1株抗根结线虫红树林放线菌的筛选与鉴定

从红树林底泥中分离筛选具有抗线虫活性的放线菌,并对活性菌株进行分类鉴定。采用稀释平板法分离放线菌,采用24孔板液体筛选模型筛选具有抗线虫活性的菌株,并对活性菌株进行形态学和生理生化特征研究,测定其16S rDNA序列并进行系统发育分析。筛选得到1株具有杀线虫活性的菌株,编号为HA11090,该菌株发酵液在稀释20倍和40倍后,抗根结线虫校正死亡率分别为70.5%和65.0%。菌株HA11090的16S rDNA序列与Saccharopolyspora hirsuta subsp.kobensis和Saccharopolyspora jiangxiensis的相似性最高,分别为99.35%和99.27%,三者在系统发育树上聚为一个分支,形态和生理生化特征分析显示,菌株HA11090与Saccharopolyspora jiangxiensis基本一致,而与S.hirsuta subsp.kobensis差异较大。鉴定菌株HA11090为Saccharopolyspora jiangxiensis,其发酵液具有较强的抗根结线虫活性,值得进一步研究。     

放线菌Snea253代谢产物及寡糖对南方根结线虫活性的影响

本文针对前期筛选获得的高效放线菌Snea253菌株代谢产物中杀线虫活性成分进行试管法和薄层层析法预试,结果显示活性物质含糖和氨基酸。贝氏皿浸没法研究了Snea253代谢产物中杀线虫活性物质和已知寡糖对南方根结线虫卵孵化和二龄幼虫J2的影响,结果表明:Snea253活性物质对南方根结线虫卵孵化的相对抑制率为84.95%,供试寡糖中除麦芽糖和蔗糖有不显著的抑制作用外,半乳糖、果糖和木糖显著促进了南方根结线虫卵孵化;乳糖、葡萄糖和山梨糖均具有促进作用,但与对照相比不显著。Snea253活性物质对J2的校正死亡率为98.21%,与各寡糖处理和对照相比差异显著;葡萄糖、乳糖和蔗糖处理J2无死亡现象,反而利于南方根结线虫J2的存活。研究结果显示普通寡糖对南方根结线虫基本无毒性,而放线菌Snea253代谢产物中氨基糖类物质则对线虫有毒性。     

蔬菜根结线虫生物防治的研究

对已筛选的蔬菜根结线病生防菌淡紫拟青霉菌（Paecileieomyeoceseieacinus）、厚壁抱予轮技菌（Verticilliumchlamydosporium）、节从孢属（ArthrobotrysSP）进行试验研究,证明这3种生防菌防效好、成本低、操作方便、可行性强、有着广阔的前景。     

烟草根结线虫生物防治研究进展*

简要介绍了烟草根结线虫生物防治生防资源收集、菌株筛选、制剂研制、生防菌相关生态学及分子标记的研究进展。     

朱砂莲果实化学成分杀南方根结线虫活性研究

为了解朱砂莲(Aristolochia tuberosa O.F.Liang et S.M.Huang)的化学成分,从其果实的甲醇提取物中分离得到4个化合物。通过波普数据分析,分别鉴定为马兜铃内酰胺W(1)、8-epidiosbulbin e acetate(2)、diosbulbin B(3)和β-sitosterol(4)。化合物1~3对南方根结线虫2龄幼虫具有不同程度的毒杀作用,尤其马兜铃内酰胺W(1)活性最好,其96 h后的LC50为119.94μg m L–1。马兜铃属植物具有开发为新型植物源杀根结线虫剂的潜在价值。     

不同AMF组合提高黄瓜抗根结线虫效果的比较

本研究旨在评价不同组合丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)抑制根结线虫和降低根结线虫病害的效应,以获得抗病作用较大的AMF组合。将由AMF光壁无梗囊霉Acaulospora laevis(Al)、摩西管柄囊霉Funneliformis mosseae(Fm)、幼套近明球囊霉Claroideoglomus etunicatum(Ce)、根内根孢囊霉Rhizophagus intraradices(Ri)、变形球囊霉Glomus versiforme(Gv)、珠状巨孢囊霉Gigaspora margarita(GIm)和全球盾巨孢囊霉Scutellospora aurigloba(SCa)组成的12个组合分别接种"津优35号"黄瓜,接种南方根结线虫Meloidogyne incognita并设置不接种对照。盆栽试验结果表明,接种AMF组合均能显著降低线虫卵囊数和每个卵囊的含卵数量,但Fm+Ri+GIm+SCa组合则增加根内二龄幼虫数、根内雌虫数和根内线虫总数;其他大多数组合能够抑制线虫的繁殖数量,其中,以Al+Fm+GIm及Fm+Ce+Gv组合抑制线虫生长发育的作用最大;除GIm+SCa组合外,其他AMF组合均能不同程度地降低线虫为害,防效达31%–83%,以Al+Fm+GIm组合处理的根结指数最低,防效最大。设施田间试验结果表明,以Al+Fm+GIm组合抗病作用最强。本研究表明,不同组合AMF防病效应差异显著,Al+Fm+Ce+Gv组合及Al+Fm+GIm是本试验条件下防治根结线虫病害效应最好的AMF组合,具有较大的应用潜力。     

根结线虫天敌真菌及其高效菌株筛选

本文报道捕食和寄生南方根结线虫(Meloidogyne incognita)和爪哇根结线虫(M.javanica)的天敌真菌6属14种。其中Arthrobotrys candida A.cladodes var.macroides,A.musiformis,Monacrosporium cionopagum,M.doedycoides,Drechmeria coniospora,Harposporium crassum,H.lilliputanum,Myzocytium humicola和Verticillum coccospora为我国新纪录种。室内测定结果表明:GACA 1B(Arthrobotrys oligospora)、GACM 102(Monacrosporium doedycoides)菌株对南方根结线虫和爪哇根结线虫均具极高的捕食率,接种线虫7天后,捕食率达98%以上,接种20天后,捕食率均达100%。     

球毛壳菌对植物病原真菌和根结线虫的生物防治潜力

球毛壳菌(Chaetomium globosum)隶属于子囊菌门、核菌纲、粪壳菌目、毛壳菌科、毛壳菌属真菌,广泛分布于空气、土壤等多种自然环境中,也是植物最常见的内生真菌之一。球毛壳菌能产生种类繁多的次级代谢产物,且其次级代谢产物具有抗真菌和杀线虫等多种生物活性,因而被制作成杀菌剂和杀线虫剂等生物农药,对植物病原真菌和根结线虫(Meloidogyne spp.)有良好的生物防治潜力。本文从球毛壳菌及其次级代谢产物抗植物病原真原菌和杀根结线虫两个方面进行综述,并探讨球毛壳菌对植物病原真菌的生防机制,为植物病原真菌及根结线虫的综合治理和新型生物农药的开发提供参考。     

Fungi and actinomycetes associated with Meloidogyne spp. eggs and females in China and their biocontrol potential

A survey was conducted to determine the microflora on eggs and females of Meloidogyne spp. collected from plant roots and infested soil in China. A total of 455 fungal isolates belonging to 24 genera and 52 isolates of actinomycetes were obtained from 28 samples from greenhouses and fields in Hainan, Yunnan, Fujian, Hebei, Shandong, and Beijing. The predominant fungal species were Paecilomyces lilacinus (49.3% of the isolates), Fusarium spp. (7.9%), Pochonia chlamydosporia (6.9%), Penicillium spp. (5.7%), Aspergillus spp. (3.2%), and Acremonium spp. (2.8%). Actinomycetes were frequently encountered (10.3%) as well. A total of 350 isolates of nematophagous fungi and actinomycetes were evaluated for their parasitism of eggs and effects on egg hatch and juvenile mortality in vitro. Pathogenicity varied among isolates, and 29.1% of isolates parasitized over 90% eggs 4 days after inoculation. Results also show that seven isolates of fungi and actinomycetes reduced egg hatch rates to less than 10% contrasted to the control of 65.8%, and three isolates killed all hatched juveniles after 7 days. Seventeen fungal isolates and four actinomycete isolates with high pathogenicity in vitro were selected to test biocontrol efficacy in the greenhouse. They reduced tomato root gall index by 13.4-58.9% compared to the no treatment control.     

The potential of Beauveria bassiana and Metarhizium anisopliae in controlling the root-knot nematode Meloidogyne incognita in tomato and cucumber

Meloidogyne incognita is one of the most important plant parasitic nematodes. This study was conducted to determine the nematicidal potential of Beauveria bassiana and Metarhizium anisopliae against M. incognita. B. bassiana and M. anisopliae spore suspensions and bio -nematicide, Purpureocillium lilacinum were applied. B. bassiana and M. anisopliae revealed considerable nematicidal activity against M. incognita in tomato and cucumber. The gall index decreased gradually from 8.0 for control to 3.2, 2.0 and 2.2 for B. bassiana, M. anisopliae and P. lilacinum in tomato, respectively. The highest reduction (%) in gall formation (control index) was calculated as 75.2 % in M. anisopliae treated group for tomato. The gall index was 7.6 for control, but decreased to 3.6, 2.0 and 2.2 for B. bassiana, M. anisopliae and P. lilacinum in cucumber, respectively. The highest control index was 71.7 % for M. anisopliae in cucumber. The number of the second instar juveniles of M. incognita was recorded as 2240 for control. However, this value reduced to 508, 332 and 328 by B. bassiana, M. anisopliae and P. lilacinum in tomato, respectively. The highest control indexes for the second instar juveniles were 85.2 % and 85.3 % for M. anisopliae and P. lilacinum in tomato, respectively. Alike, the highest control indexes were 84.9 % and 85.7 % for the same applications in cucumber, respectively. B. bassiana and M. anisopliae displayed also positive effect on the number of leaves, dry and fresh root weights of tomato. The results showed that B. bassiana and M. anisopliae can be considered as an alternative.     

Comparative analysis of two 14-3-3 homologues and their expression pattern in the root-knot nematode Meloidogyne incognita

14-3-3 proteins are highly conserved ubiquitous proteins found in all eukaryotic organisms. They are involved in various cellular processes including signal transduction, cell-cycle control, apoptosis, stress response and cytoskeleton organisation. We report here the cloning of two genes encoding 14-3-3 isoforms from the plant parasitic root-knot nematode Meloidogyne incognita, together with an analysis of their expression. Both genes were shown to be transcribed in unhatched second stage larvae, infective second stage larvae, adult males and females. The Mi-14-3-3-a gene was shown to be specifically transcribed in the germinal primordium of infective larvae, whereas Mi-14-3-3-b was transcribed in the dorsal oesophageal gland in larvae of this stage. The MI-14-3-3-B protein was identified by mass spectrometry in in vitro-induced stylet secretions from infective larvae. The stability and distribution of MI-14-3-3 proteins in host plant cells was assessed after stable expression of the corresponding genes in tobacco BY2 cells.     

Control of root-knot nematode,Meloidogyne incognita and reniform nematode,Rotylenchulus reniformis on pigeonpea and linseed

Abstract

Pigeonpea (Cajanus cajan) and linseed (Linum usitatissimum) are susceptible to Meloidogyne incognita and Rotylenchulus reniformis nematodes. Reduction in different growth parameters (length and weight of plant, number of pods), bulk density of pigeonpea stem, oil content of linseed, chlorophyll content of leaf and water absorption of roots caused by M. incognita and R. reniformis were statistically significant. Similar effects were also observed in plants raised from seeds soaked in different concentrations of water soluble fractions (WSF) of rice polish and pyridoxine solutions, however, the reductions were of a comparatively lesser extent. Higher concentrations of the solutions were more effective when compared to lower ones and pyridoxine was more beneficial than WSF for improving plant growth and reducing disease incidence.     

Overexpression of glutamate decarboxylase in transgenic tobacco plants confers resistance to the northern root-knot nematode

Previous research suggests that the endogenous synthesis of gamma-aminobutyrate (GABA), a naturally occurring inhibitory neurotransmitter, serves as a plant defense mechanism against invertebrate pests. Here, we tested the hypothesis that elevated GABA levels in engineered tobacco confer resistance to the northern root nematode (Meloidogyne hapla). This nematode species was chosen because of its sedentary nature and economic importance in Canada. We derived nine phenotypically normal, homozygous lines of transgenic tobacco (Nicotiana tabacum L.), which contain one or two copies of a full-length, chimeric tobacco glutamate decarboxylase (GAD) cDNA or a mutant version that lacks the autoinhibitory calmodulin-binding domain, under the control of a chimeric octopine synthase/mannopine synthase promoter. Regardless of experimental protocol, uninfected transgenic lines consistently contained higher GABA concentrations than wild-type controls. Growth chamber trials revealed that 9–12 weeks after inoculation of tobacco transplants with the northern root-knot nematode, mature plants of five lines possessed significantly fewer egg masses on the root surface when the data were expressed on both root and root fresh weight bases. Therefore, it can be concluded that constitutive transgenic expression of GAD conferred resistance against the root-knot nematode in phenotypically normal tobacco plants, probably via a GABA-based mechanism.     

来自西双版纳3种有毒植物的免培养与纯培养放线菌多样性及生物活性

【背景】由于土壤放线菌中获得新化合物日益困难,抗生素滥用使致病菌耐药性不断增加,人们转向研究植物内生放线菌以期发现新化合物。【目的】探究西双版纳热带雨林有毒植物内生放线菌的多样性,为开发新药提供具有潜在生物活性的菌株。【方法】通过Illumina Hi Seq高通量测序和纯培养方法分析箭毒木、八角枫、马缨丹3种有毒植物的内生放线菌群落结构组成,利用纸片扩散法筛选抑菌活性,通过PCR扩增检测7类化合物合成基因。【结果】高通量测序的多样性分析和群落结构分析得出:3种有毒植物在门分类水平检测出古菌域的2个门、细菌域的18个门和暂定的Rsa HF231、WD272门;在属分类水平检测出30个属的放线菌,八角枫和马缨丹的微生物群落结构比箭毒木更丰富。纯培养分离获得11个属34株菌,分离自箭毒木的菌株比八角枫和马缨丹的菌株更多,而且大多数高通量检测出的菌种不能通过纯培养获得。抑菌活性检测结果显示:抗菌活性作用明显的菌株以链霉菌属为主。链霉菌属的NRPS和PKS基因的检出率明显高于其他化合物合成基因。【结论】有毒植物内生放线菌多样性非常丰富。有毒植物内生放线菌具有合成次生代谢产物的潜力,可以为生物农药及抗生素开发提供丰富的菌种资源。     

Ecotypes of the model legume Lotus japonicus vary in their interaction phenotypes with the root-knot nematode Meloidogyne incognita

BACKGROUND AND AIMS: Knowledge of host factors affecting plant-nematode interactions is scarce. Here, relevant interaction phenotypes between a nodulating model host, Lotus japonicus, and the endoparasitic root-knot nematode Meloidogyne incognita are assessed via a genetic screen. METHODS: Within an alpha experimental design, 4-week-old replicate plants from 60 L. japonicus ecotypes were inoculated with 1000 nematodes from a single egg mass population, and evaluated for galling and nematode egg masses 6 weeks after inoculation. KEY RESULTS: Statistical analysis of data for 57 ecotypes showed that ecotype susceptibilities ranged from 3.5 to 406 galls per root, and correlated strongly (r = 0.8, P < 0.001, log scale) with nematode reproduction (ranging from 0.6 to 34.5 egg masses per root). Some ecotypes, however, showed a significant discrepancy between disease severity and nematode reproduction. Necrosis and developmental malformations were observed in other infected ecotypes. CONCLUSIONS: The first evidence is provided of significant variability in the interactions between L. japonicus and root-knot nematodes that may have further implications for the genetic dissection and characterization of host pathways involved in nematode parasitism and, possibly, in microbial symbiosis.     

新疆泥火山产酶嗜盐放线菌的筛选及多样性

[目的]了解新疆乌苏泥火山嗜盐放线菌及其产酶功能多样性.[方法]分别采用含有5%与10%NaCl的5种分离培养基,稀释平板涂布法对泥火山土壤样品进行分离;利用五种筛选培养基定性检测酶活性;在形态特征、耐盐性实验及16S rDNA基因测序的基础上进行系统发育学分析.[结果]获得嗜盐放线菌43株,极端嗜盐放线菌3株.4株嗜盐放线菌产脂肪酶,30株产半乳糖苷酶,27株产淀粉酶,6株产酯酶,4株产纤维素酶,1株同时产4种酶.系统发育学分析结果表明其中24株为拟诺卡氏菌属(Nocardiopsis),1株为链霉菌属(Streptomyces).产两种酶的菌株10006与Nocardiopsis exhalans(AY03600)相似性为96.64%(小于97%),可能是潜在的新种.[结论]本研究表明新疆乌苏泥火山中存在大量的产半乳糖苷酶及淀粉酶的嗜盐放线菌,所分离到的拟诺卡氏菌属产酶多样性比较高,并且潜藏着新的微生物资源.     

Piriformospora indica promotes cucumber tolerance against Root-knot nematode by modulating photosynthesis and innate responsive genes

Root Knot Nematode (RKN, Meloidogyne incognita) is one of the greatest damaging soil pathogens causes severe yield losses in cucumber and many other economic crops. Here, we evaluated the potential antagonistic effect of the root mutualistic fungus Piriformospora indica against RKN and their impact on vegetative growth, yield, photosynthesis, endogenous salicylic acid (SA) and its responsive genes. Our results showed that P. indica dramatically decreased the damage on shoot and root architecture of cucumber plants, which consequently enhanced yield of infested plants. Likewise, P. indica colonization clearly improved the chlorophyll content and delimited the negative impact of RNK on photosynthesis. Moreover, P. indica colonization exhibited a significant reduction of different vital nematological parameters such as soil larva density, amount of eggs/eggmass, eggmasses, females and amount of galls at cucumber roots. Additionally, the results showed that SA level was significantly increased generally in the roots of all treatments especially in plants infested with RKN alone as compared to control. This suggests that P. indica promoting SA levels in host cucumber plant roots to antagonize the RKN and alleviate severity damages occurred in its roots. This higher levels of SA in cucumber roots was consistent with the higher expressional levels of SA pathway genes PR1 and PR3. Furthermore, P. indica colonization reduces PR1, PR3 and increased NPR1 in roots of RKN infested cucumber plants when compared to non-colonized plants. Interestingly, our in vitro results showed that direct application of P. indica suspension against the J2s exhibited a significant increase in mortality ratio. Our results collectively suggest that P. indica promoting morphological, physiological and SA levels that might together play a major important role to alleviate the adverse impact of RKN in cucumber.     

RAPD linkage map of the genomic region encompassing the root-knot nematode (Meloidogyne javanica) resistance locus in carrot

Inheritance studies have indicated that resistance to the root-knot nematode (Meloidogyne javanica) in carrot inbred line ’Brasilia-1252’ is controlled by the action of one or two (duplicated) dominant gene(s) located at a single genomic region (designated the Mj-1 locus). A systematic search for randomly amplified polymorphic DNA (RAPD) markers linked to Mj-1 was carried out using bulked segregant analysis (BSA). Altogether 1000 ten-mer primers were screened with 69.1% displaying scorable amplicons. A total of approximately 2400 RAPD bands were examined. Four reproducible markers (OP-C2₁₇₀₀, OP-Q6₅₀₀, OP-U12₇₀₀, and OP-AL15₅₀₀) were identified, in coupling-phase linkage, flanking the Mj-1 region. The genetic distances between RAPD markers and the Mj-1 locus, estimated using an F₂ progeny of 412 individuals from ’Brasilia 1252’×’B6274’, ranged from 0.8 to 5.7 cM . The two closest flanking markers (OP-Q6₅₀₀ and OP-AL15₅₀₀) encompassed a region of 2.7 cM . The frequency of these RAPD loci was evaluated in 121 accessions of a broad-based carrot germplasm collection. Only five entries (all resistant to M. javanica and genetically related to ’Brasilia 1252’) exhibited the simultaneous presence of all four markers. An advanced line derived from the same cross, susceptible to M. javanica but relatively resistant to another root-knot nematode species (M. incognita), did not share three of the closest markers. These results suggest that at least some genes controlling resistance to M. incognita and M. javanica in ’Brasilia 1252’ reside at distinct loci. The low number of markers suggests a reduced amount of genetic divergence between the parental lines at the region surrounding the target locus. Nevertheless, the low rate of recombination indicated these markers could be useful landmarks for positional cloning of the resistance gene(s). These RAPD markers could also be used to increase the Mj-1 frequency during recurrent selection cycles and in backcrossing programs to minimize ’linkage drag’ in elite lines employed for the development of resistant F₁ hybrids. Received: 22 June 1999 / Accepted: 6 July 1999