海南岛象耳豆根结线虫的种类鉴定及其rDNA ITS序列分析

【背景】植物根结线虫病是世界性分布的土传病害,常造成农作物的重大经济损失。目前分布于热带亚热带区域的象耳豆根结线虫,由于其致病性和分子特征以及与植物互作关系的独特性,被认为是一种对农作物具有潜在危害性的重要病原根结线虫,因而引起国内外植物寄生线虫学者的广泛关注。【方法】用形态学、同工酶技术和分子生物学技术相结合的方法,对从海南岛农作物上采集到的10个根结线虫纯化种群进行分类鉴定。【结果】象耳豆根结线虫在海南岛大面积栽培的10种农作物和南药植物,包括黄瓜、南瓜、苦瓜、丝瓜、葫芦瓜、辣椒、番石榴、海巴戟、沈香和丁香上均有寄生,其形态学、酯酶表型和mtDNA PCR扩增产物均有别于常见的根结线虫种类;用引物18S和28S扩增象耳豆根结线虫种群的rDNA ITS区序列,并对其进行克隆、测序和比对分析,结果表明,象耳豆根结线虫4HBJ种群分别与南方根结线虫、爪哇根结线虫和花生根结线虫的同源性均仅为88%左右。【结论与意义】本文准确鉴定了象耳豆根结线虫,首次阐述其对海南岛多种农作物的致害性;阐释了象耳豆根结线虫的形态和分子特征,并明确了其与3种常见根结线虫的系统发育关系。本研究对今后进一步开展象耳豆根结线虫的基础研究和防治工作具有重要参考价值。     

海南岛象耳豆根结线虫的种类鉴定及其rDNA-ITS序列分析

【背景】植物根结线虫病是世界性分布的土传病害,常造成农作物的重大经济损失。目前分布于热带亚热带区域的象耳豆根结线虫,由于其致病性和分子特征以及与植物互作关系的独特性,被认为是一种对农作物具有潜在危害性的重要病原根结线虫,因而引起国内外植物寄生线虫学者的广泛关注。【方法】用形态学、同工酶技术和分子生物学技术相结合的方法,对从海南岛农作物上采集到的10个根结线虫纯化种群进行分类鉴定。【结果】象耳豆根结线虫在海南岛大面积栽培的10种农作物和南药植物,包括黄瓜、南瓜、苦瓜、丝瓜、葫芦瓜、辣椒、番石榴、海巴戟、沈香和丁香上均有寄生,其形态学、酯酶表型和mtDNA-PCR扩增产物均有别于常见的根结线虫种类;用引物18S和28S扩增象耳豆根结线虫种群的rDNA-ITS区序列,并对其进行克隆、测序和比对分析,结果表明,象耳豆根结线虫4HBJ种群分别与南方根结线虫、爪哇根结线虫和花生根结线虫的同源性均仅为88%左右。【结论与意义】本文准确鉴定了象耳豆根结线虫,首次阐述其对海南岛多种农作物的致害性;阐释了象耳豆根结线虫的形态和分子特征,并明确了其与3种常见根结线虫的系统发育关系。本研究对今后进一步开展象耳豆根结线虫的基础研究和防治工作具有重要参考价值。     

基于16S rDNA基因的谷斑皮蠹PCR检测技术

【目的】谷斑皮蠹是一种重要的检疫性害虫,在新疆周边多个国家分布,口岸检疫人员多次从进境货物中截获谷斑皮蠹,该虫对新疆的农业生产极具威胁。【方法】以谷斑皮蠹的16S rDNA基因为靶序列,用昆虫通用引物对4种供试皮蠹进行PCR扩增,将扩增产物进行克隆和测序,用生物软件设计检测谷斑皮蠹的特异性引物与探针。【结果】设计的特异性引物(TG-SNP-F/TG-SNP-R)及所建立的常规PCR方法能有效检测出谷斑皮蠹,其扩增产物的片段大小为250 bp,灵敏度为3 ng·μL~(-1)。设计的特异性引物(TG-F/TG-R)和探针(TG-probe),以及所建立的实时荧光PCR方法,对谷斑皮蠹的检测特异性强,灵敏度达0.8 fg·μL~(-1)。【结论】建立的常规PCR方法和实时荧光PCR检测方法能够对谷斑皮蠹进行准确鉴定,为口岸检疫人员检测进境货物中携带的谷斑皮蠹提供技术支持。     

M60家族肽酶参与调控蜡质芽胞杆菌AR156防治南方根结线虫病的作用机制

【背景】南方根结线虫(Meloidogyne incognita)寄主范围广泛,能侵染茄科、豆科、葫芦科等蔬菜,对世界农业生产造成了严重危害,每年造成的损失可高达数百亿美元。蜡质芽胞杆菌(Bacillus cereus) AR156是一种革兰氏阳性杆状细菌,对于南方根结线虫引起的土传病害有较好的防治效果,并且已经完成了全基因组测序。【目的】了解AR156对南方根结线虫的生防机理,寻找与生防功能相关的基因,进行遗传功能基因的分析。【方法】构建蜡质芽胞杆菌AR156 miniTn10随机插入突变体文库,通过对南方根结线虫致死率和温室防病试验进行筛选,找到与AR156生防能力相关的突变体,鉴定相关功能基因,并对产蛋白酶活性、定殖能力、生物膜形成和游动性等方面进行检测。【结果】与AR156野生型相比,转座子插入位点在M60家族肽酶的突变体BC41产蛋白酶活性下降,植物根围定殖能力减弱,生物膜形成能力和游动性受到影响,从而导致其防治南方根结线虫的能力降低。【结论】通过对生防相关功能基因的分析,初步明确M60家族肽酶在蜡质芽胞杆菌AR156防治南方根结线虫的过程中发挥重要作用。     

植物青枯病菌环介导等温扩增快速检测技术研究

为实现植物青枯病的早期诊断,需要建立一种适于田间快速便捷检测青枯病菌的方法。以细胞色素C基因为靶标设计一套特异性引物,建立了植物青枯病的LAMP检测方法。此方法最低检测极限为1 pg,可在1 h内完成,不依赖昂贵复杂的仪器,结果可经肉眼观察。利用此方法,在人工接种发病的茄子、番茄、花生、芝麻和凹头苋茎部浸出液和马铃薯病薯块茎组织液中均检测出青枯病菌的存在,尤其适用于田间疑似罹病的芝麻、花生、番茄、马铃薯和甘薯等植株的检测,且LAMP法的检出率远高于PCR法。应用LAMP技术检测青枯病菌快速高效、特异性强、灵敏度高,操作简单,适于在基层推广运用。     

环介导等温扩增技术在食品中痢疾志贺氏菌快速检测

【目的】探讨、优化基于环介导等温扩增技术(LAMP)快速检测常规食品中感染性痢疾志贺氏菌的方法。【方法】在NCBI数据库中搜索获取志贺氏菌的特异性基因高度保守区,设计3对LAMP反应引物,建立、优化该LAMP可视化检测方法,并评价其特异性、灵敏度,同时与PCR检测方法和传统检测方法对比,进行结果统计分析。【结果】5株志贺氏菌标准菌株样品均检测为阳性,11株非志贺氏菌标准菌株样品均检测为阴性,无交叉反应。最低检验限为1.6×101 CFU/反应(或1.6×101 CFU/m L),且经比较,LAMP检测灵敏度比PCR检测高出1个数量级。通过对161份实际样品和人工污染样品进行检测,LAMP检测与传统方法检测结果具有较高的一致性。【结论】LAMP具有检测过程快速简便、检测结果稳定、可靠的特点,适用于对常规食品中感染性痢疾志贺氏菌的高效、快速检测需求。     

一种快速检测桉树焦枯病菌(Cylindrocladium scoparium)的LAMP体系的建立及应用

【目的】建立桉树焦枯病快速有效的环介导等温扩增反应LAMP,检测技术。【方法】以桉树焦枯病原菌Cylindrocladium scoparium为研究对象,利用Primer Explorer V4.0设计软件,针对C. scoparium的beta-tubulin gene特异保守区域设计了一套LAMP特异性引物组(内引物FIP/BIP,外引物F3/B3,环引物LooPF/LooPB),优化并建立了桉树焦枯病原菌的LAMP快速检测体系。【结果】LAMP反应体系为(50 μL)：Bst DNA polymerase (8 U) 1 μL;甜菜碱溶液(5 mol/L) 3.0 μL;dNTPs (2.5 mmol/L) 3.5 μL;10×Thermopol Ⅱ 2.5 μL;MgCl2 (100 mmol/L) 2 μL;FIP/BIP (40 μmol/L)各1 μL;F3/B3 (10 μmol/L)各0.5 μL;LooPF/LooPB (10 μmol/L)各1 μL;DNA模板2 μL;用ddH2O补足体积至50 μL。反应程序为：65 °C水浴锅中反应45 min,90 °C灭活5 min结束反应。特异性检测结果显示,供试的12个样本菌株LAMP产物可以采用肉眼观察、紫外灯照射、添加荧光染料SYBR Green I以及电泳检测条带4种不同的形式予以区分。DNA灵敏度检测结果显示,建立的LAMP体系检测灵敏度可达到40 μg/L,完全符合田间检测的要求。野外田间时效检测结果显示,无论是利用紫外检测还是电泳检测均可成功地检测出各地区不同生理小种的桉树焦枯病原菌C. scoparium,且检测效果明显。【结论】该LAMP检测是一项能够作为野外基层田间检测的重要技术。     

水霉菌环介导等温扩增检测方法的建立

【目的】建立一种快速简单检测水霉病病原菌的方法。【方法】针对水霉菌ITS区基因序列设计4条特异性引物,包括两条外引物和两条内引物,优化反应条件,观察检测结果。对该方法的特异性和敏感性进行研究。【结果】建立了环介导等温扩增技术检测水霉菌的方法,确定了其最适反应条件。该方法能够检测到浓度低至103个/mL的水霉菌孢子,其灵敏度是普通PCR方法的100倍。【结论】建立的检测水霉菌的LAMP技术,具有操作简便快速等特点,可用做特异性水霉及其孢子的快速鉴定。     

大豆疫霉菌(Phytophthora sojae)无毒基因Avr1a、Avr1k及Avr3a的分子鉴定

【目的】为大豆疫霉菌(Phytophthora sojae)无毒基因Avr1a、Avr1k和Avr3a快速分子检测提供方法,也为P.sojae其它无毒基因的快速分子检测研究提供依据。【方法】依据GenBank中公布的P.sojae无毒基因Avr1a、Avr1k和Avr3a的序列设计引物,分别筛选出特异性引物,在PCR反应体系和扩增条件优化基础上,对已经接种鉴定过无毒基因Avr1a、Avr1k和Avr3a的86株P.sojae进行PCR检测,建立一套P.sojae无毒基因Avr1a、Avr1k和Avr3a的特异性检测体系。将分子鉴定和接种鉴定结果进行比对,将扩增出的真阳性条带和假阳性条带分别进行胶回收和克隆测序,测序结果分别与3个无毒基因的原序列比对,判定分子标记方法是否适于Avr1a、Avr1k和Avr3a的快速检测。【结果】筛选出的特异性引物均能从含有对应无毒基因的菌株中扩增出约550 bp的条带。Avr1a、Avr1k和Avr3a的分子鉴定及接种鉴定结果符合率依次为45.3%、84.9%和97.7%。3个无毒基因的真阳性条带序列与原序列一致性均达97%以上,Avr1a的假阳性条带与原序列一致性在80%左右,其余2个基因的都在30%以下。【结论】利用Avr1a、Avr1k和Avr3a基因序列分别设计引物建立的检测体系可以用于Avr3a的快速检测,不适于Avr1a的快速检测,是否适合Avr1k的快速检测尚不清楚。     

环介导等温扩增联合横向流动试纸条可视化检测志贺氏菌

【目的】将环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)联合应用,建立一种可应用于志贺氏菌快速检测的LAMP-LFD技术。【方法】以福氏志贺氏菌的侵袭性质粒抗原H(ipa H)基因为检测靶标设计3对特异性引物(其中上游内引物Sfl-ipa H-FIP由生物素标记),进行LAMP反应;同时设计1条异硫氰酸荧光素(FITC)标记的探针Sfl-ipa H-HP,与获得的LAMP产物进行特异性杂交,杂交产物经LFD完成检测。【结果】优化后的LAMP反应条件为63°C 40 min,加上LFD结果判读共需50 min。LAMP-LFD方法能够特异性检测出福氏志贺氏菌,而对肠炎沙门氏菌等其它4种导致腹泻的致病菌和创伤弧菌等5种常见食物源性致病菌,以及4株不同大肠杆菌的检测结果呈阴性。该方法针对福氏志贺氏菌的检测灵敏度为1.0×10~2 CFU/m L或4 CFU/反应,针对人工污染鲤鱼肠组织的检测灵敏度是5.0×10~2 CFU/m L,是以LAMP外引物Sfl-ipa H-F3/Sfl-ipa H-B3的常规PCR方法的100倍。【结论】建立的LAMP-LFD技术具有操作简单、检测快速准确、检测成本低等优点,有望在志贺氏菌的常规监测和即时检测中被普及使用。     

Comprehensive report on the prevalence of root-knot nematodes in the Poonch division of Azad Jammu and Kashmir,Pakistan

The present study documents the root-knot nematodes (RKN) fauna of the Poonch division in Azad Jammu and Kashmir infecting vegetables. An overall prevalence of 40% of RKN was recorded. Of the four districts investigated, maximum prevalence was recorded in district Poonch with 59%, followed by Sudhnuti with 58%. The lowest prevalence of RKN was found in districts Bagh (29%) and Haveli (33%). Out of 15 vegetables investigated, RKN was found on five crops. The highest prevalence of 37.8% was recorded on okra, followed by 31.3% on cucumber and 17.5% on tomato. RKN was less prevalent on eggplant (8.3%) and beans (7.7%). Three RKN species, that is Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria, were found infecting the hosts. M. javanica was found to be the most prevalent followed by M. incognita and M. arenaria. This trend was found in all the districts. Overall prevalence of M. javanica as sole population was 9% and that of M. incognita was 2%. Meloidogyne arenaria was not found in any of the fields as sole population. The prevalence of M. incognita with M. javanica or M. arenaria as mixed populations was 8% and 5%, respectively, and that of M. javanica with M. arenaria was 4%. Similarly, all the three species prevailed as mixed populations in 12% of the fields in the division. The severity of RKN infections, measured as galling index, was found to be variable within each infected field (GI 2–9). Identification of RKN species was based on the morphology of perineal patterns and confirmed by molecular SCAR and CO1 makers based identification. In conclusion, RKN were distributed in the Poonch division and M. javanica was predominant. Cucumber, okra, tomato and eggplant were severely attacked by these nematodes warranting the adoption of stringent control strategies for their management.     

Characterization of the <Emphasis Type="Italic">RMja</Emphasis> gene for resistance to root-knot nematodes in almond: spectrum,location, and interest for <Emphasis Type="Italic">Prunus</Emphasis> breeding

In Prunus spp., resistance genes to root-knot nematodes (RKN), Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica, and Meloidogyne floridensis, confer either a complete spectrum, e.g., the Ma and Rjap genes in Myrobalan and Japanese plums (subgenus Prunophora), respectively, or a more restricted spectrum, e.g., the RMia gene (M. arenaria + M. incognita) in peach (subgenus Amygdalus). We report here characterization data of the RMja gene from the almond Alnem1, another Amygdalus source. The study of its spectrum is hampered by the inability of almond to be propagated by cuttings; we overcame this problem by using F1 and BC1 crosses with previously genotyped Myrobalan plums that conferred their rooting ability to hybrids for simultaneous evaluation to different RKN. As expected from a homozygous dominant resistance, BC1 progenies of Alnem1 segregated for resistance to M. javanica but were uniformly susceptible to M. incognita and M. floridensis, demonstrating that RMja controlled M. javanica but not M. incognita nor M. floridensis. SSR markers covering the Prunus reference map placed RMja on LG7 in the same region as Ma and Rjap and thus showed its independence from the RMia gene (LG2) of the botanically closer peach. The spectrum of this gene allows the theoretical construction of interspecific rootstocks, Myrobalan plum × (almond × peach), which cumulate RMja with Ma and RMia and are protected from each of the predominant RKN affecting Prunus, i.e., M. arenaria, M. incognita, and M. javanica, by at least two genes. This pyramiding strategy should offer to rootstock material an unprecedented guarantee of durable RKN resistance.     

Interaction of vesicular arbuscular mycorrhiza with root knot nematodes in tomato

Summary The interaction between the VA mycorrhizal fungus,Glomus fasciculatus and the root-knot nematodes,Meloidogyne incognita andM. javanica, and their effects on the growth and phosphorus nutrition of tomato was studied in a red sandy loam soil of pH 6.0. Inoculation of tomato roots with root-knot nematodes enhanced infection and spore production byG. fasciculatus. Inoculation of tomato plants withG. fasciculatus significantly reduced the number and size of the root-knot galls produced byM. incognita andM. javanica. Inoculation withG. fasciculatus although improved plant growth and its total phosphorus content compared to the uninoculated plants, the difference were not statistically significant.     

Species-Specific Restriction Site Polymorphism in Root-knot Nematode Mitochondrial DNA

Research was initiated to physically characterize the mitochondrial genomes of several Meloidogyne spp. and host-races, to address questions regarding their systematics and dispersal, and to assess the possibility of developing molecular diagnostics for these nematodes. Techniques were developed for purification and rapid detection of mitochondrial DNA from root-knot nematodes. Mitochondrial DNAs among Meloidogyne spp. were demonstrated to exhibit extensive divergence. The potential for using the rapidly diverging mitochondrial genomes as a diagnostic assay for M. incognita, M. hapla, M. arenaria, and M. javanica is discussed.     

Comparative Studies on Root Invasion,Root Galling,and Fecundity of Three Meloidogyne spp. on a Susceptible Tobacco Cultivar

Root invasion, root galling, and fecundity of Meloidogyne javanica, M. arenaria, and M. incognita on tobacco was compared in greenhouse and controlled environment experiments. Significantly more M. javanica than M. arenaria or M. incognita larvae were found in tobacco roots at 2, 4, and 6 d after inoculation. Eight days after inoculation there were significantly more M. arenaria and M. javanica than M. incognita larvae. Ten days after inoculation no significant differences were found among the three Meloidogyne species inside the roots. Galls induced by a single larva or several larvae of M. javanica were significantly larger than galls induced by M. incognita: M. arenaria galls were intermediate in size. Only slight differences in numbers of egg masses or numbers of eggs produced by the three Meloidogyne species were observed up to 35 d after inoculation.     

Penetration of Susceptible and Resistant Tobacco Cultivars by Meloidogyne Juveniles

Rates of penetration of Meloidogyne incognita, M. arenaria, and M. javanica into tobacco cultivars NC2326 (susceptible to all three species) and K399 (resistant to M. incognita) and a breeding line that had been selected for resistance to M. incognita were compared. Meloidogyne incognita penetrated NC2326 rapidly during the first 24 hours after inoculation. Numbers of M. incognita continued to increase gradually through the 14-day experiment. Higher numbers of M. incognita were observed in the roots of K399 during the first 24 hours than were observed in NC2326. The number of M. incognita in K399 peaked 4 days after inoculation, then declined rapidly as the nematodes that were unable to establish a feeding site left the root or died. Numbers of M. incognita in the breeding line followed the same pattern as with K399, but in lower numbers. Numbers of M. arenaria showed little difference between cultivars until 7 days after inoculation, then numbers increased in NC2326. Numbers of M. javanica fluctuated in all cultivars, resulting in patterns of root population different from those observed for M. incognita or M. arenaria. Resistance to M. incognita appears to be expressed primarily as an inability to establish a feeding site rather than as a barrier to penetration. Some resistance to M. arenaria may also be present in K399 and the breeding line.     

Comparison of Sequences from the Ribosomal DNA Intergenic Region of Meloidogyne mayaguensis and Other Major Tropical Root-Knot Nematodes

The unusual arrangement of the 5S ribosomal gene within the intergenic sequence (IGS) of the ribosomal cistron, previously reported for Meloidogyne arenaria, was also found in the ribosomal DNA of two other economically important species of tropical root-knot nematodes, M, incognita and M. javanica. This arrangement also was found in M. hapla, which is important in temperate regions, and M. mayaguensis, a virulent species of concern in West Africa. Amplification of the region between the 5S and 18S genes by PCR yielded products of three different sizes such that M. mayaguensis could be readily differentiated from the other species in this study. This product can be amplified from single juveniles, females, or egg masses. The sequences obtained in this region for one line of each of M. incognita, M. arenaria, and M. javanica were very similar, reflecting the close relationships of these lineages. The M. mayaguensis sequence for this region had a number of small deletions and insertions of various sizes, including possible sequence duplications.     

Identification of Sources of Resistance to Four Species of Root-knot Nematodes in Tobacco

Resistance to the southern root-knot nematode, Meloidogyne incognita races 1 and 3, has been identified, incorporated, and deployed into commercial cultivars of tobacco, Nicotiana tabacum. Cultivars with resistance to other economically important root-knot nematode species attacking tobacco, M. arenaria, M. hapla, M. javanica, and other host-specific races of M. incognita, are not available in the United States. Twenty-eight tobacco genotypes of diverse origin and two standard cultivars, NC 2326 (susceptible) and Speight G 28 (resistant to M. incognita races 1 and 3), were screened for resistance to eight root-knot nematode populations of North Carolina origin. Based on root gall indices at 8 to 12 weeks after inoculation, all genotypes except NC 2326 and Okinawa were resistant to M. arenaria race 1, and races 1 and 3 of M. incognita. Except for slight root galling, genotypes resistant to M. arenaria race 1 responded similarly to races 1 and 3 of M. incognita. All genotypes except NC 2326, Okinawa, and Speight G 28 showed resistance to M. javanica. Okinawa, while supporting lower reproduction of M. javanica than NC 2326, was rated as moderately susceptible. Tobacco breeding lines 81-R-617A, 81-RL- 2K, SA 1213, SA 1214, SA 1223, and SA 1224 were resistant to M. arenaria race 2, and thus may be used as sources of resistance to this pathogen. No resistance to M. hapla and only moderate resistance to races 2 and 4 of M. incognita were found in any of the tobacco genotypes. Under natural field infestations of M. arenaria race 2, nematode development on resistant tobacco breeding lines 81-RL-2K, SA 1214, and SA 1215 was similar to a susceptible cultivar with some nematicide treatments; however, quantity and quality of yield were inferior compared to K 326 plus nematicides.     

Nucleotide Substitution Patterning within the Meloidogyne rDNA D3 Region and Its Evolutionary Implications

Evolutionary relationships based on nucleotide variation within the D3 26S rDNA region were examined among acollection of seven Meloidogyne hapla isolates and seven isolates of M. arenaria, M. incognita, and M. javanica. Using D3A and D3B primers, a 350-bp region was PCR amplified from genomic DNA and double-stranded nucleotide sequence obtained. Phylogenetic analyses using three independent clustering methods all provided support for a division between the automictic M. hapla and the apomictic M. arenaria, M. incognita, and M. javanica. A nucleotide sequence character distinguishing M. hapla from the three apomictic species was a 3-bp insertion within the interior of the D3 region. The three apomictic species shared a common D3 haplotype, suggesting a recent branching. Single M. hapla individuals contained two different haplotypes, differentiated by a Sau3AI restriction site polymorphism. Isolates of M. javanica appeared to have only one haplotype, while M. incognita and M. arenaria maintained more than one haplotype in an isolate.     

Comparisons of Isozyme Phenotypes in Five Meloidogyne spp. with Isoelectric Focusing

Meloidogyne incognita race 1, M. javanica, M. arenaria race 1, M. hapla, and an undescribed Meloidogyne sp. were analyzed by comparing isozyme phenotypes of esterase, malate dehydrogenase, phosphoglucomutase, isocitrate dehydrogenase, and α-glycerophosphate dehydrogenase. Isozyme phenotypes were obtained from single mature females by isoelectric focusing electrophoresis. Of these five isozymes, only esterase and phosphoglucomutase could be used to separate all five Meloidogyne spp.; however, the single esterase electromorphs were similar for M. incognita and M. hapla. Yet when both nematodes were run on the same gel, differences in their esterase phenotypes were detectable. Isozyme phenotypes from the other three isozymes revealed a great deal of similarity among M. incognita, M. javanica, M. arenaria, and the undescribed Meloidogyne sp.