A novel source of the cardiac glycoside digoxin from the endophytic fungus Epicoccum nigrum: isolation, characterization, production enhancement by gamma irradiation mutagenesis and anticancer activity evaluation.

AIMS: Different endophytic fungi were isolated and screened for their digoxin-producing ability. Strain improvement and different culture conditions were studied for more effective production of digoxin. METHODS AND RESULTS: Among the isolated fungi, an isolate produced digoxin in a concentration of 2·07 mg l-1 . The digoxin-producing fungal isolate was identified as Epicoccum nigrum Link according to the morphological features and phylogenetic analyses. The potentiality of the fungal strain for production enhancement of digoxin was performed by gamma radiation mutagenesis. Gamma irradiation dose of 1000 Gy intensified the digoxin yield by five-fold. Using this dose, a stable mutant strain with improved digoxin productivity was isolated and the stability for digoxin production was followed up across four successive generations. In the effort to increase digoxin magnitude, selection of the proper cultivation medium, addition of some elicitors to the most proper medium and several physical fermentation conditions were tested. Fermentation process carried out in malt extract autolysate medium (pH 6·5) supplemented by methyl jasmonate and inoculated with 2 ml of 6-day-old culture and incubated at 25°C for 10 days stimulated the highest production of digoxin to attain 50·14 mg l-1 . Moreover, cytotoxicity of digoxin separated from the fungal culture was tested against five different cancer cell lines. Based on the MTT assay, digoxin inhibited the proliferation of the five different cancer cell lines and the recorded 50% inhibitory concentration ranged from 10·76 to 35·14 µg ml-1 . CONCLUSIONS: This is the first report on the production and enhancement of digoxin using fungal fermentation as a new and alternate source with high productivity. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings offer new and alternate sources with excellent biotechnological potential for digoxin production by fungal fermentation. Moreover, digoxin proved to be a promising anticancer agent whose anticancer potential should be assessed in prospective cancer therapy.

Adaptation of subpopulations of the Norway spruce needle endophyte Lophodermium piceae to the temperature regime.

Lophodermium piceae represents the most common Norway spruce needle endophyte. The aim of this study was to find out whether subpopulations of L. piceae in climatically different environments (in which Norway spruce occurs natively) are adapted to local thermal conditions. L. piceae's ability for thermal adaptation was investigated by determining growth rates of 163 isolates in vitro at four different temperatures: 2, 6, 20 and 25 °C. Isolates were obtained between 1995 and 2010 from apparently healthy needles sampled in Finland, Poland, Switzerland, Italy and southeastern Siberia. The sampling sites represent seven climatically distinct locations. Results were evaluated in relation to the age and geographic origin of the isolate, in addition to the highest and lowest average monthly temperature of the sampling location. We found a significant correlation between the growth rate and the age of the isolate at 25 °C. Variation in growth rates between subpopulations was low compared to within subpopulations. Only at 2 °C did statistically significant differences between the average growth rates of subpopulations emerge. These results suggest that L. piceae covers the whole distribution area of Norway spruce but that generally the thermal reaction norm of its subpopulations has not changed according to local temperature ranges, despite high contrast in thermal conditions across this vast area. Therefore, it would appear that the thermal environment is not a crucial factor in assessing the fitness of this fungal species within the native range of Norway spruce.

Physiological consequences of gamma ray irradiation in tall fescue with elimination potential of Epichloë fungal endophyte.

Perennial plants and their associated microorganisms grow in the areas that may be contaminated with long-lived gamma-emitting radionuclides. This will induce gamma stress response in plants and their accompanying microorganisms. The present work investigated the growth and physiological responses of Epichloe endophyte infected tall fescue to gamma radiation, as well as whether the endophyte could persist and infect the host plant once exposed to gamma radiation. Seeds of Iranian native genotype of 75B+ of tall fescue were exposed to different doses, including 5.0, 10.0, 15.0, 20.0, 30.0 and 40.0 krad of gamma ray from a 60Co source. Irradiated and unirradiated seeds were sown in pots and grown under controlled conditions in the greenhouse. The growth and physiological parameters associated with plant tolerance to oxidative stress of host plants, as well as endophytic infection frequency (% of plants infected) and intensity (mean number of endophytic hyphae per the field of view), were examined in 3 months-old seedlings. The results indicated that all gamma radiation doses (except 5.0 kr) significantly reduced the height and survival percentage of the host plant. Days to the emergence of seedling increased gradually as gamma doses rose. A dose-rate dependent induction was seen for photosynthetic pigments and proline content. Malondialdehyde (MDA) content grew with elevation of irradiation doses. Depending on the dose and time, the activities of antioxidant enzymes in the host plant responded differently to gamma radiation. Gamma radiation altered the enzyme activities with sever decline in SOD and CAT activities. However, it had barely any effect on in APX and POD activities. The results also revealed that the persistence and intensity of endophyte were affected after gamma-ray irradiation. The initial percentage of tall fescue seeds infected with the endophyte was 91% in un-irradiated seeds. Presence of the viable endophyte started to decline significantly (23%) at 5.0 kr of gamma radiation. A dramatic reduction in the presence and intensity of endophyte occurred at 10.0 to 40.0 kr intensities. Gamma radiation × trait (GT)-biplot analysis indicated positive correlations between the endophyte symbiosis and antioxidant enzyme activities. Also, negative correlations were observed between the endophyte and MDA content in the host plant. Our results suggest that radiation stress (doses over 5.0 kr) caused reduction in the growth and antioxidant enzyme activities of the host plant that accompanied by a dramatic reduction in the persistence and intensity of endophyte fungi. Our findings have provided the basic information for future studies on the effect of gamma irradiation on the interaction between endophytic fungi and its host plant.

Production of fumonisins by endophytic strains of Tolypocladium cylindrosporum and its relation to fungal virus infection.

Fumonisins were first discovered in Fusarium verticillioides, a fungus associated to disease and asymptomatic infections in maize. Afterwards, other fungal taxa have been found to produce fumonisins. The entomopathogenic ascomycete Tolypocladium cylindrosporum has been isolated from soil and also as an endophyte from leaves of grasses. The objectives of this work were to determine the in vitro production of fumonisin B (FB) mycotoxins and the immunosuppressive compound cyclosporine A (CyA) in several strains of T. cylindrosporum, and to examine the effect of fungal virus infection and temperature in FB production. FB1 was detected in 30% of the strains, ranging from 0.16 to 5.52 µg cm-2 in solid media, and FB2 was detected in 78% of the strains, ranging from 0.764 to 40.92 µg cm-2. CyA was not detected in any strain. The mean FB2 concentration of the endophytic strain Tc37W was three times greater (p < 0.05) than that of any other strain. Up to 34% more of FB2 was detected in strains infected by the virus TcV3 than in the corresponding virus-free versions. The effect of temperature on FB2 content was interactively significantly dependent on fungal strain and growth medium; in the YES medium, the FB2 of virus-infected strains Tc37-1V and Tc37W increased by 67 and 16%, respectively, at 26 °C as compared to 20 °C. The FB concentration in some fungal strains was similar to that in fungi associated to food and feed intoxications.