Quality of floral stems of lisianthus ( Eustoma grandiflorum Raf.) inoculated with Bacillus subtilis and Glomus intraradices

Lisianthus ( Eustoma g randiflorum ) is an ornamental species used as a potted plant or cut flower, its popularity is due to the diversity of colors, number of flower buds, and shelf life. Nevertheless, during the first phases of development, problems such as foliar chlorosis and root diseases affects most cultivars, causing poor growth, thin stems, and few flowers. The use of plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) improved plant growth as these microbes colonize the plant system root. Therefore, in order to provide better conditions for the stem development, the aim of this work was to evaluate the individual and combined effect of Bacillus subtilis (PGPR) and Glomus intraradices (AMF) on the growth and postharvest quality of the stems of lisianthus cv. Mariachi. Then commercial product Alubión-X ( Bacillus subtilis (PGPR) and mycorrhizal fungus ( Glomus intraradices ) were used. The variables evaluated were stem height and diameter, foliar area, leaves number and in postharvest, buds number, open and diameter of flowers and stem dry weight. The results showed a significant effect of the inoculation of G. intraradices on the size (66.92 cm) of the stem, as well as the combination of B. subtilis + G. intraradices (65.51 cm) compared to the control (36.9 cm). The number of buds and open flowers of the stems treated with G. intraradices were 33.35 and 23.9 respectively significantly higher than the control. G. intraradices alone is the best option for applying to lisianthus, when compared to applying with B. Subtilis.


Introduction
Lisianthus (Eustoma grandiflorum) is a non-traditional floral species that has gained popularity in recent years, due to the diversity of colors, number of flower buds, and durability of the flower stem (Castillo-González et al., 2017).During the first phases of development, problems such as foliar chlorosis, growth retardation, or an under developed root system may occur, as a result of the conditions of the culture medium, pH, incidence of root diseases caused by Fusarium avenaceum, F. solani, or presence of root galls associated with the nematode Meloidogyne sp.(Neves et al., 2017;Xiao et al., 2018).The incidence of root diseases caused by Fusarium spp., Pythium spp, Rhizoctonia sp.among others, affects most cultivars of this species, causing poor growth, thin stems, and few flowers (Mcgovern, 2018).
In the rhizosphere, there is a wide variety of microorganisms with high beneficial microbial activity (plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF)) that can improve plant growth as these microbes colonize the plant system root increasing its yield (Sagar et al., 2021).The predominant bacteria identified as PGPR include the genera Bacillus and Pseudomonas.The mechanisms they use to promote plant growth are: (1) biofertilization (for example, biological fixation of atmospheric nitrogen, phosphate solubilization, siderophore production, and exopolysaccharide production); (2) phytostimulation (production of indole acetic acid, gibberellins, cytokinins, and ethylene); and (3) biological control (induction of systemic resistance, competition for iron, nutrients, and space, production of antibiotics, lytic enzymes, hydrogen cyanide, and volatile compounds) as well as resistance to heavy-metal, drought or temperature stress (Noumavo et al., 2016;Xie et al., 2020).
Several important species of the genus Bacillus (B. cereus, B. pocheonensis, B. circulans, B. amyloliquefaciens, and B. subtilis) have shown the ability to increase crop yields, quality, and plant health.Also, Bacillus genus can stimulate plant growth through the synthesis and secretion of phytohormones (auxins or cytokinins), organic volatiles, and even the activation of the compounds production that reinforce plant immunity (e.g., jasmonic acid, salicylic acid, and phytoalexins); additionally, improves the bioavailability of Fe and P and increases tolerance to water stress in ornamental plants modifying hormone levels (Nordstedt and Jones, 2020;Barros et al., 2018).On the other hand, this bacterium synthesizes and secrete antibiotics or other compounds (belong to polyketides, heterocyclic nitrogenous compounds, and lipopeptides) which have broad-spectrum action by the inhibition of phytopathogenic organisms or, by the activation of the induced systemic response (ISR), a mechanism by which the plant activates its defense systems against pathogen infection (Sohrabi et al., 2020;Kenawy et al., 2019).
Meanwhile, the arbuscular mycorrhizal fungi (AMF) -Glomus mosseae (Nicol.& Gerd.)Gerd.& Trappe, and G. intraradices (Schenck & Smith) -have proven their effectiveness in different ornamental species.For example, Soroa et al. (2003) studied the effect of the application of arbuscular mycorrhizal fungi (AMF) and plant growthpromoting rhizobacteria (PGPR) on some growth and yield variables of Gerbera jamesonii cv.Bolus, their results showed that Glomus fasciculatum inoculation which increased the diameter of the flowers by 27.9%, accelerated the beginning of flowering (50 days before), and increased yield compared to the control.Khandan-Mirkohi et al. (2015) evaluated the inoculation of lisanthus with Glomus mosseae (Nicol.& Gerd.)Gerd.& Trappe and G. intraradices, showing a significant reduction in the number of days to flowering, an increase in the length and number of floral stems and flowers, diameter and fresh weight of flowers per plant, and a reduction in the requirements of external phosphorus.
The combination of PGPR and FMA also has a positive effect.When B. subtilis and P. fluorescens were combined with AMF in bean roots, Mohamed et al. (2019) noticed an increase in the activity of chitinase, peroxidase, and polyphenol oxidase enzymes, reducing the Sclerotium rolfsii infection, and showing their capacity as disease control bioagents, possibly as a consequence of the production of antifungal compounds, the increase of root cell lignification, and the removal of high Fe concentrations from the medium, as well as the limitation of fungal growth.Nevertheless, Cai et al., (2021) evaluated the effect of plant symbiotic microbes in tomato plants, and the results showed that depending of the combination, the microbes may or may not promote the plant development, for instance the height of the plant inoculated with Bacillus subtilis was 50.5 cm, or with Trichoderma harzianum 53.2 cm, and combined the height remained in 50.5 cm, nevertheless with Rhizophagus intraradices + B. subtilis + Trichoderma harzianum the plant height was significantly higher (60.5 cm).
Therefore, in order to provide better conditions for improving growth, the aim of this work was to evaluate the individual and combined effect of Bacillus subtilis (PGPR) and Glomus intraradices (FMA) on the development and postharvest quality of the stems of lisianthus cv.Mariachi.

Experiment conditions
The experiment was established in a greenhouse with a milky plastic cover (25% shade), located at 19° 28'-19° 36' N and 98° 47'-98° 55' W, and an altitude of 2,250 m.The climate is temperate with rains in summer, the average annual temperature is 15 °C, and the average annual precipitation is 772 mm.The conditions inside the greenhouse during the period of plant growth (November-July) were monitored with temperature and humidity sensors (HOBO ® Data logger Onset ® U12-012).The minimum and maximum monthly temperatures were 10.6 and 33.1 °C, respectively; relative humidity ranged from 33.1% to 91.1%; light conditions ranged from 900 to 8,000 µmol m 2 s -1 of photosynthetically active radiation (PAR), between noon and 4 pm at the plant level.
The commercial product Alubión-X (Bacillus subtilis (PGPR)) was used as biofertilizer, while the mycorrhizal fungus (Glomus intraradices) was isolated from the rhizosphere of Dahlia x hybrida and multiplied in wheat (Triticum aestivum L.) seedlings.The harvest was carried out after eight months and the root staining (0.05% trypan blue) was determined using the Phillips and Hayman (1970) method, while the percentage of the root mycorrhizal colonization was measured by the intersection of quadrants and the values were expressed as a percentage.The number of spores was evaluated by separating the spores with 44-, 325-and 400-µm sieves by the method of Gerdemann and Nicolson (1963).The stained root longitudinal segments were examined with a microscope (American Optical, USA) at 100X.
Biofertilizers inoculation: The treatments were: T1=B.subtilis; T2=G.intraradices; T3= B. subtilis + G. intraradices; and T4=Control.The commercial product Alubión-X (Bacillus subtilis) was applied manually at 1x10 7 CFU at the base of the stem of the plants using an atomizer, 15 and 30 days after transplantation (ddt).Meanwhile the inoculation of G. mosseae was carried out by transplanting the plants and adding 350 spores (10 g of substrate) to the root system of each plant, in order to assure the inoculation.

Variables evaluated
Plant height was measured every two months with a graduated ruler from the base of the stem to the apical part.Stem diameter was measured every two months with a digital vernier (Caldi-6MO, Truper, U. S. A.) at 10 cm from the base.Leaf number and leaf area were determined every two months with a leaf area integrator (Licor, Model 3100 Area Meter®).
The floral stems were harvested after the second flower was completely open and they were randomly placed in glasses with 300 mL of tap water for postharvest evaluation.The postharvest variables evaluated were: number of flower buds, open flowers, and the diameter of the third flower.After harvest, the dry weights of the root, stem, and leaves were recorded by drying the tissues at 70 °C in an oven (Thermo Scientific Model No. 3471-1), until a constant weight was reached.

Statistical analysis
Five monitorings were carried out (November 5, January 5, March 5, May 5, and July 5) and 20 repetitions were taken for each variable (1 plant=1 repetition).The generalized additive model (GAM) was applied to analyze the possible non-linear behavior between each growth variable and time.The model does not subject the data to the torture of transformation to meet a requirement of a linear relationship between the response variable and an explanatory variable (time).The model divides the data into fragments defined by several points (knots) and fits a polynomial regression in each data subset that, to analyze the effect of treatments, is defined as: g(μ)=Ti+f(x)+ϵ_ij; i=1,2,…,k;j=1,2,…,rep.
Where Ti is the i-th treatment of a total of k treatments, f is a smoothing function (i.e., the polynomial by parts), and compare the curves of growth variables, comparing all pairs of curves, through a permutation test of the difference between two growth curves.The quality variables were subject to the Kruskal and Wallis model which analyzes the effect of the treatments.

Results and Discussion
G. intraradices had high percentage of colonization and sporulation (87.5 %) in lisianthus, structures typical of plant-AMF symbioses such as hyphae, vesicles and arbuscules were present in all screened roots at each location (Figure 1).
According to the analysis of growth curves based on estimates derived from the GAM model, the treatments with G. intraradices and the B. subtilis + G. intraradices combination significantly increased plant height (Figure 2).
The control treatment plants were the smallest.The symbiosis established by arbuscular mycorrhizal fungi (AMF) with most plant species manages to increase their development, because it facilitates the absorption of nutrients (mainly phosphorus) and increases plant vigor.Factors such as the species of mycorrhizae, dose, and time of inoculation are important to obtain greater benefits.If the AMF infection units are sufficient, they can contribute to guarantee an appropriate colonization rate.In addition to the development stage of the infection, the time when plants are inoculated can be critical: the earlier the inoculation, the greater the benefits for the plant (Meir et al., 2010).For example, Rubí-Arriaga et al. ( 2012) inoculated G. fasciculatum and B. subtilis in Lilium sp. which resulted in an increase in height and diameter of the stem, as well as greater dry weight of the vegetative aerial part of the inoculated plants, in relation to the control plants.Adding phosphorus produced higher quality flowers than control and improved plant growth.While some studies suggest that mycorrhizal fungi and soil bacteria may compete for carbon in the rhizosphere, other studies indicate that plant growth promotion by mycorrhizal fungi may counteract this effect and actually stimulate bacterial activity in the soil rhizosphere (Sagar et al., 2021).
Depending on the strain, PGPR improve plant growth through the production of phytohormone precursors; however, they also facilitate the absorption of nutrients or produce substances that repel pathogens.The colonization of PGPR plants can be highly specific to certain plant species, which could explain why, in this case, B. subtilis alone only had a lower effect than G. intraradices (Table 1).The root colonization and symbiotic interaction formation depends on several factors such as the composition of the exudates, the development stage, environment (such as soil type, temperature, moisture, pH, and nutrient availability) and biofilm formation (Lucke et al., 2020).
The differences between the diameter and the leaf area of the stems become more evident from the third evaluation onwards; the combined treatment of B. subtilis + G. intraradices results in thicker stems and a greater leaf area (Figure 3, Table 1).*Different letters in each column show significant differences.HSD= honest significance difference and CV: coefficient of variation (%).n=20.
Evaluations performed every two months: November (initial), January (1), March (2), May (3), and July (4).One of the functions of AMF is to improve the availability of phosphorus in the soil by solubilizing its inorganic forms or by mineralizing organic phosphorus.
Therefore, the more colonized the root system is, the plant will make a more efficient use of nutrients and accumulate more dry matter (Table 2).* Different letters in each column show significant differences.HSD= honest significance difference and CV: coefficient of variation (%).n=20.
Synergistic interaction between AMF and PGPR most of the times, effects positively the plant growth than to single inoculation (Nanjundappa et al., 2019;Mohamed et al., 2019), but the positive result of the positive effect will depend on the species of microorganism, host plant, and environment (Cai et al., 2021).On the other hand, Cruz-Crespo et al. (2020) showed that foliar fertilization with Humifert (N=2.0,P=1.0, and K=1.0 g L -1 ) during the growth and development of lisianthus ´Flamenco purple´ result in significantly wider stems than the fertilization with Bayfolan Forte ® (N = 0.33, P = 0.24 and K = 0.18 g L -1 ), showing the response of lisianthus stem to higher nutrient doses.Likewise, the combined B. subtilis + G. intraradices treatment increased significantly the diameter of the stems, foliar area and number of leaves compared to the control treatment, as a probably greater absorption of macro and micronutrients (Table 2).
Arbuscular mycorrhizal fungi search for soil nutrients with higher efficiency, as a result of their greater surface-volume ratio.Therefore, they penetrate deeper into the soil and extract water with higher efficiency; consequently, plants inoculated with FMA grow faster.Azcón et al. (1992) showed that Lactuca sativa L. plants inoculated with Glomus mosseae or G. fasciculatum had greater leaf area, fresh weight of leaves, and photosynthetic activity than control plants, regardless of the source of nitrogen fertilization.But Garmedia and Mangas (2012) did not get a positive nutritional effect of mycorrhizal inoculation in cut roses leaves, probably due to the low root colonization percentages reached.In our study from the second evaluation, the size of the stems increases more than 40% in all treatments, except for the control (25%).Finally, as the last evaluation approached, the size of the stems increased more than 70% and consequently increasing its leaf area.Figure 3 shows a significant increase in foliar area for G. intraradices (2,342 cm 2 ) and for the B. subtilis + G. intraradices combination (2,902 cm 2 ), compared with the Bacillus treatment and control, which reached 1,487 and 479 cm 2 respectively.
The number of leaves increased significantly from the second evaluation.The lisianthus leaves are turgid with an intense green color, symbiosis indirectly stimulates photosynthesis to supply the plant energetic balance.More leaves represent greater photosynthetic activity and consequently a greater reserve of carbohydrates -which is useful to maintain the respiratory intensity of the stem, once it has been cut.

Quality evaluation
Unlike the growth variables, the mean and the median of the quality variables had very close values, which implies almost symmetrical distributions.Likewise, the quality variables of the stems responded better to the inoculation by G. intraradices and to the B. subtilis + G. intraradices combination.Sagar et al. (2021), describe that the dual inoculation of PGPR and AMF enhances nutrient uptake and productivity of several crops compared to a single inoculation in both normal and stressed environments.Positively interacting PGPR + AMF combination is an efficient and cost-effective recipe for improving plant growth.Lisianthus stems are characterized by thick stems and a large number of flower buds, although not all of them develop and open (Figure 4).The number of flower buds and open flowers were 50 % greater and the flower diameter was significantly wider in the entire floral stem in the G. intraradices (8.23 cm) and B. subtilis + G. intraradices treatments (7.58 cm) compared to B. subtilis and the control treatments (5.88 and 4.17 cm respectively).In addition, the dry weight of the stems was considerably higher for G. intraradices (3.73 g plant -1 ) compared to 1.62 g plant -1 of the control treatment, which indicates a higher carbohydrate reserve -a determining factor in the development of the bud and flower opening (Figure 5).Cavasini et al. (2018) recorded a >50% reduction in carbohydrate reserves in lisianthus buds, from 20.74 mg 100 g -1 (harvest) to 9.85 mg 100 g -1 (day 17), which decrease during the harvest and postharvest stage, so bigger and wider stems represent more reserves for postharvest (Norikoshi et al., 2016).

Conclusions
The growth and development of the lisianthus plants was stimulated by the inoculation of Glomus intraradices and the B. subtilis + G. intraradices combination, being Glomus by itself more efficient in stimulating the absorption of nutrients that was reflected in thicker and taller stems, more dry weight that means higher carbohydrate reserves for flower opening.So we can assume that the nutrients absorption in lisianthus was more efficient for the arbuscular mycorrhizal fungi (AMF) than for Bacillus a plant growthpromoting rhizobacteria.Then in order to minimize costs, applying only Glomus intraradices would be enough.

Figure 1 .
Figure 1.a) Typical vesicles of mycorrhizal fungi, b) Example of arbuscule of arbuscular mycorrhizal fungi, and c) Root colonization of G. intraradices in lisianthus.The roots were stained with trypan blue (100X).