Trifolitoxin production by Rhizobium leguminosarum bv. trifolii T24 was observed in three sterile soils. The three soils differed in texture and fertility. Two of these soils were silt loams while ...the third soil was a silty sand. One of the silt loams was amended with manure and composted. The amount of trifolitoxin in these soils was determined by bioassay after inoculation with T24. The largest amount of trifolitoxin in each soil was observed at 48 h after inoculation; after 48 h, trifolitoxin declined. The amended soil contained 5-10 fold more trifolitoxin than did the unamended soils. The production of trifolitoxin in soil by T24 was verified in two ways: (i) T24 inoculated soils contain a substance which inhibits a trifolitoxin-sensitive strain of Rhizobium but not a trifolitoxin-resistant strain; and (ii) sterile soils inoculated with a Tn5 mutant of T24 that lacks trifolitoxin production (T24::Tn5(1)) did not contain any detectable trifolitoxin activity during the 4 days of the experiment. These data demonstrate that trifolitoxin is produced by T24 in unamended as well as amended soils, although much larger amounts are detected in the amended soil.
Two methods were developed for the detection of altered ureide metabolism in legume nodules. Both techniques are based on the positive correlation between the presence of high xanthine dehydrogenase ...(EC 1.2.1.37) specific activity in nodules and the ability of those nodules to produce the ureides, allantoin and allantoic acid. In the first method, nodulated legumes are treated for 2 weeks with a soil drench of allopurinol. After allopurinol treatment, leaves of N
2
-fed, ureide-producing legumes, soybean, cowpea, and lima bean, became very chlorotic. Leaves of KNO
3
−
or NH
4
Cl-fed ureide-producing legumes were unaffected by the allopurinol treatment. Leaves of the amide-producing legumes, alfalfa, clover, peak, and lupin, were unaffected by the allopurinol treatment with N
2
, KNO
3
, or NH
4
Cl as nitrogen source. These experiments showed that long-term allopurinol treatments are useful in differentiating between ureide- and amide-producing legumes when effectively nodulated. A second method was developed for the rapid, qualitative estimation of xanthine dehydrogenase activity in legume nodules. This method utilizes pterin, an alternate substrate for xanthine dehydrogenase. Xanthine dehydrogenase hydroxylates pterin in the presence of NAD
+
to produce isoxanthopterin. When exposed to long wave ultraviolet light (365 nanometers), isoxanthopterin emits blue fluorescence. When nodules of ureide-producing legumes were sliced in half and placed in microtiter plate wells containing NAD
+
and pterin, isoxanthopterin was observed after 6 hours of incubation at room temperature. Allopurinol prevented isoxanthopterin production. When slices of amide-producing legume nodules were placed in wells with pterin and NAD
+
, no blue fluorescence was observed. The production of NADH by xanthine dehydrogenase does not interfere with the fluorescence of isoxanthopterin. These observations agree with the high specific activity of xanthine dehydrogenase in nodules of ureide-producing legumes and the low activity measured in amide-producing nodules. The wild soybean,
Glycine soja
Sieb. and Zucc., was examined for ureide synthesis. Stems of wild soybean plants had a high ureide abundance with N
2
as sole nitrogen source when nodulated with either
Rhizobium fredii
or
Bradyrhizobium japonicum.
Ureide abundance declined when nitrate or ammonium was added to the nutrient solution. Nodule slices of these plants produced isoxanthopterin when incubated with pterin. Nodule crude extracts of
G. soja
had high levels of xanthine dehydrogenase activity. Both
Glycine max
and
G. soja
plants were found to produce ureides when plants were inoculated with fast-growing
R. fredii.
The two methods described here can be used to discriminate ureide producers from amide producers as well as detect nitrogen-fixing legumes which have altered ureide metabolism. A nodulated legume that lacks xanthine dehydrogenase activity as demonstrated by the pterin assay cannot produce ureides since ureide synthesis has been shown to require xanthine dehydrogenase activity both
in vivo
and
in vitro.
A nodulated legume that remains green during allopurinol treatment also lacks ureide synthesis since the leaves of ureide-producing legumes are very chlorotic following allopurinol treatment.
The distribution of xanthine dehydrogenase throughout the soybean plant as well as the intercellular localization of xanthine dehydrogenase within soybean nodules was determined. Polyclonal ...antibodies against purified xanthine dehydrogenase were prepared and used in an enzymelinked immunosorbent assay to determine whether xanthine dehydrogenase is a nodule-specific protein. This immunological assay showed that xanthine dehydrogenase is present in far greater concentration in the nodule than in any other plant organ. Immunodiffusion tests showed that anti-soybean nodule xanthine dehydrogenase would cross-react with nodule crude extracts from the ureide producers, soybean, cowpea, and lima bean, but would not cross-react with those of the amide producers, alfalfa and lupine. A crude extract from pea nodules cross-reacted slightly with anti-soybean xanthine dehydrogenase. Anti-soybean xanthine dehydrogenase did not cross-react with buttermilk xanthine oxidase either by enzyme-linked immunosorbent assay or by immunodiffusion test.
Fresh nodule sections from the ureide-producers, soybean, cowpea, and lima bean, all stained positively for xanthine dehydrogenase. The substrate-dependent stain was inhibited by allopurinol and was observed only in the infected nodule cells of these species. Nodules from the amideproducers, alfalfa and white lupine, did not stain for xanthine dehydrogenase.
Rhizobium leguminosarum
bv.
trifolii
T24 is ineffective in symbiotic nitrogen fixation, produces a potent antibiotic (referred to here as trifolitoxin) that is bacteriostatic to certain
Rhizobium
...strains, and is very competitive for clover root nodulation (EA Schwinghamer, RP Belkengren 1968 Arch Mikrobiol 64: 130-145). The primary objective of this work was to demonstrate the roles of nodulation and trifolitoxin production in the expression of nodulation competitiveness by T24. Unlike wildtype T24, transposon mutants of T24 lacking trifolitoxin production were unable to decrease clover nodulation by an effective, trifolitoxin-sensitive strain of
R. leguminosarum
bv.
trifolii.
A non-nodulating transposon mutant of T24 prevented clover nodulation by a trifolitoxin-sensitive
R. leguminosarum
bv.
trifolii
when co-inoculated with a T24 mutant lacking trifolitoxin production. Neither mutant alone prevented nodulation by the trifolitoxin-sensitive strain. These results demonstrate that trifolitoxin production and nodulation are required for the expression of nodulation competitiveness by strain T24. A trifolitoxin-sensitive strain of
R. meliloti
did not nodulate alfalfa when co-inoculated with T24 and a trifolitoxin-resistant strain of
R. meliloti.
Thus, a trifolitoxin-producing strain was useful in regulating nodule occupancy on a legume host other than clover. Trifolitoxin production was constitutive in both minimal and enriched media. Trifolitoxin was found to inhibit the growth of 95% of all strains of
R. leguminosarum
bvs.
trifolii, viceae,
and
phaseoli
tested. Strains of all 13 biotypes of
R. leguminosarum
bv.
trifolii
were inhibited by trifolitoxin. Three strains of
R. fredii
were also inhibited. Strain T24 ineffectively nodulated 46 clover species, did not nodulate
Trifolium ambiguum,
and induced partially effective nodules on
Trifolium micranthum.
Since T24 produced partially effective nodules on
T. micranthum
and since a trifolitoxin-minus mutant of T24 induced ineffective nodules, trifolitoxin production is not the cause of the symbiotic ineffectiveness of T24.
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