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Hoʻohana pinepine ʻia nā fungicides i ka wā o ka pua ʻana o nā hua lāʻau a hiki ke hoʻoweliweli i nā mea hoʻopulapula pepeke. Eia nō naʻe, ʻaʻole i ʻike nui ʻia e pili ana i ke ʻano o ka pane ʻana o nā mea hoʻopulapula ʻaʻole pi (e laʻa, nā pi hoʻokahi, ʻo Osmia cornifrons) i nā fungicides pili a me nā systemic i hoʻohana pinepine ʻia ma nā ʻāpala i ka wā o ka pua ʻana. Hoʻopaʻa kēia ʻokoʻa ʻike i nā hoʻoholo hoʻoponopono e hoʻoholo ana i nā ʻano palekana a me ka manawa o ka pīpī ʻana i ka fungicide. Ua loiloi mākou i nā hopena o ʻelua mau fungicides pili (captan a me mancozeb) a me ʻehā mau fungicides interlayer/phytosystem (ciprocycline, myclobutanil, pyrostrobin a me trifloxystrobin). Nā hopena ma ka loaʻa ʻana o ke kaumaha larval, ke ola ʻana, ka lakio wahine a me ka ʻokoʻa bacteria. Ua hana ʻia ka loiloi me ka hoʻohana ʻana i kahi bioassay waha mau loa kahi i mālama ʻia ai ka pollen i ʻekolu mau mahele e pili ana i ka mahele i ʻōlelo ʻia no ka hoʻohana ʻana i ke kahua (1X), ka hapalua mahele (0.5X) a me ka mahele haʻahaʻa (0.1X). ʻO nā mahele āpau o mancozeb a me pyritisoline ua hōʻemi nui i ke kaumaha o ke kino a me ke ola larval. A laila ua hoʻonohonoho mākou i ka gene 16S e wehewehe i ka bacteriome larval o mancozeb, ka fungicide kuleana no ka make kiʻekiʻe loa. Ua ʻike mākou ua emi nui ka ʻokoʻa o ka bacteria a me ka nui o nā larvae i hānai ʻia i ka pollen i mālama ʻia me ka mancozeb. Hōʻike nā hopena o kā mākou keʻena hoʻokolohua he mea ʻino loa ka pīpī ʻana i kekahi o kēia mau fungicides i ka wā pua i ke olakino o O. cornifrons. He mea pili kēia ʻike no nā hoʻoholo hoʻokele e hiki mai ana e pili ana i ka hoʻohana mau ʻana i nā huahana pale lāʻau hua a lawelawe ia ma ke ʻano he kumu no nā kaʻina hana hoʻoponopono e pili ana i ka pale ʻana i nā pollinators.
Ua hoʻolauna ʻia ka pi mason hoʻokahi ʻo Osmia cornifrons (Hymenoptera: Megachilidae) i ʻAmelika Hui Pū ʻIa mai Iapana mai i ka hopena o nā makahiki 1970 a me ka hoʻomaka ʻana o nā makahiki 1980, a ua hoʻokani ka ʻano i kahi kuleana pollinator koʻikoʻi i nā kaiaolaola i mālama ʻia mai ia manawa. ʻO nā heluna kanaka kūlohelohe o kēia pi he ʻāpana o kahi 50 mau ʻano pi hihiu e hoʻokō ana i nā pi e pollinate i nā māla ʻalemona a me nā ʻāpala ma ʻAmelika Hui Pū ʻIa2,3. Ke kū nei nā pi Mason i nā pilikia he nui, me ka fragmentation habitat, pathogens, a me nā pesticides3,4. Ma waena o nā insecticides, hoʻemi nā fungicides i ka loaʻa ʻana o ka ikehu, ka ʻai ʻana5 a me ke kūlana kino6,7. ʻOiai ke hōʻike nei nā noiʻi hou ua hoʻopili pololei ʻia ke olakino o nā pi Mason e nā microorganisms commensal a me ectobactic, 8,9 no ka mea hiki i ka bacteria a me nā fungi ke hoʻopili i ka meaʻai a me nā pane pale, ke hoʻomaka wale nei e aʻo ʻia nā hopena o ka hōʻike ʻana o ka fungicide i ka ʻokoʻa microbial o nā pi Mason.
Hoʻopili ʻia nā fungicides o nā hopena like ʻole (hoʻopili a me ka ʻōnaehana) i loko o nā māla huaʻai ma mua a i ka wā o ka pua ʻana e mālama i nā maʻi e like me ka ʻōpala ʻāpala, ka pala ʻawaʻawa, ka pala ʻeleʻele a me ka pauka mildew10,11. Manaʻo ʻia nā fungicides he mea ʻino ʻole i nā pollinators, no laila ua ʻōlelo ʻia lākou i nā māla i ka wā pua; ʻIke nui ʻia ka hōʻike ʻana a me ka ʻai ʻana i kēia mau fungicides e nā pi, ʻoiai he ʻāpana ia o ke kaʻina hana hoʻopaʻa inoa pesticide e ka US Environmental Protection Agency a me nā keʻena hoʻoponopono aupuni ʻē aʻe he nui12,13,14. Eia nō naʻe, ʻaʻole ʻike nui ʻia nā hopena o nā fungicides ma nā mea ʻaʻole pi no ka mea ʻaʻole koi ʻia ma lalo o nā ʻaelike ʻae kūʻai aku ma ʻAmelika Hui Pū ʻIa15. Eia kekahi, ʻaʻohe mau protocol maʻamau no ka hoʻāʻo ʻana i nā pi hoʻokahi16,17, a ʻo ka mālama ʻana i nā kolone e hāʻawi ana i nā pi no ka hoʻāʻo ʻana he mea paʻakikī18. Ke hana nui ʻia nei nā hoʻokolohua o nā pi i mālama ʻia ma ʻEulopa a me USA e aʻo i nā hopena o nā pesticides ma nā pi hihiu, a ua hoʻomohala ʻia nā protocol maʻamau no O. cornifrons19.
He mau monocytes nā pi pepeiaohao a hoʻohana ʻia i ke kālepa ʻana i nā mea kanu carp ma ke ʻano he mea hoʻohui a pani paha no nā pi meli. Puka mai kēia mau pi ma waena o Malaki a me ʻApelila, me nā kāne precocious e puka mai ana i ʻekolu a ʻehā mau lā ma mua o nā wahine. Ma hope o ka male ʻana, hōʻiliʻili ikaika ka wahine i ka pollen a me ka nectar e hāʻawi i kahi moʻo o nā cell brood i loko o ka lua pūnana tubular (kūlohelohe a hana ʻia paha)1,20. Hoʻomoe ʻia nā hua ma luna o ka pollen i loko o nā cell; a laila kūkulu ka wahine i kahi pā lepo ma mua o ka hoʻomākaukau ʻana i ka cell aʻe. Hoʻopuni ʻia nā larvae instar mua i loko o ka chorion a hānai i nā wai embryonic. Mai ka lua a hiki i ka instar ʻelima (prepupa), hānai nā larvae i ka pollen22. Ke pau loa ka lako pollen, hana nā larvae i nā cocoons, pupate a puka mai ma ke ʻano he mau mākua i loko o ka lumi brood like, maʻamau i ka hopena o ke kauwela20,23. Puka mai nā mākua i ka pūnāwai aʻe. Pili ke ola ʻana o ka pākeke me ka loaʻa ʻana o ka ikehu net (loaʻa ke kaumaha) ma muli o ka ʻai ʻana. No laila, ʻo ke ʻano o ka meaʻai o ka pollen, a me nā mea ʻē aʻe e like me ke aniau a i ʻole ka ʻike ʻana i nā pesticides, nā mea hoʻoholo o ke ola a me ke olakino24.
Hiki i nā insecticides a me nā fungicides i hoʻopili ʻia ma mua o ka pua ʻana ke neʻe i loko o ke kahe koko o ka mea kanu i nā kekelē like ʻole, mai ka translaminar (e laʻa, hiki ke neʻe mai ka ʻili o luna o nā lau a i ka ʻili o lalo, e like me kekahi mau fungicides) 25 a hiki i nā hopena ʻōnaehana maoli. , hiki ke komo i ka lei aliʻi mai nā aʻa, hiki ke komo i ka nectar o nā pua ʻāpala 26, kahi e hiki ai iā lākou ke pepehi i ka O. cornifrons makua 27. Hoʻopili pū kekahi mau pesticides i loko o ka pollen, e hoʻopilikia ana i ka ulu ʻana o nā larvae maile a make lākou 19. Ua hōʻike ʻia nā haʻawina ʻē aʻe e hiki i kekahi mau fungicides ke hoʻololi nui i ke ʻano o ka pūnana o nā ʻano pili ʻo O. lignaria 28. Eia kekahi, ua hōʻike nā haʻawina hale hana a me nā kahua e hoʻohālike ana i nā hiʻohiʻona hōʻike pesticide (me nā fungicides) he hopena maikaʻi ʻole nā pesticides i ka physiology 22 morphology 29 a me ke ola ʻana o nā pi meli a me kekahi mau pi hoʻokahi. ʻO nā ʻano fungicidal sprays i hoʻopili pololei ʻia i nā pua hāmama i ka wā pua e hoʻohaumia i ka pollen i hōʻiliʻili ʻia e nā pākeke no ka ulu ʻana o ka larval, nā hopena o ia mea e pono e aʻo ʻia 30.
Ua ʻike nui ʻia ua hoʻopili ʻia ka ulu ʻana o ka larva e nā kaiāulu pollen a me nā microbial o ka ʻōnaehana hoʻoheheʻe. Hoʻopili ka microbiome o ka meli i nā palena e like me ke kaumaha o ke kino31, nā loli metabolic22 a me ka hiki ke loaʻa i nā pathogens32. Ua nānā nā haʻawina mua i ka mana o ke kahua hoʻomohala, nā meaʻai, a me ke kaiapuni ma ka microbiome o nā pi hoʻokahi. Ua hōʻike kēia mau haʻawina i nā ʻano like i ke ʻano a me ka nui o nā microbiomes larval a me pollen33, a me nā genera bacteria maʻamau ʻo Pseudomonas a me Delftia, i waena o nā ʻano pi hoʻokahi. Eia nō naʻe, ʻoiai ua pili nā fungicides me nā hoʻolālā e pale ai i ke olakino o ka pi, ʻaʻole i ʻimi ʻia nā hopena o nā fungicides ma ka microbiota larval ma o ka hōʻike pololei ʻana i ka waha.
Ua hoʻāʻo kēia haʻawina i nā hopena o nā mahele maoli o ʻeono mau fungicides i hoʻohana pinepine ʻia i hoʻopaʻa inoa ʻia no ka hoʻohana ʻana ma nā hua lāʻau ma ʻAmelika Hui Pū ʻIa, me nā fungicides pili a me nā systemic i hāʻawi waha ʻia i nā larvae moth hornworm kulina mai ka meaʻai haumia. Ua ʻike mākou ua hōʻemi nā fungicides pili a me nā systemic i ka pi i ka loaʻa ʻana o ke kaumaha o ke kino a me ka hoʻonui ʻana i ka make, me nā hopena koʻikoʻi loa e pili ana me ka mancozeb a me ka pyrithiopide. A laila ua hoʻohālikelike mākou i ka ʻokoʻa microbial o nā larvae i hānai ʻia ma ka ʻai pollen i mālama ʻia me ka mancozeb me nā mea i hānai ʻia ma ka ʻai kaohi. Kūkākūkā mākou i nā ʻano hana hiki ke hoʻokumu i ka make a me nā hopena no nā polokalamu hoʻokele pest a me pollinator i hoʻohui ʻia (IPPM)36.
Ua loaʻa nā O. cornifrons makua e noho ana i loko o nā cocoons mai ke Fruit Research Center, Biglerville, PA, a mālama ʻia ma −3 a 2°C (± 0.3°C). Ma mua o ka hoʻokolohua (600 cocoons i ka huina). I Mei 2022, ua hoʻoili ʻia he 100 mau O. cornifrons cocoons i kēlā me kēia lā i loko o nā kīʻaha plastik (50 cocoons no ke kīʻaha, DI 5 cm × 15 cm ka lōʻihi) a ua kau ʻia nā kāwele holoi i loko o nā kīʻaha e hoʻolaha i ka wehe ʻana a hāʻawi i kahi substrate nau, e hōʻemi ana i ke kaumaha ma luna o nā pi pōhaku37. E kau i ʻelua kīʻaha plastik me nā cocoons i loko o kahi pā pepeke (30 × 30 × 30 cm, BugDorm MegaView Science Co. Ltd., Taiwan) me nā mea hānai 10 ml me ka 50% sucrose solution a mālama no ʻehā lā e hōʻoia i ka pani ʻana a me ka male ʻana. 23°C, ka haʻahaʻa pili 60%, photoperiod 10 l (haʻahaʻa haʻahaʻa): 14 lā. Ua hoʻokuʻu ʻia he 100 mau wahine a me nā kāne i hoʻopili ʻia i kēlā me kēia kakahiaka no nā lā ʻeono (100 i kēlā me kēia lā) i loko o ʻelua mau pūnana hana i ka wā o ka pua ʻāpala kiʻekiʻe (pūnana pahele: laulā 33.66 × kiʻekiʻe 30.48 × lōʻihi 46.99 cm; Kiʻi Hoʻohui 1). Ua waiho ʻia ma ka Pennsylvania State Arboretum, kokoke i ka cherry (Prunus cerasus 'Eubank' Sweet Cherry Pie™), peach (Prunus persica 'Contender'), Prunus persica 'PF 27A' Flamin Fury®), pea (Pyrus perifolia 'Olympic', Pyrus perifolia 'Shinko', Pyrus perifolia 'Shinseiki'), lāʻau ʻāpala coronaria (Malus coronaria) a me nā ʻano lāʻau ʻāpala he nui (Malus coronaria, Malus), lāʻau ʻāpala kūloko 'Co-op 30′ Enterprise™, lāʻau ʻāpala Malus 'Co-Op 31′ Winecrisp™, begonia 'Freedom', Begonia 'Golden Delicious', Begonia 'Nova Spy'). Hoʻokomo ʻia kēlā me kēia hale manu plastik polū ma luna o ʻelua pahu lāʻau. Aia i loko o kēlā me kēia pahu pūnana he 800 mau ʻōmole pepa kraft hakahaka (ʻōwili hāmama, 0.8 cm ID × 15 cm L) (Jonesville Paper Tube Co., Michigan) i hoʻokomo ʻia i loko o nā ʻōmole cellophane opaque (0.7 OD e nānā i nā plug plastik (T-1X plugs) e hāʻawi i nā wahi pūnana.
Ua kū pono nā pahu pūnana ʻelua i ka hikina a ua uhi ʻia me ka pā māla plastik ʻōmaʻomaʻo (Everbilt model #889250EB12, nui o ka puka 5 × 5 cm, 0.95 m × 100 m) i mea e pale ai i ke komo ʻana o nā ʻiole a me nā manu a kau ʻia ma luna o ka ʻili lepo ma ka ʻaoʻao o nā pahu lepo pahu pūnana. Pahu pūnana (Kiʻi Hoʻohui 1a). Ua hōʻiliʻili ʻia nā hua manu kulina i kēlā me kēia lā ma ka hōʻiliʻili ʻana i 30 mau ʻōmole mai nā pūnana a lawe iā lākou i ka hale hana. Me ka hoʻohana ʻana i nā ʻokiʻoki, e hana i kahi ʻoki ma ka hopena o ka ʻōmole, a laila e wehe i ka ʻōmole wili e hōʻike i nā ʻōpū hoʻohua. Ua wehe ʻia nā hua manu pākahi a me kā lākou pollen me ka hoʻohana ʻana i kahi spatula kūlou (Microslide tool kit, BioQuip Products Inc., Kaleponi). Ua hoʻoulu ʻia nā hua manu ma ka pepa kānana pulu a waiho ʻia i loko o kahi kīʻaha Petri no 2 mau hola ma mua o ka hoʻohana ʻia ʻana i kā mākou mau hoʻokolohua (Kiʻi Hoʻohui 1b-d).
Ma ke keʻena hoʻokolohua, ua loiloi mākou i ka ʻawahia waha o ʻeono mau fungicides i hoʻopili ʻia ma mua a i ka wā o ka pua ʻāpala ma ʻekolu mau ʻano (0.1X, 0.5X, a me 1X, kahi ʻo 1X ka māka i hoʻopili ʻia no 100 galani o ka wai/ʻeka. ʻO ka nui o ke kahua kiʻekiʻe = ka nui ma ke kula). , Papa 1). Ua hana hou ʻia kēlā me kēia ʻano i 16 mau manawa (n = 16). ʻElua mau fungicides pili (Papa S1: mancozeb 2696.14 ppm a me captan 2875.88 ppm) a me ʻehā mau fungicides systemic (Papa S1: pyrithiostrobin 250.14 ppm; trifloxystrobin 110.06 ppm; myclobutanil azole 75 .12 ppm; cyprodinil 280.845 ppm) ka ʻawahia i nā huaʻai, nā mea kanu a me nā mea kanu hoʻonaninani. Ua hoʻohuihui mākou i ka pollen me ka hoʻohana ʻana i kahi mea wili, hoʻoili i 0.20 g i kahi lua (24-well Falcon Plate), a hoʻohui a hui pū i 1 μL o ka hopena fungicide e hana i ka pollen pyramidal me nā lua hohonu 1 mm kahi i waiho ʻia ai nā hua. E kau me ka hoʻohana ʻana i kahi spatula mini (Kiʻi Hoʻohui 1c, d). Ua mālama ʻia nā papa Falcon ma ka mahana o ka lumi (25°C) a me 70% ka haʻahaʻa pili. Ua hoʻohālikelike mākou iā lākou me nā larvae kaohi i hānai ʻia i kahi ʻai pollen homogeneous i mālama ʻia me ka wai maʻemaʻe. Ua hoʻopaʻa mākou i ka make a ana i ke kaumaha o ka larval i kēlā me kēia lā ʻē aʻe a hiki i ka wā i hiki ai i nā larvae i ka makahiki prepupal me ka hoʻohana ʻana i kahi kaulike analytical (Fisher Scientific, pololei = 0.0001 g). ʻO ka hope, ua loiloi ʻia ka lakio wahine ma ka wehe ʻana i ka cocoon ma hope o 2.5 mau mahina.
Ua unuhi ʻia ka DNA mai nā larvae O. cornifrons holoʻokoʻa (n = 3 no kēlā me kēia kūlana lapaʻau, pollen i mālama ʻia me ka mancozeb a me ka pollen i mālama ʻole ʻia) a ua hana mākou i nā loiloi ʻokoʻa microbial ma kēia mau laʻana, ʻoiai no ka mea ma ka mancozeb ka make kiʻekiʻe loa i ʻike ʻia i loko o nā larvae. e loaʻa ana iā MnZn. Ua hoʻonui ʻia ka DNA, hoʻomaʻemaʻe ʻia me ka hoʻohana ʻana i ka DNAZymoBIOMICS®-96 MagBead DNA kit (Zymo Research, Irvine, CA), a ua hoʻonohonoho ʻia (600 mau pōʻaiapuni) ma kahi Illumina® MiSeq™ me ka hoʻohana ʻana i ka v3 kit. Ua hana ʻia ka hoʻonohonoho ʻana i kuhikuhi ʻia o nā genes RNA ribosomal 16S bacteria me ka hoʻohana ʻana i ka Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA) me ka hoʻohana ʻana i nā primers e kuhikuhi ana i ka ʻāpana V3-V4 o ka gene 16S rRNA. Eia kekahi, ua hana ʻia ka hoʻonohonoho ʻana 18S me ka hoʻohana ʻana i ka 10% PhiX inclusion, a ua hana ʻia ka hoʻonui ʻana me ka hoʻohana ʻana i ka hui primer 18S001 a me NS4.
Hoʻokomo a hana i nā heluhelu i hui pū ʻia39 me ka hoʻohana ʻana i ka pipeline QIIME2 (v2022.11.1). Ua ʻoki ʻia a hoʻohui ʻia kēia mau heluhelu, a ua wehe ʻia nā moʻo chimeric me ka hoʻohana ʻana i ka plugin DADA2 ma QIIME2 (qiime dada2 noise pairing)40. Ua hana ʻia nā haʻawina papa 16S a me 18S me ka hoʻohana ʻana i ka plugin object classifier Classify-sklearn a me ka artifact silva-138-99-nb-classifier i aʻo mua ʻia.
Ua nānā ʻia nā ʻikepili hoʻokolohua āpau no ka normality (Shapiro-Wilks) a me ka homogeneity o nā variances (hoʻāʻo ʻo Levene). No ka mea, ʻaʻole i hoʻokō ka ʻikepili i nā kuhiakau o ka loiloi parametric a ʻaʻole i hoʻokō ka hoʻololi ʻana i nā koena, ua hana mākou i kahi ANOVA ʻelua ala nonparametric (Kruskal-Wallis) me ʻelua mau mea [manawa (ʻekolu-phase 2, 5, a me 8 mau lā manawa) a me ka fungicide] e loiloi i ka hopena o ka mālama ʻana ma ke kaumaha hou o ka larval, a laila ua hana ʻia nā hoʻohālikelike pairwise nonparametric post hoc me ka hoʻohana ʻana i ka hoʻāʻo Wilcoxon. Ua hoʻohana mākou i kahi kumu hoʻohālike linear generalized (GLM) me kahi hoʻolaha Poisson e hoʻohālikelike i nā hopena o nā fungicides i ke ola ʻana ma waena o ʻekolu mau ʻano fungicide41,42. No ka loiloi nui ʻokoʻa, ua hoʻohiolo ʻia ka helu o nā ʻano amplicon sequence (ASVs) ma ka pae genus. Ua hana ʻia nā hoʻohālikelike o ka nui like ʻole ma waena o nā hui e hoʻohana ana i ka 16S (pae genus) a me ka nui pili 18S me ka hoʻohana ʻana i kahi kumu hoʻohui laulā no ke kūlana, ka unahi, a me ke ʻano (GAMLSS) me nā hoʻolaha ʻohana beta zero-inflated (BEZI), i hoʻohālikelike ʻia ma kahi macro . ma Microbiome R43 (v1.1). 1). Wehe i nā ʻano mitochondrial a me chloroplast ma mua o ka nānā ʻana i ka ʻokoʻa. Ma muli o nā pae taxonomic like ʻole o 18S, ʻo ka pae haʻahaʻa loa o kēlā me kēia taxon wale nō i hoʻohana ʻia no nā loiloi ʻokoʻa. Ua hana ʻia nā loiloi helu āpau me ka hoʻohana ʻana iā R (v. 3.4.3., papahana CRAN) (Team 2013).
ʻO ka hōʻike ʻana i ka mancozeb, pyrithiostrobin, a me ka trifloxystrobin ua hōʻemi nui i ka piʻi ʻana o ke kaumaha o ke kino ma O. cornifrons (Kiʻi 1). Ua ʻike mau ʻia kēia mau hopena no nā lāʻau ʻekolu i loiloi ʻia (Kiʻi 1a–c). ʻAʻole i hōʻemi nui ʻo Cyclostrobin lāua ʻo myclobutanil i ke kaumaha o nā ʻenuhe.
ʻO ke kaumaha hou awelika o nā ʻōpū borer kumu i ana ʻia ma ʻekolu mau manawa ma lalo o ʻehā mau lāʻau lapaʻau ʻai (hānai pollen homogeneous + fungicide: kaohi, 0.1X, 0.5X a me 1X mau lāʻau). (a) Haʻahaʻa ka nui (0.1X): manawa mua (lā 1): χ2: 30.99, DF = 6; P < 0.0001, manawa ʻelua (lā 5): 22.83, DF = 0.0009; manawa ʻekolu; manawa (lā 8): χ2: 28.39, DF = 6; (b) hapalua ka nui (0.5X): manawa mua (lā 1): χ2: 35.67, DF = 6; P < 0.0001, manawa ʻelua (lā hoʻokahi). ): χ2: 15.98, DF = 6; P = 0.0090; kiko manawa ʻekolu (lā 8) χ2: 16.47, DF = 6; (c) Kahi a i ʻole ka mahele piha (1X): kiko manawa mua (lā 1) χ2: 20.64, P = 6; P = 0.0326, kiko manawa ʻelua (lā 5): χ2: 22.83, DF = 6; P = 0.0009; kiko manawa ʻekolu (lā 8): χ2: 28.39, DF = 6; ka nānā ʻana o ka ʻokoʻa nonparametric. Hōʻike nā pā i ka awelika ± SE o nā hoʻohālikelike pālua (α = 0.05) (n = 16) *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.
Ma ka mahele haʻahaʻa loa (0.1X), ua hoʻemi ʻia ke kaumaha o ke kino o ka larva ma 60% me ka trifloxystrobin, 49% me ka mancozeb, 48% me ka myclobutanil, a me 46% me ka pyrithistrobin (Kiʻi 1a). I ka wā i hōʻike ʻia ai i ka hapalua o ka mahele kahua (0.5X), ua hoʻemi ʻia ke kaumaha o ke kino o ka larva mancozeb ma 86%, pyrithiostrobin ma 52% a me trifloxystrobin ma 50% (Kiʻi 1b). ʻO ka mahele kahua piha (1X) o ka mancozeb i hoʻemi i ke kaumaha o ka larva ma 82%, pyrithiostrobin ma 70%, a me trifloxystrobin, myclobutanil a me sangard ma kahi o 30% (Kiʻi 1c).
ʻOi aku ka nui o ka make ma waena o nā larvae i hānai ʻia i ka pollen i mālama ʻia me ka mancozeb, a ukali ʻia e ka pyrithiostrobin a me ka trifloxystrobin. Ua piʻi ka make me ka hoʻonui ʻana i nā dosis o ka mancozeb a me ka pyritisoline (Kiʻi 2; Papa 2). Eia naʻe, ua piʻi iki ka make o ka corn borer i ka piʻi ʻana o ka nui o ka trifloxystrobin; ʻaʻole i hoʻonui nui ʻo cyprodinil lāua ʻo captan i ka make i hoʻohālikelike ʻia me nā lāʻau lapaʻau kaohi.
Ua hoʻohālikelike ʻia ka make ʻana o nā ʻōpū lele borer ma hope o ka ʻai ʻana i ka pollen i mālama ʻia me nā fungicides ʻeono like ʻole. ʻOi aku ka maʻalahi o Mancozeb lāua ʻo pentopyramide i ka hōʻike waha ʻana i nā ilo kulina (GLM: χ = 29.45, DF = 20, P = 0.0059) (laina, pali = 0.29, P < 0.001; pali = 0.24, P < 0.00)).
Ma ka awelika, ma waena o nā lāʻau lapaʻau āpau, he 39.05% o nā maʻi he wahine a he 60.95% he kāne. Ma waena o nā lāʻau lapaʻau kaohi, ʻo ka hapa o nā wahine he 40% ma nā haʻawina haʻahaʻa (0.1X) a me ka hapalua (0.5X), a me 30% ma nā haʻawina kahua (1X). Ma ka 0.1X dose, ma waena o nā larvae i hānai ʻia me ka pollen i mālama ʻia me mancozeb a me myclobutanil, he 33.33% o nā pākeke he wahine, 22% o nā pākeke he wahine, 44% o nā larvae makua he wahine, 44% o nā larvae makua he wahine. wahine, 41% o nā larvae makua he wahine, a ʻo nā kaohi he 31% (Kiʻi 3a). Ma ka 0.5 manawa o ka mahele lāʻau, 33% o nā ilo makua ma ka hui mancozeb a me pyrithiostrobin he wahine, 36% ma ka hui trifloxystrobin, 41% ma ka hui myclobutanil, a me 46% ma ka hui cyprostrobin. ʻO kēia helu he 53% ma ka hui. ma ka hui captan a me 38% ma ka hui kaohi (Kiʻi 3b). Ma ka mahele lāʻau 1X, 30% o ka hui mancozeb he wahine, 36% o ka hui pyrithiostrobin, 44% o ka hui trifloxystrobin, 38% o ka hui myclobutanil, 50% o ka hui kaohi he wahine - 38.5% (Kiʻi 3c).
Pakeneka o nā borers wahine a me ke kāne ma hope o ka hōʻike ʻana i ka fungicide pae larval. (a) Ka nui haʻahaʻa (0.1X). (b) Ka hapa nui (0.5X). (c) Ka nui o ke kahua a i ʻole ka nui piha (1X).
Ua hōʻike ka loiloi moʻo 16S ua ʻokoʻa ka hui bacteria ma waena o nā larvae i hānai ʻia me ka pollen i mālama ʻia me ka mancozeb a me nā larvae i hānai ʻia me ka pollen i mālama ʻole ʻia (Kiʻi 4a). ʻOi aku ka kiʻekiʻe o ka helu microbial o nā larvae i mālama ʻole ʻia i hānai ʻia i ka pollen ma mua o nā larvae i hānai ʻia i ka pollen i mālama ʻia me ka mancozeb (Kiʻi 4b). ʻOiai ʻaʻole koʻikoʻi ka ʻokoʻa i ʻike ʻia ma ka waiwai ma waena o nā hui, ua haʻahaʻa loa ia ma mua o ka mea i ʻike ʻia no nā larvae e hānai ana i ka pollen i mālama ʻole ʻia (Kiʻi 4c). Ua hōʻike ka nui o ka microbiota o nā larvae i hānai ʻia i ka pollen kaohi ʻoi aku ka ʻokoʻa ma mua o nā larvae i hānai ʻia i nā larvae i mālama ʻia me ka mancozeb (Kiʻi 5a). Ua hōʻike ka loiloi wehewehe i ke alo o 28 genera i nā laʻana kaohi a me nā laʻana mancozeb i mālama ʻia (Kiʻi 5b). ʻAʻole hōʻike ka loiloi e hoʻohana ana i ka 18S sequencing i nā ʻokoʻa koʻikoʻi (Kiʻi Hoʻohui 2).
Ua hoʻohālikelike ʻia nā ʻikepili SAV e pili ana i nā moʻo 16S me ka waiwai o Shannon a me ka waiwai i ʻike ʻia ma ka pae phylum. (a) Ka nānā ʻana i ka hoʻonohonoho kumu (PCoA) e pili ana i ka hoʻonohonoho kaiāulu microbial holoʻokoʻa i loko o nā larvae i hānai ʻole ʻia i ka pollen a i ʻole ka mana (uliuli) a me ka mancozeb-fed (ʻalani). Hōʻike kēlā me kēia kiko ʻikepili i kahi laʻana kaʻawale. Ua helu ʻia ʻo PCoA me ka hoʻohana ʻana i ka mamao Bray-Curtis o ka hoʻolaha multivariate t. Hōʻike nā ovals i ka pae hilinaʻi 80%. (b) Boxplot, ʻikepili waiwai Shannon maka (nā kiko) a me c. Waiwai i ʻike ʻia. Hōʻike nā boxplots i nā pahu no ka laina waena, ka laulā interquartile (IQR), a me 1.5 × IQR (n = 3).
ʻO ka haku mele ʻana o nā kaiāulu microbial o nā larvae i hānai ʻia ma ka pollen i mālama ʻia a mālama ʻole ʻia e ka mancozeb. (a) Ka nui o nā heluhelu genera microbial i loko o nā larvae. (b) Palapala wela o nā kaiāulu microbial i ʻike ʻia. Delftia (odds ratio (OR) = 0.67, P = 0.0030) a me Pseudomonas (OR = 0.3, P = 0.0074), Microbacterium (OR = 0.75, P = 0.0617) (OR = 1.5, P = 0.0060); Hoʻohui ʻia nā lālani palapala wela me ka hoʻohana ʻana i ka mamao correlation a me ka pilina awelika.
Hōʻike kā mākou mau hopena i ka hoʻolaha waha ʻana i nā fungicides pili (mancozeb) a me nā systemic (pyrostrobin a me trifloxystrobin), i hoʻohana nui ʻia i ka wā pua, ua hōʻemi nui i ka piʻi ʻana o ke kaumaha a me ka hoʻonui ʻana i ka make ʻana o nā larvae maize. Eia kekahi, ua hōʻemi nui ka mancozeb i ka ʻokoʻa a me ka waiwai o ka microbiome i ka wā prepupal. ʻO Myclobutanil, kahi fungicide systemic ʻē aʻe, ua hōʻemi nui i ka piʻi ʻana o ke kaumaha o ke kino larval ma nā dosis ʻekolu. Ua ʻike ʻia kēia hopena ma ka lua (lā 5) a me ke kolu (lā 8) mau manawa. I ka hoʻohālikelike ʻana, ʻaʻole i hōʻemi nui ka cyprodinil lāua ʻo captan i ka piʻi ʻana o ke kaumaha a i ʻole ke ola ʻana i hoʻohālikelike ʻia me ka hui kaohi. I ko mākou ʻike, ʻo kēia hana ka mea mua e hoʻoholo ai i nā hopena o nā helu mahina ʻai o nā fungicides like ʻole i hoʻohana ʻia e pale i nā mea kanu kulina ma o ka hōʻike pololei ʻana i ka pollen.
Ua hōʻemi nui nā lāʻau lapaʻau fungicide āpau i ka piʻi ʻana o ke kaumaha o ke kino i hoʻohālikelike ʻia me nā lāʻau lapaʻau kaohi. ʻO Mancozeb ka hopena nui loa i ka piʻi ʻana o ke kaumaha o ke kino larval me ka emi ʻana o 51%, ukali ʻia e pyrithiostrobin. Eia nō naʻe, ʻaʻole i hōʻike ʻia nā haʻawina ʻē aʻe i nā hopena maikaʻi ʻole o nā dosis kula o nā fungicides ma nā pae larval44. ʻOiai ua hōʻike ʻia nā biocides dithiocarbamate he haʻahaʻa ka ʻawaʻawa koʻikoʻi45, hiki i ka ethylene bisdithiocarbamates (EBDCS) e like me mancozeb ke hoʻohaʻahaʻa i ka urea ethylene sulfide. Ma muli o kona mau hopena mutagenic i nā holoholona ʻē aʻe, hiki i kēia huahana degradation ke kuleana no nā hopena i ʻike ʻia46,47. Ua hōʻike ʻia nā haʻawina ma mua ua hoʻopili ʻia ka hoʻokumu ʻia ʻana o ka ethylene thiourea e nā mea e like me ke kiʻekiʻe o ka mahana48, nā pae haʻahaʻa49 a me ka lōʻihi o ka mālama ʻana i ka huahana50. Hiki i nā kūlana mālama kūpono no nā biocides ke hōʻemi i kēia mau hopena ʻaoʻao. Eia kekahi, ua hōʻike ka European Food Safety Authority i ka hopohopo e pili ana i ka ʻawaʻawa o ka pyrithiopide, kahi i hōʻike ʻia he carcinogenic i nā ʻōnaehana digestive o nā holoholona ʻē aʻe51.
ʻO ka lawelawe waha ʻana o ka mancozeb, pyrithiostrobin, a me ka trifloxystrobin e hoʻonui i ka make o nā ʻōpū borer kulina. I ka hoʻohālikelike ʻana, ʻaʻohe hopena o ka myclobutanil, ciprocycline a me ka captan i ka make. ʻOkoʻa kēia mau hopena mai nā hopena o Ladurner et al.52, nāna i hōʻike ua hōʻemi nui ʻo captan i ke ola ʻana o ka O. lignaria makua a me Apis mellifera L. (Hymenoptera, Apisidae). Eia kekahi, ua ʻike ʻia nā fungicides e like me ka captan a me ka boscalid e hoʻoulu ai i ka make ʻana o ka larval52,53,54 a hoʻololi paha i ke ʻano hānai55. Hiki i kēia mau loli ke hoʻopilikia i ka maikaʻi o ka meaʻai o ka pollen a me ka loaʻa ʻana o ka ikehu o ke kahua larval. Ua kūlike ka make i ʻike ʻia ma ka hui kaohi me nā haʻawina ʻē aʻe 56,57.
ʻO ka lakio kāne makemake i ka wahine i ʻike ʻia i kā mākou hana e wehewehe ʻia e nā mea e like me ka lawa ʻole o ka male ʻana a me nā kūlana ʻino o ka wā pua, e like me ka mea i hōʻike mua ʻia no O. cornuta e Vicens lāua ʻo Bosch. ʻOiai he ʻehā mau lā o nā wahine a me nā kāne i kā mākou noiʻi e male ai (kahi manawa i manaʻo ʻia he lawa no ka male kūleʻa), ua hoʻemi mākou i ka ikaika o ka mālamalama e hōʻemi i ke kaumaha. Eia nō naʻe, hiki i kēia hoʻololi ke hoʻopilikia me ka manaʻo ʻole i ke kaʻina male61. Eia kekahi, ʻike nā pi i kekahi mau lā o ka wā ʻino, me ka ua a me nā mahana haʻahaʻa (<5°C), hiki ke hoʻopilikia maikaʻi ʻole i ka holomua o ka male4,23.
ʻOiai ua kālele kā mākou noiʻi ʻana i ka microbiome larval holoʻokoʻa, hāʻawi kā mākou mau hopena i ka ʻike i nā pilina hiki ke loaʻa ma waena o nā kaiāulu bacteria e koʻikoʻi paha i ka meaʻai pi a me ka hōʻike ʻana i ka fungicide. No ka laʻana, ʻo nā larvae i hānai ʻia i ka pollen i mālama ʻia me ka mancozeb ua hoʻemi nui ʻia ke ʻano o ke kaiāulu microbial a me ka nui i hoʻohālikelike ʻia me nā larvae i hānai ʻia i ka pollen i mālama ʻole ʻia. I nā larvae e ʻai ana i ka pollen i mālama ʻole ʻia, ʻo nā hui bacteria Proteobacteria a me Actinobacteria ka poʻokela a he aerobic a facultatively aerobic paha. ʻIke ʻia nā bacteria Delft, e pili pinepine ana me nā ʻano pi hoʻokahi, he hana antibiotic, e hōʻike ana i kahi kuleana pale e kūʻē i nā pathogens. ʻO kekahi ʻano bacteria ʻē aʻe, ʻo Pseudomonas, ua nui i nā larvae i hānai ʻia i ka pollen i mālama ʻole ʻia, akā ua emi nui i nā larvae i mālama ʻia me ka mancozeb. Kākoʻo kā mākou mau hopena i nā noiʻi mua e ʻike ana iā Pseudomonas ʻo ia kekahi o nā genera nui loa ma O. bicornis35 a me nā wasps hoʻokahi ʻē aʻe34. ʻOiai ʻaʻole i aʻo ʻia nā hōʻike hoʻokolohua no ke kuleana o Pseudomonas i ke olakino o O. cornifrons, ua hōʻike ʻia kēia bacteria e hoʻolaha i ka synthesis o nā mea ʻawahia pale i loko o ka beetle Paederus fuscipes a hoʻolaha i ka metabolism arginine ma vitro 35, 65. Hōʻike kēia mau nānā ʻana i kahi kuleana kūpono i ka pale ʻana o ka viral a me ka bacteria i ka wā hoʻomohala o ka larvae O. cornifrons. ʻO Microbacterium kekahi ʻano ʻē aʻe i ʻike ʻia i kā mākou noiʻi i hōʻike ʻia he nui nā helu i loko o nā larvae lele koa ʻeleʻele ma lalo o nā kūlana pōloli66. I loko o nā larvae O. cornifrons, hiki i nā microbacteria ke kōkua i ke kaulike a me ke kūpaʻa o ka microbiome gut ma lalo o nā kūlana kaumaha. Eia kekahi, loaʻa ʻo Rhodococcus i loko o nā larvae O. cornifrons a ua ʻike ʻia no kona mau hiki ke detoxification67. Loaʻa pū kēia ʻano ma ka ʻōpū o A. florea, akā ma ka nui haʻahaʻa loa68. Hōʻike kā mākou mau hopena i ke alo o nā ʻano like ʻole o ka genetic ma nā taxa microbial he nui e hiki ke hoʻololi i nā kaʻina hana metabolic i loko o nā larvae. Eia naʻe, pono ka ʻike maikaʻi ʻana i ka ʻokoʻa hana o O. cornifrons.
I ka hōʻuluʻulu manaʻo, hōʻike nā hopena ua hōʻemi ka mancozeb, pyrithiostrobin, a me trifloxystrobin i ka piʻi ʻana o ke kaumaha o ke kino a me ka hoʻonui ʻana i ka make ʻana o nā larvae borer kulina. ʻOiai ke ulu nei ka hopohopo e pili ana i nā hopena o nā fungicides i nā pollinators, aia kahi pono e hoʻomaopopo maikaʻi i nā hopena o nā metabolites koena o kēia mau hui. Hiki ke hoʻokomo ʻia kēia mau hopena i loko o nā ʻōlelo paipai no nā papahana hoʻokele pollinator i hoʻohui ʻia e kōkua i nā mahiʻai e pale i ka hoʻohana ʻana i kekahi mau fungicides ma mua a i ka wā o ka pua ʻana o nā lāʻau hua ma ke koho ʻana i nā fungicides a me ka hoʻololi ʻana i ka manawa o ka noi ʻana, a i ʻole ma ka paipai ʻana i ka hoʻohana ʻana i nā koho ʻē aʻe i emi iki ka ʻino 36. He mea nui kēia ʻike no ka hoʻomohala ʻana i nā ʻōlelo paipai. ma ka hoʻohana ʻana i ka pesticide, e like me ka hoʻoponopono ʻana i nā papahana pīpī e kū nei a me ka hoʻololi ʻana i ka manawa pīpī i ke koho ʻana i nā fungicides a i ʻole ka hoʻolaha ʻana i ka hoʻohana ʻana i nā koho ʻē aʻe i emi iki ka weliweli. Pono ka noiʻi hou aku i nā hopena maikaʻi ʻole o nā fungicides ma ka lakio wahine, ke ʻano hānai, ka microbiome gut, a me nā ʻano molekala e pili ana i ka pohō kaumaha a me ka make o ka borer kulina.
Ua waiho ʻia ka ʻikepili kumu 1, 2 a me 3 ma nā Kiʻi 1 a me 2 i loko o ka waihona ʻikepili figshare DOI: https://doi.org/10.6084/m9.figshare.24996245 a me https://doi.org/10.6084/m9. figshare.24996233. Loaʻa nā moʻo i kālailai ʻia i ke aʻo ʻana o kēia manawa (Nā Kiʻi 4, 5) i loko o ka waihona NCBI SRA ma lalo o ka helu komo PRJNA1023565.
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