At the highest level of NLS induction (2 x 10-7 to 4 x 10-7 M 2,4-D) over 70% of plants subjected to 2,4-D without Rhizobium inoculation developed NLS (at 2 x 10-7 M, 6.0 ±1.5 NLS per plant), and more than 50% for plants inoculated with Rhizobium ANU843p(Tp2) (at 2 x 10-7 M, 13.9 ±2.6 NLS per plant)(Table 2). However, as yet the data are insufficient to say whether rhizobia had an influence on the percentage of plants with NLS and numbers of NLS.
The levels of 2,4-D required to induce NLS were at levels that inhibit normal plant growth. Within the same window of 2,4-D concentration that induced NLS, there was a steady reduction in lateral root numbers (Table 3). Levels of 2,4-D lower than 9 x 10-8 M had no effect compared to controls, whereas levels of 2,4-D higher than 9 x 10-7 M killed all plants (Table 1).
In order to visualize the presence of bacteria, Rhizobium ANU843 carrying the GUS (6-glucuronidase) gene (ANU843(Tp2)) was inoculated to the seedlings. The GUS enzyme hydrolises X-gluc (5-bromo-4-chloro-3indolyl B-glucuronide) forming a blue indigo dye. Because GUS activity is lacking in this rice variety, the presence of bacteria can be determined by blue staining. Nearly 100% of the NLS were colonized by rhizobia and structures such as lateral roots and root tips were also stained blue (Table 4). Rhizobia colonised the surface cells of the root and the surface and inner tissues of the NLS.
The development of NLS is most likely the result of the initiation of multiple meristems stimulated by accumulation of 2,4-D where lateral roots emergence. The development of multiple meristems dividing in several directions results in round or elongate-shaped NLS (Ridge et al. 1993).
The plant tests presented here were done without the addition of nitrate, and further tests are needed to find a balance between induction of NLS, health of the plant, and the ability of free-living nitrogen fixation organisms to contribute to the dry weight of the plant.
We conclude that Rhizobium leguminosarum bv trifolii ANU843p(Tp2) is able to colonise the surface of the root and invade NLS structures induced by 2,4-D application. If 2,4-D is to be used on plants to induce NLS as colonisation sites for nitrogen-fixing organisms, systemic treatment of the plants must be carefully applied to both avoid reduction in healthy growth and to provide the levels of 2,4-D required to induce the NLS. An alternative strategy would be to insert genes into the appropriate bacteria so that they would apply (by secretion) the NLS-inducing substance directly at the NLS-susceptible site, and thus not affect the whole plant.
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Effects on plant growth |
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10-8 to 9 x 10-8 M 10-7 2 x 10-7 4 x 10-7 5 x 10-7 6 x 10-7 8 x 10-7 9 x 10-7 to 2 x 10-6 |
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no effect compared to control slight effects to plant growth root length and lateral root numbers reduced stunted roots; lateral root numbers reduced stunted roots; very few lateral roots very stunted roots; some plant death most plants dead; few of the living plants had roots that penetrated the agar all plants dead at an early stage |
Table 2. NLS on plants at different levels of 2,4-D Percentage of plants with NLS / number of NLS per plant. 2,4-D No rhizobia (n) ANU843(Tp2) (n) 0 0 / 0 (18) 0 / 0 (22) 10-7 M 22 / 5.5 ±2.1 (9) 12 / 3.0 ±0.4 (16) 2 x 10-7 73 / 6.0 ±1.5 (15) 27 / 13.9 ±2.6 (30) 4 x 10-7 52 / 5.4 ±1.6 (29) 50 / 9.3 ±1.1 (36) 5 x 10-7 43 / 2.2 ±0.3 (14) 18 / 6.5 ±0.9 (22) 6 x 10-7 10 / 5.3 ±1.0 (21) 23 / 3.5 ±0.4 (17) 8 x 10-7 0 / 0 (13) no data Only plants with NLS were counted for the mean. ± = standard error. Table 3. Number of lateral roots per plant at different levels of 2,4-D Percentage of plants with lateral roots / number of lateral roots per plant. 2,4-D No rhizobia (n) ANU843(Tp2) (n) 0 100 / 119.5 ±16.27 (18) 100 / 102.2 ±15.8 (22) 10-7 M 100 / 67.8 ±17.3 (9) 100 / 34.3 ±10.1 (16) 2 x 10-7 72 / 17.7 ±3.8 (25) 63 / 18.1 ±3.6 (30) 4 x 10-7 80 / 13.5 ±3.2 (29) 50 / 11.1 ±4.8 (12) 5 x 10-7 50 / 8.5 ±1.7 (14) 41 / 4.7 ±0.7 (22) 6 x 10-7 33 / 4.3 ±0.9 (21) 12 / 4.5 ±0.9 (17) 8 x 10-7 30 / 2.0 ±0.3 (13) no data Only plants with lateral roots were counted for the mean. ± = standard error. Table 4. Percentage of GUS staining of different parts of the root. 2,4-D primary roots root tips lateral roots NLS 0 93 71 96 - 10-7 M 86 78 85 100 2 x 10-7 78 65 63 99 4 x 10-7 66 54 66 83 5 x 10-7 59 52 71 100 6 x 10-7 75 70 57 100 8 x 10-7 58 53 77 i.d. i.d. = insufficient dataFurther reading
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