Infestans DNA Research Articles

Integrated Sensing Chip for Ultrasensitive Label-Free Detection of the Products of Loop-Mediated Isothermal Amplification.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that has been widely used for the detection of pathogens in many organisms. Current LAMP-based sensors usually require the LAMP products to be labeled in order for them to be detected. Here, we present a novel label-free LAMP chip, which consists of a nanopore thin-film sensor embedded inside a LAMP reaction chamber. A fraction of LAMP primers is immobilized on the sensor surface, allowing the LAMP products to be synthesized and bound to the sensor surface via immobilized primers. After the LAMP reaction components are removed from the reaction chamber, the amplified LAMP products bound to the sensor surface give rise to significantly increased transducing signals, which can be measured by a portable optical spectrometer through an optical fiber probe. As a demonstration, we used the LAMP chip to detect the causal agent of late blight, Phytophthora infestans, which is one of the most devastating plant pathogens and poses a major threat to sustainable crop production worldwide. We show that this chip can detect as low as 1 fg/μL of P. infestans DNA in 30 min, which corresponds to an attomolar level of 1.6 × 10-6 attomole/μL and is at least 10 times more sensitive than the currently available methods. This label-free sensing technology holds great promise to open up a new avenue for ultrasensitive, highly specific, rapid, and cost-effective point-of-care diagnostics of plant, animal, human, and foodborne pathogens.

Phenotypic and Genotypic Analysis of the Population of Phytophthora infestans in Bangladesh Between 2014 and 2019

In Bangladesh, the third largest producer of potatoes in Asia, late blight, caused by Phytophthora infestans, is the major constraint to production. Nevertheless, there is a lack of published information on the pathogen population. A collection of 69 isolates obtained from samples of infected potato foliage collected in 2018–2019 varied significantly in their aggressiveness to detached potato leaflets and tuber slices of cv. Diamant. On leaflets, most isolates were highly aggressive, colonizing more than half the surface in 7 days. On tuber slices, while some isolates were highly aggressive, those collected in 2019 were less so. There was a significant effect of the locations from which the isolates were obtained on aggressiveness to both leaflets and tuber slices, but aggressiveness to leaflets and to tuber slices was not significantly correlated. All isolates were A2 mating type and either intermediate (38%) or resistant (62%) in sensitivity to the fungicide metalaxyl. A subset of 24 isolates all had mitochondrial DNA haplotype Ia.Samples of P. infestans DNA (124) from infected potato foliage obtained from Bangladesh (2014–2019) were genotyped by 12-plex SSR. The vast majority (95%) were assigned to EU_13_A2, which was present in every year in which samples were collected. Numerous EU_13_A2 variants were identified, many specific to Bangladesh. Six samples (from 2017 to 2019) had genotypes distinct from EU_13_A2 and with no close match to known European lineages. It is concluded that the current P. infestans population of Bangladesh is dominated by the aggressive lineage EU_13_A2, also dominant in India and Pakistan. The implications of this for control of late blight are discussed.

Rapid Extraction of Plant Nucleic Acids by Microneedle Patch for In-Field Detection of Plant Pathogens.

Plant diseases pose a significant threat to global food security. Molecular diagnosis currently plays a crucial role in mitigating the negative impacts of plant diseases by accurately identifying the disease-causing pathogens and revealing their genotypes. However, current molecular assays are constrained to the laboratory because of the cumbersome protocols involved in plant nucleic acid extraction. To streamline this, we have developed a polymeric microneedle (MN) patch-based nucleic acid extraction method, which can be applied to various plant tissues and easily performed in field settings without using bulky laboratory equipment. The MN patch instantly isolates both host and pathogen's DNA and RNA from plant leaves by two simple steps: press and rinse with a buffer solution or nuclease-free water. The MN-extracted DNA and RNA are purification-free and directly applicable to downstream molecular assays such as polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Here, we describe the fabrication procedures of the MN patch and demonstrate the application of the MN method by extracting Phytophthora infestans DNA and tomato spotted wilt virus (TSWV) RNA from infected tomato leaves. After MN extraction, we directly utilize the MN-extracted nucleic acid samples to run PCR, RT-PCR, LAMP, or RT-LAMP reactions to amplify various biomarker genes, such as the ribulose-bisphosphate carboxylase (rbcL) gene of host tomato DNA, internal transcribed spacer (ITS) region of P. infestans DNA, and nucleocapsid (N) gene of TSWV RNA. Furthermore, this simple and rapid nucleic acid method can be integrated with portable nucleic acid amplification platforms such as smartphone-based microscopy devices to achieve "sample-to-answer" detection of plant pathogens directly in the field.

Assessing the Use of DNA Detection Platforms Combined with Passive Wind-Powered Spore Traps for Early Surveillance of Potato and Tomato Late Blight in Canada.

Quantitative PCR (qPCR), loop-mediated amplification (LAMP), and lateral flow strip-based recombinase polymerase amplification (RPA-LFS) assays were assessed for early detection of Phytophthora infestans, the global causal agent of potato and tomato late blight, on passive wind-powered spore traps known as Spornados. Spore traps were deployed in potato and tomato fields during the 2018, 2019, and 2020 growing seasons in the provinces of Alberta, British Columbia, Manitoba, Prince Edward Island, and Ontario. All assays used DNA extracts from Spornado cassette membranes targeting the P. infestans nuclear ribosomal internal transcribed spacer. A total of 1,003 Spornado samples were qPCR tested, yielding 115 positive samples for P. infestans spores. In further assessment of these samples, LAMP detected P. infestans in 108 (93.9%) of 115 qPCR positive samples, and RPA-LFS detected it in 103 (89.6%). None of the assays showed cross-reaction with other Phytophthora species or pathogenic fungi known to infect potato and tomato. The qPCR detected ≤1 fg of P. infestans DNA, and LAMP and RPA-LFS amplified 10 fg in as little as 10 min. All assays detected P. infestans before the first report of late blight symptoms in commercial potato or tomato fields within each region or province. The combination of Spornado passive samplers with qPCR, LAMP, or RPA-LFS proved a valuable spore trapping system for early surveillance of late blight in potato and tomato. Both LAMP and RPA-LFS showed potential as alternative approaches to qPCR for in-field monitoring of P. infestans.

Quantification of Phytophthora infestans population densities and their changes in potato field soil using real-time PCR

Tuber infection of Phytophthora infestans often occurs at harvest. However, it is difficult to accurately estimate the population densities of P. infestans in soil, especially Japanese soil. In the present study, P. infestans DNA was extracted from soil samples using a modified CTAB-bead method and quantified using real-time PCR to accurately, rapidly and easily estimate the P. infestans population densities in upland soils in Japan. P. infestans was well quantified in eleven types of soil samples, including nine types of upland soils in Japan, that were artificially inoculated with a zoosporangia suspension. The amounts of P. infestans DNA estimated by the real-time PCR were proportional to the inoculum densities. In the non-controlled experimental potato field, P. infestans population densities in soil corresponded to the development of symptoms and were correlated with the number of lesions on the potato foliage. These results imply that the proposed real-time PCR assay is suitable for the estimation or monitoring of P. infestans population densities in upland soils in Japan. The population densities at the ridge bottoms were larger than those at any other location in commercial potato fields. These results were similar to those of a previous report using a bioassay. Moreover, a correlation between DNA quantity and inoculum potential was observed. In conclusion, the real-time PCR assay developed in this study is suitable for indirect estimation of the inoculum potential of P. infestans.

Peptide-Based Identification of Phytophthora Isolates and Phytophthora Detection in Planta.

Phytophthora is arguably one of the most damaging genera of plant pathogens. This pathogen is well suited to transmission via the international plant trade, and globalization has been promoting its spread since the 19th century. Early detection is essential for reducing its economic and ecological impact. Here, a shotgun proteomics approach was utilized for Phytophthora analysis. The collection of 37 Phytophthora isolates representing 12 different species was screened for species-specific peptide patterns. Next, Phytophthora proteins were detected in planta, employing model plants Solanum tuberosum and Hordeum vulgare. Although the evolutionarily conserved sequences represented more than 10% of the host proteome and limited the pathogen detection, the comparison between qPCR and protein data highlighted more than 300 protein markers, which correlated positively with the amount of P. infestans DNA. Finally, the analysis of P. palmivora response in barley revealed significant alterations in plant metabolism. These changes included enzymes of cell wall metabolism, ROS production, and proteins involved in trafficking. The observed root-specific attenuation in stress–response mechanisms, including the biosynthesis of jasmonates, ethylene and polyamines, and an accumulation of serotonin, provided the first insight into molecular mechanisms behind this particular biotic interaction.

Rapid detection of potato late blight using a loop-mediated isothermal amplification assay

Potato late blight caused by Phytophthora infestans is one of the most destructive plant diseases that threaten global food security. Early and effective diagnosis of P. infestans is required before disease management decisions are made. Here, we developed a quick protocol to detect P. infestans based on a loop-mediated isothermal amplification (LAMP) assay. The P. infestansspecific multiple copy DNA sequences (PiSMC), a transposon-like element, provides an ideal target for molecular detection of this pathogen. We designed highly specific and sensitive primers allowing effective LAMP detection of the pathogen at 64°C in 70 min. In the validation assay, all 15 P. infestans isolates collected from China, Europe and South America could be positively detected, but results of the other 9 Phytophthora species infecting different plants, fungal and bacterial plant pathogens tested were negative. The detection limit of this assay is 1 pg P. infestans DNA. Moreover, the LAMP-PiSMC assay is able to detect P. infestans from infected leaves, tubers and soil. Taken together, this study reports the development of a specific and sensitive LAMP-PiSMC assay for early diagnosis of potato late blight.

DNA Extraction from Plant Leaves Using a Microneedle Patch.

Isolation of high-quality DNA from infected plant specimens is an essential step for the molecular detection of plant pathogens. However, DNA isolation from plant cells surrounded by rigid polysaccharide cell walls involves complicated steps and requires benchtop laboratory equipment. As a result, plant DNA extraction is currently confined to well-equipped laboratories and sample preparation has become one of the major hurdles for on-site molecular detection of plant pathogens. To overcome this hurdle, a simple DNA extraction method from plant leaf tissues has been developed. A microneedle (MN) patch made of polyvinyl alcohol (PVA) can isolate plant or pathogenic DNA from different plant species within a minute. During DNA extraction, the polymeric MN patch penetrates into plant leaf tissues and breaks rigid plant cell walls to isolate intracellular DNA. The extracted DNA is polymerase chain reaction (PCR) amplifiable without additional purification. This minimally invasive method has successfully extracted Phytophthora infestans DNA from infected tomato leaves. Moreover, the MN patch could be used to isolate DNA from other plant pathogens directly in the field. Thus, it has great potential to become a rapid, on-site sample preparation technique for plant pathogen detection. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Microneedle patch-based DNA extraction Support Protocol 1: Microneedle patch fabrication Support Protocol 2: Real-time PCR amplification of microneedle patch extracted DNA.

Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans.

To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA. Two sets of loop-mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross-reacted with the closely related species Phytophthora nicotianae. Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross-react with P. nicotianae. The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis and P. phaseoli. Cross-reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions. Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity. This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.

Development of a quantitative real‐time PCR assay for Phytophthora infestans and its applicability to leaf, tuber and soil samples

A sensitive real‐time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non‐potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogens P. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestans oospore detection are explored and tested experimentally.

Development of a species‐specific and sensitive detection assay for Phytophthora infestans and its application for monitoring of inoculum in tubers and soil

A specific and sensitive PCR assay for the detection of Phytophthora infestans, the cause of late blight of potato, in soil and plant tissues was developed. A P. infestans‐specific primer pair (INF FW2 and INF REV) was designed by comparing the aligned sequences of rDNA internal transcribed spacer regions of most of the known Phytophthora species. PCR amplification of P. infestans DNA with primers INF FW2 and INF REV generated a 613 bp product, and species specificity was demonstrated against DNA from nine other Phytophthora species and seven potato‐blemish pathogens. In a single‐round PCR assay, 0·5 pg pure P. infestans DNA was detectable. Sensitivity was increased to 5 fg DNA in a nested PCR assay using Peronsporales‐specific‐primers in the first round. As few as two sporangia or four zoospores of P. infestans could be detected using the nested assay. Procedures are described for detection of P. infestans in leaves, stem and seed potato tubers before expression of symptoms. A soil assay in which 10 oospores per 0·5 g soil were detectable was developed and validated using samples of field soil. The PCR assay was used to examine the long‐term survival of sexual (oospores) and asexual (sporangia and mycelium) inoculum of P. infestans in leaf material buried in a replicated experiment under natural field conditions. Oospores were consistently detected using the PCR assay up to 24 months (total length of the study) after burial in soil, whereas the sporangial inoculum was detected for only 12 months after burial. Sporangial inoculum was shown to be nonviable using a baiting assay, whereas leaf material containing oospores remained viable up to 24 months after burial.

Enhanced Polymerase Chain Reaction Methods for Detecting and Quantifying Phytophthora infestans in Plants

ABSTRACT Sensitive and specific primer sets for polymerase chain reaction (PCR) for Phytophthora infestans, the oomycete that causes late blight of potato and tomato, were developed based on families of highly repeated DNA. The performance of these primers was compared to those developed previously for the internal transcribed spacer (ITS) of ribosomal DNA. The detection limit using the new primers is 10 fg of P. infestans DNA, or 0.02 nuclei. This is about 100 times more sensitive than ITS-directed primers. Nested polymerase chain reaction (PCR) allows the measurement of down to 0.1 fg of DNA using the new primers. To enhance the reliability of diagnostic assays, an internal positive control was developed using an amplification mimic. The mimic also served as a competitor for quantitative PCR, which was used to assess the growth of P. infestans in resistant and susceptible tomato. A key dimension of this study was that two laboratories independently checked the specificity and sensitivity of each primer set; differences were noted that should be considered when PCR is adopted for diagnostic applications in any system.

A linear RNA replicon from the oomycete Phytophthora infestans.

An unusual RNA element was discovered in an isolate of the oomyceteous fungus Phytophthora infestans. The RNA exists predominantly as single-stranded molecules of about 625 nucleotides with complementary strands present at a ratio of approximately 130:1. Gel mobility and PCR assays indicated that the element was linear. The RNA appeared to be an autonomous element, since P. infestans DNA did not contain cross-hybridizing sequences. Standard methods for virus purification yielded no evidence for encapsidation of the RNA, or for other virus particles in the isolate bearing the replicon. The replicon contained polyU and polyA tracts at its 5' and 3' termini, respectively, with a central region that had a GC content of 47%, and lacked obvious ORFs. Two-thirds of the replicon co-purified with nuclei, at about 200 copies per nucleus, while one-third resided in a cytoplasmic but non-mitochondrial location. Maternal inheritance was observed in sexual crosses, with a few exceptions. The replicon was not widely distributed throughout the species and had little effect on growth or pathogenicity. The data suggest that the RNA is best characterized as a novel linear RNA plasmid.

Families of repeated DNA in the oomycete Phytophthora infestans and their distribution within the genus.

The abundance, genomic organization, species distribution, and structure of 33 distinct families of repetitive DNA in Phytophthora are described. The families were identified by screening a library of Phytophthora infestans DNA for repetitive sequences. These was subsequently characterized within 26 species distributed within each of the six taxonomic groups traditionally defined within the genus. Some repeat elements were specific to P. infestans and its close relative, Phytophthora mirabilis, while other repeated sequences were present in most species. The distribution of the DNA families did not conform to the traditional taxonomic groups used for the genus. Characterization of the repeated sequences in P. infestans indicated that they included both dispersed and tandemly repeated elements, with copy numbers ranging from 70 to 8400 per haploid genome. In total, these repeats were estimated to represent 51% of the nuclear genome of P. infestans. Reverse transcriptase motifs were detected in seven of the repeat families that were widely distributed throughout the genus.

Isolation of putative pathogenicity genes of the potato late blight fungus Phytophthora infestans by differential hybridization of a genomic library

Plant pathogens produce pathogenicity factors which enable them to parasitize and colonize their host. A strategy for identifying pathogenicity factors involves the isolation and characterization of genes encoding these factors. Potential pathogenicity genes are genes whose expression is induced during pathogenesis. In order to isolate such genes of the late blight Fungus Phytophthora infestans , a genomic library of P. infestans DNA was differentially hybridized using labelled first strand cDNA probes synthesized on mRNA isolated from P. infestans infected potato leaves and on mRNA isolated from the fungus grown on a basic medium in culture. In total, nine distinct in planta induced ( ipi) genes were selected. Expression studies revealed that the mRNA levels of seven of these genes, ipiA, ipi C, ipiD, ipiJ1, ipiJ2, ipiN and ipi Q increased 5-10 fold during colonization. The two other genes, ipi B and ipiO, showed a transient expression pattern with the highest mRNA levels in the early stages of infection.