Evaluation of Cryptosporidium parvum genotyping techniques.

We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are not Cryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.     

Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.     

Pantoea ananatis: an unconventional plant pathogen

Pantoea ananatis causes disease symptoms in a wide range of economically important agricultural crops and forest tree species worldwide. It is regarded as an emerging pathogen based on the increasing number of reports of diseases occurring on previously unrecorded hosts in different parts of the world. Its unconventional nature lies in the fact that, unlike the majority of plant pathogenic microbes, P. ananatis is capable of infecting humans and occurs in diverse ecological niches, such as part of a bacterial community contaminating aviation jet fuel tanks and contributing to growth promotion in potato and pepper.
Taxonomy: Bacteria; Gammaproteobacteria ; family Enterobacteriaceae ; genus Pantoea.
Microbiological properties: Gram-negative; facultatively anaerobic; most strains are motile and produce a yellow pigment in culture; indole positive.
Biology: Pantoea ananatis is a common epiphyte; it also occurs endophytically in hosts where it has been reported to cause disease symptoms and in hosts where no such symptoms have been described. Some strains are ice-nucleating, a feature which has been used as a biological control mechanism against some insect pests of agricultural crops and by the food industry.
Disease symptoms: Pantoea ananatis infects both monocotyledonous and dicotyledonous plants. The symptoms are diverse depending on the host infected, and include leaf blotches and spots, die-back, and stalk, fruit and bulb rot.
Biological control agent: Pantoea ananatis has both antifungal and antibacterial properties. These characteristics have the potential of being exploited by biological control specialists.     

A gravity model for the spread of a pollinator-borne plant pathogen

Many pathogens of plants are transmitted by arthropod vectors whose movement between individual hosts is influenced by foraging behavior. Insect foraging has been shown to depend on both the quality of hosts and the distances between hosts. Given the spatial distribution of host plants and individual variation in quality, vector foraging patterns may therefore produce predictable variation in exposure to pathogens. We develop a "gravity" model to describe the spatial spread of a vector-borne plant pathogen from underlying models of insect foraging in response to host quality using the pollinator-borne smut fungus Microbotryum violaceum as a case study. We fit the model to spatially explicit time series of M. violaceum transmission in replicate experimental plots of the white campion Silene latifolia. The gravity model provides a better fit than a mean field model or a model with only distance-dependent transmission. The results highlight the importance of active vector foraging in generating spatial patterns of disease incidence and for pathogen-mediated selection for floral traits.     

Application of fluorescence-based resistance gene analog analysis for genotyping plant genetic resources

Conventional approaches for detecting disease resistance gene analogs (RGAs) in plants are based on agarose gels or on polyacrylamide gel electrophoresis (PAGE) in combination with silver staining or radioactive labeling. A modified method for RGA analysis has been developed by using fluorescence-labeled primers for PCR amplifications. The amplified fragments are detected by denaturing PAGE using an automated laser fluorescence DNA sequencer and analyzed by fragment analysis software. This technique is not limited to specific plant species and is suitable for high-throughput genotyping plant genetic resources. We demonstrate here the efficiency of this method for comparison of RGA patterns in diverse plant species and for genotyping of natural populations of the wheat progenitor, Triticum dicoccoides. Revisions requested 8 October 2004; Revisions received 15 November 2004     

Target region amplification polymorphism: A novel marker technique for plant genotyping

The advent of large-scale DNA sequencing technology has generated a tremendous amount of sequence information for many important organisms. We have developed a rapid and efficient PCR-based technique, which uses bioinformatics tools and expressed sequence tag (EST) database information to generate polymorphic markers around targeted candidate gene sequences. This target region amplification polymorphism (TRAP) technique uses 2 primers of 18 nucleotides to generate markers. One of the primers, the fixed primer, is designed from the targeted EST sequence in the database; the second primer, the arbitrary primer, is an arbitrary sequence with either an AT-or GC-rich core to anneal with an intron or exon, respectively. PCR amplification is run for the first 5 cycles with an annealing temperature of 35°C, followed by 35 cycles with an annealing temperature of 50°C. For different plant species, each PCR reaction can generate as many as 50 scorable fragments with sizes ranging from 50–900 bp when separated on a 6.5% polyacrylamide sequencing gel. The TRAP technique should be useful in genotyping germplasm collections and in tagging genes governing desirable agronomic traits of crop plants.     

Tracks for traffic: microtubules in the plant pathogen Ustilago maydis

Pathogenic development of the corn smut fungus Ustilago maydis depends on the ability of the hypha to grow invasively. Extended hyphal growth and mitosis require microtubules, as revealed by recent studies on the microtubule cytoskeleton. Surprisingly, hyphal tip growth involves only two out of 10 kinesins. Kinesin-3 is responsible for tip-directed (anterograde) endosome motility of early endosomes, which are thought to support hyphal elongation by apical membrane recycling. In addition, kinesin-3, together with kinesin-1 and myosin-5, appear to deliver secretory vesicles to the hyphal tip. Kinesin-1 also affects endosome motility by targeting cytoplasmic dynein to microtubule plus ends. This plus-end localization of dynein is essential for cell body-directed (retrograde) endosome motility, but also allows force generation during spindle elongation in mitosis. Furthermore, kinesin-1 and dynein participate in the organization of the microtubule array, thereby building their own network of tracks for intracellular motility. The recent progress in understanding microtubule-based processes in U. maydis has revealed an unexpected complexity of motor functions essential for the virulence of this pathogen. Further studies on structural and regulatory requirements for motor activity should help identify novel targets for fungicide development.     

Detecting single nucleotide polymorphisms using DNA arrays for plant pathogen diagnosis

The lack of a rapid and reliable means for routine pathogen identification has been one of the main limitations in plant disease management, and has pushed the development of culture-independent, molecular approaches. Currently, DNA array technology is the most suitable technique for high-throughput detection and identification, as well as quantification, of multiple pathogens in a single assay. Closely related pathogens that may have completely different host ranges or pathogenicity often differ in only a single to a few base pairs in genes that may be targeted for identification. Therefore, the ability to discriminate single nucleotide polymorphisms (SNPs) should be pursued in any diagnostic assay. In this paper, we demonstrate the utility of DNA array technology to detect SNPs while accounting for specific criteria such as the position of the mismatch, the sequence of the oligonucleotide, and the length and amount of labeled amplicons that are hybridized. When disregarding mismatches at the extreme ends of the oligonucleotides, cross hybridization to single mismatch oligonucleotides is rare when processing environmental samples that contain genetic material from unknown sources. In addition to plant pathology, this study is relevant for any field of research where DNA arrays are used to detect mutations or polymorphisms, ranging from human diagnostics to environmental microbiology and microbial ecology.     

The development of a transformation system for the dimorphic plant pathogen Holleya sinecauda based on Ashbya gossypii DNA elements

We have developed a transformation system for the dimorphic plant pathogenic fungus Holleya sinecauda based on an electroporation protocol used for the closely related filamentous fungus Ashbya gossypii. DNA-mediated transformation of the dominant selection marker kanMX generated H. sinecauda transformants that were resistant to the antibiotic drug G418/geneticin. Freely replicating plasmids could be established in H. sinecauda using an A. gossypii autonomously replicating sequence (ARS) element, whereas Saccharomyces cerevisiae ARS elements, which are functional in A. gossypii, were not functional in H. sinecauda. In addition, centromeric DNA of A. gossypii stabilized the maintenance of plasmids in H. sinecauda under non-selective conditions. We isolated a fragment of the HsLEU2 gene and used this locus for targeted integration of kanMX3, consisting of the kanMX gene flanked by direct repeats. This allowed the construction of a Hsleu2 strain which became G418 sensitive after direct repeat-induced marker excision. The Hsleu2 strain can be complemented by the ScLEU2 gene. Finally, we constructed high- and low-copy shuttle vectors for H. sinecauda.     

Modification of coevolved insect–plant interactions by an exotic plant pathogen

New Perspectives is intended to allow the communication of comments, viewpoints, and speculative interpretation of issues in ecology pertinent to entomology. Comments, viewpoints, or suggestions arising from published papers intended to fuel discussion and debate are also welcome. Contributions should be as concise as possible, normally not exceeding two thousand words. Formal research reports will not be acceptable, but summarised novel data, suitably supported by statistics, may be allowed.     

Proteomics of a plant pathogen: Agrobacterium tumefaciens

Agrobacterium tumefaciens is an important plant pathogen which belongs to the α-proteobacteria. In addition, it has served as the main tool for plant molecular genetics. Here we focus on three major aspects: (i) proteomic mapping, (ii) the use of proteomics for the understanding of the response of A. tumefaciens to changes in environmental conditions and (iii) the analysis of the changes in genome expression following interaction with the host. These studies convey a global outlook on the functional genomics of A. tumefaciens and help to understand the physiology of this important organism.     

A soil-borne generalist pathogen regulates complex plant interactions

Background and aims

Seeds are inhabited by diverse bacterial and fungal taxa whose colonization patterns are little understood. We hypothesized, however, that specific niches within seeds host microbes.

Methods

In this study, the putative presence of bacteria, inhabiting the seed endosphere of an angiosperm, the melon Cucumis melo reticulatus group cv. ‘Dulce’, was examined by scanning electron microscopy (SEM) and confocal laser-scanning microscopy coupled with double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH).

Results

SEM images showed microbial-like structures in different tissues and FISH revealed endophytic bacteria colonizing the outer and inner seed parts, on perisperm/endosperm envelope, inside the cotyledons as parts of the embryo, and, to a lesser extent, inside embryonic hypocotyl-root axis tissues. Alphaproteobacteria were shown to inhabit the seed coat and the envelope surrounding the embryonic hypocotyl-root tissues, but could not be seen in the cotyledons, whereas Betaproteobacteria were only detected in the outer seed coat. Some Gammaproteobacteria were also seen in the outer seed coat, but were mainly visualized in the cotyledons with a few inside the seed’s embryonic hypocotyl-root tissues, among other bacteria. Firmicutes were visualized inside the seed coat, but mostly inside the cotyledon tissues, on the perisperm/endosperm envelope and inside the embryonic hypocotyl-root axis tissues. Microscopy revealed Actinobacteria inside the inner and outer seed coat and inside the embryonic parts such as cotyledons, with a few inside the hypocotyl-root axis.

Conclusions

This is the first demonstration of niches for the most active groups of bacteria inhabiting different seed tissues of an angiosperm.

     

Global switches and fine-tuning-ABA modulates plant pathogen defense

Plants are obliged to defend themselves against a wide range of biotic and abiotic stresses. Complex regulatory signaling networks mount an appropriate defense response depending on the type of stress that is perceived. In response to abiotic stresses such as drought, cold, and salinity, the function of abscisic acid (ABA) is well documented: elevation of plant ABA levels and activation of ABA-responsive signaling result in regulation of stomatal aperture and expression of stress-responsive genes. In response to pathogens, the role of ABA is more obscure and is a research topic that has long been overlooked. This article aims to evaluate and review the reported modes of ABA action on pathogen defense and highlight recent advances in deciphering the complex role of ABA in plant-pathogen interactions. The proposed mechanisms responsible for positive or negative effects of ABA on pathogen defense are discussed, as well as the regulation of ABA signaling and in planta ABA concentrations by beneficial and pathogenic microorganisms. In addition, the fast-growing number of reports that characterize antagonistic and synergistic interactions between abiotic and biotic stress responses point to ABA as an essential component in integrating and fine-tuning abiotic and biotic stress-response signaling networks.     

Disrupting the transmission of a vector-borne plant pathogen

Approaches to control vector-borne diseases rarely focus on the interface between vector and microbial pathogen, but strategies aimed at disrupting the interactions required for transmission may lead to reductions in disease spread. We tested if the vector transmission of the plant-pathogenic bacterium Xylella fastidiosa was affected by three groups of molecules: lectins, carbohydrates, and antibodies. Although not comprehensively characterized, it is known that X. fastidiosa adhesins bind to carbohydrates, and that these interactions are important for initial cell attachment to vectors, which is required for bacterial transmission from host to host. Lectins with affinity to substrates expected to occur on the cuticular surface of vectors colonized by X. fastidiosa, such as wheat germ agglutinin, resulted in statistically significant reductions in transmission rate, as did carbohydrates with N-acetylglucosamine residues. Presumably, lectins bound to receptors on the vector required for cell adhesion/colonization, while carbohydrate-saturated adhesins on X. fastidiosa's cell surface. Furthermore, antibodies against X. fastidiosa whole cells, gum, and afimbrial adhesins also resulted in transmission blockage. However, no treatment resulted in the complete abolishment of transmission, suggesting that this is a complex biological process. This work illustrates the potential to block the transmission of vector-borne pathogens without directly affecting either organism.     

Molecular markers for plant breeding: comparisons of RFLP and RAPD genotyping costs

Three molecular marker protocols, chemiluminescent restriction fragment length polymorphisms (c-RFLPs), radioactivity-based restriction fragment length polymorphisms (r-RFLPs), and randomly amplified DNA polymorphisms (RAPDs) were compared in terms of cost and time efficiency. Estimates of cost of supplies and time requirements were obtained from simulations of maize (Zea mays L.) genotyping experiments utilizing protocols currently in use. The increase in total cost with increasing numbers of individuals genotyped and markers analyzed is higher for RAPDs than for RFLPs. RAPDs were generally found to be more cost and time efficient for studies involving small sample sizes, while RFLPs have the advantage for larger sample sizes. Because of the shorter exposure times involved, c-RFLPs require less time than r-RFLPs to obtain a given amount of information. Variations in the protocols, such as number of re-uses of Southern blots or cost of Taq DNA polymerase per reaction of amplification, also affect the relative merits of RAPDs and RFLPs. Two examples were analyzed where molecular markers are used: a germ plasm survey and quantitative trait loci (QTL) mapping in a segregating population. No protocol was found to be the most cost and time efficient over the entire range of sample sizes and number of marker loci studied.