Hongjuan Huang

Mutations and amplification of EPSPS gene confer resistance to glyphosate in goosegrass (Eleusine indica)

AbstractMain conclusionField-evolved resistance of goosegrass to glyphosate is due to double or single mutation inEPSPS, or amplification ofEPSPSleads to increased transcription and protein levels. Glyphosate has been used widely in the south of China. The high selection pressure from glyphosate use has led to the evolution of resistance to glyphosate in weeds. We investigated the molecular mechanisms of three recently discovered glyphosate-resistant Eleusine indica populations (R1, R2 and R3). The results showed that R1 and R2 had double Thr102Ile and Pro106Ser mutation and a single mutation of Pro106Leu in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, respectively. Escherichia coli containing the mutated EPSPS genes was tolerant to glyphosate. EPSPS activity in R1 and R2 plants was higher than in the sensitive plants. There was no amino acid substitution in EPSPS gene in R3. However, expression of EPSPS in R3 plants was higher than in glyphosate-susceptible (S) population (13.8-fold) after glyphosate treatment. EPSPS enzyme activity in both R3 and S plants was inhibited by glyphosate, while shikimate accumulation in R3 was significantly lower than for the S population. Further analysis revealed that the genome of R3 contained 28.3-fold more copies of the EPSPS gene than that of susceptible population. EPSPS expression was positively correlated with copy number of EPSPS. In conclusion, mutation of the EPSPS gene and increased EPSPS expression are part of the molecular mechanisms of resistance to glyphosate in Eleusine indica.

Rapid and Effective Methods for Breaking Seed Dormancy in Buffalobur (Solanum rostratum)

Abstract The spread of buffalobur in China poses a serious threat to existing ecosystems, and control and eradication of this species have become increasingly important. Studies were carried out to ascertain the seed production, morphological characterization, dormancy behavior, and methods for breaking dormancy of buffalobur. The results showed that a single buffalobur plant could produce 1,600 to 43,800 seeds with an average weight of 3.0 mg. Average seed length, width, and thickness were 2.5, 2.0, and 1.0 mm, respectively. Newly ripened buffalobur seeds were innately dormant and exhibited combinational dormancy, which involves a hard seed coat (physical dormancy, PY), a partial dormant embryo (physiological dormancy, PD), and a dark requirement to germinate. PY of buffalobur seeds could be broken by dehusking or acid scarification by 14 M H2SO4 for 15 min, with germination rates of 55 or 50%, respectively. PD was effectively broken by KNO3 or gibberellic acid (GA3). The optimum concentration for KNO3 was between 20 and 40 mM, which resulted in over 70% seed germination. When presoaked with GA3 at 30 C in dark for 24 h, maximum germination (> 98%) was obtained at 2.4 mM, the corresponding germination speed (85%) and germination index (16) were also highest at this concentration. Synergistic effects were observed in seed germination when H2SO4 and GA3 were combined. The most rapid and effective combination in breaking dormancy was when the seeds were immersed in H2SO4 (14 M) for 20 min and presoaked with 2.4 mM GA3 for 24 h. Germination index for this combination was over 35, and 95% of the seeds germinated within 7 d. Knowledge gained in this study will be useful in increasing germination of buffalobur and facilitating further laboratory studies. Nomenclature: Buffalobur, Solanum rostratum Dunal SOLCU

Factors Affecting Buffalobur (Solanum rostratum) Seed Germination and Seedling Emergence

Abstract Buffalobur is a noxious and invasive weed species native to North America. The influence of environmental factors on seed germination and seedling emergence of buffalobur were evaluated in laboratory and greenhouse experiments. The germination of buffalobur seeds occurred at temperatures ranging from 12.5 to 45 C, with optimum germination attained between 25 and 35 C. Buffalobur seeds germinated equally well under both a 14-h photoperiod and continuous darkness; however, prolonged light exposure (≥ 16 h) significantly inhibited the seed germination. Buffalobur seed is rather tolerant to low water potential and high salt stress, as germination was 28 and 52% at osmotic potentials of −1.1 MPa and salinity level of 160 mM, respectively. Medium pH has no significant effect on seed germination; germination was greater than 95% over a broad pH range from 3 to 10. Seedling emergence was higher (85%) for seeds buried at a soil depth of 2 cm than for those placed on the soil surface (32%), but no seedlings emerged when burial depth reached 8 cm. Knowledge of germination biology of buffalobur obtained in this study will be useful in predicting the potential distribution area and developing effective management strategies for this species. Nomenclature: Buffalobur, Solanum rostratum Dunal SOLCU.

Investigating the mechanisms of glyphosate resistance in goosegrass (Eleusine indica (L.) Gaertn.) by RNA sequencing technology

Summary Glyphosate is an important non‐selective herbicide that is in common use worldwide. However, evolved glyphosate‐resistant (GR) weeds significantly affect crop yields. Unfortunately, the mechanisms underlying resistance in GR weeds, such as goosegrass (Eleusine indica (L.) Gaertn.), an annual weed found worldwide, have not been fully elucidated. In this study, transcriptome analysis was conducted to further assess the potential mechanisms of glyphosate resistance in goosegrass. The RNA sequencing libraries generated 24 597 462 clean reads. De novo assembly analysis produced 48 852 UniGenes with an average length of 847 bp. All UniGenes were annotated using seven databases. Sixteen candidate differentially expressed genes selected by digital gene expression analysis were validated by quantitative real‐time PCR (qRT‐PCR). Among these UniGenes, the EPSPS and PFK genes were constitutively up‐regulated in resistant (R) individuals and showed a higher copy number than that in susceptible (S) individuals. The expressions of four UniGenes relevant to photosynthesis were inhibited by glyphosate in S individuals, and this toxic response was confirmed by gas exchange analysis. Two UniGenes annotated as glutathione transferase (GST) were constitutively up‐regulated in R individuals, and were induced by glyphosate both in R and S. In addition, the GST activities in R individuals were higher than in S. Our research confirmed that two UniGenes (PFK, EPSPS) were strongly associated with target resistance, and two GST‐annotated UniGenes may play a role in metabolic glyphosate resistance in goosegrass. Significance Statement Glyphosate‐resistant weeds affect crop yields. Here we used RNA‐seq to explore mechanisms of resistance in glyphosate‐resistant populations of goosegrass. In addition to the expected target of glyphosate, EPSP synthase, increased expression of phosphofructokinase and of glutathione S‐ transferases were strongly associated with glyphosate resistance, suggesting that they might play a role in metabolic resistance.

Characterization of glyphosate-resistant goosegrass（Eleusine indica） populations in China

Abstract Goosegrass is a worst grass weed in orchards and turf. The increased use of glyphosate for goosegrass control has led to the occurrence of many resistant populations. Although glyphosate has been used to control weeds for the past 30 years in China, few reports are available on glyphosate-resistant (GR) googegrass. In this study, we determined the GR level of 14 goosegrass populations from Chengdu and Guangzhou, China. Glyphosate only controlled 3.1 and 25.0% of the populations SL5 and SL1, respectively, at the dose of 1 680 g acid equivalent (ae) ha −1 at 14 days after treatment (DAT). In contrast, the susceptible population (XD1) was completely (100%) controlled. The resistant index (RI) of SL5 and SL1 were 5.1 and 4.5, and the RI for SL2, SL3 and ZC1 were 4.2, 3.2 and 2.6, respectively. The RI for other populations was range from 1.8 to 2.5. Under the dose of glyphosate at 1 640 g ae ha −1 at 10 DAT, shikimate accumulation in susceptible population XD1 was 17.6 and 16.4 times higher than SL5 and SL1, respectively. And the chlorophyll content in the plant leaf of populations SL1, SL2 and SL5 were decreased slightly ranging from 22.6 to 28.0. These results confirmed that the SL1, SL2, SL3, ZC1 and SL5 populations had evolved moderate resistance to glyphosate. This is the first report for the GR goosegrass populations confirmed in Chengdu, China.

Target-site basis for resistance to imazethapyr in redroot amaranth (Amaranthus retroflexus L.).

Experiments were conducted to confirm imazethapyr resistance in redroot amaranth (Amaranthus retroflexus L.) and study the target-site based mechanism for the resistance. Whole-plant response experiments revealed that the resistant (R) population exhibited 19.16 fold resistance to imazethapyr compared with the susceptible (S) population. In vitro ALS activity assay demonstrated that the imazethapyr I50 value of the R population was 21.33 times greater than that of the S population. However, qRT-PCR analysis revealed that there is no difference in ALS gene expression between the R and S populations. Sequence analysis revealed an Asp-376-Glu substitution in ALS in the R population. In order to verify that the imazethapyr resistance was conferred by Asp-376-Glu mutation, the ALS-R and ALS-S genes were fused to the CaMV 35S promoter and introduced into Arabidopsis respectively. The expression of ALS-R in transgenic Arabidopsis plants exhibited 13.79 fold resistance to imazethapyr compared to ALS-S transgenic Arabidopsis.

Tribenuron-Methyl Resistant Flixweed(Descurainia sophia)

Abstract Flixweed seeds were collected from suspected winter wheat fields and remote hillside in Shaanxi Province, China, their sensitivities to tribenuron-methyl were evaluated in the greenhouse. Results revealed that biotype S was susceptible to tribenuron, and its GR 50 was 0.23 g a.i. ha −1 , whereas biotypes R1, R2, R3, and R4 were resistant to the tribenuron, and their GR 50 were 161.99, 79.70, 439.80, and 312.30, respectively. Biotypes R1 and R2 showed moderate resistance with resistant indices (RI) of 615.23 and 302.7, respectively. Whereas biotypes R3 and R4 showed high resistance to the herbicide with RI values as high as 1 670.34 and 1186.10, respectively.

Selection of relatively exact reference genes for gene expression studies in goosegrass ( Eleusine indica ) under herbicide stress

Goosegrass (Eleusine indica) is one of the most serious annual grassy weeds worldwide, and its evolved herbicide-resistant populations are more difficult to control. Quantitative real-time PCR (qPCR) is a common technique for investigating the resistance mechanism; however, there is as yet no report on the systematic selection of stable reference genes for goosegrass. This study proposed to test the expression stability of 9 candidate reference genes in goosegrass in different tissues and developmental stages and under stress from three types of herbicide. The results show that for different developmental stages and organs (control), eukaryotic initiation factor 4 A (eIF-4) is the most stable reference gene. Chloroplast acetolactate synthase (ALS) is the most stable reference gene under glyphosate stress. Under glufosinate stress, eIF-4 is the best reference gene. Ubiquitin-conjugating enzyme (UCE) is the most stable reference gene under quizalofop-p-ethyl stress. The gene eIF-4 is the recommended reference gene for goosegrass under the stress of all three herbicides. Moreover, pairwise analysis showed that seven reference genes were sufficient to normalize the gene expression data under three herbicides treatment. This study provides a list of reliable reference genes for transcript normalization in goosegrass, which will facilitate resistance mechanism studies in this weed species.

Molecular basis of resistance to imazethapyr in redroot pigweed (Amaranthus retroflexus L.) populations from China.

Three putative resistant Amaranthus retroflexus L. populations were collected in Heilongjiang province in China. Whole plant bioassays indicated high resistance (RI > 10) to imazethapyr in the three populations. In vitro acetolactate synthase (ALS) assays revealed that ALS from populations H3, H17 and H39 was less sensitive to imazethapyr inhibition compared to the susceptible population H76. The half-maximal inhibitory concentration (I50) values for H3, H17 and H39 were 14.83, 15.27 and 268 times greater, respectively, than that of the susceptible population H76. Three nucleotide mutations resulted in three known resistance-endowing amino acid substitutions, Ala-205-Val, Trp-574-Leu and Ser-653-Thr in the three resistant populations respectively. Therefore, ALS target-site mutations in resistant A. retroflexus could be responsible for imazethapyr resistance.

Characterization of Eleusine indica with gene mutation or amplification in EPSPS to glyphosate

The evolution of weed-resistant species threatens the sustainable use of glyphosate, which is the most important herbicide widely used in agriculture worldwide. Moreover, the high glyphosate resistance (>180-fold based on LD50) of Eleusine indica found in Malaysia, which carries a double mutation in its 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), made the control of this species more difficult. By contrast, the same species carrying the same double mutation in EPSPS (T102I+P106S) but found in China only shows a resistance level of not more than 14-fold based on GR50. The resistance level of this population is four times higher than that of the population carrying a single mutation (P106L). Although the members of this population survive under a high glyphosate dosage of 10,080gaeha-1, their growth was significantly inhibited by glyphosate under the recommend dose (840gaeha-1), where in the fresh weight was 85.4% of the control. EPSPS expression, relative copy number, and EPSPS activity in this population were similar to those of the susceptible population. In addition, the expression of two glutathione transferase (GST) genes (GST-U8 and GST-23) and the enzyme activity of the GST in this population did not significantly differ from those of the susceptible population. This finding is important in elucidating the resistance of the naturally evolved glyphosate-resistant (GR) weed species carrying a double mutation in EPSPS to glyphosate.