Detection of Parietaria Mottle Virus by RT-qPCR: An Emerging Virus Native of Mediterranean Area That Undermine Tomato and Pepper Production in Southern Italy

Parietaria mottle virus (PMoV) is considered an emerging virus in many countries of the Mediterranean basin, especially on tomato and pepper crops. Symptoms on tomato leaves and fruits can be easily confused with those induced by cucumber mosaic virus (CMV) with necrogenic satellite RNA (CMV-satRNA), tomato spotted wilt virus (TSWV) or tomato mosaic virus (ToMV). Mixed infection of these viruses has been also reported in some tomato cultivars, with an increase in the complexity of the symptoms and severity of the disease. Although a specific serum and riboprobes have been produced, nowadays no sensitive diagnostic methods are available for the rapid PMoV detection. Here, we have developed a RT-qPCR assay with the aim to establish a more sensitive and specific method for PMoV detection. Specific primers and TaqMan probe were designed and in silico tested with all PMoV isolates available in GenBank. Moreover, this method was evaluated on tomato naturally infected samples from Sicily region (Italy). Results obtained showed that the RT-qPCR assay developed in this work is extremely sensitive, in fact, it is able to detect as few as 10 PMoV RNA copies in tomato total RNA; moreover, it will be a particularly valuable tool for early detection of PMoV. Furthermore, the analyzes on field samples show how this pathogen is increasingly present in tomato crops in the last years, helping to undermine the Italian horticultural sector

First Report of Tomato Leaf Curl New Delhi Virus Causing Yellow Leaf Curl of Pepper in Europe

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus (family Geminiviridae) with two circular ssDNA genome components (DNA-A and DNA-B), is transmitted in a circulative nonpropagative manner by the whitefly Bemisia tabaci (Gennadius). Although it was first reported in Asia on tomato and other solanaceous crops such as eggplant, potato, and chilli pepper in the Mediterranean basin, this virus was mainly detected on cucurbits and only sporadically on tomato and on two wild solanaceous species, Datura stramonium L. and Solanum nigrum L. (Juárez et al. 2019). In 2018, separate surveys were carried out in protected cultivations of sweet pepper (Capsicum annuum L.) in two Italian regions: Lazio and Campania. The greenhouses were in areas with high density of B. tabaci and where ToLCNDV outbreaks occurred on cucurbits since 2016 (Panno et al. 2019). Some plants showing symptoms of yellowing and leaf curling were found in both regions, whereas fruit symptoms were neither observed nor reported by farmers. This disease syndrome, known as yellow leaf curl disease (YLCD), can be caused in pepper by several begomoviruses, as reported recently in a review listing the viruses causing YLCD in peppers in Thailand (Chiemsombat et al. 2018). Symptomatic leaves were collected during late summer 2018 from different pepper plants as well as from the neighboring zucchini cultivations, showing the typical symptomatology induced by ToLCNDV. Total DNA was extracted (DNeasy Plant Mini kit, Qiagen, Germany), and the presence of ToLCNDV was ascertained by PCR with the specific primers ToLCNDV-CP1 and ToLCNDV-CP2 (Panno et al. 2019; Parrella et al. 2018). ToLCNDV infection was further ascertained in three symptomatic leaf samples from Campania by using specific ToLCNDV ImmunoStrips (Agdia, Elkhart, IN). Successively, one symptomatic pepper sample from each greenhouse was selected and amplified by rolling circle amplification technique (RCA; Inoue-Nagata et al. 2004). The amplicons were cloned, and the DNA-A and DNA-B were full-length sequenced. The sequences were deposited in GenBank NCBI database (MK732932 DNA-A and MK732933 DNA-B, pepper sample from Campania; MK756106 DNA-A and MK756107 DNA-B, pepper sample from Lazio). The RCA analysis was performed also on a ToLCNDV-infected zucchini sample collected in the same area in Lazio region (MK756108 DNA-A and MK756109 DNA-B). The analysis of the ToLCNDV sequences showed a low level of genetic variability between the two pepper isolates from Lazio and Campania regions (rate of substitutions: 0.016 for DNA-A and 0.023 for DNA-B). A high genetic similarity was recorded between the zucchini isolate and both the pepper isolates from Campania (0.019 for DNA-A and 0.023 for DNA-B) and Lazio (0.003 for both DNA-A and B). The three characterized isolates showed a high sequence homology also with both the DNA-A (MH577751 from a melon isolate) and DNA-B (MH577673 from a zucchini isolate) of the ToLCNDV-ES genotype (Fortes et al. 2016), which differed in 15 and 13 nucleotide substitutions from pepper sample from Lazio, 29 and 51 substitutions from Campania sample, and 10 and 5 substitutions from zucchini sample. High homology was also identified compared with the other Spanish isolates collected since the first appearance of the virus (2014) and to the Tunisian (2015) and Moroccan (2018) isolates, confirming the hypothesis that the Mediterranean population of ToLCNDV is highly conserved (Juárez et al. 2019). To our knowledge, this is the first report of ToLCNDV infection on pepper in Europe and indicates that sweet pepper could also act as a reservoir of the virus for further spread to other solanaceous plants and cucurbits

Curvas de maturação de cultivares de sorgo sacarino.

Nos últimos anos houve um aumento expressivo no interesse pelo sorgo sacarino como cultura alternativa e complementar à cana-de-açúcar na obtenção de etanol como biocombustível. Devido a este fato, existe a necessidade de se analisar as propriedades do sorgo afim de se atender às demandas da agroindustria. No presente trabalho buscou-se desenvolver e analisar curvas de maturação para três cultivares de sorgo sacarino em oito epocas de amostragem. Os resultados mostram que houve diferença significativa para cultivares e épocas de colheita para todas as variáveis analisadas (p≤0,01). Quanto aos teores de açúcares, a cultivar BRS508 apresentou as maiores médias para as variáveis ART, ATR e AT. As cultivares XBSW80147 e Sugargraze apresentaram menor período de utilização industrial e menores teores de açúcares redutores totais e açúcares totais recuperáveis

Avaliação do bagaço de biomassa de genótipos de sorgo sacarino para a produção de etanol celulósico.

Neste trabalho avaliou-se 4 genótipos de sorgo sacarino, BRS 506; 508; 509 e 511 para produção de etanol

Filter preconditioning enables representative scaled-down modelling of filter capacity and viral clearance by mitigating the impact of virus spike impurities

Endogenous and adventitious virus removal by size-exclusion membrane filtration is a critical dedicated step in an overall viral clearance strategy employed by biologics manufacturers as required by industry regulators. However, the addition of impurities from virus spike preparations used in validation studies can significantly reduce filter capacity, resulting in an oversized and suboptimal virus filtration step. The hydraulic filter performance and virus retention observed in conventional scaled-downed validation models may not necessarily represent performance observed during process development, nor be predictive of manufacturing performance. Using filter flow decay as a relevant processing endpoint, an alternative and more comprehensive approach to virus filter validation has been developed to overcome the limitations imposed by virus spike impurities. With a model feedstream, we have demonstrated comparable virus removal using the conventional virus spiking approach and a complementary preconditioned virus challenge. Similar to a currently accepted method used in the validation of sterilizing-grade filters, this method entails processing non-spiked feed to a volumetric throughput target, followed by processing virus-spiked feed to a final flow decay endpoint to determine viral clearance. This comprehensive approach yields predictive virus retention data under protein-dominant fouling conditions that better model the hydraulic performance of the manufacturing-scale virus filtration operation