Identification of Aspergillus flavus isolates as potential biocontrol agents of aflatoxin contamination in crops.

Aspergillus flavus, a haploid organism found worldwide in a variety of crops, including maize, cottonseed, almond, pistachio, and peanut, causes substantial and recurrent worldwide economic liabilities. This filamentous fungus produces aflatoxins (AFLs) B1 and B2, which are among the most carcinogenic compounds from nature, acutely hepatotoxic and immunosuppressive. Recent efforts to reduce AFL contamination in crops have focused on the use of nonaflatoxigenic A. flavus strains as biological control agents. Such agents are applied to soil to competitively exclude native AFL strains from crops and thereby reduce AFL contamination. Because the possibility of genetic recombination in A. flavus could influence the stability of biocontrol strains with the production of novel AFL phenotypes, this article assesses the diversity of vegetative compatibility reactions in isolates of A. flavus to identify heterokaryon self-incompatible (HSI) strains among nonaflatoxigenic isolates, which would be used as biological controls of AFL contamination in crops. Nitrate nonutilizing (nit) mutants were recovered from 25 A. flavus isolates, and based on vegetative complementation between nit mutants and on the microscopic examination of the number of hyphal fusions, five nonaflatoxigenic (6, 7, 9 to 11) and two nontoxigenic (8 and 12) isolates of A. flavus were phenotypically characterized as HSI. Because the number of hyphal fusions is reduced in HSI strains, impairing both heterokaryon formation and the genetic exchanges with aflatoxigenic strains, the HSI isolates characterized here, especially isolates 8 and 12, are potential agents for reducing AFL contamination in crops.

Induced mitotic homologous recombination by the babesicide imidocarb dipropionate in Aspergillus nidulans diploid cells.

Imidocarb dipropionate (IMD) is a chemotherapeutic agent prescribed for the treatment and control of babesiosis; it is known to be a nucleic acid synthesis inhibitor. Although it is an effective babesicide, there are reports of persistent IMD residues retained at high levels in edible tissues of cattle, swine and sheep, raising concerns about potential effects on humans. Since the carcinogenic potential of a chemical compound can be assessed through its effect on the homologous recombination, we investigated whether IMD is recombinogenic in Aspergillus nidulans diploid cells and whether it is capable of inducing homozygosis in genes that were previously heterozygous. This analysis was done with a homozygotization assay applied to a heterozygous diploid strain of A. nidulans. IMD used at non-toxic concentrations (2.5 to 10.0 µM) was recombinogenic, demonstrated by homozygotization indices higher than 2.0 for diploid markers. A diploid homozygous for genetic markers from chromosomes I and II was also produced. Since DNA replication blockers that induce DNA strand breaks have been classified as potent inducers of homologous recombination, the recombinogenic potential of IMD may be due to induction of recombinational repair.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean.

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Asexual recombination in a uvsH mutant of Aspergillus nidulans.

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.

Genotoxicity (mitotic recombination) of the cancer chemotherapeutic agents cisplatin and cytosine arabinoside in Aspergillus nidulans.

Cisplatin (cis-diamminedichloroplatinum, cis-DDP) and cytosine arabinoside (ara-C) are anticancer drugs used in the treatment of human cancer. The two chemotherapeutic drugs were tested in current research for their recombinogenic potential in diploid cells of Aspergillus nidulans. Non-cytotoxic concentrations of ara-C (0.4 and 0.8 microM) and cis-DDP (1.5, 3.0 and 6.0 microM) were strong recombinagens in A. nidulans UT448//A757 diploid strain, which induced homozygosis of recessive genetic markers, previously present in heterozygous condition. Drugs significantly increased homozygosity index (HI) values for five nutritional genetic markers when compared with those determined in the absence of anticancer drugs. Since mitotic recombination is a mechanism leading to malignant growth through loss of heterozygosity at tumor-suppressor loci, ara-C and cis-DDP may be characterized as secondary promoters of malignant neoplasia in diagnosed cancer patients, after chemotherapy treatment.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Asexual Recombination in a uvsH Mutant of Aspergillus nidulans

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.

Chlorhexidine digluconate induces mitotic recombination in diploid cells of Aspergillus nidulans.

UNLABELLED: Chlorhexidine digluconate (1,1'-hexamethylene-bis[(5-p-clorophenyl)-biguanide]) is a bisbiguanidine antiseptic, used to decrease plaque formation and to control periodontal diseases. The determination of the frequency of mitotic crossing-over constitutes a very important method for detecting carcinogenic agents. OBJECTIVE: The recombinogenic potential of chlorhexidine digluconate was evaluated on Aspergillus nidulans by the production of cells homozygous for the following nutritional markers: riboA1, pabaA124, biA1, methA17 and pyroA4. METHOD: A. nidulans was exposed to three concentrations of chlorhexidine digluconate (1, 5, and 10 microM). RESULTS: Inhibition of colony development, conidiophore morphological alteration (cytotoxic effect), and the recombinogenic effect, indicated by homozygotization index (HI) values higher than 2.0, were observed for all concentrations of chlorhexidine digluconate. A homozygous pyro+//pyro+ diploid strain and a diploid homozygous for the recessive w gene were isolated from UT448//A757 diploid treated with chlorhexidine digluconate, emphasazing its recombinogenic potential. CONCLUSION: Although, beneficial effects of chlorhexidine, as an antiseptic agent, are reported in the literature, our results revealed that chlorhexidine digluconate, at less levels lowered those used clinically, caused toxic and recombinogenic effects on diploid A. nidulans strain.

Loss of heterozygosity by mitotic recombination in diploid strain of Aspergillus nidulans in response to castor oil plant detergent

A recombinação somática em células diplóides heterozigotas pode atuar como agente promotor de neoplasias por induzir homozigose de genes deletéreos. Por meio desse processo, genes supressores de tumores podem ser completamente suprimidos em células recombinantes. O presente trabalho avaliou a genotoxicidade do detergente derivado do óleo da semente da mamona (Ricinus communis) em células diplóides heterozigotas do fungo filamentoso Aspergillus nidulans. Trabalhos anteriores avaliaram a aplicação dessa solução no tratamento de canais radiculares como líquido irrigador. O potencial recombinagênico desse composto foi estudado pela origem de células homozigotas para os marcadores nutricionais: riboA1, pabaA124, biA1, metA17 e piroA4. A solução, diluída em 1:40, 1:20 e 1:10, induziu alterações morfológicas e atraso no desenvolvimento dos conidióforos da linhagem UT448//UT196 e aumento nas freqüências de recombinação mitótica. Embora trabalhos anteriores relatem a atividade antimicrobiana da solução em estudo, nossos resultados evidenciam a citotoxicidade e o potencial recombinagênico dessa substância.

Loss of heterozygosity by mitotic recombination in diploid strain of Aspergillus nidulans in response to castor oil plant detergent.

Somatic recombination in heterozygous diploid cells may be a promotional agent of neoplasms by inducing homozygosity of defective genes. Tumor suppressor genes may in this way be completely suppressed in recombinant cells. In this work, the genotoxic effects of detergent derived from the castor oil plant (Ricinus communis) in heterozygous diploid cells of Aspergillus nidulans are evaluated. Previous studies have evaluated the application of this substance in endodontic treatments as an irrigating solution. The recombinogenic potential of the compound has been studied through the production of homozygous cells for nutritional markers riboA1, pabaA124, biA1, methA17, and pyroA4. Detergent was diluted to 1:10, 1:20, and 1:40, and morphologic alterations, delay in conidiophore development, and mitotic recombination occurrence were reported for the three dilutions. Although past studies have demonstrated the antimicrobial action of the detergent under analysis, our results revealed its cytotoxic effects and recombinogenic potential.

Recombinagenic effect of cryptolepine in uvsH+//uvsH+ and uvsH//uvsH diploid strains of Aspergillus nidulans.

Recombinagenic potential of the alkaloid cryptolepine was evaluated in two diploid strains of Aspergillus nidulans--a wild type strain (uvsH+//uvsH+) and a DNA-repair-deficient one (uvsH//uvsH). Treatment of both strains with cryptolepine failed to alter colony growth in culture; its recombinagenic potential was determined by the homozygotization index (in which events of mitotic exchange may cause expression of genes previously masked by the dominant allele). Mitotic crossing-overs were induced by 7 and 14 mg/L doses of cryptolepine in both diploid A. nidulans strains.

Avaliaçäo do potencial recombinogênico do antibiótico danofloxacina em células diplóides de Apergillus nidulans / Evaluation of the recombinogenic activity of danofloxacin in diploid cells of Aspergillus nidulans

Estudou-se o potencial recombinogênico da danofloxacina, novo antimicrobiano pertencente ao grupo das 4-fluoroquinolonas e de uso exclusivo em medicina veterinária, no fungo filamentoso Aspergillus nidulans. A linhagem mestra UTl96 e o mutante Z1 foram utilizados para formar o diplóide Z1/UT196. Conídios desse diplóide foram inoculados em placas de Petri contendo meio mínimo suplementado com 2,5, 5,0 e 10,0 µg/ml de danofloxacina. As placas foram incubadas por cinco dias a 37§C. Segregantes mitóticos foram isolados das colônias tratadas com o antimicrobiano e as análises de seus fenótipos evidenciaram o efeito recombinogênico da danofloxacin na dose de 10,0 µg/ml. Recombinantes para vários intervalos dos cromossomos I e II foram identificados entre os segregantes analisados

Avaliação do potencial recombinogênico do antibiótico danofloxacina em células diplóides de Apergillus nidulans / Evaluation of the recombinogenic activity of danofloxacin in diploid cells of Aspergillus nidulans

Estudou-se o potencial recombinogênico da danofloxacina, novo antimicrobiano pertencente ao grupo das 4-fluoroquinolonas e de uso exclusivo em medicina veterinária, no fungo filamentoso Aspergillus nidulans. A linhagem mestra UTl96 e o mutante Z1 foram utilizados para formar o diplóide Z1/UT196. Conídios desse diplóide foram inoculados em placas de Petri contendo meio mínimo suplementado com 2,5, 5,0 e 10,0 µg/ml de danofloxacina. As placas foram incubadas por cinco dias a 37§C. Segregantes mitóticos foram isolados das colônias tratadas com o antimicrobiano e as análises de seus fenótipos evidenciaram o efeito recombinogênico da danofloxacin na dose de 10,0 µg/ml. Recombinantes para vários intervalos dos cromossomos I e II foram identificados entre os segregantes analisados (AU)

The aim of this work was to demonstrate the recombinogenic effect of danofloxacin, a new 4-fluoroquinolone antimicrobial used only in veterinary medicine, in the filamentous fungi Aspergillus nidulans. The UT196 master strain and the Z1 mutant were used to produce the Z1//UT196 diploid strain. Conidia of this diploid strain were inoculated in Petri dishes containing selective medium supplemented with 2.5, 5.0 or 10.0 mg/ml danofloxacin. The plates were incubated for five days at 37 ºC. Mitotic segregants were isolated from the diploid colonies and phenotypes of the segregants were analyzed. Inoculation of 10.0 mg/ml danofloxacin was shown to be positive in inducing somatic crossing-over in diploid cells of A. nidulans. An increase in the mitotic recombination frequencies was observed in several linkage-intervals of chromosomes I and II from Z1//UT196 diploid strain. (AU)