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Identification and Characterization of Gliocladium viride Isolated from Mushroom Fly Infested Oak Log Beds Used for Shiitake Cultivation
Mycobiology
Abstract and Figures
A green mold species that has not previously been reported in Korea was isolated from oak log beds used for shiitake (Lentinula edodes) cultivation that were infested by mushroom flies. In this study, we identify the mold species as Gliocladium viride (an anamorph of Hypocrea lutea) and describe its mycological properties. The fungus was cottony on both potato dextrose agar (PDA) and Czapek yeast extract agar (CYA), but was colored white on PDA and became yellowish green and brown on CYA. Mycelial growth on PDA attained a diameter of 73 mm at 30℃ after 5 days. The fungus grew faster on malt extract agar (> 80 mm, 5 days at 25℃) compared to CYA and PDA (< 68 mm, 5 days at 25℃). Penicillate conidiophores of the fungus are hyaline, smooth walled, branching above typically in four stages, and 120~240 µm in length. Club-shaped or slender phialides are formed on the metulae. Conidia of the fungus were ovate and elliptic, yellowish brown and green, and 2.5~3.0 µm × 1.8~2.3 µm in size. Typically, slimy conidia are formed in a mass and colored brown to dark green to almost black. The internal transcribed spacer rDNA and translation elongation factor 1 alpha gene sequences of the fungus isolated here show 99% identity with previously identified G. viride strains.
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... Trichoderma, Diatrype, and Hypoxylon are problematic ascomycete genera during shiitake cultivation in Korea [11]. Recently, several green mold species of Trichoderma and Gliocladium genera associated with mushroom fly damaged shiitake during cultivation on oak logs in the domestic environment [12][13][14]. In a previous survey work of greenhouse diseases in shiitake mushroom, we found a new disease of brown rot caused by the basidiomycete yeast Cryptococcus pseudolongus [15]. ...
... The C. pseudolongus DUCC 4014 inoculated and non-inoculated (control) fruit bodies were also subject to scanning electron microscopy (SEM) to observe comparatively the microstructures of the surface layer and the edge of the mushroom cap and the basidia and basidiospores developed and aligned on gills. For SEM image analysis, the subject samples were prefixed with 1% osmium tetroxide, prepared as described by Kim et al. [12], and observed by SEM (S-4300 Hitachi, Tokyo, Japan). ...
Recently, Cryptococcus pseudolongus has been reported as a new pathogen of shiitake (Lentinula edodes). However, its pathological properties are not much known. To further understand its impact on the mushroom, we investigated the pathogen’s interactions with the mycelium of shiitake, histopathological properties, host range, and sensitivity to diverse antifungal agents. The strain C. pseudolongus DUCC 4014 inhibited the mycelial growth of L. edodes strain (cultivar Sanjo 701ho) and caused browning in the mycelia confronted with the yeast on PDA. Spray inoculation of the yeast caused an abnormal browning symptom on the cap and/or gills of three shiitake cultivars grown on sawdust media in vinyl bags. Scanning electron microscopic images of the abnormally browned parts of shiitake fruit body illustrated that mushroom tissues were loosed and dispersed in the middle and edge of the cap and the arrangement of basidiospores borne on basidia in the gills was disturbed compared to those of normal shiitake fruit body. Spray inoculation also led to developing abnormal browning on the harvested fruit body, indicating C. pseudolongus could be a problem during mushroom storage. But the yeast was not able to induce abnormal browning on mushrooms of Pleurotus ferulae, Pleurotus fostreatus, and Agaricus bisporus. But it induced browning only on button mushroom (A. bisporus) when they were inoculated after wounding. Tests with 16 kinds of fungicides revealed that the cell growth of C. pseudolongus could be inhibited by benzalkonium chloride at MIC 7 μg/ml and benomyl at MIC 3 μg/ml.
... These sticks were kept as intact as possible to avoid secondary contamination due to breakage. Cultures were obtained by aseptically transferring the contaminated substrates of collected stick specimens onto potato dextrose agar (PDA) following the method of Kim et al. (2010) (Supplementary Figure S1). The isolation frequency (%) for Trichoderma species was calculated: the number of isolates per species/total number isolates × 100. ...
Introduction
The contamination of Trichoderma species causing green mold in substrates poses a significant obstacle to the global production of Lentinula edodes, adversely impacting both yield and quality of fruiting bodies. However, the diversity of Trichoderma species in the contaminated substrates of L. edodes (CSL) in China is not clear. The purpose of this study was to assess the biodiversity of Trichoderma species in CSL, and their interactions with L. edodes.
Methods
A comprehensive two-year investigation of the biodiversity of Trichoderma species in CSL was conducted with 150 samples collected from four provinces of China. Trichoderma strains were isolated and identified based on integrated studies of phenotypic and molecular data. Resistance of L. edodes to the dominant Trichoderma species was evaluated in dual culture in vitro.
Results
A total of 90 isolates were obtained and identified as 14 different Trichoderma species, including six new species named as Trichoderma caespitosus, T. macrochlamydospora, T. notatum, T. pingquanense, T. subvermifimicola, and T. tongzhouense, among which, T. atroviride, T. macrochlamydospora and T. subvermifimicola were identified as dominant species in the CSL. Meanwhile, three known species, namely, T. auriculariae, T. paraviridescens and T. subviride were isolated from CSL for the first time in the world, and T. paratroviride was firstly reported to be associated with L. edodes in China. Notebly, the in vitro evaluation of L. edodes resistance to dominant Trichoderma species showed strains of L. edodes generally possess poor resistance to Trichoderma contamination with L. edodes strain SX8 relatively higher resistant.
Discussion
This study systematically investigated the diversity of Trichoderma species in the contaminated substrate of L. edodes, and a total of 31 species so far have been reported, indicating that green mold contaminated substrates of edible fungi were undoubtedly a biodiversity hotspot of Trichoderma species. Results in this study will provide deeper insight into the genus Trichoderma and lay a strong foundation for scientific management of the Trichoderma contamination in L. edodes cultivation.
... Specimens were separately collected from contaminated substrate of edible fungi in North China from 2020 to 2022 (Table S1), and strains were isolated following the method of a previous study [11]. The ex-type strains were deposited in the culture collection of Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences (JZB culture collection). ...
Trichoderma is known worldwide as biocontrol agents of plant diseases, producers of enzymes and antibiotics, and competitive contaminants of edible fungi. In this investigation of contaminated substrates of edible fungi from North China, 39 strains belonging to 10 Trichoderma species isolated from four kinds of edible fungi were obtained, and three novel species belonging to the Harzianum clade were isolated from the contaminated substrates of Auricularia heimuer and Pholiota adipose. They were recognized based on integrated studies of phenotypic features, culture characteristics, and molecular analyses of RNA polymerase II subunit B and translation elongation factor 1-α genes. Trichoderma auriculariae was strongly supported as a separate lineage and differed from T. vermifimicola due to its larger conidia. Trichoderma miyunense was closely related to T. ganodermatigerum but differed due to its smaller conidia and higher optimum mycelial growth temperature. As a separate lineage, T. pholiotae was distinct from T. guizhouense and T. pseudoasiaticum due to its higher optimum mycelial growth temperature and larger conidia. This study extends the understanding of Trichoderma spp. contaminating substrates of edible fungi and updates knowledge of species diversity in the group.
... produced a lot of conidia that can spread easily, they became detrimental fungi in oak wood mushroom cultivation (Davidse, 1986;Eveleigh, 1985). In Korea, T. citirnoviride and Gliocladium viride (Current name: Trichoderma deliquescens) were reported to new recorded pathogens to oak wood mushroom (Kim et al., 2010;Kim et al., 2012b). In 2016, the damage rate of oak wood mushroom production by green mold disease of Trichoderma spp. ...
Some species of the Trichoderma genus are reported as the major problem in oak wood mushroom production in Korea. In spite of economic loss by the fungi, scientific information on airborne Trichoderma species is not much available. To generate information for disease management development we analyzed airborne Trichoderma. A total of 1,063 fungal isolates were purely obtained from indoor air sampling of cultivation houses used for oak wood mushroom using sawdust media. Among the obtained isolates, 248 isolates were identified as Trichoderma fungi including T. harzianum, T. atroviride, T. citrinoviride, and T. pseudokoningii, by morphological and molecular analysis. T. harzianum was dominant among the four identified species. All the four Trichoderma species grew fast on solid nutrient media tested (potato dextrose agar [PDA], malt extract agar [MEA], Czapek's Dox + yeast extract agar [CYA] and cornmeal dextrose agar). Compact mycelia growth and mass spore production were better on PDA and CYA. In addition, T. harzianum and T. citrinoviride formed greenish and yellowish mycelium and spores on PDA and CYA. Greenish and yellowish pigment was saturated into PDA only by T. pseudokoningii. These four Trichoderma species could produce extracellular enzymes of sawdust substrate degradation such as β-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease. Their mycelia inhibited the growth of oak wood mushroom mycelia of two tested cultivars on dual culture assay. Among of eleven antifungal agents tested, benomyl was the best to inhibit the growth of the four Trichoderma species. Our results demonstrate that the airborne Trichoderma fungi need to be properly managed in the cultivation houses for safe mushroom production.
... es la presencia de conidióforos y fiálides semejantes a las de Penicillium; estos conidióforos son erectos y poseen ramificaciones en la parte superior. Kim et al. (2010) destacan que sus fiálides son ramificadas y al final de ellas se encuentran los conidios con morfología esférica y en grupos, formando una bola. Estas características descritas por dichos autores concuerdan con lo observado bajo el microscopio de Gliocladium sp. ...
Algunos géneros fúngicos han sido utilizados para el control de agentes causantes de enfermedades en plantas, por lo que su actividad representa una alternativa para la disminución e incluso la sustitución de químicos sintéticos. Existen hongos antagonistas ampliamente estudiados, como Trichoderma sp., que concurre con otros hongos que poseen un alto potencial como agentes biocontroladores, entre ellos se destaca Gliocladium sp. En este estudio se pretende establecer la capacidad antagonista que tiene Gliocladium contra el patógeno causante de la pudrición blanca de la cebolla, Sclerotium cepivorum. Se logró aislar Gliocladium sp. a partir de una muestra de suelo dedicada al cultivo de cebolla (Allium cepa L.), se subcultivó hasta obtener un cultivo axénico y se realizaron cultivos duales en placas con el hongo y el patógeno. Se determinó que el porcentaje de inhibición del crecimiento radial (PICR) promedio obtenido a los ocho días de exposición (53,66%) no presentaba diferencias significativas (p=0,19) con el obtenido a los 15 días (54,20%) y, dadas las características de inhibición, se presume que el mecanismo de control presentado por la cepa de Gliocladium sp. utilizada responde a la producción de enzimas, metabolitos y posiblemente una mezcla de compuestos orgánicos volátiles capaces de inhibir el crecimiento y desarrollo de S. cepivorum aun sin que sus hifas entren en contacto.
Mushrooms are important organisms because of their human nutritional and medicinal value. With the expansion of the cultivation of edible mushrooms, fungal diseases have become a major problem in limiting their production. Numerous fungi can cause mushroom deformation or rots. In this publication we report on fungal diseases found during Morchella cultivation in China, with emphasis on morphology and phylogeny to characterise species. The key findings include 1) establishment of a new family Albomorchellophilaceae in Hypocreales, and a novel monotypic genus Albomorchellophila with the type species A. morchellae. Divergence time estimates indicate that Albomorchellophilaceae diverged from its sister family Calcarisporiaceae at ca. 105 (92–120) MYA; 2) the phylogeny and morphology of the family Pseudodiploosporeaceae (Hypocreales) is revised. The family contains a single genus Pseudodiploospora. Intraspecific genetic analyses of Pseudodiploospora longispora reveals significant base differences within strains, especially in the regions of protein-coding genes RPB 2 and TEF; 3) four fungicolous taxa, i.e., Cylindrodendrum alicantinum, Hypomyces aurantius, Hypomyces rosellus, and Trichothecium roseum, are reported as putative pathogens on cultivated morels for the first time. In addition, the previously reported pathogens of morels, Clonostachys rosea, Clonostachys solani, Hypomyces odoratus, and Pseudodiploospora longispora are also detailed in their symptoms and morphology; 4) the phylogeny and morphology of “Zelopaecilomyces” previously placed within Pseudodiploosporeaceae are re-assessed. “Zelopaecilomyces” is proved to be introduced through a chimerism of gene fragments sourced from two distinct organisms. Consequently, it is recommended that “Zelopaecilomyces” should not be recognised due to the mixed up molecular data in phylogeny and a lack of support from morphological evidence. Furthermore, this study discusses the voucher specimen Paecilomyces penicillatus (CBS 448.69), which may contain two mixed taxa, i.e., Pseudodiploospora longispora and a member of Penicillium. Publications on pathogenic fungi of cultivated mushrooms is sporadically, which leads to a lack of understanding of causal agents. As a follow up to the diseases of morel cultivation, we also review the fungal diseases of cultivated mushrooms reported over the last four decades. More than 130 pathogens affect the growth and development of the main cultivated mushrooms. The taxonomic diversity of these pathogens is high, distributed in 58 genera, 40 families, 20 orders, 12 classes and six phyla. The host infected are from Ascomycota to Basidiomycota, mainly being reported from Agaricus bisporus, Cordyceps militaris, Morchella spp., and Pleurotus spp. This study not only enriches our current knowledge on the diversity of pathogens of cultivated mushrooms, especially morels, but also recognizes the importance of some taxa as potential pathogens. Taxonomic investigation and accurate identification are initial and key steps to understanding pathogen-mushroom interactions, and will result in better disease management strategies in the mushroom industry.
This chapter presents a literature overview about the green mould disease of cultivated mushrooms caused by Trichoderma species, a problem which may result in serious economic damage due to substantial losses of crop yield in mushroom production. The first major green mould epidemic struck the production of white button mushroom (Agaricus bisporus) in Northern Ireland in 1985, but since then, the problem has occurred in a series of further countries. Outbreaks appear to be primarily caused by two subspecies of Trichoderma aggressivum. Trichoderma green mould infection has also been observed in many countries in cultivated oyster mushroom (Pleurotus ostreatus), where the main pathogens have been described as T. pleuroticola and T. pleuroti. Moreover, various further Trichoderma species have been reported to cause green mould problems in the production of shiitake (Lentinula edodes), as well as less frequently produced other mushrooms like Ganoderma species and Cyclocybe aegerita.Components of mushroom cultivation substrata, the air, vehicles, equipment, contaminated clothing and animal vectors are among the potential sources of green mould infections. General symptoms of green mould generally appear in the mushroom cultivation substratum as large, greenish spots, but the causal agents may also colonise the surface of fruiting bodies. Green mould species compete efficiently for nutrients and space in the cultivation substratum; in addition, they produce extracellular enzymes, toxic secondary metabolites and volatile organic compounds that can lead to drastic crop losses.Species-specific primers are now available for the effective monitoring of Agaricus-, Pleurotus- and Lentinula pathogenic Trichoderma species. Control measures include pasteurisation, the use of disinfectants, chemical treatments, biological control by natural products or the use of antagonistic bacteria and fungi, as well as the breeding and cultivation of resistant mushroom cultivars.KeywordsWhite button mushroom
Agaricus bisporus
Shiitake
Lentinula edodes
Oyster mushroom
Pleurotus ostreatus
Ganoderma
Cyclocybe aegerita
Green mould disease
Trichoderma
Previously, severe green mould infections could be attributed mainly to Trichoderma aggressivum Samuels & W. Gams, as well as T. pleuroti S.H. Yu & M.S. Park and T. pleuroticola S.H. Yu & M.S. Park in the case of Agaricus bisporus (J.E. Lange) Imbach (button mushroom) and Pleurotus ostreatus (Jacq.) P. Kumm. (oyster mushroom), respectively. The purpose of our study was the examination of green mould agents deriving from the growing facilities of button mushroom, oyster mushroom and shiitake (Lentinula edodes (Berk.) Pegler) located in various countries of Europe, and initially classified into the Trichoderma harzianum Rifai species complex (THSC). Species identification was carried out using the multilocus sequence typing analysis of the internal transcribed spacer regions, as well as translation elongation factor 1-alpha, calmodulin and RNA polymerase B subunit II gene sequences. In vitro confrontation assays were applied to test the aggressiveness of the isolates towards mushrooms, while the effect of commercial fungicides on the growth of the strains was examined by the macrodilution method. Six Trichoderma species, namely T. afroharzianum P. Chaverri, F.B. Rocha, Degenkolb & Druzhin., T. atrobrunneum F.B. Rocha, P. Chaverri & Jaklitsch, T. guizhouense Q.R. Li, McKenzie & Yong Wang, T. harzianum sensu stricto, T. pollinicola F. Liu & L. Cai and T. simmonsii P. Chaverri, F.B. Rocha, Samuels, Degenkolb & Jaklitsch were detected in the different samples, with T. harzianum, T. pollinicola and T. simmonsii being the most aggressive. Prochloraz was found to have strong in vitro inhibitory effect on mycelial growth on most strains, however, T. simmonsii isolates showed remarkable tolerance to it. Our data suggest that T. harzianum and T. simmonsii may also be considered as potential causal agents of mushroom green mould.
Plant viruses pose a serious threat to agricultural production and incur
enormous costs to growers each year, both directly, in the form of yield and quality
loss, and indirectly, in the forms of time and funds spent on scouting and disease
management. Virus diseases cause plants crops losses annually in average of US$
60 billion. Accordingly, viral diseases need to be controlled for the sustainable agriculture and in order to maintain the quality and abundance of food production. Moreover, recently agriculture suffered from different problems such as; the contentious
changes in climatic factors globally, increasing the numbers of pathogens per year
and appearance of anew pests. Unfortunately, chemical control has negative impact
on the environment and on human health as well in addition it creates an imbalance
in the microbial biodiversity, which may be unfavorable to the activity of the beneficial organisms and may lead to the development of pathogens-resistant strains. The
most important thing is that agricultural sustainability should be supported by ecofriendly approaches such as discovery of new biocontrol agents capable to control the
plant viral diseases. To achieve this was inevitable to use the plant growth-promoting
microbes as effective biocontrol agents against plant viruses will hold the greatest
promise and is considered a pillar of integrated viral diseases management. We argue
that the use of growth-promoting microbes will preserve sustainable agriculture as
well as a clean environment free from pollution, which will be benefiting for both
the farmer and the consumer. So far, there are no such pesticides at the local level,
while at the international level there may be one or two products. We succeeded to
control some viruses infect potato by using the filtrates of seven Bacillus spp. mixed
with nanoclay, but the product is still under research and development.
Plant fungal diseases are the most destructive diseases where the fungal pathogens attack many economic crops causing yield losses, which affect directly many countries’ economy. The great Irish Famine in 19th century was due to potato (a great portion of Irish diets) was attacked by an oomycete pathogen Phytophthora infestans causing late blight disease which destroyed the potato crop for several years (1845–1852). Since this date the plant fungal diseases have a great attention from the researchers. Control of fungal diseases using different fungicides has dangerous effects on human beings as well as animals by precipitating in the plant tissues and then transfer to human and animals causing many health complications. Hence, the biological control of plant pathogenic fungi became the most important issue, due to the chemical risk to control the fungal diseases. From 1990’s the importance of using microorganisms was increased as biocontrol agents to decrease the chemical uses and their hazardous for human and animal health topics. In this chapter, using of different microorganism as biological control agents of plant fungal diseases were reviewed, as well as using chemicals in controlling fungal diseases and their effects on plants, environment and common health impacts.
The systematics of species of Hypocrea with green ascospores and their Trichoderma anamorphs is presented. Multiple phenotypic characters were analysed, including teleomorph and anamorph, as well as colony morphology and growth rates at various temperatures. In addition, phylogenetic analyses of two genes, the RNA polymerase II subunit (RPB2) and translation elongation factor 1-alpha (EF-1α), were performed. These analyses revealed that species of Hypocrea with green ascospores and Trichoderma anamorphs are derived from within Hypocrea but do not form a monophyletic group. Therefore, Creopus and Chromocrea, genera formerly segregated from Hypocrea only based on their coloured ascospores, are considered synonyms of Hypocrea. The present study showed that phenotypic characters alone are generally not helpful in understanding phylogenetic relationships in this group of organisms, because teleomorph characters are generally highly conserved and anamorph characters tend to be morphologically divergent within monophyletic lineages or clades. The species concept used here for Hypocrea/Trichoderma is based on a combination of phenotypic and genotypic characteristics. In this study 40 species of Hypocrea/Trichoderma having green ascospores are described and illustrated. Dichotomous keys to the species are given. The following species are treated (names in bold are new species or new combinations): H. albocornea, H. atrogelatinosa, H. aureoviridis/T. aureoviride, H. candida/T. candidum, H. catoptron/T. catoptron, H. centristerilis, H. ceracea/T. ceraceum, H. ceramica/T. ceramicum, H. chlorospora/T. chlorosporum, H. chromosperma/T. chromospermum, H. cinnamomea/T. cinnamomeum, H. clusiae, H. cornea, H. costaricensis, H. crassa/T. crassum, H. cremea/T. cremeum, H. cuneispora/T. cuneisporum, H. estonica/T. estonicum, H. gelatinosa/T. gelatinosum, H. gyrosa, H. lixii/T. harzianum, H. macrospora, H. melanomagna/T. melanomagnum, H. nigrovirens/T. nigrovirens, H. phyllostachydis/T. phyllostachydis, H. rugulosa, H. sinuosa/T. sinuosum, H. spinulosa, H. straminea/T. stramineum, H. strictipilosa/T. strictipile, H. substipitata, H. sulawesensis, H. surrotunda/T. surrotundum, H. tawa/T. tawa, H. thailandica/T. thailandicum, H. thelephoricola/T. thelephoricola, H. tuberosa, H. velenovskyi, H. virens/T. virens, and H. virescentiflava. The following species are excluded: H. andinogelatinosa, H. dacrymycella, H. dichromospora, H. palmicola, H. pseudogelatinosa, H. subalbocornea, H. subatrogelatinosa, H. tropicosinensis, H. viscidula, H. viridis, and Chromocrea leucostroma.
Trichoderma spp. is the cause of green mold, a disorder that affects cultivated mushrooms. The aims of the study were to establish whether improvement of mushroom resistance to Trichoderma aggressivum could be obtained by inducing reaction mechanisms before contact with the pathogen and whether this ability was species or strain dependent. Twenty nine isolates of Agaricus bisporus, 29 isolates of Lentinula edodes and 18 isolates of Pleurotus spp. were studied. The effect of T. harzianum metabolites on mycelial growth of these isolates was evaluated on YMEA (yeast, malt extract and agar), supplemented or not with Lysing Enzymes from T. harzianum (Sigma®, L1412). Mycelial growth generally was affected by Lysing Enzymes, but some L. edodes and Pleurotus spp. adapted to Lysing Enzymes. When mycelium was taken from a first culture with Lysing Enzymes and placed on YMEA with Lysing Enzymes for a second culture, their growth rate was not different from those of the controls. In the case of A. bisporus, only partial adaptation was obtained with a few isolates. The effect of adaptation to Lysing Enzymes on resistance to T. aggressivum was assayed for one strain of each group. Trichoderma aggressivum was exposed to the margin of 5- to 9-day-old mushroom colonies. Agaricus bisporus produced brown droplets, and T. aggressivum overgrew its mycelium. Lentinula edodes and P. ostreatus produced brown lines blocking the progression of T. harzianum, both on YMEA and YMEA plus Lysing Enzymes. The line was visible after 3 d on YMEA and after only 2 d on YMEA plus Lysing Enzymes. Improvement in the resistance to antagonists by introduction of some of their metabolites to the culture medium is a method for mushroom protection.
A rapid, sensitive, and simple method was developed to detect the sapstain fungi Ophiostoma piceae and O. quercus in stained wood. By using microwave heating for DNA extraction and PCR with internal transcribed spacer-derived-specific primers, detection was feasible within 4 h, even with DNA obtained from a single synnema. This method can easily be extended for the detection of other wood-inhabiting fungi.
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
A total of 179 isolates of Trichoderma spp. were collected from oyster mushroom substrates in Korea. On the basis of morphological and cultural characteristics, Trichoderma isolates were divided into seven groups, namely T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum, T. virens, and two unidentified species, referred to as Trichoderma sp. 1 and 2. The predominant species was Trichoderma sp. 2 (n=86) followed by Trichoderma sp. 1 (n=52). Trichoderma sp. 1 and 2 were morphologically distinct not only from the other species of Trichoderma reported but also from each other in the characteristics such as mycelial growth rate, colony appearance, shape of conidia and conidiophores and branching pattern of phialides, although branching pattern of phialides of Trichoderma sp. 1 was similar to that of T. harzianum. In virulence test, the degree for compost colonization of Trichoderma sp. 2 was significantly greater than that of the other Trichoderma species. Trichoderma sp. 2 was found to be the main cause of green mold disease in oyster mushroom production. More work including molecular characterization is needed to confirm the species of Trichoderma sp. 1 and 2.
Some simple formulae were obtained which enable us to estimate evolutionary distances in terms of the number of nucleotide substitutions (and, also, the evolutionary rates when the divergence times are known). In comparing a pair of nucleotide sequences, we distinguish two types of differences; if homologous sites are occupied by different nucleotide bases but both are purines or both pyrimidines, the difference is called type I (or "transition" type), while, if one of the two is a purine and the other is a pyrimidine, the difference is called type II (or "transversion" type). Letting P and Q be respectively the fractions of nucleotide sites showing type I and type II differences between two sequences compared, then the evolutionary distance per site is K = -(1/2) ln [(1-2P-Q) square root of 1-2Q]. The evolutionary rate per year is then given by k = K/(2T), where T is the time since the divergence of the two sequences. If only the third codon positions are compared, the synonymous component of the evolutionary base substitutions per site is estimated by K'S = -(1/2) ln (1-2P-Q). Also, formulae for standard errors were obtained. Some examples were worked out using reported globin sequences to show that synonymous substitutions occur at much higher rates than amino acid-altering substitutions in evolution.
Two novel metabolites with potential antifeedant activity were isolated from cultures of the fungus Trichoderma citrinoviride strain ITEM 4484 grown in solid-state fermentation on sterile rice kernels. The producing strain was identified at species level by sequence analysis of the internal transcribed spacer regions ITS-1 and ITS-2 of the nuclear rDNA and a fragment of the translation elongation factor gene TEF-1alpha. Fractionation by column chromatography and TLC of the culture organic extract, followed by feeding preference tests on the aphid pest Schizaphis graminum (Rondani), allowed the purification of 5.8 and 8.9 mg/kg of culture of two bioactive metabolites, which were named citrantifidiene and citrantifidiol ( 1 and 2). Citrantifidiene and citrantifidiol, whose structures were determined by spectroscopic methods (NMR and MS) are a symmetrical disubstituted hexa-1,3-dienyl ester of acetic acid and a tetrasubstituted cyclohexane-1,3-diol, respectively. The pure metabolites influenced the feeding preference of S. graminum restraining individuals from feeding on wheat leaves dipped in 5% aqueous methanol solution containing 0.57 mg/mL of citrantifidiene or 0.91 mg/mL of citrantifidiol.
PCR protocols: a guide to methods and applications
- TJ White
- T Bruns
- S Lee
- J Taylor
Sur un Gliocladium nouveau
- L Matruchot