Pseudomonas Chlororaphis Subsp

Rosas S.B. International Biology Review, vol. 1, issue 3, December 2017 Page 1 of 19

REVIEW ARTICLE

Pseudomonas chlororaphis subsp. aurantiaca SR1: isolated from rhizosphere and its return as inoculant. A review Susana B. Rosas Affiliation: Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Campus Universitario, Ruta 36, Km 601, 5800 Río Cuarto, Cba, Argentina Corresponding e-mail address: [email protected]

Abstract Most pseudomonas isolated from the plant rhizosphere favor the growth of plants through direct and indirect mechanisms. These bacteria produce phytohormones that promote plant growth and produce secondary metabolites that inhibit the growth of pathogenic bacteria and fungi, ensuring crop health. The present review is a compilation on the characterization of Pseudomonas aurantiaca SR1 and its role in the promotion of growth on several crops and its capacity to produce secondary metabolites involved in the control of pathogenic fungi. Pseudomonas aurantiaca SR1, a subspecies of Pseudomonas chlororaphis, produces IAA, HCN, siderophores, phenazines, and quorum, among other abilities. It has shown antifungal activity against several pathogenic strains, among them, Fusarium, Pythium, Rhizoctonia and Sclerotium spp. The SR1 aurantiaca strain has been shown to be a plant growth promoter for various crops, such as alfalfa, wheat, soybean, maize, carob, sugar cane, as well as promoting germination and obtaining vigorous and healthy seedlings. Pseudomonas chlororaphis subsp. aurantiaca SR1 is currently marketed as PSA liquid from BIAGRO-BAYER Laboratory. Keywords: Pseudomonas aurantiaca SR1, growth promotion, biocontrol, inoculant, crops, seedling

1. Introduction The interactions between the plants and the Natural agricultural ecosystems depend microorganisms have undoubtedly big directly on beneficial microorganisms that effects on the development of humanity, are present in the soil rhizosphere and help given that man for his survival needs to crops to reach higher productivities. The increase every day the agricultural beneficial microorganisms of the rhizosphere productions for nutritive ends, between are important determinants of plant health others. and soil fertility because of their At the global level, phytopathogenic fungi participation in several key processes such as are the most economically important group those involved in the biological control of in terms of frequency of appearance and plant pathogens, nutrient cycling and damage that they can cause. The damage seedling establishment [1]. they cause not only refers to the loss of

Copyright 2017 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Rosas S.B. International Biology Review, vol. 1, issue 3, December 2017 Page 2 of 19 economic production, but also to the losses least partially, the practices used by new of biological production [2] and have caused methods. havoc and major social changes. But Although PGPRs have been classified agricultural diseases, although arising from a according to their mechanism to promote mode of interaction between plants and plant growth or to control pathogens, they microorganisms, are not the only example of exert their beneficial effect by employing a such interactions. A large number of combination of mechanisms to stimulate beneficial relationships make a tremendous growth and maintain plant health [6]. importance for the development of sustainable agriculture. Moreover, through Fluorescent pseudomonades have received beneficial associations between plants and the most attention as candidates for microorganisms, attempts are made to biocontrol agents and biofertilizer due to displace the diseases that affect plants. their ability to colonize surfaces and internal tissues of roots and stems at high densities. The rhizosphere is called the zone adjacent These bacteria can compete successfully to the root system of plants influenced by with soil microorganisms. They are well root exudates [3].The rhizosphere is a site of known for promoting the growth of several intense and complex microbiological plants due to their ability to produce activity. It is an absolutely dynamic indoleacetic acid (IAA) and to solubilize environment due to the constant change in phosphate [7, 8, 9]. Pseudomonas also have the root structures, the exudate profile of the a great capacity to produce secondary root and the balance between the different antifungal metabolites. It is known that more microbial communities. The promotion of than twenty species of Pseudomonas plant growth mediated by PGPR (Plant synthesize more than 100 antibiotics and growth promoting rhizobacteria) is caused aromatic antibiotics [10]. Some well known by the interaction of the entire microbial antibiotics are phenazine-1-carboxylic acid community in the rhizosphere through the (PCA), phenazine-1-carbo-xamide (PCN), production of various substances [4]. In 2,4-diacetyl-phloroglucinol (Phl), pyro- general, PGPR promote plant growth cyanine, 2-aceta-midophenol, pyrrolnitrine, directly either facilitating the acquisition of pyoluteorin [11]. resources (nitrogen, phosphorus and minerals) or modulating hormone levels Siderophores include salicylic acid, plant or indirectly inhibiting deleterious piocheline and pioverdine, which chelate effects of various pathogens on growth and ions and contribute to disease control by development of the plants in the form of limiting the availability of trace minerals biological control agents [5]. Losses in plant required for microbial growth [12]. production caused by fungal diseases are an important economic factor to be considered. 2. Pseudomonas chlororaphis subsp. Historically, control over plant diseases has aurantiaca SR1 been carried out through the use of resistant varieties and agronomic practices, including P. chlororaphis subsp. Aurantiaca SR1 the application of synthetic fungicides and (GenBank accession number GU734089) other agrochemicals. However, ecological was isolated from the rhizosphere of soybean problems arose with risks to human health, in the area of Río Cuarto, Córdoba, contamination of water and selection of Argentina [13, 14]. It was initially classified pathogens with chemical resistance as P. aurantiaca by using the BIOLOG associated with the use of agrochemicals. (Biolog Inc., Hayward, CA) system [15] and This raises the urgent need to replace, at by amplification and sequencing of a partial fragment from the 16S rDNA gene. The

Copyright 2017 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Rosas S.B. International Biology Review, vol. 1, issue 3, December 2017 Page 3 of 19 species Pseudomonas aurantiaca was and pltC, genes encoding the production of reclassified as P.chlororaphis subsp. pyrrolnitrin (PRN) and pyoluteorin (PLT), aurantiaca 16]. respectively, were performed as described by It produces siderophores, behaves as an De Souza and Raaijmakers [24]. On the endophyte and is capable of promoting plant other hand, detection of hcnAB genes growth through mechanisms that involve (involved in the biosynthesis of HCN phytohormone-like substances [17]. For synthetase) was performed by PCR using the instance, SR1 shows the ability to produce primers PM2-F5′-TGCGGCATGGGCGC indole 3-acetic acid (16,5 ug. ml-1). In ATTGCTGCCTGG3′) and PM2-R (5′- addition, strain SR1 is able to colonize the CGCTCTTGATCTGCAATTGCAGGC-3′) [25]. As a result, fragments of the predicted root-system of several crops and behaves as an excellent growth promoter in wheat, size for PCA, PRN and HCN were amplified alfalfa, soybean, sugarcane, corn [18, 19, 20] from the DNA of strain SR1. maintaining appropriate population densities 2.1. Pigment production by P. aurantiaca in the rhizospheric area [21]. Its production in different culture media capacity for chitinases has been evaluated, proving that it does not present this property, The carbon sources normally found in root nor does it present conserved secretion exudates have a differential influence on the protein genes type III although spectrum of antibiotics produced by the polyacrylamide gels have been shown to be strains involved in biological control [26, 27, active in secretion of extracellular. The 28]. It is important to note that many other electrophoretic profiles of the LPS show a variables affect biological control, some of model similar to that of the pseudomonas which reside particularly in soil genus with a multiband profile. In addition, physicochemical characteristics such as the ability to fix N2 in P. aurantiaca SR1 moisture, temperature, pH, soil texture and free life was studied, obtaining a positive mineral nutrients; hence the importance of ARA at 24, 48 and 72 hours of incubation the in vitro study of the effects of the under microaerobiosis conditions. The strain chemical composition of the culture medium produces signals Acil type Homoserine and growth conditions on the antifungal lactones (AHL's) [22]. activity. Strain SR1 inhibits a wide range of Macrophomina phaseolina, due to its high phytopathogenic fungal species including sensitivity to P. aurantiaca, was selected to Macrophomina phaseolina, Rhizoctonia spp. evaluate the biocontrol in the media used for T11, Fusarium spp., Alternaria spp., the detection of antifungal activity and the Pythium spp., Sclerotinia minor and correlation with the presence of pigment. Sclerotium rolfsii [15]. In addition, strain The presence of pigment and a marked SR1 is able to colonize the root-system of inhibition (greater than 60%) of fungal several crops, maintaining appropriate growth in TSA medium with presence of population densities in the rhizosphere area SRI was observed. This percentage [21]. Recently, PCR assays were carried out inhibition was maintained when 5.5 mM to detect phlD and phz, genes involved in the glucose was added to the medium but was biosynthesis of 2,4-diacetylphloroglucinol reduced to 30% when added 27.5 mM and (DAPG) and phenazine-1-carboxylic acid 5% when 55.5 mM glucose was added. (PCA), respectively, in strain SR1 through the use of primers and protocols described The addition of FeCl3 revealed a decrease in by Raaijmakers et al.[23]. Also, PCR assays pigment color intensity as well as in involving the specific primers to detect prnD biological activity, where percentages of

Copyright 2017 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Rosas S.B. International Biology Review, vol. 1, issue 3, December 2017 Page 4 of 19 inhibition were maintained between 20 and strains tested was observed, which allows to 30%. In the YEM medium, in which no infer that the pigment produced by P. pigment was produced, the inhibitory auriantiaca SR1 does not interfere with the activity did not exceed 30%, being clearly normal development of other bacteria; These lower to the one observed in the TSA data may reflect that the strain in a medium. The addition of glucose to the rhizosphere environment would not affect YEM medium did not modify the the development of beneficial micro- antagonistic capacity of P. aurantiaca SR1. organisms such as those that play a major The antifungal activity was not observed in role in the rhizobium-legume symbiosis. minimal medium alone or supplemented with yeast extract. The addition of tryptone 3. Effect of radical exudates on pigment allowed to observe an important antifungal induction activity, with approximately 60 % of Exudates of soybean, extracted at different inhibition of the growth of Macrophomina times of the development of the seedling, phaseolina and appearance of pigment. were used, in sterile form, to supplement When the minimal medium was plates of cultures with minimal medium, supplemented with different amino acids, no where Pseudomonas aurantiaca does not pigment production was observed. However, form pigment, in order to observe if they are inhibition of about 20% was detected when able to trigger the formation of said supplemented with methionine, histidine and compound. tryptophan. Fungal inhibition was not Early tests indicate that exudates do not observed with the addition of arginine. induce pigment formation. The addition of mannitol and sucrose The test of biocontrol activity with reduced the biocontrol capacity to 17% and phytopathogenic fungi is necessary for the 7% respectively. tests to be complemented since it has been It was observed that P. aurantiaca produced observed that there is a fraction that is not pigment at the tested temperatures (4, 10, 16, part of the pigment that shows biocontrol 18, 28 and 32°C). It seems that the capacity. temperature does not affect pigment production and biocontrol capacity. 4. Production of rhizosphere pigment It was shown that the greater antagonistic In rhizospheric soil Pseudomonas effect of P. aurantiaca is associated with the aurantiaca, previously pelleted with soybean production of a pigment and is affected seed, was able to produce pigment and to when the media is supplemented with establish effectively on root and rhizospheric different carbon sources, with glucose being area. one of the most influential carbohydrates in the biological action. In the presence of The extraction of pigment was carried out continuous light, pigment is not produced in using soil agarized with the same protocol the culture media that promote it, although used for the plate extractions. already produced it is not affected. The TLC chromatography detected the same High temperatures, extreme pH and the pattern of bands as the pigment under dark action of proteases did not affect antifungal conditions, with the band at Rfs 0.35 and activity. 0.37 corresponding to those of antifungal activity. In the medium supplemented with the pigment, the growth of all the rhizospheric

5. Effect of inoculation with strain SR1 on determines the effect on germination and agronomically important crops development of the plant. The optimal concentration to inoculate is between 106 and P. aurantiaca SR1 has been inoculated in 7 -1 several crops and growth promotion in these 10 CFU g of the bioformulation [22]. crops has been reported [ 17, 18, 19, 20, 21, Finally, we also studied strain SR1 in co- 22, 29]. In order to evaluate its growth inoculation with Sinorhizobium meliloti promotion effect in greenhouse and field strain 3DOh13 to determine their effects on conditions, P. aurantiaca SR1 was nodulation and growth of alfalfa plants [17]. formulated as inoculant and applied on seeds The inoculant was prepared by mixing strain at the sowing time. SR1 and S. meliloti 3DOh13 in a 1:1 ratio (v v-1). The optical cell density at 600 nm 5.1. Alfalfa (Medicago sativa) (OD600) was 0.25, which corresponded to approximately 6.6 × 108 CFUml-1 of S. In vitro nodulation tests showed that alfalfa meliloti 3DOh13 and 6.3 × 108 CFU ml-1 of seeds pelleted with the formulated inoculant P. aurantiaca SR1. of Pseudomonas aurantiaca (109 CFU g-1) inhibited the development of the plant while SR1, when inoculated alone, stimulated being co-inoculated with Sinorhizobium shoot and root length of alfalfa by 82 and meliloti 3DOh13, inhibition decreased 57%, respectively, compared to control showing excellent root development and plants. Co-inoculation of strain SR1 and S. greater number of nodules than the control meliloti 3DOh13 stimulated shoot and root treatment. When the tests were carried out in length of alfalfa by 140 and 96%, pots supplemented with soil: sand: perlite (2: respectively, as compared to control. 1: 1) the first effect observed when the Additionally, co-inoculation of alfalfa seeds alfalfa seed was pelleted with the with strain SR1 and S. meliloti 3DOh13 Pseudomonas inoculant is attenuated. The caused a significant increase in dry weight of results indicate that it is the actual shoot and root (Table 1). concentration of the inoculant that

Table 1. Effect of co-inoculation with P. aurantiaca SR1and S. meliloti 3DOh13 on alfalfa growth. Means with different letters in the same column differ significantly at P ≤0.05 (Bonferroni test)

Shoot Root Shoot length Root length Treatment dry weight dry Weight (cm) (cm) (mg) (mg) Control 3.4c 7.5c 4c 4c N2 Control 5.3c 9.7c 5c 5b S. meliloti 3DOh13 7.0a 13.2b 26a 9b

P. aurantiaca SR1 6.2b 11.8b 19b 3c

Co- 8.2a 14.7a 29a 14a inoculation

Finally, co-inoculation significantly with SR1 in unfertilized soil; (5) seeds enhanced nodulation and total N content, inoculated with SR1 in soil fertilized with compared to inoculation with S. meliloti the 100% dose; (6) seeds inoculated with 3DOh13 alone or uninoculated control. SR1 in soil fertilized with the 50% dose. Biological nitrogen fixation is the most Seeds were inoculated with a formulation manufactured by Laboratorios Biagro S.A. important biochemical process after 9 -1 photosynthesis. It can contribute substantial containing strain SR1 at 10 CFU g of peat. amounts of N2 to plants and soil, reducing Briefly, 40 g inoculant, 20 g S2 adherent the need for industrial fertilizers [30, 31]. (Laboratorios Biagro S.A.), and 5 g cell protector S1 (Laboratorios Biagro S.A.) The use of PGPR in combination with were mixed in 80 ml of water. Then, 12 g of rhizobia is an interesting alternative to this mixture was added to 1 kg wheat seeds facilitate and optimize the nitrogen fixation to obtain a colony count of 105 CFU g-1 of legume crops [32, 33, 34 ,35 ]. The effects seeds. of PGPR can influence the activity of rhizobia to compete with indigenous Growth and yield parameters were recorded populations for nodulation [36].The effects at the growth stages termed 1.5 (5 leaves), of PGPR co-inoculated in legume symbiosis 3.0 (tillering), and 11.4 (ripe for harvest) include increases in nodule numbers and/or (Feekes International Scale—Large 1954). At Feekes 1.5, the number of seedlings nodule weight and in some cases increased 2 nitrogen fixation or N accumulation [35]. A emerging per m was evaluated. At Feekes 1.5 and 3.0, shoot length, root length, variety of mechanisms have been proposed 3 for the observed responses of symbiotic number of tillers, root volume (cm ), shoot legumes to co-inoculation of PGPR, and root dry weight (72 h at 60 ºC) were assessed. Yield parameters evaluated were: including phytohormonal root growth -1 stimulation [37]. The production of kg ha , weight of 1,000 grains, number of indoleacetic acid is of particular interest, spikes per plant, and number of grains per since this phytohormone stimulates root spike. lengthening and increased density of both Inoculation had no effect on emergence of root and lateral roots [38]. As roots are the plants, as compared to control. On the other starting point for nodule formation, hand, the number of plants per m2 was increased growth may result in more higher for inoculation treatments than for rhizobial colonization sites [39]. fertilization without inoculation. Increases in mean shoot length (14%) were observed for 5.2. Wheat (Triticum aestivum L.) the inoculated/unfertilized treatment and for In these studies, was evaluated the effect of fertilization with a 50% dose (8%) during inoculating wheat seeds with strain SR1 on Feekes 1.5, compared to control plants. By plant growth, under field conditions [18, 19, comparison, a 60% increase in shoot length, 40]. relative to control plants, was observed during Feekes 3.0 in plants inoculated and Six treatments were performed: (1) fertilized with a 100% dose. Plants uninoculated seeds in unfertilized soil inoculated with strain SR1 showed increases (control); (2) uninoculated seeds in soil -1 -1 between 47 and 78% in root length during fertilized with 80 kg ha of urea - 60 kg ha Feekes 1.5 and between 65 and 75% during of diammonium phosphate (100% dose); (3) Feekes 3.0, compared to control. Also, root uninoculated seeds in soil fertilized with 40 -1 -1 volume significantly increased during kg ha of urea - 30 kg ha of diammonium Feekes 1.5 after inoculation and fertilization phosphate (50% dose); (4) seeds inoculated with the 100% dose (Table 2).

Table 2. Root length and root volume of wheat plants during Feekes 1.5 and 3.0 Values in each column with different letters are significantly different according to the LSD test (P<0.05) Root length Root volume Treatment (cm) (ml) 1.5 3.0 1.5 3.0 Control 101b 204b 0.9c 2.7b Uninoculated seeds in soil fertilized with the 87b 357a 1.2b 4.2a 100% dose Uninoculated seeds in soil fertilized with the 159a 235b 1.2b 2.4b 50% dose Seeds inoculated with SR1 in unfertilized soil 160a 339a 1.7a 4.4a Seeds inoculated with SR1 in soil fertilized 181a 346a 2.0a 3.9a with the 100% dose Seeds inoculated with SR1 in soil fertilized 150a 359a 1.6a 3.6a with the 50% dose

All mean values of shoot dry weight from control. Throughout Feekes 1.5 and Feekes inoculation and/or fertilization treatments 3.0, root dry weight was significantly were higher than those of control plants increased by inoculation with strain SR1 during Feekes 1.5, but differences were not alone, as compared to control (Table 3). In significant. The higher mean value was addition, the number of tillers increased obtained after inoculating and fertilizing between 31 and 50 % at Feekes 3.0 in with the 50% dose, which increased shoot inoculated plants, with or without dry weight by 64 mg when compared to fertilization, compared to control plants.

Table 3. Shoot and root dry weight of wheat plants during Feekes 1.5 and 3.0 Values in each column with different letters are significantly different according to the LSD test (P<0.05)

Dry weight Dry weight (mg) at Feekes (mg) at Feekes Treatment 1.5 3.0 Shoot Root Shoot Root Control 198a 80d 790c 326c Uninoculated seeds in soil 217a 128bcd 1,510a 498ab fertilized with the 100% dose Uninoculated seeds in soil 228a 109cd 1,080bc 377bc fertilized with the 50% dose Seeds inoculated with SR1 in 223a 190a 1,310ab 536a unfertilized soil Seeds inoculated with SR1 in soil fertilized with the 100% 252a 183ab 1,310ab 420abc dose Seeds inoculated with SR1 in soil fertilized with the 50% 262a 156c 1,370ab 512a dose

When considering the yield parameters, the values for inoculation treatments were value of kg ha-1 was significantly higher in always higher than for control. The highest plants inoculated with SR1 and fertilized value was observed after inoculation and with a 50% dose, as compared to control. fertilization with the 50% dose (40% more Regarding number of grains per spike, than the control) (Table 4).

Table 4. Parameters of wheat yield Values in each column with different letters are significantly different according to the LSD test (P<0.05)

Yield Number of grains per Treatment (kg ha-1) spike Control 2,005b 32c Uninoculated seeds in soil fertilized with the 100% 2,169ab 39b dose Uninoculated seeds in soil fertilized with the 50% dose 2,264ab 40b Seeds inoculated with SR1 in unfertilized soil 2,249ab 42b Seeds inoculated with SR1 in soil fertilized with the 1,776b 41b 100% dose Seeds inoculated with SR1 in soil fertilized with the 2,641a 45a 50% dose

There are few reports on the contribution of 5.3. Soybean (Glycine max L. Merril) inoculation of wheat seeds with This study investigated the ability of the SR1 Pseudomonas strains for improving plant strain to promote legume growth and growth and yield under field conditions. For biocontrol capacity over the pathogenic instance, Shaharoona et al [41] tested several fungus Macrophomina phaseolina. Pseudomonas spp. strains in the field to determine their efficacy to increase growth Treatment of soybean seeds with strain SR1 and yield of this crop plant. Their results was studied to determine the effect of revealed that all of the strains significantly inoculation on plant growth, under increased plant height compared to greenhouse conditions. At the present time, uninoculated control. Strain P. fluorescens soybean is the most important oleaginous biotype F caused the maximum increase seed worldwide. In Argentina, soybean (16%). This strain also significantly cultivation was introduced in the 1970’s and increased the number of grains per spike it has been characterized by an incredible (11.7% more than the uninoculated control). rate of adoption and growth. Indeed, Another strain, P. fluorescens biotype G Argentina is one the main exporters of increased the number of tillers per m2 by soybean flour (27% of the world exports) as 9%, compared to uninoculated control well as soybean oil (30% of the world plants. The maximum increase in 1,000- exports) [42]. grain weight was recorded with P. Soybean seeds were inoculated with a peat- fluorescens (ACC50) (34% higher than the based formulation prepared and packed by uninoculated control). They also reported Laboratorios Biagro S.A. containing strain that inoculation with strain P. fluorescens SR1 at 2.4 x 109 CFU g-1 peat. Then, plastic (ACC50) increased grain yield by 39% when pots were filled with sterile soil and 4 compared to the uninoculated control. inoculated seeds were placed into the soil

Copyright 2017 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Rosas S.B. International Biology Review, vol. 1, issue 3, December 2017 Page 9 of 19 surface in each pot. The four treatments arranged in a completely randomized design were: (1) uninoculated seeds (control); (2) with five replicates per treatment. 7 seeds inoculated with strain SR1 at 10 CFU SR1 at 10-9 CFU g-1 enhanced shoot length g-1; (3) seeds inoculated with SR1 at 108 -1 by 31%, as compared to control plants. CFU g ; (4) seeds inoculated with SR1 at There were no significant differences in root 109 CFU g-1. The inoculant containing 108 7 -1 length. Although there were no significant and 10 CFU g was obtained by diluting the differences among the three inoculation original formulation with sterile peat. Pots doses for shoot dry weight, the optimum were incubated in a greenhouse. Shoot and inoculation dose proved to be 108 CFU g-1. root length as well as shoot and root dry Compared to control plants, SR1 at 108 CFU weight (120 h at 60 ºC) was recorded from -1 g increased shoot and root dry weight of each treatment after 25 days. Pots were inoculated soybean plants by 53 and 14%, respectively (Table 5).

Table 5. Soybean growth parameters Values in each column with different letters are significantly different according to the Scheffé test (P<0.05)

Shoot Root Shoot Root Treatments length length dry weight dry weight (cm) (cm) (mg) (mg) Control 34.2b 8.1a 430a 140a Seeds inoculated with SR1 at 107 41.2b 6.3a 500a 160a CFU g-1 Seeds inoculated with SR1 at 108 41.7b 8.5a 660a 160a CFU g-1 Seeds inoculated with SR1 at 109 45.0a 6.6a 420a 110b CFU g-1

In addition, we evaluated strain SR1 for strain SR1, inoculated at 107 CFU g-1, control of Macrophomina phaseolina (Tassi) increased shoot and root length by 277 and Goid. in soybean, under greenhouse 290%, and shoot and root dry weight by 275 conditions. Macrophomina (the cause of and 375%, respectively. Results suggest that charcoal rot, dry root rot and damping-off of strain SR1 provides effective control of M. many crop plants) is one of the most Phaseolina and that it might be applied as a destructive plant pathogenic fungal genera. It biological control agent to protect soybean prevails in the tropics and sub-tropics, plants from this phytopathogen. inciting diseases in a wide range of hosts Wahyudi et al.[45] isolated Pseudomonas [43]. Significant yield losses of soybean are spp. from the rhizosphere of soybean and reported every year due to charcoal rot tested them for promotion of seed growth. fungus M. Phaseolina (Tassi) Goid. [44]. As a result, they found that two isolates During biocontrol assays, soybean seeds (Crb-44 and 63) exhibited promoting activity were inoculated with strain SR1 prior to for all of the measured parameters (length of planting into infested soil. Growth para- primary root, shoot length and number of meters of soybean plants were recorded after lateral roots). Also, they reported that other 25 days. Compared to pathogen controls, 15 Pseudomonas isolates showed promotion

Copyright 2017 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Rosas S.B. International Biology Review, vol. 1, issue 3, December 2017 Page 10 of 19 of soybean seed growth at varying degrees. Egamberdiyeva et al. [48] reported on the After their experiments, they concluded that effect of a Pseudomonas fluorescens strain, the Pseudomonas spp. isolates could be termed PsIA12, and three Pantoea applied as inoculants of soybean plants agglomerans strains (370320, 020315 and because of their excellent growth promotion 050309) on the growth of maize in the field. and biocontrol activities. Inoculation with these bacterial strains was found to significantly increase the root and 5.4. Maize (Zea mays L.) shoot growth of maize grown in loamy sand The application of a SR1 formulation on at 16 ºC. Also, K content was significantly maize seeds allowed us to evaluate its increased in all treatments. More recently, effectiveness as maize growth promoter in Naveed et al. [49] assessed the performance the field [40]. For these experiments, plots of an organic fertilizer and three were arranged in a completely randomized Pseudomonas strains prepared as bio- design, with four replicates of 156 m2 for fertilizers for improving growth and yield of each treatment. Two treatments were maize in the field. Their results revealed that included: 1. Seeds inoculated with strain application of bio-fertilizers significantly SR1 and 2. Uninoculated seeds. Soil was improved the growth and yield of this crop. fertilized with 100 kg ha-1 of diammonium Indeed, plant height increased between 4 and phosphate at the sowing time and 100 kg ha-1 9% after inoculation with the bio-fertilizers of urea during V7-8 stages for both and only 2% after treatment with the organic treatments. fertilizer, compared to control. Similarly, they observed that total biomass was Length and dry weight of shoot were enhanced between 21 and 39% by bio- determined during the V2, V5, V13, R3 and fertilizers and 11.4% by the organic R6 phenological stages. In addition, the fertilizer, compared to control plants. The following parameters were recorded during increases obtained for grain yield (t ha-1) the first stages (V2 and V5): root length, root were 14.2% by the organic fertilizer and surface [46] and root volume [47], weight of -1 between 21 and 30% by treatment with the 1,000 grains and grain yield (kg ha ) were bio-fertilizers. Finally, bio-fertilization evaluated at the harvest time. caused increases between 14 and 19% in During V2 and V5, the beneficial effect of 1,000 grain weight, as compared to control inoculation with strain SR1 was evidenced at plants. the root system level. Root length increased 28% during V2 and 32% during V5 in 5.5. Carob tree (Prosopis L) inoculated plants. Similar results were Within a plan of reforestation and protection obtained with root volume (42% and 36%, of regional species was worked with respectively) and root surface (39% and different clones of Prosopis (mesquite). 34%, respectively). Shoot dry weight Prosopis clones are susceptible to Fusarium determinations indicated that inoculation spp. and within the Environmental Program with strain SR1 impacted favorably during where the project was developed, Santiago the whole cycle of the crop. For instance, we del Estero, Argentina, it was decided to observed a 22% increase in shoot dry weight displace the use of agrochemicals and to use during stage R3, as compared to control plant growth promoting microorganisms plants. Such beneficial effect was also (PGPR), including Pseudomonas SRI, which observed for yield parameters. To illustrate, produce hormones and have biocontrol the weight of 1,000 grains and grain yield capacity[50]. Pseudomonas aurantiaca SR1 (kg ha-1) were 11 and 20% higher in inoculated plants.

Copyright 2017 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Rosas S.B. International Biology Review, vol. 1, issue 3, December 2017 Page 11 of 19 was formulated as an inoculant for this It is inferred that Pseudomonas aurantiaca purpose. acts as a biological control agent, either by Six easy-rooting Prosopis: 3 clones antibiosis and/or ISR (induction of systemic (B2F17A2, B5F9A2, B6F5A2) and 3 resistance) and is a producer of substances difficult root clones (B7F6A4, B1F5A3, similar to phytohormones such as IAA. In B2F4A3) were used. All clones were tests performed with Azospirillum, there was susceptible to Fusarium sp. For rooting, the no rooting capacity greater than with clones were treated with Ac Indol butyric, Pseudomonas, except clone B2F17A2. Ac Naphthalene Acetic and Thiamine-HCl 5.6. Sugarcane (Saccharum officinarum L) as a chemical treatment and inoculated with Pseudomonas aurantiaca as a biological Micropropagation is a technique of in vitro treatment (UFC 107g-1 of inoculant). culture of vegetal tissues that allows the obtaining of seed sugarcane of quality. The The parameters evaluated were the number seedlings obtained were acclimatized in of roots, maximum long root, percentage of greenhouses, optimizing the management of rooting and sanity. sanitation, environmental and nutritional Rooting percentage: There was no conditions, in order to adapt to ex vitro significant difference in Pseudomonas conditions. treatment in the easy rooting clones. In Sugarcane was of importance in the NOA difficult rooting clones, 30% higher rooting (Argentine Northwest), representing more is observed when clones are treated with than 13% of GDP. The area cultivated in Pseudomonas. Tucumán, Argentina was 242,000 ha [51]. The authors conjecture that this is a Plants of sugarcane (Saccharum officinarum hormonal balance that favors rooting in L. cvs CP 65-357 and TUC 77-42) from certain clones. Hard rooting clones contain vitroplants were inoculated with an endogenous group of lower hormones, the Pseudomonas aurantiaca SR1 (inoculant optimal concentration is brought about by formulated by BIAGRO SA, stage bacteria. experimentation) in Tucumán, Argentina. In the presence of excess hormones, there is Significant differences were observed in no response or inhibition occurs. stem length and total weight in the variety CP 65-357 and varTuc 77-42, relative to As for the number of roots, the same anterior profile is observed, accentuated in clone Control. In both varieties was also observed B1F52A3, when treated with Pseudomonas. a higher production of roots. As for the length of the root the same In biocontrol experiments carried out in response is observed. When the clones were vitro, Pseudomonas aurantiaca SR1 subjected to chemical treatment, although controlled Fusarium subglutinans up to 60% three of them achieve rooting, they only of micellar development which would have a 5% survival due to the attack of indicate that the pigment produced would Fusarium spp. have antibiotic action, siderophore and The treatment of the same with induction of systemic resistance, which confirms the antagonistic effect of this Pseudomonas aurantiaca allowed the rooting of some clones and the protection of bacterium on different pathogenic fungi, the fungal attack in 70%. Easy-to-root clones including Fusarium spp.[15, 52 ] In vivo, a are more susceptible to Fusarium attack. high health index was observed corroborating the in vitro effect. Fusarium subglutinans was isolated in the first year,

Copyright 2017 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Rosas S.B. International Biology Review, vol. 1, issue 3, December 2017 Page 12 of 19 and in the second year Fusarium of roots, their weight and length at the subglutinans, Pythium spp. and Rhizoctonia concentration used, effect already com- spp., were controlled. The quality of the mented. At a concentration of 105 CFU ml-1, plants given by the increase of fresh weight Pseudomonas and Azospirillum spp. increase of plants, development of adventitious roots the number of roots and shoots without and length of roots, would be due to the affecting other parameters action of substances of type auxins in In the field trial of 2012 that was affected by addition to other hormones that significantly a high and severe incidence of white rust and affect the growth of them. excessive precipitation, it was observed that The inoculation with Pseudomonas treatment with Azospirillum spp. produced aurantiaca SR1 in sugarcane plants obtained the highest weight and quality of bulbs. by tissue culture in vitro improves survival, However, inoculation with Pseudomonas total weight of plants and decreases the aurantiaca increased yield by ensuring incidence of diseases caused by Fusarium garlic health [54]. subglutinans, the most frequent pathogen. 5.8. Pepper and tomato seedlings There are differences between sugarcane genotypes in response to the action of The use of quality seedling is a precondition Pseudomonas aurantiaca SR1. As for to increase yield and quality of vegetable growth promotion, comparative experiments crops. Liquid fertilizers including nitrogen were performed with Azospirillum are used during seedling production. brasilense, both bacteria show a similar However, much of the fertilizer applied promoter behavior, superior to the control. contributes potentially to the contamination [53] of surface water, producing detrimental environmental effects. The use of plant 5.7. Garlic (Allium sativum) growth promoting rhizobacteria (PGPR) Over the years 2012/13, white garlic was with the goal of improving nutrients planted under field conditions, with and availability for plants has been suggested to without inoculation of Pseudomonas be important and necessary for agriculture fluorescens, Pseudomonas aurantiaca SR1 [55]. Plant growth promoting rhizobacteria and Azospirillum spp. in the concentration of have been used for the production of 1 x 109 CFUml-1, to observe the effect of the seedlings. application of these commercial There are important positive effects of biofertilizers on the ontogeny of the plant. bacteria on the yield and growth of In laboratory and greenhouse, two trials vegetables such as, rocket, tomatoes, were carried out: one on the effect produced cucumber, cauliflower, pepper, potato, by the aqueous extract of garlic on the radish and lettuce [56, 57, 58, 59, 60, 61, 62] growth of the bacteria used and another on but little is known about the mechanism(s) the effect of the inoculation on the shoot and involved in their effect on seedling the development of the roots of the plants. performance [62, 63]. The production of The results indicate that concentrated vegetable crops with planting transplants is aqueous extract of garlic does not inhibit the recently preferred by growers as the time growth of the strains used and that compared required for cultivation is reduced and also to the control but affects pigment production provides a low seed cost to establish a good in Pseudomonas aurantiaca SR1. and homogeneous planting of vegetables, a Pseudomonas fluorescens and Pseudomonas more efficient use of fertilizers and irrigation aurantiaca cause a decrease in the number water during the early stages of growth. The use of a quality transplant is important to

Copyright 2017 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Rosas S.B. International Biology Review, vol. 1, issue 3, December 2017 Page 13 of 19 achieve uniform growth of plants, increase values between 70 and 78% of germination. the yield and quality of horticultural crops Despite these differences in germination and effectively protect against environmental onset until the seventh day, there were no stress, insect diseases and pathogens. significant differences between treatments. Pepper (Capsicum annuum L.) has a great At 14 days, the seedlings were transplanted demand in the international market and its and inoculated again with the same dose. success is that it is a crop with several The height of the seedlings at 20 days was consumption destinations: fresh evaluated from the beginning, with a slight consumption, universal condiment and preponderance of the inoculated treatments, canned. Among the conditions required to which was accentuated as a function of time increase production and yield of this crop is with a final result at 40 days, in which a to achieve a high percentage of germination highly significant statistical difference was and obtaining vigorous seedlings [64]. defined for this treatment. Greater In order to increase and accelerate the seed development in shoots and roots was also germination process and to obtain a high observed. production and homogeneity of seedlings, Similar results were obtained with tomato pepper and tomato seeds were inoculated seeds, where germination was more uniform with SR1 strain at a concentration of but higher in the inoculated treatment. 107CFU ml-1 to observe their effect on Pseudomonas increased the biomass of germination. Early germination was tomato plants and roots (42 and 35% observed in both plants, being highly respectively). These seedlings were more significant in the case of pepper, with very vigorous than those that were not inoculated. slow germination and emergence, in which a certain germination energy disparity can be 5.8.1. Development of pepper inoculated observed in the same lot. Several substances on soils infested with Fusarium have been tested to optimize germination, moniliforme with KNO3 (0.2%) as recommended by Pepper seeds were inoculated with ISTA [65]. According to International Seed Pseudomonas aurantiaca SR1 (107, 108, 109 Testing Association standards [66], the first CFU g-1 in soil infested with Fusarium count of germinated pepper seeds should be moniliforme (104, 105, 106 spores g-1 soil) to done at 7 days and the final count at 14 days observe the biocontrol capacity of the after the start of treatment for the percentage proposed strain. At 15 days of sowing, the of total germination. Thus, the statistical fresh weight and dry weight of the stem and analysis of the mean comparison between roots were determined. treatments was made with the data obtained 7 -1 during this period, in which no significant Seeds inoculated at a dose of 10 CFU g differences were obtained between the Pseudomonas showed improvements over control in soils infested with 106 spores g-1. treatments, in terms of the number of 4 5 germinated seeds. However, it was observed Spore concentrations 10 and 10 produced that with the inoculation of the SR1 strain, a no damage to seedlings higher final percentage of germinated seeds was obtained. The germinated pepper seeds 6. Conclusions (36%) with Pseudomonas aurantiaca were The plant genotype affects the response to detected three days after the test, presenting PGPR inoculation because it affects root statistically significant differences in relation colonization by introduced bacteria as well to water control (23%) and KNO3 (6%). On as the total population size of the microbial the seventh day, the treatments reached

Copyright 2017 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Rosas S.B. International Biology Review, vol. 1, issue 3, December 2017 Page 14 of 19 communities in the plant and probably also reasonable alternative for wheat production, affects the composition of those with a minimization of negative communities [67]. The effect of the environmental impacts. Pseudomonas introduction of PGPRs in the rhizospheric chlororaphis subsp. aurantiaca SR1 community has not been intensively studied, demonstrated sufficient merit that a since many experiments have been carried commercial formulation containing the SR1 out under gnotobiotic or greenhouse strain, called PSA liquid, is currently conditions. However, recent studies strongly registered with the National Agricultural suggest that increases in plant growth can be Health Service (SENASA) of Argentina for attributed to changes in the microbial the promotion of wheat growth. PSA Liquid community of the rhizosphere due to the was produced by Laboratorios Biagro S.A., presence of PGPR microorganisms currently acquired by Bayer. inoculated in soils [68]. There is extensive evidence and research in 7. Acknowledgement the literature indicating that PGPR This work was supported by grants from organisms can be a true success story in Secretaría de Ciencia y Técnica, Universidad sustainable agriculture. In fact, through their Nacional de Río Cuarto (Córdoba, numerous direct or indirect mechanisms of Argentina), Agencia Nacional de Promoción action, PGPRs can allow a significant Científica y Tecnológica (Secretaría de reduction in the use of chemical pesticides Ciencia y Técnica de la Nación) and Consejo and fertilizers. These beneficial events that Nacional de Investigaciones Científicas y produce biological control of diseases and Técnicas (CONICET, Argentina), BIAGRO pests, promoting plant growth, increasing S. A. crop yields, and improving quality can take place simultaneously or sequentially. The age of the plant and the chemical, physical 8. References and biological properties of the soil will 1. Jeffries,P., S. Gianinazzi, S. Perotto, K. greatly influence the result of PGPR Turnau, J.M. Barea. The contribution of inoculation. arbuscular mycorrhizal fungi in Among strains of P. aurantiaca, SR1 had sustainable maintenance of plant and soil been extensively studied for the promotion fertility. BiolFertil Soils. 2003; 37: 1-16. of plant growth with different cultures at 2. Agrios, G. N. PlantPathology. Fifth field level [28, 69, 70]. edition. California, USA: Elsevier Pseudomonas chlororaphis subsp. Academic Press; 2005 aurantiaca SR1 colonized the root system of 3. Kloepper, J.W. and M.N. Schroth.M.N. the tested crops, persisted at appropriate Plant growth promoting rhizobacteria on population densities in the rhizosphere area radishes. In : Station de Pathologie and showed a significant effect of plant Vegetale et Phyto-bacteriologie, ed. growth which was reflected in yield. In Proceedings of the 4th international general, the promoter effect was observed on conference on plant pathogenic bacteria the growth parameters in all the phenological II Tours, 1978: p. 879-882 stages of the cultures. A relevant finding was 4. Kloepper, J.W. and M.N. Schroth. Plant that wheat plants, after inoculation with SR1, growth-promoting rhizobacteria and showed higher yields with lower fertilization plant growth under gnotobiotic doses than those applied conventionally. conditions. Phytopathology. 1981; 71: This described the potential use of SR1 as a 642-644.

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