Vapor heat treatment against Planococcus lilacinus Cockerell (Hemiptera:Pseudococcidae) on dragon fruit.

BACKGROUND: The coffee mealybug Planococcus lilacinus Cockerell, is an invasive pest that infests dragon fruit [Selenicereus undatus (Haworth) D.R. Hunt], that may require a phytosanitary treatment to meet quarantine requirements. In this study, vapor heat treatment was conducted to disinfest P. lilacinus on dragon fruit and the quality of dragon fruit was evaluated thereafter. RESULTS: Adult female P. lilacinus was the most heat-tolerant stage at 47, 48 and 49 °C. The death kinetic model and probit model were used to predict LT99.9968 at the three temperatures. The treatment times predicted by the kinetic model that could effectively disinfest adult female P. lilacinus females were 120.84, 78.06 and 67.96 min at 47, 48 and 49 °C, respectively. In a confirmatory test of vapor heat treatment at 49 °C for 70 min, complete mortality was recorded for 33 195 adult females infesting 148 dragon fruits; thus, the efficacy level of disinfestation was 99.9910% at 95% confidence level. The quality of dragon fruit generally was not affected by heat treatment, fruit firmness was increased remarkably and respiration rate was significantly decreased. CONCLUSION: Our result indicates that vapor heat treatment at 49 °C for a duration of 70 min is an effective phytosanitary treatment for control of P. lilacinus on dragon fruit that minimally impacts fruit quality. © 2021 Society of Chemical Industry.

Evaluation of Reference Genes for Quantitative Reverse Transcription Polymerase Chain Reaction in Bactrocera dorsalis (Diptera: Tephritidae) Subjected to Various Phytosanitary Treatments.

Bactrocera dorsalis is a major pest that causes serious damage to many fruits. Although phytosanitary treatment methods have been developed for Bactrocera control, there is a lack of information related to the gene expression pattern of B. dorsalis subjected to phytosanitary treatment conditions. Prior to quantitative reverse transcription polymerase chain reaction analysis of the most stable reference genes in B. dorsalis (Diptera: Tephritidae), B. dorsalis third-instar larvae were exposed to various phytosanitary treatments; seven candidate reference genes (18S, G6PDH, GAPDH, RPL-13, RPL-32, RPS-3, and α-Tub) were amplified and their expression stabilities were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Different reference genes were found under different stress conditions. G6PDH was the most stable gene after heat treatment. After cold treatment, α-Tub exhibited the highest expression stability. G6PDH expression stability was the highest after fumigation with methyl bromide. RPL-32 showed the highest expression stability after irradiation treatment. Collectively, RefFinder analysis results revealed G6PDH and RPL-32 as the most suitable genes for analyzing phytosanitary treatment in B. dorsalis. This study provides an experimental basis for further gene expression analyses in B. dorsalis subjected to various phytosanitary treatments, which can aid in the development of novel phytosanitary treatments against insect pests.

Radioprotective Effects on Late Third-Instar Bactrocera dorsalis (Diptera: Tephritidae) Larvae in Low-Oxygen Atmospheres.

Ionizing radiation creates free radicals, the effect of which is enhanced by the presence of oxygen; a low oxygen level produces radioprotective effects for insects compared with irradiation in ambient air. Modified (controlled) atmosphere packaging is used for maintaining quality and shelf-life extension; therefore, treatment efficacy may be affected, and there is a need to determine the critical O2 levels that may cause radioprotective effects. Late third-instar Bactrocera dorsalis (Hendel) larvae were irradiated in bags filled with ambient or low-oxygen air (0%, 2%, 4%, 6%, 8% O2) and were exposed to radiation doses of 8 to 64 Gy with intervals of 8 Gy. Efficacy was measured by the prevention of adult emergence. Dose-response data on mortality (failure of adult emergence) were analyzed via two-way ANOVA (analysis of variance), ANCOVA (analysis of covariance), and probit regression. The difference in radiotolerance was only significant in 0% O2 atmospheres through two-way ANOVA; therefore, the 95% confidence limits (CLs) of lethal dose ratios at LD99 were used to determine significant differences between treatments at different O2 levels. The differences in radiotolerance were significant in 0% and 2% O2 but insignificant in 4%, 6%, and 8% O2 environments when compared with radiation in ambient air. The critical threshold of radioprotective effects for late third-instar B. dorsalis larvae is an O2 level of ≥4% and <6%, but a maximum radiation dose of 14 Gy can compensate for this effect during phytosanitary irradiation treatment.

The mitochondrial genome of Leptopeza flavipes (Diptera: Empididae).

The dance fly Leptopeza flavipes belongs to the subfamily Ocydromiinae of Empididae. The mitogenome (GenBank accession number: MT610901) of L. flavipes was sequenced, the new representative of the mitogenome of the subfamily. The nearly complete mitogenome is 15,267 bp totally, consisting of 13 protein-coding genes, 2 rRNAs and 22 transfer RNAs. All genes have similar locations and strands with that of other published species of Empididae. The nucleotide composition biases toward A and T, which together made up 78.1％of the entirety. Bayesian inference analysis strongly supported the monophyly of Empidoidea, Empididae and Dolichopodidae. It is clear that the phylogenetic relationship within Empidoidea: (Dolichopodinae + Neurigoninae) + ((Empidinae + (Trichopezinae + Oreogetoninae)) + Ocydromiinae) in this study.

Rapid detection of the pathogenic fungi causing blackleg of Brassica napus using a portable real-time fluorescence detector.

The fungus Leptosphaeria maculans leading to Phoma stem canker (blackleg) of Brassica napus (oilseed rape, canola) produces the phytotoxin sirodesmin PL, which is responsible for major yield losses of oilseed rape worldwide. Polymerase chain reaction (PCR) remains the gold standard diagnostic tool for L. maculans, but the required expensive equipment and long time make it inappropriate for fast field test. Herein, a portable system for rapid assaying L. maculans and L. biglobosa is designed around recombinase polymerase amplification (RPA) with fluorescent probe as the signal indicator, which allowed the real-time assay of amplification performed on a portable device between 37 and 42 °C. The time needed to observe the positive reaction results is controlled within 30 min. The proposed assay system is a good choice for on-site disease screening of oilseed rape plant where rapid detection is valuable, including port quarantine, agriculture quality testing, and pathogen spreading control.

The mitochondrial genome of Empis separata (Diptera: Empididae).

The dance fly Empis separata belongs to the subfamily Empidinae of Empididae. The mitogenome (GenBank accession number: MK993569) of E. separata was sequenced, the new representative of the mitogenome of the subfamily. The complete mitogenome is 14,961 bp totally, consisting of 13 protein-coding genes, two rRNAs, and 22 transfer RNAs. All genes have the similar locations and strands with that of other published species of Empididae. The nucleotide composition biases toward A and T, which together made up 76.3% of the entirety. Bayesian inference analysis strongly supported the monophyly of Empidoidea, Empididae, and Dolichopodidae. This result also suggested that Empidinae is the sister group to the clade of Trichopezinae.

Pheromone-binding proteins in the Asian gypsy moth females, Lymantria dispar, recognizing the sex pheromone and plant volatiles.

Lepidopterans are known to have different pheromone-binding proteins with differential expression patterns that facilitate specific signal transduction of semiochemicals. Two PBPs of the Asian gypsy moth, Lymantria dispar, were reported to express in both females and males, but their physiological functions were unknown. Results showed that LdisPBP1 and LdisPBP2 were expressed in the sensilla trichodea of males and the s. trichodea and s. basiconica of females. When LdisPBP1 gene was targeted by RNA interference (RNAi) in males, the expression of LdisPBP1 and LdisPBP2 decreased by 69 and 76%, respectively, and when LdisPBP2 gene was targeted by RNAi, they decreased by 60 and 42%, respectively. In females, after treatment with LdisPBP1 dsRNA, LdisPBP1 and LdisPBP2 levels were reduced by 26 and 69%, respectively, and LdisPBP2 dsRNA reduced the relative expression of them by 4 and 62%, respectively. The expression of LdisPBP1 and LdisPBP2 was interdependent. Electroantennogram (EAG) recordings showed that LdisPBPs participate in the recognition of the sex pheromone in males, and the sex pheromone and plant volatiles in females. The function of LdisPBPs represents the sex-specific roles.

Development of Chloroplast Genomic Resources for Oryza Species Discrimination.

Rice is the most important crop in the world as the staple food for over half of the population. The wild species of Oryza represent an enormous gene pool for genetic improvement of rice cultivars. Accurate and rapid identification of these species is critical for effective utilization of the wild rice germplasm. In this study, we developed valuable chloroplast molecular markers by comparing the chloroplast genomes for species identification. Four chloroplast genomes of Oryza were newly sequenced on the Illumina HiSeq platform and other 14 Oryza species chloroplast genomes from Genbank were simultaneously taken into consideration for comparative analyses. Among 18 Oryza chloroplast genomes, five variable regions (rps16-trnQ, trnTEYD, psbE-petL, rpoC2 and rbcL-accD) were detected for DNA barcodes, in addition to differences in simple sequence repeats (SSR) and repeat sequences. The highest species resolution (72.22%) was provided by rpoC2 and rbcL-accD with distance-based methods. Three-marker combinations (rps16-trnQ + trnTEYD + rbcL-accD, rps16-trnQ + trnTEYD + rpoC2 and rpoC2 + trnTEYD + psbE-petL) showed the best species resolution (100%). Phylogenetic analysis based on the chloroplast genome provided the best resolution of Oryza. In the comparison of chloroplast genomes in this study, identification of the most variable regions and assessment of the focal regions of divergence were efficient in developing species-specific DNA barcodes. Based on evaluation of the chloroplast genomic resources, we conclude that chloroplast genome sequences are a reliable and valuable molecular marker for exploring the wild rice genetic resource in rice improvement.

Chloroplast Genomic Resource of Paris for Species Discrimination.

Paris is famous in China for its medicinal value and has been included in the Chinese Pharmacopoeia. Inaccurate identification of these species could confound their effective exploration, conservation, and domestication. Due to the plasticity of the morphological characteristics, correct identification among Paris species remains problematic. In this regard, we report the complete chloroplast genome of P. thibetica and P. rugosa to develop highly variable molecular markers. Comparing three chloroplast genomes, we sought out the most variable regions to develop the best cpDNA barcodes for Paris. The size of Paris chloroplast genome ranged from 162,708 to 163,200 bp. A total of 134 genes comprising 81 protein coding genes, 45 tRNA genes and 8 rRNA genes were observed in all three chloroplast genomes. Eight rapidly evolving regions were detected, as well as the difference of simple sequence repeats (SSR) and repeat sequence. Two regions of the coding gene ycf1, ycf1a and ycf1b, evolved the quickest and were proposed as core barcodes for Paris. The complete chloroplast genome sequences provide more integrated and adequate information for better understanding the phylogenetic pattern and improving efficient discrimination during species identification.

Identification of odorant binding proteins in Carpomya vesuviana and their binding affinity to the male-borne semiochemicals and host plant volatiles.

Carpomya vesuviana (Costa; Diptera: Tephritidae) is an agricultural pest that causes serious damage to jujube fruits. However, the mechanism of olfaction, which is critical for host identification, is not well understood in this pest. In this study, we have identified for the first time five protein types involved in the olfactory signal transduction of C. vesuviana by using transcriptome sequencing. These include 6 odorant-binding proteins (OBPs), 15 odorant receptors (ORs), 22 gustatory receptors (GRs), 2 chemosensory proteins (CSPs), and 2 sensory neuron membrane proteins (SNMPs). Amino acids alignment and phylogenetic analysis showed that all 6 OBPs have a signal peptide at their respective N-termini with four OBPs belonging with the classic OBPs, and OBP2 and OBP5 belonging to the Minus-C family. OBP3 clustered with the OBP83a/83b clade, which comprised pheromone binding protein related proteins (PBPRPs). Moreover, volatiles from C. vesuviana adults and its host plants were collected and identified by using solid phase microextraction (SPME) and gas-chromatography/mass spectrometry (GC/MS). The results indicated that male adults emitted nonanal, and five other compounds, caryophyllene, chamigrene, camphene, (Z)-3-hexen-1-ol acetate, and ocimene were identified in the fruits of jujubes. Electroantennogram (EAG) assays revealed that adult C. vesuviana responded to all six compounds along with two additional pheromones (geranyl acetate and α-farnesene) from other tephritids and the values ranged from 0.50mV to 1.26mV. To further explore the interaction between OBPs and volatiles, competitive binding assays were carried out. The results showed that only CvesOBP2 had binding affinity to (Z)-3-hexen-1-ol acetate. OBP5 and OBP6 exhibited broad spectrum binding to compounds with relatively low molecular weights, and OBP1 and OBP4 had some affinity to caryophyllene and chamigrene. However, OBP3 exhibited relatively high binding affinity to α-farnesene. The findings of this study provide insights into the olfactory mechanisms and the potential functions of OBPs in the olfactory reception pathway in C. vesuviana. The OBPs identified in this study could be used as potential targets to develop attractants to monitor this insect pest for effective pest control.

The complete chloroplast genome of traditional Chinese medical plants Paris polyphylla var. yunnanensis.

Paris polyphylla var. yunnanensis is a perennial medical plant widely used in traditional Chinese medicine. Here, we report the complete chloroplast genome of P. polyphylla var. yunnanensis. The genome is 157 675 bp in length including a small single-copy region (SSC, 18 319 bp) and a large single-copy region (LSC, 84 108 bp) separated by a pair of inverted repeats (IRs, 27 624 bp). The genome contained 115 genes, including 81 protein-coding genes, 4 ribosomal RNA genes, and 30 tRNA genes. Among these genes, 13 harbored a single intron and 2 contained a couple of introns. The overall G + C content of the cpDNA is 37.4%, while the corresponding values of the LSC, SSC, and IR regions are 35.71%, 31.43%, and 41.87%, respectively. A Maximum-likelihood phylogenetic analysis suggested that genus Trillium, Paris, Fritillaria, and Lilium were strongly supported as monophyletic and the P. polyphylla var. yunnanensis is closely related to Trillium.

Structural and functional difference of pheromone binding proteins in discriminating chemicals in the gypsy moth, Lymantria dispar.

Pheromone-binding proteins (PBPs) of the gypsy moth, Lymantria dispar L., play an important role in olfaction. Here structures of PBPs were first built by Homology Modeling, and each model of PBPs had seven α-helices and a large hydrophobic cavity including 25 residues for PBP1 and 30 residues for PBP2. Three potential semiochemicals were first screened by CDOCKER program based on the PBP models and chemical database. These chemicals were Palmitic acid n-butyl ester (Pal), Bis(3,4-epoxycyclohexylmethyl) adipate (Bis), L-trans-epoxysuccinyl-isoleucyl-proline methyl ester propylamide (CA-074). The analysis of chemicals docking the proteins showed one hydrogen bond was established between the residues Lys94 and (+)-Disparlure ((+)-D), and л-л interactions were present between Phe36 of PBP1 and (+)-D. The Lys94 of PBP1 formed two and three hydrogen bonds with Bis and CA-074, respectively. There was no residue of PBP2 interacting with these four chemicals except Bis forming one hydrogen bond with Lys121. After simulating the conformational changes of LdisPBPs at pH7.3 and 5.5 by constant pH molecular dynamics simulation in implicit solvent, the N-terminal sequences of PBPs was unfolded, only having five α-helices, and PBP2 had larger binding pocket at 7.3 than PBP1. To investigate the changes of α-helices at different pH, far-UV and near-UV circular dichroism showed PBPs consist of α-helices, and the tertiary structures of PBP1 and PBP2 were influenced at pH7.3 and 5.5. The fluorescence binding assay indicated that PBP1 and PBP2 have similarly binding affinity to (+)-D at pH 5.5 and 7.3, respectively. At pH 5.5, the dissociation constant of the complex between PBP1 and 2-decyl-1-oxaspiro [2.2] pentane (OXP1) was 0.68 ± 0.01 µM, for (+)-D was 5.32 ± 0.11 µM, while PBP2 with OXP1 and (+)-D were 1.88 ± 0.02 µM and 5.54 ± 0.04 µM, respectively. Three chemicals screened had higher affinity to PBP1 than (+)-D except Pal at pH5.5, and had lower affinity than (+)-D at pH7.3. To PBP2, these chemicals had lower affinity than the sex pheromone except Bis at pH 5.5 and pH 7.3. Only PBP1 had higher affinity with Sal than the sex pheromone at pH 5.5. Therefore, the structures of PBP1 and PBP2 had different changes at pH5.5 and 7.3, showing different affinity to chemicals. This study helps understanding the role of PBPs as well as in developing more efficient chemicals for pest control.