Effect of Pseudomonas putida on chrysanthemum growth under greenhouse and field conditions

Chrysanthemum production stands out in ornamental plants market and several techniques can improve the production of this plant. Among them, is the use of beneficial microorganisms such as plant growth promoting rhizobacteria (PGPR); however, the use of PGPR has not been explored enough so far in chrysanthemum production, especially in field conditions. With regards to rhizobacteria, the group, Pseudomonas, can be explored as a good alternative to improve plant development. Hence, the objective of the present study was to evaluate the effect of PGPR on seedlings and, on the field experiments, plants development and the impact on rhizosphere soil. The strains of Pseudomonas putida IAC-RBcr5 and IAC-RBcr2 were able to improve chrysanthemum shoot biomass up to 40%. In addition, the plants treated with these strains had increased number of flowers, which is a desirable feature for the flower market. This is the first study to demonstrate the effect of P. putida strains on chrysanthemum seedlings and cut flowers grown in tropical field conditions.


INTRODUCTION
The flower market in Brazil is growing, between 10 and 15%, and generates nearly $ 800 million per year (CNA, 2017).The chrysanthemum plants (Chrysanthemum morifolium L.) have a wide variety of inflorescences types, with great durability, different formats, and can be commercialized in vessels or as cut flower (Lopes et al., 2004).Research to generate knowledge and technologies to improve chrysanthemum cultivation are conducted including the management of organic and mineral fertilization (Ji et al., 2017), alternative substrates as pathogen suppressive (Pinto et al., 2013), and the improvement of irrigation contributing to the development of plants (Kirnak 2016).Furthermore, application of beneficial microorganisms may generate positive impact on chrysanthemum production.
Among the beneficial microorganisms, there is a group of bacteria living in the soil under the roots that increases the growth of plants, called plant growth promoting rhizobacteria (PGPR) (Kloepper and Schroth, 1978).Represented by a wide variety of rhizosphere bacteria, the use of those microorganisms is a viable alternative and meets the new expectations of the consumer market, by adding quality to the products and decreasing the use of pesticides (Gosal et al., 2017).Plant growth improvement can be triggered by PGPR through different mechanisms or by a combination of different factors, such as auxin indole-3-acetic acid (IAA) production, hydrogen cyanide production (HCN), phosphate solubilization and biological nitrogen fixation (Kielak et al., 2016;D'Agostino et al. 2017).A specific group of rhizobacteria widely studied is the Pseudomonas group that can promote the growth of several plants and decrease the damage caused by pathogens (Khabbaz et al., 2015;Rodríguez-Blanco et al., 2015).However, little is known about the effect of those growth promoters on ornamental plants, such as chrysanthemum, especially in field conditions.
There are several microorganisms recommended as inoculants with plant growth improvement and antagonistic properties (Meena et al., 2017).Regarding chrysanthemum culture, few studies indicate potential use of rhizobacteria as inoculant.For example, chrysanthemum treated with Pseudomonas fluorescens resulted in higher plant weight and number of flowers (Göre and Altin, 2006).Recently, researches showed the same effect in chrysanthemum plants treated with Pseudomonas spp. or Bacillus spp. or a mixture of both microorganisms that resulted in maximum of fresh and dry weight and flower yield (Kumari et al., 2016;Hanudin et al., 2017).The rhizobacteria-chrysanthemum interaction can have effect on the plant growth and also in the biological control of some pathogens.A mixture of rhizobacteria such as Pseudomonas, Bacillus, Azotobacter, inoculated on chrysanthemum reduce the white rust incidence (Hanudin et al., 2017) and the combined inoculation of fungi Glomus mosseae and the rhizobacteria Pseudomonas putida can mitigate phytoplasma damage (D'Amelio et al., 2011).Although, these researches present promissory use of rhizobacteria Pseudomonas as inoculant, there are no studies on the effect of Pseudomonas strains on chrysanthemum seedlings and on plants cultivated in the field conditions, as well as soil quality indicators such as microbial biomass and basal respiration, especially in tropical soil in Brazil.Based on this, this study aimed to evaluate the effect of P. putida strains on chrysanthemum growth in the field conditions and on soil biological properties.The hypothesis was that P. putida strains would have positive impact on the soil quality indicators, improving the plant biomass and increasing the number of chrysanthemum flowers.

Traits of P. putida strains and inocula preparation
The nine strains tested (belonging to Agronomic Institute of Campinas IAC, Brazil, culture collection) were isolated from healthy lettuce, chrysanthemum, rucola and maize rhizosphere.All P. putida strains were producers of growth promoting metabolites and have already been reported as plant growth promoting rhizobacteria in lettuce, in previous study by Cipriano et al. (2016).The characteristics of the strains are shown in Table 1.
Bacterial strains were grown on King's B (KB) (King et al., 1954) liquid medium and incubated at 28°C for 24 h.At the end of the Cipriano and Freitas 303 interval, the suspensions were centrifuged (13.000 xg for 10 min), suspended in MgSO4.7H2O(0.01 mol.L -1 ) and final concentration adjusted to 10 8 CFU mL -1 .

Location and experimental design
The experiments were conducted in two parts.The nine strains tested in this work were evaluated in three different experiments.In Experiment 1 (September to December, 2012), three strains were tested with and without root hormone (indolic-3-butyric acid -IBA), in a completely randomized design in the factorial scheme 2 x 3 (with or without IBA x three strains -IAC-RBal3, IAC-RBcr2 and IAC-RBcr5).In Experiment 2 (October 2012 to January 2013), four strains were evaluated in the factorial scheme 2 x 4 (with or without IBA x four strains -IAC-RBcr1, IAC-RBal2, IAC-RBal1 and IAC-RBcr3).In Experiment 3 (February to May 2013), three treatments were evaluated in the factorial scheme 2 x 3 (with or without IBA x two strains -IAC-RBmi1, IAC-RBcr4 and the third treatment composed by the mixture of the others two strains, named Mix).

Part 1
The seedlings were prepared with plants cutting (5 cm of lenth), obtained from pattern plants (Zembla variety).After plants were treated with IBA (IBA talc -2000 mg.mL -1 ) they were placed in trays (63 cells seedlings trays) with substrate (mixture of rice straw and pine bark, disinfected in a boiler machine).Rhizobacterial suspension (4 mL/each plant) was used to inoculate seedlings lap and seedlings of control were drenched with 0.01 mol L -1 MgSO4.7H2Oinstead of bacterial suspension.The same procedure was also applied to treatments without IAB.After three days, Chlorothalonil and Triazole fungicides were applied, according to the manufacturer's recommendations.The seedlings remained for 14 days in a greenhouse under artificial illumination (artificial illumination for 15 min every 3 h at night).After that, half of the plants were harvested and the remaining ones were treated once again with the strains and transferred to the field of São Pedro company for part 2 of the experiments.

Part 2
The soil of the field was previously fertilized before transplanting the seedlings, wherein the organic fertilizer BioBokashi was incorporated into the soil, according to the manufacturer's instructions.This organic fertilizer (composed of bone meal, fish and rock carbonized cereals, and molasses) is routinely used for chrysanthemum production in Brazil.After the seedlings transplantation to the field, the fertilization with organic fertilizer was performed 15, 30 and 45 days later, by aspersing Sulfammo 11 ® (18-0-18), at the same intervals as already described.Plants were also treated with fungicides Cercobim 700WP (thiophanate-methyl), Table 1.Pseudomonas putida strains able (+) or not able (-) to produce indole-3-acetic acid (IAA), hydrogen cyanide (HCN) and phosphate solubilization (PS), and 16S RNA accession numbers deposited in GenBank, according to Cipriano et al. (2016).

Strain Host plant origin PGP traits Accession number
NT Dithane (mancozeb), acaricide/fungicide Score (difenoconazole) and biological fungicide Trichoderma harzianum Ecotrich ® , following the manufacturers' recommendations.The plants remained in the field for 75 days, and at the end of this period, they were harvested and samples of soil were collected.The plant parameters evaluated were shoot and roots dry masses, and number of flowers.Soil quality indicators analyses were also evaluated: microbial biomass carbon (MBC) was determined by the fumigation-extraction (Vance et al. 1987), and basal respiration (BR), determined by quantification of CO2 released during soil incubation according to Alef (1995) methodology.The metabolic quotient (qCO2) was obtained by the ratio of BR and MBC.Throughout all the experiments, each treatment consisted of five replicates completely randomized, each replicate contained five plants.The data were submitted to analysis of variance by the Scott-Knott test at 5% probability using the statistical software Sisvar (Ferreira, 2008).It is worth noting that, except the inoculation of rhizobacteria, all management adopted in the experiments was the same as chrysanthemum farmers' in Brazil.

Greenhouse and field experiments-plant growth promotion
In Experiment 1, the strains inoculated had valuable effect on growth promotion on seedlings and cut plants (Table 2).The strains IAC-RBcr 2 and IAC-RBcr5 improved the shoot dry mass (16 and 18%, respectively) of the seedlings without IBA; regarding the roots, the strains IAC-RBal3 and again the strain IAC-RBcr5 improved the growth independent of the treatment with hormone (up to 23 and 30% respectively).According to the cut plants data, the plants with IBA treated with the rhizobacteria IAC-RBcr2 and IAC-RBcr5 yielded the highest values for shoot dry mass (40 and 29%, respectively).The strain IAC-RBcr2 also improved the root dry mass of roots without IBA (38%).
In Experiment 2, the strains had no effect on the shoot dry mass of the seedlings.Regarding the roots, with IBA, the plants treated with IAC-RBal1, IAC-RBal2 and IAC-RBcr3 obtained lower dry matter of roots.With regards to the shoots of the cut plants, the strains IAC-RBal2, IAC-RBal1 and IAC-RBcr3 without IBA and IAC-RBcr1 and IAC-RBal2 with IBA showed lower dry mass.The strains IAC-RBal1 improved the root growth of the plants without IBA (48%) and IAC-RBcr1 improved the root growth of the plants without and with IBA (38 and 31%).
In Experiment 3, the shoots of the seedlings treated with IAC-RBcr4 and Mix (composed by strains IAC-RBcr4 and IAC-RBmi1) without IBA and IAC-RBmi1 with IBA resulted in lower dry mass.Moreover, cut plants with IBA and treated with the strains IAC-RBmi1 and IAC-RBcr4 obtained the highest values for shoot dry biomass (20 and 28%).

Soil microbial activity-rhizospheric soil
The results of rhizosphere soil showed that inoculation of the strains interfered in the soil microbial activity (Table 3).In experiment 1, according the MBC values, there was a significant difference between control treatments with and without IBA, and MBC values of the treatments without IBA were higher than those with IBA.The BR values indicated that the treatment without hormone IAC-RBcr5 and with hormone, IAC-RBal3 and IAC-RBcr2, were lower than the control.The results obtained by the MBC and BR analysis generated the qCO 2 data revealing no changes in this variable due the strains inoculation, in comparison with the control treatment.In Experiment 2, there were significant differences in the MBC values due the inoculation of the strain IAC-RBal2 resulting in lower values in treatments with and without IBA.The BR values were higher in the treatments without IBA in plants treated with the strains IAC-RBal2, IAC-RBal1 and IAC-RBcr3.The BR values were higher in the treatments with IBA, in plants treated with strains IAC-RBcr1, IAC-RBal1 and IAC-RBcr3.According to the qCO 2 results, only the control treatment without IBA had lower value.
In Experiment 3, the two strains tested, IAC-RBmi1 and IAC-RBcr4, and Mix improved the CBM in the treatment with IBA.The BR was higher in the rhizosphere treated with strains IAC-RBmi1 independent of the hormone treatment.The strains had no impact on the qCO 2 data.

Flower evaluation-cut plants
In Experiment 1 (Figure 1a), the plants without IBA treated with strains IAC-RBcr2 and IAC-RBcr5 obtained more flowers than the other treatments, including the control treatment.The plants with IBA and treated with the strains IAC-RB al3 and IAC-RBcr5 also had more flowers than the other treatments.
The plants without IBA, in Experiment 2, treated with IAC-RBal2 and IAC-RBcr3 produced more flower than the other treatments (Figure 1b).In plants with IBA, the plants treated with IAC-RBal1 and IAC-RBcr3 produced more flowers.In Experiment 3, the rhizobacteria inoculation did not improve the flowers numbers (Figure 1c).

Effect of rhizobacterial strains on plant biomass
The strains evaluated in the present study had already been tested in a previous work on lettuce seedlings and field conditions (Cipriano et al., 2016) and plant growth promoting characteristics are listed in Table 1.The principal results of the present research are summarized in Table 4, indicating the promising use of Pseudomonas trains in chrysanthemum production in seedlings and cutting plants phases.Interestingly, the Mix treatment (Experiment 3) was the only one that had no effect on the Control 420 plant growth and flower number.The rhizobacteria strains used in this mixture had already been tested in combination with others Pseudomonas strains on lettuce seedlings and adult plants revealing the strain IAC-RBcr4 as a promising inoculant for that culture when applied alone, but not when applied in combination with other strains (Cipriano et al., 2016).The data of the present study is in agreement with that of Cipriano et al. (2016) indicating that our strain (IAC-RBcr4 P. putida) can improve plant growth when applied alone, but not in combination with others Pseudomonas strains.Several studies have reported that the mixture of different rhizobacteria strains, from different genera, can improve the plant growth, but rhizobacteria from the same genera, do not have the same benefit.For example, the mixture of Pseudomonas fluorescens, Azotobacter chroococcum, Bacillus subtilis, Trichoderma sp. and Paecilomyces sp.promoted chrysanthemum plant growth (Hanidum et al., 2017).The mixture of Pseudomonas strains is not commonly evaluated on chrysanthemum growth.Otherwise, pseudomonad combinations increase the yield of wheat plants (Pierson and Weller, 1994).The strains, IAC-RBcr1 and IAC-RBmi1 evaluated in the present study were tested previously in combination with other strains and improved the shoot growth of lettuce cultivated in field conditions (Cipriano et al., 2016).Based on these observation, it is concluded that certain combinations of strains can benefit the plant growth or not, as observed by Pierson and Weller (1994).As known, this is the first study on the effect of the interaction of P. putida strains mixture with chrysanthemum on seedlings and under field cultivation (cut plants).The strains IAC-RBal1and IAC-RBcr1 (Table 2, Experiment 2) were capable of improving the root development of plants cultivated on field untreated with IAB, and the strains IAC-RBcr1 also improved the root growth of plants treated with IBA.It can be hypothesized that benefit triggered by IAC-RBal1 (Table 1) can be related to the strain characteristics; this strain is IAA producer and able to solubilize P, which can improve the root growth and provide soluble phosphate to the plant (Ibañez et al., 2014;Oteino et al., 2015).The strain IAC-RBcr1 also improved the root growth, even though this strain do not produce any of the compounds related to plant growth promotion investigated (Table 1).The strains inoculation resulted in higher values of microbial biomass carbon (MBC), basal respiration (BR) in some treatments (Table 3).Based on this, it can be assumed that the strains may have improved the abundance and activity of microorganisms according to the MBC and RB data, respectively.These results can be related specially to the strains IAC-RBcr1 and IAC-RBal1 inoculated, which consequently increased the MBC and BR resulting in root growth improvement.Here, it is hypothesized that these strains were able to improve the quality indicator, such as MBC and RB, and improve the beneficial microorganisms resulting in root plant growth.Otherwise, this result did not alter the metabolic quotient (qCO 2 ).The current findings are in agreement with other studies which observed no impact on microbial community activity according to the same analyses (Fließbach et al., 2009;Cipriano et al., 2016).Moreover, in some cases, the inoculation of rhizobacteria does not result in greater plant growth; however, it may increase the MBC, nutrients in the plant and reduce the effects of disturbances in plants under adverse conditions (Lau and Lennon, 2012).The inoculation of beneficial microorganisms may increase the CBM values of soil under roots influence and benefit the host plant, improving the soil quality, root improvement and phosphorus availability (Prasanna et al., 2013).
The most promising result regarding plant growth promotion was obtained with the strains evaluated in the first experiment (Table 2), and all them had a good impact on plants cultivated with seedlings and/or cutting plants on field.At seedlings phase, the strain IAC-RBcral3 improved the root growth, independent of the hormone treatment, and IACRB-cr2 improved the shoot growth of plants without IBA.The plants gained more biomass when treated with IAC-RBcr5 strain, resulting in improvement of total plant growth (shoot and root), without IBA and also the root of plants with IBA.At cutting plants phase, the strains IAC-RBcr2 and IAC-RBcr5 again affected plant growth, especially, the shoot.The highest shoot growth suggests plants with better visual architecture for marketing (Neto et al., 2015), a major factor in the flower market.According to the microbial soil quality indicators, the lower BR in soil that received the strains indicated lower microbial activity in those treatments.
The effect of rhizobacteria inoculation on plant growth and soil quality can vary according the soil microbiota and plant development.The plants were harvested and the soil samples were collected 75 days after strains inoculation.So, in this period (75 days), probably the endogenous microbiota was restored in the soil and did not let the strains interferer anymore with plant development.The findings are in agreement with some studies which also observed this behavior pattern for rhizobacteria strains inoculated in field conditions (Schreiter et al., 2014;Cipriano et al., 2016).

Flowers improvement due to Pseudomonas inoculation
Six different strains (IAC-RBal3, IAC-RBcr2, IAC-RBcr5, IAC-RBal2, IAC-RBal1 and IAC-RBcr3) improved the chrysanthemum number flowers per plant (Table 4).Previous study also showed an increase of chrysanthemum flower, due the Pseudomonas strains inoculation (Kumari et al., 2016).The findings are in the same line with that of Kumari et al. (2016), although these authors did not characterize their strains regarding plant growth promoting traits.In the present study, the strains IAC-RBcr5 and IAC-RBal1, which improved the flowers number, are known as IAA producers (Table 1).IAA is one of the most auxin produced and released by rhizobacteria which can interfere with plants growth process and regulate root growth and flower development (Patel and Saraf, 2017).There is scarce information on the effect of Pseudomonas strains on chrysanthemum flower development, but other study revealed that the inoculation of Pseudomonas strains with arbuscular mycorrhizal fungi (AMF) induced the earlier flower production, probably due the hormone production and photosynthate which have an indirect influence on flowering time (Bona et al., 2015).The current data suggest that these rhizobacteria can improve the numbers of flowers of chrysanthemum cutting plants, at least in tropical soils, according the management done by chrysanthemum producers.

Conclusion
In the present work, knowledge on chrysanthemum-Pseudomonas interaction regarding seedling and cut plants cultivated in field conditions were broadened.Moreover, the findings led to accepting the initial hypothesis that Pseudomonas strains are able to improve the chrysanthemum plant growth and increase the number of flowers, without negative impact on soil quality.
The experiments were conducted with the same management used by local chrysanthemum producers, in order to approach as much as possible, the use of rhizobacteria inoculants for their production.Improvement of seedling production is necessary because of the importance of this stage for producers working with seedlings production, such as the producers of chrysanthemum seedlings in Brazil.It is known that poorly formed seedlings will give rise to plants with production below their genetic potential (Bezerra Neto et al., 2005).In addition, a plant obtained in the seedling phase must have high vigor, showy leaves, with sufficient amounts of roots to adapt to field conditions.
The strains, IAC-RBcr5 and IAC-RBcr2 evaluated in the present work improved the chrysanthemum production up to 40% (shoot dry mass) under field conditions and improved the number of flowers per plant.Here, it is reported for the first time, important findings regarding chrysanthemum market by using Pseudomonas on seedlings phase and field conditions in tropical soils, to increase the plant growth and number of flowers.

Figure 1 .
Figure 1.Effect of Pseudomonas putida strains on number of chrysanthemum flowers.*Bars for each treatment, with the same small or large letters represent values that are not significantly different according to Scott-Knott test (P≤0.05).

Table 2 .
Pseudomonas putida strains and their effect on chrysanthemum plants (seedlings and cutting plants).Data shows shoot and root dry mass (g/plant).The data with bold letters represent the best result obtained.
Aa*Mean values followed by the same small or large letters within a line or column represent values that are not significantly different according to Scott-Knott test (P≤0.05).

Table 3 .
Microbial biomass carbon (MBC), basal respiration (BR) and metabolic quotient (qCO2) from soils of chrysanthemum plants treated with P. putida strains.The data with bold letters represent the best result obtained.

Table 4 .
Pseudomonas putida strains and their effect on chrysanthemum plants at different growth stages (seedlings and adult plants).Dry mass increase (+) or no dry mass increase (-) and higher number of flowers (+) or not (-).