Parasitica Strains Research Articles

Characterization of an endopolygalacturonase produced by the chestnut blight fungus

Extracellular endopolygalacturonase was produced by Cryphonectria parasitica strain Ep 155 (ATCC No. 38755) in culture media amended with 1 % sodium polypectate. Production of the enzyme was stimulated dramatically by 1 % glucose, or 0·25 % yeast extract and 0·75 % malt extract. The enzyme was purified 244-fold to apparent homogeneity by ultrafiltration, cation exchange chromatography on CM-cellulose, and gel filtration on Sephadex G-75. Its molecular weight was approximately 42 kDa as estimated by SDS-PAGE and gel filtration, and its isoelectric point was about pH 8·0. The pH optimum of the enzyme was pH 4·5-5·0, and its optimum temperature was 40 °C. The K m and V max of the enzyme were 0·22 mg ml −1 with polygalacturonic acid as substrate, and 0·241 mmol reducing group min −1 mg −1 protein, respectively. The enzyme acted in an endo fashion; a 50% reduction in viscosity of polygalacturonic acid resulted in hydrolysis of fewer than 2·5 % of the glycosidic bonds.

CDNA-Derived Hypovirus RNA in Transformed Chestnut Blight Fungus Is Spliced and Trimmed of Vector Nucleotides

Unencapsidated double-stranded viral RNAs belonging to the genus Hypovirus attenuate virulence of the chestnut blight fungus, Cryphonectria parasitica . A full-length cDNA clone of hypovirus CHV1-713 double-stranded RNA was recently shown to be infectious when introduced into the C. parasitica genome by DNA-mediated transformation. In this study, we show that the viral RNA derived from the chromosomally integrated cDNA copy is trimmed of extraneous vector nucleotide sequences. The cDNA-derived viral RNA was also found to contain a 73-bp deletion located within the 5′-noncoding leader sequence as a result of a pre-mRNA splicing event. Implications of these results are discussed in terms of hypovirus RNA replication and anticipated field studies involving engineered hypovirulent C. parasitica strains.

Variability in genome organization of the zygomycete Parasitella parasitica.

In addition to conventional methods for the identification of fungi, molecular techniques at the DNA level are increasingly being employed. In order to check the validity of such experimental approaches, we have analyzed the well-defined species Parasitella parasitica, which belongs to the family Mucoraceae (Mucorales, Zygometes). The seven strains of this species, which are available from international strain collections, were analyzed by several molecular methods: restriction fragment length polymorphism analysis (RFLP), the random primer-dependent polymerase chain reaction (RAPD-PCR), and electrophoretic karyotyping. Unexpectedly, these strains are highly diverse at the molecular level. By these techniques they can be divided consistently into two different groups. Nevertheless, all seven strains belong to a single species. They show no morphological differences and sexual spores (zygospores) were found in all possible combinations either within or between the two groups. Southern-blot analysis of genomic DNA of all P. parasitica strains with RAPD-PCR-derived labelled probes shows the existence of repetitive elements characteristic for only one group of P. parasitica. In addition, chromosome sizes, which were separated by rotating-field electrophoresis, were highly divergent, and ranged from 3 to 6.5 Mb in one group and between 2 and 4.5 Mb in the other. The RAPD-PCR patterns also discriminate both groups of P. parasitica. However, they are very similar if strains of a single group are compared. Therefore, we propose that the determination of fungal species by molecular techniques should be vetted at least by morphological and physiological parameters and, whenever possible, by mating experiments.

Mitotic stability and nuclear inheritance of integrated viral cDNA in engineered hypovirulent strains of the chestnut blight fungus.

Transmissible hypovirulence is a novel form of biological control in which virulence of a fungal pathogen is attenuated by an endogenous RNA virus. The feasibility of engineering hypovirulence was recently demonstrated by transformation of the chestnut blight fungus, Cryphonectria parasitica, with a full-length cDNA copy of a hypovirulence-associated viral RNA. Engineered hypovirulent transformants were found to contain both a chromsomally integrated cDNA copy of the viral genome and a resurrected cytoplasmically replicating double-stranded RNA form. We now report stable maintenance of integrated viral cDNA through repeated rounds of asexual sporulation and passages on host plant tissue. We also demonstrate stable nuclear inheritance of the integrated viral cDNA and resurrection of the cytoplasmic viral double-stranded RNA form in progeny resulting from the mating of an engineered hypovirulent C. parasitica strain and a vegetatively incompatible virulent strain. Mitotic stability of the viral cDNA ensures highly efficient transmission of the hypovirulence phenotype through conidia. Meiotic transmission, a mode not observed for natural hypovirulent strains, introduces virus into ascospore progeny representing a spectrum of vegetative compatibility groups, thereby circumventing barriers to anastomosis-mediated transmission imposed by the fungal vegetative incompatibility system. These transmission properties significantly enhance the potential of engineered hypovirulent C. parasitica strains as effective biocontrol agents.

Hypovirulence of Chestnut Blight Fungus Conferred by an Infectious Viral cDNA

Strains of the chestnut blight fungus Cryphonectria parasitica that contain viral double-stranded RNAs often exhibit reduced virulence. Such hypovirulent strains act as biocontrol agents by virtue of their ability to convert virulent strains to hypovirulence after anastomosis. Transformation of virulent C. parasitica strains with a full-length complementary DNA copy of a hypovirulence-associated viral RNA conferred the complete hypovirulence phenotype. Cytoplasmic double-stranded RNA was resurrected from the chromosomally integrated complementary DNA copy and was able to convert compatible virulent strains to hypovirulence. These results establish viral double-stranded RNA as the casual agent of hypovirulence and demonstrate the feasibility of engineering hypovirulent fungal strains.

A viral gene confers hypovirulence-associated traits to the chestnut blight fungus.

A viral double-stranded (ds)RNA associated with reduced virulence (hypovirulence) and the accompanying biological control of the chestnut blight fungus, Cryphonectria parasitica, was shown recently to contain two contiguous coding domains designated ORF A and ORF B. We report here that transformation of an isogenic virulent, dsRNA-free C. parasitica strain with a cDNA copy of ORF A conferred traits similar to those exhibited by the dsRNA-containing hypovirulent strain: characteristics included reduced pigmentation, reduced laccase accumulation and suppressed conidiation. However virulence was not reduced, indicating an apparent uncoupling of associated traits from hypovirulence. These results establish a direct cause and effect relationship between a viral dsRNA genetic element present in a hypovirulent C. parasitica strain and specific phenotypic traits. They demonstrate further that these traits are not the result of a general reaction of the fungus to the presence of the replicating viral RNA, but are caused by a specific viral coding domain.

A Rapid Method for Testing the Virulence ofCryphonectria parasiticaUsing Excised Bark and Wood of American Chestnut

A rapid and reproducible method for testing the virulence of Cryptonectria parasitica using American chestnut (Castanea dentata) was developed. Bark- and wood-tissue samples excised from American chestnut tree stems were inoculated with virulent and hypovirulent C. parasitica strains. Samples were incubated at 25 C for 4 days, and virulence was assessed as the area of bark and wood tissue having brown, necrotic cells. Hypovirulent strains damaged an area 2.16 cm 2 or less of each tissue sample, while virulent strains consistently damaged a greater area (3.62-3.67 cm 2 ) []

Molecular Analysis of the Laccase Gene from the Chestnut Blight Fungus and Selective Suppression of Its Expression in an Isogenic Hypovirulent Strain

The gene encoding laccase in the chestnut blight fungus, Cryphonectria parasitica, has been cloned and characterized. The predicted C. parasitica laccase amino acid sequence (591 aa) was 57% identical to the Neurospora crassa laccase sequence and contained four potential copper-binding regions that are conserved in a number of copper-binding proteins. Treatment of a virulent C. parasitica strain with 3 microM cycloheximide resulted in a marked increase in laccase mRNA accumulation, whereas identical treatment of an isogenic strain that contained a hypovirulence-associated virus failed to significantly increase laccase mRNA levels. In contrast, the accumulation of mRNAs encoding beta-tubulin, actin, or glyceraldehyde-3-phosphate dehydrogenase was not appreciably altered by either the presence of a hypovirulence-associated virus or treatment with cycloheximide. These results provide evidence that the expression of a specific fungal gene encoding a known protein product is selectively modulated by a hypovirulence-associated virus.

Growth and Stromata Production of Hypovirulent and Virulent Strains of Cryphonectria Parasitica on Dead Quercus Rubra and Acer Rubrum

Dead red oak (Quercus rubra) and red maple trees (Acer rubrum) that had been girdled were inoculated with one virulent and six hypovirulent strains of Cryphonectria parasitica. Bark samples were removed from specific points on the trees and fungi from them were subsequently cultured to determine the potential for survival and growth of the C. parasitica strains. All seven strains survived up to ten months after inoculation on red oak. Virulent strain 5-9-IB was recovered from oak bark plugs with a higher frequency than any of the hypovirulent strains but produced low numbers of stromata. Hy? povirulent strains that produced the highest rates of stromata on dead red oaks were more frequently reisolated. Survival of all seven strains on red maple stems was very poor. Seven months after stem inoculations, approximately 0.5% ofthe bark plug samples yielded reisolations of C. parasitica. Only strain Ep 88 produced a few stromata during one sampling date on two red maple trees. Survival of hypovirulent strains of Crypho? nectria parasitica (Murr.) Barr on different tree species may be essential for control ofthe chest? nut blight organism. The first collection of the fungus on a host other than American chestnut (Castanea dentata [Marsh.] Borkh.) was ob? tained by J. Franklin Collins at Martic Forge,

Growth and Stromata Production of Hypovirulent and Virulent Strains of Cryphonectria parasitica on Dead Quercus rubra and Acer rubrum

Dead red oak (Quercus rubra) and red maple trees (Acer rubrum) that had been girdled were inoculated with one virulent and six hypovirulent strains of Cryphonectria parasitica. Bark samples were removed from specific points on the trees and fungi from them were subsequently cultured to determine the potential for survival and growth of the C. parasitica strains. All seven strains survived up to ten months after inoculation on red oak. Virulent strain 5–9-1B was recovered from oak bark plugs with a higher frequency than any of the hypovirulent strains but produced low numbers of stromata. Hypovirulent strains that produced the highest rates of stromata on dead red oaks were more frequently reisolated. Survival of all seven strains on red maple stems was very poor. Seven months after stem inoculations, approximately 0.5% of the bark plug samples yielded reisolations of C. parasitica. Only strain Ep 88 produced a few stromata during one sampling date on two red maple trees.

The ultrastructure of hyphal anastomoses between vegetatively compatible and incompatible virulent and hypovirulent strains of Cryphonectria parasitica

European hypovirulent (dsRNA-containing) Cryphonectria parasitica strain Ep-50 was paired individually with West Virginia virulent (dsRNA-free) strains Ep 15-7-7 (vegetatively compatible) and EP 7-5-1 (vegetatively incompatible) on cellophane membranes. Four to six hours after anastomoses formed, the strains were preserved using freeze-substitution and observed using transmission electron microscopy. Hyphal anastomoses between Ep-50 and Ep 15-7-7 showed complete cytoplasmic continuity, with microtubules and mitochondria extending through the fusion aperture. Spherical, membrane-bound virus-like particles, measuring 50–90 nm in diameter, were located in the Ep-50 hypha, the Ep 15-7-7 hypha, and the short anastomosis bridge between them. All anastomoses between the compatible strains involved a hyphal peg that grew toward a swelling that developed on the receiving hypha. Fusion took place between the swelling and the lateral wall of the peg. Anastomoses between the incompatible strains showed cellular collapse and cytoplasmic degeneration that extended away from the anastomosis area in hyphae of both strains. Because of this, vegetative incompatibility would seem to be a formidable barrier to hypovirulence conversion and biocontrol of C. parasitica. Key words: Endothia parasitica, hyphal fusion, virus-like particles, hypovirulence conversion.

Virus-like particles in hyphae and conidia of European hypovirulent (dsRNA-containing) strains of Cryphonectria parasitica

Hyphae and germinating conidia of European hypovirulent (dsRNA-containing) Cryphonectria parasitica strain Ep-50 and virulent (dsRNA-free) strain Ep-67 (isogenic derivative from Ep-50), and hyphae only of European hypovirulent (dsRNA-containing) strains Ep-4 and Euro-7 were freeze-substituted and examined for the presence of virus-like particles (VLPs) using transmission electron microscopy. Spherical, membrane-bounded VLPs, measuring 50–90 nm in diameter, were located in hyphae and conidia of hypovirulent strain Ep-50, but not virulent strain Ep-67. Hyphae of hypovirulent strains Ep-4 and Euro-7 contained VLPs similar to those found in Ep-50. The VLPs occurred in aggregates surrounded by rough endoplasmic reticulum or more rarely, scattered throughout the cytoplasm; most possessed an electron-dense core. Results of Bernhard's regressive staining technique and lipid extraction cytochemistry suggested that the particles consisted of RNA surrounded by a lipid membrane. A unique Golgi body was associated with the formation of VLPs in hypovirulent strain Ep-50. The VLPs do not resemble typical fungal viruses and may be the end product of a defense response on the part of the fungal host designed to wall off foreign nucleic acid. Key words: Endothia parasitica, mycovirus, defense response, hypovirulence, viroid, fungal Golgi.

Conversion to Curative Morphology in Endothia parasitica and Its Restriction by Vegetative Compatibility

Endothia parasitica strains in some vegetative compatibility (v-c) groups barrage weakly when their mycelia meet on agar media. The morphology associated with Italian white curative (hypovirulent) strains of this fungus was used as a marker to detect cytoplasmic transfer between weakly-barraging strains. The (cytoplasmic) determinants for curative morphology were rapidly transferred between most weakly-barraging strains, but transferred infrequently or not at all between strongly-barraging strains.

Conversion to Curative Morphology inEndothia Parasiticaand its Restriction by Vegetative Compatibility

Endothia parasitica strains in some vegetative compatibility (v-c) groups barrage weakly when their mycelia meet on agar media. The morphology associated with Italian white curative (hypovirulent) strains of this fungus was used as a marker to detect cytoplasmic transfer between weakly-barraging strains. The (cytoplasmic) determinants for curative morphology were rapidly transferred between most weakly-barraging strains, but transferred infrequently or not at all between strongly-barraging strains.