Biological control of chestnut blight with hypovirulence : a critical analysis

Most hypovirulence in the chestnut blight fungus, Cryphonectria parasitica, is associated with infection by fungal viruses in the family Hypoviridae. Hypovirulence has controlled chestnut blight well in some locations in Europe and in Michigan in the United States. In contrast, with few exceptions, biological control has failed almost completely in eastern North America. Therapeutic treatment of individual cankers is successful in most cases, but the success of hypovirulence at the population level depends on the natural spread of viruses. Characteristics of three interacting trophic levels (virus, fungus, and tree), plus the environment, determine the success or failure of hypovirulence. Vegetative incompatibility restricts virus transmission, but this factor alone is a poor predictor of biological control. Any factor reducing the rate of chestnut blight epidemics enhances hypovirus invasion. Overall, however, not enough is understood about the epidemiological dynamics of this system to determine the crucial factors regulating the establishment of hypovirulence in chestnut forests

Outcrossing and diversity of vegetative compatibility types in populations of Eutypa lata from grapevine

The sources of inoculum of Eutypa dieback of grapevines, caused by Eutypa lata, are not fully understood. Ascospores are thought to be the main source, but in populations with low disease incidence conidia may be important. Earlier studies of this disease in Italy and Germany did not report sexual structures (perithecia) of E. lata on grapevines, leaving open the possibility that populations of this fungus are clonal in some areas. However, we found perithecia in 24% and 43% of symptomatic vines in vineyards in Italy and Germany, respectively. Spatial patterns of symptomatic vines in the German vineyard were significantly aggregated but the aggregation was not strong, as might be expected if the fungus dispersed by conidia or mycelium on pruning tools. In contrast, vines with stromata and perithecia were not significantly aggregated in space. Vegetative compatibility (vc) types segregated among ascospore progeny in all perithecia sampled from both vineyards, indicating that this fungus consistently out-crosses in these populations. Finally, we found a high diversity of vc types in both vineyards, where isolates from different vines (with one exception) had unique vc types. These results reject the hypothesis that clonal reproduction of E. lata is epidemiologically significant, and strongly suggests that ascospores of E. lata are an important source of inoculum. Furthermore, E. lata's mating system is characterized by outcrossing in nature

Distribution and diversity of vegetative compatibility types in subpopulations of Cryphonectria parasitica in Italy

Using a medium that discriminated with high resolution, 20 vegetative compatibility (vc) types were detected among a sample of 716 isolates of Cryphonectria parasitica from 11 widely separated subpopulations throughout Italy. Each isolate was assigned unambiguously to a single vc type; no isolates were compatible with more than one vc type. Most isolates (85%) were in four common vc types; the other 16 vc types were represented by only a few isolates each. Vegetative compatibility type frequencies were markedly different between northern and southern Italy. Three of the most common vc types (I-1, I-2 and I-5) were found mostly in the North, where diversity was greatest, while two others (I-10 and I-12) were primarily found in the South

Identification and spread of Fomitiporia punctata associated with wood decay of grapevine showing symptoms of Esca disease

ABSTRACT A full understanding of the pathology of esca, a chronic disease of grapevines, has been problematic, in part because the identity of the pathogen (or pathogens) has been difficult to determine. The wood decay symptoms of esca have been most often associated with Phellinus igniarius or Fomitiporia punctata. However, Koch's postulates have not been completely fulfilled because symptoms take many years to develop. The goal of this study was to determine the identity and mode of spread of basidiomycetes associated with wood decay in vines showing esca symptoms in Italian vineyards. Vineyards were intensively studied for the presence of basidiocarps, and mycelium was isolated from symptomatic vines. Fruiting bodies were identified by morphology, while mycelial isolates were identified by restriction fragment length polymorphism analysis of the internal transcribed spacer region of the nuclear ribosomal RNA gene cluster. Fomitiporia punctata fruiting bodies and mycelium were associated with approximately 50% of the vines showing esca symptoms in two vineyards; P. igniarius was not found in any samples. Fruiting bodies of F. punctata were found in five of six vineyards examined, but at low frequencies except in one vineyard. The diversity of somatic incompatibility types was very high; isolates from almost every vine had different somatic incompatibility types. With few exceptions, symptomatic and dead vines were not spatially aggregated within 12 vineyards. The combination of diverse somatic incompatibility types and lack of spatial aggregations are not consistent with the hypothesis that the disease is spread clonally through roots or by pruning tools. The correct identity of basidiomycetes associated with wood decay of vines with esca symptoms is important for understanding the epidemiology of this disease because F. punctata is found commonly on many woody hosts in Europe, which may represent a potential inoculum source for this disease

Genetic differentiation between two subpopulations of Erysiphe necator in Italy

Erysiphe necator (syn. Uncinula necator) overwinters both as ascospores in cleistothecia and as mycelium in dormant buds of grapevines. Shoots developing from infected buds are known as \u2018flag shoots\u2019. We showed in a previous study that a flag shoot subpopulation of E. necator deviates from a strictly clonal or strictly randomly mating mode of reproduction. In this study we expand the investigation to a subpopulation overwintering strictly as ascospores with the objectives of: i) analyse its multilocus genetic structure, and ii) determine whether there is genetic differentiation between the two subpopulations. Finally, the two subpopulations, as defined by biological traits, were also compared using genetic markers developed to distinguish flag shoot and ascospore fungal biotypes, as defined in early studies. Two vineyards of Tuscany (central Italy), were intensively sampled for two years for flag shoots and for ascospore infections at the same time early in the epidemic. Isolates of the two mating types were found in 1:1 ratios in both subpopulations. Genotypic diversity, based on ISSR markers, was high in both years. Multilocus analysis of the population structure was not consistent with the hypothesis of random mating in either subpopulation. The two subpopulations were genetically differentiated within years based on ISSR markers. In the vineyard where the fungus overwinters as mycelium in dormant buds the subpopulation comprised both flag shoot and ascospore biotypes as shown by both sets of markers. By contrast, the subpopulation overwintering as ascospores comprised almost exclusively isolates of the ascospore biotype. These results indicate that the E. necator population in Tuscany is genetically divided in subpopulations in the two geographic areas considered. Moreover, E. necator may reproduce both clonally and sexually in both subpopulations, with restricted gene flow be-tween them. However, genetic differentiation did not correlate strictly with the overwintering strategies of the fungus because both biotypes were found within flag shoot isolates

Spatial and genetic analysis of a flag shoot subpopulation of Erysiphe necator in italy

Erysiphe necator overwinters both as ascospores in cleistothecia and as mycelium in dormant buds of grapevines. Shoots developing from infected buds early in the growing season are covered with dense mycelium and are known as \u201cflag shoots\u201d. Combining epidemiological and Cortesi et al. Phytopathology, page 2 genetic analyses, the objective of this study was to analyze the spatial and genetic structure of a flag shoot subpopulation of E. necator, as a way to assess the contribution of flag shoots as primary inoculum and to determine if the flag shoot subpopulations are clonal, with only one mating type. One vineyard in Tuscany, Italy, was surveyed intensively for flag shoots for eight years; isolations of E. necator were made from flag shoots for five years. We observed distinct disease foci developing around flag shoots early in epidemics, demonstrating a steep dispersal gradient of conidia and the importance of flag shoots as primary inoculum sources. Flag shoots were spatially aggregated within and between years, most likely as a result of short-distance dispersal of conidia from flags early in the season when dormant buds for the next year\u2019s shoots are formed and are susceptible to infection. The two mating types were found in 1:1 ratios in this flag shoot subpopulations. Genotypic diversity, based on ISSR markers, was high in all years, with only two haplotypes occurring twice, and subpopulations were genetically differentiated between years. Similarities between haplotypes were not spatially autocorrelated. One multilocus analysis of population structure is consistent with the hypothesis of random mating, but another is not. These results are not consistent with expectations for either a strictly clonal or strictly randomly mating flag shoot subpopulation. Instead, the hypothesis that the flag shoot subpopulation of E. necator may reproduce both clonally and sexually needs further testing

Mixed mating in natural populations of the chestnut blight fungus, Cryphonectria parasitica

As in plants, fungi exhibit wide variation in reproductive strategies and mating systems. Although most sexually reproducing fungi are either predominantly outcrossing or predominantly selfing, there are some notable exceptions. The haploid, ascomycete chestnut blight pathogen, Cryphonectria parasitica, has previously been shown to have a mixed mating system in one population in USA. In this report, we show that both selfing and outcrossing occur in 10 additional populations of C. parasitica sampled from Japan, Italy, Switzerland and USA. Progeny arrays from each population were assayed for segregation at vegetative incompatibility (vic) and DNA fingerprinting loci. Outcrossing rates (t\u2c6 m) were estimated as the proportion of progeny arrays showing segregation at one or more loci, corrected by the probability of nondetection of outcrossing (a# ). Estimates of t\u2c6 m varied from 0.74 to 0.97, with the lowest rates consistently detected in USA populations (0.74\u20130.78). Five populations (four in USA and one in Italy) had t\u2c6 m significantly less than 1, supporting the conclusion that these populations exhibit mixed mating. The underlying causes of variation in outcrossing rates among populations of C. parasitica are not known, but we speculate that \u2013 as in plants \u2013 outcrossing is a function of ecological, demographic and genetic factors

Persistence and spatial autocorrelation of clones of Erysiphe necator overwintering as mycelium in dormant buds in an isolated vineyard in northern Italy

The population structure of the grape powdery mildew fungus, Erysiphe necator (formerly Uncinula necator), has been hypothesized to vary from being clonal to highly diverse and recombining. We report here on the structure of an E. necator population sampled during a 4-year period from an isolated vineyard in northern Italy (Voghera, Pavia Province). We obtained 54 isolates of E. necator that overwintered asexually as mycelium in grapevine buds and caused severe symptoms on the emerging shoots, known as flag shoots. All isolates were genotyped for mating type, four multilocus polymerase chain reaction (PCR)-based markers (a total of 64 loci were scored), and two single-copy loci designed to identify genetic subgroups in E. necator. All isolates had the same mating type and single-locus alleles that correlate to isolates from flag shoots in other areas. Only 2 of the 64 loci scored from multilocus markers were polymorphic; 46 of the 54 isolates had the same multilocus haplotype. Seven isolates had a second haplotype that was recovered over 3 years, and only a single isolate was found with a third haplotype. Both variant haplotypes differed from the main clonal haplotype by single loci. Spatial autocorrelation analyses showed that vines with flag shoots were not aggregated within years, but they were aggregated between consecutive years. These results demonstrate that this subpopulation of E. necator on flag shoots is composed of a single clonal lineage that has persisted for at least 4 years. We speculate that the lack of diversity in the flag shoot subpopulation in this vineyard is the result of restricted immigration from surrounding areas and genetic drift operating through founder effects and periodic bottlenecks. We propose a model that integrates epidemiology and population genetics to explain the variation observed in genetic structure of E. necator flag shoot subpopulations from different vineyards or viticultural regions