Analysis of Cassava Brown Streak Disease in Rwanda: Incidence, Dissemination, Genetic Diversity, and Innovative Mitigation Strategies.

Cassava brown streak disease (CBSD), caused by Cassava brown streak ipomoviruses (CBSIs), namely Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), poses a significant threat to global food security. It particularly jeopardizes the food security of tropical Africa, where approximately 450 million people rely heavily on cassava as a staple food and vital income source. In Rwanda, CBSD has rapidly spread since its first report in 2009, with the incidence rising from 18.5% in 2012 to 69% in 2014. This widespread outbreak has resulted in severe consequences, including a shortage of planting materials and a 73% decline in cassava yields. CBSD is primarily transmitted to a longer distance through infected cuttings and, to a shorter distance, by the white fly vector. Infected plants exhibit symptoms on the leaves, stem, and, very importantly, on the storage roots, rendering them unsuitable for consumption.
In response to the CBSD outbreak, the government of Rwanda and researchers have joined forces to combat the burden of CBSD. Notably, they started to import and distribute tolerant cassava planting materials to farmers in 2015. Given that infected cuttings are a significant transmission mode for CBSD, it is imperative to break the cycle of disease transmission and minimize the risk of CBSD spread and its consequences by ensuring the availability of healthy planting materials. In this context, in vitro virus cleaning approaches were applied to combat the virus build-up effect over multiple cycles due to cassava's vegetative nature. However, these approaches take time and are often too costly for subsistence crops. Furthermore, studies have been conducted to assess the prevalence and diversity of CBSD causative agents in Rwanda, albeit with a focus limited to partial coat protein, and different breeding research projects have been initiated. These initiatives were reflecting important investments to mitigate the impact of CBSD while acknowledging the need for further extensive research to tackle the disease comprehensively.
In this regard, the first goal of this thesis was to conduct a countrywide cassava seed system survey to determine CBSD status following interventions and the risk factors that may contribute to its continued spread in Rwanda. To achieve this, 130 cassava farmers were interviewed across 13 major cassava-growing districts and their fields were visited to evaluate disease incidence. Leaf samples were collected and analyzed using RT-PCR (reverse transcription polymerase chain reaction) to confirm CBSIs infection. The findings revealed that CBSD has spread in all surveyed districts, and the overall incidence was 35.3%, with UCBSV being the most common, accounting for 61% of the infections. Several key risk factors that could contribute to the spread of the disease in Rwanda were also identified, including the source of planting materials, geographical location, knowledge of disease transmission, and disease management practices. These findings highlight the need to develop a robust seed system and train farmers to increase awareness and skills to mitigate the spread and impact of CBSD in cassava farming communities.
Recognizing the pivotal role of robust diagnostic tools in fortifying the seed system, the second goal of this thesis was to investigate the genetic diversity of CBSD-causing agents in Rwanda by analyzing whole genomes with innovative methods to provide valuable insights into the evolutionary patterns of CBSIs in Rwanda. High-throughput sequencing (HTS) technologies were applied on 13 pooled samples (corresponding to 13 surveyed districts), enabling us to obtain comprehensive genomic data. Through HTS data analysis, 12 nearly complete consensus genomes of UCBSV were successfully reconstructed. Phylogenetic analysis of these genomes revealed a remarkable reduction in genetic diversity, with a maximum of 0.8% nucleotide divergence between the genomes. Further investigation beyond the consensus sequences utilizing the combination of fixation index (FST) calculation and Principal Component Analysis (PCA) based on SNPs patterns unveiled three distinct UCBSV haplotypes exhibiting geographic clustering. Interestingly, the distribution of haplotype two (H2) was found to be associated with one of the CBSD tolerant cultivars widely distributed to farmers, "NAROCAS1". In addition, HTS allowed the assembly of the partial genome of Manihot esculenta-associated virus 1(MEaV-1) for the first time in Rwanda. Identifying distinct UCBSV haplotypes and their geographic distribution represents the first study in Rwanda, marking a significant advancement into the local patterns of UCBSV evolution, facilitating a better understanding of the disease's spread, and developing targeted control strategies.
Considering that the current main CBSD management relies on the distribution of tolerant cultivars susceptible to viral buildup effect, the third objective of this thesis was to transform existing in vitro virus cleaning methods into practical farmer-friendly approaches at the greenhouse and field levels toward CBSD mitigation. The present study assessed the effectiveness of combining greenhouse thermotherapy with chemotherapy and field chemotherapy, employing salicylic acid (SA) and Benzothiadiazole (BTH) on CBSIs-infected cuttings. The results revealed a remarkable reduction in viral loads, especially when combining thermotherapy with SA at 50 mg/L and thermotherapy with BTH at 50 mg/L, which exhibited the most substantial reduction compared to other treatments. Additionally, a significant decrease in the severity of CBSD root symptoms through field chemotherapy was observed among treated plants. These findings highlight the potential effectiveness of these combined approaches in mitigating the impact of CBSD and offer promising avenues for disease management in cassava. Furthermore, RNA sequencing on uninfected cassava plants exogenously treated with SA and BTH was conducted to investigate their impact on the cassava transcriptome. It was revealed that SA and BTH deregulate numerous cassava genes, including genes with potential involvement in plant defense, such as transcription factors (e.g., WRKY), Leucine Rich Repeat (LRR) Protein, Heat shock Protein (HSP), Mitogen-Activated Protein Kinase (MAPK), Cytochrome P450, and ethylene-responsive genes. The gene ontology (GO) enrichment analysis revealed that hormone signaling, defense response, response to stress, and regulation of transcription were among the enriched GO, suggesting their potential role in viral host response.
Overall, this thesis contributed significantly to understanding and managing CBSD, providing valuable knowledge for sustainable cassava farming in Rwanda. The countrywide farmers and cassava fields survey provided crucial findings on CBSD status and risk factors, emphasizing the urgency of a robust seed system and farmers' training. The high-resolution investigation into UCBSV genetic diversity through an innovative approach shed light on its evolutionary patterns and geographic distribution, offering insights for targeted control measures. Finally, transforming in vitro virus cleaning methods into greenhouse and field approaches showcased promising results in reducing CBSD severity and viral loads, supported by identifying potential defense-related cassava genes.
The present thesis can also serve as the basis for future research. Notably, the innovative approach to characterizing genetic diversity could be applied to study other crucial plant viruses. It is also worth investigating CBSV diversity and the factors driving the evolution of CBSIs in Rwanda. Furthermore, future research is needed to optimize the efficacy of greenhouse and field CBSD mitigation approaches and delve into the specific functions of genes regulated by SA and BTH. Moreover, assessing the impact of chemotherapy on the environment and microbiome and analyzing large-scale cost-benefit viability would provide invaluable insights. These avenues of exploration will undoubtedly contribute to a deeper understanding of virus management strategies and bolster efforts to safeguard plant health and agricultural productivity.