Evaluation of spectrophotometric and HPLC methods for shikimic acid determination in plants: models in glyphosate-resistant and -susceptible crops

Endogenous shikimic acid determinations are routinely used to assess the efficacy of glyphosate in plants. Numerous analytical methods exist in the public domain for the detection of shikimic acid, yet the most commonly cited comprise spectrophotometric and high-pressure liquid chromatography (HPLC) methods. This paper compares an HPLC and two spectrophotometric methods (Spec 1 and Spec 2) and assesses the effectiveness in the detection of shikimic acid in the tissues of glyphosate-treated plants. Furthermore, the study evaluates the versatility of two acid-based shikimic acid extraction methods and assesses the longevity of plant extract samples under different storage conditions. Finally, Spec 1 and Spec 2 are further characterized with respect to (1) the capacity to discern between shikimic acid and chemically related alicyclic hydroxy acids, (2) the stability of the chromophore (t1/2), (3) the detection limits, and (4) the cost and simplicity of undertaking the analytical procedure. Overall, spectrophotometric methods were more cost-effective and simpler to execute yet provided a narrower detection limit compared to HPLC. All three methods were specific to shikimic acid and detected the compound in the tissues of glyphosate-susceptible crops, increasing exponentially in concentration within 24 h of glyphosate application and plateauing at approximately 72 h. Spec 1 estimated more shikimic acid in identical plant extract samples compared to Spec 2 and, likewise, HPLC detection was more effective than spectrophotometric determinations. Given the unprecedented global adoption of glyphosate-resistant crops and concomitant use of glyphosate, an effective and accurate assessment of glyphosate efficacy is important. Endogenous shikimic acid determinations are instrumental in corroborating the efficacy of glyphosate and therefore have numerous applications in herbicide research and related areas of science as well as resolving many commercial issues as a consequence of glyphosate utilization.     

Inhibitory effects of gossypol, gossypolone, and apogossypolone on a collection of economically important filamentous fungi

Racemic gossypol and its related derivatives gossypolone and apogossypolone demonstrated significant growth inhibition against a diverse collection of filamentous fungi that included Aspergillus flavus, Aspergillus parasiticus, Aspergillus alliaceus, Aspergillus fumigatus, Fusarium graminearum, Fusarium moniliforme, Penicillium chrysogenum, Penicillium corylophilum, and Stachybotrys atra. The compounds were tested in a Czapek agar medium at a concentration of 100 μg/mL. Racemic gossypol and apogossypolone inhibited growth by up to 95%, whereas gossypolone effected 100% growth inhibition in all fungal isolates tested except A. flavus. Growth inhibition was variable during the observed time period for all tested fungi capable of growth in these treatment conditions. Gossypolone demonstrated significant aflatoxin biosynthesis inhibition in A. flavus AF13 (B(1), 76% inhibition). Apogossypolone was the most potent aflatoxin inhibitor, showing greater than 90% inhibition against A. flavus and greater than 65% inhibition against A. parasiticus (B(1), 67%; G(1), 68%). Gossypol was an ineffectual inhibitor of aflatoxin biosynthesis in both A. flavus and A. parasiticus. Both gossypol and apogossypolone demonstrated significant inhibition of ochratoxin A production (47%; 91%, respectively) in cultures of A. alliaceus.     

Growout of Pacific White Shrimp, Litopenaeus vannamei, Stocked into Production Ponds at Three Different Ages

This study was designed to determine the production characteristics of the Pacific white shrimp, Litopenaeus vannamei, stocked into grow‐out ponds at three different sizes and ages. To meet this goal, three groups of postlarvae (PL) were obtained. The first group was placed in a nursery system for 21 d (N21), the second for 14 d (N14), and the third was stocked directly into ponds (DS). Shrimp from each nursery treatment (three tanks per treatment) were pooled and then subdivided for stocking into four replicate 0.1 ha ponds per treatment, another four ponds were stocked directly (DS) with PL₈. All 12 ponds were stocked on the same day at a density of approximately 35 PL/m², and cultured over a 16‐wk period and then drain harvested. After harvest, mean average weights (15.4, 16.9, and 14.9 g), survivals (63, 62, and 64%), FCRs (2.7, 2.5, and 2.7), and average yields (3592, 4005, and 3374 kg/ha) were determined for N21, N14, and DS, respectively. No significant (P > 0.05) differences were observed among treatments. Regardless of nursing time, nursed juveniles did not differ significantly in production characteristics from shrimp stocked directly from the hatchery.     

A novel P106L mutation in EPSPS and an unknown mechanism(s) act additively to confer resistance to glyphosate in a South African Lolium rigidum population

Glyphosate resistance evolution in weeds is a growing problem in world agriculture. Here, we have investigated the mechanism(s) of glyphosate resistance in a Lolium rigidum population (DAG1) from South Africa. Nucleotide sequencing revealed the existence of at least three EPSPS homologues in the L. rigidum genome and identified a novel proline 106 to leucine substitution (P106L) in 52% DAG1 individuals. This mutation conferred a 1.7-fold resistance increase to glyphosate at the whole plant level. Additionally, a 3.1-fold resistance increase, not linked to metabolism or translocation, was estimated between wild-type P106-DAG1 and P106-STDS sensitive plants. Point accepted mutation analysis suggested that other amino acid substitutions at EPSPS position 106 are likely to be found in nature besides the P106/S/A/T/L point mutations reported to date. This study highlights the importance of minor mechanisms acting additively to confer significant levels of resistance to commercial field rates of glyphosate in weed populations subjected to high selection pressure.     

Fragmentation Status of Tall-Tussock Grassland Relicts in the Flooding Pampa,Argentina

Since European settlement vast areas of the tall tussock grassland dominated by Paspalum quadrifarium Lam. and Paspalum exaltatum J. Presl (“pajonal” grassland) in the Flooding Pampa of Argentina were converted to croplands and short grasslands. With the use of Landsat satellite images, we analyzed current (1998–2000) cover and spatial integrity of the pajonal community, and compared it with a vegetation map made 50 yr ago (1956–1960). Six categories of land cover were adopted: crops, sown pastures, short grassland, pajonal, wetlands, and anthropogenic areas. With the use of metrics from FRAGSTATS, landscape pattern and composition were analyzed at two scales: 1) regionally, by comparing two edaphic domain areas with relatively low and high restrictions for agriculture (low-restriction domain [LRD] and high-restriction domain [HRD], respectively); and 2) at landscape scale, by comparing ten 22 500-ha randomly selected areas (landscapes) within each edaphic domain. Current relative cover of pajonal grassland (2 173 600 ha) was 32.5%, and similar values were obtained within each edaphic domain. However, the number of pajonal patches was higher and their mean patch size, the Euclidean nearest-neighbor distance among patches (degree of isolation), and their border regularity were lower in the LRD than in the HRD. At landscape scale, the mean size of pajonal patches diminished with the percent of agricultural land within both edaphic domains. The isolation among pajonal patches increased with percent of agricultural land in the HRD, whereas no relationship between the isolation of pajonal patches and percent of agriculture was found in the LRD. As suggested by comparison with past vegetation, current pajonal status mostly results from replacement of pajonal grassland by short grassland types, cultivated pastures, and annual crops (52% and 44% of previously occupied areas in LRD and HRD, respectively), but some expansion of pajonal grassland was also observed (10% and 4% of previously unoccupied areas in LRD and HRD, respectively).     

Herbicide-resistant crops and weed resistance to herbicides

The adoption of genetically modified (GM) crops has increased dramatically during the last 3 years, and currently over 52 million hectares of GM crops are planted world-wide. Approximately 41 million hectares of GM crops planted are herbicide-resistant crops, which includes an estimated 33.3 million hectares of herbicide-resistant soybean. Herbicide-resistant maize, canola, cotton and soybean accounted for 77% of the GM crop hectares in 2001. However, sugarbeet, wheat, and as many as 14 other crops have transgenic herbicide-resistant cultivars that may be commercially available in the near future. There are many risks associated with the production of GM and herbicide-resistant crops, including problems with grain contamination, segregation and introgression of herbicide-resistant traits, marketplace acceptance and an increased reliance on herbicides for weed control. The latter issue is represented in the occurrence of weed population shifts, the evolution of herbicide-resistant weed populations and herbicide-resistant crops becoming volunteer weeds. Another issue is the ecological impact that simple weed management programs based on herbicide-resistant crops have on weed communities. Asiatic dayflower (Commelina cumminus L) common lambsquarters (Chenopodium album L) and wild buckwheat (Polygonum convolvulus L) are reported to be increasing in prominence in some agroecosystems due to the simple and significant selection pressure brought to bear by herbicide-resistant crops and the concomitant use of the herbicide. Finally, evolution of herbicide-resistant weed populations attributable to the herbicide-resistant crop/herbicide program has been observed. Examples of herbicide-resistant weeds include populations of horseweed (Conyza canadensis (L) Cronq) resistant to N-(phosphonomethyl)glycine (glyphosate). An important question is whether or not these problems represent significant economic issues for future agriculture.     

Differential response of Amaranthus tuberculatus (Moq ex DC) JD Sauer to glyphosate

Midwest USA farmers have reported inconsistent control of Amaranthus tuberculatus (= rudis) (Moq ex DC) JD Sauer by glyphosate in glyphosate-resistant crops. The potential of selection for decreased A tuberculatus sensitivity to glyphosate was therefore investigated in a reportedly resistant Everly, IA population (P0-EV). Evaluation of six A tuberculatus populations from the Midwest USA estimated a seedling baseline sensitivity of 2.15 mM glyphosate. Based on these results, three generations of divergent recurrent selection were implemented on P0-EV to isolate resistant and susceptible populations. A seedling assay was developed to screen large amounts of seeds and thus expedite the selection process. Whole-plant and seedling rate responses of P0-EV and a known pristine A tuberculatus population from Paint Creek, OH (P0-WT) identified no significant difference in response to glyphosate; however, greater phenotypic variance was ostensibly evident in P0-EV. The first recurrent generation selected for resistance at 3.2 mM glyphosate (RS1-R) had a 5.9- and 1.7-fold resistance increase at the seedling and whole-plant levels, respectively, compared with the susceptible generation selected at 32 microM glyphosate. After three cycles of recurrent selection, 14.6-fold difference in resistance at the seedling level and 3.1-fold difference at the whole-plant level were observed when comparing the populations selected for resistance (RS3-R) and susceptibility (RS3-S). Overall, recurrent selection increased the frequency of resistant individuals and decreased the variability to glyphosate at the population level. Nevertheless, variability for glyphosate resistance was still evident in RS3-R. Results herein suggested that A tuberculatus is inherently variable to glyphosate and that selection decreased the sensitivity to glyphosate. We purport that evolved glyphosate resistance in A tuberculatus may require multiple cycles of selection under field conditions. Historic estimated use of glyphosate alludes to the evolution of tolerant weed populations.     

Changes in Biochemical Compositions and Quality of White Shrimp (Litopenaeus vannamei) During Storage

ABSTRACT

To understand biochemical characteristics, storage stability, and freshness indicators of white shrimp (Litopenaeus vannamei), changes in extractable nitrogenous compounds, microbial count, and sensory rating of white shrimp during storage at 25 and 4°C were investigated. Free amino acids showed a slow increase during storage at 25°C, but no obvious change was found at 4°C. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were found at initial stage and decreased rapidly after storage. Both inosine 5′-monophosphate (IMP) and adenosine monophosphate (AMP) increased and then decreased during storage. Inosine, hypoxanthine, and the K-value gradually increased with time. The levels of total volatile basic nitrogen (TVB-N), NH₃, and trimethylamine (TMA) of white shrimp increased with storage time at 25 and 4°C. The TVB-N, NH₃, TMA, inosine, hypoxanthine, and K-value could be considered as freshness indicators of white shrimp during storage. However, the total plate count did not corroborate the acceptability recommended limits for white shrimp during storage. The sensory evaluation, associated with TVB-N, TMA, and K-value, showed the quality was unacceptable after 6 h storage at 25°C and 7 days at 4°C.     

The Influence of Artemia and Algal Supplements during the Nursery Phase of Rearing Pacific White Shrimp, Litopenaeus vannamei

A 21‐d nursery trial was conducted to evaluate various food supplements on growth and survival of postlarval (PL) Litopenaeus vannamei. Each of four treatments was provided with an equal quantity of a dried commercial feed throughout the study. Three treatments received algae paste (Thalassiosira weissflogii) supplemented every 3 d. These include F, commercial feed plus algae; FAr3, commercial feed plus algae plus Artemia every other day during the first 7 d; and FAr7, commercial feed plus algae plus Artemia every day during the first 7 d. The fourth treatment served as control (FNA); it relied only on the commercial feed plus naturally occurring algae. At the conclusion of the nursery period, there were no significant differences in survival or feed conversion ration for PL nursed in the various treatments. Artemia did have some effect in that PL receiving Artemia supplement for 3 d (FAr3) were significantly larger than those that did not. Algal paste in itself had no significant effect. Overall, results suggest an advantage to supplementing dried feed with Artemia for at least 3 d during the first week of nursery culture but little advantage to the use of a diatom paste as a food supplement.