Pine nematode shine keɓe ƙaura endoparasite wanda aka sani yana haifar da mummunar asarar tattalin arziƙi a cikin yanayin gandun daji na Pine. Binciken na yanzu yana duba ayyukan nematicidal na halogenated indoles akan Pine nematodes da tsarin aikinsu. Ayyukan nematicidal na 5-iodoindole da avermectin (madaidaicin iko) akan pine nematodes sun kasance iri ɗaya kuma suna da girma a ƙananan ƙira (10 μg/mL). 5-iodoindole ya rage yawan haihuwa, aikin haihuwa, mace-macen amfrayo da tsutsa, da halayen locomotor. Hanyoyin hulɗar kwayoyin halitta na ligands tare da invertebrate-takamaiman glutamate-gated chloride tashar masu karɓa suna goyan bayan ra'ayi cewa 5-iodoindole, kamar avermectin, yana ɗaure tam zuwa wurin mai karɓa mai aiki. 5-Iodoindole kuma ya haifar da nakasar dabi'a iri-iri a cikin nematodes, gami da rugujewar gaɓoɓin gaɓoɓin gaɓoɓi da ƙanƙantar ɓarna. Wadannan sakamakon sun nuna cewa vacuoles na iya taka rawa a cikin mutuwar nematode methylation. Mahimmanci, 5-iodoindole bai kasance mai guba ga nau'in shuka ba (kabeji da radish). Don haka, wannan binciken ya nuna cewa aikace-aikacen iodoindole a ƙarƙashin yanayin muhalli na iya sarrafa raunin pine wilt.
nematode itacen Pine (Bursaphelenchus xylophilus) na cikin nematodes na itacen pine (PWN), nematodes na ƙaura na endoparasitic da aka sani suna haifar da mummunar lalacewar muhalli ga yanayin dajin Pine1. Ciwon pine wilt (PWD) wanda itacen pine nematode ya haifar yana zama matsala mai tsanani a nahiyoyi da dama, ciki har da Asiya da Turai, kuma a Arewacin Amirka, nematode yana lalata nau'in pine da aka gabatar1,2. Rugujewar bishiyar Pine babbar matsala ce ta tattalin arziki, kuma hasashen yaduwarsa a duniya yana da damuwa3. Mafi yawan nau'in pine suna kaiwa hari ta hanyar nematode: Pinus densiflora, Pinus sylvestris, Pinus thunbergii, Pinus koraiensis, Pinus thunbergii, Pinus thunbergii, da Pinus radiata4. Pine nematode wata cuta ce mai tsanani da za ta iya kashe bishiyar Pine a cikin makonni ko watanni na kamuwa da cuta. Bugu da kari, barkewar cutar pine nematode ya zama ruwan dare a cikin halittu daban-daban, don haka an kafa sarƙoƙin kamuwa da cuta mai tsayi1.
Bursaphelenchus xylophilus shine keɓewar shuka-parasitic nematode na dangin Aphelenchoidea da clade 102.5. Nematode yana ciyar da fungi kuma yana haifuwa a cikin kyallen itace na bishiyar pine, yana haɓaka zuwa matakai huɗu daban-daban: L1, L2, L3, L4 da mutum mai girma1,6. A ƙarƙashin yanayin ƙarancin abinci, Pine nematode yana shiga cikin wani yanki na musamman na tsutsa - dauer, wanda ke lalata vector ɗin sa - ƙwararrun Pine (Monochamus alternatus) kuma ana tura shi zuwa bishiyar Pine mai lafiya. A cikin rundunonin lafiya, nematodes suna yin ƙaura da sauri ta cikin kyallen jikin shuka kuma suna ciyar da sel parenchymatous, wanda ke haifar da yawan halayen haɓakar hankali, pine wilting da mutuwa a cikin shekara guda bayan kamuwa da cuta1,7,8.
Ikon nazarin halittu na pine nematodes ya daɗe yana zama ƙalubale, tare da matakan keɓewa tun daga ƙarni na 20. Dabarun na yanzu don sarrafa nematodes na Pine da farko sun ƙunshi jiyya na sinadarai, gami da fumigation na itace da dasa nematicides cikin kututturan bishiya. Mafi yawan amfani da nematicides sune avermectin da avermectin benzoate, waɗanda ke cikin dangin avermectin. Waɗannan sinadarai masu tsada suna da matuƙar tasiri a kan nau'ikan nematode da yawa kuma ana ɗaukarsu lafiyayye9. Duk da haka, ana sa ran yin amfani da waɗannan nematicides akai-akai zai haifar da matsin lamba wanda kusan zai haifar da bayyanar pine nematodes, kamar yadda aka nuna ga kwari da yawa, irin su Leptinotarsa decemlineata, Plutella xylostella da nematodes Trichostrongylus colubriformis da Ostertagia sun ci gaba da juriya a hankali,1. Don haka, ana buƙatar tsarin juriya akai-akai da yin nazari akai-akai kuma ana bincikar nematicides a ci gaba da nemo madaidaicin matakan da ke da tsada, masu dacewa da muhalli don sarrafa PVD. A cikin 'yan shekarun nan, yawancin mawallafa sun ba da shawarar yin amfani da tsantsa daga tsire-tsire, mai mahimmanci da masu canzawa a matsayin masu kula da nematode13,14,15,16.
Kwanan nan mun nuna aikin nematicidal na indole, kwayar siginar siginar intercellular da interkingdom, a cikin Caenorhabditis elegans 17. Indole shine siginar intracellular da ke yaduwa a cikin ilimin halittu na microbial, yana sarrafa ayyuka masu yawa waɗanda ke shafar ilimin halittar jiki, ƙwayar cuta, kwanciyar hankali na plasmid, juriya na miyagun ƙwayoyi, haɓakar biofilm, da virulence 18, 19. Ba a yi nazarin ayyukan indole da abubuwan da suka samo asali daga sauran nematodes masu cutarwa ba. A cikin wannan binciken, mun bincika ayyukan nematicidal na 34 indoles a kan Pine nematodes kuma mun bayyana tsarin aikin 5-iodoindole mafi ƙarfi ta hanyar amfani da microscopy, ɗaukar hoto na lokaci-lokaci, da gwaje-gwajen docking na kwayoyin halitta, da kuma tantance tasirinsa mai guba a kan tsire-tsire ta amfani da gwajin ƙwayar iri.
A baya an ba da rahoton babban taro (> 1.0 mM) na indole yana da tasirin nematicidal akan nematodes17. Bayan jiyya na B. xylophilus (matakan rayuwa masu haɗuwa) tare da indole ko 33 daban-daban na indole a cikin 1 mM, an auna mutuwar B. xylophilus ta hanyar kirga rayayyun nematodes masu rai da matattu a cikin kulawa da ƙungiyoyi masu kulawa. Indoles guda biyar sun nuna mahimman ayyukan nematidal; Rayuwar ƙungiyar kulawa da ba a kula da ita ba shine 95 ± 7% bayan 24 h. Daga cikin indoles 34 da aka gwada, 5-iodoindole da 4-fluoroindole a 1 mM sun haifar da mutuwar 100%, yayin da 5,6-difluoroindigo, methylindole-7-carboxylate, da 7-iodoindole ya haifar da kusan 50% mace-mace (Table 1).
Tasirin 5-iodoindole akan samuwar vacuole da metabolism na itacen Pine nematode. (A) Tasirin avermectin da 5-iodoindole akan manya nematodes maza, (B) L1 matakin nematode qwai da (C) metabolism na B. xylophilus, (i) vacuoles ba a lura a 0 h, jiyya ya haifar da (ii) vacuoles, (iii) tarawa na vacuoles da yawa, (ivfusions), vacuoles na vacuoles da yawa, vacuoles, vacuoles da kumburi. (vi) samuwar giant vacuoles. Jajayen kiban suna nuna kumburin vacuoles, kibiyoyi masu shuɗi suna nuna haɗuwar vacuoles kuma baƙaƙen kibau suna nuna ƙaton vacuoles. Ma'aunin Sikeli = 50 μm.
Bugu da ƙari, wannan binciken ya kuma bayyana tsarin tsarin da ake yi na mutuwar methane a cikin pine nematodes (Figure 4C). Mutuwar Methanogenic shine nau'in mutuwar tantanin halitta mara apoptotic wanda ke da alaƙa da tarin fitattun ƙwayoyin cytoplasmic vacuoles27. Lalacewar yanayin halittar da aka gani a cikin nematodes na Pine sun bayyana suna da alaƙa da tsarin mutuwar methane. Binciken ƙananan ƙwayoyin cuta a lokuta daban-daban ya nuna cewa an kafa ƙaƙƙarfan vacuoles bayan 20 h na fallasa zuwa 5-iodoindole (0.1 mM). An lura da ƙwayoyin microscopic bayan 8 h na jiyya, kuma adadin su ya karu bayan sa'o'i 12. An lura da yawa manyan vacuoles bayan sa'o'i 14. Yawancin fused vacuoles an bayyana a fili bayan 12-16 h na jiyya, yana nuna cewa haɗuwar vacuole shine tushen tsarin mutuwa na methanogenic. Bayan sa'o'i 20, an sami ɗimbin giant vacuoles a cikin tsutsa. Waɗannan abubuwan lura suna wakiltar rahoton farko na metuosis a cikin C. elegans.
A cikin tsutsotsi na 5-iodoindole, an kuma lura da tarawar vacuole da rupture (Fig. 5), kamar yadda aka nuna ta hanyar tsutsa tsutsa da sakin vacuole a cikin yanayi. Hakanan an lura da rushewar vacuole a cikin membrane na eggshell, wanda yawanci L2 ke kiyaye shi yayin ƙyanƙyashe (Ƙarin Hoton S2). Wadannan abubuwan lura suna goyan bayan shigar da tarin ruwa da gazawar osmoregulatory, da kuma raunin da zai iya jujjuyawa tantanin halitta (RCI), a cikin tsarin samar da vacuole da suppuration (Fig. 5).
Yin hasashen rawar aidin a cikin samuwar vacuole da aka lura, mun bincika aikin nematidal na sodium iodide (NaI) da potassium iodide (KI). Duk da haka, a ƙididdigewa (0.1, 0.5 ko 1 mM), ba su shafi ko dai nematode tsira ba ko samuwar vacuole (Ƙarin Hoton S5), ko da yake 1 mM KI yana da ɗan tasiri na nematicidal. A gefe guda, 7-iodoindole (1 ko 2 mM), kamar 5-iodoindole, ya haifar da ɓarna da yawa da nakasar tsarin (Ƙarin Fig. S6). Iodoindoles guda biyu sun nuna halaye iri ɗaya a cikin pine nematodes, yayin da NaI da KI ba su yi ba. Abin sha'awa shine, indole bai haifar da samuwar vacuole a cikin B. xylophilus ba a ƙididdigar da aka gwada (bayanan da ba a nuna ba). Don haka, sakamakon ya tabbatar da cewa hadaddun indole-iodine yana da alhakin vacuolization da metabolism na B. xylophilus.
Daga cikin indoles da aka gwada don aikin nematicidal, 5-iodoindole yana da mafi girman zamewar -5.89 kcal / mol, sannan 7-iodoindole (-4.48 kcal / mol), 4-fluoroindole (-4.33), da indole (-4.03) (Figure 6). Ƙarfin kashin baya na hydrogen bonding na 5-iodoindole zuwa leucine 218 yana tabbatar da ɗaurin sa, yayin da duk sauran abubuwan da aka samo asali na indole suna ɗaure zuwa serine 260 ta hanyar sarkar hydrogen ta gefe. Daga cikin sauran nau'ikan iodoindoles, 2-iodoindole yana da ƙimar ɗauri na -5.248 kcal / mol, wanda shine saboda babban haɗin hydrogen tare da leucine 218. Sauran abubuwan da aka sani sun haɗa da 3-iodoindole (-4.3 kcal / mol), 4-iodoindole (-4.0 kcal/mol.6 kcal/mol). (Ƙarin Hoto S8). Yawancin halogenated indoles da indole kanta, ban da 5-iodoindole da 2-iodoindole, suna samar da haɗin gwiwa tare da serine 260. Gaskiyar cewa haɗin gwiwar hydrogen tare da leucine 218 yana nuna alamar haɓakaccen ligand mai karɓa, kamar yadda aka lura ga ivermectin (Ƙarin Fig. S5-iodoindoin) wanda ya tabbatar da haka. ivermectin, ɗaure tam zuwa wurin aiki na mai karɓar GluCL ta hanyar leucine 218 (Fig. 6 da Ƙarin Hoto S8). Muna ba da shawarar cewa ana buƙatar wannan ɗaurin don kula da tsarin buɗaɗɗen buɗaɗɗen GluCL da kuma cewa ta hanyar ɗaurewa sosai zuwa wurin aiki na mai karɓar GluCL, 5-iodoindole, 2-iodoindole, avermectin da ivermectin don haka kiyaye tashar ion a buɗe kuma ba da izinin ɗaukar ruwa.
Docking na kwayoyin halitta na indole da halogenated indole zuwa GluCL. Hanyoyin dauri na (A) indole, (B) 4-fluoroindole, (C) 7-iodoindole, da (D) 5-iodoindole ligands zuwa wurin aiki na GluCL. Ana wakilta sunadarin da kintinkiri, kuma ana nuna haɗin haɗin hydrogen na baya a matsayin layin dige-dige rawaya. (A'), (B'), (C'), da (D') suna nuna ma'amalar ma'amalar ma'auni tare da ragowar amino acid da ke kewaye, kuma ana nuna haɗin sarkar hydrogen ta gefen kibiyoyi masu dige-dige.
An gudanar da gwaje-gwaje don kimanta tasirin mai guba na 5-iodoindole akan germination na kabeji da radish tsaba. 5-iodoindole (0.05 ko 0.1 mM) ko avermectin (10 μg/mL) ba su da wani tasiri ko rashin tasiri a farkon germination da fitowar shuka (Hoto 7). Bugu da ƙari, ba a sami wani gagarumin bambanci tsakanin yawan germination na sarrafawar da ba a kula da su ba da kuma tsaba da aka yi da 5-iodoindole ko avermectin. Tasiri a kan elongation taproot da adadin tushen tushen da aka kafa ba shi da mahimmanci, kodayake 1 mM (sau 10 maida hankali mai aiki) na 5-iodoindole ya ɗan jinkirta ci gaban tushen gefe. Waɗannan sakamakon suna nuna cewa 5-iodoindole ba mai guba ba ne ga sel shuka kuma baya tsoma baki tare da tsarin ci gaban shuka a ƙididdigar da aka yi nazari.
Tasirin 5-iodoindole akan germination iri. Germination, sprouting da a kaikaice rooting na B. oleracea da R. raphanistrum tsaba a kan Murashige da Skoog agar matsakaici tare da ko ba tare da avermectin ko 5-iodoindole. An rubuta germination bayan kwanaki 3 na shiryawa a 22 ° C.
Wannan binciken ya ba da rahoton lokuta da yawa na kashe nematode ta hanyar indoles. Mahimmanci, wannan shine rahoton farko na iodoindole yana haifar da methylation (tsari da ke haifar da tarawar ƙananan ƙwayoyin cuta waɗanda sannu a hankali ke haɗuwa cikin giant vacuoles, ƙarshe yana haifar da fashewar membrane da mutuwa) a cikin alluran pine, tare da iodoindole yana nuna mahimman kaddarorin nematicidal kama da na kasuwanci nematicide avermectin.
An ba da rahoton Indoles a baya don aiwatar da ayyuka masu yawa na sigina a cikin prokaryotes da eukaryotes, gami da hanawa / samuwar biofilm, tsirar ƙwayoyin cuta, da cututtukan cututtuka19,32,33,34. Kwanan nan, yuwuwar tasirin warkewa na indoles halogenated, indole alkaloids, da abubuwan da suka samo asali na indole na semisynthetic sun jawo sha'awar bincike mai yawa35,36,37. Misali, an nuna indoles mai halogenated don kashe Escherichia coli da kuma Staphylococcus aureus cell37. Bugu da kari, yana da sha'awar kimiyance don yin nazarin ingancin da aka saba da shi a kan wasu nau'in, Genera, da mulkoki don cimma nasarar wannan buri.
Anan, muna ba da shawarar wani tsari don 5-iodoindole-induced mutuwa a cikin C. elegans dangane da raunin da ya faru (RCI) da methylation (Figures 4C da 5). Canje-canje na edematous irin su kumbura da raguwar vacuolar sune alamun RCI da methylation, wanda aka bayyana azaman giant vacuoles a cikin cytoplasm48,49. RCI yana tsoma baki tare da samar da makamashi ta hanyar rage samar da ATP, haifar da gazawar famfo na ATPase, ko rushe membranes cell da haifar da hanzari na Na +, Ca2 +, da ruwa50,51,52. Intracytoplasmic vacuoles suna tasowa a cikin ƙwayoyin dabbobi sakamakon tarin ruwa a cikin cytoplasm saboda kwararar Ca2+ da ruwa53. Abin sha'awa shine, wannan tsari na lalata kwayoyin halitta yana canzawa idan lalacewar ta kasance na wucin gadi kuma sel sun fara samar da ATP na wani ɗan lokaci, amma idan lalacewar ta ci gaba ko ta ci gaba, kwayoyin sun mutu.
Halin phenotype na methylation wanda 5-iodoindole ya haifar a cikin B. xylophilus na iya zama saboda kasancewar iodine da rarraba kwayoyin halitta, tun da 7-iodoindole yana da ƙarancin hanawa akan B. xylophilus fiye da 5-iodoindole (Table 1 da Ƙarin Hoto S6). Waɗannan sakamakon sun yi daidai da nazarin Maltese et al. (2014), wanda ya ba da rahoton cewa juyawa na pyridyl nitrogen moiety a cikin indole daga para- zuwa meta-matsakaicin soke vacuolization, hana girma, da cytotoxicity a cikin U251 Kwayoyin, bayar da shawarar cewa hulɗar kwayoyin tare da wani takamaiman wurin aiki a cikin furotin yana da mahimmanci27,44,45. Ma'amala tsakanin indole ko halogenated indoles da masu karɓa na GluCL da aka lura a cikin wannan binciken kuma suna goyan bayan wannan ra'ayi, kamar yadda aka samo 5- da 2-iodoindole don ɗaure masu karɓar GluCL fiye da sauran indoles da aka bincika (Hoto 6 da Ƙarin Hoto S8). Iodin a matsayi na biyu ko na biyar na indole an samo shi don ɗaure zuwa leucine 218 na mai karɓar GluCL ta hanyar haɗin hydrogen na kashin baya, yayin da sauran indoles na halogenated da indole da kanta suna samar da haɗin gwiwar sarkar hydrogen mai rauni tare da serine 260 (Hoto 6). Don haka muna hasashen cewa gano halogen yana taka muhimmiyar rawa wajen shigar da lalatawar vacuolar, yayin da ɗaure mai ƙarfi na 5-iodoindole ya sa tashar ion ta buɗe, ta haka yana ba da damar kwararar ruwa da sauri da fashewar vacuole. Koyaya, cikakken tsarin aikin 5-iodoindole ya rage don ƙayyade.
Kafin aikace-aikacen aikace-aikacen 5-iodoindole, yakamata a bincika tasirinsa mai guba akan tsire-tsire. Gwaje-gwajen haɓakar iri ɗin mu sun nuna cewa 5-iodoindole ba shi da wani mummunan tasiri a kan germination iri ko hanyoyin ci gaba na gaba a ƙididdigar da aka yi nazari (Hoto 7). Don haka, wannan binciken ya ba da tushe don amfani da 5-iodoindole a cikin yanayin muhalli don sarrafa cutarwar pine nematodes ga bishiyar pine.
Rahotannin da suka gabata sun nuna cewa maganin indole na wakiltar wata hanyar da za ta iya magance matsalar juriyar ƙwayoyin cuta da ci gaban ciwon daji55. Bugu da kari, indoles suna da maganin kashe kwayoyin cuta, anticancer, antioxidant, anti-inflammatory, antidiabetic, antiviral, antiproliferative da antituberculosis kuma yana iya zama tushe mai ban sha'awa ga ci gaban miyagun ƙwayoyi56,57. Wannan binciken ya nuna a karon farko yiwuwar amfani da iodine a matsayin maganin antiparasitic da anthelmintic.
An gano Avermectin shekaru talatin da suka wuce kuma ya lashe kyautar Nobel a cikin 2015, kuma amfani da shi azaman maganin anthelmintic yana ci gaba da gudana. Koyaya, saboda saurin haɓaka juriya ga avermectins a cikin nematodes da kwari, ana buƙatar madadin, rahusa, da dabarun abokantaka na muhalli don sarrafa kamuwa da cutar PWN a cikin bishiyoyin Pine. Wannan binciken kuma ya ba da rahoton tsarin da 5-iodoindole ke kashe pine nematodes da kuma cewa 5-iodoindole yana da ƙarancin guba ga ƙwayoyin shuka, wanda ke buɗe kyakkyawan fata don aikace-aikacen kasuwanci na gaba.
Kwamitin Da'a na Jami'ar Yeungnam, Gyeongsan, Koriya ne ya amince da dukkan gwaje-gwajen, kuma an yi hanyoyin ne bisa ka'idojin Kwamitin Da'a na Jami'ar Yeungnam.
An gudanar da gwaje-gwajen ƙwai ta hanyar amfani da hanyoyin da aka kafa43. Don tantance ƙimar ƙyanƙyashe (HR), nematodes manya masu shekaru 1 (kimanin mata 100 da maza 100) an tura su zuwa jita-jita na Petri mai ɗauke da naman gwari kuma an bar su suyi girma na sa'o'i 24. An ware ƙwai kuma an bi da su tare da 5-iodoindole (0.05 mM da 0.1 mM) ko avermectin (10 μg/ml) a matsayin dakatarwa a cikin ruwa maras kyau. Wadannan dakatarwar (500 μl; kusan ƙwai 100) an tura su zuwa rijiyoyin farantin al'adun nama mai rijiyar 24 kuma an sanya su a 22 ° C. An ƙididdige L2 bayan sa'o'i 24 na shiryawa amma an ɗauke su a matsayin matattu idan sel ba su motsa ba lokacin da aka motsa su da wayar platinum mai kyau. An gudanar da wannan gwaji a matakai biyu, kowanne tare da maimaita shida. An haɗa bayanan daga gwaje-gwajen biyu kuma an gabatar da su. An ƙididdige yawan adadin HR kamar haka:
An tantance mace-macen tsutsa ta amfani da hanyoyin da aka inganta a baya. An tattara ƙwai na Nematode kuma an haɗa embryos ta hanyar ƙyanƙyashe a cikin ruwa maras kyau don samar da matakan L2. An yi amfani da tsutsa masu aiki tare (kimanin nematodes 500) tare da 5-iodoindole (0.05 mM da 0.1 mM) ko avermectin (10 μg/ml) kuma an girma a kan faranti na B. cinerea Petri. Bayan sa'o'i 48 na shiryawa a 22 ° C, an tattara nematodes a cikin ruwa maras kyau kuma an bincika kasancewar matakan L2, L3, da L4. Kasancewar matakan L3 da L4 sun nuna canjin tsutsa, yayin da kasancewar matakin L2 bai nuna canji ba. An samo hotuna ta amfani da iRiS™ Tsarin Hoto na Dijital. An gudanar da wannan gwaji a matakai biyu, kowanne tare da maimaita shida. An haɗa bayanan daga gwaje-gwajen biyu kuma an gabatar da su.
An kiyasta yawan guba na 5-iodoindole da avermectin zuwa tsaba ta hanyar amfani da gwaje-gwajen germination akan faranti na Murashige da Skoog agar.62 B. Oleracea da R. raphanistrum tsaba an fara tsoma su a cikin ruwa mai tsabta mai tsabta don kwana ɗaya, an wanke tare da 1 ml 100% ethanol, 1 ml na kasuwanci tare da 3% hypochlorite. Minti 15, kuma an wanke sau biyar tare da ruwa mai tsabta 1 ml. An dasa tsaba masu haifuwa a kan faranti na germination mai ɗauke da 0.86 g/l (0.2X) Murashage da Skoog matsakaici da 0.7% agar bacteriological tare da ko ba tare da 5-iodoindole ko avermectin ba. An sanya faranti a zafin jiki na 22 ° C, kuma an ɗauki hotuna bayan kwanaki 3 na shiryawa. An gudanar da wannan gwaji a matakai biyu, kowanne daga cikinsu yana da maimaitawa guda shida.
Lokacin aikawa: Fabrairu-26-2025