Elimination of grapevine fleck virus and grapevine rupestris stem pitting-associated virus from Vitis vinifera 87-1 by ribavirin combined with thermotherapy

Vitis vinifera 87-1 plants infected by grapevine fleck virus(GFkV) and grapevine rupestris stem pitting-associated virus(GRSPaV) were used as the plant materials for virus elimination treatment. This study evaluated the effects of ribavirin at different concentrations(15 and 25 μg mL^(–1);R15 and R25, respectively), thermotherapy(37°C;T), and the combination of ribavirin and thermotherapy(R15+T and R25+T) on eliminating viruses from grapevine plants in vitro. Both R15 and R25 had phytotoxic effects and weakened plant growth. Thermotherapy positively affected the growth of grapevine plants. Plant height was significantly greater in T, R15+T, and R25+T than in CK, R15 and R25. The proportion of dead plants after T, R15+T, and R25+T was 51.4, 11.4, and 8.6%, respectively. The survival rates of regenerated plants after all treatments were >68.0%. Ribavirin concentration and treatment time were related to the regeneration of shoot tips and elimination efficiencies of the two viruses. The survival rates of plants after R15+T for 30, 40, and 50 days were 97.3, 90.7, and 74.4%, respectively. The elimination rates of GRSPaV from plants in the three time quantum were 55.6, 84.6, and 93.8%, respectively. The elimination rate of GFkV was 23.9% higher in R25(35/44) than in R15(25/45), and that of GRSPaV was 7.0% higher in R25 than in R15. The combination of thermotherapy and chemotherapy was found to have a positive effect on the eradication of GFkV and GRSPaV, and R25+T for 50 days was able to completely eliminate the two viruses from in vitro grapevines.

Effect of Enhanced Ultraviolet-B Radiation on Photosynthetic Pigments and Flavonoids in the Leaves of Grapevine

[Objective] The study aimed at investigating the influence of enhanced UV-B radiation on photosynthesis of grapevine.[Method] The seedlings of Cabernet sauvignon were treated with different intensities of UV-B radiation under outdoor conditions,and then the contents of photosynthetic pigments and flavonoids in leaves were determined by measuring the absorbance of leaves extracts at 663,645,470 and 300 nm,respectively.[Result] The content of photosynthetic pigments in the leaves of grapevine obviously increased with time under the treatments of different enhanced UV-B radiation.Compared with the control,the chlorophyll a,chlorophyll b,total chlorophyll and carotenoid were obviously increased by 5%,2%,4% and 3% in the enhanced UV-B radiation treatment of 10.8 μW/cm2（T1）,and in the treatment of 25.6 μW/cm2（T2） the corresponding levels were subsequently increased by 11%,9%,10% and 7% with a significant increase in the content of chlorophyll a.On the other hand,the flavonoids content in the leaves of grapevine were obviously increased by 13%,9% in T1 and T2.[Conclusion] The grapevine has strong adaptability to UV-B radiation,and appropriate enhanced UV-B radiation couldn＇t decrease the photosynthesis of grapevine leaves.

A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types

To develop a rapid and high-sensitivity method for detection of grapevine virus E(GVE),a SYBR Green based real-time fluorescence quantitative RT-PCR method(RT-qPCR)was established.This method could be used to detect GVE specifically,and the sensitivity was about 100 times greater than conventional RT-PCR.An excellent linear correlation(R=0.997)and a high amplification efficiency(E=97.5%)were obtained from the standard curve of this method.Reproducibility tests revealed that the coefficients of variation in the intra-and inter-assay results were 0.31-1.03%and 0.82--262%,respectively,indicating a good reproduiblity.The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types.The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR.The detection rates in spring,summer,autumn and winter increased gradually.Samples in autumn and winter were best for detection,and the detection rates of most samples were 80-100%,which were 10 to 40%higher than conventional RT-PCR.In general,old petioles and branches were the best tissues for GVE detection.The detection rates of these samples in each season were all 100%,which were 20 to 40%higher than conventional RT-PCR.The second highest rates were in the old leaf,with detection rates for RT-qPCR of 80-100%in all seasons,which were 20 to 40%higher than conventional RT-PCR.GVE could be difficultly detected in young leaves by conventional RT-PCR,and the detection rates were only 0-50%,while by RT-qPCR the rates could increase to 0--80%.A total of 33 out of 363 samples(belonging to 68 cultivars)from 20 regions in China were detected to be positive by RT-qPCR(9.1%),which was more than twice the rate of the conventional RT-PCR(3.9%).

Advancements in plant regeneration and genetic transformation of grapevine(Vitis spp.)

Grapevine(Vitis spp.)is one of the most economically important fruit crops worldwide,and there is considerable interest in improving its major agronomic and enological traits in response to ever-changing agricultural environments and consumer demands.Molecular genetic techniques in particular,associated with rapid technological advancements,provide an attractive alternative to conventional breeding approaches for developing new grapevine varieties with enhanced yield performance,quality,stress tolerance and disease resistance.To date,several grapevine varieties have been transformed with genes associated with diverse functions through biolistic bombardment and/or Agrobacterium-mediated transformation,and transgenic grape lines have been obtained using established regeneration systems.Nevertheless,a wide range of factors,including genotype,explant source and culture medium,have been shown to affect the efficiency of plant regeneration.Moreover,the selection and use of acceptor materials,bacterial strain and cell density,selectable markers and selection methods also influence transformation efficiency.This paper provides an overview of recent advances in grapevine regeneration and genetic transformation and in-depth discussion of the major limiting factors,and discusses promising future strategies to develop robust plant regeneration and genetic transformation in grapevine.

Somatic Embryogenesis in Iberian Grapevine (Vitis vinifera) Cultivars Using Carpels as Initial Explants: Protocol Establishment and Histological Evaluation

Trincadeira and Aragonez are two important grapevine cultivars in the Iberian Peninsula, used for high quality red wines production. Both cultivars are strongly affected by fungal diseases, with consequent high loses on plant productivity and fruit quality. A successful protocol for plant regeneration via somatic embryogenesis (SE) was established for both cultivars allowing further plant improvement based on gene transfer technology. Several factors were evaluated during the three different phases which characterize an SE plant regeneration protocol. The culture room temperature during the induction phase, a parameter usually accepted as standard by most researchers, proved in these trials to significantly affect the embryo induction rates. Concerning embryo conversion, it was specially affected by the embryo developmental stage, by the intensity and duration of the chilling treatment and by the supplementation of conversion culture medium with activated charcoal. The responses obtained, both for induction and conversion, proved to be highly genotype dependent. Calli structure, as well as embryo integrity, was histologically observed, allowing to characterize embryonic and non-embryonic masses and to identify abnormalities on embryo development.

Versatile physiological functions of the Nudix hydrolase family in berry development and stress response in grapevine

Nudix hydrolases are widely distributed across all classes of organisms and provide the potential capacity to hydrolyze a wide range of organic pyrophosphates. Although Nudix hydrolases are involved in plant detoxification processes in response to abiotic and biotic stresses, the biological functions of Nudix hydrolases remain largely unclear in grapevine.In the present study, a total of 25 putative grapevine Nudix hydrolases(VvNUDXs) were identified by bioinformatics analysis and classified into eight subfamilies based to their preferred substrates. Both tandem and segmental duplications were responsible for the evolution and expansion of the NUDX gene family in grapevine. To investigate the regulatory roles of VvNUDX genes during growth and development, as well as in response to abiotic and biotic stresses in grapevine, the expression patterns were revealed in publicly available microarray data. The spatial and temporal expression patterns of the VvNUDX genes indicated that they might play important roles in multiple developmental processes. Transcriptome and qRT-PCR analyses showed that ten VvNUDX genes were specifically expressed in grapevine berries, suggesting potential roles in grapevine berry development. Expression and phylogenetic analyses demonstrated that VvNUDX1 and VvNUDX3 might be involved in terpenoid biosynthesis in grapevine. Furthermore,most VvNUDX genes active toward the ADP-ribose/NADH showed different patterns in response to various abiotic and biotic stresses, such as salinity and drought, as well as different types of biotic treatments, such as Erysiphe necator,Bois Noir phytoplasma and leaf-roll-associated virus-3(GLRaV-3). These results indicated that VvNUDX genes were associated with plant detoxification processes in response to abiotic and biotic stresses, and regulate the disease immunity and resistance pathways. The information obtained here may provide good opportunities to explore the physiological functions of VvNUDX genes in berry development and stress response networks in grapevine.

Identification of the DEAD-box RNA helicase family members in grapevine reveals that VviDEADRH25a confers tolerance to drought stress

Grapevine growing areas are increasingly affected by drought,which has greatly limited global wine production and quality.DEAD-box is one of the largest subfamilies of the RNA helicase family,and its members play key roles in the growth and development of plants and their stress responses.Previous studies have shown the potential of DEAD-box genes in the drought stress responses of Arabidopsis and tomato,rice,and other crop species.However,information about DEAD-box genes in grapevine remains limited.In this report,a total of 40 DEAD-box genes were identified in grapevine and their protein sequence characteristics and gene structures were analyzed.By comparing the expression profiles of VviDEADRHs in response to drought stress in different grapevine varieties,nine candidate genes(VviDEADRH10c,-13,-22,-25a,-25b,-33,-34,-36,and-39)were screened based on expression profiling data.Combined with qRTPCR results,Vvi DEADRH25a was selected for functional verification.Heterologous overexpression of Vvi DEADRH25a in Arabidopsis showed the transgenic plants were more sensitive to drought stress than the control.Both electrolyte permeability and malondialdehyde content were significantly increased in transgenic plants,whereas the chlorophyll content and superoxide dismutase(SOD),peroxidase(POD),catalase(CAT),and ascorbate peroxidase(APX)enzyme activities were significantly decreased.Furthermore,VviDEADRH25a-overexpressing plants showed down-regulated expression levels of several drought stress-related marker genes,namely At COR15a,At RD29A,At ERD15,and At P5CS1,which indicated that they participated in the drought stress response.In summary,this study provides new insights into the structure,evolution,and participation of DEAD-box RNA helicase genes in the response to drought stress in grapevines.

Establishment of a Rapid Propagation Protocol of GLRaV-3,GFKV,and GRSPa Free Grapevine Rootstocks

Nursery plant propagation by grafting has been widely used in modern viticulture to minimize the damage caused by biotic and abiotic stresses.In grapevine(Vitis spp.),an effective way to control disease damage is to provide producers and growers with pathogen-free stock plants.In this study,five grapevine rootstock varieties,‘SO4’,‘101-14’,‘5BB’,‘110R’and‘1103P’,were selected as explants to establish an in vitro culture protocol,and three species of grapevine viruses were tested by real-time RT-PCR.The results showed that MS medium supplemented with 0.2 mg/L IBA,1.0 mg/L 6-BA,0.5 mg/L KT,4.0 mg/L adenine for culture initiation,and WPM supplemented with 0.2 mg/L IBA for subculture were suitable for all five rootstock varieties,with multiplication coefficients ranging from 1.6 to 4.4.Virus testing showed that single RT-PCR was more effective for detecting the three viruses compared to double or triple RT-PCR.Only plantlets free from the aforementioned viruses were retained for subculture.Plantlets were hardened at room temperature under natural lighting in Hoagland solution for 2 weeks and transplanted to pots filled with mixed media in a greenhouse.This protocol is applicable for rapid propagation of the five grapevine rootstock varieties and can be used for commercial production of virus-free grapevine stocks.

Wild grapevine(Vitis vinifera subsp.sylvestris)in the Hyrcanian relict forests of northern Iran:an overview of current taxonomy,ecology and palaeorecords

Due to severe anthropogenic impacts on lowland and submontane zones of the Hyrcanian forests of northern Iran, wild grapevine(Vitis vinifera subsp. sylvestris), a sporadically distributed woody liana, is currently considered an endangered species. Using data from the literature and 34 studied populations, herbarium assessments and nine palynological sites, we provide an overview of its taxonomy,distribution and ecology in the first part of the investigation.The separation of the two subspecies, namely V. sylvestris subsp. anebophylla and V. sylvestris subsp. trichophylla(sensu Flora Iranica), based on their leaf indumentums, could not be confirmed by our examination of herbarium materials and field observations. Indumentum of the leaves is a result of leaf polymorphism in different Vitis specimens and can be strongly influenced by environmental conditions. Vitis vinifera subsp. sylvestris grows in a wide range of habitats including wetlands, seasonal stream sides in closed forests,alluvial beds of large rivers, sand dune shrublands and forested wetlands(alder forests). Parrotia persica and Carpinus betulus were the most frequent host species in the studied populations. In the Hyrcanian region, no pollen record of Vitis older than the Mid-or Late-Holocene has yet been established. Despite the intrinsic problem in pollen identification by normal(i.e., light) microscopy of wild from cultivated grapevines, the significant values and persistent occurrence of Vitis pollen since the Mid-Holocene(before the Bronze Age) in the Hyrcanian pollen records may imply the onset of viticulture in low-to mid-elevation sites in the region. This represents an argument to consider the Hyrcanian region as a possible domestication center for V. vinifera.However, the question of wild versus cultivated origin of grapevines in the Hyrcanian pollen records and the possible date of its domestication and/or cultivation will remain open until further palynological studies are undertaken.

Effects of root restriction on nitrogen and gene expression levels in nitrogen metabolism in Jumeigui grapevines(Vitis vinifera L.×Vitis labrusca L.)

To decipher the relationship between the inhibited shoot growth and expression pattern of key enzymes in nitrogen metabolism under root restriction, the effects of root restriction on diurnal variation of expression of nitrate reductase （NR）, nitrite reductase （NiR）, glutamine synthetase （GSI-1, GS1-2, GS2） and glutamate synthase （Fd-GOGAT, NADH-GOGAT） genes and nitrogen levels were evaluated in two-year-old Jumeigui grapevines （Vitis vinifera L.xVitis labrusca L.） when significant differences in shoot growth were observed between treatments at expansion stage （22 days after anthesis）. Grapevines were planted in root-restricting pits as root restriction and in an unrestricted field as the control. Results showed that root restriction significantly reduced shoot growth, but promoted the growth of white roots and fibrous brown roots and improved the fruit quality. （NO3-＋NO2-）-N concentration in all plant parts, NH4＋-N concentration in white roots and total N concentration in leaves and brown roots were significantly reduced under root restriction. Gene expression analysis revealed that mRNA levels of genes related to the GS1/NADH-GOGAT pathway were lower in root-restricted than in control petioles, whereas genes involved in the GS2/Fd-GOGAT pathway were up-regulated under root restriction. Root restriction also resulted in downregulation of genes involved in nitrogen metabolism in leaves, especially at 10：00, while transcript levels of all these genes were enhanced in root-restricted white and brown roots at most time points. This organ-dependent response contributed to the alteration in NO3- reduction and NH4＋ assimilation under root restriction, leading to less NO3- transported from roots and then assimilated in root-restricted leaves. Therefore, this study implied that shoot growth inhibition in grapevines under root restriction is closely associated with down-regulation of gene expression in nitrogen metabolism in leaves.

Neopestalotiopsis eucalypti,a causal agent of grapevine shoot rot in cutting nurseries in China

Grapevine(Vitis vinifera L.)is an economically important fruit crop in the world,and China ranks first in the production of grapes with approximately 15%of the world’s total yield.However,diseases that cause the death of grapevine shoots pose a severe threat to the production of grapes.In this study,the fungus Neopestalotiopsis eucalypti was identified as a causal pathogen of grapevine shoot rot based on the morphology of conidia and a phylogenetic analysis.The phylogenetic analysis was performed with three isolates based on the combined sequence of internal transcribed spacer(ITS)region of ribosomal DNA,part of the translation elongation factor 1-alpha(Tef)and theβ-tubulin(Tub2)genes.The three isolates were all identified as N.eucalypti.Pathogenicity tests of the three fungal isolates were conducted on grapevines shoots in vitro and in vivo.The results showed that all three fungal isolates caused severe rot lesions on the inoculated grapevine shoots,and N.eucalypti was re-isolated from the inoculated grapevine shoots.Therefore,N.eucalypti was confirmed as a causal agent of the grapevine shoot rot.This is the first report of N.eucalypti causing grapevine shoot disease in China.

Verification of the stomatal conductance of Nebbiolo grapevine

Wine is one of the most important Italian export products,and Nebbiolo is one of the most respected Italian grapes. In the summer of 2007,a measurement campaign was carried out in a Nebbiolo vineyard located in Vezza d’Alba,near Cuneo,Italy. Using a gauge of trade gases and some other instruments,we recorded the stomatal conductance and also some physiological parameters useful for estimating the dependence of stomatal conductance on environmental variables. The goal of this experiment was improving the parameterization of grapevine evapotranspiration through the assessment of the stomatal conductance and,in particular,of the functional dependence of the stomatal conductance on the following variables:the photosynthetically active radiation,the atmospheric temperature,the atmospheric moisture deficit,and the carbon dioxide concentration. The observations allowed us to check and,in some cases,to adapt the existing general parameterizations found in literature. The results showed some significant differences with the existing parameterizations concerning the atmospheric temperature,the atmospheric moisture deficit,and the carbon dioxide concentration. The parameterizations obtained in this experiment,although referring to a specific plant and site(namely the Nebbiolo at Vezza d’Alba),could allow assessment of the best environmental conditions under which the Nebbiolo grapevine production is the best,and in future could be tested for other grapevines or climates.

Characterization of an Early Berry Development Grapevine Somatic Variant (Vitis labrusca L. cv. Isabel Precoce)

Over the past 10 years significant advances have been made towards the description of genetics and molecular mechanisms controlling grapevine berry growth. Regardless of this, many aspects of early fruit morphogenesis and its development control remain to be elucidated. In an attempt to understand gene expression patterns associated with the berry growth development, the contrasting phenotype between the cv. Isabel (Vitis labrusca L.) and its early berry development mutant “Isabel Precoce” has been explored by a candidate gene approach. “Isabel Precoce” (Vitis labrusca L.) was confirmed as an EDV (Essentially Derived Variety) of Isabel, with a 30-35-day reduction in the berry growth phase when compared to the wild type and thus, it constitutes an informative model to investigate many aspects of fruit growth and development. Phenotypic analysis showed that “Isabel Precoce” develops fruits that are smaller in diameter and volume despite of following similar development kinetics. The expression of many genes associated with plant growth and development (MIKCC-type MADS box genes), sugar transport and with the control of flavonoid biosynthetic pathway have been evaluated. The majority of the genes presented a remarkably similar transcription profile. However, a higher induction of transcript accumulation for some genes has been observed in the “Isabel Precoce” genetic background.

Rt-PCR Analysis and Evolutionary Relationship of Some Hungarian <i>Grapevine leafroll associated virus</i>1 and 3 Isolates

Hungarian isolates of Grapevine leafroll associated virus 1 and 3 (GLRaV-1, GLRaV-3) were tested using serological (DAS-ELISA) and molecular (RT-PCR) methods. Five hundred bp long PCR products of the part of HSP70 gene of one serologically positive GLRaV-1 and four GLRaV-3 isolates were sequenced. These sequences were applied for phylogenetic analysis and compared to foreign virus isolates of NCBI GenBank. Phylogenetic analysis of GLRaV-1 HSP70 gene supported the earlier results that it could be divided into two clusters: E and A. The Hungarian isolate 6.4.1 belonged to the group E. This isolate showed the highest homology with the AY754914 isolate from the Czech Republic. GLRaV-3 sequence data could cluster five groups. Hungarian 2.2;3.5 and 4.2 isolates were estimated belonging to the group II. The 1.4 isolate from the same vineyard as 2.2 varied in sequence data so it belonged to the other, IV. variant group with two South African, two Austrian and a Syrah isolate. According to the phylogenetic analysis, two variant groups occurred in Hungary. These isolates related with each other, but showed higher similarity of foreign counties. In some cases, they were similar to isolates of the neighbour countries such as Slovakia and Austria. It could be supposed that mainly the exchange of virus infected propagation materials caused the dissemination of GLRaV isolates.

Characterization of the Fungi Involved with Grapevine Trunk Diseases in Castilla-La Mancha Region,Spain

Grapevine trunk diseases (GTDs) are currently one of the most important problems for grapevine plant and wine industry, causing important economic cost in Castilla-La Mancha, Spain. Traditionally, GTDs were associated with diseases present in mature grapevines, however in recent years the incidence of young vine decline has been increasing. In this work, from 2009 to 2016, 250 young grapevine plants have been analysed by fungal isolation and characterization. In addition, during 2017, 32 mature plants showing decline symptoms were analysed. Both kinds of plants were analysed in order to identify the main fungal trunk pathogens present in Castilla-La Mancha region. As a result, it was possible to isolate species associated with black-foot disease (Cylindrocarpon-like anamorphs), esca and Petri disease (Phaeomoniella chlamydospora and Phaeoacremonium spp.), and Botryosphaeria dieback. All these pathogens were isolated from young and mature plants, this fact could suggest a common origin. The lack of effective control measures for these diseases makes it necessary to implement an integrated disease management strategy that provides an efficient approach to reduce infections in nurseries and get better results in infected vineyards.

Mass production, fermentation, formulation of <i>Pseudomonas putida</i>for controlling of die back and phomopsis diseases on grapevine

The export oriented agricultural and horticultural crops depends on the export of residue free produce and has created a great potential and demand for the incorporation of biopesticides in crop protection. PGPR with wide scope for commercialization includes Pseudomonas putida. Pseudomonas putida was effective in reducing die back caused by a fungus Botryodiplodia theobromae and phomopsis or arm death, caused by a fungus, Phomopsis viticola of grapevine in vitro and in vivo. Pseudomonas putida showed optimum Siderophore pseudobactin production at 72 h, and growth peak at 120 h. glycerol as a source of carbon and yeast as a source of nitrogen Pseudomonas putida was formulated using polysaccharide dextran starch, which led to the elongation of conservation and the effectiveness of the biofungicide. Pseudomonas putida was very effective as biocontrol agents to reduce the die back and phomopsis diseases on grapevine. The obtained results showed that both bacterial treatments significantly increased the growth parameters as well as fruits dry weights and yield of grapevine.

谈谈grapevine

《新英汉词典》grapevine一词的释义为:(1)葡萄藤;(2)谣言,传闻,小道新闻。这两个词义似乎没有什么关系,那么,它们是如何联系在一起的呢? 追根溯源,grapevine本来是“葡萄藤”,其新义的产生恐怕要首先归功于电报发明家塞缪尔·莫尔斯(Samuel Morse)。莫尔斯原是一个美国画家,后来改行,立志从事电报的发明研究。为了完成用电传递信息的伟大使命,

Success Through the Grapevine

How one wine importer has become dedicated to supplying China with superior quality South African wine WINE from Africa? To some Chinese wine drinkers,this is a strange concept.In fact,there are vineyards all over Africa.But it is down south in the Cape region of South Africa where the continent’s, and even the world’s,finest wines are produced in the eyes of Si Yang,a South African

Nitrogen application regulates antioxidant capacity and flavonoid metabolism,especially quercetin,in grape seedlings under salt stress

Salt stress is a typical abiotic stress in plants that causes slow growth,stunting,and reduced yield and fruit quality.Fertilization is necessary to ensure proper crop growth.However,the effect of fertilization on salt tolerance in grapevine is unclear.In this study,we investigated the effect of nitrogen fertilizer(0.01 and 0.1 mol L^(-1)NH_(4)NO_(3))application on the salt(200 mmol L^(-1)NaCl)tolerance of grapevine based on physiological indices,and transcriptomic and metabolomic analyses.The results revealed that 0.01 mol L^(-1)NH_(4)NO_(3) supplementation significantly reduced the accumulation of superoxide anion(O_(2)^(-)·),enhanced the activities of superoxide dismutase(SOD)and peroxidase(POD),and improved the levels of ascorbic acid(AsA)and glutathione(GSH)in grape leaves compared to salt treatment alone.Specifically,joint transcriptome and metabolome analyses showed that the differentially expressed genes(DEGs)and differentially accumulated metabolites(DAMs)were significantly enriched in the flavonoid biosynthesis pathway(ko00941)and the flavone and flavonol biosynthesis pathway(ko00944).In particular,the relative content of quercetin(C00389)was markedly regulated by salt and nitrogen.Further analysis revealed that exogenous foliar application of quercetin improved the SOD and POD activities,increased the AsA and GSH contents,and reduced the H_(2)O_(2) and O_(2)^(-)·contents.Meanwhile,10 hub DEGs,which had high Pearson correlations(R^(2)>0.9)with quercetin,were repressed by nitrogen.In conclusion,all the results indicated that moderate nitrogen and quercetin application under salt stress enhanced the antioxidant system defense response,thus providing a new perspective for improving salt tolerance in grapes.

Identif ication of a hypersensitive response core peptide of HrpZ and its role in increasing grape downy mildew resistance

Harpins play a key role in inducing disease resistance in crops,and identifying their core functional regions and establishing a system for their efficient expression would be very valuable.In this study,large amounts of soluble fusion proteins of harpin HrpZ and its subpeptides were obtained via the optimized induction conditions(28℃ with 0.5 mmol·L^(-1) IPTG for 6 h)in Escherichia coli BL21(DE3).Hypersensitive response(HR)assays demonstrated that the C-terminal 66 aa of HrpZ(HrpZ_C_2_2)elicited a strong HR in tobacco(Nicotiana benthamiana)and grape(Flame Seedless)leaves.Additionally,treatment with HrpZ,and particularly HrpZ_C_2_2,significantly reduced the disease incidence and severity index of field vine leaves and those inoculated with downy mildew.The determination of the physiological parameters indicated that HrpZ,and especially HrpZ_C_2_2,improved the photosynthesis-and chlorophyll fluorescence-related parameters,enhanced the activity of defense-related enzymes,including SOD,POD,CAT and PAL,and increased the H_(2)O_(2) level.Collectively,we efficiently expressed a core peptide of HrpZ and elucidated its strong ability to elicit a HR and resistance to downy mildew.This research provides insight into understanding the structure and function of HrpZ and will advance the application of HrpZ_C_2_2 to increase the resistance of grapevine to downy mildew.