Pear Resistance Research Articles

Contrasting genetic variation and positive selection followed the divergence of NBS-encoding genes in Asian and European pears

BackgroundThe NBS disease-related gene family coordinates the inherent immune system in plants in response to pathogen infections. Previous studies have identified NBS-encoding genes in Pyrus bretschneideri (‘Dangshansuli’, an Asian pear) and Pyrus communis (‘Bartlett’, a European pear) genomes, but the patterns of genetic variation and selection pressure on these genes during pear domestication have remained unsolved.ResultsIn this study, 338 and 412 NBS-encoding genes were identified from Asian and European pear genomes. This difference between the two pear species was the result of proximal duplications. About 15.79% orthologous gene pairs had Ka/Ks ratio more than one, indicating two pear species undergo strong positive selection after the divergence of Asian and European pear. We identified 21 and 15 NBS-encoding genes under fire blight and black spot disease-related QTL, respectively, suggesting their importance in disease resistance. Domestication caused decreased nucleotide diversity across NBS genes in Asian cultivars (cultivated 6.23E-03; wild 6.47E-03), but opposite trend (cultivated 6.48E-03; wild 5.91E-03) appeared in European pears. Many NBS-encoding coding regions showed Ka/Ks ratio of greater than 1, indicating the role of positive selection in shaping diversity of NBS-encoding genes in pear. Furthermore, we detected 295 and 122 significantly different SNPs between wild and domesticated accessions in Asian and European pear populations. Two NBS genes (Pbr025269.1 and Pbr019876.1) with significantly different SNPs showed >5x upregulation between wild and cultivated pear accessions, and > 2x upregulation in Pyrus calleryana after inoculation with Alternaria alternata. We propose that positively selected and significantly different SNPs of an NBS-encoding gene (Pbr025269.1) regulate gene expression differences in the wild and cultivated groups, which may affect resistance in pear against A. alternata.ConclusionProximal duplication mainly led to the different number of NBS-encoding genes in P. bretschneideri and P. communis genomes. The patterns of genetic diversity and positive selection pressure differed between Asian and European pear populations, most likely due to their independent domestication events. This analysis helps us understand the evolution, diversity, and selection pressure in the NBS-encoding gene family in Asian and European populations, and provides opportunities to study mechanisms of disease resistance in pear.

Methyl jasmonate regulates protective enzyme activities to improve resistance to Venturia nashicola in pear (Pyrus bretschneideri Rehd.)

Pear scab caused by Venturia nashicola is a devastating disease responsible for huge economic losses to the pear industry. The enhancement of pear resistance to scab by methyl jasmonate (MeJA) was evaluated in the susceptible cultivar ‘Xuehua’ and the resistant cultivar ‘Huangguan’ of Chinese pear (Pyrus bretschneideri Rehd.). The regulatory mechanism of MeJA was analyzed by measuring the activities of four protective enzymes and expression levels of corresponding genes. The results showed that MeJA inhibited the growth of V. nashicola on oat agar medium. The in vitro growth-inhibitory effect of MeJA was enhanced with increasing MeJA concentration over the range 0.05–2 mmol L− 1, and the difference was significant at 0.1 mmol L− 1 or higher concentration levels (p < 0.05). Except peroxidase, all the other enzymes exhibited higher activities in ‘Huangguan’ pear than in ‘Xuehua’ pear after inoculation. Treatment with 0.1 mmol L− 1 MeJA substantially enhanced all the protective enzyme activities in inoculated leaves of the two pear cultivars, with increased expression of related genes. These results indicate that MeJA improves resistance of pear to scab by increasing protective enzyme activities.

Susceptibility, heterosis and heterobeltiosis evaluations for fire blight resistance in pear

Susceptibility, heterosis and heterobeltiosis evaluations for fire blight resistance in pear

Inheritance pattern of fire blight resistance in pear

Fire blight caused by Erwinia amylovora is a serious disease that directly affects pear agriculture and production around the world. The most effective way to struggle with disease is develop new varieties and rootstocks. In this study, for a better understanding of the genetic base of fire blight such as heritability of disease resistance, breeding values of parents and maternal effect were investigated on F1 hybrid pear population. According to results, disease resistance shows polygenic inheritance and multiple factors were effective in heredity. Resistance to disease is transmitted to next generations by both parents. However, the influence of the female parent on inheritance is much greater than pollinator. ‘Magness’ and ‘Mustafa Bey’ varieties were found more effective for the transmission of disease resistance as female parents to next progenies. Similar results were obtained from ‘Kiefer’ and ‘Limon’ as pollinator. In addition to the importance of the parents exclusively in disease resistance, gene interactions between the parents also had a significant effect on this matter. Heritability of fire blight that was calculated from the non-additive genes was 23%, likewise 32% was calculated from additive genes. Broad-sense heritability of fire blight, representing the sum of this two situations was 55%. Maternal gene effects were greater than the pollinator and maternal effect had a 7% effect on the disease resistance, indicating that the cytoplasmic gene effects had important roles in the resistance. Obtained results will form basis for the development of new varieties that become resistant to fire blight by hybridization.

A QTL detected in an interspecific pear population confers stable fire blight resistance across different environments and genetic backgrounds

Fire blight, caused by the bacterium Erwinia amylovora (Burrill) Winslow et al., is one of the most serious diseases of pear. The development of pear cultivars with a durable resistance is extremely important for effective control of fire blight and is a key objective of most pear breeding programs throughout the world. We phenotyped seedlings from the interspecific pear population PEAR3 (PremP003, P. × bretschneideri × P. communis) × ‘Moonglow’ (P. communis) for fire blight resistance at two different geographic locations, in France and New Zealand, respectively, employing two local E. amylovora isolates. Using a genetic map constructed with single nucleotide polymorphism (SNP) and microsatellite (SSR) markers previously developed for this segregating population, we detected a major quantitative trait locus (QTL) on linkage group (LG)2 of ‘Moonglow’ (R 2 = 12.9–34.4 %), which was stable in both environments. We demonstrated that this QTL co-localizes with another major QTL for fire blight resistance previously detected in ‘Harrow Sweet’ and that the two favorable (i.e., resistant) alleles were not identical by descent. We also identified some smaller effect (R 2 = 8.1–14.8 %) QTLs derived from the susceptible parent PEAR3. We propose SNP and SSR markers linked to the large effect QTL on LG2 as candidates for marker-assisted breeding for fire blight resistance in pear.

Identification of a QTL for psylla resistance in pear via genome scanning approach

Most current breeding programs in pear are not only aimed to improve the fruit quality, but also resistance to pathogens and pests, such as psylla (Cacopsylla pyri). For this purpose seedlings derived from the crossing ‘NY10353’בDoyenne du Comice’ were grafted on quince and tested for resistance to psylla in a tunnel, considering as parameters both the number of adults (antixenosis) and of nymphs (antibiosis) found on each plant. A “Genome Scanning Approach” was then performed on 14 seedlings selected among those showing the highest degree of resistance. This approach made it possible to identify a putative QTL on chromosome 17 of the resistant parent ‘NY10353’, that was further confirmed by the analysis of the entire population. The presence of this QTL was stable in all the three years of phenotyping by using as phenotypic parameter the number of nymphs. No associations were found on the susceptible parent ‘Doyenne du Comice’. Molecular markers located in this region represent a first tool to setup a MAS approach to select new pear genotypes resistant to psylla.

Genetic mapping and pyramiding of two new pear scab resistance QTLs

Scab is one of the major fungal diseases infecting pear trees, causing the greatest economic losses. Identifying and pyramiding scab resistance factors should help in breeding new resistant pear cultivars. We have identified and mapped two new pear resistance loci against the fungal pathogen Venturia pirina. The first locus, mapped both as a major gene and as a QTL, is located on linkage group (LG) 01 of the hybrid P3480, deriving from the European pear cultivar ‘Wilder.’ It colocalizes with the Vnk resistance gene found in the Asian pear cultivar ‘Kinchaku’ against Venturia nashicola. A second locus, mapped as a QTL, is located on LG04 of the interspecific cultivar ‘Euras.’ In a small ‘Euras’ × P3480 progeny, seven seedlings carrying the resistant alleles at both loci have been selected using SSR markers.

Candidate Resistant Genes of Sand Pear (Pyrus pyrifolia Nakai) to Alternaria alternata Revealed by Transcriptome Sequencing.

Pear black spot (PBS) disease, which is caused by Alternaria alternata (Aa), is one of the most serious diseases affecting sand pear (Pyrus pyrifolia Nakai) cultivation worldwide. To investigate the defense mechanisms of sand pear in response to Aa, the transcriptome of a sand pear germplasm with differential resistance to Aa was analyzed using Illumina paired-end sequencing. Four libraries derived from PBS-resistant and PBS-susceptible sand pear leaves were characterized through inoculation or mock-inoculation. In total, 20.5 Gbp of sequence data and 101,632,565 reads were generated, representing 44717 genes. Approximately 66% of the genes or sequenced reads could be aligned to the pear reference genome. A large number (5213) of differentially expressed genes related to PBS resistance were obtained; 34 microsatellites were detected in these genes, and 28 genes were found to be closely related to PBS resistance. Using a transcriptome analysis in response to PBS inoculation and comparison analysis to the PHI database, 4 genes (Pbr039001, Pbr001627, Pbr025080 and Pbr023112) were considered to be promising candidates for sand pear resistance to PBS. This study provides insight into changes in the transcriptome of sand pear in response to PBS infection, and the findings have improved our understanding of the resistance mechanism of sand pear to PBS and will facilitate future gene discovery and functional genome studies of sand pear.

Preparatory work for the pest categorisation of three bacteria listed in the annexes of the Council directive 2000/29/EC Erwinia amylovora Winslow et al. Xylophilus ampelinus (Panagopoulos) Willems et al. Clavibacter michiganensis subsp. insidiosus(McCulloch) Davis et al

Preparatory work for the pest categorisation of three bacteria listed in the annexes of the Council directive 2000/29/EC Erwinia amylovora Winslow et al. Xylophilus ampelinus (Panagopoulos) Willems et al. Clavibacter michiganensis subsp. insidiosus(McCulloch) Davis et al

Inheritance of fire blight resistance in Asian &amp;lt;i&amp;gt;Pyrus&amp;lt;/i&amp;gt; species

Since several Asian pear species are considered to be potential source of fire blight resistance, we crossed “Doyenne 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 “Doyenne 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 “Doyenne 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.

Isolation, characterization and expression analysis of resistance gene candidates in pear ( Pyrus spp.)

According to the conservative regions of the nucleotide-binding site and leucine-rich repeat domain (NBS–LRR) in resistance genes (R-gene), a homology-based cloning method was used to isolate disease resistance gene candidates (RGCs) from six species of pear, including Pyrus betulaefolia, P. bretschneideri, P. pyrifolia, P. ussriensis, P. sinkiangensis, P. communis and their interspecific hybrids. Approximately 100 disease resistance gene candidates from 39 cultivars in these pear species were identified. Among these RGC, 98 of genomic sequences which could be translated into polypeptides without stop codons, while the other two sequences presented multiple stop codons. The deduced amino acid sequences of the 98 RGC displayed high diversity, ranging from 20 to 100%, and could be divided into 17 distinct RGC families ( RGC 1– RGC 17) based on the phylogenetic analysis with the threshold of 68% identity. The 98 RGC contained both the toll interleukin receptor (TIR) group and non-toll interleukin receptor (non-TIR) group. RGC families RGC 16 and RGC 17 belonged to non-TIR group, the other 15 RGC families were classified to TIR group. Sequences analysis indicated that there was a strong identity of these RGCs to the known R-gene and RGC from other plants. In each RGC family, one representative RGC was selected to test the expression profile at transcriptional level, and all the 17 selected RGCs could be detected at mRNA level. In response to Venturia nashicola inoculation or salicylic acid (SA) treatment, the expression level of RGC Pb-Zs1 (RGC 1) from pear scab-resistant cultivar ‘Zaosu’ pear changed while the expression level of RGC Pb-Dshs2 (RGC 8) from pear scab-susceptible pear cultivar ‘Dangshansu’ seemed less affected. These RGCs isolated in this study could help understand the R-gene in pear species and will serve as a potential resource for future improvement of disease resistance in pear.

Molecular cloning and expression analysis of genes related to phosphatidic acid synthesis in Japanese pear leaves inoculated with Venturia nashicola

Phosphatidic acid, which can be generated by the action of phospholipase D (PLD) and by the combined action of phospholipase C (PLC) and diacylglycerol kinase (DGK), is a plant defense signal. To identify the role of the PLD, PLC, and DGK genes encoding these enzymes in pear resistance to the scab fungus Venturianashicola, we report the cloning of these genes and analysis of their expression in inoculated pear leaves. The results showed that PLD mRNA increased in the leaves of the susceptible pear cv. Kousui 1 day after inoculation (dai), then decreased to the basal level. In resistant pear cv. Kinchaku, PLD mRNA did not change significantly except for a small increase at 2 and 3 dai. However, the level of PLD mRNA in Kinchaku was higher than in Kousui 2 dai. The level of PLC mRNA significantly increased in cvs. Kousui and Kinchaku by 1 dai, peaked at day 2, then decreased to the basal level. Thus, the gene expression pattern did not differ between the two cultivars. PLC gene expression was also induced by environmental stress. The DGK gene seemed to be constitutively expressed and was not induced by inoculation in either the susceptible or resistant cultivars. Together, this data showed that both PLD and PLC are possibly involved in a defense response to V. nashicola in Japanese pear leaves, and the PLD gene seems to play a more important role than does the PLC gene.

Expression of a bacterial effector, harpin N, causes increased resistance to fire blight in Pyrus communis

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. In order to create novel mechanisms for fire blight resistance in pear, we have generated transgenic pears expressing the elicitor harpin Nea from Erwinia amylovora under the control of the constitutive promoter CaMV35S. The transient expression of hrpNEa in pear cells did not provoke any apparent damage. Therefore, stable constitutive expression of hrpNEa was studied in seventeen transgenic clones of the very susceptible cultivar “Passe Crassane.” Most transgenic clones displayed significant reduction of susceptibility to fire blight in vitro when inoculated by E. amylovora, which was positively correlated to their degree of expression of the transgene hrpNEa. These results indicate that ectopic expression of a bacterial elicitor such as harpin Nea is a promising way to improve pear resistance to fire blight.

Activation of three pathogen-inducible promoters of tobacco in transgenic pear (Pyrus communis L.) after abiotic and biotic elicitation.

In order to improve pear resistance against fire blight caused by Erwinia amylovora, a search for promoters driving high-level expression of transgenes specifically in response to this bacterial pathogen has been undertaken. We have examined the ability of hsr203J, str246C and sgd24 tobacco (Nicotiana tabacum L.) promoters to drive expression of the uidA reporter gene in pear. Transgenic pear clones were obtained by Agrobacterium tumefaciens-mediated transformation. Beta-glucuronidase activity was determined quantitatively and qualitatively in these plants grown in vitro using fluorometric and histochemical assays and compared to cauliflower mosaic virus (CaMV) 35S promoter-driven activity. The hsr203J promoter appeared to be very weakly activated following inoculation in pear, which is the converse of the situation in tobacco. The str246C promoter was rapidly activated in pear during compatible and incompatible interactions, by wounding and following the application of several elicitors (capsicein, cryptogein, harpin, salicylic acid and jasmonic acid). The sgd24 promoter, a deletion derivative of str246C, exhibited a low level of expression after bacterial inoculation, was weakly activated by wounding and elicitors, and was not activated by phytohormones (salicylic acid and jasmonic acid). Interestingly, the sgd24 promoter was locally activated in pear, whereas the str246C promoter was activated systemically from the infection site. Taken together, these data show that, although the s tr246C and sgd24 promoters are less active than the CaMV35S promoter in pear, their pathogen-responsiveness would permit them to be used to drive the expression of transgenes to promote bacterial disease resistance.

Pear transformed with a lytic peptide gene for disease control affects nontarget organism, pear psylla (Homoptera: Psyllidae).

The biology and behavior of pear psylla, Cacopsylla pyricola Foerster, on a transgenic clone of 'Bartlett' pear, Pyrus communis L., containing a synthetic antimicrobial gene, D5C1, was compared with that of a nontransgenic parental clone to determine whether there were any nontarget effects. The gene construct also contained the marker gene nptII (aminoglycoside 3'-phosphotransferase II) that encodes for antibiotic resistance to identify transformed plants. The purpose of the original transformation was to enhance pear resistance to the bacterial disease fireblight caused by Erwinia amylovora (Burr.) Winslow et al. The biology and behavior of pear psylla on a transgenic clone were compared with a nontransgenic parental pear clone in short- (< or = 7-d) and long-term (32-d) studies. Short-term studies indicated pear psylla adults preferred to settle and oviposit, and nymphs fed more and developed slightly faster, on transgenic pear compared with nontransgenic pear. In contrast, a long-term study on psylla colony development showed considerably fewer eggs, nymphs, and adults were produced on transgenic pear. Although adults reared on transgenic pear did not have weight affected, females produced fewer eggs and nymphal hatch was significantly reduced on the transgenic pear clone. Our results suggest that pear psylla biology and behavior are initially enhanced on this transgenic pear clone. However, chronic exposure of psylla populations to transformed pear plants that express the nptII marker and lytic peptide genes had detrimental effects on pear psylla reproductive biology. Field studies would be required to determine the specific effects of each gene on pear psylla biology and behavior and whether these effects would be expressed under natural conditions. The four-fold reduction in psylla population levels that resulted on this disease resistant transgenic pear line would be an added benefit to a pear integrated pest management (IPM) program. Overall, this study demonstrates that genetically altering plants to control one particular organism can have unintentional yet beneficial effects against other nontarget pest organisms in agricultural crops.

COMPARISON OF DISEASE PROGRESSION OF FIRE BLIGHT ON APPLE AND PEAR CULTIVARS

Three pear and three apple cultivars with different susceptibility to fire blight were used in this experiment. Artificial inoculations were carried out on orchard trees during 1991 in south-west Germany. Disease assessment was made over time to obtain disease progress curves. Symptomp development on pear cultivars was faster than on apple cultivars and reached to a higher asymptote. The monocyclic experimentation with Erwinia amylovora on pear and apple shoots resulted in disease progress curves that fit well by the monomolecular growth model. In this experiment Ymax, Yo and r were decreasing together with the area under the disease progress curve (AUDPC) as resistance was increasing in cultivars. The longest latent period was associated with resistant cultivars. Because AUDPC summarizes plant disease development, it should be used in the screening programs as one of the criteria for determining the varietal level of resistance in relation to standard cultivars. We suggest this epidemiological approach, especially in the later stages of the screening program where a few number of varieties are used, for blight resistance in pear and apple breeding programs. From this more accurate determination of relative resistance, selections and better comparisons of varieties for resistance to blight will result.

Estimation of Heritability and Combining Ability for Fire Blight Resistance in Pear1

Abstract Heritability estimates for fire blight resistance in pear were obtained by regressing progeny means on midparental phenotypes. Approximately half of the variability in resistance in pear was additive (h2 = 0.52), but there was also evidence for nonadditive genetic effects compatible with a proposed qualitative gene for sensitivity. A method was established to estimate relative average combining ability for fire blight resistance. Progeny means of individual parents were adjusted to the grand progeny mean of 8 intercrossed testers based on common progeny.

Relationship between Fire Blight Resistance and Fruit Quality in Pear1

Abstract Phenotypic correlations were estimated between 7 fruit quality characters and resistance to fire blight, caused by Erwinia amylovora (Burrill) Winslow et al., in crosses involving various species of pear (Pyrus spp.). Correlations were generally negative, small, and usually not significantly different from zero. Some significant negative correlations were found between fruit russett and appearance with fire blight resistance within crosses involving P. communis L. and hybrids of P. communis × P. pyrifolia (Burm.) Nakai, but none exceeded −0.30. Low correlations between fruit quality and fire blight resistance in progeny of diverse crosses indicate no genetic impediments to selection for combined high quality and blight resistance in pear.