Isolation of resistance gene analogs from grapevine resistant and susceptible to downy mildew and anthracnose

Downy mildew and anthracnose are major diseases of the grapevine (Vitis spp.) cultivars grown in Thailand. Due to the deleterious effects of fungicides frequently used for disease management in grapevine, disease resistance genes have been sought after with the ultimate goal of developing new cultivars with improved disease resistance levels. In this study, nucleotide-binding site (NBS)-leucine rich repeat (LRR) class of resistance gene analogs (RGAs) were cloned by PCR amplification using degenerate primers specific to P-loop and GLPL, conserved regions of NBS. Ninety-one clones containing putative RGA sequences were obtained from a downy mildew and anthracnose resistant hybrid ‘NY88.0507.01’ and a susceptible cultivar ‘Black Queen’. These cloned sequences were subdivided into 14 groups based on their nucleotide sequence similarity of 90% or greater. BLASTx of fourteen selected clones showed the highest amino acid sequence similarity with known NBS-LRR proteins or putative resistance (R) protein candidates. Multiple alignments of these representative RGAs with 5 known R proteins revealed conserved P-loop, kinase-2, RNBS and GLPL motifs which are typical components of the NBS-LRR proteins. Four RGAs had at least 40% identity with known R proteins. Phylogenetic analysis demonstrated that the representative RGAs from both resistant and susceptible grapevines dispersed along the phylogram on the two major branches of either TIR (Drosophila Toll and mammalian Interleukin-1 Receptor) or non-TIR type of the NBS-LRR proteins.     

Induction of resistance in Yali pear (Pyrus bretschneideri Rehd.) fruit against postharvest diseases by acibenzolar-S-methyl sprays on trees during fruit growth

The objective of this work was to evaluate how disease resistance in harvested fruit of Yali pears (Pyrus bretschneideri Rehd.) may be affected by acibenzolar-S-methyl (ASM) sprays on trees. Results showed that disease incidence and lesion diameter in mature pears from trees sprayed with ASM for three times during growth and inoculated with Penicillium expansum or Alternaria alternata after harvest were 27.9 and 42.7%, or 29.1 and 23.4% lower, respectively, than in control fruit 17 days after inoculation. Mature fruit from ASM-treated trees exhibited higher activities of defense enzymes including phenylalanine ammonia-lyase (PAL), chitinase and β-1,3-glucanase than in control at harvest. In young pears, activities of peroxidase, polyphenoloxidase, PAL, chitinase and β-1,3-glucanase were significantly enhanced by ASM after first spray on trees. The ASM spray also significantly increased H₂O₂ level and glutathione reductase activity, but reduced activities of catalase and ascorbate peroxidase in young pears. The study indicated that enhancement of disease resistance in harvested Yali pear fruit could be the result of multiple effects of several factors related to plant defenses induced by ASM sprays on trees during fruit growth. Application of ASM in the field holds great promise for controlling postharvest diseases of the fruit.     

Genetic relationships of pear cultivars in Xinjiang,China, as measured by RAPD markers

Summary

Thirteen native pear species have been identified in China, of which P. armeniacaefolia Yu and P. sinkiangensis Yu are specific to Xinjiang. P. armeniacaefolia grows wild and a few cultivars have been assigned to this species. Cultivars of P. sinkiangensis have been suspected to be of hybrid origin involving P. communis L. and P. bretschneideri Rehd. In this study, traditional pear cultivars in Xinjiang were evaluated using RAPD markers and compared with representatives of Occidental pear species, cultivars of P. communis and East Asian pear accessions. The combination of 72 pear accessions and 20 selected primers produced 231 scorable polymorphic RAPD bands, of which some were specific to certain species. Five main groups of pear accessions could be distinguished from UPGMA analysis: 1) P. xerophila Yu, its relatives and one cultivar of P. ussuriensis Max., 2) cultivars of P. sinkiangensis, 3) cultivars of P. pyrifolia Nakai and P. bretschneideri, 4) wild Occidental species, cultivars of P. communis and P. armeniacaefolia, and 5) hybrids between P. communis and Chinese or Japanese pear cultivars. The result of PCA generally agrees with that based on UPGMA. Based on RAPD data, some cultivars traditionally classified as P. bretschneideri should be assigned to P. sinkiangensis. Some heirloom cultivars assigned to P. communis were found to be of hybrid origin involving the Chinese white pear (P. bretschneideri) or sand pear (P. pyrifolia). Our results confirmed that P. sinkiangensis is of hybrid origin and at least P. communis, P. armeniacaefolia and Chinese white pears or sand pears have been involved. A further study is needed to understand how pear species and cultivars in Xinjiang are related to those originated from countries in Central Asia.     

Identification of S-alleles in pear (Pyrus communis L.) cv. ‘Rocha’ and other European cultivars

In this work we report the cloning and identification of S-RNase alleles responsible for gametophytic self-incompatibility (GSI) of ‘Rocha’ pear and of 13 other European pear cultivars that might be used as its pollinators. Partial sequences of S-RNase alleles were amplified by PCR with specific primers hybridising in conserved regions of previously identified S-RNase alleles of Pyrus communis, cloned and sequenced and the S-genotype of eight pear cultivars was fully determined. Three cultivars (‘General Léclerc’ (SqSl), ‘Tosca’ (SbSl) and ‘Alexandrine Douillard’ (SbSk)) shared no S-alleles with ‘Rocha’ (SaSe) and shall be totally compatible with this cultivar. None of the cultivars analysed showed an identical amplification pattern to the one observed in ‘Rocha’, so the other cultivars shall be at least semi-compatible. One new allele was identified in P. communis cv. ‘Beurré d’Avril’ (designated as St). The determination of both S-RNase alleles of cvs ‘Rocha’, ‘Beurré Precoce Morettini’ (SeSk) and ‘Tosca’ and the identification of one S-RNase allele in cvs ‘Carapinheira’ (Sb), ‘Amêndoa’ (Se), ‘Pérola’ (Sk) and ‘Beurré d’Avril’ (St) are important contributions for the effort recently developed worldwide to establish groups of sexual compatibility among European pears.     

Study of the structure and biosynthetic pathway of lignin in stone cells of pear

Pear stone cell content is one of key determinants of fruit quality. Stone cells form by the deposition of lignin on primary cell walls, following secondary thickening of cell walls. Studies of the structure and metabolic pathway of pear lignin are rare. Stone cell and lignin content in the pulp of Pyrus bretschneideri cv. Dangshan Su were determined during fruit development. Lignin was extracted, purified and analyzed by ultraviolet (UV) light, Fourier-transform infrared (FTIR) spectroscopy and ¹H nuclear magnetic resonance (¹H NMR) spectroscopy. Intermediates of lignin biosynthesis were detected by high-performance liquid chromatography (HPLC). The results show that lignin content increases initially and decreases afterwards during fruit development, peaking twice at day 47 and day 63 after flowering. The lignin peaks precede both peaks of stone cell content (day 51 after flowering) and at the point at which sclereids reach their maximum diameter (day 67 after flowering). The milled wood lignin in pulp was identified as guaiacyl-syringyl-lignin by spectroscopic analyses. In the FTIR spectra, the peak intensity ratio of A₁₂₆₉/A₁₂₂₇ was 1.25. HPLC analyses detected cinnamic acid and p-coumaric acid, but not caffeic acid or ferulic acid. In conclusion, during pear fruit development, lignin is first biosynthesized, and subsequently deposited on cell walls to form stone cells and sclereids. The biosynthesis of guaiacyl-syringyl- lignin in pear pulp may involve a phenylpropane-type metabolic pathway from phenylalanine to cinnamic acid and acyl-CoA ester, as pear pulp lignin contains more guaiacyl units than syringyl units.     

Syrian pear (Pyrus syriaca) as a pollinator for European pear (Pyrus communis) cultivars

In Israel four European pear cultivars are grown: ‘Spadona’ is the main cultivar and ‘Coscia’, ‘Gentile’ and ‘Spadochina’ are its pollinators. However, molecular S-genotyping revealed that ‘Spadona’ is semi-compatible with its three pollinators. This explains, at least in part, the relatively low pear yield in Israel. The Syrian pear (Pyrus syriaca) grows wild in Israel and blooms intensively, overlapping the blooming of the cultivated European pears. Cross-fertilization between Syrian pear and ‘Spadona’ was shown to be efficient suggesting that Syrian pear might be a potent pollinator for ‘Spadona’. Twenty-six Syrian pear seedlings, from different sites in north-east Israel were S-genotyped identifying 11 that are fully compatible with the four European pear varieties cultivated in Israel. By this screening, 24 different S-RNases were cloned; ten of them are new, whereas the other fourteen had been identified previously. In addition, seedlings of two wild pear species were also S-genotyped. Two seedlings from Pyrus betulifolia and one from Pyrus korshinskii were found to be genetically compatible with the four European pear cultivars. From these seedlings four S-RNases were cloned, two are new, one had been cloned previously and one was identical to an S-RNase allele cloned from Syrian pear in this work.     

梨20个品种S基因型的鉴定及新S-RNases基因克隆

 为了鉴定我国梨品种和一些野生类型个体的S基因型,应用S-RNase特异PCR扩增、克隆和测序,对其S-RNases基因核苷酸序列进行分析,鉴定了20个梨品种和野生类型个体的S基因型。起源于我国的'奥连'(S_pS₃₂)、'吊蛋'(S_dS_e)、'沙疙瘩'（S₃₆S_d）品种和杏叶梨的一个类型（S₂₂S_c）个体中存在西洋梨的S-RNase基因,证明S-RNase基因分化是在东方梨种群和西方梨种群的各个种形成之前。在秋子梨的'麦梨'、'内蒙古山梨'中发现了2个新S-RNases基因,命名为S₄₀-、S₄₁-RNase（DQ903313、DQ988687）。S₄₀-和S₄₁-RNase基因推导的部分氨基酸序列分别与苹果属S₁₁-和S₆-RNase的同源性为100%和94.4%,这表明S-RNase的存在可能在梨属和苹果属形成之前。     

Functional changes of retinal ganglion cells in rd1 mice during midterm of retinal pigmentosa

AIM: To investigate how the function of retinal ganglion cells (RGCs) change in the midterm of retinal pigmentosa (RP) in rd1 mice (a transgenic animal model of RP). METHODS: The action potentials from multiple RGCs in rd1 mice at postnatal 20 d (P20) or normal C57 mice (control) were simultaneously recorded by multi-electrode array recording. The functional changes of surviving ganglion cells were evaluated by comparing spontaneous and light-evoked activities of RGCs between rd1 and control mice. The extent of photoreceptor degeneration was verified by immunohistochemical staining. RESULTS: Immunohistochemistry results showed the thickness of the retinal photoreceptor layer of rd1 mice was significantly lower than that in normal mice at P20. According to the light response properties, we classified ganglion cells into 6 subgroups: ON sustained, ON transient, ON-OFF sustained, ON-OFF transient, OFF sustained and OFF transient RGCs, with a very tiny percentage of OFF sustained RGCs (1.0%~3.1%). The percentage of RGCs remaining light responsive in rd1 mice was significantly lower than that in C57 mice. The average spontaneous spiking rate for rd1 RGCs was overall significantly increased compared to that in C57 cells, whereas different RGC types had different changes. The light-induced responses and light sensitivities of all types of RGCs in rd1 mice were both significantly lower than those in C57 mice. CONCLUSION: The photoreceptors of rd1 mice are severely degenerated in the midterm of retinal degeneration. The functions of RGCs in rd1 mice in the midterm of degeneration decay obviously, with variance in different RGCs types.     

Relationship between inbreeding coefficients and plant height of 1-year-old seedlings in crosses among Japanese pear (Pyrus pyrifolia Nakai) cultivars/selections

Plant height, a vigor trait, in 1-year-old seedlings made from Japanese pear (Pyrus pyrifolia) cultivars/selections was measured using 994 individuals from 29 families. The family mean of plant height was negatively correlated (r = −0.72**) to the inbreeding coefficients (F). The regression of the family mean (Fm) on the F value (Fm = 130 − 104F) showed that inbreeding depressions were 8%, 20%, and 40% for F = 0.1, 0.25, and 0.5, respectively. According to the regression, the family mean at F = 0 was estimated at 130 cm. These results showed that the vigor was greatly influenced by inbreeding in Japanese pear. Within-family variances, the genetic segregation of offspring in a family, differed according to family. The proportions of offspring with plant height above 130 cm (estimated Fm for F = 0) were extremely low, i.e., 0–17% for 0.5 ≤ F < 0.60 and 0–8% for F = 0.75.     

Antimicrobial,Antioxidant, and Antimutagenic Activities of Five Turkish Pear Cultivars

The pear (Pyrus spp.) is one of the most important fruits consumed in daily life. The aim of this study was to determine the total phenolic and ascorbic acid contents, as well as the antimicrobial, antioxidant, and antimutagenic activities, of various pear cultivars. The fruits of five pear (Pyrus communis L.) cultivars (‘Deveci’, ‘Kizil’, ‘Egirsah’, ‘Gugum’, and ‘Banda’) were used in this study. It was determined that the peel and pulp of the ‘Kizil’ pear had the highest total phenolic content (402.5?mg/100?g and 215.2?mg/100?g, respectively), while those of the ‘Banda’ pear had the lowest total phenolic content (326?mg/100?g and 126.1?mg/100?g, respectively). Additionally, the ‘Kizil’ pear showed the highest antioxidant capacity in the 2,2’-azino-bis(3-ethylbenzothiazoline-6-6-sulphonic acid) (ABTS) and ferric-reducing ability assay (FRAP) (1.72 μmol TE/g FW and 161.25?μmol Fe II/g FW, respectively) and the highest ascorbic acid content (16.02?mg/100?g). The ‘Banda’ pear showed the highest antibacterial activity against the test bacteria. However, none of the pear extracts displayed antifungal activity. While all doses of the ‘Kizil’, ‘Gugum’, and ‘Banda’ pear extracts used in this study, except 80?μL/plate, exhibited antimutagenic activities, only the lowest dose (10?μL/plate) of the ‘Deveci’ pear extract showed the antimutagenic activity against induced mutagenesis in the Salmonella typhimurium TA 98 strain. Consequently, the five Turkish pear cultivars used in this study exhibited different levels of antimicrobial, antioxidant, and antimutagenic activities.     

Effect of silicon on antioxidant and stomatal response of two grapevine (Vitis vinifera L.) rootstocks grown in boron toxic,saline and boron toxic-saline soil

We investigated individual and combined effects of B toxicity and salinity in the presence or absence of silicon on the shoot growth, concentrations of sodium (Na), chloride (Cl), boron (B) and silicon (Si), and stomatal resistance (SR), lipid peroxidation (MDA), proline accumulation, H₂O₂ accumulation and the activities of major antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT and ascorbate peroxidase, APX) activity grapevine rootstocks of 41B (V. Vinifera × V. Berlandieri) and 1103P (V. Berlandieri × V. Rupestris). Applied Si counteracted the deleterious effects of salinity and boron toxicity on shoot growth by lowering the accumulation of Na in 1103P, and B and Cl in the both rootstocks. Stomatal resistance, MDA, and the concentrations of H₂O₂ and proline were higher in the plants grown in conditions of B toxicity, salinity and their combination while applied Si lowered these parameters. Lowering SOD and CAT but increasing APX, Si treatment significantly affected the enzyme activities of both rootstocks. Based on the present work, it can be concluded that Si alleviates the adverse effects of salinity, B toxicity and combined salinity-B toxicity on grapevine rootstocks by preventing both oxidative membrane damage and translocation of Na and B from root to shoots and/or soil to plant, and also lowering the phytotoxic effects of Na and B within plant tissues. When considering the antioxidative response and membrane systems, it was concluded that the rootstock 1103P was responsive to Si under stress conditions. To our knowledge, this is the first report that Si improves the combined salt and B tolerance of grapevine grown under saline, B toxic, and B toxic and saline conditions which describes membrane related parameters and antioxidant responses.     

Identification of organic acid-related genes and their expression profiles in two pear (Pyrus pyrifolia) cultivars with difference in predominant acid type at fruit ripening stage

‘Yandangxueli’ is a pear cultivar with predominant citric acid in the ripe fruit, different from most of pear cultivars such as ‘Gengtouqing’ in which malic acid is the predominant acid type. It was found that ‘Yandangxueli’ accumulated citric acid for three times against that in ‘Gengtouqing’ at fruit ripening stage. To investigate the mechanism of citric acid accumulation in ‘Yandangxueli’, organic acids content, gene expression and enzyme activity were studied in both cultivars. Five genes, Pp:mtCs, Pp:cyAco, Pp:cyIdh, Pp:mtMdh and Pp:cyMe which encoded citric synthase (CS), cytosolic aconitase (cyACO), NADP-dependent isocitrate dehydrogenase (NADP-IDH), NAD-dependent malate dehydrogenase (NAD-MDH) and NADP-dependent malic enzyme (NADP-ME) respectively, were identified from pear fruit. Their expression profiles and the corresponding enzyme activities were determined throughout fruit development in both cultivars. Results from these enzymes indicated that there were no strict relationship between gene expression, enzyme activity and citric acid accumulation. Expression analysis for two Py:vVAtp genes encoding vacuolar H⁺-ATPase A subunit and one Py:vVpp gene encoding Vacuolar H⁺-pyrophosphatase showed that they were all with up-regulated expression at the later development stage of ‘Yandangxueli’ but with down-regulated expression in ‘Gengtouqing’. Therefore, it is concluded that the different ability in citric acid transportation and storage might be involved in the high citric acid content in ‘Yandangxueli’.     

Evaluation of strains of Poncirus trifoliata and trifoliate orange hybrids for resistance to Phytophthora root rot

Ten strains of Poncirus trifoliata (L.) Raf. and 6 trifoliate orange hybrids were evaluated for resistance to root rot caused by Phytophthora nicotianae B. de Haan var. parasitica (Dast.) Waterh. A high degree of resistance was shown by Rubidoux trifoliate and Trifoliate orange (Srirampur). Trifoliate orange hybrids, in general, were less resistant than P. trifoliata strains. The usefulness of different parameters in the assessment of root damage are briefly discussed.     

Defense response of tomato fruit at different maturity stages to salicylic acid and ethephon

In order to elucidate whether fruit maturity stage influence the induced resistance of exogenous elicitors in tomato and the involved mechanisms, we investigated the defense responses of tomato fruits against Botrytis cinerea, ethylene production and internal quality following treatments of fruit with salicylic acid (SA) or ethephon (ET) at mature green (MG) and breaker (BR). SA significantly suppressed decay and disease incidence in tomato fruits at both MG and BR stages, along with higher expression level of PR1 gene after 2 days of treatment. All fruits treated by SA had lower contents of ethylene and lycopene. The ET-treated fruit at both maturity stages showed lower disease incidence and higher level of PR2 and PR3 expression compared with the control fruit. ET treatment significantly enhanced ethylene and lycopene contents, and accelerated fruit ripening. Our results suggest that SA and ET induced disease resistance in fruits by mediating the expression of different pathogenesis-related genes and have different effects on fruit ripening, which in turn influences the disease resistance of tomato fruits.     

不同梨品种对梨黑斑病抗性差异研究

对17个梨栽培品种的黑斑病田间抗性进行了研究.结果表明:17个梨品种可分为抗、中抗、感、高感4个抗性类群,不同抗性群间的抗性差异明显;爱宕、早蜜、翠冠和新竺水4个品种表现抗病,大果水晶表现高度感病.     

Novel applications of motif-directed profiling to identify disease resistance genes in plants

Background

Molecular profiling of gene families is a versatile tool to study diversity between individual genomes in sexual crosses and germplasm. Nucleotide binding site (NBS) profiling, in particular, targets conserved nucleotide binding site-encoding sequences of resistance gene analogs (RGAs), and is widely used to identify molecular markers for disease resistance (R) genes.

Results

In this study, we used NBS profiling to identify genome-wide locations of RGA clusters in the genome of potato clone RH. Positions of RGAs in the potato RH and DM genomes that were generated using profiling and genome sequencing, respectively, were compared. Largely overlapping results, but also interesting discrepancies, were found. Due to the clustering of RGAs, several parts of the genome are overexposed while others remain underexposed using NBS profiling. It is shown how the profiling of other gene families, i.e. protein kinases and different protein domain-coding sequences (i.e., TIR), can be used to achieve a better marker distribution. The power of profiling techniques is further illustrated using RGA cluster-directed profiling in a population of Solanum berthaultii. Multiple different paralogous RGAs within the Rpi-ber cluster could be genetically distinguished. Finally, an adaptation of the profiling protocol was made that allowed the parallel sequencing of profiling fragments using next generation sequencing. The types of RGAs that were tagged in this next-generation profiling approach largely overlapped with classical gel-based profiling. As a potential application of next-generation profiling, we showed how the R gene family associated with late blight resistance in the SH*RH population could be identified using a bulked segregant approach.

Conclusions

In this study, we provide a comprehensive overview of previously described and novel profiling primers and their genomic targets in potato through genetic mapping and comparative genomics. Furthermore, it is shown how genome-wide or fine mapping can be pursued by choosing different sets of profiling primers. A protocol for next-generation profiling is provided and will form the basis for novel applications. Using the current overview of genomic targets, a rational choice can be made for profiling primers to be employed.

     

DNA sequencing reveals false positives during the detection of aster yellows phytoplasmas in leafhoppers

During the summer of 2001 and 2002, 850 and 865 carrot plants and 926 and 2584 leafhoppers associated with aster yellow (AY)-type disease were collected from five fields in Manitoba, Canada. DNA was extracted from 999 individual leafhoppers and 381 leaf tissues from both apparently healthy and AY-like infected carrot plants. All DNA samples were examined by PCR for the presence of phytoplasmas using three universal primer pairs P1/P6, P1/P7 and R16F2n/R2 derived from phytoplasma rDNA sequences. DNA amplification with these three primer pairs generated the expected amplification products of 1.7, 1.5 and 1.2 kb, respectively. Diluted PCR products obtained using universal primer pair P1/P6 were nested with R16F2n/R16R2n. The latter set of primers amplified DNA samples from 92 carrot plants and 83 leafhopper samples. In order to assess the diversity among insect and plant phytoplasmas, nested PCR products from all 92 carrot and 83 leafhopper samples were subjected to RFLP analysis using restriction endonucleases KpnI, MseI, and HhaI. This RFLP analysis showed similar patterns among carrot and leafhopper samples. Phytoplasmas detected in most samples belonged to the subgroup 16Sr-IA. To understand why the R16F2n/R16R2n of primers did not amplify the PCR product obtained from the first PCR in the remaining samples, four PCR products of P1/P6 from two plants (representing 16Sr-IA and 16Sr-IB) and two leafhopper samples that did not amplify with nested PCR, were used for DNA sequencing. ¹ The BLAST analysis of the obtained sequences showed that the PCR amplicons from the two carrot samples precisely matched with the GenBank sequences of known phytoplasmas. Alignments of these two sequences have shown very slight variations (transition/transversion ratio mean of 0.539) that would correspond to the minor differences at the 16S level between the 16Sr-IA and 16Sr-IB phytoplasma subgroups. The sequences of PCR products obtained from the two insect samples had similarity (>98%) with the sequences of phytoplasma in carrot except that their length differed from the carrot samples by 6 bp. They actually matched bacterial sequences from the GenBank, indicating that a single PCR using P1/P6 was not enough to detect phytoplasma in leafhoppers.