Protective action of salicylic acid against bean yellow mosaic virus infection in Vicia faba leaves

In this study, morphological, ultrastructural and physiological modifications of faba bean (Vicia faba cv Giza 461) leaves in response to bean yellow mosaic virus (BYMV) infection and salicylic acid (SA) treatments were examined. Under BYMV stress, leaves showed symptoms including severe mosaic, mottling, crinkling, size reduction and deformations. Three weeks after virus inoculation, photosynthetic rate, pigment contents and transpiration rate were significantly reduced in response to BYMV infection.

Ultrastructural investigations of BYMV-infected leaves demonstrated that most chloroplasts with increased stromal area became spherical in shape and some lost their envelopes, either partially or totally. The internal structures of chloroplast, grana and thylakoids were dilated. Two kinds of inclusions were detected in BYMV-infected leaves: straight or slightly curved bands sometimes coiled or looped at the end, and electron opaque crystals with varied shapes. BYMV-infected cells showed lower chloroplast number in comparison to the control.

Spraying of SA on faba bean leaves helped to reduce or prevent the harmful effects produced after virus infection. Application of 100 μM SA three days before inoculation restored the metabolism of infected leaves to the levels of healthy controls. SA treatment improved plant health by increasing the photosynthesis rates, pigment contents and levels of other parameters studied similar to control values.

Moreover, SA treatment increased plant resistance against BYMV. This was observed through induction of chloroplast number, reduction in percentage of infected plants, decrease in disease severity and virus concentration of plants treated with SA prior to BYMV inoculation. Cells of SA-treated samples showed well-developed chloroplasts with many starch grains and well-organized cell organelles.

The present results provide an overview of the negative effects on faba bean leaves due to BYMV infection from physiological and subcellular perspectives. Also, a role of SA involved in induction of resistance against BYMV infection in bean plants is discussed.     

Salicylic acid-, but not cytokinin-induced, resistance to WClMV is associated with increased expression of SA-dependent resistance genes in Phaseolus vulgaris

Two-week-old Phaseolus vulgaris plants, wick-fed with 1 mmol/L salicylic acid (SA) or 50 nmol/L dihydrozeatin (DHZ), showed partial inhibition of the accumulation of white clover mosaic virus (WClMV) in infected primary leaves. This inhibition was measured as a decrease in the accumulation of both viral mRNA and viral coat protein, especially at the early stages of infection. Salicylic acid treatment resulted in moderately increased expression of phenylalanine ammonia lyase (PAL), NPR1, PR1 and HSP70 genes that participate in resistance to pathogens in plants. In contrast, DHZ treatments did not induce significant changes in expression of these genes. The expression of the P. vulgaris alternative oxidase (AOX) gene homolog, an enzyme implicated in plant resistance to viruses, showed low constitutive expression during the first 11 days post-infection and was not affected by either SA or DHZ. It appears that, while SA induced the NPR1-PR1 pathogen defense pathway genes, both SA and DHZ may use a different pathway to induce resistance to WClMV infection in P. vulgaris plants.     

Modifications of antioxidant activity and protein composition of bean leaf due to <Emphasis Type="Italic">Bean yellow mosaic virus</Emphasis> infection and salicylic acid treatments

The antioxidant status as well as protein composition of faba bean leaves infected with Bean yellow mosaic virus (BYMV) and the effect of salicylic acid application was examined in this paper. Some modifications in the antioxidant status were observed by changing some antioxidant enzymes activities and contents of antioxidant metabolites. BYMV-infected leaves revealed POD, CAT, APX and SOD induced activities while SA treatments could inhibit POD, CAT activities but induced SOD activity. The enzyme activities seemed to be SA concentration dependant. Higher H₂O₂ and MDA concentrations were recorded in virus-infected leaves than those of the corresponding controls while treatment with SA followed by virus inoculation caused lowering of MDA concentration and reducing the damage due to lipid peroxidation. Moreover, because of virus infection and/or SA treatments, an increase in the amounts of phenolics and flavonoids was noticed. As compared to the control, BYMV infection or SA application caused pronounced increase in the antioxidant activity of leaf extracts detected by DPPH assay, indicating an increase in the amounts of antioxidant compounds occurred. To test the protein composition, the contents of each protein fractions (soluble, insoluble and total) were analyzed and the change in protein patterns was visualized using SDS-PAGE. The BYMV-infected bean leaves had protein contents higher than the control indicating accumulation of pathogenesis-related proteins. Moreover, spraying SA with or without virus inoculation could accumulate soluble, insoluble and total proteins and the pattern of increase was in accordance with SA concentration. Alterations in protein patterns were observed in faba bean leaves (Vicia faba cv Giza 461) in response to BYMV infection and SA treatments. Because of BYMV infection and SA treatments, the protein profiles showed new bands in comparison to the control. Some polypeptides were highly accumulated in treatments of SA followed by virus inoculation. Changing antioxidant status and accumulation of some antioxidant metabolites as well as the pronounced alterations in the protein composition indicate a kind of plant response against pathogen invasion and in case of SA treatment it is considered a way by which a defence response was initiated and/or activated.     

Proteomic analysis of salicylic acid induced resistance to Mungbean Yellow Mosaic India Virus in Vigna mungo

The role of salicylic acid (SA) in inducing resistance to MYMIV infection in Vigna mungo has been elucidated by proteomics. Twenty-nine proteins identified by MALDI-TOF/TOF, predicted to be involved in stress responses, metabolism, photosynthesis, transport and signal transduction, showed increased abundance upon SA treatment. Susceptible plants showed characteristic yellow mosaic symptoms upon MYMIV infection. A concentration dependent decrease in physiological symptoms associated with MYMIV was observed upon exogenous SA treatment prior to viral inoculation; and no visible symptom was observed at 100 μM SA. SA treatment stimulated SOD and GPX activity and inhibited CAT activity thus preventing ROS mediated damage. Significant increase in chlorophyll, protein, carbohydrate, phenolic content and H(2)O(2) were observed. Involvement of calmodulin for transmission of defense signal by SA is suggested. A metabolic reprogramming leading to enhanced synthesis of proteins involved in primary and secondary metabolisms is necessary for SA mediated resistance to MYMIV. Identification of proteins showing increased abundance, involved in photosynthetic process is a significant finding which restores virus-induced degradation of the photosynthetic apparatus and provides enhanced metabolites required for repartition of resources towards defense.     

Serological diagnosis and host range studies of important viral diseases of a few cucurbitaceous crops in Maharashtra,India

Around 39 well characterised viruses affect cucurbits crops in developing countries and their viral diversity may be the consequence for genetic and ecological diversity of their hosts. Indeed, cucurbits are grown in variety of climatic, environmental and agricultural conditions, and this may provide more or less favourable conditions for the specific viruses or their hosts. The presence of various viral diseases caused by different viruses in Maharashtra was studied from the infected samples collected from cucurbits and melons during the survey conducted in 2009–2010 in different locations. The virus isolates collected from various cucurbitaceous crops were established and their host ranges were studied by sap transmission. The study revealed Cucumber Mosaic Virus (CMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV) and Cucumber green mottle mosaic virus infections predominately found in Nashik region, and Watermelon bud necrosis virus (WBNV), CMV, ZYMV, WMV and Watermelon silver mottle virus (WSMoV) infections in Aurangabad and Paithan regions. In Sangamner region, the crop was mostly affected by WBNV, ZYMV and WSMoV, and CMV was found only in Sillod region. The protocols for performing sap transmission tests in assay hosts were standardised for ZYMV, CMV and WBNV. Using direct antigen-coating enzyme-linked immunosorbent assay, of all the plant parts, young leaves were found to have high concentration of virus and suitable for virus detection in screening programmes. CMV and ZYMV was found to have high concentration of virus and suitable for virus detection in screening programmes.     

THE EFFECT OF INFECTION WITH BEET YELLOWS AND BEET MOSAIC VIRUSES ON THE CARBOHYDRATE CONTENT OF SUGAR-BEET LEAVES, AND ON TRANSLOCATION

The loss of total carbohydrate (sugars and starch) per cent of residual dry matter (dry matter less total carbohydrate) during a period of darkness from leaves of sugar-beet plants infected with yellows virus was as great as that from the leaves of healthy plants. The conclusion of previous workers, based on the results of the Sachs iodine test for starch and the occurrence of 'phloem gummosis' in infected plants, that starch accumulates in infected leaves because translocation is prevented by blockage of the sieve-tubes, is therefore incorrect.
Older leaves of infected plants had a higher content of reducing sugars and sucrose, and usually but not invariably of starch, both at the beginning and end of the dark period, than comparable leaves of healthy plants. By far the greater part of the increase was in reducing sugars. In leaves taken in late September from infected plants growing in the field, 20 % or more of the total dry matter was present as reducing sugars. The reducing sugars in both healthy and yellows-infected leaves were shown by paper chromatography to be glucose and fructose in approximately equal amounts.
Accumulation of carbohydrate in infected leaves is probably not a passive consequence of differences in carbohydrate production, distribution and utilization, but is attributable to changes in the physiology of the cells of the leaf.
The carbohydrate content of sugar-beet leaves was little affected by infection with beet mosaic virus.
Yellows-infected leaves had a lower water content per cent of fresh weight than healthy leaves. This was accounted for by the higher carbohydrate content of infected leaves, for the ratio of water: residual dry matter was not affected by infection or was slightly reduced. This implies that hydration was independent of carbohydrate content.     

Occurrence, distribution and relative incidence of mosaic viruses infecting field--grown squash in Tehran province, Iran

Squash (Cucurbita pepo) belongs to Cucurbitaceae family. Every year Cucurbitaceae are planted world wide. They are one of the most important economic crops. Cucurbitaceae are threatened by viruses. Many viruses damage the plants of this family. Since nine viruses have been reported on squash from Iran. In this survey, during 2002--2003, to determine the distribution of Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV), 466 samples were collected from squash field in Tehran province. Infected plants showing symptoms such as: mosaic, yellowing, deformation, shoestring of leaves and fruit deformation and yield reduction. Distribution of CMV, ZYMV and WMV were determined by DAS-ELISA. Thepercentage of ZYMV, WMV and CMV were 35.6, 26.1 and 25.1% respectively. Triple infection (CMV+ZYMV+WMV) were found in 6.4% of samples. ZYMV were found the most frequently the viruses. This is the first report of WMV on squash in Tehran province.     

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.     

Disease Interactions in a Shared Host Plant: Effects of Pre-Existing Viral Infection on Cucurbit Plant Defense Responses and Resistance to Bacterial Wilt Disease

Both biotic and abiotic stressors can elicit broad-spectrum plant resistance against subsequent pathogen challenges. However, we currently have little understanding of how such effects influence broader aspects of disease ecology and epidemiology in natural environments where plants interact with multiple antagonists simultaneously. In previous work, we have shown that healthy wild gourd plants (Cucurbita pepo ssp. texana) contract a fatal bacterial wilt infection (caused by Erwinia tracheiphila) at significantly higher rates than plants infected with Zucchini yellow mosaic virus (ZYMV). We recently reported evidence that this pattern is explained, at least in part, by reduced visitation of ZYMV-infected plants by the cucumber beetle vectors of E. tracheiphila. Here we examine whether ZYMV-infection may also directly elicit plant resistance to subsequent E. tracheiphila infection. In laboratory studies, we assayed the induction of key phytohormones (SA and JA) in single and mixed infections of these pathogens, as well as in response to the feeding of A. vittatum cucumber beetles on healthy and infected plants. We also tracked the incidence and progression of wilt disease symptoms in plants with prior ZYMV infections. Our results indicate that ZYMV-infection slightly delays the progression of wilt symptoms, but does not significantly reduce E. tracheiphila infection success. This observation supports the hypothesis that reduced rates of wilt disease in ZYMV-infected plants reflect reduced visitation by beetle vectors. We also documented consistently strong SA responses to ZYMV infection, but limited responses to E. tracheiphila in the absence of ZYMV, suggesting that the latter pathogen may effectively evade or suppress plant defenses, although we observed no evidence of antagonistic cross-talk between SA and JA signaling pathways. We did, however, document effects of E. tracheiphila on induced responses to herbivory that may influence host-plant quality for (and hence pathogen acquisition by) cucumber beetles.     

Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus,zucchini yellow mosaic virus,and watermelon mosaic virus-2 is resistant to these three viruses in the field

Cantaloupe line CZW-30 containing coat protein gene constructs of cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV-2) was investigated in the field over two consecutive years for resistance to infections by CMV, ZYMV, and/or WMV-2. Resistance was evaluated under high disease pressure achieved by mechanical inoculations and/or natural challenge inoculations by indigenous aphid vectors. Across five different trials, homozygous plants were highly resistant in that they never developed systemic symptoms as did the nontransformed plants but showed few symptomatic leaves confined close to the vine tips. Hemizygous plants exhibited a significant delay (2–3 weeks) in the onset of disease compared to control plants but had systemic symptoms 9–10 weeks after transplanting to the field. Importantly, ELISA data revealed that transgenic plants reduced the incidence of mixed infections. Only 8% of the homozygous and 33% of the hemizygous plants were infected by two or three viruses while 99% of the nontransformed plants were mixed infected. This performance is of epidemiological significance. In addition, control plants were severely stunted (44% reduction in shoot length) and had poor fruit yield (62% loss) compared to transgenic plants, and most of their fruits (60%) were unmarketable. Remarkably, hemizygous plants yielded 7.4 times more marketable fruits than control plants, thus suggesting a potential commercial performance. This is the first report on extensive field trials designed to assess the resistance to mixed infection by CMV, ZYMV, and WMV-2, and to evaluate the yield of commercial quality cantaloupes that are genetically engineered.     

Cucumber mosaic virus movement protein affects sugar metabolism and transport in tobacco and melon plants

In addition to its influence on plasmodesmal function, tobacco mosaic virus movement protein (TMV‐MP) causes an alteration in carbon metabolism in source leaves and in resource partitioning among the various plant organs. The present study was aimed at characterizing the influence of cucumber mosaic virus (CMV)‐MP on carbohydrate metabolism and transport in both tobacco and melon plants. Transgenic tobacco plants expressing the CMV‐MP had reduced levels of soluble sugars and starch in their source leaves and a significantly reduced root‐to‐shoot ratio in comparison with control plants. A novel virus‐vector system was employed to express the CMV‐coat protein (CP), the CMV‐MP or the TMV‐MP in melon plants. This set of experiments indicated that the viral MPs cause a significant elevation in the proportion of sucrose in the phloem sap collected from petioles of source leaves, whereas this sugar was at very low levels or even absent from the sap of control melon plants. The mode by which the CMV‐MP exerts its effect on phloem‐sap sugar composition is discussed in terms of possible alterations in the mechanism of phloem loading.     

Activation of a diverse set of genes during the tobacco resistance response to TMV is independent of salicylic acid; induction of a subset is also ethylene independent

Through differential screening of a cDNA library, we cloned six groups of genes that are expressed relatively early in the inoculated leaves of tobacco resisting infection by tobacco mosaic virus (TMV). Induction of all these genes was subsequently detected in the uninoculated leaves; thus, their expression is associated with the development of both local and systemic acquired resistance. Exogenously applied salicylic acid (SA) was observed to induce these genes transiently. However, analyses with transgenic NahG plants, which are unable to accumulate SA, demonstrated that expression of these genes in TMV-inoculated leaves is mediated via an SA-independent pathway. Because the expression kinetics of these genes differ from those associated with the well-characterized pathogenesis-related protein (PR-1) and phenylalanine ammonia-lyase (PAL) genes, we propose that they belong to a group which we designate SIS, for SA-independent, systemically induced genes. Interestingly, the expression of several SIS genes in the uninoculated leaves of TMV-infected NahG plants was delayed and/or reduced, raising the possibility that SA is involved in activating some of these genes in systemic tissue. Most of the SIS genes were induced by exogenous ethylene. However, analyses of infected NahG plants treated with ethylene action and/or synthesis inhibitors indicated that the TMV-induced expression of several SIS genes is independent of ethylene as well as SA.     

Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose—phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose—phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)^N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.     

Cucumber mosaic virus infection affects sugar transport in melon plants

Viral infection often affects carbon assimilation and metabolism in host plants. To better understand the effect of cucumber mosaic virus (CMV) infection on sugar transport, carbohydrate levels and the amounts of the various sugars in the phloem sap were determined in infected melon (Cucumis melo L.) plants. Source leaves infected with CMV were characterized by high concentrations of reducing sugars and relatively low starch levels. The altered level of carbohydrates was accompanied by increased respiration and decreased net photosynthetic rates in the infected leaves. Although stachyose was the predominant sugar in phloem sap collected from petioles of control leaves, sucrose (Suc) was a major sugar in the phloem sap of infected leaves. Moreover, analyses of the newly fixed (14)CO(2) revealed a high proportion of radioactive Suc in the phloem sap of infected leaves 60 min post-labeling. The alteration in phloem sap sugar composition was found in source, but not old, leaves. Moreover, elevations in Suc concentration were also evident in source leaves that did not exhibit symptoms or contain detectable amounts of virus particles. The mode by which CMV infection may cause alterations in sugar transport is discussed in terms of the mechanism by which sugars are loaded into the phloem of cucurbit plants.     

Changes in physiology and biochemistry of mottle streak virus infected finger millet plants

A significant reduction in the growth parameters viz., plant height, number of tillers, number of productive tillers, leaves, leaf area, 1000 grain weight and grain yield were observed in the mottle streak virus infected finger millet plants compared to healthy finger millet plants. The germination and vigour of seedlings from the seeds of infected plants were reduced. Physiological changes in finger millet as a result of virus infection were investigated. The chlorophyll pigments ‘a’ and ‘b’ as well as total chlorophyll were reduced due to mottle streak infection. The virus infection led to increased total sugar, starch, soluble protein and phenol contents. The mineral metabolism of infected plants showed a reduction in nitrogen, phosphorus, potassium, magnesium, calcium and iron.     

Virus-induced changes in the subcellular distribution of glutathione precursors in Cucurbita pepo (L.)

Changes in glutathione contents occur in plants during environmental stress situations, such as pathogen attack, as the formation of reactive oxygen species leads to the activation of the antioxidative defence system. As glutathione is synthesized out of its constituents cysteine, glycine, and glutamate the availability of these components will limit glutathione synthesis in plants especially during stress situations and therefore the ability of the plant to fight oxidative stress. To gain a deeper insight into possible limitations of glutathione synthesis during pathogen attack the present investigations were aimed to study how the subcellular distribution of glutathione precursors correlates with the subcellular distribution of glutathione during virus attack in plants. Selective antibodies against cysteine, glutamate, and glycine were used to study the impact of Zucchini yellow mosaic virus (ZYMV) infection on glutathione precursor contents within different cell compartments of cells from Cucurbita pepo (L.) plants with the transmission electron microscope (TEM). Generally, levels of cysteine and glutamate were found to be strongly decreased in most cell compartments of younger and older leaves including glutathione-producing cell compartments such as plastids and the cytosol. The strongest decrease of cysteine was found in plastids (- 54 %) and mitochondria (- 51 %) of younger leaves and in vacuoles (- 37 %) and plastids (- 29 %) of older leaves. The strongest decrease of glutamate in younger leaves occurred in peroxisomes (- 67 %) and nuclei (- 58 %) and in peroxisomes (- 64 %) and plastids (- 52 %) of the older ones. Glycine levels were found to be strongly decreased (- 63 % in mitochondria and - 53 % in plastids) in most cell compartments of older leaves and strongly increased (about 50 % in plastids and peroxisomes) in all cell compartments of the younger ones. These results indicate that low glycine contents in the older leaves were responsible for low levels of glutathione in these organs during ZYMV infection rather than limited amounts of cysteine or glutamate. Glutathione precursors were virtually absent in cell walls and intercellular spaces and play therefore no important role during ZYMV attack in the apoplast. While glutamate was absent in vacuoles, elevated levels of glycine (up to 30 %) and decreased cysteine contents (up to - 37 %) were observed in vacuoles during ZYMV infection. The impact of the present results on the current knowledge about glutathione synthesis and degradation on the cellular level during ZYMV infection are discussed.