Divergent Fungal Community Dynamics of Thuja sutchuenensis in Arid Environments.

Thuja sutchuenensis Franch., an endangered species sparsely distributed in the mountainous and arid regions of southwest China, faces the critical challenge of adapting to these harsh conditions. Understanding the plant's strategies for survival and the precise roles played by soil fungal communities in this adaptation remains an area of limited knowledge. Our investigation centers on the fungal communities associated with T. sutchuenensis and their interactions with soil water content. Notably, we identified unique fungal communities in the low soil moisture group, and these communities exhibited lower coverage but higher phylogenetic diversity (PD), Chao1, and Shannon indices compared to other groups. Network analysis revealed a modular structure within the fungal communities, with key hub nodes identified, particularly in the arid group. This group demonstrated higher levels of soil saprotrophs and ectomycorrhizal fungi and a reduced presence of plant pathogens. Linear discriminant analysis highlighted the significance of genera such as Russula, Myxotrichaceae, and Sebacina, emphasizing their roles in supporting the plant in arid environments. Random forest analysis indicated that soil moisture content emerged as the primary driver in determining fungal composition and diversity and contributed to the variables of several fungal genera. Collectively, this study provides valuable insights into the fungal communities associated with T. sutchuenensis, shedding light on their adaptation to extreme arid conditions.

Effects of mixed substrates of different agricultural and forestry residues on the cutting seedlings of Thuja sutchuenensis.

To screen environment-friendly seedling cultivation substrates which could replace peat and with less cost, we compared the effects of different agricultural and forestry residue mixed substrates on cutting propagation of Thuja sutchuenensis, in an experiment following randomized block design. There were five types of mixed substrates, including peat + vermiculite + perlite (T1), edible mushroom residue (EMR) + vermiculite + perlite (T2), carbo-nized rice husk (CRH) + vermiculite + perlite (T3), EMR + slag + sawdust (T4) and CRH + EMR + slag (T5). The results showed that the bulk density of T3 was the lowest, followed by T2, which significantly differed from other mixed substrates. The non-capillary porosity of T2 was significantly greater than that of T1, while the capillary porosity and the total porosity of T2 was lower than T1 and T3, respectively. T2 had the highest contents of total nitrogen, total phosphorus, total potassium, alkali-hydrolyzed nitrogen, available phosphorus, substrate moisture and the highest pH, which differed significantly from other mixed substrates in most chemical indicators. The membership function values of rooting rate and growth indicators of cuttings with different mixed substrates were in order of T2 > T3 > T1> T5 > T4. Most indicators with larger grey relation values were physical indicators. The top five indicators were capillary water capacity, total potassium, field water capacity, maximum water capacity, and total porosity, with both capillary water capacity and total potassium content ranking first. In general, the physicochemical properties, rooting rate, and growth characteristics of cuttings under T2 were better than those of other mixed substrates. The capillary water capacity and total potassium were the main factors affecting rooting and growth of cuttings. At the early stage of cutting, the physical properties of mixed substrate had greater effect on rooting rate and growth of cuttings than the chemical properties. Overall, our results suggested that T2 should be preferred in the cutting propagation of T. sutchuenensis.

Complete mitochondrial genome of Thuja sutchuenensis and its implications on evolutionary analysis of complex mitogenome architecture in Cupressaceae.

BACKGROUND: The complex physical structure and abundant repeat sequences make it difficult to assemble the mitogenomes of seed plants, especially gymnosperms. Only approximately 33 mitogenomes of gymnosperms have been reported. However, as the most widely distributed and the second largest family among gymnosperms, Cupressaceae has only six assembled mitogenomes, including five draft mitogenomes and one complete mitogenome, which has greatly hindered the understanding of mitogenome evolution within this large family, even gymnosperms. RESULTS: In this study, we assembled and validated the complete mitogenome of Thuja sutchuenensis, with a size of 2.4 Mb. Multiple sequence units constituted its complex structure, which can be reduced to three linear contigs and one small circular contig. The analysis of repeat sequences indicated that the numbers of simple sequence repeats increased during the evolutionary history of gymnosperms, and the mitogenome of Thuja sutchuenensis harboured abundant extra-long repeats (more than 5 kb). Additionally, the longest repeat sequence identified in these seven gymnosperms also came from the mitogenome of Thuja sutchuenensis, with a length of up to 47 kb. The analysis of colinear blocks and gene clusters both revealed that the orders of mitochondrial genes within gymnosperms was not conserved. The comparative analysis showed that only four tRNAs were shared by seven gymnosperms, namely, trnD-GUC, trnE-UUC, trnI-CAU and trnY-GUA. Furthermore, four genes have undergone potential positive selection in most gymnosperm species, namely, atp8, ccmB, mttB and sdh4. CONCLUSION: We successfully assembled the second complete mitogenome within Cupressaceae and verified that it consisted of multiple sequence units. Our study also indicated that abundant long repeats may contribute to the generation of the complex conformation of the mitogenome of Thuja sutchuenensis. The investigation of Thuja sutchuenensis's mitogenome in our study provides new insight into further understanding the complex mitogenome architecture within gymnosperms.

Contrasting Responses of Multispatial Soil Fungal Communities of Thuja sutchuenensis Franch., an Extremely Endangered Conifer in Southwestern China.

Thuja sutchuenensis Franch. is an endangered species in southwest China, distributed sporadically in mountainous areas. Soil property and soil fungal community play a crucial role in plant growth and survival. Nevertheless, understanding soil properties and the soil fungal community in the areas where T. sutchuenensis is distributed is extremely limited. Hence, this study collected a total of 180 soil samples from five altitudinal distribution areas (altitudinal gradients) and three vertical depths throughout four horizontal distances from the base of each tree. The results found that altitudinal gradients and vertical depths altered soil properties, including pH, organic matter content, water content, total nitrogen, phosphorus, and potassium, and available nitrogen, phosphorus, and potassium. The fungal alpha diversity indexes (Chao1 and Shannon) and beta diversity were dramatically decreased with elevation. In addition, high altitudes (2,119 m) harbored the highest relative abundance of ectomycorrhizal fungi (27.57%) and the lowest relative abundance of plant-pathogenic fungi (1.81%). Meanwhile, we identified a series of fungal communities, such as Tomentella, Piloderma, Cortinarius, Sebacina, and Boletaceae, that play an essential role in the survival of T. sutchuenensis. The correlation analysis and random forest model identified that water content and total phosphorus showed strong relationships with fungal characteristics and were the primary variables for Zygomycota and Rozellomycota. Collectively, the findings of this integrated analysis provide profound insights into understanding the contrasting responses of T. sutchuenensis soil fungal communities and provide a theoretical basis for T. sutchuenensis habitat restoration and species conservation from multispatial perspectives. IMPORTANCE The present study highlights the importance of fungal communities in an endangered plant, T. sutchuenensis. Comparative analysis of soil samples in nearly all extant T. sutchuenensis populations identified that soil properties, especially soil nutrients, might play critical roles in the survival of T. sutchuenensis. Our findings prove that a series of fungal communities (e.g., Tomentella, Piloderma, and Cortinarius) could be key indicators for T. sutchuenensis survival. In addition, this is the first time that large-scale soil property and fungal community investigations have been carried out in southwest China, offering important values for exploring the distribution pattern of regional soil microorganisms. Collectively, our findings display a holistic picture of soil microbiome and environmental factors associated with T. sutchuenensis.

Comparative Analyses of Rhizosphere Bacteria Along an Elevational Gradient of Thuja sutchuenensis.

Thuja sutchuenensis Franch. is an endangered species in southwestern China, primarily distributed in 800-2,100 m of inaccessible mountainous areas. Rhizosphere soil physicochemical properties and bacterial communities play an essential role in managing plant growth and survival. Nonetheless, the study investigating rhizosphere soil properties and bacterial communities of T. sutchuenensis is limited. The present study investigated soil properties, including soil pH, organic matter, water content, nitrogen, phosphorus, and potassium contents, and bacterial communities in nearly all extant T. sutchuenensis populations at five elevational gradients. Our results demonstrated that the increase in elevation decreased rhizosphere and bulk soil phosphorus content but increased potassium content. In addition, the elevational gradient was the dominant driver for the community composition differentiation of soil bacterial community. Proteobacteria and Acidobacteria were the dominant bacterial phyla distributed in the rhizosphere and bulk soils. Co-occurrence network analysis identified key genera, including Bradyrhizobium, Acidicapsa, Catenulispora, and Singulisphaera, that displayed densely connected interactions with many genera in the rhizosphere soil. The dominant KEGG functional pathways of the rhizosphere bacteria included ABC transporters, butanoate metabolism, and methane metabolism. Further correlation analysis found that soil phosphorus and potassium were the dominant drivers for the diversity of soil bacteria, which were distinctively contributed to the phylum of Planctomycetes and the genera of Blastopirellula, Planctomycetes, and Singulisphaera. Collectively, this comprehensive study generated multi-dimensional perspectives for understanding the soil bacterial community structures of T. sutchuenensis, and provided valuable findings for species conservation at large-scale views.

Thujasutchins N and O, two new compounds from the stems and roots of Thuja sutchuenensis.

Two novel secondary metabolites, including one thujopsene-type sesquiterpene designated thujasutchin N (1) and one norlignan named thujasutchin O (2) were obtained from the ethanolic extract of the stems and roots of Thuja sutchuenensis. Among them, thujasutchin O (2) represents the first example of lignan sharing a unique carbon-reduced skeleton with novel acetal functionality. Their structures were unambiguously determined by means of extensively spectroscopic analysis including UV, IR, HRESIMS, NMR, and single crystal X-ray diffraction. Both of the isolates were evaluated for their in vitro cytotoxic and antibacterial activities.

Chloroplast and Nuclear Genetic Diversity Explain the Limited Distribution of Endangered and Endemic Thuja sutchuenensis in China.

Narrow-ranged species face challenges from natural disasters and human activities, and to address why species distributes only in a limited region is of great significance. Here we investigated the genetic diversity, gene flow, and genetic differentiation in six wild and three cultivated populations of Thuja sutchuenensis, a species that survive only in the Daba mountain chain, using chloroplast simple sequence repeats (cpSSR) and nuclear restriction site-associated DNA sequencing (nRAD-seq). Wild T. sutchuenensis populations were from a common ancestral population at 203 ka, indicating they reached the Daba mountain chain before the start of population contraction at the Last Interglacial (LIG, ∼120-140 ka). T. sutchuenensis populations showed relatively high chloroplast but low nuclear genetic diversity. The genetic differentiation of nRAD-seq in any pairwise comparisons were low, while the cpSSR genetic differentiation values varied with pairwise comparisons of populations. High gene flow and low genetic differentiation resulted in a weak isolation-by-distance effect. The genetic diversity and differentiation of T. sutchuenensis explained its survival in the Daba mountain chain, while its narrow ecological niche from the relatively isolated and unique environment in the "refugia" limited its distribution.

Chloroplast genome of an extremely endangered conifer Thuja sutchuenensis Franch.: gene organization, comparative and phylogenetic analysis.

Thuja sutchuenensis is a critically endangered tertiary relict species of Cupressaceae from southwestern China. We sequenced the complete chloroplast (cp) genome of T. sutchuenensis, showing the genome content of 129,776 bp, 118 unique genes including 82 unique protein-coding genes, 32 tRNA genes, and 4 rRNA genes. The genome structures, gene order, and GC content are similar to other typical gymnosperm cp genomes. Thirty-eight simple sequence repeats were identified in the T. sutchuenensis cp genome. We also found an apparent inversion between trnT and psbK between genera Thuja and Thujopsis. In addition, positive selection signals were detected in seven genes with high Ka/Ks ratios. The reconstructed phylogeny based on locally collinear blocks of cp genomes among 21 gymnosperms species is similar to previous inferences. We also inferred a Late-Miocene divergence between T. sutchuenensis and T. standishii, according to the dating of ~ 11.05 Mya by cp genomes. These results will be helpful for future studies of Cupressaceae phylogeny as well as studies in population genetics, systematics, and cp genetic engineering.

Antibacterial sesquiterpenes from the stems and roots of Thuja sutchuenensis.

Eight new sesquiterpenes with diverse skeletons involving four cuparenes, denominated thujasutchins F-I (1-4), one eudesmane and one cedrol, named thujasutchin J (5) and thujasutchin K (6), as well as two thujopsenes thujasutchins L-M (7-8) together with three known congener compounds (9-11) were isolated from EtOAc soluble fraction of ethanolic extract of the stems and roots of Thuja sutchuenensis. Their structures including absolute configurations were unambiguously established by extensive interpretation of the NMR and mass spectroscopic data, X-ray diffractions, and ECD measurements powered by molecular calculations. The biological assays disclosed that 5 and 9 displayed potent inhibitory effect against Staphylococcus. aureus (CMCC 26003), methicillin-resistant Staphylococcus aureus (JCSC 4744), Bacillus cereus (ATCC 10876), and Staphylococcus epidermidis (ATCC 12228) with MICs ranging from 6.25 to 25 µg/mL.

Chemical constituents of the trunks and roots of Thuja sutchuenensis.

Five new compounds including two stilbenes, designated thujasutchins A (1) and B (2), two phenolic compounds namely thujasutchins C (3) and D (4), as well as one sesquiterpene thujasutchin E (5), were isolated from the 95% ethanolic extract from the trunks and roots of Thuja sutchuenensis. Their structures were determined by means of extensively spectroscopic analysis including UV, IR, HRESIMS, 1H and 13C NMR (COSY, HSQC, HMBC). Moreover, compounds 1, 3-5 were evaluated for in vitro cytotoxic activities against SF-268, MCF-7, HepG-2, and A549 tumor cell lines.