Diversity and nature of microbial endophytes associated with southern African Oxalis

Jooste, Michelle (2019-03)

Thesis (PhD)--Stellenbosch University, 2019.


ENGLISH ABSTRACT: Symbioses between plants and micro-organisms have profound influences on biodiversity, ecosystem structure and functioning and patterns of evolution. The Greater Cape Floristic Region (Cape) of southern Africa is global biodiversity hotspot, and is renowned for its diverse and extremely rich flora. At least some of this remarkable diversity has been attributed to abiotic factors such as palaeoclimatic stability, reliable seasonal water availability, geographical gradients and diverse soil types. Cape soils contain some of the lowest nitrogen and phosphorus levels measured globally though, and these may be limiting factors in plant growth. Despite the obvious importance of plant microbial endophytes, the role of such associations in generating and maintaining plant diversity has largely been neglected to date. The Cape is also renowned for the most diverse geophyte flora in the world, including 2100 species from 20 families. Despite this, the role of plant-microbial interactions has not yet been confirmed in any Cape geophyte lineage. Cape Oxalis (Oxalidaceae) is recognised as the sixth largest plant lineage and the largest geophytic genus in the Cape. Although widespread in southern Africa, Oxalis has undergone extensive radiation in the Cape and currently includes more than 200 known species. Members occur across a vast range of environments, but are well-represented in the nutrient poor and/or drought prone habitats of the Cape. The evolutionary success of this genus in the Cape may partly be attributed to various unique life history traits (geophytic habit, winter flowering, variable seed strategies), but this is still poorly understood. Cape Oxalis species are highly unusual in terms of their seed germination strategies. Although dormant seeds represent the ancestral state in Oxalis, approximately 60% of the lineage has exendospermous seeds that lack a dormancy period, a mechanism known as recalcitrance. The morphological gulf between these strategies (and potential intermediate morphologies) has been poorly quantified, with questions regarding their ecological function and evolution still unanswered. I hypothesized that the binary classification of seed germination strategies (dormancy and recalcitrance) oversimplifies seed physiology and morphology of Cape Oxalis. Here I identified three physiological germination strategies (supported by morphology and phenology), in a system where the ancestral dormant state has evolved towards a maximally recalcitrant peak among Cape Oxalis. Additionally, a mosaic of intermediate character states is reflected in extant taxa. Recalcitrance and intermediate germination strategies are rare among angiosperms (11% of species). Insights gained from studying Cape Oxalis as an ideal model system has promoted our understanding of the evolution of the recalcitrant germination strategy, among Oxalis and angiosperms in general. Recalcitrant and some intermediate Oxalis seeds are metabolically active when shed, which enables them to germinate, establish and reach maturity much more rapidly than dormant seeds. The majority of these recalcitrant species also display a strategy of inverse germination relative to other angiosperm seedlings, where cotyledons and the first foliar leaf develop rapidly, relative to the hypocotyl, root hairs and roots that subsequently emerge. This is a remarkable phenomenon where seedlings are capable of rapid growth and development temporarily, without well-established roots to supply the seedling with nutrients. Furthermore, 70% of the recalcitrant Oxalis species (and a few intermediate species) produce large amounts of (often acidic) mucilage around the base of the hypocotyl of seedlings. The mucilage secreted by developing recalcitrant seedlings could both include growth promoting endophytes and serve as a potential attractant to plant growth promoting micro-organisms from the soil environment. As a first step towards exploring the inter-organismal associations of Cape Oxalis, I thus studied intra-plant, intra- and inter-species, and inter-site microbe richness and community composition of rhizosphere and endosphere microbes associated with Oxalis hosts. Overall, 46 culturable bacterial and 39 culturable fungal morphotypes were associated with host plants (regardless of seed germination strategy). The endophytic microbial richness and composition changed according to the surrounding environment. The most common and frequently encountered bacterial endophytes included members from the genus Bacillus Cohn - a group well-known for various plant-growth promoting properties. A surprisingly diverse collection of bacterial and fungal endophytes was also commonly found in the reproductive and vegetative propagules of all hosts. Next culture-independent 16S metabarcoding was conducted to document non-culturable bacterial endophytes associated with Oxalis. Despite various caveats associated with this approach, significant insights into the diversity of bacterial endosymbionts associated with Cape Oxalis host plants were gained. Putative genus-level identification revealed bacterial taxa from 118 genera, as well as various uncultured bacteria, which collectively belong to 79 families, 39 orders and 19 classes from eight bacterial phyla. Metabarcoding results confirmed the presence of six out of nine bacterial genera identified with culture-dependent techniques. Even though bacterial endophyte species identities could not yet be confirmed, the majority of these genera include various well-known plant endophytes with strong growth promoting and nitrogen-fixing abilities. Filter exclusion experiments were conducted to determine if endophytes were vertically-transmitted to seeds, determine if mucilage plays a role to actively attract microbes from the soil and to assess microbial richness isolated from the mucilage of Oxalis seedlings. Fluorescent microscopy was implemented in order to visualize endophytic bacteria in cryo-sectioned seeds. I report evidence for a novel, vertically transmitted symbiosis between communities of nitrogen-fixing and plant growth-promoting Bacillus endophytes and selected Oxalis hosts. Three common nitrogen-fixing Bacillus species have known oxalotrophic properties and appear to be housed inside specialised cavities (containing oxalates) within the plant body and seeds. The discovery of vertical transmission and potential benefits to both host and endophyte suggest a particularly tight mutualism in the Oxalis-endophyte system. This discovery suggests unexpected ways in which these geophytes might avoid nitrogen deficiency, and suggest that vertically-inherited mutualisms could be impacting plant survival in nutrient-depleted environments such as the Cape. Knowledge of vertical transmission of nitrogen-fixing and oxalotrophic bacteria among angiosperms may have far-reaching conservation, agricultural and economic applications.

AFRIKAANSE OPSOMMING: Simbiose tussen plante en mikro-organismes het verrykende effekte op biodiversiteit, ekosisteem-struktuur en -funksionering en patrone van evolusie. Die Groter Kaapse Floristies Streek (Kaap) van suider Afrika is „n globale biodiversiteit brandpunt, en is bekend vir sy diverse en besonder ryk flora. Ten minste sommige van hierdie merkwaardige diversteit is al toegeskryf aan abiotiese faktore soos paleoklimatologiese stabiliteit, betroubare seisoenale beskikbaarheid van water, geografiese gradiënte en diverse grondtipes. Kaapse gronde bevat egter van die laagste globaal-gemete stikstof- en fosfaat-vlakke, en dit mag groeibeperkend wees. Ten spyte van die duidelike belang van plantmikrobiese endofiete, is die rol wat sulke assosiasies speel in die generasie en behoud van plant diversiteit steeds swak ondersoek. Die Kaap is bekend vir die mees diverse geofiet-flora in die wêreld, en sluit 2100 spesies in 20 families in. Ten spyte hiervan is die rol van plantmikrobiese interaksies nog nie bevestig vir enige Kaapse geofiet-ontwikkelingslyn nie. Oxalis (Oxalidaceae) word gereken as die sesde grootse plantontwikkelingslyn en die grootste geofietiese genus in die Kaap. Alhoewel wydverspreid in suider Afrika, het Oxalis ekstensief gediversifiseer in die Kaap, en sluit tans meer as 200 bekende spesies in. Lede kom in ʼn diverse reeks omgewings voor, maar is goed verteenwoordig in die voedingstof arm en/of droogte geteisterde habitatte in die Kaap. Die evolusionêre sukses van die genus in die Kaap mag deels toegeskryf word aan verkeie unieke lewens kenmerke (geofitiese groeivorm, winter blomtyd, varierende saadstrategieë), maar dit word nog swak verstaan. Kaapse Oxalis is besonder ongewoon in terme van hulle saadontkiemingsstrategieë. Alhoewel rustende sade die voorouerlike staat in Oxalis verteenwoordig, produseer 60% van die ontwikkelinglyn eksendosperme sade sonder ʼn rusperiode, ʼn meganisme bekend as nie-rustende sade. Die morfologiese gaping tussen strategieë (en potensiële intermediêre morfologiese vorms) is swak gekwantifiseer, met vrae rakende die ekologiese funksie en evolusie steeds onbeantwoord. Ek stel die hipotese dat die binêre klassifikasie van saadontkiemings-strategieë (rustend en nie-rustend) saadfisiologie en -morfologie van Kaapse Oxalis oorvereenvoudig. Ek het drie fisiologiese ontkiemingsstrategieë geïdentifiseer (gerugsteun deur morfologie en fenologie), in „n sisteem waar die voorouerlike rustende staat geëvolueer het na „n maksimaal nie-rustende piek binne Kaapse Oxalis. Addisioneel word ʼn mosaïek van intermediere kenmerkstate in bestaande taksa gereflekteer. Nie-rustende en intermediêre ontkiemingsstrategieë is skaars tussen angiosperme (11% van spesies). Insigte verkry deur die studie van Kaapse Oxalis is ʼn ideale modelsisteem wat ons begrip van die evolusie van die nie-rustende ontkiemingstrategie van Oxalis en angiosperme in die algemeen, versterk. Nie-rustende en sommige intermediêre Oxalis sade is metabolies aktief wanneer hulle vrygestel word, wat hulle instaat stel om baie vinniger as rustende sade te ontkiem, vestig en volwassenheid te bereik. Die meerderheid van hierdie nie-rustende spesies het ook „n omgekeerde onkiemingsvolgorde relatief tot ander angiosperm saailinge, waar die saadlobbe en die eerste loofblare vinnig ontwikkel relatief tot die hipokotiel, wortelhare en wortels wat later eers verskyn. Hierdie is „n merkwaardige verskynsel waar saailinge instaat is om vinnig te groei en ontwikkel, terwyl hulle tydelik sonder goed ontwikkelde wortels bestaan wat hulle van voedingstowwe kan voorsien. Verder produseer 70% van die nie-rustende Oxalis species (en „n paar intermediêre spesies) groot hoeveelhede (dikwels suur) slym aan die basis van die hipokotiel van hulle saailinge. Die slym wat deur die onwikkelende nie-rustende saailinge afgeskei word kan groei-bevorderende endofiete insluit, en kan ook as „n potensiële aantrekking dien vir plantgroei-bevorderinde mikro-organismes uit die grond omgewing. As „n eerste stap in die ondersoek van inter-organismiese assosiasies van Kaapse Oxalis het ek die intra-plant, intra- en inter-spesie en inter-lokaliteit mikrobe rykheid en gemeenskapssamestelling van risosfeer en endosfeer mikrobe wat met Oxalis gashere geassosieer is bestudeer. In totaal is 46 kweekbare bakterië en 39 kweekbare fungus morfotipes met die gasheerplante geassosieer (ongeag die ontkiemingstrategie). Die endofitiese mikrobiese rykheid en samestelling het verander saam met die omringende omgewing. Die mees algemene en mees dikwels geisoleerde bakteriese endofiete het lede van die genus Bacillus Cohn ingesluit – „n groep wat goed bekend is vir sy groei-bevorderlike eienskappe. „n Verrassend diverse groep bakteriële en fungus endofiete is ook dikwels in die reproduktiewe en vegetatiewe plantdele van alle gashere aangetref. Volgende is kultuur-onafhanklike 16S-strepieskode tegnieke uitgevoer om die nie-kweekbare bakteriese endofiete wat met Oxalis geassosieer word, te dokumenteer. Ten spyte van verskeie voorbehoude wat met hierdie benadering gepaard gaan, is beduidende insigte in die diversiteit van bakteriese endosimbionte van Oxalis gashere aan die lig gebring. Voorlopige genusvlak-identifikasie het bakteriële taxa van 118 genera, sowel as verskeie onbekende bakterieë, wat gesamentlik aan 79 families, 39 ordes en 19 klasse van agt bakterieë-filums behoort. Hierdie resultate bevestig die teenwoordigheid van ses van die nege bakteriese genera wat met kultuurafhanklike tegnieke geïdentifiseer is. Alhoewel identifikasies van bakteriese endofiet spesies nog nie bevestig kon word nie, sluit die meerderheid van hierdie genera verskeie bekende plantendofiete in, met sterk groei-bevorderende en stikstofvasleggingssvermoëns. Filter-uitsluitingseksperimente is uitgevoer om te bepaal of die endofiete vertikaal na sade toe oorgedra word, om vas te stel of die slym „n aktiewe rol speel om mikrobe aan te trek uit die grond en om die mikrobiese rykheid binne die slym van Oxalis saailinge te bepaal. Fluorisensie mikroskopie is gebruik om die endofitiese bakterië in sade te visualiseer wat met krioseksie gesny is. Ek rapporteer bewyse vir „n nuwe, vertikaal-oorgedraagde simbiose tussen gemeenskappe van stikstofbindende en plantgroei-bevorderende Bacillus endofiete en geselekteerde Oxalis gasheer spesies. Die algemene stikstofbindende Bacillus spesies het bekende oksalotrofiese eienskappe en dit lyk asof hulle gehuisves word binne gespesialiseerd holtes (wat oksalate bevat) binne die plantliggaam en sade. Die ontdekking van vertikale oordrag en potensiële voordele vir beide gasheer en endofiet suggureer „n besonder nou mutualisme in die Oxalis-endofiet sisteem. Die ontdekking stel onverwagte wyses voor waarop hierdie geofiete stikstof-tekorte mag vermy, en suggureer dat vertikaal oorgeerfde mutualismes mag impakteer op plantoorlewing in voedingstof-arm omgewings soos die Kaap. Kennis van vertikale oordrag van stikstofbindende en oxalotrofiese bakterië tussen angiosperme mag verrykende gevolge vir bewaring, landbou en ekonomiese toepassings hê.

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