Understanding citrus replant disease in South Africa with the aim of developing a Methyl Bromide free management strategy

Swart, Laurika (2018-03)

Thesis (MScAgric)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Citrus fruits are one of the most important fruit crops after deciduous fruit and vegetables cultivated in South Africa. The Citrus industry in South Africa, is seen as the third largest horticultural industry and is considered one of the most important horticultural crops due to its economic export value and local consumption. South Africa is currently the eleventh largest citrus producer in the world, and second largest in the Southern hemisphere. The growth of citrus production and the development of new cultivars in South Africa has become a priority that growers establish orchards on sites where citrus has been cultivated for many years. With the establishment of a new orchard symptoms associated with replant disease have been observed on these newly planted citrus trees like in many other parts of the world. The casual agents associated with citrus replant disease in South Africa have been regarded as the citrus nematode, Tylenchulus semipenetrans and the soilborne pathogens, Phytophthora nicotianae and Phytophthora citrophthora. Symptoms can be characterised by the appearance of small leaves, the formation of gummosis and trees showing low vigour with short internodes. Previous studies conducted in South Africa on citrus replant disease did not focus on the characterisation of fungal organisms present in replant sites that may play a role in replant disease. Therefore, the aim of this study was determining whether the basis of citrus replant disease is biotic or abiotic. This study also aimed to identify the exact pathogens involved and investigating the molecular characterisation of oomycetes. Phylogenetic analyses of Fusarium spp. associated with replant soils was conducted to determine the diversity of Fusaria associated with citrus in South Africa. Soil samples was collected from four orchards, aged between 37 and 47 in two major citrus producing areas. Representative soil samples from each orchard was used for nematode extraction and revealed that both juvenile and female citrus nematodes (T. semipenetrans) was present in the soil. The soil was subjected to six different treatments. The treatments included steam sterilisation, a 20% soil dilution, a mefenoxam, difenoconazole and cadusafos drench treatment and an untreated control. After treating the orchard soil two Carrizo citrange seedlings was planted per pot and left to grow for seven months in a glasshouse. Prior to planting and at trial evaluation the seedling length and weight was determined to compare the growth of the seedlings. Statistical analyses based on the growth response of citrus, revealed that all the treatments to some extent led to an increase in seedling growth. The steam sterilisation treatment showed to have the best effect on seedling growth eliminating pathogens in the soil, followed by the untreated control, mefenoxam, cadusafos, difenoconazole and soil dilution treatment. None of the biocide treatments indicated the involvement of specific biological agents targeted by each biocide. Isolations from the roots of the control bioassay seedlings in all orchards showed that isolates belonging to the ‘Fusarium solani’ species complex (FSSC) dominated in all orchards. Fusarium oxysporum species complex (FOSC) isolates and oomycetes (Phytophthora nicotianae, Phytophthora citrophthora and Pythium irregulare) were also associated with the citrus roots at more or less similar frequencies within each orchard. Identification of Fusarium spp. was based on the phylogenetic analyses of the translation elongation factor 1-alpha (TEF) and RNA polymerase II second largest subunit RPB2 gene region. Phylogeny of the FSSC isolates showed that the citrus isolates grouped into four clades including a Neocosmospora solani clade (25 isolates), Neocosmospora croci clade (one isolate), an unnamed Fusarium spp. clade (13 Isolates) with F. falciformis as the most related known Fusarium spp., and another clade (one isolate) containing an unnamed Fusarium species. The citrus FOSC isolates grouped within the F. oxysporum phylogenetic species II. The FOSC citrus isolates were furthermore polyphyletic and distributed among two subclades, previously designated as Clade 3 (11 isolates) and Clade 4 (two Isolates). This study showed that the growth response (weight and length increases) of the seedlings in the bioassay are biological in nature. Phytophthora nicotianae, Phytophthora citrophthora, Pythium irregulare, Fusarium spp. within the FSSC and FOSC as well as the citrus nematode Tylenchulus semipenetrans are all shown to be associated with citrus tree roots and replant soil. The management strategies indicate that the applications could be possible substitutes for methyl bromide soil fumigation in orchards earmarked for replant. But ultimately the effective prevention of citrus replant disease using non-methyl bromide fumigation is dependent on knowing what is present in the soil and making the correct decisions based on this knowledge.

AFRIKAANSE OPSOMMING: Sitrus word gesien as een van die belangrikste vrugte naas sagtevrugte en groente wat in Suid-Afrika verbou word. Die Sitrusbedryf in Suid-Afrika word beskou as die derde grootste hortologie bedryf en word beskou as een van die belangrikste hortologiese gewasse, weens die ekonomiese uitvoerwaarde en plaaslike verbruik. Suid-Afrika is tans die elfde grootste sitrusprodusent ter wêreld en die tweede grootste in die Suidelike Halfrond. Die groei van sitrusproduksie en die ontwikkeling van nuwe kultivars in Suid-Afrika het 'n prioriteit geword dat produsente nuwe boorde op vooraf gevestigde sitrus boorde herbou en hervestig. Met die vestiging van 'n nuwe boord word simptome waargeneem wat algemeen geassosieer word met herplant siekte op nuut geplante sitrus bome. Die patogene wat algemeen geassosieer word met sitrus herplant in Suid-Afrika word beskou as die sitrus nematode, Tylenchulus semipenetrans en die grond gedraagde patogene, Phytophthora nicotianae en Phytophthora citrophthora. Simptome word gekenmerk deur die voorkoms van klein blare, die teenwoordigheid van gom op die bas en lote asook lae groeikrag met kort internodes. Vorige herplant studies was onvolledig in die karakterisering van herplant patogene teenwoordig in die grond. Hierdie studie het ook daarop gemik om die presiese patogene betrokke by herplant te identifiseer en die molekulêre karakterisering van oomycete te ondersoek. Filogenetiese analise van Fusarium spp. is uitgevoer om die diversiteit van Fusaria wat met sitrus in Suid-Africa geassosieer word te bepaal. Grond- en wortel monsters is van vier boorde in twee sitrus produserende gebiede, tussen die ouderdom van 37 en 47 versamel. Verteenwoordigende grondmonsters van elke boord is gebruik vir nematode-ekstraksie en het aan die lig gebring dat beide onvolwasse en vroulike volwasse sitrus nematodes (T. semipenetrans) in die grond teenwoordig was. Die grond is verder gebruik waar ses verskillende behandelings toegedien is. Die behandelings sluit in stroomsterilisasie, 'n 20% grondverdunning, en 'n mefenoxam-, difenoconasol- en cadusafos drenkbehandeling en ‘n onbehandelde beheer. Na die behandeling van die grond is dit in potte geplaas, waar twee Carrizo citrange saailinge per pot geplant is en vir sewe maande gelaat is om te groei in ‘n glashuis. Voor plant en tydens proefevaluering is die saailinglengte en gewig bepaal om die groei van die saailinge oor die tydperk van sewe maande te vergelyk. Statistiese ontledings gebaseer op die groeireaksie van sitrusplantjies, het aangetoon dat al die behandelings tot 'n mate gelei het tot 'n toename in saailinggroei. Die stoom behandeling het getoon om die beste uitwerking op saailingsgroei te hê wat patogene in die grond uitskakel, gevolg deur die onbehandelde beheer, mefenoxam, cadusafos, difenoconasol en grondverdunning. Geen biologiese agente is deur die teenwoordigheid van chemiese middel aangeteken nie. Wortel-isolasie vanuit die onbehandelde grond het aan die lig gebring dat oomycete (Phytophthora nicotianae, Phytophthora citrophthora and Pythium irregulare) en Fusarium spesies wat deel uitmaak van die Fusarium solani spesies kompleks (FSSK) en Fusarium oxysporum spesies kompleks (FOSK) geassosieer word met herplant grond- en sitruswortels. Identifikasie van Fusarium spp. was gebaseer op die filogenetiese ontledings van die translasie verlenging fakor 1-alfa (TEF) en RNA polimerase II tweede grootste subeenheid (RPB2) geen streek. Filogenetiese analise van die FSSK isolate het getoon dat sitrus isolate in vier afsondelike klade groepeer. Hierdie klade verteenwoordig die, Neososmospora solani (25 isolate), Neocosmospora croci (een isolaat), ‘n onbenoemde Fusarium spp. (13 isolate) met F. falciformis as die mees naverwante spesie en ‘n ander naamlose Fusarium klade (een isolaat). Die FOSC sitrus-isolate was verder polifileties en versprei onder twee subklades, voorheen aangewys as Klade 3 (11 isolate) en Klade 4 (two Isolates). Hierdie studie het geoon dat die groeireaksie van die saalinge biologies van aard is. Phytophthora nicotianae, Phytophthora citrophthora, Pythium irregulare en Fusarium spp. binne die FSSK en FOSK sowel as die sitrus aalwurm Tylenchulus semipenetrans word almal geassosieer met sitrus wortels en herplant grond. Die bestuurstrategieë in hierdie studie dui daarop dat die toepassings moontlike plaasvervangers vir metiel bromied grondberoking kan wees. Effektiewe voorkoming van sitrus herplant siektes wat van nie-metiel bromied beroking gebruik maak, is afhanklik daarvan om te weet wat in die grond teenwoordig is en om die korrekte besluit te neem op grond van hierdie kennis.

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