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Yeasts and other culturable microorganisms associated with the nickel hyperaccumulator Berkheya coddii and its insect herbivore, Chrysolina clathrata

Postma, Ferdinand (2013-03)

Thesis (MSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: The heterogeneity and distribution of elements on earth is one of the key drivers that shape the biotic processes in any given environment. What we may consider as anomalies in an environment’s element composition often drives biological adaptation and speciation. Serpentine environments provide dramatic examples of the effect soil element composition has on life. The elevated heavy metal concentrations in these environments resulted in the adaptation of plants and insects endemic to these areas. Physiological adaptations of Berkheya coddii and its insect herbivore, Chrysolina clathrata, allowed them to exploit the nickel (Ni) rich serpentine soil of the Barberton Greenstone Belt. One of the driving forces behind these adaptations may involve interactions with microorganisms. However, the microbiology of serpentine environments is relatively unknown. In the current study we aimed to identify microorganisms that may have symbiotic relationships with C. clathrata and its diet, the herbaceous Ni hyperaccumulating plant B. coddii. Culture techniques were used to isolate bacteria and fungi from plants and the faeces of beetles that were reared under laboratory conditions. The identity of isolates was determined using morphology and molecular techniques. Several genera of filamentous fungi (Alternaria, Aspergillus, Bipolaris, Cladosporium, Epicoccum, Fusarium, and Penicillium), yeasts (Cryptococcus, Meyerozyma, and Rhodotorula), and endophytic bacteria (Bacillus and Lysinibacillus) were isolated from the leaves of B. coddii. Yeast species, representing the genera Candida, Cryptococcus, Debaryomyces, Meyerozyma and Wickerhamomyces were isolated from faeces of the beetles. The minimum inhibitory concentration (MIC) of Ni was determined for all isolates. The endophytic bacteria, filamentous fungi, Candida intermedia, Cryptococcus flavescens and Meyerozyma guilliermondii showed notable Ni resistance. The Ni resistant yeast strains were isolated from the faeces of the beetles where the yeasts were in close contact with Ni ions. A strain of M. guilliermondii was also found on leaves of B. coddii but this strain had lower resistance to Ni and occurred in much lower numbers than the faecal strain. Therefore, it seems that the gut of the beetle selects for Ni resistant yeasts. The role of the yeasts occurring in the gut of the beetle may be to metabolize waste products of the beetle or aid in the sequestration of Ni. Nitrogenous metabolic waste products are usually excreted by terrestrial insects as uric acid and/ or urea. Results obtained by UPLC-MS and colorimetry confirmed that uric acid and urea were present in the faeces of C. clathrata. Strains of the yeast species M. guilliermondii, C. flavescens and W. anomalus isolated from the beetle faeces used uric acid as sole carbon and nitrogen source. A strain of M. guilliermondii isolated from the faeces of C. clathrata sequestered Ni from an aqueous solution. Concluded from these findings, yeasts in the gut of C. clathrata, may play a role in the recycling of nitrogen and may play a role in the reduction of Ni toxicity in the insect.

AFRIKAANSE OPSOMMING: Die heterogeniteit en verspreiding van elemente op aarde is een van die sleutel drywers wat die biotiese prosesse vorm in enige gegewe omgewing. Wat ons as abnormaal mag beskou in ‘n omgewing se element samestelling is dikwels die dryfveer van biologiese aanpassing en selfs spesiëring. Serpentynsteen-omgewings verskaf dramatiese voorbeelde van die effek van grond element samestelling op lewe. Die hoë swaarmetaal konsentrasies in hierdie omgewings het gelei tot die aanpassing van plante en insekte tot so ‘n mate dat hul endemies geword het aan die omgewings. Fisiologiese aanpassings van Berkheya coddii en sy insek herbivoor, Chrysolina clathrata, stel hierdie organismes instaat om die nikkel (Ni) -ryke serpentynsteengrond van die Barberton Groensteen Belt te benut. Een van die dryfkragte agter hierdie aanpassings kan moontlik die interaksies met mikroörganismes wees, maar die mikrobiologie van serpentynsteen-omgewings is relatief onbekend. Die doel van hierdie studie was om mikroörganismes te identifiseer wat simbiotiese verwantskappe kan hê met C. clathrata en die insek se voedingsbron, die kruidagtige Ni hiperakkumulerende plant B. coddii. Kultuur tegnieke is gebruik om fungi en bakterieë van plante en insekte te isoleer en in die laboratorium aan te kweek. Die identiteit van die isolate is met morfologiese en molekulêre tegnieke bepaal. Verskeie genera filamentagtige fungi (Alternaria, Aspergillus, Bipolaris, Cladosporium, Epicoccum, Fusarium, en Penicillium), giste (Cryptococcus, Meyerozyma, en Rhodotorula), en endofitiese bakterieë (Bacillus en Lysinibacillus) is vanaf die blare van B. coddii geïsoleer. Giste verteenwoordigend van die genera Candida, Cryptococcus, Debaryomyces, Meyerozyma en Wickerhamomyces is ook vanuit die insek se faeces geïsoleer. Die minimum inhiberende Ni konsentrasie van elke isolaat is bepaal. Die endofitiese bakterieë, filamentagtige fungi, Candida intermedia, Cryptococcus flavescens en Meyerozyma guilliermondii het almal weerstandbiedenheid teen Ni getoon. Die Ni weerstandbiedende gis stamme is vanuit die insek se faeces geïsoleer waar hierdie giste in noue kontak met Ni ione was. ‘n Stam van M. guilliermondii is ook op die blare van B. coddii gevind maar hierdie stam het laer Ni weerstand gehad en het ook in baie laer getalle voorgekom as die stam wat in die faeces gevind is. Dit wil dus voorkom of die dermkanaal van C. clathrata selekteer vir Ni weerstandbiedende giste. Die rol van giste in die dermkanaal van C. clathrata kan moontlik wees om afvalprodukte van die insek te metaboliseer of om te help met die sekwestrering van Ni. Stikstofbevattende afvalprodukte, geproduseer tydens stikstof metabolisme, word dikwels deur terresetriële insekte as uriensuur en/of ureum uitgesky. Resultate van UPLC-MS en kolorimetrie dui op die teenwoordigheid van uriensuur en ureum in die feces van C. clathrata. Stamme van die gis spesies M. guilliermondii, C. flavescens and W. anomalus kon uriensuur as ‘n enigste koolstof en stikstofbron benut. ‘n Stam van Meyerozyma guilliermondii was in staat om Ni vanuit ‘n waterige oplossing te sekwestreer. Hierdie waarnemings dui daarop dat giste binne die dermkanaal van C. clathrata moontlik ‘n rol kan speel in die sirkulering van stikstof en ook bydra tot die verlaging van Ni toksisiteit in die insek.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/79816
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