Cassava Manihot esculenta Crantz (Euphorbiaceae), is a source of food for more than 700 million people in developing countries and is cultivated in estimated global area of 18.6 million hectares with total annual production of 238 million tonnes. Diseases however, take a substantial toll of yield, with CMD being the most important disease and major constraint for cassava production in Tanzania and Africa. The disease causes an estimated loss of over US$ 14 million per annum.
A study was undertaken in 2006/2007 to investigate the influence of satellite DNA molecules on severity of cassava begomoviruses and the breakdown of resistance to cassava mosaic disease (CMD) in Tanzania. The goal was to appraise the nature of resistance to CMD in indigenous and improved cassava cultivars in the presence of resistance-breaking satellites. Three specific aims were earmarked: to identify and characterize cassava mosaic virus isolates and satellite DNA molecules in major cassava growing areas of Tanzania; to screen cassava cultivars for resistance to begomoviruses in presence and absence of the satellite DNA molecules; and to determine the nature of interaction between begomovirus DNAs and Satellite DNA molecules in Nicotiana benthamiana.
To achieve these aims, a survey was done in the major cassava growing areas of Tanzania to investigate occurrence of cassava mosaic begomoviruses and associated satellites namely, SatDNA-II and SatDNA-III. Stems from plants showing CMD symptoms were collected from field. The stems were re-planted in screenhouse to study more about the symptoms. Symptomatic leaves from sprouting cuttings were collected for DNA extraction to be used in two downstream assays - amplification of EACMV, ACMV, SatDNA-II and SatDNA-III by polymerase chain reaction (PCR) and sequencing.
In another experiment to evaluate cassava cultivars for resistance to CMD in presence of satellites, stem cuttings of the classical CMD-resistant cultivars were planted in greenhouse. Infectious clones of EACMV-TZ and EACMV-UG2 comprising both DNA-A and DNA-B components of bipartite begomoviruses (EACMV-TZ and EACMV-UG2) as well as infectious clones of SatDNA-II and SatDNA-III were bombarded onto the greenhouse cassava plants using a gene gun. Emerging disease symptoms on inoculated plants were scored using standard procedure. Total nucleic acid extraction from the inoculated plants was done and PCR was performed to amplify AC1 and βC1 genes as well as full length SatDNA-II and SatDNA-III. Southern blot analysis was performed to determine the presence of AC1, βC1, SatDNA-II and SatDNA-III on the DNA.
In order to study interaction between cassava mosaic begomovirus (EACMV-TZ) and satellites, infectious clones of EACMV-TZ (DNA-A and DNA-B) and that of SatDNA-II and SatDNA-III were used. The clones (DNAs) were used to infect Nicotiana benthamiana by abrasion. Inoculated plants were covered with a plastic dome and placed in insect-free growth chamber for symptom development, which were scored on a standard scale of 1 to 5. Total DNA was extracted from the N. benthamiana leaves and used for Southern blot analysis.
Results from the field survey showed that disease incidences varied from 60 to 90% in the Lake Victoria Zone and from 10 to 90% in the Eastern Zone. Cultivar Lyongo had the highest disease symptom severity in the Lake Victoria Zone while in the Eastern zone plants with high severity levels were from cvs Maiza and Tabora. In the screenhouse, some sprouted cuttings remained healthy up to 16 days after planting (DAP) and others recovered from the disease. Reversion was also observed in some cultivars. Using PCR, East African cassava mosaic Tanzania virus (EACMV-TZ) was amplified from 72.8% of tested samples while African cassava mosaic virus (ACMV) was amplified from 4.3%. Five percent of plants had dual infection of the two viruses. While ACMV was detected in samples collected from Lake Victoria, EACMV-TZ was mostly found on samples from the Eastern zone. Sequencing showed the presence of two new virus isolates: EACMV-TZ [TZ113] and EACMV-TZ [TZ108]. Seventy five percent of plants, which showed reversion of symptoms, contained SatDNA-II. It was found that full length SatDNA-II occurred in both zones, while SatDNA-III was exclusive to the Lake Zone. Multiple DNA bands were noted in PCR agarose gels, more so in SatDNA-II than SatDNA-III. For SatDNA-II, the multiple bands were more evident for samples collected from Eastern zone than for those from the Lake Zone. Using primers based on expressed sequence tags (EST-primers) for SatDNA-II (895 bp) and SatDNA-III (306 bp), genome integrated forms of the satellites were amplified from 68% and 71.17% of samples, respectively. Thirty percent of the samples showed co-infection of the satellites. While EST-primers for detection of the integrated forms of SatDNA-III produced single bands on gels, those of SatDNA-II still produced additional bands, most noteworthy being the closely spaced „double bands‟. Upon sequencing, the satellite DNA isolates showed similarity with sequences deposited in the genebank and bearing accession numbers AY836366 and AY836367 for SatDNA-II and SatDNA-III isolates, respectively. Alignment reports (Clustal W) revealed presence of GC-rich regions, TATA protein binding motifs (TATAAAT) and CAAT boxes as well as poly (A) signal. GC-rich regions in SatDNA-II were mostly trinucleotides (CGC) and hexanucleotides (CCGCCG) while in SatDNA-III the regions were trinucleotides (CGC) and pentanucleotides (CCGCC).
Following biolistic inoculation, five-week scoring for the symptoms showed that plants from cvs AR37-92, CR27-24 and AR16-3 remained symptomless while plants from cv T200 were symptomatic. PCR amplification of βC1 gene five weeks post inoculation (wpi) gave PCR products in 19.6% of the samples while AC1 was amplified from only two plants. Full-length SatDNA-II was amplified from 70% of DNA samples, mostly from plants in which a begomovirus was co-inoculated with SatDNA-II. Amplification of full-length SatDNA-III from bombarded plants was unsuccessful. Amplification of integrated fragments of SatDNA-II from bombarded plants using EST-primers gave a PCR product in 93.7% of the samples. PCR amplification of the fragments from DNAs extracted from plants of cvs AR17-5 and CR27-24 previously inoculated with EACMV-TZ + SatDNA-II and EACMV-UG2 + SatDNA-III, respectively, gave closely spaced bands on 13% of the DNA samples. Amplification of integrated forms of SatDNA-III gave bands in 52.4% of samples. Probing for full-length SatDNA-II, SatDNA-III and AC1 from DNAs extracted from plants pre-inoculated with these DNAs using DIG- labeled probes gave hybridization signals in 60%, 83% and 68% of the samples, respectively. Further analysis of the signals in the context of screening suggested that cvs AR37-92 and AR37-96 were highly resistant to CMD while cv AR40-10 was susceptible.
In the interaction experiment, Nicotiana benthamiana plants inoculated with an infectious clone of EACMV-TZ developed moderate CMD symptoms 7 days post inoculation (dpi) with symptoms consisting of leaf distortion and moderate stunting of plants. There were also plants which recovered from the symptoms by 35 dpi. Plants inoculated with EACMV-TZ + SatDNA-II produced similar symptoms with N. benthamiana plants developing symptoms 7 dpi that became severe by 14 dpi and without recovery even after 35 dpi. Very severe symptoms were also observed when N. benthamiana plants were inoculated with EACMV-TZ + SatDNA-II + SatDNA-III. Plants inoculated with SatDNA-II or SatDNA-III alone remained asymptomatic even after 35 dpi. Southern blot analysis showed clear increase in DNA accumulation when EACMV-TZ was inoculated together with both SatDNA-II and SatDNA-III as compared to when EACMV-TZ was inoculated alone or with SatDNA-II only and probed with EACMV-TZ.
In conclusion, symptom recovery and reversion of symptoms in screenhouse plants is associated with virus resistance. There is a wide occurrence of satellites (SatDNA-II and SatDNA-III) across the sampled regions consistent with distribution of their helper cassava begomoviruses. The satellites are of a wider occurrence and diversity in Eastern zone than elsewhere in the country. The occurrence of SatDNA-III was not confined to the Lake zone as previously thought. There is evidence for satellite sequence integration into host plant genome, a further indication that the satellites are wider spread in cassava germplasm than earlier conceptualized. In few instances, both SatDNA-II and SatDNA-III isolates co-existed in the same plant though its effect on symptom enhancement could not be immediately established. The observed recovery in screening studies is thought to result from resistance introduced in the plant materials involved. Since labeled probes for satellites that were used in hybridization had been prepared from satellite sequences considered to be integrated, the hybridization signals did not depend on whether the leaf samples were picked from symptomatic or asymptomatic plants. From the study, three observations clearly suggest that SatDNA-II and SatDNA-III are biologically functional and that their effects on host plants are distinctly different. The study has demonstrated enhanced cassava begomovirus symptoms in N. benthamiana in the presence of satellite DNA molecules. This is the first detailed study undertaken to highlight the occurrence and role played by satellite DNA molecules in breaking the resistance to CMD of cassava cultivars grown in Tanzania.
Keywords: Cassava mosaic disease, Cassava mosaic begomoviruses, Satellite DNA molecules, Tanzania.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/12427 |
| Date | 14 February 2013 |
| Creators | Ndomba, Osmund Aureus |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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