Citrus Exocortis Research Articles

Effects of resistance of Eremocitrus glauca and Microcitrus australis to viroid infection: replication, accumulation and long‐distance movement of six citrus viroids

In studies to identify genotypes resistant to infection with citrus viroids, Eremocitrus glauca and Microcitrus australis were selected because their evolution in their habitat in Australia and New Guinea may have led to the selection of unusual traits. The movement and accumulation of Citrus exocortis viroid (CEVd), Hop stunt viroid, Citrus bent leaf viroid, Citrus dwarfing viroid, Citrus bark cracking viroid and Citrus viroid V (CVd‐V) in self‐rooted as well as in graft‐ propagated E. glauca and M. australis plants was assessed by northern hybridization, RT‐PCR and by topworking to the sensitive selection 861‐S1 of Etrog citron. In both plant species the inoculated viroids were undetectable unless these plants were grafted to a susceptible Citrus partner, the rough lemon rootstock and/or the topworked Etrog citron, which acted as viroid sources. The results obtained indicate that M. australis and in particular E. glauca are poor viroid hosts in which viroid replication/accumulation does not occur or is extremely inefficient. However, viroid downward and upward movement to grafted Citrus partners in which viroid replication and accumulation occurs efficiently was not impaired. Eremocitrus glauca and M. australis showed differences regarding their properties as viroid hosts, but for both species CEVd seemed to have the lowest affinity among the viroid species tested and CVd‐V the highest. Even though E. glauca and M. australis do not appear to be truly resistant to viroid infection, they are interesting genotypes for further characterization of the mechanisms involved in viroid infection.

Viroids in Tahiti Lime Scions Showing Bark Cracking Symptoms

The so-called quebra-galho clone of Tahiti lime is very popular in Brazil, because the small size of the trees is suitable for high-density plantings. These Tahiti lime trees are easily recognized because they show bark-cracking symptoms, that have been claimed to be associated with infection. Viroid infection of three quebra-galho Tahiti lime trees from a farm near Araraquara in the state of Sao Paulo, Brazil, was assessed by Northern-blot hybridization using viroid-specific probes. Similarly, eight clones of Tahiti lime from different origins and available at Topara Nursery, near Chincha, Peru, were also tested for viroids. The four clones that displayed characteristic bark-cracking symptoms were found to be infected with Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) and Citrus viroid III (CVd-III), whereas the clones that did not show bark-cracking symptoms were either viroid-free or infected only with CVd-III. A study is being conducted to establish if viroids, and in particular CEVd, are indeed the cause of quebra-galho bark cracking symptoms. Preliminary observations indicated that 2-yr old CEVd-infected lime trees at Moncada, Spain showed cracks.

Viroid species associated with the bark-cracking phenotype of 'Tahiti' acid lime in the State of São Paulo, Brazil

Viroids have been used as "graft transmissible dwarfing agents" (GTDA) in several countries, mainly to reduce growth of citrus trees, thus increasing their density in orchards. In the State of Sao Paulo, Brazil, plants of the acid lime 'Tahiti' are usually grafted with a complex of GTDA, presumably viroids. The aim of the present work was the identification and molecular characterization of the viroids infecting trees of acid lime 'Tahiti' displaying "Quebra galho" (bark-cracking). Viroids were identified and characterized by biological indexing in 'Etrog' citron, Northern-blot hybridization, RT-PCR, cloning and complete sequencing of the RNA genomes. Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) and Citrus dwarfing viroid (CDVd) were found in different combinations. Although we have not been able to infer a direct relationship between the agronomical performance and symptom severity with the presence of a specific viroid or viroid combination, the differences in the severity of "Quebra-galho" symptoms among different trees is probably associated with the presence (or absence) of CEVd, with its interaction with other viroids perhaps determining the different phenotypes observed in the field.

Metabolic response of tomato leaves upon different plant–pathogen interactions

Plants utilise various defence mechanisms against their potential biotic stressing agents such as viroids, viruses, bacteria or fungi and abiotic environmental challenges. Among them metabolic alteration is a common response in both compatible and incompatible plant-pathogen interactions. However, the identification of metabolic changes associated with defence response is not an easy task due to the complexity of the metabolome and the plant response. To address the problem of metabolic complexity, a metabolomics approach was employed in this study. To identify a wide range of pathogen (citrus exocortis viroid, CEVd, or Pseudomonas syringae pv. tomato)-induced metabolites of tomato using metabolomics. Nuclear magnetic resonance (NMR) spectroscopy in combination with multivariate data analysis were performed to analyse the metabolic changes implicated in plant-pathogen interaction. NMR-based metabolomics of crude extracts allowed the identification of different metabolites implicated in the systemic (viroid) and hypersensitive response (bacteria) in plant-pathogen interactions. While glycosylated gentisic acid was the most important induced metabolite in the viroid infection, phenylpropanoids and a flavonoid (rutin) were found to be associated with bacterial infection. NMR metabolomics is a potent platform to analyse the compounds involved in different plant infections. A broad response to different pathogenic infections was revealed at metabolomic levels in the plant. Also, metabolic specificity against each pathogen was observed.

Molecular characterization of a member of a new species of grapevine viroid

Currently, viroids of five species are known to infect grapevine trees: Hop stunt viroid (HSVd-g) [1], Citrus exocortis viroid (CEVd-g) [2], Australian grapevine viroid (AGVd) [3], Grapevine yellow speckle viroid 1 (GYSVd-1) and Grapevine yellow speckle viroid 2 (GYSVd-2) [4–6]. These five grapevine viroid species have been divided into three genera based on sequence homologies of their central conserved regions (CCRs) [7]. GYSVd-1, GYSVd-2, and AGVd are classified in the genus Apscaviroid, whereas HSVd-g [8] and CEVd-g are classified in the genera Hostuviroid and Pospiviroid, respectively [9]. Mixed infections involving these viroids in cultivated grapevines are common, and in general, grapevine viroids produce very few, if any, disease symptoms. In the absence of manifested disease symptoms, these viroids are able to replicate in a host unnoticed. Of the five grapevine viroids identified, GYSVd-1 and GYSVd-2 are associated with disease symptoms such as yellow speckle, which was originally identified under hot greenhouse conditions in Australia [4, 5, 10]. Taylor and Woodham first described yellow speckle (YS) disease in Australia, and it is still the only grapevine disease known to be caused by viroids [10]. GYSVd-1, which is approximately 367 nucleotides (nts) in length, was first isolated from the grapevine cultivar ‘Cabernet Franc’, showing typical symptoms of YS disease. From this cultivar, a single circular RNA was isolated by two-dimensional electrophoresis [4]. GYSVd-2, which is approximately 363 nts in length, was first purified from the grapevine cultivar ‘Kyoto’, which was grafted onto the ‘Dogridge’ rootstock and also exhibited symptoms of YS disease [5]. So far, GYSVd-2 has only been reported in Australia and China [5, 6], and the molecular characterization of GYSVd-2 has been described [11]. Here, we report the characterization of a member of a species of grapevine viroid isolated from China. We call it Chinese grapevine viroid (CGVd) in this report and suggest tentatively naming it Grapevine yellow speckle viroid (GYSVd-3) based on the results of sequence, phylogenetic analysis and molecular characterization.

First Report of Citrus bent leaf viroid and Citrus dwarfing viroid from Citrus in Punjab, Pakistan

Pakistan is among the top 10 citrus-producing countries of the world and the leader in Kinnow mandarin production with production concentrated in the province of Punjab, which produces more than 96% of the total citrus crop. To evaluate the presence and distribution of citrus viroids in this area, 34 samples were collected in September 2008 from citrus orchards in the Sargodha, Bhalwal, and Faisalabad areas of Punjab, including 15 'Mosambi' and two 'Bloodred' sweet oranges (Citrus sinensis), eight 'Kinnow' and four 'Feutrell Early' mandarins (C. reticulata), three 'Jatti Khatti' rough lemon (C. jambhiri), and two grapefruit (C. paradisi), which showed stunting, bark scaling, and cracking symptoms on the rootstock which was either citrange (Poncirus trifoliata × C. sinensis) or sweet lime (C. limetta). Infected budwood from these trees was grafted onto indicator plants of Arizona 861-S-1 'Etrog citron' (C. medica) budded on rough lemon rootstock, and after 3 months, the citron showed typical viroid symptoms of mild epinasty and leaf roll with 23 of the 34 samples. A one-step multiplex reverse transcription (RT)-PCR assay (3) was used to detect simultaneously Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd). On the basis of amplification of the appropriately sized DNA, CEVd, CBLVd, HSVd, and CDVd were detected in 12, 8, 31, and 17 samples, respectively, whereas CBCVd was not detected. Twenty-three of 34 infected samples harbored more than one viroid species and one had four viroids. Budwood from 11 trees did not induce viroid symptoms on Etrog citron. Two of these trees were infected with CBLVd only and nine with HSVd only. Four primer pairs were used to amplify the full sequences of CEVd, CBLVd, HSVd, and CDVd by RT-PCR (2), which were cloned by standard methods. Sequences of three cDNA clones each of CEVd (Nos. FJ773253, FJ773254, and FJ773255), CBLVd (Nos. FJ773262, FJ773263, and FJ773267), HSVd (Nos. FJ773268, FJ773269, and FJ773271), and CDVd (Nos. FJ773274, FJ773275, and FJ773276) were deposited in GenBank. BLAST analysis showed that these nucleotide sequences had greater than 97% nucleotide identity to the most similar genome sequences in GenBank. One of the HSVd sequences, FJ773271, presented the cachexia determinants. To our knowledge, this is the first report of CBLVd and CDVd in Pakistan (1). These results indicate the need for proper indexing of mother trees and a virus-free propagation scheme to create healthy budwood sources in Pakistan.

Effect of a Field-Source Mixture of Citrus Viroids on the Performance of 'Nules' Clementine and 'Navelina' Sweet Orange Trees Grafted on Carrizo Citrange.

A field-source mixture of citrus viroids was characterized and shown to contain Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). Sequencing results showed that: (i) CEVd contained the PL and PR characteristic of class A variants; (ii) HSVd was a noncachexia variant; (iii) CBLVd was related to CVd-Ia variants; (iv) CDVd was a mixture of two types (CVd-IIIa and CVd-IIIb) of variants. The presence of the same type of variants in inoculated clementine (Citrus clementina 'Nules') and sweet orange (C. sinensis 'Navelina') trees on Carrizo citrange (Poncirus trifoliata × C. sinensis) rootstocks was confirmed. The effect of infection was determined by assessing the performance of infected and noninfected trees growing in the field. Infection resulted in small trees with reduced canopy, yielding a reduced crop. Fruit characteristics were also affected: (i) clementine and sweet orange fruits from infected trees were larger than those from noninfected trees; (ii) clementine fruits from infected trees differed in shape from those of noninfected trees; (iii) sweet orange fruits from infected trees had maturity indexes and juice contents higher than those from noninfected trees; (iv) in both species, the density of the juice, the amount of soluble solids, and the acidity of the fruits from infected trees were lower than those of fruits from noninfected trees. Infected trees had a poorly developed root system with fibrous roots containing fewer amyloplasts than noninfected trees. The results of an in vitro assay on the induction and development of roots in cultured explants are discussed.

Effect of citrus hosts on the generation, maintenance and evolutionary fate of genetic variability of citrus exocortis viroid

Citrus exocortis viroid (CEVd) populations are composed of closely related haplotypes whose frequencies in the population result from the equilibrium between mutation, selection and genetic drift. The genetic diversity of CEVd populations infecting different citrus hosts was studied by comparing populations recovered from infected trifoliate orange and sour orange seedling trees after 10 years of evolution, with the ancestral population maintained for the same period in the original host, Etrog citron. Furthermore, populations isolated from these trifoliate orange and sour orange trees were transmitted back to Etrog citron plants and the evolution of their mutant spectra was studied. The results indicate that (i) the amount and composition of the within-plant genetic diversity generated varies between these two hosts and is markedly different from that which is characteristic of the original Etrog citron host and (ii) the genetic diversity found after transmitting back to Etrog citron is indistinguishable from that which is characteristic of the ancestral Etrog citron population, regardless of the citrus plant from which the evolved populations were isolated. The relationship between the CEVd populations from Etrog citron and trifoliate orange, both sensitive hosts, and those from sour orange, which is a tolerant host, is discussed.

Occurrence and identification of citrus viroids from Sudan.

The Sudanese citrus industry is mainly based on oldline cultivars of grapefruit, sweet orange and willow leaf mandarin, grafted almost exclusively on sour orange. Infectionwith graft-transmissible agents, viroids in particular, may have deleterious effects on the productivity of these citrus species and, more importantly, limits the choice of rootstocks should sour orange be replaced by alternative rootstocks because of tristeza disease. Three cv. Foster pink grapefruit sources and one local selection of Nuri-16, a local sweet orange selection, proved to be infected by several viroids, including Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the causal agents of exocortis and cachexia, respectively. Cloning and sequencing of four HSVd isolates showed that in addition to the characteristic variants containing the “cachexia expression motif” or the “non-cachexia expression motif”, other variants were also present. To secure additional information, samples from 24 citrus species and cultivars were collected in 2008 from several citrus-growing regions and analyzed for the presence of viroids. The results confirmed that CEVd, HSVd, Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd), but not Citrus viroid V (CVd-V), are widely spread throughout the surveyed areas. None of the collected samples was found to be viroid-free. The relationships between viroid infection and “gummy bark” and “Kassala disease”, two affections of unknown etiology, but with cachexia-like gum impregnations in the bark, are discussed.

Citrus exocortis viroid transmission through commercially-distributed seeds of Impatiens and Verbena plants

Surveys of Impatiens and Verbena species in local nurseries in Fredericton, Canada and Verbena species in New Delhi, India showed widespread infection of Citrus exocortis viroid (CEVd) in vegetatively-propagated and seed-grown plants. To determine viroid seed transmission, samples of eight varieties of Impatiens and 11 varieties of Verbena were obtained from four commercial sources. All 19 samples collected contained viroid infection irrespective of variety. The presence of viroid in non-germinated seed was 21%, while the transmission rate in seedlings was 66% in Impatiens walleriana in 2006. Following 2 years of seed storage, the respective figures were 6% and 26%. Similarly, in Verbena x hybrida the presence of viroid in seed was 13% in 2006 with a seed-transmission rate in seedlings of 28%, while the respective figures after 2 years of storage were 5% and 45%.

Real time RT‐PCR assay for quantitative detection of Citrus viroid III in plant tissues

A rapid and sensitive real time reverse transcription‐PCR (RT‐PCR) assay based on SYBR Green I chemistry was developed for the quantitative detection of Citrus viroid III (CVd‐III) in citrus samples. CVd‐III titre was determined at different times in green bark of sour orange, Troyer citrange, trifoliate orange and alemow seedlings inoculated with a CVd‐IIIb source. Ten weeks after inoculation the viroid was detected in the four species, without substantial differences in viroid titre among them. Nine weeks later an overall increase of viroid titre was observed. The copy number of CVd‐III in sour orange and Troyer citrange was monitored up to 52 weeks after inoculation and a further increase of viroid titre was observed at 35 weeks. For validation purposes, field samples were tested from 58 citrus trees with mixed infections of CVd‐III, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), as well as from healthy controls. Based on the sensitivity (100%), specificity (96·7%), accuracy (99·2%) and repeatability (Cohen's kappa index 0·98) of the assay, it is suggested that its employment in breeding programmes would be helpful in the evaluation of host resistance and viroid accumulation in plants.

A rapid one-step multiplex RT-PCR assay for the simultaneous detection of five citrus viroids in China

Citrus plants are natural hosts of five viroid species and large numbers of sequence variants. In this paper a simple and sensitive one step multiplex RT-PCR protocol with an internal control was utilised to simultaneously detect and differentiate five citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid-III (CVd-III) and Citrus viroid-IV (CVd-IV). In addition, a micro and rapid total nucleic acid extraction method was developed and the protocol applied to evaluate the occurrence and distribution of citrus viroids in China.

Preliminary Studies on Species and Distribution of Citrus Viroids in China

Citrus viroids are the small but economically important RNA pathogens. For investigating their occurrence and distribution in China, 65 viroid samples collected from 8 major citrus cultivated regions were evaluated using one-step or multiplex one-step RT-PCR and biological indexing for specifically detection of Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid-III (CVd-III) and Citrus viroid-IV (CVd-IV). The results showed that there were at least 4 kinds of citrus viroids (CEVd, CBLVd, HSVd, and CVd-III) on citrus trees in China. Most of the infected citrus plants harbored more than one viroid species, and two plants were infected with up to 4 citrus viroids. Sweet orange was more frequently infected by viroids than other citrus varieties. It is the preliminary report on the species and distribution of citrus viroids in China.

Monomeric Linear RNA of Citrus Exocortis Viroid Resulting from Processing In Vivo Has 5′-Phosphomonoester and 3′-Hydroxyl Termini: Implications for the RNase and RNA Ligase Involved in Replication

Members of the family Pospiviroidae, like Citrus exocortis viroid (CEVd), replicate through an RNA-based asymmetric rolling-circle mechanism in which oligomeric plus-strand [(+)] RNA intermediates are cleaved to monomeric linear (ml) RNA and then circularized. Here we show, by rapid amplification of 5' and 3' cDNA ends and in vitro ligation assays, that ml CEVd (+) RNA resulting from cleavage of a dimeric transcript transgenically expressed in Arabidopsis thaliana contains 5'-phosphomonoester and 3'-hydroxyl termini. The nature of these termini and the double-stranded structure previously proposed as the substrate for cleavage in vivo suggest that a type III RNase catalyzes cleavage and an RNA ligase distinct from tRNA ligase promotes circularization.

First Report of Solanum jasminoides Infected by Citrus exocortis viroid in Germany and the Netherlands and Tomato apical stunt viroid in Belgium and Germany.

Recent identifications of Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd) in Solanum jasminoides (3,4) prompted the testing of this plant species for infections with other pospiviroids. From autumn of 2006 to spring of 2007, samples from symptomless plants of S. jasminoides were collected in Belgium (3 samples ranging from 75 to 150 plants), Germany (3 samples ranging from 1 to 200 plants), and the Netherlands (3 samples ranging from 2 to 200 plants). Samples were tested for pospiviroids by reverse transcription (RT)-PCR assays using the Pospi1-FW/RE and Vid-FW/RE (2) and PSTV-Nb-FW (5'-ggatccccggggaaacctgga-3')/RE (5'-ggatccctgaagcgctcctcc-3') primer sets. Each set amplifies several but not all pospiviroids. The first and last primer sets amplified PCR products from six samples. The full-length genomes of all six isolates were amplified using primer pairs CEVd-FW1/RE1 (1) and CEVd-FW2 (5'-gtgctcacctgaccctgcagg-3')/RE2 (5'-accacaggaacctcaagaaag-3'), which are fully complementary to both Citrus exocortis viroid (CEVd) and Tomato apical stunt viroid (TASVd). Sequence analysis of the PCR products identified CEVd from two samples each from Germany and the Netherlands and TASVd from one sample each from Germany and Belgium (plants were imported from Israel). Although the sequences of the different CEVd isolates from S. jasminoides were not identical, all exhibited more than 95% identity with a CEVd isolate from Vicia faba (GenBank Accession No. EF494687). Both TASVd sequences were identical and showed 99.2% identity to a TASVd isolate from tomato (GenBank Accession No. AY 062121). Two nucleotide sequences of CEVd were submitted to the NCBI GenBank (Accession Nos. EU094207 and EU094208). The two other CEVd sequences and the TASVd sequence were submitted to the EMBL Nucleotide Sequence Database as Accession Nos. AM774356, AM774357, and AM777161. In addition to identification from S. jasminoides by sequence analysis, TASVd infection in the S. jasminoides sample from Germany and CEVd in one sample from the Netherlands was confirmed by mechanical inoculation to tomato followed by RT-PCR using the two CEVd-FW/RE primer pairs and analysis of the sequenced PCR product. Infection by CEVd and TASVd was also confirmed in the German samples by Northern hybridization and TASVd was confirmed in the Belgian sample by return-polyacrylamide gel electrophoresis. To our knowledge, these are the first reports of CEVd and TASVd in S. jasminoides. The viroids do not reduce the quality of S. jasminoides plants; however, the infected plants may act as infection sources for other crops.

Occurrence of Four Citrus Viroids in Chongqing, China

Chongqing Municipality, located in the superior citrus belt of the upper-middle Yangtze River, is one of the most important citrus-producing areas in China. A survey was performed to evaluate the occurrence and distribution of citrus viroids in this area, where Poncirus trifoliata is the main rootstock. From 2002 to 2006, 72 samples of sweet oranges (Citrus sinensis), lemons (C. jambhiri), mandarins (C. reticulata), and mandarin hybrids, which showed stunting, bark scaling, and cracking symptoms on the rootstock, were collected and graft inoculated into Arizona 861-S1 Etrog citron (C. medica) on rough lemon rootstock and maintained in a greenhouse at 28 to 32°C. Fifty-one of the seventy-two samples were cultivars imported from abroad, and the remaining samples were all local cultivars. Sixty samples induced symptoms typical of citrus viroids on the Etrog indicator plants. To identify the causal agent(s), a one-step reverse transcription (RT)-PCR protocol, using five primer pairs (1) targeting the complete genome sequences, was used to detect Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) (2). All 72 samples were infected by citrus viroids. Sixty-five and thirty-five of the seventy-two samples were positive for HSVd and CVd-III, respectively. CEVd and CBLVd were found, respectively, in 20 and 11 of 72 samples, whereas CVd-IV was not detected. Of 72 samples, 12 without typical symptoms on Etrog citrons were infected by HSVd and CBLVd. Nearly all (70 of 72) infected citrus plants harbored more than one viroid species, and two plants were both infected by CEVd, CBLVd, HSVd, and CVd-III. RT-PCR products were purified and ligated into pGEM T-Easy Vector (Promega, Madison, WI) and three clones for each of the four viroid species were sequenced and deposited in GenBank: CEVd (Accession Nos. EU382202, U382203, and EU382204); CBLVd (Accession Nos. EU382211, EU382212, and EU382213); HSVd (Accession Nos. EU382208, EU382209, and EU382210); and CVd-III (Accession Nos. EU382205, EU382206, and EU382207). BLAST analysis showed that these nucleotide sequences had greater than 94% nucleotide identity to the most similar genome sequences in GenBank. Sweet orange was more frequently infected by viroids than the other citrus cultivars. To our knowledge, this is the first report of CBLVd, HSVd, and CVd-III in Chongqing and the first report of CBLVd in China. The high incidence of citrus viroids in Chongqing necessitates rapid development of a system of propagation and testing to reduce the incidence of viroids and the associated loss of citrus production.

Pest risk assessment made by France on Citrus exocortis viroid (CEVd) considered by France as harmful in French overseas department of Réunion - Scientific Opinion of the Panel on Plant Health

Pest risk assessment made by France on Citrus exocortis viroid (CEVd) considered by France as harmful in French overseas department of Réunion - Scientific Opinion of the Panel on Plant Health

First report of pospiviroids infecting ornamentals in the Netherlands: Citrus exocortis viroid in Verbena sp., Potato spindle tuber viroid in Brugmansia suaveolens and Solanum jasminoides, and Tomato apical stunt viroid in Cestrum sp.

First report of pospiviroids infecting ornamentals in the Netherlands: <i>Citrus exocortis viroid</i> in <i>Verbena</i> sp., <i>Potato spindle tuber viroid</i> in <i>Brugmansia suaveolens </i>and<i> Solanum jasminoides</i>, and<i> Tomato apical stunt viroid</i> in <i>Cestrum</i> sp.

Rapid in vitro microindexing of viroids in citrus

A new laboratory technique combining shoot‐tip grafting in vitro and biological indexing on indicator plants was explored for the detection of citrus exocortis and related viroids. Τhree in vitro laboratory methods were used and compared with the classical biological method. With the classical in vivo method, diagnosis is based on the expression of symptoms on indicators 11–14 weeks after inoculation. In contrast, with the first in vitro method, microindexing in vitro of citron seedlings by graft inoculation, diagnosis was possible 12 days after inoculation; with the second method, microindexing in vitro of citron cuttings by graft inoculation, 20 days after inoculation; and with the third method, microindexing in vitro of citron cuttings by injection inoculation, 40 days after inoculation. Inoculated Etrog citron plantlets grown in vitro and tested by RT‐PCR showed the same viroid content as the source plants. Of the three in vitro viroid indexing methods, microindexing on cuttings by grafting was easier and more reliable than microindexing either on seedlings or on cuttings by injection.

Field performance of "marsh seedless" grapefruit on trifoliate orange inoculated with viroids in Brazil

Some viroids reduce citrus tree growth and may be used for tree size control aiming the establishment of orchards with close tree spacing that may provide higher productivity than conventional ones. To study the effects of citrus viroids inoculation on vegetative growth, yield and fruit quality of 'Marsh Seedless' grapefruit (Citrus paradisi Macf.) grafted on trifoliate orange [Poncirus trifoliata (L.) Raf.], an experiment was set up in January 1991, in Bebedouro, São Paulo State, Brazil. The experimental design was randomized blocks with four treatments with two plants per plot: viroid isolates Citrus Exocortis Viroid (CEVd) + Hop stunt viroid (HSVd - CVd-II, a non cachexia variant) + Citrus III viroid (CVd-III) and Hop stunt viroid (HSVd - CVd-II, a non cachexia variant) + Citrus III viroid (CVd-III) and controls: two healthy buds (control), and no grafting (absolute control). Inoculation was done in the field, six months after planting by bud grafting. Both isolates reduced tree growth (trunk diameter, plant height, canopy diameter and volume). Trees not inoculated yielded better (average of eleven harvests) than inoculated ones but the productivity was the same after 150 months. Fruit quality was affected by viroids inoculation but not in a restrictive way. The use of such severe dwarfing isolates for high density plantings of grapefruit on trifoliate orange rootstock is not recommended.