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Phosphite sensitivity of phytophthora cinnamomi and methods for quantifying phosphite from avocado roots

Jing, Ma (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Phytophthora root rot caused by Phytophthora cinnamomi threatens the production of avocado worldwide, but the disease can be effectively managed using phosphonates. The mode of action of phosphonates is controversial and can include a direct fungistatic action and/or an indirect action involving host defence responses. In South Africa, in vitro radial growth inhibition studies, which can be indicative of a direct mode of action, have only been conducted on isolates collected in one orchard in previous studies, more than a decade ago. In in vitro studies, phosphate in the test medium can influence the in vitro toxicity of phosphite (H2PO3-), but this has not been studied in large P. cinnamomi populations. The in vivo phosphite sensitivity of P. cinnamomi isolates in avocado, which is indicative of host defence responses, has only been investigated in two non-peer reviewed studies in South Africa. Quantification of phosphite in roots is required for elucidating the mode of action of phosphonates, but no commercial analytical laboratory in South Africa can conduct these analyses due to the lack of a validated analytical method. The current study optimized a liquid chromatography-mass spectrometry (LC-MS/MS) method for quantification of phosphite, the breakdown product of phosphonates in plants, using avocado roots collected in orchards. Phosphite recovery rates were good (78 - 124%) and the precision was excellent with the percentage coefficient of variation (CV%) being between 1.9 to 9.7. Although the incurred sample reanalyses (ISR) precision for the method was unacceptable for samples with phosphite concentrations lower than 27 μg/gDW (~6.75 μg/gFW), it was acceptable for samples with higher phosphite concentrations (0.4 and 11 CV%; 0.6 - 21% difference [%DF]). The other investigated analytical methods, ion chromatography and an enzymatic fluorescent assay, were unreliable due to unacceptable ISR precision values. The in vitro phosphite (H2PO3-) sensitivities of 42 P. cinnamomi isolates from avocado in South Africa were investigated, as influenced by phosphate (HPO42-), using radial growth inhibition assays. Based on the response of isolates to all the evaluated phosphite concentrations (30 and 100 μg/ml) and phosphate concentrations in the test medium (1, 7 and 15 mM), the isolates could be grouped into a sensitive (11.04 - 89.21% inhibition), intermediate (11.26 - 66.75% inhibition) and tolerant group (3.9 - 19.09% inhibition). The inhibition of isolates as influenced by phosphate concentration was dependent on the phosphite sensitivity of isolates. In general, inhibition by phosphite for the sensitive and intermediate groups decreased as phosphate concentration increased, whereas inhibition of the tolerant group was not influenced significantly by phosphate at a phosphite concentration of 30 μg/ml. The in vivo sensitivities of one isolate from each of the sensitive and tolerant groups were investigated using an excised root bioassay. The two isolates responded similar to all phosphonate treatments, but the tolerant isolate tended to be more virulent, making it difficult to differentiate between phosphite- and virulence responses. The roots from seedlings that received phosphonate treatments that yielded root phosphite concentrations of 9.82 μg/gFW or higher, resulted in significant control. The only exception was one treatment, in one of two experiments, which contained 1.92 μg/gFW that unexpectedly caused significant control. Increasing phosphite root concentrations from 9.82 to 19.30 μg/gFW did not significantly improve control. Both isolates were inhibited to a greater extent (> 40%) in vivo than in vitro. Altogether, the data supports the involvement of host defence responses in suppression of P. cinnnamomi by phosphite. The study has improved our knowledge on the in vitro and in vivo response of P. cinnamomi isolates from avocado to phosphite, and consequently its mode of action. The data suggested that the mechanism of action is most likely host defence induction. Phosphonates thus seem to be true resistance inducing crop protection products. However, further trials are required to proof this hypothesis, since limited data and some controversial data were obtained in the current study. The in vivo data provided preliminary indications that in phosphonate application trials root phosphite concentrations should be above 10 μg/gFW to suppress P. cinnamomi. However, more in vivo trials must be conducted to confirm this. The developed LC-MS/MS method, is the only method available for root phosphite quantifications in South Africa, and is crucial for investigating the P. cinnamomi-avocado-phosphonate system.

AFRIKAANSE OPSOMMING: Phytophthora wortelvrot, veroorsaak deur Phytophthora cinnamomi, bedreig die produksie van avokado wêreldwyd, maar die siekte kan effektief deur die gebruik van fosfonate bestuur word. Die werkingswyse van fosfonate is kontroversieël en kan ‘n direkte fungistatiese aksie en/of ‘n indirekte aksie, wat gasheer verdedigingsreaksies betrek, insluit. In Suid-Afrika, is in vitro radiale groei-inhibisie studies, wat aanduidend van ‘n direkte werkingswyse kan wees, slegs op isolate wat uit een boord versamel is, in vorige studies, meer as ‘n dekade terug, uitgevoer. In in vitro studies kan fosfaat in die toetsmedium die in vitro toksisiteit van fosfiet (H2PO3-) beïnvloed, maar dit is nog nie in groot P. cinnamomi populasies bestudeer nie. Die in vivo fosfiet sensitiwiteit van P. cinnamomi isolate in avokado, wat aanduidend van gasheer verdedigingsreaksies is, is nog slegs in twee nie-eweknie beoordeelde studies in Suid-Afrika ondersoek. Kwantifisering van fosfiet in wortels word benodig ten einde die werkingswyse van fosfonate vas te stel, maar geen kommersiële analitiese laboratorium in Suid-Afrika kan hierdie analises doen nie weens die gebrek aan ‘n gevalideerde analitiese metode. Die huidige studie het ‘n vloeistof kromatografie-massa spektrometrie (LC-MS/MS) metode geoptimiseer vir kwantifisering van fosfiet, die afbreekproduk van fosfonate in plante, deur gebruik te maak van avokado wortels wat in boorde versamel is. Fosfiet terugwintempo’s was goed (78 - 124%) en die akkuraatheid was uitstekend met die persentasie van koeffisiënt van variasie (CV%), tussen 1.9 tot 9.7. Hoewel die ‘incurred sample reanalyses’ (ISR) akkuraatheid vir die metode onaanvaarbaar was vir monsters met fosfiet konsentrasies laer as 27 μg/gDW (~6.75 μg/gFW), was dit aanvaarbaar vir monsters met hoër konsentrasies (0.4 en 11 CV%; 0.6 - 21% verskil [%DF]). Die ander analitiese metodes wat ondersoek is, ioon kromatografie en ‘n ensiem fluoresserende toetse, was nie betroubaar nie weens onaanvaarbare ISR akkuraatheid. Die in vitro fosfiet (H2PO3-) sensitiwiteite van 42 P. cinnamomi isolate vanaf avokado in Suid-Afrika, is ondersoek, soos beïnvloed deur fosfaat (HPO42-), deur die gebruik van radiale groei-inhibisie studies. Gebaseer op die reaksie van isolate op al die geëvalueerde fosfiet konsentrasies (30 en 100 μg/ml) en fosfaat konsentrasies in die toetsmedium (1, 7 en 15 mM), kon die isolate in ‘n sensitiewe (11.04 - 89.21% inhibisie), intermediêre (11.26 - 66.75% inhibisie) en bestande groep (3.9 - 19.09% inhibisie) gegroepeer word. Die inhibisie van isolate, soos beïnvloed deur fosfaat konsentrasie, was afhanklik van die fosfiet sensitiwiteit van isolate. Oor die algemeen het inhibisie deur fosfiet vir die sensitiewe en intermediêre groepe afgeneem soos wat fosfaat konsentrasie toegeneem het, terwyl inhibisie van die bestande groep nie betekenisvol deur fosfaat, by ‘n fosfiet konsentrasie van 30 μg/ml, beïnvloed is nie. Die in vivo sensitiwiteite van een isolaat van elk van die sensitief en bestande groepe, is deur die gebruik van ‘n uitgesnyde wortel bio-toets ondersoek. Die twee isolate het soortgelyk op alle fosfonaat behandelings gereageer, maar die bestande isolaat neig meer virulent te wees, wat dit moeilik gemaak het om tussen fosfiet en virulensie reaksies te onderskei. Die wortels vanaf saailinge wat fosfonaat behandelings ontvang het, het wortel fosfiet konsentrasies van 9.82 μg/gFW of hoër gelewer, wat tot betekenisvolle beheer gelei het. Die enigste uitsondering was een behandeling, in een van twee eksperimente, wat 1.92 μg/gFW bevat het, wat onverwags betekenisvolle beheer gegee het. ‘n Toename in wortel fosfiet konsentrasies van 9.82 tot 19.30 μg/gFW het nie beheer betekenisvol verbeter nie. Beide isolate is tot ‘n groter mate (> 40%) in vivo as in vitro geïnhibeer. Altesaam ondersteun die data die betrokkenheid van gasheer verdedigingsreaksies in die onderdrukking van P. cinnnamomi deur fosfiet. Die studie het ons kennis oor die in vitro en in vivo reaksie van P. cinnamomi isolate vanaf avokado teenoor fosfiet verbeter, en gevolglik sy werkingswyse. Die data dui daarop dat die werkingswyse gasheer verdedigingsinduksie is. Fosfonate blyk dus ware weerstand induserende gewasbeskermingsprodukte te wees. Verdere proewe word egter benodig ten einde die hipotese te bewys, aangesien beperkte data en ‘n mate van kontroversiële data tydens die huidige studie, verkry is. Die in vivo data het voorlopige aanduidings verskaf dat in fosfonaat toedieningsproewe, wortel fosfiet konsentrasies bó 10 μg/gFW moet wees ten einde P. cinnamomi te onderdruk. Meer in vivo proewe moet egter uitgevoer word om dit te bewys. Die ontwikkelde LC-MS/MS metode is die enigste metode beskikbaar vir wortel fosfiet kwantifiserings in Suid-Afrika, en is noodsaaklik ten einde die P. cinnamomi-avokado-fosfonaatsisteem te ondersoek.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98308
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