Cauliflower Mosaic Virus TAV, a Plant Virus Protein That Functions like Ribonuclease H1 and is Cytotoxic to Glioma Cells

Recent comparisons between plant and animal viruses reveal many common principles that underlie how all viruses express their genetic material, amplify their genomes, and link virion assembly with replication. Cauliflower mosaic virus (CaMV) is not infectious for human beings. Here, we show that CaMV transactivator/viroplasmin protein (TAV) shares sequence similarity with and behaves like the human ribonuclease H1 (RNase H1) in reducing DNA/RNA hybrids detected with S9.6 antibody in HEK293T cells. We showed that TAV is clearly expressed in the cytosol and in the nuclei of transiently transfected human cells, similar to its distribution in plants. TAV also showed remarkable cytotoxic effects in U251 human glioma cells in vitro. *ese characteristics pave the way for future analysis on the use of the plant virus protein TAV, as an alternative to human RNAse H1 during gene therapy in human cells

Studies of sustainable strategies to control phytopathogen agents

Choosing natural enemies to suppress pest population has been for a long the key of biological control. Overtime the term biological control has also been applied to the use of suppressive soils, bio-disinfection and biopesticides. Biological control agents (BCA) and natural compounds, extracted or fermented from various sources, are the resources for containing phytopathogens. BCA can act through direct antagonism mechanisms or inducing hypovirulence of the pathogen. The first part of the thesis focused on mycoviruses infecting phytopathogenic fungi belonging to the genus Fusarium. The development of new approaches capable of faster dissecting the virome of filamentous fungi samples was performed. The semiconductor-based sequencer Ion Torrent™ and the nanopore-based sequencer MinION have been exploited to analyze DNA and RNA referable to viral genomes. Comparison with GeneBank accessions and sequence analysis allowed to identify more than 40 putative viral species, some of these mycovirus genera have been studied as inducers of hypovirulence in several phytopathogenic fungi, therefore future works will focus on the comparison of the morphology and physiology of the fungal strain infected and cured by the viruses identified and their possible use as a biocontrol agent. In a second part of the thesis the potential of botanical pesticides has been evaluated for the biocontrol of phloem limited phytopathogens such as phytoplasmas. The only active compounds able to control phytoplasmas are the antibiotic oxytetracyclines and in vitro direct and fast screening of new antimicrobials compounds on media is almost impossible due to the difficulty to culture phytoplasmas. For this reason, a simple and reliable screening method was developed to evaluate the effects of antimicrobials directly on phytoplasmas by an “ex-vivo” approach. Using scanning electron microscopy (SEM) in parallel with molecular tools (ddRT-PCR), the direct activity of tetracyclines on phytoplasma cells was verified, identifying also a promising compound showing similar activity

Molecular characterization of the two postharvest biological control agents Aureobasidium pullulans L1 and L8

L1 and L8 are Aureobasidium pullulans strains previously shown to effectively act as biocontrol agents (BCA) against fruit postharvest pathogens. Here, L1 and L8 strains were tested in vivo against Monilinia laxa of peaches showing the ability to reduce the brown rot incidence by 95% and by 80% respectively. To develop future bioformulation and allow commercial use of these biocontrol agents (BCAs) it is necessary to develop specific identification tools. Since A. pullulans strains share very similar morphological features and are not distinguishable by visual analysis, the RAPD (Random Amplified Polymorphic DNA) technique was applied to genomic DNA of L1 and L8 and of other 40 different A. pullulans strains, and SCAR markers were developed from a L1 and L8 specific RAPD fragment of 984 bp. These primers amplify a fragment of 137 bp exclusively in L1 and L8, and can be used to distinguish the two yeasts strains from other A. pullulans strains with high specificity. The droplet digital PCR, used to determine the precise limit of detection (LOD), showed that these markers allow to detect up to 43 and 215 cells of L1 and L8 respectively

Molecular characterization of the two postharvest biological control agents Aureobasidium pullulans L1 and L8

L1 and L8 are Aureobasidium pullulans strains previously shown to effectively act as biocontrol agents (BCA) against fruit postharvest pathogens. Here, L1 and L8 strains were tested in vivo against Monilinia laxa of peaches showing the ability to reduce the brown rot incidence by 95% and by 80% respectively. To develop future bioformulation and allow commercial use of these biocontrol agents (BCAs) it is necessary to develop specific identification tools. Since A. pullulans strains share very similar morphological features and are not distinguishable by visual analysis, the RAPD (Random Amplified Polymorphic DNA) technique was applied to genomic DNA of L1 and L8 and of other 40 different A. pullulans strains, and SCAR markers were developed from a L1 and L8 specific RAPD fragment of 984 bp. These primers amplify a fragment of 137 bp exclusively in L1 and L8, and can be used to distinguish the two yeasts strains from other A. pullulans strains with high specificity. The droplet digital PCR, used to determine the precise limit of detection (LOD), showed that these markers allow to detect up to 43 and 215 cells of L1 and L8 respectively

Not the one, but the only one: about Cannabis cryptic virus in plants showing ‘hemp streak’ disease symptoms

Interveinal chlorosis and leaf margin wrinkling are widespread symptoms of Cannabis sativa. They are traditionally attributed to the so-called hemp streak virus (HSV), but its existence has not been demonstrated yet. To our knowledge, no molecular investigation has so far been performed in order to identify the causal agent of this symptomatology, we therefore decided to use traditional and molecular virology techniques to better characterize symptoms and pursue the etiological agent. No pathogenic virus was found by using targeted PCR reactions and by RNA sequencing, whereas we were able to detect the Cannabis cryptic virus (CanCV) with both techniques. We, therefore, developed an RT-qPCR assay based on a CanCV-specific TaqMan probe and applied it to a wide range of symptomatic and symptomless plants, using a two-step (for quantification), or a one-step (for fast detection) protocol. Both symptoms and the virus were only shown to be transmitted vertically and did not pass via mechanical inoculation or grafting, though we could not find any cause-effect correlation between them. In fact, the virus was found in all the tested hemp samples, and its abundance varied greatly between different accessions and individuals, independently from the presence and severity of symptoms. The suggestion that hemp streak is caused by a virus is therefore questioned. Some abiotic stresses seem to play a role in triggering the symptoms but this aspect needs further investigation. For breeding purposes, a selection of parental plants based on the absence of symptoms proved to be efficient in containment of the disease

Geographic Distribution, Genetic Variability and Biological Properties of Rice Orange Leaf Phytoplasma in Southeast Asia

Rice orange leaf phytoplasma (ROLP) causes clear orange to yellowish leaf discoloration and severe stunting in rice seedlings. The ecological and biological characteristics of ROLP are largely unknown because the disease has not widely caused serious problems in rice cultivated areas, thereby leading to the low accumulation of research data. However, in the past decade, the disease became a threat to rice production, particularly in South China and India; it has also been recognised in other Asian countries, such as Vietnam, Thailand and the Philippines. Here, we observed the occurrence of ROLP in paddies of the Southeast Asian counties (Cambodia, Vietnam and the Philippines) and found that the isolates in the Philippines and Vietnam were monophyletic, while those in India, Thailand and Cambodia were more diverse, suggesting their potential origins. In Cambodia, it was revealed that following polymerase chain reaction (PCR) detection, the known ROLP-insect vectors, N. virescens Distant and Recilia dorsalis Motchulsky, were ROLP-positive, indicating their roles in pathogen dispersal. Moreover, fluorescent and scanning electron microscopy revealed the intensive accumulation of the phytoplasma in phloem tissues and massive accumulation of storage starch in vascular bundle sheath and parenchyma. Altogether, this study illustrated the genetic variability of global ROLP isolates and the pathogen’s biological impact on rice tissue

Not the one, but the only one: about Cannabis cryptic virus in plants showing ‘hemp streak’ disease symptoms

Interveinal chlorosis and leaf margin wrinkling are widespread symptoms of Cannabis sativa. They are traditionally attributed to the so-called hemp streak virus (HSV), but its existence has not been demonstrated yet. To our knowledge, no molecular investigation has so far been performed in order to identify the causal agent of this symptomatology, we therefore decided to use traditional and molecular virology techniques to better characterize symptoms and pursue the etiological agent. No pathogenic virus was found by using targeted PCR reactions and by RNA sequencing, whereas we were able to detect the Cannabis cryptic virus (CanCV) with both techniques. We, therefore, developed an RT-qPCR assay based on a CanCV-specific TaqMan probe and applied it to a wide range of symptomatic and symptomless plants, using a two-step (for quantification), or a one-step (for fast detection) protocol. Both symptoms and the virus were only shown to be transmitted vertically and did not pass via mechanical inoculation or grafting, though we could not find any cause-effect correlation between them. In fact, the virus was found in all the tested hemp samples, and its abundance varied greatly between different accessions and individuals, independently from the presence and severity of symptoms. The suggestion that hemp streak is caused by a virus is therefore questioned. Some abiotic stresses seem to play a role in triggering the symptoms but this aspect needs further investigation. For breeding purposes, a selection of parental plants based on the absence of symptoms proved to be efficient in containment of the disease