•First protocol for absolute quantification of cassava brown streak viruses.•Standard templates for specific absolute quantification of CBSVs generated.•Acceptable standard curves for specific ...absolute quantification of CBSVs prepared.•Screening efficiency for CBSD-resistance sources will be greatly improved.
Cassava brown streak disease (CBSD) is the most important virus disease of cassava and a major food security threat in Africa. Yearly economic losses of up to $100 million USD have been attributed to CBSD. The lack of information on plant-virus interactions has restricted progress in breeding for CBSD resistance. Virus quantification is becoming a major tool for the quick and reliable assessment of plant host resistance. Therefore, a protocol for specific absolute quantification of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) was developed. CBSV and UCBSV coat protein (CP) specific standard templates: CBSV (pFer2, 826bp) and UCBSV (pUF1-R1-1, 732) respectively were generated and maintained in a TA cloning vector. These were used to construct standard curves using a TaqMan qPCR assay. Standard curves with acceptable amplification efficiencies (90–105%) and coefficients of determination (R2) greater than 0.99 were obtained. Infected cassava plants were sampled from a screenhouse and the field and used to validate this assay. Results obtained by testing several screenhouse and field samples revealed consistent absolute quantification assays for different CBSV and UCBSV isolates. This study presents the first protocol for absolute quantification of CBSVs and is expected to accelerate screening for CBSD resistance and hence breeding for CBSD resistance. The use of the method presented here should improve the clarity of virus quantification data as the results obtained are not influenced by varietal, host, seasonal or environmental conditions. Screening efficiency will also be greatly improved as there is no need for the use of reference genes consequently allowing for a larger number of samples to be analyzed. This will increase experimental precision in a timely and cost effective manner.
Potato virus Y (PVY) is the most economically important potato virus, therefore extensive research is focusing on elucidation of its interaction with the host. To obtain repeatable results, strict ...standardization of research methods is crucial. Mechanical inoculation by rubbing sap from a PVY infected plant onto the leaf surface together with a fine abrasive powder is the most convenient way of experimental transmission of PVY to host plants. However, factors determining reproducibility of this process need to be determined. In the present study, it was shown that higher titre of the virus in the inoculum resulted in faster increase of PVYNTN RNA titre in the inoculated leaves, as well as in faster translocation of PVYNTN from inoculated leaves into upper non-inoculated leaves. The final titre of PVYNTN RNA in upper non-inoculated leaves was independent of the virus titre in the inoculum. In addition, the occurrence of the disease symptoms was followed and the dependence to the titre of the virus in the inoculum was observed.
N6 methylation of adenosine (m6A) was recently discovered to play a role in regulating the life cycle of various viruses by modifying viral and host RNAs. However, different studies on m6A effects on ...the same or different viruses have revealed contradictory roles for m6A in the viral life cycle. In this study, we sought to define the role of m6A on infection by rice black streaked dwarf virus (RBSDV), a double‐stranded RNA virus, of its vector small brown planthopper (SBPH). Infection by RBSDV decreased the level of m6A in midgut cells of SBPHs. We then cloned two genes (LsMETTL3 and LsMETTL14) that encode m6A RNA methyltransferase in SBPHs. After interference with expression of the two genes, the titre of RBSDV in the midgut cells of SBPHs increased significantly, suggesting that m6A levels were negatively correlated with virus replication. More importantly, our results revealed that m6A modification might be the epigenetic mechanism that regulates RBSDV replication in its insect vector and maintains a certain virus threshold required for persistent transmission.
N6 methylation of adenosine modification might be the epigenetic mechanism that regulates rice black streaked dwarf virus replication in its insect vector and maintains a certain virus threshold for persistent transmission.
Growth kinetics of a Vero cells adapted Bangladeshi strain of
peste des petits ruminants
virus was studied in Vero cells to determine maximum virus yield. One-step growth curve was formulated after ...determining virus in both supernatant (CFV) and cell lysate (CAV) at different time categories by microtitre plate titration in Vero cells and the viral presence was confirmed by real-time RT-PCR. The virus was first detected in both the supernatants and cell pellets at 12 hpi. The virus titre reached its plateau at 72 hpi. Maximum virus titre of CAV was 6.2 log
10
TCID
50
/ml and that of CFV was 5.2 log
10
TCID
50
/ml at 72 hpi. After that, the titer gradually declined, but maintained at 4.5 log
10
TCID
50
/ml in case of CAV and 4.2 log
10
TCID
50
/ml in case of CFV at 96 hpi. It was concluded that the optimum time point for harvesting Vero cell culture is 72 hpi.
The begomovirus Tomato severe rugose virus (ToSRV) and the crinivirus Tomato chlorosis virus (ToCV), in single and co‐infections, are very common in tomato crops in Brazil. Both viruses are ...transmitted by the whitefly Bemisia tabaciMEAM1 (biotype B). The objective of this study was to analyse the interaction between ToSRV and ToCV in tomato plants of cultivars Santa Clara and Kada. Plants at 15, 30 and 45 days after emergence were inoculated with 30 viruliferous B. tabaci per plant. The following treatments were compared: plants inoculated with ToSRV, ToCV, ToSRV + ToCV, and healthy (control). The interaction between these viruses was analysed by measuring the virus titre by qPCR and the fresh and dry weights of the aerial parts of the tomato plants. Based on two independent assays, no significant effects for co‐infection of ToSRV and ToCV on virus titres and plant development were observed compared to single infections. The dry weight of tomato plants of both cultivars infected with ToSRV, ToCV, or co‐infected did not differ significantly. However, the dry weight of Santa Clara tomato plants infected with ToSRV, ToCV and ToSRV + ToCV showed mean reductions of 21.5%, 25.5% and 32%, respectively, compared to healthy plants, and mean reductions for Kada were 31.7%, 37.5% and 38%, respectively.
Potato virus Y (PVY) is the most economically important potato virus, therefore extensive research is focusing on elucidation of its interaction with the host. To obtain repeatable results, strict ...standardization of research methods is crucial. Mechanical inoculation by rubbing sap from a PVY infected plant onto the leaf surface together with a fine abrasive powder is the most convenient way of experimental transmission of PVY to host plants. However, factors determining reproducibility of this process need to be determined. In the present study, it was shown that higher titre of the virus in the inoculum resulted in faster increase of PVYNTN RNA titre in the inoculated leaves, as well as in faster translocation of PVYNTN from inoculated leaves into upper non-inoculated leaves. The final titre of PVYNTN RNA in upper non-inoculated leaves was independent of the virus titre in the inoculum. In addition, the occurrence of the disease symptoms was followed and the dependence to the titre of the virus in the inoculum was observed.
Cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), ranks among the top seven biological threats to global food security. ...The disease poses a significant threat to cassava production in East and Central Africa (ECA). In Uganda, overall CBSD incidence increased by c. 20% since it re‐emerged in 2004, and the disease persistently reduces cassava yields and storage root qualities. The spread of CBSD has been studied spatially in fields in different agroecologies. However, within‐host distribution and accumulation of CBSV and UCBSV in naturally infected cassava plants is unknown. Therefore, within‐host CBSV and UCBSV distribution was studied to correlate CBSD symptoms with virus titre in organs of infected cassava. Leaf, stem and storage root samples, with and without symptoms, were collected from 10 genotypes of field‐grown cassava. Presence of CBSV and UCBSV was detected by RT‐PCR and virus levels determined by qRT‐PCR. CBSV was present in 100% of CBSD samples with symptoms, with 45·3% positive for presence of both CBSV and UCBSV. Tolerant cassava genotypes were infected with CBSV alone and accumulated higher titre in roots than in aerial organs. Susceptible genotypes were co‐infected with CBSV and UCBSV and exhibited variation in virus titre in each organ. Across genotypes, virus titre was lowest in the youngest leaves and highest in mature non‐senescing leaves. This information provides insight into the relationship between CBSV, UCBSV and their cassava host, and is valuable for CBSD resistance breeding, epidemiology studies and CBSD control.
In sugarcane (
Saccharum
spp. hybrids) cultivation, viral diseases pose a great challenge across the globe. Yellow leaf (YL) disease is one of the important viral diseases caused by
Sugarcane yellow ...leaf virus
(ScYLV), a positive-sense ssRNA virus, genus
Polerovirus
, family
Solemoviridae.
The disease symptoms appear in later stages of crop growth during grand growth to maturity phase with intense midrib yellowing in the abaxial leaf surface. At present, this disease is managed through tissue (meristem) culture and healthy seed nurseries in India. However, the virus-free plants are infected quickly by secondary inoculum from aphid vectors in the field, which necessitates the importance of developing YL-resistant varieties. We screened about 600–625 sugarcane parental clones to identify true YL resistance based on 0–5 disease rating scale since 2015 and categorised them as resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible. Leaf samples were collected from all these categories of plants during 2018–20 for the viral titre estimation through absolute quantification method (qRT-PCR assay). The viral load was invariably high in all categories of susceptible samples that ranged from 4.40 × 10
2
to 8.429 × 10
6
, whereas in YL-free asymptomatic clones, the viral load ranged from 82.35 ± 5.90 to 5.121 × 10
4
. The results clearly indicated that highest viral titre of 10
5
–10
7
copies was present in all the susceptible clones irrespective of their disease severity grades. Our results clearly established that about 22.85% of apparently resistant sugarcane clones remained free from YL symptoms with significantly low ScYLV titre although we could not find a significant correlation between virus titre and symptom expression. The identified resistant parents will serve as sources of YL resistance to develop virus resistant sugarcane varieties.
Turnip yellows virus (TuYV), is one of the most important pathogens of oilseed rape, which has caused enormous yield losses in all growing regions of the world in recent years. Therefore, there is a ...need for resistant varieties for sustainable crop protection. We have investigated the resistance of known varieties and newly developed advanced-breeding lines of oilseed rape to TuYV in greenhouse and field trials. We have analysed the TuYV titre of individual genotypes inoculated with the virus using viruliferous aphids
. The genotypes 'DK Temptation' and 'Rescator' had the lowest and highest virus titres, respectively, and were used as resistant and susceptible models for comparative analyses with other genotypes. In the greenhouse, the best results were obtained with the genotypes 'OP-8143 DH' (2.94 × 10
copies), OP-BN-72 (3.29 × 10
copies), 'Navajo' (3.58 × 10
copies) and 'SG-C 21215' (4.09 × 10
copies), which reached virus titres about 2 times higher than the minimum virus concentration measured in 'DK Temptation' (1.80 × 10
copies). In the field trials, the genotypes 'Navajo' (3.39 × 10
copies), 'OP-8148 DH' (4.44 × 10
copies), 'SG-C 21215' (6.80 × 10
copies) and OP-8480 (7.19 × 10
copies) had the lowest virus titres and reached about 3 times the virus titre of DK Temptation (2.54 × 10
copies). Both trials showed that at least two commercial varieties (e.g., DK Temptation, Navajo) and three advanced breeding lines (e.g., OP-8143 DH, OP-BN-72, SG-C 21215) had low titres of the virus after TuYV infection. This indicates a high level of resistance to TuYV in 'Navajo' or the newly developed breeding lines and the basis of resistance is probably different from R54 (as in 'DK Temptation'). Furthermore, the greenhouse trials together with RT -qPCR-based virus titre analysis could be a cost-effective and efficient method to assess the level of resistance of a given genotype to TuYV infection compared to the field trials. However, further research is needed to identify the underlying mechanisms causing this difference in susceptibility.
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