Isiokwu a bụ akụkụ nke isiokwu nyocha ahụ bụ "Ịkwalite iguzogide agwa ndị na-akpata ọrịa na ụmụ ahụhụ", lee isiokwu ise niile.
Ihe na-akpata ọrịa fungal necrosis Sclerotinia sclerotiorum (Lib.) de Bary na-eji usoro dị iche iche iji bute ọrịa osisi dị iche iche. Ọmụmụ ihe a na-atụ aro iji diamine L-ornithine, amino acid na-abụghị protein nke na-akpali mmepụta nke amino acid ndị ọzọ dị mkpa, dị ka usoro njikwa ọzọ iji melite mmeghachi omume molekul, physiological na biochemical nke Phaseolus vulgaris L. na ebu ọcha nke Pseudomonas sclerotiorum kpatara. Nnwale In vitro gosiri na L-ornithine gbochiri uto mycelium nke S. pyrenoidosa nke ukwuu n'ụzọ dabere na dose. Ọzọkwa, ọ nwere ike ibelata oke ibu ọcha n'okpuru ọnọdụ griin haus. Ọzọkwa, L-ornithine kpaliri uto nke osisi ndị a gwọrọ, na-egosi na mkpokọta L-ornithine a nwalere abụghị ihe na-egbu egbu nye osisi ndị a gwọrọ. Tinyere nke ahụ, L-ornithine mere ka ngosipụta nke antioxidants ndị na-abụghị enzymatic (phenolics na flavonoids niile na-agbaze) na antioxidants enzymatic (catalase (CAT), peroxidase (POX), na polyphenol oxidase (PPO)) dịkwuo mma, ma mụbaa ngosipụta nke mkpụrụ ndụ ihe nketa atọ metụtara antioxidant (PvCAT1, PvSOD, na PvGR). Ọzọkwa, na nyocha silico kpughere ọnụnọ nke protein oxaloacetate acetylhydrolase (SsOAH) na mkpụrụ ndụ ihe nketa S. sclerotiorum, nke yiri nke ukwuu na protein oxaloacetate acetylhydrolase (SsOAH) nke Aspergillus fijiensis (AfOAH) na Penicillium sp. (PlOAH) n'ihe gbasara nyocha ọrụ, ngalaba echekwara, na topology. N'ụzọ na-akpali mmasị, mgbakwunye nke L-ornithine na potato dextrose broth (PDB) mebere belatara ngosipụta nke mkpụrụ ndụ ihe nketa SsOAH na S. sclerotiorum mycelia nke ukwuu. N'otu aka ahụ, itinye L-ornithine n'èzí belatara ngosipụta nke mkpụrụ ndụ ihe nketa SsOAH na mycelia fungal nke a na-anakọta site na osisi e gwọrọ agwọ. N'ikpeazụ, itinye L-ornithine belatara nke ukwuu mmịpụta oxalic acid na ma akwụkwọ PDB ọkara na nke butere ọrịa. Na mmechi, L-ornithine na-arụ ọrụ dị mkpa n'ịnọgide na-enwe ọnọdụ redox yana ime ka nzaghachi nchebe nke osisi ndị butere ọrịa dịkwuo mma. Nsonaazụ nke ọmụmụ ihe a nwere ike inye aka n'ịmepụta ụzọ ọhụrụ, nke na-adịghị emerụ gburugburu ebe obibi iji chịkwaa ebu ọcha ma belata mmetụta ya na mmepụta agwa na ihe ọkụkụ ndị ọzọ.
Ebu ọcha, nke ero necrotrophic Sclerotinia sclerotiorum (Lib.) de Bary kpatara, bụ ọrịa na-emebi ihe, nke na-ebelata mkpụrụ nke na-etinye nnukwu ihe iyi egwu nye mmepụta agwa zuru ụwa ọnụ (Phaseolus vulgaris L.) (Bolton et al., 2006). Sclerotinia sclerotiorum bụ otu n'ime nje ndị na-akpata ọrịa fungal kachasị sie ike ịchịkwa, yana ọtụtụ ụdị osisi 600 na ikike ịme ka anụ ahụ ndị ọbịa dị ngwa ngwa n'ụzọ na-abụghị nke a kapịrị ọnụ (Liang na Rollins, 2018). N'ọnọdụ na-adịghị mma, ọ na-agafe oge dị oke mkpa nke usoro ndụ ya, na-anọgide na-ehi ụra ruo ogologo oge dị ka ihe owuwu ojii, siri ike, dị ka mkpụrụ a na-akpọ 'sclerotia' n'ime ala ma ọ bụ dị ka uto ọcha, na-adịghị mma na mycelium ma ọ bụ pith nke osisi ndị butere ọrịa (Schwartz et al., 2005). S. sclerotiorum nwere ike ịmepụta sclerotia, nke na-enye ya ohere ịdị ndụ n'ubi ndị nwere nje ruo ogologo oge ma nọgide na-arịa ọrịa (Schwartz et al., 2005). Sclerotia bara ụba na nri, nwere ike ịnọgide na ala ruo ogologo oge, ma jee ozi dị ka inoculum mbụ maka ọrịa ndị na-esote (Schwartz et al., 2005). N'ọnọdụ dị mma, sclerotia na-eto ma na-emepụta spores ikuku nke nwere ike ibute ọrịa n'akụkụ osisi niile dị n'elu ala, gụnyere mana ejedebeghị na okooko osisi, ogwe osisi, ma ọ bụ pods (Schwartz et al., 2005).
Sclerotinia sclerotiorum na-eji usoro dị iche iche iji bute ọrịa site na mkpụrụ ndụ ihe nketa ya, nke gụnyere usoro ihe omume ndị a haziri ahazi site na mkpụrụ ndụ ihe nketa sclerotial ruo na mmepe nke ihe mgbaàmà. Na mbụ, S. sclerotiorum na-emepụta spores a kwụsịrị (a na-akpọ ascospores) site na ihe ndị dị ka ero a na-akpọ apothecia, nke na-aghọ ikuku ma na-etolite ghọọ sclerotia na-anaghị agagharị na ihe mkpofu osisi ndị nwere ọrịa (Bolton et al., 2006). Ero ahụ na-ewepụta oxalic acid, ihe na-akpata ọrịa, iji chịkwaa pH nke mgbidi mkpụrụ ndụ ihe nketa, kwalite mmebi enzymatic na mwakpo anụ ahụ (Hegedus na Rimmer, 2005), ma gbochie mgbawa oxidative nke osisi nnabata ahụ. Usoro acidification a na-eme ka mgbidi mkpụrụ ndụ ihe nketa ahụ ghara ịdị ike, na-enye gburugburu ebe dị mma maka ọrụ nkịtị na nke dị irè nke enzymes fungal na-emebi mgbidi mkpụrụ ndụ ihe nketa (CWDEs), na-enye ohere ka nje ahụ merie ihe mgbochi anụ ahụ ma banye n'ime anụ ahụ nnabata ahụ (Marciano et al., 1983). Ozugbo e tinyere ya n'ime, S. sclerotiorum na-ewepụta ọtụtụ CWDE, dị ka polygalacturonase na cellulase, nke na-eme ka mgbasa ya dị mfe n'ime anụ ahụ ndị nwere ọrịa ma na-akpata necrosis anụ ahụ. Ọganihu nke mmerụ ahụ na ute hyphal na-eduga na mgbaàmà njirimara nke ebu ọcha (Hegedus na Rimmer, 2005). Ka ọ dị ugbu a, osisi ndị ọbịa na-amata ụkpụrụ molekul metụtara nje (PAMPs) site na ndị na-anabata njirimara ụkpụrụ (PRRs), na-akpali usoro ihe omume mgbaàmà nke na-eme ka nzaghachi nchebe rụọ ọrụ.
N'agbanyeghị ọtụtụ iri afọ nke mgbalị iji chịkwaa ọrịa, ụkọ nke nje bacteria na-eguzogide ọgwụ zuru oke ka dị na agwa, dịka ọ dị na ihe ọkụkụ ndị ọzọ na-azụ ahịa, n'ihi nguzogide, ndụ, na mgbanwe nke nje bacteria. Ya mere, njikwa ọrịa na-esiri ike nke ukwuu ma chọọ atụmatụ agbakwunyere, nke nwere ọtụtụ akụkụ nke gụnyere njikọta nke omume omenala, njikwa ihe ndị dị ndụ, na ọgwụ nje kemịkalụ (O'Sullivan et al., 2021). Njikwa kemịkalụ nke ebu ọcha bụ nke kachasị dị irè n'ihi na ọgwụ nje, mgbe etinyere ya nke ọma na n'oge kwesịrị ekwesị, nwere ike ijikwa mgbasa nke ọrịa ahụ nke ọma, belata oke ọrịa, ma belata mfu mkpụrụ. Agbanyeghị, iji oke na ịdabere na ọgwụ nje nwere ike ibute mpụta nke ụdị S. sclerotiorum na-eguzogide ọgwụ ma na-emetụta ihe ndị na-abụghị ihe mgbaru ọsọ, ahụike ala, na ịdị mma mmiri (Le Cointe et al., 2016; Ceresini et al., 2024). Ya mere, ịchọta ihe ndị ọzọ na-adịghị ize ndụ na gburugburu ebe obibi aghọwo ihe kacha mkpa.
Polyamines (PAs), dị ka putrescine, spermidine, spermine, na cadaverine, nwere ike ịbụ ihe ọzọ dị mma maka igbochi nje ndị na-efe efe site na osisi, si otú a belata ojiji nke ọgwụ fungicides dị ize ndụ kpamkpam ma ọ bụ akụkụ ya (Nehela et al., 2024; Yi et al., 2024). N'ime osisi ndị dị elu, PAs na-etinye aka na ọtụtụ usoro physiological gụnyere, mana ejedebeghị na, nkewa sel, ọdịiche, na nzaghachi nye nrụgide abiotic na biotic (Killiny na Nehela, 2020). Ha nwere ike ịrụ ọrụ dị ka ihe ndị na-egbochi ọrịa, nyere aka na-ekpochapụ ụdị oxygen reactive (ROS), na-enwe redox homeostasis (Nehela na Killiny, 2023), na-akpali mkpụrụ ndụ ihe nketa metụtara nchekwa (Romero et al., 2018), na-achịkwa ụzọ dị iche iche nke metabolic (Nehela na Killiny, 2023), na-agbanwe phytohormones endogenous (Nehela na Killiny, 2019), na-eguzobe iguzogide sistemụ enwetara (SAR), ma na-achịkwa mmekọrịta osisi na nje (Nehela na Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). Ọ dị mkpa ịmara na usoro na ọrụ kpọmkwem nke PAs na nchekwa osisi dị iche iche dabere na ụdị osisi, nje bacteria, na ọnọdụ gburugburu ebe obibi. PA kachasị ukwuu na osisi bụ ihe sitere na polyamine L-ornithine dị mkpa (Killiny na Nehela, 2020).
L-ornithine na-arụ ọrụ dị iche iche n'uto na mmepe osisi. Dịka ọmụmaatụ, ọmụmụ ihe ndị gara aga egosila na na osikapa (Oryza sativa), ornithine nwere ike ịbụ njikọ ya na imegharị nitrogen (Liu et al., 2018), mmepụta osikapa, ịdị mma na isi (Lu et al., 2020), na nzaghachi nrụgide mmiri (Yang et al., 2000). Ọzọkwa, itinye L-ornithine n'èzí na-eme ka nnabata nke ụkọ mmiri dịkwuo mma na shuga beet (Beta vulgaris) (Hussein et al., 2019) ma belata nrụgide nnu na yabasị (Allium Cepa) (Çavuşoǧlu na Çavuşoǧlu, 2021) na cashew (Anacardium occidentale) osisi (da Rocha et al., 2012). Ọrụ L-ornithine nwere ike ịrụ na nchekwa nrụgide abiotic nwere ike ịbụ n'ihi itinye aka ya na nchịkọta proline na osisi a gwọrọ. Dịka ọmụmaatụ, a kọọla na mbụ na mkpụrụ ndụ ihe nketa metụtara metabolism proline, dịka ornithine delta aminotransferase (delta-OAT) na proline dehydrogenase (ProDH1 na ProDH2), na-etinye aka na nchekwa Nicotiana benthamiana na Arabidopsis thaliana megide ụdị Pseudomonas syringae na-abụghị nke ndị ọbịa (Senthil-Kumar na Mysore, 2012). N'aka nke ọzọ, a chọrọ fungal ornithine decarboxylase (ODC) maka uto nje (Singh et al., 2020). Isetịpụ ODC nke Fusarium oxysporum f. sp. lycopersici site na host-induced gene silencing (HIGS) mere ka iguzogide osisi tomato sikwuo ike nke ukwuu (Singh et al., 2020). Agbanyeghị, a kabeghị amụ ọrụ nke itinye exogenous ornithine megide nrụgide biotic dị ka phytopathogens. Ihe kacha mkpa bụ na mmetụta nke ornithine nwere n'igbochi ọrịa na ihe ndị metụtara ya na ihe ndị metụtara ya ka bụ ihe a na-amaghị nke ọma.
Ịghọta mgbagwoju anya nke ọrịa S. sclerotiorum nke legumes dị mkpa maka mmepe nke atụmatụ njikwa dị irè. N'ọmụmụ ihe a, anyị bu n'obi ịchọpụta ọrụ diamine L-ornithine nwere ike ịrụ dị ka ihe dị mkpa n'ịkwalite usoro nchebe na iguzogide osisi legume megide ọrịa Sclerotinia sclerotiorum. Anyị na-eche na, na mgbakwunye na ime ka nzaghachi nchebe nke osisi ndị nwere ọrịa dịkwuo mma, L-ornithine na-arụkwa ọrụ dị mkpa n'ịnọgide na-enwe ọnọdụ redox. Anyị na-atụ aro na mmetụta L-ornithine nwere ike inwe metụtara nhazi nke usoro nchebe antioxidant enzymatic na nke na-abụghị enzymatic na nnyonye anya na ihe ndị na-akpata ọrịa fungal/virulence na protein ndị metụtara ya. Ọrụ abụọ a nke L-ornithine na-eme ka ọ bụrụ onye a ga-ekwe nkwa maka atụmatụ na-adịgide adịgide iji belata mmetụta nke ebu ọcha ma melite iguzogide nke ihe ọkụkụ legume nkịtị na nje fungal a dị ike. Nsonaazụ nke ọmụmụ ihe a nwere ike inye aka na mmepe nke ụzọ ọhụrụ, nke na-adịghị ize ndụ gburugburu ebe obibi iji chịkwaa ebu ọcha ma belata mmetụta ya na mmepụta legume.
N'ọmụmụ ihe a, e jiri ụdị agwa nkịtị dị mfe ịzụ ahịa, Giza 3 (Phaseolus vulgaris L. cv. Giza 3), mee ihe nnwale. Ngalaba Nnyocha Legume, Field Crops Research Institute (FCRI), Agricultural Research Center (ARC), Egypt nyere mkpụrụ dị mma. A kụrụ mkpụrụ ise n'ime ite plastik (dayameta dị n'ime 35 cm, omimi 50 cm) nke jupụtara na ala nwere ọrịa S. sclerotiorum n'okpuru ọnọdụ griin haus (25 ± 2 °C, iru mmiri dị nso 75 ± 1%, ìhè awa 8 / awa 16 gbara ọchịchịrị). Mgbe ụbọchị 7-10 gachara ịgha mkpụrụ (DPS), a kpụchara mkpụrụ ndị ahụ ka ha hapụ naanị mkpụrụ abụọ nwere uto otu ebe na akwụkwọ atọ gbasaara nke ọma n'ime ite ọ bụla. A na-agba mmiri osisi niile a kụrụ n'ime ite otu ugboro n'izu abụọ ọ bụla ma mee ka ha dị ọcha kwa ọnwa na ọnụego akwadoro maka ụdị a.
Iji kwadebe mkpokọta L-ornithinediamine 500 mg/L (nke a makwaara dị ka (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Germany), e buru ụzọ gbazee 50 mg na 100 ml nke mmiri a gwakọtara agwakọta. E mesịa gwakọta ihe ngwọta ngwaahịa ahụ ma jiri ya mee ihe na nnwale ndị sochirinụ. N'ụzọ dị mkpirikpi, a nwalere usoro isii nke mkpokọta L-ornithine (12.5, 25, 50, 75, 100, na 125 mg/L) na vitro. Na mgbakwunye, ejiri mmiri a gwakọtara agwakọta dị ka njikwa na-adịghị mma (Mock) na ntụ ntụ mmiri mmiri "Rizolex-T" 50% nke azụmaahịa (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Egypt) mee ihe dị ka njikwa dị mma. A nwalere ọgwụ nje "Rizolex-T" n'ime vitro na njupụta ise (2, 4, 6, 8 na 10 mg/L).
E si n'ugbo azụmahịa chịkọta ihe atụ nke ogwe agwa na mkpụrụ osisi ndị a na-ahụkarị nke na-egosi ihe mgbaàmà nke ebu ọcha (ọnụọgụ ọrịa: 10–30%). Ọ bụ ezie na a chọpụtara ọtụtụ ihe ọkụkụ ndị butere ọrịa site na ụdị/ụdị dị iche iche (ụdị ahịa a na-ahụ anya bụ Giza 3), ndị ọzọ, karịsịa ndị e si n'ahịa mpaghara nweta, bụ ụdị ndị a na-amaghị. E buru ụzọ jiri mmiri sodium hypochlorite 0.5% sachaa ihe ndị butere ọrịa ahụ ruo nkeji atọ, wee sachaa ha ọtụtụ ugboro na mmiri a gwakọtara agwakọta ma jiri akwụkwọ nzacha dị ọcha hichaa ha iji wepụ mmiri gabigara ókè. E mesịa, a bepụ akụkụ ahụ ndị butere ọrịa ahụ n'ime obere iberibe site na anụ ahụ dị n'etiti (n'etiti anụ ahụ dị mma na nke butere ọrịa), kụọ ha na poteto dextrose agar (PDA) ma kụọ ha na okpomọkụ 25 ± 2 °C na ìhè awa 12/awa 12 ọchịchịrị maka ụbọchị ise iji kpalite mmepe sclerotia. E jikwa usoro mycelial tip mee ihe iji sachaa ihe ndị sitere na ero sitere na omenala agwakọtara ma ọ bụ ndị merụrụ ahụ. E bu ụzọ chọpụta ihe e ji agwọ ero a dị ọcha dabere na njirimara omenala ya, wee gosi na ọ bụ S. sclerotiorum dabere na njirimara ndị dị obere. N'ikpeazụ, a nwalere ihe niile e ji agwọ ero a dị ọcha maka ọrịa na ụdị agwa a na-ahụkarị nke a na-akpọ Giza 3 iji mezuo ebumnuche Koch.
Tinyere nke ahụ, e gosikwara na S. sclerotiorum isolate kachasị emerụ ahụ (isolate #3) dabere na usoro nhazi nke spacer dị n'ime (ITS) dịka White et al., 1990 si kọwaa; Baturo-Ciesniewska et al., 2017. N'ụzọ dị mkpirikpi, a kụrụ ihe ndị a na-esi n'ime ofe dextrose potato (PDB) ma tinye ha na okpomọkụ 25 ± 2 °C ruo ụbọchị 5-7. E wee chịkọta mycelium fungal, sachaa ya site na cheesecloth, saa ya ugboro abụọ na mmiri dị ọcha, ma jiri akwụkwọ nzacha dị ọcha kpọọ ya. E wepụtara DNA genomic site na iji Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). E jiri usoro praịmarị pụrụ iche ITS1/ITS4 (TCCGTAGGTGAACCTGCGG TCCTCCGTTTATTGATATGC; nha a tụrụ anya ya: 540 bp) (Baturo-Ciesniewska et al., 2017). E tinyere ngwaahịa PCR e mere ka ha dị ọcha maka usoro nhazi (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). E jiri usoro nhazi Sanger mee usoro nhazi nke ITS rDNA n'akụkụ abụọ. E jiri usoro ajụjụ ndị a chịkọtara tụnyere data kachasị ọhụrụ na GenBank na National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/) site na iji ngwanrọ BLASTn. A tụnyere usoro ajụjụ a na ụdị S. sclerotiorum iri abụọ ndị ọzọ e si na data kachasị ọhụrụ dị na NCBI GenBank (Ntinye Tebụl S1) jiri ClustalW mee ihe na Ngwugwu Nyocha Mkpụrụ Ndụ Ihe Ndị Dị Ndụ (MEGA-11; ụdị 11) (Kumar et al., 2024). E mere nyocha evolushọn site na iji usoro ohere kachasị elu na ụdị mgbanwe nucleotide nke a na-agbanwe agbanwe n'oge (Nei na Kumar, 2000). E gosiri osisi nwere ohere kachasị elu maka ndekọ. A na-ahọrọ osisi mbụ maka ọchụchọ heuristic site na ịhọrọ osisi nwere ohere dị elu n'etiti osisi agbata obi na-ejikọta (NJ) (Kumar et al., 2024) na osisi parsimony kachasị elu (MP). E ji matrix anya abụọ gbakọọ osisi NJ site na iji ụdị oge izugbe a na-agbanwe agbanwe (Nei na Kumar, 2000).
E jiri usoro mgbasa agar chọpụta ọrụ nje bacteria nke L-ornithine na ọgwụ nje bacteria "Rizolex-T" n'ime vitro. Usoro: Were oke kwesịrị ekwesị nke ihe ngwọta L-ornithine (500 mg/L) ma gwakọta ya nke ọma na 10 ml nke ihe oriri PDA iji kwadebe ihe ngwọta nwere oke ikpeazụ nke 12.5, 25, 50, 75, 100 na 125 mg/L, n'otu n'otu. Ejiri oke ise nke ihe na-egbu egbu "Rizolex-T" (2, 4, 6, 8 na 10 mg/L) na mmiri a gwakọtara agwakọta mee ihe dị ka njikwa. Mgbe ihe ngwọta ahụ siri ike, e bufere ihe mkpuchi mycelium ọhụrụ nke ọdịbendị Sclerotinia sclerotiorum, dayameta 4 mm, na etiti efere Petri ma kụọ ya na 25±2°C ruo mgbe mycelium kpuchiri efere Petri niile, mgbe nke ahụ gasịrị, e dekọrọ uto fungal ahụ. Gbakọọ pasentị mgbochi nke uto radial nke S. sclerotiorum site na iji usoro 1:
A nwalere nnwale ahụ ugboro abụọ, ya na nnwale isii maka otu njikwa/nnwale ọ bụla na ite ise (osisi abụọ kwa ite) maka nnwale bayọlọji ọ bụla. A tụlere nnwale bayọlọji ọ bụla ugboro abụọ (mmegharị teknụzụ abụọ) iji hụ na nsonaazụ nnwale ahụ ziri ezi, ntụkwasị obi na imegharị ya. Na mgbakwunye, ejiri nyocha regression probit gbakọọ njupụta mgbochi ọkara kacha elu (IC50) na IC99 (Prentice, 1976).
Iji chọpụta ike L-ornithine nwere n'okpuru ọnọdụ griin haus, e mere nnwale ite abụọ n'usoro. N'ụzọ dị mkpirikpi, e ji ala ụrọ na ájá dị ọcha jupụta ite ndị ahụ (3:1) ma gbaa ha ọgwụ S. sclerotiorum ọhụrụ. Nke mbụ, e ji S. sclerotiorum (isolate #3) kụọ ihe kachasị njọ site n'ịbe otu sclerotium n'ọkara, tinye ya ihu ala na PDA ma tinye ya na 25°C n'ọchịchịrị na-adịgide adịgide (awa 24) ruo ụbọchị anọ iji kpalite uto mycelium. E wee wepụta plọg agar anọ nke dayameta 5 mm site na nsọtụ ụzọ ma gbaa 100 g nke ngwakọta amịkọrọ nke ọka wit na osikapa (1:1, v/v) ọgwụ ma tinye karama niile na 25 ± 2°C n'okpuru ìhè awa 12/oge ọchịchịrị awa 12 maka ụbọchị ise iji kpalite mmepe sclerotia. E gwakọtara ihe dị n'ime karama niile nke ọma iji hụ na ha dị n'otu tupu etinye ala. Mgbe ahụ, a na-etinye 100 g nke ngwakọta bran na-amịpụta n'ime ite ọ bụla iji hụ na nje ndị na-akpata ọrịa na-apụta mgbe niile. A na-agba ite ndị a gbara ọgwụ mmiri mmiri iji mee ka uto ero ahụ ka mma ma tinye ha n'ime ụlọ okpomọkụ ruo ụbọchị asaa.
A kụrụ mkpụrụ ise nke ụdị Giza 3 n'ime ite ọ bụla. Maka ite ndị e ji L-ornithine na ọgwụ nje Rizolex-T gwọọ, a na-ebu ụzọ tinye mkpụrụ ndị a gbanyere mkpọrọgwụ ruo awa abụọ n'ime mmiri mmiri nke ngwakọta abụọ ahụ na mkpokọta IC99 ikpeazụ nke ihe dị ka 250 mg/L na 50 mg/L, n'otu n'otu, wee kpoo ha n'ikuku ruo otu awa tupu a kụọ ha. N'aka nke ọzọ, a na-etinye mkpụrụ ndị ahụ n'ime mmiri a gbanyere mkpọrọgwụ dị ka ihe na-adịghị mma. Mgbe ụbọchị iri gasịrị, tupu mmiri mbụ a gbaa ha mmiri, a na-ewepụ mkpụrụ ndị ahụ, na-ahapụ naanị mkpụrụ abụọ dị ọcha n'ime ite ọ bụla. Na mgbakwunye, iji hụ na ọrịa S. sclerotiorum butere, a na-egbutu osisi agwa n'otu oge mmepe (ụbọchị iri) n'ebe abụọ dị iche iche site na iji scalpel a gbanyere mkpọrọgwụ ma tinye ihe dị ka 0.5 g nke ngwakọta bran na-agba agba n'ime ọnya ọ bụla, wee soro ya mmiri dị elu iji kpalite ọrịa na mmepe ọrịa na osisi niile a gbanyere mkpọrọgwụ. E merụrụ ụlọ ọrụ ndị na-achịkwa ihe ndị a n'otu aka ahụ, e tinyekwara ngwakọta bran a na-adịghị amị amị (0.5 g) n'ime ọnya ahụ ma debe ya n'okpuru oke iru mmiri iji mee ka gburugburu ebe obibi dị mma maka mmepe ọrịa ma hụ na otu ndị na-agwọ ọrịa ahụ na-adị n'otu.
Usoro ọgwụgwọ: A na-agba mkpụrụ osisi agwa mmiri site na iji mmiri mmiri L-ornithine (250 mg/l) ma ọ bụ ọgwụ nje Rizolex-T (50 mg/l) site n'ịgba mmiri n'ala, wee mee ọgwụgwọ ahụ ugboro atọ n'ime ụbọchị iri. A na-agba mmiri mmiri 500 ml nke mmiri a gwakọtara agwakọta nke a gwọrọ n'ime placebo. A na-eme ọgwụgwọ niile n'okpuru ọnọdụ griin haus (25 ± 2°C, iru mmiri 75 ± 1%, na oge foto nke ìhè awa 8/awa 16 gbara ọchịchịrị). A na-agba ite niile mmiri n'ime izu abụọ ma na-agwọ ha kwa ọnwa site na iji fatịlaịza NPK kwesịrị ekwesị (20-20-20, yana 3.6% sọlfọ na microelements TE; Zain Seeds, Egypt) na mkpokọta nke 3-4 g/l site na ịgba akwụkwọ dịka ndụmọdụ maka ụdị dị iche iche na ntuziaka onye nrụpụta si dị. Ọ gwụla ma e kwuru ihe ọzọ, a na-anakọta akwụkwọ ndị toro eto nke ọma (akwụkwọ nke abụọ na nke atọ site n'elu) site na mkpụrụ ndụ ọ bụla n'ime awa 72 mgbe ọgwụgwọ gasịrị (hpt), mee ka ha dị n'otu, chịkọta ha ma chekwaa ha na -80 °C maka nyocha ndị ọzọ gụnyere, mana ejedebeghị na, in situ histochemical localization nke ihe ngosi nrụgide oxidative, lipid peroxidation, enzymes na non-enzymatic antioxidants na ngosipụta mkpụrụ ndụ ihe nketa.
A tụlere ike ọrịa ebu ọcha kwa izu ụbọchị iri abụọ na otu mgbe a gbasịrị ya ọgwụ (dpi) site na iji nha nke 1–9 (Tebụl Mgbakwunye S2) dabere na nha Petzoldt na Dickson (1996) nke Teran et al. (2006) gbanwere. N'ụzọ dị mkpirikpi, a lere anya na ogwe na alaka nke osisi agwa site na mmalite nke ịgba ọgwụ iji soro ọganihu nke mmerụ ahụ n'akụkụ internodes na nodes. A tụziri anya nke mmerụ ahụ site na ebe a gbanyere ya ọgwụ ruo ebe kachasị anya n'akụkụ ogwe ma ọ bụ alaka ahụ ma kesaa akara nke 1–9 dabere na ebe mmerụ ahụ dị, ebe (1) gosiri na enweghị ọrịa a na-ahụ anya nso ebe a gbanyere ya ọgwụ na (2–9) gosiri mmụba nwayọ nwayọ na nha mmerụ ahụ na ọganihu n'akụkụ nodes/internodes (Tebụl Mgbakwunye S2). Mgbe ahụ, a gbanwere ike ọrịa ebu ọcha ka ọ bụrụ pasentị site na iji usoro 2:
Tinyere nke a, e jiri usoro (Shaner na Finney, 1977) gbakọọ mpaghara dị n'okpuru usoro mmepe ọrịa (AUDPC) nke e mere n'oge na-adịbeghị anya maka ire ere ọcha nke agwa nkịtị (Chauhan et al., 2020) site na iji usoro 3:
Ebe Yi = oke ọrịa n'oge ti, Yi+1 = oke ọrịa n'oge ọzọ ti+1, ti = oge nha mbụ (n'ime ụbọchị), ti+1 = oge nha ọzọ (n'ime ụbọchị), n = mkpokọta ọnụọgụ oge ma ọ bụ ebe nlele. E dekọrọ paramita uto osisi agwa gụnyere ịdị elu osisi (cm), ọnụọgụ alaka osisi ọ bụla, na ọnụọgụ akwụkwọ osisi ọ bụla kwa osisi kwa ụbọchị ruo ụbọchị iri abụọ na otu na ihe ndị dị ndụ niile.
N'ime ihe ndị e ji mee ihe n'ime ihe ndị dị ndụ ọ bụla, a chịkọtara ihe atụ akwụkwọ (akwụkwọ nke abụọ na nke atọ nke e mepụtara nke ọma site n'elu) n'ụbọchị nke iri anọ na ise mgbe e gwọchara ya (ụbọchị iri na ise ka e mesịrị ọgwụgwọ ikpeazụ). Ihe ndị e ji mee ihe n'ime ihe ndị dị ndụ ọ bụla nwere ite ise (osisi abụọ n'ime ite ọ bụla). E jiri ihe dị ka 500 mg nke anụ ahụ a gwepịara egwepịa wepụta pigments photosynthetic (chlorophyll a, chlorophyll b na carotenoids) site na iji 80% acetone na 4 °C n'ọchịchịrị. Mgbe awa iri abụọ na anọ gachara, a kpụchara ihe ndị ahụ ma chịkọta ihe ndị dị n'elu ihe ndị ahụ iji chọpụta ọdịnaya chlorophyll a, chlorophyll b na carotenoid site na iji UV-160A spectrophotometer (Shimadzu Corporation, Japan) dịka usoro (Lichtenthaler, 1987) si dị site n'ịtụ nha mmiri na ogologo mmiri atọ dị iche iche (A470, A646 na A663 nm). N'ikpeazụ, a gbakọrọ ọdịnaya nke pigments photosynthetic site na iji usoro ndị a 4–6 nke Lichtenthaler (1987) kọwara.
Mgbe awa iri asaa na abụọ gachara ọgwụgwọ (hpt), a nakọtara akwụkwọ (akwụkwọ nke abụọ na nke atọ mepụtara nke ọma site n'elu) site na ihe ọ bụla e mepụtara na bayoloji maka ebe a na-akpọ in situ histochemical location nke hydrogen peroxide (H2O2) na superoxide anion (O2•−). Ihe ọ bụla e mepụtara na bayoloji nwere ite ise (osisi abụọ kwa ite). A nyochachara ihe ọ bụla e mepụtara na bayoloji n'ime abụọ (ihe e mepụtara na teknụzụ abụọ) iji hụ na usoro ahụ ziri ezi, ntụkwasị obi na imegharị ya. Ejiri 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Germany) ma ọ bụ nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Germany) chọpụta H2O2 na O2•−, n'otu n'otu, na-agbaso usoro Romero-Puertas et al. (2004) na Adam et al. (1989) kọwara ya na obere mgbanwe. Maka ebe H2O2 dị na situ, e tinyere 0.1% DAB n'ime oghere dị na Tris buffer 10 mM (pH 7.8) n'ime oghere dị na ụlọ ahụ, wee tinye ya n'ọkụ ruo nkeji iri isii. E tinyere 0.15% (v/v) TCA n'ime ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) n'ime oghere dị na 4:1 (v/v) wee gosi ya ìhè ruo mgbe ha gbara ọchịchịrị. N'otu aka ahụ, e tinyere 10 mM potassium phosphate buffer (pH 7.8) nke nwere 0.1 w/v% HBT n'ime oghere dị na histochemical maka ebe O2•− dị na situ. E tinyere 0.1% DAB n'ime oghere dị na ụlọ ahụ ruo nkeji iri abụọ, wee tee ya ọcha dịka ọ dị n'elu, wee tee ya ọkụ ruo mgbe ntụpọ acha anụnụ anụnụ/violet gbara ọchịchịrị pụtara. E jiri ụdị Fiji nke ngwugwu nhazi onyonyo ImageJ (http://fiji.sc; abanyela na 7 Maachị 2024) nyochaa ike nke agba aja aja (dịka ihe ngosi H2O2) ma ọ bụ acha anụnụ anụnụ-violet (dịka ihe ngosi O2•−).
A chọpụtara Malondialdehyde (MDA; dị ka ihe nrịbama nke lipid peroxidation) dịka usoro Du na Bramlage (1992) si dị, ma nwee obere mgbanwe. A chịkọtara akwụkwọ sitere na mkpụrụ ndụ ọ bụla (akwụkwọ nke abụọ na nke atọ mepụtara nke ọma site n'elu) awa 72 mgbe ọgwụgwọ gasịrị (hpt). Mkpụrụ ndụ ọ bụla nwere ite ise (osisi abụọ kwa ite). A nyochachara mkpụrụ ndụ ọ bụla n'ime abụọ (mkpụrụ teknụzụ abụọ) iji hụ na usoro ahụ ziri ezi, ntụkwasị obi na ịmụgharị ọzọ. N'ụzọ dị mkpirikpi, ejiri 0.5 g nke anụ ahụ akwụkwọ ala mee ihe maka mwepụta MDA site na 20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) nke nwere 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). E mechara chọpụta ihe dị n'ime ihe na-eme ka mmiri dị n'elu mmiri site n'ịtụle mmiri na 532 na 600 nm site na iji UV-160A spectrophotometer (Shimadzu Corporation, Japan) wee gosipụta ya dị ka nmol g−1 FW.
Maka inyocha ihe ndị na-egbochi antioxidants na-abụghị enzymatic na enzymatic, a na-anakọta akwụkwọ (akwụkwọ nke abụọ na nke atọ nke mepụtara nke ọma site n'elu) site na ihe ndị na-akpata kemịkalụ nke ọ bụla n'ime awa 72 mgbe ọgwụgwọ gasịrị (hpt). Ihe ndị na-akpata kemịkalụ nke ọ bụla nwere ite ise (osisi abụọ n'ime ite ọ bụla). A tụlere ihe ndị na-akpata kemịkalụ nke ọ bụla n'ime ihe ndị a na-eme ugboro ugboro (ihe nlele teknụzụ abụọ). A na-eji nitrogen mmiri gwerie akwụkwọ abụọ ma jiri ya mee ihe ozugbo maka ikpebi ihe ndị na-akpata antioxidants na enzymatic na-abụghị enzymatic, amino acid zuru oke, ọdịnaya proline, ngosipụta mkpụrụ ndụ ihe nketa, na nhazi oxalate.
E ji Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) chọpụta phenolic niile a na-agbaze agbaze, ma jiri obere mgbanwe nke usoro Kahkonen et al. (1999) kọwara mee ihe. N'ụzọ dị mkpirikpi, e jiri 20 ml 80% methanol ewepụta ihe dị ka 0.1 g nke anụ ahụ e ji otu ihe mee n'ọchịchịrị ruo awa 24, e wee chịkọta ihe fọdụrụ mgbe e tinyere ya n'ime centrifugation. E jiri 0.1 ml nke ihe nlele ahụ gwakọta ya na 0.5 ml Folin-Ciocalteu reagent (10%), maa jijiji ruo sekọnd 30 wee hapụ ya n'ọchịchịrị maka nkeji 5. E mesịa, etinyere 0.5 ml nke 20% sodium carbonate solution (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) na tube ọ bụla, gwakọta ya nke ọma ma tinye ya n'ime okpomọkụ ụlọ n'ọchịchịrị maka awa 1. Mgbe a tụchara ya, a tụrụ mmịpụta nke ngwakọta mmeghachi omume na 765 nm site na iji UV-160A spectrophotometer (Shimadzu Corporation, Japan). E jiri usoro nhazi gallic acid (Fisher Scientific, Hampton, NH, USA) chọpụta mkpokọta phenols na-agbaze na ihe ndị a na-ewepụta ma gosipụta ya dị ka milligrams nke gallic acid nke hà nhata kwa gram nke ibu ọhụrụ (mg GAE g-1 ibu ọhụrụ).
A chọpụtara ọdịnaya flavonoid zuru oke dịka usoro Djeridane et al. (2006) si dị, ma nwee obere mgbanwe. N'ụzọ dị mkpirikpi, a gwakọtara 0.3 ml nke methanol dị n'elu na 0.3 ml nke 5% aluminum chloride solution (AlCl3; Fisher Scientific, Hampton, NH, USA), wee kpalie ya nke ọma wee kụọ ya na okpomọkụ ụlọ maka nkeji ise, wee tinye 0.3 ml nke 10% potassium acetate solution (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), gwakọtara ya nke ọma ma kụọ ya na okpomọkụ ụlọ maka nkeji iri atọ n'ọchịchịrị. Mgbe a kụọ ya, a tụrụ mmịpụta nke ngwakọta mmeghachi omume na 430 nm site na iji UV-160A spectrophotometer (Shimadzu Corporation, Japan). E jiri usoro nhazi rutin (TCI America, Portland, OR, USA) chọpụta mkpokọta flavonoids na-agbaze na ihe nlele ahụ wee gosipụta ya dị ka milligrams nke rutin nhata kwa gram nke ibu ọhụrụ (mg RE g-1 ibu ọhụrụ).
E ji ihe e ji emezi ihe na-eme ka ihe dị n'ime akwụkwọ agwa (Thermo Scientific Chemicals, Waltham, MA, USA) chọpụta ihe dị n'ime amino acid n'efu nke akwụkwọ agwa site na iji usoro Yokoyama na Hiramatsu (2003) tụrụ aro ma Sun et al. (2006) gbanwee ya. N'ụzọ dị mkpirikpi, e jiri pH 5.4 buffer wepụta 0.1 g nke anụ ahụ ala, e mekwara 200 μL nke ninhydrin (2%) na 200 μL nke pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), tinye ya n'ime mmiri ịsa ahụ ruo nkeji iri atọ, wee jụọ oyi ma tụọ ya na 580 nm site na iji UV-160A spectrophotometer (Shimadzu Corporation, Japan). N'aka nke ọzọ, e jiri usoro Bates chọpụta proline (Bates et al., 1973). E jiri 3% sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) wepụta Proline, mgbe e mechara centrifugation, e tinyere 0.5 ml nke ihe na-esi n'elu mmiri na 1 ml glacial acetic acid (Fisher Scientific, Hampton, NH, USA) na ninhydrin reagent, tinye ya na 90°C maka nkeji iri anọ na ise, wee jụọ oyi ma tụọ ya na 520 nm site na iji otu spectrophotometer ahụ dị n'elu. E jiri usoro nhazi glycine na proline (Sigma-Aldrich, St Louis, MO, USA) chọpụta amino acid na proline zuru oke na ihe ewepụtara n'akwụkwọ, ma gosipụta ya dị ka mg/g ibu ọhụrụ.
Iji chọpụta ọrụ enzymatic nke enzymes antioxidant, e jiri 3 ml nke 50 mM Tris buffer (pH 7.8) nke nwere 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) na 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA) wepụta ihe dị ka 500 mg nke anụ ahụ e jikọtara ọnụ, nke e tinyere na 10,000 × g maka nkeji iri abụọ n'okpuru friji (4 °C), wee chịkọta ihe ndị na-adịghị emerụ ahụ (ihe e si na enzyme wepụ) (El-Nagar et al., 2023; Osman et al., 2023). E mechara meghachi omume na Catalase (CAT) site na iji 2 ml nke 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) na 100 μl nke 269 mM H2O2 ngwọta iji chọpụta ọrụ enzymatic ya dịka usoro Aebi (1984) si dị, ya na obere mgbanwe (El-Nagar et al., 2023; Osman et al., 2023). E jiri usoro Harrach et al. (2009) chọpụta ọrụ enzymatic Guaiacol-dependent peroxidase (POX). (2008) site na obere mgbanwe (El-Nagar et al., 2023; Osman et al., 2023) na ọrụ enzymatic nke polyphenol oxidase (PPO) ka achọpụtara mgbe mmeghachi omume ya na 2.2 ml nke 100 mM sodium phosphate buffer (pH 6.0), 100 μl nke guaiacol (TCI chemicals, Portland, OR, USA) na 100 μl nke 12 mM H2O2. E gbanwere usoro a ntakịrị site na (El-Nagar et al., 2023; Osman et al., 2023). Emere nnwale ahụ mgbe mmeghachi omume ya na 3 ml nke ngwọta catechol (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) nke a kwadebere ọhụrụ na 0.1 M phosphate buffer (pH 6.0). A tụrụ ọrụ CAT site n'ileba anya na mmebi nke H2O2 na 240 nm (A240), a tụrụ ọrụ POX site na ileba anya na mmụba nke nnabata na 436 nm (A436), a tụkwara ọrụ PPO site na ịdekọ mgbanwe nnabata na 495 nm (A495) kwa sekọnd 30 maka nkeji 3 site na iji spectrophotometer UV-160A (Shimadzu, Japan).
E jiri RT-PCR oge a chọpụta ọkwa nke mkpụrụ ndụ ihe nketa atọ metụtara antioxidant, gụnyere peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), na glutathione reductase (PvGR; GenBank Accession No. KY195009.1), na akwụkwọ agwa (akwụkwọ nke abụọ na nke atọ mepụtara nke ọma site n'elu) awa 72 ka ọgwụgwọ ikpeazụ gasịrị. N'ụzọ dị mkpirikpi, e kewapụrụ RNA site na iji Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) dịka usoro onye nrụpụta si dị. Mgbe ahụ, e ji TOP script™ cDNA Synthesis Kit mepụta cDNA dịka ntuziaka onye nrụpụta si dị. E depụtara usoro praịmarị nke mkpụrụ ndụ ihe nketa atọ dị n'elu na Tebụl Mgbakwunye S3. E jiri PvActin-3 (nọmba njikọta GenBank: XM_068616709.1) mee ihe dị ka mkpụrụ ndụ ihe nketa na-elekọta ụlọ, e jikwa usoro 2-ΔΔCT gbakọọ ngosipụta mkpụrụ ndụ ihe nketa dị n'etiti ha (Livak na Schmittgen, 2001). E gosipụtara nkwụsi ike nke actin n'okpuru nrụgide biotic (mmekọrịta na-ekwekọghị ekwekọ n'etiti mkpụrụ osisi nkịtị na ero anthracnose Colletotrichum lindemuthianum) na nrụgide abiotic (oké ọkọchị, nnu, obere okpomọkụ) (Borges et al., 2012).
Anyị mere nyocha nke mkpụrụ ndụ ihe nketa nke protein oxaloacetate acetylhydrolase (OAH) na S. sclerotiorum site na iji ngwa BLAST protein (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). N'ụzọ dị mkpirikpi, anyị jiri OAH sitere na Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; nọmba nnweta GenBank XP_040799428.1; amino acid 342) na Penicillium lagena (PlOAH; taxide: 94218; nọmba nnweta GenBank XP_056833920.1; amino acid 316) dị ka usoro ajụjụ iji maapụ protein homologous na S. sclerotiorum (taxide: 5180). E mere BLASTp megide data mkpụrụ ndụ ihe nketa S. sclerotiorum kachasị ọhụrụ dị na GenBank na weebụsaịtị National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Tinyere nke ahụ, e jiri usoro kachasị dị na MEGA11 (Tamura et al., 2021) na ụdị JTT matrix dabere na ya (Jones et al., 1992) chọpụta mkpụrụ ndụ ihe nketa OAH nke e buru amụma site na S. sclerotiorum na usoro ajụjụ site na iji Ngwaọrụ Nhazi Dabere na Mgbochi (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos na Agarwala, 2007). Tinyere nke ahụ, e jikọtara usoro amino acid kacha mma nke SsOAH sitere na S. sclerotiorum na usoro ajụjụ (AfOAH na PlOAH) (Larkin et al., 2007) site na iji ClustalW (http://www.genome.jp/tools-bin/clustalw), e jikwa ngwaọrụ ESPript (ụdị 3.0; https://espript.ibcp.fr/ESPript/ESPript/index.php) hụ mpaghara echekwara na nhazi ahụ.
Ọzọkwa, e kewara ngalaba ndị a na-atụ anya ọrụ na ebe echekwara nke S. sclerotiorum SsOAH n'ime ezinụlọ dị iche iche site na iji ngwa InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). N'ikpeazụ, e ji Protein Homology/Analogy Recognition Engine mee ihe nlereanya nhazi atọ (3D) nke S. sclerotiorum SsOAH buru amụma (Phyre2 server version 2.0; http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) (Kelley et al., 2015) ma kwado ya site na iji sava SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). E jiri ngwugwu UCSF-Chimera (mbipụta 1.15; https://www.cgl.ucsf.edu/chimera/) mee ihe ngosi nke usoro atọ e buru amụma (ụdị PDB).
E jiri PCR fluorescence nke oge a chọpụta ọkwa ntụgharị nke oxaloacetate acetylhydrolase (SsOAH; nọmba nnweta GenBank: XM_001590428.1) na mycelia nke Sclerotinia sclerotiorum. N'ụzọ dị mkpirikpi, a gbara S. sclerotiorum ọgwụ n'ime karama nwere PDB ma tinye ya na incubator na-ama jijiji (ụdị: I2400, New Brunswick Scientific Co., Edison, NJ, USA) na 25 ± 2 °C maka awa 24 na 150 rpm na n'ọchịchịrị mgbe niile (awa 24) iji kpalite uto mycelium. Mgbe nke ahụ gasịrị, a gwọrọ mkpụrụ ndụ ndị ahụ na L-ornithine na ọgwụ nje Rizolex-T na njupụta IC50 ikpeazụ (ihe dị ka 40 na 3.2 mg/L, n'otu n'otu) wee kụọ ha maka awa 24 ọzọ n'okpuru ọnọdụ ndị ahụ. Mgbe a kpọchiri ya, a kpụchara ihe ọkụkụ ndị ahụ na 2500 rpm maka nkeji ise, a chịkọtakwara ihe ndị dị n'elu (mycelium fungal) maka nyocha mkpụrụ ndụ ihe nketa. N'otu aka ahụ, a chịkọtara mycelium fungal na awa 0, 24, 48, 72, 96, na 120 mgbe ọrịa gasịrị site na osisi ndị butere ọrịa nke kpụrụ ebu ọcha na mycelium owu n'elu anụ ahụ ndị butere ọrịa. E wepụtara RNA na mycelium fungal wee mepụta cDNA dịka akọwara n'elu. E depụtara usoro praịmarị maka SsOAH na Tebụl Mgbakwunye S3. E jiri SsActin (nọmba nnweta GenBank: XM_001589919.1) dị ka mkpụrụ ndụ ihe nketa na-elekọta ụlọ, a gbakọọkwa ngosipụta mkpụrụ ndụ ihe nketa site na iji usoro 2-ΔΔCT (Livak na Schmittgen, 2001).
A chọpụtara oxalic acid n'ime ofe poteto dextrose (PDB) na ihe nlele osisi nwere nje fungal Sclerotinia sclerotiorum dịka usoro Xu na Zhang (2000) si dị, ma nwee obere mgbanwe. N'ụzọ dị mkpirikpi, a gbara ọgwụ S. sclerotiorum isolates n'ime karama nwere PDB wee kụọ ya n'ime ihe na-ama jijiji (ụdị I2400, New Brunswick Scientific Co., Edison, NJ, USA) na 150 rpm na 25 ± 2 °C maka ụbọchị 3-5 n'ọchịchịrị mgbe niile (awa 24) iji kpalite uto mycelium. Mgbe a gbanyechara ya, a na-ebu ụzọ jiri akwụkwọ nzacha Whatman #1 wee kpụọ ya na 2500 rpm maka nkeji 5 iji wepụ mycelium fọdụrụnụ. A chịkọtara ihe na-ekpuchi ya ma chekwaa ya na 4°C maka mkpebi ọnụọgụgụ nke oxalate. Maka nkwadebe nke ihe nlele osisi, a na-ewepụta ihe dị ka 0.1 g nke iberibe anụ ahụ osisi ugboro atọ na mmiri a gwakọtara agwakọta (2 ml oge ọ bụla). E wee jiri centrifuge tụọ ihe nlele ndị ahụ na 2500 rpm maka nkeji ise, e jiri akwụkwọ nzacha Whatman Nke 1 wee chịkọta ha maka nyocha ọzọ.
Maka nyocha ọnụọgụgụ nke oxalic acid, a kwadebere ngwakọta mmeghachi omume ahụ n'ime tube a na-egbochi iko n'usoro ndị a: 0.2 ml nke ihe nlele (ma ọ bụ PDB culture filtrate ma ọ bụ oxalic acid standard solution), 0.11 ml nke bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), 0.198 ml nke 1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) na 0.176 ml nke 100 mM potassium dichromate (K2Cr2O7; kemịkalụ TCI, Portland, OR, USA), wee gwakọta ngwọta ahụ ruo 4.8 ml na mmiri a gwakọtara agwakọta nke ọma ma tinye ya ozugbo na mmiri ịsa ahụ 60 °C. Mgbe nkeji iri gachara, a kwụsịrị mmeghachi omume ahụ site na ịgbakwunye 0.5 ml nke sodium hydroxide solution (NaOH; 0.75 M). A tụrụ mmịpụta (A600) nke ngwakọta mmeghachi omume na 600 nm site na iji UV-160 spectrophotometer (Shimadzu Corporation, Japan). E jiri PDB na mmiri a gwakọtara agwakọta mee ihe dị ka njikwa maka nha nke nzacha ọdịbendị na ihe nlele osisi, n'otu n'otu. Oke oxalic acid dị na nzacha ọdịbendị, nke a na-egosipụta dị ka microgram nke oxalic acid kwa millilita nke PDB medium (μg.mL−1), na na ihe ewepụtara akwụkwọ, nke a na-egosipụta dị ka microgram nke oxalic acid kwa gram nke ibu ọhụrụ (μg.g−1 FW), ka ejiri usoro nhazi oxalic acid (Thermo Fisher Scientific Chemicals, Waltham, MA, USA) chọpụta.
N'ọmụmụ ihe a dum, e mere nnwale niile n'usoro zuru oke (CRD) site na iji usoro nhazi zuru oke (CRD) mee ihe, ya na usoro nhazi isii nke ihe ndị dị ndụ kwa ọgwụgwọ na ite ise nke ihe ndị dị ndụ kwa (osisi abụọ kwa ite) ọ gwụla ma e kwuru ihe ọzọ. E nyochachara ihe ndị dị ndụ n'usoro nhazi abụọ (ihe ndị dị ndụ abụọ). E jiri usoro nhazi abụọ nke ihe ndị dị ndụ lelee otu nnwale ahụ mana e jighị ha mee ihe na nyocha ọnụọgụgụ iji zere ihe ndị dị adị. E jiri usoro nyocha nke ihe ndị dị ndụ mee nyocha ọnụọgụgụ site na iji nyocha nke ihe ndị dị iche (ANOVA) mee nyocha ọnụọgụgụ site na iji nyocha nke ihe ndị dị iche (HSD) wee soro ule Tukey-Kramer nke nwere nnukwu ọdịiche (HSD) (p ≤ 0.05). Maka nnwale in vitro, e jiri usoro probit mee ihe gbakọọ ụkpụrụ IC50 na IC99, a gbakọọkwa oge ntụkwasị obi 95%.
E si n'ubi soybean dị iche iche chịkọta ihe anọ e si n'ubi soybean dị iche iche chịkọta na El Ghabiya Governorate, Ijipt. Na PDA medium, ihe niile e si n'ime ya mepụta mycelium ọcha na-acha ude nke na-aghọ ọcha owu ngwa ngwa (Foto 1A) wee bụrụ beige ma ọ bụ aja aja na ọkwa sclerotium. Sclerotia na-abụkarị oke, oji, gburugburu ma ọ bụ ọdịdị na-adịghị mma, ogologo 5.2 ruo 7.7 mm na dayameta 3.4 ruo 5.3 mm (Foto 1B). Ọ bụ ezie na ihe anọ e si n'ime ya mepụtala ụdị sclerotia n'akụkụ nke ihe ọkụkụ mgbe ụbọchị 10-12 nke nkụchi na 25 ± 2 °C (Foto 1A), ọnụọgụ sclerotia kwa efere dị iche nke ukwuu n'etiti ha (P < 0.001), ebe ihe atọ e si n'ime ya nwere ọnụọgụ sclerotia kachasị elu (32.33 ± 1.53 sclerotia kwa efere; Foto 1C). N'otu aka ahụ, isolate #3 mepụtara ọtụtụ oxalic acid na PDB karịa isolate ndị ọzọ (3.33 ± 0.49 μg.mL−1; Fig. 1D). Isolate #3 gosipụtara njirimara morphological na microscopic nke ero phytopathogenic Sclerotinia sclerotiorum. Dịka ọmụmaatụ, na PDA, colonies nke isolate #3 toro ngwa ngwa, ha dị ọcha ude (Foto 1A), beige azụ ma ọ bụ salmon dị nro odo-agba aja aja, ma chọọ ụbọchị 6-7 na 25 ± 2°C iji kpuchie elu nke efere dayameta 9 cm. Dabere na njirimara morphological na microscopic dị n'elu, achọpụtara isolate #3 dị ka Sclerotinia sclerotiorum.
Foto 1. Àgwà na ihe ndị na-akpata ọrịa nke ihe ndị na-akpata ọrịa S. sclerotiorum sitere na ihe ọkụkụ legume nkịtị. (A) Mmụba Mycelial nke ihe ndị na-akpata ọrịa S. sclerotiorum anọ na PDA, (B) sclerotia nke ihe ndị na-akpata ọrịa S. sclerotiorum anọ, (C) ọnụọgụgụ nke ihe ndị na-akpata ọrịa S (kwa efere), (D) ihe ndị na-akpata ọrịa oxalic acid na PDB (μg.mL−1), na (E) oke ọrịa (%) nke ihe ndị na-akpata ọrịa S. sclerotiorum anọ na ụdị legume azụmahịa dị mfe na Giza 3 n'okpuru ọnọdụ griin haus. Ụkpụrụ ndị a na-anọchite anya nkezi ± SD nke ihe ndị na-akpata ọrịa ise (n = 5). Mkpụrụ akwụkwọ dị iche iche na-egosi ọdịiche dị mkpa n'etiti ọgwụgwọ (p < 0.05). (F–H) Ihe mgbaàmà ebu ọcha a na-ahụkarị pụtara na mkpuru osisi na siliques dị n'elu ala, n'otu n'otu, ụbọchị iri mgbe a gbasịrị ya na ihe ndị na-akpata ọrịa #3 (dpi). (I) E jiri usoro ohere kachasị elu mee nyocha evolushọn nke mpaghara spacer dị n'ime nke S. sclerotiorum isolate #3 ma tụnyere ya na ụdị ntụaka iri abụọ e nwetara site na nchekwa data National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). Ọnụọgụ dị n'elu ahịrị ndị a na-akpọ clustering na-egosi mkpuchi mpaghara (%), ọnụọgụgụ ndị dị n'okpuru ahịrị ndị a na-akpọ clustering na-egosi ogologo alaka ahụ.
Ọzọkwa, iji gosi na ọrịa ahụ dị, e jiri ọgwụ anọ e nwetara site na S. sclerotiorum gbaa ọgwụ mgbochi maka ụdị agwa azụmaahịa Giza 3 n'okpuru ọnọdụ griin haus, nke kwekọrọ na ihe Koch's postulates (Foto 1E). Ọ bụ ezie na ihe niile e nwetara site na fungal bụ ihe na-akpata ọrịa ma nwee ike ibute ọrịa agwa akwụkwọ ndụ akwụkwọ ndụ (cv. Giza 3), na-akpata mgbaàmà ebu ọcha nkịtị n'akụkụ niile dị n'elu ala (Foto 1F), ọkachasị na ogwe osisi (Foto 1G) na pods (Foto 1H) na ụbọchị iri mgbe a gbasịrị ya (dpi), isolate 3 bụ ihe kachasị ike na nnwale abụọ nọọrọ onwe ha. Isolate 3 nwere oke ọrịa kachasị elu (%) na osisi agwa (24.0 ± 4.0, 58.0 ± 2.0, na 76.7 ± 3.1 na ụbọchị 7, 14, na 21 mgbe ọrịa gasịrị, n'otu n'otu; Foto 1F).
E gosikwara na njirimara nke S. sclerotiorum isolate #3 kacha emerụ ahụ dabere na usoro nhazi nke spacer dị n'ime (ITS) (Foto 1I). Nyocha phylogenetic n'etiti isolate #3 na ụdị ntụaka 20 gosiri nnukwu myirịta (>99%) n'etiti ha. Ọ dị mkpa ịmara na S. sclerotiorum isolate #3 (533 bp) nwere nnukwu myirịta na American S. sclerotiorum isolate LPM36 nke e kewapụrụ site na mkpụrụ pea akọrọ (nọmba ntinye GenBank MK896659.1; 540 bp) na S. sclerotiorum isolate nke China YKY211 (nọmba ntinye GenBank OR206374.1; 548 bp), nke na-akpata ire ere osisi violet (Matthiola incana), nke a na-ejikọta ha iche iche n'elu dendrogram (Foto 1I). E tinyela usoro ọhụrụ a n'ime nchekwa data NCBI ma kpọọ ya "Sclerotinia sclerotiorum - isolate YN-25" (nọmba nnweta GenBank PV202792). A pụrụ ịhụ na isolate 3 bụ isolate kachasị emerụ ahụ; ya mere, a họọrọ isolate a maka ọmụmụ ihe na nnwale niile sochirinụ.
A nyochachara ọrụ nje bacteria nke diamine L-ornithine (Sigma-Aldrich, Darmstadt, Germany) na njupụta dị iche iche (12.5, 25, 50, 75, 100 na 125 mg/L) megide S. sclerotiorum isolate 3 na vitro. Ọ dị mkpa ịmara na L-ornithine nwere mmetụta nje bacteria ma jiri nwayọọ nwayọọ gbochie uto radial nke S. sclerotiorum hyphae n'ụzọ dabere na dose (Foto 2A, B). Na njupụta kachasị elu a nwalere (125 mg/L), L-ornithine gosipụtara ọnụego mgbochi uto mycelium kachasị elu (99.62 ± 0.27%; Foto 2B), nke hà nhata na ọgwụ nje Rizolex-T (ọnụọgụ mgbochi 99.45 ± 0.39%; Foto 2C) na njupụta kachasị elu a nwalere (10 mg/L), na-egosi arụmọrụ yiri nke ahụ.
Foto nke 2. Ọrụ nje bacteria nke L-ornithine n'ime vitro megide Sclerotinia sclerotiorum. (A) Ntụnyere ọrụ nje bacteria nke ọtụtụ L-ornithine dị iche iche megide S. sclerotiorum na ọgwụ nje fungal Rizolex-T (10 mg/L). (B, C) Ọnụego mgbochi (%) nke uto mycelium S. sclerotiorum mgbe ọgwụgwọ gasịrị na ọtụtụ L-ornithine dị iche iche (12.5, 25, 50, 75, 100 na 125 mg/L) ma ọ bụ Rizolex-T (2, 4, 6, 8 na 10 mg/L), n'otu n'otu. Ụkpụrụ na-anọchite anya nkezi ± SD nke ihe ndị ọzọ dị ndụ ise (n = 5). Mkpụrụ akwụkwọ dị iche iche na-egosi ọdịiche ọnụọgụgụ dị n'etiti ọgwụgwọ (p < 0.05). (D, E) Nyocha nhazi ihe atụ nke L-ornithine na ọgwụ nje fungal azụmahịa Rizolex-T, n'otu n'otu. A na-egosi ahịrị mgbanwe nlereanya probit dị ka ahịrị anụnụ anụnụ siri ike, a na-egosikwa oge ntụkwasị obi (95%) dị ka ahịrị uhie gbara ọkpụrụkpụ.
Tinyere nke a, e mere nyocha mgbanwe probit ma gosipụta atụmatụ ndị kwekọrọ na Tebụl 1 na Foto 2D,E. N'ụzọ dị mkpirikpi, uru slope a na-anabata (y = 2.92x − 4.67) na ọnụ ọgụgụ dị mkpa metụtara ya (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 na p < 0.0001; Foto 2D) nke L-ornithine gosiri mmụba ọrụ antifungal megide S. sclerotiorum ma e jiri ya tụnyere ọgwụ nje Rizolex-T nke na-ere ahịa (y = 1.96x − 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 na p < 0.0001) (Tebụl 1).
Tebụl 1. Uru nke ọkara oke mgbochi (IC50) na IC99 (mg/l) nke L-ornithine na ọgwụ fungicidal "Rizolex-T" megide S. sclerotiorum.
N'ozuzu ya, L-ornithine (250 mg/L) belatara mmepe na oke ibu nke ebu ọcha na osisi agwa a na-agwọkarị ma e jiri ya tụnyere osisi ndị nwere S. sclerotiorum na-anaghị agwọ ọrịa (njikwa; Foto 3A). N'ụzọ dị mkpirikpi, ọ bụ ezie na oke ọrịa nke osisi ndị na-achịkwa ọrịa ndị a na-agwọghị na-amụba nwayọ nwayọ (52.67 ± 1.53, 83.21 ± 2.61, na 92.33 ± 3.06%), L-ornithine belatara oke ọrịa ahụ (%) n'oge nnwale ahụ (8.97 ± 0.15, 18.00 ± 1.00, na 26.36 ± 3.07) na ụbọchị 7, 14, na 21 mgbe ọgwụgwọ gasịrị (dpt), n'otu n'otu (Foto 3A). N'otu aka ahụ, mgbe ejiri 250 mg/L L-ornithine gwọọ osisi agwa ndị S. sclerotiorum butere, mpaghara dị n'okpuru usoro mmepe ọrịa (AUDPC) belatara site na 1274.33 ± 33.13 na njikwa a na-agwọghị ruo 281.03 ± 7.95, nke dị ntakịrị ala karịa nke njikwa dị mma 50 mg/L Rizolex-T fungicide (183.61 ± 7.71; Fig. 3B). A hụrụ otu usoro ahụ na nnwale nke abụọ.
Foto 3. Mmetụta nke itinye L-ornithine n'èzí na mmepe nke ire ere ọcha nke agwa nkịtị nke Sclerotinia sclerotiorum kpatara n'okpuru ọnọdụ griinhaus. (A) Usoro ọganihu nke ebu ọcha nke agwa nkịtị mgbe ejiri 250 mg/L L-ornithine gwọọ ya. (B) Mpaghara dị n'okpuru usoro ọganihu ọrịa (AUDPC) nke ebu ọcha nke agwa nkịtị mgbe ejiri L-ornithine gwọọ ya. Ụkpụrụ na-anọchite anya nkezi ± SD nke ihe ndị ọzọ mejupụtara ndụ ise (n = 5). Mkpuru akwụkwọ dị iche iche na-egosi ọdịiche dị mkpa n'etiti ọgwụgwọ (p < 0.05).
N'itinye 250 mg/L L-ornithine n'èzí, o ji nwayọọ nwayọọ mụbaa elu osisi (Foto 4A), ọnụọgụ alaka n'otu osisi (Foto 4B), na ọnụọgụ akwụkwọ n'otu osisi (Foto 4C) mgbe ụbọchị 42 gachara. Ọ bụ ezie na ọgwụ nje Rizolex-T (50 mg/L) nke a na-ere n'ahịa nwere mmetụta kachasị ukwuu na ihe niile a na-eme n'ihe gbasara nri, itinye 250 mg/L L-ornithine n'èzí nwere mmetụta kachasị ukwuu nke abụọ ma e jiri ya tụnyere njikwa ndị a na-anaghị agwọ ọrịa (Foto 4A–C). N'aka nke ọzọ, ọgwụgwọ L-ornithine enweghị mmetụta dị ukwuu na ọdịnaya nke pigments photosynthetic chlorophyll a (Foto 4D) na chlorophyll b (Foto 4E), mana ọ mụbara ntakịrị ọdịnaya carotenoid zuru oke (0.56 ± 0.03 mg/g fr wt) ma e jiri ya tụnyere njikwa na-adịghị mma (0.44 ± 0.02 mg/g fr wt) na njikwa dị mma (0.46 ± 0.02 mg/g fr wt; Fig. 4F). N'ozuzu, nsonaazụ ndị a na-egosi na L-ornithine abụghị phytotoxic nye legumes a gwọrọ ma nwee ike ịkpalite uto ha.
Foto 4. Mmetụta nke itinye L-ornithine n'èzí na njirimara uto na agba fotosynthetic nke akwụkwọ agwa ndị butere Sclerotinia sclerotiorum n'okpuru ọnọdụ griin haus. (A) Ogologo osisi (cm), (B) Ọnụọgụ alaka kwa osisi, (C) Ọnụọgụ akwụkwọ kwa osisi, (D) Chlorophyll a ọdịnaya (mg g-1 fr wt), (E) Chlorophyll b ọdịnaya (mg g-1 fr wt), (F) Ngụkọta ọdịnaya carotenoid (mg g-1 fr wt). Ụkpụrụ bụ nkezi ± SD nke ihe ndị ọzọ dị ndụ ise (n = 5). Mkpụrụ akwụkwọ dị iche iche na-egosi ọdịiche dị mkpa n'etiti ọgwụgwọ (p < 0.05).
N'ime ebe a na-akpọ "in situ histochemical determination" nke ụdị oxygen reactive (ROS; nke a na-akpọ hydrogen peroxide [H2O2]) na free radicals (nke a na-akpọ superoxide anions [O2•−]) gosiri na itinye L-ornithine (250 mg/L) n'èzí belatara mkpokọta H2O2 (96.05 ± 5.33 nmol.g−1 FW; Fig. 5A) na O2•− (32.69 ± 8.56 nmol.g−1 FW; Fig. 5B) ma e jiri ya tụnyere mkpokọta nke osisi abụọ ahụ a na-agwọghị (173.31 ± 12.06 na 149.35 ± 7.94 nmol.g−1 FW, n'otu n'otu) na osisi e ji 50 mg/L nke ọgwụ nje Rizolex-T (170.12 ± 9.50 na 157.00 ± 7.81 nmol.g−1 fr wt gwọọ, n'otu n'otu) n'ime awa 72. Oke ọkwa H2O2 na O2•− nke a chịkọtara n'okpuru hpt (Foto 5A, B). N'otu aka ahụ, nnwale malondialdehyde (MDA) nke dabeere na TCA gosiri na osisi agwa ndị nwere S. sclerotiorum gbakọtara ọkwa MDA dị elu (113.48 ± 10.02 nmol.g fr wt) n'akwụkwọ ha (Foto 5C). Agbanyeghị, itinye L-ornithine n'èzí belatara nke ukwuu lipid peroxidation dịka egosiri site na mbelata ọdịnaya MDA na osisi ndị a gwọrọ (33.08 ± 4.00 nmol.g fr wt).
Foto nke 5. Mmetụta nke itinye L-ornithine n'èzí na ihe nrịbama dị mkpa nke nrụgide oxidative na usoro nchebe antioxidant na-abụghị enzymatic na akwụkwọ agwa ndị butere S. sclerotiorum na awa 72 mgbe ọrịa gasịrị n'okpuru ọnọdụ griinhaus. (A) Hydrogen peroxide (H2O2; nmol g−1 FW) na 72 hpt, (B) anion superoxide (O2•−; nmol g−1 FW) na 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) na 72 hpt, (D) phenols na-agbaze (mg GAE g−1 FW) na 72 hpt, (E) flavonoids na-agbaze (mg RE g−1 FW) na 72 hpt, (F) amino acid na-agbaze (mg g−1 FW) na 72 hpt, na (G) ọdịnaya proline (mg g−1 FW) na 72 hpt. Ụkpụrụ na-anọchite anya nkezi ± ọkọlọtọ nkewa (nkezi ± SD) nke ihe ndị mejupụtara ihe ndị dị ndụ ise (n = 5). Mkpụrụ akwụkwọ dị iche iche na-egosi ọdịiche dị mkpa n'etiti ọgwụgwọ (p < 0.05).