Table of Content

20 July 2022, Volume 31 Issue 7

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Estimation of aboveground biomass in Menyuan grassland based on Landsat 8 and random forest approach

Yi-han ZHAO, Meng-jing HOU, Qi-sheng FENG, Hong-yuan GAO, Tian-gang LIANG, Jin-sheng HE, Da-wen QIAN

2022, 31(7):  1-14.  DOI: 10.11686/cyxb2021454

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Remote sensing monitoring of above-ground biomass （AGB） can quickly and objectively evaluate the growth status of grassland， which is important for ecological environment evaluation and grassland resource utilization. To improve the accuracy of remote sensing estimation of grassland AGB， a single factor regression model and a random forest （RF） model were constructed based on the vegetation index calculated from Landsat-8 OLI images from Menyuan County， Qinghai Province， to determine the best AGB remote sensing estimation model. The spatial distribution in the study area from 2019 to 2021 was inverted. The results were as follows： 1） Among 29 single factor regression models of vegetation indices evaluated， the correlation was high between AGB and five vegetation indices （normalized difference vegetation index， NDVI； red-blue NDVI， RBNDVI； green normalized difference vegetation index， GNVDI； modified simple ratio， MSR； transformed vegetation index， TVI）， with R² values above 0.49. NDVI had the highest accuracy with an R² of 0.50 and a root mean square error （RMSE） of 702.89 kg·ha^-1. 2） In the RF model， the R² of the model built before variable screening was 0.61， and the RMSE was 621.14 kg·ha^-1. After the variable screening， the model accuracy was improved slightly； the R² was 0.62 and the RMSE is unchanged. The accuracy of the two models was better than that of the single-factor optimal regression model. Compared with the single-factor optimal regression model， the R² was increased by 0.12 and the RMSE was decreased by 80.95 kg·ha^-1. 3） The spatial distribution of AGB was higher in the northwest and lower in the southeast in Menyuan County. The biomass distribution was high in the central part of the county and lower towards the county boundaries. From 2019 to 2021， the total annual natural grassland yield in the county ranged from 4.2827×10⁴ to 8.9776×10⁴ t， and the average AGB ranged from 1063.49 to 1484.82 kg·ha^-1. Alpine meadow is the main category of grassland， with yield ranging from 4.0825×10⁴ to 5.6653×10⁴ t^{from 2019 to 2021. The average AGB ranged from 1060.38 to 1471.94 kg·ha-1. The average AGB of montane meadows ranged from 1036.81 to 1637.43 kg·ha-1. The average AGB of temperate steppe ranged from 1198.72 to 1786.63 kg·ha-1.}

Morphological adaptation strategies of Rumex hanus planted in saline-alkali land of arid areas

Zhi-xin YANG, Xu ZHENG, Lai-bao CHEN, Yong-xin YU, Feng-hua ZHANG, Lu-hua LI, Jia-ping WANG

2022, 31(7):  15-27.  DOI: 10.11686/cyxb2021231

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The aim of this study was to clarify the root morphological adaptation strategies of Rumex hanus in response to salt-alkali stress. The profile excavation method was used to collect the roots of R. hanus in saline-alkali soil at Shihezi city， Xinjiang Province and the root distribution and morphological characteristics of R. hanus in different months were analyzed and the root morphological distribution and the adaptation strategy in the saline-alkali environment were explored. It was found that the roots of R. hanus significantly reduced soil electrical conductivity around the root system， and this effect was significantly correlated with root length and root biomass. Root length， root biomass and root volume showed a decreasing pattern with increase in soil depth. From July to October， the root volume grew the fastest in the 0-20 cm soil depth， increasing by 71.26%. The roots at a horizontal distance of 25-35 cm from the plant grew slowly and less densely compared with those within a 5-25 cm radius of the plant. In this way new root growth was concentrated in low conductivity soil closer to the plant， avoiding high-salt areas and improving nutrient absorption. Fine roots （d≤2 mm） accounted for more than 90% of the total root length. In summary， the roots of R. hanus exhibited a morphological adaptation strategy to resist stress and enhance their resistance to saline-alkali soils. The main phenomenon was that the roots more strongly colonized a soil zone within a 25 cm radius of the plant where soil conductivity was lower to approximately 40 cm soil depth. In this zone roots were the most dense and rapidly increased their length from 0-15 cm horizontally to the plant. The results illustrate one available strategy for configuration of salt-tolerant plants for saline-alkali soil in arid regions and potentially provide a foundation for the development of new management practices.

Response of root-soil friction characteristics of three common grasses to soil water content in purple soil bunds

Feng-ling GAN, Jie WEI, Sha-sha LI

2022, 31(7):  28-37.  DOI: 10.11686/cyxb2021185

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This study aimed toelucidate the reinforcement effect of grass roots on purple soil bunds. Three grasses commonly found growing on purple soil bunds （Echinochloa crusgalli， Digitaria sanguinalis and Eleusine indica） were studied. Soil is a material that cannot withstand tension but can withstand pressure， while the root system within the soil can withstand tension to some extent. When external force is applied， the contact interface between the root system causes roots and soil to dislocate each other and generate frictional resistance and aspects of this root-soil interaction were measured for roots artificially packed into specially constructed test apparatuses. Root systems were harvested from the field and manually packed into the test apparatuses. Parameters measured included： resistance to shear force under vertical load （5 pressures ranging from 25 to 150 kPa）， maximum force （N） to pull packed roots out of the test apparatuses， and tensile strength （MPa） of the roots in the test conditions. Tests were conducted under five soil water contents （5%， 10%， 15%， 20% and 25%） and the root-soil friction characteristics of the three grasses were examined by direct-shear force tests and ‘pull-out’ （extraction） force tests. In addition， the mechanisms of interaction between grass roots and soil during the friction test process were analyzed. It was found that： 1） There was a significant difference between the three grasses in extraction friction force （P<0.01）. The maximum pulling force of E. indica was on average 1.18 times greater than that of D. sanguinalis which in turn was 1.14 times higher than that of E. crusgalli. Similarly， the tensile strength of E. indica was on average 1.30 times higher than that of D. sanguinalis which in turn was 1.10 times higher than that of E. crusgalli. 2） The root-soil friction characteristics of the three tested grasses showed significant differences across the five different soil water contents tested （P<0.05）. At a soil water content of 15%-20%， the cohesion of the tested grass root-soil interface was relatively lower than at the other soil water contents， while the friction coefficient tended to be a maximised. In addition， the maximum extraction force and tensile strength of root-soil interface occurred at a soil water content of 15%. 3） The constitutive relationship between the shear strength at the root-soil interface and vertical load can be described by a hyperbolic model that satisfies the Mohr-Coulomb criterion. The shear strength of the root-soil interface reached a maximum at a soil water content of 20%-25%. 4） Under the same vertical load and soil water content， the shear strength of the goosegrassroot-soil interface was significantly higher than that of D. sanguinalis and E. crusgalli （P<0.05）. It can thus be concluded that among the three tested grasses herbs， E. indica had the most significant effect on enhancing soil shear strength， followed by D. sanguinalis and E. crusgalli. These results provide scientific data to assist with selection of grass species for soil-fixation to protect purple soil bunds.

Relationship between plant functional traits and rhizosphere bacterial community structure of two Caragana species

Yuan-yuan LI, Ting-ting XU, Zhe AI, Zhao-na ZHOU, Fei MA

2022, 31(7):  38-49.  DOI: 10.11686/cyxb2021202

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In this study， we collected soil samples from Caragana liouana and Caragana microphylla across different geographical populations and the diversity and composition of soil bacterial community were compared and analyzed using Illumina MiSeq high-throughput sequencing technology. Our objective was to test the relationships between plant functional traits and the rhizosphere soil bacterial community， in anticipation this information might provide insight into microbiological mechanisms of Caragana adaptation to drought environments. Results showed that 22 phyla， 62 classes， 131 orders， 185 families and 304 genera were identified from all rhizosphere soils. At the phylum level， the dominant taxa were Proteobacteria， Acidobacteria， Bacteroidetes and Actinobacteria. Rhizobium， Sphingomonas， RB41 and Bacillus were predominant genera. Although the soil bacterial community diversity （Chao1 index， ACE index， Shannon index and Simpson index） showed no significant difference between C. liouana and C. microphylla， a principal coordinate analysis demonstrated that there were significant differences between them in the bacterial community structure. A Mantel test indicated a significant positive relationship between plant functional traits and bacterial community composition of the rhizosphere soil， and furthermore， based on the redundancy analysis， plant height， leaf dry matter content， specific leaf area， leaf thickness and leaf length were the dominant factors associated with changes in the rhizosphere bacterial community structure. Overall， the present results indicate that there are significant differences between C. liouana and C. microphylla in the structure of their soil bacterial communities， and the bacterial community structure of the rhizosphere soil is strongly linked to plant functional traits across the geographical populations.

Screening of rhizosphere growth promoting bacteria and their application in tailings improvement

Yi-chao CHEN, Xiao-ying SUN, Zhi-jie XIE, Pan ZHOU, Lu ZHANG, Xue-li GAO, Dong LI, Xiao-feng LIU

2022, 31(7):  50-63.  DOI: 10.11686/cyxb2021233

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This research was aimed at developing economic and effective methods which have strong applicability and adoption prospects for tailings improvement and vegetation restoration using plant growth-promoting rhizobacteria （PGPR）. PGPR from tailings primary environments were screened and a single factor pot experiment with a control and eight treatments was conducted to test their effects on seed germination and growth of Achillea millefolium. The treatments were： an assembly of mixed PGPR （B）， biochar （C）， manure （F） and their combinations ［BC， BF， CF， a compound treatment of B， C and F （BⅠCF）， and BⅡCF （the same as BⅠCF， but with doubled mixed PGPR content）］. Measurements included rhizosphere microbial community composition， tailings nutrients， and for plant tissues， enzyme activities and the total amount and distribution of tailings nutrients including total nitrogen and available phosphorus and potassium. The screened PGPR comprised KSB1 （Pseudomonas sp.）， KSB2 （Pseudomonas sp.）， KSB7 （Pseudomonas sp.） and KSB21 （Rhizobium sp.） selected for their ability to fix nitrogen， dissolve phosphorus， hydrolyze potassium， synthesize indoleacetic acid or secrete 1-aminocyclopropane carboxylic acid deaminase. The pot experiment showed that： 1） The abundance of Pseudomonas and Rhizobium in the rhizosphere environment and the phosphatase activity in tailings can be increased by inoculating mixed PGPR. 2） The effect on tailings nutrients and the growth of A. millefolium of adding biochar alone， was not obvious. 3） The application of manure promoted the growth of A. millefolium， enhanced the activities of tailings enzymes， and significantly increased the abundance of Cyanobacteria （P<0.01）， the total amount of nitrogen in the potted system， and the total amount of available phosphorus and potassium. 4） Compared with F alone， both the BF and CF treatments further improved various measures of A. millefolium growth and tailings enzyme activity. In addition， the total nitrogen in the potted system in the BF treatment increased by 11.18%， and the total available phosphorus and potassium increased by 22.54% and 22.00% respectively， while the total available potassium only increased by 8.32% in CF. 5） The various measures for BICF and BIICF treatments were generally higher than other treatments. Compared with BICF， BIICF had significantly improved indicators including alkaline hydrolysable nitrogen， phosphatase activity and A. millefolium biomass and the total nitrogen in the pot system increased by 9.54%， the total potassium content of the A. millefolium plant tissues increased by 19.12%， and the relative abundances of Cyanobacteria and Pseudomonas were increased by 51.96% and 13.62%， respectively. 6） Under these experimental conditions， neither inoculating with mixed PGPR nor adding biochar alone resulted in any significant effect on tailings improvement or vegetation restoration； manure played a key role in improving the nutrient supply of tailings and promoting the growth of A. millefolium.This work indicates the benefits of mixed PGPR and biochar are directly related to the application of manure. The BIICF treatment is effective in tailings improvement and the mixed PGPR at least play the role in enhancing nitrogen fixation， phosphorus solubilization and potassium solubilization， and strengthening the potassium absorption and utilization ability of A. millefolium.

Degradation of carbendazim in orchard white clover silage and its effect on the microbial fermentative community

Jian-zhen GE, Wen-hui FU, Lu ZHANG, Bao-jun LIN, Shuai ZHAO, Ma-ga-weng BAI, Jian-cun KOU

2022, 31(7):  64-75.  DOI: 10.11686/cyxb2021197

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This research investigated the degradation of carbendazim fungicide residues in white clover （Trifolium repens） silage cut within an orchard and the effect of carbendazim residues on the diversity of the fermentative microbial community， to explore options for the use of cover plants with pesticide residues in orchards. Different carbendazim concentrations （2.0， 2.5， 3.0 g·L^-1，_{with a water spray as Control） were sprayed on white clover prior to ensiling. The silage was harvested after 60 days fermentation at room temperature. Samples were taken before ensiling， on the 3rd day and on the 60th day after ensiling to study the diversity and composition of the microbial fermentative community using Miseq high-throughput sequencing technology. The degradation rate of carbendazim during was found to be up to 59.6%， and the higher the application rate， the higher the degradation rate. Carbendazim treatment significantly increased the content of lactic acid， acetic acid， and propionic acid in white clover silage during fermentation. For days 0， 3 and 60 of ensiling， lactic acid increased by 47.55%， 63.24%， 71.08%， respectively； acetic acid increased by 24.49%， 44.90%， 46.94%， respectively， and propionic acid increased by 66.67%， 187.50%， and 250.00%， respectively. These changes enhanced the retention of nutrients in the clover silage. High-throughput sequencing results showed that spraying carbendazim significantly changed the composition of white clover silage bacterial community and increased the abundance and diversity of the silage flora. After spraying carbendazim， the abundance of Lactococcus， Weissella， Pantoea， Rosenbergiella， Pseudomonas， Stenotrophomonas， Enterobacter， and Bacillus increased and these changes not conducive to good silage fermentation. However， with the passage of time after ensiling， the abundance of Stenotrophomonas， Enterobacter， Bacillus and other flora decreased. In summary， carbendazim pesticide increased the number of microorganisms beneficial to silage fermentation， and altered the abundance of bacteria harmful to silage fermentation. Carbendazim spraying also increased the diversity of carbendazim-degrading bacteria and the silage microorganisms. The residual amounts of carbendazim in silage were higher than the maximum residue of this fungicide stipulated for feed crops by the European Food Safety Agency and hence the silage did not meet the feed standards. These results provide test data on the level of carbendazim residues remaining in herbage after ensiling following spraying and will assist in the development of safe practices for the use of orchard cover plants.}

Effects of wilting and additives on the fermentation quality and dominant microbial genera in Napier grass silage at different temperatures

Jing TIAN, Xiang YIN, Yang FAN, Xin-qin LI, Jian-guo ZHANG

2022, 31(7):  76-84.  DOI: 10.11686/cyxb2021245

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Due to the moist， humid climate in South China， the moisture content of harvested Napier grass is high， and the fermentation quality of silage， when ensiled directly， is poor. In this study， Napier grass was wilted （moisture content decreased to 75.11% from 83.19%） and 10% corn flour or 2% sucrose was added， to explore the fermentation characteristics and microbial fermentative community during ensiling at different temperatures （20， 30 and 40 ℃） for 60 days. It was found that temperature had significant effects on the fermentation quality of Napier grass silages. Wilting decreased the water-soluble carbohydrate content of pre-ensiled Napier grass and the fermentation quality of silages， especially when ensiled at 30 ℃， as indicated by a high pH of 5.85， low lactic acid content of 1.56% DM， an acetic acid content of 2.10% DM， and an NH₃-N content of 25.09% TN. The acetic acid production of both the untreated Control and silages with additives increased with increase in ensiling temperature. The gas production of wilted silages ensiled at 20 and 30 ℃ was significantly higher than that at 40 ℃ or for unwilted silages. In addition， the bacterial diversity of Napier grass before ensiling was higher than its silage. After ensiling， the microorganisms found on Napier grass were dominated by the genera Enterobacter and Lactococcus， especially when ensiled at 20 ℃. The relative abundance of Enterococcus in wilted silage at 20 and 30 ℃ was higher than at 40 ℃. In summary， adding corn flour and sucrose can decrease the negative influence of environmental temperature while wilting under humid conditions did not have a positive effect on the fermentation quality of Napier grass silage in South China.

Effect of a rumen cellulolytic microbial consortium on the degradation of structural carbohydrate in sterile rice straw silage

Jun-feng LI, Jie ZHAO, Xiao-yue TANG, Tong-tong DAI, Dong DONG, Cheng ZONG, Tao SHAO

2022, 31(7):  85-95.  DOI: 10.11686/cyxb2021261

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In order to improve the fermentation quality of rice （Oryza sativa） straw silage， this study aimed to isolate a facultative anaerobic cellulolytic microbial consortium （i.e. a group of two or more rumen bacteria working symbiotically） from the rumens of black-and-white dairy cows and further evaluate its effect on the fermentation quality and structural carbohydrates degradation of rice straw silage. The cellulolytic microbial consortium （M6） was screened from rumen contents by continuous enrichment cultivation， Congo red staining， acid-resistant induction screening， secondary screening through filter paper degradation， and enzyme activity assay. Rice straw was treated with following： 1） Natural fermentation of non-irradiated rice straw （CK）； 2） Irradiated rice straw ensiled directly （IRR）； 3） Irradiated rice straw ensiled with combined lactic acid bacteria （CLAB）； 4） Irradiated rice straw ensiled with the microbial consortium （M6）. Each treatment was sampled randomly after 3， 6， 15， 45， 60 and 90 days of ensiling for laboratory analysis. It was found that the pH of M6 silages was lower than that of other silages after 60 days of ensiling， and reached the minimum （4.62） on day-60 of ensiling. After 45 days of ensiling， the lactic acid content of M6 silage was significantly （P<0.05） higher than that of other silages and reached the maximum （23.90 g?kg^-1 DM） on the day-90 of ensiling. After 15 days of ensiling， the contents of neutral detergent fiber （NDF）， acid detergent fiber （ADF） and cellulose in M6 silages were significantly （P<0.05） lower than those of other silages， and were lowest on day-90 of ensiling. After 60 days of ensiling， the IRR and M6 silages maintained higher levels of water-soluble carbohydrate content than other treatments， followed by the CLAB silage. In summary， the microbial consortium M6 is effective in degrading cellulose and promoting lactic acid fermentation of rice straw silage. The addition of this cellulolytic microbial consortium effectively improved the fermentation quality of rice straw. This work provides a theoretical basis for the development and application of a silage additive containing the M6 microbial consortium.

Solid-state fermentation with Trichoderma koningii improves nutritional value of tea dregs

Yi-yan CUI, Jia-zhou LI, Zhi-mei TIAN, Dun DENG, Hui-jie LU, Zhi-chang LIU, Ting RONG, Xian-yong MA

2022, 31(7):  96-110.  DOI: 10.11686/cyxb2021260

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The purpose of this experiment was to study the solid-state fermentation of tea dregs （TDs） by Trichoderma koningii to improve the nutritional value of TDs， and identify the optimal fermentation conditions. A series of single factor experiments was used to optimize the substrate ratio （TDs∶corn meal=6∶4， 7∶3， 8∶2， 9∶1）， solid∶liquid ratio （3∶7， 4∶6， 5∶5， 6∶4， 7∶3）， inoculation amount （2%， 4%， 6%， 8%， 10%）， fermentation temperature （25， 28， 31， 34， 37 ℃）， and fermentation time （0， 2， 4， 6， 8， 10， 14， 22 d） in fermented TDs. The optimum fermentation conditions of TDs were identified by an L9 （3⁴） orthogonal test with substrate ratio， fermentation temperature， inoculation amount， and fermentation time. The contents of crude protein， ether extract， reducing sugar， flavonoids， saponins， and caffeine of different fermentation combinations were determined， and the multivariate scores of different groups were calculated. After determining the optimal conditions， a comparative experiment was carried out to compare the nutrients， active substances， and free amino acid contents of the TDs before and after fermentation. It was found that： 1） In the single factor tests， the multivariate scores of the following were the highest： substrate ratio 7∶3， solid∶liquid ratio 5∶5， inoculation amount 8%， fermentation temperature 31 ℃， and fermentation time 6 d. 2） The orthogonal test indicated that a substrate ratio of 7.0∶2.5， inoculation of 7% （1×10⁷ spore·mL^-1）， and a solid-liquid ratio of 5∶5 at 31 ℃ for 6 or 7 d would produce the optimal result. 3） Compared with unfermented TDs， the contents of crude protein， reducing sugar， flavonoids， caffeine， various free amino acids， essential amino acids∶total amino acids and flavor amino acids∶total amino acids of fermented TDs （under optimal conditions） were significantly increased （P<0.05）， the saponin content was significantly decreased （P<0.001）， and the ether extract content had no significant difference between fermented or unfermented TDs （P>0.05）. No significant differences in nutrients or levels of active compounds were detected in fermented TDs for 6 or 7 d （P>0.05）. In brief， the solid-state fermentation by T. koningii can improve nutritional value in TDs.

Genome-wide identification and expression profile analysis of the CPP gene family in Medicago truncatula

Jiao-yang TIAN, Qiu-xia WANG, Shu-wen ZHENG, Wen-xian LIU

2022, 31(7):  111-121.  DOI: 10.11686/cyxb2021215

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The CPP （cysteine-rich polycomb-like protein） gene family is one category of small transcription factors occurring in a wide range of organisms， excepting yeasts and prokaryotes， and which play an important role in plant growth and development and in response to stress processes. The CPP gene family has been identified and studied in many species but so far no research in the model legume species Medicago truncatula has been reported. In this study， 9 CPP genes were identified in M. truncatula by bioinformatics. By constructing phylogenetic trees with the CPP protein sequences of three species， CPP genes could be divided into three categories. The genetic relationship between MtCPP members and soybean was closer. Conserved domain analysis showed that MtCPP transcription factors all had 1-2 CXC conserved domains. Chromosome mapping analysis found that 9 CPP genes were distributed on 6 chromosomes， and two pairs of paralogous genes were identified， which both derived from fragment duplication events. Combining these results with the analysis of gene chip expression database of M. truncatula， the expression of MtCPP genes had certain temporal and spatial specificity. MtCPP gene promoters contain a large number of cis acting elements related to hormone signalling and stress， MtCPP2，MtCPP5， MtCPP6 and MtCPP7 genes have expression characteristics of response to drought， and MtCPP2 and MtCPP8 genes may be involved in salt stress response. This study provides a research foundation for further functional analysis and screening of MtCPP genes involved in stress resistance in alfalfa.

Analysis of antioxidant enzyme protection systems and gene expression differences in two Xinjiang bermudagrass genotypes with contrasting drought resistance

Ling-shuang ZENG, Pei-ying LI, Zong-jiu SUN, Xiao-fan SUN

2022, 31(7):  122-132.  DOI: 10.11686/cyxb2021178

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Xinjiang bermudagrass ecotypes have well-developed rhizomes and slender stolons， and exhibit extensive genetic variation in drought tolerance. In this study drought tolerant （C138） and drought-sensitive （C32） bermudagrass genotypes were exposed to natural drought for 7 days followed by 3 days of rehydration treatment and the differences in physiological response and gene expression levels of the antioxidant enzyme systems under drought stress and rehydration were evaluated. It was found that drought stress reduced the apparent quality of bermudagrass lawn， relative leaf water content and chlorophyll content， increased electrical conductivity and malondialdehyde （MDA） content， and these responses were expressed more significantly in C32 than in C138. With increased duration of drought exposure， the levels of O₂^·-， H₂O₂， and MDA in bermudagrass leaves increased significantly， and C32 increased more significantly than C138， while activities of superoxide dismutase （SOD）， peroxidase （POD） and catalase （CAT） initially increased and then decreased， and ascorbate peroxidase （APX） activities increased. Drought-resistant C138 and non-drought-resistant C32 showed high-levels of expression of antioxidant genes such as SOD， POD， APX， DHAR and GPX， while non-drought-resistant C32 showed the highest lipid peroxidation and the lowest antioxidant enzyme activity and gene expression levels. In the process of rehydration， POD activity plays a vital role in improving antioxidant capacity and removing reactive oxygen species. Drought stress induces the up-regulation of the expression of antioxidant enzymes and related genes， enhances the antioxidant defense in cells， helps bermudagrass maintain cell membrane stability， delays the dehydration of leaves， and improves the bermudagrass drought resistance ability.

Effects of alfalfa （Medicago sativa） proportion on growth and physiological characteristics of cold resistance in mixtures with Elymus nutans

Cai-ting LIU, Li-ping MAO, Ayixiemu, Ying-wen YU, Yu-ying SHEN

2022, 31(7):  133-143.  DOI: 10.11686/cyxb2021351

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A pot experiment was conducted in controlled conditions to assess the growth and physiological characteristics of cold tolerance of alfalfa （Medicago sativa， MS） in mixed planting with Elymus nutans. Four mixture proportions of alfalfa and E. nutans were tested （100%， 70%， 50% and 30% alfalfa， designated MS_100%， MS_70%， MS_50% and MS_30%， respectively）. After germination and establishment at 25 ℃ for 45 days， all treatment pots were moved to 15 ℃ for 10 days， then to 5 ℃ for another 10 days. The growth and physiological parameters of alfalfa in the four planting ratios with E. nutans under two temperature regimes were analyzed. For the growth characteristics， at 15 ℃， the leaflet width and length of MS_70% was the lowest. The aboveground biomass， alfalfa underground biomass and community biomass of MS_50% were the highest. The highest root collar diameter was recorded in MS_50%， 42.8% and 46.8% higher than MS_100% and MS_70%， respectively. At 5 ℃， the aboveground biomass， alfalfa underground biomass and community biomass was again the highest under MS_50%， while MS_50% and MS_30% treatments showed superior performance and cold tolerance for plant height， branch number， root collar diameter and taproot length. For the physiological characteristics， the roots showed more hardiness than leaves. The soluble sugar content in roots of the planting ratios MS_70%， MS_50% and MS_30% （13.1%， 25.2% and 14.0%， respectively at 15 ℃） was higher than for MS_100% （4.28%）. The proline content of alfalfa under MS_30% （32.9% at 15 ℃） was higher than that under MS_100% （3.81 mg·g^-1）. The highest water soluble sugar content in alfalfa roots was measured in MS_50%，_{and the soluble protein and proline contents of plants in MS50% and MS30% planting ratios were higher than those under MS100% and MS70% at 5 ℃. In summary， multivariate evaluation of alfalfa growth physiological characteristics and the screening of the optimal mixed proportion indicated that mixed planting with E. nutans can improve alfalfa growth and physiological adaptability to low temperature. Based on these results， an alfalfa planting ratio of 30% to 50% with E. nutans of alfalfa mixed proportion is optimal for the establishment of alfalfa-grass mixed grassland in the study area.}

An evaluation of drought resistance of seven Poaceous forages

Tong JI, Qi JIANG, Zhan-jun WANG, Bo JI

2022, 31(7):  144-156.  DOI: 10.11686/cyxb2021017

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Soil moisture deficit is a key factor restricting agricultural production and establishment of grassland vegetation in Ningxia， China. The identification of drought-resistant pasture species is of great significance to the region’s agricultural and pastoral production and ecological restoration of grassland vegetation. This research evaluated the growth performance and feed quality of seven Poaceous forages to determine drought resistance in arid areas. The grass species and lines evaluated were： Agropyron mongolicum-Ningxia， Agropyron mongolicum-Inner Mongolia， Agropyron desertorum， Agropyron cristatum， Agropyron trachycaulum， Bouteloua gracilis and Elymus sibiricus. The trade-off between seven agronomic traits （including green leaf number and plant height） and soil water content was quantitatively analyzed， using the root mean square deviation and soil water content as model variables. In this way， the soil water content needed to maintain normal plant growth was determined by quantile regression. A multivariate score derived from physiological indexes of plants under drought stress was used to evaluate the drought resistance of the tested forages. It was found that： 1） The higher the degree of drought stress， the greater the negative impact on the chlorophyll and plant height of forage； 2） The degree of tolerance to drought stress and the response range differed for different grass forages. In particular， the soil water content thresholds for adequate green leaf number in A. trachycaulum and B.gracilis were 9.1% and 9.7%， respectively. The soil water contents for which maintaining normal plant height growth attained the threshold for A. trachycaulum， B. gracilis and A. mongolicum-Ningxia were， respectively， 4.6%， 5.6% and 13.0%. The quantile regression models of other forages failed to pass the significance test. 3） Through evaluation and analysis of the seven physiological indexes， the drought resistance of the seven tested perennial grasses was ranked as follows： A. mongolicum-Ningxia>B. gracilis>A. trachycaulum>A. mongolicum-Inner Mongolia>A. desertorum>A. cristatum>E. sibiricus. Based on their multivariate score for drought resistance traits and tolerance threshold to soil moisture deficit， A. trachycaulum and B. gracilis were selected as recommended forages in moderately arid areas.

Effects of planting density and nitrogen application on forage yield， quality and nitrogen use efficiency in a maize/forage soybean intercropping system

Zi-wei JIANG, Gui-yu LIU, Hao-yun AN, Wei SHI, Sheng-hua CHANG, Cheng ZHANG, Qian-min JIA, Fu-jiang HOU

2022, 31(7):  157-171.  DOI: 10.11686/cyxb2021251

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A field experiment involving maize （Zea mays）/forage soybean （Glycine max） intercropping was conducted in the Hexi irrigation district in 2019 and 2020. Three planting densities of 7.5×10⁴ （D₁）， 9.0×10⁴ （D₂） and 10.5×10⁴ plant·ha^-1 （D₃）， and four nitrogen treatments of 0 （N₁）， 120 （N₂）， 240 （N₃） and 360 kg·ha^-1 （N₄） were set up to explore the effect of planting density and nitrogen application on forage yield， quality and nitrogen utilization. It was found that the hay yield and yield of crude protein of silage maize and forage soybean under the D₂ and D₃ treatments were significantly higher than those under the D₁ treatment， and the hay yield and yield of crude protein of silage maize， and the total hay yield under N₃ and N₄ treatments were significantly higher than those under N₂ and N₁ treatments. Among all treatments， D₂N₃ had the highest hay yield of 36.16 and 30.31 t·ha^-1 in 2019 and 2020， respectively. With increase in planting density， the contents of crude protein and crude fat of silage maize and forage soybean， and the total crude protein content and crude fat content showed a downward trend， while the contents of crude ash， neutral detergent fiber and acid detergent fiber increased. The total crude protein content and crude fat content increased while the contents of crude ash， neutral detergent fiber and acid detergent fiber decreased with the increase of nitrogen application rate. The total nitrogen content， nitrogen uptake and nitrogen use efficiency of the D₂ treatment were significantly higher than those of D₃， and the D₂ treatment had the highest agronomic efficiency of nitrogen use. The total nitrogen content and nitrogen uptake of N₂， N₃ and N₄ treatments were significantly higher than that of N₁， and the agronomic efficiency and nitrogen use efficiency of N₃ treatment were significantly higher than that of N₄. Among all treatments， D₂N₃ had the highest nitrogen use efficiency， which was 1.41 and 0.86 kg·kg^-1 in 2019 and 2020， respectively. Therefore， based on these results the D₂N₃ treatment is a recommended field management regime for silage maize/forage soybean intercropping systems in the Hexi irrigation area， and we advocate its promotion and adoption.

Leaf structure and photosynthetic characteristics of two species of Hesperis

Meng-yu DONG, Jin-xin WANG, Meng WU, Zi-yao ZHOU, Shun CHENG, Yan-hui LI

2022, 31(7):  172-184.  DOI: 10.11686/cyxb2021425

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This research studied the functional leaf morphology， anatomical structure， pigment content， chlorophyll fluorescence and photosynthetic parameters of Hesperis matronalis and wild-type Hesperis sibirica from two localities （Wuling Mountain and Saihanba） in Hebei Province. The relationships between leaf structure and photosynthetic physiology of the two species of Hesperis were explored using correlation and redundancy analysis methods. It was found that： 1） There were intraspecific differences in leaf structure and photosynthetic physiology of the two wild-type H.sibirica collections. The H. sibirica from Wuling Mountain had comparatively greater leaf thickness （LT）， larger leaf area （LA）， thicker palisade parenchyma thickness （PPT）， richer pigment content， and higher net photosynthetic rate （P_n）. The H. sibirica from Saihanba had comparatively high leaf stomatal density （SD）， greater sponge parenchyma thickness （SPT）， increased diameters of vessel and sieve tubes （VD and STD）， high activity parameters for the PSⅡ reaction center， and strong transpiration rate （T_r）. H. matronalis leaves had high epidermal trichome density （ETD）， a high ratio of palisade parenchyma thickness to sponge parenchyma thickness （PPT∶SPT）， rich pigment content， and high P_n and water use efficiency （WUE）， when compared with values for H. sibirica. 2） There was a strong correlation between the leaf structure and the photosynthetic physiological parameters of the two species of Hesperis. Important links between leaf structure and photosynthetic physiological differences and were identified by redundancy analysis， involving the parameters PPT∶SPT， SPT， ETD， spongy ratio， LA， VD， PPT and VBD. Among these， the effect of PPT∶SPT （P<0.05） was mathematically largest and provided the strongest single-trait association with differences in the ecological adaptability and resource acquisition of the two Hesperis species. Overall， the two species of Hesperis performed well at the sites where they were introduced and showed promise for development and utilization as garden plants. The PPT∶SPT could be used as a reference indicator trait for screening high-light-efficiency germplasm lines of Hesperis.

Effects of seed priming on cuticular wax and resistance of sweet sorghum

Lu-hua YAO, Cai QI, Jian-feng YANG, Yan-jun GUO

2022, 31(7):  185-196.  DOI: 10.11686/cyxb2021487

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Seed priming is an efficient method to improve plant tolerance and yield. However， it is still not clear whether the deposition of plant cuticular wax is altered by seed priming thus improving plant resistance to some kinds of stress. In this study conducted in 2020， seeds of sweet sorghum （Sorghum bicolor ‘Dochna’） were primed with 15% polyethylene glycol （PEG）， 150 mg·L^-1 salicylic acid （SA）， 20 mg·L^-1 abscisic acid （ABA）， 5 mg·L^-1 gibberellin （GA） or water （control）， and subsequently sorghum plants at four growth stages （seedling， jointing， heading and maturity） were analyzed for total wax content， leaf water loss rate and chlorophyll extraction rate. The seeds were harvested and reseeded in 2021， and the transgenerational effects of seed priming on cuticular wax were measured. It was found that， compared to the control， the contents of total wax at different growth stages were significantly influenced， with 57.6%， 50.8% and 80.3% increases observed at the seedling stage for PEG-， SA- and ABA-priming. The contents of alkanes were increased by 58.7%， 35.5% and 36.5% （P<0.05 in each case） at seedling， heading， and maturity stages for ABA-priming. Furthermore， the relative abundances of C₂₇， C₂₉， and C₃₃ alkanes， C₂₈-C₃₂ aldehydes， and C₂₈ alcohols， were also improved. Meanwhile， seed priming increased the leaf relative water content but reduced the water loss rate and chlorophyll extraction rate. We concluded that PEG-， SA- and ABA-priming all influenced the deposition of cuticular wax， contributing to an improvement in cuticular resistance. Importantly， the effects of seed priming on wax synthesis had transgenerational “memory”， indicating a potential new method for production of “resistant” seed lines.

Non-destructive identification of artificially aged alfalfa seeds using multispectral imaging analysis

Xue-meng WANG, Xin HE, Han ZHANG, Rui SONG, Pei-sheng MAO, Shan-gang JIA

2022, 31(7):  197-208.  DOI: 10.11686/cyxb2021198

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Aging during the storage of seeds reduces seed vitality and causes serious economic losses to the agricultural industry， and has become one of the biggest factors involved in decreased seed vigor. Distinction between aged and viable seeds is of high importance in alfalfa seed planting and production， but the existing methods are time-consuming or destructive. Therefore， a rapid and non-destructive screening method to distinguish aged and viable seeds is not only very necessary in seed testing and the alfalfa seed industry， but also potentially useful in alfalfa seed research. In this study， we collected data of both morphological features and spectral traits of alfalfa seeds using multispectral imaging （MSI） technology. Then， we evaluated three multivariate analysis methods： linear discriminant analysis （LDA）， support vector machines （SVM） and normalized canonical discriminant analysis （nCDA）， to classify seeds artificially aged for 0， 3， 6 and 14 days， and predict viable seeds which could germinate. It was found that the mean light reflectance at 470-660 nm differed significantly between non-aged and aged seeds. The LDA model based on a “hold-out method” provided accuracies of 93.0%-97.7% in distinguishing aged seeds from non?aged seeds， and 75.3%-91.7% in distinguishing the different groups of aged seeds. Corresponding values for the SVM model were a little lower， being 92.4%-94.9% and 68.7%-78.8%， respectively. The nCDA model also exhibited achieved aged seed discrimination with an accuracy of 88.0%-98.0%. Finally， viable seeds could be distinguished from dead seeds in all the categories of aged seeds， with accuracies of 98.7% and 92.1% in LDA and SVM analysis， respectively， while the accuracy of nCDA in predicting the germination of aged seeds ranged from 90% to 99%. This study showed that MSI could successfully distinguish aged seeds， and also predict germination of seeds. In summary， we demonstrated a nondestructive， rapid and high-throughput approach to screen both aged and viable seeds in alfalfa， and showed that MSI together with multivariate analysis is promising as a new tool for application in seed testing and field planting of alfalfa seeds.

Effects of milkvetch （Astragalus sinicus） decomposition leachates on germination and seedling growth of goosegrass （Eleusine indica）

Si-lin LIU, Jun-xue CHEN, Yang YANG, Zhong-wen CHEN, Ling-ling LU, Ying-hui MU

2022, 31(7):  209-219.  DOI: 10.11686/cyxb2021188

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This experiment investigated the allelopathic effects of leachate from milkvetch （Astragalus sinicus） decay on seed germination and seedling growth of goosegrass （Eleusine indica）. The experiment treatments comprised different decomposition durations and different milkvetch decomposition leachate concentrations applied to seeds of goosegrass， and six physiological indexes such as germination rate were determined. The activities of protective enzymes and the contents of proline （Pro） and malondialdehyde （MDA） in the leaves of goosegrass were determined after root irrigation with different concentrations （2%， 5%， 30% and 80%） of milkvetch decomposition leachates. It was found that for a given concentration of decomposition leachates， the inhibition effect was more pronounced with greater decomposition duration. At low concentrations of decomposition leachate， a hormesis effect was observed. When the decomposition duration exceeded 2 days and the decomposition leachate concentration was 80% or more， seed germination was completely inhibited. Superoxide dismutase activity was decreased with increased leachate concentration. Peroxidase activity was lower at leachate concentrations below 30%， and higher at concentrations greater than 30%. Catalase activity was increased with increasing leachate concentration up to 30% and was decreased as leachate concentrations reached 80%. When the concentration of decomposition leachate applied was greater than 2%， the content of MDA， Pro and H₂O₂ in goosegrass leaves increased with the increase of leachate concentration. In conclusion， the decomposition leachate of milkvetch has significant inhibitory effects on the seed germination and seedling growth of goosegrass. It is concluded that milkvetch decomposition leachate has the potential to inhibit weed growth. This study provides reference information for research into allelopathically active substances in milkvetch.

Effects of calcium salt stress on seed germination and seedling physiological characteristics of three Pueraria lobata germplasm lines

Wen-hui XIE, Li-juan HUANG, Li-li ZHAO, Lei-ting WANG, Wen-wu ZHAO

2022, 31(7):  220-233.  DOI: 10.11686/cyxb2021229

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This research investigated the adaptation mechanism of Pueraria lobata to the calcium-rich karst environment. P. lobata germplasm lines were sourced from Australia （AUS）， Jiangsu （JS） and Hunan （HN）. To investigate the effects of excess calcium （Ca） stress on seed germination， seedling growth， osmoregulatory substances， antioxidant enzyme systems and endogenous plant hormones， seeds of the three P. lobata germplasm lines were germinated at different concentrations （0， 50， 100， 150 and 200 mmol·L^-1） of CaCl₂. It was found that seed germination rate， germination potential， germination index and vigor index of the AUS and JS germplasm lines were significantly reduced and seed germination rate， germination speed and seedling vigor were significantly inhibited with increasing calcium salt concentration （P<0.05）， while seed germination rate， germination potential and germination index of Hunan germplasm were increased at 100 mmol·L^-1 CaCl₂ compared to other concentrations. With increasing CaCl₂ concentration： i） The leaf length， leaf width， plant height and biomass of seedlings of the three P. lobata germplasm lines were significantly reduced； ii） Malondialdehyde （MDA） and relative conductivity of seedlings of the three P. lobata germplasm were significantly increased； iii） Proline （Pro）， soluble sugar content and superoxide dismutase （SOD） and catalase （CAT） activities of leaves were initially increased and then decreased； iv） The leaf peroxidase （POD） activity increased and then decreased in AUS and HN germplasm， while in JS germplasm POD activity showed a significant increase； v） The ascorbate peroxidase （APX） activity increased significantly in HN and JS germplasm， while AUS germplasm showed an initial increase and then decreased； vi） The leaf ABA， IAA and GA₃ contents initially increased and then decreased in all three P. lobata germplasm lines， while zeatin did so for AUS and HN， but not JS germplasm. The seed germination and seedling growth of the three P. lobata germplasm lines were not significantly inhibited by 0-50 mmol·L^-1 CaCl₂ stress， but were significantly inhibited by 150-200 mmol·L^-1 CaCl₂ stress， while seeds of HN germplasm relied on seed dormancy to resist CaCl₂ stress. Hence it was concluded that P. lobata seedlings are tolerant to low CaCl₂ stress. When stressed with medium to high calcium salt concentration， seedlings of P. lobata actively regulated their physiological metabolism by increasing their own osmotic substance content， enhancing antioxidant enzyme activity， increasing some hormones， and changing the biomass of aboveground and underground parts to adapt to high calcium salt environments. Using affiliation function and principal component analysis， all the indicators， except for Pro content and APX activity， were considered to evaluate the salt tolerance of the three P. lobata germplasm lines， and the salt tolerance ranking of the three germplasm lines was JS>HN>AUS.