Excised Leaf Assay Research Articles

Genome-wide association study of Senegalese sorghum seedlings responding to a Texas isolate of Colletotrichum sublineola

Colletotrichum sublineola is a destructive fungal pathogen that causes anthracnose in sorghum. Senegalese sorghum germplasm is currently being considered as an option of sources for genetic resistance. In a recent study, Senegalese sorghum accessions were evaluated for response to a mixture of Texas isolates of C. sublineola at the 8-leaf stage in the greenhouse. As a comparison, 159 Senegalese sorghum accessions at the 1-leaf developmental stage were evaluated against a single Texas isolate of C. sublineola (FSP53) using an excised-leaf assay. A genome-wide association study (GWAS) was conducted based on the phenotypic data acquired to discover genetic variation associated with response to C. sublineola using 193,727 single nucleotide polymorphisms (SNPs) throughout the genome. Sorghum seedlings tended to be more resistant when compared with sorghum plants inoculated at the 8-leaf stage in the greenhouse in previous experiments. Based on the highest score evaluated in the 1-leaf developmental stage excised leaf assay for each accession, 16 accessions were labeled as susceptible. GWAS identified the SNP locus S01_72868925 that is associated with protein kinase domain // Leucine rich repeat N-terminal domain at a level of confidence that surpassed Bonferroni correction. Along with the SNP locus S01_72868925, other top SNP loci were also associated with genes that are known to play critical roles in plant defense or plant stress responses.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

An Excised Leaf Assay to Measure Acquisition of 'Candidatus Liberibacter asiaticus' by Psyllids Associated with Citrus Huanglongbing Disease.

Huanglongbing, or citrus greening disease, is the most serious disease of citrus worldwide and is associated with plant infection by 'Candidatus Liberibacter asiaticus' (CLas) and other Liberibacter species. CLas is transmitted by Diaphorina citri, the Asian citrus psyllid, in a circulative propagative manner. Circulative propagative transmission is a complex process comprising at least three steps: movement of the pathogen into vector tissues, translocation and replication of the pathogen within the vector host, and pathogen inoculation of a new host by the vector. In this work, we describe an excised leaf CLas acquisition assay, which enables precise measurements of CLas acquisition by D. citri in a streamlined laboratory assay. Briefly, healthy fourth and fifth instar D. citri nymphs acquire CLas from excised CLas-positive leaves, where the insects also complete their developmental cycle. CLas titer in the resulting adults is measured using quantitative PCR and CLas-specific 16S rRNA gene primers. We observed positive correlations between CLas titer in each leaf replicate and the CLas titer that developed in the insects after acquisition (rs = 0.78; P = 0.0002). This simple assay could be used to detect CLas acquisition phenotypes and their underlying genotypes, facilitate assessment of plant factors that impact acquisition, and screen for compounds that interfere with CLas acquisition by delivering these compounds through the excised leaf.

Effect of Leaf Age, Leaf Segments and Surface Treatments on Pathogenicity Levels of Colletotrichum sublineola in Sorghum and Johnson Grass

Colletotrichum sublineola is a casual pathogen of sorghum anthracnose. Sorghum pathologists often need to conduct evaluations for anthracnose resistance in large scale which are expensive and labor intensive. As a solution, an excised-leaf assay has been used, but whether or not leaf age, position or region affects pathogenicity scores has not previously been evaluated. Essentially, in an excised-leaf assay, is response to C. sublineola over all or part of a leaf blade identical? To get an answer, three sorghum and one Johnson grass cultivars were tested. The top five leaves were inoculated at the apex, mid-leaf and base of each leaf blade. Results show nearly no effect of leaf age to pathogenicity level within the top five leaves. Furthermore, in order to evaluate any protective role of leaf wax to C. sublineola, the wax was disrupted by simply wiping the leaf surface by a thumb, or as an alternative method, leaf surface tension was reduced by submerging leaves into 2% TWEEN 20 before inoculation. Compared to control, wiped leaves increased pathogenicity scores on the leaf blade and midrib in two of three sorghum cultivars, but 2% TWEEN 20-treated leaves had only minimal changes in pathogenicity level compared to controls.

Leaf angle distribution in Johnsongrass, leaf thickness in sorghum and Johnsongrass, and association with response to Colletotrichum sublineola

Basal leaf angle distribution was surveyed in twenty-one Johnsongrass cultivars near the end of the vegetative stage. The angles increased from the top to the bottom leaves, and compared to cultivated grain sorghums, the average angle was larger in Johnsongrass. When basal leaf angle distribution data were correlated with pathogenicity test data from excised-leaf assays for three isolates of Colletotrichum sublineola, the results showed a weak positive correlation between basal leaf angle and pathogenicity level in Johnsongrass. In order to investigate a protective role of leaf thickness to C. sublineola, leaf thickness was measured in three sorghum cultivars and one Johnsongrass cultivar at the 8-leaf-stage. Leaf thickness near the apex, near the base, and half-way between the two points were measured in the top four leaves of each plant. Thickness of leaf blade and midrib were recorded separately. Using an excised-leaf-assay, the three points were inoculated with C. sublineola, and pathogenicity level was recorded 4-days-post-inoculation. Results showed strong negative correlations between leaf midrib thickness and pathogenicity level in sorghum and Johnsongrass but not in leaf blades.

Metabolic Pathway of Topramezone in Multiple-Resistant Waterhemp (Amaranthus tuberculatus) Differs From Naturally Tolerant Maize.

Waterhemp [Amaranthus tuberculatus (Moq.) Sauer] is a problematic dicot weed in maize, soybean, and cotton production in the United States. Waterhemp has evolved resistance to several commercial herbicides that inhibit the 4-hydroxyphenylpyruvate-dioxygenase (HPPD) enzyme in sensitive dicots, and research to date has shown that HPPD-inhibitor resistance is conferred by rapid oxidative metabolism of the parent compound in resistant populations. Mesotrione and tembotrione (both triketones) have been used exclusively to study HPPD-inhibitor resistance mechanisms in waterhemp and a related species, A. palmeri (S. Wats.), but the commercial HPPD inhibitor topramezone (a pyrazolone) has not been investigated from a mechanistic standpoint despite numerous reports of cross-resistance in the field and greenhouse. The first objective of our research was to determine if two multiple herbicide-resistant (MHR) waterhemp populations (named NEB and SIR) metabolize topramezone more rapidly than two HPPD inhibitor-sensitive waterhemp populations (named SEN and ACR). Our second objective was to determine if initial topramezone metabolite(s) detected in MHR waterhemp are qualitatively different than those formed in maize. An excised leaf assay and whole-plant study investigated initial rates of topramezone metabolism (<24 h) and identified topramezone metabolites at 48 hours after treatment (HAT), respectively, in the four waterhemp populations and maize. Results indicated both MHR waterhemp populations metabolized more topramezone than the sensitive (SEN) population at 6 HAT, while only the SIR population metabolized more topramezone than SEN at 24 HAT. Maize metabolized more topramezone than any waterhemp population at each time point examined. LC-MS analysis of topramezone metabolites at 48 HAT showed maize primarily formed desmethyl and benzoic acid metabolites, as expected based on published reports, whereas SIR formed two putative hydroxylated metabolites. Subsequent LC-MS/MS analyses identified both hydroxytopramezone metabolites in SIR as different hydroxylation products of the isoxazole ring, which were also present in maize 48 HAT but at very low levels. These results indicate that SIR initially metabolizes and detoxifies topramezone in a different manner than tolerant maize.

JoVE - Year in Review: 2015

In January 2015, JoVE Developmental Biology became the newest addition to the JoVE family, and featured a live-imaging protocol for visualizing the developing Drosophila eye. In February, JoVE Medicine showed state-of-the-art cranial ultrasound imaging in neonates. Advances in this technique have improved its diagnostic value, allowing timely therapeutic intervention. In March, JoVE Environment featured dendroecology, the science of using tree rings to study ecological effects of specific environmental factors. In April, JoVE Biology examined a mode of bacterial motility called swarming with a two-phase swarm assay. that can be used to study various aspects of bacterial growth. In May, JoVE Chemistry described the self-assembly of single-stranded DNA tiles into complex 2D shapes. This demonstrated the principles of DNA tiling as an approach for making programmable nanostructures. June 2015 marked the release of JoVE's 100th issue, and in JoVE Behavior, we featured protocols for assessing cognition, including a method of quantifying learning in young infants by tracking leg movements. In July, JoVE Medicine presented a method for measuring halitosis in dogs by taking breath samples, and performing gas chromatography. This assay could be used in trials of dog food formulated to fight bad breath in our canine companions. In August, JoVE Bioengineering contained a protocol for bioengineering kidney tissues. Kidneys are decellularized by perfusion, and repopulated with human renal cells, This promising technology can lead to kidney grafts made from a patient's own cells. In September, JoVE Environment examined herbicide resistance, which threatens commercial crop production. An excised leaf assay determines the rates of herbicide metabolism, which is useful for studying the metabolic basis of resistance. In October, JoVE Neuroscience featured a method for interfacing 3D neuronal cultures to micro-electrode arrays. The complex 3D assemblies closely approximate in vivo neural networks. November's edition of JoVE Behavior showcased the natural human ability to acquire new motor skills, which is central in a structured rehabilitation program for amputees as they learn how to use multifunctional prosthetics. In December, JoVE Immunology & Infection prepared us for the impending flu season with an easy protocol for sampling influenza in pigs. Because swine are important hosts for flu viruses, this method helps to monitor virus evolution and currently circulating strains. This Year in Review was just a sampling of more than 900 video-articles that JoVE published in 2015. Browse the JoVE archives for thousands of other videos, and come back each week to see brand-new material in JoVE: The Journal of Visualized Experiments.

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and –sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay.

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and -sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

September 2015 - This Month in JoVE: Measuring Greenhouse Gases and Herbicide Resistance, Bioengineering Bone, and Analyzing Neuromuscular Control with Virtual Reality

Here's a look at what's coming up in the September 2015 issue of JoVE: The Journal of Visualized Experiments. In JoVE Environment, we've had a pretty warm summer in the northern hemisphere, so we can't help but think about all the greenhouse gases in our atmosphere that keep our planet nice and warm. These days, livestock are a major source of greenhouse gas emissions, especially methane. Livestock contribute anywhere from 7 to 18% of global emissions-depending on who you ask. Naturally, climate scientists are interested in controlling enteric methane from livestock. This requires accurate and precise measurement techniques, so Hristov et al. present a method using the Automated Head-Chamber System (AHCS), which monitors methane (CH4) and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals (e.g., cattle). This system produces repeatable and accurate emission results, and can help efforts to curb greenhouse gas emissions. In JoVE Environment, we're also concerned about herbicide resistance in weeds, which poses a serious threat to food and fiber production. Plants can gain resistance to herbicides through genetic mutations or through enhanced activity of enzymes that metabolize and detoxify chemicals. In order to determine rates of herbicide metabolism in certain weeds, Ma et al. developed an excised leaf assay, which allows them to take experiments from the greenhouse into the lab so they can perform radiolabeled herbicide experiments more efficiently. As more and more plants become resistant to herbicides, this method will be useful for investigating the metabolic basis of herbicide resistance. In JoVE Bioengineering, one of the ultimate goals of tissue engineering is to regenerate large bone defects using synthetic scaffolds or templates. Most available scaffolds don't allow appreciable cell infiltration or distribution to achieve significant bone regeneration, so Oh et al. have developed a highly porous bone-like template with micro-channels and nanopores. Cells can be seeded into the biogenic templates through capillary action, and grow to confluence within days. This concept can help advance the repair of large bone defects. In JoVE Behavior, the human neuromuscular junction can be studied using a variety of noninvasive techniques, including electromyography (EMG), 3D motion capture, and transcranial magnetic stimulation. This month, Talkington et al. add the unique aspect of virtual reality to this test battery, and describe a customizable circuit that synchronizes data sampled from all of these techniques. You've just had a sneak peek of the September 2015 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

Susceptibility of four Eucalyptus host species for the development of Thaumastocoris peregrinus Carpintero and Dellapé (Hemiptera: Thaumastocoridae)

Thaumastocoris peregrinus is a serious sap-sucking pest of Eucalyptus species in many important forest growing regions worldwide. This invasive insect may not only cause enormous economic losses to the eucalypt forest industry but also negatively affect urban trees in public spaces. We conducted laboratory experiments to evaluate the susceptibility of four Eucalyptus species for the development of T. peregrinus. Three of the tested Eucalyptus species are economically important for the forestry industry worldwide and the other is a popular amenity tree in New Zealand. The development of eggs, nymphs and adults was evaluated in excised leaf assays. Eucalyptus nitens and Eucalyptus nicholii were identified as suitable host plants. The adult leaf type of E. nitens was shown to be critical for successful feeding. Despite initiating feeding T. peregrinus did not survive beyond 1st instar nymph on either Eucalyptus fastigata or Eucalyptus regnans. Accordingly, our study contributes evidence that there may be a lower risk to species within the subgenus Monocalyptus from T. peregrinus damage. These are positive findings for the forestry sector as E. regnans and E. fastigata are both valued Eucalyptus species grown predominantly for pulp in commercial forestry in New Zealand and worldwide.

New Excised-Leaf Assay Method to Test Inoculativity of Asian Citrus Psyllid (Hemiptera: Psyllidae) With &lt;I&gt;Candidatus&lt;/I&gt; Liberibacter asiaticus Associated With Citrus Huanglongbing Disease

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the primary vector of Candidatus Liberibacter asiaticus (Las) associated with huanglongbing, or citrus greening, the most devastating citrus (Citrus spp.) disease worldwide. Here, we developed a new "excised-leaf assay" that can speed up Las-inoculativity tests on Asian citrus psyllid from the current 3-12 mo (when using whole citrus seedlings for inoculation) to only 2-3 wk. Young adults of Asian citrus psyllid that had been reared on Las-infected plants were caged on excised healthy sweet orange [Citrus sinensis (L.) Osbeck] leaves for a 1-2-wk inoculation access periods (IAP), and then both psyllids and leaves were tested later by quantitative polymerase chain reaction (PCR). When single adults were tested per leaf, percentages of Las-positive leaves averaged 2-6% by using HLBaspr primers and 10-20% by using the more sensitive LJ900 primers. Higher proportions of Las-positive leaves were obtained with 1) higher densities of inoculating psyllids (5-10 adults per leaf), 2) longer IAPs, and 3) incubation of leaves for 1 wk postinoculation before PCR. Logistic regression analysis indicated a positive correlation between Las titer in Asian citrus psyllid adults tested singly and the probability of detecting Las in the inoculated leaves, correlations that can be very useful in epidemiological studies. Comparison between excised leaves and whole seedlings, inoculated consecutively for 1 wk each by one or a group of psyllids, indicated no significant difference between Las detection in excised leaves or whole plants. This new excised-leaf assay method saves considerable time, materials, and greenhouse space, and it may enhance vector relation and epidemiological studies on Las and potentially other Liberibacter spp. associated with huanglongbing disease.

Enantioselective Demethylation of Nicotine as a Mechanism for Variable Nornicotine Composition in Tobacco Leaf

Nicotine and its N-demethylation product nornicotine are two important alkaloids in Nicotiana tabacum L. (tobacco). Both nicotine and nornicotine have two stereoisomers that differ from each other at 2'-C position on the pyrrolidine ring. (S)-Nicotine is the predominant form in the tobacco leaf, whereas the (R)-enantiomer only accounts for ∼0.2% of the total nicotine pool. Despite considerable past efforts, a comprehensive understanding of the factors responsible for generating an elevated and variable enantiomer fraction of nornicotine (EF(nnic) of 0.04 to 0.75) from the consistently low EF observed for nicotine has been lacking. The objective of this study was to determine potential roles of enantioselective demethylation in the formation of the nornicotine EF. Recombinant CYP82E4, CYP82E5v2, and CYP82E10, three known tobacco nicotine demethylases, were expressed in yeast and assayed for their enantioselectivities in vitro. Recombinant CYP82E4, CYP82E5v2, and CYP82E10 demethylated (R)-nicotine 3-, 10-, and 10-fold faster than (S)-nicotine, respectively. The combined enantioselective properties of the three nicotine demethylases can reasonably account for the nornicotine composition observed in tobacco leaves, which was confirmed in planta. Collectively, our studies suggest that an enantioselective mechanism facilitates the maintenance of a reduced (R)-nicotine pool and, depending on the relative abundances of the three nicotine demethylase enzymes, can confer a high (R)-enantiomer percentage within the nornicotine fraction of the leaf.

Low-Level Copper Plus Chitosan Applications Provide Protection Against Late Blight of Potato

The organic grower can manage potato late blight, caused by Phytophthora infestans (Mont.) de Bary, with natural copper compounds such as copper sulfate pentahydrate, copper oxide, or copper hydroxide. However, recent environmental concerns about copper residues by the USDA's National Organic Program indicate a need to control late blight with reduced copper levels. This report describes a strategy for disease control using lower levels of copper sulfate pentahydrate in combination with a chitosan sticker and complexing agent. In excised leaf assays, this combination provided moderate control of late blight and protection against copper-related leaf yellowing at copper levels approximately 40 fold lower than those recommended for a commercial fungicide with copper hydroxide as the active ingredient. Accepted for publication 23 February 2006. Published 6 April 2006.

Identification of five physiological races of blackspot, Diplocarpon rosae, Wolf on roses

AbstractDifferentiation into physiological races of Diplocarpon rosae, which causes blackspot on wild and cultivated roses, was investigated with single conidial isolates of the pathogen. Infection experiments with a simple excised leaf assay demonstrated differential interactions between single isolates and a set of 10 test rose genotypes. Differential reactions could be observed among rose varieties as well as between and within a wild rose species. Accordingly, five different physiological races can be identified among the 15 isolates tested. A breeding line and one genotype of the wild species Rosa wichuraiana were found to be resistant against all isolates tested. These data will be employed in further investigations on the genetics of blackspot resistance in roses and for resistance breeding programmes.

Pyricularia Leaf Spot: A New Disease of Ornamental Plants of the Family Marantaceae.

A severe leaf spot disease incited by the fungus Pyricularia oryzae was identified on Ctenanthe oppenheimiana and C. setosa "Greystar." Primary symptoms on young leaves consisted of individual circular to slightly irregular pinpoint spots with white necrotic centers zonated by narrow brown-yellow halos. On mature leaves, extended necrotic areas resembling those caused by phytotoxicity were formed. Artificially inoculated leaves with a spore suspension of the isolated fungus from the above case showed symptoms after 3 to 4 days' incubation at 25°C and high humidity. Fungal isolates obtained from Ctenanthe plants of Brazilian origin were found to be highly pathogenic on various plants within the family Marantaceae when they were tested by an excised leaf assay method. By contrast, P. oryzae isolates obtained from rice plants grown in Greece caused either hypersensitivity or immune response symptoms in various Marantaceae. Analysis of esterase and lactate dehydrogenase isozymes showed different banding patterns for rice and Ctenanthe isolates of P. oryzae. Conditions of prolonged leaf wetness combined with prevailing high temperature and humidity favored the epidemic appearance of the Pyricularia leaf spot disease on glasshouse-grown plants during the summer months of 1995 in Greece.

Variation in performance of western flower thrips populations on a susceptible and a partially resistant chrysanthemum cultivar

Variation in host plant performance among populations of a phytophagous insect pest is a potential threat to the durability of host plant resistance. Aggressive biotypes may overcome the protective properties of formerly resistant cultivars. Therefore, it is of interest to study such variation in breeding programs for host plant resistance to insects. In the present study, the performance of ten populations of Frankliniella occidentalis, the western flower thrips, was determined on a susceptible and a partially resistant cultivar of chrysanthemum, Dendranthema grandiflora. Damage, reproduction, and adult survival were determined using an excised leaf assay. Significant differences between the two cultivars and among the ten populations were found for all three characteristics. In general, damage, reproduction and adult survival were reduced on the resistant cultivar when compared to the susceptible cultivar. Some populations showed, in comparison to the reference population from the Netherlands, much higher damage and reproduction on one or both chrysanthemum cultivars. But also in these populations performance on the resistant cultivar was poor compared to the susceptible cultivar.

Effects of snowdrop lectin (GNA) delivered via artificial diet and transgenic plants on the development of tomato moth ( Lacanobia oleracea) larvae in laboratory and glasshouse trials

The effects of snowdrop lectin ( Galanthus nivalis agglutinin, GNA) on Lacanobia oleracea larval growth, development, consumption, and survival, were examined by 3 distinct bioassay methods. Larvae were reared on artificial diet containing GNA at 2% (w/w) dietary protein; on excised leaves of transgenic potato expressing GNA at approx. 0.07% of total soluble proteins; and on transgenic potato plants expressing GNA at approx. 0.6% of total soluble proteins in glasshouse trials. Significant effects on larval growth were observed with all three treatments. At 21 days after hatch mean larval biomass was reduced by 32 and 23%, in the artificial diet and excised leaf bioassays respectively. In glasshouse trials a 48% reduction in insect biomass per plant was observed after 35 days. The artificial diet and excised leaf assays also showed that GNA significantly slowed larval development as assessed by instar duration. GNA caused a 59% overall reduction in mean daily consumption in the artificial diet assay, and a significant reduction in leaf damage in glasshouse trials. However, prolonged compensatory feeding by larvae in the excised leaf assay resulted in their consuming 15% more total leaf material than the control group. Adaptation to low levels of GNA, in terms of biomass recovery and compensatory feeding, was observed within one larval generation in the detached leaf assay. No significant effects of GNA on larval survival were observed in the artificial diet and detached leaf bioassays, whereas survival was decreased by approx. 40% in the glasshouse bioassay. The assays show that the insecticidal effects of GNA can be observed both in vitro when fed in artificial diet and in planta, and can be demonstrated in the glasshouse as well as under growth cabinet conditions.

Biosynthesis of ecdysone and ecdysone phosphates in spinach

The polar ecdysteroid conjugate, ecdysone phosphate (2β,3β,14α,22 R,25-pentahydroxy-7-en-6-one-3- phosphate) was identified in excised first leaves of spinach, where it is endogenously produced during 20- hydroxyecdysone biosynthesis. Radiolabelled [ 14C]ecdysone phosphate was isolated from several excised leaf assays and was hydrolysed with wheat germ acid phosphatase to yield [ 14C]ecdysone. Incorporation of ecdysone into excised first leaves followed by 32P exposure produced a compound with 32P activity, with chromatographic properties identical to those of the isolated [ 14C]ecdysone phosphate and upon hydrolysis released ecdysone. In spinach first leaves with active ecdysteroid biosynthesis, ecdysone is present at 0.004% of the total free ecdysteroid and contained 6% of the total radioactivity from [2- 14C]mevalonic acid (MVA). These biosynthetically active tissues also produce radiolabelled lathosterol, ecdysone-3-phosphate and 20-hydroxyecdysone. In biosynthetically inactive tissue (immature apical organs) no radiolabelled lathosterol, ecdysone-3-phosphate, ecdysone or 20-hydroxyecdysone was produced from [2- 14C]MVA despite an active biosynthesis of C 29-sterols. Several intermediate and end product ecdysteroids, when incorporated into excised first leaves of spinach produced conjugates which were readily cleaved by wheat germ acid phosphatase. The ecdysteroid pathway appears to be regulated by the presence of ecdysteroid substrates.

Ecdysteroid biosynthesis during the ontogeny of spinach leaves

During the growth and development of spinach ( Spinacia oleracea), phytoecdysteroid biosynthesis is observed later in the ontogeny of cotyledons, first and second leaves, whereas early in leaf ontogeny no biosynthesis of phytoecdysteroid is observed. An excised leaf assay for incorporation of various substrates [2- 14C]mevalonic acid (MVA), [4- 14C]cholesterol, [2- 14C]acetate and [22,23- 3H]α-ecdysone into phytoecdysteroids was developed. All four radiolabelled substrates can be incorporated into the ecdysteroids of spinach (20-hydroxyecdysone and polypodine B). The [2- 14C]MVA is apparently incorporated into the C 27-sterol lathosterol (5α-cholest-7-en-3β-ol) prior to radiolabel incorporation into spinach ecdysteroids. Leaves early in their ontogeny fail to produce radiolabelled lathosterol or ecdysteroids from [2- 14C]MVA in spite of an active C 29-sterol biosynthesis from [2- 14C]MVA. Conversion of [22,23- 3H]α-ecdysone into radiolabelled spinach ecdysteroids parallels the incorporation of [2- 14C]MVA into these ecdysteroids with leaf ontogeny. Lathosterol was structurally identified as a component of mature spinach leaves accounting for approximately 4% of the total sterol composition. The spinach excised leaf assay provides a model system for the study of ecdysteroid biosynthesis in plants.