Evaluation of a Chemical Method for Assay of Helminthosporium maydis Race T Toxin

A chemical test reported by Karr et al. (Plant Physiol. 55:727) to assay for host-specific toxin produced by Helminthosporium maydis race T was evaluated. Preparations from culture filtrates of both race T isolates, containing host-specific toxin, and similar preparations from race O isolates, containing no detectable host-specific toxin, gave positive reactions in the chemical assay. Also, preparations containing active or inactive toxin gave equal responses in this test. The procedure does not provide a reliable method for assaying H. maydis race T toxin.     

Studies of the Toxicity of Helminthosporium maydis

Isolates of Helminthosporium maydis from blighted corn were tested for toxicity in mice, rats, swine, rabbits, microorganisms, and tissue culture. Extracts of grains, mycelia, and culture supernatant fluids killed mice on intraperitoneal (ip) injection, but were nontoxic on administration by mouth to swine. The toxin was partially purified and appears to be a glycophospholipid. Histopathological examination revealed that the toxin acted as a severe irritant on ip injection, causing death in laboratory animals. In skin tests with rabbits, considerable exudation occurred, rather than necrosis.     

Induction of Stomatal Closure by Helminthosporium maydis Pathotoxin

Helminthosporium maydis pathotoxin caused a rapid inhibition of photosynthesis in whole leaves of maize having Texas male-sterile cytoplasm but not in leaves having normal cytoplasm. Electron transport, phosphorylation, and proton uptake activities of isolated chloroplast lamellae, from either normal or Texas male-sterile cytoplasm leaves, were unaffected by addition of toxin to the reaction mixture. The toxin was found to have a direct effect on stomatal functioning. Rates of transpiration were inhibited in treated leaves at times slightly preceding the observed effects on photosynthesis. In studies with isolated epidermal peels from Texas male-sterile cytoplasm maize leaves, the toxin inhibited light-induced K⁺ uptake by guard cells. All effects of the toxin on Texas male-sterile cytoplasm maize tissues were found to be similar to the mode of action of abscisic acid.     

Force Required to Detach Conidia of Helminthosporium maydis

The force required to break the conidium-conidiophore attachment in Helminthosporium maydis was measured by centrifugation and by a small jet of air. The force which removed half the spores from their stalks was found by both methods to be about 1 × 10⁻² dynes. The corresponding speed of the air jet was about 10 m/sec. Although spores are removed over a range of applied force, most of this spread is accounted for by the variation in spore size. Therefore, the apparent strength of the attachment of conidia to conidiophores, though relatively large, is surprisingly uniform.     

利用组织培养技术选育玉米抗小叶斑病突变体

大叶斑病和小叶斑病是玉米最严重的病害,选育抗大、小叶斑病优良自交系及单交种是提高玉米产量的重要措施。Ｇｅｎｇｅｎｂａｃｈ等以玉米Ａ６１９ｃｍｓＴ愈伤组织为材料,筛选出对小叶斑病毒素具有抗性的愈伤组织与再生植株［１,２］,开辟了玉米抗病育种的新途径。...     

Carbohydrate involvement during infection of maize by Helminthosporium maydis

Germination and germ tube length of Helminthosporium maydis conidia did not exhibit much difference on fixed decolourized and living green leaves. However, appressoria, penetrations and colonizations were much less on decolourized host leaves and were enhanced significantly when sugars were added in the infection court. Few leached conidia germinated on the decolourized host leaves and appressoria, penetrations and colonizations effected on them by leached conidia were almost negligible. The presence of exogenous sugars and leaf leachates enabled the leached conidia to accomplish some penetrations and colonizations. Carbohydrate content of decolourized leaves and leached conidia was much less than the green leaves and non-leached conidia, respectively. Carbohydrates accumulated at the infection sites/green islands which also exhibited higher chlorophyll content.     

Effect of Thiamine on Ethanol and Pyruvate Production in Helminthosporium maydis

Growth of the fungus Helminthosporium maydis race T in a basal glucose-l-asparagine liquid medium, pH 5, is inhibited by thiamine-HCl. Analysis of the media for organic acids reveals that the extracellular pyruvate concentration decreases as the thiamine-HCl concentration of the medium increases. Extracellular ethanol, in contrast to pyruvate, increases in concentration as the thiamine-HCl concentration of the medium increases under both aerobic and anaerobic conditions.The changes in ethanol and pyruvate levels in the presence of thiamine-HCl occur via a thiamine-mediated increase in the activity of pyruvate decarboxylase but not alcohol dehydrogenase. This increase in pyruvate decarboxylase activity appears to be due to an increase in the quantity of enzyme present rather than an activation of pre-existing enzyme. Whereas thiamine-pyrophosphate stimulates pyruvate decarboxylase activity in vitro, thiamine-HCl has no effect. Neither thiamine derivative affects alcohol dehydrogenase activity. The increase in pyruvate decarboxylase activity which accompanies an increase in the thiamine-HCl concentration of the medium is correlated with a decrease in the level of intracellular pyruvate.     

The possible involvement of cytokinins in the pathogenicity of Helminthosporium maydis

Green islands/infection sites recorded higher cytokinin activity than surrounding tissue as well as non-inoculated tissue. This activity in infected areas increased with time of incubation while in tissue surrounding the green islands and non-inoculated tissue, cytokinin activity decreased with time of incubation. The culture filtrate extracts of H. maydis had cytokinin activity which increased with growth of the fungus. Cytokinin activity of thin-layer Chromatographic fractions from tissue and culture filtrate extracts revealed that a major portion of the activity was confined to Rf zone 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. Presence of zeatin and zeatin riboside in tissue and culture filtrates was confirmed by high performance liquid chromatography. Cytokinin substances, such as zeatin and zeatin riboside, increase at infection sites with growth of the pathogen suggesting they may be involved in the pathogenicity of H. maydis on maize.     

Histopathological changes in susceptible corn inoculated with Helminthosporium maydis race O

Seedlings of a susceptible inbred line of male-fertile corn were inoculated with conidia of Helminthosporium maydis race O. Histological and ultrastructural observations of mesophyll, bundle sheath and phloem were made over a period of 8 days. Histological observations at 1 day revealed that lesions were comprised of several dead mesophyll cells bordered by a pair of vascular bundles. By 3 days lesions had developed their characteristic appearance caused by mesophyll collapse and had increased to a width of 10–12 bundles. At the ultrastructural level, the first signs of mesophyll cell change were rupture of the tonoplast and swelling of the mitochondrial matrix followed by a disintegration of the cytoplasm and swelling of the chloroplast stroma. Following these changes the cytoplasm became filled with an electron dense material and the plasmalemma ruptured leaving only partial remnants of chloroplasts as recognizable organelles. All of these changes occurred by 1 day. Bundle sheath cells were more resistant and intact cells could be observed in 3-day-old lesions. Phloem showed signs of degeneration by 1 day with distortion of the sieve-tube element membranes and disintegration of the companion cell cytoplasm. By 4 days the phloem had disintegrated.     

Inhibition of Ornithine Decarboxylase and Growth of the Fungus Helminthosporium maydis

α-dl-Difluoromethylornithine (DFMO), a specific enzyme-activated inhibitor of ornithine decarboxylase, at 0.5 to 2.0 millimolar significantly inhibited mycelial growth and especially sporulation of Helminthosporium maydis in the dark; its inhibitory effect on sporulation was greatly increased under light conditions. Putrescine at 0.25 millimolar fully prevented the inhibitory effects of DFMO; the inhibition caused by the latter could not be prevented by cadaverine or CaCl₂. α-dl-Difluoromethylarginine, a specific enzyme-activated inhibitor of arginine decarboxylase, at 0.1 to 2.0 millimolar had a weak inhibitory effect on the fungus. The effect was not dependent on the inhibitor concentration and there was no detectable arginine decarboxylase activity in the fungus.     

Helminthosporium maydis T Toxin Increased Membrane Permeability to Ca in Susceptible Corn Mitochondria

Though Helminthosporium maydis race T (HmT) toxin decreased active Ca(2+) uptake into mitochondria isolated from susceptible (T) but not resistant (N) corn (Kimber, Sze, 1984 Plant Physiol 74: 804-809 the mode of toxin action is not understood. This study shows that HmT toxin or A23187 (a Ca(2+) ionophore) dissipated a Ca(2+) gradient in T mitochondria. However, HmT toxin had no effect on Ca(2+) gradients in N mitochondria or microsomal vesicles from T or N corn. The results suggest that HmT toxin increased membrane permeability to Ca(2+) in mitochondria of T corn specifically.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

Mitochondrial Interaction with Helminthosporium maydis Race T Toxin: Blocking by Dicyclohexylcarbodiimide

Treatment of mitochondria isolated from Texas male sterile cytoplasmcorn (T mitochondria) with high concentrations of dicyclohexylcarbodiimide(DCCD) (140 nmol DCCD mg^–1 mitochondrial protein) completelyand immediately inhibited T mitochondrial swelling by Helminthosporiummaydis Race T toxin (HmT toxin). In order to obtain a specificinteraction between DCCD and the ATPase complex T mitochondriawere incubated with lower DCCD concentrations (1–5 nmolDCCD mg^–1 mitochondrial protein) for up to 8 h at 4 °C.After 8 h incubation in the presence of 3.75 nmol DCCD mg^–1mitochondrial protein, toxin-induced swelling was decreasedby 69%. Specificity of DCCD action upon the ATPase complex wasconfirmed by (1) SDS gel electrophoresis and fluorographic analysesof proteins from [¹⁴C]-DCCD-treated T mitochondria and immunoprecipitatesand (2) physiological experiments showing that DCCD exertednone of its other documented effects. These data suggest thatHmT toxin interacts with the ATPase complex of T mitochondriaeither at or near the DCCD-binding protein within the membranesector of the complex. Key words: Zea mays L., Helminthosporium maydis, Mitochondria     

Studies on the Host-Specific Pathotoxins Produced by Helminthosporium maydis,race T

Two host-specific pathotoxins, Band 1- and Band 2-toxins, were isolated from the toxin complex obtained from the culture broth and mycelial mat of Helminthosporium maydis, race T, the fungus causing Southern corn blight disease. Chemical and spectrometric studies showed them to be polyketo-polyhydroxy compounds with C₄₁ carbon chains. Band 2-toxin is identical to Band 1-toxin except that Band 2 toxin has an additional hydroxyl group in place of one of the keto-group of Band 1-toxin.     

Inheritance of resistance to Helminthosporium maydis blight in maize (Zea mays L.)

Summary The nature and mode of inheritance of resistance to Helminthosporium maydis blight was investigated in two maize varieties, RbU-W and DIC. The study of F₁, F₂, and reciprocal backcross populations of crosses between these two varieties on the one hand and two susceptible varieties, UVE and ZPSc-58c on the other, revealed that resistance in the two varieties is monogenic recessive. The genes for resistance in the two varieties are allelic. Resistance was shown to be a lesion-type and measurements revealed that it operated through reduced lesion size and lesion number.     

The Toxins of Helminthosporium maydis (Race T) : A Colorimetric Determination of the Toxins, Their Appearance in Culture and in Infected Plants

Host-specific toxins produced by Helminthosporium maydis, race T, are measured quantitatively by a chemical assay procedure involving reaction of the toxins with a sulfuric acidacetic anhydride reagent and measurement of the absorbance of the product at 330 nm. The assay was shown to measure total toxin concentrations after only limited fractionation of the culture medium. Using the assay it was possible to show that the highest amount of toxin per gram of fungus mycelium occurs early in the growth cycle of H. maydis. Toxins I, II, and V are the predominant toxins at these early times both in culture and in infected corn and wheat varieties. Some chromatographic and spectral properties of toxin V, a previously unreported toxin, are described. Since toxin V appears in culture prior to toxins I, II, III and IV, a precursor-product relationship can be suggested.