Selection and Inheritance of Tomato Resistance against Ralstonia solanacearum

Ralstonia solanacearum is a plant pathogen causes wilting which is a major obstacle in the cultivation of tomato plants. In plant breeding, knowledge of the source of resistance genes and inheritance patterns is important in the development of bacterial wilt resistant varieties. This study aimed to obtain bacterial wilt resistant lines and to find out the inheritance pattern of tomato resistance to bacterial wilt. Selection of resistant plant involved the selected breeding lines from irradiation and crossing collections of the Genetic Laboratory, Faculty of Agriculture, Universitas Gadjah Mada. Introduced lines of H-7996 and F 1 Permata and Timoti were used as a control. H-7996 as resistant parents and GM2 as susceptible parents, and their offspring include F 1 GM2 x H-7996, F 1 reciprocal, F 2 , Back Cross 1 (F 1 x GM2), and Back Cross 2 (F 1 x H-7996) used in testing inheritance patterns. Inoculation was carried out 1 week after planting by pouring 100 ml of water suspension of R. solanacarum (10 8 cfu/ml) on the roots. Completely Randomized Design (CRD) was used in this experiment. The scoring observation was carried out every week for one month. This study showed that Permata as a control was the most resistant, while Timoti and H-7996 were medium resistant. The CLN, G6, G8, and G7 lines were susceptible medium, yet only G8 and G7 with the smallest percentage of disease intensity and not significantly different than Timoti. The resistance gene to bacterial wilt on H-7996 was controlled by genes in the cell nucleus with additive-dominant gene action. Resistance to bacteria has a moderate level of heritability.


INTRODUCTION
Tomato (Solanum lycopersicum) is one of important fruit vegetables in the world.
Tomato are not only used as fruit or vegetables but also as industrial raw materials. One of the obstacle in tomato production is bacterial wilt caused by Ralstonia solanacearum (Champoiseau & Momol, 2009). R. solanacearum is soil-borne pathogens (Hayward, 1991) become endemic in an area with attacks reached 71% (Sikirou et al., 2017). R. solanacearum infects the plant through a natural opening or wound in the root, producing putrescine as a virulent pathogenic metabolite (Lowe-Power et al., 2017), and colonize xylem. The bacteria cells cause blockage of xylem and inhibit the absorption of nutrients and water, hence the plants wilt (Arwiyanto, 2014). Bacterial wilt reduce tomato production by up to 91% (Yuliar et al., 2015).
The control techniques, such as physical, chemical, biological and technical culture have been applied (Nawangsih, 2005). The control technique which is easier, cheaper, and environmentally friendly is the use of resistant varieties. In Indonesia, the number of resistant varieties to bacterial wilt is slightly available. A study conducted by Rao et al. (1975) using 23 varieties from the USA and Philippines, only found one resistant variety to R. solanacearum isolates from India. This finding showed that geographical differences determine the pathogenicity of pathogens and the specific resistant characteristics of varieties.
Resources of resistance to R. solanacearum have been found, yet some are specific to certain races and only tomato cultivars of H-7996 have the most stable characteristic of resistance. That cultivar has a high survival rate up to 97% in 12 locations of 11 countries in Asia, America, and Australia. This cultivar is widely used as a subject to study resistance genes against R. solanacearum (Wang et al., 2013). In Indonesia, H-7996 has been used by Arwiyanto et al. (2015) as a rootstock, suppress bacterial growth, to reduce the incidence of wilt and thus better production. Breeding scheme of resistant tomatoes to bacteria is necessitated. The source of resistant genes, knowledge of inheritance schemes, type of resistance, and the mechanism of tomato resistance to bacteria have been mastered by plant breeders (Seah et al., 2007), in order to the breeding of resistant varieties is in the right direction. The use of selected germplasm, resulting in good characteristics, yet it is needed to be assessed for resistance to R. solanacearum. Therefore, this study aimed to obtain bacteria resistant lines and to find out their inheritance and dominance schemes.

MATERIALS AND METHODS
The lines used for screening resistance to R. solanacearum were from Gamma-ray Timoti, and Permata was used as a resistant variety.
Cultivars for testing inheritance patterns of resistance to bacteria used were H-7996 as resistant cultivars and GM2 as susceptible cultivars. Parents of GM2 and H-7996 were crossed to produce F1 GM2xH-7996 and the reciprocal crossing (H-7996xGM2). Then some F1 seeds were planted together with both parents and the rest was stored for the next crop season. F1 plants were partially left to self-pollinate to produce F2 seeds and some were crossed to both parents to produce backcross (BC) offspring. Backcross 1 (BC1.1) was F1xGM2 and backcross 2 (BC1.2) was F1xH-7996. After harvesting, the seeds were sown in sterile media. Planting material used to estimate the number of controlling genes and interactions between genes was carried out by planting parents, F1 and reciprocates (BC1.1, and BC1.2), F2. The number of parent plants and F1 was 30 plants, BC1.1, BC1.2 were 150 plants, and F2 were 250 plants, respectively. R. solanacearum used was race 1, biovar 3, phyllotype 1 collection of Phytopathology Laboratory was obtained from endemic areas of Seyegan, Sleman, the Special Region of Yogyakarta. Bacteria was grown on YPGA (Yeast Peptone Glucose Agar, 5 g yeast extract, peptone 10 g, glucose 10 g, agar 15 g, distilled water 1.000 ml). Inoculation was carried out on plants have 5 leaves or 7 days after transplanting by drenching 100 ml of water suspension with a density of 1 x 10 8 cfu/ml in the root area of the plant (Kim et al., 2016).
Disease symptoms were observed every 7 days for 28 days after inoculation. The Classification of resistance used the mean method and disease intensity. Lines have a mean score of < 2 were categorized as resistant (R), 2 ≤ x ≤ 3 was medium resistant (MR), and > 3 was susceptible (S) (Kim et al., 2016). Calculation of disease intensity used formula 1 (Arwiyanto et al., 1994). Classifying the level of resistance to bacterial wilt based on disease intensity referred to Table 1 T-test was used to analyze the maternal effect (Singh & Chaudary, 1977). Estimation of the heritability value used the following formula (Warner, 1952): The estimation of variant above was obtained from the calculation of the tested population variants (Mather & Jinks, 1982). According to Stansfield (1991), if the value of heritability (h 2 ) < 0.2 was categorized as low, 0.2 < h 2 <0.5 was medium, and h 2 > 0.5 was high.

RESULTS AND DISCUSSION
The results of scoring variants analysis of wilt and disease intensity showed that there were differences in resistance between the tested lines/cultivars. Based on wilting score and disease intensity showed that all further generation lines from crosses were susceptible to bacterial wilt (Table 2), because tomato Gondol, as parents, has no source of resistance genes. Gondol tomatoes are special commercial tomatoes, yet very susceptible to bacterial wilt (Harjadi & Halim, 1980), hence lines from their offspring segregation were also According to Hai et al. (2008), H-7996 was resistant to Pss186 (race 1, biovar 4) and Pss4 (race 1 biovar 3), but high susceptible to Pss190 (race 1 biovar 4). R. solanacearum was a complex species because they are heterogeneous species (Arwiyanto, 2014). According to Lebeau et al. (2011), tomato plants have a high susceptibility to the diversity of R.
solanacearum strains. Therefore, specific resistance to certain strains was present.
In the other hand, Laeshita & Arwiyanto (2017) stated that H-7996 cultivars are medium susceptible despite using the same line. This was probably due to using a different inoculation method. Laeshita & Arwiyanto (2017) used artificial wound to root during inoculation, but this study did not use that method. In this study, no wound was carried out because the conditions were expected like conditions in the field. According to Hayward (1991), the symptoms of wilting caused by infections by R. solanacearum are influenced by pathogenic strains, inoculation methods, and environmental factors such as temperature.
The screening results revealed that the lines of the UGM Genetic Laboratory collection had a lower resistance compared to H-7996 and resistant controls. Furthermore, H-7996 is used as a parent to analyze gene action from the bacterial resistance. H-7996 is a pure strain hence can be used as a line to study inheritance resistance and has been widely used. The susceptible line used as parents was GM2. The results of reciprocal testing (Table   3) showed that F1 resistance and reciprocity were not significantly different. This indicates resistance to bacterial wilting is controlled by genes in the cell nucleus and is not affected by cytoplasmic of female parents. The number of controlling genes and inheritance patterns was estimated by using a combined scale test of the population of parents, F1, F2 and reciprocal ( Figure 1). The controlling gene action that set a trait can be estimated based on the distribution of the frequency of these characteristics in the F2 population. Scoring distribution in population F2 showed the presence of two peaks leading to resistance and susceptibility. According to Acquaah (2007), this result indicated that the gene controlling resistance to bacterial wilt is controlled by major genes.
Chi-square test (Table 4) showed the ratio of 3:1 and was not significantly different at the level of α=5%. A 3:1 comparison indicated that the controlling gene of this characteristic is a single gene and the resistant gene is a recessive gene. The distribution of scoring BC1.1 (Figure 1) showed that the formation of one peak with a susceptible nature to R. solanacearum. If the dominant assumption was completed, then crossed F1 (Aa) x susceptible parent (AA) will get BC1.1 100% susceptible. In this study, BC1.1 (F1 x GM2) showed the results as hypothesized, which was almost 100% susceptible. Assumptions on crosses BC1.2: F1 (Aa) x parent resistant (aa), will get a ratio of 1 Aa (susceptible): 1 aa (resistant). In this study, a comparison of 2.5 susceptible: 1 resistant was resulted from crossing and different as hypothesized. It is suspected that there was inter-locus interaction or epistasis.
Estimating genetic parameters using three parameters used the assumption that the controlling gene was a major gene, thus only additives (AA, aa) and dominance (Aa). Based on the combined scale test method (  Opena (1994) stated that two backcrosses with resistant parents were enough to get a good level of bacterial wilt resistance.
Based on this study, it was concluded that the resistance control gene H-7996 against R. solanacearum isolates from Seyegan, Sleman (race 1, biovar 3, phyllotype 1) was the major gene with perfect dominance. The results obtained from this study were similar with Wang et al. (2000), using the strain Pss4 (race 1, biovar 3), reported that resistant genes were controlled by major genes, but when using strains GMI8271 were controlled by polygenic. Grimault (1995) and Thakur et al. (2004) reported that the controlling gene for bacterial wilt was controlled by a single recessive. Grimault (1995) conducted a field test using strain 8217 (race 1, biovar 1) stated that the resistant gene from H-7996 was controlled by a single gene.
This differences indicated that the gene controlling resistance to the bacteria R.
solanacearum varies depending on the specific strains. These findings showed that there was a gene-for-gene concept for resistance to R. solanacearum.

CONCLUSION
The G7