Tritroofiliste suhete rakendamine jätkusuutliku taimekaitse strateegia leidmiseks rapsile

Oilseed rape is one of the most important cruciferous crops grown in Europe and other parts with a temperate climate. Today, conventional agricultural practices rely heavily on chemical inputs, which have negative side effects on the environment and pesticides also lose their efficiency. This study investigated 1) the attractiveness and impact plant species and nitrogen fertilisation have on M. aeneus and its parasitoids and 2) the impact of nitrogen fertilisation on volatile organic compounds’ emission. The aim was to gain knowledge on how to manipulate M. aeneus’ abundance through their preferences. The feeding and oviposition preferences of M. aeneus and its parasitoids were studied on plants from the genus Brassica, Sinapis, Eruca, and Raphanus (Capparales: Brassicaceae). The results showed that although M. aeneus oviposit and/or feed on all studied plant species, their attractiveness differed. During the green-yellow bud stage S. alba and B. nigra were the most attractive. For oviposition, species of the genus Brassica were preferred over S. alba and E. sativa but not over R. sativus. The larval abundance was generally higher on B. napus although, depending on year the attractiveness of B. nigra was almost the same or even higher. The plant species also influenced the number of buds used for oviposition by M. aeneus. The clutch size was smaller on R. sativus compared to Brassica species. Larval survival rate was studied on all plant species but dead larvae were found only on R. sativus and E. sativa. The attractiveness of R. sativus was comparable to B. napus and one third of larvae failed to survive. The influence of plant species was also confirmed for third trophic level, parasitoids, and their species composition. The percentage of parasitized larvae was equal or higher than on B. napus on all plant species but E. sativa and R. sativus. Four species of parasitoid were found: Tersilochus heterocerus, Phradis morionellus, Diospilus capito and one unidentified species with low occurrence (<1%). Fertilisation also had impact on both pest and parasitoids, probably via VOC emission, which also differed with N fertilisation levels. Larval abundance was the highest on moderate and high nitrogen levels and the lowest at on zero fertilisation level which was attractive to parasitoids. The highest emission rates were for acetic acid, 3-carene, α-pineen and bensoealdehüüdi and emission rates of several VOCs increased with nitrogen fertilisation. The abundance of M. aeneus larvae was positively correlated with emission rates of 3-carene, limonene and indole while β-pinene, (E,E)-α-farnesene, benzaldehyde, methylbenzoate linalool were inhibitory for M. aeneus oviposition. These results show probabilities to use environmental friendly pest management systems for M. aeneus, to collect larvae to certain areas on the field where the trap crop can be destroyed in case of high pest abundance. By using mechanical destruction, it can also be used in organic farming. Brassica nigra and S. alba have potential for conventional trap cropping to prevent damage caused by overwintered M. aeneus adults. The lethal effect of R. sativus on M. aeneus was shown for the first time. Brassica nigra was attractive for oviposition which can be used to influence the next generation. The biological control-assisted trap cropping on B. napus can be promoted by using B. nigra and B. juncea, but also B. rapa and S. alba. By increasing the abundance and species diversity of parasitoids the damage caused by M. aeneus could be kept below economic threshold levels therefore decrease environment pollution. Such precautionary methods can be used in integrated and organic farming. Pest and parasitoid abundance could also be manipulated by optimising nitrogen fertilisation. By using moderate nitrogen input the pressure on the environment could be reduced and the crop might become less attractive to the pest.Raps (Brassica napus L.) on oluline parasvöötmes kasvatatav ristõieline õlikultuur, mille kasvupindala on järjest suurenenud nii Eestis kui ka Euroopas. Rapsikahjurite tõrje põhineb sünteetilistel pestitsiididel, mis aga on hävitava toimega mitte ainult kahjuritele vaid ka kasulikele organismidele. Lisaks sellele väheneb elurikkus põllumajandusmaastikul, võib kaasneda keskkonna saastatus ja majanduslik konkurentsivõime. Kõigest sellest lähtuvalt on alternatiivsete keskkonnasõbralike tõrjestrateegiate, näiteks püüniskultuuride, väljatöötamine väga oluline. Käesoleva töö käigus uuriti 1) erinevate taimeliikide atraktiivust naeri-hiilamardikale ja tema parasitoididele; 2) lämmastikväetiste erinevate koguste mõju naeri-hiilamardika arvukusele ja parasiteerituse tasemele talirapsil; 3) lämmastikväetiste mõju talirapsi lenduvate orgaaniliste ühendite (VOC) emisioonile. Töö eesmärgiks oli uurida naeri-hiilamardika toitumis- ja munemiseelistusi, et nendega manipuleerides leida sobivad kultuurid, mida saaks rakendada püüniskultuuride strateegias. Naeri-hiilamardika toitumis- ja munemiseelistusi uuriti seitsmel ristõielisel taimeliigil perekondadest kapsasrohi (Brassica), sinep (Sinapis), võõrkapsas (Eruca) ja rõigas (Raphanus) (Capparales: Brassicaceae). Töö tulemusena selgus, et kuigi naeri-hiilamardikas toitus ja/või munes suuremal või vähemal määral kõikidel katsetaime liikidel, oli taimeliikidel ja nende erinevatel kasvufaasidel atraktiivsuses suuri erinevusi. Punga staadiumis olid mardikatele atraktiivsed valge ja must sinep, seevastu munemiseks eelistati kapsasrohu perekonda kuuluvaid taimeliike, kuid atraktiivne oli ka õlirõigas. Munakurnade suurus erines taimeliigiti olles väiksem õlirõikal võrreldes kapsasrohu perekonna liikidega. Hiilamardikad eelistasid muneda mustale sinepile, rapsile ja rüpsile. Hukkunud naeri-hiilamardika vastseid leiti põld-võõrkapsa ja õlirõika pungades, viimasel liigil ei suutnud ligi kolmandik vastsetest kestuda ja hukkusid esimeses kasvustaadiumis. Seega saaks neid taimi potentsiaalselt kasutada surmava püüniskultuurina. Töö tulemusena selgus, et kahjurite peremeestaimeliigid mõjutavad ka parasitoidide munemiskäitumist ja liigilist koosseisu. Vastsete parasiteerituse tase oli rapsiga võrreldes samaväärne või kõrgem kõigil taimeliikidel peale põld-võõrkapsa ja õlirõika. Leiti nelja liiki parasitoide: Tersilochus heterocerus, Phradis morionellus ja Diospilus capito ja üks vähearvukas määramata liik. Ka lämmastikuga väetamise erinevad kogused mõjutasid kahjurit ja parasitoide, tõenäoliselt VOC emisiooni kaudu, mida samuti mõjutas väetustase. Kõige rohkem vastseid leiti kontrollvariandilt (0 kg/ha) ja kõrge väetustasemega (160 kg/ha) katsevariandilt, kontrollvariant oli ka atraktiivseim parasitoididele. Enim emiteerisid rapsitaimed 3-kareeni, α-pineeni ja bensoealdehüüdi. Naeri-hiilamardika vastsete arv korreleerus mitmete lenduvate ühendite eritumisega: kõrgema 3-kareeni, indooli ja limoneeni eritamisel oli vastsete arvukus taime kohta suurem, samas kui β-pineiini, (E,E)-α-farneseeni, metüülbensoaadi, bensoealdehüüdi ja linalooli kõrgem eritamine mõjus naeri-hiilamardika munemisaktiivsusele pärssivalt. Tulemused näitavad, et naeri-hiilamardika toitumis- ja munemiskäitumist saab mõjutada keskkonnasõbralikke tõrjevõtteid kasutades. Meelitades kahjureid püüniskultuuri aladele saab neid kõrge arvukuse korral hävitada insektitsiididega või mehaaniliselt (kultuuri sisse kündes), viimane strateegia sobib ka mahepõllumajanduses kasutamiseks. Potentsiaalseteks püüniskultuurideks mardikate valmikutele on näiteks valge ja must sinep. Viimane oli ka atraktiivne munemiseks, järelikult saab selle kaudu reguleerida kahjuri järgeva põlvkonna arvukust. Esmakordselt leiti, et õlirõigas võib olla surmav naeri-hiilamardika vastsele. Lisaks selgus, et nii mustal sinepil ja kapsasrohul, kui ka rüpsil ning valgel sinepil on potentsiaali naeri-hiilamardika bioloogilist kontrolli suurendavate püüniskultuuridena, suurendades parasitoidide arvukust ja liigilist mitmekesisust. Naeri-hiilamardika arvukust kui ka nende looduslikke vaenlasi on võimalik mõjutada optimeerides rapsi väetamist lämmastikuga. Soovitav on kasutada keskmisi lämmastikunorme, mis võimaldab vähendada ka keskkonnakoormust ja muudab rapsi kahjurile vähematraktiivseks ning ei ületa majanduslikku tõrjekriteeriumit. Ennetaval tõrjel baseeruv kahjurite regulatsioon on kasutatav nii integreeritud kui ka mahetootmises

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

Targeting a coatomer protein complex-I gene via RNA interference results in effective lethality in the pollen beetle Brassicogethes aeneus

he pollen beetle Brassicogethes aeneus is a serious pest of oilseed rape (Brassica napus) in Europe. Management of this pest has grown difficult due to B. aeneus's development of resistance to pyrethroid insecticides, as well as the pressure to establish control strategies that minimise the impact on nontarget organisms. RNA interference represents a nucleotide sequence-based, and thus potentially species-specific, approach to agricultural pest control. The present study examined the efficacy of targeting the coatomer gene coatomer subunit alpha (alpha COP), via both microinjection and dietary exposure to exogenous complementary dsRNA, on alpha COP-silencing and subsequent mortality in B. aeneus. Beetles injected with dsRNA targeting alpha COP (at 0.14 mu g/mg) showed 88% and 100% mortality at 6 and 10 days post-injection, respectively; where by the same time after dietary exposure, 43%-89% mortality was observed in the 3 mu g dsRNA/mu L treatment, though the effect was concentration-dependent. Thus, the effect was significant for both delivery routes. In working towards RNA-based management of B. aeneus, future studies should include alpha COP as a target of interest

Põllumajandusmaa mitmekesisus

Põllumajandusmaastiku kasutamise intensiivistumine on põhjustanud drastilise bioloogilise mitmekesisuse vähenemise ja pool-looduslike elupaikade kadumise (Butchart jt, 2010; Geiger jt, 2010). Oluliselt on suurenenud põldude pindala ja kasvanud erinevate agrokemikaalide kasutus. Põllumajanduse intensiivistumine on toonud kaasa elurikkuse ja põllumajandusmaastiku mitmekesisuse vähenemise, eriti just erinevate pool-looduslike alade ja koosluste kadumise tõttu (Benton jt, 2003; Geiger jt, 2010; Robinson ja Sutherland, 2002; Tscharntke jt, 2005). Pool-looduslike ja mitteharitavate alade kadumine lihtsustab radikaalselt maastikku ja vähendab ökosüsteemi funktsioneerimist, mille tõttu väheneb ka ökosüsteemi teenuste pakkumine (Cardinale jt, 2012). Põllumajanduse vaatevinklist on olulisemateks biootilisteks ökosüsteemi teenusteks tolmeldamine ja kahjuritõrje. Tolmeldamisest sõltub 1400 peamisest põllukultuurist koguni 80%. Näiteks rapsi saak ja ka kogutulu suurenes kuni 40% kui põldudel tegutses arvukalt tolmeldajaid, samas kui pestitsiidide kasutamine vähendas oluliselt nii tolmeldajate arvukust kui ka saagi suurust (Catarino jt, 2019). Mitmekesiste õistaimedega rohuribade lisamine nisupõllule vähendas viljakukkede poolt tehtavat kahju koguni 61%, kahjuri vastsete arvukus langes 40% jäädes allapoole majandusliku tõrjekriteeriumi lävendit ning seega vähendas pestitsiidide kulu (Tschumi jt, 2016). Teisalt leitakse ka, et ökosüsteemi teenustele ei ole õiglane anda rahalist vääringut ja lisaks pole see praktikas kergesti teostatav (Sall jt, 2012). Saamaks osa neist ökosüsteemiteenustest, mida pakuvad tolmeldajad ja röövtoidulised lülijalgsed tuleb neile tagada vajalik keskkond kogu elutsükli läbimiseks. Paljud lülijalgsed vajavad mitmekesist elupaika, sest eri kasvustaadiumitel võivad olla väga erinevad nõudmised/vajadused elupaigale. Nii näiteks vajavad tolmeldajad lisaks pesitsuspaikadele ka toidubaasi ja talvituskohti ning röövtoidulised putukad lisaks toiduallikale ka varje-, elu- ja talvituspaiku. Põllumajandusmaastik, kus domineerivad lühiajalised kultuurid ja puuduvad pool-looduslikud alad ei suuda toetada tootmist oluliste ökosüsteemiteenustega, sest pidev mullaharimine ja agrotehniliste tööde läbiviimine hävitab kasurite varje-, toitumis-, talvitumis- ja elupaiku, samuti ei paku suured monokultuursed põllud stabiilset toiduallikat kogu vegetatsiooniperioodi vältel (Burns, 2014). Pool-looduslikel kooslustel on kriitiline roll nii taime- kui ka loomapopulatsioonide alalhoidmisel ja agroökosüsteemide funktsioneerimisel (Tscharntke jt, 2012). Just pool-looduslikud kooslused pakuvad kasulikele lülijalgsetele, varje-, elu- ja talvitumispaiku ning varustavad neid toiduallikatega väljaspool põllukultuuride õitsemisaega, mistõttu nad suudavad põllumajandusmaastikul põllukultuure tolmeldada ja kahjureid hävitada (Holland jt, 2016). Kuna tegemist on keerulise ning väga paljusid faktoreid ja muutujaid sisaldava uurimisteemaga, siis on maastiku mitmekesisuse ja ökosüsteemiteenuste pakkumise seosete uurimustes saadud ka väga erinevaid tulemusi. Näiteks on leitud, et kasulikele lülijalgsetele ebasobiv põllumajanduspraktika (künd, laialdane pestitsiidide ja sünteetiliste väetiste kasutamine) vähendab pool-looduslike alade positiivset mõju või on pool-looduslike alade hulk liiga väike ja nende paiknemine maastikul ebasoodne kasurite toetamiseks (Rusch jt, 2011; Thies ja Tscharntke, 1999). Põllumajandusmaastikul asuvad pool-looduslikud alad on olulised ka taime- ja loomapopulatsioonide säilitamisel, seetõttu on neil oluline roll elurikkuse säilitamisel ja ka ökosüsteemide funktsioneerimisel (Grashof-Bokdam ja Langevelde, 2005; Tscharntke jt, 2012)

Blue stem weevil (Ceutorhynchus sulcicollis) – a potential threat to oilseed rape production

Oilseed rape (Brassica napus) has numerous insect pests, some of which are stem-miners. Currently, blue stem weevil (Ceutorhynchus sulcicollis) is not considered a pest of oilseed rape. In the present study, a total of 60 (30 untreated, 30 insecticide-treated) oilseed rape plants were dissected; and stem-mining larvae were collected, and subsequently allowed to pupate in soil. After pupation, all emerged adult weevils were identified as either blue stem weevil (C. sulcicollis) or cabbage stem weevil (Ceutorhynchus pallidactylus). We report that under favourable conditions C. sulcicollis was capable of reaching pest status, and was significantly more abundant than C. pallidactylus, indicating a critical need for future studies regarding C. sulcicollis

Spatiotemporal distancing of crops reduces pest pressure while maintaining conservation biocontrol in oilseed rape

BACKGROUNDAgricultural landscapes provide resources for arthropod pests as well as their natural enemies. To develop integrated pest management (IPM) practices, it is important to understand how spatiotemporal location influences crop colonization and damage severity. We performed a 3-year (2016-2018) field experiment in winter oilseed rape (OSR, Brassica napus) fields in Estonia, where half of the fields were within 500 m of the location of the previous year's winter OSR field and half were outside this zone. We investigated how distance from the previous year's OSR crop influences the infestation and parasitism rates of two of its most important pests: the pollen beetle (Brassicogethes aeneus) and the cabbage seed weevil (Ceutorhynchus obstrictus). RESULTSWhen the distance from the previous year's OSR crop was >500 m, we recorded significantly reduced pest pressure by both B. aeneus and C. obstrictus in the study fields. Biocontrol of both pests, provided by parasitic wasps, was high in each study year and commonly not affected by distance. Mean parasitism rates of B. aeneus were >31%, occasionally reaching >70%; for C. obstrictus, mean parasitism was >46%, reaching up to 79%, thereby providing effective biocontrol for both pest species. CONCLUSIONSpatiotemporal separation of OSR fields can reduce pest pressure without resulting in reduced parasitism of OSR pests. This supports a spatiotemporal field separation concept as an effective and sustainable technique for IPM in OSR

Evaluating the effect of seven plant essential oils on pollen beetle (Brassicogethes aeneus) survival and mobility

Plant essential oils (EOs) represent an area of interest for managing agricultural pests. We examined the insecticidal efficacy of seven plant EOs on the survival and mobility of the pollen beetle Brassicogethes aeneus, a primary pest of oilseed rape (Brassica napus) in Europe. Topical dosing tests showed the EO of the inner bark of Cinnamomum verum (cinnamon) to be the most effective EO examined in our study. Subsequent bioassays, exposing B. aeneus to oilseed rape plant surfaces sprayed with various concentrations of C. verum EO, additionally suggested a concentration threshold at which this EO may significantly control B. aeneus populations. We suggest that further studies on B. aeneus examine the effect of certain pure compounds associated with the most promising EOs (individually as well as in binary combinations) in order to reveal optimal chemical composition and ratios to exploit within a B. aeneus management framework

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