Development and evaluation of efficient diagnostic tools for Cassava mosaic and Cassava brown streak diseases

Rajabu, Cyprian Aloyce
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Cassava (Manihot esculenta Crantz) is affected by two major viral diseases, namely Cassava brown streak disease (CBSD) and Cassava mosaic disease (CMD). Accurate and efficient detection and identification of plant viruses are fundamental aspects of virus diagnosis leading to sustainable disease management. In the present study I describe two techniques, the first based on a single tube duplex and multiplex polymerase chain reaction (m-PCR), developed for simultaneous detection of African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV) and East African cassava mosaic Malawi virus (EACMMV), and second, a technique based on Restriction Fragment Length Polymorphism (RFLP) analysis of Reverse Transcribed (RT) -PCR amplified Cassava brown streak viruses species, Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV). In this work, the single tube duplex and multiplex PCR for simultaneous detection of the four cassava mosaic begomoviruses (CMBs) was developed successfully. Four primer pairs were designed from published DNA-A component sequences targeting specific amplification of the four cassava mosaic begomoviruses (CMBs). Evaluation of the primers sensitivity in serially diluted virus samples revealed that the new primers amplified their target virus to a dilution of 10-4 and 10-3 for uniplex and multiplex PCR respectively. Developed multiplex assay enabled specific amplification of the viruses in producing 950, 503, 435 and 260 base pairs (bp) for ACMV, EACMMV, EACMCV and EACMZV respectively in single and mixed infections of CBSVs. Analysis of 172 field samples from Kenya, Malawi, Mozambique, Rwanda, Tanzania and Zambia detected both single and mixed infections, results which were proved by analysis of the sequenced amplicons. Second, a technique based on 2 Restriction Fragment Length Polymorphism (RFLP) analysis of RT-PCR amplified cassava brown streak viruses, Cassava brown streak virus (CBSV) and cassava brown streak Uganda virus (CBSUV), was performed. A degenerate primer amplifying 785 bp of the coat protein gene (CP) of CBSV and CBSUV was also designed. Two restriction endonucleases, HindIII and EcoR1 (identified by a software package, Vector NTI® Express v1.0 from Life Technologies/Invitrogen), which produce different fragments upon digestion of RT-PCR amplicons from CBSV and CBSUV, were used to distinguish the two viruses RFLP analysis using EcoRI has no site in CBSV producing one fragment (785 bp), two fragments (525 bp and 224 bp) for CBSUV and three fragments (785, 525 and 224 bp) for the mixed infections. On the other hand, HindIII has no site in CBSUV producing one fragment (785 bp), three fragments (437 bp, 267 bp and 81 bp) were produced for CBSV, and four fragments (785, 437, 267 and 81 bp) for CBSV and CBSUV mixed infections. In both multiplex and RFLP analyses, results from the sequenced PCR/RT-PCR amplicons agreed with sequence identities of the respective published virus species. Experience from using developed multiplex and RFLP techniques show that time was saved and amount of reagents used were reduced. RFLPs confirmed the presence of CBSV and CBSUV in RT-PCR amplicons without requirement for sequencing. Additionally, modified protocols from Dellaporta et al. (1983) and Chang et al. (1993), were used to extract DNA and RNA respectively from dry and fresh cassava leaves with comparable results. I also demonstrated a method of collecting and preserving cassava leaf samples to retain their integrity during storage for a period of over one month. The two diagnostic tools can be used routinely in germplasm indexing, disease surveillance, and disease monitoring programs 3 Problem Statement and Rationale In east and southern Africa, cassava (Manihot esculenta Crantz) is one of the leading crops in terms of production and has become an important source of income to households and small-scale farmers. However, the production across the region is greatly affected by Cassava mosaic disease (CMD) and Cassava brown streak disease (CBSD). Reports from different authors (Gibson. 1996; Ogbe et al., 1996; Legg et al., 1999; Fondong et al., 2000; Bisimwa et al., 2012) have reported the occurrence of CMD in different countries in the SSA. In Tanzania, CMD has been reported from many locations. Comprehensive characterization by Ndunguru et al. (2005) showed seven cassava mosaic geminiviruses species occur in Tanzania. Mbanzibwa et al. (2009a) reported prevalence of two potyvirus species causing CBSD in the Lake Victoria basin and along the coastal belt of Indian Ocean. A countrywide survey of all major cassava-growing areas in Kenya by Bull et al. (2006) reported presence of six CMG species with novel begomoviruses and a new recombinant strain of EACMV, demonstrating increasing diversity and geographical distribution of CMGs. Similarly, recent reemergence of CBSD has been reported in many districts in Uganda (Alicai et al., 2007) as well as from Malawi (Winter et al., 2010), Kenya (Mware et al., 2009) and Rwanda (Shirima et al., 2012). No reports of occurrence of CBSD have been reported from Zambia. With the current development of more robust diagnostic tools such as RT-PCR and real-time PCR, the diagnosis of CMD and CBSD has also improved in many cassava- producing countries. Similarly, the challenges to obtain more sensitive broad-spectrum cost-effective diagnostic tools also increase. This is evident following discovery of more 4 virus species causing CMD and CBSD (Mbanzibwa et al., 2009a and Winter et al., 2010) which can easily be overlooked. In the field the co-infections of many CMBs and CBSVs is common. Therefore, it will require several tools to detect the multiple infections using the diagnostic tools currently available. Thus, development of efficient and affordable diagnostic tools for simultaneous detection and identification of CMBs and CBSVs is vital and will have a significant impact on development and implementation of cassava virus disease management. Diagnostics will be used for disease monitoring in cassava multiplication plots production and distribution of disease- free cassava planting materials. Therefore, this research make use of the available sequence information in the database for both CMBs and CBSVs to develop sensitive tools for the simultaneous detection of four species of cassava begomoviruses namely: African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV) and East African cassava Mosaic Zanzibar Virus (EACMZV) using multiplex PCR. Also identification and differentiation of two species of Cassava brown streak viruses namely Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV) by RT-PCR/RFLP approach. This study generated knowledge and new tools that will enhance the diagnosis of both CMD and CBSD. The tools will facilitate deployment of virus-indexed cassava planting materials within the region.