Utility of cotyledon and detached leaf assays for assessing root reactions of lucerne to Phytophthora root rot caused by Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne production but it can be managed through the use of resistant cultivars. Current resistance screening methods, using mature plants or post-emergence seedling assays, are costly and time consuming. The use of zoospore inoculum on detached leaves and intact cotyledons as an assay for plant resistance was assessed using genetically defined segregating populations. The detached leaf assay was a reproducible test, but this test could not be used for accurately predicting root ratings. The cotyledon tests using zoospores gave results at the population level that were indicative of the root responses of 19 cultivars and lines tested. The cotyledon reaction of individual plants also showed a strong association with root response. The cotyledon test, while not completely predictive of mature root responses, allowed the selection of Phytophthora resistant plants at a higher frequency than could be achieved by random selection.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy (R) or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to I infected in 800 samples with pepper but never detecting more than I infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Localization and movement of mineral oil in plants by fluorescence and confocal microscopy

Fluorescence and confocal laser scanning microscopy were explored to investigate the movement and localization of mineral oils in citrus. In a laboratory experiment, fluorescence microscopy observation indicated that when a 'narrow' distillation fraction of an nC23 horticultural mineral oil was applied to adaxial and opposing abaxial leaf surfaces of potted orange [Citrus x aurantium L. (Sapindales: Rutaceae)] trees, oil penetrated steadily into treated leaves and, subsequently, moved to untreated petioles of the leaves and adjacent untreated stems. In another experiment, confocal laser scanning microscopy was used to visualize the penetration into, and the subsequent cellular distribution of, an nC24 agricultural mineral oil in C. trifoliata L. seedlings. Oil droplets penetrated or diffused into plants via both stomata and the cuticle of leaves and stems, and then moved within intercellular spaces and into various cells including phloem and xylem. Oil accumulated in droplets in intercellular spaces and within cells near the cell membrane. Oil entered cells without visibly damaging membranes or causing cell death. In a field experiment with mature orange trees, droplets of an nC23 horticultural mineral oil were observed, by fluorescence microscopy, in phloem sieve elements in spring flush growth produced 4-5 months and 16-17 months after the trees were sprayed with oil. These results suggest that movement of mineral oil in plants is both apoplastic via intercellular spaces and symplastic via plasmodesmata. The putative pattern of the translocation of mineral oil in plants and its relevance to oil-induced chronic phytotoxicity are discussed.

Leaf water relations of Eucalyptus cloeziana and Eucalyptus argophloia in response to water deficit

Leaf water relations responses to limited water supply were determined in 7-month-old plants of a dry inland provenance of Eucalyptus argophloia Blakely and in a humid coastal provenance (Gympie) and a dry inland provenance (Hungry Hills) of Eucalyptus cloeziana F. Muell. Each provenance of E. cloeziana exhibited a lower relative water content at the turgor loss point, a lower apoplastic water content, a smaller ratio of dry mass to turgid mass and a lower bulk modulus of elasticity than the single provenance of E. argophloia. Osmotic potential at full turgor and water potential at the turgor loss point were significantly lower in E. argophloia and the inland provenance of E. cloeziana than in the coastal provenance of E. cloeziana. There was limited osmotic adjustment in response to soil drying in E. cloeziana, but not in E. argophloia. Between-species differences in water relations parameters were larger than those between the E. cloeziana provenances. Both E. cloeziana provenances maintained turgor under moderate water stress through a combination of osmotic and elastic adjustments. Eucalyptus argophloia had more rigid cell walls and reached lower water potentials with less reduction in relative water content than either of the E. cloeziana provenances, thereby enabling it to extract water from dryer soils.

Effect of ionic strength and clay mineralogy on Na-Ca exchange and the SAR-ESP relationship

The relationship between sodium adsorption ratio (SAR) and exchangeable sodium percentage (ESP) for all soils has traditionally been assumed to be similar to that developed by the United States Salinity Laboratory (USSL) in 1954. However, under certain conditions, this relationship has been shown not to be constant, but to vary with both ionic strength and clay mineralogy. We conducted a detailed experiment to determine the effect of ionic strength on the Na+-Ca2+ exchange of four clay minerals (kaolinite, illite, pyrophyllite, and montmorillonite), with results related to the diffuse double-layer (DDL) model. Clays in which external exchange sites dominated (kaolinite and pyrophyllite) tended to show an overall preference for Na+, with the magnitude of this preference increasing with decreasing ESP. For these external surfaces, increases in ionic strength were found to increase preference for Na+. Although illite (2:1 non-expanding mineral) was expected to be dominated by external surfaces, this clay displayed an overall preference for Ca2+, possibly indicating the opening of quasicrystals and the formation of internal exchange surfaces. For the expanding 2:1 clay, montmorillonite, Na+-Ca2+ exchange varied due to the formation of quasicrystals (and internal exchange surfaces) from individual clay platelets. At small ionic strength and large ESP, the clay platelets dispersed and were dominated by external exchange surfaces (displaying preference for Na+). However, as ionic strength increased and ESP decreased, quasicrystals (and internal exchange surfaces) formed, and preference for Ca2+ increased. Therefore, the relationship between SAR and ESP is not constant and should be determined directly for the soil of interest.

Waterlogging and fire impacts on nitrogen availability and utilization in a subtropical wet heathland (wallum)

Protein, amino acids and ammonium were the main forms of soluble soil nitrogen in the soil solution of a subtropical heathland (wallum). After fire, soil ammonium and nitrate increased 90- and 60-fold, respectively. Despite this increase in nitrate availability after fire, wallum species exhibited uniformly low nitrate reductase activities and low leaf and xylem nitrate, During waterlogging soil amino acids increased, particularly gamma-aminobutyric acid (GABA) which accounted for over 50% of amino nitrogen. Non-mycorrhizal wallum species were significantly (P < 0.05) N-15-enriched (0.3-4.3 parts per thousand) compared to species with mycorrhizal associations (ericoid-type, ecto-, va-mycorrhizal) which were strongly depleted in N-15 (-6.3 to -1.8 parts per thousand). Lignotubers and roots had delta(15)N signatures similar to that of the leaves of respective species. The exceptions were fine roots of ecto-, ecto/va-, and ericoid type mycorrhizal species which were enriched in N-15 (0.1-2 4 parts per thousand). The delta(15)N signatures of delta(15)N(total soil N) and delta(15)N(soil NH4+) were in the range 3.7-4.5 parts per thousand, whereas delta(15)N(soil NO3-) was significantly (P < 0.05) more enriched in N-15 (9.2-9.8 parts per thousand). It is proposed that there is discrimination against N-15 during transfer of nitrogen from fungal to plant partner. Roots of selected species incorporated nitrogen sources in the order of preference: ammonium > glycine > nitrate. The exception were proteoid roots of Hakea (Proteaceae) which incorporated equal amounts of glycine and ammonium.

Use of confocal scanning laser microscopy to measure the concentrations of aerial and penetrative hyphae during growth of Rhizopus oligosporus on a solid surface

In order to develop a method for use in investigations of spatial biomass distribution in solid-state fermentation systems, confocal scanning laser microscopy was used to determine the concentrations of aerial and penetrative biomass against height and depth above and below the substrate surface, during growth of Rhizopus oligosporus on potato dextrose agar. Penetrative hyphae had penetrated to a depth of 0.445 cm by 64 h and showed rhizoid morphology, in which the maximum biomass concentration, of 4.45 mg dry wt cm(-3), occurred at a depth of 0.075 cm. For aerial biomass the maximum density of 39.54 mg dry wt(-3) occurred at the substrate surface. For both aerial and penetrative biomass, there were two distinct regions in which the biomass concentration decayed exponentially with distance from the surface. For aerial biomass, the first exponential decay region was up to 0.1 cm height. The second region above the height of 0.1 cm corresponded to that in which sporangiophores dominated. This work lays the foundation for deeper studies into what controls the growth of fungal hyphae above and below the surfaces of solid substrates. (C) Wiley Periodicals, Inc.

Growth of papaya nodal and rooted shoot cultures as affected by ACC, STS, culture vessel size and incubation conditions

Nodal shoot cultures of 'Clone 003', a selected Australian papaya cultivar, were cultured on modified De Fossard medium supplemented with chemicals that either promote ethylene evolution or inhibit action while in culture. Nodal shoot cultures grown in the presence of 1-aminocyclopropane carboxylic acid (ACC, 1.0 mM) resulted in a significant reduction in percent fresh and dry weights, shoot length, leaf area, petiole length and chlorophyll content, but leaf development was significantly increased. In contrast, nodal cultures grown in the presence of silver thiosulphate (STS, 0.5 mM) significantly produced the highest percentage of fresh and dry weights, shoot length, leaf production, leaf area expansion, petiole length and leaf chlorophyll content. Nodal cultures and rooted whole plantlets placed in medium-sized (125 mL) culture vessels had significantly better growth than those cultures placed in small (70 mL) or in large (250 mL) vessels. Cultures grown in medium-sized vessels had higher fresh and dry weights, longer shoots, more leaves and larger leaf area than those cultures placed in smaller or larger vessels. Similarly, values for said growth parameters and for chlorophyll content of the nodal and rooted whole plantlets were higher when they were incubated under high light intensity of 120 mumol m(-2)s(-1) at a prevailing temperature of either 20+/-1 C or 25+/-1 C.

Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B(RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8(+) T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-gamma-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.

HPGR model verification and scale-up

This paper summarises test results that were used to validate a model and scale-up procedure of the high pressure grinding roll (HPGR) which was developed at the JKMRC by Morrell et al. [Morrell, Lim, Tondo, David,1996. Modelling the high pressure grinding rolls. In: Mining Technology Conference, pp. 169-176.]. Verification of the model is based on results from four data sets that describe the performance of three industrial scale units fitted with both studded and smooth roll surfaces. The industrial units are currently in operation within the diamond mining industry and are represented by De Beers, BHP Billiton and Rio Tinto. Ore samples from the De Beers and BHP Billiton operations were sent to the JKMRC for ore characterisation and HPGR laboratory-scale tests. Rio Tinto contributed an historical data set of tests completed during a previous research project. The results conclude that the modelling of the HPGR process has matured to a point where the model may be used to evaluate new and to optimise existing comminution circuits. The model prediction of product size distribution is good and has been found to be strongly dependent of the characteristics of the material being tested. The prediction of throughput and corresponding power draw (based on throughput) is sensitive to inconsistent gap/diameter ratios observed between laboratory-scale tests and full-scale operations. (C) 2004 Elsevier Ltd. All rights reserved.

Physiological responses to water stress in Eucalyptus cloeziana and E-argophloia seedlings

Effects of water stress duration and intensity on gas exchange and leaf water potential were investigated in 7-month-old seedlings of a humid coastal provenance (Gympie) and a dry inland (Hungry Hills) provenance of E. cloeziana F. Muell. and in a dry inland (Chinchilla) provenance of E. argophloia Blakely supplied with 100% (T-100), 70% (T-70), 50% (T-50) of their water requirements, or were watered only after they were wilted at dawn (T-0). Seedlings of E. argophloia had the highest midday net photosynthetic rate (A), stomata] conductance (g(s)), stomatal density and predawn leaf water potential (Psi(pd)) in all treatments. The E. cloeziana provenances did not differ in these attributes. The T-70 and T-50 treatments caused reductions in A of 30% in E. argophloia, and 55% in the E. cloeziana provenances. Under the T-0 treatment, E. argophloia maintained higher rates of gas exchange at all levels of water stress than E. cloeziana provenances. The estimates of Psi(pd) and midday water potential (Psi(md)) at which plants remained wilted overnight were respectively: -2.7 and -4.1 MPa for E. cloeziana (humid), -2.8 and -4.0 MPa for E. cloeziana (dry) and, -3.7 and -4.9 MPa for E. argophloia. Following stress relief, both A and g(s) recovered more quickly in E. argophloia and in the dry provenance of E. cloeziana than in the humid provenance. We conclude that E. argophloia is more drought tolerant and has a potential for cultivation in the humid and semi humid climates, whilst E. cloeziana has greater potential in the humid subtropical climates.

Variation in hydraulic conductivity of mangroves: influence of species, salinity, and nitrogen and phosphorus availability

We investigated how species identity and variation in salinity and nutrient availability influence the hydraulic conductivity of mangroves. Using a fertilization study of two species in Florida, we found that stem hydraulic conductivity expressed on a leaf area basis (K-leaf) was significantly different among species of differing salinity tolerance, but was not significantly altered by enrichment with limiting nutrients. Reviewing data from two additional sites (Panama and Belize), we found an overall pattern of declining leaf-specific hydraulic conductivity (K-leaf) with increasing salinity. Over three sites, a general pattern emerges, indicating that native stem hydraulic conductivity (K-h) and K-leaf are less sensitive to nitrogen (N) fertilization when N limits growth, but more sensitive to phosphorus (P) fertilization when P limits growth. Processes leading to growth enhancement with N fertilization are probably associated with changes in allocation to leaf area and photosynthetic processes, whereas water uptake and transport processes could be more limiting when P limits growth. These findings suggest that whereas salinity and species identity place broad bounds on hydraulic conductivity, the effects of nutrient availability modulate hydraulic conductivity and growth in complex ways.