Genetic variation in the foliar pathogen Dothistroma septospora and relationship to toxin production.

Angie Dale, MSc Candidate

Purpose and Management Implications:

Dothistroma septosporum (= pini) is a fungal pathogen belonging to the group ascomycota. It is an important pathogen on many species of pine worldwide. The fungus colonizes pine needles, and as a part of its life cycle produces dothistromin, a mycotoxin believed to be responsible for the disease, resulting in red banding on the needles, and eventually causing death of the needle and defoliation of the tree.
Dothistroma septosporum has been causing problems in exotic pine plantations in many countries of the Southern Hemisphere since the late 1950s (Gibson, 1972). Outbreaks of the disease followed the widespread planting of exotic Pinus radiata monocultures in large-scale forestry operations (Gibson, 1972). Dothistroma septosporum has been found in some countries of the Northern Hemisphere, but past outbreaks have not been as severe or caused as much concern as they have in the south.

Dothistroma septospora was identified on pine species in British Columbia in the mid 1960s (Funk & Parker, 1966, Parker & Collis, 1966). The fungus was found on three native pine species and on six exotic species (Parker and Collis, 1966). The teleomorph was identified on foliage from several British Columbia localities in 1964 and 1965 (Funk and Parker, 1966). Early studies describe the teleomorph and the life cycle in British Columbia (Funk and Parker, 1966). Further studies on the disease in British Columbia are limited, because it has not been a major cause for concern as outbreaks have been small, have gone unnoticed or have been kept under control by natural factors such as weather.

More recently there has been a large outbreak of Dothistroma septosporum in Northwest British Columbia that has been causing a lot of concern due to the extent of the outbreak as well as the severity, which includes stands with almost 100% mortality (Woods, 2003). Over 90% of lodgepole pine plantations have suffered some damage in the current outbreak. The damage ranges in these plantations from low levels of infection to nearly 100% mortality in some (Woods, 2003). In central and Northern British Columbia, lodgepole pine (Pinus contorta) is the major species of pine, and is economically important as it is the main species planted in reforestation and is one of the major species harvested. The financial impacts of this type of disease outbreak can be quite severe. In many cases, these severely infected stands have previously reached free to grow standards and responsibility of the land has been returned to the government. Now these plantations are becoming under stocked and must be reforested and managed (Woods, verbal communication).

Several factors may be contributing to the extent and severity of the outbreak. These factors include conducive weather patterns, - warm, moist summers and cool wet falls- (Woods et al., 2005), current forest practices leading to an over abundance of host, or a decrease in vigor of the host, and a more virulent strain of the pathogen itself (Woods, 2003).

The current disease outbreak in British Columbia is unique due to the severity and extent, as well as the host being a native Pinus species. These circumstances have not been previously encountered in northern countries. The favourable conditions for disease, as well as the existence of the teleomorph in British Columbia, may have led to a case where sexual reproduction has allowed for rapid evolution of the pathogen population. Rapid evolution may have given rise to a more virulent strain of the fungus itself. Knowing the dynamics of the pathogen populations in respect to how it changes with ecosystem differences, what conditions cause rapid evolution, and how it spreads, aids forest managers and silviculture planning. Ecosystem characteristics conducive to disease spread can be avoided for lodgepole pine, or other low risk stategies can be employed to reduce disease severity and spread. Therefore, the purpose of this study is to explore the population genetic structure of Dothistroma septospora populations in Northwest British Columbia, to relate population structure to the contributions of reproductive strategies, forest types and to current forest practices, and to relate population genetic structure to toxin producing abilities of the pathogen.

Methodology Overview:

Foliar samples were collected from lodgepole pine showing signs of infection by Dothistroma septosporum. Trees were sampled from approximately 24 sites, with eight sites in each of three subzones. Sites were chosen in both unmanaged stands and in plantations.

Sixteen trees separated by 30 meters were sampled in each site. The sample design was systematic with a random start. One sample was taken from each tree and on select sites, three samples were taken from each tree. One within the lower branches, one mid tree, and one in the mid to upper foliage. This was done to look at the genetic variation of the fungus within the tree.

In order to make comparisons with international populations, genetic data will be sent to international laboratories, specifically the University of Pretoria in South Africa, where a global population study is currently taking place. As well, the consensus sequence for the ITS region from our local populations will be compared to ITS sequences of other Dothistroma septosporum and pini isolates located globally. International ITS sequences will be obtained from online from the National Center for Biotechnology Information through Genbank.

Needles showing symptoms and fruiting bodies of Dothistroma needle blight were surface sterilised and placed on water agar. After two to three days, a conidial mass from one fruiting body was scooped up using a dissecting needle, and streak plated on a new dish of water agar. After the conidia start to germinate, an individual colony originating from one conidia, will be isolated onto dothistroma medium consisting of 5% (w/v) malt extract, 2.3% (w/v) nutrient agar (Bradshaw et. al., 2000). The single colony will be allowed to grow up to fill a petri plate. Cultures will be maintained at room temperature (18 - 20 degrees Celsius).

To measure growth rate, two plugs with a diameter of 5 mm will be extracted from each original isolation plate and transferred onto new Petri plates containing dothistroma medium. From these plates, measurements of two diameters at 90 degrees will be made periodically to obtain growth rates. Qualitative assessments will be made for toxin production where a code will be given for no toxin (0), some toxin leaching into the agar around the colony (1), agar half covered with red toxin (2) and all of agar covered in red toxin (3). Growth rates will be calculated with a linear regression, and growth rates will be compared for isolates between sites and between subzones with an ANOVA.

DNA will be extracted using a phenol-chloroform method modified from that developed by Al-Samarrai and Schmid, (2000). DNA will be extracted from tissue grown either on agar, or in liquid growth media. Modifications to the protocol developed from Al-Samarrai and Schmid (2000) include a second cleaning with chloroform after the addition of phenol and chloroform, followed by the addition of lithium chloride to a final concentration of 0.1M prior to the first ethanol precipitation, and sodium chloride to a final concentration of 0.2 M prior to the second ethanol precipitation.

DNA will be amplified using a polymerase chain reaction following methods of Gangley and Bradshaw (2001). Microsatellite loci will be tested to measure variation in genetic diversity. Some primers are already available for use with Dothistroma septosporum, and using microsatellites will enable us to better compare our results to results from other studies that have been done or are underway. Gangley and Bradshaw (2001) have developed primers for five loci. Two of the primers have two alleles, two primers with three alleles, and one primer with four alleles. Microsatellite PCR products will be multiplexed and viewed on an Bec CEQ 8000 DNA sequencer.

If there is little to no variation in the microsatellite loci amplified by these primers, then sequencing of the ITS region between the nuclear 18S and 5.8S rRNA genes will be looked at as an alternative method. Methods of Bradshaw et al. (2000) or Barnes et al. (2004) will be looked at for suitability if necessary. Eight individuals will initially be sequenced to determine the level of variation.

The second method being used to measure genetic diversity is amplified fragment length polymorphism (AFLP) using methods developed by Vos et al., 1995. Amplified fragment length polymorphism is a technique for generating DNA fingerprints using random fragments of genomic DNA. It couples restriction fragment length polymorphism with polymerase chain reaction to generate unique banding patterns for genetically distinct individuals. AFLP is a useful method that requires no prior sequence knowledge and the number of fragments can be manipulated by selection of specific primer sets. They have some advantage over other methods in their reliability due to stringent PCR conditions (Vos et. al., 1995).

Restriction and ligation reactions will follow the methods of Vos et al. (1995). DNA will be subjected to restriction digestions using the enzymes Eco RI and Mse 1 and after 3 hours at 37 degrees Celsius, adapters, T4 DNA ligase, and ligase buffer will be added to the reaction and incubation will continue overnight at 16 degrees Celsius.

DNA fragments will then be amplified using PCR with pre amplification primers that correspond to the adapters used in the restriction reaction and taq polymerase that is active at lower temperatures. The initial step in the PCR conditions includes a 72-degree step for 2 minutes, followed by 30 cycles of 94 degrees for 30 seconds, 56 degrees for 30 seconds and 72 degrees for 2 minutes. This is followed by a 60-degree step for 10 minutes. All other conditions follow those of Vos et al. (1995). The pre amplification is followed by a selective amplification in which the primers have one to three selective nucleotides at the ends. A GC and a GA selective primer will be tested with the pre Mse primer. Further primer pairs will be tested as needed to obtain enough informative polymorphic bands for the study. Primers will not be labeled, all other conditions follow those of Vos et al. (1995). AFLP products will be viewed on a 2.5% super fine resolution Amersham agarose.

Rigorous testing of repeatability will be done to ensure that data is reliable. Several samples will be taken through the digestion and ligation reactions three times each. The products from those will each be amplified two to four times in both the pre amplification and selective amplification steps. Data will be compared to ensure that each step in the procedure is producing reliable and repeatable results. The number of samples tested will depend on how reliable the data appears to be.

The program GeneScan will be used to score and measure the bands present for each individual. The data will then be exported to an excel file. Data will be converted into a file with each individual as one case, and data will consist of zeros representing no band and ones representing a band present for each of the alleles detected. Data analysis will be done using a data analysis package designed for population genetic analysis. An analysis of molecular variance (AMOVA) will be performed and genetic diversity will be partitioned into within population and among population diversity. Significant differences will be assessed at = 0.05. F-statistics (Wright, 1951) will be calculated to look at population differentiation, specifically Fst which relates subpopulations to the total population. A neighbour-joining tree will be generated to analyse and visually represent population divergence for the Dothistroma septosporum populations in Northwest BC.

Genetic distances will be compared to geography to determine if relationships exist due to proximity, geographical barriers, or river systems. The relationship between clones and genetic distances may infer the pattern of spread as a result of forest management practises. This will determine whether spread occurred mainly from existing stands to plantations or from plantations to natural stands.

The level of clonality will be assessed by looking at how often a clone was found and at which sites. Likewise, the contribution of sexual reproduction will be assessed by the number of genetically distinct strains found.

For variation in toxin production, 12 isolates will be grown in liquid media and the filtrate will collected and sent to Dr. Rosie Bradshaw's lab in New Zealand. Dothistromin will be extracted and quantified. The isolates will be chosen from a site that has qualitatively been shown to produce higher levels of toxin and from a site that shows low levels of toxin production. The amount of toxin produced from the isolates will then be compared with an ANOVA to determine if the isolates from one site produce more toxin than the isolates from another site. These results will be compared to growth rates and genetic data to determine if any relationships exist.

Project Scope and Regional Applicability:

This project will look at genetic diversity of the pathogen and how that diversity changes with different environments as well as how it changes with forest management. Changes in dynamics of pathogen populations can be used to predict how the pathogen will react under different environments which will allow forest practitioners to manage for lower disease incidence.

The regional applicability is throughout the range of lodgepole pine, specifically northwest British Columbia.

Interim Conclusions:

Genetic diversity has been found using the AFLP technique both within and between populations (Figure 1). This confirms that sexual reproduction is occurring in the northwest British Columbia populations. A comparison of the ITS sequence looked at in eight individuals throughout the study sites show 100% sequence identity. In comparison with the global population, the northwest British Columbia populations show 100% similarity with Dothistroma septosporum, and 99% similarity with Dothistroma pini. A small subset of individuals looked at using the mircosatellite DNA primers are 100% monomorphic, indicating these populations have not evolved from one another long enough for differences to have arisen.

Literature Cited:

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Barnes, I., Crous, P., Wingfield, B. and Wingfield, M. 2004. Multigene phylogenies reveal that red band needle blight of Pinus is caused by two distinct species of Dothistroma, D. septosporum and D. pini. Studies in Mycology 50: 551-565.

Bradshaw, R., Ganley, R. Jones, W. and Dyer, P. 2000. High levels of dothistromin toxin produced by the forest pathogen Dothistroma pini. Mycological Research 104: 325-332.

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Woods, A. 2003. Species diversity and forest health in northwest British Columbia. The Forestry Chronicle. 79:892-897.

Woods, A., Coates, D.K. & Hamann, A. (2005) Is an Unprecedented Dothistroma Needle Blight Epidemic Related to Climate Change? Bioscience 55: 761-769.

Wright, S. (1951) The genetical structure of populations. Ann. Eugen (Lond.) 1: 323-334.