Genetic Variation in the Foliar Pathogen Dothistroma septosporum and Relationship to Toxin Production

Transcription

1 Genetic Variation in the Foliar Pathogen Dothistroma septosporum and Relationship to Toxin Production Literature Review Prepared by Angie Dale and Kathy Lewis 1.0 Geographic distribution and history Dothistroma septosporum (Dorog.) M. Morelet (= D. septospora = D. pini) has been reported as causing defoliation in exotic Pinus radiata plantations in many countries in the southern hemisphere since the late 1950 s, and possibly earlier. Outbreaks of the fungus followed the widespread planting of exotic pine monocultures throughout countries in the Southern Hemisphere. In Africa, the fungus was a noteworthy pathogen in Tanzania in the late 1950s, and spread to Kenya in the sixties and by the middle of the sixties, had spread across east Africa (Gibson, 1972). Observations (Gibson et. al., 1964, cited from Gibson, 1972) suggest that the fungus was well established in central Africa since the early forties. In Chile, Pinus radiata was adopted for large-scale forestry in the 1930s (Gibson, 1972) and in 1965, Dothistroma septosporum was identified as the causal agent of a severe foliage disease by Dubin and Staley (1966). Evidence also suggested that the fungus was established several years prior to its identification as Dothistroma septosporum (Dubin, 1967, cited from Gibson, 1972). Loss in other countries of South America was minimal as Pinus plantations were not of appreciable size (Gibson, 1972). Dothistroma septosporum first made its appearance in New Zealand near Tokorua in 1964 and spread to the southeast and southwest by 1966 (Gilmour, 1966, cited from Gibson, 1972). The FSP Project Y051203, Lewis -1-

2 fungus also spread north and was a severe problem by 1967; it was also suggested that the fungus was present several years prior (Gilmour, 1966, cited from Gibson, 1972). D. septosporum also made its appearance throughout locations in the northern hemisphere in these time periods. Collections showed the fungus to be present in Idaho in the United States in 1917, Illinois in 1938 (Thyr & Shaw, 1964), and 1941 (Hulbary, 1941), and in Kansas, 1951 (Rogerson, 1951). Hulbary described the fungus (1941) proposing the name Dothistroma pini and assigning previous conspecific collections to the new species. Thyr and Shaw (1964) also assigned previously collected conspecific samples to this species and further divided the species into varieties based on conidial length differences found in the western United States (longer conidia, D. pini var. lineare) and central United States (shorter conidia, D. pini var. pini). Work by Ivory (1967) proposed a third variety with intermediete conidial lengths (D. pini var. keniensis). Reports of the fungus also exist from several other locales in the United States (Cobb & Miller, 1968). The fungus is also distributed throughout Canada, European and Asian countries, but lower temperatures and humidity may keep it from reaching epidemic levels in the latter two (Evans, 1984). In Canada and the United States, the disease not only occurs on non indigenous Pinus species, it also appears to be endemic to native species, and may be indigenous to these regions (Evans, 1984). Evans (1984) suggests that the fungus may have originated from the cloud forest regions in Central America and it remains a possibility it came to Canada and the USA from an earlier introduction from Central America (Evans, 1984). FSP Project Y051203, Lewis -2-

3 2.0 History of pathogen in BC Dothistroma septosporum was first identified as the cause of a serious foliage disease in British Columbia in 1964 (Parker & Collis, 1966). It was identified on both exotic pine species, Pinus radiata, P. muricata, P. pinaster, P. nigra var. calabrica, P. contorta x banksiana, and P. echinata x taeda on Vancouver Island, as well as throughout the province on some native pine species including P. contorta, P. monticola, and P. ponderosa (Parker & Collis, 1966). The severity of infection was worse in non-native pine species on Vancouver Island, with 60% mortality in eight plantations over three years including one seven acre plantation with 100% mortality (Parker & Collis, 1966). The fungus was identified throughout the province on native pine species, but infection was light, with the worse cases on two natural stands of Pinus contorta, one in Cinema, just south of Prince George, and one in Southern Vancouver Island (Parker & Collis, 1966). Other random surveys showed the fungus to be present in Kamloops, Nelson, Prince George, Prince Rupert, and Vancouver (Parker & Collis, 1966). During surveys done throughout British Columbia in 1964 and 1965, the teleomorph was identified in necrotic tissue of both exotic and native pine trees infected by D. septosporum (Funk & Parker, 1966). The ascostromata gave rise to cultures morphologically identical to cultures obtained from conidia. The ascospores were produced in late spring, early summer about a month after the conidia were produced, and production of ascospores lasted about a month (Funk & Parker, 1966). Since these initial studies, the fungus has been identified as a common forest pathogen in British Columbia (Hunt, 1995) and has been identified as the cause of a small but intense outbreak of FSP Project Y051203, Lewis -3-

4 needle blight in the mid 1980 s (Federal Insect and Disease Survey of Canada, Prince Rupert Region, 1984, unpublished data, cited from Woods, 2003). The fungus has now been identified as a major cause for concern due to the extent of outbreaks in Northwest British Columbia (Woods, 2003). Formal studies of population genetic structure or toxin production have not been done in British Columbia, presumably because the fungus has not caused serious damage and outbreaks have been small in the past (Lewis, 2004 personal communication). 3.0 The disease triangle A commonly used model to explain disease severity or the amount of disease is the disease triangle (Agrios, 1997). The disease triangle represents the three components needed for a disease outbreak. These include environmental conditions, host and pathogen. The factors that make up each of these three components all contribute to disease severity. The more favorable each factor is to disease, the greater the severity of the outbreak (Agrios, 1997). 3.1 Factors contributing to disease severity There are several factors that may affect the severity of outbreaks of Dothistroma septosporum. These include current forest management practices that affect the host, population structure or genetics of the host, favorable climate attributes, landscape structure that can affect local climate, and characteristics of the host including population genetic structure, life cycle, and virulence. A combination of a few or many of these attributes could create favorable conditions for disease epidemics. FSP Project Y051203, Lewis -4-

5 Pine is a very versatile species, with a rapid growth rate and an ability to tolerate many different habitats, which has lead to the widespread planting of many pine species worldwide. On a global scale, this has led to non-indigenous species growing in new habitats. Non-indigenous species can be susceptible to new pathogens that they have not coevolved with. More locally, establishment of provenances that are not well adapted to local biophysical environment can lead to more numerous and severe outbreaks. In many instances, and specifically in northwest BC, forest management practices have led to a shift in forest composition (Woods, 2003) from a mix of species, to dominance by species favored in the forest industry for attributes such as an increased rate of growth, success in a large range of environments, and marketability. Within the Kispiox Timber Supply Area (TSA) there has been a substantial change in forest composition (Woods, 2003). The Interior Cedar Hemlock biogeoclimatic zone dominates the Kispiox valley. Natural old growth stands (twenty-one years or older) in the area consist largely of western hemlock and subalpine fir, compared to young (less than 20 years old) primarily managed and reforested stands, dominated by lodgepole pine and interior spruce (Woods, 2003). This shift in forest composition has led to an over abundance of host, often in a monoculture style plantation, closely situated to one another, which facilitates spread of the pathogen. The genetic structure of the host population can affect outbreak severity. Well-adapted hosts can reduce disease severity due to genetic or phenotypic resistance. Low genetic diversity in the host population can be indicative of a highly adapted population; however, if a new pathogen is FSP Project Y051203, Lewis -5-

6 introduced, the host population may not be able to resist attack or evolve fast enough to develop resistance. The environment also plays an important role in disease outbreak. A set of favorable environmental conditions often precedes a large epidemic of many insect or pathogen outbreaks. Dothistroma septosporum is adapted to warm moist environments without temperature extremes. Studies by Peterson (1973) show that there is little to no germination of the conidia in times of low humidity and high temperatures, even if lots of conidia have previously been released. He also found times of infection and symptom development varied with temperature extremes. Water is also a key feature in a favorable environment, both moisture in the air, which can be found in close proximity to a water body, and moisture due to rainfall. Water is needed to induce germination and water is needed for dispersal of the conidia (Peterson, 1973). Air movement, which is governed by local topography, may also play a role in disease severity. Air movement and topography can create areas that are drier, or more humid, as well as cold air or warm air pockets. The third component in the disease triangle is the pathogen itself. Many factors can be present in a pathogen or in the pathogen population that can contribute to disease severity. These include large inoculum loads, greater virulence, and the pathogen population s evolutionary potential. Greater virulence can come from attributes of the pathogen such as an ability to overcome host defenses, the ability to produce toxin, and the quantity produced. The evolutionary potential is a result of factors such as mutation rates, effective population size, gene flow, natural selection, and reproduction system (McDonald and Linde, 2002). FSP Project Y051203, Lewis -6-

7 4.0 The Disease Cycle Dothistroma septosporum carries out its life cycle on the needles of many species of Pinus (Gibson, 1972). It spreads mainly via conidiospores, which are produced in an asexual fruiting body or stromata (Gibson, 1972). The stromata are produced from within the pine needle and upon maturation, erupt out of the epidermis of the pine needle. In moist conditions the stromata mature and rainfall causes the masses of conidiospores to rupture out (Peterson, 1973). These conidia are spread via a splash dispersal mechanism (Hulbary, 1941, Rogerson, 1951, Gibson, 1972). Infection of the pine needles takes place via germ tubes produced by the fungus, which penetrate the needle through the stomata (Gadgil, 1967, Ivory, 1970, Peterson, 1966). The fungal mycelium grows inter and intracellularly in the mesophyll tissue of the host, and is confined to necrotic tissue (Hulbary, 1941, Gadgil, 1967, Bradshaw, 2004). Upon host cell death, red bands are produced and stromata start to appear. Stromata mature and produce conidia one to two years after initial infection (Bradshaw, 2004). In Southern British Columbia, stromata rupture the epidermis in April and May, and about a month later in the interior of the province (Funk & Parker, 1966). Conidia are produced and released throughout the summer months. Ascospore production follows in early June and continues for about a month (Funk & Parker, 1966). The production of spores from late spring until late summer is common throughout the range of the fungus, and some production can also occur in the early spring months and late summer or fall months (Gilmour, 1981, Bradshaw, 2004). FSP Project Y051203, Lewis -7-

8 Symptoms of Dothistroma blight first appear as water soaked lesions on needles of the pine tree, followed by the appearance of red bands. The red banding is due to a mycotoxin, Dothistromin, produced during initial growth stages of the fungus (Shain & Franich, 1981). Black stromata appear within the red bands, and appear to erupt from the epidermis of the pine needle upon maturation. The needles eventually die and often drop prematurely. Infection can result in severe defoliation leaving trees with only a tuft of living needles at the ends of branches near the top of the tree (Thyr & Shaw, 1964). The pattern of infection on a tree can be attributed to dispersal by rain, where older needles located on the lower branches may have dropped or appear more severely affected, while the upper needles remain green or less symptomatic (Funk and Parker, 1966). Infection by Dothistroma septosporum can range in severity and result in reduced growth rates or wood yields (Van Der Pas, 1981) or in severe cases, infection can lead to death of the tree (Gibson, 1974, Woods, 2003). 4.1 Life cycle asexual vs sexual reproduction Life cycles of fungi vary between taxonomic groups ranging from almost completely asexual in most imperfect fungi, to cycles alternating between sexual and asexual (Agrios, 1997). The contribution of sexual reproduction in Dothistroma septosporum ranges. In most countries in the southern hemisphere where Dothistroma septosporum has long been an important forest pathogen, only the anamorph has been reported (Evans, 1984, Bradshaw, 2004). Favorable conditions may result in low selection pressures and a reduced need for a sexual reproduction cycle (Evans, 1984). The lower frequency of the teleomorph, and the shorter release period of ascospores suggests that primary dispersal of the fungus is through the asexual conidia (Cobb et FSP Project Y051203, Lewis -8-

9 al., 1969, Karadzic, 1989). Evans (1984) studied the varying morphology of the fungus and reproductive structures and suggests that it is the conidia that adapt to prevailing environmental conditions. The importance of the ascospores in the life cycle of the fungus is unknown at this time. The ascospores may play a role in dispersal during unfavorable environmental conditions. Evans (1984) collected samples from Central America in which earlier collections were composed of primarily the teleomorph and later collections were composed of primarily the anamorph. The ascospores are also produced for a shorter period during the summer months, while the conidia are produced over a longer period both prior to and following the period of production for ascospores (Funk and Parker, 1966, Karadzic, 1989) indicating a possible adaptation to temperature. The most important benefit of sexual reproduction is the generation of new genetypes which increases the ability of a population to adapt to changes in the environment, both biological and physical. Environments that the pathogen is indigenous to may impose more selective pressure on the population because the host is adapted to the environment. In environments such as these, a sexual stage may be of more importance to the pathogen. 5.0 Genetic Structure of Fungal Populations Genetic variation can directly affect a pathogen population s evolutionary potential. A fit host will pose a barrier to survival for a pathogen, and the pathogen must be able to overcome that barrier. Virulence is a measure of a pathogen s ability to cause disease in the host. If a pathogen is unable to cause disease in a host, then that pathogen is unlikely to be able to survive, or FSP Project Y051203, Lewis -9-

10 reproduce. It can therefore be considered unfit. The better a pathogen is at infecting a host and using the host nutrients for survival and reproduction, the more fit a pathogen will be. The genetic mechanisms that control virulence should be under positive selection in order for the pathogen population to continue to overcome resistance mechanisms in host populations. An available source of genes and alleles (present in populations with high diversity) coupled with recombination allows a population to adapt under selective pressure as genes are be shuffled and may produce more fit combinations. If these genes or combinations are not already present in the population, then a new source of variation is required for the population to survive. Sources of new variation include gene flow from other populations, spontaneous mutation, sexual recombination, or somatic hybridisation (Burdon, 1993). The most important source of genotypic variation in any population is sexual recombination. Sexual recombination causes the reassortment of alleles, genes, or combinations of both. It plays an important role in species that rely upon a sexual cycle to generate variation for adaptation. Diversity studies done on Puccinia graminis, a basidiomycete causing stem rust on grains, showed an ever declining number of strains present after the eradication of barberry which was the pathogen s host for the sexual stage. Average number of strains fell from seventeen to ten in ten years and down to seven over a period of seventy years (Roelfs and Groth, 1980, Alexander et al., 1984, cited in Burdon, 1993). Whereas sexual reproduction is important for genetic diversity, asexual reproduction is often important for spread. It is often coupled with sexual reproduction in a cyclic pattern. It has been found in many cases that populations of fungi will reproduce asexually and clones will spread very rapidly to colonise an entire host population, followed by a sexual reproduction cycle FSP Project Y051203, Lewis -10-

11 producing spores with an ability to over-winter with more success (Kohn, 1995). During the following season new clones spread rapidly, and these are genetically different from the clones in the previous season. This type of cycle has been found in Mycosphaerella graminicola, an ascomycete causing a foliar disease in wheat (Chen et al., 1994), and in Phytophythora infestans, an oomycete causing late blight on potatoes and tomatoes (Fry et al., 1992) (cited from Kohn, 1995). Still other fungi produce asexual spores to resist a harsh environment and clones will persist year after year as in the case with Sclerotinia sclerotiorum, an ascomycete causing potato stem rot (Kohn, 1995). The imperfect fungi or Deuteromycetes a have no known sexual stage and the Ascomycetes only rarely produce sexual ascospores in nature (Agrios, 1997). These fungi completely utilise asexual reproduction as a means of spread on host populations. Fungi that have adapted to this sort of life cycle can exist quite well without sexual reproduction. In the case with Dothistroma septosporum, the sexual cycle may be very important in overcoming less than favourable disease conditions, but energy is not wasted under conditions where the pathogen is quite successful without it. All of these strategies enable fungal pathogens to adapt very well to both the environment and the host. Reproductive strategies can influence the genetic structure of the pathogen population. Asexually reproducing populations will display low genetic diversity with increased levels of clonality (Chen and McDonald, 1996). Other signals indicating asexual reproduction include gametic phase disequilibrium and fewer genotypes (Chen and McDonald, 1996 and Borchardt et al., 1998). Randomly mating, sexually reproducing populations will display increased genetic FSP Project Y051203, Lewis -11-

12 diversity with many genotypes and display a population structure more reminiscent of panmixia (Chen and McDonald, 1996, Kohn, 1995). Little or no gametic phase disequilibrium should be present (Borchardt et al., 1998). The degree to which a population shows either of these two types of population structure will be affected by the contributions of both the asexual and sexual cycles to the next generation (Chen and McDonald, 1996). Contributions of sexual and asexual reproduction may be affected by compatible sexual strains, and environmental conditions (Leung et al., 1993, cited in Chen and McDonald, 1996). The cyclic nature and timing of some fungi that utilise both reproductive strategies may also affect population structure (Kohn, 1995). Sexual reproduction has both advantages and disadvantages associated with it. While it can provide a new genotypes through the re-assortment of alleles (Burdon, 1993) it may break up successful combinations of genes (Anderson et al., 1992). New variation is important in a species ability to colonise new niches or to adapt in a changing environment (Anderson et al., 1992). Fungi have relatively small genomes compared to plants and animals, roughly 6-10 times the size of Escherichia coli (Anderson et al., 1992). For some species or populations, the advantage of genetic variation may not outweigh the advantage of keeping successful combinations of genes together. Asexually reproducing fungi can continuously pass a successful genome on to the next generation without the costs associated with recombination. Host colonisation can occur rapidly and successfully. If the host is not resistant to the majority of strains of pathogen, natural FSP Project Y051203, Lewis -12-

13 selection on successful pathogen gene complexes should be purifying. In a situation such as this, selection would favour asexual reproduction cycles over sexual reproduction cycles. New sources of variation may not be favoured, and mutation rates should be low. Fungi with mixed reproductive strategies may have the most optimal adaptive advantage (McDonald and Linde, 2002). Sexual recombination will produce many new strains that can be tested out on the host population. Natural selection will favour the strains that are best able to overcome host resistance. Asexual reproduction keeps the fittest combinations of alleles together and amplifies the number of individuals possessing those fit combinations. If dispersal is efficient and gene flow high in the pathogen population, the fittest individuals can then be spread over a large range and cause an epidemic (McDonald and Linde, 2002). 6.0 Current Studies on Population Structure of Dothistroma Few studies have been undertaken to determine population genetic structure of Dothistroma septosporum, or the role that is plays in spread of the disease or virulence. In New Zealand, Hirst et al (1999) detected no genetic diversity of the pathogen within New Zealand using random amplified polymorphic DNA and random amplified microsatellites. This is attributed to the possible introduction of only one strain of the fungus that has since been spreading asexually ever since. This study raised questions about the hazard of introductions of new strains of the pathogen into New Zealand (Hirst, 1999, Ganley & Bradshaw, 2001). To assist with detection and identification of strains, Ganley and Bradshaw (2001) developed a DNA profile system based on microsatellite loci that was able to distinguish between isolates from a total of eight countries. FSP Project Y051203, Lewis -13-

14 Barnes et al. (2004) explored the phylogenetic relationship between isolates from different countries and used DNA sequence data to determine the validity of separating the fungus into different varieties. Their results indicate that there are two distinct phylogenetic lineages of the pathogen, but no genetic basis for the separation of the fungus into varieties. They suggest that there is one distinct lineage (D. pini) that is pathogenic on pine in the north central United States, and a second distinct lineage (D. septosporum) that has a world wide distribution. There was one isolate tested from Canada that belonged to the second lineage (D. septosporum). Bradshaw et al. (2002) also found differences in the ITS sequence that separated the central US strains out from the others. 7.0 Dothistromin Toxin Dothistroma septosporum produces the toxin dothistromin. This toxin has been shown to cause the red band needle blight symptoms in pine needles (Shain & Franich, 1981) and to elicit host responses (Franich et al., 1986). Pathogen isolates appear to vary in their abilities to produce the toxin which may depend on factors such as environment, instability of an individual, or to the toxin producing ability of the individuals (Bradshaw et al., 2000). Bradshaw et al. (2002) found that D. septosporum strains from the German Alps as well as some of the isolates from central USA were able to produce extremely high levels of the toxin. This increased ability may be due to an adaptation to the colder climate in the higher altitudes, a relatively recent outbreak (Maschning & Pehl, 1994, cited from Bradshaw et al., 2002), or the presence of the teleomorph (Bradshaw et al., 2002). FSP Project Y051203, Lewis -14-

15 Bradshaw et al. (2002) also found that an individual isolated from New Zealand in 1969 produced higher levels of dothistromin than individuals recently isolated. Based on findings that show no genetic differentiation among the New Zealand isolates, they suggest that toxin producing ability may change with time, possibly due to a loss of fitness without the presence of the teleomorph. These findings reveal the importance of programs to control the spread of such a widespread pathogen. Introductions of more strains could lead to the teleomorph and the problem could potentially be amplified. The dothistromin toxin is also of interest due to its toxicity to humans. Ferguson et al. (1986) have shown that the toxin causes chromosome abnormalities in human lymphocytes and Skinnider et al. (1989) found an increase in frequency of sister chromatid exchange. Also of interest is that the biosynthetic pathway leading to the production of dothistromin is very similar to the pathway that leads to aflatoxin production (Bradshaw et al., 2002). Studies on the toxin are important both for developing methods of control of this pathogen, but also for the development of safety protocols for forest workers (Bradshaw et al., 2002). 8.0 Forest Management The economic impacts of a foliar disease can range from negligible to quite high in forest plantations. Low levels of defoliation may reduce vigor to a small extent, but the trees will recover in the following growing seasons. Increased levels of defoliation can be more severe. Successive years of defoliation of a tree causes a decrease in photosynthates translocated to the roots which can cause a reduction in overall growth (van der PAS, 1981). Repeated defoliation events can also affect the form of a tree where there is a decrease in the base of the stem and FSP Project Y051203, Lewis -15-

16 overall less stem taper (van der PAS, 1981). Reduced tree growth translates into reduced wood yields. In Pinus radiata plantations in New Zealand, this growth loss has been estimated at 10-25% periodic increment growth loss (New, 1989). Repeated severe defoliation events can also lead to death of the host trees. When the host trees are part of a forest plantation, considerable amounts of money must be spent to assess the level of damage, monitor the situation, and to reforest the landscape (Woods, 2003). Management costs also increase when money must be spent to develop and implement control programs to combat continuous moderate to high levels of pathogen activity. 8.1 Methods of Control Currently, the disease is managed in New Zealand by the aerial application of copper fungicides (Bradshaw, 2004), which can be costly and have detrimental effects on the environment (Devey et al., 2004). Reduction in disease is also obtained through breeding programs for Dothistroma septosporum resistant pine strains. Resistance can be obtained by reforesting with naturally resistant pine strains, or with provenances that show resistance (Bradshaw, 2004). Quantitative traits can be selected for through long term breeding programs, or by selecting individuals that have a genetic marker associated with quantitative resistance (Devey et al., 2004). Breeding programs have been underway in New Zealand for some time now and have been effective in reducing overall losses (Bradshaw, 2004). FSP Project Y051203, Lewis -16-

17 Literature Cited Agrios, G.N. (1997). Plant Pathology fourth edition. Academic Press, San Diego California. pp 635. Alexander, H. M., Roelfs, A. P., & Groth, J. V. (1984) Pathogenicity associations in Puccinia graminis f. sp. tritici in the United States. Phytopathology 74: Anderson, J. B., Kohn, L. M., & Leslie, J. F. (1992) Genetic mechanisms in fungal adaptation, pp in The fungal community: Its organization and role in the ecosystem, edited by G. C. Carroll and D. T. Wicklow. Dekker, New York. Barnes, I., Crous, P.W., Wingfield, B.D., & Wingfield, M.J. (2004). Multigene phylogenies reveal that red band needle blight of Pinus is caused by two distinct species of Dothistroma, D. septosproum and D. pini. Studies in Mycology 50: Borchardt, D. S., Welz, H. G., & Geiger, H. H. (1998) Genetic structure of Setosphaeria turcica populations in tropical and temperate climates. Phytopathology 88: Bradshaw, R.E., Gangley, R.J., Jones, W.T., & Dyer, P.S. (2000). High levels of dothistromin toxin produced by the forest pathogen Dothistroma pini. Mycological Research 104: Bradshaw, R.E. (2004). Dothistroma (red-band) needle blight of pines and the dothistromin toxin: a review. Forest Pathology 34: Bradshaw, R. Bhatnagar, D., Ganley, R., Gillman, C., Monahan, B. & Seconi, J. (2002) Dothistroma pini, a forest pathogen, contains homologs of aflatoxin biosynthetic pathway genes. Applied And Environmental Microbiology 68: Burdon, J. J. (1993) Genetic variation in pathogen populations and its implications for adaptation to host resistance. In Durability of disease resistance, Edited by Jacobs Th, & Parlevliet, J. E. Kluwer Academic Publishers, Netherlands. Chen, R. S., Boeger, J. M., & McDonald, B. A. (1994) Genetic stability in a population of a plant pathogenic fungus over time. Molecular Ecology 3: Chen, R.S., McDonald, B.A. (1996). Sexual reproduction plays a major role in the genetic structure of populations of the fungus Mycosphaerella graminicola. Genetics 142: Cobb, F.W., & Miller, D.R. (1968) Hosts and geographic distribution of Scirrhia pini The cause of red band needle blight in California. Journal of Forestry 66: Cobb, F., Uhrenholdt, B., Krohn, R. (1969) Epidemiology of Dothistroma pini needle blight on Pinus radiata. Phytopathology 59: Devey, M.E., Groom, K.A., Nolan, M.F., Bell, J.C., Dudzinski, M.J., Old, K.M., Matheson, A.C. & Moran, G.F. (2004) Detection and verification of quantitative trait loci for resistance to Dothistroma needle blight in Pinus radiata. Theory of Applied Genetics 108: Dubin, H.J., Staley, J.M. (1966) Dothistroma pini on Pinus radiata in Chile. Plant Disease Reporter 50: 280. Dubin, H. J. (1967) Preliminary information about Dothistroma blight in Chile. Congress of the International Union of Forest Research Organization XIV 5: Evans, H. C. (1984). The genus Mycosphaerella and its anamorphs Cercoseptoria, Dothistroma and Lecanosticta on pines. CMI Mycological Paper no 153. Surrey, UK: Commonwealth Mycological Institute. FSP Project Y051203, Lewis -17-

18 Ferguson, L. R., Parslow, M. I., & McLarin, J.A. (1986) Chromosome damage by dothistromin in human peripheral blood lymphocyte cultures: a comparison with aflatoxin B 1. Mutation research 170: Franich, R. A., Carson, M. J., & Carson, S. D. (1986) Synthesis and accumulation of benzoic acid in Pinus radiata needles in response to tissue injury by dothistromin, and correlation with resistance of P. radiata families to Dothistroma pini. Physiological and Molecular Plant Pathology 28: Fry, W.E., Goodwin, S.B., Matuszak, J.M., Spielman, L.J., & Milgroom, M. G. (1992) Population genetics and intercontinental migrations of Phytophthora infestans. Annual Review of Phytopathology 30: Funk, A., & Parker, A.K. (1966) Scirrhia pini N. sp., the perfect state of Dothistroma pini Hulbary. Canadian Journal of Botany 44: Gadgil, P. D. (1967) Infection of Pinus radiata needles by Dothistroma pini. New Zealand Journal of Botany 5: Gadgil, P. D. (1970) Survival of Dothistroma pini on fallen needles of Pinus radiata. New Zealand Journal of Botany 8: Ganley, R. J. & Bradshaw, R. E. (2001) Rapid identification of polymorphic microsatellite loci in a forest pathogen, Dothistroma pini, using anchored PCR. Mycological research 105: Gibson, I. A. S., Christiansen, P., Munga, F. (1964) First observations in Kenya on a foliage disease of pines caused by Dothistroma pini Hulbary. Commonwealth Forest Review 45: Gibson, I. A. S. (1972) Dothistroma Blight of Pinus radiata. Annual Review of Phytopathology 10: Gibson, I. A. S. (1974) Impact and control of dothistroma blight of pines. European Journal of Forest Pathology. 4: Gilmour, J. W. (1966) The pathology of forest trees in New Zealand. The fungal, bacterial and algal pathogens. Technical paper Forest Research Institute (New Zealand) 48. Gilmour, J.W. (1981) The effect of season on infection of Pinus radiata by Dothistroma pini. European Journal of Forest Pathology 11: Hirst, P., Richardson, T., Carson, S. and Bradshaw, R. (1999) Dothistroma pini genetic diversity is low in New Zealand. New Zealand Journal of Forest Science 29: Hulbary, R. L. (1941) A needle blight of Austrian pine. Illinois Natural History Survey Bulletin 21: Hunt, R. S. (1995) Common pine needle casts and blights in the Pacific region. Forest Pest Leaflet 43, Natural Resources Canada Ivory, M.H. (1967) A new variety of Dothistroma pini in Kenya. Transactions of the British Mycological Society 50: Ivory, M.H. (1970) Dothistroma needle blight of Pinus radiata in Kenya. A study of infection and blight resistance. Ph.D. Thesis. University of London Karadzic, D. (1989) Scirrhia pini Funk et Parker. Life cycle of the fungus in plantations of Pinus nigra Arn. in Serbia. European Journal of Forest Pathology 19: Kinloch, B. B. Jr., Westfall, R. D., White, E. E., Gitzendanner, M. A., Dupper, G. E., Foord, B. M., & Hodgskiss, P. D. (1998) Genetics of Cronartium ribicola. IV. Population structure in western North America. Canadian Journal of Botany 76: FSP Project Y051203, Lewis -18-

19 Kohn, L. M. (1995) The clonal dynamic in wild and agricultural plant-pathogen populations. Canadian Journal of Botany 73(supplement 1): S1231-S1240. Leung, H., Nelson, R. J., & Leach, J. E. (1993) Population structure of plant pathogenic fungi and bacteria. pp In Advances in Plant Pathology, Vol 10, edited by J. H. Andrews and I. C. Tommerup. Academic Press, New York. Maschning, E., Pehl, L. (1994) Threat to native Pinus mugo by Dothistroma. AFZ. Allgemeine Forst Zeitschrift 49: McDonald, B. A., & Linde, C. (2002) Pathogen population genetics, evolutionary potential, and durable resistance. Annual Review of Phytopathology 40: New, D. (1989) Forest health an industry perspective of the risks to New Zealand s plantations. New Zealand Journal of Forestry Science 19: Parker, A.K., & Collis, D.G. (1966) Dothistroma needle blight of pines in British Columbia. Forestry Chronicle 42: Peterson, G. W. (1966) Penetration and infection of Austrian and ponderosa pines by Dothistroma pini. Phytopathology 56: Peterson, G. W. (1973) Infection of Austrian and ponderosa pines by Dothistroma pini in eastern Nebraska. Phytopathology 63: Roelfs, A. P. & Groth, j. V. (1980) A comparison of virulence phenotypes in wheat stem rust populations reproducing sexually and asexually. Phytopathology 70: Rogerson, C.T. (1953) Kansas mycological notes, Transactions of the Kansas Academy of Science 56: Shain, L. & Franich, R. A. (1981) Induction of dothistroma blight symptoms with dothistromin. Physiological Plant Pathology 19: Skinnider, L., Stoessl, A. & Wang, J. (1989) Increased frequency of sister-chromatid exchange induced by dothistromin in CHO cells and human lymphocytes. Mutation research 222: Thyr, B.D., Shaw, C.G. III. (1964) Identity of the fungus causing red band disease on pines. Mycologia 56: van der PAS, J. B. (1981) Reduced early growth rates of Pinus radiata caused by dothistroma pini. New Zealand Journal of Forestry Science 11: Woods, A.J. (2003). Species diversity and forest health in northwest British Columbia. The Forestry Chronicle 79, FSP Project Y051203, Lewis -19-