Figure 1: Increase in salt concentration decreases the absorbancy (reduce bacterial growth)
Abstract: Rhizobacteria,
being soil microorganisms are confronted with fluctuating osmotic pressures
of the rhizosphere. Rhizobium is an important microbe because of
its impact and interaction with the host plant. Changes in salt concentration
affect the growth and functioning of Rhizobium. In the present study,
the effects of varying salt concentrations from 0.2M to 0.00625M are reported.
Rhizobium is capable of osmoadaptation; it can tolerate high salt
concentrations, the growth tends to be inversely proportional to salt concentration.
In other words, growth decreases with increasing salt concentration. Rhizobial
growth is more abundant at lower salt concentrations ranging from 0.00625M
to 0.0125M.
Introduction:
Rhizobia are soil bacteria which display symbiotic interactions with specific legume hosts. Most of these bacteria are very sensitive to a soil water deficit, which adversely affects their dinitrogen fixation capacity and hence the productivity of the whole legume plant (Miller, 1996). It has been estimated that 23% of agricultural soils are affected by problems related to high salinity. Most crops are sensitive to relatively low levels of salinity, and, in the case of legumes, there is an additional problem because not only the plant but also the symbiotic bacteria are sensitive to salinity both at the free living stage and during the symbiotic process (Lloret, et al. 1995). Rhizosphere bacteria may benefit from access to the rich nutrient supplies offered by root exudates, from opportunities to establish mutualistic or symbiotic relationships, and from protection from soil dessiccation afforded by mucigel. At the same time, rhizosphere bacteria risk exposure to a variety of hazards that include plant defense responses, competition with other rhizosphere occupants, and exposure to toxic substances exuded from plant roots. Furthermore, rhizosphere bacteria encounter relatively high osmotic conditions (Miller, 1996). During personal communication with Annie Anthraper, I have learned that if salt concentrations are raised above Leucaena leucocephala's normal habitat, its plant biomass decreases and at lower salt concentrations, the nodules associated with the plant are more numerous but reduced in size. This conclusion correlates with the results of the present experiment, which show an increase in Rhizobial growth with a decrease in salt concentration. Rhizobium is studied in this experiment to determine the effects of different salt concentrations on the organism, the results of which might also help to observe the related effects on the interaction between plant and Rhizobium. This study addresses the question "does salt stress affect the Rhizobium's ability to grow and function normally?"
Studies have revealed that salt stress causes an alteration
of root hair curling, a reduction in the number of rhizobia attached to
root hair, and a decrease in nodule size. Also, the amount of nitrogen fixed
per unit weight of nodules also declines with salt stress (Miller, 1996).
Materials and Methods:
Rhizobia were grown on an enriched and selective medium identified and streaked for isolation. The enriched medium for Rhizobial growth consisted of the following: 5g of mannitol sugar, 0.25g of K2HPO4, 0.1g of MgSO4.7H2O, 2g of CaCO3, 0.05g of NaCl, 0.2g of yeast extract, 7.5g of Agar. The above ingredients were dissolved in 500 ml of distilled water and the pH was maintained between 6.8 and 7.0. The medium was then autoclaved. After isolation of pure cultures, seven sets (8 per set) of enriched medium petriplates were prepared with the same nutrients as above but with different salt concentrations respectively: 0.2 M, 0.15 M, 0.10 M, 0.05 M, 0.025 M, 0.0125 M, 0.00625 M. The eighth set was a control set with no additional salt (other then the minute amounts required by the microbe). The nutrient agar, with different salt concentrations, was autoclaved before being plated and inoculated. A bacterial suspension (one loopfull of bacteria from the pure isolates of Rhizobium in 1 ml of sterile water) was prepared and a lawn was made on eight sets (8 per set) of petriplates. The same procedure was followed for eight sets of broth tubes containing the same nutrients. To each set of tubes (20 ml each, 250 mls total) salt was added according to its respective concentration: 2.92g [0.2], 2.175g [0.15], 1.46g [0.10], 0.725g [0.05], 0.365g [0.025], 0.1825g [0.0125], 0.0913g [0.00625]. Bacteria (1 loopfull) were inoculated into each tube. Both the petriplates and broth tubes were incubated at room temperature and were allowed to grow for 2-4 weeks. After 4 weeks of incubation, the tubes were checked for absorbance using a spectrophotometer. The plates were simply observed for heavy or light growth.
To determine the effect of different salt treatments, Kruskal Wallis's analysis of variance was performed. Newman - Keuls multiple comparison was conducted as a post hoc analysis to determine differences among particular salt treatments.
Results:
Table 1: Different salt concentrations and its effects on Absorbance
| 0.2 M | ||
| 0.15 M | ||
| 0.1 M | ||
| 0.05 M | ||
| 0.025 M | ||
| 0.0125 M | ||
| 0.00625 M | ||
| Control |
Table 1 shows the mean of eight samples for each of
the eight different treatments. The table indicates that with decreasing
salt concentrations there is an increase in the absorbance mean.
Table 2: Newman-Keuls multiple comparison test performed on data in table 1.
| 3 [0.1M] | |||
| 2 [0.15M] | |||
| 1 [0.2M] | |||
| 5 [0.025M] | |||
| control | |||
| 4 [0.05M] | |||
| 7 [0.00625M] | |||
| 6 [0.0125M] |
Two tailed p<0.000001
Significance level for multiple comparisons: = 0.05
In conducting Newman-Keuls multiple comparisons, the
salt stress shows a significant effect on Rhizobial growth, samples 1, 2,
3 concentrations [0.2, 0.15, 0.1] overlap one another, and they are indistinguishable.
Control and sample 5 [0.025] overlap, whereas samples 4, 6, 7 [0.05, 0.0125,
0.00625 M] are totally different and do not overlap with any of the other
treatments.
Table 3: Type of growth on nutrient agar plates with different salt concentrations
| Control | Heavy growth |
| 0.2 | Very light growth |
| 0.1 | Light growth |
| 0.15 | Light growth |
| 0.05 | Heavy growth |
| 0.025 | Medium growth |
| 0.0125 | Very heavy growth |
| 0.00625 | Heavy growth |
Table 3 indicates the kind of growth on nutrient agar
plates. Rhizobium prefers lower salt concentration and grows heavily,
whereas growth at higher salt concentrations is light.
Discussion:
After performing a Kruskal-Wallis analysis and a Newman-Keuls
multiple comparison test on the data, the conclusion can be made that treatment
of Rhizobium with different salt concentration has a significant
effect on its growth (two tailed p<0.000001). As was mentioned in the
introduction, it has an ability to function normally with reduced nodule
size. Figure 1 indicates a gradual increase in Rhizobial growth, due to
which the absorbancy increases with decreasing salt concentration. Sample
5 [0.025], is the only sample that seems to be out of proportion; if excluded,
we see a gradual increase (from control to 0.0125) and then a gradual decrease
(from 0.05 to 0.2) in Rhizobial growth. Table 2 provides greater explanation
as to which sample is identical in terms of response to different salt stress.
The mean rank values in Table 2 for samples 1, 2, and 3 [0.2, 0.15, 0.10]
are very similar to one another, denoting that responses to concentrations
0.2M, 0.15M, 0.1M were not very different; they overlap. The mean rank value
in Table 2 jumps up for concentrations 0.025 M and is closer to the control
mean rank value; thus samples 5 [0.025] and control are indistinguishable.
Samples 4, 7, and 6 are not only higher in mean values from the rest of
the values but they also show no overlaps. Results indicate significant
differences in the mean values, and the treatment of Rhizobium with
different salt concentrations has an effect on its growth. As described
in the materials and methods, nutrient agar plates with different salt concentrations
were also inoculated, but it was hard to carry out a quantitative analysis
of the growth except for heavy, medium or light (shown in Table 3).
Figure 1: Increase in salt concentration decreases the absorbancy (reduce
bacterial growth)
A halotolerant strain of Rhizobium has been isolated from nodules of a melilotus plant growing in a salt marsh in Donana National Park in the southwestern region of Spain. This strain is able to grow at NaCl concentrations of up to 500 mM (Lloret, et al, 1995). The results of the present study also show the capability of Rhizobium to grow at 200 mM salt concentration, but the growth is more abundant at lower salt concentrations.
Previous research has shown that changes in osmotic pressures, concentrations, and pH, etc. changes the structure of Lipopolysaccharide of bacteria in response to salt stress, and that Rhizobia accumulate several compatible solutes to overcome the osmotic stress induced by salt. An example of this feature is Ectoine, which exhibits osmoprotective properties without being accumulated (Talibart, et al, 1994). The findings of this study may help in understanding the effect of changing osmotic pressure on the nitrogen fixing capacity of Rhizobium, which in turn effects the growth of the plant involved in symbiosis. There are other questions that still remain unanswered: "are rhizobial osmoadaptive responses integrated with those of plants?" If Rhizobium is halotolerant, what causes the plant biomass to decline?"
Acknowledgents:
I would like to thank Dr. Dubois, Dr. Howard, Dr.
Mathis for excellent guidance and support in this research and their encouragement
and interest. I would like to also thank Annie Anthraper and Mindy for their
friendship and assistance in this research project. It is a privilidge to
work with such supportive professors and researchers.
References:
1. Miller, K. J., and J. M. Wood. 1996. Osmoadaptation
by rhizosphere bacteria. Annual Review of Microbiology. 50:101-136p.
2. Lloret J., and et al. 1995. Ionic stress
and osmotic pressure induce different alterations in the LPS of a Rhizomelilte
strain. Applied and Environmental Microbiology. 61(10): 3701-3705p.
3. Talibart R., and et al. 1994. Osmoadaptation in Rhizobia: ectoine induced salt tolerance. Journal of Bacteriology. 176 (17): 5211-5217p.