Ko te tuhinga nei he wāhanga o te kaupapa rangahau "Te whakapai ake i te ātete o ngā pīni ki ngā mate me ngā pepeke", tirohia ngā tuhinga e 5 katoa
Ko te pūtake o te mate harore o ngā tipu ka whakamahia e Sclerotinia sclerotiorum (Lib.) de Bary tētahi rautaki maha-taumata hei pangia ngā tipu manaaki rerekē. E whakaaro ana tēnei rangahau ki te whakamahi i te diamine L-ornithine, he waikawa amino kore-pūmua e whakaoho ana i te hanganga o ētahi atu waikawa amino nui, hei rautaki whakahaere rerekē hei whakarei ake i ngā urupare ngota, ā-tinana me te matū koiora o Phaseolus vulgaris L. ki te harore mā i puta mai i te Pseudomonas sclerotiorum. I whakaatuhia e ngā whakamātautau in vitro he nui te aukati a te L-ornithine i te tipu mycelial o S. pyrenoidosa i runga i te horopeta. I tua atu, ka taea e ia te whakaiti nui i te kaha o te harore mā i raro i ngā āhuatanga kati kōtuhi. I tua atu, i whakaoho a L-ornithine i te tipu o ngā tipu i rongoatia, e tohu ana kāore ngā kukū o te L-ornithine i whakamatautauria he paitini ki ngā tipu i rongoatia. I tua atu, i whakarei ake te L-ornithine i te whakaaturanga o ngā antioxidants kore-enzymatic (ngā phenolics me ngā flavonoids wairewa katoa) me ngā antioxidants enzymatic (catalase (CAT), peroxidase (POX), me te polyphenol oxidase (PPO)), ā, i whakanui ake i te whakaaturanga o ngā ira e toru e pā ana ki te antioxidant (PvCAT1, PvSOD, me PvGR). I tua atu, i whakaatuhia e te tātaritanga in silico te aroaro o te pūmua oxaloacetate acetylhydrolase (SsOAH) i roto i te ira S. sclerotiorum, he tino rite ki ngā pūmua oxaloacetate acetylhydrolase (SsOAH) o Aspergillus fijiensis (AfOAH) me Penicillium sp. (PlOAH) e pā ana ki te tātaritanga mahi, ngā rohe tiakina, me te āhua o te tinana. He mea whakamīharo, ko te tāpiri o te L-ornithine ki te wai hupa dextrose rīwai (PDB) i tino whakaiti i te whakaaturanga o te ira SsOAH i roto i te mycelia S. sclerotiorum. Waihoki, ko te whakamahinga o waho o te L-ornithine i tino whakaiti i te whakaaturanga o te ira SsOAH i roto i ngā mycelia harore i kohia mai i ngā tipu i rongoatia. Hei whakamutunga, ko te whakamahinga o te L-ornithine i tino whakaiti i te whakaputanga waikawa oxalic i roto i te reo PDB me ngā rau kua pangia. Hei whakamutunga, he mahi nui tā te L-ornithine ki te pupuri i te mana redox me te whakarei ake i te urupare parepare o ngā tipu kua pangia. Tērā pea ka āwhina ngā hua o tēnei rangahau ki te whakawhanake i ngā tikanga auaha, pai ki te taiao hei whakahaere i te harore mā me te whakaiti i tōna pānga ki te whakaputa pīni me ētahi atu hua.
Ko te harore mā, i puta mai i te harore necrotrophic Sclerotinia sclerotiorum (Lib.) de Bary, he mate kino, he mate whakaiti hua e tūkino ana i te whakaputanga pīni (Phaseolus vulgaris L.) o te ao (Bolton et al., 2006). Ko te Sclerotinia sclerotiorum tētahi o ngā mate harore tipu oneone tino uaua ki te whakahaere, me te whānuitanga o ngā momo tipu neke atu i te 600, me te kaha ki te whakangaro tere i ngā kiko o te tipu i roto i te huarahi kore-motuhake (Liang and Rollins, 2018). I raro i ngā āhuatanga kino, ka pāngia e tētahi wāhanga nui o tōna huringa ora, ka noho moe mō ngā wā roa hei hanganga pango, mārō, rite ki te kākano e kiia nei ko te 'sclerotia' i roto i te oneone, hei tipu mā, puhoi rānei i roto i te mycelium, i te pūtake rānei o ngā tipu pangia (Schwartz et al., 2005). Ka taea e S. sclerotiorum te hanga sclerotia, e āhei ai ia ki te ora i roto i ngā mara kua pangia mō te wā roa, ā, ka mau tonu i te wā e mate ana (Schwartz et al., 2005). He nui ngā matūkai o te sclerotia, ka taea te noho roa ki roto i te oneone, ā, koia te pūtake matua mō ngā mate urutā o muri mai (Schwartz et al., 2005). I raro i ngā āhuatanga pai, ka pihi ake te sclerotia, ka whakaputa i ngā pua rererangi ka taea te pangia ngā wāhanga katoa o runga ake o te whenua o te tipu, tae atu ki ngā putiputi, ngā kakau, ngā pū rānei (Schwartz et al., 2005).
Ka whakamahia e Sclerotinia sclerotiorum tētahi rautaki maha-taumata hei pangia ngā tipu manaaki, tae atu ki tētahi raupapa o ngā kaupapa whakarite mai i te whakatipu sclerotial ki te whanaketanga o ngā tohu. I te tīmatanga, ka puta mai i a S. sclerotiorum ngā pua iri (e kiia nei ko ngā ascospores) mai i ngā hanganga rite ki te harore e kiia nei ko te apothecia, ka rere ki te rangi ka whanake hei sclerotia kore-nekeneke i runga i ngā toenga tipu kua pangia (Bolton et al., 2006). Kātahi ka tukuna e te harore te waikawa oxalic, he mea kaha ki te patu i ngā tipu, hei whakahaere i te pH o te pakitara pūtau tipu, hei whakatairanga i te pirau whākōkī me te whakaekenga kiko (Hegedus and Rimmer, 2005), me te pehi i te pahūtanga waikura o te tipu manaaki. Ka ngoikore te pakitara pūtau tipu i tēnei tukanga waikawa, ka whakarato i tētahi taiao pai mō te mahi noa me te whai hua o ngā whākōkī whakangaro pakitara pūtau harore (CWDEs), ka taea ai e te pathogen te wikitoria i te arai tinana me te uru ki roto i ngā kiko manaaki (Marciano et al., 1983). Kia uru atu ki roto, ka tukuna e S. sclerotiorum he maha o ngā CWDE, pērā i te polygalacturonase me te cellulase, e āwhina ana i tōna horapa ki roto i ngā kiko kua pangia, ā, ka puta te pirau o ngā kiko. Ko te haere whakamua o ngā whara me ngā whāriki hyphal ka arahi ki ngā tohu motuhake o te harore mā (Hegedus and Rimmer, 2005). I taua wā, ka kite ngā tipu manaaki i ngā tauira ngota ngota e pā ana ki te pathogen (PAMP) mā roto i ngā kaiwhakawhiwhi tohu tauira (PRR), ka whakaoho i te raupapa o ngā kaupapa tohu e whakahohe ana i ngā urupare parepare.
Ahakoa ngā tekau tau o ngā mahi whakahaere mate, kei te noho tonu te korenga o te ira-pūmua ātete rawaka i roto i te pīni, pērā i ētahi atu hua arumoni, nā te ātete, te oranga, me te urutau o te pathogen. Nō reira, he tino uaua te whakahaere mate, ā, me whai rautaki whakauru, maha-āhuatanga e uru ana ki te whakakotahitanga o ngā mahi ahurea, te whakahaere koiora, me ngā patu harore matū (O'Sullivan et al., 2021). Ko te whakahaere matū o te harore mā te mea tino whai hua nā te mea, ina whakamahia tika, i te wā tika hoki, ka taea e ngā patu harore te whakahaere i te horapa o te mate, te whakaiti i te kaha o te mate, me te whakaiti i ngā ngaronga hua. Heoi, ko te whakamahinga nui me te whakawhirinaki nui ki ngā patu harore ka arahi ki te putanga mai o ngā momo ātete o S. sclerotiorum, ā, ka pā kino ki ngā rauropi kāore i te whainga, te hauora o te oneone, me te kounga o te wai (Le Cointe et al., 2016; Ceresini et al., 2024). Nō reira, ko te kimi i ngā huarahi kē atu e pai ana ki te taiao kua noho hei kaupapa matua.
Ko ngā polyamines (PA), pērā i te putrescine, te spermidine, te spermine, me te cadaverine, ka taea te whakamahi hei huarahi kē atu ki ngā mate tipu e ahu mai ana i te oneone, ā, ka whakaitihia katoatia, ka whakaitihia rānei te whakamahinga o ngā patu harore matū kino (Nehela et al., 2024; Yi et al., 2024). I roto i ngā tipu teitei ake, ka whai wāhi ngā PA ki ngā tukanga ā-tinana maha, tae atu ki te wehenga pūtau, te rerekētanga, me te urupare ki ngā ahotea kore-ora me te koiora (Killiny and Nehela, 2020). Ka taea e rātou te mahi hei antioxidants, te āwhina ki te kohikohi i ngā momo hāora tauhohe (ROS), te pupuri i te homeostasis redox (Nehela and Killiny, 2023), te whakaoho i ngā ira e pā ana ki te parepare (Romero et al., 2018), te whakahaere i ngā ara pākia (Nehela and Killiny, 2023), te whakarerekē i ngā phytohormones taketake (Nehela and Killiny, 2019), te whakatū i te ātete whiwhi pūnaha (SAR), me te whakahaere i ngā taunekeneke tipu-patogen (Nehela and Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). He mea tika kia mōhiotia ko ngā tikanga me ngā mahi motuhake a ngā PA i roto i te parepare tipu he rerekē i runga i ngā momo tipu, ngā pathogens, me ngā āhuatanga taiao. Ko te PA tino nui i roto i ngā tipu ka hangaia mai i te polyamine L-ornithine nui (Killiny and Nehela, 2020).
He maha ngā mahi a te L-ornithine i roto i te tipu me te whanaketanga o ngā tipu. Hei tauira, kua whakaatuhia e ngā rangahau o mua i roto i te raihi (Oryza sativa), tera pea ka hono te ornithine ki te hangarua hauota (Liu et al., 2018), te hua raihi, te kounga me te kakara (Lu et al., 2020), me te urupare ki te ahotea wai (Yang et al., 2000). I tua atu, ko te whakamahinga o te L-ornithine i waho i tino whakarei ake i te manawanui ki te tauraki i roto i te pīti huka (Beta vulgaris) (Hussein et al., 2019) me te whakaiti i te ahotea tote i roto i te riki (Allium Cepa) (Çavuşoǧlu me Çavuşoǧlu, 2021) me ngā tipu kāhew (Anacardium occidentale) (da Rocha et al., 2012). Ko te mahi pea a te L-ornithine i roto i te parenga ahotea abiotic tera pea nā tōna whai wāhi ki te kohikohi proline i roto i ngā tipu kua rongoatia. Hei tauira, kua pūrongohia i mua ko ngā ira e pā ana ki te pākia o te proline, pērā i te ornithine delta aminotransferase (delta-OAT) me ngā ira proline dehydrogenase (ProDH1 me ProDH2), e whai wāhi ana ki te tiaki i a Nicotiana benthamiana me Arabidopsis thaliana mai i ngā momo Pseudomonas syringae ehara i te manaaki (Senthil-Kumar me Mysore, 2012). I tētahi atu taha, e hiahiatia ana te fungal ornithine decarboxylase (ODC) mō te tipu o ngā pathogens (Singh et al., 2020). Ko te arotahi ki te ODC o Fusarium oxysporum f. sp. lycopersici mā te host-induced gene silencing (HIGS) i tino whakanui ake i te ātete o ngā tipu tōmato ki te Fusarium wilting (Singh et al., 2020). Heoi, kāore anō kia tino rangahaua te tūranga pea o te tono ornithine o waho ki ngā ahotea koiora pērā i ngā phytopathogens. Ko te mea nui ake, ko ngā pānga o te ornithine ki te ātete ki ngā mate me ngā āhuatanga matū koiora me te ā-tinana e pā ana, kāore anō kia tūhuratia.
He mea nui te mārama ki te uaua o te pangia e te S. sclerotiorum i ngā pīni hei whakawhanake i ngā rautaki whakahaere whai hua. I roto i tēnei rangahau, i whai mātou ki te tautuhi i te tūranga pea o te diamine L-ornithine hei mea matua ki te whakarei ake i ngā tikanga parepare me te ātete o ngā tipu pīni ki te pangia e te Sclerotinia sclerotiorum. E whakapae ana mātou, i tua atu i te whakarei ake i ngā urupare parepare o ngā tipu kua pangia, he tūranga nui anō hoki tā te L-ornithine ki te pupuri i te mana redox. E whakaaro ana mātou ko ngā pānga pea o te L-ornithine e pā ana ki te whakahaere i ngā tikanga parepare antioxidant enzymatic me te kore-enzymatic me te pokanoa ki ngā āhuatanga pathogenicity/virulence harore me ngā pūmua e pā ana. Mā tēnei mahi takirua o te L-ornithine ka waiho hei kaitono whai hua mō tētahi rautaki pumau hei whakaiti i te pānga o te harore mā me te whakarei ake i te ātete o ngā hua pīni noa ki tēnei pathogen harore kaha. Mā ngā hua o tēnei rangahau ka āwhina pea ki te whakawhanake i ngā tikanga auaha, hoa taiao hei whakahaere i te harore mā me te whakaiti i tōna pānga ki te whakaputa pīni.
I roto i tēnei rangahau, i whakamahia he momo pīni noa, he momo arumoni ngoikore, ko Giza 3 (Phaseolus vulgaris L. cv. Giza 3), hei rauemi whakamātautau. Nā te Tari Rangahau Legume, te Whare Rangahau Hua Whenua (FCRI), te Pokapū Rangahau Ahuwhenua (ARC), Īhipa i tuku mai ngā purapura hauora. E rima ngā purapura i whakatōkia ki roto i ngā kohua kirihou (te whānui o roto 35 cm, te hohonu 50 cm) i whakakīia ki te oneone i pangia e te S. sclerotiorum i raro i ngā āhuatanga kati kōtuhi (25 ± 2 °C, te haumākū whanaunga 75 ± 1%, 8 h mārama/16 h pōuri). I te 7–10 rā i muri i te whakatō (DPS), i whakangāwarihia ngā tipu kia rua noa ngā tipu e tipu ōrite ana, e toru ngā rau kua whānui katoa i roto i ia kohua. I whakamakukuhia ngā tipu katoa i roto i ngā kohua kotahi i ia rua wiki, ā, i whakawairakauhia ia marama i te tere kua tūtohutia mō te momo kua hoatu.
Hei whakarite i te kukū 500 mg/L o te L-ornithinediamine (e mōhiotia ana ko te waikawa (+)-(S)-2,5-diaminopentanoic; Sigma-Aldrich, Darmstadt, Tiamana), i whakarewaina tuatahitia te 50 mg ki roto i te 100 mL o te wai whakapūkara. Kātahi ka whakapūkaratia te otinga taketake, ā, ka whakamahia i roto i ngā whakamātautau i muri mai. I roto i te poto, e ono ngā raupapa kukū L-ornithine (12.5, 25, 50, 75, 100, me te 125 mg/L) i whakamatautauria i roto i te vitro. Hei tāpiri, i whakamahia te wai whakapūkara hei mana whakahaere kino (Mock) ā, i whakamahia te paura maku 50% o te patu harore arumoni "Rizolex-T" (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Ihipa) hei mana whakahaere pai. I whakamātauria te patu harore arumoni "Rizolex-T" i roto i te vitro i ngā kukū e rima (2, 4, 6, 8 me te 10 mg/L).
I kohia ngā tauira o ngā kakau pīni me ngā pūtu e whakaatu ana i ngā tohu noa o te harore mā (te tere o te whakaekenga: 10–30%) mai i ngā pāmu arumoni. Ahakoa ko te nuinga o ngā tipu pangia i tautuhia e ngā momo/momo (momo arumoni ngoikore Giza 3), ko ētahi atu, inā koa ko ērā i riro mai i ngā mākete o te rohe, he momo kāore i te mōhiotia. Ko ngā rauemi pangia i kohia i tuatahitia i te mata i horoia ki te otinga konutai hypochlorite 0.5% mō te 3 meneti, kātahi ka horoia kia maha ngā wā ki te wai whakapūkara, ā, ka murua kia maroke ki te pepa tātari hei tango i te wai nui. Kātahi ka tapahia ngā whekau pangia kia iti mai i te kiko waenga (i waenganui i ngā kiko hauora me ngā kiko pangia), ka whakatōkia ki runga i te agar dextrose rīwai (PDA) ā, ka whakatōkia ki te 25 ± 2 °C me te huringa mārama 12 hāora/pōuri 12 hāora mō te 5 rā hei whakaoho i te hanganga sclerotia. I whakamahia hoki te tikanga pito mycelial hei pure i ngā wehenga harore mai i ngā ahurea whakauru, poke rānei. I tautuhia tuatahitia te wehenga harore kua purea i runga i ōna āhuatanga āhua ahurea, kātahi ka whakaūtia ko S. sclerotiorum i runga i ngā āhuatanga moroiti. Hei whakamutunga, i whakamatautauria ngā momo pīni parakore katoa mō te mate pāwera ki te momo pīni noa ngoikore o Giza 3 kia tutuki ai ngā paerewa a Koch.
Hei tāpiritanga, ko te momo S. sclerotiorum tino whakaeke (momo #3) i whakaūtia anō i runga i te raupapatanga ā-roto transcribed spacer (ITS) e ai ki tā White et al., 1990; Baturo-Ciesniewska et al., 2017 i whakaahua. I roto i te poto, i whakatipuria ngā momo ki roto i te hupa rīwai dextrose (PDB) ā, i whaowhia ki te 25 ± 2 °C mō ngā rā 5–7. Kātahi ka kohia te mycelium harore, ka tātarihia mā te kakahu tīhi, ka horoia kia rua ngā wā ki te wai parakore, ā, ka whakamarokehia ki te pepa tātari parakore. I wehea te DNA ira mā te whakamahi i te Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Kātahi ka whakanuia te rohe rDNA ITS mā te whakamahi i te takirua parai motuhake ITS1/ITS4 (TCCGTAGGTGAACCTGCGG TCCTCCGCTTATTGATATGC; rahi e tumanakohia ana: 540 bp) (Baturo-Ciesniewska et al., 2017). I tukuna ngā hua PCR kua purea mō te raupapatanga (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). I raupapahia ngā raupapatanga rDNA ITS i ngā taha e rua mā te whakamahi i te tikanga raupapatanga Sanger. Kātahi ka whakaritea ngā raupapatanga patai kua huihuia ki ngā raraunga hou i GenBank me te National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/) mā te whakamahi i te pūmanawa BLASTn. I whakaritea te raupapatanga patai ki ētahi atu momo/momo S. sclerotiorum e 20 i tangohia mai i ngā raraunga hou i NCBI GenBank (Ripanga Tāpiri S1) mā te whakamahi i a ClustalW i roto i te Mōkihi Tātari Ira Whakawhanaketanga Molecular (MEGA-11; putanga 11) (Kumar et al., 2024). I whakahaerehia te tātari whanaketanga mā te whakamahi i te tikanga tūponotanga nui me te tauira whakakapinga nucleotide whānui ka taea te huri i te wā (Nei and Kumar, 2000). Ka whakaaturia te rākau me te tūponotanga-takirua teitei rawa atu. Ka tīpakohia te rākau tuatahi mō te rapunga heuristic mā te whiriwhiri i te rākau me te tūponotanga-takirua teitei ake i waenga i te rākau hono-tata (NJ) (Kumar et al., 2024) me te rākau parsimony nui rawa atu (MP). I hangaia te rākau NJ mā te whakamahi i tētahi matihiko tawhiti takirua i tatauhia mā te whakamahi i te tauira whānui ka taea te huri i te wā (Nei and Kumar, 2000).
I whakatauhia te kaha patu huakita o te L-ornithine me te patu huakita "Rizolex-T" i roto i te vitro mā te tikanga horapa agar. Tikanga: Tangohia te nui e tika ana o te otinga rakau o te L-ornithine (500 mg/L) ka whakaranua kia pai ki te 10 ml o te matūkai PDA hei whakarite i ngā otinga me ngā kukū whakamutunga o te 12.5, 25, 50, 75, 100 me te 125 mg/L, ia. E rima ngā kukū o te patu harore "Rizolex-T" (2, 4, 6, 8 me te 10 mg/L) me te wai whakamāori i whakamahia hei mana whakahaere. I muri i te whakapakeketanga o te rauemi, ka whakawhitia he mono mycelial hou o te ahurea Sclerotinia sclerotiorum, 4 mm te whānui, ki waenganui o te rihi Petri, ā, ka whakatipuria ki te 25±2°C tae noa ki te kapi katoa o te mycelium i te rihi Petri whakahaere, ā, muri iho ka tuhia te tipu harore. Tātaihia te ōrau aukati o te tipu radial o S. sclerotiorum mā te whakamahi i te whārite 1:
E rua ngā wā i whakahokia ai te whakamātautau, me ngā tāruarua koiora e ono mō ia rōpū mana whakahaere/whakamātautau me ngā kohua e rima (e rua ngā tipu mō ia kohua) mō ia tāruarua koiora. E rua ngā wā i tātarihia ai ia tāruarua koiora (e rua ngā tāruarua hangarau) hei whakarite i te tika, te pono me te āheinga ki te tāruarua i ngā hua whakamātautau. Hei tāpiri, i whakamahia te tātari whakatauira probit hei tatau i te kukū aukati haurua-mōrahi (IC50) me te IC99 (Prentice, 1976).
Hei aromatawai i te kaha o te L-ornithine i raro i ngā āhuatanga kati kōtuhi, e rua ngā whakamātautau kohua i whakahaerehia. I roto i te poto, i whakakīia ngā kohua ki te oneone uku-onepu kua whakahoromatatia (3:1) ā, i werohia ki te ahurea hou o te S. sclerotiorum. Tuatahi, ko te wehenga tino whakaeke o te S. sclerotiorum (wehenga #3) i whakahoromatatia mā te tapahi i te sclerotium kia rua, ka whakatakotoria ki raro ki runga i te PDA, ā, ka whakatōkia ki te 25°C i roto i te pōuri tonu (24 hāora) mō ngā rā e 4 hei whakaoho i te tipu mycelial. Kātahi ka tangohia ngā mono agar e whā, he 5 mm te whānui, mai i te taha mua, ā, ka werohia ki te 100 karamu o te ranunga witi me te raihi parakore (1:1, v/v) ā, ka whakatōkia ngā pounamu katoa ki te 25 ± 2 °C i raro i te huringa mārama 12 hāora/pōuri 12 hāora mō ngā rā e 5 hei whakaoho i te hanganga sclerotia. I whakaranua pai ngā ihirangi o ngā pounamu katoa kia rite ai te ōritetanga i mua i te tāpiri oneone. Kātahi ka tāpirihia he 100 karamu o te ranunga parai e noho ana ki ia kohua hei whakarite kia mau tonu te kukū o ngā mate pāwera. I whakamakukuhia ngā kohua i werohia kia tere ai te tipu o te harore, ā, i whakanohoia ki roto i ngā āhuatanga kati kōtuhi mō ngā rā e whitu.
E rima ngā kākano o te momo Giza 3 i whakatōkia ki roto i ia kohua. Mō ngā kohua i rongoatia ki te L-ornithine me te harore Rizolex-T, i rumakina tuatahitia ngā kākano kua whakahoromatatia mō ngā hāora e rua ki roto i te wai o ngā matū e rua me te kukū IC99 whakamutunga o te 250 mg/L me te 50 mg/L, ia, kātahi ka whakamarokehia ki te hau mō te kotahi hāora i mua i te whakatō. I tētahi atu taha, i rumakina ngā kākano ki te wai whakahoromatatia hei mana whakahaere kino. I muri i ngā rā 10, i mua i te whakamakuku tuatahi, i whakangawaritia ngā tipu, kia rua noa ngā tipu kua ma i roto i ia kohua. Hei tāpiri, hei whakarite kia pangia e te S. sclerotiorum, i tapahia ngā kakau pīni i te wā whanaketanga kotahi (10 rā) ki ngā wāhi rerekē e rua mā te whakamahi i te maripi kua whakahoromatatia, ā, tata ki te 0.5 karamu o te ranunga paraka e noho ana i roto i ia patunga, ā, whai muri ko te haumākū teitei hei whakaoho i te mate me te whanaketanga o te mate i roto i ngā tipu katoa i werohia. I rite anō te whara o ngā tipu mana whakahaere, ā, i whakatakotoria he ranunga paraka kore-whakatō (0.5 karamu) ki roto i te patunga, ā, i puritia kia makuku rawa hei whakatauira i te taiao mō te whanaketanga o te mate, me te whakarite kia ōrite te āhua i waenga i ngā rōpū maimoatanga.
Tikanga maimoatanga: I whakamakukuhia ngā tipu pīni ki te 500 ml o te wairewa L-ornithine (250 mg/l) te rongoā patu harore rānei Rizolex-T (50 mg/l) mā te whakamakuku i te oneone, kātahi ka whakahokia te maimoatanga kia toru ngā wā i ia wā, kia 10 ngā rā. Ko ngā mana whakahaere i rongoatia ki te placebo i whakamakukuhia ki te 500 ml o te wai whakapūkara. I mahia ngā maimoatanga katoa i raro i ngā āhuatanga kati kōtuhi (25 ± 2°C, 75 ± 1% te haumākū whanaunga, me te wā mārama o te 8 hāora/16 hāora pōuri). I whakamakukuhia ngā kohua katoa i ia rua wiki, ā, i rongoatia ia marama ki te tongi NPK taurite (20-20-20, me te 3.6% whanariki me ngā huānga moroiti TE; Zain Seeds, Ihipa) i te kukū o te 3–4 g/l mā te rehu rau i runga i ngā taunakitanga mō te momo motuhake me ngā tohutohu a te kaihanga. Ki te kore e kīia tētahi atu mea, ko ngā rau kua tino whānui (rau tuarua me te tuatoru mai i runga) i kohia mai i ia tārua koiora i te 72 hāora i muri i te maimoatanga (hpt), i whakaranua, i whakaemihia, ā, i rongoatia ki te -80 °C mō ētahi atu tātaritanga tae atu ki, engari kāore i te herea ki, te taunga histochemical in situ o ngā tohu ahotea waikura, te waikura lipid, ngā antioxidants enzymatic me te kore-enzymatic me te whakaaturanga ira.
I arotakehia te kaha o te mate harore mā i ia wiki i muri i te werohanga (dpi) mā te whakamahi i te tauine 1–9 (Ripanga Tāpiri S2) i runga i te tauine Petzoldt me Dickson (1996) i whakarerekētia e Teran et al. (2006). I roto i te poto, i tirohia ngā kakau me ngā manga o ngā tipu pīni mai i te wāhi i werohangahia ai hei whai i te haere whakamua o ngā whara i ngā taha me ngā pona. Kātahi ka inehia te tawhiti o te whara mai i te wāhi i werohangahia ai ki te wāhi tawhiti rawa atu i te taha o te kakau, te manga rānei, ā, ka tohua he kaute 1–9 i runga i te wāhi o te whara, i reira (1) i tohu kāore he mate e kitea ana i te taha o te wāhi i werohangahia ai, ā, (2–9) i tohu i te piki haere haere o te rahi o te whara me te haere whakamua i ngā taha/ngā taha (Ripanga Tāpiri S2). Kātahi ka hurihia te kaha o te mate harore mā ki te ōrau mā te whakamahi i te tātai 2:
Hei tāpiritanga, i tatauhia te horahanga i raro i te pihi haere whakamua o te mate (AUDPC) mā te whakamahi i te tātai (Shaner and Finney, 1977), i urutauhia tata nei mō te pirau mā o te pīni noa (Chauhan et al., 2020) mā te whakamahi i te whārite 3:
Ko Yi = te kaha o te mate i te wā ti, Yi+1 = te kaha o te mate i te wā e whai ake nei ti+1, ti = te wā o te ine tuatahi (i roto i ngā rā), ti+1 = te wā o te ine e whai ake nei (i roto i ngā rā), n = te tapeke o ngā wā, ngā wāhi tirotiro rānei. I tuhia ngā tawhā tipu o te tipu pīni tae atu ki te teitei o te tipu (cm), te maha o ngā manga mō ia tipu, me te maha o ngā rau mō ia tipu ia wiki mō ngā rā 21 i roto i ngā tauira koiora katoa.
I roto i ia tāruarua koiora, i kohia ngā tauira rau (tuarua me te tuatoru o ngā rau kua tino whanake mai i runga) i te rā 45 i muri i te maimoatanga (15 ngā rā i muri i te maimoatanga whakamutunga). E rima ngā kohua i roto i ia tāruarua koiora (e rua ngā tipu mō ia kohua). Tata ki te 500 mg o te kiko i kuruhia i whakamahia mō te tango i ngā tae whakaahua (chlorophyll a, chlorophyll b me ngā carotenoids) mā te whakamahi i te 80% acetone i te 4 °C i te pōuri. I muri i te 24 hāora, i whakatōngia ngā tauira, ā, i kohia te wai i roto i te ipu hei whakatau i ngā ihirangi o te chlorophyll a, chlorophyll b me te carotenoid mā te whakamahi i te ine UV-160A spectrophotometer (Shimadzu Corporation, Japan) e ai ki te tikanga o (Lichtenthaler, 1987) mā te ine i te mimiti i ngā roangaru rerekē e toru (A470, A646 me te A663 nm). Hei whakamutunga, i tatauhia te ihirangi o ngā tae whakaahua mā te whakamahi i ngā tātai e whai ake nei 4–6 i whakaahuahia e Lichtenthaler (1987).
I te 72 hāora i muri i te maimoatanga (hpt), i kohia ngā rau (te tuarua me te tuatoru o ngā rau kua tino whanake mai i runga) mai i ia tauira koiora hei kimi i te wāhi histochemical o te hauwai peroxide (H2O2) me te anion superoxide (O2•−). E rima ngā kohua i roto i ia tauira koiora (e rua ngā tipu mō ia kohua). I tātarihia ia tauira koiora i roto i ngā tāruarua (e rua ngā tāruarua hangarau) hei whakarite i te tika, te pono me te āheinga ki te tāruarua i te tikanga. I whakatauhia te H2O2 me te O2•− mā te whakamahi i te 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Tiamana) te nitroblue tetrazolium rānei (NBT; Sigma-Aldrich, Darmstadt, Tiamana), e whai ana i ngā tikanga i whakaahuahia e Romero-Puertas et al. (2004) me Adam et al. (1989) me ngā whakarerekētanga iti. Mō te kimi i te H2O2 i roto i te wāhi tūturu, i whakatōkia ngā pepa iti ki te 0.1% DAB i roto i te 10 mM Tris buffer (pH 7.8) mā te korehau, kātahi ka whakatōkia i te pāmahana rūma i roto i te mārama mō te 60 meneti. I whakamākūkūhia ngā pepa iti ki roto i te 0.15% (v/v) TCA i roto i te 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) kātahi ka whakaaturia ki te mārama tae noa ki te pōuri. Waihoki, i whakatōkia ngā takirere mā te korehau ki te 10 mM potassium phosphate buffer (pH 7.8) kei roto ko te 0.1 w/v % HBT hei kimi i te O2•− i roto i te wāhi tūturu. I whakatōkia ngā pepa iti ki roto i te mārama i te pāmahana rūma mō te 20 meneti, kātahi ka whakamākūkūhia pērā i runga ake nei, kātahi ka whakamāramahia kia puta rā anō ngā ira kikorangi/papura pouri. I aromatawaihia te kaha o te tae parauri (hei tohu H2O2) te tae kikorangi-puru rānei (hei tohu O2•−) mā te whakamahi i te putanga Whītī o te kete tukatuka whakaahua ImageJ (http://fiji.sc; i tirohia i te 7 o Poutū-te-rangi 2024).
I whakatauhia te malondialdehyde (MDA; hei tohu mō te peroxidation lipid) i runga anō i te tikanga a Du rāua ko Bramlage (1992) me ngā whakarerekētanga iti. I kohia ngā rau mai i ia tauira koiora (tuarua me te tuatoru o ngā rau kua tino whanake mai i runga) 72 hāora i muri i te maimoatanga (hpt). E rima ngā kohua i roto i ia tauira koiora (e rua ngā tipu mō ia kohua). I tātarihia ia tauira koiora i roto i ngā tāruarua (e rua ngā tauira hangarau) hei whakarite i te tika, te pono me te taea o te tāruarua o te tikanga. I roto i te poto, i whakamahia te 0.5 karamu o te kiko rau whenua mō te tango MDA me te 20% waikawa trichloroacetic (TCA; MilliporeSigma, Burlington, MA, USA) kei roto ko te 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). Kātahi ka whakatauhia te nui o te MDA i roto i te waikura mā te ine i te mimiti i te 532 me te 600 nm mā te whakamahi i te inenga UV-160A (Shimadzu Corporation, Hapani) kātahi ka whakaatuhia hei nmol g−1 FW.
Hei aromatawai i ngā ārai mate kore-whākōkī me ngā ārai mate whākōkī, i kohia ngā rau (te tuarua me te tuatoru o ngā rau kua tino whanake mai i runga) mai i ia tauira koiora i te 72 hāora i muri i te maimoatanga (hpt). E rima ngā kohua i roto i ia tauira koiora (e rua ngā tipu mō ia kohua). I tātarihia ia tauira koiora i roto i ngā tāruarua (e rua ngā tauira hangarau). E rua ngā rau i hurihia ki te hauota wai, ā, i whakamahia tika hei whakatau i ngā ārai mate whākōkī me ngā ārai mate kore-whākōkī, te tapeke o ngā waikawa amino, te ihirangi proline, te whakaaturanga ira, me te ine o te oxalate.
I whakatauhia ngā phenolics wairewa katoa mā te whakamahi i te matū Folin-Ciocalteu (Sigma-Aldrich, St. Louis, MO, USA) me ngā whakarerekētanga iti o te tikanga i whakaahuahia e Kahkonen et al. (1999). I roto i te poto, tata ki te 0.1 karamu o te kiko rau kua whakaranua i tangohia me te 20 ml 80% methanol i roto i te pōuri mō te 24 hāora, ā, i kohia te wai i raro i te pehanga i muri i te centrifugation. I konatunatua te 0.1 ml o te tauira tango ki te 0.5 ml o te matū Folin-Ciocalteu (10%), i rūrūtia mō te 30 hēkona, ā, i waiho ki te pōuri mō te 5 meneti. Kātahi ka tāpirihia te 0.5 ml o te otinga konutai kāponāte 20% (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) ki ia ngongo, i konatunatua kia pai, ā, i whaowhia ki te pāmahana rūma i roto i te pōuri mō te 1 hāora. I muri i te whakatōnga, i inehia te mimiti o te ranunga tauhohenga i te 765 nm mā te whakamahi i te inenga UV-160A (Shimadzu Corporation, Hapani). I whakatauhia te kukū o ngā phenol wairewa katoa i roto i ngā tangohanga tauira mā te whakamahi i te kōpiko whakatikatika waikawa gallic (Fisher Scientific, Hampton, NH, USA) ā, i whakaaturia hei mirikaramu o te waikawa gallic ōrite mō ia karamu o te taumaha hou (mg GAE g-1 taumaha hou).
I whakatauhia te nui o te flavonoid wairewa katoa i runga i te tikanga a Djeridane et al. (2006) me ngā whakarerekētanga iti. I roto i te poto, i konatunatua te 0.3 ml o te tangohanga methanol i runga ake nei ki te 0.3 ml o te otinga konumohe pūhaumāota 5% (AlCl3; Fisher Scientific, Hampton, NH, USA), i whakaohokia kaha, kātahi ka whaowhia i te pāmahana rūma mō te 5 meneti, ā, i muri mai ka tāpirihia te 0.3 ml o te otinga pāhare pākawa 10% (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), i whakaranua kia tino pai, ā, i whaowhia i te pāmahana rūma mō te 30 meneti i roto i te pōuri. I muri i te whaowhia, i inehia te mimiti o te ranunga tauhohenga i te 430 nm mā te whakamahi i te inenga UV-160A (Shimadzu Corporation, Japan). I whakatauhia te kukū o ngā flavonoid wairewa katoa i roto i ngā tangohanga tauira mā te whakamahi i te pihi whakatikatika rutin (TCI America, Portland, OR, USA) kātahi ka whakaatuhia hei mirikaramu o te rutin ōrite mō ia karamu o te taumaha hou (mg RE g-1 taumaha hou).
I whakatauhia te katoa o ngā waikawa amino kore utu o ngā rau pīni mā te whakamahi i tētahi matū ninhydrin kua whakarerekētia (Thermo Scientific Chemicals, Waltham, MA, USA) i runga i te tikanga i whakatakotoria e Yokoyama rāua ko Hiramatsu (2003) me te whakarerekētanga e Sun et al. (2006). I roto i te poto, i tangohia te 0.1 karamu o te kiko whenua ki te pH 5.4 buffer, ā, i tauhohehia te 200 μL o te supernatant ki te 200 μL o te ninhydrin (2%) me te 200 μL o te pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), i whaowhia ki roto i te kaukau wai kohua mō te 30 meneti, kātahi ka whakamataohia, ka inehia i te 580 nm mā te whakamahi i tētahi spectrophotometer UV-160A (Shimadzu Corporation, Japan). I tētahi atu taha, i whakatauhia te proline mā te tikanga Bates (Bates et al., 1973). I tangohia te proline ki te 3% waikawa sulfosalicylic (Thermo Scientific Chemicals, Waltham, MA, USA) ā, i muri i te centrifugation, i konatunatua te 0.5 ml o te supernatant ki te 1 ml waikawa acetic glacial (Fisher Scientific, Hampton, NH, USA) me te ninhydrin reagent, i whaowhia ki te 90°C mō te 45 meneti, i whakamataohia, ā, i inehia ki te 520 nm mā te whakamahi i te spectrophotometer kotahi i runga ake nei. I whakatauhia te tapeke o ngā waikawa amino kore utu me te proline i roto i ngā tangohanga rau mā te whakamahi i ngā ānau whakatikatika glycine me te proline (Sigma-Aldrich, St Louis, MO, USA), ia, ā, i whakaaturia hei mg/g taumaha hou.
Hei whakatau i te kaha whākōkī o ngā whākōkī ārai-mate, tata ki te 500 mg o te kiko kua whakaranua i tangohia me te 3 ml o te 50 mM Tris buffer (pH 7.8) kei roto ko te 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) me te 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), i whakatōngia ki te 10,000 × g mō te 20 meneti i raro i te pouaka whakamātao (4 °C), ā, i kohia te waikeri (tangohanga whākōkī mata) (El-Nagar et al., 2023; Osman et al., 2023). Kātahi ka tauhohehia te Catalase (CAT) ki te 2 ml o te 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) me te 100 μl o te 269 mM H2O2 otinga hei whakatau i tōna ngohe whākōkī e ai ki te tikanga a Aebi (1984) me ngā whakarerekētanga iti (El-Nagar et al., 2023; Osman et al., 2023). I whakatauhia te ngohe whākōkī o te Guaiacol-dependent peroxidase (POX) mā te whakamahi i te tikanga a Harrach et al. (2009). (2008) me ngā whakarerekētanga iti (El-Nagar et al., 2023; Osman et al., 2023) ā, i whakatauhia te ngohe whākōkī o te polyphenol oxidase (PPO) i muri i te tauhohenga ki te 2.2 ml o te 100 mM konutai phosphate buffer (pH 6.0), 100 μl o te guaiacol (TCI chemicals, Portland, OR, USA) me te 100 μl o te 12 mM H2O2. I whakarerekētia paku te tikanga mai i (El-Nagar et al., 2023; Osman et al., 2023). I whakahaerehia te whakamātautau i muri i te tauhohenga ki te 3 ml o te otinga catechol (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) i takaia houtia ki roto i te 0.1 M phosphate buffer (pH 6.0). I inehia te ngohe CAT mā te aroturuki i te pirau o te H2O2 i te 240 nm (A240), i inehia te ngohe POX mā te aroturuki i te pikinga o te mimiti i te 436 nm (A436), ā, i inehia te ngohe PPO mā te tuhi i ngā pikinga mimiti i te 495 nm (A495) ia 30 hēkona mō te 3 meneti mā te whakamahi i te inenga UV-160A (Shimadzu, Hapani).
I whakamahia te RT-PCR wā-tūturu hei kimi i ngā taumata tuhinga o ngā ira e toru e pā ana ki te antioxidant, tae atu ki te peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), te superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), me te glutathione reductase (PvGR; GenBank Accession No. KY195009.1), i roto i ngā rau pīni (te tuarua me te tuatoru o ngā rau kua tino whanake mai i runga) 72 hāora i muri i te maimoatanga whakamutunga. I roto i te poto, i wehea te RNA mā te whakamahi i te Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, Haina) e ai ki te kawa a te kaihanga. Kātahi ka hangaia te cDNA mā te whakamahi i te TOP script™ cDNA Synthesis Kit e ai ki ngā tohutohu a te kaihanga. Kei roto i te Ripanga Tāpiri S3 ngā raupapatanga tuatahi o ngā ira e toru o runga ake nei. I whakamahia te PvActin-3 (tau urunga GenBank: XM_068616709.1) hei ira tiaki whare, ā, i tatauhia te whakaaturanga ira whanaunga mā te whakamahi i te tikanga 2-ΔΔCT (Livak and Schmittgen, 2001). I whakaaturia te pumau o te Actin i raro i te ahotea koiora (te taunekeneke kore hototahi i waenga i ngā pīni noa me te harore anthracnose Colletotrichum lindemuthianum) me te ahotea abiotic (te tauraki, te tote, te pāmahana iti) (Borges et al., 2012).
I te tuatahi i whakahaerehia e mātou he tātaritanga in silico whānui-ira o ngā pūmua oxaloacetate acetylhydrolase (OAH) i roto i te S. sclerotiorum mā te whakamahi i te taputapu BLAST pūmua-pūmua (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). I roto i te poto, i whakamahia e mātou te OAH mai i Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; tau urunga GenBank XP_040799428.1; 342 waikawa amino) me te Penicillium lagena (PlOAH; taxide: 94218; tau urunga GenBank XP_056833920.1; 316 waikawa amino) hei raupapatanga patai hei mahere i te pūmua ōrite i roto i te S. sclerotiorum (taxide: 5180). I whakahaerehia te BLASTp ki ngā raraunga ira S. sclerotiorum hou rawa atu i roto i te GenBank i te paetukutuku National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Hei tāpiritanga, ko te ira OAH i matapaetia mai i S. sclerotiorum (SsOAH) me te tātari whanaketanga me te rākau whakawhanaungatanga o AfOAH mai i A. fijiensis CBS 313.89 me PlOAH mai i P. lagena i whakatauria mā te whakamahi i te tikanga tūponotanga mōrahi i roto i te MEGA11 (Tamura et al., 2021) me te tauira e hangai ana ki te matrix JTT (Jones et al., 1992). I whakakotahihia te rākau whakawhanaungatanga me te tātari whakarārangi maha o ngā raupapatanga pūmua o ngā ira OAH katoa i matapaetia (SsOAH) mai i S. sclerotiorum me te raupapatanga patai mā te whakamahi i te Constraint-Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos and Agarwala, 2007). Hei tāpiritanga, ko ngā raupapatanga waikawa amino tino ōrite o SsOAH mai i S. sclerotiorum i whakaritea ki ngā raupapatanga patai (AfOAH me PlOAH) (Larkin et al., 2007) mā te whakamahi i a ClustalW (http://www.genome.jp/tools-bin/clustalw), ā, ko ngā rohe kua tiakina i roto i te whakaritenga i tirohia mā te whakamahi i te taputapu ESPript (putanga 3.0; https://espript.ibcp.fr/ESPript/ESPript/index.php).
I tua atu, ko ngā rohe mahi tohu me ngā wāhi tiakina o S. sclerotiorum SsOAH i whakarōpūtia kia rite ki ngā whānau rerekē mā te whakamahi i te taputapu InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Hei whakamutunga, ko te whakatauira hanganga toru-ahu (3D) o te S. sclerotiorum SsOAH i matapaetia i mahia mā te whakamahi i te Protein Homology/Analogy Recognition Engine (Phyre2 server version 2.0; http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) (Kelley et al., 2015) ā, i whakamanahia mā te whakamahi i te tūmau SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). I whakaatuhia ngā hanganga toru-ahu i matapaetia (te whakatakotoranga PDB) mā te whakamahi i te mōkihi UCSF-Chimera (putanga 1.15; https://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004).
I whakamahia te PCR fluorescence wā-tūturu tauanga hei whakatau i te taumata whakawhiti o te oxaloacetate acetylhydrolase (SsOAH; GenBank accession number: XM_001590428.1) i roto i te mycelia o Sclerotinia sclerotiorum. I roto i te poto, i werohia a S. sclerotiorum ki roto i tētahi pounamu kei roto ko te PDB, ā, i whakanohoia ki roto i tētahi incubator wiri (tauira: I2400, New Brunswick Scientific Co., Edison, NJ, USA) i te 25 ± 2 °C mō te 24 hāora i te 150 rpm me te pōuri tonu (24 hāora) hei whakaoho i te tipu mycelial. I muri iho, i rongoatia ngā pūtau ki te L-ornithine me te harore Rizolex-T i ngā kukū IC50 whakamutunga (tata ki te 40 me te 3.2 mg/L, ia) kātahi ka whakatōkia mō tētahi atu 24 hāora i raro i ngā āhuatanga ōrite. I muri i te whakatōnga, i whakatōngia ngā ahurea i te 2500 rpm mō te 5 meneti, ā, i kohia te waikura (te mycelium harore) hei tātari i te whakaaturanga ira. Waihoki, i kohia te mycelium harore i te 0, 24, 48, 72, 96, me te 120 hāora i muri i te pangia mai i ngā tipu pangia kua hanga he harore mā me te mycelium miro i runga i te mata o ngā kiko pangia. I tangohia te RNA mai i te mycelium harore, kātahi ka hangaia te cDNA e ai ki te whakaahuatanga i runga ake nei. Kei roto i te Ripanga Tāpiri S3 ngā raupapatanga tuatahi mō te SsOAH. I whakamahia te SsActin (tau whakauru GenBank: XM_001589919.1) hei ira tiaki whare, ā, i tatauhia te whakaaturanga ira whanaunga mā te whakamahi i te tikanga 2-ΔΔCT (Livak and Schmittgen, 2001).
I kitea te waikawa oxalic i roto i te hupa dextrose rīwai (PDB) me ngā tauira tipu kei roto te mate harore Sclerotinia sclerotiorum e ai ki te tikanga a Xu rāua ko Zhang (2000) me ngā whakarerekētanga iti. I roto i te poto, i werohia ngā wehenga S. sclerotiorum ki roto i ngā pounamu kei roto te PDB, kātahi ka whakatipuria ki roto i te incubator wiri (tauira I2400, New Brunswick Scientific Co., Edison, NJ, USA) i te 150 rpm i te 25 ± 2 °C mō ngā rā 3–5 i roto i te pōuri tonu (24 h) hei whakaoho i te tipu mycelial. I muri i te whakatipuranga, i tātarihia tuatahitia te ahurea harore mā te pepa tātari Whatman #1, kātahi ka whakatōngia i te 2500 rpm mō te 5 min hei tango i te mycelium toenga. I kohia te supernatant, ā, i rongoatia ki te 4°C hei whakatau i te nui o te oxalate. Mō te whakarite i ngā tauira tipu, tata ki te 0.1 g o ngā kongakonga kiko tipu i tangohia kia toru ngā wā me te wai whakakore (2 ml ia wā). Kātahi ka porotītahatia ngā tauira i te 2500 rpm mō te 5 meneti, kātahi ka tātarihia te wai i roto i te pepa tātari Whatman Nama 1, ā, ka kohia hei tātaritanga anō.
Mō te tātaritanga rahinga o te waikawa oxalic, i whakaritea te ranunga tauhohenga i roto i tētahi ngongo karāhe kua āraia i te raupapa e whai ake nei: 0.2 ml o te tauira (te tātari ahurea PDB rānei, te otinga paerewa waikawa oxalic rānei), 0.11 ml o te kikorangi bromophenol (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), 0.198 ml o te waikawa whanariki 1 M (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) me te 0.176 ml o te 100 mM pāhare pākawa dichromate (K2Cr2O7; TCI chemicals, Portland, OR, USA), kātahi ka whakaranua te otinga ki te 4.8 ml ki te wai kua whakakorea, ka whakaranua kia kaha, ā, ka whakanohoia tonutia ki roto i te kaukau wai 60 °C. I muri i te 10 meneti, ka mutu te tauhohenga mā te tāpiri i te 0.5 ml o te otinga hauwai konutai (NaOH; 0.75 M). I inehia te mimiti (A600) o te ranunga tauhohenga i te 600 nm mā te whakamahi i te inenga UV-160 (Shimadzu Corporation, Hapani). I whakamahia te PDB me te wai whakapūkara hei mana whakahaere mō te ine i ngā tātari ahurea me ngā tauira tipu. Ko ngā kukū waikawa oxalic i roto i ngā tātari ahurea, i whakaaturia hei maikorokaramu o te waikawa oxalic ia miririta o te reo PDB (μg.mL−1), me ngā tangohanga rau, i whakaaturia hei maikorokaramu o te waikawa oxalic ia karamu o te taumaha hou (μg.g−1 FW), i whakatauhia mā te whakamahi i te pihi whakatikatika waikawa oxalic (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
I roto i te rangahau, i hangaia ngā whakamātautau katoa i roto i tētahi hoahoa matapōkere katoa (CRD) me ngā tāruarua koiora e ono mō ia maimoatanga me ngā kohua e rima mō ia tāruarua koiora (e rua ngā tipu mō ia kohua) mēnā kāore i te kīia tētahi atu mea. I tātarihia ngā tāruarua koiora i roto i ngā tāruarua (e rua ngā tāruarua hangarau). I whakamahia ngā tāruarua hangarau hei tirotiro i te āheinga tāruarua o taua whakamātautau engari kāore i whakamahia i roto i te tātaritanga tatauranga hei karo i ngā tāruarua rūpahu. I tātarihia ngā raraunga i runga i te tatauranga mā te whakamahi i te tātaritanga rerekētanga (ANOVA) ā, i muri mai ko te whakamātautau rerekētanga tino nui a Tukey-Kramer (HSD) (p ≤ 0.05). Mō ngā whakamātautau in vitro, i tatauhia ngā uara IC50 me IC99 mā te whakamahi i te tauira probit, ā, i tatauhia ngā wā whakawhirinaki 95%.
E whā ngā momo motuhake i kohia mai i ngā māra pīni soya rerekē i te Kāwanatanga o El Ghabiya, i Ihipa. I runga i te pāpāho PDA, i puta mai i ngā momo motuhake katoa he mycelium mā kirīmi i huri wawe ki te mā miro (Pikitia 1A) kātahi ka parauri, ka parauri rānei i te wāhanga sclerotium. He matotoru, he mangu, he porowhita, he koretake rānei te āhua o ngā sclerotia, 5.2 ki te 7.7 mm te roa, ā, 3.4 ki te 5.3 mm te whānui (Pikitia 1B). Ahakoa i puta mai i ngā momo motuhake e whā he tauira sclerotia taha i te taha o te pāpāho ahurea i muri i ngā rā 10–12 o te whakatipu i te 25 ± 2 °C (Pikitia 1A), he tino rerekē te maha o ngā sclerotia mō ia pereti i waenga i a rātou (P < 0.001), ko te momo motuhake 3 te mea nui rawa atu o ngā sclerotia (32.33 ± 1.53 sclerotia mō ia pereti; Pikitia 1C). Waihoki, he nui ake te waikawa oxalic i whakaputaina e te wehenga #3 i roto i te PDB i ērā atu wehenga (3.33 ± 0.49 μg.mL−1; Pikitia 1D). I whakaatuhia e te wehenga #3 ngā āhuatanga āhua me ngā āhuatanga moroiti noa o te harore phytopathogenic Sclerotinia sclerotiorum. Hei tauira, i runga i te PDA, i tere te tipu o ngā koroni o te wehenga #3, he mā kirīmi (Pikitia 1A), he parauri parauri whakamuri, he parauri māwhero rānei, ā, e 6-7 ngā rā i hiahiatia i te 25 ± 2°C hei kapi katoa i te mata o te pereti 9 cm te whānui. I runga i ngā āhuatanga āhua me ngā āhuatanga moroiti i runga ake nei, i tautuhia te wehenga #3 ko Sclerotinia sclerotiorum.
Pikitia 1. Ngā āhuatanga me te mate pāwera o ngā momo S. sclerotiorum mai i ngā hua pīni noa. (A) Te tipu mycelial o ngā momo S. sclerotiorum e whā i runga i te reo PDA, (B) sclerotia o ngā momo S. sclerotiorum e whā, (C) te maha o ngā sclerotia (mō ia pereti), (D) te hunanga waikawa oxalic i runga i te reo PDB (μg.mL−1), me te (E) te kaha o te mate (%) o ngā momo S. sclerotiorum e whā i runga i te momo pīni arumoni Giza 3 i raro i ngā āhuatanga kati kōtuhi. Ko ngā uara e tohu ana i te toharite ± SD o ngā tāruarua koiora e rima (n = 5). Ko ngā reta rerekē e tohu ana i ngā rerekētanga nui i waenga i ngā maimoatanga (p < 0.05). (F–H) I puta ngā tohu harore mā noa i runga i ngā kakau me ngā silique i runga ake i te whenua, ia, 10 ngā rā i muri i te werohanga ki te momo #3 (dpi). (I) I whakahaerehia te tātari whanaketanga o te rohe ā-roto o te whakawātea whakawhiti (ITS) o te momo S. sclerotiorum #3 mā te whakamahi i te tikanga tūponotanga mōrahi, ā, i whakaritea ki ngā momo/momo tohutoro 20 i riro mai i te pātengi raraunga o te National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). Ko ngā tau kei runga ake i ngā rārangi whakarōpū e tohu ana i te kapi rohe (%), ā, ko ngā tau kei raro iho i ngā rārangi whakarōpū e tohu ana i te roa o te peka.
Hei whakaū i te mate, e whā ngā momo S. sclerotiorum i whiwhihia i whakamahia hei werohanga i te momo pīni arumoni ngoikore o Giza 3 i raro i ngā āhuatanga kati kōtuhi, he rite ki ngā whakatau a Koch (Pikitia 1E). Ahakoa he mate ngā momo harore katoa i whiwhihia, ā, ka taea te pangia te pīni matomato (cv. Giza 3), ka puta ngā tohu harore mā i runga i ngā wāhanga katoa o runga ake i te whenua (Pikitia 1F), inā koa i runga i ngā kakau (Pikitia 1G) me ngā pū (Pikitia 1H) i te 10 rā i muri i te werohanga (dpi), ko te momo 3 te momo tino kaha i roto i ngā whakamātautau motuhake e rua. Ko te momo 3 te tino kaha o te mate (%) i runga i ngā tipu pīni (24.0 ± 4.0, 58.0 ± 2.0, me te 76.7 ± 3.1 i te 7, 14, me te 21 rā i muri i te pangia; Pikitia 1F).
I whakaūhia anō te tautuhi i te momo S. sclerotiorum tino whakaekenga #3 i runga i te raupapatanga ā-roto o te whakawātea whakawhiti (ITS) (Pikitia 1I). Ko te tātaritanga phylogenetic i waenga i te momo #3 me ngā momo/momo tohutoro 20 i whakaatu i te ōritetanga teitei (>99%) i waenga i a rātou. He mea tika kia mōhio ko te momo S. sclerotiorum #3 (533 bp) he ōritetanga teitei ki te momo S. sclerotiorum o Amerika LPM36 i wehea mai i ngā purapura pī maroke (nama urunga GenBank MK896659.1; 540 bp) me te momo S. sclerotiorum Hainamana YKY211 (nama urunga GenBank OR206374.1; 548 bp), e puta ai te pirau kakau waioleti (Matthiola incana), e whakarōpūhia ana ēnei katoa i te tihi o te dendrogram (Pikitia 1I). Kua whakatakotoria te raupapatanga hou ki roto i te pātengi raraunga NCBI, ā, kua tapaina ko "Sclerotinia sclerotiorum – isolate YN-25" (nama urunga GenBank PV202792). Ka kitea ko te isolate 3 te isolate tino whakaeke; nō reira, i whiriwhiria tēnei isolate hei rangahau i roto i ngā whakamātautau katoa i muri mai.
I tirotirohia te kaha patu huakita o te diamine L-ornithine (Sigma-Aldrich, Darmstadt, Tiamana) i ngā kukū rerekē (12.5, 25, 50, 75, 100 me te 125 mg/L) ki te S. sclerotiorum isolate 3 i roto i te vitro. He mea nui kia mōhiotia ko te L-ornithine he pānga patu huakita, ā, i āta aukati i te tipu radial o te S. sclerotiorum hyphae i runga i te tikanga e whakawhirinaki ana ki te horopeta (Pikitia 2A, B). I te kukū teitei rawa i whakamatautauria (125 mg/L), i whakaatu te L-ornithine i te tere aukati tipu mycelial teitei rawa (99.62 ± 0.27%; Pikitia 2B), he rite ki te harore harore arumoni Rizolex-T (tere aukati 99.45 ± 0.39%; Pikitia 2C) i te kukū teitei rawa i whakamatautauria (10 mg/L), e tohu ana i te whai huatanga ōrite.
Pikitia 2. Te kaha patu huakita in vitro o te L-ornithine ki te Sclerotinia sclerotiorum. (A) Te whakataurite i te kaha patu huakita o ngā kukū rerekē o te L-ornithine ki te S. sclerotiorum me te patu harore arumoni Rizolex-T (10 mg/L). (B, C) Te tere aukati (%) o te tipu mycelial S. sclerotiorum i muri i te maimoatanga me ngā kukū rerekē o te L-ornithine (12.5, 25, 50, 75, 100 me te 125 mg/L) Rizolex-T rānei (2, 4, 6, 8 me te 10 mg/L), ia. Ko ngā uara e tohu ana i te toharite ± SD o ngā tāruarua koiora e rima (n = 5). Ko ngā reta rerekē e tohu ana i ngā rerekētanga tatauranga i waenga i ngā maimoatanga (p < 0.05). (D, E) Te tātari whakatauira tauira Probit o te L-ornithine me te patu harore arumoni Rizolex-T, ia. Ka whakaaturia te rārangi whakatauira tauira probit hei rārangi kikorangi totoka, ā, ko te wā whakawhirinaki (95%) ka whakaaturia hei rārangi whero pītiti.
Hei tāpiritanga, i whakahaerehia te tātaritanga whakatauira probit, ā, e whakaaturia ana ngā kauwhata e rite ana i roto i te Ripanga 1 me ngā Pikitia 2D,E. I roto i te poto, ko te uara pikinga e whakaaetia ana (y = 2.92x − 4.67) me ngā tatauranga nui e pā ana (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 me te p < 0.0001; Pikitia 2D) o te L-ornithine i tohu i te kaha ake o te mahi harore ki a S. sclerotiorum i whakaritea ki te harore arumoni Rizolex-T (y = 1.96x − 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 me te p < 0.0001) (Ripanga 1).
Ripanga 1. Ngā uara o te kukū aukati haurua-mōrahi (IC50) me te IC99 (mg/l) o te L-ornithine me te patu harore arumoni "Rizolex-T" ki te S. sclerotiorum.
I te nuinga, i whakaitihia e te L-ornithine (250 mg/L) te whanaketanga me te kaha o te harore mā i runga i ngā tipu pīni noa i rongoatia, ki te whakataurite ki ngā tipu i pangia e te S. sclerotiorum kāore i rongoatia (mana; Pikitia 3A). I ​​roto i te poto, ahakoa i piki haere te kaha o te mate o ngā tipu mana i pangia kāore i rongoatia (52.67 ± 1.53, 83.21 ± 2.61, me te 92.33 ± 3.06%), i whakaitihia e te L-ornithine te kaha o te mate (%) puta noa i te whakamātautau (8.97 ± 0.15, 18.00 ± 1.00, me te 26.36 ± 3.07) i te 7, 14, me te 21 rā i muri i te maimoatanga (dpt), ia (Pikitia 3A). Waihoki, i te wā i rongoātia ai ngā tipu pīni i pangia e te S. sclerotiorum ki te 250 mg/L L-ornithine, ka heke te horahanga i raro i te pihi haere whakamua o te mate (AUDPC) mai i te 1274.33 ± 33.13 i te mana whakahaere kāore i rongoātia ki te 281.03 ± 7.95, he iti iho i tērā o te mana whakahaere pai 50 mg/L Rizolex-T harore (183.61 ± 7.71; Pikitia 3B). I kitea anō te ia o taua ia i te whakamātautau tuarua.
Pikitia 3. Te pānga o te tono o waho o te L-ornithine ki te whanaketanga o te pirau mā o te pīni noa i puta mai i te Sclerotinia sclerotiorum i raro i ngā āhuatanga kati kōtuhi. (A) Te kōpiko haere o te mate o te harore mā o te pīni noa i muri i te maimoatanga ki te 250 mg/L L-ornithine. (B) Te horahanga i raro i te kōpiko haere o te mate (AUDPC) o te harore mā o te pīni noa i muri i te maimoatanga ki te L-ornithine. Ko ngā uara e tohu ana i te toharite ± SD o ngā tāruarua koiora e rima (n = 5). Ko ngā reta rerekē e tohu ana i ngā rerekētanga nui i waenga i ngā maimoatanga (p < 0.05).
I muri i ngā rā 42, ka piki haere te teitei o te tipu mā te whakamahi i te 250 mg/L L-ornithine mā te whakamahi i waho (Pikitia 4A), te maha o ngā manga mō ia tipu (Pikitia 4B), me te maha o ngā rau mō ia tipu (Pikitia 4C). Ahakoa ko te harore patu huakita arumoni Rizolex-T (50 mg/L) te mea nui rawa atu te pānga ki ngā tawhā kai katoa i akohia, ko te whakamahi i te 250 mg/L L-ornithine mā te whakamahi i waho te tuarua o ngā pānga nui rawa atu ki ngā mana kāore i rongoātia (Pikitia 4A–C). I tētahi atu taha, kāore he pānga nui o te maimoatanga L-ornithine ki te ihirangi o ngā tae whakaahua chlorophyll a (Pikitia 4D) me te chlorophyll b (Pikitia 4E), engari i paku piki ake te ihirangi carotenoid katoa (0.56 ± 0.03 mg/g fr wt) ki te whakamahi i te mana kino (0.44 ± 0.02 mg/g fr wt) me te mana pai (0.46 ± 0.02 mg/g fr wt; Pikitia 4F). I te nuinga, e whakaatu ana ēnei hua kāore te L-ornithine e paitini ki ngā pīni kua rongoatia, ā, ka taea hoki e ia te whakaoho i tō rātou tipu.
Pikitia 4. Te pānga o te tono L-ornithine o waho ki ngā āhuatanga tipu me ngā tae whakaahua o ngā rau pīni kua pangia e te Sclerotinia sclerotiorum i raro i ngā āhuatanga kati kōtuhi. (A) Teitei o te tipu (cm), (B) Te maha o ngā manga mō ia tipu, (C) Te maha o ngā rau mō ia tipu, (D) Te nui o te chlorophyll a (mg g-1 fr wt), (E) Te nui o te chlorophyll b (mg g-1 fr wt), (F) Te katoa o te carotenoid (mg g-1 fr wt). Ko ngā uara ko te toharite ± SD o ngā tāruarua koiora e rima (n = 5). Ko ngā reta rerekē e tohu ana i ngā rerekētanga nui i waenga i ngā maimoatanga (p < 0.05).
I whakaatuhia e te tirotiro matū in situ o ngā momo hāora tauhohe (ROS; i whakaatuhia hei hauwai peroxide [H2O2]) me ngā rauropi kore utu (i whakaatuhia hei anions superoxide [O2•−]) ko te tono o waho o te L-ornithine (250 mg/L) i whakaiti nui i te kohikohinga o te H2O2 (96.05 ± 5.33 nmol.g−1 FW; Pikitia 5A) me te O2•− (32.69 ± 8.56 nmol.g−1 FW; Pikitia 5B) ki te whakataurite ki te kohikohinga o ngā tipu pangia kāore i rongoātia (173.31 ± 12.06 me te 149.35 ± 7.94 nmol.g−1 FW, ia) me ngā tipu i rongoātia ki te 50 mg/L o te harore patu huakita arumoni Rizolex-T (170.12 ± 9.50 me te 157.00 ± 7.81 nmol.g−1 fr wt, ia) i te 72 hāora. He nui ngā taumata o te H2O2 me te O2•− i kohia i raro i te hpt (Pikitia 5A, B). Waihoki, i whakaatuhia e te whakamātautau malondialdehyde (MDA) e ahu mai ana i te TCA he nui ake ngā taumata o te MDA i kohia e ngā tipu pīni i pangia e te S. sclerotiorum (113.48 ± 10.02 nmol.g fr wt) i roto i ō rātou rau (Pikitia 5C). Heoi, ko te whakamahinga o te L-ornithine i waho i tino whakaiti i te lipid peroxidation e whakaatuhia ana e te hekenga o te ihirangi MDA i roto i ngā tipu i rongoatia (33.08 ± 4.00 nmol.g fr wt).
Pikitia 5. Te pānga o te tono L-ornithine o waho ki ngā tohu matua o te ahotea waikura me ngā tikanga parepare ārai kore-enzymatic i roto i ngā rau pīni i pangia e te S. sclerotiorum i te 72 hāora i muri i te pangia i raro i ngā āhuatanga kati kōtuhi. (A) Te hauwai peroxide (H2O2; nmol g−1 FW) i te 72 hpt, (B) te anion superoxide (O2•−; nmol g−1 FW) i te 72 hpt, (C) te malondialdehyde (MDA; nmol g−1 FW) i te 72 hpt, (D) ngā phenol wairewa katoa (mg GAE g−1 FW) i te 72 hpt, (E) ngā flavonoid wairewa katoa (mg RE g−1 FW) i te 72 hpt, (F) ngā waikawa amino kore utu katoa (mg g−1 FW) i te 72 hpt, me te (G) te ihirangi proline (mg g−1 FW) i te 72 hpt. Ko ngā uara e tohu ana i te toharite ± paerewa rerekētanga (toharite ± SD) o ngā tāruarua koiora e 5 (n = 5). Ko ngā reta rerekē e tohu ana i ngā rerekētanga hiranga tatauranga i waenga i ngā maimoatanga (p < 0.05).