Hypovirulent Strains Research Articles

Conidia of hypovirulent strains of Cryphonectriaparasitica differ in their potential for biocontrol of chestnut blight

Conidia from eight hypovirulent strains of Cryphonectriaparasitica (Murr.) Barr were suspended in water and applied to the bark of American chestnut (Castaneadentata (Marsh.) Borkh.) trees. Cankers were initiated with mycelium of virulent strains isogenic to the applied conidia. Hypovirulent strains differed significantly in the ability of their conidia to putatively convert isogenic virulent strains in cankers, as indicated by the recovery of cultures with hypovirulent morphology from challenged cankers. A tank mix of conidia from two hypovirulent strains in different conversion groups putatively converted significantly more cankers initiated by isogenic virulent strains than did separate applications of conidia from these strains, particularly when the second application was delayed by 2 weeks. Cankers initiated on excised stems with virulent strain Ep 155 (American Type Culture Collection 38755) yielded cultures with hypovirulent morphology after application of conidia from isogenic hypovirulent strain Ep 780 at densities as low as 50 and 700 conidia/cm2 for inoculations sealed or not sealed with latex caulk, respectively.

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.

Movement of cytoplasmic hypovirulence agents in chestnut blight cankers

Chestnut blight cankers, initiated with virulent methionine auxotrophic strains of Cryphonectria parasitica, were challenged by placing one or two discs of agar and mycelium of prototrophic hypovirulent strains at their base. Movement of hypovirulence agents was confirmed by recovery of an isolate that exhibited the typical morphology of the introduced hypovirulent strain on media with methionine but little or no growth on media without methionine. Cultures from bark showed that the hypovirulent agent moved through mycelium around the circumference of cankers within 3 weeks when the virulent and challenging hypovirulent were vegetatively compatible. Conversion of the mycelium in the canker interior proceeded more slowly. Conversion of mycelium also was delayed, but eventually occurred, when virulent and hypovirulent inocula differed in vegetative compatibility. Conidia, however, continued to yield virulent, methionine-requiring cultures up to 65 weeks after challenge, even though underlying bark frequently yielded hypovirulent, methionine-requiring cultures. This disinclination of hypovirulent agents to enter the asexual apparatus in cankers may contribute substantially to the observed ineffective dissemination of hypovirulence in the natural range of American chestnut. Exploitation of those hypovirulent agents that can enter conidia in cankers may enhance the biological control of chestnut blight by hypovirulence. Key words: biological control, Cryphonectria parasitica, Endothia parasitica, Castanea dentata.

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.

Biological control of chestnut blight: an example of virus-mediated attenuation of fungal pathogenesis.

Environmental concerns have focused attention on natural forms of disease control as potentially safe and effective alternatives to chemical pesticides. This has led to increased efforts to develop control strategies that rely on natural predators and parasites or that involve genetically engineered microbial pest control agents. This review deals with a natural form of biological control in which the virulence of a fungal pathogen is attenuated by an endogenous viral RNA genetic element: the phenomenon of transmissible hypovirulence in the chestnut blight fungus, Cryphonectria parasitica. Recent progress in the molecular characterization of a hypovirulence-associated viral RNA has provided an emerging view of the genetic organization and basic expression strategy of this class of genetic elements. Several lines of evidence now suggest that specific hypovirulence-associated virus-encoded gene products selectively modulate the expression of subsets of fungal genes and the activity of specific regulatory pathways. The construction of an infectious cDNA clone of a hypovirulence-associated viral RNA represents a major advancement that provides exciting new opportunities for examining the molecular basis of transmissible hypovirulence and for engineering hypovirulent strains for improved biocontrol. These developments have significantly improved the prospects of using this system to identify molecular determinants of virulence and elucidate signal transduction pathways involved in pathogenic responses. In addition, novel approaches are now available for extending the application of transmissible hypovirulence for management of chestnut blight and possibly other fungal diseases.

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.

Inoculation trials with hypovirulent strains of Cryphonectria parasitica1

AbstractHealing cankers were obtained in some artificial inoculation tests with combination of four hypovirulent strains of Cryphonectria parasitica which sporulated on the cankers.

The contribution of defective RNAs to the complexity of viral-encoded double-stranded RNA populations present in hypovirulent strains of the chestnut blight fungus Cryphonectria parasitica.

Hypovirulent strain EP713 of the chestnut blight fungus Cryphonectria (Endothia) parasitica harbors a family of viral encoded double-stranded (ds) RNAs thought to be responsible for the hypovirulence phenotype. These include L-dsRNA, described in the accompanying paper (Shapira et al., 1991); several prominent species in the estimated size range of 8 to 10 kb, referred to here as M-dsRNAs; and several smaller species designated S-dsRNAs which range in size from approximately 0.6 to 1.7 kb. The characterization of the M- and S-dsRNA species is the subject of this report. Results from polymerase chain reaction mapping and molecular hybridization analysis indicate that the M- and S-dsRNA species are internally deleted forms of L-dsRNA. Three different S-dsRNA species were cloned and sequenced. Each species contained a single deletion breakpoint and retained either 149, 155 or 156 bp of the terminus corresponding to the 5'-end of the coding strand and 440, 447 or 449 bp of the other terminus. Two of the S-dsRNA species contained, within the boundaries of the breakpoint, additional sequence information consisting of 42 bp or 95 bp that appeared to be unrelated to the L-dsRNA sequence. These results demonstrate that defective RNAs contribute significantly to the complexity of dsRNA populations found in hypovirulent strains of C. parasitica and provide a first approximation of the location of cis-acting signals involved in their replication.

Virus-like genetic organization and expression strategy for a double-stranded RNA genetic element associated with biological control of chestnut blight.

The complete nucleotide sequence of the largest double-stranded (ds) RNA present in hypovirulent strain EP713 of the chestnut blight pathogen, Cryphonectria parasitica, was determined and the predicted genetic organization was confirmed by translational mapping analysis. The deduced RNA sequence was 12 712 bp in length, excluding the terminal poly(A):poly(U) homopolymer domain. The strand terminating with 3'-poly(A) contained two contiguous large open reading frames (ORF A and ORF B) beginning at nucleotide residues 496-498 and extending to nucleotide positions 11 859-11 861. The junction between ORF A and ORF B consisted of the sequence 5'-UAAUG-3', where UAA served as the termination codon for ORF A and AUG was the 5'-proximal initiation codon within ORF B. ORF A (622 codons in length, excluding the termination codon) was recently shown to encode two polypeptides, p29 and p40, which were generated from a nascent polyprotein by an autocatalytic event mediated by p29 (Choi et al., 1991). A similar autocatalytic event was observed during in vitro translation of ORF B (3165 codons in length) resulting in the release of a 48 kd polypeptide from the amino-terminal portion of the ORF B-encoded polyprotein. These results are discussed in terms of the opportunities they provide for elucidating the molecular basis of transmissible hypovirulence and possible origins of hypovirulence-associated dsRNAs.

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.

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,

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.

Cotranslational autoproteolysis involved in gene expression from a double-stranded RNA genetic element associated with hypovirulence of the chestnut blight fungus.

The genetic information responsible for reduced virulence (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica is thought to reside on cytoplasmically replicating double-stranded RNA (dsRNA) molecules. Cell-free translation of synthetic transcripts corresponding to the 5'-terminal 2640 nucleotides of the sense strand of the large dsRNA present in C. parasitica hypovirulent strain EP713 yielded two polypeptides with apparent molecular masses of 29 and 40 kDa. Nucleotide sequence analysis indicated that p29 and p40 were encoded by a single large open reading frame. The coding regions for p29 and p40 were mapped to nonoverlapping portions of the 5'- and 3'-terminal domains of the open reading frame, respectively. Kinetic analysis and in vitro translation studies with chimeric transcripts indicated that p29 is autocatalytically released from a nascent polyprotein during translation. Microsequence analysis of the amino terminus of radiolabeled p40 indicated that cleavage occurred between Gly-248 and Gly-249, consistent with translational mapping analysis. Examination of the p29 amino acid sequence revealed similarity to the Potyvirus-encoded cysteine-type proteinase HC-Pro. These results indicate the types of mechanism that operate during gene expression by hypovirulence-associated dsRNA genetic elements.

Recovery of Additional Flat mutants of Cryphonectria parasitica

Additional mutants with the phenotype have been recovered from hypovirulent (dsRNA containing) strains of the chestnut blight fungus, Cryphonectria parasitica. The nuclear type of these strains was one that has been used in many laboratory and field tests and crossing experiments. Singleconidial isolates were recovered, and some of these were crossed with a standard strain. As reported previously, the phenotype segregated among the progeny as if controlled by a single nuclear gene, and single-ascospore isolates with flat phenotypes were not virulent in American chestnut trees.

The effect of Cryphonectria parasitica on water relations of American chestnut

Chestnut blight has commonly been regarded as a phloem disease due to conspicuous stem cankers that result from infection by the fungal pathogen Cryphonectria (Endothia) parasitica. Stomatal conductance (g s), transpiration rate (E) and leaf water potential were measured throughout the day on leaves distal to naturally-occurring virulent (sunken bark with abundant stromata) or hypovirulent (swollen bark lacking stromata) cankers and cankers induced by inoculation with virulent or hypovirulent strains of C. parasitica. Relative to control stems, hydraulic conductivity (K h), g s and E were reduced significantly (α = 0·05) for both natural virulent cankers, and cankers that were induced by a virulent strain. These effects were less pronounced for both natural and induced hypovirulent cankers. Isolation experiments revealed that the percentage of xylem tissue chips yielding C. parasitica was greater for virulent than for hypovirulent cankers. The data provide evidence that the localized presence of C. parasitica in cankers of American chestnut results in stomatal closure, possibly as a direct result of xylem dysfunction.

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.

Improved Chestnut Tree Condition Maintained in Two Connecticut Plots after Treatments with Hypovirulent Strains of the Chestnut Blight Fungus

Abstract In two forest plots, competing trees were killed or cut around stump sprouts of American chestnut trees. Chestnut blight cankers were treated with hypovirulent (dsRNA-containing) strains at least once a year for 4 years to control the blight disease. Nine years after the first treatment, abnormal cankers were found as far as 75 and 55 meters from the edges of the treatment areas, and strains isolated from these cankers contained dsRNA molecules. The dsRNA was found in strains with vegetative compatibility types different from those of the treatment strains, indicating that dsRNA had spread to other strains and had been maintained in the pathogen population. Trees in areas with abnormal cankers had more live stems per sprout clump and more cankers per live stem than trees in a comparison area with a similar harvest history, and more than trees in an undisturbed forest area. For. Sci. 36(1):113-124.