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Miguel A Gomezlim - One of the best experts on this subject based on the ideXlab platform.
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efficient transformation of Mycosphaerella Fijiensis by underwater shock waves
Journal of Microbiological Methods, 2015Co-Authors: Lina Escobartovar, Denis Maganaortiz, Francisco Fernandez, Mauricio Guzmanquesada, Jorge A Sandovalfernandez, Elizabeth Ortizvazquez, Achim M Loske, Miguel A GomezlimAbstract: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.
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comparative analysis of the in vitro and in planta secretomes from Mycosphaerella Fijiensis isolates
Fungal Biology, 2015Co-Authors: Lina Escobartovar, Mauricio Guzmanquesada, Jorge A Sandovalfernandez, Miguel A GomezlimAbstract:Abstract 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.
Monica Hofte - One of the best experts on this subject based on the ideXlab platform.
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a green fluorescent protein transformed Mycosphaerella Fijiensis strain shows increased aggressiveness on banana
Australasian Plant Pathology, 2012Co-Authors: Orelvis Portal, Mayra Acostasuarez, Barbara Ocana, Wilhelm Schafer, Elio Jimenez, Monica HofteAbstract:The fungal pathogen Mycosphaerella Fijiensis, causal agent of black leaf streak disease of bananas and plantains, was transformed with a green fluorescent protein-carrying construct by using a restriction enzyme-mediated integration methodology. A quantitative polymerase chain reaction was adapted to estimate transgene copy number and pathogenicity assays with three banana genotypes with dissimilar reactions to M. Fijiensis infection were performed to characterize the transformants. Transgene insertion varied from one to five copies per genome among four random selected transformants. All M. Fijiensis strains produced typical symptoms of the black leaf streak disease on the three banana genotypes assayed. Interestingly, the GFP-18 transformant showed increased aggressiveness on susceptible ‘Grande naine’ and resistant ‘Yangambi km5’ plants demonstrating that mutation events in M. Fijiensis can increase virulence.
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analysis of expressed sequence tags derived from a compatible Mycosphaerella Fijiensis banana interaction
Plant Cell Reports, 2011Co-Authors: Orelvis Portal, Mayra Acostasuarez, Barbara Ocana, Elio Jimenez, Yovanny Izquierdo, David De Vleesschauwer, Aminael Sanchezrodriguez, Milady Mendozarodriguez, Monica HofteAbstract:Mycosphaerella Fijiensis, a hemibiotrophic fungus, is the causal agent of black leaf streak disease, the most serious foliar disease of bananas and plantains. To analyze the compatible interaction of M. Fijiensis with Musa spp., a suppression subtractive hybridization (SSH) cDNA library was constructed to identify transcripts induced at late stages of infection in the host and the pathogen. In addition, a full-length cDNA library was created from the same mRNA starting material as the SSH library. The SSH procedure was effective in identifying specific genes predicted to be involved in plant–fungal interactions and new information was obtained mainly about genes and pathways activated in the plant. Several plant genes predicted to be involved in the synthesis of phenylpropanoids and detoxification compounds were identified, as well as pathogenesis-related proteins that could be involved in the plant response against M. Fijiensis infection. At late stages of infection, jasmonic acid and ethylene signaling transduction pathways appear to be active, which corresponds with the necrotrophic life style of M. Fijiensis. Quantitative PCR experiments revealed that antifungal genes encoding PR proteins and GDSL-like lipase are only transiently induced 30 days post inoculation (dpi), indicating that the fungus is probably actively repressing plant defense. The only fungal gene found was induced 37 dpi and encodes UDP-glucose pyrophosphorylase, an enzyme involved in the biosynthesis of trehalose. Trehalose biosynthesis was probably induced in response to prior activation of plant antifungal genes and may act as an osmoprotectant against membrane damage.
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an efficient method for the extraction of high quality fungal total rna to study the Mycosphaerella Fijiensis musa spp interaction
Molecular Biotechnology, 2008Co-Authors: Aminael Sanchezrodriguez, Orelvis Portal, Barbara Ocana, Elio Jimenez, Luis Rojas, Milady Mendoza, Mayra Acosta, Monica HofteAbstract:Efficient RNA isolation is a prerequisite for gene expression studies and it has an increasingly important role in the study of plant–fungal pathogen interactions. However, RNA isolation is difficult in filamentous fungi. These organisms are notorious for their rigid cell walls and the presence of high levels of carbohydrates, excreted from the fungal cells during submerged growth, which interferes with the extraction procedures. Although many commercial kits are already available for RNA isolation, they do not provide, in most cases, enough amount of pure RNA to be used in upstream applications. In the present work, we propose an easy and efficient protocol for isolating total RNA from the filamentous fungus Mycosphaerella Fijiensis, the most important foliar pathogen of Musa spp. varieties worldwide. In addition, we applied the proposed protocol to the isolation of total RNA from banana leaves infected with the pathogen. Our methodology was developed based on the SDS method with modifications including a carbohydrate precipitation step. The protocol resulted in high-quality total RNA, from fungal mycelium grown in PDB medium and infected banana leaves, suitable for further molecular studies. The proposed methodology is also applicable to the ascomycete fungus Passalora fulva (syn. Cladosporum fulvum).
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identificacion de secuencias que se expresan diferencialmente en la interaccion no compatible musa acuminata Mycosphaerella Fijiensis morelet
Biotecnología Vegetal, 2008Co-Authors: Milady Mendozarodriguez, Orelvis Portal, Mayra Acostasuarez, Monica Hofte, Berkis Roque, Aminael Sanchezrodriguez, Elio JimenezAbstract:Black leaf streak disease caused by Mycosphaerella Fijiensis Morelet is considered the most destructive and costly foliar disease of bananas and plantain. An important step for the elucidation of molecular mechanisms of the disease resistance is to know about genes involved in plant defense response to pathogens. Identification of differentially expressed sequences in resistant genotype ‘Calcutta 4’ ( Musa acuminata , AA) at an early stage of infection with M. Fijiensis (6 to 12 days post inoculation) from a suppressed subtractive library (SSH) was carried out. A number of 63 ESTs, which include 42 singletons and 21 contigs were obtained by assembling 97 sequences with CAP3 algorithm. Identification of sequences according to their homology with sequences stated at protein non-redundant database GenBank, allowed to gather them in six functional categories: protein destiny (1.6%), oxidative stress (4.8%), metabolism (6.3%), energy production (6.3%), unknown function (38.1%) and without homology (42.8%). Results obtained will contribute to a better understanding of pathosystem, which allow the design of new strategies related to genetic improvement of banana. Keywords: Black leaf streak disease, expressed sequence tags, functional gene classification, Musa spp.,subtractive library
R C Ploetz - One of the best experts on this subject based on the ideXlab platform.
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evaluation of banana hybrids for tolerance to black leaf streak Mycosphaerella Fijiensis morelet in puerto rico
Crop Protection, 2013Co-Authors: Brian M Irish, Ricardo Goenaga, C Rios, J Chavarriacarvajal, R C PloetzAbstract:Abstract In Puerto Rico, bananas (including plantains) are important agricultural commodities; their combined production totaled over 158,000 tons in 2011. Black leaf streak (BLS) and Sigatoka leaf spot diseases, caused by Mycosphaerella Fijiensis and Mycosphaerella musicola, respectively, are responsible for significant losses of this crop, due to the high susceptibility of the most important cultivars. Diploid, triploid and tetraploid hybrids were introduced from international breeding programs for evaluation in Isabela, Puerto Rico. Accessions were established in the field in a randomized complete block design and were evaluated over two cropping cycles (2007–2010) for response to BLS and agronomic traits. Significant differences (P = 0.05) in BLS severity were observed among accessions throughout both crop cycles and were most pronounced at harvest. When averaged across production cycles, severity indices at harvest ranged from very resistant (20% of the leaf surface affected) for ‘FHIA 02’ to extremely susceptible (97%) for ‘Grand Nain’. Yield attributes varied widely among the accessions, including mean bunch weights (6.9–41.0 kg), numbers of hands per bunch (6.6–13.4), and the numbers of fruit per bunch (57.0–239.2). Several accessions, mainly from the Fundacion Hondurena de Investigacion Agricola (FHIA), were BLS resistant and had short pseudostems, and large bunches. They could potentially replace susceptible cultivars in commercial production or play roles in a nascent organic market.
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Mycosphaerella Fijiensis causal agent of black sigatoka of musa spp found in puerto rico and identified by polymerase chain reaction
Plant Disease, 2006Co-Authors: Brian M Irish, R Goenaga, R C PloetzAbstract:Black Sigatoka, also known as black leaf streak, is caused by Mycosphaerella Fijiensis Morelet (anamorph Pseudocercospora Fijiensis (Morelet) Deighton). It is the most significant disease of bananas and plantains (Musa spp.) because most of the economically important cultivars of exported and staple commodities are highly susceptible. The Caribbean is one of the few regions of the world where black Sigatoka is not widespread. Black Sigatoka has been reported in the Bahamas, Cuba, Hispaniola, and Jamaica (2). Yellow Sigatoka, caused by M. musicola Leach (anamorph P. musae (Zimm.) Deighton), has been recognized in Puerto Rico since 1938-1939 (3). In August 2004, symptoms resembling black Sigatoka were first observed in Anasco, Puerto Rico by extension personnel from the University of Puerto Rico. Since black and yellow Sigatoka produce similar disease symptoms, a survey was conducted in the western banana- and plantain-production region of Puerto Rico to confirm the presence of black Sigatoka. Leaf samples were collected from production fields near the towns of Las Marias, Maricao, and Anasco. Single-ascospore isolates were recovered using the discharge technique from moistened pseudothecia in necrotic lesions that were inverted over water agar, and ascospores were transferred to potato dextrose agar. The isolates were subcultured in potato dextrose broth for mycelium production. DNA was isolated from mycelium with the FastDNA kit (Q-Biogen, Irvine, CA) for 19 isolates. Internal transcribed spacer as well as the 5.8s rDNA regions were polymerase chain reaction amplified with primers specific to M. Fijiensis or M. musicola (1). Amplification products (˜1,100 bp) were observed for 18 of the 19 isolates, 6 of which were M. Fijiensis and the remaining 12 were M. musicola, while the positive controls for both species were also amplified with the respective primer pairs. M. Fijiensis was recovered from production fields close to all three towns. The source of M. Fijiensis in Puerto Rico is unclear, but it may have originated from introduced leaf material and/or wind dispersed ascospores from neighboring countries. The presence of black Sigatoka in Puerto Rico will most likely increase production costs where fungicide applications will be needed to maintain yields. The USDA-ARS, Tropical Agriculture Research Station is the official Musa spp. germplasm repository for the National Plant Germplasm System. As such, efforts are underway to introduce and evaluate black Sigatoka disease-resistant clones that can satisfy local and export market criteria. References: (1) A. Johnasen. Detection of Sigatoka leaf spot pathogens of banana by the polymerase chain reaction. Chatman, UK, Natural Resource Institute, 1997. (2) R. C. Ploetz. Plant Dis. 88:772, 2004. (3) R. H. Stover. Trop. Agric. Trinidad. 39:327, 1962.
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first report of black sigatoka of banana caused by Mycosphaerella Fijiensis on grand bahama island
Plant Disease, 2004Co-Authors: R C PloetzAbstract:Black Sigatoka, which is also known as black leaf streak, is caused by Mycosphaerella Fijiensis (anamorph Pseudocercospora [formerly Paracercospora) Fijiensis]). It is the most important disease of commercially produced banana (Musa spp.) and also has a major impact on production for local consumption. Although the disease occurs throughout the humid tropics, it has been reported in the Caribbean from only Cuba, Hispaniola, and Jamaica (1). In February 2004, black Sigatoka was observed at two isolated and widely separated sites on Grand Bahama island (26.7°N, 78.5°W and 26.7°N, 78°W) on cvs. Silk AAB and Williams AAA, and a French Horn AAB plantain. Symptoms included wet, dark brown streaks on the adaxial leaf surface, 1 to 2 × 10 mm, with chlorotic haloes. Lesions enlarged to 5 × 20 mm and developed tan, necrotic centers; large, blackened, water-soaked areas that resulted from the coalescence of streaks were rare. The disease was confirmed by observing the following characteristics of P. Fijiensis in necrotic lesions on preserved leaf specimens: simple conidiophores with a broadened base and one to several septa, straight to variously bent cercosporoid conidia as much as 100 μm long with two to several septa, and a conspicuously thickened scar at the base. Both plantings were several years old and new planting material that could have been infested with the pathogen had not been introduced since their establishment. Symptoms were not severe and were distributed sporadically in both locations. The disease was not observed at the only other large planting of banana on the island (26.6°N, 78.6°W). The sporadic and apparently new infestations of two of three banana plantings on the island suggest that the pathogen may have arrived recently via natural means, possibly from neighboring Florida (2). In contrast, black Sigatoka appears to have spread to other islands in the Caribbean via infested propagation materials (1). To my knowledge, this is the first report of black Sigatoka in the Bahamas, and with a previous report from Bhutan (1), represents the northernmost spread of this important disease. References: (1) J. Carlier et al. Pages 37-79 in: Diseases of Banana, Abaca and Enset. D. R. Jones, ed. CABI Publishing. Wallingford, UK, 2000. (2) R. C. Ploetz and X. Mourichon. Plant Dis. 83:300, 1999.
Blondy Cantocanche - One of the best experts on this subject based on the ideXlab platform.
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genome wide in silico identification of gpi proteins in Mycosphaerella Fijiensis and transcriptional analysis of two gpi anchored β 1 3 glucanosyltransferases
Mycologia, 2013Co-Authors: Nuvia Kantunmoreno, Cecilia Monica Rodriguezgarcia, Leticia Perazaecheverria, Ignacio Islasflores, Rosa Grijalvaarango, Miguel Tzecsima, Andrew C James, Roberto Vazquezeuan, Jorge H Ramirezprado, Blondy CantocancheAbstract:The hemibiotrophic fungus Mycosphaerella Fijiensis is the causal agent of black Sigatoka (BS), the most devastating foliar disease in banana (Musa spp.) worldwide. Little is known about genes that are important during M. Fijiensis-Musa sp. interaction. The fungal cell wall is an attractive area of study because it is essential for maintenance of cellular homeostasis and it is the most external structure in the fungal cell and therefore mediates the interaction of the pathogen with the host. In this manuscript we describe the in silico identification of glycosyl phosphatidylinositol-protein (GPI) family in M. Fijiensis, and the analysis of two b-1,3-glucanosyltrans- ferases (Gas), selected by homology with fungal pathogenicity factors. Potential roles in pathogenesis were evaluated through analyzing expression during different stages of black Sigatoka disease, comparing expression data with BS symptoms and fungal biomass inside leaves. Real-time quantitative RT-PCR showed nearly constant expression of MfGAS1 with slightly increases (about threefold) in conidia and at speck- necrotrophic stage during banana-pathogen interac- tion. Conversely, MfGAS2 expression was increased during biotrophy (about seven times) and reached a maximum at speck (about 23 times) followed by a
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the in vitro secretome of Mycosphaerella Fijiensis induces cell death in banana leaves
Plant Physiology and Biochemistry, 2011Co-Authors: Jose Chucuc, Carlos A Cruzcruz, Cecilia Monica Rodriguezgarcia, Leticia Perazaecheverria, Blondy Cantocanche, Andrew Jameskay, Ligia Britoargaez, Miguel Tzecsima, Santy Perazaecheverria, Luis M PenarodriguezAbstract:The hemibiotrophic filamentous fungus Mycosphaerella Fijiensis causes the banana foliar disease known as black Sigatoka, responsible for major worldwide losses in the banana fruit industry. In this work the in vitro secretome of M. Fijiensis was characterized. Native and denaturant polyacrylamide gel protease assays showed the M. Fijiensis secretome contains protease activity capable of degrading gelatin. Necrotic lesions on leaves were produced by application of the in vitro secretome to the surface of one black Sigatoka-resistant banana wild species, one susceptible cultivar and the non-host plant Carica papaya. To distinguish if necrosis by the secretome is produced by phytotoxins or proteins, the latter ones were precipitated with ammonium sulfate and applied in native or denatured forms onto leaves of the same three plant species. Proteins applied in both preparations were able to produce necrotic lesions. Application of Pronase, a commercial bacterial protease suggested that the necrosis was, at least in part, caused by protease activity from the M. Fijiensis secretome. The ability to cause necrotic lesions between M. Fijiensis secreted- and ammonium sulfate-precipitated proteins, and purified lipophilic or hydrophilic phytotoxins, was compared. The results suggested that leaf necrosis arises from the combined action of non-host specific hydrolytic activities from the secreted proteins and the action of phytotoxins. This is the first characterization of the M. Fijiensis protein secretome produced in vitro but, more importantly, it is also the first time the M. Fijiensis secretome has been shown to contain virulence factors capable of causing necrosis to its natural host.
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isolation of retro transcribed rna from in vitro Mycosphaerella Fijiensis infected banana leaves
Genetics and Molecular Research, 2010Co-Authors: Cecilia Monica Rodriguezgarcia, Leticia Perazaecheverria, Blondy Cantocanche, Ignacio Islasflores, Rosa GrijalvaarangoAbstract:High polyphenol and polysaccharide levels in plant tissues such as banana fruit and leaves constitute a significant challenge to the extraction of sufficient amounts of high-quality RNA required for cDNA library synthesis and molecular analysis. To determine their comparative effectiveness at eliminating polyphenols, polysaccharides and proteins, three protocols for RNA extraction from in vitro banana plantlet leaves were tested: Concert(TM) Plant RNA isolation kit, a small-scale protocol based on Valderrama-Chairez, and a modified version of the Valderrama-Chairez protocol. RNA quantity and purity were evaluated by UV-spectrophotometry using DEPC-treated water and Tris-HCl, pH 7.5. Purity was greater using Tris-HCl. The Concert(TM) Plant protocol produced the poorest quality RNA. Reverse transcription into cDNAs from RNA isolated from in vitro banana plantlet leaves infected with Mycosphaerella Fijiensis using the modified Valderrama-Chairez protocol, followed by PCR using primers designed against gamma-actin from banana and M. Fijiensis, yielded products of the anticipated size. In addition, this protocol reduced the processing time, lowered costs, used less expensive equipment, and could be used for other plants that have the same problems with high polyphenol and polysaccharide levels.
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isolation and characterization of the mating type locus of Mycosphaerella Fijiensis the causal agent of black leaf streak disease of banana
Molecular Plant Pathology, 2007Co-Authors: Laura Condeferraez, Blondy Cantocanche, Andrew C James, G H J Kema, Cees Waalwijk, P W Crous, Edwin C A AbelnAbstract:SUMMARY Idiomorphs mat1-1 and mat1-2 from Mycosphaerella Fijiensis, the causal agent of black leaf streak disease of banana, were isolated. Degenerate oligos were used to amplify the HMG box of the mat1-2 idiomorph from M. Fijiensis, showing homology with the HMG box of Mycosphaerella graminicola. Using a DNA walking strategy, anchored on the DNA lyase gene towards the HMG box, a 9-kb-long region of mat1-2 was obtained. A 5-kb fragment from the mat1-1 region was obtained by long-range PCR using primers on the flanking regions, which have close to 100% identity between both idiomorphs. High-identity (77‐ 89%), inverted regions within both idiomorphs were found, which suggest unique inversion events, which have not been found before, and that could have been significant in the evolution of this species. The predicted genes showed the conserved introns in both idiomorphs as well as an additional intron within the alpha box. The implications for the evolution of species in the Mycosphaerella complex on banana are discussed.
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extraction of high quality melanin free rna from Mycosphaerella Fijiensis for cdna preparation
Molecular Biotechnology, 2006Co-Authors: Ignacio Islasflores, Blondy Cantocanche, Leticia Perazaecheverria, Cecilia Monica RodriguezgarciaAbstract:High-quality RNA preparations are critical for further applications such as reverse transcriptase-polymerase chain reaction (RT-PCR) transcript amplifications, and elaboration of cDNA and expressed sequence tag libraries. Melanins are phenolic compounds present in many fungi and apparently play key roles in fungi pathogenesis and survival. However, during RNA extraction these compounds constitute a significant challenge to extraction of substantial quantities of high-quality RNA, and consequently to preparation of cDNA libraries. No method currently exists for RNA extraction from Mycosphaerella Fijiensis that produces high quantities of melanin-free RNA. This fungus is the most important pathogen of cultivated Musa sp. varieties. A comparison is made between results obtained from the Trizol and RNeasy protocols for RNA extraction, two commercially available methods commonly used to obtain RNA from various sources. An improved methodology is described that allows isolation of intact RNA and elimination of melanins from M Fijiensis mycelium. RNA quality is evaluated by electrophoresis in formaldehyde-agarose gels, RT into cDNAs, and subsequent PCR amplification using primers designed against actin and β-tubulin from fungi.
W Schuh - One of the best experts on this subject based on the ideXlab platform.
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effects of leaf wetness duration and temperature on development of black sigatoka disease on banana infected by Mycosphaerella Fijiensis var difformis
Phytopathology, 1992Co-Authors: L H Jacome, W SchuhAbstract:Severity of Black Sigatoka disease on banana leaves infected by either conidia or ascospores from two isolates of Mycosphaerella Fijiensis var. difformis was influenced by temperature and leaf wetness duration. A film of water on the leaf surface was required for ascospore infection. Conidial infection was observed at a leaf wetness of 0-18 h. Initial foliar symptoms of Black Sigatoka disease required an incubation period of 14 days on leaves subjected to 18 h of leaf wetness after inoculation. Symptom expression was delayed 7-14 days at lower levels of leaf wetness. Disease severity increased with increasing leaf wetness duration (...)
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effect of temperature and relative humidity on germination and germ tube development of Mycosphaerella Fijiensis var difformis
Phytopathology, 1991Co-Authors: L H Jacome, W Schuh, R E StevensonAbstract:Conidia (three isolates) and ascospores (one isolate) of Mycosphaerella Fijiensis var. difformis germinated at 20-35 C. Germination followed a quadratic response function on temperature (R 2 =0.90-0.98), with an estimated optimum at 26.5 C. Maximum germination was observed in free-water and decreased at lower relative humidities (RH). Conidia germinated over a wider range of RH (92-100%) than ascospores (98-100%).Ascospores germinated earlier than conidia. The highest number of germ tubes per conidium was at 25 C for two isolates and at 30 C for the third isolate in free-water after 24 h (...)
