Review Article ISSN: 0976-7126 CODEN (USA): IJPLCP Batabyal , 12(1):17-25, 2021

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Azospirillum: Diversity, Distribution, and Biotechnology Applications Biswajit Batabyal* Consultant Microbiologist, Serum Analysis Center Pvt. Ltd., Kolkata, (W.B) - India

Article info Abstract The genus comprises plant-growth-promoting Received: 11/12/2020 (PGPB), which have been broadly studied. The benefits to plants by inoculation with Azospirillum have been primarily attributed to its Revised: 23/12/2020 capacity to fix atmospheric , but also to its capacity to synthesize phytohormones, in particular indole-3-acetic acid. Recently, an increasing number of studies has attributed an important role Accepted: 21/01/2021 of Azospirillum in conferring to plants tolerance of abiotic and biotic stresses, which may be mediated by phytohormones acting as signaling © IJPLS molecules. Tolerance of biotic stresses is controlled by mechanisms of induced systemic resistance, mediated by increased levels of www.ijplsjournal.com phytohormones in the jasmonic acid/ethylene pathway, independent of salicylic acid (SA), whereas in the systemic acquired resistance—a mechanism previously studied with phytopathogens—it is controlled by intermediate levels of SA. Both mechanisms are related to the NPR1 protein, acting as a co-activator in the induction of defense genes. Azospirillum can also promote plant growth by mechanisms of tolerance of abiotic stresses, named as induced systemic tolerance, mediated by , osmotic adjustment, production of phytohormones, and defense strategies such as the expression of pathogenesis-related genes. The mechanisms triggered by Azospirillum in plants can help in the search for more-sustainable agricultural practices and possibly reveal the use of PGPB as a major strategy to mitigate the effects of biotic and abiotic stresses on agricultural productivity. The development of cultivars with improved nitrogen use efficiency (NUE) together with the application of plant growth-promoting bacteria is considered one of the main strategies for reduction of fertilizers use. Although Azospirillum strains used in commercial inoculants formulations presents diastrophic activity, it has been reported that their ability to produce phytohormones plays a pivotal role in plant growth-promotion, leading to a general recommendation of its use in association with regular N-fertilizer doses. In addition, a high variability in the effectiveness of Azospirillum inoculants is still reported under field conditions, contributing to the adoption of the inoculation technology as an additional management practice rather than its use as an alternative practice to the use of chemical inputs in agriculture. Keywords: Azospirillum; Diversity; Distribution; Applications

Introduction bacterium [4]. A. brasilense is able to fix nitrogen Azospirillum is a Gram-negative, microaerophilic, in the presence of low oxygen levels, making it a non-fermentative and nitrogen-fixing bacterial microaerobic . Originally isolated from genus from the family of . [1, nitrogen poor soils in the Netherlands in 1925, 2] Azospirillum bacteria can promote plant growth [3] . is a well studied, nitrogen-fixing (diazotroph), genetically *Corresponding Author tractable, Gram-negative, alpha-proteobacterium E.Mail : [email protected]

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It is widely found in the of grasses horizontally acquired [17]. Up until now, around the world where it confers plant growth 16 Azospirillum have been characterized; promotion [5, 6]. however complete genomic sequences of Whether growth promotion occurs through direct only Azospirillum brasilense, Azospirillum nitrogen flux from the bacteria to the plant or lipoferum, Azospirillum sp. B510, and a draft through hormone regulation is debated [7, 8].The of Azospirillum amazonense genome have been two most commonly studied strains are Sp7 published [18]. (ATCC 29145) and Sp245. Azospirillum amazonense was found to be The genome of A. brasilense Sp245 has been associated with the roots and of sequenced and is 7Mbp in size and spread across several grasses including sugar-cane, , 7 chromosomes. The high GC content (70%) sorghum, and rice revealing a broad ecological makes it challenging to engineer. [9] Sp245 can distribution in Brazil. Studies revealed that A. be transformed with OriV origin of amazonense is phylogenetically closer replication through conjugation and elect to Rhodospirillum centenum and Azospirillum roporation. The strain is natively resistant to irakense than to A. brasilense. Unlike both spectinomycin and ampicillin antibiotics. Ka other Azospirillum strains, A. amazonense can namycin resistance is used as a selectable grow in the presence of sucrose as sole carbon marker. A. brasilense has a high evolutionary source and is also better adapted to soil acidity, adaptation rate driven by codon mutation which offers the bacterium additional advantages and transposon hopping. for colonization of plant root tissue in acid A strain originally classified as environments [19, 20]. Moreover, A. fauriae was reclassified as A. brasilense. It was amazonense genomic analyses revealed the first isolated from a hand wound of a woman in presence of genes not commonly distributed in Hawaii in 1971, and was named for Yvonne Faur other Azospirillum species such as those "for her contributions to public health responsible for the utilization of salicin as carbon bacteriology and, specifically, for her contribution source (similar to A. irakense) and a gene cluster to the recognition of pink-pigmented bacteria [10, (RubisCO) implicated in carbon fixation. 11]. However, our understanding of phytohormone The genus Azospirillum comprises free-living, production in A. amazonense is still incomplete. nitrogen-fixing bacteria that are known as plant The genomic plasticity of A. amazonense is growth-promoting rhizobacteria (PGPR), which probably related to the versatile gene repertoire can colonize, by adhesion, the root surface or the present in the genome of this bacterium intercellular spaces of the host plant roots. The suggesting that horizontal gene transfer may have potential role of the PGPR in association with an impact on the adaptation and evolution of this economically important cereals and other grasses species. Gene organization and phylogenetic is to promote plant growth by several mechanisms analysis demonstrated that genes coding for including and phytohormone proteins responsible for the nitrogen fixation production [12]. Several species process, carbon fixation (RubisCOs), and of Azospirillum are able to secrete phytohormones molecular hydrogen oxidation (hydrogenases) is such as auxins, gibberellins, cytokinins, and nitric more closely related to Rhizobiales members than oxide as signals of plant growth promotion [13, to related species. 14]. Azospirillum genomes, as previously The importance of A. amazonense genetic suggested for various strains, are larger and are variability, an in silico comparative genomic comprised of multiple replicons indicating a analysis using subtractive hybridization was potential for genome plasticity [15]. Genomic performed using total coding sequences (CDS) rearrangements can occur spontaneously where from A. amazonense to compare with genomes of replicons can be lost upon the formation of new closely related bacteria. The analysis of conserved megaplasmids [16, 17]. Moreover, genome and specific A. amazonense coding sequences sequencing of some Azospirillum species revealed indicated features that distinguished A. that significant part of the genome has been amazonense from other Azospirillum species.

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Furthermore, the specific interesting features bacterial broth for producing liquid inoculants, or related to phytohormone production may provide obtaining dehydrated cells that may be several cues to establish A. amazonense pathways incorporated in a solid or liquid carrier [25, 26, for auxin biosynthesis. and 27]. Liquid inoculants simplify both the Inoculation technology with plant growth- industrial production and the field application, promoting bacteria (PGPB) has been presented although compared with solid formulations, such worldwide as an important tool for reaching as peat- or polymer-based inoculants, bacteria in sustainability in agriculture due to its low liquid inoculants appear to be more sensitive to environmental and production costs compared stressful conditions and can exhibit decreased with industrial inputs. However, different from viability when used on seeds or soil [28]. symbiotic relationships, where plant-bacteria Effectiveness of PGPB inoculants has been interactions have been widely exploited and are improved by immobilization of inoculant cells in relatively well understood, the associative polymeric carriers, such as alginate and starch interactions driven by PGPB are discreet and foam [29, 30]. Thus, the actual demand is for elicited by factors that have only recently started improved liquid inoculants formulations, which to be clarified [21,22]. It is not difficult to realize are replacing peat-based inoculants and currently that the broad adoption of PGPB inoculation as comprise ~80% of doses sold for soybean crops in regular agricultural practice is somehow impaired Brazil [31]. While the technical criteria required by the lack of scientific knowledge regarding the to produce high-performance peat-based ecology, physiology and biochemistry of inoculants are well defined, these same criteria are associative plant-bacteria interactions. Although treated as industrial secrets or proprietary feasible, the replacement of chemical inputs in information for liquid inoculants. Even commercial agriculture with bioproducts considering the presence of high- developed from and based on the rational performance Azospirillum-based liquid inoculants exploitation of plant-microbe natural on the market, the information regarding the relationships, such as nutrient-provider bacteria in composition of these inoculants is mostly limited substitution for soluble fertilizers, or biocontrol to that presented on the product label, which agents in substitution for pesticides, remains a increases the difficultly of conducting a thorough major challenge [23]. In this sense, efforts to scientific analysis of the role of each ingredient in strengthen inoculation technology in non- the formulation and its effects on the final quality leguminous crops with PGPB need to incorporate of the product applied in the field [24]. This may a broader understanding of the determinants of be best exemplified by inoculants formulations bacterial rhizocompetence and competitiveness using the PGPB Azospirillum brasilense, which necessary to successfully achieve a plant-PGPB are prepared from different bacterial strains and interaction. In the same way, one must consider are available in a wide variety of commercial the physiological status of the inoculated products worldwide; variations in their microorganisms such that high viability is performance under field conditions are still maintained under adverse conditions found in soil reported. and/or during storage. Such challenges are The diazotroph A. brasilense is considered a magnified if one considers the identification of model PGPB, and a great amount of information PGPB with high biotechnological potential in regarding the physiology of its growth and distinct phylogenetic clusters, and the low development has been published [32]. However, it probability of finding conditions to produce high- is unclear when this available knowledge is in fact quality inoculants that can be universally applied applied in the inoculants industry. Quality control for any PGPB [24]. assessments of agricultural inoculants are Commercial inoculants carrying PGPB are commonly defined by the presence of generally available as dry or liquid formulations contaminants and the population density of viable of different organic and/or inorganic materials, cells in the final product throughout its shelf-life, which may be prepared with cells grown in a and determining any information about the liquid culture medium or via the direct use of physiological state of the inoculants PGPB strain

International Journal of Pharmacy & Life Sciences Volume 12 Issue 1: Jan. 2021 19 Review Article ISSN: 0976-7126 CODEN (USA): IJPLCP Batabyal , 12(1):17-25, 2021 in a commercial product is not required. To growth conditions were defined to lead this consider inoculants production solely in terms of bacterium to increase its biopolymer contents. The the yield of microbial biomass from a specific importance of the cellular content of PHB and strain has the potential to produce poor-quality EPS on improving the viability of A. inoculants, which plays against the broad adoption brasilense was evaluated by a short-period assay of PGPB inoculation as an alternative to carried out under greenhouse conditions. Bacterial conventional agronomical practices. To biomass produced under the conditions defined so implement this new paradigm in modern on was used to produce liquid inoculants, which agriculture, commercial inoculants formulations were applied on seeds or topdressing in maize should be produced with bacterial cells at a high plants; the results of these treatments were population density and in high physiological state compared with the performance of a peat to enable them to face adverse conditions that inoculants applied on seeds. occur both during storage (shelf life) and at the Projections of population increases, especially in time of its use (on seeds and in soil) [24,28]. It is developing countries, as well as of life expectancy not difficult to realize that the broad adoption by worldwide, imply greater needs for food and feed. the industrial and regulatory agencies of the To achieve higher productivity, agriculture is available PGPB physiology knowledge, as well as being intensified, mainly with monocultures the implementation of research studies aimed to highly dependent on increased chemical inputs, better understand the physiological characteristics including pesticides and fertilizers [33, 34]. for which no such knowledge is yet available, However, to ensure long-term food production, should result in better inoculants with higher field we must develop sustainable agricultural performance. practices, based on conservationist practices, to At least in Brazil, the inoculation of cereals with achieve economic returns for farmers, but with diazotrophic PGPB, such as maize inoculation stability in long-term production and minimal with A. brasilense, is considered an additional adverse impact on the environment [35]. In this practice and its adoption occurs under the context, the use of microbial inoculants plays a application of regular amounts of N fertilizer that key role, and we may say that we are starting a can exceed 200 kg ha−1. Concerns leading to the “microgreen revolution.” distrust of the natural nitrogen inputs provided by The nomenclature “plant-growth-promoting diazotrophic PGPB substituting at least part of the bacteria (PGPBs)” has been increasingly used for nitrogen demand in commercial crops reflect the bacteria able to promote plant growth by a variety lack of scientific information available for the of individual or combined mechanisms. By this commercial inoculants formulations. In addition, definition, rhizobia—studied and used in there is a huge variability in inoculation efficiency commercial inoculants for more than a century— as a result of the use of low-quality inoculants [25, are also PGPBs. Undoubtedly, besides rhizobia, 28]. Maize inoculants are mainly applied over the most studied and used PGPB is Azospirillum , seeds before sowing, which is an additional encompassing bacteria with a remarkable capacity practice that presents risks of lowering the to benefit a range of plant species [36,37,38, 39, germination rate if the seeds are mechanically and 40] damaged during this process. Furthermore, The genus was first reported by inoculating seeds places the bacteria in contact Beijerinck (1925), [41] and decades later with the pesticides and agrochemicals commonly reclassified as Azospirillum , because of its ability found covering commercial seeds, which are then to fix atmospheric nitrogen (N2), discovered and sown close to the fertilizers applied in the soil. reported by the group of Dr. Johanna Döbereiner Intending to determine whether conditioning the in Brazil, in the 1970s [4]. After the discovery PGPB A. brasilense Ab-V5 to accumulate high that Azospirillum was diazotrophic, several studies amounts of polyhydroxybutyrate (PHB) and evaluated its capacity to fix N2 and to replace N- exopolysaccharides (EPS) during its growth leads fertilizers when associated with grasses [42], to an improved inoculants performance in the including sugarcane ( Saccharum spp.), grain field, a culture medium was developed, and crops such as maize ( Zea mays L.), wheat

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(Triticum aestivum L.), and rice ( Oryza sativa L.), resistance to infection by pathogens, a mechanism pastures such as Brachiaria (= Uruchloa ), among known as induction of systemic resistance (ISR) others [32, 39, 40, 43, 44, 45]. Twenty species [68]. Due to the several mechanisms reported to of Azospirillum (DSMZ 2018) have been promote plant growth, Bashan and De-Bashan described so far, but A. brasilense and A. (2010) [36] proposed the “theory of multiple lipoferum have been the subjects of the highest mechanisms” in which the bacterium acts in a numbers of physiological and genetic studies [46, cumulative or sequential pattern of effects, 47]. resulting from mechanisms occurring Beneficial results have been obtained consistently simultaneously or consecutively. In this review with Azospirillum applied to a variety of crops we will give emphasis to the mechanisms [40, 48, 49] in dozens of commercial inoculants of Azospirillum that can improve plant tolerance worldwide [50]. Intriguingly, although the of biotic and abiotic stresses. Brazilian research group headed by Dr. Conclusion Döbereiner contributed to dozens of studies Azospirillum is currently one of the most broadly with Azospirillum [46, 51 and 52], it was only in studied and commercially employed PGPB. 2009 that the first commercial inoculant Previous studies with Azospirillum emphasize its containing A. brasilense started to be capacity of fixing atmospheric N2, followed by commercialized in the country [37, 38]; however, benefits in promoting plant growth via synthesis more than 3 million doses of inoculants are now of phytohormones. More recently, it has been applied annually by farmers, for inoculation both shown that the benefits should be extended to the of non-legumes and for co-inoculation of capacity of some Azospirillum strains to protect legumes. plants from biotic stresses, triggering ISR defense Although the most prevalent reported benefit mechanisms, and from abiotic stresses, through of Azospirillum has been its capacity of fixing N2, IST. The mechanisms discussed in this review of an increasing number of studies describe other tolerance of abiotic and biotic stresses promoted properties that imply growth-promotion. One by inoculation of Azospirillum in plants, main property of Azospirillum relies on the encompassing detoxification of oxidative stress, synthesis of phytohormones and other ISR and IST. The mechanisms that PGPB use to compounds, including auxins [53], cytokinins [6], cope with biotic and abiotic stresses vary with the gibberellins [54], abscisic acid [55], ethylene [56], plant species and cultivar and with the bacterial and salicylic acid [57]. Phytohormones greatly species and strains, and also depend on the affect root growth, resulting in improvements in phytopathogen and the intensity of the abiotic uptake of moisture and nutrients [58]. stress. Further studies to elucidate the mechanisms Some Azospirillum strains can solubilize of action of PGPB—as well as of the response of inorganic phosphorus, making it more readily plants to stresses—are of fundamental importance available to the plants and resulting in higher for understanding the potential and increasing the yields [59]. There are also reports use of PGPB as an important and sustainable of Azospirillum helping in the mitigation of strategy to mitigate the effects of biotic and abiotic stresses, such as salinity and drought [60, abiotic stresses in agriculture. 61, 62], by triggering induced systemic tolerance References (IST). Azospirillum has also been reported to help 1. Naveen Kumar, Arora . Plant Microbes in the mitigation of excessive compost and heavy Symbiosis: Applied Facets. Springer; 2014. metals [36, 63]. Another important feature 2. Fabricio Dario, Cassán; Yaacov, Okon; of Azospirillum is related to biological control of Cecilia M., Creus. Handbook for plant pathogens [64,65, 66 and 67], enabled by the Azospirillum : Technical Issues and Protocols. synthesis of siderophores, and limiting the Springer; 2015. availability of iron (Fe) to phytopathogens [67], or 3. Elena I., Katsy. Plasticity in plant-growth- causing alterations in the metabolism of the host promoting and phytopathogenic bacteria. New plant, including the synthesis of a variety of York, NY: Imprint: Springer; 2014. secondary metabolites that increase plant

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Cite this article as: Batabyal B. (2021). Azospirillum : Diversity, Distribution, and Biotechnology Applications, Int. J. of Pharm. &

Life Sci. , 12(1): 17-25.

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