Lettuce Production under Reduced Levels of N-fertilizer in the Presence of Plant Growth-promoting Bacillus spp. Bacteria

Lettuce consumption has increased worldwide due to the recognition of its nutritional value and to the consumers’ search for healthier foods. Increased consumption must be coupled with more efficient production, demanding more nutrients. However, the utilization of chemical inputs in horticulture is decreasing, and biological approaches, such as the inoculation with plant growth-promoting bacteria (PGPB), are becoming more popular. Species of Bacillus have long been known promote the growth of plants, including lettuce, in agricultural soils, but little is known about their potential under Brazilian conditions. We have tested the ability of B. subtilis, B. pumilus, and B. amyloliquefaciens, carried in single or combined pre-commercial inoculants, to act as PGPB for field-grown lettuce plants of cultivar Elisa. In our experiments, the PGPB were challenged to promote plant growth when the recommended dose of nitrogen (N) fertilizer was reduced by 50%. Our results demonstrated that all species, either alone or combined, were able to improve plant vigor, the number of leaves, and the average diameter and weight of the lettuce heads, under the test conditions. Average gains in plant vigor and head weight due to all species were of the order of 50% relative to the control (no N fertilizer) treatment and, in most cases, gains due to the presence of the bacteria were superior to those promoted by the full (100%) N fertilization. This is the first report of positive utilization of pre-commercial products containing Bacillus for lettuce growth in Brazil.

contributing directly or indirectly to plant growth 14 . Species of the genus Bacillus have been implicated in plant growth promotion, for example, by means of the production of phytohormones, nutrient solubilization and mobilization, production of antibiotics, and induction of plant systemic resistance to pathogens [15][16][17][18] . It is believed that the growth-promoting effects of Bacillus spp. result from the combined action of different mechanisms. The species B. amyloliquefaciens, B. pumilus, and B. subtilis are probably the best studied PGPB species of the genus 14 .
One strain (KPS46) of B. amyloliquefaciens that produces auxin, surfactin, and extracellular proteins has successfully enhanced the growth of soybean [Glycine max (L.) Merr.] plants 19 . Other authors observed that inoculation of soybean seeds with B. pumilus resulted in enhanced plant growth and increased seed protein yield 20 . Saleem et al. 21 postulated that 1-aminocyclopropane-1carboxylate (ACC) deaminase-producing strains of B. pumilus may also act as PGPB because the enzyme helps regulate ethylene production by plants under certain stress conditions, thus helping to sustain plant growth. Furthermore, B. pumilus strain YSPMK11 was active against the fungal pathogen Sclerotinia scletotiorum that causes stalk rot in cauliflower (Brassica oleracea var. botrytis L.) and which is pathogenic to many other host plants 22 .
B. subtilis strain 21-1 has been shown to act both as plant growth promoter and disease suppressor in association with four crops, Chinese cabbage (Brassica rapa subsp. pekinensis) cucumber (Cucumis sativus L.), tomato (Lycopersicon esculentum Miller), and lettuce grown under two different soil conditions 23 . In fact, B. subtilis has been demonstrated experimentally to act as PGPB by an array of mechanisms, such as competitive colonization of the rhizosphere, production and secretion of antibiotics, induction of host systemic resistance to pathogens, production of cytokinin-like substances that promote root growth, resulting in increased plant vigor and yield, disease escape, and stress tolerance 24 .
Even though bacteria of the genus Bacillus are successfully employed as PGPB for various crops in other countries, very little is known about that in Brazil, where the technology of utilization of PGPB other than rhizobia for legumes is still incipient. In this study, we evaluated the ability of strains of B. amyloliquefaciens, B. pumilus, and B. subtilis, prepared as single inoculants or as a mixture of the three species, to promote the growth of field-grown lettuce.

MATERIALS AND METHODS
A collection of 20 bacterial isolates obtained from the soil of a private farm in the town of Lapa (25° 46' 11" S, 49° 42' 57" W, Altitude 908m, Atlantic Forest biome), State of Parana, Brazil was screened for the ability to promote the growth of roots of lettuce seedlings. Four promising isolates were selected for further studies. The isolates are currently deposited at the Culture Collection of Diazotrophic and Plant Growth-Promoting Bacteria of Embrapa Soja (WFCC Collection # 1213, WDCM Collection # 1054).
The four selected isolates were characterized by rep-PCR fingerprinting 25 and sequencing of the 16S rRNA gene. Briefly, genomic DNAs of the four isolates were extracted with Axyprep Bacterial Genomic DNA Miniprep kit (Axygen ® , USA). For the rep-PCR analysis, the DNA of each of the isolates was amplified with the BOX-A1R primer (Invitrogen ® Life Technologies ® , Brazil) using the procedures described elsewhere 26 . The PCR reactions were performed on an Eppendorf ® Mastercycler Gradient (Hamburg, Germany). Amplified fragments were separated through electrophoreses at 120 V for 7 h, in gels of 1.5% (w:v) agarose. The gels were stained with ethidium bromide and were visualized and photographed under UV light using a digital camera.
The cluster analysis was performed with the software Bionumerics ® 7.5 (Applied Mathematics, Sint-Martens-Latem, Belgium), using the UPGMA algorithm 27 and the Jaccard coefficient 28 with 1.0% tolerance.
Field experiments were carried out in three cropping seasons, 2016, 2016/2017, and 2017/2018, at six experimental sites located at the Estacao Experimental Agricola Campos Gerais -EEACG, and one experimental site in a private farm, all in the town of Palmeira, State of Parana, Brazil, with geographic coordinates of 25° 26' 9" S and 50° 01' 01" W, at an altitude of 929 m above sea level. The climate of the region is classified as Cfb (temperate humid with temperate summer), according to K‫צ‬pen 31 .
The soil of the experimental areas is classified as Cambisol. Twenty to 30 days before  Table 2.
Prior to inoculation, 300 ml of each product were diluted to 1,000 L with water, in order to make the final volume necessary to inoculate one hectare, according to the manufacturer's instructions. For inoculation, trays with seedlings were soaked in a volume of diluted inoculant proportional to the area of the tray until the substrate was saturated. Seedlings were then transplanted to the definitive beds and the remaining diluted inoculant was sprayed over the seedlings with the aid of a CO 2 -pressurized precision sprayer for agronomic experimentation, with a constant pressure of 35 lb in -2 and equipped with an XR 110 02 fan nozzle. Seedlings of the treatments that did not receive bacteria were treated the same way, but with plain water. In the plots, seedlings were planted in a 0.3 m x 0.3 m arrangement, making up a total of 11.1 plants m -2 . Plots received either sprinkler or drip irrigation, as necessary.
Treatments consisted of i) non-inoculated, no N fertilizer control (T1), ii) non-inoculated, with 50% of the recommended N fertilizer (T2), iii) non-inoculated, with 100% of the recommended N fertilizer (T3), and iv) inoculated, with 50% of the recommended N fertilizer (T4). The reason for reducing the amount of N fertilizer by 50% in the presence of the inoculants is because Brazilian authorities require that any product that is to be employed in agriculture as plant growth promoter must be able to replace, at least, part of some nutrient. All experiments were set up in a completely randomized block design with six replicates. NT -the products were not tested at those sites When the crop reached the BBCH 49 32 growth stage, eight to ten plants from the central area of each plot were harvested for evaluations. Plant vigor was rated on a scale of 1 (reduced vigor, highly defoliated, unrecoverable plants with compromised nutrition) to 10 (excellent vigor, full of leaves with accentuated growth, appearance as expected for the cultivar). In addition, the number of leaves per plant and the diameter and average weight of the lettuce heads were determined.
All data were subjected to an analysis of homogeneity of the variances and an ANOVA was performed. When significant differences were detected by the ANOVA, treatment means were compared by the Duncan test at p < 0.05. All statistical analyses were performed using the SASM -agri 33 software.

Genetic characterization of the strains
DNA profiles obtained in the BOX-PCR analysis indicated that each strain was unique ( Fig. 1). High genetic diversity between the strains was highlighted, such that in the dendrogram they were joined at a final level of similarity of less 35% (Fig. 1).

Inoculation with Bacillus subtilis (Product A)
Seedling inoculation with B. subtilis significantly increased (50%) plant vigor over the control (T1) treatment in all four experiments (Table 3). In two out of the four experiments, B. subtilis-inoculated plants (T4) were significantly more vigorous than those that received only 50% of the recommended dose of N fertilizer (T2), whereas in all four experiments they were not significantly different from plants that received 100% of the recommended dose of N fertilizer (T3; Table 3).
All treatments significantly increased the number of leaves relative to the T1 treatment, and in two out of the four experiments T4 induced the production of significantly more (23%) leaves than T2 or T3 (Table 3). All treatments significantly increased the diameter of the lettuce heads over T1, and T4 was significantly superior to T2 and T3 in three and two out of the four experiments, respectively ( Table 3). The average weight of the lettuce heads was positively and significantly influenced by all treatments relative to T1 in all four experiments (Table 3). Lettuce heads produced by T4 were significantly (49%) heavier than those form the T2 and T3 treatments in all four and three out of the four experiments, respectively (Table 3). On average, mean values of all parameters evaluated were highest for the T4 treatment (Table 3).

Inoculation with Bacillus pumilus (Product B)
The presence of B. pumilus in the inoculant resulted in significant increases (54%) in plant vigor over T1 in all four experiments (Table  4). In addition, significant increases in plant vigor due to the presence of B. pumilus over T2 and T3 were observed in two out of the four experiments ( Table 4). The number of leaves was significantly   Journal of Pure and Applied Microbiology increased by all treatments relative to T1 in all four experiments, and T4 induced the production of significantly more (26%) leaves than T2 and T3 in three and two out of the four experiments, respectively ( Table 4).
The diameter of the lettuce heads was significantly increased by all treatments relative to T1 in all but one experiment (Table 4). Once again, T4 induced the production of significantly wider heads than T2 alone, and in three out of the four experiments the results promoted by T4 were equivalent to those promoted by T3 (Table 4). All treatments produced heavier lettuce heads than T1 in all four experiments (Table 4). T4 produced heavier heads over T2 in all four, and over T3 in three out of the four experiments (Table 4). On average, mean values of all parameters were highest for the T4 treatment than for T2 or T3 (Table 4).

Inoculation with Bacillus amyloliquefaciens (Product C)
B. amyloliquefaciens significantly increased plant vigor relative to T1 (51%) in all four experiments, and relative to T2 in two out of the four experiments; no differences relative to T3 were observed ( Table 5). The number of leaves was significantly increased by all treatments, relative to T1, in all four experiments, and T4 (27% increase) was superior to T2 or T3 in three out of the four experiments ( Table 5).
The diameter of the lettuce heads was significantly increased by all treatments relative to T1 in all but one of the experiments; in addition, T4 was superior to T2 in three, and to T3 in two out of the four experiments, respectively (Table 4). All treatments induced the production of heavier lettuce heads relative to T1, and positive significant effects of T4 could be observed over T2 in all four experiments, whereas T4 was superior to T3 in three out of the four experiments ( Table 5). The mean values of all parameters evaluated were highest in response to the T4 treatment, when compared to T2 or T3 (Table 5).

Inoculation with Bacillus subtilis, B. pumilus, and B. amyloliquefacients (Product D)
The triple inoculant, consisting of the mixture of the three species of Bacillus, was effective to significantly (39% increase) improve plant vigor over T1 in all four experiments (Table 6). When compared to T2 or T3, significant differences were observed in only one experiment, probably due to a higher degree of variability among data ( Table 6). All treatments significantly improved the number of leaves relative to T1 in three out of the four experiments, and T4 was significantly better than T2 or T3 in two of them ( Table 6).
The diameter of the lettuce heads was significantly improved by the inoculant relative to T1; however, in most cases no significant differences were detected between T4 and T2 or T3 alone ( Table 6). All treatments resulted in significantly heavier lettuce heads in three out of the four experiments, in comparison to T1 (Table  6). T4 was significantly superior to T3 in two, and superior to T2 alone in all four experiments, respectively (Table 6). On average, mean values of all parameters were highest in response to T4 than all other treatments.
Overall, the parameters that were most positively influenced by all inoculants tested were plant vigor and the average weight of the lettuce heads.

DISCUSSION AND CONCLUSION
Modern agriculture is challenged to be able to feed an ever-increasing population with quantity and quality products. In order to attain these objectives, intensification of agricultural practices has become necessary, frequently compromising the environment 34 . In recent years, efforts have been made to reduce chemical inputs into horticultural systems, with the concomitant utilization of soil microorganisms with varying modes of interaction with plants 35 . Such microorganisms are collectively known as plant growth-promoting bacteria (PGPB), acting to increase nutrient-and water-use efficiency and uptake capacity, and to alleviate biotic stresses 36 .
We have evaluated the ability of the species Bacillus subtilis (strain CCTB04), B. pumilus (strain CCTB05), and B. amyloliquefaciens (strains CCTB06 and CCTB09), either as single inoculants or a mixture of the three species, to promote the growth and production of lettuce under reduced N fertilization. In our experiments, all isolated species, as well as their mixture, contributed to improve all parameters evaluated relative to the controls, and the gains obtained with inoculation of the PGPB were superior to those obtained when the full dose of N fertilizer was applied. The  bacteria were especially efficient to improve plant vigor and the weight of the lettuce heads, both with an average 49% increase over the control. One strain of B. subtilis (BS 21-1) has been previously demonstrated to improve plant height and leaf width of four different crops, including lettuce, and to help suppress soft rot of Chinese cabbage and lettuce, when plants were challenged by pathogen inoculation 23 . We have not detected any disease incidence in our experiments, but the positive responses of plant growth, expressed as increased plant vigor, numbers of leaves and average weight of lettuce heads could be attributed to the strain of B. subtilis we have employed. Other authors 37 have verified an average 30% increase in the weight of lettuce heads produced with inoculation of B. subtilis, which was associated with the production and secretion of cytokinin and auxin by the tested strain. We have not evaluated the production of plant-growth hormones by our strain, but since this biochemical trait is common among strains of B. subtilis 38 , the increases we observed in our experiments may be related to it.
Gains in plant vigor and average head weight upon inoculation with B. pumilus were slightly higher than for B. subtilis. These results may be related to the ability of strain CCTB 3203 to produce indole-acetic acid, which is not produced by B. subtilis strain CCTB 2720 (data not shown). Furthermore, Adesemoye et al. 39 have demonstrated that one strain (T4) of B. pumilus is able to increase N uptake by tomato plants that received only 80% of the recommended dose of N fertilizer. We have not determined the N content in the leaves of our plants, but our results strongly suggest that the strain we employed was able to stimulate N uptake by the lettuce plants even when only 50% of the N fertilizer dose was applied, as the presence of the PGPB improved the results over the treatment with 50% N alone. In addition, strains of B. pumilus that produce the enzyme ACC-deaminase have been shown to sustain root and plant growth and development due to the inhibition of ethylene synthesis by the plants under stress conditions 21 . We have not determined if strain CCTB05 produces ACC-deaminase, but if we consider that reduced levels of an important nutrient such as N in the soil represent an abiotic stress for the plant, it is reasonable to speculate that the positive results we observed may have been due to the enzymatic activity of the bacteria, especially since ACC-deaminase-producing strains of Pseudomonas fluorescens have been sown to perform better as PGPB under reduced levels of soil nitrate 40 .
Inoculation with B. amyloliquefaciens also resulted in gains over 50% in plant vigor and average head weight over the control. Earlier studies 19 demonstrated that this species produced indoles, lipopeptides and proteins that stimulated soybean growth both in the laboratory and in the greenhouse. Since this species produces auxins 17 , which are known to favor the development of secondary roots and root hairs, improving water-and nutrient-uptake and plant growth 41 , the positive results we observed may be due to hormone production by the strain we used. Furthermore, strains of B. amyloliquefaciens have been implicated in the induction of plant systemic resistance to pathogens 42 . Since lettuce is quite susceptible to foliar diseases and no measures for disease control have been adopted in our experiments, it is possible that the presence of the inoculated bacteria may have helped plants suppress any casual incidence of pathogens.
The combination of all three species of Bacillus also promoted gains in plant vigor and average head weight. Mixed inoculants containing bacilli have been successfully employed in experiments before. For example, Adesemoye et al. 43 have successfully employed a mixture of B. pumilus, B. amyloliquefaciens and mycorrhizal fungus that allowed a 25% reduction in the rate of fertilization for tomato. As mentioned before 14 , several species of the genus Bacillus have been implicated in plant growth promotion by a diversified array of mechanisms. For example, B. subtilis has 4 -5% of its genome devoted to producing antimicrobial compounds 38 . B. pumilus and B. amyloliquefaciens have also been shown to produce substances with antimicrobial activity 45, 46 . The antimicrobial activity of these species may account for protection against pathogens and the presence of two or more species in the inoculants may bring complementary effects.
In our experiments, all three species of Bacillus tested, as well as their mixture were effective to promote growth of lettuce, with a concomitant 50% reduction in the amount of Journal of Pure and Applied Microbiology N fertilizer applied to the crop. This is the first report of such data in Brazil, supporting the recommendation of the products for lettuce inoculation.