Study On The Root-associated Fungi Of Pinus Tabuliformis In Pine Wilt Disease Damaged And Undamaged Areas

Pine wilt disease, caused by pine wood nematode (Bursaphelenchus xylophilus), isthreating the pine forest resources, ecological environments and the natural landscape in ourcountry. Pine wilt disease has caused so big economic and ecological losses that it has beenlisted as the first of the forest disease. It is very urgent to find the effective method to controlpine wilt disease. Uasually, there is an interaction between the root microorganisms and theplant disease. Some researches have showed that pine wilt disease can be influenced by theectomycorrhizal fungi (ECMF). In order to recover the relationships between pine wiltdisease and the root-associated fungi of pine tree systematically, this thesis studied the activityof root tips, colonization rates of ECMF and dark septate endophytes (DSE) at the roots ofPinus tabuliformis in pine wilt disease damaged and undamaged areas located in Zhashuidistrict, Shangluo city, Shaanxi province. The fungal community structure and diversity of thetwo sample areas were analyzed by the isolates and identification of the culturable fungi inthe root of P. tabuliformis. The isolated fungi were then inoculated to and re-isolated from theseedling of P. tabuliformis, and their influences on the growth of pine seedlings also wereinvestigated. In addition, the biological characteristics and culture conditions of the dominantspecies were studied and optimized, respectively. This research is the first report about therelationship between DSE and pine wilt disease. Further research is required to make clear theeffect of these isolates on the resistance of pine tree against pine wilt disease.1. Fungal colonization characteristics at the root of P. tabuliformis in pine wilt diseasedamaged and undamaged areasAccording to the results, not only the ratio of active root tips and ectomycorrhizal tips,but also the colonization rate of ECMF were significantly higher in pine wilt diseaseundamaged area than those in damaged area (p<0.05), while the colonization rate of DSE(p<0.05) and the density of microsclerotium were lower than damaged area. The colonizationrates of ECMF were significantly higher (p<0.05) than those of DSE in both of the twosample areas. It is suggested that pine wilt disease affected the root activity, ectomycorrhizal development, colonization of ECMF and DSE.2. Culturable fungal structures and diversities at the root of P. tabuliformis in pine wiltdisease damaged and undamaged areasBased on the morphological and molecular characteristics of131isolates, they werefinally identified as23species. Among them, the dominant fungi were DSE, especiallyPhialocephala fortinii and Cryptosporiopsis ericae. The two sample areas exhibited obviousdifferences in root fungal community structures and diversities. In the pine wilt diseaseundamaged area, the richness (17) and Shannon-Wiener biodiversity index (2.012) of rootfungal community, as well as the relative abundance of ECMF (8%) were higher than those indamaged area (11,1.198and1.6%, respectively), but the relative abundance of DSE (70%)was lower than that in damaged area (82.7%). And5species were shared in the two sampleareas. Above all, it is suggested that the outbreak of pine wilt disease affected the fungalcommunity structure and diversity in the root of P. tabuliformis.3. Reinoculation of isolates to P. tabuliformis seedlingsEight DSE (Phialophora mustea, Exophiala salmonis, Paraphoma chrysanthemicola, P.fortinii, Cadophora finlandia, C. ericae, Oidiodendron maius, Trichocladium opacum) andtwo ECMF (Lycoperdon perlatum, Ceriporia lacerate) isolates were successfully reinoculatedinto pine seedlings with a colonization rate ranged from34.0%to96.0%. They all formed thetipical characteristic of DSE and ECMF in the root of pine seedlings, respectively. However,four DSE isolates (P. mustea, E. salmonis, P. chrysanthemicola and P. fortinii) also formedthe structures similar to typical ECM--mantle. These fungi were all re-isolated from the rootsof tested pine seedings, suggesting they are the ECMF and DSE of P. tabuliformis.4. Influences of isolates on P. tabuliformis seedlingsThe heights, stem diameters, shoot and root dry weights of pine seedlings inoculatedwith above isolates were all higher than those of non-inoculated control. Stem diameters ofpine seedlings inoculated with E. salmonis and P. fortinii were significantly higher thannon-inoculated control, and the shoot dry weights of pine seedlings inoculated with P. mustea,E. salmonis, P. chrysanthemicola, P. fortinii and C. finlandia were obviously higher thancontrol (p<0.05). Shoot dry weight of pine seedlings inoculated with P. fortinii was175%higher than non-inoculated control. It is obvious P. fortinii can improve the growth andbiomass of pine seedlings more greatly.5. The growth characteristics and culture conditions of the dominant species P. fortiniiIn this study, P. fortinii is the dominant species and can improve the growth of pineseedlings, so its growth characteristics and culture conditions were studied and optimized.The results showed that the best growth of P. fortinii was exhibited at20°C,140rpm and pH 3in conditions of temperatures (20,25,30,35°C), pH (3,4,5,6,7) and agitation speed (80,100,120,140rpm). Under the conditions of different carbon sources (glucose, maltose,lactose, soluble starch, mannitol) and nitrogen sources ((NH4)2HPO4, NH4NO3, KNO3,peptone, malt resinoid), the optimum carbon and nitrogen sources of P. fortinii were lactoseand malt resinoid on solid medium and soluble starch and peptone on liquid medium,respectively. The growth and colony characteristics of P. fortinii were not easy to be affectedby carbon sources, however, the color, texture and size of its colonies were influenced moregreatly by nitrogen sources.