Interactions between transgenic trees and mycorrhizal and pathogenic fungi

The development of biotechnology techniques in plant breeding and the new commercial applications have raised public and scientific concerns about the safety of genetically modified (GM) crops and trees. To find out the feasibility of these new technologies in the breeding of commercially important Finnish hardwood species and to estimate the ecological risks of the produced transgenic plants, the experiments of this study have been conducted as a part of a larger project focusing on the risk assessment of GM-trees. Transgenic Betula pendula and Populus trees were produced via Agrobacterium mediated transformation. Stilbene synthase (STS) gene from pine (Pinus sylvestris) and chitinase gene from sugar beet (Beta vulgaris) were transferred to (hybrid) aspen and birch, respectively, to improve disease resistance against fungal pathogens. To modify lignin biosynthesis, a 4-coumarate:coenzyme A ligase (4CL) gene fragment in antisense orientation was introduced into two birch clones. In in vitro test, one transgenic aspen line expressing pine STS gene showed increased resistance to decay fungus Phellinus tremulae. In the field, chitinase transgenic birch lines were more susceptible to leaf spot (Pyrenopeziza betulicola) than the non-transgenic control clone while the resistance against birch rust (Melampsoridium betulinum) was improved. No changes in the content or composition of lignin were detected in the 4CL antisense birch lines. In order to evaluate the ecological effects of the produced GM trees on non-target organisms, an in vitro mycorrhiza experiment with Paxillus involutus and a decomposition experiment in the field were performed. The expression of a transgenic chitinase did not disturb the establishment of mycorrhizal symbiosis between birch and P. involutus in vitro. 4CL antisense transformed birch lines showed retarded root growth but were able to form normal ectomycorrhizal associations with the mycorrhizal fungus in vitro. 4CL lines also showed normal litter decomposition. Unexpected growth reductions resulting from the gene transformation were observed in chitinase transgenic and 4CL antisense birch lines. These results indicate that genetic engineering can provide a tool in increasing disease resistance in Finnish tree species. More extensive data with several ectomycorrhizal species is needed to evaluate the consequences of transgene expression on beneficial plant-fungus symbioses. The potential pleiotropic effects of the transgene should also be taken into account when considering the safety of transgenic trees.

Combining Plant Pathogenic Fungi and the Leaf-Mining Fly, Hydrellia pakistanae, Increases Damage to Hydrilla

Four fungal species, F71PJ Acremonium sp., F531 Cylindrocarpon sp., F542, Botrytis sp., and F964 Fusarium culmorum [Wm. G. Sm.] Sacc. were recovered from hydrilla [ Hydrilla verticillata (L. f.) Royle] shoots or from soil and water surrounding hydrilla growing in ponds and lakes in Florida and shown to be capable of killing hydrilla in a bioassay. The isolates were tested singly and in combination with the leaf-mining fly, Hydrellia pakistanae (Diptera: Ephydridae), for their capability to kill or severely damage hydrilla in a bioassay.

A simple inhibition coefficient for quantifying potency of biocontrol agents against plant-pathogenic fungi

Microbial interactions depend on a range of biotic and environmental variables, and are both dynamic and unpredictable. For some purposes, and under defined conditions, it is nevertheless imperative to evaluate the inhibitory efficacy of microbes, such as those with potential as biocontrol agents. We selected six, phylogenetically diverse microbes to determine their ability to inhibit the ascomycete Fusarium
coeruleum, a soil-dwelling pathogen of potato tubers that causes the storage disease dry rot. Interaction assays, where colony development was quantified (for both fungal pathogen and potential control agents), were therefore carried out on solid media. The key parameters that contributed to, and were indicative of, inhibitory efficacy were identified as: fungal growth-rates (i) prior to contact with the biocontrol
agent and (ii) if/once contact with the biocontrol agent was established (i.e. in the zone of mixed
culture), and (iii) the ultimate distance traveled by the fungal mycelium. It was clear that there was no correlation between zones of fungal inhibition and the overall reduction in the extent of fungal colony development. An inhibition coefficient was devised which incorporated the potential contributions of distal inhibition of fungal growth-rate; prevention of mycelium development in the vicinity of the biocontrol
agent; and ability to inhibit plant-pathogen growth-rate in the zone of mixed culture (in a ratio of 2:2:1). The values derived were 84.2 for Bacillus subtilis (QST 713), 74.0 for Bacillus sp. (JC12GB42), 30.7 for Pichia anomala (J121), 19.3 for Pantoea agglomerans (JC12GB34), 13.9 for Pantoea sp. (S09:T:12), and
21.9 (indicating a promotion of fungal growth) for bacterial strain (JC12GB54). This inhibition coefficient, with a theoretical maximum of 100, was consistent with the extent of F. coeruleum-colony development (i.e. area, in cm2) and assays of these biocontrol agents carried out previously against Fusarium
spp., and other fungi. These findings are discussed in relation to the dynamics and inherent complexity of natural ecosystems, and the need to adapt models for use under specific sets of conditions.

Stress tolerance and virulence of insect-pathogenic fungi are determined by environmental conditions during conidial formation

The virulence to insects and tolerance to heat and UV-B radiation of conidia of entomopathogenic fungi are greatly influenced by physical, chemical, and nutritional conditions during mycelial growth. This is evidenced, for example, by the stress phenotypes of Metarhizium robertsii produced on various substrates. Conidia from minimal medium (Czapek's medium without sucrose), complex medium, and insect (Lepidoptera and Coleoptera) cadavers had high, moderate, and poor tolerance to UV-B radiation, respectively. Furthermore, conidia from minimal medium germinated faster and had increased heat tolerance and were more virulent to insects than those from complex medium. Low water-activity or alkaline culture conditions also resulted in production of conidia with high tolerance to heat or UV-B radiation. Conidia produced on complex media exhibited lower stress tolerance, whereas those from complex media supplemented with NaCl or KCl (to reduce water activity) were more tolerant to heat and UV-B than those from the unmodified complex medium. Osmotic and nutritive stresses resulted in production of conidia with a robust stress phenotype, but also were associated with low conidial yield. Physical conditions such as growth under illumination, hypoxic conditions, and heat shock before conidial production also induced both higher UV-B and heat tolerance; but conidial production was not decreased. In conclusion, physical and chemical parameters, as well as nutrition source, can induce great variability in conidial tolerance to stress for entomopathogenic fungi. Implications are discussed in relation to the ecology of entomopathogenic fungi in the field, and to their use for biological control. This review will cover recent technologies on improving stress tolerance of entomopathogenic fungi for biological control of insects.

Potential pathogenic fungi assessment through molecular biology in cork industry

Portugal has been the world leader in the cork sector in terms of exports, employing ten thousands of workers. In this working activity, the permanent contact with cork may lead to the exposure to fungi, raising concerns as potential occupational hazards in cork industry. The application of molecular tools is crucial in this setting, since fungal species with faster growth rates may hide other species with clinical relevance, such as species belonging to P. glabrum and A. fumigatus complexes. A study was developed aiming at assessing fungal contamination due to Aspergillus fumigatus complex and Penicillium glabrum complex by molecular methods in three cork industries in the outskirt of Lisbon city.

Melanin as a virulence factor of Paracoccidioides brasiliensis and other dimorphic pathogenic fungi: a minireview

Melanin pigments are substances produced by a broad variety of pathogenic microorganisms, including bacteria, fungi, and helminths. Microbes predominantly produce melanin pigment via tyrosinases, laccases, catecholases, and the polyketide synthase pathway. In fungi, melanin is deposited in the cell wall and cytoplasm, and melanin particles (""ghosts"") can be isolated from these fungi that have the same size and shape of the original cells. Melanin has been reported in several human pathogenic dimorphic fungi including Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii. Melanization appears to contribute to virulence by reducing the susceptibility of melanized fungi to host defense mechanisms and antifungal drugs.

Evaluation of pathogenic fungi occurrence in traumatogenic structures of freshwater fish

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Clues to the presence of pathogenic fungi in certain environments

The presence of various pathogenic fungi in rather unsuspected hosts and environments has always attracted the attention of the scientific community. Reports on the putative role of animals in fungal infections of humans bear important consequences on public health as well as on the understanding of fungal ecology. Fungi are ubiquitous in nature and their great capacity for adaptation allows them to survive and indeed, to thrive, in plants, trees and other natural substrata. Nonetheless, we are just beginning to learn the significance that these diverse fungal habitats have on the increasing number of immunosuppressed individuals. The accidental or permanent presence of fungi in animals, plants, soils and watercourses should not be taken too lightly because they constitute the source where potential pathogens will be contracted. If those fungal habitats that carry the largest risks of exposure could be defined, if seasonal variations in the production of infectious propagules could be determined, and if their mode of transmission were to be assessed, it would be possible to develop protective measures in order to avoid human infection. Additionally, unsuspected avenues for the exploration of fungal survival strategies would be opened, thus enhancing our capacity to react properly to their advancing limits. This paper explores several ecological connections between human pathogenic fungi and certain animals, trees, waterways and degraded organic materials. The occurrence of such connections in highly endemic areas will hopefully furnish more precise clues to fungal habitats and allow the design of control programs aimed at avoiding human infection.

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or internal proteins are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

Seasonality of pathogenic fungi in mites of rubber tree plantations adjacent to fragments of Cerrado

Fungos são os patógenos mais freqüentemente observados em populações de ácaros, auxiliando o controle destes em diferentes culturas. Vinte e cinco amostras de folhas foram coletadas de cultivos de seringueiras vizinhas a dois fragmentos de vegetação de Cerrado. em cada seringal, 25 plantas foram selecionadas para coletas e sete folhas ao redor da copa foram coletadas até a altura de sete a oito metros do solo. Aproximadamente 250 indivíduos de Calacarus heveae Feres, Phyllocoptruta seringueirae Feres e Tenuipalpus heveae Baker, coletados aleatoriamente, foram montados. Hirsutella thompsonii Fisher foi observado nas três espécies de ácaros e T. heveae foi a espécie mais infectada. Os maiores níveis de infecção ocorreram de novembro a fevereiro (estação chuvosa). Na estação seca, os níveis de infecção ficaram abaixo dos 5%. H. thompsonii tem potencial para ser usado como micoacaricida na estação chuvosa.

Interaction of pathogenic fungi with host cells: Molecular and cellular approaches

This review provides an overview of several molecular and cellular approaches that are likely to supply insights into the host-fungus interaction. Fungi present intra- and/or extracellular host-parasite interfaces, the parasitism phenomenon being dependent on complementary surface molecules. The entry of the pathogen into the host cell is initiated by the fungus adhering to the cell surface, which generates an uptake signal that may induce its cytoplasmatic internalization. Furthermore, microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. on the other hand, integrins mediate the tight adhesion of cells to the ECM at sites referred to as focal adhesions and also play a role in cell signaling. The phosphorylation process is an important mechanism of cell signaling and regulation; it has been implicated recently in defense strategies against a variety of pathogens that alter host-signaling pathways in order to facilitate their invasion and survival within host cells. The study of signal transduction pathways in virulent fungi is especially important in view of their putative role in the regulation of pathogenicity. This review discusses fungal adherence, changes in cytoskeletal organization and signal transduction in relation to host-fungus interaction. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Antifungal Activity of Decyl Gallate against Several Species of Pathogenic Fungi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Secondary metabolites from Spirotropis longifolia (DC) Baill and their antifungal activity against human pathogenic fungi

A phytochemical study of the ethyl acetate extract of the roots and adventitious roots of Spirotropis longifolia, a monodominant tree species of the Guianan rainforest, has allowed the isolation of three compounds: 2- hydroxy-8,9-methylenedioxy-2',2'-dimethylpyrano-[5',6':4,3]-6a-prenyl-[6aS,11aS]-pterocarpan (spirotropin A), 2-hydroxy-8,9-methylenedioxy-2',2'-dimethy1-3',4'-dihydropyrano-[5',6':4,3]-6a-prenyl-(6aS,11aS]-pterocarpan (spirotropin B), and 5,7-dihydroxy-6.8-dipreny1-2 ''''.2 ''''-dimethylpyrano[5 '''',6 '''': 3',4]-isoflavone (spirotropone). In addition, 10 known compounds, trans-oxyresveratrol, trans-resveratrol, piceatannol, daidzein, genistein, isoprunetin, lupeol, latifolol, gnetin D and gnetin E, were also isolated. These compounds were evaluated for their antifungal activity and their cytotoxicity, and their structures were established by 1D and 2D NMR, HRMS, CD and optical rotation measurements. (C) 2011 Elsevier Ltd. All rights reserved.

Synthetic molecular mimics of naturally occurring cyclopentenones exhibit antifungal activity towards pathogenic fungi

The naturally occurring reactive electrophilic species 12-oxo-phytodienoic acid (12-oxo-PDA) is a potent antifungal agent, whereas the plant growth regulator jasmonic acid, which is synthesized from 12-oxo-PDA, is ineffective. To address what structural features of the molecule endow it with antifungal activity, we synthesized a series of molecular mimics of 12-oxo-PDA varying in the length of the alkyl chain at its C-4 ring position. The octyl analogue (4-octyl cyclopentenone) was the most effective at suppressing spore germination and subsequent mycelial growth of a range of fungal pathogens and was particularly effective against Cladosporium herbarum and Botrytis cinerea, with minimum fungicidal concentrations in the range 100-200 µM. Introduction of a carboxyl group to the end of the chain, mimicking natural fatty acids, markedly reduced antifungal efficacy. Electrolyte leakage, indicative of membrane perturbation, was evident in both C. herbarum and B. cinerea exposed to 4-octyl cyclopentenone. Lipid composition analysis of the fungal spores revealed that those species with a high oil content, namely Fusarium oxysporum and Alternaria brassicicola, were less sensitive to 4-octyl cyclopentenone. The comparable hydrophobicity of 4-octyl cyclopentenone and 12-oxo-PDA accounts for the similar spore suppression activity of these two compounds. The relative ease of synthesis of 4-octyl cyclopentenone makes it an attractive compound for potential use as an antifungal agent. © 2011 SGM.