Response Of Fungal Community Structure Associated With Hair Roots Of Rhododendron Fortunei To Environmental Changes

Mycorrhiza is a mutualistic symbiosis spawned by the roots of higher plants and soil fungi.The hairy roots of most Ericaceae plants can form a special type of endomycorrhiza,named Ericoid mycorrhizas（ERM）.ERM has positive roles in improving host plant nutrition,regulating plant metabolism,and enhancing plant stress resistance,etc.In recent decades,the old population of Rhododendron fortunei Lindl.in Huading National Forest Park（or Huading Mountain）in Tiantai Mountain,Zhejiang Province,has been gradually declining,with reduced flowering and sparse branches,which may be related to the changes of the accompanying species and their soil environment.From2019 to 2021,in the hot summer,we collected hair roots of R.fortunei at altitude between 900 and 1100m,from different age groups（young,middle-aged and old,with ground diameters at about 15 cm,25～30 cm,and>45 cm,respectively）and different habitats（with Indocalamus tessellatus（a kind of bamboo）,with Camellia sinensis（tea plant）,and without shrubs as control）.The composition of fungal communities associated with hair roots of R.fortunei was analyzed by Illumina Mi Seq technology,one kind of high throughput sequencing;the microstructure of roots of R.fortunei,I.tessellatus and C.sinensis was observed by acetic acid and ink staining;the mass ratio of hair roots and mycorrhizal infection of R.fortunei in different aged hosts and different habitats were compared.At the same time,the soil around these roots of the R.fortunei samples was collected to determine the composition of various chemical elements,where we determine the total C and total N by Vario EL III elementanalyzer,total P by Molybdenum antimony anti colorimetry,NH₄⁺-N by Sodium salicylate method,NO₃^--N by Na OH-sulfuric acid hydrazine process,available P by NH₄F-HCl method and p H value by potentiometric method,and C/N and N/P was calculated.All efforts are dedicated to analyze the effects of the accompanying species and environmental changes to R.fortunei.The main results were as follows:（1）Given the difference of the accompanying species,there was no significant difference on p H and NH₄⁺-N（P>0.05）in soil,however other parameters had extremely significant differences among various habitats（P≤0.01）.（2）The related parameters of litter under the canopy of the three habitats were also analyzed,and the total N,N/P and C/N in soil with C.sinensis were significantly higher than those in the habitats without shrubs and I.tessellatus.（3）There were typical ERM structures in the hair roots of R.fortunei with different ages and habitats.Less obvious vesicles and arbuscular structure in the roots of C.sinensis and I.tessellatus was also observed as arbuscular mycorrhiza.The mass ratio of hair roots and mycorrhizal infection rate of R.fortunei vulgaris in the old R.fortunei were extremely significantly lower in the associated habitat of I.tessellatus than other habitats and hosts of other ages in the same habitat（P≤0.01）.（4）The fungal species associated with hair roots of R.fortunei in Huading mountain were highly diversified,mainly consisting of Ascomycota and Basidiomycota.The putative ERM fungal orders—Helotiales and Sebacinales were dominant,the former showed significantly positive correlation with NO₃^--N and the available P in soil,while the latter showed negative correlation with NO₃^--N.The ERM fungal species with the highest relative abundance were Oidiodendron maius and unclassified Helotiales of Ascomycota,as well as Serendipitacea sp.and Serendipita sp.of Basidiomycota,and they showed more relations with soil factors.（5）In habitats without shrubs,the relative abundance of Helotiales and Sebacinales were the highest among the middle-aged hosts and lowest among the old hosts.Helotiales decreased but Sebacinales increased with host ages.（6）With I.tessellatus habitats,the variation of the relative abundances of Helotiales and Sebacinales in their fungal communities with ages were opposite to those in habitats without shrubs.And the relative abundances of the two fungal orders（Helotiales and Sebacinales）were significantly negatively correlated（P≤0.01）.（7）With C.sinensis habitats,the relative abundance of ERMF associated with hair roots of R.fortunei generally decreased in the communities.The relative abundance of O.maius showed opposite trends with C.sinensis and without shrubs with increasing host ages.Some potential pathogenic fungi（such as Fusarium concentricum,Didymella rosea and Gibberella avenacea）saw significant increase in the hair roots of young R.fortunei.（8）For two consecutive years,the comparison of the root fungal communities of the middle-aged R.fortunei found that the composition of the root fungal community was relatively stable,especially the ERM fungi.However,with the change of weather constituent,the structure of fungal community associated with hair roots of R.fortunei showed a certain dynamic change.The composition and structure of fungal community in the hair roots of R.fortunei in Huading Mountain were affected by the changes of accompanying species（especially the expansion of I.tessellatus）.Different age groups of R.fortunei showed different responses to the changes of other woody species and soil factors.Compared with the hosts of young and middle-aged R.fortunei,development of hair root,mycorrhizal infection,and ERM fungal composition and structure on the old R.fortunei were affected more heavily by changes with I.tessellatus expansion and their habitat’s variation.With I.tessellatus expansion,the stunted development and the change of mycorrhizal composition of R.fortunei under the ground may be one of the main reasons for the decline of them above the ground.We suggest that to protect the sustainable development of the precious R.fortunei population in Huading Mountain one must control the expansion of I.tessellatus by wiping them out or by introducing herbivores.