Content removed from thesis due to copyright restrictions: Schwelm, A., Barron, N. J., Zhang, S. & Bradshaw, R. E. (in press). Early expression of aflatoxin-like dothistromin genes in the forest pathogen Dothistroma septosporum. / Dothistroma septosporum causes pine needle blight, a foliar disease currently causing epidemics in the Northern hemisphere. D. septosporum synthesizes dothistromin, a mycotoxin similar in structure to the aflatoxin (AF) precursor versicolorin B. Orthologs of AF genes, required for the biosynthesis of dothistromin, have been identified along with others that are speculated to be involved in the same pathway. The dothistromin genes are located on a mini-chromosome in Dothistroma septosporum but, in contrast to AF genes, not in a continuous cluster. The aim of this study was to increase knowledge of the biological role of dothistromin, which was previously a suspected pathogenicity factor. To identify putative roles of dothistromin, the dothistromin gene expression was investigated and green fluorescence protein (GFP) reporter gene strains of D. septosporum were developed. Expression analyses of dothistromin genes revealed co-regulation. More surprisingly, dothistromin is produced at an early stage of growth and gene expression is highest during exponential growth. This is fundamentally different to the late exponential/stationary phase expression usually seen with secondary metabolites such as AF. Strains with a dothistromin gene (dotA) promoter-regulated GFP confirmed early expression of the toxin genes, even in spores and germtubes. Parallel studies with transformants containing a GFP-DotA fusion protein suggest spatial organization of dothistromin biosynthesis in intracellular vesicles. The early expression of dothistromin genes led to the hypotheses that dothistromin is either required in the early stage of the plant/fungi interaction, or for inhibiting the growth of competing fungi. Constitutive GFP strains helped to determine that dothistromin is not a pathogenicity factor. However, a putative role of dothistromin in competition with other fungi, including pine-colonizing species, was detected, supporting the second hypothesis. It was shown that dothistromin-producing strains appear to have a competitive advantage which is lacking in dothistromin-deficient strains. However, some competitors were not affected and have potential as biocontrol agents. In summary, this work has led to the discovery of an unusual pattern of regulation of a secondary metabolite, has made substantial progress in identifying the biological role of dothistromin, and has indicated potential for biocontrol of Dothistroma needle blight.

Thesis (MScFor (Forest and Wood Science))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Mid rotation fertiliser application is generally practised in forestry to enhance nutrient availability in areas were soils are impoverished and do not provide sufficient nutrients for high productivity. Generally speaking there is great potential for mid rotation fertiliser in pine plantations, but stand and site characteristics such as water availability, soil depth, stand density and available nutrients need to be considered before fertiliser treatments are implemented. Foliar nutrient analyses were used to estimate nutrient availability. These levels were measured throughout the study and were used to implement specific fertiliser treatments that would boost nutrient availability. Treatments consisted of an unfertilised control, a single fertiliser application (1F) and two fertiliser applications over two consecutive years (2F). Leaf area index (LAI) and stem volume increment were measured in order to evaluate its influence on growth efficiency. LAI was estimated using the gap fraction method with the use of a ceptometer. Volume increment was calculated with diameter and height measurements. Basal area was calculated by means of diameter measurements. These growth responses were used to determine the effect of increased nutrient availability and although increases were found in LAI, volume increment, basal area increment and growth efficiency, none were significant. The lack of significance may be due to relatively large variations in other factors such as stand density and initial volume of the experimental plots. The 18 month monitoring period apparently did not allow complete reaction time to increased nutrient availability and limited our understanding of the responses somewhat. Despite this, the magnitude of some growth responses was large as nutrient ratios in the foliage increased to levels within the norms range. Increases in current annual volume increment (CAI) of 3.48 m3 ha-1 a-1 and 3.62 m3 ha-1 a-1 in 1F plots at Grabouw and La Motte plantations indicated that it may be economically feasible to fertilise at mid rotation age as the NPV and IRR increased over a projected 25 year rotation. The Grabouw site had the most significant response with regards to CAI in 2F treatment with a mean volume increment of 5.43 m3 ha-1 a-1. The mechanism of the response was examined further by taking water availability and soil characteristics into account. The seasonal climatic effect (length of the moisture growing season) had a significant influence on the response to fertilisation. / AFRIKAANSE OPSOMMING: Die toediening van mid-rotasie bemesting word algemeen in bosbou toegepas om voedingstofbeskikbaarheid te verhoog in areas waar voedingstowwe onvoldoende is vir hoë produktiwiteit. Daar is oor die algemeen ruim potensiaal vir mid-rotasie bemesting in denne plantasies, maar eienskappe soos waterbeskikbaarheid, gronddiepte, opstanddigtheid en beskikbaarheid van voedingstowwe moet in ag geneem word voor optimum bemestingtoedienings bepaal kan word. Blaaranalise is gebruik om voedingstofbeskikbaarheid in plantasies te skat. Hierdie voedingstofvlakke is deurgans gemeet en is gebruik om spesifieke bemestingsbehandelings te implementeer wat voedingstofbeskikbaarheid kon opstoot. Behandelings het bestaan uit ‘n onbemeste kontrole, ‘n eenmalige kunsmistoediening (1F) en twee kunsmistoedienings in opeenvolgende jare (2F). Blaar oppervlak indeks en toename in stamvolume is gemeet om die invloed daarvan op die effektiwiteit van groei te bepaal. Blaaroppervlakindeks is bepaal deur middel van die gapingfraksie metode met behulp van ‘n stralingsmeter. Toename in volume is bereken met stamdeursnee en hoogte meetings. Basale oppervlakte is bereken deur middel van deursnee metings. Hierdie groeireaksies is gebruik om die effek van verbeterde voedingstofbeskikbaarheid te bepaal. Al die groeireaksies het toegeneem maar was nie statisties beduidend nie. Die gebrek aan beduidende toename kan toegeskryf word aan variasies in opstandsdigtheid en oorspronklike volume van die bome in die navorsingspersele. Die toetstydperk van 18 maande het moontlik nie genoeg tyd gegee vir die bome om op die toename in voedingstofbeskikbaarheid te reageer nie. ‘n Goeie groeirespons is wel waargeneem waar die voedingstofverhoudings in die naalde aanvaarbare norme bereik het. Die toename in volume aanwas van tussen 3.48m3 ha-1 a-1 en 3.62 m3 ha-1 a-1 in 1F persele by Grabouw en La Motte plantasies het aangedui dat dit ekonomies lewensvatbaar is om op mid-rotasie ouderdom bemesting toe te dien aangesien die netto teenswoordige waarde en die interne opbrengs koers toegeneem het op ‘n geprojekteerde 25 jaar rotasie. Die persele op Grabouw plantasie het die mees beduidende respons getoon met betrekking tot huidige jaarlikse aanwas (5.43 m3 ha-1 a-1 in die 2F perseel). Die meganisme van die respons is verder nagevors met inagneming van waterbeskikbaarheid en grondeienskappe. Die seisoenale klimaatseffek (lengte van die vog-groeiseisoen) het ‘n beduidende impak op die respons tot bemesting.

Submitted by Maximiliano Kawahata Pagliarini null (max.pagliarini@gmail.com) on 2016-07-19T18:22:23Z No. of bitstreams: 1 Tese_Maximiliano_Pagliarini_PPGAgronomia_UNESP.pdf: 2759397 bytes, checksum: 252905ebd212e708c5070c9c9f59d79a (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-20T18:24:06Z (GMT) No. of bitstreams: 1 pagliarini_mk_dr_ilha.pdf: 2759397 bytes, checksum: 252905ebd212e708c5070c9c9f59d79a (MD5) / Made available in DSpace on 2016-07-20T18:24:06Z (GMT). No. of bitstreams: 1 pagliarini_mk_dr_ilha.pdf: 2759397 bytes, checksum: 252905ebd212e708c5070c9c9f59d79a (MD5) Previous issue date: 2016-05-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Espécies exóticas de Pinus foram introduzidas no Brasil para promoverem o crescimento socioeconômico do país e ajudar na redução da pressão causada pelo uso de florestas nativas Com a crescente demanda por essas espécies, pesquisas em melhoramento genético tem aumentado na busca de novos germoplasma mais produtivos em menor tempo. Duas espécies foram utilizadas no presente trabalho: Pinus elliottii Engelm. var. elliottii e Pinus radiata D. Don. A primeira parte do trabalho teve a finalidade de identificar a estabilidade, a adaptabilidade, a produtividade e os parâmetros genéticos, além do ganho de seleção e diversidade genética em progênies de polinização aberta de segunda geração de P. elliottii var. elliottii considerando os caracteres fenotípicos. Foram estabelecidos dois testes, um em Ponta Grossa-PR com 24 progênies e outro em Ribeirão Branco-SP com 44 progênies visando identificar os genótipos mais produtivos para áreas de plantio comercial em ambos locais. Foi observada variação significativa (p<0,01) entre as progênies para os caracteres de crescimento e alguns caracteres de forma. Os altos coeficientes de variação genética para volume de madeira (14,31% a 16,24% - Ribeirão Branco e 31,78% a 33,77% - Ponta Grossa) e herdabilidade (0,10 a 0,15 – Ribeirão Branco e 0,36 a 0,48 – Ponta Grossa) mostraram baixa influência do ambiente na variação fenotípica, o que é importante para a predição do ganho genético mediante a seleção e confirmam potencial genético em ambos os locais, especialmente Ponta Grossa. O efeito da interação genótipo x ambiente é simples. As progênies plantadas em um local poderão também ser plantadas no outro. Dentre essas as C-197, C-189-1, C-084-2 e C-032-2 são indicadas para plantações tanto na região estudada do estado de São Paulo quanto do Paraná. Apesar de um número maior de progênies em Ribeirão Branco, constatou-se o mesmo número de agrupamentos de progênies pelo método UPGMA e de otimização de Tocher em ambos os testes. Existe diversidade genética entre as progênies de P. elliottii. Para programas de melhoramento, recomenda-se o cruzamento entre progênies de grupos divergentes para aumentar a variação genética, e consequentemente, o ganho genético nas gerações subsequentes, sem esquecer de se levar em consideração a performance do caráter de interesse. O objetivo do trabalho em P. radiata foi relacionar os resultados de características da madeira obtidas a partir de dois métodos Pilodyn e SilviScan visando validar uma metodologia eficiente para fenotipagem de um maior número de amostras. Um teste com 30 progênies de P. radiata foi estabelecido em Flynn na Austrália. As características avaliadas foram densidade da madeira, o ângulo microfibrilar e o módulo de elasticidade. A correlação genética e fenotípica entre os caracteres da madeira obtidas a partir dos dois métodos e a herdabilidade individual no sentido restrito foram estimadas. Os dados de Pilodyn apresentaram alta herdabilidade e alta correlação genética e fenotípica entre densidade de madeira e moderada com ângulo microfibrilar e módulo de elasticidade. Os resultados confirmam que o Pylodyn é um efetivo método indireto e rápido para avaliação de parâmetros genéticos para caracteres de qualidade madeira em P. radiata. / Exotic forest species have been introduced in Brazil in order to promote improvements in socioeconomic development and help to reduce the pressure caused to native forests. With growing demand for these species, research on genetic improvement has increased to find new, more productive germplasm and preferably in less time. Two species were used in the study: slash pine (Pinus elliottii Engelm. var. elliottii) and radiata pine (Pinus radiata D. Don). The first part of the study had the purpose to identify the stability, adaptability, productivity and genetic parameters, in addition to selection gain and genetic divergence in slash pine open pollinated second generation progenies considering phenotypic trait. Two tests were established, one in Ponta Grossa-PR with 24 progenies and one in Ribeirão Branco-SP with 44 progenies, both in Brazil, to identify the most productive genotypes for commercial planting areas in both sites. There was significant variation (p<0.01) among progenies for growth and form traits. The high coefficients of genetic variation for wood volume (14.31% to 16.24% - Ribeirão Branco-SP and 31.78% to 33.77% - Ponta Grossa-PR) and heritability (0.10 to 0.15 – Ribeirão Branco-SP and 0.36 to 0.48 – Ponta Grossa-PR) have shown low environmental influence on phenotypic variation, which is important for the prediction of genetic gain by selecting and confirming genetic potential in both places, especially Ponta Grossa. The effect of genotype x environment interaction is simple. Progenies planted in one site can also be planted in the other. Among these C-197, C-189-1, C-084-2 and C-032-2 progenies are suitable for plantations in both studied region of São Paulo and Paraná. Although larger number of progenies in Ribeirão Branco, it was found the same number of clusters through UPGMA and Tocher methods in both tests. There is genetic diversity among slash pine progenies. For breeding programs, it is recommended to cross progenies between different groups to increase genetic variation, and consequently the genetic gain in subsequent generations, not forgetting to take into account the performance of interest trait. The objective of the study in Radiata pine was relate wood quality traits obtained from two methods Pilodyn and SilviScan to validate an efficient phenotyping methodology for a greater number of samples. A test with 30 progenies of Radiata pine was established in Flynn Australia. The evaluated traits were wood density, microfibril ange and modulus of elasticity. Genetic and phenotypic correlation between traits of wood quality obtained from two methods and narrow-sense individual heritability were estimated. The Pilodyn data showed high heritability and high genetic and phenotypic correlation between wood density and moderate with microfibril angle and modulus of elasticity. The results confirm that the Pylodyn is an effective indirect and rapid method for evaluation of genetic parameters for wood quality traits in Radiata pine.

The chemical elicitor methyl jasmonate (MeJA) could replace insecticides in Europe and Asia for protection of young conifers against the pine weevil (Hylobius abietis). This thesis mainly focuses on the effects of MeJA treatment on chemical defenses of conifers from seedling batches with documented field resistance. Tissues of three pine species and one spruce species, with various treatments, were here extracted in hexane, whereafter volatile contents of tissues were separated and analyzed by 2D GC-MS. Induced responses of seedlings of Maritime pine (Pinus pinaster) and Monterey pine (Pinus radiata) to the folivore pine processionary (Thaumetopoea pityocampa), and the phloem-feeder H. abietis, have been studied. Amounts of mono- and sesqui-terpenes (and also non-volatile resin) in conifer tissues (needles and phloem) were less induced by T. pityocampa than by H. abietis. The MeJA-treated seedlings of Scots pine (Pinus sylvestris) changed their composition of phloem monoterpenes (induction of (-)-β-pinene), and were better protected in field than the seedlings of Norway spruce (Picea abies), which increased their total amounts of monoterpenes. Orientation bioassays with H. abietis showed deterrent effects of (-)-β-pinene, (+)-3-carene, (-)-bornyl acetate and 1,8-cineole. Conversely, (-)-α-pinene (induced in P. abies but not in P. sylvestris) was non-deterrent. MeJA-treated seedlings fed on by H. abietis contained higher amounts of the H. abietis antifeedant 2-phenylethanol, in tissues and emissions. Phloem of control seedlings instead induced the (+)-α-pinene, which is one of the enantiomers of the H. abietis attractant α-pinene. In response to MeJA treatment, the relative amounts of the (+)-α-pinene increased in the phloem of  P. radiata, while it decreased in P. pinaster phloem. The preference of H. abietis in the field for P. radiata before P. pinaster may be explained by these changes in enantiomers of the H. abietis attractant α-pinene. / Barrträd är viktiga för skogsindustrin. I Sverige består 57% av ytan av produktiv skogsmark, och av dessa träd utgörs 80% av gran och tall. Som försvar mot insekter och sjukdomar, producerar barrträden kådämnen. För unga barrträd i Europa och Asien är snytbaggen (Hylobius abietis) en allvarlig skadegörare, som orsakar skogsnäringen stora kostnader. För att skydda små gran- och tallplantor före utplanteringen, skulle växthormonet metyljasmonat (MeJA) kunna användas. I denna avhandling har effekter av MeJA-behandling och insektsangrepp undersökts på de två viktigaste barrträden i Sverige, tall och gran (Pinus sylvestris och Picea abies) och på två arter i medelhavsområdet (Pinus radiata och Pinus pinaster). De kemiska försvarsämnena i barrträd har analyserats för att utvärdera effekterna av MeJA-behandling. Vävnader från barrträd med olika behandling har extraherats i organiska lösningsmedel och dofterna från plantorna har insamlats genom fastfas-mikroextraktion. De flyktiga ämnena har separerats och identifierats med hjälp av gaskromatografi och masspektrometri (GC-MS). Dessutom har optiskt aktiva doftämnen separerats med hjälp av en tvådimensionell GC-MS. Plantornas olika försvarsreaktioner har studerats vid angrepp av snytbaggen, som är en floemätare, och av tallfjärilslarv (Thaumetopoea pityocampa), som huvudsakligen äter barr. De lättflyktiga ämnena (mono- och seskviterpener) och de icke-flyktiga ämnena (kådsyror) ökade mer i stam och barr från angrepp av H. abietis än av T. pityocampa. Sammansättning av monoterpener ändrades i de MeJA-behandlade tallplantorna i Sverige, specifikt ökade (-)-β-pinene i stamfloemet. Tallplantorna var bättre skyddade i fält än granplantorna, där den totala mängden monoterpener ökade. (-)-β-Pinene, (+)-3-carene, (-)-bornyl acetate och 1,8-cineole visade sig ha en negativ effekt på snytbaggens orientering mot talldoft i orienteringstester. En av monoterpenerna, (-)-α-pinene, som av MeJA-behandlingen inducerats i granen, men inte i tallen, hade ingen negativ effekt på snytbaggen. MeJA-behandlade tallplantor som angripits av snytbaggen, innehöll större mängder av snytbagge-äthämmaren 2-fenyletanol, både i vävnader och i emissioner. Stam från kontrollplantor inducerade istället (+)-α-pinene, vilken är en av de optiska isomererna av snytbagge-attrahenten α-pinene. För de spanska arterna, som svar på MeJA-behandling, ökade de lättflyktigaste terpenerna i barren hos P. radiata men inte hos P. pinaster, vilken redan innehöll en hög terpenhalt. De relativa mängderna av (+)-α-pinene ökade också i stammen hos P. radiata, medan de minskade i P. pinaster. I fält väljer snytbaggen att gå till P. radiata- framför P. pinaster-plantor, vilket kan vara kopplat till de skillnader i α-pinene-isomerer, vilka här har observerats. / <p>QC 20160304</p>

Leaky buildings are those that show elevated moisture contents of the framing timber, which can subsequently lead to the establishment of fungal and bacterial decay. Prior to this study, the causative agents of the decay in these leaky buildings were unknown, though it was suspected to be one or more species of decay fungi. Therefore, the overall goal of this multi-disciplinary PhD thesis research was to determine the causative agents of decay in leaky buildings of New Zealand in an effort to develop solutions for both their remediation and future prevention. Use of molecular biology methodology and classical mycological techniques based on morphology enabled identification of decay fungi from framing timber and air samples of leaky New Zealand buildings and provided insight into relative importance based on isolation frequency. In most cases, fungi colonising Pinus radiata D. Don were isolated to produce pure cultures. Mycelia from these cultures on agar media were collected to extract DNA. To identify the fungi to the species level, polymerase chain reaction (PCR) with fungal specific DNA primer pairs were performed followed by DNA sequencing of the internal transcribed spacer (ITS) region. Identification was by BLAST (Basic Local Alignment Search Tool) search on sequences in known GenBanks. In total, 421 samples from leaky buildings were processed, predominately untreated P. radiata decayed framing timber and also fibre cement boards and building paper. From these, sixty-eight fungal identifications were made. The only taxa that were isolated with significant frequency were identified as 4 basidiomycete species, as follows, along with the number of times they were isolated from the 421 samples: • Gloeophyllum sepiarium (Wulf.: Fr.) Karst. 13x • Oligoporus placenta (Fries 1865) Gilb. In Ryv.1985 11x • Antrodia sinuosa (Fr.) Karst. 8x • Gloeophyllum trabeum (Fr.) Murr. 4x Although these species were identified repeatedly, in total they represent less than 10% of the total samples and, therefore, it is concluded that the leaky building decay samples represent high fungal biodiversity. An aerial spore study of internal air, wall cavity air and exterior air of leaky buildings was carried out using a Merck MAS-100 instrument which collects spores directly onto selective media plates. Viable fungal aerial spores were detected at every sampling location tested at the leaky buildings, by the criteria of culturing, with a highest mean of 3714 colony-forming units (CFU) per cubic metre found in the cavities of water-damaged walls. This aerial spore study in conjunction with isolation from decayed wood samples from the same leaky buildings enabled identification of G. sepiarium and A. sinuosa at the same test site. The use of carboxymethylcellulose medium further demonstrated the presence of potential cellulose-degrading fungi within and around the location. Overall, the combination of direct sampling of timber and air sampling proved useful for detection of fungal species variability at a multi-unit building. Four decay fungi isolated from New Zealand leaky buildings and two standard control decay fungi (Coniophora puteana and Serpula lacrymans) were submitted to laboratory wood block testing to determine the effectiveness of currently used wood framing preservatives under laboratory conditions before and after a standard leaching regime. P. radiata blocks were treated with water based boron copper azole and solvent based IPBC propiconazole plus tebuconazole (1:1) preservatives and exposed to the basidiomycetes for 12 weeks. Mass loss for the fungal decay-infected samples was recorded of up to 55% for preservative-treated samples, up to 62% mass loss for leached samples and up to 58% mass loss for un-preservative treated samples. Additionally, well defined dosage responses and approximate toxic thresholds were obtained for all preservatives tested. Results suggested that the minimum IPBC retention specified by Hazard Class 1.2 of NZS3640:2003 (0.025% m/m) is on the low side, and demonstrated after the 2 week leaching regime complete loss of efficacy of boron at 0.4% m/m boric acid equivalent (BAE). This PhD research gave a first overview of fungi occurring in New Zealand leaky buildings, and it demonstrated the following key aspects of wood preservation: 1. The isolated test fungus Antrodia sinuosa was more difficult to control with propiconazole plus tebuconazole at retention 0.007% m/m than the known tolerant fungus Oligoporus placenta; 2. Boron at Hazard Class 1.2 retention of 0.4% m/m BAE was not toxic to Oligoporus placenta; 3. Serpula lacrymans exhibited tolerance to the highest retention of 0.06 %m/m tebuconazole plus propiconazole; and 4. Gloeophyllum species appeared susceptible to all wood preservatives. In order to correlate fungal colonisation and wood decay, colonised wood blocks were studied using light microscopy (LM) and field- emission scanning electron microscopy (FE-SEM). Microscopic observations of P. radiata wood blocks following a standard wood decay test of twelve weeks of fungal colonisation by Serpula lacrymans, Antrodia sinuosa, Oligoporus placenta and Gloeophyllum sepiarium revealed that the two microscopic techniques employed were complementary by allowing features such as pit membranes, chlamydospores or S3/S2 compound middle lamella interface to be photographed in greater detail, allowing for more precise analyses and interpretation of key findings, as follows: 1. Brown rot fungi directly target their apical growth towards degraded pit apetures; 2. Reliance on light microscopy and observed birefringence as a tool to record changes in cell wall crystallinity associated with brown rot decay alone could be misleading; 3. Presence of fine (≤ 1 m) to wide (≥ 3.5 m) bore-hole and hyphal size ranges, and nearly unchanged cell wall thickness of all wood/test fungal combinations, confirmed active decay at moderate to late stages; 4. Some ray parenchyma cells for Antrodia sinuosa, Oligoporus placenta and Gloeophyllum sepiarium colonised blocks were intact throughout late stages of decay, outlining that they were not preferentially degraded early in the brown rot decay process, and 5. Presence of bore-holes, clamp and medallion clamp formation and resting spores (chlamydospores and arthrospores) are fungal specific, can aid in their differentiation and identification, and should be recorded during wood decay studies, as especially resting spores are an important factor when planning remediation strategies. In summary, this PhD thesis research provided the first comprehensive investigation into the biodiversity of fungi from leaky New Zealand buildings, identified the dominant species and presented details about their micromorphology and their decay patterns. It also demonstrated substantial differences in efficacy of preservative formulations currently (December 2008) approved for framing treatments in New Zealand and possible deficiencies where framing may be subjected to severe leaching. This study also provided the first comparative analyses of viable fungal aerial spores between leaky wall cavities and the surrounding air environment. Subsequently, this research added to the knowledge of the decay fungal species diversity in and around New Zealand leaky buildings, outlined their capabilities to degrade treated and un-treated P. radiata framing timber and illustrated the efficacy of New Zealand approved wood preservatives for their potential as remedial treatment and future prevention.

Aboveground and belowground ectomycorrhizal (ECM) communities associated with different age classes of the exotic plantation species Pinus radiata were investigated over the course of two years in the North Island of New Zealand. ECM species were identified with a combined approach of morphological and molecular (restriction fragment length polymorphism (RFLP) and DNA sequencing) analysis. ECM species richness and diversity of a nursery in Rotorua, and stands of different ages (1, 2, 8, 15 and 26 yrs of age at time of final assessment) in Kaingaroa Forest, were assessed above- and belowground; furthermore, the correlation between the above- and belowground ECM communities was assessed. It was found that the overall and stand specific species richness and diversity of ECM fungi associated with the exotic host tree in New Zealand were low compared to similar forests in the Northern Hemisphere but similar to other exotic plantations in the Southern Hemisphere. Over the course of this study, 18 ECM species were observed aboveground and 19 ECM species belowground. With the aid of molecular analysis the identities of Laccaria proxima and Inocybe sindonia were clarified. In the aboveground study, five species were found associated with P. radiata that were previously not reported with this host in New Zealand (Inocybe sindonia, Lactarius rufus, Lycoperdon gunii, Rhizopogon pseudoroseolus and Wilcoxina mikolae). Belowground, the species Psudotomentella sp., P. tristis, R. luteorubescens, Tomentella sp., Wilcoxina mikolae were found as new associates of P. radiata in New Zealand, additionally nine ECM types were found that could not be identified with molecular analysis. There was little correlation between the species fruiting and the species colonising root tips. Only seven species were found in common between the above- and belowground communities, furthermore the dominant species aboveground were not observed in the belowground ECM communities. The influence of host age on the above- and belowground ECM communities of different age classes of P. radiata plantations was investigated. The aboveground species richness increased from the nursery to the oldest age group investigated (26 yrs), while diversity increased to the 15 yr old age group and decreased slightly to the oldest stand. A clear sequence of ECM species changes was observed to be related to stand age with a growing complexity over the chronosequence. The belowground ECM communities showed a different picture and richness and diversity initially decreased from the nursery to the outplanting but increased thereafter. Belowground no change in ECM composition that was directly related to the age of the host was observed, but two distinct groups of ECM species were found – a 'young' and a 'plantation forest' group, with the respective discriminating species being Rhizopogon rubescens and Type unknown Basidiomycete/Amanita muscaria. Another aspect of the study was the fate of the nursery ECM species in the outplanting and the arrival of non-nursery species. The ECM communities of seedlings in the nursery were investigated in 2006 and these seedlings were followed up over eight assessments in the field for one year, furthermore data from the 1-, 2 and 8 yr old plantation stands was analysed. It was found that the nursery species do survive the first year of outplanting and are dominant in the first year. The first non-nursery species occurred six months after outplanting but was only in minor abundance. Nursery ECM were dominant for two years after the seedlings were planted, and were completely replaced after seven years. Rhizopogon rubescens was found to be the most persistent and dominant species in the outplanting, facilitating the successful establishment of the seedlings in the plantation forest.

Interactions between plant roots and soil microorganisms in the rhizosphere are critical for plant growth. However, understanding of precisely how root exudates influence the diversity and activity of rhizosphere microorganisms is limited. The main objective of this study was to investigate the effect of radiata pine (Pinus radiata) root exudates on rhizosphere soil microbial communities, with an emphasis on the role of low molecular weight organic anions. The study involved the development and validation of new methods for investigating rhizosphere processes in a purpose-built facility. This included development of an in situ sampling technique using an anion exchange membrane strip to collect a range of organic anions exuded from radiata pine roots grown in large-scale rhizotrons. These included tartarate, quinate, formate, malate, malonate, shikimate, lactate, acetate, maleate, citrate, succinate and fumarate. Soil microbial activity and diversity were determined using dehydrogenase activity and denaturing gradient gel electrophoresis. Links between organic anions in root exudates and rhizosphere soil microbial community structures were investigated by comparing wild type and genetically modified radiata pine trees which were grown in rhizotrons for 10 months. As expected, there was considerable temporal and spatial variability in the amounts and composition of organic anions collected, and there were no consistent or significant differences determined between the two tree lines. Significant differences in rhizosphere microbial communities were detected between wild type and genetically modified pine trees; however, they were inconsistent throughout the experiment. The shifts in microbial communities could have been related to changes in exudate production and composition. Based on results from the main rhizotron experiment, a microcosm study was carried out to investigate the influence of selected pine root exudate sugars (glucose, sucrose and fructose) and organic anions (quinate, lactate and maleate) on soil microbial activity and diversity. Soil microbial activity increased up to 3-fold in all of the sugar and organic anion treatments compared to the control, except for a mixture of sugars and maleate where it decreased. The corresponding impacts on soil microbial diversity were assessed using denaturing gradient gel electrophoresis and 16S rRNA phylochips. Addition of the exudate compounds had a dramatic impact on the composition and diversity of the soil microbial community. A large number of bacterial taxa (88 to 1043) responded positively to the presence of exudate compounds, although some taxa (12 to 24) responded negatively. Organic anions had a greater impact on microbial communities than sugars, which indicated that they may have important roles in rhizosphere ecology of radiata pine. In addition, a diverse range of potentially beneficial bacterial taxa were detected in soil amended with organic anions, indicating specific regulation of rhizosphere microbial communities by root exudates. This project highlighted the considerable challenges and difficulties involved in detailed investigation of in situ rhizosphere processes. Nonetheless, the findings of this study represent a significant contribution to advancing understanding of relationships between root exudates and soil microbial diversity, which will be further enhanced by refinement and application of the specific methodologies and techniques developed.

The general goal of this research was to understand the agronomic and physiological changes of a lucerne crop in distinct physical radiation environments and to verify the potential of lucerne to grow under shaded conditions. To achieve this, the research was conducted in four main steps: (i) firstly, experimental data collection in the field using two artificial shade materials (shade cloth and wooden slats) under inigated and non-irrigated conditions; (ii) a second experiment with data collection in a typical temperate dryland agroforestry area under non-irrigated conditions; (iii) generation of a light interception sub-model suitable for shaded crops and (iv) a linkage between the light interception sub-model and a canopy photosynthesis model for agroforestry use. In experiments 1 and 2, lucerne crop was exposed to 6 different light regimes: full sunlight (FS), shade cloth (FS+CL), wooden slats (FS+SL), trees (T), trees+cloth (T +CL) and trees+slats (T+SL). The FS+SL structure produced a physical radiation environment (radiation transmission, radiation periodicity and spectral composition) that was similar to that observed in the agroforestry site (f). The mean annual photosynthetic photon flux density (PPFD) was 41 % under the FS+CL, 44% under FS+SL and 48% under T compared with FS in clear sky conditions. Plants were exposed to an intermittent (sun/shade) regime under both FS+SL and T, whereas under FS+CL the shaded light regime was continuous. The red to far-red (RIFR) ratio measured during the shade period under the slats was 0.74 and under the trees was 0.64. However, R/FR ratio increased to 1.26 and 1.23 during the illuminated period under FS+SL and T, respectively, and these were equivalent to the ratio of 1.28 observed under the FS+CL and 1.31 in FS. The radiation use efficiency (RUE) of shoots increased under the 5 shaded treatments compared with full sunlight. The pattern of radiation interception was unchanged by radiation flux, periodicity and spectral composition and all treatments had a mean extinction coefficient of 0.82. However, the magnitude of the decrease in canopy growth was less than those in PPFD transmissivity. The mean lucerne annual dry matter (DM) yield was 17.5 t ha⁻¹ in FS and 10 t ha⁻¹ under the FS+CL, FS+SL and T regimes. This declined to 3.4 t DM ha⁻¹ under T+CL (22% PPFD transmissvity) and 4.1 t DM ha⁻¹ under T+SL (23% transmissivity). A similar pattern of response was observed for leaf net photosynthesis (Pn) rates under the shade treatments compared with full sun. In addition, spectral changes observed under the trees and slats affected plant motphology by increasing the number of long stems, stem height and internode length compared with full sunlight. Thus, there were two main explanations for the increase in RUE under shade compared with full sun: (i) preferential partition of assimilates to shoot rather than root growth and/or (ii) leaves under shade were still operating at an efficient part of the photosynthetic light curve. The changes proposed for the canopy Pn model were appropriate to simulate the radiation environment of an agroforestry system. However, the model underestimated DM yields under the continuous and intermittent shade regimes. These were considered to be mainly associated with plant factors, such as overestimation in maintenance respiration and partitioning between shoots and roots in shade and the intermittency light effect on leaf Pn rates. Further investigation in these topics must be addressed to accurately predict crop yield in agroforestry areas. Overall, the lucerne crop responded typically as a sun-adapted plant under shade. It was concluded that lucerne yield potential to grow under intermediate shade was superior to most of C3 pastures previously promoted in the literature.

A series of glasshouse and laboratory experiments was carried out to enable comparison of two woody perennial legumes, gorse and broom, with other legumes, nodulated non-legumes and other biological nitrogen fixing systems. Both species had distinct juvenile phases in which broom closely resembled herbaceous species in appearance, but adult plants of both species bore little resemblance to each other or to other legume species. Nodule development was similar to that of other legumes, but mature nodules exhibited structural adaptations to longevity - meristematic activity, a well developed vascular system and numerous cytoplasmic granules in cortical cells. Acetylene reduction and ¹⁵N₂ fixation continued for much longer following excision than has been observed in other legumes. In all experiments, broom nodules exhibited higher rates of acetylene reduction and nitrogen fixation than did gorse nodules. The first detectable product of nitrogen fixation in excised nodules - ammonia - was rapidly incorporated into amide and ∝ amino groups and another unidentified fraction. The principle free amino acid in nodules and sap was asparagine. Its preponderance increased as plants aged. Whole nodulated plants and excised nodules of both species exhibited a relatively low temperature optimum for growth and nitrogen fixation (22°C). They were very sensitive to elevated temperatures. Results indicated that gorse and broom have relatively low light requirements. When aeration was sufficient, combined nitrogen had little effect on growth of nodulated plants. Nodulation in both species was reduced by increasing amounts of combined nitrogen. High levels (100 mg/1) of nitrate and ammonia caused considerable inhibition of nitrogen function. Both species showed large responses to phosphate, but were able to grow and fix nitrogen when supplied with low amounts of phosphate. Boron deficiency reduced nitrogen fixation. Nodulation was increased to compensate for this. Considerable amounts of nitrogen can be contributed to the ecosystem in gorse end broom litter. Direct transfer between gorse or broom and Pinus radiate is likely to be small and may be masked by competition for other nutrients. These findings are discussed with respect to the use of gorse and broom to overcome nitrogen deficiency in reafforestation on the Moutere Gravels, in Nelson, N.Z.

We are currently faced with the global challenge of conserving biological diversity while also increasing food production to meet the demands of a growing human population. Land-use change, primarily resulting from conversion to production land, is currently the leading cause of biodiversity loss. This occurs through habitat loss, fragmentation of remaining natural habitats, and resulting edge effects. Land-sparing and land-sharing approaches have been discussed as alternative ways to engineer landscapes to mitigate biodiversity loss while meeting production objectives. However, these represent extremes on a continuum of real-world landscapes, and it will be important to understand the mechanisms by which adjacent land use affects natural remnant ecosystems in order to make local land-management decisions that achieve conservation, as well as production, objectives. This thesis investigates the impact of juxtaposing production and natural forest on the community-wide interactions between lepidopteran herbivores and their parasitoids, as mediated by parasitoid spillover between habitats. The first and overarching objective was to determine whether herbivore productivity drives asymmetrical spillover of predators and parasitoids, primarily from managed to natural habitats, and whether this spillover alters trophic interactions in the recipient habitat. The study of trophic interactions at a community level requires understanding of both direct and indirect interactions. However, community-level indirect interactions are generally difficult to predict and measure, and these have therefore remained understudied. Apparent competition is an indirect interaction mechanism thought to be very important in structuring host-parasitoid assemblages. However, this is known primarily from studies of single species pairs, and its community-wide impacts are less clear. Therefore, my second objective was to determine whether apparent competition could be predicted for all species pairs within an herbivore assemblage, based on a measure of parasitoid overlap. My third objective was to determine whether certain host or parasitoid species traits can predict the involvement of those species in apparent competition. My key findings were that there is a net spillover of generalist predators and parasitoids from plantation to native forest, and that for generalists, this depends on herbivore abundance in the plantation forest. Herbivore populations across the edge were linked by shared parasitoids in apparent competition. Consequently, an experimental reduction of herbivore density in the plantation forest changed parasitism rates in the natural forest, as predicted based on parasitoid overlap. Finally, several host and parasitoid traits were identified that can predict the degree to which host or parasitoid species will be involved in apparent competition, a finding which may have extensive application in biological control, as well as in predicting spillover edge effects. Overall, this work suggests that asymmetrical spillover between production and natural habitats occurs in relation to productivity differences, with greater movement of predators and parasitoids in the managed-to-natural forest direction. The degree to which this affected species interactions has implications for landscape design to achieve conservation objectives in production landscapes.