Inheritance of fire blight resistance in Asian Pyrus species

Kamila L. Bokszczanin, Andrzej A. Przybyla, Malgorzata Schollenberger, Dariusz Gozdowski, Wieslaw Madry, Slawomir Odziemkowski

Department of Experimental Design and Bioinformatics, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Warsaw, Poland.
Department of Plant Genetics, Breeding, and Biotechnology, Warsaw University of Life Sciences, Warsaw, Poland.
Department of Plant Pathology, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences, Warsaw, Poland.
Department of Pomology, Warsaw University of Life Sciences, Warsaw, Poland.

DOI: 10.4236/ojgen.2012.22016                

Abstract

Since several Asian pear species are considered to be potential source of fire blight resistance, we crossed “Doyenné du Comice”, the susceptible European cultivar, with four Asian pear species. The aim of the study was to establish the level of resistance of each genotype and the mode of transmission of fire blight resistance to each F₁ full-sib progeny. The best sources of resistance were P. ussuriensis 18 and P. ussuriensis var. ovoidea 8 ranked to resistant and highly resistant, respectively. Although pear resistance to fire blight is suggested to be polygenic, distribution of phenotypes in “Doyenné du Comice” × P. ussuriensis var. ovoidea 8 hybrid family suggests the possibility of monogenic inheritance with the dominance of resistance derived from P. ussuriensis var. ovoidea 8. Polygenic inheritance of pear resistance to fire blight was identified in cross combinations of “Doyenné du Comice” with P. pyrifolia 6, and contributed by the major gene, with P. ussuriensis 18 and P. calleryana 12. Transgressive segregation was observed within the progenies of susceptible, moderately susceptible and resistant parents.

Keywords

“DoyennÉ Du Comice”; Asian Pear Species; Fire Blight Resistance; Pear Interspecific Hybrids; Polygenic Resistance; Monogenic Resistance; Transgression

1. INTRODUCTION

Fire blight is the most devastating disease of pears and apples caused by the bacterium Erwinia amylovora (Burrill) Winslow et al. Symptoms of the disease were noticed as early as 1780 in New York [1]. The bacterial etiology of the disease was recognized in the early 80’s of the 19^th century by the pioneer of plant pathology Thomas J. Burrill. In 1871 he reported the results connected with peach and pear diseases near Cobden, Illinois, where he found first symptoms of fire blight [2]. In 1885 Arthur [3] published his classic paper on the completion of Koch’s postulates with the bacterium, thus proving beyond doubt that fire blight was indeed caused by this organism. Erwinia amylovora is considered to be the first proven bacterial plant pathogen [4,5].

The principal and most susceptible hosts are in the subtribe Pyrinae (formerly Pomoideae) of the family Rosaceae. The pathogen is considered to be a quarantine organism and its introduction is prohibited by almost all countries. All plant organs except seeds are considered to be potential source of disseminating this pathogen. All the above and underground parts of hosts can be infected by this pathogen. It can make a considerably reduced yield.

Although fire blight has been known for more than 200 years there is no adequate chemical or other treatment for the elimination of this pathogen from plant material without destroying the plant tissues. The treatment with antibiotic streptomycin is forbidden in the EU.

The most important pear commercial cultivars originnate from P. communis and are susceptible to fire blight. The resistance in P. communis is relatively rare with only 5% - 10% being rated as at least moderately resistant [6-8]. In the European pear cultivars different levels of susceptibility to fire blight are observed. The most resistant main pear cultivars grown in the world are, among others, ‘Harrow Sweet’ and ‘Conference’, and the most susceptible ‘Doyenné du Comice’ [9]. Out of 287 cultivars named prior to 1920, only 11% are resistant or highly resistant and out of 113 cultivars released between 1920 and 1978, about one-third were reported to be predominantly resistant [10]. The US National Clonal Germplasm Repository (NCGR) Pyrus collection possesses more than 160 pear clones identified as being highly to moderately resistant to fire blight. Fire blight resistant accessions include 22 Asian cultivars, 80 European and hybrid cultivars, 2 ornamental cultivars and 61 rootstock and species selections [11].

Breeding for pear resistance to fire blight began in the nineteenth century after the introduction of Japanese sand pear (P. pyrifolia) to the eastern part of the United States [12]. The first fire blight resistant pear cultivar was ‘Patten’ released from Iowa Experimental Station after the introduction of Chinese pear P. ussuriensis [13]. Old dessert cultivars most consistently rated as resistant are ‘Alexander Lucas’, ‘Tyson’, ‘Seckel’, and ‘Maxine’ [14]. Resistant cultivars developed by later breeding are ‘Harrow Delight’, ‘Harrow Sweet’, ‘Honeysweet’ and ‘Magness’. ‘Gourmet’ and ‘Honeysweet’ are fire blight resistant interspecific hybrids of P. communis × P. ussuriensis parentage developed for the northern plains of North America where cold hardiness is essential. All other European, circum-Mediterranean, and mid-Asian species are generally susceptible [14]. A high level of resistance occurs in higher frequency among the East Asian pear species. As the sources of resistance van der Zwet and Keil [15] ranked in descending order based on their degree of fire blight resistance: P. ussuriensis, P. calleryana, P. betulaefolia, P. pyrifolia, and P. communis. Hartman [4] listed resistant individuals within P. calleryana, P. betulaefolia, P. phaeocarpa, P. faurieri, and P. variolosa. Since the pathogen is not known to occur in Asia, it is proposed that fire blight resistance in Asian species evolved independently of the fire blight pathogen.

The strategy of reducing the host susceptibility to fire blight infection is especially valuable for Integrated Fruit Production (IFP). Prior to 2002, no information on molecular genetics of pear existed. Since then a few genetic maps based on molecular markers have been described [16-18] as well as the first report on quantitative trait loci (QTLs) linked to fire blight resistance genes in LG 2, 4, and 9 of resistant cv ‘Harrow Sweet’ was made by Dondini et al. [19]. For the first time a putative QTL for fire blight resistance in P. ussuriensis 18 linkage group 11 and putative QTL in linkage group 4 of susceptible ‘Doyenné du Comice’ were reported [20]. Genetic resistances are more often identified in wild species, botanical varieties or obsolete cultivars with mediocre appearance and quality. The use of wild species is very problematic because of its extremely bad fruit quality and small fruit size. Since biotechnology tools can help to solve the transmission of resistance genes into established cultivars, a pear breeding program with Asian pear species as the sources of fire blight resistance was initiated in 2003 at Warsaw University of Life Sciences, Department of Pomology.

The objective of the present study was to determine the levels of fire blight resistance in Asian pear species, European pear cultivar ‘Doyenné du Comice’ and the hybrid families as well as to evaluate the transmission of resistance to F₁ full-sib progeny.

2. MATERIALS AND METHODS

2.1. Plant Materials and Crosses

In order to obtain F₁ full-sib progeny, interspecific crosses were performed using the cultivar ‘Doyenné du Comice’ originated from P. communis, susceptible to fire blightfemale component and the following accessions of Asian pear species: P. calleryana 12, P. pyrifolia 6, P. pyrifolia 19, P. ussuriensis 18, and P. ussuriensis var. ovoidea 8— pollen donors. Hand-pollination method was employed with flower emasculation at balloon stage by tearing away the calyx cup pinching it with the fingernails. Crosses were carried out in May 2003-2007 in the Wilanow orchard of the Department of Pomology, Warsaw University of Life Sciences. To enlarge progeny, crosses of ‘Doyenné du Comice’ with Asian pear species were repeated for few consecutive years, i.e. for P. calleryana 12 in 2004 and 2005, for P. pyrifolia 6 in 2003-2006, for P. pyrifolia 19 in 2004 and 2005, for P. ussuriensis 18 in 2003-2007, and for P. ussuriensis var. ovoidea 8 in 2003- 2006. Seedlings obtained in the current year served for one-year observations.

2.2. Artificial Shoot Inoculation

The greenhouse-resistance assessments of the seedlings and parental forms were performed in the summer of 2004-2009 at Warsaw University of Life Sciences, Department of Pomology, in cooperation with the Department of Plant Pathology.

Parental forms were grafted in ten replicates on the potted seedlings of P. communis L. subsp. caucasica (Fed.) Browicz. At the time of vegetation from each scion two shoots were developed. Seedlings were not grafted. At the turn of June and July, according to method used by Bell et al. [21], two youngest leaves of 50 cm tall shoots were dissected with scissors dipped in the inoculum of E. amylovora (Burrill) Winslow et al., highly virulent strain 691 (10⁸ cfu·ml^–1), isolated from ‘Šampion’ apple cv in 1998 at Research Institute of Pomology and Floriculture, Skierniewice, Poland. In order to provide high humidity conditions after inoculation the plants were covered with plastic bags for 24 hours. During 4 weeks of evaluation the following mean values were maintained: 27˚C (day), 21˚C (night) and 55% air humidity.

Starting one week after inoculation and continuing for four consecutive weeks the plants were scored for the progression of the disease. The fire blight lesion length and total length of the current season’s growth of the inoculated shoots were measured. The severity of infection was expressed as the length of the fire blight lesion (necrosis) as a percentage of the overall shoot length after 1, 2, 3 and 4 weeks after inoculation. In the case of grafted plants the measurements were averaged over all replicates. The level of resistance of parental forms was established by employing Gardner scale [22]: 0% - 10% highly resistant, 10% - 30% resistant, 30% - 50% moderately susceptible, 50% - 90% susceptible, and 90% - 100% highly susceptible. Each next class starts above the upper limit of the previous one. In order to obtain accurate results on the transmission of the studied trait, the distribution of individuals in each F₁ full-sib progeny was investigated in ten classes of resistance: 0% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, 90% - 100%. These classes correspond to the classes of Gardner, i.e. 0% - 10% highly resistant, 10% - 30% resistant etc.

2.3. Statistical Analysis

The data for the fire blight infection recorded on each offspring resulted from interspecific crosses were subjected to statistical analysis. Descriptive statistics were used separately for each hybrid family. Mean, median, minimum and maximum value of necrosis, lower and upper quartile, standard deviation, coefficient of variation, and skewness were computed.

Pearson’s chi-square test was chosen to check if resistance distribution in each seedling progeny depends on parental combination.

The Kolmogorov-Smirnov one-sample test was used to test the hypothesis about the normal distribution of phenotypes in each hybrid family.

The shape of the observed non-normal, continuous distribution of phenotypes in ‘Doyenné du Comice’ × P. ussuriensis var. ovoidea 8 progeny was analyzed with Gaussian mixture simulation.

The data for the fire blight severity recorded on 5 progenies and 6 parents for 4 measurement times were analyzed using two stage cluster analysis. At the first stage the variables were values for individual plants for each cross separately (from 12 to 155 depending on cross). Groups of plants with similar pattern of fire blight infection were distinguished within each hybrid family. At the second stage mean values for distinguished groups and mean values for parents were used for cluster analysis for all five crosses together. Classification of genotypes and groups of genotypes into homogenous groups was carried out using Ward’s method of cluster analysis. The squared Euclidean distance was used as the dissimilarity measure between genotypes. The analyses were conducted using Statistica 7.1 package, StatSoft 2005.

3. RESULTS

Both the number of individuals obtained from each cross of ‘Doyenné du Comice’ with Asian pear species, and the distribution of these individuals in classes of resistance in each F₁ progeny are given in Table 1.

It was found, that the levels of disease severity depended very much on cross combination (p < 0.0001). The probability of the occurrence of observed numbers of seedlings in classes of resistance at the assumption of independence between the infection level and cross combination would be much less than 0.001%.

Among the tested genotypes the highest level of resistance showed botanical ussurian pear variety—P. ussuriensis var. ovoidea 8 with a mean lesion length of 2.34%, which corresponds to a highly resistant class. Both P. ussuriensis 18 and P. pyrifolia 6 were classified as resistant and ranked in the 2^nd class (with the lesion length of 15.87% and 24.10%, respectively). In the 3^rd class (moderately susceptible) the following were ranked: P. calleryana 12, P. pyrifolia 19, and ‘Doyenné du Comice’ with the severity of infection expressed by 31.67%, 43.43% and 49.41% respectively (Table 2).

Different levels of resistance were observed between genotypes of the same species like between P. pyrifolia 6 and P. pyrifolia 19 as well as between P. ussuriensis 18 and P. ussuriensis var. ovoidea 8 (Table 2).

To characterize the distribution of phenotypes in different interspecific crosses the mean, median, minimum and maximum value of necrosis, lower and upper quartile, standard deviation, coefficient of variation, and skewness were taken into account (Table 3). In the interspecific cross ‘Doyenné du Comice’ × P. pyrifolia 19 the highest mean susceptibility value and the lowest coefficient of variation were observed (Table 3, Figure 1). Half of the progeny was ranked to susceptible and highly susceptible

Table 1. Distribution of individuals in each F₁ progeny according to percentage of lesion length.

Table 2. Level of resistance to fire blight of ‘Doyenné du Comice’ and Asian pear species (average from 2008-2009).

Table 3. Mean, median, minimum and maximum value of necrosis, lower and upper quartile, standard deviation, coefficient of variation, and skewness in hybrid families.

           

Figure 1. Distribution of F₁ full-sib progeny in classes of fire blight (Erwinia amylovora) resistance. Arrows indicate range of parental fire blight necrosis (♀, female; ♂, male). Results of the Kolmogorov-Smirnov one-sample test for normality are given under the title of each histogram.

class with the lesion length not less than 63%. In a quarter of the progeny there was necrosis equal to or greater than 86.5% (Table 3). Seedlings distribution appeared to be not continuous (gradually presented), since there were no genotypes with 30% - 40% and 60% - 70% of the severity of fire blight infection (Figure 1). Resistant seedlings were not observed due to either a low number of seedlings or lack of valuable alleles in the male parent.

On the contrary, in ‘Doyenné du Comice’ × P. calleryana 12 population susceptible seedlings were not observed. The lowest mean value of both lesion length and standard deviation were noted. In three quarter of the progeny necrosis did not exceed 27% and in half—21% (Table 3). The most numerous class was resistant with the lesion length ranged from 10% - 30% (Figure 1). It is a result of either a low number of seedlings or the presence of valuable resistance alleles in P. calleryana 12.

The lowest skewness equal to 0.47 and phenotypic mean value of 50.50 were attributed to ‘Doyenné du Comice’ × P. pyrifolia 6 cross combination implicating the symmetric distribution of resistance/susceptibility trait. It is also expressed by the median value closed to the mean since necrosis evaluated in one half of the progeny was reported to be equal to or greater than 46% (Table 3). In comparison to the remaining hybrid families, there occurred the greatest number of seedlings classified as highly susceptible with 100% of the disease severity. The average difference between plant necrosis and the mean amounted to 24 (Table 3). Phenotypic values were relatively evenly distributed on both sides of the mean, resembling bell-shaped distribution characteristic for polygenic traits. The hypothesis that the respective distribution is normal was confirmed by the KolmogorovSmirnov one-sample test (Figure 1).

Only in the interspecific crosses of ‘Doyenné du Comice’ × P. ussuriensis 18 and ‘Doyenné du Comice’ × P. ussuriensis var. ovoidea 8 highly resistant seedlings with no symptoms were observed –12 and 21 individuals respectively. Moreover half of the progeny appeared to be infected with necrosis equal to or less than 22% (Table 3).

In the case of ‘Doyenné du Comice’ × P. ussuriensis 18 the greatest positive skew of the distribution was observed with the mass of the distribution concentrated on the left of the figure. Predominant number of individuals was ranked to the resistant class with the lesion length ranging from 21% - 30% (Figure 1). In a quarter of the progeny necrosis did not exceed 14.5% (Table 3). This interspecific cross transmitted resistance to 65% of its offspring ranked to two resistant classes ranged from 0% - 30% of fire blight severity.

‘Doyenné du Comice’ × P. ussuriensis var. ovoidea 8 is the sole interspecific cross where the most numerous 1^st class of resistance was noted (Figure 1). A quarter of the progeny was ranked as highly resistant with necrosis equal to or less than 9.5%. Individuals determined to be moderately susceptible to highly resistant constituted three quarter of the progeny with the lesion length less than 43%. It was found that the coefficient of variation was the highest when compared to the rest of families with a standard deviation only about 15% below the mean (Table 3). Non-normal, but still a continuous distribution of phenotypes was observed (Figure 1). ‘Doyenné du Comice’ × P. ussuriensis var. ovoidea 8 cross resulted in the transmission of the highest level of resistance to their offspring, with 58% of the offspring in the 1^st highly resistant and 2^nd resistant class, presumably as an outcome of monogenic inheritance. Simulation analysis revealed Gaussian mixture distribution of phenotypes (Figure 2), observed also by Layne et al. [23] and described and illustrated by Allard [24] as attributed to monogenic inheritance with dominance for resistance and 50 percent heritability.

In all studied hybrid families, transgressive individuals more resistant or more susceptible than their respective parents were observed (Figure 1).

Figure 3 presents major trend patterns of the fire blight severity across 4 time measurements for hybrid family groups obtained by cluster analysis (three groups within each family) and mean values of parent necrosis (%). At the second stage of cluster analysis four clusters of genotypes (groups of crosses and parents) were distinguished (Figure 4). The most resistant was cluster 1, where there was only one parent i.e. P. ussuriensis var. ovoidea 8. The mean percentage of necrosis in fourth measurement for P. ussuriensis var. ovoidea 8 was equal to 2.3% (Table 4). Groups of the following crosses: ‘Doyenné du Comice’ × P. ussuriensis var. ovoidea 8; ‘Doyenné du Comice’ × P. calleryana 12 and ‘Doyenné du Comice’ × P. ussuriensis 18 belonged to the first

Conflicts of Interest

The authors declare no conflicts of interest.

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