First report of Leek yellow stripe virus, Onion yellow dwarf virus, and the putative allium polerovirus A in elephant garlic (Allium ampeloprasum) in Brazil.

Five elephant garlic plants (Allium ampeloprasum L.) showing leaf symptoms of chlorotic streaks and mosaic (Figure 1A and B) were collected, in September 2021, in an experimental area in municipality of Rio do Sul (27°11'07"S, 49°39'39"W), State of Santa Catarina, Brazil. Total RNA was extracted using TRIzol® reagent (Invitrogen, USA), according to the manufacturer's instructions to investigate viral infection. The RNA from all five plants were pooled into a single sample for cDNA library construction with the TruSeq Stranded Total RNA with Ribo-Zero Plant (Illumina) kit, which was then sequenced on the Illumina HiSeq2500 platform (Proteimax Biotechnology LTDA). After high throughput sequencing (HTS), 49 million raw reads (each 151nt) were generated. They were trimmed with the BBduk tool and de novo assembled with the Tadpole assembler tool (Geneious Software version 2022). A total of 28,345 contigs were generated and searched against the NCBI virus genome database using BLASTn and BLASTx, with positive results for two potyviruses, leek yellow stripe virus (LYSV), onion yellow dwarf virus (OYDV), and the putative polerovirus allium polerovirus A (APVA). The trimmed reads were mapped with the BBmap tool (Bushell 2014), using reference sequences for LYSV (NC_004011), OYDV (NC_005029), and APVA isolate Won (MH898527). A total of 806,060 reads were mapped, resulting in the nearly complete genome of LYSV (isolate RDS22-2, 10,268 bp, ON565071), which shared the highest (89.41%) nucleotide (nt) identity with LYSV isolate MG (KP258216). The nearly complete genome of OYDV (isolate RDS22-1, 10,519 bp, ON565070) was assembled using 311,467 reads, being 90.21% nt identical to OYDV isolate G-118 (KF632714). The APVA genome (isolate RDS22-3, 4,367 bp, ON565072, Figure 1C) was assembled from 116,303 reads and it shared the highest (90.73%) nt identity with APVA isolate Won. Subsequently, each sample was RT-PCR screened separately for potyviruses and poleroviruses, using the generic primer pairs NIb2F/NIb3R (Zheng et al., 2010) and Pol-G-F/Pol-G-R (Knierim et al., 2010), respectively. Amplified DNA fragments with approximately 350 bp and 1000 bp were obtained for potyviruses and poleroviruses, respectively, and were sent for Sanger sequencing (ACTGene, Alvorada, Brazil). The Sanger derived partial sequences shared 98 to 100% nt identities with corresponding HTS-derived sequences. The most common virus was LYSV, which was found in three of the five tested samples, whereas OYDV and APVA were only found in one sample each. The plants were also screened with specific primers for each virus, and none of the samples revealed mixed infections. Elephant garlic is primarily utilized for industrial garlic production in several countries, and it is now being researched in Brazil for the same purpose. It can be observed from this study that elephant garlic is susceptible to two of the most common viruses in garlic (LYSV and OYDV), which must be considered in the future while developing resistant varieties or in using thermotherapy and shoot tip/meristem culture to recover virus-free cultivars. LYSV and OYDV have already been described in Brazil infecting Allium sativum (Kitajima 2020). The only complete APVA sequence available is from China (Isolate Won), but no further characterization of the virus has been performed and published. The occurrence of this virus in Brazil highlights the importance of further research to obtain a more robust virus characterization.

First report of maize rayado fino virus in corn fields in Santa Catarina, Brazil.

Maize (Zea mays L.) is the main cereal food of humans and animals in Brazil. In 2020 and 2021, a severe infestation of corn leafhoppers (Dalbulus maidis; Hemiptera: Cicadellidae) was observed in Santa Catarina State (South of Brazil). Subsequently, symptoms of chlorotic stripes limited in leaf veins started to appear in maize plants. Given the similarity of symptoms and the presence of high populations of corn leafhoppers in corn production areas, 30 plants in reproductive stage showing systemic symptoms were collected in summer and autumn from commercial fields of five municipalities in Santa Catarina: Campos Novos (27°23'18.0"S, 51°12'52.7"W), Lages (27°47'17.8"S, 50°18'16.9"W), Mafra (26°06'42"S, 49°48'25"W), Fraiburgo (27°01'36"S, 50°55'19"W), and Abelardo Luz (26°34'02"S, 52°20'02"W). The young leaves of these samples were used for molecular analyses targeting the maize rayado fino virus (MRFV; Tymoviridae: Marafivirus). Total nucleic acids were extracted using TRIzol® (Invitrogen, USA), following the manufacturer's instructions. These were used as a template for cDNA synthesis with the enzyme MMLV-RT (Promega, USA), following the manufacturer's instructions. The polymerase chain reaction (PCR) was performed using Gotaq® DNA polymerase (Promega, USA) and MRFV-09/MRFV-10 primers (Hammond et al. 1997). All PCR products were subjected to electrophoresis in 1% agarose gel and were visualized under ultraviolet light. Twenty-eight of the 30 tested plants were MRFV-positive, showing a fragment with an expected size of ~633 bp. To confirm our results, all MRFV-positive samples were sent for sequencing (GenBank accession numbers OM763708 - OM763710 and ON730784 - ON730806) and submitted to BLASTn search (https://blast.ncbi.nlm.nih.gov/Blast.cgi), resulting in identities ranging from 96.21% to 99.21% with the isolate "Brazil 26" of MRFV, which was detected in 2005 in São Paulo, Brazil (GenBank accession nº: AF186178) (Hammond and Bedendo 2005). A second set of primers was used to validate the first PCR, confirming MRFV infection (data not shown).Moreover, whitish streaks and leaf reddening were observed on the leaves of some plants; therefore, the identification for phytoplasmas (Candidatus Phytoplasma asteris) and spiroplasmas (Spiroplasma kunkelii) from the corn stunt complex was performed. For this, previously extracted nucleic acids from each sample were used as templates for a multiplex PCR using the primers CSSR6/CSSF2 and R16F2n/R16R2 (Gundersen and Lee 1996; Barros et al. 2001). Two plants were infected with only spiroplasma, 17 samples were infected with Spiroplasma and MRFV, and three samples were infected by these three pathogens. An increasing incidence of corn stunt has been observed in commercial fields in Santa Catarina in recent years. Mollicutes are commonly found and mostly studied as causal agents of corn stunt disease. On the contrary, despite being present in Brazil since the 1970s, the virus is less studied because its contribution to the corn stunt complex is still unknown (Hammond and Bedendo 2001). In this report, indications that the virus is expanding to different regions in southern Brazil were observed, which raises an opportunity for further evaluation and its consideration in monitoring programs. Moreover, to the best of our knowledge, this is the first report of MRFV in Santa Catarina, Brazil.

Establishing the normal ranges of female and male anal canal and rectal wall vascularity with color Doppler anorectal ultrasonography / Estabelecendo padrões normais da vascularização do canal anal e da parede retal em indivíduos do sexo masculino e feminino com ultrassom anorretal com Doppler colorido

ABSTRACT Study objectives: To evaluate blood supply in the anal canal, rectal wall and mesorectal fat of men and women, using color Doppler endorectal sonography to establish normal ranges for vascular parameters. Methods: A prospective cross-sectional study conducted at a tertiary-care hospital recruited asymptomatic volunteers (≤50 years). Vascularity percentage and index were calculated for defined regions. Results: Vascularity percentage and index were significantly higher in the puborectalis, mid-level external and upper internal anal sphincter compared to the low anal canal; these parameters were higher in men than in women at upper and middle levels of the inner anal canal structures. At mid-level, vascularity was greater in the external compared to the internal anal sphincter in both sexes; however, at the upper level it was greater in the puborectalis compared to the internal anal sphincter in women. Vascularity was greater in the rectal wall compared to the mesorectal fat, with no difference between middle and lower levels. Conclusions: Blood supply is highest at upper levels of the anal canal; however, inner structures are better irrigated in men. Moreover, the rectal wall is better irrigated than the mesorectal fat. Establishing normal ranges may permit future comparisons of the studied structures in disease states as well as the hormonal and age related changes.

RESUMO Objetivo: Avaliar vascularização do canal anal, parede retal e gordura mesorretal em homens e mulheres, usando ultrassonografia endorretal com Doppler colorido para estabelecer parâmetros vasculares de normalidade. Métodos: Estudo prospectivo transversal incluindo voluntários assintomáticos com até 50 anos. Medidos os valores da porcentagem e do índice de vascularização foram calculados para regiões escolhidas. Resultados: Valores da porcentagem e do índice foram significativamente maiores no puborretal, esfíncter externo (canal anal médio) e o esfíncter interno (canal anal superior) comparado ao canal anal inferior; esses parâmetros foram maiores em homens que em mulheres no canal anal médio e alto. No médio, a vascularização foi maior no esfíncter externo comparado ao interno em ambos os sexos; contudo, no canal anal superior, foi maior no puborretal comparado ao esfíncter anal interno em mulheres. A vascularização foi maior na parede retal comparada à gordura mesorretal, sem diferenças entre os níveis. Conclusões: O suprimento sanguíneo é maior nos níveis altos do canal anal. As estruturas internas são mais irrigadas nos homens. A parede retal é mais irrigada que a gordura mesorretal. Os parâmetros vasculares estabelecidos permitirá futuras comparações das estruturas estudadas nos casos de doenças, assim como, alterações que ocorrem com a idade e mudanças hormonais.

Immobilization and characterisation of a lipase from a new source, Bacillus sp. ITP-001.

A new source of lipase from Bacillus sp. ITP-001 was immobilized by physical adsorption on the polymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. The support and immobilized lipase were characterised, compared to the lyophilised lipase, with regard to the specific surface area, adsorption-desorption isotherms, pore volume (V(p)) and size (dp) by nitrogen adsorption, differential scanning calorimetry, thermogravimetric analysis, chemical composition analysis, Fourier transform infrared spectroscopy and biochemical properties. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0, whereas the optimum pH for the free enzyme was at pH 7.0; the optimum temperature of activity was 80 and 37 °C for the free and immobilized enzyme, respectively. The inactivation rate constant for the immobilized enzyme at 37 °C was 0.0038 h⁻¹ and the half-life was 182.41 h. The kinetic parameters obtained for the immobilized enzyme gave a Michaelis-Menten constant (K(m)) of 49.10 mM and a maximum reaction velocity (V(max)) of 205.03 U/g. Furthermore, the reuse of the lipase immobilized by adsorption allowed us to observe that it could be reused for 10 successive cycles, duration of each cycle (1 h), maintaining 33 % of the initial activity.