AJCS 18(07):434-441 (2024) ISSN:1835-2707

https://doi.org/10.21475/ajcs.24.18.07.pne-170

Influence of pre-inoculation of soybean seeds with

Bradyrhizobium

15

days before sowing

Wilson Story Venancio¹, Eduardo Gilberto Dallago¹, Gislaine Martins

2

Keywords: Pre-sowing; Rhizobia;

Glycine max

; symbiosis; Nitrogen

fixation; Crop yield.

Abbreviations: CFU_colony-forming unit, BNF_biological nitrogen

fixation, N_nitrogen, NNMR_number of nodules on the main root,

DMNMR_dry mass of nodules on the main root, FMS_fresh mass of

Introduction

In Brazil, sowing soybeans (

Glycine max

without nitrogen fertilizers, owing to its biological nitrogen fixation

(BNF) character, reducing production costs and increasing grain

production (Hungria et al., 2007). Nitrogen (N) is essential for

soybeans, needing approximately 80 kg of N to produce 1000 kg of

grain (Kaschuk et al., 2016). N sources are nitrogen fertilizers and

fertilization, and environmental conditions (Obserson et al., 2007;

Anghinoni et al., 2017; Hungria et al., 2007; Campo et al., 2009; Zilli

et al., 2010).

Some

(formerly

and studied.

B. diazoefficiens

also forms nodules on other plants like

mung bean and siratro (Sprent et al., 2017; Piromyou et al., 2021).

Inoculating seeds before marketing or sowing (pre-inoculation) has

been practiced for decades for crops like soybeans (Deaker et al.,

2004; Herridge, 2008; Santos et al., 2019). This provides uniform

Bradyrhizobium

populations on seeds due to active ingredients and

solvents (Campo et al., 2009). This can lead to fewer and smaller

nodules, though some fungicides do not significantly affect

nodulation (Andrés et al., 1998; Bikrol et al., 2005).

Using a cell protector in seed treatment can protect bacteria and

extend their survival from treatment to planting.

Table 1. Number of nodules on the main root (NNMR), dry mass of

Means followed by the same letter in the column do not differ from

each other using the Duncan test at 10% probability.

Coefficient of variation in percentage.

Experiments in Brazil showed soybeans could be inoculated up to five

days before sowing (Campo and Hungria, 2007), and with a

protectant, it may be extended up to 30 (Araujo et al., 2017) or 60

days (Machineski et al., 2018).

2015).

Results

Symbiotic efficiency

The effectiveness of treatments based on the number of nodules on

the main root (NNMR), dry mass of nodules on the main root (DMNR),

fresh and dry mass of the shoot (FMS and DMS), and the Falker

chlorophyll index (FCI) in four regions (Table 1).

In Palmeira-PR, Biofix Protec (T4) applied 15 days before planting

In Araguari-MG, the commercial product (T3) had higher averages of

77.39 nodules and 288.0 mg DMNR compared to other treatments.

Biofix Protec (T4) also showed significant

increases compared to T2 and T1. FMS did not significantly vary

among T4, T3, and T2, but T3 and T2 differed from T1. No significant

differences were observed in DMAP and FCI among T4, T3, and T2,

but they were superior to T1.

thousand grain weight (TGW), nitrogen content, and total N content in

grains are shown in Table 2. In Palmeira-PR, Biofix Protec (T4) resulted

in 3546.13 kg.ha

, an 8.5% yield gain, statistically superior to T1 and

T3 but not different from T2. No significant differences were found in

TGW, with N content ranging from 5.43% to 5.71%.

In Itapira-SP, Biofix Protec (T4) and the commercial product (T3) were

equal, with gains of 11% and 12%, respectively, significantly differing

from T2 and T1. TGW varied from 130.53 to 136.47 grams, with Biofix

Protec (T4) resulting in 5.48% N in grains and 212.51 kg.ha

-1

N, significantly different from other treatments.

Table 2. Product factors (productivity and weight of a thousand grains

– TGW), relative production gain in percentage, percentage of nitrogen

in grains and total nitrogen content in the different regions evaluated

during 2021/2022 agricultural year.

1. Means followed by the same letter in the column do not differ from

each other using the Duncan test at 10% probability.

2. Variation coefficient in percentage.

In Catalão-GO, productivity was equal for T4, T3, and T2, with gains

compared to T1, with averages ranging from 231.75 to 238.56 kg.ha

Discussion

In the four different edaphoclimatic regions, nodule formation on

main roots varied by location. Initial rhizobia population assessments

in Palmeira-PR and Itapira-SP influenced nodulation in control

high

Bradyrhizobium

cell counts, as shown in Cerrados and Paraná

experiments (Vargas et al., 1994). In Araguari-MG and Catalão-GO, no

rhizobia were found despite a history of soybean rotation. T1 (control

without inoculation) showed less nodulation and lower dry mass, while

seed inoculation treatments showed efficient nodulation.

Based on this context, in Palmeira-PR the treatment with Biofix Protec

(T4) was significantly equal to the commercial product inoculated on

the day of sowing (T3) and statistically superior to the nitrogen

treatment (T2). The same was

higher than T2 and T1. In Catalão-GO, the results for Biofix Protec in

pre-inoculation (T4) were highly efficient, significantly differentiating

from the other treatments used. These results directly reflected the

final yield of the crop, as according to Câmara (2000), soybean plants

with a large number of nodules at flowering present sufficient

conditions to obtain high levels of fixed nitrogen and, consequently,

high grain yield.

In Palmeira-PR, Araguari-MG, and Catalão-GO, nodule numbers

In Itapira-SP, the average number of nodules was lower than that

found in other trials. However, the application of Biofix Protec (T4) did

not statistically differ from the commercial product (T3). It is known

that crop nodulation may vary according to soil pH, vegetative stage

and environmental conditions in which the plants are located.

According to Silva et al. (2002), to have an efficient development of

bacteria, the soil must have a pH of 6.5, with the soil pH in Itapira-SP

being around 5.8.

Table 3. Description of biological products, additives, protectors and

varying soil types and climatic conditions. In Palmeira, PR, the soil was

Alitic Haplic Cambisol with silty clay loam texture, under a Cfb climatic

classification. Itapira, SP, featured Dystrophic Red-Yellow Latosol with

clay texture, classified as Cfa. Araguari, MG, had Dystrophic Red

Latosol with sandy clay texture, categorized as Aw. Catalão, GO, had

Dystrophic Haplic Cambisol with loamy sand texture, also classified as

Aw. The trials were conducted in different edaphoclimatic regions to

evaluate the efficiency of the product across varying soil types and

approved by Normative Instruction DAS/MAP 30/2010 of

11/12/2010, published in the D.O.U. of 11/17/2010. It consists of

inoculating serial dilutions of the samples, in specific test plants

(soybeans), grown under aseptic conditions, evaluating the formation

of nodules (NMP Technique – Most Probable Number).

The management and fertilization used in soybean cultivation during

the trials was carried out in order to meet the cultivation needs for

each region (Table 4). Other cultural treatments, such as applications

of herbicides, fungicides and insecticides, were carried out according

to the needs of the crop (Seixas et al., 2020).

Treatments

50% was applied at planting and 50% was applied at flowering;

Treatment 3:

Commercial seed inoculation (50 mL/50 kg of seeds),

formulated with the bacteria

Bradyrhizobium japonicum

strains SEMIA

5079 and 5080, at concentration of 5x10

9

-1

, following the

technical recommendation on the label, applied on the sowing day;

Treatment 4:

Seed inoculation with Biofix Protec (100 mL/50 kg of

seeds), a liquid inoculant made of the bacteria

Bradyrhizobium

japonicum

– SEMIA 5079 and

Bradyrhizobium diazoefficiens

– SEMIA

5080, at a final concentration of 5x10

CFU mL

following products: cell protector Protec (0.5 mL/kg), Potenzial TS (0.2

mL/kg), and CoMo Platinum (100 mL/ha), applied 15 days before

planting.

A description of treatments is also presented in Table 5.

After treatment and complete drying, the seeds were placed in a paper

container, stored in a dry place, protected from light, with

temperatures between 20 and 24 °C and air humidity below 70%.

Evaluated characters and statistical analysis

Five plants with intact roots were collected from the central area of

each plot, immediately before flowering (30 to 40 days after

emergence), being evaluated the number of nodules on the main root

The plants collected were weighed in order to determine the fresh

mass of shoots (FMS) and after that, they were dried in an oven at 65

ºC, until they reached constant weight for determination of the dry

mass of shoots (DMS). The data was expressed in grams per plant.

Grain yield (GY) was expressed in Kg ha

thousand grains (WTG) in grams, with data corrected to 13% moisture.

The N content in the grains was evaluated by the ABC Foundation

laboratory, which used the proposed method by Dumas (AOAC, 2005).

The results obtained were subjected to analysis of variance using the F

test, and the difference between means, when significant, was

compared using the Duncan test at a 10% probability level, using the

Sasm – Agri software (Canteri et al., 2001).

Conclusions

The Biofix Protec inoculant pre-inoculated 15 days before sowing (T4)

in the soybean crop, increased nodulation in the main root and the dry

mass of nodules, being, therefore, statistically equal to or greater than

superior to the commercial standard inoculant (T3), which was

inoculated on the day of sowing. Pre-inoculation of the Biofix Protec

inoculant 15 days before sowing, using strains of

Bradyrhizobium

diazoefficiens

- SEMIA

5079 and specific formulation, combined with protective agents and

chemical seed treatments, is as effective as the inoculation carried out

at the time of sowing, as it did not result in a reduction in plant

nodulation, as well as crop productivity. These results confirm the

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