Use of Bacillus spp . as growth promoter in carrot crop

Growth promoting rhizobacteria may increase the yield of some crops. Several microbial products that stimulate plant growth have been marketed. Therefore, the objective of this study was to evaluate the effect of bacteria from Bacillus genera on the production of commercial carrot roots (Daucus carota L.) in field conditions. The following isolates: SF 264 (Bacillus spp.), SF 268 (Bacillus spp.) and commercial formulations based on SF 202 (Rizos®, B. subtilis), SF 266 (Quartz®, B. methylotrophicus) and SF 267 (Onix®, B. methylotrophicus) were evaluated in four experiments conducted in commercial fields of carrot production in the municipality of Rio Paranaíba, Minas Gerais Brazil. Each plot was 6 m long and 1.75 m wide (10.5 m 2 ) including four double rows. An additional treatment containing only water was the control treatment. The experiment was designed as randomized blocks with five replications. The products Rizos ® , Quartz ® and Onix ® containing Bacillus spp. increased the production of commercial carrots roots of all cultivars and sites. The SF 268 and SF 264 isolates were efficient only in two and three experiments, respectively.


INTRODUCTION
In Brazil, carrot is among the five main garden crops grown and 80 % of the total production supplies Brazilian domestic market.The Southeast, Northeast, and South regions are the largest producers of this root crop.The State of Minas Gerais stands out in the production of this vegetable crop and the Alto Paranaiba region is one of the main producers.Yield potential of the crop is from 100 to 120 t ha -1 ; however, the Brazilian average is much lower around 33 tons ha -1 (Embrapa, 2010).Free-living bacteria are found in the rhizosphere of plants and parts of them are known as Plant Growth Promoting Rhizobacteria -PGPR (Alves, 2007).Pseudomonas, Bacillus, Azospirillum, Agrobacterium and Azotobacte have been reported as PGPR, being Pseudomonas and Bacillus the PGPR widely reported.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License rhizobacteria in commercially cultivated crops is worldwide known in onion (Harthmann et al., 2010), potato (Sottero et al., 2006) and tomato Mena-Violante and Olalde-Portugal, 2007).However, this potential is not yet known in several crops, such as carrot (Konusny- Andreani et al., 2014).
Carrot marketing depends on the roots pattern that considers the length, diameter and the defects absence (Ceasaminas, 2015).Thus, the application of PGPR may increase the production of commercial roots, resulting in lower waste and consequently higher profitability to farmers.Bacillus spp. in special is easily grown in liquid culture media with low cost, which make easier its mass production in industrial fermenters.Furthermore, they produce resistant endospores, which increase the shelf life of products and integrated use with chemical products (Lanna Filho, 2010).
Therefore, the aim of this study was to evaluate the efficiency of five Bacillus spp.isolates in increasing the production of commercial roots of carrot under field conditions.

Carrot cultivars
The cultivar Brasilia, developed by EMBRAPA, was used in two sites.In the two other sites the cultivars were Suprema Max ® (Isla Sementes Ltda, Porto Alegre, Brasil) and Juliana ® (Seminis Vegetable Seeds, St. Louis, EUA), respectively.

Soil and sites characterization
The chemical and physical characteristics of the soils are presented in the Table 1.
All isolates were prepared in liquid culture media and formulated as a protocol used in the company.The concentration of the isolates SF 264, SF 266, SF 267 and SF 268 was 1 x 10 9 colony forming units (cfu) per mL, while the concentration of SF 202 was 5 x 109 cfu mL -1 .
At 15 days after planting, the isolate suspensions were applied on the plants using a CO2 -pressurized backpack sprayer furnished with three 110-02 nozzles working in constant pressure of 2.2 bar, spaced at 0.5 m.The dose and volume of the spray solution were 4 L ha -1 and 200 L ha -1 , respectively.Immediately after the application of the bacterial isolates, each experimental area was irrigated at 5 mm water depth in order to facilitate the bacteria propagules percolation in the soil profile.The sprays were done after 3 pm.
Each plot was 6 m long and 1.75 m wide (10.5 m 2 ), including four double rows.An additional treatment containing only water composed the control treatment.The experiment was designed as randomized blocks.Crop management was carried out following the pattern adopted by the growers, including irrigation, topdressing fertilizations, pest management and rough-hewing.

Yield evaluation
The harvest was carried out at 105 days after planting.The fresh weight of commercial roots of carrot was evaluated in 1 m 2 , located at the center of each plot (useful plot).All roots in the useful plots were classified according to the carrot standard classification of a Brazilian program for commercial standards and horticultural packaging improving (Ceasaminas, 2015).It was considered only the roots with a length between 14 and 26 cm without defects.Later the selected roots were weighed using a digital scale.

Statistical analysis
The data were submitted to variance analysis (F test, P = 0.05) and the means compared by Scott-Knott test (P = 0.05) using the software SISVAR 5.1 Build 72 (Ferreira, 2007).

RESULTS AND DISCUSSION
The bacteria isolates increased the production of commercial roots of the cultivar Juliana in both sites (Table 2).For the cultivar Suprema Max, the SF 267, SF 266, SF 264 and SF 202, even in different groups, increased the carrot production.For the cultivar Juliana, yields with SF 202, SF 266 and SF 267 application were 10.40; 12.48 and 23.32 higher than the control.
The SF 267, SF 266 and SF 202 isolates of B. methylotrophicus are soil nitrifying bacteria, which means, they may convert ammonia to nitrite and later nitrite to nitrate which is a readily assimilable nitrogen form by plant roots (Zhang et al., 2012).This characteristic may explain the outstanding performance observed for SF 266 and SF 267 isolates to increase the production of commercial roots of carrot.Furthermore, Yan et al. (2011) and Dev Sharma et al. (2013) reported in several studies that B. methylotrophicus isolates produced antagonistic metabolites against several pathogenic bacteria and fungi.Andreani et al. (2014) evaluated the effect of rhizobacteria isolates from Crotalaria spectabilis on the development of carrot (cultivar Nantes), in greenhouse conditions, through seed microbiolization with bacterial suspensions.In this study the bacterial isolates could not be identified.They were merely coded as UCCBj-CE's.The UCCBj-CE 04, UCCBj-CE 11, UCCBj-CE 17 and UCCBj-CE 18 isolates induced higher productivity in carrot crop.
According to Lanna Filho (2010), the growth-promotion induced by B. subtilis may be a consequence of increasing nitrogen fixation, nutrients solubilization, hormone synthesis and soil conditions improvement, besides indirect benefits by environmental suppression against pathogens.Additionally, the beneficial association provides an increase in physiological metabolites that unleash the root system sensitivity to external conditions, which may improve the nutrient uptake (Manjula and Podile, 2005).Isolates of B. subtilis also has the ability to induce hormonal regulation in plants, as reported by Tsavkelova et al. (2006), Persello-Cartieaux et al. (2003) and Lanna Filho (2010) controlling the root growth by auxin, gibberellin and cytokinin synthesis.It may also explain the responses of B. subtilis isolates in all experiments.Besides the genetic differences among isolates and their possible influence on root colonization, the interaction between isolates and carrots cultivar may also influenced the plant growth (Romeiro et al., 2005;Choudhary et al., 2007).
For the cultivar Brasilia, all isolates increased the carrot production.The SF 264 isolate in the cultivar Juliana and the SF 268 in the cultivar Suprema Max and Juliana did not have the same beneficial effect.It is possible that the production and root exudates composition among cultivars are different; therefore, the SF 268 and SF 264 isolates have not been able to colonize efficiently the rhizosphere of Suprema Max ® and Juliana ® cultivars, and consequently act as growth promoter.
When a microorganism is introduced into a new environment, it must overcome the competition with other microorganisms (microbiostase), in order to develop and perform its ecological functions (Tsavkelova et al., 2006).There are reports that show the high specificity of growth -promoting bacteria to colonize specific hosts.This specificity was not observed in the present study.Raasch et al. (2013) stated that eucalyptus production with Rizolyptus® (B.subtilis) varied according to the tested clones, suggesting a high specificity of root colonization regarding the host.Mello et al. (2002) reported the lack of specificity of the C210, ENF10, ENF16 and RAB9 isolates, obtained from cabbage, beans and radish, respectively, regarding growth promotion in seedlings of pineapple micropropagation.In another study, isolates of C116 (Bacillus pumilus) and C25 (Bacillus thuringiensis subvar.kenyae), from cabbage were effective in promoting the growth of lettuce seedlings (Gomes et al., 2003).
Besides the specificity of bacterial isolates to the host, stands out the differences in the soil of rooting for rhizobacteria colonization, once abiotic factors (pH, nutrient availability, moisture retention, aeration etc.) and biotic (qualitative and quantitative composition of the microbiota and others) may favor or not the colonization, survival and beneficial activity of rhizobacteria (Mafia et al., 2009).
Such variables may explain the difference in the production of commercial carrot when using a same bacterial isolate and in different experiments, i.e. under different environmental conditions.This was observed for the cultivar Brasilia -site 1, where the SF 267 isolate (B.methylotrophicus, Onix ® ) was used, the fresh weight was 6.41 kg while in the site 2 it was 8.51 kg.
In the cultivation fields of the cultivar Suprema Max and Juliana, the potassium content in the soil was around 40% lower than the other fields as highlighted in the Table 1.There may be some relationship between potassium and phosphorus contents in the soil and the capacity of the SF 268 isolate colonize the rhizosphere of carrot plants and promote the plant growth.Moreover, the SF 267, SF 266 and SF 202 isolates do not seem to depend on the presence of high amounts of these nutrients in the soil, considering that some PGPR solubilize phosphates and/or provide potassium to the plants (Gomes et al., 2003;Viruel et al., 2014).Further studies are recommended to investigate whether the beneficial effect of the SF 267, SF 266 and SF 202 isolates in increasing the production of commercial carrot roots is due to their action as nutrients solubilizers in the soil.

Conclusions
The application of Bacillus spp., present in the products Rizos®, Quartz® and Onix®, enhanced the yield of commercial carrot roots.

Table 1 .
Chemical and physical characterization of commercial fields with carrot cultivation.

Table 2 .
Fresh weight of commercial carrot roots (kg m -2 ) after different applications of Bacillus spp.isolates.Means followed by the same letter, in the columns, are not different at 5% probability by Scott-Knott test.CV: coefficient of variation.