应用克隆文库研究玉米根系AMF多样性方法的建立

以玉米根系为例,建立植物根系丛枝菌根18SrRNA基因克隆文库构建的方法。CTAB法提取的丛枝菌根真菌总DNA能够满足后续PCR扩增的要求。运用真菌引物GeoA2/Geo11及AMF特异引物AM1、AM2、AM3/NS31进行巢式PCR扩增,获得的产物连接转化入大肠杆菌构建18SrRNA部分基因克隆文库。通过限制性内切酶Hinf I、TaqI对随机挑选的50个克隆子进行ARDRA(Amplified Ribosomal DNA Restriction Analysis)分析,共获得11个差异图谱。每个图谱选择1个代表测序,结果表明,3个序列为玉米自身未知基因,其余8个都为Clomus属序列。文库评价统计结果发现,AMF克隆文库的Coverage值为72.4%,Rarefaction曲线趋于平缓,所建立文库的容量较为充足,能较全面地代表玉米根系AMF的多样性。     

Molecular characterization and glomalin production of arbuscular mycorrhizal fungi colonizing a heavy metal polluted ash disposal island, downtown Venice

In this work the arbuscular mycorrhizal (AM) fungal communities colonizing a polluted ash dump island, downtown Venice, were studied by using a multimodal approach. The island, Sacca San Biagio, was covered with a thick layer of municipal solid waste residues produced by an incinerator operating from 1973, to 1984. Such residues contained high levels of heavy metals (Cu, Pb and Zn). We characterized the AMF communities present in soils on Sacca San Biagio island by using molecular methods. Nine AM fungal sequence types were detected in the roots of three plant species, representative of the dominant flora, by using partial SSU ribosomal RNA genes. The most abundant sequence types corresponded to Glomus intraradices/Glomus fasciculatum, and to Glo18, a sequence detected so far only in planta. Two sequences were new to science. Glomalin-related soil protein (GRSP), extracted from rhizosphere soil of dominant plant species, ranged from 1.6 to 2.3 mg g⁻¹. The occurrence of an active AM fungal community able to live in such harsh environment was evinced by the correlation between mycorrhizal colonization and GRSP content.     

丛枝菌根真菌对君迁子贮藏营养及抗冻性的影响

研究了3种丛枝菌根真菌Glomus mosseae(Nicol. & Gerd.)Gerd. & Trappe,Glomus intraradices Schenck& Smith和Glomus versiforme(Karsten) Berch接种对君迁子生长、贮藏营养和抗冻性的影响。结果表明,接种丛枝菌根真菌提高了枝干木质部和韧皮部的贮藏营养水平,木质部和韧皮部淀粉、韧皮部可溶性糖含量极显著高于对照,木质部可溶性糖、全氮含量显著高于对照;同时,接种后极显著提高了君迁子1年生苗的抗冻性,这与贮藏营养水平的提高相一致;接种后显著促进了君迁子苗木的生长,苗高、二次枝数、地上部干重、鲜重极显著高于对照,苗高提高了24 5%~50 7%,二次枝数增加了2 14~2 37条,地上部干重为对照的2 9~4 4倍。     

两种AMF对巨菌草根际土壤Cd生物可利用性以及Cd积累的影响

利用盆栽巨菌草(Pennisetum sp.)实验,研究了不同土壤镉(Cd)浓度(T0:空白;T1:5 mg·kg~(-1);T2:10 mg·kg~(-1);T3:15 mg·kg~(-1))条件下,接种两种丛枝菌根真菌(Arbuscular mycorrhizal fungi,AMF)[摩西斗管囊霉(Funneliformis mosseae,Fm)和根内根孢囊霉(Rhizophagus intraradices,Ri)]后土壤中Cd的生物有效性、巨菌草生物量、巨菌草Cd积累量等的变化。结果表明:与不施加菌剂(CK)相比,接种AMF显著降低了土壤中Cd的生物可利用性,在5、10、15 mg·kg~(-1)处理下,接种Fm和Ri后可交换态Cd分别降低了18.65%、20.51%、6.53%和12.54%、16.64%、6.66%;在10、15 mg·kg~(-1)处理下,接种Fm和Ri,植物地上部分生物量分别增加了20.98%、36.94%和36.54%、43.88%,地下部分生物量增加了14.31%、21.79%和25.78%、12.83%。接种AMF显著提高了巨菌草对Cd的吸收能力,其中在5 mg·kg~(-1)处理下接种Ri,巨菌草的重金属富集系数(BCF)最高,达到0.77,由于植物地上、地下部分Cd的含量均增加,巨菌草的Cd转移系数(TF)并没有显著变化。     

外源磷与AMF对间作玉米种植红壤无机磷形态的影响

通过三室隔网分室盆栽模拟试验,研究了分室不同磷(P)源[无机磷(磷酸二氢钾)和有机磷(大豆卵磷脂),P添加量均为50 mg·kg-1]添加和根室接种不同丛枝菌根真菌(AMF)[Glomus mosseae(GM)、Glomus etunicatum(GE)]对间作玉米种植红壤无机磷形态的影响。结果表明:无论接种与否,间作处理使根室土壤有效磷含量显著降低,说明间作能够促进玉米植株对土壤有效磷的吸收,且有效磷的耗竭从根际土壤开始。除OP50-单作玉米处理的Org-P外,接种AMF均一定程度增加了各形态无机磷含量。此外,根室土壤有效磷的主要组分为Ca2-P、Al-P和Org-P,其中Org-P与土壤有效磷有着极显著的负相关关系。     

Above- and belowground responses to tree thinning depend on the treatment of tree debris

Mechanical mastication is increasingly prescribed for wildfire mitigation, yet little is known about the ecological impacts of this fuels treatment. Mastication shreds trees into woodchips as an alternative to tree thinning and burning the resulting slash, which can create soil disturbances that favor exotic plants. Previous research on mastication has not simultaneously considered both the responses of soil organisms and understory plant communities. We compared mastication to slash pile burning (both 6-months and 2.5-years post-treatment) and untreated controls in pinyon–juniper (Pinus edulis–Juniperus osteosperma) woodland and measured soil properties, arbuscular mycorrhizal fungi (AMF) and understory plant composition. Our results showed that slash pile burns had severely degraded soil properties and low AMF abundance and richness compared to untreated or mastication plots. Pile burns were dominated by exotic plant species and had approximately 6× less understory plant abundance and richness than untreated plots. Only two variables differed between mastication and untreated plots 6-months post-treatment: mastication had lower soil temperature and higher soil moisture. Mastication plots 2.5-years post-treatment had more plant cover and richness than untreated plots or pile burns, although non-native Bromus tectorum cover was also greater and AMF spore richness was lower than untreated plots. The structural equation model (SEM) we developed showed that plant cover strongly influenced AMF abundance (0.50) and both plant cover (0.36) and AMF (0.31) positively influenced soil stability. In the short-term, mastication is a preferable method as it creates fewer disturbances than pile burning; however long-term impacts of mastication need further study as this practice could affect native plant communities. Our results suggest that the manner in which woody debris is treated following tree thinning has an important influence on soil stability and native plant biodiversity.     

巴西大西洋森林中巴西松地面根部显示丛枝菌根真菌高定殖率和多样性

Almost 30 different arbuscularmycorrhizal fungi (AMF)species, distributed in different genera such as Glomus, Acaulospora,Scutellospora,Entrophospora,Ambispora,Kuklospora,Gigaspora,and Archeospora, have been identified in the root zone of Araucaria angustifolia, known as Brazil Pine. During our AMF survey in this ecosystem, our attention was called to the presence of many superficially growing Araucaria roots. Our hypothesis was that these roots were colonized with AMF because of the presence of AMF spores in organic material aboveground. Samples of these superficial roots and the organic substrate they were growing on were evaluated for their mycorrhizal status. DNA was extracted from the AMF colonized superficial roots and submitted to polymerase chain reaction (PCR) amplification using the NS31-AM1 primer pair, followed by cloning and sequencing. We found that the root colonization percentages were between 31% and 52%, and the number of AMF spores in the substrate ranged from 27 to 164 spores per 50 g dry substrate.The phylogenetic analyses and tree construction using maximum parsimony (MP) and neighbor-joining (NJ) methods identified 13 different species of the phylum Glomeromycota belonging to the genera Glomus, Funneliformis, Rhizophagus, Gigaspora, Acaulospora,and Archaeospora, and five isolates were identified only at the genus level. To our knowledge, this is the first report on Araucaria angustifolia with roots growing aboveground, producing runner roots that develop on dead tree trunks and organic material. The higher colonization of the aboveground roots than those commonly found in belowground Araucaria roots suggests that they may present active metabolic uptakeof nutrients.     

丛枝菌根真菌(AMF)对玉米/蚕豆和玉米/稗草互作体系植物生长的反馈调节

【目的】丛枝菌根真菌(arbuscular mycorrhizal fungi,简称AMF)对农田生态系统中作物的生长起着重要作用。不同宿主植物和作物种植方式对土壤中菌根真菌具有一定的选择性,从而影响菌根真菌对后茬植物生长的反馈效应。间作体系是农业生产中增产增效的一种重要的种植模式,明确AMF在间作体系中对植物生长的反馈作用,对理解间作体系地上-地下相互作用具有重要的意义。【方法】本试验采用三种常见的AMF(Funneliformis mosseae、Rhizophagus intraradices和Claroideoglomus claroideum)混合接种剂,在单作(玉米,蚕豆,稗草)和间作(玉米/蚕豆和玉米/稗草)条件下,通过两个阶段(AMF驯化及反馈)的反馈试验,模拟研究了三种植物在单作和间作中的反馈强度及AMF的调节作用。【结果】在驯化阶段,玉米/稗草间作体系中,与单作相比,间作玉米地上部生物量减少了64.0%,间作稗草地上部生物量增加了47.8%。玉米/蚕豆间作体系中,与单作相比,两者作物地上部生物量增量分别达21.7%和38.3%。反馈阶段中,单作时,与灭菌处理相比接种AMF后玉米、稗草和蚕豆的地上部生物量分别增加602.3%、 80.6%和21.1%; 间作时,与灭菌处理相比接种AMF后玉米地上部生物量平均增加613.1%,稗草增加80.7%, 蚕豆增加21.4%。单作玉米存在负到零反馈作用,与灭菌处理相比,接种AMF后玉米负反馈作用减弱至零反馈作用。从单作到间作,玉米的反馈作用由负变为零到正反馈作用。【结论】玉米和稗草之间是竞争关系,玉米处于竞争弱势稗草处于竞争优势。玉米对AMF的响应最为强烈,其次是稗草,最后是蚕豆。接种AMF或与其它植物间作后均减弱玉米的负反馈作用,表明丛枝菌根真菌可通过减弱单作玉米的负反馈实现间作增产。     

丛枝菌根真菌对番茄苗期生长及矿质营养吸收的作用

以烘干粉碎后的秸秆与灭菌后的土壤配制成栽培基质,接种5种丛枝菌根菌种接种剂进行番茄栽培试验。结果表明,各种丛枝菌根共生微生物接种后对番茄幼苗生长发育和矿质元素吸收有很好的改善和促进作用,特别是对P、K吸收作用显著,对番茄干质量有明显增加作用。通过对番茄菌根侵染情况分析发现根部丛枝菌根侵染率(F)以Glomusmosseae-1和G.mosseae-2最高,可达30%以上,而丛枝菌根形成率(a)则以G.mosseae-2最高,促进番茄生长效果最好的菌种亦为G.mosseae-2。     

水分胁迫下丛枝菌根真菌对紫穗槐生长和抗旱性的影响

采用盆栽试验研究了水分胁迫下接种丛枝菌根真菌(AMF)对紫穗槐生长和抗旱性的影响。结果表明:不同 水分条件下,接种植株的株高、根系活力、地上和地下干质量高于对照;水分胁迫下菌根侵染率显著高于正常供水 下菌根侵染率;接种AMF 可以显著增加不同胁迫时期叶片脯氨酸、可溶性糖和可溶性蛋白的积累,尤其到胁迫后 期,中度胁迫和重度胁迫下接种植株脯氨酸含量大幅升高;接种AMF 可以显著提高抗氧化酶活性,并且使其维持 在一个较高水平。随着土壤相对含水量下降,Fv / Fm 降低,接种植株始终高于对照;随着水分胁迫时间的延长, ETR、qL 在中期下降,对照植株后期不能回升到原有水平,而接种植株可以恢复到接近前期水平;不同水分条件下, 接种植株NPQ 始终低于对照。研究表明:干旱条件下,接种AMF 可以稳定叶绿素荧光参数,并且通过提高根系活 力、植株体内渗透调节物质含量和抗氧化酶的活性来促进紫穗槐生长,提高抗旱性。