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    20 August 2019, Volume 28 Issue 8
    Ecological stoichiometry characteristics of forest litter and its influencing factors in Qinghai Province
    WANG Xin, LUO Xue-ping, ZI Hong-biao, YANG Wen-gao, HU Lei, WANG Chang-ting
    2019, 28(8):  1-14.  DOI: 10.11686/cyxb2018495
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    Litter plays an important role in regulating the nutrient cycle between plants and soil, and maintaining C, N, and P balances in forest ecosystems. This research aimed to clarify the nutrient content of litter from various sources and the environmental factors influencing the different nutrient content in litter under different forest types (coniferous forest and broadleaf forest) in forest ecosystems in Qinghai Province. This study examined coniferous forest and broadleaf forest litter samples from 21 counties in Qinghai Province, and explored the changes in litter C, N, P stoichiometric characteristics in different forest types with altitudes, as well as the influencing factors. It was found that: 1) The contents of litter C and N, the ratio of C∶P and N∶P in the coniferous forests were higher than that in the broadleaf forests, while the litter P content was lower, across the altitude gradient; 2) The content of litter N, the ratio of C∶P and N∶P in the coniferous forests increased linearly with increasing altitude, while the litter P content and the ratio of C∶N decreased with altitude; 3) For both the coniferous and broadleaf forests, significant correlations were found among plant factors (litter biomass, nutrient content and the C, N, P stoichiometric characteristics) and longitude, latitude, gradient, canopy density, plant community height, shadow area (except the litter P content and the C∶P ratio in the broadleaf forests); 4) The above results showed that the litter C, N, and P contents were generally low in the coniferous and broadleaf forests, which were limited by the N content (N∶P<14). Meanwhile in the high-altitude areas the coniferous forests were also limited by P content (14<N∶P<16); 5) Environmental factors (latitude, altitude, gradient, etc.) directly affected the litter biomass, nutrient content and ecological stoichiometric characteristics. In summary, the nutrient content of litter is affected by the forest type, resulting in significant differences in ecological stoichiometry. At the same time, geographic factors (latitude, altitude, slope, among others) were also significantly correlated with nutrient content. Therefore, the changes in litter stoichiometry and factors affecting those changes should be taken into consideration in order to formulate ecologically sound forest management and utilization measures.
    Fertilization effect of alfalfa high yield field and its recommended fertilizer application in northwest drought irrigated area
    YU Tie-feng, LIU Xiao-jing, WU Yong, KUAI Jia-lin
    2019, 28(8):  15-27.  DOI: 10.11686/cyxb2018503
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    The comparative importance of nitrogen, phosphorus and potassium fertilizer addition in alfalfa forage production, was studied using an incomplete orthogonal regression design. The forage yield and total protein yield of alfalfa were included in a fertilizer effect model in this study. It was found that the contribution of N, P and K to the yield of a two year old alfalfa stand was K>P>N, and the contribution to the yield of three year old alfalfa was P>K>N, while the interaction effect in two and three year old alfalfa stands ranked N-P>N-K>P-K. For total protein yield the corresponding rankings were N>K>P, and N>P>K, respectively, for two and three year old alfalfa stands, indicating that nitrogen is the key limiting factor in alfalfa protein production. For total protein yield, the N-P interaction and the N-K interaction effects were significantly greater than that of the P-K interaction. By using the frequency analysis method, the optimal fertilization amounts of N, P and K were calculated as 56.27-67.51 kg·ha-1, 77.69-90.48 kg·ha-1 and 76.43-87.18 kg·ha-1, respectively, when the target dry matter (DM) yield of two year old alfalfa is higher than 17522 kg·ha-1 ; as 46.75-57.66 kg·ha-1, 80.15-92.28 kg·ha-1 and 57.79-69.74 kg·ha-1, respectively, when the target DM yield of three year old alfalfa is higher than 19234 kg·ha-1; as 66.35-77.48 kg·ha-1, 79.34-98.87 kg·ha-1 and 73.68-85.38 kg·ha-1, respectively, when the target total protein yield of two year old alfalfa is higher than 2116 kg·ha-1; and 68.44-79.50 kg·ha-1, 72.74-85.96 kg·ha-1 and 50.68-61.61 kg·ha-1, respectively, when the target total protein yield of three year old alfalfa is higher than 2656 kg·ha-1.
    Effects of different crop rotations on alfalfa yield and soil quality in the Jiang-huai area
    LI Zheng-yan, XU Zhi-ming, SHI Shang-li, HE Chun-gui
    2019, 28(8):  28-39.  DOI: 10.11686/cyxb2019007
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    In order to determine the effects of different crop rotations on alfalfa yield and soil quality in the Jiang-huai area, an experiment was set up with the following three crop rotation treatments: (i) a Control (CK) with four years of continuous monoculture alfalfa, then replanted for one year of measurement; (ii) a 3-year sequence of alfalfa-maize-alfalfa (T1); and (iii) a 3-year sequence of alfalfa-sorghum-alfalfa (T2). Traditional methods were used to determine the alfalfa yield and physical and chemical properties of the soil in different soil depths. Enzyme activities in rhizosphere soil were determined using proprietary analytical kits, and the diversity of the bacterial community in the rhizosphere soil was determined by Illumina Miseq analysis. Incorporation of maize and sorghum into the rotation significantly improved alfalfa yield in the final measurement year. Specifically, alfalfa yield in T1 and T2 was, respectively, 1.27 and 1.13 times higher than that in the continuous alfalfa (CK) treatment. Maize rotation (T1) significantly (P<0.05) increased soil total nitrogen, while inclusion of sorghum in the rotation (T2) significantly (P<0.05) increased soil pH and organic matter. Both maize and sorghum rotations significantly (P<0.05) increased the soil available potassium content. Sorghum rotation (T2) significantly (P<0.05) increased soil pH and soil organic matter content. T1 and T2 treatments reduced soil bulk density; the sorghum rotation more so than the maize rotation. Compared with alfalfa continuous cropping, catalase and urease activities in soils of the maize and sorghum rotations were significantly (P<0.05) higher than those in CK-treatment soil. The Illumina Miseq analysis indicated that the soil bacterial community diversity was significantly higher in T1 and T2 soils than in CK, and the OTU count was, respectively, 1.25 and 1.39 times higher than that in the continuous alfalfa CK treatment. In addition, the Shannon, Chao, and Sobs diversity indices in T2 soil were significantly higher than in CK (P<0.05). The relative abundance of Proteobacteria and Firmicutes in CK soil was significantly higher than that in T1 and T2 soils, while the relative abundance of Chloroflexi and Planctomycetes in CK soil was significantly lower than in the T1 and T2 soils. It was concluded that the maize and sorghum crop rotations improved soil fertility and soil enzyme activities, stabilized bacterial community structure and thus increased the alfalfa yield.
    Yield and nutritional value of silage maize varieties in Ningxia Yellow River irrigation area
    LIN Yu-fan, ZHU Hong-fu, WANG Li-hui, ZHANG Gui-jie
    2019, 28(8):  40-48.  DOI: 10.11686/cyxb2018820
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    The aim of this research was to identify suitable varieties of Zea mays for silage production in the Ningxia Yellow River irrigation area. A randomized block design was used, and data collected on yield performance, nutrient composition, in vitro digestibility of dry matter and in vitro digestibility of neutral detergent fiber, for five foreign varieties of Z. mays bred for silage (CLO131, CLO1312, CLO133, CLO134 and CLO135) and one Chinese silage variety of Z. mays (Jingang Silage 50, JGS50) as the control variety (CK). All tested varieties had normal growth and no major pest or disease outbreaks occurred. The mean plant height of JGS50 was 300.3 cm, while plant height of the introduced varieties ranged from 254.3 cm (CLO133) to 291.7 cm (CLO132), but these differences were non-significant (P>0.05). The content of crude protein, neutral detergent fiber and starch differed significantly (P<0.05) among varieties, with CLO135 having the highest crude protein content (9.23%), and CLO134 having the lowest (8.38%). The neutral detergent fiber contents of CLO132, CLO134 and CLO135 were all lower than 30%; the variety with the highest starch content was CLO135 (43.11%). There were no significant differences (P>0.05) among varieties in the acid detergent fiber, ether extract, crude ash and lignin levels. The calcium content differed significantly between varieties (P<0.05), and the calcium content of the control variety was 0.44% higher than the other varieties. The in vitro dry matter digestibility (IVDMD) and neutral detergent fiber digestibility (IVNDFD) of CLO135 were 89.08% and 58.17%, respectively, and did not differ among varieties (P>0.05). Considering all measured parameters, the suitability ranking of the six tested Z. mays silage cultivars for maize silage production in the Ningxia Yellow River irrigation district was CLO135>CLO134>CLO131>CLO132>CLO133>CK. CLO135 showed the highest overall potential and therefore can be recommended as the preferred variety for silage maize growers in the Ningxia Yellow River irrigation area.
    A comparison of 21 varieties of silage maize in Henan Province
    LIU Xiao, WANG Bo, ZHU Xiao-yan, GUO Xiao-jie, WANG Cheng-zhang, LI De-feng
    2019, 28(8):  49-60.  DOI: 10.11686/cyxb2018539
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    The aim of this research was to select silage maize varieties suitable for planting in Henan province. A field experiment was set up with 21 varieties and five replications in a random design. Total herbage dry matter yield, grain yield, quality, and lodging resistance were recorded, and relationships among agronomic characters were examined. The results showed that: 1) The herbage dry matter yields and grain yields of Jingke silage 932, and Weike 106 varieties were the best. These two varieties also had better lodging resistance than other varieties. 2) In terms of quality, the crude protein (CP), ether extract (EE), and starch contents of Weike 106 and Jingke silage 932 varieties were higher, while neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were lower than other varieties, indicating their forage quality was also good. 3) There were positive, very significant correlations between herbage dry matter yield and the green leaf number, the green leaf weight, the plant height, the diameter and weight of stem, and the diameter and weight of the ear. Grain yield was found to have a very significant positive correlation with leaf number, leaf weight and stem weight, and ear weight. There was a positive, very significant correlation between total herbage dry matter yield and grain yield. Therefore, high grain yield can be obtained by selecting varieties with good quality of green leaves, larger stem diameter, larger ear size and greater plant height. The CP content was positively correlated with the contents of EE and starch. Considering all measured traits: herbage dry matter yield, grain yield, forage quality lodging resistance and other agronomic traits, the performance of Jingke silage 932 and Weike 106 varieties was superior to other varieties tested, and these two varieties can be recommended for whole crop silage and maize grain production in Henan province.
    Effects on the storage quality of dry corn stalk of adding cabbage wastes and organic acid
    REN Hai-wei, SUN An-qi, REN Jun-le, LI Zhi-zhong, FAN Wen-guang, WANG Yu, LIU Fei-fei, SHEN Jia-li, SUN Wen-bin
    2019, 28(8):  61-71.  DOI: 10.11686/cyxb2018244
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    Based on the physicochemical complementarity between dry corn stalk (DCS) and cabbage waste (CW), this experiment investigated the effects of the addition of cabbage wastes on the storage quality of dry corn stalk. The effects of adding formic acid to DCS with CW were also tested. The microbial community diversity during storage was investigated using Miseq high throughput sequencing technology. Three materials were prepared for testing of their storage fermentation reactions: (i) DCS with no additives (SE); (ii) DCS mixed with CW (ME); (iii) DCS mixed with CW and with the addition of formic acid (FB). The prepared materials were stored at 18±1 ℃ for 60 days, to undergo a fermentation reaction similar to silage making. The dynamic changes in the chemical composition, fermentation quality and microbial community diversity were analyzed at 30-day intervals. It was found that dry matter loss and pH value of the ME and FB materials were significantly (P<0.05) lower than in the SE material, and that longer-term storage of 60 d was beneficial through reduction of acid detergent lignin (ADL) content. The microbial community data indicated a greater diversity and abundance of lactic acid bacteria in ME and FB than in SE materials, with the more important genera identified being Lactobacillus, Paralactobacillus, Carnobacterium, Lactococcus, Pediococcus and Enterococcus. The main spoilage bacterium identified during storage was Enterobacter, and the abundance of Enterobacter in FB was significantly lower than in SE material. In summary, addition of CW to DCS reduced the loss of dry matter during fermentation and storage, and improved the structure of the microbial community. The application of formic acid during mixed storage of DCA and CW can further assist lignocellulosic breakdown.
    Effect of different turf-bed soil ratios on the traffic resistance of ‘Yangjiang’ bermudagrass sports turf
    LI Dan-dan, ZONG Jun-qin, GUO Hai-lin, CHEN Jing-bo, ZHANG You-hai, ZHOU Zhen-dong, LIU Jian-xiu
    2019, 28(8):  72-83.  DOI: 10.11686/cyxb2018451
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    The effects of simulated trampling on turf physical characteristics and quality, turfgrass growth, root morphology, root spatial distribution, were measured for two consecutive years in a field experiment, using the nationally registered Bermudagrass variety ‘Yangjiang’ with different turf bed ratios [river sand∶yellow sand] of 0∶100, 20∶80, 40∶60, 60∶40, 80∶20, 100∶0 (control). It was found that simulated trampling increased soil bulk density, soil compaction and soil shear strength, and decreased soil total porosity. The compaction-related changes in physical properties of the experimental turf-beds caused a change in root morphology reflected in deeper expansion of the root system, but reduced above-ground growth of ‘Yangjiang’ Bermudagrass, including decreased dry weight of clippings and verdure, a decline in turf quality score, but increased above-ground density of turf. Compared with a pure river sand turf-bed, inclusion of yellow-sand in the turf-bed decreased soil compaction, promoted root growth and rooting depth, increased turf density, dry weight of turf clippings and turf verdure, and enhanced the traffic resistance of the turf. The beneficial effects of including yellow sand in the turf-bed were optimized with a 60%∶40% ratio of river sand∶yellow sand. Therefore, 60% river sand and 40% yellow-sand is the recommended optimum ratio for construction of turf-beds.
    Responses of AMF and PGPR to Festuca elata under phenanthrene and pyrene stress
    LI Wen-bin, NING Chu-han, LI Wei, LI Feng, GUO Shao-xia
    2019, 28(8):  84-94.  DOI: 10.11686/cyxb2018510
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    The purpose of the present study was to evaluate mechanisms (and their efficiency) for degradation of residues of phenanthrene and pyrene by arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR). A total of 16 treatments were designed involving Festuca elata, with or without inoculation with AMF Glomus versiforme (Gv), PGPR Pseudomonas fluorescens (Ps2-6), or Gv+Ps2-6, and grown soil with 0 (Control), 50, 100 or 150 mg·kg-1 added polycyclic aromatic hydrocarbons (PAHs) (phenanthrene∶pyrene=1∶1 in weight). The results showed that inoculation with AMF, PGPR or AMF+PGPR significantly increased the biomass and mycorrhizal infection rate of tall fescue, enhanced photosynthesis and chlorophyll content of plants, and significantly increased the physiological activity of plants. At 100 mg·kg-1 phenanthrene and pyrene, the chlorophyll a, b and total chlorophyll content in the leaves of tall fescue were increased by 57.7%, 41.7% and 51.8%, respectively, compared with the Control, and the net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci) and gas pore conductivity (Gs) were increased by 70.6%, 100.0%, 4.5% and 78.6%, respectively; the maximum photochemical efficiency (Fv/Fm) and PSⅡ potential activity (Fv/Fo) were increased by 2.2% and 8.8%, respectively. The activities of superoxide dismutase (SOD), catalase (CAT), soluble sugar and proline were, respectively, 1.6, 1.5, 2.3 and 2.7 times those of the Control; the malondialdehyde content was decreased by 46.0%, and the height and fresh weight were increased by 63.0% and 69.6%. Inoculation with PGPR increased AMF infection rate and mycorrhizal dependency. Under the test conditions, double inoculation with a Gv+Ps2-6 treatment increased the content of photosynthetic pigments and antioxidant capacity of Festuca elata, enhanced photosynthetic capacity, reduced the level of membrane lipid peroxidation, and significantly promoted plant growth.
    Alleviation of drought stress and the physiological mechanisms in tobacco seedlings treated with exogenous melatonin
    LIU Ling, LI Dong, MA Yi-lin, WANG Li-jun, ZHAO Shi-min, ZHOU Jun-xue, SHEN Hong-tao, WANG Yan-fang
    2019, 28(8):  95-105.  DOI: 10.11686/cyxb2019098
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    This experiment explored the role of exogenous melatonin (MT) in enhancing the resistance of tobacco seedlings to drought stress. Moderate drought stress was simulated using 15% polyethylene glycol (PEG-6000), and the drought-tolerant variety ‘Yuyan No.6’ and drought-sensitive variety ‘Yuyan No.10’ were tested for their response following foliar-spraying with MT at concentrations of 0, 0.1, 0.2, and 0.4 mmol·L-1. Parameters measured included seedling growth, chlorophyll content, photosynthetic characteristics, chlorophyll fluorescence, antioxidant enzyme activities and reactive oxygen metabolism of the tobacco seedlings. It was found that MT application significantly increased total root length, total root surface area, average root diameter and root tip number; enhanced both shoot and root fresh weight of tobacco seedlings under drought stress; increased chlorophyll content, net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), water use efficiency (WUE), maximal PSⅡ quantum yield (Fv/Fm), PSⅡ actual photochemical efficiency (ΦPSⅡ) and photochemical quenching coefficient (qP); and decreased the enhancement of intercellular carbon dioxide concentration (Ci) and non-photochemical quenching coefficient (NPQ). Moreover, the stomatal and non-stomatal limitations to photosynthetic ability were effectively reduced by exogenous MT in seedlings under drought stress. The application of MT enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT); and reduced malondialdehyde (MDA) content and $O^{-·}_{2}$ generation rate. The changes in drought-sensitive ‘Yuyan No.10’ were greater than those in drought-tolerant ‘Yuyan No.6’. Across the range of exogenous MT concentration tested, the seedling growth indexes of the two tobacco varieties showed stimulation at lower rates and inhibition at higher rates. Foliar application with 0.2 mmol·L-1 MT produced the largest growth promotion effects and drought stress alleviation. In summary, MT application at the appropriate concentration positively stimulated seedlings to capture and convert solar energy, thus promoting seedling photosynthesis and growth. Exogenous MT also enhanced the adaptability of seedlings to drought stress by improving antioxidant capacity and reducing oxidative damage. The positive effect of MT was more obvious for drought-sensitive variety ‘Yuyan No.10’.
    Effect of high temperature shock on actual and potential competitiveness of Zoysia tenuifolia turf invaded by Imperata koenigii
    WANG Zhi-yu, LI Ying, LIU Jin-ping, WU De, GOU Rong
    2019, 28(8):  106-118.  DOI: 10.11686/cyxb2018490
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    This research investigated the problem of warm season Zoysia tenuifolia turf degradation caused by invading Imperata koenigii in the subtropical region of China. Pots 30 cm in diameter were prepared containing the two species in a replacement series; 9∶1, 8∶2, 7∶3, 6∶4, 5∶5 Z. tenuifoliaI. koenigii. The Z. tenuifolia was planted first and after it had formed 2-3 true leaves, the rhizomes of I. koenigii were inserted into Z. tenuifolia turf. After 40 days of normal culture, 3 replicate pots of each of the replacement series mixtures were placed into each of three controlled temperature chambers at 40, 42 and 44 ℃, for 2 hours each day over a period of 30 days, to provide a ‘high temperature shock’. Various parameters, including underground and aboveground relative yield, total relative yield, attack power and competition balance index, plant height, tiller number, leaf area, chlorophyll and physiological active substance, among others, were were determined on the 71st day. Subsequently, ‘resilience’ data such as photo-assimilate levels, regeneration capacity and resistance physiology were also collected. The aim was to study the effect of the high temperature shock on actual and potential competitiveness of Z. tenuifolia when challenged by I. koenigii invasion. It was found that: 1) High temperature shock was detrimental to Z. tenuifolia, and resulted in decline in the aboveground: below-ground relative yield, and a decline in competitiveness of Z. tenuifolia against I. koenigii. The high temperature shock increased the antagonism between the aerial parts of the two species. Invasion by I. koenigii had a greater effect on relative yield of Z. tenuifolia than high temperature shock. The synergistic effect of greater than 30% I. koenigii invasion and high temperature shock, sharply decreased the aboveground relative yield of Z. tenuifolia compared with underground yield. 2) Aboveground and underground attack force index and competition balance index for Z. tenuifolia were all at their maximum with a 42 ℃ heat shock. High temperature shock over 42 ℃ significantly reduced the attack power and competition balance index of Z. tenuifolia, and increased same indexes for I. koenigii. Only at 40 and 42 ℃, and with 20% I. koenigii invasion or less, was the competitiveness of Z. tenuifolia superior. At 44 ℃ or with 30% or greater presence of I. koenigii, the aboveground and underground attack power of I. koenigii were superior to Z. tenuifolia, as reflected by invasion and expansion of I. koenigii in the artificial turves. 3) High temperature shock increased height and tiller number of I. koenigii, and high temperature over 42 ℃ limited tiller number of Z. tenuifolia. The invasion of 30% I. koenigii or more, inhibited tillering but promoted height of Z. tenuifolia (P<0.05). High temperature shock also reduced the number of leaves and leaf area of Z. tenuifolia and increased these two traits in I. koenigii. Invasion by 10% or more I. koenigii reduced leaf area of Z. tenuifolia, and invasion by 30% or more I. koenigii reduced leaf thickness and leaf number of Z. tenuifolia. 4) High temperature shock and invasion of I. koenigii significantly affected the content of chlorophyll, superoxide dismutase, peroxidase, catalase, malondialdehyde and proline of Z. tenuifolia, indicating that the physiological response of Z. tenuifolia was more susceptible to high temperature shock than that of I. koenigii. 5) The influence of high temperature shock on morphology, physiology, relative yield and attack power of Z. tenuifolia was greater than the influence on the same indexes of I. koenigii. Invasion by I. koenigii caused significant damage to Z. tenuifolia turf. The synergistic effect of high temperature and I. koenigii invasion reduced the actual competitiveness of Z. tenuifolia and limited its potential competitiveness. Therefore, the basic requirement for conservating Z. tenuifolia turf is to control the proportion of invading I. koenigii to less than 20% in summer.
    Physiological characteristics of Psathyrostachys juncea at seed germination and seedling growth stages under salt stress
    LI Zhen, YUN Lan, SHI Zi-ying, WANG Jun, ZHANG Chen, GUO Hong-yu, SHENG Yu
    2019, 28(8):  119-129.  DOI: 10.11686/cyxb2018430
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    Salt tolerance of Psathyrostachys juncea at the germination and seedling growth stages was evaluated using 6 different accessions planted in saline-alkali soil. Seeds of the 6 accessions were treated with 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8% and 2.0% NaCl solutions, and pure water was used as the Control. The germination rate, germination potential, root length and shoot lengths were measured under salt stress. The seedlings germinated under the high 1.8% and 2.0% NaCl solution concentrations were transplanted into nursery pots. After these transplanted seedlings reached the three-leaf stage, 0.6% NaCl solution was added to the growth media, representing medium salt stress. Leaf length was measured. The soluble sugar, free proline, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and other antioxidant enzyme activity and malondialdehyde (MDA) contents were also measured. The general salt tolerance of the six P. juncea accessions at the germination and seedling growth stages was evaluated by subordinate function analysis. The results showed that germination rate, germination potential, seedling vigor index and relative growth rate all decreased with increase in salt concentration. The contents of free proline, soluble sugar, MDA and relative electric conductivity of leaves were increased by exposure to medium salt stress in seedling stage. The changes in SOD, POD and CAT activities varied in different materials. Principal component analysis showed that germination related indexes under salt stress were the key indicators of P. juncea salt tolerance. Antioxidant enzyme activity and MDA level were also identified as salt-tolerance indicators by the second principal component.
    Allelopathic effects of Stellera chamaejasme on seed germination and seedling growth of alfalfa and two forage grasses
    LIU Ya-jing, MENG Zhong-ju, DANG Xiao-hong, SONG Wen-juan, ZHAI Bo
    2019, 28(8):  130-138.  DOI: 10.11686/cyxb2019122
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    This research aimed to provide technical information relevant to the restoration of degraded grassland. We used a petri-dish method to study allelopathic effects of an extract of Stellera chamaejasme on the seed germination and seedling growth of three forage plant species: Medicago sativa, Elymus dahuricus and Agropyron cristatum. It was found that: 1) The germination rate of M. sativa seeds was inhibited in the presence of S. chamaejasme extract. With addition of 125 g·L-1 of extract germination was deceased by 33.56% compared to CK. However, with the two grass species E. dahuricus and A. cristatum, addition of S. chamaejasme extract at low rates promoted germination, and addition at higher rates was inhibitory. Compared to CK, with addition of 25 g·L-1 S. chamaejasme extract, the germination rate of the seeds of E. dahuricus was increased by 19.76%, and the germination rate of A. cristatum seeds was increased by 45.91%. 2) The seedling height of M. sativa was increased when exposed to S. chamaejasme extract concentrations ≤50 g·L-1 and was decreased when exposed to extract concentration ≥75 g·L-1, while the seedling height of E. dahuricus was increased when the extract concentration was ≤25 g·L-1, and decreased at concentrations ≥50 g·L-1. Finally, S. chamaejasme extract had an inhibitory effect on the root length of A. cristatum, with the root length decreased by 78.21% compared to CK. In summary, the seed germination and seedling growth of all three forage plant species tested were inhibited by treatment with S. chamaejasme extract, and the inhibition strength was enhanced with increasing concentration of the extract.
    Effects of an arbuscular mycorrhizal fungus and a rhizobium species on Medicago sativa wilt and Fusarium oxysporum root rot
    WANG Xiao-yu, DING Ting-ting, LI Yan-zhong, DUAN Ting-yu
    2019, 28(8):  139-149.  DOI: 10.11686/cyxb2018453
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    In this research, a pot experiment was conducted to test the effects of Funnelliformis mosseae, an arbuscular mycorrhizal (AM) fungus and the rhizobium species Sinorhizobium medicae on the wilt and root rot disease of alfalfa (Medicago sativa) caused by Fusarium oxysporum, to evaluate the biocontrol potential of the two symbiotic microorganisms. The experiment design was a 23 factorial with 8 treatments, involving alfalfa plants with or without inoculation with AM, rhizobium, and the Fusarium pathogen. AM fungi reduced the incidence of wilting and root rot in alfalfa by 80.13% (P<0.05). The interaction of AM fungi and rhizobia promoted the absorption of N and P and the synthesis of chlorophyll in the alfalfa plants. The chlorophyll content (SPAD value), shoot N content, shoot P content and shoot dry weight of AM+rhizobium treatments were, respectively, 61.85%, 26.5 fold, 54.3 fold and 23.4 fold higher than those of the control treatment (P<0.01). AM fungi promoted the formation of alfalfa root nodules, the number of which was on average 111.73% higher than in the control plants. The interaction of AM fungi and rhizobia reduced disease incidence of alfalfa, and significantly improved alfalfa root biochemical indicators. Compared with the control plants, the superoxide dismutase activity, β-1, 3-glucanase activity, the concentration of jasmonic acid and of lignin in alfalfa roots were increased, respectively, by 41.05%, 33.9%, 23.32% and 16.92% (P<0.05), while the concentrations of ABA, NO, H2O2 and malondialdehyde were reduced, respectively, by 26.68%, 9.63%, 25.26%, and 24.57%. In conclusion, the synergistic of F. mosseae and S. medicae can effectively reduce the occurrence of wilt and root rot disease caused by F. oxysporum in alfalfa. The two microorganisms have potential to be used as biocontrol agents to reduce alfalfa Fusarium wilt and root rot disease.
    Isolation and identification of Epichloё sp. infecting Stipa aliena in alpine meadows of the Qinghai-Tibet Plateau
    BAO Gen-sheng, YU Yong-fei, LI Chun-jie
    2019, 28(8):  150-160.  DOI: 10.11686/cyxb2018470
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    Stipa aliena is one of the important native plant species and a dominant grass species in alpine meadows of the Qinghai-Tibet Plateau. There has been little study of the endophyte(s) of S. aliena; therefore, in order to determine colonisation morphology and taxonomic status of the endophytes of this grass, samples of S. aliena were isolated from two different geographical populations, and the morphological features of endophyte strains found were observed. In addition, three housekeeper genes (tub, actin, tef) were selected for amplification of specific gene fragments in order to construct phylogenetic trees for each housekeeper gene for the endophyte species recovered. The endophyte morphological traits, such as colonisation morphology, hyphal growth rate, spore morphology and size were consistent with the recovered endophytes being Epichloё sp. Phylogenetic trees for tub and actin indicated that endophytes isolated from S. aliena had a close relationship with Epichloё gansuensis and Epichloё sylvatica; however, phylogenetic trees for tef showed that endophytes isolated from S. aliena were closely related to E. gansuensis and E. inebrians. Therefore, the endophytes of S. aliena were preliminarily identified as E. gansuensis from China (Xinjiang), E. inebrians from China (Gansu) and E. sylvatica from Europe. Our results suggest that the host-specificity might not be strongly developed in endophytes infecting S. aliena, and that different Epichloё species have independently developed a symbiotic relationship with the host grass S. aliena.
    Antifungal activity and identification of soil bacteria from the rhizosphere of Stipa plants in alpine grassland of Qinghai
    LIU Xue-er, MA Jin-feng, YANG Cheng-de, LI Tong-hua
    2019, 28(8):  161-169.  DOI: 10.11686/cyxb2018504
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    Soil bacteria were isolated from the rhizosphere of Stipa plants in alpine grassland of Qinghai province, China. Antifungal activity of the isolated bacteria was determined using the plate confrontation method for Alternaria solani, Fusarium avenaceum, Phoma foveata and Colletotrichum coccodes, and the bacteria were identified by 16S rDNA sequence analysis. The results showed that 23 strains of bacteria were isolated, and 17 of these strains could be sub-cultured. The soil bacteria count was between 6.4×106 and 36×106 cfu·g-1. Four bacterial strains (17.39% of isolated taxa) designated 4T1, 5T1, 10T1 and 10T3 had an antagonistic effect on more than one tested fungus, and the degree of inhibition was between 46.68% and 75.71%. Strain 10T1 had a notably strong antifungal effect, with the inhibition levels to A. solani, F. avenaceum, P. foveata and C. coccodes being 67.14%, 68.57%, 61.43% and 75.71%, respectively. The size of this strain ranged between 3.79 μm×0.85 μm and 10.50 μm×2.37 μm and the organism is Gram-positive, and rod-shaped. Combining information about culture characteristics, morphological characteristics and 16S rDNA sequence analysis, the 17 strains of soil bacteria isolated were identified as belonging to 7 genera and 12 species, of which Bacillus was the dominant genus. Of the strains isolated, 8 strains (47.05% of isolated strains), were identified as Bacillus. In summary, several isolated soil bacteria from the rhizosphere of Stipa plants in the alpine grassland of Qinghai, displayed significant antifungal activity and could potentially provide microbial resources for the development of biocontrol agents.
    Soil bacterial community changes across a degradation gradient in alpine meadow grasslands in the central Qilian Mountains
    LI Hai-yun, YAO Tuo, MA Ya-chun, ZHANG Hui-rong, LU Xiao-wen, YANG Xiao-lei, XIA Dong-hui, ZHANG Jian-gui, GAO Ya-min
    2019, 28(8):  170-179.  DOI: 10.11686/cyxb2018245
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    The objective of this research was to explore the distribution characteristics of soil bacterial communities in alpine meadows with different degrees of degradation in the central Qilian mountains. Illumina HiSeq PE250 high-throughput sequencing technology was used to study the bacterial communities in soils of grasslands with light, moderate and severe degradation. The relationships between soil bacterial communities, soil enzyme activities, and soil physiochemical characteristics were also analyzed. It was found that: with increased intensity of degradation, vegetation cover, height, aboveground biomass and diversity index were all significantly decreased (P<0.05), while the soil enzyme activities and soil physiochemical characteristics generally showed significant decline (P<0.05). The high throughput sequencing identified 257971 effective sequences, 219017 high quality sequences and 2004 operational taxonomic units. The three categories of grassland ranked in order: lightly degraded>moderately degraded>severely degraded, for abundance of soil bacterial communities, while the diversity index ranked lightly degraded>severely degraded>moderately degraded grassland. Analysis of Beta diversity showed that the differences among all plots ranked lightly degraded>severely degraded>moderately degraded grassland. The main feature of change in soil bacterial communities across the three categories of grassland degradation was a change in microbial biomass, and the effect on bacterial community structure was not obvious. Actinobacteria, Firmicutes, Acidobacteria, and Proteobacteria were the dominant phyla at all three levels of degradation in the tested grassland soils, with the proportion of the total bacterial population from these phyla being 77.25%, 84.27% and 78.66%, in mildly, moderately and severely degraded soils, respectively. Lactococcus was found to be a dominant genus in all three soil degradation categories, and the corresponding proportion of this genus in the total population was 14.29%, 38.84% and 7.39%, respectively. Redundancy analysis showed that soil enzyme activities and soil physiochemical characteristics differed based on the distribution of bacterial communities, and that the soil pH is the main driving factor affecting the distribution of soil bacterial communities. The degradation-linked changes in soil bacterial communities in alpine meadow grassland in the central Qilian mountains were strongly linked to the soil enzyme activities and the soil physiochemical characteristics.
    Cloning and expression analysis of the K+ channel gene AvAKT1 in Apocynum venetum
    XIA Zeng-run, WANG Wen-ying, LIU Ya-qi, WANG Suo-min
    2019, 28(8):  180-189.  DOI: 10.11686/cyxb2018456
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    Apocynum venetum is a typical K+-efficient species with notably high K+ uptake and utilization efficiency under K+-deficiency conditions. Furthermore, A. venetum can maintain high plant tissue K+ contents and K+/Na+ ratio through enhancement of the capacity for selective absorption and transport of K+ under salinity or drought conditions. In order to investigate the underlying molecular mechanisms, a potassium channel gene, designated AvAKT1, was isolated from the root of A. venetum using RT-PCR and RACE methodologies. The full cDNA sequence was 2906 bp in length, encoding 899 amino acid residues. The deduced amino acids of AvAKT1 exhibited all the structural features shared by other plant Shaker-like K+-channel family members, including six transmembrane helices, a K+-selective pore-forming domain, a cyclic nucleotide-binding domain (cNBD), an ankyrin-related domain (ANKY) and a domain rich in hydrophobic and acidic residues (KHA). The phylogenetic analysis showed that AvAKT1 belonged to Group Ⅰ (AKT1-subfamily) in the Shaker-like K+ channel family, and formed a clade with the closest relation to the dicotyledon AKT1 homologue NtAKT1 from Nicotiana tabacum. Real-time fluorescent quantitative PCR suggested that AvAKT1 is expressed especially in roots. Furthermore, AvAKT1 was induced strongly by supplying of 5 mmol·L-1 K+ in the medium. The expression level of AvAKT1 was significantly increased within a short time (e.g. 6 h) under -0.2 MPa osmotic stress or 25 mmol·L-1 NaCl treatment. These results indicate that AvAKT1 is probably involved in the process of low affinity K+ absorption, and might play an effective role in response to salinity or drought in A. venetum.
    Construction of a genetic linkage map for tetraploid potato based on SRAP markers
    ZHANG Ming-fei, YU Zhuo, YU Xiao-xia, LI Jing-wei, MA Yan-hong, WU Guo-fang, WANG Ying
    2019, 28(8):  190-199.  DOI: 10.11686/cyxb2019230
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    This research aimed to lay a foundation for molecular assisted breeding of potato through location of quantitative trait loci (QTL) for important traits such as high starch content, high dry matter content and yield of potato tubers. The mapping population comprised 182 F1 genotypes from a cross of YSP-4×MIN-021 tetraploid hybrid potato lines. We constructed biparental genetic linkage maps based on sequence related amplified polymorphism (SRAP) molecular makers. A total of 36 out of 357 suitable SRAP primers were selected, the genomic DNA of the 182 F1 genotypes and their parents was amplified, and a total of 598 SRAP markers was obtained. The chi-square test showed that there were 127 distorted markers, of which 59 were in the maternal linkage group, 68 were in the paternal linkage group, and the segregation distortion ratio was less than 30%, which conformed to the requirements for genetic mapping. The two parental SRAP genetic maps of tetraploid potato were established using the software package Join Map 4.0. The maternal map included 274 markers, had a total length of 1572.2 cM with an average interval of 5.74 cM, and the length of the linkage groups ranged from 14.03 cM to 214.44 cM. The paternal map included 324 markers, had a total length of 1932.23 cM with an average interval of 5.96 cM, and the length of the linkage groups ranged from 93.65 cM to 242.06 cM.
    Cloning and functional analysis of the MtNSN1 gene in Medicago truncatula
    ZHANG Zhi-qi, WANG Zhen, ZHANG Tie-jun, LONG Rui-cai, YANG Qing-chuan, KANG Jun-mei
    2019, 28(8):  200-208.  DOI: 10.11686/cyxb2019145
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    Nucleostemin (NS), a nucleolar GTP-binding protein widely present in animals, is involved in embryogenesis and cell proliferation, which are heavily dependent on cell cycling. This research investigated the expression pattern of MtNSN1 and the function in plant growth and development in Medicago truncatula. The full-length cDNA of MtNSN1 (2193 bp) was cloned revealing an open reading frame of 1800 bp encoding 599 amino acids of molecular weight 133.7 kDa. Phylogenetic analysis showed that MtNSN1 is related to NSN1 in Glycine max with a homology of 66.67%. The highest transcript level of MtNSN1 was detected in flowers, followed by roots, and the lowest in leaves. In situ hybridization showed that was mainly expressed in the shoot apical meristem and the nearby leaf primordia and emerging leaves. Transgenic plants overexpressing MtNSN1 had longer primary roots, and the transgenic plants possessed more leaves than the non-transgenic ones. Real-time quantitative PCR analysis indicated that five cell cycling marker genes were significantly up-regulated in plants overexpressing MtNSN1. Treatment with the genotoxic agent bleomycin demonstrated that the length of the primary roots of plants overexpressing MtNSN1 was longer than that of the non-transgenic control plants. These findings suggest that MtNSN1 may be involved in the maintenance of the shoot of Medicago truncatula, which is crucial for the development of both underground and aerial organs. In addition, ectopic expression of MtNSN1 altered the transcriptional level of the cell cycling genes.
    Selection of different explants from hops and establishment of a regeneration system
    YANG Ke, CHEN Yi-you, WANG Jun-cheng, YAO Li-rong, MENG Ya-xiong, MA Xiao-le, LI Bao-chun, SI Er-jing, WANG Hua-jun
    2019, 28(8):  209-217.  DOI: 10.11686/cyxb2019160
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    The aim of this research was to study the effects of different explants and hormone combinations on callus induction, differentiation and regeneration of hops, and finally to establish a stable and efficient hop plant regeneration system. Five different hop genotypes with semi-lignified shoots were selected, and the optimal explant donors were screened by cutting rooting and germination. The root tip, axillary bud and leaves of the hop genotypes were used as explants to study the effects of different exogenous hormone combinations on callus induction, adventitious bud differentiation and rooting. It was found that a genotype ‘PJ274’ had a rooting rate of up to 85% under the test growth conditions, and the germination rate of PJ274 material in hydroponic liquid of the ‘Japanese garden’ formula was as high as 40%, making PJ274 the most suitable among the tested genotypes for producing hop explants. Comparing the root tip, axillary bud and leaf as tissues for explant-induced callus, we found that axillary bud to be optimal with an induction rate of 51.11 %. The optimum culture conditions for callus induction using hop axillary buds were: 6-Benzylaminopurine (6-BA) 0.1 mg·L-1+IAA (indole-3-acetic acid) 1.0 mg·L-1+2,4-dichlorophenoxyacetic acid (2,4-D) 2.0 mg·L-1, and with this formulation callus induction rate reached 86.67%. The differentiation rate of axillary bud callus was achieved using 6-BA 1.0 mg·L-1+1-naphthaleneacetic acid) 0.2 mg·L-1+2,4-D 2.0 mg·L-1, with up to 66.67% success. The optimal hormoned regime for promotion of callus root differentiation of hops was 6-BA 0.1 mg·L-1+indole-3-butytric acid 1.0 mg·L-1, with a maximum rooting rate of 66.67% achieved. The test tube seedlings were transplanted after refining, and the survival rate was 80%. With these protocols, a high-output regeneration system for hops, using axillary buds as explants, was successfully established, so laying a foundation for the preservation of hop germplasm resources, tissue culture and rapid propagation, and genetically engineered breeding.