Seed treatment with Bacillus subtilis or indol butyric acid: germination and early development of bean seedlings/Tratamiento de semillas con Bacillus subtilis o acido indol-butirico: germinacion y crecimiento inicial de plantulas de frijol/Tratamento de sementes com Bacillus subtilis ou acido indol-butirico: germinacao e crescimento inicial de plantulas de feijoeiro.
Microrganisms are extremely important in the control of their own environment and in affecting the plant metabolism in a complex way (Bloemberg and Lugtenberg, 2001). The vast majority of the works about plant growth-promoting rhizobacteria (PGPR), consider these phenomena as due to a indirect effect associated with biological control of secondary pathogens (Coelho et al., 2007; Ashrafuzzaman et al., 2009). However, aspects as germination and seedling growth are affected by PGPR (Kloepper et al., 2004) even in stress conditions such as salinity (Mishra et al., 2010, Naz et al., 2009) or low temperature (Khan and Patel, 2007). PGPR also act in the plant growth by affecting nutrient absorption, which implies in a more rational use of fertilizers (Adesemoye et al. 2008; Vessey, 2003).
According to Yasdani et al. (2009) PGPR use and phosphorus solubilizer bacteria may be responsible for an annual decrease up to 50% in fertilizers use, without affecting growth and corn production. For Ashrafuzzaman et al. (2009) the use of PGPR was efficient to improve the germination and rice growth, due to the auxin production increment and making the phosphorus more soluble, which in crops of high expressive importance for human feeding may be a new approach.
Plant growth regulators are substances that in low concentrations are able to affect the plant physiological processes. Their production as secondary microorganism metabolites, in the soil, is directly linked to substrates, including plant exudates and other residues, which may be affected by environmental conditions as salinity and oxygen concentration (Radwan et al., 2005; Ashrafuzzaman et al., 2009).
In some works (Khalid et al., 2004; Radwan et al., 2005) it was clear that plant growth increase by use of PGPR was related to the production of auxins, which are responsible for cellular growth, shoot and root elongation, fruit development, apical dominance and abscission delay (Taiz and Zeiger, 2004). Among the auxins, indol acetic acid (IAA) is the most studied one, and is also produced by PGPR (Khalid et al., 2004; Khan and Patel, 2007; Ashrafuzzaman et al., 2009; Naz et al., 2009; Calvo et al. 2010; Mishra et al., 2010). Bacillus subtilis produced IAA and IBA (indol butiric acid) as a response to soybean exudates (Araujo et al., 2005) and increased growth in soybean, corn and cotton (Araujo, 2008); in this case, seed inoculation with PGPR exhibited the advantage of an interaction with the plant along its whole cycle.
Common beans (Phaseolus vulgaris L.), a highly consumed grain in Brazil, is normally grown by low yield farmers, with low input of fertilizers and pesticides, witch leads to low productivity. Brazil, though, produces around 3,3 x [10.sup.6] tons of common beans, with a mean yield of 882.6kg x [ha.sup.-1], being the second largest producer of beans (CONAB, 2010); however, in irrigated areas, it can reach up to 3000kg x [ha.sup.-1]. This high production is directly linked to the fact that beans are a basic food of the population and one of the main sources of protein in the diet (EMBRAPA, 2004).
However, the bean plant is sensitive to water deficit after sowing. Values of -0.15MPa in the soil induce the first symptoms of deficiency in the primary leaves, and at -0.35MPa germination and cell elongation may be drastically reduced. On the other hand, in lab conditions, germination was still observed at a simulated water deficiency of up to -0,9MPa (Machado Neto et al., 2006, Custodio et al, 2009, Coelho et al., 2010). Common bean is cultivated in marginal soils, with fertility problems related to nitrogen and phosphorus deficit (Barbosa Filho et al., 2003) and yield reduction due to sanitary problems (Sartorato et al., 2003), from sowing to harvest. This means increasing agrochemicals use. PGPR should be an alternative for the rational production of this crop, minimizing several impacts of modern technologies on the environment.
The objective of this work was the study bean seed inoculation with Bacillus subtilis and to compare this treatment with the use of increasing doses of indolbutyric acid, during the germination and initial growth of bean seedlings.
Material and Methods
The work consisted of two experiments. The first one was carried out in the laboratory and the second in a greenhouse. The bean seeds used in both experiments were Phaseolus vulgaris cv Perola, a carioca type, with normal cycle, semi erect, indeterminate growth, with a 100 seeds mass of 23-25g (EMBRAPA, 2004). Used seeds were the ones retained in a 6.3mm circular holes sieve.
Immediately before sowing, the seeds were treated with 5ml of indol-butyric acid (IBA) solution (0, 0.35, 0.7, 1.4, 2.8 and 5.6mg x [ml.sup.-1]) per kg of seed for setting up the treatment (0, 1.75, 3.5, 7.0, 14 and 28mg x [kg.sup.-1] seed). IBA was diluted in 3ml ethanol for the highest concentration and the volume completed to 10ml with water. The low concentration solutions were obtained by dilutions with a 30% ethanol solution (Remans et al., 2008). One treatment with B. subtilis, PRBS-1 (accession number AY504952, NCBI; Araujo et al, 2005), in the concentration of [10.sup.9] cells/g as recommended by Araujo (2008), was used in the proportion of 10g inoculant per kg of seed. For a higher adhesion seeds were previously wetted with a 10% sucrose solution (Khalid et al., 2004) using a dose of 5ml. Two controls, one treated with 30% ethanol and a second with non treated seeds were used.
In the lab experiment, all treatments were used, with four replications per treatment, containing 50 seeds each, enveloped by three paper sheets, two as base and one as cover. They were rolled and placed in polyethylene bags and wetted with water 2.5 times their weight. Germination was carried out at 25[degrees]C, and evaluated at seven days after sowing, counting normal, strong, weak and abnormal seedlings as well as dead seeds (BRASIL, 2009; Nakagawa, 1999). The results were expressed as percentages. Seedling performance was evaluated by a germination test; shoot and root were separated, placed in paper bags and dried at 60[degrees]C for 48h. Dried materials, were let to cool down in desiccators, and weighted in an analytical balance with a precision of 0.001g (Nakagawa, 1999). The relation between shoot and root dry weights was determined.
Based on germination data obtained in the laboratory, an experiment was conducted in the greenhouse, in which soil was conditioned in pots and seeds from the control, AIB treated (7, 14 and 28mg x [kg.sup.-1]) and inoculated with B. subtilis treatments were chosen, based on their lab performance. As there were no differences between seeds treated or not with ethanol (Tables I and II) the latter was omitted. The pots, of 8 liters capacity, were filled with agricultural soil collected in the 0-20cm layer of a Distroferric Red Argisoil (a sandy loam soil). The soil was air dried and passed through a sieve with 2mm mesh. Soil samples were taken for characterizing chemical attributes and granulometry, with the following results: pH (Ca[Cl.sub.2] 1mol[l.sup.-1]) 5.1; organic matter 11g x [dm.sup.-3] or 0,92%; [P.sub.resin] 10mg x [dm.sup.-3] or 10ppm; H+Al 17m[mol.sub.c] x [dm.sup.-3]; K 1,9m[mol.sub.c] x [dm.sup.-3]; Ca 18m[mol.sub.c] x [dm.sup.-3]; Mg 7m[mol.sub.c] x [dm.sup.-3]; SB (sum of bases) 27m[mol.sub.c] x [dm.sup.-3]; CEC (cation exchange capacity) 44 m[mol.sub.c] x [dm.sup.-3]; base saturation 62%; sand 740g x [kg.sup.-1]; silt 80g x [kg.sup.-1]; and clay 180g x [kg.sup.-1]. Field capacity on non structured (sieved) soil was determined at -0.03MPa in the Richards extractor, and the value obtained was 165g x [kg.sup.-1] of water. Dolomitic limestone was added to the sieved soil to elevate its base saturation to 70%. After liming, the soil was maintained in plastic bags for 20 days with moisture content close to field capacity. Four pots with 50 seeds per treatment were used.
Daily counts were made to calculate the maximum percentage of emergence and the emergence speed index (ESI; Nakagawa, 1999) in each treatment. At 18 days after sowing, plants were harvested, washed in a sieve and split into canopy and root. They were dried at 60[degrees]C for 48h to obtain the dry matter of root, shoot and their ratio.
In the laboratory, the experimental design used was completely randomized with eight treatments and four replications. The greenhouse experiment was conducted in a randomized block design with four blocks and five treatments per block. The percentage data were transformed to arcsine [(X/100).sup.1/2]. The F test was applied for variance analysis; when this was significant, polynomial regression for levels of IBA (quantitative treatments) was used to analyze and determine significant equations with lower polynomial degree and higher determination coefficient ([R.sup.2]). Means of all treatments (IBA concentrations and Bacillus use) were compared by Scott-Knott test (p<0.05), which is a method for grouping means, distinguishing results without ambiguity (Bhering et al., 2008) as for example Tukey's test. The SISVAR software was used (Ferreira, 2008).
Results and Discussion
Analysis of variance of the first experiment (Table I) showed that germination, vigor classification, root length, shoot dry mass, root dry mass, total dry mass and root/shoot ratio were significant by the F test for both treatments, either for the qualitative and quantitative data (IBA), indicating the need to compare means in the first case and a regression analysis in the second.
Germination and vigor classification were higher in the seeds inoculated with B. subtilis and treated with the higher auxin concentrations (IBA) in the laboratory test (Table II). This was also observed in rice (Ashrafuzzaman et al., 2009), chickpea (Khan and Patel, 2007, Mishra et al., 2010), soybean (Naz et al., 2009) and a perennial crop (Pinus; Kloepper et al., 2004)
Treatments with B. subtilis and auxin did not increase root and shoot dry mass compared to control (Table III). Root dry mass was higher with the highest dose of IBA, and was not enhanced by inoculation with B. subtilis. However, seed inoculation produced higher seedling total mass, which was more influenced by the shoot than by the root mass (Table III). The root/shoot values were <1 for most of the treatments. The highest and unique value >1 (1.32) resulted from the treatment with auxin at 28mg x [kg.sup.-1] seed. The lowest value (0.21) was observed in the treatment with B. subtilis (Table III). The root/shoot ratio indicates reserve allocation from the cotyledons to the different organs. In this case, auxin (IBA) induced the highest mass transfer to root development at shoot expense at the highest dose, while the bacteria induced greater mass allocation to shoot. According to weber et al. (2000) plant growth provided by diazotrophic bacteria can be attributed mainly to the plant production of growth regulator substances; however, PGPR can change dry matter allocation, root morphology and biomass increase, enabling plants to better exploit soil volume and nutrient absorption (Malik et al., 1997). Although the inoculation with B. subtilis in the lab, on paper substrate, promoted the bean seedling overall development, the ratio root/shoot did not indicate preferential allocation of biomass to root growth.
An analysis of auxin doses (IBA) indicated that seed germination and vigor classification responded to the increase in the concentration of hormone. Each 1mg x [kg.sup.-1] increase in IBA concentration led to an increase of 0.87% and 0.78% in the germination and vigour classification (graphs not shown), according to the equations Y=73.3+0.87x with a determination coefficient [R.sup.2] = 0.7868 for germination and Y=63.8+0.78x, [R.sup.2] of 0.7343, for vigour classification, both significant at 1% by the F Test, with the equation coefficients also significant at 1% by the same test. Root length did not respond to increase in the IBA doses tested (Figure 1a).
Shoot and total dry mass showed the maximum level (shoot maximum growth) in a concentration of 17.81mg x [kg.sup.-1] (Figure 1b) and seedling total dry mass in 27.12mg x [kg.sup.-1] (Figure 1d). Root dry weight indicated that the increase of each 1mg in the growth regulator concentration led to an increase of 0.036g root dry magnitude (Figure 1c). In the root/shoot analysis (Figure 2), the linear regression showed that each 1mg increase in IBA, under laboratory conditions, led to an increase of 0.026 in the R/S, indicating that the IBA is an auxin acting in the rooting (Castro and Alvarenga, 2001).
Analysis of variance of the greenhouse experiment (Table IV) indicated that the variables emergence, emergence speed index, shoot dry mass, root dry mass, total dry matter and root/shoot ratio were significant accordin to the F test for both treatments for the quantitative and qualitative data (IBA), indicating the need for comparison of means in the first case and in the second a regression analysis.
In greenhouse conditions, treatment with B. subtilis showed similar results to the highest concentration of IBA on the emergence speed index and emergence, superior to other treatments (Table V). The emergence of small plants in the control treatment may have been due to limiting factors in the soil, such as the presence of pathogens, since the seeds had not been previously treated with fungicides. Treatments with some organisms may protect the plants by synthesizing compounds that are beneficial to the seedling either by turning the seedling stronger or by secreting molecules that may have adverse effects on the pathogens.
In relation to the shoot dry mass, treatment with B. subtilis and IBA at concentrations of 14 and 28mg x [kg.sup.-1] had similar results, superior to the control. In the evaluation of root dry mass, treatment with the bacterium and the IBA, at 7 and 14mg x [kg.sup.-1] proved to be superior to other treatments. In the plant total dry mass, the control showed lower performance (Table VI). As for the root/ shoot ratio, treatment with B. subtilis promoted a more balanced relationship with the nearest one, indicating proportionality in root growth and shoot. This result differs from that obtained in the laboratory test, in which the biological treatment produced the lowest root/shoot ratio among the treatments (0.21). This could be because the B. subtillis in the soil may have to counterattack other microorganisms, producing less auxin or having it diluted in the soil, which also decreases the amount of auxin instead of promoting root growth. B. subtillis also produces antibiotics what could improve the resistance of the seedlings to soil born pathogens (Araujo et al, 2005).
The regression studies for the concentration of IBA applied to the seeds in the greenhouse test showed that IBA increased the emergence and the emergence speed index of the beans linearly (graphs not shown). For each 1mg x [kg.sup.-1] increase in the concentration of the growth regulator there was an increase of 1.39% in the emergence of beans, according to the equation Y= 28.6 +1.39x, with coefficient of determination [R.sup.2]= 0.8423, and 1.12 at emergence speed index, according to the equation Y= 12.75 +1.24x, [R.sup.2] = 0.9649, being the equations and coefficients significant at 1% by F test.
The IBA treatment induced greater growth of shoots up to a concentration of 24.12mg x [kg.sup.-1] seed, calculated as the maximum (Figure 3a). Root growth occurred up to a maximum at the concentration of 15.37mg x [kg.sup.-1] seed (Figure 3b). The concentration of 17.19mg x [kg.sup.-1] seed of IBA solution was calculated as the maximum for the production of total dry mass (Figure 3c). However, the ratio root/shoot reached a maximum with 13.67mg of auxin per [kg.sup.-1] of seed (Figure 3d).
The effects of plant growth regulators on beans have been demonstrated to be positive for plant development (Vieira and Castro, 2001). In this paper it was noted that seed treatment with synthetic auxin in larger doses provided an increase of germination and plant development in the laboratory and greenhouse. It was also observed that different doses of auxin employed in the treatment modified the root/shoot ratio. This effect is clearer in the quadratic fitting found with the increasing doses of the growth regulator (Figure 3d). It is well known that the effects of auxin in root development can change from negative to positive with increasing doses of growth regulator (Taiz and Zeiger, 2004).
The seed treatment with B. subtilis led to an increase in germination and plant development. This result may be associated with the indirect beneficial effect of rhizobacteria that, besides the direct promotion of growth, also have control effects on plant pathogens (Araujo et al., 2005). The rhizobacteria performance on beans development confirmed what was found by Araujo (2008), who observed gains in developing soybean, corn and cotton when the same bacterial strain was seed innoculated. Lazzaretti and Melo (2005) working with inoculation of B. subtilis in beans, also concluded that the use of rhizobacteria is a promising technique to increase root nodulation and to promote growth of bean plants.
The production of auxin by the same strain of B. subtilis used in this work was proved in the lab, in a study with soybean seeds (Araujo et al., 2005). On the other hand, a survey of PGPR isolated from wheat rhizosphere showed that the amount of indole compounds released by the rhizobacteria in the culture medium under aseptic conditions ranged from 1.8 to 24.8mg x [l.sup.-1] (Khalid et al., 2004). Performance of B. subtilis in this paper was similar to that found by seed treatment with higher doses of plant growth regulator in the shoot dry mass production, emergence and emergence speed index, which could likely prove the production of regulatory growth substances in the interaction with the beans, and in pot conditions the results were more promising than those observed in the laboratory. Probably, in contact with the soil, the bacteria found better conditions for growth and their interaction with bean roots was stronger than in the laboratory, where paper was used as substrate for germination.
Summarizing, B. subtilis was beneficial for crop establishment (emergence and seedling vigor) and also provided increases in plant growth comparable to the higher levels of IBA in this phase, which also influenced the bean germination and proved to be efficient both for shoot and root growth. At higher doses auxin increased, in the lab, shoot growth at the expense of the root system, and lower doses in the field promoted root growth in shoot detriment. The benefits derived from the interaction between bean and B. subtillis can be extended for the whole cycle of the plant. On the other hand, IBA effects were ephemeral and could be observed only when the substance was still present in the seed or seedling.
Bacillus subtilis should be used as bean inoculant as it promotes a better growth in the early plant stages.
Adesemoye AO, Torbert HA, Kloepper JW (2008) Enhanced plant nutrient use efficiency with PGPR and AMF in an integrated nutrient management system. Can. J. Microbiol. 54: 876-886.
Araujo FF (2008) Inoculacao de sementes com Bacillus subtilis, formulado com farinha de ostra e desenvolvimento de milho, soja e algodao. Cienc. Agrotec. 32: 340-346.
Araujo FF, Henning AA, Hungria M (2005) Phytohormones and antibiotics produced by Bacillus subtilis and their effects on seed pathogenic fungi and on soybean root development. World J. Microbiol. Biotechnol. 27:1639-1645.
Ashrafuzzaman M, Hossen FA, Ismail MR, Hoque MA, Islam MZ, Shahidullah SM, Meon S (2009) Efficiency of plant growth-promoting rhizobacteria (PGPR) for the enhancement of rice growth. Afr. J. Biotechnol. 8: 1247-1252.
Barbosa Filho MP, Fageria NK, Silva OF (2003) Calagem e adubacao. In Cultivo do Feijoeiro Comum. EMBRAPA Arroz e Feijao. Goiania, Brazil. http://sistemasdeproducao. cnptia.embrapa.br/FontesHTML/Feijao/CultivodoFeijoeiro/ adubacao.htm (Cons. 01/31/2010).
Bhering LL, Cruz CD, Vasconcelos ES, Ferreira A, Resende Jr MFR (2008) Alternative methodology for Scott-Knott test. Crop Breed. App. Bio technol. 8: 9-16.
Bloemberg GV, Lugtenberg BJJ (2001) Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Curr Opin. Plant Biol. 4: 343-350.
BRASIL (2009) Regras para Analises de Sementes. SNAD/ DNDV/CLAV. Ministerio da Agricultura. Brasilia, Brazil. 399 pp.
Calvo P, Ormeno-Orrillo E, Martinez-Romero E, Zuniga D (2010) Characterization of Bacillus isolates of potato rhizozphere from andean soils of Peru and their potential PGPR characteristics. Braz. J. Microbiol. 41: 899906.
Castro AHF, Alvarenga AA (2001) Influencia do acido indol-3-butirico no crescimento inicial de plantas de confrei (Symphytum officina le L.). Cienc. Agrotec. 25: 96-101.
Coelho DLM, Agostini EAT, Guaberto LM, Machado Neto NB, Custodio CC (2010) Estresse hidrico com diferentes osmoticos em sementes de feijao e expressao diferencial de proteinas durante a germinacao. Acta Scient. Agron. 32: 491-499.
Coelho LF, Freitas SS, Melo AMT, Ambrosano GMB (2007) Inte racao de bacterias fluorescentes do genero Pseudomonas e de Bacillus spp. com a rizosfera de diferentes plantas. Rev. Bras. Cienc. Solo 31: 1413-1420.
CONAB (2010) Acompanhamento da Safra Brasileira: Graos. Safra 2009/2010. Oitavo levantamento. Maio 2010. Companhia Nacional de Abastecimento. Brasilia, Brazil. 45 pp.
Custodio CC, Salomao GR, Machado Neto NB (2009) Estresse hidrico na germinacao e vigor de sementes de feijao submetidas a diferentes solucoes osmoticas. Rev. Cienc. Agr. 40: 617-623.
EMBRAPA (2004) Feijao, Sustento Arrancado. Produgao Agricola Mundial. FAOSTAT.: http:// sistemasdeproducao.cnptia.embrapa.br/FontesHTML/Feijao/ FeijaoIrrigadoNoroesteMG/in dex.htm (Cons. 08/10/2007).
Ferreira DF (2008) SISVAR: um programa para analises e ensino de estatistica. Rev. Symp. 6: 36-41.
Khalid A, Arshad M, Zahir ZA (2004) Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat. J. App. Microbiol. 96: 473-480.
Khan M, Patel CB (2007) Plant growth effect of Bacillus firmus strain NARS1 isolated from central Himalayan region of India on Cicer arietinum at low temperature. Afr. Crop Sci. Conf. Proc. 8: 1179-1181.
Kloepper JW, Ryu C-M, Zhang S (2004) Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathol. 94: 1259-1266.
Lazzaretti E, Melo IS (2005) Influencia de Bacillus subtilis na Promogao de Crescimento de Plantas e Nodulagao de Raizes de Feijoeiro. Boletim de Pesquisa e Desenvolvimento N[degrees] 28. Embrapa Ambiente. Jaguariuna, Brazil. 15 pp.
Machado Neto NB, Custodio CC, Costa PR, Dona FL (2006) Deficiencia hidrica induzida por diferentes agentes osmoticos na germinacao e vigor de sementes de feijao. Rev. Bras. Sem. 28: 142-148.
Malik KA, Bilal R, Mehnaz S, Rasul G, Mirza MS, Ali S (1997) Association of nitrogen fixing, plant-growth-promoting rhizobacteria (PGPR) with kallar grass and rice. Plant and Soil 194: 37-44
Mishra M, Kumar U, Mishra PK, Prakash V (2010) Efficiency of plant growth promoting rhizobacteria for the enhancement of Cicer arietinum L. growth and germination under salinity. Adv. Biol. Res. 4: 92-96.
Nakagawa J (1999) Testes de vigor baseados nos desempenhos das plantulas. In Krzyzanowski FC, Vieira RD, Franca Neto JB (Eds) Vigor de Sementes: Conceitos e Testes. Abrates. Londrina, Brazil. pp. 2.1-2.24.
Naz I, Bano A, Ul-Hassan T (2009) Isolation of phytohormones producing plant growth promoting rhizobacteria from weeds growing in Khewra salt range, Pakistan and their implication in providing salt tolerance to Glycine max L. Afr. J Biotechnol. 8: 5762-6766.
Radwan TEE, Mohamed ZK, Reis VM (2005) Aeracao e adicao de sais na producao de acido indol-acetico por bacterias diazotroficas. Pes. Agropec. Bras. 40: 997-1004.
Remans R, Beebe S, Blair M, Manrique G, Tovar E, Rao I, Croonenborghs A, Torres-Gutierrez R, El-Howeity M, Mich iels J, Vanderleyden J (2008)
Physiological and genetic anal ysis of root responsiveness to auxin-producing plant growth-promoting bacteria in common bean (Phaseolus vulgaris L.) Plant Soil 302: 149-161.
Sartorato A, Rava CA, Faria JC (2003) Doencas e metodos de controle. In Cultivo do Feijoeiro Comum. EMBRAPA Arroz e Feijao. Goiania, Brazil. http://sistemasdeproducao.cnptia.embrapa.br/FontesHTML/ Feijao/CultivodoFeijoeiro/doencas.htm (Cons. 01/31-2010).
Taiz L, Zeiger E (2004) Fisiologia Vegetal. 3rd ed. Artmed. Porto Alegre, Brazil. 719 pp.
Vessey JK (2003) Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 225: 571-586.
Vieira EL, Castro PRC (2001) Efei to de bioestimulante na germinacao de sementes, vigor das plantulas, crescimento radicular e produtividade de soja. Rev. Bras. Sem. 23: 222-228.
Weber OB, Baldani JI, Dobereiner J (2000) Bacterias diazotroficas em mudas de bananeira. Pesq. Agrope. Bras. 35: 2277-2285.
Yazdani M, Bahmanyar MA, Pir dashti H, Esmaili MA (2009) Effect of phosphate solubilization microorganisms (PSM) and plant growth promoting rhizobacteria (PGPR) on yield and yield components of corn (Zea mays L.). World Acad. Sci. Eng. Techn. 49: 90-92.
Received: 10/22/2011. Modified: 11/21/2012. Accepted: 04/04/2013
Ceci Castilho Custodio. Doctor in Seed Production and Technology, Universidade do Oeste Paulista (UNOESTE), Brazil, Professor, UNOESTE, Brazil. e-mail: email@example.com
Fabio Fernando de Araujo. Doctor in Microbiology, UNOESTE, Brazil. Professor, UNOESTE, Brazil.
Aline Manholer Ribeiro. Agronomical Engineer, UNOESTE, Brazil.
Nilton vieira de Souza Filho. Agronomical Engineer, UNOESTE, Brazil.
Nelson Barbosa Machado-Neto. Doctor in Plant Biology and Biotechnology, UNOESTE, Brazil. Professor, Agronomical Engineer, UNOESTE, Brazil. UNOESTE, Brazil. Address: Faculdade de Ciencias Agrarias, UNOESTE. Rod. Raposo Tava res, Km 572, CEP 19067175, Presidente Prudente--SP, Brazil. e-mail: firstname.lastname@example.org
TABLE I VARIANCE ANALYSIS OF GERMINATION (G), VIGOR CLASSIFICATION (CV), SHOOT (SDM), ROOT (RDM) AND TOTAL (TDM) DRY MASS AND ROOT/SHOOT RATE (R/S) OF COMMON BEAN SEEDS TREATED WITH DIFFERENT IBA CONCENTRATIONS AND B. subtilis AFTER SEVEN DAYS OF SOWING IN THE LAB Mean square F test for all treatments Variation G CV CR SDM (g) source Treatment 389.42 ** 369.55 ** 4.631 ** 0.5204 ** Residue 63.66 60.21 0.7734 0.0174 VC (%) 9.58 10.94 10.06 11.23 Mean square F test for all treatments Treatment 426.27 ** 365.07 ** 3.419 * 0.2083 ** Residue 65.55 49.67 0.837 0.017 VC (%) 9.98 9.95 10.95 12.32 Mean square F test for all treatments Variation RDM (g) TDM (g) R/S rate source Treatment 0.5733 ** 1.1856 ** 0.3927 ** Residue 0.0101 0.0327 0.0164 VC (%) 13.52 9.43 19.22 Mean square F test for all treatments Treatment 0.6686 ** 1.436 ** 0.3367 ** Residue 0.0033 0.0213 0.0193 VC (%) 7.02 7.70 18.26 *, ** significant at 5% and 1% by the F test, respectively. VC: variance coefficient. TABLE II GERMINATION (G) AND SEED VIGOR CLASSIFICATION (VC) OF BEAN SEEDS TREATED WITH DIFFERENT CONCENTRATION OF IBA (mg x [kg.sup.-1] seed) AND B. subtilis (10g x [kg.sup.-1] seed) AFTER SEVEN DAYS OF SOWING IN THE LAB Treatment G (%) CV (%) B. subtilis 88 a (1) 81 a 0 76 b 64,5 b 1.75 69 b 61 b Indol-butyric acid 3.5 76 b 70,5 b 7 77 b 64 b 14 93 a 82 a 28 94 a 83 a Control 71 b 62 b (1) Means followed by the same letter are not statistically different by Scott-Knott (p >0.05). TABLE III ROOT LENGTH (RL), SHOOT (SDM), ROOT (RDM) AND TOTAL (TDM) DRY MASS AND ROOT/SHOOT RATE (R/S) OF BEAN SEEDS TREATED WITH DIFFERENT CONCENTRATIONS OF IBA (mg x [kg.sup.-1] seed) AND B. subtilis (10g x [kg.sup.-1] seed) AT SEVEN DAYS AFTER SOWING IN THE LAB Treatment RL (cm) SDM (g) RDM (g) TDM (g) R/S rate B. subtilis 10.18 a 1.92 a 0.42 c 2.35 a 0.21 c 0 9.22 a 0.91 b 0.57 c 1.48 b 0.63 b 1.75 7.99 b 0.91 b 0.52 c 1.42 b 0.58 b 3.5 8.60 b 1.04 a 0.61 b 1.65 b 0.59 b Indol- 7 7.62 b 0.91 b 0.56 c 1.47 b 0.62 b butyric 14 9.51 a 1.48 a 1.20 b 2.69 a 0.81 b acid 28 7.14 b 1.15 a 1.47 a 2.62 a 1.32 a Control 9.65 a 1.05 a 0.58 c 1.62 b 0.54 b Means followed by the same letters are not significant by the Scott-Knott test. TABLE IV SUMMARY OF ANALYSIS OF VARIANCE OF THE EMERGENCE (E) EMERGENCE SPEED INDEX (ESI), SHOOT DRY MASS (SDM), ROOT DRY MASS (RDM), TOTAL DRY MASS (TDM) AND ROOT / SHOOT (R/S) RATE OF BEAN SEEDS TREATED WITH DIFFERENT CONCENTRATIONS OF IBA AND B. SUBTILIS AT SEVEN DAYS AFTER SOWING IN THE GREENHOUSE Variation source Mean square F Test for all treatments E ESI RDM Treatment 2352.70 ** 1087.77 ** 94.864 * Block 2259.40 * 1152.47 ** 100.39 * Residue 382.56 103.24 20.26 VC(%) 26.29 22.10 20.35 Mean square F Test for all treatments Treatment 1310.91 * 749.43 ** 113.80 * Block 3019.58 ** 1301.34 ** 97.30 * Residue 249.13 70.18 21.36 VC(%) 24.60 21.58 24.15 Variation source Mean square F Test for all treatments TDM R/S rate Treatment 245.24 * 0.600 * Block 299.23 ** 0.3402 (ns) Residue 49.48 0.1505 VC(%) 22.42 28.42 Mean square F Test for all treatments Treatment 249.78 * 0.7945 * Block 357.01 ** 0.2795 (ns) Residue 36.76 0.1991 VC(%) 20.32 23.49 *, ** significant at 5% and 1% by the F test, respectively. VC: variance coefficient. TABLE V SEEDLING EMERGENCE (E) AND EMERGENCE SPEED INDEX (ESI) OF BEANS TREATED WITH DIFFERENT CONCENTRATIONS OF INDOL-BUTYRIC ACID (mg x [kg.sup.-1] seed) AND BACILLUS SUBTILIS (10g x [kg.sup.-1] seed) AFTER 18 DAYS OF SOWING Treatment E (%) ESI B. subtilis 87 a 52.15 a Indol-butyric acid 7 49 b 24.18 b 14 43 b 28.27 b 28 68 a 43.45 a Control 24 b 10.19 c Means followed by the same letters are not significant by Scott-Knott Test at 5%. TABLE VI SHOOT DRY MASS (SDM), ROOT DRY MASS (RDM), TOTAL DRY MASS (TDM) AND ROOT/SHOOT RATIO (R/S) OF BEAN SEEDLINGS TREATED WITH DIFFERENT CONCENTRATIONS OF IBA (mg x [kg.sup.-1] seed) AND B. subtilis (10 x g [kg.sup.-1] seed) AT 18 DAYS AFTER SOWING IN POTS Treatment SDM (g) RDM (g) TDM (g) R/S B. subtilis 14.59 a 13.91 a 28.51 a 0.95 b Indol-butyric acid 7 8.99 b 14.29 a 23.29 a 1.44 a 14 11.59 a 15.41 a 27.00 a 1.38 a 28 12.90 a 8.06 b 20.96 a 0.68 b Control 4.64 b 4.10 b 8.75 b 0.68 b Means followed by same letters are not statistically different by the Scott-Knott test at 5%.