A suite of novel promoters and terminators for plant biotechnology II. The pPLEX series for use in monocots

A suite of plant expression vectors (pPLEX), constructed from the gene regulation signals from subterranean clover stunt virus (SCSV) genome, has previously been used in dicot transformation for a variety of applications in plant biotechnology. To assess their use for the transformation of monocots, a number of modifications were made to the basic vector series and assessed in rice. In their unmodified forms, the SCSV promoters directed low levels of gene expression, however, insertion of an intron between the promoter and the transgene open reading frame (analogous to the rice actin and maize ubiquitin promoter systems) increased transgene expression 50-fold. The expression patterns from the intron-modified SCSV (segments 4 and 7) promoters were very similar to those directed by the actin or ubiquitin promoters. All promoter systems investigated directed expression that appeared to be constitutive within leaf tissue, and localised to the epidermal and vascular tissues of the root. The pPLEX vectors described here are an important counterpart to the dicot pPLEX series and have the potential to be useful in monocot research and biotechnology.

Expression patterns of vascular-specific promoters RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants

The expression patterns of GUS fusion constructs driven by the Agrobacterium rhizogenes RolC and the maize Sh (Shrunken: sucrose synthase-1) promoters were examined in transgenic potatoes (cv. Atlantic). RolC drove high-level gene expression in phloem tissue, bundle sheath cells and vascular parenchyma, but not in xylem or non-vascular tissues. Sh expression was exclusively confined to phloem tissue. Potato leafroll luteovirus (PLRV) replicates only in phloem tissues, and we show that when RolC is used to drive expression of the PLRV coat protein gene, virus-resistant lines can be obtained. In contrast, no significant resistance was observed when the Sh promoter was used.

Nucleopolyhedrovirus introduction in Australia

Nucleopolyhedrovirus (NPV) has become an integral part of integrated pest management (IPM) in many Australian agricultural and horticultural crops. This is the culmination of years of work conducted by researchers at the Queensland Department of Primary Industries and Fisheries (QDPI&F) and Ag Biotech Australia Pty Ltd. In the early 1970’s researchers at QDPI&F identified and isolated a virus in Helicoverpa armigera populations in the field. This NPV was extensively studied and shown to be highly specific to Helicoverpa and Heliothis species. Further work showed that when used appropriately the virus could be used effectively to manage these insects in crops such as sorghum, cotton, chickpea and sweet corn. A similar virus was first commercially produced in the USA in the 1970’s. This product, Elcar®, was introduced into Australia in the late 1970’s by Shell Chemicals with limited success. A major factor contributing to the poor adoption of Elcar was the concurrent enormous success of the synthetic pyrethroids. The importance of integrated pest management was probably also not widely accepted at that time. Gradual development of insect resistance to synthetic pyrethroids and other synthetic insecticides in Australia and the increased awareness of the importance of IPM meant that researchers once again turned their attentions to environmentally friendly pest management tools such NPV and beneficial insects. In the 1990’s a company called Rhone-Poulenc registered an NPV for use in Australian sorghum, chickpea and cotton. This product, Gemstar®, was imported from the USA. In 2000 Ag Biotech Australia established an in-vivo production facility in Australia to produce commercial volumes of a product similar to the imported product. This product was branded, ViVUS®, and was first registered and sold commercially in Australia in 2003. The initial production of ViVUS used a virus identical to the American product but replicating it in an Australian Helicoverpa species, H. armigera. Subsequent research collaboration between QDPI&F and Ag Biotech reinvigorated interest in the local virus strain. This was purified and the production system adapted to produce it on a commercial scale. This new version of ViVUS, which was branded ViVUS Gold®, was first registered and sold commercially in 2004. Widespread insect resistance to insecticides and a greater understanding of integrated pest management is leading to increased adoption of technologies such NPV in Australian agriculture.

The dominant 55kDa allergen of the subtropical Bahia grass (Paspalum notatum) pollen is a group 13 pollen allergen, Pas n 13

Bahia grass, Paspalum notatum, is an important pollen allergen source with a long season of pollination and wide distribution in subtropical and temperate regions. We aimed to characterize the 55. kDa allergen of Bahia grass pollen (BaGP) and ascertain its clinical importance. BaGP extract was separated by 2D-PAGE and immunoblotted with serum IgE of a grass pollen-allergic patient. The amino-terminal protein sequence of the predominant allergen isoform at 55. kDa had similarity with the group 13 allergens of Timothy grass and maize pollen, Phl p 13 and Zea m 13. Four sequences obtained by rapid amplification of the allergen cDNA ends represented multiple isoforms of Pas n 13. The predicted full length cDNA for Pas n 13 encoded a 423 amino acid glycoprotein including a signal peptide of 28 residues and with a predicted pI of 7.0. Tandem mass spectrometry of tryptic peptides of 2D gel spots identified peptides specific to the deduced amino acid sequence for each of the four Pas n 13 cDNA, representing 47% of the predicted mature protein sequence of Pas n 13. There was 80.6% and 72.6% amino acid identity with Zea m 13 and Phl p 13, respectively. Reactivity with a Phl p 13-specific monoclonal antibody AF6 supported designation of this allergen as Pas n 13. The allergen was purified from BaGP extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. Purified Pas n 13 reacted with serum IgE of 34 of 71 (48%) grass pollen-allergic patients and specifically inhibited IgE reactivity with the 55. kDa band of BaGP for two grass pollen-allergic donors. Four isoforms of Pas n 13 from pI 6.3-7.8 had IgE-reactivity with grass pollen allergic sera. The allergenic activity of purified Pas n 13 was demonstrated by activation of basophils from whole blood of three grass pollen-allergic donors tested but not control donors. Pas n 13 is thus a clinically relevant pollen allergen of the subtropical Bahia grass likely to be important in eliciting seasonal allergic rhinitis and asthma in grass pollen-allergic patients.

Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen

Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the β-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29 kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.

The nutritional management of acute diarrhea in young infants : effect of carbohydrate ingested

To compare the efficacy of a low-lactose hy-drolyzed milk formula, a lactose-free corn syrup-based milk formula, and a standard lactose-containing formula during refeeding after rehydration in infants with gastroenteritis, 135 patients older than 2 years were studied by randomized trial. Clearly demonstrated disadvantages in terms of early weight loss and longer duration of diarrhea were observed with the lactose-based formula compared with early weight gains on both the low-lactose formulae, and thus the lactose-containing formula was discontinued after 91 patients. The early weight loss with the lactose-containing formula was statistically significantly related to the degree of relative (rehydrated) underweight. The two low-lactose formulae were further compared in the remaining 44 patients. Early weight gain (48 h) was sig-nificantly greater with the lactose-hydrolyzed formula compared with the corn syrup-based formula, but no statistically significant differences were observed in duration of diarrhea, energy intake, treatment failures, or late weight gain. We conclude that the routine use of a low-lactose formula during refeeding after rehydration in infants with gastroenteritis may have some advantages in underweight infants and toddlers in whom it is important to prevent further weight loss. © 1994 Raven Press Ltd, New York.

Mycotoxins and Fungal Damage in Maize Harvested during 1982 in Far North Queensland

A survey for mycotoxins and fungal damage in maize (Zea mays L.) grown during 1982 in Far North Queensland is reported. This season had a rainfall distribution which was typical for the reglon. The 293 samples examined came from 11 1 farms in eight maize-growing districts. The samples were first subjected to rapid screening tests for fungal damage. Aflatoxins B1, B2, G1, G2 ochratoxin A, T-2 toxin, and sterigmatocystin were not detected, but zearalenone was found in 85% of the samples. The concentrations of zearalenone were correlated with the extent of Gibberella zeae cob rot as indicated by the proportion (up to 2%) of kernels in each sample having a reddish-purple discoloration. In four samples the zearalenone concentration exceeded 1 mg kg-1, but the mean ¦ s.d. (n = 293) concentration in all samples was 0.17 ¦ 0.225 mg kg-1. Concentrations were highest in districts with the highest rainfall during the period of maize growth.

Effect of irrigation amounts applied with subsurface drip irrigation on corn evapotranspiration, yield, water use efficiency, and dry matter production in a semiarid climate

Quantifying the local crop response to irrigation is important for establishing adequate irrigation management strategies. This study evaluated the effect of irrigation applied with subsurface drip irrigation on field corn (Zea mays L.) evapotranspiration (ETc), yield, water use efficiencies (WUE = yield/ETc, and IWUE = yield/irrigation), and dry matter production in the semiarid climate of west central Nebraska. Eight treatments were imposed with irrigation amounts ranging from 53 to 356 mm in 2005 and from 22 to 226 mm in 2006. A soil water balance approach (based on FAO-56) was used to estimate daily soil water and ETc. Treatments resulted in seasonal ETc of 580-663 mm and 466-656 mm in 2005 and 2006, respectively. Yields among treatments differed by as much as 22% in 2005 and 52% in 2006. In both seasons, irrigation significantly affected yields, which increased with irrigation up to a point where irrigation became excessive. Distinct relationships were obtained each season. Yields increased linearly with seasonal ETc (R 2 = 0.89) and ETc/ETp (R 2 = 0.87) (ETp = ETc with no water stress). The yield response factor (ky), which indicates the relative reduction in yield to relative reduction in ETc, averaged 1.58 over the two seasons. WUE increased non-linearly with seasonal ETc and with yield. WUE was more sensitive to irrigation during the drier 2006 season, compared with 2005. Both seasons, IWUE decreased sharply with irrigation. Irrigation significantly affected dry matter production and partitioning into the different plant components (grain, cob, and stover). On average, the grain accounted for the majority of the above-ground plant dry mass (≈59%), followed by the stover (≈33%) and the cob (≈8%). The dry mass of the plant and that of each plant component tended to increase with seasonal ETc. The good relationships obtained in the study between crop performance indicators and seasonal ETc demonstrate that accurate estimates of ETc on a daily and seasonal basis can be valuable for making tactical in-season irrigation management decisions and for strategic irrigation planning and management.

Relative contributions of light interception and radiation use efficiency to the reduction of maize productivity under cold temperatures

Maize (Zea mays L.) is a chill-susceptible crop cultivated in northern latitude environments. The detrimental effects of cold on growth and photosynthetic activity have long been established. However, a general overview of how important these processes are with respect to the reduction of productivity reported in the field is still lacking. In this study, a model-assisted approach was used to dissect variations in productivity under suboptimal temperatures and quantify the relative contributions of light interception (PARc) and radiation use efficiency (RUE) from emergence to flowering. A combination of architectural and light transfer models was used to calculate light interception in three field experiments with two cold-tolerant lines and at two sowing dates. Model assessment confirmed that the approach was suitable to infer light interception. Biomass production was strongly affected by early sowings. RUE was identified as the main cause of biomass reduction during cold events. Furthermore, PARc explained most of the variability observed at flowering, its relative contributions being more or less important according to the climate experienced. Cold temperatures resulted in lower PARc, mainly because final leaf length and width were significantly reduced for all leaves emerging after the first cold occurrence. These results confirm that virtual plants can be useful as fine phenotyping tools. A scheme of action of cold on leaf expansion, light interception and radiation use efficiency is discussed with a view towards helping breeders define relevant selection criteria. This paper originates from a presentation at the 5th International Workshop on Functional–Structural Plant Models, Napier, New Zealand, November 2007.

Soil properties and crop yields in a dryland Vertisol sown with cotton-based crop rotations

Information on the effects of growing cotton (Gossypium hirsutum L.)-based crop rotations on soil quality of dryland Vertisols is sparse. The objective of this study was to quantify the effects of growing cereal and leguminous crops in rotation with dryland cotton on physical and chemical properties of a grey Vertisol near Warra, SE Queensland, Australia. The experimental treatments, selected after consultations with local cotton growers, were continuous cotton (T1), cotton-sorghum (Sorghum bicolor (L.) Moench.) (T2), cotton-wheat (Triticum aestivum L.) double cropped (T3), cotton-chickpea (Cicer arietinum L.) double cropped followed by wheat (T4) and cotton-wheat (T5). From 1993 to 1996 land preparation was by chisel ploughing to about 0.2 m followed by two to four cultivations with a Gyral tyne cultivator. Thereafter all crops were sown with zero tillage except for cultivation with a chisel plough to about 0.07-0.1 m after cotton picking to control heliothis moth pupae. Soil was sampled from 1996 to 2004 and physical (air-filled porosity of oven-dried soil, an indicator of soil compaction; plastic limit; linear shrinkage; dispersion index) and chemical (pH in 0.01 M CaCl2, organic carbon, exchangeable Ca, Mg, K and Na contents) properties measured. Crop rotation affected soil properties only with respect to exchangeable Na content and air-filled porosity. In the surface 0.15 m during 2000 and 2001 lowest air-filled porosity occurred with T1 (average of 34.6 m3/100 m3) and the highest with T3 (average of 38.9 m3/100 m3). Air-filled porosity decreased in the same depth between 1997 and 1998 from 45.0 to 36.1 m3/100 m3, presumably due to smearing and compaction caused by shallow cultivation in wet soil. In the subsoil, T1 and T2 frequently had lower air-filled porosity values in comparison with T3, T4 and T5, particularly during the early stages of the experiment, although values under T1 increased subsequently. In general, compaction was less under rotations which included a wheat crop (T3, T4, T5). For example, average air-filled porosity (in m3/100 m3) in the 0.15-0.30 m depth from 1996 to 1999 was 19.8 with both T1 and T2, and 21.2 with T3, 21.1 with T4 and 21.5 with T5. From 2000 to 2004, average air-filled porosity (in m3/100 m3) in the same depth was 21.3 with T1, 19.0 with T2, 19.8 with T3, 20.0 with T4 and 20.5 with T5. The rotation which included chickpea (T4) resulted in the lowest exchangeable Na content, although differences among rotations were small. Where only a cereal crop with a fibrous root system was sown in rotation with cotton (T2, T3, T5) linear shrinkage in the 0.45-0.60 m depth was lower than in rotations, which included tap-rooted crops such as chickpea (T4) or continuous cotton (T1). Dispersion index and organic carbon decreased, and plastic limit increased with time. Soil organic carbon stocks decreased at a rate of 1.2 Mg/ha/year. Lowest average cotton lint yield occurred with T2 (0.54 Mg/ha) and highest wheat yield with T3 (2.8 Mg/ha). Rotations which include a wheat crop are more likely to result in better soil structure and cotton lint yield than cotton-sorghum or continuous cotton.

Effect of timing of a deficit-irrigation allocation on corn evapotranspiration, yield, water use efficiency and dry mass

Water regulations have decreased irrigation water supplies in Nebraska and some other areas of the USA Great Plains. When available water is not enough to meet crop water requirements during the entire growing cycle, it becomes critical to know the proper irrigation timing that would maximize yields and profits. This study evaluated the effect of timing of a deficit-irrigation allocation (150 mm) on crop evapotranspiration (ETc), yield, water use efficiency (WUE = yield/ETc), irrigation water use efficiency (IWUE = yield/irrigation), and dry mass (DM) of corn (Zea mays L.) irrigated with subsurface drip irrigation in the semiarid climate of North Platte, NE. During 2005 and 2006, a total of sixteen irrigation treatments (eight each year) were evaluated, which received different percentages of the water allocation during July, August, and September. During both years, all treatments resulted in no crop stress during the vegetative period and stress during the reproductive stages, which affected ETc, DM, yield, WUE and IWUE. Among treatments, ETc varied by 7.2 and 18.8%; yield by 17 and 33%; WUE by 12 and 22%, and IWUE by 18 and 33% in 2005 and 2006, respectively. Yield and WUE both increased linearly with ETc and with ETc/ETp (ETp = seasonal ETc with no water stress), and WUE increased linearly with yield. The yield response factor (ky) averaged 1.50 over the two seasons. Irrigation timing affected the DM of the plant, grain, and cob, but not that of the stover. It also affected the percent of DM partitioned to the grain (harvest index), which increased linearly with ETc and averaged 56.2% over the two seasons, but did not affect the percent allocated to the cob or stover. Irrigation applied in July had the highest positive coefficient of determination (R2) with yield. This high positive correlation decreased considerably for irrigation applied in August, and became negative for irrigation applied in September. The best positive correlation between the soil water deficit factor (Ks) and yield occurred during weeks 12-14 from crop emergence, during the "milk" and "dough" growth stages. Yield was poorly correlated to stress during weeks 15 and 16, and the correlation became negative after week 17. Dividing the 150 mm allocation about evenly among July, August and September was a good strategy resulting in the highest yields in 2005, but not in 2006. Applying a larger proportion of the allocation in July was a good strategy during both years, and the opposite resulted when applying a large proportion of the allocation in September. The different results obtained between years indicate that flexible irrigation scheduling techniques should be adopted, rather than relying on fixed timing strategies.

A comparison of alternative plant mixes for conservation bio-control by native beneficial arthropods in vegetable cropping systems in Queensland Australia.

Cucurbit crops host a range of serious sap-sucking insect pests, including silverleaf whitefly (SLW) and aphids, which potentially represent considerable risk to the Australian horticulture industry. These pests are extremely polyphagous with a wide host range. Chemical control is made difficult due to resistance and pollution, and other side-effects are associated with insecticide use. Consequently, there is much interest in maximising the role of biological control in the management of these sap-sucking insect pests. This study aimed to evaluate companion cropping alongside cucurbit crops in a tropical setting as a means to increase the populations of beneficial insects and spiders so as to control the major sap-sucking insect pests. The Population of beneficial and harmful insects, with a focus on SLW and aphids, and other invertebrates were sampled weekly oil four different crops which could be used for habitat manipulation: Goodbug Mix (GBM; a proprietary seed Mixture including self-sowing annual and perennial herbaceous flower species); lablab (Lablab purpureus L. Sweet); lucerne (Medicago sativa L.); and niger (Guizotia abyssinica (L.f.) Cass.). Lablab hosted the highest numbers of beneficial insects (larvae and adults of lacewing (Mallada signata (Schneider)), ladybird beetles (Coccinella transversalis Fabricius) and spiders) while GBM hosted the highest numbers of European bees (Apis mellifera Linnaeus) and spiders. Lucerne and niger showed little promise in hosting beneficial insects, but lucerne hosted significantly more spiders (double the numbers) than niger. Lucerne hosted significantly more of the harmful insect species of aphids (Aphis gossypii (Glover)) and Myzus persicae (Sulzer)) and heliothis (Heliothis armigera Hubner). Niger hosted significantly more vegetable weevils (Listroderes difficillis (Germar)) than the other three species. Therefore, lablab and GBM appear to be viable options to grow within cucurbits or as field boundary crops to attract and increase beneficial insects and spiders for the control of sap-sucking insect pests. Use of these bio-control strategies affords the opportunity to minimise pesticide usage and the risks associated with pollution.

Analysis and Modelling of the Effects of Water Stress on Maize Growth and Yield in Dryland Conditions.

It is essential to provide experimental evidence and reliable predictions of the effects of water stress on crop production in the drier, less predictable environments. A field experiment undertaken in southeast Queensland, Australia with three water regimes (fully irrigated, rainfed and irrigated until late canopy expansion followed by rainfed) was used to compare effects of water stress on crop production in two maize (Zea mays L.) cultivars (Pioneer 34N43 and Pioneer 31H50). Water stress affected growth and yield more in Pioneer 34N43 than in Pioneer 31H50. A crop model APSIM-Maize, after having been calibrated for the two cultivars, was used to simulate maize growth and development under water stress. The predictions on leaf area index (LAI) dynamics, biomass growth and grain yield under rain fed and irrigated followed by rain fed treatments was reasonable, indicating that stress indices used by APSIM-Maize produced appropriate adjustments to crop growth and development in response to water stress. This study shows that Pioneer 31H50 is less sensitive to water stress and thus a preferred cultivar in dryland conditions, and that it is feasible to provide sound predictions and risk assessment for crop production in drier, more variable conditions using the APSIM-Maize model.

Temporal genetic structure patterns in tropical maize populations under reciprocal recurrent selection.

In order to investigate the effect of long term recurrent selection on the pattern of gene diversity, thirty randomly-selected individuals from the progenitors (p) and four selection cycles (C0, C3, C6 and C11) were sampled for DNA analysis from the tropical maize (Zea mays L.) breeding populations, Atherton 1 (AT1) and Atherton 2 (AT2). Fifteen polymorphic Simple Sequence Repeat markers amplified a total of 284 and 257 alleles in AT1 and AT2 populations, respectively. Reductions in the number of alleles were observed at advanced selection cycles. About 11 and 12% of the alleles in AT1 and AT2 populations respectively, were near to fixation. However, a higher number of alleles (37% in AT1 and 33% in AT2) were close to extinction. Fisher's exact test and analysis of molecular variance (AMOVA) showed significant population differentiations. Gene diversity estimates and AMOVA revealed increased genetic differentiations at the expense of loss of heterozygosity. Population differentiations were mainly due to fixation of complementary alleles at a locus in the two breeding populations. The estimates of effective population at an advanced selection cycle were close to the population size predicted by the breeding method.

Commercial products from bio-active extractives in cypress milling residues.

Extractive components obtained from milling residues of white cypress were studied for chemical identity and bioactivity with a view to developing a commercial use for these components, thus increasing the value of the residues and improving the economics of cypress sawn wood production. Extracts obtained by solvent or steam extraction techniques from cypress sawdust were each fractionated by a range of techniques into groups of similar compounds. Crude extracts and fractions were screened against a range of agricultural pests and diseases, including two fungi, subterranean termites, fruit spotting bugs, two-spotted mites, thrips, heliothis, banana scab moths, silverleaf whiteflies, cattle tick adults and larvae, and ruminant gastrointestinal nematodes. Additional screening was undertaken where encouraging results were achieved, for two-spotted mites, thrips, silverleaf whiteflies, cattle tick adults and ruminant gastrointestinal nematodes. After considering degrees of efficacy against, and economic importance of, the agricultural pests, and likely production costs of extracts and fractions, the crude extract (oil) produced by steam distillation was chosen for further study against silverleaf whitefly. A useful degree of control was achievable when this oil was applied to tomato or eggplant at 0.1%, with much less harmful effects on a beneficial insect. Activity of the oil against silverleaf whitefly was undiminished 3.5 years after it was generated. There was little benefit from supplementing the extract with co-formulated paraffinic oil. From the steam distilled oil, fifty-five compounds were characterised, thirty-five compounds representing 92.478 % of the oil, with guaiol (20.8%) and citronellic acid (15.9%) most abundant. These two compounds, and a group of oxygenated compounds containing bulnesol and a range of eudesmols, were found to account for most of the activity against silverleaf whitefly. This application was recommended for first progression to commercialisation.