Ratoon stunting disease
- The incidence and effects of RSD in the South African sugar industry
- Causal organism
- Symptoms of RSD
- Effect on growth of sugarcane
- Methods of diagnosis
- Microscopic diagnosis of RSD
- Immunoassays to diagnose RSD
- DNA-based diagnostic methods
- Transmission and control of RSD
- Planting material
- Why is RSD still prevalent in most sugarcane industries?
Initial surveys indicated that approximately 30% of fields in the southern regions of the industry were infected with RSD (Bailey and Fox, 1984). The use of RSD-free planting material from an established nursery is therefore important in controlling the disease (Steindl, 1961; Egan, 1980; Roach, 1987).
Screening for resistance to diseases
- Essential criteria for successful disease screening
- Disease screening in the South African sugar industry
- Stability of resistance
- The effect of disease screening on the breeding programme
In the early stages of the selection program, genotypes are exposed to the common diseases at the various selection sites in KwaZulu Natal. In a later study, they found that quantitative estimates of the number of colonized vascular bundles (cvb) obtained with TBIA were highly correlated with the relative densities of the pathogen population in adjacent tissue as determined by counting the number of fluorescent antibody stained cells (Harrison and Davis). , 1990).
Introduction
Development of a laboratory technique for counting vascular bundles colonized by Leifsonia xyli susbp xyli.
Materials and methods
- Production of L. xyli subsp xyli polyclonal antiserum
- Isolation of L. xyli subsp m
- Immunisation of rabbits
- Testing the specificity of the L. xyli subsp xyli antiserum
- Sample preparation
- Silver staining
- Western blotting ,
- Enzyme linked-immunosorbant assay
- Preparation of immunoglobulin
- Tissue blot immunoassay
- Tissue blot procedure
- Immunoassay procedure
- Analysis of data 33
- Isolation and culture of L. xyli subsp xyli
- Antiserum production
- Specificity of antisera using EeL hybridisation and EB-EIA
- Optimisation of the tissue blot immunoassay
- Effect of stalk age on the proportion of cvb
- Effect of internode position and variety on the proportion of cvb
- Effect of variety on the extent of colonisation
The blot was then incubated for one minute in enhanced chemiluminescence reagents (Amersham, Buckinghamshire) before being exposed to autoradiographic film (Hyperfilm-ECL) for five minutes. Membranes were allowed to air-dry before the impressions of the tissue sections on the membrane were examined with a stereomicroscope (Zeiss, West Germany) at 40× magnification. Individual bands were detected by ECl hybridization in broth culture samples of L.
This would be the first investigation of differences in the extent of colonization in a range of South African cultivars. The average proportion of cvb in all cultivars in the first and second internode was higher than in the third and fourth internode, although these differences were not significant (Figure 3.7). In cultivars N12, N14 and N17, there was little variability in the proportion of cvb from internodes one to four (Figure 3.8).
There was also a significant difference in the proportion of CVB in internode three compared to the other internodes in N22. This study also found that the degree of variability in the second internode was generally low in most varieties tested.
Conclusions
1994) showed that TBIA was effective in differentiating cultivars based on the incidence and severity of RSD infection. Data from preliminary experiments reported in this chapter showed that there were significant differences. Furthermore, the classification of cultivars based on the extent of colonization corresponded in most cases to the classification based on yield loss in previous trials (see 2.1.2), with N17, N14 and NC0376 being the most susceptible and N23 and N12 being the most resistant.
N22 was ranked as the most resistant variety in the yield loss experiment, but TBIA results showed it to be more susceptible than N12 and N23. In subsequent studies described in this thesis, only older stems in the mud were sampled, and then the second lowest internode was selected for TBIA. TBIA was able to distinguish a highly susceptible cultivar such as N17 from N23 and N12, two cultivars known to show higher resistance to RSD under most environmental conditions.
Based on the information in this chapter, further investigation into the use of TBIA for RSD screening in the South African sugar industry was warranted. Relationship between the extent of colonization of sugarcane by Leifsoniaxili subspxyli and the rate of spread of ratoon stunting disease.
Introduction ~
Materials and methods
- Establishment of field trial
- Choice of varieties
- Layout of trial
- Preparation of planting material
- Sampling and laboratory analysis
- Assessment of RSD infection 49
Seed canes from the twelve varieties were cut into single-bud setts (SBS), treated with hot water (HWT) for 2 hours at 50°C and planted in seedling trays to produce transplants for the trial. The trial was planted in October 1998 and all transplants survived transfer to the field. The trial was harvested manually and at each harvest three cane cutters were assigned to the trial, with each cutter responsible for harvesting one replicate.
At harvest, each row was systematically cut, starting from the infested control litter, through the N14 spreading section and continuing through the non-inoculated plants to the end of the row. The oldest stem was plucked from each uninoculated mud and marked by row number and petiole position along the row. Sections of spreader N14 were sampled after the first harvest to assess colonization rates, but were not sampled in the second crop of ratoon.
Diagnosis of RSD and the proportion of colonized vascular bundles (cvb) were determined using TBIA on the single stem samples according to the procedure described in Chapter 3. This was used to estimate the rate of spread of RSD along the row, where each stem sample represented 0 .5m of the row.
Results and discussion
- RSD status in the plant crop
- RSD in the inoculated control plots
- RSD in the inoculated spreader plots
- RSD in the uninoculated rows
- RSD status in the inoculated stools in the first and second ratoon crops
- Populations of L. xvii subsp m in spreader plots of N14
- Populations of L. xvii subsp m in inoculated controls
- RSD status of stools planted after the spreader plots in the first and second
- Rate of spread (RSD incidence) and severity of RSD along the
- Relationship between RSD incidence and severity
This may explain the negative result in one of the inoculated control plots of N12 in the first ratoon. The false negative results in the N12 and N24 control plots would have affected the mean proportion of cvb. The proportion of cvb in the first two stools along the rows of N14 and N22 was higher than N28, but the total colonization in these two variants was lower.
The percentage of cvb in the first bench after the distribution section was higher in the sensitive standard NC0376. The extent of colonization was particularly high in the first two stools after the two harvests, but decreased steadily along the row. The percentage of cvb along the row was generally lower in N27 and N31 in the second ratoon.
Rather, the mean incidence of RSD in the three replicates was plotted against the mean percentage of cvb in each variety in the first and second ratoon cultures. The incidence and severity of RSD along the row and in the inoculated control plots showed that N14 and N22 were the most susceptible of the varieties tested.
Conclusions. 62
They stated that the inoculum levels in the distribution plots could influence the rate of spread. It is therefore unlikely that this affected the severity and rate of spread of RSD in N22. Although this could play a role when comparing results from trials at different locations, it is unlikely to have had an effect in this trial where all varieties were exposed to the same conditions and the soil was uniform.
Although this should be considered a limitation, it is important to identify those varieties that support high populations of L. The fact that the correlation between the proportion of cvb and the average RSD incidence was highly significant suggests that greater spread occurred in those varieties that were more extensively colonized by L. The effect of RSD on the yield of six sugarcane varieties and relationship with degree of colonization by Leifsonia xyli subsp xyli.
They are therefore not suitable for use in a routine disease screening program where large numbers of breeds are required to be assessed. Using this method it is possible to routinely screen up to approximately 1,000 genotypes each season (Comstock et al., 2001).
Materials and methods
- Establishment and management of trial
- Layout and planting of trial
- Preparation of seedcane
- Harvesting of trial ,
- Sampling and laboratory analysis
- RSD diagnosis using EB-EIA
- Assessment of RSD incidence and severity using TBIA
- Assessments of yield and cane quality
- Analysis of data
TBIA was used as the only method of RSD diagnosis in the first ratoon harvest. The stem population in the healthy plots of N12 in the plant crop was the highest of the varieties included in the trial. The overall sugarcane yield in the healthy plots of the crop was satisfactory with an average of 75 t/ha after 12.5 months (Table 5.3).
RSD caused a reduction in sugarcane yield (tonnes/ha) in five of the six varieties in the crop and in all varieties in the first ratoon (Table 5.2). In the South African sugar industry, sugarcane transplants are produced as an alternative method of propagation (Anon, 1988; Anon, 1990). Relationship between resistance to Clavibacter xyli subsp xyli colonization in sugarcane and the spread of field rat disease.
Introduction of a serological method for the large-scale diagnosis of ratoon stunting disease in the South African sugar industry. Susceptibility to ratoon stunting disease in the Saccharum complex and feasibility of breeding for resistance.
Results and disucussion ;
- RSD incidence in the plant and first ratoon crops ;
- Effect of RSD on germination and certain yield components
- Stalk characteristics
- Effect of RSD on cane and sucrose yield
- Effect of RSD on cane yield of six varieties
- Estimated recoverable crystal
- Cane quality. 76
- Relationship between yield loss and the extent of colonisation
Conclusions
Screening for resistance to ratoon stunting disease in Florida. · Proceedings of the International Association of Sugarcane Technologists. Comparison of diagnostic techniques for determining the incidence of ratoon stunting disease of sugarcane in Florida. In situ localization and morphology of the bacterium associated with ratoon stunting disease of sugarcane.
Establishment and management of trial '" 82
- Layout of trial ' · ,
- Preparation of planting material
The twelve varieties selected for this trial were the same as those included in the field trial investigating the prevalence of RSD in different varieties (Chapter 4). Therefore, comparisons can be made between the extent of colonization observed in control plots inoculated in the field trial in Chapter 4 and that of transplants inoculated in this greenhouse trial. Seed cane for the experiment was obtained from a propagation plot established with healthy seed cane that was treated for two hours at 50°C before planting.
The leaves of the transplants were clipped every other week to encourage the development of more vigorous primary shoots.
Testing for RSD using TBIA.
Analysis of data
Results and discussion
- Germination
- TBIA for detecting L. xyli subsp xyli in sugarcane transplants
- RSD incidence in the different varieties
- Extent of colonisation in transplants of different varieties
Due to the large number of vascular bundles in the transplants, only those colonized by L. This therefore gave an indication of the number, rather than the proportion, of cvb in each transplant. Since more than 1,000 genotypes are tested with TBIA in the USA each year, only the number of cvb per stem (Comstock et al., 1995).
The results of this greenhouse trial were highly variable from one replicate to another when comparing the number of CVB in the different varieties. 86 The controls did not respond as expected, with N14 being in the group with the lowest colonization rate and N12 among the most severely infected groups. N27 had the lowest CVB rate of all breeds, mainly due to the higher number of healthy transplants.
The maximum number of cvbs recorded in this variety was 25, which is one less than that recorded in N14. At ten weeks, the grafts may have been too young to distinguish between the different cultivars in terms of the degree of colonization.
Conclusions ·
Effect of rat blight on yield and yield components of sugarcane under rainfed conditions. The incidence and effects of sugarcane blight disease in southern and central Africa. Mechanisms of infection of sugarcane by the bacterium C/avibacter xyli subsp xyli.
Influence of sugarcane cultivar susceptibility to the spread of ratoon stunting disease by a mechanical harvester. Distribution of Clavibacter xyli subsp xyli in stems of sugarcane cultivars differing in ratoon disease resistance. Single-mud plots for estimating relative yield losses due to stunting disease of sugarcane.
Spread and increase of sugarcane stunting disease and comparison of disease detection methods.
