In vitro and In vivo Effects of Some Fungicides against the Chickpea Blight Pathogen, Ascochyta rabiei

The effects of various fungicides on mycelial growth and spore germination of Ascochyta rabiei were determined by incorporating them into potato dextrose agar and measuring colony diameter and observing colony growth and spore germination at 20 ± 2°C. Eight fungicides prevented spore germination of the pathogen at concentrations of 0.125–2 μg/ml, three hindered mycelial growth at 2–4 μg/ml and seven failed to inhibit mycelial growth even at 128 μg/ml. The reference fungicide for the pathogen, chlorothalonil, stopped conidial germination at low rates but did not prevent mycelial growth at 128 μg/ml. Thirteen fungicides were tested against seed infections of the pathogen, and benomyl + thiram, carbendazim and carbendazim + chlorothalonil seed treatments gave more than 85% inhibition on both vacuum‐infiltrated and naturally infected seeds. Coating the seeds with polymers did not increase the effectiveness of fungicides. Three fungicides; (azoxystrobin, chlorothalonil and mancozeb), gave the highest protection in the field but protection decreased with increased inoculum pressure. Addition of humic acid to fungicide suspensions did not affect their performance.     

Assessing in vitro efficacy of certain fungicides to control Sclerotinia sclerotiorum in peanut

The fungal pathogen Sclerotinia sclerotiorum is responsible for Sclerotinia blight in several crops around the world, including peanut. This study was conducted under laboratory conditions to determine the effects of four registered fungicides, Propulse?, Fontelis^®, Omega^® and Endura^® on mycelial growth and pigmentation, as well as sclerotia and oxalic acid production on a growth medium modified with a fungicide and on the pathogenicity of S. sclerotiorum on leaflets detached from Valencia peanut. Propulse, Omega and Fontelis inhibited mycelial growth of S. sclerotiorum, while, mycelial growth on a modified support with Endura was similar to the control treatment. All fungicides, except Endura, inhibited the production of oxalic acid. Pigmentation of the mycelium was observed in both the control and endura treatments. Sclerotia production was observed only in the control treatment. With the exception of Endura, all fungicides were effective in controlling the development of lesions on Valencia peanut leaflets.     

Studies on the Acid-protease of Paecilomyces varioti Bainier TPR-220

The crystalline acid-protease of Paecilomyces varioti Bainier TPR-220 is most active toward casein as substrate, at pH 3.0 and 60°C, and stable at pH 3.0 to 6.0 below 40°C. The enzyme decomposes protein molecules into smaller fragments than pepsin does and is inhibited by p-chloromercuri-benzoate, monoiodoacetate, sodium lauryl sulfate, iodine, potassium permanganate, N-bromosuccinimide, bacitracin, nitrofurylacrylamide, and Hg⁺ ion, but affected neither by metal ion except Hg⁺ ion, nor metal chelating agent, soy bean trypsin inhibitor, potato-protease inhibitior, cysteine, diiso-propylfluorophosphate, cyanogen bromide, and heparin. The presence of Ca⁺⁺, Co⁺⁺, Cu⁺⁺, Mg⁺⁺, Sr⁺⁺, and Zn⁺⁺ ions prevents heat inactivation of the enzyme.     

Efficacy of benzimidazole and related fungicides against Rhizoctonia solani and R. bataticola

Five formulations of four benzimidazole derived fungicides, carbendazim, benomyl, thiophanate methyl and methyl 4-[2-(2-dimethylamino acetamide) phenyl]-3-thioallophanate were compared for their toxicity towards two pathogenic isolates of Rhizoctonia solani and three of R. bataticola. The isolates of two fungi showed significant differences in mycelial growth inhibition by the five fungicides. Benomyl and carbendazim were most inhibitory to all isolates of both fungi while the sesame isolate of R. bataticola was least sensitive to all fungicides. Disease control (90%) was obtained with low concentrations of benomyl against root rot of cowpea caused by R. solani, and with thiophanate methyl against root rot of sesame and sunflower, and leaf blight of mung bean caused by R. bataticola. The spread of stalk-end rot of sunflower heads was best checked with a spray of thiophanate methyl. The results suggest that benzimidazole fungicides having similar toxophores act differently for disease control in different host-parasite combinations.     

Pathogenic stability of Alternaria longipes (Ell. & Ev.) Mason subjected to different methods of isolation,storage and inoculum production

Summary The pathogenic stability ofA. longipes was greatest when the composition of the medium promoted maximum sporulation and minimal mycelial proliferation.A Whatman No. 17 filter paper disc saturated with an 0.1 % dextrose infusion medium from carrots and potatoes minimised mycelial proliferation, and promoted rapid and extensive spore production in two to four days at 25° C. Approximately 75% of the cultural period on 2% PDA was devoted to mycelial proliferation. The difference in extent of mycelial growth in the filter paper and standard methods was apparently instrumental in eliminating a decline in pathogenicity when using the former method. Weekly mycelial subculturing on 2% PDA caused rapid drop in pathogenicity and a total loss of pathogenicity and sporulative ability between the 62nd and 76th day.The use of a modified filter paper method for large scale inoculum production for greenhouse and field variety trials is discussed.     

Sensitivity of Clonostachys rosea and Trichoderma spp. as Potential Biocontrol Agents to Pesticides

Clonostachys rosea 47 (CR47), Trichoderma atroviride 59 (TA59), T. atroviride 312 (TA312), Trichoderma harzianum 24 (TH24), Trichoderma longibrachiatum 9 (TL9), T. longibrachiatum 144 (TL144) and Trichoderma viride 15 (TV15) were tested to evaluate their in vitro sensitivity towards five fungicides (carboxin, guazatine, prochloraz, thiram and triticonazole) and four herbicides (chlorsulfuron, chlorotoluron, flufenacet and pendimethalin). All antagonists showed low sensitivity to carboxin and thiram and high sensitivity to prochloraz. For mycelial radial growth, TV15 was highly sensitive to guazatine, prochloraz and triticonazole and TH24 moderately insensitive to carboxin, guazatine and thiram. For conidial germination TL144 was the most sensitive to the fungicides, for mycelial radial growth and conidial germination CR47 was the least sensitive. None of the antagonists showed any mycelial radial growth inhibition in presence of the herbicides at field dose, except for TL144. Most antagonists did not show any conidial germination inhibition by the herbicides. The in vitro toxicity of prochloraz, guazatine and triticonazole towards the antagonists was confirmed by light and scanning electron microscope showing hyphal disruptions and extrusion of cytoplasmic content. A mixture of CR47 and/or TA312 with carboxin, thiram and triticonazole, applied to wheat seeds, was able to control Fusarium culmorum artificially inoculated to wheat seedlings in growth chambers. In the field, the antagonists applied along with triticonazole or thiram, at 1/10 of the field dose to seeds naturally infected by F. culmorum, gave a disease control comparable to that induced by triticonazole at full field dose. Our results demonstrate how an integration of microorganisms with pesticides makes the control of wheat foot rot possible.     

Inorganic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase in mung beans and its activation by D-fructose 1,6-bisphosphate and D-glucose 1, 6-bisphosphate

Inorganic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase was detected in extracts of mung bean sprouts, the first such detection in C₃ plants. The enzyme had an absolute requirement for a divalent metal (Mg⁺⁺) as well as for D-fructose 6-phosphate and inorganic pyrophosphate. An examination of anomalous kinetics revealed that the enzyme was activated by a product of the reaction, D-fructose 1,6-bisphosphate; micromolar concentrations of this effector increased the activity of the enzyme about 20-fold. D-Glucose 1,6-bisphosphate at higher concentrations could substitute for D-fructose 1,6-bisphosphate as an activator, but not as a substrate in the reverse reaction. The enzyme was fully active under conditions wherein ATP: D-fructose-6-phosphate 1-phosphotransferase from the same source was inhibited >99% (e.g., in the presence of 10 μM phosphoenolpyruvate).     

Comparison of fungicides for the control of Rhizoctonia solani causing datnping-off of mung bean (Phaseohis aureus)

Of 41 fungicides tested in the laboratory, copper carbonate, copper sulphate, mercuric chloride, Agrosan GN, quintozene, kasugamycin, carboxin, pyracar-bolid, carbendazim, chloroneb, benomyl, Ohric, RH 893 (2-n-octyl-4-isothiazole-3-one) and Terrazole were most inhibitory to the mycelial growth of Rhizoctonia solani on Czapek's agar plates and had EC₅₀ values of less than 1 μg a.i./ml, while copper oxychloride, Udonkor, zineb, ziram, F 319 (3-hydroxy-5-methyl isoxazole) and anilazine were much less toxic, ziram being least inhibitory with an EC₅₀ of 214 μg a.i./ml. Of 17 fungicides tested in the greenhouse as seed treatments, thiabendazole, carbendazim, benomyl, thiophanate-methyl, dichlozoline and Ohric gave 80–90% control of damping-off of mung bean seedlings. A single soil drench with thiophanate-methyl and two drenches with benomyl gave about 90% disease control, More seedlings with R. solani infection survived when thiophanate-methyl was used as a post-inoculation soil drench than when benomyl or chloroneb were used.     

Production of α-galactosidase by a novel actinomycete <Emphasis Type="Italic">Streptomyces griseoloalbus</Emphasis> and its application in soymilk hydrolysis

α-Galactosidase production by a newly isolated actinomycete Streptomyces griseoloalbus under submerged fermentation was investigated. The influence of initial pH of medium, incubation temperature, inoculum age and inoculum size on α-galactosidase formation was studied. Various carbon sources were supplemented in the medium to study their effect on enzyme production. The influence of the concentration of locust bean gum on enzyme production also was optimized. Optimization of process parameters resulted in a highest α-galactosidase activity of 20.4 U/ml. The highest α-galactosidase activity was obtained when the fermentation medium with initial pH 6.0 and containing 1% locust bean gum as growth substrate was inoculated with 10% (v/v) of 72 h grown inoculum and incubated at 30°C. The hydrolysis of flatulence-causing oligosaccharides in soymilk by the enzyme was also investigated. Thin layer chromatographic analysis of enzyme-treated soymilk samples showed the complete hydrolysis of soy oligosaccharides liberating galactose, the final product.     

Calcium, asparagine and cAMP are required for ornithine decarboxylase activation in intact Chinese hamster ovary cells.

Asparagine specifically activated ornithine decarboxylase activity 5–7 fold by 7–8 h in confluent cultures maintained with a salts/glucose medium. When dibutyryl cAMP was added with asparagine, a 40–50 fold stimulation of ornithine decarboxylase activity was produced. Ornithine decarboxylase activation in the salts/glucose medium was not sensitive to actinomycin D. Omission of Ca⁺⁺ and Mg⁺⁺ from the medium abolished the ability of asparagine and/or dibutyryl cAMP to stimulate enzyme activity. Calcium was essential for the asparagine and dibutyryl cAMP mediated stimulation of ornithine decarboxylase activity.     

Some factors affecting the control of Rhizoctonia solani by systemic and non-systemic fungicides

In laboratory tests, Rhizoctonia solani grew best on agar at 30 ^oC and pH 5-5. Mycelial growth was strongly inhibited by benomyl, chloroneb and quintozene, less so by thiophanate-methyl. The optimum temperature for inhibition was 25 ^oC for quintozene; the other three fungicides gave greatest inhibition at the lowest temperature tested (20 ^oC). Benomyl and thiophanate-methyl were most inhibitory at pH 7–8, chloroneb and quintozene at pH 5–6. In pot trials using mung bean, long melon, egg-plant, common pea and sugar beet, R. solani caused maximum disease at 20 ^oC and in wet and alkaline soils. As seed treatments, benomyl and thiophanate-methyl gave optimum control at 20 ^oC and pH 7-6; chloroneb and quintozene were most effective at 30 ^oC and pH 5-4. All four fungicides gave maximum control on plants growing in wet sandy loam. Comparisons of host effects showed that, on all the four hosts tested, thiophanate-methyl wettable powder at 0.25% (0.175% a.i.) gave 90% control, chloroneb w.p. at 0.3% (0.195% a-i-) gave 80% control on mung bean and sugar beet, benomyl w.p. at 0.3% (0.15% a.i.) was satisfactory on mung bean, egg-plant and sugar beet but not on long melon, quintozene w.p. at 0.3 % (0.225 % a-i-) gave effective control on sugar beet only. The senior author is grateful to the Council of Scientific and Industrial Research, New Delhi, India, for the award of a Research Fellowship for undertaking these studies. Thanks are due to Dr S. D. Gupta and Mr S. L. Verma for determining the soil characteristics.     

Biosynthesis of pure araban and xylan

A crude particulate enzyme preparation from mung bean shoots partially freed from sugar transferases synthesized pure araban from UDP-l-¹⁴C-arabinose. The preparation thus allowed to study some properties of the UDP-arabinose transferase which was shown to require 7 mM Mn²⁺ and pH 6–6·5 for optimal activity. Pure xylan was synthesized from UDP-d-¹⁴C-xylose if a mixture of 0·06% Triton X100 and 35 mM EDTA was added to the crude enzyme preparation. In contrast to the UDP-arabinose transferase the UDP-xylose transferase does not require bivalent metal ions.     

Effect of magnesium and calcium ions on blocking of synaptic transmission by adenosine in isolated rat hippocampal slices

Dependence of the inhibitory action of adenosine on the extracellular composite EPSP on the concentrations of Mg and Ca cations in the medium was investigated in isolated slices of rat hippocampusin vitro. Extracellular EPSPs were derived in the region of apical dendrites of pyramidal cells in area CA₁ during stimulation of Schaffer's collaterals. The blocking action of bivalent cations (an increase in Mg⁺⁺ or a decrease in Ca⁺⁺) developed almost five times more slowly than the action of adenosine. An increase in the external Mg⁺⁺ concentration potentiated whereas a decrease weakened the inhibitory action of adenosine. Ca⁺⁺ ions had the opposite effect. Antagonistic relations were exhibited between Mg⁺⁺ and Ca⁺⁺ ions. Analysis of dose-response curves for adenosine showed that during a simultaneous increase in the extracellular Ca⁺⁺ and decrease in Mg⁺⁺ concentrations, not only was the maximal effect of adenosine reduced, but so also was its binding constant with the receptor. The results suggest that antagonism between Ca cations and adenosine is mixed in character — both competitive and noncompetitive. The possible mechanism of the inhibitory action of adenosine on synaptic transmission and the role of bivalent cations in this process are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 532–539, July–August, 1984.     

Sudden death syndrome of soybean in South America is caused by four species of Fusarium: Fusarium brasiliense sp. nov., F. cuneirostrum sp. nov., F. tucumaniae, and F. virguliforme

Soybean sudden death syndrome (SDS) pathogens and dry bean root-rot pathogens were studied taxonomically, phylogenetically, and pathologically. Detailed phenotypic comparisons of macro- and microscopic features and phylogenetic analyses of multilocus DNA sequence data, including those on the nuclear ribosomal intergenic spacer region and the single copy nuclear gene translation elongation factor 1-a, indicated that they comprised five distinct species of Fusarium. Two new species causing soybean SDS in Brazil, F. brasiliense and F. cuneirostrum, are formally described. Fusarium cuneirostrum is responsible for soybean SDS in Brazil and dry bean or mung bean root-rot in the United States, Canada, and Japan. Strains of each species, including F. cuneirostrum isolates from dry bean and mung bean and F. phaseoli isolates from dry bean, were inoculated on soybean cultivar Pioneer 9492RR to determine their pathogenicity. Although intraspecific variation in pathogenicity was observed, all the species were able to induce typical SDS symptoms on soybean plants in the artificial inoculation tests. Comparisons of the key diagnostic morphological features reveal that all five species can be diagnosed using conidial morphology.     

Induction of germ-tube formation by Candida albicans in amino acid liquid synthetic medium at 25°C

Candida albicans (3153A) was found to exhibit extensive germ-tube and mycelial development at 25°C when transferred from amino acid synthetic medium at pH 6 to medium of pH 7. Significant germ-tube formation was detectable after approximately 8 h and in all experimental treatments, the peaks of maximal germination occurred at approximately 40–44 h. Such a transition was not only dependent on the initial pH of the medium but also on the glucose concentration and inoculum size. The optimum initial glucose concentration and inoculum size for maximal germ-tube development was 1.25% and 2×10⁶ cells ml^–1 respectively and above or below these values the extent of germ-tube formation was greatly reduced.     

Purification and Characterization of Invertase from Lactobacillus reuteri CRL 1100

The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45°C over a wide pH range (4.5–7.0) with maximum activity at pH 6.0 and 37°C. The invertase activity was significantly inhibited by bivalent metal ions (Ca⁺⁺, Cu⁺⁺, Cd⁺⁺, and Hg⁺⁺), β-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified 32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K _m and V_max values for sucrose were 6.66 mM and 0.028 μmol/min, respectively. An invertase is purified and characterized for the first time in Lactobacillus, and it proved to be a β-fructofuranosidase. Received: 13 August 1999 / Accepted: 15 September 1999     

Ecto-ATPase

Summary An ecto-adenosine triphosphatase (E.C. 3.6.1.4 ATP-phosphohydrolase) is shown to be localised on the outer surface of varieties of cell membrane. The enzyme is different from the ATPase involved in biological energy transduction and ion transport mechanism. The characteristic of the enzyme lies in having a very broad substrate specificity and is inhibited by EDTA and higher concentration of ATP. The enzyme is dependent on bivalent metal ions, Mg⁺⁺ or Ca⁺⁺ for its optimum activity. The enzyme is highly sensitive to SH-reagents but insensitive to inhibitors of mitochondrial ATPase or Na⁺−K⁺-ATPase. The possible functions of the enzyme in being oriented outside the cell membrane is discussed.     

Cloning and sequencing of V-ATPase subunit d from mung bean and its function in passive proton transport

We have previously shown that vacuolar H+-ATPase subcomplex V_o from mung bean contains subunit d, however, its sequence and function were unknown. In the present study, we report the cloning and recombinant over expression of subunit d from mung bean in E. coli. To study the function of subunit d, two vacuolar H+-ATPase subcomplexes V_o from mung bean were purified-one containing subunits a and c(c’,c”) and the other containing subunits a, c(c’,c”) and d. After reconstitution of the purified V_o subcomplexes into liposomes, the proton translocation was studied. Our results show that the V_o subcomplex in the absence of subunit d is a passive proton channel, while the V_o subcomplex in the presence of the subunit d is not. Taken together, our data supports the conclusion that the subunit d of the plant vacuolar H⁺-ATPase from mung bean is positioned at the central stalk and involved in the proton translocation across the tonoplast membrane.     

The persistence in soil of the fungicidal seed dressings captan and thiram

The persistence in soil of captan and thiram was investigated by means of a technique in which the fungicidal content of soil was assayed by incubating plugs of soil containing fungicide on agar plates seeded with spores of Myrothecium verrucaria and measuring the diameter of the zone of fungal inhibition that was produced. When the fungicides were well distributed in soil they showed extremely low persistence, both fungicides having a half-life of between 1 and 2 days. In contrast, when the fungicides were added to soil in the form of dressings on the surface of glass beads they persisted well in soil, little change from their initial concentration occurring even after 21 days. These results suggest that captan and thiram persist far longer in soil when localized in high concentrations than when uniformly distributed through soil. If a glass bead is regarded as a reasonable simulation of a seed these results help to explain the effectiveness of these fungicides as seed dressings despite their apparently low persistence in soil.     

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule,N-cadherin

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2mM Ca⁺⁺ or 0.2 mM Ca⁺⁺, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca⁺⁺, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca⁺⁺ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca⁺⁺ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca⁺⁺ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca⁺⁺ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca⁺⁺, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca⁺⁺. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca⁺⁺ levels had similar effects as lowering the Ca⁺⁺ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.