Nyatia nye numekuku ƒe tanya si nye “Mɔli ƒe tsitretsitsi ɖe dɔlékuiwo kple nudzodzoeviwo ŋu ƒe nyonyome” ƒe akpa aɖe, kpɔ nyati 5 la katã
Nusi hea fungal plant disease necrosis Sclerotinia sclerotiorum (Lib.) de Bary zãa mɔnu si le ɖoɖo vovovowo nu tsɔ xɔa dɔ le numiemie vovovo siwo xɔa dɔlékuiwo ŋu. Numekuku sia do susua ɖa be woazã diamine L-ornithine, si nye amino acid si menye protein o si doa ŋusẽ amino acid vevi bubuwo ƒe wɔwɔme, abe dɔdzikpɔkpɔmɔnu bubu ene be woatsɔ ado Phaseolus vulgaris L. ƒe molecule, ŋutilã kple nugbagbeŋusẽ ƒe ŋuɖoɖo ɖe aŋɔ ɣi si Pseudomonas sclerotiorum hena vɛ ŋu ɖe ŋgɔ. Dodokpɔ siwo wowɔ le vitro ɖee fia be L-ornithine xe mɔ ɖe S. pyrenoidosa ƒe mycelial tsitsi nu vevie le mɔ si nɔ te ɖe agbɔsɔsɔme si wozã dzi nu. Gawu la, ate ŋu aɖe aŋɔ ɣi ƒe sesẽ dzi akpɔtɔ ŋutɔ le dzoxɔxɔ ƒe nɔnɔmewo me. Gawu la, L-ornithine do ŋusẽ numiemie siwo ŋu wowɔ atike ɖo la ƒe tsitsi, si ɖee fia be L-ornithine ƒe agbɔsɔsɔ si wodo kpɔ la menye numiemie siwo ŋu wowɔ atike ɖo o. Tsɔ kpe ɖe eŋu la, L-ornithine na antioxidants siwo menye enzyme o (phenolics kple flavonoids siwo katã me wodea tsii) kple enzymatic antioxidants (catalase (CAT), peroxidase (POX), kple polyphenol oxidase (PPO)) ƒe ɖeɖefia dzi ɖe edzi, eye wòdzi domenyiŋusẽfianu etɔ̃ siwo do ƒome kple antioxidant (PvCAT1, PvSOD, kple PvGR) ƒe ɖeɖefia ɖe edzi. Gawu la, in silico numekuku ɖee fia be protein si wosusu be enye oxaloacetate acetylhydrolase (SsOAH) le S. sclerotiorum ƒe domenyiŋusẽfianu me, si ɖi oxaloacetate acetylhydrolase (SsOAH) protein siwo le Aspergillus fijiensis (AfOAH) kple Penicillium sp. (PLOAH) le dɔwɔwɔ ŋuti numekuku, domenyinyi siwo wodzra ɖo, kple anyigba ƒe nɔnɔme gome. Enyo be míade dzesii be L-ornithine tsɔtsɔ kpe ɖe mɔli dextrose broth (PDB) medium ŋu ɖe SsOAH domenyiŋusẽfianu ƒe ɖeɖefia dzi kpɔtɔ ŋutɔ le S. sclerotiorum mycelia me. Nenema ke L-ornithine zazã le gota ɖe SsOAH domenyiŋusẽfianu ƒe ɖeɖefia dzi kpɔtɔ ŋutɔ le fungi mycelia siwo woƒo ƒu tso numiemie siwo ŋu wowɔ atike ɖo me. Mlɔeba la, L-ornithine zazã ɖe oxalic acid ƒe dodo dzi kpɔtɔ ŋutɔ le PDB medium kple aŋgba siwo ŋu dɔlélea le siaa me. Le nyataƒoƒo me la, L-ornithine wɔa akpa vevi aɖe le redox ƒe nɔnɔmea me léle ɖe asi me eye wòdoa numiemie siwo ŋu dɔlélea le ƒe ametakpɔkpɔ ƒe ŋuɖoɖo ɖe ŋgɔ hã. Numekuku sia me tsonu ate ŋu akpe ɖe ame ŋu le mɔnu yeye siwo megblẽa nu le nutome ŋu o toto vɛ be woatsɔ aɖu aŋɔ ɣiwo dzi eye woaɖe ŋusẽ si wòkpɔna ɖe gbewo wɔwɔ kple nuku bubuwo dzi dzi akpɔtɔ.
Aŋɔ ɣi, si tso lãvi si woyɔna be necrotrophic fungus Sclerotinia sclerotiorum (Lib.) de Bary gbɔ la nye dɔléle si gblẽa nu ŋutɔ, si ɖea nukuwo ƒe ŋeŋe dzi kpɔtɔna, si nye afɔku gã aɖe na xexeame katã ƒe gbe (Phaseolus vulgaris L.) wɔwɔ (Bolton et al., 2006). Sclerotinia sclerotiorum nye fungi numiemie ƒe dɔlékui siwo dzi ɖuɖu sesẽ wu la dometɔ ɖeka, eye numiemie ƒomevi siwo wu 600 ƒe amedzroxɔƒe keke ta eye ŋutete le esi be wòawɔ macerate kabakaba le amedzroa ƒe lãkusiwo ŋu le mɔ si menye tẽ o nu (Liang kple Rollins, 2018). Le nɔnɔme madeamedziwo me la, etoa eƒe agbenɔnɔ ƒe akpa vevi aɖe me, enɔa alɔ̃ dɔm hena ɣeyiɣi didi abe xɔ yibɔ, sesẽ, si le abe nuku ene si woyɔna be ‘sclerotia’ le anyigba me alo abe tsitsi ɣi, si me fu le le mycelium alo stem pith me le numiemie siwo ŋu dɔlélea le me (Schwartz et al., 2005). S. sclerotiorum te ŋu wɔa sclerotia, si wɔnɛ be wònɔa agbe le agble siwo me dɔlélea le me ɣeyiɣi didi eye wònɔa anyi le dɔléleɣi (Schwartz et al., 2005). Nunyiame geɖe le Sclerotia me, ateŋu anɔ anyigba me ɣeyiɣi didi, eye wònye atike gbãtɔ na dɔlékui siwo kplɔe ɖo (Schwartz et al., 2005). Le nɔnɔme nyuiwo me la, sclerotia miena eye wowɔa spores siwo le ya me siwo ateŋu axɔ numiemiea ƒe akpa siwo katã le anyigba dzi, siwo dometɔ aɖewoe nye seƒoƒowo, kewo, alo aŋgbawo gake menye woawo koe o (Schwartz et al., 2005).
Sclerotinia sclerotiorum zãa mɔnu si le ɖoɖo vovovowo nu tsɔ xɔa dɔ le numiemie siwo xɔe ŋu, si lɔ nudzɔdzɔ vovovo siwo wɔ ɖeka tso sclerotial ƒe miemi dzi va ɖo dzesiwo ƒe dzɔdzɔ dzi ɖe eme. Le gɔmedzedzea me la, S. sclerotiorum wɔa spores siwo le tsia dzi (siwo woyɔna be ascospores) tso nusiwo le abe lãmi ene siwo woyɔna be apothecia me, siwo va zua ya me eye wozua sclerotia siwo meʋãna o le numiemie ƒe gbeɖuɖɔ siwo ŋu dɔlélea le dzi (Bolton et al., 2006). Emegbe lãvi la ɖea oxalic acid, si nye dɔlékuiwutike, ɖa be wòakpɔ numiemie ƒe lãmenugbagbeviwo ƒe gli ƒe pH dzi, ado enzymatic ƒe gbegblẽ kple lãmenugbagbeviwo ƒe amedzidzedze ɖe ŋgɔ (Hegedus kple Rimmer, 2005), eye wòaxe mɔ ɖe numiemie si xɔe ƒe oxidative burst nu. Acidification ƒe ɖoɖo sia gbɔdzɔa numiemie ƒe lãmenugbagbeviwo ƒe gli, si naa nɔnɔme nyui aɖe na fungal cell wall degrading enzymes (CWDEs) ƒe dɔwɔwɔ nyuie eye wòwɔa dɔ nyuie, si wɔnɛ be dɔlékui la te ŋu ɖua ŋutilã me mɔxenu dzi eye wògena ɖe amedzroa ƒe lãkusiwo me (Marciano et al., 1983). Ne wonya ge ɖe S. sclerotiorum me ko la, eɖea CWDE geɖe, abe polygalacturonase kple cellulase ene, si nana wòkakana ɖe lãkusi siwo ŋu dɔlélea le me bɔbɔe eye wònana lãmenugbagbeviwo ƒe kuku. Abi kple hyphal mats ƒe ŋgɔyiyi hea dzesi siwo ɖe dzesi le aŋɔ ɣi ŋu vɛ (Hegedus kple Rimmer, 2005). Le ɣeyiɣi ma ke me la, numiemie siwo xɔa dɔlékuiwo dea dzesi molecule ƒe nɔnɔme siwo do ƒome kple dɔlékuiwo (PAMPs) to pattern recognition receptors (PRRs) dzi, si hea dzesiwo ƒe nudzɔdzɔ vovovowo vɛ siwo nana ametakpɔnu ƒe ŋuɖoɖowo wɔa dɔ mlɔeba.
Togbɔ be wodze agbagba ƒe bla nanewoe nye sia be woaɖu dɔlélewo dzi hã la, dɔlékuiwutike si te ŋu nɔa te ɖe dɔlékuiwo nu nyuie ƒe anyimanɔmanɔ gakpɔtɔ le gbe me abe alesi wòle le agblemenuku bubu siwo wodzrana me ene, le dɔlékuia ƒe dɔlélenutsiŋutete, eƒe agbetsitsi, kple eƒe tɔtrɔ ɖe nɔnɔmewo ŋu ta. Eyata dɔlélewo dzikpɔkpɔ sesẽ ŋutɔ eye wòbia aɖaŋu si wowɔ ɖekae, si me go geɖe le, si me dekɔnuwo ƒe nuwɔnawo, nugbagbewo dzi ɖuɖu, kple atike siwo wua fungi ƒe ƒuƒoƒo le (O’Sullivan et al., 2021). Atikewo zazã atsɔ aɖu aŋɔ ɣi dzie wɔa dɔ wu elabena ne wozã fungicides nyuie eye wozãe le ɣeyiɣi nyuitɔ dzi la, ate ŋu aɖu dɔlélea ƒe kaka dzi nyuie, aɖe dɔlékuiwo ƒe sesẽ dzi akpɔtɔ, eye wòaɖe nukuwo ƒe bublu dzi akpɔtɔ. Ke hã, fungicides zazã fũ kple ŋuɖoɖo ɖe fungicide ŋu akpa ateŋu ana S. sclerotiorum ƒe ƒomevi siwo te ŋu nɔa te ɖe dɔlékuiwo nu nado eye wòakpɔ ŋusẽ gbegblẽ ɖe nugbagbe siwo menye taɖodzinu o, anyigba ƒe lãmesẽ, kple tsi ƒe nyonyome dzi (Le Cointe et al., 2016; Ceresini et al., 2024). Eyata mɔnu bubu siwo megblẽa nu le nutome ŋu o didi va zu nu vevitɔ kekeake.
Polyamines (PAs), abe putrescine, spermidine, spermine, kple cadaverine, ateŋu anye mɔnu bubu siwo ŋugbe wodo ɖe numiemie ƒe dɔlékui siwo tso anyigba me ŋu, si ana woaɖe atike siwo me afɔku le siwo wua fungi zazã dzi akpɔtɔ keŋkeŋ alo woƒe akpa aɖe (Nehela et al., 2024; Yi et al., 2024). Le numiemie siwo kɔkɔ wu me la, PAwo kpɔa gome le ŋutilã ƒe dɔwɔwɔ geɖe me siwo dometɔ aɖewoe nye, gake menye woawo koe o, lãmenugbagbeviwo ƒe mama, vovototodedeameme, kple ŋuɖoɖo ɖe abiotic kple biotic nuteɖeamedziwo ŋu (Killiny kple Nehela, 2020). Woateŋu awɔ dɔ abe antioxidants ene, akpe ɖe wo ŋu woaɖe oxygen ƒomevi siwo wɔa dɔ (ROS), alé redox homeostasis me ɖe asi (Nehela kple Killiny, 2023), ahe domenyiŋusẽfianu siwo do ƒome kple ametakpɔkpɔ (Romero et al., 2018), aɖɔ lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe mɔnu vovovowo ɖo (Nehela kple Killiny, 2023), atrɔ asi le domenyiŋusẽfianu siwo le lãmenugbagbeviwo me (Nehela kple Killiny, 2023) ŋu. 2019), ɖoa dɔlélenutsiŋutete si woxɔ le ɖoɖo nu (SAR), eye woɖɔa numiemie kple dɔlékui ƒe kadodo ɖo (Nehela kple Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). Enyo be míade dzesii be mɔnu tɔxɛwo kple akpa siwo PAwo wɔna le numiemiewo takpɔkpɔ me toa vovo le numiemie ƒomeviwo, dɔlékuiwo, kple nutome ƒe nɔnɔmewo nu. PA si bɔ wu le numiemiewo me la nye biosynthesized tso polyamine vevi L-ornithine (Killiny kple Nehela, 2020).
L-ornithine wɔa akpa geɖe le numiemiewo ƒe tsitsi kple woƒe tsitsi me. Le kpɔɖeŋu me, numekuku siwo wowɔ va yi ɖee fia be le bli (Oryza sativa) me la, ornithine ateŋu ado ƒome kple nitrogen gbugbɔgawɔ (Liu et al., 2018), bli ƒe kutsetse, eƒe nyonyome kple ʋeʋẽ (Lu et al., 2020), kple tsi ƒe nuteɖeamedzi ƒe ŋuɖoɖo (Yang et al., 2000). Gawu la, L-ornithine zazã le gota na kuɖiɖi dzi ɖuɖu dzi ɖe edzi ŋutɔ le sukli-ku (Beta vulgaris) me (Hussein et al., 2019) eye wòɖe dze ƒe nuteɖeamedzi dzi kpɔtɔ le sabala (Allium Cepa) (Çavuşoǧlu kple Çavuşoǧlu, 2021) kple cashew (Anacardium occidentale) me . numiemiewo (da Rocha kple ehatiwo, 2012). Akpa si L-ornithine ate ŋu awɔ le abiotic stress defense me ate ŋu atso eƒe gomekpɔkpɔ le proline ƒe ƒuƒoƒo me le numiemie siwo ŋu wowɔ atike ɖo me gbɔ. Le kpɔɖeŋu me, woka nya ta va yi be domenyiŋusẽfianu siwo do ƒome kple proline ƒe asitɔtrɔ le lãmenugbagbeviwo ŋu, abe ornithine delta aminotransferase (delta-OAT) kple proline dehydrogenase (ProDH1 kple ProDH2) domenyiŋusẽfianuwo ene, kpɔa gome le Nicotiana benthamiana kple Arabidopsis thaliana takpɔkpɔ me tso Pseudomonas syringae ƒomevi siwo menye amedzro o (Senthil-Kumar kple... Mysore, ƒe 2012). Le go bubu me la, fungal ornithine decarboxylase (ODC) hiã na dɔlékuiwo ƒe tsitsi (Singh et al., 2020). Fusarium oxysporum ƒe ODC ƒe taɖodzinu f. sp. lycopersici to host-induced gene silencing (HIGS) dzi na tomatos numiemiewo ƒe tsitretsitsi ɖe Fusarium ƒe ƒuƒu ŋu dzi ɖe edzi ŋutɔ (Singh et al., 2020). Gake womesrɔ̃ nu tso akpa si ornithine si tso gota ate ŋu awɔ ɖe nugbagbewo ƒe nuteɖeamedziwo abe phytopathogens ene ŋu nyuie o. Vevietɔ wu la, womeku nu me le ŋusẽ si ornithine kpɔna ɖe dɔlélenutsiŋutete dzi kple nugbagbeŋusẽ kple ŋutilã me nudzɔdzɔ siwo do ƒome kplii ŋu le akpa gãtɔ me o.
S. sclerotiorum ƒe dɔlékui si le mɔli me ƒe sesẽ gɔmesese le vevie na mɔxexeɖedɔléle nu ƒe mɔnu nyuiwo toto vɛ. Le numekuku sia me la, míeɖoe be míade dzesi akpa si diamine L-ornithine ate ŋu awɔ abe nu vevi aɖe si ana mɔli numiemiewo ƒe ametakpɔkpɔ ƒe mɔnuwo kple tsitretsitsi ɖe Sclerotinia sclerotiorum dɔlékui ŋu nadzi ɖe edzi me. Míesusui be, tsɔ kpe ɖe numiemie siwo ŋu dɔlélea le ƒe ametakpɔkpɔ ƒe ŋuɖoɖowo dodo ɖe ŋgɔ ŋu la, L-ornithine hã wɔa akpa vevi aɖe le redox ƒe nɔnɔmea me léle ɖe asi me. Míeɖoe be nusiwo ate ŋu agblẽ le L-ornithine ŋu la do ƒome kple enzymatic kple non-enzymatic antioxidant defense mechanisms ƒe ɖoɖowɔwɔ kple nudede fungal pathogenicity/virulence factors kple protein siwo do ƒome kplii. L-ornithine ƒe dɔwɔwɔ eve sia na wònye amesi ŋugbe wodo na aɖaŋu si anɔ anyi ɖaa be woatsɔ aɖe aŋɔ ɣi ƒe ŋusẽkpɔɖeamedzi dzi akpɔtɔ eye wòana mɔli ƒe agblemenuku siwo bɔ ƒe tsitretsitsi ɖe fungi dɔlékui sesẽ sia ŋu nadzi ɖe edzi. Numekuku si míele wɔwɔm fifia me tsonu ate ŋu akpe ɖe ame ŋu le mɔnu yeye siwo megblẽa nu le nutome ŋu o toto vɛ be woatsɔ aɖu aŋɔ ɣiwo dzi eye woaɖe ŋusẽ si wòkpɔna ɖe mɔli wɔwɔ dzi dzi akpɔtɔ.
Le numekuku sia me la, wozã gbe bɔbɔe ƒomevi aɖe si woate ŋu adzra bɔbɔe, si nye Giza 3 (Phaseolus vulgaris L. cv. Giza 3), abe dodokpɔnu ene. Wona nuku siwo le lãmesẽ me dɔmenyotɔe tso Mɔli Ŋuti Numekuku Dɔwɔƒe, Agblemenukuwo Ŋuti Numekuku Dɔwɔƒe (FCRI), Agbledede Ŋuti Numekuku Dɔwɔƒe (ARC), Egipte gbɔ. Woƒã nuku atɔ̃ ɖe plastic zewo (ememe ƒe didime 35 cm, goglome 50 cm) siwo me wotsɔ anyigba si me S. sclerotiorum dɔlékui le yɔ fũ le dzoxɔxɔ ƒe nɔnɔmewo me (25 ± 2 °C, fafa si sɔ 75 ± 1%, 8 h kekeli/16 h viviti). Le ŋkeke 7–10 le nukuwo ƒaƒã (DPS) megbe la, woɖea nukuawo ɖa be nuku eve siwo tsi ɖekae kple aŋgba etɔ̃ siwo keke bliboe koe susɔ ɖe ze ɖesiaɖe me. Wodea tsi numiemie siwo katã le ze me zi ɖeka le kwasiɖa eve ɖesiaɖe me eye wodoa vi ɣleti sia ɣleti le agbɔsɔsɔ si wokafu na ƒomevi si wona wo nu.
Be woawɔ L-ornithinediamine (si wogayɔna be (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Germany) ƒe agbɔsɔsɔme 500 mg/L la, wotsɔa 50 mg dea tsi si me dɔlékui mele o ƒe mL 100 me gbã. Emegbe woɖea tsi si le stock solution la me eye wozãnɛ le dodokpɔ siwo kplɔe ɖo me. Kpuie ko la, wodo L-ornithine ƒe agbɔsɔsɔ ade siwo kplɔ wo nɔewo ɖo (12.5, 25, 50, 75, 100, kple 125 mg/L) kpɔ le vitro. Gakpe ɖe eŋu la, wozã tsi si woɖe tso tsi si me dɔlékui mele o abe atike si woatsɔ axe mɔ ɖe dɔlékuiwo nu (Mock) eye wozã fungicide si wodzrana “Rizolex-T” 50% si woate ŋu atsɔ tsii (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Egypt) abe atike si woatsɔ axe mɔ ɖe fungi nu nyuie ene. Wodo fungicide si wodzrana “Rizolex-T” kpɔ le vitro le agbɔsɔsɔ atɔ̃ me (2, 4, 6, 8 kple 10 mg/L).
Woxɔ gbegãdzi ƒe ke kple aŋgba siwo bɔ siwo ɖea aŋɔ ɣi ƒe dzesi tɔxɛwo fiana ƒe kpɔɖeŋuwo (dɔlékuiwo ƒe agbɔsɔsɔ: 10–30%) tso agble siwo wodzrana le asitsaƒewo me. Togbɔ be wode dzesi numiemie siwo ŋu dɔlélea le ƒe akpa gãtɔ to ƒomevi/ƒomevi (ƒomevi si woate ŋu axɔ le asitsatsa me Giza 3) dzi hã la, bubuwo, vevietɔ esiwo wokpɔ tso nutoa me asiwo me, nye ƒomevi siwo womenya o. Wotsɔ sodium hypochlorite solution 0.5% tsɔ wu dɔlékui siwo woƒo ƒu la gbã hena aɖabaƒoƒo 3, emegbe wotsɔ tsi si me dɔlékui mele o klɔ wo zi geɖe eye wotsɔ filter paper si me dɔlékui mele o tutu wo woƒu be woaɖe tsi si sɔ gbɔ ɖe wo me. Emegbe wolã ŋutinu siwo ŋu dɔlélea le la ɖe akpa suewo me tso titina lãkusi me (le lãkusi siwo le lãmesẽ me kple esiwo ŋu dɔlélea le dome), wode wo ɖe mɔli dextrose agar (PDA) me eye wotsɔ wo de dzoxɔxɔ 25 ± 2 °C me kple kekeli ƒe tsatsam gaƒoƒo 12/gaƒoƒo 12 hena ŋkeke 5 be wòaʋã sclerotia ƒe wɔwɔ. Wozã mycelial tip mɔnu hã tsɔ kɔ fungi siwo woɖe ɖe vovo tso dekɔnu siwo wotsaka alo esiwo me dɔlékuiwo le me ŋu. Wonɔ te ɖe eƒe dekɔnu ƒe nɔnɔme ƒe nɔnɔmewo dzi de dzesi fungi si woɖe ɖe vovo si ŋu wokɔ la gbã eye emegbe woɖo kpe edzi be enye S. sclerotiorum le nusuekpɔmɔ̃ ƒe nɔnɔmewo nu. Mlɔeba la, wodo lã siwo katã woɖe ɖe vovo siwo ŋu wokɔ la kpɔ be dɔlékuiwo ate ŋu ahe wo vɛ hã le gbe si bɔ si woate ŋu axɔ bɔbɔe si nye Giza 3 dzi be woawɔ ɖe Koch ƒe nukpɔsusuwo dzi.
Gakpe ɖe eŋu la, wogaɖo kpe S. sclerotiorum si woɖe ɖe vovo (si woɖe ɖe vovo #3) si xɔa ame me wu dzi le ememe transcribed spacer (ITS) ƒe ɖoɖowɔwɔ dzi abe alesi White et al., 1990 ɖɔe ene; Baturo-Ciesniewska et al., 2017. Kpuie ko la, wode lã siwo woɖe ɖe vovo la ɖe mɔli dextrose broth (PDB) me eye wotsɔ wo de dzoxɔxɔ 25 ± 2 °C me hena ŋkeke 5–7. Emegbe woƒo fungi mycelium nu ƒu, woɖee to cheesecloth me, woklɔe zi eve kple tsi si me dɔlékui mele o, eye wotsɔ filter paper si me dɔlékui mele o ƒu gbe. Wozã Quick-DNATM Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah kple ehatiwo, 2022, 2024) tsɔ ɖe domenyiŋusẽfianu ƒe DNA ɖe vovo. Emegbe wodo ŋusẽ ITS rDNA nutoa to primer eve tɔxɛ ITS1/ITS4 (TCCGTAGGTGAACCTGCGG TCCTCCGCTTATTGATATGC; lolome si wokpɔ mɔ na: 540 bp) zazã me ( Baturo-Ciesniewska et al., 2017 ). Wotsɔ PCR-nu siwo ŋu wokɔ la ɖo ɖa be woawɔ ɖoɖo ɖe wo nɔewo yome (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). Wotsɔ Sanger ƒe ɖoɖowɔwɔ ƒe mɔnu ɖo ITS rDNA ƒe ɖoɖowo ɖe ɖoɖo nu le mɔ eve nu. Emegbe wotsɔ nyabiase siwo woƒo ƒu la sɔ kple nyatakaka yeye siwo le GenBank kple Dukɔa ƒe Nu gbagbewo Ŋuti Nyatakaka Dɔwɔƒe (NCBI, http://www.ncbi.nlm.nih.gov/gene/) to BLASTn kɔmpiuta dɔwɔɖoɖo zazã me. Wotsɔ biabia ƒe ɖoɖoa sɔ kple S. sclerotiorum ƒomevi/ɖe vovo 20 bubu siwo woxɔ tso nyatakaka yeyetɔ kekeake siwo le NCBI GenBank (Kpekpeɖeŋu Tabla S1) me to ClustalW zazã me le Molecular Evolutionary Genetics Analysis Package (MEGA-11; version 11) (Kumar et al., 2024). Wowɔ nɔnɔmetɔtrɔ ƒe numekuku to mɔnu si nye maximum likelihood kple general time-reversible nucleotide substitution model (Nei kple Kumar, 2000) zazã me. Woɖe ati si ƒe log-likelihood sɔ gbɔ wu la fia. Wotiaa ati gbãtɔ na heuristic didi to ati si ƒe log-likelihood si lolo wu la tiatia me le neighbor-joining (NJ) tree (Kumar et al., 2024) kple maximum parsimony (MP) tree dome. Wozã didime ƒe matriki si wobu akɔnta le ɣeyiɣi ƒe tɔtrɔ ƒe kpɔɖeŋu si wozãna le mɔ gbadza nu zazã me tsɔ tu NJ ati la (Nei kple Kumar, 2000).
Woto agar kaka mɔnu dzi kpɔ L-ornithine kple dɔlékuiwutike “Rizolex-T” ƒe dɔlékuiwutike ƒe dɔwɔwɔ le vitro. Mɔnu: Tsɔ L-ornithine ƒe stock solution (500 mg/L) ƒe agbɔsɔsɔ si sɔ eye nàtsakae nyuie kple PDA nunyiame ƒe ʋeʋẽ lilili 10 atsɔ adzra tsi siwo ƒe agbɔsɔsɔme mamlɛtɔ nye 12.5, 25, 50, 75, 100 kple 125 mg/L ɖo. Wozã fungicide “Rizolex-T” (2, 4, 6, 8 kple 10 mg/L) kple tsi si me dɔlékui mele o ƒe agbɔsɔsɔ atɔ̃ abe atike si woatsɔ axe mɔ ɖe enu ene. Esi nusi wotsɔ wɔe la sesẽ vɔ la, wotsɔ Sclerotinia sclerotiorum culture ƒe mycelial plug si wodzra yeyee, si ƒe didime nye mm 4, yi Petri-gba la titina eye wode agble ɖe edzi le 25±2°C vaseɖe esime mycelium la tsyɔ Petri-nuɖugba bliboa dzi, eye le ema megbe la, woŋlɔ fungi ƒe tsitsi ɖi. Bu akɔnta le S. sclerotiorum ƒe tsitsi le radial ƒe mɔxexeɖedɔléle nu ƒe alafa memamã ŋu to sɔsɔŋusẽ 1 zazã me:
Wogbugbɔ do dodokpɔa wɔ zi eve, eye wotsɔ nugbagbe ade siwo wogbugbɔ wɔ na ƒuƒoƒo ɖesiaɖe si dzi woɖu/dodokpɔ kple ze atɔ̃ (numiemie eve le ze ɖesiaɖe me) na nugbagbewo ƒe gbugbɔgawɔ ɖesiaɖe. Woda nugbagbewo ƒe gbugbɔgawɔ ɖesiaɖe kpɔ zi eve (mɔ̃ɖaŋununya ƒe gbugbɔgawɔ eve) be woakpɔ egbɔ be dodokpɔa me tsonuawo de pɛpɛpɛ, kakaɖedzi le wo ŋu eye woate ŋu agbugbɔ wo awɔ. Gakpe ɖe eŋu la, wozã probit regression analysis tsɔ bu akɔnta le mɔxexeɖedɔléle ƒe agbɔsɔsɔme si sɔ gbɔ wu afã (IC50) kple IC99 (Prentice, 1976).
Be woada L-ornithine ƒe ŋutete kpɔ le dzoxɔxɔ ƒe nɔnɔmewo me la, wowɔ ze dodokpɔ eve siwo kplɔ wo nɔewo ɖo. Kpuie ko la, wotsɔ anyikpe-ke si me dɔlékui mele o (3:1) yɔ zewo me eye wotsɔ S. sclerotiorum ƒe dekɔnu si wodzra ɖo yeyee do atike na wo. Gbã la, wode S. sclerotiorum si woɖe ɖe vovo si xɔa ame me wu (si woɖe ɖe vovo #3) to sclerotium ɖeka lãɖeɖe ɖe afã me, tsɔe ɖo ŋkume ɖe PDA dzi eye wotsɔe de dzoxɔxɔ 25°C me le viviti me ɣesiaɣi (gaƒoƒo 24) hena ŋkeke 4 be wòaʋã mycelial ƒe tsitsi. Emegbe woɖe agar plug ene siwo ƒe didime nye mm 5 tso ŋgɔgbekpa dzi eye wotsɔ lu kple bli ƒe ʋeʋẽ si me dɔlékui mele o ƒe g 100 de wo me (1:1, v/v) eye wotsɔ flaskwo katã de dzoxɔxɔ 25 ± 2 °C me le 12 h kekeli/12 h viviti ƒe tsatsam me hena ŋkeke 5 be woaʋã sclerotia ƒe wɔwɔ. Wotsaka nusiwo le nugoeawo katã me nyuie be woakpɔ egbɔ be wowɔ ɖeka hafi tsɔ anyigba kpe ɖe eŋu. Emegbe wotsɔa colonizing bran tsakatsaka g 100 dea ze ɖesiaɖe me be woakpɔ egbɔ be dɔlékuiwo sɔ gbɔ ɖe wo me ɣesiaɣi. Wode tsi ze siwo wodo atike na la be fungiwo natsi eye wotsɔ wo de dzoxɔxɔ ƒe nɔnɔmewo me ŋkeke 7 sɔŋ.
Emegbe woƒãa Giza 3 ƒomevi la ƒe nuku atɔ̃ ɖe ze ɖesiaɖe me. Le ze siwo wotsɔ L-ornithine kple fungicide Rizolex-T da dɔe gome la, wotsɔ nuku siwo me dɔlékui mele o de tsi si me atike eveawo le me gaƒoƒo eve gbã eye IC99 ƒe agbɔsɔsɔ mamlɛtɔ anɔ abe 250 mg/L kple 50 mg/L ene, eye emegbe wotsɔ ya ƒu gbe gaƒoƒo ɖeka hafi woƒã wo. Le go bubu me la, wotsɔ nukuawo de tsi si me dɔlékui mele o me be woatsɔ axe mɔ ɖe nukuawo nu. Le ŋkeke 10 megbe, hafi woade tsi wo zi gbãtɔ la, woɖea nukuawo ɖa, eye nuku eve siwo le dzadzɛ koe susɔ ɖe ze ɖesiaɖe me. Gakpe ɖe eŋu la, be woakpɔ egbɔ be woxɔ S. sclerotiorum la, wotsɔ lãgbalẽgolo si me dɔlékui mele o lã bean numiemie ƒe ke siwo le tsitsi ƒe afɔɖeɖe ɖeka me (ŋkeke 10) le teƒe vovovo eve eye wotsɔ colonizing bran tsakatsaka si ade 0.5 g de abi ɖesiaɖe me, eye wotsɔ fafa gã aɖe kplɔe ɖo be wòaʋã dɔlékui kple dɔléle ƒe dzɔdzɔ le numiemie siwo katã wodo atike na me. Wode abi numiemie siwo dzi woɖuna ŋu nenema ke eye wotsɔ aɖuɖɔ si me dɔlékui mele o, si wometsɔ lãgbalẽ wɔe o ƒe tsakatsaka si sɔ (0.5 g) de abi la me eye wolée ɖe te le fafa gã me be woatsɔ awɔ nutoa me ƒe kpɔɖeŋu hena dɔléle ƒe dzɔdzɔ eye woakpɔ egbɔ be wowɔ ɖeka le atikewɔhawo dome.
Atikewɔwɔ ƒe mɔnu: Wotsɔ L-ornithine (250 mg/l) alo fungicide Rizolex-T (50 mg/l) ƒe tsi 500 ml de tsi gbe ƒe nukuwo to tsidede anyigbaa me, emegbe wogbugbɔ trɔ asi le eŋu zi etɔ̃ le ŋkeke 10 ƒe domedome. Wotsɔ tsi si woɖe tso tsi si me dɔlékui mele o ml 500 de tsi amesiwo wotsɔ placebo da dɔe lae. Wowɔ dɔdamɔnuawo katã le dzoxɔxɔ ƒe nɔnɔmewo me (25 ± 2°C, 75 ± 1% ƒe fafa si sɔ, kple fotoɖeɣi si nye 8 h kekeli/16 h viviti). Wode tsi zeawo katã kwasiɖa eve ɖesiaɖe eye wotsɔa NPK ami si da sɔ (20-20-20, si me sulfur kple TE microelements 3.6% le; Zain Seeds, Egypt) le agbɔsɔsɔ si nye 3–4 g/l me to aŋgbawo sisi me le aɖaŋuɖoɖo siwo woɖo na ƒomevi tɔxɛa kple ewɔla ƒe mɔfiamewo nu. Negbe ɖe wogblɔe bubui ko la, woƒo aŋgba tsitsi siwo keke bliboe (aŋgba 2 lia kple 3 lia tso etame) nu ƒu tso nugbagbe ƒe aŋgba ɖesiaɖe si wogbugbɔ wɔ le 72 h le atikewɔwɔ vɔ megbe (hpt), wowɔ wo ɖekae, woƒo wo nu ƒu eye wodzra wo ɖo ɖe -80 °C hena numekuku bubuwo siwo dometɔ aɖewoe nye, gake menye woawo koe o, in situ histochemical localization of oxidative stress indicators, lipid peroxidation, enzymatic and non-enzymatic antioxidants kple domenyiŋusẽfianu ƒe ɖeɖefia.
Woda aŋɔ ɣi ƒe dɔlékui ƒe sesẽme kpɔ kwasiɖa sia kwasiɖa le ŋkeke 21 le atike dodo vɔ megbe (dpi) to dzidzenu si nye 1–9 (Kpekpeɖeŋu Tabla S2) si wotu ɖe Petzoldt kple Dickson ƒe dzidzenu (1996) si Teran et al. (ƒe 2006). Kpuie ko la, wodo gbegãdzi numiemiewo ƒe kewo kple alɔwo kpɔ tso afisi wodo atike na wo le be woakplɔ abiawo ƒe ŋgɔyiyi le lãkusiwo kple lãkusiwo dome ɖo. Emegbe wodzidze abi la ƒe didime tso afisi wodo atike ɖo vaseɖe teƒe si didi wu le ke alo alɔ la ŋu eye wotsɔ dzesi 1–9 na wo le afisi abi la le nu, afisi (1) ɖee fia be dɔlékui aɖeke si woate ŋu akpɔ le afisi te ɖe afisi wodo atike le ŋu eye (2–9) ɖee fia be abi ƒe lolome kple ŋgɔyiyi vivivi le nodes/internodes dzi (Kpekpeɖeŋu Tabla S2). Emegbe wotrɔ aŋɔ ɣi ƒe dɔlékui ƒe sesẽme wòzu alafa memamã to mɔnu 2 zazã me:
Gakpe ɖe eŋu la, wobu teƒe si le dɔlélea ƒe ŋgɔyiyi ƒe fli (AUDPC) te to mɔfiame (Shaner kple Finney, 1977) zazã me, si wotrɔ asi le nyitsɔ laa na gbe bɔbɔ ƒe gbegblẽ ɣi (Chauhan et al., 2020) to sɔsɔŋusẽ 3 zazã me:
Afisi Yi = dɔlélea ƒe sesẽme le ɣeyiɣi ti me, Yi+1 = dɔlélea ƒe sesẽme le ɣeyiɣi si kplɔe ɖo me ti+1, ti = ɣeyiɣi si wodzidzee zi gbãtɔ (le ŋkekewo me), ti+1 = ɣeyiɣi si wodzidzee si kplɔe ɖo (le ŋkekewo me), n = ɣeyiɣiwo alo ŋkuléleɖenuŋu teƒewo ƒe xexlẽme bliboa. Woŋlɔ bean numiemie ƒe tsitsi ƒe nɔnɔme siwo dometɔ aɖewoe nye numiemie ƒe kɔkɔme (cm), alɔ ƒe xexlẽme le numiemie ɖeka me, kple aŋgba ƒe xexlẽme ɖe numiemie ɖeka me kwasiɖa sia kwasiɖa hena ŋkeke 21 le nugbagbe siwo katã wogbugbɔ wɔ me.
Le nugbagbewo ƒe wɔwɔme ɖesiaɖe me la, woƒo aŋgba ƒe kpɔɖeŋuwo (aŋgba siwo tsi bliboe evelia kple etɔ̃lia tso etame) nu ƒu le ŋkeke 45 lia dzi le atikewɔwɔ vɔ megbe (ŋkeke 15 le atikewɔwɔ mamlɛtɔ megbe). Nu gbagbe siwo wogbugbɔ wɔ la dometɔ ɖesiaɖe nye ze atɔ̃ (numiemie eve le ze ɖeka me). Wozã lãmenugbagbevi siwo wogbã la ƒe mg 500 lɔƒo tsɔ ɖe keklẽŋusẽ ƒe amadedewo (chlorophyll a, chlorophyll b kple carotenoids) ɖa to acetone 80% zazã me le 4 °C me le viviti me. Le gaƒoƒo 24 megbe la, woɖe kpɔɖeŋuawo le centrifuged me eye woƒo tsi si le edzi la nu ƒu hena chlorophyll a, chlorophyll b kple carotenoid ƒe agbɔsɔsɔme nyanya to colorimetrically to UV-160A spectrophotometer (Shimadzu Corporation, Japan) zazã me le mɔnu si (Lichtenthaler, 1987) wɔ nu to alesi wodzidzea absorbance le wavelength vovovo etɔ̃ me (A470, A646 kple A663 nm). Mlɔeba la, wozã nɔnɔmetata siwo gbɔna 4–6 siwo ŋu Lichtenthaler (1987) ƒo nu tsoe tsɔ bu akɔnta le keklẽŋusẽ ƒe amadedewo me.
Le gaƒoƒo 72 le atikewɔwɔ vɔ megbe (hpt) la, woƒo aŋgbawo (aŋgba evelia kple etɔ̃lia siwo tsi bliboe tso etame) nu ƒu tso nugbagbewo ƒe wɔwɔme ɖesiaɖe me hena in situ histochemical localization of hydrogen peroxide (H2O2) kple superoxide anion (O2•−). Nu gbagbe siwo wogbugbɔ wɔ la dometɔ ɖesiaɖe nye ze atɔ̃ (numiemie eve le ze ɖeka me). Woda nugbagbewo ƒe gbugbɔgawɔ ɖesiaɖe kpɔ zi eve (mɔ̃ɖaŋununya ƒe gbugbɔgawɔ eve) be woakpɔ egbɔ be mɔnua de pɛpɛpɛ, kakaɖedzi le eŋu eye woate ŋu agbugbɔe awɔ. Wozã 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Germany) alo nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Germany), tsɔ de dzesi H2O2 kple O2•−, le mɔnu siwo Romero-Puertas et al. (2004) kple Adam kple exɔlɔ̃wo. (1989) kple tɔtrɔ suesuesuewo. Le histochemical localization of H2O2 in situ, wotsɔ vacuum de agbalẽviawo me kple 0.1% DAB le 10 mM Tris buffer (pH 7.8) me eye emegbe wotsɔ wo de xɔme ƒe dzoxɔxɔ me le kekeli me hena 60 min. Wotsɔ 0.15% (v/v) TCA le 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) me ble agbalẽviawo eye emegbe wotsɔ wo de kekeli me vaseɖe esime wodo viviti. Nenema ke wotsɔ vacuum de valvɔwo me kple 10 mM potassium phosphate buffer (pH 7.8) si me 0.1 w/v % HBT le hena histochemical localization of O2•− in situ. Wotsɔ agbalẽviawo de kekeli me le xɔme ƒe dzoxɔxɔ nu hena miniti 20, emegbe wotsɔ wo ɣie abe alesi míegblɔe ɖe etame ene, eye emegbe woklẽ wo vaseɖe esime teƒe blɔ/violet vivitiwo dze. Wozã nɔnɔmetata ŋuti dɔwɔwɔ ƒe agbalẽvi si nye ImageJ (http://fiji.sc; wokpɔe le March 7, 2024) ƒe Fiji tɔ tsɔ da amadede si nye brown (abe H2O2 ƒe dzesi ene) alo blue-violet (abe O2•− dzesi ene) ƒe sesẽme kpɔ.
Wokpɔ malondialdehyde (MDA; abe lipid peroxidation ƒe dzesi ene) le Du kple Bramlage (1992) ƒe mɔnu nu kple tɔtrɔ sue aɖewo. Woƒo aŋgba siwo tso nugbagbe ƒe aŋgba ɖesiaɖe si wogbugbɔ wɔ (aŋgba evelia kple etɔ̃lia siwo tsi bliboe tso etame) nu ƒu le gaƒoƒo 72 le atikewɔwɔ vɔ megbe (hpt). Nu gbagbe ɖesiaɖe si wogbugbɔ wɔ la lɔ ze atɔ̃ (numiemie eve le ze ɖeka me) ɖe eme. Woda nugbagbewo ƒe gbugbɔgawɔ ɖesiaɖe kpɔ zi eve (mɔ̃ɖaŋununya ƒe gbugbɔgawɔ eve) be woakpɔ egbɔ be mɔnua de pɛpɛpɛ, kakaɖedzi le eŋu eye woate ŋu agbugbɔe awɔ. Kpuie ko la, wozã aŋgba ƒe lãkusi si wotu 0.5 g tsɔ ɖe MDA ɖa kple 20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, U.S.A.) si me butylated hydroxytoluene 0.01% (BHT; Sigma-Aldrich, St. Louis, MO, U.S.A.) le. Emegbe wotsɔ amadededzidzenu de dzesi MDA si le tsi si le tsia dzi me to alesi wodzidzea alesi wòxɔa tsii le 532 kple 600 nm to UV-160A spectrophotometer (Shimadzu Corporation, Japan) zazã me eye emegbe woɖee fia abe nmol g−1 FW ene.
Le non-enzymatic kple enzymatic antioxidants ƒe dodokpɔ me la, woƒo aŋgbawo (aŋgba evelia kple etɔ̃lia siwo tsi bliboe tso etame) nu ƒu tso nugbagbewo ƒe wɔwɔme ɖesiaɖe me le 72 h le atikewɔwɔ vɔ megbe (hpt). Nu gbagbe siwo wogbugbɔ wɔ la dometɔ ɖesiaɖe nye ze atɔ̃ (numiemie eve le ze ɖeka me). Woda nugbagbewo ƒe kpɔɖeŋu ɖesiaɖe kpɔ zi eve (mɔ̃ɖaŋununya ƒe kpɔɖeŋu eve). Wotsɔ nitrogen si nye tsi tu aŋgba eve eye wozã wo tẽ tsɔ nya nusiwo tsia dzoxɔxɔ nu siwo me enzyme kple esiwo menye enzyme o, amino acid bliboa, proline ƒe agbɔsɔsɔ, domenyiŋusẽfianu ƒe ɖeɖefia, kple oxalate ƒe agbɔsɔsɔme nyanya.
Wozã Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) tsɔ de dzesi phenolics siwo katã me wodea tsii eye wotrɔ asi le mɔnu si ŋu Kahkonen et al. (ƒe 1999). Kpuie ko la, woɖe aŋgba ƒe lãkusi si wowɔ ɖekae si ade 0.1 g kple 20 ml 80% methanol le viviti me hena gaƒoƒo 24 eye woƒo tsi si le edzi la nu ƒu le centrifugation megbe. Wotsɔ 0.1 ml ƒe kpɔɖeŋu si woɖe tso eme la tsaka kple 0.5 ml Folin-Ciocalteu reagent (10%), woʋuʋui hena s 30 eye wogblẽe ɖe viviti me hena miniti 5. Emegbe wotsɔ 20% sodium carbonate solution (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) 0.5 ml de ʋuƒo ɖesiaɖe me, wotsakae nyuie eye wotsɔe de xɔme ƒe dzoxɔxɔ me le viviti me hena gaƒoƒo 1. Le vidzidzi megbe la, wodzidze nusiwo wotsɔ tsakae ƒe xɔxlɔ̃ le 765 nm to UV-160A spectrophotometer (Shimadzu Corporation, Japan) zazã me. Wozã gallic acid calibration curve (Fisher Scientific, Hampton, NH, USA) tsɔ de dzesi phenol siwo katã me wodea tsii ƒe agbɔsɔsɔme le kpɔɖeŋu me tsiwo me eye woɖee fia abe gallic acid miligram siwo sɔ kple gram ɖeka le kpekpeme yeye ƒe gram ɖeka me (mg GAE g-1 fresh weight).
Wokpɔ flavonoid si me wodea tsii ƒe agbɔsɔsɔ bliboa le Djeridane et al. (2006) kple tɔtrɔ sue aɖewo. Kpuie ko la, wotsɔ methanol si le etame ƒe ml 0.3 tsaka kple 0.3 ml si nye 5% aluminum chloride solution (AlCl3; Fisher Scientific, Hampton, NH, USA), woʋuʋui sesĩe eye emegbe wotsɔe de xɔme ƒe dzoxɔxɔ me hena miniti 5, eye wotsɔ 0.3 ml si nye 10% potassium acetate solution (Al-Gomhoria) kpe ɖe eŋu Atikewo kple Atikewɔnuwo, Cairo, Egypt), wotsaka nyuie eye wotsɔe de xɔme ƒe dzoxɔxɔ me hena miniti 30 le viviti me. Le vidzidzi megbe la, wodzidze nusiwo wotsɔ tsakae ƒe xɔxlɔ̃ le 430 nm to UV-160A spectrophotometer (Shimadzu Corporation, Japan) zazã me. Wozã rutin calibration curve (TCI America, Portland, OR, USA) tsɔ de dzesi flavonoids siwo katã me wodea tsii ƒe agbɔsɔsɔme le kpɔɖeŋu me tsiwo me eye emegbe woɖee fia abe rutin miligram siwo sɔ kple gram ɖeka ƒe kpekpeme yeye (mg RE g-1 fresh weight) ene.
Wozã ninhydrin reagent si wogbugbɔ trɔ (Thermo Scientific Chemicals, Waltham, MA, USA) si wotu ɖe mɔnu si Yokoyama kple Hiramatsu (2003) do ɖa dzi eye Sun et al. (ƒe 2006). Kpuie ko la, wotsɔ pH 5.4 buffer ɖe anyigba 0.1 g, eye wotsɔ 200 μL si le tsia dzi la wɔ dɔ kple 200 μL ninhydrin (2%) kple 200 μL pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), wotsɔe de tsi dzodzoe ƒe tsilele me hena miniti 30, emegbe wofae eye wodzidzee le 580 nm to UV-160A ƒe keklẽŋusẽdzidzenu (Shimadzu Corporation, Japan) zazã me. Le go bubu me la, wotsɔ Bates ƒe mɔnu de dzesi proline (Bates et al., 1973). Wotsɔ 3% sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) ɖe proline eye le centrifugation megbe la, wotsɔ tsi si le edzi ƒe 0.5 ml tsaka kple 1 ml glacial acetic acid (Fisher Scientific, Hampton, NH, USA) kple ninhydrin reagent, wotsɔe de dzoxɔxɔ 90°C me hena miniti 45, wofae eye wodzidzee le 520 nm zã spectrophotometer si le etame la ke. Wozã glycine kple proline calibration curves (Sigma-Aldrich, St Louis, MO, USA) tsɔ de dzesi amino acid siwo katã le ablɔɖe me kple proline siwo le aŋgba me tsiwo me, eye woɖee fia abe mg/g ƒe kpekpeme yeye ene.
Be woanya antioxidant enzymes ƒe enzyme ƒe dɔwɔwɔ la, wotsɔ 50 mM Tris buffer (pH 7.8) ml 3 si me 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) kple 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), wotsɔ centrifuged le 10,000 × g hena miniti 20 le nufamɔ̃ (4 °C) te, eye woƒo tsi si le edzi (crude enzyme extract) nu ƒu (El-Nagar et al., 2023; Osman et al., 2023). Emegbe wotsɔ Catalase (CAT) wɔ dɔ kple 2 ml 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) kple 100 μl 269 mM H2O2 solution tsɔ de dzesi eƒe enzymatic dɔwɔwɔ le Aebi (1984) ƒe mɔnu nu kple tɔtrɔ suewo (El-Nagar et al., 2023; Osman kple ame bubuwo, 2023). Wozã Harrach et al. ƒe mɔnu tsɔ de dzesi Guaiacol-dependent peroxidase (POX) enzymatic dɔwɔwɔ. (ƒe 2009). (2008) kple tɔtrɔ suesuesuewo (El-Nagar et al., 2023; Osman et al., 2023) eye wokpɔ polyphenol oxidase (PPO) ƒe enzyme ƒe dɔwɔwɔ le dɔwɔwɔ kple 2.2 ml 100 mM sodium phosphate buffer (pH 6.0), 100 μl guaiacol (TCI chemicals, Portland, OR, ) megbe. USA) kple 100 μl ƒe 12 mM H2O2. Wotrɔ mɔnu la vie tso (El-Nagar et al., 2023; Osman et al., 2023). Wowɔ dodokpɔa le catechol solution 3 ml (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) si wodzra ɖo yeyee le 0.1 M phosphate buffer (pH 6.0) me ƒe dɔwɔwɔ vɔ megbe. Wodzidze CAT ƒe dɔwɔwɔ to ŋkuléle ɖe H2O2 ƒe gbegblẽ ŋu le 240 nm (A240), wodzidze POX ƒe dɔwɔwɔ to ŋkuléle ɖe absorbance ƒe dzidziɖedzi ŋu le 436 nm (A436), eye wodzidze PPO ƒe dɔwɔwɔ to absorbance ƒe tɔtrɔwo ŋɔŋlɔ ɖi le 495 nm (A495) s 30 ɖesiaɖe hena miniti 3 zazã UV-160A zazã me keklẽŋusẽdzidzenu (Shimadzu, Japan).
Wozã ɣeyiɣi ŋutɔŋutɔ me RT-PCR tsɔ de dzesi domenyiŋusẽfianu etɔ̃ siwo do ƒome kple antioxidant ƒe nuŋɔŋlɔ ƒe agbɔsɔsɔ, siwo dometɔ aɖewoe nye peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), kple glutathione reductase (PvGR; GenBank Accession No. KY195009.1), le bean aŋgbawo me (agba evelia kple etɔ̃lia si tsi bliboe tso etame) 72 h le atikewɔwɔ mamlɛtɔ megbe. Kpuie ko la, wozã Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) tsɔ ɖe RNA ɖe vovo le amesi wɔe ƒe ɖoɖo nu. Emegbe wozã TOP scriptTM cDNA Synthesis Kit tsɔ wɔ cDNA le amesi wɔe ƒe mɔfiamewo nu. Woŋlɔ domenyiŋusẽfianu etɔ̃ siwo le etame ƒe gɔmedzedze ƒe ɖoɖowo ɖe Kpekpeɖeŋu Tabla S3 me. Wozã PvActin-3 (GenBank accession number: XM_068616709.1) abe aƒeme domenyiŋusẽfianu ene eye wobu domenyiŋusẽfianu ƒe ɖeɖefia si sɔ kple wo nɔewo to 2-ΔΔCT mɔnu zazã me (Livak kple Schmittgen, 2001). Woɖe actin ƒe liƒo le nugbagbewo ƒe nuteɖeamedzi te (kadodo si mesɔ o le mɔli bɔbɔewo kple anthracnose fungus Colletotrichum lindemuthiaanum dome) kple abiotic nuteɖeamedzi (kuɖiɖi, dze, dzoxɔxɔ si bɔbɔ) te (Borges et al., 2012).
Míewɔ domenyiŋusẽfianu bliboa ƒe silico numekuku le oxaloacetate acetylhydrolase (OAH) proteinwo ŋu le S. sclerotiorum me gbã to protein-protein BLAST dɔwɔnu (BLASTp 2.15.0+) zazã me (Altschul et al., 1997, 2005). Kpuie ko la, míezã OAH si tso Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; GenBank ƒe xexlẽdzesi XP_040799428.1; amino acid 342) kple Penicillium lagena (PlOAH; taxide: 94218; GenBank ƒe ŋkɔxɔxɔ ƒe xexlẽdzesi XP_056833920.1; Wowɔ BLASTp ɖe S. sclerotiorum ƒe domenyiŋusẽfianu ŋuti nyatakaka siwo wokpɔ nyitsɔ laa le GenBank dzi le Dukɔa ƒe Nu gbagbewo Ŋuti Nyatakaka Dɔwɔƒe (NCBI) ƒe nyatakakadzraɖoƒe, http://www.ncbi.nlm.nih.gov/gene/.
Gakpe ɖe eŋu la, wozã mɔnu si nye maximum likelihood le MEGA11 (Tamura et al., 2021) kple JTT matrix-based model (Jones et al., ƒe 1992). Wotsɔ dzidzime ƒe ati la ƒo ƒu kple protein ƒe ɖoɖo vovovowo me dzodzro le OAH domenyiŋusẽfianu siwo katã wogblɔ ɖi (SsOAH) tso S. sclerotiorum me kple nyabiase ƒe ɖoɖo si wozã Constraint-Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos kple Agarwala, Ƒe 2007 me). Gakpe ɖe eŋu la, wotsɔ SsOAH ƒe amino acid ƒe ɖoɖo siwo sɔ nyuie wu tso S. sclerotiorum me sɔ kple nyabiase ƒe ɖoɖowo (AfOAH kple PlOAH) (Larkin et al., 2007) to ClustalW (http://www.genome.jp/tools-bin/clustalw) zazã me, eye wotsɔ ESPript dɔwɔnu (version 3.0; https://espript.ibcp.fr/ESPript/ESPript/index.php) Ƒe 1999 ƒe ɣleti gbãtɔ me.
Gawu la, woma S. sclerotiorum SsOAH ƒe dɔwɔwɔ teƒenɔlawo ƒe domenyinyi siwo wogblɔ ɖi kple teƒe siwo wodzra ɖo la ɖe ƒome vovovowo me le nuwɔwɔ aduadu me to InterPro dɔwɔnu (https://www.ebi.ac.uk/interpro/) zazã me (Blum et al., 2021). Mlɔeba la, wowɔ akpa etɔ̃ (3D) ƒe wɔwɔme ƒe kpɔɖeŋuwɔwɔ na S. sclerotiorum SsOAH si wogblɔ ɖi la to Protein Homology/Analogy Recognition Engine (Phyre2 server version 2.0; http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) zazã me eye wozãe tsɔ da asi ɖe edzi SWISS-MODEL dɔdzikpɔla (https://swissmodel.expasy.org/) (Biasini kple ehatiwo, 2014). Wotsɔ UCSF-Chimera package (version 1.15; https://www.cgl.ucsf.edu/chimera/ ) (Pettersen et al., 2004) tsɔ kpɔ akpa etɔ̃ ƒe xɔtuɖoɖo siwo wogblɔ ɖi (PDB ƒe nɔnɔme) le susu me le nuwɔwɔ aduadu me.
Wozã agbɔsɔsɔme ŋutɔŋutɔ ƒe keklẽŋusẽ PCR tsɔ de dzesi oxaloacetate acetylhydrolase (SsOAH; GenBank accession number: XM_001590428.1) ƒe nuŋɔŋlɔ ƒe agbɔsɔsɔ le Sclerotinia sclerotiorum ƒe mycelia me. Kpuie ko la, wodo S. sclerotiorum ɖe nugoe si me PDB le me eye wotsɔe de vidzidɔ si le ʋuʋum (kpɔɖeŋu: I2400, New Brunswick Scientific Co., Edison, NJ, USA) me le dzoxɔxɔ 25 ± 2 °C me hena gaƒoƒo 24 le 150 rpm kple viviti me ɣesiaɣi (gaƒoƒo 24) be wòaʋã mycelial ƒe tsitsi. Emegbe la, wotsɔ L-ornithine kple fungicide Rizolex-T da dɔ na lãmenugbagbeviawo le IC50 ƒe agbɔsɔsɔ mamlɛawo me (anɔ abe 40 kple 3.2 mg/L ene) eye emegbe wode wo agble gaƒoƒo 24 bubu le nɔnɔme mawo ke me. Le woƒe vidzidzi megbe la, wotsɔ centrifuged dekɔnuawo le 2500 rpm hena miniti 5 eye woƒo tsi si le wo dzi (fungal mycelium) nu ƒu hena domenyiŋusẽfianu ƒe ɖeɖefia me dzodzro. Nenema ke woƒo fungal mycelium nu ƒu le 0, 24, 48, 72, 96, kple 120 h le dɔlélea xɔxɔ vɔ megbe tso numiemie siwo ŋu dɔlélea le siwo wɔ aŋɔ ɣi kple kotoku me mycelium le lãkusi siwo ŋu dɔlélea le ƒe anyime. Woɖe RNA tso fungi mycelium me eye emegbe wowɔ cDNA abe alesi míegblɔe ɖe etame ene. Woŋlɔ gɔmedzenuwo ƒe ɖoɖo siwo wowɔ na SsOAH ɖe Kpekpeɖeŋu Tabla S3 me. Wozã SsActin (GenBank accession number: XM_001589919.1) abe aƒedzikpɔkpɔ ƒe domenyiŋusẽfianu ene, eye wobu domenyiŋusẽfianu ƒe ɖeɖefia le ƒomedodo me to 2-ΔΔCT mɔnu zazã me (Livak kple Schmittgen, 2001).
Wokpɔ oxalic acid le mɔli dextrose broth (PDB) kple numiemie ƒe kpɔɖeŋu siwo me fungal dɔlékui Sclerotinia sclerotiorum le me le Xu kple Zhang (2000) ƒe mɔnu nu kple tɔtrɔ sue aɖewo. Kpuie ko la, wodo S. sclerotiorum siwo woɖe ɖe vovo ɖe nugoe siwo me PDB le me eye emegbe wodo wo ɖe vidzidɔ si le ʋuʋum (kpɔɖeŋu I2400, New Brunswick Scientific Co., Edison, NJ, USA) me le 150 rpm me le 25 ± 2 °C hena ŋkeke 3–5 le viviti me ɣesiaɣi (gaƒoƒo 24) be woado ŋusẽ mycelial ƒe tsitsi. Le vidzidzi megbe la, woɖea fungi dekɔnua to Whatman #1 filter paper me gbã eye emegbe wotsɔa centrifuged le 2500 rpm me hena miniti 5 be woaɖe mycelium si susɔ la ɖa. Woƒo tsi si le edzi la nu ƒu eye wodzrae ɖo ɖe dzoxɔxɔ 4°C me hena oxalate ƒe agbɔsɔsɔme nyanya bubuwo. Le numiemie ƒe kpɔɖeŋuwo dzadzraɖo me la, woɖe numiemie ƒe lãkusi kakɛ siwo ade 0.1 g zi etɔ̃ kple tsi si woɖe tso tsi me (2 ml ɣesiaɣi). Emegbe wotsɔa centrifuged na kpɔɖeŋuawo le 2500 rpm hena miniti 5, woɖe tsi si le edzi la ƒuƒlu to Whatman No. 1 filter paper me eye woƒo wo nu ƒu hena numekuku bubuwo wɔwɔ.
Le oxalic acid ƒe agbɔsɔsɔme me dzodzro me la, wodzra nusi wotsɔ tsakae ɖo le glass stoppered tube me le ɖoɖo si gbɔna nu: 0.2 ml ƒe kpɔɖeŋu (alo PDB culture filtrate alo oxalic acid standard solution), 0.11 ml ƒe bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), 0.198 ml ƒe 1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) kple potassium dichromate 100 mM (K2Cr2O7; TCI chemicals, Portland, OR, USA) 0.176 ml, eye emegbe wotsɔa tsi si woɖe tso tsi si woɖe tso tsia me la ƒo tsi si woɖe tso eme la wòde mililita 4.8, wotsakae sesĩe eye wotsɔe de tsi si ƒe dzoxɔxɔ nye 60 °C me enumake le tsi. Le miniti 10 megbe la, wotɔ te nuwɔna la to sodium hydroxide solution (NaOH; 0.75 M) 0.5 ml dede eme me. Wodzidze nusiwo wotsɔ tsakae ƒe absorbance (A600) le 600 nm to UV-160 spectrophotometer (Shimadzu Corporation, Japan) zazã me. Wozã PDB kple tsi si woɖe tso tsi me abe atike siwo woatsɔ akpɔ dekɔnu me tsiwo kple numiemiewo ƒe kpɔɖeŋuwo ƒe agbɔsɔsɔme dzi ene. Wozã oxalic acid ƒe agbɔsɔsɔme si woɖe fia le dekɔnu me tsiwo me, si woɖe fia abe oxalic acid microgram le mililita ɖeka me le PDB medium (μg.mL−1), kple le aŋgba me tsiwo me, si woɖe fia abe oxalic acid microgram le gram ɖeka me le kpekpeme yeye me (μg.g−1 FW) me, to oxalic acid calibration curve (Thermo Fisher Scientific Chemicals, Waltham, MA, ) zazã me. USA).
Le numekukua katã me la, wowɔ dodokpɔawo katã le ɖoɖo si wowɔ le vome keŋkeŋ (CRD) nu eye wowɔ nugbagbe ade siwo wogbugbɔ wɔ le atikewɔwɔ ɖesiaɖe me kple ze atɔ̃ le nugbagbevi siwo wogbugbɔ wɔ ɖesiaɖe me (numiemie eve le ze ɖeka me) negbe ɖe wogblɔe bubui hafi. Woda nugbagbe siwo wogbugbɔ wɔ la kpɔ zi eve (mɔ̃ɖaŋununya ƒe nɔnɔmetata eve). Wozã mɔ̃ɖaŋununya ƒe nɔnɔmetata siwo wogbugbɔ wɔ tsɔ lé ŋku ɖe dodokpɔ ma ke ƒe gbugbɔgawɔ ŋu gake womezã wo le akɔntabubu me dzodzro me be woaƒo asa na alakpa gbugbɔgawɔ o. Wozã akɔntabubu le vovototowo me dzodzro (ANOVA) zazã me eye wotsɔ Tukey-Kramer honestly significant difference (HSD) dodokpɔ (p ≤ 0.05) kplɔe ɖo. Le dodokpɔ siwo wowɔ le vitro me la, wobu IC50 kple IC99 ƒe asixɔxɔwo to probit ƒe kpɔɖeŋu zazã me eye wobu kakaɖedzi ƒe dometsotso 95%.
Woƒo lã ene siwo woɖe ɖe vovo la nu ƒu tso soya-gble vovovowo me le El Ghabiya Nutome, Egipte. Le PDA medium dzi la, nusiwo katã woɖe ɖe vovo la na mycelium ɣi si le abe kreme ene si trɔna kabakaba abe kotoku ɣi ene (Nɔnɔmetata 1A) eye emegbe wòzua beige alo brown le sclerotium ƒe afɔɖeɖea me. Zi geɖe la, sclerotia nyea ʋuʋudedi, yibɔ, goglo alo esi ƒe nɔnɔme mesɔ o, didime nyea milimeta 5.2 va ɖo 7.7 eye wòkeke milimeta 3.4 va ɖo 5.3 (Nɔnɔmetata 1B). Togbɔ be lã ene siwo woɖe ɖe vovo la va tu sclerotia ƒe nɔnɔme si le axadzi le agblemenukua ƒe go kemɛ dzi le ŋkeke 10–12 ƒe vidzidzi le 25 ± 2 °C megbe (Fig. 1A) hã la, sclerotia ƒe xexlẽme le agba ɖeka me to vovo ŋutɔ le wo dome (P < 0.001), eye isolate 3 ye nye sclerotia ƒe xexlẽme si sɔ gbɔ wu (32.33 ± 1.53 sclerotia le ɖesiaɖe me agbalẽvi 1C). Nenema ke nusi woɖe ɖe vovo #3 na oxalic acid geɖe le PDB me wu esiwo woɖe ɖe vovo (3.33 ± 0.49 μg.mL−1; Fig. 1D). Isolate #3 ɖe nɔnɔme kple nusuekpɔmɔ̃ ƒe nɔnɔme tɔxɛwo fia le phytopathogenic fungus Sclerotinia sclerotiorum ŋu. Le kpɔɖeŋu me. Wonɔ te ɖe nɔnɔme kple nusuekpɔmɔ̃ ƒe nɔnɔme siwo le etame dzi de dzesi nusi woɖe ɖe vovo #3 be enye Sclerotinia sclerotiorum.
Figure 1. S. sclerotiorum siwo woɖe ɖe vovo tso mɔli ƒe agblemenuku siwo bɔ me ƒe nɔnɔmewo kple dɔlékuiwo xɔxɔ. (A) S. sclerotiorum ene siwo woɖe ɖe vovo le PDA medium dzi ƒe mycelial tsitsi, (B) S. sclerotiorum ene siwo woɖe ɖe vovo ƒe sclerotia, (C) sclerotia ƒe xexlẽme (le agba ɖeka me), (D) oxalic acid ƒe dodo le PDB medium dzi (μg.mL−1), kple (E) dɔléle ƒe sesẽme (%) le S. sclerotiorum ɖekaɖeka ene siwo woɖe ɖe vovo le asitsatsa me legume si woate ŋu axɔ bɔbɔe me cultivar Giza 3 le dzoxɔxɔ ƒe nɔnɔmewo me. Asixɔxɔwo tsi tre ɖi na nugbagbevi atɔ̃ siwo wogbugbɔ wɔ (n = 5) ƒe mama dedie ± SD. Ŋɔŋlɔdzesi vovovowo ɖe vovototo siwo ɖe dzesi le akɔntabubu nu le atikewɔwɔwo dome fia (p < 0.05). (F–H) Aŋɔ ɣi ƒe dzesi siwo bɔ la dze le atikutsetse siwo le anyigba dzi kple siliques dzi, le ŋkeke 10 le atike si woɖe ɖe vovo #3 (dpi) dodo vɔ megbe. (I) Wowɔ nɔnɔmetɔtrɔ ƒe numekuku le S. sclerotiorum isolate #3 ƒe ememe transcribed spacer (ITS) nuto me to mɔnu si nye maximum likelihood zazã me eye wotsɔe sɔ kple reference isolates/strains 20 siwo woxɔ tso National Center for Biotechnology Information (NCBI) ƒe nyatakakadzraɖoƒe (https://www.ncbi.nlm.nih.gov/). Xexlẽdzesi siwo le ƒuƒoƒo ƒe fliawo tame la fia nuto si me wole (%), eye xexlẽdzesi siwo le ƒuƒoƒo ƒe fliawo te fia alɔdzea ƒe didime.
Gawu la, be woaɖo kpe dɔlékuiwo ƒe dzɔdzɔ dzi la, wozã S. sclerotiorum ƒe lã ene siwo woɖe ɖe vovo tsɔ do atike na asitsatsa ƒe gbe ƒomevi si woate ŋu axɔ bɔbɔe si nye Giza 3 le dzoxɔxɔ ƒe nɔnɔmewo me, si sɔ kple Koch ƒe nukpɔsusuwo (Fig. 1E). Togbɔ be fungi siwo katã woɖe ɖe vovo siwo wokpɔ la nye dɔlékuiwo eye woate ŋu axɔ dɔ le gbe dama (cv. Giza 3) ŋu, si ahe aŋɔ ɣi ƒe dzesi siwo bɔ ɖe akpa siwo katã le anyigba dzi (Fig. 1F), vevietɔ le kewo (Fig. 1G) kple pods (Fig. 1H) dzi le ŋkeke 10 le atike dodo vɔ megbe (dpi) hã la, isolate 3 ye nye esi woɖe ɖe vovo si wɔ adã wu le dodokpɔ eve siwo wowɔ le wo ɖokui si me. Isolate 3 ye nye dɔlélea ƒe sesẽme wu (%) le bean numiemiewo dzi (24.0 ± 4.0, 58.0 ± 2.0, kple 76.7 ± 3.1 le ŋkeke 7, 14, kple 21 le dɔlélea xɔxɔ vɔ megbe, le wo nɔewo yome; Nɔnɔmetata 1F).
Wogaɖo kpe S. sclerotiorum si woɖe ɖe vovo #3 si dzea ame dzi wu ƒe dzedzeme dzi le ememe transcribed spacer (ITS) ƒe ɖoɖowɔwɔ dzi (Fig. 1I). Dzɔdzɔmeŋusẽŋununya me dzodzro le isolate #3 kple 20 reference isolates/strains dome ɖee fia be woɖi wo nɔewo ŋutɔ (>99%). Edze be míade dzesii be S. sclerotiorum si woɖe ɖe vovo #3 (533 bp) sɔ kple Amerikatɔwo ƒe S. sclerotiorum si woɖe ɖe vovo LPM36 si woɖe ɖe vovo tso pea nuku ƒuƒuwo me (GenBank accession number MK896659.1; 540 bp) kple Chinatɔwo ƒe S. sclerotiorum isolate YKY211 (GenBank accession number OR206374.1; Wotsɔ ɖoɖo yeyea da ɖe NCBI ƒe nyatakakadzraɖoƒea eye wotsɔ ŋkɔ nɛ be “Sclerotinia sclerotiorum – isolate YN-25” (GenBank accession number PV202792). Woate ŋu akpɔe be isolate 3 ye nye isolate si xɔa ame me wu; eyata wotia nusi woɖe ɖe vovo sia be woasrɔ̃ nu tso eŋu le dodokpɔ siwo katã kplɔe ɖo me.
Woku nu me le diamine L-ornithine (Sigma-Aldrich, Darmstadt, Germany) ƒe dɔlékuiwutike ƒe dɔwɔwɔ le agbɔsɔsɔ vovovowo me (12.5, 25, 50, 75, 100 kple 125 mg/L) ɖe S. sclerotiorum isolate 3 ŋu le vitro. Enyo be míade dzesii be L-ornithine wɔ dɔlékuiwutike ƒe ŋusẽ eye vivivi la, exe mɔ ɖe S. sclerotiorum hyphae ƒe tsitsi le radial nu le mɔ si nɔ te ɖe agbɔsɔsɔme si wozã dzi (Nɔnɔmetata 2A, B). Le agbɔsɔsɔ si wodo kpɔ wu (125 mg/L) me la, L-ornithine ɖe mycelial ƒe tsitsi ƒe mɔxexeɖedɔléle nu ƒe agbɔsɔsɔme si lolo wu fia (99.62 ± 0.27%; Nɔnɔmetata 2B), si sɔ kple fungicide si wodzrana Rizolex-T (si xea mɔ na fungicide 99.45 ± 0.39%; Nɔnɔmetata 2C) le agbɔsɔsɔ gãtɔ si wodo kpɔ me (10 mg/L), si fia be esɔ nenema dɔwɔwɔ nyuie.
Nɔnɔmetata 2. L-ornithine ƒe dɔlékuiwutike ƒe dɔwɔwɔ le vitro ɖe Sclerotinia sclerotiorum ŋu. (A) L-ornithine ƒe agbɔsɔsɔ vovovowo ƒe dɔlékuiwutike ƒe dɔwɔwɔ ɖe S. sclerotiorum ŋu tsɔtsɔ sɔ kple fungicide si wodzrana Rizolex-T (10 mg/L). (B, C) S. sclerotiorum ƒe mycelial ƒe tsitsi ƒe mɔxexe ƒe agbɔsɔsɔme (%) le atikewɔwɔ kple L-ornithine (12.5, 25, 50, 75, 100 kple 125 mg/L) alo Rizolex-T (2, 4, 6, 8 kple 10 mg/L) ƒe agbɔsɔsɔ vovovowo megbe. Asixɔxɔwo tsi tre ɖi na nugbagbevi atɔ̃ siwo wogbugbɔ wɔ (n = 5) ƒe mama dedie ± SD. Ŋɔŋlɔdzesi vovovowo fia akɔntabubu ƒe vovototo siwo le atikewɔwɔwo dome (p < 0.05). (D, E) Probit model regression numekuku le L-ornithine kple asitsatsa fungicide Rizolex-T, ɖe wo nɔewo yome. Woɖe probit model regression line fia abe fli blɔ sesẽ ene, eye woɖe kakaɖedzi ƒe dometsotso (95%) fia abe fli dzĩ si wotso ene.
Gakpe ɖe eŋu la, wowɔ probit regression analysis eye woɖe plot siwo sɔ la fia le Table 1 kple Figures 2D,E me. Kpuie ko la, L-ornithine ƒe togbɛ ƒe asixɔxɔ si dzi woda asi ɖo (y = 2.92x − 4.67) kple akɔntabubu vevi siwo do ƒome kplii (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 kple p < 0.0001; Nɔnɔmetata 2D) ɖee fia be dɔlékuiwutike ƒe dɔwɔwɔ dzi ɖe edzi ɖe S. sclerotiorum ŋu ne wotsɔe sɔ kple fungicide si wodzrana Rizolex-T (y = 1.96x − 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 kple p < 0.0001) (Tabla 1).
Table 1. Asixɔxɔ siwo le L-ornithine kple asitsatsa ƒe fungicide “Rizolex-T” ƒe mɔxexeɖedɔlékui ƒe agbɔsɔsɔ si sɔ gbɔ wu (IC50) kple IC99 (mg/l) ŋu ɖe S. sclerotiorum ŋu.
Le blibo me la, L-ornithine (250 mg/L) ɖe aŋɔ ɣi ƒe tsitsi kple eƒe sesẽ dzi kpɔtɔ ŋutɔ le gbe bɔbɔ ƒe numiemie siwo ŋu wowɔ atike ɖo dzi ne wotsɔe sɔ kple numiemie siwo ŋu S. sclerotiorum dɔlékui le si womewɔ atike na o (dziɖuɖu; Nɔnɔmetata 3A). Kpuie ko la, togbɔ be dɔlélea ƒe sesẽme dzi ɖe edzi vivivi le numiemie siwo ŋu dɔlélea mele o ŋu (52.67 ± 1.53, 83.21 ± 2.61, kple 92.33 ± 3.06%) hã la, L-ornithine ɖe dɔlélea ƒe sesẽ dzi kpɔtɔ ŋutɔ (%) le dodokpɔa katã me (8.97 ± 0.15, 18.00 ± 1.00, kple 26.36 ± 3.07) le ŋkeke 7, 14, kple 21 le atikewɔwɔ vɔ megbe (dpt), le wo nɔewo yome (Nɔnɔmetata 3A). Nenema ke esi wotsɔ L-ornithine 250 mg/L da gbe na gbegãdzi numiemie siwo ŋu S. sclerotiorum dɔlékui le la, teƒe si le dɔlélea ƒe ŋgɔyiyi ƒe fli (AUDPC) te dzi ɖe kpɔtɔ tso 1274.33 ± 33.13 le atike si womewɔ atike na o me va ɖo 281.03 ± 7.95, si bɔbɔ vie wu esi ŋu dɔlélea le 50 mg/L Rizolex-T fungicide (183.61 ± 7.71; Nɔnɔmetata 3B). Wokpɔ nɔnɔme ma ke le dodokpɔ evelia me.
Fig. 3. Ŋusẽ si L-ornithine zazã le egodo kpɔna ɖe gbe bɔbɔe ƒe gbegblẽ ɣi si Sclerotinia sclerotiorum hena vɛ le dzoxɔxɔ ƒe nɔnɔmewo me ƒe tsitsi dzi. (A) Dɔléle ƒe ŋgɔyiyi ƒe fli si le gbe ɣi si nye common bean ƒe atikewɔwɔ vɔ megbe kple 250 mg/L L-ornithine. (B) Nuto si le dɔlélea ƒe ŋgɔyiyi ƒe fli (AUDPC) te le gbe ɣi ƒe aŋɔ si woyɔna be common bean le atikewɔwɔ kple L-ornithine megbe. Asixɔxɔwo tsi tre ɖi na nugbagbevi atɔ̃ siwo wogbugbɔ wɔ (n = 5) ƒe mama dedie ± SD. Ŋɔŋlɔdzesi vovovowo fia vovototo siwo ɖe dzesi le akɔntabubu nu le atikewɔwɔwo dome (p < 0.05).
L-ornithine 250 mg/L zazã le gota na numiemie ƒe kɔkɔme dzi ɖe edzi vivivi (Nɔnɔmetata 4A), alɔdzewo ƒe xexlẽme le numiemie ɖeka me (Nɔnɔmetata 4B), kple aŋgbawo ƒe xexlẽme ɖe numiemie ɖeka dzi (Nɔnɔmetata 4C) le ŋkeke 42 megbe. Togbɔ be fungicide si wodzrana le asi me si nye Rizolex-T (50 mg/L) kpɔ ŋusẽ gãtɔ ɖe nunyiame ƒe nɔnɔme siwo katã ŋu wosrɔ̃ nu tsoe dzi hã la, L-ornithine 250 mg/L zazã le gotae kpɔ ŋusẽ gã evelia wu ne wotsɔe sɔ kple esiwo womewɔ atike na o (Fig. 4A–C). Le go bubu me la, L-ornithine zazã mekpɔ ŋusẽ gã aɖeke ɖe keklẽŋusẽ ƒe amadede siwo nye chlorophyll a (Nɔnɔmetata 4D) kple chlorophyll b (Nɔnɔmetata 4E) ƒe agbɔsɔsɔ dzi o, gake edzi carotenoid ƒe agbɔsɔsɔ bliboa ɖe edzi vie (0.56 ± 0.03 mg/g fr wt) ne wotsɔe sɔ kple esi womekpɔ nyuie o (0.44 ± 0.02 mg/g fr wt) kple dzikpɔkpɔ nyuie (0.46 ± 0.02 mg/g fr wt; Nɔnɔm. 4F). Le nusianu me la, emetsonu siawo ɖee fia be L-ornithine menye numiemie na mɔli siwo ŋu wowɔ atike ɖo o eye ate ŋu ana woƒe tsitsi gɔ̃ hã.
Fig. 4. Ŋusẽ si exogenous L-ornithine zazã kpɔna ɖe tsitsi ƒe nɔnɔmewo kple keklẽŋusẽ ƒe amadedewo dzi le bean aŋgba siwo ŋu Sclerotinia sclerotiorum dɔlékui le le dzoxɔxɔ ƒe nɔnɔmewo me. (A) Numiemie ƒe kɔkɔme (cm), (B) Alɔwo ƒe xexlẽme le numiemie ɖeka me, (C) Agba ƒe xexlẽme le numiemie ɖeka me, (D) Chlorophyll a ƒe agbɔsɔsɔ (mg g-1 fr wt), (E) Chlorophyll b ƒe agbɔsɔsɔ (mg g-1 fr wt), (F) Carotenoid ƒe agbɔsɔsɔ bliboa (mg g-1 fr wt). Asixɔxɔwo nye mamã dedie ± SD na nugbagbe atɔ̃ siwo wogbugbɔ wɔ (n = 5). Ŋɔŋlɔdzesi vovovowo ɖe vovototo siwo ɖe dzesi le akɔntabubu nu le atikewɔwɔwo dome fia (p < 0.05).
In situ histochemical localization of reactive oxygen species (ROS; woɖee fia abe hydrogen peroxide [H2O2]) kple free radicals (si woɖena fiana abe superoxide anions [O2•−]) ɖee fia be exogenous zazã le L-ornithine (250 mg/L) ɖe H2O2 ƒe ƒuƒoƒo dzi kpɔtɔ ŋutɔ (96.05 ± 5.33 nmol.g−1 FW; Fig. 5A) kple O2•− (32.69 ± 8.56 nmol.g−1 FW; Fig. 5B) ne wotsɔe sɔ kple numiemie siwo ŋu dɔlélea mele o (173.31 ± 12.06 kple 149.35 ± 7.94 nmol.g−1 FW, le wo nɔewo yome) kple numiemie siwo wotsɔ 50 mg/L si nye fungicide si wodzrana da dɔe ƒe ƒuƒoƒo Rizolex-T (170.12 ± 9.50 kple 157.00 ± 7.81 nmol.g−1 fr wt, le wo nɔewo yome) le gaƒoƒo 72 me. H2O2 kple O2•− ƒe agbɔsɔsɔ gbogbo aɖewo ƒo ƒu ɖe hpt te (Fig. 5A, B). Nenema ke TCA-based malondialdehyde (MDA) dodokpɔ ɖee fia be S. sclerotiorum-dɔlékui bean numiemiewo ƒo MDA ƒe agbɔsɔsɔ si lolo wu (113.48 ± 10.02 nmol.g fr wt) nu ƒu ɖe woƒe aŋgbawo me (Fig. 5C). Ke hã, L-ornithine zazã le gota ɖe lipid peroxidation dzi kpɔtɔ ŋutɔ abe alesi MDA ƒe agbɔsɔsɔ dzi ɖe kpɔtɔ le numiemie siwo ŋu wowɔ atike ɖo me ene (33.08 ± 4.00 nmol.g fr wt).
Fig. 5. Ŋusẽ si exogenous L-ornithine zazã kpɔna ɖe oxidative stress kple non-enzymatic antioxidant defense mechanisms ƒe dzesi veviwo dzi le bean aŋgba siwo ŋu S. sclerotiorum le le 72 h le dɔlélea xɔxɔ vɔ megbe le dzoxɔxɔ ƒe nɔnɔmewo me. (A) Haidrodzin peroxide (H2O2; nmol g−1 FW) le 72 hpt, (B) superoxide anion (O2•−; nmol g−1 FW) le 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) le 72 hpt, (D) phenol siwo katã me wodea tsii (mg GAE g−1 FW) le 72 hpt, (E) flavonoid siwo katã me wodea tsii (mg RE g−1 FW) le 72 hpt me, (F) amino acid siwo katã me wodea tsii o (mg g−1 FW) le 72 hpt me, kple (G) proline ƒe agbɔsɔsɔ (mg g−1 FW) le 72 hpt me. Asixɔxɔwo tsi tre ɖi na nugbagbevi 5 siwo wogbugbɔ wɔ (n = 5) ƒe mama dedie ± vovototo si woɖo ɖi (mamã dedie ± SD). Ŋɔŋlɔdzesi vovovowo ɖe vovototo siwo ɖe dzesi le akɔntabubu nu le atikewɔwɔwo dome fia (p < 0.05).