Radócz, LászlóBoukhili, Mariem2023-05-172023-05-172023-06-30https://hdl.handle.net/2437/353736Chestnut blight canker is a serious disease caused by the Ascomycota fungus Cryphonectria parasitica. It started from China to North America and it was quickly spread to many other countries all over the world. The European Epidemic was first found in Italy in 1938 and spread throughout Europe, eventually reaching neighboring nations: France, Spain, Turkey, Greece and many other countries. The pathogen has spread to a lot of chestnut orchards, resulting in the loss of more than half of Castanea sativa spp. In Hungary, Castanea sativa is an important tree, cultivated due to its economic impact. The tree is preferred because it has a good nutrition value and usually used as wood resources as well as animal feed. Unfortunately, the introduction of the disease in 1960 leads to a loss of about 50% of orchard trees. In neighbouring countries, particularly in Slovakia, this situation has also been acknowledged. This disease may be spreading so quickly within the region as well as between countries, for that Cryphonectria parasitica was considered a quarantine species in order to limit its introduction into healthy countries and to reduce the growth of the fungus in a contaminated area. Concerning the eradication of the disease, there were no positive results from cutting and burning of the contaminated trees. Chemical treatment was not always possible due to the lack of chemical spectrum availability and due to the size of chestnut trees. The large size of the tree limit the efficiency of chemical application. Hence, biological control using anatogonistic microorganisms could be an efficient treatment against chestnut blight canker. In order to induce resistance to Cryphonectria parasitica, Bacillus subtilis was applied to healthy chestnut trees. Furthermore, Bacillus subtilis is helping plants that are infected by this fungus to recover so fast. In several additional studies, the mycelia growth of C. parasitica has been significantly reduced by Trichoderma spp. Naturally, C. parasitica has two forms: hypovirulent and virulent form. Based on the morphology, hypovirulent strains show white growth mycelia however, reddish to orange mycelia with high pigmentation are shown in the case of virulent isolates. Genetically, the presence of the mycovirus make the difference between the two forms. Hypovirulent isolates are always recognized as positive strand RNA virus hence dsRNA is limited to the cytoplasm of hypovirulent isolates. Based on this, 44% of samples are hypovirulent and 56% are virulent. FG2, FG5, FG6 and FG7 are the hypovirulent strains however FG3, FG4, FG8, FG10, and FG12 present the virulent ones. Biological control by using hypovirulence was used successfully in the control of chestnut blight canker. Several researchers have confirmed that due to the presence of hypovirus, hypoviruelnt strains have the potential to attenuate the virulent effect of C. parasitica going to the total elimination of its virulence and this is through its conversion resulting of the acquisition of dsRNA. Hence hypovirulent isolates have the potential to transfer their genetic material (dsRNA) to the virulent samples in order to convert all virulent isolates into hypovirulent ones. This conversion can be acquired under certain conditions, the most crucial among which is the vegetative compatibility between strains. Vegetative compatibility refers to the total fusion of hyphae between samples which allowed the transmission of dsRNA from hypovirulent to virulent isolate. Barrier zone can be seen in case of vegetative incompatibility and it refers to the death of cells between incompatible strains. Genetically, vegetative compatible strains share the same alleles at every locus. The recombination of vegetative incompatible genes during the sexual reproduction is the main cause of high VC-type diversity. In Europe, huge distribution of VC types were found in different regions. The most frequent VC type in Eastern Europe is EU-12, same VC type were found in Southern Europe such as Italy and Greece. EU-1, EU-2 and EU-5 are dominant in France, Spain and Switzerland (ROBIN – HEINIGER, 2001). EU-11 was the most frequent VC-type in Portugal, followed by EU-12 however EU-66 was the less frequent (BRAGANÇA et al., 2007). In Hungary, according to RADOCZ (2001), the most common VC type is EU-12. In our research EU-12 was recognized in both sites. In Farkasgyepű, 2 VC type were found: EU-1 and EU-12 and in Nagykanizsa 4 VC type were noted: EU-1, EU12, EU3 and EU-20. Low genetic diversity is noted comparing to the findings of RADÓCZ (2001). Low genetic diversity of VC type can be explained by the dominance of asexual reproduction between VC loci, inoculation of new samples through biological control or introduction of new groups through mutation. Both sites have not been treated before, for that reason, asexual reproduction should be the main cause of the low rate of genetic diversity. The low genetic diversity is required for biological control using hypoviruelnce. For that the success of this method in the given area is promoted. To confirm the hypovirulence and the conversion of different strains. Inoculation on apple fruit was done. The results obtained confirm that hypoviruelnt and converted strains (except C3) gave the same result as the control samples: no necrotic growth was induced. However, the virulent strains induced a lesion necrotic growth and a significant growth was noted 10 days after inoculation. According to the formula of relative virulence given by DING et al. (2007), FG3, FG4 and FG10 are the most virulent strains. FG8 and FG12 are medium virulent strains. These results were confirmed by statistical analysis using MINITAB, 2020. The converted C3: FG2 × FG10, morphologically, shows white growth mycelia and based on the microscope observation a hyphae fusion was recognized. Hence, the conversion was noted however, during inoculation the converted C3 has induced a small necrotic growth and this effect was different to the hypovirulent and the other converted strains. These results have been explained by the fact that FG10 has received the dsRNA from the hypovirulent FG2, however, its conversion was slow for that the necrotic growth was detected (ANAGNOSTAKIS – DAY, 1979). Positive results are expected for field inoculation. As a conclusion, we can confirm that biological control in Hungary is possible due the low genetic diversity of VC type. Hypovirulence can exist naturally, and this is the situation in Farkasgyepű and Nagykanisza. Conversion is possible between compatible fungal sample and this is based on the transmission of dsRNA between isolates. Conversion can be rapid (less than 48h) or slow (going for some months/years). In order to confirm that samples showing white mycelia are the hypovirulent ones. dsRNA extraction was done for FG2 and NF11. The results based on the gel electrophoresis confirmed the presence of the genetic material. Based on these findings it was confirmed that the white mycelia is the basic phenotype of hypoviruelnt strain and the dsRNA is strictly related to the hypovirulent ones.50enbiological controlCryphonectria parasiticachestnut blight diseasehypovirulencevirulencechestnutDEENK Témalista::KörnyezettudományokHozzáférhető a 2022 decemberi felsőoktatási törvénymódosítás értelmében.