Assessment of the pathogenicity of fusarium euwallaceae to grapevine and deciduous fruit trees in South Africa and its rapid detection in woody tissues

Date
2022-04
Journal Title
Journal ISSN
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Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: A myriad of different tree species in South Africa are under threat from the invasive woodboring beetle, Euwallacea fornicatus (Coleoptera: Curculionidae: Scolytinae), the polyphagous shot hole borer, PSHB. While constructing galleries in the wood of hosts these beetles release spores of a mutualistic fungus, Fusarium euwallaceae (Hypocreales; Nectriaceae).The fungus colonises the xylem tissues and acts as the primary food source for the beetle, but colonisation can lead to Fusarium dieback disease in susceptible hosts. Many economically important fruit tree species have been identified as possible hosts; however, no assessments have specifically tested the pathogenicity of F. euwallaceae towards these. The work presented in this thesis set out to evaluate the susceptibility of different deciduous fruit trees (plum, nectarine and apple) and grapevine to F. euwallaceae. The effect of branch diameter and increased inoculum load on the rate of lesion development was also assessed. Fusarium euwallaceae was pathogenic to all fruit tree species and cultivars tested but no evidence of disease development was recorded in grapevine. There were no significant differences in virulence between the different isolates. There was also no evidence that differences in branch diameter or differences in the number of inoculation points on a branch can affect the growth rate of F. euwallaceae. For monitoring, the presence of PSHB is often determined by positive identification of F. euwallaceae without collection of the beetle, particularly when beetles are not able to establish viable colonies. Standard approaches to identify F. euwallaceae are costly and labour- and time intensive as the fungus needs to be isolated and cultivated from freshly collected material, the DNA needs to be extracted and purified, and the DNA needs to be sequenced for a marker that can be used to identify it. In this thesis a faster, more accessible, and cheaper tool for the identification of F. euwallaceae in both fresh and dried wood samples which increases monitoring capacity when resources are limited is presented. A species-specific marker was identified from literature and an optimized PCR protocol for the identification of F. euwallaceae was developed that removes requirements to rear the fungus, purify its DNA and to sequence a DNA marker. This protocol was tested on eleven different hosts, all but one of which that produced positive results in at least one of the replicates. Amplification was not possible in one of the hosts likely due to the high concentration of PCR inhibitory compounds. In cases like this, a secondary measure based on the protocol developed here can be used where fungal isolates are first obtained from diseased woody tissues and these subjected to the rapid detection protocol. Amplification of F. euwallaceae DNA using this approach had a 100% reproducibility rate.
AFRIKAANSE OPSOMMING: 'n Magdom verskillende boomspesies in Suid Afrika is bedreig deur die indringer stamboorder, Euwallacea fornicatus (Coleoptera: Curcilonidae: Scolutinae), die polifage stompkopboorder , (PSHB). Terwyl die kewer gate deur die hout boor van gashere stel dit spore van sy mutualistiese swam, Fusarium euwallaceae (Hypocreales; Nectriaceae), vry. Die swam koloniseer die xileem weevsel en dien as die primêre voedingsbron vir die kewer, maar kolonisering kan lei tot Fusarium afsterwe siekte in vatbare gashere. Baie boom spesies wat ekonomies belangrik is, is al identifiseer as moontlike gashere; maar geen assesserings wat spesefiek die patogenisiteit van F. euwallaceae op hierdie gashere toets was al uitgevoer nie. Die werk wat in hierdie tesis aangebied word stel om die vatbaarheid van verskillende sagtevrugtebome (pruim, nektarien en apple) en wingerdstokke aan F. euwallaceae te evalueer. Die effek wat tak diameter en toegeneemde inokulum lading het op die koers van letsel groei was ook geassesseer. Fusarium euwallaceae was patogenies tot alle vrugteboom spesies en kultivars wat getoets is, maar geen bewyse van siekte ontwikkeling was waargeneem in wingerd nie. Daar was geen verskille in virulensie tussen verskillende isolate gevind wat betekenisvol was nie. Daar was ook geen bewyse dat verskille in tak diameter of verskille in die nommer van inokulasiepunte die koers van F. euwallaceae groei affekteer nie. Vir monitering, is die teenwoordigheid van PSHB vas gestel deur positiewe identifiikasie van F. euwallaceae sonder versameling van die beesie, veral wanneer beesies nie lewensvatbare kolonies vestig nie. Standaard benaderings om F. euwallaceae te identifiseer is duur en arbeids- en tyd intensief omdat die swam eers geisoleer en gekweek moet word vanaf vars versamelde plant material, dan word DNA ekstraksie uitgevoer en DNA word gesuiwer. Daarna moet volgordebapaling uitgevoer word met ‘n merker wat kan gebruik word om F. euwallaceae te identifiseer. In hierdie tesis word ‘n vinniger, meer toeganklikke en goedkoper metode vir die identifikasie van F. euwallaceae in beide vars en gedroogte plantmateriaal aangebied. ‘n Spesies-spesefieke merker was identifiseer vanaf literatuur en ‘n geoptimiseerde PCR protokol was ontwikkel vir die identifikasie van F. euwallaceae wat die vereistes verwyder om die swam eers te kweek dan DNA ekstraksie- en DNA merker volgordebepaling uit te voer. Hierdie protokol was getoets op elf verskillende gashere, waarvan almal behalwe een positiewe resultate in ten minste een van vyf replikate positiewe resultate gegee het. Versterking was nie moontlik in een van die gashere nie, waarskynlik omdat daar hoë konsentrasies van inihiberende verbindings teenwoordig is. In gevalle soos hierdie is daar ‘n sekondêre benadering wat gebasseer is op die protokol wat hier ontwikkel is; die vinnige opsprong protokol kan toegepas word op swam isolate wat vooraf verkry is vanaf siek houtagtige weevsels. Versterking van F. euwallaceae DNA met hierdie benadering het ‘n 100% reproduseerbaarheid.
Description
Thesis (MScConsEcol)--Stellenbosch University, 2022.
Keywords
Polyphagous Shothole Borer -- South Africa, Grapevine -- Fungal diseases, Fusarium euwallaceae -- Classification, Pathogenicity, Fruit trees -- South Africa, Fusarium diseases of plants -- Genetic aspects, Fruit -- Diseases and pests -- Control, UCTD
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