Relationship Between Biological Control, Incidence of Hypovirulence, and Diversity of Vegetative Compatibility Types of Cryphonectria parasitica in France

ABSTRACT In France, chestnut blight, caused by Cryphonectria parasitica, has been controlled since 1974 in orchards, but never in coppice forests, by releasing hypovirulent strains infected with CHV1 hypovirus. We tested the hypothesis that this biological control (BC) has lead to a decrease in blight severity, spread of hypovirulence, and change in C. parasitica populations. The low severity of chestnut blight was confirmed in the six regions studied (subdivided into zones). The remission of cankers was associated with the presence of white isolates presumed to be hypovirulent. These two parameters were also correlated, at the zonal level, to the frequency of sites where BC was used. However, the estimates of the natural background level of hypovirulence, independent of BC, ranged from 4% in forests in Dordogne to 60% in orchards in Lozère. Differences in the rate of hypovirulent isolates among regions were consistent with the diversity of vegetative compatibility (VC) types in populations of C. parasitica. The highest VC-type diversity and mean allelic diversity for known vegetative incompatibility (vic) genes were observed in Dordogne. We showed that the current diversity of VC types in populations of C. parasitica was lower than in 1981. We found 30 VC types among 1,113 isolates of C. parasitica. Ten VC types were incompatible with known EU testers, suggesting that one additional vic gene or allele at one of the six vic loci known should be present in Europe.     

Population Structure and Disease Development of Cryphonectria parasitica in European Chestnut Forests in the Presence of Natural Hypovirulence

ABSTRACT The Cryphonectria parasitica populations in two 6-year-old European chestnut (Castanea sativa) coppices were investigated in southern Switzerland over a period of 4 years. Occurrence of white isolates indicating an infection with Cryphonectria hypovirus, vegetative compatibility groups (VCGs), hypovirulence conversion capacity, and mating types were used to characterize the populations. Sampling of randomly chosen cankers in the first year yielded 59% white isolates in one and 40% in the other population. The distribution of the VCGs and mating types was similar among white and orange isolates, indicating a homogeneous infection of the two populations by the hypovirus. Fourteen VCGs were found in the first population, 16 VCGs in the second. Altogether, 21 VCGs were determined. The same three VCGs dominated in both populations, comprising more than 60% of all isolates. Several VCGs were represented only by white isolates. Five of the six most common VCGs were clustered in two hypovirulence conversion groups, with almost 100% hypovirus transmission within each cluster. Repeated sampling of the same cankers in 1990, 1992, and 1994 did not reveal an increase of white isolates. The portion of blighted stems rose from 37% to about 60% in both plots within 4 years. In this time, chestnut blight killed 15% and competition an additional 21% of the sprouts. Predominantly, sprouts with low diameters at breast height were killed. The growth rate of new cankers was high in their first year and decreased gradually in the following years. A role of hypovirulence in the decline of disease severity was evident since (i) cankers yielding white isolates grew slower and killed considerably fewer sprouts than cankers with orange isolates; and (ii) the majority of the cankers yielded white isolates at least once during the 4-year observation period.     

The effect of Cryphonectria parasitica on water relations of American chestnut

Chestnut blight has commonly been regarded as a phloem disease due to conspicuous stem cankers that result from infection by the fungal pathogen Cryphonectria (Endothia) parasitica. Stomatal conductance (g_s), transpiration rate (E) and leaf water potential were measured throughout the day on leaves distal to naturally-occurring virulent (sunken bark with abundant stromata) or hypovirulent (swollen bark lacking stromata) cankers and cankers induced by inoculation with virulent or hypovirulent strains of C. parasitica. Relative to control stems, hydraulic conductivity (K_h), g_s and E were reduced significantly (α = 0·05) for both natural virulent cankers, and cankers that were induced by a virulent strain. These effects were less pronounced for both natural and induced hypovirulent cankers. Isolation experiments revealed that the percentage of xylem tissue chips yielding C. parasitica was greater for virulent than for hypovirulent cankers. The data provide evidence that the localized presence of C. parasitica in cankers of American chestnut results in stomatal closure, possibly as a direct result of xylem dysfunction.     

Seasonal patterns of dispersal of ascospores of Cryphonectria parasitica (chestnut blight)

Infected barks of chestnut blight cankers, caused by Cryphonectria parasitica , were collected from a naturally infected orchard and incubated at different temperatures. Cankers started to discharge ascospores about a week after incubation at 15–25°C; most ascospores were collected at 20 and 25°C. When incubated at 5, 10 or 30°C, only a few cankers released a small number of ascospores and only during the later stages of incubation. However, the rate of formation of perithecia was not affected by the incubation temperature. The number of airborne ascospores was monitored using a volumetric spore trap in a chestnut orchard during 1996 and 1997. In both years, the number of ascospores trapped daily varied greatly, but in general it increased sharply from March onwards, reached a peak in May, and then declined steeply. There was a significant correlation between daily counts of ascospores and air temperature. Time-series transfer function (TF) analysis showed a positive association of the daily number of ascospores with increasing temperature, rain events and wet/humid conditions. In general, values predicted by the TF model agreed well with the observed pattern. However, a multiple regression equation based on TF analysis failed to provide a satisfactory prediction of the daily number of ascospores.     

Low diversity of vegetative compatibility types and mating type of Cryphonectria parasitica in the southern Balkans

The diversity of vegetative compatibility (vc) types and mating type was estimated in populations of the chestnut blight fungus, Cryphonectria parasitica , throughout Macedonia and from selected areas in Greece. Nearly all of the 786 isolates (94%) from Macedonia were in a single vc type, EU-12; all 379 isolates from Greece were EU-12. Only six of 20 populations in Macedonia had more than one vc type. The diversity of vc types in the most diverse populations of Macedonia was comparable with the least diverse populations found previously in Italy. All but six of the 313 isolates assayed had the same mating type, MAT-1 , and no perithecia of Cryphonectria parasitica were observed in any population. These results lead to the conclusion that sexual reproduction does not occur in these populations. The lack of vc type diversity may indicate a high potential for the spread of hypoviruses and successful biological control with transmissible hypovirulence. However, if sexual reproduction should occur in Macedonian populations, up to 32 vc types would be possible by recombination among vegetative incompatibility loci.     

Factors influencing growth,sporulation and virus transfer in <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> isolates from Castilla and León (Spain)

Biological control with Cryphonectria hypovirus CHV1 of the chestnut blight, caused by the fungus Cryphonectria parasitica, has reduced the impact of the disease in Europe. The virus reduces the virulence of the fungus so that it causes non-lethal cankers, thus enabling the chestnut trees to overcome the disease. The virus can be transmitted horizontally by hyphal anastomosis or vertically to the conidia. In this study, we investigated growth and sporulation of the fungus as well as rates of horizontal transmission of the virus at different temperatures. We used fungal isolates of the vegetative compatibility types (vc types) that are most prevalent in Castilla and León (central northern Spain) to evaluate the effects of fungal strain on the parameters tested. In addition, we infected four isolates of C. parasitica with hypovirus subtypes CHV1-F1 and CHV1-I, to determine the influence of virus subtype on growth, sporulation and virus transfer. We assessed growth of fungal colonies and horizontal transmission of the virus at 15 °C and 25 °C. Colony growth was affected by an interaction between fungal isolates included in vc type EU1 or EU11 and virus at both 15 °C and 25 °C. However, horizontal transmission of the virus was only influenced by the fungal genotype of isolates included in vc type EU1 or EU11, and spore production was only affected by the virus subtype. Vertical transmission was also influenced by the fungal isolate and virus subtype. Growth of the fungal isolates varied depending on the virus subtype with which they were infected. This supports the theory that fungal host and virus subtype influence transmission and dissemination of hypovirulence. The fungal genotype affects colony growth and horizontal transmission of the virus. It is common to expect a good dissemination of the hypovirus with a low vc type diversity but the selection of the best combination of hypovirus and fungal isolate is crucial for the success of biological control not only for small areas but in larger chestnut populations as well.     

Elevated mitochondrial alternative oxidase activity in dsRNA-free, hypovirulent isolates of Cryphonectria parasitica

In Cryphonectria parasitica, the causal agent of chestnut blight, hypovirulence is commonly associated with the presence of cytoplasmically transmissible, double-stranded RNA (dsRNA). Recently, hypovirulent isolates of C. parasitica that do not have detectable levels of dsRNA have been obtained from healing cankers on chestnut trees. In these isolates, most of the respiratory activity is cyanide-resistant and salicylhydroxamate-sensitive, indicating that the mitochondrial alternative oxidase was induced. In contrast, the respiration of virulent isolates from the same trees and most of the dsRNA-containing hypovirulent isolates from a variety of geographical locations was cyanide sensitive. In all of these isolates, the alternative oxidase could be induced by growing them in the presence of chloramphenicol, an inhibitor of mitochondrial protein synthesis. The attenuated virulence and altered respiratory phenotypes could be transferred from the dsRNA-free hypovirulent isolates to virulent strains through hyphal anastomosis. Some of the dsRNA-free hypovirulent isolates grew more slowly than virulent strains. These results indicate that, in Cryphonectria, cytoplasmically transmitted hypovirulence may have more than one cause, and that mitochondrial mutations sometimes may be involved in the generation of this phenotype.     

Hypovirus Transmission to Ascospore Progeny by Field-Released Transgenic Hypovirulent Strains of Cryphonectria parasitica

ABSTRACT Strains of the chestnut blight fungus, Cryphonectria parasitica, have been genetically engineered to contain an integrated full-length cDNA copy of the prototypic virulence-attenuating hypovirus CHV1-EP713. Unlike natural hypovirulent C. parasitica strains, these transgenic hypovirulent strains are able to transmit virus to ascospore progeny under laboratory conditions. This ability provides the potential to circumvent barriers to cytoplasmic virus transmission imposed by the fungal vegetative incompatibility system. During July 1994, transgenic hypovirulent strains were introduced into a Connecticut forest site (Biotechnology Permit 94-010-01). Subsequent analysis of the release site confirmed hypovirus transmission from transgenic hypovirulent strains to ascospore progeny under field conditions. Additionally, it was possible to recover transgenic hypovirulent strains from the test site as long as 2 years after the limited, single-season release. Evidence also was obtained for cytoplasmic transmission of transgenic cDNA-derived hypovirus RNA, including transmission to mycelia of a virulent C. parasitica canker after treatment with conidia of a transgenic strain. Finally, a transgenic hypovirulent strain was recovered from a superficial canker formed on an untreated chestnut tree. Genetic characteristics of the recovered strain suggested that the canker was initiated by an ascospore progeny derived from a cross involving an input transgenic hypovirulent strain. The durability of a molecular marker for field-released cDNA-derived hypovirus RNA is discussed.     

Characterization ofCryphonectria parasitica isolates collected from Aydın province in Turkey

The major chestnut-growing areas in Aydın Province, which yield nearly 35% of Turkey’s production, were examined for the presence of chestnut blight and bark samples were collected from the canker formations. Generally virulent type cankers were observed. Of 31 villages, 22 were found to be contaminated with the disease. A total of 97Cryphonectria parasitica (Murrill) M.E. Barr. isolates were characterized for the presence of potential hypovirulence by looking at cultural characteristics on media and virulence in cv. ‘Golden Delicious’ apple fruit. The isolates were placed into four groups based on pigmentation and abundance of pycnidia formation. Isolates produced lesions of various sizes on apple fruits but lesion size was not correlated with cultural characteristics typical to hypovirulent isolates. Among 97 isolates, ten possessing the characteristics associated with hypovirulence were assayed for the presence of dsRNA. Among these isolates only one, coded M1-3, was found to contain dsRNA. No dsRNA was detected in the single conidium isolates derived from M1-3. All of the isolates were screened for vegetative compatibility and two European vc types, EU-1 and EU-12, were detected. This low vc-type diversity in the region may indicate a high potential for spread of transmissible hypovirulence. http://www.phytoparasitica.org posting May 30, 2008.     

Genetic diversity of Cryphonectria parasitica causing chestnut blight in South Tyrol (northern Italy)

European Journal of Plant Pathology - European chestnut (Castanea sativa) is threatened by the invasive fungus Cryphonectria parasitica, which causes chestnut blight. The virulence of the fungus...     

Diversity of vegetative compatibility types and mating types of Cryphonectria parasitica in Slovenia and occurrence of associated Cryphonectria hypovirus 1

Cryphonectria parasitica, the causal agent of chestnut blight, has been present in Slovenia since at least 1950. To improve understanding of its diversity, 254 isolates of the fungus from 11 Slovenian populations were sampled. Fifteen vegetative compatibility (vc) types were identified. The dominant vc type was EU‐13, comprising 40·1% of all isolates tested, followed by EU‐1 (19·7%), EU‐2 (12·2%) and EU‐12 (9%). The vc type diversity in the most diverse population sampled in Slovenia was higher than in the populations found previously in northern Italy and Croatia. Both mating types and perithecia were observed in surveyed populations. Natural hypovirulence was found in six out of seven populations tested, with frequencies ranging from 72·2% in the population sampled near the Croatian border to 11·1% in the population sampled near the Austrian border. All identified hypoviral isolates (21) belonged to the Italian subtype of Cryphonectria hypovirus 1 and were closely related to the hypoviruses found in other European countries. Despite the high vc type diversity, incidence of hypovirulence was also high, indicating widespread natural biological control of the disease.     

Detection of Eutypa lata and Eutypella vitis in Grapevine by Nested Multiplex Polymerase Chain Reaction

ABSTRACT Two fungi were isolated from grapevines in Michigan vineyards with Eutypa dieback symptoms: Eutypa lata and Eutypella vitis. These fungi are difficult to distinguish morphologically but are genetically distinct as determined by sequencing of the internal transcribed spacer (ITS) regions. The ITS regions of 25 Eutypa lata and 15 Eutypella vitis isolates were sequenced. Eutypa lata sequences were more variable than those of Eutypella vitis. Polymerase chain reaction (PCR) primers were designed for each species and evaluated against isolates of both fungi as well as 11 closely related Diatrypaceous fungi and 23 isolates of other fungi representing various pathogenic, saprophytic, and endophytic genera on grape and other small fruit crops. The primers were specific for their intended species. A nested multiplex PCR protocol was developed and used to successfully detect these fungi in wood samples from cankers with and without stromata from naturally infected vines as well as in artificially inoculated, potted canes. The primers developed in this study will assist in our abilities to diagnose and study the roles of Eutypa lata and Eutypella vitis in Eutypa dieback development.     

栗疫病菌侵染板栗枝条的显微观察

栗疫病是一种严重危害栗属植物的病害。为了明确栗疫病菌侵染板栗枝条的过程及侵染的关键时间点,本研究利用病理组织切片技术、显微镜和扫描电镜技术对栗疫病菌侵染板栗枝条的过程进行了观察。结果表明:接种栗疫病菌后0～5 h,菌丝先降解枝条表皮,进行横向营养生长的同时沿着伤口纵向侵染,为进入皮层做准备;接种后6 h病菌开始在表皮定殖,并侵入皮层;接种后9 h在皮层可观察到侵染性菌丝沿着细胞间隙向相邻细胞延伸;接种后12 h栗疫病菌侵入韧皮部,在皮层的侵染面积扩大。随着侵染程度加深,皮层、韧皮部等处细胞被菌丝降解,最终在形成层附近聚集。接菌后9 h为栗疫病菌侵染板栗枝条的关键时间点。     

Pathogenicity of Marssonina betulae and other fungi on birch

Marssonina betulae , Discula betulina , Melanconium bicolor and Fusarium avenaceum were inoculated onto shoots of 1- and 2-year-old seedlings of Betula pendula and B. pubescens and symptom development monitored over several seasons. Marssonina betulae caused disease on B. pendula , but not on B. pubescens . On B. pendula symptoms included discrete lesions, which often girdled, causing dieback of inoculated leading shoots, and the development of secondary sunken cankers on the main stems, which were usually centred around a dead sideshoot. Cankers on the main stems expanded during subsequent growing and dormant seasons, and often coalesced, girdling stems and causing the death of some seedlings. All isolates of M. betulae caused disease on B. pendula and conidia were able to infect young shoots in early flush without requiring a wound. Discula betulina caused lesions and dieback on B. pendula and B. pubescens within 3 months of inoculation, but disease did not progress thereafter and all inoculated seedlings recovered. Melanconium bicolor and F. avenaceum caused very little disease on either birch species. This study showed that M. betulae is an aggressive pathogen on B. pendula , causing sunken stem cankers and progressive crown dieback, which are symptoms commonly observed on young, planted birch at field sites across Scotland.     

Cryphonectria parasitica vegetative compatibility type analysis of populations in south-western France and northern Spain

A comprehensive study of the population biology of Cryphonectria parasitica , the causal agent of chestnut blight, is required to understand the spread of the epidemic in Europe and its natural regulation by the Cryphonectria hypovirus-1 (CHV-1). With this objective in mind, the diversity in vegetative compatibility (vc) types and mating types of C. parasitica populations was assessed in 43 chestnut sites in western France and northern Spain. Isolates were sampled in three regions along the Pyrénées mountains (Hautes Pyrénées, Pyrénées Atlantiques and Navarra) and in two regions north of the Pyrénées (Landes and Dordogne). There were 61 vc types observed in 682 isolates sampled. Ninety-five isolates (14%) could not be assigned to any one of the previously known European vc types. The finding of 47 incompatible vc types confirmed that the genetics of vegetative compatibility could not be totally accounted for by six diallelic vic genes. The two idiomorphs of the mating type gene were detected in all regions, indicating that sexual reproduction could occur within and between vc types. In all regions except Dordogne, C. parasitica populations were dominated either by the vc type EU-66 or EU-72. Neither vc type has been reported elsewhere in Europe, which suggests that two different introductions of C. parasitica have occurred in the study area. In Dordogne, populations were dominated by EU-33 and EU-2, and to a lesser extent EU-66 and EU-72. The low diversity in vc types for most of the C. parasitica populations provides good opportunities for natural regulation in forest coppices and for biocontrol with CHV-1 in orchards.     

First report of the canker pathogen Endothia gyrosa on Eucalyptus in South Africa

During country-wide surveys of Eucalyptus plantations for Cryphonectria canker, cankers distinctly different from those usually associated with Cryphonectria cubensis were observed. These cankers were less severe and were exemplified by cracked and slightly swollen areas on the bark. Endothia gyrosa , a well-known pathogen of woody plants including Eucalyptus spp., was consistently associated with these cankers. The pathogen is easily distinguished from C. cubensis by the presence of orange-brown stromata and non-septate ascospores. Inoculations on Eucalyptus grandis resulted in lesions similar to those observed on naturally infected trees. The disease associated with E. gyrosa is widespread in South Africa, and research is required to establish control strategies.     

Molecular mapping of resistance to blight in an interspecific cross in the genus castanea

ABSTRACT A three-generation American chestnut x Chinese chestnut pedigree was used to construct a genetic linkage map for chestnut and to investigate the control of resistance to Endothia parasitica (chestnut blight fungus). DNA genotypes for 241 polymorphic markers (eight isozymes, 17 restriction fragment length polymorphisms [RFLPs], and 216 random amplified polymorphic DNAs [RAPDs]) were assayed on an F(2) family consisting of 102 individuals. Of these markers, 196 were segregating as expected and, subsequently, used for primary linkage mapping. Two isozymes, 12 RFLPs, and 170 RAPDs were mapped to 12 linkage groups spanning a total genetic distance of 530.1 Kosambi centimorgans. F(2) plants were evaluated for a response to E. parasitica infection by directly inoculating them with two unique fungal isolates and measuring canker expansion over a period of 3.5 months. Results were compared with the marker genotype data, thereby identifying genomic regions significantly associated with a resistance response. Single-marker or nonsimultaneous analyses of variance identified seven genomic regions that appear to have an effect on host response. Multiple-marker or simultaneous models suggest that three of these regions have a significant effect on host response, together explaining as much as 42.2% of the total variation for canker size. At each of the three putative resistance loci, alleles derived from the Chinese chestnut grandparent were associated with smaller canker size, or higher levels of resistance.     

Modeling Spatial Characteristics in the Biological Control of Fungi at Leaf Scale: Competitive Substrate Colonization by Botrytis cinerea and the Saprophytic Antagonist Ulocladium atrum

ABSTRACT A spatially explicit model describing saprophytic colonization of dead cyclamen leaf tissue by the plant-pathogenic fungus Botrytis cinerea and the saprophytic fungal antagonist Ulocladium atrum was constructed. Both fungi explore the leaf and utilize the resources it provides. Leaf tissue is represented by a two-dimensional grid of square grid cells. Fungal competition within grid cells is modeled using Lotka-Volterra equations. Spatial expansion into neighboring grid cells is assumed proportional to the mycelial density gradient between donor and receptor cell. Established fungal biomass is immobile. Radial growth rates of B. cinerea and U. atrum in dead cyclamen leaf tissue were measured to determine parameters describing the spatial dynamics of the fungi. At temperatures from 5 to 25 degrees C, B. cinerea colonies expanded twice as rapidly as U. atrum colonies. In practical biological control, the slower colonization of space by U. atrum thus needs to be compensated by a sufficiently dense and even distribution of conidia on the leaf. Simulation results confirm the importance of spatial expansion to the outcome of the competitive interaction between B. cinerea and U. atrum at leaf scale. A sensitivity analysis further emphasized the importance of a uniform high density cover of vital U. atrum conidia on target leaves.     

Molecular characterization of Endothia gyrosa isolates from Eucalyptus in South Africa and Australia

Endothia gyrosa is a canker pathogen best known as the causal agent of pin oak blight in North America, and causes cankers on other woody hosts such as Castanea spp. and Liquidambar spp. In South Africa, Australia and Tasmania, a fungus identified as E. gyrosa has been recorded on Eucalyptus spp. Some morphological differences exist between the North American fungus and the isolates from Eucalyptus . Phylogenetic relationships between E. gyrosa from North America and E. gyrosa from South Africa and Australia, as well as that of the related fungi Cryphonectria parasitica and C. cubensis , were studied using PCR-based restriction fragment length polymorphism (RFLP) and sequences of the internal transcribed spacer (ITS) region of the rRNA operon. Endothia gyrosa isolates from South Africa produced the same RFLP banding patterns as those from Australia, which differed markedly from North American isolates of E. gyrosa . In a phylogram based on the DNA sequences, the Australian and South African isolates of E. gyrosa resided in a single, well resolved clade, distinct from North American isolates. Isolates of C. parasitica grouped in the same clade as the South African and Australian isolates of E. gyrosa , but C. cubensis was distantly related to them. The molecular data suggest that the E. gyrosa isolates from South Africa and Australia represent a distinct taxon, and probably belong to the genus Cryphonectria .     

稻曲病菌的形态学观察研究

 利用光学和电子显微镜对稻曲病菌的有性子座和不同来源的繁殖体进行了形态学观察。子囊壳单层环状埋生于子座外层,顶端突出子座表面而形成许多乳突结构。子囊发育成熟后,子囊壳壁的顶端可自行消解,露出里面的子囊。子囊孢子无色单胞,线状。厚垣孢子圆形或稍椭圆,黄褐色至黑褐色,胞壁厚密,外表有许多疣状突起。子囊孢子和厚垣孢子萌发都产生次生分生孢子。这些次生分生孢子与液体培养获得的分生孢子在大小和形态特征上是一样的,它们都是薄壁分生孢子。薄壁分生孢子卵圆形或长圆形,大小为2.6~8μm×2~5μm,无色透明,外表光滑。薄壁分生孢子萌发直接形成新一代薄壁分生孢子,此繁殖过程可不断重复进行。