草假单胞菌HT1在蚕豆根和茎的定殖特性及对内生细菌多样性的影响

[背景] 关于蚕豆内生生防细菌的定殖规律及其对内生细菌微生物多样性的研究鲜有报道。[目的] 明确草假单胞菌HT1在蚕豆的定殖特性以及对根茎部内生细菌群落多样性的影响。[方法] 采用抗生素标记法测定菌株HT1的定殖特性,利用高通量测序技术分析该菌灌根处理与对照处理蚕豆根茎部内生细菌的群落多样性。[结果] 菌株HT1的定殖数量为根>茎>叶,呈先升后降的趋势,根部、茎部、叶部在第7天达到最大值,在第83天仍能检测到标记菌株。灌根处理降低了内生细菌的丰富度,提高了根内生细菌物种多样性;灌根处理组根部厚壁菌门、放线菌门、拟杆菌门丰度显著提高,茎部变形菌门的相对丰度有所升高但无显著影响;在属水平上,灌根处理后,根部如Romboutsia、Mitsuaria、乳杆菌属、德沃斯氏菌属等属的相对丰度明显升高,茎部假单胞菌、Muribaculaceae、Elstera、鞘氨醇单胞菌属等属的相对丰度明显升高。[结论] HT1菌株能在蚕豆植株体内定殖达83 d以上,并且可以影响蚕豆植株内部的微生物群落结构组成,提高相关微生物的相对丰度。     

基因标记枯草芽孢杆菌BS-68A在黄瓜上定殖

目的:野生型枯草芽孢杆菌Bacillus subtilisBS-68能有效地防治由Pythium spp.和Fusarium oxporum引起的黄瓜立枯病和枯萎病。为了探究该菌株的生防机制,利用该菌株的黄绿荧光蛋白基因和氯霉素抗性基因标记菌株BS-68A研究其在黄瓜植株各个部位的定殖能力、种群动态和在根围的分布。方法和结果:用基因标记菌株发酵液分别对黄瓜种子进行浸种和浇穴处理,播种后30d,该菌能在黄瓜根部和茎基部定殖,不能在茎部和叶部定殖。浸种处理,该菌在茎基部的种群数量为3.1×104cfu/株,大于根部的种群数量4.1×102cfu/株;浇穴处理,该菌在茎基部的种群数量8.0×103cfu/株,低于根部的种群数量2.5×104cfu/株。     

内生拮抗细菌在哈密瓜植株体内的传导定殖和促生作用研究

采用抗生素标记的方法研究了内生拮抗细菌P38和B167菌株在哈密瓜植株体内的定殖动态和对植株生长的影响.结果表明,接种方法显著影响P38菌株在植株体内的定殖和传导,并以浸种处理最佳,蘸根和灌根处理次之,喷叶处理最差;浸种可使P38菌株在根、茎、叶中良好传导和稳定定殖,随着植株的生长,根内菌量呈下降趋势,而茎、叶内的含菌量先上升后下降;P38菌株还具有促进哈密瓜种子萌发和植株生长的作用.B167菌株只在根内定殖,在体内的扩展性较差,不能进入叶片;它对植株的生长表现出一定的抑制作用.     

生防菌AS818抗药性标记标在大豆根限定殖

通过60Coγ-射线(1.0×104rad)对生防菌株链霉菌AS818孢子悬液(106/mL)进行诱变,得到抗链霉素(＞50μg/mL)突变株5株.经诱导,RL-4抗链霉素水平达到10μg/mL.标记株RL-4在1%水琼脂(WA)和无菌土中培养的大豆根际定殖趋势有所不同.RL-4在1%WA培养的大豆根表的定殖量呈逐步上升趋势.无菌土中种植的大豆根际和根表的检测表明RL-4可在无菌土中大豆根际短期定殖,在根际第1周数量降低了100倍,以后数量开始逐渐上升,第3周达到高峰,数量比第1周增加了3个数量级,但在第4周其数量又开始下降;在根表RL-4数量逐步下降,到第4周已检测不到RL-4的存在.组织印记法检测发现标记株RL-4可以在无菌培养的大豆根表定殖并沿根分布,但却不能定殖于根内.     

luxAB基因标记的K2116L菌株在棉花根际中的定殖

采用三亲本杂交法将发光酶luxAB基因转移进棉花根际促生菌芽胞杆菌K2116菌株中,获得标记菌株K2116L.标记菌株连续传代15次均未发生质粒丢失现象,表明标记菌株具有较好的遗传稳定性;K2116L菌株的生长及其释钾能力未受到标记质粒的影响.K2116L菌株在灭菌和非灭菌的黄褐土、黄潮土和红壤3种土壤中均能长期存活;在灭菌土壤中的数量稍高于非灭菌土壤;在3种土壤中的数量依次为:黄褐土＞黄潮土＞红壤;在不同土壤中,K2116L菌株具有与土著菌株进行空间和营养竞争的能力.采用根盒试验追踪标记菌株在棉花根部的定殖动态,棉花播种12d时标记菌株在0～2、2～4 cm深度根际土壤定殖密度达到最大;18 d时在4 cm以下的深度达到最高水平.棉花播种18 d时标记菌株在所有深度的根表均达到最高定殖水平,0～2 cm根段定殖密度为1.76×106cfu·g-1,8cm以下根段达到1.6×105cfu·g-1.补充营养后,根际和根表标记的菌株数量均有明显上升.试验数据显示,随着根的生长标记菌株不断向根尖方向扩散.     

两株香蕉枯萎病拮抗菌在香蕉体内的定殖

本研究将2株香蕉枯萎病拮抗细菌0202和1112分别涂布于含有不同浓度的利福平培养基上,筛选出抗药性标记菌株0202-r和1112-r.将香蕉枯萎病菌Foc4接种于香蕉植株根部,3 d后再将抗药性标记菌株接种在同一部位或位点,于不同时间测定拮抗细菌在根际、根内、球茎和假茎的定殖情况.研究结果显示,将拮抗细菌接种香蕉根部1～3 d后,在植株根际土壤内存在大量拮抗细菌,而根内、球茎和假茎内存在少量拮抗细菌;接种7～14 d后,在植株根内、球茎和假茎内定殖的拮抗细菌增多,并达到最大量;在接种14～21 d后,在植株根内、球茎和假茎内定殖的拮抗细菌量急剧下降;在接种28～35 d后,在植株根内、球茎和假茎内定殖的拮抗细菌量稍有回升.     

巨大芽胞杆菌luxAB标记菌株的根际定殖研究

通过三亲本杂交方法成功地用发光酶基因luxAB标记巨大芽胞杆菌ATCC14581,所获得的标记菌株ATCC14581-L在不同的条件下能稳定发光.将该标记菌株制成微生物接种剂,并利用土壤微缩系统将其接种小麦进一步研究它在小麦根际的定殖动态和散布规律.结果发现,ATCC14581-L在灭菌土壤中的定殖水平高于不灭菌土壤,在垂直方向上主要的定殖在0～7cm根段间,且随深度增加而降低.ATCC14581-L在小麦种后第7d之前就已达到最高定殖水平,在初始接种量为3.40×107cfu/g根情况下,第7d时灭菌土壤处理的根际菌数为2.54×105cfu/g根,而不灭菌土壤的根际菌数为8.87×104cfu/g根;随着时间的增长,定殖数量明显降低.     

硅酸盐细菌NBT菌株在小麦根际定殖的初步研究

对硅酸盐细菌NBT菌株进行了耐药性标记,得到稳定的链霉素抗性标记菌NBT菌株,采用选择性培养基分离计数,通过琼脂平板和盆栽试验,研究了标记菌NBT在小麦根际的定殖动态及影响因素。结果表明,在灭菌土盆栽中,播种后9d左右NBT菌株在小麦根际的定殖水平达最高(1．4×10^8cfu·g^-1根土),播种后54d左右趋向稳定,NBT菌株细胞数量为2．4×10^3cfu·g^-1根土;未灭菌土盆栽中,播种后9d左右NBT菌株的定殖水平达最高(3．8×10^8cfu·g^-1根土),60d左右趋向稳定,菌数为3．1×10^3cfu·g^-1根土,牛物和非牛物因素对NBT菌株定殖小麦根系有影响。     

生防菌AS818抗药性标记株在大豆根际定殖

通过₆₀Co 射线 (1 .0× 1 0 ⁴ rad)对生防菌株链霉菌AS81 8孢子悬液 (1 0 6/mL)进行诱变 ,得到抗链霉素 (>5 0μg/mL)突变株 5株。经诱导 ,RL 4抗链霉素水平达到 1 0 0μg/mL。标记株RL 4在 1 %水琼脂 (WA)和无菌土中培养的大豆根际定殖趋势有所不同。RL 4在1 %WA培养的大豆根表的定殖量呈逐步上升趋势。无菌土中种植的大豆根际和根表的检测表明 :RL 4可在无菌土中大豆根际短期定殖 ,在根际第 1     

辣椒内生细菌BS-1和BS-2在植物体内的定殖及鉴定

以抗利福平为标记,用浸种、涂叶和灌根方法接种,测定菌株在植物体内的定殖。结果表明,来自辣椒体内的BS_2和BS_1菌株不仅可在辣椒体内定殖,也可在番茄、茄子、黄瓜、甜瓜、西瓜、丝瓜、小白菜等植物体内定殖,BS_2菌株还可在水稻、小麦及豇豆等植物体内定殖,BS_2菌株的内生定殖宿主范围比BS_1菌株的广;另外BS_2菌株可在辣椒和白菜体内较长期定殖。用常规方法、Biolog及16S rRNA序列比较,两菌株鉴定为枯草芽杆菌内生亚种（Bacillus subtilis subsp. endophyticus）。     

Assessing the pathogenic effect ofFusarium, Geosmithia andOphiostoma fungi from broad-leaved trees

Phytopathogenic effect of Geosmithia pallida, G. langdonii, Ophiostoma grandicarpum, O. querci, two isolates of O. piceae, and two isolates of Fusarium solani was compared using plant growth test (stem and root length of garden cress plants seeded on mycelium-covered potato carrot agar); Ophiostoma spp. and F. solani were isolated from oak, Geosmithia spp. from galleries of Scolytus intricatus on beech. All fungi inhibited more the root elongation than that of stems. F. solani led to plant collapse after briefly stimulating the growth of stem and in one case also root. G. langdonii inhibited stem and root growth to 20% and led to plant collapse. G. pallida inhibited root growth to 25% whereas stem growth was almost unimpaired. Ophiostoma spp. reduced stem growth to approximately 60-80% and root growth to 25-60%. O. piceae and O. querci caused plant collapse after 15-20 d.     

木薯蔗糖合成、转运与块根淀粉积累关系研究

该研究以淀粉含量不同的两个木薯品种(辐选01和华南124)为材料,通过测定各品种不同生育期叶、茎和根的蔗糖含量及块根淀粉含量,分析了蔗糖合成、转运和块根淀粉积累过程的相关性.结果表明:与华南124相比,在整个生育期内辐选01叶、茎的蔗糖含量均较高,块根蔗糖含量在块根膨大初期以前高于华南124,块根膨大初期以后则相反.在木薯的整个生育期,与辐选01相比,华南124的淀粉合成量和淀粉合成速率均较低.叶和茎蔗糖含量的变化规律与淀粉合成速率的变化规律相反,即块根淀粉积累明显加快时叶和茎的蔗糖含量略呈下降趋势,而块根淀粉合成减慢时叶、茎的蔗糖含量又开始上升.随着生育期的延后,块根蔗糖含量越来越低.在块根形成初期,蔗糖含量最高的组织部位为块根,其次为茎秆,最低的是叶片;而在块根成熟期时则相反,即蔗糖含量最高的部位是叶片,其次为茎杆,块根的蔗糖含量最小.相关性分析结果表明,木薯叶片蔗糖含量与块根淀粉含量呈显著的正相关;茎秆蔗糖含量与块根淀粉积累量呈不显著的正相关;块根蔗糖含量与淀粉积累量呈显著的负相关.由此可见,木薯叶、茎和根蔗糖与块根淀粉积累过程密切相关,其中叶片合成蔗糖的能力与块根利用蔗糖的能力在淀粉的积累过程中发挥关键作用.该研究结果为木薯的生产选育与高效栽培提供了理论依据.     

Spatial peculiarities of rhizoplane colonization by microscopic fungi

Spatial peculiarities in the colonization of the tomato, cucumber, and barley rhizoplanes by microscopic fungi were studied. The apical zone of roots was colonized with a limited number of R strategists (the order Mucorales, Fusarium sp., Aspergillus niger, and Mycelia sterilia). The fungal population of the root hairs and the basal zone of roots was 2- to 3-fold more dense due to the prevalence of K strategists. Fusaria, Fusarium oxysporum in particular, colonized roots in earlier terms than the genera Trichoderma, Penicillium, Gliocladium, and others. The F. oxysporum population was at a maximum in the rhizoplane zone nearest the root tip.     

Root colonization of faba bean (Vicia faba L.) and pea (Pisum sativum L.) by Rhizobium leguminosarum bv. viciae in the presence of nitrate-nitrogen

There is a lack of knowledge concerning the effect of nitrate-nitrogen (NO3(-)-N) at levels known to inhibit nodule formation and functioning on root colonization of dinitrogen-fixing legumes. Firstly, this study investigated potential differences between Rhizobium leguminosarum bv. viciae 175F9 and its bioluminescent-labeled strain 175F9.lux on root colonization of faba bean (Vicia faba L.) and pea (Pisum sativum L.). These two strains similarly colonized the roots of both hosts. Secondly, this study evaluated the effects of 0 and 10 mol x m(-3) NO3(-)-N on root colonization of faba bean and pea by strain 175F9.lux, over time. Averaged over both hosts and harvest dates, the presence of NO3(-)-N increased the rhizobial population and the root length colonized. In addition, our results showed that bioluminescence activity increased from 7 to 14 days after sowing and was not correlated to rhizobial population. Finally, to demonstrate that an increase in bioluminescence activity was not an indirect effect of nitrate on R. leguminosarum bv. viciae 175F9.lux, this study investigated the effects of increasing carbon (mannitol) and nitrogen (NO3(-)-N) concentrations on the rhizobial population and bioluminescence activity. The carbon source was more important than the nitrogen source to increase the rhizobial population and bioluminescence activity, which increased with increasing mannitol concentration, but not with increasing nitrate concentration. Results from this study demonstrated that NO3(-)-N increased rhizobial population, especially for faba bean, and the length of root colonized.     

The insect-pathogenic fungus Metarhizium robertsii (Clavicipitaceae) is also an endophyte that stimulates plant root development

? Premise of the study: The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. Metarhizium robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M. robertsii were examined after inoculation with fungal conidia. ? Methods: We used light and confocal microscopy to ascertain the plant endophytic association with GFP-expressing M. robertsii. Root lengths, root hair density, and lateral roots emerged were also observed. ? Key results: Initially, M. robertsii conidia adhered to, germinated on, and colonized roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M. robertsii on the root (within 1-2 d). Plants inoculated with M. robertsii ΔMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M. robertsii did not stimulate root hair proliferation. Longer-term (60 d) associations showed that M. robertsii endophytically colonized cortical cells within bean roots. Metarhizium appeared as a mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. ? Conclusions: These results suggest that M. robertsii is not only rhizosphere competent but also displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs.     

Salicylic acid accumulates in the roots and hypocotyl after inoculation of cucumber leaves with Colletotrichum lagenarium

Increased amounts of salicylic acid (SA) were detected in the roots and hypocotyl of cucumber plants (Cucumis sativus) using high-performance liquid chromatography following inoculation of the leaves with the anthracnose pathogen, Colletotrichum lagenarium. The concentrations of SA in the internodes immediately below the infected leaves increased to more than 1microg/g fresh weight. In contrast, the concentrations of SA in stems distant from, or above the infected leaves increased to 100-300ng/g. An increase in SA levels was observed in the upper stem 2d after inoculation, followed by the hypocotyl with an increase detected 4d after inoculation. An initial increase in the SA levels was detected in the stem, followed by an increase in SA levels in the root from a basal level of approximately 300ng/g to more than 1microg/g. The increased level of SA in the lower leaves was less than 100ng/g. These results indicate that the levels of SA in the hypocotyl and root increased significantly following inoculation of the leaves with a microorganism capable of inducing SAR.     

Endophytic fungal diversity of 2 sand dune wild legumes from the southwest coast of India

Endophytic fungi of 3 age classes (seeds, seedlings, and mature plants) and 5 tissue classes (cotyledons, seed coats, roots, stems, and leaves) of coastal sand dune legumes Canavalia cathartica and Canavalia maritima were assessed by plating surface-sterilized segments on malt extract agar. Forty-six fungal taxa comprising 6 ascomycetes, 33 mitosporic fungi, 2 zygomycetes, and 5 sterile morphospecies were recovered. There was no significant difference in the colonization frequency of endophytes between plant species (p = 0.4098, Student's t test). Among the age classes, endophytic fungi colonized over 90% of seedlings and mature plants. Similarly, among tissue classes, endophytic fungi colonized over 90% of root, stem, and leaf segments. Diversity and richness of endophytic fungi were higher in C. cathartica than in C. maritima. Rarefaction curves revealed a "higher expected number of species" in mature plants of C. cathartica and seedlings of C. maritima, whereas it was highest in leaves of both plant species. The most dominant endophyte, Chaetomium globosum, colonized over 50% of the root, stem, and leaf segments of C. maritima and over 50% of the root segments of C. cathartica. The colonization frequency of C. globosum was found to be 5%-12.5% in seeds and increased up to 40%-64.4% in seedlings or mature plants. Halosarpheia sp. was the only marine fungus recovered among the endophytes.     

Initial stages in the morphogenesis of nitrogen-fixing stem nodules of Sesbania rostrata.

Morphogenesis of stem nodules in Sesbania rostrata was studied over a period of 6 days after inoculation with an appropriate species of Rhizobium. Nodulation sites were initially slightly raised, circular areas 0.3 to 0.6 mm in diameter and 4 to 5 mm apart in vertical rows along the length of the stem. Each site was underlaid by an adventitious root primordium. A site became susceptible to infection by a specific Rhizobium sp. when the root primordium broke through the epidermis, leaving a fissure. Rhizobia multiplied within this fissure and colonized the exposed intercellular spaces. The infection extended inward as narrow, branched intercellular threads moved into a cortical meristematic zone, where cell division was initiated, and invagination of infection thread branches into adjacent plant cells followed. Rhizobia were released into the plant cells and surrounded immediately by plant membrane. Intracellular rhizobia divided actively, leading to bacteroid-filled cells. Infected areas enlarged and coalesced as the nodule matured.