Application of 1-aminoocyclopropanecarboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii.
Elimination of rhizobitoxine production in B. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. The symbiotic interactions between a legume and brady rhizobia result in a unique, nitrogen-fixing plant organ, the nodule. Recent studies have shown that the phytohormone ethylene inhibits nodule formation in some legumes 8 , 9 , 16 , 24 , Application of 1-aminoocyclopropanecarboxylic acid ACC , a precursor of ethylene, inhibits nodulation in Medicago truncatula Rhizobitoxine [2-amino 2-aminohydropropoxy - trans -butenoic acid] is an ethylene synthesis inhibitor that is produced by the legume symbiont Bradyrhizobium elkanii 15 , 17 — 19 , 22 , It is thought that production of this compound enhances nodulation of the host legume because of its inhibitory effect on ethylene synthesis.
However, some reports have shown that there is not a significant difference in nodule number between plants inoculated with B. Recently, Duodu et al. Although these findings do not seem to be consistent with the hypothesis that rhizobitoxine has a positive effect on nodulation, the inconsistency can be explained by differences in the ethylene sensitivity of nodulation among leguminous species; nodulation of G.
The inconsistency could also result from differences in the abilities of the strains used in the experiments to produce rhizobitoxine; strain USDA61 is a weak producer of rhizobitoxine In addition to G. Although the effect of ethylene on nodulation has been studied in many leguminous host plants so far, the effect of ethylene in M. In general, B.
These results led us to investigate the role of rhizobitoxine production on the nodulation and competitiveness of B. Siratro M. Siratro seeds were obtained from Yukijirushi Shubyo Co.
Hokkaido, Japan. Bradyrhizobium strains were maintained in HM medium 4 containing 0. Escherichia coli strains were maintained in Luria-Bertani medium Before inoculation, Bradyrhizobium strains were cultured in HM medium containing 0.
One milliliter of the bacterial suspension was inoculated onto 2-day-old seedlings in sterile growth pouches.
To see if production of ethylene in M. It is thought that applying ACC to plants increases the ethylene level because this compound is a precursor of ethylene.
The nutrient solutions containing ACC were added to plant roots just after inoculation and every day during plant growth. Siratro seedlings that received a nitrogen-free nutrient solution that did not contain ACC were used as controls.
For a time course study of nodulation, nodules were counted by counting the visible nodules with diameters greater than 0. Student's t test was used to assess the statistical significance of differences in nodule number at a confidence level of 0. Ethylene synthesis was measured by the method of Suganuma et al.
The ethylene concentration in the container was measured by using a model GC-7A gas chromatograph Shimadzu, Tokyo, Japan equipped with a flame ionization detector and a Porapak Q column 2. We calculated the rate of ethylene synthesis by using the concentrations obtained. These results suggest that inoculation with B.
These results indicate that ACC treatment increased ethylene synthesis in M.
The number of nodules formed in the presence of ACC 8 days after inoculation and later were significantly less than the numbers of nodules formed in the absence of ACC Fig.
This finding suggests that in M. Effect of ACC on nodulation of M. Siratro inoculated with B.
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Each point represents the mean number of nodules per plant. The bars indicate standard errors.
Nine plants were treated and assessed for each data point. Asterisks indicate significant differences in mean nodule number between treatments at a confidence level of 0.
The rtxA gene reportedly is responsible for rhizobitoxine production by B. This gene encodes two regions similar to a rat serine:pyruvate aminotransferase and a yeast O -acetylhomoserine sulfhydrolase 27 , One of the clones obtained contained a 2,bp insert whose sequence was This plasmid was designated pCR Plasmid pCR The plasmid obtained was designated pUC Plasmid pUC These cloning experiments were performed by using E.
Construction of rhizobitoxine production-deficient mutant RTS2. RTS2 was derived from B. The vector region of pSUP is 7. Cells of the E.
Kanamycin- and polymyxin B-resistant colonies were selected and maintained in HM medium containing mg of kanamycin per liter and were used for Southern analysis.
The probes used in the Southern analysis were the 3. One of the appropriate strains, a kanamycin-resistant mutant that was designated RTS2 and was obtained from USDA94, was used in this study.
Rhizobitoxine concentrations in cultures were determined as described by Yasuta et al.
The limit of the detection was 0. Inoculation with mutant RTS2 did not induce foliar chlorosis in G. Lee data not shown , a finding similar to a previous finding for a rhizobitoxine mutant of B. Because the single-crossover mutant RTS2 did not produce rhizobitoxine, at least one additional gene downstream of rtxA and in the same operon may be required for rhizobitoxine production. Ethylene synthesis in plant roots was measured as described above by examining plants on days 6 and 23 after inoculation.
On days 6 and 23 after inoculation in the absence of ACC, the rates of ethylene synthesis in siratro roots inoculated with USDA94 were less than the rates of ethylene synthesis in uninoculated control roots Fig. When siratro was inoculated with RTS2, the ethylene synthesis rate was greater than the rate of synthesis in roots inoculated with USDA94 and equivalent to the rate of synthesis in uninoculated roots. The lack of rhizobitoxine production by B. Rhizobitoxine is produced in nodules and is transported to the roots and shoots 17 , Because the number of nodules on day 23 after inoculation with USDA94 was about 13 times more than the number of nodules on day 6 after inoculation data not shown , the increase in nodule number should have resulted in a higher concentration of rhizobitoxine in plants inoculated with B.
Effects of B. Plants were inoculated with B. For each treatment, the mean ethylene evolution rate was determined 6 days after inoculation 15 plants and 23 days after inoculation 9 plants.
ND, not detected. The effect of rhizobitoxine-deficient strain RTS2 on nodulation of siratro was also examined. When we compared inoculation with mutant RTS2 and inoculation with wild-type strain USDA94, we found no difference in the percentages of nodulated plants that had the first visible nodules Fig.
Bradyrhizobium elkanii pdf to word
The same trend was observed for the percentages of nodulated plants having the first large nodules data not shown. These results suggest that a lack of rhizobitoxine production in B.
A lack of rhizobitoxine production did not affect emergence of the first nodules, but the numbers of nodules were significantly different over time after inoculation when the isogenic variants of USDA94 were used Fig. When the numbers of nodules were compared, the numbers of visible nodules on siratro roots after inoculation with the different isogenic variants were not different before day 6 after inoculation.
From 8 days after inoculation, however, the numbers of visible nodules on siratro roots inoculated with RTS2 were less than the numbers of visible nodules on roots USDA Fewer nodules after inoculation with RTS2 were also observed when the numbers of large nodules were compared data not shown.
These results suggest that rhizobitoxine production by B. A similar effect of rhizobitoxine production was described by Duodu et al.
The reason s for the delayed effect of rhizobitoxine production on the number of siratro nodules is not clear. One possible explanation is that a higher concentration of rhizobitoxine in plants is necessary before there is a visible effect on nodulation.
This seems logical because there was no difference in the time of appearance of the first nodules when inoculation with the rhizobitoxine mutant and inoculation with the wild type were compared Fig. The conclusion that rhizobitoxine production by B. The latter data might have resulted from differences in the ability to produce rhizobitoxine; Xiong and Fuhrmann 39 reported that USDA94 produced more rhizobitoxine than USDA61 produced in plants.
Nodulation of M. Each point represents a mean based on six independent experiments. A Time course for percentages of nodulated plants. B Time course for number of nodules per plant. The asterisks indicate significant differences in mean numbers of nodules between the treatments at a confidence level of 0.
To investigate competitiveness for nodulation in the wild-type and rhizobitoxine-deficient strains, mTn 5 SS gusA20 37 was used to label B.
Recently, gusA -marked transposons, including mTn 5 SS gusA20 , have been developed 37 , and using these transposons has some advantages over other techniques 2 , 3 , 6 , 13 , 30 , The gusA marking system results in marked Brady rhizobium cells with competitive ability indistinguishable from the competitive ability of the parent cells; this makes screening for the competitive ability of strains of interest simple and rapid In this study, 3 weeks after coinoculation nodules whose diameters were greater than 1 mm were harvested and used in a GUS assay performed as described by Yuhashi et al.
Each of the harvested nodules was cut in half with a razor blade. Samples were fixed with 0. When uniform GUS activity was observed in the infected region of a nodule, the nodule was considered occupied only by a gusA -marked strain.