Luis Galipienso

Low genetic variation between isolates of Citrus leaf blotch virus from different host species and of different geographical origins

The population structure and genetic diversity of Citrus leaf blotch virus (CLBV) were estimated by single-strand conformation polymorphism and nucleotide sequence analyses of two genomic regions located within the replicase (R) and the coat protein (C) genes. Analysis of 30 cDNA clones of each genomic region from two CLBV isolates showed that both isolates contained a predominant haplotype and others closely related. Analysis of 37 CLBV Spanish field isolates showed low genetic diversity (0.0041 and 0.0018 for genomic regions R and C, respectively). Comparison of 14 CLBV isolates from Spain, Japan, USA, France and Australia showed genetic diversities of 0.0318 (R) and 0.0209 (C), respectively. No correlation was found between genetic distance and geographical origin or host species of the isolates. The ratio between nonsynonymous and synonymous substitutions was the lowest found in a plant virus, indicating a strong negative selective pressure in both genomic regions.

Evolutionary analysis of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) causes severe economic losses in many crops worldwide and often overcomes resistant cultivars used for disease control. Comparison of nucleotide and amino acid sequences suggested that tomato resistance conferred by the gene Sw-5 can be overcome by the amino acid substitution C to Y at position 118 (C118Y) or T120N in the TSWV movement protein, NSm. Phylogenetic analysis revealed that substitution C118Y has occurred independently three times in the studied isolates by convergent evolution, whereas the substitution T120N was a unique event. Analysis of rates of non-synonymous and synonymous changes at individual codons showed that substitution C118Y was positively selected.

Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat

Summary. Citrus leaf blotch virus (CLBV) was purified from leaves of Nagami kumquat SRA-153 that showed bud union crease when propagated on Troyer citrange. Virions were filamentous particles (960 × 14 nm) containing a 42 kDa protein and a single-stranded RNA (ssRNA) of about 9,000 nt (Mr 3 × 106). Infected tissue contained three species of double-stranded RNA (dsRNA) of Mr 6, 4.5 and 3.4 ×  106. The nucleotide sequence of several complementary DNA (cDNA) clones showed significant similarities with replication-related proteins from plant filamentous viruses in several genera. A digoxigenin-labelled probe from one of these cDNA clones hybridised in Northern blots with ssRNA from virions and with the three dsRNA species, suggesting that the ssRNA is the genomic RNA of the virus, the largest dsRNA is its replicative form, and the two smaller dsRNAs probably replicative forms of 5′ co-terminal subgenomic RNAs. CLBV was also detected in several citrus cultivars from Spain and Japan including Navelina sweet orange field trees propagated on Troyer citrange showing bud union crease; however, no virus could be detected in other citrus trees with similar symptoms. This indicates that CLBV is not restricted to kumquat SRA-153, but its involvement in causing the bud union disorder remains unclear.

Detection, discrimination and absolute quantitation of Tomato spotted wilt virus isolates using real time RT-PCR with TaqMan(®)MGB probes.

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan(®)MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidentalis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.

Simultaneous detection of six RNA plant viruses affecting tomato crops using a single digoxigenin-labelled polyprobe

A polyprobe for the simultaneous detection by non-isotopic molecular hybridisation has been developed to detect any of the following six viruses causing important economic losses in tomato crops: Tomato spotted wilt virus, Tomato mosaic virus, Pepino mosaic virus, Cucumber mosaic virus, Potato Y virus and Parietaria mottle virus. The polyprobe detected all six viruses with similar sensitivity to that obtained using individual riboprobes. In addition, we evaluated the possible use of the tissue-printing as a sample preparation technique applied to routine diagnosis of tomato plants with the polyprobe.

Detection of citrus leaf blotch virus using digoxigenin-labeled cDNA probes and RT-PCR

Citrus leaf blotch virus (CLBV) was detected by dot-blot hybridization (DBH), and tissue print hybridization (TPH) and by one-step RT–PCR in citrus plants growing both in the greenhouse and in the field. DBH with digoxigenin-labeled cDNA probes allowed CLBV detection in dsRNA-rich and total RNA preparations equivalent to 5 and 0.1 mg of infected tissue, respectively. DBH gave intense signals with RNA extracts from young bark, tender shoots and young leaves, whereas the best hybridization signals with TPH were obtained using tender shoots and young leaf petioles. One-step RT–PCR was 10-fold more sensitive than DBH and amplification was obtained with all infected tissues. CLBV was readily detected in young leaves of infected Eureka lemon, Marsh grapefruit, Nules clementine, Navelina orange and Nagami kumquat in the greenhouse, using either hybridization or RT–PCR, but not in leaves of Pineapple sweet orange. Detection in field trees was less consistent and was only achieved by RT–PCR and DBH. CLBV was detected by DBH and RT–PCR in different citrus varieties from several geographic areas showing bud union crease on trifoliate rootstocks, but not in neighbor trees with the same symptoms or in other varieties showing bud union crease on those rootstocks. Failure to detect CLBV in trees with bud union crease could be due to low virus titer or uneven distribution within the plant. Alternatively, a different agent could be involved in causing bud union crease.

Complete nucleotide sequence of a Spanish isolate of Parietaria mottle virus infecting tomato.

The genome of a Spanish isolate of Parietaria mottle virus (PMoV) obtained from tomato (strain PMoV-T) was completely sequenced. Protein motifs conserved for RNA viruses were identified: the p1 protein contained a metyltransferase domain in its N-terminal half and a triphosphatase/helicase domain in its C-terminal half, the p2 protein contained a RNA polymerase domain; the 3a protein contained a RNA-binding domain with α-helix and β-sheet secondary structures. In addition, stem-loop structures with potential capacity of protein interactions were predicted on the untranslated terminal regions. Comparison with the other sequenced PMoV isolate showed nucleotide identities of 93, 90, and 93% for genomic RNAs 1, 2 and 3, respectively, and amino acid identities ranging from 88 to 97% for the different proteins. A cytosine deletion was detected at position 1,366 of RNA 3, involving a start codon for the coat protein (CP) gene different from the other PMoV isolate, resulting in a CP 16 amino acids shorter. Comparison of synonymous and nonsynonymous mutations revealed different selective constraints along the genome.

Sequence analysis within the RNA 3 of seven Spanish tomato isolates of Parietaria mottle virus (PMoV-T) reveals important structural differences with the parietaria isolates (PMoV)

The genomic regions encoding the putative movement protein (MP), coat protein (CP) and intergenic region (IGR) of seven Spanish isolates of the Parietaria mottle virus which infects tomato plants (PMoV-T) were sequenced. Values for the genetic diversity of the PMoV-T isolates were 0.056, 0.047 and 0.013 for the CP, MP genes and IGR, respectively. Nucleotide and amino acid sequence comparison of the seven PMoV-T isolates with those of PMoV revealed significant differences. All of them had a cytosine deletion at position 1366, also confirmed in an Italian tomato isolate, which involves a start codon for the CP gene different from that for the PMoV sequence, resulting in a CP 16 amino acids shorter than the PMoV CP. The certainty of a cytosine deletion only associated to the tomato isolates or the possibility of a mistake in the PMoV published sequence are the two hypotheses that could explain this difference. Structural motifs highly conserved in Ilarviruses were identified in PMoV-T MP and CP. A stable hairpin structure is proposed for IGR, by the initiation site for subgenomic RNA 4 synthesis. Phylogenetic analysis of CP and MP amino acid sequences showed that Spanish PMoV-T isolates form a separate group from PMoV and other members of the Ilarvirus genus. Comparative analysis with different PMoV isolates including tomato isolates from other regions and isolates from different hosts are necessary to confirm this differentiation.

Fast detection of Southern tomato virus by one-step transcription loop-mediated isothermal amplification (RT-LAMP)

Southern tomato virus (STV) is a double stranded RNA (dsRNA) virus belonging to genus Amalgavirus (family Amalgamaviridae) which has been detected in tomato plants showing stunting, fruit discoloration and size reduction. A one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of STV in total RNA or sap extracts (obtained just by grinding in buffer) from STV-infected tomato plants by using a set of three primers pairs which were designed to the sequence of the STV putative coat protein. Amplification products were visualized by gel electrophoresis or direct staining of DNA. The sensitivity of RT-LAMP was identical to that of the conventional RT-PCR and less affected by the presence of polymerase inhibitors. STV was detected by RT-LAMP in different tomato tissues, i.e. leaves, roots, fruits and seeds. Also the virus was successfully detected by RT-LAMP from sap extracts obtained from field tomato plants whereas conventional RT-PCR did not. Results of this work show that RT-LAMP is a specific, rapid and cheap procedure to detect STV and it could be implemented on field surveys and sanitation programs.

Characterisation of several heterogeneous species of defective RNAs derived from RNA 3 of cucumber mosaic virus

Summary.Preparations of double-stranded RNAs (dsRNAs) extracted from Nicotiana tabacum cv Xanthi plants infected with a subgroup IB isolate of Cucumber mosaic virus (CMV) were found to contain a heterogeneous population of defective RNAs (D-RNAs) derived from RNA 3. Characterised D-RNAs ranged in size from 1.5 to 1.9 kb and were derived either by a single in-frame deletion within the 3a or 3b genes or by means of double in-frame deletions within both genes. Also, northern blot hybridisation showed two other types of RNA derived from RNA 3: (a) RNA species of ca. 0.7 kb containing the 3′-terminus but lacking the 5′-terminus, which could be 3′-coterminal subgenomic of D-RNAs derived from the 3b gene and (b) RNA species of unknown origin of ca. 0.8 kb containing the 5′-terminus but lacking the 3′-terminus.