利用有序差异显示技术克隆受稻瘟病菌诱导表达的水稻cDNA的研究

利用有序差异显示技术(ODD)分析受稻瘟病菌诱导表达的水稻基因,通过反式RNA印迹进行辅助筛选,获得了37个在接种稻瘟病菌后表达量增强的水稻cDNA克隆.采用RNA印迹对其中5个克隆在接种稻瘟病菌后的表达分析表明,这些克隆在抗病以及感病的水稻株系中都具有诱导表达的特点.根据序列同源性分析,与这些克隆序列相应的同源基因可能涉及抑制病原菌生长、清除真菌毒素、传递抗病信号以及调节宿主生理状态等几方面的功能.     

Clonal Variation in Morphology of Populus Root Systems Following Irrigation with Landfill Leachate or Water during 2 Years of Establishment

Increased municipal solid waste generation in North America has prompted the use of Populus for phytoremediation of waste waters including landfill leachate. Populus species and hybrids are ideal for such applications because of their high water usage rates, fast growth, and extensive root systems. Adventitious rooting (i.e., lateral rooting from primordia and basal rooting from callus) of Populus is important for phytotechnologies to ensure successful plantation establishment with genotypes that thrive when irrigated with highly variable or specific contaminants. We evaluated differences in root system morphology following establishment with high-salinity municipal solid waste landfill leachate or uncontaminated well water (control). Populus clones (NC13460, NC14018, NC14104, NC14106, DM115, DN5, NM2, and NM6) were irrigated during 2005 and 2006 in northern Wisconsin, USA and tested for differences in morphology of lateral and basal root types, as well as fine (0–2 mm diameter), small (2–5 mm), and coarse (>5 mm) roots. Across treatments and clones, trees averaged five roots per root type. Leachate-irrigated trees had 87% (lateral) and 105% (basal) as many roots as those irrigated with water. Leachate-irrigated trees had 96% as many fine roots as watering with irrigation water, whereas trees with leachate had 112% (small) and 88% (coarse) as many roots versus water. Despite root necrosis and regrowth in 23% of the trees, leachate irrigation did not negatively affect root diameter or dry mass. Given that adequate rooting is necessary for plantation establishment, leachate and similar waste waters are viable irrigation and fertilization sources of Populus crops used as feedstocks for biofuels, bioenergy, and bioproducts.     

Effect of epiphytes on the extent of necrotic injuries of resistant and susceptible poplar clones infected with Dothichiza populea

Poplar cuttings of a resistant clone, Populus ‘Grandis’, and susceptible clones, Populus nigra ‘Italica’ and Populus ‘Robusta’, were infected with the pathogenic fungus Dothichiza populea alone, or with the pathogen and one of five strains of epiphytes antagonistic towards it (in vitro), isolated from poplar bark. The extent of injury was examined for 28 days after infection by determining the length of necrotic patches and their area as expressed in per cent of the total area of a cutting or the area of necrotic injuries caused by the pathogen alone.All the poplar cuttings of both the resistant and susceptible clones became diseased when infected with the pathogen alone. Surprisingly enough, however, the least affected clone was the susceptible P. ‘Robusta’, in which necrotic injuries covered 28% of the total area, as against 40% and 70% in the resistant P. ‘Grandis’ and the susceptible P. nigra ‘Italica’, respectively.When the cuttings were infected simultaneously with Dothichiza populea and its antagonistic epiphytes, the diseased area in the resistant clone diminished by as much as two-thirds, and in the susceptible P. nigra ‘Italica’, by one-third in comparison with the area affected by the pathogen alone. In turn, in the susceptible P. ‘Robusta’ the introduction of three out of five epiphytes stimulated the growth of the pathogenic fungus producing on average a double increase in the necrotic area. The differences in the response of the pathogen to the presence of epiphytes recorded in the susceptible clones indicate a marked influence of the plant on the nature of interactions between its epiphytic microflora and the pathogen.     

Enhanced resistance to the poplar fungal pathogen, Septoria musiva, in hybrid poplar clones transformed with genes encoding antimicrobial peptides

Plasmids, pCA1 and pCWEA1, carrying antimicrobial peptide gene(s), Ac-AMP1.2 and ESF12, were used to transform hybrid poplar clones Ogy and NM6. Peptide Ac-AMP1.2 is an analog of Ac-AMP1 which is one of the smallest chitin-binding proteins. Synthetic peptide ESF12 mimics the amphipathic -helix found in magainins. Transgene mRNA was detected in the transformed plants. When evaluated for resistance to hybrid poplar pathogen Septoria musiva with an in vitro leaf disk assay, the transformed Ogy plants showed significantly increased pathogen resistance as compared to the untransformed Ogy.     

Biomass and Genotype × Environment Interactions of Populus Energy Crops in the Midwestern United States

Using Populus feedstocks for biofuels, bioenergy, and bioproducts is becoming economically feasible as global fossil fuel prices increase. Maximizing Populus biomass production across regional landscapes largely depends on understanding genotype × environment interactions, given broad genetic variation at strategic (genomic group) and operational (clone) levels. A regional network of Populus field tests was established in the Midwest USA in 1995, 1997, and 2000 to assess relative productivity of 187 clones grown at Westport, Minnesota (45.7° N, 95.2° W); Waseca, Minnesota (only 2000; 44.1° N, 93.5° W); Arlington, Wisconsin (43.3° N, 89.4° W); and Ames, Iowa (42.0° N, 93.6° W). We evaluated biomass potential throughout plantation development and identified clones with yield substantially greater than commercial controls (Eugenei, NM6). For each site, biomass ranges (Mg ha^?1 year^?1) of the best six clones were: Westport: 2.3 to 3.9 (5 years), 8.0 to 10.1 (8 years), and 8.9 to 11.3 (10 years); Waseca: 10.4 to 13.4 (7 years); Arlington: 5.1 to 7.1 (3 years), 14.8 to 20.9 (6 years), and 16.1 to 21.1 (8 years); and Ames: 4.3 to 5.3 (4 years), 11.1 to 20.9 (7 years), and 14.3 to 24.5 (9 years). Mean biomass of the best three clones was 1.4 to 2.7 times greater than controls as trees developed at Westport (1995, 1997) and Waseca 2000. Genotype × environment interactions governed biomass production, with clone–mean rank correlations across sites ranging from 0.29 to 0.81. We identified generalist genotypes (e.g. Crandon, NC14105, NM2) with elevated biomass across the region and specialists (e.g. 7300501, 80X01015, and NC14103) with exceptional biomass at specific locations.     

Genetic and environmental influences on root development in cuttings of selected <Emphasis Type="Italic">Salix</Emphasis> and <Emphasis Type="Italic">Populus</Emphasis> clones – a greenhouse experiment

Aims

This study investigated how genetic determination of adventitious root development compared in experimental hybrid and parental Salix and Populus clones, and how soil bulk density influenced root development.

Methods

Cuttings of 11 Salix clones and 10 Populus clones were grown in pots with water, a low bulk density soil and a high bulk density soil for 4 (water) or 10 weeks (soils). Parameters relating to root development were measured.

Results

Root initiation, total root length (RL), and dry mass (DM), as well as root: shoot relationships in Salix clones exceeded that of Populus clones in all media. For Salix clones RL and DM were highest in S. matsudana?×?pentandra and for Populus clones RL and DM were generally higher in hybrid clones having P. trichocarpa parentage. Mean RL and DM for all clones were generally greater in the low bulk density soil than in the high bulk density soil. There were a greater proportion of thinner roots in the low bulk density soil than in the high bulk density soil.

Conclusions

There were significant differences in root initiation, RL, and DM among clones within each genus. Increasing soil bulk density significantly reduced root development in both Salix and Populus clones. Evaluating cutting root development in pot trials could be a useful clone selection tool in willow and poplar breeding.

     

Comparison of Compatible and Incompatible Response of Potato to Meloidogyne incognita

One susceptible (D6) and two resistant (E2 and N4) clones of Solanum sparsipilum × (S. phureja × haploid of S. tuberosum) were used to study the responses of potato roots and tubers to race 1 of Meloidogyne incognita (Kofoid &White) Chitwood. The compatible response was characterized by rapid penetration of large numbers of second-stage juveniles (J2) into roots, cessation of root growth, and occasional curving of root tips. The life cycle of M. incognita in the susceptible clone was completed in 25 days at 23-28 C. The incompatible response was characterized by penetration of fewer J2 into roots, necrosis of feeding sites within 2-7 days, and lack of nematode development. There were no differences in response of tubers from resistant and susceptible clones to nematode infection. Small numbers of J2 were detected in tubers, but they did not develop.     

Identification of the determinants of host resistantce and pathogenicity in interactions between Alternaria linicola Groves & Skolko and Linum Usitatissiumum L. accessions using multivariate analyses

Multivariate analysis of variance (MANOVA) and canonical variates analysis (CVA) were used to examine differences in host plant resistance and pathogen behaviour in interactions between Altemaria linicola and three genotypes of Linum usitatissimum, previously identified as susceptible, moderately resistant and resistant to the pathogen. Significant differences in pathogen development were found among the Linum accessions at 18, 24, and 40 h after inoculation. At 18 h after inoculation attempted penetration by the pathogen was relatively rare on all three accessions and canonical variates analysis revealed that overall differences among accessions resulted from large differences with respect to a small number of variables associated with successful penetration on the most susceptible accession. At later times after inoculation, when attempted penetration was more common, overall differences among accessions were found to result from smaller absolute differences among a group of variables which characterised the early colonisation of the host tissue. The results from these investigations are discussed in relation to recent research on the ecology of the pathogen and the importance of the timing of host responses to infection in determining host plant resistance.     

Stomatal Conductance and Sulfur Uptake of Five Clones of Populus tremuloides Exposed to Sulfur Dioxide

Plants of five clones of Populus tremuloides Michx. were exposed to 0, 0.2 or 0.5 microliter per liter SO₂ for 8 hours in controlled environment chambers. In the absence of the pollutant, two pollution-resistant clones maintained consistently lower daytime diffusive conductance (LDC) than did a highly susceptible clone or two moderately resistant clones. Differences in LDC among the latter three clones were not significant. At 0.2 microliter per liter SO₂, LDC decreased in the susceptible clone after 8 hours fumigation while the LDC of the other clones was not affected. Fumigation with 0.5 microliter per liter SO₂ decreased LDC of all five clones during the fumigation. Rates of recovery following fumigation varied with the clone, but the LDC of all clones had returned to control values by the beginning of the night following fumigation. Night LDC was higher in the susceptible clone than in the other clones. Fumigation for 16 hours (14 hours day + 2 hours night) with 0.4 microliter per liter SO₂ decreased night LDC by half. Sulfur uptake studies generally confirmed the results of the conductance measurements. The results show that stomatal conductance is important in determining relative susceptibility of the clones to pollution stress.     

Variations in the Phenolic Contents of Cocoyam Clones in Correlation to Resistance to Pythium Myriotylum

Phenolic compounds from leaves and roots of infected and healthy cocoyam clones resistant (RO1075), tolerant (RO1043), and susceptible (RO1157) to Pythium myriotylum were quantified and tested for their in vitro fungitoxicity on the causal agent of the cocoyam root rot disease. All clones, infected or not, have phenolic compounds showing fungitoxic activity. The phenolic content of the tolerant and susceptible clones is less than that observed in the resistant one meanwhile in the resistant clone RO1075, a large increase in phenolic content is observed particularly in the roots during attack by pathogen.     

Effect of epiphytes on the extent of necrotic injuries of resistant and susceptible poplar clones infected with Dothichiza populea.

Poplar cuttings of a resistant clone, Populus ‘Grandis’, and susceptible clones, Populus nigra ‘Italica’ and Populus ‘Robusta’, were infected with the pathogenic fungus Dothichiza populea alone, or with the pathogen and one of five strains of epiphytes antagonistic towards it (in vitro), isolated from poplar bark. The extent of injury was examined for 28 days after infection by determining the length of necrotic patches and their area as expressed in per cent of the total area of a cutting or the area of necrotic injuries caused by the pathogen alone.

All the poplar cuttings of both the resistant and susceptible clones became diseased when infected with the pathogen alone. Surprisingly enough, however, the least affected clone was the susceptible P. ‘Robusta’, in which necrotic injuries covered 28% of the total area, as against 40% and 70% in the resistant P. ‘Grandis’ and the susceptible P. nigra ‘Italica’, respectively.

When the cuttings were infected simultaneously with Dothichiza populea and its antagonistic epiphytes, the diseased area in the resistant clone diminished by as much as two-thirds, and in the susceptible P. nigra ‘Italica’, by one-third in comparison with the area affected by the pathogen alone. In turn, in the susceptible P. ‘Robusta’ the introduction of three out of five epiphytes stimulated the growth of the pathogenic fungus producing on average a double increase in the necrotic area. The differences in the response of the pathogen to the presence of epiphytes recorded in the susceptible clones indicate a marked influence of the plant on the nature of interactions between its epiphytic microflora and the pathogen.     

Role of hydrogen peroxide and ascorbate-glutathione pathway in host resistance to bacterial wilt of eggplant

Ralstonia solanacearum, a soil-borne bacterium causes bacterial wilt, is a lethal disease of eggplant (Solanum melongena L.). However, the first line of defense mechanism of R. solanacearum infection remains unclear. The present study focused on the role of induced H₂O₂, defense-related enzymes of ascorbate-glutathione pathway variations in resistant and susceptible cultivars of eggplant under biotic stress. Fifteen cultivars of eggplant were screened for bacterial wilt resistance, and the concentration of antioxidant enzymes were estimated upon infection with R. solanacearum. A quantitative real-time PCR was also carried out to study the expression of defense genes. The concentration of H₂O₂ in the pathogen inoculated seedlings was two folds higher at 12 h after pathogen inoculation compared to control. Antioxidant enzymes of ascorbate-glutathione pathway were rapidly increased in resistant cultivars followed by susceptible and highly susceptible cultivars upon pathogen inoculation. The enzyme activity of ascorbate-glutathione pathway correlates by amplification of their defense genes along with pathogenesis-related protein-1a (PR-1a). The expressions of defense genes increased 2.5?3.5 folds in resistant eggplant cultivars after pathogen inoculation. The biochemical and molecular markers provided an insight to understand the first line of defense responses in eggplant cultivars upon inoculation with the pathogen.     

The infection processes of Sclerotinia sclerotiorum in cotyledon tissue of a resistant and a susceptible genotype of Brassica napus

Background and Aims

Sclerotinia sclerotiorum can attack >400 plant species worldwide. Very few studies have investigated host–pathogen interactions at the plant surface and cellular level in resistant genotypes of oilseed rape/canola (Brassica napus).

Methods

Infection processes of S. sclerotiorum were examined on two B. napus genotypes, one resistant cultivar ‘Charlton’ and one susceptible ‘RQ001-02M2’ by light and scanning electron microscopy from 2 h to 8 d post-inoculation (dpi).

Key Results

The resistant ‘Charlton’ impeded fungal growth at 1, 2 and 3 dpi, suppressed formation of appresoria and infection cushions, caused extrusion of protoplast from hyphal cells and produced a hypersensitive reaction. At 8 dpi, whilst in ‘Charlton’ pathogen invasion was mainly confined to the upper epidermis, in the susceptible ‘RQ001-02M2’, colonization up to the spongy mesophyll cells was evident. Calcium oxalate crystals were found in the upper epidermis and in palisade cells in susceptible ‘RQ001-02M2’ at 6 dpi, and throughout leaf tissues at 8 dpi. In resistant ‘Charlton’, crystals were not observed at 6 dpi, whereas at 8 dpi they were mainly confined to the upper epidermis. Starch deposits were also more prevalent in ‘RQ001-02M2’.

Conclusions

This study demonstrates for the first time at the cellular level that resistance to S. sclerotiorum in B. napus is a result of retardation of pathogen development, both on the plant surface and within host tissues. The resistance mechanisms identified in this study will be useful for engineering disease-resistant genotypes and for developing markers for screening for resistance against this pathogen.     

Identification of Sources of Resistance to Four Species of Root-knot Nematodes in Tobacco

Resistance to the southern root-knot nematode, Meloidogyne incognita races 1 and 3, has been identified, incorporated, and deployed into commercial cultivars of tobacco, Nicotiana tabacum. Cultivars with resistance to other economically important root-knot nematode species attacking tobacco, M. arenaria, M. hapla, M. javanica, and other host-specific races of M. incognita, are not available in the United States. Twenty-eight tobacco genotypes of diverse origin and two standard cultivars, NC 2326 (susceptible) and Speight G 28 (resistant to M. incognita races 1 and 3), were screened for resistance to eight root-knot nematode populations of North Carolina origin. Based on root gall indices at 8 to 12 weeks after inoculation, all genotypes except NC 2326 and Okinawa were resistant to M. arenaria race 1, and races 1 and 3 of M. incognita. Except for slight root galling, genotypes resistant to M. arenaria race 1 responded similarly to races 1 and 3 of M. incognita. All genotypes except NC 2326, Okinawa, and Speight G 28 showed resistance to M. javanica. Okinawa, while supporting lower reproduction of M. javanica than NC 2326, was rated as moderately susceptible. Tobacco breeding lines 81-R-617A, 81-RL- 2K, SA 1213, SA 1214, SA 1223, and SA 1224 were resistant to M. arenaria race 2, and thus may be used as sources of resistance to this pathogen. No resistance to M. hapla and only moderate resistance to races 2 and 4 of M. incognita were found in any of the tobacco genotypes. Under natural field infestations of M. arenaria race 2, nematode development on resistant tobacco breeding lines 81-RL-2K, SA 1214, and SA 1215 was similar to a susceptible cultivar with some nematicide treatments; however, quantity and quality of yield were inferior compared to K 326 plus nematicides.     

Time-course study of the accumulation of hydroxyproline-rich glycoproteins in root cells of susceptible and resistant tomato plants infected byFusarium oxysporum f. sp.radicis-lycopersici

The accumulation of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants is thought to be involved in the defense response to pathogens. An antiserum raised against deglycosylated HRGPs from melon was used for studying the subcellular localization of these glycoproteins in susceptible and resistant tomato (Lycopersicon esculentum Mill.) root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici. A time-course of HRGP accumulation revealed that these glycoproteins increased earlier and to a higher extent in resistant than in susceptible cultivars. In the compatible interaction, increase in HRGPs was largely correlated with pathogen invasion and appeared to occur as a result of wall damage. In the incompatible interaction, HRGPs accumulated in the walls of uninvaded cells, thus indicating a possible role in the protection against fungal penetration. The occurrence of substantial amounts of HRGPs in papillae, known to be physical barriers formed in response to infection, and in intercellular spaces provides additional support to the concept that such glycoproteins play an important role in disease resistance.     

Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene

A cDNA clone of a wheat germin-like oxalate oxidase (OxO) gene regulated by the constitutive CaMV 35S promoter was expressed in a hybrid poplar clone, Populus × euramericana (`Ogy'). Previous studies showed that OxO is likely to play an important role in several aspects of plant development, stress response, and defense against pathogens. In order to study this wheat oxalate oxidase gene in woody plants, the expression of this gene and the functions of the encoded enzyme were examined in vitro and in vivo in transgenic `Ogy'. The enzyme activity in the transformed `Ogy' was visualized by histochemical assays and in SDS-polyacrylamide gels. It was found that the wheat OxO gene is expressed in leaves, stems, and roots of the transgenic `Ogy' plants and the encoded enzyme is able to break down oxalic acid. Transgenic `Ogy' leaves were more tolerant to oxalic acid as well as more effective in increasing the pH in an oxalic acid solution when compared to untransformed controls. In addition, when leaf disks from `Ogy' plants were inoculated with conidia of the poplar pathogenic fungus Septoria musiva, which produces oxalic acid, the OxO-transformed plants were more resistant than the untransformed controls.     

Local and systemic production of nitric oxide in tomato responses to powdery mildew infection

Various genetic and physiological aspects of resistance of Lycopersicon spp. to Oidium neolycopersici have been reported, but limited information is available on the molecular background of the plant–pathogen interaction. This article reports the changes in nitric oxide (NO) production in three Lycopersicon spp. genotypes which show different levels of resistance to tomato powdery mildew. NO production was determined in plant leaf extracts of L. esculentum cv. Amateur (susceptible), L. chmielewskii (moderately resistant) and L. hirsutum f. glabratum (highly resistant) by the oxyhaemoglobin method during 216 h post-inoculation. A specific, two-phase increase in NO production was observed in the extracts of infected leaves of moderately and highly resistant genotypes. Moreover, transmission of a systemic response throughout the plant was observed as an increase in NO production within tissues of uninoculated leaves. The results suggest that arginine-dependent enzyme activity was probably the main source of NO in tomato tissues, which was inhibited by competitive reversible and irreversible inhibitors of animal NO synthase, but not by a plant nitrate reductase inhibitor. In resistant tomato genotypes, increased NO production was localized in infected tissues by confocal laser scanning microscopy using the fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. NO production observed in the extracts from pathogen conidia, together with elevated NO production localized in developing pathogen hyphae, demonstrates a complex role of NO in plant–pathogen interactions. Our results are discussed with regard to a possible role of increased NO production in pathogens during pathogenesis, as well as local and systemic plant defence mechanisms.