转Bt基因“克螟稻”秸秆还田对稻田厌氧微生物种群和酶活性的影响

采用改良亨盖特技术,通过最大或然数法(mostprobablenumber,MPN法)及固体滚管法,系统研究了转Bt基因克螟稻秸秆对淹水条件下稻田土壤厌氧微生物种群的影响;同时采用比色法,研究其对土壤酶活性的影响。结果表明,克螟稻秸秆对水田土壤反硝化细菌和产甲烷细菌种群有显著的抑制作用(p0.05);对厌氧发酵细菌种群有显著的刺激作用(P0.05);而对厌氧固氮细菌种群有刺激作用但差异未达到显著水平(p0.05)。克螟稻秸秆还田在短期内可显著提高土壤纤维素酶、磷酸酶活性(p0.05),但对脱氢酶活性有明显抑制作用(p0.05)。     

cry1Ac/cpti转基因大米水稻土中微生物群落的多样性研究

Two types of cry1Ac/cpti transgenic rice(GM1 and GM2)and their parental non-cry1Ac/cpti rice(CK1 and CK2)were planted in the field at Wufeng,Fujian Province,China for four years to investigate the influence of genetically modified rice on diversity of bacterial and fungal community in the paddy soil.The community composition and abundance of bacteria or fungi in the paddy soil were assessed at different growth stages of rice by denaturing gradient gel electrophoresis and real-time polymerase chain reaction based on 16S rRNA gene or SSU rRNA gene in the 4th year after the experimental establishment.The composition of bacterial or fungal community changed during rice growth,while no significant differences were observed between the fields cultivated with GM1and CK1,or between the fields cultivated with GM2 and CK2 in either bacterial or fungal community composition.The copy numbers of bacterial 16S rRNA gene in the soils with CK1,CK2,GM1 and GM2 ranged from 5.64×1011to 6.89×1011copies g-1dry soil at rice growth stages,and those of fungal SSU rRNA gene from 5.24×108to 8.68×108copies g-1dry soil.There were no marked differences in the copies of bacterial 16S rRNA gene or fungal SSU rRNA gene between CK1 and GM1 or between CK2 and GM2at any growth stage of rice.Planting cry1Ac/cpti transgenic rice had no significant effect on composition and abundance of bacterial and fungal community in paddy soil during the rice growing season at least in the short term.     

Soil microbial biomass and bacterial and fungal community structures responses to long-term fertilization in paddy soils

Purpose

Long-term fertilization can influence soil biological properties and relevant soil ecological processes with implications for sustainable agriculture. This study determined the effects of long-term (>25 years) no fertilizer (CK), chemical fertilizers (NPK) and NPK combined with rice straw residues (NPKS) on soil bacterial and fungal community structures and corresponding changes in soil quality.

Materials and methods

Soil samples were collected from a long-term field site in Wangcheng County established in 1981 in subtropical China between mid summer and early autumn of 2009. Terminal restriction fragment length polymorphism (T-RFLP) and the real-time quantitative polymerase chain reaction (real-time qPCR) of bacterial and fungal community and microbial biomass (MB-C, -N and -P) were analyzed.

Results and discussion

Redundancy analysis of the T-RFLP data indicated that fertilization management modified and selected microbial populations. Of the measured soil physiochemical properties, soil organic carbon was the most dominant factors influencing bacterial and fungal communities. The bacterial and fungal diversity and abundance all showed increasing trends over time (>25 years) coupling with the increasing in SOC, total N, available N, total P, and Olsen P in the fertilized soils. Compared to chemical fertilizer, NPKS resulted in the greater richness and biodiversity of the total microbial community, soil organic C, total N, MB-C, -N and -P. The high biodiversity of microbial populations in NPKS was a clear indication of good soil quality, and also indicated higher substrate use efficiency and better soil nutrient supplementation. Otherwise, unfertilized treatment may have a soil P limitation as indicated by the high soil microbial biomass N: P ratio.

Conclusion

Our results suggest that NPKS could be recommended as a method of increasing the sustainability of paddy soil ecosystems.     

Linking microbial community to soil water-stable aggregation during crop residue decomposition

The dynamics of soil water-stable aggregation (WSA) following organic matter (OM) addition are controlled by microbial activity, which in turn is influenced by carbon substrate quality and mineral N availability. However, the role of microbial communities in determining WSA at different stages of OM decomposition remains largely unknown. This study aimed at evaluating the role of microbial communities in WSA during OM decomposition as affected by mineral N. In a 35-day incubation experiment, we studied the decomposition of two high-C/N crop residues (miscanthus, C/N = 311.3; and wheat, C/N = 125.6) applied at 4 g C kg⁻¹ dry soil with or without mineral N addition (120 mg N kg⁻¹ dry soil). Microbial characteristics were measured at day 0, 7, and 35 of the experiment, and related to previous results of WSA. Early increase in WSA (at 7 days) was related to an overall increase of the microbial biomass (MBC) with wheat residues showing higher values in MBC and WSA than miscanthus. In the intermediate stage of decomposition (from day 7 to 35), the dynamics of WSA were more associated with the dynamics of microbial polysaccharides and greatly influenced by mineral N addition. Mineral N addition resulted in a decrease or leveling off of WSA whereas it increased in its absence. We suggest that opportunistic bacterial populations stimulated by N addition may have consumed binding agents which decreased WSA or prevented its increase. To the contrary, microbial polysaccharide production was high when no mineral N was added which led to the higher WSA in the late stage of decomposition in this treatment. The late stage of decomposition was associated with a particular fungal community also influenced by the mineral N treatment. We suggest that WSA dynamics in the late stage of decomposition can be considered as a « narrow process³ where the structure of the microbial community plays a greater role than during the initial stages.     

转Bt基因棉叶对土壤微生物多样性的影响

应用Biolog方法研究了转Bt基因棉粉碎叶腐解对土壤微生物群落结构功能多样性的影响。取腐解10d、25d、40d、55d、70d土样分析土壤微生物群落多样性指数及土壤微生物对聚合物、胺类、氨基酸、糖、羧酸和其他类碳源利用情况。结果表明:在腐解过程中,转Bt基因棉粉碎叶土壤微生物群落丰富度下降,群落多样性显著降低,而群落优势集中性明显提高;转Bt基因棉粉碎叶影响了土壤微生物群落对碳源的利用程度,表现为可显著增加对糖类、胺类和氨基酸类碳源的利用,初期显著降低对羧酸类碳源的利用,对聚合物类和其他类碳源的利用率无显著影响;主成分分析表明转Bt基因棉粉碎叶对土壤微生物群落原有结构功能影响具有持续性。     

Microbial community responsible for the decomposition of rice straw in a paddy field: estimation by phospholipid fatty acid analysis

To estimate the microbial communities responsible for rice straw decomposition in paddy field, phospholipid fatty acid (PLFA) composition of leaf sheaths and blades was analyzed during the decomposition of both residues under upland conditions after harvest and under flooded conditions at the time of transplanting of rice plants. In addition, rice straw that had been placed in the field under upland conditions (November to April) was taken out in spring, and placed again in the same field under flooded conditions at the time of transplanting. High proportions of the branched-chain PLFAs were observed under flooded conditions (June to September); the proportions of straight mono-unsaturated and straight poly-unsaturated PLFAs were high under upland conditions in the winter season for 4 months. The dominant PLFAs in straight mono-unsaturated, straight poly-unsaturated and branched-chain PLFA groups were 18:19, 18:17 and 16:17c, 18:26c and i15:0, i17:0 and ai15:0, respectively, under both upland and flooded conditions. These findings indicated the important roles of Gram-negative bacteria and fungi under upland conditions and of Gram-positive bacteria and anaerobic Gram-negative bacteria under flooded conditions. Cluster analysis of PLFA composition showed the difference of community structure of microbiota in rice straw between upland and flooded conditions. In addition principal component analysis revealed the difference between leaf sheaths and blades under upland conditions and indicated that the content of straight unsaturated PLFAs (sheaths > blades) characterized their community structures.     

Soil microbial community responses to sulfadiazine-contaminated manure in different soil microhabitats

Veterinary antibiotics such as sulfadiazine (SDZ) are applied with manure to agricultural soil. Antimicrobial effects of SDZ on soil microbial community structures and functions were reported for homogenized bulk soils. In contrast, field soil is structured. The resulting microhabitats are often hot spots that account for most of the microbial activity and contain strains of different antibiotic sensitivity or resilience. We therefore hypothesize that effects of SDZ are different in diverse soil microhabitats. We combined the results of laboratory and field experiments that evaluated the fate of SDZ and the response of the microbial community in rhizosphere, earthworm burrow, and soil macroaggregate microhabitats. Microbial communities were characterized by phenotypic phospholipid fatty acid (PLFA) and genotypic 16S rRNA gene patterns (DGGE) and other methods. Data was evaluated by principle component analyses followed by two-way ANOVA with post-hoc tests. Extractable SDZ concentrations in rhizosphere soil were not clearly different and varied by a factor 0.7–1.2 from those in bulk soil. In contrast to bulk soil, the extractable SDZ content was two-fold larger in earthworm burrows, which are characterized by a more hydrophobic organic matter along the burrow surface. Also, extractable SDZ was larger by up to factor 2.6 in the macroaggregate surface soil. The rhizosphere effect clearly increased the microbial biomass. Nonetheless, in the 10 mg SDZ kg⁻¹ treatment, the biomass deceased by about 20% to the level of uncontaminated bulk soil. SDZ contamination lowered the total PLFA concentrations by 14% in the rhizosphere and 3% in bulk soil of the field experiment. Structural shifts represented by Pseudomonas DGGE data were larger in SDZ-contaminated earthworm burrows compared to bulk soils. In the laboratory experiment, a functional shift was indicated by a four-fold reduced acid phosphatase activity in SDZ-contaminated burrows compared to bulk soil. Structural and functional shifts after SDZ contamination were larger by a factor of 2.5 in the soil macroaggregate surface versus interior, but this relation reversed over the long-term under field conditions. Overall, the combined effects of soil microhabitat, microbial community composition, and exposure to SDZ influenced the microbial susceptibility towards antibiotics under laboratory and field conditions.     

Detritivorous earthworms modify microbial community structure and accelerate plant residue decomposition

The impact of detritivorous earthworms (Eisenia andrei) on microbial community structure and function in grape marc, a lignocellulosic enriched plant residue, was investigated in a mesocosm experiment. Analysis of carbon and nitrogen pools was also carried out in order to evaluate how changes in microbial communities affect plant residue decomposition. The grape marc was completely processed after fifteen days as a result of the high density of earthworms present and the rapid gut transit time. Eisenia andrei had a large impact on the structure of the microbial community, as revealed by phospholipid fatty acid analysis. Earthworm activity reduced the abundance of both bacterial (except for Gram-negative bacteria) and fungal PLFA biomarkers relative to the control values. Decreases in microbial activity and in protease and cellulase activities were also attributable to the presence of earthworms. Moreover, earthworms strongly modified the substrate utilization patterns of microbial communities, as revealed by BIOLOG analysis. The presence of earthworms led to an increase in the utilization of some amino acids and polymers, which reached a higher substrate diversity value than that in the control mesocosm. The differences in microbial communities were accompanied by a reduction in the total C content and the labile C pool, relative to the control, although there were no significant differences in either cellulose or hemicellulose contents. However, total N content increased in both mesocosms – with and without earthworms – and the concentration of NH₄⁺ was also enhanced by earthworm activity. The results indicate that detritivorous earthworms play a key role in decomposing fresh plant residues in the short term via their intensive interactions with microbial communities.     

Biodegradation of Cry1Ab protein from Bt transgenic rice in aerobic and flooded paddy soils

Degradation of Cry1Ab protein from Bt transgenic rice was examined under both aerobic and flooded conditions in five paddy soils and in aqueous solutions. The hydrolysis rate of Cry1Ab protein in aqueous solutions was correlated inversely with the solution pH in the range of 4.0 to 8.0, and positively with the initial concentration of Cry1Ab protein. Rapid degradation of Cry1Ab protein occurred in paddy soils under aerobic conditions, with half-lives ranging from 19.6 to 41.3 d. The degradation was mostly biotic and not related to any specific soil property. Degradation of the Cry1Ab protein was significantly prolonged under flooded conditions compared with aerobic conditions, with half-lives extended to 45.9 to 141 d. These results suggest that the toxin protein, when introduced into a paddy field upon harvest, will probably undergo rapid removal after the field is drained and exposed to aerobic conditions.     

Soil microbial community responses to dairy manure or ammonium nitrate applications

Soil management practices that result in increased soil C also impact soil microbial biomass and community structure. In this study, the effects of dairy manure applications and inorganic N fertilizer on microbial biomass and microbial community composition were determined. Treatments examined were a control with no nutrient additions (CT), ammonium nitrate at 218 kg N ha⁻¹ (AN), and manure N rates of 252 kg manure-N ha⁻¹ (LM) and 504 kg manure-N ha⁻¹ (HM). All plots were no-till cropped to silage corn (Zea mays, L. Merr) followed by a Crimson clover (Trifolium incarnatum, L.)/annual ryegrass (Lolium multiflorum, Lam.) winter cover crop. Treatments were applied yearly, with two-thirds of the N applied in late April or early May, and the remainder applied in September. Soil samples (0–5, 5–10, and 10–15 cm) were taken in March 1996, prior to the spring nutrient application. Polar lipid fatty acid (PLFA) analysis was used to assess changes in microbial biomass and community structure. Significantly greater soil C, N and microbial biomass in the 0–5 cm depth were observed under both manure treatments than in the CT and AN treatments. There was also a definable shift in the microbial community composition of the surface soils (0–5cm). Typical Gram-negative bacteria PLFA biomarkers were 15 and 27% higher in the LM and HM treatments than in the control. The AN treatment resulted in a 15% decrease in these PLFA compared with the control. Factor analysis of the polar lipid fatty acid profiles from all treatments revealed that the two manure amendments were correlated and could be described by a single factor comprised of typical Gram-negative bacterial biomarkers. The AN treatments from all three depths were also correlated and were described by a second factor comprised of typical Gram-positive bacterial biomarkers. These results demonstrate that soil management practices, such as manuring, that result in accumulations of organic carbon will result in increased microbial biomass and changes in community structure.     

转基因水稻秸秆还田对土壤硝化反硝化微生物群落的影响

转基因作物可能通过根系分泌物和植株残体组成的改变及外源基因的转移释放令土壤微生物群落产生变化,影响土壤微生物的生态功能。氨氧化细菌和反硝化细菌是驱动土壤硝化和反硝化过程的关键微生物,其群落结构的变化直接关系土壤氮素的转化与利用。本研究利用荧光定量PCR和PCR-DGGE技术分析了转cry1Ac/cpti双价抗虫基因水稻‘Kf8’秸秆还田降解过程中,土壤氨氧化细菌和反硝化细菌群落丰度与组成的变化,探讨转基因水稻是否存在影响稻田土壤氮素转化与N2O排放的可能。结果显示:无论是氨氧化细菌amo A基因还是反硝化细菌nirS基因,其丰度在转基因水稻‘Kf8’与非转基因水稻‘Mh86’的秸秆还田土壤中都没有显著差异;转基因水稻‘Kf8’和非转基因水稻‘Mh86’秸秆还田降解过程中0~10 cm土层中的amo A基因丰度均显著高于10~20 cm及20~30 cm土层(P0.05);各深度土层中的nirS基因丰度均存在随秸秆还田时间延长而增加的趋势。水稻秸秆还田降解过程中,转基因水稻‘Kf8’的土壤氨氧化细菌和反硝化细菌的群落多样性指数及组成,均与非转基因水稻‘Mh86’没有显著差异。相关分析结果表明土壤氨氧化细菌和反硝化细菌群落组成均与水稻秸秆还田时间存在显著相关性(P=0.002),反硝化细菌群落组成还与土层深度显著相关(P=0.024)。本研究表明转cry1Ac/cpti抗虫基因水稻秸秆还田对稻田土壤硝化和反硝化关键微生物群落不会产生明显影响。就土壤微生物群落而言,转cry1Ac/cpti抗虫基因水稻秸秆还田不存在影响土壤氮素转化与N2O排放的可能。     

Decomposition of Bacillus thuringiensis (Bt) transgenic rice residues (straw and roots) in paddy fields

Background, aim, and scope  

Genetic modification of commercial crops may affect their decomposition and nutrient cycling processes in agricultural ecosystems. Intensive rice cultivation under partially submerged conditions (paddy rice) is an important and widespread cropping system, particularly in the tropics, yet there is little data on the decomposition of Bt rice residue under field conditions. We investigated straw and root decomposition of rice modified to express the Cry1Ab protein of Bacillus thuringiensis (Bt) to kill lepidopteran pests, compared with a parental non-Bt isoline. The objective of this study was to assess the possible impacts of cry gene transformation of rice on residue decomposition under intensive rice cultivation with long period of submergence.     

植物残体分解过程中微生物群落变化影响因素研究进展

植物残体是土壤有机质的重要来源,研究分解植物残体的微生物群落结构及其演替规律日益受到重视。本文综述了影响植物残体分解过程中微生物群落结构和功能变化的3个主要因素:植物残体的性质、土壤和气候环境因素、农艺措施,这些因素通过影响微生物本身的活性和植物残体分解过程中化学组成的变化从而导致微生物群落的变化,同时植物残体腐解过程中微生物群落存在明显的演替现象。以上因素的影响并不是孤立的,而是相互联系和制约的。未来针对野外田间条件下植物残体的分解过程,仍需深入研究关键微生物群落的演替规律以及不同影响因素的交互作用机制。     

Eukaryotic communities associated with the decomposition of rice straw compost in a Japanese rice paddy field estimated by DGGE analysis

To estimate the succession and phylogenetic composition of the eukaryotic communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analysis targeting 18S rDNA followed by sequencing was conducted in a Japanese paddy field. The eukaryotic communities in rice straw compost incorporated into the flooded paddy field were influenced by the mid-season drainage and mainly composed of fungi (Ascomycota, Zygomycota, and Chytridiomycota) and protozoa (Ciliophora, Euglyphida, and Dactylopodida), most of which existed continuously during the cultivation period of paddy rice. The results indicated that these eukaryotic members were associated with the decomposition of rice straw compost in paddy field soil directly or indirectly.     

The microbial community composition changes rapidly in the early stages of decomposition of wheat residue

It is generally accepted that during the early stages of residue decomposition, easily available compounds are decomposed, leading to a relative increase in more recalcitrant compounds in the later stages of decomposition and that these changes in substrate availability are associated with changes in microbial community composition. However most studies on residue decomposition are conducted over several weeks or months; little is known about the changes in microbial community composition in the first weeks of decomposition. To address this knowledge gap, we incubated wheat residues inoculated with a microbial suspension in mesh bags buried in sand for 30 days, with sampling on days 0, 2, 4, 6, 8, 10, 15, 20, 25 and 30. Of the C added with the residues, 10, 18 and 25% had been respired on days 10, 20 and 30, respectively. The sum of PLFAs (phospholipid fatty acids), as an indicator of microbial biomass, increased strongly in the first 4 days and then decreased. The concentration of bacterial fatty acids was maximal on days 2 and 4, whereas the concentration of fungal fatty acids peaked on day 15. Microbial community composition (based on PLFA patterns) changed rapidly, with significant changes in the first 8 days and from day 8 to day 20. There were no significant changes in microbial community composition after day 20. The concentration of water-soluble C decreased strongly in the first 8 days, suggesting that the rapid changes in microbial community in this period are related to the changes in water-soluble C. Residue C chemistry, assessed by ¹³C NMR spectroscopy, changed little during the incubation period. This study showed that microbial community composition in decomposing residues changes rapidly in the first 1-2 weeks, which is, at least partly, the result of competition for the easily available compounds in the water-soluble fraction. After depletion of the water-soluble compounds, the microbial community composition changes more slowly.     

转CryIAb水稻对稻田杂草群落组成及多样性的影响

尽管基因工程技术可以增加作物产量, 但转基因作物是否对农田生态产生影响受到广泛关注。本研究通过田间定位试验, 应用群落生态学方法研究了转CryIAb基因抗虫水稻"Mfb"连续2年在传统栽培和半野生条件下对稻田杂草群落组成及多样性的影响。调查结果显示: 转CryIAb基因稻"Mfb"与非转基因稻"明恢86"田间杂草种类没有显著差异。稻田杂草的频度和密度与栽培方式有关, 半野生稻田杂草的频度和密度显著高于传统稻田, 但相同栽培条件下, 转基因稻"Mfb"与非转基因稻"明恢86"田间杂草频度和密度在整个生长期内均无显著差异。半野生稻田物种丰富度(Sr)指数明显大于传统稻田; 相同栽培条件下, 相同生长时期抗虫转基因水稻"Mfb"与其非转基因对照"明恢86"对稻田杂草群落丰富度的影响差异不显著。稻田杂草群落优势度(D)、均匀度(J)以及多样性(H)各处理、各生长时期内转基因稻与非转基因稻相比均没有显著差异。稻田杂草Shannon-Wiener多样性指数变化无明显规律, 相同栽培方式相同生长期的抗虫转基因水稻"Mfb"与其非转基因对照"明恢86"的Shannon-wiener指数差异不显著。综合上述分析, 转CryIAb基因抗虫稻对稻田杂草群落的组成及多样性没有显著影响。     

Comparison of transgenic Bt rice and its non‐Bt counterpart in adaptability to nitrogen deficiency

Resistances of newly bred Bacillus thuringiensis (Bt) crops have been dramatically improved because of the effective and high expression of Bt protein in the plant. However, poorer adaptabilities to environmental stresses were observed in some Bt crops compared to their non‐Bt counterparts. The biological reasons for the poorer adaptabilities were still unclear. A nitrogen (N) deficiency experiment was conducted to investigate variations in growth and physiology characteristics of a newly bred Bt rice [Oryza sativa L. line MH63 (Cry2A*)] compared to its non‐Bt counterpart MH63. MH63 (Cry2A*) showed lower grain yields and lower biomass under low N levels compared to MH63. Earlier leaf senescence associated with disorder in protein metabolism was observed in MH63 (Cry2A*) when the N concentration was lower than 13.50 mg g^?1 in MH63 (Cry2A*) leaves and the ratio of Bt protein to soluble protein (BT : SP) was higher than 2203 μg g^?1 in MH63 (Cry2A*) leaves. The lower grain yield, the lower biomass and the earlier leaf senescence associated with disorder in protein metabolism in MH63 (Cry2A*) were correlated to the high BT : SP in MH63 (Cry2A*) leaves. The results suggest that MH63 (Cry2A*) has a poorer adaptability to N deficiency compared to its non‐Bt counterpart MH63. This poorer adaptability might be related to the high Bt protein expression in MH63 (Cry2A*).     

Community structure of bacteria and fungi responsible for rice straw decomposition in a paddy field estimated by PCR-RFLP analysis

Rice straw including leaf sheaths and blades put in nylon mesh bags was placed in the plow layer of a Japanese paddy field after harvest under upland conditions and after transplanting of rice seedlings under flooded conditions. In addition, rice straw that was decomposed under the upland conditions during the off-crop season in winter was placed again in soil at the time of transplanting. The materials were collected periodically to analyze the community structure of the bacteria and fungi responsible for rice straw decomposition by PCR-RFLP analysis. The PCR products with 27f and 1492r primers designed for bacterial 16S rDNA and with EF3 and EF4 primers designed for fungal 18S rDNA were digested with four restriction endonucleases (Hinf I, Sau3A I, Hae III, EeoR I). Bacterial communities in the decomposing rice straw were different from each other between upland and flooded conditions, between leaf sheaths and blades, and between straw samples with and without decomposition under upland conditions during the off-crop season. Fungal communities in the decomposing rice straw were also different between the leaf sheaths and blades under upland soil conditions. Score plots of bacterial and fungal communities in the principal component analysis were separated from the plot of the straw materials along with the duration of the placement, indicating the succession of bacterial and fungal communities in decomposing rice straw with time.     

稻田甲烷传输的研究进展

稻田生态系统中CH4 排放是由土壤中CH4 产生、氧化和向大气传输这3个过程相互作用的结果,CH4传输主要通过液相扩散、冒气泡和植株传输3种方式。这3种途径的相对强弱取决于水稻植株通气组织的完善与否以及水稻品种、种植密度和温度的季节变化等。但大量研究表明,稻田土壤中产生的CH4 绝大部分通过植株通气组织排放到大气中。在应用稳定性碳同位素方法研究水稻植株向大气传输CH4的过程时发现,在传输过程也会发生同位素分馏,据现有文献报道,CH4传输的同位素分馏系数 e传输 有两种计算方法,获得的结果也比较接近,为 -18‰ ~ -9‰。但研究方法还存在一些缺陷,可能对结果的准确性产生影响。此外有关稻田CH4传输在模型的建立方面还比较缺乏。