Väetamise, põllukultuuri ning sordi mõju taimejuurte seente kooslustele

A thesis for applying for the degree of Doctor of Philosophy in Agricultural Sciences.Väitekiri filosoofiadoktori kraadi taotlemiseks põllumajanduse erialal.As the human population increases exponentially, intensification of agriculture is inevitable. Thus, there is an ongoing need for further research towards sustainable agriculture. Soil is a habitat with great microbial diversity. Many of these microorganisms interact with plants, promoting plant growth, improving nutrient uptake, and making plants less susceptible to pathogens. Therefore, it is important to study microorganisms and their coexistence with plants. The aim of this thesis was to study the root fungal community structure across different fertilisation treatments, crop species and cultivars. Fertilisation treatment had no effect on overall root fungal community composition and richness. However, changes occurred when pathogenic and mutualistic fungi were studied separately. Both manure amendment and alternative organic fertilisation decreased mutualists and increased pathogen richness in comparison to mineral fertilisation. Fertilisation is not the only factor affecting the root mycobiome; the results of this thesis suggest crop species as the most important factor. When the effects of fertilisation treatment and crop species were studied together, the effect of crop species was greater than both fertilisation treatment and the species−treatment interaction. Species richness and diversity were highest in wheat roots and lowest in potato roots. Crops species was the main factor determining differences in root fungal community composition. These results suggest that the root mycobiome is selectively recruited from the soil and related to host-plant physiological or morphological traits. In addition, potato cultivars differed in root fungal community composition and richness, again indicating the importance of host genotype in structuring the root mycobiome. This thesis improves our knowledge of how agricultural practices can shape root fungal community structure. This expertise is important for both scientists and agricultural practitioners. Understanding fungal communities allows us to use them to our advantage, and offers a promising strategy for improving soil quality and crop productivity. Without a fundamental understanding of how fungi respond to different agricultural practices at the field scale, it is not possible to develop these mycobiome-based sustainable farming practices.Põllumajandus on inimkonna peamine toiduallikas ning rahvaarvu ja ressursinõudluse kasvades on looduslike alade muutmine põllumaaks paratamatu. Seega on oluline leida jätkusuutlikke põllumajandusstrateegiad, millel oleks keskkonnale võimalikult väike mõju. Muld on elupaigaks tohutule hulgale mikroorganismidele nagu näiteks seened, bakterid ja nematoodid. Paljud neist mikroorganismidest on vastastikmõjus taimedega, soodustades taime kasvu, parandades toitainete omastamist ning muutes taimi patogeenidele vähem vastuvõtlikuks. Seetõttu on oluline uurida mikroorganisme ja nende kooseksisteerimist taimedega. Käesolevas töös uuriti, kuidas mõjutab väetamine, põllukultuur ja põllukultuuri sort juurtega seotud seenekooslusi. Lisaks üldisele seenekooslusele hinnati uuritavate tegurite mõju ka patogeensetele ja mutualistlikele seentele. Doktoritöö tulemused näitasid, et orgaanilise sõnniku lisamine mineraalsele väetisele ei mõjutanud juurtega seotud seenekooslusi. Samas täheldati muutusi patogeenide ja arbuskulaar-mükorriissete seente esinemises. Ent väetamine ei ole ainus tegur, mis võib mõjutada seenekooslusi. Antud doktoritöö tulemused näitasid, et olulisem mõjutaja võib olla hoopis uuritav põllukultuur ise. Kui väetamise ja põllukultuuri mõju uuriti samas mudelis, siis taime liigi mõju prevaleeris väetamise üle. Nii üldine, patogeenide kui ka arbuskulaarse-mükoriisa liigirikkus ja mitmekesisus oli suurim nisu juurtes ning madalaim kartuli juurtes. Lisaks oli kõikide põllukultuuride juurtes erinev seente liigiline koosseis. Lahknevused võivad olla tingitud nii taime füsioloogilisestest kui ka morfoloogilistest erinevustest. Lisaks erines seente liigiline koosseis kartulisortide juurtes. Ka see viitab, et peremeesorganismi genotüübil on oluline roll kujundamaks taime juure seenekooslust. Antud uurimistöö tulemused parandavad teadmisi põllumajanduse mõjust seenekooslustele. Teadmine, kuidas põllumajandus mõjutab seenekooslusi, on kasulik nii teadlastele kui põllumajandustootjatele, kuna see võimaldab paljutõotavat strateegiat nii mulla kvaliteedi kui ka taimede tootlikkuse parandamiseks. Ent ilma põhimõttelise teadmiseta, kuidas seened reageerivad erinevatele häiringutele lokaalsel tasandil, pole võimalik selliseid jätkusuutlikke meetodeid arendada.Publication of this thesis is supported by the Estonian University of Life Sciences

The indigenous arbuscular mycorrhizal fungal colonisation potential in potato roots is affected by agricultural treatments

ArticlesThere is an urgent need to develop novel approaches to enhance sustainable agriculture while not reducing cr op yields. Arbuscular mycorrhizal (AM) fungi establish symbiotic associations with most crop plants improving plant performance and soil health. This study investigated the extent of colonisation of potato roots by indigenous AM fungi in the arable soil under conventional and organic farming systems. Potato roots had greater AM fungal colonisation levels under organic than conventional farming, though in general, root colonisation levels were extremely low in both farming systems . Pota to root AM fungal colonisation was lower with higher soil P content and higher with higher annual C input. Trap plant root AM fungal colonisation was considerably higher than in field potato roots and showed that soil mycorrhizal inoculum potential was hig her in organic than in conventional farming. Thus, the positive impact of manure application in organic fields to the potato AM fungal colonisation can be explained by previous higher total annual C fresh organic matter input and lower soil P content under treatment. Furthermore, the natural AM fungal abundance in the soil was sufficient to colonise trap plant roots, suggesting a low mycorrhizal dependence of the studied potato cultivar

First evidence of bud feeding-induced RNAi in a crop pest via exogenous application of dsRNA

An ecologically sustainable strategy for managing the pollen beetle Brassicogethes aeneus, a key pest of oilseed rape (Brassica napus) in Europe, is greatly needed. Gene silencing via RNA interference, through sprayed applications of target-specific double-stranded RNA, represents a potential alternative to conventional insecticides. We used dsRNA designed to target a vital gene in this pollen beetle species and allowed the beetles to feed on dsRNA-coated oilseed rape buds. We observed a significant silencing of the target gene; and this was followed by a significant, albeit delayed, reduction in pollen beetle survival rate. Further experiments are necessary in order to better understand the potential for developing a dsRNA-spray approach to pollen beetle management. Spray-induced gene silencing (SIGS) is a potential strategy for agricultural pest management, whereby nucleotide sequence-specific double-stranded RNA (dsRNA) can be sprayed onto a crop; the desired effect being a consumption of dsRNA by the target pest, and subsequent gene silencing-induced mortality. Nucleotide sequence-specificity is the basis for dsRNA's perceived biosafety. A biosafe approach to pollen beetle (Brassicogethes aeneus) management in oilseed rape (Brassica napus) agroecosystems is needed. We examined the potential for SIGS in B. aeneus, via bud feeding, a field-relevant dsRNA exposure route. Oilseed rape buds were uniformly treated with dsRNA designed to target alpha COP in B. aeneus. Our model control dsRNA (dsGFP) remained detectable on buds throughout the entire 3 d exposure period. When applied at 5 mu g/mu L, ds alpha COP induced significant alpha COP silencing 3 d after dietary exposure to buds treated with this ds alpha COP concentration. We also observed a trend of increased alpha COP silencing with increasing concentrations of ds alpha COP at both 3 and 6 d. Furthermore, we observed a marginally significant and significant reduction in B. aeneus survival at 10 and 15 d, respectively. Our results suggest potential for developing a SIGS approach to B. aeneus management-though further experiments are needed to more fully understand this potential

FungalTraits:A user-friendly traits database of fungi and fungus-like stramenopiles

The cryptic lifestyle of most fungi necessitates molecular identification of the guild in environmental studies. Over the past decades, rapid development and affordability of molecular tools have tremendously improved insights of the fungal diversity in all ecosystems and habitats. Yet, in spite of the progress of molecular methods, knowledge about functional properties of the fungal taxa is vague and interpretation of environmental studies in an ecologically meaningful manner remains challenging. In order to facilitate functional assignments and ecological interpretation of environmental studies we introduce a user friendly traits and character database FungalTraits operating at genus and species hypothesis levels. Combining the information from previous efforts such as FUNGuild and Fun(Fun) together with involvement of expert knowledge, we reannotated 10,210 and 151 fungal and Stramenopila genera, respectively. This resulted in a stand-alone spreadsheet dataset covering 17 lifestyle related traits of fungal and Stramenopila genera, designed for rapid functional assignments of environmental studies. In order to assign the trait states to fungal species hypotheses, the scientific community of experts manually categorised and assigned available trait information to 697,413 fungal ITS sequences. On the basis of those sequences we were able to summarise trait and host information into 92,623 fungal species hypotheses at 1% dissimilarity threshold

Examining spray-induced gene silencing for pollen beetle control

Pollen beetles are a transcontinental threat to oilseed rape cultivation. Conventional insecticide use has resulted in resistance development, and can furthermore lead to losses of non-target organisms, including those that contribute to natural biological control of pollen beetle populations. Biosafe alternatives to synthetic pesticides must be explored. Double-stranded RNA (dsRNA) targeting pollen beetle, by the RNA interference (RNAi) mode of action, has been previously demonstrated under laboratory conditions. The present study examined, under a whole plant context, the potential for spray-induced gene silencing (SIGS) in pollen beetle. We confirmed translocation of sprayed dsRNA to later-emerging, non-sprayed dietary tissues of oilseed rape, as well as infiltration of sprayed dsRNA to the inner tissues of reproductive buds (pollen beetle's food source). No effect on survival was observed. The inefficacy of SIGS observed here highlights the requirement for more effective SIGS approaches to pollen beetle management. The translocation of sprayed dsRNA within oilseed rape plants, and the non-significant yet detectable effect on target mRNA downregulation, together suggest potential for future optimization of a SIGS approach against pollen beetle

Anther-Feeding-Induced RNAi in Brassicogethes aeneus Larvae

The biosafety aspect of applying double-stranded RNA (dsRNA) in crop pest management is rooted in dsRNA's mode of action, which displays nucleotide sequence-specificity to a particular region of a messenger RNA (mRNA), against which the insecticidal dsRNA is designed. This prominent and promising class of insecticides therefore has the potential to target a single pest species while conferring negligible effect on non-target organisms. Recent studies examining the effect of target-specific dsRNA in adults of the pollen beetle Brassicogethes aeneus, a major pest of oilseed rape (Brassica napus) crops in Europe, suggest the potential for developing a gene-silencing approach within integrated B. aeneus management. The present study examines the efficacy of target-specific dsRNA on target-mRNA silencing, and subsequent gene-silencing-induced mortality, in B. aeneus larvae, as this life stage represents a critical target for achieving optimal integrated B. aeneus control. Treatment applications occurred via feeding on dsRNA-treated anthers for 3 d. We observed variable gene-silencing efficacy, all target treatments having a significant or marginally significant effect after 3 d of dsRNA feeding, with greater variability at 6 d. These results further validated significant gene-silencing-induced mortality observed for one of the target treatments. Moreover, gene-silencing-induced mortality occurred at a quicker rate in B. aeneus larvae compared to what has been previously observed in B. aeneus adults. Finally, we consider refinements that must be made to B. aeneus larval bioassay setups to promote and strengthen future larval studies regarding this important crop pest species

Highly Clonal Structure and Abundance of One Haplotype Characterise the Diplodia sapinea Populations in Europe and Western Asia

Diplodia sapinea is a cosmopolitan endophyte and opportunistic pathogen having occurred on several conifer species in Europe for at least 200 years. In Europe, disease outbreaks have increased on several Pinus spp. in the last few decades. In this study, the genetic structure of the European and western Asian D. sapinea population were investigated using 13 microsatellite markers. In total, 425 isolates from 15 countries were analysed. A high clonal fraction and low genetic distance between most subpopulations was found. One single haplotype dominates the European population, being represented by 45.3% of all isolates and found in nearly all investigated countries. Three genetically distinct subpopulations were found: Central/North European, Italian and Georgian. The recently detected subpopulations of D. sapinea in northern Europe (Estonia) share several haplotypes with the German subpopulation. The northern European subpopulations (Latvia, Estonia and Finland) show relatively high genetic diversity compared to those in central Europe suggesting either that the fungus has existed in the North in an asymptomatic/endophytic mode for a long time or that it has spread recently by multiple introductions. Considerable genetic diversity was found even among isolates of a single tree as 16 isolates from a single tree resulted in lower clonal fraction index than most subpopulations in Europe, which might reflect cryptic sexual proliferation. According to currently published allelic patterns, D. sapinea most likely originates from North America or from some unsampled population in Asia or central America. In order to enable the detection of endophytic or latent infections of planting stock by D. sapinea, new species-specific PCR primers (DiSapi-F and Diplo-R) were designed. During the search for Diplodia isolates across the world for species specific primer development, we identified D. africana in California, USA, and in the Canary Islands, which are the first records of this species in North America and in Spain.publishedVersio

Examining Phenotypic Traits Contributing to the Spread in Northern European Potato Crops of EU_41_A2, a New Clonal Lineage of Phytophthora infestans

International audienceUntil recently, genotypes of Phytophthora infestans were regionally distributed in Europe, with populations in western Europe being dominated by clonal lineages and those in northern Europe being genetically diverse because of frequent sexual reproduction. However, since 2013 a new clonal lineage (EU_41_A2) has successfully established itself and expanded in the sexually recombining P. infestans populations of northern Europe. The objective of this study was to study phenotypic traits of the new clonal lineage of P. infestans, which may explain its successful establishment and expansion within sexually recombining populations. Fungicide sensitivity, aggressiveness, and virulence profiles of isolates of EU_41_A2 were analyzed and compared with those of the local sexual populations from Denmark, Norway, and Estonia. None of the phenotypic data obtained from the isolates collected from Denmark, Estonia, and Norway independently explained the invasive success of EU_41_A2 within sexual Nordic populations. Therefore, we hypothesize that the expansion of this new genotype could result from a combination of fitness traits and more favorable environmental conditions that have emerged in response to climate change