Genome-wide association study of cassava brown streak disease resistance in cassava germplasm conserved in South America.

Cassava (Manihot esculenta Crantz) is a vital carbohydrate source for over 800 million people globally, yet its production in East Africa is severely affected by cassava brown streak disease (CBSD). Genebanks, through ex-situ conservation, play a pivotal role in preserving crop diversity, providing crucial resources for breeding resilient and disease-resistant crops. This study genotyped 234 South American cassava accessions conserved at the CIAT genebank, previously phenotyped for CBSD resistance by an independent group, to perform a genome-wide association analysis (GWAS) to identify genetic variants associated with CBSD resistance. Our GWAS identified 35 single nucleotide polymorphism (SNP) markers distributed across various chromosomes, associated with disease severity or the presence/absence of viral infection. Markers were annotated within or near genes previously identified with functions related to pathogen recognition and immune response activation. Using the SNP candidates, we screened the world's largest cassava collection for accessions with a higher frequency of favorable genotypes, proposing 35 accessions with potential resistance to CBSD. Our results provide insights into the genetics of CBSD resistance and highlight the importance of genetic resources to equip breeders with the raw materials needed to develop new crop varieties resistant to pests and diseases.

Cleaning cassava genotypes infected with cassava frogskin disease via in vitro shoot tip culture.

This study aimed to develop a methodology for eliminating cassava frogskin disease (CFSD) from in vitro shoot tip culture by associating thermotherapy and tetracycline. Cuttings from different accessions (BGM0232, BGM0315, BGM0464, BGM584, BGM0841, and BGM1342), infected with CFSD according to visual inspection of the disease symptoms, were used for cleaning. To verify the absence of other diseases, the plants were indexed for Cassava common mosaic virus - CsCMV (by ELISA) and Cassava vein mosaic virus - CsVMV (by polymerase chain reaction, PCR), proving that the accessions were free of these viruses, except for BGM0315 and BGM0464, which were infected with CsVMV. Subsequently, the cuttings were submitted to different tetracycline concentrations for 3 min, and then subjected to thermotherapy under different temperatures (35°, 38°, 40°, 45°, and 55°C). Shoots of 2 cm were harvested, and their surfaces were sterilized in a laminar flow chamber. Subsequently, the shoot tips of different sizes were removed (0.2, 0.4, 0.5, and 1.0 mm) for inoculation in a culture medium with tetracycline at the same concentrations in which the cuttings were dipped. After 60 days of cultivation, the explants were transferred to a multiplication medium without antibiotics. Thirty days after the transfer, the viability of the regenerated plants was evaluated, which were then acclimatized for 70 days in a greenhouse and transferred to the field. After 7 months, a visual analysis of the symptomatic roots and a PCR analysis were held to prove the elimination of CFSD and CsVMV from the accessions infected with these viruses (BGM0315 and BGM0464), respectively. Most of the treatments resulted in 100% cleaning of CFSD-infected plants. From accessions that were also infected with CsVMV, only 2% of the plants remained infected, also demonstrating the cleaning efficiency of this protocol for this disease.

Resolution of cassava-infecting alphaflexiviruses: Molecular and biological characterization of a novel group of potexviruses lacking the TGB3 gene.

Several potexviruses (Family Alphaflexiviridae) have been reported infecting cassava (Manihot esculenta Crantz) in the Americas. They were isolated from severely diseased plants during the last 30-40 years and include: Cassava common mosaic virus (CsCMV), Cassava Caribbean mosaic virus (CsCaMV), Cassava Colombian symptomless virus (CsCSV) and Cassava virus X (CsVX). However, their definitive classification as distinct species remains unresolved for several reasons, including the lack of sequence data and unavailability of samples from original isolates. This complicates disease diagnostics, cassava germplasm exchange certification, evaluation of virus cleaning protocols and epidemiological studies. Furthermore, a recently detected novel alphaflexivirus, indicates that cassava-infecting potexviruses may be more diverse. To solve the identity of these viruses, we started indexing samples from different parts of Colombia using different sets of PCR primers, antisera available and inoculation to indicator plants. Results show that there are three major phylogenetic groups of potexviruses infecting cassava, and they correspond to CsCMV, CsVX and the newly identified Cassava new alphaflexivirus (CsNAV). Bioassays and sequence analysis established that isolates of CsNAV and CsVX cause latent infections in different cassava landraces, they are not efficiently transmitted to the indicator plant Nicotiana benthamiana and they lack the gene 3 of the conserved potexviral 'triple gene block' (TGB). In contrast, all isolates of CsCMV (which have a characteristic potexvirus genome arrangement) caused Cassava Common Mosaic Disease (CCMD) in single infections and were efficiently transmitted to N. benthamiana. Although phylogenetic analysis of the replicase sequence placed CsNAV and CsVX as members of the Potexvirus genus, their distinct genome arrangement and biological characteristics suggest they can be considered as members of a separate taxonomic group.

Crop losses in Brazilian cassava varieties induced by the Cassava common mosaic virus

Despite the widespread distribution of the Cassava common mosaic virus (CsCMV) in Brazil, little is known about the losses it causes in yield. The effect of CsCMV on different varieties was evaluated by reference to several agronomic traits. Four field trials were established in 2012/2013 and 2013/2014 using six varieties of cassava. Following mechanical inoculation with CsCMV, the presence of the virus was confirmed using the ELISA assay. The evaluated traits were plant height (PH), dry matter content (DMC), harvest index (HI), aerial part yield (APY), root yield (RoY), and starch yield (StY) in both inoculated and non-inoculated plants. Overall, the presence of the virus contributed little to the reduction in PH, HI, and DMC across the varieties, with PH being significantly reduced by 9.2 and 7.0 % in the BGM0212 and BRS Kiriris varieties, respectively. In contrast, APY, RoY, and StY were reduced by 30.2, 29.3, and 30.0 %, in the virus-infected plants respectively. While the BRS Kiriris and BRS Jari varieties suffered the highest reductions overall and were considered highly susceptible to CsCMV, none of the traits suffered reductions in the inoculated BRS Formosa plants. Although RoY and StY were reduced in inoculated plants of BRS Tapioqueira, crop yield for this variety was the highest. Thus, BRS Formosa and BRS Tapioqueira exhibited tolerance against CsCMV, which warrants further investigation.(AU)

Crop losses in Brazilian cassava varieties induced by the Cassava common mosaic virus

Despite the widespread distribution of the Cassava common mosaic virus (CsCMV) in Brazil, little is known about the losses it causes in yield. The effect of CsCMV on different varieties was evaluated by reference to several agronomic traits. Four field trials were established in 2012/2013 and 2013/2014 using six varieties of cassava. Following mechanical inoculation with CsCMV, the presence of the virus was confirmed using the ELISA assay. The evaluated traits were plant height (PH), dry matter content (DMC), harvest index (HI), aerial part yield (APY), root yield (RoY), and starch yield (StY) in both inoculated and non-inoculated plants. Overall, the presence of the virus contributed little to the reduction in PH, HI, and DMC across the varieties, with PH being significantly reduced by 9.2 and 7.0 % in the BGM0212 and BRS Kiriris varieties, respectively. In contrast, APY, RoY, and StY were reduced by 30.2, 29.3, and 30.0 %, in the virus-infected plants respectively. While the BRS Kiriris and BRS Jari varieties suffered the highest reductions overall and were considered highly susceptible to CsCMV, none of the traits suffered reductions in the inoculated BRS Formosa plants. Although RoY and StY were reduced in inoculated plants of BRS Tapioqueira, crop yield for this variety was the highest. Thus, BRS Formosa and BRS Tapioqueira exhibited tolerance against CsCMV, which warrants further investigation.

Molecular-assisted selection for resistance to cassava mosaic disease in Manihot esculenta Crantz

The geminivirus complex known as cassava mosaic disease (CMD) is one of the most devastating viruses for cassava (Manihot esculenta Crantz). The aim of this study was to use molecular-assisted selection (MAS) to identify CMD-resistant accessions and ascertain promising crosses with elite Brazilian varieties. One thousand two hundred twenty-four accessions were genotyped using five molecular markers (NS169, NS158, SSRY028, SSRY040 and RME1) that were associated with resistance to CMD, along with 402 SNPs (single-nucleotide polymorphism). The promising crosses were identified using a discriminant analysis of main component (DAPC), and the matrix of genomic relationship was estimated with SNP markers. The CMD1 gene, previously described in M. glaziovii, was not found in M. esculenta. In contrast, the CMD2 gene was found in 5, 4 and 5 % of cassava accessions, with flanking markers NS169+RME1, NS158+RME1 and SSRY28+RME1, respectively. Only seven accessions presented all markers linked to the CMD resistance. The DAPC of the seven accessions along with 17 elite cassava varieties led to the formation of three divergent clusters. Potential sources of resistance to CMD were divided into two groups, while the elite varieties were distributed into three groups. The low estimates of the genomic relationship (ranging from -0.167 to 0.681 with an average of 0.076) contributed to the success in identifying contrasting genotypes. The use of MAS in countries where CMD is a quarantine disease constitutes a successful strategy not only for identifying the resistant accessions but also for determining the promising crosses.(AU)

Molecular-assisted selection for resistance to cassava mosaic disease in Manihot esculenta Crantz

The geminivirus complex known as cassava mosaic disease (CMD) is one of the most devastating viruses for cassava (Manihot esculenta Crantz). The aim of this study was to use molecular-assisted selection (MAS) to identify CMD-resistant accessions and ascertain promising crosses with elite Brazilian varieties. One thousand two hundred twenty-four accessions were genotyped using five molecular markers (NS169, NS158, SSRY028, SSRY040 and RME1) that were associated with resistance to CMD, along with 402 SNPs (single-nucleotide polymorphism). The promising crosses were identified using a discriminant analysis of main component (DAPC), and the matrix of genomic relationship was estimated with SNP markers. The CMD1 gene, previously described in M. glaziovii, was not found in M. esculenta. In contrast, the CMD2 gene was found in 5, 4 and 5 % of cassava accessions, with flanking markers NS169+RME1, NS158+RME1 and SSRY28+RME1, respectively. Only seven accessions presented all markers linked to the CMD resistance. The DAPC of the seven accessions along with 17 elite cassava varieties led to the formation of three divergent clusters. Potential sources of resistance to CMD were divided into two groups, while the elite varieties were distributed into three groups. The low estimates of the genomic relationship (ranging from -0.167 to 0.681 with an average of 0.076) contributed to the success in identifying contrasting genotypes. The use of MAS in countries where CMD is a quarantine disease constitutes a successful strategy not only for identifying the resistant accessions but also for determining the promising crosses.

Unraveling complex viral infections in cassava (Manihot esculenta Crantz) from Colombia.

In the Americas, different disease symptoms have been reported in cassava including leaf mosaics, vein clearings, mottles, ring spots, leaf distortions and undeveloped and deformed storage roots. Some viruses have been identified and associated with these symptoms while others have been reported in symptomless plants or latent infections. We observed that reoviruses associated with severe root symptoms (RS) of Cassava Frogskin Disease (CFSD) are not associated with leaf symptoms (LS) observed in the cassava indicator plant 'Secundina'. Neither were these LS associated with the previously characterized Cassava common mosaic virus, Cassava virus X, Cassava vein mosaic virus or phytoplasma, suggesting the presence of additional pathogens. In order to explain LS observed in cassava we used a combination of biological, serological and molecular tests. Here, we report three newly described viruses belonging to the families Secoviridae, Alphaflexiviridae and Luteoviridae found in cassava plants showing severe RS associated with CFSD. All tested plants were infected by a mix of viruses that induced distinct LS in 'Secundina'. Out of the three newly described viruses, a member of family Secoviridae could experimentally induce LS in single infection. Our results confirm the common occurrence of complex viral infections in cassava field-collected since the 1980s.

Tropical Whitefly IPM Project.

The Tropical Whitefly IPM Project (TWFP) is an initiative of the Systemwide IPM Programme of the Consultative Group on International Agricultural Research (CGIAR), financed by the Department for International Development (DFID) of the United Kingdom, the Danish International Development Agency (DANIDA), the United States Department of Agriculture (USDA) and Agency for International Development (USAID), the Australian Centre for International Agricultural Research (ACIAR), and the New Zealand Agency for International Development (NZAID), to manage whitefly pests and whitefly-transmitted viruses in the Tropics. Participating CGIAR and other international centers include the Centre for International Tropical Agriculture (CIAT); the International Institute of Tropical Agriculture (IITA); The International Potato Centre (CIP); the Asian Vegetable Research and Development Centre (AVRDC); and the International Centre of Insect Physiology and Ecology (ICIPE), in close collaboration with the National Resources Institute (NRI-UK); national agricultural research institutions; agricultural universities; and advanced agricultural research laboratories in Africa, Asia, Europe, the Pacific Region, and the Americas. The TWFP was launched in 1996 as five separate but closely linked subprojects targeting: (1) Bemisia tabaci as a vector of viruses affecting cassava and sweet potato in sub-Saharan Africa (IITA, NRI, CIP, CIAT); (2) B. tabaci as a vector of viruses in mixed cropping systems of Mexico, Central America, and the Caribbean (CIAT); (3) B. tabaci as a vector of viruses in mixed cropping systems of eastern and southern Africa (ICIPE, AVRDC); (4) B. tabaci as a vector of viruses in mixed cropping systems of Southeast Asia (AVRDC); (5) Trialeurodes vaporariorum as a pest in mixed cropping systems of the Andean highlands (CIAT); and (6) whiteflies as pests of cassava in South America (CIAT). Diagnostic surveys conducted in Phase I (1997-2000) clearly showed that the two main whitefly pests in the Tropics are B. tabaci and, in the highlands, T. vaporariorum. Other whitefly species investigated by the TWFP included B. afer and Aleurotrachelus socialis. B. tabaci is the main vector of plant viruses inducing African cassava mosaic disease, sweet potato virus disease, bean golden mosaic disease, and many other diseases of horticultural crops, mainly tomato, hot and sweet peppers, squash, melon, and several other cucurbits. Genetic resistance was identified as the most important component of an IPM programme, followed by phytosanitary, cultural and legal measures. The two most important factors contributing to whitefly/virus epidemics were shown to be pesticide abuse and the use of virus-infected planting materials. Biological control is only effective in cropping systems with minimal or rational use of insecticides, and should be considered only as a complementary IPM strategy. Farmer education and technical assistance are considered the most critical steps toward the implementation of sustainable and economically viable IPM strategies in tropical countries affected by whitefly pests and whitefly-transmitted viruses.

Serial analysis of gene expression (SAGE) of host-plant resistance to the cassava mosaic disease (CMD).

Cassava mosaic disease (CMD) is a viral disease of the important tropical staple crop cassava (Manihot esculenta) and preferred management involves use of host-plant resistance. The best available resistance is controlled by a single dominant gene. Serial analysis of gene expression (SAGE) was used to analyze the gene expression pattern in a bulk of 40 each of CMD resistant and susceptible genotypes drawn from a gene mapping progeny. Messenger RNA used for the SAGE analysis came from plants that were exposed to heavy disease pressure over a period of 2 years in the field. A total of 12,786 tags were studied, divided into 5733 and 7053 tags from the resistant and susceptible genotypes, respectively. Tag annotation was by PCR amplification using the tag sequence as sense primer and 4000 cassava ESTs generated from the bulk of CMD resistant genotypes. Annotation of more than 30 differentially expressed tags revealed several genes expressed during systemic acquired resistance (SAR) in plants and other genes involved in cell-to-cell and cytoplasm-to-nucleus virus trafficking. Differential expression of the most abundantly expressed tag, corresponding to a beta-tubulin gene, was confirmed by Northern Analysis. RFLP analysis of the tags in the parents and bulks of the CMD mapping progeny revealed only one tag, a WRKY transcription factor, associated with the region bearing the dominant CMD gene.

Characterization of cassava common mosaic virus and a defective RNA species.

The genome of cassava common mosaic potexvirus (CsCMV) has been sequenced from cDNA clones and consists of 6376 nucleotides (nt). A 76 nt untranslated region (UTR) at the 5' terminus was followed by ORF1 which potentially encodes a protein of 1449 amino acids (aa). ORFs 2, 3, and 4 were predicted to encode proteins of 231, 112 and 97 aa, respectively. ORF5 potentially encodes a 229 aa protein of 25 kDa that is similar to the coat proteins of other potexviruses. The 3'-terminal UTR of 114 nt was followed by a poly(A) tail. The genomic organization of the CsCMV genome is similar to that of other potexviruses. A cDNA clone that was apparently obtained from a defective RNA species contained both the 5' and 3' UTRs and an ORF that potentially encodes the first 263 aa of ORF1 and the last 33 aa of the coat protein. Defective RNA species were found both in purified preparations of the virus and in total nucleic acid isolated from CsCMV-infected plants.

Characterization of cassava vein mosaic virus: a distinct plant pararetrovirus.

Cassava vein mosaic virus (CVMV) was found to be widespread throughout the north-eastern region of Brazil. The complete sequence of CVMV was determined, and the genome was 8158 bp in size. A cytosolic initiator methionine tRNA (tRNA met1)-binding site that probably acts as a primer for minus-strand synthesis was present. The genome contained five open reading frames that potentially encode proteins with predicted molecular masses of 186 kDa, 9 kDa, 77 kDa, 24 kDa and 26 kDa. The putative 186 kDa protein had regions with similarity to the zinc finger-like RNA-binding domain that is a common element in the capsid proteins and similarity to the intercellular transport domain of the plant pararetroviruses. The predicted 77 kDa protein had regions with similarity to aspartic proteases, reverse transcriptase and RNase H of pararetroviruses. This gene order was confirmed by the amplification of similar PCR products from total DNA extracted from CVMV-infected cassava plants. The genomic organization of CVMV was different from the organization of either the caulimoviruses or badnaviruses. In comparisons of the regions with the reverse transcriptase motif, CVMV was grouped between the caulimoviruses and badnaviruses. It appears that CVMV is distinct from the other well-characterized plant pararetroviruses.