SEGS-1 a cassava genomic sequence increases the severity of African cassava mosaic virus infection in Arabidopsis thaliana.

Cassava is a major crop in Sub-Saharan Africa, where it is grown primarily by smallholder farmers. Cassava production is constrained by Cassava mosaic disease (CMD), which is caused by a complex of cassava mosaic begomoviruses (CMBs). A previous study showed that SEGS-1 (sequences enhancing geminivirus symptoms), which occurs in the cassava genome and as episomes during viral infection, enhances CMD symptoms and breaks resistance in cassava. We report here that SEGS-1 also increases viral disease severity in Arabidopsis thaliana plants that are co-inoculated with African cassava mosaic virus (ACMV) and SEGS-1 sequences. Viral disease was also enhanced in Arabidopsis plants carrying a SEGS-1 transgene when inoculated with ACMV alone. Unlike cassava, no SEGS-1 episomal DNA was detected in the transgenic Arabidopsis plants during ACMV infection. Studies using Nicotiana tabacum suspension cells showed that co-transfection of SEGS-1 sequences with an ACMV replicon increases viral DNA accumulation in the absence of viral movement. Together, these results demonstrated that SEGS-1 can function in a heterologous host to increase disease severity. Moreover, SEGS-1 is active in a host genomic context, indicating that SEGS-1 episomes are not required for disease enhancement.

Begomovirus Tomato Leaf Curl New Delhi Virus Is Seedborne but Not Seed Transmitted in Melon.

Seed transmission can be of considerable relevance to the dissemination of plant viruses in nature and for their prevalence and perpetuation. Long-distance spread of isolates of the begomovirus species Tomato leaf curl New Delhi virus (genus Begomovirus, family Geminiviridae) has recently occurred from Asia to the Middle East and the Mediterranean Basin. Here, we investigated the possible transmission by melon (Cucumis melo L.) seeds of a tomato leaf curl New Delhi virus (ToLCNDV) isolate of the "Spain" strain widely distributed in the Mediterranean area as an alternative mechanism for long-distance spread. PCR amplification detection of ToLCNDV in floral parts and mature seeds of melon plants reveals that this virus is seedborne. "Seedborne" is defined as the ability of a virus to be carried through seeds, which does not necessarily lead to transmission to the next generation. Treatment with a chemical disinfectant significantly reduced the detectable virus associated with melon seeds, suggesting ToLCNDV contamination of the external portion of the seed coat. Also, when the internal fraction of the mature seed (seed cotyledons + embryo) was analyzed by quantitative PCR amplification, ToLCNDV was detectable at low levels, suggesting the potential for viral contamination or infection of the internal portions of seed. However, grow-out studies conducted with melon progeny plants germinated from mature seeds collected from ToLCNDV-infected plants and evaluated at early (1-leaf) or at late (20-leaf) growth stages did not support the transmission of ToLCNDV from seeds to offspring.

Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of 'Candidatus Liberibacter Solanacearum' by qPCR.

'Candidatus Liberibacter solanacearum' (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in Solanaceae and Apiaceae crops. CaLsol detection in psyllids often requires insect destruction, preventing a subsequent morphological identification. In this work, we have assessed the influence on the detection of CaLsol by PCR in Bactericera trigonica (Hemiptera: Psyllidae), of four specimen preparations (entire body, ground, cut-off head, and punctured abdomen) and seven DNA extraction methods (PBS suspension, squashing on membrane, CTAB, Chelex, TRIsureTM, HotSHOT, and DNeasy®). DNA yield and purity ratios, time consumption, cost, and residues generated were also evaluated. Optimum results were obtained through grinding, but it is suggested that destructive procedures are not essential in order to detect CaLsol. Although CaLsol was detected by qPCR with DNA obtained by the different procedures, HotSHOT was the most sensitive method. In terms of time consumption and cost, squashed on membrane, HotSHOT, and PBS were the fastest, while HotSHOT and PBS were the cheapest. In summary, HotSHOT was accurate, fast, simple, and sufficiently sensitive to detect this bacterium within the vector. Additionally, cross-contamination with CaLsol was assessed in the ethanol solutions where B. trigonica specimens were usually collected and preserved. CaLsol-free psyllids were CaLsol-positive after incubation with CaLsol-positive specimens. This work provides a valuable guide when choosing a method to detect CaLsol in vectors according to the purpose of the study.

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses.

Cassava mosaic disease (CMD), which is caused by single-stranded DNA begomoviruses, severely limits cassava production across Africa. A previous study showed that CMD symptom severity and viral DNA accumulation increase in cassava in the presence of a DNA sequence designated SEGS-2 (sequence enhancing geminivirus symptoms). We report here that when SEGS-2 is coinoculated with African cassava mosaic virus (ACMV) onto Arabidopsis thaliana, viral symptoms increase. Transgenic Arabidopsis with an integrated copy of SEGS-2 inoculated with ACMV also display increased symptom severity and viral DNA levels. Moreover, SEGS-2 enables Cabbage leaf curl virus (CaLCuV) to infect a geminivirus-resistant Arabidopsis thaliana accession. Although SEGS-2 is related to cassava genomic sequences, an earlier study showed that it occurs as episomes and is packaged into virions in CMD-infected cassava and viruliferous whiteflies. We identified SEGS-2 episomes in SEGS-2 transgenic Arabidopsis. The episomes occur as both double-stranded and single-stranded DNA, with the single-stranded form packaged into virions. In addition, SEGS-2 episomes replicate in tobacco protoplasts in the presence, but not the absence, of ACMV DNA-A. SEGS-2 episomes contain a SEGS-2 derived promoter and an open reading frame with the potential to encode a 75-amino acid protein. An ATG mutation at the beginning of the SEGS-2 coding region does not enhance ACMV infection in A. thaliana. Together, the results established that SEGS-2 is a new type of begomovirus satellite that enhances viral disease through the action of an SEGS-2-encoded protein that may also be encoded by the cassava genome. IMPORTANCE Cassava is an important root crop in the developing world and a food and income crop for more than 300 million African farmers. Cassava is rising in global importance and trade as the demands for biofuels and commercial starch increase. More than half of the world's cassava is produced in Africa, where it is primarily grown by smallholder farmers, many of whom are from the poorest villages. Although cassava can grow under high temperature, drought, and poor soil conditions, its production is severely limited by viral diseases. Cassava mosaic disease (CMD) is one of the most important viral diseases of cassava and can cause up to 100% yield losses. We provide evidence that SEGS-2, which was originally isolated from cassava crops displaying severe and atypical CMD symptoms in Tanzanian fields, is a novel begomovirus satellite that can compromise the development of durable CMD resistance.

Revisiting Seed Transmission of the Type Strain of Tomato yellow leaf curl virus in Tomato Plants.

Isolates of the Tomato yellow leaf curl virus (TYLCV) species (genus Begomovirus, family Geminiviridae) infect tomato crops worldwide, causing severe economic damage. Members of the whitefly Bemisia tabaci sibling species group are the vector of begomoviruses, including TYLCV. However, transmission of isolates of the type strain (Israel [IL]) of TYLCV (TYLCV-IL) by tomato seed has recently been reported based on infections occurring in Korea. Because of the consequences of this finding on the epidemiology and control of the disease caused by TYLCV and on the seed market, it was considered essential to revisit and expand those results to other tomato-growing areas. TYLCV DNA content was detected in tomato and Nicotiana benthamiana seed collected from plants naturally or experimentally infected with TYLCV-IL, supporting its seedborne nature. The TYLCV-IL replication detected in tomato and N. benthamiana flower reproductive organs demonstrated close association of this virus with the seed during maturation. However, the significant reduction of TYLCV DNA load after surface disinfections of tomato seed suggests that most of the virus is located externally, as contaminant of the seed coat. Transmission assays, carried out with seven tomato genotypes and more than 3,000 tomato plants, revealed no evidence of seed transmission from "surface-disinfected" or untreated seed for two Mediterranean isolates of TYLCV-IL. Similar results were also obtained for seed collected from TYLCV-IL-infected N. benthamiana plants. The results support the conclusion that TYLCV-IL is seedborne but is not seed transmitted in tomato or N. benthamiana, suggesting that transmission through seed is not a general property of TYLCV.

Two Novel DNAs That Enhance Symptoms and Overcome CMD2 Resistance to Cassava Mosaic Disease.

UNLABELLED: Cassava mosaic begomoviruses (CMBs) cause cassava mosaic disease (CMD) across Africa and the Indian subcontinent. Like all members of the geminivirus family, CMBs have small, circular single-stranded DNA genomes. We report here the discovery of two novel DNA sequences, designated SEGS-1 and SEGS-2 (forsequencesenhancinggeminivirussymptoms), that enhance symptoms and break resistance to CMD. The SEGS are characterized by GC-rich regions and the absence of long open reading frames. Both SEGS enhanced CMD symptoms in cassava (Manihot esculentaCrantz) when coinoculated withAfrican cassava mosaic virus(ACMV),East African cassava mosaic Cameroon virus(EACMCV), orEast African cassava mosaic virus-Uganda(EACMV-UG). SEGS-1 also overcame resistance of a cassava landrace carrying the CMD2 resistance locus when coinoculated with EACMV-UG. Episomal forms of both SEGS were detected in CMB-infected cassava but not in healthy cassava. SEGS-2 episomes were also found in virions and whiteflies. SEGS-1 has no homology to geminiviruses or their associated satellites, but the cassava genome contains a sequence that is 99% identical to full-length SEGS-1. The cassava genome also includes three sequences with 84 to 89% identity to SEGS-2 that together encompass all of SEGS-2 except for a 52-bp region, which includes the episomal junction and a 26-bp sequence related to alphasatellite replication origins. These results suggest that SEGS-1 is derived from the cassava genome and facilitates CMB infection as an integrated copy and/or an episome, while SEGS-2 was originally from the cassava genome but now is encapsidated into virions and transmitted as an episome by whiteflies. IMPORTANCE: Cassava is a major crop in the developing world, with its production in Africa being second only to maize. CMD is one of the most important diseases of cassava and a serious constraint to production across Africa. CMD2 is a major CMD resistance locus that has been deployed in many cassava cultivars through large-scale breeding programs. In recent years, severe, atypical CMD symptoms have been observed occasionally on resistant cultivars, some of which carry the CMD2 locus, in African fields. In this report, we identified and characterized two DNA sequences, SEGS-1 and SEGS-2, which produce similar symptoms when coinoculated with cassava mosaic begomoviruses onto a susceptible cultivar or a CMD2-resistant landrace. The ability of SEGS-1 to overcome CMD2 resistance and the transmission of SEGS-2 by whiteflies has major implications for the long-term durability of CMD2 resistance and underscore the need for alternative sources of resistance in cassava.

Antibacterial properties of phenolic triterpenoids against Staphylococcus epidermidis.

Two phenolic triterpenoids, pristimerol (30 µg/mL) and 8- EPI-6-deoxoblepharodol (20 µg/mL), obtained by catalytic reduction of pristimerin, exhibited bacteriostatic action against Staphylococcus epidermidis. This activity was not dependent on the inoculum size and the growth phase although it showed a stronger effect when cells were growing actively. Addition of phenolic triterpenoids to S. epidermidis cultures in the log-phase of growth led to an inhibitory effect on incorporation and uptake of radiolabeled precursors thymidine, uridine, leucine, and N-acetyl-glucosamine after 30 min of treatment. Furthermore, a clear release of UV-absorbing material and leakage of intracellular potassium were also detected. These findings, coupled with the high lipophilicity of these molecules, shown by high ClogP values, suggest that 8-EPI and pristimerol are able to interact within the lipid bilayer and as a consequence cause functional alterations on the cytoplasmic membrane of S. epidermidis cells.

Antimicrobial activity of 6-oxophenolic triterpenoids. Mode of action against Bacillus subtilis.

Zeylasteral and demethylzeylasteral are 6-oxophenolic triterpenoids isolated from the root of Maytenus blepharodes, which have antimicrobial activity against Gram-positive bacteria and the yeast Candida albicans. The time-kill curves for zeylasteral and demethylzeylasteral at concentrations twice their MICs, against Bacillus subtilis showed that the colony forming units were reduced in 3-log10 and > 4-log10 respectively. This reduction was dependent on inoculum size and the growth phase of cells, and was greater when the compounds were incorporated in the exponential phase, indicating a bacteriolytic effect. Treatment with both agents, particularly with zeylasteral (20 microg/mL) caused a reduction of optical density at 550 nm. With regard to the synthesis of DNA, RNA, protein and cell wall, the compounds exhibited the fastest inhibition against cell wall synthesis. Thus, the predisposition to lysis, the morphological changes seen by microscopy, and the complete inhibition in the incorporation the N-acetyl-d-[1 - 14C]glucosamine, suggest that the phenolic compounds compromise the cell wall synthesis and/or cytoplasmic membrane.