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

Specific amino acids of Olive mild mosaic virus coat protein are involved in transmission by Olpidium brassicae

Abstract: Transmission of Olive mild mosaic virus (OMMV) is facilitated by Olpidium brassicae (Wor.) Dang. An OMMV mutant (OMMVL11) containing two changes in the coat protein (CP), asparagine to tyrosine at position 189 and alanine to threonine at position 216, has been shown not to be Olpidium brassicae-transmissible owing to inefficient attachment of virions to zoospores. In this study, these amino acid changes were separately introduced into the OMMV genome through site-directed mutagenesis, and the asparagine-to-tyrosine change was shown to be largely responsible for the loss of transmission. Analysis of the structure of OMMV CP by comparative modelling approaches showed that this change is located in the interior of the virus particle and the alanine-to-threonine change is exposed on the surface. The asparagine-to-tyrosine change may indirectly affect attachment via changes in the conformation of viral CP subunits, altering the receptor binding site and thus preventing binding to the fungal zoospore

Detection and Quantification of <em>Fusarium</em> spp. (<em>F. oxysporum</em>, <em>F. verticillioides</em>, <em>F. graminearum</em>) and <em>Magnaporthiopsis maydis</em> in Maize Using Real-Time PCR Targeting the ITS Region

Fusarium spp. and Magnaporthiopsis maydis are soil-inhabiting fungi and respectively the causal agents of fusarium ear rot and late wilt, two important diseases that can affect maize, one of the most important cereal crops worldwide. Here, we present two sensitive real-time PCR TaqMan MGB (Minor Groove Binder) assays that detect and discriminate several Fusarium spp. (F. oxysporum, F. verticillioides, and F. graminearum) from M. maydis. The method is based on selective real-time qPCR amplification of the internal transcribed spacer (ITS) region and allows the quantification of the fungi. The applicability of this newly developed TaqMan methodology was demonstrated in a field experiment through the screening of potentially infected maize roots, revealing a high specificity and proving to be a suitable tool to ascertain Fusarium spp. and M. maydis infection in maize. Its high sensitivity makes it very efficient for the early diagnosis of the diseases and also for certification purposes. Thus, qPCR through the use of TaqMan probes is here proposed as a promising tool for specific identification and quantification of these soil-borne fungal pathogens known to cause disease on a large number of crops

Tomato Response to <i>Fusarium</i> spp. Infection under Field Conditions: Study of Potential Genes Involved

Tomato is one of the most important horticultural crops in the world and is severely affected by Fusarium diseases. To successfully manage these diseases, new insights on the expression of plant–pathogen interaction genes involved in immunity responses to Fusarium spp. infection are required. The aim of this study was to assess the level of infection of Fusarium spp. in field tomato samples and to evaluate the differential expression of target genes involved in plant–pathogen interactions in groups presenting different infection levels. Our study was able to detect Fusarium spp. in 16 from a total of 20 samples, proving the effectiveness of the primer set designed in the ITS region for its detection, and allowed the identification of two main different species complexes: Fusarium oxysporum and Fusarium incarnatum-equiseti. Results demonstrated that the level of infection positively influenced the expression of the transcription factor WRKY41 and the CBEF (calcium-binding EF hand family protein) genes, involved in plant innate resistance to pathogens. To the best of our knowledge, this is the first time that the expression of tomato defense-related gene expression is studied in response to Fusarium infection under natural field conditions. We highlight the importance of these studies for the identification of candidate genes to incorporate new sources of resistance in tomato and achieve sustainable plant disease management

Metagenomic Assessment Unravels Fungal Microbiota Associated to Grapevine Trunk Diseases

Grapevine trunk diseases (GTDs) are among the most important problems that affect the longevity and productivity of vineyards in all the major growing regions of the world. They are slow-progression diseases caused by several wood-inhabiting fungi with similar life cycles and epidemiology. The simultaneous presence of multiple trunk pathogens in a single plant together with the inconsistent GTDs symptoms expression, their isolation in asymptomatic plants, and the absence of effective treatments make these diseases extremely complex to identify and eradicate. Aiming to gain a better knowledge of GTDs and search sustainable alternatives to limit their development, the present work studied the fungal community structure associated with GTDs symptomatic and asymptomatic grapevines, following a metagenomic approach. Two important cultivars from the Alentejo region with different levels of susceptibility to GTDs were selected, namely, ‘Alicante Bouschet’ and ‘Trincadeira’. Deep sequencing of fungal-directed ITS1 amplicon led to the detection of 258 taxa, including 10 fungi previously described as responsible for GTDs. Symptomatic plants exhibited a lower abundance of GTDs-associated fungi, although with significantly higher diversity of those pathogens. Our results demonstrated that trunk diseases symptoms are intensified by a set of multiple GTDs-associated fungi on the same plant. The composition of fungal endophytic communities was significantly different according to the symptomatology and it was not affected by the cultivar. This study opens new perspectives in the study of GTDs-associated fungi and their relation to the symptomatology in grapevines

Tomato Response to Fusarium spp. Infection under Field Conditions: Study of Potential Genes Involved

Tomato is one of the most important horticultural crops in the world and is severely affected by Fusarium diseases. To successfully manage these diseases, new insights on the expression of plant&ndash;pathogen interaction genes involved in immunity responses to Fusarium spp. infection are required. The aim of this study was to assess the level of infection of Fusarium spp. in field tomato samples and to evaluate the differential expression of target genes involved in plant&ndash;pathogen interactions in groups presenting different infection levels. Our study was able to detect Fusarium spp. in 16 from a total of 20 samples, proving the effectiveness of the primer set designed in the ITS region for its detection, and allowed the identification of two main different species complexes: Fusarium oxysporum and Fusarium incarnatum-equiseti. Results demonstrated that the level of infection positively influenced the expression of the transcription factor WRKY41 and the CBEF (calcium-binding EF hand family protein) genes, involved in plant innate resistance to pathogens. To the best of our knowledge, this is the first time that the expression of tomato defense-related gene expression is studied in response to Fusarium infection under natural field conditions. We highlight the importance of these studies for the identification of candidate genes to incorporate new sources of resistance in tomato and achieve sustainable plant disease management

OMMV and TNV-D isolates present in single and in double infected plant samples and Genbank sequences used in coat protein variability analysis.

<p>* Number of clones that present the same sequence are between brackets.</p><p>OMMV and TNV-D isolates present in single and in double infected plant samples and Genbank sequences used in coat protein variability analysis.</p

Multiple sequence alignment of the deduced amino acid sequences of TNV-D variants analysed: WT (TNV-D^H, NC_003487.1), G5R, F13I, S17T, A23N, S129P, N227D, C230R, A244S, E261K.

<p>Multiple sequence alignment of the deduced amino acid sequences of TNV-D variants analysed: WT (TNV-D^H, NC_003487.1), G5R, F13I, S17T, A23N, S129P, N227D, C230R, A244S, E261K.</p