Microsatellite markers for the grapevine pathogen, Eutypa lata

We isolated and characterized nine polymorphic microsatellite markers for Eutypa lata, a fungal pathogen responsible for Eutypa dieback of grapevine, in populations from two California vineyards (24 isolates per vineyard). Allele frequency ranged from two to 11 alleles per locus and haploid gene diversity ranged from 0.33 to 0.83. All samples comprised unique haplotypes. Our results suggest that there is sufficient allelic polymorphism to estimate fine-scale spatial structure, and to identify possible sources of inoculum from habitats outside of vineyards.     

Phenolic and heterocyclic metabolite profiles of the grapevine pathogen Eutypa lata

The ascomycete Eutypa lata is the causative agent of eutypa dieback in grapevines, a serious economic problem in major wine grape producing areas. In order to develop a predictive, non-destructive assay for early detection of fungal infection, the phenolic metabolite profiles of 11 strains of E. lata grown on four different artificial growth media were analyzed by HPLC and their variability compared with growth on Cabernet Sauvignon grapevine wood and wood extracts. Six compounds were generally produced in significant amounts, namely eutypinol, eulatachromene, and eutypine and its benzofuran cyclization product, together with siccayne and eulatinol. The two most widely distributed and abundant metabolites were eutypinol and eulatachromene, which were present in 8 of the strains grown on grapewood aqueous extract fortified with sucrose. Metabolite production on grapevine extract was greatly enhanced relative to the artificial media, indicating that this native substrate provides optimal conditions and a more representative profile of the metabolites produced in the natural disease state. The primary metabolites were tested in a grapeleaf disc bioassay to establish their relative toxicity. Neither eutypinol nor siccayne were phytotoxic; eulatachromene, eulatinol, eutypine, and the benzofuran exhibited necrotic effects in the bioassay. The results indicate that eutypa dieback may be caused by several E. lata metabolites rather than a single compound.     

Characterizing the production of a wild-type and benomyl-resistant Fusarium lateritium for biocontrol of Eutypa lata on grapevine

Benomyl-resistant (BR) and wild-type (WT) strains of Fusarium lateritium were examined for their tolerance to benomyl on potato dextrose agar (PDA) containing benomyl and control of the Eutypa lata in grapevine bioassays. The WT strain grew on PDA containing 1 μg/ml benomyl at 13, 26 and 29°C. The BR strain grew on PDA containing 10 μg/ml benomyl at 4°C, on PDA containing 100 μg/ml benomyl at 29°C, and on PDA containing 1000 μg/ml benomyl at 13 and 26°C. The BR strain was also able to colonize grapevine segments and control E. lata in the presence of 1000 μg/ml benomyl. Both strains were amenable to production via liquid fermentation and both achieved 100% control of E. lata in grapevine bioassays. Neither the duration of fermentation nor incubation temperature during grapevine bioassays influenced the efficacy of either strain against E. lata. The results suggest that application of BR F. lateritium alone or in combination with benomyl may provide good control of E. lata. Journal of Industrial Microbiology & Biotechnology (2001) 26, 151–155. Received 22 December 1999/ Accepted in revised form 20 October 2000     

Influence of temperature and nutritional requirements for mycelial growth of Eutypa lata, a vineyard pathogenic fungus

Eutypa dieback (dying arm disease, eutypiosis) is a very devastating disease in many grape-producing areas around the world. This vascular disease is induced by the ascomycete Eutypa lata Pers. Fr. Tul & C. Tul. invading the trunk by pruning wounds. The environmental factors and the nutritional requirements regulating fungus development are yet poorly known. This work shows that the isolated strain of E. lata was able to grow in a large temperature range (2-30 degrees C). However, a higher temperature (35 degrees C) presented inhibitory effects on mycelial growth. E. lata was able to use various osidic molecules (C5, C6, C12, C18, C24, and starch); showing thus a large adaptation to the carbon source supplied. As nitrogen source, it used salts and numerous natural amino acids. A significant result was obtained with cysteine presenting obvious antifungal properties. This effect can further be used with the aim of setting up a curative treatment of the disease.     

Susceptibility of pruning wounds to grapevine trunk disease pathogens Eutypa lata and Diplodia seriata in three climatic conditions in Australia

The grapevine trunk diseases Eutypa and Botryosphaeria dieback, caused by fungal species that infect pruning wounds, are a threat to vineyard longevity worldwide. This study evaluated the susceptibility of grapevine pruning wounds in three climatic regions of Australia. In field trials, wounds were made early, mid- and late winter, and inoculated with spores of Eutypa lata or Diplodia seriata at various times, from 1 to 112 days after pruning. For both pathogens, wounds were highly susceptible immediately after pruning, followed by a rapid decrease in susceptibility over the next 14 days in McLaren Vale and Adelaide Hills, South Australia, whereas the period of susceptibility was longer in Big Rivers, New South Wales, where high natural disease pressure of D. seriata confounded results. In the Adelaide Hills, delaying pruning to late winter may reduce the risk of infection by E. lata. A detached cane assay confirmed that the duration of susceptibility of six commonly grown cultivars to E. lata infection was similar.     

Enantiomeric oxidation of organic sulfides by the filamentous fungi Botrytis cinerea,Eutypa lata and Trichoderma viride

The biotransformations of a series of substituted sulfides were carried out with the filamentous fungi Botrytis cinerea, Eutypa lata and Trichoderma viride. Several products underwent microbial oxidation of sulfide to sulfoxide with medium to high enantiomeric purity. With regard to sulfoxide enantioselectivity, the (R)-enantiomer was favoured in biotransformations by T. viride and E. lata while the (S)-enantiomer was favoured in those by B. cinerea. A minor amount of sulfone product was also obtained.     

Quantitative cell-based protein degradation assays to identify and classify drugs that target the ubiquitin-proteasome system

We have generated a set of dual-reporter human cell lines and devised a chase protocol to quantify proteasomal degradation of a ubiquitin fusion degradation (UFD) substrate, a ubiquitin ligase CRL2(VHL) substrate, and a ubiquitin-independent substrate. Well characterized inhibitors that target different aspects of the ubiquitin-proteasome system can be distinguished by their distinctive patterns of substrate stabilization, enabling assignment of test compounds as inhibitors of the proteasome, ubiquitin chain formation or perception, CRL activity, or the UFD-p97 pathway. We confirmed that degradation of the UFD but not the CRL2(VHL) or ubiquitin-independent substrates depends on p97 activity. We optimized our suite of assays to establish conditions suitable for high-throughput screening and then validated their performance by screening against 160 cell-permeable protein kinase inhibitors. This screen identified Syk inhibitor III as an irreversible p97/vasolin containing protein inhibitor (IC(50) = 1.7 μM) that acts through Cys-522 within the D2 ATPase domain. Our work establishes a high-throughput screening-compatible pipeline for identification and classification of small molecules, cDNAs, or siRNAs that target components of the ubiquitin-proteasome system.     

Polymorphisms in grapevine DNA detected by the RAPD PCR technique

A sensitivie, reproducible technique is described for detecting polymorphisms between the nuclear DNA of grapevine isolates using the RAPD PCR technique. Unique fingerprints of a number of cultivars were readily distinguished by using either single primers or mixtures of two primers. The method will be used to provide a databank of fingerprints for the rapid identification of grapevine cultivars, and to develop phylogenetic relationships. It will also be extended and modified in an attempt to detect polymorphisms between DNAs of clonal selections of individual cultivars.     

Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5' nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5' nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% +/- 9.8% to 73.4% +/- 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% +/- 1.92% and 8.11% +/- 4.12%, respectively, versus 0.15% +/- 0.11% and 1.38% +/- 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

Volatiles that encode host-plant quality in the grapevine moth

Plant volatiles are signals used by herbivorous insects to locate host plants and select oviposition sites. Whether such volatiles are used as indicators of plant quality by adult insects in search of host plants has been rarely tested. We tested whether volatiles indicate plant quality by studying the oviposition of the grapevine moth Lobesia botrana on the grapevine plant Vitis vinifera. Host plants were infected with a variety of microorganisms, and larval fitness was correlated to the infected state of the substrate. Our results show an oviposition preference for volatiles that is significantly correlated with the fitness of the substrate. The chemical profiles of the bouquets from each V. vinifera–microorganism system are clearly differentiated in a PCA analysis. Both the volatile signal and the quality of the plant as larval food were affected by the introduction of microorganisms. Our study represents a broad approach to the study of plant–insect interactions by considering not only the direct effect of the plant but also the effect of plant–microorganism interactions on insect population dynamics.     

Quantitative PCR assays for mouse enteric flora reveal strain-dependent differences in composition that are influenced by the microenvironment

The mammalian gastrointestinal (GI) tract is inhabited by over a hundred species of symbiotic bacteria. Differences among individuals in the composition of the GI flora may contribute to variation in in vivo experimental analyses and disease susceptibility. To investigate potential interindividual differences in GI flora composition, we developed real-time quantitative PCR-based assays for the detection of the eight members of the Altered Schaedler Flora (ASF) as representative members of different bacterial niches within the mammalian GI tract. Quantitative and reproducible strain-specific variations in the numbers of the ASF members were observed across 23 different barrier-housed inbred mouse strains, suggesting that the ASF assays can be used as sentinels for changes in GI flora composition. A significant cage effect was also detected. Isogenic mice that cohabited at weaning, whether from the same or different litters, showed little variation in ASF profiles. Conversely, litters split among different cages at weaning showed divergence in ASF profiles after three weeks. Individual ASF profiles, once established, were highly stable over time in the absence of environmental perturbation. Furthermore, cohabitation of different inbred strains maintained most of the interstrain variation in the GI flora, supporting a role of host genetics in determining GI flora composition.     

Infection assays in Arabidopsis reveal candidate effectors from the poplar rust fungus that promote susceptibility to bacteria and oomycete pathogens

Fungi of the Pucciniales order cause rust diseases which, altogether, affect thousands of plant species worldwide and pose a major threat to several crops. How rust effectors—virulence proteins delivered into infected tissues to modulate host functions—contribute to pathogen virulence remains poorly understood. Melampsora larici‐populina is a devastating and widespread rust pathogen of poplar, and its genome encodes 1184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria, we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial (Pseudomonas syringae) and oomycete (Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of green fluorescent protein‐tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth.     

PCR enrichment techniques to identify the diet of predators

The increasing sensitivity of PCR has meant that in the last two decades PCR has emerged as a major tool in diet studies, enabling us to refine our understanding of trophic links and to elucidate the diets of predators whose prey is as yet uncharacterized. The achievements and methods of PCR-based diet studies have been reviewed several times, but here we review an important development in the field: the use of PCR enrichment techniques to promote the amplification of prey DNA over that of the predator. We first discuss the success of using group-specific primers either in parallel single reactions or in multiplex reactions. We then concentrate on the more recent use of PCR enrichment techniques such as restriction enzyme digests, peptide nucleic acid clamping, DNA blocking and laser capture microdissection. We also survey the vast literature on enrichment techniques in clinical biology, to ascertain the pitfalls of enrichment techniques and what refinements have yielded some highly sensitive methods. We find that while there are several new approaches to enrichment, peptide nucleic acid clamping and DNA blocking are generally sufficient techniques for the characterization of diets of predators and highlight the most important considerations of the approach.     

Gain of function assays identify non- rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity

This study tested the morphogenetic potential of 15 open reading frames of the TL-DNA of Agrobacterium rhizogenes strain HRI. These open reading frames were expressed individually under the control of the 35S RNA promoter in transgenic tobacco plants ( Nicotiana tabacum L.). Expression of three T-DNA loci, ORF3n, ORF8 and ORF13, alters plant morphogenesis or the response of transgenic tissues to plant hormones. ORF3n transgenic plants are characterized by retarded flowering, altered internode elongation, altered leaf shape and, in particular, leaf tip necrosis. ORF3n and ORF8 expression reduces the sensitivity to auxin and cytokinin in combination or auxin alone. Tetracycline-dependent expression of ORF13 overcomes a selection of low levels of expression during plant regeneration and reveals a strong inhibitory effect of the ORF13 gene product on cell division and cell elongation. We conclude that the A. rhizogenes TL-DNA harbors genetic information that is important for pathogenicity apart from the well studied rol genes. We propose that these genes play mainly a negative regulatory role during pathogenesis. Moreover, these loci might be relevant to successful infections in specific host plants.     

Predicting the sensitivity and specificity of published real-time PCR assays

Background

In recent years real-time PCR has become a leading technique for nucleic acid detection and quantification. These assays have the potential to greatly enhance efficiency in the clinical laboratory. Choice of primer and probe sequences is critical for accurate diagnosis in the clinic, yet current primer/probe signature design strategies are limited, and signature evaluation methods are lacking.

Methods

We assessed the quality of a signature by predicting the number of true positive, false positive and false negative hits against all available public sequence data. We found real-time PCR signatures described in recent literature and used a BLAST search based approach to collect all hits to the primer-probe combinations that should be amplified by real-time PCR chemistry. We then compared our hits with the sequences in the NCBI taxonomy tree that the signature was designed to detect.

Results

We found that many published signatures have high specificity (almost no false positives) but low sensitivity (high false negative rate). Where high sensitivity is needed, we offer a revised methodology for signature design which may designate that multiple signatures are required to detect all sequenced strains. We use this methodology to produce new signatures that are predicted to have higher sensitivity and specificity.

Conclusion

We show that current methods for real-time PCR assay design have unacceptably low sensitivities for most clinical applications. Additionally, as new sequence data becomes available, old assays must be reassessed and redesigned. A standard protocol for both generating and assessing the quality of these assays is therefore of great value. Real-time PCR has the capacity to greatly improve clinical diagnostics. The improved assay design and evaluation methods presented herein will expedite adoption of this technique in the clinical lab.     

First evidences that the ectomycorrhizal fungus Paxillus involutus mobilizes nitrogen and carbon from saprotrophic fungus necromass

Fungal succession in rotting wood shows a surprising abundance of ectomycorrhizal (EM) fungi during the late decomposition stages. To better understand the links between EM fungi and saprotrophic fungi, we investigated the potential capacities of the EM fungus Paxillus involutus to mobilize nutrients from necromass of Postia placenta, a wood rot fungus, and to transfer these elements to its host tree. In this aim, we used pure cultures of P. involutus in the presence of labelled Postia necromass (¹⁵N/¹³C) as nutrient source, and a monoxenic mycorrhized pine experiment composed of labelled Postia necromass and P. involutus culture in interaction with pine seedlings. The isotopic labelling was measured in both experiments. In pure culture, P. involutus was able to mobilize N, but C as well, from the Postia necromass. In the symbiotic interaction experiment, we measured high ¹⁵N enrichments in all plant and fungal compartments. Interestingly, ¹³C remains mainly in the mycelium and mycorrhizas, demonstrating that the EM fungus transferred essentially N from the necromass to the tree. These observations reveal that fungal organic matter could represent a significant N source for EM fungi and trees, but also a C source for mycorrhizal fungi, including in symbiotic lifestyle.