Symbiotic Characteristics Between Pinus Massoniana And Suillus Bovinus

Ectomycorrhizas(ECM)are symbiotic associations between woody plant roots and fungi that form specific structures and functions.The beneficial effects of ECM are mainly due to the enhanced nutrient and water acquisition of the host,as well as the alleviation of host damage derived from varies stresses,which is of great important for the stability of forest ecosystems.Moreover,there are many groups ofECM fungi that could produce edible sporocarps,which are of great economic value.So far,the study of ECM mainly focuses on its growth-promoting and stress-resistant effects on host plants,whereas the symbiotic characteristics of most ECM(especially the process of symbiotic establishment)are still unknown,which largely limits the application of those ECM fungi with both economic and ecological values.By means of morphological,molecular biological,and ecological techniques,the symbiotic characteristics between Pinus massoniana and its dominant ECM fungus-Suillus bovinus,as well as the phenology of Suillus bovinus sporocarps were investigated.The main results are as follow:1.Suillus bovinus isolate obtained from the sporocarp could be well cultured with PDA,MMN and Pachlewski(Pach)media,among which MMN was the optimum.No spores were produced in pure culture.The isolate produced white colony on MMN medium,andits hyphae have septaand few clamp connections.The aseptic seedlings of Pinus massoniana were prepared combining seed surface sterilization procedures and subsequence substrate cultivation.The optimal procedures for seed surface sterilization consists of a preliminary wash of seeds with 0.01% Tween 20,soak for 2h with 0.5% KMn O4,1h with 0.01% Tween 20 and 0.5% carbendazim mixture,and followed by 15 min in the mixture of streptomycin sulfate(200mg/L)and gentamicin(100mg/L),and the vermiculite was most suitable for seedling growth.2.The morphological characteristics during the symbiosis between Pinus massoniana and Suillus bovinus were carefully studied under aseptic condition.There were two stages emerged chronologically during the symbiosis establishment.(1)During the pre-infection stage(before physical contact),the volatile organic compounds and exudates emitted by Suillus bovinus could promote root growth and lateral root formation of Pinus massoniana.(2)The symbiotic stage could be furtherly divided into three periods.The contact period started from four days after inoculation,and the fungus began to adhere to the roots and the appressorium was formed.By the day of seven,hyphae began to penetrate the roots,and the colonization period started.After that and till 28 days post inoculation,the Hartig net and the mantle formation period was present,and the swelled mycorrhized root tips were formed.60 days postinoculation,the typical dichotomous branching ECM were emerged.The Hartig net and the mantle developed simultaneously,but the Hartig net was matured a little bit earlier.3.RNAseq techniquewas employed to screen the differential expressed genes(DEGs)in Pinus massoniana inoculated with Suillus bovinus.213 DEGs were detected,with 109 genes up-regulated and 104 genes down-regulated.GO enrichment analysis revealed that the DEGs were mainly localized in cytoplasm and cytoplasmic part,and involving biological processes such as protein metabolism,cellular nitrogen compounds biosynthesis and organic nitrogen compounds metabolism.The results of KEGG metabolic pathway analysis further indicated that the early stage of the establishment of the symbiosis between Pinus massoniana and Suillus bovinus was involved in a series of biological processes such as phenylpropanoid biosynthesis,plant hormone signal transduction,plant-pathogen interaction pathway and glycerolipid metabolism pathway.4.The ectomycorrhizas formed by the Suillus bovinus with Pinus massoniana roots is usually coralloid in shape with dichotomous branches,and had smooth surface without emanating hyphae but with rhizomorph;The sporcarps formed by this fungi has long fruiting seasons and considerable production under the Pinus massoniana forest.Sporocarp productivity increased as the temperature or precipitation increased and it decreased as the temperature or precipitation decreased.In summary,the presented results shed new lights onthe symbiotic mechanisms between Pinus massoniana and Suillus bovinus,and provide knowledge for future mycosilviculture of edible sporocarps in the forests of China.