Race 15B Research Articles

The inheritance of stem rust resistance in Thatcher wheat

Thatcher was the predominant wheat (Triticum aestivum L.) cultivar on the Canadian prairies in the 1950s. Until race 15B (TMH) of stem rust (Puccinia graminis pers. f. sp. tritici Eriks. & Henn.) became widespread, Thatcher had good resistance to stem rust, but was susceptible to leaf rust (P. recondita f. sp. tritici Rob. ex Desm.). Although genes for stem rust resistance have been identified in Thatcher, the inheritance of its resistance has never been fully understood. The objective of this research was to attempt to elucidate the inheritance of the resistance of Thatcher and to determine why it had a reputation as a poor parent for rust resistance. Over a period of 40 yr, crosses and backcrosses to a susceptible genotype and two sets of single seed descent (SSD) lines were studied. The second set of SSD lines was tested with isolates of six races of stem rust to which Thatcher is resistant. The data showed that Thatcher is a very heterogenous cultivar with individual plants differing widely in the genes for stem rust resistance that they carry. The inheritance of rust resistance varied greatly from race to race and was often quite complex. Either complementary genes or a gene plus a suppressor appeared to condition resistance to one race. Most genes gave resistance to only one race. The presence of Sr5, which Thatcher is known to have obtained from Kanred, was confirmed. Most of its many additional genes probably came from Iumillo durum wheat. Key words: Stem rust, Thatcher wheat, single seed descent

Inheritance of Resistance to Stem Rust in a Selection of the Wheat Cultivar ‘Waldron’1

The hard red spring wheat (Triticum aestivum L.) cultivar ‘Waldron’ is resistant to North American strains of stem rust (Puccinia graminis f. sp. tritici). The inheritance of resistance in a selection of Waldron (Wdr‐sel) was studied to determine the number of genes affecting reaction to 15 cultures of the stem rust fungus. F2 plants from Wdr‐sell/‘Little Club’ were progeny tested by inoculating families of F3 seedlings. Infection types were used to classify each F2 family into homozygous susceptible (S), segregating resistant and susceptible seedlings in a monogenic ratio (Seg), and inseparable (I). Segregation ratios were tested by chi‐square for goodness of fit to ratios of 4n‐2n‐1 (1):2n(Seg):l(S), where n is the number of segregating genes for resistance. Wdr‐sel had one gene for reaction to culture A‐14NW (race 29, HNH) and two genes for reaction to cultures 72‐14‐5 (race 11‐32‐113, RPQ), 72‐41spl (race 15B, TLM), WM‐1 (race 18, LBC), Gb121 (race 121, DCM), 370c (race 151, QFL), Or9e (race 9, JBC), Orllc (race 151, QBC), 41‐21spl (race 15, TBC), 72.00 (race 11‐32‐113, RTQ), A‐1 (race 151, QSH), A‐5 (race 156, MBC; one F1 family), and 75‐86spl (race 151, QSH; one F1 family). One F1 family gave trigenic segregations with cultures WM‐1, Or9e, Orllc, 41‐21sp1, and A‐5. Data for reactions to culture A‐15 (race 17, HNQ) were a poor fit to a digenic ratio and were inconclusive; however, the data were a better fit to a digenic ratio than to a monogenic or trigenic ratio. Wdr‐sel had four genes for resistance to culture 111‐SS2 (race 111, LCB).Paired comparisons of reactions to the different cultures were inconclusive but were consistent with the hypothesis that the gene for resistance to culture A‐14NW also conditioned resistance to the other cultures, and that the same two genes conditioned resistance to 13 of the cultures.

INHERITANCE OF RESISTANCE TO STEM RUST RACES 15B AND 56 IN EIGHT CULTIVARS OF COMMON WHEAT

Eight cultivars of common wheat showing good stem rust resistance in International Spring Wheat Rust Nurseries were selected. They were backcrossed to the susceptible cultivars Marquis and Little Club and the inheritance of their resistance to races 15B and 56 was studied. The major genes carried by the cultivars had all been identified previously. The cultivars and the major genes carried by them were as follows: Andes Sib (Sr8, Sr9?), Colotana (Sr8), Giza-Hegazy (Sr7a, Sr9a or b) Huascav (Sr9d), S.A. 112/52 (Sr9d), Bonza Selection (Sr6), Kuntur (Sr6, Sr7a) and Maribal Marina (Sr7a). In addition, four of the cultivars carried one or more genes giving very moderate resistance to one or both of the races.

The physiology of host–parasite relations. XIX. Further observations on nucleoprotein changes in wheat leaf nuclei during rust infection

Cytophotometric measurements of DNA (Feulgen) and protein-bound lysine and arginine (fluorodinitrobenzene-Sakaguchi) were made on nuclei in mesophyll cells of infected and uninfected leaves of Little Club wheat at intervals of 2 to 4 days up to 13 days after inoculation with uredospores of race 15B of the stem rust fungus (Puccinia graminis tritici Erikss. and Henn.). No change in host DNA was found within 6 days after inoculation, but there were marked decreases in protein-bound lysine and arginine as early as 2 days after inoculation. The DNA/lysine and DNA/arginine ratios were higher in rust-affected host nuclei, but infection did not alter the ratio of protein-bound lysine to arginine. In another series of measurements it was shown that rust infection caused striking increases in the volume and the RNA and protein contents of host nucleoli. These changes persisted even in advanced infections.All the results are consistent with our earlier observations and indicate that profound changes in nuclear metabolism are induced by infection with the rust fungus.

The effect of rust infection on DNA, RNA, and protein in nuclei of Khapli wheat leaves

The effects were determined of infection with Race 15B of the stem rust fungus on the levels of nuclear DNA (Feulgen), RNA (azure B), histone (fast green pH 8.1), and total protein (fast green pH 2.0) and on the size of the nuclei in the mesophyll cells of the primary leaf of Khapli wheat (rust reaction type 1). Nuclear size, RNA, and total protein content increased and histone content decreased by 3 days after inoculation. DNA content decreased from 4 days after inoculation. These results for the resistant variety Khapli are similar to those reported earlier for the susceptible variety, Little Club, but the observed changes occur more rapidly after inoculation than they do in Little Club.

THE UTILIZATION OF AMMONIUM CHLORIDE-15N BY UREDOSPORES OF WHEAT STEM RUST

When uredospores of wheat stem rust, Puccinia graminis van tritici (race 15B), were incubated with a 3 mM solution of ammonium chloride-15N, a significant amount of nitrogen 15 was converted into organic nitrogen. Most of this organic nitrogen 15 was found in the ethanol and water extracts, with lesser amounts in the buffer and in extracted spores.Amino acids extracted from the spores all contained excess nitrogen 15. Nitrogen 15 from the inorganic source was diluted by factors of 1.7 and 2.7 in free aspartic and glutamic acids respectively; these amino acids were the most heavily labeled with the isotope. Proline was the most weakly labeled amino acid, the nitrogen 15 being diluted by a factor of 102. Good incorporation of nitrogen 15 into glutamic acid compared to simultaneous poor incorporation into the biochemically related amino acid, proline, parallels previous observations made during carbon 14 experiments with rust uredospores.Fourteen "bound" amino acids were isolated after acid hydrolysis of extracted spores. All contained nitrogen 15, the dilution of the added ammonia nitrogen ranging from 96 for glutamic acid to 7660 for proline.The results are taken as evidence that uredospores of wheat stem rust can incorporate ammonium nitrogen into free amino acids and into proteins.

Inheritance of Stem Rust Resistance in Triticum aestivum ssp. vulgare ‘Reliance’ and P.I. 94701 of Triticum durum1

Seedling resistance to physiologic race 111 (culture 111‐SS2) of Puccinia graminis f. sp. tritici was conditioned by two and three independent genes, respectively, in the durum wheat P.I. 94701 and the common wheat Reliance. The infection type was lower when P.I. 94701 genes for resistance were combined in a single plant than when they occurred singly. One of the P.I. 94701 genes also conditioned moderate resistance to physiologic race 15B (culture 15B‐SS1). Low infection types masked high infection types in plants carrying two or three genes for resistance from Reliance.

THE METABOLISM OF ACETATE BY WHEAT STEM RUST UREDOSPORES.

When uredospores of Puccinia graminis var. tritici (race 15B) were incubated at pH 6.2 in phosphate buffer containing either acetate-1-C14or -2-C14, about 12% of the radioactivity was removed from the solution in a period of 3 hours. Respired carbon dioxide contained about 45% and 22% of the carbon-14 taken up as acetate-1-C14and acetate-2-C14, respectively. Incorporation of carbon-14 into spore components was considerably higher with acetate-2-C14than with acetate-1-C14. With either tracer most of the radioactivity in water-soluble spore materials was accounted for in amino acids and neutral substances. Glutamic acid was particularly radioactive and accounted for about 40% of the radioactivity in the amino acid fraction. Incorporation of carbon-14 into the glutamic acid skeleton was consistent with the view that both the tricarboxylic acid cycle and the glyoxalate cycle were functioning.

PREPARATION AND PROPERTIES OF A MITOCHONDRIAL FRACTION FROM WHEAT STEM RUST UREDOSPORES

Ungerminated uredospores of Puccinia graminis f. sp. tritici Erikss. & Henn. race 15B were disrupted in a buffered sucrose serum albumin solution, using a "Teflon" pestle homogenizer and glass beads 80 μ in diameter. A particulate fraction was sedimented between 2000 and 30,000 × g and its oxidation of Krebs' cycle acids measured manometrically. Endogenous respiration of washed preparations was low or negligible. Oxygen uptake was observed with succinate (40 to 60 μl/hour mg protein), α-ketoglutarate, malate, citrate, and isocitrate (10 to 30 μl/hour mg protein) but not with fumarate or pyruvate. Succinate oxidation was sensitive to heat, cyanide, and malonate.Aspects of the extraction procedure were examined for effects on yield and activity. Serum albumin in the grinding medium, very low grinding speed, and short grinding time favored high activity per mg protein. Glass beads increased the yield with the "Teflon" pestle while an all-glass homogenizer gave preparations of low activity.Oxidatively active particle suspensions reduced Janus green B and were shown by electron microscopy to consist of vesicles and some mitochondria.

ENZYMATIC PRODUCTION OF (+) threo-9,10-DIHYDROXYOCTADECANOIC ACID IN THE SPORES OF PLANT RUSTS

Evidence is presented to show that an epoxide-hydrolyzing enzyme system is present in the uredospores of Puccinia graminis Pers. f. sp. tritici Erikss. and Henn. race 15B and in the teliospores of Gymnosporangium clavariiforme (Pers.) DC. Incubation of the spores results in a partial conversion of cis-9,10-epoxy-octadecanoic acid, which is present in the spore oil as a glyceride, to (+) threo-9,10-dihydroxyoctadecanoic acid.

ENZYMATIC PRODUCTION OF (+) threo-9,10-DIHYDROXYOCTADECANOIC ACID IN THE SPORES OF PLANT RUSTS

Evidence is presented to show that an epoxide-hydrolyzing enzyme system is present in the uredospores of Puccinia graminis Pers. f. sp. tritici Erikss. and Henn. race 15B and in the teliospores of Gymnosporangium clavariiforme (Pers.) DC. Incubation of the spores results in a partial conversion of cis-9,10-epoxy-octadecanoic acid, which is present in the spore oil as a glyceride, to (+) threo-9,10-dihydroxyoctadecanoic acid.

THE METABOLISM OF VALERATE-2-C14BY UREDOSPORES OF WHEAT STEM RUST

When uredospores of Puccinia graminis var. tritici race 15B were shaken in a medium containing M/30 phosphate buffer, pH 6.2, and valerate-2-C14, about 88% of the radioactivity was removed from the buffer solution in a period of 3 hours. About 40% of the carbon-14 taken from the buffer was found in a water-soluble extract of the spores and about 15% was respired as carbon dioxide. The result is compared with an earlier report that carbon 1 of valerate is more extensively released as carbon dioxide and less extensively incorporated into spore components. Glutamic acid, glutamine, γ-aminobutyric acid, and alanine of high specific activity were isolated. It was estimated from partial degradation that more than one-half of the carbon-14 of glutamic acid occurred in position 4 and that carbon 5 was very weakly labelled. Citric acid was also of high specific activity and was labelled predominantly in the internal carbons.It is concluded that respiring rust spores utilize externally supplied valerate by β-oxidation, which releases carbons 1 and 2 in a form which is metabolized as acetate by the tricarboxylic acid cycle.

THE METABOLISM OF VALERATE-2-C14BY UREDOSPORES OF WHEAT STEM RUST

When uredospores of Puccinia graminis var. tritici race 15B were shaken in a medium containing M/30 phosphate buffer, pH 6.2, and valerate-2-C14, about 88% of the radioactivity was removed from the buffer solution in a period of 3 hours. About 40% of the carbon-14 taken from the buffer was found in a water-soluble extract of the spores and about 15% was respired as carbon dioxide. The result is compared with an earlier report that carbon 1 of valerate is more extensively released as carbon dioxide and less extensively incorporated into spore components. Glutamic acid, glutamine, γ-aminobutyric acid, and alanine of high specific activity were isolated. It was estimated from partial degradation that more than one-half of the carbon-14 of glutamic acid occurred in position 4 and that carbon 5 was very weakly labelled. Citric acid was also of high specific activity and was labelled predominantly in the internal carbons.It is concluded that respiring rust spores utilize externally supplied valerate by β-oxidation, which releases carbons 1 and 2 in a form which is metabolized as acetate by the tricarboxylic acid cycle.

THE PHYSIOLOGY OF HOST-PARASITE RELATIONS: XII. A CYTOPHOTOMETRIC STUDY OF THE DISTRIBUTION OF DNA AND RNA IN RUST-INFECTED LEAVES

Uninfected and rusted leaves of Little Club wheat (reaction type 4) were sectioned at intervals up to 15 days after inoculation with Puccinia graminis tritici Erikss. and Henn., race 15B. Infection caused a decrease in the size of the chloroplasts, an increase in size followed by a collapse of the nuclei in the mesophyll cells and an increase in the size of the nucleoli. These effects also occurred, but more rapidly, in infected leaves of Khapli wheat (rust reaction type 1). They were most pronounced in the tissue beneath uredia. Cytophotometry of nuclei in Little Club by the 'two-wavelength' method after staining with the Feulgen reagent showed that there was no increase in the level of DNA in enlarged nuclei at 6 days after inoculation; collapse of the nuclei at 15 days after inoculation was accompanied by the loss of 60% of the DNA originally present. Cytophotometry of nucleoli in nuclei in mesophyll cells stained with azure B after selective removal of DNA with DNase showed that nucleolar enlargement was accompanied by a significant (P = 0.05) increase in RNA. The fungal nuclei stained with the Feulgen reagent, but not after treatment with DNase. The cytoplasm in fungal hyphae, haustorial mother cells and haustoria stained heavily with azure B, but not after treatment with RNase. The absorption spectrum of the stained mycelium was characteristic of the RNA - azure B complex. The results are discussed.

THE PHYSIOLOGY OF HOST–PARASITE RELATIONS: XI. THE EFFECT OF STEM RUST ON THE PHOSPHATE FRACTIONS IN WHEAT LEAVES

Inorganic, organic, and various organic phosphate fractions were determined in uninfected and infected first seedling leaves of Little Club wheat at various stages between 6 to 18 days after incubation with Puccinia graminis tritici Erikss. and Henn. (Race 15B). Inorganic (Pi), organic (Po), and total P per unit fresh weight increased as uninfected leaves aged and the levels of Pi were much more readily altered by changes in nutritional status and environmental conditions than the levels of Po. Infection increased total P and the highest concentrations occurred at the loci of rust pustules. Since the concentration of P was lower in uninfected portions of infected leaves than in comparable parts of uninfected leaves or in rust pustules, it appeared that P was transported into the pustules from uninfected parts of the leaf. Both Pi and Po were increased by infection, but the Pi/Po ratio was lowered. Acid-soluble bound P, easily hydrolyzable P, lipid P, RNA-P, and residual organic P were all increased by infection.When leaf pieces were infiltrated with and then floated on 10−4 M 2,4-dinitrophenol for 24 hours, the levels of all organic phosphate fractions were lowered and Pi was increased, both in uninfected and infected leaves. Dinitrophenol raised the Pi/Po ratio about 30% in uninfected and about 100% in infected leaves.The results militate against the view that the increase in respiration rate caused by infection is due to the uncoupling of oxidative phosphorylation.

BIOASSAY OF ANTIBIOTIC P-9 BY THE DIRECT RESPONSE OF STEM RUST OF WHEAT

A simple medium was found for the production of antibiotic P-9 used in experiments for the control of stem rust of wheat.A rust-infection bioassay method for the estimation of the potency of different production lots of filtrate was developed; potency was measured as the proportionate inhibition of stem rust (Puccinia graminis Pers. f. sp. tritici Erikss. & Henn. race 15B) infection on wheat seedlings in relation to log concentration of the material under test. The data from the bioassays took the form of a sigmoid curve when the concentrations were plotted arithmetically and of a straight line when they were plotted logarithmically. The slope of a weighted mean regression line based on 11 assays was regarded tentatively as characterizing pure antibiotic P-9.When the data of the 11 assays were adjusted for differences in potency between assays, the weighted mean regression line was statistically significant at the 1% point of probability. It is hoped that the slope of this line characterizes the pure antibiotic P-9 sufficiently well to be a useful tool in estimations of the relative purity of fractions obtained in the progressive purification of the antibiotic. Regression lines that differ from it substantially in slope may be suspected of influence from some extraneous factor.

THE INHERITANCE OF RUST RESISTANCE: IX. THE INHERITANCE OF RESISTANCE TO RACES 15B AND 56 OF STEM RUST IN THE WHEAT VARIETY KHAPSTEIN

The inheritance of resistance to races 15B and 56 of stem rust was studied in the variety Khapstein which obtained its resistance from Khapli emmer. Khapstein was found to carry gene Sr7 which controls resistance to race 15B and two additional genes, one conditioning a type 2 reaction to race 56 and a 2+ – 3 reaction to race 15B, and a second controlling a striking, grey necrosis around pustules produced by race 56. The two have been designated Sr13 and Sr14 respectively.

Effect of short-chain fatty acids on respiration of wheat stem rust uredospores.

Fatty acids added to a suspension of wheat stem rust uredospores (Puccinia graminis var. tritici, race 15B) stimulate respiration. When compared on the basis of equal molar concentration, the utilization of oxygen by spores from wheat grown in the field or in the greenhouse is stimulated by short-chain fatty acids as follows: acetate < propionate < butyrate < valerate. The order for butyrate and valerate is reversed with spores from plants grown under artificial light. Radiotracer experiments indicate that the amount of respired carbon dioxide derived from the carboxyl carbon of the fatty acid added increases markedly with increase in chain length. The addition of exogenous acetate stimulated conversion of spore carbon to carbon dioxide, whereas valerate replaces, in part, spore material as a source of respiratory carbon. Valerate-1-C14 is almost 4 times as effective as acetate-1-C14 for labelling spore material, but the ratio of carbon-14 respired to that incorporated is 2.6 for valerate-1-C14 as compared with 1.5 for acetate-1-C14.

THE PHYSIOLOGY OF HOST–PARASITE RELATIONS: VIII. EFFECTS OF RUST INFECTION ON ASCORBIC ACID ANDGLUTATHIONE IN WHEAT LEAVES

First seedling leaves of Little Club (susceptible), Khapli I (moderately susceptible), and Khapli (resistant) wheats were inoculated with Puccinia graminis tritici Erikss. and Henn. (Race 15B). Ascorbic (AA) and dehydroascorbic (DHA) acids and glutathione (GSH) were measured at 1–2 day intervals after inoculation until after sporulation. After infection, AA and DHA increased in Little Club and decreased in Khapli. In Khapli the DHA/AA ratio rose sharply 6–8 days after inoculation; in Little Club a similar rise occurred 15–16 days after inoculation. In Khapli I and Little Club, but not in Khapli, GSH increased sharply when sporulation occurred. AA was not detected in uredospores, but DHA was. Oxidized glutathione was not detected in healthy or infected leaves but was present in uredospores. The results are discussed briefly in relation to the changes in auxin content and redox potentials caused by rust infection.

Studies on the metabolism of valerate-1-C14 by uredospores of wheat stem rust.

When uredospores of Puccinia graminis var. tritici race 15B were shaken in a medium containing M/30 phosphate buffer, pH 6.2, and valerate-1-C14, 97% of the radioactivity was removed from the solution in a period of 3 hours. Fifty-five per cent of the carbon-14 was released as carbon dioxide, and 42% was incorporated into the spores. Carbon-14 was found in many cellular components but the water-soluble fraction accounted for 48% of the tracer in the spores. About two thirds of the water-soluble carbon-14 was in a fraction containing amino acids, amides, and peptides, with glutamic acid, glutamine, and γ-aminobutyric acid being highly radioactive. Carbon-5 of glutamic acid and carbon-1 of γ-aminobutyric acid were particularly radioactive. In addition carbon-1 of glutamic acid was appreciably radioactive. The results are consistent with the view that γ-aminobutyric acid was formed by decarboxylation of glutamic acid and that glutamic acid became labelled as a result of β-oxidation of the valerate-1-C14 to yield acetate-1-C14 which in turn was metabolized by the tricarboxylic acid cycle.