scholarly journals Development of a LAMP-Based Molecular Species Diagnosis Method for Four Major Agricultural Pests in the Genus Spodoptera (Lepidoptera: Noctuidae)

Insects ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 883
Author(s):  
Hwa Yeun Nam ◽  
Ju Hyeon Kim ◽  
Si Hyeock Lee ◽  
David G. Heckel ◽  
Juil Kim
Keyword(s):  
Agricultural Research ◽  
Tissue Sample ◽  
Lamp Assay ◽  
Molecular Diagnostic ◽  
Amplification Efficiency ◽  
Agricultural Pests ◽  
Specific Sequence ◽  
Wide Range ◽  
Nonspecific Amplification ◽  
Species Specific

Molecular-based species identification tools are helpful to identify tiny insect and lepidopteran pests that show morphological similarities in the larval stage and are essential for quarantine as well as agricultural research. Here, we focused on four major Spodoptera pests: S. exigua, S. frugiperda, S. litura, and S. littoralis. S. exigua and S. litura mitochondrial genome sequences were newly identified and species-specific sequence regions were identified in the cytochrome c oxidase subunit II and III regions. Species primers were designed and applied in loop-mediated isothermal amplification (LAMP) and PCR to identify Korean field-collected or overseas samples. The optimal incubation conditions for LAMP were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency for S. exigua, and the nonspecific amplification for other species. The LAMP assay could detect a wide range of DNA concentrations, with the range 1 ng–1 pg in dependence of four LAMP primers. The DNA-releasing technique, without DNA extraction, in the LAMP assay involved larval or adult tissue sample incubation at 95 °C for 5 min. The entire process takes approximately 70 min. This new molecular diagnostic method is simple and accurate, with application in the field and laboratory and for monitoring and ecological studies.

scholarly journals Development of a simple and accurate molecular tool for Spodoptera frugiperda species identification using LAMP

2020 ◽  
Author(s):  
Juil Kim ◽  
Hwa Yeun Nam ◽  
Min Kwon ◽  
Hyun Ju Kim ◽  
Hwi-Jong Yi ◽  
...  
Keyword(s):  
Spodoptera Frugiperda ◽  
Native Species ◽  
Tissue Sample ◽  
Lamp Assay ◽  
Molecular Diagnostic ◽  
Amplification Efficiency ◽  
Incubation Condition ◽  
Unique Region ◽  
Dna Concentration ◽  
Wide Range

ABSTRACTThe fall armyworm, Spodoptera frugiperda is a native species in the Americas. However, nowadays it is one of the most serious invasive lepidopteran pests in African and Asian countries. S. frugiperda has been spread very quickly after the first outbreak was reported in many countries. Based on mt genome sequence alignment, S. frugiperda specific sequence region was identified in tRNAs coding region between NADH dehydrogenase, ND3 and ND5. By using this unique region, species diagnostic primers were designed and applied in LAMP (lamp loop mediated isothermal amplification) assay as well as conventional PCR to identify the field-collected samples of S. frugiperda. Optimal incubation condition of LAMP assay was 61°C for 90 minutes with 4 LAMP primers, and additional loop primer increased the amplification efficiency. Also, wide range of DNA concentration responded in LAMP assay and minimum detectable DNA concentration was 10 pg. This LAMP assay was also applied in DNA releasing technique from larval and adult sample, without DNA extraction, 95°C incubation for five minutes of the tissue sample. This new molecular diagnostic method is easy to use and accurate. It possibly applied in intensive field monitoring of S. frugiperda and its ecological studies.

scholarly journals Development of a species diagnostic molecular tool for an invasive pest, Mythimna loreyi using LAMP

2020 ◽  
Author(s):  
Hwa Yeun Nam ◽  
Min Kwon ◽  
Hyun Ju Kim ◽  
Juil Kim
Keyword(s):  
Integrated Management ◽  
Lamp Assay ◽  
Invasive Pest ◽  
Similar Species ◽  
Coding Region ◽  
Specific Sequence ◽  
Incubation Condition ◽  
Dna Concentration ◽  
Invasive Pests ◽  
Species Specific

AbstractThe Mythimna loreyi (Duponchel) is one of the well-known a noctuid pest in Africa, Australia, and many Asian countries. This species has recently emerged as an invasive pest of some cereal crops in Korea. However, it is extremely difficult to identify the morphologically similar species, Mythimna separate, which occur at the cornfield in the larvae stage. Therefore, it is hard to accurately investigate invasive pests. In this study, the LAMP assay was developed for rapid, simple, effective species identification. By analyzing the mt genome, the species-specific sequence was found at the coding region of the NADH dehydrogenase subunit 5 gene. Based on this unique sequence, four LAMP primers and two loop primers were designed. The F3 and B3 primers were able to diagnose species-specific in general and multiplex PCR, and specifically reacted within the inner primers in LAMP assay. The optimal incubation condition of the LAMP assay was 61 □ for 60 minutes with four LAMP primers, though additional loop primer, BF and LF, did not significantly shorten the amplification time. The broad range of DNA concentration was workable in LAMP assay, in which the minimum detectable DNA concentration was 100 pg. Here, DNA releasing method was applied which took five minutes of incubation at 95 □ without the DNA extraction process, and only some pieces of tissue from larvae and adult samples were needed. The incidence of invasive pests is gradually diversifying, therefore, this simple and accurate LAMP assay possibly applied in the intensive field monitoring for the invasive pests and integrated management of Mythimna loreyi.

scholarly journals Development of a Multiplex Assay for Genus- and Species-Specific Detection of Phytophthora Based on Differences in Mitochondrial Gene Order

2014 ◽  
Vol 104 (7) ◽  
pp. 733-748 ◽  
Author(s):  
Guillaume J. Bilodeau ◽  
Frank N. Martin ◽  
Michael D. Coffey ◽  
Cheryl L. Blomquist
Keyword(s):  
Mitochondrial Gene ◽  
Taqman Probe ◽  
Reference Sequence ◽  
Molecular Diagnostic ◽  
Specific Detection ◽  
Taqman Probes ◽  
Phytophthora Spp ◽  
Wide Range ◽  
Detection Capabilities ◽  
Species Specific

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed based on the high copy sequences of the mitochondrial DNA utilizing gene orders that were highly conserved in the genus Phytophthora but different in the related genus Pythium and plants to reduce the importance of highly controlled annealing temperatures for specificity. An amplification primer pair designed from conserved regions of the atp9 and nad9 genes produced an amplicon of ≈340 bp specific for the Phytophthora spp. tested. The TaqMan probe for the genus-specific Phytophthora test was designed from a conserved portion of the atp9 gene whereas variable intergenic spacer sequences were used for designing the species-specific TaqMan probes. Specific probes were developed for 13 species and the P. citricola species complex. In silico analysis suggests that species-specific probes could be developed for at least 70 additional described and provisional species; the use of locked nucleic acids in TaqMan probes should expand this list. A second locus spanning three tRNAs (trnM-trnP-trnM) was also evaluated for genus-specific detection capabilities. At 206 bp, it was not as useful for systematic development of a broad range of species-specific probes as the larger 340-bp amplicon. All markers were validated against a test panel that included 87 Phytophthora spp., 14 provisional Phytophthora spp., 29 Pythium spp., 1 Phytopythium sp., and 39 plant species. Species-specific probes were validated further against a range of geographically diverse isolates to ensure uniformity of detection at an intraspecific level, as well as with other species having high levels of sequence similarity to ensure specificity. Both diagnostic assays were also validated against 130 environmental samples from a range of hosts. The only limitation observed was that primers for the 340 bp atp9-nad9 locus did not amplify Phytophthora bisheria or P. frigida. The identification of species present in a sample can be determined without the need for culturing by sequencing the genus-specific amplicon and comparing that with a reference sequence database of known Phytophthora spp.

Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

scholarly journals Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

3 Biotech ◽  
2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Domenico Rizzo ◽  
Nicola Luchi ◽  
Daniele Da Lio ◽  
Linda Bartolini ◽  
Francesco Nugnes ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Loop Mediated Isothermal Amplification ◽  
Larval Stages ◽  
Longhorn Beetle ◽  
Rapid Monitoring ◽  
Major Pest ◽  
Wood Borer

AbstractThe red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

scholarly journals The draft genome sequence of the grove snail Cepaea nemoralis

2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Suzanne V Saenko ◽  
Dick S J Groenenberg ◽  
Angus Davison ◽  
Menno Schilthuizen
Keyword(s):  
Genome Sequence ◽  
Draft Genome ◽  
Agricultural Pests ◽  
Shell Color ◽  
Protein Coding ◽  
Cepaea Nemoralis ◽  
Edible Species ◽  
Wide Range ◽  
In Captivity ◽  
Long Read

Abstract Studies on the shell color and banding polymorphism of the grove snail Cepaea nemoralis and the sister taxon Cepaea hortensis have provided compelling evidence for the fundamental role of natural selection in promoting and maintaining intraspecific variation. More recently, Cepaea has been the focus of citizen science projects on shell color evolution in relation to climate change and urbanization. C. nemoralis is particularly useful for studies on the genetics of shell polymorphism and the evolution of “supergenes,” as well as evo-devo studies of shell biomineralization, because it is relatively easily maintained in captivity. However, an absence of genomic resources for C. nemoralis has generally hindered detailed genetic and molecular investigations. We therefore generated ∼23× coverage long-read data for the ∼3.5 Gb genome, and produced a draft assembly composed of 28,537 contigs with the N50 length of 333 kb. Genome completeness, estimated by BUSCO using the metazoa dataset, was 91%. Repetitive regions cover over 77% of the genome. A total of 43,519 protein-coding genes were predicted in the assembled genome, and 97.3% of these were functionally annotated from either sequence homology or protein signature searches. This first assembled and annotated genome sequence for a helicoid snail, a large group that includes edible species, agricultural pests, and parasite hosts, will be a core resource for identifying the loci that determine the shell polymorphism, as well as in a wide range of analyses in evolutionary and developmental biology, and snail biology in general.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

A species-specific satellite DNA from the entomopathogenic nematode Heterorhabditis indicus

Genome ◽  
1998 ◽  
Vol 41 (2) ◽  
pp. 148-153 ◽  
Author(s):  
Monique Abadon ◽  
Eric Grenier ◽  
Christian Laumond ◽  
Pierre Abad
Keyword(s):  
Dna Sequence ◽  
Satellite Dna ◽  
Tandem Repeats ◽  
Sequence Data ◽  
Entomopathogenic Nematode ◽  
Consensus Sequence ◽  
Repeated Sequence ◽  
Nucleotide Sequence Analysis ◽  
Specific Sequence ◽  
Species Specific

An AluI satellite DNA family has been cloned from the entomopathogenic nematode Heterorhabditis indicus. This repeated sequence appears to be an unusually abundant satellite DNA, since it constitutes about 45% of the H. indicus genome. The consensus sequence is 174 nucleotides long and has an A + T content of 56%, with the presence of direct and inverted repeat clusters. DNA sequence data reveal that monomers are quite homogeneous. Such homogeneity suggests that some mechanism is acting to maintain the homogeneity of this satellite DNA, despite its abundance, or that this repeated sequence could have appeared recently in the genome of H. indicus. Hybridization analysis of genomic DNAs from different Heterorhabditis species shows that this satellite DNA sequence is specific to the H. indicus genome. Considering the species specificity and the high copy number of this AluI satellite DNA sequence, it could provide a rapid and powerful tool for identifying H. indicus strains.Key words: AluI repeated DNA, tandem repeats, species-specific sequence, nucleotide sequence analysis.

scholarly journals Potential impacts of climate change on Mongolia’s plant protection status quo

2018 ◽  
Vol 24 (02) ◽  
pp. 26-32
Author(s):  
Batkhuyag B ◽  
Batnaran Kh
Keyword(s):  
Climate Change ◽  
Climate Modeling ◽  
Plant Protection ◽  
Current Trend ◽  
Animal Husbandry ◽  
Distribution Patterns ◽  
Agricultural Pests ◽  
Migratory Locust ◽  
Colorado Beetle ◽  
Wide Range

Mongolia’s 2030 Sustainable Development Vision set a goal to be a self-sufficient in grain, potatoes and vegetables by 2030. However, Mongolia’s pastoral animal husbandry and rain-fed agriculture are extremely sensitive to climate change. The Asian migratory locust is considered as the most harmful grasshopper in the world. Until 1970th, these locust’s distribution areas in Mongolia were confined to oasis of Gobi deserts. A study on Asian migratory locust in Russia predicts distribution and formation of new permanent habitats of the locust in Chita oblast, Krasnoyarsk territory and Republic of Tyva. The Colorado beetle is one of the world’s most infamous invasive species due to its rapid adaptation to a wide range of ecological conditions and ability to disperse long distances. The climate modeling of Colorado beetle showed that with current trend, the beetle will expand its distribution into the most eastern and north-eastern regions of the Russian Federation. In China, the Colorado beetle was first detected in Xinjiang in 1993 and subsequently spread eastward. In China the Colorado beetle is currently expanding its areas at rate of 25 kms year (12-45 kms/year). Both species’ distribution patterns in neighboring countries show eventual establishment of permanent habitats around Mongolia. Their invasion to Mongolia will threaten country’s food security due to direct destruction of cereal and potato crops, and increased application of highly toxic pesticides. In light of these threats, Mongolia should start taking serious preventive measures by increasing surveillance and dedicated risk assessment studies for potential agricultural pests and diseases.

scholarly journals Mycorrhizal Communities and Isotope Signatures in Two Partially Mycoheterotrophic Orchids

2021 ◽  
Vol 12 ◽  
Author(s):  
Hans Jacquemyn ◽  
Rein Brys ◽  
Michael Waud ◽  
Alexandra Evans ◽  
Tomáš Figura ◽  
...  
Keyword(s):  
Isotopic Signatures ◽  
Carbon And Nitrogen ◽  
Wide Range ◽  
Terrestrial Orchids ◽  
Significant Enrichment ◽  
Mycoheterotrophic Plants ◽  
C And N ◽  
Species Specific ◽  
Epipactis Helleborine ◽  
Isotope Analyses

Partial mycoheterotrophy, the ability of plants to obtain carbon from fungi throughout their life cycle in combination with photosynthesis, appears to be more common within the Plant Kingdom than previously anticipated. Recent studies using stable isotope analyses have indicated that isotope signatures in partially mycoheterotrophic plants vary widely among species, but the relative contributions of family- or species-specific characteristics and the identity of the fungal symbionts to the observed differences remain unclear. Here, we investigated in detail mycorrhizal communities and isotopic signatures in four co-occurring terrestrial orchids (Platanthera chlorantha, Epipactis helleborine, E. neglecta and the mycoheterotrophic Neottia nidus-avis). All investigated species were mycorrhizal generalists (i.e., associated with a large number of fungi simultaneously), but mycorrhizal communities differed significantly between species. Mycorrhizal communities associating with the two Epipactis species consisted of a wide range of fungi belonging to different families, whereas P. chlorantha and N. nidus-avis associated mainly with Ceratobasidiaceae and Sebacinaceae species, respectively. Isotopic signatures differed significantly between both Epipactis species, with E. helleborine showing near autotrophic behavior and E. neglecta showing significant enrichment in both carbon and nitrogen. No significant differences in photosynthesis and stomatal conductance were observed between the two partially mycoheterotrophic orchids, despite significant differences in isotopic signatures. Our results demonstrate that partially mycoheterotrophic orchids of the genus Epipactis formed mycorrhizas with a wide diversity of fungi from different fungal families, but variation in mycorrhizal community composition was not related to isotope signatures and thus transfer of C and N to the plant. We conclude that the observed differences in isotope signatures between E. helleborine and E. neglecta cannot solely be explained by differences in mycorrhizal communities, but most likely reflect a combination of inherent physiological differences and differences in mycorrhizal communities.

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