Detection, identification and functional characterisation of plant and microbial volatile organic compounds with inhibitory activity against two plant pathogens

Volatile organic compounds (VOCs) play crucial ecological roles in interactions among organisms. For example, plant VOCs can act as a powerful deterrent of herbivore insects and pathogens or they can act as resistance inducers to stimulate plant defences. Likewise, bioactive VOCs can be emitted by beneficial microorganisms and they may potentially act as key molecules in the microbe-microbe and plant-microbe communications. However, scarce information is available concerning the role of VOCs produced by grapevine (Vitis vinifera) plants and beneficial bacteria belonging to the Lysobacter genus in defence mechanisms against two important phytopathogenic oomycetes, namely Plasmopara viticola and Phytophthora infestans, which are the causal agents of grapevine downy mildew and potato late blight, respectively. The major objectives of this PhD thesis were the detection, identification and the functional characterization of VOCs from Vitis spp. and Lysobacter spp., in order to better understand their role in plant-microbe and microbe-microbe communications and to identify new active molecules from natural origin to control phytopathogens. In particular, VOCs from resistant and susceptible grapevine genotypes were identified following P. viticola inoculation and their effect as toxic molecules against downy mildew was explored (publications 1 and 2). Likewise, VOCs produced by Lysobacter spp. were identified and characterised, in order to identify microbial VOCs able to inhibit P. infestans growth (publication 3). In order to reach these goals, a headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS) and proton transfer reaction time of flight-mass spectrometry (PTR-ToF-MS) have been used. Two downy mildew resistant hybrids (SO4 and Kober 5BB) and the susceptible V. vinifera cultivar Pinot noir were analysed in vitro using PTR-ToF-MS. We found that P. viticola inoculation resulted in a significant increase monoterpene and sesquiterpene emission by resistant genotypes (SO4 and Kober 5BB) and not by the susceptible cultivar (Vitis vinifera Pinot noir; publication 1). Grapevine VOCs were further identified by HS-SPME/GC-MS using greenhouse-grown plants. The four resistant genotypes tested (BC4, Kober 5BB, SO4 and Solaris) showed significantly increased production of VOCs after P. viticola inoculation under greenhouse conditions. Conversely, no significant emission of volatile terpenes was detected from Pinot noir plants after P. viticola inoculation, suggesting that VOCs of resistant genotypes could play an important role in grapevine resistance against downy mildew. The chemical structures of P. viticola-induced VOCs were identified by retention index and the GC-MS spectrum evaluation and VOCs potentially involved in the grapevine resistance were selected according to their emission profiles. Pure compounds were tested against P. viticola by leaf disk assays and different experiments were set up, in order to elucidate the efficacy of pure VOCs both in a liquid suspension of P. viticola sporangia and after application via the gas phase. These experiments revealed six (2-phenylethanol, β-caryophyllene, β-selinene, trans-2-pentenal, 2-ethylfuran, and β-cyclocitral) and four VOCs (2-phenylethanol, trans-2-pentenal, 2-ethylfuran, and β-cyclocitral) which impaired downy mildew symptoms after direct application of liquid suspension and after treatment with VOC enriched air (without direct contact with the leaf tissue), respectively. With these results we demonstrated that VOCs produced by resistant grapevine genotypes are related to post-infection mechanisms and may contribute to grapevine resistance against P. viticola by inhibition of pathogen development (publication 2). In the second part of the PhD project, the volatilome of Lysobacter spp. was characterised for its inhibitory activity against the soil pathogen P. infestans (publication 3). The effect of VOCs emitted by Lysobacter strains was demonstrated in vitro by dual-culture assay and profiles were characterised by HS-SPME/GC-MS and PTR-ToF-MS analysis. Interestingly, the biocontrol activity and VOC profiles of Lysobacter spp. depended on the bacterial growth media. In particular, VOCs with inhibitory properties (pyrazines, pyrrole and decanal) were mainly emitted by Lysobacter type strains grown on a protein-rich medium, demonstrating the importance of the culture medium composition to optimise the biocontrol efficacy of Lysobacter spp. against plant pathogens. In summary, the presented thesis showed that both analytical chemistry techniques used (PTR-ToF-MS and HS-SPME/GC-MS) can be employed synergistically to detect and identify VOCs from different biological matrixes such as leaf tissue or bacterial cultures. The presented thesis also suggested that VOCs contribute to grapevine resistance and they can effectively be used to control economically important plant pathogens such as P. viticola. Furthermore, results generated in this work indicate that nutrient availability may affect the aggressiveness of Lysobacter spp. in the soil to maximise biocontrol efficacy against P. infestans. However, further metabolomic and transcriptomic analyses are required to investigate the VOC-mediated plant defence mechanisms and to characterize metabolic changes and VOC emissions of Lysobacter spp. grown in soil condition

Host dispersal shapes the population structure of a tick-borne bacterial pathogen

Birds are hosts for several zoonotic pathogens. Because of their high mobility, especially of longdistance migrants, birds can disperse these pathogens, affecting their distribution and phylogeography. We focused on Borrelia burgdorferi sensu lato, which includes the causative agents of Lyme borreliosis, as an example for tick-borne pathogens, to address the role of birds as propagation hosts of zoonotic agents at a large geographical scale. We collected ticks from passerine birds in 11 European countries. B. burgdorferi s.l. prevalence in Ixodes spp. was 37% and increased with latitude. The fieldfare Turdus pilaris and the blackbird T. merula carried ticks with the highest Borrelia prevalence (92 and 58%, respectively), whereas robin Erithacus rubecula ticks were the least infected (3.8%). Borrelia garinii was the most prevalent genospecies (61%), followed by B. valaisiana (24%), B. afzelii (9%), B. turdi (5%) and B. lusitaniae (0.5%). A novel Borrelia genospecies "Candidatus Borrelia aligera" was also detected. Multilocus sequence typing (MLST) analysis of B. garinii isolates together with the global collection of B. garinii genotypes obtained from the Borrelia MLST public database revealed that: (a) there was little overlap among genotypes from different continents, (b) there was no geographical structuring within Europe, and (c) there was no evident association pattern detectable among B. garinii genotypes from ticks feeding on birds, questing ticks or human isolates. These findings strengthen the hypothesis that the population structure and evolutionary biology of tick-borne pathogens are shaped by their host associations and the movement patterns of these hosts.Peer reviewe

Az ellés lefolyásának objektív vizsgálata non stop kamera megfigyeléssel, valamint annak hatása az involúcióra

A tejelő szarvasmarhák fertilitását alapvetően meghatározza a takarmányozás és az ellést követő involúciós időszak lezajlása. Az involúció egészséges lefolyásának elősegítésével csökkenthető a két ellés közti idő, ami gazdaságosabbá teszi a tejtermelést. Az involúcióra meghatározó hatással van az ellés lefolyása és körülményei, amit objektíven csak folyamatos megfigyeléssel lehet értékelni. Vizsgálataimban két magyarországi, közepes tejtermelésű tehenészetben 2-2 hónapon keresztül videofelvételek segítségével elemeztem az ellések lefolyását (segítségnyújtás alkalmazása és módja) és az ellett állatok szaporodásbiológiai adatait (magzatburok-visszatartás előfordulása, méhkezelések aránya, termékenyítés ideje és eredménye, magzatvesztés) a vemhesülésig követtem. Az adatokat Fischer egzakt teszt és t-próba segítségével elemeztem. Az ellés során és azt követően nyerhető szaporodási adatok telepenként több paraméter tekintetében eltértek, ami technológiai különbségekre utalhat. Egyik telepen sem volt kimutatható összefüggés a borjú születési súlya és a segítségnyújtás ténye között. A segítséggel ellett állatok esetében szignifikánsan több idő telt el a vemhesülésig (p=0.005), valamint a különböző fokú segítségnyújtások között a magzatburok-visszatartás aránya is eltért (p=0.02). Ellető szerkezet segítségével ellett tehenek esetében szignifikánsan nagyobb volt a méhkezelések aránya (p=0.03), és a magzatburok-visszatartás előfordulása (p=0.04), valamint gyakoribb volt az első sikeres termékenyítést követően a magzatvesztések előfordulása (p=0.02). Összességében tehát elmondható, hogy a nem megfelelő időben és körülmények között végzett segítségnyújtások negatív hatással vannak a későbbi reprodukciós eredményekre. Különösen igaz ez az ellető szerkezet esetében, aminek használata következtében a tehenek fertilitása lényegesen csökkenhet. A segítségnyújtások számának mérséklésével, illetve indokolt esetben a szakma szabályai szerint történő alkalmazással több szaporodásbiológiai probléma előfordulása is csökkenthető, aminek hatására az állományok fertilitási eredményei javíthatók.Reproduction performance of dairy cattles is basically determined by nutrition and the events of the postpartum involution period. Providing normal uterine involution can reduce the number of open days, what provides improving efficiency and economy of milk production Conditions of parturition which affects involution sorely can be observed by an automatic camera system. In this study we investigated the parturition conditions (calving assistance/ method) for 2-2 months with a camera and then analyzed the reproduction performance data (incidence of retained fetal membranes, number of uterine treatments, conception rate, and late embryonic loss and intervals from calving to first service) of two Holstein Friesian dairy herds. Data were analyzed using t-tests and Fischer’s exact test. Data from the two herds around parturition were different, indicated that there are differences in the herding technology. Assistance during parturition had no association with higher calf birth weight. Type of calving assistance was significantly associated with retained fetal membranes (p=0.02). Assisted calving was associated with increased number of days from calving to first service (p=0.005). The use of calving aid methods – for example a calving jack – were associated with frequency of the uterine treatments (p=0.03), retained fetal membranes (p=0.04), and incidence of late embryonic loss (p=0.02). Improper timed calving assistance and/or conditions could impair reproductive performance. This impact is more relevant when traction is performed with a calving jack, because of the reduced fertility of the cows. Decrease the frequency of calving assistance, or giving a proper assistance if it’s necessary, fertility problems of dairy cattles could be greatly reduced

The role of birds in the eco epidemiology of tick-borne pathogens Literature review

SUMMARY In this review the authors summarize the epidemiological role of tick infestation of birds, in the context of the recent scientific results. Birds play an important role in the transportation of ticks and tick-borne pathogens, some of which may pose a risk to humans and domestic animals. Two host-generalist tick species, Ixodes ricinus and Haemaphysalis concinna occur more often on birds in Central Europe, than ornitophilic tick species. The prevalence of tick infestation among birds may depend on several factors, such as migrating strategy (short or long distance migrants), feeding level (on the ground or above), climatic factors and also the pathogens living in ticks. It is known that during the spring migration birds arriving from the south may carry developmental stages of exotic ticks (e.g. Hyalomma species) and if these immature ticks are able to moult to adults, they can feed on domestic animals. Bird ticks may carry viruses (e.g. TBE virus), bacteria (mostly Borrelia spp., Anaplasma spp. and Rickettsia spp.) and parasi tes (Babesia spp.) which could represent potential threat to humans. Ticks of migratory songbirds could carry exotic pathogens, as well. The knowledge on tick-borne pathogens in bird tissues is limited, but Anaplasma phagocytophilum, Coxiella burnetii and Rickettsia spp. have been shown to be present in the blood of birds. In this way, it has been demonstrated, that avian hosts can be potential reservoirs of bacteria. Therefore it is important to monitor birds and their ticks, especially because birds are the most common representatives of wildlife near human communities and animal farms, so they can easily pose a risk to human and animal health

Evidence for host specificity of Theileria capreoli genotypes in cervids

Data on the prevalence of piroplasms in buffaloes and large game animal species are lacking from several central European countries. Therefore, to investigate the presence of Babesia/Theileria DNA in these hosts, 239 blood and 270 spleen samples were taken from cervids (red, fallow, and roe deer), as well as from water buffaloes, mouflons, and wild boars in southwestern Hungary, followed by DNA extraction and molecular analysis for piroplasms. All samples from buffaloes and wild boars were PCR negative. Based on spleen samples, the prevalence of piroplasms was significantly higher in red deer (41.7%) than in fallow deer (23.5%). Two genotypes of Theileria capreoli were identified, which showed significant association with their host species (i.e. genotype "capreoli-CE1" was exclusively found in roe deer, whereas red and fallow deer harbored only genotype "elaphi-CE1"). Genotype "elaphi-CE1" of T. capreoli was also detected in one mouflon. No Babesia spp. were identified. In conclusion, in the evaluated region, genotypes of T. capreoli show host-associations among cervids, and at least one of these genotypes may infect mouflons