PASTURE SCIENCE

Increase in the development of Pueraria phaseoloides (tropical Kudzu) due to tolerant acid rhizobia under acidity and low fertility conditions

0000-0002-5760-816XI. Hernández^{1  *}  , P. R. Rosales¹  , P. J. González¹  , J. F. Ramírez²  , María C. Nápoles¹  , Reneé Pérez¹ 

¹Instituto Nacional de Ciencias Agrícolas. Gaveta postal 1, San José de las Lajas, Mayabeque, CP 32 700.

²Instituto de Investigaciones de Pastos y Forrajes, Avenida Independencia, km 81/2 Boyeros, La Habana, Cuba.

Abstract

The effect of acid-tolerant rhizobia isolates on growth, nodulation and yield of tropical kudzu, grown under acidity conditions, was determined. By measuring the diameter of the colony and the number of viable cells, the pH tolerance of three Bradyrhizobium strains: K2, 1_2 and 2_4 was established under acidity conditions. Two inoculation tests of tropical kudzu were carried out: one under controlled conditions, and another in the field. The effect of acidity on nodulation, growth and yield of crop was determined. A simple classification ANOVA and a two-factor arrangement were used in the statistical analysis. The three strains of rhizobia were tolerant at pH 4.5. This condition did not decrease its dispersion in the Petri dish, the diameter of the colonies or the number of viable cells. The acidity did not decrease the nodulation or the growth of kudzu plants, inoculated with K2 and 1_2 under controlled conditions. The inoculation of kudzu with rhizobia isolates produced effectiveness in the biological fixation of nitrogen, more than 90 % of the total nodules, more than 75 % of the aerial dry mass yield, and more than 45 % of the nitrogen content of the aerial biomass of plants, under field conditions. The elaboration and application of inoculants based on K2 and 1_2 would contribute to reducing the use of mineral fertilizers in soils affected by acidity.

Key words: Bradyrhizobium; stress; forages; growth

Introduction

Approximately 50 % of the world's tropical soils are affected by acidity, a phenomenon that causes a major problem in world agricultural production (^{Vitorello 2005} and ^{Toledo 2016}). In Cuba, more than 30 % are affected by this limitation (^{Hernández et al. 2015}). One of the main factors that promote soil acidity is the indiscriminate use of acidifying mineral fertilizers (^{Goulding 2016}).

The knowledge about the symbiosis established by diazotrophic bacteria, such as rhizobia, with leguminous plants has allowed the elaboration and use of biofertilizers made from these microorganisms, a practice that helps to reduce the application of mineral fertilizers (^{Saldaña 2017}).

However, the soils acidity constitutes a limiting factor for establishing the interaction between legumes and rhizobia (^{Reeve et al. 2006}). The low pH levels decrease the survival of these bacteria and the establishment of the plant. In addition, the processes of infection, nodulation and biological nitrogen fixation (BNF) are affected (^{Graham and Vance 2003}).

Pueraria phaseoloides (tropical kudzú) is a forage legume that, in association with grasses, allows raising the protein level in the animals diet, and increasing the forage quality and digestibility (^{Carvalho da Paz et al. 2016}). The inoculation of this crop with rhizobia strains, competitive and specific, promotes their growth, provides nitrogen to the soil and favors the accompanying and successive crops (^{Sarr et al. 2016}).

The inoculation of tropical kudzú with rhizobia that have a certain acidity tolerance is an interesting practice that could improve the establishment of the crop in soils affected by acidity.

The objective of this study was to determine the effect of acid-tolerant rhizobia isolates on growth, nodulation and yield of tropical kudzú, grown under acidity conditions.

Materials and Methods

Three Bradyrhizobium isolates (K2, 1_2 and 2_4) were used, from nodules of tropical kudzu (^{Hernández et al. 2013}). To obtain pre-inoculums in 250 mL Erlenmeyers bottles, with 50 mL of the same liquid medium, a loop of the isolates, stored at 4 ^oC in test tubes with mannitol yeast agar (MY medium) (^{Vincent 1970}) and pH 6.8 was used. The cell concentration of cultures was fitted to 1x10⁸ cells mL^-1.

In the inoculation tests of the three bacterial isolates, under controlled culture conditions and in field conditions, tropical kudzu seeds were used, donated by the Estación Experimental de Pastos y Forrajes (EEPF) of Cascajal, Villa Clara. For the test under controlled conditions, the seeds were superficially disinfected with 70 % ethanol for 5 min. and scarified with 98 % sulfuric acid for 10 min. They were immersed in 25 % (v/v) sodium hypochlorite for 15 min. Then, they were washed ten times with sterile distilled water, under aseptic conditions. The seeds were placed on plates containing water agar (0.75 %) (m/v) and incubated at 28 ^oC in the darkness for 72 h.

Classification of bacterial isolates according to their tolerance to acidity. A loop of the bacterial cultures was grown in triplicate on plates containing solid MY medium, with pH 4.5 and 6.8. The culture technique described by de ^{Oliveira and Magalhães (1999)} was applied for this purpose (figure 1). Based on the acidity levels of the medium, this technique allows the bacteria to be classified as sensitive, moderately tolerant or tolerant, according to the areas of the plate that cover with their growth. In the experiment, the growth of the isolates at pH 6.8 was used as a control.

Figure 1 Culture technique proposed by de ^{Oliveira and Magalhães (1999)} to classify rhizobia, according to the level of acidity they tolerate in the culture medium:growth area 1 (a loop rubbing several times in both directions), area 2 (four loops only in one direction), area 3 and 4 (same as area 2, but in other regions of the Petri dish). The scoring intervals for the rhizobia classification were: sensitive (1.00-2.00), moderately tolerant (2.01-3.00) and tolerant (3.01-4.00). 

Plates were incubated at 28^oC for 10 d and the areas covered by bacterial growth were monitored every two days. In addition, the colony diameter of the isolates was measured under both pH conditions, at 6 and 10 d of incubation. The diameter values of five colonies were taken in each of the replications per treatment.

Effect of pH on cell viability. Pre-inoculums of the three rhizobia isolates were used for inoculation in 250 mL Erlenmeyers bottles, containing 50 mL of MY medium at pH 4.5 and 6.8, at a rate of 8 % (v/v). The cultures were maintained under agitation conditions at 150 rpm for 56 hours and at a temperature of 28°C. A completely randomized design with three repetitions was used for each isolate and pH condition.

The concentration of viable cells (CFU mL^-1) in the logarithmic or exponential growth phase (56 h of culture) was determined by the serial dilution method (10-4-10-6). The samples were cultured by dissemination in plates with solid MY medium at pH 6.8 and the plates were incubated at 28°C for 10 d.

In vitro nodulation test. The disinfected, scarified and pre-germinated kudzu seeds with emerging roots (1-2 cm) were placed in glass bottles with 50 mL of semi-solid ^{Norris and Date (1976)} medium, at a rate of one seed per bottle and were inoculated with 1 mL of the isolates. Inoculants similar to those used in the test of the effect of pH on cell viability were used and ten plants per treatment were established in a completely random design.

The plants grew under controlled conditions, with photoperiod of 16 h daylight/8 h of darkness, at a day/night temperature of 26/22 ^oC and relative humidity of 7 %. Four weeks after inoculation it was determined: number of nodules in main root (NNmr), total nodulation (TN), number of effective nodules in the main root (NNemr), total (NteN) and dry mass of total nodules (DMtN) (g). The effectiveness of nodules was determined by observing a reddish coloration inside the nodules, characteristic of the leghemoglobin protein (^{Singh and Varma 2017}). The dissection of nodules was made with stainless steel scalpel blades. The root length (cm), dry mass of the aerial part (DMA) (g) and dry mass of root (DMR) (g) were also determined.

Inoculation test in the field. The experiment was carried out in the Estación Experimental de Pastos y Forrajes (EEPF) of Cascajal, in Villa Clara, in a petroferric ferruginous nodular gleysol soil (^{Hernández et al. 2015}). This soil has strongly acidic pH. The content of organic matter (OM), assimilable phosphorus (P₂O₅), calcium (Ca²⁺), magnesium (Mg²⁺), sodium (Na⁺), potassium (K⁺) and cationic exchange capacity (CEC) are low (^{Paneque et al. 2010}) (table 1).

Table 1 Chemical characteristics of the soil (0-20 cm deep) petroferric ferruginous nodular gleysol 

pH H2O	OM (%)	P2O5 (mg 100 g^-1)	Exchangeable cations (cmolc kg^-1)
			Ca^2+	Mg^2+	Na^+	K^+	CEC
4.7±0.2	2.50±0.09	1.3±0.3	3.51±0.12	1.10±0.03	0.08±0.01	0.11±0.02	4.80±0.16

The confidence intervals of each parameter are shown (Tukey, α =0. 05)

The soil preparation was carried out in a conventional way by means of ploughing, light harrow, crossing, harrowing and furrowing. No fertilizer or fertility enhancing products were applied. Plots with a total area of 21 m² and a calculation area of 14 m² were used (six rows 5 m long, 70 cm apart). Four treatments were established: one without inoculation and the remaining three with rhizobia isolates, all distributed in a random block design, with four replications.

The inoculation of kudzu seeds with the rhizobia isolates was carried out using the seed covering method at the time of sowing, at a rate of 200 mL of inoculum for every 50 kg of seeds. The kudzu was sown in rows, with doses of 8 kg of total seed ha^-1 (2 kg of pure germinable seed per ha).

At 120 d after sowing, samples from 10 plants of each plot were taken and the total number of nodules and the percentage of total effective nodules were determined. The aerial part of the plants was placed in an oven at 70 ^oC, for 72 h and the ADM was determined. The percentage of ADM (% ADM) was calculated by the relation between the ADM of the control treatment, without inoculation, and the inoculated.

The yield in aerial dry mass of the plants (YADM, t ha^-1) was determined 180 d after sowing, from the equation: YinADM = (green mass x% ADM)/100. The percentage of nitrogen content of the aerial biomass in 200 g of green mass was evaluated by colorimetric methods (^{Paneque et al. 2010}). The percentage of increase of this variable in the inoculated treatments with respect to the control without inoculation was also evaluated.

Statistical analysis. The data from the acidity tolerance tests in the culture medium were subjected to the normality test (Bartlett test) and variance homogeneity (Kormogorov-Smirnov test). Simple classification variance analysis was applied. The Tukey (P <0.05) means comparison test was used to discriminate differences between the means (^{Sigarroa 1985}).

A bifactorial arrangement was used with the inoculation test data under controlled conditions and the strain factors with three levels (1_2, 2_4 and K2) and pH with two (6.8 and 4.5) were taken into account. The data were processed similar to that described above, regarding the test of normality and homogeneity of variance. The SPSS 21 program was used for statistical processing (^{Gray and Kinnear 2012})

The results of the experiment under field conditions were processed by analysis of variance. The Tukey's multiple comparison test was used (^{Sigarroa 1985}) in cases there were significant differences between treatments.

At average values of the petroferric ferruginous nodular gleysol soil of the EEPF Cascajal, Villa Clara, was estimated the confidence interval of means at α = 0.05 (^{Payton et al. 2000}). All results were showed in the SigmaPlot program (2001).

Results

Tolerance and multiplication of rhizobia isolates under acidity conditions. Rhizobia isolates takes up to the fourth growth area in the solid ML medium, with pH 4.5, at six incubation days, so they are microorganisms tolerant to this acidity condition (^{dos Santos Hara and Oliveira 2005}). This performance occurred at four days for the isolated 2_4, at six for 1_2, and at eight days for K2. It took place in the same culture medium with pH 6.8 (figure 2).

Figure 2 Scoring intervals, according to the growth areas of rhizobia isolates in the Petri dish (pH 4.5 and 6.8) during 10 d of incubation. 

In the medium with pH 4.5, isolate 2_4 grew to the third growth area after four days of incubation. The remaining two isolates grew to the second area in the same interval. The K2 isolate took longer to occupy up to the fourth growth zone in the MY medium, with pH 6.8 (8 d) than in the medium with pH 4.5 (6 d).

The three rhizobia isolates produced small colonies, smaller than 1 mm, in MY medium (pH 4.5 and 6.8) at 10 d of incubation (figure 3).

Figure 3 Colony diameter (mm) of rhizobia isolates at 6 and 10 d of incubation (pH 4.5 and 6.8). 

There were no significant differences between the diameter of the colonies produced by the isolate 1_2 in the medium with pH 4.5 with respect to those formed with pH 6.8, at six and 10 d of incubation. At six days, the rest of the isolates formed colonies that were significantly higher at pH 6.8 than at pH 4.5. However, at 10 d, there were no significant differences between the isolates, nor between the colonies of the same isolate under different pH conditions.

In the effect of the pH of the medium on the multiplication of the three rhizobia isolates (figure 4), isolate 2_4 had a CFU number mL^-1 at pH 4.5, which was significantly higher compared to those quantified at pH 6.8. The two remaining isolates showed a similar amount of viable cells under both pH conditions. There wereno significant differences between the three isolates in each of the pH conditions studied.

Figure 4 Number of viable cells of rhizobia isolates in MY medium (pH 4.5 and 6.8) 

Positive effect of rhizobia isolates on the nodulation, growth and yield of tropical kudzu plants, grown under acidity conditions. The analysis of the data from the inoculation test under controlled conditions allowed to detect the influence of each factor separately (strain and pH) (table 2) and the interaction of both factors together, on the nodulation and growth of tropical kudzu plants (table 3).

Table 2 Independent effect of strain and pH factors on the nodulation and growth of tropical kudzu plants 

Treatments	Nten	DMtN	Root length (cm)	ADM (g)	RDM (g)
Strain
K2	3.65^a	0.0044^a	8.02^c	0.0411^b	0.0061^a
1_2	3.15^b	0.0037^b	9.09^a	0.0383^c	0.0058^b
2_4	2.95^c	0.0036^c	8.86^b	0.0424^a	0.0054^c
F-Test	*	***	*	***	***
SE±	0.42	0.0005	0.38	0.0025	0.0003
pH
6.8	3.67^a	0.0041^a	8.98^a	0.042^a	0.0059^a
4.5	2.84^b	0.0038^b	8.33^b	0.040^b	0.0056^b
F-Test	*	***	*	***	***
SE±	0.33	0.0004	0.31	0.0021	0.0003

NteN, number of total effective nodules; DMtN, dry mass of total nodules; ADM, aerial dry mass; RDM, root dry mass, Same letters do not significantly differ (Tukey P < 0. 05, n=10),

Table 3 Combined effect of strain and pH factors on the nodulation of tropical kudzu.  

(Strain*pH)	NNmr	NtN	NeNmr
K2, pH 6,8	3.30^a	3.80^a	3.00^ab
K2, pH 4,5	3.44^a	4.11^a	3.33^ab
1_2, pH 6.8	3.50^a	4.00^a	3.40^a
1_2, pH 4.5	2.10^ab	2.60^ab	1.90^ab
2_4, pH 6.8	3.30^a	4.10^a	3.20^ab
2_4, pH 4.5	0.90^b	1.40^b	1.50^b
SE±	0.61*	0.76*	0.64*

NNmr number of nodules in main root, NtN number of total nodules, NeNmr number of effective nodules in the main root.

Same letters do not significantly differ (Tukey P < 0.05 n=10),

The strain factor influenced all the variables that were evaluated for nodulation and growth of tropical kudzu plants (table 2). The strain K2 stands out, with significant differences with respect to the rest of the strains, except in the root length and the ADM, variables where 1_2 and 2_4 highlight, respectively.

The pH factor, when was independently analyzed (table 2), allowed to identify the negative effect of the acidity of the medium on the nodulation and growth of kudzu plants. However, in the analysis of the influence of both factors (pH and strain) (table 3) on the NNmr, TN and NENmr there were no significant differences between the plants inoculated with K2 and with 1_2 in pH 4.5 and 6.8. In the rest of variables there was no interaction between both factors.

The field condition test showed that rhizobia isolates produced a significant increase in the number of total nodules, ADM and nitrogen content in the aerial biomass of kudzu plants, grown in an acid petroferric ferruginous nodular Gleysol soil, in Cascajal, Villa Clara (table 4).

Table 4  Effect of rhizobia isolates on the nodulation and growth of tropical kudzu plants, grown in acid petroferric ferruginous nodular Gleysol soil, in Cascajal, Villa Clara. 

Treatments	Nodulation		ADM (t ha^-1)	% increase in ADM (t ha^-1)	Nitrogen content in aerial biomass (%)	% increase in nitrogen content in aerial biomass
	TN	NteN (%)
Control without inoculation	21 ^c	31	4.03 ^c	-	2.19 ^b	-
K2	53 ^a	93	7.27 ^a	80.4	3.28 ^a	49.77
1_2	50 ^a	95	7.11 ^a	76.43	3.33 ^a	52.05
2_4	32 ^b	68	5.19 ^b	28.78	2.52 ^b	15.07
SE±	2**	-	0.23**	-	0.04**	-

NtN number of total nodules, NteN number of total effective nodules, ADM aerial dry mass.

Same letters do not significantly differ (Tukey P < 0.05, n=10).

More than 60 % of total nodules were effective in BNF in plants inoculated with rhizobia isolates. K2 and 1_2 highlighted with more than 90 %. Both isolates also produced an increase of more than 75 % of the yield of the aerial dry mass, and more than 45 % of the nitrogen content of the aerial biomass of the kudzu plants.

Discussion

The use of biofertilizers based on rhizobia strains, adapted to stressful conditions in the soil, such as acidity, is a biotechnological practice that offers a solution to improve agricultural production. The isolation, characterization and use of these bacteria in cultures of economic importance, especially those affected by abiotic factors as aggressive as the acidity of the soil, is increasingly important. This research allowed identifying potentialities in rhizobia strains, such as promising microorganisms for the inoculation of tropical kudzu in Cuban acid soils.

The three isolates, Bradyrhizobium K2, 1_2 and 2_4, showed slow growth in the culture medium (figure 2) and formed colonies smaller than 1 mm in diameter (figure 3). These characteristics have been previously reported for the Bradyrhizobium genus (^{Wang and Martínez-Romero 2001} and ^{da Costa et al. 2017}).

The soils acidity constitutes a factor that limits bacterial growth (^{Turan et al. 2010}). However, the three isolates studied in this research showed tolerance to acidic pH. The isolates grew in all areas of the Petri dishes where they were inoculated (figure 2). The acidity condition did not affect the diameter of their colonies (figure 3) and the number of viable cells during the exponential growth phase either (figure 4). Bradyrhizobium strains, equally tolerant to acid pH, have been recently identified (^{Helene et al. 2017} and ^{Jang et al. 2018}).

The three bacterial isolates excrete base to the culture medium (^{Hernández et al.2013}). This characteristic is also representative of the Bradyrhizobium genus (^{Wang and Martínez-Romero 2001}) and has been interpreted as one of the acidity tolerance strategies (^{Graham et al. 1994}), which allows neutralizing the acidic pH and consuming nutrients from the medium. The production of bases could explain the performance of the three rhizobia isolates in the MY medium with pH 4.5.

The isolates 2_4 and K2 showed performances that suggest greater adaptation to acidity conditions (pH 4.5) than in mediums close to neutrality (pH 6.8). In general, they occupied more areas in the Petri dishes under acidic conditions than at pH 6.8. In addition, 2_4 had a higher concentration of viable cells at pH 4.5. Similar results with the K2 isolate were previously obtained (^{Pérez 2010}). Both isolates, in addition to the basic excretion, could have other mechanisms that allow them to remain viable and multiply in acidity conditions in the medium. These mechanisms could be the accumulation of internal buffers, the synthesis of proteins of acid shock, the exclusion of protons, the synthesis of surface lipopolysaccharides and the decrease in the permeability of the cytoplasmic membrane (^{Geddes et al. 2014}).

The physicochemical characteristics of the soils of Cascajal in Villa Clara, mainly its acidity, constitute a decisive factor in the selection of rhizobia populations that reside there (^{Morón et al. 2005}). The mechanisms of tolerance to this condition allow them to maintain intracellular pH between 7.2 and 7.5 to survive in free life way (^{Madigan et al. 2011}) and establish symbiosis with the legumes resident in these soils. The adaptation of rhizobia to acidity requires the joint collaboration of various central functions of metabolism, such as the expression of membrane proteins, related to respiration and changes in the central metabolism of carbon and lipids (^{Draghi et al. 2016}).

As part of this research, inoculation tests of rhizobia isolates were carried out in tropical kudzu seeds under acidity conditions. In the experiment, under controlled conditions, the analysis of the pH factor separately allowed to conclude that acidity decreased nodulation and plant growth. ^{Plá and Cobos-Porras (2015)} reported the negative effect of acidity on the formation of nodules. However, the joint analysis of the strain and pH factors allowed concluding that K2 and 1_2, when inoculated under acidity conditions, managed to counteract the negative effect of acidity on the nodulation of plants (table 3). The acid tolerance of both isolates (figures 2, 3 and 4), perhaps due to their ability to produce base to the medium (^{Hernández et al. 2013}), would neutralize the acidity in the rhizosphere environment of kudzu plants.

This mechanism would allow adequate colonization, nodule formation and subsequent BNF, and would favor N to the kudzu plant for their growth. Therefore, the plant would be better prepared to be establishing under acidity conditions. There were no significant differences in any of the plant growth variables, when the pH and strain factors were analyzed together. These results could support the proposed hypothesis.

The field inoculation test showed the positive effect of rhizobia isolates on effective nodulation, BNF and ADM, as well as on the nitrogen content of the aerial biomass of kudzu plants. Similar results were obtained in this same crop (^{González et al. 2016}) and in canavalia (^{Martín et al. 2015}).

Many legumes require neutral or slightly acidic pH for growth and nodulation. pH values, less than 5.5, generally causes problems in the plant establishment and, consequently, in the BNF (^{Bordeleau and Prévost 1994}). However, the inoculation of kudzu plants with the three rhizobia isolates considerably increased the number of total effective nodules in the acidic petroferric ferruginous nodular Gleysol soil, in Cascajal area (table 4). The acidity tolerance of rhizobia isolates that were used in this test could have been a determining factor for the establishment of an effective symbiosis between the kudzu plants and the bacteria.

A greater number of effective nodules for BNF in leguminous plants allow the increase of nitrogen content. This element is used for the formation of new structures that, in turn, it is an increase in plant growth (^{Gardner et al. 2017}). The increase in the percentages of the nitrogen content in the aerial biomass and of the aerial dry mass of the plants inoculated with the isolates, is clear evidence of this phenomenon

There were nodes in the roots of the plants that were used as a negative control of the experiment. This suggests the presence of compatible rhizobia on the soil in the Cascajal area. However, this nodulation, and the effects that were found in the growth of the plants, were lower to that found in the inoculated plants. The use of more efficient strains and the application of higher concentration of bacteria offer advantages in colonization during root emission.

The rhizobia isolates used in this research were selected in previous researches, as promising for the inoculation of tropical kudzu. This selection was based on some physiological characteristics, such as the use of various carbon sources, the production of polyhydroxybutyrates (PHB) and the nodulation of this forage legume (^{Hernández et al. 2013}).

These strains, in addition to the advantages they have for the form of application on the seed and its tolerance to acidity, have several characteristics that allow them to compete with other soil bacteria during the process of colonization and formation of nodules in kudzu plants. The inoculation of legumes with competitive and specific strains considerably increases the efficiency of rhizobia-legume symbiosis and BNF (^{Andrews and Andrews 2017}).

It is concluded that the use of tolerant acid rhizobia for the inoculation of tropical kudzu, grown under acidity conditions, guarantees the successful establishment of symbiosis. The isolates of Bradyrhizobium K2, 1_2 and 2_4, not only allow an effective nodulation in the BNF, but also increase these variables and the performance of kudzu under acidity conditions in the field. These results constitute an attractive proposal to validate inoculants based on these bacterial isolates in other acidic soils of the country.

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Received: April 22, 2019; Accepted: October 24, 2019

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