低温对黄瓜花叶病毒CMV-BG株系2b基因多态性的影响

 本文将黄瓜花叶病毒CMV-BG株系侵染普通烟草的不同单株后,放在不同温度下培养,30d后对接种植株症状进行对比观察,并对2b基因表达进行了检测及其序列测定分析。结果表明:被CMV-BG株系侵染的植株在低温条件下,植株新生的大部分叶片严重畸形,而CMV-BG株系2b基因的表达与不同温度条件没有相关性;CMV2b基因序列与源分离物CMV-BG序列相比发生了较明显的变异,它们作为一组序列与分离物SF/GF/SY等的相似性甚至高于源BG序列;此外,尽管来自2组植物中的病毒序列变化均未表现正选择(positive selection),但来自低温培养的侵染植株的2b基因的多态性明显高于常温条件培养的植株,发生非同义突变的位点数目也高于同义突变的位点数目。因此认为,低温有助于加强CMV-BG株系2b基因的多态性。     

海南红麻曲叶病的病原检测与鉴定

 红麻曲叶病是2012年在海南省海口市发现的一种新病害,病株表现为叶片向上卷曲、叶脉肿大、叶脉变深绿色等症状。PCR检测结果显示,该病样中均存在菜豆金色花叶病毒属病毒。基因克隆及序列分析结果表明,该病毒分离物(HN08)基因组仅含A组分(DNA-A),其全长为2 738 nt,与木尔坦棉花曲叶病毒(CLCuMuV)各分离物的相似性均大于89.0 %,其中与中国各分离物的相似性均大于99.0 %。该病毒分离物也伴随有β卫星分子,其全长为1 346 nt,与CLCuMuV各分离物伴随的β卫星分子(CLCuMuB)序列相似性大于83.0 %,其中与分离物Fz1的序列相似性最高,为99.7 %。构建了HN08 DNA-A及其β卫星分子侵染性克隆,通过农杆菌注射接种红麻,接种后30 d,HN08 DNA-A及其β卫星分子混合接种的红麻植株新出叶片开始产生曲叶症状;接种后60 d,二者混合接种的植株大部分叶片表现为严重的曲叶症状,且与田间自然病株症状相同,而二者各自单独接种的红麻植株没有产生明显的症状。PCR及Southern blot检测进一步证实这些症状是由HN08 DNA-A及其β卫星分子的共同侵染引起的。因此,海南红麻曲叶病是由CLCuMuV及其伴随的β卫星分子(CLCuMuB)共同侵染引起的。本文首次报道了红麻是CLCuMuV自然新寄主。     

侵染木薯的两种单组分菜豆金色花叶病毒属病毒及卫星分子基因组结构特征分析

为明确木薯(Manihot esculenta Crantz)植株叶片皱缩、畸形是否由菜豆金色花叶病毒属病毒侵染引起,从云南省红河州田间采集具有疑似感染症状的木薯植株叶片样品,应用菜豆金色花叶病毒属病毒简并引物、种专化性引物及卫星分子引物进行PCR扩增、克隆,通过测序分析其核苷酸序列特征并对其进行系统进化分析.结果 显...     

高温激活RNA沉默介导的心叶烟抗病毒防御反应

 在真核细胞内,RNA沉默是一种保守的抵抗病毒及外源基因等寄生分子的防御机制。温度可以明显地影响植物体与病毒之间的相互作用。在常温状态下,常常会伴随病毒病的爆发,但在较高温度下,症状则会减轻,甚至有些感病植株新生叶片可以较快地恢复健康。目前这类现象的内在机制还不完全清楚。本文通过定量分析不同温度下马铃薯X病毒(Potato virus X,PVX)和马铃薯Y病毒(Potato virus Y,PVY)侵染的心叶烟(Nicotiana glutinosa)植株体内病毒蛋白含量,结合症状观察,确定30℃左右是PVX和PVY在心叶烟上引起症状消失即发生系统性RNA沉默的临界温度,此时的感病植株上部新生展开叶片呈现无症状态,下部已发病叶片症状不变。在常温时,由病毒激发的RNA沉默会受到抑制,病毒来源的小分子干涉RNA (small interfering RNAs,siRNAs)含量极少或根本检测不到,植株对于病毒的敏感性较强。当温度达到30或32℃时,感病植株下部症状保持叶片中,病毒起源的siRNAs含量明显增加,而上部新生健康叶片中却检测不到siRNAs的存在。这说明高温激活了RNA沉默介导的植物抗病毒防御反应,导致病毒症状减轻甚至消失。     

巴西首次在马铃薯上报道番茄褪绿病毒

2011年6月,在巴西Goiás州Cristalina县的一个商业种植园中发现一些马铃薯植株的叶片特别是老叶表现叶片卷曲、脉间褪绿等症状,并且该种植园B型烟粉虱(Bemisia tabaci)发生严重。从表现症状的植株上采集4个块茎样品,用于检测马铃薯植株是否受到以下4种病毒的侵染:番茄褪绿病毒(Tomato chloro-     

病毒载体介导的基因沉默体系对朱顶红褪绿环斑病毒NSm基因的沉默效应

为探讨烟草脆裂病毒（tobacco rattle virus,TRV）载体介导的基因沉默技术对朱顶红褪绿环斑病毒（Hippeastrum chlorotic ringspot virus,HCRV）运动蛋白基因NSm的沉默效应,以本氏烟Nicotiana tabacum为材料,构建靶向HCRV NSm序列的基因沉默表达载体pTRV2-NSm,经农杆菌Agrobacterium介导侵染烟苗,检测其侵染效率,人工接种HCRV到沉默表达载体处理过的烟苗,通过观察发病症状、统计病情并应用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术检测TRV介导的基因沉默体系对HCRV侵染的沉默效应。结果表明,pTRV2-NSm沉默表达载体对本氏烟植株的侵染率达到95.00%,并且对本氏烟的生长无明显影响;与对照相比,经沉默载体处理的烟草植株接种HCRV后发病率显著降低,在接种后14 d发病率下降了90个百分点,病毒积累量明显下降,随着时间延长pTRV2-NSm对病毒的抑制效果持续升高,在接种后14 d对病毒病的防治效果最高,达93.13%;qRT-PCR检测发...     

壳寡糖诱导对烟草体内TMV-CP基因表达的抑制作用

 采用实时荧光定量PCR方法, 研究了壳寡糖诱导对烟草体内TMV-CP基因表达量的影响。结果表明, 壳寡糖诱导后烟草表达了对TMV侵染的系统获得抗病性, 植株显症推迟4~7 d, 症状减轻, 平均严重度比对照降低了82.9%。同时烟草叶片中CP基因的表达量显著降低。壳寡糖诱导处理的植株在接种病毒时(0 h), CP基因的表达量(拷贝数/2μL)为0.918, 接种后168 h增加到167.730。而在不诱导对照植株内, CP基因的表达量在0 h为1.218, 接种后168 h猛增到648.623。换言之, 不处理对照的表达量在此期间增长了532.53倍, 而壳寡糖诱导处理植株中仅增长了182.71倍。试验证明壳寡糖诱导的系统获得抗病性强烈抑制了烟草体内TMV-CP基因的表达。     

从进境的蝴蝶兰上检出齿兰环斑病毒

齿兰环斑病毒 (OdontoglossumringspotvirusORSV)是兰花上的主要病毒 ,可侵染多种兰科植物 ,在卡特兰和建兰上危害尤为严重。在蝴蝶兰上 ,叶表现轻花叶 ,花畸形或发育不良 ,严重影响花的数量和品质。 2 0 0 2年9月 ,我们从来自台湾的蝴蝶兰中发现可疑症状植株 ,经透射电镜观察和血清学检测为齿兰环斑病毒。1　材料和方法1 1　材料进境的蝴蝶兰苗 ,花叶坏死症状 ( 1号样品 )、没有症状的 ( 2号样品 )取叶片进行检测 ;2 %的磷钨酸 (厦门大学化学系提供 ) ;血清学试剂和阳性对照由国家质检总局动物检所提供 ;本实验室保存的无病毒蝴蝶兰植株…     

菠萝凋萎病的发生因素及防治技术

2月中旬琼海市中原一带由于阴雨、低温的影响，常较大面积发生菠萝凋萎病，发病株率 50％左右，重病园高达 90％。经济损失一般 25％～ 30％。　　菠萝凋萎病又名菠萝根腐病，当地叫“菠萝瘟”，此病在海南、广东、广西、福建及台湾等地均有发生。1症状　　地上部症状表现为发病初期基部叶片变黄发红，皱缩，失去光泽，叶缘向内卷曲，以后叶尖干枯，叶片凋萎，植株停止生长。随着病情发展，由根尖腐烂发展到根系的部分或全部腐烂，植株枯死。病原，多数人认为病害是由菠萝粉蚧传带的病毒侵染所致。2发病条件　　1)气候因素。一般高温、干旱…     

转基因烟草中PVYN CP基因沉默及其介导的抗病性受温度的调控

 本研究以转不可翻译的马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)烟草的T₃代植株为材料,在获得高度抗病植株并证明转基因植株的抗病性是由RNA沉默介导的基础上,采用Northern杂交及ELISA检测病毒的方法,分析了温度对转基因烟草中RNA沉默以及转基因植株抗病水平的影响。结果表明,低温可以改变转基因植株中已发生的RNA沉默和转基因植株的抗病状态。在15℃低温下生长的转基因植株,转基因产生的RNA沉默被抑制,转基因植株失去了对PVY^N高度抗病的特性,表现感病症状;而在25℃或以上高温(30℃、35℃)下生长的转基因植株,转基因产生的RNA沉默没有发生被抑制的现象,转基因植株对PVY^N病毒的侵染仍保持高度抗病性。     

重组黄瓜花叶病毒2b基因PVX侵染性载体的稳定性分析

 本研究利用北京甘蓝CMV分离物(CMV-BG)全长2b基因,构建了含其正义和反义序列的PVX侵染性载体pVX2bS和pVX2bAS,并进行了它们与PVX侵染性载体间的共侵染稳定性实验。结果表明:pVX2bS和pVX2bAS能侵染本氏烟、SR1、Sam sun NN烟草、Shepody马铃薯和中蔬5号番茄,与pSfinx相比,表现不同程度的症状延迟效应;pVX2bS和pVX2bAS在22℃/16℃(昼/夜温度)条件下,接种SR1烟草45d仍可检测到重组载体的存在;pVX2bS和pVX2bAS菌液混合接种15d后只能检测到正义或反义一种载体的随机表达,二者交互接种,后接种的一方不能表达。     

温度对胡瓜新小绥螨生殖力及钠钾泵α亚基基因表达的影响

胡瓜新小绥螨Neoseiulus cucumeris（Oudemans）广泛地用于蓟马类害虫的防治、控制及管理。本研究克隆了1条胡瓜新小绥螨钠钾泵α亚基基因序列,分析了温度对雌螨生殖力及钠钾泵α亚基基因表达的影响。在15、20、25和30℃下,胡瓜新小绥螨在25℃时生殖力最优。在上述温度条件下胁迫48 h,雌螨钠钾泵α亚基在15和30℃下表达量显著上升,能量代谢活跃。表达量越多,表明能量消耗越大,投入生殖的能量就越少,结合生殖力研究结果,二者一致。高温或低温均会降低胡瓜新小绥螨的生殖力,且高温影响更为严重。     

多粘类芽胞杆菌LB-9的诱变及抑菌防病效果研究

为获得对烟草疫霉菌生防效果更佳的拮抗菌株,对多粘类芽胞杆菌LB-9进行基因诱变。利用原生质体电转化法,构建了菌株LB-9的Tn-5转座插入突变体库,获得约2000个转化子,通过平板对峙法筛选出对烟草疫霉菌拮抗能力增强且效果稳定的突变菌株3个,其中编号为A13的突变菌株对烟草疫霉菌的抑制效果达到80%以上,与原始菌株LB-9的抑菌率相比提高了20%以上。采用室内盆栽接种法研究了正突变菌株A13对烟草黑胫病的防控效果,并利用实时荧光定量PCR法初步分析了其诱导烟株体内抗病相关基因的表达。结果表明,正突变菌株A13处理烟株7 d后,对烟草黑胫病的防效高于原始菌株LB-9,防效可达80%以上,同时可诱导烟株体内不同信号通路中抗性相关基因的上调表达。本研究为多粘类芽胞杆菌插入诱变育种方法研究提供了理论支撑,对烟草黑胫病的生物防治具有重要意义。     

Effect of temperature on resistance to Potato virus Y in potato cultivars carrying the resistance gene Rychc

The effect of cultivation temperatures on the resistance reaction to three Potato virus Y strains (PVY^O, PVY^N and PVY^NTN) in potato cultivars carrying Ry_chc was examined. When potato plants carrying Ry_chc were cultivated at 22 °C, a few small necrotic spots developed on inoculated leaves by 5 days after mechanical inoculation (dpi), and systemic infection of a few symptomless plants was confirmed at 28 dpi by IC‐RT‐PCR. At 28 °C, distinct necrotic spots developed on inoculated leaves by 5 dpi, and systemic symptoms occasionally appeared at 28 dpi. Thus, high temperature weakens Ry_chc‐conferred resistance. However, the incidence of systemic infection and the titre of virus in resistant cultivars at 28 °C were lower than in a susceptible cultivar. In graft inoculation under high summer temperatures, some plants developed necrosis on the leaves and stem, but PVY was barely detected by RT‐PCR in leaves on potato carrying Ry_chc. When seedlings from progeny tubers of plants that were inoculated with PVY and grown in a greenhouse at >30 °C in the daytime were examined by ELISA and IC‐RT‐PCR, PVY was not detected in cultivars carrying Ry_chc. These results show that Ry_chc confers an extreme resistance to PVY strains occurring in Japan.     

Sap flow characteristics and physiological adjustments of two dominant tree species in pure and mixed plantations in the semi-arid Loess Plateau of China

It is essential to understand the water consumption characteristics and physiological adjustments of tree species under drought conditions, as well as the effects of pure and mixed plantations on these characteristics in semi-arid regions. In this study, the normalized sap flow (SF_n), leaf water potential, stomatal conductance (g_s), and photosynthetic rate (P_r) were monitored for two dominant species, i.e., Pinus tabuliformis and Hippophae rhamnoides, in both pure and mixed plantations in a semi-arid region of Chinese Loess Plateau. A threshold-delay model showed that the lower rainfall thresholds (R^L) for P. tabuliformis and H. rhamnoides in pure plantations were 9.6 and 11.0 mm, respectively, and the time lags (τ) after rainfall were 1.15 and 1.76 d for corresponding species, respectively. The results indicated that P. tabuliformis was more sensitive to rainfall pulse than H. rhamnoides. In addition, strong stomatal control allowed P. tabuliformis to experience low g_s and P_r in response to drought, while maintaining a high midday leaf water potential (Ψ_m). However, H. rhamnoides maintained high g_s and P_r at a low Ψ_m expense. Therefore, P. tabuliformis and H. rhamnoides can be considered as isohydric and anisohydric species, respectively. In mixed plantation, the values of R^L for P. tabuliformis and H. rhamnoides were 6.5 and 8.9 mm, respectively; and the values of τ were 0.86 and 1.61 d for corresponding species, respectively, which implied that mixed afforestation enhanced the rainfall pulse sensitivity for both two species, especially for P. tabuliformis. In addition, mixed afforestation significantly reduced SF_n, g_s, and P_r for P. tabuliformis (P<0.05), while maintaining a high leaf water potential status. However, no significant effect of mixed afforestation of H. rhamnoides was observed at the expense of leaf water potential status in response to drought. Although inconsistent physiological responses were adopted by these species, the altered water consumption characteristics, especially for P. tabuliformis indicated that the mixed afforestation requires further investigation.     

陕西省小麦白粉菌群体毒性结构及遗传多样性

 为明确陕西省小麦白粉菌群体的毒性频率和遗传多样性,利用34个含有已知抗白粉病基因的小麦品种(系)和5对多态性ISSR分子标记,分别对2016年渭南、西安、咸阳、宝鸡、汉中和安康等6市的15个乡镇160个小麦白粉菌单孢子堆菌株进行毒性频率分析。结果显示:供试小麦白粉菌群体对Pm1、Pm2、Pm3b、Pm3c、Pm3e、Pm3f、Pm6、Pm7、Pm8、Pm19和Pm1+2+19的毒性频率在60%~100%之间,表明这些抗性基因已丧失抗性,在生产上已经丧失利用价值,对Pm4b、Pm24、Pm2+6、Pm2+Mld、Pm2+6+?、Pm4b+Mli、Pm"Era"、Pm"XBD"和Pm21的毒性频率低于20%,表明这些抗性基因抗性良好,可在生产中利用。选取其中93个单孢子堆菌株进行遗传多样性分析,结果表明白粉菌地理群体间遗传距离在0.020 4~0.103 7之间,其中宝鸡和渭南群体的遗传距离最近,汉中和咸阳群体的遗传距离最远。群体间遗传变异占总体变异的12.82%,群体内遗传变异占87.12%,表明遗传变异主要来自于群体内。Mantel Test分析表明,小麦白粉菌群体间遗传距离与地理距离相关性不大。     

<Emphasis Type="Italic">In planta</Emphasis> multiplication and graft transmission of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus’ revealed by Real-Time PCR

The bacterium Candidatus Liberibacter asiaticus (CLas) is associated with huanglongbing (HLB) in citrus in many countries. Despite the fact that many characteristics of the disease are known, the rate of multiplication of the bacterium within an infected tree is still poorly understood. To study this feature, we used the quantitative Real-Time polymerase chain reaction (Q-PCR) assay to follow and to quantify the multiplication of CLas in grafted infected young sweet orange plants. The rate of infection by grafting reached 100% at 120 days post-inoculation (dpi) showing that grafting could easily transmit CLas. A well-adjusted linear regression equation describing the bacterial growth in planta was obtained independently with measurements taken using repeated sampling in the same plant or different plants through the analysed period. The bacterial population, measured as copy number (CN) of the 16S rDNA target gene g⁻¹ of tissue, increased 10,000 times from 10³ at 30 dpi to approximately 10⁸ CN at 240 dpi indicating that CLas multiplication was fastest in young citrus plants. We observed a direct relationship between the concentration of pathogen and the expression of symptoms. Yellowed leaves or shoots, are commonly the first observed symptom of HLB, and were present in trees with a low amount of bacteria (10⁵ CN g⁻¹). Blotchy mottle symptoms were observed in trees with 10⁷ CN g⁻¹ of bacteria after 180 dpi. Buds taken from infected, but non-symptomatic branches were grafted on Rangpur lime and resulted in transmission rates ranging from 10 to 60%.