Thesis (MSc (Plant Pathology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Dry core rot (DCR) and wet core rot (WCR) are among some of the most important postharvest diseases of apples in South Africa. Mouldy core (MC) is also a symptom associated with the core region of apples, but it is not of economical importance since apple tissue surrounding the core region is not affected as is the case with DCR and WCR. The incidence of core rots in harvested fruits can be as high as 12%, but in general ranges from 3 to 8%. Infections and losses can also occur during fruit handling in pack houses and during storage. Additionally, yield losses also occur prior to harvest within orchards due to premature fruit drop of core rot affected fruits. The incidence of core rot diseases in apples differ among apple cultivars, with most Red Delicious varieties being susceptible to the development of core rots, whereas core rots have rarely been reported in other cultivars such as Granny Smith. The etiology and epidemiology of WCR and DCR are poorly understood. Although many fungal genera have been associated with the diseases, small-spored Alternaria species are mainly associated with DCR, whereas Penicillium species including P. roquefortii, P. expansum and P. funiculosum have mainly been associated with WCR. Dry core rot infections have long been known to occur pre-harvest, whereas WCR is primarily known as a post-harvest disease where infections take place during fruit handling in pack houses. Recently, Tarsonemus mites have also been indicated as being a potential role player in the etiology of core rot diseases. The mites have been hypothesised to carry pathogen spores into the core region of apples, and they may also possibly cause small wounds that facilitate pathogen entry. In South Africa, apple growers have recently reported WCR as being present prior to harvest, which has not been reported previously. Therefore, the first aim of the study was to investigate the incidence, as well as the causal agent/s of pre-harvest WCR. The incidence of WCR ranged from 0% to 1.7% in eleven orchards, and was in general lower than that of DCR (0.4% to 6%). Isolation studies from eight internal positions in WCR apples showed that Penicillium was the predominant fungal genus in most of the positions, including the lesion area. Morphological and molecular characterisation of Penicillium isolates from WCR showed that P. 2 ramulosum prov. nom. was the main species isolated from lesions, as well as other isolation positions. However, this species was also the main species isolated from DCR, MC and asymptomatic apples. Penicillium expansum was only isolated at low frequencies from WCR and DCR apples. Other Pencillium species that were occasionally isolated included P. glabrum, P. chloroloma, P. chermisinum and a putative new species with closest affinity to P. dendriticum (P. species aff. dendriticum) on a DNA nucleotide sequence basis. Pathogenicity and virulence studies using three different inoculation methods showed that P. expansum was the most virulent species, followed by P. species aff. dendriticum. The P. ramulosum prov. nom. isolates varied in their virulence, but were all considered to have low virulence. The role of Tarsonemus mites in the etiology and epidemiology of core rot diseases is poorly understood, and therefore the second aim of the study was to investigate some of these aspects. The specific aims of the study were to (1) investigate the ecology of Tarsonemus mites in Red Delicious and Granny Smith orchards during different apple developmental stages, (2) determine if there is a significant association of Tarsonemus mites with diseased (WCR and DCR) fruits and (3) determine if potential core rot pathogenic fungi are associated with the mites. Tarsonemus mites were found in all of the investigated apple developmental stages (buds, blossoms, 4cm diameter fruit, mature fruit and mummies), having the highest incidence in mummies and mature fruits from Red Delicious and Granny Smith orchards. In Red Delicious fruits the Tarsonemus mites were found within the core and/or calyx tube, whereas in Granny Smith fruits the mites were restricted to the calyx tube. In Red Delicious fruits there was a significant association between dry core rot as well as total core rot (wet- and dry-core rot) with the presence of mites in the core, as well as total mites (mites in core and calyx tubes). Fungal isolation studies from the Tarsonemus mites showed that they carried potential core rot fungal pathogens within the genera Penicillium and Alternaria. The Penicillium species isolated from the mites included two of the most virulent WCR species, P. expansum and P. species aff. dendriticum. / AFRIKAANSE OPSOMMING: Droë kernvrot and nat kernvrot is van die belangrikste na-oes siektes van appels in Suid- Afrika. Beskimmelde kern word ook met die kern van appels geassosieer, maar hierdie toestand is egter nie van ekonomiese belang nie, aangesien die weefsel rondom die kern nie geaffekteer word soos in die geval van nat- en droë kernvrot nie. Die voorkoms van kernvrot in vrugte na oes, kan vlakke van tot 12% bereik, maar oor die algemeen is die voorkoms tussen 3 en 8%. Infeksie en verliese kan ook voorkom gedurende die hantering en verpakking van vrugte in pakhuise en gedurende storing. Addisionele verliese in opbrengs kan ook voor-oes voorkom in boorde. Dit is te wyte aan voortydige vrugval van appels wat besmet is met kernvrot. Die voorkoms van kernvrot by appels verskil tussen kultivars. Meeste van die “Red Delicious” variëteite is vatbaar vir die ontwikkeling van kernvrot. Die toestand is egter skaars by ander kultivars soos Granny Smith. Die etiologie en epidemiologie van nat- en droë kernvrot word nie goed verstaan nie. ‘n Groot aantal swamgenera is al met kernvrot geassosieer. Klein-spoor Alternaria spesies word hoofsaaklik met droë kernvrot geassosieer en Penicillium spesies, insluitende P. roquefortii, P. expansum en P. funiculosum, word meestal met nat kernvrot geassosieer. Dit is lank reeds bekend dat droë kernvrot as voor-oes siekte kan voorkom, maar nat kernvrot is algemeen bekend as na-oes siekte waar infeksie tydens vrughantering en verpakking plaasvind. Daar is onlangs aangedui dat Tarsonemus myte potensiële rolspelers in die etiologie van kernvrot is. Hipoteties is die myte in staat om spore van die patogene in die kern van die appels in te dra, asook om klein wonde te veroorsaak wat infeksie deur patogene vergemaklik. In Suid-Afrika is nat kernvrot wat voor-oes in die boorde ontstaan onlangs deur boere aangemeld; hierdie toestand is nog nie op ‘n vorige geleentheid aangemeld nie. Die eerste doelwit van hierdie studie was dus om die voorkoms en veroorsakende organisme/s van voor-oes nat kernvrot te ondersoek. Die voorkoms van nat kernvrot was tussen 0 en 1.7% in elf boorde en was oor die algemeen laer as die voorkoms van droë kernvrot (0.4 tot 6%). Isolasiestudies uit agt interne posisies van nat kernvrot appels het getoon dat Penicillium die dominante swamgenus in die meeste posisies was, insluitend die letsels. Morfologiese en molekulêre karakterisering van 4 Penicillium isolate uit nat kernvrot letsels het aangedui dat P. ramulosum prov. nom. die spesie is wat die meeste geïsoleer is vanuit die letsels, asook ander isolasie posisies. Dié spesie was egter ook die mees algemene spesie wat uit nat- en droë kernvrot, asimptomatiese appels en appels wat slegs swamgroei in die kern gehad het, geïsoleer is. Penicillium expansum was ook in lae getalle uit nat- en droë kernvrotletsels geïsoleer. Ander Penicillium spesies wat ook soms geïsoleer is, sluit P. glabrum, P. chloroloma, P. chermisinum, asook ‘n moontlik nuwe spesie wat op DNA volgorde basis die naaste aan P. dendriticum (P. spesie aff. dendriticum) is. Studies wat patogenesiteit en virulensie van die isolate ondersoek het, is ook uitgevoer deur gebruik te maak van drie verskillende inokulasie metodes. Die studies het aangedui dat P. expansum die mees virulente spesie is, gevolg deur P. spesie aff. dendriticum. Die P. ramulosum prov. nom. isolate het variasie in virulensie getoon maar is oor die algemeen aanvaar om minder virulent te wees. Die rol van Tarsonemus myte in die etiologie en epidemiologie van kernvrot word nie goed verstaan nie en dus was die tweede doelwit van die studie om sommige van dié aspekte te ondersoek. Die spesifieke doelwitte was (1) om die ekologie van die Tarsonemus myte in “Red Delicious” en Granny Smith boorde tydens verskillende ontwikkelingstadiums van die appels te ondersoek, (2) om te bepaal of daar ‘n betekenisvolle assosiasie van Tarsonemus myte met siek (nat- en droë kernvrot) vrugte bestaan en (3) om te bepaal of potensiële kernvrot patogeniese swamme geassosieer is met die myte. Tarsonemus myte is gevind in al die ontwikkelingstadiums (knoppies, bloeisels, 4 sentimeter deursnee vrugte, volwasse vrugte en mummies) van appels wat ondersoek is. Die hoogste voorkoms van myte was in die mummies en volwasse vrugte van “Red Delicious”, asook Granny Smith kultivars gevind. In “Red Delicious” vrugte is myte in die kern en/of kaliksbuis gevind, maar in die Granny Smith vrugte was die myte tot die kaliksbuis beperk. In “Red Delicious” vrugte was daar ‘n betekenisvolle assosiasie tussen droë kernvrot, asook totale kernvrot (nat en droë kernvrot) met die teenwoordigheid van myte in die kern, asook totale myte (myte in die kern en kaliksbuis). Swam isolasiestudies vanaf die Tarsonemus myte het aangetoon dat potensiële kernvrot swampatogene in die genera Penicillium en Alternaria wel by die myte teenwoordig was. Die Penicillium spesies wat vanaf die myte geïsoleer is het twee van die mees virulente nat kernvrot spesies ingesluit, nl. P. expansum en P. spesie aff. dendriticum.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study looked at the incidence, etiology and epidemiology of core rot of apples in orchards situated in the Western Cape, South Africa. Core rot is a post-harvest disease, with three symptoms, namely mouldy core (MC), dry core rot (DCR) and wet core rot (WCR). These symptoms are caused by various pathogenic fungi, including Alternaria and Penicillium. Although MC is not economically important, DCR and WCR are, as they affect the flesh of the fruit. Core rot occurs worldwide in susceptible apple cultivars such as ‘Starking’ and ‘Red Delicious’. These cultivars have a wider, open calyx tube which results in an open core area. In South Africa, core rot of apples are important post-harvest diseases and losses of between 5 and 12% occur in apple cultivars. An in depth literature search was done on core rot including literature on each core rot symptom, the genuses Alternaria and Penicillium, molecular identification and techniques, disease incidence and its economic importance, various inoculum sources, pathogenicity of core rot organisms and integrated management of core rot. This study included two research chapters, with seven objectives, namely, to 1, determine the incidence of core rot in apples from commercial orchards both pre- and post-harvest; 2, to identify the causal organisms associated with core rot symptoms; 3, to identify potential sources of inoculum of core rot pathogens and determine whether there is synergism between Alternaria and Tarsonemus mites associated with core rot; and 4, to determine whether the fungicide Bellis®, used a full bloom application, can be used to manage core rot in South Africa; 5, to identify the species of Alternaria and Penicillium sampled from core rot symptomatic fruit and inoculum sources (air, apple mummies and mites), using morphological and molecular methods; 6, to compare Penicillium species isolated from pre- and post-harvest WCR symptomatic fruit, using molecular species identification methods and 7, to compare and to select the most reliable pathogenicity test for use in future research. The total decay incidence for Ceres is considerably higher than the previous losses indicated in literature. Pre-harvest core rot, which was confirmed by previous studies, had a higher incidence of each core rot symptom than previously indicated. The two most frequently isolated causal organisms were Alternaria and Penicillium. Other organisms isolated and then identified from the symptoms were Fusarium, Cladosporium, Epicoccum, Ulocladium, Stemphylium, Phoma, Botryosphaeria, Botrytis, Trichoderma, Verticillium, Paecilomyces and Gliocladium. Three inoculum sources, air, mummies and mites, were regarded as potential sources of infection for core rot. During this study the sources of infection were verified and core rot causing organisms were isolated from these sources. Alternaria was isolated from air inoculum samples, but was not found on the other two sources. This dismissed the hypothesis that there was a possible synergism between Alternaria and Tarsonemus mites. Penicillium species were isolated from all three sources, more frequently from the mummies and mites. Bellis® was applied three times during the bloom period. The subsequent results showed a significant difference between the control and Bellis® treated treatments with the treated fruit having a significant higher incidence than the controlled fruit. No control was observed with this result and managing core rot with only Bellis® is not advisable. Alternaria species were identified using the following genetic loci, ITS, OPA1-3, 2-1 and 10-2 as well as endoPG. Isolates from pre- and post-harvest symptoms and air inoculum were identified using each of the genetic loci. Alternaria arborescens was one of the species that was identified. The other isolates obtained were A. alternata, A. tenuissima, A. gaisen, A. dumosa, A. turkisafria and A. perangusta. Separating combined species was not possible. Another molecular technique, ISSR, was used to identify Alternaria species. This technique, after multiple re-runs, did not give consistent results and species could not be identified. Penicillium species were identified using the genetic loci ITS for isolates collected from pre- and post-harvest symptoms and inoculum sources. Thirteen clades were identified, including the species P. ramulosum, P. sp. (aff. cecidicola), P. sp (aff. dendriticum), P. expansum, P. paneum, P. solitum, P. crustosum , P. brevicompactum, P. novae-zeelandiae, P. glabrum and P. rugulosum. Penicillium expansum and P. ramulosum had the highest distribution between the isolates. Pre- and post-harvest WCR isolates were identified using the partial beta-tubulin PCR-RFLP method, and comparing different banding-patterns. The species identified using this method were P. expansum, P. ramulosum, P. sp. (aff. cecidicola), P. sp (aff. dendriticum), P. rugulosum, P. chermesinum and P. glabrum. Penicillium ramulosum and P. expansum had the highest incidence with P. ramulosum occurring more frequently pre-harvest than post-harvest and P. expansum occurring more frequently post-harvest. Five methods, previously published, were compared to select the most reliable pathogenicity test. The methods included surface wounding of an apple with colonised toothpicks, surface wound inoculated with a pipette, inoculation of an open mesoderm core cavity, deep and non-wounding of apple fruit with colonised toothpicks. The surface wounding with a colonised toothpick gave the most reliable results and can be used in industry as a pathogenicity test for Alternaria in apples. This study contributed to our understanding on the incidence and etiology of core rot in the Western Cape as well as in identifying inoculum sources from where infection can take place in the orchard. The results for the fungicide trial were not as anticipated and more research is required on selecting fungicides for the control of core rot in South African orchards. Although molecular techniques reduce the time in identifying fungal species, it is costly and mistakes can occur due to contamination. Identification of species can be incorrect when using a Genbank as the sequence information may be incorrect. Molecular techniques, though a good tool in identifying species, should be combined with morphological characteristics to ensure more accurate results. / AFRIKAANSE OPSOMMING: Hierdie studie het gekyk na die insidensie, etiologie en epidemiologie van kernvrot in appels vanuit boorde in die Wes-Kaap, Suid-Afrika. Kernvrot is ‘n na-oes iekte, met drie simptome, naamlik beskimmelde kern, droë kernvrot en nat kernvrot. Hierdie simptome word veroorsaak deur verskillende patogeniese swamme, insluitend Alternaria en Penicillium. Alhoewel beskimmelde kern nie ekonomies belangrik is nie, is droë en nat kernvrot wel belangrik, omdat hulle die vrug se vlees affekteer. Kernvrot kom wêreldwyd voor in vatbare kultivars soos ‘Starking’ en ‘Red Delicious’. Hierdie kultivars het ‘n wye, oop kelkbuis wat ‘n oop kern area veroorsaak. In Suid-Afrika is kernvrot van appels ‘n belangrike na-oes siekte en verliese tussen 5 en 12% kom voor in appel kultivars. ‘n In diepte literatuurstudie is gedoen omtrent kernvrot, insluitend literatuur omtrent elke kernvrot simptoom, die genera Alternaria en Penicillium, molekulêre identifikasie en tegnieke, siekte insidensie en sy ekonomiese impakte, verskillende inokulum bronne, patogenisiteit van kernvrot organismes en die geïntegreerde bestuur van kernvrot. Hierdie studie sluit in twee navorsings hoofstukke met sewe doelwitte, naamlik om 1, te bepaal wat die insidensie van kernvrot in appels is vanuit kommersiële boorde vir beide voor en na-oes; 2, om veroorsakende organismses wat met kernvrot simptome geassosieër is te identifiseer; 3, om potensiële inokulum bronne van kernvrot patogene te identifiseer en te bepaal of daar ‘n sinergisme tussen Alternaria en Tarsonemus myte, wat geassosieër is met kernvrot, is; 4, om te bepaal of die fungisied Bellis®, gebruik as ‘n volblom toediening, gebruik kan word om kernvrot in Suid-Afrika te beheer; 5, om die Alternaria en Penicillium spesies wat uit simptomatiese kernvrot vrugte en inokulum bronne geïsoleer is te identifiseer; 6, om Penicillium spesies, wat uit voor en na-oes nat kernvrot simptome geïsoleer is, te vergelyk deur gebruik te maak van molekulêre spesies identifiserings metodes en 7, om die betroubaarste patogenisiteits toets te vergelyk en selekteer vir toekomstige gebruik. Die totale bederfde insidensie vir Ceres is heelwat hoër as die vorige verliese wat aangedui is in literatuur. Vooroes kernvrot, wat deur vorige studies bevestig is, het ‘n hoër insidensie vir elke kernvrot simptoom gehad as wat voorheen aangedui is. Die twee geïsoleerde veroorsakende organismes wat die meeste voorgekom het was Alternaria en Penicillium. Ander organismes wat geïsoleer en geïdentifiseer is vanuit die simptome was Fusarium, Cladosporium, Epicoccum, Ulocladium, Stemphylium, Phoma, Botryosphaeria, Botrytis, Trichoderma, Verticillium, Paecilomyces en Gliocladium. Drie inokulum bronne, lug, gemummifiseerde vrugte en myte, is geag as potensiële bronne van infeksie vir kernvrot. Gedurende hierdie studie is hierdie bronne bevestig en kernvrot veroorsakende organismes is uit die bronne geïsoleer. Alternaria is geïsoleer vanuit die lug inokulum monsters, maar is nie geïsoleer vanuit die ander twee bronne nie. Dus die hipotese dat daar ‘n sinergisme tussen Alternaria en Tarsonemus myte is, is verwerp. Penicillium spesies is geïsoleer vanuit al drie bronne, maar meer gereeld vanuit die gemummifiseerde vrugte en die myte. Bellis® is drie keer gedurende die bot toegedien. Die daaropvolgende resultate het ‘n betekenisvolle verskil tussen die kontrole en Bellis® beheerde behandelings getoon, met die behandelde vrugte wat ‘n betekenisvolle hoër insidensie gehad het as die kontrole vrugte. Geen beheer is waargeneem nie en beheer van kernvrot met net Bellis® word nie aanbeveel nie. Alternaria spesies is geïdentifiseer deur die volgende genetise lokusse, ITS, OPA1-3, 2-1 en 10-1, asook endoPG. Isolate van voor en na-oes simptome en lug inokulum is geïdentifiseer deur elk van die genetiese lokusse. Alternaria arborescens is een van die spesies wat geïdentifiseer is. Ander isolate wat verkry is, was A. alternata, A. tenuissima, A. gaisen, A. dumosa, A. turkisafria and A. perangusta. Om gekombineerde spesies te skei was nie moontlik nie. ‘n Ander molekulêre tegniek, ISSR, was gebruik om Alternaria spesies te identifiseer. Hierdie tegniek, na menigte probeerslae, het nie konsekwente resultate gegee nie en spesies kon nie hiermee geïdentifiseer word nie. Penicillium spesies, versamel vanuit voor en na-oes simptome en inokulum bronne, is geïdentifiseer deur die genetiese lokus ITS. Dertien ‘clades’ is geïdentifiseer, insluitend die spesies P. ramulosum, P. sp. (aff. cecidicola), P. sp (aff. dendriticum), P. expansum, P. paneum, P. solitum, P. crustosum , P. brevicompactum, P. novae-zeelandiae, P. glabrum en P. rugulosum. Penicillium expansum en P. ramulosum het die hoogste distribusie tussen die isolate. Voor en na-oes nat kernvrot isolate is geïdentifiseer deur die deels beta-tubulin PCR-RFLP metode, en verskillende band patrone te vergelyk. Die spesies geïdentifiseer deur hierdie metode is P. expansum, P. ramulosum, P. sp. (aff. cecidicola), P. sp (aff. dendriticum), P. rugulosum, P. chermesinum en P. glabrum. Penicillium ramulosum en P. expansum het die hoogste insidensie gehad met P. ramulosum wat meer dikwels vooroes voorkom en P. expansum wat meer dikwels na-oes voorkom. Vyf metodes, wat voorheen gepubliseer is, is vergelyk om die betroubaarste patogenisiteits toets te selekteer. Die metodes sluit in die oppervlak wond van ‘n appel met ‘n gekoloniseerde tandestokkie, oppervlak wond geïnokuleer met ‘n pipette, inokulasie van ‘n oop mesoderm kern area, diep besering en nie-besering van die appel met gekoloniseerde tandestokkies. Die oppervlak besering met ‘n gekoloniseerde tandestokkie het die betroubaarste resultate gegee en kan in die industrie gebruik word as ‘n patogenisiteits toets vir Alternaria in appels. Hierdie studie het bygedra tot ons kennis van die insidensie en etiologie van kernvrot in die Wes-Kaap sowel as die identifisering van die inokulum bronne, van waar die infeksie in die boord kan plaasvind. Die resultate vir die fungisied proef was nie wat ons verwag het nie en meer navorsing word benodig om fungisiede te selekteer vir die beheer van kernvrot in Suid-Afrikaanse boorde. Alhoewel molekulêre tegnieke die tyd verminder om ‘n swam spesie te identifiseer, is dit wel duur en foute kan voorkom as gevolg van kontaminasie. Identifikasie van spesies kan verkeerd wees indien Genbank gebruik is, omdat die informasie daar nie altyd korrek is nie. Molekulêre tegnieke, alhoewel ‘n goeie manier om spesies te identifiseer, moet gekombineer word met morfologiese karakter eienskappe om akurate resultate te verseker.