These results suggest that the ihpCP lines, with no evidence of infection, allow virus movement to the susceptible tissues .Therefore, we are currently performing a new assay grafting the susceptible nontransgenic and CP-line scions before CPsV infection, giving kinetic advantage to PTGS establishment.Leprosis is a complex pathosystem that involves an atypical virus, Citrus leprosis virus C , an unusual mite vector, Brevipalpus sp., and host plants belonging to at least 18 botanical families. CiLV-C causes only localized lesions and does not spread systemically within its hosts, originally thought to be restricted to Citrus sp. Most of what was accepted about leprosis until the early 2000’s was shown to be inaccurate. Exactly ten years ago the first sequences of Citrus leprosis virus Cwere obtained and used for the development of an RT-PCR-based method for the diagnosis of leprosis. Since then, much has been done by our and other research groups and more light has been shed onto the pathosystem. Data obtained during the last decade will be discussed in the conference; mainly on CiLV-C taxonomy , variability , serological detection , and biological and molecular relationships with other viruses, with its mite vector and with its natural and experimental hosts, now expanded to dozens of plant species. Ultrastructural studies made on viruliferous Brevipalpus phoenicisdetected presumed Citrus leprosis vírus Cparticles between membranes at the basal part of the caeca, dorsal podocephalic gland and adjacent cells. Their viral nature was confirmed by in situ immunogold labeling using an antibody against the p29 protein of CiLV-C,mobile grow rack the putative capsid gene. No evidence of viral replication was obtained and it was concluded that CiLV-C circulates but does not replicate in the mite.
A meticulous anatomical study was made parallel to this study, to map the sites where CiLV-C was present within the mite. However, how the vírus enters, circulates and exits the mite body is still an open question. For feeding, it is believed that B. phoenicis uses the stylet to perforate the epidermis to reach parenchymal cells below,and injects saliva for a predigestion of the cell content, when CiLV-C must be injected into the host cell. Then the stylet is withdrawn and the cell sap is sucked with the help of cell turgor and the pharyngeal pump, and is delivered through the esophagus to the ventriculus and midgut. This process is being monitored by an adaptation of the EPG . The graphs obtained are being interpreted and tentatively associated with different phases of the feeding process. Using common bean as indicator plant, a series of parameters .Citrus leprosis, transmitted by the tenuipalpid mite Brevipalpus phoenicis, is caused by Citrus leprosis virus C , the type member of the genus Cilevirus. The disease is responsible for losses of ~US$ 80 million every year to the Brazilian citrus industry due to the localized lesions it induces on leaves, stems and fruits, and to severe die back and fruit drop it can cause. The disease occurs in several countries in South and Central America and is spreading towards the north of the Continent, having reached Mexico. Preliminary, unpublished data on the variability of the putative movement protein and replicase-associated protein genes suggest very low genetic variability among isolates. Additionally, the only three complete genome sequences of CiLV-C available in the GenBank, two from Brazilian isolates and one from a Panamanian isolate of the virus, share sequence identities of 99%. Altogether, these data suggest low divergence amongst isolates. However, since Brazil is likely to be the center or origin of this virus, we decided to determine the variability of the p29 ORF of 22 CiLV-C isolates from nine Brazilian states and one isolate from Argentina. RT-PCR products were cloned, sequenced and compared among them and with the GenBank sequences of the virus. Overall, p29 sequence identity ranged from 98% to 100%, with the exception of one isolate from São José do Rio Preto, São Paulo State. The sequence identity of this isolate ranged between 85% and 86% when compared to the other 25 sequences available. This shows that, even though CiLV-C variability may be considered low, there are isolates with significantly higher variability infecting citrus in Brazil.
It is even possible that other species of cilevirus causing leprosis-like symptoms are present in the country, as recently reported in Colombia. The ecology of plant pathogens of perennial crops is impacted by the long-lived nature of their immobile hosts. Over time, host plants are subject to changes to the genotype structure of pathogen populations that may impact disease spread and management practices; examples include the local adaptation of more fit genotypes, or the introduction of novel genotypes from geographically distant areas via human movement of infected plant material or insect vectors. We studied the genotype mixture of Xylella fastidiosa populations causing disease in sweet orange and coffee crops in Brazil at multiple scales, using fast-evolving molecular markers . Results show that populations of this bacterial plant pathogen were regionally isolated, as were the hosts. Independently of host and geographic origin the data suggest that the populations evolved locally and were not the result of migration. At a smaller spatial scale , results suggest that X. fastidiosa isolates within plants originated from a shared common ancestor, indicating that despite the long-term exposure of trees to infection, infection occurred only once, even though the vector visited the plant multiple times. It is possible that systemically infected trees are less susceptible to new invasions by competing X. fastidiosa genotypes. In summary, new insights to the ecology of this economically important plant pathogen were obtained by sampling populations at different spatial scales and from two different hosts. Xylella fastidiosa is a plant pathogenic bacterium that causes diseases in many different crops. The mechanism of pathogenicity of this bacterium is associated with its capacity to colonize and form a biofilm in the xylem vessels of host plants. There is no method to control this pathogen in field. In this study, we investigated the inhibitory effect of N-Acetylcysteine , a cysteine analogue used mainly to treat human diseases, on X. fastidiosa under different experimental conditions. Concentrations of NAC over 1 mg/mL reduce bacterial adhesion to glass surfaces, biofilm formation and the amount of EPS. The minimal inhibitory concentration of NAC was 6 mg/mL. NAC was supplied to infected plants in hydroponics, by fertigation, and adsorbed to organic fertilizer . HPLC analysis indicated that plants absorbed NAC at concentrations of 0.48 and 2.4 mg/mL, but not at 6 mg/mL. Sweet orange plants with typical CVC symptoms and treated with NAC in hydroponic solutions showed clear symptom remission and reduced the bacterial population to less than 5% compared with untreated plants, as analyzed by quantitative PCR and bacterial isolation.
Fertigation and NAC-fertilizer experiments were done to simulate a condition closer to that normally used in the field. For both, significant symptom remission and reduced bacterial replication rate were observed. Using NAC-Fertilizer the lag for resurgence of symptoms on leaves after interruption of the treatment was increased to around eight months. This is the first report on the effect of NAC as an anti-bacterial agent against a phytopathogenic bacterium. The use of NAC in agriculture might be a new and sustainable strategy for controlling plant pathogen bacteria. A prophage is bacteriophage DNA integrated into a bacterial chromosome or existing as a plasmid inside the bacterial cell. It provides important biological traits of bacteria that may involve virulence,ebb and flow tables environmental adaptation, strain specification and genome evolution. Genome sequence analyses indicate that both Candidatus Liberibacter asiaticus and Spiroplasma citri harbor prophages. Ca. L. asiaticus is associated with citrus Huanglongbing and S. citri causes citrus stubborn disease . Ca. L. asiaticus is not culturable in vitro. S. citri is culturable but the process is highly challenging. For these reasons, knowledge of the biology of the two bacteria is very limited. To study the prophage diversity of Ca. L. asiaticus in China where HLB has been endemic for over 100 years, 12 consecutive open reading frames in a prophage of a Florida Ca. L. asiaticus isolate were selected and primers were synthesized. Using these primers with PCR, 150 Ca. L. asiaticus samples from southern China were examined. At least three prophage types were detected, indicating a possible rich pool of Ca. L. asiaticus prophage in southern China. Since S. citri was culturable, pure culture of nine S. citri strains were obtained and whole genome sequences were generated. Two prophage/phage genes were selected and their abundance in each of the 9 whole genome sequences was estimated. Copy number of one gene varied from 13 to 154. Copy number of the other gene varied from 11 to 56. These results suggest that in addition to the chromosomal form, prophages of S. citri may also exist in extrachromosomal forms. Overall, these studies demonstrate that prophages are important constituents of both Ca. L. asiaticus and S. citri. More information on prophages are needed for better understanding of the two fastidious prokaryotes important to world citrus industry.The impact of the COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 was foreshadowed by earlier epidemics of new or re-emerging diseases such as SARS , influenza , Middle East Respiratory Syndrome , Ebola , and Zika affecting localized regions . These events showed that novel and well-known viral diseases alike can pose a threat to global health. In 2014, an article published in Nature Medicine stated that the Ebola outbreak should have been “a wake-up call to the research and pharmaceutical communities, and to federal governments, of the continuing need to invest resources in the study and cure of emerging infectious diseases” . Recommendations and even new regulations have been implemented to reduce the risk of zoonotic viral infections , but the extent to which these recommendations are applied and enforced on a regional and, more importantly, local level remains unclear. Furthermore, most vaccine programs for SARS, MERS, and Zika are still awaiting the fulfillment of clinical trials, sometimes more than 5 years after their initiation, due to the lack of patients .
In light of this situation, and despite the call to action, the SARS-CoV-2 pandemic has resulted in nearly 20 million infections and more than 700,000 deaths at the time of writing based on the Johns Hopkins University Hospital global database.1 The economic impact of the pandemic is difficult to assess, but support programs are likely to cost more than €4 trillion in the United States and EU alone. Given the immense impact at both the personal and economic levels, this review considers how the plant-based production of recombinant proteins can contribute to a global response in such an emergency scenario. Several recent publications describe in broad terms how plant-made countermeasures against SARSCoV-2 can contribute to the global COVID-19 response . This review will focus primarily on process development, manufacturing considerations, and evolving regulations to identify gaps and research needs, as well as regulatory processes and/or infrastructure investments that can help to build a more resilient pandemic response system. We first highlight the technical capabilities of plants, such as the speed of transient expression, making them attractive as a first-line response to counter pandemics, and then we discuss the regulatory pathway for plant-made pharmaceuticals in more detail. Next, we briefly present the types of plant-derived proteins that are relevant for the prevention, treatment, or diagnosis of disease. This sets the stage for our assessment of the requirements in terms of production costs and capacity to mount a coherent response to a pandemic, given currently available infrastructure and the intellectual property landscape. We conclude by comparing plant-based expression with conventional cell culture and highlight where investments are needed to adequately respond to pandemic diseases in the future. Due to the quickly evolving information about the pandemic, our statements are supported in some instances by data obtained from web sites . Accordingly, the scientific reliability has to be treated with caution in these cases.The development of a protein-based vaccine, therapeutic, or diagnostic reagent for a novel disease requires the screening of numerous expression cassettes, for example, to identify suitable regulatory elements that achieve high levels of product accumulation, a sub-cellular compartment that ensures product integrity, as well as different product candidates to identify the most active and most amenable to manufacturing in plants .