Recombination hotspots and host susceptibility modulate the adaptive value of recombination during maize streak virus evolution

Background Maize streak virus -strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae), the maize-adapted strain of MSV that causes maize streak disease throughout sub-Saharan Africa, probably arose between 100 and 200 years ago via homologous recombination between two MSV strains adapted to wild grasses. MSV recombination experiments and analyses of natural MSV recombination patterns have revealed that this recombination event entailed the exchange of the movement protein - coat protein gene cassette, bounded by the two genomic regions most prone to recombination in mastrevirus genomes; the first surrounding the virion-strand origin of replication, and the second around the interface between the coat protein gene and the short intergenic region. Therefore, aside from the likely adaptive advantages presented by a modular exchange of this cassette, these specific breakpoints may have been largely predetermined by the underlying mechanisms of mastrevirus recombination. To investigate this hypothesis, we constructed artificial, low-fitness, reciprocal chimaeric MSV genomes using alternating genomic segments from two MSV strains; a grass-adapted MSV-B, and a maize-adapted MSV-A. Between them, each pair of reciprocal chimaeric genomes represented all of the genetic material required to reconstruct - via recombination - the highly maize-adapted MSV-A genotype, MSV-MatA. We then co-infected a selection of differentially MSV-resistant maize genotypes with pairs of reciprocal chimaeras to determine the efficiency with which recombination would give rise to high-fitness progeny genomes resembling MSV-MatA. Results Recombinants resembling MSV-MatA invariably arose in all of our experiments. However, the accuracy and efficiency with which the MSV-MatA genotype was recovered across all replicates of each experiment depended on the MSV susceptibility of the maize genotypes used and the precise positions - in relation to known recombination hotspots - of the breakpoints required to re-create MSV-MatA. Although the MSV-sensitive maize genotype gave rise to the greatest variety of recombinants, the measured fitness of each of these recombinants correlated with their similarity to MSV-MatA. Conclusions The mechanistic predispositions of different MSV genomic regions to recombination can strongly influence the accessibility of high-fitness MSV recombinants. The frequency with which the fittest recombinant MSV genomes arise also correlates directly with the escalating selection pressures imposed by increasingly MSV-resistant maize hosts.

A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay

Centre for High-Performance Computing, Rosebank, Cape Town, South Africa Maize streak disease, caused by the A strain of the African endemic geminivirus, maize streak mastrevirus (MSV-A), threatens the food security and livelihoods of subsistence farmers throughout sub-Saharan Africa. Using a well-established transient expression assay, this study investigated the potential of a spliceable-intron hairpin RNA (hpRNA) approach to interfere with MSV replication. Two strategies were explored: (i) an inverted repeat of a 662 bp region of the MSV replication-associated protein gene (rep), which is essential for virus replication and is therefore a good target for post-transcriptional gene silencing; and (ii) an inverted repeat of the viral long intergenic region (LIR), considered for its potential to trigger transcriptional silencing of the viral promoter region. After co-bombardment of cultured maize cells with each construct and an infectious partial dimer of the cognate virus genome (MSV-Kom), followed by viral replicativeform-specific PCR, it was clear that, whilst the hairpin rep construct (pHPrepDI662) completely inhibited MSV replication, the LIR hairpin construct was ineffective in this regard. In addition, pHPrepDI662 inhibited or reduced replication of six MSV-A genotypes representing the entire breadth of known MSV-A diversity. Further investigation by real-time PCR revealed that the pHPrepDI662 inverted repeat was 22-fold more effective at reducing virus replication than a construct containing the sense copy, whilst the antisense copy had no effect on replication when compared with the wild type. This is the first indication that an hpRNA strategy targeting MSV rep has the potential to protect transgenic. © 2011 SGM.

Moisture and tensile strength properties of starch-sugar cane nanofibre films

There is an increasing need for biodegradable, environmentally friendly plastics to replace the petroleum-based non-degradable plastics which litter and pollute the environment. Starch-based plastic film composites are becoming a popular alternative because of their low cost, biodegradability, the abundance of starch, and ease with which starch-based films can be chemically modified. This paper reports on the results of using sugar cane bagasse nanofibres to improve the physicochemical properties of starch-based polymers. The addition of bagasse nanofibre (2.5, 5, 10 or 20 wt%) to (modified) potato starch (‘Soluble starch’) reduced the moisture uptake by up to 17 % at 58 % relative humidity (RH). The film’s tensile strength and Young’s Modulus increased by up to 100 % and 200 % with 10 wt% and 20 wt% nanofibre respectively at 58% RH. The tensile strain reduced by up to 70 % at 20 wt% fibre loading. These results indicate that addition of sugar cane bagasse nanofibres significantly improved the properties of starch-based plastic films

Preparation and characterization of composites from starch with sugarcane bagasse nanofibres

This paper reports on the results of using unbleached sugar cane bagasse nanofibres (average diameter 26.5 nm; aspect ratio 247 assuming a dry fibre density of 1,500 kg/m3) to improve the physico-chemical properties of starch-based films. The addition of bagasse nanofibres (2.5 to 20 wt%) to modified potato starch (i.e. soluble starch) reduced the moisture uptake by up to 17 % at 58 % relative humidity. The film’s tensile strength and Young’s modulus increased by up to 100 % (3.1 to 6.2 MPa) and 300 % (66.3 to 198.3 MPa) respectively with 10 and 20 wt% fibre addition. However, the strain at yield dropped by 50 % for the film containing 10 wt% fibre. Models for composite materials were used to account for the strong interactions between the nanofibres and the starch matrix. The storage and loss moduli as well as the glass transition temperature (Tg) obtained from dynamic mechanical thermal analysis, were increased with the starch-nanofibre films indicating decreased starch chain mobility due to the interacting effect of the nanofibres. Evidence of the existence of strong interactions between the starch matrix and the nanofibres was revealed from detailed Fourier transform infra-red and scanning electron microscopic evaluation.

Inducible resistance to maize streak virus

Maize streak virus (MSV), which causes maize streak disease (MSD), is the major viral pathogenic constraint on maize production in Africa. Type member of the Mastrevirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic maize lines constitutively expressing ‘‘dominant negative mutant’’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.

Developing starch-based polymer composites

This project aim was to replace petroleum-based plastic packaging materials that pollute the environment, with biodegradable starch-based polymer composites. It was demonstrated that untreated sugar cane bagasse microfibres and unbleached nanofibres significantly improved the physical, mechanical and chemical properties of starch films, while thermal extrusion of starch with alcohol improved the stiffness and the addition of aconitic acid cross-linked the film making it moisture resistant and extensible.

Studies on glass transition and starch re-crystallization in wheat bread during staling using electrical impedance spectroscopy

Bread staling is a very complex phenomenon that is not yet completely understood. The present work explains how the electrical impedance spectroscopy technique can be utilized to investigate the effect of staling on the physicochemical properties of wheat bread during storage. An instrument based on electrical impedance spectroscopy technique is developed to study the electrical properties of wheat bread both at its crumb and crust with the help of designed multi-channel ring electrodes. Electrical impedance behavior, mainly capacitance and resistance, of wheat bread at crust and crumb during storage (up to 120 h) is investigated. The variation in capacitance showed the glass transition phenomenon at room temperature in bread crust after 96 h of storage with 18% of moisture in it. The resistance changes at bread crumb showed the starch recrystallization during staling.

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.

Effective Use in Livestock Feeds of Mouldy and Weather-damaged Grain Containing Mycotoxins-Case Histories and Economic Assessments Pertaining to Pig and Poultry Industries of Queensland

Mould growth in field crops or stored grain reduces starch and lipid content, with consequent increases in fibre, and an overall reduction in digestible energy; palatability is often adversely affected. If these factors are allowed for, and mycotoxin concentrations are low, there are sound economic reasons for using this cheaper grain. Mycotoxins are common in stock feed but their effects on animal productivity are usually slight because either the concentration is too low or the animal is tolerant to the toxin. In Australia, aflatoxins occur in peanut by-products and in maize and sorghum if the grain is moist when stored. Zearalenone is found in maize and in sorghum and wheat in wetter regions. Nivalenol and deoxynivalenol are found in maize and wheat but at concentrations that rarely affect pigs, with chickens and cattle being even more tolerant. Other mycotoxins including cyclopiazonic acid, T-2 toxin, cytochalasins and tenuazonic acid are produced by Australian fungi in culture but are not found to be significant grain contaminants. Extremely mouldy sorghum containing Alternaria and Fusarium mycotoxins decreased feed conversion in pigs and chickens by up to 14%. However, E moniliforme- and Diplodia maydis-infected maize produced only slight reductions in feed intake by pigs and Ustilago- infected barley produced no ill effects. Use of these grains would substantially increase profits if the grain can be purchased cheaply.

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.

Population ecology of Heteronyx piceus (Coleoptera: Scarabaeidae) in a peanut/maize cropping system

Large larval populations of the scarabaeid beetle Heteronyx piceus Blanchard that occur under peanuts, but not maize, in the South Burnett region of Australia are the result of a high rate and prolonged period of egg production by females feeding on peanut foliage. Heteronyx piceus is a relatively sedentary species and movement of females between adjacent fields is low. Populations of H. piceus varied markedly with landscape position. High larval populations are more likely (1 in 4 chance) to be encountered on the ‘scrub’ soils in the upper parts of the landscape than in the ‘forest’ soils in the lower half (1 in 20 chance), indicating that soil type/landscape position is a key risk factor in assessing the need for management intervention. The studies indicate that, because of the species' sedentary nature, the most meaningful population entity for management of H. piceus is the individual field, rather than the whole-farm or the region. The implications of this population ecology for management of the pest are discussed in relation to control strategies.

Modelling Leaf Production and Crop Development in Maize (Zea mays L.) After Tassel Initiation Under Diverse Conditions of Temperature and Photoperiod

Prediction of the initiation, appearance and emergence of leaves is critically important to the success of simulation models of crop canopy development and some aspects of crop ontogeny. Data on leaf number and crop ontogeny were collected on five cultivars of maize differing widely in maturity and genetic background grown under natural and extended photoperiods, and planted on seven sowing dates from October 1993 to March 1994 at Gatton, South-east Queensland. The same temperature coefficients were established for crop ontogeny before silking, and the rates of leaf initiation, leaf tip appearance and full leaf expansion, the base, optimum and maximum temperatures for each being 8, 34 and 40 degrees C. After silking, the base temperature for ontogeny was 0 degrees C, but the optimum and maximum temperatures remained unchanged. The rates of leaf initiation, appearance of leaf tips and full leaf expansion varied in a relatively narrow range across sowing times and photoperiod treatments, with average values of 0.040 leaves (degrees Cd)-1, 0.021 leaves (degrees Cd)-1, and 0.019 leaves (degrees Cd)-1, respectively. The relationships developed in this study provided satisfactory predictions of leaf number and crop ontogeny (tassel initiation to silking, emergence to silking and silking to physiological maturity) when assessed using independent data from Gatton (South eastern Queensland), Katherine and Douglas Daly (Northern Territory), Walkamin (North Queensland) and Kununurra (Western Australia).

Short-term responses of leaf growth rate to water deficit scale up to whole-plant and crop levels: An integrated modelling approach in maize.

Physiological and genetic studies of leaf growth often focus on short-term responses, leaving a gap to whole-plant models that predict biomass accumulation, transpiration and yield at crop scale. To bridge this gap, we developed a model that combines an existing model of leaf 6 expansion in response to short-term environmental variations with a model coordinating the development of all leaves of a plant. The latter was based on: (1) rates of leaf initiation, appearance and end of elongation measured in field experiments; and (2) the hypothesis of an independence of the growth between leaves. The resulting whole-plant leaf model was integrated into the generic crop model APSIM which provided dynamic feedback of environmental conditions to the leaf model and allowed simulation of crop growth at canopy level. The model was tested in 12 field situations with contrasting temperature, evaporative demand and soil water status. In observed and simulated data, high evaporative demand reduced leaf area at the whole-plant level, and short water deficits affected only leaves developing during the stress, either visible or still hidden in the whorl. The model adequately simulated whole-plant profiles of leaf area with a single set of parameters that applied to the same hybrid in all experiments. It was also suitable to predict biomass accumulation and yield of a similar hybrid grown in different conditions. This model extends to field conditions existing knowledge of the environmental controls of leaf elongation, and can be used to simulate how their genetic controls flow through to yield.

Fungal colonization of sago starch in Papua New Guinea

Sago starch is an important source of dietary carbohydrates in lowland Papua New Guinea. Over the past 30 years there have been sporadic reports of severe illness following consumption of sago starch. A common assumption is that fungal metabolites might be associated with the illness, leading to the need for a more thorough investigation of the mycoflora of sago starch. Sago starch was collected from areas of high sago consumption in Papua New Guinea for fungal analysis (69 samples). Storage methods and duration were recorded at the time of collection and pH on arrival at the laboratory. Yeasts were isolated from all samples except two, ranging from 1.2 × 103 to 8.3 × 107 cfu/g. Moulds were isolated from 65 of the 69 samples, ranging from 1.0 × 102 to 3.0 × 106 cfu/g. Of 44 samples tested for ergosterol content, 42 samples showed the presence of fungal biomass. Statistical analyses indicated that sago starch stored for greater than five weeks yielded significantly higher ergosterol content and higher numbers of moulds than sago stored for less than five weeks. The method of storage was also shown to influence mould numbers with storage in natural woven fibre containers returning significantly greater numbers than present in other storage methods tested. Potentially mycotoxigenic genera of moulds including Aspergillus and Penicillium were commonly isolated from sago starch, and as such storage factors that influence the growth of these and other filamentous fungi might contribute to the safety of traditional sago starch in PNG.