Iyo nkuru iri mu ciyumviro c’ubushakashatsi “Gutuma ibiterwa vy’ibitoke bishobora guhangana n’ibitera indwara n’ibikoko bitera indwara”, raba ingingo 5 zose
Igitera indwara y’ibimera vy’ibiherere ku mubiri yitwa Sclerotinia sclerotiorum (Lib.) de Bary gikoresha ubuhinga bufise inzira nyinshi kugira ngo kirwaze ibimera bitandukanye bivyakira. Iyi nyigisho isaba gukoresha diamine L-ornithine, aside amino idafise poroteyine ivyura ugukorana kw’izindi aside amino zihambaye, nk’uburyo bundi bwo kuyicungera kugira ngo yongere inyishu z’ibihimba vy’umubiri, z’umubiri n’iz’ibinyabuzima z’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko vy’ibikoko. Ivyagezwe vyakozwe mu bipimo vy’amaraso vyerekanye ko L-ornithine ibuza cane ukura kw’imiceli ya S. pyrenoidosa mu buryo bushingiye ku rugero rw’imiti. Vyongeye, vyoshobora kugabanya cane uburemere bw’ibikoko vyera iyo umuntu ari mu nzu y’ivyatsi. Ikindi kandi, L-ornithine yaratumye ibitegwa vyavuwe bikura, ivyo bikaba vyerekana ko ivyiyumviro vya L-ornithine vyageragejwe bitagira ubumara ku bitegwa vyavuwe. Ikindi, L-ornithine yaratumye habaho uguserura kw’ibinyabuzima bitagira intungamubiri (ibinyabuzima vyose bishobora gusenyuka n’ibinyabuzima bita flavonoïdes) n’ibinyabuzima bitagira intungamubiri (catalase (CAT), peroxidase (POX), na polyphenol oxidase (PPO)), kandi yongera guseruka kw’ibinyabuzima bitatu bifitaniye isano n’ibinyabuzima, PGRvD, PGRvD). Ikindi kandi, mu gusesangura kwa silico, vyagaragaje ko hariho poroteyine yitwa SsOAH mu genome ya S. sclerotiorum, iyo poroteyine ikaba yari isa cane n’iyi Aspergillium splacilliOAH (SsOAH). (PlOAH) mu bijanye n’isesengura ry’ibikorwa, ivyicaro vyazigamye, n’ivy’ubuhinga. Igitangaje ni uko kwongerako L-ornithine ku mutobe w’ibitoke (PDB) vyagabanyije cane ukugaragara kw’umugera wa SsOAH mu mycelia ya S. sclerotiorum. Na vyo nyene, gukoresha L-ornithine mu buryo bw’inyuma vyagabanuye cane ukugaragara kw’umugera wa SsOAH mu bimera vy’ibiherere ku mubiri vyakoranijwe mu bimera vyavuwe. Ubwa nyuma, gukoresha L-ornithine vyagabanuye cane ivyimburwa vy’aside oxalic mu mababi yo hagati ya PDB no mu mababi yanduye. Mu gusozera, L-ornithine irafise uruhara runini mu kubungabunga ikibanza ca redox hamwe no kwongerera ubushobozi bwo kwikingira ibimera vyanduye. Ivyo bizova muri iri koraniro bishobora gufasha mu gutegura uburyo bushasha, butagira ingaruka ku bidukikije bwo kugenzura ibikoko vyera no kugabanya ingaruka bigira ku bijanye n’uburimyi bw’ibiharage n’ibindi bihingwa.
Igikoko c’umukara, giterwa n’igikoko citwa Sclerotinia sclerotiorum (Lib.) de Bary, ni indwara itera ubwoba, igabanya umwimbu, ikaba itera ingorane nyinshi ku bijanye n’umwimbu w’ibiharage (Phaseolus vulgaris L.) kw’isi yose (Bolton n’abandi, 2006). Sclerotinia sclerotiorum ni kimwe mu bitera indwara z’ibimera biva mw’ivu bigoye cane gucungera, gifise ubwoko bwinshi bw’ibimera burenga 600 kandi gishobora gucapura ningoga utugingo ngengabuzima tw’ibimera mu buryo butaboneka (Liang na Rollins, 2018). Mu bihe bitari vyiza, iraca mu gihe gikomeye c’ubuzima bwayo, ikaguma isinziriye igihe kirekire nk’ibintu vyirabura, bikomeye, bimeze nk’imbuto vyitwa ‘sclerotia’ mw’ivu canke nk’ibintu vyera, bimera nk’imbuto mu mycelium canke mu gitereko c’ibimera vyanduye (5.0). S. sclerotiorum irashobora guhingura sclerotia, ivyo bikaba bituma ishobora kubaho mu mirima yanduye igihe kirekire kandi igakomeza kubaho mu gihe c’ingwara (Schwartz n’abandi, 2005). Sclerotia zifise ingaburo nyinshi, zishobora kuguma mw’ivu igihe kirekire, kandi zikora nk’intanga y’intango y’indwara zikurikira (Schwartz n’abandi, 2005). Iyo ibintu bimeze neza, sclerotia iramera maze igatanga ibikoko bishobora kwanduza ibice vyose vyo hejuru y’ubutaka vy’igitegwa, harimwo ariko bitagarukiye ku mashurwe, ku nkoko canke ku bihimba vy’umubiri (Schwartz et al., 2005).
Sclerotinia sclerotiorum ikoresha ubuhinga bw’inzira nyinshi kugira ngo yanduze ibiterwa vyayikira, bikaba birimwo urutonde rw’ibintu bihuye kuva ku gukura kw’ibimera gushika ku gutera kw’ibimenyetso. Mu ntango, S. sclerotiorum ivyara ivyatsi bimanitse (vyitwa ascospores) biva mu bihimba bimeze nk’ibizinu vyitwa apothecia, bica bigenda mu kirere bikaja mu bihimba vy’umubiri bitagendagenda ku bisigazwa vy’ibimera vyanduye (Bolton n’abandi, 2006). Ico gikoko gica gisohora aside oxalic, ikintu gitera ubumara, kugira ngo kigenzure pH y’uruhome rw’ingingo z’igitegwa, gitere imbere mu gusenyuka kw’intungamubiri n’ugutera kw’imitsi (Hegedus na Rimmer, 2005), no guhagarika uguturika kw’igitegwa cakira. Uwo murongo wo guhindura aside utuma uruhome rw’ingingo z’igitegwa rugoyagoya, bigatuma haba ahantu heza ho gukora neza kandi neza kw’ibinyabuzima bishobora kwonona uruhome rw’ingingo z’ibimera (CWDEs), bikaba bituma umugera ushobora gutsinda inzitizi y’umubiri maze ukaja mu bihimba vy’umubiri (Marciano n’abandi, 1983). Iyo S. sclerotiorum imaze kwinjira, isohora umubare w’ibihimba vy’umubiri bita CWDE, nka polygalacturonase na cellulase, ivyo bikaba bituma ishobora gukwiragira mu bihimba vy’umubiri vyanduye, bigatuma n’ibihimba vy’umubiri bipfa. Ugutera imbere kw’ibisebe n’ibihimba vy’umubiri bitera ibimenyetso vy’ibikoko vyera (Hegedus na Rimmer, 2005). Hagati aho, ibiterwa vyakira abashitsi biramenya imiterere y’ibihimba vy’umubiri bifitaniye isano n’ibitera indwara (PAMPs) biciye ku bihimba vy’umubiri bimenya imiterere y’ibihimba vy’umubiri (PRRs), bikaba bituma haba urutonde rw’ibintu bitanga ibimenyetso bishobora gutuma amaherezo umuntu agira inyishu zo kwikingira.
Naho haciye imyaka mirongo hakozwe utwigoro two kurwanya indwara, ubukene bw’imigera ihagije ishobora guhangana n’indwara buracariho mu bitunguru, nk’uko biri mu bindi bihingwa vy’ubudandaji, kubera ukuntu iyo ndwara ishobora guhangana n’indwara, ishobora kurokoka be n’uko ishobora guhindura uko umuntu ameze. Gucungera indwara rero biragoye cane kandi bisaba ingamba zihurikiye hamwe, zifise imice myinshi, zirimwo uguhuza imigenzo y’imico kama, ukugenzura ibinyabuzima, n’imiti yica ibikoko (O’Sullivan n’abandi, 2021). Gukingira ibikoko vyera ni vyo bikora neza cane kubera ko imiti yica ibikoko, iyo ikoreshejwe neza kandi ku gihe kibereye, ishobora gukingira neza ukwiragira kw’iyo ndwara, igagabanya uburemere bw’indwara, igagabanya n’ugutakaza umwimbu. Ariko rero, gukoresha cane no kwizigira cane imiti yica ibiherere ku mubiri birashobora gutuma habaho imigera ya S. sclerotiorum ishobora guhangana n’indwara kandi bikagira ingaruka mbi ku binyabuzima bitagira intumbero, ku buzima bw’ubutaka, no ku buzima bw’amazi (Le Cointe n’abandi, 2016; Ceresini n’abandi, 2024). Ku bw’ivyo, kurondera ubundi buryo bwo gutorera umuti ibidukikije vyacitse ikintu gihambaye cane.
Ivyo bita polyamines (PAs), nka putrescine, spermidine, spermine, na cadaverine, birashobora gukora nk’ubundi buryo bwiza bwo kurwanya indwara ziterwa n’ibimera ziterwa n’ubutaka, gutyo bikagabanya burundu canke igice c’ikoreshwa ry’imiti y’ibicurane itera akaga (Nehela n’abandi, 2024; Yi20 n’abandi, 2 ). Mu bimera vy’imbere, PAs zigira uruhara mu bikorwa vyinshi vy’umubiri harimwo, ariko bitagarukiye kuri, ugucapura kw’ingingo, ugutandukanya, no kwishura ku mibabaro y’ibinyabuzima n’ibinyabuzima (Killiny na Nehela, 2020). Bishobora gukora nk’ibitera indwara, bifasha gukuraho ubwoko bw’umwuka w’ikirere (ROS), kubungabunga ubuzima bw’umubiri (Nehela na Killiny, 2023), gutuma habaho ivyiyumviro bijanye n’ukwikingira (Romero n’abandi, 2018), gutunganya inzira zitandukanye z’imibiri (Nehela na Killiny, 2023). Killiny, 2019), gushinga ubushobozi bwo guhangana n’indwara (SAR), no gutunganya imigenderanire hagati y’ibimera n’ibitera indwara (Nehela na Killiny, 2020; Asija n’abandi, 2022; Czerwoniec, 2022). Birabereye kumenya ko uburyo bwihariye n’uruhara rwa PA mu kwikingira ibitegwa bitandukanye bivanye n’ubwoko bw’ibitegwa, ibitera indwara, n’ibidukikije. PA igwiriye mu bimera ni iyo guhingura ibintu biva mu bihimba vy’umubiri vy’ingenzi vyitwa L-ornithine (Killiny na Nehela, 2020).
L-ornithine irafise uruhara rwinshi mu gukura no gutera imbere kw’ibimera. Nk’akarorero, inyigisho za kera zerekanye ko mu muceri (Oryza sativa), ornithine ishobora gufatanya n’ugusubira gukoresha azote (Liu n’abandi, 2018), umwimbu w’umuceri, uburyo n’akamoto (Lu n’abandi, 2020), n’inyishu y’imibabaro y’amazi (Yang n’abandi, 2000). Ikindi kandi, gukoresha L-ornithine mu buryo bw’inyuma vyatumye habaho ukwihanganira amapfa mu bihimba vy’isukari (Beta vulgaris) (Hussein n’abandi, 2019) kandi bigabanya umubabaro w’umunyu mu bitunguru (Allium Cepa) ( Rocha n’abandi, mu mwaka w’2012). Uruhara rwa L-ornithine mu kwikingira imibabaro y’ibinyabuzima rushobora kuba ruterwa n’uruhara rwayo mu kwirundanira kwa proline mu bimera vyavuwe. Nk’akarorero, utugingo ngengabuzima tujanye n’uguhinduka kw’ibinyabuzima bita proline, nk’utugingo ngengabuzima dukorana n’ibinyabuzima (delta-OAT) be n’utugingo ngengabuzima dukorana n’ibinyabuzima (ProDH1 na ProDH2), vyari vyaravuzwe mbere ko bifise uruhara mu gukingira ibikoko bita Nicotiana benthamiana na Arabidopsis thaliana ku bikoko bitagira umubiri bita Pse Misori, mu 2012). Ku rundi ruhande, umuti w’ibikoko bita ornithine decarboxylase (ODC) urakenewe kugira ngo ibikoko bitera indwara bikure (Singh n’abandi, 2020). Gutera ODC y’umugera wa Fusarium f. sp. lycopersici biciye mu guceceka kw’imitsi y’umubiri (HIGS) vyatumye ibiterwa vy’itomate bishobora guhangana n’indwara ya Fusarium wilt (Singh n’abandi, 2020). Ariko rero, uruhara rushobora kubaho rw’ugukoresha ornithine y’inyuma ku bibazo vy’ibinyabuzima nk’ibitera indwara z’ibimera ntirwari bwarize neza. Ikiruta vyose, ingaruka z’imiti yitwa ornithine ku bushobozi bwo kurwanya indwara be n’ibintu bifitaniye isano n’ivyo bihimba vy’umubiri n’ivyo mu mubiri, ahanini ntibiramenyekana.
Gutahura uburemere bw’indwara ya S. sclerotiorum mu bihingwa vy’ibitoke ni ikintu gihambaye mu gutegura ingamba nziza zo kuzicungera. Muri iki cigwa, twari dufise intumbero yo kumenya uruhara rushobora kuba rwa diamine L-ornithine nk’ikintu nyamukuru co kwongereza uburyo bwo kwikingira no guhangana n’indwara ya Sclerotinia sclerotiorum. Twiyumvira ko, uretse kwongerera ubushobozi bwo kwikingira ibiterwa vyanduye, L-ornithine na yo nyene ifise uruhara runini mu kubungabunga ikibanza ca redox. Turasaba ko ingaruka zishobora guterwa na L-ornithine zifitaniye isano n’ugutunganya uburyo bwo kwikingira indwara ziterwa n’indwara ziterwa n’indwara ziterwa n’indwara ziterwa n’ibiherere ku mubiri be n’ibindi bitera indwara ziterwa n’indwara ziterwa n’indwara. Ukwo gukora kabiri kwa L-ornithine gutuma iba umukandida mwiza w’ingene yogabanya ingaruka z’ibikoko vyera no kwongera ubushobozi bw’ibiterwa vy’ibitoke rusangi bwo kurwanya iyo ndwara ikomeye y’ibikoko. Ivyo bizova muri iri koraniro bishobora gufasha mu gutegura uburyo bushasha, butagira ingaruka ku bidukikije bwo kugenzura ibikoko vyera no kugabanya ingaruka bigira ku bijanye n’uguhingura ibitoke.
Muri iri koraniro, hakoreshejwe ubwoko bw’ibiharage rusangi bushobora gufatwa n’ubudandaji, ari bwo Giza 3 (Phaseolus vulgaris L. cv. Giza 3), nk’ibikoresho vyo kugerageza. Imbuto zifise amagara meza zatanzwe n’ubugwaneza n’Igisata c’ubushakashatsi ku bihingwa vy’ibitoke, Ikigo c’ubushakashatsi ku bihingwa vyo mu mirima (FCRI), Ikigo c’ubushakashatsi ku vy’uburimyi (ARC), mu Misiri. Imbuto zitanu zabibwe mu nkono z’ipulasitike (uburebure bw’imbere 35 cm, uburebure 50 cm) zuzuye ubutaka bwanduye S. sclerotiorum mu bihe vy’ivyatsi (25 ± 2 °C, ubukonje buringaniye 75 ± 1%, umuco w’amasaha 8/umwiza w’amasaha 16). Imisi 7–10 inyuma yo kubiba (DPS), ingemwe zaragabanywa kugira ngo zisige ingemwe zibiri gusa zikura kimwe n’amababi atatu yagutse neza mu nkono imwe imwe. Ibiterwa vyose vyari mu bibindi vyavomerwa rimwe mu ndwi zibiri, bigaterwa ifumbire buri kwezi ku rugero rwategekanijwe ku bwoko bwatanzwe.
Kugira ngo hategurwe umugera wa L-ornithinediamine (uzwi kandi kw’izina rya (+)-(S)-2,5-diaminopentanoïque; Sigma-Aldrich, Darmstadt, mu Budagi), 50 mg zabanje guterwa mu mazi 100 mL y’amazi atagiramwo ubumara. Ivyo bihimba vy’umubiri vyaciye bivanwa maze bikoreshwa mu ngeragezwa zakurikiyeho. Muri make, urutonde rutandatu rw’ibipimo vya L-ornithine (12,5, 25, 50, 75, 100, na 125 mg/L) rwageragejwe mu bikoresho vy’ubushakashatsi. Ikindi, amazi ayunguruwe atagiramwo ubumara yarakoreshejwe nk’ikigereranyo kibi (Mock) n’umuti w’ubudandaji “Rizolex-T” 50% w’ifu ishobora gutonyanga (toclofos-methyl 20% + thiram 30%; nk’ubugenzuzi bwiza. Umuti w’ubudandaji “Rizolex-T” warageragejwe mu bipimo vy’ibikoko ku bipimo bitanu (2, 4, 6, 8 na 10 mg/L).
Ivyitegererezo vy’ibiti vy’ibiharage rusangi n’ibitoke vyerekana ibimenyetso bimenyerewe vy’ibikoko vyera (igipimo c’ibikoko: 10–30%) vyakuwe mu mirima y’ubudandaji. Naho vyinshi mu bikoresho vy’ibimera vyanduye vyamenyekanye hakurikijwe ubwoko/ubwoko (ubwoko bw’ubudandaji bushobora kwandura Giza 3), ibindi, cane cane ivyo vyaronswe mu masoko yo mu karere, vyari vy’ubwoko butazwi. Ivyo bikoresho vyakwegeranijwe vyanduye vyabanje gusukurwa ku ruhande n’umuti w’amazi 0,5% wa sodium hypochlorite mu minota 3, hanyuma bisukurwa incuro nyinshi n’amazi asukuye atagiramwo ubumara, hanyuma bihanagurwa n’impapuro z’iyunguruzo zitagira ubumara kugira ngo amazi arenze gukuraho. Ivyo bihimba vyanduye vyaciye bitemwa mu bice bitobito bivuye mu nyama zo hagati (hagati y’ingingo zifise amagara meza n’izanduye), biterwa ku gikoresho citwa potato dextrose agar (PDA) hanyuma biterwa ku bushuhe bwa 25 ± 2 °C n’umuco w’amasaha 12/umwiza w’amasaha 12 mu kiringo c’imisi 5 kugira ngo bivyure sclerotia . Uburyo bwa mycelium tip bwakoreshejwe kandi mu gusukura ibiherere ku mubiri bivanze canke vyanduye. Ico gikoko c’ibiherere ku mubiri casukuwe carabanje kumenyekana bishingiye ku mico kama yaco, hanyuma cemezwa ko ari S. sclerotiorum bishingiye ku bimenyetso vy’imikorosikopi. Ubwa nyuma, ivyimburwa vyose vyasukuwe vyageragejwe ku bijanye n’ubushobozi bwo gutera indwara ku bwoko bw’ibiharage rusangi Giza 3 kugira ngo bihure n’ivyo Koch yashizeho.
Ikindi, igihimba ca S. sclerotiorum gitera cane (isolate #3) carasubiye kwemezwa bishingiye ku rutonde rw’ibice vy’imbere (ITS) nk’uko vyadondowe na White n’abandi, 1990; Baturo-Ciesniewska n’abandi, 2017. Muri make, ivyatsi vyihariye vyatewe mu mutobe w’ibirayi (PDB) hanyuma biterwa ku bushuhe bwa 25 ± 2 °C imisi 5–7. Hanyuma, imicelium y’ibiherere ku mubiri yarakorakoranywa, ikayungururwa biciye mu mpuzu y’umuceri, ikayozwa incuro zibiri n’amazi atagiramwo ubumara, hanyuma ikayumwa n’impapuro zitagiramwo ubumara. ADN y’umubiri yatanzwe hakoreshejwe igikoresho co gutegura ibiherere ku mubiri (Kuramae-Izioka, 1997; Atallah n’abandi, 2022, 2024). Akarere ka rDNA ya ITS rero karakomejwe hakoreshejwe umugwi w’intango wihariye ITS1/ITS4 ( Ivyo bikoresho vya PCR vyasukuwe vyashikirijwe kugira ngo bikurikiranywe (Ishirahamwe ry’ubuhinga bwa none ry’i Beijing Aoke Dingsheng). Ivyiyumviro vya ITS rDNA vyakurikiranywe mu buryo bubiri hakoreshejwe uburyo bwo gukurikiranya bwa Sanger. Ivyo bibazo vyakoranijwe vyaciye bigereranywa n’amakuru ya nyuma yo muri GenBank no mu kigo c’igihugu c’amakuru y’ubuhinga bwa none (NCBI, http://www.ncbi.nlm.nih.gov/gene/) hakoreshejwe porogarama ya BLASTn. Urutonde rw’ibibazo rwagereranywa n’izindi nzira 20 za S. sclerotiorum zaronswe mu makuru ya nyuma yo muri NCBI GenBank (Imbonerahamwe y’inyongera S1) hakoreshejwe ClustalW mu gitabu c’isesengura ry’ivy’ivyaduka (MEGA-11; verisiyo 11) (Kumar n’abandi, 20). Isesengurwa ry’ubwihindurize ryakozwe hakoreshejwe uburyo bw’ibishoboka vyinshi n’uburyo rusangi bwo gusubirira nucleotide bushobora gusubirwamwo igihe (Nei na Kumar, 2000). Igiti gifise ubushobozi bwo gutera ibiti vyinshi cane ni co kirerekanwa. Igiti c’intango c’ugushakashaka kw’ubuhinga gitorwa mu guhitamwo igiti gifise ubushobozi bwo gutera ivyatsi hagati y’igiti c’ugufatanya n’ababanyi (NJ) (Kumar et al., 2024) n’igiti c’ugushiramwo ibintu vyinshi (MP). Igiti ca NJ cubatswe hakoreshejwe urutonde rw’urugendo rw’ababiri babiri rwaharuwe hakoreshejwe uburyo rusangi bwo gusubira inyuma bw’igihe (Nei na Kumar, 2000).
Ibikorwa vy’ukurwanya imigera vya L-ornithine n’umuti wica imigera “Rizolex-T” vyamenyekanye mu buryo bw’ugukwiragiza agar. Uburyo: Fata umubare ukwiye w’umuti w’ububiko wa L-ornithine (500 mg/L) uwuvange neza na militiro 10 z’umuti w’ingaburo wa PDA kugira ngo utegure umuti ufise ivyiyumviro vy’iherezo vy’imiti 12,5, 25, 50, 75, 100 na 125 mg/L, uko bikurikirana. Ivyiyumviro bitanu vy’umuti wica ibikoko “Rizolex-T” (2, 4, 6, 8 na 10 mg/L) n’amazi ayunguruwe atagiramwo ubumara vyakoreshejwe nk’ikigereranyo. Inyuma y’aho iyo nzira ikomeye, igipfukisho c’imicelium c’umuco wa Sclerotinia sclerotiorum, gifise uburebure bwa milimetero 4, carashizwe hagati y’igitereko ca Petri, giterwa ku bushuhe bwa 25±2°C gushika iyo micelium ipfutse igitereko cose ca Petri co kugenzura, hanyuma haca handikwa ukura kw’ibiherere ku mubiri. Harura ijana kw’ijana ry’ukubuza ukukura kw’imirongo ya S. sclerotiorum ukoresheje ingano ya 1:
Iryo gerageza ryasubiwemwo incuro zibiri, hakaba hariho ibisubizo bitandatu vy’ibinyabuzima ku mugwi wose w’abagenzuzi/igerageza n’inkono zitanu (ibiterwa bibiri ku nkono) ku bisubizo vy’ibinyabuzima vyose. Igisubizo cose c’ibinyabuzima carasuzumwe incuro zibiri (ibisubizo bibiri vy’ubuhinga) kugira ngo hamenyekane ko ivyavuye mu ngeragezwa ari ukuri, ko vyizewe kandi ko bishobora gusubirwamwo. Ikindi, hakoreshejwe isesengura ry’ugusubira inyuma kw’ibiharuro vy’ibiharuro vy’ibiharuro vy’ibiharuro vy’ibiharuro vy’ibiharuro vy’ibiharuro (IC50) na IC99 (Prentice, 1976).
Kugira ngo hasuzumwe ubushobozi bwa L-ornithine mu bihe vy’ivyatsi bitoto, hakozwe igerageza ribiri rikurikirana ry’inkono. Muri make, inkono zari zuzuye ubutaka bw’ibumba n’umusenyi butagiramwo ubumara (3:1) hanyuma zicandwa n’umuco wa S. sclerotiorum wari uherutse gutegurwa. Ica mbere, igihimba c’umubiri gitera cane ca S. sclerotiorum (igihimba #3) caratewe mu guca igihimba kimwe c’umubiri mu bice bibiri, kigashirwa ahantu hasi kuri PDA hanyuma kigaterwa ku 25°C mu mwiza uhoraho (amasaha 24) imisi 4 kugira ngo kivyure ukura kw’imitsi. Hanyuma hafatwa ibipfukisho bine vy’uburebure bwa milimetero 5 ku ruhande rw’imbere maze bicangwa n’amagarama 100 y’uruvange rw’ingano n’umuceri (1:1, v/v) hanyuma amafishi yose aterwa ku bushuhe bwa 25 ± 2 °C munsi y’ingendo y’umuco w’amasaha 12/umwiza w’amasaha 12 kugira ngo 5. Ivyo vyari mu bikoresho vyose vyavangwa neza kugira ngo bibe bimwe imbere y’uko bongerako ivu. Hanyuma, 100 g y’umuvango w’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke. Ivyo bikono vyaciwe incanco vyavomerwa kugira ngo bikoreshe ukukura kw’ibiherere ku mubiri, hanyuma bishirwa mu nzu y’ivyatsi bitoto imisi 7.
Mu nkono imwe imwe haciye habibwa imbuto zitanu z’ubwoko bwa Giza 3. Ku nkono zavuwe na L-ornithine n’umuti wica ibikoko Rizolex-T, imbuto zari zatewe ubumara zabanje guterwa amasaha abiri mu mazi y’ivyo bihimba bibiri bifise IC99 ya nyuma y’agaciro kagera kuri 250 mg/L na 50 mg/L, hanyuma zigaterwa mu kirere imbere y’uko ziyumwa mu kirere mu kiringo c’isaha imwe. Ku rundi ruhande, imbuto zari zishizwe mu mazi atagiramwo ubumara nk’ikigereranyo kibi. Haciye imisi 10, imbere y’uko bavomera ubwa mbere, izo mbuto zarazigabanywa, hasigaye gusa ingemwe zibiri zisukuye mu nkono imwe imwe. Ikindi, kugira ngo umuntu ashobore kwandura S. sclerotiorum, inkomoko z’ibitoke ziri mu rwego rumwe rw’iterambere (imisi 10) zaciwe ahantu habiri hatandukanye hakoreshejwe igikoresho co gucapura gifise ubumara kandi hafi 0,5 g y’umuvango w’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vy’ibitoke vyashizwe mu gikomere cose, hanyuma hakurikirwa ugucandaga kw’ubukonje bwinshi kugira ngo indwara zose zitere imbere. Ibiterwa vyo kugenzura na vyo nyene vyakomeretse kandi umubare ungana (0,5 g) w’umuvango w’ibitoke bitagiramwo ubumara, bitagiramwo ubukoloni vyashizwe mu gikomere maze bikaguma mu bukonje bwinshi kugira ngo bigereranye ibidukikije kugira ngo indwara zitere imbere no kugira ngo habeho uguhuza hagati y’imigwi y’abavuzi.
Uburyo bwo kuvura: Intete z’ibiharage zavomerwa n’imiliyoni 500 z’umunyu w’amazi wa L-ornithine (250 mg/l) canke umuti wica ibicurane Rizolex-T (50 mg/l) mu kuvomera ubutaka, hanyuma iyo miti igasubirwamwo incuro zitatu mu kiringo c’imisi 10. Abagenzuzi bavuwe n’imiti y’ibitera indwara bavomerwa n’amazi 500 ml y’amazi atagiramwo ubumara. Ivyo bikoresho vyose vyakozwe mu nzu y’ivyatsi (25 ± 2°C, 75 ± 1% y’ubukonje, n’igihe c’umuco c’amasaha 8/umwiza w’amasaha 16). Inkono zose zavomerwa buri ndwi zibiri kandi zigavugwa buri kwezi n’ifumbire ya NPK iri ku rugero rwiza (20-20-20, ifise 3,6% y’ikibiriti n’ibintu bitobito vya TE; Zain Seeds, Misiri) ku rugero rwa 3–4 g/l hakoreshejwe gutera amababi hakurikijwe impanuro z’ubwoko bwihariye n’ifumbire yihariye’. Keretse iyo bivuzwe ukundi, amababi akuze yagutse neza (amababi ya 2 n’aya 3 kuva hejuru) yaronkejwe muri buri nkuru y’ibinyabuzima ku masaha 72 inyuma y’ugutera (hpt), ahurizwa hamwe, akoranywa kandi abikwa ku -80 °C kugira ngo hakorwe ibindi bipimo harimwo, ariko bitagarukiye kuri, ibimenyetso vy’imibabaro y’umubiri, ivy’imibabaro y’umubiri, n’ibitera indwara bitagira enzyme be n’uguserura ivy’uruvyaro.
Uburemere bw’indwara y’ibikoko vyera bwasuzumwa buri ndwi imisi 21 inyuma y’ugucandagwa (dpi) hakoreshejwe urugero rwa 1–9 (Imbonerahamwe y’inyongera S2) rushingiye ku rugero rwa Petzoldt na Dickson (1996) rwahinduwe na Teran n’abandi. (2006). Muri make, inkomoko n’amashami y’ibiterwa vy’ibiharage vyasuzumwe guhera aho vyatewe kugira ngo hakurikiranwe ingene ibisebe bigenda bigenda bigenda bigenda ku nzira y’ibihimba vy’umubiri n’ibihimba vy’umubiri. Urugendo rw’igisebe kuva aho cacanzwe gushika aho kiri kure cane ku nzira y’uruti canke ishami rwaciye rupimwa maze hashirwako amanota 1–9 hakurikijwe aho igisebe cari, aho (1) vyerekana ko ata ndwara iboneka hafi y’aho igisebe cacanzwe na (2–9) vyerekana ko ubunini bw’ibisebe bugenda buratera imbere buhoro buhoro a/interuS noudes na S2). Uburemere bw'indwara y'ibicurane vyera bwahinduwe mu ijanisha hakoreshejwe uburyo bwa 2:
Ikindi, uburebure buri munsi y’umurongo w’iterambere ry’ingwara (AUDPC) bwaharuwe hakoreshejwe ubuhinga (Shaner na Finney, 1977), bwaherutse guhindurwa ku bijanye n’ukubora kwera kw’ibiharage rusangi (Chauhan n’abandi, 2020) hakoreshejwe ingano ya 3:
Aho Yi = uburemere bw’ingwara ku gihe ti, Yi+1 = uburemere bw’ingwara ku gihe gikurikira ti+1, ti = igihe co gupima ubwa mbere (mu misi), ti+1 = igihe co gupima gikurikira (mu misi), n = umubare wose w’ibihe canke ibihe vyo kwihweza. Ivyerekeye ukura kw’igitegwa c’ibitoke harimwo uburebure bw’igitegwa (cm), umubare w’amashami ku gitegwa, n’umubare w’amababi ku gitegwa vyanditswe buri ndwi mu misi 21 mu bisubizo vyose vy’ibinyabuzima.
Mu nkuru yose y’ibinyabuzima, ivyerekanwa vy’amababi (amababi ya kabiri n’aya gatatu yateye imbere neza kuva hejuru) vyakwegeranijwe ku musi ugira 45 inyuma y’ugutera (imisi 15 inyuma y’ugutera kwa nyuma). Igishushanyo cose c’ibinyabuzima cari gifise inkono zitanu (ibiterwa bibiri ku nkono). Nk’imigera 500 y’ivyo bihimba vyajanjaguwe vyakoreshejwe mu gukuraho amabara y’izuba (klorofile a, klorofile b na carotenoïdes) hakoreshejwe 80% vy’acetone ku bushuhe bwa 4 °C mu mwiza. Inyuma y’amasaha 24, ivyerekanwa vyaciwe mu gitereko c’amazi maze amazi yari hejuru y’ivyo bipimo arakusanywa kugira ngo hamenyekane ibirimwo klorofile a, klorofile b n’ivyo bita carotenoïde hakoreshejwe ubuhinga bwo gupima ibara ry’umuco UV-160A (Shimadzu Corporation, Ubuyapani) hakurikijwe uburyo bwa (Lichtebaring the30 at71) uburebure bw’imipfunda (A470, A646 na A663 nm). Ubwa nyuma, ibirimwo amabara y’izuba vyaharuwe hakoreshejwe amafomula akurikira 4–6 yadondowe na Lichtenthaler (1987).
Ku masaha 72 inyuma y’ugutera (hpt), amababi (amababi ya kabiri n’aya gatatu yateye imbere neza kuva hejuru) yarakoranijwe muri buri nkuru y’ibinyabuzima kugira ngo haboneke in situ histochimical localisation y’ivyuma vy’amazi (H2O2) n’ivyuma vy’amazi (O2•−). Igishushanyo cose c’ibinyabuzima cari gifise inkono zitanu (ibiterwa bibiri ku nkono). Igisubizo cose c’ibinyabuzima carasuzumwe mu bice bibiri (ibisubizo bibiri vy’ubuhinga) kugira ngo hamenyekane ukuri, ukwizigirwa n’uburyo bwo gusubiramwo. H2O2 na O2•− vyamenyekanye hakoreshejwe 0,1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Ubudage) canke tetrazolium y’ubururu bw’umukara (NBT; (2004) na Adamu n’abandi. (1989) n’ibintu bikeyi vyahinduwe. Kugira ngo H2O2 iboneke mu kibanza cayo, amababi yashizwe mu mwuka w’ubusa n’ibice 0,1% vya DAB mu 10 mM Tris buffer (pH 7,8) hanyuma bishirwa mu bushuhe bwo mu cumba mu muco iminota 60. Amababi yashizwe mu 0,15% (v/v) TCA mu 4:1 (v/v) ethanol:chloroforme (Ikigo c’imiti n’ibikoresho vy’ubuvuzi ca Al-Gomhoria, Cairo, Misiri) hanyuma ashirwa mu muco gushika ahindutse umwijima. Na vyo nyene, amavalve yari yashizwe mu mwuka w’ubusa n’amazi y’ipotasiyumu 10 mM (pH 7,8) arimwo 0,1 w/v % HBT kugira ngo O2•− ishobore gushirwa mu bihimba vy’umubiri. Amababi yashizwe mu muco mu bushuhe bwo mu cumba iminota 20, hanyuma arahindurwa ibara nk’uko biri hejuru, hanyuma aramurikwa gushika aho amabara y’ubururu bwijimye/ibara ry’umuhondo abonetse. Uburemere bw’ibara ry’umuhondo (nk’ikimenyetso ca H2O2) canke ibara ry’ubururu-violet (nk’ikimenyetso ca O2•−) ryasuzumwe hakoreshejwe verisiyo y’i Fiji y’ibara ry’ibara ry’amashusho ImageJ (http://fiji.sc; ryabonetse ku wa 7 Ntwarante 2024).
Malondialdehyde (MDA; nk’ikimenyetso c’uguhinduka kw’amaraso) yamenyekanye hakurikijwe uburyo bwa Du na Bramlage (1992) hahinduwe gatoyi. Amababi avuye muri buri nkuru y’ibinyabuzima (amababi ya kabiri n’aya gatatu yateye imbere neza avuye hejuru) yaronkejwe amasaha 72 inyuma y’ugutera (hpt). Igishushanyo cose c’ibinyabuzima carimwo inkono zitanu (ibiterwa bibiri ku nkono). Igisubizo cose c’ibinyabuzima carasuzumwe mu bice bibiri (ibisubizo bibiri vy’ubuhinga) kugira ngo hamenyekane ukuri, ukwizigirwa n’uburyo bwo gusubiramwo. Mu magambo make, 0,5 g y’amababi y’ivu yakoreshejwe mu gukuraho MDA n’aside trichloroacetique 20% (TCA; MilliporeSigma, Burlington, MA, USA) irimwo 0,01% y’amazi y’ivyatsi (BHT; Sigma-Aldrich, St. Ivyo MDA iri mu mazi yari hejuru y’amazi vyaciye bimenyekana hakoreshejwe ubuhinga bwo gupima ibara ry’amazi ku 532 na 600 nm hakoreshejwe igikoresho co gupima ibara ry’umuco UV-160A (Shimadzu Corporation, Ubuyapani) hanyuma bigaragazwa nk’nmol g−1 FW.
Kugira ngo hasuzumwe ivyitwa antioxydants bitagira enzymes n’ivy’intungamubiri, amababi (amababi ya kabiri n’aya gatatu yateye imbere neza kuva hejuru) yaronkejwe ku nkuru yose y’ibinyabuzima ku masaha 72 inyuma y’ugutera (hpt). Igishushanyo cose c’ibinyabuzima cari gifise inkono zitanu (ibiterwa bibiri ku nkono). Icogereranyo cose c’ibinyabuzima carasuzumwe mu bice bibiri (ivyitegererezo bibiri vy’ubuhinga). Amababi abiri yarasezwe n’azote y’amazi maze akoreshwa ata guca ku ruhande mu kumenya ivyitwa enzymes n’ibitagira enzymes, aside amino zose hamwe, ibirimwo proline, ukugaragara kw’imitsi, n’ingero ya oxalate.
Ivyuma vyose bishobora gusenyuka vyamenyekanye hakoreshejwe igikoresho ca Folin-Ciocalteu (Sigma-Aldrich, St. (1999). Muri make, hafi 0,1 g y’ibihimba vy’amababi vy’umubiri bimwe bimwe vyakuwemwo 20 ml 80% ya methanol mu mwijima mu kiringo c’amasaha 24 hanyuma ivyuma vyari hejuru y’ivyo bihimba vy’umubiri bikuwemwo inyuma yo gucapura. 0,1 ml y’ivyo bihimba vy’umubiri vyavanzwe na 0,5 ml y’ivyo bihimba vy’umubiri bita Folin-Ciocalteu (10%), bizunguruka amasegonda 30 hanyuma bisigara mu mwijima iminota 5. Hanyuma 0,5 ml y’umunyu w’ivyatsi bifise 20% (Na2CO3; Ishirahamwe ry’imiti n’ibikoresho vy’ubuvuzi rya Al-Gomhoria, Cairo, Misiri) yongerwa ku nzira imwe imwe, ivangwa neza hanyuma iterwa mu bushuhe bwo mu cumba mu mwiza mu kiringo c’isaha 1. Inyuma y’ugutera, ubushobozi bwo gufata amazi bw’ivyo bivanze bwagereranywa ku 765 nm hakoreshejwe igikoresho co gupima umuco w’izuba UV-160A (Shimadzu Corporation, Ubuyapani). Igitigiri c’ibinyabuzima vyose bishobora gusenyuka mu bihimba vy’umubiri vyamenyekanye hakoreshejwe ubuhinga bwo gupima aside ya gallique (Fisher Scientific, Hampton, NH, USA) kandi kigaragazwa nk’imiligramu y’aside ya gallique ingana n’igarama imwe y’uburemere bushasha (mg GAE g-1 uburemere bushasha).
Igitigiri cose c’ivyo bita flavonoïdes bishobora gusenyuka vyamenyekanye hakurikijwe uburyo bwa Djeridane n’abandi. (2006) harimwo n’ibintu bikeyi vyahinduwe. Muri make, 0,3 ml y’ivyo bihimba vy’umubiri bivugwa haruguru vyavanzwe na 0,3 ml y’umunyu w’aluminium 5% (AlCl3; Fisher Scientific, Hampton, NH, USA), bivugururwa cane hanyuma bishirwa mu bushuhe bwo mu cumba mu minota 5, hanyuma hakurikirwa n’umunyu wa mssiuml10. (Imiti n’ibikoresho vy’ubuvuzi vya Al-Gomhoria, Cairo, Misiri), bivanze neza bigaterwa mu bushuhe bwo mu cumba iminota 30 mu mwiza. Inyuma y’ugutera, ubushobozi bwo gufata amazi bw’ivyo bivanze bwagereranywa ku 430 nm hakoreshejwe igikoresho co gupima umuco w’izuba UV-160A (Shimadzu Corporation, Ubuyapani). Igitigiri c’ibinyabuzima vyose bishobora gusenyuka mu bihimba vy’umubiri vyamenyekanye hakoreshejwe ubuhinga bwo gupima rutini (TCI America, Portland, OR, USA) hanyuma bigaragazwa nk’imiligramu y’urutini ingana na gramme y’uburemere bushasha (mg RE g-1 uburemere bushasha).
Igitigiri cose c’acides aminés y’ubuntu kiri mu mababi y’ibitoke camenyekanye hakoreshejwe igikoresho cahinduwe c’ivyuma bita ninhydrin (Thermo Scientific Chemicals, Waltham, MA, USA) gishingiye ku buryo bwashikirijwe na Yokoyama na Hiramatsu (2003) kandi gihindurwa na Sun n’abandi. (2006). Mu magambo make, 0,1 g y’umubiri wo hasi yakuwemwo n’umuti wo gukingira pH 5,4, hanyuma 200 μL y’ivyo bihimba vy’umubiri vyari hejuru y’ivyo bihimba vy’umubiri vyashizwemwo 200 μL ya ninhydrine (2%) na 200 μL ya piridine (10%; bipimwa ku 580 nm hakoreshejwe igikoresho co gupima umuco w’izuba UV-160A (Ishirahamwe Shimadzu, mu Buyapani). Ku rundi ruhande, proline yamenyekanye hakoreshejwe uburyo bwa Bates (Bates n’abandi, 1973). Proline yavanywe n’aside sulfosalicylique 3% (Thermo Scientific Chemicals, Waltham, MA, USA) hanyuma inyuma yo gucapura, 0,5 ml y’ivyo bihimba vy’umubiri vyavanzwe n’imiliyoni 1 y’aside acetique y’urubura (Fisher Scientific, Hampton, NH, USA) n’ivyo gupima ninhydrin ku min°4d, incu ku 520 nm hakoreshejwe igipimo c’umuco nk’ico kiri hejuru. Igitigiri cose c’acides aminés n’ivyo bita proline biri mu bihimba vy’amababi vyamenyekanye hakoreshejwe ubuhinga bwo gupima glycine na proline (Sigma-Aldrich, St Louis, MO, USA), hakurikijwe uko bigenda, kandi bigaragazwa nk’uburemere bushasha bwa mg/g.
Kugira ngo hamenyekane igikorwa c’intungamubiri zifasha umubiri guhangana n’indwara, hafi 500 mg y’imitsi y’umubiri ihurikiye hamwe yarakuwemwo militiro 3 z’umuti witwa Tris buffer 50 mM (pH 7,8) urimwo 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA), vyashizwe mu 10.000 × g mu minota 20 mu gitereko (4 °C), hanyuma ivyari hejuru (ivy’intungamubiri bitagiramwo ivyatsi) birakorakoranywa (El-Nagar n’abandi, 2023; Osman n’abandi, 2023). Catalase (CAT) yaciye ikoreshwa n’imiti 2 ml y’umunyu w’amazi y’umunyu 0,1 M (pH 6,5; Sigma-Aldrich, St. 2023; Osman n’abandi, 2023). Ibikorwa vy’indwara y’umutima (POX) vyamenyekanye hakoreshejwe uburyo bwa Harrach n’abandi. (2009). (2008) n’ibintu bikeyi vyahinduwe (El-Nagar n’abandi, 2023; Osman n’abandi, 2023) n’igikorwa c’indwara y’ibinyabuzima (PPO) vyamenyekanye inyuma y’ugukorana n’imiti 2,2 y’umuti w’ibinyabuzima (pH 6,0l). Portland, OR, muri Leta Zunze Ubumwe za Amerika) n’amazi 100 μl y’amazi 12 mM. Ubwo buryo bwahinduwe gatoyi buva kuri (El-Nagar n’abandi, 2023; Osman n’abandi, 2023). Iryo suzuma ryakozwe inyuma y’ugukorana n’imiti 3 y’umunyu w’ivyatsi bita catechol (0,01 M) wateguwe vuba mu mazi y’ivyatsi bita phosphate 0,1 M (pH 6,0). Ibikorwa vya CAT vyagereranywa mu kugenzura ukubora kwa H2O2 ku 240 nm (A240), ibikorwa vya POX vyagereranywa mu kugenzura ukwiyongera kw’ugufata ku 436 nm (A436), igikorwa ca PPO na co cagereranywa mu kwandika uguhinduka kw’ugufata ku 495 nm buri min U30 a igikoresho co gupima umuco (Shimadzu, mu Buyapani).
RT-PCR y’igihe nyaco yakoreshejwe kugira ngo hamenyekane ingero z’inyandiko z’imitsi itatu ijana n’imiti irwanya indwara, harimwo n’imitsi y’umubiri (PvCAT1; Accession No. KY195009.1), mu mababi y’ibiharage (amababi ya kabiri n’aya gatatu akuze neza kuva hejuru) 72 h inyuma y’ugutera kwa nyuma. Muri make, ARN yatanzwe hakoreshejwe igikoresho co gukura ARN yose hamwe citwa Simply P (Cat. No. BSC52S1; BioFlux, Biori Technology, Ubushinwa) hakurikijwe amategeko y’uwayikoze. Hanyuma, cDNA yarakozwe hakoreshejwe igikoresho co gukora cDNA citwa TOP scriptTM cDNA Synthesis Kit hakurikijwe amabwirizwa y’uwayikoze. Urutonde rw’ivya mbere vy’ivyo bihimba bitatu biri hejuru biri mu ndome y’inyongera S3. PvActin-3 (inomero y’ukwinjira muri GenBank: XM_068616709.1) yakoreshejwe nk’umugera wo mu nzu kandi ivyerekanwa vy’umugera bihuye vyaharuwe hakoreshejwe uburyo bwa 2-ΔΔCT (Livak na Schmittgen, 2001). Ugushikama kwa actin mu gihe c’imibabaro y’ibinyabuzima (ugukorana kudahuye hagati y’ibinyabuzima bisanzwe n’igikoko c’indwara y’ibicurane Colletotrichum lindemuthianum) n’imibabaro y’ibinyabuzima (amapfa, umunyu, ubushuhe buke) vyarerekanwe (Borges n’abandi, 2012).
Twabanje gukora isesengura ry’ibihimba vy’umubiri vy’ibihimba vy’umubiri vy’ibihimba vy’umubiri vy’ibihimba vy’umubiri (OAH) mu bihimba vy’umubiri bita S. sclerotiorum dukoresheje igikoresho c’ibihimba vy’umubiri (BLASTp 2.15.0+) (Altschul n’abandi, 1997, 2005). Muri make, twakoresheje OAH ivuye mu giti citwa Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; inomero y’ugushirwa mu gisata ca GenBank XP_040799428.1; aside amino 342) na Penicillium lagena (PlOAH2; taxide1bank:99). XP_056833920.1; 316 aside amino) nk’urutonde rw’ibibazo kugira ngo umuntu ashobore gukora ikarita y’ivyo bita proteine ​​bihuye n’ivyo bita S. sclerotiorum (taxide: 5180). BLASTp yakozwe ku makuru y’ivy’umubiri wa S. sclerotiorum aherutse kuboneka muri GenBank ku rubuga rwa interineti rw’Ikigo c’Igihugu c’Amakuru y’Ikoranabuhanga ry’Ibinyabuzima (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Ikindi, umugera wa OAH wari waravuzwe imbere y’igihe uvuye muri S. sclerotiorum (SsOAH) n’isesengura ry’iterambere n’igiti c’ubuhinga bwa AfOAH uvuye muri A. fijiensis CBS 313.89 na PlOAH uvuye muri P. lagena vyashizwe ahabona hakoreshejwe uburyo bushingiye ku bishoboka vyinshi muri JTamura11, 11. n’akarorero (Jones n’abandi, 1992). Igiti c’ubuhinga bwa none cari gifatanijwe n’isesengura ry’urutonde rw’ibinyabuzima vyinshi vy’urutonde rw’ibinyabuzima vyose vya OAH (SsOAH) vyavuzwe imbere y’igihe bivuye muri S. sclerotiorum be n’urutonde rw’ibibazo hakoreshejwe igikoresho co gutunganya ibintu bishingiye ku nzitizi (COBALT; na Agarwala, mu mwaka w’2007). Ikindi, urutonde rw’acides aminés rwo mu bwoko bwa SsOAH rwo muri S. sclerotiorum rwahuye neza cane rwari rwahuye n’urutonde rw’ibibazo (AfOAH na PlOAH) (Larkin n’abandi, 2007) hakoreshejwe ClustalW (http://www.genome.jp/tools-bin n’akarere k’ubuhinga bwa none). Igikoresho ca ESPript (verisiyo 3.0;
Ikindi kandi, ivyicaro vy’ibikorwa vyavuzwe mbere n’ibibanza vyazigamye vya S. sclerotiorum SsOAH vyashizwe mu miryango itandukanye hakoreshejwe igikoresho ca InterPro (https://www.ebi.ac.uk/interpro/) (Blum n’abandi, 2021). Ubwa nyuma, igishushanyo c’imiterere y’ibinyabuzima bitatu (3D) vy’ivyo bikoko vya S. sclerotiorum SsOAH vyari vyaravuzwe vyakozwe hakoreshejwe ubuhinga bwo kumenya ivy’ibinyabuzima bihuye (Phyre2 server version 2.0; 2015) kandi vyemezwa hakoreshejwe umukozi wa SWISS-MODEL (Biasini n’abandi, 2014). Ivyo bishushanyo vy’ingero zitatu vyari vyaravuzwe (uburyo bwa PDB) vyabonetse mu buryo bukorana hakoreshejwe ubuhinga bwa UCSF-Chimera (uburyo bwa 1.15; https://www.cgl.ucsf.edu/chimera/ ) (Pettersen n’abandi, 2004).
Ivyiyumviro vy’ubuhinga bwa none vyakoreshejwe kugira ngo hamenyekane urugero rw’inyandiko y’ivyo bita oxaloacetate acetylhydrolase (SsOAH; inomero y’ivyo bita GenBank: XM_001590428.1) mu bihimba vy’umubiri vyitwa Sclerotinia sclerotiorum. Muri make, S. sclerotiorum yaciwe mu gitereko kirimwo PDB hanyuma ishirwa mu gikoresho co gutera ivyatsi (ikigereranyo: I2400, New Brunswick Scientific Co., Edison, NJ, USA) ku bushuhe bwa 25 ± 2 °C mu kiringo c’amasaha 24 ku 150 rpm no mu mwijima uhoraho) kugira ngo ivyure g. Hanyuma, izo nsoro zavuwe n’umuti L-ornithine be n’umuti wica ibikoko Rizolex-T ku rugero rwa nyuma rwa IC50 (hafi 40 na 3,2 mg/L, hakurikijwe) hanyuma ziterwa mu yindi masaha 24 mu bihe nk’ivyo nyene. Inyuma y’ugutera, ivyo bihingwa vyaciwe mu nzira ku 2500 rpm mu minota 5 hanyuma ivyari hejuru (mycélium fungal) birakusanywa kugira ngo bisuzumwe ukuntu ivyamwa vy’umubiri bigaragara. Na vyo nyene, imicelium y’ibiherere ku mubiri yakoranijwe ku masaha 0, 24, 48, 72, 96, na 120 inyuma y’aho umuntu arwaye, ivuye mu bimera vyanduye vyari vyarateye ibikoko vyera n’imicelium y’ipampa ku ruhande rw’ibihimba vyanduye. ARN yavanywe mu mycélium y’ibiherere ku mubiri hanyuma cDNA irakorwa nk’uko vyavuzwe haruguru. Ivyiyumviro vy'intango vya SsOAH biri mu ndome y'inyongera S3. SsActin (inomero y’ukwinjira muri GenBank: XM_001589919.1) yakoreshejwe nk’umugera wo mu nzu, kandi ivyerekanwa vy’umugera bihuye vyaharuwe hakoreshejwe uburyo bwa 2-ΔΔCT (Livak na Schmittgen, 2001).
Asidi ya oxalic yamenyekanye mu mutobe w’ibirayi (PDB) no mu bipimo vy’ibimera birimwo umugera w’ibiherere ku mubiri Sclerotinia sclerotiorum hakurikijwe uburyo bwa Xu na Zhang (2000) hahinduwe gatoyi. Mu magambo make, ivyimburwa vya S. sclerotiorum vyaciwe mu bikoresho vy’ugutera ivyatsi birimwo PDB hanyuma biterwa mu gikoresho co gutera ivyatsi binyiganyiga (ikigereranyo I2400, New Brunswick Scientific Co., Edison, NJ, USA) ku 150 rpm ku bushuhe bwa 25 ± 2 °C (imisi 3–5 mu gihe c’ubushuhe buhoraho). Inyuma y’ugutera, umuco w’ibiherere ku mubiri wabanje gucungururwa biciye ku mpapuro z’icunguruzo za Whatman #1 hanyuma ucungurwa ku 2500 rpm mu minota 5 kugira ngo hakurwemwo mycelium yasigaye. Ivyo bihimba vy’umubiri vyari vyuzuye vyakwegeranijwe bibikwa ku bushuhe bwa 4°C kugira ngo haboneke ibindi bihimba vy’umubiri vyitwa oxalate. Ku bijanye no gutegura ivyerekanwa vy’ibimera, hafi 0,1 g y’ibice vy’imitsi y’ibimera vyakuwe incuro zitatu hakoreshejwe amazi ayunguruwe (2 ml igihe cose). Ivyo bipimo vyaciye bishirwa mu gipimo c’umuriro ku 2500 rpm mu minota 5, ivyo bihimba vyari hejuru y’ivyo bihimba vyumye bicunzwe biciye ku mpapuro z’iyunguruzo za Whatman No. 1 hanyuma bikusanywa kugira ngo bisuzumwe.
Kugira ngo hakorwe isesengura ry’ingero z’aside oxalique, umuvango w’inyishu wateguwe mu gitereko c’ibirahuri gipfutse mu buryo bukurikira: 0,2 ml y’ivyafashwe (canke umunyuro w’umuco wa PDB canke umunyuro w’aside oxalique), 0,11 ml y’ubururu bwa bromophenol (BPB, 1 mM; Fisher Chemical, PA1 m0l of Pittsburgh. aside (H2SO4; Ishirahamwe ry’imiti n’ibikoresho vy’ubuvuzi rya Al-Gomhoria, Cairo, Misiri) n’imiliyoni 0,176 y’amazi y’ipotasiyumu 100 mM (K2Cr2O7; imiti ya TCI, Portland, OR, USA), hanyuma iyo ntungamubiri irashirwa mu mazi y’amazi y’amazi y’amazi y’amazi y’amazi y’amazi y’amazi y’amazi y’amazi y’amazi y’amazi y’amazi. mu mazi yo kwoga afise ubushuhe bwa 60 °C. Inyuma y’iminota 10, iyo nzira yahagaritswe mu kwongerako 0,5 ml y’umunyu w’amazi y’ivyatsi (NaOH; 0,75 M). Ivyo bimenyetso vy’ugufata (A600) vy’ivyo bivanze vyagereranywa ku 600 nm hakoreshejwe igikoresho co gupima umuco w’izuba UV-160 (Shimadzu Corporation, Ubuyapani). PDB n’amazi ayunguruwe vyakoreshejwe nk’ibigenzura mu gupima igitigiri c’ibiyunguruwe n’ibimera, uko bikurikirana. Ivyiyumviro vy’aside oxalique mu biyunguruzo vy’imico kama, vyerekanywe nk’imikorogarama z’aside oxalique kuri militiro y’umuti wa PDB (μg.mL−1), no mu bihimba vy’amababi, vyerekanywe nk’imikorogarama z’aside oxalique kuri garama y’uburemere bushasha (μg.g.g−1 FW), vyamenyekanye hakoreshejwe ubuhinga bwa curvetham aside oxalic, The Walific Slibration, MA, muri Leta Zunze Ubumwe za Amerika).
Mu gihe cose c’itohoza, ivyageragejwe vyose vyakozwe mu buryo bushitse (CRD) harimwo ibisubizo bitandatu vy’ibinyabuzima ku buvuzi n’inkono zitanu ku bisubizo vy’ibinyabuzima (ibiterwa bibiri ku nkono) kiretse iyo bivuzwe ukundi. Ivyiyumviro vy’ibinyabuzima vyasuzumwe mu bice bibiri (ivyigwa bibiri vy’ubuhinga). Ivyiyumviro vy’ubuhinga vyakoreshejwe mu kugenzura ko igerageza rimwe rishobora gusubirwamwo ariko ntivyakoreshejwe mu gusesangura imibare kugira ngo ntihagire ivyigwa vy’ibinyoma. Amakuru yasuzumwe mu mibare hakoreshejwe isesengura ry’ugutandukanya (ANOVA) hakurikijwe ikigeragezo ca Tukey-Kramer c’itandukaniro ry’ukuri (HSD) (p ≤ 0.05). Ku bijanye n’igerageza ry’in vitro, agaciro ka IC50 na IC99 karaharuwe hakoreshejwe uburyo bwa probit kandi 95% vy’ibihe vy’ukwizigira vyaharuwe.
Ivyiyumviro bine vy’ibitoke vyatoranijwe mu mirima itandukanye y’ibitoke vya soya muri Guverinera ya El Ghabiya, muri Misiri. Ku gikoresho ca PDA, ivyatsi vyose vyari vyihariye vyavyaye mycelium yera nk’iyitwa cream yihuta guhinduka yera nk’ipampa (Ishusho 1A) hanyuma ihinduka beige canke brown ku rwego rwa sclerotium. Sclerotia akenshi ziba zikomeye, zirabura, zifise uburebure bw’umubumbe canke zidasanzwe, zifise uburebure bwa milimetero 5,2 gushika kuri 7,7 n’uburebure bwa milimetero 3,4 gushika kuri 5,3 (Ishusho 1B). Naho ivyimburwa bine vyateye imbere mu bijanye n’ugutera ivyatsi ku mpera y’ivyo bihimba vy’umubiri inyuma y’imisi 10–12 vy’ugutera ku bushuhe bwa 25 ± 2 °C (Ishusho 1A), igitigiri c’ibihimba vy’umubiri ku gipande cari gitandukanye cane muri vyo (P < 0.001), ivyatsi vyihariye vyari vyinshi cane (P < 0.001). 1,53 sclerotia ku isahani; Na vyo nyene, igice ca #3 caravuyemwo aside oxalique nyinshi muri PDB kuruta ibindi bice (3,33 ± 0,49 μg.mL−1; ishusho 1D). Igishushanyo #3 cerekanye ibiranga imiterere n’ibiranga imiterere y’igikoko gitera indwara z’ibimera Sclerotinia sclerotiorum. Nk’akarorero, kuri PDA, amakoloni y’isolate #3 yarakuze ningoga, yari yera nk’iy’umukara (Ishusho 1A), yari ihindutse beige canke umuhondo w’umuhondo w’umukara, kandi vyasaba imisi 6-7 ku bushuhe bwa 25 ± 2°C kugira ngo bipfuke vyose ku ruhande rw’isahani y’uburebure bwa cm 9. Hashingiwe ku bimenyetso vy’imiterere n’ivy’imikorosikopi vyavuzwe haruguru, igice ca #3 caramenyekanye ko ari Sclerotinia sclerotiorum.
Igishushanyo 1. Ibirango n’ubushobozi bwo gutera indwara bwa S. sclerotiorum bwitandukanijwe n’ibiterwa vy’ibitoke bisanzwe. (A) Ukukura kw’imitsi y’ibikoko bine vya S. sclerotiorum ku bikoresho vya PDA, (B) ivyimburwa vy’ibikoko bine vya S. sclerotiorum, (C) umubare w’ibihimba vy’umubiri (ku gipande), (D) ugusohoka kw’aside oxalic ku bikoresho vya PDB (μg.mL−1), na (E) uburemere bw’indwara S. ubwoko bw’ibitoke vy’ubudandaji bishobora gufatwa Giza 3 mu bihe vy’ivyatsi bitotahaye. Ivyiza bigereranya umubare usanzwe ± SD w’ibisubizo bitanu vy’ibinyabuzima (n = 5). Inyuguti zitandukanye zirerekana itandukaniro rikomeye hagati y’imiti (p < 0.05). (F–H) Ibimenyetso vy’ububi bwera vyabonetse ku bihimba vyo hejuru y’ubutaka no ku bihimba vy’umubiri, hakurikijwe imisi 10 inyuma y’ugucandagwa n’isolate #3 (dpi). (I) Isesengura ry’iterambere ry’akarere k’imbere mu gice c’imbere (ITS) c’igice ca S. sclerotiorum #3 cakozwe hakoreshejwe uburyo bw’ibishoboka vyinshi kandi kigereranywa n’igice 20 c’ibimenyetso vyaronswe mu kigo c’igihugu c’amakuru y’ubuhinga bwa none (NCBI) (httbih.gonivwwwm.). Imibare iri hejuru y’imirongo y’amatsinda yerekana akarere gapfutse (%), imibare iri musi y’imirongo y’amatsinda yerekana uburebure bw’ishami.
Ikindi kandi, kugira ngo bemeze ko iyo ndwara ishobora gutera indwara, hakoreshejwe ibice bine vya S. sclerotiorum vyaronswe mu gucandaga ubwoko bw’ibiharage vy’ubudandaji bishobora kwandura Giza 3 mu bihe vy’ivyatsi bitoto, ivyo bikaba bihuye n’ivyo Koch yashizeho (Ishusho 1E). Naho ibiherere ku mubiri vyose vyabonetse vyari bitera indwara kandi bishobora kwanduza ibitoke vy’icatsi kibisi (cv. Giza 3), bikaba bitera ibimenyetso vy’ibikoko vyera ku bice vyose biri hejuru y’ubutaka (Ishusho 1F), cane cane ku bihimba vy’umubiri (Ishusho 1G) no ku bihimba vy’umubiri (Ishusho 1H dapiys), post the inoculation (Ishusho 1H dapiys) yari 1000. aggressive isolate mu ngeragezwa zibiri zigenga. Isolate 3 yari ifise uburemere bwinshi bw’ingwara (%) ku bihingwa vy’ibitoke (24,0 ± 4,0, 58,0 ± 2,0, na 76,7 ± 3,1 ku misi 7, 14, na 21 inyuma y’aho umuntu afatiye iyo ndwara; Igishushanyo 1F).
Kumenya S. sclerotiorum isolate #3 itera cane vyaremejwe kandi bishingiye ku rutonde rw’ibice vy’imbere (ITS) (Ishusho 1I). Isesengurwa ry’ubuhinga bw’ivyaduka hagati y’ivyatsi #3 n’ivyatsi/imiryango 20 vyerekanye ko hariho ugusa cane (>99%) hagati yavyo. Birabereye kumenya ko S. sclerotiorum #3 (533 bp) isa cane n’iyi Amerika S. sclerotiorum LPM36 itandukanijwe n’imbuto z’ibitunguru vyumye (inomero y’ugushiramwo GenBank MK896659.1; 540 bp) n’umugera wa S. OR206374.1; 548 bp), ivyo bikaba bituma igiti c’umukara (Matthiola incana) kibora, ivyo vyose bikaba bishizwe hamwe bitandukanye hejuru y’igiti c’umukara (Ishusho 1I). Ivyo bihimba bishasha vyashizwe mu rutonde rw’ibiharuro vya NCBI, vyiswe “Sclerotinia sclerotiorum – isolate YN-25” (inomero y’ugushiramwo amakuru ya GenBank PV202792). Birashobora kubonwa ko isolate 3 ari yo isolate itera cane; rero, iyo isolate yaratowe kugira ngo yigishwe mu ngeragezwa zose zakurikiye.
Ibikorwa vyo kurwanya imigera y’umugera wa S. Biragaragara ko L-ornithine yagira ingaruka zo kurwanya imigera kandi buhoro buhoro igahagarika ukukura kw’imigera ya S. sclerotiorum mu buryo bushingiye ku rugero (Ishusho 2A, B). Ku rugero rwo hejuru rwageragejwe (125 mg/L), L-ornithine yerekanye urugero rwo guhagarika ukukura kw’imitsi (99,62 ± 0,27%; Igishushanyo 2B), rwari rungana n’umuti w’ubudandaji Rizolex-T wageragejwe) (urugero rwo kubuza gukura rwo hejuru 99.2% the Igishushanyo ±0.45 (10 mg/L), vyerekana ko bikora nk’ivyo nyene.
Igishushanyo 2. Ibikorwa vy’imigera ya L-ornithine ku ndwara y’ibicurane. (A) Kugereranya ibikorwa vy’ukurwanya imigera vy’ibice bitandukanye vy’umuti wa L-ornithine ku ndwara ya S. sclerotiorum n’umuti w’ubudandaji witwa Rizolex-T (10 mg/L). (B, C) Igitigiri c’uguhagarika (%) c’ukukura kw’imitsi ya S. sclerotiorum inyuma yo guvuzwa n’imiti itandukanye y’imiti yitwa L-ornithine (12,5, 25, 50, 75, 100 na 125 mg/L) canke Rizolex-T (2, 4, 6, 8 na 10 mgly). Ivyiza bigereranya umubare usanzwe ± SD w’ibisubizo bitanu vy’ibinyabuzima (n = 5). Inyuguti zitandukanye zigaragaza itandukaniro ry’imibare hagati y’imiti (p < 0.05). (D, E) Isesengura ry’ivy’ugusubira inyuma ry’umuti wa L-ornithine n’umuti w’ubudandaji witwa Rizolex-T, uko bikurikirana. Umurongo w’ugusubira inyuma w’akarorero ka probit werekanwa nk’umurongo w’ubururu bukomeye, kandi igihe co kwizigira (95%) kigaragazwa nk’umurongo w’umutuku w’agacavutu.
Ikindi, hakozwe isesengura ry’ihinduka ry’ibintu (probit regression) kandi ibishushanyo bihuye n’ivyo vyerekanywe mu ndome ya 1 no mu bishushanyo 2D,E. Mu magambo make, agaciro k’ugutemba kwemewe (y = 2,92x − 4,67) n’imibare ihambaye ijana (Cox & Snell R2 = 0,3709, Nagelkerke R2 = 0,4998 na p < 0,0001; Igishushanyo 2D) c’umuti wo kurwanya ibicurane vyerekanye ko umuti wo kurwanya ibicurane ushobora gutera imbere. umuti wica ibikoko Rizolex-T (y = 1,96x − 0,99, Cox na Snell R2 = 0,1242, Nagelkerke R2 = 0,1708 na p < 0,0001) (Imbonerahamwe 1).
Imbonerahamwe 1. Agaciro k’igice c’uburemere bw’ubumara (IC50) na IC99 (mg/l) bwa L-ornithine n’umuti w’ubudandaji “Rizolex-T” wo kurwanya S. sclerotiorum.
Muri rusangi, L-ornithine (250 mg/L) yagabanyije cane ugukura n’uburemere bw’ibikoko vyera ku bihingwa vy’ibiharage rusangi vyavuwe ugereranyije n’ibihingwa vyandujwe na S. sclerotiorum bitavuwe (ugucungera; igishushanyo 3A). Muri make, naho uburemere bw’ingwara bw’ibimera bigenzurwa vyanduye bitavuwe bwagenda buragwira buhoro buhoro (52,67 ± 1,53, 83,21 ± 2,61, na 92,33 ± 3,06%), L-ornithine yaragabanyije cane uburemere bw’ingwara (%) mu gihe cose c’igerageza, ±8,019. 1.00, na 26.36 ± 3.07) ku misi 7, 14, na 21 inyuma y’uguvugwa (dpt), hakurikijwe (Ishusho 3A). Na vyo nyene, igihe ibiterwa vy’ibiharage vyandujwe na S. sclerotiorum vyavuzwa n’imiti 250 mg/L L-ornithine, uburebure buri munsi y’umurongo w’iterambere ry’ingwara (AUDPC) bwagabanutse buva kuri 1274,33 ± 33,13 mu bihingwa bitavuwe buja kuri 281,03 ± 7,95, ivyo bikaba vyari hasi y’ivyo bihimba vy’umubiri bitavuwe. Igitera ibikoko citwa Rizolex-T (183,61 ± 7,71; ishusho 3B). Ivyo nyene ni vyo vyabonetse mu ngeragezwa ya kabiri.
Igishushanyo 3. Ingaruka z’ugukoresha L-ornithine bivuye hanze ku gutera kw’ukubora kwera kw’ibitoke bisanzwe bitewe na Sclerotinia sclerotiorum mu bihe vy’ivyatsi bitotahaye. (A) Igishushanyo c’ugutera kw’indwara y’ibikoko vyera vy’ibitoke bisanzwe inyuma yo kuvuzwa n’imiti 250 mg/L ya L-ornithine. (B) Ubutaka buri munsi y’umurongo w’iterambere ry’ingwara (AUDPC) bw’ibikoko vyera vy’ibitoke bisanzwe inyuma yo kuvuzwa na L-ornithine. Ivyiza bigereranya umubare usanzwe ± SD w’ibisubizo bitanu vy’ibinyabuzima (n = 5). Inyuguti zitandukanye zigaragaza itandukaniro rikomeye hagati y’imiti (p < 0.05).
Gukoresha 250 mg/L L-ornithine mu buryo bw’inyuma vyatumye buhoro buhoro uburebure bw’igitegwa bugenda burakura (Ishusho 4A), umubare w’amashami ku gitegwa (Ishusho 4B), n’umubare w’amababi ku gitegwa (Ishusho 4C) inyuma y’imisi 42. Naho umuti w’ubudandaji wo kwica ibicurane Rizolex-T (50 mg/L) wari ufise ingaruka nyinshi ku mirongo yose y’ingaburo yigishijwe, gukoresha 250 mg/L L-ornithine mu buryo bw’inyuma ni vyo vyari bifise ingaruka nyinshi kuruta ibindi vyose ugereranyije n’ivyo bikoresho bitavuwe (Ishusho 4A–C). Ku rundi ruhande, ubuvuzi bwa L-ornithine ntibwagize ingaruka zikomeye ku birimwo ivy’ibara ry’umubiri ryitwa chlorophylle a (Ishusho 4D) na chlorophylle b (Ishusho 4E), ariko bwarongereje gatoyi ivy’ibara ry’umubiri ryose ryitwa carotenoïde (0,56 ± 0,03 mg2 w2 ±03 mg2 fr wt) ugereranyije n’ivyo bihimba vy’umubiri bikora nabi (0.0. ivyiza (0,46 ± 0,02 mg/g fr wt; ishusho 4F). Muri rusangi, ivyo bimenyetso vyerekana ko L-ornithine atari ubumara bw’ibimera ku bihingwa vy’ibitoke vyavuwe kandi ko mbere bishobora no gutuma bikura.
Igishushanyo 4. Ingaruka z’ugukoresha L-ornithine ivuye hanze ku biranga ukura n’ibara ry’amababi y’ibiharage vyandujwe na Sclerotinia sclerotiorum mu bihe vy’ivyatsi bitotahaye. (A) Uburebure bw’igitegwa (cm), (B) Umubare w’amashami ku gitegwa, (C) Umubare w’amababi ku gitegwa, (D) Ivyo bita klorofile a (mg g-1 fr wt), (E) Ivyo bita klorofile b (mg g-1 fr wt), (F) Ivyo bita carotenoïde vyose hamwe (mg g-1 fr). Ivyiza ni umubare usanzwe ± SD w’ibisubizo bitanu vy’ibinyabuzima (n = 5). Inyuguti zitandukanye zirerekana itandukaniro rikomeye hagati y’imiti (p < 0.05).
Ivyigwa vyerekanye ko gukoresha L-ornithine (250 mg/L) biva hanze vyagabanyije cane ukwirundanira kwa H2O (H2O2]) n’ivy’imitsi y’umubiri (igaragazwa nk’umuyagankuba w’amazi [O2•−]) vyagaragaje ko gukoresha L-ornithine (250 mg/L) bigabanywa cane n’ukwirundanira kwa H2O (H2O2). nmol.g−1 FW; ishusho 5A) na O2•− (32,69 ± 8,56 nmol.g−1 FW; ishusho 5B) ugereranyije n’urugero rw’ibimera vyose bibiri vyanduye bitavuwe (173,31 ± 12,06) n’ibimera 149,35 ± 7,94 nmol. mg/L y’umuti w’ubudandaji Rizolex-T (170,12 ± 9,50 na 157,00 ± 7,81 nmol.g−1 fr wt, hakurikijwe) ku masaha 72. Ivyiyumviro vyinshi vya H2O2 na O2•− vyirundanijwe munsi ya hpt (Ishusho 5A, B). Na vyo nyene, igerageza ry’ivyo bihimba vy’umubiri bishingiye kuri TCA ryerekanye ko ibiterwa vy’ibiharage vyandujwe na S. sclerotiorum vyirundanije ivyimburwa vyinshi vy’ivyo bihimba vy’umubiri (113,48 ± 10,02 nmol.g fr wt) mu mababi yavyo (Ishusho 5C). Ariko rero, gukoresha L-ornithine mu buryo bw’inyuma vyagabanuye cane peroxydation y’amaraso nk’uko vyemezwa n’ukugabanya ivyimburwa vya MDA mu bimera vyavuwe (33,08 ± 4,00 nmol.g fr wt).
Ingaruka z’ugukoresha L-ornithine ku bimenyetso bikuru vy’imibabaro y’imitsi n’uburyo bwo kwikingira imiti idatera imitsi mu mababi y’ibiharage yandujwe na S. sclerotiorum amasaha 72 inyuma y’aho yandukiye mu bihe vy’ivyatsi. (A) Ivyuma vy’amazi (H2O2; nmol g−1 FW) ku 72 hpt, (B) anion y’amazi (O2•−; nmol g−1 FW) ku 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) ku 72 hpt, (D) umubare wose w’amazi (Fg−1 FW) (E) ivyimburwa vyose bishobora gusenyuka (mg RE g−1 FW) ku 72 hpt, (F) ivyimburwa vyose vy’umubiri bishobora gusenyuka (mg g−1 FW) ku 72 hpt, na (G) ivyimburwa vya proline (mg g−1 FW) ku 72 hpt. Ivyiza bigereranya umubare usanzwe ± ukunyuranya kw’itegeko (umubare usanzwe ± SD) w’ibisubizo 5 vy’ibinyabuzima (n = 5). Inyuguti zitandukanye zirerekana itandukaniro rikomeye hagati y’imiti (p < 0.05).