Temperate Cereals Research Articles

Genes of the RAV Family Control Heading Date and Carpel Development in Rice.

In plants, correct formation of reproductive organs is critical for successful seedset and perpetuation of the species. Plants have evolved different molecular mechanisms to coordinate flower and seed development at the proper time of the year. Among the plant-specific RELATED TO ABI3 AND VP1 (RAV) family of transcription factors, only TEMPRANILLO1 (TEM1) and TEM2 have been shown to affect reproductive development in Arabidopsis (Arabidopsis thaliana). They negatively regulate the floral transition through direct repression of FLOWERING LOCUS T and GIBBERELLIN 3-OXIDASE1/2, encoding major components of the florigen. Here we identify RAV genes from rice (Oryza sativa), and unravel their regulatory roles in key steps of reproductive development. Our data strongly suggest that, like TEMs, OsRAV9/OsTEM1 has a conserved function as a repressor of photoperiodic flowering upstream of the floral activators OsMADS14 and Hd3a, through a mechanism reminiscent of that one underlying floral transition in temperate cereals. Furthermore, OsRAV11 and OsRAV12 may have acquired a new function in the differentiation of the carpel and the control of seed size, acting downstream of floral homeotic factors. Alternatively, this function may have been lost in Arabidopsis. Our data reveal conservation of RAV gene function in the regulation of flowering time in monocotyledonous and dicotyledonous plants, but also unveil roles in the development of rice gynoecium.

A Genome Assembly of the Barley 'Transformation Reference' Cultivar Golden Promise.

Barley (Hordeum vulgare) is one of the most important crops worldwide and is also considered a research model for the large-genome small grain temperate cereals. Despite genomic resources improving all the time, they are limited for the cv. Golden Promise, the most efficient genotype for genetic transformation. We have developed a barley cv. Golden Promise reference assembly integrating Illumina paired-end reads, long mate-pair reads, Dovetail Chicago in vitro proximity ligation libraries and chromosome conformation capture sequencing (Hi-C) libraries into a contiguous reference assembly. The assembled genome of 7 chromosomes and 4.13Gb in size, has a super-scaffold N50 after Chicago libraries of 4.14Mb and contains only 2.2% gaps. Using BUSCO (benchmarking universal single copy orthologous genes) as evaluation the genome assembly contains 95.2% of complete and single copy genes from the plant database. A high-quality Golden Promise reference assembly will be useful and utilized by the whole barley research community but will prove particularly useful for CRISPR-Cas9 experiments.

Ppd-H1 integrates drought stress signals to control spike development and flowering time in barley.

Drought impairs growth and spike development, and is therefore a major cause of yield losses in the temperate cereals barley and wheat. Here, we show that the photoperiod response gene PHOTOPERIOD-H1 (Ppd-H1) interacts with drought stress signals to modulate spike development. We tested the effects of a continuous mild and a transient severe drought stress on developmental timing and spike development in spring barley cultivars with a natural mutation in ppd-H1 and derived introgression lines carrying the wild-type Ppd-H1 allele from wild barley. Mild drought reduced the spikelet number and delayed floral development in spring cultivars but not in the introgression lines with a wild-type Ppd-H1 allele. Similarly, drought-triggered reductions in plant height, and tiller and spike number were more pronounced in the parental lines compared with the introgression lines. Transient severe stress halted growth and floral development; upon rewatering, introgression lines, but not the spring cultivars, accelerated development so that control and stressed plants flowered almost simultaneously. These genetic differences in development were correlated with a differential down-regulation of the flowering promotors FLOWERING LOCUS T1 and the BARLEY MADS-box genes BM3 and BM8. Our findings therefore demonstrate that Ppd-H1 affects developmental plasticity in response to drought in barley.

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.

The contribution of nitrogen fixed by annual legume pastures to the productivity of wheat in two contrasting Mediterranean environments in central Chile

Annual legume pastures exhibit high resilience and productivity in Mediterranean climate regions and can be an important source of nitrogen (N) for temperate cereals in integrated livestock production systems. This work seeks to evaluate the inputs of N fixed by annual legume mixtures (composed of 3–4 species and cultivars) grown for one, two or three years in a cropping sequence, and their contribution to soil N availability, N uptake and the grain yield of wheat, and compares this with the cereal rotation of oats and wheat with and without N fertilizer. Two field experiments comprising annual legume-wheat rotations were carried out in two contrasting Mediterranean environments in central Chile, the interior dryland in the eastern part of the coastal mountain rage, and the Andean foothills, over four growing seasons (2008–2011). At both experimental sites two mixtures of annual legumes were used in rotation with wheat. The duration of the pasture phase was between one and three years. In the interior dryland, the shoot DM of mixture M1 (T. subterraneum + M. polymorpha + T. michelianum) was higher than M2 (T. subterraneum + B. penicilius + O. compressus), and in the Andean foothills M4 (T. subterraneum + T. vesiculosum + O. compressus) was more productive than M3 (two cv. of T. subterraneum + T. incarnatum). The amount of N2 fixed by the legume mixtures, estimated by the 15N natural abundance technique, ranged between 28 and 87 kg N ha−1 in the interior dryland, and between 176 and 385 kg N ha−1 in the Andean foothills, representing respectively around 25 and 23 kg N ha−1 fixed for every Mg of shoot DM accumulated by the annual legumes. Wheat grain yields following legume mixtures represented 53–104 % of the 3.39 Mg ha−1 produced after oats with N fertilizer in the interior dryland, and between 68 and 101 % of 6 Mg ha−1 attained after oats in the Andean foothills, in 2011. N use efficiency (NUE) of wheat was significantly (P ≤ 0.05) higher in legume-based crop rotation than after oats with or without N fertilizer, at both sites. Legume mixtures also improved the N uptake (NUPE) and N utilization (NUtE) efficiencies of wheat compared to those observed after oats with N fertilizer, at both sites. Important differences among legume mixtures and the duration of the pasture phase were observed at both sites.

Progressive refinement of the karyotyping of Brachypodium genomes.

Brachypodium distachyon is a weedy grass species that is firmly established as a model for the comparative and functional genomics of temperate cereals and grasses. Its simple, nuclear genome of five chromosomes contrasts it with other relatives of the genus with different, and usually higher, basic chromosome numbers and ploidy levels. This variation in karyotypic structure affords the possibility of reconstructing evolutionary pathways that have shaped the genome structure of extant species. This Tansley insight documents how key refinements in molecular cytogenetic approaches, from simple fluorescence in situ hybridization to comparative chromosome barcoding, have enabled genome structure studies and yielded valuable information about the drivers of karyotypic reorganization and evolution in the model grass genus Brachypodium.

The Effect of Ambient Temperature on Brachypodium distachyon Development.

Due to climate change, the effect of temperature on crops has become a global concern. It has been reported that minor changes in temperature can cause large decreases in crop yield. While not a crop, the model Brachypodium distachyon can help to efficiently investigate ambient temperature responses of temperate grasses, which include wheat and barley. Here, we use different accessions to explore the effect of ambient temperature on Brachypodium phenology. We recorded leaf initiation, heading time, leaf and branch number at heading, seed set time, seed weight, seed size, seed dormancy, and seed germination at different temperatures. We found that warmer temperatures promote leaf initiation so that leaf number at heading is positively correlated to temperature. Heading time is not correlated to temperature but accessions show an optimal temperature at which heading is earliest. Cool temperatures prolong seed maturation which increases seed weight. The progeny seeds of plants grown at these cool ambient temperatures show stronger dormancy, while imbibition of seeds at low temperature improves germination. Among all developmental stages, it is the duration of seed maturation that is most sensitive to temperature. The results we found reveal that temperature responses in Brachypodium are highly conserved with temperate cereals, which makes Brachypodium a good model to explore temperature responsive pathways in temperate grasses.

Cas Endonuclease Technology-A Quantum Leap in the Advancement of Barley and Wheat Genetic Engineering.

Domestication and breeding have created productive crops that are adapted to the climatic conditions of their growing regions. Initially, this process solely relied on the frequent occurrence of spontaneous mutations and the recombination of resultant gene variants. Later, treatments with ionizing radiation or mutagenic chemicals facilitated dramatically increased mutation rates, which remarkably extended the genetic diversity of crop plants. However, a major drawback of conventionally induced mutagenesis is that genetic alterations occur simultaneously across the whole genome and at very high numbers per individual plant. By contrast, the newly emerging Cas endonuclease technology allows for the induction of mutations at user-defined positions in the plant genome. In fundamental and breeding-oriented research, this opens up unprecedented opportunities for the elucidation of gene functions and the targeted improvement of plant performance. This review covers historical aspects of the development of customizable endonucleases, information on the mechanisms of targeted genome modification, as well as hitherto reported applications of Cas endonuclease technology in barley and wheat that are the agronomically most important members of the temperate cereals. Finally, current trends in the further development of this technology and some ensuing future opportunities for research and biotechnological application are presented.

An Efficient System for Ds Transposon Tagging in Brachypodium distachyon.

Transposon tagging is a powerful tool that has been widely applied in several species for insertional mutagenesis in plants. Several efforts have aimed to create transfer-DNA (T-DNA) insertional mutant populations in Brachypodium distachyon, a monocot plant used as a model system to study temperate cereals, but there has been a lack of research aimed at using transposon strategies. Here, we describe the application of a maize (Zea mays) Dissociation (Ds) transposon tagging system in B distachyon The 35S::AcTPase cassette and Ds element were constructed within the same T-DNA and transformed into B distachyon plants. The Ds element was readily transposed to other chromosomes or to the same chromosome under the function of Activator (Ac) transposase. Through homologous chromosome synapsis, recombination, and segregation, the Ds element separated from the Ac element. We selected stable Ds-only plants using G418 and GFP assays and analyzed 241 T0 lines, some of which were highly efficient at producing Ds-only progeny. Through thermal asymmetric interlaced PCR, we isolated 710 independent Ds flanking sequences from Ds-only plants. Furthermore, we identified a large collection of mutants with visible developmental phenotypes via this transposon tagging system. The system is relatively simple and rapid in comparison to traditional T-DNA insertion strategies, because once efficiency lines are obtained they can be reused to generate more lines from nontransposed plants without the use of time-consuming tissue culture steps.

Genome size variation among natural populations of Brachypodium distachyon and B. hybridum collected from different regions of Turkey

Brachypodium distachyon (L.) P.Beauv. is a model grass species that is useful for studying temperate cereal, forage, and energy crops. In this study we aimed to determine the genome size and species identity in the collections of the B. distachyon complex that included samples from 56 different locations in Turkey, applying chromosome counting and nuclear genome size evaluation by flow cytometry method (FCM). Moreover, the study examined whether diverse geographical conditions at the collecting sites influence the genome size. Chromosomal analysis revealed that the number of B. distachyon (x = 5, 2n = 10) genotypes was 48 and the number of B. hybridum (x = 5 + 10, 2n = 30) ones was 11. No genotypes of B. stacei were found. FCM analyses revealed that the minimum and maximum genome size of B. distachyon was 0.732 and 0.752 pg2C?1, respectively. The mean genome size of B. distachyon was 0.743 pg2C?1. The mean genome size of B. hybridum was 1.431 pg2C?1 with the minimum 1.417 pg2C?1 and maximum 1.451 pg2C?1. Intraspecific variation in the genome size was identified for both species. It was determined that geographical origin (localization, altitude) had a statistically significant effect on genome size in B. distachyon. In addition, a negative correlation was found between altitude and genome size in this species. On the other hand, localization and altitude did not have any statistically significant effect on genome size in B. hybridum.

Beyond heading time: FT-like genes and spike development in cereals.

This article comments on: Shaw LM, Lyu B, Turner R, Li C, Chen F, Han X, Fu D, Dubcovsky J. 2018. FLOWERING LOCUS T2 regulates spike development and fertility in temperate cereals. Journal of Experimental Botany 70, 193–204 .

Identification of novel C-repeat binding factor (CBF) genes in rye (Secale cereale L.) and expression studies

Although rye is one of the most cold-tolerant species among temperate cereals, its huge and complex genome has prevented us from identifying agronomically useful genes. However, advances in high-throughput sequencing technology are making it increasingly possible to investigate its genome. The C-repeat binding factor (CBF) gene family controls cold tolerance in plants and its members are well conserved among eudicots and monocots, among which there are diverse homologs. Despite its large genome, only a small number of CBF genes have been identified in rye. In this study, we explored high-throughput sequencing data of the rye genome and identified 12 novel CBF genes. Sequence analyses revealed that these genes contain signature sequences of the CBF family. Chromosomal localization of the genes by PCR using wheat–rye addition lines showed that most of these are located on the long arm of chromosome 5, but also on the long arm of chromosomes 2 and 6. On the basis of comparative analyses of CBF family members in the Triticeae, CBF proteins were divided into several groups according to phylogenetic relationship and conserved motifs. Light is essential to fully induce CBF gene expression and there is specificity in the response to different types of abiotic stresses in ScCBF genes. The results of our study will assist investigations of CBF genes in the Triticeae and the mechanism of cold tolerance through the CBF-dependent pathway in plants.

FLOWERING LOCUS T2 regulates spike development and fertility in temperate cereals.

FLOWERING LOCUS T2 (FT2) is the closest paralog of the FT1 flowering gene in the temperate grasses. Here we show that overexpression of FT2 in Brachypodium distachyon and barley results in precocious flowering and reduced spikelet number, while down-regulation by RNA interference results in delayed flowering and a reduced percentage of filled florets. Similarly, truncation mutations of FT2 homeologs in tetraploid wheat delayed flowering (2-4 d) and reduced fertility. The wheat ft2 mutants also showed a significant increase in the number of spikelets per spike, with a longer spike development period potentially contributing to the delayed heading time. In the wheat leaves, FT2 was expressed later than FT1, suggesting a relatively smaller role for FT2 in the initiation of the reproductive phase. FT2 transcripts were detected in the shoot apical meristem and increased during early spike development. Transversal sections of the developing spike showed the highest FT2 transcript levels in the distal part, where new spikelets are formed. Our results suggest that, in wheat, FT2 plays an important role in spike development and fertility and a limited role in the timing of the transition between the vegetative and reproductive shoot apical meristem.

RNAi-suppression of barley caffeic acid O-methyltransferase modifies lignin despite redundancy in the gene family.

Summary Caffeic acid O‐methyltransferase (COMT), the lignin biosynthesis gene modified in many brown‐midrib high‐digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl‐to‐guaiacyl ratio was reduced by ~50%, the 5‐hydroxyguaiacyl (5‐OH‐G) unit incorporated into lignin at 10‐–15‐fold higher levels than normal, and the amount of p‐coumaric acid ester‐linked to cell walls was reduced by ~50%. No brown‐midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown‐midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.

Dissecting the chromosomal composition of mutagen-induced micronuclei in Brachypodium distachyon using multicolour FISH.

Background and Aims Brachypodium distachyon (Brachypodium) is a model species for temperate cereals and other economically important grasses. Its favourable cytogenetic features and advanced molecular infrastructure make it a good model for understanding the mechanisms of instability of plant genomes after mutagenic treatment. The aim of this study was to qualitatively and quantitatively assess the composition and origin of micronuclei arising from genomic fracture, and to detect possible ‘hot spots’ for mutagen-induced DNA breaks.MethodsSeeds of Brachypodium were treated with maleic hydrazide (MH) or X-rays. The structure of mutagen-induced micronuclei was analysed in root-tip meristematic cells using multicolour fluorescence in situ hybridization (mcFISH) with various repetitive (5S rDNA, 25S rDNA, telomeric, centromeric) and low-repeat [small and large pools of bacterial artificial chromosome (BAC) clones specific for chromosome Bd1] DNA sequences.Key ResultsThe majority of micronuclei derive from large, acentric fragments. X-rays caused more interstitial DNA breaks than MH. Double-strand breaks rarely occurred in distal chromosome regions. Bd1 contributed to the formation of more mutagen-induced micronuclei than expected from random chromosome involvement.ConclusionsmcFISH with chromosome-specific BAC clones offers insight into micronuclei composition, in so far as it allows their origin and formation to be determined more specifically. A reliable assay for micronuclei composition is crucial for the development of modern genotoxicity tests using plant cells. The combination of mutagenic treatments and well-developed cytomolecular resources in Brachypodium make this model species very promising for plant mutagenesis research.

Expression of a rice soluble starch synthase gene in transgenic wheat improves the grain yield under heat stress conditions

Wheat (Triticum aestivum L.) is a temperate cereal with an optimum temperature range of 15–22°C during the grain filling stage. Heat stress is one of the major environmental constraints for wheat production worldwide. Temperatures above 25°C during the grain filling stage significantly reduced wheat yield and quality. This reduction was reported due to the inactivation of the soluble starch synthase, a key heat-labile enzyme in starch transformation of wheat endosperm. To improve wheat productivity under heat stress, the rice soluble starch synthase I, under the control of either a constitutive promoter or an endosperm-specific promoter, was expressed in wheat and the transgenic lines were monitored for expression and the effects on yield-related traits. The results showed that the transgenic wheat events expressed rice soluble starch synthase I at a high level after four generations, and transgenic plants produced grains of greater weight during heat stress. Under heat stress conditions, the thousand kernel weight increased 21–34% in T2 and T3 transgenic plants compared to the non-transgenic control plants. In addition, the photosynthetic duration of transgenic wheat was longer than in non-transgenic controls. This study demonstrated that the engineering of a heat tolerant soluble starch synthase gene can be a potential strategy to improve wheat yield under heat stress conditions.

Interacting Environmental Stress Factors Affects Targeted Metabolomic Profiles in Stored Natural Wheat and That Inoculated with F. graminearum.

Changes in environmental stress impact on secondary metabolite (SM) production profiles. Few studies have examined targeted SM production patterns in relation to interacting environmental conditions in stored cereals. The objectives were to examine the effect of water activity (aw; 0.95–0.90) x temperature (10–25 °C) on SM production on naturally contaminated stored wheat and that inoculated with Fusarium graminearum. Samples were analysed using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) on (a) total number of known SMs, (b) their concentrations and (c) changes under environmental stress. 24 Fusarium metabolites were quantified. Interestingly, statistical differences (ChisSq., p < 0.001) were observed in the number of SMs produced under different sets of interacting environmental conditions. The dominant metabolites in natural stored grain were deoxynivalenol (DON) and nivalenol (NIV) followed by a range of enniatins (A, A1, B, B1), apicidin and DON-3-glucoside at 10 °C. Increasing temperature promoted the biosynthesis of other SMs such as aurofusarin, moniliformin, zearalenone (ZEN) and their derivatives. Natural wheat + F. graminearum inoculation resulted in a significant increase in the number of metabolites produced (ChisSq., p < 0.001). For ZEN and its derivatives, more was produced under cooler storage conditions. Fusarin C was enhanced in contrast to that for the enniatin group. The relative ratios of certain groups of targeted SM changed with environmental stress. Both temperature and aw affected the amounts of metabolites present, especially of DON and ZEN. This study suggests that the dominant SMs produced in stored temperate cereals are the mycotoxins for which legislation exists. However, there are changes in the ratios of key metabolites which could influence the relative contamination with individual compounds. Thus, in the future, under more extreme environmental stresses, different dominant SMs may be formed which could make present legislation out of step with the future contamination which might occur.

Natural variation and genetic make-up of leaf blade area in spring barley

Key messageGWAS analysis for leaf blade area (LA) revealed intriguing genomic regions associated with putatively novel QTL and known plant stature-related phytohormone and sugar-related genes.Despite long-standing studies in the morpho-physiological characters of leaf blade area (LA) in cereal crops, advanced genetic studies to explore its natural variation are lacking. The importance of modifying LA in improving cereal grain yield and the genes controlling leaf traits have been well studied in rice but not in temperate cereals. To better understand the natural genetic variation of LA at four developmental stages, main culm LA was measured from 215 worldwide spring barleys including 92 photoperiod-sensitive accessions [PHOTOPERIOD RESPONSE LOCUS 1 (Ppd-H1)] and 123 accessions with reduced photoperiod sensitivity (ppd-H1) locus under controlled greenhouse conditions (long-day; 16/8 h; ~ 20/~ 16 °C day/night). The LA of Ppd-H1-carrying accessions was always smaller than in ppd-H1-carrying accessions. We found that nine SNPs from the Ppd-H1 gene were present in the collection of which marker 9 (M9; G/T in the CCT-domain) showed the most significant and consistent effect on LA at all studied developmental stages. Genome-wide association scans (GWAS) showed that the accessions carrying the ppd-H1 allele T/M9 (late heading) possessed more genetic variation in LA than the Ppd-H1 group carrying G/M9 (early heading). Several QTL with major effects on LA variation were found close to plant stature-related heading time, phytohormone- and sugar-related genes. The results provide evidence that natural variation of LA is an important source for improving grain yield, adaptation and canopy architecture of temperate cereals.

SeedStor: A Germplasm Information Management System and Public Database.

SeedStor (https://www.seedstor.ac.uk) acts as the publicly available database for the seed collections held by the Germplasm Resources Unit (GRU) based at the John Innes Centre, Norwich, UK. The GRU is a national capability supported by the Biotechnology and Biological Sciences Research Council (BBSRC). The GRU curates germplasm collections of a range of temperate cereal, legume and Brassica crops and their associated wild relatives, as well as precise genetic stocks, near-isogenic lines and mapping populations. With >35,000 accessions, the GRU forms part of the UK’s plant conservation contribution to the Multilateral System (MLS) of the International Treaty for Plant Genetic Resources for Food and Agriculture (ITPGRFA) for wheat, barley, oat and pea. SeedStor is a fully searchable system that allows our various collections to be browsed species by species through to complicated multipart phenotype criteria-driven queries. The results from these searches can be downloaded for later analysis or used to order germplasm via our shopping cart. The user community for SeedStor is the plant science research community, plant breeders, specialist growers, hobby farmers and amateur gardeners, and educationalists. Furthermore, SeedStor is much more than a database; it has been developed to act internally as a Germplasm Information Management System that allows team members to track and process germplasm requests, determine regeneration priorities, handle cost recovery and Material Transfer Agreement paperwork, manage the Seed Store holdings and easily report on a wide range of the aforementioned tasks.

Comparison of dry matter losses and aflatoxin B1 contamination of paddy and brown rice stored naturally or after inoculation with Aspergillus flavus at different environmental conditions

The objective of this study was to compare the effect of different storage moisture conditions (0.70, 0.85, 0.90 and 0.95 water activity, aw) and temperatures (20, 25, 30 °C) on (a) respiration rates (R) and dry matter loss (DML) of paddy and brown rice and (b) quantify aflatoxin B1 (AFB1) production by isolates of Aspergillus flavus from the rice samples and (c) inoculation of both rice types with A. flavus under these storage conditions on R, DML and AFB1 contamination. There was an increase in temporal CO2 production with wetter and warmer conditions in naturally contaminated rice. Higher R and consequently, % DML, were generally found in the brown rice (21%) while in paddy rice this was only up to 3.5% DML. From both rice types, 15 (83.3%) of 18 A. flavus isolates produced detectable levels of AFB1 in a range 2.5–1979.6 μg/kg. There was an increase in DML in both rice types inoculated with A. flavus as temperature and aw were increased. Interestingly very little AFB1 was detected in paddy rice, but significant contamination occurred in the brown rice. The %DML in the control and A. flavus inoculated rice increased with temperature and aw at both 25 and 30 °C from 1-2% to 15–20% DML at 30 °C and 0.95 aw. All the inoculated rice samples had AFB1 levels above the EU legislative limits for contamination in other temperate cereals and products derived from cereals (=2 μg/kg). Even samples with % DML as low as 0.2% had AFB1 contamination levels twice the limits for other cereals. These results suggest that the mycotoxin contamination risk in staple commodities like rice, is influenced by whether the rice is processed or not, and that measurement of R rates can be used to predict the relative risk of AFB1 contamination in such staple commodities.