Necrotrophic Alternaria alternata induces EXECUTER 1(EX1)/2-dependent singlet oxygen (1O2) bursts, leading to plant cell death, with jasmonic acid (JA) acting as a key signal transducer downstream of EX1/2-mediated signaling. Salicylic acid (SA), a crucial defense hormone, is known to respond to pathogen invasion and activate defense gene expression. Previous studies emphasized the importance of SA in A. alternata-induced necrosis in the light of the increased susceptibility of SA-deficient transgenic Arabidopsis NahG to A. alternata. In this study, we investigated the role of SA in A. alternata-triggered 1O2 signaling in Arabidopsis. We found that EX1/2 deficiency did not alter SA levels in Arabidopsis infected with A. alternata, indicating that SA signaling regulates A. alternata-induced pathogenesis through an EX1/2-independent pathway. Exogenous SA application and increased endogenous SA in the ssi2-2 mutant enhanced resistance but inhibited JA production. Conversely, SA signaling deficiency in the eds1 and pad4 mutants increased susceptibility and elevated JA levels. In conclusion, SA enhances Arabidopsis defense against A. alternata via an EX1/2-independent 1O2 signal pathway and antagonizes JA biosynthesis.

Plant resistant induction is considered as a promising strategy for protecting crops against extreme high temperature (HT). However, a high-throughput method to accurately estimate the capacity of plant resistance inducers (PRIs) for HT resistance has not been developed. Here, we present a simple approach using fast chlorophyll fluorescence kinetics in Arabidopsis leaf discs to assess PRI efficacy in inducing HT resistance. Both 2-amino-3-methylhexanoic acid (AMHA) and salicylic acid (SA) significantly alleviated the temperature-dependent increase in the K-peak of the OJIP curve and variations in amplitude of heat-responsive JIP-test parameters within the elevated-temperature range of 25-42 degrees C. The PIABS (performance index on absorption basis) and W-K (relative variable fluorescence at the K-step to the amplitude F-J - F-O) as two classical heat-responsive characteristic parameters were used to produce a novel hypersensitive parameter HT sensitivity indicator, PIABS/W-K (named H-s). Based on the correlation of logH(s) with elevated temperatures, a model for quantifying the capacity of HT-resistance induction (called C-i) by AMHA or SA was established. A three-grade classification according to the C-i value was proposed as low (0< C-i <= 1 degrees C), moderate (1 degrees C < C-i <= 2 degrees C), and high res(i)stance (C-i > 2 degrees C). AMHA at 1 mu M and SA at 100 mu M had C-i values of 2.49 degrees C and 4.09 degrees C in Arabidopsis plants, respectively, associated with their high level of HT resistance induction. Additionally, the EC50 derived from the relative stimulation ratio (K-c) was also introduced as a quantitative index for measuring the ability of AMHA and SA to induce HT resistance. The EC50 value of AMHA is about 0.1 mu M in Arabidopsis and 0.35 mu M in tomato, being much lower than that of SA (approximately 63 mu M in Arabidopsis). Thus, AMHA is a more potent plant inducer than SA. The model was validated through additional experimental evidence, demonstrating its reliability and applicability. This study provides an expeditious high-throughput method for screening promising PRI candidates.

The tetramic acid moiety is a pivotal structural unit in numerous natural products. As an analogue of the simplest tetramic acid compound tenuazonic acid (TeA), sec-pentyl-TeA (S-TeA) exhibits double herbicidal activity of TeA. Here, this work identifies S-TeA as a novel natural product synthesized by Alternaria alternata and three other filamentous fungi. Chiral analysis confirm the absolute configuration of natural S-TeA as (5S, 6S). Configuration-bioactivity studies reveal that natural (5S, 6S)-S-TeA is the eutomer and possesses the highest herbicidal activity among all tested diastereomers. Biosynthetic analyses demonstrate that threonine is the precursor to S-TeA, beginning with the production of 2-amino-3-methylhexanoic acid (AMHA) via eight enzymes from the branched-chain amino acid (BCAA) biosynthetic pathway, including threonine deaminase, 2-isopropylmalate synthase (IPMS), 3-isopropylmalate dehydratase (IPMDH), isopropylmalate dehydrogenase (ISMD), acetolactate synthase, ketol-acid reductoisomerase, dihydroxy acid dehydratase, and BCAA aminotransferase. Subsequently, AMHA undergoes acetylation and cyclization by non-ribosomal peptide synthetases to form S-TeA. Distinct differences in the biosynthetic pathways of S-TeA and TeA are identified. In vitro studies confirm the critical roles of three unique enzymes IPMS, IPMDH, and ISMD in S-TeA biosynthesis, which are absent in TeA biosynthesis. These findings provide a solid basis for developing S-TeA as a natural product herbicide.

The mycotoxin tenuazonic acid (TeA) inhibits photosynthesis and is expected to be developed as a bioherbicide to control Ageratina adenophora that is one of the most serious invasive alien plants in China. New leaves sprouting from A. adenophora at low temperatures (LT) in early spring are less sensitive to TeA compared to those growing in summer. However, the molecular mechanism of LT-caused decrease in the susceptibility of A. adenophora to TeA is unclear. In this study, three singlet oxygen-responsive genes (SORGs) and three jasmonic acid responsive genes (JARGs) were cloned to further probe the role of singlet oxygen (1O2) signaling during TeA-induced disease development in A. adenophora leaves exposed to LT. TeA triggered chloroplast-derived 1 O 2 production as a result of photosystem II (PSII) photoinhibition during leaf lesion formation in A. adenophora. Moreover, TeA indeed induced the expression of SORGs and JARGs as well as a high level of JA generation, activating the 1 O 2 signaling pathway in A. adenophora. LT (12 degrees C) pretreatment can cause PSII photoinhibition and increase the SORG AaAAA-ATPase expression level in A. adenophora leaves, meaning that 1 O 2 signaling was activated by LT. Thus TeA led to less increase of the SORGs and JARGs expression and JA level in plants pretreated by LT compared with non-pretreated plants, although both of them had the same level of 1 O 2 production after TeA treatment. It was concluded that the low susceptibility to TeA of A. adenophora subjected to LT can be attributed to the occurrence of 1 O 2 acclimation.

BACKGROUNDBioherbicides are becoming more attractive as safe weed control tools towards sustainable agriculture. Natural products constitute an important source chemicals and chemical leads for discovery and development of novel pesticide target sites. Citrinin is a bioactive compound produced by fungi of the genera Penicillium and Aspergillus. However, its physiological-biochemical mechanism as a phytotoxin remains unclear. RESULTSCitrinin causes visible leaf lesions on Ageratina adenophora similar to those produced by the commercial herbicide bromoxynil. Phytotoxicity bioassay tests using 24 plant species confirmed that citrinin has a broad activity spectrum and therefore has potential as a bioherbicide. Based on chlorophyll fluorescence studies, citrinin mainly blocks PSII electron flow beyond plastoquinone Q(A) at the acceptor side, resulting in the inactivation of PSII reaction centers. Furthermore, molecular modeling of citrinin docking to the A. adenophora D1 protein suggests that it binds to the plastoquinone Q(B) site by a hydrogen bond between the O1 hydroxy oxygen atom of citrinin and the histidine 215 of the D1 protein, the same way as classical phenolic PSII herbicides do. Finally, 32 new citrinin derivatives were designed and sorted according to free energies on the basis of the molecular model of an interaction between the citrinin molecule and the D1 protein. Five of the modeled compounds had much higher ligand binding affinity within the D1 protein compared with lead compound citrinin. CONCLUSIONCitrinin is a novel natural PSII inhibitor that has the potential to be developed into a bioherbicide or utilized as a lead compound for discovery of new derivatives with high herbicidal potency. (c) 2023 Society of Chemical Industry.

2-Amino-3-methylhexanoic acid (AMHA) was synthetized as a non-natural amino acid more than 70 years ago; however, its possible function as an inducer of plant resistance has not been reported. Plant resistance inducers, also known as plant elicitors, are becoming a novel and important development direction in crop protection and pest management. We found that free AMHA accumulated in the mycelia but not in fermentation broths of four fungal species, Magnaporthe oryzae and three Alternaria spp. We unequivocally confirmed that AMHA is a naturally occurring endogenous (2S, 3S)-α-amino acid, based on isolation, purification and structural analyses. Further experiments demonstrated that AMHA has potent activity-enhancing resistance against extreme temperature stresses in several plant species. It is also highly active against fungal, bacterial and viral diseases by inducing plant resistance. AMHA pretreatment strongly protected wheat against powdery mildew, Arabidopsis against Pseudomonas syringae DC3000 and tobacco against Tomato spotted wilt virus. AMHA exhibits a great potential to become a unique natural elicitor protecting plants against biotic and abiotic stresses.

INTRODUCTION:Computer-aided design has become an important tool to develop novel pesticides based on natural lead compounds. Tenuazonic acid (TeA), a typical representative of the natural tetramic acid family, was patented as a potential bioherbicide. However, its herbicidal efficacy is still not up to the ideal standard of commercial products.OBJECTIVES:We aim to find new TeA's derivatives with improved potency.METHODS:Molecular docking was used to build ligand-acceptor interaction models, design and screen new derivatives. Phytotoxicity, oxygen evolution rate, chlorophyll fluorescence and herbicidal efficacy were determined to estimate biological activity of compounds.RESULTS:With the aid of a constructed molecular model of natural lead molecule TeA binding to the QB site in Arabidopsis D1 protein, a series of derivatives differing in the alkyl side chain were designed and ranked according to free energies. All compounds are stabilized by hydrogen bonding interactions between their carbonyl oxygen O2 and D1-Gly256 residue; moreover, hydrogen bond distance is the most important factor for maintaining high binding affinity. Among 54 newly designed derivatives, D6, D13 and D27 with better affinities than TeA were screened out and synthesized to evaluate their photosynthetic inhibitory activity and herbicidal efficacy. Analysis of structure-activity relationship indicated that D6 and D13 with sec-pentyl and sec-hexyl side chains, respectively, were about twice more inhibitory of PSII activity and effective as herbicide than TeA with a sec-butyl side chain.CONCLUSION:D6 and D13 are promising compounds to develop TeA-derived novel PSII herbicides with superior performance.

BACKGROUND Tea, one of the most important commercial crops on earth, is strongly affected by weeds on productivity and quality. Bioherbicides are shedding new light on weed control in tea gardens in an economical and safe manner. RESULTS A pathogenic strain SYNJC-2-2 was isolated from diseased leaves of a noxious weed, goosegrass (Eleusine indica), from a tea garden in Zhejiang Province, China. It was identified as the fungal species Bipolaris bicolor based on the morphological characteristics and phylogenetic analysis. The potential of the B. bicolor strain SYNJC-2-2 as a bioherbicide was assessed by determining its efficacy to control weeds and selectivity to crops, its infection process and the influence of environmental conditions on conidial production and germination. The ED90 (effective dose of conidia resulting in 90 disease index) of SYNJC-2-2 on goosegrass was 2 x 10(4) conidia mL(-1). Additionally, three Poaceae weeds, Setaria viridis, Microstegium vimineum and Pennisetum alopecuroides, were also extremely susceptible to SYNJC-2-2. SYNJC-2-2 was safe to 14 out of 17 crop species in nine families, especially tea plants. Conidial germination, hyphal growth and appressorial formation occurred within 3 to 6 h on goosegrass leaves. Hyphae invaded leaf tissues mainly through epidermal cell junctions and cracks, causing cell death and necrotic lesions within 2 days on inoculated leaves and killing goosegrass plants within 7 days. Furthermore, SYNJC-2-2 has a strong adaptability to environmental variables and high conidial production capacity on goosegrass juice agar media. CONCLUSION Bipolaris bicolor strain SYNJC-2-2 has the potential to be developed as a bioherbicide for controlling goosegrass, especially in tea gardens.

Angiosperms are believed to have emerged initially in the tropics and expanded their distribution range poleward through diverse mechanisms, for example polyploidization-driven cold tolerance evolution. Reversed expansion from temperate to pan-tropic climates through a polyploidization-driven shift in heat tolerance remains largely unknown. Here, we found autopolyploidy in relation to the global expansion of Solidago canadensis from its temperate-climate native range in North American to hot-summer climate in an introduced range. Our cytogeographical study of 2,062 accessions from 471 locations worldwide demonstrates that ploidy levels correlate negatively with latitude and positively with average temperature. An isotherm-dependent shift of the climate niches at the threshold of 20 degrees-24 degrees C between geo-cytotypes can be attributed mainly to autopolyploidy-driven differentiation of heat tolerance; only polyploids and not diploids are able to complete sexual reproduction, germinate, and grow in the hot-summer climate of low latitudes. Ploidy-dependent fertility appears to play a key role in the hot-summer introduced range in the northern hemisphere through both pre-adaptation and rapid post-introduction adaptive evolution of delayed flowering and improved heat tolerance during embryo development. The MaxEnt model predicts continued expansion of this plant species under global change. These results provide new insights into the mechanisms governing autopolyploidy-driven backward range expansion of plant species from temperate origins.

Bromacil was introduced at the beginning of the 1960s for PRE and early POST control of grasses and broadleaf weeds, particularly in citrus (Rutaceae spp.) orchards and pineapple [Ananas comosus (L.) Merr.] plantations as well as in noncultivated areas. Both the acidic form of bromacil and its lithium salt are highly soluble in water; the herbicide is moderately to highly persistent in the soil with a half-life from 60 d to 8 mo and is prone to percolate in the soil and reach groundwater. In Costa Rica, bromacil was registered for both citrus and pineapple, but in recent years its major use has been in pineapple. An average of 60,000 kg of active bromacil per year were imported before its banning in 2017. Pineapple is grown in more than 40,000 ha; the recommended rate of bromacil was 1.6 to 3.2 kg ha(-1). In a survey conducted by the National University between 2001 and 2004, bromacil was the most frequently found pesticide, at levels between 0.5 and 20 mu g L-1, in water springs and wells in the pineapple-growing area of the Caribbean side of Costa Rica. Further studies conducted more recently also documented the presence of bromacil in the ground and surface water in areas where pineapple is planted. The local standard for the quality of drinking water of 2015 established maximum acceptable values of 0.1 mu g L-1 and 0.5 mu g L-1 for a single pesticide and for the sum of all pesticides present, respectively, but it was amended for bromacil to comply with requirements determined by the Constitutional Court to "non-detectable by method." This paper provides an account of the scientific and administrative considerations for the banning of bromacil that occurred on May 24, 2017.

Water is one of the main dispersal agents of plant seeds and influences plant population dynamics and community structure. Although a large proportion of paddy fields are subjected to irrigation, limited research has addressed weed seed dispersal via water in rice-based systems. In this study, safranin T-dyed Beckmannia syzigachne seeds were released and their movement was tracked to characterize their dispersal dynamics via water in an irrigation canal and in a rice field. B. syzigachne seeds, floating on the water surface, moved from their release point at the canal entrance via irrigation water along the irrigation canal. A well-simulated Gaussian plume model indicated that the seed dispersal of B. syzigachne conformed to a leptokurtic distribution, and the peak dispersal kernel showed that seeds moved along the irrigation water flow to approximately 800-1000 km away from the release point 36 h after release into the canal. When seeds were released in the center of a flooded field, a total of 60% of the released seeds were water-dispersed to the northwest corner by the southeast prevailing monsoon wind within 72 h. The well-fitted Gauss 2D-model illustrated the widening of the seed distribution range with time and dispersal pattern with an accumulation center in the field. Our study is the first to show that buoyant weed seeds are mainly dispersed by irrigation water in both the canal and field and that the water dispersal of seeds influences the spatial deposition and distribution of the weed seed bank and strongly affects weed occurrence patterns in irrigated fields.

Pollen-mediated transgenic flow of herbicide resistance occurs bidirectionally between transgenic cultivated rice and weedy rice. The potential risk of weedy traits introgressing into hybrid rice has been underestimated and is poorly understood. In this study, two glufosinate-resistant transgenic rice varieties, hybrid rice (F-1), and their succeeding generations (F-2-F-4) were planted for 3 years in field plots free of weedy rice adjacent to experimental weedy-rice fields. Weedy-rice-like (feral) plants that were both glufosinate-resistant and had red-pericarp seed were initially found only among the F-3 generations of the two glufosinate-resistant transgenic hybrid cultivars. The composite fitness (an index based on eight productivity and weediness traits) of the feral progeny was significantly higher than that of the glufosinate-resistant transgenic hybrid (the original female parent of the feral progeny) under monoculture common garden conditions. The hybrid rice progeny segregated into individuals of variable height and extended flowering. The hybrid rice F-2 generations had higher outcrossing rates by pollen reception (0.96-1.65%) than their progenitors (0.07-0.98%). The results show that herbicide-resistant weedy rice can rapidly arise by pollen-mediated gene flow from weedy to transgenic hybrid rice, and their segregating pollen-receptive progeny pose a greater agroecological risk than transgenic varieties. The safety assessment and management regulations for transgenic hybrid rice should take into account the risk of bidirectional gene flow.

The effects of four phytotoxins usnic acid (UA), salicylic acid (SA), cinnamic acid (CA) and benzoic acid (BA) on photosynthesis of Chlamydomonas reinhardtii were studied in vivo to identify and localise their initial action sites on two photosystems. Our experimental evidence shows that the four phytotoxins have multiple targets in chloroplasts, which mainly lie in photosystem II (PSII), not photosystem I (PSI). They share an original action site by blocking electron transport beyond QA (primary plastoquinone acceptor) at PSII acceptor side since a fast increase of the J-step level is the greatest change in chlorophyll a fluorescence induction kinetics OJIP in C. reinhardtii cells treated with the phytotoxins. UA decreases photosynthetic activity by reducing O2 evolution rate, interrupting PSII electron transport at both the donor and acceptor sides, inactivating the PSII reaction centers (RCs), reducing the content of chlorophylls and carotenoids, destroying the conformation of antenna pigment assemblies, and casuing the degradation of D1/D2 proteins. SA damage to photosynthetic machinery is mainly attributed to inhibition of PSII electron transport beyond QA at the acceptor side, inactivation of the PSII RCs, reduction of chlorophyll content, digestion of thylakoid ploypeptides and destabilization of thylakoid membranes. Both CA and BA affect the photosynthetic process by decreasing PSII electron transport efficiency at the acceptor side and the amount of active PSII RCs. Besides, the initial cause of BA-inhibiting photosynthesis is also assocaited with the O2 evolution rate and the disconnection of some antenna molecules from PSII RCs.

Liriope spicata (Thunb.) Lour has a unique LsEPSPS structure contributing to the highest-ever-recognized natural glyphosate tolerance. The transformed LsEPSPS confers increased glyphosate resistance to E. coli and A. thaliana. However, the increased glyphosate-resistance level is not high enough to be of commercial value. Therefore, LsEPSPS was subjected to error-prone PCR to screen mutant EPSPS genes capable of endowing higher resistance levels. A mutant designated as ELs-EPSPS having five mutated amino acids (37Val, 67Asn, 277Ser, 351Gly and 422Gly) was selected for its ability to confer improved resistance to glyphosate. Expression of ELs-EPSPS in recombinant E. coli BL21 (DE3) strains enhanced resistance to glyphosate in comparison to both the LsEPSPS-transformed and -untransformed controls. Furthermore, transgenic ELs-EPSPS A. thaliana was about 5.4 fold and 2-fold resistance to glyphosate compared with the wild-type and the Ls-EPSPS-transgenic plants, respectively. Therefore, the mutated ELs-EPSPS gene has potential value for has potential for the development of glyphosate-resistant crops.

A toxin-deficient mutant strain, HP001 mutant of Alternaria alternata, whose mycelium is unable to infect its host, produces little tenuazonic acid (TeA) toxin. How TeA plays a role in initiating host infection by A. alternata remains unclear. In this research we use Imaging-PAM based on chlorophyll fluorescence parameters and transmission electron microscopy to explore the role of TeA toxin during the infection process of A. alternata. Photosystem II damage began even before wild type mycelium infected the leaves of its host, croftonweed (Ageratina adenophora). Compared with the wild type, HP001 mutant produces morphologically different colonies, hyphae with thinner cell walls, has higher reactive oxygen species (ROS) content and lower peroxidase activity, and fails to form appressoria on the host surface. Adding TeA toxin allows the mutant to partially recover these characters and more closely resemble the wild type. Additionally, we found that the mutant is able to elicit disease symptoms when its mycelium is placed on leaves whose epidermis has been manually removed, which indicates that TeA may be determinant in the fungus recognition of its plant host. Lack of TeA toxin appears responsible for the loss of pathogenicity of the HP001 mutant. As a key virulence factor, TeA toxin not only damages the host plant but also is involved in maintaining ROS content, host recognition, inducing appressoria to infect the host and for allowing completion of the infection process.

BACKGROUND:Weedy rice, as one of the worst paddy field weeds worldwide, bears vigorous seedlings and dominantly competes with cultivated rice causing serious crop yield losses. To elucidate the causes of its stronger seedling vigour endowing its dominant competition with cultivated rice, comparative studies on seedling growth characteristics were conducted among six weedy rice biotypes and the two indica and japonica cultivars Shanyou-63 (SY-63) and Zhendao-8 (ZD-8), respectively, in the greenhouse.RESULTS:Weedy rice emerged 2 to 3 days earlier, rapidly grew 1.3-1.7 cm taller daily, produced more secondary adventitious roots and greater aboveground fresh biomass than cultivated rice. Moreover, weedy rice exhibited greater photosynthetic pigment content, net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, and chlorophyll fluorescence kinetic parameters. An enhanced overall photosynthetic activity in weedy rices was attributed to the combined action of a larger antenna, more active reaction centres and higher quantum yield for electron transfer beyond QA .CONCLUSIONS:Enhanced photosynthesis of weedy rice at the seedling stage should be the main factor for leading to strong competitive dominance over cultivated rice. © 2016 Society of Chemical Industry.

MAIN CONCLUSION:A combination of unique EPSPS structure and increased gene copy number and expression contribute to natural glyphosate tolerance in three lilyturf species. A few plants are naturally tolerant to glyphosate, the most widely used non-selective herbicide worldwide. Here, the basis for natural tolerance to glyphosate in three lilyturf species, Ophiopogon japonicus (OJ), Liriope spicata (LS), and Liriope platyphylla (LP), is characterized. These species tolerate glyphosate at about five times the commercially recommended field dose. They share three unique amino acids in their 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that affect glyphosate binding. These correspond to Asp71Met, Ala112Ile, and Val201Met amino acid variations compared to 231 other published plant EPSPS amino acid sequences. There was also a common deletion at 91 of a highly conserved glutamic acid. Glyphosate-treated lilyturf plants accumulated little shikimic acid but had significantly higher levels of EPSPS mRNA than initially expressed in the control. The IC50 of LsEPSPS was 14.0 µM compared to the 5.1 µM of Arabidopsis thaliana. The higher K m and K i values of LsEPSPS kinetics showed that LsEPSPS had lower substrate binding affinity to glyphosate. Overexpression of LsEPSPS in the recombinant E. coli BL21 (DE3) strain enhanced its tolerance to glyphosate. Both OJ and LS had two copies of the EPSPS gene, while LP had three copies. Therefore, a combination of unique EPSPS structure and increased gene copy number and expression contribute to natural glyphosate tolerance in the three lilyturf species.

Glyphosate is a commonly applied herbicide in coffee plantations. Because of its non-selective mode of action it can damage the crop exposed through spray drift. Therefore, it is of interest to study glyphosate fate in coffee plants. The aim of this study was to develop an analytical method for accurate and precise quantification of glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) at trace levels in coffee leaves using liquid chromatography with single-quadrupole mass spectrometry detection. The method is based on a two-step solid phase extraction (SPE) with an intermediate derivatization reaction using 9-fluorenylmethylchloroformate (FMOC). An isotope dilution method was used to account for matrix effects and to enhance the confidence in analyte identification. The limit of quantification (LOQ) for glyphosate and AMPA in coffee leaves was 41 and 111 μg kg−1 dry weight, respectively. For the method optimization a design of experiments (DOE) approach was used. The sample clean-up procedure can be simplified for the analysis of less challenging matrices, for laboratories having a tandem mass spectrometry detector and for cases in which quantification limits above 0.1 mg kg−1 are acceptable, which is often the case for glyphosate. The method is robust, possesses high identification confidence, while being suitable for most commercial and academic laboratories. All leaf samples from five coffee fields analyzed (n=21) contained glyphosate, while AMPA was absent. The simplified clean-up procedure was successfully validated for coffee leaves, rice, black beans and river water.

Weedy rice infests paddy fields worldwide at an alarmingly increasing rate. There is substantial evidence indicating that many weedy rice forms originated from or are closely related to cultivated rice. There is suspicion that the outbreak of weedy rice in China may be related to widely grown hybrid rice due to its heterosis and the diversity of its progeny, but this notion remains unsupported by direct evidence. We screened weedy rice accessions by both genetic and molecular marker tests for the cytoplasmic male sterility (CMS) genes (Wild abortive, WA, and Boro type, BT) most widely used in the production of indica and japonica three-line hybrid rice as a diagnostic trait of direct parenthood. Sixteen weedy rice accessions of the 358 tested (4.5%) contained the CMS-WA gene; none contained the CMS-BT gene. These 16 accessions represent weedy rices recently evolved from maternal hybrid rice derivatives, given the primarily maternal inheritance of this trait. Our results provide key direct evidence that hybrid rice can be involved in the evolution of some weedy rice accessions, but is not a primary factor in the recent outbreak of weedy rice in China.