Barokun in ye ɲinini barokun dɔ ye min tɔgɔ ye ko “Ka sɛnɛfɛnw ni fɛnɲɛnamafagalanw kɛlɛli ɲɛ”, barokun 5 bɛɛ lajɛ
Funteni jiri bana nekɔrɔsi sababuyalan Sclerotinia sclerotiorum (Lib.) de Bary bɛ baara kɛ ni fɛɛrɛ caman ye walasa ka jatigila jiri suguya caman bana. Nin ɲinini in b’a jira ko diyamini L-ornithine, n’o ye amino acide ye min tɛ farikolojɔli ye, n’o bɛ amino acide nafama wɛrɛw labɛnni lawuli, o ye furakɛli fɛɛrɛ wɛrɛ ye walasa ka Phaseolus vulgaris L. ka jaabi molekiyɔmu, farikolo ani biyokimiki ka jaabiw bonya ka ɲɛsin nɔgɔ finman ma min bɛ sɔrɔ Pseudomonas sclerotiorum fɛ. Kɔrɔbɔli minnu kɛra in vitro la, olu y’a jira ko L-ornithine ye S. pyrenoidosa ka misɛti falenni bali kosɛbɛ cogo la min bɛ bɔ a tata hakɛ la. Ka fara o kan, a bɛ se ka dɔ bɔ kosɛbɛ nɔgɔ finman juguya la serili cogoyaw la. Ka fara o kan, L-ornithine ye jiri furakɛlenw falenni lawuli, o b’a jira ko L-ornithine hakɛ sɛgɛsɛgɛlenw tun tɛ jiriw baga ye minnu furakɛra. Ka fara o kan, L-ornithine ye dɔ fara farikolo tangalanw jirali kan minnu tɛ anzimu ye (fenolikiw ni flavonoidw bɛɛ lajɛlen) ani farikolo tangalanw (catalase (CAT), peroxidase (POX), ani polyphenol oxidase (PPO)), ani ka dɔ fara farikolo tangalanw ni ɲɔgɔn cɛ jamu saba jirali kan (PvCAT1, PvSOD, ani PvGR). Ka fara o kan, in silico sɛgɛsɛgɛli y’a jira ko oxaloacetate acetylhydrolase (SsOAH) furakisɛ dɔ bɛ S. sclerotiorum jamu kɔnɔ, o ni Aspergillus fijiensis (AfOAH) ani Penicillium sp. (PLOAH) baarakɛcogo sɛgɛsɛgɛli siratigɛ la, yɔrɔ maralenw, ani topidiya siratigɛ la. A nafa ka bon kosɛbɛ ka a dɔn ko L-ornithine farali pɔmitɛri dextrose (PDB) fɛnɲɛnamafagalan kan, o ye dɔ bɔ kosɛbɛ SsOAH jamu jirali la S. sclerotiorum mycelia kɔnɔ. O cogo kelen na, L-ornithine tali kɛnɛma, o ye dɔ bɔ kosɛbɛ SsOAH jamu jirali la ɲɛgɛnw na minnu lajɛra jiri furakɛlenw na. A laban na, L-ornithine tali ye asidi ɔkisaliki bɔli dɔgɔya kosɛbɛ PDB fɛnɲɛnamafagalanw ni furabulu banakisɛw bɛɛ la. Kuncɛli la, L-ornithine jɔyɔrɔ ka bon kosɛbɛ redox cogoya sabatili la ka fara jiri banakisɛw ka lafasali jaabi bonya kan. Nin ɲinini in jaabiw bɛ se ka dɛmɛ don fɛɛrɛ kuraw labɛnni na, minnu tɛ sigida lakana, walasa ka nɔgɔ finmanw kɛlɛ ani k’a nɔ nɔgɔya shɛfanw dilanni ni sɛnɛfɛn wɛrɛw la.
Nɔgɔ finman, min bɛ sɔrɔ ɲɛgɛnɛsira la min bɛ wele ko Sclerotinia sclerotiorum (Lib.) de Bary, o ye bana ye min bɛ tiɲɛni kɛ, min bɛ sɔrɔ dɔgɔya, min bɛ faratiba lase diɲɛ shɛfan (Phaseolus vulgaris L.) sɔrɔli ma (Bolton et al., 2006). Sclerotinia sclerotiorum ye bɔgɔ kɔnɔ ɲɛgɛnw banakisɛw dɔ ye min kɛlɛli ka gɛlɛn kosɛbɛ, a jatigila ka ca ni jiri suguya 600 ni kɔ ye ani a bɛ se ka jatigila farikolo yɔrɔw sɔgɔ teliya la cogo la min tɛ kɛrɛnkɛrɛnnen ye (Liang ni Rollins, 2018). Ni a kɛra cogo jugu la, a bɛ tɛmɛ a ka ɲɛnamaya kɛcogo gɛlɛnba dɔ fɛ, a bɛ to sunɔgɔ la waati jan kɔnɔ i n’a fɔ fɛn nɛrɛma, gɛlɛn, kisɛ ɲɔgɔnna minnu bɛ wele ko ‘sclerotia’ dugukolo kɔnɔ walima i n’a fɔ jirisun finmanw, nɛrɛmuguma minnu bɛ falen jiriw la minnu bananen don misɛli walima u jirisunba kɔnɔ (Schwartz et al., 2005). S. sclerotiorum bɛ se ka sɔgɔsɔgɔninjɛ dilan, o b’a to a bɛ se ka balo forow la banakisɛ bɛ minnu na waati jan kɔnɔ ani ka to bana waati la (Schwartz et al., 2005). Sklerotiw nafa ka bon balofɛnw na, u bɛ se ka to dugukolo kɔnɔ waati jan kɔnɔ, wa u bɛ kɛ boloci fɔlɔ ye banakisɛw nataw la (Schwartz et al., 2005). Ni cogoya ɲuman don, sklerotiw bɛ falen ka fiɲɛbɔlanw bɔ minnu bɛ se ka jiri sanfɛyɔrɔw bɛɛ minɛ, hali a falenw, a jiribolow walima a jiribolow (Schwartz et al., 2005).
Sclerotinia sclerotiorum bɛ fɛɛrɛ caman kɛ walasa ka banakisɛ don a jatigila jiriw la, o bɛ kɛ ni ko caman ye minnu bɛ kɛ ɲɔgɔn fɛ k’a ta sklerotiya falenni na ka se bana taamasiɲɛw sɔrɔli ma. A daminɛ na, S. sclerotiorum bɛ sɔgɔsɔgɔninjɛ jɛgɛlenw (minnu bɛ wele ko ascospores) bɔ ɲɛgɛnw na minnu bɛ wele ko apothecia, olu bɛ kɛ fiɲɛ fɛ ka kɛ skleroti ye min tɛ lamaga jiriw nɔgɔ banakisɛw kan (Bolton et al., 2006). O kɔfɛ, ɲɛgɛn bɛ asidi ɔkisaliki bɔ, o min ye banakisɛfagalan ye, walasa ka jiri selilɛri kogo pH kɔlɔsi, ka anzimu tiɲɛni ni farikolo yɔrɔw binkanni sabati (Hegedus ni Rimmer, 2005), ani ka jatigila jiri ka oksidan bɔli bali. O asidi kɛcogo in bɛ jiriw ka selilɛri kogo fanga dɔgɔya, ka sigida ɲuman di ɲɛgɛnw ka selilɛri kogo tiɲɛbagaw (CWDE) baara kɛcogo ɲuman ni baarakɛcogo ɲuman ma, o bɛ kɛ sababu ye banakisɛ in ka se sɔrɔ farikolo barika kan ani ka don jatigila farikolo yɔrɔw la (Marciano et al., 1983). Ni S. sclerotiorum donna a kɔnɔ, a bɛ CWDE caman bɔ, i n’a fɔ poligalacturonase ani cellulase, olu bɛ a jɛnsɛnni nɔgɔya farikolo yɔrɔw la minnu bananen don ani ka farikolo yɔrɔw faga. Joginda ni hyphal mats ka taa ɲɛ, o bɛ na ni nɔgɔ finman taamasiɲɛw taamasiɲɛw ye (Hegedus ni Rimmer, 2005). O waati kelen na, jatigila jiriw bɛ banakisɛw ni ɲɔgɔn cɛ molekiyɔmu cogoyaw (PAMP) dɔn misali dɔnni fɛɛrɛw fɛ (PRR), o bɛ taamasiɲɛw kɛcogo caman daminɛ minnu laban bɛ lafasali jaabiw lawuli.
Hali ni banaw kɛlɛli cɛsiriw kɛra san tan caman kɔnɔ, banakisɛfagalan minnu bɛ se ka bana kɛlɛ, olu dɛsɛ bɛ to shɛfan na, i n’a fɔ jago sɛnɛfɛn tɔw, k’a sababu kɛ banakisɛ in kɛlɛli ye, a ka ɲɛnamaya ani a ka se ka ladamu. O la, banaw kunbɛnni ka gɛlɛn kosɛbɛ, wa a bɛ fɛɛrɛ jɛlen de wajibiya, min bɛ kɛ ni fɛɛrɛ caman ye, min kɔnɔ, laadala walew, fɛnɲɛnamaw kɛlɛli, ani ɲɛgɛnw fagalanw faralen don ɲɔgɔn kan (O’Sullivan et al., 2021). Nɔgɔ finmanw kɛlɛli ni furakisɛw ye, o de ka fisa kosɛbɛ barisa ɲɛgɛnɛsiraw, ni u kɛra ka ɲɛ ani waati bɛnnen na, u bɛ se ka bana jɛnsɛnni kunbɛn ka ɲɛ, ka banakisɛw juguya dɔgɔya, ka sɔrɔ dɔgɔya. Nka, ni u tali kojugu ani ka u jigi da ɲɛgɛnɛsiraw kan kojugu, o bɛ se ka kɛ sababu ye ka S. sclerotiorum suguyaw bɔ kɛnɛ kan minnu bɛ se ka bana kɛlɛ, ka nɔ jugu bila fɛnɲɛnamaw la minnu tɛ laɲini ye, dugukolo kɛnɛya, ani jiko la (Le Cointe et al., 2016; Ceresini et al., 2024). O la, fɛɛrɛ wɛrɛw sɔrɔli minnu tɛ sigida lakana, o kɛra ko fɔlɔ ye.
Poliyaminiw (PAs), i n’a fɔ putrecine, spermidine, spermine, ani cadaverine, olu bɛ se ka kɛ fura wɛrɛw ye minnu bɛ se ka kɛ fura wɛrɛw ye minnu bɛ se ka kɛ sababu ye ka jiriw banakisɛw kɛlɛ minnu bɛ sɔrɔ dugukolo kɔnɔ, o la, u bɛ dɔ bɔ pewu walima a yɔrɔ dɔ la, ka dɔ bɔ farikolojidɛsɛ fura faratilenw tali la (Nehela et al., 2024; Yi et al., 2024). Fɛnɲɛnɛma sanfɛtaw la, PAw sen bɛ farikoloɲɛnajɛ kɛcogo caman na i n’a fɔ, nka u dan tɛ, selilu tilali, danfara, ani jaabi di abiyɔtiki ni biyotiki degunw ma (Killiny ni Nehela, 2020). U bɛ se ka kɛ farikolo tangalanw ye, ka dɛmɛ don ka oksizɛni suguya minnu bɛ se ka kɛ (ROS), ka redox homeostasis (redox homeostasis) mara (Nehela ni Killiny, 2023), ka jamu minnu bɛ tali kɛ lafasali la (Romero et al., 2018), ka farikoloɲɛnajɛ sira suguya caman labɛn (Nehela ni Killiny, 2023), ka fitohormones endogènes (Nehela ni Killiny, 2023) sɛmɛntiya (Nehela ni Killiny, 2019), ka farikolo tangacogo sɔrɔlen (SAR) sigi sen kan, ani ka jiriw ni banakisɛw ka jɛɲɔgɔnyaw ɲɛnabɔ (Nehela ni Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). A ka kan ka kɔlɔsi ko PAw ka fɛɛrɛ kɛrɛnkɛrɛnnenw n’u jɔyɔrɔw jiriw lakanani na, olu bɛ danfara ka kɛɲɛ ni jiri suguyaw ye, banakisɛw ani sigida cogoyaw. PA min ka ca jiriw la, o bɛ kɛ biyosintɛzi ye ka bɔ poliyamini L-ɔrnitini nafama na (Killiny ni Nehela, 2020).
L-ornithine jɔyɔrɔ ka ca jiriw falenni ni u yiriwali la. Misali la, kalan tɛmɛnenw y’a jira ko tiga la (Oryza sativa), ɔrinitini bɛ se ka kɛ sababu ye ka azote lasegin (Liu et al., 2018), tiga sɔrɔta, a jogo ni a kasa (Lu et al., 2020), ani ji degun jaabi (Yang et al., 2000). Ka fara o kan, L-ornithine tali kɛnɛma ye ja muɲuni bonya kosɛbɛ sukaro (Beta vulgaris) la (Hussein et al., 2019) ani ka kɔgɔ degun nɔgɔya soja (Allium Cepa) (Çavuşoǧlu ani Çavuşoǧlu, 2021) ani kafe (Anacardium occidentale) la jiriw (da Rocha ni a ɲɔgɔnnaw, 2012). L-ornithine jɔyɔrɔ min bɛ se ka kɛ degun abiyɔtikiw lafasali la, o bɛ se ka kɛ sababu ye a sen don porolini dalajɛli la jiri furakɛlenw na. Misali la, jamu minnu bɛ tali kɛ porolini farikoloɲɛnajɛ la, i n’a fɔ ɔrnitini delta aminotransferase (delta-OAT) ani porolini dehidrojɛnazi (ProDH1 ani ProDH2) jamu, a fɔra ka tɛmɛ ko olu sen bɛ Nicotiana benthamiana ni Arabidopsis thaliana lafasali la ka ɲɛsin Pseudomonas syringae suguyaw ma minnu tɛ jatigila ye (Senthil-Kumar ani Misɔri, san 2012). Faan wɛrɛ fɛ, funu ornithine decarboxylase (ODC) de ka kan banakisɛw falenni na (Singh et al., 2020). Fusarium oxysporum f. ka ODC laɲini. sp. lycopersici via host-induced gene silencing (HIGS) ye tomati jiriw ka se ka Fusarium wilt kɛlɛ kosɛbɛ (Singh et al., 2020). Nka, ɔrinitini tali kɛnɛma jɔyɔrɔ bɛ se ka kɛ biyotiki degunw na i n’a fɔ fitopathogènes, o ma sɛgɛsɛgɛ ka ɲɛ. Min nafa ka bon kosɛbɛ, o ye ko ɔrinitini nɔ minnu bɛ banaw kɛlɛli la ani a ni a ɲɔgɔnna biyokimiki ni farikoloɲɛnajɛ ko minnu bɛ kɛ, olu fanba ma sɛgɛsɛgɛ fɔlɔ.
S. sclerotiorum banakisɛ ka gɛlɛya faamuyali min bɛ sɔrɔ shɔw la, o nafa ka bon walasa ka a kɛlɛcogo ɲumanw labɛn. Nin kalan in kɔnɔ, an y’a ɲini ka diyamine L-ornithine jɔyɔrɔ dɔn min bɛ se ka kɛ sababu ye ka sɛnɛfɛnw tangacogo n’u ka se ka Sclerotinia sclerotiorum banakisɛ kɛlɛ. An bɛ hakilina ta ko, ka fara jiri banakisɛw ka lafasali jaabiw bonya kan, L-ornithine fana jɔyɔrɔ ka bon redox cogoya sabatili la. An b’a jira ko L-ornithine nɔ minnu bɛ se ka sɔrɔ, olu bɛ tali kɛ farikolo tangalanw lafasali fɛɛrɛw labɛnni na minnu bɛ sɔrɔ anzimu ni anzimu tɛ minnu na ani ka dankari kɛ ɲɛgɛnw ka banakisɛfagalanw/banakisɛfagalanw ni u ni ɲɔgɔn cɛ farikolojɔlifɛnw na. Nin L-ornithine ka baarakɛcogo fila in b’a kɛ kandida ye min bɛ layidu ta fɛɛrɛ sabatilen dɔ la walasa ka nɔgɔ finman nɔw nɔgɔya ani ka sɛnɛfɛnw sɛnɛ minnu bɛ kɛ tuma bɛɛ, olu ka se ka banakisɛfagalan barikama in kɛlɛ. Nin ɲinini in jaabiw bɛ se ka dɛmɛ don fɛɛrɛ kuraw labɛnni na, minnu tɛ sigida lakana, walasa ka nɔgɔ finmanw kɛlɛ ani k’a nɔ nɔgɔya sɛnɛfɛnw bɔli la.
Nin kalan in kɔnɔ, shɛfan suguya dɔ min bɛ se ka sɔrɔ jago la, n’o ye Giza 3 (Phaseolus vulgaris L. cv. Giza 3) ye, o kɛra k’a kɛ k’a lajɛ. Kisɛ kɛnɛmanw dira ni ɲumanya ye ka bɔ sɛnɛfɛnw ɲinini baarada fɛ, foro sɛnɛfɛnw ɲinini baarada (FCRI), sɛnɛko ɲininikɛyɔrɔ (ARC), Misira. Kisɛ duuru danna plastiki dagaw kɔnɔ (kɔnɔna cɛmancɛ ye santimɛtɛrɛ 35 ye, a juguman ye santimɛtɛrɛ 50 ye) minnu falen don S. sclerotiorum banakisɛ bɛ dugukolo la, kɛnɛyaso cogoyaw la (25 ± 2 °C, nɛnɛ hakɛ 75 ± 1%, lɛrɛ 8 yeelen/lɛrɛ 16 dibi). Tile 7–10 danni kɔfɛ (DPS), jirisunw tun bɛ fin walasa ka jirisun fila dɔrɔn to minnu falen bɛ cogo kelen na ani furabulu saba minnu bonyalen don kosɛbɛ daga kelen-kelen bɛɛ kɔnɔ. Ji tun bɛ kɛ daga kɔnɔ jiriw bɛɛ la siɲɛ kelen dɔgɔkun fila o dɔgɔkun fila, ka balo kɛ kalo o kalo ni hakɛ fɔlen ye suguya dilen na.
Walasa ka L-ornithinediamine (a bɛ fɔ fana ko asidi (+)-(S)-2,5-diaminopentanoïque; Sigma-Aldrich, Darmstadt, Alimanjamana) hakɛ miligaramu 500/L labɛn, aw bɛ miligaramu 50 kɛ fɔlɔ ji wulilen mililitiri 100 na. O kɔfɛ, stock solution tun bɛ dilan ka sɔrɔ ka baara kɛ ni a ye kɔrɔbɔli nataw la. Kuma surun na, L-ornithine hakɛ sigiyɔrɔma wɔɔrɔ (miligaramu 12,5, 25, 50, 75, 100 ani 125/L) sɛgɛsɛgɛra in vitro. Ka fara o kan, ji dilanlen sterili kɛra fura ye min tɛ se ka furakɛ (Mock) ani jagokɛla ka funufagala “Rizolex-T” 50% mugu min bɛ se ka jigin (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Egypt) kɛra fura ye min bɛ se ka furakɛ. Jagokɛla ka ɲɛgɛnɛsiralabanaw “Rizolex-T” sɛgɛsɛgɛra in vitro la, a hakɛ duuru (2, 4, 6, 8 ani 10 mg/L).
Shɔ jirisunw ni jirisunw minnu bɛ sɔrɔ tuma caman na, n’u bɛ nɔgɔ finman taamasiɲɛw jira (banakisɛ sɔrɔli hakɛ: 10–30%), olu misaliw lajɛra jagokɛyɔrɔw la. Hali ni jiriw fɛn minnu banana, olu fanba dɔnna suguya/suguya fɛ (jagokɛla suguya min bɛ se ka bana sɔrɔ Giza 3), dɔw fana, kɛrɛnkɛrɛnnenya la minnu sɔrɔla sigida suguw la, olu tun ye suguya dɔnbaliw ye. Banakisɛfagalan minnu lajɛra, olu fɔlɔ kɛra banakisɛfagalan ye sanfɛ ni hipokuloriti sodiyɔmu jilama 0,5% ye miniti 3 kɔnɔ, ka sɔrɔ ka u ko siɲɛ caman ni ji wulilen sterili ye ka u ko ka u ja ni filɛtiri papiye sterili ye walasa ka ji tɛmɛnen bɔ. O kɔfɛ, farikolo yɔrɔ banakisɛw tigɛra ka kɛ yɔrɔ misɛnninw ye ka bɔ cɛmancɛ la (yɔrɔ kɛnɛmanw ni farikolo yɔrɔ banakisɛlenw cɛ), ka u falen pɔmitɛri dekstrose agar (PDA) fɛnɲɛnamafagalan kan ka u bila 25 ± 2 °C la ni yeelen ye lɛri 12/lɛrɛ 12 dibi la tile 5 kɔnɔ walasa ka sklerose (sklerotie) sɔrɔli lawuli. Miseli tip fɛɛrɛ fana kɛra ka ɲɛgɛnw bɔlenw saniya ka bɔ sɛnɛfɛnw na minnu ɲagaminen don walima minnu nɔgɔlen don. Funteni bɔlen saniyalen in fɔlɔla ka dɔn ka da a ka laadala cogoyaw kan, o kɔfɛ, a jirala ko a ye S. sclerotiorum ye ka da a kan ka da a ka fɛn fitininw kan. A laban na, isolati saniyalenw bɛɛ sɛgɛsɛgɛra banakisɛ sɔrɔli la shɛfan suguya caman kan min bɛ se ka bana sɔrɔ Giza 3 walasa ka Koch ka hakilinaw dafa.
Ka fara o kan, S. sclerotiorum isolate min bɛ don kosɛbɛ (isolate #3) o sabatira ka t’a fɛ ka da internal transcribed spacer (ITS) sinsinni kan i n’a fɔ White ni a ɲɔgɔnnaw y’a ɲɛfɔ cogo min na, Baturo-Ciesniewska et al., 2017. Kuma surun na, fɛn minnu bɔra ɲɔgɔn na, olu tun bɛ sɛnɛ pɔmitɛri dekistrose (PDB) kɔnɔ ka u bila 25 ± 2 °C la tile 5–7 kɔnɔ. O kɔfɛ, ɲɛgɛnɛsiraw lajɛra, ka u sama foromazi fini na, ka u ko siɲɛ fila ni ji sterili ye, ka u ja ni filɛri papiye sterili ye. ADN jamu bɔra ɲɔgɔn na ni Quick-DNATM Fungal/Bacterial Miniprep Kit ye ​​(Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). O kɔfɛ, ITS rDNA yɔrɔ bonyalen don ni primer fila kɛrɛnkɛrɛnnen ITS1/ITS4 ye (TCCGTAGGTGAACCTGCGG TCCTCCGCTTATTGATATGC; hakɛ makɔnɔnen: 540 bp) ( Baturo-Ciesniewska et al., 2017 ). PCR fura saniyalenw bilala ka kɛ ɲɔgɔndan ye (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). ITS rDNA sinsinniw kɛra ɲɔgɔn kɔ sira fila fɛ ni Sanger sinsinni fɛɛrɛ ye. O kɔfɛ, ɲininkaliw sigiyɔrɔma minnu lajɛra, olu sumalen don ni kunnafoni labanw ye minnu bɛ GenBank kɔnɔ ani jamana ka biyoteknoloji kunnafoniko baarada la (NCBI, http://www.ncbi.nlm.nih.gov/gene/) ni porogaramu BLASTn ye. Ɲininkali kɛcogo sumalen don ni S. sclerotiorum suguya/isolate 20 wɛrɛw ye minnu sɔrɔla kunnafonidilan labanw na NCBI GenBank kɔnɔ (Taabolo S1 faralen ɲɔgɔn kan) ni ClustalW ye Molecular Evolutionary Genetics Analysis Package kɔnɔ (MEGA-11; version 11) (Kumar et al., 2024). Ɲɛnabɔli sɛgɛsɛgɛli kɛra ni fɛɛrɛ ye min bɛ se ka kɛ kosɛbɛ ani ni nukilitɔn caman cili modɛli ye min bɛ se ka segin waati kɔnɔ (Nei ni Kumar, 2000). Jiri min ka log-likelihood ka ca, o jiralen don. Jiri fɔlɔ min bɛ kɛ ka ɲɛsin heuristic ɲinini ma, o bɛ sugandi ni jiri sugandili ye min ka log-likelihood ka bon sigiɲɔgɔn-joining (NJ) jiri (Kumar et al., 2024) ni maximum parsimony (MP) jiri cɛ. NJ jiri in jɔra ni yɔrɔjan matiriyali fila-fila ye min jatebɔra ni waati-ko-segincogo caman ye (Nei ni Kumar, 2000).
L-ornithine ni banakisɛfagalan “Rizolex-T” ka banakisɛfagalan baara jateminɛna in vitro ni agar diffusion fɛɛrɛ ye. Fɛɛrɛ : Aw bɛ L-ornithine (miligaramu 500/L) hakɛ bɛnnen ta ka a ɲagami ka ɲɛ ni PDA balolan mililitiri 10 ye walasa ka furajiw labɛn minnu hakɛ laban ye miligaramu 12,5, 25, 50, 75, 100 ani 125 ye. Fɛnɲɛnamafagalan “Rizolex-T” (miligaramu 2, 4, 6, 8 ani 10/L) hakɛ duuru ni ji dilanlen sterili kɛra a kunbɛncogo ye. O fɛnɲɛnamafagalan jɔlen kɔfɛ, Sclerotinia sclerotiorum culture ka miseli dakun kura labɛnna, min janya ye milimɛtɛrɛ 4 ye, o bilala Petri minɛn cɛmancɛ la, ka a sɛnɛ 25±2°C la fo ka se misɛli ka Petri minɛn kɔrɔlen bɛɛ datugu, o kɔfɛ, ɲɛgɛnw falenni sɛbɛnna. S. sclerotiorum ka bonya radiyali balili kɛmɛsarada la jatebɔ kɛ ni daɲɛ 1 ye:
Kɔrɔbɔli in kɛra siɲɛ fila, ni fɛnɲɛnamafagalan wɔɔrɔ ye minnu bɛ kɛ kɔrɔsili/kɔrɔbɔli kulu kelen-kelen bɛɛ la ani daga duuru (jiri fila daga kelen kɔnɔ) fɛnɲɛnamafagalan kelen-kelen bɛɛ la. Biologique replication kelen-kelen bɛɛ sɛgɛsɛgɛra siɲɛ fila (férique replication fila) walasa ka a jira ko kɔrɔbɔli jaabiw bɛ se ka kɛ tiɲɛ ye, ka da u kan ani ka se ka segin u cogo kɔrɔ la. Ka fara o kan, probit kɔsegin sɛgɛsɛgɛli kɛra ka jatebɔ kɛ ni balili hakɛ tilancɛ hakɛ ye (IC50) ani IC99 (Prentice, 1976).
Walasa ka L-ornithine seko jateminɛ kɛnɛyaso kɔnɔ, daga kɔnɔ k’a lajɛ fila tugu-tugu ɲɔgɔn na. Kuma surun na, dagaw tun bɛ fa ni bɔgɔ-cɛncɛn bɔgɔ sterilizelen ye (3:1) ka sɔrɔ ka u boloci ni S. sclerotiorum sɛnɛfɛn labɛnni kura ye. Fɔlɔ, S. sclerotiorum isolati min bɛ don kosɛbɛ (isolate #3) o sɛnɛna ni sclerotium kelen tigɛ ye tilayɔrɔ fila la, k’a bila a ɲɛda duguma PDA kan, ka a bila 25°C la dibi la tuma bɛɛ (lɛrɛ 24) tile 4 kɔnɔ walasa ka misɛli falenni lawuli. O kɔfɛ, agar plug naani minnu janya ye milimɛtɛrɛ 5 ye, olu bɔra ɲɛfɛla la ka u boloci ni malo ni tigamugu ɲagaminen sterili garamu 100 ye (1:1, v/v) ani ka fiyɛlifɛnw bɛɛ bila 25 ± 2°C la lɛrɛ 12 yeelen/lɛrɛ 12 dibi la tile 5 kɔnɔ walasa ka sklerotiya sɔrɔli lawuli. Filɛriw bɛɛ kɔnɔfɛnw tun bɛ ɲagami kosɛbɛ walasa u ka kɛ kelen ye sani u ka bɔgɔ fara u kan. O kɔfɛ, kolosinsinnan ɲagaminen garamu 100 farala daga kelen-kelen bɛɛ kan walasa banakisɛw ka to ka caya. Ji donna daga minnu bolocilen don, olu jiginna walasa ka ɲɛgɛnw falenni baara, ka u bila jirisunw na tile 7 kɔnɔ.
O kɔfɛ, Giza 3 suguya kisɛ duuru danna daga kelen-kelen bɛɛ kɔnɔ. Ni daga minnu furakɛra ni L-ornithine ni Rizolex-T ye, olu ye kisɛ sterilizelenw su fɔlɔ fo lɛri fila o furakisɛ fila ninnu jilama la ni IC99 hakɛ laban ye miligaramu 250/L ni miligaramu 50/L ɲɔgɔn ye, o kɔfɛ, u bɛ ja fiɲɛ na lɛri kelen kɔnɔ ka sɔrɔ ka dan. Faan wɛrɛ fɛ, kisɛw tun bɛ su ji dilanlen na min tɛ se ka furakɛ, k’a kɛ a kɛlɛcogo jugu ye. Tile 10 tɛmɛnen kɔ, sani ji fɔlɔ ka kɛ, jirisunw tun bɛ fin ka bɔ, ka jirisun fila dɔrɔn to daga kelen-kelen bɛɛ kɔnɔ. Ka fara o kan, walasa ka S. sclerotiorum banakisɛ sɔrɔ, shɛfan jiribolo minnu bɛ yiriwali waati kelen na (tile 10) olu tigɛra yɔrɔ fila la minnu tɛ kelen ye ni sɔgɔsɔgɔninjɛ sterili ye ani ka koloni sɔgɔlen ɲagaminen garamu 0,5 ɲɔgɔn bila joginda kelen-kelen bɛɛ la, o kɔfɛ, nɛnɛba kɛra walasa ka banakisɛw ni banaw lawuli jiri bolocilenw bɛɛ la. Kɔlɔsili jiriw joginna o cogo kelen na ani ka sɔgɔsɔgɔninjɛ ɲagaminen sterili, kolonibali hakɛ kelen (garamu 0,5) bila joginda kɔnɔ, ka mara nɛnɛba kɔnɔ walasa ka sigida ladege banaw sɔrɔli kama ani ka bɛnkan sabati furakɛli kuluw ni ɲɔgɔn cɛ.
Furakɛli fɛɛrɛ : Aw bɛ shɛfan kisɛw ji kɛ ni L-ornithine (miligaramu 250/l) jilama mililitiri 500 ye walima ni ɲɛgɛnɛsira Rizolex-T (miligaramu 50/l) jilama ye, ni aw ye dugukolo ji kɛ, o kɔfɛ, aw bɛ segin furakɛli kan siɲɛ saba tile 10 cɛ. Kɔlɔsilikɛla minnu furakɛra ni furakisɛ ye, olu jiginna ni ji dilanlen mililitiri 500 ye. Furakɛli bɛɛ kɛra serili cogoyaw la (25 ± 2°C, 75 ± 1% nɛnɛ hakɛ, ani fotota waati ye lɛrɛ 8 yeelen/lɛrɛ 16 dibi ye). Ji tun bɛ daga bɛɛ la dɔgɔkun fila o dɔgɔkun fila, ka furakɛli kɛ kalo o kalo ni NPK bagaji balannen ye (20-20-20, ni kisɛkisɛ ni TE mikroelements 3,6% ye; Zain Seeds, Egypt) ni a hakɛ ye garamu 3–4 ye l/l kɔnɔ ni furabuluw fiyɛli ye ka kɛɲɛ ni suguya kɛrɛnkɛrɛnnen ka laadilikanw ye ani a dilannikɛla ka laadilikanw. Fo n’a fɔra cogo wɛrɛ la, furabulu kɔgɔlen minnu falen don kosɛbɛ (bulu 2nan ni 3nan ka bɔ sanfɛ) olu lajɛra ka bɔ fɛnɲɛnamaw ka ɲɔgɔn falen kelen-kelen bɛɛ la lɛrɛ 72 furakɛli kɔfɛ (hpt), ka u kɛ kelen ye, ka u fara ɲɔgɔn kan ani ka u mara -80 °C la walasa ka sɛgɛsɛgɛli wɛrɛw kɛ i n’a fɔ, nka a dan tɛ o la, oksidan degun jiralanw sigiyɔrɔmali in situchimique, lipid peroxidation, enzymatique et non-enzymatique farikolo tangalanw ni jeninida jiracogo.
Nɔgɔ finman banakisɛw fanga jateminɛna dɔgɔkun o dɔgɔkun tile 21 boloci kɔfɛ (dpi) ni sɛgɛsɛgɛli ye min ye 1–9 ye (Taabolo S2 faralen ɲɔgɔn kan) min sinsinnen bɛ Petzoldt ni Dickson ka sɛgɛsɛgɛli kan (1996) min sɛmɛntiyalen don Teran ni a ɲɔgɔnnaw fɛ. (2006) la. Kuma surun na, shɛfan jiriw buluw ni u bolow sɛgɛsɛgɛra k’a daminɛ boloci yɔrɔ la walasa ka tugu joginda taabolo kɔ u ni ɲɔgɔn cɛ. O kɔfɛ, joginda janya ka bɔ boloci yɔrɔ la ka taa a bila yɔrɔ jan na, a jiribolo walima a bolo la, o jateminɛna ka sɔrɔ ka 1–9 sɔrɔ ka da joginda sigiyɔrɔ kan, o yɔrɔ la (1) y’a jira ko banakisɛ yelen si tɛ boloci yɔrɔ kɛrɛfɛ ani (2–9) y’a jira ko joginda hakɛ bɛ bonya dɔɔni dɔɔni ani ka taa ɲɛ node/internodes sira fɛ (Taabolo faralen S2). O kɔfɛ, nɔgɔ finman banakisɛ fanga jiginna ka kɛ kɛmɛsarada la ni formula 2 ye:
Ka fara o kan, yɔrɔ min bɛ bana taabolo kurulen (AUDPC) jukɔrɔ, o jate kɛra ni formula ye (Shaner ni Finney, 1977), min bɛrɛbɛnna kɔsa in na shɛfan jɛlen toli finman kama (Chauhan et al., 2020) ni daɲɛ 3 ye:
Yɔrɔ min na Yi = bana juguya waati ti la, Yi+1 = bana juguya waati nata la ti+1, ti = sumani fɔlɔ waati (tile kɔnɔ), ti+1 = sumani nata waati (tile kɔnɔ), n = waati bɛɛ hakɛ walima kɔlɔsili yɔrɔw. Shɔ jiri falencogo jateminɛw i n’a fɔ jiri janya (santimɛtɛrɛ), jiribolo hakɛ jiri kelen na, ani furabulu hakɛ jiri kelen na, olu sɛbɛnna dɔgɔkun o dɔgɔkun tile 21 kɔnɔ fɛnɲɛnamaw ka ɲɔgɔndan bɛɛ la.
Biologique replicate kelen-kelen bɛɛ la, furabuluw tacogo (bulu filanan ni sabanan minnu falen don kosɛbɛ ka bɔ sanfɛ) olu lajɛra tile 45 furakɛli kɔfɛ (tile 15 furakɛli laban kɔfɛ). Biologique replicate kelen-kelen bɛɛ tun ye daga duuru ye (jiri fila bɛ daga kelen kɔnɔ). O fɛnɲɛnamafagalan miligaramu 500 ɲɔgɔn kɛra ka yeelenbɔlanw (kulorofili a, kulorofili b ani karotɛnoyi) bɔ ni asetɔni 80% ye 4°C la dibi la. Lɛrɛ 24 kɔfɛ, u ye fɛnw kɛ santirifugu ye ka u sanfɛla lajɛ walasa ka kulorofili a, kulorofili b ani karotɛnoyi hakɛ dɔn kolorimɛtiri la ni UV-160A spektrofotomɛtiri ye (Shimadzu Corporation, Japon) ka kɛɲɛ ni (Lichtenthaler, 1987) ka fɛɛrɛ ye, ni u minɛni sumani ye jikuru saba danfaralenw na (A470, A646 ani A663 nm). A laban na, yeelenbɔlanw kɔnɔfɛnw jatebɔ kɛra ni nin formula 4–6 ninnu ye minnu ɲɛfɔra Lichtenthaler fɛ (1987).
Furakɛli kɔfɛ lɛrɛ 72 kɔnɔ (hpt), furabuluw (bulu filanan ni sabanan minnu falen don kosɛbɛ ka bɔ sanfɛ) lajɛra ka bɔ fɛnɲɛnamaw ɲɔgɔnna kelen-kelen bɛɛ la walasa ka perokisidi idɔrɔji (H2O2) ni aniyoni surɔkisidi (O2•−) sigiyɔrɔmali histochimique in situ kɛ. Biologique replicate kelen-kelen bɛɛ tun ye daga duuru ye (jiri fila bɛ daga kelen kɔnɔ). Biologique replicate kelen-kelen bɛɛ sɛgɛsɛgɛra siɲɛ fila (fὲɛrɛko lasegincogo fila) walasa ka fɛɛrɛ in tiɲɛni, dannaya ani a lasegincogo Lajɛ. H2O2 ni O2•− jateminɛna ni 0,1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Alimanjamana) walima nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Alimanjamana) ye, ka kɛɲɛ ni fɛɛrɛw ye minnu ɲɛfɔra Romero-Puertas ni a ɲɔgɔnnaw fɛ. (2004) ani Adama ni a ɲɔgɔnnaw. (1989) ni fɛn misɛnninw b’u la. Walasa ka H2O2 sigiyɔrɔma histochimique sɔrɔ yɔrɔ la, sɛbɛnfuraw donna ni 0,1% DAB ye ni fɛnɲɛnamafagalan ye Tris tampon 10 mM kɔnɔ (pH 7,8) ka sɔrɔ ka u bila so funteni na yeelen na miniti 60 kɔnɔ. Sɛbɛnw jɛlen don TCA 0,15% (v/v) la etanɔli:kulorofɔrimu 4:1 (v/v) la (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) ka sɔrɔ ka u bila yeelen na fo u ka dibi. O cogo kelen na, valvew donna ni fɛnɲɛnamafagalan ye ni 10 mM potasiyɔmu fositɛri tampon ye (pH 7,8) min kɔnɔ 0,1 w/v % HBT bɛ sɔrɔ walasa ka O2•− sigiyɔrɔma histochimique kɛ yɔrɔ la. Aw bɛ sɛbɛndenninw bila yeelen na so funteni na miniti 20 kɔnɔ, o kɔ aw bɛ u jɛya i n’a fɔ a fɔra cogo min na sanfɛ, ka sɔrɔ ka yeelen bɔ fo ka se bulu/violet dibimanw ma. O kulɛri min sɔrɔla, n’o ye bulama ye (i n’a fɔ H2O2 jiralan) walima bulu-violet (i n’a fɔ O2•− jiralan) o fanga jateminɛna ni ja baarakɛcogo pake ImageJ (http://fiji.sc; a sɔrɔla Marisikalo tile 7 san 2024) ka Fidji ta ye.
Malondialdehyde (MDA; i n’a fɔ lipid peroxidation taamasiɲɛ) jateminɛna ka kɛɲɛ ni Du ni Bramlage (1992) ka fɛɛrɛ ye ni fɛn dɔw sɛmɛntiyalen don. Furaw minnu bɔra fɛnɲɛnamaw ka ɲɔgɔn falen kelen-kelen bɛɛ la (bulu filanan ni sabanan minnu falen don kosɛbɛ ka bɔ sanfɛ) olu lajɛra lɛrɛ 72 furakɛli kɔfɛ (hpt). Biologique replicate kelen-kelen bɛɛ tun bɛ ni daga duuru ye (jiri fila bɛ daga kelen kɔnɔ). Biologique replicate kelen-kelen bɛɛ sɛgɛsɛgɛra siɲɛ fila (fὲɛrɛko lasegincogo fila) walasa ka fɛɛrɛ in tiɲɛni, dannaya ani a lasegincogo Lajɛ. Kuma surun na, furabulu mugulen garamu 0,5 kɛra ka MDA bɔli kɛ ni asidi trikuloroasetiki 20% ye (TCA; MilliporeSigma, Burlington, MA, Etazuni) min kɔnɔ, 0,01% butilɛti hidroksitoluyɛni (BHT; Sigma-Aldrich, St. Louis, MO, Etazuni) bɛ min na. O kɔfɛ, MDA hakɛ min bɛ ji sanfɛla la, o jateminɛna ni kolorimɛtiri ye ni a minɛcogo sumani ye 532 ani 600 nm la ni UV-160A spectrophotomètre (Shimadzu Corporation, Japon) ye, o kɔfɛ, a jirala ni nmol g−1 FW ye.
Walasa ka farikolo tangalanw jateminɛ minnu tɛ anzimu ye ani minnu bɛ kɛ ni anzimu ye, furabuluw (bulu filanan ni sabanan minnu falen don kosɛbɛ ka bɔ sanfɛ) lajɛra ka bɔ fɛnɲɛnamaw ka ɲɔgɔn falen kelen-kelen bɛɛ la lɛrɛ 72 furakɛli kɔfɛ (hpt). Biologique replicate kelen-kelen bɛɛ tun ye daga duuru ye (jiri fila bɛ daga kelen kɔnɔ). Biologique sample kelen-kelen bɛɛ sɛgɛsɛgɛra siɲɛ fila (sɛgɛsɛgɛli fɛɛrɛ fila). Fura fila mugulen don ni azote jilama ye, ka baara kɛ ni u ye k’a ɲɛsin farikolo tangalanw ma minnu bɛ sɔrɔ anzimu ni anzimu tɛ minnu na, asidi aminikiw bɛɛ lajɛlen, porolini hakɛ, jeninida jirali, ani ɔkisalati hakɛ jateminɛ.
Fenɔli minnu bɛ se ka wuli, olu bɛɛ lajɛlen jateminɛna ni Folin-Ciocalteu reagent ye (Sigma-Aldrich, St. Louis, MO, USA) ni fɛn dɔw sɛmɛntiyalen don fɛɛrɛ la min ɲɛfɔra Kahkonen ni a ɲɔgɔnnaw fɛ. (1999) la. Kuma surun na, furabulu kelen-kelen garamu 0,1 ɲɔgɔn bɔra ni mɛtanɔli 80% mililitiri 20 ye dibi la fo lɛri 24 ani ka a sanfɛla lajɛ santirifugare kɔfɛ. Aw bɛ fɛnɲɛnamafagalan mililitiri 0,1 ɲagami ni Folin-Ciocalteu reagent mililitiri 0,5 ye (10%), ka a lamaga fo s 30 ka sɔrɔ ka a bila dibi la miniti 5 kɔnɔ. O kɔfɛ, karɔbɔni sodiyɔmu 20% jilama mililitiri 0,5 (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) bɛ fara tubabufura kelen-kelen bɛɛ kan, ka a ɲagami ka ɲɛ ka a bila so funteni na dibi la fo lɛri 1. Kɔnɔnafili kɔfɛ, farikoloɲɛnajɛ ɲagaminen in minɛni hakɛ jateminɛna 765 nm la ni UV-160A spectrophotomètre ye (Shimadzu Corporation, Japon). Fenɔli minnu bɛ se ka wuli, olu bɛɛ hakɛ jateminɛna sɛgɛsɛgɛli kɛlenw na ni asidi galiki jateminɛcogo ye (Fisher Scientific, Hampton, NH, Etazuni) ani ka a jira ni asidi galiki miligaramu ye min bɛ bɛn asidi galiki ma min bɛ bɛn girinya kura garamu kelen ma (mg GAE g-1 girinya kura).
Flavonoid soluble hakɛ bɛɛ lajɛlen jateminɛna ka kɛɲɛ ni Djeridane ni a ɲɔgɔnnaw ka fɛɛrɛ ye. (2006) ni fɛn dɔw b’u la. Kuma surun na, sanfɛ mɛtanɔli bɔlen mililitiri 0,3 ɲagamina ni kuloridi aluminiyɔmu 5% jilama mililitiri 0,3 ye (AlCl3; Fisher Scientific, Hampton, NH, USA), ka a wuli ni fanga ye ka sɔrɔ ka a bila so funteni na miniti 5 kɔnɔ, o kɔfɛ, aw bɛ potasiyɔmu asetati jilama 10% mililitiri 0,3 fara a kan (Al-Gomhoria Furakɛminɛnw ni furakɛlifɛnw, Kayira, Misira), u ɲagaminen don kosɛbɛ ka u bila so funteni na miniti 30 dibi la. Kɔnɔnafili kɔfɛ, farikoloɲɛnajɛ ɲagaminen in minɛni hakɛ jateminɛna 430 nm la ni UV-160A spectrophotomètre ye (Shimadzu Corporation, Japon). Flavonoids solubles totales hakɛ min bɛ sample extracts kɔnɔ, o hakɛ jateminɛna ni rutin calibration curve (TCI America, Portland, OR, USA) ye, o kɔfɛ, a jirala ni rutin miligaramu ye min bɛ bɛn rutin ma min bɛ bɛn girinya kura garamu kelen ma (mg RE g-1 fresh weight).
Shɔ buluw kɔnɔ asidi aminikiw bɛɛ lajɛlen hakɛ jateminɛna ni ninhidrini sɛgɛsɛgɛlikɛlan ye min sɛmɛntiyalen don (Thermo Scientific Chemicals, Waltham, MA, Etazuni) ka da fɛɛrɛ kan min fɔra Yokoyama ni Hiramatsu fɛ (2003) ani ka Sun ni a ɲɔgɔnnaw ka fɛɛrɛ sɛmɛntiya. (2006) la. Kuma surun na, dugukolo yɔrɔ garamu 0,1 bɔra ni pH 5,4 tampon ye, ka sɔrɔ ka jisuma 200 μL kɛ ni ninhidrini 200 μL (2%) ani piridini 200 μL ye (10%; Spectrum Chemical, New Brunswick, NJ, USA), ka a bila ji tobilen na miniti 30 kɔnɔ, ka sɔrɔ ka a nɛnɛ ka suman fo ka se a dan na 580 nm ni UV-160A spektrofotomɛtiri ye (Shimadzu Corporation, Japon). Faan wɛrɛ fɛ, porolini (proline) tun bɛ sɔrɔ Bates ka fɛɛrɛ fɛ (Bates et al., 1973). Proline bɔra ni asidi sulfosalicylic 3% ye (Thermo Scientific Chemicals, Waltham, MA, USA) ka sɔrɔ ka santirifugare kɛ, a sanfɛla mililitiri 0,5 ɲagamina ni asidi asetiki glacial mililitiri 1 ye (Fisher Scientific, Hampton, NH, USA) ani ninhydrin reagent, ka a bila 90°C la miniti 45 kɔnɔ, ka a nɛnɛ ka a suman 520 nm ni spektrofotomɛtiri kelen ye i n’a fɔ sanfɛ. Asidi aminiki hɔrɔnmaw ni porolini bɛɛ lajɛlen minnu bɛ furabuluw la, olu jateminɛna ni glycine ni proline calibration curves (Sigma-Aldrich, St Louis, MO, USA) ye, ka u jira ni miligaramu/g girinya kura ye.
Walasa ka farikolo tangalanw ka anzimu baara dɔn, farikolo yɔrɔ kelen miligaramu 500 ɲɔgɔn bɔra ni Tris tampon mililitiri 3 ye (pH 7,8) min kɔnɔ EDTA-Na2 mililitiri 1 (Sigma-Aldrich, St. Louis, MO, Etazuni) ani polivinilipirolidɔni 7,5% (PVP; Sigma-Aldrich, St. Louis, MO, USA), a kɛra santirifugare la 10.000 × g la miniti 20 kɔnɔ nɛnɛ na (4 °C), ka sɔrɔ ka ji min bɛ a sanfɛ (anzyme crude extract) lajɛ (El-Nagar et al., 2023; Osman et al., 2023). O kɔfɛ, katalase (CAT) kɛra ni fositɛri sodiyɔmu tampon 0,1 M mililitiri 2 ye (pH 6,5; Sigma-Aldrich, St. Louis, MO, USA) ani 269 mM H2O2 jilama 100 μl walasa k’a ka anzimu baara dɔn ka kɛɲɛ ni Aebi (1984) ka fɛɛrɛ ye ni fɛn dɔw b’a la (El-Nagar et al., 2023; Osman ni a ɲɔgɔnnaw, 2023). Guaiacol-dependent peroxidase (POX) anzimu baarakɛcogo jateminɛna ni Harrach ni a ɲɔgɔnnaw ka fɛɛrɛ ye. (2009) la. (2008) ni fɛn misɛnninw b’a la (El-Nagar ni a ɲɔgɔnnaw, 2023; Osman ni a ɲɔgɔnnaw, 2023) ani polifenɔli ɔkisidazi (PPO) ka anzimu baara dɔnna ni a kɛra ni mililitiri 2,2 ye ni tampon sodiyɔmu fosifate 100 mM (pH 6,0), guayacol 100 μl (TCI kemikaliw, Portland, OR, USA) ani 100 μl 12 mM H2O2. Fɛɛrɛ in sɛmɛntiyalen don dɔɔnin ka bɔ (El-Nagar ni a ɲɔgɔnnaw, 2023; Osman ni a ɲɔgɔnnaw, 2023). Sɛgɛsɛgɛli kɛra ni katekɔli jilama mililitiri 3 ye (Thermo Scientific Chemicals, Waltham, MA, USA) (0,01 M) min labɛnna kokura fositɛri tampon 0,1 M (pH 6,0) kɔnɔ, o kɛlen kɔfɛ. CAT baara suman ni H2O2 tiɲɛni kɔlɔsili ye 240 nm (A240), POX baara suman ni absorbance caya kɔlɔsili ye 436 nm (A436), ani PPO baara suman ni absorbance fluctuations sɛbɛnni ye 495 nm (A495) s 30 o miniti 3 kɔnɔ ni UV-160A ye spektrofotomɛtiri (Shimadzu, Japon).
RT-PCR kɛra waati yɛrɛ la walasa ka jamu saba minnu bɛ tali kɛ farikolo tangalanw na, olu ka sɛbɛnni hakɛw dɔn, i n’a fɔ peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), ani glutathione reductase (PvGR; GenBank Accession No. KY195009.1), shɛfan buluw la (bulu filanan ni sabanan minnu falen don kosɛbɛ ka bɔ sanfɛ) lɛrɛ 72 furakɛli laban kɔfɛ. Kuma surun na, ARN bɔra ɲɔgɔn na ni Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) ye ka kɛɲɛ ni a dilabaga ka sariya ye. O kɔfɛ, ADNc dilanna ni TOP scriptTM cDNA Synthesis Kit ye ​​ka kɛɲɛ ni a dilabaga ka laadilikanw ye. Sanfɛla jamu saba ninnu ka primer sequences bɛ sɔrɔ Tablo supplémentaire S3 kɔnɔ. PvActin-3 (GenBank accession number: XM_068616709.1) kɛra du ladonni jamu ye ani jamu jiracogo danfaralen jate kɛra ni 2-ΔΔCT fɛɛrɛ ye (Livak ni Schmittgen, 2001). Aktini sabatili biyotiki degun kɔrɔ (ɲɔgɔndɛmɛ min tɛ bɛn shɔ caman ni antracnose ɲɛgɛn Colletotrichum lindemuthianum cɛ) ani degun abiyɔtiki (ja, kɔgɔ, funteni dɔgɔyali) jirala (Borges et al., 2012).
An ye a daminɛ na, an ye oxaloacetate acetylhydrolase (OAH) furakisɛw sɛgɛsɛgɛli kɛ jamu bɛɛ kɔnɔ S. sclerotiorum kɔnɔ ni poroteyini-poroteyini BLAST baarakɛminɛn ye (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Kuma surun na, an ye OAH kɛ ka bɔ Aspergillus fijiensis CBS 313.89 (AfOAH; taksidi: 1191702; GenBank doncogo nimɔrɔ XP_040799428.1; asidi aminiki 342) ani Penisiliyɔmu lajɛna (PlOAH; taksidi: 94218; GenBank doncogo nimɔrɔ XP_056833920.1; BLASTp kɛra ka kɛɲɛ ni S. sclerotiorum ka jamu kunnafoniw ye minnu sɔrɔla kɔsa in na GenBank kɔnɔ, jamana ka kunnafoniko baarada (NCBI) ka siti kan, http://www.ncbi.nlm.nih.gov/gene/.
Ka fara o kan, OAH jamu min fɔra ka kɔrɔ ka bɔ S. sclerotiorum (SsOAH) la ani AfOAH ka sɛgɛsɛgɛli ni jamu jiri min bɔra A. fijiensis CBS 313.89 la ani PlOAH min bɔra P. lagena la, olu jateminɛna ni fɛɛrɛ ye min bɛ se ka kɛ kosɛbɛ MEGA11 kɔnɔ (Tamura et al., 2021) ani JTT matrix-based model (Jones et al., san 1992). Jiri suguya in farala ɲɔgɔn kan ni OAH jamu (SsOAH) minnu kofɔra ka ɲɛ, olu bɛɛ ka farikolojɔli caman sɛgɛsɛgɛli ye ka bɔ S. sclerotiorum la ani ɲininkali sinsinni ni Constraint-Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos ani Agarwala, 2007 sàn). Ka fara o kan, SsOAH ka amino asidi sinsinni minnu bɛ bɛn kosɛbɛ ka bɔ S. sclerotiorum la, olu ni ɲininkali sinsinniw bɛ bɛn ɲɔgɔn ma (AfOAH ni PlOAH) (Larkin et al., 2007) ni ClustalW ye (http://www.genome.jp/tools-bin/clustalw), ani yɔrɔ minnu maralen bɛ ɲɔgɔndan na, olu jirala ni ESPript baarakɛminɛn ye (version 3.0 https://espript.ibcp.fr/ESPript/ESPript/index.php) Kalanjɛ ni sɛbɛnni: kalanjɛ ni sɛbɛnni gafew.
Ka fara o kan, S. sclerotiorum SsOAH ka baarakɛyɔrɔw jiracogo minnu fɔra ka kɔrɔ ani minnu maralen don, olu tilalen don ɲɔgɔn fɛ denbayaw kɔnɔ minnu tɛ kelen ye ni InterPro baarakɛminɛn ye (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). A laban na, S. sclerotiorum SsOAH min fɔra ka kɔrɔ, o sigicogo misali saba (3D) kɛra ni Protein Homology/Analogy Recognition Engine ye (Phyre2 server version 2.0; http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) (Kelley et al., 2015) ani a tiɲɛna ni baara ye SWISS-MODEL baarakɛminɛn (https://swissmodel.expasy.org/) (Biasini ni a ɲɔgɔnnaw, 2014). Fɛn saba sigicogo minnu fɔra ka kɔrɔ (PDB cogoya) olu jirala ɲɔgɔn fɛ ni UCSF-Chimera pake ye (version 1.15; https://www.cgl.ucsf.edu/chimera/ ) (Pettersen et al., 2004).
PCR quantitative real-time fluorescence PCR kɛra ka oxaloacetate acetylhydrolase (SsOAH; GenBank accession number: XM_001590428.1) ka sɛbɛnni hakɛ dɔn Sclerotinia sclerotiorum ka miseli la. Kuma surun na, S. sclerotiorum boloci kɛra fiyɛlilan kɔnɔ PDB bɛ min kɔnɔ, ka bila inkubatɛri yɛrɛyɛrɛ kɔnɔ (modɛli: I2400, New Brunswick Scientific Co., Edison, NJ, Etazuni) 25 ± 2 °C la lɛrɛ 24 kɔnɔ ni 150 ye miniti kɔnɔ ani dibi la tuma bɛɛ (lɛrɛ 24) walasa ka misɛti falenni lawuli. O kɔfɛ, farikolokisɛw furakɛra ni L-ornithine ni funufagala Rizolex-T ye IC50 hakɛ laban na (miligaramu 40 ni miligaramu 3,2 ɲɔgɔn/L ɲɔgɔn) ka sɔrɔ ka u falen fo lɛri 24 wɛrɛ o cogo kelen na. U bilalen kɔfɛ, sɛnɛfɛnw kɛra santirifuge la ni 2500 ye miniti 5 kɔnɔ miniti 5 kɔnɔ ani ka ji min bɛ sanfɛ (misɛli funteni) lajɛ walasa ka jeninida jiracogo sɛgɛsɛgɛ. O cogo kelen na, ɲɛgɛnɛsiraw lajɛra lɛrɛ 0, 24, 48, 72, 96 ani 120 banakisɛ sɔrɔlen kɔfɛ ka bɔ jiri banakisɛw la minnu tun ye nɔgɔ finman ni misɛli cotony kɛ farikolo yɔrɔw kan minnu banakisɛ bɛ u la. ARN bɔra funu misɛli la ka sɔrɔ ka ADNc dilan i n’a fɔ a ɲɛfɔlen don cogo min sanfɛ. SsOAH ka primer sinsinniw bɛ sɔrɔ Tablo supplémentaire S3 kɔnɔ. SsActin (GenBank accession number: XM_001589919.1) kɛra du ladonni jamu ye, ani jamu jiracogo danfaralen jatebɔ kɛra ni 2-ΔΔCT fɛɛrɛ ye (Livak ni Schmittgen, 2001).
Asidi ɔkisaliki sɔrɔla pɔmitɛri dekistrosi shɔ (PDB) la ani jiriw la minnu kɔnɔ ɲɛgɛnɛsira banakisɛ Sclerotinia sclerotiorum bɛ sɔrɔ ka kɛɲɛ ni Xu ni Zhang (2000) ka fɛɛrɛ ye ni fɛn dɔw sɛmɛntiyalen don. Kuma surun na, S. sclerotiorum isolates boloci kɛra fiyɛlilanw kɔnɔ PDB bɛ minnu na, o kɔfɛ, u falenna inkubatɛri yɛrɛyɛrɛli la (model I2400, New Brunswick Scientific Co., Edison, NJ, USA) ni 150 rpm ye 25 ± 2 °C la tile 3–5 dibi la tuma bɛɛ (lɛrɛ 24) walasa ka misɛli falenni lawuli. Kɔnɔnafili kɔfɛ, ɲɛgɛnɛsiraw falenna fɔlɔ ka tɛmɛ Whatman #1 filɛri papiye fɛ, ka sɔrɔ ka a kɛ santirifugu la ni 2500 ye miniti kɔnɔ miniti 5 kɔnɔ walasa ka misɛti tolenw bɔ. Ji min bɛ sanfɛ, o lajɛra ka mara 4°C la walasa ka oxalate hakɛ dɔn ka taa a fɛ. Walasa ka jiriw tacogo labɛn, jiriw farikolo yɔrɔw garamu 0,1 ɲɔgɔn bɔra siɲɛ saba ni ji wulilen ye (mililitiri 2 o siɲɛ kelen-kelen bɛɛ). O kɔfɛ, u bɛ centrifugé ni 2500 rpm ye miniti 5 kɔnɔ, ka ji min bɛ sanfɛ, o jalen don ka filɛri kɛ Whatman No. 1 filɛri papier fɛ ka sɔrɔ ka u lajɛ walasa ka sɛgɛsɛgɛli wɛrɛw kɛ.
Walasa ka asidi ɔkisaliki hakɛ jateminɛ, a kɛcogo ɲagaminen labɛnna tubabukan na min datugulen don ni galasi ye nin cogo la: mililitiri 0,2 ye sɛgɛsɛgɛli kɛ (walima PDB culture filtrate walima asidi ɔkisaliki jilama), mililitiri 0,11 bromofenol bulu (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, Etazuni), mililitiri 0,198 asidi sulfuriki 1 M (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) ani potasiyɔmu dichromate mililitiri 0,176 (K2Cr2O7; TCI chemicals, Portland, OR, USA) mililitiri 0,176, o kɔfɛ, aw bɛ furaji kɛ ka a kɛ mililitiri 4,8 ye ni ji wulilen ye, ka a ɲagami ni fanga ye ka a bila ji la min janya ye 60°C ye ka ko. Miniti 10 kɔfɛ, aw bɛ a kɛcogo jɔ ni aw ye mililitiri 0,5 kɛ a la ni idɔrɔkisidi sodiyɔmu jilama (NaOH; 0,75 M) ye. O waleya ɲagaminen ka absorbance (A600) suman na 600 nm la ni UV-160 spectrophotometer ye (Shimadzu Corporation, Japon). PDB ni ji dilanlen kɛra kɔrɔsili ye sɛnɛfɛnw hakɛ jateminɛni na ani jiriw tacogo. Asidi ɔkisaliki hakɛ minnu bɛ sɛnɛfɛnw filɛriw kɔnɔ, n’olu jiralen don ni asidi ɔkisaliki mikrogaramu ye mililitiri kelen na PDB fɛnɲɛnamafagalan na (μg.mL−1), ani furabulu bɔlenw na, minnu jiralen don ni asidi ɔkisaliki mikrogaramu ye garamu kura girinya kura kɔnɔ (μg.g−1 FW), olu jateminɛna ni asidi ɔkisaliki hakɛ jateminɛcogo ye (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
Kalan bɛɛ kɔnɔna na, k’a lajɛ bɛɛ kɛra cogo la min kɛra ni mɔgɔw ye pewu (CRD) ni fɛnɲɛnamafagalan wɔɔrɔ ye furakɛli kelen kɔnɔ ani daga duuru fɛnɲɛnamafagalan kelen na (jiri fila bɛ kɛ daga kelen kɔnɔ) fo n’a fɔra cogo wɛrɛ la. Biologique replications (ɲɛnamaya ɲɔgɔnna caman kɛlenw) sɛgɛsɛgɛra siɲɛ fila (sɛgɛsɛgɛli fila kɛra fɛɛrɛko siratigɛ la). Fɛɛrɛko seginkanni kɛra walasa ka o kɔrɔbɔli kelen in lasegincogo lajɛ nka u ma kɛ jatebɔ sɛgɛsɛgɛli la walasa ka seginkanni nkalonmaw bali. Kunnafoni sɛgɛsɛgɛli kɛra ni jateminɛ ye ni sɛgɛsɛgɛli ye (ANOVA) o kɔfɛ Tukey-Kramer honestly significant difference (HSD) test (p ≤ 0.05). Ka ɲɛsin sɛgɛsɛgɛliw ma minnu kɛra in vitro kɔnɔ, IC50 ni IC99 hakɛw jatera ni probit modɛli ye ani dannaya danfara 95% jatera.
Isolati naani bɛɛ lajɛlen lajɛra ka bɔ soja foro suguya wɛrɛw la El Ghabiya mara la, Misira jamana na. PDA medium kan, isolates bɛɛ ye miseli finman kreman bɔ min bɛ teliya ka kɛ coton fin ye (Ja 1A) ka sɔrɔ ka kɛ beige walima brown ye sclérotium taabolo la. A ka ca a la, sɔgɔsɔgɔninjɛ bɛ kɛ fɛn ye min ka bon, u ye nɛrɛmuguma ye, u bɛ kɛ sɛrɛkili ye walima u cogoya tɛ kelen ye, u janya bɛ se milimɛtɛrɛ 5,2 fo 7,7 ma ani u cɛmancɛ bɛ kɛ milimɛtɛrɛ 3,4 fo 5,3 ye (Jaa 1B). Hali ni isolati naani ye sɔgɔsɔgɔninjɛ cogoya danmadɔ sɔrɔ sɛnɛfɛn da la, tile 10–12 kɛlen kɔ ka u bila 25 ± 2 °C la (Ja 1A), skleroti hakɛ min bɛ plati kelen kɔnɔ, o tun tɛ kelen ye kosɛbɛ u ni ɲɔgɔn cɛ (P < 0,001), ni skleroti 3 ye skleroti hakɛ cayalenba ye (32,33 ± 1,53 skleroti kelen na plati 1C). O cogo kelen na, isolati #3 ye asidi ɔkisaliki caman bɔ PDB la ka tɛmɛ isolati tɔw kan (3,33 ± 0,49 μg.mL−1; Jaa 1D). Isolate #3 ye jiriturubanakisɛ min bɛ wele ko Sclerotinia sclerotiorum, o cogoya ni a mikroskɔpi cogoya jira. Misali la. Ka da sanfɛla cogoya ni mikroskɔpi jogo kan, isolate #3 jirala ko Sclerotinia sclerotiorum.
Jaa 1. S. sclerotiorum minnu bɔra sɛnɛfɛnw na minnu bɛ sɔrɔ sɛnɛfɛnw na minnu bɛ kɛ tuma caman na, olu jogo ni u banakisɛ sɔrɔli. (A) S. sclerotiorum isolates naani ka misɛli falenni PDA medium kan, (B) S. sclerotiorum isolates naani ka sclerotie, (C) scleroties hakɛ (plate kelen kɔnɔ), (D) asidi oxalic bɔli PDB medium kan (μg.mL−1), ani (E) S. sclerotiorum isolates naani ka bana juguya (%) jagokɛla ka legume susceptible kan sɛnɛfɛn Giza 3 sɛnɛcogo la. Nafaw bɛ ± SD jira fɛnɲɛnamaw ka ɲɔgɔnkanu duuru la (n = 5). Sɛbɛn suguya wɛrɛw bɛ danfara jira jateminɛ na furakɛli ni ɲɔgɔn cɛ (p < 0,05). (F–H) Nɔgɔ finman taamasiɲɛw bɛ ye dugukolo sanfɛ jiriw ni silisiw kan, u kelen-kelen bɛɛ, tile 10 boloci kɔfɛ ni isolate #3 (dpi) ye. (I) S. sclerotiorum isolate #3 kɔnɔna na, yɔrɔ min sɛbɛnnen don (ITS) yɔrɔ sɛgɛsɛgɛli kɛra ni sɛgɛsɛgɛli kɛcogo ye min bɛ se ka kɛ kosɛbɛ, ka sɔrɔ ka a suma ni 20 ye minnu bɛ tali kɛ fɛnw na minnu bɛ tali kɛ ɲɔgɔn na/suguya minnu sɔrɔla jamana ka kunnafonidilan na (NCBI) (https://www.ncbi.nlm.nih.gov/). Jateden minnu bɛ kulusi sinsinni sanfɛ, olu bɛ marabolo dafalen jira (%), jateden minnu bɛ kulusi sinsinni jukɔrɔ, olu bɛ bolofara janya jira.
Ka fara o kan, walasa ka banakisɛ sɔrɔli dafa, S. sclerotiorum isolates naani sɔrɔlaw kɛra ka jagokɛla ka shɛfan suguya Giza 3 min bɛ se ka bana sɔrɔ, o boloci kɛ serili cogoyaw la, o bɛ bɛn Koch ka hakilinaw ma (Ja 1E). Hali ni ɲɛgɛnɛsira minnu sɔrɔla, olu bɛɛ tun ye banakisɛw ye, wa u tun bɛ se ka shɛfan jɛman (cv. Giza 3) bana, ka kɛ sababu ye ka nɔgɔ finman taamasiɲɛw jira dugukolo sanfɛyɔrɔw bɛɛ la (Ja 1F), kɛrɛnkɛrɛnnenya la jirisunw kan (Ja 1G) ani jirisunw kan (Ja 1H) tile 10 boloci kɔfɛ (dpi), fɛnɲɛnamafagalan 3 de tun ye fɛnɲɛnamafagalan ye min tun bɛ kɛ ni fanga ye ka tɛmɛ tɔw bɛɛ kan, k’a sɔrɔ u tun bɛ kɛ yɛrɛmahɔrɔnyalen fila la. Isolati 3 de tun ye bana juguya hakɛ cayalenba ye (%) shɛfan jiriw kan (24,0 ± 4,0, 58,0 ± 2,0, ani 76,7 ± 3,1 tile 7, 14 ani 21 banakisɛ sɔrɔlen kɔfɛ, o cogo kelen na; Jaa 1F).
S. sclerotiorum isolate #3 min bɛ don kosɛbɛ, o dɔnni sabatira ka t’a fɛ ka da kɔnɔna sɛbɛnnikɛlan (ITS) sinsinni kan (Ja 1I). Fɛnɲɛnɛma-sɛgɛsɛgɛli isolate #3 ni 20 reference isolates/strains cɛ, o y’a jira ko ɲɔgɔndɛmɛba bɛ u ni ɲɔgɔn cɛ (>99%). A ka kan ka kɔlɔsi ko S. sclerotiorum isolate #3 (533 bp) ni Ameriki S. sclerotiorum isolate LPM36 min bɔra pea kisɛ jalenw na (GenBank accession number MK896659.1; 540 bp) ani Sinuwa S. sclerotiorum isolate YKY211 (GenBank accession number) bɔlen don ɲɔgɔn fɛ kosɛbɛ OR206374.1; O ɲɔgɔndan kura in bilala NCBI ka kunnafonidilan kɔnɔ, wa a tɔgɔ dara ko “Sclerotinia sclerotiorum – isolate YN-25” (GenBank ka don nimɔrɔ PV202792). A bɛ se ka ye ko isolate 3 ye isolate ye min bɛ mɔgɔ minɛ ka tɛmɛ tɔw bɛɛ kan; o de kama, nin isolate in sugandira walasa ka kalan kɛ kɔrɔbɔli nataw bɛɛ la.
Diamine L-ornithine (Sigma-Aldrich, Darmstadt, Allemagne) ka banakisɛfagalan baara kɛcogo sɛgɛsɛgɛra a hakɛ danfaralenw na (12,5, 25, 50, 75, 100 ani 125 mg/L) S. sclerotiorum isolate 3 kan, o sɛgɛsɛgɛra in vitro. A kɔlɔsira ko L-ornithine ye banakisɛfagalanw kɛ ani ka S. sclerotiorum hyphae ka bonyali bali dɔɔni dɔɔni ka kɛɲɛ ni a tata hakɛ ye (Jaa 2A, B). Ni a hakɛ sɛgɛsɛgɛra ka caya (miligaramu 125/L), L-ornithine ye misɛli bonya balili hakɛ caman jira (99,62 ± 0,27%; Jaa 2B), o ni jagokɛla ka ɲɛgɛnɛsiralabanaw Rizolex-T bɛ bɛn (a balili hakɛ 99,45 ± 0,39%; Jaa 2C) hakɛ min sɛgɛsɛgɛra ka caya (miligaramu 10/L), o b’a jira ko o bɛ tali kɛ ɲɔgɔn na nafa sɔrɔli.
Jaa 2. L-ornithine ka banakisɛfagalanw ka baara in vitro (in vitro) ka ɲɛsin Sclerotinia sclerotiorum ma. (A) L-ornithine hakɛ danfaralenw ka banakisɛfagalan baara sumalen S. sclerotiorum kan ni jagokɛla ka ɲɛgɛnɛsiralabanaw Rizolex-T (miligaramu 10/L) ye. (B, C) S. sclerotiorum misɛli bonya balili hakɛ (%) furakɛli kɔfɛ ni L-ornithine (12,5, 25, 50, 75, 100 ani 125 mg/L) walima Rizolex-T (2, 4, 6, 8 ani 10 mg/L) hakɛ danfaralenw ye. Nafaw bɛ ± SD jira fɛnɲɛnamaw ka ɲɔgɔnkanu duuru la (n = 5). Sɛbɛnni danfaralenw bɛ danfara jira jateminɛw la furakɛliw ni ɲɔgɔn cɛ (p < 0,05). (D, E) Probit model regression analysis of L-ornithine ni jagokɛla ka ɲɛgɛnɛsiralabanaw Rizolex-T, olu kelen-kelen bɛɛ. Probit model regression line jiralen don i n’a fɔ layini bulu solide, ani dannaya cɛmancɛ (95%) jiralen don i n’a fɔ layini bilenman tigɛlen.
Ka fara o kan, probit kɔsegin sɛgɛsɛgɛli kɛra ani jatebɔ minnu bɛ bɛn o ma, olu jiralen bɛ jatebɔsen 1 na ani ja 2D,E kɔnɔ. Kuma surun na, L-ornithine ka jiginni hakɛ min bɛ sɔn (y = 2,92x − 4,67) ani jateminɛ nafama minnu bɛ tali kɛ a la (Cox & Snell R2 = 0,3709, Nagelkerke R2 = 0,4998 ani p < 0,0001; Jaa 2D) o y’a jira ko ɲɛgɛnɛsiraw kɛlɛli baara cayara S. sclerotiorum kan ni a ni jagokɛla ka ɲɛgɛnɛsiraw ta kɛra ɲɔgɔn fɛ Rizolex-T (y = 1,96x − 0,99, Cox & Snell R2 = 0,1242, Nagelkerke R2 = 0,1708 ani p < 0,0001) (Taabolo 1).
Tablo 1. L-ornithine ni jagokɛla ka ɲɛgɛnɛsiralabanaw “Rizolex-T” hakɛ tilancɛ hakɛ dantɛmɛnen (IC50) ani IC99 (mg/l) nafaw S. sclerotiorum kan.
Kuma bɛɛ la, L-ornithine (250 mg/L) ye dɔ bɔ kosɛbɛ nɔgɔ finman sɔrɔli n’a juguya la shɛfan jiriw la minnu furakɛra, ni i y’a suma ni S. sclerotiorum banakisɛ bɛ jiri minnu furakɛbali ye (kɔlɔsili; Jaa 3A). Kuma surun na, hali ni banakisɛ jugumanba minnu ma furakɛ, olu ka bana juguya cayara dɔɔni dɔɔni (52,67 ± 1,53, 83,21 ± 2,61, ani 92,33 ± 3,06%), L-ornithine ye bana juguya dɔgɔya kosɛbɛ (%) kɔlɔsili bɛɛ kɔnɔ (8,97 ± 0,15, 18,00 ± 1,00, ani 26,36 ± 3,07) tile 7, 14 ani 21 furakɛli kɔfɛ (dpt), o cogo kelen na (Jaa 3A). O cogo kelen na, ni S. sclerotiorum banakisɛ bɛ shɛfan jiri minnu na, olu furakɛra ni L-ornithine miligaramu 250 ye/L, bana taabolo kurulen (AUDPC) jukɔrɔ yɔrɔ dɔgɔyara ka bɔ 1274,33 ± 33,13 la furakɛlibali la ka se 281,03 ± 7,95 ma, o min tun ka dɔgɔn dɔɔni ka tɛmɛ kɔrɔlen ɲuman ta kan, miligaramu 50/L Rizolex-T funufagalan (183,61 ± 7,71; Jaa 3B). O cogoya kelen de Kɔlɔsira kɔrɔbɔli filanan na.
Jaa 3. L-ornithine tali kɛnɛma nɔ min bɛ sɔrɔ shɛfan gansan toli finman yiriwali la min bɛ sɔrɔ Sclerotinia sclerotiorum fɛ serili cogoyaw la. (A) Bana taabolo kurulen min bɛ sɔrɔ shɔ finman na furakɛli kɔfɛ ni L-ornithine miligaramu 250/L ye. (B) Yɔrɔ min bɛ bana taabolo kuru (AUDPC) jukɔrɔ, shɛfan gansan nɔgɔ finman furakɛli kɔfɛ ni L-ornithine ye. Nafaw bɛ ± SD jira fɛnɲɛnamaw ka ɲɔgɔnkanu duuru la (n = 5). Sɛbɛnni danfaralenw bɛ danfara jira jateminɛ siratigɛ la furakɛliw ni ɲɔgɔn cɛ (p < 0,05).
L-ornithine miligaramu 250/L kɛli kɛnɛma, o bɛ dɔ fara jiri janya kan dɔɔni dɔɔni (Ja 4A), ka dɔ fara a bolow hakɛ kan jiri kelen na (Ja 4B), ani ka dɔ fara furabulu hakɛ kan jiri kelen na (Ja 4C) tile 42 kɔfɛ. Hali ni jagokɛla ka ɲɛgɛnɛsiralabanaw Rizolex-T (miligaramu 50/L) de ye nɔba bila balocogo bɛɛ la minnu sɛgɛsɛgɛra, L-ornithine miligaramu 250/L kɛli kɛnɛma, o de kɛra nɔba filanan ye ni i ye a suma ni furakɛli kɛlenw ye (Ja 4A–C). Faan wɛrɛ fɛ, L-ornithine furakɛli ma nɔba bila kɛnɛyaji ɲɛgɛnw na minnu bɛ wele ko kulorofili a (Ja 4D) ani kulorofili b (Ja 4E), nka a ye dɔ fara karotɛni hakɛ bɛɛ kan dɔɔni (0,56 ± 0,03 mg/g fr wt) ni i ye a suma ni kɔrɔlen juguman ye (0,44 ± 0,02 mg/g fr wt) ani kunbɛncogo ɲuman (0,46 ± 0,02 mg/g fr wt; Jaa 4F). Kuma bɛɛ la, nin jaabi ninnu b’a jira ko L-ornithine tɛ jirituru ye sɛnɛfɛnw na minnu furakɛra, wa a bɛ se ka u falen yɛrɛ.
Jaa 4. L-ornithine tali kɛnɛma nɔ min bɛ sɔrɔ shɛfan buluw falencogo n’u ɲɛda ɲɛgɛnw na minnu banakisɛ bɛ Sclerotinia sclerotiorum la serili cogoyaw la. (A) Jiri janya (cm), (B) Bolo hakɛ jiri kelen na, (C) Fura hakɛ jiri kelen na, (D) Kulorofili a hakɛ (mg g-1 fr wt), (E) Kulorofili b hakɛ (mg g-1 fr wt), (F) Karotɛnoyi hakɛ bɛɛ lajɛlen (mg g-1 fr wt). Nafaw ye ± SD ye biologique replicate duuru la (n = 5). Sɛbɛn suguya wɛrɛw bɛ danfara jira jateminɛ na furakɛli ni ɲɔgɔn cɛ (p < 0,05).
Oksizɛni suguya minnu bɛ se ka baara kɛ (ROS; a jiralen don ni perokisidi idɔrɔji [H2O2]) ani radikali hɔrɔnyalenw (a jiralen don ni superoxyde aniions [O2•−]) ka sigiyɔrɔmali in situ ye, o y’a jira ko L-ornithine (250 mg/L) tali kɛnɛma, o ye H2O2 dalajɛli dɔgɔya kosɛbɛ (96,05 ± 5,33 nmol.g−1 FW Jaa 5A) ani O2•− (32,69 ± 8,56 nmol.g−1 FW; Jaa 5B) ni i y’a suma ni jiri bananen fila bɛɛ lajɛlen dalajɛli ye minnu ma furakɛ (173,31 ± 12,06 ani 149,35 ± 7,94 nmol.g−1 FW, o cogo kelen na) ani jiri minnu furakɛra ni jagokɛla ka funtenifagalan miligaramu 50/L ye Rizolex-T (170,12 ± 9,50 ani 157,00 ± 7,81 nmol.g−1 fr wt, o cogo kelen na) ni lɛrɛ 72 ye. H2O2 ni O2•− hakɛ caman bɛ dalajɛ hpt jukɔrɔ (Ja 5A, B). O cogo kelen na, TCA-based malondialdehyde (MDA) sɛgɛsɛgɛli y’a jira ko S. sclerotiorum banakisɛ bɛ shɛfan jiri minnu na, olu ye MDA hakɛ caman dalajɛ u buluw la (113,48 ± 10,02 nmol.g fr wt (Ja 5C). Nka, L-ornithine tali kɛnɛma ye tulumafɛnw bɔli dɔgɔya kosɛbɛ i n’a fɔ a jiralen bɛ cogo min na MDA hakɛ dɔgɔyali fɛ jiri furakɛlenw na (33,08 ± 4,00 nmol.g fr wt).
Jaa 5. L-ornithine tali kɛnɛma nɔ min bɛ sɔrɔ oksidan degun taamasiɲɛbaw la ani farikolo tangacogo minnu tɛ anzimu ye shɛfan buluw la minnu banakisɛ bɛ S. sclerotiorum la lɛrɛ 72 kɔnɔ banakisɛ sɔrɔlen kɔfɛ serili cogoyaw la. (A) Idɔrɔzi perokisidi (H2O2; nmol g−1 FW) ni 72 hpt, (B) superoxyde aniyoni (O2•−; nmol g−1 FW) ni 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) ni 72 hpt, (D) fenɔli wulilenw bɛɛ lajɛlen (mg GAE g−1 FW) ni 72 hpt, (E) flavonoids solubles totales (mg RE g−1 FW) ni 72 hpt, (F) amino acides libres totales (mg g−1 FW) ni 72 hpt, ani (G) proline hakɛ (mg g−1 FW) ni 72 hpt. Nafaw bɛ ± deviation standard (kɛrɛnkɛrɛnnenya ± SD) jira 5 biologique replicates (n = 5). Sɛbɛn suguya wɛrɛw bɛ danfara jira jateminɛ na furakɛli ni ɲɔgɔn cɛ (p < 0,05).