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

1990 ◽  
Vol 68 (1) ◽  
pp. 90-101 ◽  
Author(s):  
Joseph R. Newhouse ◽  
William L. MacDonald ◽  
Harvey C. Hoch
Keyword(s):  
Defense Response ◽  
Lipid Membrane ◽  
Virulent Strain ◽  
Lipid Extraction ◽  
Staining Technique ◽  
Cryphonectria Parasitica ◽  
Fungal Host ◽  
Virus Like Particles ◽  
Hypovirulent Strain ◽  
Endothia 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.

scholarly journals The Morphology and the Biological Control of Cryphonectria parasitica

2012 ◽  
Vol 69 (1) ◽  
Author(s):  
Carmen Emilia PUIA ◽  
Daniela Andreea GRIGORESCU ◽  
Raluca Vasilica MICLEA
Keyword(s):  
Biological Control ◽  
Virulent Strain ◽  
Morphological Characteristics ◽  
Castanea Sativa ◽  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Dual Culture ◽  
Natural Form ◽  
Hypovirulent Strain ◽  
Endothia Parasitica

Cryphonectria parasitica  (Murr.) Bar [syn. Endothia parasitica (Murr. And.] (anamorf: Endothiella sp .) is the causal agent of chestnut bark disease or chestnut blight, disease which produced great damages throughout the world, for example, in Europe, the European chestnut tree ( Castanea sativa (P.) Mill) was heavily affected. Environmental concerns have focused attention on natural forms of disease control as an effective alternative to chemical pesticides. In the chestnut blight fungus, Cryphonectria parasitica 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 hypovirulent strain. In our researches we picked samples of chestnut bark from different areas in Maramures county. We’ve isolated the fungus on PDA medium and we’ve studied the morphological characteristics of the usual virulent strain and we looked for a possible hypovirulent strain in order to study its capacity for biological control. The fungus develops in the bark and in cambium where forms a yellowish or brownish stroma and produces both conidia and ascospores. The pycnidia stromata break through the lenticels producing conidia and later in the same stroma develop the perithecia which produce ascospores. Both strains of the fungus were found in the research area. The hypovirulent strain had a slower development, showed no sporu lation and pigmentation “white cultural strain” and was tested in vitro for the capacity to convert the virulent isolates by dual culture tests.

Characterization of the chestnut blight canker and the localization and isolation of the pathogen Cryphonectria parasitica

1989 ◽  
Vol 67 (12) ◽  
pp. 3600-3607 ◽  
Author(s):  
Patricia S. McManus ◽  
Frank W. Ewers ◽  
Dennis W. Fulbright
Keyword(s):  
Transverse Section ◽  
Virulent Strain ◽  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Growth Rings ◽  
Double Stranded Rna ◽  
Naturally Occurring ◽  
Xylem Tissue ◽  
Hypovirulent Strain

Naturally occurring chestnut blight cankers on Castanea dentata were categorized as virulent (sunken bark with abundant stromata) or hypovirulent (swollen bark lacking stromata). In transverse section, xylem tissue of virulent cankers was discolored and largely nonconductive. Hypovirulent cankers had anomalous secondary xylem tissue that was light in color and conducted safranin dye, although xylem of the main stem axes of hypovirulent cankers was discolored. In naturally occurring cankers, no correlation was found between canker morphology and the presence or absence of double-stranded RNA in the inciting fungus (Cryphonectria parasitica). However, a virulent strain lacking double-stranded RNA induced cankers that resembled natural virulent cankers, and inoculation with a known hypovirulent strain containing double-stranded RNA resulted in swollen cankers that resembled natural hypovirulent cankers. Cryphonectria parasitica was isolated from all growth rings of virulent cankers and from the discolored xylem tissue of hypovirulent cankers but not from the conductive anomalous xylem tissue of hypovirulent cankers. Hyphae were observed via fluorescence microscopy in the xylem of natural and induced virulent cankers but not in hypovirulent cankers, whether natural or induced by artificial means.

Movement of cytoplasmic hypovirulence agents in chestnut blight cankers

1992 ◽  
Vol 70 (3) ◽  
pp. 557-561 ◽  
Author(s):  
L. Shain ◽  
J. B. Miller
Keyword(s):  
Biological Control ◽  
Key Words ◽  
Cryphonectria Parasitica ◽  
Castanea Dentata ◽  
American Chestnut ◽  
Chestnut Blight ◽  
Typical Morphology ◽  
Natural Range ◽  
Hypovirulent Strain ◽  
Endothia Parasitica

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.

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

1991 ◽  
Vol 69 (3) ◽  
pp. 602-614 ◽  
Author(s):  
Joseph R. Newhouse ◽  
William L. MacDonald
Keyword(s):  
Freeze Substitution ◽  
Lateral Wall ◽  
Cryphonectria Parasitica ◽  
Hyphal Fusion ◽  
Virus Like Particles ◽  
Transmission Electron ◽  
Membrane Bound ◽  
Endothia Parasitica ◽  
Cytoplasmic Continuity ◽  
Free Strains

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.

scholarly journals ORF Ι of Mycovirus SsNSRV-1 is Associated with Debilitating Symptoms of Sclerotinia sclerotiorum

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 456
Author(s):  
Zhixiao Gao ◽  
Junyan Wu ◽  
Daohong Jiang ◽  
Jiatao Xie ◽  
Jiasen Cheng ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Protein Biosynthesis ◽  
Secretory Proteins ◽  
Viral Mrna ◽  
Fungal Host ◽  
Reading Frame ◽  
Mutant Strains ◽  
Hypovirulent Strain ◽  
Viral Proliferation ◽  
Host Genes

We previously identified Sclerotinia sclerotiorum negative-stranded virus 1 (SsNSRV-1), the first (−) ssRNA mycovirus, associated with hypovirulence of its fungal host Sclerotinia sclerotiorum. In this study, functional analysis of Open Reading Frame Ι (ORF Ι) of SsNSRV-1 was performed. The integration and expression of ORF Ι led to defects in hyphal tips, vegetative growth, and virulence of the mutant strains of S. sclerotiorum. Further, differentially expressed genes (DEGs) responding to the expression of ORF Ι were identified by transcriptome analysis. In all, 686 DEGs consisted of 267 up-regulated genes and 419 down-regulated genes. DEGs reprogramed by ORF Ι were relevant to secretory proteins, pathogenicity, transcription, transmembrane transport, protein biosynthesis, modification, and metabolism. Alternative splicing was also detected in all mutant strains, but not in hypovirulent strain AH98, which was co-infected by SsNSRV-1 and Sclerotinia sclerotiorum hypovirus 1 (SsHV-1). Thus, the integrity of SsNSRV-1 genome may be necessary to protect viral mRNA from splicing and inactivation by the host. Taken together, the results suggested that protein ORF Ι could regulate the transcription, translation, and modification of host genes in order to facilitate viral proliferation and reduce the virulence of the host. Therefore, ORF Ι may be a potential gene used for the prevention of S. sclerotiorum.

scholarly journals Characterization of Hypovirus-Derived Small RNAs Generated in the Chestnut Blight Fungus by an Inducible DCL-2-Dependent Pathway

2008 ◽  
Vol 82 (6) ◽  
pp. 2613-2619 ◽  
Author(s):  
Xuemin Zhang ◽  
Gert C. Segers ◽  
Qihong Sun ◽  
Fuyou Deng ◽  
Donald L. Nuss
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Fungal Host ◽  
Chestnut Blight Fungus ◽  
Mutant Strains ◽  
Suppressor Of Rna Silencing ◽  
Dependent Pathway

ABSTRACT The disruption of one of two dicer genes, dcl-2, of the chestnut blight fungus Cryphonectria parasitica was recently shown to increase susceptibility to mycovirus infection (G. C. Segers, X. Zhang, F. Deng, Q. Sun, and D. L. Nuss, Proc. Natl. Acad. Sci. USA 104:12902-12906, 2007). We now report the accumulation of virus-derived small RNAs (vsRNAs) in hypovirus CHV1-EP713-infected wild-type and dicer gene dcl-1 mutant C. parasitica strains but not in hypovirus-infected dcl-2 mutant and dcl-1 dcl-2 double-mutant strains. The CHV1-EP713 vsRNAs were produced from both the positive and negative viral RNA strands at a ratio of 3:2 in a nonrandom distribution along the viral genome. We also show that C. parasitica responds to hypovirus and mycoreovirus infections with a significant increase (12- to 20-fold) in dcl-2 expression while the expression of dcl-1 is increased only modestly (2-fold). The expression of dcl-2 is further increased (∼35-fold) following infection with a hypovirus CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene, dcl-2. They also reveal that dcl-2 expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing.

scholarly journals Hypovirus Papain-Like Protease p29 Suppresses RNA Silencing in the Natural Fungal Host and in a Heterologous Plant System

2006 ◽  
Vol 5 (6) ◽  
pp. 896-904 ◽  
Author(s):  
Gerrit C. Segers ◽  
Rene van Wezel ◽  
Xuemei Zhang ◽  
Yiguo Hong ◽  
Donald L. Nuss
Keyword(s):  
Rna Silencing ◽  
Defense Mechanism ◽  
Fluorescent Protein ◽  
Cryphonectria Parasitica ◽  
Fungal Host ◽  
Chestnut Blight Fungus ◽  
Systemic Spread ◽  
Green Fluorescent ◽  
Fungal Viruses ◽  
Suppressor Of Rna Silencing

ABSTRACT Virulence-attenuating hypoviruses of the species Cryphonectria hypovirus 1 (CHV1) encode a papain-like protease, p29, that shares similarities with the potyvirus-encoded suppressor of RNA silencing HC-Pro. We now report that hypovirus CHV1-EP713-encoded p29 can suppress RNA silencing in the natural host, the chestnut blight fungus Cryphonectria parasitica. Hairpin RNA-triggered silencing was suppressed in C. parasitica strains expressing p29, and transformation of a transgenic green fluorescent protein (GFP)-silenced strain with p29 resulted in an increased number of transformants with elevated GFP expression levels. The CHV1-EP713 p29 protein was also shown to suppress both virus-induced and agroinfiltration-induced RNA silencing and systemic spread of silencing in GFP-expressing transgenic Nicotiana benthamiana line 16c plants. The demonstration that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral defense mechanism. The observation that a phylogenetically conserved protein of related plant and fungal viruses functions as a suppressor of RNA silencing in both fungi and plants indicates a level of conservation of the mechanisms underlying RNA silencing in these two groups of organisms.

scholarly journals New finds of Cryphonectria parasitica and the first record of chestnut blight on red oak Quercus rubra L. in the Czech Republic

2012 ◽  
Vol 51 (No. 6) ◽  
pp. 256-258 ◽  
Author(s):  
P. Haltofová ◽  
L. Jankovský ◽  
D. Palovčíková
Keyword(s):  
Czech Republic ◽  
Castanea Sativa ◽  
First Record ◽  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Red Oak ◽  
The Czech Republic ◽  
Sweet Chestnut ◽  
New Localities ◽  
Endothia Parasitica

The causal agent of the chestnut blight, the fungus Cryphonectria parasitica (Murrill) M. E. Barr (syn. Endothia parasitica (Murrill) P. J. Anderson et H. W. Anderson), was found out at new localities in the Czech Republic. The chestnut blight was observed for the first time in the Czech Republic in 2002. Two new localities were discovered in southern Moravia in May and June 2004. The disease was identified both on the sweet chestnut (Castanea sativa Mill.) and on the red oak (Quercus rubraL.). Infected trees were treated according to the order of the State Phytosanitary Administration of the Czech Republic. 

scholarly journals Influence of Cryphonectria parasitica Basal Cankers on Radial Growth of Scarlet Oak in Pennsylvania

Plant Disease ◽  
1997 ◽  
Vol 81 (4) ◽  
pp. 369-373 ◽  
Author(s):  
D. D. Davis ◽  
Mary L. Torsello ◽  
James R. McClenahen
Keyword(s):  
Fungal Infection ◽  
Radial Growth ◽  
Cryphonectria Parasitica ◽  
Breast Height ◽  
Host Pathogen ◽  
Natural Range ◽  
Endothia Parasitica

Basal cankers induced by Cryphonectria parasitica (=Endothia parasitica) occur throughout the natural range of scarlet oak (Quercus coccinea) within Pennsylvania. Because colonization of scarlet oak is thought to be a long-term host/pathogen relationship, analyses were conducted to investigate effects of basal cankers on radial growth (expressed as area increment) at 1.4 m, breast height (BH). Moderately cankered oaks grew more slowly at BH than did noncankered trees. However, severely cankered scarlet oaks generally exhibited greater radial growth at BH than did noncankered trees. This apparent anomaly likely reflects stimulated growth at BH due to the fungal infection.

scholarly journals Complete genome sequence of Mycoreovirus-1/Cp9B21, a member of a novel genus within the family Reoviridae, isolated from the chestnut blight fungus Cryphonectria parasitica

2004 ◽  
Vol 85 (11) ◽  
pp. 3437-3448 ◽  
Author(s):  
Nobuhiro Suzuki ◽  
S. Supyani ◽  
Kazuyuki Maruyama ◽  
Bradley I. Hillman
Keyword(s):  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Virus Family ◽  
Chestnut Blight Fungus ◽  
Choice Pattern ◽  
The Family ◽  
Isoelectric Points ◽  
Large Virus ◽  
Hypovirulent Strain ◽  
Cap Analysis

Mycoreovirus 1 (MYRV-1) is the type species of the newly described genus Mycoreovirus of the large virus family Reoviridae. The virus was isolated from a hypovirulent strain (9B21) of the chestnut blight fungus, Cryphonectria parasitica. A previous study showed that double-shelled particles introduced to fungal spheroplasts resulted in stably infected colonies. Of the 11 double-stranded RNA genomic segments (S1–S11), the three largest (S1–S3) were sequenced previously and shown to have moderate levels of similarity to the homologous segments of mammal-pathogenic coltiviruses (Eyach virus and Colorado tick fever virus) and another fungus-infecting reovirus, Mycoreovirus 3 of Rosellinia necatrix strain W370 (MYRV-3/RnW370). The sequences of the remaining segments (S4–S11) are reported here. All of the segments have single ORFs on their positive strands and the terminal sequences 5′-GAUCA----GCAGUCA-3′ are conserved among currently and previously sequenced segments. Oligo-cap analysis showed that the positive strands of the genomic segments are capped, whereas the negative strands are not. Similarities among the four evolutionarily related viruses include low or moderate levels of amino acid sequence identity (14·7–34·2 %) and isoelectric points among equivalent polypeptides, e.g. proteins encoded by segments S4 and S5 of the four viruses. Phylogenetic analysis indicated that MYRV-1/Cp9B21 is related more closely to MYRV-3/RnW370 than to the coltiviruses. An interesting dissimilarity is found in codon-choice pattern among the four viruses, i.e. MYRV-1/Cp9B21 segments have a lower frequency of [XYG+XYC] than corresponding segments of the other viruses, suggesting a possible adjustment of virus codon usage to their host environments.

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