棉花黄萎病拮抗细菌HMB-1005鉴定及定殖

以大丽轮枝菌(Verticillium dahliae)为指示菌,对从棉田土壤中已经分离筛选到的细菌HMB-1005进行形态、19项生理生化特征及16S rDNA试验,结果表明,HMB-1005为解淀粉芽孢杆菌(Bacillus amyloliquefaciens);通过灭菌土和非灭菌土进行盆栽试验,拮抗菌HMB-1005芽孢液浸种、土壤全部混菌和部分混菌3种处理,经过5个月的盆栽试验表明,拮抗细菌HMB-1005能够在灭菌土和非灭菌土中定殖,且定殖数量达106cfu/g土左右,并仍能保持较高的抑菌活性。通过对棉苗根内细菌的分离,证实拮抗细菌HMB-1005能在棉花根内定殖,数量达到103cfu/g。实验结果显示,灭菌土的拮抗菌数高于非灭菌土,并且3种处理方式差异不明显,在实际生产应用中,建议使用浸种方法。     

拮抗菌防治西瓜枯萎病的试验

从西瓜、黄瓜、柑桔等作物根际中分离到的B11等芽孢杆菌的哈茨木霉T31、粘帚霉G1等拮抗真菌，对防治西瓜枯萎病菌有一定抑制作用。经田间防治试验，T31、G1和B11单个菌的田间防效在35％－50％左右；施用T31、G1、B11的混合拮抗菌和采取的一些措施（有机肥作基肥，拮抗菌与杀菌霸混施）对西瓜枯萎病有更好的防治作用，防效达60％－85％。     

拮抗菌强化的生物有机肥对西瓜枯萎病的防治作用

由西瓜专化型尖孢镰刀菌(Fusarium oxysporum f.sp.niveum)引起的西瓜枯萎病是导致西瓜生产毁灭性损失的土传病害,当前对该病尚无有效的防治措施.为了探索该病的生物防治效果,本研究从土壤中分离筛选西瓜枯萎病的拮抗菌,制成生物有机肥,通过温室盆栽试验检验防病效果,并对与拮抗相关的拮抗菌葡聚糖酶进行分子生物学检测.从不同土壤中分离纯化到对西瓜枯萎病菌有潜在拮抗作用的细菌172株,通过平板对峙法筛选出抑菌率在60％以上的拮抗细菌13株,从中挑选出2株抑菌率最高的菌株Cy5和CR38,分别用其与已腐熟的有机肥制成生物有机肥BIO5和BIO38.盆栽试验结果表明,BIO5在防病和促进西瓜生长方面表现优于BIO38.与对照相比,BIO5和BIO38对西瓜枯萎病的相对防治率分别为75％和25％.BIO5处理植株的株高、地上部鲜重、地上部干重分别比对照增加64.8％、63.0％和50％.施用生物有机肥还能显著改变根际土壤的微生物组成.BIO5处理根际土壤的细菌和枯草芽孢杆菌数量分别比对照增加48.5％和61.1％,真菌和尖孢镰刀菌的数量比对照分别下降52.1％和70.2％.分子生物学分析表明,菌株Cy5属于Paenibacillus jamilae菌株,并含有类似于P.polymyxa的β-1,3-1,4-葡聚糖酶.本研究结果说明,拮抗菌强化的生物有机肥对西瓜枯萎病有防治潜力.     

有关细菌根部定殖测量的几个重要问题

在有益细菌与宿主植物的相互作用过程中，引入细菌能否在根部定殖至关重要。因此，测量细菌在根部分殖的水平已成为根圈微生物学研究的常规内容。但在测量细菌根部定殖时，必须首先给予根部定殖一个明确的概念和定义，真正意义上的根部定殖者是指那些在竞争性环境中（如自然田间土壤中）能在根部定殖的细菌。所以选择的模式检验系统应是非灭菌的。引入细菌的回收方法决定于实验目的，测量根外定殖、根内定殖或是全部，要求采用不同的     

生物有机肥对香蕉植株生长和香蕉枯萎病防治的研究

通过盆栽试验,研究了生物有机肥(BIO)对香蕉植株生长和香蕉枯萎病防治效果的影响。结果表明,1)香蕉植株移栽45 d后,施用BIO的处理香蕉植株地上部鲜重、干重、株高和假茎围分别比对照增加了31.9%~93.8%、5.9%~43.1%、43.9%~62.9%和12.3%~18.5%。2)香蕉苗移栽25 d时,施用BIO的香蕉植株根尖几丁质酶活性提高了2.64~12.88个酶活单位,显著高于对照;营养钵育苗施用BIO的处理,香蕉植株根尖β-1,3-葡聚糖酶活性显著地高于营养钵育苗未施BIO的处理,是其酶活的5.22~7.22倍。施用BIO的处理香蕉植株根尖和茎基部中胼胝质含量分别只有对照的59.7%~77.9%和47.6%~68.9%,显著低于对照处理。3)营养钵育苗施用BIO的处理,根际的细菌数量和放线菌数量分别比对照增加了1.56~1.76倍和1.11~1.55倍,而根际真菌数量却减少了19.2%~52.2%,根际尖孢镰刀菌的数量也大大减少。4)营养钵育苗施用BIO的处理,到香蕉植株移栽45 d时,香蕉植株枯萎病的病情指数未超过2.4,比对照降低47.8%~56.5%。上述结果表明,采用营养钵育苗和移栽时都施用BIO的方法种植香蕉,能显著地促进香蕉植株生长,有效地降低香蕉枯萎病的发病指数。     

香蕉种植园土壤管理防控枯萎病的研究进展

香蕉枯萎病在全球香蕉种植区广泛分布,给香蕉产业带来了严重打击,其防控技术研究成为香蕉生产领域关注的热点,目前的防控措施主要有种植抗病品种、化学防治、生物防治、轮作。香蕉枯萎病作为一种土传真菌病害,蕉地土壤管理贯穿整个防控过程。文章重点介绍了近年来通过向土壤施用抗菌剂、添加生物菌剂、科学施肥以及轮作对香蕉枯萎病防控效果的研究进展,以期为香蕉枯萎病综合防控的深入研究提供参考。抗菌剂多菌灵对香蕉枯萎病菌的抑制率达到82.9%,解淀粉芽孢菌将香蕉枯萎病的发病率降至15%以下,生物菌肥将香蕉枯萎病的发病率降至10%以下,香蕉与甘蔗、韭菜、辣椒轮作将香蕉枯萎病的发病率降至5%以下,四种方法对香蕉枯萎病的防控均表现出良好效果。     

短小芽孢杆菌BX-4抗生素标记及定殖效果研究

为研究短小芽孢杆菌BX-4在作物根际的定殖及防病效果,通过浓度梯度法用氨苄青霉素对其进行了抗性标记,并通过番茄盆栽试验研究了其在根际土壤中的定殖规律。结果表明,筛选出的突变体菌株BX-4＇能够耐受浓度为200 μg·mL^-1的氨苄青霉素,并且具有耐药和遗传双重稳定性;应用试验显示该突变体菌株能成功在番茄根际定殖,接种20 d后根际土壤中存活数量达到最高值1.34×10^8 cfu·g^-1干土,以后逐渐下降,到50 d时趋于稳定;筛选的突变体菌株对番茄青枯病具有明显的防治效果,防效达37.9%-50.9%。短小芽孢杆菌BX-4在作物根部的定殖规律为揭示其生防机理及应用该菌提供了科学根据。     

巨大芽胞杆菌WY4的GFP标记及其在小白菜上的定殖

解磷菌(phosphate solubilizing bacteria,PSB)可以通过提高土壤有效磷含量而增加作物产量,目前已有许多解磷菌被分离并应用于农业生产中,但关于解磷菌在植物根际中的定殖情况仍缺乏系统性的研究.WY4为本实验室前期从小白菜(Brassica chinensis)根际分离得到的一株高效解磷菌,本研究利用绿色荧光蛋白(green fluorescent protein,GFP)标记技术研究了WY4在小白菜根际及土壤中的定殖规律.与原菌株相比,GFP标记对菌株WY4-GFP生长及解磷活性具有较小影响,同时在促进小白菜生长上WY4-GFP与WY4无显著性差异;WY4-GFP具有持久的定殖能力(接种21d的自然土及30 d的小白菜根际土中,WY4-GFP的定殖数量分别为105和104 CFU/g左右),同时随时间的增加WY4-GFP在土壤中定殖数量逐渐减少;WY4-GFP在小白菜根冠及分生区大量定殖,在伸长区及侧根根毛处数量较少,同时表皮细胞间隙上也有较多的标记菌株.研究结果表明,WY4-GFP在小白菜根际及土壤中具有良好的定殖能力,这为后期深入研究解磷菌与植物间的关系提供了重要参考.     

防病促生枯萎病拮抗菌“98-Ⅰ”对4种枯萎病防治效果研究

经 8年系统试验研究从 330个细菌菌株中筛选出 1株防病促生枯萎病拮抗菌“98 Ⅰ” ,经鉴定属蜡质芽孢杆菌。该菌对黄瓜枯萎病、西瓜枯萎病、青椒枯萎病和番茄枯萎病 4种土传病害均有显著防治效果 ,其平皿孢子萌发抑制率分别为 79 2 %、75 1%、72 3%和 95 7% ,且该菌对多种蔬菜有促生和促进种子发芽功效。     

连作香蕉园生态熏蒸剂的筛选及其对土壤微生物群落结构的影响

通过对多年连作后发香蕉枯萎病土壤进行熏蒸处理,应用传统涂布计数法,结合变性凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)技术,较为全面地研究了不同熏蒸剂对病原菌的杀灭效果以及对土壤微生物区系的影响,为连作蕉园的可持续栽培提供了理论依据。试验通过研究石灰、碳铵、石灰碳铵联用、二氧化氯、次氯酸钠、过氧化氢和氨水处理对水体病原菌孢子、土壤中尖孢镰刀菌的杀灭作用,逐步筛选出适合连作蕉园灌溉水和土壤的熏蒸剂种类。结果表明:上述熏蒸剂对水体或土壤中尖孢镰刀菌均具有杀灭作用,土壤中氨水、石灰碳铵联用处理效果更加显著,被选择为发病蕉园土壤熏蒸剂。不同用量氨水、石灰碳铵联用处理熏蒸土壤,随着熏蒸剂施用量的增加,土壤中尖孢镰刀菌数量下降幅度逐渐增大;两种熏蒸剂对土壤中可培养总细菌和总真菌均有杀灭效果,随着用量的增加,总真菌数量下降幅度显著高于总细菌。DGGE结果显示,氨水、石灰碳铵联用处理对土壤中总真菌种类的影响也显著高于总细菌,随着熏蒸剂用量的增加,总细菌有少许条带的增加或减少,总真菌条带丰富度急剧减少。与对照相比,各熏蒸剂处理后总细菌物种多样性指数均有所降低,随着用量的增加,总体呈下降趋势,在氨水、石灰碳铵联用用量分别为2.67 ml/kg和(3.33+1.67)g/kg时各项指数最高。综上,所有熏蒸剂均能作为水体病原菌杀灭剂,其中氨水、石灰碳铵联用是良好的连作蕉园土壤熏蒸剂,最适用量分别为2.67 ml/kg、(3.33+1.67)g/kg。     

枯萎病菌侵染后香蕉乙烯形成酶基因MaA CO及乙烯响应因子MaERF1表达水平分析

香蕉与枯萎病互作机理研究一直是近年来研究的热点,目前对枯萎病菌的致病机理及香蕉的感、抗病机理还不清楚。本文利用前期表达谱分析结果克隆了乙烯合成及调控途径中的关键酶基因乙烯形成酶基因（MaACO）和乙烯响应因子基因（MaERF1）,通过RT-PCR检测,对这两个基因在感、抗病香蕉种质中的表达水平进行研究。结果表明,MaACO和MaERF1基因对机械损伤非常敏感,尤其在损伤初期（3h）的表达水平远高于后期;对于枯萎病菌侵染处理的植株,尤其在感染初期（3h）MaACO和MaERF1的表达水平在抗性植株中的表达量均比感病植株中的低;抗性种质中乙烯途径可能受到抑制,香蕉的感病性可能与侵染初期对乙烯信号的敏感性相关。本研究为利用乙烯抑制剂进行香蕉抗枯萎病新方法的研究奠定了基础。     

Effects of cultivation of preceding plants on the development of Fusarium wilt of cucumber and daikon

The effects of the cultivation of preceding plants on the inhibition of Fusarium wilts, vessel diseases of cucumber (Cucumis sativus) caused by Fusarium oxysporum f. sp. cucumerinum (FOC) and yellows of daikon (Raphanus sativus var. longipinnatus) caused by F. oxysporum f. sp. raphani (FOR), were examined. When the cucumber or daikon plant was cultivated as a succeeding plant in the pots where the soils had been inoculated with a spore suspension of FOe or FOR and then various preceding plants had been cultivated, the percentage of disease of the succeeding plants was lower in some plots than in the control plot without preceding plants. Spinach (Spinacia oleracea, cv. Atlas) and burdock (Arctium lappa, cv. Takinogawa) inhibited significantly the vessel disease caused by FOe. Thrnip (Brassica rapa, Parental Line Nou No.1) and spinach (S. oleracea, cv. Try) suppressed the yellows caused by FOR. When the number of FOC or FOR was estimated by the recovery using Komada med.ium and by testing their pathogenicity to the host plant, cucumber or daikon, the numbers of both were not reduced in the plots even where these plants had been cultivated initially. The soil extract from the pots, where preceding plants had been cultivated, did not affect the conidia of FOC and FOR. In both FOC and FOR experiments, the cultivation of preceding plants increased the number of bacteria in soil. Additionally, in the FOR experiment, preceding plants, turnip and spinach, increased the number of F. oxysporum (FOX) except for FOR and decreased the proportion of FOR to FOX. It was suggested that these preceding plants might affect the pathogens of Fusarium wilt indirectly by the changes in the numbers and flora of soil microorganisms.     

海南省香蕉枯萎病病原菌的分离鉴定及1号、4号小种的生物学特性

香蕉枯萎病是目前国内和世界很多地区香蕉种植区的一种毁灭性病害,目前尚未找到有效的防治方法。本文采集了海南省不同地区香蕉枯萎病的病原菌,通过经典病理学与分子生物学的方法进行了鉴定,此外还优化了快速检测的方法。结果表明,通过接种巴西香蕉和粉蕉进行致病性检测以及分离菌rDNA—ITS测序结果,明确鉴定出供试的香蕉分离菌株为4号生理小种,粉蕉分离菌株为1号生理小种。为了对1号、4号生理小种有更多的了解,本文还对1号、4号小种菌株生物学特性进行了测定,确定了影响菌丝生长的最佳条件。结果显示,pH5时最适合1号、4号生理小种的菌丝生长,适合菌丝生长的温度为19-28℃之间,温度达到37℃时菌丝不能生长,碳源对菌丝的生长影响不大。这些方法和数据可以有利于田间香蕉枯萎病病害的检测和最终确定,生物学特性的测定可以为耕作技术的改进和农药的研发提供参考依据。     

Combining ability of yield and yield components among Fusarium oxysporum f.sp. strigae-compatible and Striga-resistant sorghum genotypes

ABSTRACT

Use of sorghum [Sorghum bicolor (L.) Moench] cultivars with partial resistance to Striga spp. and Fusarium oxysporum f.sp. strigae (FOS) represents a novel strategy to control Striga. This study aimed to identify the nature of gene action controlling grain yield and yield components and to select promising sorghum crosses possessing both FOS compatibility and Striga resistance, along with good combining ability effects. One-hundred hybrids, developed from pairwise matings among 10 FOS compatible, high-yielding female lines and 10 Striga-resistant male lines, were evaluated with and without FOS inoculation. The F₁s were field evaluated at three locations in Tanzania known for their severe Striga infestation, using an alpha lattice design with two replications. General (GCA) and specific combining ability (SCA) variances were significant for grain yield per plant, hundred-seed weight, plant height, flowering time and the number of Striga plants. The study demonstrated FOS inoculation to be an effective means of controlling Striga. Families 675?×?672, AS435?×?3993 and 4643?×?AS436 displaying large SCA effects for grain yield, and 4567?×?AS429, 3424?×?AS430 and 3424?×?AS436 with small SCA effects for Striga counts should be useful genetic resources for breeding and integrated Striga management.     

Effect of soil sterilisation on biological control of Fusarium oxysporum f. sp. lycopersici and Meloidogyne javanica by antagonistic fungi and organic amendment in tomato crop

One of the major challenges in the application of biological control agents into the soil is their inability to withstand competition from natural microflora. In this study a management programme involving fungal biological control agents Trichoderma harzianum (TH) and Purpureocillium lilacinium (PL), and the organic amendment neem was carried out for the control of Meloidogyne javanica and Fusarium oxysporum f. sp. lycopersici (FOL). The experiment was carried out in sterile and non-sterile soil in order to test the efficacy of biological control in natural soil conditions. This experiment was repeated twice. A Wilcoxon's Signed-Rank test indicated there were no significant (P?<?0.05) differences in fusarium wilt control in sterile and non sterile soils in the treatments PL neem and TH neem, but there was significantly (P?<?0.05) higher control of M. javanica in the same treatments in sterile soils compared to non sterile soils. The combinations of fungal antagonists and neem were effective in non sterile as in sterile soils in the control of fusarium wilt, while being less effective in the control of M. javanica. Thus the biological control agents can be very effective in the control of fusarium wilt in natural conditions where the soil is not sterilised, but be less effective in the control of M. javanica in the same conditions. There is therefore potential of integrated management of fusarium wilt and root-knot nematodes (RKN) by these biological control agents in natural soil field conditions as an alternative to using chemicals.     

一株拮抗香蕉枯萎镰刀菌稀有放线菌的分离及鉴定

本研究采用苯酚、干热、SDS和超声波四种不同的预处理方法从五指山原始林区采集的土样中选择性分离得到的702株稀有放线菌,共筛选出4株具有拮抗香蕉枯萎镰刀菌活性的菌株,经过复筛,获得一株高活性的拮抗菌210-1-61。通过对其进行形态鉴定、生理生化特征鉴定和16SrDNA序列分析,结果表明该菌株与M．pattaloongensis JCM12833^T进化关系最近,其16SrDNA序列同源性最高,达98．89％;其形态与生理生化特性也与小单孢菌属M．pattaloongensis JCM12833^T很接近。此外,我们还构建了与该菌株同源性较高的模式菌株16SrDNA序列的聚类分析图,结果发现该菌株与Mpattaloongensis JCM12833^T稳定单独构成一个分支,二者亲缘关系最近,初步鉴定该菌株为Micrombonospora.pattaloongensis。本研究为今后探讨小单孢菌属稀有放线菌对香蕉尖孢镰刀菌具有拈抗活性提供研究实验材料。     

硝/铵营养对香蕉枯萎病尖孢镰刀菌生长的影响

通过室内平板培养,研究了不同硝/铵配比的氮源,以及不同的pH对香蕉枯萎病尖孢镰刀菌生长的影响。结果表明: 1）在所有不同硝/铵配比处理中,低pH（pH=4）均抑制尖孢镰刀菌的生长。在相同pH值条件下,100%铵态氮处理中尖孢镰刀菌的生长受到明显抑制,其菌落直径均小于4 cm; 2）在不同浓度铵处理后,尖孢镰刀菌的生长在铵态氮大于5 mmol/L时受到强烈的抑制; 3）通过模拟植物细胞壁被尖孢镰刀菌侵染并穿透的过程中发现,尖孢镰刀菌在100%铵态氮处理下不能穿透赛璐膜。本研究结果说明,铵态氮能够控制香蕉尖孢镰刀菌的生长,并抑制其侵染穿透寄主细胞壁。     

香蕉根围土壤尖孢镰刀菌与养分特征及相关性

拟通过分析香蕉种植土壤中尖孢镰刀菌数量与养分含量,阐明香蕉枯萎病发生流行时土壤中尖孢镰刀菌与养分的特征及其相互关系,为香蕉枯萎病的防控提供一定的理论依据。在我国及老挝15块香蕉园中分别随机采集9个罹患枯萎病与9个未患病植株根围土壤样品,应用实时荧光定量PCR与土壤农化分析技术,测定了270个土壤样品中尖孢镰刀菌数量与养分含量。结果表明,患病香蕉根围土壤中尖孢镰刀菌的数量显著高于未患病土壤,每克干土中lg转化后的平均拷贝数为5.5;而患病与未患病植株土壤各养分指标之间几乎无显著差异。各养分指标在土壤中含量分布规律各不相同,其中仅有机质与全磷的含量呈正态分布,土壤pH、全氮及锌的含量呈偏正态分布。蒙特尔检验结果表明,土壤整体养分含量与尖孢镰刀菌数量显著相关。各养分与尖孢镰刀菌的斯皮尔曼相关性分析进一步发现,土壤pH、铁、锰、铜、锌的含量与土壤中尖孢镰刀菌的数量呈显著负相关关系。此外,不同酸性程度及不同有机质含量土壤中尖孢镰刀菌与各养分的相关性分析发现,在弱酸性蕉园土壤（pH > 6.0）中,土壤尖孢镰刀菌的数量与铁、锰、铜的含量显著负相关;而在有机质含量未严重缺乏（SOM > 1%）的土壤中,土壤尖孢镰刀菌的数量与土壤pH及铁、锰、铜、锌的含量呈现显著负相关关系。在我国及老挝香蕉产区蕉园土壤普遍呈现酸化趋势,患病香蕉植株根围土壤中尖孢镰刀菌数量上升,而尖孢镰刀菌与土壤pH及微量元素,尤其是铁、锰、铜、锌的含量呈负相关关系。     

Effects of Arbuscular Mycorrhizal Colonization on Microbial Community in Rhizosphere Soil and Fusarium Wilt Disease in Tomato

Fusarium wilt is caused by soil-borne pathogen Fusarium oxysporum. Tomato (Lycopersicon esculentum Mill.) is susceptible to Fusarium oxysporum f. sp. lycopersici race 1 and was infected with wilt disease. A pot experiment was conducted to investigate effects of inoculating arbuscular mycorrhizal (AM) fungus (Glomus etunicatium) on the microbial community in the rhizosphere soil and Fusarium wilt in tomato (cv. Oogatafukuju). The results indicated that AM fungal inoculation suppressed the Fusarium number in the rhizosphere soil of tomato and decreased the Fusarium wilt disease index. Compared to the control, AM fungal inoculation increased the actinomycete number but increased bacterial number. Bacterial and fungal numbers were high but actinomycetes number was low when tomato basal stems became discolored brown. Fusarium inoculation significantly suppressed development of AM colonization and decreased polyphenol oxidase (PPO) activity in leaves and roots of tomato. Inoculation with AM fungi and Fusarium maintained high PPO activity in leaves and roots. The AM colonization increased root growth of tomato, whereas Fusarium inoculation had no significant effect on tomato growth. These findings suggest that because AM fungal inoculation changes microbial communities and enhances PPO activity, it should suppress occurrence of Fusarium wilt in tomato.