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Selection of effectiveness of rhizobia nodulating Macroptillium atropurpureum under salt stress

Raúl Carlos López Sánchez, Bettina Eichler-Löbermann1, Raúl Campos Posada2, Gloria Campos-Posada2 and Ernesto Gomez Padilla

Plant Biotechnology Studies Center, Agriculture Science Faculty, University of Granma. Carretera Manzanillo, km 17, Bayamo 85100, Granma, Cuba
rlopezs@udg.co.cu
1 Agronomy and Crop Science, University of Rostock, Justus-von Liebig-Weg 6 18059 Rostock, Germany
2 Universidad Autónoma de Coahuila, Barranquilla sin número, Colonia Guadalupe, Monclova, Coahuila, CP 25730, México

Abstract

The response of Macroptillium atropurpureum to inoc­ulation with rizobia native isolates and commercial rhizobia strain was studied under salt stress. The study was carried out under semi controlled conditions in green­house as well as under field conditions. The effects of two rhizobia isolations (Jd 10 and Jd 11) and one commercial rhizobia strain (1030) were com­pared to control and mineral N fertilization taking into account yield and nitrogen fixation parameters. Under semi controlled conditions, the best results for yield and nitro­gen fixation parameters were found for the native rhizobia isolate (Jd10). In this treatment, together with the N fertilizer treatment, the highest root and shoot weights and N uptakes were found. Furthermore, the Jd 10 treatment showed the highest K:Na ratio, the highest ureide contents, highest values of chlorophyll a and b in the shoot, and the best index of inoculation effectiveness. In the field experi­ments, the Jd 10 rhizobia isolate treatment had positive effects on all the parameters in compari­son to other treatments. The results emphasized the great potential of rhizobia isolate Jd10 in symbiosis with M. atropurpureum on salt affected soils.

Key words: inoculation, legumes, nitrogen fixation, symbiosis


Introduction

In Cuba about 42 % of soils used for pasture production are affected by salinity, which causes considerable decreases in pasture yields and animal production. Some of the alternatives to improve the fertility of these soils are the use of salt tolerant legume species, as well as the use of salt tolerant microorganisms (López et al 2007).

Salinity is considered a significant factor that affects crop production and agricultural sustainability in arid and semiarid regions of the world. Salt tolerance in plants is a complex phenomenon that involves morphological and developmental changes, as well as, physiological and biochemical processes. Salinity disrupts cell function, through the toxic effects of specific ions and by osmotic effects, or both (Munns 2005).

The identification and use of plants adapted to saline environments is very important for raising the productivity of crops in these areas. For this purpose, cultivation of legumes is recommended for the rehabilitation of arid saline soil. This solution is not only likely to make abandoned soils productive but will also ensure the conservation and improvement of the environment (Zahran 2010).

The rehabilitation of these degraded lands is limited to two possibilities: first, the exploitation of native plants to arid environments and second, devising efficient systems to use limited saline water resources either by preventing its unproductive evaporation loss (Tomar et al 2003).

Annual pasture legumes and the naturally-growing annual herb legumes include salt-tolerant species, which are usually adapted to increased soil salinity. Al-Sherif et al (2004) and Zahran et al (2007) reported the existence of Melilotus indicus and Melilotus intertexta in salt-affected lands of Egypt. Some annual pasture legumes (e.g., Melilotus siculus and Medicago polymorpha). persist in saline soils (ECe > 8 ds.m-1) of Australia (Boschma et al 2008; Nichols et al 2008).

Macroptillium atropurpureum is used for soil conservation and as a cover crop, fallow crop, or as a forage crop sown with upland rice in tropical regions (FAO 1991). Its value as a protein bank for dry season feeding is reduced by its tendency to drop its leaves under very dry conditions. It tolerates drier conditions and poorer soils, can produce seed in both spring and autumn, easier to establish than either glycine or Desmodium, has resistance/tolerance to root knot nematodes, and is moderately tolerant to amnemus weevil larvae, can be used by cattle in the warmer months, or left as a standover to help fill late autumn/winter feed gaps. Competitive against weeds and efficient fixation of nitrogen. (Zahran 2010).

In Cuba, there have been several studies about the behavior of pasture legumes in tropical conditions but not present results on the response of this species to salt stress in symbiosis with rizhobia isolations and strains. In the present work the symbiotic efficiency of rhizobia isolation and a commercial strain in controlled and field conditions in M. atropurpureum under salt stress conditions is investigated.


Materials and methods

Rhizobia

Two native rhizobia isolated from root nodules of M. atropurpureum growing in salt affected soils, as well as one commercial strain obtained from the National Forage and Pastures Institute, Havana, Cuba were used (Table 1).

The roots of M. atropurpureum (siratro) growing in salt affected soils, after harvesting were soon transported to the laboratory in plastic bags. The roots of plants were thoroughly washed and the nodules were severed and sterilized in 95% ethanol for 5 s and 3% H2O 2 for 5 min. Each nodule was crushed and the content of the nodule was transferred onto a petri dish with yeast-extract mannitol agar (YEMA) (Vincent 1970; Somasegaran and Hoben 1985). Petri dishes were incubated at 28 °C until typical colonies of rhizobia appeared. Single colonies were marked and checked for purity by repeated streaking on YEMA medium (Vincent 1970) and verifying a single type of colony morphology, absorption of Congo Red (0.00125 mg kg-1) and a uniform Gram-stain reaction, cetolactase test; PGA growth test and acid/alkaline reaction were evaluated on YEMA containing bromothymol blue (0.00125 mg kg-1) as an indicator (Alberton et al 2006). The infectivity test was carried out.

Table 1. Rhizobia used in the experiments

Rhizobia

Procedence

Isolation Jd10

20°19´ N and 76°33´ O

Isolation Jd11

20°19´ N and 76°33´ O

Strain 1030

Commercial strain

Culture conditions

Rhizobia strain and isolations were grown in 250 mL Erlenmeyer flasks containing 100 mL of yeast extract mannitol broth at 28 °C for 6 days on a rotary shaker at 160 rpm (Vincent 1970).

M. atropurpureum

Seeds were surface-sterilized with 95% ethanol for 5 min, transferred into hydrogen peroxide solution of 3% for 5 min, and rinsed 6 times with sterile water (Atici et al 2005). The germinated seeds in both experiments were treated with 2 ml of the bacterial cultures.

Plant material

M. atropurpureum seeds were obtained from Forage and Pastures National Institute; Havana; Cuba. The experiments were established under semi controlled conditions in a greenhouse as well as under field conditions. The clay soil used as growth medium had a pH of 7.5, 5 % of organic matter, low amounts of plant available phosphate (P2O5: 0.45 mg. 100g-1) and potassium (K2O: 4.40 mg.100 g -1) (extracted in 0.05m sulphuric acid), but high amounts of sodium (Na: 5 mg.100 g-1 and come from salt affected areas). All soil tests followed the methods described by MINAGRI (1981) according the international standards.

Greenhouse experiment

A randomized design with five replications was established in a greenhouse with the following treatments: (1) -N-I (without N fertilizer and without rhizobia); (2) +N-I (fertilized with 150 kg ha-1 , N); (3) Rhizobia isolation Jd 10; (4) Rhizobia strain 1030; and (5) Rhizobia isolation Jd 11. Ten plants per treatment were cultivated in pots with 4 kg of saline soil. N was applied at five moments of vegetative period before flowering, corresponding 30 kg ha-1 to each one. After 120 days, fresh leaves were harvested and chlorophyll a and b contents were measured by the method of Lichtenthaler and Wellburn (1981). Plants were harvested; root and shoot fractions were separated and nodules number were determined. Shoot samples were dried at 65 °C for 48 h. After drying, the dry weight of the nodules (DMN), the dry weight of aboveground biomass (DWP), and the dry weight of roots (DWR) were determined. Furthermore, total nitrogen (Nt) (Kjeldahl method), ureide content (after Boddey et al 1987) and the Na and K content of the plants were colorimetrically determined after dry ashing. The nitrogen response index (IRN) and the inoculation effectiveness index (IEI) were calculated using the following equations:

Where (+N) and (-N) represent the treatments with and without nitrogen, and (+I) and (-I) represent the inoculated and non-inoculated treatments, respectively (CIAT 988).

Field experiment

The experiment was carried out in two consecutive years from September till January. The plants were sowing with density of 8 kg per hectare and 20 plant per treatment were tested. Four different treatments in three replications were investigated in the field experiment: (1) -N-I (without N fertilizer and without rhizobia inoculation); (2) +N-I (fertilized with 150 kg ha-1 N); (3) Rhizobia isolate Jd 10; and (4) Rhizobia strain 1030. 150 days after sowing, 10 plants were harvested and the yield and N fixation parameters were measured, as described for the greenhouse experiment.

Statistical analysis

The data obtained were statistically analysed using the analysis of variance. The means were compared by the DUNCAN multiple range test (Duncan 1955) using the STATISTICA software (StatSoft 2011).


Results

Morpho-physiological characterization of rizhobia isolations

The rizobia isolated from M. atropurpureum showed variability in growth speed in YEM media, as well as alcali and acid production and differences in colonies morphology. M. atropurpureum has the ability to nodulate with a wide range of rhizobia strains; this could be the cause of the isolations with different features. (CIAT 1991)

Table 2. Morpho-physiological traits of rhizobia isolated from Macroptillium atropurpureum.

Isolations

Colony
diameter

Colony
morphology

Gram
reaction

Congo red
absortion

Growth

Jd10

Undefined

Aqueous

-

-

Slow

Jd11

2,0 and < 0,5

Creamy

-

-

Fast

 

Strains

Alcali
production

Acid
production

Cetolactase
test

Infectivity

PGA growth
test

Jd10

+

-

-

+

-

Jd11

-

+

-

+

-

Greenhouse experiment
Shoot and root dry weight

The N supply as well as the inoculation with rhizobia isolation Jd 10 and rhizobia strain 1030 showed increases in shoot and root biomass accumulation compared to the control without nitrogen and rhizobia isolation Jd11. The inoculation with the Jd10 isolation showed a higher shoot dry weight compared to the inoculation treatments. No differences were observed in root dry weight between both strains. (Figure 1).

Figure 1. Effect of N fertilization (+N-I), rhizobia isolation (Jd10 and Jd11) and rhizobia strain (1030) on shoot and root dry biomass
accumulation of M. atropurpureum. Different letters indicate significant (p<0.05) differences between treatments.
Nodule number and nodule dry weight

The inoculation with Jd10 isolation produced the highest values for both nodulation parameters (Figure 2). The presence of nodulation in the control without nitrogen could be caused by the native rhizobia in soil, but the low effectiveness didn’t allow to obtain high shoot and rooting biomass yields.

Figure 2. Effect of N fertilization (+N-I), rhizobia isolation (Jd10 and Jd11) and rhizobia strain (1030)) on nodule number and nodule
dry weight of M. atropurpureum. Different letters indicate significant (p<0.05) differences between treatments
Na and K accumulation

Shoot tissue Na concentrations were significantly decreased when plants were fertilized with N or inoculated with the rhizobia isolation Jd10 and strain 1030, while the opposite was observed for K. For the K/Na ratio, the treatment with N fertilization and inoculation (Jd10 and 1030) was not significantly different to the control with N fertilizer (Figure 3). The results show that the strategies of M. atropurpureum for salt tolerant are salt exclusion and limitation of K+ loss, but there is also strong relation between effective strains and salt exclusion capacity.

Figure 3. Effect of N fertilization (+N-I), rhizobia isolation (Jd10 and Jd11) and rhizobia strain (1030) on Na and K accumulation and on the K:Na ratio
in the aboveground biomass of M. atropurpureum. Different letters indicate significant (p<0.05) differences between treatments.
Chlorophyll content

The chlorophyll content (a and b) shows significant differences between the different treatments. Inoculation with Jd10 yielded the highest chlorophyll a content, while the chlorophyll b content was highest in the control treatment with N addition, followed by the treatment with Jd10 (Figure 4).

Figure 4. Effect of N fertilization (+N-I), rhizobia isolation (Jd10 and Jd11) and rhizobia strain (1030) on ureides accumulation, amount of
chlorophyll a and b of M. atropurpureum. Different letters indicate significant (p<0.05) differences between treatments.
Total N content, ureides accumulation, nitrogen response index (IRN) and inoculation effectiveness index (IEI).

N accumulation was highest in plants inoculated with the rhizobia isolation Jd10 in comparison to the other inoculation treatments as a result of abun­dant and effective nodulation and more than four times the N content of the plants inoculated with rhizobia isolation Jd11 (Figure 5). The rhizobia isolation Jd10 also showed the highest IEI, but IRN was similar for all inoculation treatments (Figure 6).

The ureide contents used as an indicator to evaluate N fixation showed the highest values for the rhizobia inoculation Jd10, indicating effective symbiosis between this isolation and M. atropurpureum. A negative effect of mineral N supply on ureide accumulation was observed for the +N treatment (Figure 5).

Figure 5. Effect of N fertilization (+N-I), rhizobia isolation (Jd10 and Jd11) and rhizobia strain (1030)) on N content and ureides
accumulation of M. atropurpureum. Different letters indicate significant (p<0.05) differences between treatments.

The poor response of 1030 strain and rhizobia isolation Jd11 (Figure 5 and 6) could be due to low effectiveness strains and high salinity. The combination of negative factors has strong influence in dry weight shoot and nodules, N content and ureides accumulation in shoot.

Figure 6. Effect of inoculation with rhizobia isolation Jd10 and rhizobia strain 1030 on nitrogen response index (IRN) and inoculation effectiveness
index (IEI) of M. atropurpureum. Different letters indicate significant (p<0.05) differences between treatments.
Field experiments

Dry matter yield and N accumulation were highest in the control treatment with addition of N, followed by the plants inoculated with the Jd10 strain. Nodules number, IEI and ureides content were higher for plants inoculated with the Jd10 strain (Picture 1), which demonstrates the effectiveness of this strain (Table 3).

Photo 1. Nodulation of M. atropurpureum with Jd10 rhizobia islation in field experiment

The lowest yield and N uptake for the control without N and without inoculation are related to the low nodule number, which is an indicator of the low presence of native rhizobia in these soils. M. atropurpureum is promiscuous for rhizobia requirements (FAO 1991), but for these conditions (salt stress) it is necessary to inoculate to obtain yield increases without necessity of N fertilizer.

Table 3. Effect of N fertilization (+N-I), rhizobia isolation Jd10 and rhizobia strain 1030 on yield, total N uptake, nodule number (Nnod), ureides accumulation, nitrogen response index (IRN) and inoculation effectiveness index (IEI) of M. atropurpureum. Different letters indicate significant (p<0.05) statistical differences of the mean.

Treatments

Yield
(t ha-1)

N uptake
(kg ha-1)

Nnod *

Ureides
(mmol kg-1 MS)

IRN

IEI

+N –I

3.12 a

7.3 a

1.41 d (2)

33.1 d

63.3

-N-I

1.30 d

2.1 d

2.23 c (5)

45.3 c

Jd10

2.86 b

6.2 b

4.58 a (21)

70.8 a

57.9 a

1030

2.11 c

4.6 c

4.21 b (18)

61.3 b

43.5 b

*Transformed data by mean √x , Original values in bracket


Discussion

According to our observations the decrease in root and shoot biomass suggests that Na+ and/or Cl- toxicities in the tissues could have inhibited root elongation, food synthesis and its translocation to the growing shoots. This might have been the cause of low root and shoot biomass in plants exposed to higher salt concentrations. This behavior has been reported in other studies involving chickpea (Cicer arietinum L.), field pea (Pisium sativum L.), faba bean (Vicia faba L.), cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris L.) (Munns 2002), (Meloni et al 2001), (Gómez et al 2014).

In salt stress conditions, inoculation with rhizobia isolation Jd10 and strain 1030 increases shoot and root biomass acumulation in M. atropurpureum while the Jd11 isolation seems to be inefficient.

Physiological mechanisms conferring exclusion that operate at the cellular and whole plant level have been described elsewhere in the literature and with particular reference to selectivity for K+ over Na + (Jeschke and Hartung 2000; Tester and Davenport 2003). There is strong correlation between salt exclusion and salt tolerance in many species (Munns and James 2003), i.e. for rice (Lee et al 2003; Zhu et al 2004) and wheat (Poustini and Siosemardeh 2004). Similarly, Cuartero et al (1992) reported that high uptake of Na+ inhibit K + accumulation. Tolerant species accumulate less Na+ and more K+ than sensitive species (Tipirdamaz and Cakirlar 1989, Padilla et al 2016, Weisany et al 2013).

Greater differences between treatments were found for chlorophyll a than for chloro­phyll b, which may be due to higher sensitivity to salt stress of chlorophyll a (Figure 4). These results differ from the results obtained by Martínez et al (1996) in Vigna unguiculata using comparable salt stress levels, who reported a larger variation in chlo­rophyll b content in response to salt stress, similar to the results obtained by O’Neill et al (2006) in corn crop.

Furthermore, the inoculated control treatment had the highest values of clorophylls and generally bacterial inoculations significantly increased above parameters compared to the uninoculated control treatment under saline conditions. These results confirm that the ability of legume species to grow and survive in saline conditions for improve when they are inoculated with salt tolerant rhizobia strains (Shamseldin and Werner 2005). Moreover, significant increases in chlorophyll content, an indication of N2 fixation (Kantar et al 2003; Oğutcu et al 2008), N percentage, and total N due to bacterial inoculation supported the hypothesis that biological N2 fixation by the Rhizobium could be responsible for the observed higher N uptake of inoculated plants under both non-saline and saline conditions (Kantar et al 2003; Oğutcu et al 2008).

Ureide accumulation under drought and salt stress has been abundantly reported for several soybean cultivars, and nitrogen fixation inhibition has been associated with high ureide levels in shoots and leaves (Serraj et al 1994). However, several reports have challenged the role of shoot ureide accumulation as a regulatory signal to inhibit nitrogen fixation, suggesting that only a rise in ureide within the stressed nodules may be involved in nitrogen fixation inhibition (King and Purcell 2005).

Recently, it has been shown that ureides play a critical role in cell protection under oxidative stress conditions, such as senescence (Brychkova et al 2008). Under such stressful situations, ureide metabolism plays an essential role in nitrogen mobilization and recycling from either protein or nucleic acid turnover (Yesbergenova et al 2005; Brychkova et al 2008).

Salt tolerance of Jd10-M. atropurpureum combination appears to be associated with stability in nodule conductance to O2 diffusion and the capacity to form nodules under salt constraint. Nodule conductance to O2 diffusion has been found to be a major factor in the inhibition of N2 fixation by salinity that severely reduces the production of legumes. (L’taief et al 2007, Vriezen et al. 2013, Kumar 2014).

Salt stress adversely affects legume production mainly due to the plant dependence on symbiotic N2 fixation for their nitrogen requirements. Some processes affected in such conditions involve the limitation of the host plant growth, the symbiotic development of root-nodule bacteria, and, finally, the nitrogen-fixation capacity. Other physiological responses, result of saline stress, consist of an accelerate greening of the nodules and lower the leghemoglobin content (Tejera et al 2006, Karmakar et al 2015)

According to Wilson (1985), this species under salt stress controlled conditions is more tolerant to glycine and presents less injury and fewer plant deaths. Few report about this species in salt affected soils are present in the international scientific literature.


Conclusions


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Received 16 January 2019; Accepted 22 January 2019; Published 1 February 2019

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