The Sigatoka Disease Complex Caused by Pseudocercospora spp. and Other Fungal Pathogens Associated with Musa spp. in Puerto Rico.

Bananas (Musa spp.) are among the world's most economically important staple food crops. The most important fungal leaf diseases of Musa spp. worldwide are caused by the Sigatoka disease complex, which comprise black Sigatoka (Pseudocercospora fijiensis), yellow Sigatoka (P. musae), and Eumusae leaf spot (P. eumusae). Considering the rapid spreading rate of black Sigatoka in Puerto Rico after its first observation in 2004, a disease survey was conducted from 2018 to 2020 to evaluate the Sigatoka disease complex on the island. Sixty-one leaf samples showing Sigatoka-like symptoms were collected throughout the island for diagnosis by molecular approaches and fungal isolation. Molecular analysis using species-specific primers for P. fijiensis, P. musae and P. eumusae detected the presence of P. fijiensis in fifty leaf samples. Thirty-eight fungal isolates were collected and identified by morphology and genomic sequencing from various nuclear genes. The analysis identified 24 isolates as P. fijiensis, while the rest of the isolates belonged to the genus Cladosporium spp. and Cladosporium-like spp. (n=5), Neocordana musae (n=2), Zasmidium spp. (n=6), and Z. musigenum (n=1). The high frequency of P. fijiensis found in leaf samples and collected isolates suggest that black Sigatoka has displaced the yellow Sigatoka (P. musae) in Puerto Rico. Accurate identification of fungal species causing foliar diseases in Musa spp. will allow the establishment of quarantine regulations and specific management approaches in Puerto Rico.

Pseudocercospora fijiensis Conidial Germination Is Dominated by Pathogenicity Factors and Effectors.

Conidia play a vital role in the survival and rapid spread of fungi. Many biological processes of conidia, such as adhesion, signal transduction, the regulation of oxidative stress, and autophagy, have been well studied. In contrast, the contribution of pathogenicity factors during the development of conidia in fungal phytopathogens has been poorly investigated. To date, few reports have centered on the pathogenicity functions of fungal phytopathogen conidia. Pseudocercospora fijiensis is a hemibiotrophic fungus and the causal agent of the black Sigatoka disease in bananas and plantains. Here, a conidial transcriptome of P. fijiensis was characterized computationally. Carbohydrates, amino acids, and lipid metabolisms presented the highest number of annotations in Gene Ontology. Common conidial functions were found, but interestingly, pathogenicity factors and effectors were also identified. Upon analysis of the resulting proteins against the Pathogen-Host Interaction (PHI) database, 754 hits were identified. WideEffHunter and EffHunter effector predictors identified 618 effectors, 265 of them were shared with the PHI database. A total of 1107 conidial functions devoted to pathogenesis were found after our analysis. Regarding the conidial effectorome, it was found to comprise 40 canonical and 578 non-canonical effectors. Effectorome characterization revealed that RXLR, LysM, and Y/F/WxC are the largest effector families in the P. fijiensis conidial effectorome. Gene Ontology classification suggests that they are involved in many biological processes and metabolisms, expanding our current knowledge of fungal effectors.

Characterization of microRNAs and Target Genes in Musa acuminata subsp. burmannicoides, var. Calcutta 4 during Interaction with Pseudocercospora musae.

Endogenous microRNAs (miRNAs) are small non-coding RNAs that perform post-transcriptional regulatory roles across diverse cellular processes, including defence responses to biotic stresses. Pseudocercospora musae, the causal agent of Sigatoka leaf spot disease in banana (Musa spp.), is an important fungal pathogen of the plant. Illumina HiSeq 2500 sequencing of small RNA libraries derived from leaf material in Musa acuminata subsp. burmannicoides, var. Calcutta 4 (resistant) after inoculation with fungal conidiospores and equivalent non-inoculated controls revealed 202 conserved miRNAs from 30 miR-families together with 24 predicted novel miRNAs. Conserved members included those from families miRNA156, miRNA166, miRNA171, miRNA396, miRNA167, miRNA172, miRNA160, miRNA164, miRNA168, miRNA159, miRNA169, miRNA393, miRNA535, miRNA482, miRNA2118, and miRNA397, all known to be involved in plant immune responses. Gene ontology (GO) analysis of gene targets indicated molecular activity terms related to defence responses that included nucleotide binding, oxidoreductase activity, and protein kinase activity. Biological process terms associated with defence included response to hormone and response to oxidative stress. DNA binding and transcription factor activity also indicated the involvement of miRNA target genes in the regulation of gene expression during defence responses. sRNA-seq expression data for miRNAs and RNAseq data for target genes were validated using stem-loop quantitative real-time PCR (qRT-PCR). For the 11 conserved miRNAs selected based on family abundance and known involvement in plant defence responses, the data revealed a frequent negative correlation of expression between miRNAs and target host genes. This examination provides novel information on miRNA-mediated host defence responses, applicable in genetic engineering for the control of Sigatoka leaf spot disease.

Transcriptome Profiling of the Resistance Response of Musa acuminata subsp. burmannicoides, var. Calcutta 4 to Pseudocercospora musae.

Banana (Musa spp.), which is one of the world's most popular and most traded fruits, is highly susceptible to pests and diseases. Pseudocercospora musae, responsible for Sigatoka leaf spot disease, is a principal fungal pathogen of Musa spp., resulting in serious economic damage to cultivars in the Cavendish subgroup. The aim of this study was to characterize genetic components of the early immune response to P. musae in Musa acuminata subsp. burmannicoides, var. Calcutta 4, a resistant wild diploid. Leaf RNA samples were extracted from Calcutta 4 three days after inoculation with fungal conidiospores, with paired-end sequencing conducted in inoculated and non-inoculated controls using lllumina HiSeq 4000 technology. Following mapping to the reference M. acuminata ssp. malaccensis var. Pahang genome, differentially expressed genes (DEGs) were identified and expression representation analyzed on the basis of gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes orthology and MapMan pathway analysis. Sequence data mapped to 29,757 gene transcript models in the reference Musa genome. A total of 1073 DEGs were identified in pathogen-inoculated cDNA libraries, in comparison to non-inoculated controls, with 32% overexpressed. GO enrichment analysis revealed common assignment to terms that included chitin binding, chitinase activity, pattern binding, oxidoreductase activity and transcription factor (TF) activity. Allocation to KEGG pathways revealed DEGs associated with environmental information processing, signaling, biosynthesis of secondary metabolites, and metabolism of terpenoids and polyketides. With 144 up-regulated DEGs potentially involved in biotic stress response pathways, including genes involved in cell wall reinforcement, PTI responses, TF regulation, phytohormone signaling and secondary metabolism, data demonstrated diverse early-stage defense responses to P. musae. With increased understanding of the defense responses occurring during the incompatible interaction in resistant Calcutta 4, these data are appropriate for the development of effective disease management approaches based on genetic improvement through introgression of candidate genes in superior cultivars.

Effective Methods Based on Distinct Learning Principles for the Analysis of Hyperspectral Images to Detect Black Sigatoka Disease.

Current chemical methods used to control plant diseases cause a negative impact on the environment and increase production costs. Accurate and early detection is vital for designing effective protection strategies for crops. We evaluate advanced distributed edge intelligence techniques with distinct learning principles for early black sigatoka disease detection using hyperspectral imaging. We discuss the learning features of the techniques used, which will help researchers improve their understanding of the required data conditions and identify a method suitable for their research needs. A set of hyperspectral images of banana leaves inoculated with a conidial suspension of black sigatoka fungus (Pseudocercospora fijiensis) was used to train and validate machine learning models. Support vector machine (SVM), multilayer perceptron (MLP), neural networks, N-way partial least square-discriminant analysis (NPLS-DA), and partial least square-penalized logistic regression (PLS-PLR) were selected due to their high predictive power. The metrics of AUC, precision, sensitivity, prediction, and F1 were used for the models' evaluation. The experimental results show that the PLS-PLR, SVM, and MLP models allow for the successful detection of black sigatoka disease with high accuracy, which positions them as robust and highly reliable HSI classification methods for the early detection of plant disease and can be used to assess chemical and biological control of phytopathogens.

Gene Expression, Histology and Histochemistry in the Interaction between Musa sp. and Pseudocercospora fijiensis.

Bananas are the main fruits responsible for feeding more than 500 million people in tropical and subtropical countries. Black Sigatoka, caused by the fungus Pseudocercospora fijiensis, is one of the most destructive disease for the crop. This fungus is mainly controlled with the use of fungicides; however, in addition to being harmful to human health, they are associated with a high cost. The development of resistant cultivars through crosses of susceptible commercial cultivars is one of the main focuses of banana breeding programs worldwide. Thus, the objective of the present study was to investigate the interaction between Musa sp. and P. fijiensis through the relative expression of candidate genes involved in the defence response to black Sigatoka in four contrasting genotypes (resistant: Calcutta 4 and Krasan Saichon; susceptible: Grand Naine and Akondro Mainty) using quantitative real-time PCR (RT-qPCR) in addition to histological and histochemical analyses to verify the defence mechanisms activated during the interaction. Differentially expressed genes (DEGs) related to the jasmonic acid and ethylene signalling pathway, GDSL-like lipases and pathogenesis-related proteins (PR-4), were identified. The number and distance between stomata were directly related to the resistance/susceptibility of each genotype. Histochemical tests showed the production of phenolic compounds and callosis as defence mechanisms activated by the resistant genotypes during the interaction process. Scanning electron microscopy (SEM) showed pathogenic structures on the leaf surface in addition to calcium oxalate crystals. The resistant genotype Krasan Saichon stood out in the analyses and has potential for use in breeding programs for resistance to black Sigatoka in banana and plantains.

Probiotic Endophytes for More Sustainable Banana Production.

Climatic factors and pathogenic fungi threaten global banana production. Moreover, bananas are being cultivated using excessive amendments of nitrogen and pesticides, which shift the microbial diversity in plants and soil. Advances in high-throughput sequencing (HTS) technologies and culture-dependent methods have provided valuable information about microbial diversity and functionality of plant-associated endophytic communities. Under stressful (biotic or abiotic) conditions, plants can recruit sets of microorganisms to alleviate specific potentially detrimental effects, a phenomenon known as "cry for help". This mechanism is likely initiated in banana plants infected by Fusarium wilt pathogen. Recently, reports demonstrated the synergistic and cumulative effects of synthetic microbial communities (SynComs) on naturally occurring plant microbiomes. Indeed, probiotic SynComs have been shown to increase plant resilience against biotic and abiotic stresses and promote growth. This review focuses on endophytic bacterial diversity and keystone taxa of banana plants. We also discuss the prospects of creating SynComs composed of endophytic bacteria that could enhance the production and sustainability of Cavendish bananas (Musa acuminata AAA), the fourth most important crop for maintaining global food security.

Genetic Improvement for Resistance to Black Sigatoka in Bananas: A Systematic Review.

Bananas are an important staple food crop in tropical and subtropical regions in Asia, sub-Saharan Africa, and Central and South America. The plant is affected by numerous diseases, with the fungal leaf disease black Sigatoka, caused by Mycosphaerella fijiensis Morelet [anamorph: Pseudocercospora fijiensis (Morelet) Deighton], considered one of the most economically important phytosanitary problem. Although the development of resistant cultivars is recognized as most effective method for long term control of the disease, the majority of today's cultivars are susceptible. In order to gain insights into this pathosystem, this first systematic literature review on the topic is presented. Utilizing six databases (PubMed Central, Web of Science, Google Academic, Springer, CAPES and Scopus Journals) searches were performed using pre-established inclusion and exclusion criteria. From a total of 3,070 published studies examined, 24 were relevant with regard to the Musa-P. fijiensis pathosystem. Relevant papers highlighted that resistant and susceptible cultivars clearly respond differently to infection by this pathogen. M. acuminata wild diploids such as Calcutta 4 and other diploid cultivars can harbor sources of resistance genes, serving as parentals for the generation of improved diploids and subsequent gene introgression in new cultivars. From the sequenced reference genome of Musa acuminata, although the function of many genes in the genome still require validation, on the basis of transcriptome, proteome and biochemical data, numerous candidate genes and molecules have been identified for further evaluation through genetic transformation and gene editing approaches. Genes identified in the resistance response have included those associated with jasmonic acid and ethylene signaling, transcription factors, phenylpropanoid pathways, antioxidants and pathogenesis-related proteins. Papers in this study also revealed gene-derived markers in Musa applicable for downstream application in marker assisted selection. The information gathered in this review furthers understanding of the immune response in Musa to the pathogen P. fijiensis and is relevant for genetic improvement programs for bananas and plantains for control of black Sigatoka.

The Mitochondrial Genome of a Plant Fungal Pathogen Pseudocercospora fijiensis (Mycosphaerellaceae), Comparative Analysis and Diversification Times of the Sigatoka Disease Complex Using Fossil Calibrated Phylogenies.

Mycosphaerellaceae is a highly diverse fungal family containing a variety of pathogens affecting many economically important crops. Mitochondria play a crucial role in fungal metabolism and in the study of fungal evolution. This study aims to: (i) describe the mitochondrial genome of Pseudocercospora fijiensis, and (ii) compare it with closely related species (Sphaerulina musiva, S. populicola, P. musae and P. eumusae) available online, paying particular attention to the Sigatoka disease's complex causal agents. The mitochondrial genome of P. fijiensis is a circular molecule of 74,089 bp containing typical genes coding for the 14 proteins related to oxidative phosphorylation, 2 rRNA genes and a set of 38 tRNAs. P. fijiensis mitogenome has two truncated cox1 copies, and bicistronic transcription of nad2-nad3 and atp6-atp8 confirmed experimentally. Comparative analysis revealed high variability in size and gene order among selected Mycosphaerellaceae mitogenomes likely to be due to rearrangements caused by mobile intron invasion. Using fossil calibrated Bayesian phylogenies, we found later diversification times for Mycosphaerellaceae (66.6 MYA) and the Sigatoka disease complex causal agents, compared to previous strict molecular clock studies. An early divergent Pseudocercospora fijiensis split from the sister species P. musae + P. eumusae 13.31 MYA while their sister group, the sister species P. eumusae and P. musae, split from their shared common ancestor in the late Miocene 8.22 MYA. This newly dated phylogeny suggests that species belonging to the Sigatoka disease complex originated after wild relatives of domesticated bananas (section Eumusae; 27.9 MYA). During this time frame, mitochondrial genomes expanded significantly, possibly due to invasions of introns into different electron transport chain genes.

A world-wide analysis of reduced sensitivity to DMI fungicides in the banana pathogen Pseudocercospora fijiensis.

BACKGROUND: Pseudocercospora fijiensis is the causal agent of the black leaf streak disease (BLSD) of banana. Bananas are important global export commodities and a major staple food. Their susceptibility to BLSD pushes disease management towards excessive fungicide use, largely relying on multisite inhibitors and sterol demethylation inhibitors (DMIs). These fungicides are ubiquitous in plant disease control, targeting the CYP51 enzyme. We examined sensitivity to DMIs in P. fijiensis field isolates collected from various major banana production zones in Colombia, Costa Rica, Dominican Republic, Ecuador, the Philippines, Guadalupe, Martinique and Cameroon and determined the underlying genetic reasons for the observed phenotypes. RESULTS: We observed a continuous range of sensitivity towards the DMI fungicides difenoconazole, epoxiconazole and propiconazole with clear cross-sensitivity. Sequence analyses of PfCYP51 in 266 isolates showed 28 independent amino acid substitutions, nine of which correlated with reduced sensitivity to DMIs. In addition to the mutations, we observed up to six insertions in the Pfcyp51 promoter. Such promoter insertions contain repeated elements with a palindromic core and correlate with the enhanced expression of Pfcyp51 and hence with reduced DMI sensitivity. Wild-type isolates from unsprayed bananas fields did not contain any promoter insertions. CONCLUSION: The presented data significantly contribute to understanding of the evolution and global distribution of DMI resistance mechanisms in P. fijiensis field populations and facilitate the prediction of different DMI efficacy. The overall reduced DMI sensitivity calls for the deployment of a wider range of solutions for sustainable control of this major banana disease. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genetic Diversity and Azole Fungicide Sensitivity in Pseudocercospora musae Field Populations in Brazil.

Pseudocercospora musae, causal agent of Sigatoka leaf spot, or yellow Sigatoka disease, is considered a major pathogen of banana (Musa spp.). Widely disseminated in Brazil, this study explored the genetic diversity in field populations of the pathogen from production areas in the Distrito Federal and the States of Bahia, Minas Gerais, and Rio Grande do Norte. Resistance to demethylation inhibitor (DMI) fungicides was also examined. For 162 isolates from 10 banana growing regions, analysis of mating type idiomorph frequency was conducted, together with estimation of genetic diversity at 15 microsatellite loci. A total of 149 haplotypes were identified across the examined populations, with an average genetic diversity of 4.06. In general, populations displayed 1:1 proportions of idiomorphs MAT1-1 and MAT1-2, providing evidence for sexual recombination. Multilocus linkage disequilibrium also indicated asexual reproduction contributing to the genetic structure of certain populations. AMOVA revealed that 86.3% of the genetic differentiation of the pathogen occurred among isolates within populations. Discriminant Analysis of Principal Components (DAPC) identified six most probable genetic groups, with no population structure associated with geographic origin or collection site. Although genetic similarity was observed among certain populations from different states, data revealed increasing genetic differentiation with increasing geographic distance, as validated by Mantel's test (r = 0.19, P < 0.001). On the basis of DMI fungicide sensitivity testing and CYP51 gene sequence polymorphism, isolates from the Distrito Federal separated into two main groups, one with generally higher EC50 values against eight DMI fungicides. A clear phenotype-to-genotype relationship was observed for isolates carrying the CYP51 alteration Y461N. Conventionally adopted fungicides for control of Sigatoka leaf spot are likely to be overcome by combined sexual and asexual reproduction mechanisms in P. musae driving genetic variability. Continued analysis of pathogen genetic diversity and monitoring of DMI sensitivity profiles of Brazilian field populations is essential for the development of integrated control strategies based on host resistance breeding and rational design of fungicide regimes.

Protein compounds of Bacillus subtilis with in vitro antifungal activity against Pseudocercospora fijiensis (Morelet).

The metabolites of Bacillus subtilis CCIBP-M27 were evaluated as an antagonist of Pseudocercospora fijiensis. The culture filtrate did not inhibit ascospore germination but significantly reduced conidial germination and mycelial growth. Through microscopic analysis, deformations were observed as vacuolization and swelling in P. fijiensis mycelia when exposed to culture filtrate during 48 h. A similar response was induced by peptide-type compounds found on Bacillus subtilis CCIBP-M27 culture filtrate. The results obtained suggest that the in vitro antifungal effect of the strain CCIBP-M27 against P. fijiensis is related to the action of diffused metabolites such as proteins or peptide substances.

Early Detection of the Fungal Banana Black Sigatoka Pathogen Pseudocercospora fijiensis by an SPR Immunosensor Method.

Black Sigatoka is a disease that occurs in banana plantations worldwide. This disease is caused by the hemibiotrophic fungus Pseudocercospora fijiensis, whose infection results in a significant reduction in both product quality and yield. Therefore, detection and identification in the early stages of this pathogen in plants could help minimize losses, as well as prevent the spread of the disease to neighboring cultures. To achieve this, a highly sensitive SPR immunosensor was developed to detect P. fijiensis in real samples of leaf extracts in early stages of the disease. A polyclonal antibody (anti-HF1), produced against HF1 (cell wall protein of P. fijiensis) was covalently immobilized on a gold-coated chip via a mixed self-assembled monolayer (SAM) of alkanethiols using the EDC/NHS method. The analytical parameters of the biosensor were established, obtaining a limit of detection of 11.7 µg mL-1, a sensitivity of 0.0021 units of reflectance per ng mL-1 and a linear response range for the antigen from 39.1 to 122 µg mL-1. No matrix effects were observed during the measurements of real leaf banana extracts by the immunosensor. To the best of our knowledge, this is the first research into the development of an SPR biosensor for the detection of P. fijiensis, which demonstrates its potential as an alternative analytical tool for in-field monitoring of black Sigatoka disease.

EFECTO DEL SOMBRÍO SOBRE LA SIGATOKA NEGRA (Mycosphaerella fijiensis MORELET) EN CULTIVO DE PLÁTANO cv HARTÓN (Musa AAB SIMMONDS)

La sigatoka negra en el cultivo del plátano es considerada como una de las enfermedades más limitante de la producción, a nivel mundial, siendo manejada con fungicidas, ocasionando contaminaciones ambientales e incremento de los costos de producción. Como estrategia no contaminante, algunos investigadores recomiendan cultivar en condiciones de sombrío, para reducir la enfermedad. El objetivo de este trabajo fue evaluar el efecto de diferentes porcentajes de sombrío en la incidencia de la enfermedad y en la producción, bajo condiciones del Caribe húmedo colombiano. En condiciones de campo, se estableció el cultivar '˜Hartón', en un diseño de bloques completos al azar y cuatro porcentajes de sombrío, 20, 35 y 50%, teniendo como testigo plantas cultivadas a libre exposición solar 100%. Las evaluaciones, se realizaron durante el periodo de floración del cultivo, cuantificando el número de hojas funcionales NHF, la hoja más joven con síntomas HMJS, la hoja más joven enferma HMJE, el índice de severidad IS y rendimiento. Los tratamientos con 20, 35 y 50% de sombrío no presentaron diferencias estadísticas entre sí; más sí, con el testigo en las variables NHF, HMJE, IS y rendimiento. Los tratamientos con sombra manifestaron menor IS de la sigatoka negra, de 13 al 15%, mientras el testigo fue del 18%; además, los rendimientos se incrementaron en 400kg ha-1. Se concluye, que el cultivo de plátano '˜Hartón', manejado a partir del 20% de sombrío, fue suficiente para reducir la severidad de la sigatoka negra e incrementar significativamente los rendimientos.

Black sigatoka in plantain cultivation is considered one of the most limiting production diseases worldwide. It is managed with fungicides, causing environmental pollution and increased production costs. As a nonpolluting strategy, some researchers recommend cultivating shade conditions to reduce the disease. The aim of this study was to evaluate the effect of different shade percentages in the incidence of disease and production under the Caribbean Colombian humid conditions. Under field conditions it was established to cultivate the '˜Harton' in a completely randomized block design and four shade percentages 20, 35 and 50%, having as control plants growing in 100% sun exposure. Evaluations were performed during the flowering period of the crop, quantifying the number of functional leaves NFL, the youngest leaf with symptoms YLS, the youngest diseased leaf YDL, the severity index SI and yield. Treatments with 20, 35 and 50% shade were not statistically different from each other; but with the witness in the NFL, YLS, SI and yield. Treatments with shade had lower SI of black sigatoka, between 13 to 15%; while the witness was 18%; also the yield increased in 400kg ha-1. It is concluded that growing '˜Harton', managed from 20% shade was enough to reduce the severity of black sigatoka and significantly increase yields.

Efficient transformation of Mycosphaerella fijiensis by underwater shock waves.

Black leaf streak disease, also known as black Sigatoka, causes dramatic losses in production of banana and plantains fruits. The disease is caused by the pathogenic fungus Mycosphaerella fijiensis (anamorph Pseudocercospora fijiensis; Mycosphaerellaceae). Genetic transformation of M. fijiensis would allow a better understanding of molecular basis of pathogenicity and design novel approaches to control the infection caused by this pathogen. However, transformation of this fungus has not been easy. We report here a protocol for genetic transformation of M. fijiensis employing underwater shock waves and intact conidia. The recombinant strains recovered showed genetic stability over >10 generations. The frequency of transformation obtained was between 75 and 150 times higher than the efficiency reported in the only article published on transformation of M. fijiensis using spheroplasts. This improvement allowed the use of a thousand times less cells than the amount employed before, avoiding the need for cumbersome successive batch cultures. Our protocol is simple, highly efficient, fast and reproducible and together with the available genomes of M. fijiensis and Musa acuminata, it offers new possibilities to study the diverse mechanisms of pathogenesis of the fungus.

Comparative analysis of the in vitro and in planta secretomes from Mycosphaerella fijiensis isolates.

Black Sigatoka, a devastating disease of bananas and plantains worldwide, is caused by the fungus Mycosphaerella fijiensis. Several banana cultivars such as 'Yangambi Km 5' and Calcutta IV, have been known to be resistant to the fungus, but the resistance has been broken in 'Yangambi Km 5' in Costa Rica. Since the resistance of this variety still persists in Mexico, the aim of this study was to compare the in vitro and in planta secretomes from two avirulent and virulent M. fijiensis isolates using proteomics and bioinformatics approaches. We aimed to identify differentially expressed proteins in fungal isolates that differ in pathogenicity and that might be responsible for breaking the resistance in 'Yangambi Km 5'. We were able to identify 90 protein spots in the secretomes of fungal isolates encoding 42 unique proteins and 35 differential spots between them. Proteins involved in carbohydrate transport and metabolism were more prevalent. Several proteases, pathogenicity-related, ROS detoxification and unknown proteins were also highly or specifically expressed by the virulent isolate in vitro or during in planta infection. An unknown protein representing a virulence factor candidate was also identified. These results demonstrated that the secretome reflects major differences between both M. fijiensis isolates.

Chemical management in fungicide sensitivity of Mycosphaerella fijiensis collected from banana fields in México.

The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L(-1) for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for ß-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L(-1) for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.

Chemical management in fungicide sensivity of Mycosphaerella fijiensis collected from banana fields in México

The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L-1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L-1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.

Chemical management in fungicide sensivity of Mycosphaerella fijiensis collected from banana fields in México

The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L-1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L-1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.(AU)

Diversidade genética de isolados de Mycosphaerella musicola obtidos de bananais do norte de Minas Gerais, Brasil por meio de marcadores RAPD / Genetic diversity of Mycosphaerella musicola isolates obtained from bananas of north of Minas Gerais, Brazil by using RAPD markers

Onze primers RAPD foram utilizados para avaliar a variabilidade genética de 31 isolados de M. musicola coletados a partir de folhas de bananeiras 'Prata Anã' e 'Nanica', cultivadas no Norte de Minas Gerais. Foram amplificados um total de 83 bandas sendo 73 polimórficas, dando uma média de 6,6 bandas polimórficas por primer. As distâncias genéticas observadas variaram de 0,56 a 0,06 entre os isolados, com distância média de 0,25. O dendrograma construído com base no método UPGMA revelou a formação de 8 grupos, não sendo observada correlação entre a diversidade genética dos isolados e as origens geográficas dos isolados avaliados.

Eleven primers RAPD were used to estimate the genetic variability between 31 isolates of M. musicola collected from 'PrataAnã' and Nanica bananas that were cultivated in northern Minas Gerais, Brazil. A total o 83 fragments were amplified, of which 73 were polymorphic, corresponding to an average of 6.6 polymorphic fragments per primer. The genetic distances ranged from 0.06 to 0.56 and the average distance of 0.21. A dendrogram constructed based on the UPGMA clustering method revealed 8 and no correlation between molecular grouping and geographical origin was observed.