A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity

Long-lasting viable fungal spores are one of the important aspects in emergence, spread and disease development of pathogenic fungi. We developed a rapid and miniaturized system using Alamar Blue (resazurin dye; 7-hydroxy-3H-phenoxazin-3-one 10-oxide) for assessing fungal spore viability, using the ascomycete Leptosphaeria maculans (causing blackleg disease on canola) as a ‘model pathogen’. The assay is dependent on the metabolic activity of viable fungal spores to convert the dark blue of resazurin (maximum absorbance 605 nm) into the pink colour of resorufin (maximum absorbance 573 nm). The Alamar Blue assay uses an optimised micro-titre based format that was far superior for determining fungal spore viability in comparison with current conventional techniques including trypan blue staining, a TC10 cellometer cell counter, or by assessing germination of the spores under the microscope. This new assay was also more rapid and reproducible than current conventional tests to detect viable spores. Viable spores could be reliably detected within two hours. The successful application of the Alamar Blue assay to measure fungal spore viability in the current study has important benefits for biosecurity operations relating to faster and more reliable confirmation of viability of potential invasive exotic fungal pathogens and in minimising any consequent disease outbreaks. The effectiveness of the Alamar Blue assay was confirmed by successfully determining the relative retention times of viable L. maculans ascospores across a range of different potential spore-carrier materials, including steel, fabric, wood, paper, rubber and leather, over a time period of eight months. To further confirm the wide applicability of the Alamar Blue assay, it was successfully applied to detect viable spores of fungal pathogens of diverse taxonomic groups, including Kabatiella caulivora, Magnaporthe oryzae and Puccinia striiformis f.sp. tritici, and also of the yeast Saccharomyces cerevisiae.     

Harmonic radar: efficacy at detecting and recovering insects on agricultural host plants

BACKGROUND: In pest management research, harmonic radar systems have been largely used to study insect movement across open or vegetation‐poor areas because the microwave signal is attenuated by the high water content of vegetation. This study evaluated whether the efficacy of this technology is sufficient to track insects in vegetative landscapes. RESULTS: Field efficacy data were collected using portable harmonic microwave radar and electronic dipole tags mounted on adults of three economically important pests: Leptinotarsa decemlineata (Say), Diabrotica virginifera virginifera (LeComte) and Conotrachelus nenuphar Herbst. Detection and recovery of tagged Colorado potato beetles, plum curculios and western corn rootworms was high within and among potato plants, moderate within apple trees and high within, but not between, corn plants respectively. The efficacy of the radar depends on the ability of the operator to move around the host, scanning for a signal ‘sightline’ with the tagged insect among plant structures. CONCLUSION: The detection rate of tagged insects by harmonic radar systems is high enough to track the walking path of pests through low row crops such as potato, tall row crops such as corn or tall but well‐separated trees of orchard‐type crops by adapting the scanning procedure to the vegetative architecture. Copyright © 2010 Crown in the right of Canada. Published by JohnWiley & Sons, Ltd     

Pathogenic Botryosphaeriaceae associated with <Emphasis Type="Italic">Mangifera indica</Emphasis> in the Kimberley Region of Western Australia

Members of the Botryosphaeriaceae, in particular Lasiodiplodia theobromae, Neofusicoccum parvum, N. mangiferum and Botryosphaeria dothidea, commonly cause stem cankers, dieback and stem end rot of mangoes worldwide. In the current study, eight taxa of Botryosphaeriaceae were identified as canker-associated fungi, pathogens, potential pathogens or endophytes of mangoes in the Kimberley, Australia. These include Neoscytalidium novaehollandiae, Ne. dimidiatum, Pseudofusicoccum adansoniae, P. ardesiacum, P. kimberleyense, Lasiodiplodia sp. 1, L. iraniensis and L. pseudotheobromae. The pathogenicity of a selection of these species toward fruit and branches was tested. All were pathogenic to mango in comparison to the control, with Lasiodiplodia spp. being the most pathogenic. It appears that either geographic isolation or the unique growing conditions in the Kimberley may have provided an effective barrier to the acquisition or establishment of known botryosphaeriaceous pathogens. Wounds caused by mechanical pruning may provide an entry point for infection, whilst severe pruning may increase plant stress.     

Trapping Cryptophlebia illepida and C. ombrodelta (Lepidoptera: Tortricidae) in macadamia in Hawaii

Abstract

Two species of Cryptophlebia found in Hawaii's macadamia orchards are active in different parts of a tree canopy. C. illepida (Butler) males were mostly caught in traps hung 1·5 m to 9.1 m from the ground; whereas C. ombrodelta (Lower) males were caught from 9.1 m to 15.2 m. A plastic, cylindrical sticky trap caught equal numbers of C. illepida males as Pherocon 1C traps but more than delta traps. A synthetic pheromone blend of 93% (Z)‐8‐dodecenyl acetate, 4% (E)‐8‐dodecenyl acetate, 1% (Z)‐8‐dodecenol, and 2% (2)‐7‐dedecenyl acetate attracted more males than other blends. No significant differences were detected between the two Cryptophlebia species in their response to the four‐component pheromone blend when traps were hung 4.6 m from the ground.     

Sensitivity of European Plasmopara viticola populations to cymoxanil

Between 1995 and 1997, 278 grape downy mildew (Plasmopara viticola) populations originating from European vineyards were characterised for their sensitivity to cymoxanil in a leaf-disc assay. The sensitivity profile revealed a wide distribution, with minimum inhibitory concentrations (MIC) ranging from 10 to more than 800 mg litre⁻¹. EC₅₀ values ranged from 1 to more than 800 mg litre⁻¹ with an average of 125 mg litre⁻¹. The sensitivity distribution was stable between 1995 and 1997. Surprisingly, populations from Portugal appeared significantly more sensitive than those from France or Italy, which could not be linked to differential cymoxanil usage in these countries. P viticola populations collected outside Europe and never exposed to cymoxanil appeared significantly more sensitive than exposed European populations, with an average EC₅₀ value of 10 mg litre⁻¹. The level of sensitivity of European P viticola populations was relatively unaffected by the number of cymoxanil applications made during a season or by the number of years of cymoxanil use. No link was found between the level of sensitivity in the leaf-disc assay and the level of performance of the cymoxanil mixtures used in the fields where the populations originated. Specific field trials conducted in Italy and Portugal have shown that the performance of cymoxanil-based mixtures remained good even on populations of the grape downy mildew fungus characterised as less sensitive in the leaf-disc assay. While there are no baseline sensitivity data for pre-commercialisation P viticola populations, the results of our study suggest that a shift in sensitivity (12.5-fold) may have occurred in some areas since introduction of cymoxanil on grapes nearly 20 years ago. Because cymoxanil is never used alone, it is difficult to determine whether or not practical resistance is occurring in European vineyards. © 1999 Society of Chemical Industry     

Occurrence of an antifungal principle in the root extract of a Bayoud — resistant date palm cultivar

An hexane extract from roots of Black Boo Stammi, a cultivar of date palm, resistant toFusarium oxysporum f. sp.albedinis, contained substances which inhibited the spore germination and the growth of the germ tubes of the three isolates of the pathogen that were tested. Extract from roots of Jihel, a susceptible cultivar, lacked these substances.Samenvatting In een hexaanextract van wortels van de dadelpalm, cv. Black Boo Stammi die resistent is tegen de Bayoudziekte, bevonden zich stoffen die de sporekieming en de groei van kiembuizen onderdrukken. Dit gold voor alle drie isolaten vanFusarium oxysporum f. sp.albedinis, die werden getoetst. In extracten van de vatbare cultivar Jihel werden de kieming en de groei niet geremd.