Genetic diversity and herbicide resistance of 15 Echinochloa crus-galli populations to quinclorac in Mekong Delta of Vietnam and Arkansas of United States.. Efficacy of Rinskor florpyr
Trang 1MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY
SUMMARY OF DOCTORAL DISSERTATION
Specialization: Plant Protection
Code: 9620112
LE DUY
STUDY ON THE RESISTANCE MECHANISM OF
BARNYARDGRASS (Echinochloa crus-galli (L.) Beauv.)
TO QUINCLORAC
IN THE MEKONG DELTA OF VIETNAM
Can Tho, 2018
Trang 2The dissertation was completed at College of Agriculture and
Applied biology, Can Tho University
Scientific supervisor:
Associate Professor PhD Nguyen Minh Chon
Reviewer 1:
Reviewer 2:
Reviewer 3:
………
The thesis is defended in front of the University Examination Council in Can Tho University Time:………… ………
Date:……….………
Further information of the thesis could be found at:
1 Learning Resource Center of Can Tho University
2 National library of Vietnam
Trang 3PUBLISHED RESEARCH
1 Duy Le, C M Nguyen, R K Mann, C N Yerkes and B V
N Kumar 2017 Genetic diversity and herbicide resistance of
15 Echinochloa crus-galli populations to quinclorac in
Mekong Delta of Vietnam and Arkansas of United States
Journal of Plant Biotechnology, 44: 472-477
2 Duy Le, C M Nguyen, R K Mann, B V N Kumar and
M Morell 2018 Efficacy of Rinskor (florpyrauxifen-benzyl
ester) on Herbicide Resistant Barnyardgrass (Echinochloa crus-galli) in Rice Fields of Mekong Delta, Vietnam Journal
of Crop Science and Biotechnology, 21: 75-81
3 Duy Le, C M Nguyen, B V N Kumar and R K Mann
2018 Weed management practices to control
herbicide-resistant Echinochloa crus galli in rice in Mekong Delta, Vietnam Research On Crops , 19: 20-27
Trang 4Chapter 1: INTRODUCTION 1.1 Problem statement
The barnyardgrass (Echinochloa crus-galli) had been
considered as the most notorious for a long time, as a C4 plant, the growth rate of barnyardgrass is far dominant to rice In addition,
Echinochloa crus-galli and Echinochloa colona can mimic the rice
appearance at the early stage of weed seedlings and make it hard to control by hand weeding Herbicide is an effective solution for controlling the barnyardgrass, however, the herbicide resistant barnyardgrass is a critical issue to the weed management in the rice field The barnyardgrass could evolve resistance to several current herbicide active ingredients Further research about herbicide resistance in barnyardgrass is critical As an outcome, the
dissertation of “Study on the resistance mechanism of barnyardgrass (Echinochloa crus-galli) to quinclorac in the Mekong Delta of Vietnam” was proposed
1.2 Objective of dissertation
(1) Evaluate the morphological variability and genetic
diversity of Echinochloa spp populations in Mekong Delta of
Vietnam
(2) Evaluate the herbicide-resistance level of Echinochloa spp
to bispyribac, penoxsulam and quinclorac in rice fields at the Mekong Delta of Vietnam
(3) Explain the biochemical mechanism and the molecular
mechanism of quinclorac-resistance in Echinochloa crus-galli
1.3 Major research topics of the dissertation
(1) Survey the rice cultivation and weed management practice
in rice fields at the Mekong Delta of Vietnam
(2) Evaluate the diversity of Echinochloa spp population in
rice fields at the Mekong Delta of Vietnam
(3) Evaluate the herbicide-resistance level of the collected
Echinochloa spp samples to bispyribac-sodium, penoxsulam and
quinclorac by dose-response screening method
Trang 5(4) Evaluate the efficacy of rinskor, a new herbicide against
the herbicide-resistant Echinochloa crus-galli to find new effective
herbicide for current herbicide-resistant barnyardgrass
(5) Use RAPD analysis to evaluate the genetic diversity of
Echinochloa crus-galli populations, and the correspondence of
quinclorac-resistance and genetic distance of Echinochloa crus-galli
populations in the Mekong delta
(6) Measure the activity of β-cyanoalanine synthase in the leaf
tissue of Echinochloa crus-galli to study the biochemical mechanism
of quinclorac-resistance in barnyardgrass
1.4 Time and location
Time of research was from 2014 to 2018
Experiment 1; 2 and 3 was carried out at households in College of Agriculture and Applied Biology, Can Tho University;
Experiment 4; 5 and 6 in Discovery Center of Dow AgroSciences in Indianapolis, Indiana State, United States of America
1.5 Novel aspects
The research has confirmed the existence of
herbicide-resistant Echinochloa spp populations in Mekong Delta of Vietnam
This research also evaluates the relationship between farmers’ weed management practice and the herbicide-resistance, therefore, the practical solutions for herbicide-resistant weed were also determined and suggested in the dissertation
The mechanisms of quinclorac-resistance in barnyardgrass were confirmed and elucidated at enzyme and molecular level, through the measurement of quinclorac detoxifying enzyme activity and its gene expression level in barnyardgrass The results establish important information for the further study about the mechanism of the herbicide-resistance in weeds
1.6 Layout
The dissertation consists of 102 pages (21 tables, 38 pictures and 202 references) In that, introduction: 3 pages, background: 33 pages; materials and methods: 18 pages; results and discussion 43 pages; conclusions and suggestions: 2 page; reference: 18 pages; appendix 42 pages for both mumber and pictures
Trang 6Chapter 2: BACKGROUND OF DISSERTATION
The genus Echinochloa includes over 250 species, and most
of these are considered as weeds The barnyardgrass (Echinochloa
spp.) is C4 monocots and originated from Europe and has been now
documented worldwide The morphology of Echinochloa spp is
highly diverse, in many cases, there are several species confused with one another In Asia, rice fields have three main barnyardgrass species, Echinochloa crus-galli, Echinocloa colona, and
Echinochloa glabrescens, which found in Vietnam
The bispyribac and penoxsulam are two popular ALS inhibitor herbicide on rice, those two herbicide is mainly used for barnyardgrass control in Vietnam Quinclorac is a synthetic auxin herbicide, and the product contains quinclorac is wide used for controlling barnyardgrass in the rice field Rinskor is a new synthetic auxin herbicide, and this herbicide is not used for weed management
in Vietnam when the dissertation started in 2014
The weed seed of 78 populations of Echinochloa spp
collected from rice field in 7 provinces of the Mekong Delta of Vietnam, the seedling from collected seed was tested with bispyribac, penoxsulam, quinclorac and rinskor to evaluating the
LD90 and herbicide resistance level To continue the study on the mechanism of resistance, 15 selected weed populations were selected for genetic distance analysis by using the RAPD method The activity of cyanoalanide synthase was analyzed to evaluate the enzyme level in leaf of 5 different weed populations under the treatment of quinclorac Finally, the expression level of mRNA of cyanoalanide synthase in 5 populations was measured by the comparative methods of 2−ΔCt, the activity of enzyme and the expression level of mRNA in the 5 populations was analyzed to find the association of two value, the result of this research was used to supporting the study of herbicide mechanism of quinclorac in barnyardgrass
Trang 7CHAPTER 3: MATERIALS AND METHODS
3.1 Experiment 1 Survey on farmer practice in rice cultivation and weed management in the Mekong Delta
Target of research: Study on the farmer practice of weed
management on the rice field in Mekong Delta of Vietnam
Barnyardgrass seed sample was collected from total 90 rice field, after germination test in greenhouse, 78 quality samples wer e selected for further experiments During the weed seed sample collecting on the field, the researcher had interviewed the field owner about the cultivation practice in the field, the questions were included 9 questions about:
- Number of rice seasons per year
- Field size that he/she is growing rice (hectare)
- Number of herbicide application per season
- Condition of water management
- Perception about the important weed species in rice field
- Most popular herbicides used for controlling barnyardgrass
- Cost for weed management per hectare per season
- Cost for herbicide per hectare per season
- Cost for hand-weeding per hectare per season
The questions were designed to get essential information about the field condition, and the weed management practice of the field owner, the questions were asked after seed sample collected, there were 17 seed samples collected without input from farmer
Statistical analysis
After cross-check, the inputs with unreliable information or unclear data were excluded from the final analysis, final answer from 71 farmers were processed for analysis
Collected data were analyzed using ANOVA with mean comparison by Tukey-Kramer test at α=0.05 One way ANOVA t-test with p<0.05 was used for analyzing the mean of field size and hand-weeding The analysis was run by statistical software JMP Pro
13 (SAS)
Trang 83.2 Experiment 2 Classification of the collected Echinochloa spp populations based on plant characteristics
Target of research
Classify the collected Echinochloa spp populations based on
reported taxonomy keys, the purpose of this research is to generate
the initial information about the diversity of Echinochloa spp
populations in Mekong Delta, before conducting further research of
herbicide-resistance mechanism in barnyardgrass (Echinochloa
crus-galli) species
The descriptions about species of Echinochloa spp
morphology of Pignatty’ key and Carretero’s key - validated by
Tabacchi et al (2006) (Appendix M), Heap (2017) and USDA
(2017) were used to identify the species of collected samples, observations of morphology are included: (1) Plant height: measure from the basal to the top leave at 56 day after germination; (2) Plant weight: the plant dry biomass (above the soil) at 56 day after germination, the plant collected and dried down under sunlight for 7 days, until the weight of plant not change; (3) Color of basal: observe the color basal of seedling, the color was red or green for collected sample; (4) Number of node: the nodes were count at 56 day after germination; (5) Panicle emerged time: the duration from germination until the first flower fully opened; (6) Grow duration: the duration from seed germination until all the seed ripen; (7) Morphology of flower: The photo of the flower and seed was taken, then used for reference of species identification; (8) In order to minimize the possibility of using incorrect species in further assays
of enzyme and molecular analysis The information of weed species was used for selection of candidate populations in the test of population genetic diversity, enzyme activity and gene expression under effect of quinclorac
Statistical analysis
Collected data were analyzed using ANOVA with mean comparison by Tukey-Kramer test at α=0.05 One way ANOVA t-test with p<0.05 was used for analyzing the means The analysis was run by JMP Pro 13 (SAS)
Trang 93.3 Experiment 3: Evaluate the herbicide-resistance level in collected Echinochloa spp populations to 3 active ingredients of bispyribac-sodium, penoxsulam and quinclorac by dose- response screening method
Target of research
Use dose-response screening method to identify the resistant and herbicide-susceptible barnyardgrass populations in Mekong Delta, and evaluate the herbicide-resistance level of those populations to 3 active ingredients of bispyribac, penoxsulam and
herbicide-quinclorac
Herbicide-resistance level evaluation
At the 3-4 leaf stage of barnyardgrass, the weed seedlings were treated by foliar application of 3 herbicides, included bispyribac (10% SC formulation), penoxsulam (2.5% OD) and quinclorac (25% SC formulation) at 6 doses range of 0.25X, 0.5X, 1.0X, 2.0X, 4.0X and 8.0X, the X dose is label dose of each herbicide products, in which bispyribac was 25 g a.i/ha, penoxsulam was 12.5 g a.i/ha, quinclorac was 250 g a.i/ ha
The experimental design was a complete randomized design (CRD), with 4 replications, one pot per replication and 10 plants per pot, the number of plants in pot was manually controlled
Herbicide application was made in a spray booth (Research track sprayer SB-8, Devries Manufacturing), pressure was calibrated
to deliver 300 L ha−1 at 140 kPa At 14 day after treatment (DAT), the mortality rate was assessed by counting the number of surviving and completely killed plants The results of dose response were used for calculating the LD90 for each population by non-linear regression model using GraphPad Prism 7.02 (San Diego, CA) software The solo-resistance and multiple-resistance of tested herbicides was calculated based on the response of weed population to the specific herbicide, the detail of calculation as described in below statistical analysis section
Trang 10on descriptions of Moss et al (2007), where control efficacy at label
dose of susceptible (S) is 81-100%; R? (suspected resistance) is 80%; RR is 36-71% and RRR is 0-35% Values of LD90 were considered to be statistically different if the respective 98% confidence interval did not overlap The correlation between candidate factors analyzed by using regression and Pearson’s correlation analysis Regression analysis and regression graph were performed by JPM Pro 13 software (SAS)
72-3.4 Experiment 4: Evaluate the efficacy of rinskor as new herbicide in herbicide resistance barnyardgrass populations Target of research
After the herbicide screening test of bispyribac, penoxsulam and quinclorac, all 78 barnyardgrass populations were treated by rinskor 2.5% at 6 doses of 0.25X, 0.5X, 1.0X, 2.0X, 4.0X and 8.0X, the 1.0X dose was 25 g a.i/ha The % mortality of weed was assessed at 14 days after application
The results of this test also served for the recommendation of effective herbicide solution for current herbicide-resistant barnyardgrass in Mekong Delta
Statistical analysis
The method for LD90 calculation and herbicide-resistance level was similar to method described in Experiment 3
Trang 113.5 Experiment 5: Compare the activity of enzyme cyanoalanine (CAS) in quinclorac-susceptible and quinclorac- resistant barnyargrass plant to study biochemical mechanism of quinclorac-resistance in barnyardgrass
β-Target of research
Plant tissue bioassay to measure the expression level of cyanoalanine from survived plants of screening test to determine the resistance level of populations to quinclorac
β-Select candidate barnyardgrass populations and herbicide treatment
Five barnyardgrass populations that exhibited most reliable herbicide-resistance or herbicide-susceptible was selected for the enzyme assay, the candidate plants at 3-4 leaf stage were treated by quinclorac at 125 g a.i/ha (half of label dose to ensure the herbicide treatment will not kill the susceptible plant during assessment) The herbicide spraying was done in a spray booth (Research track sprayer SB-8, Devries Manufacturing), pressure was calibrated
to deliver 300 L ha−1 at 140 kPa The indica rice variety IR50404 at 3-4 leaf stage was also treated by quinclorac, the rice sample was used as reference sample for the enzyme analysis, one quinclorac-resistant population and one quinclorac-susceptible population originated from rice field of Arkansas State of United of America was used as comparison for the populations in the study
Determine CAS activity in leaf tissue
The activity of CAS enzyme in barnyardgrass leaf tissue was
analyzed based on described method of Chon et al (2008):
- One gram of fresh leaves from 4 plants in each population were collected at 1 hour and 3 days after herbicide treatment
- Leaf samples were pooled and frozen rapidly in liquid nitrogen before being homogenized with TissueLyzer II (Qigen), plant tissue was homogenized at 30 strokes per second for 1 minute, this process was repeated 2 times after switching orientation to ensure uniform mixing and tissue particle size
- Centrifuged at 10,000 g for 10 min at 4oC
Trang 12- Then 2.5 mL of 100 M Tris buffer (pH=8.5) was added into microtubes containing homogenized 1 g of homogenized leaf tissue
- The supernatant was used for enzyme assay
- 100 mM Tris buffer (pH=8.5) was used to make 2 solutions of
25 mM NaCN and 5mM L-cysteine
- NaCN and L-cysteine, supernatants, were equilibrated for 10 minutes at 30oC, mixed with 0.5 ml of enzyme extract, 2 ml of NaCN, 2 ml of L-cysteine in a sealed tube and incubated at
30oC for 60 min
- After the incubation, 1 ml aliquot of solution was transferred
to a microfuge tube that contained 0.1 ml of 0.03 M FeCl3 in
1.2 N HCl followed by 0.1 ml of 0.02 M
N,N-dimethyl-p-phenylenediamine sulfate in 7.2 N HCl
- Samples were left in dark for 20 min and then centrifuged at
1020 g for 5 minutes to precipitate proteins
- The reduction of methylene blue by hydrogen sulfide was used to determine the level of CAS activity in leaf tissue
- Enzyme activity was determined by reading absorbance of methylene blue at A650 nm in a microplate reader and Na2S was used as the standard reference
- Enzyme activity was expressed in nmol H2S/g fresh weight/min Each biotype contained 4 biological replicates
Statistical analysis
The enzyme assay was conducted as completely randomized designs, with 4 replications per population Data from 4 repeated tests were pooled for analysis, the Tukey-Kramer Test was used for multiple mean comparison at α=0.05 The tests was conducted using JPM Pro 13 (SAS) statistical software
3.6 Experiment 6: Identify genetic variation among
quinclorac-resistant and quinclorac-susceptible Echinochloa crus-galli
populations in the Mekong Delta
Target of research
The Random amplified polymorphic DNA (RAPD) technique was used to evaluate the genetic variability between different
Trang 13populations to provide value information of the popularity and the distribution pattern of quinclorac-resistant weed in Mekong Delta
Plant materials
From the results of morphology study, seeds of 15
Echinochloa spp populations were selected, the selection based on
the weed group and quinclorac-resistance level, four populations of each group were selected, only choose the populations exhibited reliable data of quinclorac-resistance or quinclorac-susceptible for the test Plant was grown in greenhouse condition, and leaves from one represented plant in each population was collected at 3-4 leaf stage
Genomic DNA extraction
Genomic DNA was isolated from leaf tissue using the DNAzol Reagent protocol (CAS No 593-84-0, Invitrogen, Thermo Scientific Corp., Waltham, MA) as followed steps:
- Fresh leaf tissue (1 g) from one represented plant in each population was collected and powdered in liquid nitrogen to prepare for DNA extraction
- Each sample was powdered by mortar and pestle, the homogenized sample (100 mg) was transferred to a microcentrifuge tube containing DNAzol (1.5 mL) and RNAse (150 µL)
- The solution was mixed and shaked for 5 min at 25oC
- Chloroform (900 µL) was added to the solution and mixed thoroughly for 5 min at 25oC
- The homogenates were centrifuged at 12,000 x g for 10 min,
and the aqueous phases were transferred to new tubes
- The aqueous phase was mixed with 100% ethanol (700 µL) to precipitate the DNA
- The tubes were inverted 6-8 times, held at room temperature (25oC) for 5 min, and then centrifuged at 5000 x g for 4 min to
pellet the DNA
- The DNA pellets were washed first in a 1:0.75 (v/v) solution
of DNAzol and 100% ethanol (900 µL) by gentle vortexing for 10 s
Trang 14- The DNAzol wash solution was then removed and 75% ethanol (700 µL, prepared with nuclease-free water) was added to the DNA pellets for a second wash step
- The mixture was centrifuged at 5000 x g for 4 min
- The ethanol wash was decanted and the pellets air dried for
1-2 min
- DNA sample was solubilized in 50 µL TE buffer, pH 8.0
- DNA concentration and purity were determined by spectrophotometric measurement via a NanoDrop instrument (Thermo Scientific Corp, USA)
Random Amplified Polymorphic DNA (RAPD) amplifications
Forty 10-base pair (bp) oligonucleotide primers (synthesized
by Integrated DNA Technology, Inc., Coralville, IA) were used for RAPD analysis
All primers were screened on the genomic DNA of the 15
Echinochloa spp populations and evaluated for repeatability, high
resolution and polymorphism The PCR master mix contained 2.7
µL ddH20, 1 µL PCR buffer (Invitrogen Cat No 18067-017), 80 µL MgCl2, 5 µM primer, 0.25 µL template DNA, 0.05 µL Taq polymerase (0.5 units), and 1 µL dNTP
The amplification protocol was carried out in a LightCycler
480 II (Roche Molecular Diagnostics, Indianapolis, IN) using the following steps:
- Initial hold at 94oC for 2 min
- Followed by 45 cycles of 94oC for 45 s (ramp rate 4.8oC/s),
- 38oC for 5 min (ramp rate of 2.5oC/s)
- 72oC for 2 min (ramp rate 4.7oC/s), and a final hold of 72oC for 7 min
- Final PCR products were analyzed by LabChip GXII (PerkinElmer, Waltham, MA)
Statistical analysis
Genetic distance and cluster analyses were conducted for 15
Echinochloa crus-galli populations using the PCR products of 6