Tuber Affect Quercus Aliena Physiology And Root-associated NirK-and NirS-type Denitrifying Bacterial Communities In The Initial Stage Of Inoculation

Tuber genus(Ascomycota,Pezizales)commonly known as truffles,are edible fungi that form symbiotic relationships with plants.Tuber melanosporum and Tuber indicum are two kinds of commercial truffles with high value.Quercus aliena,a dominant tree species in southwest China with well-developed root system,is excellent host plant specie for truffles.The bacteria in rhizosphere soils of host plants mycorrhized by Tuber(ectomycorrhizosphere soils)are different from rhizosphere soils of plant without Tuber patner.Nir S-and nirK-type denitrifying bacteria as the main driver of the denitrification process affect the growth of truffles and hosts by transforming nitrogen in soils,however,the distribution of nirK-and nirS-type denitrifying bacteria in ectomycorrhizosphere soils is unclear.Moreover,there is little information on the Quercus aliena infected by Tuber species.Here,we cultivated Quercus aliena seedlings inoculated with T.melanosporum or T.indicum in the greenhouse for 6 months and then determined the morphological and physiological indices of Q.aliena at different stages(0,2,4,and 6 months post-inoculation).Furthermore,high-throughput sequencing technology was used to analyze the nirK-and nirS-denitrifying bacterial communities in the rhizosphere soil of cultivated Q.aliena at 0 and 6 months post-inoculation.Main resultes of this work are as follows:(1)The ectomycorrhizae of both T.melanosporum and T.indicum were observable four months after inoculation,and the ectomycorrhizae in every Q.aliena seedling with T.indicum inoculation was more than that with T.melanosporum inoculation.The morphological characteristics of the two mycorrhizal fungi were similar,the head was enlarged,and the root tip was milky,the base is light yellow,yellow brown,translucent,mostly bifurcate and non-branched rod-like,with a small amount of epimycorrhizal hyphae standing on the surface of mycorrhizal fungi.After aging,the color of mycorrhizal fungi becomes dark brown,black brown,only the apex is translucent,the number of epimycorrhizal mycorrhizal fungi increases significantly,and the length of the 2.0～3.0 mm,and the diameter ranges from 0.4～0.5 mm.The mantle consists of 6～10 layers of irregular thick-walled cells with a thickness of about 30～50 μm.Most of the cells are irregular.The mycorrhizal tip mantle cells are mosaic liked horseshoe.(2)The morphological index of Q.aliena seedlings inoculated with T.melanosporum or T.indicum were not significantly different from that of Q.aliena seedlings without Tuber inoculation after 6 months cultivation,which may indicated that the inoculation of T.melanosporum and T.indicum won’t altere the morphology of Q.aliena.(3)The difference of root activity and superoxide activities of Q.aliena among the three treatments was not significant.However,the peroxidase activity in root tissues was significantly higer in root tissue of Q.aliena inoculated with T.melanosporum(P(27)0.05).Thess results illustrated that the peroxidase activity and in root tissues were improved by the T.melanosporum while the root activity and superoxide activities wouldn’t be affected by T.melanosporum or T.indicum inoculation.(4)The nitrate nitrogen content in the ectomycorrhizosphere soil was significantly greater than that in the control soil,moreover,nitrate nitrogen content in the ectomycorrhizosphere soil with T.melanosporum inoculation was significantly higher than that with T.indicum inoculation(P(27)0.05),which may imply that the nitrate nitrogen content of the ectomycorrhizosphere soil was improved more significantly than that of the control soil,with the effect of the T.melanosporum being stronger.(5)The nirK-and nirS-denitrifying bacteria analysis indicated that the richness and diversity of the nirK-and nirS-denitrifying bacteria in rhizosphere soil were significantly increased after colonization by T.melanosporum and particularly T.indicum.The most abundant nirK-and nirS-denitrifying bacteria in ectomycorrhizosphere soil with T.indicum included Pseudogulbenkiania,Bosea,Rhizobium,Ochrobactrum,Mesorhizobium,Bradyrhizobium,and Cupriavidus,while the dominant nirK-and nirS-denitrifying bacteria in T.melanosporum ectomycorrhizosphere soil included Rhizobium,Agrobacterium,Pseudomonas,Ensifer,and Dechlorospirillum.This study revealed that T.melanosporum and T.indicum had remarkable effects on Q.aliena seedlings,both on their physiological indices and the nirK-and nirS-denitrifying bacteria in rhizosphere soil,as well as the soil properties,laying a theoretical foundation for the cultivation of Q.aliena as a truffle host plant.