Methods to quantify Bacillus simplex-based inoculant and its effect as a seed treatment on field-grown corn and soybean in Brazil

Abstract:

Growth-promoting bacteria in agriculture have become an important tool to improve crop performance and productivity in the face of climate change and deteriorating soil conditions. Bacillus simplex is a recently developed active ingredient for the growth promotion of corn and soybean in Brazil. This study compared three methods to quantify B. simplex colony-forming units in the inoculant product and evaluated the treatment effects of four different concentrations of a B. simplex-based inoculant on corn and soybean root and shoot dry weight, the Normalized Difference Vegetation Index (NDVI), and yield. Field trials were performed at four different locations for each crop, in Mato Grosso do Sul and Paraná for corn, and in Mato Grosso do Sul, Minas Gerais, and Paraná for soybean. The performance of B. simplex was compared to an Azospirillum brasilense-based inoculant, a polymer seed treatment, and untreated controls. The results showed that the official MAPA method for quantifying microbes in inoculants recovered the highest number of B. simplex colonies. However, all three evaluated quantification methods recovered over 100 million colony-forming units per mL (10⁸ CFU.mL^-1). The field results showed that the B. simplex inoculant generally increased corn and soybean yields as much or more as the A. brasilense product and that the polymer seed treatment had no impact on yield. The treatment effect on root and shoot weight, and NDVI, was inconsistent. This research shows that B. simplex is quantifiable with three different methods and that it can improve yield in corn and soy. The Bacillus simplex-based inoculant has the potential to become widely used in Brazil.

Index terms:
bioinputs; biological product; inoculant; plant growth-promoting; seed treatment.

Resumo:

As bactérias promotoras de crescimento na agricultura tornaram-se uma ferramenta importante para melhorar o desempenho e a produtividade das culturas em face das mudanças climáticas e da deterioração das condições do solo. Bacillus simplex é um ingrediente ativo recentemente desenvolvido para a promoção do crescimento de milho e soja no Brasil. Este estudo comparou três protocolos para quantificar unidades formadoras de colônias de B. simplex no produto inoculante e também os efeitos do tratamento de sementes com quatro concentrações do inoculante à base de B. simplex sobre o peso seco da raiz e da parte aérea, o Índice de Vegetação por Diferença Normalizada (NDVI) e a produtividade de plantas de milho e soja. Ensaios de campo foram realizados em quatro locais diferentes para cada cultura, em Mato Grosso do Sul e Paraná para milho, e em Mato Grosso do Sul, Minas Gerais e Paraná para soja. O desempenho das quatro concentrações de B. simplex foi comparado a um inoculante à base de Azospirillum brasilense, a um tratamento de sementes com polímero e também ao controle não tratado. Os resultados mostraram que o método oficial MAPA para quantificação de microrganismos em inoculantes recuperou o maior número de colônias de B. simplex. No entanto, todos os três métodos de quantificação avaliados recuperaram mais de 100 milhões de unidades formadoras de colônias por mL (10⁸ UFC.mL^-1). Os resultados de campo mostraram que o inoculante de B. simplex, em geral, aumentou a produtividade de milho e soja tanto ou mais quanto o produto de A. brasilense e que o tratamento de sementes com polímero não teve impacto na produtividade. O efeito do tratamento sobre o peso da raiz e da parte aérea, e NDVI, foi inconsistente. Esta pesquisa mostrou que B. simplex é quantificável pelos três métodos avaliados e que pode aumentar a produtividade de milho e soja. O inoculante à base de Bacillus simplex tem potencial para se tornar amplamente utilizado no Brasil.

Termos para indexação:
bioinsumo; produto biológico; inoculante; promotor de crescimento de plantas; tratamento de sementes

INTRODUCTION

The intense agricultural practices in many parts of the world have resulted in the unsustainable degradation of soils involving loss of organic matter, the release of greenhouse gases, erosion, and the excessive application of fertilizers (Kopittke et al., 2019). In addition, climate change has contributed to the frequency, increase, and severity of many biotic and abiotic stresses, mainly due to high temperatures and droughts, which drastically reduce productivity (Teixeira et al., 2013). Among the sustainable approaches to mitigating adverse impacts of soil degradation and climate change in agriculture is using plant growth-promoting microorganisms (Etesami and Maheshwari, 2018). Different microbes present plant growth-promoting properties (Pandey et al., 2019), among which Bacillus species are among the most-studied bacteria (Tiwari et al., 2019). Beneficial Bacillus and relatives are associated with plants as root endophytes (Santoyo et al., 2016), rhizoplane (Cavaglieri et al., 2009), and rhizosphere inhabitants (Lugtenberg and Kamilova, 2009). These microorganisms exhibit a variety of mechanisms involved in the plant growth-promotion (Ibanhes-Neto et al., 2021; Tiwari et al., 2019), including nitrogen fixation and phosphorus solubilization (Elkoca et al., 2007), production of siderophores for iron acquisition (Kushwaha et al., 2020), 1-amynocyclopropane-1-carboxylate deaminase (ACC deaminase) involved in the alleviation of drought stress (Gowtham et al., 2020), phytohormones promoting plant growth (Martínez-Viveros et al., 2010), and antibiotics, catabolic enzymes and other strategies to control plant pathogens (Choudhary and Johri, 2009; Schönbichler et al., 2020). Plant growth promotion may manifest in increased yield, root and shoot growth, overall plant health as measured by the normalized difference vegetation index (NDVI), and by other phenotypic changes (Tiwari et al., 2019; Witkowicz et al., 2021).

In Brazil, products of a certain degree of purity containing living organisms capable of providing benefits to plant growth are classified as inoculants. Bacillus species such as B. licheniformis (Akhtar et al., 2020), B. subtilis (Lee et al., 2014; Hashem et al., 2019), B. amyloliquefaciens (Kim et al., 2017; Ngalimat et al., 2021) and B. simplex (Barneix et al., 2005; Schwartz et al., 2013) are among well-known growth promoters that add value to crops and have been used in the country. Brazil stands out as a global leader in using Bradyrhizobium, which supplies almost the entire nitrogen demand for soybean cultivation in Brazilian agriculture (Campo et al., 2009; Hungria et al., 2015). Another commonly used nitrogen-fixing inoculant for non-leguminous crops in Brazil contains Azospirillum brasilense as the active ingredient (Santos et al., 2021).

Seed inoculation is one of the ways biological inoculants can be deployed in the field (Santos et al., 2021). Microbes added to the seeds rapidly colonize the rhizosphere upon germination and confer benefits to the plants from the beginning of crop establishment (McQuilken et al., 1998). Bacillus species are Gram-positive, spore-forming bacteria that can survive in dehydrated conditions, including on seed, longer than other bacteria (Schisler et al., 2004). This translates to extended shelf life and added flexibility in the planting process.

Current Brazilian regulations for registering microbial inoculant products require a demonstration of the product’s efficacy under field conditions. Still, they do not specify any reference methods to quantify the viable cell count of Bacillus species in the product (MAPA, 2010). However, such methods are essential for the assessment of product quality.

Modern seed-coating technology uniformly applies a wide range of active ingredients onto crop seeds at desired dosages to facilitate sowing and enhance crop performance (Afzal et al., 2020). Polymers are an essential component of seed-coating, assure adherence to the seed and improve plantability.

The aimes of this study were: (i) to compare the efficacy of three methods to quantify Bacillus simplex strain SYM00260; (ii) to assess the field performance of a B. simplex strain SYM00260 inoculant as a seed treatment in corn and soybean on yield, root and shoot weight, and NDVI; and (iii), to compare the B. simplex strain SYM00260 field performance to a commercial Azospirillum brasilense seed inoculant and a polymer seed treatment.

MATERIALS AND METHODS

The Bacillus simplex strain SYM00260, registered as an inoculant by Indigo Brazil Agriculture Ltda in Brazil (registration number: SP004627-2.000001), was used in this study.

Comparison of quantification methods for Bacillus simplex in formulated products

Method 1: This method was based on an EMBRAPA protocol for quantifying Bacillus subtilis and Bacillus licheniformis from formulated products (Embrapa, 2012) with modification of point 7.2, where the product was measured by volume. The concentration of viable B. simplex cells was assessed by performing a series of 10-fold sequential dilutions in 10 mL solution volumes and a total dilution factor of 10^-7. Peptone water (Merck) was used as the diluent. After each transfer, dilutions were homogenized on a shaker three times for 1 minute, sonicated without heating for 5 minutes, homogenized on a shaker for 1 minute, incubated in a double boiler at 80 ± 2 °C for 12 minutes, and cooled in ice water for 10 seconds. From the final dilution, 0.1 mL aliquots were transferred to five Petri plates containing nutrient agar (Merck) culture medium, and spread with a Drigalski loop. The plates were inverted after two minutes and incubated at 37 ± 2 °C for 17-20 h. Serial dilutions were repeated a total of three times.

Method 2: This method is based on a MAPA quantification protocol for Bradyrhizobium-containing formulated products (MAPA, 2010), and is the officially recognized method for quantification of microbial organisms from inoculants. Serial dilutions involved 10-fold sequential dilutions in 10 mL solution volumes and a total dilution factor of at least 10^-5, such that colony forming unit (CFU) counts from plating of 0.1 mL aliquots onto a standard-sized Petri dish reached 30 to 300 CFU. The diluent was sterile saline solution (NaCl 0.85%), and the growth medium for CFU counting was yeast mannitol agar (YMA) + Congo Red. A Drigalski loop was used for spreading, and inoculated plates were incubated at 29 ± 1 °C for 5 days. Two replication series, Series A and B, were prepared for each dilution, and CFU counts were averaged between corresponding series A and B plates. Serial dilutions were repeated a total of three times.

Method 3: This method comprised 10-fold sequential dilutions, a total dilution factor of 10^-7, 1 mL solution volumes, 1x phosphate buffered saline (PBS) as the diluent and tryptone soy agar (TSA) culture medium for CFU counting. Product aliquots of 500 µL were transferred to 2 mL deep well microplates, incubated at 65 °C for 15 ± 2 minutes in a double bath, mixed by shaking, followed by serial dilution. Dilutions were homogenized by pipetting up and down five times. Aliquots of 100 µL from the final dilutions were spread onto TSA Petri dishes in three replications using a Drigalski’s loop and allowed to soak into the medium. Petri dishes were inverted and incubated at 30 ± 2 °C for 17-24 hours. Serial dilutions were repeated three times.

Negative controls were included for all methods and consisted of two plates inoculated with the respective diluent. The positive control was Bacillus subtilis strain AGR 013.3-B acquired from the André Tosello Collection (code CCT 2576 - ATCC 6051), using the recommended dilution suggested by the supplier.

Counting of B. simplex colonies was performed after the incubation period described for each method. The descriptions from Rosenberg et al. (2016) and Parte et al. (2020) were used for the morphological characterization of the colonies.

The data were analyzed through a linear mixed-effects model conducted in the R 3.6.3 software package (R Core Team, 2021) and the lmerTest v3.1-1 package (Kuznetsova et al., 2017). Fisher’s least significant difference (LSD) method was used to assess whether the mean colony-forming unit (CFU) counts recovered by each quantification method were equal.

Assessment of Bacillus simplex performance in the field

Corn (Zea mays L.) and soybean (Glycine max (L.) Merrill) seeds were sown at a total of 4 localities for each crop in January 2018. The sowing locations, hybrid/variety, geographic coordinates of each field trial, germination and purity of the seed lots, soil type, and climate classification are described in Table 1. Hybrids/varieties were selected for each crop based on location and because they are commercially relevant for each region. The experiment was not designed to compare differences between hybrids/varieties. All experimental fields were located in experimental agronomic research areas. The soil chemical and granulometric characteristics of the field sites are shown in Table 2.

Table 1
Crops, hybrids or varieties, germination and purity, locality, geographical coordinates, climate, and soil types of the experiments.

Crop	Hybrid/Varieties- Company	Germination/Purity (%)	Locality/State	Geographical coordinates/Climate (Köppen)	Type of soil
Corn	Status Viptera- Syngenta	99/100	Ponta Grossa - Paraná (PR)	25°10’11.61” S, 51°11′17.22” E/ Oceanic climate (Cfb)	Sandy clay loam
Corn	Supremo Viptera- Syngenta	97/100	Imbituva - Paraná (PR)	25°9’23.41” S, 50°32′57.68” E/ Oceanic climate (Cfb)	Sandy clay
Corn	Supremo Viptera- Syngenta	97/100	Lapa - Paraná (PR)	25°44’30.72” S, 49°48′16.91” E/ Oceanic climate (Cfb)	Sandy clay loam
Corn	Supremo Viptera- Syngenta	97/100	Dourados - Mato Grosso do Sul (MS)	22°05’23.08” S, 55°08′22.27” E/ Tropical monsoon climate (Am)	Clay
Soybean	Brasmax Garra IPRO- Brasmax	98/100	Imbituva - Paraná (PR)	25°9’20.13” S, 50°32′57.09” E/ Oceanic climate (Cfb)	Sandy clay
Soybean	Brasmax Garra IPRO- Brasmax	98/100	Uberlândia - Minas Gerais (MG)	19°2’24.21” S, 48°11′50.83” E/ Tropical savana climate (Aw)	Clay
Soybean	Monsoy 5947 IPRO- Bayer	95/100	Lapa - Paraná (PR)	25°44’33.62” S, 49°48′19.62” E/ Oceanic climate (Cfb)	Sandy clay loam
Soybean	Monsoy 5947 IPRO- Bayer	95/100	Dourados - Mato Grosso do Sul (MS)	22°05’24.69” S, 55°08′24.23” E/ Tropical monsoon climate (Am)	Clay

Table 2
The soil chemical and granulometric analysis of the experimental localities in the top 0 -20 cm before installation of trials.

Localities	Chemical Analysis									Granulometry
	P	MO	pH	H+Al	Al	K	Ca	Mg	CTC	Clay	Silt	Sand
	(mg.m-3)	(g.dm-3)	(CaCl2)	(mmolc.dm-3)						(g.kg-1)
Ponta Grossa - PR	52	48	4.8	86	0.6	7.2	53	17	163	351	188	461
Imbituva - PR	43	37	6.2	13	N.D	1.8	115	21	151	390	66	544
Lapa - PR	17	36	4.6	65	3.7	4.1	18	8	95	230	156	614
Dourados - MS	59	26	5.0	38	N.D	7.4	34	13	92	584	125	291
Uberlândia - MG	31	23	5.0	30	N.D	3.8	21	8	63	499	157	344

The experimental design was a randomized complete block design with seven treatments and four replications. Each plot had a total area of 24 m² (3.0 m x 8.0 m). Analysis and results presented here focus on comparing the performance of seeds treated with different microbial products versus untreated controls.

Soybean seed was base-treated with Cruiser 350 FS® (thiamethoxam 350 g.L^-1) at a rate of 100 mL.100 kg^-1 of seeds, Maxim Advanced® (metalaxyl-m 20 g.L^-1 + thiabendazole 150 g.L^-1 + fludioxonil 25 g.L^-1) at a rate of 50 mL.100 kg^-1 of seeds, and Fortenza 600 FS® (cyantraniliprole 600 g.L^-1) at a rate of 30 mL.100 kg^-1 of seeds. Corn seed was base treated with Cruiser 350 FS® at 120 mL.100 kg^-1 of seeds and Fortenza 600 FS® at 40 mL.100 kg^-1 of seeds. After treatment with the base chemical products, all soybean seed was inoculated with the product Rizoliq TOP® (Bradyrhizobium japonicum 6 x 10⁹ CFU.mL^-1) at a rate of 100 mL.50 kg^-1 of seeds, and left to dry in the shade at room temperature. After the Bradyrhizobium treatment, part of the seeds was treated with Azototal® (Azospirillum brasilense 2x10¹¹ CFU.mL^-1) at a rate of 100 mL.50 kg^-1 for soybean, and 100 mL.60 kg^-1 for corn. The remaining seeds were treated with the Bacillus simplex strain SYM00260 inoculant at the inoculum concentrations of 5x10⁴, 5x10⁵, 5x10⁶, and 5x10⁷ CFU.mL^-1. A BIOCROMA® polymer was subsequently applied at a rate of 1 mL.kg^-1 for soybean and corn, and the seeds were immediately sown. The amount of B. simplex inoculum used was 9 mL.kg^-1 for soybean and 6 mL.kg^-1 for corn.

Seed treatments were performed in plastic bags with 1 kg of seeds per bag, and mixed by hand using circular movements to simulate industrial seed treatments centrifuges according to the methodology proposed by Nunes (2005). Seed was treated separately at each field trial location. Non-inoculated controls without B. simplex and without BIOCROMA, and non-inoculated controls without B. simplex but with BIOCROMA, were used in each trial.

Soybean planting density was 300,000 seeds.ha^-1, and the spacing between rows was 0.50 m in Imbituva, Lapa and Uberlândia. In Dourados, the spacing between rows was 0.45 m. The average population density was 264,744 plants.ha^-1. A base fertilization of 130 kg.ha^-1 of 04-30-10 N-P-K based on soil analyses (Table 2), was applied in-furrow at each trial immediately before sowing. Corn planting populations were 70,000 plants.ha^-1 and the row spacing was 0.50 m, with an average population density of 69,761 plants.ha^-1. The base fertilization was 300 kg.ha^-1 of 04-30-10 N-P-K with urea coverage of 350 kg.ha^-1.

For soybean, weed control was performed with Zapp Qi 620® (glyphosate potassium 620 g.L^-1). For pest control, the insecticides Engeo Pleno® (thiamethoxam 141 g.L^-1+ lambda-cyhalothrin 106 g.L^-1), Tiger 100 EC® (pyriproxifen 100 g.L^-1 + xylene 800 g.L^-1), and Oberon® (spiromesifen 240 g.L^-1) were used. Disease control was carried out with the fungicides Unizeb Gold® (mancozeb 750 g.L^-1), Fox® (trifloxystrobin 150 g.L^-1 + prothioconazole 175 g.L^-1), Previnil® (chlorothalonil 720 g.L^-1), Orkestra SC® (fluzapyroxad 167 g.L^-1 + pyraclostrobin 333 g.L^-1), Ativum® (epoxiconazole 50 g.L^-1 + fluxapyroxad 50 g.L^-1 + pyraclostrobin 81 g.L^-1) and Sphere max® (trifloxystrobin 375 g.L^-1 + cyproconazole 160 g.L^-1).

For corn, weed control was carried out with the application of Primoleo® (atrazine 400 g.L^-1) and Soberan® (tembotrione 420 g.L^-1). Pest control was carried out with the application of Engeo Pleno® (thiamethoxam 141 g.L^-1 + lambda-cyhalothrin 106 g.L^-1) and Ampligo® (lambda-cyhalothrin 50 g.L^-1 + chlorantraniliprole 100 g.L^-1). Disease control was performed with Opera Ultra® (pyraclostrobin 80 g.L^-1 + metconazole 130 g.L^-1) and Fox® (trifloxystrobin 150 g.L^-1 + prothioconazole 175 g.L^-1).

At the phenological stage V10 for soybean and corn, the Normalized Difference Vegetation Index (NDVI) was determined using a portable GreenSeeker® device on two central rows in each plot. Thirty-five to 50 days after sowing (DAS), five plants were collected randomly from each plot for root and shoot biomass evaluation. Plant materials were oven-dried at 65 °C for four days when the plants were weighed.

Soybean and corn yield measurements were made on a 5 m² area in the center of each plot. Plants were harvested manually with a sickle and processed using a threshing machine. Seeds were cleaned and weighed, and grain yield was estimated after correcting seed weights to 13% moisture.

Data were analyzed separately for each cultivar/hybrid at each location using analysis of variance (ANOVA). The Scott-Knott test was used to compare means in cases where the ANOVA F test detected statistical significance. Statistical analyses were performed using SASM-Agri® (Canteri et al., 2001).

RESULTS AND DISCUSSION

There were significant differences in colony counts between the three methods in this work (Figure 1). Method 2 recovered the highest number of B. simplex colonies with approximately 8x10⁸ CFU.mL^-1, followed by Methods 1 and 3 with about 2x10⁸ CFU.mL^-1. All three methods provided counts of over 100 million colony-forming units per mL (10⁸ CFU.mL^-1).

Figure 1
Mean colony-forming unit (CFU) counts of Bacillus simplex strain SYM00260 using three different quantification methods. Bars followed by different letters differ by the Scott-Knott Test at 5% probability. The data were analyzed using a linear mixed effects model, and LSD multiple comparisons of the number of colonies between the three methods described in the text.

The three methods compared in this work have been widely used to quantify different types of bacteria. None of the methods involve the use of any selective media. Both YMA culture media used in Method 2, and NA and TSA media used in Methods 1 and 3, respectively, provided nutrients necessary for growth of B. simplex. All methods were reproducible and consistent, although they differed from each other in some points described below.

Method 1 was developed to quantify B. subtilis and B. licheniformis - based products on the Brazilian market. The main disadvantage of this Method is that it requires a relatively high number of replications and dilutions, including two initial tubes per product, two serial dilutions from each tube, and five replications, for 20 dishes per sample. The results using Method 1 were similar to Method 3, which was less labor-intensive.

Method 2 is the official method used in Brazil for quantifying microbes classified as inoculants. By this method, a relatively rich medium is required for counting slow-growing bacteria such as Bradyrhizobium, which is not the case for the fast-growing bacteria like Bacillus. It is also relevant to point out that the use of the Congo Red dye in the medium to help distinguish rhizobia and contaminants is not convenient for Bacillus. This dye turns the Bacillus colonies slightly pink and less well-defined and may cause misinterpretation by the analyst. Another disadvantage of Method 2 is that it takes at least five days for the quantification of Bradyrhizobium. As Bacillus species grow faster, this relatively long incubation period for Bradyrhizobium may lead to quantification mistakes. Removal of the Congo Red dye should be investigated to adapt this method for counting Bacillus-based products.

Method 3 recovered a smaller number of colonies but the colonies were larger than the ones observed in the other two methods. Vieira and Nahas (2000) found similar results when evaluating Bacillus spp. isolates from soil samples. Method 3 has two significant advantages. First, it uses a TSA culture medium, which, due to its transparency, facilitates the distinction between B. simplex colonies and contaminants based on colony morphology. Second, it employs deep well plates for high-throughput analysis, which reduces costs and execution time. Method 3 is satisfactory for CFU determination of the B. simplex inoculant that has a minimum guaranteed CFU content of 9x10⁷.

Results showed that B. simplex strain SYM00260 significantly increased corn and soybean yield compared to the two non-inoculated controls, one with the BIOCROMA® polymer and the other without the BIOCROMA® polymer. The exception was soybean in Dourados (Tables 3-6), where a water deficit in the grain filling phase compromised productivity.

For corn, one to several B. simplex concentrations resulted in a significant yield increase at each location (Tables 1, 2). In Ponta Grossa, a yield increase of up to 26% was observed corresponding to an additional 35 bags hectare (60 kg.bag^-1) (Table 3). In Imbituva, yield increased by up to 23% or 23 bags.ha^-1 (Table 3), and in Lapa by up to 24% or 24 bags.ha^-1 (Table 4). In Dourados, only the highest B. simplex inoculation rate of 5x10⁷ CFU.mL^-1 resulted in a significant yield increase of 24% or 29 bags.ha^-1 (Table 4). For the remaining response variables, including root dry weight, shoot dry weight, and NDVI, significant differences between the B. simplex treatments and the non-inoculated controls were only observed at some locations: for root dry weight in Imbutiva (Table 3) and Dourados (Table 4); for shoot dry weight in Ponta Grossa, Imbituva (Table 3), and Dourados (Table 4); and for NDVI in Dourados (Table 4). Dourados was the only location where for all variables, at least one B. simplex concentration was significant (Table 4). For the A. brasilense treatment, yield increase was equal to the maximum B. simplex concentration as in Ponta Grossa, Imbituva, and Lapa, or significantly lower as in Dourados (Tables 3 and 4).

Table 3
Mean values of variables root dry weight, shoot dry weight, NDVI, and yield used to assess the efficacy of corn seed treatment with Bacilllus simplex and Azospirillum brasiliense in field experiments with hybrids Status and Supremo in Ponta Grossa and Imbituva.

Treatment a	Status								Supremo
	Ponta Grossa								Imbituva
	Root dry weight		Shoot dry weight		NDVI		Yield		Root dry weight		Shoot dry weight		NDVI		Yield
	(g)		(g)		(N)		(kg.ha-1)		(g)		(g)		(N)		(kg.ha-1)
Non-inoculated	148	a	263	b	76	a	8028	b	2228	a	248	b	81	a	5865	b
Non-inoculated + Polymer	185	a	217	b	77	a	8230	b	188	b	202	b	82	a	5635	b
Ab 2x108 CFU.mL-1	231	a	285	b	80	a	9178	a	3149	a	350	a	81	a	6463	a
Bs 5x104 CFU.mL-1	200	a	287	b	77	a	7981	b	239	a	263	b	82	a	7224	a
Bs 5x105 CFU.mL-1	142	a	351	a	77	a	10136	a	145	b	251	b	82	a	6582	a
Bs 5x106 CFU.mL-1	161	a	314	a	77	a	9644	a	245	a	263	b	82	a	6927	a
Bs 5x107 CFU.mL-1	183	a	275	b	79	a	8978	a	245	a	290	b	83	a	7028	a
CV (%)	29		14		3		7		23		12		2		12

• ^a
  Means (n = 4) followed by different letters on the same column are significantly different (p ≤ 0.05, Scott-Knott test). Legend: Ab=Azospirillum brasilense, Bs=Bacillus simplex, CV=coefficient of variation.

Table 4
Mean values of root dry weight, shoot dry weight, NDVI, and yield used to assess the efficacy of corn seed treatment with Bacilllus simplex and Azospirillum brasiliense in field experiments with hybrid Supremo in Lapa and Dourados.

Treatment a	Supremo								Supremo
	Lapa								Dourados
	Root dry weight		Shoot dry weight		NDVI		Yield		Root dry weight		Shoot dry weight		NDVI		Yield
	(g)		(g)		(N)		(kg.ha-1)		(g)		(g)		(N)		(kg.ha-1)
Non-inoculated	230	a	253	a	81	b	5979	b	109	b	109	b	80	d	7312	b
Non-inoculated + Polymer	222	a	231	a	82	b	6214	b	110	b	111	b	80	d	6964	b
Ab 2x108 CFU.mL-1	226	a	258	a	84	a	6827	a	131	a	124	b	83	c	6906	b
Bs 5x104 CFU.mL-1	231	a	258	a	83	a	7399	a	112	b	109	b	84	b	6296	b
Bs 5x105 CFU.mL-1	257	a	268	a	83	a	6436	b	121	b	125	b	83	b	7556	b
Bs 5x106 CFU.mL-1	250	a	278	a	84	a	6792	a	145	a	121	b	85	a	7299	b
Bs 5x107 CFU.mL-1	276	a	287	a	84	a	7284	a	151	a	144	a	85	a	9043	a
CV (%)	15		11		1		7		10		9		1		10

• ^a
  Means (n = 4) followed by different letters on the same column are significantly different (p ≤ 0.05, Scott-Knott test). Legend: Ab=Azospirillum brasilense, Bs=Bacillus simplex, CV=coefficient of variation.

In soybean, B. simplex triggered a significant yield increase at most locations (Tables 5 and 6). In Imbituva, yield increased by up to 16% (7 bags.ha^-1) (Table 5); in Uberlândia by up to 16% (7 bags.ha^-1) (Table 5); in Lapa by up to 22% (8 bags.ha^-1); and in Dourados there was no significant difference in terms of yield between any of the B. simplex concentrations and the non-inoculated controls (Table 6). For the remaining response variables, significant differences were only observed for some of the B. simplex concentrations for shoot dry weight in Dourados (Table 6) and for NDVI in Imbituva (Table 5) and Dourados (Table 6). For the A. brasilense treatment, yield increase was equal to B. simplex in Lapa (Table 6), or yield was significantly lower as in Imbituva and Uberlândia (Table 5). There was no difference between the A. brasilense treatment, the B. simplex treatments, and the non-inoculated controls in Dourados (Table 6).

Table 5
Mean values of root dry weight, shoot dry weight, NDVI, and yield used to assess the efficacy of soybean seed treatment with Bacilllus simplex and Azospirillum brasiliense in field experiments with cultivar BMX in Imbituva and Uberlândia.

Treatment a	BMX								BMX
	Imbituva								Uberlândia
	Root dry weight		Shoot dry weight		NDVI		Yield		Root dry weight		Shoot dry weight		NDVI		Yield
	(g)		(g)		(N)		(kg.ha-1)		(g)		(g)		(N)		(kg.ha-1)
Non-inoculated	3	a	31	a	81	c	2562	b	4	a	30	a	83	a	2643	b
Non-inoculated + Polymer	3	a	31	a	79	d	2351	b	4	a	31	a	82	a	2717	b
Ab 2x108 CFU.mL-1	2	a	33	a	81	c	2581	b	3	a	26	a	82	a	2655	b
Bs 1x104 CFU.mL-1	3	a	36	a	79	d	2528	b	4	a	30	a	83	a	2946	a
Bs 1x105 CFU.mL-1	3	a	40	a	84	b	2859	a	4	a	30	a	84	a	3079	a
Bs 1x106 CFU.mL-1	3	a	37	a	86	a	2832	a	4	a	31	a	82	a	2933	a
Bs 1x107 CFU.mL-1	3	a	35	a	86	a	2985	a	4	a	31	a	83	a	2846	a
CV (%)	15		17		1		11		18		16		1		7

• ^a
  Means (n = 4) followed by different letters on the same column are significantly different (p ≤ 0.05, Scott-Knott test). Legend: Ab=Azospirillum brasilense, Bs=Bacillus simplex, CV=coefficient of variation.

Table 6
Mean values of root dry weight, shoot dry weight, NDVI, and yield used to assess the efficacy of soybean seed treatment with Bacillus simplex and Azospirillum brasiliense in field experiments with cultivar Monsoy in Lapa and Dourados.

Treatment a	Monsoy								Monsoy
	Lapa								Dourados
	Root dry weight		Shoot dry weight		NDVI		Yield		Root dry weight		Shoot dry weight		NDVI		Yield
	(g)		(g)		(N)		(kg.ha-1)		(g)		(g)		(N)		(kg ha-1)
Non-inoculated	3	a	38	a	85	b	2317	b	4	a	5	b	72	d	1306	a
Non-inoculated + Polymer	3	a	29	a	85	b	2291	b	4	a	5	b	73	c	1315	a
Ab 2x108 CFU.mL-1	3	a	38	a	88	a	2677	a	4	a	6	b	74	c	1193	a
Bs 1x104 CFU.mL-1	3	a	31	a	87	a	2819	a	4	a	7	a	75	b	1373	a
Bs 1x105 CFU.mL-1	3	a	43	a	87	a	2660	a	4	a	7	a	75	b	1363	a
Bs 1x106 CFU.mL-1	3	a	33	a	87	a	2684	a	4	a	7	a	76	a	1419	a
Bs 1x107 CFU.mL-1	3	a	43	a	88	a	2665	a	5	a	8	a	78	a	1431	a
CV (%)	12		20		1		9		17		6		1		8

• ^a
  Means (n = 4) followed by different letters on the same column are significantly different (p ≤ 0.05, Scott-Knott test). Legend: Ab=Azospirillum brasilense, Bs=Bacillus simplex, CV=coefficient of variation.

Yield increase was observed in both corn and soybean at B. simplex strain SYM00260 inoculum concentrations of 5x10⁴ CFU.mL^-1 or above except in Dourados for soybean, demonstrating that B. simplex is stable and effective even at lower concentrations across a range of different environmental conditions. Higher B. simplex inoculum concentrations may be beneficial under high-stress situations such as those encountered in Dourados, where overall yield was low for soybean and corn crops, likely due to a combination of late planting and lower rainfall in the final grain filling phase. Even so, corn yield in Dourados for the B. simplex treatment was significantly increased by up to 24% or 1731 kg.ha^-1.

The impact of Bacillus simplex on corn and soybean root and shoot dry weight and NDVI was also investigated, but none of which was consistently impacted by the B. simplex treatments. Root and shoot dry weight, and NDVI, are commonly evaluated because they reflect plant vigor tied to higher photosynthetic activity, potentially translating into increased yield. The increase in the green area assessed by NDVI was significant for some B. simplex inoculum concentrations in five of the eight locations. Shoot dry weight was also significantly elevated at three of the eight locations for certain concentrations. Root dry weight increased significantly at the highest B. simplex inoculum concentration in Dourados. This shows that under certain situations, Bacillus simplex, in addition to yield, can also positively impact other key traits. The fact that simultaneous positive impact on all investigated traits was inconsistent, has been found with other inoculants (Hassen and Labuschagne, 2010).

Azospirillum in Brazil has been used as seed inoculant to increase nitrogen fixation in soybean together with Bradyrhizobium (Hungria et al., 2015) or without Bradyrhizobium in corn. The yield increase of the B. simplex inoculant was similar to the A. brasilense product, except for corn in Dourados, and soybean in Imbituva and Uberlândia, where only the B. simplex inoculant resulted in a significant yield increase.

The BIOCROMA polymer treatment did, in general, have no effect as compared to the untreated control. This was expected, as polymers are designed not to impact seed treatments or germination (Afzal et al., 2020).

Beneficial impacts on plants by Bacillus and related genera are well documented in the literature. In corn, Lima et al. (2011) observed a higher yield and overall improved plant development upon the use of B. subtilis. In soybean, Tavanti et al. (2019); and Araújo and Hungria (1999) reported increased yield using soybean seeds treated with B. subtilis. Paenibacillus strains isolated from the wheat rhizosphere and applied to soybean, maize, and wheat, showed significant increases in crop growth compared to the control (Akinrinlola et al., 2018). The application of Bacillus-based products has many benefits for plant growth and contributes to increasing crop yields and soil fertility (García-Fraile et al., 2015). Bacillus species can convert the complex form of essential nutrients, such as phosphorus and nitrogen in the soil, to available forms that can be taken up by plant roots (Kuan et al., 2016). Some species have the nifH gene and produce nitrogenase, which can fix atmospheric nitrogen and make it available to plants, thereby enhancing plant growth and yield (Ding et al., 2005; Szilagyi-Zecchin et al., 2014).

In this work, before B. simplex inoculation, standard chemical treatments were applied to corn and soybean seed, and a Bradyrhizobium inoculant to soybean seed, as is common in Brazil. It has been shown that the co-inoculation of compatible microorganisms can benefit Bradyrhizobium nodulation by increasing nitrogen fixation (Santos et al., 2019). Bacillus species are compatible with other beneficial microorganisms when applied as plant growth promoters to plants. Wu et al. (2005) reported improved growth and uptake of nitrogen, phosphorus, and potassium by corn plants when inoculated with B. megaterium and B. mucilaginosus. Schwartz et al. (2013) noticed a change in pea root architecture, nodule, and nodule size when co-inoculating B. simplex and Rhizobium leguminosarum. Bacillus subtilis and Bradyrhizobium co-inoculation also increased germination rate, plant height, dry root weight, and the number of nodules in soybean (Petkar et al., 2018). Azospirillum species in Brazil have been used as co-inoculants with Bradyrhizobium species in soybean (Hungria et al., 2015) or on their own in corn, aiming to increase nitrogen fixation.

The standard chemical base treatments applied to the seed did not appear to negatively affect B. simplex. This shows that the integration of B. simplex into the prevailing seed treatment regimen can be done easily for additional productivity gain.

CONCLUSIONS

The three methods are suitable for quantifying Bacillus simplex colony-forming units (CFU) in the inoculant product. In field trials, the B. simplex inoculant increased corn yield by up to 26% corresponding to an additional 2100 kg.ha^-1. Soybean yield was increased up to 22% or 500 kg.ha^-1. The B. simplex inoculant had no consistent effect on shoot and shoot weight, and NDVI. Yield increase of the B. simplex and Azospirillum brasilense inoculants was comparable. The polymer seed treatment had no impact on yield.

ACKNOWLEDGMENTS

We want to thank reviewers for valuable suggestions and Zhanshan Dong for help with statistical analyses of the Bacillus simplex quantification data.

REFERENCES

• AFZAL I., JAVED T., AMIRKHANI M., TAYLOR A.G. Modern seed technology: seed coating delivery systems for enhancing seed and crop performance. Agriculture, v.10, n.526, p1-20, 2020. https://doi.org/10.3390/agriculture10110526
  » https://doi.org/10.3390/agriculture10110526
• AKHTAR, S.S.; AMBY, D.B.; HEGELUND, J.N.; FIMOGNARI, L.; GROßKINSKY, D.K.; WESTERGAARD, J.C.; MÜLLER, R.; MOELBAK, L.; LIU, F.; ROITSCH, T. Bacillus licheniformis FMCH001 increases water use efficiency via growth stimulation in both normal and drought conditions. Frontiers in Plant Science, v.11, p.297, 2020. https://www.frontiersin.org/articles/10.3389/fpls.2020.00297/full
  » https://www.frontiersin.org/articles/10.3389/fpls.2020.00297/full
• AKINRINLOLA, R.J.; YUEN, G.Y.; DRIJBER, R.A.; ADESEMOYE, A. O. Evaluation of Bacillus strains for plant growth promotion and predictability of efficacy by in vitro physiological traits. International Journal of Microbiology, v.2018, p.5686874. 2018. https://www.hindawi.com/journals/ijmicro/2018/5686874/
  » https://www.hindawi.com/journals/ijmicro/2018/5686874/
• ARAÚJO, F.F.; HUNGRIA, M. Nodulação e rendimento de soja co-infectada com Bacillus subtilis e Bradyrhizobium japonicum / Bradyrhizobium elkanii Pesquisa Agropecuária Brasileira, v.34, n.9, p.1633-43, 1999. https://doi.org/10.1590/S0100-204X1999000900014
  » https://doi.org/10.1590/S0100-204X1999000900014
• BARNEIX, A.; SAUBIDET, M.; FATTA, N.; KADE, M. Effect of rhizobacteria on growth and grain protein in wheat. Agronomy for Sustainable Development, v.25, n.4, p.505-511, 2005. https://www.agronomy-journal.org/articles/agro/abs/2005/04/a5022/a5022.html
  » https://www.agronomy-journal.org/articles/agro/abs/2005/04/a5022/a5022.html
• CAMPO, R.J.; ARAUJO, R.S.; HUNGRIA, M. Nitrogen fixation with the soybean crop in Brazil: compatibility between seed treatment with fungicides and bradyrhizobial inoculants. Symbiosis, v.48, n.1, p.154-163, 2009. https://link.springer.com/article/10.1007/BF03179994
  » https://link.springer.com/article/10.1007/BF03179994
• CANTERI, M.G.; ALTHAUS, R.A.; DAS VIRGENS FILHO, J.S.; GIGLIOTI, E.; GODOY, C.V. SASM-AGRI-Sistema para análise e separação de médias em experimentos agrícolas pelos métodos Scott-Knott, Tukey e Duncan. Revista Brasileira de Agrocomputação, v.1, n.2, p.18-24, 2001. https://www.alice.cnptia.embrapa.br/bitstream/doc/512901/1/SASMAGRI.pdf
  » https://www.alice.cnptia.embrapa.br/bitstream/doc/512901/1/SASMAGRI.pdf
• CAVAGLIERI, L.; ORLANDO, J.; ETCHEVERRY, M. Rhizosphere microbial community structure at different maize plant growth stages and root locations. Microbiological Research, v.164, n.4, p.391-399, 2009. https://www.sciencedirect.com/science/article/pii/S0944501307000596
  » https://www.sciencedirect.com/science/article/pii/S0944501307000596
• CHOUDHARY, D.K.; JOHRI, B.N. Interactions of Bacillus spp. and plants-with special reference to induced systemic resistance (ISR). Microbiological Research , v.164, n.5, p.493-513, 2009. https://www.sciencedirect.com/science/article/pii/S0944501308000566
  » https://www.sciencedirect.com/science/article/pii/S0944501308000566
• DING, Y.; WANG, J.; LIU, Y.; CHEN, S. Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region. Journal of Applied Microbiology, v.99, n.5, p.1271-1281, 2005. https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2672.2005.02738.x
  » https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2672.2005.02738.x
• ELKOCA, E.; KANTAR, F.; SAHIN, F. Influence of nitrogen fixing and phosphorus solubilizing bacteria on the nodulation, plant growth, and yield of chickpea. Journal of Plant Nutrition, v.31, n.1, p.157-171, 2007. https://www.tandfonline.com/doi/abs/10.1080/01904160701742097
  » https://www.tandfonline.com/doi/abs/10.1080/01904160701742097
• EMBRAPA. Empresa Brasileira de Pesquisa Agropecuária. Metodologia para o controle de qualidade de produtos biológicos a base de Bacillus In: Quantificação e Identificação de Bacillus subtilis e Bacillus licheniformis: Curso Teórico Pratico: Embrapa Meio Ambiente 25 e 26 de abril de 2012. https://www.cnpma.embrapa.br/down_site/forum/2012/bacillus/ApostilaCursoBacillus2012.pdf
  » https://www.cnpma.embrapa.br/down_site/forum/2012/bacillus/ApostilaCursoBacillus2012.pdf
• ETESAMI, H.; MAHESHWARI, D.K. Use of plant growth promoting rhizobacteria (PGPRs) with multiple plant growth promoting traits in stress agriculture: Action mechanisms and future prospects. Ecotoxicology and Environmental Safety, v.156, p.225-246, 2018. https://www.sciencedirect.com/science/article/abs/pii/S0147651318301921
  » https://www.sciencedirect.com/science/article/abs/pii/S0147651318301921
• GARCÍA-FRAILE, P.; MENÉNDEZ, E.; RIVAS, R. Role of bacterial biofertilizers in agriculture and forestry. AIMS Bioengineering, v.2, n.3, p.183-205, 2015. http://www.aimspress.com/article/10.3934/bioeng.2015.3.183
  » http://www.aimspress.com/article/10.3934/bioeng.2015.3.183
• GOWTHAM, H.; SINGH, B.; MURALI, M.; SHILPA, N.; PRASAD, M.; AIYAZ, M.; AMRUTHESH, K.; NIRANJANA, S. Induction of drought tolerance in tomato upon the application of ACC deaminase producing plant growth promoting rhizobacterium Bacillus subtilis Rhizo SF 48. Microbiological Research , v.234, p.126422. 2020. https://www.sciencedirect.com/science/article/pii/S0944501319309048
  » https://www.sciencedirect.com/science/article/pii/S0944501319309048
• HASHEM, A.; TABASSUM, B.; ABD_ALLAH, E.F. Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi Journal of Biological Sciences, v.26, n.6, p.1291-1297, 2019. https://www.sciencedirect.com/science/article/pii/S1319562X19300890
  » https://www.sciencedirect.com/science/article/pii/S1319562X19300890
• HASSEN, A.I.; LABUSCHAGNE, N. Root colonization and growth enhancement in wheat and tomato by rhizobacteria isolated from the rhizoplane of grasses. World Journal of Microbiology and Biotechnology, v.26, n.10, p.1837-1846, 2010. https://link.springer.com/article/10.1007/s11274-010-0365-z
  » https://link.springer.com/article/10.1007/s11274-010-0365-z
• HUNGRIA, M.; NOGUEIRA, M.A.; ARAUJO, R.S. Soybean seed co-inoculation with Bradyrhizobium spp. and Azospirillum brasilense: a new biotechnological tool to improve yield and sustainability. American Journal of Plant Sciences, v.6, p.811-817, 2015. https://www.alice.cnptia.embrapa.br/handle/doc/1012889
  » https://www.alice.cnptia.embrapa.br/handle/doc/1012889
• IBANHES-NETO, H.F; SILVA, A.C.; SUMIDA C.H.; GOUVEIA, M. S.; PELLIZZARO V.; TAKAHASHI L.S.A. Physiological potential of green bean seeds treated with Bacillus subtilis Journal of Seed Science, v.43, e202143016, 2021. https://www.scielo.br/j/jss/a/jssz7xBgvdm47SzCzBqbp9v/
  » https://www.scielo.br/j/jss/a/jssz7xBgvdm47SzCzBqbp9v/
• KIM, M.J.; RADHAKRISHNAN, R.; KANG, S.M.; YOU, Y.-H.; JEONG, E.-J.; KIM, J.-G.; LEE, I.-J. Plant growth promoting effect of Bacillus amyloliquefaciens, H-2-5 on crop plants and influence on physiological changes in soybean under soil salinity. Physiology and Molecular Biology of Plants, v.23, n.3, p.571-580. 2017. https://link.springer.com/article/10.1007/s12298-017-0449-4
  » https://link.springer.com/article/10.1007/s12298-017-0449-4
• KOPITTKE, P.M.; MENZIES, N.W.; WANG, P.; MCKENNA, B.A.; LOMBI, E. Soil and the intensification of agriculture for global food security. Environment International, v.132, p.105078. 2019. https://www.sciencedirect.com/science/article/pii/S0160412019315855
  » https://www.sciencedirect.com/science/article/pii/S0160412019315855
• KUAN, K.B.; OTHMAN, R.; ABDUL RAHIM, K.; SHAMSUDDIN, Z.H. Plant growth-promoting rhizobacteria inoculation to enhance vegetative growth, nitrogen fixation and nitrogen remobilisation of maize under greenhouse conditions. PloS ONE, v.11, n.3, p.e0152478, 2016. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152478
  » https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152478
• KUSHWAHA, P.; KASHYAP, P.L.; SRIVASTAVA, A.K.; TIWARI, R.K. Plant growth promoting and antifungal activity in endophytic Bacillus strains from pearl millet (Pennisetum glaucum). Brazilian Journal of Microbiology, v.51, n.1, p.229-241. 2020. https://link.springer.com/article/10.1007/s42770-019-00172-5
  » https://link.springer.com/article/10.1007/s42770-019-00172-5
• KUZNETSOVA, A.; BROCKHOFF, P.B.; CHRISTENSEN, R.H. lmerTest package: tests in linear mixed effects models. Journal of Statistical Software, v.82, n.1, p.1-26, 2017. http://statistik-jstat.uibk.ac.at/article/view/v082i13
  » http://statistik-jstat.uibk.ac.at/article/view/v082i13
• LEE, S.W.; LEE, S.H.; BALARAJU, K.; PARK, K.S.; NAM, K.-W.; PARK, J.-W.; PARK, K. Growth promotion and induced disease suppression of four vegetable crops by a selected plant growth-promoting rhizobacteria (PGPR) strain Bacillus subtilis 21-1 under two different soil conditions. Acta Physiologiae Plantarum, v.36, n.6, p.1353-1362, 2014. https://link.springer.com/article/10.1007/s11738-014-1514-z
  » https://link.springer.com/article/10.1007/s11738-014-1514-z
• LIMA, F.F.; NUNES, L.A.; DO VB FIGUEIREDO, M.; ARAÚJO, F.F.; LIMA, L.M.; ARAÚJO, A.S. Bacillus subtilis e adubação nitrogenada na produtividade do milho. Brasileira de Ciências Agrárias, v.6, n.4, p.657-661. 2011. https://www.redalyc.org/pdf/1190/119021237016.pdf
  » https://www.redalyc.org/pdf/1190/119021237016.pdf
• LUGTENBERG, B.; KAMILOVA, F. Plant-growth-promoting rhizobacteria. Annu Rev Microbiol v.63, p.541-556. 2009. https://www.annualreviews.org/doi/pdf/10.1146/annurev.micro.62.081307.162918
  » https://www.annualreviews.org/doi/pdf/10.1146/annurev.micro.62.081307.162918
• MAPA. Ministério da Agricultura, Pecuária e Abastecimento. Instrução Normativa N ^o 30, de 12/11/2010 http://sistemasweb.agricultura.gov.br/sislegis/loginAction.do?method=exibirTela
  » http://sistemasweb.agricultura.gov.br/sislegis/loginAction.do?method=exibirTela
• MARTÍNEZ-VIVEROS, O.; JORQUERA, M.; CROWLEY, D.; GAJARDO, G.; MORA, M. Mechanisms and practical considerations involved in plant growth promotion by rhizobacteria. Journal of Soil Science and Plant Nutrition, v.10, n.3, p.293-319. 2010.
• MCQUILKEN, M.P.; HALMER, P.; RHODES, D.J. Application of microorganisms to seeds. In: Formulation of Microbial Biopesticides Springer. 1998. p.255-285.
• NGALIMAT, M.S.; YAHAYA, R.S.R.; BAHARUDIN, M.M.A.; YAMINUDIN, S.M.; KARIM, M.; AHMAD, S.A.; SABRI, S. A review on the biotechnological applications of the operational group Bacillus amyloliquefaciens Microorganisms, v.9, n.3, p.614, 2021. https://www.mdpi.com/2076-2607/9/3/614
  » https://www.mdpi.com/2076-2607/9/3/614
• NUNES, J.C. Tratamento de semente - qualidade e fatores que podem afetar a sua performance em laboratório. Syngenta Proteção de Cultivos Ltda, 2005. 16p.
• PANDEY, A.; TRIPATHI, A.; SRIVASTAVA, P.; CHOUDHARY, K.K.; DIKSHIT, A. Plant growth-promoting microorganisms in sustainable agriculture In Role of Plant Growth Promoting Microorganisms In: Sustainable Agriculture and Nanotechnology Woodhead Publishing. 2019. p.1-19.
• PARTE, A.C.; CARBASSE, J.S.; MEIER-KOLTHOFF, J.P.; REIMER, L.C.; GÖKER, M. List of prokaryotic names with standing in nomenclature (LPSN) moves to the DSMZ. International Journal of Systematic and Evolutionary Microbiology, v.70, n.11, p.5607, 2020. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723251/
  » https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723251/
• PETKAR, V.; DESHMUKH, T.; JADHAV, A. Effect of dual inoculation of Bacillus subtilis and Bradyrhizobium japonicum on growth parameters of soybean (Glycine max L.). International Journal of Current Microbiology and Applied Sciences, v.7, p.563-567, 2018. https://www.ijcmas.com/7-10-2018/V.V.%20Petkar,%20et%20al.pdf
  » https://www.ijcmas.com/7-10-2018/V.V.%20Petkar,%20et%20al.pdf
• R CORE TEAM. R: A language and environment for statistical computing R Foundation for Statistical Computing, Vienna, Austria. https://www.r-project.org/
  » https://www.r-project.org/
• ROSENBERG, G.; STEINBERG, N.; OPPENHEIMER-SHAANAN, Y.; OLENDER, T.; DORON, S.; BEN-ARI, J.; SIROTA-MADI, A.; BLOOM-ACKERMANN, Z.; KOLODKIN-GAL, I. Not so simple, not so subtle: the interspecies competition between Bacillus simplex and Bacillus subtilis and its impact on the evolution of biofilms. NPJ Biofilms and Microbiomes v.2, n.1, p.1-11, 2016. https://www.nature.com/articles/npjbiofilms201527
  » https://www.nature.com/articles/npjbiofilms201527
• SANTOS, M.S.; NOGUEIRA, M.A.; HUNGRIA, M. Microbial inoculants: reviewing the past, discussing the present and previewing an outstanding future for the use of beneficial bacteria in agriculture. AMB Express, v.9, n.1, p.1-22, 2019. https://link.springer.com/article/10.1186/s13568-019-0932-0
  » https://link.springer.com/article/10.1186/s13568-019-0932-0
• SANTOS, M.S.; NOGUEIRA, M.A.; HUNGRIA, M. Outstanding impact of Azospirillum brasilense strains Ab-V5 and Ab-V6 on the Brazilian agriculture: Lessons that farmers are receptive to adopt new microbial inoculants. Revista Brasileira de Ciência do Solo, v.45, 2021. https://www.scielo.br/j/rbcs/a/CdtxRfpzKD8Y7DxBmXQfYgf/abstract/?lang=en
  » https://www.scielo.br/j/rbcs/a/CdtxRfpzKD8Y7DxBmXQfYgf/abstract/?lang=en
• SANTOYO, G.; MORENO-HAGELSIEB, G.; DEL CARMEN OROZCO-MOSQUEDA, M.; GLICK, B.R. Plant growth-promoting bacterial endophytes. Microbiological Research , v.183, p.92-99, 2016. https://www.sciencedirect.com/science/article/pii/S094450131530029X
  » https://www.sciencedirect.com/science/article/pii/S094450131530029X
• SHISLER, D.; SLININGER, P.; BEHLE, R.; JACKSON, M. Formulation of Bacillus spp. for biological control of plant diseases. Phytopathology, v.94, n.11, p.1267-1271, 2004. https://apsjournals.apsnet.org/doi/epdf/10.1094/PHYTO.2004.94.11.1267
  » https://apsjournals.apsnet.org/doi/epdf/10.1094/PHYTO.2004.94.11.1267
• SCHÖNBICHLER, A.; DÍAZ-MORENO, S.M.; SRIVASTAVA, V.; MCKEE, L.S. Exploring the potential for fungal antagonism and cell wall attack by Bacillus subtilis natto Frontiers in Microbiology, v.11, p.521, 2020. https://www.frontiersin.org/articles/10.3389/fmicb.2020.00521/full
  » https://www.frontiersin.org/articles/10.3389/fmicb.2020.00521/full
• SCHWARTZ, A.R.; ORTIZ, I.; MAYMON, M.; HERBOLD, C.W.; FUJISHIGE, N.A.; VIJANDERAN, J.A.; VILLELLA, W.; HANAMOTO, K.; DIENER, A.; SANDERS, E.R. Bacillus simplex-a little known PGPB with anti-fungal activity-alters pea legume root architecture and nodule morphology when coinoculated with Rhizobium leguminosarum bv. viciae Agronomy, v.3, n.4, p.595-620, 2013. https://www.mdpi.com/2073-4395/3/4/595
  » https://www.mdpi.com/2073-4395/3/4/595
• SZILAGYI-ZECCHIN, V.J.; IKEDA, A.C.; HUNGRIA, M.; ADAMOSKI, D.; KAVA-CORDEIRO, V.; GLIENKE, C.; GALLI-TERASAWA, L.V. Identification and characterization of endophytic bacteria from corn (Zea mays L.) roots with biotechnological potential in agriculture. AMB Express , v.4, n.1, p.1-9, 2014. https://amb-express.springeropen.com/articles/10.1186/s13568-014-0026-y
  » https://amb-express.springeropen.com/articles/10.1186/s13568-014-0026-y
• TAVANTI, T.R.; TAVANTI, R.F.; GALINDO, F.S.; SIMÕES, I.; DAMETO, L.S.; SÁ, M.E.DE. Yield and quality of soybean seeds inoculated with Bacillus subtilis strains. Revista Brasileira de Engenharia Agrícola e Ambiental, v.24, p.65-71, 2019. https://www.scielo.br/j/rbeaa/a/BM7r9xKwmfx8h4zr6t7qDWF/?lang=en
  » https://www.scielo.br/j/rbeaa/a/BM7r9xKwmfx8h4zr6t7qDWF/?lang=en
• TEIXEIRA, E.I.; FISCHER, G.; VAN VELTHUIZEN, H.; WALTER, C.; EWERT, F. Global hot-spots of heat stress on agricultural crops due to climate change. Agricultural and Forest Meteorology, v.170, p.206-215, 2013. https://www.sciencedirect.com/science/article/abs/pii/S0168192311002784
  » https://www.sciencedirect.com/science/article/abs/pii/S0168192311002784
• TIWARI, S.; PRASAD, V.; LATA, C. Bacillus: Plant growth promoting bacteria for sustainable agriculture and environment. In New and Future Developments in Microbial Biotechnology and Bioengineering Elsevier. 2019. p.43-55. https://www.sciencedirect.com/science/article/pii/B9780444641915000031
  » https://www.sciencedirect.com/science/article/pii/B9780444641915000031
• VIEIRA, F.C.S.; NAHAS, E. Quantificação de bactérias totais e esporuladas no solo. Scientia Agricola, v.57, p.539-545, 2000. https://www.scielo.br/j/sa/a/tSZxyXp7MVR9mY65Nq7zfGz/?format=pdf⟨=pt
  » https://www.scielo.br/j/sa/a/tSZxyXp7MVR9mY65Nq7zfGz/?format=pdf⟨=pt
• WITKOWICZ, R.; SKRZYPEK, E.; GLEŃ-KAROLCZYK, K.; KRUPA, M.; BIEL, W.; CHŁOPICKA, J.; GALANTY, A. Effects of application of plant growth promoters, biological control agents and microbial soil additives on photosynthetic efficiency, canopy vegetation indices and yield of common buckwheat (Fagopyrum esculentum Moench). Biological Agriculture & Horticulture, v.37, n.4, p.234-251, 2021. https://www.tandfonline.com/doi/abs/10.1080/01448765.2021.1918579
  » https://www.tandfonline.com/doi/abs/10.1080/01448765.2021.1918579
• WU, S.; CAO, Z.; LI, Z.; CHEUNG, K.; WONG, M.H. Effects of biofertilizer containing N-fixer, P and K solubilizers and AM fungi on maize growth: a greenhouse trial. Geoderma, v.125, n.1-2, p.155-166, 2005. https://www.sciencedirect.com/science/article/abs/pii/S0016706104001922
  » https://www.sciencedirect.com/science/article/abs/pii/S0016706104001922

Publication Dates

• Publication in this collection
  14 Oct 2022
• Date of issue
  2022

History

• Received
  22 Apr 2022
• Accepted
  28 Aug 2022