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Ma fungicides amagwiritsidwa ntchito nthawi zambiri pakupanga maluwa ndipo amatha kuwopseza tizilombo toyambitsa matenda.Komabe, ndi zochepa zomwe zimadziwika za momwe tizilombo toyambitsa matenda omwe si njuchi (mwachitsanzo, njuchi zokhala paokha, Osmia cornifrons) zimayankhira kukhudzana ndi mankhwala opha tizilombo omwe amagwiritsidwa ntchito pa maapulo panthawi ya maluwa.Kusiyana kwa chidziwitsoku kumachepetsa zisankho zamalamulo zomwe zimatsimikizira madera otetezeka komanso nthawi yopopera mankhwala ndi fungicide.Tinaona zotsatira za mankhwala opha tizilombo awiri (captan ndi mancozeb) ndi ma interlayer/phytosystem fungicides anayi (ciprocycline, myclobutanil, pyrostrobin ndi trifloxystrobin).Zotsatira pa kulemera kwa mphutsi, kupulumuka, chiŵerengero cha kugonana ndi kusiyana kwa mabakiteriya.Kuunikiraku kunachitika pogwiritsa ntchito kuyesa kwapakamwa kosatha komwe mungu adathandizidwa m'miyeso itatu kutengera mlingo womwe ukulimbikitsidwa kuti agwiritse ntchito kumunda (1X), theka la mlingo (0.5X) ndi mlingo wochepa (0.1X).Mlingo wonse wa mancozeb ndi pyritisoline umachepetsa kwambiri kulemera kwa thupi komanso kupulumuka kwa mphutsi.Kenako tidasanja jini ya 16S kuti iwonetse bacteriome ya mphutsi ya mancozeb, mankhwala opha bowa omwe amachititsa kuti anthu azifa kwambiri.Tinapeza kuti kusiyanasiyana kwa mabakiteriya ndi kuchuluka kwake kunachepetsedwa kwambiri mu mphutsi zomwe zimadyetsedwa ndi mungu wopangidwa ndi mancozeb.Zotsatira zathu za labotale zikuwonetsa kuti kupopera mankhwala ena a fungicides panthawi yamaluwa kumawononga kwambiri thanzi la O. cornifrons.Izi ndi zofunika pazisankho zamtsogolo zokhuza kagwiritsidwe ntchito kosasunthika kwa zinthu zoteteza mitengo yazipatso ndipo zimagwira ntchito ngati maziko owongolera omwe amateteza tizilombo toyambitsa matenda.
Njuchi yomanga yokhayokha yotchedwa Osmia cornifrons (Hymenoptera: Megachilidae) inabweretsedwa ku United States kuchokera ku Japan chakumapeto kwa zaka za m'ma 1970 ndi kumayambiriro kwa zaka za m'ma 1980, ndipo zamoyozi zakhala zikugwira ntchito yofunika kwambiri yoyendetsa mungu poyang'anira zachilengedwe kuyambira pamenepo.Mitundu yodziwika bwino ya njuchi imeneyi ndi mbali ya mitundu pafupifupi 50 ya njuchi zakutchire zomwe zimayenderana ndi njuchi zomwe zimapatsa mungu m'minda ya zipatso za amondi ndi maapulo ku United States2,3.Njuchi za Mason zimakumana ndi zovuta zambiri, kuphatikizapo kugawikana kwa malo, tizilombo toyambitsa matenda, ndi mankhwala ophera tizilombo3,4.Pakati pa mankhwala ophera tizilombo, ma fungicides amachepetsa mphamvu, kufunafuna chakudya5 komanso kukonza thupi6,7.Ngakhale kafukufuku waposachedwa akuwonetsa kuti thanzi la njuchi za Mason limakhudzidwa mwachindunji ndi tizilombo tating'onoting'ono ta commensal ndi ectobactic, 8,9 chifukwa mabakiteriya ndi bowa amatha kukhudza zakudya komanso mayankho a chitetezo chamthupi, zotsatira za kuwonekera kwa fungicide pamitundu yosiyanasiyana ya njuchi za Mason zikungoyamba kumene. anaphunzira.
Mankhwala opha tizilombo toyambitsa matenda osiyanasiyana (okhudzana ndi machitidwe) amawapopera m'minda ya zipatso asanatuluke ndi maluwa kuti athetse matenda monga nkhanambo ya apulo, zowola zowawa, zowola za bulauni ndi powdery mildew10,11.Ma fungicides amawonedwa kuti alibe vuto kwa olima mungu, chifukwa chake amalimbikitsidwa kwa wamaluwa nthawi yamaluwa;Kuwonekera ndi kuyamwa kwa fungicides ndi njuchi ndizodziwika bwino, chifukwa ndi gawo la ndondomeko yolembetsa mankhwala ophera tizilombo ndi bungwe la US Environmental Protection Agency ndi mabungwe ena ambiri olamulira dziko12,13,14.Komabe, zotsatira za mankhwala ophera njuchi kwa omwe si njuchi sizidziwika chifukwa sizifunikira pansi pa mapangano ovomerezeka ku United States15.Kuphatikiza apo, nthawi zambiri palibe njira zoyeserera zoyeserera njuchi imodzi16,17, ndikusunga madera omwe amapereka njuchi kuti ziyesedwe ndizovuta18.Mayesero a njuchi zoyang'aniridwa zosiyanasiyana akuchitidwa mochulukira ku Ulaya ndi ku USA kuti aphunzire zotsatira za mankhwala ophera tizilombo ku njuchi zakutchire, ndipo ndondomeko zovomerezeka zapangidwa posachedwapa za O. cornifrons19.
Njuchi za nyanga ndi ma monocyte ndipo zimagwiritsidwa ntchito pogulitsa mbewu za carp monga chowonjezera kapena m'malo mwa njuchi za uchi.Njuchi zimenezi zimamera pakati pa Marichi ndi Epulo, ndipo zazimuna zolusa zimatuluka masiku atatu kapena anayi zazikazi zisanachitike.Pambuyo pa makwerero, yaikazi mwachangu imasonkhanitsa mungu ndi timadzi tokoma kuti tipereke maselo angapo a ana mkati mwa chisa cha tubular (chachilengedwe kapena chochita kupanga)1,20.Mazira amaikidwa pa mungu mkati mwa maselo;yaikazi kenako imamanga khoma ladongo isanakonzekere selo lotsatira.Mphutsi zoyamba za instar zimatsekeredwa mu chorion ndipo zimadya madzi a embryonic.Kuyambira wachiwiri mpaka wachisanu (prepupa), mphutsi zimadya mungu22.Mungu ukatha, mphutsi zimapanga zikwa, mphutsi ndipo zimatuluka m'chipinda chimodzi cha ana, nthawi zambiri kumapeto kwa chirimwe20,23.Akuluakulu amatuluka masika wotsatira.Kupulumuka kwa akulu kumalumikizidwa ndi kupindula kwamphamvu (kulemera) kutengera kudya.Choncho, ubwino wa thanzi la mungu, komanso zinthu zina monga nyengo kapena kukhudzana ndi mankhwala ophera tizilombo, ndizo zomwe zimakhudza moyo ndi thanzi24.
Mankhwala ophera tizilombo ndi ma fungicide omwe amagwiritsidwa ntchito asanatuluke maluwa amatha kusuntha m'mitsempha ya chomera kupita kumadera osiyanasiyana, kuchokera ku translaminar (mwachitsanzo, kusuntha kuchokera pamwamba pamasamba kupita kumunsi, monga ma fungicides) 25 kupita ku zotsatira zenizeni., yomwe imatha kulowa mu korona kuchokera ku mizu, imatha kulowa mu timadzi tokoma tamaluwa a apulo26, komwe imatha kupha wamkulu O. cornifrons27.Mankhwala ena amalowanso mungu, zomwe zimakhudza kukula kwa mphutsi za chimanga ndikupangitsa kufa kwawo19.Kafukufuku wina wasonyeza kuti mankhwala ena ophera bowa amatha kusintha kwambiri zisa zamtundu wa O. lignaria28.Kuonjezera apo, kafukufuku wa labotale ndi m'munda wofanana ndi zochitika zokhudzana ndi mankhwala ophera tizilombo (kuphatikizapo mankhwala ophera tizilombo) awonetsa kuti mankhwala ophera tizilombo amakhudza momwe physiology 22 morphology 29 imawonongera moyo ndi kupulumuka kwa njuchi ndi njuchi zina zokhala paokha.Kupopera mankhwala osiyanasiyana opha mphutsi omwe amagwiritsidwa ntchito potsegula maluwa nthawi yamaluwa amatha kuwononga mungu wotengedwa ndi akuluakulu kuti mphutsi ikule, zotsatira zake ziyenera kuwerengedwa30.
Zikudziwika kuti kukula kwa mphutsi kumakhudzidwa ndi midzi ya mungu ndi tizilombo toyambitsa matenda.The honeybee microbiome imakhudza magawo monga thupi31, kusintha kwa kagayidwe kachakudya22 komanso kutengeka ndi tizilombo toyambitsa matenda32.Kafukufuku wam'mbuyo adawona momwe njuchi zimakhudzira chitukuko, zakudya, komanso chilengedwe pazamoyo za njuchi zokhala paokha.Kafukufukuyu adawonetsa kufanana kwa kapangidwe kake ndi kuchuluka kwa ma microbiomes a larval ndi mungu33, komanso mtundu wa bakiteriya wodziwika bwino wa Pseudomonas ndi Delftia, pakati pa mitundu ya njuchi zokhala paokha.Komabe, ngakhale kuti ma fungicides akhala akugwirizana ndi njira zotetezera thanzi la njuchi, zotsatira za fungicides pa larval microbiota kudzera m'kamwa mwachindunji zimakhalabe zosawerengeka.
Kafukufukuyu adayesa zotsatira za Mlingo wapadziko lonse wamankhwala asanu ndi limodzi omwe amagwiritsidwa ntchito kwambiri omwe amalembetsedwa kuti agwiritsidwe ntchito pazipatso zamitengo ku United States, kuphatikiza mankhwala opha tizilombo toyambitsa matenda omwe amaperekedwa pakamwa ku mphutsi za njenjete za chimanga kuchokera ku chakudya choyipitsidwa.Tidapeza kuti kukhudzana ndi ma fungicides amthupi kumachepetsa kunenepa kwa njuchi ndikuwonjezera kufa, ndi zotsatira zoyipa kwambiri zomwe zimagwirizanitsidwa ndi mancozeb ndi pyrithiopide.Kenako tidafanizira mitundu yosiyana siyana ya mphutsi zomwe zimadyetsedwa ndi mungu wopangidwa ndi mancozeb ndi zomwe zimadyetsedwa pazakudya zowongolera.Timakambilana za njira zomwe zingayambitse kufa ndi zotsatirapo za pulogalamu yophatikizika ya tizirombo ndi pollinator (IPPM)36.
Achikulire O. cornifrons overwintering in cocoons anapezedwa kuchokera ku Fruit Research Center, Biglerville, PA, ndipo amasungidwa pa -3 mpaka 2 ° C (±0.3 ° C).Asanayambe kuyesera (600 cocoons okwana).Mu Meyi 2022, zikwa 100 za O. cornifrons zimasamutsidwa tsiku lililonse mu makapu apulasitiki (zikwa 50 pa kapu, DI 5 cm × 15 cm utali) ndipo zopukuta zidayikidwa mkati mwa makapu kulimbikitsa kutsegula ndikupereka gawo lotafuna, kuchepetsa kupsinjika pamwala. njuchi37 .Ikani makapu awiri apulasitiki okhala ndi zikwa mu khola la tizilombo (30 × 30 × 30 cm, BugDorm MegaView Science Co.23°C, chinyezi wachibale 60%, photoperiod 10 l (otsika kwambiri): masiku 14.100 mated akazi ndi amuna anamasulidwa m'mawa uliwonse kwa masiku asanu ndi limodzi (100 patsiku) mu zisa ziwiri yokumba pa pachimake apulo maluwa (msampha chisa: m'lifupi 33.66 × kutalika 30.48 × kutalika 46.99 cm; Supplementary Chithunzi 1).Zoyikidwa ku Pennsylvania State Arboretum, pafupi ndi chitumbuwa (Prunus cerasus 'Eubank' Sweet Cherry Pie™), pichesi (Prunus persica 'Contender'), Prunus persica 'PF 27A' Flamin Fury®), peyala (Pyrus perifolia 'Olympic', Pyrus perifolia 'Shinko', Pyrus perifolia 'Shinseiki'), mtengo wa apulosi wa coronaria (Malus coronaria) ndi mitundu yambiri yamitengo ya maapulo (Malus coronaria, Malus), mtengo wapakhomo 'Co-op 30' Enterprise™, Malus apulo mtengo 'Co- Op 31′ Winecrisp™, begonia 'Freedom', Begonia 'Golden Delicious', Begonia 'Nova Spy').Nyumba iliyonse ya mbalame ya pulasitiki ya buluu imakhala pamwamba pa mabokosi awiri amatabwa.Bokosi lililonse lachisa linali ndi machubu 800 opanda mapepala a kraft (otseguka, 0.8 cm ID × 15 cm L) (Jonesville Paper Tube Co., Michigan) olowetsedwa m'machubu opaque cellophane (0.7 OD onani Mapulagi apulasitiki (mapulagi a T-1X) amapereka malo okhala .
Onse zisa mabokosi anayang'ana kum'mawa ndipo anali yokutidwa ndi wobiriwira pulasitiki mpanda munda (Everbilt chitsanzo #889250EB12, kutsegula kukula 5 × 5 cm, 0.95 m × 100 m) kuteteza makoswe ndi mbalame mwayi ndi kuikidwa pa nthaka pamwamba pafupi chisa bokosi nthaka. mabokosi.Nest box (Chithunzi chowonjezera 1a).Mazira a chimanga ankasonkhanitsidwa tsiku lililonse potola machubu 30 ku zisa ndi kuwatengera ku labotale.Pogwiritsa ntchito lumo, dulani kumapeto kwa chubu, kenako masulani chubu chozungulira kuti muwonetse ma cell a ana.Mazira amodzi ndi mungu wawo adachotsedwa pogwiritsa ntchito spatula yopindika (Microslide tool kit, BioQuip Products Inc., California).Mazirawo adakulungidwa pa pepala lonyowa la fyuluta ndikuyikidwa mu mbale ya Petri kwa maola a 2 asanagwiritsidwe ntchito pazoyesera zathu (Supplementary Figure 1b-d).
Mu labotale, tidayesa kawopsedwe wapakamwa wa mankhwala opha fungicide asanu ndi limodzi omwe adagwiritsidwa ntchito asanatuluke komanso pomwe apulo adaphuka pamiyeso itatu (0.1X, 0.5X, ndi 1X, pomwe 1X ndi chizindikiro chomwe chimayikidwa pa malita 100 amadzi/ekala. m'munda)., Table 1).Kukonzekera kulikonse kunabwerezedwa nthawi 16 (n = 16).Ma fungicides awiri okhudzana (Table S1: mancozeb 2696.14 ppm ndi captan 2875.88 ppm) ndi ma fungicides anayi amtundu uliwonse (Table S1: pyrithiostrobin 250.14 ppm; trifloxystrobin 110.06 ppm; myclobupp5m8 5ppm; 2 azole 5pp5 m) kawopsedwe ku zipatso, ndiwo zamasamba ndi zokongoletsa mbewu .Timasakaniza mungu pogwiritsa ntchito chopukusira, kusuntha 0,20 g ku chitsime (24-chitsime Falcon Plate), ndikuwonjezera ndi kusakaniza 1 μL ya fungicide solution kuti tipange mungu wa pyramidal ndi zitsime zakuya za 1 mm momwe mazirawo anayikidwa.Ikani pogwiritsa ntchito spatula yaying'ono (Chithunzi chowonjezera 1c,d).Mbalame za Falcon zimasungidwa kutentha (25 ° C) ndi 70% chinyezi.Tidawayerekeza ndi mphutsi zowongolera zomwe zimadyetsedwa ndi zakudya zokhala ndi mungu wothira madzi oyera.Tidajambulitsa zakufa ndikuyesa kulemera kwa mphutsi tsiku lililonse mpaka mphutsi zikafika zaka za prepupal pogwiritsa ntchito kusanthula (Fisher Scientific, accuracy = 0.0001 g).Pomaliza, chiŵerengero cha kugonana chinayesedwa potsegula chikwa pambuyo pa miyezi 2.5.
DNA inatengedwa ku mphutsi zonse za O. cornifrons (n = 3 pa chikhalidwe chamankhwala, mungu wothiridwa ndi mancozeb ndi wosachiritsidwa) ndipo tinasanthula mitundu yosiyanasiyana ya tizilombo pazitsanzozi, makamaka chifukwa mu mancozeb kufa kwakukulu kunawonedwa mu mphutsi.kulandira MnZn.DNA idakulitsidwa, kuyeretsedwa pogwiritsa ntchito DNAZymoBIOMICS®-96 MagBead DNA kit (Zymo Research, Irvine, CA), ndikutsatiridwa (mizungu 600) pa Illumina® MiSeq™ pogwiritsa ntchito zida za v3.Kutsata kotsatana kwa mabakiteriya a 16S ribosomal RNA majini kunachitika pogwiritsa ntchito Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA) pogwiritsa ntchito zoyambira zomwe zimayang'ana dera la V3-V4 la jini ya 16S rRNA.Kuphatikiza apo, kutsatizana kwa 18S kunachitika pogwiritsa ntchito 10% kuphatikizidwa kwa PhiX, ndipo kukulitsa kunachitika pogwiritsa ntchito primer 18S001 ndi NS4.
Lowetsani ndikuphatikiza zowerengera39 pogwiritsa ntchito payipi ya QIIME2 (v2022.11.1).Zowerengera izi zidakonzedwa ndikuphatikizidwa, ndipo zotsatizana za chimeric zidachotsedwa pogwiritsa ntchito pulogalamu yowonjezera ya DADA2 mu QIIME2 (qiime dada2 noise pairing)40.Ntchito zamakalasi a 16S ndi 18S zidachitidwa pogwiritsa ntchito pulagi ya classifier Classify-sklearn ndi silva-138-99-nb-classifier yophunzitsidwa kale.
Deta yonse yoyesera idayang'aniridwa kuti ikhale yachibadwa (Shapiro-Wilks) ndi homogeneity of variances (mayeso a Levene).Chifukwa deta sichinagwirizane ndi malingaliro a kusanthula kwa parametric ndipo kusinthaku sikunakwaniritsire zotsalirazo, tinachita ANOVA (Kruskal-Wallis) yopanda njira ziwiri ndi zinthu ziwiri [nthawi (atatu-gawo 2, 5, ndi tsiku la 8). nthawi) ndi fungicide] kuti awone momwe chithandizo chimakhudzira kulemera kwa mphutsi, kenako kufananitsa kwapawiri kwapawiri kunachitika pogwiritsa ntchito mayeso a Wilcoxon.Tinagwiritsa ntchito chitsanzo chodziwika bwino (GLM) ndi kugawa kwa Poisson kuti tifanizire zotsatira za fungicides pa kupulumuka pamagulu atatu a fungicide41,42.Pakuwunika kwachulukidwe kosiyanasiyana, kuchuluka kwa ma amplicon sequence variants (ASVs) kudagwa pamlingo wamtundu.Kuyerekeza kwa kuchuluka kwa kusiyana pakati pa magulu omwe amagwiritsa ntchito 16S (genus level) ndi 18S kuchuluka kwachibale kunachitika pogwiritsa ntchito njira yowonjezera yowonjezera ya udindo, sikelo, ndi mawonekedwe (GAMLSS) ndi magawo a mabanja a beta zero-inflated (BEZI), omwe adapangidwa molingana ndi macro. .mu Microbiome R43 (v1.1).1).Chotsani mitundu ya mitochondrial ndi chloroplast musanawunike mosiyanasiyana.Chifukwa cha milingo yosiyanasiyana ya taxonomic ya 18S, gawo lotsika kwambiri la taxon iliyonse ndilomwe limagwiritsidwa ntchito posanthula mosiyanasiyana.Kusanthula konse kwa ziwerengero kunachitika pogwiritsa ntchito R (v. 3.4.3., CRAN project) (Timu 2013).
Kuwonetsedwa kwa mancozeb, pyrithiostrobin, ndi trifloxystrobin kunachepetsa kwambiri kulemera kwa thupi mu O. cornifrons (Mkuyu 1).Zotsatirazi zinkawonedwa nthawi zonse pamiyeso yonse itatu yomwe inayesedwa (mkuyu 1a-c).Cyclostrobin ndi myclobutanil sizinachepetse kwambiri kulemera kwa mphutsi.
Avereji yolemera mwatsopano ya mphutsi za stem borer zoyezedwa katatu pansi pa zakudya zinayi (homogeneous mungu chakudya + fungicide: control, 0.1X, 0.5X ndi 1X mlingo).(a) Mlingo wochepa (0.1X): nthawi yoyamba (tsiku 1): χ2: 30.99, DF = 6;P <0.0001, mfundo yachiwiri (tsiku 5): 22.83, DF = 0.0009;kachitatu;mfundo (tsiku 8): χ2: 28.39, DF = 6;(b) theka la mlingo (0.5X): nthawi yoyamba (tsiku 1): χ2: 35.67, DF = 6;P <0.0001, nthawi yachiwiri mfundo (tsiku loyamba).): χ2: 15.98, DF = 6;P = 0.0090;nthawi yachitatu (tsiku 8) χ2: 16.47, DF = 6;(c) Malo kapena mlingo wathunthu (1X): nthawi yoyamba (tsiku 1) χ2: 20.64, P = 6;P = 0.0326, mfundo yachiwiri (tsiku 5): χ2: 22.83, DF = 6;P = 0.0009;nthawi yachitatu (tsiku 8): χ2: 28.39, DF = 6;nonparametric kusanthula kwa kusiyana.Mipiringidzo imayimira ± SE ya kufananitsa awiriwiri (α = 0.05) (n = 16) *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.
Pa mlingo wotsika kwambiri (0.1X), kulemera kwa mphutsi kunachepetsedwa ndi 60% ndi trifloxystrobin, 49% ndi mancozeb, 48% ndi myclobutanil, ndi 46% ndi pyrithistrobin (mkuyu 1a).Atakumana ndi theka la mlingo wa kumunda (0.5X), kulemera kwa thupi la mphutsi za mancozeb kunachepetsedwa ndi 86%, pyrithiostrobin ndi 52% ndi trifloxystrobin ndi 50% (mkuyu. 1b).Mlingo wathunthu (1X) wa mancozeb umachepetsa kulemera kwa mphutsi ndi 82%, pyrithiostrobin ndi 70%, ndi trifloxystrobin, myclobutanil ndi sangard pafupifupi 30% (mkuyu 1c).
Kufa kunali kwakukulu pakati pa mphutsi zodyetsedwa ndi mungu wothira mancozeb, kutsatiridwa ndi pyrithiostrobin ndi trifloxystrobin.Kufa kumawonjezeka ndi kuchuluka kwa mancozeb ndi pyritisoline (mkuyu 2; Gulu 2).Komabe, kufa kwa mbewu za chimanga kunangowonjezereka pang'ono pamene kuchuluka kwa trifloxystrobin kumawonjezeka;cyprodinil ndi captan sizinawonjezere kwambiri kufa poyerekeza ndi mankhwala owongolera.
Kufa kwa mphutsi za ntchentche kunayerekezedwa pambuyo pa kumeza mungu payekhapayekha wothandizidwa ndi fungicides sikisi.Mancozeb ndi pentopyramide anali okhudzidwa kwambiri ndi mphutsi zamkamwa (GLM: χ = 29.45, DF = 20, P = 0.0059) (mzere, otsetsereka = 0.29, P <0.001; otsetsereka = 0.24, P) <0.0.
Pafupifupi, pamankhwala onse, 39.05% ya odwala anali akazi ndipo 60.95% anali amuna.Pakati pamankhwala owongolera, gawo la amayi linali 40% m'maphunziro a mlingo wochepa (0.1X) ndi theka la mlingo (0.5X), ndi 30% m'maphunziro a mlingo wa kumunda (1X).Pa mlingo wa 0.1X, pakati pa mphutsi zodyetsedwa ndi mungu zomwe zimathandizidwa ndi mancozeb ndi myclobutanil, 33.33% ya akuluakulu anali akazi, 22% akuluakulu anali akazi, 44% a mphutsi zazikulu zinali zazikazi, 44% za mphutsi zazikulu zinali zazikazi.chachikazi, 41% ya mphutsi zazikulu zinali zazikazi, ndipo zowongolera zinali 31% (mkuyu 3a).Pa nthawi ya 0,5 mlingo, 33% ya nyongolotsi zazikulu mu gulu la mancozeb ndi pyrithiostrobin zinali zazikazi, 36% mu gulu la trifloxystrobin, 41% mu gulu la myclobutanil, ndi 46% mu gulu la cyprostrobin.Chiwerengerochi chinali 53% pagulu.mu gulu la captan ndi 38% mu gulu lolamulira (mkuyu 3b).Pa mlingo wa 1X, 30% ya gulu la mancozeb linali akazi, 36% ya gulu la pyrithiostrobin, 44% ya gulu la trifloxystrobin, 38% ya gulu la myclobutanil, 50% ya gulu lolamulira linali akazi - 38.5% (mkuyu 3c) .
Peresenti ya mbozi zazikazi ndi zazimuna pambuyo pa kukhudzana ndi fungicide.(a) Mlingo wochepa (0.1X).(b) Mlingo wa theka (0.5X).(c) Mlingo wamunda kapena mlingo wathunthu (1X).
Kusanthula kwa 16S kunawonetsa kuti gulu la bakiteriya limasiyana pakati pa mphutsi zodyetsedwa ndi mungu wopangidwa ndi mancozeb ndi mphutsi zomwe zimadyetsedwa ndi mungu wosachiritsidwa (mkuyu 4a).Mlozera wa tizilombo tating'onoting'ono ta mphutsi zosagwiritsidwa ntchito zodyetsedwa ndi mungu unali wapamwamba kuposa wa mphutsi zomwe zimadyetsedwa ndi mungu wopangidwa ndi mancozeb (mkuyu 4b).Ngakhale kuti kusiyana komwe kunawonedwa mu chuma pakati pa magulu sikunali kofunikira kwambiri, kunali kochepa kwambiri kusiyana ndi komwe mphutsi zimadya pa mungu wosachiritsidwa (mkuyu 4c).Kuchuluka kwa mphutsi kunawonetsa kuti tizilombo toyambitsa matenda timene timadyetsedwa ndi mungu wowongolera zinali zosiyana kwambiri ndi mphutsi zomwe zimadyetsedwa ndi mphutsi zothandizidwa ndi mancozeb (mkuyu 5a).Kusanthula kofotokozera kunawonetsa kukhalapo kwa 28 genera mu ulamuliro ndi zitsanzo za mancozeb (mkuyu 5b).c Kusanthula pogwiritsa ntchito kutsatizana kwa 18S sikunawonetse kusiyana kwakukulu (Chithunzi chowonjezera 2).
Mbiri ya SAV yotengera kutsata kwa 16S idayerekezedwa ndi kulemera kwa Shannon ndikuwona kulemera pamlingo wa phylum.(a) Principal coordinate analysis (PCoA) yotengera ma microbial community structure mu mungu wosadyetsedwa kapena control (buluu) ndi mphutsi zodyetsedwa ndi mancozeb (lalanje).Deta iliyonse imayimira chitsanzo chosiyana.PCoA idawerengedwa pogwiritsa ntchito mtunda wa Bray-Curtis wa kugawa kwa multivariate t.Ovals amaimira 80% yodalirika.(b) Boxplot, data yazachuma ya Shannon (mfundo) ndi c.Chuma chowoneka.Mabokosi amawonetsa mabokosi a mzere wapakati, interquartile range (IQR), ndi 1.5 × IQR (n = 3).
Mapangidwe a tizilombo toyambitsa matenda a mphutsi zodyetsedwa ndi mungu wothiridwa ndi mancozeb komanso wosachiritsidwa.(a) Kuchuluka kwamtundu wa tizilombo tating'onoting'ono timawerengedwa mu mphutsi.(b) Mapu otentha a magulu odziwika a tizilombo.Delftia (chiŵerengero chazovuta (OR) = 0.67, P = 0.0030) ndi Pseudomonas (OR = 0.3, P = 0.0074), Microbacterium (OR = 0.75, P = 0.0617) (OR = 1.5, P = 0.0060);Mizere yamapu otentha amalumikizidwa pogwiritsa ntchito mtunda wolumikizana komanso kulumikizana kwapakati.
Zotsatira zathu zikuwonetsa kuti kukhudzana kwapakamwa ndi mancozeb (mancozeb) ndi systemic (pyrostrobin ndi trifloxystrobin) fungicides, omwe amagwiritsidwa ntchito kwambiri panthawi yamaluwa, amachepetsa kwambiri kuwonda komanso kufa kwa mphutsi za chimanga.Kuphatikiza apo, mancozeb adachepetsa kwambiri kusiyanasiyana komanso kuchuluka kwa ma microbiome panthawi ya prepupal.Myclobutanil, mankhwala ena opha tizilombo toyambitsa matenda, adachepetsa kwambiri kulemera kwa mphutsi pamilingo yonse itatu.Zotsatirazi zidawonekera pa nthawi yachiwiri (tsiku la 5) ndi lachitatu (tsiku la 8).Mosiyana ndi zimenezi, cyprodinil ndi captan sizinachepetse kwambiri kulemera kapena kupulumuka poyerekeza ndi gulu lolamulira.Kudziwa kwathu, ntchitoyi ndi yoyamba kudziwa zotsatira za mitengo yamitundu yosiyanasiyana ya fungicide yomwe imagwiritsidwa ntchito poteteza mbewu za chimanga kudzera muvuto lachindunji.
Mankhwala onse a fungicide amachepetsa kwambiri kulemera kwa thupi poyerekeza ndi kuwongolera.Mancozeb idakhudza kwambiri kulemera kwa mphutsi ndikuchepetsa pafupifupi 51%, kutsatiridwa ndi pyrithiostrobin.Komabe, kafukufuku wina sananenepo zoyipa zomwe zimachitika m'munda wamankhwala a fungicides pamagawo amphutsi44.Ngakhale ma biocides a dithiocarbamate awonetsedwa kuti ali ndi chiwopsezo chochepa kwambiri45, ethylene bisdithiocarbamates (EBDCS) monga mancozeb amatha kutsika kukhala urea ethylene sulfide.Chifukwa cha zotsatira zake za mutagenic mu nyama zina, chinthu chowonongekachi chikhoza kukhala ndi zotsatira zowoneka46,47.Kafukufuku wam'mbuyomu awonetsa kuti mapangidwe a ethylene thiourea amakhudzidwa ndi zinthu monga kutentha kwakukulu48, milingo ya chinyezi49 ndi kutalika kwa kusungirako zinthu50.Malo oyenera osungiramo ma biocides amatha kuchepetsa zovuta izi.Kuphatikiza apo, European Food Safety Authority yawonetsa kukhudzidwa ndi kawopsedwe ka pyrithiopide, komwe kwawonetsedwa kuti ndi carcinogenic ku machitidwe am'mimba a nyama zina51.
Kugwiritsa ntchito pakamwa mancozeb, pyrithiostrobin, ndi trifloxystrobin kumawonjezera kufa kwa mphutsi za chimanga.Mosiyana ndi zimenezi, myclobutanil, ciprocycline ndi captan sizinakhudze imfa.Zotsatirazi zimasiyana ndi za Ladurner et al.52, omwe adawonetsa kuti captan adachepetsa kwambiri kupulumuka kwa wamkulu O. lignaria ndi Apis mellifera L. (Hymenoptera, Apisidae).Kuphatikiza apo, mankhwala opha fungicides monga captan ndi boscalid apezeka kuti amayambitsa kufa kwa mphutsi52,53,54 kapena kusintha khalidwe la kudyetsa55.Zosinthazi, zimatha kukhudza thanzi la mungu ndipo pamapeto pake kupindula kwamphamvu kwa siteji ya mphutsi.Kufa komwe kunawonedwa mu gulu lolamulira kunali kogwirizana ndi maphunziro ena 56,57.
Chiŵerengero cha kugonana kokomera amuna chomwe chawonedwa mu ntchito yathu chikhoza kufotokozedwa ndi zinthu monga kusakwanira kwa makwerero ndi nyengo yoipa pa nthawi ya maluwa, monga momwe adanenera kale O. cornuta ndi Vicens ndi Bosch.Ngakhale kuti akazi ndi amuna mu phunziro lathu anali ndi masiku anayi okwatirana (nthawi yomwe nthawi zambiri imatengedwa kuti ndi yokwanira kuti tikweretse bwino), tinachepetsa dala kuwala kuti tichepetse nkhawa.Komabe, kusinthidwaku kukhoza kusokoneza mwadala njira yokweretsa61.Kuphatikiza apo, njuchi zimakumana ndi nyengo yoyipa kwa masiku angapo, kuphatikiza mvula komanso kutentha pang'ono (<5°C), zomwe zingasokonezenso kupambana kwa makwerero4,23.
Ngakhale kuti phunziro lathu linayang'ana pa microbiome yonse ya larval, zotsatira zathu zimapereka chidziwitso pa maubwenzi omwe angakhalepo pakati pa magulu a mabakiteriya omwe angakhale ovuta kwambiri pa zakudya za njuchi ndi kuwonetsa fungicide.Mwachitsanzo, mphutsi zomwe zimadyetsedwa ndi mungu wa mancozeb zidachepetsa kwambiri mapangidwe a tizilombo toyambitsa matenda komanso kuchuluka kwake poyerekeza ndi mphutsi zomwe zimadyetsedwa mungu wosachiritsidwa.Mphutsi zomwe zimadya mungu wosachiritsidwa, magulu a mabakiteriya a Proteobacteria ndi Actinobacteria anali otchuka ndipo makamaka anali aerobic kapena facultatively aerobic.Mabakiteriya a Delft, omwe nthawi zambiri amagwirizanitsidwa ndi mitundu ya njuchi zodziyimira pawokha, amadziwika kuti ali ndi zochita zowononga maantibayotiki, zomwe zikuwonetsa momwe angatetezere tizilombo toyambitsa matenda.Mtundu wina wa bakiteriya, Pseudomonas, unali wochuluka mu mphutsi zomwe zimadyetsedwa ndi mungu wosachiritsidwa, koma zinachepetsedwa kwambiri mu mphutsi zothandizidwa ndi mancozeb.Zotsatira zathu zimathandizira maphunziro am'mbuyomu ozindikiritsa Pseudomonas ngati amodzi mwamagulu ochulukirapo mu O. bicornis35 ndi mavu ena okha34.Ngakhale kuti umboni woyesera wa ntchito ya Pseudomonas pa thanzi la O. cornifrons sunaphunzirepo, bakiteriya iyi yasonyezedwa kuti imalimbikitsa kaphatikizidwe ka poizoni wotetezera mu kachilomboka ka kachilomboka Paederus fuscipes ndikulimbikitsa kagayidwe ka arginine mu vitro 35, 65. ntchito yomwe ingatheke pachitetezo cha ma virus ndi mabakiteriya panthawi yakukula kwa mphutsi za O. cornifrons.Microbacterium ndi mtundu wina womwe wadziwika mu kafukufuku wathu womwe umadziwika kuti umapezeka mu mphutsi zankhondo zakuda pansi pa njala66.Mu mphutsi za O. cornifrons, tizilombo toyambitsa matenda tizilombo toyambitsa matenda tingathandize kuti matumbo a microbiome asamawonongeke komanso kuti azikhala olimba.Kuonjezera apo, Rhodococcus imapezeka mu mphutsi za O. cornifrons ndipo imadziwika ndi mphamvu zake zochotsa poizoni67.Mtundu uwu umapezekanso m'matumbo a A. florea, koma mochepa kwambiri68.Zotsatira zathu zikuwonetsa kukhalapo kwa mitundu yosiyanasiyana ya ma genetic pamitundu ingapo ya ma microbial taxa yomwe imatha kusintha kagayidwe kachakudya mu mphutsi.Komabe, kumvetsetsa bwino kwa magwiridwe antchito a O. cornifron ndikofunikira.
Mwachidule, zotsatira zikuwonetsa kuti mancozeb, pyrithiostrobin, ndi trifloxystrobin amachepetsa kulemera kwa thupi komanso kufa kwa mphutsi za chimanga.Ngakhale pali nkhawa yomwe ikukula chifukwa cha zotsatira za fungicides pa pollinators, pakufunika kumvetsetsa bwino zotsatira za metabolites otsalira a mankhwalawa.Zotsatirazi zitha kuphatikizidwa m'malingaliro a mapulogalamu ophatikizika a kasamalidwe ka mungu wothandiza alimi kupewa kugwiritsa ntchito mankhwala ena ophera bowa mitengo yazipatso isanayambe kutulutsa maluwa posankha mankhwala opha mafangasi ndi kusinthasintha nthawi yogwiritsidwa ntchito, kapena kulimbikitsa kugwiritsa ntchito njira zina zosavulaza 36. Izi ndizofunikira pakukhazikitsa malingaliro.pakugwiritsa ntchito mankhwala ophera tizilombo, monga kusintha mapulogalamu omwe alipo komanso kusintha nthawi yopopera posankha mankhwala ophera tizilombo kapena kulimbikitsa kugwiritsa ntchito njira zina zosaopsa kwambiri.Kafukufuku wowonjezereka akufunika pazovuta za mankhwala ophera fungal pa kuchuluka kwa kugonana, kadyedwe kake, matumbo a microbiome, ndi njira zama cell zomwe zimayambitsa kuchepa kwa thupi ndi kufa kwa borer.
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Nthawi yotumiza: May-14-2024