Biological Control Of Chestnut Blight Research Articles

Various Population Structures of Cryphonectria parasitica in Cáceres (Spain) Determine the Feasibility of the Biological Control of Chestnut Blight with Hypovirulent Strains

Chestnut blight, caused by Cryphonectria parasitica, is a severe disease that may be biologically controlled by the use of hypovirulent strains, but the diversity of the pathogen population affects biocontrol feasibility. Villuercas–Ibores–Jara, the Jerte Valley and La Vera are the main chestnut production districts in Cáceres (central-western Spain). The purpose of this study is to determine the Vegetative Compatibility Groups (VCGs) and mating types in these districts and to identify hypovirulent isolates to be used in biological control. The VCGs were determined by the merging/barrage response; PCR tests were used for the mating type determination and CHV-1 hypovirus detection. In total, 40 sites were surveyed and 269 isolates were obtained, most of them (227) from the Villuercas–Ibores–Jara district, where EU11 was the predominant VCG (88.1%) with EU1 (6.6%) and EU12 (4.4%) being also present. In the Jerte Valley and La Vera, EU1 (61.9%), EU11 (11.9%) and EU12 (11.9%) were the predominant VCGs. Both mating types were detected (48% MAT-1; 50% MAT-2) and in general, only one mating type was found in each site. The presence of Cryphonectria hypovirus 1 (CHV-1), subtype I, was identified in only one isolate (VCG EU11) from Villuercas–Ibores–Jara. The characteristics of the C. parasitica population in this district and the occurrence of CHV-1 hypovirus support the potential of successful biological control in Villuercas–Ibores–Jara using hypovirulent strains, while in the Jerte Valley and La Vera only preventive measures are recommended.

Identification and characterization of hypovirus-infected Cryphonectriaparasiticaisolates from biological control plots in İzmir, Kütahya, and Sinop

In a pilot study, a biological control with hypovirus-infected Cryphonectria parasitica was applied in 3 study plots in Turkey, in June 2013. The aim of this study was to evaluate the success of the applied biological control by verifying the establishment of the released hypovirus (CHV-1) strains using molecular markers. C. parasitica isolates were sampled from cankers at 3 different time points: before the biological control treatments (April 2013), and 5 months (October 2013) and 11 months (May 2014) after the treatment. In total, 255 C. parasitica isolates were recovered and characterized. First, the culture morphology and vegetative compatibility type of these isolates were assessed. Next, the presence of hypoviruses in white isolates was checked by RNA extraction and subsequent RTPCR. Finally, a sequence analysis was performed to compare the hypoviruses to the released biological control hypoviruses by examining single nucleotide markers and reconstructing their phylogenetic relationship. The study sites in İzmir and Kütahya were found to be free of hypoviruses prior to the biological control, whereas in Sinop, the occurrence of hypoviruses of the Italian subtype was observed. Reisolations of the treated cankers and subsequent molecular analysis resulted in the detection of the released biocontrol hypovirus strains in all 3 study sites. The reisolated hypoviruses in İzmir and Kütahya originated from either one of the released biocontrol strains. In Sinop, both natural and artificially introduced hypoviruses were detected. Our study showed that the released biological control hypoviruses persisted in the treated cankers, which is promising for the biological control of chestnut blight in Turkey.

Genetic Diversity of Cryphonectria hypovirus 1, a Biocontrol Agent of Chestnut Blight, in Croatia and Slovenia.

Transmissible hypovirulence associated with Cryphonectria hypovirus 1 (CHV1) has been used for biological control of chestnut blight, devastating disease of chestnut caused by the fungus Cryphonectria parasitica. The main aims of this study were to provide molecular characterization of CHV1 from Croatia and Slovenia and to reveal its genetic variability, phylogeny, and diversification of populations. Fifty-one CHV1 haplotypes were detected among 54 partially sequenced CHV1 isolates, all belonging to Italian subtype (I). Diversity was mainly generated by point mutations while evidence of recombination was not found. The level of conservation over analyzed parts of ORF-A proteins p29 and p40 varied, but functional sites were highly conserved. Phylogenetic analysis revealed close relatedness and intermixing of Croatian and Slovenian CHV1 populations. Our CHV1 isolates were also related to Swiss and Bosnian hypoviruses supporting previously suggested course of CHV1 invasion in Europe. Overall, this study indicates that phylogeny of CHV1 subtype I in Europe is complex and characterized with frequent point mutations resulting in many closely related variants of the virus. Possible association between variations within CHV1 ORF-A and growth of the hypovirulent fungal isolates is tested and presented.

A multiplexed genotyping assay to determine vegetative incompatibility and mating type in Cryphonectria parasitica

The highly destructive chestnut blight disease can be successfully controlled by infecting the virulent strain of C. parasitica with the hyperparasitic mycovirus CHV-1. The artificial application of the virus-induced hypovirulence, however, requires that the vegetative compatibility (vc) diversity in the target C. parasitica population be determined beforehand. Conventional vc type determination by pairing the unknown isolate with tester strains of known vc types is time consuming and not always reliable. A genotyping assay was developed that uses DNA from C. parasitica isolates and discriminates the six known di-allelic vic loci and the two mating idiomorphs in two fluorescence-labeled multiplex PCRs. Validation was conducted by successfully genotyping the 74 known European vc tester strains and a conventionally characterized natural population of C. parasitica. Cross-species amplification tests with three Cryphonectria species (C. japonica, C. naterciae, and C. radicalis) revealed a high amplification specificity for C. parasitica, while the analytical sensitivity of the assay was established at 20 pg per reaction of genomic DNA. In conclusion, this fluorescent multiplex genotyping assay offers a simple and high-throughput tool for the characterization of the vegetative compatibility and mating type of C. parasitica at population level, which is particularly important for the application of mycovirus-mediated biological control of chestnut blight.

Characterization of the Papain-Like Protease p29 of the Hypovirus CHV1-CN280 in Its Natural Host Fungus Cryphonectria parasitica and Nonhost Fungus Magnaporthe oryzae.

Cryphonectria hypovirus 1 strain CN280 (CHV1-CN280) was isolated from North China and exhibited typical hypovirulence-associated traits. We previously reported that CHV1-CN280 was more aggressive and had a higher horizontal transmission ability between Cryphonectria parasitica isolates belonging to different vegetative compatibility groups than two other CHV1 hypoviruses (namely, CHV1-EP713 and CHV1-Euro7), thus displaying greater potential for biological control of chestnut blight. The genome sequence of CHV1-CN280 shared approximately 70% identity with three other hypoviruses (CHV1-EP713, CHV1-Euro7, and CHV1-EP721). The coding region for p29, a papain-like protease encoded by CHV1-CN280 hypovirus, displayed an average of only approximately 60% amino acid identity among them, while the identity between the other three CHV1 isolates was higher than 89%. Protease p29 acted as a virus-encoded determinant responsible for altering fungal host phenotypes in other CHV1 isolates. In this study, the impacts of CHV1-CN280 p29 expression in virus-free C. parasitica were investigated. CHV1-CN280 p29 expression in C. parasitica resulted in significantly reduced sporulation, pigmentation, extracellular laccase activities, and pathogenicity, which is consistent with previous investigations. Subsequently, the potential of CHV1-CN280 p29 as a viral determinant responsible for suppression of host phenotypes in other phytopathogenic fungi such as Magnaporthe oryzae, the causal agent of rice blast disease, was discussed. However, heterologous expression of p29 in M. oryzae induced the opposite effect on sporulation, extracellular laccase activities, and pathogenicity; had no significant effect on pigmentation and mycelial growth; and contributed to extracellular peroxidase activities, suggesting that CHV1-CN280 p29 may disturb a unique regulatory pathway in C. parasitica, rather than a basic regulatory pathway conserved in diverse range of fungi. Alternatively, CHV1-CN280 p29-mediated modulation of fungal phenotypes may be facilitated by the specific interaction between p29 and a special fungal-host component, which exists only with C. parasitica but not M. oryzae.

Use of biocontrol agents as potential tools in the management of chestnut blight

Castanea sativa is a widespread and important multi-purpose tree in the Mediterranean area. Recently, intensive infestation of gall wasp decreased the production of chestnut, and makes the plants more susceptible to Cryphonectria parasitica, the causal agent of chestnut blight. Generally, biological control of chestnut blight is through natural spread or artificial release of hypovirulent strains of C. parasitica. However, this approach is not always successful. The aim here was to evaluate alternative approaches for control chestnut blight based on application of antagonistic microorganisms. In-vitro antagonistic activities of fungal species Trichoderma and Penicillium were tested after in vitro isolation from chestnut bark. In dual cultures, these antagonistic fungi consistently reduced growth of C. parasitica. Three commercial formulations based on Trichoderma spp., Glomus spp. and Bacillus subtilis were tested at three doses, both in vitro and directly applied to cuttings of chestnut stems as co-inoculations and as separated inoculations with C. parasitica mycelium plugs. With co-inoculation, B. subtilis showed the best performance against chestnut blight, with necrotic areas reduced by >70%. In separated inoculations, B. subtilis reduced the size of cankers on chestnut stems, by 29%–67%, as also for Trichoderma spp., by 36%–65%, and Glomus spp., by 31%–63%. Application of commercial formulations based on antagonistic microorganisms are effective tools in management of chestnut blight.

Low genetic diversity but frequent sexual reproduction of the chestnut blight fungus Cryphonectria parasitica in Azerbaijan

SummaryChestnut blight was first recorded in Azerbaijan on the native European chestnut (Castanea sativa) in 2004, and since then, the disease is expanding in the country. In this study, chestnut blight was detected in seven of eight forest districts that were inspected. To characterize the local population structure of the chestnut blight fungus, 199 Cryphonectria parasitica isolates were collected and assessed for vc type, mating type and microsatellite genotype. Among these isolates, one dominant vc type was detected comprising 93% of the isolates. Six additional vc types were identified among the other isolates. The microsatellite analysis revealed a very similar pattern with 96.5% of the isolates belonging to the same multilocus genotype. Both mating types of C. parasitica were present in all seven districts with a mating type ratio not different from 1:1 in five districts. In accordance with the occurrence of both mating types, sexual fruiting bodies (perithecia) of C. parasitica were found in all districts with an overall prevalence on 20.6% of the cankers. The dominant vc type and microsatellite genotype in Azerbaijan do not occur in Europe, but have previously been found to be widespread in neighbouring Georgia. Our study reveals that sexual reproduction in the invasive C. parasitica population in Azerbaijan is frequent, although the population shows a low genetic diversity. This could favour the biological control of chestnut blight using hypovirulence, which so far does not seem to be present in Azerbaijan.

Occurrence of hypovirus-infected Cryphonectria parasitica isolates in northern Spain: an encouraging situation for biological control of chestnut blight in Asturian forests

The presence of hypovirulent (hypovirus-infected) isolates of the chestnut blight fungus Cryphonectria parasitica in Asturias, northern Spain, is first reported by using both morphological and molecular assessments. Hypovirulence was detected in 16 of the 69 councils where the fungus was previously observed. In total 30 of the 863 C. parasitica isolates analyzed were found to be hypovirus-infected. The isolates belonged to the two main vegetative compatibility (vc) types (EU-1 and EU-13) of the region. Asturian hypoviruses were genetically characterized by sequencing of ORFA and ORFB specific regions of the hypoviral RNA. The results show that 70% of the investigated hypovirulent isolates contained the Spanish subtype (subtype E) while 30% isolates the German subtype (D) of Cryphonectria hypovirus 1 (CHV-1). The presence of hypovirulent isolates compatible with the dominant vc types and the low vc diversity in this region favours the application of biological control. An initial disease management effort could be applied by using the hypovirus of subtype D, whereas further research related to the biocontrol properties of the subtype E hypovirus should be conducted.

Natural dissemination of hypovirulent Cryphonectria parasiticastrain used for biological control of chestnut blight

Chestnut blight, caused by Cryphonectria parasitica Murr. Barr, is the most serious disease of chestnut trees in Turkey on which several studies related to biological control were conducted. The aim of this study was to determine the cause of the occurrence of healing cankers on untreated chestnut trees and the role of carriers in the spread of hypovirulence by laboratory tests, and to discover the effect of some environmental factors (temperature and relative humidity) on the natural spreading of hypovirulence. The study was carried out in a chestnut grove where biological control studies of the disease had been performed in previous years. Bark samples were collected from healed cankers and 388 possibly hypovirulent isolates were obtained. Two of them that were white converted virulent isolates to hypovirulent ones, but others developed yellowish cream-colored mycelia and did not convert virulent isolates. dsRNA analysis was performed on randomly selected possible hypovirulent isolates collected from healed cankers. After the virus concentration had increased, dsRNA-positive reactions were obtained for 56.73% of the isolates. Thirty-one possible hypovirulent isolates from Formicidae and Gastropoda did not convert virulent isolates to hypovirulent ones. dsRNA analysis was performed on these isolates and 61.29% of them containing dsRNA were observed in the invertebrates collected from cankers that had healed from hypovirulence. It was also observed that there was no significant difference among the date of average temperatures and date of relative humidity from 3 different altitudes. It was determined that the reason for the occurrence of the healed cankers on untreated trees was the natural dissemination of hypovirulence, ants and snails might have played a role in the natural dissemination of hypovirulence, and healed cankers mostly occurred on the western sides of shaded slopes.

New formulation and delivery method ofCryphonectria parasiticafor biological control of chestnut blight

This study aimed to develop a new formulation of Cryphonectria parasitica hypovirulent mycelium suitable for inoculations of tall trees from the ground. Cryphonectria parasitica hypovirulent strains are widely used for biological control of chestnut blight. However, it is often inconsistent and ineffective not only for biological reasons but also because the current manual application of hypovirulent strains on adult plants is difficult, time-consuming and expensive. Here, we propose an improved formulation and more effective mode of application of hypovirulent strains, which could boost chestnut blight biocontrol. The Cp 4.2H hypovirulent strain was formulated as mycelium discs with polyethylene glycol and hydroxypropyl methylcellulose, loaded into lead-free pellets that are used as carriers to inoculate cankers on chestnut stems by shooting. The formulation of mycelium did not hamper its viability which was stable, with an estimated shelf life of 72days at 6±1°C. The inoculum effectiveness was confirmed ex planta and in planta in a small-scale pilot study in field, where formulated mycelium discs of hypovirulent strain Cp 4.2H were inoculated by airgun shot method into the chestnut bark. In planta, Cp 4.2H was recovered in 37% of bark samples taken around the inoculated points 1year after the treatment. We demonstrated that the proposed airgun shooting inoculation method of C. parasitica hypovirulent strain formulated as mycelium discs is suitable for treatment of adult chestnut trees. The proposed method could be a valid alternative to the traditional manual technique of chestnut biocontrol. The main advantages are the cost-effectiveness and the ease to treat high-positioned, otherwise unreachable cankers both in orchards and forests.

Biological control of chestnut blight in Croatia: an interaction between host sweet chestnut, its pathogen Cryphonectria parasitica and the biocontrol agent Cryphonectria hypovirus 1.

Chestnut blight, caused by the fungus Cryphonectria parasitica, is a severe chestnut disease that can be controlled with naturally occurring hypoviruses in many areas of Europe. The aim of this research was to measure the effect of different Cryphonectria hypovirus 1 (CHV1) strains on the growth of the fungal host and select strains that could potentially be used for human-mediated biocontrol in forests and orchards, and to investigate whether and how chestnut-fungus-virus interactions affect the development and growth of the lesion area on cut stems. Two Croatian CHV1 strains (CR23 and M56/1) were selected as potential biocontrol agents. The sequencing of CHV1/ORF-A showed that both of these virus strains belonged to the Italian subtype of CHV1. In vitro transfection of selected virus strains from hypovirulent to genetically diverse virus-free fungal isolates and subsequent inoculation of all virus/fungus combinations on stems of genetically diverse sweet chestnut trees revealed that Croatian virus strain CR23 had an equally hypovirulent effect on the host as the strong French strain CHV1-EP713, while M56/1 had a weaker effect. Furthermore, it was shown that in some cases the same hypovirus/fungus combinations induced various degrees of canker development on different chestnut genotypes. Some CHV1 strains belonging to the Italian subtype have similar hypovirulent effects on C. parasitica to those belonging to the French subtype. Furthermore, chestnut susceptibility and recovery could be influenced by the response of chestnut trees to particular hypovirulent C. parasitica isolates, and virus-fungus-chestnut interactions could have significant implications for the success of chestnut blight biocontrol. © 2016 Society of Chemical Industry.

Soluble material secreted from Penicillium chrysogenum isolate exhibits antifungal activity against Cryphonectria parasitica- the causative agent of the American Chestnut Blight

The American chestnut (Castanea dentata) was once the dominant canopy tree along the eastern region of the United States. Cryphonectria parasitica, the causative agent of chestnut blight, was introduced from Asia in the early 1900's, and obliterated the chestnut population within 50 years. We sought to identify environmental microbes capable of producing factors that were fungicidal or inhibited growth of C. parasitica in the hopes developing a biological control of chestnut blight. We isolated a filamentous fungus that significantly inhibited the growth of C. parasitica upon co-cultivation. Extracellular fractions of this fungal isolate prevented C. parasitica growth, indicating that a potential fungicide was produced by the novel isolate. Sequence analysis of 18S rRNA identified this inhibitory fungus as Penicillium chrysogenum. Furthermore, these extracellular fractions were tested as treatments for blight in vivo using chestnut saplings. Scarred saplings that were treated with the P. chrysogenum extracellular fractions healed subjectively better than those without treatment when inoculated with C. parasitica. These data suggest that material secreted by P. chrysogenum could be used as a treatment for the American chestnut blight. This work may assist the reclamation of the American chestnut in association with breeding programs and blight attenuation. Specifically, treatment of small groves under the right conditions may allow them to remain blight free. Future work will explore the mechanism of action and specific target of the extracellular fraction.

Controlo biológico do cancro do castanheiro: Deteção, identificação e caracterização do Hypovirus - CHV1

The chestnut blight caused by the fungus Cryphonectria parasitica is considered a major cause of the decline and disappearance of chestnut trees across Europe. C. parasitica is an ascomycete fungus (Diaporthales) that is native to eastern Asia. Infection of chestnut plants with this fungus is typically associated with extensive necrosis (cankers) of the bark on stems and branches, resulting in the subsequent death of the tree. Hypovirulence is a specific method for biological control of Chestnut Blight, it is an infection of C. parasitica with Cryphonectria hypovirus 1 (CHV1), a unencapsidate dsRNA virus of the genus Hypovirus. Biological control with hypovirulent strains is considered an efficient method to control the disease and improve the host plant recovery. The field application needs laboratorial production of compatible hypovirulent strain and introduction by inoculation at the margin of active cankers. In this work it was intended to identify and characterize the hypovirus with potential ability to integrate biological control programmes against Chestnut Blight. Using molecular techniques 11 hypovirus with hypovirulence potential were identified on white isolates of C. parasitica isolated on cankers from northeast of Portugal.

Multiple introductions and recombination in Cryphonectria hypovirus 1: perspective for a sustainable biological control of chestnut blight.

Cryphonectria hypovirus 1 (CHV1) is a mycovirus which decreases the virulence of its fungal host Cryphonectria parasitica, the causal agent of chestnut blight recently introduced in Europe. The understanding of the evolutionary processes which have shaped CHV1 populations in Europe is required to develop a sustainable biocontrol strategy targeting chestnut blight and effective in European chestnut forests. To retrace the evolutionary history of CHV1, we analyzed sequences from two genomic regions on a collection of 55 CHV1 strains from France and northern Spain, two countries where multiple introductions of C. parasitica occurred. Several recombination events and variable selection pressures contributed to CHV1 evolution, agreeing with a non-clock-like diversification rate. These two mechanisms may be at the origin of CHV1 population diversity observed in western Europe. Considering the actual prevalence of CHV1 and its association with host genotypes, multiple introductions of CHV1 may have occurred in Europe, some of them directly from Asia and some of them through North America. Although some viral strains remained with low frequency in their introduction area, multiple infections might have allowed homologous recombination within parental sequences. Some of these recombinant lineages are associated with the spread of CHV1 in European regions.

Thyreophagus corticalis as a vector of hypovirulence in Cryphonectria parasitica in chestnut stands

The natural spread of hypovirulence in Cryphonectria parasitica (Murr.) Barr. occurs in chestnut (Castanea sativa Mill) stands and orchards in Italy and other European countries, leading to spontaneous recovery of the diseased trees. Little is known about how hypovirulence spreads in chestnut stands but various corticolous mite species frequently detected on chestnut cankers could be one of the many factors playing a role in the spread. Artificial virulent cankers created in inoculation field tests and treated with Thyreophagus corticalis (Acari, Sarcoptiformes, Acaridae) raised on hypovirulent cultures showed similar growth to those treated with mycelia of the hypovirulent strain over 18 months of inoculation. Cultures re-isolated from virulent cankers treated with mites were found to contain hypovirus like those derived from pairings of virulent and hypovirulent strains. Viral dsRNA could be carried externally and/or ingested by mites from the hypovirulent mycelia and then transmitted to the mycelia of virulent strains, causing their conversion. In a laboratory study, all fecal pellets collected from mites reared on hypovirulent and virulent strains grown on semi-selective media gave rise to colonies of C. parasitica with similar morphological characters and virulence to the original cultures. Field inoculation of stump sprouts with the resulting colonies revealed that mite digestive tract passage did not alter the virulence of the studied strains. These results are of interest for the biological control of chestnut blight.

Characterization of hypovirulent isolates of the chestnut blight fungus, Cryphonectria parasitica from the Marmara and Black Sea regions of Turkey

Chestnut blight caused by the introduced fungus Cryphonectria parasitica has been responsible for the decline of Castanea sativa in Turkey since the 1960s. In this study, 72 C. parasitica isolates were recovered from the Marmara and Black Sea regions of Turkey showing white or cream-coloured culture morphology and were subjected to various tests to determine if they were infected by Cryphonectria hypovirus 1 (CHV-1). The vast majority of the isolates (69 out of 72) were vc type EU-1. Both mating types were found among a subsample of the isolates. The hypovirus was detected in 55 isolates by dsRNA extraction and/or virus specific RT-PCR on total RNA extracts. All but one isolates showed no or only weak phenol oxidase activity on agar medium containing tannic acid, typical of CHV-1 infected isolates. Through sequencing of a specific region of the hypovirus genome, we found that 24 hypovirus isolates belonged to the CHV-1 subtype I and six to the CHV-1 subtype F2. The distribution of the two CHV-1 subtypes in Turkey showed a clear geographic pattern. CHV-1 subtype I was only detected in the Marmara and western Black Sea region, whereas subtype F2 was restricted to the eastern part of the Black Sea region. The effectiveness of 23 hypovirulent isolates was tested against a virulent isolate on 2–3 years old chestnut sprouts. Ten hypovirulent isolates, all infected by CHV-1 subtype I, prevented canker development by more than 80 % suggesting that they might be suitable for biological control of chestnut blight in Turkey.

Variation in virulence of Cryphonectria hypovirus 1 in Macedonia

SummaryWe investigated variation in virulence of Cryphonectria hypovirus 1 (CHV‐1) to the chestnut blight fungus, Cryphonectria parasitica, in Macedonia by inoculating chestnut stems in the field. We inoculated trees with two isolates of C. parasitica, each infected with one of five isolates of CHV‐1, four of which were the same for both fungal isolates. Two virus isolates, [Sk28] and [Sk47], were significantly more virulent than the others when compared in the same fungal host isolates, as measured by reduced canker growth and increased callus formation. Mycelial growth rate in vitro was weakly correlated to canker growth or callus formation and is therefore not a reliable predictor for virulence. We found significant fungus × virus interactions for canker growth and callus formation, which seems due mainly to one virus isolate. Significant interactions were not expected because the two fungal host isolates are members of the same clone that is dominant in Macedonia and most of southeastern Europe. Phenotypic variation for response to viruses, therefore, is greater than variation revealed by the genetic markers used to define clones. More than half of the trees inoculated with virus‐free controls were dead within 2 years, and the 30% still alive after 5 years had cankers with extensive callus formation, indicating that natural virus transmission had occurred after inoculation. In contrast, only 2% of the trees inoculated with virus‐infected isolates were dead after 5 years. Hypoviruses naturally occurring in Macedonia reduce canker development and tree mortality similarly to those in other parts of southern Europe, and therefore, may have good potential for biological control of chestnut blight.

Biological control of chestnut canker, caused by Cryphonectria parasitica, by antagonistic organisms and hypovirulent isolates

Biological control of chestnut blight was investigated by using 3 hypovirulent isolates of Cryphonectria parasitica, 5 Trichoderma sp., 4 Penicillium sp., and 4 Bacillus sp. isolates. Hypovirulent isolates and antagonistic organisms were obtained from samples collected from the Black Sea region of Turkey, in 2008 and 2009. Effectiveness of the hypovirulent isolates and antagonistic microorganisms was tested on 3-year-old chestnut saplings. In the tests, bark disks of 6 mm were removed from the stem bases and culture disks of the virulent isolate of C. parasitica were first placed into the hole and then the hypovirulent isolate or the antagonistic fungi. Bacillus strains were applied as bacterial suspensions of 10^{10} cell mL^{-1} to the holes; then the virulent isolate of C. parasitica was placed. Evaluations were made by measuring the canker lengths in 3 periods and effectiveness was given as percent inhibition (PI) of the treatments. Antagonistic microorganisms yielded varying PI values in 3 time periods and the highest rate of inhibition (68%) was obtained from the Trichoderma sp. isolate T - 2. A Penicillium sp. isolate (P - 3) and 2 Bacillus spp. isolates (B - z and B - b) also provided 30%, 40%, and 31% disease inhibition, respectively, 58 days after the inoculation. Effectiveness of the hypovirulent isolates varied depending on the virulent isolates and the hypovirulent isolate Z - 1 provided 59% inhibition against the most virulent isolate K - 19, while it gave 32% inhibition against the less aggressive isolate K - 44. The other hypovirulent isolate Ba - 6 also inhibited the canker development of the virulent isolate by 42%.

ACTUAL STATUS OF BIOLOGICAL CONTROL STUDIES ON CHESTNUT BLIGHT IN TURKEY

This article reports the results of a long-term study conducted in province Manisa, Ege region, on biological control of chestnut blight (caused by Cryphonectria parasitica) by using hypovirulent strains and on their natural spread in the stand between the years 2002- 2009. Since hypovirulent strains were not found in the chestnut orchards of the region, hypovirulent isolates from the Marmara region which belong to vc type EU-1 and MAT-1 mating type were used in the study. The virulent strains isolated from the infected trees in the research area have revealed that they also belong to vc type EU-1 and MAT-1 mating type. After determination of the canker dimensions and types on the young chestnut trees, the introduced hypovirulent strain was inoculated into the holes around these cankers and the dimensional changes of the diseased areas in these trees were measured at monthly intervals and recorded. It was observed that most of the cankers treated with the hypovirulent strain on chestnut trees were healed between 2004 and 2005. The hypovirulent strain could be re-isolated from the healing cankers. Five years after inoculation, 45 new healed cankers were determined on 31 trees in the research area between 2007 and 2009, showing that hypovirulence disseminates naturally in the chestnut stand.

CHESTNUT BLIGHT AND EVALUATION OF THE FEASIBILITY OF ITS BIOLOGICAL CONTROL IN THE AYDIN PROVINCE, TURKEY BY USING HYPOVIRULENCE

Aydm is the leading chestnut producing province of Turkey and supplies approximately one-third of the Turkish production. However, the nut production and the survival of the chestnut trees is under the threat of chestnut blight. So far the limited efforts applied by chestnut growers to control the disease through their own practices have been insufficient to decrease its spread and to prevent its introduction into new disease-free areas. On the contrary, the disease spread was encouraged by the sylvicultural and harvesting mistreatments. In Aydm Province out of 31 chestnut-producing villages, 22 of them were found to be contaminated with chestnut blight. Among all C. parasitica isolates obtained from the cankers of blighted trees, only one exhibited dsRNA, but its association with hypovirulence has not been established yet. Two vc types, the EU-1 and EU-12, were identified. EU-1 was the most widespread vc type. The low vc diversity provides a good potential for hypovirulence related biological control of chestnut blight in the province. In addition, the low vc diversity might be an indication of lack of sexual reproduction of C. parasitica. Mating type assays on C. parasitica isolates showed that both mating types were present in the province, however, the MAT-1:MAT-2 ratio was significantly skewed from 1:1 in each village, which might be another evidence of lack of sexual recombination. No perithecia were encountered in nature. These results emphasize the opportunity to control the disease through biological control by introducing appropriate hypovirulent strains. But it should be remembered that in some villages of Aydm Province the presence of EU-1 and EU-12 from opposite mating types (MAT-1 and MAT-2) may create a possibility that the fungus may initiate sexual reproduction and this may result in the emergence of new vc groups.