Author: Sestras, Adriana F
Date published: June 1, 2011
(ProQuest: ... denotes formulae omitted.)
Apple scab, caused by Venturia inaequalis (Cke.) Wint., and powdery mildew, caused by Podosphaera leucotricha (Ell. et Everh.) SaIm., are the main diseases that cause most damage in apple cultures (Janick et al., 1996; Dunemann et al., 2007; HoIb, 2009; Mitre et al, 2010). The two fungal diseases can be controlled through treatments, but with considerable difficulties and costs, and with long-term consequences and negative effects both on the environment and on the quality of fruits (Berriea and Xua, 2003; HoIb, 2009). In addition, depending on climatic conditions, the effectiveness of fungicide treatments is relative (Koller et al., 2004; Jamar et al, 2010; HoIb, 2009). The creation of resistant cultivars to apple scab and powdery mildew attack is a one of the main goals in apple breeding programmes (Janick et al., 1996; Pitera, 2000, 2003; Bus et al, 2002, 2005; Sestras, 2004; Dunemann et al, 2007). Also, new resistant apple cultivars are necessary for organic growing and integrated fruit production, since many of the highly appreciated commercial cultivars are very susceptible to apple scab and powdery mildew attack (Sandskär and Gustafsson, 2004; Sestras, 2003a, 2003b; Mitre et al, 2010). Creating new apple cultivars largely depends on the availability of sufficient genetic diversity, while apple breeding has eroded in time the genetic base of domestic cultivars. The genetic diversity of apple trees is nowadays limited due to the previously use of domestic cultivars in breeding (Vanwynsberghe, 2006). In addition, apple cultivars used in modem commercial orchards are nearly all genetically based on several relatively disease susceptible ancestors like Golden Delicious, Cox Orange, Jonathan, Mcintosh, Red Delicious and James Grieve (Bannier, 2011). One of the most popular methods, reckon to induce a wide genetic diversity, to enable a wider selection base and to create new resistant cultivars, is interspecific hybridization. Interspecific hybridization was previously used on different Malus species in order to induce resistance to apple scab (e.g. M. floribunda, M. atrosanguinea, M. boccata, M. micromalus, M. prunifolia, etc.) as well as to induce resistance to powdery mildew (e.g. M. zumi, M. robusta, M. sargentii, M. boccata jackii etc.) (Janick et al, 1996; Sestras, 2004). The morphological, biochemical and molecular variation within wild apple species indicates that the starting point in selection of domesticated apples could have come directly from wild apple, without the involvement of other species (Harris et al, 2002). The inheritance of resistance to apple scab and powdery mildew attack is twofold, monogenic and polygenic, as different genetic sources are being known and recommended as genitors in apple breeding programs (Visser et al, 1974; Janick et al, 1996). The use of M. floribunda specie in the creation of the first cultivar with genetic resistance to apple scab, on which PRI series has further been developed (based on Vf gene), is a relevant example in this sense (Crosby et al, 1992; Janick et al, 1996; Janick, 2002). The monogenic resistance has the advantage that it could be more easily incorporated in a cultivar compared to polygenic resistance, while its main disadvantage consists in the apparition of new pathogenic virulent strains of Venturia inaequalis (Cke) Wint. (strains with which it needs to fight). The polygenic resistance is considered more durable compared to the monogenic resistance due to the complexity of the involved mechanisms (Janick et al, 1996; Sestras, 2004). The efficiency of selection out of the offsprings obtained by artificial hybridization is dependent on the heritability of the valuable traits of genitors. Therefore, this study aimed to analyze the resistance heritability to apple scab and powdery mildew attack, starting from the premise that these features have a polygenic inheritance, respectively that the transmission and manifestation of the seedlings resistance are governed by polygenes. In addition, there was analysed the possibility to increase genetic diversity in apple using wild species as genitors, in order to broad genetic enrichment, necessary for selection of new cultivars resistant to apple scab and powdery mildew attack.
Materials and methods
The response to apple scab and powdery mildew attack of 1650 Fi hybrids was analysed at the Fruit Research Station Cluj-Napoca, Romania. The offsprings were obtained through crosses among Malus species and apple cultivars, belonging to fifteen hybrid combinations. The following wild species of Malus were used as male and/or female testers (Bojñansky and Fargasová, 2007; Sestras, 2004): M. coronaria, a North America specie, also known as sweet crab-apple or garland crab; M. floribunda, also known as Japanese flowering crab-apple, originated from Japan and East Asia, being very well known by apple breeders as monogenic source of resistance to apple scab (Vf gene); M. niedzwetzkyana, found in the fruit and nut forests of Central Asia, are considered rare apple trees; M. zumi, also known as O-zumi or Zumi Crabapple, is native to Europe and Asia; M. prunifolia, also known as pear-leaf or plum-leaf crab-apple or Chinese apple, ornamental tree native to China. The following cultivars were used as male and/or female testers (see Table 1): Cluj 218/2, Frumos de Voinesti, Golden Delicious, Jonathan, Reinette Baumann, and Rosu de Cluj. Among these, Jonathan and Rosu de Cluj are very susceptible to powdery mildew attack, while Golden Delicious and Jonathan are susceptible to apple scab (Sestras, 2003a, 2003b).
The seedlings were framed in five cyclic models of hybridization, in which each specie used as maternal or paternal tester was crossed with minimum two (M. coronaria as maternal tester) and maximum four (M. niedzwetzkyana as paternal tester) cultivars, used as genitors. The number of progenies per combination varied from 31 (Cluj 218/2 × M. floribunda) up to 142 (Reinette Baumann × M. floribunda) (Table 1).
Apple scab caused by the fungus Venturia inaequalis is the most important disease of apple worldwide (Bowen et al, 2011). This disease can cause extensive losses (70% or greater) where humid, cool weather occurs during the spring months. It can be seen frequently on leaves, petioles, blossoms, sepals, fruit, and pedicels. Powdery mildew occurs wherever apples are grown and may be a very serious disease, as many apple cultivars are susceptible to its attack (Sestras, 2003b). The fungus can affect leaves, buds, growing tips and fruit, and lead to leaf drop and dieback. Both apple scab and powdery mildew are threats to apple foliage and fruits every year. When severe attacks occurs over several years, the defoliation can seriously weaken trees, reducing the productivity, fruits qualities and survival capability of fruit trees, as well as the aesthetic value of ornamentals.
The response of hybrids to diseases attack was assessed on three years interval, on hybrids of seven to nine years old, in natural conditions of infection, without fungicide treatments. Each year, scab and powdery mildew incidence was assessed two times, in the first decade of July and August, based on scab attack on leaves and powdery mildew on shoots. A scale of 0 to 5 was used, following the standard diagram corresponding to an Attack Index or Attack Degree - AD%, whereby (Sestras, 2003a, 2003b): 0 = no attack (AD% = 0); 1 = very weak attack (AD%=0.1-1); 2 = weak attack (ADM. 1-5.0); 3 = medium attack (AD%=5.1-15); 4 = strong attack (AD%=15.1-20); 5 = very powerful attack (AD%>20.1).
The statistical analysis of experiments data was carried out by applying the ANOVA test. The data were summarized as means and standard deviations for each cyclic hybridization. The genetic analysis of the families involved the decomposition of variances for each hybrid cyclic siblings (half-sib), which were grouped thought siblings having a common hereditary basis, derived from the common parent (mother or father). Calculation of the broad sense heritability (H2) and of the narrow sense heritability (h2) was based on the variances of inter-families and intra-families of siblings, the proportion of common genes, respectively the degree of relatedness, considering equal to 25%, or 1/4 (Falconer and Mackay, 19%; Souza et al, 2000; Gatti et al, 2005). Broad sense heritability and narrow-sense heritability were computed by the classical model, H^sup ...,
where σ^sub G^^sup 2^ is the genotypic variance; σ^sub G^^sup 2^ is the phenotypic variance; O032 is the additive variance (Holland et al, 2003; Piepho and Möhring, 2007; Lu and Myers, 201 1). Heritability in narrow sense was used to predict the response to selection (Falconer and Mackay, 1996) and to the expected selection response (R). The Coefficient of Genetic Variability (CGV%), Genetic Gain (GG or AG) and expected selection Response (R) were computed as follows:
..., where: σ = square root of the total variance among families; σ^sub G^ = square root of the genotypic variance among families; X = mean of trait (mean of marks for apple scab or powdery mildew response of F^sub 1^ hybrids).
... where: k or i = selection intensity (considered 2.06 for the top 5%); σ^sub P^ = square root of the phenotypic variance among families or populations represented by F^sub 1^ hybrids from each cyclic combination of half-siblings (Gatti et al, 2005). The expected selection response (R) was estimated considering the half-sib family selection method described by Nyquist ..., where: ... = phenotypic variance among families or populations represented by F^sub 1^ hybrids from each cyclic combination of half-siblings.
Results and Discussion
Response of F^sub 1^ hybrids to apple scab attack
The top-three hybrid combinations with the lowest average for apple scab attack derived from the following crosses: M. coronaria × Reinette Baumann, Frumos de Voinesti × M. niedzwetzkyana and M. zumi × Golden Delicious (Table 2). Top- three hybrids in regards of higher mean of rates for scab attack were: Cluj 218/2 × M. floribunda and Frumos de Voinesti × M. floribunda combination. Between the five cyclic hybridizations, the mean of marks for apple scab attack was quite dispersing, with limits ranging from 0.69 (M. coronaria used as maternal tester) to 3.09 (M. floribunda used as paternal tester). The results showed that M. coronaria used as mother genitor transmitted by crossing with two cultivars (Jonathan and Reinette Baumann) a valuable response to apple scab attack to their seedlings. M. floribunda specie used as father genitor transmitted to their progenies susceptibility to scab. M. prunifolia, M. niedzwetzkyana and M. zumi passed the intermediate response to scab to their descendants, compared with M. coronaria and M. floribunda. The obtained results when apple scab was investigated in F1 offspring proved to be different in some cases compared to already published results. The most unexpected result consists in the high average obtained by the offsprings which have M. floribunda as parental genitor. Thus, the progenies of M. floribunda used in the analysed hybridisations had a substantially different reaction to apple scab attack, compared to the Clone 821, most frequently used as source for scab resistance, due to dominant Vf gene (Koller et al, 1994; Tartarini, 1996). The result does not rule out the possibility that M. floribunda specie used to hold the hereditary endowment major resistance gene Vf and Fi hybrids who have not inherited it have been particularly susceptible to scab so the result is a high average grade for the attack. Obviously, in combinations with M. floribunda specie, there is the possibility that some hybrids to have a good resistance to scab, due to monogenic resistance induced by the existence of Vf gene in their genome, or due to a combined strength given by the association of Vf gene with polygenes.The offsprings resulted from M. niedzwetzkyana and M. prunifolia species used as paternal testers and Rosu Cluj used as maternal genitor proved to be the ones with the highest mean of marks, and thus with a high rate of susceptibility to scab. The result is surprising, as Rosu de Cluj is known to have a certain tolerance to scab (Sestras, 2004; Quamme et al 2003) due to polygenic resistance to apple scab attack. The descendents resulted from the combinations in which Golden Delicious, a cultivar susceptibility to scab (Sestras, 2004), participated as genitor, proved to have a small average of marks for scab attack: M. zumi ? Golden Delicious (0.58 ± 0.18) and Golden Delicious ? M. prunifolia (1.00 ± 0.25). Even if Malus species have the advantage of transmitting to their decedents the diseases resistance genes, this is unlikely to happen when interspecific crosses are used to create new cultivars. This could be explained by the dominant transmission of the rustic characters from the wild genitors (Sestras et al, 2010).
Furthermore, the genetic analysis is difficult to be carried out on apple since a long time is needed for reliable apple breeding programmes. For this reason, quantitative genetic principles have not been extensively applied in apple breeding (Noiton and Shelboume, 1992). In addition, very few quantitative genetic studies have been conducted on apple, because the work to-date has been mainly devoted to field selection within progenies, rather than analysing wellconstructed mating designs (Durel et al., 1998). The present study allows us to calculate some important genetic parameters (Table 3). The broad sense heritability coefficient proved to have homogenous values among the investigated cyclic hybridizations, with values from 0.685 (M. zumi used as maternal tester) to 0.887 (M. niedzwetzkyana used as paternal tester). The results showed that all species used as genitors transmitted to their progenies, with satisfactory reliability, their response to apple scab attack. Coefficients of heritability in the broad sense means a majority response which shares the genotype effect in the manifestation of reactions of the seedlings to disease, but also a significant contribution of the environmental factors, according to species participation as maternal or paternal genitor. With one exception, the analysis of the heritability coefficients in narrow sense (h^sup 2^) revealed that the non-additive effects are more important than additive effects. The exception is represented by the cyclic combination of M. coronaria used as maternal tester. Therefore, except of the M. coronaria descendants, the narrow sense heritability coefficients varied from 0.159 (M. zumi used as maternal tester) to 0.278 (M. floribunda used as paternal tester). The highest share of polygenic additive contribution to transmission of seedlings resistance to the apple scab attack occurred at M. coronaria. This was the only case in the experiment in which the additive effects were superior compared to the genetic effects of dominance and epistasis, which presented a stronger contribution in the manifestation of the character in the other cyclic combinations. Coefficients of genetic variability proved to have high values in all hybrid cyclic combinations, showing the existence of hybrids with a broad response to apple scab attack, especially among the descendants of M. floribunda (28.1%). Therefore, the CGV values indicate that it is virtually possible to identify hybrid offsprings with a proper response to disease in any cyclic combination. The chances to obtain a population of hybrids with the desired response to apple scab proved to be higher when M. coronaria and M. prunifolia are used as genitors in interspecific hybridization, especially compared to M. zumi (this is sustained by the values of genetic gain). The highest value of the expected selection response was noticed among M. prunifolia descendants, offering a good background for an efficient selection for scab resistant individuals.
Response OfF1 hybrids to powdery mildew attack
Compared with the average score recorded for the scab attack, inside the hybrid combinations, powdery mildew attack showed smaller oscillations, ranging from 0.42 to 2.02 (Table 4). The smallest average value of the marks, identifying the hybrids with the best response to the attack, was recorded in the same combination in which the average score for scab attack was the lowest, M. coronaria x Reinette Baumann respectively (see Table 4). The highest average grade was 2.02 and was recorded in two variants, both having Rosu de Cluj as maternal genitor, in combination with M. niedzwetzkyana and M. prunifolia used as testers. This sensitive response of the offsprings could be explained by the cultivars influences, Rosu de Cluj being known as sensitive to powdery mildew (Sestras et a/., 2010). The mean of marks for powdery mildew attack was quite close, ranging from 0.85 to 1.71, among the five cyclic hybridizations. The lower limit was noted where the specie M. coronaria was used as maternal tester, in its cyclic combinations being registered the lowest value of marks for apple scab. Opposite, the M. prunifolia used as paternal tester gave birth to the most powerful attacked descendants (Table 4). The general combining ability, as well as the specific combining ability effects, proved to influence the transmission of the traits (Bus et al, 2005; Hampson et al, 2009; Soltanloo et al, 2010) as it was also noticed in cyclic hybridisations with M. niedzwetzkyana as paternal tester. Here, two combinations presented large differences as mean of marks for powdery mildew attack, strongly depending by the parental formula and therefore by the cultivars that participated in hybridization with the rustic specie. The Fi hybrids belonging to Cluj 218/2 ? M. niedzwetzkyana proved to have distinct significant negative differences (0.77) compared to the mean (1.36), while seedlings derived from Rosu de Cluj × M. niedzwetzkyana proved to have highly significant positive differences. M. coronaria has a very valuable polygenic background for assuring the resistance to Podosphaera leucotricha since the mean of marks correlates with broad sense heritability and narrow sense heritability (Table 5). Very low values of narrow sense heritability are surprising for several combinations, especially for those where M. floribunda, M. zumi and M. prunifolia (between 0.012 and 0.071) were used as testers. Moreover, also the genetic gain and expected selection response for the studied trait proved to be roughly insignificant in these cases. It does not mean that for these species, as for other wild ones, hybridizations could not be significant in the creation of new cultivars that carry economically important characteristics (Harris et al, 2002), including powdery mildew resistance. The results do not exclude the possibility that these species, when used as genitors, could produce descendents with favourable response (tolerant or even resistant) against powdery mildew attack, due to epistasis and dominance effects of polygene. There were identified phenotypic correlations statistically assured in three out of five cyclic combinations (Figure 1) when the responses to apple scab and powdery mildew attack of F^sub 1^ interspecific hybrids were investigated. The strongest correlation proved to be among the descendents of M. coronaria used as maternal tester (r = 0.709). The M. coronaria wild specie proved to be able to transmit to its offsprings both the resistance to apple scab and powdery mildew attacks. The response to scab and powdery mildew attack on seedlings belonging to M. zumi and M. niedzwetzkyana proved to be also assured at a significance of 0.5%. On seedlings belonging to M. floribunda and M. prunifolia the coefficient of correlations for the response to scab and powdery mildew attack had values close to the significance level. These results sustained the use of wild apple species as suitable resources of genes for both resistance to scab and powdery mildew attacks (Harris et al, 2002). Elite individuals resistant or tolerant to apple scab and powdery mildew attack and with other superior characteristics (including acceptable fruit size and valuable taste for dessert apple) have been identified among the F) offsprings, but the intensity of selection had relative small values (Sestras et aU 2010). Even if the seedlings had prevalent inherited rustic characteristics from the wild species, the elite individuals selected could significantly reduce the backcrossing procedure with regard to the use of the small-fruited wild Malus species that carry several undesirable agronomic traits.
Considerations regarding the use of interspecific hybridisations to increase genetic diversity for inducing resistance to diseases attack on apple
In conclusion, the large variability created through artificial interspecific hybridisations and the reckon values of genetic gain and expected selection response, showed that the chances to obtain a population of hybrids that have a valuable response to apple scab and powdery mildew attack are considerable. Interspecific hybridization also remains a useful method for increasing genetic diversity in apple, offering a good background for an efficient selection for resistant descendants to diseases. In some cyclic hybridization, genetic gain and expected selection response could be downright spectacular, like in offspring populations belonging to M. coronaria used as maternal genitor, regarding the response to powdery mildew attack. Even in such situations, the result depends on the specific combining ability of genitors, respectively on the parental formula, the inexistence of hybridisation barriers for interspecific crosses with Malus × domestica, ploidy etc. (Korban, 1986; Vanwynsberghe, 2006; Sestras et al, 2010). Whether that the genetic base of modem apple breeding programmes is too narrow to guarantee breeding progress in the future (Noiton and Alspach, 1996) or not (Vanwynsberghe, 2006), wild apple species remain of interest because they comprise a potential valuable genetic resource for the continuous improvement of the crops, respectively for sources of genes that control desirable traits in apple. According to available data on this particular case, coefficients of heritability in the broad sense means a majority shares the genotype in manifestation of resistance or susceptibility to apple scab and powdery mildew (Bus et al, 2002), but also a significant contribution of the environmental factors, according to the species participation as maternal or paternal tester. Even if the studied traits showed a strong genetic determinism, the analysis of the heritability coefficients in narrow sense revealed that not always the additive effects played the most important role, but non-additive effects. Depending on the parental formula, in some cases the additive effects were inferior compared to genetic effects of dominance and epistasis, which could present a stronger contribution to the manifestation of seedlings response to diseases attack. Finally, among populations of interspecific hybrids, because of the polygenic effects of dominance and epistasis that express themselves and the manifestation of other characters, e.g. small fruits, low quality of fruits, etc., the selection is generally more effective in the direction of ornamental forms with small fruit, like wild apple crabs, then dessert apple (Sestras et al, 2010). As such, interspecific hybrids have very low chances to becoming dessert cultivars, therefore, they should be included in programs for future improvements through direct hybridization or "modified backcross", according to classical models (Crosby et al, 1992; Janick, 2002). These hybrids can be used to achieve new generations of seedlings, needed to enrich the genetic future background for selections, or for recurrent selection as a new strategy, respectively for combining phenotypic selection with MAS selection (Stankiewicz et al., 2002; Oraguzie, 2003; Oraguzie et al, 2004; Sestras et al, 2009, 2010; Marie et al, 2010; Kumar et al, 2010; Bus et al, 2010), in order to obtain new genotypes for ornamental or dessert apple.
This study was financed by the National Council for Higher Education Research (CNCSIS), the Executive Unit for the Funding of Higher Education and Academic Research (UEFISCSU), Romania, project PCE-IDEI, no. 1105, code CNCSIS 1499.
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Adriana F. Sestras1, Doni Pamfíl2, Catalina Dan2, Sorana D. Bolboaca3, Lorentz Jäntschi4, Radu E. Sestras*2
1 Fruit Research Station, 3 Horticultorilor Str., 400457 Cluj-Napoca, Romania; firstname.lastname@example.org
2 University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, 3-5 Manastur Str., 400372 Cluj-Napoca, Romania; email@example.com, firstname.lastname@example.org
3 Iuliu Hatieganu University of Medicine and Pharmacy Cluj-Napoca, 13 EmU Isac, 400023 Cluj-Napoca, Romania; email@example.com
4 Technical University of Cluj-Napoca, 103-105 Mundi Bvd, 400641 Cluj-Napoca, Romania; firstname.lastname@example.org
* Corresponding author: email@example.com