Plant Pathogen

Conventional methods to detect and assess the viability of plant pathogenic bacteria are usually based on plating assays or serological techniques. Plating assays provide information about the number of viable cells, expressed as colony-forming units, but are time-consuming and laborious. Serological methods, such as the enzyme-linked immunosorbent assay (ELISA) and immunofluorescence microscopy (IF), can be performed in a shorter timespan than most plating assays, but they do not discriminate between live and dead cells. Flow cytometry (FCM) in combination with fluorescent probe technology is a rapid, sensitive, and quantitative technique to detect microorganisms and assess their viability. Quantitative information on the presence and viability of plant pathogenic microorganisms is valuable for risk assessment regarding disease transmission and disease development. FCM has been applied successfully in the fields of food microbiology, veterinary science, and medical research to dete...

Plant pathogen resistance is mediated by a large repertoire of resistance (R) genes, which are often clustered in the genome and show a high degree of genetic variation. Here, we show that anArabidopsis thaliana R-gene cluster is also subject to epigenetic variation. We describe a heritable but metastable epigenetic variant bal that overexpresses the R-like geneAt4g16890 from a gene cluster on Chromosome 4. The balvariant and Arabidopsis transgenics overexpressing theAt4g16890 gene are dwarfed and constitutively activate the salicylic acid (SA)-dependent defense response pathway. Overexpression of a related R-like gene also occurs in the ssi1(suppressor of SA insensitivity1) background, suggesting that ssi1 is mechanistically related to bal.

The utility of polymorphism analysis was determined for differentiation of the following subspecies of the Gram-positive plant pathogenic bacterium, Clavibacter michiganensis: C. m. subsp. michiganensis, C. m. subsp. sepedonicus, C. m. subsp. insidiosus C. m. subsp. nebraskensis, and C. m. subsp. tessellarius. Specific primers designed for amplification of the housekeeping genes recA, rpoB, and rpoD generated 827-, 1037-, and 862-bp DNA fragments, respectively. PCR products obtained from 40 C. michiganensis strains were analysed using RFLP with four restriction endonucleases, and those PCR products with specific RFLP patterns were sequenced. The genotypes discriminated after PCR–RFLP were specific for each subspecies and also allowed for differentiation of C. m. subsp. michiganensis strains. Sequence analysis of the recA, rpoB, and rpoD gene fragments also distinguished C. michiganensis subspecies and was useful for phylogenetic analysis of all subspecies. For rapid, inexpensive, and effective differentiation of the five subspecies in this research, we recommend the amplification of recA and/or rpoD gene fragments and digestion of the PCR products with the restriction endonuclease FnuDII.

Colletotrichum musae is an important pathogen causing banana anthracnose. The type material (K) had no conidia or sclerotia, and DNA could not be extracted from a darkened area of the herbarium sample. This sample thus provides few characters to delimit this species from other closely related taxa in the “gloeosporioides” species complex. An epitype is therefore designated for C. musae

Abstract: Wilt is an important disease of brinjal crop causing significant reduction in yield. In present study, the pathogenic fungus was isolated from infected plant parts and identified based on morphological and cultural characters as Fusarium solani f. sp. melongenae. ...

Twenty-two microsatellite loci were characterized in the soilborne plant pathogenic 8 fungus Verticillium dahliae by analysis of the genome sequence. All loci were 9 polymorphic in at least two of three populations of V. dahliae from lettuce, spinach and 10 tomato. These loci were useful in genotyping isolates and highlighting differences in 11 genetic diversity among the three tested populations.

Hemileia vastatrix is a biotrophic fungus, causing coffee leaf rust in all coffee growing countries, leading to serious social and economic problems. Gene expression studies may have a key role unravelling the transcriptomics of this pathogen during interaction with the plant host. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the golden standard for gene expression analysis, although an accurate normalisation is essential for adequate conclusions. Reference genes are often used for this purpose, but the stability of their expression levels requires validation under experimental conditions. Moreover, pathogenic fungi undergo important biomass variations along their infection process in planta, which raises the need for an adequate method to further normalise the proportion of fungal cDNA in the total plant and fungus cDNA pool. In this work, the expression profiles of seven reference genes [glyceraldehyde-3-phosphate dehydrogenase (GADPH), elongation factor (EF-1), Beta tubulin (β-tubulin), cytochrome c oxidase subunit III (Cyt III), cytochrome b (Cyt b), Hv00099, and 40S ribosomal protein (40S_Rib)] were analysed across 28 samples, obtained in vitro (germinated uredospores and appressoria) and in planta (post-penetration fungal growth phases). Gene stability was assessed using the statistical algorithms incorporated in geNorm and NormFinder tools. Cyt b, 40S_Rib, and Hv00099 were the most stable genes for the in vitro dataset, while 40S_Rib, GADPH, and Cyt III were the most stable in planta. For the combined datasets (in vitro and in planta), 40S_Rib, GADPH, and Hv00099 were selected as the most stable. Subsequent expression analysis for a gene encoding an alpha subunit of a heterotrimeric G-protein showed that the reference genes selected for the combined dataset do not differ significantly from those selected specifically for the in vitro and in planta datasets. Our study provides tools for correct validation of reference genes in obligate biotrophic plant pathogens, as well as the basis for RT-qPCR studies in H. vastatrix.► Reference genes were selected for Hemileia vastatrix in planta RT-qPCR studies. ► Gene stability was assessed using geNorm and NormFinder programmes. ► 40S_Rib, GADPH, and Hv00099 were the most stable reference genes. ► An extra correction step enabled normalisation of fungal biomass in planta.

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 103 P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.

Siderophores produced by rhizosphere bacteria may enhance plant growth by increasing the availability of Fe near the root or by inhibiting the colonization of roots by plant pathogens or other harmful bacteria. To examine the populations of siderophore-producing bacteria colonizing the roots of two grass species that differed in their susceptibility to Fe deficiency, we inoculated serial dilutions of root samples onto chrome azurol S (CAS) agar and several other selective and non-selective culture meida. CAS agar effectively differentiated bacteria that were capable of excreting large amounts of siderophore, but the composition of the medium limited its usefulness for ecological studies. A large proportion (71–79%) of the bacterial population that grew on a non-selective medium (tryptic soy agar) failed to grow on CAS agar, and several isolates that showed no sign of siderophore production on CAS agar produced siderophore in liquid culture. Similar populations of siderophore-producing bacteria were observed on roots of St. Augustine grass, which frequently exhibits Fe chlorosis, and bermuda grass, which does not. Roots of both grasses were colonized by bacteria that produced siderophore in vitro at concentrations ranging from 100 to 230 μM. The CAS assay solution was also used to compare siderophore production by Pseudomonas fluorescens Q6, an isolate from bermuda grass, and by P. putida B 10, a plant growth-promoting pseudomonad. P. fluorescens Q6 produced 2.4 times more siderophore in vitro than P. putida B 10.

The survival of Sclerotinia sclerotiorum, to a large extent depends on the production and viabilty of sclerotia. Reduction in the production of sclerotia is essential in the control of the pathogen. A local Trichoderma harzianum isolate and two Bacillus amyloliquefaciens isolates were evaluated in vitro and in vivo as potential biological control agents (BCAs) against S. sclerotiorum. The study showed that T. harzianum and B. amyloliquefaciens inhibited the growth and production of mycelia and sclerotia. The local isolates, T. harzianum and B. amyloliquefaciens appeared to exhibit mycoparasitism and antibiosis, respectively, in the in vitro study. As antagonists, these isolates protected over 80% of tomato, squash and eggplant seedlings inoculated with S. sclerotiorum. The efficacy of T. harzianum and B. amyloliquefaciens compared with two commercial products, PlantShield and SoilGard in the control of S. sclerotiorum was similar or slightly lower depending on the crop plant.

The highly divergent characters of morphology, ecology, and biology in the Helotiales make it one of the most problematic groups in traditional classification and molecular phylogeny. Sequences of three rDNA regions, SSU, LSU, and 5.8S rDNA, were generated for 50 helotialean fungi, representing 11 out of 13 families in the current classification. Data sets with different compositions were assembled, and parsimony and Bayesian analyses were performed. The phylogenetic distribution of lifestyle and ecological factors was assessed. Plant endophytism is distributed across multiple clades in the Leotiomycetes. Our results suggest that (1) the inclusion of LSU rDNA and a wider taxon sampling greatly improves resolution of the Helotiales phylogeny, however, the usefulness of rDNA in resolving the deep relationships within the Leotiomycetes is limited; (2) a new class Geoglossomycetes, including Geoglossum, Trichoglossum, and Sarcoleotia, is the basal lineage of the Leotiomyceta; (3) the Le...

Roots serve a multitude of functions in plants including anchorage, acquisition of nutrients and water, and production of exudates with growth regulatory properties. The root–soil interface, or rhizosphere, is the site of greatest biological and chemical activity within the soil matrix. Plant growth-promoting rhizobacteria (PGPR) are known to influence plant health by controlling plant pathogens or via direct enhancement of plant development in the laboratory and in greenhouse experiments. Unfortunately, however, results in the field have been less consistent. The colonization of roots by inoculated bacteria is an important step in the interaction between beneficial bacteria and the host plant. However, colonization is a complex phenomenon influenced by many biotic and abiotic parameters, some of which are only now apparent. Monitoring fate and metabolic activity of microbial inoculants as well as their impact on rhizosphere and soil microbial communities are needed to guarantee safe and reliable application, independent of whether they are genetically modified or not. The first and most crucial prerequisite for effective use of PGPRs is that strain identity and activity are continuously confirmed. A combination of both classical and molecular techniques must be perfected for more effective monitoring of inoculants strain (both genetically modified and unmodified) after release into the soil. Recent developments in techniques for studying rhizobacterial communities and detection and tracking systems of inoculated bacteria are important in future application and assessment of effectiveness and consistent performance of microbial inoculants in crop production and protection.

The family Xanthomonadaceae is a wide-spread family of bacteria belonging to the gamma subdivision of the Gram-negative proteobacteria, including the two plant-pathogenic genera Xanthomonas and Xylella, and the related genus Stenotrophomonas. Adhesion is a widely conserved virulence mechanism among Gram-negative bacteria, no matter whether they are human, animal or plant pathogens, since attachment to the host tissue is one of the key early steps of the bacterial infection process. Bacterial attachment to surfaces is mediated by surface structures that are anchored in the bacterial outer membrane and cover a broad group of fimbrial and non-fimbrial structures, commonly known as adhesins. In this chapter, we discuss recent findings on candidate adhesins of plant-pathogenic Xanthomonadaceae, including polysaccharidic (lipopolysaccharides, exopolysaccharides) and proteineous structures (chaperone/usher pili, type IV pili, autotransporters, two-partner-secreted and other outer membrane adhesins), their involvement in the formation of biofilms and their mode of regulation via quorum sensing. We then compare the arsenals of adhesins among different Xanthomonas strains and evaluate their mode of selection. Finally, we summarize the sparse knowledge on specific adhesin receptors in plants and the possible role of RGD motifs in binding to integrin-like plant molecules.