Genetic diversity among Botryosphaeria isolates and their correlation with cell wall-lytic enzyme production

Nine isolates of Botryosphaeria spp. were evaluated for their growth and the production of cell wall-lytic enzymes (laccase, pectinase and beta-1,3-glucanase) when grown on basal medium in the absence and presence of the laccase inducer, veratryl alcohol (VA). The genetic relationship among the nine isolates collected from different host plants was determined by RAPD analyses. ITS sequence analysis showed eight closely related isolates classified as Botryosphaeria rhodina, and one isolate classified as Botryosphaeria ribis. RAPD analysis resolved the isolates into three main clusters based upon levels of laccase and beta-1,3-glucanase activity. There appears to be no correlation between pectinase production and genetic diversity among the nine isolates. However, the strain characterized as B. ribis, positioned out of the main cluster, was found to be the highest producer of pectinases in the presence of VA.

Nove isolados de Botryosphaeria spp foram avaliados quanto ao crescimento e produção de enzimas líticas da parede celular (lacase, pectinase e beta-1,3-glucanase) quando cultivados em meio basal na ausência e presença do indutor de lacase álcool veratrílico (VA). As relações genéticas entre os nove isolados coletados de diferentes plantas hospedeiras foram determinadas por RAPD. A análise das seqüências de nucleotídeos da região ITS mostrou oito isolados estreitamente relacionados, os quais foram classificados como Botryosphaeria rhodina e um isolado como Botryosphaeria ribis. A análise por RAPD agrupou os isolados em três grupos principais condizentes com os níveis de atividades de lacase e beta-1,3-glucanase. Nenhuma correlação foi detectada entre a produção de pectinase e a diversidade genética nos nove isolados. Entretanto, a linhagem caracterizada como B. ribis, posicionada fora dos grupos principais, se mostrou maior produtora de pectinase na presença de álcool veratrílico.

Ethylene inhibits aflatoxin biosynthesis in Aspergillus parasiticus grown on peanuts.

The filamentous fungi Aspergillus parasiticus and Aspergillus flavus synthesize aflatoxins when they grow on a variety of susceptible food and feed crops. These mycotoxins are among the most carcinogenic naturally occurring compounds known and they pose significant health risks to humans and animals. We previously demonstrated that ethylene and CO2 act alone and together to reduce aflatoxin synthesis by A. parasiticus grown on laboratory media. To demonstrate the potential efficacy of treatment of stored seeds and grains with these gases, we tested ethylene and CO2 for ability to inhibit aflatoxin accumulation on Georgia Green peanuts stored for up to 5 days. We demonstrated an inverse relationship between A. parasiticus spore inoculum size and the level of toxin accumulation. We showed that ethylene inhibits aflatoxin synthesis in a dose-dependent manner on peanuts; CO2 also inhibits aflatoxin synthesis over a narrow dose range. Treatments had no discernable effect on mold growth. These observations support further exploration of this technology to reduce aflatoxin contamination of susceptible crops in the field and during storage.

Ethylene modulates development and toxin biosynthesis in aspergillus possibly via an ethylene sensor-mediated signaling pathway.

Ethylene, a biologically active natural compound, inhibited aflatoxin accumulation by Aspergillus parasiticus on a solid growth medium in a dose-dependent manner at concentrations of 0.1 to 150 ppm. The activity of the nor-1 promoter (an early aflatoxin gene) was reduced to nondetectable levels by similar quantities of ethylene, suggesting that the inhibitory effect on toxin synthesis occurred, at least in part, at the level of transcription. The inhibitory effect of ethylene on aflatoxin accumulation was also observed when A. parasiticus was grown on raw peanuts. Under similar growth conditions and doses, ethylene strongly inhibited development of asci and ascospores in Aspergillus nidulans, with no detectable effect on Hülle cell formation, conidiation, or sterigmatocystin accumulation. During early growth, A. parasiticus and A. nidulans produced ethylene with approximately twofold higher quantities measured in continuous light than in the dark. 1-Methylcyclopropene (an inhibitor of ethylene receptors in plants), light, CO2, temperature, and growth medium composition altered the effect of ethylene on A. nidulans and A. parasiticus. These observations are consistent with the existence of an ethylene sensor molecule that mediates the function of an ethylene-responsive signaling pathway(s) in Aspergillus.

Evidence that MRas1 and MRas3 proteins are associated with distinct cellular functions during growth and morphogenesis in the fungus Mucor racemosus.

The filamentous fungus Mucor racemosus provides a simple and unique model system for defining the function of individual ras genes in a gene family which is closely related to mammalian ras genes. The current study was designed to investigate the role of Mras1 and Mras3 in different stages of fungal morphogenesis, including sporangiospore germination, sporulation, and dimorphic transitions. The overall patterns of Mras1 and Mras3 transcript and protein accumulation were markedly different but, in general, transcripts and proteins were present at low levels during spherical growth and their accumulated level increased severalfold during polar growth (germ tube emergence and elongation). In contrast to Mras1, relatively high levels of Mras3 transcript accumulated during sporulation and MRas3 protein accumulated in sporangiospores. Transformation of M. racemosus with an activated allele of Mras3 reduced growth rate during aerobic sporangiospore germination, while a dominant-negative allele of Mras3 caused a 40% decrease in viable asexual spores. An activated allele of Mras1 increased growth rate during sporangiospore germination but neither activated nor dominant-negative alleles of Mras1 affected total number of asexual spores. Expression of MRas3 and MRas1 proteins appear to be subject to different regulatory mechanisms: exogenous dibutyryl-cAMP and fusidienol caused a strong repression of the level of MRas3 protein (but not MRas1) concurrent with the inhibition of polar growth. Differential posttranslational modification and intracellular localization of MRas1 and MRas3 proteins were also observed. The data strongly suggest that Mras3 and Mras1 play different roles in regulation of cell growth and morphogenesis in Mucor.

Lovastatin triggers an apoptosis-like cell death process in the fungus Mucor racemosus.

The filamentous dimorphic fungus Mucor racemosus possesses three ras genes, Mras1, 2, and 3, whose expression is correlated to morphogenesis of the fungus. Lovastatin, an indirect inhibitor of protein prenylation, altered the processing of MRas1 protein, blocked the accumulation of MRas3 protein, and caused the MRas1/p20 protein complex to disappear in M. racemosus. Concurrently it arrested sporangiospore germination, decreased growth rate, caused a loss of cell viability accompanied by cell shrinkage, increased cell density and cytoplasm condensation, and triggered DNA fragmentation, resulting in nucleosomes and nucleosome multimers. The specific morphological and biochemical events seen in Mucor cell death, particularly DNA fragmentation, resemble the best known characteristics of classical apoptosis in mammalian cells and prompted us to classify lovastatin-induced cell death as an apoptosis-like process. Lovastatin did not cause cell death in a leucine auxotroph of Mucor grown in YNB minimal medium, conditions which support only spherical growth during spore germination. Exogenous dibutyryl-cAMP initiated morphogenesis from hyphal (polar) growth to yeast-like (spherical) growth during spore germination and strongly prevented cell death which resulted from lovastatin treatment. Wortmannin added together with dibutyryl-cAMP showed a synergistic effect in the prevention of fungal cell death. These data suggest that the regulation of lovastatin-induced cell death in Mucor requires a signal transduction pathway(s) involving cAMP whose function is specific to a particular developmental stage.

c-erbA alpha/T3R and RARs control commitment of hematopoietic self-renewing progenitor cells to apoptosis or differentiation and are antagonized by the v-erbA oncogene.

In AEV-transformed erythroleukemic cells the v-erbA gene product is likely to antagonize the function of triiodothyronine (T3) and retinoic acid (RA) receptors and thereby to block cell differentiation. We have thus investigated the effects of T3 and RA on normal early erythrocytic progenitor cells. Here we show: (1) that either RA or T3 play an essential role during the early commitment to erythrocytic differentiation, (2) that both T3 and RA induce death by apoptosis and a strong inhibition of self-renewal in progenitor cells grown in the absence of differentiation-inducing agents and (3) that the v-erbA oncogene renders erythrocytic progenitor cells insensitive to apoptosis and to self-renewal inhibition induced by RA or T3. The behaviour of a non-transforming mutant of v-erbA suggests that this v-erbA-induced protection is related to its transforming potential.