Taxonomy and biocontrol potential of a new species of <Emphasis Type="Italic">Trichoderma</Emphasis> from the Amazon basin of South America

The new species Trichoderma ovalisporum is described and its biocontrol potential against Crinipellis species is analyzed. Using partial nuclear translation elongation factor 1α (EF-1α) and partial nuclear actin gene intron and exon sequences, T. ovalisporum is identified as a member of Trichoderma sect. Trichoderma and as a close relative of T. koningii-like taxa that have ellipsoidal to oblong, smooth conidia. Sequences of the internal transcribed spacer regions 1 and 2 (ITS) of the nuclear ribosomal gene cluster did not resolve the phylogeny of T. ovalisporum and its closest relatives. Trichoderma ovalisporum is morphologically similar to T. koningii, Hypocrea stilbohypoxyli, and three as yet undescribed Trichoderma taxa. It differs from its close relatives in having smaller, ovoidal conidia and in its fast rate of growth at 30 °C. Trichoderma ovalisporum was isolated twice: once from witches’ broom (Crinipellis perniciosa)-infected tissue of a liana (Banisteriopsis caapi, Malpighiaceae) collected in Ecuador. The second isolation was from the healthy bole of a mature tree of Theobroma grandiflorum (cupuaçu, Malvaceae) collected in Brazil (Pará). The liana isolate reinfected and was reisolated from meristematic tissues of seedlings of Theobroma cacao, and inhibited radial growth of the frosty pod rot pathogen (Crinipellis roreri) in vitro. It also persisted on the surface, and within the tissues, of cocoa pods in the field for at least 10 weeks.     

Molecular characterisation of fungal endophytic morphospecies associated with the indigenous forest tree, Theobroma gileri, in Ecuador

Fungal endophytes were isolated from healthy stems and pods of Theobroma gileri, an alternative host of the frosty pod rot pathogen of cacao. Non-sporulating isolates were grouped into 46 different morphological species according to their colony morphology. Many of these morphospecies were assumed to be basidiomycetes and, therefore, were of particular interest. Basidiomycetous endophytes have received far less attention than ascomycetes and also have potential as biological control agents of the basidiomycetous pathogens of T. cacao: Moniliophthora roreri (frosty pod rot pathogen) and M. perniciosa (witches' broom disease). The morphospecies were further characterised by molecular analyses. Amplification of the nuLSU was undertaken for phylogenetic placement of these non-sporulating cultures and revealed a total of 31 different taxa of which 15 were basidiomycetes belonging to the class Agaricomycetes, and 16 ascomycetes primarily belonging to the Sordariomycetes.     

Creation of BAC genomic resources for cocoa (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) for physical mapping of RGA containing BAC clones

We have constructed and validated the first cocoa (Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp (palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.Communicated by J.W. Snape     

Quality control by leaf-cutting ants: evidence from communities of endophytic fungi in foraged and rejected vegetation

Leaf-cutting ants of the genera Acromyrmex and Atta forage vegetation for incorporation into their mutualistic fungal gardens. However, the presence of certain endophytic fungi in this predominantly leaf-based material could affect the fungal garden and thus the choice of material by the ants. The present study was conducted to document the endophytic fungal communities occurring in the vegetation being transported by workers of Atta laevigata into their nests and to compare this community structure with that of the vegetative material subsequently rejected from the nests. We found considerable diversity in the fungi isolated. Acremonium, Cylindrocladium, Drechslera, Epicoccum, Fusarium, Trichoderma, Ulocladium and two unidentified morphospecies were significantly more common in rejected compared with foraged material, and some of these genera include mycoparasites, which could represent a threat to the fungal gardens. Conversely, Colletotrichum, Pestalotiopsis, Phomopsis, Xylaria and an unidentified morphospecies were more common in carried compared with rejected material. The possibility that ants have a ‘quality-control’ mechanism based on the presence of antagonistic fungal endophytes is discussed, as is the potential use of these fungi as biocontrol agents against Attini pests.     

Characterization of microsatellites in the fungal plant pathogen Crinipellis perniciosa

Microsatellite markers of Crinipellis perniciosa, with three and four repeats, were developed from sequence database and evaluated for their usefulness in detecting genetic polymorphism. Thirty‐three primers produced unambiguous amplification products of 28 microsatellite‐containing loci and 14 microsatellite‐like polymorphic loci, with two to seven alleles at each locus. Three loci were useful to distinguish isolates from different biotypes and isolates from different countries. Amplification of the markers in the closely related fungi Moniliophthora roreri indicates that their usefulness in population's studies may go beyond the present study of the C. perniciosa and may have applications in population genetics of M. roreri.     

Moniliophthora roreri,causal agent of cacao frosty pod rot

Taxonomy : Moniliophthora roreri (Cif.) H.C. Evans et al. 1978 ; Phylum Basidiomycota; Class Agaricomycetes; Order Agaricales; Family Marasmiaceae; Genus Moniliophthora. Biology : Moniliophthora roreri attacks Theobroma and Herrania species causing frosty pod rot. Theobroma cacao (cacao) is the host of major economic concern. Moniliophthora roreri is a hemibiotroph with a long biotrophic phase (45–90 days). Spore masses, of apparent asexual origin, are produced on the pod surface after initiation of the necrotrophic phase. Spores are spread by wind, rain and human activity. Symptoms of the biotrophic phase can include necrotic flecks and, in some cases, pod malformation, but pods otherwise remain asymptomatic. Relationship to Moniliophthora perniciosa : Moniliophthora roreri and Moniliophthora perniciosa, causal agent of witches’ broom disease of cacao, are closely related. Their genomes are similar, including many of the genes they carry which are considered to be important in the disease process. Moniliophthora perniciosa, also a hemibiotroph, has a typical basidiomycete lifestyle and morphology, forming clamp connections and producing mushrooms. Basidiospores infect meristematic tissues including flower cushions, stem tips and pods. Moniliophthora roreri does not form clamp connections or mushrooms and infects pods only. Both pathogens are limited to the Western Hemisphere and are a threat to cacao production around the world. Agronomic importance : Disease losses caused by frosty pod rot can reach 90% and result in field abandonment. Moniliophthora roreri remains in the invasive phase in the Western Hemisphere, not having reached Brazil, some islands within the Caribbean and a few specific regions within otherwise invaded countries. Disease management : The disease can be managed by a combination of cultural (for example, maintenance of tree height and removal of infected pods) and chemical methods. These methods benefit from regional application, but can be cost prohibitive. Breeding for disease resistance offers the greatest potential for frosty pod rot management and new tolerant materials are becoming available.     

Witches' broom disease of cocoa Crinipellis perniciosa) in Ecuador

Two pathotypes of Crinipellis perniciosa were found in both eastern and western Ecuador. The cocoa strain was highly pathogenic to species of the genera Theobrotna and Herrania and caused the witches' broom disease. The liana strain was weakly or non-pathogenic to cocoa and its allies; it is endemic in the forests of Ecuador on lianes, shrubs and trees and is possibly parasitic in these tissues. The taxonomic position of the fungus is discussed.     

Witches' broom disease of cocoa (Crinipettis perniciosa) in Ecuador.

Inoculation of cocoa with basidiospores of the witches' broom fungus Crinipellis perniciosa showed that stem, petiole and pulvinus tissues of unhardened flushes and also exposed wound surfaces were susceptible to infection in addition to the growing tip. Inoculation of flower cushions and young pods showed that carrot-shaped pods were usually the result of fertilisation of systemically infected flowers. In the absence of fertilisation such flowers produced parthenocarpic pods or chirimoyas. Transmis-sion of the fungus via infected beans was demonstrated and this may be a potent method of long-distance dispersal.     

Application of chemical and biological agents for the management of frosty pod rot (Moniliophthora roreri) in Costa Rican cocoa (Theobroma cacao)

This article describes two field trials carried out at La Lola, Costa Rica, to assess control measures against frosty pod rot of cocoa (Theobroma cacao) caused by Moniliophthora (Crinipellis) roreri. In the first, factorial, trial the control agents were applied using motorised mistblowers (MMs) and hydraulic sprayers fitted with a narrow angle cone nozzle. There was an interaction between agents and application methods; together with previous application data for the most active fungicide (copper hydroxide), these trials indicate that best yields are achieved with sprays that maximise deposits on pods. We describe the droplet size spectra produced by a Stihl SR400 MM under a range of conditions because this has become the standard method of fungicide application in this series of trials at La Lola. The factor that had the largest effect on droplet size spectrum was the presence or the absence of a detachable baffle plate in front of the air‐shear nozzle. In both trials described here, MMs were fitted with baffle plates, a formulation pump and restrictor transmitting 550 mL min^?1 to deliver an estimated equivalent of 190 L ha^?1. Copper hydroxide as prophylactic applications at 1500 g a.i. ha^?1 have, to date, shown the most consistent (but incomplete) improvement in healthy pod yield. Use of copper fungicides may be cost effective when farm‐gate cocoa prices exceed approximately $1.25 kg^?1. In these trials, isolates of the hyperparasitic fungi Clonostachys byssicola and Trichoderma asperellum and two off‐patent triazole fungicides (bitertanol and triadimenol) made no significant improvement to healthy yields. The systemic oxathiin fungicide flutolanil, at a dosage of 300 g a.i. ha^?1, appears to protect pods substantially at early stages but gives proportionately less control of M. roreri than copper at later stages of pod development.     

Mycoparasitism as a mechanism of Trichoderma-mediated suppression of plant diseases

Trichoderma spp. are widely used as plant disease biocontrol agents in agriculture. Mycoparasitism, which is an ancestral trait of Trichoderma, is one of the most important mechanisms of reducing the pathogen inocula. Mycoparasitism is a complex physiological process that should be viewed in the broad perspective of microbial competition, and involves the production of enzymes and secondary metabolites. Trichoderma spp. have traditionally been viewed as necrotrophic mycoparasites; however, there are evidences that, at least in some instances, they behave as hemibiotrophs, causing minor damage to the host cell wall and having an intracellular existence in the host cell for a significant period. In this review, we cover different aspects of Trichoderma as mycoparasites, ranging from evolution to genomics and interactions with “non-target” fungi.     

The Endophytic Mycoflora of Bark,Leaf, and Stem Tissues of <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss (Neem) from Varanasi (India)

A systematic study was made of the endophytes of Azadirachta indica A. Juss (the neem tree) growing in several of its natural habitats in India. A total of 233 isolates of endophytic fungi representing 18 fungal taxa were obtained from segments of bark, stem, and leaves of this tree. Hyphomycetes (62.2%) were the most prevalent followed by the Coelomycetes (27.4%) and Mycelia Sterilia (7.7%). As mathematically determined, the maximum species richness and frequency of colonization of endophytes appeared in leaf segments rather than stem and bark tissues from each location. Endophytic colonization frequency was also greater in leaves (45.5%) than bark (31.5%). The leaf samples from all locations were nearly constant in their endophytic composition, whereas bark samples showed maximum diversity at different locations. Inter-site comparisons for endophytic diversity, however, were not significantly different with Loc1 and Loc2 having a maximum of 66.67% J _c. The smallest similarity was between Loc2 and Loc3 of 54.17% J _c. The dominant endophytic fungi isolated were Phomopsis oblonga, Cladosporium cladosporioides, Pestalotiopsis sp., Trichoderma sp, and Aspergillus sp. Genera such as Periconia, Stenella, and Drechslera are reported here for the first time as endophytes from this host plant. This report illustrates the value of sampling different tissues of a given plant in several locations to obtain the greatest species diversity of endophytes. The rich and sizeable collection of endophytic fungi from this specific plant may represent a unique source of one or more of the interesting and useful bioactive compounds normally associated with A. indica such as the azadirachtins and related tetranortriterpenoids.     

Diversity of fungal endophytes in some medicinal plants of Himachal Pradesh,India

Fungal endophytes were isolated from surface sterilised leaf segments of five medicinal plants collected from Mandi district, Himachal Pradesh, India. A total of 373 fungal strains belonging to 15 fungal genera and 18 species, Aspergillus niger, A. flavus, A. clavatus, A. variecolor, Penicillium chrsogenum, Alternaria alternata, Curvularia lunata, Haplosporium sp., Phoma sp., Nigrospora sp., Colletotrichum sp., Cladosporium sp., Stemphylium sp., Fusarium sp., Geotrichum sp., Phomopsis sp., Trichoderma sp., Rhizopus sp. and some sterile mycelium were isolated from all the plants. The relative frequency, isolation rate and colonisation rates of endophytes were used to study the endophytic diversity. The results showed that the highest colonisation rate (93.05%) was observed in Adhathoda vasica, while it was 91.66% in Ocimum sanctum, 85% in Viola odorata, 82.81% Cannabis sativa and lowest (61.11%) in Withania somnifera. Moreover, reading the richness and diversity of the endophytic fungi, the highest was obtained for O. sanctum, W. somnifera and C. sativa having eight species each, while lowest (6 and 4) was obtained from A. vasica and V. odorata, respectively. As the role of endophytic organisms in defensive mechanisms of plants is now well established, the present study is an important step to find new and interesting endophytes among the medicinal plants.     

入侵植物紫茎泽兰叶内生真菌多样性研究

外来入侵植物通常要和当地的生物发生相互关系并对当地生态系统产生影响。为表明叶内生真菌是否有利于紫茎泽兰（Eupatorium adenophorum）的入侵力,首次调查了其叶内生真菌多样性。利用过夜冰冻组织法和常规的麦芽汁琼脂平板培养法,分别于3、6月份从昆明西山、金殿种群分离得到312个菌株,根据其ITS基因差异,分为77个可操作分类单元（OTUs）,系统发育地位分布在Agaricomycetes、Dothideomycetes、Sordariomycetes和 Pezizomycetes 4个纲。在低阶分类上这些真菌可分为19个属,优势属为链格孢属（Alternaria）（25.09%）和炭疽菌属（Colletotrichum）（10.80%）。有2.09%的菌株属于未知真菌。相比较,6月份的紫茎泽兰种群比3月份具有较高的内生真菌多样性。结果表明,入侵植物紫茎泽兰叶内生真菌非常丰富,这些真菌是否对紫茎泽兰的入侵力具有影响值得今后深入研究。     

Glyphosate and Previous Crop Residue Effect on Deleterious and Beneficial Soil-borne Fungi from a Peanut–Corn–Soybean Rotations

Bean seedlings (Phaseolus vulgaris L.) were transplanted to soil with corn previous crop residue, peanut previous crop residue and no agricultural soil, and treated with a range of glyphosate concentrations. Trichoderma, Gliocladium, Fusarium and Pythium soil‐borne fungi populations were monitored during 24 days after glyphosate treatment to study the glyphosate and previous crop residue effects on these populations. In addition, those genera of soil‐borne fungi were tested to study in vitro toxicity to glyphosate. Independently of glyphosate concentration, the highest population of Trichoderma spp. and Gliocladium spp. were registered on soil with previous corn residue. Fusarium and Pythium populations increased proportionally to the increment of glyphosate concentration. No effect of glyphosate was founded on Trichoderma and Gliocladium populations. The in vitro study results indicated an inhibitory effect of glyphosate on mycelial grown of the most studied soil‐borne fungi.     

Diverse non-mycorrhizal fungal endophytes inhabiting an epiphytic,medicinal orchid (<Emphasis Type="Italic">Dendrobium nobile</Emphasis>): estimation and characterization

Although the terrestrial and temperate orchids–fungal biology have been largely explored, knowledge of tropical epiphytic orchids–fungus relationships, especially on the ecological roles imparted by non-mycorrhizal fungal endophytes, is less known. Exploitation of the endophytic fungal mycobiota residing in epiphytic orchid plants may be of great importance to further elucidate the fungal ecology in this special habitat as well as developing new approaches for orchid conversations. The composition of fungal endophytes associated with leaves, stems and roots of an epiphytic orchid (Dendrobium nobile), a famous Chinese traditional medicinal plant, was investigated. Microscopic imaging, culture-dependant method and molecular phylogeny were used to estimate their entity and diversity. Totally, there were 172 isolates, at least 14 fungal genera and 33 different morphospecies recovered from 288 samples. Ascomycetes, coelomycetes and hyphomycetes were three major fungal groups. There were higher overall colonization and isolation rates of endophytic fungi from leaves than from other tissues. Guignardia mangiferae was the dominant fungal species within leaves; while the endophytic Xylariaceae were frequently observed in all plant tissues; Colletotrichum, Phomopsis and Fusarium were also frequently observed. Phylogenetic analysis based on ITS gene revealed the high diversity of Xylariacea fungi and relatively diverse of non-Xylariacea fungi. Some potentially promising beneficial fungi such as Clonostachys rosea and Trichoderma chlorosporum were found in roots. This is the first report concerning above-ground and below-ground endophytic fungi community of an epiphytic medicinal orchid, suggesting the ubiquitous distribution of non-mycorrhizal fungal endophytes in orchid plants together with heterogeneity and tissue specificity of the endophyte assemblage. Possible physiological functions played by these fungal endophytes and their potential applications are also discussed briefly. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mycobiota of the giant oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis> (Thunberg, 1787) (Bivalvia) from the Peter the Great Bay of the Sea of Japan

Mycological investigation of the Pacific (giant) oyster Crassostrea gigas (Thunberg, 1793) (Bivalvia) from the Peter the Great Bay of the Sea of Japan was carried out. The taxonomic composition of filamentous fungi associated with C. gigas was studied. The taxonomic composition of the fungi associated with the giant oyster included 22 species of filamentous fungi of which 17 species were identified. The latter belonged to six genera: Alternaria, Aspergillus, Botrytis, Fusarium, Penicillium, and Trichoderma. The distribution of filamentous fungi in the internal organs of the bivalve mollusk was studied.     

Cultivable fungal community associated with the tropical orchid Dichaea andina

The orchid–fungus relationship has been studied since the discovery that the minute seeds of orchids depend on fungi to support the germination process. With the aim of describing the biodiversity of cultivable endophytic and mycorrhizal fungi from the orchid Dichaea andina, we isolated pure fungal cultures from its roots and identified them by sequencing the internal transcribed spacer. We recorded 22 fungal operational taxonomic units belonging to eight orders of Ascomycota: Eurotiales, Hypocreales, Xylariales, Helotiales, Boliniales, Chaetothyriales, Chaetosphaeriales and Pleosporales. The only Basidiomycota isolated belonged to the genus Ceratobasidium from the order Cantharellales, whose members are known as orchid mycorrhizal fungi. At the genus level, we identified 16 genera, the most common of which were Byssochlamys, Camarops, Trichoderma, Cladophialophora, Fusarium and Xylaria; some of them had been reported previously as orchid endophytes. The relevance of endophytic fungi to their hosts is still unclear, but this widely distributed interaction deserves further investigation.     

A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa,the causal agent of witches' broom disease of cocoa

A Trichoderma harzianum isolate (1051), which was able to antagonize in field the phytopathogen Crinipellis perniciosa, the causal agent of witches' broom disease of cocoa, produces several hydrolytic enzymes. A chitinase, with molecular mass of about 37 kDA, which was secreted by the Trichoderma in the culture medium containing chitin, was partially purified by gel filtration followed by hydrophobic chromatography. The optimal pH and temperature for chitin hydrolysis by the partially purified enzyme were 4.0 and 37 °C, respectively. Chitobiose, laminarin, cellulosic substrates including aryl-glucosides, xylan, starch and -galactomannan were not hydrolysed by the enzyme. Remarkably, the partially purified enzyme drastically affected the cell wall of the phytopathogen C. perniciosa in vitro.     

Fungal and plant gene expression during the colonization of cacao seedlings by endophytic isolates of four Trichoderma species

Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases of Theobroma cacao (cacao). Gene expression was studied during the interaction between cacao seedlings and four endophytic Trichoderma isolates, T. ovalisporum-DIS 70a, T. hamatum-DIS 219b, T. harzianum-DIS 219f, and Trichoderma sp.-DIS 172ai. Isolates DIS 70a, DIS 219b, and DIS 219f were mycoparasitic on the pathogen Moniliophthora roreri, and DIS 172ai produced metabolites that inhibited growth of M. roreri in culture. ESTs (116) responsive to endophytic colonization of cacao were identified using differential display and their expression analyzed using macroarrays. Nineteen cacao ESTs and 17 Trichoderma ESTs were chosen for real-time quantitative PCR analysis. Seven cacao ESTs were induced during colonization by the Trichoderma isolates. These included putative genes for ornithine decarboxylase (P1), GST-like proteins (P4), zinc finger protein (P13), wound-induced protein (P26), EF-calcium-binding protein (P29), carbohydrate oxidase (P59), and an unknown protein (U4). Two plant ESTs, extensin-like protein (P12) and major intrinsic protein (P31), were repressed due to colonization. The plant gene expression profile was dependent on the Trichoderma isolate colonizing the cacao seedling. The fungal ESTs induced in colonized cacao seedlings also varied with the Trichoderma isolate used. The most highly induced fungal ESTs were putative glucosyl hydrolase family 2 (F3), glucosyl hydrolase family 7 (F7), serine protease (F11), and alcohol oxidase (F19). The pattern of altered gene expression suggests a complex system of genetic cross talk occurs between the cacao tree and Trichoderma isolates during the establishment of the endophytic association.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

湖北烟草内生真菌生物多样性和种群结构分析

【目的】研究传统药用植物烟草(Nicotiana tabacum L.)内生真菌的丰富度,揭示其种群多样性和群落结构,为烟草内生真菌资源的有效利用奠定基础。【方法】采用组织分离法进行烟草内生真菌的分离,通过形态学和分子生物学相结合的方法进行菌株分类鉴定,以香农多样性指数及相对分离频率反映内生真菌物种多样性及分布规律。【结果】从不同组织部位、不同生长时期、不同海拔样地的健康烟草中共分离获得539株内生真菌,根据r DNA-ITS系统发育分析鉴定为31属73种,香农多样性指数为2.78,曲霉属Aspergillus和镰孢属Fusarium为优势菌群,其相对分离频率分别为22.63%和12.99%。其分布规律表现为茎部内生真菌的多样性高于叶部和根部;随着生育期的延长,内生真菌多样性逐步增多;随着海拔高度升高,内生真菌的种类和数量呈现降低的趋势。【结论】烟草内生真菌具有丰富的生物多样性,其分布表现出组织、生长时期、海拔高度专化性。阐明内生真菌在烟草中的分布规律,可以为烟草内生真菌在农业生产领域的开发应用提供科学依据。