Refinement of the Citrus Tristeza Virus Resistance Gene (Ctv) Positional Map in Poncirus trifoliata and Generation of Transgenic Grapefruit (Citrus paradisi) Plant Lines with Candidate Resistance Genes in this Region

Citrus tristeza virus (CTV) is a major pathogen of Citrus. A single dominant gene Ctv present in the trifoliate relative of Citrus, Poncirus trifoliata confers broad spectrum resistance against CTV. Refinement of genetic maps has delimited this gene to a 121 kb region, comprising of ten candidate Ctv resistance genes. The ten candidate genes were individually cloned in Agrobacterium based binary vector and transformed into three CTV susceptible grapefruit varieties. Two of the candidate R-genes, R-2 and R-3 are exclusively expressed in transgenic plants and in Poncirus trifoliata, while five other genes are also expressed in non-transformed Citrus controls. Northern blotting with a CTV derived probe for assessment of infection in virus inoculated plants over a span of three growth periods, each comprising of six to eight weeks, indicates either an absence of initiation of infection or it’s slow spread in R-2 plant lines or an initial appearance of infection and it’s subsequent obliteration in some R-1 and R-4 plant lines. Limited genome walk up- and downstream form R-1 gene, based on it’s 100% sequence identity between Poncirus and Citrus, indicates promoter identity of 92% between the two varieties. Further upstream and downstream sequencing indicates the presence of an O-methyl transferase and a Copia like gene respectively in Citrus instead of the amino acid transporter like gene upstream and a sugar transporter like gene downstream in Poncirus. The possibility of recombinations in the resistance locus of Citrus and the need for consistent monitoring for virus infection and gene expression in the transgenic Citrus trees is discussed. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

High temperatures are not responsible for lack of infection of citrus roots by Phytophthora citrophthora during the summer,but suppressive soil microorganisms may inhibit infection by P. citrophthora

In regions with a mediterranean climate such as southern California, P. citrophthora infection of citrus does not normally occur during the summer. However, in this study, P. citrophthora was isolated abundantly from alternative hosts planted in an infested citrus grove, in the middle of the summer. Citrus root infection from P. citrophthora did occur in this study but it was extremely low. Citrus seedlings grown in autoclaved soil from Pauma Valley, artificially infested with P. citrophthora, contained significantly higher levels of infection than seedlings grown in infested, non-autoclaved, soil or in part autoclaved, part non-autoclaved, infested soil. This suggests that the Pauma Valley soil contains microorganisms naturally suppressive to P. citrophthora.The low occurrence of P. citrophthora infection of citrus roots during the summer may be partially due to soil microorganisms associated with the citrus host and not the inability of P. citrophthora to grow well at the soil temperatures found during the summer in southern California.     

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.     

Detection and Inoculation of Peanut Witches' Broom Phytoplasma (16SrII‐A) and Periwinkle Leaf Yellowing Phytoplasma (16SrI‐B) in Citrus Cultivars in Taiwan

To clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S‐Sr and R16F2n/R16R2. The HLB‐diseased citrus trees with typical HLB symptoms showed a high detection of 89.7% (322/359) of HLB‐Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB‐affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co‐infected with HLB‐Las. The phytoplasma alone was also detected in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP‐DL) by graft inoculation with phytoplasma‐infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches' broom (PnWB) phytoplasma (16SrII‐A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI‐B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma‐infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI‐B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII‐A).     

Distribution of Phytophthora citrophthora R.E. Sm. and E.H. Sm. and P. parasitica Dastur within citrus orchards in Kerman province,Iran

In this research, distribution of Phytophthora species were determined in Kerman Province (Bam, Shahdad and Arzuiyeh) during 2004–2007. The Phytophthora species were isolated from infected root, crown and soil. Root and crown pieces were washed and cultured on a CMA-PARPH medium. The isolation from infected soil was performed by bating method using citrus leaves. It was identified by morphological and several physiological characteristics. Distribution of species was determined by recording the number of isolates recovered from samples from each city. In this study, from 220 soil samples collected from 52 citrus orchards, 80 isolates of Phytophthora were recovered. Among of all isolates of Phytophthora, P. parasitica and P. citrophthora were the most important species of causal agent of gummosis on Citrus sp. Distribution of P. citrophthora was highest in Arzuiyeh and lowest in Bam and Shahdad cities, while distribution of P. parasitica was highest in Bam and Shahdad cities. The majority of soil samples contained only P. parasitica, but a few of percentage samples containing P. citrophthora alone and both of fungi in cites samples.     

利用DNA片段测序方法探究枳属和富民枳的分类地位

富民枳(Poncirus polyandra)属于芸香科(Rutaceae)枳属(Poncirus Raf.).自发表以来,分类地位一直备受争议,其中在Flora of China中认为富民枳为柑橘杂交种(Poncirus polyandra),把枳属归并于柑橘属(Citrus).该研究选取枳属的富民枳、枳(Ponci...     

Development of Species-Specific InDel Markers in Citrus

Citrus taxonomy is complex owing to the existence of a wide range of species: Poncirus is used mainly for rootstock; Fortunella produces small fruit and edible pericarp; and Citrus comprises the most widespread fruit crop species worldwide. Rapidly increasing genome resources from different citrus species facilitate the development of convenient and genome-wide molecular markers that can be applied to both inter- and intra-species analyses. In this study, by comparing the genome sequences of four citrus species, a set of 1958 InDels were identified and 453 candidate InDels were converted into PCR-based markers. Among these candidate InDels, 268 (65%) exhibited length polymorphisms from 30 bp to 200 bp when applied to seven species from the genera Poncirus, Fortunella and Citrus. Seven InDel markers exhibited high intraspecific polymorphisms in a natural pummelo population. The results showed that the InDel markers are effective for both inter- and intra-specific variation and identification analyses. These InDel markers are expected to be applied to germplasm identification, phylogenetic analysis, genetic diversity evaluation and marker-assisted breeding in citrus.     

Pathological categorization of the stem‐pitting and mild strains Citrus tristeza virus in Taiwan and their genomic analysis

Citrus tristeza virus (CTV), the causal agent of tristeza disease, causes the devastating diseases worldwide. In Taiwan, complex cultivars and long‐term infection by CTV result in more than 90% of infected citrus trees, but local strain identification and classification are still incomplete. Here, six CTV strains were categorized by grafting onto eight citrus cultivars and the pathological characteristics of the stem‐pitting mild strains were identified. After 6 months of inoculation, the pummelo stem‐pitting severe strain (CTV‐Pum/SP/T1) only caused severe symptoms in Wentan pummelo (WP) and the mild strain (CTV‐Pum/M/T5) was symptomless in every cultivar; the sweet orange (SO) stem‐pitting severe strain (CTV‐SwO/SP/T7) affected SO, WP and Ponkan mandarin (PM), and the mild strain (CTV‐SwO/M/T51) caused no symptoms in SO except for WP; the mandarin stem‐pitting severe strain (CTV‐Man/SP/T46) caused severe impacts in PM, WP and Eureka lemon, whereas the mild strain (CTV‐Man/M/T2) only caused severe stem‐pitting in WP. The full‐length sequencing of both pummelo stem‐pitting strains and phylogenetic analysis revealed that CTV‐Pum/SP/T1 and CTV‐Pum/M/T5 were related to the HA18‐9 and HA16‐5 strains from Hawaii, respectively. Moreover, recombination analysis revealed that TCT repeat sequences existed at open reading frame 1a in both the CTV‐Pum/SP/T1 and the T36 strains from the United States, indicating that the possible evolution relationship between two regions. Furthermore, improved universal and specific primer pairs were designed for more specific, sensitive detection to meet the needs for quarantine and early prevention. The understanding of strain pathogenicity and genomic analysis provided further characterization of each strain and enabled practical challenge inoculation against CTV disease.     

Identification and genomic distribution of<Emphasis Type="Italic"> gypsy</Emphasis> like retrotransposons in<Emphasis Type="Italic"> Citrus</Emphasis> and<Emphasis Type="Italic"> Poncirus</Emphasis>

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.Communicated by C. Möllers     

Chemotaxonomic Study of Citrus,Poncirus and Fortunella Genotypes Based on Peel Oil Volatile Compounds - Deciphering the Genetic Origin of Mangshanyegan (Citrus nobilis Lauriro)

Volatile profiles yielded from gas chromatography-mass spectrometry (GC-MS) analysis provide abundant information not only for metabolism-related research, but also for chemotaxonomy. To study the chemotaxonomy of Mangshanyegan, its volatile profiles of fruit and leaf and those of 29 other genotypes of Citrus, Poncirus, and Fortunella were subjected to phylogenetic analyses. Results showed that 145 identified (including 64 tentatively identified) and 15 unidentified volatile compounds were detected from their peel oils. The phylogenetic analysis of peel oils based on hierarchical cluster analysis (HCA) demonstrated a good agreement with the Swingle taxonomy system, in which the three genera of Citrus, Poncirus, and Fortunella were almost completely separated. As to Citrus, HCA indicated that Citrophorum, Cephalocitrus, and Sinocitrus fell into three subgroups, respectively. Also, it revealed that Mangshanyegan contain volatile compounds similar to those from pummelo, though it is genetically believed to be a mandarin. These results were further supported by the principal component analysis of the peel oils and the HCA results of volatile profiles of leaves in the study.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

Phylogenetic relationships of <Emphasis Type="Italic">Citrus</Emphasis> and its relatives based on <Emphasis Type="Italic">rbcL</Emphasis> gene sequences

We sequenced the rbcL genes of 64 accessions from 24 genera of Citrus relatives and analyzed them by neighbor-joining and maximum parsimony methods. Both trees supported Swingle and Reece’s (1967) treatment of the subfamily Aurantioideae as monophyletic. However, the trees did not support Swingle and Reece’s treatment of tribes and subtribes. The subgenera Citrus and Papeda were not clustered clearly. The analysis associated the Fortunella group with mandarin, Poncirus with Citrus ichangensis, Severinia buxifolia with Atalantia ceylanica, Microcitrus with Eremocitrus and Citrus micrantha, and Hesperethusa crenulata with Citropsis. Furthermore, Atalantia species showed polytomy. The classification of Swingle and Reece should be reviewed.     

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.     

Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding

The long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus‐based vector (clbvINpr‐AtFT and clbvINpr‐CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4–6 months, with no alteration of the plant architecture, leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr‐AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.     

Pathogenicity of four Phytophthora Species on Wild Cherry and Italian Alder Seedlings

Inoculation tests were carried out in the greenhouse on wild cherry (Prunus avium) and Italian alder (Alnus cordata) seedlings, to determine their susceptibility to certain Phytophthora species (P. citrophthora, P. alni, P. megasperma and P. cinnamomi) that are commonly present in the soil. Host susceptibility was evaluated in accordance with a disease index, with the lesion length after stem inoculation, and with a root system disease index. Wild cherry was found to be highly susceptible to P. citrophthora, and was also found to be susceptible to P. alni, although to a lesser extent. Italian alder was very susceptible to P. alni, but had only low susceptibility to P. citrophthora. The other Phytophthora species caused only modest symptoms. The danger to alder and wild cherry is all the greater because these trees not only share the same pathogens, but also commonly planted together in mixed stands. The results will now have to be confirmed by using a more natural inoculation method.