不同供氮条件下水稻的化感抑草作用与资源竞争分析

为探讨化感水稻在不同氮素水平下的生物干扰现象,提出了一种新的测试方法———化感竞争分离法(allelopathyandcompetitionseparation basedbioassay,ACS),成功地区分了在稻/稗共生系统中水稻化感作用与资源竞争及其在不同N条件下的反应特性.结果表明,水稻PI312777具有较强的生物干扰能力.在不同N水平下,其资源竞争能力较强且表现稳定,但化感作用潜力则随供N水平的下降而明显增强.水稻Lemont不具化感作用潜力,在供N水平正常或充足条件下,其资源竞争能力减弱,但在N胁迫下却明显增强.这是在环境资源贫乏时,稻/稗共生系统中生态位竞争加剧的结果.     

Genomic analysis of allelopathic response to low nitrogen and barnyardgrass competition in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To explore the molecular mechanism of allelopathic rice in response to low nitrogen (N) supply or accompanying weed stress, allelopathic rice PI 312777 and its counterpart Lemont were grown under low N supply or co-cultured with barnyardgrass [Echinochloa crus-galli (L.) Beauv.] in hydroponics. The suppression subtractive hybridization (SSH) technique was employed to isolate the up-regulated genes in the treated rice accession. The results indicated that the expression of the genes associated with N utilization was significantly up-regulated in allelopathic rice PI 312777, and the higher efficiency of N uptake and its utilization were also detected in PI 312777 than that in Lemont when the two rice accessions were exposed to low N supply. This result suggested that the allelopathic rice had higher ability to adapt to low N stress than its non-allelopathic counterpart. However, a different response was observed when the allelopathic rice was exposed to accompanying weed (barnyardgrass) co-cultured in full Hoagland solution (normal N supply). It showed that the expression of the genes associated with allelochemical synthesis and its detoxification were all up-regulated in the allelopathic rice when co-cultured with the target weed under normal N supply. The results suggested that the allelopathic rice should be a better competitor in the rice-weed co-culture system, which could be attributed to increasing de novo biosynthesis and detoxification of allelochemicals in rice, consequently resulting in enhanced allelopathic effect on the target and preventing the autotoxicity in this process. These findings suggested that the accompanying weed, barnyardgrass is not only the stressful factor, but also one of the triggers in activating allelopathy in rice. This implies that the allelopathic rice is sensible of the existing target in chemical communication.     

低钾胁迫对水稻(Oryza sativa L.)化感潜力变化的影响

研究以国际公认的化感水稻P1312777和非化感水稻Lemont为供体,稗草（Echinochloa cru-galli L.）为受体,采用稻／稗共培体系,研究低钾胁迫对水稻化感潜力变化的影响及其机制。受体稗草的形态指标分析结果表明,低钾胁迫促使化感水稻P1312777对共培稗草的根长、株高和干重的抑制率均升高,增幅远大于非化感水稻Lemont。受体稗草生理生化指标分析结果表明,低钾胁迫下化感与非化感水稻对受体稗草保护酶系（SOD、POD、CAT）及根系活力的抑制作用增强,但化感水稻P1312777比非化感水稻Lemont的抑制程度大,且达极显著差异。实时荧光定量PCR分析结果表明,低钾胁迫下,化感水稻P1312777根部与叶部中酚类代谢的关键酶——苯丙氨酸解氨酶、肉桂酸-4-羟化酶、羟化酶、O-甲基转移酶的基因均上调表达,而非化感水稻根部相应酶均下调表达,叶部除苯丙氨酸解氨酶上调,其余酶也下调表达。而萜类代谢途径关键酶——HMG—CoA还原酶、角鲨烯合酶、单萜烯环化酶、倍半萜烯环化酶、二萜烯环化酶的基因,在两种水稻根部中呈现出相同或相似的表达方式（上调或下调）,即HMG—CoA还原酶上调表达,角鲨烯合酶、单萜烯环化酶、倍半萜烯环化酶、二萜烯环化酶下调表达;而在水稻叶部,非化感水稻Lmont相应酶基因表达方式仍然不变,化感水稻P1312777除了角鲨烯合酶下调表达,其余4个酶均上调表达。水稻根系分泌物中酚类物质的HPLC分析结果表明,低钾胁迫下,化感水稻P1312777根系分泌物中,所检出的酚酸类物质总量是正常营养条件下的2．30倍,而非化感水稻Lemont则是正常营养条件下的0．91倍。综合分析认为低钾胁迫下,化感水稻P1312777抑草能力增强主要是由于酚类代谢途径关键酶基因表达上调,导致酚类代谢途径旺盛,分泌出更多的酚类物质,进而破坏受体稗草保护酶系统,抑制了稗草的正常生长。     

Analysis of gene expressions associated with increased allelopathy in rice (Oryza sativa L.) induced by exogenous salicylic acid

The defense characteristics of allelopathic rice accession PI312777 and its counterpart Lemont induced by exogenous salicylic acid (SA) to suppress troublesome weed barnyardgrass (BYG) were investigated using the methods of suppression subtractive hybridization (SSH) and real-time fluorescence quantitative PCR (qRT-PCR). The results showed that exogenous SA could induce the allelopathic effect of rice on BYG and this inducible defense was SA dose-respondent and treatment time-dependent. PI312777 exhibited higher inhibitory effect than Lemont on BYG after treated with different concentrations of SA. The activities of cell protective enzymes including SOD, POD and CAT in the BYG plants co-cultured with PI312777 treated by SA were highly depressed compared with the control (co-cultured with rice without SA-treatment). Similar but lower depression on these enzymes except for CAT was also observed in the BYG plants when co-cultured with Lemont treated by SA. It is therefore suggested that allelopathic rice should be more sensitive than non-allelopathic rice to exogenous SA. Seventeen genes induced by SA were obtained by SSH analysis from PI312777. These genes encode receptor-kinase proteins, ubiquitin carrier proteins, proteins related to phenylpropanoid metabolism, antioxidant related proteins and some growth-mediating proteins. The differential expressions of these genes were validated in part by qRT-PCR in the two rice accessions. Our work elucidated that allelopathic rice possesses an active chemical defense and auto-detoxifying enzyme system such as the up-regulated enzymes involved in de novo biosynthesis of phenolic allelochemicals and the glutathione-S-transferase (GST) associated with xenobiotic detoxification.     

不同胁迫条件下化感与非化感水稻PAL多基因家族的差异表达

水稻苯丙氨酸解氨酶(PAL)调控酚酸类化感物质的合成代谢。编码PAL的基因是一个基因家族,包含至少11个基因成员,并受不同环境条件的调控。为了明确PAL基因家族中调控水稻化感作用的特定基因成员,本研究运用实时荧光定量PCR技术分析了低氮及稗草胁迫条件下强化感水稻PI312777与非化感水稻Lemont中根系的11个PAL成员基因的表达差异。结果表明,低氮和稗草胁迫条件下,PI312777和Lemont中的 PAL4和PAL10均不表达,其余9个PAL基因成员发生了不同程度的表达变化。其中,PAL11均上调表达,其分别在低氮处理和稗草胁迫的PI312777中上调3.29倍和1.07倍,而在相同处理下的Lemont中上调3.92倍和1.08倍;PAL3和PAL9则仅在低氮和稗草胁迫条件下的PI312777中上调表达,低氮胁迫分别为1.83倍和2.66倍,稗草胁迫为1.46倍和2.65倍;而这两个基因在相同处理下的Lemont中表达下调,低氮胁迫下调1.05和1.24倍,稗草胁迫下调1.14和1.16倍,推测PAL3和PAL9可能与胁迫初期调控水稻化感作用有关。     

不同化感潜力水稻(Oryza sativa L.)苗期的氮素养分效率及相关基因的表达

为了阐明养分水平引起水稻(Oryza sativa L.)化感抑草潜力变化的生理生态机制,研究了不同N素营养处理下,不同化感潜力水稻苗期对N素营养逆境的响应特性及N素养分效率的差异,并运用实时荧光定量RT-PCR技术(FQ-PCR)检测与N素代谢和次生代谢关键酶的基因表达.结果表明:弱化感水稻品种Lemont对N素营养胁迫较敏感,强化感水稻品种PI312777对资源波动的适应性较强,N素养分效率较高.FQ-PCR分析结果显示,在低N条件下Lemont中的亚硝酸还原酶基因(nir),谷氨酰胺合成酶基因(gs)相对表达量均有不同幅度的下调, PI312777分别下调了1.2倍和1.4倍,而Lemont分别下调了3.0倍和1.8倍, Lemont下调的幅度分别是PI312777的2.5倍和1.3倍,但对于苯丙氨酸解氨酶基因(pal)与3-羟基-3甲基戊二酰辅酶A还原酶基因(hmgr)而言, PI312777叶组织中的pal和hmgr均上调表达,与对照相比上调了6.0倍和1.6倍,而Lemont中对应的基因均下调表达,分别下调了1.3倍和6.8倍,佐证了上述差异的分子生态学特性.     

Identification and comparative analysis of microRNAs in barnyardgrass (Echinochloa crus‐galli) in response to rice allelopathy

Rice allelopathy is a hot topic in the field of allelopathy, and behaviour of donor allelopathic rice has been well documented. However, few study addresses response of receiver barnyardgrass (BYG). We found that expression of miRNAs relevant to plant hormone signal transduction, nucleotide excision repair and the peroxisome proliferator‐activated receptor and p53 signalling pathways was enhanced in BYG co‐cultured with the allelopathic rice cultivar PI312777, the expression levels of these miRNAs in BYG plants were positively correlated with allelopathic potential of the co‐cultured rice varieties. Treatment of BYG plants with rice‐produced phenolic acids also increased miRNA expression in BYG, while treatment with rice‐produced terpenoids had no obvious effect on miRNA expression. In the hydroponic system, the largest number of Myxococcus sp. was found in the growth medium containing rice with the highest allelopathic potential. The addition of phenolic acids in the hydroponic medium also increased the number of Myxococcus sp. More interestingly, inoculation with Myxococcus xanthus significantly increased miRNA expression in the treated BYG. Jointed treatments of ferulic acid and M. xanthus led to strongest growth inhibition of BYG. The results suggest that there exist involvement of Myxococcus sp. and mediation of miRNA expression in rice allelopathy against BYG.     

不同水层和密度条件下化感作用水稻对无芒稗的干扰控制作用

不同水层下水稻对无芒稗的干扰控制作用研究表明,水稻化感作用品种吓一跳、谷梅2号和中156对无芒稗的株高抑制作用比无化感作用品种秀水63和春江11呈显著性差异．随盆栽水稻密度增加,水稻对无芒稗的干扰控制作用加大．在每盆4株栽植密度下,水稻化感作用品种鸡早籼、谷梅2号显著抑制无芒稗植株株高;在每盆32株密度时,水稻化感作用品种吓一跳、PI312777、TN1等对受控无芒稗的干扰控制与无化感作用品种春江11相比,呈显著性水平．     

水稻品种化感潜力的双重评价与筛选

以初筛获得的7份水稻品种为材料,用特征次生物质标记法在HPLC上测定化感指数AI值并结合田间小区试验进行双重评价与筛选,得到3份既具高化感指数又具较高田间抗草活性的水稻化感品种(系),它们是:我国台湾品种I-Kung-Pao、Parahainakoru和大陆品种HB-1。它们对无芒稗根长抑制率分别达57%、64%、55%,均超过50%;其化感指数分别是0.61、0.56、0.59,均与美国化感潜力品种PI312777的化感指数0.59相近;结果表明,水稻品种I-Kung-Pao、Parahainakoru和HB-1是我国宝贵的化感品种资源。     

外源水杨酸调控水稻化感抑草作用及其分子生理特性

为了探讨外源水杨酸（SA）调控水稻化感抑草效应的可行性,研究了不同浓度的外源SA对强化感水稻PI312777抑草效应的影响及其生理生化特性,并运用实时定量RT-PCR(FQ-PCR)技术检测SA介导的关键酶苯丙氨酸解氨酶基因（ZB8）的相对表达量.结果表明：外源SA能够诱导水稻化感抑草效应增强,而且这种诱导效应与SA的浓度和处理时间相关.叶面喷施SA后,PI312777对稗草的抑制率显著提高,其根系活力、超氧化物歧化酶（SOD）和过氧化物酶（POD）活性增强,过氧化氢酶（CAT）活性降低;而伴生杂草稗草的相应生理指标的变化趋势则相反.PI312777植株中的苯丙氨酸解氨酶（PAL）活性增强,总酚含量升高.0.2 mmol·L^-1的SA诱导水稻化感抑草效应最显著,该浓度下目的基因ZB8的相对表达丰度随处理时间先上调后下调,在24 h达到表达高峰.     

Response and relation of allantoin production in different rice cultivars to competing barnyardgrass

The production of secondary metabolites and biomass of plants may be altered by coexistence and competition. Rice has coexisted with barnyardgrass in paddy systems for centuries and represents a model system for understanding plant–plant interactions. Despite increasing knowledge of allelochemicals produced and released by rice involved in coexistence of these two species, relatively little is known about the role and production of beneficial chemicals of rice in response to competing barnyardgrass. The purpose of this study was to quantify the growth-stimulating allantoin (5-ureidohydantoin) and its effect on barnyardgrass growth in a rice–barnyardgrass system. In this system, rice produced and released allantoin but barnyardgrass did not. The production of allantoin by rice responded to competition from barnyardgrass, and its concentration varied among rice cultivars tested. At 1:1 barnyardgrass and rice mixture proportion, allantoin concentration in allelopathic cultivars was obviously lowered by competing barnyardgrass, while no significant variation of allantoin concentration was observed in non-allelopathic cultivars. Barnyardgrass biomass was reduced in mixed-culture with rice cultivars, in particular, allelopathic cultivars. There was a positive relationship between allantoin concentration in rice cultivars and barnyardgrass biomass. Furthermore, allantoin stimulated the growth of barnyardgrass once released from rice or added to soil. The results suggest that rice plants of allelopathic cultivars appear to be able to detect the presence of competing barnyardgrass and respond by decreasing production of growth-stimulating allantoin, regulating the growth of barnyardgrass. In this manner, given rice cultivars may alleviate the competition of barnyardgrass and provide greater benefit to their own growth.     

水稻化感生物测试方法的比较及应用

选用8个化感作用潜力各异的水稻品种(系)：PI312777、Lemont、Moroberekan、IAC25、IACA7、IAC120、Batatais、Iguape Cateto为供试材料,以田间伴生稗草为受体材料,比较分析了目前较为通用的3种生物测试方法：琼脂迟播共培法(RSA)、石英砂迟播共培法(RSS)和根系分泌物培养法(SRE)对水稻化感潜力的测试效果．结果表明,RSA法应用于测试水稻化感作用潜力的效果最好,RSS法和SRE法的测试结果虽较为一致,但两者都存在不足．据此,运用RSA法对57个水稻材料进行了化感抗草种质资源的初步筛选,从中获得5个强化感作用潜力的水稻材料：Iguape Cateto、PI312777、Azucena、Taichung Native 1和IAC25．     

Impact of allelopathic rice seedlings on rhizospheric microbial populations and their functional diversity

Field performance of rice allelopathic potential is indirectly regulated by the microflora in the rhizosphere. The present study aimed to investigate the dynamics of microbial populations and their functional diversities in the seedling rhizospheres of rice cultivars with varied allelopathic activities by employing agar plate bioassay, fumigation and BIOLOG analysis. Rice cultivars significantly affected the microbial carbon content in their associated rhizospheric soil. The microbial carbon contents were ranked in a decreasing order as Iguape Cateto (441.0 mg·kg^–1) > IAC47 (389.7 mg·kg^–1) > PI312777 (333.2 mg·kg^–1) > Lemont (283.8 mg·kg^–1) with the nil-rice control soil of 129.3 mg·kg^–1. Similarly, the respiration rate of the soils was 1.404, 1.019, 0.671 and 0.488 μgC·g^–1· h^–1 for PI312777, Iguape Cateto, IAC47 and Lemont, respectively. The respiration rate was only 0.304 μ gC·g^–1·h^–1 for the control soil. The microbial flora in the rhizospheric soil of different rice cultivars was dominated by bacteria (58.4%–65.6%), followed by actinomycete (32.2%–39.4%) and fungi (2.2%–2.8%). BIOLOG analysis showed that the value of Average Well Color Development (AWCD) differed significantly among rice cultivars. It was always the highest in the rhizospheric soil of the strongly allelopathic rice cv. PI312777, and the lowest in the rhizospheric soil of the poorly allelopathic rice cv. Lemont. The AWCD value reached the maximum in all the sampled soils after 144 hours of incubation. The AWCD values from the rhizospheric soils of PI312777, IAC47, Iguape Cateto and Lemont were 1.89, 1.79, 1.60 and 1.43 times higher than that of the control soil. Principal Component Analysis (PCA) identified 3 principal component factors (PCF) in relation to carbon sources, accounting for 70.1%, 11.3% and 7.0% of the variation, respectively. 19 categories of carbon sources were significantly positively correlated to the 3 principal components. Phenolic acids, carbohydrates, amino acids and amides were significantly correlated to the principal component 1, phenolic acids, carbohydrates and fatty acids to the principal component 2, and carbohydrates and hydroxylic acids to the principal component 3. Amino acids and amides were the two main carbon sources separating the 3 principal component factors. In addition, the total microbial population in the rhizospheric soil was significantly positively correlated with AWCD, microbial biomass carbon, microbial respiration and Shannon index. There was a significantly positive correlation between the total microbial population and the inhibition rate (IR) on the root length of lettuce owing to the different allelopathic activities of the rice cultivars. These results suggest that changes in microbial population, activity and functional diversity in the rhizospheres are highly cultivar-dependent. These changes might play an important role in governing the rice allelopathic activity in the field.     

不同化感水稻品种根系分泌物中萜类化合物的差异分析

以强化感水稻品种PI312777和弱化感水稻品种Lemont为供试材料,运用“循环法”收集供试水稻经土壤和石英沙介质后的根系分泌物,并经乙醚萃取,所获萃取物经气相色谱 质谱联用仪（GC-MS）测试,检索谱图库确定化合物.结果表明,乙醚萃取物中检测到含氧萜类化合物,如柠檬烯氧化物、柠檬烯二氧化物、香芹酮氧化物、香芹醇、雪松醇等,不同品种间含氧萜类化合物种类相似,但在不同介质中的存留方式和数量存在差异.文中还就不同培养方式和品种间萜类化合物的差异作了进一步讨论.     

不同氮素供应下水稻酚类物质代谢关键酶基因差异表达

运用实时荧光定量PCR技术探讨了不同供氮条件下强化感与弱化感水稻苯丙烷代谢途径中9个关键酶基因的表达差异。结果表明,与正常氮素供应相比,低氮胁迫引起强化感水稻‘P1312777’中与酚类代谢途径相关的9个关键酶基因表达量均上调,表达量增幅在1.9～5.4倍之间,且以PAL基因上调倍数最大。而弱化感水稻‘Lemont’则相反,只有2个基因(苯丙氨酸裂解酶基因和肉桂酰CoA基因)表达上调,但上调倍数分别是强化感水稻对应的基因的22%和74%,其余的7个基因表达均下调,降幅在29%～72%之间,表明低氮胁迫诱发的水稻化感抑草能力增强与其体内酚类物质合成代谢增强有关。     

化感水稻根际微生物类群及酶活性变化

以化感水稻PI312777（PI）和非化感水稻Lemont（LE）为材料,分别测定不同水稻叶龄期（3～7叶期）根际微生物区系变化及根际土壤酶活性.结果表明,化感水稻明显影响土壤根际微生物类群及相关酶活性.化感水稻PI根际细菌、放线菌、固氮菌的数量高于非化感水稻LE,增幅分别在11.2%～28.3%、40%～78.6%和111.5%～173.9%之间,而真菌数量低于非化感水稻LE,最高仅为其值的25.5％,说明化感水稻PI对绝大多数细菌、放线菌、固氮菌生长有促进作用,对一些真菌生长有抑制作用.进一步分析表明,化感水稻PI对氨化细菌、亚硝酸细菌、硝酸细菌、好气性固氮菌、好气性纤维素分解菌、硫化细菌的生长具有促进作用,其中以氨化细菌、好气性固氮菌的更为明显,最低增幅分别为53.7％和57.6％;而对反硫化细菌、反硝化细菌生长有抑制作用,其值最高分别为非化感水稻的54.2％和50.6％.此外,化感水稻PI根系分泌物对脲酶、磷酸酶、蔗糖酶的活性具有促进作用,而对过氧化氢酶则呈抑制作用.     

相同遗传背景不同植株形态水稻等基因系与杂草的竞争及化感作用

利用相同遗传背景不同株形水稻浙辐802等基因系为材料,研究了IG1、IG4、IG25和IG26与无芒稗的竞争和化感作用潜力,探索了二者之间的相互关系.结果表明,IG1和IG4的植株高度和分蘖角度显著大于IG25和IG26,但它们的根系活力却显著小于IG25和IG26.IG1和IG4对无芒稗生物量干重抑制率分别达到15%和17%,显著高于IG26的64%,表明水稻与无芒稗竞争力与水稻植株高度和分蘖角度成正比,而与水稻根系活力成反比.IG1叶片水浸提液对无芒稗根长抑制率为77.6%,显著高于非化感对照品种秀水63,而植株形态相近的IG4的化感作用却未达到显著水平,显示在相同遗传背景下水稻化感作用强弱与水稻株型无相关性.     

低磷营养胁迫下水稻化感抑草潜力的变化特性及其作用机理

在低磷(0.5 mg·L^-1) 营养胁迫下,运用生理生化方法分析了化感水稻PI312777（PI）与非化感水稻Lemont (Le) 对稗草抑制作用潜力的变化特性及其内在机理.结果表明,在低磷营养胁迫下,化感水稻品种PI对受体稗草根干重的抑制能力明显提高,在处理后的5 、10和15 d,其对稗草地下部干重的抑制率分别增加了5.64%、3.89%和12.13%,增加幅度比非化感水稻品种Le显著.生理生化分析结果表明,与正常营养条件相比,用低磷营养下生长的化感水稻PI的根系分泌物处理稗草5、10和15 d,受体稗草叶片中POD活性的促进率分别提高了20.19%、15.47%和6.68%,吲哚乙酸氧化酶活性的促进率分别提高了18.08%、17.71%和12.50%,硝酸还原酶活性的抑制率分别增加了13.89%、18.60%和2.10%. 在低磷营养胁迫下,化感水稻通过抑制受体植物的硝酸还原酶活性,影响其对氮营养的吸收,同时显著提高了吲哚乙酸氧化酶活性,减缓了受体稗草的生长速度,提高了其抑草作用潜力.     

水稻对稗草的化感作用研究

用差时播种共培法的改进方法对138份稻种资源进行了化感作用抑稗评价。结果表明,江西的青困二号、江苏的吓一跳、安徽的鸡早籼、江西的赣早籼37、陕西的商糯1号、IRRI的1R68465-2-3-2、韩国的水原92对稗草表现出较强的化感抑制作用。盆栽研究表明,谷梅2号、中156对稗草的抑制作用显著大于对照,而化感作用材料TN1对稗草的抑制作用与无化感作用材料秀水63无显著差异,中156的抑制作用强与其株高与秀水63有显著差异有关,而谷梅2号对稗草的抑制作用强主要在于其本身具有化感作用特性。     

水稻和稗草共生土壤微生物生物量碳及酶活性的变化

以稻田稗草、化感水稻PI312777和普通水稻辽粳9为试材,研究了田间稗草和水稻1∶1共生条件下,土壤微生物生物量碳及脱氢酶、脲酶和转化酶活性的变化.结果表明：在稗草 的干扰下,化感水稻PI312777根区土壤微生物生物量碳含量比单作减少了 50.52％（P<0.01）,而行间土壤微生物生物量碳含量增加;普通水稻辽粳9根区土壤 微生物生物量碳含量比单作减少了38.99％（P<0.01）,但其行间土壤微生物生物量碳含量无明显变化.两个水稻品种根区土壤脱氢酶活性均被显著抑制（P<0.05）,下降率都在20％以上;PI312777根区土壤脲酶和转化酶活性均被显著促进（P<0.01）;而辽粳9根区土壤转化酶活性也被显著抑制（P<0.01）,但脲酶活性无明显变化.化感水稻根区土壤微生物生物量碳含量的显著减少及脲酶、转化酶活性的增加是其化感特性的表现,表明土壤微生物和酶均参与了水稻和稗草的种间作用,化感水稻具有抗稗草干扰的明显优势.