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Environmental Impact of Using Phlebiopsis gigantea in Stump Treatment Against Heterobasidion annosum sensu lato and Screening Root Endophytes to Identify Other Novel Control Agents

Approximately 15% of the spruce trees felled in Finland are rotten and thus commercially less valuable. The majority of this conifer wood decay is due to the root and butt rot pathogen Heterobasidion annosum sensu lato. Extensive logging of conifer forests has changed the environment into favouring this pathogen in stands where it originally has been rare. The proportion of diseased forest stands and associated production losses are expected to increase in the foreseeable future due to year-round logging. The saprotrophic fungus Phlebiopsis gigantea has for several years been used as a biocontrol agent against H. annosum s.l. in spruce and pine stumps. The intensive use of a biocontrol, P. gigantea, might disturb the microbial communities in wood stumps. Understanding the consequences on other wood microbes of the long-term application of P. gigantea to living wood tissues would be important for gaining knowledge of the biocontrol effect on the microbial environment in the stumps. The first objective of this thesis was to evaluate the impact of the only biocontrol agent used against root and butt rot fungus (H. annosum s.l.) on other resident microflora of Norway spruce stumps. The application of P. gigantea cannot control already existing infection of H. annosum sensu lato. This fact necessitated exploratory isolation studies of fungal root endohytes to screen and identify other potentially novel bioagents that can be deployed for the biocontrol of the conifer pathogen.

To find out whether the P. gigantea treatment impacts the overall diversity of other non-target stump microbes we used the 454- pyrosequencing approach. Samples were collected from forest sites previously pre-treated with P. gigantea either one, six or 13 years ago, DNA was isolated and the PCR products of fungal internal transcribed spacer (ITS) and bacterial 16S of ribosomal DNA, regions were pyrosequenced. Similarly samples were collected from untreated stumps within the same forest site over the same period of time. The results revealed that initial application of the biocontrol agent influenced the fungal species composition, but the overall fungal diversity was not affected and no statistical differences were observed between treated and non-treated stumps in the mycobiota. The biocontrol treatment significantly decreased the initial bacterial richness in the stumps, but the bacterial community gradually recovered and the negative effect of P. gigantea was attenuated. Our results 13 years after "Rotstop" application to the stumps supports the continued use of P. gigantea for stump pre-treatment in Finnish forests against H. annosum s. lato. Compared to other control methods (chemical, stump removal, no control), the use of the "Rotstop" strain in the clear-cutting areas to decrease the occurrence of H. annosum s.l. is recommended when loggings are performed during summertime to reduce the basidiospore dispersal of this pathogen. Biocontrol application seems to currently have the smallest negative impact on the environment. However, comprehensive studies regarding longer-term monitoring from the same site are still needed to prove that the adverse initial impacts of P. gigantea are attenuated over time.

In parallel to the above studies, I further explored the potential of finding other novel biocontrol agents for use in managing the disease caused by the root rot pathogen Heterobasidion parviporum. This necessitated isolation studies of fungal root endophytes from forestry sites such as pristine mires and drained peatlands where the spread of H. annosum s.l. species have not been commonly reported. The reasons why H. annosum s.l. are not commonly observed in peatland still remains unclear. A possible reason for the suppression of H. annosum s.l. in peatlands is the diverse microbial community and their antifungal substances. Draining of pristine mires is likely to change the water balance of the sites, possibly transforming the microbial communities in plant roots, which might facilitate the spreading of the pathogen (H. annosum s.l.). Consequently, I sampled non-mycorrhizal P. abies roots and isolated endophytes from a pristine mire, a drained peatland and mineral soil and investigated the potential inhibitory effect of a subset on the root rot pathogen H. parviporum. Protecting young seedlings using endophytic fungi during the early stages of establishment under field conditions was a primary consideration. Additionally, the metabolites secreted by the selected root endophytes were extracted and the inhibitory effects on these pathogenic fungi were assayed. The root endophytes identified as Cryptosporiopsis sp. and Phialocephala sp. were able to form inhibition zones in paired cultures with the phytopathogenic fungi. Secreted metabolites from the endophytes also had similar inhibitory effects. The secreted metabolites were further chemically analysed using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). The higher numbers of unique metabolites were observed within Cryptosporiopsis sp., further suggesting that the stronger inhibitory effect observed could be due to acquisition of a more diverse metabolite pool. The findings in this thesis revealed no significant difference between the fungal isolate frequencies or diversity at the various site habitats. However, these results were based only on cultivated endophytes, which may have led to an undersampling of the fungal community and a combination of various methods (direct sequencing of DNA and cultural methods) should be applied in future research. Diversity studies of root endophytes in different environments provide additional knowledge about their community and host interactions, but we still know little about their precise functional role in the roots of their hosts. Overall, this thesis provides an initial insight into the major fungal root endophytes of pristine mires, mineral soil and drained peatlands. A subset of the endophytes with potential inhibitory effects on the growth of H. parviporum and other phytopathogenic fungi were identified. The secreted metabolites from these endophytes were also found to possess inhibitory properties. The best way to achieve the set objectives of integrated pest management (IPM) policy would be the exploration of biotechnology application of these beneficial endophytes as inoculants to facilitate plant protection.

The new possibilities to protect the valuable seedling roots from root rot pathogens are vital in the foreseeable future in forestry. The biocontrol capability of inhibitory root endophytes documented in this study could serve as an alternative method to restrict and manage the disease caused by the root rot pathogen H. annosum s. lato. Root endophytes might serve as the first root protectors of young seedlings after planting, especially on most contaminated clear-cut sites. This may potentially enhance seedling survival during the most critical years against root rot pathogens. Further studies on this are on-going. Molecular biology is becoming an increasingly important tool in forestry for tackling forest ecology and pathology problems. These new methods and accumulated information provides a better understanding of complex interactions in forest ecology and provide possibilities to go deeper into molecular forest pathology applications in practical forestry. Presently, we still do not fully understand the environmental consequences in the application of a single strain of biocontrol agent P. gigantea to freshly cut stump surfaces. The biological and antagonistic activity against non-target microbes in principle implies a potential environmental risk. A deeper understanding on the mechanism of action of P. gigantea would be of importance for the further improvement and management of the biocontrol effect. To accomplish this, long-term follow up trials and basic research are still needed. This would form the basis for the development of environmentally friendly and sustainable management strategies for forestry.

M.Sc. Eeva-Liisa Terhonen, (MMM, Metsänhoitaja) will defend the doctoral dissertation entitled "Environmental Impact of Using Phlebiopsis gigantea in Stump Treatment Against Heterobasidion annosum sensu lato and Screening Root Endophytes to Identify Other Novel Control Agents" in the Faculty of Agriculture and Forestry, University of Helsinki, on 4th September 2015 at 12:00.

The public examination will take place at the following address: B-talo, Sali B6, Latokartanonkaari 9.

Professor Paolo Capretti, University of Florence, will serve as the opponent, and Professor Fred Asiegbu as the custos.

The dissertation will be published in the series YEB. The dissertation is also available in electronic form through the E-thesis service.