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African Crop Science Journal, Vol. 9, No. 1, March 2001, pp. 9-16
M. O. Osiru, P. R. Rubaihayo and A. F. Opio1
Code Number: CS01029 ABSTRACT Bacterial wilt (Ralstonia solanacearum), known to attack over 450 plant
species in the tropics and subtropics, is a devastating disease limiting tomato
production in Uganda and worldwide. Two bacterial wilt resistant, two susceptible
and two mildly resistant tomato cultivars were crossed in a half diallel at
Kabanyolo, Uganda. Parents and F1's were root inoculated and data collected
on bacterial wilt intensity at 3 day intervals 6-21 days after inoculation and
analysed for bacterial wilt reactions. Combining ability results indicated that
both general combining ability (G.C.A.) and specific combining ability (S.C.A.)
effects were significant for bacterial wilt resistance, indicative of both additive
and non-additive gene actions. However, GCA was found to be six times as large
as SCA indicating the predominance of additive gene effects in bacterial wilt
resistance. Cultivars MT55, MT74, MT15 and MT164 showed negative G.C.A values
indicative of good sources of resistance to bacterial wilt. Hybridisation of
parents (MT55, MT74, MT15 and MT164) followed by selection in segregating populations
might yield inbred progeny with resistance greater than that of parents. Joint
regression analysis revealed an additive-dominance model for bacterial wilt
resistance with no evidence of epistasis. The results further revealed that
resistance to bacterial wilt is controlled by two genes. The implication of
these results to selecting for bacterial wilt 'resistance' in potato are discussed. RÉSUMÉ La bactériose (Ralstonia solanacearum), reconnue pour attaquer plus de 450 espèces de plantes dans les régions sous tropicales et tropicales, est une maladie dévastatrice limitant la production de la tomate en Uganda et dans le monde entier. Deux cultivars de tomate résistants, deux sensibles et deux moyennement résistants ont été croisés dans un demi diallèle à Kabanyolo, Uganda. Les parents et les F1 ont été inoculés au nivea des racines et des données ont été collectées sur l' intensité de la barctériose à un intervale de 3 jours , 6-21 jours après inoculation et analysées pour la réaction à la bactériose. Les résults d' efficacité à la combinaison ont montré que les effets de l' efficacité générale à la combinaison (G.C.A.) et de l'efficacité spécifique à la combinaison (S.C.A.) étaient significatifs pour la résistance à la bactériose suggérant des actions additives et non-additives des gènes. Cependant, G.C.A. était six fois plus importante que SCA indiquant la prédominance des effets additifs des gènes. Les cultivars MT55, MT74, MT15, et MT164 ont eu des valeurs négatives de G.C.A. montrant une bonne source de résistance à la bactériose. L'hybridation des parents (MT55, MT74, MT15 et MT164) suivie d'une sélection dans les populations ségrégantes pourraient aboutir à des hybrides ayant une résistance supérieure à celle des parents. L'analyse de la régression a montré un modèle additif-dominance pour la résistance à la bactériose sans évidence de l' épistasie. Encore les résultats ont montré que la résistance à la bactériose est contrôlée par deux gènes. Les implications de ces résultats dans la sélection pour la résistance à la bactériose chez la pomme de terre sont discutées. Mots Clés: Effets addititifs, diallèle, G.C.A., S.C.A., Lycopersicon esculentum, Solanum tuberosum. INTRODUCTION Tomato (Lycopersicon esculentum L.) growing is one of the most promising areas for horticultural expansion and development in Uganda. The crop is grown in virtually all districts of the country, and it is estimated that by the year 2007, tomato production will increase to 130,586 tonnes (Anon, 1994) but currently, tomato production is severely limited by Ralstonia solanacearum syn. Pseudomonas solanacearum E.F. Smith, a soil- borne pathogen and causal agent of bacterial wilt (Low, 1997; Tumwine, 1999). The causal pathogen, reported to attack over 450 plant species (Prior et al., 1998), has a wide host range including potato (Solanum tuberosum L.), eggplant (Solanum melongena L.), groundnut (Arachis hypogaea L.), banana (Musa spp.), pepper (Capsicum spp.), tobacco (Nicotiana tobacum L.) and tomato (Wang et al., 1998). Protective measures have proven ineffective for its control since the bacterium resides in the host plant xylem, has a large host range and is soil-borne (Grimault et al., 1993). Additionally, control methods such as chemical control and soil fumigation are not financially practical for resource-poor farmers. Under these circumstances, the only alternative is the use of host plant resistance (Wang et al., 1998). Genetic improvement for host resistance is the most important component of any integrated control against tomato bacterial wilt (Liao et al., 1998) and the use of resistant varieties has been reported to be the most effective and practical method to control bacterial wilt (Grimault et al., 1994). Unlike in potato, where the search for bacterial wilt resistance and its use has been characterised with much frustration and little success (Mehan et al., 1994), many tomato cultivars have been developed with useful levels of resistance for certain environments (Gomes et al., 1998). However, there is still difficulty in obtaining cultivars with stable resistance under conditions of high temperature and humidity in the lowland tropics (Hayward, 1991). Varieties bred for resistance in one area frequently do not sustain that resistance when transferred elsewhere due to the complex nature of the resistance itself and the strong host x pathogen x environment interaction, partly the cause of failure to achieve stable resistance (Hanson et al., 1996). Tikoo et al. (1983) and Scot et al. (1992) reported that the mode of inheritance to bacterial wilt resistance was dependent on particular lines under study and methods of evaluation. Genetic experiments with North Carolina and Hawaii bacterial wilt resistant tomato germplasm indicated that resistance was conditioned by recessive genes and largely inherited in a polygenic fashion (Acosta et al., 1964). Genetic and biochemical evidence also suggest that R. solanacearum most likely evolved with several wild hosts in geographically isolated areas with the present day crop plants like tomato and potato being chance victims when introduced into infested areas (Charkrabarti et al., 1992). This complex non-specific resistance is further hampered by a highly heterogenous pathogen capable of creating variability with such high frequency, that most workers now refer to R. solanacearum sub-groupings (biovars, races) as separate pathogens. Therefore, in any breeding programme we are dealing with a group of related pathogens, but not with a single homogenous species. There is need to identify specific pathovars of R. solanacearum to which resistance in Uganda is required. A diallel analysis can be used to provide information on the nature and amount of genetic parameters, general and specific combining abilities of parents which can be translated into genetical components such as additive and dominance variance with certain assumptions (Griffing, 1956). In the study reported in this paper, attempts were made to gain some insight on the mode of inheritance of resistance to the bacterial wilt disease in six tomato cultivars under Ugandan conditions. The study also aimed to identify possible breeding methods to aid development of tomato cultivars resistant to the bacterial wilt disease in the country and the implications for potato bacterial wilt resistance breeding in Uganda. MATERIALS AND METHODS Six tomato cultivars, MT 74 and MT 55 (resistant), MT 164 and MT 15 (mildly resistant), MT 86 and MT 29 (susceptible), selected from previous screening experiments in bacterial wilt infested fields at Kabanyolo, Uganda were crossed in a half diallel in a screenhouse in February 1999. The parents and F1's were inoculated by the root inoculation method of Winstead and Kelman (1952). Ten plants in each cross were uprooted and roots washed clean of soil and trimmed using sterile scissors along one side and were inoculated by pouring 10 ml of bacterial suspension (1x108 cfu), obtained photospectrometrically, onto the injured roots at the three leaf stage. The inoculated plants were re-planted in plastic pots with sterilised soil and grown in a screenhouse. For the first 2 days after re-planting, plants were kept under a polythene chamber to maintain a high relative humidity. Observation of wilt symptoms and wilt intensity were made 6-21 days following inoculation. Disease ratings were done using a 1-5 scale where; 1= no symptoms, 2= one leaf wilted at the inoculation point, 3= two to three leaves wilted, 4= four or more leaves wilted, 5= whole plant wilted (dead plant) (Winstead and Kelman, 1952). Similarly, the wilt intensity was calculated at 3 day intervals from 6-21 days after inoculation according to Winstead and Kelman, (1952) formula, I = σ (n1 x v1) x 100 V x N
Where, I= wilt intensity (%), n1= Number of plants with respective disease rating, v1= Disease scale (1-5), N= Total number of plants observed, and V= the highest disease scale (Winstead and Kelman, 1952). The data were analysed using the combining ability analysis of Griffing (1956) and the diallel graphical analysis of Hayman (1954) and Jinks (1956) by the diallel cross computer programme (Christie et al., 1988) and Mstat-C statistical package. RESULTS AND DISCUSSION The results of combining ability analysis for bacterial wilt intensity on six tomato cultivars are presented in Table 1. Significant (P<0.001) differences were observed in both the general combining ability (G.C.A.) and the specific combining ability (S.C.A.) effects for bacterial wilt intensity indicating the significant contribution of additive gene effects and dominance effects to the inheritance of resistance to bacterial wilt (Hanson et al., 1998). Liao et al, (1990) reported that although both G.C.A and S.C.A. were important for resistance to bacterial wilt, G.C.A. was more important in Arachis hypogea. Contradicting results were, however, observed by Pham and Schmiediche (1993) in potato where non-additive mode of inheritance of bacterial wilt was more important than the additive mode. In our study, the general combining ability components were about six times as large as that of specific combining ability, suggesting the dominant role of additive gene effects (Baker, 1978) in bacterial wilt inheritance. The G.C.A. effects of the cultivars are presented in Table 2. Parents MT 55, MT 74, MT 15 and MT 164 showed negative G.C.A. effects, indicating a high gene frequency for bacterial wilt resistance (Christie and Shattuck, 1992) and hence good sources of resistance to bacterial wilt. The results indicated that hybridisation of these parents (MT 55, MT 74, MT 15, MT 164) followed by selection in segregating populations would yield inbred progeny with resistance greater than that of the parents (Hanson et al., 1998). MT 29 and MT 86 had positive G.C.A. estimates suggestive of a low gene frequency (Gevers and Lake, 1994). Cultivar MT 74 showed the most negative G.C.A. effects suggesting that it would be the best parent in a resistance breeding programme while Parent MT 29 would be the worst parent. Specific combining ability effects for each of the 15 individual crosses are presented in Table 3. The results showed significant positive S.C.A. effects between parents MT55 x MT 15, and MT 74 x MT 15 for bacterial wilt intensity, suggesting that these specific crosses would be useful to breeders in improvement of tomato bacterial wilt resistance (Jatasra, 1980). These results suggested that those parents (MT74, MT15 and MT55) most resistant to bacterial wilt, were the best general combiners for bacterial wilt resistance. Such lines could be used to improve resistance to bacterial wilt. The results of the variance (Vr), covariance (Wr) graphical analysis of Hayman (1954) and Jinks (1956), which assumed homozygous parents, diploid segregation, no reciprocal differences, no epistasis or multiple alleles and uncorrelated gene distributions are presented in Table 4. The Wr-Vr analysis provided a test for epistasis and indicated that this factor was non-significant in resistance to bacterial wilt. The results indicated that the values were constant over the arrays, suggesting again, that additive effects were more important than non-additive effects in the inheritance of resistance to bacterial wilt and that the additive dominance model for genetic variability was valid. The results of a joint regression analysis of Wr on Vr (Table 4) showed a regression coefficient which was significantly (P≤0.01) different from 0 but was not significantly different from 1 indicating the predominance of additive and dominance gene actions (Christie and Shattuck, 1988). Wang et al. (1998) suggested that bacterial wilt resistance in tomato was inherited as a polygenic character with several loci playing a quantitative role in the ability of a plant to withstand pathogen attack. The estimates of the components of variation proportion of gene effects and heritability are presented in Table 5. Standard errors were calculated using specific multipliers according to Hayman (1954). Components of variation due to the additive effects were found to be significant (P<0.05) suggesting the preponderance of additive gene effects for the inheritance of resistance to bacterial wilt (Christie and Shattuck, 1992). Mean covariance of additive and dominance effects over all arrays (F) was not significantly different from 0 indicating unequal allelic distribution among the parents (Christie and Shattuck, 1988). The results indicated an F value of 3296.9 for bacterial wilt resistance, suggesting an excess of dominant alleles (Christie et al., 1988). The estimate of the average degree of dominance provided by H1/D_ (0.647) was suggestive of partial dominance. Similar results were reported by Christie et al. (1988). The results of the coefficient of correlation between the parental order of dominance (Wr + Vr) and the parental mean Y (0.1745) showed that most recessive genes were positive (Table 5). The positive correlation indicated that positive alleles (those increasing the mean of parents) were recessive, and that most negative alleles (those causing lower means) were dominant (Table 5). Liao et al. (1990) reported bacterial wilt in groundnut as a partially dominant character. Both narrow sense and broad sense heritability estimates were medium indicating that bacterial wilt has a high proportion of total variance contributed by the phenotypic variance. Heritability estimates indicate to what extent selection is likely to be effective (Dudley and Moll, 1969). These values show that a sufficient proportion of additive genetic variance was available in the population under study, confirmed by the large additive variance estimates. The result of the estimate of effective factors (1.7 genes) is presented in Table 5. The estimate of effective factors obtained in this manner was likely to underestimate the number of genes affecting the trait under consideration, since the estimates of the actual number of genes requires no linkage, no epistasis, and all loci displaying dominance (Christie and Shattuck, 1992). These results indicated a two-gene model for bacterial wilt inheritance. This is in agreement with Acosta et al. (1964) who suggested that a two-gene model with epistasis adequately explained the observed segregation of the bacterial wilt stock from North Carolina. Pham and Schmiediche (1993) reported that both additive and non-additive gene actions weresignificant in the inheritance of resistance to bacterial wilt. Wang et al. (1998), however, reported that resistance in tomato is inherited as a polygenic character with several loci playing a quantitative role in the ability of a plant to withstand pathogen attack. Implications for potato improvement. Bacterial wilt resistance in potato could be due to as many as 10 major genes and its nature is highly complex (Rowe and Sequeira, 1970), unlike in tomato where probably only 2-4 genes are involved in the resistance (Acosta et al., 1964). More recent evidence (Chakrabarti et al., 1992) shows that resistance is apparently polygenic and quantitative in nature involving genes with major and minor effects and even non-additive gene actions (epistasis) probably play a role in determining resistance. In addition, present day potato cultivars have higher ploidy levels and heterozygosity which pose a formidable problem to a successful breeding programme. A source of resistance to R. solanacearum approaching immunity is highly desirable for a potato-breeding programme. It has been demonstrated that bacterial wilt resistance in potato is strain and temperature specific (French and Lindo, 1982) and the so-called resistant cultivars are in fact wilt tolerant, since they have been found to harbor a large population of R. solanacearum in vivo (Grimault, 1994). The level of resistance found in S. tuberosum to R. solanacearum is relatively low. However, this might be compared with that first discovered in tobacco and tomatoes (Nielson and Haynes, 1959). Robinson (1968) postulated that the frequent breakdown of vertical resistance in potato was due to the appearance of vertical pathotypes in R. solanacearum. After much frustration in many potato breeding experiments, screening efforts are now placed on detection of clones with a high degree of tolerance and not resistance (Charkrabarti et al., 1992). Hence, although it is difficult at present to obtain a major gene-like resistance in potato, there is need for breeding strategists to aim at strengthening the basic constitution of the host potato plant by either pooling sources from different partially resistant Solanum spp. into a single population (Schemidiche, 1986) or by use of non-conventional methods. However, for the farmer in Uganda and other developing countries, use of tolerant varieties coupled with integrated disease management (IDM) remain the only means of control for the bacterial wilt pathogen on the cultivated potato. CONCLUSION The results indicated that resistance to bacterial wilt in tomato is controlled by 2 genes. Inheritance of resistance and its expression were complex with involvement of both additive and non-additive gene actions. However, the relative magnitudes of additive and non-additive gene actions indicated that additive gene effects are more important in bacterial wilt resistance. The results, therefore, suggested that breeding methods such as recurrent selection that exploit additive genetic effects would be useful in incorporating resistance to bacterial wilt in susceptible tomato lines. Hybridisation of parents MT55, MT74, MT15, and MT164 followed by selection in segregating populations would yield inbred progeny with resistance greater than that of parents. Immune potato cultivars are still unavailable and use of integrated disease management remains the primary control strategy for control of the wilt pathogen on this crop. ACKNOWLEDGEMENT This study was funded by the United States Agency for International Development (USAID). REFERENCES
TABLE 1. Mean squares for combining ability analysis for bacterial wilt intensities on six tomato lines
**- Significantly different at P ≤0.001
TABLE 2. Estimates of general combining ability effects for bacterial wilt intensities on six tomato parents
MT- Makerere tomato accession code
TABLE 3. Specific combining ability effects for parents and their F1 hybrids involved in a six parent diallel cross at MUARIK
TABLE 4. Analysis of variance for Wr + Vr values and joint regression
*significant at P<0.01, ns-not significant
TABLE 5. Components of variation of tomato resistance to R. solanacearum
*significant at P<0.05, ns-not significant
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