The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways

Citrus tristeza virus (CTV) induces in the field the decline and death of citrus varieties grafted on sour orange (SO) rootstock, which has forced the use of alternative decline‐tolerant rootstocks in affected countries, despite the highly desirable agronomic features of the SO rootstock. Declining citrus plants display phloem necrosis below the bud union. In addition, SO is minimally susceptible to CTV compared with other citrus varieties, suggesting partial resistance of SO to CTV. Here, by silencing different citrus genes with a Citrus leaf blotch virus‐based vector, we have examined the implication of the RNA silencing and salicylic acid (SA) defence pathways in the resistance of SO to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, associated with these defence pathways, enhanced virus spread and accumulation in SO plants in comparison with non‐silenced controls, whereas silencing of the genes NPR3/NPR4, associated with the hypersensitive response, produced a slight decrease in CTV accumulation and reduced stunting of SO grafted on CTV‐infected rough lemon plants. We also found that the CTV RNA silencing suppressors p20 and p23 also suppress the SA signalling defence, with the suppressor activity being higher in the most virulent isolates.     

Sensitive and Specific Digoxigenin-labelled RNA Probes for Routine Detection of Citrus tristeza virus by Dot-blot Hybridization

A non‐radioactive dot‐blot hybridization assay for the successful detection of Citrus tristeza virus (CTV) RNA in total nucleic acid extracts of infected citrus was developed. Two digoxigenin (DIG)‐labelled minus‐sense riboprobes, complementary to the coat protein gene sequence of a Chinese and an Apulian CTV isolate were synthesized. Several citrus tissues were evaluated as optimal virus source and leaf petioles were found appropriate material for reliable detection. The hybridization assay showed a detection limit corresponding to 0.2 mg of fresh infected tissue. The riboprobes allowed CTV detection in isolates from different geographical areas, grown in the screenhouse or in the field, resulting in similar hybridization patterns. The infected trees were tested during different seasons with positive results, although from July to August most of the samples gave a weaker hybridization signal, compared to other seasons. The high sensitivity and reliability of the molecular hybridization assay described make it a good alternative to serological methods for CTV detection.     

Citrus tristeza virus replicates and forms infectious virions in protoplasts of resistant citrus relatives

Citrus tristeza virus (CTV) is the most economically important viral disease of citrus worldwide. Cultivars with improved CTV tolerance or resistance are needed to manage CTV-induced diseases. The citrus relatives Poncirus trifoliata (L.) Raf., Swinglea glutinosa (Blanco) Merr., and Severinia buxifolia (Poir) Ten. are potential sources of CTV resistance, but their resistance mechanisms are poorly characterized. As a first step to examine the mechanisms of resistance to CTV in these citrus relatives and selected Citrus × Poncirus hybrids, it was necessary to develop methods for protoplast isolation and viral inoculation to allow examination of CTV multiplication in this range of citrus varieties and relatives. Leaf and/or cultured cell protoplasts were isolated and inoculated with four biologically distinct CTV isolates. Northern-blot hybridization analyses for progeny RNAs and immuno-electron microscopy assays for newly produced virions showed that CTV replicated and produced infectious particles in protoplasts from all of the resistant plants tested. These results suggest that resistance to CTV observed at the plant level results from a lack of virus movement and/or some induced resistance response, rather than lack of viral multiplication at the cellular level.     

In vitro production of Indian citrus ringspot virus (ICRSV) free Kinnow plants employing thermotherapy coupled with shoot tip grafting

Indian citrus ringspot virus (ICRSV) is known to cause serious disease problem in Kinnow (Citrus nobilis Lour × C. deliciosa Tenora) plants. This work reports the elimination of ICRSV by using thermotherapy coupled with shoot tip grafting in vitro. Nodal segments from infected mother plants (indexed by indirect ELISA and RT-PCR) were treated both in water bath and moist hot air at different temperatures viz. 40, 45 and 50°C for 30, 60 and 120 min and cultured on MS medium containing 2-iP (1 mg/l) and malt extract (800 mg/l). Shoot tips were excised from the nodal sprouts and grafted on to rough lemon (C. jambhiri) rootstock under aseptic conditions. Water bath treatment was found to be more effective as compared to moist hot air treatment as maximum number of ICRSV free plants (36.84%) were obtained by grafting the tips (0.7 mm) taken from the nodal segments treated at 50°C in water bath for 2 h. In an alternate treatment regime, 1-year-old infected plants were kept at various temperatures viz.36, 38 and 40°C in a thermotherapy chamber. Maximum of 60% ICRSV free plants were obtained by grafting the tips (0.7 mm) from the plants placed at 40°C followed by the plants placed at 38°C (59.09%) and the least was observed in case of the plants placed at 36°C (40.74%). Only those plants/plantlets were considered virus free, which showed negative reaction both in Indirect ELISA and RT-PCR.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

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.     

Interference Between Tobacco necrosis virus and Turnip crinkle virus in Nicotiana benthamiana

Mixed infections of Nicotiana benthamiana plants by Tobacco necrosis virus (TNV) and Turnip crinkle virus (TCV) exhibited an interference interaction. Accumulation of TNV (+)RNA as well as capsid protein in mixed infection were considerably lower than that of singly infected plants. There were also a slight reduction in the levels of TCV (+)RNA and capsid protein in doubly infected plants, which displayed the concentration of both viruses decreased in dually infected plants. Tissue immunoblot analysis of systemic N. benthamiana leaves infected by TNV and TCV singly or doubly showed the interference between the two viruses in situ, which exhibited the decrease of both viruses in doubly infected leaves although the distribution of them did not change remarkably. These results were consistent with the hybridization analysis of viral genomic RNA and coat protein. Both cross‐protection test and mixed infection of the two viruses confirmed TCV had relatively stronger interference to the infection of TNV. Interference infection by TNV and TCV induced higher increase in the levels of cytochrome pathway respiration and alternative pathway respiration in host plants, especially the latter. Interference often occurred in different strains of one kind of virus or two different closely related viruses in one genus. Our results showed that interference could also occur in different viruses belonging to different genera.     

Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem‐associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression

Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23‐kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N‐terminal 157‐amino‐acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV‐like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23‐derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem‐specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV‐specific symptoms (vein clearing and necrosis, and stem pitting), but not the non‐specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem‐specific accumulation of the p23Δ158–209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N‐terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.     

Production of Polyclonal Antibodies to the Recombinant Coat Protein of Citrus tristeza virus and their Effectiveness for Virus Detection

Citrus tristeza virus (CTV) is distributed worldwide and causes the most economically important virus diseases of citrus. Enzyme‐linked immunosorbent assay (ELISA) and/or immunoprinting have become an indispensable tools for large‐scale diagnosis of CTV worldwide. Several CTV detection kits are commercially available, based on either polyclonal or monoclonal antibodies developed against purified virus preparations. We have developed polyclonal antibodies to recombinant p25 CTV coat proteins (rCP) and determined their effectiveness for both trapping and as the intermediate antibody in double‐antibody sandwich indirect (DASI) ELISA. The p25 coat protein gene of three CTV isolates was amplified by RT‐PCR and further cloned and expressed in Escherichia coli cells. The rCP was injected into rabbits and goats for antibody production. Western blotting assays with the rCP CTV‐specific antibodies reacted positively with the homologous and heterologous rCP of the three CTV isolates and with the corresponding native coat protein present in crude sap extracts of CTV‐infected citrus tissue, but not with extracts from healthy tissue. The rCP antibodies from goat and rabbit reacted as both plate trapping and intermediate antibodies in DASI‐ELISA, discriminating healthy and CTV‐infected citrus, with optical density (OD₄₀₅) values in the range of 0.151–2.415 for CTV‐infected samples and less than 0.100 for healthy tissue. Commercially available anti‐CTV antibodies were used as a reference. Previous reports indicate that antibodies developed to recombinant antigens, including those of CTV, may not be functional for trapping the target antigens under non‐denaturing conditions. Our results showed the feasibility of CTV antibodies developed to the rCP for use as both trapping and intermediate antibodies in DASI‐ELISA, when the recombinant antigen was fractioned with polyacrylamide electrophoresis gel and further extensively dialysed against phosphate buffer saline prior to its use as immunogen.     

Settling preferences of the whitefly vector Bemisia tabaci on infected plants varies with virus family and transmission mode

Virus infection may change not only the host‐plant phenotypic (morphological and physiological) characteristics, but can also modify the behavior of their insect vector in a mutualistic or rather antagonistic manner, to promote their spread to new hosts. Viruses differ in their modes of transmission and depend on vector behavior for successful spread. Here, we investigated the effects of the semi‐persistently transmitted Tomato chlorosis virus (ToCV, Crinivirus) and the persistent circulative Tomato severe rugose virus (ToSRV, Begomovirus) on alighting preferences and arrestment behavior of their whitefly vector Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Middle East Asia Minor 1 (MEAM1) on tomato plants (Solanum lycopersicum L. cv. Santa Clara, Solanaceae). The vector alighting preferences between infected and uninfected plants in choice assays were apparently influenced by the presence of ToCV and ToSRV in the whiteflies or by their previous exposure to infected plants. The observed changes in vector behavior do not seem to benefit the spread of ToCV: non‐viruliferous insects clearly preferred mock‐inoculated plants, whereas ToCV‐viruliferous insects landed on mock‐inoculated and ToCV‐infected plants, indicating a partial change in insect behavior – ToCV was able to directly affect the preference of its vector B. tabaci, but this change in insect behavior did not affect the virus spread because viruliferous insects landed on mock‐inoculated and infected plants indistinctly. In contrast, ToSRV‐viruliferous insects preferred to land on mock‐inoculated plants, a behavior that increases the probability of spread to new host plants. In the arresting behavior assay, the majority of the insects remained on mock‐inoculated plants when released on them. A greater number of insects moved toward mock‐inoculated plants when initially released on ToCV‐ or ToSRV‐infected plants, suggesting that these viruses may repel or reduce the nutritional quality of the host plants for B. tabaci MEAM1.     

Comparison of shoot tip culture and cryotherapy for eradication of ampeloviruses associated with Pineapple mealybug wilt in wild varieties

The Pineapple mealybug wilt-associated virus (PMWaV) provokes a disease that causes considerable losses to pineapple growers due to the reduced leaf tissue turgescence, resulting in leaf necrosis and death in severe cases. In this work, we describe the comparison of shoot tip culture and cryotherapy methods to eradicate the pineapple wilt disease-associated ampeloviruses. Plants from the accessions Ananas comosus var. comosus (AGB-009), var. bracteatus (AGB-119), and var. parguazensis (AGB-376) were indexed by RT-PCR, confirming mixed infections of PMWaV-1, PMWaV-2, and PMWaV-3. The accessions were cultured in vitro and their shoot tips treated by cryotherapy following a droplet vitrification protocol. The regeneration rate from shoot tip culture was 93% for AGB-376 and 100% for the other two accessions. After freezing, AGB-376 had 100% regeneration subsequent to exposure to PVS2 for 45 min, followed by 95% for AGB-009, while for AGB-119 the optimal exposure time was 60 min, with plant regeneration from nearly 80% of the shoot tips. For the accessions AGB-009 and AGB-376, all the recovered plants were virus free by the two methods, while 50% of the plants from accession AGB-119 remained infected. These results indicate that shoot tip culture alone or in association with cryotherapy is a promising routine method for virus removal from pineapple plant tissues and is useful to ensure that backup reserves of pineapple germplasm, conserved by in vitro bank and cryobank, are formed with virus-free plants.