He article hi research topic “Legumes te natna hrik leh rulhut laka invenna tihchangtlun” tih a ni a, article 5 te hi en vek rawh
Fungal plant disease necrosis thlentu agent Sclerotinia sclerotiorum (Lib.) de Bary hian multi-tiered strategy hmangin host plant hrang hrang a kai thin. He zirchianna hian Pseudomonas sclerotiorum vanga Phaseolus vulgaris L.-in white mold a neih avanga a molecular, physiological leh biochemical response tihchak nan management strategy dang atan non-protein amino acid, amino acid pawimawh dangte siam chhuahna tichaktu diamine L-ornithine hman a rawt a ni. In vitro experiment-ah chuan L-ornithine hian S. pyrenoidosa mycelial growth chu dose-dependent takin nasa takin a titawp tih hmuhchhuah a ni. Chubakah, greenhouse condition hnuaia white mold natna nasa takin a tihziaawm thei bawk. Chubakah, L-ornithine hian thlai enkawl tawhte chu a tipung a, hei hian L-ornithine concentration test tawhte chu enkawl tawhte tan phytotoxic a ni lo tih a tilang bawk. Hei bakah hian L-ornithine hian non-enzymatic antioxidants (total soluble phenolics leh flavonoids) leh enzymatic antioxidants (catalase (CAT), peroxidase (POX), leh polyphenol oxidase (PPO) te expression a tichak a, antioxidant nena inzawm gene pathum (PvCAT1, PvSOD, leh PvGR) expression a tipung bawk. Chubakah, in silico analysis-ah chuan S. sclerotiorum genome-ah hian putative oxaloacetate acetylhydrolase (SsOAH) protein a awm tih hmuhchhuah a ni a, hei hi Aspergillus fijiensis (AfOAH) leh Penicillium sp. (PLOAH) chu functional analysis, conserved domains, leh topology lam hawiin a hmang a ni. Ngaihven awm tak chu, potato dextrose broth (PDB) medium-a L-ornithine dah belh hian S. sclerotiorum mycelia-a SsOAH gene expression nasa takin a tihhniam a ni. Chutiang bawkin, L-ornithine exogenous application hian treated plant atanga fungal mycelia lakkhawmah SsOAH gene expression nasa takin a tihhniam bawk. A tawp berah chuan L-ornithine hman hian PDB medium leh infected leaves-ah te oxalic acid secretion nasa takin a ti tlem a ni. Thutawp atan chuan L-ornithine hian redox status vawng reng bakah hri kai thlai te defense response tihchakna kawngah pawh hmun pawimawh tak a chang a ni. He zirchianna result hian white mold control nan leh bean production leh thlai dang a nghawng dan tihziaawmna tur kawng thar, boruak tichhe lo siam chhuahna kawngah a pui thei ang.
White mold, necrotrophic fungus Sclerotinia sclerotiorum (Lib.) de Bary avanga lo awm hi natna rapthlak tak, rah chhuah tlem thei tak a ni a, khawvel pum huapa bean (Phaseolus vulgaris L.) siamchhuahna atana hlauhawm tak a ni (Bolton et al., 2006). Sclerotinia sclerotiorum hi leia fungal plant pathogens control harsa ber pawl a ni a, host range zau tak, thlai chi hrang hrang 600 chuang a nei a, host tissue te chu non-specific takin rang taka macerate theihna a nei bawk (Liang and Rollins, 2018). Thil awmdan \ha lo tak hnuaiah chuan a nun kawng pawimawh tak a paltlang a, leia ‘sclerotia’ an tih, chi dum, khauh, chi ang chi emaw, hri kai thlai mycelium emaw stem pith-a var, fluffy growth angin hun rei tak chhung a muhil a ni (Schwartz et al., 2005). S. sclerotiorum hian sclerotia a siam thei a, hei hian natna kai field-ah hun rei tak a dam thei a, natna a awm laiin a awm reng thei bawk (Schwartz et al., 2005). Sclerotia hi ei tur tha tak tak a ni a, leia rei tak a awm thei a, a hnu lama natna hrik kai tur inoculum hmasa ber a ni (Schwartz et al., 2005). Thil awm dan tha takah chuan sclerotia te hi an lo chhuak a, boruak atanga spore an siam chhuak a, chu chuan lei chunglam hmun zawng zawngah a kai thei a, pangpar, hnah emaw, a hnah te pawh a huam tel lo (Schwartz et al., 2005).
Sclerotinia sclerotiorum hian a host plant te hi a kai theih nan multi-tiered strategy a hmang a, sclerotial germination atanga symptom development thlengin thil thleng inzawmkhawm hrang hrang a awm a ni. A tirah chuan S. sclerotiorum hian mushroom ang chi apothecia an tih atang hian suspended spore (ascospores an tih) a siam chhuak a, chu chu boruakah a lo chang a, natna vei thlai bawlhhlawh chungah non-motile sclerotia-ah a lo chang ta a ni (Bolton et al., 2006). Chumi hnuah chuan fungus hian oxalic acid, virulence factor a tichhuak a, chu chuan plant cell wall pH a control a, enzymatic degradation leh tissue invasion a tichak a (Hegedus and Rimmer, 2005), host plant oxidative burst a titawp bawk. He acidification process hian plant cell wall a tichak lo a, fungal cell wall degrading enzymes (CWDEs) te hnathawh dan pangngai leh tha taka hman theihna tur boruak tha tak a siam a, chu chuan natna hrik chu taksa inkharkhip chu hnehin host tissue chhungah a lut thei a ni (Marciano et al., 1983). S. sclerotiorum hian a luh hnuah CWDE engemaw zat a tichhuak a, chungte chu polygalacturonase leh cellulase te an ni a, chungte chuan natna hrik kai tissue-ah a darh awlsam a, tissue necrosis a thlen bawk. Lesions leh hyphal mats te hmasawnna hian white mold characteristic symptoms a thlen thin (Hegedus and Rimmer, 2005). Hetihlai hian host plant te hian pattern recognition receptor (PRRs) hmangin pathogen-associated molecular patterns (PAMPs) an hre thei a, hei hian signaling event hrang hrang a tichhuak a, a tawpah chuan defense response a tichak ta a ni.
Kum sawm tam tak chhung natna dona kawnga hmalakna awm mah se, bean-ah pawh, sumdawnna atana thlai dang ang bawkin, natna hrik a do theihna, a dam khawchhuah theihna leh a insiamrem theihna avang hian germplasm do thei tur tling tak a tlachham reng a ni. Chuvangin natna enkawl hi a harsa hle a, culture hrang hrang, biological control, leh chemical fungicides te inzawmkhawm, integrated, multifaceted strategy a mamawh a ni (O’Sullivan et al., 2021). White mold chemical control hi a hlawk ber a, a chhan chu fungicides hi a dik leh a hun taka hman a nih chuan natna darh zel tur chu tha takin a control thei a, infection nasatzia a ti tlem thei a, yield loss a ti tlem thei bawk. Mahse, fungicides hman tam lutuk leh rinchhan lutuk hian S. sclerotiorum chi hrang hrang do thei lo a lo chhuak thei a, non-target organisms, lei hriselna, leh tui quality te a nghawng tha lo thei a ni (Le Cointe et al., 2016; Ceresini et al., 2024). Chuvangin, boruak tichhe lo tur kawng dang zawn chu thil pawimawh ber a lo ni ta a ni.
Polyamines (PAs), putrescine, spermidine, spermine, leh cadaverine te hi leia thlai natna hrik laka thil dang beisei awm tak angin a thawk thei a, chu chuan chemical fungicide hlauhawm tak tak hmanna chu a ti tlem vek emaw, a then emaw a ti tlem thei a ni (Nehela et al., 2024; Yi et al., 2024). Thlai sang zawkah chuan PA te hi physiological process tam takah an inrawlh a, chung zingah chuan cell inthen darh, inthliarna, leh abiotic leh biotic stress laka chhanna te pawh a tel (Killiny and Nehela, 2020). Antioxidant angin an thawk thei a, reactive oxygen species (ROS) scavenge-ah an pui thei a, redox homeostasis an vawng reng thei bawk (Nehela leh Killiny, 2023), defense-related genes an induce thei bawk (Romero et al., 2018), metabolic pathway hrang hrang an regulate thei bawk (Nehela and Killiny, 2023), endogenous phytohormones an modulate thei bawk (Nehela leh Killiny, 2023). 2019), systemic acquired resistance (SAR) an din a, plant-pathogen inzawmna an tidanglam bawk (Nehela leh Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). Hriat tur chu PA te hian thlai humhalhna kawnga an hnathawh dan leh an chanvo bik hi thlai chi hrang hrang, natna hrik, leh boruak dinhmun a zirin a inang lo hle. Thlaiah PA tam ber chu a pawimawh ber polyamine L-ornithine atanga biosynthesized a ni (Killiny and Nehela, 2020).
L-ornithine hian thlai than leh thanna kawngah hmun hrang hrang a chang a. Entirnan, zirchianna hmasaah chuan buhfai (Oryza sativa)-ah chuan ornithine hi nitrogen recycling (Liu et al., 2018), buh thar chhuah, quality leh aroma (Lu et al., 2020), leh tui stress response (Yang et al., 2000) nen a inzawm thei tih hmuhchhuah a ni. Chubakah, exogenous application of L-ornithine hian sugar beet (Beta vulgaris) (Hussein et al., 2019)-ah ruahtui tlak lohna a tichak hle a, onion (Allium Cepa) (Çavuşoǧlu leh Çavuşoǧlu, 2021) leh cashew (Anacardium occidentale)-ah pawh salt stress a tiziaawm bawk. thlai chi hrang hrang (da Rocha leh a thawhpuiten, 2012). Abiotic stress defense-a L-ornithine hian a chanvo a neih theih chhan chu treated plant-a proline accumulation-a a inrawlh vang a ni thei. Entirnan, proline metabolism nena inzawm genes, ornithine delta aminotransferase (delta-OAT) leh proline dehydrogenase (ProDH1 leh ProDH2) genes te hi a hmain Nicotiana benthamiana leh Arabidopsis thaliana te hi non-host Pseudomonas syringae strain (Senthil-Kumar leh Mysore, 2012-ah a ziak a ni). A lehlamah chuan natna hrik thanna atan fungal ornithine decarboxylase (ODC) a ngai a (Singh et al., 2020). Fusarium oxysporum-a ODC target-na f. sp. lycopersici via host-induced gene silencing (HIGS) hian tomato thingte chu Fusarium wilt laka an invenna chu nasa takin a tichak a ni (Singh et al., 2020). Mahse, exogenous ornithine application hian biotic stress, phytopathogens ang chi laka a chanvo awm thei hi zirchian a la ni lo. Chu aia pawimawh zawk chu, ornithine hian natna laka invenna a nghawng dan leh a kaihhnawih biochemical leh physiological phenomena te hi a tam zawk chu zirchian a la ni lo.
S. sclerotiorum infection of legumes hi a buaithlakzia hriatthiam hi control strategy tha tak siam nan a pawimawh hle. He zirchiannaah hian diamine L-ornithine hian Sclerotinia sclerotiorum infection laka legume plant-te defense mechanism leh resistance tihchakna atana thil pawimawh tak a nih theih dan tur hriatchhuah kan tum a ni. Kan hypothesis chu, natna vei thlai te defense response tihchak bakah hian L-ornithine hian redox status vawng reng turin hmun pawimawh tak a chang bawk. L-ornithine hian nghawng a neih theih dan hi enzymatic leh non-enzymatic antioxidant defense mechanisms regulation leh fungal pathogenicity/virulence factors leh a kaihhnawih proteins te tihbuai nen a inzawm tih kan rawt a ni. Hetianga L-ornithine hnathawh dual functionality hian white mold-in nghawng a neih tihziaawmna tur leh common legume crops-te’n he fungal pathogen chak tak laka an do theihna tihpunna tur strategy nghet tak siamna atana candidate beisei awm tak a ni. Tun tuma kan zirchianna result hian white mold control nan leh legume production-a a nghawng tihziaawmna tur kawng thar, boruak tichhe lo siam chhuahna kawngah a pui thei ang.
He zirchiannaah hian common bean chi, sumdawnna atana hlauhawm tak, Giza 3 (Phaseolus vulgaris L. cv. Giza 3) chu experimental material atan hman a ni. Thlai hrisel tak tak chu Legume Research Department, Field Crops Research Institute (FCRI), Agricultural Research Center (ARC), Egypt ten khawngaihin an pe a ni. Greenhouse condition (25 ± 2 °C, relative humidity 75 ± 1%, 8 h light/16 h dark) hnuaiah plastic pot (inner diameter 35 cm, depth 50 cm) S. sclerotiorum-in a kai leia khat)-ah thlai chi panga phun a ni. Thlai chin atanga ni 7–10 (DPS)-ah chuan thlai chi chu tihthianghlim a ni a, chu chuan chi hnih chauh, a thang inang tlang leh hnah zau tak pathum chauh chu bawm khatah a awm ta a ni. Pot-a dah zawng zawng chu kar hnih danah vawi khat tui pek vek a ni a, thla tin a chi pek zat atana ruahman rate-in fertilized pek vek a ni.
L-ornithinediamine ((+)-(S)-2,5-diaminopentanoic acid tia hriat bawk; Sigma-Aldrich, Darmstadt, Germany) 500 mg/L concentration siam tur chuan 50 mg chu sterile distilled water 100 mL-ah a hmin hmasa a. Chumi hnuah stock solution chu diluted a ni a, a hnua experiment neihnaah hman a ni. A tawi zawngin, L-ornithine concentration series paruk (12.5, 25, 50, 75, 100, leh 125 mg/L) chu in vitro-ah test a ni. Hei bakah hian sterile distilled water chu negative control (Mock) atan hman a ni a, sumdawnna atana hman tur fungicide “Rizolex-T” 50% wettable powder (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Egypt) chu positive control atan hman a ni bawk. Commercial fungicide “Rizolex-T” chu in vitro-ah concentration panga (2, 4, 6, 8 leh 10 mg/L)-ah test a ni.
Commercial farm atanga common bean stem leh pods, white mold symptoms tlangpui (infestation rate: 10–30%) nei sample lakkhawm a ni. Thlai chi hrang hrang hri kai tam zawk hi chi/chi hrang hrang (susceptible commercial variety Giza 3) hmanga hriat chhuah ni mah se, a dangte, a bik takin tualchhung market atanga lak chhuah te chu chi hriat loh a ni. Infected materials lakkhawmte chu 0.5% sodium hypochlorite solution hmangin minute 3 chhung surface disinfected hmasak a ni a, chutah chuan sterile distilled water hmangin vawi tam tak silfai a ni a, sterile filter paper hmangin wiped dry a ni a, tui tam lutuk chu paih chhuah a ni. Chumi hnuah chuan natna vei taksa pengte chu middle tissue (tissue hrisel leh hri kai inkar) atangin a te tein an tan a, potato dextrose agar (PDA) medium-ah culture a ni a, 25 ± 2 °C-ah 12 h light/12 h dark cycle-ah ni 5 chhung incubate a ni a, sclerotia siam a tichak a ni. Mycelial tip method hmang hian fungal isolates te chu mixed emaw contaminated culture atanga tihthianghlim nan an hmang bawk. Fungal isolate thianghlim chu a culture morphological characteristics atanga hriatchhuah hmasak a ni a, chumi hnuah microscopic features hmangin S. sclerotiorum a nih thu finfiah a ni. A tawpah chuan purified isolates zawng zawng chu Koch-a postulates zawm turin susceptible common bean cultivar Giza 3-ah pathogenicity test vek a ni.
Chu bakah, White et al., 1990-in an sawi angin internal transcribed spacer (ITS) sequencing hmangin S. sclerotiorum isolate (isolate #3) invasive ber chu an nemnghet lehzual a; Baturo-Ciesniewska et al., 2017. A tawi zawngin, isolates te chu potato dextrose broth (PDB) ah culture a ni a, 25 ± 2 °C ah ni 5–7 chhung an incubate a. Chumi hnuah fungal mycelium chu an la khawm a, cheesecloth hmangin an filter a, sterile water hmangin vawi hnih an silfai a, sterile filter paper hmangin an vawt bawk. Genomic DNA chu Quick-DNATM Fungal/Bacterial Miniprep Kit hmangin an la chhuak a (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Chumi hnuah ITS rDNA region chu specific primer pair ITS1/ITS4 (TCCGTAGGTGAACCTGCGG TCCTCCGCTTATTGATATGC; beisei zat: 540 bp) hmangin amplified a ni ( Baturo-Ciesniewska et al., 2017 ). PCR thil tihthianghlim tawhte chu sequencing (Beijing Aoke Dingsheng Biotechnology Co., Ltd.) atan an thehlut a ni. ITS rDNA sequence te chu Sanger sequencing method hmangin bidirectional-in an sequence a. Chumi hnuah chuan assembled query sequences te chu BLASTn software hmangin GenBank leh National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/)-a data thar ber ber nen khaikhin a ni. Query sequence hi NCBI GenBank (Supplementary Table S1)-a data thar ber atanga S. sclerotiorum strain/isolate dang 20 lakchhuah nen Molecular Evolutionary Genetics Analysis Package (MEGA-11; version 11)-a ClustalW hmangin an khaikhin a (Kumar et al., 2024). Evolutionary analysis chu maximum likelihood method leh general time-reversible nucleotide substitution model hmangin an ti a (Nei leh Kumar, 2000). Log-likelihood sang ber thing chu a lang. Heuristic search atana initial tree chu neighbor-joining (NJ) tree (Kumar et al., 2024) leh maximum parsimony (MP) tree inkara log-likelihood sang zawk nei thing thlan atanga thlan a ni. NJ thing hi general time-reversible model hmanga chhut pairwise distance matrix hmanga siam a ni (Nei and Kumar, 2000).
L-ornithine leh bactericide “Rizolex-T” te antibacterial activity chu in vitro ah agar diffusion method hmangin an zirchiang a. Thiltih dan: L-ornithine stock solution (500 mg/L) a tling tawk la la, PDA nutrient medium 10 ml nen uluk takin chawhpawlh la, final concentration 12.5, 25, 50, 75, 100 leh 125 mg/L nei solution siam rawh. Control atan fungicide “Rizolex-T” (2, 4, 6, 8 leh 10 mg/L) leh sterile distilled water concentration panga hman a ni. Medium chu a solidified hnuah Sclerotinia sclerotiorum culture mycelial plug siam thar, 4 mm diameter chu Petri dish lai takah dahin 25±2°C-ah mycelium-in control Petri dish pumpui a khuh thlengin culture a ni a, chumi hnuah fungal thanna chu record a ni. Equation 1 hmangin S. sclerotiorum radial growth percentage inhibition chu chhut rawh:
Experiment hi vawi hnih tih leh a ni a, control/experimental group tin tan biological replicate paruk leh biological replicate tin tan pot panga (pot khatah plant pahnih) dah a ni. Biological replicate tin hi vawi hnih (technical replicate pahnih) an zirchiang a, chu chuan experimental result dik tak, rintlak leh siam chhuah theih a nih leh nih loh enfiah a ni. Chu bakah, probit regression analysis hmangin half-maximal inhibitory concentration (IC50) leh IC99 te chu chhut a ni bawk (Prentice, 1976).
Greenhouse condition hnuaia L-ornithine thiltihtheihna tehna atan pot experiment vawi hnih a zawnin neih a ni. A tawi zawngin, pot-te chu sterilized clay-sand soil (3:1)-in an khat a, S. sclerotiorum culture siam thar nen an inoculate a. A hmasa berin S. sclerotiorum isolate invasive ber (isolate #3) chu sclerotium pakhat chu a chanveah then a, PDA-ah hmai hnuai lam dahin, 25°C-ah ni 4 chhung thim reng reng (24 h) chhung incubate-in mycelial growth tichak turin culture a ni. Chumi hnuah a hma lam atanga 5 mm diameter agar plug pali lakchhuah niin, buhfai leh buhfai chi (1:1, v/v) sterile mixture 100 g-in inoculate a ni a, flask zawng zawng chu 25 ± 2 °C-ah 12 h light/12 h dark cycle hnuaiah ni 5 chhung incubate a ni a, sclerotia siam chhuah a tichak a ni. Flask zawng zawng chhunga thil awmte chu lei dah hmain homogeneity a awm theih nan uluk takin an pawlh vek a ni. Tichuan, colonizing bran mixture 100 g chu pot tinah dahin, natna hrik awm zat a awm reng theih nan. Inoculated pot te chu fungal growth tichak turin tui pek a ni a, greenhouse condition-ah ni 7 chhung dah a ni.
Chumi hnuah Giza 3 chi panga chu bêl khatah an tuh a. L-ornithine leh fungicide Rizolex-T hmanga enkawl bawmte tan chuan thlai sterilized chu compound pahnih tui solution-ah darkar hnih chhung dah hmasak a ni a, a tawp berah chuan IC99 concentration chu 250 mg/L leh 50 mg/L vel a ni a, chutah chuan thlai chin hmain darkar khat chhung air-dried a ni. A lehlamah chuan a chi chu negative control atan sterile distilled water-ah an hnim a. Ni 10 hnuah tui pek hmasak ber hmain thlai chi chu an thim chhuak a, bêl khatah thlai chi thianghlim pahnih chauh a awm a ni. Tin, S. sclerotiorum kai theih nan, bean plant stems te chu a lo thanglian chhoh dan inang (ni 10) ah hmun hrang hrang pahnih ah sterilized scalpel hmangin an tan a, hliam tinah colonizing bran mixture 0.5 g vel dah a ni a, chu chu inoculated plant zawng zawngah infection leh natna lo awm tur tichak turin humidity sang takin dah a ni bawk. Control plant-te pawh chutiang bawkin hliam tuar an ni a, hliam chhungah hian sterile, uncolonized bran mixture zat inang (0.5 g) dahin, natna lo awm theihna tur boruak simulate nan leh treatment group hrang hrangte inmilna tur atan humidity sang takah dah a ni.
Treatment method: Bean seedlings chu L-ornithine (250 mg/l) emaw fungicide Rizolex-T (50 mg/l) emaw aqueous solution 500 ml hmangin leiah tui pek a ni a, chutah chuan ni 10 inkar ah vawi thum enkawl leh a ni. Placebo hmanga enkawl control te chu sterile distilled water 500 ml hmanga tui pek an ni. Treatment zawng zawng hi greenhouse condition (25 ± 2°C, 75 ± 1% relative humidity, leh photoperiod 8 h light/16 h dark) hnuaiah an ti vek a ni. Pot zawng zawng chu kar hnih danah tui pek vek a ni a, thla tin balanced NPK fertilizer (20-20-20, 3.6% sulfur leh TE microelements awmna; Zain Seeds, Egypt) hmangin 3–4 g/l concentration-ah foliar spraying hmangin a chi bik atana rawtna leh siamtute thupek angin enkawl a ni. A danglamna a awm loh chuan, biological replicate tin a\angin fully expanded mature leaves (2nd leh 3rd leaves from top) chu 72 h post-treatment (hpt)-ah lakkhawm a ni a, homogenized, pooled leh -80 °C-ah dah a ni a, analysis dang neih belh a ni a, chung zingah chuan in situ histochemical localization of oxidative stress indicators, lipid peroxidation, enzymatic leh non-enzymatic te pawh a tel a ni antioxidants leh gene expression te a awm bawk.
White mold infection intensity hi inoculation hnu ni 21 (dpi) chhungin kar tin scale 1–9 (Supplementary Table S2) hmangin Teran et al.-te siam danglam Petzoldt and Dickson scale (1996) hmanga teh a ni. (2006) a ni. A tawi zawngin, bean thing hnah leh a hnah te chu inoculation point atanga tan in internode leh node-a lesions kal zel dan zawm turin an enfiah a. Chumi hnuah chuan inoculation point atanga stem emaw branch emaw a hla ber thlenga lesion hlat zawng chu teh a ni a, lesion awmna hmun a zirin score 1–9 pek a ni a, chutah chuan (1) inoculation point bulah infection hmuh theih a awm lo tih a tarlang a, (2–9) chuan lesion size a pung zauh zauh tih a tarlang a, nodes/internodes zawh zawngin a kal zel tih a tarlang bawk (Supplementary Table S2). Chumi hnuah chuan white mold infection intensity chu formula 2 hmangin percentage-ah an chantir a:
Chu bakah, natna kal zel tur curve (AUDPC) hnuaia area chu formula (Shaner and Finney, 1977) hmangin chhut a ni a, tun hnaiah common bean white rot atan (Chauhan et al., 2020) equation 3 hmangin an siam danglam a ni:
Yi = hun ti-a natna nasat dan, Yi+1 = a hun leh ti+1-a natna nasat dan, ti = tehna hmasa ber hun (ni-a), ti+1 = tehna leh hun (ni-a), n = hun bi emaw enfiahna hmun zawng zawng zat. Bean plant growth parameters te chu plant san zawng (cm), thlai khata branch awm zat, leh thlai khata hnah awm zat te chu kar tin ni 21 chhung biological replicate zawng zawngah record vek a ni.
Biological replicate tinah hian hnah sample (a chung atanga hnah puitling pahnihna leh pathumna) chu enkawl hnu ni 45-ah (enkawl hnuhnung ber atanga ni 15) ah lakkhawm a ni. Biological replicate tin hi pot panga (pot khatah thlai pahnih) a awm a. Tissue chi hrang hrang 500 mg vel chu 80% acetone hmangin 4 °C-ah thimah hmangin photosynthetic pigment (chlorophyll a, chlorophyll b leh carotenoids) lakchhuah nan hman a ni. 24 h hnuah sample te chu centrifuge-in supernatant chu wavelength hrang hrang pathum-a absorbance tehna hmangin UV-160A spectrophotometer (Shimadzu Corporation, Japan) hmangin colorimetrically-in chlorophyll a, chlorophyll b leh carotenoid awm zat hriat nan an la khawm a ni (A470, A646 leh A663 nm) te an ni. A tawp berah chuan photosynthetic pigment awm zat chu a hnuaia formula 4–6 Lichtenthaler (1987)-a sawi hmangin chhut a ni.
72 h post-treatment (hpt)-ah biological replicate tin atangin hnah (a chung atanga hnah puitling pahnihna leh pathumna) chu in situ histochemical localization of hydrogen peroxide (H2O2) leh superoxide anion (O2•−) atan lakkhawm a ni. Biological replicate tin hi pot panga (pot khatah thlai pahnih) a awm a. Biological replicate tinte chu duplicate (technical replicate pahnih)-in an zirchiang a, chu chu a dikna, rintlak leh siam chhuah theih a nih leh nih loh enfiah a ni. H2O2 leh O2•− te chu 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Germany) emaw nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Germany) hmangin an chhut a, Romero-Puertas et al. (2004) leh Adam leh a thawhpuiten an ziak a ni. (1989) te chuan siamthatna tenau te an nei bawk. H2O2 in situ histochemical localization atan leaflet te chu 10 mM Tris buffer (pH 7.8) ah 0.1% DAB hmangin vacuum infiltrated a ni a, chutah chuan room temperature-ah light-ah 60 min incubate a ni. Leaflet te chu 0.15% (v/v) TCA in 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) ah bleach a ni a, chutah chuan a thim thlengin eng hnuaiah dah a ni. Chutiang bawkin valve te chu O2•− in situ histochemical localization atan 0.1 w/v % HBT awmna 10 mM potassium phosphate buffer (pH 7.8) hmangin vacuum infiltrated an ni. Leaflet te chu room temperature-ah light-ah min 20 chhung an incubate a, a chunga kan sawi ang khan bleach an ni a, chutah chuan dark blue/violet spot a lo lang thlengin an eng a ni. A rah chhuah brown (H2O2 indicator angin) emaw blue-violet (O2•− indicator angin) rawng intensity chu image processing package ImageJ (http://fiji.sc; accessed 7 March 2024) Fiji version hmangin an zirchiang a ni.
Du leh Bramlage (1992) te tih dan angin malondialdehyde (MDA; lipid peroxidation chhinchhiahna angin) chu siam danglam tlem te nen an chhut a. Biological replicate tin atanga hnah (a chung atanga hnah puitling pahnihna leh pathumna) chu 72 h post-treatment (hpt) ah lakkhawm a ni. Biological replicate pakhatah hian pot panga (pot khatah thlai pahnih) a awm a. Biological replicate tinte chu duplicate (technical replicate pahnih)-in an zirchiang a, chu chu a dikna, rintlak leh siam chhuah theih a nih leh nih loh enfiah a ni. A tawi zawngin, ground leaf tissue 0.5 g chu MDA lakchhuah nan 20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA) awmna hmangin hman a ni. Chumi hnuah supernatant-a MDA awm zat chu UV-160A spectrophotometer (Shimadzu Corporation, Japan) hmangin 532 leh 600 nm-a absorbance tehna hmangin colorimetrically-in an chhut a, chutah chuan nmol g−1 FW angin an tarlang a ni.
Non-enzymatic leh enzymatic antioxidants tehna atan chuan biological replicate tin atang hian hnah (a chung atanga hnah lo piang chhuak pahnihna leh pathumna) chu 72 h post-treatment (hpt) ah lakkhawm a ni. Biological replicate tin hi pot panga (pot khatah thlai pahnih) a awm a. Biological sample tin chu duplicate-in (technical sample pahnih) an zirchiang a. A hnah pahnih chu liquid nitrogen hmangin an hrual a, enzymatic leh non-enzymatic antioxidants, total amino acids, proline content, gene expression, leh oxalate quantification te hriat nan direct-in an hmang a ni.
Total soluble phenolics chu Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) hmangin Kahkonen et al. (1999) a ni. A tawi zawngin, homogenized leaf tissue 0.1 g vel chu 20 ml 80% methanol hmangin thimah 24 h chhung lakchhuah a ni a, supernatant chu centrifugation hnuah lakkhawm a ni. Sample extract 0.1 ml chu 0.5 ml Folin-Ciocalteu reagent (10%) nen chawhpawlh a, 30 s chhung vawt a, thimah min 5 chhung dah a ni. Tichuan tube tinah 20% sodium carbonate solution (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) 0.5 ml dahin, uluk takin an pawlh a, room temperature-ah thimah 1 h chhung an incubate a. Incubation hnuah UV-160A spectrophotometer (Shimadzu Corporation, Japan) hmangin 765 nm-ah reaction mixture absorbance an teh a. Sample extract-a total soluble phenols concentration chu gallic acid calibration curve (Fisher Scientific, Hampton, NH, USA) hmangin an chhut a, gram khatah gallic acid equivalent milligrams (mg GAE g-1 fresh weight) angin an tarlang a ni.
Total soluble flavonoid content chu Djeridane et al. (2006) te chuan siamthatna tlem te te nen. A tawi zawngin, a chunga kan sawi tak methanol extract 0.3 ml chu 5% aluminum chloride solution (AlCl3; Fisher Scientific, Hampton, NH, USA) 0.3 ml nen chawhpawlh a ni a, chak takin kan hrual a, chutah chuan room temperature-ah min 5 chhung dah a ni a, chumi hnuah 10% potassium acetate solution (Al-Gomhoria) 0.3 ml dah a ni Pharmaceuticals and Medical Supplies, Cairo, Egypt), uluk taka pawlh a, room temperature-ah thimah min 30 chhung dah a ni. Incubation hnuah UV-160A spectrophotometer (Shimadzu Corporation, Japan) hmangin 430 nm-ah reaction mixture absorbance an teh a. Sample extract-a total soluble flavonoids concentration chu rutin calibration curve (TCI America, Portland, OR, USA) hmangin an chhut a, chutah chuan gram khatah rutin equivalent milligrams (mg RE g-1 fresh weight) angin an tarlang a ni.
Bean hnah a free amino acid awm zawng zawng chu Yokoyama leh Hiramatsu (2003) te rawtna hmanga siam danglam ninhydrin reagent (Thermo Scientific Chemicals, Waltham, MA, USA) hmangin an chhut a, Sun et al. (2006) a ni. A tawi zawngin, ground tissue 0.1 g chu pH 5.4 buffer hmangin lakchhuah a ni a, supernatant 200 μL chu ninhydrin 200 μL (2%) leh pyridine 200 μL (10%; Spectrum Chemical, New Brunswick, NJ, USA) nen reacted a ni a, tui lum bath-ah min 30 chhung incubate a ni a, chutah chuan a lum a, chutah chuan teh a ni 580 nm-ah UV-160A spectrophotometer (Shimadzu Corporation, Japan) hmangin a tlan thei a ni. A lehlamah chuan proline chu Bates method hmangin an chhut a (Bates et al., 1973). Proline chu 3% sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) hmangin lakchhuah a ni a, centrifugation hnuah supernatant 0.5 ml chu 1 ml glacial acetic acid (Fisher Scientific, Hampton, NH, USA) leh ninhydrin reagent nen chawhpawlh a ni a, 90°C-ah min 45 chhung incubate a ni a, a lum a, a teh a ni 520 nm-ah a chunga kan sawi ang chiah spectrophotometer hmangin a tlan thei. Leaf extracts-a free amino acid leh proline zawng zawng chu glycine leh proline calibration curve (Sigma-Aldrich, St Louis, MO, USA) hmangin an chhut a, mg/g fresh weight angin an tarlang bawk.
Antioxidant enzymes enzymatic activity hriat theih nan homogenized tissue 500 mg vel chu 50 mM Tris buffer (pH 7.8) 3 ml 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) leh 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, 100) awmna hmangin lakchhuah a ni. USA), 10,000 × g-ah refrigeration (4 °C) hnuaiah min 20 chhung centrifuge-in, a chunglam (crude enzyme extract) chu an la khawm a (El-Nagar et al., 2023; Osman et al., 2023). Chumi hnuah Catalase (CAT) chu 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) 2 ml leh 269 mM H2O2 solution 100 μl nen Aebi (1984)-a tih dan angin a enzymatic activity hriat theih nan siam danglam tlem (El-Nagar et al., 2023; Osman leh a thawhpuiten 2023). Guaiacol-dependent peroxidase (POX) enzymatic activity chu Harrach et al. (2009) a ni. (2008) te chuan siamthatna tenau (El-Nagar et al., 2023; Osman et al., 2023) hmangin polyphenol oxidase (PPO) enzymatic activity chu 100 mM sodium phosphate buffer (pH 6.0) 2.2 ml, guaiacol 100 μl (TCI chemicals, Portland, OR, 2023) nena an inrem hnuah an zirchiang a ni. USA) leh 12 mM H2O2 100 μl a ni. A hman dan chu (El-Nagar et al., 2023; Osman et al., 2023) atang hian tlem a siam danglam a ni. Assay hi 0.1 M phosphate buffer (pH 6.0)-a siam thar catechol solution (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) 3 ml hmanga reaction hnuah tih a ni. CAT activity chu H2O2 decomposition 240 nm (A240)-a enfiahin an teh a, POX activity chu 436 nm-a absorbance tihpun dan enfiahin (A436) an teh a, PPO activity chu UV-160A hmangin 30 s danah 495 nm (A495)-ah absorbance fluctuation record-in min 3 chhung teh a ni spectrophotometer (Shimadzu, Japan)-ah a awm a ni.
Real-time RT-PCR hmangin antioxidant nena inzawm gene pathum transcript level an hmuchhuak a, chung zingah chuan peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), leh glutathione reductase (PvGR; GenBank Accession No. KY195009.1), bean hnah (a chung atanga hnah puitling pahnihna leh pathumna)-ah enkawl hnuhnung ber atanga darkar 72-ah. A tawi zawngin, RNA chu siamtu protocol angin Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) hmangin an isolate a. Tichuan, siamtu thupek angin TOP scriptTM cDNA Synthesis Kit hmangin cDNA siam a ni. A chunga gene pathumte primer sequence chu Supplementary Table S3-ah tarlan a ni. Housekeeping gene atan PvActin-3 (GenBank accession number: XM_068616709.1) hman a ni a, relative gene expression chu 2-ΔΔCT method hmangin chhut a ni (Livak and Schmittgen, 2001). Biotic stress hnuaia actin stability (common legumes leh anthracnose fungus Colletotrichum lindemuthianum inkara inzawmna inmil lo) leh abiotic stress (drought, salinity, low temperature) te chu hmuhchhuah a ni (Borges et al., 2012).
A tirah chuan protein-protein BLAST tool (BLASTp 2.15.0+) hmangin S. sclerotiorum-a oxaloacetate acetylhydrolase (OAH) proteins te genome-wide in silico analysis kan nei a (Altschul et al., 1997, 2005). A tawi zawngin, Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; GenBank accession number XP_040799428.1; 342 amino acids) leh Penicillium lagena (PlOAH; taxide: 94218; GenBank accession number) atanga OAH kan hmang a XP_056833920.1; BLASTp hi National Center for Biotechnology Information (NCBI) website, http://www.ncbi.nlm.nih.gov/gene/-a GenBank-a S. sclerotiorum genome data hmuh chhuah hnuhnung berte nen an khaikhin a ni.
Chu bakah, S. sclerotiorum (SsOAH) atanga predicted OAH gene leh A. fijiensis CBS 313.89 atanga AfOAH evolutionary analysis leh phylogenetic tree leh P. lagena atanga PlOAH te chu MEGA11-a maximum likelihood method hmangin (Tamura et al., 2021) leh JTT matrix-based model (Jones et..) hmangin infer a ni bawk al., 1992-ah a ziak bawk). Phylogenetic tree hi S. sclerotiorum atanga predicted OAH genes (SsOAH) zawng zawng protein sequence multiple alignment analysis leh Constraint-Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos leh Agarwala, 2007 a ni). Chu bakah, S. sclerotiorum atanga SsOAH amino acid sequence inmil ber chu ClustalW (http://www.genome.jp/tools-bin/clustalw) hmangin query sequence (AfOAH leh PlOAH) (Larkin et al., 2007) nen align a ni a, alignment-a conserved region te chu ESPript tool (version 3.0 A rilru a hah lutuk chuan a rilru a buai em em a.
Chubakah, S. sclerotiorum SsOAH predicted functional representative domains leh conserved sites te chu InterPro tool (https://www.ebi.ac.uk/interpro/) hmangin chhungkaw hrang hrangah interactive takin an thliar hrang a (Blum et al., 2021). A tawp berah chuan S. sclerotiorum SsOAH sawi lawk tawhte chu Protein Homology/Analogy Recognition Engine (Phyre2 server version 2.0; http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) hmangin three-dimensional (3D) structure modeling an ti a (Kelley et al., 2015) hmangin an validate a ni SWISS-MODEL server (https://swissmodel.expasy.org/) hmanga siam a ni (Biasini leh a thawhpuiten, 2014). Three-dimensional structures (PDB format) sawi lawk te chu UCSF-Chimera package (version 1.15; https://www.cgl.ucsf.edu/chimera/ ) hmangin interactive takin an entir a (Pettersen et al., 2004).
Sclerotinia sclerotiorum mycelia-a oxaloacetate acetylhydrolase (SsOAH; GenBank accession number: XM_001590428.1) transcriptional level hriat nan quantitative real-time fluorescence PCR hman a ni. A tawi zawngin, S. sclerotiorum chu PDB awmna flask-ah inoculate a ni a, shaking incubator (model: I2400, New Brunswick Scientific Co., Edison, NJ, USA)-ah 25 ± 2 °C-ah 24 h chhung 150 rpm-ah leh thim reng reng (24 h)-ah mycelial growth tichak turin dah a ni. Chumi hnuah chuan cell te chu L-ornithine leh fungicide Rizolex-T hmangin final IC50 concentration (40 leh 3.2 mg/L vel, a hnuaia mi ang hian)-ah treat a ni a, chumi hnuah condition ang chiah hnuaiah 24 h dang culture leh a ni. Incubation hnuah cultures te chu 2500 rpm ah min 5 chhung centrifuged a ni a, supernatant (fungal mycelium) chu gene expression analysis atan lakkhawm a ni. Chutiang bawkin, hrik kai hnu darkar 0, 24, 48, 72, 96, leh 120-ah fungal mycelium chu hri kai tissue chunglama white mold leh cottony mycelium siam tawh atanga lakkhawm a ni. Fungal mycelium atang hian RNA lakchhuah a ni a, chutah chuan a chunga kan sawi tawh ang khan cDNA siam a ni. SsOAH atana primer sequence te chu Supplementary Table S3-ah tarlan a ni. Housekeeping gene atan SsActin (GenBank accession number: XM_001589919.1) hman a ni a, relative gene expression chu 2-ΔΔCT method hmangin chhut a ni (Livak and Schmittgen, 2001).
Xu leh Zhang (2000) te tih dan angin potato dextrose broth (PDB) leh thlai sample-a fungal pathogen Sclerotinia sclerotiorum awmnaah Oxalic acid chu Xu leh Zhang (2000) te tih dan angin siam danglam tlem a ni. A tawi zawngin, S. sclerotiorum isolates te chu PDB awmna flask-ah inoculate a ni a, chu chu shaking incubator (model I2400, New Brunswick Scientific Co., Edison, NJ, USA)-ah 150 rpm-ah 25 ± 2 °C-ah ni 3–5 chhung thim reng reng (24 h)-ah mycelial growth tichak turin culture a ni. Incubation hnuah fungal culture chu Whatman #1 filter paper hmangin filter hmasain 2500 rpm-ah min 5 chhung centrifuged a ni a, residual mycelium chu paih chhuah a ni. Supernatant chu an la khawm a, 4°C-ah dahin oxalate quantitative determination dang neih belh a ni. Thlai sample siam nan hian thlai tissue fragment 0.1 g vel chu distilled water hmangin vawi thum (a vawi khatah 2 ml) lakchhuah a ni. Chumi hnuah sample te chu 2500 rpm-ah min 5 chhung centrifuged a ni a, supernatant chu Whatman No. 1 filter paper hmangin dry filtered niin, analysis dang neih belh turin an la khawm a ni.
Oxalic acid quantitative analysis atan chuan reaction mixture chu glass stoppered tube-ah hetiang hian siam a ni: sample 0.2 ml (or PDB culture filtrate emaw oxalic acid standard solution), bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA) 0.11 ml, sulfuric acid 1 M 0.198 ml (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) leh 100 mM potassium dichromate (K2Cr2O7; TCI chemicals, Portland, OR, USA) 0.176 ml te dah a ni a, chutah chuan distilled water hmangin 4.8 ml-ah tihthianghlim a ni a, chak takin a pawlh a, tui 60 °C-ah dah nghal a ni inbual. Min 10 hnuah sodium hydroxide solution (NaOH; 0.75 M) 0.5 ml dahin reaction chu tihtawp a ni. Reaction mixture absorbance (A600) chu UV-160 spectrophotometer (Shimadzu Corporation, Japan) hmangin 600 nm-ah an teh a. Culture filtrate leh plant sample quantification atan PDB leh distilled water te chu control atan hman a ni. Culture filtrate-a oxalic acid concentration, PDB medium milliliter khata oxalic acid microgram (μg.mL−1) anga tarlan leh, leaf extract-a oxalic acid microgram gram khata fresh weight (μg.g−1 FW) anga tarlan te chu oxalic acid calibration curve hmangin an chhut a (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
He zirchianna chhung zawng hian experiment zawng zawng hi completely randomized design (CRD)-a design a ni a, treatment khatah biological replicate paruk leh biological replicate khatah pot panga (pot khatah plant pahnih) a awm a, a danglamna a awm loh chuan. Biological replicate te chu duplicate-in (technical replicate pahnih) an zirchiang a. Technical replicate hmangin experiment khata reproducibility enfiah a ni a, mahse statistical analysis-ah chuan spurious replicate awm loh nan hman a ni lo. Data hi analysis of variance (ANOVA) hmangin statistically an zirchiang a, chumi hnuah Tukey-Kramer honestly significant difference (HSD) test (p ≤ 0.05) hmangin an zirchiang leh a ni. In vitro experiment atan chuan probit model hmangin IC50 leh IC99 value te chu chhut a ni a, 95% confidence interval te chu chhut a ni.
Egypt khawpui El Ghabiya Governorate-a soybean field hrang hrang aṭangin isolates pali lakkhawm a ni. PDA medium-ah chuan isolates zawng zawng hian creamy white mycelium an siam chhuak a, chu chu rang takin cottony white-ah a chang (Figure 1A) a, chutah chuan sclerotium stage-ah beige emaw brown emaw a lo ni ta a ni. Sclerotia hi a tlangpuiin a dense, dum, spherical emaw irregular emaw a ni a, 5.2 atanga 7.7 mm a sei leh 3.4 atanga 5.3 mm a sei a ni (Figure 1B). Isolates pali chuan ni 10–12 chhung 25 ± 2 °C-a an incubation hnuah culture medium sir lamah sclerotia marginal pattern nei mah se (Fig. 1A), plate khata sclerotia awm zat chu an zingah a danglam hle (P < 0.001), isolate 3-ah chuan sclerotia awm tam ber (32.33 ± 1.53 sclerotia per plate-ah hian a awm a; Chutiang bawkin isolate #3 hian isolate dang aiin PDB-ah oxalic acid a siam tam zawk (3.33 ± 0.49 μg.mL−1; Fig. 1D). Isolate #3 hian phytopathogenic fungus Sclerotinia sclerotiorum te hi a pianphung leh microscopic characteristic pangngai a lantir a. Entirnan, PDA-ah chuan isolate #3 colony chu a lo thang chak hle a, creamy white (Figure 1A), reverse beige emaw light salmon yellow-brown emaw a ni a, 9 cm diameter plate chunglam khuh vek turin 25 ± 2°C-ah ni 6-7 chhung a ngai a ni. A chunga kan sawi tak morphological leh microscopic characteristics te atanga chhut chuan isolate #3 chu Sclerotinia sclerotiorum a ni tih hmuhchhuah a ni.
Figure 1. Legume thlai chi hrang hrang atanga S. sclerotiorum isolates te mizia leh natna thlen thei. (A) PDA medium-a S. sclerotiorum isolates pali mycelial growth, (B) S. sclerotiorum isolates pali sclerotia, (C) sclerotia awm zat (plate khatah), (D) PDB medium-a oxalic acid secretion (μg.mL−1), leh (E) S. sclerotiorum isolates pali-a natna nasat dan (%), sumdawnna atana hman theih legume-a natna nasat dan (%) cultivar Giza 3 chu greenhouse dinhmun hnuaiah a awm. Value hian biological replicate panga (n = 5) mean ± SD a entir a ni. Letter hrang hrang hian enkawlna hrang hrangah statistically significant danglamna a tarlang (p < 0.05). (F–H) Isolate #3 (dpi) hmanga inoculation hnu ni 10 hnuah lei chunglam stem leh siliques-ah te typical white mold symptoms a lang a. (I) S. sclerotiorum isolate #3-a internal transcribed spacer (ITS) region evolutionary analysis chu maximum likelihood method hmangin an ti a, National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/) atanga reference isolates/strain 20 hmuhchhuah nen khaikhin a ni. Clustering line chunga number awmte hian region coverage (%) a tarlang a, clustering line hnuaia number awmte hian branch sei zawng a tarlang bawk.
Chubakah, natna thlentu a nihna finfiah nan S. sclerotiorum isolates pali hmuh chhuah hmangin greenhouse condition-ah sumdawnna atana hman tur bean chi khat Giza 3 chu inoculate a ni a, hei hi Koch-a postulate nen a inmil hle (Fig. 1E). Fungal isolates hmuh chhuah zawng zawng hi natna thlentu ni mah se, green bean (cv. Giza 3) chu kai thei a, lei chunglam hmun zawng zawngah (Fig. 1F), a bik takin stems (Fig. 1G) leh pods (Fig. 1H)-ah te inoculation hnu ni 10 (dpi)-ah typical white mold symptoms thlentu ni mah se, independent experiment pahnih-ah isolate 3 hi isolate nasa ber a ni. Isolate 3 hian bean thlaiah natna nasat ber (%) a nei a (24.0 ± 4.0, 58.0 ± 2.0, leh 76.7 ± 3.1 chu hri kai hnu ni 7, 14 leh 21-ah a ni a; Figure 1F).
S. sclerotiorum isolate #3 invasive ber hriatchhuahna chu internal transcribed spacer (ITS) sequencing hmangin an nemnghet lehzual a (Fig. 1I). Isolate #3 leh reference isolates/strain 20 te phylogenetic analysis-ah chuan an in ang lo hle (>99%). Hriat tur chu S. sclerotiorum isolate #3 (533 bp) hi American S. sclerotiorum isolate LPM36, dry pea chi atanga lakchhuah (GenBank accession number MK896659.1; 540 bp) leh Chinese S. sclerotiorum isolate YKY211 (GenBank accession number) nen hian a inang hle a ni OR206374.1;548 bp), chu chuan violet (Matthiola incana) stem rot a thlen a, chung zawng zawng chu dendrogram chung lamah a hranin group-ah dah vek a ni (Figure 1I). Sequence thar hi NCBI database-ah dah a ni a, a hmingah “Sclerotinia sclerotiorum – isolate YN-25” (GenBank accession number PV202792) tih a ni. Isolate 3 hi isolate invasive ber a ni tih a hriat theih a; chuvangin, he isolate hi a hnu lama experiment zawng zawnga zir chianna atan thlan a ni.
Diamine L-ornithine (Sigma-Aldrich, Darmstadt, Germany) concentration hrang hrang (12.5, 25, 50, 75, 100 leh 125 mg/L)-a S. sclerotiorum isolate 3 laka antibacterial activity chu in vitro-ah an zirchiang a. Hriat tur pawimawh tak chu L-ornithine hian antibacterial effect a nei a, zawi zawiin S. sclerotiorum hyphae radial growth chu dose-dependent takin a titawp a ni (Figure 2A, B). Concentration test sang ber (125 mg/L)-ah chuan L-ornithine hian mycelial growth inhibition rate sang ber (99.62 ± 0.27%; Figure 2B) a lantir a, hei hi commercial fungicide Rizolex-T (inhibition rate 99.45 ± 0.39%; Figure 2C) nen a inang a, concentration test sang ber (10 mg/L)-ah a inang tih a tilang a, hei hian a inang chiah chiah a ni a thatna (efficacy) a ni.
Figure 2. L-ornithine hian Sclerotinia sclerotiorum laka in vitro-a antibacterial activity a neih dan. (A) S. sclerotiorum laka L-ornithine concentration hrang hrangte antibacterial activity leh sumdawnna atana hman tur fungicide Rizolex-T (10 mg/L) nena khaikhin. (B, C) L-ornithine (12.5, 25, 50, 75, 100 leh 125 mg/L) emaw Rizolex-T (2, 4, 6, 8 leh 10 mg/L) emaw concentration hrang hrang hmanga enkawl hnua S. sclerotiorum mycelial growth inhibition rate (%). Value hian biological replicate panga (n = 5) mean ± SD a entir a ni. Letter hrang hrang hian enkawlna hrang hrang statistical difference a tarlang a (p < 0.05). (D, E) L-ornithine leh sumdawnna atana hman tur fungicide Rizolex-T te chu Probit model regression analysis hmangin an enfiah a. Probit model regression line chu solid blue line anga tarlan a ni a, confidence interval (95%) chu dashed red line anga tarlan a ni bawk.
Hei bakah hian probit regression analysis tih a ni a, a inmil plot te chu Table 1 leh Figure 2D,E-ah te tarlan a ni bawk. A tawi zawngin, L-ornithine-a slope value pawmtlak (y = 2.92x − 4.67) leh a kaihhnawih statistics pawimawh tak tak (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 leh p < 0.0001; Figure 2D) chuan sumdawnna atana hman tur fungicide nena khaikhin chuan S. sclerotiorum laka antifungal activity a tichak tih a tilang a ni Rizolex-T (y = 1.96x − 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 leh p < 0.0001) (Table 1) a ni.
Table 1. S. sclerotiorum laka L-ornithine leh sumdawnna atana hman tur fungicide “Rizolex-T” half-maximum inhibitory concentration (IC50) leh IC99 (mg/l) value te.
A pum puiin, L-ornithine (250 mg/L) hian S. sclerotiorum natna vei thlai enkawl lohte nena khaikhin chuan common bean thing enkawl tawhteah white mold lo awm leh a nasatzia chu nasa takin a tihtlem a ni (control; Figure 3A). A tawi zawngin, enkawl loh hri kai control plant-te natna nasatzia chu a pung zauh zauh (52.67 ± 1.53, 83.21 ± 2.61, leh 92.33 ± 3.06%) mah se, L-ornithine hian experiment chhung zawngin natna nasatzia chu nasa takin a tihhniam (%) (8.97 ± 0.15, 18.00 ± 1.00, leh 26.36 ± 3.07) a ni a, enkawl hnu ni 7, 14 leh 21 (dpt)-ah te a ni (Figure 3A). Chutiang bawkin S. sclerotiorum vei bean thingte chu 250 mg/L L-ornithine hmanga enkawl an nih chuan enkawl loh control-ah natna kal zel tur curve (AUDPC) hnuaia area chu 1274.33 ± 33.13 aṭangin 281.03 ± 7.95-ah a tlahniam a, hei hi positive control 50 mg/L aiin a hniam deuh hlek a ni Rizolex-T fungicide (183.61 ± 7.71; Fig. 3B) hmanga siam a ni. Experiment pahnihnaah pawh hetiang bawk hian a awm tih hmuhchhuah a ni.
Fig. 3. Greenhouse condition hnuaia Sclerotinia sclerotiorum vanga common bean white rot lo awm theihna tura L-ornithine exogenous application-in nghawng a neih dan. (A) Common bean white mold 250 mg/L L-ornithine hmanga enkawl hnua natna kal zel dan curve. (B) L-ornithine hmanga enkawl hnua common bean white mold natna zual zel (AUDPC) hnuaia area. Value hian biological replicate panga (n = 5) mean ± SD a entir a ni. Letter hrang hrang hian enkawlna hrang hrang statistically significant danglamna a tarlang a (p < 0.05).
Exogenous application of 250 mg/L L-ornithine hian ni 42 hnuah thlai san zawng (Fig. 4A), thlai pakhata hnah awm zat (Fig. 4B), leh thlai pakhata hnah awm zat (Fig. 4C) te chu a tisang zauh zauh a ni. Commercial fungicide Rizolex-T (50 mg/L) hian nutritional parameter zirchian zawng zawngah nghawng lian ber a neih laiin, 250 mg/L L-ornithine exogenous application chuan enkawl loh control-te nena khaikhin chuan nghawng lian ber pahnihna a nei thung (Figs. 4A–C). A lehlamah chuan L-ornithine hmanga enkawlna hian photosynthetic pigment chlorophyll a (Fig. 4D) leh chlorophyll b (Fig. 4E) te zingah nghawng lian tham a nei lo a, mahse negative control (0.44 ± 0.02 mg/g fr wt) leh positive control (0.46 ± 0.02 mg/g fr wt; Fig. 4F) a ni. A pum puiin heng results te hian L-ornithine hi treated legumes tan phytotoxic a ni lo tih a tilang a, an thanlenna pawh a tichak thei bawk.
Fig. 4. Greenhouse condition hnuaia Sclerotinia sclerotiorum kai bean hnah te thanlen dan leh photosynthetic pigment te exogenous L-ornithine hmannain a nghawng dan. (A) Thlai san zawng (cm), (B) Thlai pakhata hnah awm zat, (C) Thlai pakhata hnah awm zat, (D) Chlorophyll a awm zat (mg g-1 fr wt), (E) Chlorophyll b awm zat (mg g-1 fr wt), (F) Carotenoid awm zawng zawng (mg g-1 fr wt). Value te hi biological replicate panga (n = 5) te mean ± SD a ni. Letter hrang hrang hian enkawlna hrang hrangah statistically significant danglamna a tarlang (p < 0.05).
Reactive oxygen species (ROS; hydrogen peroxide [H2O2] anga tarlan) leh free radicals (superoxide anions [O2•−] anga tarlan) te in situ histochemical localization-ah chuan L-ornithine (250 mg/L) exogenous-a hman chuan H2O2 (96.05 ± 5.33 nmol.g−1 FW Fig. 5A) leh O2•− (32.69 ± 8.56 nmol.g−1 FW; Fig. 5B) te chu enkawl loh thlai pahnih (173.31 ± 12.06 leh 149.35 ± 7.94 nmol.g−1 FW, a hnuaia mi ang hian) leh sumdawnna atana hman tur fungicide 50 mg/L hmanga enkawl thlai te an punkhawm dan nen khaikhin a ni Rizolex-T (170.12 ± 9.50 leh 157.00 ± 7.81 nmol.g−1 fr wt, a hnuaia mi ang hian) chu 72 h. Hpt hnuaiah hian H2O2 leh O2•− level sang tak a awm khawm a (Fig. 5A, B). Chutiang bawkin TCA-based malondialdehyde (MDA) assay hmangin S. sclerotiorum kai bean thlai te chuan an hnah ah MDA level sang zawk (113.48 ± 10.02 nmol.g fr wt) an khawlkhawm tih hmuhchhuah a ni (Fig. 5C). Mahse, exogenous application of L-ornithine hian lipid peroxidation nasa takin a tihtlem a, hei hi treated plant-a MDA content tlahniam (33.08 ± 4.00 nmol.g fr wt) atanga a lan dan a ni.
Fig. 5. Greenhouse condition hnuaia S. sclerotiorum natna vei bean hnah-a oxidative stress leh non-enzymatic antioxidant defense mechanism chhinchhiahna lian ber berte chunga exogenous L-ornithine hmannain nghawng a neih dan. (A) Hydrogen peroxide (H2O2; nmol g−1 FW) 72 hpt-ah, (B) superoxide anion (O2•−; nmol g−1 FW) 72 hpt-ah, (C) malondialdehyde (MDA; nmol g−1 FW) 72 hpt-ah, (D) total soluble phenols (mg GAE g−1 FW) 72 hpt-ah, (E) 72 hpt-a soluble flavonoids zawng zawng (mg RE g−1 FW), (F) 72 hpt-a free amino acid zawng zawng (mg g−1 FW), leh (G) 72 hpt-a proline awm zat (mg g−1 FW) te. Value te hian biological replicate 5 (n = 5) te mean ± standard deviation (mean ± SD) a entir a ni. Letter hrang hrang hian enkawlna hrang hrangah statistically significant danglamna a tarlang (p < 0.05).