dsRNA-containing Strains Research Articles

EFFECTS OF BRANCH SIZE AND PATHOGEN VIRULENCE ON CANKER DEVELOPMENT AND BRANCH MORTALITY

Effective biological control using dsRNA requires that chestnuts infected with dsRNA-containing strains of the chestnut blight fungus (Cryphonectria parasitica) have higher survival than trees infected with dsRNA-free strains. The influence of branch size on canker development and branch mortality was investigated in an American chestnut stand in Michigan. Comparisons among cankers initiated naturally (after wounding), with dsRNA-free and dsRNA-containing C. parasitica revealed that larger branches are more likely to have callused cankers than smaller branches. Larger branches also are more likely to survive independent of the presence/absence of dsRNA. The presence of dsRNA reduces canker growth rates and may delay mortality of larger branches but has no impact on survival of small branches (<2 cm in diameter). These results suggest that inoculating larger branches and trees with dsRNA-containing C. parasitica may produce effective biological control while the survival of smaller individuals will not be enhanced by treatment.

Transmission of Cryphonectria hypovirus to protect chestnut trees from chestnut blight disease

The effects of hypovirus transmission in Cryphonectria parasitica were tested by co-culture of Cryphonectria hypovirus (CHV) donor strains with recipient strains. Virulence of the CHV-containing strains was determined by inoculation into cut chestnut stems in the laboratory and into chestnut trees in the field. The viral double-stranded RNA (dsRNA) transmission rate between C. parasitica strains on agar medium in the laboratory ranged from 0% to 100% depending upon the strain combination. The results of our laboratory and field inoculation experiments indicated that the virulence of six laboratory-infected hypovirulent strains (LH), 12 dsRNA donor strains (H), six pairs of hypovirulent strains (H) mixed with wild-type strains (V), and six pairs of laboratory-infected hypovirulent strains (LH) mixed with dsRNA free isolates derived from dsRNA-containing strains (LV) was lower than that of virulent recipient strains (V) alone. One year after co-inoculation in the field, most wild-type strains were converted by the hypovirulent strains, even those strains that were not converted in the laboratory. The results suggest that there is better than expected potential for biological control of chestnut blight disease.

Double-stranded RNAs associated with Fusarium proliferatum mitochondria

Many fungi harbor double-stranded (ds) RNA molecules, which can have phenotypic effects such as hypovirulence, altered colony morphology, and pigmentation. In some species of Fusarium, dsRNA molecules are found in every strain examined. We examined 100 F. proliferatum strains collected primarily from maize and sorghum in the United States, but found only four that carried dsRNAs. Each strain harbored a distinct set of dsRNAs, which ranged in size from approximately 0.7–3.1 kb. A single dsRNA band was observed from one strain, but multiple bands were observed from the other three. The strains with multiple dsRNAs transmitted these dsRNAs as sets at a high frequency (≥ 97 %) to vegetatively produced microconidia, but the single dsRNA of the fourth strain was only rarely (≤ 3 %) transmitted in this manner. None of these dsRNAs could be transmitted through sexual crosses in which the dsRNA-containing strain served as the male parent. Transmission through the female parent could not be tested as the field strains and dsRNA-free derivatives of these strains were female sterile. The dsRNAs from the strains with multiple dsRNAs were present in and protected against ribonuclease A digestion in crude mitochondrial preparations. The high transmission rate to single-conidiospore cultures, the lack of transmission through the male parent of sexual crosses, and the protection against ribonuclease A digestion are all consistent with a mitochondrial localization of the dsRNAs from the strains carrying multiple dsRNAs. dsRNAs often function as viruses in fungi, and the three F. proliferatum strains reported here join strains of Ophiostoma novo-ulmi, Rhizoctonia solani, and Cryphonectria parasitica as the only fungi known to carry dsRNAs associated with the mitochondria.

Isolation and characterization ofCryphonectria parasiticamutants that mimic a specific effect of hypovirulence-associated dsRNA on laccase activity

Hypovirulent, double-stranded (ds) RNA-containing strains of the chestnut blight fungus Cryphonectria parasitica were previously shown to produce less laccase activity than virulent strains when grown on malt extract agar containing tannic acid (Bavendamm reaction). Three mutants that lacked this specific laccase activity were selected after UV mutagenesis of a dsRNA-free, wild-type strain. Complementation tests and sexual crosses showed that all mutations were recessive, two were allelic (lacR1-1 and lacR1-2), and one (lacR2) was nonallelic. No linkage was detected between the two loci. None of the known C. parasitica laccases (LAC1, LAC2, and LAC3) was substantially reduced in the lacR1 mutants. The lacR2 mutant, in contrast, produced about 10-fold less extracellular LAC1 and LAC3 activities than the wild-type strain. Intracellular LAC2 was reduced to about 50% in this mutant. These results suggest a role for both LAC1 and LAC3 in the Bavendamm reaction. The three mutations had no significant effect on fungal virulence, pigmentation, and sporulation, all phenotypes that were suppressed in an isogenic dsRNA-containing strain. Key words: Endothia parasitica, genetics, hypovirulence, dsRNA, chestnut blight.

The comparison of oxalic acid production in vivo and in vitro by virulent and hypovirulent Cryphonectria (Endothia) parasitica

The relation between oxalic acid production in liquid broth and in infected apple tissue during virulence assays was determined for Michigan and Italian strains of Cryphonectria parasitica with or without cytoplasmic double-stranded RNA (dsRNA). Two strains without dsRNA and considered virulent produced more oxalic acid in broth than did their dsRNA-containing near-isogenic counterparts. In contrast, five dsRNA-containing strains produced more oxalic acid per gram of infected apple fruit than did the dsRNA-free strains even though the lesion size produced by most of the dsRNA-containing strains was relatively small when compared to their dsRNA-free counterparts. One dsRNA-containing isolate (M14), judged virulent, produced a relatively large lesion in the apple fruit virulence assay, but oxalic acid production by this strain was similar to that of dsRNA-free virulent strains in both culture and apple fruit. Thus, the presence of dsRNA did not consistently confer avirulence, and the amount of oxalic acid production was positively associated with virulence only in broth, not in diseased plant tissue.

Transcription of naked double-stranded RNA molecules in a fraction containing large vesicles plus mitochondria from the basidiomycete Agrocybe aegerita

Actinomycin D-resistant RNA synthesis was detected in the cellular (VM) fraction which contains large vesicles plus mitochondria from Agrocybe aegerita. It involved the RNAase-resistant complex of naked (unencapsidated) double-stranded RNAs (dsRNAs). The RNA polymerase assay showed [α-32P]UTP incorporation in dsRNAs of a size corresponding to the previously described naked M-dsRNAs and in single-stranded RNAs of related size, which are assumed to act as intermediaries in the replicative cycle of the dsRNA molecules. Moreover, the incorporation of ribonucleotides into large mitochondrial dsDNA fragments was detected in the dsRNA-containing strain. This result favours a mitochondrial location for the naked dsRNAs and their associated polymerizing enzyme(s). No replication of the encapsidated large L-dsRNA viral genome was detected in the VM fraction, supporting the hypothesis that the two types of dsRNAs observed in this A. aegerita strain are independent entities.

Physical and genetic characterization of linear DNA plasmids from the heterothallic yeast Saccharomycopsis crataegensis.

Five strains of the heterothallic yeast Saccharomycopsis crataegensis have been previously shown to contain DNA and/or RNA plasmidlike molecules (Shepherd et al. 1987). Three DNA plasmids, designated pScrl-1, -2 and -3, were found in strain NRRL Y-5902, while two were identified in each of NRRL strains Y-5903 and Y-5904. DNA plasmids were not identified in S. crataegensis strains Y-5910 or YB-192. Four S. crataegensis strains (Y-5903, Y-5904, Y-5910 and YB-192) were also shown to possess double-stranded RNA (dsRNA) molecules not found in strain Y-5902 (Shepherd et al. 1987). Hybridization studies now demonstrate the DNA plasmids in Y-5903 and Y-5904 to be highly homologous to their respective size counterparts (pScrl-1 and pScrl-2) in Y-5902 and to show some homology to pScrl-3. Restriction endonuclease mapping studies confirm the linear nature of each plasmid and establish identical restriction maps for a 1.4 kilobase (kb) region in pScrl-2 and -3. This 1.4 kb region accounts for the hybridization homology of pScrl-2 and pScrl-3 noted by Shepherd et al. (1987) and for homology of the plasmids of Y-5903 and Y-5904 to pScrl-3 of Y-5902. The pScrl plasmids show no homology to the dsRNA molecules of S. crataegensis, the 2 microM circular DNA of Staccharomyces cerevisiae, the 'killer' plasmids of Kluyveromyces lactis, or the linear DNA plasmids of Pichia inositovora. In crosses between linear DNA plasmid-containing and dsRNA-containing strains, only progeny containing the pScrl plasmids were recovered. Poor spore viability and a lack of complete tetrad recovery limited the extent of the analysis, but the findings suggest a cytoplasmic mode of inheritance for these linear DNAs.

Regulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA

Transmissible hypovirulence of the chestnut blight fungus, Cryphonectria parasitica, is associated with cytoplasmic double-stranded-RNA (dsRNA) viruses. The fungal laccase has attracted interest because its activity is reduced in hypovirulent dsRNA-containing strains. A laccase cDNA clone was isolated by screening a cDNA expression library with antibodies against the purified extracellular laccase. The amino acid sequence deduced from part of the cDNA clone revealed high homology to other fungal laccases, especially to the Neurospora crassa laccase. A major laccase transcript 2.3 kb in length was detected in Northern (RNA) blots. In liquid culture, extracellular laccase activity was reduced by about 75% in the hypovirulent (dsRNA-free) strain EP155/2. In contrast, production of biomass was not affected by the dsRNA. Northern blot analysis indicated that dsRNA down regulates laccase biosynthesis by reducing laccase mRNA accumulation. The laccase gene is one of several developmentally regulated genes affected by the presence of dsRNA.

Mycelium Formation and Calcium Oxalate Production by dsRNA-Free Virulent and dsRNA-Containing Hypovirulent Strains of Cryphonectria parasitica

Mycelium formation and oxalate production by two dsRNA-free, virulent strains and three dsRNA-containing, hypo virulent strains of Cryphonectria (Endothia) parasitica were evaluated after 10 days incubation on agar media containing either 0.010 M or 0.025 M calcium acetate, or no calcium acetate. The dsRNA-containing strains EP 31–2P and EP-752 consistently produced the least mycelium while the dsRNA-free strain EP-155 produced the most mycelium on these three media. Addition of 0.010 M calcium acetate did not affect the amount of growth by strain EP-155 or strain EP-752, enhanced growth of the dsRNA-free strain EP-42, and reduced growth of the dsRNA-containing strains EP 17–8B and EP 31–2P. Growth of all strains was reduced on the medium containing 0.025 M calcium acetate. Strain EP-155 produced the greatest total oxalate on these media, strains EP-42 and EP 17–8B produced significantly less, and dsRNA-containing strains, EP 31–2P and EP-752, produced the least. Formation of oxalate by C. parasitica may have a regulatory role in calcium metabolism and/or oxalic acid production.

Mycelium Formation and Calcium Oxalate Production By Dsrna-Free Virulent and Dsrna-Containing Hypovirulent Strains ofCryphonectria Parasitica

Mycelium formation and oxalate production by two dsRNA-free, virulent strains and three dsRNAcontaining, hypovirulent strains of Cryphonectria (Endothia) parasitica were evaluated after 10 days incubation on agar media containing either 0.010 M or 0.025 M calcium acetate, or no calcium acetate. The dsRNA-containing strains EP 31-2P and EP-752 consistently produced the least mycelium while the dsRNA-free strain EP-155 produced the most mycelium on these three media. Addition of 0.010 M calcium acetate did not affect the amount of growth by strain EP-155 or strain EP-752, enhanced growth ofthe dsRNA-free strain EP-42, and reduced growth ofthe dsRNA-containing strains EP 178B and EP 31-2P. Growth of all strains was reduced on the medium containing 0.025 M calcium acetate. Strain EP-155 produced the greatest total oxalate on these media, strains EP-42 and EP 178B produced significantly less, and dsRNA-containing strains, EP 31-2P and EP-752, produced the least. Formation of oxalate by C. parasitica may have a regulatory role in calcium metabolism and/or oxalic acid production.

Control of plant pathogens with viruses and related agents

Cytoplasmically transmissible agents causing diseases of plant pathogenic fungi characterized by reductions in pathogenicity, ability to form sexual and asexual spores, spore viability and growth rate, are often associated with the presence of one or more specific segments of virus-like double-stranded RNA (dsRNA). In Italy, hypovirulent dsRNA-containing strains of the chestnut blight fungus,Endothia(Cryphonectria)parasitica, have become predominant in many areas where blight is no longer a serious problem. dsRNA-containing strains of other pathogens, with various degrees of debilitation, survive in natural populations but have not become predominant or resulted in any great reduction in disease. Examples include the Dutch elm disease fungus,Ophiostoma(Ceratocystis)ulmi, and the wheat take-all fungus,Gaeumannomyces graminisvar.tritici. Successful biological control of such pathogens could probably be achieved, however, if methods could be developed to suppress the loss of dsRNA that occurs during the sexual and other stages of their life cycles, and to suppress the vegetative incompatibility reactions that reduce the cytoplasmic transmission of dsRNA. Systemic infection with attenuated strains of plant viruses can protect plants from later infection by virulent strains of the same or closely related viruses. Despite some notable successes, e.g. control of citrus tristeza and tomato mosaic viruses, such ‘cross-protection’ has not been widely applied because of the cost and difficulty of application, and caution about the widespread distribution of infectious agents in the environment. These problems could be overcome if cross-protection could be achieved by the expression of a single viral gene rather than infection with intact virus, and consideration of possible mechanisms of cross protection suggests novel ways of producing virus-resistant plants.