Feral rice from introgression of weedy rice genes into transgenic herbicide-resistant hybrid-rice progeny.

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 (F1), and their succeeding generations (F2-F4) 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 F3 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 F2 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 agro-ecological risk than transgenic varieties. The safety assessment and management regulations for transgenic hybrid rice should take into account the risk of bidirectional gene flow.

Error-prone PCR mutation of Ls-EPSPS gene from Liriope spicata conferring to its enhanced glyphosate-resistance.

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.

Multiple mechanism confers natural tolerance of three lilyturf species to glyphosate.

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 spray drift in Coffea arabica - sensitivity of coffee plants and possible use of shikimic acid as a biomarker for glyphosate exposure.

Glyphosate is widely used in coffee plantations to control weeds. Lacking selectivity, glyphosate spray drift is suspected to cause adverse effects in coffee plants. Symptoms caused by glyphosate can be similar to those produced by other stress factors. However, shikimic acid accumulation should be a useful biomarker for glyphosate exposure as shown for other crops. The aim of this study was to assess the sensitivity of coffee plants towards glyphosate on different biological response variables and to evaluate the use of shikimic acid as biomarker. Dose-response experiments yielded ED50 values (50% effect dose) in the range of 38-550 ga.e.ha(-1) depending on the quantitative or qualitative variable monitored. The frequency of plants showing symptoms was the most sensitive variable. The best sampling time for shikimic acid accumulation was 1-2 weeks after glyphosate application, depending on experimental conditions. The highest shikimic acid accumulation was observed in young leaves. Shikimic acid is a suitable biomarker for a glyphosate exposure in coffee, using only young leaves for the analysis. Young coffee plants are susceptible to glyphosate damage. If symptoms are absent the risk of severe crop damage or yield loss is low.

Action of the fungal compound citrinin, a bioherbicide candidate, on photosystem II.

BACKGROUND: Bioherbicides 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. RESULTS: Citrinin 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 QA 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 QB 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. CONCLUSION: Citrinin 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. © 2023 Society of Chemical Industry.

A fungal Bipolaris bicolor strain as a potential bioherbicide for goosegrass (Eleusine indica) control.

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 × 104 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.

Structure-based ligand design and discovery of novel tenuazonic acid derivatives with high herbicidal activity.

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.

Comparative phytotoxicity of usnic acid, salicylic acid, cinnamic acid and benzoic acid on photosynthetic apparatus of Chlamydomonas reinhardtii.

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.

Enhanced photosynthesis endows seedling growth vigour contributing to the competitive dominance of weedy rice over cultivated rice.

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.

TeA is a key virulence factor for Alternaria alternata (Fr.) Keissler infection of its host.

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.

Analysis of glyphosate and aminomethylphosphonic acid in leaves from Coffea arabica using high performance liquid chromatography with quadrupole mass spectrometry detection.

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.

Cytoplasmic-genetic male sterility gene provides direct evidence for some hybrid rice recently evolving into weedy rice.

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.

A strategy to provide long-term control of weedy rice while mitigating herbicide resistance transgene flow, and its potential use for other crops with related weeds.

Transgenic herbicide-resistant rice is needed to control weeds that have evolved herbicide resistance, as well as for the weedy (feral, red) rice problem, which has been exacerbated by shifting to direct seeding throughout the world-firstly in Europe and the Americas, and now in Asia, as well as in parts of Africa. Transplanting had been the major method of weedy rice control. Experience with imidazolinone-resistant rice shows that gene flow to weedy rice is rapid, negating the utility of the technology. Transgenic technologies are available that can contain herbicide resistance within the crop (cleistogamy, male sterility, targeting to chloroplast genome, etc.), but such technologies are leaky. Mitigation technologies tandemly couple (genetically link) the gene of choice (herbicide resistance) with mitigation genes that are neutral or good for the crop, but render hybrids with weedy rice and their offspring unfit to compete. Mitigation genes confer traits such as non-shattering, dwarfism, no secondary dormancy and herbicide sensitivity. It is proposed to use glyphosate and glufosinate resistances separately as genes of choice, and glufosinate, glyphosate and bentazone susceptibilities as mitigating genes, with a six-season rotation where each stage kills transgenic crop volunteers and transgenic crop x weed hybrids from the previous season.