Seed dormancy allows weedy rice (Oryza sp.) to persist in rice production systems. Weedy and wild relatives of rice (Oryza sativa L.) exhibit different levels of dormancy, which allows them to escape weed management tactics, increasing the potential for flowering synchronization, and therefore gene flow, between weedy Oryza sp. and cultivated rice. In this study, we determined the genetic diversity and divergence of representative dormant and nondormant weedy Oryza sp. groups from Arkansas. Twenty-five simple sequence repeat markers closely associated with seed dormancy were used. Four populations were included: dormant blackhull, dormant strawhull, nondormant blackhull, and nondormant strawhull. The overall gene diversity was 0.355, indicating considerable genetic variation among populations in these dormancy-related loci. Gene diversity among blackhull populations (0.398) was higher than among strawhull populations (0.245). Higher genetic diversity was also observed within and among dormant populations than in nondormant populations. Cluster analysis of 16 accessions, based on Nei’s genetic distance, showed four clusters. Clusters I, III, and IV consisted of only blackhull accessions, whereas Cluster II comprised only strawhull accessions. These four clusters did not separate cleanly into dormant and nondormant populations, indicating that not all markers were tightly linked to dormancy. The strawhull groups were most distant from blackhull weedy Oryza sp. groups. These data indicate complex genetic control of the dormancy trait, as dormant individuals exhibited higher genetic diversity than nondormant individuals. Seed-dormancy trait contributes to population structure of weedy Oryza sp., but this influence is less than that of hull color. Markers unique to the dormant populations are good candidates for follow-up studies on the control of seed dormancy in weedy Oryza sp.

Chemical options for weed control in commercial cowpea production are limited. Repeated long-term use of the acetolactate synthase (ALS) inhibitor, imazethapyr, has resulted in selection for ALS-resistant populations of Palmer amaranth. Experiments were conducted at Bixby, OK, and Kibler, AR, from 2001 to 2003 to evaluate the tolerance of cowpea cultivars and advanced breeding lines to fomesafen, a potential alternative for controlling ALS-resistant Palmer amaranth and other problematic broadleaf weeds. Eight commercial cultivars and 42 advanced breeding lines were entered in the preliminary screening, using 0.84 kg/ha fomesafen. Six breeding lines were selected for the first replicated trial and three (00-582, 00-584, and 00-609) were advanced to across-location experiments. Fomesafen doses of 0, 0.17, 0.34, and 0.67 kg/ha were tested across locations. ‘Early Scarlet’ was used as commercial standard. The advanced lines had equal or higher yield potential (1,182 to 1,936 kg/ha) than Early Scarlet (1,108 kg/ha) across locations. Of the cultivars tested, line 00-609 was the best yielder, whereas 00-584 had the highest tolerance to fomesafen. At the commercial fomesafen rate of 0.34 kg/ha, 00-584 had higher yield (974 and 1,735 kg/ha, respectively, at Bixby, OK, and Kibler, AR) than the nontreated, weed-free, Early Scarlet. Thus, fomesafen can be used on the tolerant line, 00-584, without reducing yield potential relative to Early Scarlet.

The commercialization of imazethapyr-resistant (Clearfield™, CL) rice in the southern United States has raised serious concerns about gene flow to red rice, producing imazethapyr-resistant red rice populations. Our objectives were to determine the impact of planting date, CL cultivars, and red rice biotypes on outcrossing rate; and to investigate the relative contribution of flowering time of CL rice and red rice biotypes, together with air temperature and relative humidity (RH), on outcrossing rate. Field experiments were conducted at Stuttgart, Rohwer, and Kibler, AR, from 2005 to 2007, at three or four planting times from mid-April to late May. ‘CL161’ (inbred cultivar) and ‘CLXL8’ (hybrid) rice were planted in nine-row plots, with red rice planted in the middle row. Twelve red rice biotypes were used. The flowering of red rice and CL rice, air temperature, and RH were recorded. Red rice seeds were collected at maturity. To estimate outcrossing rate, resistance to imazethapyr was evaluated in subsequent years and confirmed using rice microsatellite markers. CLXL8 rice flowered 2 to 4 d earlier than CL161 rice, and flowering was completed within 1 wk in all plantings. The flowering duration of most red rice biotypes ranged from 4 to 17 d. Flowering synchrony of red rice biotypes and CL rice ranged from 0 to 100% at different plantings. In general, CLXL8 had greater flowering overlap and higher outcrossing rate with red rice than did CL161 rice. The outcrossing rate of red rice biotypes ranged from 0 to 0.21% and 0 to 1.26% with CL161 and CLXL8 rice, respectively. The outcrossing rate differed within each planting date (P < 0.05). Outcrossing was generally lower in mid-May and late May than in mid-April and late April planting times. Flowering synchrony and outcrossing rate were not correlated (r2 < 0.01). Outcrossing with CL161 was primarily influenced by red rice biotype. A minimum air temperature of > 24 C in the evening also favors outcrossing with CL161. With CLXL8 rice, outcrossing was most affected by RH. When RH was < 54%, outcrossing was less (0.12%) than when RH was ≥ 54% (0.38%). With CLXL8 rice, a minimum RH of ≥ 54%, from mid-morning to noon, increased outcrossing with red rice. To fully understand the interaction effects of these factors on outcrossing with red rice, controlled experiments are needed.

Cultivated rice yield losses due to red rice infestation vary by cultivar, red rice density, and duration of interference. The competition effects of red rice could be influenced further by emergence characteristics, red rice biotype, and planting time of cultivated rice. We aimed to characterize the emergence of red rice biotypes at different planting dates and evaluate the effect of red rice biotype, rice cultivar, and planting date on cultivated rice yield loss. Field experiments were conducted at the Southeast Research and Extension Center, Rohwer, AR, and at the Arkansas Rice Research and Extension Center, Stuttgart, AR, in the summer of 2005 and 2006. The experimental design was a split-split plot with three or four replications. Planting time, ClearfieldTM (CL) rice cultivar, and red rice biotype were the main plot, subplot, and sub-subplot factors, respectively. There were three planting times from mid-April to mid-May at 2-wk intervals. CL rice cultivars, CL161 and hybrid CLXL8, and 12 red rice biotypes were planted. The emergence rate and coefficient of uniformity of germination differed among some red rice biotypes within a planting time. Planting date affected the emergence characteristics of red rice biotypes. The mean emergence rate of red rice was 0.043 d−1 in the mid-April planting and 0.058 d−1 in the late April planting. For the mid-April planting, 50% of red rice biotypes emerged in 20 ± 2 d compared with 15 ± 2 d for CL rice cultivars. Yield losses due to red rice biotypes generally increased in later planting dates, up to 49%. Yield losses due to interference from red rice biotypes ranged from 14 to 45% and 6 to 35% in CL161 and CLXL8, respectively. Cultivated rice became less competitive with red rice in later plantings, resulting in higher yield losses.

Experiments were conducted to determine the inheritance of resistance in crosses between imazethapyr-resistant rice and red rice. Past experiments on red rice control, using the Clearfield rice technology, resulted in outcrossing between Clearfield rice and Stuttgart strawhull red rice. The F2 generation of these spontaneous crosses were characterized with respect to inheritance of imazethapyr resistance, leaf color and leaf pubescence, and seed shattering, pubescence, color, and size. Agronomic traits of hybrids were also observed in relation to their parents. To determine the segregation of resistance among F2 phenotypes, the response of three- to four-leaf plants to imazethapyr was scored 3 wk after application as resistant (R, no imazethapyr symptoms), susceptible (S, death of plants), or intermediate (I, stunted plants). R, I, and S phenotypes segregated in a 1:2:1 ratio in the F2 generation. Two- or three-gene inheritance was documented for leaf and seed characteristics. A wide range in onset of flowering (70 to 130 d after planting) was observed in F2 families, although 6% of the plants did not flower during the growing season. F2 plants were taller and had more tillers than any of their parents. Resistance to imazethapyr is associated with a single, incompletely dominant allele.

Two red rice accessions from Arkansas have been found to be resistant to the labeled rate of imazethapyr, which is used to control red rice in ClearfieldTM rice. Full-length amplification of the acetolactate synthase (ALS) gene in imazethapyr-resistant red rice revealed a coding sequence of 1,935 base pairs, which is the same as that of the cultivated rice. Coding sequences were generated from four red rice accessions collected from different geographical regions in Arkansas, consisting of accessions that were either resistant or susceptible to imazethapyr. Nucleotide sequence alignments identified six base polymorphisms, three of which resulted in amino acid substitutions in the ALS gene. One amino acid substitution, Gly654Glu, involves a residue required for imazethapyr binding to the ALS. The other substitution, Val669Met, implies conformational changes in the ALS structure that enhances binding of thiamine diphosphate, an ALS cofactor. These novel amino acid substitutions first reported for ALS-resistant red rice accessions support the hypothesis that ALS-resistant red rice can evolve with sustained herbicide selection pressure. Thus, it behooves growers to integrate the Cleafield rice technology with other tools to achieve a successful, long-term weed management program.

The introduction of Clearfield (CL) rice cultivars resistant to imidazolinone herbicides, acetolactate synthase (ALS) inhibitors, has raised concerns of gene flow to weedy rice genotypes collectively called “red rice” that infest rice-growing areas in the southern United States. This experiment was conducted to study hybridization between CL rice and red rice using simple sequence repeats (SSR) markers, identify mutations in the ALS gene of imazethapyr-resistant red rice, and to detect the introgression of the ALS-resistant gene from CL rice into red rice. Natural outcrossing experiments between CL rice and strawhull (SH) red rice were set up in Stuttgart, AR, in 2002 and 2003. Putative red rice hybrids were detected among volunteer plants in the following year. Hybridization was confirmed using SSR markers, and introgression of the resistant ALS gene from CL rice to red rice was detected by ALS gene sequencing. The ALS gene sequences of U.S. rice cultivars ‘Bengal’ and ‘Cypress’, SH red rice, CL rice (CL161), and imazethapyr-resistant red rice/CL rice hybrids were compared. Nucleotide sequences of the ALS gene from the rice cultivars were identical. Three point mutations were present in the SH red rice ALS gene coding region relative to Bengal/Cypress. One of these resulted in the substitution of Asp630 for Glu630. The ALS gene sequences of confirmed hybrids were identical to that of the herbicide-resistant pollen source, CL161. We identified four ALS gene mutations in the herbicide-resistant red rice hybrids relative to the susceptible rice cultivars. One point mutation, resulting in a substitution of Ser653 with Asn, was linked to ALS resistance in callus tissue derived from a Kinmaze rice line from Japan. The other three mutations (Ser186—Pro, Lys416—Glu, and Leu662—Pro) are novel. This experiment confirmed that gene flow from imidazolinone-resistant rice resulted in herbicide-resistant red rice plants.

The transfer of the imazethapyr-resistant gene from Clearfield™ (CL) rice to red rice is an ecological risk. Flowering synchronization and genetic compatibility between cultivated rice and red rice could influence gene transfer. We examined the (1) variability in maximum outcrossing rate between 12 red rice biotypes and ‘CL161’ rice during their peak flowering overlap in the field and (2) genetic compatibility of red rice biotypes with CL161 rice. Experiments were conducted at Stuttgart, AR, and Fayetteville, AR, from 2005 to 2007. To evaluate the flowering synchrony of red rice and CL161 rice as well as its impact on outcrossing rate, field experiments were conducted at four planting times from early April to late May. The red rice biotypes were planted in the middle row of nine-row CL161 plots and flowering was monitored. Outcrosses were evaluated in subsequent years by herbicide response and simple-sequence-repeat marker assays. To determine compatibility, manual crosses were performed between 12 red rice biotypes and CL161 rice in the greenhouse. The flowering duration of all red rice types ranged from 5 to 16 d after the onset of flowering in contrast to 6 d in CL161 rice. Ten of the twelve types of red rice had ≥ 70% overlap in flowering time with CL161 rice in at least one planting date. The maximum field outcrossing rate between red rice biotypes and CL161 ranged from 0.03 to 0.25%. The field outcrossing rate between red rice biotypes differed (P < 0.01), but flowering synchronization was not directly related to outcrossing rate. Manual crosses resulted in seed sets of 49 to 94%. The majority of red rice biotypes had similar compatibility with CL161 rice. Thus, other factors must contribute to hybridization rates in the field. Follow-up experiments should investigate other plant factors and environmental influence on hybridization rate.

Red rice is a major weed problem in rice production of the southern United States and other rice-producing countries. One hundred thirty red rice accessions from 26 rice-growing counties in Arkansas were tested for tolerance to imazethapyr in seed- and whole-plant response bioassays. The red rice accessions were compared with imazethapyr-resistant (ClearfieldTM) rice cultivars (‘CL121’, ‘CL161’, and ‘CL-XL8’) and conventional rice cultivars (‘Bengal’, ‘Dongjin’, ‘Drew’, and ‘Wells’). Red rice accessions 79, 84, and 118 showed 17-, 48-, and 37-fold more tolerance to imazethapyr, respectively, than the standard susceptible red rice accession (82) in whole-plant bioassays. The imazethapyr-resistant rice cultivars, CL121, CL161, and CL-XL8 were 41-, >177-, and 48-fold more resistant to imazethapyr, respectively than the susceptible standard. The imazethapyr-tolerant red rice and ClearfieldTM cultivars were generally cross tolerant to other acetolactate synthase (ALS; EC 4.1.3.18) inhibiting herbicides such as imazapyr, imazaquin, imazamox, and pyrithiobac. The tolerance level of red rice or rice to imidazolinone herbicides was highest with imazaquin and lowest with imazapyr. The imazethapyr-tolerant red rice accessions and ClearfieldTM rice were susceptible to glufosinate and glyphosate. The ALS enzyme of tolerant red rice accessions was less sensitive to imazethapyr than the susceptible standard, but tolerance at the enzyme level was less than at the whole-plant level. Therefore, tolerance of red rice to imazethapyr may involve other mechanisms besides an insensitive target site. We learned that a few imazethapyr-tolerant red rice populations existed probably before ClearfieldTM rice was introduced, supporting the hypothesis that evolution of herbicide-resistant red rice populations can happen with intensive herbicide selection pressure.