No evidence for early fitness penalty in glyphosate‐resistant biotypes of Conyza canadensis: Common garden experiments in the absence of glyphosate

Strong selection from herbicides has led to the rapid evolution of herbicide‐resistant weeds, greatly complicating weed management efforts worldwide. In particular, overreliance on glyphosate, the active ingredient in RoundUp^®, has spurred the evolution of resistance to this herbicide in ≥40 species. Previously, we reported that Conyza canadensis (horseweed) has evolved extreme resistance to glyphosate, surviving at 40× the original 1× effective dosage. Here, we tested for underlying fitness effects of glyphosate resistance to better understand whether resistance could persist indefinitely in this self‐pollinating, annual weed. We sampled seeds from a single maternal plant (“biotype”) at each of 26 horseweed populations in Iowa, representing nine susceptible biotypes (S), eight with low‐level resistance (LR), and nine with extreme resistance (ER). In 2016 and 2017, we compared early growth rates and bolting dates of these biotypes in common garden experiments at two sites near Ames, Iowa. Nested ANOVAs showed that, as a group, ER biotypes attained similar or larger rosette size after 6 weeks compared to S or LR biotypes, which were similar to each other in size. Also, ER biotypes bolted 1–2 weeks earlier than S or LR biotypes. These fitness‐related traits also varied among biotypes within the same resistance category, and time to bolting was inversely correlated with rosette size across all biotypes. Disease symptoms affected 40% of all plants in 2016 and 78% in 2017, so we did not attempt to measure lifetime fecundity. In both years, the frequency of disease symptoms was greatest in S biotypes and similar in LR versus ER biotypes. Overall, our findings indicate there are no early growth penalty and possibly no lifetime fitness penalty associated with glyphosate resistance, including extremely strong resistance. We conclude that glyphosate resistance is likely to persist in horseweed populations, with or without continued selection pressure from exposure to glyphosate.     

Phytochemical Investigation of Cuscuta campestris Yunck. Stem Extract and Evaluation of Its Bioherbicidal Effect on Amaranthus retroflexus L. and Portulaca oleracea L

This study focused on characterizing chemically and evaluating in vitro allelopathic and bioherbicidal potential of secondary metabolites extracted from the stem of Cuscuta campestris in seed germination, early seedling growth and early plant growth of Amaranthus retroflexus and Portulaca oleracea. The combined effects of stem extract and a reduced dose of herbicide metribuzin were also examined. Plant extract contained 17 phenolic compounds and the most abundant phenols were flavonoids: quercetin, (+)-catechin, daidzin, luteolin, and rutin. The seeds of P. oleracea were less sensitive than the seeds of A. retroflexus. The seed bioassay confirmed the inhibitory effect of stem extract on germination and early growth of both weed seedlings at concentrations of 0.75 % and 1 %, and a minor inhibitory effect in the plant bioassay. On the other hand, a synergy of C. campestris stem extract and metribuzin was revealed, as their combination achieved better results in the control of both weed species. Based on obtained data C. campestris stem extract could be a potential source of natural-based weed control molecules.     

Competition at the soybean V6 stage affects root morphology and biochemical composition

• The experiment was conducted in the 2016/17 crop season in a greenhouse at Passo Fundo University, Brazil. We hypothesised that the morphological characteristics and biochemical and anatomical composition of soybean roots and shoots, when competing with weeds during different growth periods, are negatively affected, so current concepts of competition between plants should also consider changes in plant roots.
• The soybean cultivar P 95R51 and horseweed (Conyza bonariensis) were used. The treatments consisted of the presence or absence of weeds during different coexistence periods of soybean with horseweed. The periods were V0–V3, V0–V6, V0–R2, V3–R6, V6–R6 and R2–R6, where V0 was the date of soybean sowing and V3, V6, R2 and R6 were phenological stages of the crop. Two fresh roots were used to examine morphological traits. Four roots were used for quantification of dry matter and secondary metabolites.
• Root length was reduced by 21%, 14% and 20% when competing with a weed in the V0–V3, V0–V6 and R2–R6 coexistence periods, respectively. Total phenol content in the V0–V6 and V0–R2 periods was reduced when plants were in competition with weeds; a similar trend was found for flavonoids in the V0–V6 period.
• Soybean–horseweed competition from crop emergence to the V6 stage, in general, affects shoot and root morphological traits and the biochemical composition of the soybean roots. The presence of horseweed at the V3, V6 and R2 stages does not negatively alter the traits evaluated. Root anatomical composition is not modified during all coexistence periods with horseweed.

     

Study on the Physiological Mechanism of Early Flowering and Low Male Fertility of Limonium bicolor Mutant vrl15

As biennial recretohalophytes, Limonium bicolor plants need 2 years to complete their life cycle. A growth habit mutant Vernalization Requirement Loss 15 (vrl15) was obtained by ion implantation. However, the biological characteristics of the mutant were unclear. In the current study, the related traits of vrl15 and some possible reasons for these traits were examined. Compared with wild type (WT), vrl15 can bolt and flower in approximately four months without vernalization. Moreover, vrl15 needed much less time to bolting and flowering than wild-type L. bicolor under different vernalization treatments. After 20 days’ vernalization, bolting vrl15 plants had 24 rosette leaves and bolting WT had 31 rosette leaves. Moreover, the pollen number per anther, the proportion of active pollen, the seed setting rate and the 1000 seed weight of vrl15 were all lower than those of WT. The soluble sugar content and soluble protein content in leaves of the vrl15 were much higher than those of WT sowed at the same time. In addition, the GA content in the leaves of bolting vrl15 was higher than that of the non-bolting WT sowed at the same time and non-bolting vrl15, whereas the contents of ABA and BR were much lower than that of the non-bolting WT. These results indicate that to some extent the increase of GA and decrease of ABA and BR content may be involved in the growth habit and male fertility alteration of mutant vrl15 of L. bicolor.

     

Transformation and segregation of GFP fluorescence and glyphosate resistance in horseweed (<Emphasis Type="Italic">Conyza canadensis</Emphasis>) hybrids

The goal of this research was to generate a breeding population of horseweed segregating for glyphosate resistance. In order to generate a marker to select between hybrids of glyphosate resistant (GR) and glyphosate susceptible (GS) horseweed, a GR horseweed accession from western Tennessee was transformed with a green fluorescent protein (GFP) transgene. The GFP marker allowed for the simple and accurate determination of GR hybrid plants by visual observation. GR plants were shown to be transgenic via the green fluorescence under UV light, and resistant to glyphosate when sprayed with the field-use-rate 0.84 kg acid equivalent ha⁻¹ of glyphosate (i.e. Roundup^TM) herbicide. An in vitro screen for glyphosate resistance in seedlings was developed, and a 5 μM glyphosate concentration was found to reduce dry weight in GS seedlings but not in GR seedlings. The GR plants containing GFP were then hand-crossed with GS plants from eastern Tennessee under greenhouse conditions, with GS plants acting as the pollen acceptor. Resulting seed was collected and germinated for GFP fluorescence screening. Seedlings that exhibited the transgenic GFP phenotype were selected as F₁ hybrids between GR and GS horseweed. Thirty GS×GR hybrids were produced on the basis of a green-fluorescent GFP phenotype of GR plants. GS×GFP/GR F₁ hybrids produced F₂ seeds, and F₂ plants were shown to segregate for GFP fluorescence and glyphosate resistance independently. Both traits segregated at a Mendelian 3:1 ratio, indicating a single gene is responsible for each phenotype.     

Growth requirements for production of stable cells of the bioherbicidal bacterium Xanthomonas campestris

Xanthomonas campestris MB245, a specific pathogen of the weedy grass Poa annua (annual bluegrass), is being developed as a bioherbicide to control this pest in turf. Nutritional and environmental factors were evaluated based on their ability to support rapid submerged culture growth and high cell yield. Temperature optima for the growth of X. campestris cells in submerged culture were between 27 and 30°C. At 30°C, optimal nutritional conditions for X. campestris growth supported generation times of 150–175 min and cell yields after 24 h growth of 1–2 × 10¹⁰ cells ml⁻¹. Media containing sucrose or glucose as the carbon source and various organic nitrogen sources supported optimal X. campestris growth and cell yield. The addition of vitamin mixtures to complex and defined media had no significant effect on growth or cell yield. The age of X. campestris cultures had a significant impact on cell survival after freeze drying. Following freeze drying, log phase cell survival (44%) was significantly lower than early and late stationary phase cell survival, 62% and 68%, respectively. Cells harvested in stationary phase, freeze dried and stored under vacuum at 4°C, showed no significant loss in viability after 6 months. Thus, high cell concentrations of the bioherbicide X. campestris can be rapidly produced in submerged culture and stabilized as freeze-dried preparations. Received 14 August 1998/ Accepted in revised form 8 October 1998     

Differential accumulation of Xanthomonas campestris pv. campestris proteins during the interaction with the host plant: Contributions of an in vivo system

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot, a highly destructive disease that affects all brassicas. This work aimed to study the interaction Xcc–Brassica oleracea using an in vivo system in an attempt to identify proteins involved in pathogenicity. We used label‐free shotgun 2D‐nanoUPLC/MS^E to analyze Xcc proteins in three conditions: in the interaction with susceptible (REK) and resistant (REU) plants and in culture medium (control condition). A model of Xcc–susceptible host interaction is proposed and shows that Xcc increases the abundance of several crucial proteins for infection and cell protection. In this study, we also confirmed the differential expression by qPCR analysis of selected genes. This is the first report showing a large‐scale identification of proteins in an in vivo host plant condition. Considering that most studies involving phytopathogens are in vitro (growth in culture medium or in plant extract), this work contributes with relevant information related to the plant–pathogen interaction in planta.     

Stimulation effect of carrageenan on enzymatic defense system of sweet basil against Cuscuta campestris infection

Sweet basil is an important medicinal plant used especially for therapeutical potentials. Sweet basil is a common host for Cuscuta campestris, which has a negative effect on infected plants. Therefore, natural friendly control of C. campestris seems to be useful. It has been shown that carrageenans can act as elicitors of plant defense responses. In this work, the effect of κ-carrageenans on protection against C. campestris and suppression of its invasion in basils were studied. Basils were sprayed with a solution of κ-carrageenan (1?g?L^?1), once a week, three times in total. Infection of basils with C. campestris was performed two days after the last carrageenan treatment. C. campestris stem and the leaves of basils were collected two weeks after C. campestris inoculation for biochemical studies. Treatment with carrageenan significantly increased shoot length and leaf area of basil and decreased C. campestris infestation by about 26%. The content of malondialdehyde, other aldehydes, hydrogen peroxide and lipoxygenase (LOX) activity increased significantly in basils parasitized by C. campestris. There were significant differences in phenylalanine ammonia lyase (PAL), catalase (CAT), superoxide dismutase (SOD) and peroxidase activity of parasitized basils by C. campestris compared with healthy basils. Carrageenan treatment of basils caused a significant increase in H₂O₂ content and the activity of PAL, CAT and SOD, but not of malondialdehyde, other aldehydes content and LOX, polyphenol oxidase (PPO) and peroxidases activity. The activated enzymatic defense system (PAL, PPO, CAT, SOD and peroxidase) in carrageenan-treated basils have a vital role in alleviating oxidative stress damage in infected plants, by removing excess reactive oxygen species and inhibiting LOX activity and lipid peroxidation that was observed in this study. Our results showed that the application of κ-carrageenan-induced beneficial effects in plants, with regard to growth stimulation and the activation of enzymatic defense system. Thus, carrageenan was recommended as a natural biostimulator for protection of plants against C. campestris.     

PHYSIOLOGICAL AND MORPHOLOGICAL MODIFICATIONS OF PLANTAGO MAJOR (PLANTAGINACEAE) IN RESPONSE TO LIGHT CONDITIONS

Physiological and morphological differences between Plantago major L. (Plantaginaceae) growing in full sunlight and shaded conditions were examined. Photosynthesis of isolated leaves was saturated by irradiance around 300 μE m^−-2 sec^−-1 and 170 μE m^−-2 sec^−-1, respectively. In contrast to previous studies of sun/shade leaf responses, initial slopes of curves from shaded plants are significantly less than those taken from full-sun plants. Within the 400–500 nm and 600–700 nm ranges, leaves 5.0 cm or longer are essentially opaque, transmitting less than 1.25% of incident light. Chlorophyll content per unit leaf area is nearly equivalent for leaves from plants growing under the two extremes in light levels. Morphometric comparisons indicate shaded plants bear fewer leaves, have less leaf overlap, lower total leaf area, and longer petioles than full-sun plants. Leaf elongation rates are lower and the duration between the emergence of successive leaves is longer in shaded plants. Computer analyses of both types of rosette morphology reveal shaded plants have an equal or greater capacity to intercept light than full-sun plants, principally because of the minimization of leaf overlap and the large variation in the deflection angles of leaves in shaded rosette morphologies. Simulations, calculated on the basis of light interception, and taking into account the transition between photosynthate-importing and -exporting leaves, predict relative growth rates for full-sun and shaded rosette morphologies that are in reasonable agreement with empirically determined leaf growth rates. However, the data indicate that significant physiological and morphological differences exist among leaves from a single rosette, and among developmentally comparable leaves from rosettes growing under different ambient light environments. Differences among leaves on a single plant must be accommodated in computerized techniques attempting to simulate light interception and its consequences on potential growth rates.     

Wind‐mediated horseweed (Conyza canadensis) gene flow: pollen emission,dispersion, and deposition

Horseweed (Conyza canadensis) is a problem weed in crop production because of its evolved resistance to glyphosate and other herbicides. Although horseweed is mainly self-pollinating, glyphosate-resistant (GR) horseweed can pollinate glyphosate-susceptible (GS) horseweed. To the best of our knowledge, however, there are no available data on horseweed pollen production, dispersion, and deposition relative to gene flow and the evolution of resistance. To help fill this knowledge gap, a 43-day field study was performed in Champaign, Illinois, USA in 2013 to characterize horseweed atmospheric pollen emission, dispersion, and deposition. Pollen concentration and deposition, coupled with atmospheric data, were measured in a source field (180 m by 46 m) and its surrounding areas up to 1 km downwind horizontally and up to 100 m vertically. The source strength (emission rate) ranged from 0 to 140 pollen grains per plant per second (1170 to 2.1×10⁶ per plant per day). For the life of the study, the estimated number of pollen grains generated from this source field was 10.5×10¹⁰ (2.3×10⁶ per plant). The release of horseweed pollen was not strongly correlated to meteorological data and may be mainly determined by horseweed physiology. Horseweed pollen reached heights of 80 to100 m, making long-distance transport possible. Normalized (by source data) pollen deposition with distance followed a negative-power exponential curve. Normalized pollen deposition was 2.5% even at 480 m downwind from the source edge. Correlation analysis showed that close to or inside the source field at lower heights (≤3 m) vertical transport was related to vertical wind speed, while horizontal pollen transport was related to horizontal wind speed. High relative humidity prevented pollen transport at greater heights (3–100 m) and longer distances (0–1000 m) from the source. This study can contribute to the understanding of how herbicide-resistance weeds or invasive plants affect ecology through wind-mediated pollination and invasion.     

Multiplication of Xanthomonas campestris pv. Phaseoli and Intercellular Enzyme Activities in Resistant and Susceptible Beans

In order to assess resistance to common bean blight, populations of two isolates of Xanthomonas campestris pv. phaseoli were monitored in leaves of two Phaseolus vulgaris breeding lines, BLT87-2 (susceptible) and OAC88-1 (partially resistant) and a resistant tepary bean accession, P. acutifolius P. I. 440795. The breeding line OAC88-1 possesses resistance to common bacterial blight which was incorporated from P. acutifolius by an interspecific cross. In susceptible, leaves, bacterial populations increased to 10⁸ CFU/g leaf at 3 wk after inoculation whereas, in resistant leaves, bacterial populations declined to 10¹ - 10³ CFU/g leaf. In partially resistant leaves the population first declined similar to that in resistant P. acutifolius but later increased, and typical bacterial blight symptoms appeared. Cellulase, protease and amylase activities were monitored in culture and intercellular leaf spaces. Only cellulase activity was, clearly related to bacterial growth in the susceptible host; other enzyme activities were variable in their relationship to host resistance and bacterial growth. Differences between strains in cellulase activity inside partially resistant leaves corresponded to their ability to secrete cellulase in culture. Measuring cellulase activity in intercellular wash fluids may be a simple and sensitive method for determining X. c. phaseoli populations in leaves.     

抗感青枯病番茄的内生细菌数量动态分析及其对青枯病的生物防治

对番茄内生细菌数量动态及其对青枯病的生物防治研究结果表明:番茄内生细菌可来源于种子内部。番茄不同生育期,内生细菌数量最多在成株期,其中抗病品种根、茎分别为24.3×104CFU/g鲜重和22.9×104CFU/g鲜重,感病品种根、茎分别为9.8×104CFU/g鲜重和13.4×104CFU/g鲜重。抗病品种中具有拮抗青枯菌的内生细菌菌株为17个,感病品种中7个。部分内生细菌具促进番茄种子萌发和防治番茄青枯病的作用,其中5R和3R内生菌株的防病效果分别达91.7%和81.3%。     

Mode of action of paraquat in leaves of paraquat-resistant Conyza canadensis (L.) Cronq

The mode of action of paraquat (1,1-dimethyl-4,4-bipyridinium) and the mechanism of resistance to it were studied in leaves of atrazine/paraquat co-resistant (R) and susceptible (S) biotypes of horseweed (Conyza canadensis) collected from Hungarian vineyards. The application of 0·5 mol m^?3 paraquat by spraying onto the surface of the leaves of intact plants in the light rapidly led to typical symptoms of paraquat action in the initial period in both biotypes, i.e. inhibition of CO₂ fixation, suppression of variable chlorophyll fluorescence (F_v), decrease of oxygen evolution and stimulation of ethane production. The inhibitory effect of paraquat in the S plants was irreversible, whereas it was transient in the R plants and those plants recovered gradually afterwards. The R plants recovered from the inhibitory effect of paraquat only in the light, and an increase in light intensity was found to have a pronounced effect on the recovery of F_v. The mechanism of resistance to paraquat in C. canadensis is discussed.     

Ontogenic variation in response of Brassica campestris L. to cadmium toxicity

Abstract

Rapeseed (Brassica campestris L.) cv Pusa Gold plants, exposed to different cadmium (Cd) levels (0, 25, 50 and 100 mg kg^?1 soil) in greenhouse, pot culture experiment, were analyzed with reference to distribution of metal, accumulation of biomass and the degree of growth stage Cd-sensitivity. A significant maximum decrease in plant biomass was observed at Cd-exposed flowering stage followed by pre-flowering and post-flowering stages. Activities of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) differentially increased; while, the concentrations of non-enzymatic antioxidants such as ascorbic acid (AsA) and glutathione (GSH) drastically decreased in plants exposed to Cd at various growth stages. However, the concentrations of GSH and AsA decreased maximally in plant groups exposed to Cd at their flowering stage. The maximum Cd-accumulation occurred in roots followed by leaves and stem. Various Cd levels inhibited also the contents of plant nutrients such as nitrogen (N), phosphorous (P), potassium (K) and sulfur (S) in leaves. The present endeavor hence concludes the existence of close relationships among growth parameters, Cd-sensitivity of phenological stages of the crop and the components of antioxidant system in rapeseed plants exposed at various growth stages.     

Expression of NO scavenging hemoglobin is involved in the timing of bolting in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Plants contain three classes of hemoglobin genes of which two, class 1 and class 2, have a structure similar to classical vertebrate globins. We investigated the effect of silencing the class 1 non-symbiotic hemoglobin gene, GLB1, and the effect of overexpression of GLB1 or the class 2 non-symbiotic hemoglobin gene, GLB2, in Arabidopsis thaliana. Lines with GLB1 silencing had a significant delay of bolting and after bolting, shoots reverted to the rosette vegetative phase by formation of aerial rosettes at lateral meristems. Lines with overexpression of GLB1 or GLB2 bolted earlier than wild type plants. By germinating the lines in a medium containing the nitric oxide (NO) donor, sodium nitroprusside (SNP), it was demonstrated that both GLB1 and GLB2 promote bolting by antagonizing the effect of NO, suggesting that non-symbiotic plant hemoglobin controls bolting by scavenging the floral transition signal molecule, NO. So far, NO scavenging has only been demonstrated for class 1 non-symbiotic hemoglobins. A direct assay in Arabidopsis leaf cells shows that GLB1 as well as the class 2 non-symbiotic hemoglobin, GLB2, scavenge NO in vivo. NO has also been demonstrated to be a growth stimulating signal with an optimum at low concentrations. It was observed that overexpression of either GLB1 or GLB2 shifts the optimum for NO growth stimulation to a higher concentration. In conclusion, we have found that expression of NO scavenging plant hemoglobin is involved in the control of bolting in Arabidopsis.     

A Method for Analysis of Meiosis in Anthers of Arabidopsis thaliana

A simple method for analysis of meiosis in anthers of Arabidopsisthaliana is described. Anthers showing a high frequency of microsporocytesundergoing meiotic divisions were obtained from buds of 18–20-d-oldplants at the rosette stage of growth just prior to bolting.All stages of meiosis were readily observed. Arabidopsis thaliana var. Landsberg erecta, meiosis, chromosomes, anthers     

Inhibition of stem growth and gibberellin production in Agrostemma githago L. by the growth retardant tetcyclacis

The effects of the new growth retardant tetcyclacis (TCY) on stem growth and endogenous gibberellin (GA) levels were investigated in the long-day rosette plant Agrostemma githago. Application of TCY (10 ml of a 5·10^-5M solution daily) to the soil suppressed stem elongation in Agrostemma grown under long-day conditions. A total of 10 g GA₁ (1 g applied on alternate days) per plant overcame the growth retardation caused by TCY.Control plants and plants treated with TCY were analyzed for endogenous GAs after exposure to nine long days. The acidic extracts were fractionated by high-performance liquid chromatography. Part of each fraction was tested in the d-5 maize bioassay, while the remainder was analyzed by combined gas chromatography-selected ion monitoring. The bioassay results indicated that the GA content of plants treated with TCY was much lower than that of untreated plants. The data obtained by gas chromatography-selected ion monitoring confirmed that the levels of seven GAs present in Agrostemma were much reduced in TCY-treated plants when compared with the levels in control plants: GA₅₃ (13%), GA₄₄ (0%), GA₁₉ (1%), GA₁₇ (33%), GA₂₀ (15%), GA₁ (4%), and epi-GA₁ (13%). These results provide evidence that TCY inhibits stem growth in Agrostemma by blocking GA biosynthesis and thus lowering the levels of endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - HPLC high-performance liquid chromatography - TCY Tetcyclacis (5-[4-chlorophenyl]-3,4,5,9,10-pentaaza-tetracyclo-5,4,1,0^2,6,0^8,11-dodeca-3,9-diene)     

Biological Activity of Purpurogallin

Purpurogallin showed antibacterial activity toward gram-positive bacteria. Strong activity against methicillin-resistant Staphylococcus aureus [minimal inhibitory concentration (MIC) against methicillin of 1600 μg/ml] was found, with MIC of 11.0/μg/ml. Purpurogallin inhibited the growth of all tested plants and decreased the chlorophyll content in the cotyledons of Brassica campestris subsp. rapa. It showed potent inhibitory activity against prolyl endopeptidase (the 50% inhibitory concentration was 1.6 × 10^?5m), unlike its analogues, hinokitiol and tropolone.