Apoplastomes of contrasting cacao genotypes to witches' broom disease reveals differential accumulation of PR proteins.

Witches' broom disease (WBD) affects cocoa trees (Theobroma cacao L.) and is caused by the fungus Moniliophthora perniciosa that grows in the apoplast in its biotrophic phase and later progresses into the tissues, causing serious losses in the production of cocoa beans. Therefore, the apoplast of T. cacao can provide important defense responses during the interaction with M. perniciosa. In this work, the protein profile of the apoplast of the T. cacao genotypes Catongo, susceptible to WBD, and CCN-51, resistant one, was evaluated. The leaves of T. cacao were collected from asymptomatic plants grown in a greenhouse (GH) and from green witches' brooms grown under field (FD) conditions for extraction of apoplastic washing fluid (AWF). AWF was used in proteomic and enzymatic analysis. A total of 14 proteins were identified in Catongo GH and six in Catongo FD, with two proteins being common, one up-accumulated, and one down-accumulated. In CCN-51, 19 proteins were identified in the GH condition and 13 in FD, with seven proteins being common, one up-accumulated, and six down-accumulated. Most proteins are related to defense and stress in both genotypes, with emphasis on pathogenesis-related proteins (PR): PR-2 (ß-1,3-glucanases), PR-3 and PR-4 (chitinases), PR-5 (thaumatine), PR-9 (peroxidases), and PR-14 (lipid transfer proteins). Furthermore, proteins from microorganisms were detected in the AWF. The enzymatic activities of PR-3 showed a significant increase (p < 0.05) in Catongo GH and PR-2 activity (p < 0.01) in CCN-51 FD. The protein profile of the T. cacao apoplastome offers insight into the defense dynamics that occur in the interaction with the fungus M. perniciosa and offers new insights in exploring future WBD control strategies.

Cocoa Apoplastome Contains Defense Proteins Against Pathogens.

The apoplast performs important functions in the plant, such as defense against stress, and compounds present form the apoplastic washing fluid (AWF). The fungus Moniliophthora perniciosa, the causal agent of witches' broom disease (WBD) in Theobroma cacao, initially colonizes the apoplast in its biotrophic phase. In this period, the fungus can remain for approximately 60 days, until it changes to its second phase, causing tissue death and consequently large loss in the production of beans. To better understand the importance of the apoplast in the T. cacao-M. perniciosa interaction, we performed the first apoplastic proteomic mapping of two contrasting genotypes for WBD resistance (CCN51-resistant and Catongo-susceptible). Based on two-dimensional gel analysis, we identified 36 proteins in CCN-51 and 15 in Catongo. We highlight PR-proteins, such as peroxidases, ß-1,3-glucanases, and chitinases. A possible candidate for a resistance marker of the CCN-51 genotype, osmotin, was identified. The antioxidative metabolism of the superoxide dismutase (SOD) enzyme showed a significant increase (P < 0.05) in the AWF of the two genotypes under field conditions (FD). T. cacao AWF inhibited the germination of M. perniciosa basidiospores (>80%), in addition to causing morphological changes. Our results shed more light on the nature of the plant's defense performed by the apoplast in the T. cacao-M. perniciosa interaction in the initial (biotrophic) phase of fungal infection and therefore make it possible to expand WBD control strategies based on the identification of potential targets for resistance markers and advance scientific knowledge of the disease.

A utilização da técnica CRISPR no melhoramento do cacau: revisão bibliográfica

Uma ferramenta de engenharia genética chamada CRISPR possibilita, além de novos recursos terapêuticos e soluções para distúrbios genéticos e doenças, o melhoramento na agricultura. O aprimoramento do CRISPR/Cas9 vem evoluindo e recentemente, dois derivados dele foram desenvolvidos, o CRISPRa e o CRISPRi. Com esses sistemas é possível a modulação gênica sem a necessidade da clivagem do local-alvo. Utilizar esses sistemas na agricultura demonstra ter um bom potencial econômico, podendo garantir uma melhor qualidade de vida à população e um meio ambiente mais sustentável, sendo úteis para diminuir o desperdício e o risco de extinção de alguns alimentos, como o cacau. O cacau é um fruto oriundo da árvore Theobroma cacao e, infelizmente, é alvo de várias doenças causadas por fitopatógenos que resultam em perdas globais, além de fatores abióticos. Dessa forma, encontramos um gene na literatura e bancos de dados, chamado aleno óxido sintase (AOS), o aumento de sua expressão, pode resultar no aumento da resistência do cacaueiro contra uma dessas doenças, a vassoura-de-bruxa provocada pelo fungo Moniliophthora perniciosa. A metodologia proposta é baseada na literatura e a descrição dos resultados condiz com as atividades práticas desenvolvidas no laboratório de Desenvolvimento e Inovação do Instituto Butantan, dentro da linha de pesquisa de edição gênica por CRISPR/Cas9. O que foi apresentado é uma hipótese de melhoramento da resistência da T. cacao contra a M. perniciosa, com o aumento da expressão do gene AOS utilizando o sistema CRISPRa.

ß-Glucosidase produced by Moniliophthora perniciosa: Characterization and application in the hydrolysis of sugarcane bagasse.

ß-Glucosidases (BGLs) belong to the group of enzymes of cellulases and act in the last stage of cellulose degradation, releasing glucose molecules, eliminating the inhibitory effect of cellobiose. This study focused on the production, characterization, and application of BGL from Moniliophthora perniciosa in the hydrolysis of pretreated sugarcane bagasse (3% NaOH + 6% Na2 SO3 ), with varying enzymatic loads and reaction times. The enzyme showed an optimum pH of 4.5 and 60°C. It was stable at all temperatures analyzed (50-90°C) and retained about 100% of its activity at 50°C after 60 min of incubation. Among the ions analyzed, BaCl2 increased BGL activity 9.04 ± 1.41 times. The maximum production of reducing sugars (89.15%) was achieved after 48 h with 10 mg of protein.

Population Structure of Moniliophthora perniciosa in the Main Cacao Producing Departments of Colombia.

The witches' broom (Moniliophthora perniciosa) is considered as one of the main threats for cacao production and, consequently, for chocolate production worldwide. In this work, the genetic diversity and population structure of M. perniciosa were analyzed for 59 isolates collected in five departments of Colombia and using 10 microsatellite markers. Analyses revealed 35 multilocus genotypes and clonal populations structure according to linkage disequilibrium analysis. One of the objectives of this study was to determine whether populations were differentiated by geographic origin or Theobroma cacao host genotype. Analysis of molecular variance, discriminant analysis of principal components, and Bruvo genetic distance suggested that the genetic structure was driven by geographic origin and not by T. cacao genotype. The results of this study were consistent with previous findings obtained in other cocoa-producing countries. Important insights were discussed regarding the dispersal patterns of the pathogen in Colombia and the genetic change of its populations because of different environmental conditions.

Functional and morphological analysis of isolates of phylloplane and rhizoplane endophytic bacteria interacting in different cocoa production systems in the Amazon.

Endophytic bacteria colonize different internal tissues of plants without damaging their cells. They can establish themselves in the same niche as other microorganisms and develop antagonistic activities against phytopathogens. There is little research on the functional and morphological characterization of these bacteria in production systems in the Amazon. Thus, the objective of this work was to functionally and morphologically characterize endophytic bacteria isolated from cocoa trees (Theobroma cacao L.) and evaluate their antagonistic potential against phytopathogens. A total of 197 endophytic bacteria isolates were obtained from leaves and roots of cocoa plants with different production systems and at different times of the year. The characterization of functional groups consisted of proteolytic, amylolytic and cellulolytic activity and ability to fix nitrogen and solubilize phosphate. Morphological diversity was evaluated mainly according to the following parameters: shape, color, size and elevation of the colony. Thirteen isolates of endophytic bacteria, selected by cluster analysis, were used to evaluate the antagonistic potential in paired trials against four species of phytopathogenic fungi. The largest amount of endophytic bacteria was isolated from the root (95.9%), in the dry season. The most expressive activities with regards to the enzyme index were amylolytic (71.9%), proteolytic (70.2%) and nitrogen fixing (38.6%), respectively. The similarity analysis formed two clusters with isolates CS R 2.4 and CS R 2.25 exhibiting 100% similarity. Five isolates displayed inhibitory activity against phytopathogenic fungi, most notably isolate TS R 2.19, which exhibited antagonistic activity against all fungi and mycelial growth inhibition rates between 25.7% and 50.7%. Understanding the interaction between endophytes in cocoa plants is important as a possible additional tool in biological control. Our studies are incipient and the first to be carried out in different cocoa production systems in the state of Pará, Brazil.

Moniliophthora perniciosa, the Causal Agent of Cacao Witches' Broom Disease Is Killed in vitro by Saccharomyces cerevisiae and Wickerhamomyces anomalus Yeasts.

Cacao plantations from South America have been afflicted with the severe fungal disease known as Witches' Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça, and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa, originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts' antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecting yeast to hypha. This evidence suggests W. anomalus cells form a network of yeast cells connecting with each other and with hyphae, supporting a possible cooperative collective killing and feeding strategy. The present results provide an initial step toward the formulation of a new eco-friendly and effective alternative for controlling cacao WBD using live yeast biocides.

The glutathione peroxidase family of Theobroma cacao: Involvement in the oxidative stress during witches' broom disease.

The glutathione peroxidases (GPXs) are enzymes which are part of the cell antioxidant system inhibiting the ROS-induced damages of membranes and proteins. In cacao (Theobroma cacao L.) genome, five GPX genes were identified. Cysteine insertion codons (UGU) were found in TcPHGPX, TcGPX2, TcGPX4, TcGPX6 and tryptophan insertion codon (UGG) in TcGPX8. Multiple alignments revealed conserved domains between TcGPXs and other plants and human GPXs. Homology modeling was performed using the Populus trichocarpa GPX5 structure as template, and the molecular modeling showed that TcGPXs have affinity with selenometionine in their active site. In silico analysis of the TcGPXs promoter region revealed the presence of conserved cis-elements related to biotic stresses and hormone responsiveness. The expression analysis of TcGPXs in cacao plantlet meristems infected by M. perniciosa showed that TcGPXs are most expressed in susceptible variety than in resistant one, mainly in disease stages in which oxidative stress and programmed cell death occurred. This data, associated with phylogenetic and location analysis suggested that TcGPXs may play a role in protecting cells from oxidative stress as a try of disease progression reduction. To our knowledge, this is the first study of the overall GPX family from T. cacao.

Potential of Yeasts as Biocontrol Agents of the Phytopathogen Causing Cacao Witches' Broom Disease: Is Microbial Warfare a Solution?

Plant diseases caused by fungal pathogens are responsible for major crop losses worldwide, with a significant socio-economic impact on the life of millions of people who depend on agriculture-exclusive economy. This is the case of the Witches' Broom Disease (WBD) affecting cacao plant and fruit in South and Central America. The severity and extent of this disease is prospected to impact the growing global chocolate market in a few decades. WBD is caused by the basidiomycete fungus Moniliophthora perniciosa. The methods used to contain the fungus mainly rely on chemical fungicides, such as copper-based compounds or azoles. Not only are these highly ineffective, but also their utilization is increasingly restricted by the cacao industry, in part because it promotes fungal resistance, in part related to consumers' health concerns and environmental awareness. Therefore, the disease is being currently tentatively controlled through phytosanitary pruning, although the full removal of infected plant material is impossible and the fungus maintains persistent inoculum in the soil, or using an endophytic fungal parasite of Moniliophthora perniciosa which production is not sustainable. The growth of Moniliophthora perniciosa was reported as being antagonized in vitro by some yeasts, which suggests that they could be used as biological control agents, suppressing the fungus multiplication and containing its spread. Concurrently, some yeast-based products are used in the protection of fruits from postharvest fungal spoilage, and the extension of diverse food products shelf-life. These successful applications suggest that yeasts can be regarded a serious alternative also in the pre-harvest management of WBD and other fungal plant diseases. Yeasts' GRAS (Generally Recognized as Safe) nature adds to their appropriateness for field application, not raising major ecological concerns as do the present more aggressive approaches. Importantly, mitigating WBD, in a sustainable manner, would predictably have a high socioeconomic impact, contributing to diminish poverty in the cacao-producing rural communities severely affected by the disease. This review discusses the importance/advantages and the challenges that such a strategy would have for WBD containment, and presents the available information on the molecular and cellular mechanisms underlying fungi antagonism by yeasts.

The selenium-binding protein of Theobroma cacao: A thermostable protein involved in the witches' broom disease resistance.

The selenium-binding proteins are known to be inducers of apoptosis in human and animals, and have been studied as target for the treatment of various types of cancer. In plants, SBP expression has been related to abiotic and biotic stress resistance. The SBP from Theobroma cacao (TcSBP) was first identified from a cocoa-Moniliophthora perniciosa cDNA library. The present study provides details on the TcSBP gene and protein structure. Multiple alignments revealed conserved domains between SBP from plants, human and archea. Homology modeling and molecular docking were performed and showed that the TcSBP has affinity to selenite in the active CSSC site. This result was confirmed by circular dichroism of the recombinant TcSBP, which also presented thermostable behavior. RT-qPCR analysis showed that TcSBP was differentially expressed in resistant vs susceptible cacao varieties inoculated by M. perniciosa and its expression was probably due to hormone induction via cis-regulating elements present in its promotor. The presence of the CSSC domain suggested that TcSBP acted by altering oxidation/reduction of proteins during H2O2 production and programmed cell death in the final stages of the witches' broom disease. To our knowledge, this is the first in silico and in vitro analysis of the SBP from cacao.

Heterologous expression of an alternative oxidase from Moniliophthora perniciosa in Saccharomyces cerevisiae: Antioxidant function and in vivo platform for the study of new drugs against witches' broom disease.

The fungus Moniliophthora perniciosa is the causal agent of witches' broom disease (WBD), one of the most devastating diseases of cacao, the chocolate tree. Many strategies to control WBD have been tested so far, including the use of agrochemicals such as the strobilurins. Strobilurins are fungicides of the QoI family, and they are used in the control of a wide array of fungal diseases in many different crops, including cereals, field crops, fruits, tree nuts, and vegetables. These drugs act by specifically inhibiting fungal respiration at the Qo site of complex III, which is a component of the main mitochondrial respiratory chain. However, M. perniciosa is resistant to this family of chemicals. It has been postulated that this resistant phenotype is, at least in part, a result of the strong ability of this fungus to counteract the oxidative stress generated by the impairment of the main mitochondrial respiratory chain, through the activation of an alternative oxidase (Mp-AOX). To test this hypothesis, we expressed functional mitochondria-localized Mp-AOX in the model yeast Saccharomyces cerevisiae. We demonstrated that heterologous expression of Mp-AOX strongly inhibits hydrogen peroxide production by mitochondria. It also diminishes the total cell amount of oxidized glutathione (GSSG), resulting in a fifty-fold higher GSH/GSSG ratio in cells expressing Mp-AOX than in wild type cells. In addition, Mp-AOX activity decreases yeast growth rate and leads to low biomass production. Therefore, we propose the use of this heterologous expression system to direct the development of new inhibitors of fungal AOX by comparing the differences in optical density of Mp-AOX-expressing cells in the presence and absence of potential AOX inhibitors. Together, our results confirm the antioxidant role of Mp-AOX and provide an in vivo platform to be used in the screening of new fungicides based on Mp-AOX inhibition.

Synthesis and testing of novel alternative oxidase (AOX) inhibitors with antifungal activity against Moniliophthora perniciosa (Stahel), the causal agent of witches' broom disease of cocoa, and other phytopathogens.

BACKGROUND: Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is the causal agent of witches' broom disease (WBD) of cocoa (Theobroma cacao L.) and a threat to the chocolate industry. The membrane-bound enzyme alternative oxidase (AOX) is critical for M. perniciosa virulence and resistance to fungicides, which has also been observed in other phytopathogens. Notably AOX is an escape mechanism from strobilurins and other respiration inhibitors, making AOX a promising target for controlling WBD and other fungal diseases. RESULTS: We present the first study aimed at developing novel fungal AOX inhibitors. N-Phenylbenzamide (NPD) derivatives were screened in the model yeast Pichia pastoris through oxygen consumption and growth measurements. The most promising AOX inhibitor (NPD 7j-41) was further characterized and displayed better activity than the classical AOX inhibitor SHAM in vitro against filamentous fugal phytopathogens, such as M. perniciosa, Sclerotinia sclerotiorum and Venturia pirina. We demonstrate that 7j-41 inhibits M. perniciosa spore germination and prevents WBD symptom appearance in infected plants. Finally, a structural model of P. pastoris AOX was created and used in ligand structure-activity relationships analyses. CONCLUSION: We present novel fungal AOX inhibitors with antifungal activity against relevant phytopathogens. We envisage the development of novel antifungal agents to secure food production. © 2018 Society of Chemical Industry.

The extraction and characterisation of a polysaccharide from Moniliophthora perniciosa CCMB 0257.

The alpha-D-glucans are worldwide acknowledged as powerful immune system stimulants found in several sources; however, the fungal-derived sources appear to respond with higher activity. The present study has investigated polysaccharide production in Moniliophthora perniciosa. The dry biomass was subjected to thermal treatment in alkaline solution after fermentation. The biopolymers dissolved in this solution were precipitated after three volumes of absolute ethanol were added to the supernatant. The pure polysaccharide MPS1 was obtained through molecular exclusion chromatography using the Sephacryl S-200 column. The HPLC-RI analysis showed that MPS1 was a glucose homopolysaccharide. Nuclear magnetic resonance (NMR) spectra indicated the α-form as the anomeric carbon configuration in glucose residue. The structure of the polysaccharide was further confirmed as (1→4)-α-D-glucan through the chemical shift of C4. The molecular weight of MPS1 was 31.0 kDa.

Major phytopathogens and strains from cocoa (Theobroma cacao L.) are differentiated by MALDI-MS lipid and/or peptide/protein profiles.

Phytopathogens are the main disease agents that promote attack of cocoa plantations in all tropical countries. The similarity of the symptoms caused by different phytopathogens makes the reliable identification of the diverse species a challenge. Correct identification is important in the monitoring and management of these pests. Here we show that matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in combination with multivariate data analysis is able to rapidly and reliably differentiate cocoa phytopathogens, namely Moniliophthora perniciosa, Phytophthora palmivora, P. capsici, P. citrophthora, P. heveae, Ceratocystis cacaofunesta, C. paradoxa, and C. fimbriata. MALDI-MS reveals unique peptide/protein and lipid profiles which differentiate these phytopathogens at the level of genus, species, and single strain coming from different hosts or cocoa tissues collected in several plantations/places. This fast methodology based on molecular biomarkers is also shown to be sufficiently reproducible and selective and therefore seems to offer a suitable tool to guide the correct application of sanitary defense approaches for infected cocoa plantations. International trading of cocoa plants and products could also be efficiently monitored by MALDI-MS. It could, for instance, prevent the entry of new phytopathogens into a country, e.g., as in the case of Moniliophthora roreri fungus that is present in all cocoa plantations of countries bordering Brazil, but that has not yet attacked Brazilian plantations. Graphical Abstract Secure identification of phytopathogens attacking cocoa plantations has been demonstrated via typical chemical profiles provided by mass spectrometric screening.

Antimicrobial properties of two novel peptides derived from Theobroma cacao osmotin.

The osmotin proteins of several plants display antifungal activity, which can play an important role in plant defense against diseases. Thus, this protein can be useful as a source for biotechnological strategies aiming to combat fungal diseases. In this work, we analyzed the antifungal activity of a cacao osmotin-like protein (TcOsm1) and of two osmotin-derived synthetic peptides with antimicrobial features, differing by five amino acids residues at the N-terminus. Antimicrobial tests showed that TcOsm1 expressed in Escherichia coli inhibits the growth of Moniliophthora perniciosa mycelium and Pichia pastoris X-33 in vitro. The TcOsm1-derived peptides, named Osm-pepA (H-RRLDRGGVWNLNVNPGTTGARVWARTK-NH2), located at R23-K49, and Osm-pepB (H-GGVWNLNVNPGTTGARVWARTK-NH2), located at G28-K49, inhibited growth of yeasts (Saccharomyces cerevisiae S288C and Pichia pastoris X-33) and spore germination of the phytopathogenic fungi Fusarium f. sp. glycines and Colletotrichum gossypi. Osm-pepA was more efficient than Osm-pepB for S. cerevisiae (MIC=40µM and MIC=127µM, respectively), as well as for P. pastoris (MIC=20µM and MIC=127µM, respectively). Furthermore, the peptides presented a biphasic performance, promoting S. cerevisiae growth in doses around 5µM and inhibiting it at higher doses. The structural model for these peptides showed that the five amino acids residues, RRLDR at Osm-pepA N-terminus, significantly affect the tertiary structure, indicating that this structure is important for the peptide antimicrobial potency. This is the first report of development of antimicrobial peptides from T. cacao. Taken together, the results indicate that the cacao osmotin and its derived peptides, herein studied, are good candidates for developing biotechnological tools aiming to control phytopathogenic fungi.

Genomic analyses and expression evaluation of thaumatin-like gene family in the cacao fungal pathogen Moniliophthora perniciosa.

Thaumatin-like proteins (TLPs) are found in diverse eukaryotes. Plant TLPs, known as Pathogenicity Related Protein (PR-5), are considered fungal inhibitors. However, genes encoding TLPs are frequently found in fungal genomes. In this work, we have identified that Moniliophthora perniciosa, a basidiomycete pathogen that causes the Witches' Broom Disease (WBD) of cacao, presents thirteen putative TLPs from which four are expressed during WBD progression. One of them is similar to small TLPs, which are present in phytopathogenic basidiomycete, such as wheat stem rust fungus Puccinia graminis. Fungi genomes annotation and phylogenetic data revealed a larger number of TLPs in basidiomycetes when comparing with ascomycetes, suggesting that these proteins could be involved in specific traits of mushroom-forming species. Based on the present data, we discuss the contribution of TLPs in the combat against fungal competitors and hypothesize a role of these proteins in M. perniciosa pathogenicity.

Apoplastic and intracellular plant sugars regulate developmental transitions in witches' broom disease of cacao.

Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.

Production, purification and characterization of a thermostable β-1,3-glucanase (laminarinase) produced by Moniliophthora perniciosa

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70 percent) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L-1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20 percent of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

A enzima glucanase de Moniliophthora perniciosa foi produzida em meio líquido e purificada a partir do sobrenadante da cultura. A metodologia de superfície de resposta (MSR) foi usada para avaliar os efeitos das variáveis, incluindo indutor (extrato de levedura) e tempo de fermentação, na atividade da glucanase de M. perniciosa detectada no meio de cultura. A enzima presente no sobrenadante foi purificada em duas etapas: precipitação com sulfato de amônio (70 por cento) e cromatografia de filtração em gel em Sephacryl S-200. A produção da enzima glucanase foi maior na concentração de 5,9 g L-1 de extrato de levedura e 13 dias de fermentação. Os resultados mostraram três diferentes isoformas (GLUI, GLUII e GLUIII) com fatores de purificação de 4,33, 1,86 e 3,03, respectivamente. O extrato enzimático parcialmente purificado mostrou um pH ótimo de 5,0 e uma temperatura ótima de 40°C. A atividade enzimática aumentou na presença de KCl em todas as concentrações estudadas. A atividade da glucanase foi maior na presença de NaCl 0,2 M. A enzima apresentou alta estabilidade térmica, perdendo apenas 10,20 por cento de sua atividade específica após 40 minutos de incubação a 90°C. Os resultados de termoestabilidade e a atividade em baixo pH mostraram que a enzima glucanase de M. perniciosa tem características promissoras para futuras aplicações industriais.

Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches' Broom disease / Leveduras micocinogênicas inibem o crescimento do fitopatógeno Moniliophthora perniciosa, o agente causador da doença Vassoura-de-bruxa

Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth.

Leveduras de frutas e de solo isoladas da Floresta Amazônica, Mata Atlântica e de uma fazenda orgânica foram selecionadas em uma triagem para atividade micocinogênica. As estirpes micocinogênicas foram posteriormente testadas frente a Moniliophthora perniciosa (syn. Crinipellis perniciosa). Uma estirpe de Dipodascus capitatus e outra de Candida sp.inibiram o crescimento deste fitopatógeno.

Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches' Broom disease.

Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth.