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.

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 Identification of Some Oomycetes by Reverse Dot Blot Hybridization

1998 ◽  
Vol 88 (3) ◽  
pp. 213-222 ◽  
Author(s):  
C. André Lévesque ◽  
Colleen E. Harlton ◽  
Arthur W. A. M. de Cock
Keyword(s):  
Phytophthora Cinnamomi ◽  
Blot Hybridization ◽  
Its Region ◽  
Phytophthora Species ◽  
Universal Primers ◽  
Dot Blot ◽  
Nylon Membrane ◽  
Species Specific ◽  
Small Aliquot ◽  
Reverse Dot Blot

An assay was developed that can identify unknown isolates of Pythium or Phytophthora species in a single hybridization. This reverse dot blot system is based on arrays of species-specific amplified fragments or oligonucleotides derived from the internal transcribed spacer (ITS) region, which are blotted as dots on a nylon membrane. By using total DNA from a sample as the template, universal primers, and digoxigenin-dUTP, the ITS was amplified and labeled simultaneously by the polymerase chain reaction (PCR). A small aliquot of the resultant labeled and amplified product was used as a probe for hybridization to a dot blot membrane that contained the immobilized species-specific oligonucleotides or amplified PCR fragments. The reverse dot blot system based on arrays of oligonucleotides showed far fewer cross-hybridizations than one based on entire amplified ITS I fragments. Unknown species can be identified simply by visualizing the positive hybridization reaction between the DNA labeled directly from the sample and the immobilized specific oligonucleotide. Currently, the assay can be used to identify Pythium aphanidermatum, P. ultimum, P. acanthicum, and Phytophthora cinnamomi. An oligonucleotide that was originally designed to identify Phytophthora hybridized to 10 of the 14 Phytophthora species tested. Another oligonucleotide designed to identify oomycetes hybridized to the 68 species tested, which represented two of the four orders of this phylum.

scholarly journals Phytophthora mediterranea sp. nov., a New Species Closely Related to Phytophthora cinnamomi from Nursery Plants of Myrtus communis in Italy

Forests ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 682
Author(s):  
Carlo Bregant ◽  
Antonio A. Mulas ◽  
Giovanni Rossetto ◽  
Antonio Deidda ◽  
Lucia Maddau ◽  
...  
Keyword(s):  
New Species ◽  
Root Rot ◽  
Phytophthora Cinnamomi ◽  
Colony Growth ◽  
Phytophthora Species ◽  
Maximum Temperature ◽  
Pathogenicity Test ◽  
Forest Nurseries ◽  
Collar And Root Rot ◽  
A New Species

Monitoring surveys of Phytophthora related diseases in four forest nurseries in Italy revealed the occurrence of fourteen Phytophthora species to be associated with collar and root rot on fourteen plants typical of Mediterranean and alpine regions. In addition, a multilocus phylogeny analysis based on nuclear ITS and ß-tubulin and mitochondrial cox1 sequences, as well as micromorphological features, supported the description of a new species belonging to the phylogenetic clade 7c, Phytophthora mediterranea sp. nov. Phytophthora mediterranea was shown to be associated with collar and root rot symptoms on myrtle seedlings. Phylogenetically, P. mediterranea is closely related to P. cinnamomi but the two species differ in 87 nucleotides in the three studied DNA regions. Morphologically P. mediterranea can be easily distinguished from P. cinnamomi on the basis of its smaller sporangia, colony growth pattern and higher optimum and maximum temperature values. Data from the pathogenicity test showed that P. mediterranea has the potential to threaten the native Mediterranean maquis vegetation. Finally, the discovery of P. cinnamomi in alpine nurseries, confirms the progressive expansion of this species towards cold environments, probably driven by climate change.

scholarly journals Development of a Co-Dominant Cleaved Amplified Polymorphic Sequences Assay for the Rapid Detection and Differentiation of Two Pathogenic Clarireedia spp. Associated with Dollar Spot in Turfgrass

Agronomy ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1489
Author(s):  
Tammy Stackhouse ◽  
Sumyya Waliullah ◽  
Alfredo D. Martinez-Espinoza ◽  
Bochra Bahri ◽  
Emran Ali
Keyword(s):  
Pcr Primers ◽  
The United States ◽  
Fungal Species ◽  
Dollar Spot ◽  
Direct Pcr ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Pure Cultures ◽  
Speed Up ◽  
Cleaved Amplified Polymorphic Sequences

Dollar spot is one of the most destructive diseases in turfgrass. The causal agents belong to the genus Clarireedia, which are known for causing necrotic, sunken spots in turfgrass that coalesce into large damaged areas. In low tolerance settings like turfgrass, it is of vital importance to rapidly detect and identify the pathogens. There are a few methods available to identify the genus Clarireedia, but none of those are rapid enough and characterize down to the species level. This study produced a co-dominant cleaved amplified polymorphic sequences (CAPS) test that differentiates between C. jacksonii and C. monteithiana, the two species that cause dollar spot disease within the United States. The calmodulin gene (CaM) was targeted to generate Clarireedia spp. specific PCR primers. The CAPS assay was optimized and tested for specificity and sensitivity using DNA extracted from pure cultures of two Clarireedia spp. and other closely related fungal species. The results showed that the newly developed primer set could amplify both species and was highly sensitive as it detected DNA concentrations as low as 0.005 ng/µL. The assay was further validated using direct PCR to speed up the diagnosis process. This drastically reduces the time needed to identify the dollar spot pathogens. The resulting assay could be used throughout turfgrass settings for a rapid and precise identification method in the US.

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.

scholarly journals The pCS20 PCR assay for Ehrlichia ruminantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the United States of America

2004 ◽  
Vol 71 (2) ◽  
Author(s):  
S.M. Mahan ◽  
B.H. Simbi ◽  
M.J. Burridge
Keyword(s):  
United States ◽  
High Specificity ◽  
Pcr Primers ◽  
The United States ◽  
Rrna Gene ◽  
The Novel ◽  
Pcr Assay ◽  
Ehrlichia Ruminantium ◽  
Cowdria Ruminantium ◽  
Specificity And Sensitivity

White-tailed deer are susceptible to heartwater (Ehrlichia [Cowdria] ruminantium infection) and are likely to suffer high mortality if the disease spreads to the United States. It is vital, therefore, to validate a highly specific and sensitive detection method for E. ruminantium infection that can be reliably used in testing white-tailed deer, which are reservoirs of antigenically or genetically related agents such as Ehrlichia chaffeensis, Anaplasma (Ehrlichia) phagocytophilum (HGE agent) and Ehrlichia ewingii. Recently, a novel but as yet unnamed ehrlichial species, the white-tailed deer ehrlichia (WTDE), has been discovered in deer populations in the United States. Although the significance of WTDE as a pathogen is unknown at present, it can be distinguished from other Ehrlichia spp. based on 16S rRNA gene sequence analysis. In this study it was differentiated from E. ruminantium by the use of the pCS20 PCR assay which has high specificity and sensitivity for the detection of E. ruminantium. This assay did not amplify DNA from the WTDE DNA samples isolated from deer resident in Florida, Georgia and Missouri, but amplified the specific 279 bp fragment from E. ruminantium DNA. The specificity of the pCS20 PCR assay for E. ruminantium was confirmed by Southern hybridization. Similarly, the 16S PCR primers (nested) that amplify a specific 405-412 bp fragment from the WTDE DNA samples, did not amplify any product from E. ruminantium DNA. This result demonstrates that it would be possible to differentiate between E. ruminantium and the novel WTDE agent found in white tailed deer by applying the two respective PCR assays followed by Southern hybridizations. Since the pCS20 PCR assay also does not amplify any DNA products from E. chaffeensis or Ehrlichia canis DNA, it is therefore the method of choice for the detection of E. ruminantium in these deer and other animal hosts.

scholarly journals Antifungal Activity of Western Australian Soil Actinomycetes against Phytophthora and Pythium Species and a Mycorrhizal Fungus, Laccaria laccata

1983 ◽  
Vol 36 (2) ◽  
pp. 191 ◽  
Author(s):  
D Keast ◽  
C Tonkin
Keyword(s):  
Antifungal Activity ◽  
Phytophthora Cinnamomi ◽  
Mycorrhizal Fungus ◽  
Organic Matter Content ◽  
Phytophthora Species ◽  
Matter Content ◽  
Soil Organic Matter Content ◽  
Soil Actinomycetes ◽  
High Degree

Soil pH, soil moisture content and soil organic matter content did not appear to influence significantly the total numbers of actinomycetes isolated from sample sites in Western Australia. However, seasonal influences exist with summer conditions leading to higher spore isolation. Substantial but non-specific antifungal activity against Phytophthora cinnamomi, P. cryptogea, P. nicotiana, Pythium proli/erum and L. laccata was detected in vitro from many of the 2367 actinomycetes isolated. Antifungal activity mayor may not occur in members of the same actinomycete group, suggesting segregation of antifungal capacity within all groups. A limited number of actinomycete groups was isolated from the rhizosphere of plants and these exhibited similar properties to their counterparts in soil or litter. Actinomycetes isolated from the rhizosphere of Pinus radiata produced a high degree of in vitro antifungal activity against the Phytophthora species but, in general, actinomycetes isolated from root surfaces exhibited antibiosis against all the fungi tested. More actinomycetes showed antifungal activity from soils where P. cinnamomi was causing dieback of jarrah and other understorey species.

Temperature-Growth Relations and Genetic Diversity of A2 Mating-Type Isolates of Phytophthora cinnamomi in Australia

1974 ◽  
Vol 22 (2) ◽  
pp. 231 ◽  
Author(s):  
CJ Shepherd ◽  
BH Pratt
Keyword(s):  
Growth Rate ◽  
First Generation ◽  
Phytophthora Cinnamomi ◽  
Phytophthora Species ◽  
Temperature Growth ◽  
Field Isolates ◽  
Place Of Origin ◽  
Successive Generations ◽  
Cardinal Temperatures ◽  
Range Of Values

Determinations of cardinal temperatures for growth on various media of 50 Australian isolates of Phytophthova cinnamomi showed that growth did not occur outside the range 5-35°C. The range of temperatures at which growth optima occurred varied according to the isolate and medium used and encompassed the whole range of values reported by overseas authors. Growth rates of 361 isolates on corn meal agar at 25°C varied within the range 4.7-10.5 mm/day. There was no correlation between optimum temperature and whether isolates were slow- or fastgrowing or their place of origin. Fast-growing isolates (6-11 mm/day) were obtained from all States, but slower-growing isolates (<6 mm/day) were obtained only from southern and western regions of Australia. Populations from different regions of Australia exhibited different growth rate parameters. The variability of mycelial isolates in culture was studied by examining differences in growth rate among replicated parent, single-zoospore, single-zoosporangium and single terminal-hyphal isolates. Extensive variation was found among first generation single-zoospore progenies of field isolates, with lesser variation among progeny of single zoosporangia, terminal hyphal cultures and second and third generation zoospore derivatives. The origin of this variation is discussed and it is suggested that field isolates are heterokaryotic, since zoospores proved to be predominantly uninucleate. When various Phytophthora species were incubated at temperatures above those at which growth was possible and then returned to 25°C, their subsequent ability to resume growth depended on the particular time-temperature combination used. Considerable variation of response was found among a number of isolates of P. cinnamomi and, following the establishment of single zoospore isolates, the potential variability of field isolates was shown to persist through successive generations of zoospore propagation. It is suggested that a cytoplasmic mechanism of inheritance may be responsible for this variation.

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.

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