The molecular mechanisms underlying disease-resistance and drought-resistance in forest trees are not well understood. Linking variation in gene expression with genetic polymorphisms and with variations in disease- and drought-resistance phenotypes can provide information about these complex traits. We used real-time quantitative polymerase chain reaction (PCR) to detect variations in the expression of 88 disease- and drought-responsive genes within an association population of 354 loblolly pine trees (Pinus taeda L.). Using association genetics approaches, we then linked 3,938 single nucleotide polymorphisms (SNPs) in candidate genes with gene expression phenotypes to identify novel disease- and drought-responsive genes. To further examine differences in gene expression induced by drought, Fusarium circinatum (responsible for pitch canker disease), and drought F. circinatum, the expression of 114 genes identified through comparative and association genetics approaches was analyzed on a subset of 24 loblolly pine trees possessing a range of pitch canker- and drought-resistance phenotypes. Significant differences in the uninduced expression of all 88 genes measured on the association population were observed among loblolly pine trees. Principal component analysis showed that some variation within the association population could be accounted for by population substructure of geographic origin. Hierarchical clustering of genes based on uninduced expression did not consistently group together functionally similar genes probably because expression was collected on unstressed stem tissue. This was supported in the smaller expression study as correlations between expression values of genes in the same functional networks were usually stronger when induced by a treatment compared with correlations between the uninduced expression of genes in the control group. Gene expression frequently changed by up to 4-fold in response to one or more treatments, but PtMYB12 was the only gene that exhibited a statistically significant change in response to treatments. ANOVA analyses of gene expression controlling for pitch canker resistance and for water use efficiency phenotypes identified differentially expressed genes suggesting that they may be contributing to these phenotypes. Finally, association genetics approaches detected 101 significant associations between SNPs in 94 candidate genes potentially involved in stress responses and 27 gene expression phenotypes.

Monterey pine (Pinus radiata), a native tree to California and two Mexican islands, is important both ecologically and economically. Outside native stands, Monterey pines are grown for landscaping in California and on plantations around the world. Pitch canker, a disease caused by the fungus Gibberella circinata Nirenberg & O’Donnell (Fusarium circinatum Nirenberg and O'Donnell) is threatening the survival of Monterey pines. The disease currently affects Monterey pines in many parts of the world including the native stands. No effective chemical or biological control is available but some Monterey pines show resistance to the disease. The purpose of this project was to develop a working protocol for producing genetic clones of the resistant pines through micropropagation. These genetic clones will be used for outplanting in places outside the native stands for ornamental and plantation purposes. This project analyzes the results of ten trials with varied parameters and bases the final protocol on the parameters used in the trial that induces the growth of new shoots. The final protocol developed in this project describes, step-by-step, the media preparation for the initiation, plant material collection, surface sterilization of plant material, plating in media and initiation of shoots on explants. The protocol calls for collecting shoot tips with hardened buds that have not yet elongated, then washing the shoot tips in sterile water with Tween 20 for 15 minutes. The shoots tips are then surface sterilized in a 50% bleach solution for 20 minutes. The explants are broken into disks (to minimize damage to the cells) by inserting the tip of a scalpel and tilting it slightly. The initiation media shown to induce growth consists of ½ strength LePoivre basal salt mixture, 5mg/L benzylaminopurine, 3% sucrose and 0.8% agar and is adjusted to a pH of 5.7, then autoclaved for 20 minutes. The explants are inserted into solidified media and incubated in a growth chamber programmed for 16 hours of light and 8 hours of dark with temperatures of 27ºC and 22ºC and light irradiance of 80µEm-2s-1. After 1 month the protocol calls for transferring the growing shoots to elongation media with full LP basal salts and transferring every month. When the number of desired shoots has been reached the forthcoming protocol for rooting can be followed.

The future resilience of Pinus radiata D. Don (Monterey pine) is dependent upon the development of a silviculture program inclusive of either preventative or management techniques for the potentially fatal pitch canker disease (Fusarium circinatum Nirenberg and O’Donnell [=F.subglutinans (Wollenw. & Reinking) Nelson et al. f. sp. Pini). As an ecologically and commercially valued species, a myriad of factors threaten the genetic resources of the geographically limited natural range. This study evaluated the effectiveness of uneven-aged forest management for regeneration success in the native, pitch canker infected Año Nuevo stand at Swanton Pacific Ranch in Davenport, California. Seedling survival and growth averages were used to evaluate the impact of the variables of gap size (0.20-hectare, 0.10-hectare, and 0.05-hectare), site-preparation treatment (pile and burn, lop and scatter), and parent tree (13 local seed sources). Pitch canker symptoms were quantified to conclude if there is indeed a range of expressed resistance according to parent tree. Statistical models (i.e. ‘mixed effect’) yielded moderately significant differences in odds of survival between site treatments, with pile and burn plots producing higher survival rates (p=0.066). No significant difference in survival was detected between gap-sizes (p=0.936 and 0.803, 0.05- and 0.10-hectare gaps respectively to 0.20-hectare reference). Significant variation was expressed (estimate=0.1219, SE=0.057) between the 13 parent trees, implicating usefulness in predicting seedling survival. ‘Mixed effect’ models only detected a significant effect from gap-size on growth measures: 0.20-hectare gaps yielded height and diameter measures significantly larger than 0.05-hectare gaps (p=0.027 and 0.0081 respectively). Contingency tables detected significant association between gap-size and number of pitch canker branch symptoms. The range of infection rates produced by the 13 parent trees did not prove significant by analysis. Pile and burn site preparation treatment increases the probability of survival for artificially regenerated seedlings and 0.20-hectare gaps yield significantly higher growth averages over 0.05-hectare gaps.

This study was a part of the international collaborative IMPACT project, which aims to address the potential threat that the pitch canker disease poses to the use of Pinus radiata D. Don in plantations in New Zealand, Australia, and Chile. A field trial of 264 seedstocks was planted adjacent to a native stand of pitch canker infected P. radiata on the central coast of California, and disease symptom development was recorded over a period of 3 years. The results did not correlate with a greenhouse study of the same seedstocks inoculated with Fusarium circinatum Nirenberg & O'Donnell, the causal agent of pitch canker. Three main types of symptoms were identified (branch flagging, pitchy buds, and chlorotic tips), and preliminary isolation analyses suggest that the disease observed is actually caused by Diplodia pinea (Desm.) Kickx. Survival analysis showed that the effect of tree genetic origin was significant to its time to disease, and that spatial location in the plantation was also significant. Average nearest neighbor analysis showed disease distribution to be significantly clustered, which also suggests that the disease is not pitch canker, but diplodia blight. This experiment illustrates the difficulty in performing naturally infected field trials when another similar-looking fungal disease is also present. It also provides data on seedstock resistance to diplodia blight, another fungal disease important to P. radiata forest managers.

Thesis (MScAgric )--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Pine pitch canker, caused by Fusarium circinatum, is a major constraint to the production of high quality timber in the Western Cape Province of South Africa. Under nursery conditions the pathogen causes a serious root and collar rot disease, and in plantations it causes stem cankers and branch die-back. In this thesis, sources of inoculum within a forestry nursery were investigated and novel approaches developed to eradicate the pathogen. Plant material was evaluated for plantation establishment in the Western Cape Province, and epidemiological studies conducted to better understand the infection, disease development, spore release and dissemination of F. circinatum in pine plantations. Fusarium circinatum was first reported in a forestry nursery in the Mpumalanga Province of South Africa in 1990, and now causes significant economic losses to most forestry nurseries in the country. The irrigation system, planting tray inserts, seeds and air at Karatara nursery were screened as potential sources of F. circinatum. Both the irrigation water and planting tray inserts were found to serve as sources of inoculum. An oxidation reduction potential (ORP)-based system, using hydrogen peroxide, was tested to sanitize irrigation water and planting tray inserts. An ORP level of 400 mV for 6 hours was effective in killing F. circinatum spores in irrigation water without being phytotoxic to pine seedlings. An ORP value of >500 mV cleaned planting tray inserts of all inoculum. Pinus radiata is the principal pine species grown in the Western Cape Province, but this species is also highly susceptible to F. circinatum. There are numerous different varieties or families of P. radiata available that can be planted, and it is known that tolerance levels between these varieties and families vary. Pinus radiata families, as well as other Pinus spp. that could potentially be planted in the Western Cape Province in future, were evaluated for tolerance to the pitch canker fungus. The P. radiata families were moderately to highly susceptible to F. circinatum. Pinus taeda was the Pinus spp. most tolerant, while P. elliottii also had good tolerance to the pathogen. None of the P. radiata families screened showed tolerance comparable to that of either P. elliottii or P. taeda. Pine pitch canker was first reported from the Tokai plantation in the Western Cape Province in 2005. In order to develop a disease management strategy for the disease in pine plantations, we investigated the effect of pruning wounds as potential entry sites for the pathogen, and how fast the disease spread under South African climatic conditions. The amount and time of sporulation was also investigated. Pitch canker increased slowly over a 3-year period, and pruning wounds appeared not to serve as primary entry points for F. circinatum. Spore quantification over a 12-month period using quantitative real-time PCR showed that inoculum was produced throughout the year, but that significantly more spores were released during the cold, rainy winter months. The role of insects in pathogen transmission and spread should be investigated in the Tokai plantation. / AFRIKAANSE OPSOMMING: Fusarium circinatum veroorsaak “pine pitch canker”. Hierdie siekte is een van die grootste beperkinge op die Suid Afrikaanse bosbou industrie. In kwekerye veroorsaak die patogeen wortelvrot en wortelkraagvrot. In plantasies veroorsaak F. circinatum kankers op die hoofstam van dennebome en die terugsterwing van sytakke. In hierdie tesis was bronne van inokulum in ‘n bosbou kwekery ondersoek. Nuwe tegnieke was gebruik om die bronne uit te wis. Plantmateriaal wat in die Wes-Kaap geplant kan word, was geëvalueer vir toleransie teen F. circinatum. Epidemiologiesie studies was uitgevoer om die infeksieproses, siekte-ontwikkeling, spoorvrystelling, en verspreiding van F. circinatum binne denneplantasies beter te verstaan. Die eerste verslag van F. circinatum in ‘n bosboukwekery was in 1990 in die Mpumalanga Provinsie. Verliese word tans veroorsaak deur die patogeen in die meeste bosbou kwekerye in Suid Afrika. Die besproeiingstelsel, planthouers en saad was getoets as potensiële bronne van inokulum. Daar was gevind dat beide die besproeiingstelsel en die planthouers dien as bronne van F. circinatum inokulum. ‘n Oksidasie reduksie potensiaal (ORP)-gebaseerde sisteem waar waterstofperoksied gebruik was, was getoets of dit die besproeingwater en planthouers kan ontsmet. ‘n ORP-vlak van 400 mV vir ‘n blootstellingstydperk van 6 ure is effektief om al die F. circinatum spore dood te maak, sonder dat dit fitotoksies is teenoor die dennesaailinge. ‘n ORP-vlak van >500 mV het die F. circinatum in die planthouers uitgewis. Pinus radiata is die belangrikste dennespesie wat geplant word in die Wes-Kaap Provinsie, maar hierdie spesie is hoogs vatbaar vir F. circinatum. Verskillende variteite van P. radiata met verskillende tolleransie vlakke bestaan. Pinus radiata families en ander spesies wat in die toekoms potensieel geplant kan word in die Wes-Kaap was geevalueer vir toleransie teen F. circinatum. Die P. radiata families was matig to hoogs vatbaar vir F. circinatum. Pinus taeda het die hoogste toleransie vlak gehad en P. elliottii was matig tolererant. Nie een van die P. radiata families wat getoets was het toleransie vlakke gehad wat vergelykbaar was met die toleransie vlakke van P. taeda of P. elliottii nie. In 2005 was F. circinatum vir die eerste keer geraporteer in die Tokai plantasie op volwasse dennebome in die Wes-Kaap Provinsie. Die rol van snoeiwonde in die infeksieproses van F. circinatum was bestudeer asook hoe vinning die siekte ontwikkel en versprei. Die hoeveelheid en tyd van sporulasie was ook bestudeer. “Pitch canker” het stadig toegeneem oor die 3-jaar moniteringsperiode en dit blyk asof snoeiwonde nie die primêre ingangspunt vir die patogeen is nie. Kwantifisering van die sporulasie van F. circinatum deur middel van qPCR het aangedui dat spore reg deur die jaar geproduseer word. Meer spore word geproduseer in die koue nat wintersmaande. Die rol van insekte in die infeksieproses en verspreiding van F. circinatum in die Tokai plantasie moet ondersoek word.