Molecular identification of fungi

The affordability of modern molecular biology tools and the availability of whole genome sequences have brought substantial improvement in research on pathogenic fungi and diagnosis of fungal infection. Molecular methods have resolved many critical aspects of mycological diagnosis by (1) providing specieslevel identification of fungi through sequencing of suitable taxonomic markers; (2) shortening of the time required for microbiological confirmation of life-threatening fungal infections; and (3) tracing the molecular epidemiology of fungal diseases. Nucleic acids-based methods are less subjective than microscopy-or culture-based methods and unaffected by fungal growth conditions, thus capable of discriminating between phenotypically undistinguishable species. This chapter focuses on the contribution of DNA-based techniques to the identification of clinically important fungi such as Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Dematiaceous fungi, Fusarium, Histoplasma, Trichosporon, Zygomycetes, and Dermatophytes. Because of their excellent performances, molecular assays are being increasingly adopted by clinical laboratories to complement conventional methods, providing new diagnostic capabilities.

El maíz es el cultivo más importante de México
de acuerdo con la superficie cultivada anualmente y su
consumo per cápita. Las pérdidas en la producción del grano
se asocian con su manipulación durante la cosecha en el
campo, almacenaje, transporte y procesamiento para el
consumo humano o animal. El grano de maíz posee una
microbiota particular de bacterias, insectos y hongos que
pueden causarle daños. Entre ellos, el género fúngico
Aspergillus y, de éste, las especies A. flavus y A. parasiticus
son las más importantes porque producen aflatoxinas que
provocan gran variedad de efectos tóxicos en seres vivos
expuestos al grano contaminado. Actualmente, las
regulaciones mexicanas establecen límites permisibles sólo
para aflatoxinas en cereales y sus productos, excluyendo
otras micotoxinas. Las condiciones de producción de maíz
en climas tropicales y subtropicales, particularmente en el
noreste de México, favorecen las infecciones por hongos
toxígenos. Por ello, es necesario la identificación e
implementación de estrategias que reduzcan la
contaminación en el grano. Entre ellas, destacan el uso de
híbridos de maíz con resistencia a sequía, plagas,
enfermedades y altos rendimientos de grano (H-436, 437,
439, 443A); manejo integrado de insectos y hongos
mediante tratamiento químico, cultural o biológico y la
modificación del procesamiento del grano para consumo
humano (nixt ama li z a c ión). Di cha s medida s,
individualmente o en conjunto, reducirán paulatinamente
los daños causados por hongos potencialmente toxígenos en
la planta de maíz y el consumidor final en México. En este
trabajo presentamos una perspectiva de la investigación actual en el tema de los hongos aflatoxigénicos en maíz con
énfasis en México, sus implicaciones en salud humana y del
ganado, las herramientas (fitopatológicas, genéticas del
hospedante y el patógeno, bioquímicas, entre otras) de
estudio del problema, así como las estrategias de manejo
integrado utilizadas, para actualizar y ponderar la
información generada a la fecha, y establecer puntos
esenciales para futuras investigaciones.

Maize is the major crop in Mexico according to
the annually cultivated area and its per capita consumption.
The losses on grain production are associated with handling
during field harvest, storage, transportation, and processing
for either human or animal consumption. The maize grain
has particular pests associated such as bacteria, insects and
fungi that can cause damage. Among them, the fungal genus
Aspergillus and particularly species A. flavus and A.
parasiticus are outstanding due to their production of
aflatoxins which cause a broad variety of toxic effects on
living organisms exposed to contaminated grains. Currently,
Mexican regulations establish maximum limits of aflatoxins
in cereal products, but other mycotoxins are excluded. The
production conditions for maize in tropical and subtropical
environments, particulary in northeastern Mexico, favor
infections by toxigen fungi. Therefore, it is necessary to
identify and implement strategies that can reduce grain
fungal contamination. Among them, outstand the use of
maize hybrids with resistance to drought, pets, diseases, and
high grain yield (H-436, 437, 439, 443A); integrated
management of insects and fungi by chemical, cultural
and/or biological treatment; and modifications of grain
processing for human consumption ('flour-making'). Such
measures will individual or together gradually reduce
damage caused by potentially toxigenic fungi to the maize
plant and ultimately to the consumer in Mexico. Here, we
present an overview of current research about aflatoxigenic
fungi in maize in Mexico, their implications on both human
and cattle health, the tools (phytopathology, genetics of both
the host and the pathogen, biochemistry, among others) to
study this problem as well as those strategies for integrated
management in order to date and to weight all data published
until now, and then, establish essential points for further
research.

The cultivation of sugarcane represents an important part in the Colom-bian economy due to the diverse applications in industries like liquor, food, paper and biofuels. Sugarcane worldwide production is affected by the presence of phytopathogenic agents, mainly filamentous fungi such as Physalospora tucumanensis (red rot disease) and Fusarium spp. To date in Colombia, Pokka boheng disease whose causal agent is the fungus

A broad range of fungi were isolated from different geographic regions and substrates and identified according to traditional and modern methods. A total of 120 different isolates were assigned to the phyla, Basidiomycota with 8 isolates, Ascomycota with 75 isolates, and “Zygomycota” with 37 isolates. Although morphological characters were able to differentiate the isolates to their phyla and in most cases to the correct genera, a combination of several methods is always recommended because characterization and identification of unknown fungal isolates is highly error-prone if relying on single methods. Sequence-based identification turned out to be reliable for most Ascomycetes and Zygomycetes. But with the ongoing questionable trend to rely on sequences as first source information for species separation, the most serious problems are the annotation problems in public reference databases, the inconsistency of described taxa, and the available reference data.

Mycetoma is a chronic granulomatous, subcutaneous disease endemic in tropical and subtropical countries. It is currently a health problem in rural areas of Africa, Asia and South America. Nine cases of mycetoma were analysed in a retrospective study. All isolates were identified by morphological features. The level of species identification was reached by molecular tools. Definitive identification of fungi was performed using sequence analysis of the ITS of the ribosomal DNA region and the ribosomal large-subunit D1/D2. Identification of actinomycetes was accomplished by the 16S rRNA gene sequence. Six unusual clinical isolates were identified: Aspergillus ustus, Cyphellophora oxyspora, Exophiala oligosperma, Madurella pseudomycetomatis, Nocardia farcinica and Nocardia wallacei. The prevalence of mycetoma in Venezuela remains unknown. This study represents the first report in the literature of mycetoma caused by unusual pathogens identified by molecular techniques.

A broad range of fungi were isolated from different geographic regions and substrates and identified according to traditional and modern methods. A total of 120 different isolates were assigned to the phyla, Basidiomycota with 8 isolates, Ascomycota with 75 isolates, and "Zygomycota" with 37 isolates. Although morphological characters were able to differentiate the isolates to their phyla and in most cases to the correct genera, a combination of several methods is always recommended because characterization and identification of unknown fungal isolates is highly error-prone if relying on single methods. Sequence-based identification turned out to be reliable for most Ascomycetes and Zygomycetes. But with the ongoing questionable trend to rely on sequences as first source information for species separation, the most serious problems are the annotation problems in public reference databases, the inconsistency of described taxa, and the available reference data.

Anaerobic fungi are phylogenetically unique and form a separate group, the Neocallimastigomycota, among the chitinous fungi. Until now six genera are described within that phylum, namely the monocentric genera Neocallimastix, Caecomyces and Piromyces as well as the polycentric genera Anaeromyces, Cyllamyces and Orpinomyces. This chapter gives a brief survey of the fascinating world of anaerobic rumen fungi, their phylogeny, and identification. The golden standards of molecular identification as well as promising alternatives will be discussed.

The aim of this study was to investigate the ligninolytic system of fungal strains isolated from Egyptian agricultural soil which are
efficient in biodegradation and mineralization of lignin. They were identified morphologically, microscopically and confirmed by
RAPD profiles. Based on PCR amplification and sequencing of the 18S rDNA gene an analytical phylogenetic tree was drawn
for species confirmation. The screening experiment revealed that Emericella nidulans, Aspergillus fumigatus, Phoma betae,
Penicillium oxalicum and Humicola grisea exhibited maximum potential for high lignin degradation. They showed higher lignin
peroxidase and laccase activities. By process optimization, enhanced lignin degradation (94 %) was achieved within 7 days
in lignin-glucose medium when compared with lignin degradation (70 %) obtained in glucose-free medium. The ligninolytic
enzymatic activities had a great potential for decolorization of chemically different synthetic dyes (Azure B, Safranin, Crystal Violet
and Malachite Green). The highest enzymatic activities and the highest decolorization rates were detected at a dye concentration
of 0.2 g/L. The dye decolorization rate significantly increased with tryptophan addition as (1 mmol/L). Humicola grisea showed
the highest decolorization rate (99 %) with azure B. Phoma beta also showed a high decolorization rate (99 % with crystal
violet and 90 % with safranin). The observed activity enhancement resulted from the protective effect of tryptophan against H2O2 inactivation.

Anaerobic fungi are phylogenetically unique and form a separate group, the Neocallimastigomycota, among the chitinous fungi. Until now six genera are described within that phylum, namely the monocentric genera Neocallimastix, Caecomyces and Piromyces as well as the polycentric genera Anaeromyces, Cyllamyces and Orpinomyces. This chapter gives a brief survey of the fascinating world of anaerobic rumen fungi, their phylogeny, and identification. The golden standards of molecular identification as well as promising alternatives will be discussed.

The cultivation of sugarcane represents an important part in the Colombian economy due to the diverse applications in industries like liquor, food, paper and biofuels. Sugarcane worldwide production is affected by the presence of phytopathogenic agents, mainly filamentous fungi such as Physalospora tucumanensis (red rot disease) and Fusarium spp. To date in Colombia, Pokka boheng disease whose causal agent is the fungus Fusarium verticillioides, has not been reported, which is why it is necessary to identify appropriately this microorganism, responsible for losses in productivity and food contamination. In order to isolate and identify the infectious agent from symptomatic tissues, disinfection and culture in liquid and solid culture mediums were performed in malt extract (2%) and yeast extract (0.2%) both liquid and solid, for 7 days. After several replicates in agar plate, a purification was made along with a morphological characterization based on the shape and color of the mycel...

To further our understanding of wood decay in living light red meranti (Shorea smithiana) trees, microscopic characteristics of the cell and cell wall degradations of S. smithiana wood in the presence of the decay fungi, the identity of the causal fungi, and the decay potential and pattern by an isolated fungus were investigated. Cell wall degradations, including cell wall thinning,