Study of multiplication of cucumber mosaic virus in susceptible and resistant Capsicum annuuum lines

A previous survey on pepper lines (Capsicum annuum L.) indicated that a susceptible cultivar, Yolo Wonder, reacted to cucumber mosaic virus (CMV) by producing a systemic yellow mosaic. By contrast, CMV caused no symptoms on lines Perennial and Vania. The virus is recoverable from the uninoculated leaves of Perennial, while in Vania CMV is restricted to the inoculated leaves. To interpret these phenomena, a comparative study on CMV multiplication rates, yield, specific infectivity and relative proportion of RNAs was made in the inoculated leaves of the three pepper varieties. The rate of CMV multiplication, as estimated by the double antibody sandwich form of enzyme-linked immu-nosorbent assay, was lower in Perennial than in Vania or Yolo Wonder. The yield of virus purified from Perennial was very low when compared with Vania or Yolo Wonder. The specific infectivity of the virus extracted from Perennial was less than that from Vania or Yolo Wonder. These results suggest that Perennial is resistant to CMV multiplication, while restriction of the virus in inoculated leaves of Vania is not due to the inhibition of the virus replication. However, polyacrylamide gel electrophoresis revealed that the RNA profiles of CMV purified from the three pepper lines were similar.     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

黄瓜花叶病毒基因组不同部分及卫星RNA介导的对植株的保护及其作用机理

根据病原物介导的对自身抗性的理论,大量开展了将CMV基因组的单个或多个片断转入植物体内的研究,从而使该植株能够抵抗或延迟受CMV的侵染,CP,RP,MP基因是CMV基因组的重要组成部分,用来转化植株取得了不同程度的抗性效果,另外有些CMV株中存在着起致弱作用的卫星RNA,直接对植株接种含卫星RNA的CMV弱毒或用卫星RNA的cDNA转化植株都会减轻CMV强毒对该植株的侵害,CMV基因组不同组分进入植物体内后,它们对植株产生保护作用的机理不同,文中分别加以阐述。     

抗烟草黄瓜花叶病毒多糖筛选及其对烟草防御酶活性的影响

[目的]筛选出对烟草黄瓜花叶病毒病有良好抑制作用的多糖并探索其对烟叶防御酶活性的影响.[方法]采用半叶法,测定了安络小皮伞多糖等21种真菌多糖在枯斑三生烟上对CMV的钝化、预防及治疗效果,并测定了抗病毒多糖处理后普通烟NC-89体内防御酶的变化.[结果]安络小皮伞多糖对CMV具有较好的钝化及预防效果,其200倍液与等量供试病毒液混合30 min后接种,钝化效果为83.41%;喷施安络小皮伞多糖200倍液24 h后接毒处理,预防效果可高达93.15%.安络小皮伞多糖对CMV防治机理的研究表明,多糖处理后烟草相关防御酶POD、PAL和PPO活性增强,其中喷施安络小皮伞多糖24 h后接毒处理的酶活增加最为显著,该处理烟苗的POD、PAL和PPO的酶活峰值分别可增加至对照的2.74、3.45和2.82倍.[结论]安络小皮伞多糖通过增强烟草体内防御酶活性而提高烟草对烟草黄瓜花叶病毒病的抗性.     

Transgenic watermelon rootstock resistant to CGMMV (cucumber green mottle mosaic virus) infection

In watermelon, grafting of seedlings to rootstocks is necessary because watermelon roots are less viable than the rootstock. Moreover, commercially important watermelon varieties require disease-resistant rootstocks to reduce total watermelon yield losses due to infection with viruses such as cucumber green mottle mosaic virus (CGMMV). Therefore, we undertook to develop a CGMMV-resistant watermelon rootstock using a cDNA encoding the CGMMV coat protein gene (CGMMV-CP), and successfully transformed a watermelon rootstock named gongdae. The transformation rate was as low as 0.1–0.3%, depending on the transformation method used (ordinary co-culture vs injection, respectively). However, watermelon transformation was reproducibly and reliably achieved using these two methods. Southern blot analysis confirmed that the CGMMV-CP gene was inserted into different locations in the genome either singly or multiple copies. Resistance testing against CGMMV showed that 10 plants among 140 T₁ plants were resistant to CGMMV infection. This is the first report of the development by genetic engineering of watermelons resistant to CGMMV infection.     

Resistance to cucumber mosaic virus in potato

Reactions to two subgroup I isolates (Fny-CMV and Pf-CMV) and two subgroup II isolates (A9-CMV and LS-CMV) of cucumber mosaic virus (CMV) were studied in three non tuber-bearing wild potato species (Solanum spp.) of the series Etuberosa, and in two tuber-bearing interspecific potato hybrids and four potato cultivars using graft-inoculation. Three classes of phenotypic reactions (susceptible, hypersensitive, extreme resistance) were observed in the tuber-bearing genotypes. Susceptible genotypes developed mosaic or severe mosaic with leaf malformation and had high CMV titres. Hypersensitive genotypes developed either top necrosis or vein necrosis and/or necrotic spots on apical leaves, and had low CMV titres. Extremely resistant genotypes had no symptoms and no CMV was detected. The hybrid 87HW13.7 (S. tuberosum×S. multidissectum) developed top necrosis specific to infection with Fny-CMV. The hybrid ‘A6’ (S. demissum×S. tuberosum cv. Aquila) was hypersensitive to all CMV isolates tested. Extreme resistance was not functional against all CMV isolates. Neither hypersensitivity nor extreme resistance were related to the CMV subgroup.     

Plant resistance to fungal diseases induced by the infection of cucumber mosaic virus attenuated by satellite RNA

Biological control agents (BCAs) composed of attenuated cucumber mosaic (CMV) and its satellite RNA for controlling CMV diseases were found to induce plant resistance to a number of fungal diseases. Tests conducted in both the field and greenhouse showed evident protective effects against fungal infections by the BCAs. Artificial inoculation with a fungal spore suspension using BCA-treated plants, satellite transgenic plants and plants infected with CMV alone indicated that the resistance to fungi was induced by the virus infection, not by the presence of satellite RNA.     

Interaction between cucumber green mottle mosaic virus MP and CP promotes virus systemic infection

The movement protein (MP) and coat protein (CP) of tobamoviruses play critical roles in viral cell-to-cell and long-distance movement, respectively. Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus. The functions of CGMMV MP and CP during viral infection remain largely unclear. Here, we show that CGMMV MP can interact with CP in vivo, and the amino acids at positions 79–128 in MP are vital for the MP–CP interaction. To confirm this finding, we mutated five conserved residues within the residue 79–128 region and six other conserved residues flanking this region, followed by in vivo interaction assays. The results showed that the conserved threonine residue at the position 107 in MP (MP^T107) is important for the MP–CP interaction. Substitution of T107 with alanine (MP^T107A) delayed CGMMV systemic infection in Nicotiana benthamiana plants, but increased CGMMV local accumulation. Substitutions of another 10 conserved residues, not responsible for the MP–CP interaction, with alanine inhibited or abolished CGMMV systemic infection, suggesting that these 10 conserved residues are possibly required for the MP movement function through a CP-independent manner. Moreover, two movement function-associated point mutants (MP^F17A and MP^D97A) failed to cause systemic infection in plants without impacting on the MP–CP interaction. Furthermore, we have found that co-expression of CGMMV MP and CP increased CP accumulation independent of the interaction. MP and CP interaction inhibits the salicylic acid-associated defence response at an early infection stage. Taken together, we propose that the suppression of host antiviral defence through the MP–CP interaction facilitates virus systemic infection.     

Biological and Molecular Characterization of Taiwanese Isolates of Cucumber mosaic virus Associated with Banana Mosaic Disease

Banana mosaic disease (BMD) caused by Cucumber mosaic virus (CMV) has become an important threat to the banana industry. We collected and characterized 10 CMV isolates associated with BMD in Taiwan and compared their biological characteristics and coat protein sequences. The isolates fell into four pathotypes on the basis of the symptoms they induce on banana, Nicotiana glutinosa and Vigna unguiculata (cowpea). Double-stranded RNA analysis revealed that the different pathotypes are not related to the presence of CMV satellite RNA. Phylogenetic analysis of worldwide CMV coat protein sequences revealed that among the currently known CMV subgroups IA, IB and II, subgroup IB is phylogenetically unresolved. Our CMV isolates form a new subgroup, IT, within subgroup I. In addition, we resolved another new CMV subgroup, IS, within subgroup I. The analysis also revealed that isolates within different subgroups can infect the banana.     

Natural resistance to cucumber mosaic virus in lupin species

Ten species of lupins (Lupinus spp.) were tested for resistance to cucumber mosaic cucumovirus (CMV) in field experiments where inoculation was by naturally-occurring aphid vectors, and in the glasshouse by sap or graft-inoculation. L. albus and six species of ‘rough-seeded’ lupins did not become infected with CMV either under intense inoculum pressure in the field or when graft-inoculated. Two L. hispanicus, 17 L. luteus and four L. mutabilis genotypes became infected with CMV in the field, but no infection was detected in L. hispanicus P26858 or seven L. luteus genotypes. CMV was detected at seed transmission rates of 0.2–16% in seedlings of infected L. luteus, differences in levels of seed transmission between genotypes being significant and relatively stable from year to year. Graft-inoculation of CMV to plants of six genotypes of L. luteus in which no infection was found in the field induced a systemic necrotic reaction suggesting that the resistance they carry is due to hypersensitivity. In L. hispanicus accessions P26849, P26853 and P26858, CMV sub-group II isolate SN caused necrotic spots in inoculated leaves without systemic movement, while sub-group I isolate SL infected them systemically without necrosis. Another sub-group I and two other sub-group II isolates behaved like SL in P26849 and P26853 but infected only inoculated leaves of P26858. This suggests that two strain specific hypersensitive resistance specificities are operating against CMV in L. hispanicus. When plants of L. luteus genotypes that gave hypersensitive reactions on graft-inoculation were inoculated with infective sap containing two sub-group I and seven sub-group II isolates, they all responded like L. hispanicus P26858. A strain group concept is proposed for CMV in lupins based on the two hypersensitive specificities found: strain group 1 represented by isolate SN which induces hypersensitivity with both specificities, strain group 2 represented by the three isolates which induced hypersensitivity only with the specificity present in L. luteus and L. hispanicus P26858, strain group 3 by as yet hypothetical isolates that induce hypersensitivity only in presence of the specificity in L. hispanicus P26849 and P26853 that responded just to isolate SN, and strain group 4 by isolate SL which overcomes both specificities. When F₂ progeny plants from crosses between hypersensitive and susceptible L. luteus parents were inoculated with isolate SN, the resistance segregated with a 3:1 ratio (hypersensitive:susceptible), suggesting that a single dominant hypersensitivity gene, Ncm-1, is responsible. As gene Ncm-1 had broad specificity and was not overcome by any of the five CMV isolates from lupins tested, it is valuable for use in breeding CMV resistant L. luteus cultivars.     

Natural resistance to cucumber mosaic virus in lupin species

Ten species of lupins (Lupinus spp.) were tested for resistance to cucumber mosaic cucumovirus (CMV) in field experiments where inoculation was by naturally-occurring aphid vectors, and in the glasshouse by sap or graft-inoculation. L. albus and six species of ‘rough-seeded’ lupins did not become infected with CMV either under intense inoculum pressure in the field or when graft-inoculated. Two L. hispanicus, 17 L. luteus and four L. mutabilis genotypes became infected with CMV in the field, but no infection was detected in L. hispanicus P26858 or seven L. luteus genotypes. CMV was detected at seed transmission rates of 0.2–16% in seedlings of infected L. luteus, differences in levels of seed transmission between genotypes being significant and relatively stable from year to year. Graft-inoculation of CMV to plants of six genotypes of L. luteus in which no infection was found in the field induced a systemic necrotic reaction suggesting that the resistance they carry is due to hypersensitivity. In L. hispanicus accessions P26849, P26853 and P26858, CMV sub-group II isolate SN caused necrotic spots in inoculated leaves without systemic movement, while sub-group I isolate SL infected them systemically without necrosis. Another sub-group I and two other sub-group II isolates behaved like SL in P26849 and P26853 but infected only inoculated leaves of P26858. This suggests that two strain specific hypersensitive resistance specificities are operating against CMV in L. hispanicus. When plants of L. luteus genotypes that gave hypersensitive reactions on graft-inoculation were inoculated with infective sap containing two sub-group I and seven sub-group II isolates, they all responded like L. hispanicus P26858. A strain group concept is proposed for CMV in lupins based on the two hypersensitive specificities found: strain group 1 represented by isolate SN which induces hypersensitivity with both specificities, strain group 2 represented by the three isolates which induced hypersensitivity only with the specificity present in L. luteus and L. hispanicus P26858, strain group 3 by as yet hypothetical isolates that induce hypersensitivity only in presence of the specificity in L. hispanicus P26849 and P26853 that responded just to isolate SN, and strain group 4 by isolate SL which overcomes both specificities. When F₂ progeny plants from crosses between hypersensitive and susceptible L. luteus parents were inoculated with isolate SN, the resistance segregated with a 3:1 ratio (hypersensitive:susceptible), suggesting that a single dominant hypersensitivity gene, Ncm-1, is responsible. As gene Ncm-1 had broad specificity and was not overcome by any of the five CMV isolates from lupins tested, it is valuable for use in breeding CMV resistant L. luteus cultivars.     

The susceptibility and response of inbred and outbred Mimulus guttatus to infection by Cucumber mosaic virus

The deleterious effects of inbreeding have been well documented, but only recently have studies begun to explore the consequences of inbreeding for important ecological interactions. We examined the effects of inbreeding on the interaction between host and pathogen using the mixed-mating Mimulus guttatus (Scrophulariaceae) and Cucumber mosaic virus (CMV). Inbred (self) and outbred M. guttatus from two California populations (M5 and M13) were rub-inoculated with CMV and compared to sham-inoculated controls. Flower production by outbred plants in host population M5 showed little effect of the inoculation treatment, but inoculation reduced flower production of inbred plants by 12%, indicating that inbreeding reduces tolerance to CMV infection. This interaction fell short of significance, however. The effects of inbreeding and CMV inoculation on biomass in M5 varied significantly across the 15 families used in this experiment, indicating genetic variation in the effect of inbreeding on resistance or tolerance to CMV. CMV infection reduced biomass in host population M13, but there were no significant interactions between virus treatment and level of inbreeding for either flower production or biomass. Enzyme linked immunosorbent assay (ELISA) was used to detect CMV in host tissues. In both populations, mean ELISA absorbance values of inoculated plants were nearly identical for self and outcross hosts, indicating equal susceptibility to CMV. In outbred plants of population M5, flower production did not change with increasing ELISA absorbance, but in inbred plants it declined, indicating reduced tolerance to CMV infection. The results from this study suggest that pathogens may become increasingly detrimental as host populations become more inbred.     

Anti-sense RNAs of cucumber mosaic virus in transgenic plants assessed for control of the virus

Three synthetic genes for the production of anti-sense RNA to different regions of the cucumber mosaic virus (CMV) genome were constructed using virus-derived double-stranded cDNA coupled to a promoter sequence from cauliflower mosaic virus. The genes were used to transform tobacco plants by a Ti plasmid vector. Transgenic plants obtained with the three constructs produced anti-sense RNA at different levels. Plants expressing each of the three anti-sense RNAs were inoculated with CMV and their sensitivity to the virus infection was compared with the non-transformed plants. Only one plant line which expressed relatively low levels of one of the anti-sense RNAs showed resistance to CMV but other plants expressing the same or the other two antisense RNAs had similar sensitivity to CMV infection as the non-transformed plants.     

Contrasting physiological responses of six eucalyptus species to water deficit

BACKGROUND AND AIMS: The genus Eucalyptus occupies a broad ecological range, forming the dominant canopy in many Australian ecosystems. Many Eucalyptus species are renowned for tolerance to aridity, yet inter-specific variation in physiological traits, particularly water relations parameters, contributing to this tolerance is weakly characterized only in a limited taxonomic range. The study tests the hypothesis that differences in the distribution of Eucalyptus species is related to cellular water relations. METHODS: Six eucalypt species originating from (1) contrasting environments for aridity and (2) diverse taxonomic groups were grown in pots and subjected to the effects of water deficit over a 10-week period. Water potential, relative water content and osmotic parameters were analysed by using pressure-volume curves and related to gas exchange, photosynthesis and biomass. KEY RESULTS: The six eucalypt species differed in response to water deficit. Most significantly, species from high rainfall environments (E. obliqua, E. rubida) and the phreatophyte (E. camaldulensis) had lower osmotic potential under water deficit via accumulation of cellular osmotica (osmotic adjustment). In contrast, species from low rainfall environments (E. cladocalyx, E. polyanthemos and E. tricarpa) had lower osmotic potential through a combination of both constitutive solutes and osmotic adjustment, combined with reductions in leaf water content. CONCLUSIONS: It is demonstrated that osmotic adjustment is a common response to water deficit in six eucalypt species. In addition, significant inter-specific variation in osmotic potential correlates with species distribution in environments where water is scarce. This provides a physiological explanation for aridity tolerance and emphasizes the need to identify osmolytes that accumulate under stress in the genus Eucalyptus.     

Differences of reconstitution process between tobacco mosaic virus and cucumber green mottle mosaic virus by synchrotron small angle X-ray scattering using low-temperature quenching

The differences of the reconstitution process of tobacco mosaic virus (TMV) and its mutant, cucumber green mottle mosaic virus (CGMMV) were investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. The reconstitution in an aqueous solution is completely stopped below 5°C. The TMV and CGMMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5°C on a series of solutions prepared by low-temperature quenching after incubation at 20°C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. The incubation of RNA and protein of CGMMV did not reconstitute at the initial reaction stages below 5 min and then began to reconstitute gradually. After 60 min, the radius of gyration for CGMMV reconstitution process reached almost the value for the initial stage of TMV reconstitution process. This is due to the fact the formation of double-layered disk in CGMMV protein is much slower than in TMV protein.     

Differential selection of genes of cucumber mosaic virus subgroups

Cucumber mosaic virus (CMV) has an extremely broad plant-host range, a large number of vector species, and a wide geographical distribution. CMV is, therefore, a model by which to understand plant virus adaptation. The selective constraints exerted on the five proteins expressed from the CMV genome were evaluated by application of newly developed maximum-likelihood algorithms to analyze sequences available in data banks. The ratio between nonsynonymous and synonymous substitution rates (omega) was used to detect positive selection on particular codon sites. Amino acid sequences were conserved with omega ranging from 0.07 to 0.60 in different proteins. However, a small proportion of amino acids in proteins 1a, 2a, and 3b, the coat protein (CP), were positively selected (omega > 1). Moreover, the evolution of the CP in the three subgroups of CMV strains revealed different selection profiles along the sequence and significantly different speed of evolution at many positions. Constraints exerted by aphid transmission, rather than plant adaptation, seemed to be responsible for these patterns of evolution in the CP.