qPCR assays for sensitive and rapid detection of Quambalaria species from plant tissues

Plant Disease ◽  
2021 ◽  
Author(s):  
Hoa Thanh Duong ◽  
Briony Williams ◽  
Dianne White ◽  
Giles E. St. J. Hardy ◽  
Treena Burgess
Keyword(s):  
Infection Process ◽  
Qpcr Assay ◽  
Plant Tissues ◽  
Rapid Diagnostics ◽  
Target Species ◽  
Natural Ecosystems ◽  
Plant Host ◽  
Its1 Region ◽  
Specificity And Sensitivity ◽  
Species Specific

Several species from the genus Quambalaria (order Microstromatales) cause diseases on eucalypts (Eucalyptus and related genera) both in plantations and natural ecosystems. We developed real-time qPCR assays to rapidly detect and distinguish five Quambalaria species.The design of the species-specific qPCR assay for each species, Q. pitereka (PIT), Q. coyrecup (COR), Q. cyanescens (CYN), Q. pusilla (PUS) and Q. eucalypti (EUC) was based on the ITS1 region, and was evaluated for specificity and sensitivity. The CYN qPCR assay could amplify as little as 1fg µl-1 from pure culture, the PIT and COR qPCR assays could amplify 10fg µl-1, while PUS and EUC qPCR assays could amplify 100 fg µl-1 of their target species. The PIT, COR and CYN qPCR assays were further validated using artificially and naturally infected samples of their plant host Corymbia calophylla. These assays will be used for rapid diagnostics and for future experiments on the infection process.

scholarly journals A qPCR Assay for the Detection of Phytophthora cinnamomi Including an mRNA Protocol Designed to Establish Propagule Viability in Environmental Samples

Plant Disease ◽  
2019 ◽  
Vol 103 (9) ◽  
pp. 2443-2450 ◽  
Author(s):  
Manisha B. Kunadiya ◽  
William D. Dunstan ◽  
Diane White ◽  
Giles E. St. J. Hardy ◽  
Andrew H. Grigg ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Phytophthora Cinnamomi ◽  
Rna Isolation ◽  
Pcr Primers ◽  
Phytophthora Species ◽  
Qpcr Assay ◽  
Natural Ecosystems ◽  
Specificity And Sensitivity ◽  
Mitochondrial Locus ◽  
Species Specific

Phytophthora cinnamomi causes root and collar rot in many plant species in natural ecosystems and horticulture. A species-specific primer and probe PCIN5 were designed based on a mitochondrial locus encoding subunit 2 of cytochrome c oxidase (cox2). Eight PCR primers, including three forward and five reverse, were designed and tested in all possible combinations. Annealing temperatures were optimized for each primer pair set to maximize both specificity and sensitivity. Each set was tested against P. cinnamomi and two closely related clade 7 species, P. parvispora and P. niederhauseri. From these tests, five primer pairs were selected based on specificity and, with a species-specific P. cinnamomi probe, used to develop quantitative real-time PCR (qPCR) assays. The specificity of the two most sensitive qPCR assays was confirmed using the genomic DNA of 29 Phytophthora isolates, including 17 isolates of 11 species from clade 7, and representative species from nine other clades (all except clade 3). The assay was able to detect as little as 150 ag of P. cinnamomi DNA and showed no cross-reaction with other Phytophthora species, except for P. parvispora, a very closely related species to P. cinnamomi, which showed late amplification at high DNA concentrations. The efficiency of the qPCR protocol was evaluated with environmental samples including roots and associated soil from plants artificially infected with P. cinnamomi. Different RNA isolation kits were tested and evaluated for their performance in the isolation of RNA from environmental samples, followed by cDNA synthesis, and qPCR assay. Finally, a protocol was recommended for determining the presence of P. cinnamomi in recalcitrant environmental samples.

scholarly journals The Coevolution of Plants and Microbes Underpins Sustainable Agriculture

2021 ◽  
Vol 9 (5) ◽  
pp. 1036
Author(s):  
Dongmei Lyu ◽  
Levini A. Msimbira ◽  
Mahtab Nazari ◽  
Mohammed Antar ◽  
Antoine Pagé ◽  
...  
Keyword(s):  
Microbial Community ◽  
Plant Growth ◽  
Stress Resistance ◽  
Mycorrhizal Fungi ◽  
Plant Tissues ◽  
Terrestrial Plants ◽  
Plant Host ◽  
Symbiotic Relationships ◽  
Wide Range ◽  
Terrestrial Environments

Terrestrial plants evolution occurred in the presence of microbes, the phytomicrobiome. The rhizosphere microbial community is the most abundant and diverse subset of the phytomicrobiome and can include both beneficial and parasitic/pathogenic microbes. Prokaryotes of the phytomicrobiome have evolved relationships with plants that range from non-dependent interactions to dependent endosymbionts. The most extreme endosymbiotic examples are the chloroplasts and mitochondria, which have become organelles and integral parts of the plant, leading to some similarity in DNA sequence between plant tissues and cyanobacteria, the prokaryotic symbiont of ancestral plants. Microbes were associated with the precursors of land plants, green algae, and helped algae transition from aquatic to terrestrial environments. In the terrestrial setting the phytomicrobiome contributes to plant growth and development by (1) establishing symbiotic relationships between plant growth-promoting microbes, including rhizobacteria and mycorrhizal fungi, (2) conferring biotic stress resistance by producing antibiotic compounds, and (3) secreting microbe-to-plant signal compounds, such as phytohormones or their analogues, that regulate aspects of plant physiology, including stress resistance. As plants have evolved, they recruited microbes to assist in the adaptation to available growing environments. Microbes serve themselves by promoting plant growth, which in turn provides microbes with nutrition (root exudates, a source of reduced carbon) and a desirable habitat (the rhizosphere or within plant tissues). The outcome of this coevolution is the diverse and metabolically rich microbial community that now exists in the rhizosphere of terrestrial plants. The holobiont, the unit made up of the phytomicrobiome and the plant host, results from this wide range of coevolved relationships. We are just beginning to appreciate the many ways in which this complex and subtle coevolution acts in agricultural systems.

Identification for adulteration of beef with chicken based on single primer-triggered isothermal amplification

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Jiao Chen ◽  
Pansong Zhang ◽  
Haixia Wang ◽  
Yanjing Shi
Keyword(s):  
Limit Of Detection ◽  
Chicken Meat ◽  
Isothermal Amplification ◽  
Work Flow ◽  
Acid Extraction ◽  
Specificity And Sensitivity ◽  
Total Dna ◽  
Meat Species ◽  
Species Specific ◽  
Single Primer

Abstract Adulteration of beef with cheap chicken has become a growing problem worldwide. In this study, a quick, single primer-triggered isothermal amplification (SAMP) combined with a fast nucleic acid extraction method was employed to detect the chicken meat in adulterated beef. Chicken from adulterated beef was identified using the chicken species-specific primer designed according to the Gallus gallus mitochondrial conserved sequences. Our SAMP method displayed good specificity and sensitivity in detecting chicken and beef meat DNA–the limit of detection (LOD) of SAMP is 0.33 pg/μL of chicken and beef total DNA and 2% w/w chicken meat in beef. The whole work flow from DNA extraction to signal detection can be finished within 1 h, fulfilling the requirement of on-site meat species identification.

Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

2005 ◽  
Vol 68 (6) ◽  
pp. 1217-1221 ◽  
Author(s):  
PAVEL KRCMAR ◽  
EVA RENCOVA
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Single Species ◽  
Absolute Quantification ◽  
Quantitative Detection ◽  
Vertebrate Species ◽  
Target Species ◽  
Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

scholarly journals DNA-based species detection capabilities using laser transmission spectroscopy

2013 ◽  
Vol 10 (78) ◽  
pp. 20120637 ◽  
Author(s):  
A. R. Mahon ◽  
M. A. Barnes ◽  
F. Li ◽  
S. P. Egan ◽  
C. E. Tanner ◽  
...  
Keyword(s):  
Invasive Species ◽  
Dna Detection ◽  
Field Application ◽  
Economic Damage ◽  
Oligonucleotide Probes ◽  
Target Species ◽  
Dreissena Bugensis ◽  
Species Detection ◽  
Transmission Spectroscopy ◽  
Species Specific

Early detection of invasive species is critical for effective biocontrol to mitigate potential ecological and economic damage. Laser transmission spectroscopy (LTS) is a powerful solution offering real-time, DNA-based species detection in the field. LTS can measure the size, shape and number of nanoparticles in a solution and was used here to detect size shifts resulting from hybridization of the polymerase chain reaction product to nanoparticles functionalized with species-specific oligonucleotide probes or with the species-specific oligonucleotide probes alone. We carried out a series of DNA detection experiments using the invasive freshwater quagga mussel ( Dreissena bugensis ) to evaluate the capability of the LTS platform for invasive species detection. Specifically, we tested LTS sensitivity to (i) DNA concentrations of a single target species, (ii) the presence of a target species within a mixed sample of other closely related species, (iii) species-specific functionalized nanoparticles versus species-specific oligonucleotide probes alone, and (iv) amplified DNA fragments versus unamplified genomic DNA. We demonstrate that LTS is a highly sensitive technique for rapid target species detection, with detection limits in the picomolar range, capable of successful identification in multispecies samples containing target and non-target species DNA. These results indicate that the LTS DNA detection platform will be useful for field application of target species. Additionally, we find that LTS detection is effective with species-specific oligonucleotide tags alone or when they are attached to polystyrene nanobeads and with both amplified and unamplified DNA, indicating that the technique may also have versatility for broader applications.

scholarly journals Multiplex End-Point PCR for the Detection of Three Species of Ophiosphaerella Causing Spring Dead Spot of Bermudagrass

Plant Disease ◽  
2019 ◽  
Vol 103 (8) ◽  
pp. 2010-2014 ◽  
Author(s):  
J. Francisco Iturralde Martinez ◽  
Francisco J. Flores ◽  
Alma R. Koch ◽  
Carla D. Garzón ◽  
Nathan R. Walker
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Temperature Cycling ◽  
Molecular Technique ◽  
Correct Identification ◽  
Internal Transcribed Spacer Region ◽  
Spring Dead Spot ◽  
End Point ◽  
Specificity And Sensitivity ◽  
Species Specific

A multiplex end-point polymerase chain reaction (PCR) assay was developed for identifying the three-fungal species in the genus Ophiosphaerella that cause spring dead spot (SDS), a devastating disease of bermudagrass. These fungi are difficult to identify by morphology because they seldom produce pseudothecia. To achieve species-specific diagnosis, three pairs of primers were designed to identify fungal isolates and detect the pathogen in infected roots. The internal transcribed spacer region, the translation elongation factor 1-α, and the RNA polymerase II second-largest subunit were selected as targets and served as templates for the design of each primer pair. To achieve uniform melting temperatures, three to five random nucleotide extensions (flaps) were added to the 5′ terminus of some of the designed specific primers. Temperature cycling conditions and PCR components were standardized to optimize specificity and sensitivity of the multiplex reaction. Primers were tested in multiplex on DNA extracted from axenic fungal cultures and from field-collected infected and uninfected roots. A distinct amplicon was produced for each Ophiosphaerella sp. tested. The DNA from Ophiosphaerella close relatives and other common bermudagrass pathogens did not amplify during the multiplex assay. Metagenomic DNA from infected bermudagrass produced species-specific amplicons while DNA extracted from noninfected roots did not. This multiplex end-point PCR approach is a sensitive and specific molecular technique that allows for correct identification of SDS-associated Ophiosphaerella spp. from field-collected roots.

scholarly journals Secondary metabolites produced by endophytic bacteria against the Root-Knot Nematode (Meloidogyne sp.)

2020 ◽  
Vol 21 (11) ◽  
Author(s):  
Vina Maulidia ◽  
Loekas Soesanto ◽  
Syamsuddin Syamsuddin ◽  
Khairan Khairan ◽  
Takahiro Hamaguchi ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Endophytic Bacteria ◽  
Psidium Guajava ◽  
Plant Tissues ◽  
Root Number ◽  
Root Knot Nematode ◽  
Plant Host ◽  
Tomato Plants ◽  
Theobroma Cacao L ◽  
Meloidogyne Sp

Abstract. Maulidia V, Soesanto L, Syamsuddin, Khairan K, Hamaguchi T, Hasegawa K, Sriwati R. 2020. Secondary metabolites produced by endophytic bacteria against the Root-Knot Nematode (Meloidogyne sp.). Biodiversitas 21: 5270-5275. Endophytic bacteria live and colonize in plant tissues without causing disease to their plant host. Among several processes, these bacteria can produce secondary metabolites that can help in the defense of plant host against pathogens. This study aimed to identify endophytic bacteria as biocontrol agents against Meloidogyne sp. in tomato plants. Six endophytic bacteria candidates from the genus Pseudomonas, Arthrobacter, Bacillus, and Serratia were isolated from Solanum Lycopersicum, Psidium guajava, Pinus merkusii, Dendrocalamus asper, Albizia chinensis, and Theobroma cacao L, respectively. The average mortality of Meloidogyne sp. by endophytic bacteria was 70,27% to 95,46%. From these, B. thuringiensis AK08 produced compounds of the secondary metabolites such as flavonoid, phenol, tannins, terpenoids, steroids, saponins, and alkaloids. The best result of the average incubation period, number of galls in the root, number of nematodes at the root, and the number of nematodes in the soil on tomato plant were shown by B. thuringiensis. The major compounds in GC-MS analysis of B. thuringiensis were cholest-5-en-3-ol (3.beta.)-carbonochloridate (25.35%). Bacillus thuringiensis not only has rules as bio-insecticide but also has nematicidal effect.

scholarly journals Reaching Natural Growth: Light Quality Effects on Plant Performance in Indoor Growth Facilities

Plants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1273
Author(s):  
Camilo Chiang ◽  
Daniel Bånkestad ◽  
Günter Hoch
Keyword(s):  
Plant Growth ◽  
Field Trial ◽  
Light Quality ◽  
Plant Traits ◽  
Principal Component ◽  
Biomass Productivity ◽  
Plant Performance ◽  
Plant Responses ◽  
Natural Ecosystems ◽  
Species Specific

To transfer experimental findings in plant research to natural ecosystems it is imperative to reach near to natural-like plant performance. Previous studies propose differences in temperature and light quantity as main sources of deviations between indoor and outdoor plant growth. With increasing implementation of light emitting diodes (LED) in plant growth facilities, light quality is yet another factor that can be optimised to prevent unnatural plant performance. We investigated the effects of different wavelength combinations in phytotrons (i.e., indoor growth chambers) on plant growth and physiology in seven different plant species from different plant functional types (herbs, grasses and trees). The results from these experiments were compared against a previous field trial with the same set of species. While different proportions of blue (B) and red (R) light were applied in the phytotrons, the mean environmental conditions (photoperiod, total radiation, red to far red ratio and day/night temperature and air humidity) from the field trial were used in the phytotrons in order to assess which wavelength combinations result in the most natural-like plant performance. Different plant traits and physiological parameters, including biomass productivity, specific leaf area (SLA), leaf pigmentation, photosynthesis under a standardised light, and the respective growing light and chlorophyll fluorescence, were measured at the end of each treatment. The exposure to different B percentages induced species-specific dose response reactions for most of the analysed parameters. Compared with intermediate B light treatments (25 and/or 35% B light), extreme R or B light enriched treatments (6% and 62% of B respectively) significantly affected the height, biomass, biomass allocation, chlorophyll content, and photosynthesis parameters, differently among species. Principal component analyses (PCA) confirmed that 6% and 62% B light quality combinations induce more extreme plant performance in most cases, indicating that light quality needs to be adjusted to mitigate unnatural plant responses under indoor conditions.

scholarly journals Allelopathic effect of black mustard tissues and root exudates on some crops and weeds

2013 ◽  
Vol 31 (1) ◽  
pp. 11-19 ◽  
Author(s):  
E Al-Sherif ◽  
A.K. Hegazy ◽  
N.H. Gomaa ◽  
M.O. Hassan
Keyword(s):  
Root Exudates ◽  
High Performance ◽  
Ethanolic Extract ◽  
Plant Tissues ◽  
Allelopathic Effect ◽  
Trifolium Alexandrinum ◽  
Target Species ◽  
Phytotoxic Effect ◽  
Black Mustard ◽  
Sisymbrium Irio

Laboratory and greenhouse experiments were conducted to evaluate the phytotoxic effect of black mustard extracts and root exudates on two crops: Trifolium alexandrinum and Triticum aestivum, and two weeds: Phalaris paradoxa and Sisymbrium irio. The seeds were treated with aqueous and ethanolic extracts and chloroform for eight days, or subjected to root exudates of just harvested mustard in a greenhouse for five weeks. High-performance liquid chromatography (HPLC) was used to quantify phytotoxins from plant tissues. Seed germination of P. paradoxa was reduced with the lowest concentration of the different extracts. However, the aqueous extract at 4% completely curtailed the germination of all the target species. In general, plant extracts had a concentration-dependent reduction of seedling growth of the target species. However, the ethanolic extract, at the lowest concentration, has stimulated the shoot length of both T. alexandrinum and T. aestivum, and the root length of the former. Mustard root exudates inhibited emergence and growth of the target species throughout the experiment. Ferulic and syringic acids were the dominant allelochemicals found when HPLC was used.

scholarly journals Application of InDel markers based on the chloroplast genome sequences for authentication and traceability of tartary and common buckwheat

2017 ◽  
Vol 35 (No. 2) ◽  
pp. 122-130 ◽  
Author(s):  
Cho Kwang-Soo ◽  
Hong Su-Young ◽  
Yun Bong-Kyoung ◽  
Won Hong-Sik ◽  
Yoon Young-Ho ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Detection Method ◽  
Processed Foods ◽  
Common Buckwheat ◽  
Genome Sequences ◽  
Specific Pcr ◽  
Indel Markers ◽  
Specificity And Sensitivity ◽  
Species Specific ◽  
Qualitative Pcr

A reliable, qualitative PCR-based detection method for the traceability and authentication of common and Tartary buckwheat was developed. Five InDel markers developed from chloroplast genome variation between the two species were applied for 96 buckwheat accessions and all accessions were easily differentiated as Tartary and common buckwheat using these markers. We also determined the sample detection limit by PCR and qPCR as 0.001 and 0.02 ng/µl, respectively. InDel markers could detect the mixture of two species flour up to 10% contamination. InDel markers were also applied to processed foods such as noodles and tea, and we found that species-specific PCR bands could be used to identify buckwheat even after processing. Hence, these InDel markers are simple with higher specificity and sensitivity and are reliable for the authentication of buckwheat processed foods.

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