A repertoire of candidate effector proteins of the fungus Ceratocystis cacaofunesta.

The genus Ceratocystis includes many phytopathogenic fungi that affect different plant species. One of these is Ceratocystis cacaofunesta, which is pathogenic to the cocoa tree and causes Ceratocystis wilt, a lethal disease for the crop. However, little is known about how this pathogen interacts with its host. The knowledge and identification of possible genes encoding effector proteins are essential to understanding this pathosystem. The present work aimed to predict genes that code effector proteins of C. cacaofunesta from a comparative analysis of the genomes of five Ceratocystis species available in databases. We performed a new genome annotation through an in-silico analysis. We analyzed the secretome and effectorome of C. cacaofunesta using the characteristics of the peptides, such as the presence of signal peptide for secretion, absence of transmembrane domain, and richness of cysteine residues. We identified 160 candidate effector proteins in the C. cacaofunesta proteome that could be classified as cytoplasmic (102) or apoplastic (58). Of the total number of candidate effector proteins, 146 were expressed, presenting an average of 206.56 transcripts per million. Our database was created using a robust bioinformatics strategy, followed by manual curation, generating information on pathogenicity-related genes involved in plant interactions, including CAZymes, hydrolases, lyases, and oxidoreductases. Comparing proteins already characterized as effectors in Sordariomycetes species revealed five groups of protein sequences homologous to C. cacaofunesta. These data provide a valuable resource for studying the infection mechanisms of these pathogens in their hosts.

Chromosomal polymorphism of the Ceratocystis fimbriata species complex in Brazil.

Ceratocystis fimbriata is an important pathogen that causes wilt in several plant species. Despite the importance of this pathogen, knowledge about its karyotypic polymorphism and genomic architecture is limited. The main objective of this study was to investigate the karyotype of isolates of the C. fimbriata species complex from different host plants and geographical origins in Brazil. First, the identity of the isolates was confirmed conducting multilocus sequence analysis (MLSA) phylogeny using ß-tubulin (TUBB), translation elongation factor 1α (TEF-1α) and mating-type (MAT1 and MAT2) gene sequences. To investigate the chromosomal polymorphism, two conditions of pulsed-field gel electrophoresis (PFGE) were used and the karyotypes of the isolates obtained. The retrotransposon-microsatellite amplified polymorphism (REMAP) molecular marker was utilized to assess the genetic variability among isolates. In the MLSA utilizing the concatenated gene sequences, Ceratocystis cacaofunesta and C. fimbriata formed separate clades, but considerable variation among C. fimbriata isolates was observed. Polymorphism in chromosome number and size was found, indicating the existence of genomic differences among isolates and occurrence of chromosomal rearrangements in the species complex. The number of chromosomes varied from seven to nine and the estimated minimum chromosome sizes were estimated to be between 2.7 and 6.0 Mbp. Small polymorphic chromosomes ranging from 1.2 to 1.8 Mbp were observed in all isolates, raising the hypothesis that they could be supernumerary chromosomes. REMAP analysis revealed a high genetic variability and that isolates from the same host tend to group together in a same cluster. Our results bring new insights into the chromosomal diversity and genome organization of the C. fimbriata complex.

Ceratocystis cacaofunesta differentially modulates the proteome in xylem-enriched tissue of cocoa genotypes with contrasting resistance to Ceratocystis wilt.

MAIN CONCLUSION: Decreased accumulation of polyphenol oxidase, H2O2 accumulation, effective regulation of programmed cell death, and a protein predicted as allergenic can play key roles in cacao defense against Ceratocystis cacaofunesta. Ceratocystis wilt, caused by the fungus Ceratocystis cacaofunesta, has destroyed millions of Theobroma cacao trees in several countries of the Americas. Through proteomics, systems biology, and enzymatic analyses of infected stems, it was possible to infer mechanisms used by resistant (TSH1188) and susceptible (CCN51) cacao genotypes during infection. Protein extraction from xylem-enriched tissue of stems inoculated with the fungus and their controls 1 day after inoculation was carried out, followed by separation through two-dimensional gel electrophoresis and identification by mass spectrometry. Enzyme activity was determined at 1, 3, 7 and 15 days after inoculation. A total of 50 differentially accumulated distinct proteins were identified in the treatments of both genotypes and were classified into 10 different categories. An interaction network between homologous proteins from Arabidospsis thaliana was generated for each genotype, using the STRING database and Cytoscape software. Primary metabolism processes were apparently repressed in both genotypes. The resistance factors suggested for genotype TSH1188 were: H2O2 accumulation, effective regulation of programmed cell death, production of phytoalexins derived from tryptophan and furanocoumarins, and participation of a predicted allergenic protein with probable ribonuclease function inhibiting the germination and propagation of the fungus. In the susceptible genotype, it is possible that its recognition and signaling mechanism through proteins from the SEC14 family is easily overcome by the pathogen. Our results will help to better understand the interaction between cacao and one of its most aggressive pathogens, to create disease control strategies.

Metabolite mass spectrometry profiling of cacao genotypes reveals contrasting resistances to Ceratocystis cacaofunesta phytopathogen.

Ceratocystis wilt is a lethal disease of cacao, and the search for resistant genotypes may provide the best way to deal with the disease. Resistance or susceptibility behavior of some cacao genotypes when infected by Ceratocystis cacaofunesta is not yet understood. Herein, we report an LC-MS metabolomic screening analysis based on high-resolution MS to obtain comprehensive metabolic profile associated with multivariate data analysis of PLS-DA, which was effective to classify CCN-51 and TSH-1188 as resistant genotypes to C. cacaofunesta fungus, while CEPEC2002 was classified as a susceptible one. Using reversed-phase LC method, electrospray interface, and high-resolution tandem MS by the quadrupole-TOF analyzer, the typical profiles of metabolites, such as phenylpropanoids, flavonoids, lipids, alkaloids, and amino acids, were obtained. Untargeted metabolite profiles were used to construct discriminant analysis by partial least squares (PLS-DA)-derived loading plots, which placed the cacao genotypes into two major clusters related to susceptible or resistant groups. Linolenic, linoleic, oleic, stearic, arachidonic, and asiatic acids were annotated metabolites of infected, susceptible, and resistant genotypes, while methyl jasmonate, jasmonic acid, hydroxylated jasmonic acid, caffeine, and theobromine were annotated as constituents of the resistant genotypes. Trends of these typical metabolites levels revealed that CCN51 is susceptible, CEPEC2002 is moderately susceptible, and TSH1188 is resistant to C. cacaofunesta. Therefore, profiles of major metabolites as screened by LC-MS offer an efficient tool to reveal the level of resistance of cacao genotypes to C. cacaofunesta present in any farm around the world.

Ceratocystis fimbriata agente causal de la enfermedad de marchitez vascular de Tectona grandis L.f. (teca) en Ecuador / Ceratocystis fimbriata causative agent of vascular wilt disease of Tectona grandis L.f. (teak) in Ecuador

Tectona grandis L.f. es una especie forestal de madera dura que, a pesar de no ser nativa de América, su plasticidad de adaptación ha permitido que en Ecuador se establezcan plantaciones intensivas con fines de exportación. Una compleja enfermedad con características de marchitez vascular está matando miles de árboles de diferentes edades en el país. Se planteó conocer el agente fitopatógeno causante de la patogénesis y muerte de los árboles. Se aislaron los hongos Fusarium sp. y Ceratocystis fimbriata Ellis & Halst. desde árboles enfermos. A nivel de invernadero se plantearon los postulados de Koch, para el efecto 30 plantas de T. grandis de cuatro meses de edad por cada tratamiento, se inocularon con los microrganismos como se describe a continuación: T1= C. fimbriata, T2 = Fusarium sp., T3 = C. fimbriata + Fusarium sp., T4 = agar-agar (control). Se empleó un diseño completo al azar (DCA) y las plantas se evaluaron a los 45 días después de inoculadas. Los tratamientos C. fimbriata, y C. fimbriata + Fusarium sp., mostraron mayores volúmenes aparentes de necrosis, con 1.52 cm3 y 1.93 cm3, y plantas muertas por la infección durante el tiempo de evaluación, mientras que Fusarium sp. mostró baja o nula patogenicidad y comportamiento similar al control, con 0.27 cm3 y 0.16 cm3 respectivamente. Estos resultados sugieren que el agente causal de la enfermedad de marchitez vascular en T. grandis es C. fimbriata y sería el primer reporte del fitopatógeno atacando teca en Ecuador. (AU)

Tectona grandisL.f. it is a hardwood forest species, which despite not being native to America, its adaptive plasticity has allowed intensive plantations to be established for export purposes in Ecuador. A complex disease with characteristics of vascular wilt is killing thousands of trees of different ages in the country. It was proposed to know the phytopathogenic agent causing the pathogenesis and death of the trees. Fusarium sp. andCeratocystis fimbriata Ellis & Halst. were isolated from diseased trees. At the greenhouselevel, Koch's postulates were proposed, for the effect 30 four month old T. grandisplants for each treatment were inoculated with the microorganisms as described below: T1 = C. fimbriata, T2 = Fusariumsp ., T3 = C. fimbriata+ Fusariumsp., T4 = agar-agar (control). A complete randomized design (DCA) was used and the plants were evaluated 45 days after inoculation. The treatments C. fimbriata, and C. fimbriata+ Fusariumsp. showed higher apparent volumes of necrosis, with 1.52 cm3and 1.93 cm3, and plants killed by the infection during the evaluation time, while Fusariumsp. showed low or no pathogenicity and behavior similar to the control, with 0.27 cm3and 0.16 cm3respectively. These results suggest that the causative agent of vascular wilt disease in T. grandisis C. fimbriataand it would be the first report of phytopathogen attacking teak in Ecuador. (AU)

Hongos fitopatógenos asociados a la enfermedad de marchitez vascular y muerte regresiva en plantaciones de Tectona grandis L. f. (teca) en el Trópico Húmedo Ecuatoriano / Phytopathogen fungi related to vascular wilt and die back in Tectona grandis L. f (teak) field in Ecuadorian Tropical Rainforest

Plantaciones de teca (Tectona grandis L.f.) en Ecuador están siendo afectadas por una compleja enfermedad de marchitez vascular y muerte regresiva, con características epidémicas, sin que hasta el momento se conozca el o los agentes causales. Se planteó describir la sintomatología de la enfermedad e identificar los hongos fitopatógenos asociados a árboles enfermos en el Trópico Húmedo Ecuatoriano mediante morfofisiología. Se seleccionaron tres plantaciones de 2, 5 y 7 años de edad, en cada una se delimitó tres parcelas de 500 m2. Se realizó la descripción sintomatológica, evaluó la incidencia y severidad de la enfermedad empleando una escala de cinco categorías. Por parcela se diseccionaron tres árboles, cuyos tejidos se llevaron al laboratorio, donde se emplearon tres estrategias metodológicas (cámara húmeda, sandwiches de zanahoria, y medio de cultivo papa-dextrosa-agar; PDA) para estimular la expresión de los fitopatógenos. En árboles enfermos se detectó clorosis, pérdida de turgencia, ápices de crecimiento secos, emisión de brotes epicormicos en el fuste, y marchitez fulminante. Se aisló e identificó Ceratocystis fimbriata Ellis & Halst., y especies de Fusarium de forma consecutiva con las tres estrategias metodológicas empleadas. La incidencia de la enfermedad fue del 16.6%, 15.2%, y 11.6% para las plantaciones de 2, 5 y 7 años, respectivamente. Los árboles enfermos en la plantación de 2 años se encontraron en las escalas 2, 4 y 5, mientras que en plantaciones de 5 y 7 años se ubicaron en las escalas 2, 3 y 5 de progreso de la enfermedad...(AU)

Teak plantations (Tectona grandis L. f.) in Ecuador are being affected by a complex disease of vascular wilt and dieback, with epidemic characteristics, without knowing the causal agent(s) so far. We proposed to describe the symptomatology of the disease and identify phytopathogenic fungi associated with diseased trees in the Ecuadorian Humid Tropic by morphophysiology. Three plantations of 2, 5 and 7 years of age were selected, in each three plots of 500 m2 were delimited. The symptomatologic description was made, evaluated the incidence and severity of the disease using a scale of five categories. By plot, three trees were dissected, whose tissues were taken to the laboratory, where three methodological strategies were used (wet chamber, carrot sandwiches, and potatodextrose-agar culture medium, PDA) to stimulate the expression of phytopathogens. In diseased trees, chlorosis, turgor loss, dry growth apices, emission of epicormic shoots in the stem, and fulminating wilt were detected. It was isolated and identified Ceratocystis fimbriata Ellis & Halst., and Fusarium species. consecutively with the three methodological strategies employed. The incidence of the disease was 16.6%, 15.2%, and 11.6% for plantations of 2, 5 and 7 years, respectively. The sick trees in the plantation of 2 years were found in scales 2, 4 and 5, while in plantations of 5 and 7 years they were located in scales 2, 3 and 5 of progress of the disease. ..(AU)