4. CALIDAD DE VIDA
4.5. Relación autocuidados y calidad de vida
This experiment tested the null hypothesis that R. leguminosarum strain ADWC3a was able to compete with naturalised rhizobia for nodule occupancy. The strain ADWC3a was used because it was the most competitive strain found in Experiment 4.2.
4.2.3.1
Experimental set up
The soil used for this experiment was a non-sterile soil collected from Lincoln University (Table 3.1). The MPN of R. leguminosarum in the soil was tested (Section 2.2.2) and was 110 cells g-1. This soil
was mixed with 10% pumice and wetted with sterile water. Fifty ml tubes were filled with 25 g of the soil/pumice mixture.
4.2.3.2
Inoculation
Broth culture of ADWC3a was prepared (Section 2.2.3.3) and the original inoculant containing ADWC3a was made (Section 2.2.3.3). To make the four treatments for this experiment, the original inoculant containing ADWC3a was diluted down to concentrations of 1 x 106 cells ml-1, 1 x 105 cells
ml-1, 1 x 104 cells ml-1, 1 x 103 cells ml-1 and 1 x 102 cells ml-1 prior to inoculation. The 50 ml tubes
containing soil were inoculated two days prior to planting. Each tube was inoculated with 2 ml of the appropriate diluted inoculant. To inoculate the soil 2 x 1 ml of the inoculant was pipetted evenly over the soil. Each treatment was replicated five times. Five tubes had no rhizobia added as a control to check for rhizobia contamination. To these tubes 2 ml of sterile water was added.
4.2.3.3
Confirmation of inoculant concentration
The inoculant treatments were plated to confirm the concentration of each. Aliquots of 100 l from the 1 x 104 – 1 x 102 cells ml-1 inoculants were spread onto three replicate YMA plates (Appendix
8.3) and incubated at 25°C in the dark for 48 hours before counting CFU’s. The number of cells ml-1
in each inoculant was calculated (Appendix 10.1) as was number of cells g-1 of soil in each tube
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4.2.3.4
Planting
‘Tribute’ white clover seeds were sterilised (Section 2.2.3.2) and then spread out on to water agar plates (1.5% (w/v), autoclaved 15 minutes at 121°C, 15 Psi) and incubated in the dark at 20 °C for three days to allow germination. Two days after inoculation, seeds were planted and randomized and plants grown and watered as required (Section 4.2.2.4).
4.2.3.5
Harvest and bacterial isolation from nodules
Six weeks post soil inoculation plants were harvested and nodules were sterilised and plated (Section 4.2.2.5).
4.2.3.6
PCR amplification of bacterial DNA using ERIC primers and genotyping
Genotyping using a rapid DNA extraction process was used to identify isolates in each of the nodules (Section 4.2.2.6). The ERIC banding pattern for ADWC3a was distinct and was not found in the control plants and, therefore the samples with identical banding patterns to this inoculant strain (ADWC3a) were assumed to be from the applied inoculant and counted.
4.2.3.7
Statistical analysis
A logistic regression model was fitted to the data with proportion of ADWC3a in the nodules related to the log of ADWC3a inoculant rate to test for the significances of increasing inoculation rate on nodule occupancy of ADWC3a. Control observations were ignored in this analysis.
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4.3
Results
4.3.1
Experiment 4.1: Carbon utilisation of seven Rhizobium leguminosarum strains
measured using γ-MicroResp
In soil from Ashley Dene, the seven strains of rhizobia formed three metabolically distinct (P≤0.05) phenotypic groups based on their differences in C utilisation, which was measured by the respiration of the strains (Figure 4.1). R. leguminosarum strains WSM1325, ADWC3b, ADSub1h and ADSub1g grouped together, with all four strains able to utilise only a few C sources. R.
leguminosarum strain ADWC3a and TA1 grouped together, with both strains able to utilise more
amino acids than sugars. R. leguminosarum strain CC275e formed a distinct group based on greater use of a range of C sources.
In the soil from Lincoln University, the seven strains of rhizobia formed three metabolically distinct (P≤0.05) phenotypic groups based on differences in C utilisation (Figure 4.2). R. leguminosarum strains WSM1325, CC275e, ADWC3b, ADSub1h and ADSub1g grouped together, with all five strains able to utilise a wide range of C sources, albeit at low amounts. R. leguminosarum strain ADWC3a formed a distinct group (singleton) based on its ability to utilise a wide range of carbon sources in high amounts. In contrast, R. leguminosarum strain TA1 formed a distinct group because of its low utilisation of all C sources. Out of the three distinct groups, the largest group contained five isolates while the other two isolates were unique in their carbon utilisation profiles. Between the two soils,
R. leguminosarum strains TA1 and CC275e were the only two strains that differed in their C
utilisation between the two soils with both utilizing more carbon sources in the Ashley Dene soil compared with the Lincoln University soil. Relative to other strains R. leguminosarum strain TA1 in the soil from Lincoln University used none of the applied carbon sources (Figure 4.2). This strain is using the carbon sources just not at significant rates compared to the rates the other strains are using the carbon sources.
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Ashley Dene
Figure 4.1 Shade plot showing similarities between the strains and their ability to utilise the carbon sources in soil from Ashley Dene. Strains with the same symbol (, , ) are not significantly different from each other based on SIMPROF testing at α=0.05. The colour gradient shows differences in absorbance (carbon utilisation) at 590 nm: warmer shades (yellow, orange, red, black; in ascending order from values of 1.5 - 3) denote higher use of the carbon source than lighter shades (green, blue white; in descending order from values of 1.5 - 0).
Lincoln University
Figure 4.2 Shade plot showing similarities between the strains and their ability to utilise the carbon sources in soil from Lincoln University. Strains with the same symbol (, , ) are not significantly different from each other based on SIMPROF testing at α=0.05. The colour gradient shows differences in absorbance (carbon utilisation) at 590 nm: warmer shades (yellow, orange, red, black; in ascending order from values of 1.5 - 3) denote higher use of the carbon source than lighter shades (green, blue white; in descending order from values of 1.5 - 0).
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