A TaqMan real-time PCR assay for Rhizoctonia cerealis and its use in wheat and soil

Rhizoctonia cerealis causes sharp eyespot in cereals and the pathogen survives as mycelia or sclerotia in soil. Real-time Polymerase Chain Reaction (qPCR) assays based on TaqMan chemistry are highly suitable for use on DNA extracted from soil. We report here the first qPCR assay for R. cerealis using TaqMan primers and a probe based on a unique Sequence Characterised Amplified Region (SCAR). The assay is highly specific and did not amplify DNA from a range of other binucleate Rhizoctonia species or isolates of anastomosis groups of Rhizoctonia solani. The high sensitivity of the assay was demonstrated in soils using a bulk DNA extraction method where 200 μg sclerotia in 50 g of soil were detected. DNA of the pathogen could also be amplified from asymptomatic wheat plants. Using the assay on soil samples from fields under different crop rotations, R. cerealis was most frequently detected in soils where wheat was grown or soil under pasture. It was detected least frequently in fields where potatoes were grown. This study demonstrates that assays derived from SCAR sequences can produce specific and sensitive qPCR assays

Molecular techniques for pathogen identification and fungus detection in the environment

Many species of fungi can cause disease in plants, animals and humans. Accurate and robust detection and quantification of fungi is essential for diagnosis, modeling and surveillance. Also direct detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great many fungi are difficult or impossible to cultivate. In the last decade, effective amplification platforms, probe development and various quantitative PCR technologies have revolutionized research on fungal detection and identification. Examples of the latest technology in fungal detection and differentiation are discussed here

The use of ARMS PCR in detection and identification of xanthomonads associated with pistachio dieback in Australia

The original publication can be found at www.springerlink.comPistachio dieback occurs in the main pistachio growing areas of Australia. Xanthomonas strains belonging to the translucens group have been identified as the causal agent of the disease and two distinct groups, A and B, have been recognised within the pathogen population. In this study, specific primers for amplification of DNA of the pathogen were developed by sequencing the Internal Transcribed Spacer (ITS) region of rDNA from strains representing groups A and B, as well as from X. translucens isolated from wheat in Australia and one Xanthomonas translucens strain from orchard floor grasses. Primers were designed for amplification of DNA sequences specific to each group and a multiplex PCR test was developed that identified and differentiated strains of each group in a single PCR assay. To determine the specificity of the primers, PCR was carried out with DNA from 65 strains of the pistachio pathogen, 31 type and reference strains of Xanthomonas, and from 191 phytobacteria commonly found in and around pistachio orchards. In the multiplex PCR, a 331 bp fragment was amplified from all strains belonging to group A and a 120 bp fragment from all strains in group B. No PCR products were obtained from the other bacteria tested except for the type strain of X. translucens pv. cerealis, which has not been found in Australia. The assay was used to detect strains from both groups of the pathogen in pistachio plant material

Suppression of Rhizoctonia solani anastomosis group 8 in Australia and its biological nature

The nature of suppression in a field soil showing a decline in the Rhizoctonia barepatch disease of wheat (causal agent Rhizoctonia solani AG-8), in a minimum tillage system in southern Australia was investigated. The suppressive characteristics of the soil could be transferred to an autoclaved or pasteurized soil by adding 10% (w/w) of the unsterilised soil. This resulted in less disease following inoculation with R. solani AG-8. No transfer of suppression was observed when non-suppressive soil from an adjacent trial was added to the autoclaved or pasteurised soil. Gamma irradiation or pasteurisation at 60, 70 or 80 degrees C for 30 min eliminated both the ability of the soil to suppress disease and also differences in the soil microflora of suppressive soil and non-suppressive soil observed in untreated or 50 degrees C steam pasteurised soils. This is the first report of biologically-based suppression of this root rotting disease of wheat caused by R. solani AG-8

The post-hatch gut microbiota development in broiler chickens

V.A. Torok, G.E. Allison, K. Ophel-Keller and R.J. Hughe

Variation in rDNA ITS sequences in Glomus mosseae and Gigaspora margarita spores from a permanent pasture

To study the genetic variability in Glomus mosseae and Gigaspora margarita sequence similarities of ITS regions of rDNA were analysed from spores collected from a permanent pasture and from pot cultures. PCR amplification with the primers ITS1 and ITS4 was performed and products were cloned and sequenced. The sequences from single spores of G. mosseae and Gi. margarita confirmed that there is variation in the ITS region in a single spore. Phenetic analysis of sequences from both species supported the morphological identification which placed the species into two separate groups. Through the analysis of these sequences an estimate of genetic diversity was derived which clearly showed that the three field spores of G. mosseae were at least 2-5 times more genetically diverse than one single spore (field) and a pool of spores (pot culture) of Gigaspora margarita. This demonstrates that a high degree of variation exists in this natural population of G. mosseae.Z.I. Antoniolli, D.P. Schachtman, K. Ophel-Keller and S.E. Smit

Naturally occurring arbuscular mycorrhizal fungi can replace direct P uptake by wheat when roots cannot access added P fertiliser

© CSIRO 2008We investigated the roles of naturally occurring arbuscular mycorrhizal (AM) fungi in phosphorus (P) uptake by wheat (Triticum aestivum L.) in a calcareous, P-fixing soil. Plants grew in a main pot containing autoclaved soil (NM) or autoclaved soil mixed with non-autoclaved soil (to provide inoculum of naturally occurring AM fungi; AM). Granular (GP; monoammonium phosphate) or fluid (FP; ammonium polyphosphate) fertilisers were applied in small compartments (PCs) within a main pot, to which either roots plus hyphae (–Mesh) or hyphae only (+Mesh) had access. Controls received no additional P (NP). Inoculated plants were well colonised by AM fungi. AM growth depressions were observed in –Mesh treatments with NP and GP, but not with FP. Neither AM growth nor P responses were observed in +Mesh treatments. AM plants had much higher P uptake than NM plants, regardless of the P and mesh treatments. Total P uptake by NM plants increased with FP in –Mesh, but was unaffected by either form of P in the +Mesh treatments. Total P uptake by AM plants was similar between –Mesh and +Mesh treatments, regardless of applied P, showing that roots plus hyphae and hyphae alone have the same ability to obtain P. Thus, hyphae can take over the roles of roots in P uptake when roots are not able to access P sources

Genetic, phenotypic and pathogenic diversity among xanthomonads isolate from pistachio ('Pistacia vera') in Australia

Repetitive extragenic palindromic polymerase chain reaction (rep-PCR), sequencing of the 16S−23S rDNA internal transcribed spacer (ITS), biochemical and physiological tests, the Biolog microplate system, polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins, and pathogenicity tests were used to characterize variability among xanthomonads isolated from pistachio trees suffering from bacterial dieback in four regions of Australia. ITS sequencing and rep-PCR revealed two distinct genotypes among the strains. The ITS sequencing suggested that the pistachio strains were closely related to Xanthomonas translucens pathovars, in particular X. translucens pv. poae. Results of physiological and biochemical tests, as well as Biolog microplate analysis and protein profiling, confirmed the existence of two groups. Furthermore, pathogenicity and host-range studies indicated that the two groups were biologically different. There was an association between the two groups and the geographical origin of the strains