Iyi nkuru ni igice cy'insanganyamatsiko y'ubushakashatsi igira iti “Kunoza ubudahangarwa bw'ibinyamisogwe ku mikorobe n'udukoko”, reba ingingo zose 5
Impamvu itera indwara y’ibimera iterwa n’udukoko two mu bwoko bwa Sclerotinia sclerotiorum (Lib.) de Bary ikoresha ingamba nyinshi zo kwanduza ibimera bitandukanye. Ubu bushakashatsi bugaragaza ikoreshwa rya diamine L-ornithine, aside amine itari poroteyine ikangura ikorwa ry’izindi aside amine z’ingenzi, nk’ubundi buryo bwo gucunga kugira ngo hongerwe uburyo Phaseolus vulgaris L. ikora ku bimera byera biterwa na Pseudomonas sclerotiorum. Ubushakashatsi bwakozwe muri vitro bwagaragaje ko L-ornithine yabujije cyane gukura kwa S. pyrenoidosa bitewe n’ingano. Byongeye kandi, ishobora kugabanya cyane ubukana bw’ibimera byera mu gihe cy’ubushyuhe. Byongeye kandi, L-ornithine yashishikarije gukura kw’ibimera byavuwe, bigaragaza ko ingano ya L-ornithine yageragejwe itari uburozi ku bimera byavuwe. Byongeye kandi, L-ornithine yongereye uburyo bwo kugaragaza antioxydants zitari enzyme (phenolics na flavonoids zose) na antioxydants za enzyme (catalase (CAT), peroxidase (POX), na polyphenol oxidase (PPO)), kandi yongera uburyo bwo kugaragaza gene eshatu zifitanye isano na antioxydants (PvCAT1, PvSOD, na PvGR). Byongeye kandi, mu isesengura rya silico byagaragaje ko hariho poroteyine ya oxaloacetate acetylhydrolase (SsOAH) muri genome ya S. sclerotiorum, isa cyane na poroteyine za oxaloacetate acetylhydrolase (SsOAH) za Aspergillus fijiensis (AfOAH) na Penicillium sp. (PlOAH) mu bijyanye n'isesengura ry'imikorere, imiterere ibungabunzwe, na topology. Igishimishije ni uko kongeramo L-ornithine mu gice cy'umutobe w'ibirayi (PDB) byagabanyije cyane uburyo gene ya SsOAH igaragara muri S. sclerotiorum mycelia. Mu buryo nk'ubwo, gukoresha L-ornithine hanze byagabanyije cyane igaragara rya gene ya SsOAH mu mikorobe yakuwe mu bimera byavuwe. Amaherezo, gukoresha L-ornithine byagabanyije cyane isohoka rya aside oxalic haba mu mababi ya PDB n'ayanduye. Muri make, L-ornithine igira uruhare runini mu kubungabunga redox ndetse no kongera uburyo bwo kwirinda ibimera byanduye. Ibisubizo by'ubu bushakashatsi bishobora gufasha mu guteza imbere uburyo bushya kandi butangiza ibidukikije bwo kurwanya imyuka yera no kugabanya ingaruka zayo ku musaruro w'ibishyimbo n'ibindi bihingwa.
Ifu y'umweru, iterwa n'ibihumyo bya necrotrophic Sclerotinia sclerotiorum (Lib.) de Bary, ni indwara mbi cyane kandi igabanya umusaruro iteje akaga gakomeye ku musaruro w'ibishyimbo ku isi (Phaseolus vulgaris L.) (Bolton et al., 2006). Sclerotinia sclerotiorum ni imwe mu ndwara ziterwa n'ibihumyo ziterwa n'ubutaka zigoranye cyane kurwanya, ifite ubwoko bwinshi bw'ibimera birenga 600 kandi ikabasha kwangiza vuba ingirangingo z'ibimera mu buryo butari bwihariye (Liang na Rollins, 2018). Mu bihe bibi, inyura mu cyiciro cy'ingenzi cy'ubuzima bwayo, iguma idasinziriye igihe kirekire nk'imiterere y'umukara, ikomeye, isa n'imbuto yitwa 'sclerotia' mu butaka cyangwa nk'ibyiza byera, byoroshye mu gice cy'imitsi cyangwa igiti cy'ibimera byanduye (Schwartz et al., 2005). S. sclerotiorum irashobora gukora sclerotia, ituma ibasha kubaho mu mirima yanduye igihe kirekire no gukomeza igihe cyose irwaye (Schwartz et al., 2005). Sclerotia ikungahaye ku ntungamubiri, ishobora kuguma mu butaka igihe kirekire, kandi ikaba intanga ngabo z'ingenzi zo kwandura nyuma yaho (Schwartz et al., 2005). Mu bihe byiza, sclerotia iramera ikanakora utubuto two mu kirere dushobora kwanduza ibice byose biri hejuru y'ubutaka by'igihingwa, harimo indabyo, amashami, cyangwa ibishishwa (Schwartz et al., 2005).
Sclerotinia sclerotiorum ikoresha ingamba nyinshi zo kwanduza ibimera byayo, zirimo urukurikirane rw'ibintu bihuzwa kuva ku kumera kwa sclerotial kugeza ku kugaragaza ibimenyetso. Mu ntangiriro, S. sclerotiorum ikora utubuto duhagaze (twitwa ascospores) duturutse mu bintu bimeze nk'ibihumyo byitwa apothecia, bihinduka sclerotia idahindagurika ku bisigazwa by'ibimera byanduye (Bolton et al., 2006). Hanyuma fungus isohora aside oxalic, ikintu gitera ubukana, kugira ngo igenzure pH y'urukuta rw'ibimera, itere imbere kwangirika kwa enzymes no kwinjira mu ngirabuzimafatizo (Hegedus na Rimmer, 2005), kandi ihagarike guturika kwa oxidative kw'ibimera byakira. Iyi nzira yo gukurura aside igabanya ubukana bw'urukuta rw'ibimera, igatanga ibidukikije byiza ku mikorere isanzwe kandi iboneye ya enzymes zangiza urukuta rw'ibihumyo (CWDEs), bigatuma indwara itera indwara irenga inkombe z'umubiri no kwinjira mu ngirabuzimafatizo z'ibimera (Marciano et al., 1983). Iyo S. sclerotiorum imaze kwinjira, isohora CWDE nyinshi, nka polygalacturonase na cellulase, byoroshya ikwirakwira ryayo mu ngingo zanduye kandi bigatera necrosis y'ingingo. Gukomeza kw'ibisebe n'udusimba tw'uruhu bitera ibimenyetso biranga imyumbati yera (Hegedus na Rimmer, 2005). Hagati aho, ibimera bicumbikiye bimenye imiterere ya molekile ifitanye isano na pathogen (PAMPs) binyuze mu buryo bwo kumenya imiterere (PRRs), bigatera urukurikirane rw'ibintu bigaragaza ibimenyetso bituma imbaraga zo kwirinda zitangira.
Nubwo hamaze imyaka myinshi hakorwa ingamba zo kurwanya indwara, ibura ry’imiti ihagije irwanya ibishyimbo riracyari mu bihingwa by’ubucuruzi, kimwe no mu bindi bihingwa by’ubucuruzi, bitewe no kudahangana n’indwara, kubaho no guhangana n’imihindagurikire yayo. Kubwibyo, kurwanya indwara biragoye cyane kandi bisaba ingamba zihuriweho kandi zirimo guhuza imigenzo gakondo, kurwanya indwara, n’imiti irwanya ibihumyo (O'Sullivan et al., 2021). Kurwanya ibihumyo by’umweru mu buryo bwa chimique ni byo byiza cyane kuko imiti irwanya ibihumyo, iyo ikoreshejwe neza kandi mu gihe gikwiye, ishobora kugenzura neza ikwirakwira ry’indwara, ikagabanya ubukana bw’ubwandu, kandi ikagabanya igihombo cy’umusaruro. Ariko, gukoresha imiti irwanya ibihumyo cyane no gukoresha imiti irwanya ibihumyo bishobora gutuma habaho ubwoko bwa S. sclerotiorum burwanya kandi bukagira ingaruka mbi ku binyabuzima bitari ibyangiritse, ku buzima bw’ubutaka, no ku bwiza bw’amazi (Le Cointe et al., 2016; Ceresini et al., 2024). Kubwibyo, gushaka ubundi buryo butangiza ibidukikije byabaye ikintu cy’ingenzi cyane.
Polyamines (PAs), nka putrescine, spermidine, spermine, na cadaverine, zishobora kuba amahitamo meza yo kurwanya indwara ziterwa n’ibimera zikomoka ku butaka, bityo bikagabanya burundu cyangwa igice cy’ikoreshwa ry’imiti irwanya udukoko twangiza ibidukikije (Nehela et al., 2024; Yi et al., 2024). Mu bimera binini, PAs igira uruhare mu bikorwa byinshi by’umubiri birimo, ariko ntibigarukira gusa ku, kwigabanyamo ibice by’uturemangingo, gutandukanya, no gusubiza ibibazo bya abiotic na biotic (Killiny na Nehela, 2020). Bishobora gukora nk'ibintu birwanya ogisijeni, bigafasha mu gukuraho ubwoko bwa ogisijeni bukora neza (ROS), kubungabunga redox homeostasis (Nehela na Killiny, 2023), gutera uturemangingo dufitanye isano n'ubwirinzi (Romero et al., 2018), kugenga inzira zitandukanye za metabolisme (Nehela na Killiny, 2023), guhindura imiterere y'imisemburo ya phytohormones (Nehela na Killiny, 2019), gushyiraho uburyo bwo kurwanya indwara (SAR), no kugenga imikoranire y'ibimera n'udukoko (Nehela na Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). Ni ngombwa kumenya ko uburyo bwihariye n'uruhare rwa PA mu kwirinda ibimera bitandukanye bitewe n'ubwoko bw'ibimera, indwara zitera indwara, n'ibidukikije. PA nyinshi cyane mu bimera ni biosynthesized iva muri polyamine L-ornithine y'ingenzi (Killiny na Nehela, 2020).
L-ornithine igira uruhare runini mu mikurire n'iterambere ry'ibimera. Urugero, ubushakashatsi bwakozwe mbere bwagaragaje ko mu muceri (Oryza sativa), ornithine ishobora kuba ifitanye isano no kongera gukoresha azote (Liu et al., 2018), umusaruro w'umuceri, ubwiza n'impumuro nziza (Lu et al., 2020), ndetse no guhangana n'ikibazo cy'amazi (Yang et al., 2000). Byongeye kandi, gukoresha L-ornithine hanze byazamuye cyane kwihanganira amapfa muri beterave y'isukari (Beta vulgaris) (Hussein et al., 2019) kandi bigabanyije ikibazo cy'umunyu mu bitunguru (Allium Cepa) (Çavuşoǧlu na Çavuşoǧlu, 2021) na cashew (Anacardium occidentale) (da Rocha et al., 2012). Uruhare rwa L-ornithine mu kurinda ikibazo cy'ibinyabuzima rushobora guterwa no kugira uruhare mu kwibumbira kwa proline mu bimera byavuwe. Urugero, jeni zijyanye na metabolism ya proline, nka ornithine delta aminotransferase (delta-OAT) na jeni za proline dehydrogenase (ProDH1 na ProDH2), zavuzwe mbere ko zigira uruhare mu kurinda Nicotiana benthamiana na Arabidopsis thaliana ku bwoko bwa Pseudomonas syringae butari ubwazo (Senthil-Kumar na Mysore, 2012). Ku rundi ruhande, fungus ornithine decarboxylase (ODC) irakenewe kugira ngo indwara zikure (Singh et al., 2020). Kwibanda kuri ODC ya Fusarium oxysporum f. sp. lycopersici binyuze mu guceceka kwa gene iterwa n'umurwayi (HIGS) byongereye cyane ubudahangarwa bw'ibimera by'inyanya kuri Fusarium wilt (Singh et al., 2020). Ariko, uruhare rushoboka rw'ikoreshwa rya ornithine yo hanze mu kurwanya stress za biotic nka phytopathogens ntirwigeze rwigwa neza. Icy'ingenzi kurushaho, ingaruka za ornithine ku kudakira indwara ndetse n'ingaruka za biochemical n'imikorere y'umubiri ziracyari zitaramenyekana cyane.
Gusobanukirwa uburyo S. sclerotiorum yandura ibinyamisogwe ni ingenzi mu guteza imbere ingamba zo kubirwanya neza. Muri ubu bushakashatsi, twari tugamije kumenya uruhare rushoboka rwa diamine L-ornithine nk'ikintu cy'ingenzi mu kongera uburyo bwo kwirinda no kurwanya ibinyamisogwe kwandura Sclerotinia sclerotiorum. Dutekereza ko, uretse kongera uburyo ibinyamisogwe byitwara mu kwirinda, L-ornithine inagira uruhare runini mu kubungabunga redox. Turasaba ko ingaruka zishobora guterwa na L-ornithine zijyanye no kugenzura uburyo bwo kwirinda antioxydants mu by’imyuka n’izitari zo mu by’imyuka ndetse no kubangamira ibintu bitera indwara/uburozi bw’ibinyamisogwe hamwe na poroteyine zijyanye nabyo. Iyi mikorere ibiri ya L-ornithine ituma iba umukandida mwiza wo gufata ingamba zirambye zo kugabanya ingaruka z’ibinyamisogwe byera no kongera ubudahangarwa bw’ibinyamisogwe bisanzwe kuri iyi ndwara ikomeye y’ibinyamisogwe. Ibisubizo by’ubu bushakashatsi bishobora gufasha mu guteza imbere uburyo bushya kandi butangiza ibidukikije bwo kurwanya ibinyamisogwe byera no kugabanya ingaruka zabyo ku musaruro w’ibinyamisogwe.
Muri ubu bushakashatsi, ubwoko bw'ibishyimbo bisanzwe bushobora kwangirika, Giza 3 (Phaseolus vulgaris L. cv. Giza 3), bwakoreshejwe nk'ibikoresho byo kugerageza. Imbuto nziza zatanzwe n'Ishami rishinzwe Ubushakashatsi ku Bihingwa byo mu Girima (FCRI), Ikigo cy'Ubushakashatsi ku Buhinzi (ARC), mu Misiri. Imbuto eshanu zatewe mu nkono za pulasitiki (umurambararo w'imbere wa cm 35, ubujyakuzimu bwa cm 50) zuzuyemo ubutaka bwanduye S. sclerotiorum mu gihe cy'ubushyuhe (25 ± 2 °C, ubushuhe 75 ± 1%, amasaha 8 y'urumuri/amasaha 16 yijimye). Nyuma y'iminsi 7-10 nyuma yo gutera (DPS), ingemwe zagabanijwe kugira ngo hasigara ingemwe ebyiri gusa zifite imikurire imwe n'amababi atatu yagutse muri buri nkono. Ibimera byose byashyizwe mu nkono byavomererwaga rimwe mu byumweru bibiri kandi bigafumbirwa buri kwezi ku gipimo cyagenwe ku bwoko bwatanzwe.
Mu gutegura igipimo cya 500 mg/L cya L-ornithinediamine (izwi kandi nka (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, mu Budage), 50 mg yabanje gushonga mu mazi ya 100 mL. Umuti wakuwemo amazi waravanzwe hanyuma ukoreshwa mu igerageza ryakurikiyeho. Muri make, urukurikirane rutandatu rw'ibipimo bya L-ornithine (12.5, 25, 50, 75, 100, na 125 mg/L) rwageragejwe muri vitro. Byongeye kandi, amazi yakuwemo amazi yakoreshejwe nk'umuti urwanya indwara (Mock) naho umuti urwanya indwara wo mu bucuruzi witwa "Rizolex-T" 50% by'ifu itose (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Intara ya Gharbia, Misiri) wakoreshejwe nk'umuti urwanya indwara. Umuti urwanya udukoko "Rizolex-T" wakorewe mu bucuruzi bwa vitro ku gipimo cya gatanu (2, 4, 6, 8 na 10 mg/L).
Ingero z'imiti n'ibishishwa by'ibishyimbo bisanzwe bigaragaza ibimenyetso bisanzwe by'ibihumyo byera (igipimo cy'ubwandu: 10–30%) byakusanyijwe mu mirima y'ubucuruzi. Nubwo ibyinshi mu bimera byanduye byagaragajwe n'ubwoko/ubwoko (ubwoko bwa Giza 3 bushobora kwanduzwa), ibindi, cyane cyane ibyavuye mu masoko yo mu gace, byari amoko atazwi. Ibikoresho byanduye byakusanyijwe byabanje gusukurwa hejuru hifashishijwe umuti wa 0.5% wa sodium hypochlorite mu gihe cy'iminota 3, hanyuma byozwa inshuro nyinshi n'amazi yanduye hanyuma bihanagurwa n'urupapuro rwanduye kugira ngo bikureho amazi arenze. Hanyuma ingingo zanduye zaciwemo uduce duto two mu gice cyo hagati (hagati y'uturemangingo tuzima n'utwanduye), bigahingwa ku gice cy'amazi cya potato dextrose agar (PDA) hanyuma bishyirwa ku bushyuhe bwa 25 ± 2 °C hakoreshejwe urumuri rw'amasaha 12/amasaha 12 mu gihe cy'iminsi 5 kugira ngo hakorwe sclerotia. Uburyo bwa mycelial tip bwakoreshejwe kandi mu gusukura fungus zometse ku miterere ivanze cyangwa yanduye. Indwara y’ibihumyo yasukuwe yabanje kumenyekana hashingiwe ku miterere yayo y’umuco hanyuma byemezwa ko ari S. sclerotiorum hashingiwe ku miterere ya mikorosikopi. Amaherezo, indwara zose zasukuwe zapimwe kugira ngo harebwe niba hari indwara ku gihingwa cy’ibishyimbo bisanzwe cya Giza 3 kugira ngo hahuzwe n’indwara za Koch.
Byongeye kandi, S. sclerotiorum isolate (isolate #3) yagaragaye cyane hashingiwe ku buryo bwo gusesengura imiterere y’uturemangingo (internal transcribed spacer (ITS) nk’uko byasobanuwe na White et al., 1990; Baturo-Ciesniewska et al., 2017. Muri make, uturemangingo twatewe mu musururu wa potato dextrose (PDB) hanyuma dushyirwa ku bushyuhe bwa 25 ± 2 °C mu gihe cy’iminsi 5-7. Hanyuma mycelium ya fungal yakusanyijwe, iyungururwa mu mwenda wa cheesecloth, yozwa kabiri n’amazi yanduye, hanyuma yumishwa n’urupapuro rwa filter rwanduye. ADN ya genomic yakuwemo hakoreshejwe Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Hanyuma agace ka ITS rDNA karazamuwe hakoreshejwe primer pair yihariye ITS1/ITS4 (TCCGTAGGTGAACCTGGCG TCCTCGCTTTATTGATATGC; ingano yitezwe: 540 bp) (Baturo-Ciesniewska et al., 2017). Ibicuruzwa bya PCR byatunganyijwe byatanzwe kugira ngo bikurikirane (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). ITS rDNA sequences zakurikiranye impande ebyiri hakoreshejwe uburyo bwa Sanger sequencing. Itsinda ry'ibibazo ryakusanyijwe ryagereranyijwe n'amakuru aheruka muri GenBank na National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/) hakoreshejwe porogaramu ya BLASTn. Urukurikirane rw'ibibazo rwagereranyijwe n'izindi 20 za S. sclerotiorum strains/isolates zakuwe mu makuru aheruka muri NCBI GenBank (Imbonerahamwe Y'inyongera S1) hakoreshejwe ClustalW muri Molecular Evolutionary Genetics Analysis Package (MEGA-11; verisiyo ya 11) (Kumar et al., 2024). Isesengura ry'ihindagurika ryakozwe hakoreshejwe uburyo bwa maximum likelihood na general time-reversible nucleotide substitution model (Nei na Kumar, 2000). Igiti gifite likelihood yo hejuru cyane cyagaragajwe. Igiti cya mbere cyo gushakisha heuristic gitoranywa hagatoranywa igiti gifite likelihood yo hejuru hagati y'igiti gihuza umuturanyi (NJ) (Kumar et al., 2024) n'igiti cya maximum parsimony (MP). Igiti cya NJ cyubatswe hakoreshejwe matrix ya distance ya pairwise yabazwe hakoreshejwe general time-reversible model (Nei na Kumar, 2000).
Uburyo L-ornithine ikora ku mikorobe n'umuti wica udukoko "Rizolex-T" byagenwe muri vitro hakoreshejwe uburyo bwa agar diffusion. Uburyo: Fata ingano ikwiye y'umuti wa L-ornithine (500 mg/L) hanyuma uvange neza na 10 ml y'intungamubiri za PDA kugira ngo utegure ibisubizo bifite ingano ya nyuma ya 12.5, 25, 50, 75, 100 na 125 mg/L, uko bikurikirana. Ingano eshanu z'umuti wica udukoko "Rizolex-T" (2, 4, 6, 8 na 10 mg/L) hamwe n'amazi yakuwemo imiti yakoreshejwe nk'uburyo bwo kugenzura. Nyuma y'uko uwo muti umaze gukomera, agace gashya ka mycelial ka Sclerotinia sclerotiorum, gafite umurambararo wa mm 4, kimuriwe hagati mu isafuriya ya Petri maze gashyirwa ku bushyuhe bwa 25±2°C kugeza igihe mycelium itwikiriye isafuriya yose ya Petri, hanyuma hagafatwa inyigo y'imiti. Kubara ijanisha ry'ikumirwa ry'ikura rya S. sclerotiorum hakoreshejwe ikigereranyo cya 1:
Igerageza ryasubiwemo kabiri, hamwe n’ibyiciro bitandatu by’ibinyabuzima kuri buri tsinda ry’igenzura/igerageza n’inkono eshanu (ibimera bibiri kuri buri nkono) kuri buri kigero cy’ibinyabuzima. Buri kigero cy’ibinyabuzima cyasesenguwe kabiri (ibyiciro bibiri by’ikoranabuhanga) kugira ngo harebwe neza, byizewe kandi bikorwe neza. Byongeye kandi, isesengura rya probit regression ryakoreshejwe mu kubara igipimo cy’inzitizi cya kabiri (IC50) na IC99 (Prentice, 1976).
Kugira ngo harebwe ubushobozi bwa L-ornithine mu gihe cy’ubushyuhe, hakozwe igerageza ry’inkono ebyiri zikurikiranye. Muri make, inkono zuzuye ubutaka bw’ibumba ryahinduwemo umuti (3:1) kandi ziterwa umuti wa S. sclerotiorum. Ubwa mbere, igice kinini cya S. sclerotiorum (isolate #3) cyatewe hakoreshejwe uburyo bwo gukata sclerotium imwe mo kabiri, ishyirwa ireba hasi kuri PDA hanyuma ishyirwa kuri 25°C mu mwijima udasibangana (amasaha 24) mu gihe cy’iminsi 4 kugira ngo hakorwe imikurire ya mycelial. Hanyuma hafashwe uduce tune twa mm 5 tw’umurambararo wa agar ku nkengero z’imbere maze duterwa garama 100 z’uruvange rw’ingano n’umuceri (1:1, v/v) maze amacupa yose ashyirwa kuri 25 ± 2 °C munsi y’umwijima w’amasaha 12 mu gihe cy’iminsi 5 kugira ngo sclerotia ikore neza. Ibikubiye mu macupa yose byavanzwe neza kugira ngo harebwe ko hari ubwumvikane mbere yo kongeramo ubutaka. Hanyuma, garama 100 z'uruvange rw'ibinyampeke byongewe muri buri nkono kugira ngo habeho ubwinshi buhoraho bw'udukoko twanduza. Inkono zateweho inshinge zaravomerewe kugira ngo zitere imikurire y'ibihumyo kandi zishyirwe mu bushyuhe bw'iminsi 7.
Imbuto eshanu zo mu bwoko bwa Giza 3 zatewe muri buri nkono. Ku nkono zavuwe na L-ornithine na Rizolex-T, imbuto zasizwe zabanje kunyobwa amasaha abiri mu gisubizo cy’amazi cy’ibinyabutabire bibiri hamwe n’icyiciro cya nyuma cya IC99 kingana na 250 mg/L na 50 mg/L, hanyuma zikanikwa mu kirere isaha imwe mbere yo kubiba. Ku rundi ruhande, imbuto zasizwe mu mazi yakuwemo nk’isuku kugira ngo zigenzure nabi. Nyuma y’iminsi 10, mbere yo kuhira bwa mbere, imbuto zasizwe, hasigara gusa imbuto ebyiri nziza muri buri nkono. Byongeye kandi, kugira ngo hamenyekane ko S. sclerotiorum yandura, amashami y’ibishyimbo ari mu cyiciro kimwe cy’imikurire (iminsi 10) yaciwe ahantu habiri hatandukanye hakoreshejwe icyuma cyasizweho isuku, hashyirwa hafi garama 0.5 z’uruvange rw’ibinyabutabire muri buri gikomere, hagakurikiraho ubushuhe bwinshi kugira ngo hakorwe ubwandu n’indwara mu bimera byose byatewe inshinge. Ibihingwa byo kurwanya indwara na byo byakomeretse kimwe, kandi ingano ingana na 0.5 g y'uruvange rw'ibinyampeke byangiritse kandi bitaravangwa n'amagufwa byashyizwe mu gikomere biguma mu bushyuhe bwinshi kugira ngo byigane ibidukikije kugira ngo indwara ikure neza kandi ikomeze kuba myiza hagati y'amatsinda avura.
Uburyo bwo kuvura: Imbuto z'ibishyimbo zavomerewe na mililitiro 500 z'amazi ya L-ornithine (250 mg/l) cyangwa umuti urwanya udukoko witwa Rizolex-T (50 mg/l) binyuze mu kuhira ubutaka, hanyuma ubuvuzi busubirwamo gatatu mu gihe cy'iminsi 10. Isuzuma ryakozwe na placebo ryavomerewe na mililitiro 500 z'amazi yakuwemo. Uburyo bwose bwo kuvura bwakozwe mu gihe cy'ubushyuhe (25 ± 2°C, 75 ± 1% y'ubushyuhe, n'igihe cy'urumuri rw'amasaha 8/amasaha 16 y'umwijima). Inkono zose zavomererwaga buri byumweru bibiri kandi buri kwezi zivurwa n'ifumbire ya NPK ingana (20-20-20, hamwe na 3.6% ya sulfur na TE microelements; Zain Seeds, Misiri) ku gipimo cya 3-4 g/l hakoreshejwe gutera imbuto hakurikijwe inama ku bwoko bwihariye n'amabwiriza y'uwakoze. Keretse byavuzwe ukundi, amababi yakuze neza (amababi ya 2 n'aya 3 uhereye hejuru) yakusanyijwe muri buri bwoko bw'ibinyabuzima nyuma y'amasaha 72 nyuma yo kuvurwa (hpt), ashyirwa hamwe, ashyirwa hamwe kandi abikwa kuri -80 °C kugira ngo hakorwe isesengura rirambuye harimo, ariko ntirigarukira gusa ku, aho ibimenyetso by'ingufu za oxidative biherereye, peroxidation y'amavuta, antioxydants za enzymatic na non-enzymatic hamwe n'igaragara ry'uturemangingo.
Ubukana bw'ubwandu bw'ibihumyo byera bwasuzumwe buri cyumweru mu minsi 21 nyuma yo gutera (dpi) hakoreshejwe igipimo cya 1-9 (Imbonerahamwe y'inyongera S2) hashingiwe ku gipimo cya Petzoldt na Dickson (1996) cyahinduwe na Teran et al. (2006). Muri make, amashami n'amashami y'ibishyimbo byasuzumwe guhera aho byatewe kugira ngo hakurikiranwe uko ibikomere bigenda ku ntera y'ibikomere ku ntera n'udupfundo. Intera y'igikomere kuva aho cyatewe kugeza ku ntera ya kure ku ruti cyangwa ishami yapimwe maze amanota ya 1-9 atangwa hashingiwe aho igikomere cyabereye, aho (1) byagaragaje ko nta bwandu bugaragara hafi y'aho cyatewe na (2-9) byagaragaje ko ingano y'igikomere n'uko gikomeza ku ntera y'udupfundo/udupfundo (Imbonerahamwe y'inyongera S2). Ubukana bw'ubwandu bw'ibihumyo byera bwahinduwe mu ijanisha hakoreshejwe formula ya 2:
Byongeye kandi, agace kari munsi y’umurongo w’impinduka z’indwara (AUDPC) kabazwe hakoreshejwe formula (Shaner na Finney, 1977), iherutse guhindurwa ku kubora kw’ibishyimbo bisanzwe (Chauhan et al., 2020) hakoreshejwe ikigereranyo cya 3:
Aho Yi = ubukana bw'indwara mu gihe cya ti, Yi + 1 = ubukana bw'indwara mu gihe gitaha ti + 1, ti = igihe cyo gupima bwa mbere (mu minsi), ti + 1 = igihe cyo gupima ubutaha (mu minsi), n = umubare wose w'igihe cyangwa aho warebye. Ibipimo by'imikurire y'ibishyimbo birimo uburebure bw'igihingwa (cm), umubare w'amashami kuri buri gihingwa, n'umubare w'amababi kuri buri gihingwa byanditswe buri cyumweru mu minsi 21 mu binyabuzima byose.
Muri buri gipimo cy’ibinyabuzima, ingero z’amababi (amababi ya kabiri n’aya gatatu yakuze neza uhereye hejuru) zakusanyijwe ku munsi wa 45 nyuma yo kuvurwa (iminsi 15 nyuma yo kuvurwa bwa nyuma). Buri gipimo cy’ibinyabuzima cyari kigizwe n’inkono eshanu (ibimera bibiri kuri buri gikombe). Hafi 500 mg by’uruti rwaciwe byakoreshejwe mu gukuramo ibara rya fotosite (chlorophyll a, chlorophyll b na carotenoids) hakoreshejwe 80% ya acetone kuri 4 °C mu mwijima. Nyuma y’amasaha 24, ingero zashyizwe muri centrifuge maze supernatant ikusanywa kugira ngo hamenyekane chlorophyll a, chlorophyll b na carotenoid mu buryo bw’amabara hakoreshejwe spectrophotometer ya UV-160A (Shimadzu Corporation, Ubuyapani) hakurikijwe uburyo bwa (Lichtenthaler, 1987) hapimwa uburyo bwo kwinjiza amazi ku bunini butatu butandukanye (A470, A646 na A663 nm). Amaherezo, ibikubiye mu mabara ya fotosinte byapimwe hakoreshejwe formula zikurikira 4-6 zasobanuwe na Lichtenthaler (1987).
Nyuma y'amasaha 72 yo kuvurwa (hpt), amababi (amababi ya kabiri n'aya gatatu yakuze neza uhereye hejuru) yakusanyijwe muri buri gipimo cy’ibinyabuzima kugira ngo hamenyekane aho hydrogen peroxide (H2O2) na superoxide anion (O2 •−). Buri gipimo cy’ibinyabuzima cyari kigizwe n'inkono eshanu (ibimera bibiri kuri buri gikono). Buri gipimo cy’ibinyabuzima cyasesenguwe mu buryo bubiri (igipimo cy’ikoranabuhanga kibiri) kugira ngo hamenyekane neza, ko uburyo bworoshye kandi bushobora kongera kuboneka. H2O2 na O2 •− byagenwe hakoreshejwe 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Ubudage) cyangwa nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Ubudage), hakurikijwe uburyo bwasobanuwe na Romero-Puertas et al. (2004) na Adam et al. (1989) hamwe n'impinduka nto. Kugira ngo H2O2 imenyekane mu buryo bwa histochemical, udupapuro twashyizwemo 0.1% DAB muri 10 mM Tris buffer (pH 7.8) hanyuma dushyirwa ku bushyuhe bw'icyumba mu rumuri mu gihe cy'iminota 60. Udupapuro twashyizwemo 0.15% (v/v) TCA muri 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) hanyuma tujya ku rumuri kugeza igihe twijimye. Mu buryo nk'ubwo, utumashini twashyizwemo 10 mM potassium phosphate buffer (pH 7.8) irimo 0.1 w/v % HBT kugira ngo O2 •− imenyekane mu buryo bwa histochemical imenyekane mu gihe cy'iminota 20. Udupapuro twashyizwemo 0.1% DAB mu rumuri mu bushyuhe bw'icyumba mu gihe cy'iminota 20, hanyuma dushyirwamo 0.25% nk'uko byavuzwe haruguru, hanyuma tukamurika kugeza igihe hagaragaye utudomo tw'ubururu bwijimye/violet. Ubukana bw'ibara ry'umukara ryavuyemo (nk'ikimenyetso cya H2O2) cyangwa ubururu-violet (nk'ikimenyetso cya O2 •−) byapimwe hakoreshejwe verisiyo ya Fiji ya paki yo gutunganya amashusho ImageJ (http://fiji.sc; yabonetse ku ya 7 Werurwe 2024).
Malondialdehyde (MDA; nk'ikimenyetso cya peroxidation y'amavuta) yagenwe hakurikijwe uburyo bwa Du na Bramlage (1992) hamwe n'impinduka nto. Amababi yavuye kuri buri gipimo cy'ibinyabuzima (amababi ya kabiri n'aya gatatu yakuze neza uhereye hejuru) yakusanyijwe amasaha 72 nyuma yo kuvurwa (hpt). Buri gipimo cy'ibinyabuzima cyari gikubiyemo inkono eshanu (ibimera bibiri kuri buri nkono). Buri gipimo cy'ibinyabuzima cyasesenguwe mu nkono ebyiri (inkono ebyiri za tekiniki) kugira ngo harebwe neza, hizewe kandi ko uburyo bwo kongera kubyazwa umusaruro. Muri make, 0.5 g y'ibibabi by'ubutaka byakoreshejwe mu gukuramo MDA hamwe na aside trichloroacetic 20% (TCA; MilliporeSigma, Burlington, MA, USA) irimo 0.01% ya hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). Hanyuma ingano ya MDA muri supernatant yagenwe hakoreshejwe ibara ry'umubare w'ingufu hakoreshejwe uburyo bwa UV-160A spectrophotometer (Shimadzu Corporation, Ubuyapani) hanyuma igaragazwa nka nmol g−1 FW.
Mu gusuzuma antioxydants zitari enzyme na enzyme, amababi (amababi ya kabiri n'aya gatatu yakuze neza uhereye hejuru) yakusanyijwe muri buri binyabuzima byakusanyijwe nyuma y'amasaha 72 nyuma yo kuvurwa (hpt). Buri binyabuzima byakusanyijwe byari bigizwe n'inkono eshanu (ibimera bibiri kuri buri kibindi). Buri gipimo cy'ibinyabuzima cyasesenguwe mu buryo bubiri (ibipimo bibiri bya tekiniki). Amababi abiri yasibwe na azote y'amazi maze akoreshwa mu buryo butaziguye mu gusuzuma antioxydants za enzymatic na enzyme, aside amine yose, ingano ya proline, imiterere ya gene, no gupima oxalate.
Fenolike zose zishobora gushonga zapimwe hakoreshejwe Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) hamwe n'impinduka nto ku buryo bwasobanuwe na Kahkonen et al. (1999). Muri make, hafi garama 0.1 y'ibibabi byakuwemo imililitiro 20 ya methanol 80% mu mwijima mu gihe cy'amasaha 24 maze supernatant ikusanywa nyuma yo gukururwa. Mililitiro 0.1 y'icyitegererezo cyavanzwe na mililitiro 0.5 ya Folin-Ciocalteu reagent (10%), izunguzwa mu gihe cy'amasegonda 30 hanyuma isigara mu mwijima mu gihe cy'iminota 5. Hanyuma mililitiro 0.5 z'umuti wa sodium carbonate 20% (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Misiri) zongewe kuri buri muyoboro, zivanze neza kandi zishyirwa ku bushyuhe bw'icyumba mu mwijima mu gihe cy'isaha 1. Nyuma yo gukubitwa, uburyo bwo kwinjiza umwuka w’uruvange rw’ibisubizo byapimwe kuri 765 nm hakoreshejwe spectrophotometer ya UV-160A (Shimadzu Corporation, mu Buyapani). Igipimo cya phenols zose zishobora gushonga mu bicuruzwa byatanzwe cyapimwe hakoreshejwe curve yo gupima aside gallic (Fisher Scientific, Hampton, NH, USA) kandi cyagaragajwe nka miligarama za aside gallic zingana na garama imwe y’uburemere bushya (mg GAE g-1 uburemere bushya).
Ingano yose ya flavonoide ishobora gushonga yapimwe hakurikijwe uburyo bwa Djeridane et al. (2006) hamwe n'impinduka nto. Muri make, 0.3 ml y'umusaruro wa methanol wavuzwe haruguru yavanzwe na 0.3 ml y'umuti wa 5% wa aluminiyumu chloride (AlCl3; Fisher Scientific, Hampton, NH, USA), iravunganywa cyane hanyuma ishyirwa mu bushyuhe bw'icyumba mu minota 5, hagakurikiraho kongeramo 0.3 ml y'umuti wa 10% wa potassium acetate (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), ivangwa neza kandi ishyirwa mu bushyuhe bw'icyumba mu minota 30 mu mwijima. Nyuma yo kuyishyira mu bwihisho, uburyo bwo kuyishyira mu bwihisho bwapimwe kuri 430 nm hakoreshejwe spectrophotometer ya UV-160A (Shimadzu Corporation, Japan). Ingano ya flavonoide zose zishobora gushonga mu bicuruzwa by'icyitegererezo yapimwe hakoreshejwe umuyoboro wa rutin calibration (TCI America, Portland, OR, USA) hanyuma igaragazwa nka miligarama za rutin zingana na garama imwe y'uburemere bushya (mg RE g-1 uburemere bushya).
Ingano yose y’aside amine y’ibishyimbo yapimwe hakoreshejwe reagent ya ninhydrine yahinduwe (Thermo Scientific Chemicals, Waltham, MA, USA) hashingiwe ku buryo bwatanzwe na Yokoyama na Hiramatsu (2003) kandi bwahinduwe na Sun et al. (2006). Muri make, 0.1 g y’uruti rw’umukungugu rwakuwemo pH 5.4 buffer, maze 200 μL y’umukungugu wa supernatant iterwa na 200 μL ya ninhydrine (2%) na 200 μL ya pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), ishyirwa mu bwogero bw’amazi abira mu gihe cy’iminota 30, hanyuma ikonjeshwa igapimwa kuri 580 nm hakoreshejwe spectrophotometer ya UV-160A (Shimadzu Corporation, Japan). Ku rundi ruhande, proline yapimwe hakoreshejwe uburyo bwa Bates (Bates et al., 1973). Proline yakuwemo aside sulfosalicylic ya 3% (Thermo Scientific Chemicals, Waltham, MA, USA) hanyuma nyuma yo gukurura, 0.5 ml ya supernatant yavanzwe na aside glacial asetic ya 1 ml (Fisher Scientific, Hampton, NH, USA) na reagent ya ninhydrin, bishyirwa kuri 90°C mu gihe cy'iminota 45, bikonjeshwa kandi bipimwe kuri 520 nm hakoreshejwe spectrophotometer imwe nk'iyavuzwe haruguru. Aside amine yose hamwe na proline mu bimera by'amababi byapimwe hakoreshejwe glycine na proline calibration curves (Sigma-Aldrich, St Louis, MO, USA), uko bikurikirana, kandi byagaragajwe nk'uburemere bushya bwa mg/g.
Kugira ngo hamenyekane imikorere ya enzymes zo kurwanya antioxidant, hakuwemo hafi 500 mg y'uturemangingo twahujwe hamwe na 3 ml ya 50 mM Tris buffer (pH 7.8) irimo 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) na 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), bishyirwa kuri centrifuge kuri 10.000 × g mu gihe cy'iminota 20 munsi y'ubukonje (4 °C), hanyuma supernatant (icyiciro cya enzyme) irakusanywa (El-Nagar et al., 2023; Osman et al., 2023). Catalase (CAT) yasubijwemo mililitiro 2 za 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) na 100 μl by'umuti wa H2O2 wa 269 mM kugira ngo hamenyekane imikorere ya enzyme hakurikijwe uburyo bwa Aebi (1984) hamwe n'impinduka nto (El-Nagar et al., 2023; Osman et al., 2023). Umurimo wa enzyme wa peroxidase (POX) ushingiye kuri Guaiacol wagenwe hakoreshejwe uburyo bwa Harrach et al. (2009). (2008) hamwe n'impinduka nto (El-Nagar et al., 2023; Osman et al., 2023) kandi imikorere ya enzyme ya polyphenol oxidase (PPO) yapimwe nyuma yo gukora isuzuma hamwe na 2.2 ml ya 100 mM sodium phosphate buffer (pH 6.0), 100 μl ya guaiacol (TCI chemicals, Portland, OR, USA) na 100 μl ya 12 mM H2O2. Ubu buryo bwahinduwe gato uhereye kuri (El-Nagar et al., 2023; Osman et al., 2023). Isuzuma ryakozwe nyuma yo gukora isuzuma hamwe na 3 ml ya catechol solution (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) nshya yateguwe muri 0.1 M phosphate buffer (pH 6.0). Igikorwa cya CAT cyapimwe hakurikiranwa uburyo H2O2 ibora kuri 240 nm (A240), igikorwa cya POX cyapimwe hakurikiranwa uburyo ubwiyongere bw'uburyo amashanyarazi ashyirwa kuri 436 nm (A436), naho igikorwa cya PPO gipimwa hakurikiranwa uburyo imihindagurikire y'uburyo amashanyarazi ashyirwa kuri 495 nm (A495) buri minota 30 mu gihe cy'iminota 3 hakoreshejwe spectrophotometer ya UV-160A (Shimadzu, Ubuyapani).
RT-PCR y'igihe nyacyo yakoreshejwe mu gusuzuma urwego rw'uturemangingo dutatu dufitanye isano na antioxydants, harimo peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), na glutathione reductase (PvGR; GenBank Accession No. KY195009.1), mu mababi y'ibishyimbo (amababi ya kabiri n'aya gatatu yakuze neza uhereye hejuru) amasaha 72 nyuma y'ubuvuzi bwa nyuma. Muri make, RNA yakuwemo hakoreshejwe Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, Ubushinwa) hakurikijwe protocole y'uruganda. Hanyuma, cDNA yakozwe hakoreshejwe TOP script™ cDNA Synthesis Kit hakurikijwe amabwiriza y'uruganda. Urutonde rw'uturemangingo dutatu twavuzwe haruguru ruri mu mbonerahamwe y'inyongera S3. PvActin-3 (Nimero yo kwinjira muri GenBank: XM_068616709.1) yakoreshejwe nk'ingirabuzimafatizo z'inzu kandi imiterere y'ingirabuzimafatizo yashyizwe mu byiciro hakoreshejwe uburyo bwa 2-ΔΔCT (Livak na Schmittgen, 2001). Guhagarara kwa Actin mu gihe cy'ingufu za biotic (imikoranire idahuye hagati y'ibinyamisogwe bisanzwe n'ibihumyo bya anthracnose Colletotrichum lindemuthianum) n'ingufu za abiotic (amapfa, umunyu, ubushyuhe buke) byagaragaye (Borges et al., 2012).
Twabanje gukora isesengura rya poroteyine ya oxaloacetate acetylhydrolase (OAH) muri S. sclerotiorum hakoreshejwe igikoresho cya poroteyine-protein BLAST (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Muri make, twakoresheje OAH yo muri Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; GenBank accession nomero XP_040799428.1; aside amine 342) na Penicillium lagena (PlOAH; taxide: 94218; GenBank accession nomero XP_056833920.1; aside amine 316) nk'uruhererekane rw'ibibazo kugira ngo dufate poroteyine ihuye muri S. sclerotiorum (taxide: 5180). BLASTp yakozwe hifashishijwe amakuru ya genome ya S. sclerotiorum aherutse kuboneka muri GenBank ku rubuga rwa interineti rw'Ikigo cy'Igihugu cy'Amakuru ya Biotechnology (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Byongeye kandi, gene ya OAH yahanuwe iturutse kuri S. sclerotiorum (SsOAH) hamwe n'isesengura ry'ihindagurika n'igiti cya phylogenetike cya AfOAH cyavuye kuri A. fijiensis CBS 313.89 na PlOAH byaturutse kuri P. lagena byafashwe hakoreshejwe uburyo bwa maximum likelihood muri MEGA11 (Tamura et al., 2021) na JTT matrix-based model (Jones et al., 1992). Igiti cya phylogenetike cyahujwe n'isesengura ry'uburyo bwinshi bwo guhuza poroteyine mu magene yose ya OAH yahanuwe (SsOAH) yavuye kuri S. sclerotiorum n'urukurikirane rw'ibibazo hakoreshejwe Igikoresho cyo Kugena Ibipimo (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos na Agarwala, 2007). Byongeye kandi, uburyo bwiza bwo guhuza aside amine ya SsOAH na S. sclerotiorum bwahujwe n'uburyo bwo gushakisha (AfOAH na PlOAH) (Larkin et al., 2007) hakoreshejwe ClustalW (http://www.genome.jp/tools-bin/clustalw), kandi uturere twabitswe mu buryo bwo gushakisha twagaragajwe hakoreshejwe igikoresho cya ESPRipt (version 3.0; https://espript.ibcp.fr/ESPRipt/ESPRipt/index.php).
Byongeye kandi, imiterere y’imikorere yahanuwe hamwe n’ahantu habitswe S. sclerotiorum SsOAH byashyizwe mu miryango itandukanye hakoreshejwe igikoresho cya InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Amaherezo, icyitegererezo cy’imiterere ya S. sclerotiorum SsOAH cyahanuwe mu buryo butatu (3D) cyakozwe hakoreshejwe poroteyine Homology/Analogy Recognition Engine (Phyre2 server verisiyo ya 2.0; http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) (Kelley et al., 2015) kandi byemezwa hakoreshejwe seriveri ya SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). Imiterere y’ibice bitatu byahanuwe (imiterere ya PDB) yagaragajwe hakoreshejwe uburyo bwo guhuza hakoreshejwe paki ya UCSF-Chimera (version 1.15; https://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004).
PCR y’ubwinshi bw’urumuri rw’igihe nyacyo yakoreshejwe mu kumenya urugero rw’inyandiko ya oxaloacetate acetylhydrolase (SsOAH; GenBank accession number: XM_001590428.1) muri mycelia ya Sclerotinia sclerotiorum. Muri make, S. sclerotiorum yatewe mu icupa ririmo PDB maze ishyirwa mu cyuma gishyushya (icyitegererezo: I2400, New Brunswick Scientific Co., Edison, NJ, USA) ku bushyuhe bwa 25 ± 2 °C mu gihe cy’amasaha 24 kuri 150 rpm no mu mwijima udashira (amasaha 24) kugira ngo itere imbaraga mu gukura kwa mycelial. Nyuma yaho, uturemangingo twavuwe na L-ornithine na Rizolex-T fungicide ku gipimo cya nyuma cya IC50 (hafi 40 na 3.2 mg/L, uko bikurikirana) hanyuma duhingwa mu gihe cy’andi masaha 24 mu bihe bimwe. Nyuma yo gukubitwa, imbuto zashyizwe mu cyuma gipima 2500 rpm mu minota 5 hanyuma hakusanywa supernatant (mycelium y'ibihumyo) kugira ngo hakoreshwe isesengura ry'imyanya y'uturemangingo. Mu buryo nk'ubwo, mycelium y'ibihumyo yakusanyijwe ku masaha 0, 24, 48, 72, 96, na 120 nyuma yo kwandura ibimera byanduye byari byakoze ifu y'umweru na mycelium y'ipamba ku buso bw'uturemangingo twanduye. RNA yakuwe muri mycelium y'ibihumyo hanyuma cDNA ikorwa nk'uko byasobanuwe haruguru. Urutonde rw'ibanze rwa SsOAH ruri mu mbonerahamwe y'inyongera S3. SsActin (nomero ya GenBank yo kwinjira: XM_001589919.1) yakoreshejwe nk'ingirabuzimafatizo, kandi igaragara ry'ingirabuzimafatizo ryabazwe hakoreshejwe uburyo bwa 2-ΔΔCT (Livak na Schmittgen, 2001).
Aside Oxalic yapimwe mu musupu w’ibirayi (PDB) no mu bimera birimo indwara ya Sclerotinia sclerotiorum hakurikijwe uburyo bwa Xu na Zhang (2000) hamwe n’impinduka nto. Muri make, S. sclerotiorum isolates yatewe mu macupa arimo PDB hanyuma iterwa mu cyuma gishyushya (icyitegererezo I2400, New Brunswick Scientific Co., Edison, NJ, USA) ku muvuduko wa 150 rpm kuri 25 ± 2 °C mu gihe cy’iminsi 3-5 mu mwijima udasibangana (amasaha 24) kugira ngo hakorwe ikura rya mycelium. Nyuma yo kuyikuramo, ubwiza bw’imiti bwabanje kuyungururwa binyuze mu mpapuro za Whatman #1 hanyuma bushyirwa kuri centrifuge kuri 2500 rpm mu gihe cy’iminota 5 kugira ngo hakuremo mycelium yasigaye. Supernatant yakusanyijwe ikabikwa kuri 4 °C kugira ngo hamenyekane neza ingano ya oxalate. Mu gutegura ingero z’ibimera, hafi garama 0.1 z’ibice by’ibimera byakuwemo gatatu n’amazi yaciwe (2 ml buri gihe). Ingero zashyizwe muri centrifuge ku muvuduko wa 2500 rpm mu gihe cy'iminota 5, supernatant yumye inyuzwa mu mpapuro za Whatman No. 1 hanyuma zikusanywa kugira ngo zikorerwe isesengura ryinshi.
Mu gusesengura aside oxalic, uruvange rw'ibisubizo rwateguwe mu muyoboro w'ikirahure mu buryo bukurikira: 0.2 ml y'icyitegererezo (cyangwa PDB culture filtrate cyangwa oxalic acid standard solution), 0.11 ml ya bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), 0.198 ml ya aside sulfuric 1 M (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) na 0.176 ml ya 100 mM potassium dichromate (K2Cr2O7; TCI chemicals, Portland, OR, USA), hanyuma umuti uvangwa na 4.8 ml n'amazi yaciwe, uvangwa cyane hanyuma uhita ushyirwa mu bwogero bw'amazi bwa 60 °C. Nyuma y'iminota 10, igisubizo cyahagaze hongerwamo 0.5 ml ya sodium hydroxide solution (NaOH; 0.75 M). Uburyo bwo kwinjiza (A600) bw'uruvange rw'ibisubizo bwapimwe kuri 600 nm hakoreshejwe spectrophotometer ya UV-160 (Shimadzu Corporation, Ubuyapani). PDB n'amazi yakuwemo byakoreshejwe nk'uburyo bwo kugenzura ibipimo by'ibice by'imbuto n'ibimera, uko bikurikirana. Igipimo cya aside oxalic mu bice by'imbuto, bigaragazwa nka mikorogarama za aside oxalic kuri mililitiro ya PDB medium (μg.mL−1), no mu bikomoka ku mababi, bigaragazwa nka mikorogarama za aside oxalic kuri garama y'uburemere bushya (μg.g−1 FW), byapimwe hakoreshejwe uburyo bwo gupima aside oxalic (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
Mu bushakashatsi bwose, igerageza ryose ryakozwe mu buryo bwa "default" (CRD) aho hakozwe kopi esheshatu z'ibinyabuzima kuri buri muti n'inkono eshanu kuri buri gikoresho cy'ibinyabuzima (ibimera bibiri kuri buri gikoresho) keretse iyo byavuzwe ukundi. Inyandiko zakozwe mu buryo bwa "biologique" zasesenguwe mu buryo bwa "double" (kopi ebyiri za tekiniki). Inyandiko zakozwe mu buryo bwa "tekinique" zakoreshejwe mu kugenzura uburyo igerageza rimwe rishobora kongera kuboneka ariko ntizakoreshejwe mu isesengura ry'ibarurishamibare kugira ngo hirindwe kopi zidasanzwe. Amakuru yasesenguwe mu buryo bwa "statistical" hakoreshejwe isesengura ry'impinduka (ANOVA) hagakurikiraho ikizamini cya Tukey-Kramer cy'impinduka zikomeye (HSD) (p ≤ 0.05). Ku igerageza rya "in vitro", agaciro ka IC50 na IC99 kabazwe hakoreshejwe uburyo bwa "probit" kandi hakorwa "intervals" ya 95%.
Hakusanyijwe ubwoko bune bw’ibimera byakuwe mu mirima itandukanye ya soya mu ntara ya El Ghabiya, mu Misiri. Ku gice cya PDA, ubwoko bwose bw’ibimera bwatanze mycelium y’umweru ihumura neza yahindukaga umweru vuba (Ishusho ya 1A) hanyuma ikagira ibara ry’umukara cyangwa umukara ku cyiciro cya sclerotium. Sclerotia akenshi iba ikomeye, yirabura, ifite imiterere y’uruziga cyangwa idahuje, ifite uburebure bwa mm 5.2 kugeza 7.7 na mm 3.4 kugeza 5.3 z’umurambararo (Ishusho ya 1B). Nubwo ubwoko bune bw’ibimera bwagize imiterere y’ibimera ku nkengero z’igice cya sclerotium nyuma y’iminsi 10-12 yo gucukura kuri 25 ± 2 °C (Ishusho ya 1A), umubare wa sclerotium kuri buri gice wari utandukanye cyane muri byo (P < 0.001), aho igice cya 3 ari cyo gifite umubare munini wa sclerotium (32.33 ± 1.53 sclerotium kuri buri gice; Ishusho ya 1C). Mu buryo nk'ubwo, isolate #3 yakuyemo aside oxalic nyinshi muri PDB kurusha izindi isolate (3.33 ± 0.49 μg.mL−1; Ishusho ya 1D). Isolate #3 yagaragaje imiterere isanzwe ya phytopathogenic fungus Sclerotinia sclerotiorum. Urugero, kuri PDA, amakoloni ya isolate #3 yakuze vuba, yari umweru mwiza (Ishusho ya 1A), salmon yoroheje cyangwa umuhondo w'umuhondo, kandi byasabye iminsi 6-7 kuri 25 ± 2°C kugira ngo ipfuke neza ubuso bw'isahani ya cm 9. Hashingiwe ku miterere ya morphological na microscopic yavuzwe haruguru, isolate #3 yagaragaye ko ari Sclerotinia sclerotiorum.
Ishusho ya 1. Ibiranga n'ingaruka z'indwara za S. sclerotiorum zikomoka ku bimera bisanzwe by'ibinyamisogwe. (A) Ikura ry'udukoko tune twa S. sclerotiorum ku buryo bwa PDA, (B) sclerotia y'udukoko tune twa S. sclerotiorum, (C) umubare wa sclerotia (kuri buri sahani), (D) isohoka rya aside oxalic ku buryo bwa PDB (μg.mL−1), na (E) ubukana bw'indwara (%) y'udukoko tune twa S. sclerotiorum ku binyamisogwe byo mu bucuruzi bya Giza 3 mu gihe cy'ubushyuhe. Ingano zigaragaza impuzandengo ya ± SD y'udukoko dutanu tw'ibinyabuzima (n = 5). Inyuguti zitandukanye zigaragaza itandukaniro rikomeye hagati y'imiti (p < 0.05). (F–H) Ibimenyetso bisanzwe by'ibihumyo byera byagaragaye ku giti cyo hejuru y'ubutaka na silique, uko bikurikirana, iminsi 10 nyuma yo gutera inshinge hamwe na isolate #3 (dpi). (I) Isesengura ry’ubwihindurize bw’agace k’inyuma k’itsinda rya S. sclerotiorum isolate #3 ryakozwe hakoreshejwe uburyo bwa maximum likelihood kandi rigereranywa n’itsinda 20 ry’itsinda ryakuwe muri database y’Ikigo cy’Igihugu cy’Amakuru ya Biotechnology (NCBI) (https://www.ncbi.nlm.nih.gov/). Imibare iri hejuru y’imirongo y’itsinda yerekana akarere gaherereyemo (%), naho imibare iri munsi y’imirongo y’itsinda igaragaza uburebure bw’ishami.
Byongeye kandi, kugira ngo hemezwe ko indwara iterwa n’iyi ndwara, hakoreshejwe udukoko tune twa S. sclerotiorum twometse mu gutera ibishyimbo byo mu bwoko bwa Giza 3 mu gihe cy’ubushyuhe, ibi bikaba bihuye n’imiterere ya Koch (Ishusho ya 1E). Nubwo udukoko twometse twometse twometse twometse twashoboraga kwanduza ibishyimbo bibisi (cv. Giza 3), bigatera ibimenyetso bisanzwe by’ibihumyo by’umweru ku bice byose biri hejuru y’ubutaka (Ishusho ya 1F), cyane cyane ku mizi (Ishusho ya 1G) no ku biti by’imbuto (Ishusho ya 1H) mu minsi 10 nyuma yo gutera (dpi), agakoko ka 3 ni ko kari gakomeye cyane mu bushakashatsi bubiri bwigenga. Agakoko ka 3 kari gafite ubukana bwinshi bw’indwara (%) ku bihingwa by’ibishyimbo (24.0 ± 4.0, 58.0 ± 2.0, na 76.7 ± 3.1 mu minsi 7, 14, na 21 nyuma yo kwandura; Ishusho ya 1F).
Kumenya S. sclerotiorum isolate #3 itera cyane byanemejwe hashingiwe ku buryo bwo guhuza imiterere y’ingirabuzimafatizo (ITS) (Ishusho ya 1I). Isesengura rya phylogenetic hagati y’ingirabuzimafatizo #3 na 20 byagaragaje isano iri hagati yazo (>99%). Ni ngombwa kumenya ko S. sclerotiorum isolate #3 (533 bp) isa cyane na S. sclerotiorum isolate LPM36 yo muri Amerika yakuwe mu mbuto z’amashaza yumye (GenBank accession nomero MK896659.1; 540 bp) na S. sclerotiorum isolate yo mu Bushinwa YKY211 (GenBank accession nomero OR206374.1; 548 bp), bitera kubora kw’igiti cya violet (Matthiola incana), byose bishyizwe mu matsinda ukwayo hejuru ya dendrogram (Ishusho ya 1I). Uruhererekane rushya rwashyizwe muri database ya NCBI rwitwa "Sclerotinia sclerotiorum - isolate YN-25" (nomero ya GenBank PV202792). Bigaragara ko isolate 3 ari yo isolate ikunze kwibasira abantu benshi; bityo, iyi isolate yatoranijwe kugira ngo ikorerweho ubushakashatsi mu bushakashatsi bwose bwakurikiyeho.
Uburyo diamine L-ornithine (Sigma-Aldrich, Darmstadt, mu Budage) ikora ku bipimo bitandukanye (12.5, 25, 50, 75, 100 na 125 mg/L) ikora ku kigero cya S. sclerotiorum isolate 3 byakorewe ubushakashatsi muri vitro. Ni ngombwa kumenya ko L-ornithine yagize ingaruka ku mikorobe kandi ikabuza buhoro buhoro gukura kwa S. sclerotiorum hyphae mu buryo bushingiye ku gipimo (Ishusho ya 2A, B). Ku gipimo kinini cyapimwe (125 mg/L), L-ornithine yagaragaje igipimo kinini cyane cyo gukumira gukura kwa mycelial (99.62 ± 0.27%; Ishusho ya 2B), kingana n'umuti urwanya udukoko wa Rizolex-T (igipimo cyo gukumira 99.45 ± 0.39%; Ishusho ya 2C) ku gipimo kinini cyapimwe (10 mg/L), bigaragaza ko imikorere isa.
Ishusho ya 2. Imikorere ya L-ornithine mu kurwanya Sclerotinia sclerotiorum irwanya bagiteri. (A) Kugereranya imikorere ya bagiteri y’ibipimo bitandukanye bya L-ornithine kuri S. sclerotiorum n’umuti urwanya fungikasi ucuruzwa Rizolex-T (10 mg/L). (B, C) Igipimo cyo kubuza (%) gukura kwa S. sclerotiorum mycelial nyuma yo kuvurwa n’ibipimo bitandukanye bya L-ornithine (12.5, 25, 50, 75, 100 na 125 mg/L) cyangwa Rizolex-T (2, 4, 6, 8 na 10 mg/L), uko bikurikirana. Ingano zigaragaza impuzandengo ± SD y’ibipimo bitanu by’ibinyabuzima (n = 5). Inyuguti zitandukanye zigaragaza itandukaniro ry’imibare hagati y’ubuvuzi (p < 0.05). (D, E) Isesengura rya probit model regression rya L-ornithine n’umuti urwanya fungikasi ucuruzwa Rizolex-T, uko bikurikirana. Umurongo wo gusubira inyuma w’icyitegererezo cya probit ugaragazwa nk'umurongo w'ubururu bukomeye, naho igihe cy'icyizere (95%) kigaragazwa nk'umurongo utukura ufite akadomo.
Byongeye kandi, isesengura rya probit regression ryakozwe kandi ibishushanyo bihuye nabyo bigaragazwa muri Table 1 na Figure 2D, E. Muri make, agaciro k’ubuso bw’imirongo (y = 2.92x - 4.67) hamwe n’imibare y’ingenzi ijyanye nabyo (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 na p < 0.0001; Figure 2D) bya L-ornithine byagaragaje ko imikorere y’imiti irwanya S. sclerotiorum yazamuwe ugereranije na Rizolex-T irwanya fungicide (y = 1.96x - 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 na p < 0.0001) (Tex 1).
Imbonerahamwe ya 1. Agaciro k'igice cy'umubare ntarengwa wo gukumira indwara (IC50) na IC99 (mg/l) bya L-ornithine n'umuti urwanya indwara ukomoka ku bimera "Rizolex-T" ukoreshwa mu kuvura S. sclerotiorum.
Muri rusange, L-ornithine (250 mg/L) yagabanyije cyane iterambere n'ubukana bw'ibihumyo byera ku bimera bisanzwe byavuwe ugereranije n'ibimera byanduye S. sclerotiorum bitavuwe (igenzura; Ishusho ya 3A). Muri make, nubwo ubukana bw'indwara y'ibimera byanduye bitavuwe byariyongereye buhoro buhoro (52.67 ± 1.53, 83.21 ± 2.61, na 92.33 ± 3.06%), L-ornithine yagabanyije cyane ubukana bw'indwara (%) mu gihe cyose cy'igerageza (8.97 ± 0.15, 18.00 ± 1.00, na 26.36 ± 3.07) ku minsi 7, 14, na 21 nyuma yo kuvurwa (dpt), uko bikurikirana (Ishusho ya 3A). Mu buryo nk'ubwo, igihe ibihingwa by'ibishyimbo byanduye S. sclerotiorum byavurwaga na 250 mg/L L-ornithine, agace kari munsi y'umurongo w'impinduka z'indwara (AUDPC) kagabanutse kava kuri 1274.33 ± 33.13 mu buryo butavuwe kagera kuri 281.03 ± 7.95, byari munsi gato y'umuti wa Rizolex-T fungicide 50 mg/L (183.61 ± 7.71; Ishusho ya 3B). Uko ni ko byagenze mu igerageza rya kabiri.
Ishusho ya 3. Ingaruka zo gukoresha L-ornithine hanze ku ihinduka ry'ububore bw'umweru bw'ibishyimbo bisanzwe buterwa na Sclerotinia sclerotiorum mu gihe cy'ubushyuhe. (A) Ingano y'indwara y'ibishyimbo byera by'ibishyimbo bisanzwe nyuma yo kuvurwa na 250 mg/L L-ornithine. (B) Agace kari munsi y'ingano y'indwara (AUDPC) y'ibishyimbo byera by'ibishyimbo bisanzwe nyuma yo kuvurwa na L-ornithine. Ingano zigaragaza impuzandengo ± SD y'ibipimo bitanu by'ibinyabuzima (n = 5). Inyuguti zitandukanye zigaragaza itandukaniro rikomeye hagati y'ubuvuzi (p < 0.05).
Gukoresha L-ornithine ya 250 mg/L L byongereye uburebure bw'igihingwa buhoro buhoro (Ishusho ya 4A), umubare w'amashami kuri buri gihingwa (Ishusho ya 4B), n'umubare w'amababi kuri buri gihingwa (Ishusho ya 4C) nyuma y'iminsi 42. Nubwo umuti urwanya udukoko witwa Rizolex-T (50 mg/L) wakoreshejwe mu bucuruzi wagize ingaruka zikomeye ku bipimo byose by'imirire byizweho, gukoresha L-ornithine ya 250 mg/L L byagize ingaruka ya kabiri nini ugereranije n'imiti yakoreshejwe mu kurwanya indwara (Ishusho ya 4A–C). Ku rundi ruhande, uburyo bwo kuvura L-ornithine nta ngaruka zikomeye bwagize ku bipimo bya chlorophyll a (Ishusho ya 4D) na chlorophyll b (Ishusho ya 4E), ariko bwongereye gato ingano ya carotenoid (0.56 ± 0.03 mg/g fr wt) ugereranije n’uburyo bwo kugenzura nabi (0.44 ± 0.02 mg/g fr wt) n’uburyo bwo kugenzura neza (0.46 ± 0.02 mg/g fr wt; Ishusho ya 4F). Muri rusange, ibi bisubizo bigaragaza ko L-ornithine idahumanya ibidukikije ku binyamisogwe byavuwe kandi ishobora no gutuma bikura.
Ishusho ya 4. Ingaruka zo gukoresha L-ornithine hanze ku miterere y'imikurire n'amabara ya photosynthetic y'amababi y'ibishyimbo yanduye Sclerotinia sclerotiorum mu gihe cy'ubushyuhe. (A) Uburebure bw'igihingwa (cm), (B) Umubare w'amashami kuri buri gihingwa, (C) Umubare w'amababi kuri buri gihingwa, (D) Chlorophyll (mg g-1 fr wt), (E) Chlorophyll (mg g-1 fr wt), (F) Igiteranyo cya carotenoid (mg g-1 fr wt). Agaciro ni impuzandengo ya ± SD y'ibipimo bitanu by'ibinyabuzima (n = 5). Inyuguti zitandukanye zigaragaza itandukaniro rikomeye hagati y'imiti (p < 0.05).
Gushyira mu mwanya wa histochemical w’ubwoko bwa ogisijeni ikora neza (ROS; igaragazwa nka hydrogen peroxide [H2O2]) na radicals yigenga (igaragazwa nka superoxide anions [O2•−]) byagaragaje ko gukoresha L-ornithine (250 mg/L) byagabanyije cyane ukwiyongera kwa H2O2 (96.05 ± 5.33 nmol.g−1 FW; Ishusho ya 5A) na O2•− (32.69 ± 8.56 nmol.g−1 FW; Ishusho ya 5B) ugereranije no kwiyongera kw’ibimera byombi byanduye bitavuwe (173.31 ± 12.06 na 149.35 ± 7.94 nmol.g−1 FW, uko bikurikirana) n’ibimera byavuwe na 50 mg/L bya Rizolex-T (170.12 ± 9.50 na 157.00 ± 7.81 nmol.g−1 fr wt, uko bikurikirana) mu masaha 72. Ingano nyinshi za H2O2 na O2 •− zakusanyije munsi ya hpt (Ishusho ya 5A, B). Mu buryo nk'ubwo, isuzuma rya malondialdehyde (MDA) rishingiye kuri TCA ryagaragaje ko ibihingwa by'ibishyimbo byanduye S. sclerotiorum byakusanyije urugero rwinshi rwa MDA (113.48 ± 10.02 nmol.g fr wt) mu mababi yabyo (Ishusho ya 5C). Ariko, gukoresha L-ornithine hanze byagabanyije cyane peroxidation y'amavuta nk'uko bigaragazwa no kugabanuka k'ingano ya MDA mu bimera byavuwe (33.08 ± 4.00 nmol.g fr wt).
Ishusho ya 5. Ingaruka zo gukoresha L-ornithine hanze ku bimenyetso by'ingenzi by'ihungabana rya ogisijeni n'uburyo bwo kwirinda antioxidant butari enzyme mu mababi y'ibishyimbo byandujwe na S. sclerotiorum mu masaha 72 nyuma yo kwandura mu gihe cy'ubushyuhe. (A) Hydrogen peroxide (H2O2; nmol g−1 FW) kuri 72 hpt, (B) superoxide anion (O2•−; nmol g−1 FW) kuri 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) kuri 72 hpt, (D) phenols zose zishobora gushonga (mg GAE g−1 FW) kuri 72 hpt, (E) flavonoids zose zishobora gushonga (mg RE g−1 FW) kuri 72 hpt, (F) aside amine zose (mg g−1 FW) kuri 72 hpt, na (G) ingano ya proline (mg g−1 FW) kuri 72 hpt. Indangagaciro zigaragaza impuzandengo ± ihindagurika risanzwe (impuzandengo ± SD) ry'ibipimo 5 by'ibinyabuzima (n = 5). Inyuguti zitandukanye zigaragaza itandukaniro rikomeye hagati y'ubuvuzi (p < 0.05).