Sengoloa sena ke karolo ea sehlooho sa lipatlisiso se reng "Ho ntlafatsa ho hanyetsa ha linaoa khahlanong le likokoana-hloko le likokoanyana", sheba lingoloa tsohle tse 5
Sesosa sa lefu la dimela la fungal necrosis Sclerotinia sclerotiorum (Lib.) de Bary se sebedisa leano la maemo a mangata ho tshwaetsa dimela tse fapaneng tse amohelang. Phuputso ena e sisinya tshebediso ya diamine L-ornithine, e leng amino acid e seng ya protheine e hlasimollang tlhahiso ya di-amino acid tse ding tsa bohlokwa, e le leano le leng la taolo ho ntlafatsa dikarabelo tsa dimolek'hule, tsa mmele le tsa biochemical tsa Phaseolus vulgaris L. ho hlobo e tshweu e bakwang ke Pseudomonas sclerotiorum. Diteko tsa in vitro di bontshitse hore L-ornithine e thibetse kgolo ya mycelial ya S. pyrenoidosa haholo ka tsela e itshetlehileng ka tekanyo. Ho feta moo, e ka fokotsa haholo boima ba hlobo e tshweu tlasa maemo a greenhouse. Ho feta moo, L-ornithine e ile ya susumetsa kgolo ya dimela tse phekotsweng, e leng se bontshang hore mahloriso a lekilweng a L-ornithine a ne a se na chefo ya phytotoxic ho dimela tse phekotsweng. Ho phaella moo, L-ornithine e ntlafalitse tlhahiso ea li-antioxidants tse seng tsa enzymatic (li-phenolics le li-flavonoid tse qhibilihang ka botlalo) le li-antioxidants tsa enzymatic (catalase (CAT), peroxidase (POX), le polyphenol oxidase (PPO)), 'me ea eketsa tlhahiso ea liphatsa tse tharo tse amanang le antioxidant (PvCAT1, PvSOD, le PvGR). Ho feta moo, tlhahlobong ea silico ho senotsoe boteng ba protheine ea oxaloacetate acetylhydrolase (SsOAH) e ka har'a genome ea S. sclerotiorum, e neng e tšoana haholo le liprotheine tsa oxaloacetate acetylhydrolase (SsOAH) tsa Aspergillus fijiensis (AfOAH) le Penicillium sp. (PlOAH) mabapi le tlhahlobo ea ts'ebetso, libaka tse bolokiloeng, le topology. Hoa thahasellisa hore ho eketsoa ha L-ornithine ho potato dextrose broth (PDB) medium ho fokolitse haholo tlhahiso ea gene ea SsOAH ho S. sclerotiorum mycelia. Ka ho tšoanang, tšebeliso ea L-ornithine ka ntle ho naha e fokolitse haholo tlhahiso ea gene ea SsOAH ho mycelia ea fungal e bokelletsoeng limela tse phekoloang. Qetellong, tšebeliso ea L-ornithine e fokolitse haholo tlhahiso ea oxalic acid makhasing a mahareng a PDB le makhasi a tšoaelitsoeng. Qetellong, L-ornithine e bapala karolo ea bohlokoa ho bolokeng boemo ba redox hammoho le ho ntlafatsa karabelo ea tšireletso ea limela tse tšoaelitsoeng. Liphetho tsa phuputso ena li ka thusa ho nts'etsapele mekhoa e mecha, e mosa tikolohong ea ho laola hlobo e tšoeu le ho fokotsa tšusumetso ea eona tlhahisong ea linaoa le lijalo tse ling.
Hlobo e tšoeu, e bakoang ke fungus e nang le necrotrophic Sclerotinia sclerotiorum (Lib.) de Bary, ke lefu le senyang, le fokotsang chai le behang kotsing e kholo tlhahisong ea linaoa tsa lefats'e (Phaseolus vulgaris L.) (Bolton et al., 2006). Sclerotinia sclerotiorum ke e 'ngoe ea likokoana-hloko tse thata ka ho fetisisa tse jang limela tsa fungus tse jang mobung ho li laola, ka mefuta e mengata ea limela tse fetang 600 le bokhoni ba ho senya lisele tsa moamoheli ka potlako ka tsela e sa tobang (Liang le Rollins, 2018). Tlas'a maemo a sa rateheng, e feta mohatong oa bohlokoa oa potoloho ea eona ea bophelo, e lula e robetse nako e telele e le meaho e metšo, e thata, e kang peo e bitsoang 'sclerotia' mobung kapa e le kholo e tšoeu, e boreleli mycelium kapa kutu ea limela tse tšoaelitsoeng (Schwartz et al., 2005). S. sclerotiorum e kgona ho bopa sclerotia, e e dumellang ho phela masimong a tshwaeditsweng nako e telele le ho tswela pele nakong ya bolwetse (Schwartz et al., 2005). Sclerotia e na le dikotla tse ngata, e ka tswela pele mobung nako e telele, mme e sebetsa e le sethibela-mafu sa mantlha bakeng sa ditshwaetso tse latelang (Schwartz et al., 2005). Tlas'a maemo a matle, sclerotia e mela mme e hlahisa di-spores tse fofang tse ka tshwaetsang dikarolo tsohle tse ka hodimo ho lefatshe tsa semela, ho kenyeletswa empa ho sa felle feela dipaleseng, dikutung, kapa di-pod (Schwartz et al., 2005).
Sclerotinia sclerotiorum e sebedisa leano la maemo a mangata ho tshwaetsa dimela tsa yona, ho kenyeletsa letoto la diketsahalo tse hokahaneng ho tloha ho meleng ha sclerotial ho isa ntshetsopeleng ya matshwao. Qalong, S. sclerotiorum e hlahisa di-spores tse emisitsweng (tse bitswang ascospores) ho tswa meahong e kang ya di-mushroom e bitswang apothecia, e fetohang moya mme e fetoha sclerotia e sa tsamayeng hodima masalla a dimela a tshwaeditsweng (Bolton et al., 2006). Fungus ebe e ntsha oxalic acid, e leng ntho e bakang ho bola, ho laola pH ya lebota la sele ya semela, ho kgothaletsa ho senyeha ha enzymatic le tlhaselo ya dinama (Hegedus le Rimmer, 2005), le ho thibela ho phatloha ha oxidative ha semela sa moamoheli. Tshebetso ena ya ho eketsa asiti e fokodisa lebota la sele ya semela, e fana ka tikoloho e ntle bakeng sa tshebetso e tlwaelehileng le e sebetsang ya dienzyme tse senyang lebota la sele ya fungal (CWDEs), e dumella pathogen ho hlola thibelo ya mmele le ho kenella dinama tse amohelang (Marciano et al., 1983). Hang ha e keneletse, S. sclerotiorum e ntša li-CWDE tse 'maloa, tse kang polygalacturonase le cellulase, tse nolofalletsang ho ata ha eona liseleng tse tšoaelitsoeng le ho baka necrosis ea lisele. Ho tsoela pele ha liso le materase a hyphal ho lebisa matšoao a ikhethang a hlobo e tšoeu (Hegedus le Rimmer, 2005). Ho sa le joalo, limela tse amohelang li lemoha mekhoa ea limolek'hule e amanang le pathogen (PAMPs) ka li-receptor tsa pattern recognition (PRRs), e leng se bakang letoto la liketsahalo tsa matšoao tse qetellang li kenya tšebetsong likarabo tsa tšireletso.
Ho sa tsotellehe boiteko ba mashome a lilemo ba ho laola mafu, khaello ea germplasm e lekaneng e hanyetsanang e ntse e le teng linaoa, joalo ka lijalo tse ling tsa khoebo, ka lebaka la ho hanyetsa, ho pholoha le ho ikamahanya le maemo ha pathogen. Ka hona, taolo ea mafu e thata haholo 'me e hloka leano le kopaneng, le nang le mahlakore a mangata le kenyeletsang motsoako oa mekhoa ea setso, taolo ea baeloji le li-fungicide tsa lik'hemik'hale (O'Sullivan et al., 2021). Taolo ea lik'hemik'hale ea hlobo e tšoeu ke eona e sebetsang ka ho fetisisa hobane li-fungicide, ha li sebelisoa ka nepo le ka nako e nepahetseng, li ka laola ho ata ha lefu ka katleho, tsa fokotsa boima ba tšoaetso, le ho fokotsa tahlehelo ea chai. Leha ho le joalo, tšebeliso e feteletseng le ho itšetleha haholo ka li-fungicide ho ka lebisa ho hlaheng ha mefuta e hanyetsanang ea S. sclerotiorum le ho ama hampe lintho tse phelang tse sa reretsoeng, bophelo bo botle ba mobu le boleng ba metsi (Le Cointe et al., 2016; Ceresini et al., 2024). Ka hona, ho fumana mekhoa e meng e sireletsang tikoloho e se e le ntho ea bohlokoa ka ho fetisisa.
Li-polyamine (li-PA), tse kang putrescine, spermidine, spermine, le cadaverine, li ka sebetsa e le mekhoa e meng e tšepisang khahlanong le likokoana-hloko tse jang limela mobung, ka hona li fokotsa ka botlalo kapa ka karolo tšebeliso ea li-fungicide tse kotsi tsa lik'hemik'hale (Nehela et al., 2024; Yi et al., 2024). Limeleng tse phahameng, li-PA li ameha lits'ebetsong tse ngata tsa 'mele ho kenyeletsoa, ​​​​empa ho sa felle feela ho, karohano ea sele, phapang, le karabelo likhatellong tsa abiotic le biotic (Killiny le Nehela, 2020). Li ka sebetsa e le li-antioxidants, tsa thusa ho tlosa mefuta ea oksijene e arabelang (ROS), tsa boloka redox homeostasis (Nehela le Killiny, 2023), tsa hlahisa liphatsa tsa lefutso tse amanang le tšireletso (Romero et al., 2018), tsa laola litsela tse fapaneng tsa metabolism (Nehela le Killiny, 2023), tsa fetola li-phytohormone tsa tlhaho (Nehela le Killiny, 2019), tsa theha khanyetso e fumanoeng ke systemic (SAR), le ho laola likamano tsa limela le likokoana-hloko (Nehela le Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). Hoa bohlokoa ho hlokomela hore mekhoa le mesebetsi e itseng ea li-PA ts'ireletsong ea limela e fapana ho latela mefuta ea limela, likokoana-hloko le maemo a tikoloho. PA e ngata haholo limela e entsoe ka biosynthesized ho tsoa ho polyamine L-ornithine ea bohlokoa (Killiny le Nehela, 2020).
L-ornithine e bapala karolo e mengata kgolong le ntshetsopeleng ya dimela. Mohlala, diphuputso tse fetileng di bontshitse hore raeseng (Oryza sativa), ornithine e ka nna ya amahanngwa le ho sebediswa hape ha naetrojene (Liu et al., 2018), tlhahiso ya raese, boleng le monko (Lu et al., 2020), le karabelo ya kgatello ya metsi (Yang et al., 2000). Ho feta moo, tshebediso ya kantle ya L-ornithine e ntlafaditse haholo mamello ya komello ho beet ya tsoekere (Beta vulgaris) (Hussein et al., 2019) mme ya fokotsa kgatello ya letswai ho onion (Allium Cepa) (Çavuşoǧlu le Çavuşoǧlu, 2021) le dimela tsa cashew (Anacardium occidentale) (da Rocha et al., 2012). Karolo e ka bang teng ya L-ornithine ho sireletseng kgatello ya abiotic e ka bakwa ke ho kenella ha yona ho bokelleng ha proline dimeleng tse phekotsweng. Mohlala, liphatsa tsa lefutso tse amanang le metabolism ea proline, tse kang liphatsa tsa lefutso tsa ornithine delta aminotransferase (delta-OAT) le proline dehydrogenase (ProDH1 le ProDH2), li kile tsa tlalehoa hore li ameha ho sireletseng Nicotiana benthamiana le Arabidopsis thaliana khahlanong le mefuta ea Pseudomonas syringae e seng moamoheli (Senthil-Kumar le Mysore, 2012). Ka lehlakoreng le leng, fungal ornithine decarboxylase (ODC) ea hlokahala bakeng sa kholo ea pathogen (Singh et al., 2020). Ho shebana le ODC ea Fusarium oxysporum f. sp. lycopersici ka ho khutsisa liphatsa tsa lefutso tse bakoang ke moamoheli (HIGS) ho matlafalitse haholo ho hanyetsa limela tsa tamati ho Fusarium wilt (Singh et al., 2020). Leha ho le joalo, karolo e ka bang teng ea ts'ebeliso ea ornithine ea kantle khahlanong le likhatello tsa biotic joalo ka phytopathogens ha e so ithutoe hantle. Habohlokoa le ho feta, litlamorao tsa ornithine ho hanyetsaneng le mafu le liketsahalo tse amanang le tsona tsa lik'hemik'hale le tsa 'mele li ntse li sa hlahlojoe haholo.
Ho utloisisa ho rarahana ha tšoaetso ea S. sclerotiorum ea linaoa ho bohlokoa bakeng sa nts'etsopele ea maano a taolo a sebetsang. Thutong ena, re ne re ikemiselitse ho khetholla karolo e ka bang teng ea diamine L-ornithine e le ntlha ea bohlokoa ho ntlafatseng mekhoa ea ts'ireletso le ho hanyetsa limela tsa linaoa tšoaetsong ea Sclerotinia sclerotiorum. Re nahana hore, ntle le ho ntlafatsa likarabo tsa ts'ireletso tsa limela tse tšoaelitsoeng, L-ornithine e boetse e bapala karolo ea bohlokoa ho bolokeng boemo ba redox. Re sisinya hore litlamorao tse ka bang teng tsa L-ornithine li amana le taolo ea mekhoa ea ts'ireletso ea antioxidant ea enzymatic le e seng ea enzymatic le tšitiso ea fungus pathogenicity/virulence factors le liprotheine tse amanang le eona. Ts'ebetso ena e habeli ea L-ornithine e etsa hore e be mokhethoa ea tšepisang bakeng sa leano le tsitsitseng la ho fokotsa tšusumetso ea hlobo e tšoeu le ho ntlafatsa ho hanyetsa ha lijalo tsa linaoa tse tloaelehileng khahlanong le pathogen ena e matla ea fungus. Liphetho tsa thuto ea hona joale li ka thusa nts'etsopele ea mekhoa e mecha, e mosa tikolohong ea ho laola hlobo e tšoeu le ho fokotsa tšusumetso ea eona tlhahisong ea linaoa.
Phuputsong ena, mofuta o kotsing oa khoebo oa linaoa tse tloaelehileng, Giza 3 (Phaseolus vulgaris L. cv. Giza 3), o sebelisitsoe e le thepa ea liteko. Lefapha la Lipatlisiso tsa Lijalo tsa Legume, Setsi sa Lipatlisiso tsa Lijalo tsa Masimo (FCRI), Setsi sa Lipatlisiso tsa Temo (ARC), Egepeta, le fane ka peo e phetseng hantle. Peo e mehlano e jetsoe ka lipitseng tsa polasetiki (bophara ba ka hare ba 35 cm, botebo ba 50 cm) e tletseng mobu o tšoaelitsoeng ke S. sclerotiorum tlas'a maemo a greenhouse (25 ± 2 °C, mongobo o lekanyelitsoeng oa 75 ± 1%, khanya ea lihora tse 8/lihora tse 16 tse lefifi). Matsatsing a 7-10 kamora ho jala (DPS), lipeo li ile tsa fokotsoa ho siea lipeo tse peli feela tse nang le kholo e tšoanang le makhasi a mararo a atolositsoeng ka botlalo ka pitseng ka 'ngoe. Limela tsohle tse ka pitseng li ile tsa nosetsoa hang ka libeke tse ling le tse ling tse peli 'me tsa nontšoa khoeli le khoeli ka sekhahla se khothaletsoang bakeng sa mofuta o fanoeng.
Ho lokisa mahloriso a 500 mg/L a L-ornithinediamine (eo hape e tsejoang e le (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Jeremane), 50 mg e ile ea qhibilihisoa ka lekhetlo la pele ka har'a 100 mL ea metsi a hloekisitsoeng a sterile. Tharollo ea setoko e ile ea qhibilihisoa 'me ea sebelisoa litekong tse latelang. Ka bokhutšoanyane, letoto le tšeletseng la mahloriso a L-ornithine (12.5, 25, 50, 75, 100, le 125 mg/L) a ile a lekoa ka har'a vitro. Ho phaella moo, metsi a hloekisitsoeng a sterile a ile a sebelisoa e le taolo e mpe (Mock) 'me phofo ea fungicide ea khoebo ea "Rizolex-T" 50% ea phofo e metsi (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Egepeta) e ile ea sebelisoa e le taolo e ntle. Sebolaya-fungi sa kgwebo sa "Rizolex-T" se lekilwe ka hara vitro ka maemo a mahlano (2, 4, 6, 8 le 10 mg/L).
Disampole tsa dikutu le di-pod tse tlwaelehileng tsa dinawa tse bontshang matshwao a tlwaelehileng a hlobo e tshweu (sekgahla sa tlhaselo: 10–30%) di ile tsa bokellwa mapolasing a kgwebo. Le hoja boholo ba dihlahiswa tsa dimela tse tshwaeditsweng di ile tsa hlwauwa ka mefuta/mefuta (mofuta wa kgwebo o kotsing ya ho tshwaetswa Giza 3), tse ding, haholoholo tse fumanweng mebarakeng ya lehae, e ne e le tsa mefuta e sa tsejweng. Dihlahiswa tse tshwaeditsweng tse bokelletsweng di ile tsa tshwaetswa dibolaya-dikokwana-hloko ka tharollo ya sodium hypochlorite ya 0.5% ka metsotso e 3, tsa ntano hlatsuwa ka makgetlo a mmalwa ka metsi a hlwekileng a hlwekileng mme tsa hlakolwa ka pampiri ya sefe e hlwekileng ho tlosa metsi a mangata. Ditho tse tshwaeditsweng di ile tsa kgaolwa dikotwana tse nyane ho tloha dinama tse bohareng (pakeng tsa dinama tse phetseng hantle le tse tshwaeditsweng), tsa lengwa hodima mokgahlelo wa potato dextrose agar (PDA) mme tsa futhumatswa mochesong wa 25 ± 2 °C ka potoloho ya lefifi ya dihora tse 12/dihora tse 12 ka matsatsi a 5 ho kgothaletsa ho thehwa ha sclerotia. Mokgwa wa ntlha ya mycelial o ile wa boela wa sebediswa ho hlwekisa di-fungus tse ikarotseng ho tswa ho dimela tse tswakilweng kapa tse silafetseng. Sekotwana sa fungus se hloekisitsoeng se ile sa hlwauwa pele ho latela dibopeho tsa sona tsa setso mme sa netefatswa hore ke S. sclerotiorum ho latela dibopeho tse nyane. Qetellong, dikotwana tsohle tse hlwekisitsweng di ile tsa lekoa bakeng sa ho baka tshwaetso ho mofuta wa dinawa tse tlwaelehileng tse kotsing wa Giza 3 ho fihlela di-postulates tsa Koch.
Ho phaella moo, S. sclerotiorum isolate e hlaselang ka ho fetisisa (isolate #3) e ile ea tiisoa hape ho latela tatellano ea internal transcribed spacer (ITS) joalo ka ha ho hlalositsoe ke White et al., 1990; Baturo-Ciesniewska et al., 2017. Ka bokhutšoanyane, li-isolate li ile tsa lengoa ka moro oa litapole dextrose (PDB) 'me tsa bolokoa mochesong oa 25 ± 2 °C ka matsatsi a 5-7. Mycelium ea fungal e ile ea bokelloa, ea sefshoa ka cheesecloth, ea hlatsuoa habeli ka metsi a hloekileng, 'me ea omisoa ka pampiri ea sefe e hloekileng. DNA ea genomic e ile ea aroloa ho sebelisoa Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Sebaka sa ITS rDNA se ile sa atolosoa ho sebelisoa para e ikhethileng ea primer ITS1/ITS4 (TCCGTAGGTGAACCTGGCG TCCTCGCTTTATTGATATGC; boholo bo lebelletsoeng: 540 bp) (Baturo-Ciesniewska et al., 2017). Lihlahisoa tsa PCR tse hloekisitsoeng li ile tsa romelloa bakeng sa tatellano (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). Tatellano ea ITS rDNA e ile ea aroloa ka tatellano ka bobeli ho sebelisoa mokhoa oa Sanger oa tatellano. Tatellano ea lipotso tse kopantsoeng e ile ea bapisoa le data ea morao-rao ho GenBank le Setsi sa Naha sa Tlhahisoleseling ea Biotechnology (NCBI, http://www.ncbi.nlm.nih.gov/gene/) ho sebelisoa software ea BLASTn. Tatellano ea lipotso e bapisitsoe le mefuta/li-isolate tse ling tse 20 tsa S. sclerotiorum tse fumanoeng ho tsoa ho data ea morao-rao ho NCBI GenBank (Table Supplementary S1) ho sebelisoa ClustalW ho Molecular Evolutionary Genetics Analysis Package (MEGA-11; mofuta oa 11) (Kumar et al., 2024). Tlhahlobo ea ho iphetola ha lintho e entsoe ho sebelisoa mokhoa oa ho ba le monyetla o moholo le mohlala oa kakaretso oa ho fetola nucleotide ka nako (Nei le Kumar, 2000). Sefate se nang le monyetla o moholo oa ho ngola se bontšoa. Sefate sa pele sa patlo ea heuristic se khethoa ka ho khetha sefate se nang le monyetla o moholo oa ho ngola pakeng tsa sefate se kopanyang baahelani (NJ) (Kumar et al., 2024) le sefate sa parsimony se phahameng ka ho fetisisa (MP). Sefate sa NJ se hahiloe ho sebelisoa matrix ea sebaka se habeli se baloang ho sebelisoa mohlala oa kakaretso oa ho fetola nako (Nei le Kumar, 2000).
Tšebetso ea ho thibela likokoana-hloko ea L-ornithine le "Rizolex-T" e ile ea fumanoa ka hare ho sejana ka mokhoa oa ho hasanya agar. Mokhoa: Nka tekanyo e loketseng ea tharollo ea L-ornithine (500 mg/L) 'me u e kopanye hantle le 10 ml ea mokelikeli oa limatlafatsi oa PDA ho lokisa litharollo tse nang le likhakanyo tsa ho qetela tsa 12.5, 25, 50, 75, 100 le 125 mg/L, ka ho latellana. Likarolo tse hlano tsa mokelikeli oa fungicide "Rizolex-T" (2, 4, 6, 8 le 10 mg/L) le metsi a hloekisitsoeng a hloekisitsoeng li ile tsa sebelisoa e le taolo. Kamora hore mokelikeli o tiee, polaka ea mycelial e sa tsoa lokisoa ea setso sa Sclerotinia sclerotiorum, bophara ba 4 mm, e ile ea fetisetsoa bohareng ba sejana sa Petri 'me ea lengoa ka 25±2°C ho fihlela mycelium e koahela sejana sohle sa Petri se laoloang, ka mor'a moo kholo ea fungus e ile ea tlalehoa. Bala peresente ea thibelo ea kholo ea radial ea S. sclerotiorum u sebelisa equation ea 1:
Teko e phetiloe habeli, ka likopi tse tšeletseng tsa baeloji bakeng sa sehlopha ka seng sa taolo/teko le lipitsa tse hlano (limela tse peli ka pitsa) bakeng sa kopi e 'ngoe le e 'ngoe ea baeloji. kopi e 'ngoe le e 'ngoe ea baeloji e hlahlobiloe habeli (likopi tse peli tsa botekgeniki) ho netefatsa ho nepahala, ho tšepahala le ho ikatisa ha liphetho tsa liteko. Ho phaella moo, tlhahlobo ea probit regression e sebelisitsoe ho bala mahloriso a thibelo ea halofo-maximal (IC50) le IC99 (Prentice, 1976).
Ho lekola bokgoni ba L-ornithine tlasa maemo a greenhouse, ho ile ha etswa diteko tse pedi tse latellanang tsa dipitsa. Ka bokgutshwanyane, dipitsa di ile tsa tlatswa ka mobu o hlwekisitsweng wa lehlabathe la letsopa (3:1) mme tsa entwa ka setso se sa tswa lokiswa sa S. sclerotiorum. Sa pele, karolo e hlaselang ka ho fetisisa ya S. sclerotiorum (e ikarolang #3) e ile ya hodiswa ka ho seha sclerotium e le nngwe ka halofo, ya e beha e shebile fatshe hodima PDA mme ya engwa ka mocheso wa 25°C lefifing le sa kgaotseng (dihora tse 24) ka matsatsi a 4 ho susumetsa kgolo ya mycelial. Dipolaka tse nne tsa agar tse bophara ba 5 mm di ile tsa nkuwa pheletsong e ka pele mme tsa entwa ka 100 g ya motswako o hlwekileng wa koro le raese bran (1:1, v/v) mme diflask tsohle di ile tsa engwa ka mocheso wa 25 ± 2 °C tlasa potoloho ya lefifi ya dihora tse 12/dihora tse 12 ka matsatsi a 5 ho kgothaletsa ho thehwa ha sclerotia. Dikahare tsa diflask tsohle di ile tsa kopanngwa hantle ho netefatsa hore ho na le ho lekana pele ho eketswa mobu. Eaba, 100 g ea motsoako oa bran o bokellanang e ile ea eketsoa pitseng e 'ngoe le e 'ngoe ho netefatsa hore ho na le likokoana-hloko tse sa khaotseng. Lipitsa tse entetsoeng li ile tsa nosetsoa ho tsosa kholo ea fungus 'me tsa beoa maemong a greenhouse matsatsi a 7.
Peo tse hlano tsa mofuta oa Giza 3 li ile tsa jaloa ka pitseng ka 'ngoe. Bakeng sa lipitsa tse phekotsoeng ka L-ornithine le Rizolex-T ea sebolaya-mafu, peo e hloekisitsoeng e ile ea qala ka ho kolobisoa ka lihora tse peli ka har'a tharollo ea metsi ea metsoako e 'meli ka khatello ea ho qetela ea IC99 ea hoo e ka bang 250 mg/L le 50 mg/L, ka ho latellana, ebe e omisoa moeeng ka hora e le 'ngoe pele e jaloa. Ka lehlakoreng le leng, peo e ile ea kolobisoa ka metsing a hloekisitsoeng a sa hloekang e le taolo e mpe. Kamora matsatsi a 10, pele ho nosetso ea pele, lipeo li ile tsa fokotsoa, ​​​​tsa siea lipeo tse peli feela tse hloekileng ka pitseng ka 'ngoe. Ho feta moo, ho netefatsa tšoaetso ea S. sclerotiorum, likutu tsa limela tsa linaoa tse boemong bo tšoanang ba nts'etsopele (matsatsi a 10) li ile tsa khaoloa libakeng tse peli tse fapaneng ho sebelisoa scalpel e hloekisitsoeng 'me hoo e ka bang 0.5 g ea motsoako oa bran o koloneang o ile oa kenngoa leqebeng ka leng, 'me ha latela mongobo o phahameng ho susumetsa tšoaetso le nts'etsopele ea mafu limela tsohle tse entetsoeng. Limela tsa taolo li ile tsa tsoa likotsi ka mokhoa o tšoanang 'me palo e lekanang (0.5 g) ea motsoako oa bran o se nang likokoana-hloko, o sa kolone e ile ea kenngoa leqebeng 'me ea bolokoa tlas'a mongobo o phahameng ho etsisa tikoloho bakeng sa nts'etsopele ea lefu le ho netefatsa botsitso lipakeng tsa lihlopha tsa kalafo.
Mokhoa oa kalafo: Lipeo tsa linaoa li ile tsa nosetsoa ka 500 ml ea tharollo ea metsi ea L-ornithine (250 mg/l) kapa Rizolex-T ea fungicide (50 mg/l) ka ho nosetsa mobu, ebe kalafo e phetoa makhetlo a mararo ka nako ea matsatsi a 10. Litaolo tse phekotsoeng ka placebo li ile tsa nosetsoa ka 500 ml ea metsi a hloekisitsoeng a sterile. Litlhare tsohle li ile tsa etsoa tlas'a maemo a greenhouse (25 ± 2°C, 75 ± 1% mongobo o amanang, le nako ea khanya ea lihora tse 8/lihora tse 16 tse lefifi). Lipitsa tsohle li ile tsa nosetsoa libeke tse peli 'me tsa phekoloa khoeli le khoeli ka manyolo a leka-lekaneng a NPK (20-20-20, ka 3.6% ea sebabole le TE microelements; Zain Seeds, Egepeta) ka bongata ba 3-4 g/l ka ho fafatsa makhasi ho latela likhothaletso tsa mofuta o itseng le litaelo tsa moetsi. Ntle le ha ho boletsoe ka tsela e 'ngoe, makhasi a butsoitseng a atolositsoeng ka botlalo (makhasi a 2 le a 3 ho tloha holimo) a bokelletsoe ho tsoa ho kopi e 'ngoe le e 'ngoe ea baeloji ka mor'a lihora tse 72 kamora kalafo (hpt), a kopantsoe ka ho lekana, a kopantsoe 'me a bolokoa ho -80 °C bakeng sa litlhahlobo tse ling ho kenyeletsoa, ​​​​empa ho sa felle feela ho, in situ histochemical localization ea matšoao a khatello ea oxidative, lipid peroxidation, li-antioxidants tsa enzymatic le tse seng enzymatic le polelo ea liphatsa tsa lefutso.
Matla a tšoaetso ea hlobo e tšoeu a ile a hlahlojoa beke le beke matsatsi a 21 kamora ho entoa (dpi) ho sebelisoa sekala sa 1-9 (Tafole e Tlatsetsang S2) ho latela sekala sa Petzoldt le Dickson (1996) se fetotsoeng ke Teran et al. (2006). Ka bokhutšoanyane, likutu le makala a limela tsa linaoa li ile tsa hlahlojoa ho qala ntlheng ea ho entoa ho latela tsoelo-pele ea liso ho latela li-internode le li-node. Sebaka sa leqeba ho tloha ntlheng ea ho entoa ho ea ntlheng e hole ka ho fetisisa ho potoloha kutu kapa lekala se ile sa lekanngoa 'me lintlha tsa 1-9 tsa abeloa ho latela sebaka sa leqeba, moo (1) ho ileng ha bontša hore ha ho na tšoaetso e bonahalang haufi le ntlha ea ho entoa 'me (2-9) ho bontšitse keketseho e butle-butle ea boholo ba leqeba le tsoelo-pele ho latela li-node/li-internode (Tafole e Tlatsetsang S2). Matla a tšoaetso ea hlobo e tšoeu a ile a fetoloa ho ba peresente ho sebelisoa mokhoa oa 2:
Ho phaella moo, sebaka se ka tlasa mothapo wa ho tswela pele ha lefu (AUDPC) se badilwe ho sebediswa foromo (Shaner le Finney, 1977), e sa tswa fetolwa bakeng sa ho bola ho hosweu ha dinawa tse tlwaelehileng (Chauhan et al., 2020) ho sebediswa equation 3:
Moo Yi = boima ba lefu ka nako ti, Yi+1 = boima ba lefu ka nako e tlang ti+1, ti = nako ea tekanyo ea pele (ka matsatsi), ti+1 = nako ea tekanyo e latelang (ka matsatsi), n = palo eohle ea lintlha tsa nako kapa lintlha tsa ho shebella. Litekanyetso tsa kholo ea limela tsa linaoa ho kenyeletsoa bolelele ba semela (cm), palo ea makala ka semela, le palo ea makhasi ka semela li tlalehiloe beke le beke matsatsi a 21 ho likopi tsohle tsa baeloji.
Ho kopi e 'ngoe le e 'ngoe ea baeloji, lisampole tsa makhasi (makhasi a bobeli le a boraro a hōlileng ka botlalo ho tloha holimo) li ile tsa bokelloa ka letsatsi la 45 kamora kalafo (matsatsi a 15 kamora kalafo ea ho qetela). kopi e 'ngoe le e 'ngoe ea baeloji e ne e entsoe ka lipitsa tse hlano (limela tse peli ka pitsa). Hoo e ka bang 500 mg ea lisele tse sithabetseng e ile ea sebelisoa bakeng sa ho ntša mebala ea photosynthetic (chlorophyll a, chlorophyll b le carotenoids) ho sebelisoa 80% acetone ho 4 °C lefifing. Kamora lihora tse 24, lisampole li ile tsa kenngoa ka centrifuge 'me supernatant ea bokelloa bakeng sa ho fumana hore na chlorophyll a, chlorophyll b le carotenoid li na le 'mala joang ho sebelisoa spectrophotometer ea UV-160A (Shimadzu Corporation, Japane) ho latela mokhoa oa (Lichtenthaler, 1987) ka ho lekanya ho monya ha maqhubu a mararo a fapaneng (A470, A646 le A663 nm). Qetellong, diteng tsa dipigment tsa photosynthetic di badilwe ho sebediswa diforomo tse latelang 4–6 tse hlalositsweng ke Lichtenthaler (1987).
Kamora lihora tse 72 kamora kalafo (hpt), makhasi (makhasi a bobeli le a boraro a holileng ka botlalo ho tloha holimo) a ile a bokelloa ho tsoa ho kopi e 'ngoe le e 'ngoe ea baeloji bakeng sa sebaka sa histochemical sa hydrogen peroxide (H2O2) le superoxide anion (O2•−). kopi e 'ngoe le e 'ngoe ea baeloji e ne e entsoe ka lipitsa tse hlano (limela tse peli ka pitsa). kopi e 'ngoe le e 'ngoe ea baeloji e ile ea hlahlojoa ka makhetlo a mabeli (kopi tse peli tsa botekgeniki) ho netefatsa ho nepahala, ho tšepahala le ho ikatisa ha mokhoa ona. H2O2 le O2•− li ile tsa fumanoa ho sebelisoa 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Jeremane) kapa nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Jeremane), ka ho latellana, ho latela mekhoa e hlalositsoeng ke Romero-Puertas et al. (2004) le Adam et al. (1989) ka liphetoho tse nyane. Bakeng sa ho fumana sebaka sa H2O2 ka har'a sebaka sa histochemical, lipampitšana li ile tsa kenngoa ka vacuum light ka 0.1% DAB ka har'a 10 mM Tris buffer (pH 7.8) ebe li kenngoa ka har'a mocheso oa kamore leseling metsotso e 60. Lipampitšana li ile tsa hloekisoa ka har'a 0.15% (v/v) TCA ka har'a 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) ebe li pepesetsoa leseling ho fihlela li fifala. Ka mokhoa o ts'oanang, li-valve li ile tsa kenngoa ka vacuum cleaner ka 10 mM potassium phosphate buffer (pH 7.8) e nang le 0.1 w/v % HBT bakeng sa ho fumana sebaka sa O2 •− ka har'a sebaka sa histochemical. Lipampitšana li ile tsa kenngoa ka har'a leseling mochesong oa kamore metsotso e 20, ebe li hloekisoa joalo ka ha ho boletsoe kaholimo, ebe li bonesoa ho fihlela matheba a lefifi a boputsoa/violet a hlaha. Matla a 'mala o sootho o hlahang (joalo ka sesupo sa H2O2) kapa 'mala o moputsoa-violet (joalo ka sesupo sa O2•−) a ile a lekoa ho sebelisoa mofuta oa Fiji oa sephutheloana sa ho sebetsana le litšoantšo ImageJ (http://fiji.sc; e fihletsoe ka la 7 Hlakubele 2024).
Malondialdehyde (MDA; e le letshwao la peroxidation ya lipid) e ile ya fumanwa ho ya ka mokgwa wa Du le Bramlage (1992) ka diphetoho tse nyane. Makhasi a tswang ho kopi e nngwe le e nngwe ya baeloji (makhasi a bobedi le a boraro a hodileng ka botlalo ho tloha hodimo) a ile a bokellwa dihora tse 72 kamora kalafo (hpt). kopi e nngwe le e nngwe ya baeloji e ne e kenyeletsa dipitsa tse hlano (dimela tse pedi ka pitsa). kopi e nngwe le e nngwe ya baeloji e ile ya sekasekwa ka kopi e le nngwe (kopi tse pedi tsa botekgeniki) ho netefatsa ho nepahala, ho tshepahala le ho ikatisa ha mokgwa. Ka bokhutshwane, 0.5 g ya dinama tse tala tsa lekhasi la mobu e sebediseditswe ho ntshwa ha MDA ka 20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) e nang le 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). Dikahare tsa MDA ka hara supernatant di ile tsa lekanyetswa ka mmala ka ho lekanya monya ho 532 le 600 nm ho sebediswa spectrophotometer ya UV-160A (Shimadzu Corporation, Japane) mme tsa hlahiswa e le nmol g−1 FW.
Bakeng sa tlhahlobo ea li-antioxidants tse seng tsa enzymatic le enzymatic, makhasi (makhasi a bobeli le a boraro a holileng ka botlalo ho tloha holimo) a ile a bokelloa ho tsoa ho kopi e 'ngoe le e 'ngoe ea baeloji ka mor'a lihora tse 72 kamora kalafo (hpt). kopi e 'ngoe le e 'ngoe ea baeloji e ne e entsoe ka lipitsa tse hlano (limela tse peli ka pitsa). Sampole e 'ngoe le e 'ngoe ea baeloji e ile ea hlahlojoa ka makhetlo a mabeli (lisampole tse peli tsa botekgeniki). Makhasi a mabeli a ile a siloa ka naetrojene e metsi 'me a sebelisoa ka kotloloho bakeng sa ho fumana li-antioxidants tsa enzymatic le tse seng tsa enzymatic, li-amino acid tsohle, litaba tsa proline, tlhahiso ea liphatsa tsa lefutso le tekanyo ea oxalate.
Li-phenolic tsohle tse qhibilihang li fumanoe ho sebelisoa reagent ea Folin-Ciocalteu (Sigma-Aldrich, St. Louis, MO, USA) ka liphetoho tse nyane tsa mokhoa o hlalositsoeng ke Kahkonen et al. (1999). Ka bokhutšoanyane, hoo e ka bang 0.1 g ea lisele tsa makhasi tse homogenized li ile tsa ntšoa ka 20 ml ea methanol ea 80% lefifing ka lihora tse 24 'me supernatant ea bokelloa ka mor'a ho centrifugation. 0.1 ml ea sampole e ntšitsoeng e ile ea kopanngoa le reagent ea Folin-Ciocalteu ea 0.5 ml (10%), ea sisinngoa metsotsoana e 30 'me ea siuoa lefifing ka metsotso e 5. Eaba 0.5 ml ea tharollo ea sodium carbonate ea 20% (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egepeta) e eketsoa ka har'a tube e 'ngoe le e 'ngoe, ea kopanngoa hantle 'me ea kenngoa mochesong oa kamore lefifing ka hora e le' ngoe. Kamora ho futhumala, ho monya ha motsoako oa karabelo ho ile ha lekanngoa ho 765 nm ho sebelisoa spectrophotometer ea UV-160A (Shimadzu Corporation, Japane). Khokahano ea li-phenol tse qhibilihang ka botlalo li-extracts tsa sampole e ile ea fumanoa ho sebelisoa curve ea calibration ea gallic acid (Fisher Scientific, Hampton, NH, USA) 'me ea hlahisoa e le li-milligrams tsa gallic acid e lekanang ka gram ea boima bo bocha (mg GAE g-1 boima bo bocha).
Kakaretso ea flavonoid e qhibilihang e ile ea fumanoa ho latela mokhoa oa Djeridane et al. (2006) ka liphetoho tse nyane. Ka bokhutšoanyane, 0.3 ml ea setlolo sa methanol se kaholimo se ile sa kopanngoa le 0.3 ml ea tharollo ea 5% ea aluminium chloride (AlCl3; Fisher Scientific, Hampton, NH, USA), sa hlohlelletsoa ka matla 'me sa beoa mochesong oa kamore metsotso e 5, sa lateloa ke ho eketsoa ha 0.3 ml ea tharollo ea 10% ea potassium acetate (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), sa tsoakoa hantle 'me sa beoa mochesong oa kamore metsotso e 30 lefifing. Kamora ho beoa, ho monngoa ha motsoako oa karabelo ho ile ha lekanngoa ho 430 nm ho sebelisoa spectrophotometer ea UV-160A (Shimadzu Corporation, Japane). Khatello ea li-flavonoid tse qhibilihang ka botlalo li-extracts tsa sampole e ile ea fumanoa ho sebelisoa sekhutlo sa rutin calibration (TCI America, Portland, OR, USA) ebe se hlahisoa e le li-milligram tsa rutin e lekanang ka gram ea boima bo bocha (mg RE g-1 boima bo bocha).
Kakaretso ea amino acid e lokolohileng ea makhasi a linaoa e fumanoe ka ho sebelisa reagent e fetotsoeng ea ninhydrin (Thermo Scientific Chemicals, Waltham, MA, USA) ho latela mokhoa o hlahisitsoeng ke Yokoyama le Hiramatsu (2003) 'me ea fetoloa ke Sun et al. (2006). Ka bokhutšoanyane, 0.1 g ea lisele tse sitsitsoeng e ile ea ntšoa ka pH 5.4 buffer, 'me 200 μL ea supernatant e ile ea arajoa ka 200 μL ea ninhydrin (2%) le 200 μL ea pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), e ile ea kenngoa ka bateng ea metsi a belang metsotso e 30, eaba e pholile 'me ea lekanngoa ho 580 nm ho sebelisoa spectrophotometer ea UV-160A (Shimadzu Corporation, Japane). Ka lehlakoreng le leng, proline e ile ea khethoa ka mokhoa oa Bates (Bates et al., 1973). Proline e ntšitsoe ka 3% sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) mme kamora ho centrifugation, 0.5 ml ea supernatant e ile ea kopanngoa le 1 ml glacial acetic acid (Fisher Scientific, Hampton, NH, USA) le ninhydrin reagent, e ile ea beoa mochesong oa 90°C metsotso e 45, ea pholisoa 'me ea lekanngoa ho 520 nm ho sebelisoa spectrophotometer e tšoanang le e kaholimo. Li-amino acid tsohle tse lokolohileng le proline tse ntšitsoeng makhasing li ile tsa fumanoa ka ho sebelisa li-curve tsa glycine le proline calibration (Sigma-Aldrich, St Louis, MO, USA), ka ho latellana, 'me tsa hlahisoa e le boima bo bocha ba mg/g.
Ho fumana ts'ebetso ea enzymatic ea li-enzyme tsa antioxidant, hoo e ka bang 500 mg ea lisele tse homogenized e ntšitsoe ka 3 ml ea 50 mM Tris buffer (pH 7.8) e nang le 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) le 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), e tšetsoeng ka centrifuge ho 10,000 × g metsotso e 20 tlas'a sehatsetsing (4 °C), 'me supernatant (extract ea enzyme e tala) e ile ea bokelloa (El-Nagar et al., 2023; Osman et al., 2023). Ka mora moo, Catalase (CAT) e ile ya arajwa ka 2 ml ya 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) le 100 μl ya tharollo ya 269 mM H2O2 ho fumana mosebetsi wa yona wa enzymatic ho ya ka mokgwa wa Aebi (1984) ka diphetoho tse nyane (El-Nagar et al., 2023; Osman et al., 2023). Mosebetsi wa enzymatic wa peroxidase (POX) o itshetlehileng ka Guaiacol o ile wa fumanoa ho sebediswa mokgwa wa Harrach et al. (2009). (2008) ka diphetoho tse nyane (El-Nagar et al., 2023; Osman et al., 2023) mme ts'ebetso ya enzymatic ya polyphenol oxidase (PPO) e ile ya fumaneha kamora karabelo ka 2.2 ml ya 100 mM sodium phosphate buffer (pH 6.0), 100 μl ya guaiacol (dikhemikhale tsa TCI, Portland, OR, USA) le 100 μl ya 12 mM H2O2. Mokgwa o fetotswe hanyane ho tloha ho (El-Nagar et al., 2023; Osman et al., 2023). Teko e entswe kamora karabelo ka 3 ml ya tharollo ya catechol (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) e sa tswa lokiswa ho 0.1 M phosphate buffer (pH 6.0). Mosebetsi oa CAT o lekantsoe ka ho beha leihlo ho bola ha H2O2 ho 240 nm (A240), mosebetsi oa POX o lekantsoe ka ho beha leihlo keketseho ea ho monya ha 436 nm (A436), 'me mosebetsi oa PPO o lekantsoe ka ho rekota liphetoho tsa ho monya ha 495 nm (A495) metsotsoana e meng le e meng e 30 ka metsotso e 3 ho sebelisoa spectrophotometer ea UV-160A (Shimadzu, Japane).
RT-PCR ea nako ea sebele e sebelisitsoe ho fumana maemo a transcript a liphatsa tsa lefutso tse tharo tse amanang le antioxidant, ho kenyeletsoa peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), le glutathione reductase (PvGR; GenBank Accession No. KY195009.1), makhasing a linaoa (makhasi a bobeli le a boraro a holileng ka botlalo ho tloha holimo) lihora tse 72 kamora kalafo ea ho qetela. Ka bokhutšoanyane, RNA e ile ea aroloa ho sebelisoa Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) ho latela protocol ea moetsi. Eaba, cDNA e entsoe ho sebelisoa TOP script™ cDNA Synthesis Kit ho latela litaelo tsa moetsi. Tatellano ea pele ea liphatsa tsa lefutso tse tharo tse kaholimo e thathamisitsoe ho Supplementary Table S3. PvActin-3 (nomoro ea kamohelo ea GenBank: XM_068616709.1) e sebelisitsoe e le gene ea tlhokomelo ea ntlo 'me polelo ea gene e amanang e baliloe ho sebelisoa mokhoa oa 2-ΔΔCT (Livak le Schmittgen, 2001). Ho tsitsa ha Actin tlas'a khatello ea biotic (tšebelisano e sa lumellaneng pakeng tsa linaoa tse tloaelehileng le fungus ea anthracnose Colletotrichum lindemuthianum) le khatello ea abiotic (komello, letsoai, mocheso o tlase) ho bontšitsoe (Borges et al., 2012).
Qalong re entse tlhahlobo ea silico ea liprotheine tsa oxaloacetate acetylhydrolase (OAH) ka har'a S. sclerotiorum ka ho sebelisa sesebelisoa sa BLAST sa protheine-protheine (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Ka bokhutšoanyane, re sebelisitse OAH ho tsoa ho Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; nomoro ea ho kenngoa ha GenBank XP_040799428.1; li-amino acid tse 342) le Penicillium lagena (PlOAH; taxide: 94218; nomoro ea ho kenngoa ha GenBank XP_056833920.1; li-amino acid tse 316) e le tatellano ea lipotso ho etsa 'mapa oa protheine e tšoanang ho S. sclerotiorum (taxide: 5180). BLASTp e entsoe khahlanong le data ea genome ea S. sclerotiorum e fumanehang morao tjena ho GenBank webosaeteng ea National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Ho phaella moo, jini ya OAH e boletsweng esale pele ho tswa ho S. sclerotiorum (SsOAH) le tlhahlobo ya ho iphetola ha dintho le sefate sa phylogenetic sa AfOAH ho tswa ho A. fijiensis CBS 313.89 le PlOAH ho tswa ho P. lagena di ile tsa hakanyetswa ho sebediswa mokgwa wa monyetla o moholo ho MEGA11 (Tamura et al., 2021) le mohlala o thehilweng hodima matrix wa JTT (Jones et al., 1992). Sefate sa phylogenetic se ile sa kopanngwa le tlhahlobo ya ho hokahanya ka bongata ditatelano tsa protheine tsa dijini tsohle tsa OAH tse boletsweng esale pele (SsOAH) ho tswa ho S. sclerotiorum le tatelano ya dipotso ho sebediswa Sesebelisoa sa Constraint-Based Alignment (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos le Agarwala, 2007). Ho phaella moo, tatellano e ntle ka ho fetisisa ya amino acid ya SSOAH ho tswa ho S. sclerotiorum e ile ya tsamaellana le tatellano ya dipotso (AfOAH le PlOAH) (Larkin et al., 2007) ho sebediswa ClustalW (http://www.genome.jp/tools-bin/clustalw), mme dibaka tse bolokilweng tse hokahaneng di ile tsa bontshwa ka ho sebedisa sesebediswa sa ESpript (mofuta wa 3.0; https://espript.ibcp.fr/ESpript/ESpript/index.php).
Ho feta moo, libaka tse boletsweng esale pele tsa boemedi ba tshebetso le dibaka tse bolokilweng tsa S. sclerotiorum SsOAH di ile tsa arolwa ka hara malapa a fapaneng ka ho sebedisa sesebediswa sa InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Qetellong, mohlala wa sebopeho sa mahlakore a mararo (3D) wa S. sclerotiorum SsOAH e boletsweng esale pele o entswe ka ho sebedisa Protein Homology/Analogy Recognition Engine (Phyre2 server version 2.0; http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) (Kelley et al., 2015) mme tsa netefatswa ka ho sebedisa seva ya SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). Dibopeho tse boletsweng esale pele tsa mahlakore a mararo (mokgwa wa PDB) di ile tsa bontshwa ka tsela e sebedisanang ho sebedisa sephutheloana sa UCSF-Chimera (mofuta wa 1.15; https://www.cgl.ucsf.edu/chimera/ ) (Pettersen et al., 2004).
PCR ea fluorescence ea nako ea sebele e sebelisitsoe ho fumana boemo ba transcriptional ba oxaloacetate acetylhydrolase (SsOAH; nomoro ea kamohelo ea GenBank: XM_001590428.1) ho mycelia ea Sclerotinia sclerotiorum. Ka bokhutšoanyane, S. sclerotiorum e ile ea entoa ka har'a flask e nang le PDB 'me ea kenngoa ka har'a incubator e thothomelang (mohlala: I2400, New Brunswick Scientific Co., Edison, NJ, USA) ho 25 ± 2 °C ka lihora tse 24 ka 150 rpm le lefifing le sa feleng (lihora tse 24) ho susumetsa kholo ea mycelial. Kamora moo, lisele li ile tsa phekoloa ka L-ornithine le Rizolex-T ea fungicide maemong a ho qetela a IC50 (hoo e ka bang 40 le 3.2 mg/L, ka ho latellana) ebe li lengoa lihora tse ling tse 24 tlas'a maemo a tšoanang. Kamora ho futhumala, dimela di ile tsa centrifugetswa ka lebelo la 2500 rpm ka metsotso e 5 mme supernatant (fungal mycelium) ya bokellwa bakeng sa tlhahlobo ya tlhahiso ya dijini. Ka ho tshwanang, fungal mycelium e ile ya bokellwa ka dihora tse 0, 24, 48, 72, 96, le 120 kamora tshwaetso ho tswa dimeleng tse tshwaeditsweng tse neng di entse hlobo e tshweu le cottony mycelium hodima dinama tse tshwaeditsweng. RNA e ile ya ntshuwa ho fungal mycelium mme cDNA ya etswa jwalo ka ha ho hlalositswe ka hodimo. Ditatelano tsa primer bakeng sa SsOAH di thathamisitswe ho Supplementary Table S3. SsActin (nomoro ya kamohelo ya GenBank: XM_001589919.1) e ile ya sebediswa e le gene ya ho hlokomela ntlo, mme polelo ya gene e amanang e ile ya balwa ho sebediswa mokgwa wa 2-ΔΔCT (Livak le Schmittgen, 2001).
Asiti ea oxalic e ile ea fumanoa ka har'a moro oa litapole oa dextrose (PDB) le lisampoleng tsa limela tse nang le pathogen ea fungal Sclerotinia sclerotiorum ho latela mokhoa oa Xu le Zhang (2000) ka liphetoho tse nyane. Ka bokhutšoanyane, li-isolates tsa S. sclerotiorum li ile tsa entoa ka har'a li-flask tse nang le PDB ebe li lengoa ka har'a incubator e sisinyehang (mohlala oa I2400, New Brunswick Scientific Co., Edison, NJ, USA) ka 150 rpm ka 25 ± 2 °C matsatsi a 3-5 lefifing le sa feleng (lihora tse 24) ho susumetsa kholo ea mycelial. Kamora ho ikatisa, setso sa fungus se ile sa sefshoa pele ka pampiri ea sefe ea Whatman #1 ebe se kenngoa ka centrifuge ka 2500 rpm metsotso e 5 ho tlosa mycelium e setseng. Supernatant e ile ea bokelloa 'me ea bolokoa ho 4°C bakeng sa ho fumana tekanyo e eketsehileng ea oxalate. Bakeng sa ho lokisa lisampole tsa limela, hoo e ka bang 0.1 g ea likotoana tsa lisele tsa limela li ile tsa ntšoa hararo ka metsi a hloekisitsoeng (2 ml nako le nako). Disampole di ile tsa kenngwa ka sentrifuge ka lebelo la 2500 rpm ka metsotso e 5, supernatant e ile ya sefshwa ka omme ka pampiri ya sefe ya Whatman No. 1 mme ya bokellwa bakeng sa tlhahlobo e eketsehileng.
Bakeng sa tlhahlobo ea bongata ba oxalic acid, motsoako oa karabelo o ile oa lokisoa ka har'a tube e koetsoeng ka khalase ka tatellano e latelang: 0.2 ml ea sampole (kapa PDB culture filtrate kapa tharollo e tloaelehileng ea oxalic acid), 0.11 ml ea bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), 0.198 ml ea 1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) le 0.176 ml ea 100 mM potassium dichromate (K2Cr2O7; TCI chemicals, Portland, OR, USA), ebe tharollo e tšeloa ho 4.8 ml ka metsi a hloekisitsoeng, e tsoakoa ka matla 'me hang-hang e beoa ka bateng ea metsi ea 60 °C. Kamora metsotso e 10, karabelo e ile ea emisoa ka ho eketsa 0.5 ml ea tharollo ea sodium hydroxide (NaOH; 0.75 M). Monyako (A600) oa motsoako oa karabelo o lekantsoe ho 600 nm ho sebelisoa spectrophotometer ea UV-160 (Shimadzu Corporation, Japane). PDB le metsi a hloekisitsoeng li sebelisitsoe e le taolo bakeng sa ho lekanya li-filtrate tsa setso le lisampole tsa limela, ka ho latellana. Maqhubu a oxalic acid ho li-filtrate tsa setso, a hlahisoang e le li-microgram tsa oxalic acid ka milliliter ea PDB medium (μg.mL−1), le ho li-extracts tsa makhasi, a hlalositsoeng e le li-microgram tsa oxalic acid ka gram ea boima bo bocha (μg.g−1 FW), a ile a fumanoa ka ho sebelisa curve ea calibration ea oxalic acid (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
Ho pholletsa le phuputso, liteko tsohle li entsoe ka moralo o sa reroang ka ho feletseng (CRD) ka likopi tse tšeletseng tsa baeloji ka kalafo le lipitsa tse hlano ka likopi tse ling tsa baeloji (limela tse peli ka pitsa) ntle le haeba ho boletsoe ka tsela e 'ngoe. Likopi tse peli tsa baeloji li hlahlobiloe ka likopi tse peli (likopi tse peli tsa botekgeniki). Likopi tse peli tsa botekgeniki li sebelisitsoe ho hlahloba ho ikatisa ha teko e tšoanang empa ha lia ka tsa sebelisoa tlhahlobong ea lipalo-palo ho qoba likopi tse fosahetseng. Lintlha li hlahlobiloe ka lipalo-palo ho sebelisoa tlhahlobo ea variance (ANOVA) e lateloang ke teko ea Tukey-Kramer e nang le phapang e kholo (HSD) (p ≤ 0.05). Bakeng sa liteko tsa in vitro, boleng ba IC50 le IC99 bo baliloe ho sebelisoa mohlala oa probit 'me likarolo tsa kholiseho tsa 95% li baliloe.
Ho ile ha bokelloa mefuta e mene e arohaneng masimong a soya a fapaneng Province of El Ghabiya, Egepeta. Ho PDA medium, mefuta yohle e arohaneng e hlahisitse mycelium e tšoeu e boreledi e ileng ya fetoha bosweu bo kang ba k'hothone kapele (Setshwantsho sa 1A) ebe e ba beige kapa sootho mohatong wa sclerotium. Hangata di-Sclerotia di teteaneng, di ntsho, di chitja kapa di sa tsitsa ka sebopeho, di bolelele ba 5.2 ho isa ho 7.7 mm le bophara ba 3.4 ho isa ho 5.3 mm (Setshwantsho sa 1B). Le hoja mefuta e mene e arohaneng e hlahisitse mohlala o moqotetsane wa sclerotia pheletsong ya medium ya temo kamora matsatsi a 10-12 a ho ikatisa ka 25 ± 2 °C (Setshwantsho sa 1A), palo ya sclerotia ka poleiti e ne e fapane haholo hara tsona (P < 0.001), ka karohano ya 3 e nang le palo e hodimo ya sclerotia (32.33 ± 1.53 sclerotia ka poleiti; Setshwantsho sa 1C). Ka ho tšoanang, isolate #3 e hlahisitse oxalic acid e ngata ho PDB ho feta isolate tse ling (3.33 ± 0.49 μg.mL−1; Setšoantšo sa 1D). Isolate #3 e bontšitse litšobotsi tse tloaelehileng tsa sebopeho le microscopic tsa fungus ea phytopathogenic Sclerotinia sclerotiorum. Mohlala, ho PDA, likolone tsa isolate #3 li ile tsa hola ka potlako, li ne li le tšoeu ka 'mala o mosehla (Setšoantšo sa 1A), li beige kapa salmon e bobebe e sootho-e sootho, 'me li ne li hloka matsatsi a 6-7 mochesong oa 25 ± 2°C ho koahela ka botlalo bokaholimo ba poleiti ea bophara ba 9 cm. Ho latela litšobotsi tse ka holimo tsa sebopeho le microscopic, isolate #3 e ile ea khetholloa e le Sclerotinia sclerotiorum.
Setšoantšo sa 1. Litšobotsi le ho baka mafu ha li-isolate tsa S. sclerotiorum tse tsoang lijalong tse tloaelehileng tsa linaoa. (A) Kholo ea mycelial ea li-isolate tse 'ne tsa S. sclerotiorum holim'a PDA, (B) sclerotia ea li-isolate tse 'ne tsa S. sclerotiorum, (C) palo ea sclerotia (ka poleiti), (D) tlhahiso ea oxalic acid holim'a PDB (μg.mL−1), le (E) boima ba lefu (%) la li-isolate tse 'ne tsa S. sclerotiorum holim'a mofuta oa linaoa tsa khoebo tse kotsing ea Giza 3 tlas'a maemo a greenhouse. Litekanyetso li emela karolelano ea ± SD ea li-replicates tse hlano tsa baeloji (n = 5). Litlhaku tse fapaneng li bontša liphapang tse bohlokoa tsa lipalo-palo lipakeng tsa mekhoa ea phekolo (p < 0.05). (F–H) Matšoao a tloaelehileng a hlobo e tšoeu a hlahile holim'a likutu tse ka holimo ho lefatše le silique, ka ho latellana, matsatsi a 10 kamora ho entoa ka isolate #3 (dpi). (I) Tlhahlobo ea ho iphetola ha lintho ea sebaka sa internal transcribed spacer (ITS) sa S. sclerotiorum isolate #3 e entsoe ho sebelisoa mokhoa oa maximum likelihood mme e bapisoa le li-isolates/mefuta e 20 ea litšupiso e fumanoeng ho tsoa polokelongtshedimosetsong ea National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). Linomoro tse kaholimo ho mela ea lihlopha li bontša sebaka seo (%) se koahetsoeng ho sona, 'me linomoro tse ka tlase ho mela ea lihlopha li bontša bolelele ba lekala.
Ho feta moo, ho netefatsa hore na ho na le tšoaetso, li-isolate tse 'ne tsa S. sclerotiorum tse fumanoeng li sebelisitsoe ho enta mofuta oa linaoa tsa khoebo tse kotsing ea ho angoa ke Giza 3 tlas'a maemo a greenhouse, e leng se lumellanang le li-postulates tsa Koch (Setšoantšo sa 1E). Leha li-isolate tsohle tsa fungal tse fumanoeng li ne li baka tšoaetso 'me li ka tšoaetsa linaoa tse tala (cv. Giza 3), li baka matšoao a tloaelehileng a hlobo e tšoeu likarolong tsohle tse ka holimo ho mobu (Setšoantšo sa 1F), haholo-holo likutung (Setšoantšo sa 1G) le li-pod (Setšoantšo sa 1H) matsatsing a 10 kamora ho enta (dpi), isolate 3 e ne e le isolate e matla ka ho fetisisa litekong tse peli tse ikemetseng. Isolate 3 e ne e e-na le bothata bo boholo ba lefu (%) limela tsa linaoa (24.0 ± 4.0, 58.0 ± 2.0, le 76.7 ± 3.1 matsatsing a 7, 14, le 21 kamora ho tšoaetsoa, ​​ka ho latellana; Setšoantšo sa 1F).
Ho hlwaya S. sclerotiorum isolate #3 e hlaselang ka ho fetisisa ho ile ha netefatsoa hape ho latela tatellano ya ka hare ya di-spacer tse ngotsweng (ITS) (Setshwantsho sa 1I). Tlhahlobo ya phylogenetic pakeng tsa di-isolate/mefuta ya di-isolate tse 20 tse tshwantshitsweng e bontshitse ho tshwana ho hoholo (>99%) pakeng tsa tsona. Ho bohlokwa ho hlokomela hore S. sclerotiorum isolate #3 (533 bp) e na le ho tshwana ho hoholo le American S. sclerotiorum isolate LPM36 e arotsweng ho dipeo tse ommeng tsa dierekisi (nomoro ya kopano ya GenBank MK896659.1; 540 bp) le China S. sclerotiorum isolate YKY211 (nomoro ya kopano ya GenBank OR206374.1; 548 bp), e bakang ho bola ha kutu ya violet (Matthiola incana), tseo kaofela di hlophiswang ka thoko hodimo ho dendrogram (Setshwantsho sa 1I). Tatellano e ncha e kentsoe polokelongtshedimosetsong ya NCBI mme e bitswa “Sclerotinia sclerotiorum – isolate YN-25” (nomoro ya kamohelo ya GenBank PV202792). Ho ka bonwa hore isolate 3 ke isolate e hlaselang ka ho fetisisa; ka hona, isolate ena e kgethilwe bakeng sa thuto ditekong tsohle tse latelang.
Tšebetso ea antibacterial ea diamine L-ornithine (Sigma-Aldrich, Darmstadt, Jeremane) maemong a fapaneng (12.5, 25, 50, 75, 100 le 125 mg/L) khahlanong le S. sclerotiorum isolate 3 e ile ea hlahlojoa ka vitro. Hoa hlokomeleha hore L-ornithine e bile le phello ea antibacterial 'me butle-butle ea thibela kholo ea radial ea S. sclerotiorum hyphae ka mokhoa o itšetlehileng ka tekanyo (Setšoantšo sa 2A, B). Maemong a holimo ka ho fetisisa a lekiloeng (125 mg/L), L-ornithine e bontšitse sekhahla se phahameng ka ho fetisisa sa thibelo ea kholo ea mycelial (99.62 ± 0.27%; Setšoantšo sa 2B), se neng se lekana le Rizolex-T ea fungicide ea khoebo (sekhahla sa thibelo 99.45 ± 0.39%; Setšoantšo sa 2C) maemong a holimo ka ho fetisisa a lekiloeng (10 mg/L), e bontšang katleho e tšoanang.
Setšoantšo sa 2. Mosebetsi oa L-ornithine oa antibacterial oa in vitro khahlanong le sclerotinia sclerotiorum. (A) Papiso ea mosebetsi oa antibacterial oa mahloriso a fapaneng a L-ornithine khahlanong le S. sclerotiorum le Rizolex-T (10 mg/L) ea fungicide ea khoebo. (B, C) Sekhahla sa thibelo (%) ea kholo ea mycelial ea S. sclerotiorum kamora kalafo ka mahloriso a fapaneng a L-ornithine (12.5, 25, 50, 75, 100 le 125 mg/L) kapa Rizolex-T (2, 4, 6, 8 le 10 mg/L), ka ho latellana. Litekanyetso li emela karolelano ea ± SD ea likopi tse hlano tsa baeloji (n = 5). Litlhaku tse fapaneng li bontša liphapang tsa lipalo-palo lipakeng tsa kalafo (p < 0.05). (D, E) Tlhahlobo ea regression ea mohlala oa probit oa L-ornithine le fungicide ea khoebo ea Rizolex-T, ka ho latellana. Mola wa ho kgutlisa mohlala wa probit o bontshwa e le mola o putswa o tiileng, mme karohano ya boitshepo (95%) e bontshwa e le mola o mofubedu o nang le di-dash.
Ho phaella moo, tlhahlobo ya probit regression e entswe mme dipolane tse tsamaellanang di bontshitswe ho Tafole ya 1 le Ditshwantsho tsa 2D,E. Ka bokhutshwane, boleng bo amohelehang ba leralla (y = 2.92x - 4.67) le dipalopalo tse amanang le tsona tsa bohlokwa (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 le p < 0.0001; Setshwantsho sa 2D) sa L-ornithine se bontshitse ts'ebetso e ntlafetseng ya antifungal kgahlanong le S. sclerotiorum ha e bapiswa le Rizolex-T ya sebolaya-dikokwana-hloko sa kgwebo (y = 1.96x - 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 le p < 0.0001) (Tafole ya 1).
Tafole ea 1. Boleng ba khatello ea halofo ea max ea thibelo (IC50) le IC99 (mg/l) ea L-ornithine le sebolaya-mafu sa khoebo sa "Rizolex-T" khahlanong le S. sclerotiorum.
Ka kakaretso, L-ornithine (250 mg/L) e fokoditse haholo ntshetsopele le boima ba hlobo e tšoeu dimeleng tsa dinawa tse tlwaelehileng tse phekotsweng ha e bapiswa le dimela tse tshwaeditsweng ke S. sclerotiorum (taolo; Setšoantšo sa 3A). Ka bokhutšoanyane, le hoja boima ba lefu la dimela tse tshwaeditsweng tse sa phekolwang bo ile ba eketseha butle-butle (52.67 ± 1.53, 83.21 ± 2.61, le 92.33 ± 3.06%), L-ornithine e fokoditse boima ba lefu (%) haholo nakong yohle ya teko (8.97 ± 0.15, 18.00 ± 1.00, le 26.36 ± 3.07) matsatsing a 7, 14, le a 21 kamora kalafo (dpt), ka ho latellana (Setšoantšo sa 3A). Ka ho tšoanang, ha limela tsa linaoa tse tšoaelitsoeng ke S. sclerotiorum li ne li phekoloa ka 250 mg/L L-ornithine, sebaka se tlas'a sekhahla sa tsoelo-pele ea lefu (AUDPC) se ile sa fokotseha ho tloha ho 1274.33 ± 33.13 taolong e sa phekoloang ho ea ho 281.03 ± 7.95, e neng e le tlase hanyane ho feta ea 50 mg/L Rizolex-T fungicide (183.61 ± 7.71; Setšoantšo sa 3B). Mokhoa o tšoanang o ile oa bonoa tekong ea bobeli.
Setšoantšo sa 3. Tšusumetso ea ts'ebeliso ea L-ornithine ka ntle ho nts'etsopele ea ho bola ho hosoeu ha linaoa tse tloaelehileng ho bakoang ke Sclerotinia sclerotiorum tlas'a maemo a greenhouse. (A) Mokokotlo oa tsoelo-pele ea lefu la hlobo e tšoeu ea linaoa tse tloaelehileng kamora kalafo ka 250 mg/L L-ornithine. (B) Sebaka se tlas'a mokokotlo oa tsoelo-pele ea lefu (AUDPC) oa hlobo e tšoeu ea linaoa tse tloaelehileng kamora kalafo ka L-ornithine. Boleng bo emela karolelano ea ± SD ea likopi tse hlano tsa baeloji (n = 5). Litlhaku tse fapaneng li bontša liphapang tse bohlokoa tsa lipalo-palo lipakeng tsa kalafo (p < 0.05).
Tšebeliso ea kantle ea 250 mg/L L-ornithine e ile ea eketsa bolelele ba semela butle-butle (Setšoantšo sa 4A), palo ea makala ka semela (Setšoantšo sa 4B), le palo ea makhasi ka semela (Setšoantšo sa 4C) kamora matsatsi a 42. Leha Rizolex-T (50 mg/L) ea sebolaya-mafu sa khoebo e bile le tšusumetso e kholo ho liparamente tsohle tsa phepo tse ithutiloeng, ts'ebeliso ea kantle ea 250 mg/L L-ornithine e bile le phello ea bobeli e kholo ha e bapisoa le taolo e sa phekoloang (Setšoantšo sa 4A–C). Ka lehlakoreng le leng, kalafo ea L-ornithine ha ea ka ea ba le tšusumetso e kholo holim'a litaba tsa li-pigment tsa photosynthetic chlorophyll a (Setšoantšo sa 4D) le chlorophyll b (Setšoantšo sa 4E), empa e ekelitse hanyane kakaretso ea carotenoid (0.56 ± 0.03 mg/g fr wt) ha e bapisoa le taolo e mpe (0.44 ± 0.02 mg/g fr wt) le taolo e ntle (0.46 ± 0.02 mg/g fr wt; Setšoantšo sa 4F). Ka kakaretso, liphetho tsena li bontša hore L-ornithine ha e na chefo ea limela tse phekotsoeng 'me e ka ba ea susumetsa kholo ea tsona.
Setšoantšo sa 4. Tšusumetso ea ts'ebeliso ea L-ornithine ea kantle ho litšobotsi tsa kholo le mebala ea photosynthetic ea makhasi a linaoa a tšoaelitsoeng ke Sclerotinia sclerotiorum tlas'a maemo a greenhouse. (A) Bolelele ba semela (cm), (B) Palo ea makala ka semela, (C) Palo ea makhasi ka semela, (D) Chlorophyll a (mg g-1 fr wt), (E) Chlorophyll b (mg g-1 fr wt), (F) Kakaretso ea carotenoid (mg g-1 fr wt). Boleng ke karolelano ea ± SD ea likopi tse hlano tsa baeloji (n = 5). Litlhaku tse fapaneng li bontša liphapang tse bohlokoa tsa lipalo-palo lipakeng tsa mekhoa ea phekolo (p < 0.05).
Sebakeng seo histochemical e leng ho sona, ho fumaneha ha mefuta ya oksijene e arabelang (ROS; e hlahiswang e le hydrogen peroxide [H2O2]) le di-free radical (tse hlahiswang e le di-anion tsa superoxide [O2•−]) ho senotse hore tshebediso ya kantle ya L-ornithine (250 mg/L) e fokoditse haholo ho bokellana ha H2O2 (96.05 ± 5.33 nmol.g−1 FW; Setshwantsho sa 5A) le O2•− (32.69 ± 8.56 nmol.g−1 FW; Setshwantsho sa 5B) ha ho bapiswa le ho bokellana ha dimela tse tshwaeditsweng tse sa phekolwang (173.31 ± 12.06 le 149.35 ± 7.94 nmol.g−1 FW, ka ho latellana) le dimela tse phekolwang ka 50 mg/L ya Rizolex-T e bolayang dikokwana-hloko tsa kgwebo (170.12 ± 9.50 le 157.00 ± 7.81 nmol.g−1 fr wt, ka ho latellana) ka lihora tse 72. Maemo a phahameng a H2O2 le O2•− a bokelletsoeng ka tlase ho hpt (Setšoantšo sa 5A, B). Ka mokhoa o ts'oanang, teko ea malondialdehyde (MDA) e thehiloeng ho TCA e bontšitse hore limela tsa linaoa tse tšoaelitsoeng ke S. sclerotiorum li bokelletse maemo a holimo a MDA (113.48 ± 10.02 nmol.g fr wt) makhasing a tsona (Setšoantšo sa 5C). Leha ho le joalo, ts'ebeliso ea kantle ea L-ornithine e fokolitse haholo peroxidation ea lipid joalo ka ha ho pakoa ke phokotso ea litaba tsa MDA limela tse phekoloang (33.08 ± 4.00 nmol.g fr wt).
Setšoantšo sa 5. Tšusumetso ea ts'ebeliso ea L-ornithine ea kantle ho matšoao a maholo a khatello ea oxidative le mekhoa ea ts'ireletso ea antioxidant e seng ea enzymatic makhasing a linaoa a tšoaelitsoeng ke S. sclerotiorum ka mor'a lihora tse 72 kamora tšoaetso tlas'a maemo a greenhouse. (A) Hydrogen peroxide (H2O2; nmol g−1 FW) ka 72 hpt, (B) superoxide anion (O2•−; nmol g−1 FW) ka 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) ka 72 hpt, (D) li-phenol tse qhibilihang ka botlalo (mg GAE g−1 FW) ka 72 hpt, (E) li-flavonoid tse qhibilihang ka botlalo (mg RE g−1 FW) ka 72 hpt, (F) li-amino acid tse lokolohileng ka botlalo (mg g−1 FW) ka 72 hpt, le (G) litaba tsa proline (mg g−1 FW) ka 72 hpt. Boleng bo emela karolelano ea ± ho kheloha ho tloaelehileng (karolelano ea ± SD) ea likopi tse 5 tsa baeloji (n = 5). Litlhaku tse fapaneng li bontša liphapang tse bohlokoa tsa lipalo-palo lipakeng tsa mekhoa ea phekolo (p < 0.05).