Basidiomycete Moniliophthora Perniciosa Research Articles

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.

Expression of the Theobroma cacao Bax-inhibitor-1 gene in tomato reduces infection by the hemibiotrophic pathogen Moniliophthora perniciosa.

Programmed cell death (PCD) plays a key role in plant responses to pathogens, determining the success of infection depending on the pathogen lifestyle and on which participant of the interaction triggers cell death. The hemibiotrophic basidiomycete Moniliophthora perniciosa is the causal agent of witches' broom disease of Theobroma cacao L. (cacao), a serious constraint for production in South America and the Caribbean. It has been hypothesized that M. perniciosa pathogenesis involves PCD, initially as a plant defence mechanism, which is diverted by the fungus to induce necrosis during the dikaryotic phase of the mycelia. Here, we evaluated whether the expression of a cacao anti-apoptotic gene would affect the incidence and severity of M. perniciosa infection using the 'Micro-Tom' (MT) tomato as a model. The cacao Bax-inhibitor-1 (TcBI-1) gene, encoding a putative basal attenuator of PCD, was constitutively expressed in MT to evaluate function. Transformants expressing TcBI-1, when treated with tunicamycin, an inducer of endoplasmic reticulum stress, showed a decrease in cell peroxidation. When the same transformants were inoculated with the necrotrophic fungal pathogens Sclerotinia sclerotiorum, Sclerotium rolfsii and Botrytis cinerea, a significant reduction in infection severity was observed, confirming TcBI-1 function. After inoculation with M. perniciosa, TcBI-1 transformant lines showed a significant reduction in disease incidence compared with MT. The overexpression of TcBI-1 appears to affect the ability of germinating spores to penetrate susceptible tissues, restoring part of the non-host resistance in MT against the S-biotype of M. perniciosa.

Protein profile and protein interaction network of Moniliophthora perniciosa basidiospores

BackgroundWitches’ broom, a disease caused by the basidiomycete Moniliophthora perniciosa, is considered to be the most important disease of the cocoa crop in Bahia, an area in the Brazilian Amazon, and also in the other countries where it is found. M. perniciosa germ tubes may penetrate into the host through intact or natural openings in the cuticle surface, in epidermis cell junctions, at the base of trichomes, or through the stomata. Despite its relevance to the fungal life cycle, basidiospore biology has not been extensively investigated. In this study, our goal was to optimize techniques for producing basidiospores for protein extraction, and to produce the first proteomics analysis map of ungerminated basidiospores. We then presented a protein interaction network by using Ustilago maydis as a model.ResultsThe average pileus area ranged from 17.35 to 211.24 mm2. The minimum and maximum productivity were 23,200 and 6,666,667 basidiospores per basidiome, respectively. The protein yield in micrograms per million basidiospores were approximately 0.161; 2.307, and 3.582 for germination times of 0, 2, and 4 h after germination, respectively. A total of 178 proteins were identified through mass spectrometry. These proteins were classified according to their molecular function and their involvement in biological processes such as cellular energy production, oxidative metabolism, stress, protein synthesis, and protein folding. Furthermore, to better understand the expression pattern, signaling, and interaction events of spore proteins, we presented an interaction network using orthologous proteins from Ustilago maydis as a model. Most of the orthologous proteins that were identified in this study were not clustered in the network, but several of them play a very important role in hypha development and branching.ConclusionsThe quantities of basidiospores 7 × 109; 5.2 × 108, and 6.7 × 108 were sufficient to obtain enough protein mass for the three 2D-PAGE replicates, for the 0, 2, and 4 h-treatments, respectively. The protein extraction method that is based on sedimentation, followed by sonication with SDS-dense buffer, and phenolic extraction, which was utilized in this study, was effective, presenting a satisfactory resolution and reproducibility for M. perniciosa basidiospores. This report constitutes the first comprehensive study of protein expression during the ungerminated stage of the M. perniciosa basidiospore. Identification of the spots observed in the reference gel enabled us to know the main molecular interactions involved in the initial metabolic processes of fungal development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0753-0) contains supplementary material, which is available to authorized users.

Differential expression of jasmonate biosynthesis genes in cacao genotypes contrasting for resistance against Moniliophthora perniciosa.

The resistance mechanism of cacao against M. perniciosa is likely to be mediated by JA/ET-signaling pathways due to the preferential TcAOS and TcSAM induction in a resistant genotype. The basidiomycete Moniliophthora perniciosa causes a serious disease in cacao (Theobroma cacao L.), and the use of resistant varieties is the only sustainable long-term solution. Cacao resistance against M. perniciosa is characterized by pathogen growth inhibition with reduced colonization and an attenuation of disease symptoms, suggesting a regulation by jasmonate (JA)/ethylene (ET) signaling pathways. The hypothesis that genes involved in JA biosynthesis would be active in the interaction of T. cacao and M. perniciosa was tested here. The cacao JA-related genes were evaluated for their relative quantitative expression in susceptible and resistant genotypes upon the exogenous application of ET, methyl-jasmonate (MJ), and salicylic acid (SA), or after M. perniciosa inoculation. MJ treatment triggered changes in the expression of genes involved in JA biosynthesis, indicating that the mechanism of positive regulation by exogenous MJ application occurs in cacao. However, a higher induction of these genes was observed in the susceptible genotype. Further, a contrast in JA-related transcriptional expression was detected between susceptible and resistant plants under M. perniciosa infection, with the induction of the allene oxide synthase gene (TcAOS), which encodes a key enzyme in the JA biosynthesis pathway in the resistant genotype. Altogether, this work provides additional evidences that the JA-dependent signaling pathway is modulating the defense response against M. perniciosa in a cacao-resistant genotype.

Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene.

Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet–C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.

The infection process of Moniliophthora perniciosa in cacao

The development of the basidiomycete Moniliophthora perniciosa in resistant and susceptible Theobroma cacao genotypes was analysed. The infection process leading to broom formation in shoot apexes was characterized by studying the kinetics of basidiospore germination, mode of penetration and colonization of the pathogen. Both resistant and susceptible cacao genotypes were inoculated with M. perniciosa and kept in the greenhouse for 90 days, explants were collected, treated for histological studies and meristematic tissues were observed by electron and light microscopy. Variation in the kinetics of germination between the cacao genotypes was detected 4 h after inoculation. The fungal penetration occurred through the star‐shaped trichome base, natural openings on the cuticular surface and stomata. Host responses between genotypes were found to be different. Compared with non‐infected plants, the swelling of all the stem tissues was evident at 60 days after inoculation. In the susceptible genotype, typical symptoms developed and fungal colonization was more intense than in resistant genotypes, which showed little or no fungal colonization. The investigations reported herein provide an important step in understanding the pattern of pre‐ and post‐penetration events of M. perniciosa in cacao genotypes with different levels of resistance to this disease.

Antimicrobial activity of fermented Theobroma cacao pod husk extract.

Theobroma cacao L. contains more than 500 different chemical compounds some of which have been traditionally used for their antioxidant, anti-carcinogenic, immunomodulatory, vasodilatory, analgesic, and antimicrobial activities. Spontaneous aerobic fermentation of cacao husks yields a crude husk extract (CHE) with antimicrobial activity. CHE was fractioned by solvent partition with polar solvent extraction or by silica gel chromatography and a total of 12 sub-fractions were analyzed for chemical composition and bioactivity. CHE was effective against the yeast Saccharomyces cerevisiae and the basidiomycete Moniliophthora perniciosa. Antibacterial activity was determined using 6 strains: Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis (Gram-positive) and Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella choleraesuis (Gram-negative). At doses up to 10 mg/mL, CHE was not effective against the Gram-positive bacteria tested but against medically important P. aeruginosa and S. choleraesuis with a minimum inhibitory concentration (MIC) of 5.0 mg/mL. Sub-fractions varied widely in activity and strongest antibacterial activity was seen with CHE8 against S. choleraesuis (MIC of 1.0 mg/mL) and CHE9 against S. epidermidis (MIC of 2.5 mg/mL). All bioactive CHE fractions contained phenols, steroids, or terpenes, but no saponins. Fraction CHE9 contained flavonoids, phenolics, steroids, and terpenes, amino acids, and alkaloids, while CHE12 had the same compounds but lacked flavonoids.

Activity of polygalacturonases from Moniliophthora perniciosa depends on fungus culture conditions and is enhanced by Theobroma cacao extracts

We report the first analysis of polygalacturonase regulation in the basidiomycete Moniliophthora perniciosa. Non-secreted and secreted polygalacturonase activity was obtained from M. perniciosa cultivated on bran-based solid medium or liquid media containing additional carbon sources or cacao extracts (infected or not by the fungus). Polygalacturonase activity assays were carried out under different temperatures and incubation periods. The best secreted polygalacturonase activity was obtained when the enzymatic assay was made at 50 °C for 10 min. Moreover, the polygalacturonase activity was enhanced when the fungus was cultivated on potato dextrose medium, in the presence of additional fermentable carbon sources, in the presence of cacao pulp or non infected cacao extracts.

Biologia de Sistemas de Pectinases do Fungo Moniliophthora perniciosa

Pectinases (pectin methylesterases [PMEs], polygalacturonases [PGs] and protopectinases) are enzymes involved in the degradation of the pectic molecule, which is the main component of the plant middle lamella. Phytopathogens produce such enzymes, which are very important for their virulence. The witches' broom disease, caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa, has committed the cacao production in South Bahia, Brazil. Moniliophthora perniciosa genome sequencing revealed the presence of homologous of pectinase genes. The objective of this work was to obtain a physical protein-protein interaction network by systems biology, generated from a M. perniciosa polygalacturonase (MpPG2). The search for Neurospora crassa MpPG2 ortholog was performed using the reciprocal Blast. The network was obtained in http://string-db.org/ with the NCU02369 ortholog and presented 683 nodes. For the network study, we used the Cytoscape software in which the following analyses were made: i) centrality which revealed 30 bottlenecks including the MpPG2, 149 hubs and 142 hubs-bottleneck; ii) modularity which allowed the identification of 6 clusters; and iii) gene ontology. The NCU10045 is a pectinesterase which interacts in the network with NCU02369-MpPG2; it was also related to other proteins such as the NCU05063 glycosyl hydrolase or with the NCU06326.1 pectate lyase-1. Thus, it was possible to suggest that pectinases and hydrolases were found in the network and interacted directly or indirectly with NCU02369-MpPG2 forming a set of enzymatic activity that may be crucial for host invasion. (Resume d'auteur)

The interaction of Theobroma cacao and Moniliophthora perniciosa, the causal agent of witches’ broom disease, during parthenocarpy

Witches’ broom disease of Theobroma cacao L. is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa. Infection of flower cushions by M. perniciosa results in parthenocarpy. Healthy and parthenocarpic immature cacao pods were obtained from seven cacao clones. Microscopic observations of parthenocarpic pods from two clones confirmed that fruits lack viable seed. Septate mycelia colonized parthenocarpic pods, but were absent from healthy pods. Parthenocarpic pods had increased concentrations of leucine, methionine, serine, phenylalanine, and valine. Major transport metabolites sucrose and asparagine were decreased by 63 and 40 %, respectively, during parthenocarpy. M. perniciosa expressed sequence tags (ESTs) related to detoxification (MpSOD2 and MpCTA1) and nutrient acquisition (MpAS, MpAK, MpATG8, MpPLY, and MpPME) were induced in parthenocarpic pods. Most M. perniciosa ESTs related to plant hormone biosynthesis were repressed (MpGAox, MpCPS, MpDES, MpGGPPS, and MpCAO) in parthenocarpic pods. RT-qPCR analysis was conducted for 54 defense-related cacao ESTs and 93 hormone-related cacao ESTs. Specific cacao ESTs related to plant defense were induced (TcPR5, TcChi4, TcThau-ICS) while others were repressed (TcPR1, TcPR6, TcP12, and TcChiB). Cacao ESTs related to GA biosynthesis (TcGA20OX1B) were repressed in parthenocarpic pods. Cacao ESTs putatively related to maintaining cytokinin (TcCKX3 and TcCKX5) and IAA (TcGH3.17a, TcGH3.1, TcARF18) homeostasis were induced in parthenocarpic pods, suggesting an attempt to regulate cytokinin and auxin concentrations. In conclusion, M. perniciosa expresses specific sets of transcripts targeting nutrient acquisition and survival while altering the host physiology without causing significant necrosis resulting in parthenocarpy. Only a general host defense response is elicited.

Evaluation of Trichoderma spp for the biocontrol of Moniliophthora perniciosa Subgroup 1441

Trichoderma species , isolated from different producer regions of cocoa (Bahia, Brazil), were evaluated as for their capacity of usage in the biocontrol of the basidiomycete Moniliophthora perniciosa subgroup 1441, which causes the witches’ broom in cocoa. The isolates of Trichoderma were evaluated through individual indices so called %AP (Antagonistic Potential to Moniliophthora perniciosa subgroup 1441), %PG (Potential Growth in vitro) and %PSPr (Potential of Spore Production on rice) These indices were evaluated together, also they were used for the determination of Biological Control Potential (%BCP) of each antagonistic specie to the evaluated pathogen. Afterwards, the ability of the antagonistic to colonize and to produce spores on sterilized dry brooms was also evaluated. Some of the isolates Trichoderma spp showed a high %AP to the pathogen and high %PG, but did not present a significant %PSPr, turning impossible the spore production for biocontrol at commercial level. Significant differences were found within the individual indices among the species and isolates of the same species of Trichoderma spp, pointing out a great genetic variability among them. Trichoderma harzianum 911 showed to have the best biocontrol potential to the pathogen when compared to the other isolates, presenting a %BCP de 91.86% (mainly by the high %AP of 97,76%) a %PSPr of 99.53%, also producing 22.67 spores x 109. mL-1 by dry broom segment. Trichoderma harzianum 911 showed to be as promising isolate for future researches on biocontrol of cocoa witches’ broom.

Searching for Moniliophthora perniciosa pathogenicity genes

The basidiomycete Moniliophthora perniciosa is the causal agent of witches’ broom disease of Theobroma cacao (cacao). Pathogenesis mechanisms of this hemibiotrophic fungus are largely unknown. An approach to identify putative pathogenicity genes is searching for sequences induced in mycelia grown under in vitro conditions. Using this approach, genes from M. perniciosa induced under limiting nitrogen and light were identified from a cDNA library enriched by suppression subtractive hybridization as potential putative pathogenicity genes. From the 159 identified unique sequences, 59 were annotated and classified by gene ontology. Two sequences were categorized as “Defence genes, Virulence, and Cell response” presumably coding for allergenic proteins, whose homologues from other fungi are inducers of animal or plant defences. Differential gene expression was evaluated by quantitative amplification of reversed transcripts (RT-qPCR) of the putative identified genes coding for the two allergenic proteins (Aspf13 and 88KD), and for the enzymes Arylsulfatase (AS); Aryl-Alcohol Oxidase; Aldo-Keto Reductase (AK); Cytochrome P450 (P450); Phenylalanine Ammonia-Lyase; and Peroxidase from mycelia grown under contrasting N concentrations. All genes were validated for differential expression, except for the putative Peroxidase. The same eight genes were analysed for expression in susceptible plants inoculated with M. perniciosa, and six were induced during the early asymptomatic stage of the disease. In infected host tissues, transcripts of 88KD and AS were found more abundant at the biotrophic phase, while those from Aspf13, AK, PAL, and P450 accumulated at the necrotrophic phase, enabling to suggest that mycelia transition from biotrophic to necrotrophic might occur earlier than currently considered. These sequences appeared to be virulence life-style genes, which encode factors or enzymes that enable invasion, colonization or intracellular survival, or manipulate host factors to benefit the pathogen’s own survival in the hostile environment.

Reactive oxygen species and autophagy play a role in survival and differentiation of the phytopathogen Moniliophthora perniciosa

The hemibiotrophic basidiomycete Moniliophthora perniciosa causes “witches’ broom disease” in cacao ( Theobroma cacao). During plant infection, M. perniciosa changes from mono to dikaryotic life form, an event which could be triggered by changes in plant nutritional offer and plant defense molecules, i.e., from high to low content of glycerol and hydrogen peroxide. We have recently shown that in vitro glycerol induces oxidative stress resistance in dikaryotic M. perniciosa. In order to understand under which conditions in parasite-plant interaction M. perniciosa changes from intercellular monokaryotic to intracellular dikaryotic growth phase we studied the role of glycerol on mutagen-induced oxidative stress resistance of basidiospores and monokaryotic hyphae; we also studied the role of H 2O 2 as a signaling molecule for in vitro dikaryotization and whether changes in nutritional offer by the plant could be compensated by inducible fungal autophagy. Mono-/dikaryotic glycerol or glucose-grown cells and basidiospores were exposed to the oxidative stress-inducing mutagens H 2O 2 and Paraquat as well as to pre-dominantly DNA damaging 4-nitroquinoline-1-oxide and UVC irradiation. Basidiospores showed highest resistance to all treatments and glycerol-grown monokaryotic hyphae were more resistant than dikaryotic hyphae. Monokaryotic cells exposed to 1 μM of H 2O 2 in glycerol-media induced formation of clamp connections within 2 days while 1 mM H 2O 2 did not within a week in the same medium; no clamp connections were formed in H 2O 2-containing glucose media within a week. Lower concentrations of H 2O 2 and glycerol, when occurring in parallel, are shown to be two signals for dikaryotization in vitro and may be also during the course of infection. Q-PCR studies of glycerol-grown dikaryotic cells exposed to oxidative stress (10 mM H 2O 2) showed high expression of Mp SOD2 and transient induction of ABC cytoplasmic membrane transporter gene Mp YOR1 and autophagy-related gene Mp ATG8. Expression of a second ABC transporter gene Mp SNQ2 was 14-fold induced after H 2O 2 exposure in glucose as compared to glycerol-grown hyphae while Mp YOR1 did not show strong variation of expression under similar conditions. Glucose-grown dikaryotic cells showed elevated expression of Mp ATG8, especially after exposure to H 2O 2 and 4-nitroquinoline-1-oxide. During different stages preceding basidiocarp formation Mp ATG8 and the two catalase-encoding genes Mp CTA1 and Mp CTT1 were expressed continuously. We have compiled our results and literature data in a model graph, which compares the in vitro and in planta development and differentiation of M. perniciosa with the help of physiological and morphological landmarks.

Identification of a second family of genes in Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao, encoding necrosis-inducing proteins similar to cerato-platanins

The hemibiotrophic basidiomycete Moniliophthora perniciosa is the causal agent of witches' broom disease in cacao. This is a dimorphic species, with monokaryotic hyphae during the biotrophic phase, which is converted to dikaryotic mycelia during the saprophytic phase. The infection in pod is characterized by the formation of hypertrophic and hyperplasic tissues in the biotrophic phase, which is followed by necrosis and complete degradation of the organ. We found at least five sequences in the fungal genome encoding putative proteins similar to cerato-platanin (CP)-like proteins, a novel class of proteins initially found in the phytopathogen Ceratocystis fimbriata. One M. perniciosa CP gene (MpCP1) was expressed in vitro and proved to have necrosis-inducing ability in tobacco and cacao leaves. The protein is present in solution as dimers and is able to recover necrosis activity after heat treatment. Transcription analysis ex planta showed that MpCP1 is more expressed in biotrophic-like mycelia than saprotrophic mycelia. The necrosis profile presented is different from that caused by M. perniciosa necrosis and ethylene-inducing proteins (MpNEPs), another family of elicitors expressed by M. perniciosa. Remarkably, a mixture of MpCP1 with MpNEP2 led to a synergistic necrosis effect very similar to that found in naturally infected plants. This is the first report of a basidiomycete presenting both NEP1-like proteins (NLPs) and CPs in its genome.

The mitochondrial genome of the phytopathogenic basidiomycete Moniliophthora perniciosa is 109 kb in size and contains a stable integrated plasmid

We present here the sequence of the mitochondrial genome of the basidiomycete phytopathogenic hemibiotrophic fungus Moniliophthora perniciosa, causal agent of the Witches' Broom Disease in Theobroma cacao. The DNA is a circular molecule of 109 103 base pairs, with 31.9 % GC, and is the largest sequenced so far. This size is due essentially to the presence of numerous non-conserved hypothetical ORFs. It contains the 14 genes coding for proteins involved in the oxidative phosphorylation, the two rRNA genes, one ORF coding for a ribosomal protein ( rps3), and a set of 26 tRNA genes that recognize codons for all amino acids. Seven homing endonucleases are located inside introns. Except atp8, all conserved known genes are in the same orientation. Phylogenetic analysis based on the cox genes agrees with the commonly accepted fungal taxonomy. An uncommon feature of this mitochondrial genome is the presence of a region that contains a set of four, relatively small, nested, inverted repeats enclosing two genes coding for polymerases with an invertron-type structure and three conserved hypothetical genes interpreted as the stable integration of a mitochondrial linear plasmid. The integration of this plasmid seems to be a recent evolutionary event that could have implications in fungal biology. This sequence is available under GenBank accession number AY376688.

Carbon source-dependent variation of acquired mutagen resistance of Moniliophthora perniciosa: Similarities in natural and artificial systems

The basidiomycete Moniliophthora perniciosa causes Witches’ Broom disease in Theobroma cacao. We studied the influence of carbon source on conditioning hyphae to oxidative stress agents (H 2O 2, paraquat, 4NQO) and to UVC, toward the goal of assessing the ability of this pathogen to avoid plant defenses involving ROS. Cells exhibited increased resistance to H 2O 2 when shifted from glucose to glycerol and from glycerol to glycerol. When exposed to paraquat, cells grown in fresh medium were always more resistant. Apparently glycerol and/or fresh media, but not old glucose media, up-regulate oxidative stress defenses in this fungus. For the mutagens UVC and 4NQO, whose prime action on DNA is not via ROS, change of carbon source did not elicit a clear change in sensitivity/resistance. These results correlate with expression of fungal genes that protect against ROS and with biochemical changes observed in infected cacao tissues, where glycerol and high amounts of ROS have been detected in green brooms.

Production of Calcium Oxalate Crystals by the Basidiomycete Moniliophthora perniciosa, the Causal Agent of Witches’ Broom Disease of Cacao

Oxalic acid has been shown as a virulence factor for some phytopathogenic fungi, removing calcium from pectin and favoring plant cell wall degradation. Recently, it was published that calcium oxalate accumulates in infected cacao tissues during the progression of Witches' Broom disease (WBD). In the present work we report that the hemibiotrophic basidiomycete Moniliophthora perniciosa, the causal agent of WBD, produces calcium oxalate crystals. These crystals were initially observed by polarized light microscopy of hyphae growing on a glass slide, apparently being secreted from the cells. The analysis was refined by Scanning electron microscopy and the compositon of the crystals was confirmed by energy-dispersive x-ray spectrometry. The production of oxalate by M. perniciosa was reinforced by the identification of a putative gene coding for oxaloacetate acetylhydrolase, which catalyzes the hydrolysis of oxaloacetate to oxalate and acetate. This gene was shown to be expressed in the biotrophic-like mycelia, which in planta occupy the intercellular middle-lamella space, a region filled with pectin. Taken together, our results suggest that oxalate production by M. perniciosa may play a role in the WBD pathogenesis mechanism.

Use of response surface methodology to examine chitinase regulation in the basidiomycete Moniliophthora perniciosa

We report here the first analysis of chitinase regulation in Moniliophthora perniciosa, the causal agent of the witches' broom disease of cacao. A multivariate statistical approach was employed to evaluate the effect of several variables, including carbon and nitrogen sources and cultivation time, on M. perniciosa non-secreted (detected in mycelium, i.e. in symplasm and cell wall) and secreted (detected in the culture medium) chitinase activities. Non-secreted chitinase activity was enhanced by peptone and chitin and repressed by glucose. Chitinase secretion was increased by yeast extract alone or in combination with other nitrogen sources, and by N-acetylglucosamine, and repressed in presence of chitin. The best cultivation times for non-secreted and secreted chitinase activities were 30 and 20 d, respectively. However, chitinase activity was always higher in the mycelium than in the culture medium, suggesting a relatively poor chitinase secretion activity. Conversely, higher mycelial growth was observed when the activity of the non-secreted chitinase was at its lowest, i.e. when the fungus was grown on glucose and yeast extract as sources of carbon and nitrogen, respectively. Conversely, the induction of non-secreted chitinase activity by chitin decreased the mycelium growth. These results suggest that the culture medium, by the induction or repression of chitinases, affected the hyphal growth. Thus, as an essential component of M. perniciosa growth, chitinases may be a potential target for strategies to control disease.

Comparative Analysis of Expressed Genes from Cacao Meristems Infected by Moniliophthora perniciosa

Witches' broom disease is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa, and is one of the most important diseases of cacao in the western hemisphere. Because very little is known about the global process of such disease development, expressed sequence tags (ESTs) were used to identify genes expressed during the Theobroma cacao-Moniliophthora perniciosa interaction. Two cDNA libraries corresponding to the resistant (RT) and susceptible (SP) cacao-M. perniciosa interactions were constructed from total RNA, using the DB SMART Creator cDNA library kit (Clontech). Clones were randomly selected, sequenced from the 5' end and analysed using bioinformatics tools including in silico analysis of the differential gene expression. A total of 6884 ESTs were generated from the RT and SP cDNA libraries. These ESTs were composed of 2585 singlets and 341 contigs for a total of 2926 non-redundant sequences. The redundancy of the libraries was low and their specificity high when compared with the few other cacao libraries already published. Sequence analysis allowed the assignment of a putative functional category for 54 % of sequences, whereas approx. 22 % of sequences corresponded to unknown function and approx. 24 % of sequences did not show any significant similarity with other proteins present in the database. Despite the similar overall distribution of the sequences in functional categories between the two libraries, qualitative differences were observed. Genes involved during the defence response to pathogen infection or in programmed cell death were identified, such as pathogenesis related-proteins, trypsin inhibitor or oxalate oxidase, and some of them showed an in silico differential expression between the resistant and the susceptible interactions. As far as is known this is the first EST resource from the cacao-M. perniciosa interaction and it is believed that it will provide a significant contribution to the understanding of the molecular mechanisms of the resistance and susceptibility of cacao to M. perniciosa, to develop strategies to control witches' broom, and as a source of polymorphism for molecular marker development and marker-assisted selection.

Characterization of necrosis and ethylene-inducing proteins (NEP) in the basidiomycete Moniliophthora perniciosa, the causal agent of witches' broom in Theobroma cacao

The hemibiotrophic basidiomycete Moniliophthora perniciosa causes witches' broom disease of Theobroma cacao. Analysis of the M. perniciosa draft genome led to the identification of three putative genes encoding necrosis and ethylene-inducing proteins (MpNEPs), which are apparently located on the same chromosome. MpNEP1 and 2 have highly similar sequences and are able to induce necrosis and ethylene emission in tobacco and cacao leaves. MpNEP1 is expressed in both biotrophic and saprotrophic mycelia, the protein behaves as an oligomer in solution and is very sensitive to temperature. MpNEP2 is expressed mainly in biotrophic mycelia, is present as a monomer in solution at low concentrations (<40 μ m) and is able to recover necrosis activity after boiling. These differences indicate that similar NEPs can have distinct physical characteristics and suggest possible complementary roles during the disease development for both proteins. This is the first report of NEP1-like proteins in a basidiomycete.