Toward routine,DNA-based detection methods for marine pests

Marine pest incursions can cause significant ongoing damage to aquaculture, biodiversity, fisheries habitat, infrastructure and social amenity. They represent a significant and ongoing economic burden. Marine pests can be introduced by several vectors including aquaculture, aquarium trading, commercial shipping, fishing, floating debris, mining activities and recreational boating. Despite the inherent risks, there is currently relatively little routine surveillance of marine pest species conducted in the majority of countries worldwide. Accurate and rapid identification of marine pest species is central to early detection and management. Traditional techniques (e.g. physical sampling and sorting), have limitations, which has motivated some progress towards the development of molecular diagnostic tools. This review provides a brief account of the techniques traditionally used for detection and describes developments in molecular-based methods for the detection and surveillance of marine pest species. Recent advances provide a platform for the development of practical, specific, sensitive and rapid diagnosis and surveillance tools for marine pests for use in effective prevention and control strategies.     

Prediction of take-all disease risk in field soils using a rapid and quantitative DNA soil assay

A rapid, routine DNA-based assay to quantify Gaeumannomyces graminis var. tritici (Ggt), the causal agent of take-all disease of cereals, has been developed and used for the prediction of take-all in a wide range of field soils. Based on the correlation of the DNA-based assay and a soil bioassay, the risk of disease development can be estimated. Ggt DNA levels of <30 pg, 30–50 pg and >50 pg in 0.1 g soil organic matter correspond to low, moderate and high levels of the disease, respectively. Limitations in the prediction of take-all, including sampling requirements to obtain representative soil samples from fields and increasing the sensitivity and the accuracy of the DNA assay, are described. The main advantage in using the DNA-based assay, in estimating the amount of Ggt inoculum in soil, is that the levels of Ggt in soil samples can be assessed rapidly and accurately. Farmers can now have soil samples assessed before sowing. The DNA result can be used to predict the potential yield loss and determine the most appropriate management options using decision support software that is currently available. This DNA technology is currently being used commercially to detect and predict take-all. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of cultural growth and in planta quantification of Didymella pinodes, Phoma koolunga and Phoma medicaginis var. pinodella, causal agents of ascochyta blight on field pea (Pisum sativum)

The causal agents of ascochyta blight on field pea in South Australia, Didymella pinodes, Phoma medicaginis var. pinodella and Phoma koolunga, are isolated from a single plant within a crop, suggesting competition for space and nutrients. Interactions among these pathogens were investigated. Diameters of colonies of D. pinodes and of P. medicaginis var. pinodella were significantly reduced on PDA amended with filtrate from broth cultures of P. koolunga as were diameters of colonies of D. pinodes on PDA amended with filtrate from P. medicaginis var. pinodella or D. pinodes. This effect was negated when cultures were transferred to unamended PDA, indicating filtrates were fungistatic instead of fungicidal. The diameter of P. koolunga colonies was not influenced by filtrate from any of the three species. When pathogens were co-inoculated in pairs onto leaves on field pea plants, the quantity of DNA of D. pinodes and of P. medicaginis var. pinodella was significantly reduced if co-inoculated with P. koolunga. The quantity of DNA of P. koolunga was not influenced by co-inoculation. When co-inoculated onto excised leaf disks on sterile water the mean lesion diameter due to D. pinodes and to P. medicaginis var. pinodella was significantly reduced if co-inoculated with P. koolunga isolate DAR78535. Lesions caused by D. pinodes were significantly reduced when inoculum was self-paired. Conversely the diameter of lesions caused by P. koolunga DAR78535 increased when self-paired or when co-inoculated with P. medicaginis var. pinodella. Unlike leaf disks on sterile water, co-inoculation had no influence on lesion size or quantity of pathogen DNA in leaf disks on water agar. Antagonism, including self-antagonism, was detected among these species, leading to reduction in lesion size and quantity of pathogen DNA. The slower growing species, P. koolunga, was not self-antagonistic, and in a few instances the effect of co-inoculation was additive or synergistic.     

Direct measurement of roots in soil for single and mixed species using a quantitative DNA-based method

Molecular techniques present a new opportunity to study roots and their interactions in soil. Extraction and quantification of species-specific DNA directly from soil allows direct identification of roots in mixed swards reducing the need for labour-intensive methods to recover and identify individual roots. DNA was extracted directly from up to 0.5 kg of soil and the presence of individual species quantified using species-specific probes with quantitative real-time PCR. A range of plant and soil factors influenced the DNA content measured in roots and it was necessary to account for these influences when converting DNA amount to root mass. The utility of the method for quantitative root studies was demonstrated in an experiment to investigate the effect of lime on root growth of acid-soil resistant and sensitive perennial grasses grown together in an aluminium-toxic soil. The root mass of an acid-soil resistant species was unaffected by lime application, whereas that of an acid-soil sensitive species was restricted by soil acidity. Molecular techniques present a promising tool for quantification of root mass directly in soil and have applications for field studies involving mixed species of plants.     

Correlative 3D‐imaging of Pipistrellus penis micromorphology: Validating quantitative microCT images with undecalcified serial ground section histomorphology

Detailed knowledge of histomorphology is a prerequisite for the understanding of function, variation, and development. In bats, as in other mammals, penis and baculum morphology are important in species discrimination and phylogenetic studies. In this study, nondestructive 3D‐microtomographic (microCT, µCT) images of bacula and iodine‐stained penes of Pipistrellus pipistrellus were correlated with light microscopic images from undecalcified surface‐stained ground sections of three of these penes of P. pipistrellus (1 juvenile). The results were then compared with µCT‐images of bacula of P. pygmaeus, P. hanaki, and P. nathusii. The Y‐shaped baculum in all studied Pipistrellus species has a proximal base with two club‐shaped branches, a long slender shaft, and a forked distal tip. The branches contain a medullary cavity of variable size, which tapers into a central canal of variable length in the proximal baculum shaft. Both are surrounded by a lamellar and a woven bone layer and contain fatty marrow and blood vessels. The distal shaft consists of woven bone only, without a vascular canal. The proximal ends of the branches are connected with the tunica albuginea of the corpora cavernosa via entheses. In the penis shaft, the corpus spongiosum‐surrounded urethra lies in a ventral grove of the corpora cavernosa, and continues in the glans under the baculum. The glans penis predominantly comprises an enlarged corpus spongiosum, which surrounds urethra and baculum. In the 12 studied juvenile and subadult P. pipistrellus specimens the proximal branches of the baculum were shorter and without marrow cavity, while shaft and distal tip appeared already fully developed. The present combination with light microscopic images from one species enabled a more reliable interpretation of histomorphological structures in the µCT‐images from all four Pipistrellus species. J. Morphol. 276:695–706, 2015. © 2015 Wiley Periodicals, Inc.