Morphological and Biochemical Characterization of Soybean Nodulating Rhizobia Indigenous to Zambia

Soybean [Glycine max (L.) Merrill] is known for nitrogen fixation by rhizobia present in the soil with which it establishes an efficient symbiosis. In Zambia, current rhizobial inoculants used in soybeans production are based on non-indigenous strains; this creates a need to isolate local strains that can be used for the development of local inoculants for soybeans in Zambian soils. This paper reports the isolation and characterization of rhizobial isolates from virgin and cultivated soils of the three agro-ecological regions of Zambia. Rhizobia were isolated using the Trap Method and characterized using selected morphological and biochemical markers. A total of 61 isolates were isolated on Yeast Extract Mannitol (YEM) agar medium. Isolates varied in colony form, color, margin and texture. From the 61 isolates from the three regions, 87 % were circular, 8 % irregular and 5 % punctuate in form with 100 % convex elevation. The isolates had 88% entire, 10% undulate and 2 % lobate colony margins with different colors – 56 % cream, 24 % white, 11 % yellow, 5 % transparent and 3 % pink. Transparent colonies were peculiar to Region I and III while pink colonies were peculiar to Region III. All isolates produced mucous, were gram negative and rod shaped, a characteristic of rhizobial cells. None of the isolates could tolerate extremes of pH (4 and 9) in growth medium but grew well at pH 6.8. All isolates utilized glucose as a source of carbon. Based on the Bromothymol Blue (BTB) assay, 59 isolates were fast growing while two isolates from cultivated soils of region II were slow growing. The fast growing 59 isolates showed an acidic reaction changing the medium from green to yellow, while the others showed an alkaline reaction. Based on the results, the 59 fast-growers could be Ensifer fredii or/and Rhizobium tropici rather than Bradyrhizobium. However, further tests to confirm these findings using ketolactose, genetic characterization and inclusion of reference strains, are still needed and are being recommended here.


Introduction
Soybean [Glycine max (L.) Merrill.] is an important legume crop that was introduced to Zambia in the 1930s, but has remained an insignificant crop among smallholder farmers and thus is grown mostly by commercial farmers.The crop is now grown by both small and large scale farmers.It is adapted to agro-ecological regions II (800 -1000 mm of rainfall) and III (above 1000 mm of rainfall) of Zambia and will grow well anywhere maize grows (Miti, 1997).Following successful infection with soybean-nodulating rhizobia, soybean forms root nodules where rhizobia symbiotically fix atmospheric nitrogen.The main soybean-nodulating rhizobia are Bradyrhizobium japonicum, Bradyrhizobium elkanii, Sinorhizobium xinjiangense, Mesorhizobium tianshanense, Ensifer fredii, and Rhizobium tropici (Shiro et al., 2013).Of these, Bradyrhizobium japonicum and Bradyrhizobium elkanii are both slow growing while Ensifer fredii, and Rhizobium tropici are fast growing (Li et al., 2011).Soybean offers a variety of possible benefits to crop production systems, diets, and incomes of its producers.In addition to being a potentially profitable cash crop, the high protein content (about 40%) in soybeans means it could also contribute to an improved nutritional status of its consumers (Dixit et al., 2011).Soybean is an economically significant crop because of its wide use for production of cooking oil, human food, stock feed and several industrial products (Miti, 1997).Soybean production also has potential agronomic benefit of rejuvenating soils; soybean canopies protect the soil from recurrent erosion, fix atmospheric nitrogen into the soil and decaying root residues improve soil fertility.This soil improvement leads to elevated levels of sustainable agriculture with minimal input requirement.
In Zambia, soybean is mostly grown for industrial purposes; for example, in the production of edible oils, soy chunks, soy milk, and soy meal.The by-product (soy cake) can be fed directly to animals or processed with other ingredients into animal feed stock.With an increase in animal population in Zambia, industrial demand for soybeans is likely to increase because it provides a cheaper source of high quality protein in feed rations (Lubungu, Burke, & Sitko, 2013.Current production of soybean in Zambia is estimated at 214,179 metric tons (CSO/MAL, 2013).This shows an increase of more than 17 times from what Tefera (2011Tefera ( ) reported in 2006. .Biological Nitrogen Fixation (BNF) is important in farming systems and can be ameliorated by simple and inexpensive inoculation procedures.Extended use of biological fertilizers maintains soil fertility of agro-ecosystems, thereby reducing the cost of chemical fertilizers borne by farmers.In Zambia, inoculants are produced by Government facilities essentially to service emerging soybean industries (Hardarson & Broughton, 2003).
While inoculation with Bradyrhizobia has been shown to improve nitrogen fixation and subsequent soybean yield (Shiro et al., 2013), the efficiency of the inoculants maybe poor if the inoculated strains cannot out-compete the indigenous ones or cannot establish an efficient symbiosis with the host plant (Mweetwa et al., 2014).Therefore, a complete understanding of the ecology of indigenous soybean-nodulating rhizobia with respect to their genetic diversity and the environmental factors associated with their localization and dominance in the soil is important.Some soils may be devoid of rhizobia (Hassen, Bopape, Rong, & Seane, 2014), contain low numbers of effective strains (Radke, 2003), or have high numbers of ineffective or partially effective strains (Herridge, 2002).As early as 1961, four indicators necessitating inoculation were outlined as being the unavailability of a related legume in the immediate cropping sequence of the field, previous unsatisfactory nodulation of the same crop, a cropping sequence of a legume followed by non-legume and land undergoing reclamation (Allen & Allen, 1961).The surest indicator for inoculation is the prevailing numbers of rhizobia in the soil.It is better to over-inoculate than to produce N-deficient crops.
In Zambia, current rhizobial inoculants used in soybeans production are based on non-indigenous strains; this creates a need to isolate local strains that can be used for the development of local inoculants for soybeans grown on Zambian soils.The ability to select elite strains indigenous and adapted to prevailing conditions and soybean cultivars relies on the assessment of the diversity of rhizospheric rhizobial communities.There is currently no known information on local strains of rhizobia nodulating soybean in different soils of Zambia.This paper reports results from a study conducted to isolate and characterize rhizobial strains infecting a common genotype of soybean in Zambia.We isolated rhizobial strains infecting Magoye, a common promiscuous genotype of soybean in Zambia and characterized these isolated strains by using morphological and biochemical markers.

Site description and Soil Sampling
Zambia is divided into three agro-ecological regions largely based on the average amount of rainfall received annually.The study was conducted on soils collected from selected sites in all the agro-ecological regions of Zambia.Soil samples were collected from Mambwe in agro-ecological region I (less than 800 mm of annual rainfall) located at S13 o 15'22.6"and E031 o 55'08.3";Chipata in agro-ecological region II (between 800 mm and 1000 mm of rainfall annually) at S13 o 38'49.8"and E032 o 34'22.3";and Luanshya in agro-ecological region III (greater than 1000 mm of rainfall annually) at S13 o 11'45.5"and E028 o 20'49.4".All the fields from which soil samples were collected had soybean grown on them in the previous growing season (2012/13).Additionally, adjacent fields that had never been cultivated (virgin) were identified and soils were collected from them likewise.
Soil sampling was done following the procedure outlined by Barker and Pilbeam (2007).Soil samples were randomly collected from various points to a depth of 20 cm.These soil sub-samples were then thoroughly mixed to make composite samples.Six composite samples were collected from each location in total (virgin and cultivated).Five kilograms of soil from each composite sample was then air-dried and sieved (2 mm) for characterization.

Rhizobia Isolation
Rhizobia were isolated from soils using the Trap method (Dubey & Maheshwari, 2006).A promiscuous soybean genotype (Magoye) was grown in pots containing five (5) kg of soil collected from the different agro-ecological regions as previously described.Pots were set up in the greenhouse in a completely randomized design (CRD) with three replicates.Five (5) healthy nodules of soybeans were collected from each plant for rhizobia isolation at eight (8) weeks after planting.Nodules were first washed thoroughly with tap water, followed by a rinse with sterile distilled water.Nodules were then immersed in 95% ethanol for 5-10 seconds and in 4 % Sodium hypochlorite for 3 minutes and then finally rinsed five times with sterile distilled water.Each sterilized nodule was then transferred into a sterile test tube containing 1 ml sterilized distilled water.Using a sterile glass rod, each nodule was crushed, after which a loopful of the crushed nodule material was streaked on the Yeast Extract Mannitol (YEM) agar.YEM was prepared from 10 g mannitol, 0.5 g di-potassium hydrogen phosphate (K 2 HPO 4 ), 0.2 g Magnesium sulphate (MgSO 4. 7H 2 O), 0.1 g Sodium chloride (NaCl), 1g yeast extract and 15g agar suspended in 1 litre distilled water (adjusted to pH 6.8 using 1 M HCl and 1 M NaOH before the addition of the agar).The plates were then incubated for 48-72 hours in the dark in an incubator at 27 °C.Single colonies were selected and re-streaked on the same medium; this process was repeated until pure single colonies were obtained.

Morphological Characterization
Isolated colonies were characterized on the basis of colony morphology-form, elevation, margin and colour (Aneja, 1996) and on the cell shape (Bisset, 1959).

Biochemical Characterization
Biochemical characteristics included: the ability of the strains to utilize glucose as a carbon source, Gram stain reaction, growth rate (fast vs. slow), mucous production and pH tolerance.Acid or alkali production from glucose indicates glucose-C source utilization.This assay was conducted by replacing mannitol in the YEM with glucose (10 g/l) and adding an indicator for pH change, Bromothymol Blue (BTB; 0.025 g/l).Plates were then incubated to observe colour change from green to blue or yellow as indicators of alkali or acid production, respectively, arising from the utilization of glucose.Gram staining was conducted to confirm that the isolates were gram negative, a characteristic of rhizobia (Bisset, 1959;Somasegaran & Hoben, 1985).To determine whether isolates were fast or slow growing, YEM agar was supplemented with BTB; isolates were classified as fast if they turned the medium yellow and slow growers if they turned the medium blue (Chen et al., 2001).Mucous production assessment was based on the appearance of isolates on YEM agar plates after the incubation of 7 days at 27 °C.;isolates were scored based on the amount of mucous (exo-polysaccharides) they had produced during the incubation period (Sayyed, Jamadar, & Patel, 2011).To analyse the tolerance of isolates to extremes of pH, YEM agar media were prepared with pH adjusted to 4.0, 6.8 and 9.0.After inoculation, the plates were kept at 27 °C and growth was observed after 72 hours.

Selected Soil Characteristics and Classification
The texture of the soils collected from regions I and II were sandy loam while those from region III were loamy sand (Table 1).Soils from Mambwe were classified as Fluvisols, which in the Food Agriculture Organisation (FAO) World Reference Base (WRB) for Soil Resources is a genetically young soil in alluvial deposits (IUSS Working Group WRB, 2007).The soils that were collected from Chipata were classified as Alisols, which are moderately weathered but strongly leached acid soils; while the soils collected from Luanshya were classified as Acrisols which are highly weathered, strongly leached and of poor fertility.
Critical values for key soil parameters are a rough guide on the fertility status.Depending on the concentration determined through laboratory analysis, the critical value might indicate whether the soil is able to supply sufficient quantities of a nutrient for crop growth (Fairhurst, 2012).Critical values also differ from crop to crop.The soil reaction (pH value) was 6.0, 5.8 and 4.8 for soils from region I, II and III, respectively (Table 1).The other parameters, SOC, available P and total N were below the critical levels in all the three regions.In this study, SOC in regions I, II and III was 47%, 72% and 55% less than the critical level of 1.5%, respectively (Table 1).Available P was 53%, 47% and 25% lower than the critical level of 15 mg/kg soil, for regions II, III and I, respectively.For all regions, N content was 67% lower than critical value.

Isolation and Characterization of Rhizobial Isolates
A total of 61 isolates from soybean nodules were obtained, of these 22, 17 and 22 were from region I, II and III, respectively.The isolates were designated using a combination of the site (district name) abbreviation, abbreviation for soil (V, Virgin: C, Cultivated), nodule number (1-5), replication number (RI to RIII) and letters A to C depending on the number of different colonies on the initial YEM agar plates to be streaked (e.g., for Mambwe district, MV1RI, MC1RI, MC1RII A).The colonies obtained from region I were all circular in form, all convex in elevation, with entire or smooth and undulate margins with cream, yellow, white or transparent colors (Table 2).Isolates obtained from region II varied from irregular, punctuate to circular in form, all colonies had convex elevation with entire, smooth margins to lobate margins.The colors of isolates were observed that is cream, yellowish and white (Table 2).In the case of colonies obtained from region III, colony morphology varied from circular, irregular to punctuate, all colonies had a convex elevation with entire or smooth margins to undulate margin and different colors of isolates were observed that is cream, yellow, white, transparent and pink (Table 2).
From the 61 isolates from the three regions, 87 % were circular, 8 % irregular and 5% punctuate in form with 100 % convex elevation.The isolates were 88 % entire, 10% undulate and 2 % lobate margins with different colors -56 % cream, 24 % white, 11 % yellow, 5 % transparent and 3 % pink.Transparent colonies were peculiar to Region I and III while pink colonies were peculiar to Region III.

Glucose-C Utilization, Gram Stain Reaction, Growth Rate, Mucous Production and Tolerance to pH Extremes of Rhizobial Isolates
After incubating the plates containing BTB at 27 ºC for 72 hours, 97 % of the isolates showed an acidic reaction, changing the medium colour from green to yellow while 3 % of the isolates changed the colour of medium from green to blue showing an alkaline reaction (Table 3; Figure 1A), an indication that all isolates could utilize glucose as a carbon source.
Table 3. Gram stain and acid/alkali reaction of the isolates from agro-ecological regions I, II and III *Based on acid/alkaline production on YEM agar + BTB at pH 6.8, 27 o C. Acid production changed colour of plate from green to yellow; alkaline production from green to blue.
The 61 rhizobial isolates were all gram negative and rod shaped as revealed by Gram's staining technique (Table 3; Figure 1B).Exo-polysaccharide (mucous) production of the isolates after 7 days of incubation is indicated in Table 4.All 61 isolates produced mucous, although the extent of production varied from low, intermediate to high.A higher percentage of isolates were high and intermediate mucous producers.+ indicates extent of mucous production after 7 days growth of isolates.
Rhizobial growth was good on medium at pH 6.8 (27 °C) (Figure 1C), however, this was not the case for pH 4.0 and 9.0 at the same temperature.

I
average generation times of 2 to 4 h and tend to depress the pH of the medium, while the slow-growers have average regeneration times of 6 h (Sadowsky et al., 1983).Bradyrhizobium japonicum and Bradyrhizobium elkanii, the species that typically nodulate soybean have been classified as slow growers.The results of this study indicate that 59 out of 61 isolates from the three agro-ecological regions were fast growers, while only 2 from region II cultivated soils were slow-growers.Based on this observation and the knowledge that soybean are also nodulated by Ensifer fredii and Rhizobium tropici which are fast growers (Li et al., 2011;Kaur et al., 2012), we speculate that the 59 isolates are in fact Ensifer fredii and/or Rhizobium tropici rather than Bradyrhizobium.Without molecular characterization, this cannot be stated with certainty but beckons further characterization of the isolates using DNA extraction and sequencing tools.The ability to grow fast has been implicated in the establishment of nodules and the subsequent nitrogen-fixing abilities of the organism.
While rhizobia are commonly reported to be Gram negative, non-spore forming and rod-shaped, earlier work suggested that they can present as weakly Gram-positive bacilli forming occasional refractile spores (Bisset, 1959).This suggests possible pleomorphism for rhizobia making it possible for them to assume this form under certain culture conditions and the Gram-negative, rod-form with polar flagella, under different culture conditions.The results of the current study indicated Gram-negative and rod-shaped cells.
Growth of rhizobia in soils is sensitive to pH; pH has in fact been shown to limit survival and persistence in soils.The results of this study show normal growth at pH 6.8, but limited growth at pH 4 and 9.These results are similar to what has already been shown by others (Kaur et al., 2012) that the best rhizobial growth is in media with pH around neutral.On the other hand, Sadowsky et al. (1983) have shown that slow-growing rhizobia such as Bradyrhizobium, can have a high level of tolerance to acid conditions (pH 4.5) while fast-growers can tolerate alkaline conditions of pH 9 and 9.5 in growth media.This observation in-vitro agrees with what has been observed in China that Ensifer (acid producers, fast-growers) are dominant in alkaline-saline soils while Bradyrhizobium japonicum (alkaline producers, slow-growers) in acid soils (Yan et al., 2014).Both of these studies, indicate that fast-growers are relatively more alkali tolerate and acid sensitive than slow growers.This attribute is particularly important when considering strains of rhizobia to be included in the development of inoculants suited for use in different regions with differing soil reaction.From our study, both fast-and slow-growers could not tolerate extremes of pH, placing them in the same group with respect to this attribute.
The ability to produce a variety of exo-polysaccharides (gum or mucous) is a characteristic of a very large number of microorganisms.This characteristic has been associated with and is characteristic of rhizobia.Results of this study indicated mucous production of all the isolates.The mucous plays a critical role in maintaining minimum moisture in the immediate environment of the microorganisms.Through the high moisture holding capacity, it prevents desiccation, and serves as a potential source of energy under conditions of paucity (Sayyed et al., 2011).Mucous produced by rhizobia has been shown to consist of glucose, maltose, rhamnose, galactose and other glucans.In rhizobia infected plant nodules, the mucous has been shown to accumulate between the peribacteroid membrane and the symbiosome membrane filling up most of the symbiosome volume (Streeter, Salminen, Whitmoyer, & Carlson, 1992).The ability to produce the mucous in-vitro is an indication that this process is independent of the plant host and that the kind of polysaccharides making up the mucous are completely dependent on the genotype of the microorganism.
In conclusion, morphological characteristics varied among the 61 isolates obtained from the three agro-ecological regions of Zambia.The colours of colonies were translucent, cream, white, pink and the uncommon yellow.Pink colonies were peculiar to region III.The elevation of all the colonies was convex.Biochemically, all Rhizobia were Gram-negative, mucous producing and acid producing with only 2 producing alkali.Isolates were fast growing with two slow growing, able to utilize glucose as a carbon source and unable to tolerate extremes of pH at 27°C.Based on results, the 59 fast-growers could be Ensifer fredii or/and Rhizobium tropici rather than Bradyrhizobium.However, further tests to confirm these findings using ketolactose, genetic characterization and an inclusion of reference strains, are still needed and are being recommended here.

Table 1 .
Properties and classification of soil from selected sites of agro-ecological I, II and III regions of Zambia

Table 2 .
Rhizobial colony morphological characteristics of isolates from cultivated and virgin soils of regions I,

Table 4 .
Mucous production of rhizobial isolates from agro-ecological regions I, II and III