MICROBIAL SYNTHESIS OF PHYTOHORMONES

Plant growth regulators (PGR) attract a lot of attention in the agro-industrial complexes of economically developed countries. Using them allows optimizing the plant metabolism in order to increase the yield and improve the quality of crops. By origin, growth regulators are divided into the following groups [1]: endogenous compounds synthesized by plants (phytohormones); products of microbial synthesis; synthetic compounds. Because growth regulators of microbial origin are similar to compounds synthesized by plants (auxins, cytokinins, gibberellins, abscisic acid), they are also called phytohormones. In agriculture growth regulators are used in stimulating seeds germination, activating vegetative growth of plants, accelerating their flowering and maturing, increasing yields, protecting against certain diseases, etc. Using those in agriculture production can significantly reduce the use of chemical plant protectors [1]. In 2015, the global market for plant growth regulators was estimated at $ 1.6 billion. From 2015 to 2020, its growth is forecasted to be 3.6% (to $ 1.91 billion) [2]. The leading PGR producers are FMC (Food Machinery Corporation), Dow (USA), Syngenta AG (Switzerland), BASF SE (Germany) and Nufarm Limited (Australia). The most marketed plant growth regulators are gibberellins, consumed in about 60 tons per year [2]. Increasing the efficiency of microbial synthesis of gibberellins [3–6] and understanding of the plant-microbial interaction mechanisms [7–9] stimulated studying the ability of various physiological and taxonomic microorganism groups to produce phytohormones [10–19], as well as the development of microbial technologies to obtain several of them [6, 20, 21]. The production of phytohormones by microorganisms was previously reviewed in 2013 [22]. This work summarizes the results of studies mainly by Ukrainian scientists, focusing on the bacterial synthesis of phytohormones included in preparations for crop production, and the methods for determining these compounds are analyzed. The purpose of this review is to summarize the current literary data on the phytohormone UDC 579.64:581.1 https://doi.org/10.15407/biotech11.01.005

Plant growth regulators (PGR) attract a lot of attention in the agro-industrial complexes of economically developed countries.Using them allows optimizing the plant metabolism in order to increase the yield and improve the quality of crops.
By origin, growth regulators are divided into the following groups [1]: endogenous compounds synthesized by plants (phytohormones); products of microbial synthesis; synthetic compounds.Because growth regulators of microbial origin are similar to compounds synthesized by plants (auxins, cytokinins, gibberellins, abscisic acid), they are also called phytohormones.
In agriculture growth regulators are used in stimulating seeds germination, activating vegetative growth of plants, accelerating their flowering and maturing, increasing yields, protecting against certain diseases, etc.Using those in agriculture production can significantly reduce the use of chemical plant protectors [1].
In 2015, the global market for plant growth regulators was estimated at $ 1.6 billion.From 2015 to 2020, its growth is forecasted to be 3.6% (to $ 1.91 billion) [2].The leading PGR producers are FMC (Food Machinery Corporation), Dow (USA), Syngenta AG (Switzerland), BASF SE (Germany) and Nufarm Limited (Australia).The most marketed plant growth regulators are gibberellins, consumed in about 60 tons per year [2].
The production of phytohormones by microorganisms was previously reviewed in 2013 [22].This work summarizes the results of studies mainly by Ukrainian scientists, focusing on the bacterial synthesis of phytohormones included in preparations for crop production, and the methods for determining these compounds are analyzed.
The purpose of this review is to summarize the current literary data on the phytohormone synthesis by microorganisms either plantassociated or not, and description of approaches to intensify the corresponding technologies of microbial phytohormone's synthesis.

The general characteristics of phytohormones
The term "hormone" was first proposed by animal physiologists Bayliss and Starling in 1904 (cited in [6]).At that time, a chemical compound was considered a hormone if during the migration with blood from one part of the body to another it caused a behavioral change.A few years later, in 1910, this term was introduced in the physiology of plants.
In 1948, after a lengthy discussion, the term "plant hormones" or "phytohormones" was established.By Thimann's definition (cited in [6]) phytohormone is an organic substance which is synthesized in trace amounts in certain parts of the plant and can be transported to other parts for the implementation of specific physiological functions.Now, the term "phytoregulators" describes both synthetic and natural organic compounds that affect plant life processes, but are not nutritional [1].
Currently, a substance is considered a phytohormone if it has the following properties (cited in [22,23]): -causes a specific physiological response; -is synthesized in a plant by one group of cells, and causes a response in another group (different places of synthesis and action); -has almost no significance in the main cell metabolism, and is used only for signal regulation; -acts in low concentration: 10 -5 -10 -12 mol/l.About five thousand compounds of plant and microbial origin, as well as artificially synthesized are known to have a regulatory effect on plants.However, no more than 50 are used in production [1].
Until recently, five types of phytohormones have been universally recognized: gibberellins, auxins, cytokinins, abscisic acid (ABA) and ethylene [1,[22][23][24].There also are hormone-like compounds of double auxincytokinin action, such as brassinosteroids and fusicoccin.Fusicoccin is synthesized by fungus Fusicoccum amygdali (parasitizing mainly on peach and almonds), and also is isolated from flowering plants.Steroid hormones brassinosteroids characterized by high biological activity, were isolated in 1979 from rapeseed pollen by American scientists.Today, more than 40 brassinosteroids have been identified, but the most physiologically effective are three of them: brassinolide, 24-epibrassinolide and homobrassinolide [1].

Phytohormones of microbial origin
The fundamental difference between plant and microbial phytohormones is that microorganisms do not need phytohormones to exist.These compounds are secondary metabolites, which are synthesized irregularly and often have undetermined physiological functions [23].
The study [10] is one of the first to research the role and biosynthesis pathways of auxins (IAA) in gram-positive phytopathogenic bacteria Rhodococcus fascians.In addition to auxins, R. fascians also produces cytokinins [30].
The ability to synthesize phytohormones (auxins, abscisic acid, cytokinins, and gibberellins) is also found in many microalgae [12,14].Although the functional role of endogenous phytohormones in microalgae remains unknown, studies conducted on Nannochloropsis oceanica suggest that it is similar to that of plants [14].

Microbial synthesis of auxins
As the most common plant hormone, auxin is well studied not only as a factor of the growth and development of vascular plants, but also as a metabolite of cyanobacteria [12,14], bacteria [11,20,24,25,29] and fungi [28,33].
Most publications are devoted to the synthesis of auxins by Rhizobacteria.As early as in 1990's it has been shown that 80% of the bacteria isolated from the rhizosphere synthesize IAA [40].
Synthesis of auxins by rhizobacteria.In [24], rhizobacterial proximity to the roots is described as follows: (1) rhizosphere: the microbes exist in the soil near the roots; (2) rhizoplane: the bacteria colonize the surface of the root; (3) endophytes live in the root tissue; (4) symbiotic nitrogen fixing bacteria include two groups: rhizobia (in symbiosis with leguminous plants) and representatives of the genus Frankia (symbionts of alder).Rhizobacteria can stimulate plant growth either directly (as a result of nitrogen fixation, phosphate solubilizing, iron ion chelating and phytohormones synthesis) or indirectly (inhibition of phytopathogens, induction of resistance to phytopathogens and abiotic stress conditions).That is why they are also called PGPR (plant growth promoting rhizobacteria) [24,41,42].Vessey [43] suggested calling the representatives of the first three groups of Rhizobacterium extracellular (extracellular PGPR, ePGPR), and the fourth -intracellular (intracellular PGPR, iPGPR).Extracellular rhizobacteria include representatives of the genera Bacillus, Pseudomonas, Erwinia, Caulobacter, Serratia, Arthrobacter, Micrococcus, F l a v o b a c t e r i u m , C h r o m o b a c t e r i u m , Agrobacterium, Hyphomycrobium.Intracellular rhizobacteria belong to the genera Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium and Allorhizobium [24].In [44], it is proposed to divide rhizobacteria into two groups: symbiotic and free-living.
In early 1990's it was found that the auxin synthesis in rhizobia increases in the presence of flavonoids secreted by the plant to start the processes of nodules formation [45].This supports the theory of the interaction between symbiotic microorganisms and plants through the excretion of phytohormones in natural conditions.
The ability to synthesize IAA is found in Klebsiella pneumoniae strains, isolated from the rhizosphere of wheat [51].Studies conducted with Klebsiella oxytoca isolated from the rhizosphere of Aspidosperma polyneuron showed that immobilized on inorganic matrices microorganisms retain or even increase the ability to synthesize IAA, while free cells gradually lose it [52].
Symbiotic and non-symbiotic nitrogen fixing bacteria of the genera Agrobacterium, Paenibacillus, Rhizobium and Azotobacter [53] are also capable of synthesizing IAA.Maximum synthesis (up to 5.23 μg/mg of biomass) was observed in Rhizobium and Paenibacillus, and if the representatives of these genera were grown together, their auxinogenic ability increased compared to that found for monocultures.
Our study [54] on the auxin's synthesis by highly effective strains of soybean rhizobia Bradyrhizobium japonicum UCM B-6023, B. japonicum UCM B-6036 and by an ineffective strain B. japonicum 604k showed that this ability does not correlate with their symbiotic activity.The strain B. japonicum 604k forms a large number of nodules with almost totally absent nitrogenase activity and synthesizes high amounts of auxins (indole-3carboxylic acid, indole-3-carbinol and indole-3-acetic acid hydrazide) but does not form IAA which would be physiologically active in plants.
It was first established in [55] that rhizospheric bacteria of the phylum Acidobacteria (representatives of the genera Granulicella and Acidicapsa) through the synthesis of IAA and iron chelating, stimulate the growth of Arabidopsis thaliana, and therefore can be considered representatives of PGPR-microbiota.
The synthesis of IAA in the presence of tryptophan is intensified because in microorganisms this amino acid is a precursor in the biosynthesis of IAA [33,39,49].Tryptophan can be transformed into IAA in three ways (Figure ): -synthesis via indole-3-pyruvic acid and indole-3-acetic aldehyde.This is the main route, typical for mushrooms and bacteria; -transformation of tryptophan to indole-3-acetic aldehyde may include an alternative pathway with the synthesis of tryptamine.This path is found in mycorrhiza fungi and cyanobacteria; -IAA formation through indole-3-acetamide.It is characteristic for phytopathogenic bacteria and fungi.
In [17,42,56,57] it is noted that the synthesis of IAA by rhizobacteria significantly increased in the presence of tryptophan in the cultivation medium.Table 1 shows indicators of the synthesis of IAA by a number of rhizobacteria depending on the presence of tryptophan in the cultivation medium.Thus, in order to establish the ability of bacteria to form IAA, virtually all researchers introduced the precursor of biosynthesis of this phytohormone to cultivation medium.
Synthesis of auxins by bacteria which are not associated with plants.Though phytohormone synthesis is one of the main factors of plant-microbe interaction, the plantassociated microbes are not necessarily the only producers of auxins.There is a lot of data supporting the auxinogenic activity of PGPRmicrobiota; however, many microbes not associated with plants also can synthesize IAA.
From twelve samples of sea water, sediment, and shrimp collected in the Egyptian coastal areas, 112 isolates belonging to the genus Streptomyces were isolated [15].The level of IAA synthesis by six most active strains was in the range of 5-50 μg/ml in the presence of tryptophan in starch-casein medium.Marine Streptomyces, in addition to

Biosynthesis pathways of from tryptophan in bacteria:
А -with indole-3-pyruvate; B -with indole-3-acetamide; C -with tryptamine; D -tryptophan pathway; E -with indole-3-acetonitrile phytohormones, synthesized metabolites with antibacterial and antifungal activity, and 28 strains showed nematocidal activity.Synthesis of IAA by fungi and yeasts.Many phytopathogenic cecidia-inducing fungi, as well as mycorrhizal fungi, are capable of synthesizing IAA.Those include representatives of the genera Taphrina, Phytophthora, Ustilago, Colletotrichum, Laccaria, Pisolithus, Amanita, Rhizopogon, Paxillus.Among the micromycetes, auxins are produced by fungi of the genera Fusarium, Rhizoctonia, Rhizopus, Absidia, Aspergillus, and Penicillium [39].It was found that Aspergillus niger synthesizes up to 128.3 mg/l of this auxin, and the production of phytohormones and fungal growth are positively influenced by the presence of gibberellin in the cultivation medium [78].
It was shown that the foliar treatment of Agrostis leaves with cells of Pythium aphanidermatum capable of auxin synthesis was accompanied by 200 times increased IAA content in leaves (up to 9760 ng/g of raw mass) and started an infectious process [79].However, the infectious process and phytohormonal activity are not always interconnected.For example, Ustilago maydis damages corn causing the formation of tumors with increased IAA concentration due to the fungal phytohormonal ability.However, mutants that do not generate this auxin still cause the development of tumors, so the infectious process is not related to IAA synthesized by the fungus [80].
Yeasts are typical inhabitants of the phyllosphere, but until in the last decade their phytohormonal activity has not been studied.The first works on the synthesis of phytohormones in yeasts (2004)(2005)(2006)) appeared immediately after their ability for endophytic development had been discovered [81,82].At present, the literature has information on mainly auxin synthesis by yeasts.
For example, Cyberlindnera (Williopsis) saturnus, isolated from the roots of maize, produce IAA [81].These yeasts were selected from 24 endophytes' species and artificially inoculated in studied corn plants.L-tryptophan, a precursor of auxin, was introduced in the soil on which the plants inoculated with C. saturnus grew.In one of the versions, L-tryptophan was not added in soil.It was established that plants inoculated with yeast grew faster than non-inoculated plants and the best growth was observed in soil with tryptophan.In 2009, strains of Rhodotorula graminis and Rhodotorula mucilaginosa, which synthesized up to 40 mg/g biomass of IAA acid, were isolated from the apexes of poplar trees [83].In 2012, scientists have researched the ability to synthesize auxins in 114 strains of yeast isolated from leaves of tropical plants [84].Thirty nine strains were found to produce phytohormones, although in different quantities (27-234 μg/ml).In 2014, the same authors isolated 158 yeast strains from sugar cane and found that 69 of them synthesized IAA.The maximum concentration (565.1 mg/l) was observed in Rhodosporidium fluviale.In these studies, tryptophan (1 g/l) was added into the cultivation medium [85].
The work [17] researched IAA synthesis by strains of Saccharomyces cerevisiae and Notes: «-» -no data; «+» -IAA concentration is not given.
Saccharomyces paradoxus, isolated from different ecosystems.All 24 studied yeast strains synthesized from 10 to 120 μg/ml of IAA in the presence of tryptophan in the medium.Growing on tryptophan-less medium, only three strains were able to synthesize IAA.In 2017, 147 strains of 46 yeast species were studied.Most of them were isolated from phyllosphere, rhizosphere, leaf bedding, soil and entomophilic flowers [23].The ability to synthetize IAA was found in 92% of the researched strains.Metschnikowia pulcherrima KBP Y-6020 and Saitozyma podzolica KBP Y-4614 synthesized the highest amount of this phytohormone (18693.3 and 22206.7 μg/ml, respectively).The levels of IAA synthesis by Rhodotorula mucilaginosa KBP Y-5419 and Candida trypodendroni KBP Y-5475 ranged 7593.3-5033.3μg/ml [23].
Nutaratat et al. [86] researched the synthesis of IAA by yeasts isolated from rice leaves and sugar cane.Of more than 1000 tested strains, only 13 produced this hormone in a concentration of 1.2 to 29.3 mg/g of biomass.The highest amount of IAA was synthesized by the strain Rhodosporidium paludigenum DMKURP301.
Ways of intensification of IAA synthesis in microorganisms.The IAA synthesis path and mechanisms of its genetic and biochemical regulation were studied in Pseudomonas mendocina BKMB 1299 [20].It was established that the synthesis of this hormone occurs by indole-3-pyruvic acid path (Figure ) involving three enzymes: tryptophan-aminotransferase, indole-3-pyruvate decarboxylase and indole-3acetaldehyde dehydrogenase.
The shikimate pathway in the studied bacteria was shown to be regulated by retroinhibition of the key enzyme 3-deoxy-Darabinoheptulosonate 7-phosphate (DAHP) synthase with two amino acids, tyrosine and tryptophan.The synthesis of this enzyme in P. mendocina BKMB 1299 was not repressed.The synthesis of tryptophan is controlled by the repression of trpE-, trpD-and trpCgenes by tryptophan, and by retro-inhibition of anthranilate synthase with the same amino acid.On the contrary, the synthesis of tryptophan-aminotransferase and indole-3-pyruvate decarboxylase is activated by tryptophan.In addition, the synthesis of tryptophan-aminotransferase is repressed by anthranilate.Using nitrosoguanidine mutagenesis and further selection of clones resistant to 5-Fluoro-dl-tryptophan (which is the toxic analogue of tryptophan) produced regulatory mutants capable of over-synthesis of IAA.The production of the hormone in the P. mendocina mutant strain 9-40 was 10 times higher than that of P. mendocina BKMB 1299.IAA over-synthesis correlated with an increase in the synthesis of key enzymes of the aromatic pathway, DAHP synthase and tryptophan synthase (twice), tryptophanaminotransferase and indole-3-pyruvate decarboxylase (approximately eight and 80 times, respectively) [20].
Then, the strains producing indole-3pyruvate decarboxylase, an enzyme involved in IAA synthesis, were genetically engineered.In order to create such IAA producing strain of P. mendocina, the ipdC gene (encoding the synthesis of indole-3-pyruvate decarboxylase) was cloned in Escherichia coli DH5 strains using pUC18 and pXcmKn12 vectors.As a result, a hybrid plasmid pTVN4 (4.5 kb) was obtained with the inserted ipdC gene of 1.7 kb.To study the expression of the ipdC gene in P. mendocina, a plasmid pAYSCD1.7 was constructed.It was stably inherited in the bacterial cells.
The presence of a plasmid with an integrated ipdC gene increased the level of synthesis of indole-3-pyruvate decarboxylase (in 5.3 times) and IAA.Thus, the P. mendocina 9-40 regulatory mutant and the recombinant strain carrying the plasmid pAYC1.7 with the integrated ipdC gene are promising for use in crop production [20].
The study [21] is a continuation of research on IAA synthesis by R. paludigenum DMKURP301 [86].This is one of the few publications in which the authors optimized the process of biosynthesis of this phytohormone.Mathematical planning of the experiment allowed increasing the concentration of the target product to 1,624 g/l.The maximum IAA synthesis was achieved under conditions of growth of DMKURP301 strain on sucrose (1%) as carbon source, corn extract (0.1%) as nitrogen source, yeast extract (1%) as growth factors, and tryptophan (0.4%) as precursor of biosynthesis.The optimum temperature was 30 С, pH 7.0, the duration of cultivation under agitation (200 rpm) was 9 days.The process was scaled: the IAA concentration was 1.627 g/l in fermenter of 2 liters [21].
However, the authors of [21] failed to exceed the levels of IAA synthesis by the bacterial strain Pantoea agglomerans PVM [87].The optimization of the cultivation conditions (in particular, the composition of the nutrient medium) increased the concentration of IAA to 2.191 g/l.The medium contained sucrose (1%) as carbon source, meats extract (8 g/l) as nitrogen source, and tryptophan (1 g/l) as the precursor of IAA biosynthesis.
Thus, the production of auxins (in particular, their physiologically active form, IAA) is characteristic of microorganisms that interact with plants, as well as of many bacteria, fungi and yeast that are nonassociated with plants.The synthesis of IAA in vitro is usually enhanced by the presence of tryptophan, a precursor of this phytohormone biosynthesis, in the cultivation medium.Representatives of the genera Streptomyces, Bacillus and Paenibacillus simultaneously with phytohormones synthesize metabolites that can be used for pest control in crop production.The progress achieved so far in increasing IAA synthesis by P. agglomerans PVM and R. paludigenum DMKURP301 means that biotechnological production of IAA is possible.
Microbial synthesis of cytokinins.Symbiotic nitrogen-fixing bacteria.Unlike auxins, there is much less data on formation of cytokinin by microorganisms, although the ability of Rhizobium leguminosarum bacteria to synthesize these phytohormones in vitro has been reported for the first time in 1970s [88].Recently, the study of cytokinin synthesis by symbiotic nitrogen-fixing bacteria of the genera Sinorhizobium, Mesorhizobium and Bradyrhizobium is associated with finding out the role of these phytohormones in nodulation [8,26,89,90].It was found in [26] that 9 strains of Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium medicae and Mesorhizobium loti synthesize 25 forms of cytokinins, some of which are methylated.The strain Bradyrhizobium sp.ORS285 also synthesizes mainly 3-methyl-thiol derivatives of trans-zeatin and N 6 -(2-isopentenyl) adenine in concentrations three orders of magnitude higher than non-methylated analogs (1000-5000 and 5-60 pmol/l, respectively) [89].However, it is noted [26,89,90] that the bacterial synthesis of cytokinins is not a prerequisite for the symbiotic relationship with a plant, and the decisive role in this process belongs to plant cytokinins [90].At the same time, our research [54] found a direct correlation between the symbiotic efficiency of rhizobial strains and the level of cytokinins synthesis.For example, highly effective strains of soybean symbionts B. japonicum UCM B-6023 and B. japonicum UCM B-6036 produced a wide range of cytokinins, mostly zeatin and transzeatin-riboside. Ineffective strain B. japonicum 604k synthesized these phytohormones in significantly smaller amounts comparable to the highly effective strains.
Other rhizobacteria.Inoculating wheat rhizosphere by strains of Bacillus sp., capable of synthesizing cytokinins, resulted in an increased content of zeatin-riboside in roots and then in stems.Strains of Bacillus licheniformis, Bacillus subtilis and Pseudomonas aeruginosa, isolated from the rhizosphere of different plants, synthesized cytokinins.The maximum concentration of phytohormones (1091.9g/ml trans-zeatin and 521 ng/ml zeatin-riboside) was achieved in the stationary phase of Bacillus licheniformis growth, which is typical of secondary microbial metabolites [91].
The endophytic strain of Bacillus amyloliquefaciens IMV B-7100, isolated from cotton, produced cytokinins in the concentration of 141 μg/g of biomass, mostly zeatin (113 μg/g biomass) [92].B. amyloliquefaciens subsp.plantarum UCM B-5113 synthesized 152.4 pcmol/l of cytokinins in 120 hours of growth in liquid LB medium [93], and in the presence of Arabidopsis thaliana root extractors under similar conditions of growth, the concentration of phytohormones increased to 295.4 pcmol/l.The authors suppose that cytokinins can stimulate synthesis of SHY2, the key regulator of growth and development of plant meristem.Pseudomonas fluorescens 6-8 improves the growth of cauliflower roots under gnotobiotic conditions [94].Investigated strain is characterized by high ability to colonize the surface of the roots due to the synthesis of cytokinins.A fundamentally new role of cytokinins of Pseudomonas fluorescens G20-18 as intermediaries in the biocontrol of phytopathogenic bacteria Pseudomonas syringae was established in [18].
There are two main ways of synthesizing cytokinins in microorganisms [39]: de novo synthesis of isopentenyl pyrophosphate and adenosine-5-monophosphate (characteristic of phytopathogenic bacteria) and the destruction of tRNA resulting in cis-zeatin produced by tRNA-isopenthenyltransferase.This path is found in phytopathogenic fungi.The process of cytokinins formation was interrupted in the mutants of Magnaporthe oryzae which lacked the gene responsible for the synthesis of tRNA-isopenthenyltransferase and they were characterized by reduced virulence [35].
The strain of phytopathogenic bacteria Rhodococcus fascians D188 synthesizes N 6 -(2isopentenyl)adenine, cis-zeatin, trans-zeatin, 2-methylthio derivatives of N 6 -(2-isopentenyl) adenine, 2-methylthio derivatives of cis-zeatin at a concentration of 3, 2.5, 0.03, 0.4 and 4.5 nM respectively [30].Synthesis of cytokinins is encoded by six genes that form the fas operon on pFiD188 plasmid.Analysis of various fas mutants, defective in one or more genes, showed that the formation of cytokinins is only one of the mechanisms of pathogenicity in this strain.
Other microorganisms.If the role of cytokinins in phytopathogenic microorganisms is clear, then the discovery of these phytohormones in the tuberculosis pathogen Mycobacterium tuberculosis in 2015 was a real surprise [16].The authors [16] suggested that cytokinins can contribute to infecting cells and serve as a kind of communicative molecule between mycobacteria to control the development of infection.
In 1980's it was found that bacteria isolated from the sea and sea sediments synthesized cytokinins in concentrations of 0.05-0.30μg/l [99].Moreover, 45-55% of bacteria isolated from the sediments were capable of synthesizing phytohormones, compared to 5-15% isolated from water.The role of bacterial cytokinins in marine ecosystems remains a controversial issue, but it is assumed that they can be associated with algal blooms of water.
Thus, the ability to synthesize cytokinins and auxins is detected in a wide range of microorganisms, not necessarily associated with plants.Until recently there were not so many publications about the production of these phytohormones, and researchers mainly studied the cis-, trans-zeatin and zeatinriboside, since these phytohormones are the most widespread in nature.However, the development of analytical methods [100,101] offered new opportunities for detecting new forms of these phytohormones [26,30].
Microbial synthesis of gibberellins.Many micromycetes are capable of synthesizing gibberellins, not only the representatives of the genus Fusarium (Gibberella), which are industrial producers of gibberellic acid [5,6,102,103].Table 3 shows data on the synthesis of gibberellins by endophytic fungi isolated from various plants [103].The level of synthesis of gibberellins in these fungi is low, but there are fungi that can synthesize from 6 to 600 ng/ml of A 4 .Synthesis of gibberellins by endophytic fungi of the genus Penicillium is one of the mechanisms that allow plants to survive under salt stress [104].Concerning the relationship between pathogenicity and phytohormonal activity, the study of growth stimulating and pathogenic strains of Fusarium culmorum showed that the latter synthesized four times less gibberellin [105].Many plant-associated phytopathogens [9,24,34,41] and freely existent [12,15] bacteria also synthesize gibberellins.Ten wild and mutant (nod, fix) strains of Rhizobium phaseoli were study in 1980s and showed that gibberellins were also synthesize by mutants unable to form nodules and fix nitrogen.So, nitrogen-fixing ability is not related to phytohormonal activity [106].
Since the gibberellic acid (A 3 ) is the first phytohormon produced by microbial synthesis, and this technology has been developing for more than 50 years, we will now consider recent approaches to improve it.

Intensification of synthesis of microbial gibberellins
Famous industrial producers of gibberellic acid are Gibberella fujikuroi and Fusarium monilforme.This phytohormone is mainly obtained by submerged fermentation [3,5,6,102,103,107,108], but recently, these compounds were produce in conditions of solid state fermentation [4,109].
Optimization of cultivation conditions for producers of gibberellic acid.Under optimal conditions for the cultivation of F. moniliforme (Egyptian local isolate), the synthesis of gibberellic acid increased by 4.3 times (up to 1.4 g/l).Such conditions are: the concentration of fructose 6%, ammonium sulfate 0.6 g/l, magnesium sulfate 1.5 g/l, potassium dihydrogen phosphate 1.0 g/l, temperature 30 C, initial pH 5.0 [101].
Optimizing the cultivation conditions of F. moniliforme M104 strain (temperature 30 C, initial pH 5.5, cultivation duration 8 days, glucose concentration in the medium 30 g/l, ammonium chloride 3 g/l) led to 4.775 g/l concentration of synthesized gibberellic acid, which is more than 5.5 times higher with indicators before optimization [102].
Cultivating F. moniliforme NCIM 1100 strain for eight days on a Caspec-Dax liquid medium with sucrose as a carbon source at 30 °C and an initial pH of 7.0 was accompanied by synthesis of almost 15 g/l of gibberellic acid.At present, this is the highest level of microbial synthesis of A 3 gibberellin [5].
Improvement of strains producing gibberellic acid.To enhance the synthesis ability, F. moniliforme strain was subjected to -irradiation ( 60 Co -radiation, sublethal dose of 6.5 kgy).Of the 28 obtained mutants, F. moniliforme -14 strain synthesized twice more gibberellic acid compared to the nonirradiated original strain [107].
In other studies, F. moniliforme (Egyptian local isolate) after -irradiation ( 60 Co -radiation, 0.5 kg) synthesized 2.36 g/l gibberellic acid, which is 1.4 times more than the original strain under similar cultivation conditions [101].
The initial pigmented strain G. fujikuroi NCIM 1019, characterized by the presence of intracellular carotenoids, was exposed to ultraviolet irradiation, resulting in a nonpigmented intermediate mutant Car-1 [3].After the UV irradiation of the Car-1 strain, the mutant Mor-1, capable of increased synthesis of gibberellic acid, was obtained.As a result of further irradiation of the Mor-1 strain, the strain Mor-25 was isolated.It was characterized by the presence of short, heavily branched hyphae.While growing in a liquid medium, the Car-1 strain formed a high-tensile culture liquid, unlike the Mor-25 strain.The concentration of gibberellic acid synthesized by the Mor-25 strain was twice higher than that generated by the Car-1 strain.The strain Mor-25 synthesized only gibberellic acid, while Car-1 also produced fusaric acid.These results are quite significant, since fusaric acid is toxic for animals and plants [3].
Improvement of producers of gibberellins A 4 and A 7 .The gibberellin A 4 is notable for its high biological activity and promotes the formation  (13.4),A 20 (1.11) and growth of fruits like apples and grapes, and vegetables (tomatoes, peas) [108].Despite the high biological activity of gibberellin A 4 , its application in crop production is limited due to the high costs.The spectrum of action of gibberellin A 7 is wider, and its biological activity in many cases is higher compared with such A 3 and A 4 .Most well-known producers synthesize a mixture of gibberellins A 4 and A 7 , in which the A 4 /A 7 ratio varies greatly.
In addition, isolating individual preparations of A 4 and A 7 from the mixture is complicated because of their very close polarity.
In 1997, the strain F. moniliforme VKPM F-446 was created.It is the first superproducer of gibberellin A 7 which forms gibberellins A 3 and A 4 in insignificant quantities.The superproducing strain was obtained by fusing protoplasts of a strain isolated from the affected rice, followed by UV irradiation treatment.The strain synthesized 400-700 mg/l of gibberellin A 7 and only 20-80 mg/l of A 4 on medium with sunflower oil (60 g/l), corn extract (35 g/l) and ammonium acetate (0.57 g/l) [110].
In other studies [108], the strain G. fujikuroi 1019 was subjected to combined mutagenesis using UV-irradiation and pravastatin (250 mg/l), which inhibits the activity of HMG-CoA reductase, involved in the formation of mevalonic acid (an intermediate of biosynthesis of gibberellins).Thus, the mutant Mor-189 was obtained, capable of synthesizing gibberellins A 3 and A 4 .That mutant synthesized mostly A 4 at pH levels of 5.5 during cultivation using glucose and wheat gluten as sources of carbon and nitrogen, respectively.The A 4 synthesis increased with glucose supplementation during the cultivation of the strain.Under such conditions, the concentration of gibberellin A 4 reached 600 mg/l, which was 84% of the total amount of A 4 and A 3 (713 mg/l) [108].
Immobilization of producer cells.F. mo nili forme -14 was immobilized by adsorption on sponge disks (2-4 mm in diameter and 18-20 mm in diameter) cut from dried Luffa fruits [107].Cultivating immobilized cells increased the concentration of A 3 to 1.9 g/l, and at initial pH 5.0 of the medium (milk permeate), up to 2.25 g/l.
Subsequent experiments showed the possibility of repeated reuse of immobilized F. moniliforme -14 cells on Luffa disks.Thus, a one-time replacement of the nutrient medium was accompanied by an increase in the concentration of gibberellic acid to 2.4 g/l on the eighth day of cultivation.
The main advantages of this technology are [107]: immobilization of cells, which allows them to live and be active for a long time; immobilization via adsorption (as opposed to inclusion in gel or covalent binding) avoids the cost of purchasing expensive gels and prevents cell release due to weak binding to carriers; the use of a non-toxic cheap and affordable natural matrix, Luffa sponge with a lot of free pores for new cells which provides stable contact surface in prolonged re-use; using whey as a substrate, which is a cheap by-product of the dairy industry.
Cultivating the immobilized in Ca-po lygalacturonate G. fujikuroi cells in a fluidized bed reactor in a medium containing glucose and ammonium chloride (carbon/nitrogen ratio 38.6), rice flour (2 g/l) at pH 5.0 and 30 C was accompanied by a synthesis of 3.9 g/l of gibberellic acid, which is three times higher compared to the values established for the suspension culture under similar conditions of cultivation [111].
Immobilized on sponge cubes F. moniliforme (Egyptian local isolate) cells in a medium based on milk permeate synthesized 1.93 g/l gibberellic acid, while free cells produced only 1.6 g/l.One-time replacement of the nutrient medium after six days of immobilized cell cultivation was accompanied by an increase in the concentration of gibberellic acid to 2.2 g/l [101].
Synthesis of gibberellic acid on industrial waste.Industrial wastes as substrates for the production of gibberellic acid are used predominantly in solid-phase cultivation.
Table 4 shows data on biosynthesis of gibberellic acid on different substrates in solid phase cultivation.According to Table 4, the highest rates of synthesis of gibberellic acid (105 g/kg) were achieved using Jatropha press cake as a substrate [5].In these studies, F. moniliforme NCIM 1100 was used as a producing strain.Jatropha press cake is a biodiesel production waste; the seed oil of this plant is transetherified into biodiesel.
Press cakes are relatively useless lignocellulosic substrate containing 15% cellulose and 30% lignin.In addition, these wastes are toxic because of the presence of phorbol ethers and require detoxification before use as animal feed.It should be noted that the level of synthesis of gibberellic acid by the strain F. moniliforme NCIM 1100 is the highest presently for solid-phase cultivation of producers [5].
In other studies [4], strains G. fujikuroi LPB 02, LPB 05, LPB 06, LPB Bca and F. moniliforme LPB 03 were used as producers of gibberellic acid in solid-phase cultivation on such industrial waste as citrus pulp, soybean bran, cane pulp, soybean and coffee bean husks, and manioc pulp.The cultivation of strains producing gibberellic acid was carried out on both mono-and mixed industrial waste.In the mixed substrates, the ratio of mono substrates was 1: 1.
The highest concentration of gibberellic acid was observed under cultivation of all strains on citrus pulp (3.1-5.7 g/kg), as well as on a mixture of citrus pulp and coffee husks (about 3 g/kg).For further research, the strain F. moniliforme LPB 03 was selected because it was characterized by the highest level of synthesis of the final product.
The following experiments showed that F. moniliforme LPB 03 inoculum cultivated on citrus pulp extract with the addition of 35 g/l sucrose, synthesized 5.9 g gibberellic acid per kg of citrus pulp at the third day of cultivation [4].Subsequently [115], the same authors found that the level of synthesis of gibberellic acid on a citrus pulp depends on the level of aeration: during the cultivation of F. moniliforme LPB 03 in a column reactor, the amount of the target product increased to 7.34 g/kg.
So, the methodology of microbial synthesis of gibberellic acid has recently developed much.If in the first technologies of submerged cultivation A 3 concentration did not exceed 0.3-0.5 g/l, now it reaches 5-15 g/l.A significant advantage of solidphase cultivation compared to the submerged fermentation is the possibility of bioconversion of industrial waste into economically valuable phytohormones.In addition, there are other advantages that make the process of solid phase cultivation commercially viable: high output of the final product, lower energy consumption, and lesser environmental impact.At the same time, the final product yield is sufficient to compensate for higher allocation costs, thereby reducing the cost of gibberellic acid.
Synthesis of phytohormones by the produ cers of surfactantas.Acinetobacter calcoaceticus ІМV В-7241, Rhodococcus erythropolis ІМV Ac-5017 and Nocardia vaccinii ІМV В-7405.In recent years, there has been evidence that some microorganisms synthesize other metabolites (enzymes, bacteriocins, polysaccharides, polyhydroxyalkanoates) simultaneously with surfactants under certain conditions of cultivation [116][117][118].The ability of strains to synthesize a complex of metabolites with a variety of biological properties greatly extends the scope of their practical application.
Our studies have shown that Rhodococcus erythropolis IMV Ac-5017 Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii IMV B-7405 have antimicrobial properties against a number of microorganisms, including phytopathogenic bacteria of genera Pseudomonas and Xanthomonas [119].Moreover, the water phase remaining after the extraction of surfactant from the supernatant of the culture liquid activated the cell growth of several phytopathogenic bacteria.Such unexpected results allowed us to assume that the producers of surfactants synthesize also other biologically active substances, in particular, phytohormones.
Table 5 shows the data on the synthesis of phytohormones by A. calcoaceticus IMV В-7241, R. erythropolis IMV Ac-5017 and N. vaccinii IMV В-7405 cultivated on various carbon substrates, including processed sunflower oil.
Presently, there are many publications about the synthesis of phytohormones by microorganisms.The cultivation media, Coffee husks and manioc pulp Optimized cultivation conditions Flasks 492.5 mg/kg [113] Wheat flour and starch Optimized cultivation conditions Flasks 4.5-5 g/kg [114] Citrus pulp Method of inoculum preparation Flasks 5.9 g/kg [4] Jatropha seed press cake Optimized cultivation conditions Flasks 105 g/kg [5] Shea nut shells Optimized cultivation conditions Flasks 1.8 mg/ml [109] Citrus pulp Levels of aeration Column reactor 7.34 g/kg [115] however, contain glucose, sucrose, dextrose, glucuronic acid, peptone, tryptone, mannitol as a source of carbon and exogenously introduced tryptophan as a precursor of auxins biosynthesis (Table 1).Our studies have shown for the first time the possibility of producing phytohormones in cheap media using toxic industrial waste as substrates (in particular, waste oil) without the addition of tryptophan.There are also some reports on the simultaneous synthesis of phytohormones and metabolites with antimicrobial properties in the literature (Table 2), but these antimicrobial metabolites are mostly antifungal (rarely nematocidal).If antibacterial they are antibiotics and consequently, in this case, the resistant forms of microorganisms may rapidly appear.The mechanism of antimicrobial activity of surfactants, unlike antibiotics, prevents the emergence of bacteria resistant to them.We have also for the time established [120] the ability of surfactant producers to synthesize phytohormones.The formation of indole-3-acetic acid by bacteria (mainly by representatives of the genus Rhodococcus), isolated from soils contaminated with hydrocarbons and heavy metals was reported only in 2016 [121].However, the ability to synthesize surfactants was determined by the emulsification index and decrease in surface tension, which turned out to be insignificantup to 60-65 mN/m (compared with 30-35 mN/m by the surfactant producers).
Ability of A. calcoaceticus IMV В-7241, R. erythropolis IMV Ac-5017 and N. vaccinii IMV В-7405 to simultaneously synthesize surfactants and phytohormones when cultivated on different substrates, including cheap industrial waste, allows developing economically profitable nonwaste technology for obtaining complex microbial preparations promising for use in plant growing.
Thus, review of the literature on the microbial synthesis of phytohormones confirms the general conclusions drawn in [23]: -many microorganisms are capable of synthesizing phytohormones of the three main groups of hormonal stimulants: auxins, cytokinins and gibberellins.Moreover, representatives of the same genus and even species are capable of synthesizing several hormones at once; -microorganisms, capable of synthesizing phytohormones, also stimulate the growth of higher plants, which is confirmed in many studies; -there is no confirmed association of phytohormonal activity with the pathogenicity of microorganisms or their epiphytic (endophytic) lifestyle; -the ability to synthesize phytohormones differs greatly not only within the same genus, but even within a species; -microorganisms synthesize phyto hormones as secondary metabolites.
In addition, there are a few reports on simultaneous synthesis of phytohormones and specific final products.This does not support the generally accepted in the biotechnological research concept of "one producer -one product" which focuses only on increasing the synthesis of the main product.
Individual literary data and our own results show the promise in creating multifunctional microbial preparations with diverse biological properties.These preparations would include a complex of biologically active substances, among them phytohormones of different chemical nature, synthesized together.A few of the preparations we have developed at the present time are available in Ukraine (Ecovital, Ecophosphoryn, Azotobacteryn-K, Rizobin, Ecoryz, Averkom-nova) [1,77].
Increasing IAA synthesis to 1.6-2 g/l by yeast R. paludigenum DMKURP301 and bacteria P. agglomerans PVM is a reason to hope that this phytohormone and gibberellic acid will be obtained by microbial synthesis in the near future.

Table 1 . Effect of exogenous tryptophan on the indole-3-acetic acid synthesis by rhizobacteria Strain Main components of cul- tivation medium IAA concentration Source With added tryptofan Without tryptofan
Note: N.d.-not determined.