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

Molekuláris járványtani vizsgálatok hazai szarvasmarhák haemoplamosisa és elhullással járó anaplasmosisa kapcsán

Haematológiai és molekuláris vizsgálatokat végeztünk szarvasmarhák hazánkban megállapított, elhullással járó anaplasmosisa kapcsán. A heveny megbetegedés öt állatnál fordult elő, amelyek közül kettő elhullott. Az anaplasma-hordozó státuszt 69 tehén esetében állapítottuk meg, ami az állomány 92%-a. Huszonnégy vérminta további vizsgálata haemoplasmák jelenlétét is felfedte, így a Mycoplasma wenyonii 22, a 'Candidatus M. haemobos' 21 állatban fordult elő. Az utóbbi két kórokozó hazai előfordulásának ez az első molekuláris igazolása

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

Evidence for host specificity of Theileria capreoli genotypes in cervids

Abstract 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

Diversity of Haemaphysalis-associated piroplasms of ruminants in Central-Eastern Europe, Hungary

BACKGROUND Increasing numbers of genetic variants are being recognized among piroplasms, but the precise taxonomical status, the tick vector and the geographical range of several species or genotypes are still unknown. Bovine piroplasmosis was reported to re-emerge in north-east Hungary. Because Theileria-infection was newly diagnosed in one cattle herd in the same region of the country, the aim of this study was to molecularly identify the relevant agent, to find its local vector tick species, and to examine the range of Babesia/Theileria spp. of ruminants in Haemaphysalis sp. ticks collected previously in Hungary. FINDINGS Blood samples were drawn on two occasions from 90 dairy cattle in northern Hungary, and ticks were collected on their pastures. In addition, questing ticks (315 Haemaphysalis inermis, 259 H. concinna and 22 H. punctata), which originated mainly in the same region of the country from 2007, were included in the study. DNA was extracted from these samples, followed by molecular analysis for piroplasms. In the cattle Theileria orientalis was identified, with 100 % sequence homology to isolates from Japan, China, South-Africa and Australia. Based on GenBank data this genotype has not been previously reported in Europe. The prevalence of infection in the herd remained almost constant in the main tick season, suggesting exposure in previous years. Retrospective analysis of ticks revealed the presence of Babesia crassa in H. inermis, for the first time in Europe and in this tick species. On the other hand, H. concinna carried five different piroplasms, including B. motasi that was also newly detected in Central-Eastern Europe and in this tick species; whereas H. punctata harboured Theileria sp. OT3, hitherto known to occur in the Mediterranean region. CONCLUSIONS Results of this study broaden the range of piroplasms that are infective for ruminants in Central-Eastern Europe. Although bovine babesiosis and theileriosis was known to occur in Hungary, molecular evidence is provided here for the first time on the presence of Babesia and/or Theileria spp. of sheep, goats and cervids in Hungary