Physiological, biochemical and molecular characterization of multiple herbicide resistance in Palmer amaranth (Amaranthus palmeri)

Doctor of PhilosophyDepartment of AgronomyMithila JugulamPalmer amaranth (Amaranthus palmeri) is one of the most aggressive, troublesome and damaging broadleaf weeds in many cropping systems including corn, soybean, cotton, and grain sorghum causing huge yield losses across the USA. As a result of extensive and intensive selection of pre- and -post emergence herbicides, Palmer amaranth has evolved resistance to multiple herbicide modes of action, microtubule-, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-, acetolactate synthase (ALS)-, photosystem II (PS II)-, hydroxyphenylpyruvate dioxygenase (HPPD)- and more recently to protoporphyrinogen oxidase (PPO)-inhibitors. A Palmer amaranth population from Kansas was found resistant to HPPD-, PS II-, and ALS-inhibitors. The overall objective of this research was to investigate the target-site and/or non-target-site resistance mechanisms in Palmer amaranth from KS (KSR) to mesotrione (HPPD-inhibitor), atrazine (PS II-inhibitor), and chlorsulfuron (ALS-inhibitor) relative to known susceptible Palmer amaranth from Mississippi (MSS) and KS (KSS). Whole plant dose-response assays showed high level of resistance in KSR to mesotrione, atrazine and chlorsulfuron. KSR was 10-18, 178-237 and >275 fold more resistant to mesotrione, atrazine, and chlorsulfuron, respectively, compared to MSS and KSS. Metabolism studies using [¹⁴C] labeled mesotrione and atrazine demonstrated non-target-site resistance to both herbicides, particularly, enhanced metabolism of [¹⁴C] mesotrione likely mediated by cytochrome P450 monooxygenases and rapid degradation of [¹⁴C] atrazine by glutathione S-transferases (GSTs). In addition, molecular and biochemical basis of mesotrione resistance was characterized by quantitative PCR (qPCR) and immunoblotting. These results showed 4-12 fold increased levels of the HPPD transcript and positively correlated with the increased HPPD protein. Sequencing of atrazine and chlorsulfuron target genes, psbA and ALS, respectively, showed interesting results. The most common mutation (serine264glycine) associated with atrazine resistance in weeds was not found in KSR. On the other hand, a well-known mutation (proline197serine) associated with chlorsulfuron resistance was found in 30% of KSR, suggesting ~70% of plants might have a non-target-site, possibly P450 mediated metabolism based resistance. Over all, KSR evolved both non-target-site and target-site based mechanisms to mesotrione and chlorsulfuron with only non-target-site based mechanism of resistance to atrazine leaving fewer options for weed control, especially in no-till crop production systems. Such multiple herbicide resistant Palmer amaranth populations are a serious threat to sustainable weed management because metabolism-based resistance may confer resistance to other herbicides and even those that are yet to be discovered. The findings of this research are novel and valuable to recommend appropriate weed management strategies in the region and should include diversified tactics to prevent evolution and spread of multiple herbicide resistance in Palmer amaranth

Systems level analysis of two-component signal transduction systems in Erwinia amylovora: Role in virulence, regulation of amylovoran biosynthesis and swarming motility

<p>Abstract</p> <p>Background</p> <p>Two-component signal transduction systems (TCSTs), consisting of a histidine kinase (HK) and a response regulator (RR), represent a major paradigm for signal transduction in prokaryotes. TCSTs play critical roles in sensing and responding to environmental conditions, and in bacterial pathogenesis. Most TCSTs in <it>Erwinia amylovora </it>have either not been identified or have not yet been studied.</p> <p>Results</p> <p>We used a systems approach to identify TCST and related signal transduction genes in the genome of <it>E. amylovora</it>. Comparative genomic analysis of TCSTs indicated that <it>E. amylovora </it>TCSTs were closely related to those of <it>Erwinia tasmaniensis</it>, a saprophytic enterobacterium isolated from apple flowers, and to other enterobacteria. Forty-six TCST genes in <it>E. amylovora </it>including 17 sensor kinases, three hybrid kinases, 20 DNA- or ligand-binding RRs, four RRs with enzymatic output domain (EAL-GGDEF proteins), and two kinases were characterized in this study. A systematic TCST gene-knockout experiment was conducted, generating a total of 59 single-, double-, and triple-mutants. Virulence assays revealed that five of these mutants were non-pathogenic on immature pear fruits. Results from phenotypic characterization and gene expression experiments indicated that several groups of TCST systems in <it>E. amylovora </it>control amylovoran biosynthesis, one of two major virulence factors in <it>E. amylovora</it>. Both negative and positive regulators of amylovoran biosynthesis were identified, indicating a complex network may control this important feature of pathogenesis. Positive (non-motile, EnvZ/OmpR), negative (hypermotile, GrrS/GrrA), and intermediate regulators for swarming motility in <it>E. amylovora </it>were also identified.</p> <p>Conclusion</p> <p>Our results demonstrated that TCSTs in <it>E. amylovora </it>played major roles in virulence on immature pear fruit and in regulating amylovoran biosynthesis and swarming motility. This suggested presence of regulatory networks governing expression of critical virulence genes in <it>E. amylovora</it>.</p

Time course of <i>HPPD</i> expression levels (a) relative to <i>CPS</i> and (b) <i>β</i>-tubulin in mesotrione-treated (105 g ai ha^-1, 0.85% w/v AMS, and 1% v/v COC) Palmer amaranth leaves under three different temperatures (low temperature, LT, 25/15°C; optimum temperature, OT, 32.5/22.5°C; and high temperature, HT, 40/30°C; 15/9 h day/night).

<p>Total RNA was extracted from a bulked sample of Palmer amaranth leaves. Data represent means of two to three biological samples. Error bars represent SE.</p

Whole-plant mesotrione dose-response of Palmer amaranth at different temperatures (low temperature, LT, 25/15°C; optimum temperature, OT, 32.5/22.5°C; and high temperature, HT, 40/30°C; 15/9 h day/night) as measured by (a) plant height 3 weeks after treatment (WAT), (b) visual injury 3 WAT, (c) leaf chlorophyll index 2 WAT, and (d) photochemical efficiency of PSII (<i>F</i>_<i>v</i><i>/F</i>_<i>m</i>) 2 WAT.

<p>Palmer amaranth plants (10 to12 cm tall, 8 to 10 leaf stage) were treated with 0, 3.28, 6.563, 13.125, 26.25, 52.5, 105, and 210 g ai ha^-1 mesotrione with 1% v/v crop oil concentrate (COC) and 0.85% w/v ammonium sulphate (AMS). Curves for height and visual injury, and chlorophyll index and <i>F</i>_<i>v</i><i>/F</i>_<i>m</i> data were fitted using three parameter log-logistic and Weibull model, respectively, as described by Knezevic et al. (2007).</p

Photographs of mesotrione-treated Palmer amaranth plants grown under (a) LT (25/15°C, day/night), (b) OT (32.5/22.5°C, day/night), and (c) HT (40/30°C, day/night) conditions (15/9 h day/night).

<p>Plant to plant variability was observed within the growth temperature and mesotrione rate. These are the representative plants for each dose and temperature. The photographs were taken 4 weeks after treatment and all photographs were taken under the same magnification.</p

Representative reverse-phase HPLC chromatograms of mesotrione metabolism in Palmer amaranth plants grown under (a) LT (25/15°C day/night), (b) OT (32.5/22.5°C day/night, and (c) HT (40/30°C day/night) conditions (15/9 h day/night) at 48 h after treatment.

<p>Peak retention time around 18.3 min represents the parent mesotrione compound. Palmer amaranth plants (10 to 12 cm tall) were treated with 8- x 2.5-μL droplets (1.6548 mM mesotrione, 0.85% w/v AMS, and 1% COC) containing 7.2 kBq of [¹⁴C] mesotrione on the upper surface of fourth and fifth youngest leaves. Numbers above the peaks represent retention time (min).</p

[¹⁴C] mesotrione absorption (a), translocation (b), total recovery (c), and translocation to treated-leaf (d), above treated-leaf (e) and below treated-leaf (f) at three different temperatures (low temperature, LT, 25/15°C; optimum temperature, OT, 32.5/22.5°C; and high temperature, HT, 40/30°C; 15/9 h day/night).

<p>The upper surface of fourth youngest leaf of Palmer amaranth plants (10 to 12 cm tall, 8 to 10 leaf stage) were treated with 4- x 2.5-μL droplets (1.6548 mM mesotrione, 0.85% w/v AMS, and 1% COC) containing 3.3 kBq of [¹⁴C] mesotrione. Significant differences (within harvest time) between the OT and LT (blue asterisks) or HT (red asterisks) plants are indicated with asterisks (*, P ≤ 0.05; **, P < 0.01). Error bars represent SE.</p