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Brazilian Journal of Biology = Revista... 2024The gall wasp, Leptocybe invasa, poses a significant global threat to Eucalyptus cultivation, by causing substantial economic losses. The objective of this study was to...
The gall wasp, Leptocybe invasa, poses a significant global threat to Eucalyptus cultivation, by causing substantial economic losses. The objective of this study was to differentiate between resistant and susceptible genotypes by morphological characteristics using image analysis based on the damage caused by the gall wasp. In addition, consensus sequences derived from transposable elements (TEs) and the genome of Eucalyptus spp. Were identified by in silico analysis. Furthermore, another objective was to discriminate Eucalyptus genotypes in response to Leptocybe invasa by conducting molecular analyses involving transposable elements and inter simple sequence markers. For image analysis, the GroundEye ® system was used to collect images of 60 leaves from six genotypes, three of which were resistant and three susceptible. Eucalyptus spp. sequences were obtained from the GenBank database by in silico analysis and pairwise alignments with TE sequences were conducted using BLASTN. Multiple sequence alignment was performed with Clustal Omega, followed by the identification of conserved regions in Jalview. A motif signature was generated using Weblogo. For molecular characterization using ISSR markers and TEs, samples of young leaves were obtained from a total of 80 Eucalyptus seedlings, of which 50 were classified as resistant and 30 as susceptible to L. invasa. It was possible to distinguish gall wasp susceptible and resistant genotypes by image analysis. In silico analysis enabled the identification of conserved regions in the Eucalyptus spp. genome, which were associated with proteins involved in secondary metabolite production, e.g., terpenes, which play a role in the response to L. invasa. The discrimination capacity of TEs and ISSR primers was demonstrated and bands were generated that could be used to identify resistant genotypes. However, increasing the number of markers required to discriminate genotypes in both cases is suggested.
Topics: Eucalyptus; Genotype; Phenotype; Animals; Wasps; Disease Resistance; Computer Simulation; Plant Diseases; DNA Transposable Elements
PubMed: 38896727
DOI: 10.1590/1519-6984.279850 -
Physiologia Plantarum 2024RNA-seq data is currently generated in numerous non-model organisms that lack a reference genome. Nevertheless, the confirmation of gene expression levels using RT-qPCR...
RNA-seq data is currently generated in numerous non-model organisms that lack a reference genome. Nevertheless, the confirmation of gene expression levels using RT-qPCR remains necessary, and the existing techniques do not seamlessly interface with the omics pipeline workflow. Developing primers for many targets by utilising orthologous genes can be a laborious, imprecise, and subjective process, particularly for plant species that are not commonly studied and do not have a known genome. We have developed a primer design tool, named PABLOG, that analyses the alignments generated from long or short RNA-seq reads and a reference orthologous gene. PABLOG scans, much like a bee searching several flowers for pollen, and presents a sorted list of potential exon-exon junction locations, ranked according to their reliability. Through computational analysis across the whole genomes of several non-model species, we demonstrate that PABLOG performs more effectively than other methods in identifying exon-exon junctions since it generates significantly fewer false-positive results. Examination of candidate regions at the gene level, in conjunction with laboratory studies, shows that the suggested primers successfully amplified particular targets in non-model plants without any presence of genomic contamination. Our tool includes a consensus sequence feature that enables the complete process of primer design, from aligning with the target gene to determining amplification parameters. The utility can be accessed via the GitHub repository located at: https://github.com/tools4plant-omics/PABLOG.
Topics: Bees; DNA Primers; Exons; Software; Animals; Genes, Plant; Genome, Plant; Computational Biology
PubMed: 38894544
DOI: 10.1111/ppl.14398 -
Foods (Basel, Switzerland) May 2024The adulteration of goat milk powder occurs frequently; cattle-derived and soybean-derived ingredients are common adulterants in goat milk powder. However,...
The adulteration of goat milk powder occurs frequently; cattle-derived and soybean-derived ingredients are common adulterants in goat milk powder. However, simultaneously and rapidly detecting cattle-derived and soybean-derived components is still a challenge. An efficient, high-throughput screening method for adulteration detection is needed. In this study, a rapid method was developed to detect the adulteration of common cattle-derived and soybean-derived components simultaneously in goat milk powder by combining the CRISPR/Cas12a system with recombinant polymerase amplification (RPA). A dual DNA extraction method was employed. Primers and crRNA for dual detection were designed and screened, and a series of condition optimizations were carried out in this experiment. The optimized assay rapidly detected cattle-derived and soybean-derived components in 40 min. The detection limits of both cattle-derived and soybean-derived components were 1% (/) for the mixed adulteration models. The established method was applied to a blind survey of 55 commercially available goat milk powder products. The results revealed that 36.36% of the samples contained cattle-derived or soybean-derived ingredients, which revealed the noticeable adulteration situation in the goat milk powder market. This study realized a fast flow of dual extraction, dual amplification, and dual detection of cattle-derived and soybean-derived components in goat milk powder for the first time. The method developed can be used for high-throughput and high-efficiency on-site primary screening of goat milk powder adulterants, and provides a technical reference for combating food adulteration.
PubMed: 38890866
DOI: 10.3390/foods13111637 -
Communications Chemistry Jun 2024Oligonucleotides are advancing as essential materials for the development of new therapeutics, artificial genes, or in storage of information applications. Hitherto, our... (Review)
Review
Oligonucleotides are advancing as essential materials for the development of new therapeutics, artificial genes, or in storage of information applications. Hitherto, our capacity to write (i.e., synthesize) oligonucleotides is not as efficient as that to read (i.e., sequencing) DNA/RNA. Alternative, biocatalytic methods for the de novo synthesis of natural or modified oligonucleotides are in dire need to circumvent the limitations of traditional synthetic approaches. This Perspective article summarizes recent progress made in controlled enzymatic synthesis, where temporary blocked nucleotides are incorporated into immobilized primers by polymerases. While robust protocols have been established for DNA, RNA or XNA synthesis is more challenging. Nevertheless, using a suitable combination of protected nucleotides and polymerase has shown promises to produce RNA oligonucleotides even though the production of long DNA/RNA/XNA sequences (>1000 nt) remains challenging. We surmise that merging ligase- and polymerase-based synthesis would help to circumvent the current shortcomings of controlled enzymatic synthesis.
PubMed: 38890393
DOI: 10.1038/s42004-024-01216-0 -
Parasites & Vectors Jun 2024Malaria transmission in Tanzania is driven by mosquitoes of the Anopheles gambiae complex and Anopheles funestus group. The latter includes An. funestus s.s., an...
BACKGROUND
Malaria transmission in Tanzania is driven by mosquitoes of the Anopheles gambiae complex and Anopheles funestus group. The latter includes An. funestus s.s., an anthropophilic vector, which is now strongly resistant to public health insecticides, and several sibling species, which remain largely understudied despite their potential as secondary vectors. This paper provides the initial results of a cross-country study of the species composition, distribution and malaria transmission potential of members of the Anopheles funestus group in Tanzania.
METHODS
Mosquitoes were collected inside homes in 12 regions across Tanzania between 2018 and 2022 using Centres for Disease Control and Prevention (CDC) light traps and Prokopack aspirators. Polymerase chain reaction (PCR) assays targeting the noncoding internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA (18S rDNA) were used to identify sibling species in the An. funestus group and presence of Plasmodium infections, respectively. Where DNA fragments failed to amplify during PCR, we sequenced the ITS2 region to identify any polymorphisms.
RESULTS
The following sibling species of the An. funestus group were found across Tanzania: An. funestus s.s. (50.3%), An. parensis (11.4%), An. rivulorum (1.1%), An. leesoni (0.3%). Sequencing of the ITS2 region in the nonamplified samples showed that polymorphisms at the priming sites of standard species-specific primers obstructed PCR amplification, although the ITS2 sequences closely matched those of An. funestus s.s., barring these polymorphisms. Of the 914 samples tested for Plasmodium infections, 11 An. funestus s.s. (1.2%), and 2 An. parensis (0.2%) individuals were confirmed positive for P. falciparum. The highest malaria transmission intensities [entomological inoculation rate (EIR)] contributed by the Funestus group were in the north-western region [108.3 infectious bites/person/year (ib/p/y)] and the south-eastern region (72.2 ib/p/y).
CONCLUSIONS
Whereas An. funestus s.s. is the dominant malaria vector in the Funestus group in Tanzania, this survey confirms the occurrence of Plasmodium-infected An. parensis, an observation previously made in at least two other occasions in the country. The findings indicate the need to better understand the ecology and vectorial capacity of this and other secondary malaria vectors in the region to improve malaria control.
Topics: Anopheles; Animals; Tanzania; Mosquito Vectors; Malaria; Humans; RNA, Ribosomal, 18S; Polymerase Chain Reaction; Female; Plasmodium; DNA, Ribosomal Spacer
PubMed: 38886827
DOI: 10.1186/s13071-024-06348-9 -
Plant Disease Jun 2024Avocado (), which is native to Latin America, is mostly planted in southwest China. In November 2021, leaf spot symptoms were observed in a nursery in Chongzuo...
Avocado (), which is native to Latin America, is mostly planted in southwest China. In November 2021, leaf spot symptoms were observed in a nursery in Chongzuo (22.2019°N, 106.4723°E), Guangxi, China. Approximately 90% of avocado seedlings in the nursery were affected. Symptomatic plant fully expanded leaves showed small brown spots that ranged from 1 to 3 mm, with a yellow halo around (Fig.1). Lesions gradually expanded and became nearly round and dark brown. Finally, leaves withered or curled. For pathogen isolation, 15 symptomatic leaves were randomly sampled from different plants of the nursery, five leaves were selected and four samples size 4×4mm were taken from each leaf and were plated on potato glucose agar. Identical fungus colonies were observed in 80% of the samples, and no bacteria were isolated. Single conidial isolation was performed. After 4 days, the colony diameter reached 74.6 mm, colonies appeared gray, and developed aerial hyphae. Conidiophores were mostly solitary with a few clustered erect or slightly curved, knee shaped, and 3.89 to 5.24 µm wide. Conidia were 39.33 -96.88 × 9.96 - 15.59 µm, slightly curved, rarely straight, light brown to yellowish brown, fusoid or navicular, and truncated at the base with 4 to 10 septa. Based on morphological and cultural characteristics, the fungus was identified as sp. (Manamgoda et al. 2014). An isolate named MP211122 was grown on Sachs' ager at 27℃ under 12-h light/dark for 1 week and consistently with Adhikari et al. (2021) no sexual from was observed. To confirm the tentative identification, genomic DNA was extracted, ITS and GAPDH gene were amplified and sequenced using primers ITS1/ITS4 and GPD/GPD2, respectively (Tan et al. 2022). The ITS sequence (GenBank ON248469) shared 100% identity with (MN215632.1), and the GAPDH sequence (ON642344) shared 99.82% identity with (MF490833.1, MK144540.1) and (MK026428.1). A maximum likelihood phylogenetic analysis based on GAPDH and ITS sequences using MEGA 7.0 revealed that the isolate clustered with with 100% bootstrap support(Fig. 2). Healthy 11-month old potted avocado seedlings from disease-free nursery were selected , the conidial suspension (1 × 10 conidia/mL) of MP211122 isolate was prepared by harvesting conidia from a 10-day-old culture on water agar. Conidia were sprayed onto young leaves of six potted plants. Three additional seedlings sprayed with sterile distilled water served as controls. All plants were covered with plastic bags for 3 days to maintain high humidity and then maintained in a greenhouse at 30℃ with a 12-h/12-h light/dark cycle. After 5 days, typical symptoms of small brown spots were observed on all inoculated leaves (Fig.3). All leaves on control plants remained asymptomatic. The reisolated fungus was morphologically identical to the original isolate used for inoculation, fulfilling Koch's postulates. This is the first report of as a pathogen causing leaf spot on avocado in China. This information will facilitate further studies, monitoring and control of the disease as accurate identification of the causal agent is a primary requisite for designing management strategies.
PubMed: 38885029
DOI: 10.1094/PDIS-12-22-2958-PDN -
Plant Disease Jun 2024Datura stramonium L.(jimson weed) is an invasive weed in agricultural fields and a medicinal plant. In April 2022, a leaf disease on D. stramonium was observed in...
Datura stramonium L.(jimson weed) is an invasive weed in agricultural fields and a medicinal plant. In April 2022, a leaf disease on D. stramonium was observed in Zhanjiang (21.17 N, 110.18 E), Guangdong province, China. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned becoming necrotic with a clear yellow halo and a white center. The disease incidence in the field was 85% (n = 50, about 1 ha). Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively, and rinsed twice with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. After 2-day incubation, grayish fungal colonies appeared on the PDA, then pure cultures were produced by transferring hyphal tips to new PDA plates. Single-spore isolation method was used to recover pure cultures for three isolates (DSAC-1, DSAC-2, and DSAC-3). The isolates were morphologically identical . They colonies were gray to brownish black. Conidiophores were branched, brown. Conidia were brown, long ellipsoid, had 4-12 transverse and 0-3 longitudinal septa; measured within 67.5-127.8 (average = 105.6) × 12.5-27.8 (average = 20.4) µm (n = 30). Apical beak was longer than conidia body. measured within 40.5-423.5 (average = 365.2) × 2.5-5.8 (average = 3.2) µm (n = 30). Based on morphological characteristics, the three isolates were identified as Alternaria crassa (Sacc.) Rands (Simmons 2007). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) to amplify internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2) and translation elongation factor (TEF1) with primers of ITS1/ITS4, GDF1/GDR1, RPB2-5F2/fRPB2-7cR, and EF-1α-F/EF-1α-R, respectively (Walther et al. 2013; Woudenberg et al. 2015; Nishikawa and Nakashima. 2020). Amplicons of the isolates were sequenced and submitted to GenBank (ITS, ON430524-ON430526; GAPDH, ON500656-ON500658; RPB2, ON500659-ON500661; TEF1, ON500662-ON500664). The sequences were 100% identical with those of Alternaria crassa strain CBS 116647 upon BLAST analysis. The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered with A. crassa (CBS 116647, CBS 116648, CBS CBS-110.38, and CBS_103.18 ). Thus, the fungus associated with leaf yellow spot on D. stramonium was identified as A. crassa. Pathogenicity tests were conducted in a greenhouse at 24 ℃-30 ℃ with 80% relative humidity using 3 isolates. Individual plants were grown in pots (n = 5, 1 month old). The unwounded leaflets were inoculated using three isolates (DSAC-1, DSAC-2, and DSAC-3). The fungal mycelium on 5 mm-diameter PDA plugs were placed faced down to the leaves. Sterile PDA was used for mock inoculated comtrols.. The test was performed three times. Disease symptoms were observed on the leaves after 7 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and was morphologically identical to the original isolates, fulfilling Koch's postulates. A. crassa was reported causing leaf spot on D. stramonium in Algeria (Nabahat et al. 2020). To our knowledge, this report is the first report of A. crassa causing leaf yellow spot on D. stramonium in China. This pathogen possesses potential biocontrol properties on the invasive weed, while this study also provides an important reference for the control of the disease of the medicinal plant.
PubMed: 38885028
DOI: 10.1094/PDIS-08-23-1494-PDN -
Plant Disease Jun 2024Psidium guajava L. is widely cultivated in southern China. In May 2021, guava scab on cv. Zhenzhu was observed in Zhanjiang (21.18° N, 110.21° E), Guangdong province,...
Psidium guajava L. is widely cultivated in southern China. In May 2021, guava scab on cv. Zhenzhu was observed in Zhanjiang (21.18° N, 110.21° E), Guangdong province, China. Guava scab was corky with ovoid or round lesions on the surfaces of green fruits. Gradually the lesions sunk. Disease incidence was estimated as 85% in 500 investigated plants in about 50 ha. Twenty diseased fruits were collected from twenty trees in the field. From each fruit the margin of the diseased tissues was cut into 2 mm × 2 mm pieces; surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, successively; and rinsed thrice with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. Thirty-four isolates were obtained. Single-spore isolation method (Liu et al. 2021) was used to recover pure cultures of three isolates (PGNC-1, PGNC-2, and PGNC-3) . The colonies were initially white with cottony aerial mycelium at 7 days on PDA. Then, these colonies form black acervular conidiomata at 10 days. Conidia were clavate to fusiform, four-septate, straight or slightly curved, and measured 15.8 to 21.2 µm × 4.5 to 6.5 µm (n = 40). The three median cells were versicolored, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (4.5 to 5.5 µm long; n = 40) and three apical appendages (19.2 to 24.5 µm long; n = 40). The morphological characteristics of the isolates were consistent with the description of Neopestalotiopsis clavispora (Maharachchikumbura et al. 2012). Molecular identification was performed using PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). Sequences were generated from the isolates using primers for the rDNA ITS (ITS1/ITS4), TEF1-α (EF1-728F/EF1-986R), and β-tubulin (T1/βt2b) loci (Maharachchikumbura et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, OQ996557 to OQ996559; TEF, OR101037 to OR101039; β-tubulin, OR100971 to OR100973). The sequences of the isolates were 100% identical to the type strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis. The isolates clustered with N. clavispora (MFLUCC12-0280 and MFLUCC12-0281). N. clavispora and Pestalotiopsis clavispora are synonyms. The pathogenicity was tested in vivo. Plants (cv. Zhenzhu) were grown ( 3 years old) in a quarantine orchard at 25 ℃ to 32 ℃ with 60 to 80% relative humidity in May 2022. Disease-free green fruits were inoculated. Sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) and sterile distilled water (control) for about 15 s before they were fixed on the wounded fruits with transparent tape. Five fruits on one plant per isolate were inoculated. Five fruits on one plant severed as control. The test was performed thrice. Disease symptoms were found on the inoculated fruits after 20 days, whereas the controls remained healthy. The pathogen was re-isolated from infected fruits and was phenotypically identical to the original isolates thus fulfilling Koch's postulates. Neopestalotiopsis or Pestalotiopsis spp. were reported to be the causal agents of guava scab in Colombia and in Hawaii (Keith et al. 2006; Solarte et al. 2018). N. clavispora has been reported to cause disease in a broad range of hosts (Ge et al. 2009; Chen et al. 2018), but not in guava. This is the first report of N. clavispora causing guava scab in China. There would be no harvest if this disease is left unmanaged.
PubMed: 38885025
DOI: 10.1094/PDIS-11-23-2357-PDN -
Plant Disease Jun 2024Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated amplification...
Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii, the causal agents of dollar spot within the continental US. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount purified DNA needed for detection was 0.05 ng µL-1. Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple and reliable detection in 1.25 hours. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection.
PubMed: 38885023
DOI: 10.1094/PDIS-12-23-2608-RE -
Biochemical Society Transactions Jun 2024Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the... (Review)
Review
Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the mechanism determining the switch between continuous transcription and premature termination to generate RNA primers for mitochondrial DNA (mtDNA) replication remains unclear. The pentatricopeptide repeat domain of mtRNAP exhibits exoribonuclease activity, which is required for the initiation of mtDNA replication in Drosophila. In this review, we explain how this exonuclease activity contributes to primer synthesis in strand-coupled mtDNA replication, and discuss how its regulation might co-ordinate mtDNA replication and transcription in both Drosophila and mammals.
Topics: DNA, Mitochondrial; DNA Replication; Animals; Mitochondria; Humans; DNA-Directed RNA Polymerases; Transcription, Genetic; Drosophila; Exoribonucleases; Drosophila melanogaster
PubMed: 38884788
DOI: 10.1042/BST20230952