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...     

酵母pac1基因介导对葡萄B病毒的抗性

为明确广谱性抗病毒基因—酵母pac1基因对葡萄B病毒(Grapevine virus B,GVB)的抗性效果,通过农杆菌介导的遗传转化,将pac1基因导入西方烟37B,对转基因植株进行PCR鉴定及Southern blot分析,通过病毒摩擦接种观察症状以及实时荧光定量RT-PCR检测植株体内病毒含量,并对转基因植株抗病性进行初步鉴定。结果表明,目的基因pac1成功导入并整合至西方烟37B基因组,共获得10个转基因株系。不同株系的T1代烟草中阳性植株比例为16.7%~72.7%,表明目的基因可成功遗传到子代。接种病毒后转基因植株普遍延迟发病,但后期症状与非转基因对照相似,其中仅1个转基因株系B6具有不表现典型症状等抗性反应。接种植株病毒含量检测中,所有转基因植株均检测到病毒存在,但表现为抗病的B6株系中病毒含量显著低于非转基因对照,表明该转基因植株虽不能完全抵抗GVB侵染,但对GVB具有耐病性。     

Virus transmission by aphids in potato crops

This paper reviews the contribution of vector activity and plant age to virus spread in potato crops. Determining which aphid species are vectors is particularly important for timing haulm destruction to minimize tuber infection by potato virus Y (PVY). Alate aphids of more than 30 species transmit PVY, and aphids such asRhopalosiphum padi, that migrate in large numbers before flights of the more efficient vector,Myzus persicae, appear to be important vectors. Differences in methodology, aphid biotypes and virus strains prevent direct comparisons between estimates of vector efficiencies obtained for aphids in different countries in north western Europe. M. persicae is also the most efficient vector of potato leafroll virus (PLRV), but some clones ofMacrosiphum euphorbiae transmit PLRV efficiently toNicotiana clevelandii and potato test plants. The removal of infected plants early in the season prevents the spread of PLRV in cool regions with limited vector activity. The proportion of aphids acquiring PLRV from infected potato plants decreases with plant age, and healthy potato plants are more resistant to infection later in the season. Severe symptoms of secondary leafroll developed on progeny plants of cv. Maris Piper derived from mother plants inoculated with PLRV in June or July of the previous year. Progeny plants derived from mother plants inoculated in August showed only mild symptoms, but the concentration of PLRV in these plants was as high as that in the plants with severe symptoms.     

High resistance to rice yellow mottle virus in two cultivated rice cultivars is correlated with failure of cell to cell movement

Rice yellow mottle virus (RYMV) accumulation in protoplasts and whole plants was investigated in two highly resistant cultivars, Tog5681 (Oryza glaberrima) and Gigante (Oryza sativa). Three susceptible cultivars, i.e. one O. glaberrima Tog5673 and two O. sativa (IR64, Ac. 2428), and a partially resistant cultivar (Azucena) were used as control. After inoculation, accumulation of coat protein (CP) and viral RNA were monitored on protoplasts, inoculated leaves, sheaths of inoculated leaves and newly infected leaves by serological and Northern blot analysis. Viral RNA accumulated to a similar extent in protoplasts from all cultivars studied. In contrast, three distinct in planta behaviors were noted. In susceptible plants (IR64, Tog5673 and Ac. 2428), there was high CP and RNA accumulation at 5 d.p.i. in whole plants, suggesting that cell to cell and vascular movements occurred before 5 d.p.i. in inoculated leaves. The second behavior concerned Azucena, which showed a delay (around 7 d.p.i.) of viral accumulation in inoculated leaves. The third behavior involved the highly resistant cultivars Tog5681 and Gigante. CP and viral RNA were not detected in these cultivars. The comparison of viral accumulation in protoplasts and plants suggested that resistance of the highly resistant cultivars Tog5681 (O. glaberrima) and Gigante (O. sativa) was not due to the inhibition of virus replication but rather to the failure of cell to cell movement.     

Nematicidal Activity of Chrysanthemum coronarium

Organic amendments and green manure are potential alternatives to the harmful chemical control means currently used against plant-parasitic nematodes. In this work, Chrysanthemum coronarium was applied to the soil as a green manure to control the root-knot nematodes Meloidogyne incognita and M. javanica. Chrysanthemum coronarium significantly reduced nematode infection of tomato roots and improved plant-top fresh weight, both in the greenhouse and in microplots. Other green manures, derived from Anthemis pseudocotula, wild chickpea (Cicer pinnatifidum), Geranium spp. and wheat, were not as effective as C. coronarium. Chrysanthemum coronarium, retained its nematicidal activity even when applied as a dried material. Only mature C. coronarium plants, in their flowering stage, exhibited nematode control activity, but the green plant parts were more effective than the flowers. An aqueous extract of C. coronarium exhibited in vitro, nematostatic activity towards M. incognita and M. javanica second-stage juveniles and inhibited their hatching from eggs and egg-masses; its nematostatic activity was expressed also against other phytonematode species such as Heterodera avenae and Pratylenchus mediterraneus, but did not affect the beneficial entomopathogenic nematode Steinernema feltiae.     

Analysis of Accumulation Patterns of <Emphasis Type="Italic">Barley yellow dwarf virus-PAV</Emphasis> (BYDV-PAV) in Two Resistant Wheat Lines

Barley yellow dwarf (BYD) is one of the main viral diseases of small-grain cereals. This disease, reported on numerous plant species of the Poaceae family, is caused by a complex of eight viral species including the species Barley Yellow Dwarf Virus-PAV (BYDV-PAV), frequently found in western Europe. Resistance sources against BYDV-PAV are scarce and only identified in perennial Triticineae. Some BYDV-resistant wheat lines have been obtained by introgressing these resistances into bread wheat germplasms. Genetic and biological characterization of the resulting lines has been undertaken. However, little information on the resistant behaviour of these lines during the early stages of the infection process is available. To evaluate the resistance of two genetically distinct resistant lines (Zhong ZH and TC14), 1740 young plantlets, belonging to susceptible reference hosts (barley cv. Express and wheat cv. Sunstar), Zhong ZH or TC14 wheat lines, were inoculated in controlled conditions with French BYDV-PAV isolates. The infection process was monitored during the first 21 days after inoculation (DAI) using a semi-quantitative ELISA. A standardized protocol including five successive samplings of leaves from all inoculated plants and the collection of plant roots at the end of the monitored period was carried out. This protocol enabled an assessment of the infection percentage and the evolution of the viral load in plants from the 7th DAI to the 21st DAI. Statistical analyses of the BYDV infection kinetics using raw ELISA data, a model of the time-dependent variation of the percentage of infected plants and the area under concentration progress curves (AUCPC) demonstrated that Zhong ZH and TC14 lines (1) reduce the development rate of the BYD disease during the first days of infection, (2) decrease the infection efficiency of BYDV-PAV isolates, in the leaves, from 98.7% for susceptible plant genotypes to 81.9% and 71.7% for Zhong ZH and TC14, respectively, (3) reduce the virus load in the leaves of infected plants and (4) are not spared from BYDV infection, as 95.1% of Zhong ZH and 90.2% of TC14 inoculated plants accumulated viral particles in roots and/or in leaves at 21 DAI. These results confirm the BYDV-partial resistant behaviour of both Zhong ZH and TC14 lines. The development rate of the disease was the single parameter that allowed the distinction between the two resistant sources present in the tested lines.     

Separation of two leafhopper populations of theCirculifer haematoceps complex on different host plants in Israel

TwoCirculifer leafhopper populations of thehaematoceps complex have been collected in large numbers in Israel for several years. One of these leafhoppers inhabited prickly saltwort (Salsola kali) plants on sandy soil on the Mediterranean coast. It has a narrow host range and was found to survive for one generation on turnip. The other leafhopper population was collected in 1986 on common goosefoot (Chenopodium album) plants in the Golan Heights. This population is polyphagous and in the laboratory reproduced well onMatthiola incana, turnip, sugar beet andChrysanthemum coronarium; however, reproduction was nil onPhaseolus vulgaris and very low onBrassica oleracea, Portulaca oleracea andCarthamus tinctorius. Contribution from the Agricultural Research Organization. No. 2502-E, 1988 series.     

地被菊CVB病毒基因的RT-PCR检测

为建立可靠、灵敏且高效的地被菊中菊花B病毒(Chrysanthemum virus B,CVB)的检测方法,利用特异性CVB基因引物,通过RT-PCR技术进行了特异性、重复性和灵敏度测试,最终利用优化的RT-PCR体系对地被菊植株感染CVB病毒情况进行了检测。结果显示,以染病地被菊植株的总RNA为模板扩增出的特异性片段编码211个氨基酸,与基因数据库中其它来源的CVB基因外壳蛋白同源性为96%~99%,可确定为菊花B病毒外壳蛋白基因;重复性和灵敏度检测中均获得了清晰的目标条带,且1 ng的总RNA即可扩增出特异性片段;对7个地被菊品种共32个植株的检测中,CVB的检出率为56.2%,检测的准确率为68.75%。表明建立的地被菊CVB基因的RT-PCR检测体系可有效用于地被菊植株感染病毒情况的鉴定。     

Tobacco streak virus in sunflower (Helianthus annuus)

Tobacco streak virus (TSV) was isolated from a plant of sunflower (Helianthus annuus) showing severe necrosis and chlorosis in the leaves. The virus was identified as TSV by serology and, to some extent, by host range. The type of symptoms varied with the host plant in which the virus had been propagated in successive transfers. Test plants inoculated with the virus propagated inNicotiana rustica produced symptoms which very much differed from those brought about by the virus from eitherN. clevelandii orChenopodium amaranticolor.The significance of the host-mediated variation in symptoms is discussed.Samenvatting Tabaksstrepenvirus (TSV) werd geïsoleerd uit zonnebloem (Helianthus annuus) die sterke necrose en chlorose van de bladeren vertoonde. De identiteit van het virus werd vastgesteld op grond van serologische reacties en, tot op zekere hoogte, de symptomatologie. Het type symptoom op de toetsplanten bleek echter sterk afhankelijk te zijn van de plant waarvan het inoculum afkomstig was. Was het virus verschillende malen achtereen vermeerderd inNicotiana rustica dan waren de symptomen op de toetsplanten zeer verschillend van die, welke werden veroorzaakt door virus vermeerderd inN. clevelandii ofChenopodium amaranticolor. De betekenis van deze door de waardplant bewerkstelligde variatie in symptomen wordt besproken.     

Effects of inoculum density,leaf wetness duration and nitrate concentration on the occurrence of lettuce leaf spot

In Ehime Prefecture, Japan, lettuce leaf spot (Septoria lactucae) caused huge losses in marketable lettuce yields. To explore potential measures to control disease outbreaks, the effects of inoculum density, leaf wetness duration and nitrate concentration on the development of leaf spot on lettuce (Lactuca sativa) were evaluated. Conidia were collected from diseased plants in an infested field by single-spore isolation and were used to inoculate potted lettuce plants with different conidial concentrations. Lesions developed on inoculated lettuce plants at inoculum concentrations from 10⁰ to 10⁶ conidia/ml. The disease was more severe when the inoculum exceeded 10² conidia/ml, and severity increased with increasing concentrations. Assessment of the relationship between disease development and the duration of postinoculation leaf wetness revealed that symptoms appeared when the inoculated plants remained wet for 12 h or longer. The number of lesions and total nitrogen content in the lettuce leaves both increased when nitrate was applied.     

Behavior and mutation of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in <Emphasis Type="Italic">Solanum toxicarium</Emphasis> grown in aseptic culture

To study the behavior and mutation of Ralstonia solanacearum in Solanum toxicarium, which is resistant to bacterial wilt, S. toxicarium was grown in aseptic culture and inoculated with R. solanacearum. Although 60%–80% of the inoculated plants were wilting after 2 to 3 days, most wilted plants had recovered by 20 days after inoculation. The pathogen was reisolated from over 98% of inoculated plant stems, but the percentage of recovery decreased the closer the isolation sites were toward the upper stem sections. Three colony types, characterized as fluidal white, nonfluidal red, and a mixture of fluidal white and nonfluidal red, were reisolated from the stems. Nonfluidal red colonies were less virulent on tomato plants than fluidal white colonies.     

Timing and extent of hypersensitive response are critical to restrict local and systemic spread of Pepper mild mottle virus in pepper containing the L 3 gene

Amino acid changes in Pepper mild mottle virus (PMMoV) coat protein (CP) that enhance, decrease, or nullify the resistance-inducing activity in Capsicum plants carrying the L ³ gene have been identified. In this study, molecular events underlying the L ³ -gene-mediated resistance were analyzed through the expression of hypersensitive response (HR)-related genes, HSR203J-Cc and HIN1-Cc, and defense-related genes, PR1-Cc and PR4b-Cc, upon infection with PMMoV CP mutants. The expression kinetics of the genes correlated with the degree of restriction of virus distribution in the inoculated leaves. The results suggest that the timing and extent of HR are critical factors to restrict virus spread both locally and systemically in L ³ -gene-mediated resistance.The nucleotide sequence data reported are available in the DDBJ/GenBank/EMBL databases under accession numbers AB162220 (HSR203J-Cc), AB162221 (HIN1-Cc), AB162222 (PR1-Cc), and AB162223 (PR4b-Cc)     

Natural infection of Chrysanthemum stunt viroid in dahlia plants

For the first time, Chrysanthemum stunt viroid (CSVd) was detected in commercial dahlia bulbs in Japan. CSVd was found in 77.2% of the tested plants (Dahlia spp.). In nucleotide sequence analysis, a CSVd variant was detected consisting of 354 nucleotides, which differed slightly from previously reported CSVd variants. The nucleotide sequence data reported is available in the DDBJ/EMBL/GenBank databases under accession number AB255879.     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.     

Interaction of Root-knot Nematodes (RKN) and the Bacterium Agrobacterium Tumefaciens in Roots of Prunus Cerasifera: Evidence of the Protective Effect of the Ma RKN Resistance Genes Against Expression of Crown Gall Symptoms

Agrobacterium tumefaciens (AT) is the causal agent of crown gall, a major problem in the family Rosaceae and particularly for Prunus spp. Crown gall symptoms result from the bacterial infection of the cells damaged mechanically at the collar or by root parasitic nematodes. Myrobalan plum (P. cerasifera) is susceptible to AT and is not a host for the root-knot nematode (RKN), M. hapla. Some clones of this plum carry single Ma resistance genes that control M. arenaria, M. incognita and M. javanica. The four above mentioned RKN and Myrobalan progenies segregating for Ma were used in experiments aimed at obtaining a better knowledge of the interaction between AT and RKN in relation to the RKN resistance genes. Prunus rooted cuttings, naturally infected with the bacterium were repotted, grown and inoculated individually with RKN. In a first experiment, Prunus plants were (i) either inoculated with 10,000 juveniles (J2s) of M. arenaria to provide a short inoculum pressure (SIP) or (ii) inoculated by association with one M. arenaria-galled tomato root system that produced a high and durable inoculum pressure of the same nematode species. Four months after RKN inoculation, plants were rated for nematode and bacterial root galling symptoms. RKN and AT galls were more numerous and more homogenous under DIP than under SIP. Nevertheless, for both inoculum regimes, AT galls were present in the RKN-susceptible clones (= carrying none of the Ma genes) and absent in the RKN-resistant clones. Subsequent experiments, conducted under DIP with M. arenaria, M. incognita, M. javanica and M. hapla, also showed, for the three first species, the presence of AT galls only in RKN-susceptible clones whereas Prunus plants inoculated with M. hapla and nematode-free controls were free of AT galls. Consequently RKN act as a wound agent in the AT infection process of Myrobalan plum only when the plant develops a compatible reaction (i.e. when it lacks the Ma resistance genes). Considering that J2s do penetrate the roots of resistant plants, the absence of crown gall symptoms on this material even under durable inoculum pressure strengthens the hypothesis that this nematode stage has a very weak effect on plant cells during the infection process. This is the first evidence of the protective effect of a RKN resistance gene against the expression of root crown gall consecutive to RKN infection. The protective effect of Ma and presumably of other RKN resistance genes against AT is a strong argument for their introgression into Prunus and other Rosaceae or bacterium-susceptible crops.     

Infection and colonization of jojoba byGanoderma lucidum

In arid conditions in India,Ganoderma lucidum (Leyss: Fr.) P. Karsten was found to cause root rot diseases in jojoba (Simmondsia chinensis (Link) Schneider) plants. In the rainy season, 10–15-year-old jojoba plants growing in the proximity of aGanoderma-infectedAcacia tortilis tree, developed disease symptoms. Twigs of affected plants started drying from the top of the branch; leaves turned yellowish brown and finally abscissed; plants dried up within 1 to 3 months. Basidiocarps developed from decaying roots near the collar region and produced colored stalks and fruiting caps. Pathogenicity of the fungus was established by keeping the infected root segments in direct contact with roots of healthy jojoba plants. Root rot symptoms were expressed within 5 months in inoculated plants subjected to moisture stress.     

Glomus intraradices, Pseudomonas alcaligenes, and Bacillus pumilus: effective agents for the control of root-rot disease complex of chickpea (Cicer arietinum L.)

The effects of Glomus intraradices, Pseudomonas alcaligenes and Bacillus pumilus on the root-rot disease complex caused by the root-knot nematode Meloidogyne incognita and the root-rot fungus Macrophomina phaseolina in chickpea was assessed by quantifying differences in the shoot dry mass, pod number, nodulation, and shoot content of chlorophyll, nitrogen, phosphorus and potassium. Inoculation of plants with G. intraradices, P. alcaligenes and B. pumilus alone and in combination significantly increased shoot dry mass, pod number, and content of chlorophyll, nitrogen, phosphorus and potassium in plants inoculated with pathogens over that in the uninoculated control plants. P. alcaligenes caused a greater increase in shoot dry mass, pod number, chlorophyll, nitrogen, phosphorus and potassium in plants with pathogens than did G. intraradices or B. pumilus. Combined application of G. intraradices, P. alcaligenes and B. pumilus to plants inoculated with pathogens caused a greater increase in shoot dry mass, pod number, nitrogen, phosphorus, and potassium than did an application of P. alcaligenes plus B. pumilus or of G. intraradices plus B. pumilus. Root colonization by G. intrardices was high when used alone, while inoculation with the pathogens reduced root colonization by G. intraradices. In the presence of P. alcaligenes and B. pumilus, root colonization by G. intraradices increased. In plants inoculated with just one antagonist, P. alcaligenes reduced galling and nematode multiplication the most, followed by G. intraradices, then B. pumilus. The greatest reduction in galling, nematode multiplication and root-rot was observed when both bacterial species and G. intraradices were applied together.