The Importance of Nitrogen-Fixing Bacteria Azotobacter chroococcum in Biological Control to Root Rot Pathogens (Review)

Intense research continues by researchers to find safe alternatives to chemical pesticides and synthetic fertilizers, and its frequent use without correct programming and guiding awareness, which has caused many environmental problems, poisoning, destruction of the ecosystem, and pollution of water, air, and agricultural soils. The use of these chemicals has also affected many living organisms, including them Humans, which motivated researchers to study and re-introduce the natural elements and beneficial microorganisms into agricultural production so that the consumer can obtain safe and healthy food free from the accumulation of harmful chemical residues. Inside the soil, which can carry out the process of converting nitrogen while it is in the state of being a gas to a state of solubility. Through this process, plants can obtain the benefit from it through the stabilization process that occurs. These bacteria perform a major work, as they work to fix nitrogen in the air This is because the plant finds it difficult to use it in its form as a gas, so it makes it in the soil in a form ready for the plant. Studies have also shown that this bacteria has many beneficial properties for the growth and protection of the plant from pathogens and the importance of this bacteria and to try to shed light on its beneficial activities for the plant. The importance of this bacteria and the role of Azotobacter chroococcum in protecting plants from root rot diseases.


Introduction
The use and spread of bacterial and fungal biofertilizers has expanded in the last two decades of the last century, as a result of the scarcity of energy sources and the consequent rise in the prices of chemical fertilizers, as well as recent trends in reducing the sources of environmental pollution and the adoption of biofertilization as one of the modern technologies to reduce the excessive use of chemical fertilizers.This is in addition to the steady increase in population and lack of food resources [1][2][3].There are microorganisms in the rhizosphere that differ in their types, numbers, and ways of living.Some of them are harmful to the plant, Harmful, as they harm the plant through several relationships such as competition, antagonism, predation, parasitism, etc.And beneficial to the plant through production of indole acetic acid IAA, gibberellins, and natural auxins, therefore, the attention of biological and agricultural scientists was directed to identify these organisms in the plant roots, because they are closely related to plant life [4][5][6].One of the most important bacterial species that encourage root growth is Azotobacter spp.They are groups of bacteria present in the rhizosphere and on the surfaces of plant roots, most of which live freely in the rhizosphere.They are very beneficial for soil and plants alike.They help reduce the concentration of heavy elements and toxic compounds and inhibit the growth of pathogens present in the soil [7].Some types are essential elements in biological control programs, it also contributes to stimulating plant growth through the production of growth regulators and the secretion of compounds and nutrients that cause increased plant growth and development [8].The surface area of the roots, the name Azotobacter consists of two syllables: Azot, which means nitrogen, which is the nitrogen element, and the word bacter, which means bacteria that converts the nitrogen element in the atmosphere into a form that can be used by a vital process called nitrogen fixation.This bacteria has several species, the most important of which is the genus Azotobacter It includes several species: A. chroococcum, A.vinelandii, A.agilis, A.armeniacus, A.beijerincki, A. nigrican, A. paspali, A. salinestris, A. tropicalis, and the most important and widespread of these species is A. chroococcum [9,10].

The Bacteria Azotobacter Chroococcum
In 1901, the Dutch microbiologist Beijerinck gave the name Azotobacter chroococcum to the bacterium, which he discovered through its growth and its ability to fix atmospheric nitrogen in food media free from it.A. chroococcum is Gram-negative, free-living, obligate aerobic, heterotrophic, dependent on organic materials as a source of carbon and energy, which is an important characteristic of the species, as it has the ability to exploit several carbohydrate sources [5,11].Its cells are large, 2 x 3.1 micrometers (mµ), multi-shaped, Pleomorphic, especially in old farms.They may be spherical, sticky, usually short or oval.In the form of chains or clusters of two or four cells, not forming spores, but forming vesicles, a capsule, or a thick outer shell called Slime, which improves its resistance to heat, drought, and harsh conditions.These vesicles germinate under appropriate conditions to give vegetative cells [5].Azotobacter is one of the most important microorganisms in the soil that has the ability to fix large amounts of atmospheric nitrogen in a non-symbiotic manner, as it fixes atmospheric nitrogen to benefit from it in building its components, and after its death and decomposition, it is transferred to the soil to be used by plants and other organisms [12].These bacteria encourage plants as a result of the conversion of nitrogen from the inactive form, that is, the elemental form (N2) to the active form (ammonium NH4+) with the help of the Nitrogenase enzyme.It also works to analyze organic matter, produce chelating compounds, and reduce ethylene and is used in biological control.These bacteria contribute Bacteria in the root zone provide protection for the plant from infection with many pathogens present in the soil through its direct and indirect effects by competing with pathogenic organisms for place and nutrients and preventing the pathogen from reaching the infection areas, and the A. chroococcum bacteria causes major mutations in the root system Including encouraging the formation of lateral roots and increasing the surface area of the root, mainly due to its secretion of auxin IAA, and these modifications are related to improving the absorption of water and nutrients by the pollinated plant.There are many factors that affect the activity and numbers of these bacteria, including the presence of organisms associated with the growth of these bacteria and the quality of the soil and root secretions, and these bacteria can rely on low-carbon substances such as sugars as an energy source for their food [13][14][15] .The benefit of A. chroococcum bacteria is not limited to the process of fixing atmospheric nitrogen only, but rather it secretes useful substances that are almost parallel to the process of biological nitrogen fixation, the most important of which are plant hormones, which are substances with stimulating or inhibitory effects on certain biochemical physiological processes in plants and microorganisms, including Indole Acetic Acid (IAA), Gibberellins, Cytokinins, and what these substances are and their important role in plant growth and development reflects the importance of these bacteria [7,16,17].These bacteria are famous for producing siderophores, which are substances with low molecular weight produced by the bacterial cell under conditions of iron deficiency in the soil or culture medium.The complexes enter the cell so that the iron is separated from the siderophore so that the latter is released to bind with the iron again, and this process leads to an increase in the readiness of the iron component of the plant and then an increase in its growth and productivity, Some of its isolates can also produce vitamin B group and they also produce precursor vitamin D and hydrogen cyanide (HCN) [18][19][20][21].A. chroococcum also produces some enzymes such as Nitrogenase, Phosphatase, Amylase, Esterase, Cellulase, Catelase, Peroxidase, Phenol oxidase [21][22][23][24].The possession of the bacterium A. chroococcum of this enzyme system made it at the forefront of the organisms that analyze organic matter in the soil and a bioremediation agent for the organic waste that it analyzes and recycles its elements and facilitates them for the plant [25][26][27][28][29][30].Several studies have shown the importance of A. chroococcum for the studied plants and their contribution to increasing the rates of germination of their seeds, standards of growth and production, quantity and quality, plant content of N.P.K and other nutrients.And other tested measurements according to the study [31][32][33][34].This bacterium is also associated with a positive interaction with other types of Plant Growth Promoting Rhizobacteria (PGPR), and several researchers have sought to invest this interference in increasing the growth and development of many economic plants [30,[35][36][37], and to determine the effect of A. chroococcum and Rhizobium leguminosarum bv.phaseoli and Bacillus megaterium in the growth parameters of two bean cultivars, it was found that the treatment of the mixture of bacterial species gave the highest significant effect on plant growth, node formation, the amount of N fixation, fresh and dry weight of the plant, the number of branches and pods, and the content of leaves of chlorophyll [38].On the other hand, some studies have demonstrated the positive interaction between the bacteria A. chroococcum and the fungus Trichoderma harzianum in increasing field crop production, In addition, the bacteria A. chroococcum has an important synergistic role with AM fungi, and the effects of this role on plant growth and development [4].

The Role of Azotobacter Chroococcum in Plant Protection from Root Rot Diseases
A. chroococcum bacteria, through its presence in the soil and around the rhizosphere, contribute to protecting the plant from infection with many soil-borne pathogens, with its direct and indirect effects on the pathogenic organisms by competing for space and nutrients and preventing the pathogen from reaching the infection areas [39].The formation of siderophores and competition for iron in the soil is one of the important means of antagonism with pathogens in the soil.In addition, it was concluded that siderophores compounds are the triggers of systemic resistance induced in plants and can play a dual role in reducing diseases by depriving pathogens of iron in the soil and inducing resistance systemic in plants [13,40,41]. A. chroococcum also produces some anti-fungal substances such as antibiotics, the most important of which are Azotobacterin, Conactine, and hydrogen cyanide (HCN), as the presence of these compounds at high concentrations inhibits pathogenic fungi [40,42,43].Research has shown, in an applied manner, the efficiency of A. chroococcum bacteria in protecting plants from infection with pathogens.The addition of the bacterium A. chroococcum to pots planted with potato tubers provided protection for the stalks, stems and out runners from infection with the pathogenic fungus Rhizoctonia solani and prevented the formation of sclerotia of the fungus on the tubers under greenhouse conditions, The treatment also increased the production significantly compared to the control treatment [44].Azotobacter bacteria were used in the biological resistance of some fungi such as Rhizoctonia, Sclerotinia, Pythium, and Fusarium.It was found that Azotobacter bacteria reduce the dry weight of Fusarium mycelium by 90-96% and Rhizoctonia by 90-96%.72-94% and Pythium by 7-95% and Sclerotinia by 100% and reduced the incidence of damping-off disease by 56% and led to an increase in the dry weight of the shoots and seeds of cucumber plants by 30% and 80%, respectively, as well as an increase in the nitrogen content by 89% [ 45,46,47].EL-Komy [48] found in a study measuring the effect of A. chroococcum and Azospirillum bacteria on root rot causes of sunflower plants, that both factors caused a significant reduction in the growth of pathogenic fungi.On the other hand, the researcher found that the bacteria tolerated the recommended doses of chemical fungicides (Topsin M, Chem Z, Ridomil, Cosid, Orthocide, 45M Dithane, Rizolex and Homai) added to seeds before planting, which indicates the possibility of using these agents integrated with Fungal pesticides.Benuzzi et al. [49] mentioned that Azotobacter is among the important genera that contribute to reducing the density of pathogenic fungi, including Fusarium, Pythium, R. solani and Aphanomyces, in addition to its important role in increasing the amount of yield.While Mali and Bodhankar [50] showed that the isolates of the bacteria A. chroococcum isolated from pistachio fields were efficient in antagonism with fungi Aspergillus flavus, A. terrus, Alternaria and F. oxysporum through the production of anti-fungal metabolites and the production of phytohormones such as IAA Gibberellins also increased growth parameters and yield of field pistachio.It has also proven the efficiency of a number of PGPRs, including A. chroococcum caused a significant reduction in the incidence of soybean seedling death disease caused by the pathogenic fungus Macrophomina phaseolina under protected and field conditions, and this was positively reflected on plant growth [51].That strains of Azotobacter spp.And Azospirillum spp.isolated from wheat plants in four different locations in Pakistan was indole acetic acid producing and has the ability to dissolve phosphorus, It positively affected the percentage of germination of wheat seeds as well as increasing the biomass and the length of the vegetative system through the effect on the pathogenic fungus when tested in the potting experiments [52].

Useful Interaction Between Bacteria and Mycorrhiza and other Microorganisms in Protecting Plants from Infection with Root Rot Pathogens
There are many microorganisms in the soil in complete proximity and coexist in different ways, and the members of these microorganisms depend on one another in obtaining certain materials for growth, but at the same time they may have harmful effects on other microorganisms, and then both harmful and beneficial effects are achieved and a result arises from them The microbial equilibrium population [53].Many overlapping effects may occur between two types of microorganisms.Odum [54] suggested the following relationships between microorganisms:  Beneficial associations: In these types of interferences, both or one of the two objects involved benefit from the interference in different ways. Harmful associations: This type of interference results in damage to all or one of the overlapping neighborhoods, and the nature and degree of damage varies. Neutralism associations: In this type, organisms overlap with each other without a clear harmful or beneficial effect appearing between them.However, such a division may not represent the ideal case, since the biological interactions that appear at a specific time may change at a later time to another type of the aforementioned interactions.The microorganisms, through their interactions, affect from one form to another the plant host as well, as the microorganism does not carry out any process that causes it to waste energy without this process benefiting it in some way, as it may be directly through benefiting from the other organism Or indirectly by improving the growth condition of the host plant that needs it.It is now clear that the growth and development of internal or external mycorrhiza fungi causes a lot of changes in the communities of microorganisms in an area around the roots, and this can result through interactions between these organisms, the most important of which is interactions with bacteria that stimulate plant growth, These bacteria can also stimulate the action of mycorrhizae and show beneficial effects, so they are always called "Mycorrhizae-Helper Microorganisms" [55].There is a close relationship between Azotobacter and mycorrhizal fungi in nature, as Azotobacter bacteria form clusters of their cells in the form of shell-like envelopes surrounding the spores of mycorrhiza fungi, as this bacterium was isolated from the surfaces of mycorrhiza spores [56].Many studies indicate that there is a positive overlap between mycorrhizal fungi and free nitrogenfixing Azotobacter bacteria.The process of biological nitrogen fixation can be inhibited or stopped due to the lack of this element, which is important for the bacteria's need for energy and is necessary in the process of synthesis of nucleic acids and nucleotides and the synthesis of phospholipids.Mycorrhiza fungi have a role in removing ammonium ions from N-fixation sites, thus prolonging the effectiveness of the Nitrogenase enzyme and a role in the fixation process.Hence, we see the importance of the overlap between these two organisms [57,58].Azotobacter also has an important role in the production of plant hormones such as auxins, gibberellins and cytokinins, which have a role in the growth of roots and plants and in improving the metabolic processes of mycorrhizae.It also increases the size of the leaves, which leads to an increase in the process of photosynthesis and provides an increase in the processing of nutrients for the internal mycorrhizae in the plant.These hormones increase the size and branching of the roots, and as long as the absorption capacity of the root system for nutrients and water is directly related to the activity of root growth or indirectly through the presence of more sites for the growth and development of mycorrhiza fungi, this can lead to an increase in the rate of nutrient absorption by the plant with integrated inoculum of mycorrhizae and nitrogen-fixing bacteria [59].Mycorrhiza fungi have an important role in bio-nitrification fixation in many leguminous plants through their contribution to knot formation and nitrogen fixation.Bagyaraj et al. [60] found an increase in the growth of soybean plants inoculated with a double inoculation of mycorrhizal fungi and rhizobia bacteria compared to untreated plants.Several studies indicated that the interaction of AM fungi with the bacterium A. chroococcum contributed positively to plant growth, increasing the percentage of mycorrhiza infection, the amount of nitrogen fixation, and the studied plant growth parameters, more than treating the plant with each agent alone [61][62][63].On the other hand, the interaction of mycorrhiza fungi with bacteria plays an important role in suppressing many root diseases [64].As [62] isolates of bacteria were isolated from the surface of mycorrhiza spores, and these bacteria are related with spores that proved to be antagonistic to Meloidogyne incognita and pathogenic soil fungi F. oxysporum and Pythium spp. on tomato plants, it was found that the integration between this bacteria and the fungus G. intraradices achieved the highest percentage of reduction in knots and the production of egg masses, as well as inhibiting fungal infection on tomato stems [65].EL-Batanony et al. [66] found that all interaction treatments between the bacterium Rhizobium leguminosarum and the mycorrhizal fungus inhibited the pathogens of R. solani, F. solani and F. oxysporum.The effectiveness of this interaction was demonstrated through positive changes in plant growth parameters and an increase in the bean plant content N.P.K.When chickpea plants were inoculated with G. intraradices alone or in combination with Pseudomonas aeruginosa and Aspergillus awamori, the integration treatment induced the highest increase in chickpea plant growth, number of pods, plant content of chlorophyll, nitrogen, phosphorus, and potassium, and reduced root-knot disease caused by M. incognita and rot disease caused by the fungus Macrophomina phaseolina [67].Some study revealed that the efficiency of some Azotobacter isolates and Glomus spp.and Gigaspora spp. as a result of a high compatibility between them, and this was evident through a significant reduction in the percentage of tomato root rot disease caused by the two pathogens R. solani and F. solani, and it improved the parameters of plant growth under the conditions of protected cultivation and the field [53,68].Some study indicated that the microbiological substances Trichodermin secreted by Trichoderma fungi and Azotobactrin secreted by Azotobacter bacteria have biological resistance to plant diseases, and were more effective than the same chemically manufactured substances [42,69].In a field experiment Somani et al. (1998) [70] studied the effect of inoculation with mycorrhizal fungus (G.fasciculatum) and each of the A.chroococcum and A. brasilense individually and with their interactions and with three fertilizer levels on the growth of the sorghum plant, and the results showed The treatment of the mixed inoculum of mycorrhiza, Azotobacter and Azospirlem gave the highest live mass of the plant and increased the rates of dry weights, the lengths of the plant.

Conclusion
Studies have also shown that this bacteria has many beneficial properties for the growth and protection of the plant from pathogens and the importance of this bacteria and to try to shed light on its beneficial activities for the plant.The importance of this bacteria and the role of Azotobacter chroococcum in protecting plants from root rot diseases.