Corn Leaf Discs Research Articles

Rolling circle transcription: A new system to produce RNA microspheres for improving RNAi efficiency in an agriculturally important lepidopteran pest (Mythimna separate)

We applied a new RNA interference (RNAi) system using rolling circle transcription (RCT) technology to generate RNA microspheres (RMS) for targeting two key chitin synthetic pathway genes [chitin synthase A (CHSA), chitin synthase B (CHSB)] in the larvae of the oriental armyworm (Mythimna separate), a RNAi-unsusceptible agriculturally important lepidopteran pest. Feeding the third-instar larvae with the RMS-CHSA- or RMS-CHSB-treated corn leaf discs suppressed the expression of CHSA by 81.7% or CHSB by 88.1%, respectively, at 72 h. The silencing of CHSA consequently affected the larval development, including the reduced body weight (54.0%) and length (41.3%), as evaluated on the 7th day, and caused significant larval mortalities (51.1%) as evaluated on the 14th day. Similar results were obtained with the larvae fed RMS-CHSB. We also compared RNAi efficiencies among different strategies: 1) two multi-target RMS [i.e., RMS-(CHSA + CHSB), RMS-CHSA + RMS-CHSB], and 2) multi-target RMS and single-target RMS (i.e., either RMS-CHSA or RMS-CHSB) and found no significant differences in RNAi efficiency. By using Cy3-labeled RMS, we confirmed that RMS can be rapidly internalized into Sf9 cells (<6 h). The rapid cellular uptake of RMS accompanied with significant RNAi efficiency through larval feeding suggests that the RCT-based RNAi system can be readily applied to study the gene functions and further developed as bio-pesticides for insect pest management. Additionally, our new RNAi system takes the advantage of the microRNA (miRNA)-mediated RNAi pathway using miRNA duplexes generated in vivo from the RMS by the target insect. The system can be used for RNAi in a wide range of insect species, including lepidopteran insects which often exhibit extremely low RNAi efficiency using other RNAi approaches.

Utilization of image-based spectral reflectance to detect herbicide resistance in glufosinate-resistant and glufosinate-susceptible plants: a proof of concept

AbstractGlufosinate is an effective postemergence herbicide, and overreliance on this herbicide for weed control is likely to increase and select for glufosinate-resistant weeds. Common assays to confirm herbicide resistance are dose–response and molecular sequencing techniques; both can require significant time, labor, unique technical equipment, and a specialized skillset to perform. As an alternative, we propose an image-based approach that uses a relatively inexpensive multispectral sensor designed for unmanned aerial vehicles to measure and quantify surface reflectance from glufosinate-treated leaf disks. Leaf disks were excised from a glufosinate-resistant and glufosinate-susceptible corn (Zea mays L.), cotton (Gossypium hirsutum L.), and soybean [Glycine max (L.) Merr.] varieties and placed into a 24-well plate containing eight different concentrations (0 to 10 mM) of glufosinate for 48 h. Multispectral images were collected after the 48-h incubation period across five discrete wave bands: blue (475 to 507 nm), green (560 to 587 nm), red (668to 682 nm), red edge (717 to 729 nm), and near infrared (842 to 899 nm). The green leaf index (GLI; a metric to measure chlorophyll content) was utilized to determine relationships between measured reflectance from the tested wave bands from the treated leaf disks and the glufosinate concentration. Clear differences of spectral reflectance were observed between the corn, cotton, and soybean leaf disks of the glufosinate-resistant and glufosinate-susceptible varieties at the 10 mM concentration for select wave bands and GLI. Leaf disks from two additional glufosinate-resistant and glufosinate-susceptible varieties of each crop were subjected to a similar assay with two concentrations: 0 and 10 mM. No differences of spectral reflectance were observed from the corn and soybean varieties in all wave bands and the GLI. The leaf disks of the glufosinate-resistant and glufosinate-susceptible cotton varieties were spectrally distinct in the green, blue, and red-edge wave bands. The results provide a basis for rapidly detecting glufosinate-resistant plants via spectral reflectance. Future research will need to determine the glufosinate concentrations, useful wave bands, and susceptible/resistant thresholds for weeds that evolve resistance.

Secondary Metabolites of Asclepias curassavica (Apocynaceae) and its Effects on Food Preference and Mortality of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Asclepias curassavica L. plant is toxic for vertebrates, and little is known about its effects on invertebrates, as well as whether its secondary metabolites have an influence on food preference and survival of insects. Thus, a study was conducted to verify the action of A. curassavica on Spodoptera frugiperda J.E.Smith. The plants were collected, dried and ground to compose a crude ethanolic extract. Food tests were carried out with and without opportunity of choice, using corn leaf discs immersed in the extract at concentrations of 1%, 2% and 4%, for assessment of food consumption (g) and preference index. Later, the extract was topically applied on second- and fifth-instar caterpillars, at concentrations of 1%, 2%, 4% and 6%, in order to observe its effect on survival. In parallel, analysis was conducted to verify the presence and measure the amount of total polyphenols and flavonoids in the extract. There was contact action between the extract and second-instar caterpillars at all concentrations, with treatment 6% causing 100% of mortality 72 hours after application. None of the treatments promoted contact action on fifth-instar caterpillars. In food tests with and without opportunity of choice, all treatments caused reductions in consumption, which classified all as phagodeterrent, with treatment 4% standing out as the least favorite. The presence of total polyphenols and flavonoids was found in the extract, presenting 58.75 ug/mL and 150.1 ug/mL, respectively. The A. curassavica extract proved promising in S. frugiperda control.

Plant Preferences of Z-Pheromone Race Ostrinia nubilalis (Lepidoptera: Crambidae) Based on Leaf Tissue Consumption Rates.

European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae), has been present in the United States for over 100 yr and documented on >200 plant species, including economically valuable crops. The reported preferred host of O. nubilalis is corn, Zea mays L. (Cyperales: Poaceae), although it is considered to be a generalist agricultural pest. Life cycles of the two pheromone races, E and Z, align with the seasonality of different agricultural plants. Since the introduction of Bt corn in 1996, overall O. nubilalis presence has declined and suggests that alternative crop plants might not be suitable hosts. We investigated plant vegetation preference of third-instar Z-race O. nubilalis for leaf disks of corn and a variety of other crops using 48 h no-choice and choice tests. Z-race larvae gained more mass on V6 non-Bt field corn leaf disks in comparison to other plant species options. Additionally, a preference for non-Bt field corn leaf disks was observed in most comparisons. Higher consumption of cucumber, Cucumis sativus L. (Cucurbitales: Cucurbitaceae), leaf disks as compared to non-Bt field corn leaf disks suggested an ability to feed on excised leaf tissues of a plant species that does not induce defenses to herbivory.

Toxicidade de extratos de Anadenanthera macrocarpa (Fabaceae: Mimosoideae) e Bougainvillea buttiana (Nyctaginaceae) para lepidópteros-praga

http://dx.doi.org/10.5007/2175-7925.2017v30n2p15O uso de extratos vegetais apresenta grande eficiência no manejo de pragas, destinados a produções agrícolas de pequeno e médio porte, por controlar uma gama muito grande de artrópodes. O objetivo desta pesquisa consiste em avaliar os efeitos tóxicos dos extratos de Anadenanthera macrocarpa (Fabaceae: Mimosoideae) e Bougainvillea buttiana (Nyctaginaceae) em Helicoverpa armigera (Hübner), Spodoptera cosmioides (Walker) e Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae). Lagartas de H. armigera e S. cosmioides foram mantidas em discos foliares de soja, enquanto S. frugiperda foi mantida em discos foliares de milho, tratados com os extratos aquoso diluídos nas concentrações de: 0, 500, 1.000, 2.500, 4.000, 5.000, 10.000 e 15.000 mg.L-1. Foram mensurados os efeitos letais e subletais dos extratos sobre os insetos. De acordo com os resultados obtidos, a concentração de 500 mg.L-1 do extrato de A. macrocarpa causou o maior percentual de mortalidade (93,33%) em H. armigera. O extrato de B. buttiana causou 86,67% e 60% de mortalidade em H. armigera (1.000 mg.L-1) e S. cosmioides (4.000 mg.L-1), respectivamente. A redução da emergência dos adultos foi o principal efeito subletal constatado em S. cosmioides e S. frugiperda. Considerando os resultados obtidos, destaca-se que os extratos vegetais utilizados apresentam-se como uma alternativa em potencial para o manejo de lepidópteros-praga.

Toxicity of Secondary Metabolites from Meliaceae Against Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

The study was carried out to evaluate the bioactivity of secondary metabolites from Trichilia pallida, Trichilia pallens, and Toona ciliata against fall armyworm Spodoptera frugiperda (J. E. Smith) larvae. The studied compounds included (+/-)-catechins, a triglyceride, and cedrelone isolated from T. ciliata branches, fruits, and stems, respectively; dammaradienol isolated from T. pallida leaves; and scopoletin isolated from T. pallens branches. The compounds' activity was evaluated through ingestion and topic treatment. Treated artificial diet was offered to first instar larvae to evaluate ingestion effect, while an application on the dorsal thoracic region of third instar larvae was used to evaluate the topic effect. Mortality was assessed daily, and larval weight was recorded after 7days for ingestion and 5days for topic application. Scopoletin and triglyceride caused low mortality rates and reduction in larval weight by ingestion, (+/-)-catechins caused larval weight reduction by ingestion, and scopoletin reduced survival by topic treatment. The most effective compound was cedrelone that affected larval survival and development mainly by ingestion. The estimated LC50, LC90, and EC50 for cedrelone were 0.0365, 0.0659, and 0.0095%, respectively. Further, cedrelone-treated corn leaf discs were offered to fourth instar larvae during 16h in choice and no-choice tests. The deterrence indexes obtained in the choice tests were 23.5 and 36.3% at concentrations of 0.0365 and 0.0659, respectively. Consumption of cedrelone-treated leaf discs at the concentration of 0.0659% was lower compared to the control in the no-choice test. Thus, cedrelone caused lethal and sublethal effects and phagodeterrence on S. frugiperda and should be further studied.

Evaluation of neem-based nanoformulations as alternative to control fall armyworm

ABSTRACT Neem (Azadirachta indica A. Juss.) based insecticides are efficient for the control of various pest species, but their low residual effect and the lack of standardized formulations are problems for field use. Nanoformulations of neem in colloidal suspension or powder, containing the polymers poly (ε-caprolactone) (PCL), poly (β-hidroxibutirate) (PHB) or poly (methyl methacrylate) (PMMA), in capsules or spheres, were developed in order to reduce biodegradation of the active neem compounds and improve their residual effect. Corn leaves treated with the nanoformulations were offered to first instar larvae of fall armyworm during 10 days, observing mortality and larval weight and comparing it to a commercial neem oil and negative controls. The residual effect of the four most efficient nanoformulations was evaluated at 1, 3 and 7 days after spraying (DAS). Feeding preference tests with dual-choice (treatment vs. control) were performed with corn leaf disks at 1, 3 and 7 after DAS, and a preference index was determined 24 hours after larvae feeding on the disks. Some nanoformulations caused mortality up to 3 DAS and sublethal effects up to 7 DAS, but none outperformed the residual effect of commercial neem oil. All treatments showed phagodeterrence at 1 DAS, but this was lost over time. The short duration of the nanoformulations' efficacy suggests that there was little or no release of neem by the nanoparticles, so more studies are needed to improve the release kinetics of the nanoformulated products.

Joint action of Beauveria bassiana and the insect growth regulators diflubenzuron and novaluron, on the migratory locust, Locusta migratoria

Beauveria bassiana (Balsamo) Vuillemin and sublethal concentrations of the insect growth regulators (IGR) diflubenzuron and novaluron were applied simultaneously and sequentially to second-instar Locusta migratoria migratorioides (Sauss.) to determine the interaction between these materials and an entomopathogenic fungus. Nymphs were fed on corn leaf disks treated with several concentrations of each IGR, and a constant dose of B. bassiana was applied topically. Additive interaction was demonstrated in all instances when second-instar nymphs were exposed to diflubenzuron or novaluron simultaneously with B. bassiana treatment, and when the fungus was applied first and IGR after 48 h. Additive interaction was also exhibited when novaluron was applied first and B. bassiana after 48 h. Antagonism was demonstrated when nymphs were fed diflubenzuron-treated corn leaves first, then after 48 h were treated with B. bassiana. The additive interaction that was observed in our experiments could still be useful to achieve efficacious levels of acridid control at low rates of an IGR along with a biological control agent such as an entomopathogenic fungus.

A rapid in vivo shikimate accumulation assay with excised leaf discs

An in vivo shikimate accumulation assay with excised leaf tissue was developed to provide a fast and reliable method for identifying glyphosate-resistant plants. The assay is based on glyphosate-induced accumulation of shikimate. There was a linear accumulation of shikimate in excised leaf discs of soybean and canola treated with 250 μM glyphosate for 48 h. The IC50 for the accumulation of shikimate in soybean and corn leaf discs was 34 and 87 μM, respectively. Leaf discs excised from glyphosate-resistant corn or soybean did not accumulate shikimate when treated with 500 μM glyphosate. Leaf discs taken from a number of field-grown plants accumulated shikimate in a glyphosate dose–dependent manner. The accumulation of shikimate was dependent on light and the age of the leaf from which the disc was taken. The assay worked either in 96-well microtiter plates or in vials, and it clearly differentiated between glyphosate-resistant and -susceptible crops in which the resistance is due to an alteration of the target site for glyphosate. The assay was simple and robust and has the potential to be used as a high throughput assay to detect glyphosate resistance in weeds.

Aggressiveness and Perennation of Isolates of Cochliobolus heterostrophus from North Carolina.

Selection occurring during the saprophytic or overwintering phase of the life cycle of Cochliobolus heterostrophus, the causal agent of southern leaf blight of maize, may be a factor in the persistence of apparently less aggressive isolates in the pathogen population. The relative aggressiveness and ability to perennate of 22 isolates of C. heterostrophus from North Carolina was measured in series of experiments. Significant differences in aggressiveness and percent perennation (overwintering survival) were observed. There was a weak but often significant negative correlation between the ability of isolates to persist on the soil surface and their aggressiveness. The ability of race O isolates to sporulate on senescent corn leaf discs was positively correlated with their aggressiveness. Selection against increased aggressiveness during overwintering does not appear sufficient by itself to counter selection for increased aggressiveness occurring during the pathogen's pathogenic phase.

Comparative Physiological Effects of Quinclorac and Auxins, and Light Involvement in Quinclorac-Induced Chlorosis in Corn Leaves

The physiological effects of the herbicide quinclorac (3,7-dichloro-8-quinolinecarboxylic acid) in corn and the involvement of light on the herbicide-induced chlorosis were investigated. Quinclorac was highly inhibitory to corn growth. However, different from the tested auxins, quinclorac showed no promotion in an auxinic activity assay (corn coleoptile elongation). Quinclorac induced significantly lower amounts of ethylene in corn coleoptile sections compared with IAA, NAA, and 2,4-D under darkness. Furthermore, quinclorac did not bind with a putative corn membrane-bound auxin receptor. Quinclorac caused chlorotic discoloration in the youngest leaf under light. When the quinclorac-treated corn leaf disks were continuously exposed to light, a significant decrease in total chlorophyll content was observed. No decrease in total chlorophyll content of the herbicide-treated leaf disks under dark conditions was detected. There was a significant correlation between light intensity and the reduction in total chlorophyll content of the leaf disks induced by quinclorac. Moreover, quinclorac considerably increased ethylene production in leaf disks under light conditions, whereas no enhancement of ethylene production in the dark was observed. 2,4-D slightly stimulated ethylene production under both the light and dark conditions. There was a high negative correlation between quinclorac-induced ethylene production and total chlorophyll content in quinclorac-treated leaf disks. These results suggest that the physiological actions of quinclorac applied to corn are not necessarily the same as 2,4-D and other auxins. Ethylene biosynthetic reaction may be closely related with the major herbicidal action of quinclorac, similar to that of 2,4-D. However, our data indicate that the quinclorac-enhanced ethylene biosynthetic reaction and the herbicide-induced chlorosis in corn leaves are light dependent.

Production of Methanol from Heat-stressed Pepper and Corn Leaf Disks

`Early Calwonder' pepper (Capsicum annuum L.) and `Jubilee' corn (Zea mays L.) leaf disks exposed to high temperature stress produced ethylene, ethane, methanol, acetaldehyde, and ethanol based on comparison of retention times during gas chromatography to authentic standards. Methanol, ethanol, and acetaldehyde were also identified by mass spectroscopy. Corn leaf disks produced lower levels of ethylene, ethane, and methanol, but more acetaldehyde and ethanol than pepper. Production of ethane, a by-product of lipid peroxidation, coincided with an increase in electrolyte leakage (EL) in pepper but not in corn. Compared with controls, pepper leaf disks infiltrated with linolenic acid evolved significantly greater amounts of ethane, acetaldehyde, and methanol and similar levels of ethanol. EL and volatile hydrocarbon production were not affected by fatty acid infiltration in corn. Infiltration of pepper leaves with buffers increasing in pH from 5.5 to 9.5 increased methanol production.

METHANOL PRODUCTION FROM HEAT-STRESSED PEPPER AND CORN LEAF DISKS

Pepper (Capsicum annuum L. `Early Calwonder') and corn (Zea mays L. `Jubilee') leaf disks exposed to high temperature stress produced ethylene, ethane, methanol, acetaldehyde, and ethanol based on comparison of retention times during gas chromatography to authentic standards. Methanol, ethanol, and acetaldehyde were also identified by mass spectroscopy. Corn leaf disks produced lower levels of ethylene, ethane and methanol, but more acetaldehyde and ethanol than pepper. Production of ethane, a by-product of lipid peroxidation, coincided with an increase in electrolyte leakage (EL) in pepper but not in corn. Compared with controls, pepper leaf disks infiltrated with linolenic acid evolved significantly greater amounts of ethane, acetaldehyde and methanol, and similar levels of ethanol. Introduction of linoleic acid did not significantly affect volatile hydrocarbon production in pepper. Electrolyte leakage and volatile hydrocarbon production were not affected by fatty acid infiltration in corn.

Feeding Stimulants for the European Corn Borer (Lepidoptera: Pyralidae): Additives to a Starch-Based Formulation for Bacillus thuringiensis

A starch formulation of Bacillus thuringiensis Berliner is being developed as a component in a pest management system to control Ostrinia nubi/alis (Hubner). Palatability is one property of starch formulations which must be investigated; this study was done to identify materials that will cause O. nubi/alis larvae to feed readily on starch granules. A two-choice behavioral bioassay was developed for evaluating phagostimulants in starch gran­ ules (without B. thuringiensis). Various materials were tested: compounds reported in the literature as feeding stimulants, the artificial diet for the species, corn foliage, corn oil, and the commercial feeding stimulant, Coax. Starch granules without additives were seldom fed upon by larvae, but it was possible to make the granules even more acceptable to larvae than corn foliage. The best feeding responses were to mixtures containing lipid, sugar, and protein. Coax was the most effective material tried; for example, in bioassays, the ratio of larvae that chose granules with Coax over those with only starch was 320:1, and the ratio of larvae choosing granules with Coax over corn leaf disks was 11.5:1. In greenhouse tests, phagostimulants increased the effectiveness of granules containing B. thuringiensis for killing O. nubilalis larvae. For example, when Coax was added to the granules, the amount of B. thuringiensis used could be reduced by 75% without reducing mortality.

Effect of ergot alkaloids from fungal endophyte-infected grasses on fall armyworm (Spodoptera frugiperda)

Ergot alkaloids produced by endophytic fungi in the tribe Balansiae (Clavicipitaceae, Ascomycetes), which infect grasses, may provide plant defense against herbivores. This study examined the effects of six ergot alkaloids on survivorship, feeding, and growth of larvae of the fall armyworm (Spodoptera frugiperda, Lepidoptera: Noctuidae), a generalist herbivore of grasses. Corn leaf disks were soaked in solutions of individual ergot alkaloids at different concentrations and presented to neonate larvae. At the highest concentrations (77-100 mg/liter) of ergonovine, ergotamine, ergocryptine, agroclavine, and elymoclavine, larval weights and/or leaf area consumed after eight days were reduced relative to controls. Lysergol had no effect on larval weights and leaf consumption at any concentration. Although active concentrations were higher than those reported from two host grasses, in vivo levels of ergot alkaloids have not been quantified for most endophyte-infected grasses. The detrimental effects on fall armyworm observed in this study suggest that ergot alkaloids could be responsible, at least in part, for the greater insect resistance of endophyte-infected grasses.

Effect of Host Plant on Cannibalism Rates by Fall Armyworm (Lepidoptera: Noctuidae) Larvae

The fall armyworm, Spodoptera frugiperda (J. E. Smith), a polyphagous pest of grasses and legumes, showed higher rates of cannibalism when feeding on red kidney bean seedlings than on corn seedlings. These differences are related to behavioral preferences. In choice tests, the larvae showed a strong preference for corn. To see if these differences in cannibalism rates could be attributed to host chemistry alone, these tests were repeated using detached corn and bean leaf disks. No differences in cannibalism rates were observed under these conditions, although cannibalism on corn was higher than in earlier experiments. In no-choice tests, the efficiency of conversion of ingested food (ECI) was higher on corn seedling foliage than on bean seedling foliage. The ECI of cannibalizing larvae that were limited to other larvae as a food source was equivalent to that on corn. Larvae were able to offset these differences in food quality to some degree by increasing relative consumption rates on beans. The relative growth rate was highest on corn, second highest on other larvae, and lowest on beans. These data provide the first nutritional indices for a normally herbivorous species on a carnivorous diet, and so are useful for ecological studies of between-species comparisons relating growth to food-source differences.

Role of calcium in ripening and senescence

Abstract In view of the possibility that senescence may be a consequence of the deterioration of cell walls and membrane compartments, calcium was studied as a possible agent which might defer senescence. Calcium treatment delayed leaf senescence in corn leaf discs. The effect of calcium on senescencse was additive to the cytokinin deferral of senescence. An increase in hydraulic permeability to [3H] water during senescence was demonstrataed and this increase was deferred by calcium; calcium plus benzyladenine was even more effective. Analysis of inorganic ions in the pericarp of nonripening rin mutant tomajç fruits and isogenic Rutgers revealed higher levels of bound Ca in rin at advanced stages of maturity. In the normal fruits, bound Ca decreased about 3‐fold during maturity. In rin fruits, no change in polygalacturonase (PG) activity was detected, whereas in Rutgers, an increase in PG activity was observed at advanced stages of maturity. Each of the measured changes associated with senescence (pigment changes, protein decrease, hydraulic permeability increase, and an increase in cell wall enzyme activity) was suppressed by calcium, and the interpretation is offered that the effects are a consequence of the calcium function in maintaining cell wall structure and membrane integrity.

Changes in specific radioactivities of corn-leaf metabolites during photosynthesis in 14CO2 and 12CO2 at normal and low oxygen

The (12)CO2- and (14)CO2-exchange of illuminated corn leaf discs were measured at normal (21%) and low (1%) oxygen. After periods of exposure to (14)CO2 or to (14)CO2 followed by (12)CO2, the discs were killed and the specific activities of some metabolites were determined. At both O2 concentrations the specific activity of 3-PGA increased and decreased rapidly during the first 5 min of (14)CO2-feeding or (12)CO2-flushing but did not equilibrate with that of the CO2 in the assimilation chamber even after 15 min. The specific activity of aspartic acid also showed bimodal kinetics during both feeding and flushing. The specific activities of 3-phosphoglyceric acid (3-PGA), aspartic acid and alanine were higher at 1% O2 than at 21% O2, but glycine and serine were lower in specific activity at 1% O2. The results are in agreement with the proposed initial fixation of CO2 into C4-dicarboxylic acids and subsequent transfer of this carbon to 3-PGA. Indirect evidence supports the idea that at 21% O2, CO2 was produced by the corn leaf discs in the light and was refixed into C4-dicarboxylic acids. At 1% O2, the photorespiratory process could also have been active although the flux of carbon through the glycolate pathway was probably smaller than at 21% O2.

Deferral of Leaf Senescence with Calcium

In view of the possibility that senescence may be a consequence of the deterioration of membrane compartments in the cells of leaves, calcium was studied as a possible agent which might defer senescence. The senescence of corn leaf discs was deferred by added calcium, and the effect was additive to the cytokinin deferral of senescence. Likewise, the senescence of Rumex leaf discs was deferred by added calcium, and the effect was additive to the gibberellin deferral of senescence. Detailed experiments with corn leaf discs established that the increase in apparent free space associated with senescence was completely prevented by calcium. An increase in hydraulic permeability during senescence was likewise demonstrated, and this increase was deferred by calcium; calcium plus benzyladenine was even more effective. Each of the measured functions of leaf senescence (chlorophyll content, protein decrease, apparent free space increase, and hydraulic permeability increase) was suppressed by calcium, and the interpretation is offered that the effects are a consequence of the calcium function in maintaining cellular membranes.

Effects of Atrazine on Chlorophyll Retention in Corn Leaf Discs

Corn(Zea maysL. ‘Dixie 18′) leaf discs were incubated in light or darkness in dilute solutions of 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine) with and without 6-furfurylamino-purine (kinetin). Kinetin decreased chlorophyll loss in light whereas atrazine had no effect. In darkness atrazine at a concentration of 5 × 10−8M increased clorophyll a retention in the absence of exogenous kinetin; but in the presence of 1.5 μg/ml of kinetin, the herbicide increased loss of chlorophyll a. Increasing the kinetin concentration to 15 μg/ml relieved the effect of 5 × 10−8M atrazine, but 10−7M atrazine increased chlorophyll loss. Higher concentrations of kinetin (150 and 1500 μg/ml) prevented all effects of atrazine. Factorial analysis revealed a highly significant interaction between atrazine and kinetin.