草地贪夜蛾和其它五种重要鳞翅目害虫蛹的形态特征比较

【目的】田间草地贪夜蛾Spodopterafrugiperda(J.E.Smith)蛹与斜纹夜蛾Spodopteralitura Fabricius、甜菜夜蛾Spodoptera exigua(Hübner)、粘虫Mythimna separata(Walker)、棉铃虫Helicoverpa armigera(Hübner)和亚洲玉米螟Ostrinia furnacalis 5种重要鳞翅目害虫蛹容易混淆。明确草地贪夜蛾与其它5种重要的鳞翅目害虫蛹的形态差异,为田间鳞翅目害虫蛹的准确鉴别和田间调查提供依据。【方法】采用体式显微镜拍照观察并分析草地贪夜蛾蛹、斜纹夜蛾蛹、甜菜夜蛾蛹、粘虫蛹、棉铃虫蛹和亚洲玉米螟蛹的外部形态特征,根据相关特征差异编制了区分草地贪夜蛾与此5种鳞翅目害虫的检索表和快速区分表。【结果】草地贪夜蛾蛹主要特征为中足末端到达下颚末端之前,触角末端到达中足末端之前;中胸背板前缘略直;第4-7腹节背面前缘着生多行粗点刻;腹部末端臀棘上着生1对基部分开呈八字形的刺。草地贪夜蛾蛹腹部末端臀棘部位与其它5种害虫均不相同,辅以附肢位置、中胸背板前缘线和腹部点刻等特征可以准确将其与斜纹夜蛾、甜菜夜蛾、粘虫、棉铃虫和亚洲玉米螟的蛹区分。斜纹夜蛾中胸背板前缘线和臀棘上的刺与草地贪夜蛾有异;甜菜夜蛾下颚与中足位置及臀棘上的刺与草地贪夜蛾有异;粘虫腹部点刻和臀棘上的刺与草地贪夜蛾有异;棉铃虫触角末端位置和腹部末端的刺与草地贪夜蛾有异;亚洲玉米螟腹足遗迹和臀棘上的刺与草地贪夜蛾有异。【结论】根据蛹的结构特征,可以区分草地贪夜蛾与斜纹夜蛾、甜菜夜蛾、粘虫、棉铃虫和亚洲玉米螟等害虫的蛹。     

四种鳞翅目害虫精氨酸激酶基因的表达谱及基于RNAi的功能分析

【目的】精氨酸激酶(arginine kinase, AK)(EC 2.7.3.3)是昆虫体内重要的磷酸原激酶(能量代谢调节因子),也是唯一能够形成有效ATP的磷酰基供体,起着与脊椎动物中肌酸激酶相同的作用。本研究旨在了解鳞翅目害虫AK基因的表达和功能。【方法】利用qRT-PCR方法测定AK基因在大螟Sesamia inferens、二化螟Chilo suppressalis、甜菜夜蛾Spodoptera exigua和斜纹夜蛾Spodoptera litura 这4种鳞翅目害虫不同发育阶段和3龄幼虫不同组织中的表达谱;通过终点法检测了这4种害虫不同发育阶段和幼虫不同组织中的AK酶活性;采用RNAi技术抑制该基因的表达并分析其功能。【结果】AK基因在大螟、二化螟、甜菜夜蛾和斜纹夜蛾这4种鳞翅目昆虫的不同发育阶段和3龄幼虫不同组织中均有表达,说明该基因的表达不具有发育时期和组织特异性。不同发育时期和3龄幼虫不同组织中AK酶活性与基因表达量变化趋势大体一致。注射以AK基因为靶标的dsRNA 6 d后,4种害虫体内AK基因的mRNA表达下降30%~50%,AK酶活性降低30%左右;14 d后幼虫的死亡率达50%左右,显著高于对照组幼虫的死亡率。【结论】AK基因在上述4种鳞翅目害虫中为组成型表达,RNAi抑制AK基因的表达可导致4种害虫的幼虫死亡,研究结果为开发以AK基因为靶标的鳞翅目害虫防治新技术提供了理论依据。     

甘蓝夜蛾和八字地老虎肌动蛋白基因的克隆与序列分析

肌动蛋白是细胞骨架微丝的主要组成成分,在肌肉收缩、细胞骨架形成、细胞移动等方面起重要作用。以鳞翅目夜蛾科昆虫甘蓝夜蛾Mamestra brassicae L.和八字地老虎Agrotis c-nigrum 3龄幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到2种昆虫的肌动蛋白的cDNA序列,甘蓝夜蛾肌动蛋白的cDNA序列含有1441个碱基,而八字地老虎肌动蛋白的cDNA序列含有1411个碱基。2种昆虫的该基因的cDNA序列均包括1个1131个碱基的开放阅读框,编码1个含376个氨基酸的蛋白。甘蓝夜蛾肌动蛋白分子量约为41.8kDa;八字地老虎肌动蛋白分子量约为41.9kDa。Prosite软件分析结果表明,甘蓝夜蛾和八字地老虎肌动蛋白氨基酸序列中存在3个肌动蛋白特征片段。GenBank数据库搜索及序列比对结果表明,甘蓝夜蛾肌动蛋白属于肌肉特异型肌动蛋白,八字地老虎肌动蛋白属于细胞质特异型肌动蛋白。2个基因的cDNA序列已经登录GenBank并获得登录号,甘蓝夜蛾肌动蛋白cDNA序列登录号为EU035314,八字地老虎肌动蛋白cDNA序列登录号为EU035315。利用RT-PCR技术在八字地老虎4龄、5龄、6龄幼虫、蛹期4个不同发育阶段和6龄期的肠道、体壁、脂肪体3种不同组织中都检测到了肌动蛋白基因在mRNA水平的表达。     

利用mtCOI PCR-RFLP技术鉴别草地贪夜蛾与其他3种形态相近昆虫

【目的】田间调查发现草地贪夜蛾与甜菜夜蛾、斜纹夜蛾、粘虫常混合发生,传统的形态学鉴定方法不能快速鉴别出该虫,当前亟需快速鉴别该虫的方法。【方法】本研究分析了草地贪夜蛾与甜菜夜蛾、斜纹夜蛾、粘虫mtCOI基因序列的酶切位点,根据目的片段设计上游引物并进行PCR-RFLP验证。【结果】草地贪夜蛾个体在mtCOI片段的556~561 bp处均存在Sbf I内切酶酶切位点,斜纹夜蛾、甜菜夜蛾、粘虫均无Sbf I酶切位点。草地贪夜蛾PCR产物经过Sbf I内切酶酶切,可出现420 bp左右的特征带,斜纹夜蛾、甜菜夜蛾、粘虫种群均不能被Sbf I内切酶酶切。【结论】基于新设计引物扩增的mtCOI片段的PCR-RFLP方法可有效鉴别草地贪夜蛾与其他3个形态相近昆虫,研究结果为草地贪夜蛾的快速鉴别提供了方法。     

Cry1C蛋白对4种棉花害虫的致死效果

【背景】苏云金芽胞杆菌(Bt)属革兰氏阳性细菌,是目前世界上应用最广泛的杀虫剂。来自Bt的Cry1Ac基因自1997年商业化以来,其产生的蛋白对棉铃虫、红铃虫等鳞翅目害虫起到了很好的控制作用。但该基因对甜菜夜蛾、斜纹夜蛾、玉米螟等害虫的控制作用较差,因此,需要寻找新的Bt基因亚型控制上述害虫。【方法】将Cry1C蛋白加到人工饲料上形成药膜后,接入低龄幼虫进行生物测定,以常规不加毒蛋白的饲料为对照,计算1、3、5、7 d后不同害虫的校正死亡率。【结果】随着Cry1C蛋白浓度的增加及时间的延长,甜菜夜蛾和斜纹夜蛾的校正死亡率逐渐升高,Cry1C浓度为10μg·m L-1时,处理7 d后,甜菜夜蛾和斜纹夜蛾的校正死亡率分别为100%和85.42%,表明甜菜夜蛾比斜纹夜蛾更敏感,而棉铃虫和玉米螟对Cry1C蛋白的敏感性较差。【结论与意义】Cry1C基因可作为控制棉田甜菜夜蛾和斜纹夜蛾的有价值的候选基因,但是对棉铃虫和玉米螟无效。该研究为筛选新的Bt基因亚型用于防治不同的棉花害虫提供了数据参考。     

利用末龄幼虫蜕和蛹壳对单头昆虫基因的无损伤检测

【目的】在昆虫基因功能等相关研究中,通常需要利用单对交配策略来筛选纯合突变品系,如何在配对前确定个体基因型同时又不对昆虫造成损伤,显得尤为重要。本文旨在探讨利用末龄幼虫蜕和蛹壳进行单头昆虫的无损伤基因检测方法。【方法】针对大小不同的3种鳞翅目昆虫斜纹夜蛾Spodoptera litura Fabricius、二点委夜蛾Athetis lepigone M?schler和小菜蛾Plutella xyllostella Linnaeus,收集末龄幼虫蜕及蛹壳,利用常规分子生物学技术进行基因组DNA提取、靶标基因PCR扩增、琼脂糖凝胶电泳检测、连接转化和单克隆测序验证。【结果】从斜纹夜蛾、二点委夜蛾末龄幼虫蜕和蛹壳提取的基因组DNA,以其为模板对GOBP1基因进行PCR扩增,产物经琼脂糖凝胶电泳检测得到单一、明显的条带,进一步经连接转化和单克隆测序得到目的序列;但由于小菜蛾末龄幼虫蜕和蛹壳太小,以同样方法提取的基因组DNA浓度太低,PCR产物经电泳检测,未能得到目的条带。【结论】对于与斜纹夜蛾和二点委夜蛾相近或更大的昆虫,可以利用单头末龄幼虫蜕或蛹壳提取基因组DNA,通过常规PCR技术克隆特定基因序列,为突变品系筛选过程中昆虫个体的无损伤基因型检测提供了方法。     

支持向量机和邻接法在夜蛾科昆虫条码研究中的应用

【背景】鳞翅目夜蛾科昆虫种类繁多,目前已经超过3．5万种,绝大多数是农林生产的主要害虫。由于多数近缘属种形态相似,难以鉴定,给农林害虫的防治工作带了很大的困难。DNA条形码技术是一种快速、准确鉴定物种的方法。支持向量机作为一种新的机器学习方法,自1995年被提出以来已经在数据分类和高维模式识别等领域取得不错的效果。【方法】将北京妙峰山采集的58种夜蛾101个样品的COI序列分成3套数据集,分别通过邻接法和支持向量机对其进行验证。【结果】通过对DNA条形码物种鉴定结果的验证表明,邻接法优于支持向量机。但DNA条形码在鉴定夜蛾科的一些近缘种上,效果不佳,如棉铃虫和烟青虫。【结论与意义】DNA条形码作为一种新兴的物种鉴定方法,在分类学上具有很高的应用价值。通过邻接法和支持向量机的比较,虽然支持向量机的成功率低于邻接法,但是其在DNA条形码中的应用是对数据问询方式的一种探索。     

不同温度下斜纹夜蛾的两性生命表

【目的】斜纹夜蛾Spodoptera litura(Fabricius)是一种鳞翅目夜蛾科食叶害虫,在不同的寄主、饲料等条件下表现出不同的生长发育特点。本实验旨在探究温度对斜纹夜蛾生长发育状况和种群参数的影响。【方法】在室内人工气候箱20±1℃,27±1℃和30±1℃(相对湿度和光周期分别为65%±10%和14L∶10D)条件下饲养斜纹夜蛾,观察和组建斜纹夜蛾在3种温度下的年龄-阶段两性生命表。【结果】斜纹夜蛾各个阶段的发育历期随温度升高变短,成虫产卵前期(adult preoviposition period,APOP)和总产卵前期(total preoviposition period,TPOP)同样表现为随温度升高而变短。此外,20±1℃处理的平均产卵量最高(1 810.36粒/雌),27±1℃处理次之(1 623.51粒/雌),30±1℃处理最低(1 084.60粒/雌)。斜纹夜蛾在不同温度条件下内禀增长率(r)、周限增长率(λ)和净增殖率(R0)有显著差异,20±1℃处理的r和λ显著低于27±1℃处理和30±1℃处理;27±1℃处理的R0显著大于20±1℃和30±1℃处理;20±1℃,27±1℃和30±1℃下平均世代周期(T)分别为52.51,30.63和26.87 d,依次减少且相互间差异显著(P0.05)。【结论】斜纹夜蛾在27±1℃和30±1℃温度条件下均表现出很强的增殖能力,该结果可为田间综合防治斜纹夜蛾提供理论依据。     

红棕灰夜蛾核型多角体病毒的研究

红棕灰夜蛾 Sarcopolia illoba(Butler)是鳞翅目夜蛾科害虫。分布于我国东北、华北、华东、华中及苏联、日本、朝鲜、印度等地。幼虫多食性,为害烟草、甘蓝、马铃薯、甜莱、大豆、豌豆、菜豆等作物。1984年在哈尔滨大豆田中,从该虫幼虫自然死虫体内分离出一株核型多角体病毒。我们对该病毒的形态学进行了观察。这种病毒在国内外尚未见报道。并对12种夜蛾科害虫:迹幽夜蛾(Discestra stigmosa)、甘蓝夜蛾(Mamestra brassicae)、黄地老虎(Scotia segetum)、小地老虎(S.ipsilon)、银纹夜蛾     

甘蓝夜蛾几丁质酶基因cDNA序列的克隆与序列分析

昆虫几丁质酶在害虫生物防治中具有很大的发展潜力。以甘蓝夜蛾Mamestra brassicae L.预蛹期幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),扩增得到其几丁质酶的cDNA序列。该序列含有2826个碱基,包括1个1689个碱基的开放阅读框,预测编码1个含562个氨基酸的多肽,分子量约为62.6kDa,等电点为5.30。推导得到的氨基酸序列含有2个N-位糖基化位点,22个O-位糖基化位点,氨基酸序列与其他昆虫,尤其是鳞翅目昆虫的几丁质酶高度同源。获得的甘蓝夜蛾几丁质酶基因cDNA序列已经登录GenBank并获得登录号FJ436415。     

杠柳新苷P和E对东方粘虫和小地老虎幼虫中肠蛋白酶活性的影响

从杀虫植物杠柳Periploca sepium Bunge根皮中分离得到的杠柳新苷P具有很高的杀虫活性, 为了探索其杀虫机理, 采用经典的昆虫蛋白酶活性测定方法, 比较研究了杠柳新苷P和无杀虫活性的杠柳新苷E对东方粘虫Mythimna separata与小地老虎Agrotis ypsilon 6龄幼虫中肠类胰蛋白酶和类胰凝乳蛋白酶活性的影响。结果表明： 对东方粘虫弱碱性类胰蛋白酶, 杠柳新苷P表现出强激活作用（酶活性为对照的3.43倍）, 激活时间可长达8 h, 而杠柳新苷E则无明显激活作用。杠柳新苷P和E对东方粘虫弱碱性类胰蛋白酶活性的影响二者差异显著（P=0.01）, 杠柳新苷P药后2, 4和8 h, 东方粘虫中肠弱碱性类胰蛋白酶的活性分别是杠柳新苷E药后的15.4, 106.8和242.7倍。酶活性测定结果还表明, 与东方粘虫相比, 小地老虎中肠类胰蛋白酶活性相对较低, 且杠柳新苷P的激活作用也较弱, 这可能是杠柳新苷P对东方粘虫具杀虫活性, 而小地老虎对其不敏感的原因之一; 另外, 杠柳新苷P和E对试虫中肠类凝乳胰蛋白酶活性均无明显影响。据此推测, 杠柳活性成分新苷P对东方粘虫中肠弱碱性类胰蛋白酶的激活作用可能是造成试虫中毒的机理之一。     

Horizontal transfer of Wolbachia between phylogenetically distant insect species by a naturally occurring mechanism

Wolbachia is a genus of alpha-proteobacteria found in obligate intracellular association with a wide variety of arthropods, including an estimated 10-20% of all insect species [1]. Wolbachia represents one of a number of recently identified 'reproductive parasites' [2] which manipulate the reproduction of their hosts in ways that enhance their own transmission [3] [4] [5] [6] [7] [8] [9]. The influence of Wolbachia infection on the dynamics of host populations has focused considerable interest on its possible role in speciation through reproductive isolation [3] [10] [11] and as an agent of biological control [2] [12] [13]. Although Wolbachia normally undergoes vertical transmission through the maternal line of its host population [14], there is compelling evidence from molecular phylogenies that extensive horizontal (intertaxon) transmission must have occurred [1] [9] [15] [16] [17]. Some of the best candidate vectors for the horizontal transmission of Wolbachia are insect parasitoids [15], which comprise around 25% of all insect species and attack arthropods from an enormous range of taxa [18]. In this study, we used both fluorescence microscopy and PCR amplification with Wolbachia-specific primers to show that Wolbachia can be transmitted to a parasitic wasp (Leptopilina boulardi) from its infected host (Drosophila simulans) and subsequently undergo diminishing vertical transmission in this novel host species. These results are, to our knowledge, the first to reveal a natural horizontal transfer route for Wolbachia between phylogenetically distant insect species.     

Horizontal transmission of the symbiotic bacterium <Emphasis Type="Italic">Asaia</Emphasis> sp. in the leafhopper <Emphasis Type="Italic">Scaphoideus titanus</Emphasis> Ball (Hemiptera: Cicadellidae)

BACKGROUND: Bacteria of the genus Asaia have been recently recognized as secondary symbionts of different sugar-feeding insects, including the leafhopper Scaphoideus titanus, vector of Flavescence dorée phytoplasmas. Asaia has been shown to be localized in S. titanus gut, salivary glands and gonoducts and to be maternally transmitted to the progeny by an egg smearing mechanism. It is currently not known whether Asaia in S. titanus is transmitted by additional routes. We performed a study to evaluate if Asaia infection is capable of horizontal transmission via co-feeding and venereal routes. RESULTS: A Gfp-tagged strain of Asaia was provided to S. titanus individuals to trace the transmission pathways of the symbiotic bacterium. Co-feeding trials showed a regular transfer of bacterial cells from donors to recipients, with a peak of frequency after 72 hours of exposure, and with concentrations of the administrated strain growing over time. Venereal transmission experiments were first carried out using infected males paired with uninfected females. In this case, female individuals acquired Gfp-labelled Asaia, with highest infection rates 72-96 hours after mating and with increasing abundance of the tagged symbiont over time. When crosses between infected females and uninfected males were conducted, the occurrence of "female to male" transmission was observed, even though the transfer occurred unevenly. CONCLUSIONS: The data presented demonstrate that the acetic acid bacterial symbiont Asaia is horizontally transmitted among S. titanus individuals both by co-feeding and venereal transmission, providing one of the few direct demonstrations of such a symbiotic transfer in Hemiptera. This study contributes to the understanding of the bacterial ecology in the insect host, and indicates that Asaia evolved multiple pathways for the colonization of S. titanus body.     

八门湾红树林土壤芽胞杆菌分离与多样性分析

【目的】了解八门湾红树林海漆林区土壤中可培养芽胞杆菌资源的多样性。【方法】采用水浴处理与直接涂布相结合的方法选择性分离土壤中的芽胞杆菌;利用16S rDNA PCR-RFLP与16S rDNA序列分析技术研究可培养芽胞杆菌资源的遗传多样性和系统发育关系。【结果】16S rDNA PCR-RFLP酶切图谱UPGMA聚类分析表明,在100%的相似性水平上,分离的155株芽胞杆菌分属21个遗传类群,显示了较为丰富的遗传多样性;由21种遗传类型代表菌株的16S rDNA序列分析结果得知,这些芽胞杆菌主要分布在Bacillaceae和Paenibacillaceae科下的Bacillus、Halobacillus、Virgibacillus和Paenibacillus 4个属,其中Bacillus为优势属;有8株芽胞杆菌的16S rDNA序列与数据库中相应模式菌株的最大相似性在95.1%-99.0%之间。【结论】八门湾红树林土壤可培养芽胞杆菌有着较为丰富的遗传多样性,并存在新的芽胞杆菌物种资源。     

Wolbachia screening in spiders and assessment of horizontal transmission between predator and prey

Recent studies have revealed that the prevalence of Wolbachia in arthropods is attributable not only to its vertical transmission, but also to its horizontal transfer. In order to assess the horizontal transmission of Wolbachia between predator and prey, arthropods belonging to 11 spider families and six insect families were collected in the same field of rice. The distribution of Wolbachia in these arthropods was detected by diagnostic PCR amplification of the wsp (Wolbachia outer surface protein gene) and 16S rDNA genes. Nurscia albofasciata Strand (Araneae: Titanoecidae), Propylea japonica Thunberg (Coleoptera: Coccinellidae), Paederus fuscipes Curtis (Coleoptera: Staphylinidae), and Nilaparvata lugens Stal (Homoptera: Delphacidae) were infected with Wolbachia. This is the first report of infection of N. albofasciata and P. fuscipes by Wolbachia. No direct evidence indicated the existence of horizontal transmission of Wolbachia between predator and prey.     

Vertical transmission of Wolbachia in Tetranychus kanzawai Kishida and Panonychus mori Yokoyama (Acari: Tetranychidae)

The vertical transmission of Wolbachia in two species of spider mite was investigated and compared. One species, Tetranychus kanzawai Kishida, was infected with a modification negative strain of Wolbachia while the other species, Panonychus mori Yokoyama, was infected with a modification positive strain. The infection showed perfect maternal transmission in the laboratory population of T. kanzawai in which Wolbachia-infected females produced infected offspring regardless of whether they mated with infected or uninfected males, and uninfected females produced Wolbachia-free progenies without regard to the infection status of their mating partners. In artificial P. mori populations initiated with 50% infected and 50% uninfected female adults, the infection frequencies among progenies increased with each generation, reaching 100% at the sixth generation in the Sendai population and after the sixth generation in the Toyama population. In another experiment, in which an artificial T. kanzawai population was composed of 50% infected and 50% uninfected female adults, the infection frequency in progeny populations increased very slowly, reaching 62.5% at the 15th generation. The difference in infection frequency in the two spider mites may be due to the different strains of Wolbachia.     

Physiological host specificity: a model using the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and microsporidia of row crop and other stalk-boring hosts

We investigated vertical and horizontal transmission as means by which entomopathogenic microsporidia may be isolated in their hosts. Ostrinia nubilalis larvae were challenged with microsporidia isolated from other stalk-boring and row crop Lepidoptera and were susceptible to seven species. Two species were horizontally transmitted. A Nosema sp. from Eoreuma loftini was transmitted among O. nubilalis larvae but not among larvae of the E. loftini host. This species was also vertically transmitted to the offspring of infected O. nubilalis females. An rDNA sequence showed the E. loftini isolate to be Nosema pyrausta, a naturally occurring species in O. nubilalis. Our results suggest that both horizontal and vertical transmission provide physiological barriers to host switching in the microsporidia, thus restricting the natural host range.     

Absolute configuration of volicitin from the regurgitant of lepidopteran caterpillars and biological activity of volicitin-related compounds

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine are known as insect-produced plant volatile elicitors. The absolute configuration of the hydroxylinolenoyl moiety of volicitin from three noctuid species, Helicoverpa armigera, Mythimna separata and Spodoptera litura, was determined to be all 17S in high enantiomeric excess. When treated with 30 pmol of (17S)- and (17R)-volicitin, corn seedlings were induced to release volatiles, there being no significant difference in the amount released between the two isomers. On the other hand, N-linolenoyl-L-glutamine was only about 30% as active as volicitin. Among several synthesized N-linolenoylamino acid conjugates, only the L-glutamine conjugate induced the emission of volatile organic compounds. These results show that the L-glutamine moiety of volicitin played a more critical role than the hydroxyl moiety, although both moieties affected the elicitor activity inducing the release of volatiles.     

The Molecular Epidemiology of the Highly Virulent ST93 Australian Community Staphylococcus aureus Strain

In Australia the PVL - positive ST93-IV [2B], colloquially known as "Queensland CA-MRSA" has become the dominant CA-MRSA clone. First described in the early 2000s, ST93-IV [2B] is associated with skin and severe invasive infections including necrotizing pneumonia. A singleton by multilocus sequence typing (MLST) eBURST analysis ST93 is distinct from other S aureus clones. To determine if the increased prevalence of ST93-IV [2B] is due to the widespread transmission of a single strain of ST93-IV [2B] the genetic relatedness of 58 S. aureus ST93 isolated throughout Australia over an extended period were studied in detail using a variety of molecular methods including pulsed-field gel electrophoresis, spa typing, MLST, microarray DNA, SCCmec typing and dru typing. Identification of the phage harbouring the lukS-PV/lukF-PV Panton Valentine leucocidin genes, detection of allelic variations in lukS-PV/lukF-PV, and quantification of LukF-PV expression was also performed. Although ST93-IV [2B] is known to have an apparent enhanced clinical virulence, the isolates harboured few known virulence determinants. All PVL-positive isolates carried the PVL-encoding phage ΦSa2USA and the lukS-PV/lukF-PV genes had the same R variant SNP profile. The isolates produced similar expression levels of LukF-PV. Although multiple rearrangements of the spa sequence have occurred, the core genome in ST93 is very stable. The emergence of ST93-MRSA is due to independent acquisitions of different dru-defined type IV and type V SCCmec elements in several spa-defined ST93-MSSA backgrounds. Rearrangement of the spa sequence in ST93-MRSA has subsequently occurred in some of these strains. Although multiple ST93-MRSA strains were characterised, little genetic diversity was identified for most isolates, with PVL-positive ST93-IVa [2B]-t202-dt10 predominant across Australia. Whether ST93-IVa [2B] t202-dt10 arose from one PVL-positive ST93-MSSA-t202, or by independent acquisitions of SCCmec-IVa [2B]-dt10 into multiple PVL-positive ST93-MSSA-t202 strains is not known.     

Identification of expressed genes during infection of Chinese cabbage (Brassica rapa subsp. pekinensis) by Plasmodiophora brassicae

Plasmodiophora brassicae is an obligate, biotrophic pathogen causing the club-root disease of crucifers. Despite its importance as a plant pathogen, little is known about P. brassicae at the molecular level as most of its life cycle takes place inside the plant host, and axenic culturing is impossible. Discovery of genes expressed during infection and gene organization are the first steps toward a better understanding of the pathogen-host interaction. Here, suppression subtractive hybridization was used to search for the P. brassicae genes expressed during plant infection. One-hundred and forty ESTs were found of which 49% proved to be P. brassicae genes. Ten novel P. brassicae genes were identified, and the genomic sequences surrounding four of the ESTs were acquired using genome walking. Alignment of the ESTs and the genomic DNA sequences confirmed that P. brassicae genes are intron rich and that the introns are small. These results show that it is possible to discover new P. brassicae genes from a mixed pool of both plant and pathogen cDNA. The results also revealed that some of the P. brassicae genes expressed in Chinese cabbage (Brassica rapa subsp. pekinensis) were identical to the genes expressed in the infection of Arabidopsis plants, indicating that these genes play an important role in P. brassicae infection.