One is enough: in vivo effective population size is dose-dependent for a plant RNA virus

Effective population size (N(e)) determines the strength of genetic drift and the frequency of co-infection by multiple genotypes, making it a key factor in viral evolution. Experimental estimates of N(e) for different plant viruses have, however, rendered diverging results. The independent action hypothesis (IAH) states that each virion has a probability of infection, and that virions act independent of one another during the infection process. A corollary of IAH is that N(e) must be dose dependent. A test of IAH for a plant virus has not been reported yet. Here we perform a test of an IAH infection model using a plant RNA virus, Tobacco etch virus (TEV) variants carrying GFP or mCherry fluorescent markers, in Nicotiana tabacum and Capsicum annuum plants. The number of primary infection foci increased linearly with dose, and was similar to a Poisson distribution. At high doses, primary infection foci containing both genotypes were found at a low frequency (<2%). The probability that a genotype that infected the inoculated leaf would systemically infect that plant was near 1, although in a few rare cases genotypes could be trapped in the inoculated leaf by being physically surrounded by the other genotype. The frequency of mixed-genotype infection could be predicted from the mean number of primary infection foci using the independent-action model. Independent action appears to hold for TEV, and N(e) is therefore dose-dependent for this plant RNA virus. The mean number of virions causing systemic infection can be very small, and approaches 1 at low doses. Dose-dependency in TEV suggests that comparison of N(e) estimates for different viruses are not very meaningful unless dose effects are taken into consideration.     

Age-related Resistance in Bell Pepper to Cucumber mosaic virus

We demonstrated the occurrence of mature plant resistance in Capsicum annuum‘Early Calwonder’ to Cucumber mosaic virus (CMV) under greenhouse conditions. When Early Calwonder plants were sown at 10 day intervals and transplanted to 10‐cm square pots, three distinct plant sizes were identified that were designated small, medium and large. Trials conducted during each season showed that CMV accumulated in inoculated leaves of all plants of each size category. All small plants (with the exception of the winter trial) developed a systemic infection that included accumulation of CMV in uninoculated leaves and severe systemic symptoms. Medium plants had a range of responses that included no systemic infection to detection of CMV in uninoculated leaves with the systemically infected plants being either symptomless or expressing only mild symptoms. None of the large plants contained detectable amounts of CMV in uninoculated leaves or developed symptoms. When plants were challenged by inoculation of leaves positioned at different locations along the stem or different numbers of leaves were inoculated, large plants continued to accumulate CMV in inoculated leaves but no systemic infection was observed. When systemic infection of large plants did occur, e.g. when CMV‐infected pepper was used as a source of inoculum, virus accumulation in uninoculated leaves was relatively low and plants remained symptomless. A time‐course study of CMV accumulation in inoculated leaves revealed no difference between small and large plants. Analyses to examine movement of CMV into the petiole of inoculated leaves and throughout the stem showed a range in the extent of infection. While all large plants contained CMV in inoculated leaves, some had no detectable amounts of virus beyond the leaf blade, whereas others contained virus throughout the length of the stem but with limited accumulation relative to controls.     

Suppression of long-distance movement of tobacco etch virus in a nonsusceptible host.

To investigate host functions involved in the tobacco etch potyvirus (TEV) infection process, a tobacco line (V20) with a strain-specific defect in supporting systemic infection was analyzed. Using a modified TEV encoding a reporter protein, beta-glucuronidase (GUS), genome amplification, cell-to-cell movement, and long-distance movement were measured in V20 and a susceptible line, Havana425. Comparable levels of TEV-GUS genome amplification were measured in inoculated protoplasts from both tobacco lines. The rates of cell-to-cell movement of virus in inoculated leaves were nearly identical in V20 and Havana425 between 48 and 72 h postinoculation. In contrast, long-distance movement from leaf to leaf was markedly restricted in V20 relative to Havana425. In situ histochemical analysis of inoculated leaves revealed that infection foci expanded radially over time, providing the potential for contact of virus with veins. Immunocytochemical analysis of V20 tissue from infection foci indicated that TEV-GUS entered the phloem parenchyma or companion cells adjacent to the sieve elements, suggesting that the block in long-distance movement was associated with entry into, or exit from, sieve elements. The genetic basis for the V20 restriction was characterized in a segregation analysis of a cross between V20 and Havana425. The heterozygous F1 progeny displayed the susceptible phenotype, indicating that the V20 restriction was a recessive trait. Segregation in the F2 progeny indicated that the restriction was likely due to the interaction of recessive genes at two nonlinked loci. These data support the hypothesis that long-distance movement requires a set of host functions that are distinct from those involved in cell-to-cell movement.     

Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants.

Tobacco etch potyvirus engineered to express the reporter protein beta-glucuronidase (TEV-GUS) was used for direct observation and quantitation of virus translocation in plants. Four TEV-GUS mutants were generated containing capsid proteins (CPs) with single amino acid substitutions (R154D and D198R), a double substitution (DR), or a deletion of part of the N-terminal domain (delta N). Each modified virus replicated as well as the parental virus in protoplasts, but was defective in cell-to-cell movement through inoculated leaves. The R154D, D198R and DR mutants were restricted essentially to single, initially infected cells. The delta N variant exhibited slow cell-to-cell movement in inoculated leaves, but was unable to move systemically due to a lack of entry into or replication in vascular-associated cells. Both cell-to-cell and systemic movement defects of each mutant were rescued in transgenic plants expressing wild-type TEV CP. Cell-to-cell movement, but not systemic movement, of the DR mutant was rescued partially in transgenic plants expressing TEV CP lacking the C-terminal domain, and in plants expressing CP from the heterologous potyvirus, potato virus Y. Despite comparable levels of accumulation of parental virus and each mutant in symptomatic tissue of TEV CP-expressing transgenic plants, virions were detected only in parental virus- and delta N mutant-infected plants, as revealed using three independent assays. These data suggest that the potyvirus CP possesses distinct, separable activities required for virion assembly, cell-to-cell movement and long-distance transport.     

Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vasculature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.     

Observations and experiments on the use of an avirulent mutant strain of tobacco mosaic virus as a means of controlling tomato mosaic

In 1973 tobacco mosaic virus (TMV) strain M II-16 was successfully used by growers in the United Kingdom to protect commercial tomato crops against the severe effects of naturally occurring strains of TMV. However, plants in many crops had mosaic leaf symptoms which were occasionally severe, so possible reasons for symptom appearance were examined. The concentration of the mutant strain in commercially produced inocula (assessed by infectivity and spectrophotometry) ranged from 28 to 1220 μg virus/ml; nevertheless all samples contained sufficient virus to infect a high percentage of inoculated tomato seedlings. Increasing the distance between the plants and the spray gun used for inoculation from 5 to 15 cm resulted in a significant decrease in the number of tomato seedlings infected. When M II-16 infected tomato plants were subsequently inoculated with each of fifty-three different isolates of TMV, none showed severe symptoms of the challenging isolates within 4 wk, although some isolates of strain o induced atypically mild leaf symptoms. In a further experiment, M II-16 infected plants showed conspicuous leaf symptoms only 7 wk after inoculation with a virulent TMV isolate. M II-16 multiplied more slowly in tomato plants and had a lower specific infectivity than a naturally occurring strain of TMV. More than 50% of plants in crops inoculated with strain M II-16 which subsequently showed conspicuous leaf mosaic contained TMV strain 1 or a form intermediate between strains o and 1. It is suggested that the production of TMV symptoms in commercial crops previously inoculated with strain M II-16 may result from an initially low level of infection, due to inefficient inoculation, which allows subsequent infection of unprotected plants by virulent strains. Incomplete protection by strain M II-16 against all naturally occurring strains may also be an important factor.     

The effect on yield in courgette and marrow of the mild strain of zucchini yellow mosaic virus used for cross-protection

The effects on yield in courgette and marrow (Cucurbita pepo) crops resulting from inoculation with the mild strain of zucchini yellow mosaic virus (ZYMV:WK), have been determined in polythene-house trials and in three years of outdoor, commercial field trials. In polythene-house trials ZYMV:WK inoculated plants were up to 10 days later in flowering than uninoculated plants and their cumulative yields were between 5% and 26% less than uninoculated plants depending on the cultivar. In most field trials cumulative yields from inoculated plants were between 4% and 38% less than uninoculated plants depending on the site and cultivar, but in one trial the yield was 7% higher from inoculated plants. In all experiments, courgette and marrow fruits harvested from ZYMV:WK inoculated plants were symptomless and indistinguishable from fruit harvested from uninoculated plants. The mild leaf symptoms induced by ZYMV:WK infection did not intensify to severe leaf symptoms and where there were natural outbreaks of severe ZYMV infection, fruits from inoculated plants remained symptomless whilst those from uninoculated plants were severely affected and unmarketable.     

Host-Specific Involvement of the HC Protein in the Long-Distance Movement of Potyviruses

Plum pox virus (PPV) is a member of the Potyvirus genus that, in nature, infects trees of the Prunus genus. Although PPV infects systemically several species of the Nicotiana genus, such as N. clevelandii and N. benthamiana, and replicates in the inoculated leaves of N. tabacum, it is unable to infect systemically the last host. The long-distance movement defect of PPV was corrected in transgenic tobacco plants expressing the 5"-terminal region of the genome of tobacco etch virus (TEV), a potyvirus that infects systemically tobacco. The fact that PPV was unable to move to upper noninoculated leaves in tobacco plants transformed with the same TEV transgene, but with a mutation in the HC protein (HC-Pro)-coding sequences, identifies the multifunctional HC-Pro as the complementing factor, and strongly suggests that a defect in an HC-Pro activity is responsible for the long-distance movement defect of PPV in tobacco. Whereas PPV HC-Pro strongly intensifies the symptoms caused by potato virus X (PVX) in the PPV systemic hosts N. clevelandii and N. benthamiana, it has no apparent effect on PVX pathogenicity in tobacco, supporting the hypothesis that long-distance movement and pathogenicity enhancement are related activities of the potyviral HC proteins. The movement defect of PPV in tobacco could also be complemented by cucumber mosaic virus in a mixed infection, demonstrating that at least some components of the long-distance machinery of the potyviruses are not strictly virus specific. A general conclusion of this work is that the HC-Pro might be a relevant factor for controlling the host range of the potyviruses.     

Effect of banana bunchy top virus infection on morphology and growth characteristics of banana

Field experiments were conducted in Oahu, Hawaii, to investigate the effects of banana bunchy top virus (BBTV) infection on growth and morphology of banana ( Musa acuminata ). The time interval between aphid inoculation of BBTV and the initial appearance of disease symptoms (i.e. incubation period) was also determined. Plants infected with BBTV showed a significant reduction in petiole size (i.e. length and distance), plant canopy and height, leaf area, pseudostem diameter and chlorophyll content compared with control plants. Growth differences between virus-infected and control plants were not observed until 40–50 days after the plants were inoculated with viruliferous aphids. Other growth parameters such as petiole width and leaf production were not statistically different between infected and control plants. The incubation period of banana bunchy top disease or appearance of symptoms ranged from 25 to 85 days after aphid inoculation. However, PCR assays provided earlier detection of BBTV in banana plants compared with visual symptoms.     

The Glycine-Glomus-Rhizobium Symbiosis : VII. Photosynthetic Nutrient-Use Efficiency in Nodulated, Mycorrhizal Soybeans

Four consecutive trifoliate leaves of 56-day-old symbiotic or nonsymbiotic soybean plants were evaluated individually for CO₂ exchange rates (CER), leaf area and dry weight, and leaf N, P, and starch concentrations. Plants had been inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae and Rhizobium japonicum, with either of the endophytes alone, or with neither at time of planting. Plants lacking one or both endophytes received N and/or P fertilizers to produce plants of equal total leaf dry weight in all four treatments. Photosynthetic P-use efficiency (CER per unit leaf P) was higher in the leaves of VAM plants than in P-fertilized plants regardless of the N source (N₂ fixation or combined N). Photosynthetic N-use efficiency was also higher in VAM than in non-VAM plants, but it was affected by the N source, with higher CER in the nodulated plants. The greatest differences in CER, starch accumulation and leaf area were found between the nonsymbiotic plants and those with both endophytes. Statistical evaluations of leaf parameters for treatment or nutrient concentration (N and P) effects between the tri-partite and the nonsymbiotic treatments showed significant changes in concentration of P, but not N, with decreasing leaf age. Both endophytes apparently enhance CO₂ fixation at N and/or P concentrations lower than those of the nonsymbiotic plants. The effects of the endophytes on CO₂ fixation were additive.     

Effects of Light Quality on the Interaction between Cucumber Mosaic Virus and Nicotiana tabacum

Light is an important environment factor controlling plant growth, development, and nutritional quality and is also one of the most important factors inducing plant defence. In this study, we assayed the potential effects of light quality on the interaction between Nicotiana tabacum and cucumber mosaic virus (CMV). Our results indicated that white light‐treated N. tabacum plants displayed obvious symptoms at early stage postinoculation, while the symptoms were significantly inhibited under red light and blue light. Western blotting and quantitative real‐time PCR (qRT‐PCR) analyses showed that blue light and red light can effectively delay the replication of CMV compared with white light. The activities of various reactive oxygen species (ROS)‐scavenging enzymes and reducing substances [reduced glutathione (GSH) and ascorbic acid (ASA)] were increased under blue light and red light. In addition, hormone measurements and qRT‐PCR analyses revealed that salicylic acid (SA)‐mediated signalling pathway plays positive role in the related regulation, and cytokinin (CTK) may also participate in them. Furthermore, we found that the formation of dark green islands (DGIs) was significantly suppressed in plants under red light and blue light at 30 days postinoculation (Dpi). However, the accumulation of virus in plants under different light conditions had no notable differences at later stage of postinoculation. Taken together, these results indicated that red light and blue light could effectively delay symptom expression and replication of CMV on N. tabacum at the relatively earlier stage postinoculation.     

Crassulacean Acid Metabolism in Two Species of Orchids Infected by Tobacco Mosaic Virus-orchid Strain and/or Cymbidium Mosaic Virus

The effect of viral infections on the Crassulacean Acid Metabohsm (CAM) was determined in two orchid species. Plants of a Sophrolaeliocattleya (SLC) commercial hybrid were inoculated with either the orchid strain of the Tabacco Mosaic Virus (TMV-O), with Cymbidium Mosaic Virus (CybMV) or with a mixture of both viruses. Plants of Epidendrum elongatum, a wild terrestrial orchid, were inoculated either with TMV-O or with TMV-O and CybMV simultaneously. Virus infected plants started to show a systemic mosaic on the leaves 3 to 4 years after inoculation, the symptoms being more appreciable in SLC than in E. elongatum. Double inoculation with TMV-O and CybMV proved to the lethal for the SLC plants. TMV-O infection caused 58 % and 24 % reduction in nocturnal leaf titratable acidity (TA) in SLC and E. elongatum, respectively. TMV-O also changed the daily pattern of leaf nonstructural carbohydrates typical of CAM plants. A 50 % decrease in TA at dawn and a mid-morning peak of sugar accumulation were measured in leaves of doubly inoculated E. elongatum plants. In SLC CybMV infection inhibited CAM and induced an accumulation of glucans in the leaves. Cytoplasmic agglomerations of CybMV, but not of TMV-O, were detected in SLC mfected cells, whereas TMV-O particles were clearly observed in the cytoplasm of E. elongatum. Infection by TMV-O or CybMV in SLC caused an increase in chloroplasts volume and distortion of the grana due to high glucan accumulation. In contrast, TMV-O infection in E. elongatum induced a lesser degree of damage in the cell ultrastructure.     

Presymptomatic and quantitative detection of Mycosphaerella graminicola development in wheat using a real-time PCR assay

Presymptomatic and accurate diagnosis of Mycosphaerella graminicola leaf blotch is desirable for the disease prediction and the timely application of fungicides. To develop a sensitive PCR assay, four specific primer pairs were designed. They were more specific than three known specific primer pairs. Three of them could detect as little as 0.5 pg M. graminicola DNA in a conventional PCR. A real-time PCR assay was applied for monitoring the disease progression in both inoculated and naturally infected wheat plants using the primer pair ST-rRNA F/R. In inoculated plants, M. graminicola DNA could be detected immediately after inoculation and a steady increase was detected before visible symptoms appeared at 8 days. The rapid growth period took place between 6 and 16 days postinoculation. In the field, the disease progression in the top three leaf layers was followed during the epidemic period. The results were significantly correlated to the disease indices (R=0.8986) and also to the number of pycnidia per leaf (R=0.9227). These suggest that the real-time PCR assay is a reliable approach for the presymptomatic and accurate detection of M. graminicola development in the field.