create new strains in which these problems are overcome . Genetic manipulation requires the development of vector-mediated transformation systems that include, first, infusion of exogenous DNA into receiver cells; second, expression of genes present on the incoming DNA; and, finally, stable preservation and replication of the inserted DNA, leading to the expression of the desired phenotypic trait ( Figure 2 ).
The development of an effective strain that can tolerate the environmental adversities and securing of the essential regulatory approval are primary obligations for the successful use of a GM inoculant as biocontrol agents . Resca et al.  genetically modified the strains of
2.3 Improvement of the bioagents via mutagenesis
Breeding of antagonists is directed to achieve effective strains for biocontrol of plant pathogens under a wide range of environmental conditions. Mutation techniques have been used to improve antagonistic potential of biocontrol agents to manage phytopathogens. Physical and chemical mutagens have been applied by many researchers to generate new biotypes . Ultraviolet light (UV), ionizing radiation, and chemicals (as genotoxic agents) can randomly induce modifications in organism’s genome. About 25 years ago, X-ray, as an active mutagen, was used to produce the first mutant strain of
Later on, fast neutrons and gamma irradiation were used successfully. The effectiveness of gamma rays significantly surpassed that of UV and X-rays. Also, mutagenic effects of chemicals in combination with UV light on
Moreover, Balasubramanian et al.  tested biocontrol efficacy of the UV mutants and wild strain of
2.3.1 Mechanism of ionizing radiation effects on bioagents
Irradiation can have direct or indirect effects on organisms. In the case of a direct hit, electromagnetic radiance and high-energy particles break chemical bonds or reactive oxygen-centered (•OH) radicals originated from the radiolysis of water. Hydroxyl radicals are very reactive and are known to interfere with the bonds between nucleic acids within a single strand or between opposite strands. An indirect effect occurs on organisms when radiation ionizes a neighboring molecule, which in turn reacts with the genetic material . Ionizing radiation effects (on microorganisms) are primarily the result of disruption of the DNA or RNA ( Figure 3 ).
Since the DNA is much larger than other molecular structures in a cell, the induced biological and chemical changes by either primary ionizing or through secondary free radical attack can prevent replication and destroy cells  or generate some genetic traits of microorganisms to have higher antagonistic potential . Therefore, the induced biological and chemical changes are related to the absorbed radiation dose . The pathogenicity of infectious organisms is diminished by irradiation. Lim et al.  showed that the expression of the virulence genes in
2.3.2 Irradiation sources
Ionizing radiation is mainly known to high-energy photons of radio nuclides (gamma rays) and X-rays from machine sources with energies up to 5 MeV and accelerated electrons with energies up to 10 MeV generated by electron accelerating machines . Gamma ray occurs in the short wavelength, with high-energy region of the spectrum, and has the greatest penetrating power. Gamma rays come from spontaneous breakdown of radionuclides including the radionuclide’s cobalt-60 (60Co) with a half-life of 5.3 years or cesium-137 (137Cs) with a half-life of 30 years . X-ray is similar to gamma radiation based on radioactive isotopic sources. Although their effects on materials are generally similar, these kinds of radiations differ in their energy spectra, angular distributions, and absorbed dose rates [36, 37]. Electrons can be produced from machines capable of accelerating electrons as light speed by means of a linear accelerator. In comparison with gamma ray and X-rays, electrons cannot penetrate very far into materials and do not have deep penetrating power. So, electrons as beta particles are usually chosen to treat the surface of materials [32, 34]. Generally, the strength of the source and the length of time a material is exposed to the ionizing energy determine the irradiation dose, measured in grays (Gy) or kilo grays (1 kGy = 1000 Gy). One gray is equal with one joule of energy absorbed in a mass of one kilogram . Based on available evidence, the safety of the irradiation technology in food industry was considered and judged acceptable. This has developed in international bodies such as the World Health Organization (WHO), the Food and Agriculture Organization (FAO), the International Atomic Energy Agency (IAEA), the and Codex Alimentarius Commission [35, 39].
Plant pathogens are a worldwide problem. A variety of approaches may be applied to control plant diseases. Beyond suitable agronomic performances, farmers frequently rely on chemicals. Environmental pollution and carcinogenic effects of pesticides are limiting factors in the success of their application. Nowadays, there are severe regulations and political pressure to remove the most dangerous chemicals from the market. So, biological control could be the best alternative against plant pathogens, and development of mutants is an important technique in strain improvement toward plant pathogen suppression, which yields reliable strains for biocontrol. Since strains bred by mutagenesis can get registration (from environmental protection agencies) more easily than strains produced by protoplast fusion and transformation or via gene cloning for field use, more attention should be paid to the mutagenic methods. In an agricultural environment, mutants are interacting and competing with various communities of microorganisms that can have intense effects on the survival and performance of the introduced mutants. Hence, before commercial application of such inoculants in an open environment, their behaviors and potential impact on ecosystems should be investigated as part of the risk assessment. Additionally, integrated treatment of irradiation and biocontrol agent has the potential as an alternative means for postharvest disease control. In fact, there is a limited dose rate for its application on postharvest diseases of fruits and vegetables. Thus, the combination of irradiation and biocontrol agent increases applied range of irradiation for postharvest control by decreasing of dose rate to which the product has been exposed. From our point of view, considering different aspects of irradiation may provide useful information for managing harmful microorganisms on crops, so that in the near future, this technique will be used as one of the most important research tools for biotechnologists, plant pathologists, and molecular biologists.
Authors want to thank Prof. Vahe Minassian from Tarbiat Modares University for critical reading of the manuscript.