Expression of an extracellular ribonuclease gene increases resistance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

     

Transmission of carrot mosaic virus by aphids

The transmission of the carrot mosaic virus (CMV) by the aphidsAcyrtJiosiphon pisum HARRÍS,Cavariela aegopodii SCOP, andMyzus persicae SULZ was proved experimentally. It was observed simultaneously that CMV has a non-persistent character. CMV can be transmitted already 2 min after acquisition feeding by the aphidsMyzus persicae SDLZ andCavariella aego-podii Scop. When the time of acquisition feeding is prolonged to 4 min, CMV is transmitted also by aphidAcyrthosiphon pisum HAREÍs. The host range of the investigated virus wasalso determined and its transmission to 8 plant species, belonging to 4 families, was achieved. On the basis of studies of the vector virus relationship and of the host range, further proof was given for the different character of the Australian Carrot motley dwarf virus, theApivm virus 1 Roland and CMV. The experiments showed that preliminary starving of the aphids for 1 h increases their ability to transmit the virus by 3–3%.     

Effects of rice resistance on the feeding behavior and subsequent virus transmission efficiency of <Emphasis Type="Italic">Laodelphax striatellus</Emphasis>

Laodelphax striatellus is an important vector of rice stripe virus (RSV). In this study, electrical penetration graph technology was applied to investigate the feeding behavior of L. striatellus associated with virus transmission. The effects of a disease-resistant variety Yandao No. 8 on the feeding behavior and subsequent virus transmission efficiency of L. striatellus were examined. The results indicate that in addition to the phloem sap ingestion phase, which was previously reported as a behavior associated with virus acquisition, phases of salivation and stylet movement were relevant to RSV acquisition by naïve L. striatellus. The duration of the non-penetration phase of naïve L. striatellus on healthy Yandao No. 8 plants was significantly longer, and the duration of sap ingestion was significantly shorter compared to those on a susceptible control. RSV acquisition rate of naïve L. striatellus on Yandao No. 8 was only 28 % of that on the susceptible control. Virus inoculation by viruliferous L. striatellus could occur during the salivation, stylet movement, and phloem sap ingestion phases. Yandao No. 8 significantly prolonged the duration of the non-penetration phase and significantly shortened the duration of sap ingestion in viruliferous L. striatellus. Virus inoculation rate of viruliferous L. striatellus feeding on healthy Yandao No. 8 was significantly lower, decreasing by 27 %, than that of the control. The mechanisms of varietal effects on the feeding behavior and virus transmission of L. striatellus are discussed. The varietal effect on virus transmission should have significance for viral disease control.     

Temperature dependent defence of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> against <Emphasis Type="Italic">Cucumber mosaic</Emphasis> virus and recovery occurs with the formation of dark green islands

Temperature is an important environmental factor controlling plant growth, development, and immune response. However, the role of temperature in plant disease resistance is still elusive. In the present study, the potential effects of temperature on the interaction between Nicotiana tabacum and Cucumber mosaic virus (CMV) were investigated. Our results indicated that N. tabacum plants displayed severe symptoms at early stage of post inoculation at high temperature (HT, 28°C), associated with higher viral replication level, more serious stress damages. By contrast, low temperature (LT, 18°C) effectively delayed the replication of CMV compared with elevated temperatures. Additionally, quantitative real-time PCR analyses revealed that lower temperatures (≤ 24°C) promote salicylic acid (SA) dependent responses, whereas higher temperatures (> 24°C) activate the genes expression of jasmonic acid (JA) pathway. Interestingly, the dark green islands (DGIs) appeared much earlier in CMV-inoculated plants grown at HT compared with those at LT and the accumulation of virus small interfering RNAs in plants were significantly up-regulated under elevated temperatures at early stage of post inoculation. Taken together, these results indicated that temperature changes had important effects on plant defence response, and different temperatures could induce different immune pathways of N. tabacum against CMV infection.     

Effective expression of the gene encoding an extracellular ribonuclease of <Emphasis Type="Italic">Zinnia elegans</Emphasis> in the SR1 <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> plants

Complementary DNA for the extracellular RNase of Zinnia elegans was cloned under control of the cauliflower mosaic virus 35S RNA constitutive promoter and transferred into the Nicotiana tabacum SR1 plants. Primary tobacco transformants were characterized by a high level of RNase activity.     

<Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> HW2 enhanced cucumber resistance against cucumber green mottle mosaic virus

Cucumber green mottle mosaic virus (CGMMV) is a major limiting factor in the production of cucumber plants worldwide. In the present study, we use plant growth-promoting rhizobacteria (PGPR) to control this virus effectively. Stenotrophomonas maltophilia HW2 was isolated from healthy cucumber root, exhibited a good biocontrol efficacy against CGMMV. Here, it is documented that 20 d after virus inoculation, the biocontrol efficacy of HW2 reached 52.61%. HW2 can effectively colonize in cucumber rhizosphere, and also promoted cucumber plants growth. We also examined the effect of HW2 on viral replication and its mechanism. Compared with the control, HW2 pre-treated plants could delay virus replication for more than 3 d and inhibit viral protein genes (CP, MP, Rep) expression in the cucumber leaf. The expression of antioxidant enzyme genes (SOD and CAT) and defense-related genes (PR1 and PR5) were quickly induced by HW2. These results suggest that HW2 induced plant defense responses to CGMMV by increasing the expression of defense response genes. We report for the first time that Stenotrophomonas maltophilia improved cucumber resistance against CGMMV, which highlights the applying of PGPR on controlling of virus diseases.     

Reactions and resistance ofChenopodium species to tobacco mosaic virus

Chenopodium species react on infection with tobacco mosaic virus by the formation of chlorotic or necrotic lesions and later by the abscission of infected leaves. A transition of local infection into the stem has been observed exceptionally inChenopodium quinoa, C. hybridum, andC. rubrum, but no systemic infection of the leaves followed. Systemic infection was demonstrated only inC. polyspermum andC. murale. The recovery of new sprouts was demonstrated in C.murale in the late chronic phase of infection.     

Morphological variation,management and domestication of ‘maguey alto’ (<Emphasis Type="Italic">Agave inaequidens</Emphasis>) and ‘maguey manso’ (<Emphasis Type="Italic">A. hookeri</Emphasis>) in Michoacán,México

Background

Agave inaequidens and A. hookeri are anciently used species for producing the fermented beverage ‘pulque’, food and fiber in central Mexico. A. inaequidens is wild and cultivated and A. hookeri only cultivated, A. inaequidens being its putative wild relative. We analysed purposes and mechanisms of artificial selection and phenotypic divergences between wild and managed populations of A. inaequidens and between them and A. hookeri, hypothesizing phenotypic divergence between wild and domesticated populations of A. inaequidens in characters associated to domestication, and that A. hookeri would be phenotypically similar to cultivated A. inaequidens.

Methods

We studied five wild and five cultivated populations of A. inaequidens, and three cultivated populations of A. hookeri. We interviewed agave managers documenting mechanisms of artificial selection, and measured 25 morphological characters. Morphological similarity and differentiation among plants and populations were analysed through multivariate methods and ANOVAs.

Results

People recognized 2–8 variants of A. inaequidens; for cultivation they select young plants collected in wild areas recognized as producing the best quality mescal agaves. Also, they collect seeds of the largest and most vigorous plants, sowing seeds in plant beds and then transplanting the most vigorous plantlets into plantations. Multivariate methods classified separately the wild and cultivated populations of A. inaequidens and these from A. hookeri, mainly because of characters related with plant and teeth size. The cultivated plants of A. inaequidens are significantly bigger with larger teeth than wild plants. A. hookeri are also significatly bigger plants with larger leaves but lower teeth density and size than A. inaequidens. Some cultivated plants of A. inaequidens were classified as A. hookeri, and nearly 10% of A. hookeri as cultivated A. inaequidens. Wild and cultivated populations of A. inaequidens differed in 13 characters, whereas A. hookeri differed in 23 characters with wild populations and only in 6 characters with cultivated populations of A. inaequidens.

Conclusions

Divergence between wild and cultivated populations of A. inaequidens reflect artificial selection. A. hookeri is similar to the cultivated A. inaequidens, which supports the hypothesis that A. hookeri could be the extreme of a domestication gradient of a species complex.

     

The Mechanical transmission and some properties of a virus disease of cow-parsnip,Heracleum sphondylium L

The described virus of cow-parsnip,Heracleum sphondylium L., was found in three ruderal localities of Greater Praha. The symptoms are manifested by decolorations which consist of bright yellow areas spreading from the centre of the leaf blade along the main veins. These symptoms appear severely in May. Under higher temperatures and in a chronic stage of infection the symptoms are more or less masked. The disease is mechanically transmissible to parsley, coriander, parsnip, dill, sowbane,Chenopodium quinoa and C.giganteum. The author failed to transmit the disease to celery, carrot, caraway and to 27 species of differential host plants, he failed in the transmission of the virus by the dodder,Cuscuta campestris YUNCK., too. Thermal inactivation point of the virus lies between 51° and 55° C. Infectivity of extracted sap was lost after 2 days at room temperature.     

Grapevine leaf tissue colonization by the fungal entomopathogen <Emphasis Type="Italic">Beauveria bassiana</Emphasis><Emphasis Type="Italic">s.l.</Emphasis> and its effect against downy mildew

A study was conducted to examine whether Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) can colonize grapevine leaf tissues and subsequently confer protection against downy mildew caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni. Following the foliar inoculation of plants with conidial suspensions of selected B. bassiana strains, colonization of leaves by the fungus was determined using culture-based and PCR techniques at different time intervals. Seven days following B. bassiana inoculation, grapevine plants were challenged with P. viticola and symptoms were assessed by calculating the disease incidence and severity. Although all tested strains were able to colonize grapevine plants, percent colonization differed significantly among strains. Disease incidence and severity were, on the other hand, significantly reduced in B. bassiana-inoculated plants compared to control plants irrespective of strain. This study is one of very few studies investigating the promising role B. bassiana could play as a plant disease antagonist.     

Rapid identification of QTLs underlying resistance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in pepper (<Emphasis Type="Italic">Capsicum frutescens</Emphasis>)

Key message

Next-generation sequencing enabled a fast discovery of QTLs controlling CMV resistant in pepper. The gene CA02g19570 as a possible candidate gene of qCmr2.1 was identified for resistance to CMV in pepper.

Abstract

Cucumber mosaic virus (CMV) is one of the most important viruses infecting pepper, but the genetic basis of CMV resistance in pepper is elusive. In this study, we identified a candidate gene for CMV resistance QTL, qCmr2.1 through SLAF-seq. Segregation analysis in F₂, BC₁ and F_2:3 populations derived from a cross between two inbred lines ‘PBC688’ (CMV-resistant) and ‘G29’ (CMV-susceptible) suggested quantitative inheritance of resistance to CMV in pepper. Genome-wide comparison of SNP profiles between the CMV-resistant and CMV-susceptible bulks constructed from an F₂ population identified two QTLs, designated as qCmr2.1 on chromosome 2 and qCmr11.1 on chromosome 11 for resistance to CMV in PBC688, which were confirmed by InDel marker-based classical QTL mapping in the F₂ population. As a major QTL, joint SLAF-seq and traditional QTL analysis delimited qCmr2.1 to a 330 kb genomic region. Two pepper genes, CA02g19570 and CA02g19600, were identified in this region, which are homologous with the genes LOC104113703, LOC104248995, LOC102603934 and LOC101248357, which were predicted to encode N-like protein associated with TMV-resistant in Solanum crops. Quantitative RT-PCR revealed higher expression levels of CA02g19570 in CMV resistance genotypes. The CA02g19600 did not exhibit obvious regularity in expression patterns. Higher relative expression levels of CA02g19570 in PBC688 and F₁ were compared with those in G29 during days after inoculation. These results provide support for CA02g19570 as a possible candidate gene of qCmr2.1 for resistance to CMV in pepper.

     

Contents of Heavy Metals in Chinese Edible Herbs: Evidence from a Case Study of Epimedii Folium

Toxic heavy metal contamination in Chinese edible herbs has raised a worldwide concern. In this study, heavy metals in Epimedii Folium, an edible medicinal plant in China, were quantitatively analyzed. Variations of heavy metals in different species, in various organs (i.e., leaves, stems, and roots), in wild-growing and cultivated plants, and in 35 market samples of Epimedii Folium, were systematically investigated. In all of Epimedium samples, Hg (mercury) was not detectable (0.00 μg/g). Four species, Epimedium pubescens, Epimedium sagittatum, Epimedium brevicornu, and Epimedium wushanense, were found to contain Cu (copper) and Pb (lead). And contents of Cu and Pb in E. brevicornu were significantly higher than those in other species (P < 0.01). In wild-growing and cultivated Epimedium plants, Cd (cadmium) and As (arsenic) were not detectable, and concentrations of Cu and Pb in wild-growing plants were significantly higher than those in cultivated plants (P < 0.01). Cd was not detectable in leaves, roots, and stems, while organ specificity was apparent in the distribution of Cu, As, and Pb. And the highest levels of Cu and Pb were observed in roots and leaves, respectively. In Chinese markets, several samples of Epimedii Folium contained excessive Cu, Cd, As, and Pb beyond the national permissible limits. In summary, there was a large variation of heavy metals among Epimedii Folium samples, and Cu and Pb were the most important heavy metals contaminating the edible medicinal plant. Application of Epimedii Folium to drug and food industries will need to focus more on toxic heavy metal contamination.     

Tissue specific expression of hepatitis B virus surface antigen in transgenic plant cells and tissue culture

The tobacco plants (Nicotiana tabacum L.) carrying the HBsAg gene controlled by (Aocs)₃AmasPmas, the hybrid promoter that includes regulatory elements of the agrobacterial octopine and mannopine synthase genes, as well as plants controlled by the same promoter and adh1, maize alcohol dehydrogenase gene intron were obtained. The presence of the adh1 gene intron did not significantly change the level of expression of the HBsAg gene in plants. The analysis of expression of hepatitis B virus surface antigen (HBs-antigen) in transformed plants expressing the HBsAg under the control of different promoters was made. The level of HBs-antigen in plants carrying the HBsAg gene controlled by (Aocs)₃AmasPmas, the hybrid agrobacterium-derived promoter, was the highest in roots and made up to 0.01% of total amount of soluble protein. The level of HBs-antigen in plants carrying the HBsAg gene controlled by the dual 35S RNA cauliflower mosaic virus promoter was the same in all organs of the plant and made up to 0.06% of the total amount of soluble protein. Hairy root and callus cultures of plants carrying the HBsAg gene and expressing the HBs-antigen were obtained.