Until recently, research on CBSD diversity/severity has largely been restricted to observations in the field on cassava plants of different age, genetic makeup, and grown in different agroecological zones with varying environmental conditions and possibly infected with different virus strains, all of which can independently or in combination influence symptom development. This made the comparison of the field observations between the various studies particularly difficult and the question of whether a severe form of CBSD is associated with the latest epidemic in Uganda has remained unanswered. Inoculation of herbaceous host plants by various researchers provided somewhat uniform conditions for symptom diversity studies [29
] but until recently no such comparison has been made with isolates from the coastal endemic and inland epidemic areas involving the two different species of CBSVs [15
]. It was particularly difficult to conclude whether the severe CBSD symptoms observed in the fields of coastal Mozambique and Tanzania [9
], for example, or the relatively milder leaf symptoms seen in Uganda (severity score of 2.0, [5
]) were due to the effect of virus isolate or the tolerance/susceptibility of the cassava varieties being grown in those regions. In order to answer these questions, experiments were carried out in controlled environmental conditions in a glasshouse using a standard range of CBSD isolates from both the endemic and epidemic regions to determine the virulence of the isolates. This was particularly relevant to understand if the new outbreaks of CBSD at high altitudes in Uganda and the lake zone areas of Tanzania were due to the prevalence of a severe form of the virus, similar to those observed during the course of the CMD pandemic in Uganda in the early 1990s.
In order to investigate this, a number of parameters were used to assess the severity levels between one epidemic and five endemic CBSD isolates including the symptoms on leaf and roots of five infected cassava varieties, the effect of virus on sprouting of cassava stem cuttings, the rate of graft transmission, and virus titres in infected leaves as well as symptom severity on herbaceous host plants. Amongst the isolates examined, the endemic isolates CBSV-[MZ:Nam1-1:07] and CBSV-[TZ:Nal3-1:07] produced the most severe symptoms with mean symptom severity scores of 3.7-3.8 on a five-point scale [9
]. In comparison, the epidemic CBSUV-[UG:Kab4-3:07] isolate produced relatively mild symptoms with a mean leaf severity score of 1.9. These differences were further confirmed upon the inspection of root symptoms in which CBSV-[MZ:Nam1-1:07] and CBSV-[TZ:Nal3-1:07] infections resulted in root necrosis in all five cassava varieties tested including the tolerant variety Kiroba, however, this was not by CBSUV-[UG:Kab4-3:07] (). The severity of CBSVs can also be estimated by their ability to affect the young growing buds of infected cassava plants [2
]. Using these earlier observations as cues, the differences in the severity levels of the epidemic and endemic isolates were further demonstrated when a significantly higher number of cuttings failed to sprout from the severe endemic isolates compared to the milder epidemic isolate. Between 22 and 26% of the cuttings failed to sprout when infected with CBSV-[MZ:Nam1-1:07] or CBSV-[TZ:Nal3-1:07] while only 4% of the cuttings were similarly affected by the infection of CBSUV-[UG:Kab4-3:07] (). These observations were further supported by the higher rates of virus transmission by grafting of the endemic severe isolates which is probably due to high virus titre (about 1000-times higher virus titre in the two severe endemic isolates CBSV-[MZ:Nam1-1:07] or CBSV-[TZ:Nal3-1:07] compared to the epidemic isolate CBSUV-[UG:Kab4-3:07]). A notable difference observed between this and earlier studies, however, is the infection of the cassava variety Albert by all isolates of this study. In graft-inoculation experiments, Winter et al. [16
] failed to infect Albert by the CBSD isolates from Kenya, Uganda, and Malawi. While the differences between these two similar studies could not be explained at this stage, these results nonetheless have great implications for developing disease management strategies since Albert once considered resistant to CBSD in Kenya, Uganda and Malawi has now been proven susceptible. In southern Tanzania, growing of Albert has been largely abandoned due to its susceptibility to the disease (R. J. Hillocks, unpublished).
The differences in symptoms were also observed on infected herbaceous hosts. Compared to the previously reported N. benthamiana
], N. clevelandii
in particular were highly susceptible to both CBSV and CBSUV in our conditions, and this could be an excellent differential host for separating severe and mild isolates. On N. clevelandii
, the severe isolates CBSV-[MZ:Nam1-1:07] and CBSV-[TZ:Nal3-1:07] produced symptoms early, caused severe stunting of infected plants, leaf necrosis, and often plant death. The remaining isolates including CBSUV-[UG:Kab4-3:07] caused various forms of leaf chlorosis, the symptoms were less severe and nonlethal (). To correlate the symptom diversity observed to the genetic diversity of CBSD isolates, the complete CP aa sequences of 24 isolates were compared with those reference sequences available in gene bank databases. Similar to the results obtained in previous studies [15
], our virus isolates grouped into two clusters based on the two described species: CBSV and CBSUV (). Based on aa sequence identities, the Ugandan epidemic isolate was highly similar to those from Kenya and Malawi, suggesting that CBSUV-[UG:Kab4-3:07] may have originated form one or both these countries.
Put together, these collective observations on symptom diversity as well as genetic differences did not indicate the association of a severe form of CBSD in Uganda. These results are indeed consistent with studies on another epidemic isolate (Namulonge) from Uganda [16
] and especially agree with field observations in which the maximum average severity recorded at the onset of CBSD in Uganda was only 2.0 [5
]. In the absence of a particularly virulent virus in Uganda, our results, however, raise serious questions as to the factors responsible for the current outbreaks of CBSD in eastern African countries. The possible explanations for this are the presence of unusually high populations of whitefly vectors (B. tabaci
) on cassava that may be responsible for the rapid spread of the virus in the field. The recent widespread introduction of CMD-resistant varieties that are particularly susceptible to CBSD or the combination of both could be a factor in disease outbreak. Recent surveys in Uganda indeed confirmed these possibilities, where more than 70% of the cassavas grown in 23 districts were CMD-resistant improved varieties, all of which are susceptible to CBSD. These varieties also support high whitefly numbers, in excess of 200 adults per top five leaves. Although such elite cassava has not been introduced in high quantities to the Lake Zone Tanzania, the high susceptibility of local land races grown in the region and the sudden development of unusually high whitefly populations on cassava there is ensuring the spread of CBSD [6
]. Identification of severe forms of CBSVs in CBSD endemic regions is particularly worrying because the spread of these isolates into areas of high whitefly population has greater potential to cause even more severe damage to cassava production than yet encountered. Our results emphasize the need for exercising strict quarantine measures for preventing further spread of CBSD between country borders and have also identified the need for developing cassava varieties with broad spectrum resistance to both viruses.