Stress tolerant rhizobia inhabiting the root nodules of Gliricidia sepium from selected locations of Anuradhapura district, Sri Lanka

The symbiotic association between Rhizobium and leguminous species plays a significant role in sustainable agricultural systems as it contributes higher amounts of fixed nitrogen to soil through biological nitrogen fixation. Gliricidia sepium is one of the most commonly cultivated agro forestry trees in the world. This leguminous tree is widely distributed throughout Sri Lanka. A very limited number of studies are available on G. sepium–Rhizobium symbiosis in Sri Lanka. The main objective of this study is to isolate and identify the stress tolerant Rhizobium sp. in G. sepium as a preliminary approach to use rhizobial strains for cross inoculation of crop legumes in order to minimize the heavy use of chemical nitrogen fertilizers. The root nodules of G. sepium were collected from seven sites in Anuradhapura district which is located in the dry zone of Sri Lanka. A total of 34 strains were tested for their tolerance to different physiological conditions namely pH, salinity, drought and temperature. Fifteen strains showed a higher tolerance among the isolated 34 strains when grown under a wide range of physiological conditions. When these 15 stress tolerant rhizobial strains were grown under extreme physiological conditions, 12 strains could survive well as they often experience adverse environmental conditions in their natural habitat. DNA fingerprinting analysis with ERIC 1R and ERIC 2R showed that the 15 strains are genetically diverse and they belong to 9 clusters at 71% similarity level.


Introduction
Biological nitrogen fixation and the symbiotic relationship between Rhizobia and legumes play a significant role in reinvigorating sustainable agricultural practices to increase world food production and provide adequate food for the growing human population (Laranjoa et al. 2014). Excessive use of industrially produced chemical N fertilizers had contributed to major health problems to mankind and hazards to the environment and biological nitrogen fixation could provide better solutions to them (Bohlool et al. 1992). Biological nitrogen fixation is a process in which atmospheric nitrogen is converted to ammonia, a plant usable form by microorganisms. This reaction is catalyzed by the enzyme nitrogenase which is found in prokaryotic microorganisms (Cheng 2008). This biological process is an efficient method of delivering nitrogen to the plants (Peoples et al. 1995). According to Burns and Hardy (1975) and Paul (1988) biological nitrogen fixation contributes 139 to 175 million tonnes of nitrogen to terrestrial eco systems per year.
The symbiotic relationship of Rhizobia with legumes acquires a higher value as it is capable of fixing 30 -40 Kg of nitrogen for every tonne of total dry mass produced by crop legumes (Peoples et al. 2009). The global attention on legumes is increasing day by day as they play a significant role in some fields such as agriculture and ecology (Zahran 1999). It is estimated that approximately about 20% of legumes have the capacity for nodulation and nitrogen fixation among 700 genera and 13000 species (Sprent and Sprent 1990). Nodule is a structure which contains all the required modifications to carry out biological nitrogen fixation successfully and they can be seen on the roots of most of the legumes (Sprent 2009)  Black pepper, Coffee and Cocoa are some of the well-known perennial crops grown in the mid country region of Sri Lanka. Gliricidia sepium is the most commonly grown leguminous tree species in these agro-forestry systems in order to provide shade for the crops (Gunaratne et al. 2000). G. sepium is a member of family Fabaceae (Lavin et al. 1991) and this plant species is utilized for several purposes other than as a shade tree such as fuel wood, crop support, green manure, fodder and also to stabilize soils and prevent erosion (Simons and Stewart 1994).
According to Jayasundara et al. (1997), G. sepium was identified as a high N fixer by confirming the experimental results submitted by several workers and it was shown that this legume species has the potential of obtaining 52% -74% of its N from the atmosphere (Awonaike et al. 1992;Liyanage et al. 1994). G. sepium has the ability to enhance soil fertility and productivity when intercropped with coconut (Liyanage et al. 1988).
Anuradhapura district is located in the North Central Province of Sri Lanka. According to the data of the Sri Lankan Department of Meteorology, The Anuradhapura district has an average annual temperature of 27.3 0 C and receives 1368 mm of average rainfall annually.
In his report on Anuradhapura district environmental profile submitted to Central Environmental Authority of Colombo Handawela (1992) indicates the soil type of this area as well-drained, reddish brown, while its pH varies from 5.0-8.0 (Renuka and Senevirathne 2017). G. sepium is widely distributed all over Sri Lanka due to its ability to grow in a wide range of soil types and also the ability to withstand common pest diseases (Chadhokar 1982

pH tolerance of Rhizobial strains
The pH tolerance of rhizobial strains was

Salinity tolerance of rhizobial strains
The tolerance of isolated rhizobial strains for salinity was analyzed by making them to grow in the medium with a series of NaCl concentrations starting from 0.1% and goes up to 3.0% (0.1%, 1.0%, 1.5%, 2.0%, 2.5% and 3.0%). There was no clearly observable growth pattern of isolated rhizobial strains in all the seven sampling sites when increasing NaCl concentration (Fig. 2). The highest growth was observed in K-a strain at 1% NaCl and the minimum growth was shown by K-b at 1.5% NaCl in Kailapathana. Almost all the strains of each site have grown well at 1.0% and 3.0% NaCl concentration levels. The three strains K-a, K-c and K-d showed substantial amounts of growth at all the NaCl concentrations ( Fig. 2 A). In the site Galkulama, Gl-a showed the maxiumum growth at 3.0% NaCl and the strain Gl-b showed the minimum growth at the same NaCl concentration. The growth of Gl-b strain has decreased on increasing NaCl concentration from 1.5% to 3.0% and its growth has changed drastically from 1.5% (0.452) to 2.0% (0.242) NaCl concentration levels ( Fig. 2 B). The highest growth was observed in Ap-c at 1.0% NaCl and the least growth was observed in Ap-e at 2.5% NaCl concentration in Anuradhapura urban area.
All the 5 strains exhibited a better growth at  (Fig. 3).
In the site Kailapathana, K-b showed the highest growth at 0.1% PEG. The least growth was observed in K-d at 0.3% PEG. The two strains Ka and K-c were survived well even at 0.4% PEG (>0.450). All the five strains could grow well at 0.1% PEG (>0.500) (Fig. 3 A). Gl-c showed the maximum growth at 0.1% PEG in Galkulama site.
The minimum growth was shown by Gl-b at 0.4% PEG. Except the three strains Gl-a, Gl-b and Gl-e, the remaining two strains Gl-c and G-d could grow considerably at 0.4% PEG (Fig. 3 B).
In the site Anuradhapura, the growth level of the five strains at 0.3% and 0.4% PEG concentrations was almost similar. The highest growth was observed in Ap-c and the least growth was observed in Ap-a. When increasing the PEG concentration, the growth of Ap-c strain Different salinity levels of the medium were obtained by changing the NaCl concentration. The growth of 34 Rhizobial strains was determined by the optical absorbance at 600 nm at 0.1% , 1.0% , 1.5% , 2.0% , 2.5% and 3.0% NaCl concentrations. There was no clear relationship between growth of Rhizobial strains and NaCl concentration. Md-e strain showed the highest growth at 1.0% NaCl among all the 34 strains.Gl-a, Ap-a,Hr-a, Hr-b, and Md-a showed a substantial growth even at 0.4% NaCl, the highest salinity level. A-Graph for the site  (Fig. 3).

K-d showed the highest growth at 45 ºC in
Kailapathana and the least growth of K-b (0.304) and K-c (0.306) were almost similar (Fig. 4 A).
All the 5 strains have grown well at 35 ºC in the site Galkulama while the highest growth was indicated by Gl-d at the same temperature. The least growth was observed in Gl-a at 40 ºC.
Almost all the five strains could survive well (>0.300) even at the highest temperature (45 ºC) (Fig. 4 B). The rhizobial strains isolated from Anuradhapura urban area showed a better growth at 35º C while Ap-a showed the highest growth at the same temperature. Ap-c strain showed the minimum growth at 40º C. All the five strains indicated a substantial growth at 35 ºC ( Fig. 4 C). The growth of Tb-d strain at 30 ºC, isolated from Thambuththegama was extremely high among all the 34 strains. Tb-c also showed a significant growth at 35 ºC (0.695) compared to other strains. A considerable growth of the strains was observed even at 45º C (Fig. 4 D).
The maximum growth was observed in Hr-e at  showed the maximum growth at 35 0 C in Medawachchiya (Fig. 4 G).

Tolerance of rhizobial strains to combination of different physiological conditions
The strains which showed a higher growth by indicating a higher absorbance value at more than two extreme physiological conditions were chosen mainly by considering the tolerance to   These 15 strains were cultured in a medium with 3.0% NaCl, 0.4% PEG and pH 8.0 and incubated at 36 0 C and the growth was determined by measuring the absorbance at 600 nm. The highest growth was indicated by Kb-d and the lowest growth was indicated by Hr-a under these extreme physiological conditions.

Genetic diversity and relationship between selected rhizobial strains
The amplified DNA fragments of the selected 15 rhizobial strains obtained through PCR were visualized using agarose gel electrophoresis. The DNA banding patterns were highly polymorphic and some of the bands were notable compared to other bands and they were common for many strains. K-a, Tb-e, Hr-a, Hr-b and Md-b showed the same banding pattern (Fig. 6). Tb-b and Hr-d and Kb-d was similar to Md-e at 57% similarity level (Fig. 7).

Discussion
The rhizobial strains inhabiting G. sepium were isolated from seven sites (Kailapathana,  DNA fingerprinting for 15 selected rhizobial strains were performed using ERIC 1R and ERIC 2R primers. DNA banding patterns were highly polymorphic. Some bands were prominent and observed in many strains. A similar banding pattern was observed in the strains K-a, Tb-e, Hr-a, Hr-b and Md-b. Many studies indicate that salt stress has adverse effects on legume-Rhizobium symbiosis and also nodule formation (Zahran 1991;Zahran and Sprent 1986). The high salinity levels tend to decrease the respiration of the nodules and production of cytosolic proteins especially, Leghemoglobin which plays an important role in BNF (Delgado et al. 1993). Rhizobium meliloti and Rhizobium leguminosarum have been identified as some of the salt tolerant rhizobial strains which belong to the genera Rhizobium as they could survive at 300 -700 mM NaCl and upto 350 mM NaCl concentrations respectively (Embalomatis et al. 1994;Breedveld et al. 1991).
Rhizobia inhabiting woody legumes such as Hr-a exhibited low levels of growth while remaining 12 strains showed a better growth as expected by indicating their tolerance to extreme physiological conditions (Fig. 5). This is because clusters. They are genetically diverse in nature (Fig. 7). The dendrogram prepared based on the inverse gel image of ERIC profiling for 15 rhizobial strains did not provide sufficient evidence to prove the relationship between the diversity of Rhizobium sp. and the sampling site.

Conclusion
The rhizobial strains isolated from G.