A Serratia marcesens Strains Involved in Cotton (<i>Gossypium hirsutum</i>) Boll Infection by a Prokaryote

A boll infection caused by non-traditional cotton pathogens was first reported to occur in the southeastern U.S. Cotton Belt (year 2000) and has since spread to Texas causing significant yield losses. This study was aimed towards investigating the verde plant bug (Creontiades signatus) link between interior boll disease in Texas, USA. Using glasshouse grown bolls, bacteria recovered from locules with disease symptoms from field-grown cotton bolls caged with the piercing-sucking C. signatus were analyzed for the capacity to inflict the disease. For pathogenicity testing, spontaneously generated rifampicin resistant (Rifr) variants were utilized to track the antibiotic resistant bacterium and deter growth of endophytic and contaminating bacteria. To simulate C. signatus feeding, a needle (31 gauge) was employed to inoculate bolls at 13 - 15 days after flower bloom. Bacterial suspensions ranged from 101 - 106 colony forming units/ml. Field infection symptoms were duplicated after two weeks of bacterial exposure. Infectious strains were best categorized as Serratia marcescens based on traditional carbon utilization and enzyme production testing, and a 99% nucleotide sequence identity of 16S ribosomal DNA. Putative S. marcescens representatives isolated from rotted bolls exposed to C. signatus were shown to reproduce field infection symptoms upon inoculation into greenhouse grown fruit. Serratia spp. can inflict disease in alfalfa, cucurbits, and sunflower. The presented data are the first to definitively show that a Serratia sp. has the capacity to infect cotton.

Evaluating host plant resistance in cotton (<i>Gossypium hirsutum</i>L.) with varying gland densities to tobacco budworm (<i>Heliothis virescens</i>F.) and bollworm (<i>Helicoverpa zea</i>Boddie) in the field and laboratory

Cotton (Gossypium hirsutum L.) produces a number of toxic terpenoid aldehyde (TA) compounds contained in epidermal glands that help protect the plant from pests and diseases. In the seed, one of these toxic compounds, gossypol, limits the use of the seed to ruminants such as dairy cows. There are breeding techniques and germplasm available to decrease gossypol in the seed, but the breeding process also needs to include methods to evaluate the plant’s ability to resist insect pests. Three approaches were used to assess resistance of cotton to herbivory from bollworm (Helicoverpa zea Boddie) and tobacco budworm (Heliothis virescens F.) including field counts, controlled field antibiosis assays and laboratory feeding tests of young field grown leaves. Results indicated that both field and laboratory evaluation could provide an assessment of the cotton host’s resistance. Measurements of terpenoid aldehydes (TAs) in the seed and the leaves, confirmed that the levels and types of TAs in the seed were not always good estimators of leaf TAs and that other TAs such as hemigossypolone and heliocides contribute to host plant resistance.

Resistance risk assessment of six pyrethroids and acephate toward the resistant adult tarnished plant bug,Lygus lineolaris

Due to rapidly developed resistance,pest management relies less on pyrethroids to control economically damaging infestations of the tarnished plant bug(TPB),Lygus lineolaris(Palisot de Beauvois)in cotton fields of Mississippi.Yet,pyrethroid resistance remains prevalent in TPB populations.This study assessed the resistance levels in adult TPB to six common pyrethroids and acephate.Resistant TBPs were collected from wild host plants in late October after harvest in the Mississippi Delta region of the United States.Based on LCso values,the field-resistant TPBs displayed higher resistance to permethrin,esfenvalerate,and bifenthrin(approximately 30 fold)and moderate resistance toα-cyhalothrin,β-cyfluthrin,5-cypermethrin,and acephate(approximately 15 fold).Further investigations showed that the inhibitors of three detoxification enzyme,triphenyl phosphate(TPP),diethyl maleate(DEM),and piperonyl butoxide(PBO)had synergistic effects on permethrin,γ-cyhalothrin,and bifenthrin in resistant TPBs.Furthermore,elevated esterase,GST,and P450 activities were significantly expressed in fieldresistant TPBs.Additionally,GST and esterase were reduced after 48 h exposure to certain pyrethroids at LCso dose.The synergistic and biochemical assays consistently indicated that P450 and esterase were involved in pyrethroid detoxification in TPBs.This study provides valuable information for the continued use of pyrethroids and acephate in controlling TPBs in cotton fields in the Mississippi Delta region of the United States.

Feeding behavior and hormoligosis associated with imidacloprid resistance in Asian citrus psyllid,Diaphorina citri

Imidacloprid is a neonicotinoid insecticide used for managing the Asian citrus psyllid,Diaphorina citri Kuwayama,which serves as vector of phytopathogens causing citrus greening.However,development of resistance to neonicotinoids among populations of D.citri has coincided with occasional control failures in the field.The objectives of this research were to(1)survey current levels of imidacloprid resistance in Florida citrus;(2)compare feeding behavior between imidacloprid-resistant and susceptible D.citri using electrical penetration graph recordings,and(3)investigate the possible amplification of insecticide hormoligosis associated with resistance.Field surveys confirmed that the susceptibility of D.citri populations to imidacloprid has decreased in commercial Florida citrus groves compared with a laboratory-susceptible population.Following 12 generations of selection,resistance to imidacloprid increased by 438 fold compared with the susceptible strain.Imidacloprid-susceptible D.citri feeding on citrus exhibited significantly more bouts associated with intercellular pathway(C),phloem penetration(D),phloem salivation(E1),and nonprobing(Np)activities than imidacloprid-resistant counterparts.However,there were no differences observed in the frequency or duration of phloem ingestion or xylem feeding between susceptible and resistant D.citri.There was no statistical difference in fecundity between resistant and susceptible strains.However,the fecundity of imidacloprid-susceptible female D.citri treated with a sublethal concentration of imidacloprid(LC_(25))increased significantly compared with controls,while such hormoligosis was less pronounced among imidacloprid-resistant psyllids.Our results suggest that imidacloprid-resistant psyllids may cease feeding sooner than susceptible counterparts following sublethal exposure to this insecticide,indicative of a behavioral resistance mechanism.

Role of DSC1 in Drosophila melanogaster synaptic activities in response to haedoxan A

Drosophila sodium channel 1 (DSC1) encodes a voltage-gated divalent cation channel that mediates neuronal excitability in insects. Previous research revealed that DSC1 knockout Drosophila melanogaster conferred different susceptibility to insecticides, which indicated the vital regulation role of DSC1 under insecticide stress. Haedoxan A (HA) is a lignan compound isolated from Phryma leptostachya, and we found that HA has excellent insecticidal activity and is worthy of further study as a botanical insecticide. Herein, we performed bioassay and electrophysiological experiments to test the biological and neural changes in the larval Drosophila with/without DSC1 knockout in response to HA. Bioassay results showed that knockout of DSC1 reduced the sensitivity to HA in both w1118 (a common wild-type strain in the laboratory) and parats1 (a pyrethroid-resistant strain) larvae. Except for parats1/DSC1−/−, electrophysiology results implicated that HA delayed the decay rate and increased the frequency of miniature excitatory junctional potentials of Drosophila from w1118, parats1, and DSC1−/− strains. Moreover, the neuromuscular synapse excitatory activities of parats1/DSC1−/− larvae were more sensitive to HA than DSC1−/− larvae, which further confirmed the functional contribution of DSC1 to neuronal excitability. Collectively, these results indicated that the DSC1 channel not only regulated the insecticidal activity of HA, but also maintained the stability of neural circuits through functional interaction with voltage-gated sodium channels. Therefore, our study provides useful information for elucidating the regulatory mechanism of DSC1 in the neural system of insects involving the action of HA derived from P. leptostachya.

Negative cross-resistance of a pyrethroid-resistant Drosophila mutant to Phryma leptostachya-derived haedoxan A

Voltage-gated sodium channels are the primary target of pyrethroid insecticides.Mutations in sodium channel confer knockdown resistance(kdr)to pyrethroids in various arthropod pests.Haedoxan A(HA)is the major insecticidal component from Phryma leptostachya.It has been shown that HA alters electrical responses at the Drosophila neuromuscular junction and modifies the gating properties of cockroach sodium channels expressed in Xenopus oocytes.However,whether sodium channel mutations that confer pyrethroid resistance also affect the action of HA is unknown.In this study,we conducted bioassays using HA and permethrin in two Drosophila melanogaster strains:w^(1118),an insecticide-susceptible strain,and para^(tsl),a pyrethroid-resistant strain due to a I265N mutation in the sodium channel,and identified a new case of negative cross-resistance(NCR)between permethrin and HA.Both para^(tsl) larvae and adults were more resistant to permethrin,as expected.However,both para^(tsl) larvae and adults were more sensitive to HA compared to w^(1118).We confirmed that the I265N mutation reduced the sensitivity to permethrin of a Drosophila sodium channel variant,DmNa_(v)22,expressed in Xenopus oocytes.Interestingly,the I265N mutation also abolished the effect of HA on sodium channels.Further characterization showed that I265 on the sodium channels is critical for the action of both pyrethroids and HA on sodium channels,pointing to an overlapping mode of action between pyrethroids and HA on the sodium channel.Overall,our results suggest an I265N-independnt mechanism(s)in para^(tsl) flies that is responsible for the NCR between permethrin and HA at the whole insect level.

Molecular cloning and comparative analysis of transcripts encoding chemosensory proteins from two plant bugs, Lygus lineolaris and Lygus hesperus

Chemosensory proteins(CSPs)are soluble carrier proteins typically characterized by a six‐helix bundle structure joined by two disulfide bridges and a conserved Cys spacing pattern(C1‐X6‐8‐C2‐X16‐21‐C3‐X2‐C4).CSPs are functionally diverse with reported roles in chemosensation,immunity,development,and resistance.To expand our molecular understanding of CSP function in plant bugs,we used recently developed transcriptomic resources for Lygus lineolaris and Lygus hesperus to identify 17 and 14 CSP‐like sequences,respectively.The Lygus CSPs are orthologous and share significant sequence identity with previously annotated CSPs.Three of the CSPs are predicted to deviate from the typical CSP structure with either five or seven helical segments rather than six.The seven helix CSP is further differentiated by an atypical C3‐X3‐C4 Cys spacing motif.Reverse transcriptase PCR‐based profiling of CSP transcript abundance in adult L.lineolaris tissues revealed broad expression for most of the CSPs with antenna specific expression limited to a subset of the CSPs.Comparative sequence analyses and homology modeling suggest that variations in the amino acids that comprise the Lygus CSP binding pockets affect the size and nature of the ligands accommodated.

Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in CrylAb-susceptible and CrylAb-resistant strains of sugarcane borer, Diatraea saccharalis

Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is re- sponsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the CrylAb resistance in D. saccharalis is associ- ated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry 1 Ab-susceptible （Cry 1 Ab- S S） and Cry 1 Ab-resistant （Cry 1 Ab-RR） strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin- like proteinases were sequenced from CrylAb-SS and CrylAb-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all fimctional motifs, includ- ing signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between CrylAb-SS and CrylAb-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between CrylAb-SS and CrylAb-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry 1 Ab-SS and CrylAb-RR strains, but the difference was not statistically significant. Data suggest that the development of CrylAb resistance in D. saccharalis was not significantly as- sociated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes.