Differential expression of defence-related proteins in Vigna unguiculata (L. Walp.) seedlings after infection with Fusarium oxysporum

This work aimed to study the induction of defence proteins in cowpea seedlings during the first days after infection with the fungi Fusarium oxysporum f. sp. cubenses and F. oxysporum f. sp. phaseoli. Cowpea seeds, after disinfection, were transferred to Petri dishes containing 0.5% agar and, after germination, were infected with a drop of a suspension containing 0.5 × 10⁴ spores ml⁻¹. Seedlings were collected at 24, 48, 72 and 96 h after infection and were dissected into leaves, hypocotyls, roots, cotyledons and teguments, which were measured and weighed for morphometric analysis. The agar medium was also analyzed. Enzymatic assays of proteic extracts yielded antimicrobial peptides detected by Western blotting. The cowpea seedlings showed a complex pattern of induction and repression of defence proteins in response to infection by both pathogens. Furthermore, morphometric analysis showed differences between infected and control seedlings. Infected samples did not at any time exhibit chitinase activity, but did exhibit different β-1,3-glucanase and peroxidase activities. Western blotting for lipid transfer protein (LTP) demonstrated its presence in all parts of the infected seedlings. Exuded proteins, also obtained from cowpea seeds in the germination medium, were separated by SDS-PAGE and tricine gel electrophoresis. The analysis showed that some proteins were exuded from moistened cowpea seeds, particularly after F. oxysporum infection.     

Effect of pre-emergence herbicide mixtures on cowpea (Vigna unguiculata (L.) Walp) at Samaru, in northern Nigeria

Field experiments were conducted in the wet seasons of 2005 and 2006 to evaluate the effect of pre-emergence application of herbicides on cowpea. A mixture of metolachlor and prometryn at 1.25+0.08 kg a.i./ha was consistent in better growth and seed yield. However, application of mixtures of metolachlor and terbutryn at 0.99+0.50 kg a.i./ha and ametryn and terbutryn at 1.60+1.00 kg a.i./ha gave lower vigour score of the crop, higher crop injury, smaller canopy spread and lower seed yield. It can be concluded that the metolachlor and prometryn mixture gave good selective weed control and can be used instead of hand weeding in cowpea in northern Nigeria.     

Conversion into bioethanol of insect (Sitophilus zeamais Motschulsky), mold (Aspergillus flavus Link) and sprout-damaged maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench)

The bioconversion into ethanol of insect (Sitophilus zeamais), mold (Aspergillus flavus) and sprout-damaged maize and sorghum was investigated. Kernel test weight losses due to insect damage in maize were almost twice compared to sorghum (18.6 vs. 10.7%). All damaged kernels lost some of the starch and increased soluble sugars, ash and crude fiber. The mold-damaged sorghum contained approximately five times more FAN compared to the control. The sprout-damaged kernels contained the highest amounts of reducing sugars prior (11 g/L) to and at the end (146.5 g/L) of liquefaction with α-amylase. Ethanol yields based on the already damaged grain indicated that sprout-damaged kernels yielded similar amounts compared to sound kernels (381.1 vs. 382.6 L/ton and 376.6 vs. 374.8 L/ton of sorghum or maize respectively). The insect-damaged maize and sorghum have reduced ethanol yields compared with the controls (29 and 23% respectively), and this negative result was mainly due to dry matter losses during the inadequate storage. Despite differences in ethanol yield, all treatments have similar conversion efficiencies (76.1–89.9%) indicating the robustness of yeast facing biotic-damaged feedstocks. This research demonstrates that the use of already damaged insect, mold or sprouted kernels is feasible and a good alternative for biorefineries.     

Effects of heat on the removal of polyphenols and in vitro protein digestibility of cowpea (Vigna unguiculata (L.) Walp.)

Simple wet heat treatments like simple boiling (atmospheric pressure, 100 °C) and pressurized boiling (higher than 100 °C) reduced the polyphenol content of mature dark red seeds of cowpea (Vigna unguiculata (L.) Walp.) cultivar UPL Cp 3 by 61 to 80% and improved in vitro protein digestibility (IVPD) by 6 to 26%. Pressurized steaming (higher than 100 °C) removed 48 to 83% of the polyphenols but increased IVPD by only 1.1 to 4.2%. Dry heat as exemplified by roasting and microwave treatment inactivated 58 to 71% of the tannins but increased IVPD by only 1%. All the heat treatments were effective in removing/inactivating polyphenols though different responses were observed with the resulting in vitro protein digestibilities.     

Analysis of host plant resistance to multiple Fusarium species associated with stalk rot disease in sorghum [Sorghum bicolor (L.) Moench]

Fusarium spp. is among the largest and most important pathogen groups that attack major grain crops including sorghum. The objectives of this study were to compare the virulence of Fusarium spp. associated with sorghum and examine the mode of resistance of genotypes to the disease. Eight Fusarium species – F. verticillioides, F. thapsinum, F. andiyazi, F. proliferatum, F. nyagamai, F. pseudoanthophilum, F. brevicatenulatum, and F. pseudonygamai – were studied using three sorghum genotypes under greenhouse condition. Three of these pathogens (F. verticillioides, F. thapsinum, and F. andiyazi) were selected for genetic analysis of resistance under field conditions. Sorghum genotypes with contrasting stalk rot reactions were selected for use in both the greenhouse and field experiments. Two weeks after flowering, plants were inoculated with liquid inoculum culture (5 × 10⁴ conidia ml⁻¹) of the different pathogens. Plants were harvested 4 weeks after inoculation and rated for disease severity on the basis of lesion length and number of nodes crossed by the lesion. Among the pathogens, F. thapsinum resulted in consistently higher disease scores in all genotypes under all environments. Likewise, genotype SC599 showed the greatest and most stable resistance as inbred as well as in hybrid combinations as shown by consistently high GCA for resistance to all pathogen species. We recommend that future screening exercises for Fusarium stalk rot resistance utilize F. thapsinum as the causal organism and include the resistant genotype SC599 as a control.     

Timing and extent of damage caused by insect pests of green gram (Vigna radiata L.) in Bangladesh

The occurrence and abundance of green gram pests were studied during the crop season of 1982. There were six Lepidoptera, four Coleoptera, two Hemiptera, two Orthoptera and one Diptera, of which the Lepidopteran Spilosoma obliqua Walk. was the most serious. Thysanoplusia orichalcea Fabr., Spodoptera litura Fabr. and Apogonia spp. were abundant during September and October, whereas S. obliqua and Ophiomyia phaseoli Coq. were represented most in October. Etiella zinckenella Treit. was abundant during the middle of October to the second week of November. Larvae of all instars of all six Lepidopteran pests were collected in the field, except for T. orichalcea and Manduca spp. with larvae of only the third and fifth instars. No larvae were found in the field of other orders of insect pests, except O.phaseoli and Epilachna dodecastigma Muls. The fifth- and sixth-instar larvae consumed more leaf material than did those of the first four instars. Larval damage potential in terms of leaf area consumed was greatest in Manduca spp. and lowest in Lamprosema diemenalis Guen. whereas Apogonia spp. were recorded as eating the greatest leaf area among adult Coleopteran pests. The natural enemies were also recorded in case of S. obliqua and L. diemenalis. Data on the biology of some of the insect pests are given and discussed briefly.     

Isolation and identification of phase II enzyme inductors obtained from black Shawaya sorghum [Sorghum bicolor (L.) Moench] bran

The antioxidant capacity as oxygen radical absorbance capacity (ORAC) and in vitro chemopreventive effect of phytochemicals extracted, partitioned and isolated from the decorticated bran of black Shawaya sorghum (Sorghum bicolor L. Moench) was studied. The chemopreventive effect was evaluated as the induction effect of quinone reductase (QR) using murine hepatoma cells Hepa 1c1c7. The crude methanol extract with an antioxidant capacity of 3.7 μmol Trolox equivalent (TE)/mg was partitioned into 120 fractions with fast centrifugal partition chromatography (FCPC). Fractions with partition coefficients between 0.1 and 0.26 exerted more than 50% of the antioxidant activity of the original crude extract and contained the highest concentration of anthocyanins. No correlation was found for ferulic acid content and ORAC or chemoprevention. A derivative of 7-methoxyapigeninidin at a concentration of 1 μg/ml increased by 31% the QR activity while the original extract reached a maximum of 27% at 40 μg/ml.     

Potential of extracts of the tropical plant Balanites aegyptiaca (L) Del. (Balanitaceae) to control the mealy bug, Maconellicoccus hirsutus (Homoptera: Pseudococcidae)

The effects of extracts of different parts of the perennial tropical plant Balanites aegyptiaca (L) Del., including various solvent extracts of roots, methanol extracts from leaves, fruits, flowers and roots, partially purified saponins obtained from its roots and a standard saponin were studied on the life cycle (adult longevity, number of eggs, crawlers, adults, weight of adults and % wax content) of a laboratory-reared parthenogenic line of the mealy bug, Maconellicoccus hirsutus (Homoptera: Pseudococcidae). Extracts derived from various parts of B. aegyptiaca (leaves, fruits, flowers, and roots in methanol) affected the life cycle of M. hirsutus with a methanol root extract being the most effective at a concentration of 500 μg ml⁻¹. Partially purified saponin of B. aegyptiaca and the commercial bark saponin extract (Sigma) from Quillaja saponaria at a concentration of 500 μg ml⁻¹ were effective in reducing the longevity of M. hirsutus. Significant reductions in oviposition by M. hirsutus were found for all the extracts at a concentration of 500 μg ml⁻¹. Extracts also affected the number of emerging crawlers, number of adults as well as the weight and wax content of emerging adults. These studies suggest that B. aegyptiaca plant extracts and saponins can be useful botanical insecticides for the protection of crops from mealy bugs.     

Storage-induced changes in einkorn (Triticum monococcum L.) and breadwheat (Triticum aestivum L. ssp. aestivum) flours

To assess the effect of ageing on alpha-amylase activity, falling number, pasting properties and SDS sedimentation volume, whole meal and white flours of einkorn (cv Monlis) and bread wheat (cv Serio) were stored in darkness at different temperatures and analysed several times up to 374 days. Pregerminated bread wheat flours (cv Blasco) were also evaluated.     

Evaluation of genetic diversity in turmeric (Curcuma longa L.) using RAPD and ISSR markers

Turmeric (Curcuma longa L.) is an industrially important plant used for production of curcumin, oleoresin and essential oil. In the present study we examined the genetic diversity among turmeric accessions from 10 different agro-climatic regions comprising 5 cultivars and 55 accessions. Two DNA-based molecular marker techniques, viz., random amplified polymorphism DNA (RAPD) and inter simple sequence repeat (ISSR) were used to assess the genetic diversity in turmeric genotypes. A total of 17 polymorphic primers (11 RAPDs and 6 ISSRs) were used in this study. RAPD analysis of 60 genotypes yielded 94 fragments of which 75 were polymorphic with an average of 6.83 polymorphic fragments per primer. Number of amplified fragments with RAPD primers ranged from 3 to 13 with the size of amplicons ranging from 230 to 3000 bp in size. The polymorphism ranged from 45 to 100 with an average of 91.4%. The 6 ISSR primers produced 66 bands across 60 genotypes of which 52 were polymorphic with an average of 8.6 polymorphic fragments per primer. The number of amplified bands varied from 1 to 14 with size of amplicons ranging from 200 to 2000 bp. The percentage of polymorphism using ISSR primers ranged from 83 to 100 with an average of 95.4%. Nei's dendrogram for 60 samples using both RAPD and ISSR markers demonstrated an extent of 62% correlation between the genetic similarity and geographical location. The result of Nei's genetic diversity (H) generated from the POP gene analysis shows relatively low genetic diversity in turmeric accessions of South eastern ghat (P7), Western undulating zone (P8) with 0.181 and 0.199 value whereas highest genetic diversity (0.257) has been observed in Western central table land (P9). Knowledge on the genetic diversity of turmeric from different agro-climatic regions can be used to future breeding programs for increased curcumin, oleoresin and essential oil production to meet the ever-increasing demand of turmeric for industrial and pharmaceutical uses.     

Modeling the impacts of contrasting nutrient and residue management practices on grain yield of sorghum (Sorghum bicolor (L.) Moench) in a semi-arid region of Ghana using APSIM

The cropping systems model APSIM (Agricultural Production Systems sIMulator) was applied to assess the response of sorghum grain yield to inorganic fertilizers applications and residue retention in diverse farmers’ management systems (homestead fields and bush farms). The model was parameterized using data collected from experiments under optimum growth conditions (limited water or nutrient stress). Independent data from field experiments with three levels of P and four levels of N fertilizers conducted at two different locations and soils were used to evaluate the model. Soil water and fertility parameters measured were used for simulations while same starting conditions were assumed for unmeasured parameters for all trials. APSIM predicted the grain yield response of sorghum to both N and P applications with an overall modified internal coefficient of efficiency of 0.64. Following model parameterization, a long-term simulation study was conducted using a stochastic weather data derived from historical weather data to assess the effects of crop residue management on grain production. A gradual decline in sorghum grain yield was simulated over the 30-year simulation period in both the homestead fields and the bush farms, with yields being much lower in the latter under farmers’ management practices. Half the amount of mineral N fertilizer used in the bush farms was needed in the homestead fields to produce the average grain yields produced on the bush farm with full fertilization, if crop residues were returned to the fields in the homestead. Year-to-year variability in grain yield was consistently higher with the removal of crop residues, irrespective of management systems. APSIM was responsive to both organic and inorganic fertilizer applications in the study area and also highlighted the essential role of crop residues and inorganic fertilizer in influencing the temporal sorghum grain production and hence the impact of farmers’ management practices on food security. This was evident in the rapid decline in soil organic carbon (SOC) accompanied by a decline in grain yield over the 30 years of cropping. The use of inorganic fertilizer and retention of crop residues (SOC) are critical for attaining food security in the study area.     

Development of STS markers and establishment of multiplex PCR for Glu-A3 alleles in common wheat (Triticum aestivum L.)

Low-molecular-weight glutenin subunits (LMW-GS) play a key role in determining the processing quality of the end-use products of common wheat. The objectives of this study were to identify genes at Glu-A3 locus, develop the STS markers, and establish multiplex PCR with the STS markers for Glu-A3 alleles. Gene-specific PCR primers were designed to amplify six near-isogenic lines (NILs) and Glenlea with different Glu-A3 alleles (a, b, c, d, e, f and g) defined by the protein electrophoretic mobility. Three Glu-A3 genes with complete coding sequence were cloned, designated as GluA3-1, GluA3-2 and GluA3-3, respectively. Seven dominant allele-specific STS (sequence tagged sites) markers were designed based on the SNPs (single nucleotide polymorphisms) among different allelic variants for the discrimination of the Glu-A3 protein alleles a, b, c, d, e, f and g. Four multiplex PCRs were established including Glu-A3b + Glu-A3f, Glu-A3d + Glu-A3f, Glu-A3d + Glu-A3g, and Glu-A3b + Glu-A3e. These markers and multiplex-PCR systems were validated on 141 CIMMYT wheat varieties and advanced lines with different Glu-A3 alleles, confirming that they can be efficiently used in marker-assisted breeding.     

Development of simple and co-dominant PCR markers to genotype puroindoline a and b alleles for grain hardness in bread wheat (Triticum aestivum L.)

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.). A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. So far, nine Pina alleles (Pina-D1a–Pina-D1b, Pina-D1k–Pina-D1q) and seventeen Pinb alleles (Pinb-D1a–Pinb-D1g, Pinb-D1p–Pinb-D1ab) have been identified in bread wheat. The major Pina and Pinb alleles identified in hard wheat cultivars are Pina-D1b, Pinb-D1b, Pinb-D1c and Pinb-D1d. In this study, a three-primer PCR system was employed to develop nine co-dominant STS markers for genotyping Pina-D1a and Pina-D1b, whereas temperature-switch (TS) PCR was used to develop six co-dominant SNP markers for genotyping the Pinb-D1a, Pinb-D1b, Pinb-D1c and Pinb-D1d alleles. These STS and TS-PCR markers were used to verify the grain hardness genotype of 100 wheat cultivars. The reliability and genotyping accuracy of TS-PCR markers were confirmed through sequencing of PCR products and a comparison with previously published results. Therefore, STS and TS-PCR markers offer a simple, cost-effective and reliable method for high-throughput genotyping Pina and Pinb alleles to select grain hardness in wheat quality breeding programs and for wheat market classification.     

The usefulness of iron bioavailability as a target trait for breeding maize (Zea mays L.) with enhanced nutritional value

Iron (Fe) deficiency is the most widespread nutritional problem, affecting as many as half of the world's population. Only a small fraction (2-15%) of Fe from plant sources is typically bioavailable, that is, available for absorption and nutritionally useful for humans. This study evaluated Fe concentration and bioavailability for three diverse sets of 12, 14 and 16 maize hybrids grown in two- or three-location trials to assess the feasibility of selecting for Fe bioavailability in breeding programs. Bioavailability of Fe, assessed using the in vitro digestion/Caco-2 cell model, varied significantly among hybrids in two of the three trials. Location effects were larger than location by genotype interaction effects, additive but not non-additive gene action was significant, and heritability estimates were mostly between 0.55 and 0.65 for Fe bioavailability estimators. Bioavailability of Fe was not associated with Fe concentration in grain or with grain yield. Negative correlation of Fe bioavailability with zinc concentration in grain for one of the three hybrid trials, and positive correlation with provitamin A concentrations in one trial were indicative of inhibitor and enhancer effects on Fe bioavailability, respectively. Although use of the Caco-2 cell model is promising, particularly because it integrates the whole meal, or food matrix effect on Fe bioavailability, the complex nature of the assay and moderate heritability of bioavailability estimators make it most suitable as an intermediate selection tool, following high throughput selection for molecular markers of Fe bioavailability, currently in development by other researchers, and preceding validation and efficacy trials with animal and human models.     

Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal

Calli were obtained from leaf, cotyledon and internode explants of in vitro-grown plants of Indian cultivar of Withania somnifera in MS medium supplemented with 2, 4-D (2.0 mg l⁻¹) and Kinetin (0.2 mg l⁻¹). The brown, semi-friable callus (500 mg FW) derived from leaf explants produced higher number of primary adventitious roots (9 roots/callus) in half strength MS medium fortified with IBA (0.5 mg l⁻¹) and NAA (0.1 mg l⁻¹). The primary adventitious roots with an inoculum mass of 15 g FW were cultured for 6 weeks in the same medium for secondary adventitious root proliferation. Elicitation of abiotic elicitor, aluminium chloride at 10 mg l⁻¹ at the end of 4 weeks culture with 4 h exposure time enhanced withanolides productivity. Under similar culture conditions, the biotic elicitor, chitosan at 100 mg l⁻¹ stimulated higher production of all withanolides when compared to aluminium chloride treatment. This is the first report on the use of callus-derived adventitious root culture for the enhanced production of withanolides upon chitosan elicitation.     

Identification and quantification of phenolic acids and anthocyanins as antioxidants in bran,embryo and endosperm of white,red and black rice kernels (Oryza sativa L.)

Whole rice grain comprising endosperm, embryo (or germ), and bran has potential beneficial health effects in addition to provision of nutrients. The distribution of phenolic acids and anthocyanins in endosperm, embryo, and bran of white, red, and black rice grains was investigated in this study. The total phenolic content (TPC) was highest in the bran, averaging 7.35 mg GAE/g and contributing 60%, 86% and 84% of phenolics in white, red and black rice. The average TPC of the embryo and endosperm were 2.79 and 0.11 mg GAE/g accounting for 17% and 23%, 4% and 10% and 7% and 9% in white, red and black rice, respectively. The antioxidant capacity determined using DPPH and ORAC displayed a trend similar to TPC. Free/conjugated phenolic acids in white, red and black rice bran accounted for 41%, 65% and 85% of total acids. Bound phenolic acids in rice bran accounted for 90% of total acids in whole grain. Cis-p-coumaric was detected in bound form in bran while cis-sinapic acid was detected in the free/conjugated form in embryo and bran. Cyanidin-3-O-glucoside and peonidin-3-O-glucoside were identified mainly in black rice bran as the total anthocyanins. Cyanindin-3-O-rutinoside was also detected in black rice bran.     

Variability in essential oil content and composition of Origanum hirtum L., Origanum onites L., Coridothymus capitatus (L.) and Satureja thymbra L. populations from the Greek island Ikaria

Plants belonging to four “oregano” plant species (Origanum hirtum L., Origanum onites L., Coridothymus capitatus L., and Satureja thymbra L.) were collected during flowering from 33 sites located in the eastern part of the Greek island of Ikaria in the Eastern Aegean during April, May and July 2008. C. capitatus and O. hirtum were mostly observed in higher altitudes, whereas O. onites and S. thymbra in lower ones. The spatial distribution of all species was depicted on a GIS map. All four species exhibited essential oil concentrations higher than those reported in earlier literature, namely O. onites 3-4.3%, S. thymbra 4-6.5%, C. capitatus 3.7-5.6% and O. hirtum 5.5-10.0% (v/w). Carvacrol was the main constituent of the essential oils of all species, followed by γ-terpinene, p-cymene and caryophyllene, while thymol was not detected. All constituents varied remarkably among the four species, with carvacrol exhibiting the lowest variation. Carvacrol content varied between 72.3 and 89.2% in O. onites; 46.5 and 58.0% in S. thymbra; 82.9 and 90.9% in C. capitatus; and 84.4 and 93.8% in O. hirtum. By applying hierarchical cluster analysis on the basis of the essential oil constituents two main groups, divided into four subgroups of the taxa were evident. The first group consisted of O. onites and S. thymbra, while the second one of C. capitatus and O. hirtum. The results are discussed in terms of topography and climatic variation.     

Study of gene effects for cotton yield and Verticillium wilt tolerance in cotton plant (Gossypium hirsutum L.)

Verticillium wilt (VW), caused by Verticillium dahliae Kleb., is a destructive disease of cotton (Gossypium hirsutum L.). The use of resistant cultivars has long been considered the most practical and effective mean of control. The aim of this work was to study the quantitative genetic basis of Verticillium wilt resistance in Upland cotton by using five genotypes and their possible crosses without reciprocals selecting simultaneously for resistance and desirable agronomic characteristics. Five cotton cultivars and 10 F1s from half-diallel crosses were analyzed for VW resistance. The seed cotton yield, the number of bolls/ plant, and boll weight were measured and Verticillium wilt index (VWI) was estimated during two crop seasons in two different sites each year always on plots with naturally infested soil. Genetic components of variance were analyzed using the Hayman model. Analysis of variance for all characters showed significant differences between genotypes, without genotype-site interaction in most cases. Both, additive genetic variance component (D) and dominance genetic variance components (H1 and H2) were presented in all characters, except for VWI. D was the most important component for boll weight and VWI. Boll weight was the most correlated character with seed cotton yield and VWI. Broad sense heritability was high for boll weight and VWI, moderate for seed cotton yield and low for bolls per plant. Narrow sense heritability was moderate for boll weight, and high for VWI.     

Effects of plant growth regulator treatments on stem vascular tissue development in linseed (Linum usitatissimum L.)

Linseed (Linum usitatissimum L.) is a widely grown source of industrial and edible oil. Other varieties of the same species (flax) are cultivated for the long, strong bast fibres of their stems. The bast fibres of linseed generally go unused, although there is growing interest in developing linseed into a dual-purpose flax from which both seed and fibre could be utilized. Towards this objective, an improved understanding is required of the role of plant growth regulators in stem and fibre development in linseed. We have tested the effects of applying varying combinations of gibberellic acid (GA₃), the auxin indole-3-acetic acid, and a GA biosynthesis inhibitor (paclobutrazol) to an elite linseed variety (CDC Bethune). Results showed that GA stimulated stem elongation, stem expansion and the proliferation, expansion, elongation and cell wall thickening of xylem fibres. The impact of GA on phloem tissues was less apparent, although GA had a positive effect on the number of bast fibres observed in stem transverse section, and GA₃ application in combination with IAA increased the thickness of bast fibre secondary walls nearly two-fold. Other than the bast fibre cell walls, IAA treatments (alone or in combination with GA₃) did not affect most aspects of linseed stem development, suggesting that the observed effects of GA were not mediated by cross-talk with IAA. The relationships defined here between GA, stem architecture, and bast fibre properties in linseed provide a useful framework for manipulation of fibre properties through breeding, biotechnology, and field treatments.     

Regional scale distribution of imidazolinone herbicide-resistant alleles in red rice (Oryza sativa L.) determined through SNP markers

Red rice is the main weed in rice paddy fields. Imidazolinone herbicides in resistant rice cultivars currently provide a unique opportunity to control red rice in large-scale rice fields. However, the continuous use of this technology has resulted in imidazolinone-resistant red rice biotypes. This study aimed to identify the mechanism of herbicide resistance and the frequency and spatial distribution of the known imidazolinone herbicide-resistant alleles in red rice. The nucleotide sequence of the ALS gene indicated that the G₆₅₄E, S₆₅₃D and A₁₂₂T mutations are present in the imidazolinone herbicide-resistant rice cultivars IRGA 422 CL, SATOR CL and PUITÁ INTA CL, respectively. This information and the nucleotide sequence surrounding these mutations were used for the development of single nucleotide polymorphism (SNP) molecular markers to identify the possible mutations that confer herbicide resistance in red rice. This analysis was carried out in a total of 481 plants from 38 populations collected as individuals that escaped control with the herbicides imazethapyr and imazapic in rice paddy fields in Southern Brazil. The G₆₅₄E mutation was the most frequent, being found in 100% and 90.9% of the populations in the 2006/2007 and 20007/2008 seasons, respectively. In addition, the S₆₅₃D and A₁₂₂T mutations were also present either alone or as double or triple mutations in some plants. Target site insensitivity is the predominant mechanism of resistance in red rice resistant to imidazolinone herbicides in Southern Brazil. The high frequency of the S₆₅₃D mutation, the same mutation responsible for the resistance in the rice cultivar largely used in Southern Brazil, indicates that gene flow is occurring from the rice cultivar to red rice. Management practices related to increasing crop sanitation and decreasing of herbicide selection pressure through crop rotation should be enforced to prevent the evolution of herbicide resistance in red rice.