Identifying Hypovirulent Isolates of<i>Cryphonectria parasitica</i>with Broad Conversion Capacity

Kuhlman, E. G.; Bhattacharyya, H. K.; Nash, Bruce; Double, M. L.; MacDonald, William L.

doi:10.1094/phyto-74-676

Cited by 30 publications

(14 citation statements)

References 8 publications

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“…Although this approach has been successful in increasing hypovirulence conversion capacity (28,29,31), it is limited by the number of different hypovirulent strains needed to convert all strains in a highly diverse vic population as found in most North American forest settings (7,17) and requires considerable effort for strain propagation and inoculum formulation. In contrast, based on the laboratory transmission results presented here, the application of two SD strains representing the vic2-1 and vic2-2 alleles should convert field strains representing all possible vic genotypic combinations of the defined six diallelic vic genetic loci.…”

Section: Discussionmentioning

confidence: 99%

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

Zhang

Nuss

2016

Proc. Natl. Acad. Sci. U.S.A.

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Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.M ycovirus infections have been reported to reduce virulence (hypovirulence) for a wide range of plant pathogenic fungi, providing potential for biological disease control (1-6). For hypovirulence to be effective, the virulence-attenuating viruses must be efficiently transmitted from infected hypovirulent strains to uninfected virulent strains (5, 7). Mycoviruses generally have evolved exclusive intracellular lifestyles (8). With very few exceptions (9), mycovirus infections cannot be initiated by exposure of uninfected hyphae to cell extracts or secretions from infected fungal isolates. Transmission is limited to intracellular mechanisms, vertical transmission through asexual spores, and horizontal transmission through anastomosis (fusion of hyphae).Horizontal mycovirus transmission to uninfected isolates of the same fungal species is complicated by nonself allorecognition genetic systems, termed "heterokaryon" or "vegetative incompatibility" (vic), which operate widely in filamentous fungi (10). Interactions between genetically distinct individuals of the same species result in an incompatible reaction that triggers localized programmed cell death (PCD), forming a line of demarcation, or barrage, along the zone of contact (10-12) and restricting cytoplasmic transmission of viruses and other cytoplasmic elements (1,(13)(14)(15).A negative correlation between vic diversity and virus transmission has been reported for several fungal hosts (1, 16), but has most extensively been demonstrated for the chestnut blight fungus Cryphonectria parasitica infected with virulence-attenuating hypoviruses (7,(17)(18)(19)(20). Genetic analyses have defined six diallelic vic genetic loci for C. parasitica (21). These loci and associated genes were recently identified at the molecular level through a comparative genomics approach (22, 23) ( Table 1). Independent gene disruption analysis of 12 genes associated with these loci (22, 23) ...

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Section: Discussionmentioning

confidence: 99%

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

Zhang

Nuss

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The field locations included Great Smoky Mountains National Park, TN (GS); Pendleton County, WV (PC); Shenandoah National Park, VA (SH); Tucker County, WV (TC); and West Salem, WI (WS). Mating type and traditional coculturing incompatibility assays to study vic diversity previously have been published for a subset of isolates from the Great Smoky Mountains National Park, TN (30), Tucker Co., WV (31,32,33), and West Salem, WI (5,16,34). Fungal isolates were cultured on Difco potato dextrose agar (PDA; BD and Co., Franklin Lakes, NJ, USA).…”

Section: Methodsmentioning

confidence: 99%

Multilocus PCR Assays Elucidate Vegetative Incompatibility Gene Profiles of Cryphonectria parasitica in the United States

Short

Double

Nuss

et al. 2015

Appl Environ Microbiol

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Chestnut blight is a devastating disease of Castanea spp. Mycoviruses that reduce virulence (hypovirulence) of the causative agent, Cryphonectria parasitica, can be used to manage chestnut blight. However, vegetative incompatibility (vic) barriers that restrict anastomosis-mediated virus transmission hamper hypovirulence efficacy. In order to effectively determine the vegetative incompatibility genetic structure of C. parasitica field populations, we have designed PCR primer sets that selectively amplify and distinguish alleles for each of the six known diallelic C. parasitica vic genetic loci. PCR assay results were validated using a panel of 64 European tester strains with genetically determined vic genotypes. Analysis of 116 C. parasitica isolates collected from five locations in the eastern United States revealed 39 unique vic genotypes and generally good agreement between PCR and tester strain coculturing assays in terms of vic diversity and genotyping. However, incongruences were observed for isolates from multiple locations and suggested that the coculturing assay can overestimate diversity at the six known vic loci. The availability of molecular tools for rapid and precise vic genotyping significantly improves the ability to predict and evaluate the efficacy of hypovirulence and related management strategies.

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“…barriers (3,29), hypoviruses were transferred indirectly via isolates in different vegetative compatibility types as described previously (3,28,29). Hypovirus transmission was confirmed by CF11 column purification of dsRNA from recipient isolates as described above.…”

Section: Methodsmentioning

confidence: 99%

Variation in Tolerance and Virulence in the Chestnut Blight Fungus-Hypovirus Interaction

Peever

Liu

Cortesi

et al. 2000

Appl Environ Microbiol

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Chestnut blight, caused by the fungus Cryphonectria parasitica, has been effectively controlled with doublestranded RNA hypoviruses in Europe for over 40 years. The marked reduction in the virulence of C. parasitica by hypoviruses is a phenomenon known as hypovirulence. This virus-fungus pathosystem has become a model system for the study of biological control of fungi with viruses. We studied variation in tolerance to hypoviruses in fungal hosts and variation in virulence among virus isolates from a local population in Italy. Tolerance is defined as the relative fitness of a fungal individual when infected with hypoviruses (compared to being uninfected); virulence is defined for each hypovirus as the reduction in fitness of fungal hosts relative to virus-free hosts. Six hypovirus-infected isolates of C. parasitica were sampled from the population, and each hypovirus was transferred into six hypovirus-free recipient isolates. The resulting 36 hypovirus-fungus combinations were used to estimate genetic variation in tolerance to hypoviruses, in hypovirus virulence, and in virus-fungus interactions. Four phenotypes were evaluated for each virus-fungus combination to estimate relative fitness: (i) sporulation, i.e., the number of asexual spores (conidia) produced; (ii) canker area on field-inoculated chestnut trees, (iii) vertical transmission of hypoviruses into conidia, and (iv) conidial germination. Two-way analysis of variance (ANOVA) revealed significant interactions (P < 0.001) between viruses and fungal isolates for sporulation and canker area but not for conidial germination or transmission. One-way ANOVA among hypoviruses (within each fungal isolate) and among fungal isolates (within each hypovirus) revealed significant genetic variation (P < 0.01) in hypovirus virulence and fungal tolerance within several fungal isolates, and hypoviruses, respectively. These interactions and the significant genetic variation in several fitness characters indicate the potential for future evolution of these characters. However, biological control is unlikely to break down due to evolution of tolerance to hypoviruses in the fungus because the magnitudes of tolerance and interactions were relatively small.Despite widespread release over many years, the efficacy of biological control agents in populations of target pests has remained relatively stable (22,25). This stability contrasts markedly to the situation with chemical pesticides, where numerous target pests have evolved resistance to a wide range of pesticides (19). In a recent review, Holt and Hochberg (25) summarized some of the hypotheses that have been proposed to explain this dichotomy. These hypotheses include inadequate relevant genetic variation in target pests, genetic constraints (trade-offs) between resistance (or tolerance) to a biological control agent and fitness, weak selection, temporally varying selection, and coevolutionary dynamics. Few of these hypotheses have been addressed experimentally in biological control systems, although genetic variation in r...

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Identifying Hypovirulent Isolates ofCryphonectria parasiticawith Broad Conversion Capacity

Cited by 30 publications

References 8 publications

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

Multilocus PCR Assays Elucidate Vegetative Incompatibility Gene Profiles of Cryphonectria parasitica in the United States

Variation in Tolerance and Virulence in the Chestnut Blight Fungus-Hypovirus Interaction

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