Ravely Casarotti Orlandelli

Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

Screening of endophytic sources of exopolysaccharides: Preliminary characterization of crude exopolysaccharide produced by submerged culture of Diaporthe sp. JF766998 under different cultivation time

Endophytic fungi have been described as producers of important bioactive compounds; however, they remain under-exploited as exopolysaccharides (EPS) sources. Therefore, this work reports on EPS production by submerged cultures of eight endophytes isolated from Piper hispidum Sw., belonging to genera Diaporthe, Marasmius, Phlebia, Phoma, Phyllosticta and Schizophyllum. After fermentation for 96 h, four endophytes secreted EPS: Diaporthe sp. JF767000, Diaporthe sp. JF766998, Diaporthe sp. JF767007 and Phoma herbarumJF766995. The EPS from Diaporthe sp. JF766998 differed statistically from the others, with a higher percentage of carbohydrate (91%) and lower amount of protein (8%). Subsequently, this fungus was grown under submerged culture for 72, 96 and 168 h (these EPS were designated EPSD1-72, EPSD1-96 and EPSD1-168) and the differences in production, monosaccharide composition and apparent molecular were compared. The EPS yields in mg/100 mL of culture medium were: 3.0 ± 0.4 (EPSD1-72), 15.4 ± 2.2 (EPSD1-96) and 14.8 ± 1.8 (EPSD1-168). The EPSD1-72 had high protein content (28.5%) and only 71% of carbohydrate; while EPSD1-96 and EPSD1-168 were composed mainly of carbohydrate (≈95 and 100%, respectively), with low protein content (≈5%) detected at 96 h. Galactose was the main monosaccharide component (30%) of EPSD1-168. Differently, EPSD1-96 was rich in glucose (51%), with molecular weight of 46.6 kDa. It is an important feature for future investigations, because glucan-rich EPS are reported as effective antitumor agents.

β-(1→3,1→6)-d-glucans produced by Diaporthe sp. endophytes: Purification, chemical characterization and antiproliferative activity against MCF-7 and HepG2-C3A cells.

This study reports the characterization and antiproliferative activity of exopolysaccharides (EPS) produced by submerged cultures of the endophytes Diaporthe sp. JF766998 and Diaporthe sp. JF767007 isolated from the medicinal plant Piper hispidum Sw. Both strains secreted a crude EPS that, upon size exclusion chromatography, showed to contain a heteropolysaccharide (galactose, glucose and mannose) and a high-molecular weight glucan. Data from methylation analysis, FTIR and NMR spectroscopy (1H, COSY, TOCSY and HSQC-DEPT) indicated that the purified glucan consisted of a main chain of glucopyranosyl β-(1→3) linkages substituted at O-6 by glucosyl residues. According to MTT assay, some treatments of both β-glucans have antiproliferative activity against human breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG2-C3A) cells after 24 and 48h of treatment, exhibiting a degree of inhibition ratio that reached the highest values at 400μg/mL: 58.0% (24h) and 74.6% (48h) for MCF-7 cells, and 61.0% (24h) and 83.3% (48h) for HepG2-C3A cells. These results represent the first reports on the characterization and antiproliferative effect of β-glucans from Diaporthe species and also expand the knowledge about bioactive polysaccharides from endophytic sources.

Toxicity study of the anti-hypertensive agent perindopril on the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) Sorokin assessed by conidia germination speed parameter

Hypertension is an important vascular disease to the global public health, since it constitutes the principal cause of death from childhood to adulthood. In order to alleviate its symptoms, the treatment is accomplished by anti-hypertensive drugs, among them, is perindopril, an angiotensin-converting enzyme (ACE) inhibitor. The entomopathogenic filamentous fungus Metarhizium anisopliae is widely used for biological control and it has been promising in toxicity studies of substances assessed by conidia germination speed parameter. This study aimed to verify the effect of different concentrations of perindopril on the conidia germination speed of the model fungus M. anisopliae , for detecting a possible toxic effect of this medicament in another eukaryote. Conidia of M. anisopliae were incubated with perindopril in concentrations of 200 and 20 μg/ml at 28°C for 12 h, sampled at 0, 6, 8, 10 and 12 h and analyzed by light microscopy. The frequency of dormant, embedded, bud and germinated conidia was counted. As a result, perindopril in concentrations of 200 and 20 μg/ml increased the germination speed of M. anisopliae conidia , when compared to the negative control (absence of perindopril). It indicates that these two concentrations of perindopril have no toxicity on M. anisopliae , considering the Bayesian analysis. Keywords : Angiotensin-converting enzyme inhibitor, vegetative development, model fungus, entomopathogen, Bayesian analysis. African Journal of Biotechnology Vol. 12(35), pp. 5452-5457+

Sensibility of the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) Sorokin to Pelargonium sidoides extract (EPs 7630 ® ) assessed by conidia germination speed parameter

Pelargonium sidoides, known as umckaloabo, is a plant originally from South Africa and its extract is used in popular medicine. The fungus Metarhizium anisopliae, an important entomopathogen used in biological control of pests, is also employed as model for the evaluation of toxicity and compatibility of different products, considering, among other parameters, conidia germination speed. Therefore, this study aimed to evaluate the sensibility of M. anisopliae var. anisopliae in the presence of the extract of P. sidoides, at a concentration of 20.625 and 2.0625 mg/ml. During incubation at 28°C, samples were collected at 0, 6, 8, 10, 12 and 24 h and analyzed by light microscopy, with observation of 300 conidia, in triplicate, for both treatments and negative controls (in a completely randomized design). This study shows that the two concentrations of P. sidoides extract tested are capable of delaying the M. anisopliae conidia germination speed, in comparison to the controls, although the germination frequency has been restored after about 12 h of conidia incubations, with did not lack conidia viability, indicating no toxicity.