Ekihandiiko n’ekicweka ky’omutwe gw’okucondooza “Okutunguura okugumisiriza kw’ebihimba aha bukooko oburikureeta endwara n’ebizimba”, reeba ebihandiiko byona 5 .
Ekirikureetaho endwara y’ebimera ebirikureeta obuhuta Sclerotinia sclerotiorum (Lib.) de Bary nikikozesa enkora y’emiringo mingi kujanjaaza ebimera ebitari bimwe na bimwe ebirikutunga abagyenyi. Okucondooza oku nikuha enteekateeka y’okukoresa diamine L-ornithine, ekirungo ekitari kya puroteyini ekirikureetaho okuteerana kw’ebindi bihingwa ebikuru, nk’omuringo ogundi gw’okureeberera okutunguura okugarukamu kw’ebicweka by’omubiri, eby’omubiri n’eby’amagara ebya Phaseolus vulgaris L. aha nshaho erikwera erikureetwa Pseoler scrootiotiorum. Okucondooza okwakozirwe omu vitro kukooreka ngu L-ornithine ekazibira munonga okukura kwa mycelium ya S. pyrenoidosa omu muringo ogurikurugiirira aha dozi. Ekindi, nikibaasa kukyendeeza munonga oburemeezi bw’ebizimba ebirikwera omu mbeera z’ebishaka. Ekindi, L-ornithine ekareetaho okukura kw’ebimera ebyabaire biine obujanjabi, ekirikworeka ngu ebicweka bya L-ornithine ebyagyeziibwe bikaba bitari by’obutwa bw’ebimera ebyabaire biine obujanjabi. Okwongyera ahari ekyo, L-ornithine ekayongyera aha kumanyisa kw‘ebintu ebitari by‘omubiri ebirikurwanisa obukooko (ebirungo by‘omubiri ebirikubaasa kusheesheka byona n‘ebiriisa by‘omubiri) n‘ebintu ebirikurwanisa obukooko oburikureeta endwara (catalase (CAT), peroxidase (POX), na polyphenol oxidase (PPO)), kandi eyongyera aha kumanyisa kw‘ebicweka bishatu ebirikurwanisa obukooko oburikureeta endwara, PGRvD, na PvSOAT). Ekindi, omu kucondooza kwa silico kukashohoza okubaho kw’ekirungo ky’omubiri ekirikuteekateekwaho kuba eky’omubiri (SsOAH) omu nkora ya S. sclerotiorum, ekyabaire nikishushana munonga n’ebiriisa by’omubiri eby’omubiri (SsOAH) ebya PenjinsiAHiOAH (SsOAH) eby’omubiri gw’omubiri gw’omukazi (SsOAH). (PlOAH) omu kureeba okushwijuma emirimo, emyanya erikubiikwa, na topology. Ekirikushemeza, okwongyera L-ornithine omu mubazi gw’ebitokye (PDB) kikakyendeeza munonga aha kureebeka kw’omubazi gwa SsOAH omu S. sclerotiorum mycelia. Omu muringo nigwo gumwe, okukoresa L-ornithine omu muringo gw’aheeru (exogenous) kukakyendeeza munonga aha kureebeka kw’omubazi gwa SsOAH omu mycelia y’ebizimba eyaabaire etorotoirwe kuruga omu bimera ebyabaire biine obujanjabi. Eky’aha muheru, okukoresa L-ornithine kukakyendeeza munonga aha kusheeshasheesha kwa oxalic acid omu mababi gombi aga PDB medium n’againe oburwaire. Omukuhendera, L-ornithine neekora omurimo muhango omu kuguma n’embeera ya redox hamwe n’okwongyera aha kugarukamu kw’okwerinda kw’ebimera ebiine oburwaire. Ebirugire omu kucondooza oku nibibaasa kuhwera omu kuhangaho emiringo mirungi, etarikushiisha eby’obuhangwa ey’okuzibira obukooko oburikwera n’okukyendeeza aha kabi akarikureeta aha kuhinga ebihimba n’ebirimwa ebindi.
Obukooko oburikwera, oburikureetwaho obukooko oburikwetwa Sclerotinia sclerotiorum (Lib.) de Bary, n’endwara erikushiisha munonga, erikukyendeeza aha nshaho kandi erikureeta akabi k’amaani aha kuhinga ebihimba (Phaseolus vulgaris L.) omu nsi yoona (Bolton et al., 2006). Sclerotinia sclerotiorum n’emwe aha ndwara z’ebimera ezirikureetwa obukooko oburikureeta omu itaka ezigumire kukyebera, ziine omubaro muhango gw’ebimera ebirikurenga 600 n’obushoboorozi bw’okurahuka kusheenya enyama z’omugyenyi omu muringo ogutari gw’omutaano (Liang na Rollins, 2018). Omu mbeera ezitari nungi, neehingura omu bwire bw’amaani omu nkora y’amagara gaayo, eguma egwejegyeire kumara obwire buraingwa nk’ebintu ebirikwiragura, ebigumire, ebiri nk’enjuma ebirikwetwa ‘sclerotia’ omu itaka nari nk’ebintu ebirikwera, ebiine obuhuta omu mycelium nari stem pith y’ebimera ebiine oburwaire (S). S. sclerotiorum neebaasa kuhangaho ebizimba by’omubiri, ekirikugibaasisa kuguma omu misiri erimu oburwaire kumara obwire buraingwa kandi ekagumaho omu bwire bw’endwara (Schwartz et al., 2005). Sclerotia zirimu ebiriisa bingi, nizibaasa kuguma omu itaka kumara obwire buraingwa, kandi nizikora nk’omubazi gw’okubanza ogw’okujanjaba oburwaire oburikukuratira (Schwartz et al., 2005). Omu mbeera nungi, sclerotia nizikura kandi zireetaho obukooko oburi omu mwanya oburikubaasa kukwata ebicweka byona eby’ekimera ebiri ahaiguru y’itaka, otwariiremu kwonka bitarikugarukira aha bimuri, ebihimba, nari ebihimba (Schwartz et al., 2005).
Sclerotinia sclerotiorum neekozesa enkora y’emiringo mingi kujanjaaza ebimera byayo ebirikugitwara, erimu ebintu ebitari bimwe na bimwe ebikwataniise kuruga aha kukura kw’ebimera bya sclerotia kuhika aha kukura kw’obumanyiso. Omukubanza, S. sclerotiorum neereetaho obukooko oburikwemerera (oburikwetwa ascospores) kuruga omu bihingwa ebiri nk’ebihimba ebirikwetwa apothecia, ebirikuhinduka omu mwanya kandi bikakura bikahinduka ebihimba ebitarikugyendera aha bihingwa ebiine oburwaire (Bolton et al., 2006). Bwanyima, ekizimba eki nikishohoza oxalic acid, ekirikureeta oburwaire, okubaasa kukyebera pH y’ekisiika ky’obutafaari bw’ebimera, kutunguura okusheenyagura kw’enzigye n’okutaahirira kw’ebicweka by’omubiri (Hegedus na Rimmer, 2005), n’okuzibira okusheeshasheesha kw’ekimera ekirikukitunga. Enkora egi ey’okuhindura asidi neekyendeeza amaani g’obutafaari bw’ebimera, erikuha omwanya murungi gw’okukora gye kandi omu muringo oguhikire ogw’enzigye ezirikushiisha obutafaari bw’ebimera (CWDEs), ekirikureetera obukooko oburikureeta endwara kusingura omuzigo gw’omubiri kandi bukataaha omu bicweka by’omubiri ebirikubutunga (Marciano et al., 1983). S. sclerotiorum ku erikuba yaaheza kutaaha omu mubiri, neeshohoza CWDE nyingi, nka polygalacturonase na cellulase, ebirikuhwera omu kujanjaara kwayo omu bicweka by’omubiri ebiine oburwaire kandi bikareetaho okusheeshasheesha kw’ebicweka by’omubiri. Okutunguuka kw‘ebizimba n‘ebizimba by‘omubiri (hyphal mats) nibireetaho obumanyiso bw‘obumanyiso bw‘ebizimba by‘omubiri ebirikwera (Hegedus na Rimmer, 2005). Omu bwire obwo, ebimera ebirikutwara abagyenyi nibimanya enkora y‘obukooko oburikureeta endwara (PAMPs) kurabira omu bikwatiraine n‘okumanya enkora (PRRs), ebirikureetaho omubaro gw‘ebintu ebirikumanyisa ebirikuhererukayo bikareetaho okugarukamu kw‘okwerinda.
N’obu haraabe hahingwireho emyaka mingi y’okurwanisa endwara, okubura obukooko oburikurwanisa endwara oburikumara nikuguma omu bihimba, nk’oku kiri omu birimwa ebindi eby’obushuubuzi, ahabw’okuzibira, okugumaho, n’okuhindura kw’obukooko oburikureeta endwara. Nahabwekyo okureeberera endwara nikigumira munonga kandi nikyetengyesa enkora erimu okuteerana, erimu emiringo mingi erimu okuteerana kw‘enkora z‘obuhangwa, okurinda ebihangirwe, n‘emibazi y‘okurwanisa obukooko (O‘Sullivan et al., 2021). Emibazi y’okurwanisa obukooko oburikwera niyo erikukirayo kukora gye ahabw’okuba emibazi y’okurwanisa obukooko obu, ku erikuba neekoresibwa gye kandi omu bwire obuhikire, neebaasa kukyebera gye okujanjaara kw’endwara, kukyendeeza oburemeezi bw’okukwatwa endwara, n’okukyendeeza aha kufeerwa ebiriisa. Kwonka okukoresa munonga n’okwesiga munonga emibazi y’okurwanisa obukooko bw’ensiri nikibaasa kureetera okurugayo kw’ebika bya S. sclerotiorum ebitarikubaasa kukwatwa endwara kandi kikwate kubi ebihangirwe ebitarikugyenderwaho, amagara g’itaka, n’omutindo gw’amaizi (Le Cointe na bagyenzi be, 2016; Ceresini na bagyenzi be, 2024). Nahabwekyo, okutunga emiringo endiijo etarikushiisha eby’obuhangwa kihindukire ekintu ky’okubanza.
Ebirungo bya polyamines (PAs), nka putrescine, spermidine, spermine, na cadaverine, nibibaasa kukora nk’ebintu ebirikuraganisa kurwanisa obukooko oburikureeta endwara z’ebimera kuruga omu itaka, obwo nibikyendeeza kimwe nari ekicweka ky’okukoresa emibazi y’akabi ey’okurwanisa obukooko oburikureeta endwara (Nehela et al., 2024; Yi20, 2 ). Omu bimera ebiri ahaiguru, PAs nizejumba omu miringo mingi y‘omubiri erimu, kwonka etarikugarukira ahari, okugabanisa obutafaari, okutaanisa, n‘okugarukamu oburemeezi bw‘ebihangirwe ebitariho n‘ebiine amagara (Killiny na Nehela, 2020). Nibibaasa kukora nk‘ebirikurwanisa obukooko oburikureeta endwara, kuhwera omu kusheenya ebika by‘omwika ebirikukora (ROS), kuguma n‘omubiri (Nehela na Killiny, 2023), kureetaho ebiriisa ebiine akakwate n‘okwerinda (Romero na bagyenzi be, 2018), kuhabura emihanda etari emwe n‘emwe y‘okuhindura eby‘okurya (Nehela na Killiny, 2023). Killiny, 2019), kutaho okuzibira okutunga endwara (SAR), n’okutegyeka enkoragana y’ebimera n’obukooko oburikureeta endwara (Nehela na Killiny, 2020; Asija na bagyenzi be, 2022; Czerwoniec, 2022). Nikirungi kumanya ngu emiringo n‘emirimo y‘omutaano eya PAs omu kurinda ebimera neetaanira kimwe kurugiirira aha kika ky‘ebimera, obukooko oburikureeta endwara, n‘embeera z‘eby‘obuhangwa. PA erikukirayo omu bimera neekorwa omu bimera kuruga omu kirungo ky’omugasho munonga ekya L-ornithine (Killiny na Nehela, 2020).
L-ornithine neekora emirimo mingi omu kukura n’okutunguuka kw’ebimera. Nk’eky’okureeberaho, okucondooza okukozirwe enyima kworekire ngu omu muceeri (Oryza sativa), ornithine neebaasa kuba neekwatagana n’okugaruzaho nitrogen (Liu na bagyenzi be, 2018), okutunga omuceeri, omutindo n’akawoowo (Lu na bagyenzi be, 2020), n’okugarukamu okuremeera kw’amaizi (Yang na bagyenzi be, 2000). Ekindi, okukoresa L-ornithine omu muringo gw‘aheeru y‘eihanga kukatunguura munonga okugumisiriza kw‘ekyanda omu bihingwa bya sukaari (Beta vulgaris) (Hussein na bagyenzi be, 2019) kandi kwakyendeeza aha buremeezi bw‘omunyugunyu omu butunguru (Allium Cepa) Rocha n’abandi, 2012). Omurimo ogurikubaasa kukorwa L-ornithine omu kwerinda okuteganisibwa kw’ebintu ebitari bikuru nigubaasa kuba nigureetwaho okwejumba kwayo omu kuteerana kwa proline omu bimera ebijanjabirwe. Nk’eky’okureeberaho, ebiriisa ebiine akakwate n’okuhindura eby’okurya omu mubiri, nk’ebiriisa ebirikukora nk’ebiriisa ebiri omu mubiri (delta-OAT) n’ebiriisa ebiri omu mubiri (ProDH1 na ProDH2), bikaba bigambirweho enyima ngu nibikora aha kurinda ebiriisa bya Nicotiana benthamiana na Arabidopsis thaliana aha Pse ezitari z’omukaikuru/omugurusi (Pse). Omu mwaka gwa 2012). Aha rubaju orundi, obukooko oburikureeta endwara (ODC) nibwetengyesa omu kukura kw’obukooko oburikureeta endwara (Singh et al., 2020). Okuta omutima aha ODC y’obukooko oburikureeta endwara f. sp. lycopersici kurabira omu kukyebera ebiriisa by’omubiri (HIGS) kikatunguura munonga okubaasa kw’ebimera by’ennyanya okurwanisa obuhuta bwa Fusarium (Singh et al., 2020). Kwonka, omurimo ogurikubaasa kukorwa omu kukozesa exogenous ornithine aha bizibu by’ebihangirwe nk’ebirikureeta endwara z’ebimera tigukakyeberwa gye. Ekikuru munonga, ebirikuruga omu ornithine aha kugumisiriza endwara n‘ebintu ebirikukwatagana n‘eby‘amagara n‘eby‘omubiri nibiguma bitacondooziibwe munonga.
Okwetegyereza oburemeezi bw‘okukwatwa S. sclerotiorum omu bihingwa by‘ebihimba ni kikuru omu kutunguura enkora nungi z‘okuzibira. Omu kucondooza oku, tukaba nitugyenderera kumanya omurimo ogurikubaasa kukorwa diamine L-ornithine nk’enshonga nkuru omu kutunguura emiringo y’okwerinda n’okuzibira ebimera by’ebihimba oburwaire bwa Sclerotinia sclerotiorum. Nituteekateeka ngu, okwiha aha kwongyera aha kugarukamu kw’okwerinda kw’ebimera ebiine oburwaire, L-ornithine nayo neekora omurimo muhango omu kuguma n’embeera ya redox. Nituteekateeka ngu ebirikubaasa kuruga omu L-ornithine nibikwatagana n‘okutebeekanisa enkora z‘okwerinda z‘enzigye n‘ezitari z‘enzigye n‘okutabura enshonga z‘okureeta endwara z‘ebizimba/obukooko bw‘ensiri n‘ebiriisa ebiine akakwate nazo. Omurimo ogu ogw’emirundi ebiri ogwa L-ornithine nigugihindura ekintu ekirikuraganisa omu nkora y’okugumizamu ey’okukyendeeza aha kabi akarikureetwaho obukooko oburikwera n’okwongyera aha kugumisiriza kw’ebirimwa by’ebihimba ebya buriijo aha bukooko obu obw’amaani oburikureeta obukooko obu. Ebirugire omu kucondooza oku nibibaasa kuhwera omu kuhangaho emiringo mirungi, etarikushiisha eby’obuhangwa ey’okuzibira obukooko oburikwera n’okukyendeeza aha kabi akarikureeta aha kuhinga ebihimba.
Omu kucondooza oku, ekika ky’ebihimba bya buriijo ekirikubaasa kukwatwaho eby’obushuubuzi, Giza 3 (Phaseolus vulgaris L. cv. Giza 3), kikakoresibwa nk’ebintu by’okugyezesa. Enjuma eziine amagara marungi zikaheebwayo n’embabazi kuruga omu kitongore ky’okucondooza aha bihingwa by’ebihingwa, ekitongore ky’okucondooza aha birimwa by’omu misiri (FCRI), ekitongore ky’okucondooza aha by’obuhingi (ARC), Misri. Enjuma itaano zikabibwa omu biyungu bya pulasitiki (omunda diamita 35 cm, oburaingwa 50 cm) ebiijwire eitaka eririmu oburwaire bwa S. sclerotiorum omu mbeera z’ekibira (25 ± 2 °C, obutagatsi oburikuhikaana 75 ± 1%, 8 h omushana/16 h omwirima). Ebiro 7–10 bwanyima y’okubyara (DPS), embibo zikaba zikyendeezibwa kusigaho embibo ibiri zonka eziine okukura okurikushushana n’ebibabi bishatu ebihikire omu buri nyungu. Ebimera byona ebyabaire biri omu biyungu bikafukirizibwa omurundi gumwe buri wiiki ibiri kandi byafukirizibwa buri kwezi aha rurengo orurikuhaburwa aha kika ekihairwe.
Okutebeekanisa ekirungo kya 500 mg/L eky’omubaro gw’ekiriisa kya L-ornithinediamine (ekirikumanyirwa nka (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Bugirimaani), 50 mg zikabanza zaasheesheka omu miriyoni 100 z’amaizi agateirwemu obutwa. Bwanyima omubazi gwa stock gukakyeberwa kandi gwakoresibwa omu kucondooza okwakuratsireho. Omu bugufu, emiringo mukaaga y‘ebicweka bya L-ornithine (12.5, 25, 50, 75, 100, na 125 mg/L) ekakyeberwa omu nkora y‘okukyebera. Okwongyera ahari ekyo, amaizi agateirwemu obutwa gakakoresibwa nk‘okukyebera okubi (Mock) kandi omubazi gw‘okurwanisa obukooko oburikureeta obuhuta “Rizolex-T” 50% obutunguru oburikubaasa kunywa amaizi (toclofos-methyl 20% + thiram 30%; nk‘okutegyeka okurungi. Omubazi gw’okurwanisa obukooko bw’ensiri ogw’obushuubuzi “Rizolex-T” gukakyeberwa omu nkora y’okukyebera aha bihingwa bitaano (2, 4, 6, 8 na 10 mg/L).
Sampuli z’ebihimba bya buriijo n’ebihimba ebirikworeka obumanyiso bw’omutaano obw’ebizimba ebirikwera (omubaro gw’okukwatwa: 10–30%) bikatorotoorwaho kuruga omu faamu z’eby’obushuubuzi. N‘obu ebimera ebingi ebyabaire biine oburwaire biraabe byamanyirwe aha kika/ekika (ekika ky‘obushuubuzi ekirikubaasa kukwatwa Giza 3), ebindi, namunonga ebyatungirwe kuruga omu butare bw‘omu kyanga, bikaba biri eby‘ebika ebitarikumanywa. Ebintu ebyarundainwe ebiine oburwaire bikabanza byakyeberwa aha mutwe n’omubazi gwa 0.5% sodium hypochlorite kumara edakiika 3, bwanyima byasiimurwa emirundi mingi n’amaizi agatarimu kamogo kandi byasiimurwa byomire n’orupapura rw’okushwijuma orutarimu kamogo kwihaho amaizi agarengire. Ebicweka ebyabaire biine oburwaire bwanyima bikasharaho obucweka bukye kuruga omu nyama z‘ahagati (ahagati y‘enyama eziine amagara marungi n‘eziine oburwaire), byakuzibwa aha nkora ya potato dextrose agar (PDA) kandi byateebwa omu 25 ± 2 °C n‘omushana gw‘eshaha 12/omwirima 12 h kumara ebiro 5 kureetaho okuhangwa kwa sclerotia . Omuringo gwa mycelial tip nagwo gukakozesibwa kushemeza ebihimba ebitaine rubaju kuruga omu buhangwa obutaine rubaju nari oburimu obukooko. Ekicweka ky’ebishaka ekishemeziibwe kikabanza kyamanyirwa aha mitwarize yaakyo ey’obuhangwa bwanyima kyahamibwa kuba S. sclerotiorum kurugiirira aha mitwarize y’ebizimba ebikye. Eky’aha muheru, isolates zoona ezishemeziibwe zikakyeberwa kureeba yaaba nizireeta endwara aha kika ky’ebihimba ekya buriijo ekirikubaasa kukwatwa Giza 3 kuhikiiriza ebiteekateeko bya Koch.
Okwongyera ahari ekyo, ekicweka kya S. sclerotiorum ekirikukirayo kutaaha omu itaka (ekicweka #3) kikagaruka kyahamibwa kurugiirira aha kucondooza kw’omunda (ITS) nk’oku kyashoboororwa White et al., 1990; Baturo-Ciesniewska na bagyenzi be, 2017. Omu bugufu, ebihimba ebyabaire bitaanisiibweho bikaba bihingwa omu nshaho y’ebitokye (PDB) kandi byabiikwa aha 25 ± 2 °C kumara ebiro 5–7. Bwanyima, mycelium y’ebizimba ekatorotoorwamu, yasendwa kurabira omu cheesecloth, yaanaabwa emirundi ebiri n’amaizi agatarimu kamogo, kandi yayomezibwa n’orupapura rw’okushwijuma orutarimu kamogo. DNA y’omubiri ekataanisibwa barikukoresa ekikwato ky’okutebeekanisa obukooko oburikwetwa Quick-DNATM (Kuramae-Izioka, 1997; Atallah n’abandi, 2022, 2024). Ekicweka kya ITS rDNA bwanyima kikagarurwamu amaani barikukoresa omubazi gw’okubanza ogw’omutaano ITS1/ITS4 (obuhango oburikuteekateekwaho: 540 bp) ( Baturo-Ciesniewska et al., 2017 ). Ebintu bya PCR ebyashemeziibwe bikaheebwayo kubaasa kukuratana (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). Ebicweka bya ITS rDNA bikakuratana omu miringo ebiri barikukoresa omuringo gw’okukuratana ogwa Sanger. Ebibuuzo ebyabaire biteeraine bikagyeragyeranisibwa n’ebihandiiko ebisya ebiri omu GenBank n’ekitongore ky’eihanga eky’amakuru g’eby’obuhangwa (NCBI, http://www.ncbi.nlm.nih.gov/gene/) barikukoresa puroguraamu ya BLASTn. Omukuratana gw’ebibuuzo gukagyeragyeranisibwa n’ebindi bihingwa 20 ebya S. sclerotiorum/ebitari bimwe na bimwe ebyarugire omu makuru agahererukireyo omu NCBI GenBank (Ekipande ky’okwongyeraho S1) barikukoresa ClustalW omu nkora y’okucondooza aha nkora y’okutunguuka kw’ebicweka by’omubiri (MEGA-11; omuringo 11) (Kumar et al., 20). Okushwijuma eby‘entunguuka kukakorwa barikukoresa omuringo gw‘okuteekateeka okurikukirayo n‘omuringo gw‘okuhindura nucleotide ogurikugaruka enyima omu bwire bwona (Nei na Kumar, 2000). Omuti ogurikukirayo obuhango gw’okuteera emiti nigworeka. Omuti gw‘okubanza ogw‘okusherura kw‘okucondooza (heuristic search) nigutooranwa omu kutoorana omuti ogurimu log-likelihood y‘ahaiguru ahagati y‘omuti gw‘okutaho omutaahi (NJ) (Kumar et al., 2024) n‘omuti gw‘okushohoza munonga (MP). Omuti gwa NJ gukozirwe barikukoresa enkora y‘okugyeragyeranisa oburaingwa bw‘abantu babiri (pairwise distance matrix) ebariirwe barikukoresa enkora y‘okugaruka enyima omu bwire bwona (Nei na Kumar, 2000).
Omurimo gw’okurwanisa obukooko bwa L-ornithine n’omubazi gw’okurwanisa obukooko “Rizolex-T” gukamanyirwa omu nkora ya vitro ahabw’omuringo gw’okujanjaaza agar. Omuringo: Twara omubaro oguhikire gw‘omubazi gwa L-ornithine (500 mg/L) kandi ogutabwire gye na miriyoni 10 z‘omubazi gwa PDA kutebeekanisa ebirungo ebiine ebiriisa by‘aha muheru bya 12.5, 25, 50, 75, 100 na 125 mg/L, nk‘oku biri. Ebicweka bitaano by’omubazi gw’okurwanisa obukooko “Rizolex-T” (2, 4, 6, 8 na 10 mg/L) n’amaizi agateirwemu obuhuta bikakoresibwa nk’okukyebera. Bwanyima y’omubazi kuhama, ekipande ky’obuhangwa bwa Sclerotinia sclerotiorum ekyatebeekanisiibwe busya, kikatwarwa ahagati y’ekikopo kya Petri kandi kyahingwa aha 25±2°C kuhitsya obu ekikopo kyakingire ekikopo kyona eky’okutegyeka Petri, bwanyima y’ekyo okukura kw’ebizimba kukahandiikwa. Barira ebicweka 10 ahari 100 eby’okuzibira okukura kwa S. sclerotiorum orikukoresa ekibaro 1:
Okugyezesa kukagarukwamu emirundi ebiri, n’ebicweka mukaaga by’ebihangwa ebiine amagara ahari buri guruupu y’okukyebera/okugyezesa n’ebiyungu bitaano (ebimera bibiri ahari buri kiyungu) ahari buri kishushani ky’ebihangwa. Buri kimwe ekirikugarukwamu omu by’obuhangwa kikakyeberwa emirundi ebiri (ebigarukwamu bibiri eby’obuhangwa) kureeba ngu ebyarugamu omu kucondooza bihikire, nibyesigwa kandi nibibaasa kugaruka kukorwa. Okwongyera ahari ekyo, okucondooza kw‘okugaruka enyima kwa probit kukakoresibwa kubara ekicweka ky‘okuzibira okukyeberwa (IC50) na IC99 (Prentice, 1976).
Okukyebera obushoboorozi bwa L-ornithine omu mbeera za greenhouse, okucondooza kw’ebiyungu bibiri okurikukuratana kukakorwa. Omu bugufu, ebiyungu bikaijurwa eitaka ry’eibumba n’omushenyi eririmu obutwa (3:1) kandi byateebwamu obuhangwa bwa S. sclerotiorum obutebeekanisiibwe busya. Ekyokubanza, ekicweka ekirikukirayo kutaaha omu itaka ekya S. sclerotiorum (ekicweka #3) kikakuzibwa barikusharaho ekicweka kimwe kya sclerotium omu bicweka bibiri, bakakitaho amaisho ahansi aha PDA kandi bakakiteeka aha 25°C omu mwirima ogutarikuhinduka (24 h) kumara ebiro 4 kubaasa kureetaho okukura kwa mycelium. Bwanyima, ebipande bina ebya agar ebya diameter ya mm 5 bikatwarwa kuruga aha rubaju orurikwebembera kandi byateebwamu 100 g y’omutaano gw’engano n’omuceeri ogutarimu kamogo (1:1, v/v) kandi ebikopo byona bikateebwa omu 25 ± 2 °C ahansi y’omuhimbo gw’omushana gw’eshaaha 12/omwirima gw’eshaaha 12 kubaasa kureetaho sc 5. Ebirimu omu bikopo byona bikaba bitaburwa gye kureeba ngu byashushana batakayongyeiremu eitaka. Bwanyima, 100 g z’omubazi gw’ebihimba ebirikukora nk’ebihimba (colonizing bran mixture) zikateebwa omu buri nyungu kureeba ngu obukooko oburikureeta endwara bwaguma nibuteerana. Ebiyungu ebyabaire biteirwemu obutwa bikafukirizibwa kubaasa kureetaho okukura kw’ebizimba kandi byateebwa omu mbeera z’ebishaka kumara ebiro 7.
Bwanyima bakabiba embibo itaano z’omuringo gwa Giza 3 omuri buri nyungu. Aha biyungu ebyateirwemu L-ornithine hamwe n’omubazi gw’okurwanisa obukooko Rizolex-T, embibo eziteirweho obukooko zikabanza zaateebwa omu maizi kumara eshaaha ibiri omu nkora y’amaizi g’ebirungo bibiri n’ekicweka kya IC99 eky’aha muheru ekyabaire kiri nka 250 mg/L na 50 mg/L, nk’oku biri, reero bwanyima zikayomezibwa omu mwanya kumara eshaaha emwe. Aha rubaju orundi, embibo zikaba ziteirwe omu maizi agatarimu kamogo nk’okukyebera okubi. Bwanyima y’ebiro 10, amaizi gatakabanza kufukirizibwa, embibo zikaba zikyendeezibwa, zisigaho embibo ibiri zonka ezishemeire omuri buri nyungu. Okwongyera ahari ekyo, kureeba ngu S. sclerotiorum yakwatwa, ebihimba by’ebihimba aha rurengo rumwe rw’okukura (ebiro 10) bikasharaho omu myanya ebiri etarikushushana barikukoresa ekikwato ky’okusharaho ekiine obutwa kandi enkumi 0.5 g z’omubazi gw’ebihimba by’obukoloni zikateebwa omu buri kihuta, bwanyima y’okuteebwamu obutagatsi bwingi kubaasa kureetaho okukura kw’endwara zoona. Ebimera by’okukyebera bikahutaazibwa omu muringo nigwo gumwe kandi omubaro gurikwingana (0.5 g) gw’omutaano gw’ebihimba ebitarimu nshonga, ebitarikukwatwaho obukoloni gukateebwa omu kihuta kandi gwaguma ahansi y’obutagatsi bwingi kugyeragyeranisa embeera y’okukura kw’endwara n’okureeba ngu hariho okutaikirizana ahagati y’ebicweka by’okujanjaba.
Omuringo gw’okujanjaba: Embibo z’ebihimba zikafukirizibwa miriyoni 500 z’omubazi gw’amaizi ogwa L-ornithine (250 mg/l) nari omubazi gw’okurwanisa obukooko Rizolex-T (50 mg/l) barikufukiriza eitaka, bwanyima okujanjaba kukagarukaho emirundi eshatu omu bwire bw’ebiro 10. Abakyebeirwe abaine obujanjabi bwa placebo bakafukirizibwa miriyoni 500 z‘amaizi agatarimu kamogo. Obujanjabi bwona bukakorwa omu mbeera z‘ekibira (25 ± 2°C, 75 ± 1% obutagatsi oburikuhikaana, n‘obwire bw‘omushana bwa 8 h/omwirima 16 h). Ebiyungu byona bikafukirizibwa buri wiiki ibiri kandi byakyeberwa buri kwezi n’ekigimusa kya NPK ekiine omutindo (20-20-20, ekirimu 3.6% sulfur na TE microelements; Zain Seeds, Egypt) aha nkora ya 3–4 g/l barikufuuhirira amababi kurugiirira aha biragiro by’omukozi n’omuringo gw’omutaano’. Okwihaho kyaba kitagambirwe, amababi agakuzire agahikire (amababi ga 2 na 3 kuruga ahaiguru) gakatorotoorwaho kuruga omu buri kimwe ekirikugarukaho omu by‘amagara aha shaaha 72 bwanyima y‘okujanjaba (hpt), gakashushanisibwa, gakateerana kandi gabiikwa aha -80 °C ahabw‘okushwijuma okurikukiraho okurimu, kwonka hatariho kugarukira ahari, omu mwanya ogw‘okuhabura kw‘okuhabura kw‘eby‘amagara, omu mwanya gw‘okuhabura kw‘eby‘amagara, histochemical. n‘ebitari by‘enzigye ebirikurwanisa obukooko n‘okumanyisa ebiri omu nkora y‘obuzaarwa.
Oburemeezi bw‘okukwatwa obukooko oburikwera bukakyeberwa buri saabiiti ebiro 21 bwanyima y‘okukyeberwa (dpi) barikukoresa omubaro gwa 1–9 (Ekipande ky‘okwongyeraho S2) kurugiirira aha mubaro gwa Petzoldt na Dickson (1996) ogwahinduirwe Teran na bagyenzi be. (2006). Omubugufu, ebihimba n’amataagi g’ebimera by’ebihimba bikakyeberwa kutandikira aha mwanya gw’okukyebera kukuratira oku ebizimba birikugyenda aha rubaju rw’ebihimba n’ebihimba. Oburaingwa bw‘ekizibu kuruga aha mwanya gw‘okukyebera kuhika aha mwanya oguri hare munonga aha rubaju rw‘omuti nari eitaagi bwanyima bukapimwa kandi obubonero bwa 1–9 bukaheebwa kurugiirira aha mwanya gw‘ekizibu, ahu (1) kyorekire ngu tihaine oburwaire oburikureebeka haihi n‘omwanya gw‘okukyebera kandi (2–9) kyorekire okweyongyera mporampora omu bugabe bw‘ekizimba noppnolong a/S Table S2). Obuhango bw'okukwatwa obukooko oburikwera bwanyima bukahindurwa omu bicweka 10 ahari 100 barikukoresa enkora ya 2:
Okwongyera ahari ekyo, omwanya oguri ahansi y‘omutindo gw‘okutunguuka kw‘endwara (AUDPC) gukabarwa barikukoresa enkora (Shaner na Finney, 1977), eyaabaire eherize kuhindurwa ahabw‘okuvunda kw‘ebihimba ebya buriijo (Chauhan et al., 2020) barikukoresa enkora ya 3:
Aha Yi = oburemeezi bw‘endwara omu bwire ti, Yi+1 = oburemeezi bw‘endwara omu bwire oburikukurataho ti+1, ti = obwire bw‘okubanza okupima (omu biro), ti+1 = obwire bw‘okupima oburikukurataho (omu biro), n = omubaro gw‘obwire bwona nari enshonga z‘okureeberaho. Ebirikumanyisa okukura kw’ebimera by’ebihimba ebirimu oburaingwa bw’ebimera (cm), omubaro gw’amataagi ahari buri kimera, n’omubaro gw’amababi ahari buri kimera bikahandiikwa buri saabiiti kumara ebiro 21 omu bihandiiko byona eby’ebihangirwe ebiine amagara.
Omuri buri nkora y‘okugarukamu ey‘ebihangirwe, eby‘okureeberaho by‘amababi (ebibabi bya kabiri n‘ebya kashatu ebikuzire omu bwijwire kuruga ahaiguru) bikatorotoorwaho aha kiro kya 45 bwanyima y‘okujanjaba (ebiro 15 bwanyima y‘okujanjaba okurikuhererukayo). Buri kishushani ky’ebihangirwe kikaba kirimu ebiyungu bitaano (ebimera bibiri ahari buri kiyungu). Nk’emitwaro 500 y’ebicweka by’omubiri ebishazirwe bikakoresibwa omu kushohoza erangi z’omushana (klorofili a, klorofili b na carotenoids) barikukoresa 80% ya asetoni ahari 4 °C omu mwirima. Bwanyima y‘eshaaha 24, eby‘okureeberaho bikateebwa omu nkora y‘okukyebera ebiriisa bya klorofiili a, klorofiili b na carotenoid omu muringo gw‘okupima erangi barikukoresa ekikwato ky‘okupima ebiri omu itaka ekya UV-160A (Shimadzu Corporation, Japan) kurugiirira aha nkora ya (Lichtebaring three8or the different at1) . oburaingwa bw’amaizi (A470, A646 na A663 nm). Eky’aha muheru, ebiri omu rangi z’omushana bikabarwa barikukoresa enkora ezi 4–6 ezishobooroirwe Lichtenthaler (1987).
Aha shaaha 72 bwanyima y’okujanjaba (hpt), amababi (amababi ga kabiri n’aga kashatu agakuzire omu bwijwire kuruga ahaiguru) gakatorotoorwa kuruga omu buri kishushani ky’ebihangirwe ahabw’okucondooza omu mwanya gw’eby’obuhangwa (in situ histochemical localization) kw’amaizi g’omubiri (H2O2) n’amaizi g’omubiri (O2•−). Buri kishushani ky’ebihangirwe kikaba kirimu ebiyungu bitaano (ebimera bibiri ahari buri kiyungu). Buri kimwe ekirikugarukwamu omu bihangirwe kikakyeberwa omu bishushani bibiri (ebigarukwamu bibiri eby’obuhangwa) kureeba ngu omuringo ogu guhikire, gwesigwa kandi nigubaasa kugaruka kukorwa. H2O2 na O2•− bikakyeberwa barikukoresa 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Bugirimaani) nari nitroblue tetrazolium (NBT; (2004) na Adamu na bagyenzi be. (1989) n’okuhinduraho okukye. Ahabw’okutaho H2O2 omu mwanya, ebipapura bikateebwa omu mwanya ogurimu 0.1% DAB omu 10 mM Tris buffer (pH 7.8) reero byateebwa omu bwire bw’obutagatsi bw’omu kishengye omu mushana kumara edakiika 60. Ebipapura bikakyeberwa omu 0.15% (v/v) TCA omu 4:1 (v/v) ethanol:klorofoomu (eby’emibazi n’eby’obujanjabi bya Al-Gomhoria, Cairo, Misri) reero byateebwa omu mushana kuhitsya obu byahindukire omwirima. Omu muringo nigwo gumwe, valvu zikaba ziteirwemu omu mwanya ogutarimu kintu kyona n’ekirungo kya potassium phosphate 10 mM (pH 7.8) ekirimu 0.1 w/v % HBT ahabw’okutaho O2•− omu mwanya. Amababi gakateebwa omu mushana aha bwire bw’obutagatsi bw’omu kishengye kumara edakiika 20, bwanyima gakakyeberwa nk’oku kiri ahaiguru, reero gakamurikwa kuhitsya obu ebicupuri bya bururu/violet ebirikwiragura byareebekire. Obuhango bw‘erangi eya brown (nk‘eky‘okureeberaho kya H2O2) nari bururu-violet (nk‘eky‘okureeberaho kya O2•−) eyarugiremu bukakyeberwa barikukoresa omuringo gwa Fiji ogw‘ekicweka ky‘okukora aha bishushani ImageJ (http://fiji.sc; kitungirwe ebiro 7 Okwa kashatu 2024).
Malondialdehyde (MDA; nk‘akamanyiso k‘okuhindura kw‘amaizi g‘omubiri) ekamanyirwa aha nkora ya Du na Bramlage (1992) n‘okuhinduraho kakye. Amababi kuruga omu buri nkora y‘ebihangirwe (amababi ga kabiri n‘aga kashatu agakuzire omu bwijwire kuruga ahaiguru) gakatorotoorwaho 72 h bwanyima y‘okujanjaba (hpt). Buri kishushani ky’ebihangirwe kikaba kirimu ebiyungu bitaano (ebimera bibiri ahari buri kiyungu). Buri kimwe ekirikugarukwamu omu bihangirwe kikakyeberwa omu bishushani bibiri (ebigarukwamu bibiri eby’obuhangwa) kureeba ngu omuringo ogu guhikire, gwesigwa kandi nigubaasa kugaruka kukorwa. Omubugufu, 0.5 g y’ebihimba by’amababi g’ahansi bikakoresibwa omu kushohora kwa MDA n’ebiriisa bya 20% (TCA; MilliporeSigma, Burlington, MA, USA) ebirimu 0.01% by’ebiriisa by’omubiri (BHT; Sigma-Aldrich, St. Ebirimu MDA omu nshaho y’ahaiguru bikarugaho byamanyirwa aha nkora y’okupima okunywanisa aha 532 na 600 nm barikukoresa ekipimo ky’okureeba ebiri omu itaka ekya UV-160A (Shimadzu Corporation, Japan) reero bwanyima byayorekwa nka nmol g−1 FW.
Okukyebera ebiriisa ebitari by’enzigye n’eby’enzigye, amababi (amababi ga kabiri n’aga kashatu agakuzire omu bwijwire kuruga ahaiguru) gakatorotoorwaho kuruga omu buri nkora y’ebihangirwe ebirikugarukaho aha shaaha 72 bwanyima y’okujanjaba (hpt). Buri kishushani ky’ebihangirwe kikaba kirimu ebiyungu bitaano (ebimera bibiri ahari buri kiyungu). Buri sampo y‘ebihangirwe ekakyeberwa omu miringo ebiri (sampo ibiri z‘eby‘obuhangwa). Amababi abiri gakasiigwa n’amaizi ga nitrogen kandi gakakoresibwa butunu omu kumanyisa ebiriisa ebirikurwanisa obukooko oburikureeta endwara n’ebitari by’enzigye, amino acids zoona, ebiri omu proline, okureebeka kw’obuzaarwa, n’okupima oxalate.
Louis, MO, USA) n’okuhinduraho kakye omu muringo ogwashobooroirwe Kahkonen na bagyenzi be. (1999). Omubugufu, enkumi 0.1 g y’ebihimba by’amababi ebirikushushana bikaihwamu na 20 ml 80% methanol omu mwirima kumara eshaaha 24 kandi ebiri ahaiguru bikatorotoorwa bwanyima y’okubikora omu nkora ya centrifugation. 0.1 ml y’ekirungo ky’eky’okureeberaho kikataburwa na 0.5 ml y’ekirungo kya Folin-Ciocalteu (10%), kyazunguzibwa kumara 30 s kandi kyarekwa omu mwirima kumara edakiika 5. Bwanyima 0.5 ml y’omubazi gwa sodium carbonate 20% (Na2CO3; Kampuni y’emibazi n’eby’obujanjabi eya Al-Gomhoria, Cairo, Egypt) ekateebwa omu buri ntuumo, yaatabura gye kandi yaateebwa omu bwire bw’obutagatsi bw’omu kishengye omu mwirima kumara eshaaha 1 h. Bwanyima y’okuziteeka omu nkora, okunywanisa kw’omutaano gw’okugarukamu kukapimwa ahari 765 nm barikukoresa ekipimo ky’erangi ekya UV-160A (Shimadzu Corporation, Japan). Okuteerana kw’ebicweka by’omubiri ebirikubaasa kusheesheka byona omu bihingwa ebirugire omu sampo bikamanyirwa aha kukoresa omubaro gw’okupima asidi ya gallic (Fisher Scientific, Hampton, NH, USA) kandi byayorekwa nka miligiramu za asidi ya gallic erikwingana na buri giraamu y’oburemeezi obusya (mg GAE g-1 oburemeezi obusya).
Omubaro gwona ogw’ebiriisa bya flavonoid ebirikubaasa kusheesheka gukamanyirwa aha nkora ya Djeridane et al. (2006) n’okuhinduraho kakye. Omu bugufu, miriyoni 0.3 z’ekirungo kya methanol eki aharuguru kikatabura miriyoni 0.3 z’omubazi gwa 5% (AlCl3; Fisher Scientific, Hampton, NH, USA), kyateebwa omu nkora y’amaani reero kyateebwa omu bwire bw’obutagatsi bw’omu kishengye kumara edakiika 5, kyakuratirwa okwongyeraho omubazi gwa mssiumta10. (Emibazi ya Al-Gomhoria n’ebintu by’okukoresa omu by’obujanjabi, Cairo, Egypt), etaburiirwe gye kandi ekateebwa omu bwire bw’obutagatsi bw’omu kishengye kumara edakiika 30 omu mwirima. Bwanyima y’okuziteeka omu nkora, okunywanisa kw’omutaano gw’okugarukamu kukapimwa ahari 430 nm barikukoresa ekipimo ky’erangi ekya UV-160A (Shimadzu Corporation, Japan). Okuteerana kwa flavonoids zoona ezirikubaasa kusheesheka omu bihingwa by’okucondooza kukamanyirwa aha kukoresa omuringo gw’okupima rutin (TCI America, Portland, OR, USA) reero bwanyima gwayorekwa nka mirigiramu z’ebirikwingana na rutin ahari buri giraamu y’oburemeezi obusya (mg RE g-1 oburemeezi obusya).
Omubaro gwona gw’ebiriisa by’obuntu ebiri omu mababi g’ebihimba gukamanyirwa aha kukozesa ekirungo kya ninhydrin ekihindwirwemu (Thermo Scientific Chemicals, Waltham, MA, USA) kurugiirira aha nkora eyateirweho Yokoyama na Hiramatsu (2003) kandi ehindwirwemu Sun na bagyenzi be. (2006). Omu bugufu, 0.1 g y‘enyama y‘ahansi ekaihwaho n‘ekirungo kya pH 5.4, kandi 200 μL y‘amaizi g‘ahaiguru gakagambwaho na 200 μL ya ninhydrin (2%) na 200 μL ya pyridine (10%; okupimwa aha 580 nm barikukoresa ekikwato ky’okupima ebiri omu mushana ekya UV-160A (ekitongore kya Shimadzu, Japan). Aha rubaju orundi, purini ekamanyirwa aha nkora ya Bates (Bates na bagyenzi be, 1973). Proline ekaruga omu 3% ya sulfosalicylic acid (emibazi ya sayansi y‘okwotsya, Waltham, MA, USA) kandi bwanyima y‘okugikyebera, miriyoni 0.5 y‘amaizi g‘ahaiguru ekataburwa na miriyoni 1 y‘amaizi g‘okukyebera (Fisher Scientific, Hampton, NH, USA) n‘ekirungo kya ninhydrin co insureed at min5ba, incu 9d. aha 520 nm orikukoresa ekipimo ky’okureeba ebiri aharuguru. Omubaro gwona ogw’ebiriisa by’obuntu n’ebiriisa ebiri omu bihingwa by’amababi bikamanyirwa aha kukozesa enkora y’okupima ebiriisa bya glycine na proline (Sigma-Aldrich, St Louis, MO, USA), nk’oku biri, kandi byayorekwa nka mg/g oburemeezi obusya.
Okubaasa kumanya emirimo y’enzigye z’enzigye ezirikurwanisa obukooko oburikureeta endwara, enkumi 500 z’ebicweka by’omubiri ebiine obutafaari oburikushushana zikaihwaho miriyoni 3 z’ekiriisa kya Tris buffer ekya 50 mM (pH 7.8) ekirimu 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA), ekakyeberwa aha 10,000 × g kumara edakiika 20 ahansi y’okufukiiriza (4 °C), kandi ebiri aha mutwe (ekirungo ky’enzigye ebitari birungi) bikatorotoorwaho (El-Nagar na bagyenzi be, 2023; Osman na bagyenzi be, 2023). Catalase (CAT) bwanyima ekagarurwamu miriyoni 2 z‘omubazi gw‘okukyebera 0.1 M (pH 6.5; Sigma-Aldrich, St. 2023; Osman n’abandi, 2023). Omurimo gw‘enzigye ezirikurugiirira ahari Guaiacol (POX) gukamanyirwa aha nkora ya Harrach na bagyenzi be. (2009). (2008) n’okuhinduraho okukye (El-Nagar na bagyenzi be, 2023; Osman na bagyenzi be, 2023) kandi emirimo y’enzigye y’ekirungo kya polyphenol oxidase (PPO) ekamanyirwa bwanyima y’okukoragana na miriyoni 2.2 z’ekirungo kya 100 mM eky’omubazi gw’okuriisa (pH 1TCI ​​μ0l), . 100 μl ya 12 mM H2O2. Enkora ekahindurwamu kakye kuruga omu (El-Nagar na bagyenzi be, 2023; Osman na bagyenzi be, 2023). Okukyebera kukakorwa bwanyima y’okukoragana na miriyoni 3 z’omubazi gwa katekoli (0.01 M) ogwatebeekanisiibwe busya omu 0.1 M y’omubazi gwa phosphate (pH 6.0). Omurimo gwa CAT gukapimwa barikureeba okuvunda kwa H2O2 ahari 240 nm (A240), omurimo gwa POX gukapimwa barikureeba okweyongyera kw‘okunywa aha 436 nm (A436), kandi omurimo gwa PPO gukapimwa barikuhandiika okuhinduka kw‘okunywa aha 495 nm buri min U30 a30) . ekipimo ky’okureeba ebiri omu itaka (Shimadzu, Japan).
RT-PCR y’obwire obwabuzima ekakoresibwa kureeba emiringo y’okuhandiika ebiri omu nkora y’obutafaari bushatu oburikukwata aha nkora y’okurwanisa obukooko oburikureeta endwara, otwariiremu n’ebiriisa ebirikukora nk’ebiriisa ebiri omu mubiri (PvCAT1; Accession No. KY195009.1), omu mababi g’ebihimba (amababi ga kabiri n’aga kashatu agahikire kuruga ahaiguru) 72 h bwanyima y’okujanjaba okurikuhererukayo. Omu bugufu, RNA ekataanisibwa barikukoresa ekikwato ky’okushohoza RNA yoona ekya Simply P (Cat. No. BSC52S1; BioFlux, Biori Technology, China) kurugiirira aha nkora y’abakozire. Bwanyima, cDNA ekakorwa barikukoresa ekikwato kya TOP scriptTM cDNA Synthesis Kit kurugiirira aha biragiro by’omukozi. Okukuratana kw’okubanza kw’ebicweka bishatu ebiri aharuguru biri omu runyiriri rw’eby’okureeberaho S3. PvActin-3 (enamba y‘okutaaha omu GenBank: XM_068616709.1) ekakoresibwa nk‘omubazi gw‘okurinda enju kandi okureebeka kw‘omubazi gw‘obuzaarwa kukabarwa barikukoresa omuringo gwa 2-ΔΔCT (Livak na Schmittgen, 2001). Okugumaho kwa actin ahansi y’okugyemeserezibwa kw’ebihangirwe (okutaikirizana okutarikuhikaana ahagati y’ebihingwa bya buriijo n’ekizimba ky’omukishaka Colletotrichum lindemuthianum) n’okugyemeserezibwa kw’ebihangirwe ebitariho (ekyanda, omunyugunyu, obutagatsi bukye) bikooreka (Borges et al., 2012).
Omu kubanza tukakora okucondooza okwa siliko okwa puroteyini z’omubiri (OAH) omu S. sclerotiorum turikukoresa ekikwato kya puroteyini-puroteyini BLAST (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Omu bugufu, tukakoresa OAH kuruga omu kimera kya Aspergillus fijiensis CBS 313.89 (AfOAH; takisiidi: 1191702; enamba y’okutaaha omu GenBank XP_040799428.1; 342 amino asidi) na Penicillium lagena (PlOAH2; enamba y’okutaaha omu Gen1bank; XP_056833920.1; 316 ebiriisa by’omubiri) nk’okukuratana kw’ebibuuzo kubaasa kuhandiika puroteyini erikushushana omu S. BLASTp ekakorwa aha makuru g’eby’obuhangwa bya S. sclerotiorum agahererukireyo kushangwa omu GenBank aha mukutu gw’empurizana ogw’ekitongore ky’eihanga eky’amakuru g’eby’obuhangwa (NCBI), http://www.
Okwongyera ahari ekyo, oruzaaro rwa OAH orutebereziibwe kuruga omu S. sclerotiorum (SsOAH) n’okushwijuma eby’entunguuka n’omuti gw’obuzaarwa bwa AfOAH kuruga omu A. fijiensis CBS 313.89 na PlOAH kuruga omu P. lagena bikashoboororwa barikukoresa omuringo gw’okubaasa kubaho okurikukirayo omu JTamura1-11. eky‘okureeberaho (Jones n‘abandi, 1992). Omuti gw’obuzaarwa gukagambwaho n’okushwijuma okurikukwatanisa emirundi mingi kw’okukuratana kwa puroteyini z’ebicweka byona ebyatebereziibwe eby’obuzaarwa bwa OAH (SsOAH) kuruga omu S. sclerotiorum n’okukuratana kw’ebibuuzo barikukoresa ekikwato ky’okugyendera aha nkora y’okuzibira (COBALT; na Agarwala, omu mwaka gwa 2007). Okwongyera ahari ekyo, okukuratana kw‘ebicweka by‘omubiri ebirikushushana gye ebya SsOAH kuruga omu S. sclerotiorum kukahikaana n‘okukuratana kw‘ebibuuzo (AfOAH na PlOAH) (Larkin na bagyenzi be, 2007) barikukoresa ClustalW (http://www.genome.jp/tools-bin n‘okurinda ekicweka), Ekikwato kya ESPript (omuringo 3.0; https://eSPript/index.php).
Ekindi, emyanya erikujwekyera emirimo eyatebereziibwe n‘emyanya erikubiikwa eya S. sclerotiorum SsOAH bikashoroorwa omu maka gatari gamwe na gamwe barikukoresa ekikwato kya InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Eky’aha muheru, okugyeragyeranisa kw’omutindo gwa S. sclerotiorum SsOAH ogwatebereziibwe omu miringo eshatu (3D) kukakorwa barikukoresa Engine y’okumanya ebiri omu puroteyini (Phyre2 server version 2.0; 2015) kandi bakahamibwa barikukoresa omukura gwa SWISS-MODEL (Biasini na bagyenzi be, 2014). Ebishushani by‘ebicweka bishatu ebyatebereziibwe (omuringo gwa PDB) bikareebeka omu nkora y‘okukoragana barikukoresa ekicweka kya UCSF-Chimera (omuringo 1.15; https://www.cgl.ucsf.edu/chimera/ ) (Pettersen na bagyenzi be, 2004).
Omubaro gw’obwire bw’amazima ogw’okukyebera PCR gukakozesebwa kumanyisa omutindo gw’okuhandiika kw’ekiriisa kya oxaloacetate asetylhydrolase (SsOAH; enamba y’okutaaha omu GenBank: XM_001590428.1) omu mycelia ya Sclerotinia sclerotiorum. Omu bugufu, S. sclerotiorum ekateebwa omu nshaho erimu PDB kandi yaateebwa omu nkora y‘okuziika (eky‘okureeberaho: I2400, New Brunswick Scientific Co., Edison, NJ, USA) aha 25 ± 2 °C kumara eshaaha 24 aha 150 rpm kandi omu mwirima ogutarikuhinduka) kureetaho okukura kw‘eshagama (24). Bwanyima y’ekyo, obutafaari bukajanjabwa na L-ornithine n’omubazi gw’okurwanisa obukooko Rizolex-T aha nkora ya IC50 ey’aha muheru (enkumi 40 na 3.2 mg/L, buri kimwe) reero bwanyima bwakuzibwa kumara eshaaha ezindi 24 omu mbeera niyo emwe. Bwanyima y’okuziteeka, obuhangwa bukateebwa omu nkora ya centrifuge aha 2500 rpm kumara edakiika 5 kandi ebiri ahaiguru (fungal mycelium) bikatorotoorwamu kubaasa kukyebera oku ebiri omu nkora y’obuzaarwa birikworekwa. Omu muringo nigwo gumwe, mycelium y’ebizimba ekatorotoorwaho aha 0, 24, 48, 72, 96, na 120 h bwanyima y’okukwatwa oburwaire kuruga omu bimera ebiine oburwaire ebyabaire bikozire obukooko oburikwera n’ebizimba bya pamba aha mutwe gw’ebicweka by’omubiri ebiine oburwaire. RNA ekaruga omu mycelium y’ebizimba reero bwanyima cDNA ekakorwa nk’oku kyashoboororwa ahaiguru. Enkuratana y‘okubanza ahabwa SsOAH eri omu runyiriri rw‘okwongyeraho S3. SsActin (enamba y‘okutaaha omu GenBank: XM_001589919.1) ekakoresibwa nk‘omubazi gw‘okureeberera enju, kandi okureebeka kw‘omubazi gw‘obuzaarwa kukabarwa barikukoresa omuringo gwa 2-ΔΔCT (Livak na Schmittgen, 2001).
Oxalic acid ekamanyirwa omu mutokye gw’ebitokye (PDB) n’ebimera ebirimu obukooko oburikureeta endwara ya Sclerotinia sclerotiorum kurugiirira aha nkora ya Xu na Zhang (2000) n’okuhinduraho kakye. Omu bugufu, S. sclerotiorum isolates zikateebwa omu bidomora ebirimu PDB reero zaateebwa omu nkora y‘okuziika (eky‘okureeberaho I2400, New Brunswick Scientific Co., Edison, NJ, USA) aha 150 rpm aha 25 ± 2 °C kumara ebiro 3–5 omu bwire bw‘okuziika g4. Bwanyima y’okuziteeka, obuhangwa bw’ebishaka bukabanza bwashwijumwa kurabira omu mpapura z’okushwijuma eza Whatman #1 reero bwakyeberwa aha 2500 rpm kumara edakiika 5 kwihaho mycelium eyasigaireho. Ebintu ebyabaire biri ahaiguru bikatorotoorwaho kandi byabiikwa aha 4°C kubaasa kugaruka kumanya omubaro gwa oxalate. Aha kutebeekanisa eby’okureeberaho by’ebimera, ebicweka by’ebicweka by’ebimera ebirikwingana 0.1 g bikaihwaho emirundi eshatu n’amaizi agateirwemu ebiriisa (miriyoni 2 buri murundi). Sampuli zikaba ziteebwa omu centrifuge aha 2500 rpm kumara edakiika 5, amaizi g’ahaiguru gakasendwa omu mpapura z’okusheneneka eza Whatman No. 1 kandi gakatorotoorwaho kubaasa kugaruka kukyeberwa.
Aha kucondooza kw’omubaro gw’ekiriisa kya oxalic acid, omutaano gw’okugarukamu gukatebeekanisibwa omu ntuumo erimu endabirwamu omu mubaro ogu: miriyoni 0.2 z’eky’okureeberaho (nari shi ekirungo ky’obuhangwa bwa PDB nari ekirungo ky’omutindo gwa oxalic acid), miriyoni 0.11 z’ekirungo kya bromophenol bururu (BPB, 1 mM; Fisher Chemical, PA1 m0l of Pittsburgh. asidi (H2SO4; Al-Gomhoria eby’emibazi n’ebintu by’okukoresa omu by’obujanjabi, Cairo, Misri) na miriyoni 0.176 z’omubazi gw’okukyebera (K2Cr2O7; emibazi ya TCI, Portland, OR, USA), reero omubazi ogwo gukakyeberwa gwaba gurimu miriyoni 4.8 z’omubazi gw’okukyebera. omu maizi g’okwoga 60 °C. Bwanyima y‘edakiika 10, okugarukamu kukakyemerezibwa obwo barikuteekamu miriyoni 0.5 z‘omubazi gw‘okunywa (NaOH; 0.75 M). Okunywa (A600) kw’omutaano gw’okugarukamu kukapimwa ahari 600 nm barikukoresa ekipimo ky’okureeba ebiri omu mushana (Shimadzu Corporation, Japan). PDB n’amaizi agateirwemu ebiriisa bikakoresibwa nk’ebintu ebirikutegyekwa okupima obuhango bw’ebihingwa ebishwijumirwe n’eby’okureeberaho by’ebimera, nk’oku biri. Ebicweka bya oxalic acid ebiri omu bihingwa by’obuhangwa, ebirikworekwa nka maikorogiramu za oxalic asidi ahari mililita y’omugati gwa PDB (μg.mL−1), n’omu bihingwa by’amababi, ebirikworekwa nka maikorogiramu za oxalic acid ahari buri giraamu y’oburemeezi obusya (μg.g.g−1 FW), bikamanyirwa aha kukozesa oxalic acid ya curvetham, The Walisher Slirmo calibration, MA, Amerika).
Omu kucondooza kwona, okucondooza kwona kukakorwa omu nkora y‘okutooranamu (CRD) erimu ebishushani mukaaga by‘ebihangwa buri kujanjaba n‘ebiyungu bitaano ahari buri kiyungu (ebimera bibiri ahari buri kiyungu) okwihaho kyaba kitagambirwe. Ebihandiiko ebigarukiremu eby‘ebihangwa bikakyeberwa omu bishushani bibiri (ebigarukwamu bibiri eby‘obuhangwa). Ebihandiiko by’okugarukamu eby’obuhangwa bikakoresibwa kukyebera okugarukaho kw’okugyezesa kumwe kwonka tibikakozesiibwe omu kucondooza kw’embariirira okwetantara okugarukamu kw’ebishuba. Ebihandiiko bikakyeberwa omu nkora y‘embariirira barikukoresa okucondooza kw‘okuhinduka (ANOVA) kukuratirwaho okukyebera kwa Tukey-Kramer okw‘amazima (HSD) (p ≤ 0.05). Aha kucondooza kwa in vitro, emihendo ya IC50 na IC99 ekabarwa barikukoresa omuringo gwa probit kandi 95% y‘obwire bw‘okwesiga bukabarwa.
Ebicweka bina ebyabaire bikyebeirwe (isolates) bikashangwa omu misiri ya soya etari emwe n'emwe omu Governor ya El Ghabiya, Misri. Aha PDA medium, isolates zoona zikareetaho mycelium erikwera nk’ekiziibu eyaabaire neehinduka juba erikwera nk’eya pamba (ekishushani 1A) reero ekahinduka beige nari brown aha rurengo rwa sclerotium. Sclerotia nizikira kuba ziri nyingi, zirikwiragura, ziri nk’enziga nari zitarikushushana, oburaingwa bwa mm 5.2 kuhika 7.7 na mm 3.4 kuhika 5.3 omu bugazi (ekishushani 1B). N’obu ebicweka bina ebyabaire biine obuhuta bw’omubiri byatungire enkora y’okusheeshasheesha aha rubaju rw’ekicweka ky’obuhangwa bwanyima y’ebiro 10–12 by’okukyebera aha 25 ± 2 °C (Fig. 1A), omubaro gw’ebicweka by’omubiri ebiine obuhuta ahari buri rubaaho gukaba nigutaanira kimwe omuribyo (P < 0.001). 1.53 ebizimba ahari buri rupapura; Omu muringo nigwo gumwe, ekicweka #3 kikareetaho oxalic acid nyingi omu PDB kukira ebindi ebicweka (3.33 ± 0.49 μg.mL−1; Ekishushani 1D). Isolate #3 ekooreka obumanyiso bw’omutaano obw’omubiri n’obumanyiso bw’ebintu ebikye eby’ekizimba ekirikureeta endwara z’ebimera Sclerotinia sclerotiorum. Nk’eky’okureeberaho, aha PDA, amakolooni g’ebiriisa #3 gakakura juba, gakaba gari ag’erangi erikwera nk’ekiziibu (ekishushani 1A), gari ga reverse beige nari light salmon yellow-brown, kandi gakaba nigayetengyesa ebiro 6-7 ahari 25 ± 2°C kubaasa kushweka kimwe aha mutwe gw’empapura z’oburaingwa bwa sentimita 9. Okurugirira aha mitwarize y’ebihangirwe n’eby’okureeberaho ebiri aharuguru, ekicweka #3 kikamanyirwa aha Sclerotinia sclerotiorum.
Ekishushani 1. Emitwarize n’oburwaire bwa S. sclerotiorum oburikuruga omu birimwa by’ebihimba ebya buriijo. (A) Okukura kwa mycelium kw’ebicweka bina ebya S. sclerotiorum ebiri aha nkora ya PDA, (B) okukura kw’ebicweka bina ebya S. sclerotiorum, (C) omubaro gw’ebicweka (buri rubaaho), (D) okushohoza kwa oxalic acid aha nkora ya PDB (μg.mL−1), na (E) okusheeshasheesha kw’endwara i. ekika ky’ebihimba by’obushuubuzi ekirikubaasa kukwatwaho Giza 3 ahansi y’embeera z’ebishaka. Emihendo neejwekyera omubaro gw‘ahagati ± SD gw‘ebicweka bitaano by‘ebihangirwe ebigarukiremu (n = 5). Enyuguta zitari zimwe na zimwe nizooreka entaaniso z‘omubaro ahagati y‘obujanjabi (p < 0.05). (F–H) Obumanyiso bw’ebizimba ebirikwera obumanyirwe bukareebeka aha bihingwa ebiri ahaiguru y’itaka n’aha siliki, nk’oku biri, ebiro 10 bwanyima y’okukyeberwa n’ekirungo kya isolate #3 (dpi). (I) Okushwijuma okutunguuka kw’ekicweka ky’omunda ekihandiikirwe (ITS) ekya S. sclerotiorum isolate #3 kukakorwa barikukoresa omuringo gw’okuteekateeka okurikukirayo kandi kwagyeragyeranisibwa n’ebicweka 20 eby’okureeberaho/ebika ebyatungirwe kuruga omu nkora y’okuhandiika amakuru g’eby’obuhangwa (NCBI/httbih.govwww.). Enamba eziri ahaiguru y‘enyuguta z‘okuteerana nizooreka oku ekicweka kirikujanjaara (%), kandi enamba eziri ahansi y‘enyuguta z‘okuteerana nizooreka oburaingwa bw‘amataagi.
Ekindi, okuhamya oburwaire obu, ebicweka bina ebya S. sclerotiorum isolates ebyatungirwe bikakoresibwa kukyebera ekika ky’ebihimba by’obushuubuzi ekirikubaasa kukwatwa endwara Giza 3 omu mbeera z’okuriisa, ekirikuhikaana n’ebiteekateeko bya Koch (Fig. 1E). N‘obu ebicweka by‘ebishaka byona ebyatungirwe byabaire biri eby‘okureeta endwara kandi birikubaasa kukwata ebihimba bya green bean (cv. Giza 3), bikareetaho obumanyiso bw‘ebizimba eby‘omutaano aha bicweka byona ebiri ahaiguru y‘itaka (Fig. 1F), namunonga aha bihimba (Fig. 1G) n‘ebihimba by‘ebihimba (Fig. 1H dapiys), aha 10. aggressive isolate omu kucondooza kubiri okwetongoire. Isolate 3 ekaba eine oburemeezi bw‘endwara oburikukirayo (%) aha bimera by‘ebihimba (24.0 ± 4.0, 58.0 ± 2.0, na 76.7 ± 3.1 aha biro 7, 14, na 21 bwanyima y‘okukwatwa endwara, nk‘oku biri; Ekishushani 1F).
Okumanyisa ekirikukirayo kutaaha omu nkora ya S. sclerotiorum isolate #3 kikongyerwa kuhamibwa kurugiirira aha kucondooza kw’omunda (ITS) (Fig. 1I). Okushwijuma kwa phylogenetic ahagati y’ekicweka #3 n’ebicweka 20 eby’okureeberaho/enganda kukooreka okushushana kwingi (>99%) ahagati yaabyo. Nikirungi kumanya ngu S. sclerotiorum #3 (533 bp) eine okushushana kwingi n’omubazi gwa America ogwa S. OR206374.1; 548 bp), ekirikureetaho okuvunda kw’ebiti bya violet (Matthiola incana), byona biteeraine omu guruupu z’omutaano aha mutwe gw’ekipande ky’ebishaka (Ekishushani 1I). Omukuratana musya ogu guteirwe omu nkora ya NCBI kandi gwaheebwa eiziina “Sclerotinia sclerotiorum – isolate YN-25” (enamba y’okutaaha omu GenBank PV202792). Nikibaasa kureebwa ngu isolate 3 niyo isolate erikukirayo kutaahirira; n’ahabw’ekyo, isolate egi ekatooranwa kwega omu kucondooza kwona okwakuratsireho.
Omurimo gw’okurwanisa obukooko oburikwetwa diamine L-ornithine (Sigma-Aldrich, Darmstadt, Germany) omu bipimo bitarikushushana (12.5, 25, 50, 75, 100 na 125 mg/L) ahari S. Nikimanyisa ngu L-ornithine ekakora ekirikurwanisa obukooko kandi mporampora ekazibira okukura kwa radial kwa S. sclerotiorum hyphae omu muringo ogurikurugiirira aha dozi (ekishushani 2A, B). Aha nkora y‘okukyebera erikukirayo obuhango (125 mg/L), L-ornithine ekooreka omubaro gw‘okuzibira okukura kw‘ebihimba (99.62 ± 0.27%; ekishushani 2B), ogwabaire nigushushana n‘omubazi gw‘okukyebera obukooko ogwa Rizolex-T ogw‘obushuubuzi (omubaro gw‘okuzibira 99.2% the Figure 0.45; (10 mg/L), ekirikworeka oburungi oburikushushana.
Ekishushani 2. Omu nkora y‘okurwanisa obukooko oburikureeta endwara ya L-ornithine omu nkora y‘okurwanisa obukooko oburikureeta endwara. (A) Okugyeragyeranisa emirimo y‘okurwanisa obukooko bw‘omubiri ey‘ebicweka by‘omubiri ebitari bimwe na bimwe ebya L-ornithine ahari S. sclerotiorum n‘omubazi gw‘okurwanisa obukooko oburikureeta endwara Rizolex-T (10 mg/L) ogw‘obushuubuzi. (B, C) Omubaro gw‘okuzibira (%) okukura kw‘ebihimba bya S. sclerotiorum bwanyima y‘okujanjaba n‘ebicweka by‘omubiri ebitarikushushana (12.5, 25, 50, 75, 100 na 125 mg/L) nari Rizolex-T (2, 4, 6, 8/10 mgly), ebirikukuratana. Emihendo neejwekyera omubaro gw‘ahagati ± SD gw‘ebicweka bitaano by‘ebihangirwe ebigarukiremu (n = 5). Enyuguta zitari zimwe na zimwe nizooreka entaaniso z‘embariirira ahagati y‘obujanjabi (p < 0.05). (D, E) Okushwijuma okugaruka enyima kw‘omuringo gwa Probit ogwa L-ornithine n‘omubazi gw‘okurwanisa obukooko oburikureeta obuhuta Rizolex-T, nk‘oku biri. Omuringo gw‘okugaruka enyima ogwa probit model nigworeka nk‘omuriro gwa bururu ogugumire, kandi omwanya gw‘okwesiga (95%) nigworeka nk‘omuriro gw‘erangi erikutukura ogurimu obubonero.
Okwongyera ahari ekyo, okucondooza kwa probit regression kukakorwa kandi puroguraamu ezirikuhikaana nizoorekwa omu kipande 1 n‘ebishushani 2D,E. Omu bugufu, omuhendo gw‘okushenda ogurikwikirizibwa (y = 2.92x − 4.67) n‘embariirira y‘omugasho erikukwatagana (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 na p < 0.0001; Ekishushani 2D) kya L-ornithine kyorekire okweyongyera kw‘okukora kw‘okurwanisa obukooko oburikureeta obuhuta. omubazi gw‘okurwanisa obukooko oburikwetwa Rizolex-T (y = 1.96x − 0.99, Cox na Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 na p < 0.0001) (Ekipande 1).
Ekipande 1. Emihendo y‘ekicweka ky‘okuzibira ekirikukirayo (IC50) na IC99 (mg/l) ya L-ornithine n‘omubazi gw‘okuguza obukooko “Rizolex-T” ogurikurwanisa S. sclerotiorum.
Okutwarira hamwe, L-ornithine (250 mg/L) ekakyendeeza munonga okukura n’oburemeezi bw’ebizimba ebirikwera aha bimera by’ebihimba ebya buriijo ebyabaire bijanjabirwe kugyeragyeranisa n’ebimera ebitajanjabirwe ebiine oburwaire bwa S. sclerotiorum (okukyebera; ekishushani 3A). Omu bugufu, n‘obu oburemeezi bw‘endwara bw‘ebimera ebiine oburwaire obutaine bujanjabi bwabaire nibweyongyera mpora mpora (52.67 ± 1.53, 83.21 ± 2.61, na 92.33 ± 3.06%), L-ornithine ekakyendeeza munonga oburemeezi bw‘endwara (%) omu kucondooza kwona, ±0.0 (8.01). 1.00, na 26.36 ± 3.07) aha biro 7, 14, na 21 bwanyima y‘okutunga obujanjabi (dpt), nk‘oku biri (ekishushani 3A). Omu muringo nigwo gumwe, ebimera by’ebihimba ebiine oburwaire bwa S. sclerotiorum ku byajanjabirwe na 250 mg/L L-ornithine, omwanya oguri ahansi y’omutindo gw’okutunguuka kw’endwara (AUDPC) gukeyongyera kuruga ahari 1274.33 ± 33.13 omu kukyebera okutaine bujanjabi kuhika ahari 281.03 ± 7.0 L ahansi y’omushana ogwo s . Omubazi gw’okurwanisa obukooko oburikwetwa Rizolex-T (183.61 ± 7.71; ekishushani 3B). Omuze nigwo gumwe gukareebeka omu kucondooza okwa kabiri.
Ekishushani 3. Ebirikuruga omu kukozesa L-ornithine kuruga aheeru aha kukura kw’okuvunda kw’ebihimba ebya buriijo okurikureetwaho Sclerotinia sclerotiorum omu mbeera z’ebishaka. (A) Okutunguuka kw‘endwara kw‘ekizimba ky‘ebihimba eby‘omutaano bwanyima y‘okujanjaba na 250 mg/L L-ornithine. (B) Omwanya oguri ahansi y‘omutindo gw‘okutunguuka kw‘endwara (AUDPC) gw‘ebizimba ebirikwera eby‘ebihimba bya buriijo bwanyima y‘okujanjaba na L-ornithine. Emihendo neejwekyera omubaro gw‘ahagati ± SD gw‘ebicweka bitaano by‘ebihangirwe ebigarukiremu (n = 5). Enyuguta zitari zimwe na zimwe nizooreka entaaniso z‘omubaro ahagati y‘obujanjabi (p < 0.05).
Okukozesa 250 mg/L L-ornithine omu muringo gw’aheeru y’ekimera kikayongyera mpora mpora oburaingwa bw’ebimera (Fig. 4A), omubaro gw’amataagi ahari buri kimera (Fig. 4B), n’omubaro gw’amababi ahari buri kimera (Fig. 4C) bwanyima y’ebiro 42. N‘obu omubazi gw‘okurwanisa obukooko oburikureeta obuhuta Rizolex-T (50 mg/L) gwabaire guine omugasho muhango aha by‘okurya byona ebyacondooziibwe, okukoresa exogenous 250 mg/L L-ornithine kukaba kuine omugasho gwa kabiri waagyeragyeranisa n‘abatarikujanjaba (ebishushani 4A–C). Aha rubaju orundi, okujanjaba kwa L-ornithine kukaba kutaine ki kwakora aha biri omu rangi z‘omushana chlorophyll a (ekishushani 4D) na chlorophyll b (ekishushani 4E), kwonka kukayongyera kakye aha biri omu carotenoid zoona (0.56 ± 0.03 mg2 ±0r w4 fr wt) kugyeragyeranisa n‘okukyebera okubi (0.00). okukyebera okurungi (0.46 ± 0.02 mg/g fr wt; ekishushani 4F). Okutwarira hamwe, ebyarugamu ebi nibyoreka ngu L-ornithine teri phytotoxic aha bihingwa ebijanjabirwe kandi neebaasa n’okureetaho okukura kwabyo.
Ekishushani 4. Ebirikuruga omu kukozesa exogenous L-ornithine aha mitwarize y’okukura n’erangi z’omushana z’ebibabi by’ebihimba ebiine oburwaire bwa Sclerotinia sclerotiorum omu mbeera z’ebishaka. (A) Oburaingwa bw’ekimera (cm), (B) Omubaro gw’amataagi ahari buri kimera, (C) Omubaro gw’ebibabi ahari buri kimera, (D) Ebirimu klorofiili a (mg g-1 fr wt), (E) Ebirimu klorofiili b (mg g-1 fr wt), (F) Ebirimu ebiriisa bya karotenoid byona (mg g-1 fr). Emihendo n‘ebiri ahagati ± SD y‘ebicweka bitaano by‘ebihangirwe ebigarukiremu (n = 5). Enyuguta zitari zimwe na zimwe nizooreka entaaniso z‘omubaro ahagati y‘obujanjabi (p < 0.05).
Omu mwanya gw‘okucondooza aha nkora y‘eby‘amagara (ROS; ebirikworekwa nk‘eby‘okurya by‘omubiri [H2O2]) n‘eby‘okurya by‘omubiri (ebirikworekwa nk‘eby‘okurya by‘omubiri [O2•−]) bikashohoza ngu okukoresa L-ornithine (250 mg/L) omu muringo gw‘aheeru y‘omubiri (250 mg/L) kukakyendeeza munonga aha kuteerana kwa H2 ±.50. nmol.g−1 FW; ekishushani 5A) na O2•− (32.69 ± 8.56 nmol.g−1 FW; ekishushani 5B) kugyeragyeranisa n’okuteerana kw’ebimera byombi ebiine oburwaire obutaine bujanjabi (173.31 ± 12.06) na 149.35 ± 7.94 nmol y’okukyebera na W. mg/L y‘omubazi gw‘okurwanisa obukooko oburikureeta obuhuta Rizolex-T (170.12 ± 9.50 na 157.00 ± 7.81 nmol.g−1 fr wt, buri kimwe) aha shaaha 72 h. Emiringo y‘ahaiguru ya H2O2 na O2•− erikuteerana ahansi ya hpt (ekishushani 5A, B). Omu muringo nigwo gumwe, okucondooza okwakozirwe aha nkora ya TCA (MDA) kukareebeka ngu ebimera by’ebihimba ebiine oburwaire bwa S. sclerotiorum bikarundaana MDA nyingi (113.48 ± 10.02 nmol.g fr wt) omu mababi gaabyo (ekishushani 5C). Kwonka, okukoresa L-ornithine omu muringo gw’aheeru y’omubiri kukakyendeeza munonga aha kusheeshasheesha kw’amaizi nk’oku kirikworekwa omu kukyendeera kw’ebiri omu MDA omu bimera ebyabaire bijanjabirwe (33.08 ± 4.00 nmol.g fr wt).
Ekishushani 5. Ekirikubaho ahabw‘okukoresa exogenous L-ornithine aha bimanyiso bikuru eby‘okuteganisibwa kw‘okwotsya n‘emiringo y‘okwerinda etari y‘enzigye omu mababi g‘ebihimba againe oburwaire bwa S. sclerotiorum aha shaaha 72 bwanyima y‘okukwatwa omu mbeera z‘okukyebera. (A) Omubazi gw’amaizi (H2O2; nmol g−1 FW) aha 72 hpt, (B) anioni y’amaani (O2•−; nmol g−1 FW) aha 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) aha 72 hpt, (D) omubaro gwona ogw’ebintu ebirikushuuma (Fg g−1 FW) (E) omubaro gwona gw‘ebiriisa ebirikusheesheka (mg RE g−1 FW) aha 72 hpt, (F) omubaro gwona gw‘ebiriisa by‘obuntu (mg g−1 FW) aha 72 hpt, na (G) ebiriisa bya purini (mg g−1 FW) aha 72 hpt. Emihendo neejwekyera omubaro gw‘ahaiguru ± okutaana kw‘omutindo (omubaro gw‘ahaiguru ± SD) gw‘ebicweka 5 by‘ebihangwa ebigarukiremu (n = 5). Enyuguta zitari zimwe na zimwe nizooreka entaaniso z‘omubaro ahagati y‘obujanjabi (p < 0.05).