Endophytic species of Induratia from coffee and carqueja plants from Brazil and its potential for the biological control of toxicogenic fungi on coffee beans by means of antimicrobial volatiles.

Several endophytic fungi have been reported to have produced bioactive metabolites. Some of them, including the Induratia species, have the capacity to emit volatile compounds with antimicrobial properties with broad spectrum against human and plant pathogens. The present study aimed to prospect the Induratia species producing volatile organic compounds (VOCs), in carqueja plants used in alternative medicine and coffee plants in Brazil. A total of 11 fungal isolates producing volatile metabolites were obtained by a parallel growth technique, using I. alba 620 as a reference strain. Phylogenetic relationships revealed the presence of at least three distinct species, I. coffeana, I. yucatanensis, and Induratia sp. SPME/GC/MS analyses of the VOCs in the headspace above the mycelium from Induratia species cultured for 10 days on PDA revealed the volatile profile emitted by I. coffeana CCF 572, I. coffeana COAD 2055, I. yucatanensis COAD 2062, and Induratia sp. COAD 2059. Volatile organic compounds produced by I. coffeana isolates presented antimicrobial activity against Aspergillus ochraceus, A. sclerotiorum, A. elegans, A. foetidus, A. flavus, A. tamari, A. tubingensis, A. sydowii, A. niger, A. caespitosus, A. versicolor, and A. expansum, sometimes by decreasing the growth rate or, mainly, by fully inhibiting colony growth. Fifty-eight percent of the target species died after 6 days of exposure to VOCs emitted by I. coffeana CCF 572. In addition, VOCs emitted by the same fungus inhibited the growth in A. ochraceus inoculated into coffee beans, which indicates that plants which have I. coffeana as an endophyte may be protected from attacks by this plant pathogen.

Arcopilus eremanthusum sp. nov. as sources of antibacterial and antioxidant metabolites.

In this study, we evaluated the antibacterial and antioxidant activities of ethyl acetate (EtOAc) extracts of Arcopilus eremanthusum sp. nov. (CML3766) isolated from E. erythopappus. The fungi were identified using the sequences of the internal transcribed spacer (ITS), large subunit (LSU), and RNA polymerase II second largest subunit (RPB2). Antibacterial activity was determined using the minimum bactericidal concentration (MBC) method. Antioxidant activity was evaluated using free radical scavenging methods with 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS•+) assays and the ß-carotene-linoleic acid system. The total phenolic compound content was measured using the Folin-Ciocalteu method. The endophytic fungal extract presented bactericidal activity, with an MBC of 2.44 and 19.5 µg/mL against Staphylococcus aureus GL 8702 and GL 5674, respectively, and 625 µg/mL for Salmonella enterica serovar Enteritidis. In addition, this fungus demonstrated an antioxidant activity of 52.30% protection in the ß-carotene method. The total concentration of phenolic compounds was 23.73 mg gallic acid equivalent (GAE)/g. Ferulic acid, trans-cinnamic acid, chlorogenic acid, catechin, vanillin, p-coumaric acid, and caffeic acid were quantified using high-performance liquid chromatography with diode array detection. The results demonstrate the potential of A. eremanthusum sp. nov. to serve as a source of antibacterial and antioxidant metabolites with possible future biotechnological applications.

Metabolites from Induratia spp. modulating key enzymes in human hemostasis.

Induratia spp. fungi have been poorly evaluated for their non-volatile secondary metabolites. In the present work, we evaluated the effects of non-volatile secondary metabolites released into the culture medium by Induratia spp. upon toxic alterations induced by Bothrops spp. venoms. B. atrox venom phospholipase was inhibited by Induratia spp. around 12 and 16%. The extracts of the two strains inhibited 12-25% of the hemolysis induced by B.moojeni venom. Thrombolysis was inhibited by 30-60% by the compounds present in both extracts. The coagulation induced by B. moojeni venom was prolonged by 26-41 s by the action of the extracts of I. coffeana. The fungal extracts did not exert any cytotoxic effect, nor did they induce any alteration in the other evaluated substrates show the potential use of non-volatile metabolites produced by the fungi evaluated as enzyme modulators, especially for proteases with a fundamental role in human hemostasis.

Endophytic Fungi Assures Tropical Forage Grass Growth by Water Stress Tolerances.

Forage plants is the base of beef and dairy cattle production. While water stress limits agricultural production worldwide, endophytic fungi can play a beneficial role for plants, such as tolerance to biotic and abiotic stresses. The objective of this work was to evaluate the effect of inoculation of the endophytic fungi Paraconiothyrium estuarinum (CML 3695, CML 3696, CML 3699) and Paraconiothyrium cyclothyrioides (CML 3697, CML 3698) on agronomic characteristics of two forage species, Brachiaria brizantha (A. Rich) Stapf. cv. Marandu and Megathyrsus maximus Jacq. cv. BRS Mombaça, under different available water capacities. The treatments simulated a long drought period (LDH) equivalent to 10% of the available water capacity (AWC) and simulated 7 (7 DH) and 14 days of drought (14 DH) without water supply. The grasses were evaluated for length and dry weight of shoots and roots. All treatments reached humidity below the permanent wilting point (PWP) and the highest variation in soil moisture was observed at 14 DH, for both grass species. The endophytic fungi promoted an average 15% increase in shoot length (SL) for B. brizantha and an increase of 34% for SL, 266% for Dry Shoot Mass (SDM), and 340% for Dry Root Mass (RDM) for M. maximus treated with P. estuarinum (CML 3699) at 7 DH. Paraconiothyrium estuarinum (CML 3699) guaranteed the highest tolerance to water deficit and sustainable growth performance to both tested grasses.

Antiviral fungal metabolites and some insights into their contribution to the current COVID-19 pandemic.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, which started in late 2019, drove the scientific community to conduct innovative research to contain the spread of the pandemic and to care for those already affected. Since then, the search for new drugs that are effective against the virus has been strengthened. Featuring a relatively low cost of production under well-defined methods of cultivation, fungi have been providing a diversity of antiviral metabolites with unprecedented chemical structures. In this review, we present viral RNA infections highlighting SARS-CoV-2 morphogenesis and the infectious cycle, the targets of known antiviral drugs, and current developments in this area such as drug repurposing. We also explored the metabolic adaptability of fungi during fermentation to produce metabolites active against RNA viruses, along with their chemical structures, and mechanisms of action. Finally, the state of the art of research on SARS-CoV-2 inhibitors of fungal origin is reported, highlighting the metabolites selected by docking studies.

Enzymatic Modulators from Induratia spp.

In the present work, ethyl acetate extracts, consisting of non-volatile compounds, from the culture of endophytic fungi isolated from coffee plants, Induratia coffeana and Induratia yucatanensis, were prospected in enzyme modulation tests that act in human hemostasis. Dry extracts of the fungi were diluted in dimethyl sulfoxide p.a. 99.9% (DMSO), and then tested. Bothrops atrox venom was used as an enzyme source and tool to induce the activities. Prior to the evaluation of the activities, incubations of the extracts with the venom were performed in the proportions 1: 0.01, 1: 0.25, 1: 0.5, and 1: 1 (venom: extract; mass: mass). The extracts of all fungi promoted a significant increase in the clotting time induced by the venom, which was even longer when the extracts were previously incubated with the citrated plasma. The activity of phospholipases A2 did not significantly change when evaluated in the presence of fungal extracts. However, the evaluated extracts inhibited proteases by 73% and 30% in the thrombolytic and caseinolytic tests, respectively. In addition, the extracts did not induce cytotoxicity on human erythrocytes when evaluated in the absence of the venom. Thus, it is possible to suggest the presence of specific interactions between molecules present in extracts of Induratia spp. and venom proteases, highlighting non-volatile metabolites as promising sources of compounds of medical and scientific interest.

Enzyme Production by Induratia spp. Isolated from Coffee Plants in Brazil

Abstract Endophytic fungi belonging to the genus Muscodor now transferred to Induratia are known producers of bioactive volatile organic compounds (VOCs) with many industrial applications. However, the members of this genus have rarely been reported to produce non-volatile metabolites including enzyme. Enzymes of the endophytes are degraders of the polysaccharides available in the host plants and the knowledge of enzyme production by Induratia spp. may provide insights into their possible biotechnological applications. The aim of this study was to evaluate the activity of amylase, cellulase, lipase, pectinase, phytase, protease, endo β-1,4 glucanase and exo β-1,4 glucanase enzymes produced by fungi of the species Induratia coffeana, Induratia yucatanensis and Induratia sp. isolated from organic coffee plants. All Induratia spp. were able to produce the extracellular enzymes cellulase, pectinase, protease, and phytase. Eight fungi were able to produce lipase and four produced amylase. The specific activity of endo β-1, 4 glucanase and exo β-1,4 glucanase enzymes were detected for 9 and 8 endophytic fungi, respectively. This work demonstrated for the first time, the array of enzymes produced by Induratia spp. isolated from Coffea arabica in organic systems in Brazil.

Spray Drying of Coloring Extracts Produced by Fungi Isolated from Brazilian Caves

Abstract Pigments produced by submerged fermentation of three filamentous fungi isolated from Brazilian caves, namely Aspergillus keveii, Penicillium flavigenum, and Epicoccum nigrum, were submitted to spray drying in presence of the adjuvants maltodextrin, modified starch or gum arabic. Yellow fine powders with low moisture content and water activity, and high color retention (> 70%) were successfully generated with a high product recovery ratio (> 50%), independently of the adjuvant used. The dried products have enhanced stability and potential to might be used as a natural colorant in food and pharmaceutical applications.

Antimicrobial activity of endophytic fungi from coffee plants / Atividade antimicrobiana de fungos endofíticos de plantas de café

Endophytic fungi are a promising source for discovery of compounds with biotechnological potential. The aim of this study was to select and identify endophytic fungi from Coffea arabica that produce volatile organic compounds (VOCs), evaluate the effect of the VOCs produced by endophytic fungi on the growth of Rhizoctonia solani, Fusarium oxysporum, Phoma sp., Botrytis cinerea, Fusarium solani, Fusarium verticillioides, Cercospora coffeicola and Pestalotia longisetula, and select endophytic fungi with potential for biological control of Aspergillus ochraceus inoculated in coffee beans and F. verticillioides inoculated in corn seeds. An isolate of Muscodor albus was used as selection tool for VOC producing fungi. Among the 400 endophytic fungi isolates, 11 were able to grow in the presence of VOCs produced by M. albus. These fungi were identified as Muscodor spp. (9) and Simplicillium sp. according to searches in UNITE database using DNA sequences of internal transcribed spacer (ITS). The VOC's produced by endophytic fungi inhibited the growth the phytopathogenic fungi with different efficacies, compared to the control. The VOCs produced by Muscodor coffeanum (COAD 1842) showed fungicidal effect against A. ochraceus on coffee beans. Six endophytic fungi completely inhibited growth of F. verticillioides inoculated in corn seeds. This study demonstrates that the volatile-compound producing endophytic fungi, isolated from Coffea arabica, are promising sources of bioactive compounds.

Fungos endofíticos são uma fonte promissora para a descoberta de compostos com potencial biotecnológico. O objetivo deste estudo foi selecionar e identificar fungos endofíticos de Coffea arabica que produzem compostos orgânicos voláteis (COVs), avaliar o efeito dos compostos orgânicos voláteis produzido por fungos endofíticos sobre o crescimento de Rhizoctonia solani, Fusarium oxysporum, Phoma sp., Botrytis cinerea, Fusarium solani, Fusarium verticillioides, Cercospora coffeicola e Pestalotia longisetula e selecionar fungos endofíticos com potencial para controle biológico de Aspergillus ochraceus inoculado em grãos de café e F. verticillioides inoculado em sementes de milho. Um isolado de Muscodor albus foi utilizado como ferramenta de seleção para fungos endofíticos produtores de COVs. Dentre os 400 fungos endofíticos isolados, 11 foram capazes de crescer na presença de COVs produzidos por M. albus. Estes fungos foram identificados como Muscodor spp. (9) e Simplicillium sp. de acordo com pesquisas na base de dados UNITE usando sequências de DNA do espaçador transcrito interno (ITS). Os COVs produzidos por fungos endofíticos inibiram o crescimento dos fungos fitopatogênicos em comparação com o controle com diferentes eficácias. Os COVs produzidos por Muscodor coffeanum (COAD 1842) apresentou efeito fungicida contra A. ochraceus em grãos de café. Seis fungos endofíticos inibiram completamente o crescimento de F. verticillioides inoculado em sementes de milho. Este estudo demonstra que os fungos endofíticos produtores de compostos voláteis isolados de Coffea arabica são fontes promissoras de compostos bioativos.

Optimization of culture conditions for tannase production by Aspergillus sp. gm4 in solid state fermentation / Otimização das condições de cultivo para produção de tanase por Aspergillus sp. gm4 em fermentação em estado sólido

Tannase is an industrially important enzyme produced by a large number of microorganisms. This study analyzed the production of tannase by Aspergillus sp. GM4 under solid-state fermentation (SSF) using different vegetable leaves (mango, jamun and coffee) and agricultural residues (coffee husks, rice husks and wheat bran). Among the substrates used jamun leaves yielded high tannase production. The Plackett-Burman design was conducted to evaluate the effects of 12 independent variables on the production of tannase under SSF using jamun leaves as substrate. Among these variables, incubation time, potassium nitrate and tannic acid had significant effects on enzyme production. A lower incubation time was fixed and supplementation with potassium nitrate and tannic acid were optimized using the Central Composite Design. The best conditions for tannase production were: incubation time of 2 days; tannic acid at 1.53% (w w-1) and potassium nitrate at 2.71% (w w- 1). After the optimization process, tannase production increased 4.65-fold, which showed that the statistical experimental design offers a practicable approach to the implementation of optimization of tannase production.

Tanase é uma enzima industrialmente importante produzida por um grande número de microrganismos. Este estudo analisou a produção de tanase por Aspergillus sp. GM4 em fermentação em estado sólido (FES) utilizando diferentes vegetais como folhas de manga, de jambolão, de café e resíduos agrícolas, como a casca de café, casca de arroz e farelo de trigo. Entre os substratos utilizados, as folhas jambolão renderam alta produção de tanase. O planejamento de Plackett-Burman foi conduzido para avaliar os efeitos de 12 variáveis independentes sobre a produção de tanase em FES usando folhas jambolão como substrato. Entre estas variáveis, tiveram efeitos significativos na produção da enzima o tempo de incubação, o nitrato de potássio e o ácido tânico. O menor tempo de incubação foi fixado e a suplementação de nitrato de potássio e ácido tânico foi otimizada utilizando o planejamento composto central rotacional. As melhores condições para a produção de tanase foram o tempo de incubação de dois dias, a concentração de ácido tânico de 1,53% (g g-1) e de nitrato de potássio 2,71% (g gw-1). Após o processo de otimização, a produção tanase aumentou 4,65 vezes, o que mostrou que o delineamento experimental foi um método viável para a otimização da produção de tanase.

Pectin lyase production by recombinant Penicillium griseoroseum strain 105.

Recombinant Penicillium griseoroseum strain 105 overproduces an extracellular pectin lyase (PL) under the transcriptional control of the strong gpdA promoter of Aspergillus nidulans. Our aim was to evaluate PL production by recombinant P. griseoroseum strain 105 in submerged fermentation system bioreactors BioFloIII and BioFloIV using 2 or 10 L working volumes under different growth conditions and to analyze the production of cellulase, polygalacturonase, pectin methylesterase, and protease. PL overproduction by recombinant P. griseoroseum strain 105 was 112 times higher than that of P. griseoroseum PG63 grown in sugarcane juice. Cellulases and proteases were not detected in the culture filtrate, and evaluation for extracellular proteins in the culture medium by SDS-PAGE showed the presence of a 36 kDa predominant band, similar to the molecular mass estimated from the nucleotide sequence of plg1 gene for PL of P. griseoroseum strain 105. This recombinant strain provides the advantage of PL production, which predominates over other extracellular proteins usually present in most commercial pectinase preparations, using sugarcane juice as a substrate of low cost.

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice.

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortusDeltawbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. DeltawbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence.

Determination of dynamic characteristics of microbiota in a fermented beverage produced by Brazilian Amerindians using culture-dependent and culture-independent methods.

Cauim is a fermented beverages prepared by Tapirapé Amerindians in Brazil from substrates such as cassava, rice, peanuts, pumpkin, cotton seed and maize. Here we study the microorganisms associated with peanut and rice fermentation using a combination of culture-dependent and -independent methods. The bacterial population varied from 7.4 to 8.4 log CFU/ml. The yeast population varied from 4.0 to 6.6 log CFU/ml. A total of 297 bacteria and yeasts strains were isolated during fermentation, with 198 bacteria and 99 yeast. The Lactobacillus genus was dominant throughout fermentation. Bacteria and yeast community dynamics during the fermentation process were monitored by PCR-DGGE analysis. Both culture-dependent and -independent methods indicated that the bacterial species L. plantarum, L. fermentum, L. paracasei and L. brevis as well as the yeast species P. guilliermondii, K. lactis, Candida sp, R. toruloides and Saccharomyces cerevisiae, were dominant during fermentation. Multivariate analysis of microorganisms during beverage fermentation showed that the microbial community changed during the fermentation process.

Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum.

The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml(-1) respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.

Morphological and molecular differentiation of the pectinase producing fungi Penicillium expansum and Penicillium griseoroseum

Two species from the genus Penicillium, Penicillium expansum and P. griseoroseum (Brasilian isolates) were characterized morphologic and molecularlly. Morphological variability was detected among isolates in regard to colony morphology and to conidia coloration. The molecular characterization was based on the RAPD markers, telomeric fingerprinting and ITS sequencing. A total of 78 RAPD primers were used and 8 presented differences in band patterns with 54 percent of the amplified polymorphic fragments. The monomorphic fragments of 600 bp (P. expansum) and 594 bp (P. griseoroseum) were amplified. The only internal transcribed spacer region variation detected between the two species was the additional six initial nucleotides. The analysis by telomeric fingerprinting showed polymorphism between both species and the chromosome minimal numbers estimated were three. The polymorphism observed in the organization of the subtelomeric region in the genome of two Penicillium species within the high homogeneous Penicillium subgenus is for the first time reported and perhaps can be employed in future phylogenetic studies.

Penicillium expansum e P. griseoroseum foram caracterizados morfológica e molecularmente. Variações na morfologia das colônias e coloração dos conídeos foram observadas entre os isolados. A caracterização molecular foi baseada em marcadores RAPD, sequenciamento da região do espaçador interno transcrito do DNA ribossomal e "fingerprinting" telomérico. Foi usado um total de 78 primers RAPD, sendo que 8 apresentaram 54 por cento de fragmentos de DNA polimórficos. Os produtos da amplificação da região ITS de P. expansum e P. griseoroseum foram de 600 e 594 pb, respectivamente. Não foi detectada nenhuma variação na seqüência de nucleotídeos dessa região, comparando-se P. expansum e P. griseoroseum, exceto em relação aos seis nucleotídeos iniciais adicionais. Observou-se a ocorrência de polimorfismo na organização da região subtelomérica no genoma destes fungos e estimou-se um número mínimo de três cromossomos para estas espécies. Este é o primeiro trabalho que descreve a existência de polimorfismo na organização da região subtelomérica do genoma de espécies de fungos pertencentes ao gênero Penicillium, altamente homogêneo, indicando uma possível utilização da abordagem empregada neste estudo para pesquisas filogenéticas futuras.

Brucella spp noncanonical LPS: structure, biosynthesis, and interaction with host immune system.

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. Several species are recognized within the genus Brucella and this classification is mainly based on the difference in pathogenicity and in host preference. Brucella strains may occur as either smooth or rough, expressing smooth LPS (S-LPS) or rough LPS (R-LPS) as major surface antigen. This bacterium possesses an unconventional non-endotoxic lipopolysaccharide that confers resistance to anti-microbial attacks and modulates the host immune response. The strains that are pathogenic for humans (B. abortus, B. suis, B. melitensis) carry a smooth LPS involved in the virulence of these bacteria. The LPS O-chain protects the bacteria from cellular cationic peptides, oxygen metabolites and complement-mediated lysis and it is a key molecule for Brucella survival and replication in the host. Here, we review i) Brucella LPS structure; ii) Brucella genome, iii) genes involved in LPS biosynthesis; iv) the interaction between LPS and innate immunity.