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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Improving pruning wound protection against grapevine trunk disease pathogens

Mutawila, Cheusi 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Grapevine trunk diseases are a cause of decline and loss of productivity in grapevines at all stages of growth. These diseases are caused by a complex of wood-inhabiting fungi that infect mainly through pruning wounds. The management of these diseases relies on wound protection to prevent infection since there are no eradicative control measures to cure infected vines. There are few or no fungicides registered for grapevine pruning wound protection in most countries, while Trichoderma biocontrol agents are often available. This study aimed at improving grapevine wound protection by Trichoderma (T.) spp. and to gain a better understanding of the factors and mechanisms involved in biocontrol. The effect of pruning time (early or late) and five timings of application of the biocontrol agent after pruning on pruning wound colonisation by T. atroviride and T. harzianum were determined. Chenin blanc and Cabernet Sauvignon vineyards were pruned in July (early) and August (late) of 2011 and 2012, and pruning wounds were treated with suspensions of the Trichoderma spp. at various times (0, 6, 24, 48 and 96 hours) after pruning. Wound colonisation was depended on the physiological state of the vine at pruning for both cultivars. However, for the 2012 season in Chenin blanc, wound colonisation was similarly high for both pruning times, which was attributed to high rainfall and humidity. Application of the biocontrol agents 6 hours after pruning consistently resulted in high wound colonisation by the Trichoderma spp. in both cultivars and pruning times. In both cultivars, pruning wound infection due to natural inoculum was higher in wounds made in late winter than those made earlier. The effect of conidial formulation in nutritional (glucose, yeast extract and urea) and bio-enhancing (chitin and cell free culture filtrates) additives, on pruning wound colonisation by T. atroviride was also investigated. Nutritional additives increased the extent of pruning wound colonisation by T. atroviride compared to the un-amended conidial suspensions in a glass house study. The additives as well as Garrison, a fungicide containing pruning wound paint, and Eco77®, a registered T. harzianum biocontrol product, were tested in field trials for wound protection from infection by Phaeomoniella (Pa.) chlamydospora. In 2011, the pathogen was inoculated a day after pruning and all the Trichoderma spp. treatments similarly reduced Pa. chlamydospora infection by 75% to 90% in Thompson Seedless, while control was less in Chenin blanc and ranged from 40% to 74%. In 2012, the trial was carried out on Chenin blanc only and the pathogen was inoculated at intervals of 1, 3 and 7 days after pruning. Wound protection by the Trichoderma treatments was highest when wounds were inoculated with Pa. chlamydospora seven days after pruning. Two conidial formulations, a culture filtrate made from a chitin based medium and a combination of yeast extract, urea and glucose, consistently enhanced biocontrol efficacy. These formulations reduced Pa. chlamydospora infection to levels similar to those of Garrison. The integration of chemical and biological wound protection could provide both immediate and long term wound protection, but is limited by the sensitivity of the biocontrol agent to fungicides. Benzimidazole resistant Trichoderma strains were generated by gamma irradiation from the wild type isolates of T. atroviride (UST1 and UST2) and T. harzianum (T77). Mutants from UST1 and UST2 were of similar biological fitness as the wild type isolates and retained their in vitro antagonistic activity against grapevine trunk pathogens, while the mutant from T77 had reduced fitness and was not antagonistic to the pathogens. The wild type, UST1, and its mutant were tested alone and in combination with thiophanate methyl and carbendazim, respectively, for their ability to prevent pruning wound infection by Pa. chlamydospora. The combination of the UST1 mutant and carbendazim was the most effective treatment and gave the highest reduction in Pa. chlamydospora infection (70% to 93% control). Grapevine cell cultures were used to compare the response of grapevines to T. atroviride and Eutypa (E.) lata as a first step to determining the importance of Trichoderma-grapevine interactions in pruning wound bio-protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis related (PR) proteins was profiled over a 48-hour period using quantitative reverse transcriptase PCR. The cell cultures responded to fungal elicitors in a hypersensitive-like response that lead to a decrease in cell viability. Fungal elicitors from both fungi triggered the same genes and caused up-regulation of phenylalanine ammonia-lyase (PAL), 4 coumaroyl Co-A ligase (CCo-A), stilbene synthase (STS), chitinase class IV (CHIT IV), PR 3 and PR 4, and a down regulation of chalcone synthase (CHS) genes. Higher expression of PAL and CHIT IV in cell cultures treated with the T. atroviride elicitor led to a significantly higher (P < 0.05) total phenolic content and chitinolytic enzyme activity of the cell cultures compared to cell cultures treated with the E. lata elicitor. The response of the cell cultures to the T. atroviride elicitor signifies that the induction of grapevine resistance may be involved in wound bio-protection. The role of secondary metabolites produced by Trichoderma spp. used in pruning wound protection was also investigated. A volatile antimicrobial compound, 6-pentyl α-pyrone (6PP), was isolated and found to be the major secondary metabolite from the T. atroviride (UST1 and UST2) and T. harzianum (T77) isolates. This metabolite was found to inhibit mycelial growth, spore and conidia germination of E. lata, Neofussicocum (N.) australe, N. parvum and Pa. chlamydospora. The production of 6PP was induced when the T. atroviride isolates were grown in a grapevine wood extract medium while for UST1, the 6PP concentration was further doubled when it was co-cultured with N. parvum. Results therefore, indicate that 6PP is involved in the Trichoderma-pathogen interactions on pruning wounds. The results of this study have provided new information in regards to the application of Trichoderma-based pruning wound products. The best time of application proved to be 6 hours post pruning. The formulation of conidial suspensions of Trichoderma spp. with nutritional additives and in protein extracts of the biocontrol agent showed potential in reducing variability of wound bio-protection. However, further research would be necessary to develop commercial products. The application of a fungicide together with Trichoderma spp. in the field holds promise to improve control, but would require further trials for possible commercialisation. This study is the first to report on grapevine host defence genes that are activated by the Trichoderma spp. used in pruning wound protection. Together with the characterisation of the major secondary metabolite produced by these Trichoderma spp., this information aids in understanding the mechanisms involved in the complex interaction between the biocontrol agent, the host and the pathogen. / AFRIKAANSE OPSOMMING: Wingerdstamsiektes veroorsaak terugsterwing en verlies aan produktiwiteit in wingerdstokke gedurende alle groeifases. Hierdie siektes word veroorsaak deur „n verskeidenheid van hout-koloniserende swamme wat die wingerdstok meestal deur snoeiwonde infekteer. Die bestuur van hierdie siektes is afhanklik van wondbeskerming om infeksie te verhoed, omdat daar geen uitwissende beheermetodes na infeksie bestaan nie. In meeste lande is daar min of geen swamdoders geregistreer vir snoeiwond beskerming, terwyl Trichoderma biobeheer agente gereëld beskikbaar is. Hierdie studie poog om wingerd wondbeskerming deur Trichoderma (T.) spp. te verbeter en „n meer volledige begrip van die faktore en meganismes betrokke by biologiese beheer te ontwikkel. Die effek van die tydsberekening van snoei (vroeg of laat) en vyf behandelingstye van die biobeheer agent na snoei op die kolonisering van snoeiwonde deur T. atroviride en T. harzianum is bepaal. Chenin blanc en Cabernet Sauvignon wingerde is gesnoei gedurende Julie (vroeg) en Augustus (laat) in 2011 en 2012, en snoeiwonde is behandel met Trichoderma spp. suspensies op verskillende tydspunte (0, 6, 24, 48 en 96 ure) na snoei. Wond-kolonisering was afhanklik van die fisiologiese toestand van die wingerdstok gedurende snoei vir albei kultivars. Gedurende die 2012 seisoen was wond-kolonisering ewe hoog vir albei snoeitye op Chenin blanc. Dit is verklaar deur hoë reënval en humiditeit gedurende daardie seisoen. Die aanwending van biobeheer agente 6 ure na snoei het konsekwent hoë kolonisering deur Trichoderma spp. tot gevolg gehad op albei kultivars en albei snoeitye. In albei kultivars is wondinfeksie as gevolg van natuurlike inokulum hoër gewees in wonde gemaak gedurende laat winter as in wonde wat vroeër in die seisoen gemaak is. Die effek van konidia formulasie in voeding (glukose, gisekstrak en urea) en bioverbetering (chitien en sel-vrye kultuurfiltraat) toevoegings op snoeiwond-kolonisering deur T. atroviride is ook ondersoek. Voeding toevoegings het die omvangs van snoeiwond-kolonisering deur T. atroviride vergroot in vergelyking met ongewysigde konidia suspensies gedurende „n glashuis studie. Die toevoegings, sowel as Garrison, „n snoeiwond verf wat „n swamdoder bevat, en Eco77®, „n geregistreerde T. harzianum biobeheer produk, is getoets in veldproewe vir wondbeskerming teen infeksie deur Phaeomoniella (Pa.) chlamydospora. In 2011 is die patogeen geïnokuleer „n dag na snoei en al die Trichoderma spp. behandelings het infeksie verminder met 75% tot 90% op Thompson Seedless. Beheer was minder suksesvol op Chenin blanc, waar slegs 40% tot 74% beheer behaal is. In 2012 is die proef uitgevoer slegs op Chenin blanc en die patogeen is geïnokuleer teen intervalle van 1, 3 en 7 dae na snoei. Wondbeskerming by die Trichoderma behandelinge was die hoogste wanneer wonde sewe dae na snoei geïnokuleer is met Pa. chlamydospora. Twee konidia formulasies, „n kultuurfiltraat wat bestaan het uit „n chitien-gebaseerde medium en „n kombinasie van gisekstrak, urea en glukose het deurlopend die effektiwiteit van biobeheer verbeter. Hierdie formulasies het Pa. chlamydospora infeksie verminder tot soortgelyke vlakke behaal deur Garrison. Die integrasie van chemiese- en biobeheer in wondbeskerming kan onmiddelike en langtermyn wondbeskerming bied, maar is beperk deur die sensitiwiteit van die biobeheer agent teen swamdoders. Benzimidazole-weerstandbiedende Trichoderma isolate is ontwikkel deur gamma-bestraling van die wilde-tipe isolate van T. atroviride (UST1 en UST2) en T. harzianum (T77). Mutante van UST1 en UST2 het soortgelyke biologiese fiksheid getoon as die wilde-tipe en het hul in vitro antagonistiese aktiwiteit teen wingerd stampatogene behou, terwyl die mutant van T77 verminderde fiksheid getoon het en nie meer antagonisties teen patogene was nie. Die wilde-tipe, UST1, en sy mutant is apart en in kombinasie met thiofanaatmetiel en carbendazim, respektiewelik, getoets vir die vermoë om snoeiwonde te beskerm teen Pa. chlamydospora. Die kombinasie van die UST1 mutant met carbendazim was die mees effektiewe behandeling en het die hoogste vermindering in Pa. chlamydospora infeksie gelewer (70 tot 93% beheer). As „n beginpunt om die belang van Trichoderma-wingerd interaksies in snoiewondbeheer te bepaal, is die invloed van T. atroviride en Eutypa (E.) lata op somatiese selkulture van wingerd vergelyk. Die effek van dié behandelings op ensieme in die fenielpropanoïedweg en patogenese-verwante (PR) proteïene is bepaal deur intydse PKR (real time PCR) van die korresponderende gene oor „n 48 uur tydperk. Die swam-afkomstige ontlokkers het „n hipersensitiewe-tipe reaksie in die selkulture ontlok, wat tot „n afname in sellewensvatbaarheid gelei het. Ontlokkers afkomstig van beide swamme het dieselfde gene aangeskakel en het induksie van fenielalanien ammoniak-liase (PAL), 4 kumaroïel Ko-A ligase (CCo-A), stilbeen sintase (STS), chitienase klas IV (CHIT IV), PR 3 en PR 4 veroorsaak en „n onderdrukking in chalkoon sintase (CHS) gene tot gevolg gehad. Hoër uitdrukking van PAL en CHIT IV in selkulture behandel met die T. atroviride ontlokker het gelei tot „n beduidende hoër (P < 0.05) totale fenoolinhoud en chitienolitiese aktiwiteit in selkulture in vergelyking met selkulture wat behandel is met die E. lata ontlokker. Die reaksie van die selkulture op die T. atroviride ontlokker dui daarop dat die induksie van wingerd weerstandbiedenheid betrokke mag wees in wond biobeheer. Die rol van sekondêre metaboliete geproduseer deur Trichoderma spp. wat gebruik word in snoeiwond beheer is ook ondersoek. „n Vlugtige antimikrobiese verbinding, 6-pentiel α-pyroon (6PP) is geïsoleer en bepaal om die hoof sekondêre metaboliet afkomstig vanuit die T. atroviride (UST1 en UST2) en T. harzianum (T77) isolate te wees. Hierdie metaboliet is betrokke by inhibisie van miselium groei, spoor en konidium ontkieming van E. lata, Neofusicoccum (N.) australe, N. parvum en Pa. chlamydospora. Die produksie van 6PP is geïnduseer deur die T. atroviride in wingerd hout ekstrak te kweek. In die geval van UST1, is die 6PP konsenstrasie verdubbel deur die isolaat met saam met N. parvum te kweek. Hierdie resultaat is „n aanduiding dat 6PP betrokke is in die Trichoderma-patogeen interaksie op snoeiwonde. Die resultate van hierdie studie het nuwe inligting met betrekking tot die aanwending van Trichoderma-gebaseerde snoeiwond produkte verskaf. Die beste tyd vir aanwending van sulke produkte was 6 ure na snoei. Die formulasie van konidia suspensies van Trichoderma spp. met voeding toevoegings en in proteïen ekstrakte van die biobeheer agent het potensiaal getoon in die vermindering van variasie in wondbeskerming deur biobeheer agente. Verdere navorsing sal nodig wees om kommersiële produkte te ontwikkel. Die aanwending van „n swamdoder saam met Trichoderma spp. in die wingerd is belowend om beheer te verbeter, maar het meer proewe nodig voor kommersialisering. Hierdie studie is die eerste om wingerd beskerming gene wat deur Trichoderma spp. geaktiveer word aan te meld. Laasgenoemde, saam met die beskrywing van die hoof sekondêre metaboliete wat deur hierdie Trichoderma spp. geproduseer word, dra by tot „n meer volledige begrip van die meganismes betrokke by die komplekse interaksie tussen die biobeheer agent, die gasheer en die patogeen.
2

Biological control of the grapevine trunk disease pathogens : pruning wound protection

Kotze, Charl 12 1900 (has links)
Thesis (MScAgric (Plant Pathology))--Stellenbosch University, 2008. / In recent years, several studies have conclusively shown that numerous pathogens, including several species in the Botryosphaeriaceae, Phomopsis, Phaeoacremonium, as well as Phaeomoniella chlamydospora and Eutypa lata, contribute to premature decline and dieback of grapevines. These pathogens have the ability to infect grapevines through pruning wounds, which leads to a wide range of symptoms developing that includes stunted growth, cankers and several types of wood necrosis. Pruning wounds stay susceptible for 2 to 16 weeks after pruning and sustained levels of pruning wound protection is therefore required. The aims of this study were to (i) evaluate the ability of several biological agents to protect pruning wounds, (ii) characterise unknown Trichoderma strains and identify their modes of action and (iii) determine the optimal time of season for biological agent application. Several biological agents were initially evaluated in a laboratory for their antagonism against trunk disease pathogens. The best performing control agents were tested in a field trial conducted on Merlot and Chenin blanc vines in the Stellenbosch region. Spurs were pruned to three buds and the fresh pruning wounds were treated with benomyl as a control treatment, Trichoderma-based commercial products, Vinevax® and Eco77®, Bacillus subtilis, and Trichoderma isolates, USPP-T1 and -T2. Seven days after treatment the pruning wounds were spray inoculated with spore suspensions of four Botryosphaeriaceae spp. (Neofusicoccum australe, N. parvum, Diplodia seriata and Lasiodiplodia theobromae), Eutypa lata, Phaeomoniella chlamydospora and Phomopsis viticola. After a period of 8 months the treatments were evaluated by isolations onto potato dextrose agar. Trichodermabased products and isolates in most cases showed equal or better efficacy than benomyl, especially USPP-T1 and -T2. Moreover, these isolates demonstrated a very good ability to colonise the wound tissue. The two uncharacterised Trichoderma isolates (USPP-T1 and USPP-T2), which were shown to be highly antagonistic toward the grapevine trunk disease pathogens, were identified by means of DNA comparison, and their ability to inhibit the mycelium growth of the trunk disease pathogens by means of volatile and non-volatile metabolite production studied. The two gene areas that were used include the internal transcribed spacers (ITS 1 and 2) and the 5.8S ribosomal RNA gene and the translation elongation factor 1 (EF). The ITS and EF sequences were aligned to published Trichoderma sequences and the percentage similarity determined and the two Trichoderma isolates were identified as Trichoderma atroviride. The volatile production of T. atroviride isolates was determined by placing an inverted Petri dish with Trichoderma on top of a dish with a pathogen isolate and then sealed with parafilm. Trichoderma isolates were grown for 2 days on PDA where after they were inverted over PDA plates containing mycelial plugs. The inhibition ranged from 23.6% for L. theobromae to 72.4% for P. viticola. Inhibition by non-volatile products was less than for the volatile inhibition. Inhibition ranged from 7.5% for N. parvum to 20.6% for L. theobromae. In the non-volatile inhibition USPP-T1 caused significantly more mycelial inhibition than USPP-T2. The timing of pruning wound treatment and subsequent penetration and colonisation of the wound site was also determined. One-year-old canes of the Shiraz and Chenin blanc cultivars were grown in a hydroponic system, pruned and spray treated with a spore suspension of Trichoderma atroviride (USPP-T1) as well as a fluorescent pigment. On intervals 1, 3, 5 and 7 days after treatment, the distal nodes were removed and dissected longitudinally. From the one half, isolations were made at various distances from the pruning surface, while the other half was observed under ultra-violet light to determine the depth of fluorescent pigment penetration. Shortly after spray-inoculation of a fresh pruning wound, Trichoderma was isolated only from the wound surface and shallow depths into the wound (2 to 5 mm). One week after inoculation, Trichoderma was isolated at 10 mm depths, and after 2 weeks, at 15 mm depths. Fluorescent pigment particles were observed to a mean depth of 6 mm, which suggests that initial isolation of Trichoderma at these depths was resultant of the physical deposition of conidia deeper into the pruning wound tissue, whereas the isolation of Trichoderma from deeper depths might be attributed to colonisation of grapevine tissue. In a vineyard trial, fluorescent pigment was spray-applied to pruning wounds of Shiraz and Chenin blanc grapevines during July and September at intervals 0, 1, 3, 7 and 14 days after pruning. One week after treatment, the distal nodes were removed and dissected longitudinally. Each half was observed under UV light and the pigment penetration measured. For Chenin blanc and Shiraz, July pruning wounds showed significant deeper penetration of the pigment than pruning wounds treated in September. Moreover, pruning wounds made in September showed pigment particles in longitudinal sections up to 1 day after pruning, whereas wounds made in July showed pigment particles up to 3 days in the xylem vessels. These findings suggest that the best time for application of a biological control agent should be within the first 24 hours after pruning.
3

The role of sucker wounds as portals for grapevine trunk pathogen infections

Makatini, Gugulethu Joy 04 1900 (has links)
Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Grapevine trunk diseases are responsible for reduced wine and table grape production world-wide. Trunk disease infections are caused by xylem-inhabiting pathogens which include species of Botryosphaeriaceae, Diatrypaceae, Hymenochaetales and Diaporthales, as well as Phaeomoniella chlamydospora and Phaeoacremonium spp. Winter pruning wounds are regarded as the main infection-sites for trunk disease pathogens. However, the role of sucker wounds as portals of trunk disease infections has been minimally investigated. Knowledge of the potential role of grapevine trunk pathogen infections that occur through sucker wounds is important for better wound protection strategies. The aim of this study was to determine the role of grapevine sucker wounds as portals of entry for trunk disease pathogens and to assess the use of Trichoderma spp. for sucker wound protection. The susceptibility of sucker wounds to different trunk disease pathogens was assessed from natural as well as artificial infections. In addition the duration of sucker wound susceptibility in the field was also ascertained. Sucker wounds were sampled from three wine and two table grape vineyards during 2011 and 2012 in the Western Cape province of South Africa. Thereafter, fungal isolations were made from 161 sucker wounds and the cultures were identified based on cultural and morphological characteristics as well as the internal transcribed spacer regions and 5.8S ribosomal RNA gene. Sixty-two percent of the wounds were naturally infected by at least one of the trunk pathogens. Phomopsis (Po.) viticola (46%; 18%), Diplodia (D.) seriata (30%; 9%) and Phaeomoniella (Ph.) chlamydospora (27%; 5%) were the most predominant trunk disease pathogens isolated from sucker wounds of field wine and table grape cultivars, respectively. Lower incidences of Phaeoacremonium aleophilum (18%), Eutypella sp. (3%), Cryptovalsa ampelina (2%), Diplodia sp. (1%) and Neofusicoccum australe (1%) were obtained, however, only from wine grapes. Sucker wounds on 1-year-old potted grapevine plants of Chardonnay cultivar were inoculated with spore suspensions of Eutypa lata, N. parvum, Pa. aleophilum, Ph. chlamydospora and Po. viticola in the glasshouse. After 4 months all the inoculated pathogens could be re-isolated at the following incidences: N. parvum (85%), Ph. chlamydospora (75%), Po. viticola (65%), Pa. aleophilum (55%) and E. lata (45%). Sucker wound susceptibility was further ascertained under field conditions on 12-year-old Cabernet Sauvignon vines by artificial inoculation of the same pathogen species. After 5 months three pathogens could be re-isolated at the following incidences: Po. viticola (65%), N. parvum (32.5%) and Ph. chlamydospora (7.5%). The duration of susceptibility of field sucker wounds to Ph. chlamydospora was assessed for a period of 4 weeks. The wounds remained susceptible for 4 weeks with a decline in susceptibility after one week. This study showed that sucker wounds are susceptible to the major trunk disease pathogens and thus could play an important role in grapevine trunk disease epidemiology. In the second part of this thesis a possible management strategy to prevent infections of sucker wounds was investigated. The use of Trichoderma (T.) harzianum against two trunk pathogens on sucker wounds was tested in the field. Additionally the sensitivity of T. harzianum and T. atroviride was tested in vitro against 16 fungicides that are used to control powdery mildew, downy mildew, Botrytis rot and Phomopsis cane and leaf spot. In October 2012, sucker wounds were made on 1-year-old wood of Cabernet Sauvignon and spray-treated with Eco-77® immediately after desuckering, and then inoculated with spore suspensions of either Ph. chlamydospora or Po. viticola after 24 hours. After 5 months, isolations were made from the sucker wounds to evaluate the efficacy of the Trichoderma treatment. Trichoderma harzianum reduced the incidence of Ph. chlamydospora by 66.65%. Although the incidence of Po. viticola was reduced by 15.37%, it was not significantly different from the control treatment. The inhibition of mycelial growth and conidial germination of T. harzianum and T. atroviride were screened against 16 fungicides. The fungicides were applied at 0, 0.25, 0.5, 1 and 2 times the recommended dosages. Systemic fungicides boscalid, metrafenone and trifloxystrobin, as well as contact fungicides quinoxyfen and meptyldinocap were least toxic to Trichoderma spp. isolates. For the conidial germination assay, boscalid, trifloxystrobin, penconazole and metrafenone (systemic) plus quinoxyfen and folpet (contact) were compatible with Trichoderma spp. These fungicides were regarded as being compatible with Trichoderma spp. isolates because they gave mean percentage inhibitions of less than 50% at all the tested dosages. Spiroxamine and pyrimethanil gave the highest mean percentage inhibitions for both mycelial inhibition and conidial germination. The findings of this study showed that T. harzianum can protect sucker wounds against Ph. chlamydospora in the field. Furthermore, some fungicides applied for the control of powdery mildew and Phomopsis cane and leaf spot can be alternatively or simultaneously applied with T. harzianum and T. atroviride, however, this will have to be verified with field trials. / AFRIKAANSE OPSOMMING: Wingerd stamsiektes is wêreldwyd verantwoordelik vir verminderde wyn- en tafeldruif produksie. Stamsiektes word veroorsaak deur patogene wat in die xileem voorkom, insluitend verskeie spesies in die Botryosphaeriaceae, Diatrypaceae, Hymenochaetales en Diaporthales, asook Phaeomoniella chlamydospora en Phaeoacremonium spp. Winter snoeiwonde word beskou as die hoof bron van infeksies vir stamsiekte patogene. Die rol van suierwonde as poorte van infeksie vir stamsiektes is nog nie goed bestudeer nie. Kennis van die potensiële rol van wingerd stamsiekte patogeen infeksies wat deur suierwonde plaasvind is belangrik vir die formulasie van beter wondbeskerming strategieë. Die mikpunt van hierdie studie was om die rol van suierwonde as ingangsportale vir wingerd stamsiekte patogene te bepaal en om die gebruik van Trichoderma spp. vir suierwond beskerming te evalueer. Die vatbaarheid van suierwonde vir verskillende stamsiekte patogene is geëvalueer vanuit natuurlike, sowel as kunsmatige infeksies. Die duur van suierwond vatbaarheid in die veld is ook bepaal. Suierwonde is versamel vanuit drie wyn- en twee tafeldruif wingerde gedurende 2011 en 2012 in die Wes Kaap provinsie van Suid Afrika. Hierna is swam isolasies gemaak vanuit 161 suierwonde en die kulture is geïdentifiseer volgens kultuur en morfologiese kenmerke, sowel as die interne transkribeerde spasieerders en 5.8S ribosomale RNA geen. Twee-en-sestig persent van die wonde was geïnfekteer deur ten minste een van die stamsiekte patogene. Phomopsis (Po.) viticola (46%; 18%), Diplodia (D.) seriata (30%; 9%) en Phaeomoniella (Ph.) chlamydospora (27%; 5%) was die mees algemene stamsiekte patogene wat, respektiewelik, vanuit die wyn- en tafeldruif kultivars verky is. Laer hoeveelhede Phaeoacremonium aleophilum (18%), Eutypella sp. (3%), Cryptovalsa ampelina (2%), Diplodia sp. (1%) en Neofusicoccum australe (1%) is verkry, en slegs vanaf wyndruiwe. Suierwonde op 1-jaar oue Chardonnay wingerdplante in potte is in die glashuis geïnokuleer met spoorsuspensies van Eutypa lata, N. parvum, Pa. aleophilum, Ph. chlamydospora en Po. viticola. Na 4 maande kon al die geïnokuleerde patogene her-isoleer word teen die volgende hoeveelhede: N. parvum (85%), Ph. chlamydospora (75%), Po. viticola (65%), Pa. aleophilum (55%) en E. lata (45%). Suierwond vatbaarheid is verder geëvalueer onder veld kondisies op 12-jaar oue Cabernet Sauvignon plante deur kunsmatige inokulasie van die selfde patogeen spesies. Na 5 maande kon drie patogene her-isoleer word teen die volgende hoeveelhede: Po. viticola (65%), N. parvum (32.5%) en Ph. chlamydospora (7.5%). Die duur van vatbaarheid van suierwonde teen Ph. chlamydospora in die veld is geevalueer oor ‘n periode van 4 weke. Die wonde het vatbaar gebly vir 4 weke met ‘n afname in vatbaarheid na ‘n week. Hierdie studie demonstreer dat suierwonde vatbaar is vir die hoof wingerd stamsiektes en dus ‘n belangrike rol in die epidemiologie van wingerd stamsiektes kan speel. In die tweede deel van hierdie tesis is ‘n moontlike bestuurs-strategie ondersoek om infeksie van suierwonde te verhoed. Die gebruik van Trichoderma (T.) harzianum teen twee stampatogene op suierwonde is getoets in die veld. Verder is die in vitro sensitiwiteit van T. harzianum en T. atroviride getoets teen 16 fungisiedes wat gebruik word in die beheer van poeieragtige meeldou, donsskimmel, Botrytis vrot en Phomopsis streepvlek. Gedurende Oktober 2012 is suierwonde gemaak op 1-jaar oue hout van Cabernet Sauvignon en onmiddelik behandel met Eco-77® na suiering. Wonde is dan geïnokuleer met spoorsuspensies van óf Ph. chlamydospora óf Po. viticola na 24 uur. Na 5 maande is isolasies gemaak vanaf suierwonde om die doeltreffendheid van van die Trichoderma behandeling te evalueer. Trichoderma harzianum het die voorkoms van Ph. chlamydospora met 66.65% verminder. Alhoewel die voorkoms van Po. viticola verminder is met 15.37%, was dit nie ‘n beduidende verskil in vergelyking met die kontrole behandeling nie. Die inhibisie van miselium groei en konidia ontkieming van T. harzianum en T. atroviride is getoets teen 16 fungisiedes. Die fungisiedes is aangewend teen 0, 0.25, 0.5, 1 en 2 keer die aanbevole dosisse. Sistemiese fungisiedes boscalid, metrafenone en trifloxystrobin, sowel as kontak fungisiedes quinoxyfen en meptyldinocap was die minste toksies teen Trichoderma spp. Gedurende die konidia ontkiemingstoets was boscalid, trifloxystrobin, penconazole en metrafenone (sistemies) en quinoxyfen en folpet (kontak) versoenbaar met Trichoderma spp. Die fungisiedes is beskou as bruikbaar met Trichoderma spp. isolate omdat hulle gemiddelde persentasie inhibisies van minder as 50% teen al die getoetste dosisse gelewer het. Spiroxamine en pyrimethanil het die hoogste gemiddelde persentasie inhibisie gelewer vir beide die miselium inhibisie en konidia ontkieming. Die bevindings van hierdie studie het gewys dat T. harzianum suierwonde kan beskerm teen Ph. chlamydospora in die veld. Verder sou sommige fungisiedes wat aangewend word vir die bestuur van poeieragtige meeldou en streepvlek moontlik alternatiewelik of gelyktydig met T. harzianum en T. atroviride aangewend word, alhowel dit met veldproewe bevestig moet word.
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Relations entre l’omycète, Pythium oligandrum, et la vigne : étude de l’induction de résistance contre un agent pathogène du bois et impact sur les communautés microbiennes colonisant la plante / Relationships between the oomycete, Pythium oligandrum, and grapevines : induced resistance against a trunk wood pathogen and impact on plant microbial communities

Yacoub, Amira 14 December 2015 (has links)
Il est actuellement estimé qu’environ 13% du vignoble français est improductif suite aux pathologies affectant le bois des ceps, la principale d’entre elles étant l’esca. Parmi les moyens de lutte mis en œuvre, le biocontrôle, via l’utilisation d’un oomycète, Pythium oligandrum, est actuellement développé pour protéger les plants de vigne contre un agent pathogène pionnier de l’esca, Phaeomoniella chlamydospora. La sélection de souches de P. oligandrum, isolées du vignoble, et produisant in vitro des quantités importantes d’une protéine élicitrice, l’oligandrine, des systèmes de défense des végétaux a d’abord été réalisée. Trois essais en serre ont montré qu’une réduction significative (40 à 50%) des nécroses dues P. chlamydospora était observée après application d’inocula de l’oomycète sur les racines des plants de vigne pied-francs Au niveau de la tige, le niveau d’expression de 22 gènes impliqués dans les mécanismes de défenses de Vitis vinifera a été mesuré par PCR quantitative et des réponses spécifiques du végétal ont été observées selon les traitements. Six gènes (protéines PR, voie des phenylpropanoïdes, oxylipines et le système d’oxydo-réduction) ont été fortement induits lorsque les plants ont été pré-inoculés par P. oligandrum puis infectés par P. chlamydospora. Afin de mettre en évidence les mécanismes spécifiques mis en place lors de cette interaction tripartite, l'analyse de la réponse transcriptomique globale de la vigne (par microarray et RNAseq), au niveau de la tige, a été réalisée chez ces plants qui manifestent une résistance induite systémique (ISR). Plusieurs gènes impliqués dans la synthèse de l’éthylène et des jasmonates sont fortement induits, chez les plants pré-traités par l’oomycète puis infectés par l’agent pathogène. Plusieurs facteurs de transcription régulant ces voies de signalisation sont également fortement induits. Suite à l’analyse des populations de messagers (mRNA) de P. chlamydospora, il a été observé que les niveaux d’expression de gènes impliqués dans la synthèse des métabolites secondaires, des facteurs de transcription impliquées dans la régulation de différentes voies chez les champignons et certaines Carbohydrates Actives enZymes étaient modulés en présence de P. oligandrum au niveau racinaire. Ces résultats montrent que la colonisation du végétal par l’oomycète, même à distance de P. chlamydospora, induit un stress indirect important chez celui-ci. Afin d’optimiser l’implantation de cet agent de biocontrôle en pépinière et au vignoble, l’aptitude de P. oligandrum à coloniser les racines de plants de vignes greffés et à les protéger contre P. chlamydospora a été étudiée. Trois portes-greffes (SO4, 3309 et 101-14) greffés sur des cépages (Cabernet Sauvignon et Sauvignon Blanc) ont été inoculés ou non par P. oligandrum. L’oomycète s’implantait sur les différents systèmes racinaires, mais en proportion variable selon les associations cépage/porte-greffe utilisées. Les analyses par empreintes moléculaires (Single Strand Conformation Polymorphism) ont montré que des microflores fongiques et bactériennes complexes et diversifiées colonisaient les feuilles et les racines, mais que l’introduction de P. oligandrum sur la plante n’induisait pas de bouleversements directs ou indirects notables au niveau de ces microflores indigènes. Une protection des jeunes plants de vigne greffés (SO4 + Cabernet Sauvignon) semble être induite par P. oligandrum contre l’agent pathogène, P. chlamydospora. / Approximately 13% of French vineyards are currently considered unproductive due to trunk diseases, mainly Esca, a particularly destructive disease that affects grapevines worldwide. Accordingly, biological control of a pathogen implicated in Esca, Phaeomoniella chlamydospora, was developed using the oomycete, Pythium oligandrum. The selection of P. oligandrum strains, isolated from vineyards, which produced in vitro large quantities of oligandrin, an elicitin-like protein inducing plant defences, was carried out. Three greenhouse assays showed that the necroses caused by P. chlamydospora were significantly reduced (40 to 50%) when P. oligandrum colonized the root system of vine cuttings. At stem level, the expression of a set of 22 genes involved in Vitis vinifera defence mechanisms was measured by quantitative PCR. Depending on the treatments employed, significant differences in grapevine responses were observed. Six of the genes (PR proteins, phenyl-propanoid pathway, oxylipins and the oxydo-reduction system) were strongly induced in plants pre-treated with P. oligandrum, and subsequently infected by P. chlamydospora. In order to characterize the mechanisms occurring during this tri-partite interaction, the global transcriptomic grapevine responses at stem level were analysed, using microarray and RNAseq, in plants in which induced systemic resistance (ISR) had taken place. Several genes involved in ethylene and jasmonate biosynthesis were strongly induced in plants that were pre-treated with P. oligandrum, and subsequently infected by P. chlamydospora. The transcription factors involved in the regulation of these signalisation pathways were also induced. Analysis of the P. chlamydospora RNA messenger (mRNA), showed that certain genes involved in secondary metabolite synthesis, transcription factors implicated in pathway regulations, and certain Carbohydrate Active enZymes, were modulated, when P. oligandrum colonised the roots. These results demonstrated that root inoculation with P. oligandrum induced indirect stress on P. chlamydospora responses. In order to promote P. oligandrum implantation in nurseries and vineyards, the capacity of this biocontrol agent to colonize the roots of grafted-plants, and to protect them against P. chlamydospora attacks, was studied. Three rootstocks (SO4, 3309 and 101-14), grafted on two scion varieties (Cabernet Sauvignon and Sauvignon Blanc), were inoculated or not with P. oligandrum. Depending on the particular scion/rootstock associations, the oomycete colonized the various root systems differently. Single Strand Conformation Polymorphism (SSCP) analyses revealed complex and diverse fungal and bacterial communities in both the rhizosphere and the phyllosphere. These microflora, which were organ-dependent, were not direcly or indirectly affected by the root inoculation of P. oligandrum. Protection of grafted vines (SO4 + Cabernet Sauvignon) was probably induced by P. oligandrum against the pathogen, P. chlamydospora.
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Lutte biologique contre un champignon pathogène impliqué dans l’esca de la vigne, par utilisation de l’oomycète Pythium oligandrum / Biological control by the oomycete, Pythium oligandrum, of a pathogenic fungus involved in esca, a grapevine trunk disease

Gerbore, Jonathan 24 October 2013 (has links)
Les recherches sur la lutte biologique (ou biocontrôle) par utilisation de micro-organismes connaissent un essor remarquable, les applications au champ étant cependant encore limitées en raison des variations d’efficacité dans la protection des plantes. Celles-ci sont souvent imputées à la non persistance des agents de biocontrôle dans la rhizosphère ou sur le végétal qu’ils sont censés protéger. Afin de réduire ce risque, une solution consiste à utiliser des micro-organismes isolés du végétal que l’on souhaite protéger. Dans le cadre de cette thèse, Pythium oligandrum, un oomycète colonisateur de la rhizosphère de nombreuses plantes dont la vigne, a été étudié pour lutter contre l’esca, une maladie du bois de la vigne pour laquelle il n’existe actuellement aucune méthode de lutte disponible. Des souches de P. oligandrum ont été isolées de la rhizosphère de ceps cultivés dans 3 régions viticoles (12 vignobles) du Bordelais présentant des sols variés : argilo-calcaire, sable-graveleux et graveleux. Les analyses des communautés fongiques et bactériennes obtenues par empreinte moléculaire (Single Strand Conformation Polymorphism) ont montré que, contrairement aux bactéries, les espèces fongiques différaient selon les régions. Des Pythium spp. aux oospores échinulées ont été isolées à partir des racines des ceps échantillonnés, avec une prédominance de P. oligandrum (séquençage de la région ITS). L’analyse des séquences des gènes codant pour le cytochrome oxydase I et une tubuline a permis de constituer 3 groupes de souches. Le séquençage d’autres gènes codant pour des protéines « élicitines-like » a indiqué que chaque souche présentait au moins un gène codant pour chacun des 2 types d’éliciteurs de P. oligandrum : l’oligandrine et les protéines de la paroi cellulaire (CWPs). Il apparaît que le type de sol et la microflore associée à la rhizosphère n’exerceraient pas une influence suffisante pour que la structure génétique des populations de P. oligandrum soient associées à un contexte tellurique particulier. En revanche, le type de porte-greffe et la méthode de désherbage (chimique ou mécanique) pourraient avoir une incidence sur la colonisation racinaire par P. oligandrum. Les relations entre P. oligandrum et les racines de la vigne ont été étudiées par analyse transcriptomique (microarray Vitis vinifera de 29 549 gènes). Les résultats obtenus montrent que de jeunes plants de vigne ont répondu à la colonisation racinaire par P. oligandrum en modifiant l’expression de gènes intervenant dans plusieurs voies métaboliques. Deux aspects a priori opposés ont été observés : P. oligandrum serait perçu comme (1) un agresseur contre lequel la plante a mis en place des réactions de défense mais en même temps, comme (2) un micro-organisme symbiotique car un certain nombre de modifications transcriptionnelles étaient similaires à celles reportées dans les interactions rhyzosphèriques symbiotiques (e.g. forte stimulation de gènes codant pour des subtilases). Un essai visant à induire chez la vigne une protection contre un champignon pathogène impliqué dans l’esca, Phaeomoniella chlamydospora, grâce à P. oligandrum, a été réalisé. La colonisation des racines par P. oligandrum a été associée à une réduction de la longueur des nécroses dues à P. chlamydospora. En adéquation avec ce résultat, l’analyse transcriptomique par RT-PCRq et microarrays a montré une surexpression de la voie de l’éthylène. Plusieurs gènes spécifiquement induits constitueraient des marqueurs de résistance qu’il conviendra de valider lors de prochaines expérimentations. / Biocontrol research based on the use of microorganisms is expanding very rapidly. However, the use of such bioncontrol agents is still too inconsistent to effectively protect plants in field applications. This phenomenon is often attributed to the non-persistence of biocontrol agents in the rhizosphere or on the plants. In order to reduce the risk of this happening, one solution consists in using microorganisms that are isolated from the plants needing protection. In this thesis, an oomycete called Pythium oligandrum, which colonizes the rhizosphere of many plants, including grapevine, was assessed for the control of esca, a grapevine trunk disease for which no control method is currently available. P. oligandrum strains have been isolated from the rhizosphere of vines cultivated in 3 wine-growing regions (12 grapevines) of Bordeaux with different types of soil: stony-sandy, silty and stony. Analyses of fungal and bacterial communities using a molecular fingerprinting method (Single Strand Conformation Polymorphism) showed that, unlike bacteria, the fungal species varied according to the sampling region. Roots of all the vines sampled were colonized by echinulated-oospore Pythium spp., with P. oligandrum strains predominating. Phylogenetic analyses based on the genes encoding the cytochrome oxidase I and one tubulin allowed these strains to be clustered into three groups. The sequencing of the elicitin-like genes, whose proteins are key components in inducing systemic resistance in plants, showed that each strain held at least one gene encoding for each of the two kinds of P. oligandrum elicitors (i.e. oligandrin and Cell Wall Proteins). Sequencing and molecular fingerprinting analyses showed thus that the type of soil and the rhizosphere microbiota did not shape the population structure of P. oligandrum. However, other factors such as the different kinds of rootstock and weeding management can also have an influence on the root colonization by P. oligandrum. The relationship between P. oligandrum and grapevine was studied using a transcriptomic approach (microarray Vitis vinifera, 29 549 genes). The results highlighted the modifications induced by young vines in response to P. oligandrum root colonization, in the genetic expression of several genes belonging to different metabolic pathways. Two aspects, that are usually opposed, were observed: P. oligandrum was perceived by the plant either (i) as a pathogen because certain defence reactions were triggered (e.g. calcium signalling, resistance genes, abscissic acid metabolism) or as (ii) a symbiotic microorganism since several transcriptional changes were similar to those reported in symbiotic interactions (e.g. induction of subtilase genes). An assay aimed at protecting grapevine against a pathogenic fungus involved in esca, and known to be responsible for wood necrosis, i.e. Phaeomoniella chlamydospora, was carried out. The root colonization by P. oligandrum was associated with a reduction in the length of necroses. In line with this result, transcriptomic analyses by microarrays and RT-qPCR showed overexpression of several genes, particularly those of the ethylene pathway. Some of these induced genes could be thus used as resistance markers, but this needs to be validated in further experiments.
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Caractérisation, criblage et mise en oeuvre de souches bactériennes issues du vignoble bordelais pour la lutte biologique contre les champignons impliqués dans la Pourriture grise et l'Esca de la vigne / Characterization, screening and implementation of bacterial strains from Bordeaux vineyards for biological control of fungal pathogens involved in Gray mold and Esca of grapevine

Haidar, Rana 11 October 2016 (has links)
Contre la pourriture grise et les maladies du bois (MdBs), qui sont des maladies cryptogamiques majeures de la vigne, la lutte biologique a un potentiel de développement considérable dans le contexte actuel de réduction des intrants chimiques en viticulture.L’objectif de cette thèse est de sélectionner et d'étudier des souches bactériennes antagonistes de Botrytis cinerea (Pourriture grise) et de deux champignons pathogènes clefs liés aux MdBs: Phaeomoniella chlamydospora et Neofusicoccum parvum. Les expériences de screening principales sont réalisées in vivo et in planta sur 46 souches bactériennes isolées dans le vignoble bordelais. Le niveau de protection par les souches antagonistes dépend significativement de la souche bactérienne, de l’espèce de champignon pathogène ciblée, du tissu ou organe végétal hôte, mais aussi pour N. parvum, du mode d’application de la souche bactérienne et, pour B. cinerea, du génotype lié aux transposons : transposa ou vacuma.Une réduction significative de 40 à 64% de la taille des nécroses dues à P. chlamydospora et/ou N. parvum est induite par trois souches bactériennes Pantoea agglomerans (S1), Paenibacillus sp. (S19) et Bacillus pumilus (S32) sur des boutures de vigne non greffées. Ces souches ont fait l'objet d'investigations approfondies pour déterminer leurs principaux modes d’action : Antibiose, production de composés volatils qui ont été identifiés et/ou induction de différents gènes de défense de la vigne.Concernant B. cinerea, les souches Enterobacter cowanii (S22), Enterobacter sp. (S23), Bacillus ginsengihumi (S38) et Bacillus sp. (S43, S46) présentent un pouvoir antagoniste important par production de composés volatils et diffusibles anti-Botrytis, ainsi que par compétition pour les nutriments par E. cowanii (S22). / Biological control of gray mold and grapevine trunk diseases (GTDs), which are major fungal diseases of grapevine, has a considerable potential development in the current context of reduction of chemical input in viticulture.The aim of this study was to select and study bacterial strains for antagonism against Botrytis cinerea, the causal agent of gray mold, and two key pathogens involved in GTDs: Phaeomoniella chlamydospora and Neofusicoccum parvum. The main screening experiments for antagonistic activity of 46 bacterial strains, isolated from Bordeaux vineyards, have been carried out under different in vivo and in planta conditions. The efficacy of protection by the antagonistic strains significantly depended on the bacterial strain, the targeted pathogen species, the host plant tissue or organ and, for N. parvum, also on the application mode of the bacterial strain and, for B. cinerea, on the transposon genotype: transposa or vacuma.A significant reduction in length of necrosis due to P. chlamydospora and/or N. parvum, ranging between 40 and 64% in non-grafted vine cuttings, resulted from three bacterial strains: Pantoea agglomerans (S1), Paenibacillus sp. (S19) and Bacillus pumilus (S32). These strains were thoroughly further investigated to determine their major modes of action by i) Antibiosis ii) production of antifungal volatile organic compounds, which have been identified, and/or iii) induction of different grapevine defense genes. Concerning B. cinerea, Enterobacter cowanii (S22), Enterobacter sp. (S23) Bacillus ginsengihumi (S38), Bacillus sp. (S43, S46) were of prime importance in the biocontrol by producing anti-Botrytis volatile and diffusible compounds or by competing for nutrients (case of E. cowanii S22).
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Sustainable Management of Grapevine Trunk Diseases in Vineyard: Deliver Biocontrol Agents and Associated Molecules

Cunha Maia Leal, Catarina Da 12 January 2023 (has links)
[ES] Las plantas de vid (Vitis vinifera L.) están expuestas a una gran variedad de patógenos. En la actualidad, las enfermedades fúngicas de la madera de la vid (GTDs) se encuentran entre los principales factores que limitan la productividad de este cultivo. Una vez las vides están infectadas, la productividad de la planta disminuye, provocando una muerte lenta o apoplética. La investigación de agentes de control biológico (BCAs) capaces de prevenir, o al menos minimizar, el impacto de las GTDs, se considera una prioridad de investigación. En esta Tesis Doctoral se caracterizó un agente de biocontrol potencial y, junto con un producto comercial biológico ya registrado, fueron probados contra varios patógenos agentes causales de GTDs, en invernadero bajo condiciones controladas, y también durante el proceso de propagación de la vid en vivero. Los resultados del análisis genómico completo de Bacillus subtilis PTA-271 muestran un sistema funcional de motilidad de enjambre, una fuerte capacidad de supervivencia y un conjunto de genes que codifican sustancias bioactivas conocidas por estimular el crecimiento o las defensas de las plantas, influir en la microbiota beneficiosa y contrarrestar la agresividad de los patógenos. Cuando Bs PTA-271 se probó contra Neofusicoccum parvum BT67 en plantas injertadas de invernadero, Bs PTA-271 y Ta SC1 demostraron que el cultivar contribuye a los efectos beneficiosos de Bs PTA-271 y Ta SC1 contra Np-Bt67. La aplicación simultánea de ambos BCAs demostró ser beneficiosa contra este patógeno en vides del cultivar Tempranillo. El análisis transcriptómico de las mismas muestras mostró ampliamente los cambios en la fisiología de la planta inducidos tanto por Bs PTA-271 como por Ta SC1 para proteger la vid ante la infección por Np-Bt67. En Chardonnay, las plantas infectadas con Np-Bt67 presentan genes sobreexpresados que están implicados en las vías de señalización del acido absicico (ABA). En Tempranillo, la infección con Np-Bt67 provoca cambios de expresión en más de 200 genes, relacionados sobre todo con la importación de aminoácidos, procesos relacionados con el cloroplasto y el fotosistema, respuestas de la planta a estímulos bióticos y biosíntesis de metabolitos secundarios. La protección de Ta SC1 en las plantas de Tempranilllo implica un mayor número de cambios, que abarcan tanto el metabolismo primario como el secundario, relacionados con cambios en las señales hormonales, como con el aicdo absicico (ABA). Durante el proceso de producción de la vid en vivero, los resultados demostraron una reducción significativa del porcentaje de plantas infectadas con los patógenos asociados a las enfermedades de decaimiento por Botryosphaeria y Pie negro en el material de vivero tratado con Ta SC1 y Bs PTA-271 respectivamente. Los tratamientos simultáneos con ambos BCAs presentaron una reducción en el porcentaje de plantas infectadas con ambos tipos de patógenos. Al probar el efecto de Bs PTA-271 y Ta SC1 en el microbioma de la rizosfera de la vid de dos suelos diferentes infectados con patógenos del pie negro, los resultados muestran que la inoculación de los BCAs parece mejorar las redes del microbioma de la rizosfera y el estado de saneamiento, sin embargo, el efecto beneficioso de los BCAs puede ser dependiente del suelo. En general, este estudio aportó nuevos conocimientos sobre el uso de uno o más BCAs contra varios patógenos asociados a las GTDs, tanto en el vivero como en vides adultas (viñedo). Además, se destacó el modo de acción de ambos BCAs en la protección de la vid. Por lo tanto, estos hallazgos proporcionan, no sólo una mejor comprensión de las interacciones entre los BCAs, la vid y los patógenos, sino que también son una fuerte contribución a una estrategia de gestión sostenible de las GTDs. / [CA] Les plantes de vinya (Vitis vinifera L.) estan exposades a una gran varietat de patògens. En l'actualitat, les malalties fúngiques de la fusta de la vinya (GTDs) es troben entre els principals factors que limiten la productivitat d'aquest cultiu. Una vegada les vinyes estan infectades, la productivitat de la planta disminueix, provocant una mort lenta o apoplética. La investigació d'agents de control biològic (BCAs) capaços de previndre, o almenys minimitzar, l'impacte de les GTDs, es considera una prioritat d'investigació. En aquesta Tesi Doctoral es va caracteritzar en profunditat un agent de biocontrol potencial i, juntament amb un producte comercial biològic ja registrat, tots dos BCAs van ser provats contra diversos patògens agents causals de GTDs, en hivernacle sota condicions controlades, i també durant el procés de propagació de la vinya en viver. Els resultats de l'anàlisi genòmica completa de Bs PTA-271 mostren un sistema funcional de motilitat d'eixam, una forta capacitat de supervivència i un conjunt de gens que codifiquen substàncies bioactivas conegudes per estimular el creixement o les defenses de les plantes, influir en la microbiota beneficiosa i contrarestar l'agressivitat dels patògens. Quan Bs PTA-271 es va provar contra Np BT67 en plantes empeltades d'hivernacle, Bs PTA-271 i Ta SC1 van demostrar que la cultivar contribueix als efectes beneficiosos de Bs PTA-271 i Ta SC1 contra Np-Bt67. L'aplicació simultània de tots dos BCAs va demostrar ser beneficiosa contra aquest patogen en vinyes del cultivar Ull de llebre. L'anàlisi transcriptómico de les mateixes mostres va mostrar àmpliament els canvis en la fisiologia de la planta induïts tant per Bs PTA-271 com per Ta SC1 per a protegir la vinya davant la infecció per Np-Bt67. En Chardonnay, les plantes infectades amb Np-Bt67 presenten gens sobreexpresados que estan implicats en les vies de senyalització de l'acidifique absicico (ABA). En Ull de llebre, la infecció amb Np-Bt67 provoca canvis d'expressió en més de 200 gens, relacionats sobretot amb la importació d'aminoàcids, processos relacionats amb el cloroplast i el fotosistema, respostes de la planta a estímuls biòtics i biosíntesis de metabòlits secundaris. La protecció de Bs PTA-271 en Chardonnay implica gens relacionats amb la biosíntesi d'ABA, les vies dels fenilpropanoides, els metabòlits secundaris, i l'estructura i organització de la paret cellular. La protecció de Ta SC1 en les plantes de Tempranilllo implica un major nombre de canvis, que abasten tant el metabolisme primari com el secundari, relacionats amb canvis en els senyals hormonals, com amb l'acid abcísic (ABA). Durant el procés de producció de la vinya en viver, els resultats van demostrar una reducció significativa del percentatge de plantes infectades amb els patògens associats a les malalties de decaïment per Botryosphaeria i Peu negre en el material de viver tractat amb Ta SC1 i Bs PTA-271 respectivament. Els tractaments simultanis amb tots dos BCAs van presentar una reducció en el percentatge de plantes infectades amb tots dos tipus de patògens. En provar l'efecte de Bs PTA-271 i Ta SC1 en el microbioma de la rizosfera de la vinya de dos sòls diferents infectats amb patògens del Peu negre, els resultats mostren que la inoculació dels BCAs sembla millorar les xarxes del microbioma de la rizosfera i l'estat de sanejament, no obstant això, l'efecte beneficiós dels BCAs pot ser dependent del sòl. En general, aquest estudi va aportar nous coneixements sobre l'ús d'un o més BCAs contra diversos patògens associats a les GTDs, tant en el viver com en vinyes adultes. A més, es va destacar la manera d'acció de tots dos BCAs en la protecció de la vinya. Per tant, aquestes troballes proporcionen, no sols una millor comprensió de les interaccions entre els BCAs, la vinya i els patògens, sinó que també són una forta contribució a una estratègia de gestió sostenible de les GTDs. / [EN] Grapevine (Vitis vinifera L.) plants are exposed to a wide variety of pathogens. Nowadays, grapevine fungal trunk diseases (GTDs) are amongst the main constraints for the productivity of this crop. Once infected, plant productivity is decreased, leading to a plant slow or apoplectic death. Investigation of biocontrol agents (BCAs) capable to forestall or at least to minimize the impact of GTDs, while being a sustainable treatment, is viewed as a research priority. One potential BCA was deeply characterized, and together with a biological commercial product, both BCAs were tested against several GTD pathogens, in greenhouse under controlled conditions, and during the grapevine propagation process. Results from the full genomic analysis of Bacillus subtilis PTA-271 (as BCA with a potential) show a functional swarming motility system, strong survival capacities and a set of genes encoding for bioactive substances known to stimulate plant growth or defenses, influence beneficial microbiota, and counteract pathogen aggressiveness. When tested against Neofusicoccum parvum Bt67 (thereafter Np-Bt67) in greenhouse cuttings, B. subtilis PTA-271 (Bs PTA-271) and T. atroviride SC1 (Ta SC1) proved that the cultivar contributes to their beneficial effects against Np-Bt67. The simultaneous application of both BCAs was further proved to be even more effective to protect Tempranillo cuttings. Moreover, the transcriptomic analysis from the same samples showed extensively the plant physiology changes induced by the pathogen but also by each BCA, Bs PTA-271 on Chardonnay and Ta SC1 on Tempranillo, to protect grapevine from Np-Bt67 infection. Thus, Chardonnay cuttings infected with Np-Bt67 showed overexpressed genes implicated on abscisic acid (ABA) biosynthesis and signaling pathways. In Tempranillo, the infection with Np-Bt67 leads to more substantial changes in gene expression, related mostly with amino acid import, chloroplast and photosystem related processes, plant responses to biotic stimulus, and biosynthesis of secondary metabolites. Protection induced by Bs PTA-271 in Chardonnay targets genes related to ABA biosynthesis, phenylpropanoid pathways and secondary metabolites, and cell wall structure/organization in relationship with carbohydrate metabolism that requires much more consideration. Protection with Ta SC1 in Tempranilllo requires a larger number of changes related to transporters, cell wall integrity and extension, cell division and pathogen induced cell death, multidirectional active proteins, and microbiome interactions. During the grapevine nursery process, the results demonstrated a significant reduction on the percentage of infected plants with Botryosphaeria dieback and Black-foot pathogens in the material treated with Ta SC1 and Bs PTA-271 respectively. The simultaneous treatments with both BCAs presented a reduction on infected plants with both Botryosphaeria dieback and Black foot pathogens. When testing the effect of Bs PTA- 271 and Ta SC1 in grapevine rhizosphere microbiome of two different soil infected with Black foot pathogens, results show that the inoculation of BCAs seems to improve the rhizosphere microbiome networks and sanitation status, however, the beneficial effect of BCAs can be soil-dependent. Moreover, as observed in the other experiments, the combination of both BCAs improves their beneficial effect in the rhizosphere microbiome. Overall, this study brought new insights on the use of one or more BCAs against several GTD pathogens, from nursery to adult grapevines. Moreover, highlighted both BCAs mode of action in grapevine protection. Thus, these findings provide, not only a better understanding of BCAs, grapevine, and pathogens interactions, but are also a strong contribution for the future development of sustainable GTDs management strategies. / Cunha Maia Leal, CD. (2022). Sustainable Management of Grapevine Trunk Diseases in Vineyard: Deliver Biocontrol Agents and Associated Molecules [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/191261

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