Sensitivity of Petri disease pathogens to hot-water treatments in vitro

Petri disease pathogens ( Phaeoacremonium spp. and Phaeomoniella chlamydospora ) are able to colonize the vessels in the xylem of grapevine propagating material. Hot-water treatment (HWT) protocols at 50°C for 30 min have been applied in grapevine nurseries to control these pathogens with variable results. The effect of HWT in vitro at higher temperatures on Pa.   chlamydospora , Phaeoacremonium aleophilum and Phaeoacremonium parasiticum isolates was determined by placing conidial suspensions and plugs of agar with mycelia in Eppendorf vials and incubated in hot water at 49, 50, 51, 52, 53 or 54°C for 30, 45 or 60 min. Conidial germination and the colony growth rate decreased with increased temperature and time combinations. Pa.   chlamydospora was more sensitive than Phaeoacremonium spp. to the experimental temperatures for all periods of time. Pa.   chlamydospora tolerated temperatures of 53°C, while Pm.   aleophilum and Pm.   parasiticum tolerated temperatures of 54°C, although the treatments above 51–52°C drastically reduced conidial germination and mycelial growth. These results highlight the need to develop HWT using temperatures above 51°C to reduce the incidence of fungal infections and ensure high-quality propagating material for grapevine growers.     

Development of microsatellite markers for the grapevine fungal pathogen Phaeomoniella chlamydospora

Twenty polymorphic microsatellite markers from microsatellite-enriched genomic DNA of the grapevine fungal pathogen, Phaeomoniella chlamydospora, were developed and characterized. The markers were used to genotype isolates from Australia and from Europe/Eurasia. The number of alleles per locus ranged from two to 11. Gene diversity per locus ranged from 0.08 to 0.63 among Australian isolates, and from 0.2 to 0.77 among isolates from Europe/Eurasia, demonstrating the suitability of these markers for population genetic studies of P. chlamydospora. Eighteen of the 20 markers also amplified a product in the closely related Phaeoacremonium aleophilum.     

Real-time PCR detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

Phaeomoniella chlamydospora and Phaeoacremonium aleophilum are the two main fungal causal agents of Petri disease and esca. Both diseases cause significant economic losses to viticulturalists. Since no curative control measures are known, proactive defensive measures must be taken. An important aspect of current research is the development of sensitive and time-saving protocols for the detection and identification of these pathogens. Real-time PCR based on the amplification of specific sequences is now being used for the identification and quantification of many infective agents. The present work reports real-time PCR protocols for identification of P. chlamydospora and P. aleophilum. Specificity was demonstrated against purified DNA from 60 P. chlamydospora isolates or 61 P. aleophilum isolates, and no amplification was obtained with 54 nontarget DNAs. The limits of detection (i.e., DNA detectable in 95% of reactions) were around 100 fg for P. chlamydospora and 50 fg for P. aleophilum. Detection was specific and sensitive for P. chlamydospora and P. aleophilum. Spores of P. chlamydospora and P. aleophilum were detected without the need for DNA purification. The established protocols detected these fungi in wood samples after DNA purification. P. chlamydospora was detectable without DNA purification and isolation in 67% of reactions. The detection of these pathogens in wood samples has great potential for use in pathogen-free certification schemes.     

PCR-based strategy to detect and identify species of Phaeoacremonium causing grapevine diseases

Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial beta-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.     

Togninia (Calosphaeriales) is confirmed as teleomorph of Phaeoacremonium by means of morphology, sexual compatibility and DNA phylogeny

Petri disease, or black goo, is a serious disease of vines in most areas where grapevines are cultivated. The predominant associated fungus is Phaeomoniella chlamydospora (Chaetothyriales). Several species of Phaeoacremonium (Pm.) also are associated, of which Pm. aleophilum is the most common. Although no teleomorph is known for Phaeoacremonium, the genus Togninia previously has been linked to phaeoacremonium-like anamorphs. To investigate the possible anamorph-teleomorph connection of Phaeoacremonium to Togninia, anamorphs of Togninia minima, T. fraxinopennsylvanica and T. novae-zealandiae morphologically were compared with Pm. aleophilum and some representative cultures were mated in all combinations. Although no interspecies mating proved fertile, matings between isolates of Pm. aleophilum produced a Togninia teleomorph within 3-4 weeks. Certain field isolates of Pm. aleophilum commonly produced the teleomorph, demonstrating that both mating types can occur in the same vine and thus also explaining the genetic diversity observed for this fungus in some vineyards. To elucidate the phylogenetic relationships among these taxa, isolates were subjected to sequence analysis of the nuclear ribosomal internal transcribed spacers (ITS1, ITS2) and the 5.8S rRNA gene, as well as portions of the translation elongation factor 1 alpha (EF-1α) gene. The generic placement of teleomorphs within Togninia (Calosphaeriales) further was confirmed via phylogenetic analyses of 18S small subunit (SSU) DNA. From these sequences, morphological and mating data, we conclude that T. minima is the teleomorph of Pm. aleophilum, and that it has a biallelic heterothallic mating system. An epitype and mating type tester strains also are designated for T. minima.     

Differences in Protein Synthesis and Peroxidase isoenzymes between recalcitrant and regenerating ProtoPlasts

The reasons for the inability of recalcitrant mesophyll protoplasts to divide and re-enter the cell cycle are unknown. Changes in protein profile, indole-3-acetic acid (IAA)-oxidase and peroxidase activities, and isoenzymes were compared in protoplasts of recalcitrant grapcvine ( Vitis vinifera ) L. cv. Sultanina) and regenerating tobacco ( Nicotiana tabacum ) L. cv. Xanthi). Using [³⁵S]-methionine. SDS-PAGE and two-dimensional separation of proteins, differences in protein profile during protoplast culture were assessed. The changes in the de novo synthesized proteins were both qualitative and quantitative between the two species. The number of proteins which changed was double in tobacco compared to grapevine protoplasts. Peroxidase and IAA-oxidase activities increased significantly in tobacco protoplasts during culture whereas in grapevine they remained low. In tobacco protoplasts. 3 and 7 basic and acidic peroxidases, respectively, were induced during protoplast culture. which were not detected in the intact leaf, whereas in grapevine no new peroxidases were induced during protoplast culture.     

单氰胺对葡萄休眠过程中冬芽水分和碳水化合物的影响

以酿酒葡萄品种'赤霞珠'和'霞多丽'为材料,对其冬芽休眠进程和休眠过程中冬芽水分和碳水化合物含量的变化进行分析.结果表明,(1)在陕西杨陵地区气候条件下,'霞多丽'、'赤霞珠'分别在12月20日和1月9日达到深度休眠,随后开始休眠解除阶段.(2)在芽休眠过程中,两个品种冬芽的总水分含量、自由水含量和淀粉含量均随休眠的加深而降低,随休眠的解除而增加,而冬芽的束缚水含量、束缚水/自由水比值、可溶性糖含量均在休眠加深阶段持续上升,在休眠解除阶段逐渐降低.(3)单氰胺处理后,两品种冬芽中自由水含量显著增加,束缚水的比例同时显著降低,且'赤霞珠'的变化幅度大于'霞多丽',但其总水分、可溶性糖和淀粉等含量在休眠过程中无显著变化.(4)葡萄冬芽中的水分含量及存在状态、可溶性糖含量和淀粉含量的变化与其休眠进程密切相关;单氰胺处理能够增加冬芽自由水含量,降低其束缚水含量,从而有效打破葡萄休眠.     

A Basic Peroxidase Isoenzyme, Marker of Resistance Against Plasmopara viticola in Grapevines, is Induced by an Elicitor from Trichoderma viride in Susceptible Grapevines

The constitutive expression of basic peroxidase isoenzymes in the Plasmopara viticola -resistant ( Vitis vinifera × Vitis rupestris) × Vitis riparia crossing and in the P. viticola -susceptible V. vinifera parent species was studied. The results illustrate that both leaves and stems of the ( V. vinifera × V. rupestris) × V. riparia crossing showed the differential expression of a basic peroxidase isoenzyme B₃ (pl = 8.9), this being almost completely absent from the P. viticola -susceptible V. vinifera parent species. To test whether the basic peroxidase isoenzyme B₃ may be considered as a molecular marker of disease resistance in Vitis spp., suspension cell cultures derived from the P. viticola -susceptible V. vinifera parent species were treated with an elicitor (cellulase Onoztika R-10) from the soil fungus Trichoderma viride , a specific and well-known elicitor of disease resistance reactions in grapevines. The results showed that treatment with the elicitor induces, simultaneously with the activation of the disease resistance mechanism, the appearance of B₃ in the cell cultures. These results suggest that the basic peroxidase isoenzyme B₃ may be considered as a marker of disease resistance in Vitis species since it is present in the P. viticola -resistant ( V. vinifera × V. rupestris) × V. riparia hybrid and is induced by the elicitor Onozuka R-10 in cell cultures of the P. viticola -susceptible Vitis vinifera parent species. This conclusion is supported by the presence of this isoenzyme in other resistant and its absence in other susceptible Vitis spp.     

Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines

Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 3' border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were undetected after 8 wk whereas viable spores still could be detected at 17 wk.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

Ecosystem productivity can be predicted from potential relative growth rate and species abundance

We show that ecosystem-specific aboveground net primary productivity (SANPP, g g⁻¹ day⁻¹, productivity on a per gram basis) can be predicted from species-level measures of potential relative growth rate (RGR_max), but only if RGR_max is weighted according to the species' relative abundance. This is in agreement with Grime's mass-ratio hypothesis. Productivity was measured in 12 sites in a French Mediterranean post-agricultural succession, while RGR_max was measured on 26 of the most abundant species from this successional sere, grown hydroponically. RGR_max was only weakly correlated ( r ² = 0.12, P  < 0.05) with field age when species abundance was not considered, but the two variables were strongly correlated ( r ² = 0.81, P  < 0.001) when the relative abundance of species in each field was taken into account. SANPP also decreased significantly with field age. This resulted in a tight relationship ( r ² = 0.77, P  < 0.001) between productivity and RGR_max weighted according to species relative biomass contribution. Our study shows that scaling-up from the potential properties of individual species is possible, and that information on potential and realized species traits can be integrated to predict ecosystem functioning.     

The evaluation of novel chromogenic substrates for the detection of lipolytic activity in clinical isolates of Staphylococcus aureus and MRSA from two European study groups

Pinellia ternata , a traditional Chinese herb that has been used in China for over 1000 years, is susceptible to a soft rot disease, which may cause major loss of yield. The use of bacteria as potential antagonists against Pectobacterium carotovorum SXR1, the causal agent of the disease on P. ternata , was evaluated. Altogether, 1107 candidate bacteria were isolated from the rhizosphere and surface-sterilized plants of P. ternata . In Petri dish tests, 55 isolates inhibited the growth of strain SXR1, and 21 of these reduced the disease development on P. ternata slices by over 50%. Four selected antagonists significantly reduced the disease incidence on tissue culture seedlings, and also prevented the disease on the transplants. Agonist P-Y2-2 yielded a good prevention level of 81.9%. The four antagonists rapidly colonized the tissue culture seedlings and transplants, whereas greater populations of the antagonists (10⁷–10⁹ CFU g⁻¹ fresh tissues) were observed in the seedlings and in the preinoculated transplants than in those inoculated during transplanting. The use of pathogen-free tissue culture seedlings pre-inoculated with antagonist may provide a strategy for production of P. ternata plantlets resistant to soft rot disease. This is the first report on the efficacy of biocontrol agents against pathogens on P. ternata .     

Occurrence of Fungal Pathogens Associated with Grapevine Nurseries and the Decline of Young Vines in Spain

The occurrence of fungal grapevine trunk pathogens associated with grapevine nurseries and the decline of young vines in Spain was determined in extensive surveys conducted in nurseries and vineyards with young plants. The presence of Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Cylindrocarpon spp., Botryosphaeria obtusa and Botryosphaeria spp. was detected in all the surveys that were carried out. This study provides evidence for the presence of these pathogens in Spanish grapevine nurseries. Cylindrocarpon spp., B. obtusa and Botryosphaeria spp., were isolated very early in the planting material production process, nevertheless, P. aleophilum and P. chlamydospora were not detected until the rootstock–scion combinations were planted in the field to develop shoots and roots during summer. The occurrence of trunk disease pathogens in symptomatic young vineyards was also high, suggesting that infected plant material might have been used. Phaeomoniella chlamydospora and P. aleophilum were the main species associated with the decline of young vines, followed by Botryosphaeria spp., Cylindrocarpon spp. and B. obtusa. This research confirmed the importance of fungal grapevine trunk pathogens associated with the decline of young vineyards in Spain and suggested the connection between the presence of these fungi in the nurseries and the disease in the fields.     

Insecticidal activity influence of destruxins on the pathogenicity of Paecilomyces javanicus against Spodoptera litura

Abstract:  The bioactivities of destruxins (dtx), depsipeptides isolated from Metarhizium anisopliae , against Spodoptera litura were tested in laboratory. For contacting toxicities, dtx-E was more effective than dtx-A and dtx-B. The LC₅₀s values of dtx-A, B and E were 197.98, 292.00 and 113.99 mg/l at 48 h after treatment, while the LT₅₀s were 42.65, 59.45 and 23.68 h at 300 mg/l. In the experiment of antifeedant activity, dtx-A, dtx-B and dtx-E at five concentrations (200, 100, 50, 25 and 12.5 mg/l) were bioassayed. Destruxins in a dose-dependent manner gave an apparent antifeedant activity. Generally, dtx-A, over dtx-B and dtx-E had the significant (P < 0.05) larger choice and no-choice antifeedant indexes (CAIs and NCAIs). At the concentration of 200 mg/l, the CAIs or NCAIs of dtx-A, dtx-B and dtx-E were 96.78, 84.93 and 85.90 or 89.75, 62.42 and 72.28 respectively. Furthermore, the synergistic activity of crude destruxin (CD) for pathogenicity of Paecilomyces javanicus strain Pj01 was detected. The LC₅₀s values of single Pj01 and the mixtures of Pj01 plus CD at 100 or 200 mg/l (Pj01-CD100 or Pj01-CD200) were respectively 474.63 × 10⁵, and 197.45 × 10⁵ or 113.11 × 10⁵ spores/ml at the fifth day after treated. Meanwhile, Pj01, Pj01-CD100 and Pj01-CD200 gave the LT₅₀s values of 6.99 day, 5.49 day and 4.21 day at 100 × 10⁵ spores/ml. Clearly, dtx decreased the values of LC₅₀ and LT₅₀ of the strain Pj01.     

A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.     

Monoterpene glycoside biosynthesis in detached grape berries grown in vitro

A procedure for the culture in vitro of isolated small berries of Vitis vinifera L. cv. Muscat of Alexandria in a Murashige and Skoog basal medium supplemented with N⁶-benzyladenine and indoleacetic acid is described. Berries developed well in culture during 60 days and tripled in size, but remained green and smaller than normal berries grown in vivo. Some callus formed on the distal end of the berry, and where major skin damage occurred, callus emerged from the cracked berries. In order to examine their biosynthetic competency, berries which were previously cultured in vitro for 60 days were incubated for 48 h in a Murashige and Skoog medium containing a [¹⁴C]-labelled water-soluble fraction. This fraction was isolated from grape berries located adjacent to a leaf that had been exposed to gaseous ¹⁴CO₂ in full sunlight for 5 h. The berries were then recultured for 48 h after which a glycosidic fraction was isolated on a C18 reversed phase column and further separated by thin layer chromatography (TLC). The major labelled band corresponded to the geranyl-β-rutinoside marker, indicating that grape berries have the ability to synthesize monoterpene glycosides. This band also consisted of other monoterpene glycosides as revealed by the gas chromatography-mass spectrometry (GC-MS) analysis of their aglycones (released by enzymatic hydrolysis).     

Bird diversity: a predictable function of satellite-derived estimates of seasonal variation in canopy light absorbance across the United States

Aim  To investigate the relationships between bird species richness derived from the North American Breeding Bird Survey and estimates of the average, minimum, and the seasonal variation in canopy light absorbance (the fraction of absorbed photosynthetically active radiation, fPAR) derived from NASA's Moderate Resolution Imaging Spectroradiometer (MODIS).
Location  Continental USA.
Methods  We describe and apply a 'dynamic habitat index' (DHI), which incorporates three components based on monthly measures of canopy light absorbance through the year. The three components are the annual sum, the minimum, and the seasonal variation in monthly fPAR, acquired at a spatial resolution of 1 km, over a 6-year period (2000–05). The capacity of these three DHI components to predict bird species richness across 84 defined ecoregions was assessed using regression models.
Results  Total bird species richness showed the highest correlation with the composite DHI [ R ² = 0.88, P  < 0.001, standard error of estimate (SE) = 8 species], followed by canopy nesters ( R ² = 0.79, P  < 0.001, SE = 3 species) and grassland species ( R ² = 0.74, P  < 0.001, SE = 1 species). Overall, the seasonal variation in fPAR, compared with the annual average fPAR, and its spatial variation across the landscape, were the components that accounted for most ( R ² = 0.55–0.88) of the observed variation in bird species richness.
Main conclusions  The strong relationship between the DHI and observed avian biodiversity suggests that seasonal and interannual variation in remotely sensed fPAR can provide an effective tool for predicting patterns of avian species richness at regional and broader scales, across the conterminous USA.     

The role of abscisic acid in the ripening of grapes

Ripening in grapes ( Vitis vinifera L. cv. Thompson seedless) was accompanied by an increase in the levels of sucrose, glucose and fructose and a decrease in the levels of acids. The activity of glucose-6-phosphatase and fructose-l–6-bisphospbatase was lower in sweet grapes as compared to sour ones. Abscisic acid (10⁻⁶ M) stimulated the gluconeogenic process in sour grapes. The levels of some gluconeogenic enzymes were also elevated in its presence. Cyclohexitnide (0.036–1.8 mM) nullified the abscisic acid effect, suggesting that this effect involves de novo protein synthesis. The incorporation of [¹⁴C]-leucine into proteins was enhanced about 80% by abscisic acid, confirming that abscisic acid promoted protein synthesis. Again, cycloheximide blocked the hormone mediated increase in the incorporation of radioactivity into proteins. The results indicate that one of the factors for sourness in certain mature ripe grapes may be that abscisic acid is not available.     

Biological control of Dutch elm disease: Bacillus thuringiensis as a potential control agent for Scolytus scolytus and S. multistriatus

J assim , H.K., F oster , H.A. & F airhurst , C.P. 1990. Biological control of Dutch elm disease: Bacillus thuringiensis as a potential control agent for Scolytus scolytus and S. multistriatus. Journal of Applied Bacteriology 69 , 563–568.
The effects of exposing fifth instar larvae of Scolytus scolytus and S. multistriatus to spore suspensions of Bacillus spp. were investigated. Bacillus thuringiensis ser 3a, 3b increased the mortality of larvae cultured on an artificial medium from approximately 20% in control cultures to over 80% in cultures exposed to the bacteria. The mortality was dose-dependent for S. multistriatus and the approximate LC₅₀ value was 2.2 times 10³ spores/ml. Different serotypes of B. thuringiensis caused different levels of mortality: H6 produced the highest mortality and H1 the lowest. Bacillus alvei and B. cereus were also pathogenic but B. megaterium was not. The results are discussed in relation to the mechanism of pathogenicity and the potential for the use of B. thuringiensis for the control of the vectors of Dutch elm disease.