Transcriptome profiles reveal response mechanisms and key role of PsNAC1 in Pinus sylvestris var. mongolica to drought stress.

BACKGROUND: Drought stress severely impedes plant growth, and only a limited number of species exhibit long-term resistance to such conditions. Pinus sylvestris var. mongolica, a dominant tree species in arid and semi-arid regions of China, exhibits strong drought resistance and plays a crucial role in the local ecosystem. However, the molecular mechanisms underlying this resistance remain poorly understood. RESULTS: Here, we conducted transcriptome sequence and physiological indicators analysis of needle samples during drought treatment and rehydration stages. De-novo assembly yielded approximately 114,152 unigenes with an N50 length of 1,363 bp. We identified 6,506 differentially expressed genes (DEGs), with the majority being concentrated in the heavy drought stage (4,529 DEGs). Functional annotation revealed enrichment of drought-related GO terms such as response to water (GO:0009415: enriched 108 genes) and response to water deprivation (GO:0009414: enriched 106 genes), as well as KEGG categories including MAPK signaling pathway (K04733: enriched 35 genes) and monoterpenoid biosynthesis (K21374: enriched 27 genes). Multiple transcription factor families and functional protein families were differentially expressed during drought treatment. Co-expression network analysis identified a potential drought regulatory network between cytochrome P450 genes (Unigene4122_c1_g1) and a core regulatory transcription factor Unigene9098_c3_g1 (PsNAC1) with highly significant expression differences. We validated PsNAC1 overexpression in Arabidopsis and demonstrated enhanced drought resistance. CONCLUSIONS: These findings provide insight into the molecular basis of drought resistance in P. sylvestris var. mongolica and lay the foundation for further exploration of its regulatory network.

Phytic acid is a new substitutable plant-derived antifungal agent for the seedling blight of Pinus sylvestris var. mongolica caused by Fusarium oxysporum.

Phytic acid (PA) is a new substitutable plant-derived antifungal agent; however, few reports have been published regarding its antifungal effects on pathogenic fungi. The present study explored the in vitro antifungal activity of PA against four phytopathogenic fungi and found that PA was the most effective at inhibiting the growth of Fusarium oxysporum. This study aimed to investigate the in vivo and in vitro antifungal activities of PA against the seedling blight of Pinus sylvestris var. mongolica caused by F. oxysporum and to determine its possible mechanism of action. The results showed that PA inhibited spore germination and mycelial growth of F. oxysporum in a concentration-dependent manner and exhibited strong inhibition when its concentration exceeded 1000 mg/L. It mainly destroyed the integrity of the cell membrane, increasing its cell membrane permeability, causing the cell contents to spill out, and impairing fungal growth. In addition, the leakage of intercellular electrolytes and soluble proteins indicated that PA used at its EC20 and EC50 increased the membrane permeability of F. oxysporum. The increase in malondialdehyde and hydrogen peroxide content confirmed that PA treatment at its EC20 and EC50 damaged the cell membrane of the pathogen. Scanning electron microscopy revealed that PA affected the morphology of mycelia, causing them to shrivel, distort, and break. Furthermore, PA significantly reduced the activities of the antioxidant-related enzymes superoxide dismutase and catalase, as well as that of the pathogenicity-related enzymes polygalacturonase, pectin lyase, and endoglucanase (EG) in F. oxysporum (P < 0.05). In particular, EG enzyme activity was maximally inhibited in F. oxysporum treated with PA at its EC50. Moreover, PA significantly inhibited the incidence of disease, and growth indices in Pinus sylvestris var. mongolica seedling blight was determined. In summary, PA has a substantial inhibitory effect on F. oxysporum. Therefore, PA could serve as a new substitutable plant-derived antifungal agent for the seedling blight of P. sylvestris var. mongolica caused by F. oxysporum.

First Report of Black Spot Needle Blight of Pinus sylvestris var. mongolica Litv. Caused by Heterotruncatella spartii in China.

Pinus sylvestris var. mongolica Litv. (Pinales: Pinaceae) is an excellent tree for soil and water conservation in Northeast China. The Honghua'erji area in Inner Mongolia is the "hometown of P. sylvestris var. mongolica", however, in recent years, coniferous diseases of P. sylvestris var. mongolica have frequently occurred here. During the investigation, it was found that some black spot needle blight had been observed in addition to the common blight caused by Sphaeropsis sapinea. From May to September 2020, black spot needle blight was found on hundreds of P. sylvestris var. mongolica trees in four forest farms, and the infection rate among the forests was 24.58 % (n=240). This disease first appeared on the upper part of the needles, and the needles then became withered and gradually showed light black spots, although they remained green. As the disease progressed, the needles eventually died and turned gray with many dark black spots. Fungal isolate named YJ-1 was obtained from infected needles of symptomatic pine trees, and a voucher specimen was deposited in Heilongjiang Province Key Laboratory of Forest Protection. Microscopic observation showed the conidia were 3-septate (4 cells) clavate spindles that measured 23.9 µm (20.8-25.9) × 5.9 µm (4.5-8.2) (n=50). The middle two cells were dark brown, and the septa were darker than the cells. Both apical and basal cells were hyaline. The apical cell had 2-4 appendages (mostly 3), and the basal cell had a truncate base (n=50). The cultural characteristics on potato dextrose agar medium were flat off-white and dense in 3-5 d. At approximately 5-7 d, the reverse side of the colony turned pale to slightly luteous. Superficial black acervuli were distributed in the center of the mature colonies after 10 d. Morphological, cultural and microscopic characteristics observed were similar of Heterotruncatella spartii (basionym: Truncatella spartii) reported by Hlaiem et al (2019). To further identify, total DNA was extracted and the internal transcribed spacer region (ITS-rDNA) was amplified by PCR using the primers ITS1/ITS4 and sequenced for BLASTn analysis and phylogenetic tree construction. The resulting 564 bp sequence (GenBank Accession No. OL662864) had 99.24% (521/525) to H. spartii MFLUCC 15-0537, with bootstrap support of at least 94% using the Neighbor-Joining algorithm by MEGA-X (Felsenstein, 1985). The fungus was identified as H. spartii based on morphology and molecular methods. A pathogenicity test was conducted by preparing a conidial suspension of 2.0 × 107 conidia/mL. The suspension was sprayed onto the needles of 20 pots of annual P. sylvestris ar. mongolica seedlings, and the control was sprayed with sterile water. Then the seedlings were placed in a constant temperature room at 25 °C. After 30 d, typical symptoms appeared on 11 inoculated needles, while the control needles remained symptomless. After 50 d, the re-isolation infection rate reached 66.7 %. The fungus present on the inoculated seedlings was morphologically identical to that originally observed on diseased pines, fulfilling Koch's postulates. The fungus was isolated from Spartium junceum for the first time and designated Truncatella spartii (Senanayake et al, 2015). It was then renamed H. spartii (Liu et al, 2019) and has been reported to infect P. pinea in Tunisia (Hlaiem et al, 2019). To our knowledge, this is the first report of H. spartii causing black spot needle blight on P. sylvestris var. mongolica in China and worldwide.

Spatial Distribution Patterns and Assembly Processes of Abundant and Rare Fungal Communities in Pinus sylvestris var. mongolica Forests.

Revealing the biogeography and community assembly mechanisms of soil microorganisms is crucial in comprehending the diversity and maintenance of Pinus sylvestris var. mongolica forests. Here, we used high-throughput sequencing techniques and null model analysis to explore the distribution patterns and assembly processes of abundant, rare, and total fungal communities in P. sylvestris var. mongolica forests based on a large-scale soil survey across northern China. Compared to the abundant and total taxa, the diversity and composition of rare taxa were found to be more strongly influenced by regional changes and environmental factors. At the level of class, abundant and total taxa were dominated by Agaricomycetes and Leotiomycetes, while Agaricomycetes and Sordariomycetes were dominant in the rare taxa. In the functional guilds, symbiotrophic fungi were advantaged in the abundant and total taxa, and saprotrophic fungi were advantaged in the rare taxa. The null model revealed that the abundant, rare, and total taxa were mainly governed by stochastic processes. However, rare taxa were more influenced by deterministic processes. Precipitation and temperature were the key drivers in regulating the balance between stochastic and deterministic processes. This study provides new insights into both the biogeographical patterns and assembly processes of soil fungi in P. sylvestris var. mongolica forests.

Fungal complexity and stability across afforestation areas in changing desert environments.

The great achievements in combating desertification are attributed to large-scale afforestation, yet we lack verification of how the stability of the fungal community changes in afforestation areas in desert environments. Here, we present the fungal network structure from different niches (root and bulk soil) of plantations of Mongolian pine, a crucial species for afforestation introduced widely in desertification regions. We assessed changes in community complexity and stability of root-associated fungi (RAF) and soil fungi (SF) among different introduction sites: the Hulunbuir Desert (HB), the Horqin Desert (HQ) and the Mu Us Desert (MU). To illuminate the complexity and stability of the fungal network, the differences in topological properties, fungal function, and vegetation and environmental factors between introduction sites were fully considered. We showed that (1) the SF networks had more nodes and edges than the RAF networks. There was a lower ratio of negative:positive cohesion of RAF networks in HB and MU. For SF but not for RAF, across the three introduction sites, a higher modularity and ratio of negative:positive cohesion indicated higher stability. (2) Ectomycorrhizal (EcM) fungi were the dominant functional group in the RAF network (especially in HQ), and were only significantly correlated with vegetation factor. There was a higher relative abundance and number of OTUs of saprophytic fungi in the SF network and they showed positive correlations with soil nutrients. (3) RAF and SF network complexity and stability showed different responses to environmental and vegetation variables. The key determinant of the complexity and stability of the SF networks in Mongolian pine plantations was soil nutrients, followed by climate conditions. The composition and structure of the RAF community was closely related to host plants. Therefore, clarifying the complexity and stability of fungal communities in afforestation areas in changing desert environments is helpful for understanding the interactions between the environment, plants and fungi.

TMT-Based Proteomic Analysis Reveals the Molecular Mechanisms of Sodium Pheophorbide A against Black Spot Needle Blight Caused by Pestalotiopsis neglecta in Pinus sylvestris var. mongolica.

Black spot needle blight is a minor disease in Mongolian Scots pine (Pinus sylvestris var. mongolica) caused by Pestalotiopsis neglecta, but it can cause economic losses in severe cases. Sodium pheophorbide a (SPA), an intermediate product of the chlorophyll metabolism pathway, is a compound with photoactivated antifungal activity, which has been previously shown to inhibit the growth of P. neglecta. In this study, SPA significantly reduced the incidence and disease index and enhanced the chlorophyll content and antioxidant enzyme activities of P. sylvestris var. mongolica. To further study the molecular mechanism of the inhibition, we conducted a comparative proteomic analysis of P. neglecta mycelia with and without SPA treatment. The cellular proteins were obtained from P. neglecta mycelial samples and subjected to a tandem mass tag (TMT)-labelling LC-MS/MS analysis. Based on the results of de novo transcriptome assembly, 613 differentially expressed proteins (DEPs) (p < 0.05) were identified, of which 360 were upregulated and 253 downregulated. The 527 annotated DEPs were classified into 50 functional groups according to Gene Ontology and linked to 256 different pathways using the Kyoto Encyclopedia of Genes and Genomes database as a reference. A joint analysis of the transcriptome and proteomics results showed that the top three pathways were Amino acid metabolism, Carbohydrate metabolism, and Lipid metabolism. These results provide new viewpoints into the molecular mechanism of the inhibition of P. neglecta by SPA at the protein level and a theoretical basis for evaluating SPA as an antifungal agent to protect forests.

Under-canopy afforestation after 10 years: assessing the potential of converting monoculture plantations into mixed stands.

Under-canopy afforestation using different tree species is a key approach in close-to-nature management to improve the structural and functional stability of plantation forests. However, current research on understory afforestation mainly focuses on the seedling stage, with limited attention to saplings or young trees. In this study, we evaluated the growth characteristics and leaf traits of 14-year-old Pinus sylvestris var. Mongolica trees under four different upper forest density (UFD) treatments: 0 trees/hm2 (canopy openness 100%, CK), 150 trees/hm2 (canopy openness 51.9%, T1), 225 trees/hm2 (canopy openness 43.2%, T2), and 300 trees/hm2 (canopy openness 28.4%, T3). We found that the survival rate of P. sylvestris in the T3 was significantly lower than in the other treatments, with a decrease of 30.2%, 18.3%, and 19.5% compared to CK, T1, and T2, respectively. The growth of P. sylvestris in the T1 treatment exhibited superior performance. Specifically, T1 showed a significant increase of 18.8%, 5.5%, and 24.1% in tree height, diameter at breast height, and crown width, respectively, compared to the CK. The mean trunk biomass ratio in the understory was significantly higher than that in full light by 15.4%, whereas the mean leaf biomass ratio was significantly lower by 12.3%. Understory P. sylvestris trees tended to allocate more biomass to the trunk at the expense of decreasing leaf biomass, which would facilitate height growth to escape the shading environment, although the promotion was relatively limited. Leaf length, leaf width, leaf area, leaf thickness, mesophyll tissue thickness, epidermis thickness, and leaf carbon content were the highest in the CK and tended to decrease with increasing UFD, indicating that a high-light environment favored leaf growth and enhanced carbon accumulation. In summary, young P. sylvestris trees adapted to moderate shading conditions created by the upper canopy, and the T1 treatment was optimal for the growth of understory P. sylvestris. This study provides insights into different adaptive strategies of young P. sylvestris trees to changes in light environment, providing practical evidence for under-canopy afforestation using light-demanding trees during pure plantation transformation.

Diversity, Community Structure, and Antagonism of Endophytic Fungi from Asymptomatic and Symptomatic Mongolian Pine Trees.

Diplodia tip blight, caused by Diplodia sapinea (=Sphaeropsis sapinea), are widely distributed in Honghuaerji, Inner Mongolia, China, causing severe damage on natural Mongolian pine (Pinus sylvestris var. mongolica). D. sapinea is an endophyte that becomes pathogenic under conditions of drought, hail damage, or temperature-associated stress. The role of the endophytic community inhabiting different pine tissues in the expression of disease is still unknown. In this study, the diversity and community structure of endophytic fungi among asymptomatic and symptomatic Mongolian pine were detected using culture-based isolation and high-throughput sequencing (HTS), and the potential antagonistic endophytes against D. sapinea were also screened. The results indicated that 198 and 235 strains of endophytic fungi were isolated from different tissues of symptomatic and asymptomatic Mongolian pine, respectively. D. sapinea was the most common endophyte isolated from the current-year needles and shoots of symptomatic trees, and Diplodia was also the most common in the HTS data. There were no significant differences in the endophytic fungal species richness among asymptomatic and symptomatic trees, but there were differences observed within specific sampled tissues. The ANOSIM analysis confirmed that the endophytic fungi community structure significantly differed between sampling tissues among symptomatic and asymptomatic Mongolian pine. Furthermore, the antagonism study revealed Penicillium fructuariae-cellae with the ability to inhibit the growth of D. sapinea in vitro, and the potential performance of this fungus, acting as biological control agent, was evaluated under greenhouse. Our findings can pave the way to a better understanding of the interactions between D. sapinea, other endophytic fungi and their hosts, and provide helpful information for more efficient disease management strategies.

[Temporal and Spatial Variations in Root-associated Fungi Associated with Pinus sylvestris var. mongolica in the Semi-arid and Dry Sub-humid Desertified Regions of Northern China].

To illuminate the ecological functions of root-associated fungi (RAF) and their interactions with host plants, we revealed the root-associated fungal diversity and community compositions of Pinus sylvestris var. mongolica involving natural forests and plantations (half-mature, nearly mature, and mature forests) in the Hulunbuir Desert, Horqin Desert, and Mu Us Desert and investigated the environmental driving factors (climatic condition and soil property). The results indicated that: 1 the diversity of RAF in the natural forests was significantly lower than that in plantations (P<0.05), and the values were highest in the Mu Us Desert. There was a distinct geographical distribution in the RAF community, but the influence of stand age was not significant (P>0.05). 2 The relative abundance of ectomycorrhizal fungi (50.49%) in natural forests was higher than that in plantations, such as Acephala, Mycena, and Suillus. The indicator genera were diverse involving the natural forests (Acephala) and plantations in the Hulunbuir Desert (Sarcodon), Horqin Desert (Russula and Calostoma), and Mu Us Desert (Geopora, Mallocybe, and Tomentella). 3 The indicator genera were mainly affected by available nitrogen content, available phosphorus content, and stand age, and few indicator genera were related to soil water content, pH, and total nitrogen content. A total of 43.25% of the variation in the RAF community was accounted for by both geographic location and environmental factors. Overall, geographic location and environmental factors shaped the spatial variation in the RAF structure and function of P. sylvestris natural forests and plantations in the semi-arid and dry sub-humid desertified regions; there were no significant temporal variations in RAF across stand ages, but the nonuniformity in fungal distribution with stand ageing cannot be ignored. The large population of symbiotic fungi in natural forests was conducive to the healthy growth of hosts; the ratio of symbiotic, saprophytic, and pathotrophic fungi varied in different plantations, and the increase in the proportion of saprophytic and pathotrophic fungi may have negative effects on the growth and health of plantations. This improved information will provide a theoretical basis for the management of P. sylvestris plantations.

Effect of Climate and Competition on Radial Growth of Pinus sylvestris var. mongolica Forest in Hulunbuir Sandy Land of Inner Mongolia, China.

(1) Background: The forest of Pinus sylvestris var. mongolica is an important semi-arid ecosystem in Hulunbuir sandy land that plays a key role in the carbon cycle and wind erosion control. It is crucial to explore the main factors affecting the radial growth of trees of P. sylvestris var. mongolica. (2) Methods: The study established the tree-ring chronology of P. sylvestris var. mongolica and analyzed the relationships among the radial growth, competition index, and climate variables using correlation analysis and a linear mixed effect model to explore the influence of competition and climate on radial growth of P. sylvestris var. mongolica. (3) Results: The results indicated that tree growth is mainly affected by the maximum average temperature (Tmax) and precipitation in June and July of the current year and that tree growth significantly decreased with increasing competition pressure. Analysis of the linear mixed effect model showed that tree age, competition intensity, self-calibrating Palmer drought severity index (scPDSI) from May to July, and vapor pressure deficit (VPD) have a significant impact on radial growth. (4) Conclusions: The competition plays a dominant role in radial growth of P. sylvestris var. mongolica compared to climate factors. This study helps to understand the growth mechanism of P. sylvestris var. mongolica forests under climate change and provides a scientific basis for effective management of semi-arid forests.

Effects of Mixed Decomposition of Pinus sylvestris var. mongolica and Morus alba Litter on Microbial Diversity.

Pinus sylvestris var. mongolica is widely planted in China as a windbreak and sand fixation tree. To improve the current situation of large-scale declines of forested areas planted as P. sylvestris var. mongolica monocultures, we investigated the biological and microbial effects of stand establishment using mixed tree species. The interactions during the mixed decomposition of the litter and leaves of different tree species are an important indicator in determining the relationships among species. In this experiment, a method of simulating the mixed decomposition of P. sylvestris var. mongolica and Morus alba litter under P. sylvestris var. mongolica forest was used to determine the total C, total N, and total P contents in the leaf litter, and the microbial structures were determined by using Illumina MiSeq high-throughput sequencing. It was found that with samples with different proportions of P. sylvestris var. mongolica and M. alba litters, the decomposition rate of P. sylvestris var. mongolica × M. alba litter was significantly higher than that of the pure P. sylvestris var. mongolica forest, and the microbial community and composition diversity of litter in a pure P. sylvestris var. mongolica forest could be significantly improved. The possibility of using M. alba as a mixed tree species to address the declines of pure P. sylvestris var. mongolica forest was verified to provide guidance for pure P. sylvestris var. mongolica forests by introducing tree species with coordinated interspecific relationships and creating a mixed forest.

Full-Length Transcriptome Sequencing-Based Analysis of Pinus sylvestris var. mongolica in Response to Sirex noctilio Venom.

Sirex noctilio is a major international quarantine pest that recently emerged in northeast China to specifically invade conifers. During female oviposition, venom is injected into the host together with its symbiotic fungus to alter the normal Pinus physiology and weaken or even kill the tree. In China, the Mongolian pine (Pinus sylvestris var. mongolica), an important wind-proof and sand-fixing species, is the unique host of S. noctilio. To explore the interplay between S. noctilio venom and Mongolian pine, we performed a transcriptome comparative analysis of a 10-year-old Mongolian pine after wounding and inoculation with S. noctilio venom. The analysis was performed at 12 h, 24 h and 72 h. PacBio ISO-seq was used and integrated with RNA-seq to construct an accurate full-length transcriptomic database. We obtained 52,963 high-precision unigenes, consisting of 48,654 (91.86%) unigenes that were BLASTed to known sequences in the public database and 4309 unigenes without any annotation information, which were presumed to be new genes. The number of differentially expressed genes (DEGs) increased with the treatment time, and the DEGs were most abundant at 72 h. A total of 706 inoculation-specific DEGs (475 upregulated and 231 downregulated) and 387 wounding-specific DEGs (183 upregulated and 204 downregulated) were identified compared with the control. Under venom stress, we identified 6 DEGs associated with reactive oxygen species (ROS) and 20 resistance genes in Mongolian pine. Overall, 52 transcription factors (TFs) were found under venom stress, 45 of which belonged to the AP2/ERF TF family and were upregulated. A total of 13 genes related to the photosystem, 3 genes related photo-regulation, and 9 TFs were identified under wounding stress. In conclusion, several novel putative genes were found in Mongolian pine by PacBio ISO seq. Meanwhile, we also identified various genes that were resistant to S. noctilio venom, such as GAPDH, GPX, CAT, FL2, CERK1, and HSP83A, etc.

Diversity of Microbial Communities of Pinus sylvestris var. mongolica at Spatial Scale.

Soil microorganisms play an indispensable role in the forest ecosystem. It is necessary to study the soil microorganisms in Pinus sylvestris var. mongolica, which is one of the afforestation species widely planted in the northern sandy region of China. We collected soil samples of P. sylvestris at large spatial scales and analyzed bacterial and fungal community composition differences using high-throughput sequencing techniques. The results showed that: (1) the richness index of different sandy lands was significantly different. The α-diversity of bacteria was the highest in Mu Us Sandy Land, and the α-diversity of fungi was the highest in Horqin Sandy Land. (2) The dominant phyla of bacteria were Actinobacteria, Proteobacteria, Chloroflexi and Acidobacteria, while the dominant phyla of fungi were Ascomycota and Basidiomycota. The relative abundance of dominant phyla was different. (3) Temperature and precipitation were the main driving factors of bacterial and fungal community change at large spatial scale. In addition, bacteria were also affected by total nitrogen, soil organic carbon and pH content; fungal community was affected by pH. The microorganisms showed obvious differences in geographical distribution, which could provide ideas for promoting sustainable management of P. sylvestris stand.

Litter mixing promoted decomposition rate through increasing diversities of phyllosphere microbial communities.

Decomposition of forest litter is an essential process for returning nutrients to the soil, which is crucial for preserving soil fertility and fostering the regular biological cycle and nutrient balance of the forest ecosystem. About 70% of the land-based forest litter is made up primarily of leaf litter. However, research on the complex effects and key determinants of leaf litter decomposition is still lacking. In this study, we examined the characteristics of nutrient release and microbial diversity structure during the decomposition of three types of litter in arid and semi-arid regions using 16S rRNA and ITS sequencing technology as well as nutrient content determination. It was revealed that the nutrient content and rate of decomposition of mixed litters were significantly different from those of single species. Following litter mixing, the richness and diversity of the microbial community on leaves significantly increased. It was determined that there was a significant correlation between bacterial diversity and content (Total N, Total P, N/P, and C/P). This study provided a theoretical framework for investigating the decomposition mechanism of mixed litters by revealing the microbial mechanism of mixed decomposition of litters from the microbial community and nutrient levels.

Effects of thinning intensity on the understory water-holding capacity of Pinus sylvestris var. mongolica plantation in the Bashang area of north China.

The complex terrain and poor climatic conditions in Bashang area of Hebei Province result in water and soil loss and geological disasters, which pose a serious threat to ecological safety in North China. In order to improve local environmental quality, barren-resistant and fast-growing tree species such as Pinus sylvestris var. mongolica and Larix gmelinii are planted with large areas. However, unreasonable plantation density will lead to inefficient utilization of rainfall and intensify the conflict between forest and water. In this study, we analyzed the effects of five thinning intensities (0, 20%, 40%, 60%, 80%) of P. sylvestris var. mongolica plantation on herbs, litter, soil and overall water-holding capacity, with the aim to provide scientific basis for management of P. sylvestris var. mongolica. The results showed that water-holding rate of herb varied from 47.7% to 90.7%, and that the water-holding capacity of herb decreased with increasing thinning intensity. When the thinning intensity was less than 40%, water-holding capacity decreased slowly, and then decreased rapidly. With the increase of thinning intensity, natural water-holding rate and maximum water-holding rate of undecomposed layer and semi-decomposed layer decreased gradually, with the effective water-holding rate being 60%>40%>20%>80%>0, and the water-holding capacity of semi-decomposed layer being better than that of undecomposed layer. The water-holding capacity of soil decreased gradually with the increases of thinning intensity. Thinning intensity less than 40% promoted water holding capacity. Under different thinning intensities, the total water-holding rate of understory was 8.3%-14.3%, with an order of 20%>0>40%>60%>80%. In view of understory all layers and overall changes, the thinning intensity at 20% in the study area could effectively improve the understory water-holding capacity and achieve better ecological benefits.

Water Use by Chinese Pine Is Less Conservative but More Closely Regulated Than in Mongolian Scots Pine in a Plantation Forest, on Sandy Soil, in a Semi-Arid Climate.

The diversity of plant water use patterns among species and ecosystems is a matter of widespread debate. In this study, Chinese pine (Pinus tabuliformis, CP) and Mongolian Scots pine (Pinus sylvestris var. mongolica, MP), which is co-exist in the shelterbelt plantations in the Horqin Sandyland in northern China, were chosen for comparison of water use traits by monitoring xylem sap flow alongside recordings of the associated environmental factors over four growing seasons. Continuous sap flux density measurements were converted into crown projected area transpiration intensity (Tr) and canopy stomatal conductance (Gs). The results indicated that MP showed a higher canopy transpiration intensity than in CP, with Tr daily means (±standard deviation) of 0.84 ± 0.36 and 0.79 ± 0.43 mm⋅d-1, respectively (p = 0.07). However, the inter-annual variability of daily Tr in MP was not significant, varying only approximately a 1.1-fold (p = 0.29), while inter-annual variation was significant for CP, with 1.24-fold variation (p < 0.01). In particular, the daily mean Tr value for CP was approximately 1.7-times higher than that of MP under favorable soil moisture conditions, with values for relative extractable soil water within the 0-1.0 m soil layer (REW) being above 0.4. However, as the soil dried out, the value of Tr for CP decreased more sharply, falling to only approximately 0.5-times the value for MP when REW fell to < 0.2. The stronger sensitivity of Tr and/or Gs to REW, together with the more sensitive response of Gs to VPD in CP, confirms that CP exhibits less conservation of soil water utilization but features a stronger ability to regulate water use. Compared with MP, CP can better adapt to the dry conditions associated with climate change.

Linkages Between Nutrient Resorption and Ecological Stoichiometry and Homeostasis Along a Chronosequence of Mongolian Pine Plantations.

Nutrient resorption is an important strategy for nutrient conservation, particularly under conditions of nutrient limitation. However, changes in nutrient resorption efficiency with stand development and the associated correlations with ecological stoichiometry and homeostasis are poorly understood. In the study, the authors measured carbon (C), nitrogen (N), and phosphorus (P) concentrations in soil and in green and senesced needles along a chronosequence of Mongolian pine (Pinus sylvestris var. mongolica) plantations (12-, 22-, 31-, 42-, 52-, and 59-year-old) in Horqin Sandy Land of China, calculated N and P resorption efficiency (NRE and PRE, respectively), and homeostasis coefficient. The authors found that soil organic C and total N concentrations increased, but soil total P and available P concentrations decreased with stand age. Green needle N concentrations and N:P ratios as well as senesced needle C:N ratios, NRE, and PRE exhibited patterns of initial increase and subsequent decline with stand age, whereas green needle C:N ratios and senesced needle N concentrations, and N:P ratios exhibited the opposite pattern. NRE was positively correlated with N concentration and N:P ratio, but negatively correlated with C:N ratio in green needles, whereas the opposite pattern was observed in senesced needles. PRE was negatively correlated with senesced needle P concentration, soil-available N concentration, and available N:P ratio. The homeostatic coefficient of N:P was greater when including all stand ages than when including only those younger than 42 years. These findings indicate that tree growth may change from tending to be N limited to tending to be P limited along the Mongolian pine plantation chronosequence. Nutrient resorption was coupled strongly to tree growth and development, whereas it played a lesser role in maintaining stoichiometric homeostasis across the plantation chronosequence. Therefore, adaptive fertilization management strategies should be applied for the sustainable development of Mongolian pine plantations.

Effects of afforestation with Pinus sylvestris var. mongolica plantations combined with enclosure management on soil microbial community.

Grazing and litter removal can alter understory structure and composition after afforestation, posing a serious threat to sustainable forest development. Enclosure is considered to be an effective measure to restore degraded forest restoration. However, little is known about the dynamics of soil nutrients and microbial communities during the forest restoration process. In the present study, the effects of Arachis hypogaea (AH), Pinus sylvestris var. mongolica (PSM) and Pinus sylvestris var. mongolica with enclosure (PSME) on soil chemical properties and soil microbial communities were studied in Zhanggutai, Liaoning Province, China. The results showed that PSME could remarkably contribute to improve soil total C, total N and total P compared to PSM and AH. Additionally, PSM could clearly increase the soil bacterial community diversity and fungal Chao1 index and ACE index. Additionally, PSME could further increase soil Chao1 index and ACE index of soil bacteria. Soil total C, total N and available N were the main factors related to soil microbial diversity. Actinobacteria and Ascomycota were the predominant bacterial and fungal phyla, respectively. Specifically, PSME could increase the relative abundances of Actinobacteria, Gemmatimonadetes, Ascomycota and Mortierellomycota and decreased the relative abundances of Acidobacteria, Chloroflexi and Basidiomycota than PSM. PSM and PSME could clearly change soil microbial communities compared with AH and PSME could remarkably shift soil fungal communities than PSM. What's more, the soil microbial community structure were affected by multiple edaphic chemical parameters. It can be seen that afforestation combined with enclosed management potentially regulate microbial properties through shifting the soil properties. This study can provide new ideas for further understanding the impact of enclosure on PSM and provide theoretical support for the management of PSM.

Effects of Dark Septate Endophytes Strain A024 on Damping-off Biocontrol, Plant Growth and the Rhizosphere Soil Enviroment of Pinus sylvestris var. mongolica Annual Seedlings.

Dark septate endophytes (DSEs) exert a vital role in promoting plant growth, improving mineral absorption, biological disease control, and enhancing plant stress resistance. The effects of dark septate endophyte strain, Phialocephala bamuru A024 on damping-off biocontrol, plant development, nutrients within the rhizosphere soil, as well as bacterial communities in the annual seedlings of P. sylvestris var. Mongolica were studied. According to our findings, following P. bamuru A024 inoculation, the damping-off disease morbidity decreased significantly compared with control, some physiological indices such as ß-1,3-glucanase, chitinase enzyme activity as well as a soluble protein and proline content in P. sylvestris var. mongolica were elevated under R. solani stress. After inoculation with P. bamuru A024, the biomass in seedlings, nutrients in soil, root structure index, together with activities of soil enzymes were remarkably up-regulated relative to control (p < 0.05). As suggested by the results of high-throughput sequencing, the microbial structure in the rhizosphere soil of the P. sylvestris var. mongolica showed significant differences (p < 0.05) after P. bamuru A024 inoculation compared to control treatment and the rhizosphere soil bacterial community structure after DSE A024 inoculation was positively correlated to the main soil nutrition indices.

Community structure and functional group of root-associated Fungi of Pinus sylvestris var. mongolica across stand ages in the Mu Us Desert.

Root-associated fungi (RAF) are an important factor affecting the host's growth, and their contribution to Pinus sylvestris var. mongolica plantation decline is substantial. Therefore, we selected three age groups of P. sylvestris plantations (26, 33, and 43 years), in the Mu Us Desert, to characterize the community structure and functional groups of RAF, identified by Illumina high-throughput sequencing and FUNGuild platform, respectively. The effects of soil properties and enzyme activities on fungal diversity and functional groups were also examined. The results indicated that (a) 805 operational taxonomic units of RAF associated with P. sylvestris belonged to six phyla and 163 genera. Diversity and richness were not significantly different in the three age groups, but community composition showed significant differences. Ascomycota and Basidiomycota dominated the fungal community, while Rhizopogon dominated in each plot. (b) The proportion of pathotrophs decreased with increasing age, while that of symbiotrophs increased sharply, which were mainly represented by ectomycorrhizal fungi. (c) Stand age and soil enzyme activity had a greater influence on fungal community composition than did soil properties, whereas environmental variables were not significantly correlated with fungal diversity and richness. Dynamics of fungal community composition and functional groups with the aging plantations reflected the growth state of P. sylvestris and were related to plantation degradation.