Cotton Leaf Discs Research Articles

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.

First Report of Tomato Yellow Leaf Curl Virus Infecting Upland Cotton (Gossypium hirsutum) in Alabama, USA.

Cotton (Gossypium hirsutum L.) is used as a non-host of tomato yellow leaf curl virus (TYLCV) (family Geminiviridae, genus Begomovirus) in many studies (Ghanim and Czosnek 2000; Legarrea et al. 2015; Zeidan and Czosnek 1991), but only one reports methods used to determine host-status (Sinisterra et al. 2005), and there is one contradictory report from China stating cotton is a host of TYLCV (Li et al. 2014). In October 2018, cotton was screened for the presence of begomoviruses in Elmore, Escambia and Macon Counties, AL, where infestations of its whitefly vector (Bemisia tabaci Genn.) occurred in August. DNA was extracted from fully expanded leaves from the upper 1/3 of the canopy using a DNeasy® Plant Mini Kit (QIAGEN, Hilden, Germany) and amplified with primers V324/C889 targeting a 575 bp coat protein fragment of begomoviruses (Brown et al. 2001). Five out of 200 cotton samples tested positive, and sequences recovered from three samples revealed 98-99% identity to TYLCV isolates in NCBI (Accession Nos. MT947801-03); sequences from the other two samples were of low quality and inconclusive. These samples were not available for additional tests, therefore, we proceeded to confirm host status using a monopartite clone of TYLCV-Israel (Reyes et al. 2013) reported in the US (Polston et al. 1999). All experiments were conducted in growth chambers with 16:8 light:dark cycle at 25.0℃ and 50% RH. Cotton seedlings (DeltaPine 1646 B2XF) at the 2-3 true leaf stage and tomatoes (Solanum lycopersicum L., var. 'Florida Lanai') at the 4 true leaf stage were agroinoculated at the stem tissue between the apical meristem and the first node (Reyes et al. 2013). Tomato served as a positive control; tomato and cotton mock inoculated with an empty vector were negative controls. A hole-punch was used to collect 4 leaf discs along midveins of the three, uppermost fully expanded leaves. DNA was extracted 28 days after inoculation as described above. A 390 bp segment of the intergenic region of TYLCV-A was amplified using primers PTYIRc287/PTYIRv21 (Nakhla et al., 1993). PCR results from agroinoculated plants confirmed (2/18) cotton plants, (5/5) tomatoes and (0/10) mock inoculated controls were infected with TYLCV. Whitefly transmission to cotton was confirmed using a leaf-disc bioassay for rapid testing (Czosnek et al. 1993). Bemisia tabaci MEAM-1 reared on eggplant (non-host of TYLCV) were placed on agroinoculated TYLCV-infected tomato/span> plants for a 96-h acquisition access period. Cohorts of 10 viruliferous B. tabaci were aspirated into 30mL cups each containing a 2.5cm healthy cotton leaf disc set in plant agar. After a 48-h inoculation access period, adults and their eggs were removed from the leaf discs. Leaf discs were held another 96-h before they were tested for TYLCV using the methods described above. TYLCV-infection was confirmed in (9/20) cotton leaf discs, demonstrating the viral load delivered by whiteflies was high enough to initiate local infection in cotton. No obvious begomovirus symptoms were observed on cotton plants in the field or laboratory. Field collection of samples was prompted by symptoms attributed to cotton leafroll dwarf virus (Avelar et al. 2017). TYLCV infection of cotton does not appear to be of economic importance. Additional information is needed to determine the frequency of infection in the field, specificity of TYLCV isolate x cotton genotype interactions leading to successful infection, and underlying causes of conflicting host-status reports in previously published studies.

The energy cost of repairing photoinactivated photosystem II: an experimental determination in cotton leaf discs.

Summary Photosystem II (PSII), which splits water molecules at minimal excess photochemical potential, is inevitably photoinactivated during photosynthesis, resulting in compromised photosynthetic efficiency unless it is repaired. The energy cost of PSII repair is currently uncertain, despite attempts to calculate it.We experimentally determined the energy cost of repairing each photoinactivated PSII in cotton (Gossypium hirsutum) leaves, which are capable of repairing PSII in darkness.As an upper limit, 24 000 adenosine triphosphate (ATP) molecules (including any guanosine triphosphate synthesized at the expense of ATP) were required to repair one entire PSII complex. Further, over a 7‐h illumination period at 526–1953 μmol photons m−2 s−1, the ATP requirement for PSII repair was on average up to 4.6% of the ATP required for the gross carbon assimilation.Each of these two measures of ATP requirement for PSII repair is two‐ to three‐fold greater than the respective reported calculated value. Possible additional energy sinks in the PSII repair cycle are discussed.

Survival, development and reproduction of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) on kaolin-treated cotton.

The mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is a cotton pest widespread in several cotton growing regions of Brazil, particularly in the semi-arid region of southwestern Bahia. The impact of kaolin on survival, development and reproduction of P. solenopsis was evaluated in the laboratory. The experiment was developed in a completely randomized design with two treatments: immature or newly emerged adults of P. solenopsis sprayed with kaolin and fed with cotton leaf discs treated with kaolin suspension (with kaolin) (T1) and immature or newly emerged adults of P. solenopsis sprayed with distilled water and fed with cotton leaf discs treated with distilled water (without kaolin) (T2). The kaolin suspension shortens the life cycle, increases the reproductive potential and population growth of the cotton mealybug, P. solenopsis and, therefore, it should be used with caution on cotton plants in regions with a history of occurrence of this pest.

Sublethal Effects of Insect Growth Regulators on Boll Weevil (Coleoptera: Curculionidae).

Immature stages of the boll weevil, Anthonomus grandis grandis Boh. (Coleoptera: Curculionidae), develop protected inside cotton fruiting structures. Therefore, the adult beetles have become the main target of insecticide applications. The use of insect growth regulators (IGRs) is recommended against immatures, even though they may also affect the survival and reproductive traits of adult insects. The present study evaluated the impact of a juvenile hormone analog (pyriproxyfen), an ecdysteroid agonist (methoxyfenozide), and a chitin biosynthesis inhibitor (lufenuron) on adult cotton boll weevils, a key cotton pest. Mated and virgin beetles were treated by feeding them contaminated squares and cotton leaf discs that were previously immersed into pyriproxyfen, methoxyfenozide, and lufenuron solutions at field-rate concentrations. After exposure, treated couples were caged onto cotton plants, and survival, fecundity, and egg viability were evaluated. The IGRs neither affected the survival nor fecundity of adult boll weevils. On the other hand, egg viability was significantly reduced by lufenuron, regardless of whether the females were treated premating or postmating or whether their pairs were either treated or untreated. However, egg viability increased as the females aged since the initial exposure date to lufenuron, indicating a potential transovarial effect of this insecticide. Our results indicate that pyriproxyfen and methoxyfenozide do not affect adult boll weevils, whereas lufenuron temporarily reduces the egg viability of this key cotton pest.

Susceptibility of boll weevil to ready-to-use insecticide mixtures

ABSTRACT: Boll weevil is the major cotton pest in Brazil, and insecticides are widely recommended against it. We determined the susceptibility of boll weevil to insecticides either in single or in mixture ready-to-use formulations, which are registered to spray cotton fields under the hypothesis that mixtures are more toxic to the target pest. Concentration-mortality curves were determined to adult species, simultaneously through dried residues and ingestion. Ten insecticide formulations were studied with five in mixture (lambda-cyhalothrin + thiamethoxam, lambda-cyhalothrin + chlorantraniliprole, thiamethoxam + chlorantraniliprole, and fenitrothion + esfenvalerate) and their five respective single formulations. Cotton leaf discs and cotyledons were dipped into insecticide dilutions prepared by diluting the commercial products into distilled water. Adult mortality was assessed 48 hours after caging adults on treated and untreated materials. The LC50s-concentrations varied from 0.004 to 0.114 g a.i./L, with a relative potency between single and mixture ones, varying from 1.37- to 29.59-fold. Furthermore, lambda-cyhalothrin and thiamethoxam in single formulation were the most toxic insecticides to boll weevil. Among insecticide mixtures, only lambda-cyhalothrin + chlorantraniliprole resulted in a synergic effect; whereas the remaining mixtures showed an antagonistic effect. Therefore, except for the mixture of lambda-cyhalothrin + chlorantraniliprole, the remaining mixtures did not enhance toxicity against the boll weevil and should be recommended only when aimed at different purposes.

A new observation on feeding behaviour of pink bollworm and its application in screening Bt-resistant population.

The pink bollworm (PBW), Pectinophora gossypiella (Saund.) does not feed on leaves in natural circumstances. We made an attempt with the first instar larva (5days old) and found it eats the leaf in the absence of other foods. Utilizing this new feeding behaviour, a simple methodology was developed for screening of resistance in PBW against Bt cotton plants. The PBW collected from BGII Bt cotton fields (BGII-resistant population) and NBAIR culture (susceptible population) were reared under laboratory conditions for two generations. Laboratory reared 5-day old larvae of PBW were released on cotton leaf discs individually for screening. The BGII-resistant and susceptible larvae fed an average leaf area of 75.52 ± 16.68 and 5.95 ± 0.93 mm2 with survival rate of 90 and 4% respectively, in BGII Bt cotton leaves. Whereas in case of non-Bt cotton (MCU13), the BGII-resistant and susceptible larvae consumed average leaf area of 114.84 ± 23.70 and 116.80 ± 24.14 mm2 with survival rate of 93.34 and 95.33%, respectively. In addition to the screening process, the larval survivors were transferred to an artificial diet after 7days of experiment and observed up to their emergence as adults. To confirm the development of resistance in PBW, sequencing of larval DNA amplicons was carried out and it revealed mutation in the cadherin gene of the BGII-resistant PBW population. Hence, the detached leaf bit feeding assay described here could be used in a simple manner for screening of resistance developed by PBW against Bt cotton and also for evaluating Bt cotton plants for their inherent resistance to PBW. This method could also be used for studying toxicity of Bt isolates by coating spore-crystal mixture on non-transgenic cotton leaf discs.

Chemical composition, toxic and development- and reproduction-inhibiting effects of some essential oils against Tetranychus urticae Koch (Acarina: Tetranychidae) as fumigants

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most serious pests of many crops both indoors and outdoors in south-western Turkey (Antalya). In the present study, essential oils (EOs) from five medicinal and aromatic plants [Mentha pulegium L. (Labiatae), Foeniculum vulgare Mill. (Umbelliferae), Pistacia terebinthus L., Schinus molle L. (Anacardiaceae) and Vitex agnus-castus L. (Verbenaceae)] were tested for their fumigant toxic and development- and reproduction-inhibiting effects against the pest under in vivo conditions. In the fumigant toxicity assays, newly emerged (0–24 h) adult females and larvae and 0-24-h-old eggs of the mite were exposed to four different concentrations of each EO for 12 h in separate desiccators (10 L) used as test chambers. In the development- and reproduction-inhibiting assays, newly emerged (0–24 h) female deutonymphs of the mite were separately exposed to a lower concentration (0.5 µL/L for M. pulegium, 2 µL/L for F. vulgare, 2.5 µL/L for P. terebinthus and 10 µL/L for V. agnus-castus and S. molle) of each EO in desiccators for 12 h, and then, 20 survivors from each EO were individually maintained on cotton leaf discs to obtain data on some parameters (adult emergence rate, number of eggs per female, egg-hatching rate, etc.) until there are no living individuals. The results from the study indicated that all the EOs had fumigant effect against the mite in varying degrees. M. pulegium was found to be the most toxic oil against all the biological stages tested (LC50 = 0.60 µL/L air for eggs, 0.60 µL/L air for larvae and 0.49 µL/L air for adult females), followed by F. vulgare (LC50 = 2.67 µL/L air for eggs and adult females, and 2.56 µL/L air for larvae). M. pulegium EO also had the highest development- and reproduction-inhibiting effect on the pest. Fecundity was reduced by 55.9% and egg hatching was inhibited by 29.9% in survivors of deutonymphs of T. urticae fumigated with M. pulegium EO at 0.5 µL/L air for 12 h. In the phytotoxicity assays with tomato, cucumber, Phaseolus and eggplant seedlings, scattered necrotic spots and slight chlorosis only on the young foliage of cucumber seedlings exposed to M. pulegium EO at the highest concentration for 12 h were visual symptoms of phytotoxicity. Overall results indicate that M. pulegium EO can be used in the management of T. urticae in greenhouses as a fumigant for both toxic and development- and reproduction-inhibiting effects.

Leaf Tissue Assay for Lepidopteran Pests of Bt Cotton

Laboratory measurements of susceptibility to Bt toxins can be a poor indicator of the ability of an insect to survive on transgenic crops. We investigated the potential of using leaf tissue for evaluating heliothine susceptibilities to two dualgene Bt cottons, Gossypium hirsutum L. A preliminary study with different cotton leaf disk combinations was performed to determine the best procedure for using cotton tissue to assay bollworm, Helicoverpa zea Boddie, and tobacco budworm, Heliothis virescens F. Neonate larvae from a laboratory colony were assayed simultaneously on leaf disks of non-Bt and two dual-gene transgenic cottons in addition to meridic diet overlaid with discriminating doses of a commerciallyformulated Bt product. The bollworm colony in the laboratory was more tolerant than tobacco budworm to the commercially-formulated Bt product. When corrected for the non-Bt check, percentage of bollworm larval mortality was 42.1 and 71.9 on leaf disks of the two dual-gene Bt cottons. No tobacco budworm survived on dualgene Bt cotton leaf disks in the study. Assays using transgenic cotton leaf disks may compliment current meridic diet-based methods by providing a linkage to insect survival on Bt cotton plants.

Acaricidal activity of juazeiro leaf extract against red spider mite in cotton plants

Abstract: The objective of this work was to evaluate the toxicity, repellency, efficiency, and phytotoxicity of the aqueous extract of juazeiro (Ziziphus joazeiro) leaves in the control of the red spider mite, Tetranychus ludeni, on cotton plant. For toxicity evaluation, T. ludeni adult females were put on cotton leaf discs sprayed with the juazeiro extract. The repellent effect was verified in arenas that contained cotton leaf discs treated or not with the aqueous extract. The control efficiency of the juazeiro extract was evaluated by the infestation of cotton plants by adult female mites, followed by spraying with the extract. Measured LC50 was 3.54% (m/v), with 76.47% mortality and repellent effect. There was control efficiency for 120 hours, with 78.02% average, without significant difference between the evaluation intervals. Cotton plants did not show phytotoxicity. The juazeiro aqueous extract shows potential as an alternative control of T. ludeni on cotton plant due to its high toxicity, repellent effect, and efficiency in causing mortality, without being phytotoxic to cotton plants.

Whole-tissue determination of the rate coefficients of photoinactivation and repair of photosystem II in cotton leaf discs based on flash-induced P700 redox kinetics

Using radioactively labelled amino acids to investigate repair of photoinactivated photosystem II (PS II) gives only a relative rate of repair, while using chlorophyll fluorescence parameters yields a repair rate coefficient for an undefined, variable location within the leaf tissue. Here, we report on a whole-tissue determination of the rate coefficient of photoinactivation k i , and that of repair k r in cotton leaf discs. The method assays functional PS II via a P700 kinetics area associated with PS I, as induced by a single-turnover, saturating flash superimposed on continuous background far-red light. The P700 kinetics area, directly proportional to the oxygen yield per single-turnover, saturating flash, was used to obtain both k i and k r . The value of k i , directly proportional to irradiance, was slightly higher when CO2 diffusion into the abaxial surface (richer in stomata) was blocked by contact with water. The value of k r , sizable in darkness, changed in the light depending on which surface was blocked by contact with water. When the abaxial surface was blocked, k r first peaked at moderate irradiance and then decreased at high irradiance. When the adaxial surface was blocked, k r first increased at low irradiance, then plateaued, before increasing markedly at high irradiance. At the highest irradiance, k r differed by an order of magnitude between the two orientations, attributable to different extents of oxidative stress affecting repair (Nishiyama et al., EMBO J 20: 5587-5594, 2001). The method is a whole-tissue, convenient determination of the rate coefficient of photoinactivation k i and that of repair k r .

Effects of botanical insecticides on the instantaneous population growth rate of Aphis gossypii Glover (Hemiptera: Aphididae) in cotton

Botanical insecticides have been studied aiming the alternative pest control. The present study investigated the effects of these insecticides on the instantaneous population growth rate (ri) of Aphis gossypii. Botanical insecticides were tested in the following concentrations: Compostonat®, Rotenat-CE® and Neempro (0, 0.50, 0.75, 1.00, 1.25, 1.50 and 1.75%); Natuneem® and Neemseto® (0, 0.25, 0.50, 0.75 and 1.00%) and essential oils of Foeniculum vulgare Mill., Cymbopogom winterianus (L.), Chenopodium ambrosioides L. and Piper aduncum L (0, 0.0125, 0.025, 0.0375 and 0.05%). Cotton leaf discs, CNPA 8H cultivar with 5 cm in diameter were immersed for 30 seconds in products broth and dried for 30 minutes. Eight replicates per concentration were used and each disc was infested with five apterous adult females of A. gossypii uniform in size and confined for 10 days. Compostonat®, Rotenat® and Neempro provided negative ri decreasing A. gossypii population. Natuneem® and Neemseto® and the essential oil of F. vulgare showed positive ri increasing the population. The coefficients of determination (R2) of regression lines ranged from 0.46 to 0.85. The essential oils of C. winterianus, C. ambrosioides and P. aduncum were not statistically significant precluding the establishment of regression lines.

Demographic parameters of two spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) on cotton

Cotton has different economic value and two spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is a serious pest in field crops around the world. The aim of this study was to assess demographic parameters of T. urticae on cotton. This study was under laboratory conditions: (28 ± 2)°C, (60 ± 5) Rh% and (L:D) (16:8) h on cotton leaf discs. Intrinsic rate of increase (rm ), net reproductive rate (R 0), finite rate of increase (λ), doubling time (DT) and generation time (T) were: 0.233 ± 0.006, 40.01 ± 3.33, 1.26 ± 0.01, 2.98 ± 0.08 and 15.86 ± 0.30. Net fertility rate, net fecundity rate and gross hatch rate calculated 53.34 ± 4.53, 57.35 ± 4.87 and 0.93 ± 0.00. The results showed that (ex ) life expectancy had a peak on day 16.25 and maximum egg per day was 9.52 on day 19. The results of our study provide the efficiency of its natural enemies in fields under environmental conditions.

Insecticide use and competition shape the genetic diversity of the aphid Aphis gossypii in a cotton-growing landscape

Field populations of the cotton aphid, Aphis gossypii Glover, are structured into geographically widespread host races. In the cotton-producing regions of West and Central Africa (WCA), two genotypes have been repeatedly detected within the cotton host race, one of which (Burk1) is prevalent (>90%) and resistant to several insecticides, as opposed to the second one (Ivo). Here, we conducted whole plant and field cage experiments to test hypotheses for such low genetic diversity, including selection from insecticide treatments, interclonal competition and adaptation to host plant, or climatic conditions. To assess the genetic diversity of immigrant aphids, alatae were trapped and collected on cotton and relay host plants (okra and roselle) in the early cropping season. Individuals were genotyped at eight specific microsatellite loci and characterized by a multilocus genotype (MLG). When independently transferred from cotton (Gossypium hirustum L.) leaf discs to whole plants (G. hirsutum and G. arboreum, roselle and okra), Ivo and Burk1 performed equally well. When concurrently transferred from cotton leaf discs to the same plant species, Ivo performed better than Burk1, indicating that competition favoured Ivo. This was also the case on G. hirsutum growing outdoors. Conversely, Burk1 prevailed when cotton plants were sprayed with insecticides. In experiments where aphids were allowed to move to neighbouring plants, Burk1 was better represented than Ivo on low-populated plants, suggesting that dispersal may be a way to avoid competition on crowded plants. Most cotton aphids collected on cotton or relay host plants in the early cropping season were Burk1 (>90%), indicating high dispersal ability and, probably reflecting high frequency on host plants from which they dispersed. In the agricultural landscape of WCA, the use of broad-range insecticides on both cotton and relay host plants has led to the prevalence of one genotype of A. gossypii resistant to different classes of insecticides. Deployment of widespread and integrated pest management strategies are needed to restore cotton aphid control.

Effects of Bacillus thuringiensis toxin Cry1Ac and cytoplasmic polyhedrosis virus of Helicoverpa armigera (Hübner) (HaCPV) on cotton bollworm (Lepidoptera: Noctuidae)

In this study, interactions on the mortality and debilitating effects between Cry1Ac, a toxic protein produced by Bacillus thuringiensis (Berliner) and HaCPV (Chinese strain) on first and third instars larvae of Helicoverpa armigera were evaluated in laboratory. When first instar was exposed to combination of Bt cotton leaf discs containing HaCPV (6 × 10 6, 1 × 10 7, and 3 × 10 7 PIB ml −1) the effect on mortality was additive, when such instar larvae exposed to combination of Cry1Ac (0.9, 2.7, or 8.1 μg g −1) and the same concentrations of HaCPV the effect on mortality was additive except for the combination of Cry1Ac (0.3 μg g −1) and HaCPV concentrations that showed synergism. When third instars of H. armigera were infected using a suspension containing both HaCPV and Cry1Ac, most combinations of them showed additive effect except for the combination of Cry1Ac (0.3 μg g −1) and HaCPV (3 × 10 7 PIB ml −1) that showed synergism. However, when they exposed to Bt cotton leaf discs and HaCPV the effect on mortality was synergism except combination of Bt cotton leaf discs and HaCPV (6 × 10 6 PIB ml −1) that showed additive. Most of the combinations are showed additive effect in the toxicity and in combinations of Cry1Ac at lowest and HaCPV at highest concentrations synergism is observed. Not only were larval growth and development delayed, but pupation and pupal weight also decreased when larvae were fed on artificial diet containing Cry1Ac and HaCPV or transgenic Bt cotton leaf discs specially in first instar.

Effects of Cry1Ac toxin of Bacillus thuringiensis and nuclear polyhedrosis virus of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) on larval mortality and pupation

In the laboratory, the percentage mortality and pupation of Helicoverpa armigera (Hübner) were investigated when larvae were exposed to Cry1Ac of Bacillus thuringiensis Berliner, nuclear polyhedrosis virus of H. armigera (HaNPV) or Cry1Ac and HaNPV together. The results revealed that interactions between Cry1Ac and HaNPV varied with bioassay method and concentration of the suspension. When larvae were infected using a suspension containing both HaNPV and Cry1Ac, most combinations of Cry1Ac (62.5, 125 and 250 microg mL(-1)) and HaNPV (1.2 x 10(6), 6.0 x 10(6) and 3.0 x 10(7) PIB mL(-1)) showed an antagonistic effect. In the bioassay procedure where larvae were force fed diet containing Cry1Ac 48 h after being infected by HaNPV, interaction between Cry1Ac (0.5, 1, 2, 4 and 8 microg mL(-1)) and HaNPV (6.0 x 10(6) and 3.0 x 10(7) PIB mL(-1)) showed an additive effect, while combinations of Cry1Ac (0.5, 1, 2 and 4 microg mL(-1)) and HaNPV (1.2 x 10(6) PIB mL(-1)) showed an antagonistic effect. In the bioassay procedure where larvae being infected by HaNPV were fed on Cry1Ac diet from neonate to death or pupation, the results suggested that Cry1Ac and HaNPV showed an additive interaction. The percentage mortality was lower in the treatment of larvae infected by transgenic Bt cotton leaf discs containing HaNPV suspension than in the treatment of larvae by conventional cotton leaf discs containing HaNPV, while the pupation rate was higher. The combination of Bt cotton and HaNPV showed antagonism. The present results showed that a combination of Cry1Ac and HaNPV usually resulted in mortality levels greater than in the case of Cry1Ac but not greater than with the virus alone.

Early activation of cell wall strengthening-related gene transcription in cotton by a Verticillium dahliae elicitor

Differential expression of genes in cultured cotton cells and leaf disks that have been challenged with a purified elicitor from Verticillium dahliae, was investigated in order to identify genes involved in the early defense response of cotton. The mRNA differential display reverse transcriptase polymerase chain reaction was used to identify differentially expressed genes 5 h after application of 50 μ gm L �1 Verticillium dahliae elicitor. Sequence analysis of selected amplicons revealed homologies with genes involved in metabolism of carbohydrate precursors for cell wall synthesis. cDNAs identified that were up-regulated after elicitor treatment coded for homologs to a UDP-N-acetylglucosamine pyrophosphorylase-like protein, a glucosyltransferase-like protein, a beta-1,4-N-acetylglucosaminyltransferase, a cellulose synthase-like protein, a 3-deoxy-D-manno-octulosonic acid transferase-like protein and a hydroxyproline-rich glycoprotein. In addition, one cell wall-related cDNA that was down-regulated after elicitor treatment, coded for a proline-rich protein family member homolog. The differential expression of the cDNAs up-regulated after the Verticillium dahliae elicitor treatment was confirmed with reverse Northern dot blots. These results indicate that metabolic reprogramming through the enhanced synthesis of carbohydrate precursors accompanies the activation of de novo cell wall synthesis. As such it is important for the understanding of early defense related responses in cotton and for their biotechnological manipulation. © 2006 SAAB. Published by Elsevier B.V. All rights reserved.

Effect of neem extract on the cotton aphid

The aphid Aphis gossypii Glover (Hemiptera: Aphididae), a harmful pest on cotton, causes direct damage, reducing plant vigor, and indirect damage by honeydew secretion and transmission of several viruses. Due to the problem of pesticide resistance, alternative techniques for chemical control, such as the use of natural insecticides, have been object of research. The effects of aqueous extracts of neem (Azadirachta indica A. Juss) seed powder on the development, survival and fecundity of A. gossypii were evaluated. Treatments consisted of neem seed powder in the concentrations of 23.8, 122.0, 410.0 and 1,410.0 mg/100 mL of distilled water. Mortality rate during the nymphal development for aphids maintained on cotton leaf discs treated with the two highest concentrations were, respectively, 60.0% and 100.0%. With the exception of the highest concentration (1,410.0 mg/100 mL), neem concentrations did not extend the aphids' development period. The net reproductive rate (R0) was of 35.0 nymphs/female for control aphids and of 0.0 nymph/female when the group of females was exposed to neem seed powder at 1,410.0 mg/100 mL since birth. The aqueous extract of neem seeds is efficient against the aphid A. gossypii, causing nymph mortality and reducing their survival period and fecundity.

Host selection of Helicoverpa armigera and H. assulta and its inheritance *

Abstract The difference in host selection between the polyphagous Helicoverpa armigera (Hübner) and the oligophagous H. assulta Quenée was examined, with cotton (Gossypium hirsutum), tomato (Lycopersicon esculentum) (hosts of H. armigera), tobacco (Nicotiana tobaccum) (host of both H. armigera and H. assulta), and bush redpepper (Capsicum frutescens) (host of H. assulta) as testing plants. A multiple-choice test was used with caged plant cuttings for adult oviposition and with leaf discs for larval feeding. A nochoice test was run for evaluating larval growth rate. The results indicated that the relationship between larval performance and adult preference of H. assulta was more conspicuous than that of H. armigera. Reciprocal hybridization between H. armigera and H. assulta followed by backcrossing of the hybrids (F1) with H. armigera was also carried out for genetic study on host selection of these two insect species. A two-choice test with cotton and bush redpepper leaf discs showed that H. armigera larvae preferred to feed on cotton, and H. assulta larvae to bush redpepper; feeding preferences of the two F1 lines were intermediate between those of their parents, but close to that of their female parent; preference indexes of backcross lines also showed that both maternal factor and chromosomal inheritance were involved in feeding selection of two Helicoverpa species.

Storage stability of Anagrapha falcifera nucleopolyhedrovirus in spray-dried formulations.

A multiply embedded nucleopolyhedrovirus isolated from Anagrapha falcifera (Kirby) (AfMNPV) can lose insecticidal activity during months of dry storage in ambient room conditions. We tested the spray-dried AfMNPV formulations after storage for up to 1 year at room temperatures for insecticidal activity against neonate Trichoplusia ni (Hübner). Experimental formulations were made using combinations of corn flours, lignin, and sucrose, and were selected based on previous work which demonstrated that these formulations resisted solar degradation in field experiments. Twelve experimental formulations (organized in three groups of four formulations) compared the effect of (1) the ratio of formulation ingredients (lignin and corn flour) to virus concentration, (2) different sources of lignin, or (3) different corn flours and sugar. Based on a single-dose plant assay with these 12 formulations, none of the formulations lost significant activity due to the drying process, when compared with the unformulated wet AfMNPV. Samples of the 12 dried formulations were stored at room (22+/-3 degrees C) and refrigerated (4 degrees C) temperatures. Insecticidal activity (LC(50)) was determined with a dosage-response assay for neonates fed on treated cotton-leaf disks. After 6 (or 9) and 12 months storage, refrigerated samples maintained insecticidal activity better than corresponding samples stored at room temperatures with LC(50)s that averaged 2.0 x 10(6) polyhedral inclusion bodies per milliliter (pibs/ml) for refrigerated samples and 5.4 x 10(6) pibs/ml for samples stored at room temperatures. Compared with unformulated stock virus stored frozen, six formulations stored at room temperature and 10 formulations stored in the refrigerator did not lose significant insecticidal activity after 1 year based on overlapping 90% confidence intervals. Changing the ratio of virus to formulation ingredients did not provide a clear trend over the range of concentrations tested, and may be less important for shelf-life of virus activity compared with formulations made with different ingredients. Two of the four formulations made with different lignins were about 15 times less active after 1 year at room temperature compared with refrigerated samples, indicating that specific formulation ingredients can affect storage stability. Formulations that contained sugar generally maintained activity during storage better than formulations without sugar. Unformulated virus stock maintained insecticidal activity (ranged from 0.20 to 2.5 x 10(6) pibs/ml) better during storage than dried formulations with LC(50)s that ranged from 0.39 to 27 x 10(6) pibs/ml. Unformulated virus stock, which is essentially a suspension of virus occlusion bodies in homogenized insect cadavers, did not lose activity when stored at refrigerated or room temperature. We believe that stability of AfMNPV insecticidal activity during storage as dry formulations is related to the general composition of the formulation and that sugar may play a critical role in maintaining insecticidal activity.