New developments in pathogen detection and identification

By the turn of the century most of the methods for amplifying the pathogen detection signal had already been developed. However, only a few were successfully incorporated into routine diagnosis schemes. At present, real time PCR became the predominant choice with signal transduction based on fluorescence generated by intercalating dyes or fluorescent resonant energy transfer. Real-time PCR is no more exclusively restricted to niches for which no alternative was feasible; its use is widening and is substituting or complementing other techniques like ELISA. On the low technology side and judging from the success in medical fields, Loop Mediated Isothermal Amplification (LAMP) appears as an interesting alternative. Convergence into a single platform led to the concept of crop-oriented diagnosis, e.g., a unique device or system that could be used in the detection of the relevant pathogens of a crop. However, PCR by itself is not enough robust to support the degree of multiplexing needed and fluorescence detection systems are limited to a reduced number of dyes that can be used simultaneously. Initial attempts to develop alternative systems relied in the use of microarrays but suffer from limited sensitivity. Low Density Arrays, in which individual PCR reactions are spatially separated and done in parallel appear an interesting option, already available for grapevine. All these assays only provide an answer regarding the presence of certain pathogens for which there is an a priori suspicion. Recently introduced high massively parallel sequencing coupled to metagenomic analysis appears to be a major breakthrough in diagnosis, enabling the non-targeted diagnosis of bacteria, virus, fungi and novel agents in a single assay. These have been implemented among others for citrus and grapevine

Epidemiological situation of Citrus tristeza virus in mainland Portugal

This study was conducted to update the occurrence and molecular variability of Citrus tristeza virus (CTV) isolates recently obtained from surveys in different orchards in mainland Portugal. The asymmetric PCR-ELISA typing method based on the coat protein (CP) gene was used to characterize CTV isolates. Most isolates of the virus found in the Algarve region, where major citrus producing zones exist, belonged to the mild phylogenetic group (GpM). The prevalence of haplotypes from this group suggests that the aphid vector Toxoptera citricidus, which is present in the northern region of the country associated to diverse severe strains, has not yet reached the Algarve. Although most of the isolates harbour haplotypes from group M, haplotypes from the remaining phylogenetic groups were also identified and characterized

First report of Citrus tristeza virus in the State Union of Serbia and Montenegro.

Citrus production in the State Union of Serbia and Montenegro has a strategic importance to the agricultural sector. Approximately 400,000 trees are now grown in the major citrus producing region, which is the Montenegrin Coastal Region. Satsuma mandarins and lemons grafted on Poncirus trifoliata are the most cultivated varieties. In December 2003, eight samples taken from the coastal region close to the towns of Bar and Ulcinj were analyzed using enzyme- linked immunosorbent assay (ELISA) with SP7 antibodies produced at Universidade do Algarve, Portugal (3). Further analysis was done using immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) targeting the entire coat protein (CP) gene (forward primer CTV1: 5(prime)- ATGGACGACGAAACAAAGAA-3(prime) and reverse primer CTV10: 5 (prime)-ATCAACGTGTGTTGAATTTCC-3(prime)). Using both techniques, seven of eight samples analyzed were found to be infected by Citrus tristeza virus (CTV), including samples from five trees that exhibited chlorosis, gummosis, and fruit deformation, and two trees that were symptomless

Técnicas biomoleculares no diagnòstico e tipificação em virologia vegetal

Tese de dout., Ciências Agrárias (Protecção de Plantas), Unidade de Ciências e Tecnologias Agrárias, Univ. do Algarve, 1994O diagnóstico é um dos principais meios de protecção de plantas em virologia vegetal. Muito frequentemente seria vantajoso poder ainda complementar o diagnóstico com informação sobre as estirpes que estão presentes num dado contexto epidemiológico. Na maior parte dos casos isto não é possível devido à inexistência de métodos apropriados de tipificação viral. Esta limitação tem também dificultado estudos de âmbito mais teórico que envolvem a identificação de estirpes presentes em populações naturais e que permitiriam melhor delinear as estratégias protecção. Num pequeno número de casos tem sido possível obter esta informação pela análise dos perfis electroforéticos obtidos pelo fracionamento de RNA bicatenário de origem viral ou, mais recentemente, a partir da análise dos genomas virais amplificados por PCR. Neste trabalho estudam-se métodos capazes de serem aplicados em condições de rotina ao diagnóstico viral e à identificação de isolamentos de vírus, desenvolvendo-se em três partes. No primeiro capítulo estuda-se a aplicabilidade da análise de RNA bicatenário ao diagnóstico viral, empregando para tal diversos vírus e hospedeiros. Verifica-se que este método é muito dependente do hospedeiro e desenvolvem-se protocolos de extracção de ácidos nucleicos que podem ser empregues com hospedeiros considerados difíceis, tais como a videira. Não são contudo protocolos que possam ser empregues em condições de rotina. Outras limitações provêm ainda da existência de RNA bicatenários endógenos que foram encontrados em vários hospedeiros. Para além destes problemas, a possibilidade de identificar estirpes depende ainda da estratégia de expressão genómica do vírus e do hospedeiro estar a vegetar em condições apropriadas para a manifestação do perfil completo de bandas. Em comparação com outros métodos que vieram a ser desenvolvidos neste trabalho, a análise de RNA bicatenário apresenta um interesse limitado quer no diagnóstico quer na tipificação viral. Poderá ter interesse como método exploratório no estudo de doenças de etiologia presumivelmente viral ainda não esclarecida, como por exemplo o mosaico da figueira, tratado neste trabalho. Esta doença aparece relacionada com um perfil electroforético composto de várias bandas de que sobressaem duas correspondentes a fragmentos de RNA bicatenário com cerca de 10-12 Kbp e 2,2 Kbp. Estas moléculas poderão estar relacionadas com a replicação de vírus de RNA possívelmente envolvido(s) na patogénese. Na segunda parte estudam-se os métodos de diagnóstico baseados na amplificação in vitro de determinadas partes do genoma viral. Deste estudo resultou um método de diagnóstico (IC/RT-PCR) de elevada sensitividade que evita a extracção de ácidos nucleicos, o que não acontecia na metodologia até então disponível. Os viriões são capturados directamente do extracto da planta mediante anticorpos adsorvidos sobre a superfície de uma fase sólida, provavelmente sofrendo uma distorção que possibilita a iii extracção e transcrição do genoma pela RTase, seguindo-se a sua amplificação por PCR e quantificação dos resultados por fluorimetria. Todo o processo pode ser efectuado numa placa de microtitulação, sendo concebível um grau de automatização equivalente ao da técnica ELISA. Este método foi objecto de um pedido de patente (Nolasco et al., 1992). A sua validade foi verificada com vírus de diversos grupos (Potyvirus, Nepovirus, Cucumovirus, Closterovirus, Luteovirus, Tobamovirus, Tospovirus) e com RNA satélites de CMV e GFLV. Num pequeno rastreio de CTV e GFLV, comprovou-se a incapacidade da técnica ELISA em detectar o vírus em várias amostras positivas por IC/RT-PCR. Por outro lado, no caso do GFLV não foi possível amplificar alguns isolamentos positivos por ELISA. Este facto foi atribuído a uma inesperada variabilidade genómica e reforça a necessidade de se efectuarem estudos de variabilidade genómica de vírus. Alternativamente à captura por anticorpos específicos, é possível capturar os genomas virais por meio de anticorpos para RNA bicatenário, o que permite alargar o âmbito desta metodologia a patogéneos desprovidos de proteína estrutural. Na terceira parte estudou-se a conjugação da amplificação genómica por IC/RTPCR com métodos de análise mutacional com vista ao desenvolvimento de métodos de tipificação genómica de vírus. Dois destes métodos, a análise de polimorfismos conformacionais monocatenários (SSCP) e de polimorfismos de locais de restrição (RSP) empregam como técnica preparativa a amplificação específica por IC/RT-PCR e foram objecto de um pedido de patente (Nolasco et al., 1993a). Um terceiro método, a Síntese Aleatória de cDNA (SAcDNA), baseia a tipificação no aproveitamento das condições pouco restritivas da transcrição reversa para iniciar, por jusante, mediante um iniciador inespecífico, um conjunto de moléculas característico do isolamento viral, que é em seguida amplificado em condições normais de restritividade. Este método foi também objecto de um pedido de patente (Nolasco et al., 1994b). São características gerais dos métodos apresentados poderem ser aplicados directamente ao extracto da planta a analisar, serem independentes do hospedeiro e, comparativamente com outros métodos, serem muito pouco laboriosos. Como modelo de aplicação empregou-se uma série de isolamentos de GFLV e CTV, grande parte mantidos em condições naturais. Obteve-se o melhor poder discriminante com SAcDNA. Para além da discriminação, a análise por RSP permitiu ainda o estudo de relações entre isolamentos e a avaliação quantitativa da diversidade genética de populações de GFLV e CTV, o que não havia sido feito prèviamente. Verificou-se que o gene da proteína do capsídeo do GFLV é altamente variável e que o distanciamento genético entre isolamentos não se relaciona com o distanciamento espacial entre as plantas, mesmo quando lado a lado. A diversificação do CTV segue um modelo distinto, relacionando-se a proximidade geográfica com a proximidade genética entre os isolamentos e não se atingindo diversidades tão elevadas como para o GFLV.Fundação Calouste Gulbenkia

Variability of a portion of RNA 3 containing the coat protein gene of Citrus variegation virus (CVV) using single-strand conformation polymorphism (SSCP)

A portion of RNA 3 of Citrus variegation virus (CVV), comprising part of the intergenic region and the coat protein (CP) gene from eight viral isolates, was amplified by RT-PCR and cloned. The clones were compared for intra and inter-isolate variations by single-strand conformation polymorphism analysis. Some of the results were compared with sequence data previously obtained. The test discriminated between clones differing in as little as 3.2% of the nucleotides. Most isolates included several variants, in some cases with a predominant pattern, which, however, could no longer be recognised in new RT-PCR products obtained 13 months later. This procedure can there-fore be used to identify and detect variations between CVV isolates. It is rapid, inexpensive and may reduce the amount of sequencing needed for comparing viral isolates

Correction to: Can bicarbonate enhance the performance of carob seedlings grown in nutrient solutions with different Fe concentrations?

Correction to: Journal of Soil Science and Plant Nutrition https://doi.org/10.1007/s42729-019-00100-4info:eu-repo/semantics/publishedVersio

Typing of Egyptian Citrus tristeza virus (CTV) isolates based on the capsid protein gene

The capsid protein gene of three Egyptian CTV isolates from two locations was amplified by immunocapture RT-PCR and analysed by single stranded conformation polymorphism and sequencing. The CTV isolates studied did not differ significantly in sequence composition and each isolate consisted of very similar haplotypes. Comparison with reference sequences from isolates elsewhere in the world showed that these haplotypes clustered very close to the severe strain T3 from Florida causing quick decline and stem pitting. Analysis of the deduced amino acid sequence showed the epitope characteristic of reactivity with the MCA13 antibody. Sequence comparison with the sequence of an Egyptian isolate (Qaha) available in the Genbank showed a distance of about 8%, suggesting that it had a different origin

Differentiation of Citrus tristeza virus (CTV) isolates by cleavase fragment length polymorphism (CFLP) analysis of the major coat protein gene

A panel of Citrus tristeza virus (CTV, genus Closterovirus, family Closteroviridae) isolates of different origins and with different biological properties were compared for polymorphisms in the major coat protein (CP) gene by cleavase fragment length polymorphism (CFLP) and single stranded conformation polymorphism (SSCP) analysis. The similarity between the CFLP patterns, which consisted of 15 to 20 bands, was estimated by the Pearson coefficient. The clustering patterns from the CFLP data were very similar to those from sequence data in an experiment with 16 cloned standards of the CP gene. By SSCP analysis on the other hand, most of the clones were not clustered in the same way. To assess the ability of CFLP to analyse biological samples, which may consist of a mixture of genomic variants, the CP gene of 12 CTV isolates was obtained directly from infected plants by immunocapture/RT-PCR and analysed. With few exceptions, the isolates were correctly clustered according to the sequences of the variants composing the isolates. In artificial mixed infections of mild and severe isolates the patterns obtained were more closely related to the severe isolate. Thus the CFLP method was an accurate method for the identification, typing and clustering of CTV isolates. The usefulness of this technique as an alternative to SSCP analysis is suggested and discussed

Genomic variability of Citrus tristeza virus (CTV) isolates introduced into Morocco

Genomic variability of the coat protein gene of Citrus tristeza virus isolates obtained from old Meyer lemon introductions in Morocco and more recent budwood introductions from Spain were studied. The coat protein gene of the virus was amplified directly from infected tissue by immunocapture RT-PCR and analysed by single stranded conformation polymorphism (SSCP) and sequencing. Each isolate consisted of several related genomic variants, typical of a quasi-species. Although SSCP analysis has only limited typing ability it could be used in an initial screening to discriminate between isolates of different origin and to analyse the genomic structure of each isolate. Sequence analysis showed that the isolates of Spanish origin were closely related to mild isolates characterised in Florida and in Portugal. The Meyer lemon isolate on the other hand was related to severe strains of Meyer lemon characterised in Florida some years ago and to other severe strains from Brasil. A knowledge of the coat protein gene sequence is useful to trace the origin of the isolates

Tomato spotted wilt virus genes expressed in antisense orientation and their ability to control virus progression in Nicotiana benthamiana

Tomato spotted wilt virus (TSWV) is a member of the Tospoviridae family and is ranked among the top ten economically important viruses in the world. The genome of TSWV consists of three linear negative-sense or ambisense RNA segments, denoted as segments L, M and S. Segment S RNA encodes the silencing suppressor NSs, and the nucleocapsid protein N. Segment M RNA encodes the cell-to-cell movement protein NSm and two glycoproteins (Gn and Gc). The TSWV is mainly transmitted by thrips and can infect a wide range of hosts, including tomatoes, an economically important crop. Thus, control measures need to be implemented to reduce the damage caused by this virus. In the present work, a TSWV isolate from Nicotiana rustica was acquired through Leibniz DSMZ Institute. The expression of antisense transcripts of the N, NSs and M viral genes in leaves of Nicotiana benthamiana was assayed for its ability to silence virus progression. For this, each construct in the binary vector pK7WG2 was co-agroinfiltrated with pK7WG2-GFP into N. benthamiana leaves, followed by inoculation with TSWV after 48h. Inoculated leaves were harvested 5 days after agroinfiltration for RNA extraction. The ability of antisense transcripts, expressed throughout the plant to control TSWV progression was also assayed using the Tobacco rattle virus viral vector (pTRV). In this case, partial sequences of the above-mentioned genes cloned into pTRV2 were expressed as antisense transcripts. New leaves were harvested 10 days after agroinfiltration of the pTRV viral vector. TSWV detection and absolute quantification was performed by a TaqMan real-time RT-PCR assay. Inoculated leaves with TSWV alone and new leaves showed a low viral titer, a result that indicates host plant resistance to TSWV infection. In both assays, TSWV accumulation was higher for constructs carrying the N or the NSs sequences than with M sequences. These studies allowed us to conclude that M gene transcripts in the antisense orientation greatly limit virus progression in N. benthamiana plants