**2. Beneficial microorganisms against stress conditions PGPR and mycorrhiza**

#### **2.1. Plant growth promoting rhizobacteria (PGPR)**

cycles in the nature have been affected negatively [1, 2], and the nutrients (specifically nitrogen (N) and phosphorus (P) were run off, which ultimately caused degradation in the environment [3, 4]. There are several underlying reasons for this situation some of which are the low use-effectiveness of fertilizers and the constant long-term use. Although there are damaging environmental effects, it is expected that the total fertilizer amounts that are used in the whole world will increase in future due to the ever-increasing world population, because there appears a need for producing more food by applying intensive agriculture, which neces-

There are two objectives in modern horticulture that contradict with each other: the need to provide food for ever-rising population of the world; and the need for minimizing the damage done to the environment, which can affect horticulture in a negative way [7]. In this respect, horticultural industry and scientists face a major sustainability challenge [8]. In the past 10 years time period, there were some innovations in the field of technology to improve the sustainability of the production systems by reducing the use of chemicals. "Biostimulants" have been proposed as an effective tool in this context. As a result of the efforts made to reduce the harmful effects of fertilizers, plant growth promoting rhizobacteria (PGPR) and/or arbuscular mycorrhizae fungi (AMF) have been proposed as complements for fertilizers. "Plant biostimulants contain substance(s) and/or microorganisms whose function when applied to plants or to rhizosphere is to stimulate natural processes to enhance nutrient uptake, effectiveness, tolerance to abiotic stress, and crop quality, with

The rhizosphere is a soil volume under the effect of plant roots. Hiltner [9] defined "rhizosphere" as a maximum microbial activity zone. The microbial population that exists in this medium is different from the population that surrounds it because of the root exudates, which act as nutrition source for microbial growth [10]. The microorganisms may exist in the rhizosphere, rhizoplane, root tissue, and/or in a specialized root structure that is named "nodule." Among the plant, soil, and microorganisms that exist in the soil medium, significant interactions were reported [11]. These significant interactions can be beneficial, neutral, and/or harmful, and may affect growth of plants [12–14]. Usually there are bacteria, algae, fungi, protozoa, and actinomycetes in the microorganisms that colonize in the roots of plants. Evidence has been presented about the enhancement of plant growth and development by applying these microbial populations [15–19]. Bacterial population, i.e., fungi include a significant portion of soil rhizosphere microflora and affect plant growth. The togetherness of fungi and plant roots (mycorrhizae), which is symbiotic life, enhances the root surface area, and this enables the plant to absorb water and nutrients from big soil volume in a more efficient manner. Two mycorrhizae (ecto- and endo-mycorrhizae) types were reported in a few plant species. The mycorrhizae increase the availability of the nutrients and water, and in addition, protect the

Agriculture is influenced greatly by the climate change; especially agriculture in tropical areas face increased stress because of natural and anthropogenic factors. In some major crops,

sitates a great amount of fertilizers [5, 6].

66 Physical Methods for Stimulation of Plant and Mushroom Development

no direct action on pests."

plant from some abiotic stresses [20, 21].

Plant growth promoting rhizobacteria (PGPR) are useful bacteria that act on some soil types and facilitate that plants grow and develop in (in)direct ways. In a direct way, fixed nitrogen, phytohormones, iron isolated by bacterial siderophores, i.e., iron-carriers, and phosphate in soluble form are given to plants. In an indirect way, phytopathogens (biocontrol) are avoided resulting in plant growth enhancement. Such functions are performed by PGPR through several enzymes (like bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase) stimulating physiological changes at molecular level. ACC has an important effect on ethylene regulation, which is a plant hormone, resulting in modified plant growth and development. Bacterial strains with ACC deaminase may eliminate negative effects caused by stress and mediated by ethylene.

It was reported that there was ACC deaminase in some Gram-negative microbial bacteria, Grampositive bacteria, rhizobia, endophytes, and fungi. It was investigated in some species of plant growth enhancing bacteria (*Agrobacterium genomovars* and *Azospirillum lipoferum*, *Alcaligenes* and *Bacillus*, *Burkholderia*, *Enterobacter*, *Methylobacterium fujisawaense*, *Pseudomonas*, *Ralstonia solanacearum*, *Rhizobium*, *Rhodococcus,* and *Sinorhizobium meliloti*, and *Variovorax paradoxus*).

The ACC of the root is metabolized into α-ketobutyrate and ammonia by the ACC deaminase. It also checks the ethylene production. If this process did not occur in this way, the growth of the plant would be inhibited via some mechanisms. If plants are treated with bacteria that have ACC deaminase, it is possible that they have extensive root growth because of less amounts of ethylene. In this way, plants may resist several stress sources. In recent years, using PGPR with ACC deaminase activity, to improve the growth of plants under stress and normal conditions, has been dealt with researchers as an interesting and new field. Also, cultivars' genetic manipulation with genes that express this enzyme has been dealt with recently by several authors. For this reason, focus must be laid on the further parts of this manuscript on late developments in this field of biotechnology.

exudates. Plants take up some IAAs that are synthesized recently, and IAAs may stimulate the cell proliferation and elongation of plants. In addition, SAM is converted into ACC by enzyme ACC synthetase stimulated by IAA. In the model of Glick et al., an important deal of ACC can be exuded from the roots or seeds of plants. It may also be taken up by soil microbes. It is also possible that it is hydrolyzed by vital microbial enzyme ACC deaminase to produce ammonia and α-ketobutyrate. This process causes that the ACC amount is reduced outside plants. In addition, the balance between internal-external ACC is kept stable via the exudation of more ACC into the rhizosphere. Soil microbial communities with ACC deaminase activity cause that plants biosynthesize more ACC than the plant could need and arouse ACC exudation from plant roots. Meanwhile, they will also provide microorganisms with nitrogen (ACC). As a result, microorganism with ACC deaminase growth is enhanced near roots of the plants. In this way, the ACC level is reduced in plants, and also, the ethylene (stress hormone) biosynthesis is inhibited. In some studies, PGPR inoculation with ACC deaminase was shown to change the endogenous ethylene levels, which ultimately lead to

Use of Some Bacteria and Mycorrhizae as Biofertilizers in Vegetable Growing and Beneficial…

http://dx.doi.org/10.5772/intechopen.76186

69

Several chemicals (aminoethoxyvinylglycine (AVG), aminooxyacetic acid (AOA), and 1-methylcyclopropene (1-MCP)) were used to reduce the ethylene level in plants. They were also used to change the sensitivity to ethylene during fruit ripening and flower wilting. In many situations, these chemical substances are not cheap, not easily obtained, and are harmful for the environment. Using PGPR in a natural soil and plant environment is more economical and feasible and is more economical friendly because PGPR includes ACC deaminase activity. In addition, it has also some other advantages like the ACC deaminase trait being more common in some PGPR species that are native to rhizosphere and have a wide variety of survival potential in rhizosphere and rhizoplane. Moreover, PGPR has some other aspects (such as auxins, gibberellins, cytokines, and/or polyamines syntheses contributing directly to plant growth). These features cause that the selection of PGPR with ACC deaminase is more

AMF were first described in the last years of nineteenth century. Albert Bernard Frank described the symbiotic associations between the plant roots and the fungi (mycorrhizae). Mycorrhizae means "fungal root." This association's basic principle is the nutrients taken up from the soil are exchanged with sugar. Lots of microorganisms form symbiosis with plants ranging on a continuous scale from parasitic to mutualistic. A typical example of these widespread mutualistic symbioses is the arbuscular mycorrhiza formed between AMF and vascular flowering plants [32]. Many scientists and mycologists researched the relations (associations) between mycorrhizae and the plants biology and their inoculation methods. This relation includes the structure of the root and mycorrhizal inoculation. Mycorrhizae are complex symbioses and the fungi produce some structures in the root. Quantification of the structures (hyphae, arbuscules, and vesicles) was standardized by the method suggested by Hungria and Vargas [33]. An arbuscular mycorrhiza has three important elements; the root, the fungal elements between the cells of the root and an extraradical mycelium in soil [34]. The most common type

variations in plant growth.

reliable than other alternatives.

**2.2. Mycorrhizae**

Data on biosynthetic pathways of ethylene production in plants enabled us to elucidate the mechanisms by which plants regulate the endogenous ethylene level for their normal growth. It has been demonstrated that S-adenosylmethionine or ACC-degrading enzymes decrease ethylene levels in an efficient manner without changing plant physiology. For this purpose, researchers investigated some enzymes that aid to decrease ethylene levels in plants. In this respect, S-adenosylmethionine (SAM) hydrolase and SAM decarboxylase were examined less with regards to ethylene regulation in plants. ACC synthase and oxidase were examined more with several plants.

The ACC deaminase, which is a pyridoxal 5-phosphate (PLP)-dependent polymeric enzyme, was first investigated in a soil bacteria species *Pseudomonas* sp. strain. Bashan et al. [29] described structure for ACC deaminase and provided an understanding about the working of sole pyridoxal-5-phosphate that depends on cyclopropane ring-opening reactions of this enzyme in *Pseudomonas* sp. It was reported in [30] that there was a wide range (>100-fold) in ACC deaminase activity level in various organisms which show high ACC deaminase activity and typically bind to some plants. In this group, there are rhizosphere, phyllosphere organisms, and endophytes, which may behave as a sink-like structure for ACC that appear as a result of stress in plants. In addition, the abovementioned show little preference for one plant over another. However, the organisms that express low deaminase may only bind to some plants. They may also be expressed solely in some tissues; and do not reduce the level of ethylene in plants; but, they prevent a localized increase in the levels of ethylene. Glick reported that there are some rhizobia and ACC deaminases.

Glick et al. [31] investigated the model of PGPR which includes ACC deaminase. They examined how a bacterial ACC deaminase with a low relation to ACC could cope with plant enzymes and ACC oxidase that has high relation with the same substrate resulting in a reduction of endogenous ethylene concentration of a plant. They claimed that biological activity of PGPR was related with ACC deaminase ACC oxidase amounts. In order for PGPR to decrease ethylene levels in plants, the level of the ACC deaminase must be minimum from 100- to 1000 fold bigger than ACC oxidase level. For this to happen, the ACC oxidase expression must not be induced.

Indole-3-acetic acid (IAA) is synthesized and excreted by PGPR. IAA is adsorbed by the surface or roots of the seeds of plants by tryptophan and some molecules in seeds or root exudates. Plants take up some IAAs that are synthesized recently, and IAAs may stimulate the cell proliferation and elongation of plants. In addition, SAM is converted into ACC by enzyme ACC synthetase stimulated by IAA. In the model of Glick et al., an important deal of ACC can be exuded from the roots or seeds of plants. It may also be taken up by soil microbes. It is also possible that it is hydrolyzed by vital microbial enzyme ACC deaminase to produce ammonia and α-ketobutyrate. This process causes that the ACC amount is reduced outside plants. In addition, the balance between internal-external ACC is kept stable via the exudation of more ACC into the rhizosphere. Soil microbial communities with ACC deaminase activity cause that plants biosynthesize more ACC than the plant could need and arouse ACC exudation from plant roots. Meanwhile, they will also provide microorganisms with nitrogen (ACC). As a result, microorganism with ACC deaminase growth is enhanced near roots of the plants. In this way, the ACC level is reduced in plants, and also, the ethylene (stress hormone) biosynthesis is inhibited. In some studies, PGPR inoculation with ACC deaminase was shown to change the endogenous ethylene levels, which ultimately lead to variations in plant growth.

Several chemicals (aminoethoxyvinylglycine (AVG), aminooxyacetic acid (AOA), and 1-methylcyclopropene (1-MCP)) were used to reduce the ethylene level in plants. They were also used to change the sensitivity to ethylene during fruit ripening and flower wilting. In many situations, these chemical substances are not cheap, not easily obtained, and are harmful for the environment. Using PGPR in a natural soil and plant environment is more economical and feasible and is more economical friendly because PGPR includes ACC deaminase activity. In addition, it has also some other advantages like the ACC deaminase trait being more common in some PGPR species that are native to rhizosphere and have a wide variety of survival potential in rhizosphere and rhizoplane. Moreover, PGPR has some other aspects (such as auxins, gibberellins, cytokines, and/or polyamines syntheses contributing directly to plant growth). These features cause that the selection of PGPR with ACC deaminase is more reliable than other alternatives.

#### **2.2. Mycorrhizae**

The ACC of the root is metabolized into α-ketobutyrate and ammonia by the ACC deaminase. It also checks the ethylene production. If this process did not occur in this way, the growth of the plant would be inhibited via some mechanisms. If plants are treated with bacteria that have ACC deaminase, it is possible that they have extensive root growth because of less amounts of ethylene. In this way, plants may resist several stress sources. In recent years, using PGPR with ACC deaminase activity, to improve the growth of plants under stress and normal conditions, has been dealt with researchers as an interesting and new field. Also, cultivars' genetic manipulation with genes that express this enzyme has been dealt with recently by several authors. For this reason, focus must be laid on the further parts of this manuscript

Data on biosynthetic pathways of ethylene production in plants enabled us to elucidate the mechanisms by which plants regulate the endogenous ethylene level for their normal growth. It has been demonstrated that S-adenosylmethionine or ACC-degrading enzymes decrease ethylene levels in an efficient manner without changing plant physiology. For this purpose, researchers investigated some enzymes that aid to decrease ethylene levels in plants. In this respect, S-adenosylmethionine (SAM) hydrolase and SAM decarboxylase were examined less with regards to ethylene regulation in plants. ACC synthase and oxidase were examined

The ACC deaminase, which is a pyridoxal 5-phosphate (PLP)-dependent polymeric enzyme, was first investigated in a soil bacteria species *Pseudomonas* sp. strain. Bashan et al. [29] described structure for ACC deaminase and provided an understanding about the working of sole pyridoxal-5-phosphate that depends on cyclopropane ring-opening reactions of this enzyme in *Pseudomonas* sp. It was reported in [30] that there was a wide range (>100-fold) in ACC deaminase activity level in various organisms which show high ACC deaminase activity and typically bind to some plants. In this group, there are rhizosphere, phyllosphere organisms, and endophytes, which may behave as a sink-like structure for ACC that appear as a result of stress in plants. In addition, the abovementioned show little preference for one plant over another. However, the organisms that express low deaminase may only bind to some plants. They may also be expressed solely in some tissues; and do not reduce the level of ethylene in plants; but, they prevent a localized increase in the levels of ethylene. Glick reported

Glick et al. [31] investigated the model of PGPR which includes ACC deaminase. They examined how a bacterial ACC deaminase with a low relation to ACC could cope with plant enzymes and ACC oxidase that has high relation with the same substrate resulting in a reduction of endogenous ethylene concentration of a plant. They claimed that biological activity of PGPR was related with ACC deaminase ACC oxidase amounts. In order for PGPR to decrease ethylene levels in plants, the level of the ACC deaminase must be minimum from 100- to 1000 fold bigger than ACC oxidase level. For this to happen, the ACC oxidase expression must not

Indole-3-acetic acid (IAA) is synthesized and excreted by PGPR. IAA is adsorbed by the surface or roots of the seeds of plants by tryptophan and some molecules in seeds or root

on late developments in this field of biotechnology.

68 Physical Methods for Stimulation of Plant and Mushroom Development

that there are some rhizobia and ACC deaminases.

more with several plants.

be induced.

AMF were first described in the last years of nineteenth century. Albert Bernard Frank described the symbiotic associations between the plant roots and the fungi (mycorrhizae). Mycorrhizae means "fungal root." This association's basic principle is the nutrients taken up from the soil are exchanged with sugar. Lots of microorganisms form symbiosis with plants ranging on a continuous scale from parasitic to mutualistic. A typical example of these widespread mutualistic symbioses is the arbuscular mycorrhiza formed between AMF and vascular flowering plants [32]. Many scientists and mycologists researched the relations (associations) between mycorrhizae and the plants biology and their inoculation methods. This relation includes the structure of the root and mycorrhizal inoculation. Mycorrhizae are complex symbioses and the fungi produce some structures in the root. Quantification of the structures (hyphae, arbuscules, and vesicles) was standardized by the method suggested by Hungria and Vargas [33]. An arbuscular mycorrhiza has three important elements; the root, the fungal elements between the cells of the root and an extraradical mycelium in soil [34]. The most common type of mycorrhizae is the arbuscular mycorrhiza occurring in about 90% of plant species infected with mycorrhiza. The most common type of mycorrhizae is the arbuscular mycorrhiza occurring in about 90% of plant species infected with mycorrhiza, approximately 83% of dicotyledons, 79% of monocot, and 100% of gymosperms. Most crop plants form mycorrhizae with the exception of the Brassicaceae (e.g., mustard, cabbage, and canola) and Chenopodiaceae (e.g., sugar beets and spinach).

fungi build up hyphopodia (appressoria) on the surface. On the other hand, a particular mycorrhizae-specific process occurs in epidermal cells underlying hyphopodia in the plant side. They constitute the pre-penetration apparatus, which is a transient intracellular structure used by the fungi to enter the root [59]. Fungal hyphae host the roots of the plant, firstly, between/through cells with linear/simple-coiled hyphae [60], and then build up high-branch hyphal structures that resemble a tree in plant cell apoplast (the arbuscules which gave the name). *Gramineae* members form vesicles rich in lipid as storage organs [61]. Parallel to the colonization of the root, fungi examines the soil around with its hyphae with which they uptake nutrients, interact with other microorganisms, and colonize roots of nearby plants of the same (or different) species. In this way, plants and their AM fungi are interrelated with each other in a network of roots and hyphae [62, 63]. They can exchange nutrients [64] or signals [65] in this way. Eventually, new chlamydospores are created in the extraradicular mycelium. The cycle of life is ended in

Use of Some Bacteria and Mycorrhizae as Biofertilizers in Vegetable Growing and Beneficial…

http://dx.doi.org/10.5772/intechopen.76186

71

**3. The most effective environmental stress factors: salinity and** 

Under saline conditions, the changes in soil-water potential cause that plant water intake is reduced as well as the nutritional and hormonal imbalance. In these conditions, proline, glycine betaine, trehalose, polyols, and similar organic solutes accumulate in the body of the plant to preserve the plant from the stress-induced effects with osmotic adjustment, with limiting water loss and diluting the toxic ion concentration [66, 68]. Such an accumulation makes it possible for the plant to maintain osmotic potential for improved water intake. For instance, proline accumulation preserves the plant by adjusting osmotic pressure and by stabilizing many functional units (e.g., complex II of the electron transport system, proteins, and enzymes [69, 70]. There are two mechanisms in which high-concentration soluble salts influence microbes: osmotic effect and specific ion effect. Osmotic potential (more negative) is increased by soluble salts and draws water out of the cells, which in turn, may kill microbes and roots via plasmolysis. Because of the low osmotic potential, it becomes more difficult for roots and microbes to eliminate water from the soil [71]. Plants, as well as microbes, can adapt to low osmotic potential through accumulating osmolytes. However, osmolyte synthesis necessitates large amounts of energy, which in turn, results in reduced growth and activity [72, 73]. Certain ions, includ-

, and HCO−3, are toxic for some plants when they are at high-concentrations [74]. In

some previous studies, it was reported that salinity decreases microbial activity and microbial biomass and changes the structure of the microbial community [75–79]. The microbial biomass is decreased by salinity. The reason for this is that osmotic stress causes drying and cell lysis [80–86]. In previous studies, it was also reported that soil respiration was reduced with the increase in the soil EC [87–89]. Gerhardson [90] reported that soil respiration was decreased by more than 50% at EC1:5Z5.0 dS m1. However, according to Glick [91], soil respiration was not correlated at a statistically significant level with EC. However, they also reported that as

EC increased, the metabolic quotient (respiration per unit biomass) also increased.

this way.

**drought**

ing Na+

, Cl−

**3.1. Salinity stress**

AM fungi consists approximately 160 species belonging to three families. Glomaceae, Gigasporaceae, and Acaulosporaceae. More than 6000 fungal species can form mycorrhizae with about 240,000 plant species. AMF plants own bigger extraradical hyphae formation and soil aggregation. They enhance tilth and excrete hydrophobic protein called "glomalin." AMF produce more stress-resistant plants during production and for landscape, they reduce the pesticide usage, they increase the more drought and nutrient tolerant plants in landscape, and they potentially higher transplanting success and faster establishment. A symbiotic association formed by fungi with roots, exchanging for functioning as an extended root system, the fungi receives carbonhydrates from the host plant [35].

Arbuscular mycorrhizae fungi (AMF), which are useful organisms, have a significant role in performance and nutrition with plant mineral intake capacity [36]. AMF symbiosis is especially significant in improving the immobile uptake and indissoluble phosphate ions in soil with the interactions with bi/trivalent cations (especially Ca2+, Fe3+, and Al3+ [37, 38]. The main function in this mutualism is the capacity of AMF in developing external hyphae networks that may extend the surface area (up to 40 times) and the explorable soil volume for nutrient intake [39] by producing enzymes and/or excreting organic substances [40]. AMF can excrete phosphatases to hydrolyze phosphate from organic P-compounds [41–43], which enhance productivity under harsh conditions (deficiency of phosphorus; [44]). The extraradical hyphae are considered significant in terms of intake of ammonium, immobile micronutrients (Cu and Zn), and some mineral cations coming from the soil (K<sup>+</sup> , Ca2+, Mg2+, and Fe 3+) [45, 46]. It was demonstrated that AMF enhance plant nutrition (biofertilizers), and interferes with the phytohormone balance of the plants, which in turn affects development of plant (bioregulators) and alleviates the influence of the environmental stresses (bioprotectors). This increases the biomass and yield, and causes shifts in some quality parameters [47].

The horticultural products have high phytochemical elements (carotenoids, flavonoids, and polyphenols) and therefore meet the desires of consumers and authors with their health/benefit influences [48]. Furthermore, AMF also bring tolerance to drought [49, 50] and salinity [51, 52], nutrient deficiency, heavy metal contamination [53] and in adverse soil pH [54, 55].

The AMF life cycle begins with asymbiotic stage (germination of the asexual chlamydospores). This depends on several physical factors (temperature and humidity). AMF retract the cytoplasm without the presence of a plant and turn to the dormant phase because they are obligate biotrophs. However, near the roots of the plants, the presymbiotic phase begins with the ramification of the primary germ tube [56]. Root exudates [57] and specific metabolites (strigolactones) may also induce this [58]. When there is a physical contact with the surface of the root, the fungi build up hyphopodia (appressoria) on the surface. On the other hand, a particular mycorrhizae-specific process occurs in epidermal cells underlying hyphopodia in the plant side. They constitute the pre-penetration apparatus, which is a transient intracellular structure used by the fungi to enter the root [59]. Fungal hyphae host the roots of the plant, firstly, between/through cells with linear/simple-coiled hyphae [60], and then build up high-branch hyphal structures that resemble a tree in plant cell apoplast (the arbuscules which gave the name). *Gramineae* members form vesicles rich in lipid as storage organs [61]. Parallel to the colonization of the root, fungi examines the soil around with its hyphae with which they uptake nutrients, interact with other microorganisms, and colonize roots of nearby plants of the same (or different) species. In this way, plants and their AM fungi are interrelated with each other in a network of roots and hyphae [62, 63]. They can exchange nutrients [64] or signals [65] in this way. Eventually, new chlamydospores are created in the extraradicular mycelium. The cycle of life is ended in this way.
