Investigation of Trichoderma species colonization of nursery grapevines from improved management of black foot

Thesis (MScAgric)--Stellenbosch University, 2019.

ENGLISH ABSTRACT: Black foot disease (BFD) is one of the main fungal diseases associated with young grapevine decline. In recent years its incidence and severity has increased significantly, affecting both nurseries and young vineyards. The pathogens purported to contribute to this disease includes species from the genera Campylocarpon, Cylindrocladiella, Dactylonectria, Ilyonectria and Thelonectria. Currently there are no chemical control measures available to manage BFD in nurseries or vineyards. Trichoderma species are well-known biocontrol agents and offers the potential to be implemented as biological control agent (BCA) against this disease. Currently, no Trichoderma product is registered for root application on grapevines in South Africa. Previous studies, applying an imported Trichoderma product, showed that the Trichoderma colonization of nursery vines was poor and the control of black foot pathogens marginal. Therefore, the aim of this study was to investigate different Trichoderma products and application methods for the improved control of BFD in grapevine nurseries. Ten Trichoderma spp. isolates were tested in vitro for their ability to inhibit the mycelial growth of four major BFD pathogens, namely Ca. fasciculare, Ca. pseudofasciculare, D. macrodidyma and I. liriodendri by means of volatile organic compounds, diffusible antibiotic compounds and direct antagonism. In most cases Trichoderma were able to inhibit the growth of BFD pathogens, though with variation in efficacy. Generally, the diffusible antibiotic compounds resulted in greater inhibition than the volatile organic compounds. For both classes of compounds tested, D. macrodidyma was found to be the most sensitive pathogen, while a number of T. atroviride isolates resulted in higher overall growth inhibition. The competitive growth study revealed all Trichoderma isolates to exert some form of antagonism towards BFD pathogens. The efficacy of T. atroviride to endophytically colonize different grapevine rootstock cultivars were evaluated on dormant rootstock shoots of five cultivars including Ramsey, Richter 99, Richter 110, US8-7 and Paulsen. The lower 5 cm of the rootstock material was soaked in a T. atroviride conidial suspension for different time periods. The rootstock material was then incubated in sterile moist chambers followed by fungal isolations. Trichoderma atroviride was able to successfully colonize all five rootstocks to a depth of 10 cm. In general did a longer soaking period not significantly increase T. atroviride colonization. In order to assess the effect of different application methods on Trichoderma colonization and control BFD, nine treatments were evaluated on nursery vines post callusing. One hundred graftlings were used per treatment, replicated five times and repeated over two seasons. In order to assess the efficacy of different Trichoderma-based products another trial was carried out on nursery vines post callusing using eight commercially produced products. One hundred graftlings were used per treatment, replicated four times and repeated over two seasons. For both trials the graftlings were uprooted after 7 months and the number of certifiable vines and total wet root mass determined. Fungal isolations were made from the xylem and pith in the basal end as well as at three sections of the roots. Subsequent Trichoderma isolates and BFD pathogens were identified based on colony morphology. In order to confirm the identify the BFD pathogens a subset of 703 isolates were selected for identification by means of genus-specific PCRs using a newly designed primer pair for the Campylocarpon genus in combination with two previously described primer pairs for Dactylonectria spp. and Ilyonectria liriodendri. The different application methods clearly showed that a newly described method of application, that consists of dipping the basal ends in a dry formulation followed by monthly soil drenches, consistently gave the highest colonization of Trichoderma. Field drenching alone was significantly less effective than the dry dip application or a combination of these treatments. Soaking of the basal ends of vines in a conidial suspension for one hour was ineffective and did not differ from the untreated control. None of the application methods resulted in significant differences between percentage certifiable vines, total wet root mass or BFD pathogen incidence. The trial evaluating different Trichoderma-based products showed products that contain isolates originating from grapevine to be the most effective in colonizing nursery vine rootstocks. In the 2016/17 season all of the products resulted in significantly lower black foot pathogen incidence in the basal ends of the vines. However, in the 2016/17 season three of the products resulted in significantly lower root mass than the untreated control, while one product resulted in significantly less certifiable vines in the 2017/18 season. When comparing tissue parts, the base of the vine and top part of roots had significantly higher Trichoderma colonization than the middle and bottom parts of the roots, while significantly less black foot pathogens were isolated from the base in comparison to the roots. Even though Trichoderma spp. were not sufficient to prevent infections by BFD pathogens, a certain degree of protection was obtained in the basal ends. The effect of the Trichoderma spp. in the nursery vines post transplanting in relation to black foot development remains to be determined. Combining existing knowledge of Trichoderma spp. as BCA with the knowledge obtained from this research will assist in optimizing the application procedure in nurseries post callusing.

AFRIKAANSE OPSOMMING: Swartvoet is een van die belangerikste swamsiektes wat geassosieer word met jong wingerd afname. Die voorkoms daarvan het onlangs toegeneem en affekteer beide kwekerye en jong wingerde. Die patogene wat verantwoordelik is vir die siekte sluit spesies van die Campylocarpon, Cylindrocladiella, Dactylonectria, Ilyonectria en Thelonectria genera in. Daar is tans geen chemiese beheermaatreëls beskikbaar om die siekte in kwekerye of wingerde te beheer nie. Trichoderma spesies is bekende biologiese beheeragente wat die potensiaal het om vir siektebeheer gebruik te word. Daar is egter geen Trichoderma produkte geregistreer vir worteltoediening op wingerde in Suid-Afrika nie. Vorige studies wat ’n ingevoerde produk ondersoek het, het bevind dat die toediening van die swam tot swak kolonisasie gelei het en net matige beheer van die siekte tot gevolg gehad het. Die doel van hierdie studie was dus om verskillende Trichoderma produkte, asook maniere van toediening te evalueer om die beheer van swartvoet te verbeter. Tien Trichoderma isolate is ondersoek vir hul vermoë om die groei van miselia van vier belangrike swartvoet patogene, naamlik Ca. fasciculare, Ca. pseudofasciculare, D. macrodidyma en I. liriodendri te inhibeer deur middel van vlugtige organsiese verbindings, oplosbare antibiotiese verbindings, asook direkte antagonisme. Trichoderma was meestal in staat om die groei van swartvoet patogene te inhibeer, maar met variasie in die doeltreffendheid daarvan. Die oplosbare antibiotiese verbindings het dikwels meer inhibisie veroorsaak as die vlugtige organiese verbindings. Dactylonectria macrodidyma was die mees sensitiewe patogeen vir beide groepe verbindings. Die kompeterende groeistudie het daarop gedui dat alle Trichoderma isolate ’n mate van antagonisme toon. Die potensiaal van T. atroviride om verskillende wingerdonderstok-kultivars endofities te koloniseer, was geëvalueer op dormante lote van vyf wingerdonderstokkultivars insluitend Ramsey, Richter 99, Richter 110, US8-7 en Paulsen. Die basis van die onderstokmateriaal was in ’n T. atroviride spoorsuspensie geweek vir verskillende periodes. Die onderstokmateriaal was daarna in steriele vogkamers geïnkubeer, waarna swamisolasies gedoen was. Trichoderma atroviride was in staat om al vyf onderstokkultivars suksesvol te koloniseer tot ’n hoogte van 10 cm. Oor die algemeen het ’n langer weekperiode nie tot hoër kolonisasie van T. atroviride gelei nie. Ten einde die effek van verskillende toedieningsmetodes op Trichoderma kolonisasie en die beheer van swartvoet te evalueer, was nege behandelinge uitgevoer op kwekeryplante na kallus. Eenhonderd geënte plante was gebruik per behandeling en vier keer herhaal oor twee seisoene. Nog ’n veldproef was uitgevoer ten einde die doeltreffendheid van agt verskillende Trichoderma-gebaseerde produkte te evalueer op kwekeryplante na kallus. Eenhonderd geënte plante was gebruik per behandeling en vyf keer herhaal oor twee seisoene. Beide proewe was uitgehaal ná sewe maande waarna die aantal sertifiseerbare plante bepaal is asook die totale nat wortelmassa. Swam isolasies was gedoen vanuit die xileem en pit in die basis van die onderstokke asook van drie gedeeltes van die wortels. Die Trichoderma isolate en swartvoet patogene wat gevolglik geïsoleer was, is geïdentifiseer op grond van hul kolonie morfologie. Ten einde die identiteit van die swartvoet patogene te bevestig was ’n subgroep van 703 isolate gekies vir identifikasie deur middel van genus-spesifieke polimerasie kettingreaksies. Dit het gebruik gemaak van een inleier stel vir die Campylocarpon genus wat nuut ontwikkel is, asook twee inleier stelle vir Dactylonectria spesies en Ilyonectria liriodendri wat voorheen beskryf is. Die proef wat verkillende toedieningsmetodes ondersoek het, het duidelik gewys dat die nuwe metode van toediening wat die doop van die basis van die wingerd onderstokke in ’n droë produk formulasie behels en dan opgevolg word deur maandelikse grond toedienings, konsekwent die hoogste kolonisasie van Trichoderma gelewer het. Veldtoediening alleen was noemenswaardig minder doeltreffend as die droë produk toediening of ’n kombinasie van hierdie behandelinge. Weking van die wingerdonderstokke in ’n spoorsuspensie vir een uur was ondoetreffend en het nie van die onbehandelde kontrole verskil nie. Geen toedieningsmetodes het gelei tot noemenswaardige verkille tussen die persentasie sertifiseerbare wingerde, totale nat wortelmassa of voorkoms van swartvoet nie. Die proef wat verskillende Trichoderma-gebaseerde produkte geëvalueer het, het gewys dat die produkte wat isolate bevat wat oorspronklik van wingerde afkomstig is, lei tot die hoogste kolonisasie van kwekery wingerdonderstokke. In die 2016/17-seisoen het al die produkte tot noemenswaardig laer swartvoet voorkoms in die basis van die onderstokke van die wingerde gelei. Drie van die produkte het egter tot noemenswaardig laer wortelmassa gelei as die onbehandelde kontrole in die 2016/17-seisoen, terwyl een produk tot noemenswaardig minder sertifiseerbare wingerde in die 2017/18-seisoen gelei het. Die verskillende weefseltipes is met mekaar vergelyk en het daarop gedui dat die basis van die onderstok van die wingerdplante en die boonste dele van die wortels noemenswaardig hoër vlakke van Trichoderma kolonisasie gehad het as die middelste en onderste dele van die wortels, terwyl noemenswaardig minder swartvoet patogene daaruit geïsoleer was. Selfs al was Trichoderma spesies nie voldoende om infeksie van swartvoet patogene te voorkom nie, het dit ’n mate van beskerming in die basis van die onderstokke gebied. Die effek van Trichoderma spesies op swartvoet patogene in kwekerywingerde nadat dit uitgeplant is, moet steeds ondersoek word. Die inligting wat versamel is in hierdie studie sal bydra tot die kennis van Trichoderma as biologiese beheeragent en sal help met die optimalisering van toedieningsprosedures van Trichoderma in die wingerdkwekeryproses.

Masters