Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium spinosum infecting cucumber in Oman

A total of 24 isolates of Pythium spinosum from cucumber obtained from five regions in Oman were characterized for genetic diversity using amplified fragment length polymorphism (AFLP) fingerprinting and three isolates from the Netherlands, South Africa and Japan were included for comparison. Isolates from Oman were also characterized for aggressiveness on cucumber seedlings and sensitivity to metalaxyl. Identity of all isolates was confirmed using sequences of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), which showed more than 99% nucleotide similarity among all isolates. Using six primer-pair combinations, AFLP fingerprinting resolved 295 AFLP markers of which 193 were polymorphic among isolates from other countries and only six were polymorphic among isolates of P. spinosum from Oman. Seven different AFLP phenotypes of P. spinosum were recovered in Oman; two of them were found to contain over 79% of isolates and one was recovered from all regions in Oman. Phenotypes from Oman showed very high (?99%) levels of genetic similarity to each other compared to moderate (mean =53%) levels of genetic similarity with phenotypes from other countries. In addition, all isolates from Oman were found to be highly sensitive to metalaxyl and all were aggressive on cucumber seedlings at 25°C. The high genetic similarity among phenotypes of P. spinosum in Oman as well as recovering two major clones across regions may suggest that P. spinosum has been recently introduced in Oman via a common source.

Isolation and expression analysis of multiple isoforms of putative farnesoic acid O-methyltransferase in several crustacean species

Farnesoic acid O-methyltransferase (FaMeT) is the enzyme responsible for the conversion of farnesoic acid (FA) to methyl farnesoate (MF) in the final step of MF synthesis. Multiple isoforms of putative FaMeT were isolated from six crustacean species belonging to the families Portunidae, Penaeidae, Scyllaridae and Parastacidae. The portunid crabs Portunus pelagicus and Scylla serrata code for three forms: short, intermediate and long. Two isoforms (short and long) were isolated from the penaeid prawns Penaeus monodon and Fenneropenaeus merguiensis. Two isoforms were also identified in the scyllarid Thenus orientalis and parastacid Cherax quadricarinatus. Putative FaMeT sequences were also amplified from the genomic DNA of P. pelagicus and compared to the putative FaMeT transcripts expressed. Each putative FaMeT cDNA isoform was represented in the genomic DNA, indicative of a multi-gene family. Various tissues from P. pelagicus were individually screened for putative FaMeT expression using PCR and fragment analysis. Each tissue type expressed all three isoforms of putative FaMeT irrespective of sex or moult stage. Protein domain analysis revealed the presence of a deduced casein kinase II phosphorylation site present only in the long isoform of putative FaMeT.

Pasteurella multocida Expresses Two Lipopolysaccharide Glycoforms Simultaneously, but Only a Single Form Is Required for Virulence: Identification of Two Acceptor-Specific Heptosyl I Transferases

Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-D-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo1 residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures.

Fishery biology and management of Protonibea diacanthus (Sciaenidae) aggregations in far Northern Cape York Peninsula waters

The sciaenid Protonibea diacanthus is a large, long-lived predatory fish of inshore northern Australian waters, which forms annual aggregations that are fished extensively by traditional (subsistence) and recreational fishers. There are now widespread concerns that the resource is being overexploited. Indigenous fishers of the Cape York Northern Peninsula Area (NPA) relate that large adult fish (up to 1500 mm total length (TL)) made up the bulk of the catch from the sciaenid aggregations until about 1994. In contrast, sexually mature P. diacanthus comprised only a small component (12 fish out of 270=4.4%) examined in a 1999–2000 sampling programme that was biased towards the largest individuals available. At 790 mm TL, the minimum size at first maturity for female P. diacanthus in this study is much smaller than the 920 mm TL reported previously in Queensland waters. Developing ovaries were observed in specimens sampled from sciaenid aggregations which formed in NPA waters between May and September 2000. However, no fish with ripe or spent gonads were found in the study, so the current timing and location of the spawning season for P. diacanthus in the region remain unknown. Food items observed in the analysis of the diet of P. diacanthus from the NPA included a variety of teleosts and invertebrates. The range of animal taxa represented in the prey items support the description of an ‘opportunistic predator’ attributed to the species. In our sampling, the stomach contents of fish caught during the time of the aggregation events did not differ from those observed at other times of the year. A total of 114 P. diacanthus were tagged and released at aggregation sites during the study period, and 3 fish (2.6%) were subsequently recaptured. The low rate of tag returns from the wild stock tagging programme, both in this study (2.6%) and from recreational fisher tag/release programmes for the sciaenid elsewhere in Queensland (6.5%), were not explained by tag loss nor mortality, given the high retention rate of tags and the zero mortality seen in tank trials. In response to the biological findings from this study, indigenous community councils of the NPA imposed a 2-year fishing moratorium for P. diacanthus. Surveys at aggregation sites in 2002 and 2003 established that much larger fish (mean size 103.5 cm TL) were again present on the grounds, albeit in very low numbers. These recent preliminary results highlight the critical need for continued monitoring and management of the P. diacanthus fishery in the NPA, if prospects for resource recovery are to be realised. The NPA initiative has provided a rare opportunity to negotiate a co-management strategy, based on scientific data and traditional knowledge, for the recovery of a cultural and economically significant fished resource.

Soil sorption-desorption of phosphorus from piggery effluent compared with inorganic sources

The leaching of phosphorus (P) within soils can be a limiting consideration for the sustainable operation of intensive livestock enterprises. Sorption curves are widely used to assist estimation of P retention, though the effect of effluent constituents on their accuracy is not well understood. We conducted a series of P-sorption-desorption batch experiments with an Oxic Haplustalf (soil 1), Haplusterts (soils 2 and 3), and a Natrustalf (soil 4). Phosphorus sources included effluent, orthophosphate-P in a matrix replicating the effluent's salt constituents (the reference solution), and an orthophosphate-P solution. Treated soils were incubated for up to 193 days before sequential desorption extraction. Effluent constituents, probably the organic or particulate components, temporarily increased the vulnerability of sorbed-P to desorption. The increase in vulnerability was removed by 2-113 days of incubation (25 degrees C). Despite vigorous extraction for 20 consecutive days, some P sorbed as part of the treatments of soils 1 and 2 was not desorbed. The increased vulnerability due to effluent constituents lasted a maximum of about one cropping season and, for all other treatments, adsorption curves overestimated vulnerability to desorption. Therefore, adsorption curves provide a conservative estimate of vulnerability to desorption where effluent is used in continued crop production in these soils.

Phosphorus nutrition and tolerance of cotton to water stress: II. Water relations, free and bound water and leaf expansion rate

In previous experiments, increased leaf-Phosphorus (P) content with increasing P supply enhanced the individual leaf expansion and water content of fresh cotton leaves in a severely drying soil. In this paper, we report on the bulk water content of leaves and its components, free and bound water, along with other measures of plant water status, in expanding cotton leaves of various ages in a drying soil with different P concentrations. The bound water in living tissue is more likely to play a major role in tolerance to abiotic stresses by maintaining the structural integrity and/or cell wall extensibility of the leaves, whilst an increased amount of free water might be able to enhance solute accumulation, leading to better osmotic adjustment and tolerance to water stress, and maintenance of the volumes of sub-cellular compartments for expansive leaf growth. There were strong correlations between leaf-P%, leaf water (total, free and bound water) and leaf expansion rate (LER) under water stress conditions in a severely drying soil. Increased soil-P enhanced the uptake of P from a drying soil, leading to increased supply of osmotically active inorganic solutes to the cells in growing leaves. This appears to have led to the accumulation of free water and more bound water, ultimately leading to increased leaf expansion rates as compared to plants in low P soil under similar water stress conditions. The greater amount of bound and free water in the high-P plants was not necessarily associated with changes in cell turgor, and appears to have maintained the cell-wall properties and extensibility under water stressed conditions in soils that are nutritionally P-deficient.

Natural enemies of Paropsis atomaria Olivier (Coleoptera: Chrysomelidae) in south-eastern Queensland eucalypt plantations

A field survey for natural enemies of Paropsis atomaria was conducted at two south-eastern Queensland Eucalyptus cloeziana plantation sites during 2004-2005. Primary egg and larval parasitoids and associated hyperparasitoids were identified to genus or species, and parasitism rates were determined throughout the season. Predators were identified to family level but their impact was not quantified. P. atomaria adults were also examined as potential hosts for parasitic mites and nematodes. An undescribed species of Neopolycystus (Pteromalidae) was the major primary egg parasitoid species reared from egg batches, parasitising half of all egg batches collected. Three hyperparasitoid species (Baeoanusia albifunicle (Encyrtidae), Neblatticida sp. (Encyrtidae) and Aphaneromella sp. (Platygasteridae) were present, representing around one-quarter to one-third of all emergent wasps; this is the first host association record for Neopolycystus-B. albifunicle. In contrast to populations of P. atomaria from the Australian Capital Territory, primary larval parasitism was very low, around 1%, and attributable only to the tachinid flies Anagonia sp. and Paropsivora sp. However, the presence of the sit-and-wait larval hyperparasitoid, Perilampus sp. (Perilampidae) was high, emerging from around 17% of tachinid pupae, with planidia infesting a further 40% of unparasitised hosts. Three species of podapolipid mites parasitised sexually mature P. atomaria adults, while no nematodes were found in this study. Spiders were the most common predators and their abundance was positively correlated with P. atomaria adult and egg numbers. Although natural enemy species composition was identical between our two study sites, significant differences in abundance and frequency were found between sites.

Thermal requirements, field mortality and population phenology modelling of Paropsis atomaria Olivier, an emergent pest in subtropical hardwood plantations

Paropsis atomaria is a recently emerged pest of eucalypt plantations in subtropical Australia. Its broad host range of at least 20 eucalypt species and wide geographical distribution provides it the potential to become a serious forestry pest both within Australia and, if accidentally introduced, overseas. Although populations of P. atomaria are genetically similar throughout its range, population dynamics differ between regions. Here, we determine temperature-dependent developmental requirements using beetles sourced from temperate and subtropical zones by calculating lower temperature thresholds, temperature-induced mortality, and day-degree requirements. We combine these data with field mortality estimates of immature life stages to produce a cohort-based model, ParopSys, using DYMEX™ that accurately predicts the timing, duration, and relative abundance of life stages in the field and number of generations in a spring–autumn (September–May) field season. Voltinism was identified as a seasonally plastic trait dependent upon environmental conditions, with two generations observed and predicted in the Australian Capital Territory, and up to four in Queensland. Lower temperature thresholds for development ranged between 4 and 9 °C, and overall development rates did not differ according to beetle origin. Total immature development time (egg–adult) was approximately 769.2 ± S.E. 127.8 DD above a lower temperature threshold of 6.4 ± S.E. 2.6 °C. ParopSys provides a basic tool enabling forest managers to use the number of generations and seasonal fluctuations in abundance of damaging life stages to estimate the pest risk of P. atomaria prior to plantation establishment, and predict the occurrence and duration of damaging life stages in the field. Additionally, by using local climatic data the pest potential of P. atomaria can be estimated to predict the risk of it establishing if accidentally introduced overseas. Improvements to ParopSys’ capability and complexity can be made as more biological data become available.

Decoration of Pasteurella multocida lipopolysaccharide with phosphocholine is important for virulence

Phosphocholine (PCho) is an important substituent of surface structures expressed by a number of bacterial pathogens. Its role in virulence has been investigated in several species, in which it has been shown to play a role in bacterial adhesion to mucosal surfaces, in resistance to antimicrobial peptides, or in sensitivity to complement-mediated killing. The lipopolysaccharide (LPS) structure of Pasteurella multocida strain Pm70, whose genome sequence is known, has recently been determined and does not contain PCho. However, LPS structures from the closely related, virulent P. multocida strains VP161 and X-73 were shown to contain PCho on their terminal galactose sugar residues. To determine if PCho was involved in the virulence of P. multocida, we used subtractive hybridization of the VP161 genome against the Pm70 genome to identify a four-gene locus (designated pcgDABC) which we show is required for the addition of the PCho residues to LPS. The proteins predicted to be encoded by pcgABC showed identity to proteins involved in choline uptake, phosphorylation, and nucleotide sugar activation of PCho. We constructed a P. multocida VP161 pcgC mutant and demonstrated that this strain produces LPS that lacks PCho on the terminal galactose residues. This pcgC mutant displayed reduced in vivo growth in a chicken infection model and was more sensitive to the chicken antimicrobial peptide fowlicidin-1 than the wild-type P. multocida strain

The distribution of Fasciola hepatica in Queensland, Australia, and the potential impact of introduced snail intermediate hosts

A survey was conducted to establish the distribution of the liver fluke, Fasciola hepatica, in the state of Queensland, Australia, and to evaluate the impact of the introduced snail intermediate hosts, Pseudosuccinia columella and Austropeplea viridis. Serum samples from a total of 5103 homebred cattle in 142 beef herds distributed throughout the state and 523 pooled milk samples from dairy herds from the state's major dairying regions were tested for antibodies to F. hepatica by ELISA. Snails were collected on infected properties around the limits of the F. hepatica distribution. F. hepatica infection was detected in 44 dairy herds and two beef herds. The distribution of infected herds indicates that F. hepatica is established only in southeast Queensland. The distribution there was patchy but the parasite was more widespread than suggested by an earlier survey. The predominant intermediate host species found along the northern limit of the distribution was P. columella. We conclude that the introduction of P. columella and A. viridis has not yet had a major impact on the distribution of F. hepatica in Queensland. However, the presence of P. columella, which is much more adaptable to tropical habitats than the native intermediate host, Austropeplea tomentosa, at the northern limit of the F. hepatica distribution suggests that there is potential for the parasite to expand its range.

Molecular characterisation, pathogenesis and fungicide sensitivity of Pythium spp. from table beet (Beta vulgaris var. vulgaris) grown in the Lockyer Valley, Queensland

Table beet production in the Lockyer Valley of south-eastern Queensland is known to be adversely affected by soilborne root disease from infection by Pythium spp. However, little is known regarding the species or genotypes that are the causal agents of both pre- and post-emergence damping off. Based on RFLP analysis with HhaI, HinfI and MboI of the PCR amplified ITS region DNA from soil and diseased plant samples, the majority of 130 Pythium isolates could be grouped into three genotypes, designated LVP A, LVP B and LVP C. These groups comprised 43, 41 and 7% of all isolates, respectively. Deoxyribonucleic acid sequence analysis of the ITS region indicated that LVP A was a strain of Pythium aphanidermatum, with greater than 99% similarity to the corresponding P. aphanidermatum sequences from the publicly accessible databases. The DNA sequences from LVP B and LVP C were most closely related to P. ultimum and P. dissotocum, respectively. Lower frequencies of other distinct isolates with unique RFLP patterns were also obtained with high levels of similarity (>97%) to P. heterothallicum, P. periplocum and genotypes of P. ultimum other than LVP B. Inoculation trials of 1- and 4-week-old beet seedlings indicated that compared with isolates of the LVP B genotype, a higher frequency of LVP A isolates caused disease. Isolates with the LVP A, LVP B and LVP C genotypes were highly sensitive to the fungicide Ridomil MZ, which suppressed radial growth on V8 agar between approximately four and thirty fold at 5 μg/mL metalaxyl and 40 μg/mL mancozeb, a concentration far lower than the recommended field application rate.

Variation in acquisition of Fiji disease virus by Perkinsiella saccharicida (Hemiptera: Delphacidae).

Fiji leaf gall, caused the Fiji disease virus (genus Fijivirus, family Reoviridae, FDV), is a serious disease of sugarcane, Saccharum officinarum L., in Australia and several other Asia-Pacific countries. In Australia FDV is transmitted only by the planthopper Perkinsiella saccharicida Kirkaldy (Hemiptera: Delphacidae), in a propagative manner. Successful transmission of FDV by single planthoppers confined to individual virus free plants is highly variable, even under controlled conditions. The research reported here addresses two possible sources of this variation: 1) gender, wing form, and life stage of the planthopper; and 2) genotype of the source plant. The acquisition of FDV by macropterous males, macropterous females, brachypterous females, and nymphs of P. saccharicida from infected plants was investigated using reverse transcription-polymerase chain reaction to diagnose FDV infection in the vector. The proportion of individuals infected with FDV was not statistically related to life stage, gender, or adult wing form of the vector. The acquisition of FDV by P. saccharicida from four cultivars of sugarcane was compared to assess the influence of plant genotype on acquisition. Those planthopper populations reared on diseased 'NCo310' plants had twice as many infected planthoppers as those reared on 'Q110', 'WD1', and 'WD2'. Therefore, variation in FDV acquisition in this system is not the result of variation in the gender, wing form and life stage of the P. saccharicida vectors. The cultivar used as the source plant to rear vector populations does affect the proportion of infected planthoppers in a population.

Resistance to the root-lesion nematode Pratylenchus thornei of Iranian landrace wheat.

Pratylenchus thornei is widespread throughout the wheat-growing regions in Australia and overseas and can cause yield losses of up to 70% in some intolerant cultivars. The most effective forms of management of P. thornei populations are crop rotation and plant breeding. There have been no wheat accessions identified as completely resistant to P. thornei, therefore breeding programs have used moderately resistant parents. The objective of the present research was to evaluate 274 Iranian landrace wheats for resistance to P. thornei and identify accessions with resistance superior to the current best resistance source (GS50a). Plants were grown in P. thornei inoculated soil under controlled conditions in a glasshouse pot experiment for 16 weeks. Ninety-two accessions found to be resistant or moderately so were retested in a second experiment. From combined analysis of these experiments, 34 accessions were identified as resistant with reproduction factors (final population per kg soil/initial inoculum rate per kg soil) <= 1. In total, 25 accessions were more resistant than GS50a, with AUS28470 significantly (P < 0.05) more resistant. The resistant Iranian landraces identified in the present study are a valuable untapped genetic pool offering improved levels of P. thornei resistance over current parents in Australian wheat-breeding programs.

Resistance to root-lesion nematodes (Pratylenchus thornei and P-neglectus) in synthetic hexaploid wheats and their durum and Aegilops tauschii parents.

Root-lesion nematodes (Pratylenchus thornei Sher and Allen and P. neglectus (Rensch) Filipijev and Schuurmans Stekhoven) cause substantial yield loss to wheat crops in the northern grain region of Australia. Resistance to P. thornei for use in wheat breeding programs was sought among synthetic hexaploid wheats (2n= 6x = 42, AABBDD) produced through hybridisations of Triticum turgidum L. subsp. durum (Desf.) Husn (2n= 4x = 28, AABB) with Aegilops tauschii Coss. (2n= 2x = 14, DD). Resistance was determined for the synthetic hexaploid wheats and their durum and Ae. tauschii parents from the numbers of nematodes in the roots of plants grown for 16 weeks in pots of pasteurised soil inoculated with P. thornei. Fifty-nine (32%) of 186 accessions of synthetic hexaploid wheats had lower numbers of nematodes than Gatcher Selection 50a (GS50a), a partially resistant bread wheat. Greater frequencies of partial resistance were present in the durum parents (72% of 39 lines having lower nematode numbers than GS50a) and in the Ae. tauschii parents (55% of 53 lines). The 59 synthetic hexaploids were re-tested in a second experiment along with their parents. In a third experiment, 11 resistant synthetic hexaploid wheats and their F-1 hybrids with Janz, a susceptible bread wheat, were tested and the F(1)s were found to give nematode counts intermediate between the respective two parents. Synthetic hexaploid wheats with higher levels of resistance resulted from hybridisations where both the durum and Ae. tauschii parents were partially resistant, rather than where only one parent was partially resistant. These results suggest that resistance to P. thornei in synthetic hexaploid wheats is polygenic, with resistances located both in the D genome from Ae. tauschii and in the A and/or B genomes from durum. Five synthetic hexaploid wheats were selected for further study on the basis of (1) a high level of resistance to P. thornei of the synthetic hexaploid wheats and of both their durum and Ae. tauschii parents, (2) being representative of both Australian and CIMMYT (International Maize and Wheat Improvement Centre) durums, and (3) being representative of the morphological subspecies and varieties of Ae. tauschii. These 5 synthetic hexaploid wheats were also shown to be resistant to P. neglectus, whereas GS50a and 2 P. thornei-resistant derivatives were quite susceptible. Results of P. thornei resistance of F(1)s and F(2)s from a half diallel of these 5 synthetic hexaploid wheats, GS50a, and Janz from another study indicate polygenic additive resistance and better general combining ability for the synthetic hexaploid wheats than for GS50a. Published molecular marker studies on a doubled haploid population between the synthetic hexaploid wheat with best general combining ability (CPI133872) and Janz have shown quantitative trait loci for resistance located in all 3 genomes. Synthetic hexaploid wheats offer a convenient way of introgressing new resistances to P. thornei and P. neglectus from both durum and Ae. tauschii into commercial bread wheats.

Metabolite mobilization in the stem galls of Parthenium hysterophorus induced by Epiblema strenuana inferred from the signatures of isotopic carbon and nitrogen and concentrations of total non-structural carbohydrates

Parthenium hysterophorus L. (Asteraceae) is a weed of national significance in Australia. Among the several arthropod agents introduced into Australia to control populations of P. hysterophorus biologically, Epiblema strenuana Walker (Lepidoptera: Tortricidae) is the most widespread and abundant agent. By intercepting the normal transport mechanisms of P. hysterophorus, the larvae of E. strenuana drain nutrients, other metabolic products, and energy, and place the host plant under intense metabolic stress. In this study, determinations of total non-structural carbohydrates (TNC) levels and carbon and nitrogen isotope ratios of fixed products in different parts of the plant tissue, including the gall, have been made to establish the function of gall as a sink for the nutrients. Values of δ13C and δ15N in galls were significantly different than those in proximal and distal stems, whereas the TNC levels were insignificant, when measured in the total population of P. hysterophorus, regardless of plant age. However, carbon, nitrogen, and TNC signatures presented significant results, when assayed in different developmental stages of P. hysterophorus. Carbon isotope ratios in galls were consistently more negative than those from the compared plant organs. Nitrogen isotope ratios in galls, on the contrary, were either similar to or less negative than the compared plant organs, especially within a single host-plant stage population (i.e., either rosette, preflowering, or flowering stage). TNC levels varied within compared plant populations. The stem distal to the gall functioned more efficiently as a nodal channel than the stem proximal to the gall, especially in the translocation of nitrogenous nutrients. Our findings indicate that the gall induced by E. strenuana functions as a sink for the assayed nutrients, although some variations have been observed in the patterns of nutrient mobilization. By creating a sink for the nutrients in the gall, E. strenuana is able to place the overall plant metabolism under stress, and this ability indicates E. strenuana has the necessary potential for use as a biological-control agent.