Comparison of the effects of salicylic acid and ethephon with virus-induced hypersensitivity and acquired resistance in tobacco

The induction of a hypersensitive reaction in Samsun NN tobacco by tobacco mosaic virus (TMV) at 20°C leads to the development of both localized and systemic acquired resistance, and is associated with the appearance of pathogenesis-related proteins (PR's) and large increases in peroxidase activity and ethylene production. Salicylic acid (SA) induced a similar resistance in treated plant parts and occasionally also in untreated upper leaves of plants of which three lower leaves had been injected. SA also induced the same four PR's, but these were confined to the treated leaves. Thus, the connection between the presence of PR's and the reduction of TMV multiplication and spread may not be direct.In contrast to TMV, SA did not stimulate ethylene production and hardly increased peroxidase activity. Induction of acquired resistance and PR's by SA developed equally well at 20°C and at 32°C. However, pricking leaves with needles moistened with the ethylene-releasing compound ethephon mimicked TMV infection in inducing acquired resistance and PR's in both treated and untreated leaves at 20°C, but not at 32°C. Ethephon increased peroxidase activity at both temperatures, but only at 20°C dit it induce changes in both the anodic and the cathodic isoenzymes that were similar to those induced as a result of TMV infection. SA induced PR's and reduced TMV multiplication in Samsun tobacco, and inhibited virus spread in Samsun NN at 32°C.These observation indicate that neither the induction of PR's, nor the development of acquired resistance is temperature-sensitive. On the other hand, the effects of ethephon are temperature-sensitive in the same way as the hypersensitive response to TMV. It can thus be hypothesized that ethylene, produced naturally during the hypersensitive reaction of tobacco to TMV, leads to the temperature-sensitive synthesis or release of a presumably benzoic acid-type compound that functions as the natural inducer of PR's and acquired resistance. Although vanillic acid has been shown to accumulate in hypersensitively reacting tobacco leaves, it produced none of the effects of SA, and thus cannot be the natural inducer.Samenvatting Inductie van een overgevoeligheidsreactie in Samsun NN-tabak door tabaksmozaïekvirus (TMV) bij 20°C leidt tot de ontwikkeling van een verworven resistentie die zowel lokaal als systemisch werkzaam is, en gaat samen met het verschijnen van pathogenesis-related proteins (PR's) en sterke toename in de activitieit van peroxidase en de produktie van ethyleen. Salicylzuur (SA) induceerde een vergelijkbare resistentie in behandelde plantedelen en af en toe ook in niet behandelde bovenbladeren van planten waarvan drie onderbladeren waren ingespoten. SA induceerde ook dezelfde vier PR's, maar deze waren beperkt tot de behandelde bladeren. Er bestaat dus geen directe samenhang tussen de aanwezigheid van PR's en de remming van de vermeerdering en uitbreiding van TMV in de plant.In tegenstelling tot TMV stimuleerde SA de ethyleenproduktie niet en verhoogde het de peroxidaseactiviteit nauwelijks. Inductie van verworven resistentie en PR's door SA trad even goed op bij 32°C als bij 20°C. Net als infectie met TMV leidde aanprikken van bladeren met naalden die gedoopt waren in een oplossing van ethefon — waaruit in het blad ethyleen vrijkomt — echter tot inductie van verworven resistentie en PR's in zowel behandelde als onbehandelde bladeren bij 20°C, maar niet bij 32°C. Ethefon verhoogde de peroxidaseactiviteit bij beide temperaturen, maar alleen bij 20°C induceerde het veranderingen in zowel de anodische als de kathodische isoënzymen die vergelijkbaar waren met die welke geïnduceerd werden als gevolg van infectie met TMV. SA induceerde PR's en verminderde de vermenigvuldiging van TMV in Samsun tabak, en remde de uitbreiding van het virus in Samsun NN bij 32°C.Deze waarnemingen tonen dat noch de inductie van PR's, noch de ontwikkeling van verworven resistentie een temperatuurgevoelig proces is. Daarentegen zijn de effecten van ethefon op dezelfde wijze temperatuurgevoelig als de overgevoeligheidsreactie op TMV. Men kan daarom veronderstellen dat ethyleen, dat op natuurlijke wijze geproduceerd wordt tijdens de overgevoeligheidsreactie van tabak op TMV, aanleiding geeft tot een temperatuurgevoelig proces, namelijk de synthese of het vrijkomen van een verbinding, vermoedelijk een benzoëzuurderivaat, dat fungeert als de natuurlijke inductor van PR's en verworven resistentie. Hoewel is aangetoond dat vanillinezuur zich ophoopt in overgevoelig reagerende tabaksbladeren, veroorzaakte deze verbinding geen enkel van de effecten van SA. Vanillinezuur kan dus niet de natuurlijke inductor zijn.     

Pelargonium flower-break and pelargonium line pattern viruses in the Netherlands; purification,antiserum preparation,serological identification,and detection in pelargonium by ELISA

Two viruses, detected frequently in the Netherlands in pelargonium, were identified by serology and test plant reactions. Antisera were prepared and an ELISA procedure was developed to detect the viruses in pelargonium.One of the viruses, PFBV-N, proved to be pelargonium flower-break virus. With the antiserum to PFBV-N, it could be detected reliably throughout the year inPelargonium zonale Springtime Irene.The other virus, PLPV-N, was serologically closely related to pelargonium line pattern virus (PLPV) and to pelargonium ring pattern virus (PRPV), as were an old virus isolate from Saturnus, collected in the Netherlands in 1971 (L128), and PLPV isolates from Yugoslavia (PLPV-Y) and Denmark (PLPV-D). There were only minor differences in host-plant reactions between the virus isolates. Based on these tests, PLPV and PRPV are considered as isolates of the same virus, for which, for practical reasons, the name pelargonium line pattern virus is proposed.PLPV could be reliably detected by ELISA inP. zonale Springtime Irene and Amanda throughout the year with only a few exceptions. InPelargonium peltatum Tavira, however, reslts were erratic due to uneven distribution of virus in the plant. Best results were obtained when petioles of fully expanded leaves were tested.     

Detection of squash mosaic virus in seeds of melon (Cucumis melo) by enzyme-linked immunosorbent assay (ELISA)

Squash mosaic virus (SqMV, comovirus) is seed-transmitted in severalCucurbitaceae. Therefore, the use of virus-free seed is important to prevent establishment of this virus in the Netherlands and to avoid spread to other countries.This study was undertaken to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of SqMV in melon seeds. An antiserum was produced to a serotype 1 isolate from melon. Two ELISA variants were investigated viz. an ELISA variant with simultaneous incubation of sample and enzyme conjugate (ELISA 1) and an ELISA variant with successive incubation of sample and enzyme conjugate (ELISA 2). The sensitivity of ELISA was tested by mixing fluor of ground infected and non-infected seeds in different proportions. SqMV was detected by both ELISA variants at dilutions of 1 160 (1 part of infected flour mixed with 159 parts of non-infected flour) or higher after a substrate incubation period of 4 h. However, ELISA 1 gave relatively higher absorbance values than ELISA 2 for nearly all dilutions. Since ELISA 1 is also faster than ELISA 2, ELISA 1 is advised for routine testing. In these test, using subsamples of 100 melon seeds SqMV is detected reliably. ELISA 1 is now used in the Netherlands for routine-indexing of melon seed lots for SqMV.Samenvatting Het pompoenemozaïekvirus gaat over met het zaad van verscheideneCucurbitaceae. Het gebruik van virusvrij zaad is belangrijk om te voorkomen dat het virus zijn intrede doet in Nederland en zich naar andere landen verspreidt.Een antiserum werd geproduceerd tegen een serotype 1 isolaat van meloen. Met behulp van dit antiserum werd een ELISA ontwikkeld om pompoenemozaïekvirus in zaden van meloen aan te tonen. Twee varianten van ELISA werden vergeleken, namelijk een variant waarbij monster en enzymconjugaat gelijktijdig geïncubeerd werden (ELISA 1) en een variant waarbij monster en enzymconjugaat na elkaar geïncubeerd werden (ELISA 2). De gevoeligheid van de ELISA varianten werd uitgetoetst door meel van zieke zaden in verschillende verhoudingen te mengen met meel van gezonde zaden. Het pompoenemozaïekvirus werd met beide ELISA varianten aangetoond in verdunningen van 1 160 (1 deel meel van zieke zaden gemengd met 159 delen meel van gezonde zaden) of hoger na 4 uur incubatie met substraat. ELISA 1 gaf doorgaans hogere extinctiewaarden dan ELISA 2 voor bijna alle verdunningen. Omdat ELISA 1 ook nog sneller is dan ELISA 2, wordt ELISA 1 aanbevolen voor routinematig gebruik. Wanneer voor routinematig gebruik 100 meloenezaden per submonster getoetst worden, kan het pompoenemozaïek virus betrouwbaar worden aangetoond. In Nederland worden momenteel per zaadpartij 20 submonsters van 100 zaden getoetst.     

A seroepidemiological study on bluetongue virus in dairy cattle in the central valley of California

A seroepidemiological study on bluetongue virus (BTV) infection in California dairy cattle was conducted to estimate the prevalence and distribution by age and season of BTV group-reactive antibodies and to look for possible associations between the presence of antibodies and cattle age or breed and farm. Between December 1985 and March 1987, a sample of cattle was tested at approximately two-month intervals for BTV group-reactive antibodies using an enzyme-linked immunosorbent assay (ELISA). Data taken during the month of December 1986 were used to evaluate possible associations between a positive antibody test and certain intrinsic (age, breed) and extrinsic (farm) factors.Univariate and multivariate statistical analyses using the -square test for associations and multiple logistic regression, respectively, were carried out for possible associations between positive antibody tests to BTV and each factor of interest. The strengths of the associations were determined using estimates of the odds ratio.Of the 3774 serum samples tested, 238 (6.3%) were from calves, 1045 (27.6%) were from heifers and 2492 (66.0%) were from cows. Seroprevalence varied from nil in calves on two occasions to over 90% on several occasions in cows. Cows consistently had higher prevalence rates than heifers or calves across all test dates (p<0.05). The seroprevalence of BTV group-reactive antibodies also showed a seasonal fluctuation, with the highest rates occurring during the warmer months of the year. These highest prevalence rates coincided with heavy activity of the known vector of BTV, Culicoides spp. Breed and farm effects were not statistically significant (p>0.05). With the exception of one farm, all cattle were of the Holstein breed, which reduced confidence in assessing any breed effect in this study. Relative estimates of the sensitivity and specificity of BTV ELISA were 87% and 100% respectively, compared to the standard agar gel immunodiffusion (AGID) test.The observations support previous findings of seasonal distribution of BTV antibodies and suggest an age relationship, whereby older cattle are more likely to be positive to BTV group-reactive antibodies than younger cattle.     

Kinetics of NDV specific antibodies in chickens—V. Analysis of frequency distributions of antibody titers against Newcastle disease virus by investigation of random samples in chicken flocks

Chicks and chickens maintained under commercial conditions were vaccinated against Newcastle Disease via drinking water. Prior and after different times of vaccination blood samples were drawn from different numbers of birds and checked for HI antibodies. The modes of distribution of the antibody titers within the random sample were assayed. The following results were obtained:

1. 1. On the basis of the distribution of the serum titers can be concluded whether the antibody level within a flock is increasing or decreasing.

◦ —A right steep asymmetry can be observed up to 20 days post vaccination.

◦ —In the phase of maximal antibody levels an almost symmetric distribution of the titers is present.

◦ —In later times (more than three weeks p. vacc.) the distribution shows a left steep asymmetry (Poisson distribution).

2. 2. A poisson distribution is also observable during the elimination of maternal antibodies of chicks until complete elimination.

3. 3. The mode of distribution of the HI titers in sera of day-old chicks correlates with the mode of distribution of the dams. Therefore, conclusions are possible from the status of the chicks to the dams and reverse.

4. 4. Factors which interfere with the mode of distribution are:

◦ —Two or more peaks after vaccination of chickens. This indicates an uneven immune response within the flock.

◦ —Distributions with several peaks may also occur if flocks are composed of day-old chicks from parent flocks with different levels of antibody titers.

外源Ca2+ 和ABA 对高温下黄瓜花药中Ca2 + 、ABA及蛋白质合成的影响

 外源Ca^{2 +}能降低高温下黄瓜单核期花药的胞内Ca^{2 +}水平并提高花粉萌发率, 而ABA的作用与此相反。外源Ca^{2 +}和ABA处理能显著提高高温下黄瓜花药内源ABA的水平, 并产生新的蛋白质。以上结果表明, 高温下外源Ca^{2 +}可影响黄瓜花药内ABA信号系统, 继而可能改变基因表达, 并获得耐热性;外源ABA亦能影响黄瓜花药内Ca^{2 +}信号系统。     

黄瓜全雌性基因连锁的AFLP和SCAR分子标记

 本研究以全雌品种‘戴多星’自交系和弱雌品种‘北京截头’自交系为双亲杂交获得F₁ ,然后得到F₂ 性型分离群体, 利用分离群体分组分析法(Bulked Segregant Analysis, BSA) 构建全雌和弱雌两个基因池, 筛选了64对AFLP选择性引物EcoR I-NN +Mse I-NNN组合, 发现EcoR I-TG +Mse I-CAC引物组合在全雌基因池中扩增出一条分子量为234 bp的特异带。经F₂ 代单株验证, 该特异条带能在全雌单株中稳定出现。以MAP MAKER (Version 310) 软件分析, 该标记与全雌性位点的连锁距离在617 cM。命名该连锁标记为TG/CAC₂₃₄。将该特异条带回收、克隆、测序, 设计特异SCAR引物, 再对F₂ 代单株基因组DNA进行扩增, 仅在全雌单株中扩增出1条分子量为166 bp 的特异带, 表明已成功地将与黄瓜全雌性连锁的AFLP标记转化为操作简便、表现稳定的SCAR标记, 该标记命名为SA₁₆₆。     

低氧胁迫下黄瓜幼苗根系多胺代谢的变化

 利用外源Spd、PAO 抑制剂研究了黄瓜幼苗根系在低氧水培时的生长状况以及内源PAs、H₂O₂ 含量和PAO活性的动态变化。结果表明, 外源PAs对低氧胁迫下黄瓜幼苗根系的生长有促进作用;低氧处理后3种PAs含量都明显上升, 外源Spd和PAO抑制剂增加了根系中的PAs含量, 降低了根系中H₂O₂ 含量, 减缓了低氧伤害; PAs为低氧胁迫的应激产物, PAO为适应酶类, 其活性随PAs含量的变化而发生变化。     

黄瓜抗霜霉病异源易位系CT201的筛选与鉴定

 将栽培黄瓜‘北京截头’ (Cucumis sativus L. , 2n = 14) 与Cucum is属双二倍体新种C. hytivus (2n = 38) 回交, 随后自交。通过霜霉病田间接种试验筛选, 从中发现抗霜霉病单株HH12825。将HH12825与C. hytivus的原始亲本‘北京截头’和野生种C. hystrix进行农艺性状对比观察, 发现此单株在叶面积、节长、果长×果径等方面介于栽培黄瓜和野生种之间, 在侧枝数、果刺颜色等方面偏向野生种。观察其花粉母细胞减数分裂过程, 发现其PMC中期Ⅰ多价体频率(56% ±8% ) 和后期Ⅰ二价体分离滞后率(72% ±5% ) 都较高, 具有染色体易位的细胞学特征。进一步利用RAPD分子标记分析, 结果在40个随机引物中找到了两个引物( E219: ACGGCGTATG和A I220: CCTGTTCCCT) 能够在HH12825中重复地扩增出原始亲本野生种C. hystrix特异DNA片断, 从分子水平上证明了其为黄瓜异源易位系, 并把此易位系命名为CT201。     

黄瓜枯萎病新型抑制蛋白L37的研究

 从土壤中分离到对黄瓜枯萎病病原菌有抑制作用的枯草芽孢杆菌(Bacillus subtilis) , 发酵产物经DEAE - 纤维素、Sephadex G-100柱层析, 分离到15 kD抑菌蛋白L37, SDS-PAGE显示纯度达到电泳纯, N - 末端氨基酸序列同源性检测结果提示可能是一种新型的抑菌蛋白。蛋白质抑菌结果表明, 对多种病原真菌具有抑制效果, 且对蛋白酶、温度和大部分有机物不敏感, 是稳定的抑菌蛋白。对病原菌菌丝抑制机理研究表明, 抑菌蛋白具有严重扭曲、断裂、阻止生长等抗生效果。应用发酵蛋白产物进行两年大田防效试验表明, 对黄瓜枯萎病防效显著, 最高达到90% , 具有显著提高产量的作用。