Objective: Analyze the seasonal differences of net ecosystem exchange (NEE) of carbon in the study area(the forest of Korean pine and broad leaved trees in Changbai Mountain) and the response of the NEE to meteorological factors, reveal the dynamic impacts of meteorological factors on the NEE at a monthly basis, and provide a theoretical guidance for regulating the carbon budget in the study area. Meanwhile, the application of temporal convolution network (TCN) in the simulation of the NEE was studied to explore a new method of carbon flux simulation. Method: Based on 3 min data obtained at the flux observation station in temperate forest of Korean pine and broad-leaved trees in Changbai Mountain from 2007 to 2010, the seasonal differences of the NEE and five meteorological factors were analyzed, and correlation between the meteorological factors and the NEE was analyzed. The random forest model was used to calculate the importance scores of factors affecting NEE, five meteorological factors with higher scores were selected as inputs to the NEE simulation: latent heat flux, sensible heat flux, air humidity above the canopy, water vapor pressure above the canopy and net radiation; five NEE models were developed respectively based on TCN, long short term memory (LSTM), artificial neural network (ANN), support vector regression (SVR) and extreme learning machine (ELM). The prediction precision and stability of different models were examined by calculating coefficient of determination (R²), mean absolute error (MAE) and root mean square error (RMSE). Result: The annual total NEE was -74.777 3 gCO₂·m^-2a^-1 revealing an overall carbon sink for the whole year, but seasonally with a carbon sink in summer and a carbon source in winter. The NEE was extremely significantly (P < 0.000 1) negatively correlated with latent heat flux, vapor pressure over canopy, net radiation and air humidity over canopy, but not significantly correlated with sensible heat flux (0.077 0). The RMSE and the R² of TCN were respectively 0.110 5 mgCO₂·m^-2s^-1 and 0.821 4, the RMSE was decreased by 0.024 8, 0.022 4, 0.022 2, 0.006 8 mgCO₂·m^-2s^-1 and the R² was increased by 0.080 6, 0.077 7, 0.068 6, 0.022 3 compared with ELM, SVR, ANN and LSTM, respectively. According to 10 tests of the above 5 models, the standard deviation of RMSE of TCN obtained by calculation was 0.000 4 mgCO₂·m^-2s^-1, which was 0.001 4, 0.001 3, 0.000 2 mgCO₂·m^-2s^-1 lower than that of ELM, ANN and LSTM, respectively. Conclusion: The NEE of the flux observation station was a carbon sink overall, with obvious seasonal differences. The NEE was extremely significantly (P < 0.000 1) negatively correlated with latent heat flux, vapor pressure above the canopy, air humidity above the canopy and net radiation, but not significant (P=0.141 8) with the sensible heat flux. The long-term NEE prediction showed good precision and strong stability by the simulation model based on the TCN, providing a feasible basis for the application of the TCN in the field of ecological simulation. The study provides a theoretical guidance for regulating the carbon budget of the forest of Korean pine and broad-leaved trees in Changbai Mountain.

Objective: Three stratification schemes were used to construct canopy coverage-aboveground biomass model of tree-shrub based on UAV data and aboveground biomass models of tree-shrub based on spectral indices of Lansat8 OLI data. Differences among stratification schemes in the precision of the aboveground tree-shrub biomass model were investigated to provide a scientific and theoretical basis for high-precision estimation of aboveground tree-shrub biomass of plantations in dryland based on remote sensing data. Method: A total of 102 plots were surveyed in Mu Us Sandy Land, information on vegetation coverage of tree, shrub and herbaceous were obtained from the high-resolution UAV images using object-oriented machine learning algorithm, and then the canopy coverage-aboveground biomass models of tree-shrub were developed with three stratification schemes (non- stratification, stratification-based two vegetation types of tree and shrub, stratification-based five tree species). Based on landsat-8 OLI images, 6 spectral indices (NDVI, RVI, MSAVI, TCG, NDMI, NIRv) were used to interpret the herbaceous coverage in combination with UAV images. Different spectral index-aboveground tree-shrub biomass models were developed using the 3 stratification schemes (non-stratification, stratification using plots with and without herbaceous vegetation, stratification using plots with three herbaceous coverage level). Finally, the precision of estimation was compared among the models with different stratification schemes. Results: The canopy coverage-aboveground tree-shrub biomass model without stratification has the worst robustness (R²=0.22, n=102) and the lowest estimation precision (RMSE=14.98 t·hm^-2). The estimation errors were reduced by 50.32% and 61.1% for the models with stratification by 2 vegetation types of tree and shrub (RMSE=7.44 t·hm^-2) and by 5 tree species (RMSE=5.82 t·hm^-2), respectively. In the spectral index-aboveground tree-shrub biomass models, NIRv had the highest precision in estimation of aboveground tree-shrub biomass (the average RMSE of the 3 stratification schemes was 7.25 t·hm^-2), and NDVI had the lowest precision (the average RMSE of the 3 stratification schemes was 9.43 t·hm^-2). The explanatory ability of different spectral indices to variation of aboveground biomass of sparse tree-shrub was ranked as follows: NIRv > NDMI > TCG > MSAVI > RVI > NDVI. The effect of woody vegetation types on precision of spectral index-aboveground tree-shrub biomass model was less than that of the canopy coverage-aboveground tree-shrub biomass model. The RMSE of the spectral index-aboveground tree-shrub biomass models was reduced by 8.13%~16.62% considering the background of herbaceous coverage, and the sensitivity of precision to such background was ranked as follow: NIRv > TCG > NDVI > MSAVI > RVI > NDMI. Conclusion: The high spatial resolution remote sensing of UAV can be used to obtain prior knowledge of tree species types and herbaceous vegetation information for sparse tree-shrub mixed forest. The woody vegetation types have a great influence on the precision of canopy coverage-aboveground biomass model in sparse tree-shrub mixed forest, and at least the two vegetation types of tree and shrub should be distinguished to ensure the estimation precision of the method to meet the practical application requirements. The stratification model schemes of NIRv-aboveground biomass model derived from Landsat-8 OLI satellite data considering herbaceous vegetation coverage is suitable for remote sensing estimation of aboveground tree-shrub biomass in a large area.

Objective: In order to improve the accuracy of biomass estimation of southern subtropical evergreen broad-leaved forest, the biomass distribution among different organs of 4 common tree species were investigated, and the models of single plant and different biomass components(aboveground and underground) were constructed for each of the 4 species. Method: Based on the historical data of forest survey of the southern subtropical evergreen broad-leaved forest in Guangzhou and its actual species composition, 4 common tree species, Castanopsis fissa, C. chinensis, Aleurites montana and Machilus chinensis were selected to measure the biomass of single plant and different components of each tree species using the harvest method, and further to construct the biomass models of each tree species. The influences of adding tree height (H) and wood density (ρ) as second independent variables in the models on the accuracy of the models were discussed. Result: The proportion of leaf biomass of the four 4 tree species accounted for 5.34%-7.28%, and the proportion was significantly lower of M. chinensis than of C. fissa (P < 0.05). The proportion of branch biomass accounted for 16.82%-24.20%, and significantly lower of M. chinensis than of C. chinensis and C. fissa (P < 0.05). The proportion of trunk biomass accounted for 47.22%-58.05%, and significantly lower of C. chinensis than of the other three species (P < 0.05). The proportion of root biomass accounted for 14.25%-22.25%, and significantly lower of C. fissa and A. montana than of C. chinensis and M. chinensis (P < 0.05). The proportion of leaf biomass of A. montana and M. chinensis decreased significantly (P < 0.01) with the increase of DBH. With the increase of DBH, the proportion of branch biomass increased, while the proportion of trunk biomass decreased of the four species. There was no significant correlation between the proportion of root biomass and DBH. The biomass model with DBH as a single independent variable had good fitting accuracy (average R²=0.947). Adding H compounded with D as one variable (D²H) in the models decreases the model fitness of aboveground and total biomass, and slightly improves the fitness of underground biomass. Including H as an independent second variable in the models increases the multiple col-linearity of the model. Adding ρ compounded with D as one variable (D²ρ) in the models decreases the model fitness of single plant and all components, but including ρ as an independent second variable in the models slightly improves the model fitness. Conclusion: The sunlight-loving tree species, A. montana, allocated more biomass to the trunk, while the shade tolerant tree species, C. chinensis, allocated more biomass to the crowns. The shade tolerant tree species have more developed roots. We suggest that the model of D as a single independent variable be used to estimate the biomass of the four species, and H is not recommended to be included in the model as the second variable. Adding ρ as the second independent variable slightly improves the fitting ability of the model. It is suggested to make a trade-off between accuracy and difficulty according to the investigation purpose in practical applications.

Objective: Prediction of the potential of carbon pool and carbon sink by China's forest biomass before 2060 is carried out in order to provide an important basis for the study and development policies on emission reduction and sink enhancement, it is of significant implications for China to allow necessary CO₂ emissions and negotiations on global climate change. Method: Based on the data from the National Forest Inventory (NFI), eight to nine major dominant tree species (groups) were selected by region to fit the Richards growth equation, the relationship between volume per hectare and age of each dominant species (group) was established by dividing China into six regions. Using China's forest management plans to project forest area by period, the forest timber volume and biomass carbon pool and carbon sink potential (excluding economic forests and bamboo forests) are estimated in China by 2060. Results: By 2030, forest timber volume will reach 20 473 million m³, an increase of 7 473 million m³ compared with 2005, and by 2060, it will reach 28 645 million m³. In view of the dynamic changes in each region, the southwest and southeast regions are the places with the fastest growth of forest timber volume in China in the future, and the areas with the greatest potential for precise improvement of forest quality, accounting for 37.68% and 21.37% of the national forest timber volume in 2060 respectively. By 2060, the existing forest carbon stocks will reach 12.12 Pg C and new plantations will add another 0.92 Pg C; the total forest biomass carbon pool will reach 13.04 Pg C, an increase of 5.47 Pg C compared to 7.57 Pg C in 2018. The forest carbon density will reach 63.96 Mg C·hm^-2 in the same period. Conclusion: Given that China's forests are currently dominated by middle- and young-aged forests and the forest area is still increasing, the carbon pool and carbon sink capacity of China's forest biomass will continue to grow in the next 40 years, with average annual increase in forest carbon sink up to 0.13 Pg C·a^-1. This indicates that China's forests have a large potential for carbon sinks. For this reason, it is necessary to further strengthen the protection and management of forest resources, reduce forest carbon losses and continue to promote large-scale greening of the national territory, in order to maintain and enhance the carbon sink capacity of China's forests and help achieve the goal of "carbon neutrality".

Objective: The purpose of this work is to develop meteorological factor acquisition system of Phyllostachys praecox stand, to obtain the relationship between CO₂ concentration and meteorological factors (temperature and humidity, etc.), to discusses the CO₂ concentration inversion model based on GA-BP neural network (abbreviated as GA-BP model), and to provide fundamental data for carbon storage, carbon sinks and carbon sequestration capacity of P. praecox stand. Method: According to the relevant principles and methods of micrometeorology and the requirements of dynamic sensing of forest carbon flux, a remote and real-time monitoring system of forest carbon flux data based on embedded system is designed. Taking the mature P. praecox stand, stand as monitored object, this system monitored the environmental data for two months (October ~ November 2019). After analyzing these data, a CO₂ concentration inversion model based on genetic classification optimization neural network is proposed. Results: According to the inversion results of GA-BP and BP inversion model, the determinative coefficient R² of the inversion results of GA-BP inversion model is 0.86, which is 7 percentage points higher than that of BP inversion model. The mean absolute error is 8.12 mg·m^-3, which is 2.79 mg·m^-3 lower than that of BP inversion model. Compared with the BP inversion model, the GA-BP inversion model has more stable inversion performance and higher inversion accuracy. Conclusion: The P. praecox stand meteorological factor acquisition system can be used to obtain relevant meteorological data. Based on the correlation between CO₂ concentration and meteorological factors (temperature and humidity, etc.), the CO₂ concentration inversion model based on GA-BP neural network can effectively invert the CO₂ concentration data in the survey region.

Objective: The study aims to investigate the patterns of latitudinal variation of soil microbial biomass carbon (MBC) and its driving forces, further to reveal the effect and mechanism of soil MBC content across forest ecosystems along the latitudinal gradients in China, providing a theoretical basis for understanding forest soil carbon turnover and sequestration in the context of global warming. Method: Ten typical forest ecosystems along the latitude gradients in China were selected to measure soil microbial biomass carbon (MBC) contents at 0-10 cm soil layer, and the correlation between MBC and abiotic factors (climate factors, soil physical and chemical properties) were conducted. Result: The MBC content of the forest soil decreased with decreasing latitude, ranging from (200.57 ± 13.99) mg·kg^-1 to (913.32 ± 39.62) mg·kg^-1. The soil MBC content and soil physical and chemical properties were positively correlated (r=0.51, P < 0.01). Highly significant positive correlation with the soil MBC content were identified in soil organic carbon content (r=0.64, P < 0.01), soil sand content (r=0.48, P < 0.01), soil hydrolase activity (r=0.48, P < 0.01), soil total phosphorus content (r=0.47, P < 0.01), pH value (r=0.43, P < 0.01) and soil total nitrogen content (r=0.31, P < 0.01). While silt content (r=0.15, P < 0.05) and soil oxidase activity (r=0.15, P < 0.05) were significantly positively correlated with MBC content. Both the mean annual temperature (r=0.31, P < 0.01), and the alkyl carbon content (r=0.21, P < 0.01) in the chemical structure of soil organic carbon were highly positively correlated with MBC content. Conclusion: Our results demonstrated a prominent decrease of MBC content along the latitude, with the main affecting factors being soil texture, soil nutrient, soil enzyme activities, and soil alkyl carbon content, followed by mean annual temperature.

Objective: This article aims to analyze the variation of phosphorus fractions in the soil of Chinese fir plantations with different stand ages, and to explore the internal relationship between the stand age and the species and relative contents of phosphorus solubilizing microbes at different classification levels, so as to provide reference for the effective use of soil phosphorus and sustainable management of plantations. Method: Five Chinese fir plantations of different ages (4-, 15-, 24-, 43- and 100-year-old) were selected in Nanping, Fujian. The modified Hedley phosphorus grading method was used to determine the phosphorus content of different grades in 0-20 cm soil layer. High-throughput sequencing technology was used to determine the phoD gene amplified by PCR to obtain the relative abundance of phosphorus solubilizing microbes at different classification levels. Result: Overall, at the same age of Chinese fir plantations, the phosphorus content of different grades was Residual-P > NaOH-P > NaHCO₃-P > H₂O-P > HCl-P. The content of H₂O-IP increased significantly with the increase of stand age (P < 0.05), and the content of NaHCO₃-IP increased and then decreased with the increase of the stand age, reaching the maximum in 43-year-old stands. As the stand age increased, the soil NaOH-P content fluctuated significantly, showing 24 year-old stands > 100-year-old stands > 43-year-old stands > 4-year-old stands > 15-year-old stands. The total amount of organic phosphorus showed an upward trend as a whole, in the order of 100-year-old stands > 24-year-old stands > 43-year-old stands > 15-year-old stands >4-year-old stands. With the increase of stand age, the content of labile phosphorus in the soil of Chinese fir plantation gradually increased. The soil phosphorus activity in young forest, middle-aged forest, and near-mature forest of Chinese fir showed that stable phosphorus > moderately labile phosphorus > labile phosphorus. The phosphorus activity of over-mature forest showed the order of stable phosphorus > labile phosphorus > moderately labile phosphorus. Overall, the abundance of phosphorus solubilizing microbes in the soil of Chinese fir forest showed 43-year-old stands > 100-year-old stands > 24-year-old stands > 15-year-old stands > 4-year-old stands, and the diversity showed 43-year-old stands > 100-year-old stands > 15-year-old stands > 24-year-old stands > 4-year-old stands. OTU Venn diagram and PCoA analysis showed that the structure and diversity of phosphorus solubilizing microbes in 15-year-old stands and 24-year-old stands were similar. The soil of 43-year-old Chinese fir plantation contained the most abundant and unique groups of phosphorus solubilizing microbes. The number of phosphorus solubilizing microbes in 4-year-old stands Chinese fir forest was the least. At the phylum classification level, the content of Proteobacteria in the soil was the highest, followed by Deinococcus-Thermus and Actinobacteria. At the genus classification level, the dominant groups were mainly Bradyrhizobium, Pseudolabrys, Pseudomonas, Deinococcus, etc. Redundancy analysis showed that there was a significantly positive correlation between relative contents of soil NaHCO₃-IP and Firmicutes, and a significant negative correlation between NaOH-IP and Actinobacteria, Gemmatimonadetes, etc. HCl-OP was significantly negatively correlated with Acidobacteria, while H₂O-OP, and NaHCO₃-IP were significantly negatively correlated with Bradyrhizobium. Conclusion: With the increase of stand age, the content of different forms of phosphorus in the soil of Chinese fir plantation varies significantly, and the level of soil labile phosphorus continuously increases. The potential supply capacity of the soil in near-mature and over-mature forests is higher than that of young and middle-aged forests. The changes in soil chemical properties affect soil microbial activity, and the increase in the abundance and diversity of phosphorus solubilizing microbes may increase the availability of soil phosphorus in Chinese fir plantations to a certain extent.

Objective: This study aimed to investigate the diversity characteristics and distribution patterns of forest soil microorganisms to provide a basis for the understanding soil microbial diversity and its response to environmental changes, as well as forest ecosystem management. Method: This study was conducted in different climate zones, including temperate coniferous forest (Kanas, Xinjiang, KNS; Ku'erdening, Xinjiang, KEDN), warm temperate coniferous and broad-leaved mixed forest (Xiaolong Mountain, Gansu), subtropical evergreen deciduous broad-leaved mixed forest (Mulinzi, Hubei), and tropical rain forest (Bawangling, Hainan). Soil samples were collected from the established six hm² permanent plots, and the distribution patterns of bacterial and fungal diversity were analyzed by using Illumina sequencing technology. Result: The results showed that the diversity of soil bacteria was the highest in the warm temperate coniferous and broad-leaved mixed forest. It exhibited a single peak distribution in the five sample plots along the climate gradient. The Shannon index of soil fungi was the highest in temperate coniferous forest, and the richness of soil fungi was the highest in tropical rain forest. The null model revealed that the soil bacterial community was mainly dominated by deterministic processes, while the fungal community was mainly dominated by stochastic processes. Canonical correlation analysis (CCA) and Mantel test indicated that soil pH (r=0.826, P < 0.001) and plant diversity (r=0.474, P < 0.001) were significantly correlated with soil bacterial and soil fungal community structure. Conclusion: There are significant differences in the diversity and community structure of forest soil bacteria and fungi in different climatic zones. Soil bacteria and fungi have different community assembly characteristics. Soil pH and plant diversity are important factors affecting forest soil microbial diversity in different climate zones.

Objective: This study aimed to investigate the changes of soil nematode community structure in Cunninghamia lanceolata plantations with different forest ages in subtropical region, so as to provide a theoretical basis for the sustainable management of C. lanceolata plantation. Method: C. lanceolata plantations at five different forest ages (5, 8, 21, 27, 40 a) were selected as research object using a space-for-time substitution approach. Soil nematodes were isolated and extracted using the platter method. The species and genera of soil nematodes were identified, and the density of total nematodes, the relative abundances of various nutritional nematode groups were determined simultaneously. The Shannon diversity (H′), the maturity indexes of plant parasities (PPI) and the Wasilewska index (WI) were calculated as well. The changes of soil nematode community structure in C. lanceolata plantations with different ages were analyzed by non-metric multidimensional scale (NMDS). Result: 1) A total of 9 872 nematodes, were isolated, belonging to 68 genera, and the dominant genus was different at the different stand ages of C. lanceolata. The genus, Meloidogyne, was predominant in all forest stands except for the young stage of C. lanceolate. 2) The total density and diversity of soil nematodes did not change significantly in the all stand developmental stages. There were differences in community structure of soil nematodes between 40-year-old stand and 5-year-old and 8-year-old stands, indicating that the development of C. lanceolata plantation had changed the structure of soil nematode community. The relative abundance of plant parasitic nematodes was the highest, followed by omnivores-predators and bacterivores, and the relative abundance of fungivores was the lowest. The relative abundance of plant parasites decreased first and then increased, reaching the lowest in 8 years. The relative abundance of fungivores was the highest in 5 years, and decreased with the increment of the stand age of C. lanceolata. The development of C. lanceolata plantation had changed the relative abundance of plant parasities as well as the density of fungivores. However, there was no significant difference in the relative abundance and density of bacterivores or omnivores-predators with the development of C. lanceolate. 3) The maturity indexes of plant parasites decreased first, reaching the lowest in 21 years, and then increased with the development of C. lanceolata. The Wasilewska indexes in 5-year-old stand were significantly higher than those in 40-year-old stand. The WI value showed a decreasing trend with the development of stand ages. The correlation analysis showed that the Wasilewska index was positively correlated with soil pH, and the density and relative abundance of parasitic nematodes were positively correlated with the soil total carbon and carbon phosphorus ratio, respectively. Conclusion: In subtropical Chinese fir plantation, plant parasitic nematodes occupy a dominant position in quantity. The long-term plantation of C. lanceolata has changed the soil nematode community structure, especially triggering a dominant role of the plant parasites, which could probably result in adverse effects on soil health and plant growth. More attention should be paid to the study of soil nematode community structure and underlying mechanisms for the sustainable management of C. lanceolata plantations in subtropical China.

Objective: This study aims to study the spatial structure of soil microbial communities and the physicochemical properties of soil in rhizosphere and non rhizosphere of Rhododendron moulmainense in Shenzhen Wutong Mountain, so as to provide theoretical support for the further study of the rhizosphere microecology of R. moulmainense. Method: The rhizosphere (R) and non rhizosphere (NR) soil of R. moulmainense were used as the research object, the soil physical and chemical indexes were measured, and the community characteristics of soil bacteria and fungi were analyzed by high-throughput sequencing technology. Results: The pH of rhizosphere and non-rhizosphere soil was slightly acidic, and the rhizosphere had significantly lower pH than non-rhizosphere (P < 0.05). The available phosphorus in rhizosphere soil was significantly higher than that in non-rhizosphere soil (P < 0.05).The organic matter, total nitrogen and alkali-hydrolyzed nitrogen in rhizosphere soil were higher than those in non-rhizosphere soil, but there was no significant difference. A total of 23 phyla and 291 species of rhizosphere bacteria and 25 phyla and 305 species of non-rhizosphere bacteria were annotated respectively. The rhizosphere fungi were annotated to 8 phyla and 520 species, and the non rhizosphere fungi were annotated to 8 phyla and 518 species. The dominant bacteria in rhizosphere and non rhizosphere were Acidobacteria, Protecbacteria, Actinobacteria, Chloroflexi, etc. The dominant fungi were Ascomycota, Basidiomycota and Mortierellomycota. Rhizosphere and non-rhizosphere dominant bacteria of R. moulmainense were mainly subgroup_2, Acidobacteriales, solidobacterales, rhizobiales, etc. The dominant fungi in rhizosphere and non rhizosphere were mainly Eurotiales, Hypocreales, Sebacinales, etc.There was no difference in the diversity index of bacteria and fungi between rhizosphere and non-rhizosphere, while Beta diversity analysis showed that there were differences in the diversity of soil bacterial and fungal community structure between rhizosphere and non-rhizosphere. The results of functional prediction analysis based on FUNGuild showed that the functions of rhizosphere and non-rhizosphere fungi were mainly Symbiotroph trophic(23.57%~33.44%), Saprotroph-Symbiotroph trophic(6.48%~7.63%), Saprotroph(19.84%~22.77%), Pathotroph-Symbiotroph trophic(0.76%~0.79%), Pathotroph-Saprotroph-Symbiotroph trophic(0.25%~0.34%), Pathotroph-Saprotroph(33.83%~38.02%)and Pathotroph trophic(5.39%~6.86%). Both rhizosphere and non-rhizosphere soils were dominated by pathological saprophytic and symbiotic vegetative fungi. Conclusion: The rhizosphere and non-rhizosphere soils are slightly acidic. There is no significant difference in the species of dominant microorganisms between rhizosphere and non-rhizosphere.The dominant bacteria are Acidobacteria and Proteobacteria, and the dominant fungi are Ascomycota and Basidiomycota. There are differences in fungal functional structure between rhizosphere and non-rhizosphere, mainly because rhizosphere symbiotic fungi are much higher than non-rhizosphere fungi.

Objective: This paper aims to study the germination regularity of R. pseudoacacia seeds with different sizesunder salt and drought stresses, and propose suggestions on the selection of suitable seed sizes and degree of the stress, in order to provide a theoretical basis for the cultivation of excellent seedlings of R. pseudoacacia. Method: The seeds of R. pseudoacacia were divided into three categories of sizes (large, medium and small) with the soil sieve, and the range of seed size, thousand grain weight and the imbibition rate of each category were determined. NaCl andpolyethylene glycol 6000 (PEG6000) (iso-osmotic conditions of 0, -0.05, -0.15, -0.30and -0.49 MPa)were prepared to simulate salt and drought stress. Seeds of different sizes were placed in the light incubator for germination test. In this way, the germination characteristics, seedling biomass and root growth of seeds of different sizes were analyzed, and the plasticity indexes of those seeds were compared. Result: 1) The imbibition rate of large seeds and medium seeds was significantly higher than that of small seeds, and the thousand grain weight of medium and small seeds were reduced by 22.01% and 61.72% respectively compared with large seeds.2)With the increase of stress degree, the germination rate, germination index and vigor index decreased significantly, and the mean germination time increased significantly. The water content, biomass, root length, root surface area, specific root length and specific root surface area of seedlings decreased significantly with the osmotic potential decreasing. Under salt stress, the germination rate, germination index, vigor index and water content of seedlings were higher than those under drought stress. The root dry weight, root shoot ratio, root length, root surface area, specific root length, and specific root surface area under salt stress were lower than those under drought stress.3)In the whole germination process, the large and medium seeds had higher germination rate, germination index, vigor index and seedling biomass compared with the small seeds, but they werenot as good as the small seeds in seedling water content and root development. In addition, seed size had significant interaction with salt and drought stress on seed germination. Compared with small seeds, large and medium seeds had higher germination ability and seedling biomass under stress conditions, though such advantage gradually decreased with the decline of osmotic potential.4)In terms of plasticity index, the total plasticity indexes of large and medium seeds were greater than that of small seeds under both salt and drought stress. The total plasticity index under salt stress was lower than that under drought stress. Conclusion: Salt and drought stresses significantly inhibit the seed germination, seedling growth and root development of R. pseudoacacia, those impacts under drought stress are more obvious than those under salt stress. Large seeds and medium seeds have obvious advantages in germination and seedling growth, but such advantages are affected by the type and degree of the stress. Therefore, both the seed size and stress conditions affect the seed germination and seedling growth of R. pseudoacacia. It is suggested to choose large and medium seeds in sowing and raising seedlings of R. pseudoacacia in arid and saline areas.

Objective: To study the characteristics of rooting anatomical structure, and to investigate and analyze the dynamic changes and effects of endogenous hormones, oxidases and nutrients in the process of adventitious root development during softwood cuttings of Eucommia ulmoides, so as to provide reference for further research on the development mechanism and regulation techniques of E. ulmoides. Method: The E. ulmoides 'Huazhong No.6' semi-lignified twigs were used as the experimental material, the development process of adventitious roots was investigated by observing the anatomical structure of its paraffin sections that were made of the basis of cuttings collected regularly; the content of IAA, GA₃, ABA and ZR of E. ulmoides was determined by using HPLC-MS/MS; the activity of IAAO, POD, PPO was determined by colorimetric method; soluble sugar, soluble protein and total nitrogen were determined by the anthrone-sulfate method, Coomassie brilliant blue method and Kjeldahl method. Result: 1) The adventitious roots primordium of E. ulmoides was differentiated from the vascular cambium parenchyma cell of bark, which belongs to the induce-to-root type of the bark. The ontogeny process of adventitious roots can be divided into four stages: induction period (0-12 d after cutting), initiation period (12-22 d after cutting), expression period (22-32 d after cutting) and elongation period (32-53 d after cutting). It can be observed that there are young white adventitious roots breaking through the cortex at the base of the cuttings during the expression period. 2) The average number of adventitious roots in the treatment group with special rooting agent of E. ulmoides can reach 25.6, which was 20 more than of the control group treated with water. The average root length was 8.41 cm, which was about 2 times of the control group; The rooting rate was 85.3%, an increase of 60%. 3)IAA had significant function in the rooting process of E. ulmoides. The activity of POD and PPO increased first, then decreased, the activity of IAAO decreased first and increased later. Nutrients accumulated before rooting, and their content decreased after rooting. Conclusion: The adventitious root formation of E. ulmoides belongs to the type induce-to-root in the bark. The dynamic changes of various physiological indicators are consistent with the development of adventitious roots. IAA is a key hormone for inducing adventitious roots. During the critical period of adventitious root formation, the increase in IAA content is the key to inducing root primordium. Meanwhile, IAAO activity decreases, POD and PPO activity increases, and the nutrients rapidly accumulate, which are closely related to rooting.

Objective: Polyamines exist widely in plants, and are involved in the regulation of plant development and stress response. S-adenosylmethionine decarboxylase (SAMDC) is a key rate-limiting enzyme in the polyamine biosynthesis pathway. In this study, the role of SAMDC in poplar 84K (Populus alba × P. glandulosa '84K') under drought stress was analyzed using SAMDC overexpressing plants. This work will lay on important foundation for the elucidation of the role of polyamines in stress tolerance. Method: The nontransgenic(control) and transgenic PagSAMDC4a overexpressing poplar 84K plants growing in soil for 3 months were used and subjected to drought treatment to analyze phenotypic and physiological changes, including polyamines content, H₂O₂ content, relative water content of leaves, water loss rate of leaves, electrolytic leakage and so on. Result: The contents of endogenous spermidine and spermine in PagSAMDC4a-OE#3, PagSAMDC4a-OE#15 and PagSAMDC4a-OE#17 transgenic poplars were significantly higher than those in nontransgenic control, and the contents of endogenous putrescine, spermidine and spermine in PagSAMDC4a-OE#17 transgenic plant were 1.95, 3.43 and 1.32 times higher than those in control, respectively. Under normal conditions, compared with nontransgenic control, leaf phenotype, leaf relative water content and electrolytic leakage of overexpressing plants had no significant differences, while the water loss rate of PagSAMDC4a-OE#15 and PagSAMDC4a-OE#17 plants were significantly lower than that of control. Under drought stress, the leaves of nontransgenic plants were wilted, the relative water content decreased by 26.44% and the electrolytic leakage increased by 27.68%. However, PagSAMDC4a-OE plants grew normally, and the relative water content of overexpressing PagSAMDC4a-OE#3, PagSAMDC4a-OE#15 and PagSAMDC4a-OE#17 plants decreased by 10.9%, 3.66% and 1.33%, respectively. The overexpressing plants had low electrolytic leakage and a small variation. Under normal conditions, H₂O₂ contents of PagSAMDC4a transgenic plants were significantly reduced. Under the drought stress, the increase of H₂O₂ content of PagSAMDC4a overexpressing plants was significantly lower than that of nontransgenic plants, and the content variation from high to low was nontransgenic plant > PagSAMDC4a-OE#3 > PagSAMDC4a-OE#15 > PagSAMDC4a-OE#17. Conclusion: Overexpression of PagSAMDC4a gene can increase endogenous polyamines and decrease H₂O₂ content in poplar 84K, which can reduce the oxidative damage caused by drought stress and effectively alleviate the water stress damage suffered by the plant leaves, contributing to relieving the sensitivity of poplar to drought stress. Therefore, SAMDC4a could affect the changes of endogenous polyamines in response to drought stress.

Objective: The WRKY transcription factor gene family members were identified in the whole genome of Phoebe bournei. Gene structure and protein sequences of them were characterized and their expression was analyzed under phosphorus deficiency. Then, PbWRKY members that were involved in phosphorus starvation response were screened. The result will give information to further study their function in the molecular mechanism of phosphorus deficiency stress and provide foundation to molecular assisted breeding for tolerance to low phosphorus in Phoebe. Method: The hidden Markov model (pfam03106) of WRKY protein sequence were used to search PbWRKY protein sequence from protein database of P. bournei. Then, the chromosome localization, gene structure, protein sequences and phylogenetic tree of PbWRKYs were analyzed by Protaram, GSDS2.0, MEGA, Batch CD-search, TBtools and ClustalX software. Tissue-specific expression was analyzed based on transcriptome sequence data of roots, phloem and cambium, xylem and mature leaves of 3-year-old P. bournei plants. And, six-month-old seedlings were planted in phosphorus deficiency solution. After 60 days, the leaves and roots of phosphorus deficiency treatment and control were harvested for phosphorus content measuring and transcriptome sequencing. The expression of PbWRKY genes under phosphorus deficiency were analyzed, and the PbWRKY genes expressed differentially were verified by qPCR. Result: 68 WRKY genes, named PbWRKY1-68, were identified in P. bournei. They were distributed on all chromosomes of P. bournei and the most were on chromosome 3. All the PbWRKYs had introns, the number of them ranged from 1 to 28. The molecular weight of them from 19.09 to 115.56 kDa. Each PbWRKY had 1 or 2 WRKY domains and a specific zinc finger motif. Based on the number of WRKY domain and the type of zinc finger structure, PbWRKYs can be divided into 3 subclasses. Subclass Ⅰ contained 2 WRKY domains and 1 C2H2 type of zinc finger, and 14 genes were included in it. There were 47 genes in subclass Ⅱ which had 1 WRKY domains and 1 C2H2 type of zinc finger. And phylogenetic analysis showed this subclass can be classified into 5 subgroups: Ⅱa, Ⅱb, Ⅱc, Ⅱd, Ⅱe, but PbWRKY37 didn't belong to any subgroup above according to the phylogenetic tree although it was a gene of subclass Ⅱ. Subclass Ⅲ contained a WRKY domain and a C2HC type of zinc finger, a total of 7 genes. Most of WRKY domain sequence in PbWRKYs were "WRKYGQK", but it was "WRKYGKK" in PbWRKY62. Tissue-specific expression analysis showed five expression patterns of PbWRKYs were found in different tissues: low expression in phloem and cambium, high expression in xylem but low expression in leaves, high expression in phloem and cambium and low expression in leaves, high expression in roots, and high expression in leaves but low expression in roots. And some of the WRKY genes which closely related had similar tissue-specific expression, suggesting their functions possibly correlated. After 60 days of phosphorus deficiency treatment, 21 PbWRKY genes expressed differently more than 2 folds in leaves and roots compared to the control. All the 21 PbWRKY genes were strongly induced in leaves, implying that they possibly participated in the transportation and distribution of phosphorus in leaves under low phosphorus condition. Among them, the expressions of PbWRKY52, PbWRKY55, PbWRKY56, PbWRKY65 and PbWRKY66 were also inhibited in roots, suggesting that they also may be involved in the transportation of phosphorus in roots simultaneously. Conclusion: There were 68 PbWRKY genes in Phoebe bournei. The WRKY domain sequence of them was very conservative and the variation was very low. 21 PbWRKY genes were involved in the response to phosphorus deficiency after 60 days of phosphorus starvation treatment, and most of them were involved in the transportation and distribution of phosphorus in leaves when plants faced phosphorus starvation. However, PbWRKY52, PbWRKY55, PbWRKY56, PbWRKY65 and PbWRKY66 possibly not only played a role in leaves, but also participated in the absorption and transportation of phosphorus in roots under phosphorus deficiency stress.

Objective: The transcriptome sequencing analysis of Paulownia fortunei terminal bud induced by short days was carried out to explore the molecular mechanism of terminal bud death, so as to provide a theoretical basis for solving the problem of Paulownia "crown big stem low" Method: Under the condition of constant temperature of 25 ℃, the one-year-old seedlings of P. fortunei were induced by short days (SD) so that the terminal buds were in three periods: growth period (SDa), growth cessation period (SDb), and terminal bud death period (SDc). Terminal buds of each period were tested for transcriptome sequencing, and fluorescent quantitative PCR (qRT-PCR) was used to verify the expression of 11 genes. Result: A total of 57.47 Gb clean data were obtained from the transcriptome sequencing of P. fortunei. A total of 44 397 differentially expressed genes were screened out by comparing the samples of three periods using the software DESeq2. Among them, 37076 genes (83.51%) were annotated into seven databases (NR, NT, GO, EggNOG, KEGG, UniProt, Pfam). KEGG pathway enrichment analysis showed that the main enrichment was in the plant hormone signaling pathway. Through the analysis of the expressed genes in the plant hormone signaling pathway of SDc during the terminal bud death stage, it was concluded that the abscisic acid (ABA) signal transduction factor ABF was down-regulated, the ethylene (ETH) signal transduction factor ERF1 was up-regulated, brassinolide(BR) hormone signal transduction of TCH4 encoding xyloglucan endoglycosyltransferase and cell cycle-specific protein gene CYCD3 were up-regulated, which were different from those in most plants during bud dormancy. The result of qRT-PCR and transcriptome sequencing had deviation, but the overall trend was consistent, which proved that transcriptome sequencing result were correct and reliable. Conclusion: The expression of ABF, ERF1, TCH4 and CYCD3 in the plant hormone signal transduction pathway may lead to the continued division and differentiation of the shoot apical meristem in SDc. At the same time, the young leaves that wrapped the apical meristem fall off, so that the terminal bud of Paulownia fortunei cannot form dormant buds and result in death.

Objective: Objective: The tung oil tree, Vernicia fordii, is suffered from the soil-borne Fusarium oxysporum f. sp. fordii, Fof-1. However its sister species, Vernicia montana, shows high resistance to the pathogen. This study aims to investigate the resistance mechanism of V.montana, so as to provide ideas for resistance breeding of V. fordii. Method: The ethyl acetate extraction method was used to extract the root metabolites of V.montana and V. fordii after infection. The ultra performance liquid chromatography and tandem mass spectrometry were used to detect the root metabolites. The Illumina HiSeqTM2000 was used to detect the changes of gene expression mode and the pathway in the roots of V.montana and V. fordii during the infection by the Fusarium wilt pathogen. The gene expression was further validated by qRT-PCR method. The data were analyzed using R soft package and other bioinformatics methods. Result: 1) Comparing with that of V. fordii, the root extract of V. Montana had an obvious inhibition effect on the growth of Fof-1. 2) After being infected, the isoflavone and flavanone, such as formononetin 7-O-glucoside (Ononin) and hesperetin 7-rutinoside (hesperidin), in the roots of V. montana were 1 000 folds higher than those in the roots of V. fordii. 3) The phenylpropanoid biosynthesis pathway, the upstream key pathway responsible for flavonoid biosynthesis, was significantly enriched in the roots of resistant V. montana upon Fof-1 infection. 4) There were four hub genes, including 4-coumarate: CoA ligase (4CL), β-D-xylosidase, β-glucosidase, and peroxidase N1 with high connection with the other 1625 genes, which were all up-expressed in the early stage of Fof-1 infection in the phenylpropanoid biosynthesis pathway. Conclusion: Based on the metabonomic and transcriptomic analyses, it is revealed that the phenylpropanoid biosynthesis pathway actively responds to Fof-1 infection in the roots of V. montna, and the oflavone and flavanone, such as on onin and hesperidin, are correspondingly produced for the resistance to Fusarium pathogen Fof-1 in the roots of V. montana.

Objective: This study was carried out to explore the influences of extraction treatments on the acoustic vibration performance of paulownia wood used for Chinese national musical instrument, with the aims to provide a theoretical basis for the improvement of wood acoustic quality, promote the improvement of acoustic quality of Chinese musical instrument products, and alleviate the increasingly scarce situation of high-quality wood resources for musical instrument soundboards. Method: Four solvents(benzyl alcohol, deionized water, dichloromethane and absolute ethanol) were used to extract paulownia wood for 15 days. Based on the free bending vibration method, the effects of extraction treatments on the acoustic vibration performance parameters such as elastic modulus E, shear modulus G, specific dynamic elastic modulus E/ρ, logarithmic attenuation coefficient σ, acoustic impedance ω, acoustic radiation quality constant R, E/G, sound transmission speed υ, transmission parameter υ/σ and acoustic conversion efficiency υ/(σ ·ρ) were measured and analyzed. The influence mechanisms of extraction treatments on acoustic vibration performances were analyzed by microstructure observation, infrared spectroscopy(FTIR) and X-ray diffraction(XRD). Result: Acoustic performance parameters of paulownia wood were changed after extracting by deionized water, dichloromethane, benzyl alcohol and absolute ethanol. The specific dynamic elastic modulus E/ρ, acoustic radiation quality constant R and sound transmission speed υ increased by 7.67%, 11.36% and 3.75%, respectively. The improvement effect of dichloromethane extraction treatment was the best, with an increase of 13.99%, 17.28% and 6.77%, respectively. The acoustic impedance ω and logarithmic attenuation coefficient σ decreased by 3.23% and 19.42%, on average. The maximum decrease was -5.03% and -23.45%, respectively, after deionized water extraction. From the perspective of energy utilization, the acoustic conversion efficiency υ/(σ ·ρ) and transmission parameter υ/σ increase by 38.89% and 29.21%, on average. The improvement effect of dichloromethane extraction treatment was the best, with an increase of 47.66% and 35.10%, respectively. The infrared spectrum curve of paulownia wood after extraction treatment did not change significantly, but the absorption peak intensity of hydroxyl and hemicellulose decreased slightly. It could be seen from the microstructure of wood that the ducts and pits of the specimens after extraction were clearer, and the porosity of wood was retained. XRD analysis showed that the extraction treatment did not change the diffraction peak position, only affected the intensity of the diffraction peak. The relative crystallinity of paulownia wood treated with benzyl alcohol, deionized water, dichloromethane and absolute ethanol increased by 4.73%, 10.25%, 3.56% and 7.66%, respectively. The change rate of dichloromethane extraction was the largest (17.42%). Conclusion: The acoustic vibration performance of paulownia wood can be improved with extraction treatment, but the degree of improvement is different with different solvent extraction treatments. Overall, the improvement effect of dichloromethane and benzyl alcohol extraction is better. Different solvents have different improvement effects on different acoustic vibration performance indexes of wood. In the future, a combination of two or more solvent extraction can be considered to improve the acoustic vibration performance of wood.

With the continuous development of archaeological technology in modern society, the number of ancient ships excavated is increasing gradually. It is important and necessary to conserve these archaeological wood artifacts timely and effectively, which are precious records of human civilization. To promote proactively the development of ancient ship conservation technology, there is a growing trend to constantly seek excellent wood finishing materials, and explore more effective and efficient wood finishing method in the practice, thereby trying to protect the important information like runes or artistic ornamentation carried by ancient ships and to avoid damaging human historical resources. At present, many wood finishing materials or method have been applied to the reinforcement and conservation of ancient ships. Under this situation, by sorting out the relevant literature of traditional and conventional method, the main aim of this study is to summarize the merits and demerits of various wood finishing method, after comparing with finishing effect. Nanocellulose, a unique and promising natural material extracted from native cellulose, has gained much attention for its utilization as an archaeological wood finishing material. At present, nanocellulose has been applied in the restoration and conservation of archaeological wood artifacts. Furthermore, there have been some exploratory studies on the finishing and reinforcement of ancient ships. Nanocellulose is a good candidate for finishing and strengthening materials of ancient ships, because of its remarkable physical properties, special surface chemistry and excellent biodegradability. Particularly, owing to working wonderfully on the wood surface, the effects of nanocellulose remaining the original appearances of ancient ships are extremely prominent. Thus, using nanocellulose to strengthen and conserve ancient ships can not only improve their stability but also remain the original appearances as much as possible, which provide a new approach to strengthen ancient ships. Nowadays, with the rapid technological development of heritage conservation, there are many research aspects about ancient ships finishing needing to be developed in the future: 1) In-depth study of the anti-oxidation, anti-corrosion and moth-proof mechanisms of the traditional treatments such as tung oil coating and lacquer coating to reveal the interactions between tung oil and urushiol polymer and the chemical components of the cell wall, and further explore the modification and technological innovation of traditional natural finishing materials. 2) The development of the anti-bacterial and anti-mildew protection of ancient wooden ships, to enhance the adaptation of ancient ships to exhibition environments. The water-soluble protective agent is urgently needed to improve the biological resistance of the ancient ships. 3) The cutting-edge research result of nanocellulose are highly inspired to ancient wood finishing. The systematical study on the sealing performances and the long-term stability of nanocellulose on different reinforced wooden substrates is needed to comprehensively evaluate the role of nanocellulose in wood coating. Especially the performance improvement of nanocellulose-modified traditional materials, such as tung oil and raw lacquer, on mechanical properties, weather resistance and pollution resistance should be focused on, and the novel necessary function of nanocellulose-based wood finishing materials, like capturing the abundant Fe³⁺ in ancient wooden ships might be futher developed. Thus, the development of nanocellulose in wood cultural relics protection should be expanded.

Xylem embolism refers to the interrupt of plant water transport function caused by external air bubbles entering the xylem water-filled conduits. Bordered pit membrane is the crucial location where xylem embolism, spread and fatigue occur. In recent years, the characteristics of bordered pit membrane have received increasing attention in the study of xylem embolism mechanisms. In order to have more comprehensive and clear understanding of the role of bordered pit membrane in the formation, spread and fatigue of xylem embolism, we firstly discussed the formation mechanism and diffusion pattern of xylem embolism in angiosperms and gymnosperms, based on relevant research on xylem embolism at home and abroad. We elucidated the "air seeding hypothesis" in xylem embolism formation and diffusion, and clarified the crucial role of the bordered pit membrane; we also reviewed the main characteristics of the structural, chemical, physical and micromechanical properties of the bordered pit membrane, as well as the interrelationships between the different characteristics. Then we summarized the important role of bordered pit membranes in different stages of xylem embolism, diffusion and fatigue. We also summarized the recent advances and problems of the key characterization method of bordered pit membrane, such as the construction of three-dimensional structure model, in-situ detection of chemical composition, and mechanical behavior of the bordered pit membrane, microflow quantification and analysis of the single pit membrane. Finally, we proposed three research topics which should be given priority in xylem embolism: 1) changes in the chemical composition, physical and mechanical properties of the bordered membrane under different moisture conditions; 2) the formation and diffusion of air bubbles at the water-solid interface between water and the three-dimensional porous structure of the bordered pit membrane; 3) structural changes of the bordered pit membrane under different moisture conditions and their effects on the efficiency of microflow. Meanwhile, it is urgent to break through the technical bottleneck of the accurate in-situ characterization of the three-dimensional structure of the bordered pit membrane, and the construction and optimization of biological models. The further investigation of the structural, chemical, physical, mechanical properties and microflow behavior of the bordered pit membrane to reveal the influences of the characteristics of the bordered pit membrane on xylem embolism would be helpful to understand the mechanisms of xylem embolism, to provide scientific basis for exploring the drought-resistant mechanisms of xylem and breeding excellent drought-tolerant germplasm resources.

Permanent fixation of wood plastic deformation has been one of the key scientific and technologic issues for wood modifications such as densification and bending. This paper reviews plastic deformation fixation via hygro-thermal treatments from the perspectives of mechanisms for plastic deformation formation and set recovery. Recent research on fixation of solid wood plastic deformation, especially the advances and challenges in wood plastic deformation fixation by hygro-thermal techniques and the associated mechanisms, are overviewed, with the objective of providing a reference for breaking through the bottleneck of hygro-thermal treatment techniques scale-up to industrial level for wood deformation fixation. Hygro-thermal treatment is the main technique to fix plastic deformation by releasing wood interior stress. Depending on the treatment method, hygro-thermal treatments are categorized into heat treatment under atmospheric pressure and heated treatment under pressurized conditions. Currently, hygrothermal treatment requires the heating temperature to reach to over 180 ℃ for the permanent fixation of wood plastic deformation. Thus, to accelerate the heat transfer rate and heat wood center to 180 ℃ can effectively shorten the time required for wood plastic deformation fixation. Research on permanent fixation of wood plastic deformation is generally carried out by hygrothermal treatment in forms of heat treatment and saturated steam treatment, and was explained by the relationships between wood internal stress release and chemical changes. Three mechanisms for hygro-thermal treatment based permanent fixation of plastic deformation have been widely acknowledged. The first one is stabilized structures caused by the intercrossing of matrix molecules, the second one refers to molecular chains scission caused relaxation of stress in microfibril and matrix, the third is that polymerization of hydrophilic wood cell wall components especially hemicellulose and the resultant retardancy of the hydrophilic wood cell wall components to re-softening. However, research on hygro-thermal treatments for wood plastic deformation fixation is actually limited to two extreme conditions: one is the heat treatment under dry conditions, by applying elevated heating temperatures and extended heating time for internal stress release; the other is the heat treatment under almost water saturated conditions, by employing saturated steam at high temperatures under high pressure, to release wood interior stress. Neither of these involves processing variables/parameters of heat and mass transfer affecting wood plastic deformation fixation and wood properties, causing a contradiction between permanent fixation of wood deformation and wood properties deterioration. Even though permanent fixation of wood fixation has been theoretically realized by hygro-thermal treatment, it has not been completely applied in industry. The main reason for this is the immaturity of theories and technology system supporting hygro-thermal treatment based permanent fixation of wood deformation. To solve the application issues in hygro-thermal treatment based permanent fixation of wood deformation, more attention should be paid to how to make the hygro-thermal treatment conditions applicable rather the conditions themselves. Thus, research on hygro-thermal treatment for permanent fixation of wood deformation from the perspectives of functions of water and heat, oxidation and their interaction, as well as wood interior stress relaxation during hygro-thermal treatment should be carried out.