Spread of white hypovirulent strains of Cryphonectria parasitica on grafted American chestnut trees exhibiting a high level of blight control

In 1982 and 1983, natural blight cankers, located in a zone extending from the ground to 183 cm on the main stem of grafted American chestnut trees, were inoculated with a mixture of dsRNA-containing, white (European) and pigmented hypovirulent strains of Cryphonectria parasitica (H-inoculated zone). In 1996, white isolates (34% of 156 C. parasitica isolates) were recovered from superficial cankers throughout the grafts and as far as 564 cm from the H-inoculated zone. Lloyd's index of patchiness value (1.36) indicated that white isolates were slightly aggregated in cankers on the grafts. Forty-five percent of 95 C. parasitica isolates, recovered 5–50 months after inoculating the grafts with pigmented virulent strain WK, were white and some converted strain WK to the white phenotype in vitro. All cankers and bark cores yielding only pigmented isolates, vegetatively compatible with virulent strain WK, had superficial necrosis 5 and 11 months after inoculation with strain WK. All white isolates of C. parasitica assayed contained a 12.7 kbp dsRNA in high concentrations, and were hypovirulent in pathogenicity trials. Twenty-eight vegetative compatibility groups were identified among 65 pigmented graft area isolates; none of 48 pigmented isolates contained dsRNA. In addition to host resistance factors, spread of white strains may be responsible for the unusually high level of blight control on the grafts.     

Epidemiological role of strains of Cryphonectria parasitica isolated from hosts other than chestnut

Cryphonectria parasitica is not common in the forests of the Emilia Romagna region of Italy on hosts other than chestnut. Three isolates with morphological cultural characteristics of C. parasitica were collected: two from infected bark of Quercits pubescens (EQ1 and EQ2) and one from Carpinus betulus (EC1). Pathogenicity tests on chestnut and crosses in vitro with strains of C. parasitica of different mating types confirmed that EQ1 and EQ2 are C. parasitica isolates, whereas EC1 seems to belong to a different species. Progeny analysis of crosses with the isolate EQ1 and a strain from chestnut showed the formation of new vegetative compatibility groups. Therefore hosts other than chestnut can contribute to increasing pathogen variability through recombination of the v-c genes.     

Cryphonectria parasitica in Portugal: diversity of vegetative compatibility types,mating types,and occurrence of hypovirulence

Cryphonectria parasitica, the causal agent of chestnut blight, has been responsible for the decline in chestnut in Portugal for the last two decades. In order to improve understanding of C. parasitica diversity, a total of 617 isolates from all affected chestnut‐growing areas in continental Portugal, Madeira and the Azores archipelagos were studied. Nine vegetative compatibility (vc) types were identified among the isolates. EU‐11 was the most widespread vc type comprising 80.2% of the isolates, followed by EU‐12 (7.1%) and EU‐66 (6.6%). Two of the Portuguese vc types could not be assigned to a known European vc type. The diversity of vc types was low in the Portuguese populations of C. parasitica, but comparable with other areas where C. parasitica was introduced recently. The frequent occurrence of perithecia and both mating types of C. parasitica indicates that sexual reproduction of the chestnut blight fungus is common in Portugal. One C. parasitica isolate from Trás‐os‐Montes showed a white culture morphology and contained dsRNA, indicating the presence of hypovirulence in this area.     

Incomplete movement of Cryphonectria hypovirus 1 within a vegetative compatibility type of Cryphonectria parasitica in natural cankers on grafted American chestnut trees

American chestnut trees, grafted in 1980 from large survivors, were inoculated in 1982 and 1983 with four white (European) hypovirulent strains of Cryphonectria parasitica, infected with C. hypovirus 1 (CHV1); this hypovirus has been shown to be capable of moving rapidly within the mycelium of a vegetative compatibility (vc) type of C. parasitica in blight cankers. Using a 49‐cell lattice plot, 17.8×17.8 cm, the spatial patterns and frequencies of white and pigmented isolates and white and pigmented vc types were investigated within superficial cankers on the grafts located outside the hypovirulent‐strain‐inoculated zone. Four of six cankers assayed contained white isolates, and three of the four had random spatial patterns of white isolates, based on join‐count statistics. Vc tests, using pigmented isolates and pigmented single‐spore colonies of white isolates, indicated that the majority of white and pigmented isolates recovered from each of two cankers assayed were in one vc type. White and pigmented lattice‐plot cells of the same vc type were frequently in contact with each other, indicating incomplete movement of CHV1 within a vc type. Nine and 10 vc types were found in the two cankers; it is hypothesized that small, white vc type areas in each canker may be a source of CHV1 transmission to the major vc types. Based on join‐count statistics, the spatial pattern of the single, major vc type in one canker was non‐random (aggregated), whereas the other canker had a random major vc type pattern. White and pigmented in vitro variants (sectors) of C. parasitica, that resemble white and pigmented in vivo variants in spatial contact and vc compatibility, were intermediate hypovirulent and virulent on forest American chestnuts, and dsRNA positive and negative, respectively. Incomplete movement of CHV1 within a vc type could be a major cause of the prevalence of pigmented isolates in superficial cankers on chestnut trees.     

Frequencies and spatial patterns of white hypovirulent and pigmented strains of Cryphonectria parasitica within blight-controlled cankers on grafted American chestnut trees 15–16 years after inoculation

The frequencies and spatial patterns of white and pigmented strains of Cryphonectria parasitica were investigated within cankers in a zone on grafted American chestnut trees inoculated with white (European) and pigmented hypovirulent strains (H-inoculated zone) 15–16 years earlier. Six 7 × 7 lattice plots (each 17.8 × 17.8 cm) were established on cankers in the H-inoculated zone of the grafts. Assays of 49 bark cores per lattice indicated that 35.3% of 306 C. parasitica isolates recovered from the six lattice plots were white. The white isolates had a random pattern, potentially favorable to biocontrol, within the highly superficial cankers, based on join-count statistics of the six lattice plots. Pigmented isolates dominated the C. parasitica population, and virulence trials on American chestnut sprouts indicated 36% of the pigmented isolates from the H-inoculated zone were hypovirulent and 27% were virulent. Most (84.3%) pigmented isolates in a bark core could not be converted to the white phenotype in vitro by white isolates in the same bark core. Five of six lattice plots had a random pattern of pigmented isolates, based on join-count statistics. The sixth lattice plot was composed of an aggregate of 36 lattice cells (area = 232 cm²) containing 12 pigmented vegetative compatability (vc) groups of C. parasitica, which were interwoven in the lattice as a mosaic of thread-like forms, blocks, or ‘islands’ 32 cm² or less in area for each vc group. Hypotheses are advanced to explain why virulent pigmented strains persist in blight-controlled cankers of the H-inoculated zone but do not kill the vascular cambium.     

Persistence of Cryphonectria hypoviruses after their release for biological control of chestnut blight in West Virginia forests

Although 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.     

Spread of Cryphonectria hypovirus1 into 45 vegetative compatibility types of Cryphonectria parasitica on grafted American chestnut trees

A mixture of hypovirulent strains of Cryphonectria parasitica, including four white (European) strains infected with Cryphonectria hypovirus1 (CHV1), was used in 1982 and 1983 to inoculate natural blight cankers located within a zone ranging from the ground to 183 cm on grafted American chestnut trees. These four white strains belonged to three vegetative compatibility (vc) types. Using pigmented, single‐spore colonies from white isolates, 48 vc types were identified among 110 white isolates recovered in 1996, 1998, and 1999 from cankers located outside the inoculated zone. Twenty‐five of the 48 white vc types consisted of two or more isolates. The 25 major white vc types were vegetatively incompatible with all four of the original white hypovirulent strains, providing evidence for spread of CHV1 but not for spread of the original inoculated strains. Forty‐five vc types represent the minimum number of `new' vc types into which CHV1 had spread. The ratio of white vc types to white isolates tested (S/N) and Shannon diversity index were 0.436 and 3.64, respectively. The spatial pattern of white vc types on the grafts was found to be non‐random (p=0.019). White single‐spore colonies of white isolates were placed into four cultural morphology (CM) groups. The two largest groups contained 37 (CM group 3) and 33 (CM group 1) isolates. Single‐spore colonies from the original, white inoculated strain, Ep 49, were classified into CM groups 3 and 1, and colonies of Ep 51 W were classified into CM group 1.     

Chestnut blight (Cryphonectria parasitica) north of the Swiss alps

Chestnut blight (Cryphonectria parasitica) was found in several stands in the Swiss plateau. At each site only one or two compatibility (vc) groups were present. The vc-group I was the most common. At a heavily infested site also young oaks (Quercus petraea) were infected.     

Characteristics of the Cryphonectria parasitica isolated from Quercus in Slovakia

The occurrence of chestnut blight (Cryphonectria parasitica) on oaks in mixed chestnut‐oak forests was studied in 2003–2008 in Slovakia. Infections on living Quercus trees were found at four of seven localities. The disease incidence on oaks ranged from 1.3% to 15.8%. The symptoms on infected oaks were similar to those on chestnut, but less conspicuous. Cankers of C. parasitica were found only on Quercus robur and Q. petraea. A total of 22 isolates of C. parasitica, all virulent, were isolated. Each site yielded only a single vc type (EU12 or EU13). Field inoculation experiments on chestnuts with seven strains of C. parasitica from oaks and an isolate from Castanea sativa showed no differences in virulence. On Quercus robur stems, the cankered area was significantly smaller than on C. sativa and the cankers developed very slowly.     

Virulence and double‐stranded RNA in Sphaeropsis sapinea

Forty wildtype isolates of Sphaeropsis sapinea were grouped into the morphotypes A and B based on previously defined differences in cultural and morphological criteria as well as restriction sites for Dde I and Bst UI endonucleases in nuclear ribosomal DNA amplicons. Thirteen of 20 type A isolates and nine of 20 type B isolates contained detectable dsRNA (55%) of different molecular weight and size. dsRNA was transmitted into conidia at a frequency of 71–100%. By selecting single conidia, dsRNA‐free subcultures were obtained from six of 22 isolates containing dsRNA. Pathogenicity tests on expanding buds of landscape trees of three species of Pinus showed highly significant statistical interactions between isolate virulence, Pinus species, and year. Pine species‐year had a profound impact on virulence. The pattern in the interactions was revealed by principal component analysis of the interaction sums of squares of the anova (Additive Main Effects and Multiplicative Interaction; AMMI). Pinus sylvestris was highly interactive in its susceptibility to S. sapinea with seasonal effects. P. nigra and P. resinosa were more stable. The interactivity analysis was used to apportion interaction to specific isolates to improve the accuracy of the estimates of virulence. Estimates of the relative virulence of isolates were predicted over five different Pinus species‐years. Isolates were ranked in virulence and interactivity using the AMMI model. This model permitted mean separation tests of the relative virulence among isolates over the combined Pinus species‐years. One isolate was identified as potentially having dsRNA‐mediated hypovirulence based on the significantly greater virulence of its isogenic, dsRNA‐free subculture, as expressed over the three Pinus species and 2 years. Type A isolates containing dsRNA ranged from stable to highly interactive and from low to high in virulence. Type B isolates containing dsRNA were similar in interactivity but virulence ranged from avirulent to moderate, seldom exceeding the mean for S. sapinea. dsRNA‐free isogenic subcultures tended not to express higher virulence than their dsRNA‐containing parent strains but often changed in interactivity. Therefore, in one year a dsRNA‐free subculture might be more virulent than its dsRNA‐containing parent. In another year the dsRNA‐free subculture might be less virulent.     

Distribution of chestnut blight and diversity of Cryphonectria parasitica in chestnut forests in Bulgaria

We surveyed chestnut stands at 18 sites in 11 locations in Bulgaria in 2005 and 2007 for the presence of chestnut blight. We found chestnut blight in seven locations (Belogradchik, Berkovitsa, Brezhani, Barziya, Govezhda, Petrich and Petrovo) but not in four others (Tsaparevo, Kresna, Dupnica and Botevgrad). We successfully isolated Cryphonectria parasitica from cankers on 606 trees with symptoms of chestnut blight and assayed them for vegetative compatibility (vc) types and mating type. Three vc types were identified among the 606 isolates; all three were among the European vc types with known vegetative incompatibility (vic) genotypes. Vc type EU‐12 was the most common, representing 80% of the isolates, and was found at all locations with blight, with the exception of Belogradchik in north‐west Bulgaria, where all isolates were vc type EU‐2. Only one population (Barziya) had more than one vc type, with a combination of EU‐12 and EU‐10 in almost equal frequencies. Similarly, the diversity of mating types was very low. All but three of 536 isolates assayed were in mating‐type MAT‐1; MAT‐2 was only found in one population in the north‐west (Berkovitsa). We inspected 671 bark samples from chestnut blight cankers with stromata of C. parasitica and found perithecia in only 33, of which 28 were from Berkovitsa where MAT‐2 was present. We did not detect hypoviruses in any of the 270 isolates screened using the standard double‐stranded RNA extraction protocol. Similar to results from previous studies in south‐eastern Europe, the diversity of vc types and mating type of C. parasitica in Bulgaria is low, and reproduction of the fungus is mainly asexual. Unfortunately, naturally occurring hypovirulence was not detected. Nevertheless, we observed a small number of superficial cankers typical of those caused by C. parasitica isolates infected with a hypovirus.     

Chestnut blight in south‐western Germany: multiple introductions of Cryphonectria parasitica and slow hypovirus spread

Cryphonectria parasitica was discovered in the sweet chestnut forests of south‐western Germany in 1992. Two main areas affected by chestnut blight were discerned, one to the east and one to the west of the Rhine valley. The occurrence of the fungal pathogen with respect to vegetative compatibility (vc) type and hypovirulence was analysed by sampling chestnut blight cankers between 1992 and 2010. Among 368 C. parasitica isolates sampled in south‐western Germany, 9 different vc types were found. East of the Rhine valley, EU‐2 is the most widespread vc type. In addition, two isolated forest areas infected with C. parasitica of the vc types EU‐14 and EU‐28 were detected. West of the Rhine valley, C. parasitica of the vc type EU‐65 was repeatedly isolated from an infection focus, the spread of which was successfully suppressed for several years by sanitation measures. Since 2003, additional outbreaks of C. parasitica belonging to the vc types EU‐2 and EU‐5 were detected in the vicinity. Several other vc types (EU‐1, EU‐12, EU‐33 and one vc type incompatible with any of the 74 European testers) were identified on isolated trees mainly in urban areas across the study area and were subsequently eradicated. The spatial and temporal distribution of the different vc types indicates at least nine different introductory events of C. parasitica into south‐western Germany. Natural hypovirulence was only found in the infection area in Baden‐Württemberg. A total of four hypovirulent isolates of the vc type EU‐2 were obtained, one in 1992 and three in recent years. The four hypoviruses were genetically closely related and belonged to the Spanish/German subtype of Cryphonectria hypovirus 1. As the different vc types in south‐western Germany occur mostly in spatially separated, single stands, the introduction of hypovirulence as biological control is expected to be effective.     

栗疫菌营养体亲和型多样性比较及野生栗栗疫菌交配型的分子检测

在北京周边县区、湖北大老岭山区以及陕西安康地区分别调查栗疫病发生情况,采集病原栗疫菌(Cryphonectria parasitica)菌株并进行营养体亲和性试验.结果表明:北京板栗栽培区和陕西安康地区野生栗栗疫病发病率较高,局部地区危害严重;湖北大老岭山区栗疫病发病率低,危害轻微.北京地区菌株群体营养体亲和型多样性指数(Shannon-Wiener's diversity index)极显著低于湖北菌株群体和陕西菌株群体,而陕西与湖北菌株群体营养体亲和型多样性指数差异不显著.随机选取湖北、陕西野生栗栗疫菌部分菌株,利用特异性引物,通过普通PCR和巢氏PCR,对其交配型进行测定,发现陕西与湖北野生栗菌株群体中均存在MAT-1与MAT-2两种交配型的菌株.通过PCR扩增,在两地的野生菌株群体里均发现同时具有两种交配型基因的菌株,其中,陕西群体此类菌株比例较大,湖北菌株比例较小     

Diversity of Cryphonectria parasitica in western Spain and identification of hypovirus‐infected isolates

Isolates of the chestnut blight fungus Cryphonectria parasitica were obtained from 44 localities in four provinces in Western Spain and characterized for vegetative compatibility (vc) types, mating types and the presence of Cryphonectria hypovirus 1 (CHV1). Among the 1232 isolates recovered from chestnut blight cankers, 11 vc types were identified: five known vc types included in EU1 to EU74 (EU1, EU11, EU12, EU28 and EU66) and six unknown vc types (CL4, CL5 CL6, CL8, CL9 and CL10). The number of vc types found per province varied between two and seven. The vc type EU11 was present in all provinces and accounted for 48.9% of all isolates. EU1 was detected in three provinces and accounted for 39.1% of the isolates. The vc types EU12, EU66, CL5 and CL6 were present in one or two provinces and comprised between 2.4% and 3% of the isolates. The other vc types were represented by only one or very few isolates. The mating type MAT‐1 was largely dominant in the provinces Leon and Avila, while both mating types MAT‐1 and MAT‐2 were found in Salamanca and Zamora. Fourteen hypovirus‐infected C. parasitica isolates were found, nine were in vc type EU1 and five in EU11, and they were detected only in the province León. All isolates analysed contained the French hypovirus subtype CHV1‐F1.     

Vegetative compatibility and conversion to hypovirulence among Italian isolates of Cryphonectria parasitica

A total of 850 virulent isolates of Cryphonectria parasitica were isolated from natural cankers on European chestnut trees growing in various Italian regions. Vegetative-compatibility (v-c) group membership was tested using the merge-barrage method. In all, 19 vegetative-compatibility groups were found, and, for each of these, a representative isolate (v-c tester) was identified; 49% of the isolates were compatible with the tester of only one v-c group, whereas 51% were compatible with the testers of more than one v-c group; 3% of the isolates showed compatibility with seven v-c groups. The coincidence between vegetative incompatibility and failure of conversion occurred in only a few cases; 42% of the converts were stable with respect to their morphological characteristics and presence of cytoplasmic ds-RNA. The results obtained in Italy suggest that, for an efficient biocontrol programme, it is preferable to evaluate the tendency of a population to be converted rather than evaluating the v-c group membership.     

Multi‐seasonal field release and spermatization trials of transgenic hypovirulent strains of Cryphonectria parasitica containing cDNA copies of hypovirus CHV1‐EP713

Three indigenous field strains of the chestnut blight fungus, Cryphonectria parasitica collected in 1996 from a test site in the Meshomasic State Forest, Connecticut, USA, were engineered to contain a chromosomally integrated full‐length infectious cDNA copy of the severe, virulence‐attenuating hypovirus CHV1‐EP713. These transgenic hypovirulent strains were introduced into the test site after it had been clear‐cut, as sprayed conidia in multiple applications over 4 years, beginning in 1997. Evidence was obtained for cytoplasmic transmission of cDNA‐derived hypovirus RNA and for recovery of transgenic strains from canker tissue of the spray‐treated trees. However, there was no indication of spread of transgenic strains or cDNA‐derived viral RNA to the control plot or outside the treatment plot. A single transgenic C. parasitica isolate was identified from a total of 156 isolates recovered from 1330 insects representing five insect orders. Only 2.6% of the 1082 C. parasitica bark isolates recovered during the course of this study were vegetatively compatible with the untransformed input strains. Transgenic ascospore progeny were produced by 40% of the perithecia collected in 1999 and by only 7.3% of the perithecia collected in 2000. A concurrent field release study performed in the Monongahela National Forest in West Virginia in 1998 and 1999 with transgenic hypovirulent strains constructed with the same CHV1‐EP713 cDNA‐containing plasmid identified seasonal application dates and delivery methods that resulted in very high levels of transgenic ascospore production. Results of the combined field release studies are discussed in terms of requirements for improved formulations and delivery methods and the importance of balancing ecological fitness and virulence attenuation.     

Diversity of vegetative compatibility types,distribution of mating types and occurrence of hypovirulence of Cryphonectria parasitica in chestnut stands in NW Spain

An extensive survey on chestnut stands in NW Spain was carried out to study the population biology of Cryphonectria parasitica in order to design future biological control programmes of chestnut blight based on the field introduction of native hypovirulent strains. Five hundred and thirty-nine Cryphonectria parasitica isolates, belonging to five populations (A Coruña, Lugo, Ourense, Pontevedra and León), were obtained. The diversity of vegetative compatibility was very low. Six vc types were found, each population showing only from 2 to 4 vc types. Two vc types were the known European testers EU1 and EU66, and the other four were undescribed vc types that were named E1, E2, E3 and E4. E1 was the dominant vc type in Lugo and Ourense populations, and EU1 in Pontevedra and León. Hypovirulent isolates (a total of 15) were only detected in the León population, all but one being compatible with the vc type EU1. Hypovirulent strains belonged to the French subtype F1 of the Cryphonectria hypovirus-1 (CHV1).     

Diversity of Cryphonectria parasitica in callused chestnut blight cankers on European and American chestnut

Infection of American and European chestnuts with the chestnut blight fungus Cryphonectria parasitica results in the formation of cankers, lesions caused by the growth of mycelia within bark tissue of the host plant. Infection of the fungus with Cryphonectria 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 hypovirulent C. parasitica and diversity of vegetative compatibility (vc) types present in calluses and randomly sampled cankers. Callused cankers on C. dentata at West Salem in the USA yielded significantly more hypovirulent C. parasitica isolates compared with four sampled populations on C. 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 of C. parasitica vc 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 in C. parasitica populations with high vc type diversity, no more than three different vc types were observed in a single callus.     

Long‐term field study of transgenic hypovirulent strains of Cryphonectria parasitica in a forest setting

Transgenic 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.