Induced resistance during the interaction pathogen x plant and the use of resistance inducers

Plants react to aggressions through different defence responses. Mechanical barriers consist in the increase of production and deposition of substances capable of containing pathogen invasion. Chemical barriers consist in the increase of concentration or activity of defence proteins and synthesis of phenolic compounds and phytoalexins. Elicitor substances have been widely used in plant disease control showing impressive results and a low impact to the environment and man. This review contains information about plant defence mechanisms and shows the use of inducers of resistance in the control of pathogens and prospects of advance towards sustainable agriculture

Biology and molecular characterization of necroviruses

Three necroviruses, family Tombusviridae, are known to infect Olea europaea L., Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) and Tobacco necrosis virus D (TNV-D), often asymptomatically. OLV-1 has a wide geographical distribution whereas that of OMMVand TNV-D is not known as their discovery is recent, dating from 2005 and 2009, respectively. OMMV has received special attention because of its origin, likely to have resulted from a recombination event between TNV-D, with which it shares a coat protein (CP) amino acid identity of 86% and OLV-1 sharing an RNA-dependent RNA polymerase amino acid identity of 91%. Due to these properties OMMV and TNV-D are indistinguishable by serology or Reverse Transcription – Polymerase Chain Reaction assays that use primers hybridizing in the CP gene region, rendering ambiguous earlier TNV-D identifications based on such tests. OLV-1 and OMMV are transmitted through the soil to roots of host plants in the absence of any vectors but the transmission rate of OMMV increases to more than double in the presence of Olpidium brassicae zoospores that vector this virus in an in vitro manner. A point mutation leading to an amino acid substitution located in an inner region of the virion is responsible for the loss of biologically assisted transmission, probably by altering the particle conformation rendering the binding site inaccessible to the zoospore receptors. The field occurrences of necrovirus multiple infections are common and increase the chances for recombination events, the epidemiological implications of which are still uncertain. The biological and molecular properties of the necroviruses identified in olive trees are the main focus of this review

A Tobacco necrosis virus D isolate from Olea europaea L.: viral characterization and coat protein sequence analysis

A virus isolated from Olea europaea L. grown in Portugal, was identified as a member of the species Tobacco necrosis virus D (TNV-D, genus Necrovirus, family Tombusviridae), based on the molecular and serological properties of the purified virus particles. The genomic region encoding the coat protein (CP) of this isolate (named GP isolate) was amplified by RT-PCR and the cDNA was cloned and sequenced. The CP gene encodes a predicted protein of 269 amino acids showing high identity (86.2%) to TNV-D coat protein sequence. Phylogenetic analysis based on necroviruses CP sequences, confirmed GP as a TNV-D isolate. The alignment with homologous TNV-D CP sequences revealed four conserved amino acids involved in Ca2+ binding as well as the plant virus icosahedral capsid protein "S" signature. Based on the determined nucleotide sequence, specific primers were designed and successfully used in RT-PCR for virus diagnosis in naturally infected olive trees

First characterization of infectious cDNA clones of Olive mild mosaic virus

Full-length cDNA clones of an Olive mild mosaic virus (OMMV) isolate were constructed in order to find infectious cDNA clones. The sequencing of three individual full-length clones revealed some differences between them. In vitro transcription of these clones was performed and the effect of spontaneous mutations in the biological behaviour of the in vitro transcripts was evaluated by symptomatology, RNA accumulation and virus replication in inoculated plants. In vitro synthesized RNA from one of these clones was found to mimic the wild-type OMMV, making it useful in future studies on protein structure and function by site directed mutagenesis of individual genes. This is the first report on constructing full-length cDNA clones of OMMV from which infectious RNAs can be transcribed in vitro

Biological and Molecular Characterization of Olive latent virus 1

Olive latent virus 1 (OLV-1) belongs to the Necrovirus genus, Tombusviridae family and is pathogenic to olive, citrus and tulip plants. It is easily mechanically transmissible to indicator plants causing necrotic lesions and can be transmitted through the soil into the plant roots in the absence of biological vectors. Infected cells contain virus aggregates, inclusions made up of excess of viral coded peptides and extensive vesiculation in the cytoplasm. The virions are isometric with ca. 30 nm, possess a monopartite single-stranded positive-sense RNA genome sized 3700 nt with 5 open reading frames (ORFs) and small inter cistronic regions. ORF 1 encodes a polypeptide with a molecular weight of 23 kDa and the read through of its amber stop codon results in ORF 1 RT that encodes the virus RNA dependent RNA polymerase with 82 kDa. ORF2 and ORF3 encode two small peptides, with 8 kDa and 6 kDa, respectively, which appear to be involved in the virus cell-to-cell movement. ORF 4 is located in the 3′-terminal and encodes a protein with 30 kDa identified as the viral coat protein. The complete genomic sequences of two well characterized OLV-1 isolates (obtained from citrus and olive) are similar, revealing an overall nucleotide sequence identity of 95%. The electrophoretic profile of the dsRNAs recovered from infected tissues exhibits three major species with ca. 3.7, 1.5, and 1.3 kbp. Application of molecular techniques based on PCR and on dot blot hybridization has been successfully used for routine diagnosis of OLV-1 infections

D etection of sweet potato virus C, sweet potato virus 2 and sweet potato feathery mottle virus in Portugal

Field sweet potato plants showing virus-like symptoms, as stunting, leaf distortion, mosaic and chlorosis, were collected in southwest Portugal and tested for the presence of four potyviruses, sweet potato virus C (SPVC), sweet potato virus 2 (SPV2), sweet potato feathery mottle virus (SPFMV), sweet potato virus G (SPVG), and the crinivirus sweet potato chlorotic stunt virus (SPCSV). DsRNA fractions were extracted from symptomatic leaves and used as templates in single and multiplex RT-PCR assays using previously described specific primers for each analyzed virus. The amplified reaction products for SPVC, SPV2 and SPFMV were of expected size, and direct sequencing of PCR products revealed that they correspond to the coat protein gene (CP) and showed 98%, 99% and 99% identity, respectively, to those viruses. Comparison of the CP genomic and amino acid sequences of the Portuguese viral isolates recovered here with those of ten other sequences of isolates obtained in different countries retrieved from the GenBank showed very few differences. The application of the RT-PCR assays revealed for the first time the presence of SPVC and SPFMV in the sweet potato crop in Portugal, the absence of SPVG and SPCSV in tested plants, as well as the occurrence of triple virus infections under field conditions

Multiplex RT-PCR for detection and identification of three necroviruses that infect olive trees

An optimized multiplex RT-PCR assay was developed to discriminate three necrovirus (Olive latent virus 1 (OLV-1), Tobacco necrosis virus D (TNV-D) and Olive mild mosaic virus (OMMV)) that infect olive trees. An olive orchard consisting of 54 trees of cv. "Galega vulgar" in the south of Portugal was surveyed. dsRNA fraction was used as template and revealed the 3 viruses, singly or in multiple infections, present in 17 out of 54 trees in the orchard. OMMV was the most frequent occurring in 15 trees, followed by OLV-1 in 12 and TNV-D in 4 plants. The results obtained showed that necrovirus- specific dsRNAs do exist in infected tissues in amounts below the resolution permitted by gel electrophoresis analysis and that the developed multiplex PCR based assay is of much higher sensitivity. The design of the specific primers described enabled, for the first time, to discriminate between OMMV and TNV-D by means of RT-PCR assays, an indispensable tool in identification, epidemiology and survey studie

Evidence of olive mild mosaic virus transmission by Olpidium brassicae

Transmission of three strains of OMMV by an Olpidium sp. was evaluated and compared. The three strains were 1) an OMMV wild type (WT) recovered from olive trees, 2) an OMMV variant (L11) obtained after 15 serial passages of single local lesions induced in Chenopodium murale plants, and 3) a construct OMMV/OMMVL11 in which the coat protein (CP) gene replaced that of the wild type. A single-sporangial culture derived from Chinese cabbage (Brassica pekinensis) used as a bait plant grown in soil of an olive orchard, was identified as Olpidium brassicae based on the size and sequence of the generated amplicon in PCR specific tests. Each of the three virus strains was soil transmitted to cabbage roots in the absence of the fungus at similar rates of 30 to 40%. Separate plant inoculation by O. brassicae zoospores incubated with each viral strain resulted in enhanced transmission of OMMV, reaching 86% of infection whereas that of the other two strains remained practically unaffected at ca. 34%. Binding assays showed that the amount of virus bound to zoospores, estimated spectrophotometrically, was 7% in the case of OMMV, and practically nil in the case of the other two viral strains. Substitution of the coat protein (CP) gene of OMMV by that of the OMMV L11 strain, drastically reduced viral transmissibility in the presence of zoospores to the level of that observed in their absence. Our data shows that OMMV soil transmission is greatly enhanced by O. brassicae zoospores and that the viral CP plays a significant role in this process, most likely by facilitating virus binding and later entrance into the host plant roots

Monoclonal antibody to Olive latent virus 1

Olive is a crop of high economic importance in Portugal. Its susceptibility to virus infections justifies research leading to accurate virus detection. Two necroviruses mechanically transmissible to herbaceous indicator plants have been isolated from symptomatic olive trees: Olive latent virus 1 (OLV-1) and Tobacco necrosis virus D (TNV-D). OLV-1 was also isolated from citrus trees in Turkey and from tulips in Japan. In this study, we attempted to prepare a monoclonal antibody (MAb) specific to OLV-1, which could be used in ELISA for accurate diagnostics of this virus

Complete nucleotide sequence of an Olive latent virus 1 isolate from olive trees

Olive latent virus 1 (OLV-1) is a necrovirus belonging to the familyTombusviridae. It is a small icosahedral plant virus, which encapsidates a single stranded positivesense RNA. This virus was first isolated from symptomless olive trees in Italy [7] and afterwards in Jordan and Portugal [10, 4]. OLV-1 was also isolated from symptomatic hosts, such as citrus trees in Turkey [11] and tulips in Japan [9]. Up to now, only one complete genome sequence of an OLV-1 citrus isolate has been determined [8]. This report describes the first full genomic sequence of an OLV-1 isolated from olive trees