Presence Of Benomyl Research Articles

Limitation of nitrogen source facilitated the production of nonmeiotic recombinants in Aspergillus nidulans.

Aspergillus nidulans is a fungal model organism extensively used in genetic approaches. It may reproduce sexually and asexually, with a well-defined parasexual cycle. The current paper demonstrates that the limitation of nitrogen source facilitates the production of A. nidulans's nonmeiotic recombinants directly from heterokaryons, without the recovery of the diploid phase. Heterokaryons formed between master strains were inoculated in sodium nitrate-low (basal medium [BM]) and sodium nitrate-rich media (minimal medium [MM]). All mitotic segregants produced by the heterokaryons were tested for their mitotic stability in the presence of benomyl, the haploidizing agent. Only mitotically stable haploid segregants were selected for subsequent analysis. Phenotypic analyses of such haploids favored the characterization of nonmeiotic recombinants. Asthe number of such recombinants was higher in BM than in MM, nitrogen limitation may have facilitated the isolation of nonmeiotic recombinants from heterokaryons by stimulating nuclear fusion still inside the heterokaryotic mycelium as a survival strategy.

Heterokaryosis and diploid formation among Brazilian isolates of Macrophomina phaseolina

Heterokaryosis is the association of genetically distinct nuclei in a common hyphal cytoplasm, and is a process involved in the generation of fungal variation. The fusion of the distinct nuclei within a heterokaryotic hypha produces a heterozygous diploid nucleus and is one of the stages of the parasexual cycle. A heterokaryon's viability depends on the isolates’ genetic constitution with regard to loci called het (heterokaryon incompatibility) or vic (vegetative incompatibility). The current study evaluates for the first time the diversity of vegetative compatibility reactions in isolates of Macrophomina phaseolina from different hosts. Complementary nit (nitrate non‐utilizing) mutants of each isolate were obtained and paired in all possible combinations. Isolates were classified in vegetative compatibility groups (VCG) according to their ability to form viable heterokaryons. Ten VCGs were identified, two of them containing two isolates, and the remainder containing a single isolate. When grown on basal medium, heterokaryons produced both (i) auxotrophic segregants exhibiting the same phenotype as the paired mutants; and (ii) a fast‐growing sector, characterized as a heterozygous diploid sector, named D653, which showed a nit+ phenotype with a growth rate similar to the original wild isolate. When grown in the presence of benomyl, a haploidizing agent, D653 produced auxotrophic haploid segregants exhibiting the nit phenotypes of the crossed mutants. The results demonstrate for the first time the ability of M. phaseolina isolates to form viable heterokaryons and heterozygous diploid nuclei, suggesting that the parasexual cycle may be an alternative source of genetic variability in this species.

SAGA/ADA complex subunit Ada2 is required for Cap1- but not Mrr1-mediated upregulation of the Candida albicans multidrug efflux pump MDR1.

Overexpression of the multidrug efflux pump MDR1 is one mechanism by which the pathogenic yeast Candida albicans develops resistance to the antifungal drug fluconazole. The constitutive upregulation of MDR1 in fluconazole-resistant, clinical C. albicans isolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activates MDR1 transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However, MDR1 expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response in C. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotes MDR1 overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 caused MDR1 overexpression in a wild-type strain but only weakly in mutants lacking ADA2. In the presence of benomyl or H2O2, compounds that induce MDR1 expression in an Mrr1- and Cap1-dependent fashion, MDR1 was upregulated with the same efficiency in wild-type and ada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to the MDR1 promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required for MDR1 overexpression in fluconazole-resistant C. albicans strains containing gain-of-function mutations in Mrr1.

Kinetic stabilization of microtubule dynamic instability by benomyl increases the nuclear transport of p53

Using time-lapse confocal microscopy and enhanced green fluorescent protein–tubulin transfected MCF-7 cells, we found that a tubulin-targeted antimitotic agent, benomyl at its half-maximal proliferation inhibitory concentration (5 μM) strongly suppressed the rate and extent of growing and shortening excursions of individual microtubules in MCF-7 cells without noticeably depolymerizing the microtubule network or decreasing the polymerized mass of tubulin. Further, benomyl treatment caused an increase in the acetylation level of microtubules suggesting that it stabilizes microtubules. Under the conditions that suppressed the dynamic instability, a sharp increase in the nuclear accumulation of p53 in MCF-7 cells was observed in the presence of benomyl. Up regulation of bax and the increased nuclear accumulation of p21 upon benomyl treatment confirmed the activation of p53. Cisplatin caused an increase in the translocation of p53 into the nucleus in the presence of lower effective concentrations of benomyl while a decrease in the nuclear accumulation of p53 was observed in the presence of high concentrations of benomyl suggesting that the stabilized microtubules assist in the nuclear transportation of p53. Furthermore, increased localization of the light chain of the minus end directed motor protein dynein was detected on the microtubules in the benomyl-treated cells indicating that the suppression of microtubule dynamics may influence the binding of dynein on the microtubules and dynein-mediated cargo transport. Together the data indicate that benomyl inhibits mitosis primarily by suppressing the dynamic instability of microtubules and support the hypothesis that the kinetic stabilization of microtubules enhances the microtubule-mediated transport of p53 into the nucleus.

Combined application of Pseudomonas fluorescens strain LRB3W1 with a low dosage of benomyl for control of cabbage yellows caused by Fusarium oxysporum f. sp. conglutinans

An antagonistic bacterium, Pseudomonas fluorescens strain LRB3W1, inhibited the fungal growth of Fusarium oxysporum f. sp. conglutinans in vitro and suppressed cabbage yellows caused by F. oxysporum f. sp. conglutinans. Under glasshouse conditions, the bacterium survived at ca. 106–107 CFU g−1 in the cabbage rhizosphere for 4 weeks after the initial application. The chemical fungicide, benomyl, did not suppress the disease severity at low concentration (1 or 10 µg mL−1). However, the disease severity was decreased by the combined application of a low dosage of benomyl with strain LRB3W1. Combined application of a low dosage of benomyl with strain LRB3W1 was more effective than treatment with the bacterium alone. The survival of strain LRB3W1 was not influenced by the presence of benomyl. This combined use of the biocontrol agent, strain LRB3W1, and a fungicide, benomyl, should be an attractive approach for suppressing cabbage yellows in sustainable agriculture because of the reduced chemical dosages needed for disease management.

Action of benzimidazole fungicides on resorcinolic lipid metabolism in rye seedlings depends on thermal and light growth conditions

The content and homologue composition of alkylresorcinols were investigated in rye seedlings ( Secale cereale L.) treated with benzimidazole fungicides and grown for 5 days under various thermal and light conditions. The fresh and dry biomasses of green and etiolated plants were greatly increased by benomyl and carbendazim at 29 °C. At 22 and 15 °C benomyl had an inhibitory action on growth of rye, whereas the effects caused in the presence of carbendazim were slightly dependent on external stimuli. On the other hand, benomyl enhanced resorcinol biosynthesis in green seedlings grown at all temperatures, while carbendazim only at 29 °C. In plants kept in the darkness, both fungicides increased content of alkylresorcinols at 29 °C and decreased its at 22 and 15 °C. The qualitative pattern of resorcinolic homologues was also significantly modified in the presence of benomyl and carbendazim and depended on other physical stimuli.

Parasexuality in asexual development mutants of Aspergillus nidulans

The parasexual cycle with parameiosis has been characterized previously by the occurrence of genetic recombination and haploidization inside heterokaryotic hyphae prior to conidial formation. The aim of current research was to characterize, through genetic and cytological analyses, an asexual development mutant strain of A. nidulans and to use it to obtain parameiotic segregants. Analyses showed the medusa phenotype of the B84 strain, whose mutant allele was mapped in the chromosome I. The heterokaryons B84(med)//G422(med+) and B84(med)//G839(brl) were formed in liquid MM+2% CM and inoculated in the appropriate selective media. Two mitotic segregant groups were obtained: aneuploids and haploid stable recombinants. Mitotic segregants, wild-types, and developmental mutants, which did not produce new visible mitotic sectors in the presence of Benomyl and which showed normal meiotic behavior during the sexual cycle, were classified as parameiotics.

Combined Use of the Biocontrol Bacterium Pseudomonas fluorescens Strain LRB3W1 with Reduced Fungicide Application for the Control of Tomato Fusarium Wilt

Pseudomonas fluorescens strain LRB3W1 inhibited the mycelial growth of Fusarium oxysporum f. sp. lycopersici and suppressed the Fusarium wilt of tomato. The chemical fungicide, benomyl, did not suppress the disease incidence at low concentrations. However, the disease incidence was decreased by the combined application of benomyl at low concentrations with strain LRB3W1. Combined application of benomyl with the bacterium was more effective than treatment with the bacterium alone. The survival of strain LRB3W1 was not influenced by the presence of benomyl. This combined use of the biocontrol bacterium, strain LRB3W1, and a fungicide, benomyl, should be an attractive approach for suppressing tomato wilt.

First Report of the Prevalence of Benzimidazole-Resistant Isolates in a Population of Cylindrocladium pauciramosum in Italy.

Cylindrocladium pauciramosum C.L. Schoch & Crous (teleomorph Calonectria pauciramosa C.L. Schoch & Crous), described as a member of the Cylindrocladium candelabrum Viégas complex (4), was recently reported from Europe (3). In southern Italy, the fungus has caused extensive losses, and chemical control measures are necessary, especially in ornamental nurseries. Several researchers have found benzimidazole fungicides to be effective for control of different species of Cylindrocladium, however, in fungicide trials conducted on myrtle plants infected by C. pauciramosum, benomyl was ineffective (2). Another study showed that mycelial growth of six isolates was completely inhibited by carbendazim at a concentration of 1 μg a.i./ml whereas, concentrations of 10, 100, and 500 μg a.i./ml did not completely inhibit growth of four isolates (1). To examine benzimidazole resistance in C. pauciramosum, 200 single-conidia isolates were tested. These were collected during 1996 and 1997 from several symptomatic hosts in different nurseries located in Sicily and Calabria and identified through morphological characteristics as well as mating-type studies with tester strains. Sensitivity to benomyl was determined by plating mycelial plugs on potato dextrose agar (PDA) amended with benomyl at 1, 10, 100, and 500 μg a.i./ml. For 20 benomyl-resistant isolates, fungal growth was also determined at the same concentrations on carbendazim-amended PDA. Sensitivity was expressed as the minimum inhibitory concentration (MIC) (the lowest fungicide concentration that completely prevented fungal growth). Isolates that did not grow on benzimidazole-amended PDA were classed as sensitive. Isolates were considered resistant to benzimidazole if MIC values were greater than 1 μg a.i./ml. Of the 200 isolates tested, 58% were resistant to benomyl. The benomyl-resistant isolates tested for carbendazim sensitivity were cross-resistant to carbendazim. Most resistant isolates grew in the presence of benomyl at 500 μg a.i./ml. On agar culture, the isolates were either the fast-growing or slow-growing type. The slow-growing phenotype appears to be related to the higher level of resistance (500 μg a.i./ml). On the basis of these data, the use of benzimidazole for the control of this pathogen should be seriously questioned. To our knowledge, this is the first report of the prevalence of benzimidazole resistance in a population of C. pauciramosum.

Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii.

We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 microm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network.

Transformation of the entomopathogenic fungusMetarhizium flavovirideto high resistance to benomyl

A Brazilian isolate of Metarhizium flavoviride, which has been developed as a biocontrol agent against the grasshopper Rhammatocerus schistocercoides, was stably transformed to be resistant to benomyl (beta-tubulin gene). Highly resistant transformants were obtained which grew in benomyl concentrations greater than 30 times (200 µg mL-1) the concentration that inhibits wild type proliferation. These transformants were mitotically stable after 20 successive transfers on non-selective media. No significant differences in conidia yield were observed between stably transformed strains and wild type (CG423). Chymoelastase (Pr1) secretion was greater in some transformants than in the wild type. In the presence of benomyl, appressoria differentiation occurred at similar rates in CG423 and transformants. However, the percentage of conidial germination in the transformants was higher than in the wild type, indicating the potential use of these transformants along with benomyl. Additionally, the resistance levels of the transformants observed in the present study demonstrate the potential use of these transformants for assessing the persistence of a particular isolate in fields without this fungicide.Key words: Metarhizium flavoviride, genetic transformation, benomyl resistance, biocontrol.

Pollen germination, tube growth and morphology, and microtubule organization after exposure to benomyl

The inhibitory effects of benomyl on pollen tube growth have received little attention, particularly at the microscopic and immunohistochemical levels. Pollen germination and tube growth in the presence of benomyl were evaluated in Tradescantia virginiana to investigate the effects of this fungicide on pollen germination rate, tube growth and morphology, and microtubule (Mt) organization. Benomyl was incorporated in germination media at 0,480, 600 or 720 mg 1−1. Inhibition of pollen germination, cytoplasmic streaming, and tube elongation were associated with benomyl treatments. Benomyl also induced abnormal pollen tube morphology and Mt organization. Compared to controls, Mts in the treated tubes were characteristically fewer in number, fragmented, sinuous and increasingly disorganized. At the two highest benomyl concentrations, Mts were considerably fewer or absent in apical/subapical regions of the pollen tubes. This work verifies that benomyl incorporated into germination media at concentrations lower than recommended field rates inhibit pollen germination and tube growth, and that the effects are associated with alterations of Mt organization.

Effects of agrochemicals and heavy metals on fast-growing rhizobia and their symbiosis with small-seeded legumes

The effect of potentially hazardous agrochemicals including fungicides, herbicides and heavy metals on symbiotic nitrogen fixation have been investigated. The substances were tested with eight rhizobial strains from three cross-inoculation groups: Rhizobium leguminosarum b.v. trifolii, R. meliloti and R. loti in pure culture studies. Bacteria were obtained from a culture collection or from soils. Sensitivity of the bacteria to the agrochemicals and heavy metals varied. None of the bacteria were tolerant to all chemicals. No difference in tolerance between cross-inoculation groups existed. Bacteria were able to multiply at concentrations of agrochemicals equal to or higher than recommended field-application rates. Heavy metals concentrations that severely inhibited growth were far lower than the highest amounts allowed under the current Commission of the European Communities' guidelines for environmental protection. Bacterial growth in presence of the agrochemicals and heavy metals, apart from glyphosate and zinc, did not influence nodulation ability of the strains. Development of uninoculated plants was inhibited at increasing concentrations of all compounds, red clover being most sensitive. Herbicides were most harmful, with injuries occurring at levels 1 10 - 1 10,000 of recommended applied concentrations. Uninoculated plants were less tolerant to agrochemicals, but were more tolerant to heavy metals compared to the bacteria. Root hair deformations similar to bacterial-induced root hair deformations were induced by bentazone, chlorsulphuron and monochlorophenoxyacetic acid on uninoculated plants. Symbiotic interactions were adversely affected by several of the agrochemicals. Bacterial-induced root hair deformations necessary for nodulation decreased in the presence of benomyl, bentazone, chlorsulphuron, fenpropimorph, mancozeb and monochlorophenoxyacetic acid. Fenpropimorph and mancozeb did not cause root hair deformations at increasing concentrations, indicating that these may inhibit nodulation under field conditions. Nodule development was inhibited at increased levels of bentazone, chlorsulphuron, glyphosate and mancozeb. Dry matter production of nodulated plants was adversely affected by bentazone and chlorsulphuron, indicating disturbances in nodule function.

Feedback control of mitosis in budding yeast.

We have investigated the feedback control that prevents cells with incompletely assembled spindles from leaving mitosis. We isolated budding yeast mutants sensitive to the anti-microtubule drug benomyl. Mitotic arrest-deficient (mad) mutants are the subclass of benomyl-sensitive mutants in which the completion of mitosis is not delayed in the presence of benomyl and that die as a consequence of their premature exit from mitosis. A number of properties of the mad mutants indicate that they are defective in the feedback control over the exit from mitosis: their killing by benomyl requires passage through mitosis; their benomyl sensitivity can be suppressed by an independent method for delaying the exit from mitosis; they have normal microtubules; and they have increased frequencies of chromosome loss. We cloned MAD2, which encodes a putative calcium-binding protein whose disruption is lethal. We discuss the role of feedback controls in coordinating events in the cell cycle.

A suppressor of a centromere DNA mutation encodes a putative protein kinase (MCK1).

A new approach to identify genes involved in Saccharomyces cerevisiae kinetochore function is discussed. A genetic screen was designed to recover extragenic dosage suppressors of a CEN DNA mutation. This method identified two suppressors, designated MCK1 and CMS2. Increased dosage of MCK1 specifically suppressed two similar CEN DNA mutations in CDEIII, but not comparably defective CEN DNA mutations in CDEI or CDEII. A strain containing a null allele of MCK1 was viable under standard growth conditions, had a cold-sensitive phenotype (conditional lethality at 11 degrees C), and grew slowly on Benomyl (a microtubule-destabilizing drug). Furthermore, when grown at 18 degrees C or in the presence of Benomyl, the null mutant exhibited a dramatic increase in the rate of mitotic chromosome loss. The allele-specific suppression and chromosome instability phenotypes suggest that MCK1 plays a role in mitotic chromosome segregation specific to CDEIII function. The MCK1 gene encodes a putative protein-serine/threonine kinase, which suggests a possible role for the MCK1 protein in regulating the activity of centromere-binding proteins by phosphorylation. MCK1 was identified and cloned independently for its involvement in the induction of meiosis and is identical to a gene that encodes a phosphotyrosyl protein with protein kinase activity.

Breeding by protoplast fusion for glucoamylase production

Protoplast fusion was carried out between two strains ofAspergillusniger 8-2, a fast growing culture and poor producer of glucoamylase enzyme andA.niger 8-7, a slow growing culture and good producer of the enzyme. The nonconidiating fused mass in presence of benomyl, produced fast growing segregants showing various combinations of the two parental gene markers. Some of the segregants produced up to 68% more glucoamylase than the better yielding parent 8-7.

Involvement of a particular species of beta-tubulin (beta 3) in conidial development in Aspergillus nidulans.

Strains of Aspergillus containing the benA22 mutation are resistant to benomyl for vegetative growth but do not produce conidia. To test whether conidiation involved an additional benomyl-sensitive tubulin (i.e., was mediated by a tubulin other than the tubulins coded for by the benA locus), a collection of mutants was produced that formed conidia in the presence of benomyl, i.e., were conidiation-resistant (CR-) mutants. We analyzed the tubulins of these CR- mutants using two-dimensional gel electrophoresis and found that the mutants lacked one species of beta-tubulin (designated beta 3). We have examined two of these mutants in detail. In crosses with strains containing wild-type tubulins, we found that the absence of the beta 3-tubulin co-segregated perfectly with the CR- phenotype. In diploids containing both the benA22 and CR- mutations, we found that the CR- phenotype was recessive and that beta 3-tubulin was present on two-dimensional gels of tubulins prepared from these diploids. In another set of crosses, these two CR- strains and seven others were first made auxotrophic for uridine and then crossed against strains that had homologously integrated a plasmid containing an incomplete internal fragment of the beta 3-tubulin gene and the pyr4 gene of Neurospora crassa (which confers uridine prototrophy on transformants). If the CR- phenotype were produced by a mutation in a gene distinct from the structural gene for beta 3-tubulin (designated the tubC gene), then crossing over should have produced some CR+ segregants among the uridine auxotrophic progeny of the second cross. All of the uridine auxotrophs from this type of cross, however, showed the CR- phenotype, suggesting that the mutation in these strains is at or closely linked to the tubC locus. The most obvious explanation of these results is that beta 3-tubulin is ordinarily used during conidiation and the presence of this species of beta-tubulin renders conidiation sensitive to benomyl. In the CR- mutants, beta 3-tubulin is absent, and in the presence of the benA22 mutation the benomyl-resistant beta 1-and/or beta 2-tubulin substitutes for beta 3 to make conidiation benomyl resistant. We discuss these results and give two models to explain the interactions between these beta-tubulin species.

Induced somatic segregation in Armillaria mellea diploids

Armillaria mellea is a bifactorially heterothallic fungus with a fertile, diploid, vegetative phase. While diploids of this fungus are readily recovered by nutritional selection from compatible and incompatible pairings of haploids, genetic analyses have been limited both because this organism does not produce fruiting bodies reliably in the laboratory and because somatic segregation occurs infrequently in diploid cultures. In this study, diploids of A. mellea heterozygous at nutritional and mating-type marker loci were treated with formaldehyde, para-fluorophenylalanine, benomyl, and ultraviolet light in order to recover somatic segregants. Of these agents, only benomyl dramatically increased the frequency of somatic segregation under the conditions used. Auxotrophic segregants were recovered from macerates of prototrophic, diploid mycelia that had been grown in the presence of benomyl. Many of these segregants differed from their diploid progenitors in mating behavior as well as in nutritional phenotype. The development of a reliable method for the recovery of somatic segregants from diploids of A. mellea permits parasexual analysis and reduces the need for in vitro production of fruiting bodies in future genetic studies.

Resistance ofCoccomyces hiemalisto Benzimidazole Fungicides

Resistance of<i>Coccomyces hiemalis</i>to Benzimidazole Fungicides

Nuclear movement is β-tubulin-dependent in Aspergillus nidulans

We have examined nuclear transport in Aspergillus nidulans to determine whether microtubles function in the movement of this organelle. Nuclear movement was found to be inhibited in germinating conidia (uninucleate asexual spores) by the microtubule inhibitor benomyl. To show that the benomyl inhibition was due to its effect on microtubules, the test was repeated with mutants which have genetic lesions in β-tubulin which produce resistance to benomyl in one case ( benA15) and super-sensitivity in another ( benA16). Nuclear movement was resistant to benomyl in the strain carrying benA15 and super-sensitive in the strain carrying benAl6. Since altered sensitivity to benomyl in these strains is specifically due to alterations in β-tubulin, these results show that β-tubulin is involved in nuclear movement. To eliminate the possibility that nuclear movement blockage was a secondary consequence of nuclear division blockage, this experiment was repeated with temperature-sensitive nuclear division mutants. At restrictive temperature, nuclear division was blocked in these mutants but nuclear movement was not. In the presence of benomyl, nuclear division and migration were blocked at permissive and restrictive temperatures. Thus nuclear division blockage alone is not sufficient to block nuclear movement. These experiments were corroborated by similar experiments on a temperature-sensitive nuclear movement mutant. Five previously isolated nonallelic temperature-sensitive nuclear movement mutants, nudA-E, were analyzed genetically and found not to be allelic to the benA (β-tubulin) tubA α-tubulin genes.