Proteomic analysis of grapevine stem in response to Xylella fastidiosa inoculation

Xylella fastidiosa (Xf) is the bacterial causal agent of Pierce’s disease (PD) as well as other economically important diseases in a number of agronomic, horticultural and ornamental plants. The objective of this research was to tentatively identify proteins that are differentially expressed in grapevines and involved in disease development or defense responses to Xf-inoculation. We comparatively analyzed proteins differentially expressed in Xf-inoculated grape stems using a pair of siblings of 9621-67 (highly susceptible) and 9621-94 (highly resistant) from a cross of Vitis rupestris × Vitis arizonica. Total proteins were extracted from the stems of uninoculated controls and Xf-inoculated plants at 1, 6, and 12 weeks after inoculation, separated by a 2D-PAGE system, and spots representing differentially expressed proteins were analyzed and tentatively identified using LC/MS/MS. Protein identification was performed using BLASTp and tBLASTn against NCBI non-redundant protein databases and EST databases, respectively. Ten tentatively identified proteins were differentially expressed at different time points after inoculation. A thaumatin-like protein and the pathogenesis-related protein 10 from both genotypes, and the 40S ribosomal protein S25 from the susceptible genotype were up-regulated in response to Xf-inoculation. Furthermore, the expression of the thaumatin-like protein increased sharply 12 weeks post-inoculation in the PD-resistant genotype only. Three heat shock proteins, 17.9 kDa class II, protein 18 and 21 were highly expressed in healthy tissues compared with those in tissues infected with Xf, and heat shock protein 21 was not detectable in the Xf-inoculated PD-susceptible genotype. In addition, a down-regulated putative ripening related protein was found in the Xf-inoculated PD-susceptible genotype. Glycoprotein and formate dehydrogenase were identified in the PD-resistant genotype and their expression was constant during plant development. A putative GTP-binding protein was down-regulated in the PD-susceptible genotype. Our results revealed that differential expression of proteins in response to Xf-inoculation was genotype and tissue development stage dependent. The specific roles of these candidate proteins in alleviation or aggravation of this disease are under investigation. The information obtained in this study will aid in the understanding of the mechanisms related to the host–pathogen interactions involved in PD.     

Induction of systemic resistance to Agrobacterium tumefaciens by endophytic bacteria in grapevine

Crown gall disease of grapevine, caused by Agrobacterium tumefaciens, often results in severe economic loss to grape production worldwide. This study demonstrated the ability of the endophytic bacteria Pseudomonas sp. Sn48 and Pantoea sp. Sa14 isolated from domesticated and wild grapevines to induce resistance in both above- and belowground tissues of grapevines infected with A. tumefaciens. Our results provide evidence that both strains can colonize roots and/or shoots. We showed that the strains Pseudomonas sp. Sn48 and Pantoea sp. Sa14 are capable of inducing stilbenic phytoalexin production in grapevine tissues and to further prime plantlets for enhanced phytoalexin production after A. tumefaciens inoculation. We also showed that in the majority of treatments, polyamine accumulation remained unchanged or slightly increased in plantlets treated with Pseudomonas sp. Sn48 and Pantoea sp. Sa14 compared with the control. Our findings indicated that the levels of polyamines remain unchanged or significantly decrease in plantlets treated with endophytic bacteria after A. tumefaciens challenge compared to the control and plantlets treated with individual endophytic bacterial strains. PR1, PR2, and PR4 gene expression levels of plantlets treated with Pseudomonas sp. Sn48 and Pantoea sp. Sa14 significantly increased after A. tumefaciens inoculation. The findings revealed the efficacy of the selected endophytic bacteria in triggering grapevine resistance against A. tumefaciens and the possible use of these strains as an alternative to chemical control methods in grapevine crown gall disease management.     

The importance of multilocus sequence typing: cautionary tales from the bacterium Xylella fastidiosa

Microbial identification methods have evolved rapidly over the last few decades. One such method is multilocus sequence typing (MLST). MLST is a powerful tool for understanding the evolutionary dynamics of pathogens and to gain insight into their genetic diversity. We illustrate the importance of accurate typing by reporting on three problems that have arisen in the study of a single bacterial species, the plant pathogen Xylella fastidiosa. Two of these were particularly serious since they concerned contamination of important research material that has had detrimental consequences for Xylella research: the contamination of DNA used in the sequencing of an X. fastidiosa genome (Ann-1) with DNA from another X. fastidiosa strain, and the unrecognized mislabeling of a strain (Temecula1) distributed from a culture collection (ATCC). We advocate the routine use of MLST to define strains maintained in culture collections and emphasize the importance of confirming the purity of DNA submitted for sequencing. We also present a third example that illustrates the value of MLST in guiding the choice of taxonomic types. Beyond these situations, there is a strong case for MLST whenever an isolate is used experimentally, especially where genotypic differences are suspected to influence the outcome.     

Analysis of resistance to Xylella fastidiosa within a hybrid population of Pera sweet orange × Murcott tangor

Resistance to Xylella fastidiosa was evaluated within a population of 20 interspecific hybrids of Pera sweet orange and Murcott tangor under greenhouse conditions. Efficiency of inoculation, multiplication of bacteria within the plants, xylem vessel morphology, and symptom expression were analysed. The rate of infection ranged from 40 to 100% (average 70%) for all genotypes analysed. Xylella fastidiosa populations ranged from log 0·59 to log 2·13 cells mg⁻¹ tissue for the resistant hybrids. These values were significantly different ( P  = 0·05) from those obtained for the tolerant (no symptoms but bacteria recovered) or susceptible (symptoms and bacteria recovered) hybrids (log 3·02 to log 4·06 cells mg⁻¹). Xylella fastidiosa was recovered from all hybrids (log 2·31 to 5·03 CFU mg⁻¹ tissue) except the resistant ones. The first foliar symptoms appeared at least 90 days post-inoculation, the time varying according to genotype. No correlation between xylem vessel morphology and disease expression was observed, indicating that the resistance was the result of a genetic response of the host. According to this hypothesis, a high broad-based heritability index for resistance was obtained (0·96) at 210 days from X. fastidiosa inoculations, using bacterial quantification by real-time PCR, which indicated that the influence of the number of bacteria was the result of genetic rather than environmental variations.     

马铃薯内生细菌的分离及环腐病拮抗菌的筛选鉴定

 本研究从大同、太原和内蒙古等地采集马铃薯块茎,分离到240株内生细菌,通过离体抑菌作用测定,共得到55株对环腐病菌有拮抗作用的菌株,占菌株总数的22.9%,抑菌圈半径最大的可达13 mm。按抑菌圈半径大小将拮抗菌分为强、中、弱三类。从中筛选出9个对环腐病等病菌具有较强拮抗作用的内生菌株进行了细菌学鉴定,结果表明:118为荧光假单胞生物型V (Pseudomonas fluorescens biovar V);110为短小芽孢杆菌(Bacillus pumilus);085为嗜热脂肪芽孢杆菌(Bacillus stearothermophilus);069为草生欧文氏菌(Erwinia herbicola);043为草莓黄单胞菌(Xanthomonas fragariae);A-10'、T3为芽孢杆菌属(Bacillus);H1-6为荧光假单胞菌(Pseudomonas fluorescens);116为短小杆菌属(Curtobacterium)。     

草莓内生细菌的分离及草莓灰霉病菌拮抗菌的筛选鉴定

本研究从健康的草莓植株内分离到54株内生细菌,其中根、茎、叶组织中分离内生细菌种群密度为2.8×101～3.95×104cfu/g鲜重不等。通过对峙试验,得到5株对草莓灰霉病菌有强拮抗作用的菌株,占所分离内生细菌总数的9.26%。来自草莓叶的SL6菌株抑菌效果最佳,抑菌半径达15mm,无菌发酵液对草莓灰霉病菌菌丝的抑制率为97.6%,具有作为生防菌的应用潜能。经形态、生理生化和16SrRNA等分析测定,并通过MEGA4方法构建其16SrDNA系统发育树,将SL6菌株鉴定为枯草芽胞杆菌(Bacillus subtilis)。     

Seven years of negative detection results confirm that Xylella fastidiosa,the causal agent of CVC,is not transmitted from seeds to seedlings

Knowledge about the mechanism of transmission of systemic pathogens of citrus species is highly important for the safe movement of citrus germplasm, management of citrus mother trees, and also production of young plants. Among systemic pathogens of citrus, Xylella fastidiosa the causal agent of the citrus variegated chlorosis (CVC), is one the most important pathogens causing decline in tree vigour and productivity. Seven-year experiments were conducted to evaluate the hypothesis of seed-to-seedling transmission of X. fastidiosa. This bacterium was found colonizing the fruit (exocarp, central axis and mesocarp) and the seed parts (seed coat and endosperm plus embryo). After 7 years of PCR assay, no positive PCR detection of X. fastidiosa was confirmed in seedlings propagated from the seeds infected with X. fastidiosa. This result demonstrates the lack of seed-to-seedling transmission of this bacterium.     

Bacterial extraction from grapevine and detection of Xylophilus ampelinus by a PCR and Microwell plate detection system*

Xylophilus ampelinus is listed as a quarantine pest in Annex II/A2 of EU Directive 2000/29. Grapevine cuttings used either as rooting or grafting material represent a primary source of inoculum because of the systemic nature of bacterial colonization. We describe a PCR-based method for the specific detection of X. ampelinus from various plant materials (trunks, woody branches, growing shoots, leaves and bleeding sap), making the detection of X. ampelinus reliable throughout the year. Bacterial cells were extracted by crushing the plant tissues and soaking them in sterile distilled water for 30–120 min at 4 °C. Bacterial cells were harvested by centrifugation at 13 000 g for 10 min through sterile paper discs, and lysed in SDS buffer. DNA was extracted by a silica-based procedure. Specific DNA amplifications were performed with a pair of primers, of which one was labelled with biotin. A digoxygenin-labelled oligonucleotide with a sequence specific for the amplicon was used as the probe. Finally, amplicons were immobilized in microplate wells coated with streptavidine, and hybridization of the digoxygenin-labelled probe was detected with alkaline phosphatase-conjugated antibiodies in a colorimetric assay. This method was sensitive, specific and allowed experiments with high throughput.     

Phenology modelling of major insect pests in fruit orchards from biological basics to decision support: the forecasting tool SOPRA*

The forecasting tool SOPRA has been developed with the objective of optimizing timing of monitoring, management and control measures of insect pests in fruit orchards in Switzerland. Applying time‐varying distributed delay approaches, phenology‐models were developed driven by solar radiation, air temperature and soil temperature on hourly basis. Relationships between temperature and stage‐specific development rates for relevant stages of the life cycles were established under controlled laboratory conditions for Dysaphis plantaginea, Hoplocampa testudinea, Cydia pomonella, Grapholita lobarzewskii, Cacopsylla pyri, Rhagoletis cerasi, Anthonomus pomorum and Adoxophyes orana. The implementation of body temperatures in the models is based on habitat selection and biophysical modelling of habitat conditions. In order to validate modelling, phenology predictions were compared with several years of independent field observations. On the basis of local weather data, the age structure of the pest populations is simulated and crucial events for management activities are announced. Through a web interface, the simulation results are made available to consultants and growers ( http://www.sopra.info ) and the latter can be applied as a decision support system for the eight major insect pests of fruit orchards in the alpine valleys and north of the Alps on local and regional scale.     

Application of a putative fatty-acid binding protein to discriminate serologically the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from other Meloidogyne species

Two major proteins, Mcf-A67 and Mcf-B66, were identified by mini two-dimensional polyacrylamide gel electrophoresis in order to distinguish the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from eight other species. These quarantine proteinic markers have been microsequenced after enzymatic digestion. The internal amino acid sequences exhibit similarities to members of a family of low molecular weight intracellular lipid-binding proteins. Moreover, to explore a simple, rapid, and inexpensive way to identify the two quarantine nematodes, dot blot hybridizations were performed using an antiserum (A67) produced from the longest amino-acid sequence of the protein Mcf-A67. Although several proteins stained on the M. chitwoodi and M. fallax western blot membranes, the two nematodes were easily distinguished from other root-knot nematodes, on dot blot assays with soluble proteins extracted from a single female. Because of its specificity and sensitivity, the use of the A67 antiserum to improve the diagnosis of the two European quarantine root-knot nematodes is discussed.     

Contribution of molecular studies to botanical epidemiology and disease modelling: grapevine downy mildew as a case-study

After being accidentally introduced from the USA at the end of the 19^th century, downy mildew caused by Plasmopara viticola (Berk. et Curt.) Berlese et De Toni became one of the most damaging diseases affecting Vitis vinifera in Europe. Downy mildew causes both direct and indirect losses and can lead to severe reduction of yield. Our understanding of the life cycle and epidemiology of P. viticola has been recently altered by molecular studies that revealed that the overwintering inoculum (i.e., the oospores) does more than initiate disease, as was previously thought. A mechanistic model was developed for predicting the entire chain of processes leading to primary infections, and this primary infection model was linked to other models of secondary infection cycles. The model for primary infections defines the length of the primary inoculum season and a seasonal oospore dose consisting of several cohorts of oospores that progressively mature. The model was evaluated by means of Bayesian analysis in both Italy and eastern Canada, and showed high sensitivity, specificity, and accuracy both for potted plants and vineyards. Fungicide applications are necessary to control downy mildew because preventive agronomic practices are not very effective, including host resistance. The use of warning systems based on weather-driven models leads to a reduction in the use and cost of chemicals and a reduction in their environmental impact.     

Biofilter:a promising tool for mitigating methane emission from manure storage

Liquid manure storage may contribute to methane(CH4) emission and this emission can be greatly reduced if appropriate management practices are applied. Biofiltration has been used in other fields for mitigating greenhouse gas(GHG) emission(e.g.,landfill) and shown promise for mitigation CH4 emission from liquid manure storage. It has been reported that biofilter was capable of reducing 80% of CH4 emissions from manure storage. The CH4 removal efficiency is influenced by many factors,including CH4 and O2 concentrations,temperature,moisture,composition of the filter bed,nutrient,and empty bed residency time(EBRT) . Biological conversion of methane of a biofilter is a slow process due to the low water solubility of methane. The residence times(EBRT) between 5 min and 5 h have been used,whereas a typical EBRT of 25 s is used for common biofilter applications. Temperature at which methanotrophic bacteria are active ranges from 10oC to 45oC. The maximum activity is found at around 30oC. The optimal filter bed water content depends on both the gas flow rate and the type of filter bed(soil,compost,etc.) and ranges from 30%-70% of the water holding capacity. Compost is the best material for filter bed. The optimal pH for methanotrophic bacteria is neutral to slightly acidic. Copper and nitrogen compounds especially nitrate are important nutrients to methanotrophic bacteria but their optimal concentrations have not been founded. Phosphorus and other elements such as potassium and manganese are reported to affect the performance of methanotrophic bacteria but need further confirmation.     

Evaluation of a new natural adjuvant obtained from locust bean gum to reduce the amount of copper necessary to control downy mildew of grapevine

Journal of Plant Diseases and Protection - Two field trials in integrated cultivation (2014 and 2016) were carried out with the aim to evaluate the efficacy and the persistence of copper...     

Computer‐assisted identification and clustering for regulated phytopathogenic bacteria: construction of a reference database and development of a computer system*

CABIQ is a specific computer‐assisted identification system for the reliable and rapid identification of the main regulated phytopathogenic bacteria. It is based on phenotypic and genomic properties of bacteria. About 500 reference strains have been used to initiate the database, including conventional phenotypic tests and the Biotype 100 (BioMérieux) galleries. The CABIQ system, with its database and reference matrices, is a guide on the tests to be done when identifying new isolates. The modules dealing with phenotypic and Biotype100 characteristics are already finalized. Results on repetitive PCR will soon be added to the system. This opens interesting perspectives for improving the reliability of computer‐assisted identification.     

The reliability of leaf bioassays for predicting disease resistance on fruit: a case study on grapevine resistance to downy and powdery mildew

This study was designed to assess the reliability of grapevine leaf bioassays for predicting disease resistance on fruit in the field. The efficacy of various grapevine quantitative trait loci (QTLs) for conferring resistance to downy and powdery mildew was evaluated in bioassays and in a 2‐year field experiment for downy mildew. The resistance genes studied were inherited from Muscadinia rotundifolia (Rpv1 and Run1) and from American Vitis species through cv. Regent (QTLRgP and QTLRgD). In bioassays, genotypes carrying Run1 blocked powdery mildew development at early stages. Genotypes combining Run1 with QTLRgP displayed no greater level of resistance. For downy mildew, genotypes carrying Rpv1 and/or QTLRgD were more resistant than the susceptible cv. Merlot, and showed a high level of leaf resistance in the field (<10% severity). Disease levels on bunches were much higher than those on leaves, with a high variability between Rpv1 genotypes (1–48%). A Bayesian decision theory framework predicted that an OIV‐452 threshold of 5 in leaf bioassays allowed accurate selection of grapevine genotypes (P = 0·83) with satisfactory disease severity on bunches. Therefore, this study validates that the use of early bioassays on leaves, as currently performed by grapevine breeders, ensures a satisfactory level of resistance to downy mildew of bunches in the field.     

Use of ITS nuclear sequences from Phelipanche aegyptiaca as a direct tool to detect single seeds of broomrape species in the soil

Broomrape (Phelipanche and Orobanche spp.) are obligate holoparasites that attack roots of almost all economically-important crops in semiarid regions of the world. Broomrape seeds are extremely small (dust-like seeds), averaging 200 to 300?μm in size and because of the miniscule seed size it is difficult to detect and confirm via conventional methods. In this study our aim was to develop a PCR-based assay specific for broomrape soil-borne seeds and sensitive enough to detect a single or few broomrape seeds in a soil sample. For this purpose, we used complementary polymerase chain reaction (PCR) primers based upon unique sequences in the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA of Phelipanche aegyptiaca. Genomic DNA was extracted from soil samples artificially infested with broomrape seeds or tissue of Phelipanche aegyptiaca Pers., Orobanche cumana Wallr. and Phelipanche crenata Forsk. and subjected to PCR analysis. Using ITS-350 primers, a specific PCR product (350?bp) was amplified and detected in all samples containing broomrape species, but was not detected in soil sample free of broomrape seeds or tissues. Additionally, the PCR-based assay was sensitive enough to detect even a single broomrape seed in the soil. As expected the universal internal control primers amplified a PCR product (555?bp) of genomic DNA extracted from soil samples with or without broomrape tissues or seeds. This diagnostic method is simple, reliable and rapid and could help for assessment of broomrape seed contamination in a crop field.     

Horizontal gene transfer of atrazine-degrading genes (atz) from Agrobacterium tumefaciens St96-4 pADP1::Tn5 to bacteria of maize-cultivated soil

The plasmid pADP1::Tn5 derived from pADP1[Atr+] carrying a Tn5 transposon conferring kanamycin and streptomycin resistances was constructed and introduced in Agrobacterium tumefaciens St96-4. This genetically modified strain was inoculated (approximately 10(8) cfu g(-1)) in potted soils planted with maize and treated or not with atrazine (1.5 mg kg(-1)). Bulk and maize rhizosphere soils were sampled 39 days after planting to look for soil indigenous bacteria that had acquired pADP1::Tn5. Four transconjugants were isolated from four different soil samples. The estimated transfer frequency of pADP1::Tn5 was 10(-4) per donor. Maize rhizosphere and atrazine treatment had no obvious effect on pADP1::Tn5 transfer frequency. The sequencing of the 16S rDNA sequences of the transconjugants revealed that they were almost identical and highly similar to that of Variovorax spp (97%). In addition, their characterization suggested that the atzA and atzB genes had been transferred from pADP1::Tn5 to the bacterial chromosome in two of the four transconjugants. These data suggest that the atz degrading genes are horizontally transferred in soil and possibly subjected to gene rearrangement.     

Spread modelling: a suitable tool to explore the role of human-mediated dispersal in the range expansion of the yellow-legged hornet in Europe

Abstract

Invasive species can spread locally on their own and can be introduced at long distance by humans. Here, we show how a spread model can be used to explore the role of humans in the range expansion of the invasive yellow-legged hornet, Vespa velutina nigrithorax, in Europe with a special focus on some islands. In 2017, the hornet distribution in France, southern Belgium, south-eastern Germany and northern Spain could largely be explained by the insect’s own dispersal while the occurrence in Portugal, Italy, the Netherlands and Great Britain likely results from human-mediated dispersal. However, in the following years, it could spread to Portugal, Italy and Great Britain also by its own means. The yellow-legged hornet has likely reached the Channel Islands by its own flight but it could hardly reach the Mediterranean islands. Hence, the infestation in Majorca likely results from an accidental introduction. When simulating human-mediated dispersal in the Mediterranean islands, the hornet density would remain relatively low anyhow. Assessing the means of dispersal is important in terms of pest management as the target is either to reduce the spread rate and the population density, or to reduce the risk of entry.     

Plant biology and other issues that relate to the management of water hyacinth: a global perspective with focus on Europe1

Water hyacinth reproduces vegetatively and sexually and seeds are numerous and long‐lived. Rates of vegetative growth are high (weight and numbers can double in less than 1 week in optimal conditions) and governed by temperature and available nutrients. The period from germination to setting seed can be less than 12 weeks. Dispersal of propagules is by flow, man and machines. Biomass of living material is high 90 to > 800 tonnes/ha and comprises around 95% water, and the weed often grows in inaccessible areas. Consequently, social, economic and environmental impacts of water hyacinth are often large and management of the problem becomes very important. However, management of water hyacinth is difficult, and utilization of this weed should not be mistaken as a control measure. Containment and eradication from a catchment may only be accomplished if the invasion is very young, small, isolated and accessible, and if the short‐term resource commitment is high. Most infestations are non‐eradicable because once noticed reaction time is too long. However, in Europe, as the weed invades new areas on the edges of its ecological range, eradication of new infestations could (should) be attempted as this is the most cost effective way to deal with the weed. In addition, pathway shut‐down and awareness campaigns should seek to reduce further new introductions, which, for spread to new regions, are mostly anthropogenic. For established populations the removal of accessible floating biomass can be accomplished using herbicides (if permitted) and by mechanical means. Both are expensive because re‐growth ensures repeated action is necessary. Biological control is a useful tool in warmer climates, less so in cooler climates. It may be more useful in cooler areas if integrated with other management techniques. Additional biological control agents are being studied to improve and broaden the value of biological control. The lack of policies to permit the use of biological control for weeds in Europe limits the ability to manage this weed.