Biological control of Fusarium spp. and other soil-borne pathogens on tree seedlings
Soil borne fungi isolated from forest areas and nurseries in North east of Scotland using baiting techniques, were identified using classical taxonomy and molecular methods (PCR amplification of ITS regions; restriction digestion; sequencing of PCR products) as Fusarium lateritium, F. tricinctum, F. sambucinum, Phytophthora cinnamomi, Pythium ultimum var. ultimum and Rhizoctonia binucleate (Ceratobasidium sp.). Virulence was tested in vitro on young seedlings of Pinus sylvestris and Alnus glutinosa, and Koch's postulates fulfilled through reisolation of the pathogens and confirmation of fungal penetration into host tissues. Root growth was measured using the Winrhizo program, and dry weights recorded. Symptoms on aerial parts were assessed using a categorical scale from 0 (healthy) to 5 (damage > 76%). Fusarium spp. caused significant different (P 0.01) symptom intensity on both host plants. However, no significant difference in root growth was found between treatments and control (P 0.05). The effects of different compost treatments on disease development in seedlings of both hosts inoculated with the same fine root pathogens was tested in the glasshouse confirming the virulence of the fungal pathogens on P. sylvestris and A. glutinosa seedlings. Although mean dry weights of P. sylvestris and A. glutinosa varied between compost treatments, differences were not significantly different. Isolation, characterization and identification of bacterial isolates, Bacillus subtilis B1, fluorescent pseudomonads B4 and B5 with antagonistic action against pathogens were also carried out. These isolates along with the known bacterial antagonists Bacillus subtilis MB600, MB205 and Pseudomonas corrugata R117 were used for biological control in vitro and in planta experiments using Alnus glutinosa or Pinus sylvestris seedlings. All bacterial isolates colonized root systems of both tree species. Higher numbers of bacterial cells were observed on roots of A. glutinosa than on P. sylvestris roots. High bacterial cell numbers were observed in plants of both tree species inoculated with fluorescent pseudomonads B4 or B5. In vitro antagonism on agar plates, indicated by inhibition in fungal colony diameter growth, was recorded for F. tricinctum, F. lateritium and F. sambucinum, Pythium ultimum var. ultimum and Phythophthora cinnamomi with all bacterial isolates tested (P 0.05). Biological control of the fine root pathogens on Pinus sylvestris and Alnus glutinosa seedlings by bacteria semi in vivo in test tubes was carried out with various responses in both tree hosts. All bacterial treatments resulted in a lower sporangium germination rate for P. ultimum var. ultimum than was found in controls (P 0.05). Effect of the bacterial isolates separately on growth and disease development in Pinus sylvestris and Alnus glutinosa seedlings inoculated with the pathogens under glasshouse conditions using autoclaved compost was tested. The bacterial isolates had various effects against the pathogens, although in most cases no significant differences were observed relative to controls. Further soil-based trials were carried out in the glasshouse to achieve control of root disease development on Pinus sylvestris and Alnus glutinosa using a combination of different antagonists, based on a mixture of the bacterial isolates used previously and Trichoderma koningii (TC6-Colombia). None of the antagonistic treatments showed a clear antagonistic effect in Pinus sylvestris against the fungal infections compared to control plants inoculated with the pathogens alone. In contrast, in Alnus glutinosa plants T. koningii co-inoculation improved plant growth in several of the growth parameter measured.