Cyanide poisoning in cattle from Dysphania glomulifera (red crumbweed): Using the internet for rapid plant identification and diagnostic advice: Case Report

A 300-strong Angus-Brahman cattle herd near Springsure, central Queensland, was being fed Acacia shirleyi (lancewood) browse during drought and crossed a 5-hectare, previously burnt area with an almost pure growth of Dysphania glomulifera subspecies glomulifera (red crumbweed) on their way to drinking water. Forty cows died of cyanide poisoning over 2 days before further access to the plant was prevented. A digital image of a plant specimen made on a flat-bed scanner and transmitted by email was used to identify D glomulifera. Specific advice on the plant's poisonous properties and management of the case was then provided by email within 2 hours of an initial telephone call by the field veterinarian to the laboratory some 600 km away. The conventional method using physical transport of a pressed dried plant specimen to confirm the identification took 5 days. D glomulifera was identified in the rumen of one of two cows necropsied. The cyanogenic potential of D glomulifera measured 4 days after collection from the site of cattle deaths was 18,600 mg HCN/kg in dry matter. The lethal dose of D glomulifera for a 420 kg cow was estimated as 150 to 190 g wet weight. The plant also contained 4.8% KNO3 equivalent in dry matter, but nitrate-nitrite poisoning was not involved in the deaths.

An identification tool for the Australian weedy Sporobolus species based on random amplified polymorphic DNA (RAPD) profiles

Based on morphological features alone, there is considerable difficulty in identifying the 5 most economically damaging weed species of Sporobolus [viz. S. pyramidalis P. Beauv., S. natalensis (Steud.) Dur and Schinz, S. fertilis (Steud.) Clayton, S. africanus (Poir.) Robyns and Tourney, and S. jacquemontii Kunth.] found in Australia. A polymerase chain reaction (PCR)-based random amplified polymorphic DNA (RAPD) technique was used to create a series of genetic markers that could positively identify the 5 major weeds from the other less damaging weedy and native Sporobolus species. In the initial RAPD profiling experiment, using arbitrarily selected primers and involving 12 species of Sporobolus, 12 genetic markers were found that, when used in combination, could consistently identify the 5 weedy species from all others. Of these 12 markers, the most diagnostic were UBC51490 for S. pyramidalis and S. natalensis; UBC43310.2000.2100 for S. fertilis and S. africanus; and ORA20850 and UBC43470 for S. jacquemontii. Species-specific markers could be found only for S. jacquemontii. In an effort to understand why there was difficulty in obtaining species-specific markers for some of the weedy species, a RAPD data matrix was created using 40 RAPD products. These 40 products amplified by 6 random primers from 45 individuals belonging to 12 species, were then subjected to numerical taxonomy and multivariate system (NTSYS pc version 1.70) analysis. The RAPD similarity matrix generated from the analysis indicated that S. pyramidalis was genetically more similar to S. natalensis than to other species of the 'S. indicus complex'. Similarly, S. jacquemontii was more similar to S. pyramidalis, and S. fertilis was more similar to S. africanus than to other species of the complex. Sporobolus pyramidalis, S. jacquemontii, S. africanus, and S. creber exhibited a low within-species genetic diversity, whereas high genetic diversity was observed within S. natalensis, S. fertilis, S. sessilis, S. elongates, and S. laxus. Cluster analysis placed all of the introduced species (major and minor weedy species) into one major cluster, with S. pyramidalis and S. natalensis in one distinct subcluster and S. fertilis and S. africanus in another. The native species formed separate clusters in the phenograms. The close genetic similarity of S. pyramidalis to S. natalensis, and S. fertilis to S. africanus may explain the difficulty in obtaining RAPD species-specific markers. The importance of these results will be within the Australian dairy and beef industries and will aid in the development of integrated management strategy for these weeds.

Identification of quantitative trait loci for resistance to two species of root-lesion nematode (Pratylenchus thornei and P. neglectus) in wheat

Pratylenchus thornei and P. neglectus are two species of root-lesion nematode that cause substantial yield losses in wheat. No commercially available wheat variety has resistance to both species. A doubled-haploid population developed from a cross between the synthetic hexaploid wheat line CPI133872 and the bread wheat Janz was used to locate and tag quantitative trait loci (QTLs) associated with resistance to both P. thornei and P. neglectus. Wheat plants were inoculated with both species of nematode in independent replicated glasshouse trials repeated over 2 years. Known locations of wheat microsatellite markers were used to construct a framework map. After an initial single-marker analysis to detect marker-trait linkages, chromosome regions associated with putative QTLs were targetted with microsatellite markers to increase map density in the chromosome regions of interest. In total, 148 wheat microsatellite markers and 21 amplified fragment length polymorphism markers were mapped. The codominant microsatellite marker Xbarc183 on the distal end of chromosome 6DS was allelic for resistance to both P. thornei and P. neglectus. The QTL were designated QRlnt.lrc-6D.1 and QRlnn.lrc-6D.1, for the 2 traits, respectively. The allele inherited from CPI133872 explained 22.0-24.2% of the phenotypic variation for P. thornei resistance, and the allele inherited from Janz accounted for 11.3-14.0% of the phenotypic variation for P. neglectus resistance. Composite interval mapping identified markers that flank a second major QTL on chromosome 6DL (QRlnt.lrc-6D.2) that explained 8.3-13.4% of the phenotypic variation for P. thornei resistance. An additional major QTL associated with P. neglectus resistance was detected on chromosome 4DS (QRlnn.lrc-4D.1) and explained a further 10.3-15.4% of the phenotypic variation. The identification and tagging of nematode resistance genes with molecular markers will allow appropriate allele combinations to be selected, which will aid the successful breeding of wheat with dual nematode resistance.

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides

Resistance against synthetic pyrethroid (SP) products for the control of cattle ticks in Australia was detected in the field in 1984, within a very short time of commercial introduction. We have identified a mutation in the domain II S4-5 linker of the para-sodium channel that is associated with resistance to SPs in the cattle tick Rhipicephalus (Boophilus) microplus from Australia. The cytosine to adenine mutation at position 190 in the R. microplus sequence AF134216, results in an amino acid substitution from leucine in the susceptible strain to isoleucine in the resistant strain. A similar mutation has been shown to confer SP resistance in the whitefly, Bemisia tabaci, but has not been described previously in ticks. A diagnostic quantitative PCR assay has been developed using allele-specific Taqman® minor groove-binding (MGB) probes. Using the assay to screen field and laboratory populations of ticks showed that homozygote allelic frequencies correlated highly with the survival percentage at the discriminating concentration of cypermethrin.

A one-tube fluorescent assay for the quarantine detection and identification of Tilletia indica and other grass bunts in wheat

A molecular assay with enhanced specificity and sensitivity has been developed to assist in the surveillance of Karnal bunt, a quarantineable disease with a significant impact on international trade. The protocol involves the release of DNA from spores, PCR amplification to enrich Tilletia-specific templates from released DNA and a five-plex, real-time PCR assay to detect, identify and distinguish T. indica and other Tilletia species (T. walkeri, T. ehrhartae, T. horrida and a group comprising T. caries, T. laevis, T. contraversa, T. bromi and T. fusca) in wheat grains. This fluorescent molecular tool has a detection sensitivity of one spore and thus bypasses the germination step, which in the current protocol is required for confirmation when only a few spores have been found in grain samples. The assay contains five dual-labelled, species-specific probes and associated species-specific primer pairs in a PCR mix in one tube. The different amplification products are detected simultaneously by five different fluorescence spectra. This specific and sensitive assay with reduced labour and reagent requirements makes it an effective and economically sustainable tool to be used in a Karnal bunt surveillance program. This protocol will also be valuable for the identification of some contaminant Tilletia sp. in wheat grains.

Mitochondrial DNA supports the identification of two endangered river sharks (Glyphis glyphis and Glyphis garricki) across northern Australia

The river sharks (genus Glyphis) are a small group of poorly known sharks occurring in tropical rivers and estuarine waters across northern Australia, south-east Asia and the subcontinent. The taxonomy of the genus has long been unclear due to very few individuals having been caught and examined, resulting in a paucity of data regarding their distribution, biology and ecology. Only recently has attention focussed on the two Australian species, G. glyphis and G. garricki. This study is a result of a rare opportunity to collate the few samples that have been collected from these species and the bull shark Carcharhinus leucas, which shares an overlapping range. These samples were analysed using the DNA barcoding approach (cox1 mitochondrial gene), compared with six other species of carcharhinids and evaluated in light of the current taxonomic classification. Nine species-specific nucleotide differences were found between G. glyphis and G. garricki and no intra-specific variation provides strong support for the separation into distinct species. Significant differences were also observed at the inter-generic level, with Glyphis forming a distinct clade from Carcharhinus. This study provides the basis for future molecular studies required to better address conservation issues confronting G. glyphis and G. garricki in Australia.

Maximising the contribution of native-range studies towards the identification and prioritisation of weed biocontrol agents

Effective study in the native range to identify potential agents underpins all efforts in classical biological control of weeds. Good agents that demonstrate both a high degree of host specificity and the potential to be damaging are a very limited resource and must therefore be carefully studied and considered. The overseas component is often operationally difficult and expensive but can contribute considerably more than a list of herbivores attacking a particular target. While the principles underlying this foreign component have been understood for some time, recently developed technologies and methods can make very significant contributions to foreign studies. Molecular and genetic characterisations of both target weed and agent organism can be increasingly employed to more accurately define the identity and phylogeny of them. Climate matching and modelling software is now available and can be utilised to better select agents for particular regions of concern. Relational databases can store collection information for analysis and future enquiry while quantification of sampling effort, employment of statistical survey methods and analysis by techniques such as rarefaction curves contribute to efficient and effective searching. Obtaining good and timely identifications for discovered agent organisms is perhaps the most serious issue confronting the modern explorer. The diminishing numbers of specialist taxonomists employed at the major museums while international and national protocols demand higher standards of identity exacerbates the issue. Genetic barcoding may provide a very useful tool to overcome this problem. Native-range work also offers under-exploited opportunities for contributing towards predicting safety, abundance and efficacy of potential agents in their target environment.

Functional dynamics of the nitrogen balance of sorghum. II. Grain filling period.

Maintenance of green leaf area during grain filling can increase grain yield of sorghum grown under terminal water limitation. This 'stay-green' trait has been related to the nitrogen (N) supply-demand balance during grain filling. This study quantifies the N demand of grain and N translocation rates from leaves and stem and explores effects of genotype and N stress on onset and rate of leaf senescence during the grain filling period. Three hybrids differing in potential height were grown at three levels of N supply under well-watered conditions. Vertical profiles of biomass, leaf area, and N% of leaves, stem and grain were measured at regular intervals. Weekly SPAD chlorophyll readings on main shoot leaves were correlated with observed specific leaf nitrogen (SLN) to derive seasonal patterns of leaf N content. For all hybrids, individual grain N demand was sink determined and was initially met through N translocation from the stem and rachis. Only if this was insufficient did leaf N translocation occur. Maximum N translocation rates from leaves and stem were dependent on their N status. However, the supply of N at canopy scale was also related to the amount of leaf area senescing at any one time. This supply-demand framework for N dynamics explained effects of N stress and genotype on the onset and rate of leaf senescence.

Functional dynamics of the nitrogen balance of sorghum: I. N demand of vegetative plant parts.

Stay-green, an important trait for grain yield of sorghum grown under water limitation, has been associated with a high leaf nitrogen content at the start of grain filling. This study quantifies the N demand of leaves and stems and explores effects of N stress on the N balance of vegetative plant parts of three sorghum hybrids differing in potential crop height. The hybrids were grown under well-watered conditions at three levels of N supply. Vertical profiles of biomass and N% of leaves and stems, together with leaf size and number, and specific leaf nitrogen (SLN), were measured at regular intervals. The hybrids had similar minimum but different critical and maximum SLN, associated with differences in leaf size and N partitioning, the latter associated with differences in plant height. N demand of expanding new leaves was represented by critical SLN, and structural stem N demand by minimum stem N%. The fraction of N partitioned to leaf blades increased under N stress. A framework for N dynamics of leaves and stems is developed that captures effects of N stress and genotype on N partitioning and on critical and maximum SLN.

Identification of genetic regions associated with black point in barley.

Black point (BP) can cause severe losses to the barley industry through downgrading and discounting of malting barley. The genetic improvement in BP resistance of barley is complex, requiring reliable screening tools, an understanding of genotype by environment interactions and an understanding of the biochemical mechanisms of melanisation involved in BP development. Thus the application of molecular markers for resistance to BP may be a useful tool for plant breeders. We have investigated the genetic regions associated with BP resistance in the barley F2 population, Valier/Binalong. Quantitative trait loci (QTLs) contributed by the resistant parent Valier, were detected on chromosomes 2HS, 2HC, 3HL, 4HL and a QTL contributed by the susceptible parent, Binalong was detected on 5HL. Three of the four QTLs were detected in two distinctly different environments. The differences observed in BP resistance between these two environments and the implications for accelerated screening are discussed. Identified SSR markers in these regions may be useful for selecting black point resistance in related breeding materials.

Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.).

The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to beta-carotene (yellow) is catalysed by lycopene beta-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene beta-cyclases (lcy-beta1 and lcy-beta2) from red (Tainung) and yellow (Hybrid 1 B) papaya cultivars. A mutation in the lcy-beta2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-beta1 and lcy-beta2 genes is similar and low in leaves, but lcy-beta2 expression increases markedly in ripe fruit. Isolation of the lcy-beta2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties.

Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractive hybridization.

Haemophilus parasuis is the causative agent of Glässer's disease. Up to now 15 serovars of H. parasuis have been identified, with significant differences existing in virulence between serovars. In this study, suppression subtractive hybridization (SSH) was used to identify the genetic difference between Nagasaki (H. parasuis serovar 5 reference strain, highly virulent) and SW114 (H. parasuis serovar 3 reference strain, non-virulent). A total of 191 clones were obtained from the SSH library. Using dot hybridization and PCR, 15 clones were identified containing fragments that were present in the Nagasaki genome while absent in the SW114 genome. Among these 15 fragments, three fragments (ssh1, ssh13, ssh15) encode cell surface-associated components; three fragments (ssh2, ssh5, ssh9) are associated with metabolism and stress response; one fragment (ssh8) is involved in assembly of fimbria and one fragment (ssh6) is a phage phi-105 ORF25-like protein. The remaining seven fragments are hypothetical proteins or unknown. Based on PCR analysis of the 15 serovar reference strains, eight fragments (ssh1, ssh2, ssh3, ssh6, ssh8, ssh10, ssh11 and ssh12) were found in three to five of most virulent serovars (1, 5, 10, 12, 13 and 14), zero to two in three moderately virulent serovars (2, 4 and 15), but absent in the low virulent serovar (8) and non-virulent serovars (3, 6, 7, 9 and 11). In vivo transcription fragments ssh1, ssh2, ssh8 and ssh12 were identified in total RNA samples extracted from experimental infected pig lung by RT-PCR. This study has provided some evidence of genetic differences between H. parasuis strains of different virulence.

DNA-based identification of Quambalaria pitereka causing severe leaf blight of Corymbia citriodora in China

Quambalaria spp. include serious plant pathogens, causing leaf and shoot blight of Corymbia and Eucalyptus spp. In this study, a disease resembling Quambalaria leaf blight was observed on young Corymbia citriodora trees in a plantation in the Guangdong Province of China. Comparisons of rDNA sequence data showed that the causal agent of the disease is Q. pitereka. This study provides the first report of Quambalaria leaf blight from China, and it is also the first time that this pathogen has been found on trees outside the native range of Eucalypts.

Identification of insects, spiders and mites in vegetable crops. Workshop Manual, 2nd edition

Accurate and confident identification of the insects, spiders and mites in vegetable crops is the first step towards successful management of pests and natural enemies. It is an essential prerequisite for crop monitoring, which is the backbone of an effective pest management program. This workshop manual and trainer's handbook were compiled as part of an insect, spider and mite identification program for Australian vegetable growers. The workshop training is designed to help growers to: • know how to collect and preserve insects for identification • be able to classify most common insects (particularly those of horticultural significance) into broad groups • appreciate the importance of these groups in pest, predator and parasite identification and management • collect and classify some insect pests, predators and parasites of horticultural importance.

Identification of insects, spiders and mites in vegetable crops: Trainer's handbook

The trainers manual provides workshop plans and sample slides for trainers wishing to conduct the 'Identification of insects, spiders and mites in vegetable crops' workshop.