Occurence of canker and woodrot pathogens in young apple trees and possible sources of inoculum

Thesis (MScAgric)--Stellenbosch University, 2017.ENGLISH ABSTRACT: Apples are one of the most important deciduous fruit crops cultivated in South Africa. The deciduous fruit industry identified a higher occurrence of young apple trees that died due to canker or wood rot diseases. The infected plant part starts to die back, rapidly killing the young tree shortly after establishment. Knowledge regarding the occurrence of canker or wood rot pathogens in healthy nursery trees in South African is lacking. Therefore, the aim of this study was to investigate the occurrence of stem canker and wood rot pathogens in young apple trees, as well as to identify possible inoculum sources which include mother material used for propagation. The sampling strategy was divided up into three phases namely diseased trees collected from 1-year-old commercial orchards, certified nursery apple trees and propagation material including scion mother block trees and rootstock layer blocks. Thirteen 1-year-old orchards, which exhibited dieback symptoms shortly after establishment were sampled (ten trees per orchard). One soil sample per orchard to a depth of 30 cm was also collected. The soil sample was collected near the roots of one of the diseased trees. A total of 480 certified nursery apple trees were collected from four nurseries to determine if seemingly healthy trees are infected with dieback pathogens. These certified trees adhere to the standards set out by the Scheme. Cankers, pruning wounds and green shoots from 310 trees in scion mother blocks were collected as well as asymptomatic 405 green shoots from rootstock layer blocks. Plant material was surface sterilised, cut open, and isolations were made from the internal vascular discolouration. Fungal cultures obtained during the study were identified to species level by DNA sequencing and phylogenetic analyses of either the ITS, β-tubulin or EF1α gene regions, within each specific taxonomic group. A total of 45 fungal species belonging to the taxonomic groups: Basidiomycetes, Botryosphaeriaceae, Diaporthales, Diatrypaceae, Dothideomycetes, as well as, Phaeoacremonium species and Truncatella angustata, were identified in this study. The species identified in the current study have all been associated with canker or wood rot symptoms found on fruit trees in other fruit growing regions of the world. Thirty-one canker and/or wood rot causing fungi found in this study are firstly reported in apples in South Africa, of which 27 species are also reported for the first time in apples worldwide. A latent infection level of 65% was found in certified nursery apple trees with the four most predominantly isolated pathogens, including Didymosphaeria rubi-ulmifolii s.l., Diplodia seriata, Diaporthe eres and Didymella pomorum. The majority of the pathogens were found from brown wood discoloration and white rot symptoms observed in the bud union and pruning wounds on the rootstock in nursery trees. The high infection rate in the bud union and pruning wound made on apple trees indicated that nursery trees got infected during the propagation process via aerial inoculum, which was present at the time of budding and pruning back. The same fungal species were found causing latent infections in nursery trees, which were later found to be diseased in newly established orchards. The soil analyses indicated that dieback of 1-year-old commercial trees was related to the stress conditions created when apple trees were established on sub-optimal soil. More soil samples should be collected in the future to confirm these findings. Green shoots of scion trees from mother blocks, from which buds are exiced for budding, presented 5% infection and the shoots from rootstock layer blocks had an infection of 21%. Thus, buds and rooted rootstock cuttings used during propagation can also contribute to infected nursery apple trees. Basidiomycete and Ascomycete fruiting structures were found on dead trees and cankers in the scion mother blocks, 1-year-old commercial orchards as well as in 1-year-old nursery blocks. These fruiting structures can contribute to the aerial inoculum present during propagation and establishment. However, spore trapping studies should be done in the nurseries, to determine the incidence and extend of the inoculum present during the propagation process in the nursery. This study has found that mother plant material can be the source of infected nursery trees and that a high percentage of certified nursery apple trees were infected with possible canker and wood rot pathogens resulting in the distribution of seemingly healthy apple trees to farmers.AFRIKAANSE OPSOMMING: Appels is een van die belangrikste sagtevrugte gewasse wat in Suid-Afrika verbou word. Die sagtevrugtebedryf het ‘n hoër voorkoms van jong appelbome geïdentifiseer wat gesterf het as gevolg van kanker- en houtverrottings siektes. Die besmette plantdeel begin terug te sterf, wat lei tot die spoedige dood kort na vestiging. Kennis oor die voorkoms van kanker en houtverottings patogene in gesertifiseerde kwekery bome in Suid-Afrika ontbreek. Die doel van hierdie studie was dus om die voorkoms van kanker en houtverottings patogene in jong appelbome te ondersoek, asook om moontlike inokulum bronne te identifiseer. Inokulum bronne wat ondersoek was sluit in moeder materiaal wat gebruik word tydens voortplanting. Die monsterneming strategie was verdeel in drie fases, meer spesifek; siek bome gevind in 1-jarige kommersiële boorde, gesertifiseerde kwekery appelbome asook voortplantingsmateriaal insluitend bostok moedermateriaal en onderstok leier blokke. Dertien 1-jarige boorde met appelbome wat terugsterf simptome kort na vestiging getoon het was gemonster (tien bome per boord). Een grondmonster per boord tot 'n diepte van 30 cm is ook ingesamel. Die grondmonster was naby die wortels van een van die siek bome in elke boord geneem. 'n Totaal van 480 gesertifiseerde kwekery appelbome is ingesamel uit vier kwekerye om te bepaal of visueel gesonde bome wel besmet is met terugsterf patogene. Hierdie gesertifiseerde bome voldoen aan die voorwaardes wat uiteengesit is deur die Skema. Kankers, snoeiwonde en groen lote van 310 bome in bostam moederblokke asook 405 asimptomatiese groen lote van onderstok leier blokke was ingesamel. Plantmateriaal is oppervlakkig gesteriliseer, oopgesny en isolasies is gemaak van die interne vaskulêre verkleuring. Swam kulture wat tydens die studie geïdentifiseer was, was tot spesie vlak geïdentifiseer deur DNS volgorde bepaling en filogenetiese analise van die ITS, β-tubulin of EF1α geen areas, binne elke spesifieke taksonomiese groep. 'n Totaal van 45 swam spesies was geïdentifiseer wat deel vorm van die taksonomiese groepe: Basidiomycetes, Botryosphaeriaceae, Diaporthales, Diatrypaceae, Dothideomycetes, asook, Phaeoacremonium spesies en Truncatella angustata. Die spesies wat in die studie geïdentifiseer was, is almal geassosieer met kanker of houtverottings simptome wat op vrugtebome in ander vrugte produserede streke in die wêreld gevind is. Vir 31 kanker en / of houtverottings swamme wat gevind was in hierdie studie is dit die eerste rapport op appelbome in Suid-Afrika, en daarvan 27 spesies ook gerapporteer vir die eerste keer op appels wêreldwyd. 'n Latente infeksie van 65% is in gesertifiseerde kwekery appelbome gevind. Die vier mees oorwegend geïsoleerde patogene sluit in; Didymosphaeria rubi-ulmifolii s.l., Diplodia seriata, Diaporthe eres en Didymella pomorum. Die meerderheid van die patogene was geïsoleer van bruin hout verkleuring en wit sagteverrot simptome wat waargeneem was in die okkulasiewond asook by die snoeiwond wat gemaak was op die onderstok van die kwekery bome. Die hoë infeksie in die okkulasiewond en snoeiwond op kwekery appelbome dui daarop aan dat kwekery bome besmet word tydens die voortplantingsproses via lug inokulum wat teenwoordig is tydens die tyd van okkuleering en terug snoei. Soortgelyke patogene was gevind wat latente infeksies in kwekery bome veroorsaak, wat ook siekte uitdrukking veroorsaak het in nuutgevestigde boorde. Die grond ontledings het getoon dat terugsterf van 1-jarige kommersiële bome verband hou met stres toestande wat veroorsaak word deur vestiging op sub-optimale grond. Meer grondmonsters moet ingesamel word in die toekoms om hierdie bevindinge te bevestig. Daar was ook gevind dat 5% van groen lote wat versamel was van bostam moederblokke latente infeksies gehad het. Onderstam leier blokke het 'n latente infeksie van 21% gehad. Dus, ogies en gewortel onderstok plante wat gebruik word tydens voortplanting kan ook bydra tot die besmetting van kwekery appelbome. Basidiomycete en Ascomycete vrugstrukture was gevind op dooie bome en kankers in die bostam moederblokke, 1-jarige kommersiële boorde asook in 1-jarige kwekery blokke. Hierdie vrug strukture kan bydra tot die lug inokulum wat teenwoordig is tydens voortplanting en vestiging. Daar moet egter spore vang studies gedoen word in die kwekerye, om die voorkoms van die inokulum te bepaal wat teenwoordig is tydens die voortplantingsproses in die kwekery. Hierdie studie het bevind dat moedermateriaal die bron van besmette kwekery bome kan wees en dat 'n hoë persentasie van gesertifiseerde kwekery appelbome besmet is met moontlike kanker en houtverottings patogene wat lei tot die verspreiding van visueel gesonde appelbome aan boere.National Research Foundation (NRF