The role of fungus Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya in ash dieback (Fraxinus spp.) in the Republic of Croatia

Obični jasen (Fraxinus excelsior L.) i poljski jasen (Fraxinus angustifolia Vahl), gospodarski i ekološki vrlo značajne vrste gorskih i nizinskih šuma Hrvatske, u posljednje vrijeme pokazuju sve izraženije simptome odumiranja. Mehanizmi koji dovode do ove pojave još nisu u potpunosti razjašnjeni iako od biotičkih čimbenika novootkriveni gljivični patogen Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya predstavlja najveću neposrednu opasnost ovim sastojinama. Provedeno istraživanje utvrđuje distribuciju i djelovanje patogene gljive H. fraxineus, te njenu sveukupnu ulogu u odumiranju vrsta Fraxinus spp. u gorskim i nizinskim područjima Hrvatske. Istražene su mogućnosti njene biološke kontrole, te rezistentnosti pojedinih genotipova poljskog jasena na djelovanje ovog patogena koje mogu dati doprinos rješavanju ove problematike. Gljivični patogen H. fraxineus prvi je put opisan u Hrvatskoj 2009. godine. Vrlo brzo se etablirao i sustavno proširio na čitav prostor areala rasprostranjenja običnog i poljskog jasena u Hrvatskoj. Bolest nije utvrđena u termofilnim zajednicama na mediteranskom području rasprostranjenja crnog jasena (Fraxinus ornus L.). Rezultati procjene oštećenosti stabala u sastojinama koreliraju s vremenima pojave plodnih tijela patogena i njegovim infekcijskim potencijalom na pojedinim lokacijama u Hrvatskoj. Kod djelovanja patogena H. fraxineus kao primarnog uzročnika sušenja jasena, prateće vrste gljiva sudjeluju u bržem progresivnom propadanju i narušavanju vitaliteta stabala jasena. Utvrđeno je da niske temperature djeluju inhibitnorno na rast izolata gljive H. fraxineus, dok visoke temperature djeluju inhibitnorno i sterilizirajuće. Temperatura od 30 °C je izazvala mortalitet 5,5% hrvatskih i čak 60% belgijskih izolata. Istraživanjem su utvrđena dva glavna morfotipa izolata H. fraxineus. Real time qPCR-om za detekciju i identifikaciju gljive H. fraxineus u tkivima jasena potvrđen je identitet svih 33 istraživana izolata kao gljivični patogen H. fraxineus. Omjeri idiomorfa MAT1:MAT2 kod hrvatskih izolata bili su 2:1, a kod belgijskih izolata 6:1. Tip idiomorfa pokazuje utjecaj vezan za geografsko podrijetlo. Mitovirus HfMV1 pozitivno utvrđeno je u 27,7% hrvatskih i 66,7% belgijskih izolata. Postoji statistički značajna razlika u rasprostranjenosti virusa između populacija, što može biti razlog pojave virulentnijih izolata u Hrvatskoj. RAMS markerima utvrđeno je postojanje dviju populacija s obzirom na genetsku varijabilnost, koje se dalje mogu razgranati na čak sedam subpopulacijskih grupa izolata. Dobivene populacije ne korespondiraju s geografskim podrijetlom izolata (Hrvatska i Belgija). Izolati H. fraxineus, ovisno o temperaturi ukazuju značajne razlike u stopi rasta in vitro što je pretpostavljeno povezano s genetskom varijabilnošću. Istraživanje provedeno na tri tipa agar hranjivih medija dokazalo je sposobnost stimulacije rasta izolata patogena s ekstraktima lišća svih autohtonih vrsta jasena. Stope rasta na različitim medijima koreliraju s osjetljivošću vrsta domaćina. Zabilježeno je da djelovanje UVB i UVA spektra svjetlosti utječe na redukciju rasta i sporulaciju gljive međutim nije uzrokovalo odumiranje izolata. Vidljivi spektar svjetlosti uzrokuje pojačanu reprodukciju gljive. U testiranjima in vitro i in vivo potvrđena je glavna uloga temperature na rast gljive i razvoj nekrotičnih lezija na biljkama u prirodi, te da rast nekroza, osim o temperaturi, ovisi i o vrsti domaćina jasena. Utvrđena je značajna osjetljivost klonova povećanjem progresije bolesti od 97% u prva četiri mjeseca od početka inokulacije, te kontinuirano slamanje svojstva rezistentnosti domaćina pojavom stope mortaliteta od 49% u razdoblju od jedne godine. Niti jedan od testiranih klonova nije ukazao na totalnu rezistentnost prema uzročniku sušenja jasena H. fraxineus, međutim nekoliko ih je pokazivalo reduciranu osjetljivost. Istraživani klonovi su klasirani u rane i kasne fenološke grupe s obzirom na početak listanja, ali i na odbacivanje lista u jesen. Klon NG03 pripada grupi ranih fenotipova listanja i pokazuje tendenciju smanjene osjetljivosti. Dobar klon za nastavak istraživanja osjetljivosti na patogena H. fraxineus s obzirom na fenologiju je klon NG31 s obzirom da ima kraći vegetacijski period i smanjenu prosječnu duljinu razvjenih nekroza kore u inokuliranim sadnicama. Utvrđeno je povećanje vrijednosti kalija i cinka kod zaraženih sadnica poljskog jasena, te smanjene vrijednosti željeza. Najtolerantniji klonovi na infekciju patogenom iz testova osjetljivosti pokazuju smanjene vrijednosti mangana, a povećane vrijednosti cinka. Hipovirulentnost kod H. fraxineus uzrokovana mitovirusom HfMV1 nije neosporno dokazana. Postoji statistički značajna redukcija promjera na svim inficiranim biljkama u odnosu na kontrolnu skupinu neinficiranih sadnica neovisno o prisustvu HfMV1. U in vitro testiranjima rasta i fruktifikacije izolata metodama primjene Allicin sredstva nakon i prije infekcije dokazana je značajna redukcija rasta i fruktifikacije patogena. U in vivo testiranjima injektiranjem Allicin sredstva u sadnice poljskog jasena u svrhu zaštite nije potpuno zaustavilo rast nekroza, ali je usporilo daljnju progresiju bolesti.Common ash (Fraxinus excelsior L.) and narrow–leaved ash (Fraxinus angustifolia Vahl), economically and ecologically very important species in mountain and lowland forests of Croatia, have recently developed more prominent decline symptoms. Specific mechanisms which cause this phenomenon are not yet identified, however the newly discovered fungal pathogen Hymenoscyphus fraxineus represents the highest direct danger for these stands. This research determined the distribution and the function of fungal pathogen Hymenoscyphus fraxineus, and its overall role in declining Fraxinus spp. in mountain and lowland regions of Croatia. Also the possibility of its biological control, and the susceptibility of seedlings to the infection of this pathogen which could give a contribution to the resolution of this rising problem. Fungal pathogen H. fraxineus was first described in Croatia in 2009. In short period was well established and systematically spread across the whole area of common and narrow-leaved ash regions. The disease is not present in the thermophilic communities on the Mediterranean prevailing area of manna ash (Fraxinus ornus L.). Damage stand assessments correlated with first pathogen infection appearance and infection potential of the pathogen on the individual locations in Croatia. The activity of the pathogen H. fraxineus as the primary cause of ash dieback was followed with other fungal species that participated in the decline and caused progressive, faster degradation of ash tree vitality. Low temperatures inhibited the growth of fungal isolates, and high temperatures inhibited and sterilised the H. fraxineus isolates. Temperature of 30 °C caused the mortality of 5.5% of Croatian and 60% of Belgian isolates. Two main morphotypes of fungal isolates H. fraxineus were obtained. Real time qPCR for detection and identification of fungus H. fraxineus in the ash tissues was used to confirm the identity of 33 isolates. The mating type ratio MAT1:MAT2 was 2:1 in Croatian isolates and 6:1 in Belgian isolates. Mating type ratio shows influence related to geographical origin of isolates. Mitovirus HfMV1 was found in 27.7% of Croatian and 66.7% of Belgian isolates. There was a significant statistical difference in virus prevalence between populations, which could be the reason of more virulent isolate prevalence in Croatia. RAMS markers were used to determine two populations according to genetic variability, which could be subdivided in 7 isolate groups. The populations do not correspond with geographical origin of the isolates (Croatia and Belgium). Isolates of H. fraxineus show significant difference in vitro in growth rate according to temperature which corresponds to genetic variability. Research on different types of agar medium with ash leaf extracts provided stimulation of pathogen isolates’ growth on all mediums with leaf extracts. Growth rates of isolates on different mediums with leaf extract correlated with the susceptibility of ash host species. Activity of UVB and UVA light spectrum was strong enough to reduce the growth and fungal sporulation but not enough to cause death. Visible light spectrum stimulated fungal reproduction battling for survival of the species. In vitro and in vivo experiments confirmed the main role of temperature on fungal growth and necrotic lesions development on seedlings in nature, and also that necrosis development, besides the temperature, was directly influenced by ash host species. Significant susceptibility of clones was established by the increase of disease progression on 97% in just first four months in the inoculation experiment, and the breakage of resistant trait was confirmed on the mortality rate of 49% after one year monitoring period. None of the tested clones showed total resistance to the pathogen H. fraxineus, but some of the clones showed reduced susceptibility. Clones were classified in early and late phenotypic groups according to leaf flushing and leaf shedding. Clone NG03 belongs to the early phenotypic group and has a tendency of reduced susceptibility. Interesting clone for the future research in susceptibility to the pathogen H. fraxineus is clone NG31 which revealed shorter vegetative period and reduced average necrosis length. Leaf analysis of the inoculated seedlings of narrow-leaved ash revealed higher values of potassium and zinc and lower values of iron. The most tolerant clones to the pathogen in the susceptibility trial, revealed lower values of manganese and higher values of zinc. Hipovirulence in H. fraxineus caused by the mitovirus HfMV1 was not indisputably proven in the test. There is statistically significant reduction of seedlings diameter growth on every infected seedling compared to control uninfected seedlings regardless of the mitovirus presence. In vitro tests of isolate growth and fructification by application methods of Allicine biological agent after and before the infection showed significant reduction in growth and fructification of the isolates is possible. In vivo tests by injection methods of Allicine agent in infected seedlings of narrow-leaved ash did not fully stopped the necrosis growth, but it slowed down the progression of the disease