ʻO ka ʻatikala kekahi ʻāpana o ke kumuhana noiʻi "Hoʻomaikaʻi i ke kūpaʻa o nā legumes i nā pathogens a me nā mea ʻino", e nānā i nā ʻatikala 5 āpau
ʻO ke kumu o ka maʻi fungal plant necrosis ʻo Sclerotinia sclerotiorum (Lib.) de Bary e hoʻohana i kahi hoʻolālā multi-tiered e hoʻopili i nā mea kanu host like ʻole. Manaʻo kēia haʻawina i ka hoʻohana ʻana i ka diamine L-ornithine, kahi waikawa amino non-protein e hoʻoulu ai i ka synthesis o nā waikawa amino koʻikoʻi ʻē aʻe, ma ke ʻano he hoʻolālā hoʻokele ʻē aʻe e hoʻoikaika i nā pane molekala, physiological a me biochemical o Phaseolus vulgaris L. i ka ʻōpala keʻokeʻo i hoʻokumu ʻia e Pseudomonas sclerotiorum. Ua hōʻike nā hoʻokolohua in vitro ua kāohi nui ʻo L-ornithine i ka ulu ʻana o ka mycelial o S. pyrenoidosa ma ke ʻano hilinaʻi i ka dose. Eia kekahi, hiki iā ia ke hōʻemi nui i ke koʻikoʻi o ka ʻōpala keʻokeʻo ma lalo o nā kūlana hale kōmahana. Eia kekahi, ua hoʻoulu ʻo L-ornithine i ka ulu ʻana o nā mea kanu i mālama ʻia, e hōʻike ana ʻaʻole phytotoxic nā ʻano i hoʻāʻo ʻia o L-ornithine i nā mea kanu i mālama ʻia. Eia kekahi, ua hoʻonui ʻo L-ornithine i ka hōʻike ʻana o nā antioxidants non-enzymatic (nā phenolics soluble a me nā flavonoids) a me nā antioxidants enzymatic (catalase (CAT), peroxidase (POX), a me ka polyphenol oxidase (PPO)), a ua hoʻonui i ka hōʻike ʻana o ʻekolu mau genes e pili ana i ka antioxidant (PvCAT1, PvSOD, a me PvGR). Eia kekahi, ua hōʻike ka loiloi silico i ke alo o kahi protein putative oxaloacetate acetylhydrolase (SsOAH) i loko o ka genome S. sclerotiorum, kahi i like loa me nā protein oxaloacetate acetylhydrolase (SsOAH) o Aspergillus fijiensis (AfOAH) a me Penicillium sp. (PlOAH) ma ke ʻano o ka loiloi hana, nā kikowaena i mālama ʻia, a me ka topology. ʻO ka mea hoihoi, ʻo ka hoʻohui ʻana o L-ornithine i ka potato dextrose broth (PDB) medium i hoʻemi nui i ka hōʻike ʻana o ka gene SsOAH ma S. sclerotiorum mycelia. Pēlā nō, ua hoʻemi nui ka hoʻopili ʻana o L-ornithine ma waho i ka hōʻike ʻana o ka gene SsOAH i loko o ka mycelia fungal i hōʻiliʻili ʻia mai nā mea kanu i mālama ʻia. ʻO ka mea hope loa, ua hoʻemi nui ka hoʻopili ʻana o L-ornithine i ka hoʻokuʻu ʻana o ka waikawa oxalic i loko o ka medium PDB a me nā lau i loaʻa i ka maʻi. I ka hopena, he kuleana koʻikoʻi ko L-ornithine i ka mālama ʻana i ke kūlana redox a me ka hoʻoikaika ʻana i ka pane pale o nā mea kanu i loaʻa i ka maʻi. Hiki i nā hopena o kēia noiʻi ke kōkua i ka hoʻomohala ʻana i nā ʻano hana hou a pili i ke kaiapuni e kāohi i ka ʻōpala keʻokeʻo a hoʻēmi i kona hopena i ka hana pīni a me nā mea kanu ʻē aʻe.
ʻO ka ʻōpala keʻokeʻo, i hoʻokumu ʻia e ka fungus necrotrophic ʻo Sclerotinia sclerotiorum (Lib.) de Bary, he maʻi ʻino loa ia, e hōʻemi ana i ka hua e hoʻoweliweli nui ana i ka hana ʻana o ka pīni honua (Phaseolus vulgaris L.) (Bolton et al., 2006). ʻO Sclerotinia sclerotiorum kekahi o nā pathogens mea kanu fungal i lawe ʻia e ka lepo e kaohi, me kahi ākea ākea o nā ʻano mea kanu ma mua o 600 a me ka hiki ke macerate koke i nā ʻiʻo kikowaena ma ke ʻano kikoʻī ʻole (Liang lāua ʻo Rollins, 2018). Ma lalo o nā kūlana maikaʻi ʻole, ke hele nei ia i kahi pae koʻikoʻi o kona pōʻaiapuni ola, e noho hiamoe ana no ka manawa lōʻihi e like me nā ʻano ʻeleʻele, paʻakikī, e like me ka hua i kapa ʻia ʻo 'sclerotia' i loko o ka lepo a i ʻole he mau ulu keʻokeʻo, fluffy i loko o ka mycelium a i ʻole ke kumu pith o nā mea kanu i loaʻa i ka maʻi (Schwartz et al., 2005). Hiki iā S. sclerotiorum ke hana i ka sclerotia, kahi e hiki ai iā ia ke ola i nā mahina i loaʻa i ka maʻi no ka manawa lōʻihi a e hoʻomau i ka wā o ka maʻi (Schwartz et al., 2005). He waiwai nui nā Sclerotia i nā meaʻai, hiki ke noho i ka lepo no ka manawa lōʻihi, a lawelawe ma ke ʻano he inoculum mua no nā maʻi ma hope (Schwartz et al., 2005). Ma lalo o nā kūlana maikaʻi, ulu ka sclerotia a hana i nā spores ea e hiki ke hoʻopili i nā ʻāpana āpau o ka mea kanu ma luna o ka honua, me ka palena ʻole i nā pua, nā kumu, a i ʻole nā ​​​​ʻōpuʻu (Schwartz et al., 2005).
Hoʻohana ʻo Sclerotinia sclerotiorum i kahi hoʻolālā multi-tiered e hoʻopili i kāna mau mea kanu kikowaena, e pili ana i kahi moʻo o nā hanana i hoʻonohonoho ʻia mai ka germination sclerotial a hiki i ka ulu ʻana o nā hōʻailona. I ka wā mua, hana ʻo S. sclerotiorum i nā spores i hoʻokuʻu ʻia (i kapa ʻia ʻo ascospores) mai nā ʻano mushroom-like i kapa ʻia ʻo apothecia, e lilo i lewa a ulu i sclerotia non-motile ma luna o nā ʻōpala mea kanu i loaʻa i ka maʻi (Bolton et al., 2006). A laila huna ka fungus i ka waikawa oxalic, kahi mea virulence, e kāohi i ka pH o ka paia cell mea kanu, e paipai i ka hoʻohaʻahaʻa enzymatic a me ka hoʻouka kaua ʻana o nā ʻiʻo (Hegedus lāua ʻo Rimmer, 2005), a kāohi i ka pahū oxidative o ka mea kanu kikowaena. Hoʻonāwaliwali kēia kaʻina hana acidification i ka paia cell mea kanu, e hāʻawi ana i kahi ʻano maikaʻi no ka hana maʻamau a me ka maikaʻi o nā enzymes fungal cell wall degrading (CWDEs), e ʻae ana i ka pathogen e lanakila ma luna o ka pale kino a komo i loko o nā ʻiʻo host (Marciano et al., 1983). Ke komo i loko, hoʻopuka ʻo S. sclerotiorum i kahi helu o nā CWDE, e like me ka polygalacturonase a me ka cellulase, ka mea e hoʻomaʻamaʻa ai i kona hoʻolaha ʻana i nā ʻiʻo i loaʻa i ka maʻi a hoʻoulu i ka necrosis o nā ʻiʻo. ʻO ka holomua o nā lesions a me nā moena hyphal e alakaʻi i nā hōʻailona hiʻona o ka ʻōpala keʻokeʻo (Hegedus lāua ʻo Rimmer, 2005). I kēia manawa, ʻike nā mea kanu kikowaena i nā ʻano molekala pili i ka pathogen (PAMPs) ma o nā mea loaʻa ʻike ʻano (PRRs), e hoʻoulu ana i kahi moʻo o nā hanana hōʻailona e hoʻāla hope i nā pane pale.
ʻOiai he mau makahiki o nā hana kaohi maʻi, ke koe nei ka nele o ka germplasm kūpaʻa kūpono i ka pī, e like me nā mea kanu kalepa ʻē aʻe, ma muli o ke kūpaʻa, ke ola ʻana, a me ka hiki ke hoʻololi i ka pathogen. No laila, he mea paʻakikī loa ka hoʻokele maʻi a koi aku i kahi hoʻolālā i hoʻohui ʻia a multifaceted e komo pū ana me ka hui pū ʻana o nā hana moʻomeheu, ka kaohi olaola, a me nā fungicides kemika (O'Sullivan et al., 2021). ʻO ka kaohi kemika o ka ʻōpala keʻokeʻo ka mea maikaʻi loa no ka mea, ke hoʻopili pololei ʻia a i ka manawa kūpono, hiki i nā fungicides ke kaohi pono i ka laha ʻana o ka maʻi, hoʻemi i ke koʻikoʻi o ka maʻi, a hoʻemi i ka pohō hua. Eia nō naʻe, ʻo ka hoʻohana nui ʻana a me ka hilinaʻi nui ʻana i nā fungicides hiki ke alakaʻi i ka puka ʻana mai o nā ʻano kūpaʻa o S. sclerotiorum a hoʻopilikia maikaʻi ʻole i nā meaola ʻaʻole i hoʻolālā ʻia, ke olakino lepo, a me ka maikaʻi o ka wai (Le Cointe et al., 2016; Ceresini et al., 2024). No laila, ua lilo ka loaʻa ʻana o nā koho ʻē aʻe e pili ana i ke kaiapuni i mea nui loa.
Hiki i nā Polyamines (PA), e like me ka putrescine, spermidine, spermine, a me cadaverine, ke lawelawe ma ke ʻano he mau koho ʻē aʻe e kūʻē i nā pathogens mea kanu i lawe ʻia e ka lepo, no laila e hōʻemi piha a hapa paha i ka hoʻohana ʻana i nā fungicides kemika weliweli (Nehela et al., 2024; Yi et al., 2024). I nā mea kanu kiʻekiʻe, pili nā PA i nā kaʻina hana physiological he nui, me ka hoʻokaʻawale ʻana o nā cell, ka hoʻokaʻawale ʻana, a me ka pane ʻana i nā pilikia abiotic a me biotic (Killiny lāua ʻo Nehela, 2020). Hiki iā lākou ke hana ma ke ʻano he antioxidants, kōkua i ka scavenge reactive oxygen species (ROS), mālama i ka redox homeostasis (Nehela lāua ʻo Killiny, 2023), hoʻoulu i nā genes e pili ana i ka pale (Romero et al., 2018), hoʻoponopono i nā ala metabolic like ʻole (Nehela lāua ʻo Killiny, 2023), hoʻololi i nā phytohormones endogenous (Nehela lāua ʻo Killiny, 2019), hoʻokumu i ka systemic acquired resistance (SAR), a hoʻoponopono i nā pilina mea kanu-pathogen (Nehela lāua ʻo Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). He mea kūpono ke hoʻomaopopo ʻana he ʻokoʻa nā ʻano hana kikoʻī a me nā kuleana o nā PA i ka pale ʻana o nā mea kanu ma muli o nā ʻano mea kanu, nā pathogens, a me nā kūlana o ke kaiapuni. ʻO ka PA nui loa i nā mea kanu ua biosynthesized ʻia mai ka polyamine L-ornithine koʻikoʻi (Killiny lāua ʻo Nehela, 2020).
He nui nā kuleana o L-ornithine i ka ulu ʻana a me ka hoʻomohala ʻana o nā mea kanu. No ka laʻana, ua hōʻike ʻia nā haʻawina mua i ka laiki (Oryza sativa), pili paha ka ornithine me ka hana hou ʻana o ka naikokene (Liu et al., 2018), ka hua laiki, ka maikaʻi a me ka ʻala (Lu et al., 2020), a me ka pane ʻana i ke kaumaha wai (Yang et al., 2000). Eia kekahi, ua hoʻonui nui ka hoʻohana ʻana o L-ornithine i waho i ka hoʻomanawanui maloʻo i ka beet kō (Beta vulgaris) (Hussein et al., 2019) a ua hoʻēmi i ke kaumaha paʻakai i ka ʻakaʻakai (Allium Cepa) (Çavuşoǧlu a me Çavuşoǧlu, 2021) a me nā mea kanu cashew (Anacardium occidentale) (da Rocha et al., 2012). ʻO ke kuleana kūpono o L-ornithine i ka pale ʻana i ke kaumaha abiotic ma muli paha o kona komo ʻana i ka hōʻiliʻili proline i nā mea kanu i mālama ʻia. No ka laʻana, ua hōʻike mua ʻia nā genes e pili ana i ka metabolism proline, e like me nā genes ornithine delta aminotransferase (delta-OAT) a me proline dehydrogenase (ProDH1 a me ProDH2), e pili ana i ka pale ʻana o Nicotiana benthamiana lāua ʻo Arabidopsis thaliana e kūʻē i nā ʻano Pseudomonas syringae ʻole (Senthil-Kumar lāua ʻo Mysore, 2012). Ma ka ʻaoʻao ʻē aʻe, pono ka fungal ornithine decarboxylase (ODC) no ka ulu ʻana o ka pathogen (Singh et al., 2020). ʻO ka hoʻopaʻa ʻana i ka ODC o Fusarium oxysporum f. sp. lycopersici ma o ka host-induced gene silencing (HIGS) i hoʻonui nui i ke kūʻē ʻana o nā mea kanu tōmato i ka Fusarium wilt (Singh et al., 2020). Eia nō naʻe, ʻaʻole i aʻo maikaʻi ʻia ke kuleana o ka noi exogenous ornithine e kūʻē i nā pilikia biotic e like me nā phytopathogens. ʻOi aku ka mea nui, ʻo ka hopena o ka ornithine i ke kūpaʻa ʻana i nā maʻi a me nā hanana biochemical a me physiological e pili ana ʻaʻole i ʻimi nui ʻia.
He mea nui ka hoʻomaopopo ʻana i ka paʻakikī o ka maʻi S. sclerotiorum o nā legumes no ka hoʻomohala ʻana i nā hoʻolālā kaohi kūpono. Ma kēia haʻawina, ua manaʻo mākou e ʻike i ke kuleana kūpono o ka diamine L-ornithine ma ke ʻano he kumu nui i ka hoʻoikaika ʻana i nā ʻano pale a me ke kūʻē ʻana o nā mea kanu legume i ka maʻi Sclerotinia sclerotiorum. Ke kuhi nei mākou, ma waho aʻe o ka hoʻoikaika ʻana i nā pane pale o nā mea kanu i loaʻa i ka maʻi, he kuleana koʻikoʻi nō hoʻi ʻo L-ornithine i ka mālama ʻana i ke kūlana redox. Ke hāpai nei mākou e pili ana nā hopena kūpono o L-ornithine i ka hoʻoponopono ʻana i nā ʻano pale antioxidant enzymatic a me non-enzymatic a me ke komo ʻana me nā kumu pathogenicity/virulence fungal a me nā protein pili. ʻO kēia hana pālua o L-ornithine e lilo ia i moho hoʻohiki no kahi hoʻolālā hoʻomau e hōʻemi i ka hopena o ka ʻōpala keʻokeʻo a hoʻonui i ke kūʻē ʻana o nā mea kanu legume maʻamau i kēia pathogen fungal ikaika. Hiki i nā hopena o ke aʻo ʻana i kēia manawa ke kōkua i ka hoʻomohala ʻana i nā ʻano hana hou a pili i ke kaiapuni e kaohi i ka ʻōpala keʻokeʻo a hōʻemi i kona hopena i ka hana legume.
Ma kēia noiʻi, ua hoʻohana ʻia kahi ʻano kūʻai maʻalahi o ka pī maʻamau, ʻo Giza 3 (Phaseolus vulgaris L. cv. Giza 3), ma ke ʻano he mea hoʻokolohua. Ua hāʻawi lokomaikaʻi ʻia nā hua olakino e ka Legume Research Department, Field Crops Research Institute (FCRI), Agricultural Research Center (ARC), ʻAigupita. Ua lūlū ʻia ʻelima mau hua i loko o nā ipu plastik (ke anawaena o loko he 35 cm, ka hohonu he 50 cm) i hoʻopiha ʻia me ka lepo i loaʻa i ka maʻi S. sclerotiorum ma lalo o nā kūlana hale kōmahana (25 ± 2 °C, ka haʻahaʻa pili 75 ± 1%, 8 h mālamalama/16 h pouli). I 7-10 mau lā ma hope o ka lūlū ʻana (DPS), ua hoʻomāmā ʻia nā mea kanu e waiho wale i ʻelua mau mea kanu me ka ulu like a me ʻekolu mau lau i hoʻonui piha ʻia i kēlā me kēia ipu. Ua hoʻoinu ʻia nā mea kanu ipu āpau i hoʻokahi manawa i kēlā me kēia ʻelua pule a hoʻomoʻa ʻia i kēlā me kēia mahina ma ka nui i ʻōlelo ʻia no ke ʻano i hāʻawi ʻia.
No ka hoʻomākaukau ʻana i kahi 500 mg/L o ka L-ornithinediamine (ʻike ʻia hoʻi ʻo (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Kelemānia), ua hoʻoheheʻe mua ʻia he 50 mg i loko o 100 mL o ka wai distilled sterile. A laila ua hoʻoheheʻe ʻia ka hopena waihona a hoʻohana ʻia i nā hoʻokolohua ma hope. I ka pōkole, ua hoʻāʻo ʻia ʻeono mau moʻo o nā L-ornithine (12.5, 25, 50, 75, 100, a me 125 mg/L) ma vitro. Eia kekahi, ua hoʻohana ʻia ka wai distilled sterile ma ke ʻano he kaohi maikaʻi ʻole (Mock) a ua hoʻohana ʻia ka fungicide kalepa "Rizolex-T" 50% pauka wettable (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, ʻAigupita) ma ke ʻano he kaohi maikaʻi. Ua hoʻāʻo ʻia ka fungicide kalepa ʻo "Rizolex-T" ma vitro ma ʻelima mau ʻano (2, 4, 6, 8 a me 10 mg/L).
Ua hōʻiliʻili ʻia nā laʻana o nā kumu pīni maʻamau a me nā ʻōpuʻu e hōʻike ana i nā hōʻailona maʻamau o ka ʻōpala keʻokeʻo (ka nui o ka infestation: 10-30%) mai nā mahiʻai kalepa. ʻOiai ua ʻike ʻia ka hapa nui o nā mea kanu i loaʻa i ka maʻi e nā ʻano/ʻano like ʻole (ʻano kalepa maʻalahi ʻo Giza 3), ʻo nā mea ʻē aʻe, ʻoi aku ka poʻe i loaʻa mai nā mākeke kūloko, he ʻano ʻike ʻole ʻia. Ua hoʻomaʻemaʻe mua ʻia nā mea i loaʻa i ka maʻi i hōʻiliʻili ʻia me ka 0.5% sodium hypochlorite solution no 3 mau minuke, a laila holoi ʻia i nā manawa he nui me ka wai distilled sterile a holoi maloʻo me ka pepa kānana sterile e wehe i ka wai keu. A laila ua ʻoki ʻia nā ʻōpū i loaʻa i ka maʻi i nā ʻāpana liʻiliʻi mai ka ʻiʻo waena (ma waena o nā ʻiʻo olakino a me nā ʻiʻo i loaʻa i ka maʻi), i mahi ʻia ma ka potato dextrose agar (PDA) medium a hoʻomoʻa ʻia ma 25 ± 2 °C me kahi pōʻaiapuni mālamalama 12 h/12 ​​h no 5 mau lā e hoʻoulu ai i ka hoʻokumu ʻana o sclerotia. Ua hoʻohana pū ʻia ke ʻano mycelial tip e hoʻomaʻemaʻe i nā isolates fungal mai nā moʻomeheu i hui pū ʻia a haumia paha. Ua ʻike mua ʻia ka isolate fungal i hoʻomaʻemaʻe ʻia ma muli o kona ʻano morphological moʻomeheu a laila ua hōʻoia ʻia ʻo S. sclerotiorum ma muli o nā hiʻohiʻona microscopic. ʻO ka mea hope loa, ua hoʻāʻo ʻia nā isolates i hoʻomaʻemaʻe ʻia no ka pathogenicity ma ka ʻano pīni maʻamau ʻo Giza 3 e hoʻokō ai i nā postulate a Koch.
Eia kekahi, ua hōʻoia hou ʻia ka isolate S. sclerotiorum invasive loa (isolate #3) ma muli o ka hoʻonohonoho ʻana o ka internal transcribed spacer (ITS) e like me ka mea i wehewehe ʻia e White et al., 1990; Baturo-Ciesniewska et al., 2017. I ka pōkole, ua mahi ʻia nā isolates i loko o ka wai dextrose potato (PDB) a hoʻomoʻa ʻia ma 25 ± 2 °C no 5-7 mau lā. A laila ua hōʻiliʻili ʻia ka mycelium fungal, kānana ʻia ma o ka cheesecloth, holoi ʻia i ʻelua manawa me ka wai sterile, a hoʻomaloʻo ʻia me ka pepa kānana sterile. Ua hoʻokaʻawale ʻia ka DNA Genomic me ka hoʻohana ʻana i ka Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). A laila ua hoʻonui ʻia ka ʻāpana ITS rDNA me ka hoʻohana ʻana i ka hui primer kikoʻī ITS1/ITS4 (TCCGTAGGTGAACCTGCGG TCCTCCGCTTATTGATATGC; ka nui i manaʻo ʻia: 540 bp) (Baturo-Ciesniewska et al., 2017). Ua waiho ʻia nā huahana PCR i hoʻomaʻemaʻe ʻia no ka hoʻonohonoho ʻana (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). Ua hoʻonohonoho ʻia nā moʻo ITS rDNA ma ke ʻano bidirectional me ka hoʻohana ʻana i ke ʻano hoʻonohonoho ʻana o Sanger. A laila ua hoʻohālikelike ʻia nā moʻo nīnau i hōʻuluʻulu ʻia me ka ʻikepili hou loa ma GenBank a me ka National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/) me ka hoʻohana ʻana i ka polokalamu BLASTn. Ua hoʻohālikelike ʻia ke kaʻina nīnau me 20 mau ʻano/isolates S. sclerotiorum ʻē aʻe i kiʻi ʻia mai ka ʻikepili hou loa ma NCBI GenBank (Papa Hoʻohui S1) me ka hoʻohana ʻana iā ClustalW ma ka Molecular Evolutionary Genetics Analysis Package (MEGA-11; mana 11) (Kumar et al., 2024). Ua hana ʻia ka loiloi evolutionary me ka hoʻohana ʻana i ke ʻano likelihood kiʻekiʻe loa a me ke kumu hoʻohālike hoʻololi nucleotide manawa-hoʻohuli nui ʻia (Nei lāua ʻo Kumar, 2000). Hōʻike ʻia ka lāʻau me ka likelihood log kiʻekiʻe loa. Ua koho ʻia ka lāʻau mua no ka ʻimi heuristic ma ke koho ʻana i ka lāʻau me ka likelihood log kiʻekiʻe ma waena o ka lāʻau hoʻohui hoalauna (NJ) (Kumar et al., 2024) a me ka lāʻau parsimony kiʻekiʻe loa (MP). Ua kūkulu ʻia ka lāʻau NJ me ka hoʻohana ʻana i kahi matrix mamao pairwise i helu ʻia me ka hoʻohana ʻana i ke kumu hoʻohālike manawa-hoʻohuli nui ʻia (Nei lāua ʻo Kumar, 2000).
Ua hoʻoholo ʻia ka hana antibacterial o L-ornithine a me ka bactericide "Rizolex-T" in vitro e ke ʻano hoʻolaha agar. ʻAno hana: E lawe i ka nui kūpono o ka hopena waihona o L-ornithine (500 mg/L) a kāwili maikaʻi me 10 ml o ka PDA nutrient medium e hoʻomākaukau i nā hopena me nā ʻano hope loa o 12.5, 25, 50, 75, 100 a me 125 mg/L, kēlā me kēia. ʻElima mau ʻano o ka fungicide "Rizolex-T" (2, 4, 6, 8 a me 10 mg/L) a me ka wai distilled sterile i hoʻohana ʻia ma ke ʻano he kaohi. Ma hope o ka paʻa ʻana o ka medium, ua hoʻoili ʻia kahi plug mycelial hou o ka moʻomeheu Sclerotinia sclerotiorum, 4 mm ke anawaena, i waenakonu o ke kīʻaha Petri a hoʻoulu ʻia ma 25±2°C a hiki i ka uhi ʻana o ka mycelium i ka pā Petri kaohi holoʻokoʻa, ma hope o ka hoʻopaʻa ʻia ʻana o ka ulu ʻana o ka fungal. E helu i ka pakeneka inhibition o ka ulu radial o S. sclerotiorum me ka hoʻohana ʻana i ka equation 1:
Ua hana hou ʻia ka hoʻokolohua i ʻelua manawa, me ʻeono mau replicates olaola no kēlā me kēia hui kaohi/hoʻokolohua a me ʻelima mau ipu (ʻelua mau mea kanu no kēlā me kēia ipu) no kēlā me kēia replicate olaola. Ua kālailai ʻia kēlā me kēia replicate olaola i ʻelua manawa (ʻelua mau replicates loea) e hōʻoia i ka pololei, ka hilinaʻi a me ka hana hou ʻana o nā hopena hoʻokolohua. Eia kekahi, ua hoʻohana ʻia ka loiloi regression probit e helu i ka hapalua-maximal inhibitory concentration (IC50) a me IC99 (Prentice, 1976).
No ka loiloi ʻana i ka hiki o L-ornithine ma lalo o nā kūlana hale kōmahana, ua hana ʻia ʻelua mau hoʻokolohua ipuhao. I ka pōkole, ua hoʻopiha ʻia nā ipuhao me ka lepo one-palolo i hoʻomaʻemaʻe ʻia (3:1) a ua hoʻokomo ʻia me kahi moʻomeheu hou o S. sclerotiorum. ʻO ka mea mua, ua hoʻoulu ʻia ka isolate invasive loa o S. sclerotiorum (isolate #3) ma ka ʻoki ʻana i hoʻokahi sclerotium i ka hapalua, e kau ana i lalo ma luna o kahi PDA a hoʻomoʻa ʻia ma 25°C i ka pouli mau (24 h) no 4 mau lā e hoʻoulu ai i ka ulu ʻana o ka mycelial. A laila ua lawe ʻia ʻehā mau plug agar 5 mm diameter mai ka ʻaoʻao mua a hoʻokomo ʻia me 100 g o kahi hui sterile o ka palaoa a me ka bran laiki (1:1, v/v) a ua hoʻomoʻa ʻia nā ʻōmole āpau ma 25 ± 2 °C ma lalo o kahi pōʻaiapuni 12 h mālamalama/12 h pouli no 5 mau lā e hoʻoulu ai i ka hoʻokumu ʻana o sclerotia. Ua hui maikaʻi ʻia nā mea o loko o nā ʻōmole āpau e hōʻoia i ka homogeneity ma mua o ka hoʻohui ʻana i ka lepo. A laila, ua hoʻohui ʻia he 100 g o ka hui ʻana o ka bran colonizing i kēlā me kēia ipuhao e hōʻoia i ka nui mau o nā pathogens. Ua hoʻoinu ʻia nā ipu i hoʻopili ʻia e hoʻoulu i ka ulu ʻana o ka fungal a waiho ʻia i loko o nā kūlana hale kōmahana no 7 mau lā.
A laila ua lūlū ʻia ʻelima mau hua o ke ʻano Giza 3 i loko o kēlā me kēia ipuhao. No nā ipuhao i mālama ʻia me L-ornithine a me ka fungicide Rizolex-T, ua hoʻomoʻa mua ʻia nā hua i hoʻomaʻemaʻe ʻia no ʻelua hola i loko o kahi wai wai o nā hui ʻelua me ka nui hope loa o IC99 ma kahi o 250 mg/L a me 50 mg/L, kēlā me kēia, a laila hoʻomaloʻo ʻia i ka ea no hoʻokahi hola ma mua o ka lūlū ʻana. Ma ka ʻaoʻao ʻē aʻe, ua hoʻomoʻa ʻia nā hua i loko o ka wai i hoʻomaʻemaʻe ʻia ma ke ʻano he kaohi maikaʻi ʻole. Ma hope o 10 mau lā, ma mua o ka hoʻoinu mua ʻana, ua hoʻomāmā ʻia nā mea kanu, e waiho ana i ʻelua mau mea kanu maʻemaʻe i loko o kēlā me kēia ipuhao. Eia kekahi, no ka hōʻoia ʻana i ka maʻi me S. sclerotiorum, ua ʻoki ʻia nā kumu o ka mea kanu pī ma ke kahua hoʻomohala like (10 mau lā) ma ʻelua mau wahi like ʻole me ka hoʻohana ʻana i kahi scalpel i hoʻomaʻemaʻe ʻia a ua kau ʻia ma kahi o 0.5 g o ka hui bran colonizing i loko o kēlā me kēia ʻeha, a ukali ʻia e ka haʻahaʻa kiʻekiʻe e hoʻoulu i ka maʻi a me ka ulu ʻana o nā maʻi i nā mea kanu i hoʻopili ʻia. Ua ʻeha like ʻia nā mea kanu kaohi a ua waiho ʻia kahi nui like (0.5 g) o ka hui ʻana o ka bran sterile, uncolonized i loko o ka ʻeha a mālama ʻia ma lalo o ka haʻahaʻa kiʻekiʻe e hoʻohālike i ke kaiapuni no ka ulu ʻana o ka maʻi a hōʻoia i ke kūlike ma waena o nā hui lapaʻau.
ʻAno lapaʻau: Ua hoʻoinu ʻia nā hua pīni me 500 ml o kahi wai hoʻoheheʻe wai o L-ornithine (250 mg/l) a i ʻole ka fungicide Rizolex-T (50 mg/l) ma ka hoʻoinu ʻana i ka lepo, a laila ua hana hou ʻia ka lapaʻau ʻekolu manawa ma kahi manawa o 10 mau lā. Ua hoʻoinu ʻia nā kaohi i mālama ʻia me ka placebo me 500 ml o ka wai i hoʻoheheʻe ʻia. Ua mālama ʻia nā lapaʻau āpau ma lalo o nā kūlana hale ʻōmaʻomaʻo (25 ± 2°C, 75 ± 1% ka haʻahaʻa pili, a me kahi photoperiod o 8 h mālamalama/16 h pouli). Ua hoʻoinu ʻia nā ipu āpau i kēlā me kēia pule ʻelua a mālama ʻia i kēlā me kēia mahina me kahi mea hoʻouluulu NPK kaulike (20-20-20, me 3.6% sulfur a me nā microelements TE; Zain Seeds, ʻAigupita) ma kahi nui o 3-4 g/l ma ka pīpī ʻana i nā foliar e like me nā ʻōlelo paipai no ke ʻano kikoʻī a me nā kuhikuhi a ka mea hana. Inā ʻaʻole i ʻōlelo ʻia, ua hōʻiliʻili ʻia nā lau makua i hoʻonui piha ʻia (nā lau 2 a me 3 mai luna) mai kēlā me kēia replicate biological ma 72 hola ma hope o ka mālama ʻana (hpt), homogenized, hōʻuluʻulu ʻia a mālama ʻia ma -80 °C no nā loiloi hou aku e pili ana, akā ʻaʻole i kaupalena ʻia i, ka localization histochemical in situ o nā hōʻailona stress oxidative, lipid peroxidation, enzymatic a me nā antioxidants non-enzymatic a me ka hōʻike ʻana o ka gene.
Ua loiloi ʻia ka ikaika o ka maʻi ʻōpala keʻokeʻo i kēlā me kēia pule he 21 mau lā ma hope o ka hoʻopili ʻana (dpi) me ka hoʻohana ʻana i kahi unahi o 1-9 (Papa Hoʻohui S2) ma muli o ka unahi Petzoldt lāua ʻo Dickson (1996) i hoʻololi ʻia e Teran et al. (2006). I ka pōkole, ua nānā ʻia nā kumu a me nā lālā o nā mea kanu pīni e hoʻomaka ana ma kahi o ka hoʻopili ʻana e hahai i ka holomua o nā ʻeha ma nā internodes a me nā nodes. A laila ua ana ʻia ka mamao o ka ʻeha mai kahi o ka hoʻopili ʻana a i kahi mamao loa ma ke kumu a i ʻole ka lālā a ua hāʻawi ʻia kahi helu o 1-9 ma muli o kahi o ka ʻeha, kahi (1) i hōʻike ʻole ai i kahi maʻi ʻike ʻia kokoke i kahi o ka hoʻopili ʻana a me (2-9) i hōʻike i ka piʻi mālie ʻana o ka nui o ka ʻeha a me ka holomua ma nā nodes/internodes (Papa Hoʻohui S2). A laila ua hoʻololi ʻia ka ikaika o ka maʻi ʻōpala keʻokeʻo i ka pakeneka me ka hoʻohana ʻana i ke ʻano 2:
Eia kekahi, ua helu ʻia ka wahi ma lalo o ke kiʻikuhi holomua o ka maʻi (AUDPC) me ka hoʻohana ʻana i ke ʻano (Shaner lāua ʻo Finney, 1977), kahi i hoʻololi hou ʻia no ka pala keʻokeʻo o ka pīni maʻamau (Chauhan et al., 2020) me ka hoʻohana ʻana i ka hoʻohālikelike 3:
Ma kahi ʻo Yi = ka nui o ka maʻi i ka manawa ti, Yi+1 = ka nui o ka maʻi i ka manawa aʻe ti+1, ti = ka manawa o ke ana mua ʻana (i nā lā), ti+1 = ka manawa o ke ana aʻe (i nā lā), n = ka huina o nā manawa a i ʻole nā ​​​​kiko nānā. Ua hoʻopaʻa ʻia nā ʻano ulu o ka mea kanu pī me ke kiʻekiʻe o ka mea kanu (cm), ka helu o nā lālā no kēlā me kēia mea kanu, a me ka helu o nā lau no kēlā me kēia mea kanu i kēlā me kēia pule no 21 mau lā ma nā replicates olaola āpau.
I kēlā me kēia kope olaola, ua hōʻiliʻili ʻia nā laʻana lau (ʻelua a me ke kolu o nā lau i ulu piha mai luna mai) i ka lā 45 ma hope o ka mālama ʻana (15 mau lā ma hope o ka mālama hope loa). ʻO kēlā me kēia kope olaola he ʻelima mau ipu (ʻelua mau mea kanu no kēlā me kēia ipu). Ma kahi o 500 mg o ka ʻiʻo i kuʻi ʻia ua hoʻohana ʻia no ka unuhi ʻana o nā pigments photosynthetic (chlorophyll a, chlorophyll b a me carotenoids) me ka hoʻohana ʻana i ka 80% acetone ma 4 °C i ka pōʻeleʻele. Ma hope o 24 hola, ua centrifuged ʻia nā laʻana a ua hōʻiliʻili ʻia ka supernatant no ka hoʻoholo ʻana i ka chlorophyll a, chlorophyll b a me nā ʻike carotenoid colorimetrically me ka hoʻohana ʻana i ka spectrophotometer UV-160A (Shimadzu Corporation, Iapana) e like me ke ʻano o (Lichtenthaler, 1987) ma ke ana ʻana i ka absorbance ma ʻekolu mau nalu like ʻole (A470, A646 a me A663 nm). ʻO ka hope loa, ua helu ʻia ka ʻike o nā pigments photosynthetic me ka hoʻohana ʻana i nā formula 4-6 i wehewehe ʻia e Lichtenthaler (1987).
Ma 72 hola ma hope o ka mālama ʻana (hpt), ua hōʻiliʻili ʻia nā lau (ʻo ka lua a me ke kolu o nā lau i ulu piha mai luna mai) mai kēlā me kēia replicate biological no ka localization histochemical in situ o ka hydrogen peroxide (H2O2) a me ka superoxide anion (O2•−). ʻO kēlā me kēia replicate biological he ʻelima mau ipu (ʻelua mau mea kanu no kēlā me kēia ipu). Ua kālailai ʻia kēlā me kēia replicate biological ma ka pālua (ʻelua mau replicates loea) e hōʻoia i ka pololei, ka hilinaʻi a me ka hana hou ʻana o ke ʻano. Ua hoʻoholo ʻia ʻo H2O2 a me O2•− me ka hoʻohana ʻana i ka 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Kelemānia) a i ʻole nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Kelemānia), kēlā me kēia, ma muli o nā ʻano i wehewehe ʻia e Romero-Puertas et al. (2004) a me Adam et al. (1989) me nā hoʻololi liʻiliʻi. No ka localization histochemical o H2O2 in situ, ua hoʻokomo ʻia nā leaflet i loko o ka vacuum me 0.1% DAB i loko o 10 mM Tris buffer (pH 7.8) a laila hoʻomoʻa ʻia i ka mahana o ka lumi i loko o ke kukui no 60 min. Ua hoʻokeʻokeʻo ʻia nā leaflet i loko o 0.15% (v/v) TCA i loko o 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) a laila hoʻokuʻu ʻia i ka mālamalama a hiki i ka wā e pouli ai. Pēlā nō, ua hoʻokomo ʻia nā valves i loko o ka vacuum me 10 mM potassium phosphate buffer (pH 7.8) nona ka 0.1 w/v % HBT no ka localization histochemical o O2•− in situ. Ua hoʻomoʻa ʻia nā leaflet i loko o ke kukui ma ka mahana o ka lumi no 20 min, a laila hoʻokeʻokeʻo ʻia e like me luna, a laila hoʻomālamalama ʻia a hiki i ka wā e ʻike ʻia ai nā kiko polū/violet ʻeleʻele. Ua loiloi ʻia ka ikaika o ke kala ʻeleʻele i loaʻa (ma ke ʻano he hōʻailona H2O2) a i ʻole polū-violet (ma ke ʻano he hōʻailona O2•−) me ka hoʻohana ʻana i ka mana Fiji o ka pūʻolo hana kiʻi ImageJ (http://fiji.sc; i komo ʻia ma 7 Malaki 2024).
Ua hoʻoholo ʻia ʻo Malondialdehyde (MDA; ma ke ʻano he māka o ka lipid peroxidation) e like me ke ʻano o Du lāua ʻo Bramlage (1992) me nā hoʻololi iki. Ua hōʻiliʻili ʻia nā lau mai kēlā me kēia replica biological (ʻo ka lua a me ke kolu o nā lau i ulu piha mai luna) 72 hola ma hope o ka mālama ʻana (hpt). Loaʻa i kēlā me kēia replica biological ʻelima mau ipu (ʻelua mau mea kanu no kēlā me kēia ipu). Ua kālailai ʻia kēlā me kēia replica biological ma ka pālua (ʻelua mau replicates loea) e hōʻoia i ka pololei, ka hilinaʻi a me ka hana hou ʻana o ke ʻano. I ka pōkole, ua hoʻohana ʻia he 0.5 g o ka ʻiʻo lau lepo no ka MDA extraction me 20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) nona ka 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). A laila ua hoʻoholo ʻia ka nui o ka MDA i loko o ka supernatant ma ke ʻano colorimetrically ma ke ana ʻana i ka absorbance ma 532 a me 600 nm me ka hoʻohana ʻana i kahi spectrophotometer UV-160A (Shimadzu Corporation, Iapana) a laila ua hōʻike ʻia ma ke ʻano he nmol g−1 FW.
No ka loiloi ʻana i nā antioxidants non-enzymatic a me enzymatic, ua hōʻiliʻili ʻia nā lau (ʻo ka lua a me ke kolu o nā lau i ulu piha mai luna mai) mai kēlā me kēia replicate biological ma 72 mau hola ma hope o ka mālama ʻana (hpt). ʻO kēlā me kēia replicate biological he ʻelima mau ipu (ʻelua mau mea kanu no kēlā me kēia ipu). Ua kālailai ʻia kēlā me kēia laʻana biological ma ke pālua (ʻelua mau laʻana loea). Ua wili ʻia ʻelua lau me ka naikokene wai a hoʻohana pololei ʻia no ka hoʻoholo ʻana i nā antioxidants enzymatic a me non-enzymatic, nā waikawa amino āpau, ka nui o ka proline, ka hōʻike ʻana o ka gene, a me ka helu ʻana o ka oxalate.
Ua hoʻoholo ʻia nā phenolics hiki ke hoʻoheheʻe ʻia me ka hoʻohana ʻana i ka reagent Folin-Ciocalteu (Sigma-Aldrich, St. Louis, MO, USA) me nā hoʻololi iki o ke ʻano i wehewehe ʻia e Kahkonen et al. (1999). I ka pōkole, ua unuhi ʻia ma kahi o 0.1 g o ka ʻiʻo lau homogenized me 20 ml 80% methanol i loko o ka pouli no 24 hola a ua hōʻiliʻili ʻia ka supernatant ma hope o ka centrifugation. Ua hui pū ʻia ʻo 0.1 ml o ka extract sample me 0.5 ml Folin-Ciocalteu reagent (10%), lulu ʻia no 30 s a waiho ʻia i loko o ka pouli no 5 min. A laila ua hoʻohui ʻia ʻo 0.5 ml o ka 20% sodium carbonate solution (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) i kēlā me kēia paipu, ua hui maikaʻi ʻia a hoʻomoʻa ʻia i ka mahana o ka lumi i loko o ka pouli no 1 hola. Ma hope o ka hoʻoulu ʻana, ua ana ʻia ka absorbance o ka hui hopena ma 765 nm me ka hoʻohana ʻana i ka spectrophotometer UV-160A (Shimadzu Corporation, Iapana). Ua hoʻoholo ʻia ka nui o nā phenols hiki ke hoʻoheheʻe ʻia i loko o nā ʻāpana laʻana me ka hoʻohana ʻana i kahi piʻo calibration gallic acid (Fisher Scientific, Hampton, NH, USA) a ua hōʻike ʻia ma ke ʻano he milligrams o ka gallic acid equivalent no ka gram o ke kaumaha hou (mg GAE g-1 kaumaha hou).
Ua hoʻoholo ʻia ka nui o ka flavonoid hiki ke hoʻoheheʻe ʻia e like me ke ʻano o Djeridane et al. (2006) me nā hoʻololi iki. I ka pōkole, ua hui pū ʻia ka 0.3 ml o ka methanol extract i luna me ka 0.3 ml o ka 5% alumini chloride solution (AlCl3; Fisher Scientific, Hampton, NH, USA), ua hoʻoulu ikaika ʻia a laila hoʻomoʻa ʻia ma ka mahana o ka lumi no 5 min, a ukali ʻia e ka hoʻohui ʻana o 0.3 ml o ka 10% potassium acetate solution (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), ua hui maikaʻi ʻia a hoʻomoʻa ʻia ma ka mahana o ka lumi no 30 min i ka pōʻeleʻele. Ma hope o ka hoʻomoʻa ʻana, ua ana ʻia ka absorbance o ka hui hopena ma 430 nm me ka hoʻohana ʻana i kahi spectrophotometer UV-160A (Shimadzu Corporation, Iapana). Ua hoʻoholo ʻia ka nui o nā flavonoids hiki ke hoʻoheheʻe ʻia i loko o nā ʻāpana laʻana me ka hoʻohana ʻana i kahi curve calibration rutin (TCI America, Portland, OR, USA) a laila ua hōʻike ʻia ma ke ʻano he milligrams o ka rutin equivalent no kēlā me kēia gram o ke kaumaha hou (mg RE g-1 kaumaha hou).
Ua hoʻoholo ʻia ka nui o ka waikawa amino manuahi o nā lau pī me ka hoʻohana ʻana i kahi reagent ninhydrin i hoʻololi ʻia (Thermo Scientific Chemicals, Waltham, MA, USA) e pili ana i ke ʻano i hāpai ʻia e Yokoyama lāua ʻo Hiramatsu (2003) a hoʻololi ʻia e Sun et al. (2006). I ka pōkole, ua unuhi ʻia he 0.1 g o ka ʻiʻo lepo me ka buffer pH 5.4, a ua hoʻopili ʻia he 200 μL o ka supernatant me 200 μL o ka ninhydrin (2%) a me 200 μL o ka pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), i hoʻomoʻa ʻia i loko o kahi ʻauʻau wai e paila ana no 30 min, a laila hoʻomaʻalili ʻia a ana ʻia ma 580 nm me ka hoʻohana ʻana i kahi spectrophotometer UV-160A (Shimadzu Corporation, Iapana). Ma ka ʻaoʻao ʻē aʻe, ua hoʻoholo ʻia ka proline e ke ʻano Bates (Bates et al., 1973). Ua unuhi ʻia ka Proline me ka 3% sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) a ma hope o ka centrifugation, ua hui pū ʻia ka 0.5 ml o ka supernatant me ka 1 ml glacial acetic acid (Fisher Scientific, Hampton, NH, USA) a me ka ninhydrin reagent, i hoʻomoʻa ʻia ma 90°C no 45 min, hoʻomaʻalili ʻia a ana ʻia ma 520 nm me ka hoʻohana ʻana i ka spectrophotometer like me luna. Ua hoʻoholo ʻia ka huina o nā waikawa amino manuahi a me ka proline i loko o nā unuhi lau me ka hoʻohana ʻana i nā glycine a me nā proline calibration curves (Sigma-Aldrich, St Louis, MO, USA), kēlā me kēia, a ua hōʻike ʻia ma ke ʻano he mg/g kaumaha hou.
No ka hoʻoholo ʻana i ka hana enzymatic o nā enzymes antioxidant, ua unuhi ʻia ma kahi o 500 mg o ka ʻiʻo homogenized me 3 ml o 50 mM Tris buffer (pH 7.8) e loaʻa ana he 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) a me 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), centrifuged ma 10,000 × g no 20 min ma lalo o ka refrigeration (4 °C), a ua hōʻiliʻili ʻia ka supernatant (crude enzyme extract) (El-Nagar et al., 2023; Osman et al., 2023). A laila ua hoʻopili ʻia ʻo Catalase (CAT) me 2 ml o 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) a me 100 μl o 269 mM H2O2 solution e hoʻoholo ai i kāna hana enzymatic e like me ke ʻano o Aebi (1984) me nā hoʻololi iki (El-Nagar et al., 2023; Osman et al., 2023). Ua hoʻoholo ʻia ka hana enzymatic Guaiacol-dependent peroxidase (POX) me ka hoʻohana ʻana i ke ʻano o Harrach et al. (2009). (2008) me nā hoʻololi liʻiliʻi (El-Nagar et al., 2023; Osman et al., 2023) a ua hoʻoholo ʻia ka hana enzymatic o ka polyphenol oxidase (PPO) ma hope o ka hopena me 2.2 ml o 100 mM sodium phosphate buffer (pH 6.0), 100 μl o guaiacol (TCI chemicals, Portland, OR, USA) a me 100 μl o 12 mM H2O2. Ua hoʻololi iki ʻia ke ʻano hana mai (El-Nagar et al., 2023; Osman et al., 2023). Ua hana ʻia ka hoʻāʻo ma hope o ka hopena me 3 ml o ka hopena catechol (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) i hoʻomākaukau hou ʻia i loko o 0.1 M phosphate buffer (pH 6.0). Ua ana ʻia ka hana CAT ma ka nānā ʻana i ka decomposition o H2O2 ma 240 nm (A240), ua ana ʻia ka hana POX ma ka nānā ʻana i ka piʻi ʻana o ka absorbance ma 436 nm (A436), a ua ana ʻia ka hana PPO ma ka hoʻopaʻa ʻana i nā fluctuations absorbance ma 495 nm (A495) i kēlā me kēia 30 s no 3 min me ka hoʻohana ʻana i ka spectrophotometer UV-160A (Shimadzu, Iapana).
Ua hoʻohana ʻia ka RT-PCR manawa maoli e ʻike i nā pae transcript o ʻekolu mau genes e pili ana i ka antioxidant, me ka peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), a me ka glutathione reductase (PvGR; GenBank Accession No. KY195009.1), i loko o nā lau pī (ʻo ka lua a me ke kolu o nā lau i hoʻomohala piha ʻia mai luna) 72 hola ma hope o ka mālama hope loa. I ka pōkole, ua hoʻokaʻawale ʻia ka RNA me ka hoʻohana ʻana i ka Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, Kina) e like me ke kaʻina hana a ka mea hana. A laila, ua synthesized ʻia ka cDNA me ka hoʻohana ʻana i ka TOP script™ cDNA Synthesis Kit e like me nā kuhikuhi a ka mea hana. Ua helu ʻia nā moʻo primer o nā genes ʻekolu ma luna ma ka Papa Hoʻohui S3. Ua hoʻohana ʻia ʻo PvActin-3 (helu komo GenBank: XM_068616709.1) ma ke ʻano he gene housekeeping a ua helu ʻia ka hōʻike ʻana o ka gene pili me ka hoʻohana ʻana i ke ʻano 2-ΔΔCT (Livak lāua ʻo Schmittgen, 2001). Ua hōʻike ʻia ke kūpaʻa o Actin ma lalo o ke kaumaha biotic (ka pilina kūpono ʻole ma waena o nā legumes maʻamau a me ka anthracnose fungus Colletotrichum lindemuthianum) a me ke kaumaha abiotic (maloʻo, paʻakai, mahana haʻahaʻa) (Borges et al., 2012).
Ua hana mua mākou i kahi loiloi genome-ākea in silico o nā protein oxaloacetate acetylhydrolase (OAH) ma S. sclerotiorum me ka hoʻohana ʻana i ka mea hana protein-protein BLAST (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). I ka pōkole, ua hoʻohana mākou iā OAH mai Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; GenBank accession number XP_040799428.1; 342 amino acids) a me Penicillium lagena (PlOAH; taxide: 94218; GenBank accession number XP_056833920.1; 316 amino acids) ma ke ʻano he mau moʻo nīnau e palapala i ka protein homologous ma S. sclerotiorum (taxide: 5180). Ua hana ʻia ʻo BLASTp e kūʻē i ka ʻikepili genome S. sclerotiorum i loaʻa hou loa ma GenBank ma ka pūnaewele National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Eia kekahi, ua kuhi ʻia ka gene OAH i wānana ʻia mai S. sclerotiorum (SsOAH) a me ka loiloi evolutionary a me ka lāʻau phylogenetic o AfOAH mai A. fijiensis CBS 313.89 a me PlOAH mai P. lagena me ka hoʻohana ʻana i ke ʻano likelihood kiʻekiʻe loa ma MEGA11 (Tamura et al., 2021) a me ke kumu hoʻohālike JTT matrix-based (Jones et al., 1992). Ua hui pū ʻia ka lāʻau phylogenetic me ka loiloi alignment lehulehu o nā moʻo protein o nā gene OAH āpau i wānana ʻia (SsOAH) mai S. sclerotiorum a me ka moʻo nīnau me ka hoʻohana ʻana i ka Constraint-Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos lāua ʻo Agarwala, 2007). Eia kekahi, ua hoʻohālikelike ʻia nā moʻo amino acid kūpono loa o SsOAH mai S. sclerotiorum me nā moʻo nīnau (AfOAH a me PlOAH) (Larkin et al., 2007) me ka hoʻohana ʻana iā ClustalW (http://www.genome.jp/tools-bin/clustalw), a ua ʻike ʻia nā ʻāpana i mālama ʻia i ke kūlike me ka hoʻohana ʻana i ka mea hana ESPript (mana 3.0; https://espript.ibcp.fr/ESPript/ESPript/index.php).
Eia kekahi, ua hoʻokaʻawale like ʻia nā kikowaena hana i wānana ʻia a me nā wahi i mālama ʻia o S. sclerotiorum SsOAH i nā ʻohana like ʻole me ka hoʻohana ʻana i ka mea hana InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). ʻO ka hope loa, ua hana ʻia ke ʻano hoʻohālike ʻekolu-dimensional (3D) o ka S. sclerotiorum SsOAH i wānana ʻia me ka hoʻohana ʻana i ka 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) a ua hōʻoia ʻia me ka hoʻohana ʻana i ke kikowaena SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). Ua ʻike maka ʻia nā ʻano hana ʻekolu-dimensional i wānana ʻia (ʻano PDB) me ka hoʻohana ʻana i ka pūʻolo UCSF-Chimera (mana 1.15; https://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004).
Ua hoʻohana ʻia ka PCR fluorescence manawa maoli quantitative e hoʻoholo i ka pae transcriptional o ka oxaloacetate acetylhydrolase (SsOAH; GenBank accession number: XM_001590428.1) i loko o ka mycelia o Sclerotinia sclerotiorum. I ka pōkole, ua hoʻokomo ʻia ʻo S. sclerotiorum i loko o kahi ʻōmole i loaʻa iā PDB a waiho ʻia i loko o kahi incubator lulu (hoʻohālike: I2400, New Brunswick Scientific Co., Edison, NJ, USA) ma 25 ± 2 °C no 24 h ma 150 rpm a i ka pouli mau (24 h) e hoʻoulu i ka ulu ʻana o ka mycelial. Ma hope iho, ua mālama ʻia nā cell me L-ornithine a me ka fungicide Rizolex-T ma nā ʻano hope loa o IC50 (ma kahi o 40 a me 3.2 mg/L, kēlā me kēia) a laila ua hoʻoulu ʻia no kekahi 24 h ma lalo o nā kūlana like. Ma hope o ka hoʻoulu ʻana, ua centrifuged ʻia nā moʻomeheu ma 2500 rpm no 5 min a ua hōʻiliʻili ʻia ka supernatant (fungal mycelium) no ka nānā ʻana i ka hōʻike ʻana o ka gene. Pēlā nō, ua hōʻiliʻili ʻia ka fungal mycelium ma 0, 24, 48, 72, 96, a me 120 h ma hope o ka maʻi ʻana mai nā mea kanu i loaʻa i ka maʻi i hana i ka ʻōpala keʻokeʻo a me ka cottony mycelium ma luna o ka ʻili o nā ʻiʻo i loaʻa i ka maʻi. Ua unuhi ʻia ka RNA mai ka fungal mycelium a laila ua synthesized ʻia ka cDNA e like me ka mea i wehewehe ʻia ma luna. Ua helu ʻia nā moʻo primer no SsOAH ma ka Papa Hoʻohui S3. Ua hoʻohana ʻia ʻo SsActin (GenBank accession number: XM_001589919.1) ma ke ʻano he gene housekeeping, a ua helu ʻia ka hōʻike ʻana o ka gene pili me ka hoʻohana ʻana i ke ʻano 2-ΔΔCT (Livak lāua ʻo Schmittgen, 2001).
Ua hoʻoholo ʻia ka waikawa Oxalic i loko o ka wai dextrose ʻuala (PDB) a me nā laʻana mea kanu e loaʻa ana ka pathogen fungal Sclerotinia sclerotiorum e like me ke ʻano o Xu lāua ʻo Zhang (2000) me nā hoʻololi iki. I ka pōkole, ua hoʻokomo ʻia nā isolates S. sclerotiorum i loko o nā ʻōmole e loaʻa ana ka PDB a laila hoʻoulu ʻia i loko o kahi incubator lulu (hoʻohālike I2400, New Brunswick Scientific Co., Edison, NJ, USA) ma 150 rpm ma 25 ± 2 °C no 3-5 mau lā i ka pouli mau (24 h) e hoʻoulu i ka ulu ʻana o ka mycelial. Ma hope o ka incubation, ua kānana mua ʻia ka moʻomeheu fungal ma o ka pepa kānana Whatman #1 a laila centrifuged ma 2500 rpm no 5 min e wehe i ke koena mycelium. Ua hōʻiliʻili ʻia ka supernatant a mālama ʻia ma 4 °C no ka hoʻoholo hou ʻana o ka oxalate. No ka hoʻomākaukau ʻana i nā laʻana mea kanu, ua unuhi ʻia ma kahi o 0.1 g o nā ʻāpana ʻiʻo mea kanu ʻekolu manawa me ka wai distilled (2 ml i kēlā me kēia manawa). A laila ua centrifuged ʻia nā laʻana ma 2500 rpm no 5 min, ua kānana maloʻo ʻia ka supernatant ma o ka pepa kānana Whatman No. 1 a hōʻiliʻili ʻia no ka nānā hou aku.
No ka loiloi quantitative o ka waikawa oxalic, ua hoʻomākaukau ʻia ka hui ʻana o ka hopena i loko o kahi paipu pani aniani ma ke ʻano penei: 0.2 ml o ka laʻana (a i ʻole ka filtrate moʻomeheu PDB a i ʻole ka hopena maʻamau oxalic acid), 0.11 ml o ka bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), 0.198 ml o ka 1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) a me 0.176 ml o 100 mM potassium dichromate (K2Cr2O7; TCI chemicals, Portland, OR, USA), a laila ua hoʻoheheʻe ʻia ka hopena i 4.8 ml me ka wai i hoʻoheheʻe ʻia, i kāwili ikaika ʻia a waiho koke ʻia i loko o ka ʻauʻau wai 60 °C. Ma hope o 10 min, ua hoʻōki ʻia ka hopena ma ka hoʻohui ʻana i 0.5 ml o ka hopena sodium hydroxide (NaOH; 0.75 M). Ua ana ʻia ka absorbance (A600) o ka hui hopena ma 600 nm me ka hoʻohana ʻana i ka spectrophotometer UV-160 (Shimadzu Corporation, Iapana). Ua hoʻohana ʻia ka PDB a me ka wai i hoʻoheheʻe ʻia ma ke ʻano he mau kaohi no ka helu ʻana o nā kānana moʻomeheu a me nā laʻana mea kanu, kēlā me kēia. ʻO nā ʻano waikawa oxalic i loko o nā kānana moʻomeheu, i hōʻike ʻia ma ke ʻano he micrograms o ka waikawa oxalic no ka milliliter o ka PDB medium (μg.mL−1), a ma nā ʻāpana lau, i hōʻike ʻia ma ke ʻano he micrograms o ka waikawa oxalic no ka gram o ke kaumaha hou (μg.g−1 FW), ua hoʻoholo ʻia me ka hoʻohana ʻana i kahi piʻo hoʻoponopono waikawa oxalic (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
Ma loko o ke aʻo ʻana, ua hoʻolālā ʻia nā hoʻokolohua āpau ma kahi hoʻolālā randomized piha (CRD) me ʻeono mau replicates biological no kēlā me kēia lāʻau lapaʻau a me ʻelima mau ipu no kēlā me kēia replicate biological (ʻelua mau mea kanu no kēlā me kēia ipu) ke ʻole i ʻōlelo ʻia. Ua kālailai ʻia nā replicates biological ma ka pālua (ʻelua mau replicates loea). Ua hoʻohana ʻia nā replicates loea e nānā i ka hana hou ʻana o ka hoʻokolohua like akā ʻaʻole i hoʻohana ʻia i ka loiloi helu e pale aku i nā replicates spurious. Ua kālailai ʻia nā ʻikepili ma ka helu me ka hoʻohana ʻana i ka loiloi o ka variance (ANOVA) a ukali ʻia e ka hoʻāʻo ʻokoʻa koʻikoʻi ʻo Tukey-Kramer (HSD) (p ≤ 0.05). No nā hoʻokolohua in vitro, ua helu ʻia nā waiwai IC50 a me IC99 me ka hoʻohana ʻana i ke kumu hoʻohālike probit a ua helu ʻia nā manawa hilinaʻi 95%.
Ua hōʻiliʻili ʻia he ʻehā mau isolates mai nā mahina ʻai soybean like ʻole ma ke Kiaʻāina ʻo El Ghabiya, ʻAigupita. Ma ka medium PDA, ua hoʻopuka nā isolates āpau i ka mycelium keʻokeʻo creamy i hoʻololi koke i ke keʻokeʻo pulupulu (Kiʻi 1A) a laila beige a brown paha ma ke kahua sclerotium. ʻO ka maʻamau, he ʻeleʻele, poepoe a ʻano ʻē paha ke ʻano o Sclerotia, 5.2 a 7.7 mm ka lōʻihi a me 3.4 a 5.3 mm ke anawaena (Kiʻi 1B). ʻOiai ua hoʻomohala ʻehā mau isolates i kahi ʻano marginal o sclerotia ma ka lihi o ka medium culture ma hope o 10-12 mau lā o ka incubation ma 25 ± 2 °C (Kiʻi 1A), ʻokoʻa loa ka helu o sclerotia no kēlā me kēia pā ma waena o lākou (P < 0.001), me ka isolate 3 ka helu kiʻekiʻe loa o sclerotia (32.33 ± 1.53 sclerotia no kēlā me kēia pā; Kiʻi 1C). Pēlā nō, ua hoʻopuka ka isolate #3 i ka waikawa oxalic i loko o PDB ma mua o nā isolates ʻē aʻe (3.33 ± 0.49 μg.mL−1; Fig. 1D). Ua hōʻike ʻo Isolate #3 i nā ʻano morphological a me microscopic maʻamau o ka fungus phytopathogenic Sclerotinia sclerotiorum. No ka laʻana, ma PDA, ua ulu wikiwiki nā kolone o ka isolate #3, he keʻokeʻo creamy (Kiʻi 1A), beige reverse a i ʻole salmon māmā melemele-brown, a pono 6-7 mau lā ma 25 ± 2°C e uhi piha i ka ʻili o kahi papa 9 cm ke anawaena. Ma muli o nā ʻano morphological a me microscopic i luna, ua ʻike ʻia ka isolate #3 ʻo Sclerotinia sclerotiorum.
Kiʻi 1. Nā ʻano a me ka pathogenicity o nā isolates S. sclerotiorum mai nā mea kanu legume maʻamau. (A) Ka ulu ʻana o ka mycelial o ʻehā mau isolates S. sclerotiorum ma ka PDA medium, (B) sclerotia o ʻehā mau isolates S. sclerotiorum, (C) ka helu o ka sclerotia (no kēlā me kēia pā), (D) ka hoʻokuʻu ʻana o ka waikawa oxalic ma ka PDB medium (μg.mL−1), a me (E) ke koʻikoʻi o ka maʻi (%) o ʻehā mau isolates S. sclerotiorum ma ka cultivar legume kalepa susceptible Giza 3 ma lalo o nā kūlana hale ʻōmaʻomaʻo. Hōʻike nā waiwai i ka awelika ± SD o ʻelima mau replicates biological (n = 5). Hōʻike nā leka like ʻole i nā ʻokoʻa koʻikoʻi ma waena o nā lāʻau lapaʻau (p < 0.05). (F–H) Ua ʻike ʻia nā hōʻailona ʻōpala keʻokeʻo maʻamau ma nā kumu a me nā siliques ma luna o ka honua, kēlā me kēia, 10 mau lā ma hope o ka inoculation me ka isolate #3 (dpi). (I) Ua hana ʻia ka loiloi hoʻomohala o ka ʻāpana internal transcribed spacer (ITS) o ka S. sclerotiorum isolate #3 me ka hoʻohana ʻana i ke ʻano likelihood kiʻekiʻe loa a ua hoʻohālikelike ʻia me 20 mau isolates/strains kuhikuhi i loaʻa mai ka waihona ʻikepili National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). Hōʻike nā helu ma luna o nā laina clustering i ka uhi ʻana o ka ʻāpana (%), a hōʻike nā helu ma lalo o nā laina clustering i ka lōʻihi o ka lālā.
Eia kekahi, no ka hōʻoia ʻana i ka pathogenicity, ua hoʻohana ʻia ʻehā mau isolates S. sclerotiorum i loaʻa e hoʻokomo i ka cultivar pī kalepa ʻo Giza 3 ma lalo o nā kūlana hale kōmahana, kahi e kūlike me nā postulates a Koch (Kiʻi 1E). ʻOiai ʻo nā isolates fungal i loaʻa he pathogenic a hiki ke hoʻopili i ka pī ʻōmaʻomaʻo (cv. Giza 3), e hana ana i nā hōʻailona ʻōpala keʻokeʻo maʻamau ma nā ʻāpana āpau ma luna o ka honua (Kiʻi 1F), ʻoi aku hoʻi ma nā kumu (Kiʻi 1G) a me nā pods (Kiʻi 1H) i 10 mau lā ma hope o ka inoculation (dpi), ʻo ka isolate 3 ka isolate ikaika loa i ʻelua mau hoʻokolohua kūʻokoʻa. ʻO ka Isolate 3 ka mea i loaʻa ka maʻi koʻikoʻi kiʻekiʻe loa (%) ma nā mea kanu pī (24.0 ± 4.0, 58.0 ± 2.0, a me 76.7 ± 3.1 ma 7, 14, a me 21 mau lā ma hope o ka maʻi, kēlā me kēia; Kiʻi 1F).
Ua hōʻoia hou ʻia ka ʻike ʻana o ka isolate S. sclerotiorum invasive loa #3 ma muli o ka internal transcribed spacer (ITS) sequencing (Kiʻi 1I). Ua hōʻike ka loiloi phylogenetic ma waena o ka isolate #3 a me 20 reference isolates/strains i ke ʻano like kiʻekiʻe (>99%) ma waena o lākou. He mea kūpono ke hoʻomaopopo ʻana he like kiʻekiʻe ka isolate S. sclerotiorum #3 (533 bp) me ka isolate ʻAmelika S. sclerotiorum LPM36 i hoʻokaʻawale ʻia mai nā hua pea maloʻo (GenBank accession number MK896659.1; 540 bp) a me ka isolate Kina S. sclerotiorum YKY211 (GenBank accession number OR206374.1; 548 bp), ka mea e hoʻoulu ai i ka pala o ke kumu violet (Matthiola incana), a ua hui pū ʻia nā mea a pau ma ka piko o ka dendrogram (Kiʻi 1I). Ua waiho ʻia ke kaʻina hou i loko o ka waihona ʻikepili NCBI a ua kapa ʻia ʻo "Sclerotinia sclerotiorum - isolate YN-25" (GenBank accession number PV202792). Hiki ke ʻike ʻia ʻo ka isolate 3 ka isolate invasive loa; no laila, ua koho ʻia kēia isolate no ke aʻo ʻana i nā hoʻokolohua ma hope.
Ua noiʻi ʻia ka hana antibacterial o ka diamine L-ornithine (Sigma-Aldrich, Darmstadt, Kelemānia) ma nā ʻano like ʻole (12.5, 25, 50, 75, 100 a me 125 mg/L) e kūʻē i ka S. sclerotiorum isolate 3 ma vitro. He mea kūpono ia e hoʻomaopopo ʻia ua hoʻokō ʻo L-ornithine i kahi hopena antibacterial a kāohi mālie i ka ulu radial o S. sclerotiorum hyphae ma ke ʻano hilinaʻi i ka lāʻau (Kiʻi 2A, B). Ma ka nui kiʻekiʻe loa i hoʻāʻo ʻia (125 mg/L), ua hōʻike ʻo L-ornithine i ka nui kiʻekiʻe loa o ka mycelial growth inhibition rate (99.62 ± 0.27%; Kiʻi 2B), i like me ka fungicide kalepa ʻo Rizolex-T (ka nui inhibition 99.45 ± 0.39%; Kiʻi 2C) ma ka nui kiʻekiʻe loa i hoʻāʻo ʻia (10 mg/L), e hōʻike ana i ka pono like.
Kiʻi 2. Hana antibacterial in vitro o L-ornithine e kūʻē iā Sclerotinia sclerotiorum. (A) Hoʻohālikelike o ka hana antibacterial o nā ʻano like ʻole o L-ornithine e kūʻē iā S. sclerotiorum me ka fungicide kalepa ʻo Rizolex-T (10 mg/L). (B, C) Ka helu pale (%) o ka ulu ʻana o ka mycelial S. sclerotiorum ma hope o ka mālama ʻana me nā ʻano like ʻole o L-ornithine (12.5, 25, 50, 75, 100 a me 125 mg/L) a i ʻole Rizolex-T (2, 4, 6, 8 a me 10 mg/L), kēlā me kēia. Hōʻike nā waiwai i ka awelika ± SD o ʻelima mau replicates biological (n = 5). Hōʻike nā leka like ʻole i nā ʻokoʻa helu ma waena o nā mālama ʻana (p < 0.05). (D, E) Ka loiloi regression kumu hoʻohālike Probit o L-ornithine a me ka fungicide kalepa ʻo Rizolex-T, kēlā me kēia. Ua hōʻike ʻia ka laina regression kumu hoʻohālike probit ma ke ʻano he laina polū paʻa, a ua hōʻike ʻia ka wā hilinaʻi (95%) ma ke ʻano he laina ʻulaʻula i kahakaha ʻia.
Eia kekahi, ua hana ʻia ka loiloi regression probit a ua hōʻike ʻia nā kiʻi like ma ka Papa 1 a me nā Kiʻi 2D, E. I ka pōkole, ʻo ka waiwai pali i ʻae ʻia (y = 2.92x − 4.67) a me nā helu helu koʻikoʻi e pili ana (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 a me p < 0.0001; Kiʻi 2D) o L-ornithine i hōʻike i kahi hana antifungal i hoʻonui ʻia e kūʻē iā S. sclerotiorum i hoʻohālikelike ʻia me ka fungicide kalepa ʻo Rizolex-T (y = 1.96x − 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 a me p < 0.0001) (Papa 1).
Papa 1. Nā waiwai o ka hapalua o ka palena kiʻekiʻe o ka inhibitory concentration (IC50) a me IC99 (mg/l) o L-ornithine a me ka fungicide kalepa "Rizolex-T" e kūʻē iā S. sclerotiorum.
Ma keʻano holoʻokoʻa, ua hōʻemi nui ka L-ornithine (250 mg/L) i ka ulu ʻana a me ke koʻikoʻi o ka ʻōpala keʻokeʻo ma nā mea kanu pīni maʻamau i mālama ʻia i hoʻohālikelike ʻia me nā mea kanu i loaʻa i ka maʻi S. sclerotiorum i mālama ʻole ʻia (mana; Kiʻi 3A). I ​​ka pōkole, ʻoiai ua piʻi mālie ka koʻikoʻi o ka maʻi o nā mea kanu kaohi i loaʻa i ka maʻi i mālama ʻole ʻia (52.67 ± 1.53, 83.21 ± 2.61, a me 92.33 ± 3.06%), ua hōʻemi nui ka L-ornithine i ke koʻikoʻi o ka maʻi (%) ma o ka hoʻokolohua (8.97 ± 0.15, 18.00 ± 1.00, a me 26.36 ± 3.07) ma 7, 14, a me 21 mau lā ma hope o ka mālama ʻana (dpt), kēlā me kēia (Kiʻi 3A). Pēlā nō, i ka wā i mālama ʻia ai nā mea kanu pī i loaʻa i ka maʻi S. sclerotiorum me 250 mg/L L-ornithine, ua emi ka ʻāpana ma lalo o ke piʻo holomua o ka maʻi (AUDPC) mai 1274.33 ± 33.13 ma ka mana i mālama ʻole ʻia a i 281.03 ± 7.95, ʻoi aku ka haʻahaʻa ma mua o ka mana maikaʻi 50 mg/L Rizolex-T fungicide (183.61 ± 7.71; Fig. 3B). Ua ʻike ʻia ke ʻano like ma ka hoʻokolohua ʻelua.
Kiʻi 3. Hopena o ka hoʻopili ʻana o L-ornithine ma waho i ka ulu ʻana o ka pala keʻokeʻo o ka pī maʻamau i hoʻokumu ʻia e Sclerotinia sclerotiorum ma lalo o nā kūlana hale kōmahana. (A) Piʻo holomua o ka maʻi o ka ʻōpala keʻokeʻo o ka pī maʻamau ma hope o ka mālama ʻana me 250 mg/L L-ornithine. (B) ʻĀpana ma lalo o ke piʻo holomua o ka maʻi (AUDPC) o ka ʻōpala keʻokeʻo o ka pī maʻamau ma hope o ka mālama ʻana me L-ornithine. Hōʻike nā waiwai i ka awelika ± SD o ʻelima mau replicates biological (n = 5). Hōʻike nā leka like ʻole i nā ʻokoʻa koʻikoʻi ma waena o nā mālama ʻana (p < 0.05).
Ua hoʻonui mālie ka hoʻopili ʻana o 250 mg/L L-ornithine ma waho i ke kiʻekiʻe o ka mea kanu (Kiʻi 4A), ka helu o nā lālā no kēlā me kēia mea kanu (Kiʻi 4B), a me ka helu o nā lau no kēlā me kēia mea kanu (Kiʻi 4C) ma hope o 42 mau lā. ʻOiai ʻo ka fungicide kalepa ʻo Rizolex-T (50 mg/L) ka hopena nui loa ma nā ʻano meaʻai āpau i aʻo ʻia, ʻo ka hoʻopili ʻana o 250 mg/L L-ornithine ma waho ka lua o ka hopena nui loa i hoʻohālikelike ʻia me nā kaohi i mālama ʻole ʻia (Kiʻi 4A–C). Ma ka ʻaoʻao ʻē aʻe, ʻaʻohe hopena koʻikoʻi o ka mālama ʻana o L-ornithine i ka nui o nā pigments photosynthetic chlorophyll a (Kiʻi 4D) a me chlorophyll b (Kiʻi 4E), akā ua hoʻonui iki i ka nui o ka carotenoid (0.56 ± 0.03 mg/g fr wt) i hoʻohālikelike ʻia me ka kaohi maikaʻi ʻole (0.44 ± 0.02 mg/g fr wt) a me ka kaohi maikaʻi (0.46 ± 0.02 mg/g fr wt; Kiʻi 4F). Ma keʻano holoʻokoʻa, hōʻike kēia mau hopena ʻaʻole phytotoxic ka L-ornithine i nā legumes i mālama ʻia a hiki ke hoʻoulu i ko lākou ulu ʻana.
Kiʻi 4. Hopena o ka noi ʻana o ka L-ornithine exogenous ma nā ʻano ulu a me nā pigments photosynthetic o nā lau pī i hoʻopilikia ʻia me Sclerotinia sclerotiorum ma lalo o nā kūlana hale kōmahana. (A) Kiʻekiʻe o ka mea kanu (cm), (B) Helu o nā lālā no kēlā me kēia mea kanu, (C) Helu o nā lau no kēlā me kēia mea kanu, (D) ʻIke Chlorophyll a (mg g-1 fr wt), (E) ʻIke Chlorophyll b (mg g-1 fr wt), (F) ʻIke carotenoid holoʻokoʻa (mg g-1 fr wt). ʻO nā waiwai ka awelika ± SD o ʻelima mau replicates biological (n = 5). Hōʻike nā leka like ʻole i nā ʻokoʻa koʻikoʻi ma waena o nā lāʻau lapaʻau (p < 0.05).
Ua hōʻike ʻia ka localization histochemical in situ o nā ʻano oxygen reactive (ROS; i hōʻike ʻia e like me ka hydrogen peroxide [H2O2]) a me nā radical manuahi (i hōʻike ʻia e like me nā anions superoxide [O2•−]) ua hōʻemi nui ka hoʻopili ʻana o L-ornithine (250 mg/L) i ka hōʻiliʻili ʻana o H2O2 (96.05 ± 5.33 nmol.g−1 FW; Fig. 5A) a me O2•− (32.69 ± 8.56 nmol.g−1 FW; Fig. 5B) i hoʻohālikelike ʻia me ka hōʻiliʻili ʻana o nā mea kanu i loaʻa i ka maʻi i mālama ʻole ʻia (173.31 ± 12.06 a me 149.35 ± 7.94 nmol.g−1 FW, kēlā me kēia) a me nā mea kanu i mālama ʻia me 50 mg/L o ka fungicide kalepa ʻo Rizolex-T (170.12 ± 9.50 a me 157.00 ± 7.81 nmol.g−1 fr wt, kēlā me kēia) ma 72 h. Ua hōʻiliʻili ʻia nā pae kiʻekiʻe o H2O2 a me O2•− ma lalo o hpt (Kiʻi 5A, B). Pēlā nō, ua hōʻike ka hoʻāʻo malondialdehyde (MDA) ma muli o TCA ua hōʻiliʻili nā mea kanu pīni i loaʻa i ka maʻi S. sclerotiorum i nā pae kiʻekiʻe o MDA (113.48 ± 10.02 nmol.g fr wt) i loko o ko lākou mau lau (Kiʻi 5C). Eia naʻe, ua hōʻemi nui ka hoʻopili ʻana o L-ornithine ma waho i ka lipid peroxidation e like me ka hōʻike ʻana o ka emi ʻana o ka nui o MDA i nā mea kanu i mālama ʻia (33.08 ± 4.00 nmol.g fr wt).
Kiʻi 5. Hopena o ka hoʻopili ʻana o ka L-ornithine exogenous ma nā māka nui o ke kaumaha oxidative a me nā ʻano pale antioxidant non-enzymatic i loko o nā lau pī i hoʻopilikia ʻia e S. sclerotiorum ma 72 hola ma hope o ka maʻi ma lalo o nā kūlana hale ʻōmaʻomaʻo. (A) Hydrogen peroxide (H2O2; nmol g−1 FW) ma 72 hpt, (B) superoxide anion (O2•−; nmol g−1 FW) ma 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) ma 72 hpt, (D) nā phenols soluble āpau (mg GAE g−1 FW) ma 72 hpt, (E) nā flavonoids soluble āpau (mg RE g−1 FW) ma 72 hpt, (F) nā waikawa amino manuahi āpau (mg g−1 FW) ma 72 hpt, a me (G) ka nui o ka proline (mg g−1 FW) ma 72 hpt. Hōʻike nā waiwai i ka awelika ± ka ʻokoʻa maʻamau (awelika ± SD) o 5 mau replicates biological (n = 5). Hōʻike nā leka like ʻole i nā ʻokoʻa koʻikoʻi ma waena o nā lāʻau lapaʻau (p < 0.05).