Assessment of inoculation techniques to evalute apple resistance to Phytophthora cactorum

Zondo, Patience Thembelihle (2001-03)

Thesis (MSc)--University of Stellenbosch, 2001.

Thesis

ENGLISH ABSTRACT: Phytophthora cactorum (Lebert & Cohn) Schrot. is the primary cause of crown, collar and root rot diseases of apple (Malus domestica Borkh.) trees worldwide. This pathogen is most destructive in commercial apple orchards under waterlogged soil conditions and has recently been identified as causing serious disease in some South African apple orchards. Crown, collar and root diseases are difficult to control because of their unpredictability and catastrophic nature. The use of resistant cultivars and rootstocks is economical and environmentally considerate. Therefore the need to develop screening techniques that will enable the selection of desirable disease resistant traits as part of an apple-breeding program in South Africa was identified. The work undertaken in this study was aimed at optimizing different techniques to test resistance. Using two direct inoculation techniques (excised stem and intact stem) the aggressiveness of lO isolates of P. cactorum on apple rootstocks was determined. The susceptibilities of five apple rootstocks were also compared. Results have shown isolate by rootstock interaction which means isolate aggressiveness was influenced by rootstocks tested. The selectivity of isolates suggests that there may be several strains of the pathogen. Population studies of the pathogen might contribute valuable information that could lead to better interpretation of results. Rootstock susceptibility was monitored in vitro throughout the season by inoculating at monthly intervals for 26-months. It was observed that during winter, rootstock susceptibility was low compared to high susceptibility during summer. These results have revealed new information regarding changes in the relative resistance of the different rootstocks over the growing season, e.g. the susceptibility pattern of rootstock MMl06 occurred 1 to -2 months later than that of other rootstocks. This finding has important implications on the way in which resistance test results are interpreted, and emphasizes the importance of not relying on point sampling. Furthermore, useful information has been acquired regarding the epidemiology of the disease with regard to "windows of susceptibility". The phenomenon of a phase shift in susceptibility of different rootstocks needs to be tested on a broader scale to assess whether it has any practical application on resistance testing. Although different inoculation techniques are applied in breeding programs, up to now there is no consensus on which technique works best for seedling selections. Since large numbers of individuals must be tested to improve the chances of detecting resistant genotypes, mass inoculations of young seedlings is a rapid way of identifying resistant individuals. Two different screening methods were tested during this study. Using the sand-bran technique, seedlings were transplanted onto inoculated soil and the root mass was used as a measure of resistance. In a second method zoospore inoculum was applied to seedlings growing in a sand:bark mixture at different concentrations and the seedlings were subjected either to water drenching or not. In both trials the aggressiveness of isolates differed significantly from each other and only higher inoculum concentrations were effective in causing disease. The age of seedlings used in tests emerged as an important factor. Seedlings under five-months-old should not be used. Drenching inoculated seedlings enhanced disease development but the production of sufficiently high numbers of zoospores was a laborious task. Thus, it is recommended that the sand-bran inoculum technique be tested with the drenching treatment for mass selection. In conclusion this study confirms the importance of both choice of isolate and choice of inoculation intervals in determining susceptibility of rootstocks to infection. In spite of the fact that stem inoculation bioassays have limited resemblance to natural disease situations, these bioassays are useful for obtaining an indication as to whether genotypes have a degree of resistance and merit further testing. For this reason refinement of the stem inoculation bioassay is worthwhile pursuing. With regard to seedling trials, both the sand-bran and the zoospore technique appear promising but refinement of these techniques is necessary in order to present a more practical way of testing large volumes of seedlings.

AFRIKAANSE OPSOMMING: Evaluering van inokulasietegnieke om weerstand teen Phytophthora cactorum in appels te evalueer: Phytophthora cactorum (Lebert & Cohn) Schrot. is die primêre oorsaak van kroon-, kraag en wortelvrot van appelbome (Malus domestica Borkh.). Dit is die mees verwoestende patogeen in kommersiële appelboorde waar daar versuipte toestande grond voorkom. P. cactorum is onlangs identifiseer as die patogeen wat ernstige kroon- en kraag-verotting in Suid Afrikaanse appelboorde veroorsaak. Kroon-, kraag- en wortelvrot is moeilik om te beheer as gevolg van die onvoorspelbaarheid en rampspoedige aard van die siekte. Die gebruik van kultivars en onderstamme wat weerstandbiedend is teen siektes en plae is omgewingsvriendelik en is ekonomies van belang, dus het die behoefte ontstaan om inokulasietegnieke te ontwikkelom weerstandige saailinge te identifiseer en te selekteer as deel van 'n appelteelprogram in Suid Afrika. Die doelwit van hierdie studie is om verskillende inokulasietegnieke te toets en te verfyn om weerstand in appelsaailinge te identifiseer. Deur gebruik te maak van twee inokulasietegnieke (die afgesnyde loot- en intakte loot tegniek), is die relatiewe aggressiwiteit van 10 isolate van P. cactorum en die vatbaarheid van vyf appelonderstamme ondersoek. Resultate het aangetoon dat die aggressiwiteit van die isolate gevarieer het na aanleiding van die onderstam wat getoets is. Die selektiwiteit van die isolate is 'n aanduiding dat daar moontlik verskeie rasse van die patogeen voorkom. Toekomstige studies op die populasiestruktuur van P. cactorum sal 'n belangrike bydrae maak tot die interpretasie van resultate oor weerstand en weerstandsteling. Die vatbaarheid van onderstamme was ook in in vitro proewe ondersoek deur maandelikse inokulasies toe te pas oor 'n tydperk van 26 maande. Dit is opgemerk dat die onderstamvatbaarheid gedurende die winter laag was in vergelyking met die somer. Nie al die onderstamme het dieselfe gereageer gedurende verskillende toetstye nie. Hierdie resultate toon aan dat die relatiewe weerstand van verskillende onderstamme oor die groeiseisoen verskil, byvoorbeeld die vatbare reaksie van die onderstam 'l\.1MI06' het een tot twee maande later voorgekom in vergelyking met ander onderstamme wat getoets is. Hierdie bevinding het belangrike implikasies op die interpretasie van weerstandstoetsing en beklemtoon die moontlike tekortkominge in enkelproefwaarnemings. Bruikbare inligting ten opsigte van die epidemiologie van die siekte is versamel wat beskryf kan word in terme van vensters van vatbaarheid wat verskil van onderstam tot onderstam. Verdere ondersoeke in die verband word aanbeveel. Hoewel verskeie inokulasietegnieke bestaan om jong saailinge vir weerstand te toets, is daar tot op hierdie stadium nog nie ooreenstemming oor die beste tegniek wat toegepas moet word om saailingseleksie te doen nie. Omdat groot getalle saailinge getoets moet tydens die seleksieproses sal massa-inokulasie van saailinge die aangewese metode wees. Twee verskillende inokulasie tegnieke is getoets in die studie. Deur gebruik te maak van die sandsemel tegniek, is saailinge geplant in geinfesteerde plantmedium, waartydens die wortelmassa van saailinge gebruik is om die reaksie op infeksie te kwantifiseer. Die soëspoor inokulasietegniek was toegepas op saailinge wat in 'n sand en basmengsel geplant is teen verskillende inokulurnkonsentrasies. 'n Waterverdrenkingsbehandeling is ook getoets. In albei hierdie proewe het die aggressiwiteit van die isolate van mekaar verskil. Slegs die hoër inokulumkonsentrasies was effektief in die ontwikkeling van die siekte. Die ouderdom van saailinge is ook uitgewys as 'n belangrike faktor wat 'n rol speel in weerstandstoetsing. Saailinge jonger as 5 maande word nie aanbeveel vir hierdie toetse nie. Verdrenking van saailinge het die voorkoms van die siekte verhoog, maar die produksie van groot getalle soëspore was 'n beperkende faktor in die uitvoering van die proef Dit word aanbeveel dat die sand-semel inokulasietegniek verder evalueer moet word onder verskeie toestande, onder andere deur dit met verdrenkinghte kombineer. Die belang van die keuse van isolaat en inokulasiedatum in bepaling van relatiewe weerstand van onderstamme teen P. cactorum is tydens die studie bevestig. Afgesien van die beperking van die staminokulasietegnieke in soverre dit verwyderd is van natuurlike infeksie, word die tegnieke aanbeveel om 'n indikasie te kry van die relatiewe weerstand van onderstamme. Beide die sand-semel en soëspoor tegnieke kan gebruik word om weerstandige saailinge te identifiseer, maar tegniese verfyning van hierdie tegnieke is nodig om saailinge in massa te evalueer.

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