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Seed size influences the promoting activity of rhizobia on plant growth, nodulation and N fixation in lima bean

O tamanho da semente influencia a atividade promotora dos rizóbios sobre o crescimento de plantas, nodulação e fixação de N em feijão-fava

ABSTRACT:

This study evaluated the activity of rhizobia isolates inoculated in large (18 mm) and small (11 mm) seeds on lima bean growth, nodulation and N fixation. Selected rhizobia isolates were compared with a reference strain CIAT899 and two controls without inoculation. Large seeds contributed for highest plant growth, nodulation and N fixation than small seeds. The isolates UFPI-59, UFPI-18 and UFPI-38 promoted the highest values of shoot and root dry weight, respectively. The isolates UFPI-32 promoted the highest values of nodule number, while UFPI-59 promoted the highest values of nodule dry weight. The isolates UFPI-38 and UFPI-59 promoted the highest accumulation of N. This study showed that seed size really influences lima bean growth, nodulation and BNF. Considering rhizobia isolates, UFPI-59, UFPI-38, and UFPI-18 contributed for plant growth, promoted better nodulation and effectiveness on biological N fixation.

Key words:
Phaseolus lunatus; N-fixers; Rhizobium; Bradyrhizobium; plant-microbes interaction

RESUMO:

Este estudo avaliou a atividade de isolados de rizóbios inoculados em sementes pequenas (11 mm) e grandes (18 mm) sobre o crescimento, nodulação e fixação de N de feijão-fava. Isolados selecionados de rizóbios foram comparados com uma estirpe de referência CIAT899 e dois controles sem inoculação. Sementes grandes contribuíram para maior crescimento de plantas, nodulação e fixação de N do que sementes pequenas. Os isolados UFPI-59, UFPI-18 e UFPI-38 promoveram os maiores valores de massa seca de parte aérea e raízes. Os isolados UFPI-32 e UFPI-59 promoveram os maiores valores de número e massa de nódulos, respectivamente. Os isolados UFPI-38 e UFPI-59 promoveram maior acúmulo de N. Este estudo mostrou que o tamanho de sementes influencia o crescimento, nodulação e fixação de N em feijão-fava. Considerando os isolados de rizóbios, UFPI-59, UFPI-38 e UFPI-18 contribuíram para o crescimento de plantas, promoveram melhor nodulação e eficiência na fixação de N.

Palavras-chave:
Phaseolus lunatus; fixadores de N; Rhizobium; Bradyrhizobium; interação plantas-microrganismos

INTRODUCTION:

Lima bean (Phaseolus lunatus L.) is an important crop for several countries, such as Peru, Colombia, Mexico, USA and Brazil (AMORIM et al., 2019AMORIM, M.R. et al. Current knowledge and future prospects of lima bean (Phaseolus lunatus)-rhizobia symbiosis. Revista Faculdad de Ciências Agrárias, v.51, n.2, p.280-288, 2019. Available From: <Available From: http://revistas.uncu.edu.ar/ojs/index.php /RFCA/article/view/2696/1960 > Accessed: Mar. 12, 2020.
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). In Brazil, it is cultivated in the Northeastern region, mainly by small farmers, in an estimated area around 37,500 ha. The main characteristics of lima bean are the high rusticity and tolerance to abiotic factors, such as drought and high temperature (ARAÚJO et al., 2017ARAÚJO, A.S.F. et al. Nodulation ability in different genotypes of Phaseolus lunatus by rhizobia from California agricultural soils. Symbiosis, v. 73, p.7-14, 2017. Available From: <Available From: https://link.springer.com/article/10.1007/s13199-016-0465-0 >. Accessed: Feb. 17, 2020. doi: 10.1007/s13199-016-0465-0.
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).

Although, important for small farmers, lima bean still has low productivity and some reasons contribute to this situation, such as the absence of commercial varieties and low availability of nutrients, mainly nitrogen (N). Nitrogen is essential to plants and its supply to lima bean could contribute for increasing the yield (LOPES et al., 2015). Importantly, lima bean can associate with N-fixing rhizobia and through biological N fixation (BNF) uptake N for its growth and yield (AMORIM et al., 2019AMORIM, M.R. et al. Current knowledge and future prospects of lima bean (Phaseolus lunatus)-rhizobia symbiosis. Revista Faculdad de Ciências Agrárias, v.51, n.2, p.280-288, 2019. Available From: <Available From: http://revistas.uncu.edu.ar/ojs/index.php /RFCA/article/view/2696/1960 > Accessed: Mar. 12, 2020.
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).

However, the efficiency of the BNF varies according to the rhizobia strains and the selection of efficient rhizobia become an important step before recommendation to seed inoculation (IRISARRI et al., 2019IRISARRI, P. et al. Selection of competitive and efficient rhizobia strains for white clover. Frontiers in Microbiology, v.10, p.768, 2019. Available From: <Available From: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6489563/pdf/fmicb-10-00768.pdf >. Accessed: Feb. 15, 2020. doi: 10.3389/fmicb.2019.00768.
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). Several strains of rhizobia are already recommended for different legumes species, such as soybean (MERKEB et al., 2016MERKEB, F. et al. Evaluation of different commercial rhizobial strains on soybean (Glycine max L.) yield at Pawe District, Northwestern Ethiopia. World Scientific News, v. 55, p. 15-26, 2016. Available From: <Available From: http://psjd.icm.edu.pl/psjd/element/bwmeta1.element.psjd-8d8691fd-5abd-46c9-b0df-a990974e435c >. Accessed: Oct. 24, 2019.
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), and cowpea (BATISTA et al., 2017BATISTA, E.R. et al. Combined inoculation of rhizobia on the cowpea development in the soil of Cerrado. Revista Ciência Agronômica, v.48, n.5, p.745-755, 2017. Available From: <Available From: http://ccarevista.ufc.br/seer/index.php/ccarevista/article/view/5200 > Accessed: Dec. 12, 2019. doi: 10.5935/1806-6690.20170087.
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). So far, lima bean does not have any recommended rhizobia for inoculation and some previous studies have reported potential rhizobia isolates (ANTUNES et al, 2011ANTUNES, J.E.L. et al. Eficiência simbiótica de isolados de rizóbio noduladores de feijão-fava (Phaseolus lunatus L.). Revista Brasileira de Ciência do Solo, v.35, n.3, p.751-757, 2011. Available From: <Available From: http://www.scielo.br/scielo.php?pid=S0100-06832011000300011&script=sci_abstract&tlng=pt > Accessed: Jan. 10, 2020. doi: 10.1590/S0100-06832011000300011.
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; COSTA NETO et al, 2017COSTA NETO, V.P. et al. Symbiotic performance, nitrogen flux and growth of lima bean (Phaseolus lunatus L.) varieties inoculated with different indigenous strains of rhizobia. Symbiosis, v.73, p.117-124, 2017. Available From: <Available From: https://link.springer.com/article/10.1007/s13199-017-0475-6 > Accessed: Nov. 12, 2019. doi: 10.1007/s13199-017-0475-6.
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). However, these studies were not conducted in soils, but under axenic conditions. Thus, it is necessary to advance the studies for selecting rhizobia under soil conditions.

Plant traits also influence nodulation and BNF in legumes. Among these traits, seed size contributes for increasing or decreasing the process of N fixation (DOBER &TBLEVINS, 1993; SINGH and WRIGHT, 2002SINGH, G.; WRIGHT, D. Effects of herbicides on nodulation and growth of two varieties of pea (Pisum sativum). Acta Agronomica Hungarica, v.50, n.3, p.337-348, 2002. Available From: <Available From: https://www.researchgate.net/publication/250006731 >. Accessed: Oct. 25, 2019. doi: 10.1556/AAgr.50.2002.3.11.
https://www.researchgate.net/publication...
; ERDEMCI et al., 2017ERDEMCI, I. et al. Effect of fertilization and seed size on nodulation, yield and yield components of Chickpea (Cicer Arietinum L.). Applied Ecology and Environmental Research, v.15, p.563-571, 2017. Available From: <Available From: https://www.researchgate.net/publication/316547498 >. Accessed: 10 Dec. 2019. doi: 10.15666/aeer/1501_563571.
https://www.researchgate.net/publication...
). According to ERDEMCI et al. (2017) seed size influences seedling vigor and plant growth and large seeds usually produce more vigorous seedlings than small ones. In a previous study with Pisum sativum, SINGH and WRIGHT (2002) reported that large seeds had a higher increase in nodulation than small ones. In lima bean, DOBERT and BLEVINS (1993DOBERT, R.C.; BLEVINS, D.G. Effect of seed size and plant growth on nodulation and nodule development in lima bean (Phaseolus lunatus L.). Plant and Soil, v.148, p.11-19, 1993. Available From: <Available From: https://link.springer.com/article/10.1007/BF02185380 >. Accessed: Nov. 20 2019.
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) found a positive correlation between seed size and nodulation. However, they did not evaluate the correlation between seeds size and rhizobia on plant growth and BNF. Thus, it remains unclear how are the interaction between rhizobia in seed size on plant growth, nodulation, and BNF of lima bean. The aim of this study was to evaluate the influence of seed size in promoting the activity of rhizobia on plant growth, nodulation and BNF in lima bean.

MATERIALS AND METHODS:

The study was conducted in a greenhouse located at the Agricultural Science Center, Federal University of Piaui, Brazil, from January to March 2019. A sandy soil was used in this study and it was collected at 0-20 cm depth. The soil presented the following physical and chemical properties: sand - 832 g kg-1, silt - 64 g kg-1, clay - 104 g kg-1, pH - 6.2, organic matter - 0.57 g kg-1, P - 1.8 mg kg-1, K - 38.9 mg kg-1, Ca - 1.06 cmolc kg-1 and Mg - 0.3 cmolc kg-1.

For the experiment, five rhizobia isolates, selected according to their high efficiency under axenic condition (ANTUNES et al., 2011ANTUNES, J.E.L. et al. Eficiência simbiótica de isolados de rizóbio noduladores de feijão-fava (Phaseolus lunatus L.). Revista Brasileira de Ciência do Solo, v.35, n.3, p.751-757, 2011. Available From: <Available From: http://www.scielo.br/scielo.php?pid=S0100-06832011000300011&script=sci_abstract&tlng=pt > Accessed: Jan. 10, 2020. doi: 10.1590/S0100-06832011000300011.
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), were inoculated in small (genotype UFPI-1120; 11 mm and 350 mg of diameter and dry weight) and large (genotype UFPI-480; 18 mm and 800 mg of diameter and dry weight) seeds of lima bean (Phaseolus lunatus L). The isolates were: UFPI-18 (Enterobacter sp.), UFPI-32 (Bradyrhizobium sp.), UFPI-38 (Rhizobium sp.), UFPI-50 (Bradyrhizobium sp.), and UFPI-59 (Bradyrhizobium sp.). These isolates were compared against a reference strain to Phaseolus (CIAT 899 - Rhizobium tropici;FLORENTINO et al., 2018FLORENTINO, L.A. et al. Inoculação e aplicação de diferentes doses de nitrogênio na cultura do feijoeiro. Revista de Ciências Agrárias, v.41, n.4, p.963-970, 2018. Available From: <Available From: http://www.scielo.mec.pt/scielo.php?script=sci_arttext&pid=S0871-018X2018000400010 >. Accessed: Aug. 20, 2020. doi: 10.19084/RCA17001.
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), and two controls without inoculation: negative (NC; without N fertilization) and positive (PC; with N fertilization).

The isolates were grown in Erlenmeyer flasks containing 50 mL of liquid culture broth (under orbital shaking at 200 rpm, 28 °C, 72 h). The bacterial growth was verified through a spectrophotometer (wavelength of 540 nm) considering a final concentration of 109 CFU mL-1. The experimental unit consisted of 64 pots (diameter 18 cm, length 16 cm) filled with 5 kg of soil (previously air-dried at room temperature and sieved in a 2 mm mesh). Before inoculation, seeds were disinfected with alcohol (70%) for 30 seconds and sodium hypochlorite (2%) for 60 seconds, being washed five times with sterile distilled water. Five seeds were sowed per pot and each one was directly inoculated with 1 mL of the suspension containing the rhizobia. The positive control (+N) received 50 mg N per pot, in accordance with N requirement by lima bean. Five days after germination, plants were thinned, leaving one plant per pot. Pots were irrigated daily with sterilized water to maintain soil moisture at 80% of field capacity.

The experiment was harvested 45 days after sowing (flowering stage). Plants were excised at the cotyledonal node to separate shoots from roots. Nodules were separated from the roots and counted to determine nodule number (NN). Afterward, nodules, shoots and roots were dried (65°C; 72 h) and weighed to determine nodules (NDW), shoot (SDW), and roots (RDW) dry weight. Total N content in shoot was estimated by Kjedahl method and the accumulation of N (AcN) in shoot was estimated by the shoot dry matter. The effectiveness of BNF was estimated comparing the inoculated treatments against negative control, being the values expressed in %, according to the expression: Effectiveness = (SDWi / SDWni) x 100, in which SDWi and SDWni are the values of shoot dry weight reported in inoculated and non-inoculated plants, respectively.

The treatments were arranged in a randomized design, under an 8 x 2 factorial scheme with four replications, corresponding to eight treatments (rhizobia and controls) and two seed sizes (small and large). The normality of data was analyzed by using the test of Shapiro-Wilk. Afterward, data were statistically analyzed by using ANOVA, and the means were compared using Scott-Knott test (p<0.05%) through the statistical program SISVAR, version 5.6 (FERREIRA, 2014FERREIRA, D. F. Sisvar: A guide for its bootstrap procedures in multiple comparisons. Ciência e Agrotecnologia, v.38, n.2, p.109-112, 2014. Available From: <Available From: https://www.scielo.br/scielo.php?pid=S1413-70542014000200001&script=sci_arttext >. Accessed: Oct. 12, 2019. doi: 10.1590/S1413-70542014000200001.
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). In addition, to compare the isolate profiles we conducted a principal component analysis biplot (PCA) based on the determined characteristics. For this, the data matrix was initially analyzed using detrended correspondent analysis (DCA) to evaluate the distribution of the data, which indicated the best-fit model PCA. Then, PCA plots were generated using the Canoco 4.5 software (BIOMETRICS, WAGENINGEN, THE NETHERLANDS).

RESULTS:

Treatments, seed size and interactions between treatments and seed size significantly influenced all variables. In general, the values of SDW were highest with large seeds than small ones (Figure 1A). The exception was CIAT899 that showed the highest SDW with small seeds. For this plant parameter, UFPI-59 promoted the highest values when inoculated in large seeds. For small seeds, UFPI-18 and UFPI-38 promoted the highest values of SDW. In contrast, the values of RDW were highest with small seeds (Figure 1B). The exception was UFPI-59 and NC that promoted the highest RDW with large seeds. For this plant parameter, UFPI-38 and UFPI-59 promoted the highest values, when inoculated in small and large seeds, respectively. Interestingly, UFPI-38 and UFPI-18, in small seeds, and UFPI-59, in large seeds, increased the root growth when compared to NC, PC and CIAT899.

Figure 1
Shoot (A) and root (B) dry weight of lima bean inoculated with rhizobia isolates in small (11 mm) and large (18 mm) seeds. Similar lower case letters comparing seeds sizes for the same treatment, and upper case letters comparing treatments for the same seeds size do not differ significantly by the Tukey test (p < 0.05).*UFPI-18 (Enterobacter sp.), UFPI-32 (Bradyrhizobium sp.), UFPI-38 (Rhizobium sp.), UFPI-50 (Bradyrhizobium sp.), and UFPI-59 (Bradyrhizobium sp.), CIAT 899 (Rhizobium tropici), NC (negative control), PC (positive control). Enviar em .tiff com pelo menos 300 dpi.

The values of NN and NDW were highest with large seeds (Figure 2). The exception was CIAT899 and PC, for NN, and CIAT899 and UFPI-32, for NDW, that presented the highest values with small seeds. The isolates UFPI-32 increased NN in both small and large seeds as compared to other treatments. For NDW, UFPI-59 and UFPI-38 promoted the highest values when inoculated in large seeds. In small seeds, UFPI-59, UFPI-32 and PC presented the highest values of SDW (Figure 2).

Figure 2
Nodule number (A) and nodule dry weight (B) in lima bean inoculated with rhizobia isolates in small (11 mm) and large (18 mm) seeds. Similar lower case letters comparing seeds sizes for the same treatment, and upper case letters comparing treatments for the same seeds size do not differ significantly by the Tukey test (p < 0.05). *UFPI-18 (Enterobacter sp.), UFPI-32 (Bradyrhizobium sp.), UFPI-38 (Rhizobium sp.), UFPI-50 (Bradyrhizobium sp.), and UFPI-59 (Bradyrhizobium sp.), CIAT 899 (Rhizobium tropici), NC (negative control), PC (positive control). Idem 1.

The highest values of AcN were reported with UFPI-38 and UFPI-59 in small and large seeds, respectively (Figure 3A). In addition, these isolates promoted higher AcN than NC, and PC. In small seeds, UFPI-38 and CIAT899 presented the highest effectiveness, while that UFPI-32, UFPI-38, UFPI-50 and UFPI-59 presented higher effectiveness when inoculated in large seeds (Figure 3B).

Figure 3
Accumulated N (A) and effectiveness (B) in lima bean inoculated with rhizobia isolates in small (11 mm) and large (18 mm) seeds. Similar lower case letters comparing seeds sizes for the same treatment, and upper case letters comparing treatments for the same seeds size do not differ significantly by the Tukey test (p < 0.05). *UFPI-18 (Enterobacter sp.), UFPI-32 (Bradyrhizobium sp.), UFPI-38 (Rhizobium sp.), UFPI-50 (Bradyrhizobium sp.), and UFPI-59 (Bradyrhizobium sp.), CIAT 899 (Rhizobium tropici), NC (negative control), PC (positive control). Idem 1.

The PCA showed the relationship between treatments and the evaluated parameters, according to seeds size (Figure 4). This analysis explained 96.8 % of the total variation of which 87.2% and 9.6% are displayed on the horizontal and vertical axes, respectively. Large seeds inoculated with rhizobia correlated with the plant and nodules parameters than small ones. The exception was the treatments NC, PC and CIAT899. Thus, when large seeds were inoculated, isolates UFPI-38 and UFPI-59 correlated with SDW, AcN, NDW and effectiveness, while UFPI-32 correlated with NN.

Figure 4
Principal component analysis biplot (PCA) based on the treatments* and the evaluated parameters (NN -Nodule number; NDW-nodule dry weight, SDW-shoot dry weight, RDW-root dry weight, AcN- Accumulated N, effectiveness) in relation to seeds size. The significance of treatments on related parameters is indicated by the arrows. *UFPI-18 (Enterobacter sp.), UFPI-32 (Bradyrhizobium sp.), UFPI-38 (Rhizobium sp.), UFPI-50 (Bradyrhizobium sp.), and UFPI-59 (Bradyrhizobium sp.), CIAT 899 (Rhizobium tropici), NC (negative control), PC (positive control). Idem 1.

DISCUSSION:

Results showed that lima bean growth, nodulation and BNF varied according to rhizobia. These results are in line with previous studies that showed differences on nodulation and symbiotic efficiency by rhizobia in soybean (BARBOSA et al., 2017BARBOSA, L.P. et al. Symbiotic efficiency and genotypic characterization of variants of Bradyrhizobium spp. in commercial inoculants for soybeans. Revista Brasileira de Ciência do Solo, v.41, e0160572, 2017. Available From: <Available From: http://www.scielo.br/pdf/rbcs/v41/0100-0683-rbcs-18069657rbcs20160572.pdf >. Accessed: Jan. 10 2020. doi: 10.1590/18069657rbcs20160572.
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) and common bean (MERCANTE et al., 2017MERCANTE, F.M. et al. New Native Rhizobia Strains for Inoculation of Common Bean in the Brazilian Savanna. Revista Brasileira de Ciência do Solo, v.41, e0150120, 2017. Available From: <Available From: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-06832017000100405 > Accessed: Oct. 12, 2019. doi: 10.1590/18069657rbcs20150120.
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). In addition, results showed that, in sandy soil with low fertility, the inoculation in lima bean should be strongly recommended. In general, the inoculation of UFPI-59, UFPI-18 and UFPI-38 promoted the highest plant growth, when inoculated in large and small seeds, as compared with NC, PC and CIAT899. The plant growth, i.e. shoots and roots dry weight, is an important parameter for selecting rhizobia to inoculation. Thus, these isolates present potential for further studies in field and recommendation as inoculant.

The nodulation, i.e. nodule number and dry weight, differed between rhizobia and seed size. The inoculation of UFPI-32 stimulated the nodule number, while that UFPI-59 increased the nodule biomass. Although, nodule number has been stimulated by inoculation of UFPI-32, this isolate did not stimulate the nodule growth as compared to UFPI-59. According to ÖĞÜTÇÜ et al. (2010ÖĞÜTÇÜ, H. et al. Effects of rhizobium strains isolated from wild chickpeas on the growth and symbiotic performance of chickpeas (Cicer arietinum L.) under salt stress. Turkish Journal of Agriculture and Forestry, v.34, p.361-371, 2010. Available From: <Available From: https://www.researchgate.net/publication/278071740 >. Accessed: Mar. 14, 2020. doi: 10.3906/tar-0904-34.
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), nodule dry weight is more reliable than nodule number as indicator of effective symbiosis since larger nodules fix more N than smaller ones. Therefore, UFPI-59 presents high potential for increasing the lima bean growth and nodulation. According to PARRA-COLMENARES and KAHN (2005PARRA-COLMENARES, A.; KAHN, M.L. Determination of nitrogen fixation effectiveness in selected Medicago truncatula isolates by measuring nitrogen isotope incorporation into pheophytin. Plant and Soil, v.270, p.159-168, 2005. Available From: <Available From: https://link.springer.com/article/10.1007/s11104-004-1308-y >. Accessed: Jan. 25, 2020. doi: 10.1007/s11104-004-1308-y.
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), nodulation and plant growth are key parameters commonly used to estimate the potential of rhizobia. In a previous study, the isolate UFPI-59 also promoted higher nodule biomass in lima bean under axenic condition (ANTUNES et al., 2011ANTUNES, J.E.L. et al. Eficiência simbiótica de isolados de rizóbio noduladores de feijão-fava (Phaseolus lunatus L.). Revista Brasileira de Ciência do Solo, v.35, n.3, p.751-757, 2011. Available From: <Available From: http://www.scielo.br/scielo.php?pid=S0100-06832011000300011&script=sci_abstract&tlng=pt > Accessed: Jan. 10, 2020. doi: 10.1590/S0100-06832011000300011.
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). Thus, results suggested that this isolate is also effective under soil condition.

The nodulation observed in uninoculated plants (NC and PC) suggested the presence of native rhizobia nodulating lima bean. However, these native rhizobia were not more effective than the isolates. The results showed that CIAT899 presented highest nodulation in small seeds and it confirmed the recommendation of this strain for Phaseolus vulgaris (common bean) which presents small seeds.

The isolate UFPI-59, that promoted highest nodule biomass, contributed to higher accumulation of N by lima bean. However, the isolate UFPI-38, that did not present highest nodulation, also contributed to higher accumulation of N. Although, UFPI-38 did not present high nodulation, this isolate promoted higher root growth by which can contribute to the high N uptake due to the increased root surface. In addition, UFPI-38 presents urease activity (CHIBEBA et al., 2020CHIBEBA, A. M. et al. Polyphasic characterization of nitrogen-fixing and co-resident bacteria in nodules of Phaseolus lunatus inoculated with soils from Piauí State, Northeast Brazil. Symbiosis, v.80, p.279-292, 2020. Available From: <Available From: https://link.springer.com/article/10.1007/s13199-020-00672-1 >. Accessed: Aug. 22, 2020. doi: 10.1007/s13199-020-00672-1.
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) and it can provide greater efficiency in the use of N by the plants (MOBLEY & HAUSINGER, 1989MOBLEY, H. L. T.; HAUSINGER, R. P. Microbiol ureases: significance, Regulation, and Molecular Characterization. Microbiology Review, v.53, n.1, p.85-108, 1989. Available From: <Available From: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC372718/ > Accessed: Aug. 23, 2020.
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). Results have shown that the inoculation of UFPI-59 and UFPI-38 potentially increased plant growth, nodulation and N fixation independent of seeds size. This finding agrees with ANTUNES et al. (2011ANTUNES, J.E.L. et al. Eficiência simbiótica de isolados de rizóbio noduladores de feijão-fava (Phaseolus lunatus L.). Revista Brasileira de Ciência do Solo, v.35, n.3, p.751-757, 2011. Available From: <Available From: http://www.scielo.br/scielo.php?pid=S0100-06832011000300011&script=sci_abstract&tlng=pt > Accessed: Jan. 10, 2020. doi: 10.1590/S0100-06832011000300011.
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) who reported a strong response of UFPI-59 on nodulation and accumulation of N in lima bean under axenic conditions. Usually, higher nodule biomass increases the accumulation of N in plants (HAMAWAKI and KANTARTZI, 2018HAMAWAKI, R.L.; KANTARTZI, S.K. Di-nitrogen fixation at the early and late growth stages of soybean. Acta Scientiarum. Agronomy, v.40, e36372, 2018. Available From: <Available From: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S180786212018000100615 >. Accessed: Oct. 15, 2019. doi: 10.4025/actasciagron.v40i1.36372.
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). Also, the isolate UFPI-59 presents protease activity and the capability of solubilize P (CHIBEBA et al., 2020), being two important biochemical properties to increase plant growth. Thus, both isolates can be selected as potential for increasing N fixation and contributing for lima bean growth.

The multivariate analysis (PCA) showed differences between large and small seeds on all parameters. It confirmed the variation found on plant growth, nodulation and BNF according to seeds size. The results confirmed UFPI-38 and UFPI-59 as more efficient rhizobia, mainly with large seeds. In addition, the highest values of parameters were reported in large seeds and it agrees with previous studies assessing the effect of seed size on plant growth and nodulation (DOBERT & BLEVINS 1993DOBERT, R.C.; BLEVINS, D.G. Effect of seed size and plant growth on nodulation and nodule development in lima bean (Phaseolus lunatus L.). Plant and Soil, v.148, p.11-19, 1993. Available From: <Available From: https://link.springer.com/article/10.1007/BF02185380 >. Accessed: Nov. 20 2019.
https://link.springer.com/article/10.100...
; SINGH and WRIGHT,2002SINGH, G.; WRIGHT, D. Effects of herbicides on nodulation and growth of two varieties of pea (Pisum sativum). Acta Agronomica Hungarica, v.50, n.3, p.337-348, 2002. Available From: <Available From: https://www.researchgate.net/publication/250006731 >. Accessed: Oct. 25, 2019. doi: 10.1556/AAgr.50.2002.3.11.
https://www.researchgate.net/publication...
; ERDEMCI et al, 2017ERDEMCI, I. et al. Effect of fertilization and seed size on nodulation, yield and yield components of Chickpea (Cicer Arietinum L.). Applied Ecology and Environmental Research, v.15, p.563-571, 2017. Available From: <Available From: https://www.researchgate.net/publication/316547498 >. Accessed: 10 Dec. 2019. doi: 10.15666/aeer/1501_563571.
https://www.researchgate.net/publication...
). Since large seeds present higher reserve content, it could promote faster emergence and more vigorous seedling (SINGH and WRIGHT, 2002). Although, this study did not evaluate seedling emergence and vigor, these factors may have contributed for highest plant growth and nodulation. Indeed, DOBERT & BLEVINS (1993) have reported an influence of seed-factors on lima bean nodulation.

Since the lima bean presents importance mainly to smallholders who use different types of seeds (PENHA et al., 2017PENHA, J.S. et al. Estimation of natural outcrossing rate and genetic diversity in Lima bean (Phaseolus lunatus L. var. lunatus) from Brazil using SSR markers: implications for conservation and breeding. Genetic Resource and Crop Evolution, v.64, p.1355-1364, 2017. Available From: <Available From: https://link.springer.com/article/10.1007/s10722-016-0441-9 >. Accessed: Aug. 25, 2020. doi: 10.1007/s10722-016-0441-9.
https://link.springer.com/article/10.100...
), this study brings important information regarding to the potential rhizobia isolate to be inoculated in small or large seeds. In addition, the practice of inoculation is important to lima bean since this crop presents low yield and, at the same time, it can contribute for decreasing the dependency of N fertilizers and increasing the environmental sustainability.

CONCLUSION:

Seed size influences plant growth, nodulation and BNF, being a key trait to determine the success for inoculation in lima bean. Thus, large seeds contributed with better lima bean performance on all evaluated parameters. Considering rhizobia isolates, UFPI-59 and UFPI-38 contributed for plant growth, nodulation and BNF. Therefore, these isolates present high potential for evaluation under field experiments.

ACKNOWLEDGEMENTS

Claudyanne do Nascimento Costa, Joao Pedro Alves Aquino and Jadson Emanuel Lopes Antunes thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for their scollarship. Angela Celis de Almeida Lopes, Marcia do Vale Barreto Figueiredo and Ademir Sergio Ferreira Araujo thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their “Productivity Research” fellowship. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil - Finance code 001.

REFERENCES

  • CR-2020-0246.R2

Publication Dates

  • Publication in this collection
    08 Jan 2021
  • Date of issue
    2021

History

  • Received
    18 Mar 2020
  • Accepted
    11 Sept 2020
  • Reviewed
    14 Oct 2020
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