Chestnut blight cankers caused by the fungal pathogen Cryphonectria parasitica on infected American chestnut trees can be invaded by hypoviruses that infect C. parasitica mycelia. Hypoviruses lower pathogen virulence (i.e. hypovirulence) and decrease the probability of stem girdling. Cankers also are susceptible to invasion by non-C parasitica fungi; however, the influence of invading fungi on disease severity is unknown. Fungi may antagonize C. parasitica growth and combine with hypovirulence to further reduce the probability of stem girdling. We conducted a survey to investigate how spatial and temporal dynamics of the fungal community within cankers correlate with canker severity and the likelihood of girdling. Cankers from six American chestnut populations were monitored from 2012 to 2016. We found that the spatial distribution of the fungal community within a canker resembled a mosaic that increased in fungal diversity over time. Cankers with increased fungal diversity were not associated with higher stem survivorship over time, compared to cankers with abundant hypovirulence in recovering chestnut populations. Fungal communities also were unstable, and cankers on surviving trees containing hypovirulence consistently were invaded by non-C. parasitica fungi. Our results suggest that canker communities are not stable, and volatility of the fungal community indicates that cankers can change quickly from less severe to more severe cankers through loss of hypovirulence. Non-C. parasitica fungi may facilitate canker expansion through greater inhibition of hypovirulent C. parasitica relative to virulent C. parasitica, which may permit virulent C. parasitica to escape hypovirus infection and resume rapid canker expansion. (C) 2020 Elsevier Ltd and British Mycological Society. All rights reserved.

Infection of American and European chestnuts with the chestnut blight fungusCryphonectria parasiticaresults in the formation of cankers, lesions caused by the growth of mycelia within bark tissue of the host plant. Infection of the fungus withCryphonectria hypovirus 1(CHV-1) results in conversion of the mycelial phenotype from virulent to hypovirulent, thus allowing production of callus around cankers as a reaction by infected trees, rendering active into inactive cankers. In this study, we sampled one USA and six European chestnut stands and assessed frequency of hypovirulentC. parasiticaand diversity of vegetative compatibility (vc) types present in calluses and randomly sampled cankers. Callused cankers onC. dentataat West Salem in the USA yielded significantly more hypovirulentC. parasiticaisolates compared with four sampled populations onC. sativa, while all six sampled European populations did not show any statistically significant differences among themselves. We observed no correlation between hypovirulence frequencies in randomly sampled cankers and calluses, as well as no correlation ofC. parasiticavc type diversity in calluses and residential populations of the fungus. Furthermore, even though we have observed calluses with more than one vc type, they do not occur regularly. Even when present inC. parasiticapopulations with high vc type diversity, no more than three different vc types were observed in a single callus.

Horizontal transmission of virulence attenuating hypoviruses of Cryphonectria parasitica is restricted by an allorecognition system termed vegetative incompatibility (vic). A super donor formulation of two engineered C. parasitica strains (SD328/SD82) with gene disruptions at four of six vic loci transmitted hypovirus to strains in the laboratory independent of vic genotype. We now report the transmission of hypovirus by the SD328/82 formulation to a diverse, natural C. parasitica population infecting American chestnut in a forest setting. Hypovirulent (HV) isolates were recovered from 94% of cankers treated with the hypovirus-infected SD328/82 formulation compared to 51% of cankers treated with a hypovirus-infected EU5/6 formulation (strains having the same vic genotypes as SD strains but lacking vic gene disruptions). Overall, the SD328/82 formulation transmitted hypovirus into more divergent vic genotypes compared to the EU5/6 formulation. These results demonstrate the SD328/82 formulation can serve as an enhanced hypovirus vector for highly divergent C. parasitica populations.

Hypovirus-infected Cryphonectria parasitica strains were introduced in a large stand of American chestnut (>4,000 individuals) in western Wisconsin (USA) to evaluate whether hypoviruses can serve as biological control agents. They were deployed by treating cankers from 1992 to 1997 and again from 2004 to 2014. After 17 years of hypovirus introductions within an area of the stand with the longest history of disease, isolation of hypovirus-infected strains increased from 55% in 1994 to 86% in 2014 from cankers that were treated. During the same period, isolation from cankers that arose on trees with treated cankers increased from 29 to 72% and from 15 to 84% for cankers on nearby trees that received no treatment. Tree survivorship over the 23-year study period for trees with treated cankers was 51% compared with 31% for trees that were not treated. Introduction of hypovirus has resulted in the regrowth of the crowns of many large-diameter trees. Putative recovery of American chestnut in this stand provides evidence that prolonged hypovirus treatment can act as a biological control when limited numbers of vegetative compatibility types of C. parasitica exist.

SummaryTransgenic hypovirulent strains of Cryphonectria parasitica, the chestnut blight fungus, engineered to contain a chromosomally integrated full‐length infectious cDNA copy of virulence‐attenuating hypoviruses, differ from natural hypovirulent strains in the ability to transmit hypoviruses to ascospore progeny and with 100% efficiency through asexual spores. We report the results of a long‐term field study that examined whether these properties result in enhanced hypovirulence establishment, dissemination and persistence under field conditions. Informed by previous field results using a severe hypovirus, this study that employed 144 American chestnut trees was designed to provide improved inoculum formulation and delivery and to include the use of a mild hypovirus isolate (less debilitating) CHV‐1/Euro7 in an attempt to increase dissemination. Isogenic transgenic hypovirulent (TG), non‐transgenic cytoplasmic hypovirulent (CH) or virus‐free virulent (V) treatment strains were applied to artificially initiated and natural C. parasitica cankers three times each year for 7 years. Reservoirs of treatment inoculum also were initiated and refreshed annually for the first 6 years of the study. Sampling of 111,000 individual ascospores from 4,500 perithecia confirmed hypovirus‐containing spermatia successfully transmitted TG hypoviruses to ascospore progeny under field conditions. Surprisingly, TG ascospore progeny were recovered 3 years after the last annual application of treatment inoculum. Repeated sampling of over 440 cankers revealed dissemination of both CH and TG hypovirulent strains. However, no significance differences in establishment or dissemination were observed for the two hypovirulent strains. The results are discussed in terms of the contribution of ascospore progeny to infection, competition by endemic virulent C. parasitica, size of inoculated trees and the biological control potential of TG hypovirulent strains.

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.

A research project to introduce hypoviruses and monitor their spread was initiated 20 years ago in an American chestnut (Castanea dentata) stand growing 900 km west of the natural range of the species in western Wisconsin (USA). During this period, CHV1 and CHV3 hypoviruses were deployed by treating cankers with the resident Cryphonectria parasitica strains that were hypovirus infected. Hypovirus spread has been assessed annually by removing and culturing small bark plugs from cankers and evaluating whether the resulting colonies were morphologically virulent or hypovirulent. A variety of fungi other than C. parasitica have been recovered from cankers during this study. In order to better understand the role of recovered fungi and their relationship over time, fungal isolates recovered in 2012 from 500 cankers were categorized by the age of the canker (based on date of discovery), appearance/morphology and location within the stand. Cankers were divided into four age categories: (1) one-year-old cankers; (2) two-to-four years-old; (3) five-to-ten years-old; and (4) eleven-to-twenty years-old. Virulent C. parasitica isolates were most frequently recovered from one-year old cankers, but recovery decreased from a high of 60% in one-year-old cankers to 43, 30 and 19%, respectively, for the three increasingly older categories. In contrast, the recovery of hypovirulent C. parasitica isolates increased from 17% in one-year-old cankers to 35% in cankers representing the oldest canker category. Isolation of Trichoderma atroviride and T. aureoviride increased as cankers aged and accounted for 42% of all the colonies recovered from the oldest cankers. Most species of Trichoderma are aggressive saprophytes; some are proven biocontrol agents. Other fungi that were isolated included Botryosphaeria sp., Didymostilbe sp., Dothiorella sp., Epicoccum nigrum, Paraconiothyrium sp., Pestalotia sp., Penicillium spinulosum, Penicillium glabrum and Umbellopsis isabellina but these represented only 4-7% of isolates among all age categories. A decrease in the recovery of virulent C. parasitica strains and a corresponding increase in recovery of hypovirulent C. parasitica strains would be expected if biological control is active. The frequency with which Trichoderma spp. have been recovered may implicate this organism as a contributor to chestnut blight biological control.

Transgenic hypovirulent (HV) strains of Cryphonectria parasitica engineered to contain a complimentary DNA (cDNA) copy of Cryphonectria hypovirus 1 (CHV1) may provide an efficient mechanism to introduce hypoviruses into genetically diverse populations of the chestnut blight fungus. However, elucidating the factors that influence infection of American chestnut (Castanea dentata) and the initiation of HV cankers is necessary to exploit their biological control potential. In this study, inoculation tests were performed to determine whether spore concentration (300 vs. 3,000 spores/inoculation) influenced the incidence of infection after seven months for transgenic HV, cytoplasmic HV and virulent conidia. Inoculations also were conducted using ascospores and mycelium of the virulent and HV strains. Growth and asexual sporulation measurements of the resulting cankers showed that spore concentrations delivered at 300 spores/site had no effect on the number of infections or on canker growth. In contrast, inoculations using 3,000 virulent conidia significantly increased the frequency of infection compared to those made with 300 virulent conidia. Asexual sporulation, however, was not observed on the surface of cankers initiated by virulent, cytoplasmic HV or transgenic HV inocula. Both the cytoplasmic and transgenic HV isolates were less effective at infecting and growing in chestnut bark when compared to the virulent isolate. Deployment of transgenic HV isolates for the biological control of C. parasitica likely provides no significant advantage over control strategies that utilize cytoplasmic HV isolates when canker initiation to establish HV cankers is the goal.

Strawberry fruit were incorporated into a potato dextrose agar (PDA) medium in an attempt to develop a medium that would promote conidiation of Cryphonectria parasitica isolates. Two other plant pathogenic fungi (Helminthosporium sativum and Fusarium solani) were also tested. Isolates were grown on PDA and strawberry-amended PDA for 14 d at which time agar plugs were removed to assess conidiation. In most instances, more conidia were produced on the strawberry-amended PDA than on unamended PDA for both virulent and hypovirulent C. parasitica isolates and for Helminthosporium sativum and Fusarium solani.

A quantitative evaluation of hypovirus treatment on the recovery of infected American chestnuts at West Salem, Wisconsin was made by monitoring 164 trees from 2006 to 2012. Survivorship of treated trees was better than untreated control trees for the period from 2008 to 2012, and the cumulative increase in survivorship was 20% by the 2012 tree census. Survivorship of trees was influenced by initial tree size regardless of treatment, with larger trees surviving better than smaller ones. Treated trees also grew faster than control trees in four of the six years. Hypovirus treated trees grew seven times faster than control trees from 2007 to 2008 and four times faster from 2010 to 2011.

To evaluate the frequency of naturally-occurring hypovirus-containing isolates in the southern Appalachian Mountains, one hundred fifty-nine cankers on one hundred thirty-nine American chestnut trees were sampled over a two-year period (2010-2011) in the Great Smoky Mountains National Park (GRSM). Trees were growing along GRSM hiking trails in Tennessee and North Carolina (USA). Cryphonectria parasitica was recovered from all cankers and from >95% of the samples. Vegetative compatibility (vc) testing identified fifty-two vc groups among the GRSM isolates using the sixty-four European Union (EU) vc testers. Isolates from one canker were putatively identified as hypovirulent, based on colony morphology, dsRNA extraction, single sporing and pathogenicity tests. These data indicate that a high level of vc diversity exists among the C. parasitica population in the GRSM and naturally-occurring hypoviruses are rare. Diversity in vc may represent significant restriction to hypovirus spread.

The recovery of chestnut from chestnut blight in Italy and Michigan largely was responsible for the resurgence in chestnut research. The observed remission of disease now has been attributed to a biological control process called hypovirulence, whereby virulent strains are debilitated as a result of infection by fungal viruses (hypoviruses). Several species of hypoviruses now are known and each may impart unique effects on Cryphonectria parasitica. Lethal infections often are controlled by introducing the appropriate hypovirus into cankers. Unfortunately, at many locations within the native range of American chestnut, a complex system of vegetative incompatibility restricts hypovirus transmission among strains. Factors like vegetative incompatibility apparently regulate the widespread establishment of hypoviruses and presumably are, in part, responsible for our inability to artificially establish hypoviruses to the extent that has occurred naturally. Some of the factors that regulate hypovirus success or failure may be discovered as part of ongoing research at an isolated Wisconsin chestnut stand. Hopefully, understanding the phenomenon of hypovirulence eventually will allow it to be employed as part of the American chestnut restoration program.

Historically, hypovirulent strains of Cryphonectria parasitica have been introduced into cankers principally by inoculating wounds made to the margin of cankers. Twelve hypovirus introduction methods were tested. Seventy-two trees were inoculated in June 2002 with an orange-pigmented virulent strain of C. parasitica (two inoculations per tree). After 11 weeks, the resulting cankers were treated with a brown-pigmented hypovirulent C. parasitica isolate using one of the following methods: (1) a non-invasive treatment where the inoculum was painted onto the canker surface; (2) an invasive treatment where the canker face was wounded with a sharp blade prior to the painting application of the hypoviruses; and, (3) a margin punch treatment where a series of wounds were made around the canker perimeter and filled with inoculum of the hypovirulent strain. Additionally, half the cankers in each treatment were covered to keep treatment inoculum moist and to protect treatment inoculum from biotic and abiotic factors. Isolates that were vegetatively compatible or incompatible with the canker inciting strain were employed. Hypovirus transmission was evaluated in November 2002 and May 2003 by culturing bark plug samples and evaluating the pigmentation and morphologies of isolates recovered. Canker dimensions also were recorded to determine if hypovirus transmission had an affect on canker expansion. (Abstract shortened by UMI.).

Heterokaryosis was recently reported in the chestnut blight fungus, Cryphonectria parasitica, in which individuals contain nuclei that are isogenic except at the mating-type locus (MAT). MAT heterokaryons were found in several natural populations, including a putatively clonal population in West Salem, Wisconsin, providing an opportunity to address the question of how heterokaryons arise. We represented relationships among RFLP fingerprint haplotypes as networks in which loop formation is considered evidence of recombination. From 1990 to 1995, this population was clonal, as indicated by a simple haplotype network without loops, and the correlation of vegetative compatibility (vc) types and mating types with haplotype lineages. By 1999, we observed loops in the haplotype network involving isolates of two vc types (WS-2 and WS-3). Isolates with haplotypes in the loops were either MAT heterokaryons, carried the opposite mating type from other isolates of the same vc type, and/or had two alleles at two or more codominant SCAR (sequence-characterized amplified region) loci. Segregation of markers and recombination were evident among single-spore isolates from one heterokaryon; these single-spore isolates had novel fingerprint haplotypes, also within the loops. In contrast, vc type WS-1, which comprises 85% of the population, was represented by a simple network with no loops, indicating a clonal lineage varying only by mutation. Almost all isolates of WS-1 had the same mating type; the exceptions were five isolates that were MAT heterokaryons. These results are consistent with the hypothesis that heterokaryons formed between vegetatively incompatible individuals, and recombination occurred by a parasexual process.

SummaryAlthough Cryphonectria hypoviruses have been relatively successful as biological control agents of chestnut blight in Europe, their success in North America has been limited. Experimental releases of hypoviruses were made in 1978–82 at two sites in West Virginia forests with high densities of regenerating chestnut trees. Cryphonectria hypovirus 1 (CHV‐1) from Europe, as well as American isolates of Cryphonectria parasitica containing CHV‐3, were used for these releases. Although most trees died during the 5‐year release period, it was not known if the hypoviruses persisted in the C. parasitica population at the two sites. When the experimental plots were revisited in 1994, few chestnut trees were found. The exception was one plot containing coppice sprouts that had grown from the root collars of the original trees. The authors intensively sampled C. parasitica from experimental plots and screened recovered isolates for double‐stranded RNA (dsRNA). None of the isolates contained CHV‐1; only six isolates contained CHV‐3, all from the plots with the coppice sprouts. CHV‐4, which occurs naturally in West Virginia forests and in two released isolates, hybridized to dsRNA from the isolates containing CHV‐3, indicating mixed infections. CHV‐4 also hybridized to dsRNA from other isolates sampled inside and outside the treated plots. In contrast to CHV‐1 and CHV‐3, however, CHV‐4 has little effect on the growth or phenotype of C. parasitica. The limited persistence of CHV‐1 and CHV‐3 may have resulted when the C. parasitica population was reduced in size due to the failure of chestnut trees to resprout because of competition from other hardwood species.