Next Article in Journal
Effects of Genotype and Culture Conditions on Microspore Embryogenesis and Plant Regeneration in Brassica Rapa ssp. Rapa L.
Next Article in Special Issue
Phylogenetic Analysis and Development of Molecular Tool for Detection of Diaporthe citri Causing Melanose Disease of Citrus
Previous Article in Journal
Molecular Basis of Root Nodule Symbiosis between Bradyrhizobium and ‘Crack-Entry’ Legume Groundnut (Arachis hypogaea L.)
Previous Article in Special Issue
Uncovering the Host Range for Maize Pathogen Magnaporthiopsis maydis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 9
Issue 2
10.3390/plants9020277
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

A PCR, qPCR, and LAMP Toolkit for the Detection of the Wheat Blast Pathogen in Seeds

by
Maud Thierry
1,2,3,†,
Axel Chatet
1,†,
Elisabeth Fournier
2,
Didier Tharreau
2,3 and
Renaud Ioos
1,*
1
ANSES Plant Health Laboratory, Mycology Unit, Domaine de Pixérécourt, Bâtiment E, F-54220 Malzeville, France
2
UMR BGPI, Montpellier University, INRAE, CIRAD, Montpellier SupAgro, 34398 Montpellier, France
3
CIRAD, UMR BGPI, F-34398 Montpellier, France
*
Author to whom correspondence should be addressed.
†
These authors contribute equally to this work.
Plants 2020, 9(2), 277; https://doi.org/10.3390/plants9020277
Submission received: 7 February 2020 / Revised: 17 February 2020 / Accepted: 18 February 2020 / Published: 21 February 2020
(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)
Versions Notes

Abstract

:
Wheat blast is a devastating disease caused by the pathogenic fungus Pyricularia oryzae. Wheat blast first emerged in South America before more recently reaching Bangladesh. Even though the pathogen can spread locally by air-dispersed spores, long-distance spread is likely to occur via infected wheat seed or grain. Wheat blast epidemics are caused by a genetic lineage of the fungus, called the Triticum lineage, only differing from the other P. oryzae lineages by less than 1% genetic divergence. In order to prevent further spread of this pathogen to other wheat-growing areas in the world, sensitive and specific detection tools are needed to test for contamination of traded seed lots by the P. oryzae Triticum lineage. In this study, we adopted a comparative genomics approach to identify new loci specific to the P. oryzae Triticum lineage and used them to design a set of new markers that can be used in conventional polymerase chain reaction (PCR), real-time PCR, or loop-mediated isothermal amplification (LAMP) for the detection of the pathogen, with improved inclusivity and specificity compared to currently available tests. A preliminary biological enrichment step of the seeds was shown to improve the sensitivity of the tests, which enabled the detection of the target at an infection rate as low as 0.25%. Combined with others, this new toolkit may be particularly beneficial in preventing the trade of contaminated seeds and in limiting the spread of the disease.

1. Introduction

Wheat blast is an emerging disease threatening global wheat production and thereby global food safety. The major agricultural impact of the disease and its rapid propagation require a robust tool for reliable and timely identification of the pathogen in order to prevent its spread and avoid further exchange of contaminated biological material at a global scale.
The pathogen responsible for wheat blast (Pyricularia oryzae Triticum lineage, synonym Magnaporthe oryzae) is capable of infecting all aerial parts of the wheat plant, but spike infection is the most common symptom observed in the field [1,2,3]. Symptoms of wheat blast include necrotic lesions on leaves, stems, grains, and partial or total bleaching of the spikes leading to sterility or empty grains. Wheat blast disease reduces grain yield and grain quality [2,4]. During major epidemics, it has caused up to 100% yield loss [5]. The disease could potentially have a significant economic impact since cereals account for about 40% of the world’s agricultural yield, with wheat coming second in 2017 after maize and before rice, and reaching more than 750 M t produced and 500 M t consumed annually by humans [6].
This devastating disease first emerged in 1985 in the state of Paraná in Brazil [7]. The disease then spread to the neighboring federal states of São Paulo and Mato Grosso do Sul in 1986, and Rio Grande do Sul in 1987, causing a decrease of 95% in the wheat crop yield of the Cerrado ecoregion [8,9,10,11]. The pathogen subsequently spread to eastern Bolivia in 1996, eastern Paraguay in 2002, and northern Argentina in 2007 [5]. It then broke out in Bangladesh in 2016 [12,13].
P. oryzae spores are mainly dispersed on short distance by the wind [14]. However, the transport of contaminated seed or grains facilitates the spread of the fungus over long distances [15]. Comparative genomic studies demonstrated that P. oryzae isolates collected from wheat in different parts of Bangladesh and isolates causing epidemics in Brazil were strongly genetically related [12,13]. The transport of grains contaminated with P. oryzae, harvested in the wheat blast epidemic zone in Brazil, likely caused the recent emergence of this pathogen in Bangladesh in 2016 [12].
P. oryzae is responsible for blast disease on numerous Poaceae species [16,17]. Phylogenetic analyses of 81 genomes of P. oryzae isolates sampled from 12 different genera of Poaceae revealed multiple divergent lineages within P. oryzae, each of which was mostly associated with one specific host plant genus [18]. The existence of these host-specific lineages revealed incipient speciation, following host jump or host range expansion of the pathogen. However, the genetic divergence (number of differences per kilobase) observed between host-specific lineages was less than 1% on the entire genome—by comparison, the genetic divergence between different species of Pyricularia (P. grisea, P. oryzae, and P. pennisetigena) is greater than 10%—and gene flow was detected between host-specific lineages [18]. P. oryzae therefore represents a single species grouping different host-specific lineages.
The majority of isolates sampled from infected wheat cluster within one of these host-specific lineages, the Triticum lineage. However, P. oryzae isolates are sometimes able to opportunistically infect a host plant different from their original host, but this causes far fewer symptoms. Several studies have recorded opportunistic infection of wheat caused by isolates belonging to the Lolium lineage of P. oryzae, which is genetically the closest host-specific lineage to the Triticum lineage. These isolates, however, were weakly aggressive on wheat during artificial inoculation tests and do not appear to be capable of causing major epidemics on this host [3,19]. The Triticum lineage of P. oryzae is therefore considered responsible for wheat blast epidemics.
Accurate and rapid methods for detecting wheat blast isolates are required to limit or prevent the spread of the pathogen in disease-free areas [5]. Misidentification of the pathogen could lead to drastic unnecessary measures, such as unjustified destruction of seeds or healthy biological material. On the other hand, a false-negative result could result in introduction of the pathogen to a new geographical area, increasing the risk of wheat blast outbreak.
However, intra-specific detection is challenging, since gene flow likely occurs regularly among lineages or subpopulations belonging to the same species. In the specific case of wheat blast, the detection method must be able to discriminate isolates responsible for wheat blast epidemics (belonging to the Triticum lineage) from isolates belonging to the other host-specific lineages of the species, but which may be capable of causing opportunistic infections on wheat plants. Host-specific lineages have identical morphology in pure culture, which does not enable the observer to differentiate them visually [20]. Pathotyping tests can be used for diagnosis but they are time-consuming and cannot identify opportunistic infections unambiguously. DNA-based detection tests are a good alternative because they allow detection at very precise taxonomic levels, provided, however, that they can identify a specific polymorphism in the targeted taxon. In the case of wheat blast, the identification of such polymorphisms is made difficult by the low genetic divergence and the gene flow between host-specific lineages [18].
Currently, several DNA-based diagnostic tests have been developed for the detection of wheat blast isolates [3,20,21]. All these tests are highly, but not perfectly, inclusive (i.e., detecting all wheat-blast strains) and/or specific (not detecting non-wheat blast isolates): none of them allows for optimal detection of the pathogen. Pieck et al. [3] and Yasuhara-Bell et al. [21] developed polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), and loop-mediated isothermal amplification (LAMP) diagnostic tests targeting the same genomic region, the MoT3 locus. This region was selected because it is highly conserved in wheat blast isolates and absent from most non-wheat blast isolates. However, some wheat blast isolates, such as the BR0032 isolate, do not include the MoT3 locus and remained undetectable using these tests. Thierry et al. [20] developed the C17 qPCR test targeting a different genomic region. This test allowed for the detection of all wheat blast isolates tested so far. However, some isolates that are not pathogenic on wheat were also detected with this test, leading to 4% false-positive results.
The objectives of this work were (i) to design primers targeting new genomic regions in order to identify polymorphisms fully specific to the Triticum lineage; (ii) to develop a toolkit of detection tests using multiple DNA amplification techniques (PCR, qPCR, and LAMP) to be suitable for any type of analysis and improving current wheat blast detection; and (iii) to verify the ability of these tests to detect the pathogen on artificially contaminated wheat grains.

2. Results

2.1. Primers Screening

2.1.1. PCR Primers

Here, 49 designed primer pairs were first screened using conventional PCR on a small DNA panel composed of five wheat-borne isolates and ten non–wheat-borne isolates. Among them, 12 primer pairs displayed full inclusivity (i.e., positive result for all wheat-borne pathogenic isolates) and specificity (no detection of non-wheat borne pathogenic isolates; Figure S1). Four of these (C45, C74, C82, and C92) were selected for further analysis because they were designed on different regions/scaffolds of the reference genome BR0032 (scaffolds 15, 17, and 44).

2.1.2. LAMP Primers

Five groups of primers were designed for a LAMP isothermal amplification. The inclusivity and specificity of the five groups of LAMP primers designed were tested on a small panel composed of three wheat-borne isolates and four non-wheat-borne isolates (Figure S2). None of the five groups of primers tested displayed full specificity and inclusivity. Two groups of primers did not amplify any of the DNA (groups 2 and 3) and two groups amplified all DNA extracts tested (groups 1 and 4). Group 5 was finally selected for further analysis because primers of this group made it possible to amplify every wheat-borne isolate in a very short time (around three minutes), even though full specificity of this primer group could not be achieved.

2.2. Primer Specificity and Inclusivity Assessment Using a Large DNA Panel

The inclusivity and specificity of the primers previously selected were assessed using a larger DNA panel of 113 or 185 strains (Table 1).

2.2.1. PCR and qPCR Primers

The inclusivity and specificity of the C45, C74, C82, and C92 primer pairs were first assessed by conventional PCR (Table 1). The C45 primers displayed the highest inclusivity and specificity and were further assessed with additional DNAs (see Table 1) by conventional PCR, and by real-time PCR combing a hydrolysis probe (Table 1).
The C45 primers were designed in the scaffold 15 of the reference genome BR0032 and targeted a single SNP positioned at the 3’ end of the forward primer. PCR and qPCR tests using these primers and probe showed 97% inclusivity. All wheat-borne isolates but one (isolate AG0102) were amplified. To validate AG0102 host spectrum, a pathogenicity test was done by inoculating a spore suspension of AG0102 on the leaves of the susceptible wheat cultivar Thésée. No symptoms were observed, questioning the virulence of this isolate on wheat. Furthermore, no amplification of AG0102 DNA was observed using either the MoT3 test [3] or the C17 test [20] (Table 1).
C45 primers displayed full specificity by conventional PCR since no non-wheat-borne isolates of P. oryzae were amplified. However, late amplifications of DNA from non-wheat-borne isolates were observed when tested in real-time PCR. Additional qPCR repetitions with these DNA samples showed that these amplifications were not fully repeatable and only occurred erratically. Finally, qPCR testing using C45 primers allowed amplification of all wheat-borne isolates except AG0102 (Ct values between 23 and 28), but 10 out of 151 non–wheat-borne isolates were sometimes amplified with late Ct values (between 35 and 39). Specificity of the primers was also challenged with DNAs from other wheat infecting fungal species such as Fusarium graminearum, Fusarium poae or Alternaria tenuissima. None of these 19 DNAs were amplified using C45 primers either by conventional PCR or qPCR.

2.2.2. LAMP Primers

LAMP primer group 5 successfully amplified DNA from all wheat-borne isolates, except the AG0102 isolate, in a very short time (between 3:37 and 4:10 min). However, as expected, full specificity could not be achieved. Six isolates (AG0067, Cd88215, CR0023, CR0057, JP0031, and JP0033) out of 151 (4%) were amplified with Ct values equivalent to the values observed with wheat-borne isolates, making them indistinguishable. Three of these six were also detected using the C17 qPCR test [20]. Late but non-repeatable amplifications could also be observed using the group 5 LAMP primers after 9 to 25 min of reaction (Table 1).

2.3. Assay Sensitivity

C45 PCR and qPCR primers: The sensitivity of PCR and qPCR primers was assessed using serial 10-fold dilutions of genomic DNA of three P. oryzae wheat-borne isolates (BR0031, BL0063, BL0023) and 10-fold dilutions of plasmidic constructions integrating the sequences targeted by the primers. C45 PCR and qPCR primers succeeded in detecting as low as 5 pg of DNA/reaction or 12 plasmidic copies/reaction for genomic DNA and plasmidic DNA, respectively. This limit of detection was validated using 16 replicates for each DNA concentration. PCR reaction efficiency was measured using the plasmidic dilution to 92% and the high R2 value (R2 = 0.9969) demonstrated good correlation between initial plasmidic DNA quantity and the Ct values.
Group 5 LAMP primers are as follows: the sensitivity of group 5 LAMP primers was assessed by serial 10-fold dilutions of initially diluted genomic samples of two P. oryzae wheat-borne isolates (BR0031 and BL0023). LAMP primers were able to amplify down to 5 pg of genomic DNA per reaction. This limit of detection was validated using five replicates.

2.4. Pathogen Detection in Contaminated Seeds

We assessed the ability of the PCR (C45 primers), qPCR (C45 primers + C45 probe), and LAMP (group No 5 primers) tests to detect the pathogen in a seed matrix. Wheat seeds were artificially contaminated with a spore suspension of the pathogen. Individual lots of 400 seeds were prepared containing 0, 1, 2, 5, 10, 15, or 50 artificially contaminated seeds mixed with non-contaminated seeds. Total DNA was extracted from each lot after blending. Before the blending step, some lots were incubated for 72 h in potato dextrose broth (PDB) media to test whether this enrichment process improved pathogen detection.
Without the incubation step, the conventional PCR succeeded in amplifying all nine replicates in seed lots containing 15 contaminated seeds or more. The real-time PCR test was less sensitive since it succeeded in amplifying all nine replicates only in seed lots containing 50 contaminated seeds or more. LAMP tests, on the other hand, failed to amplify DNA for any of the seed lots tested.
The incubation step greatly improved detection in all tests. After 72 h of incubation, the PCR test, as well as the qPCR test, were able to detect the pathogen for all replicates in seed lots containing only one contaminated seed. The LAMP primers allowed for the detection of the pathogen after an enrichment step, for all replicates, in lots containing two or more contaminated seeds in less than five min (Table 2).
Additionally, no non-specific late amplifications were obtained on seed lots highly contaminated by the BR0079 isolate (sampled on Eleusine indica). Seed lots containing 20 and 50 seeds contaminated with BR0079 spores did not yield a PCR or qPCR signal even after an incubation period of 72 h (Table 3).

3. Discussion

In this study, we designed primers targeting polymorphisms located in yet unexploited genomic regions in order to find a method with greater specificity for the P. oryzae Triticum lineage over the currently existing tests [3,20,21]. Screening these primers highlighted the C45 pair. The specificity of this pair is based on a single substitution located at the 720791 position of scaffold 15 in the BR0032 reference genome [22]. The isolates belonging to the Triticum lineage display a “C” at this position, while this nucleotide is substituted by a “G” in other lineages. In our conditions, this single substitution allowed the C45 pair to amplify the DNA of all the isolates sampled on wheat included in this study, with the exception of isolate AG0102 whose pathogenicity on wheat was questionable on the basis of our experiments. In addition, no amplification of DNA from P. oryzae isolates sampled from other Poaceae was found using conventional PCR, which demonstrates a higher level of specificity compared to the assays already described in the literature.
The use of these primers supplemented with a hydrolysis fluorescent probe enabled the development of a qPCR PCR test. Real-time PCR has two major advantages compared to conventional PCR: (i) it enables quantification of the pathogen in the samples tested and (ii) it reduces the turnaround time for analysis and offers reaction in close tubes, thus reducing the risk of self-contamination. The C45 qPCR test showed a similar level of inclusivity as conventional PCR (all isolates from wheat were amplified with the exception of AG0102). However, late but non-repeatable amplifications were observed for ten non-target isolates, with Ct higher than 35.5. The specificity of the targeted polymorphism is not called into question by these results. Importantly, the strong shift of Ct values between amplification of isolates sampled from wheat and the non-specific amplifications clearly demonstrates a difference in their nucleotide sequence. One explanation would be that, over time, a partial degradation of the primers could suppress the single nucleotide carrying the specificity and lead to these late amplifications during the amplification cycle. Late amplification obtained using the C45 qPCR test should be validated using other previously developed tests (MoT3: [3], C17: [20]) or by sequencing the locus. Clearly, a combination of several tests makes it possible to validate the results because different genomic regions are targeted by these tests, leading to different patterns of amplification (i.e., different false-positive or false-negative results).
The single substitution targeted by C45 did not allow the development of fully specific LAMP primers. The LAMP primers (group No 5) targeted another genomic region located at the 42,8000 position of the BR0032 scaffold 15. This test detected all isolates sampled on wheat, except isolate AG0102, in a very short time (<4 min). However, specificity assessed on 185 DNA samples from different P. oryzae isolates showed several non-specific amplifications. Six isolates (AG0067, Cd88215, CR0023, CR0057, JP0031, and JP0033) led to amplifications at similar times as the target Triticum isolates, between three and four min. As observed by Thierry et al. [20], some polymorphisms highly specific of the Triticum lineage identified on the scaffold 15 of the BR0032 isolate can still be shared with some isolates sampled on other grasses, like isolates CR0023, CR0057, and JP0033 [20]. Later, 22 other non-specific amplifications were observed after eight min of amplification. As for qPCR, late amplifications may be yielded by LAMP with DNA of these isolates due to rare unspecific bindings of the primers. In order to eliminate these late non-specific amplifications, the LAMP PCR run can be stopped at eight min without impacting the sensitivity of the test. However, the six non-specific amplifications observed between three and four min will not be eliminated by these modifications. Nonetheless, positive results can be confirmed with the C45 PCR or qPCR tests, which do not amplify DNA from these non-target isolates. The LAMP group 5 test could be used as a quick pre-screening, providing a result within eight min. If the result is positive on all replicates, then confirmation by PCR or qPCR should be done.
Knowing the sensitivity of a test is essential to validate the method. The limit of detection was very low for the three tests. The C45 PCR and qPCR tests as well as the LAMP group 5 test were able to detect as little as 5 pg of DNA per PCR reaction. The sensitivity of the C45 PCR and qPCR tests was also evaluated using plasmid DNA. The limit of detection obtained for both tests was 12 plasmid copies of the target DNA per PCR tube.
Detection of the pathogen on wheat grains is essential to validate the health status of seeds before trade and export or import. However, amplification inhibitors may be present in seeds and impact the performance of detection tests [23]. An enrichment phase in fungal biomass is often used to overcome these barriers [23,24]. In our study, only the C45 PCR and qPCR tests enabled repeatable amplification without an enrichment phase. Surprisingly, the C45 PCR test showed better sensitivity when used in a seed matrix (detection of 15 contaminated seeds per 400-seed lot for all replicates) than the qPCR test (detection of 50 contaminated seeds per 400-seed lot). The addition of a simple enrichment phase consisting of the incubation of grains for 72 h in a rich medium greatly improved the sensitivity of all the tests. Both the C45 PCR and qPCR tests allowed for systematic detection of the pathogen in a batch of 400 seeds containing only one artificially contaminated seed, which represents a 0.25% infection rate. The LAMP group 5 test allowed for systematic detection of the pathogen in lots containing two contaminated seeds after the enrichment phase.
In order to use the detection tests developed in this study to test for the presence of P. oryzae isolates belonging to the Triticum lineage on wheat seeds, lots of 400 seeds should be incubated for 72 h in a culture medium (PDB) prior to testing. DNA extracted from these grains will then serve as a template for detection tests. The detection method used may be selected depending on the financial, material, time and labor resources available. However, we recommend to first use fully inclusive detection tests, which do not generate false negatives on the isolates tested to date (C17, C45 PCR, C45 qPCR, and LAMP group 5 tests). The conventional C45 PCR test, however, is the only one that displayed perfect specificity for the isolates tested. Given the high genetic proximity of the host-specific lineages as well as the potential gene flow between these lineages, all positive results must be validated by the use of another test to avoid costly and unjustified quarantine measures, such as the complimentary approach proposed for the deadly Fusarium oxysporum f. sp. cubense race 4 on banana trees [25].
This study provides a new toolkit for the efficient detection of wheat-blast causal pathogen in seed, and therefore contributes to limit the spread of this destructive disease. The different types of techniques proposed may be of great help for laboratories in charge of phytosanitary controls and will allow to adapt to different levels of equipment.

4. Materials and Methods

4.1. Biological Materials

4.1.1. P. Oryzae DNA

A total of 185 P. oryzae strains were screened to assess the specificity and the inclusivity of the diagnostic tests designed in this study. Among these P. oryzae isolates, 34 were sampled from field-infected wheat (Triticum sp.) and 151 were sampled from 28 different Poaceae genera. Isolates sampled from wheat are referred to as wheat-borne isolates in this article. Isolates were sampled in various countries, in order to cover the genetic diversity of the pathogen as much as possible and sent as DNA extracts to our lab. Included were 19 isolates belonging to nine other fungal species pathogenic on wheat (Table S1). All DNA concentrations were measured with a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA) and adjusted to 0.5 ng/µL by dilution with a Tris EDTA 1X buffer. The amplifiability of all DNA was verified by the FungiQuant real-time PCR assay, which targeted a conserved 351 bp region of the fungal 18S rRNA gene [26].

4.1.2. Positive Control Plasmids

Positive controls for the C45 PCR and qPCR detection tests were prepared by cloning the target amplicon in a plasmid. Plasmids are considered to be stable, homogeneous during pipetting, easily quantifiable, and producible in virtually unlimited quantities. The genomic region targeted by the C45 primer pair was amplified by PCR and the size of the amplicon checked on an electrophoresis gel. The amplicon was inserted into the pCR4-TOPO vector following the TOPO TA cloning kit protocol (Invitrogen) and used to transform TOPO10 chemically competent bacteria (Escherichia coli), according to the manufacturer’s instructions. After culturing the clones at 37 °C and selecting clones with fragment integration by qPCR, the plasmids were purified using a Nucleospin® plasmid kit (Macherey-Nagel, Düren, Germany). The plasmid solution was used as a positive control for all PCR and qPCR reactions using the c45 couple.

4.2. Wheat Seed Inoculation and DNA Extraction

The wheat-borne isolate BL0092 was cultured on potato dextrose agar (PDA) medium at 23 °C, under day-night alternation (12 h/12 h). After seven days of culture, fungal spores were collected by adding 5 mL of sterile water to the Petri dish and scratching the mycelium to harvest the spores. The resulting suspension was filtered to remove mycelium fragments and retain only the conidia. Finally, the concentration of the suspension was calibrated using a haemocytometer at 200 spores/µL (supplemented by two drops of Tween 20)) and used to artificially inoculate wheat seeds of the susceptible wheat variety Filon. These seeds were previously disinfected by dipping for 10 min in a 1.5% active chlorine solution. The inoculation was carried out by depositing 10 μL of spore solution on individual grains and drying them overnight in a sterile atmosphere at room temperature. Multiple lots of 400 seeds in total but containing different proportions of contaminated seeds were created (0, 1, 2, 5, 10, 15, 20, or 50 artificially contaminated seeds per lot). Fungal enrichment was carried out by incubating the 400-grain lots in a sterile Petri dish containing 15 mL of potato dextrose broth (PDB) media for 72 h at room temperature (23 °C). Grinding of incubated or non-incubated seed lots was carried out with 30 mL of PDB using a Microtron™ MB 550 Laboratory Mixer (Kinematica™) until a homogenous milky mixture was obtained. For each mixture, three samples of 500 μL each were collected using a truncated 1 mL cone and used for DNA extraction using a Nucleospin® Plant II extraction kit (Macherey-Nagel), following the manufacturer’s instructions. To serve as a negative control, the same spore inoculation protocol was performed with the BR0079 isolate sampled from Eleusine indica, and the DNA of two seed lots containing 20 and 50 inoculated seeds, respectively, were extracted after a fungal enrichment step.

4.3. Primer Design

PCR and qPCR: The primers and probes were designed to target Triticum-specific polymorphisms identified by comparison of 76 P. oryzae genomes, including 20 genomes assigned to the Triticum lineage. Here, we studied some of the polymorphisms highlighted but not exploited in the publication by Thierry et al. [20], as well as new polymorphisms further identified. Primers and probes for PCR and qPCR were designed using Geneious 11.1.2 software. The specific polymorphisms of the Triticum lineage were positioned at the 3′ end of the forward and reverse primers to maximize the specificity of the primers. The forward primer of the C45 pair was additionally purified by high performance liquid chromatography (HPLC) by the manufacturer to ensure the integrity of the primer, whose specificity is based on a single nucleotide located at the 3′ end of the forward primer. Primer sequences of the C45 test (PCR or qPCR) were C45-forward: 5′-TCTTTCACTCCTCCGAAAGAC-3′ and C45-reverse: 5′-GTATAGCTGGGTATCTTGGTAGAC-3′, whereas the combined hydrolysis probe in qPCR was C45-probe: 5′-FAM-TGCCCTCATCAAAACCTGCAGCCAT-BHQ1-3′.
LAMP: The four primers required for each LAMP reaction including the two external primers F3 and B3, and the two internal primers FIP (F1c + TTTT + F2) and BIP (B1c + TTTT + B2), were designed with the PrimerExplorer V5 online tool (https://primerexplorer.jp/e/). Sequences of the primers used for the group No.5 LAMP test were: No.5-F3: 5′-TGCGTGATCAACGAATGGC-3′; No.5-B3: 5′-CGGAAGCAAACTCTGCGATT-3′; No.5-FIP: 5′-GCAAGATGCCTACCGTGGGGTTTTCTGGGTTCCCCTCACCAT-3′; No.5-BIP: 5′-TCGTAAGGAGCATGAAGGGCTGTTTTGCGGACGGACATACGTAGT-3′.
All the primers used in this study were custom synthesized by Eurogentec. The list of all the candidate primers is available in Table S2.

4.4. Primer Screening

4.4.1. PCR Primers

All primer pairs designed were screened using a small DNA panel from 15 isolates including five isolates sampled from Triticum (BR0086, BR0036, AG0103, BL0017 and BL0093), three from Oryza (CH1120, FR0013 and BR0019), four from Lolium (AG0064, CHW, PL2-1 and CR0057), one from Eleusine (IN0113), one from Echinochloa (CR0023), and one from Eriochloa (JP0033). The PCR reactions for the screening were carried out in qPCR conditions, without a hydrolysis probe, using a LightCycler® 480 Probes Master (Roche), with the following reaction mixture: 1X Roche Kit qPCR premix, 0.3 μM of forward and reverse primers, 2 μL of template DNA (0.5 ng/μL), and ultrapure water for a final volume of 20 μL. The PCR reaction was carried out in a LightCycler® 480 II thermal cycler (Roche). The PCR cycle was (i) an initial denaturation step at 95 °C for 10 min; (ii) 40 denaturation cycles at 95 °C for 15 s and hybridization-synthesis at 62 °C for 55 s; and (iii) a final elongation at 62 °C for 10 min.

4.4.2. LAMP Primers

The small DNA panel used to screen LAMP primers was composed of seven DNA including the DNA of isolates sampled from Triticum (BR0036, BR0031 and BR0088), Eriochloa (JP0033), Bromus (AG0061), Eleusine (IN0113), and Lolium (CR0057). The reaction mix and amplification cycles used for this screening are described in the “PCR, qPCR, and LAMP amplifications” section below.

4.5. PCR, qPCR, and LAMP Amplifications

4.5.1. Conventional PCR

After screening primer pairs using a small subset of DNAs, inclusivity and specificity of the remaining primers were tested on all 185 strains with the following conditions. The conventional PCR mix included 1X polymerase buffer, 5 mM MgCl2, 0.25 mM of each dNTP, 0.025 U/µL of HGS Diamond Taq® DNA polymerase (Eurogentec, Seraing, Belgium), 0.3 µM of each forward and reverse primer, 2 µL of DNA matrix (0.5 ng/µL), and ultrapure water to reach a final 20 µL volume. The amplification cycle was 10 min of initial denaturation at 95 °C, followed by 40 cycles each composed of 30 s of denaturation at 95 °C, 30 s of hybridization at 65 °C and 45 s of extension at 72 °C, and a final extension at 65 °C for 10 min. PCR amplifications were visualized after a one-hour electrophoresis at 110 volts on a 1.5% agarose gel. Conventional PCR runs were carried out in a LightCycler® 480 II thermal cycler (Roche).

4.5.2. Real-Time PCR

The qPCR reactions were carried out on a RotorGene 4.4.1 (Qiagen, Hilden, Germany) using the following reaction mix: 1X No-ROX mastermix (Eurogentec), 0.3 µM of forward and reverse primers, 0.1 µM of hydrolysis fluorescent probe, 2 µL DNA matrix (0.5 ng/µL), and ultrapure water to reach a final volume of 20 µL. The qPCR amplification cycle included (i) a preliminary UNG glycosylase activation step at 50 °C for 2 min, (ii) an initial denaturation step at 95 °C for 10 min, and (iii) 40 cycles composed of 15 s of denaturation at 95 °C, and 55 s of hybridization-synthesis at 65 °C. The detection threshold was manually fixed at 0.02.

4.5.3. LAMP

LAMP reactions were carried out using the following mix: 0.2 µM of each external primer (F3 et B3), 0.8 µM of each internal primer (FIP et BIP), 1X Isothermal Master Mix ISO-001 (OptiGene, Horsham, UK), 2 µL of DNA matrix (0.5 ng/µL), and ultrapure water to reach a final volume of 25 µL. LAMP being an isothermal amplification technique, amplifications were performed using a RotorGene 4.4.1thermocycler (Qiagen) whose temperature was set at 65 °C for 30 min. The detection threshold was manually fixed at 0.02.

Supplementary Materials

The following are available online at https://www.mdpi.com/2223-7747/9/2/277/s1, Figure S1: PCR primer screening using a small panel. Green: specific amplification; red: non-specific amplification; white: no amplification; Figure S2: LAMP primer screening using a small panel. Green: specific amplification; red: non-specific amplification (min); Table S1: List of DNA used in this study; Table S2: List of candidate primers and probes used in this study.

Author Contributions

Conceptualization, M.T., E.F., D.T. and R.I.; Methodology, M.T., A.C., E.F., D.T. and R.I.; Validation, D.T. and R.I.; Formal analysis, M.T. and A.C.; Investigation, M.T. and A.C.; Resources, D.T. and R.I.; Data curation, M.T. and A.C.; Writing—original draft preparation, M.T., E.F., D.T. and R.I.; Writing—review and editing, M.T., A.C., E.F., D.T. and R.I.; Visualization, R.I.; Supervision, D.T and R.I.; Project administration, D.T and R.I.; Funding acquisition, D.T and R.I. All authors have read and agree to the published version of the manuscript.

Funding

This research received no external funding

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Urashima, A.S.; Igarashi, S.; Kato, H. Host range, mating type, and fertility of Pyricularia grisea from wheat in Brazil. Plant Dis. 1993, 77, 1211–1216. [Google Scholar] [CrossRef]
  2. Islam, M.T.; Kim, K.-H.; Choi, J. Wheat blast in Bangladesh: The current situation and future Impacts. Plant Pathol. J. 2019, 35, 1–10. [Google Scholar] [PubMed]
  3. Pieck, M.L.; Ruck, A.; Farman, M.L.; Peterson, G.L.; Stack, J.P.; Valent, B.; Pedley, K.F. Genomics-based marker discovery and diagnostic assay development for wheat blast. Plant Dis. 2017, 101, 103–109. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Urashima, A.S.; Lavorent, N.A.; Goulart, A.C.P.; Mehta, Y.R. Resistance spectra of wheat cultivars and virulence diversity of Magnaporthe grisea isolates in Brazil. Fitopatol. Bras. 2004, 29, 511–518. [Google Scholar] [CrossRef] [Green Version]
  5. Cruz, C.D.; Valent, B. Wheat blast disease: Danger on the move. Trop. Plant Pathol. 2017, 42, 210–222. [Google Scholar] [CrossRef] [Green Version]
  6. FAOSTAT. Available online: http://www.fao.org/faostat/fr/#home (accessed on 3 February 2020).
  7. Igarashi, S.; Utiamada, C.M.; Kasuma, A.H.; Lopez, R.S. Pyricularia em trigo. 1. Ocorrência de Pyricularia sp. no estado do Paraná. Fitopatol. Bras. 1986, 11, 351–352. [Google Scholar]
  8. Anjos, J.; Da Silva, D.B.; D’Avila Charchar, M.J.; Rodrigues, G.C. Occurrence of blast fungus (Pyricularia grisea) on wheat and rye in the savanna region of central Brazil. Pesq. Agropec. Bras. 1996, 31, 79–82. [Google Scholar]
  9. Goulart, A. Incidência da brusone (Pyricularia oryzae) em diferentes cultivares de trigo (Triticum aestivum) em condições de campo. Fitopatol. Bras. 1992, 17, 321–325. [Google Scholar]
  10. Goulart, A.C.P.; Sousa, P.G.; Urashima, A.S. Damages in wheat caused by infection of Pyricularia grisea. Summa Phytopathol. 2007, 33, 358–363. [Google Scholar] [CrossRef] [Green Version]
  11. Piccinini, E.C.; Fernandes, J.M.C. Ocorrencia da brusone (Pyricularia oryzae) em lavouras comerciais de trigo (Triticum aestivum) no estado do Rio Grande do Sul, Brazil. Fitopatol. Bras. 1990, 14, 125. [Google Scholar]
  12. Islam, M.T.; Croll, D.; Gladieux, P.; Soanes, D.M.; Persoons, A.; Bhattacharjee, P.; Hossain, M.S.; Gupta, D.R.; Rahman, M.M.; Mahboob, M.G.; et al. Emergence of wheat blast in Bangladesh was caused by a South American lineage of Magnaporthe oryzae. BMC Biol. 2016, 14, 84. [Google Scholar] [CrossRef] [Green Version]
  13. Malaker, P.K.; Barma, N.C.D.; Tiwari, T.P.; Collis, W.J.; Duveiller, E.; Singh, P.K.; Joshi, A.K.; Singh, R.P.; Braun, H.-J.; Peterson, G.L.; et al. First report of wheat blast caused by Magnaporthe oryzae pathotype triticum in Bangladesh. Plant Dis. 2016, 100, 2330. [Google Scholar] [CrossRef]
  14. Urashima, A.S.; Leite, S.F.; Galbieri, R. Eficiência da disseminação aérea em Pyricularia grisea. Summa Phytopathol. 2007, 33, 275–279. [Google Scholar] [CrossRef] [Green Version]
  15. Gomes, D.P.; Rocha, V.S.; Rocha, J.R.D.A.S.D.C.; Pereira, O.L.; Souza, M.A.D. Potential of transmission of Pyricularia graminis-tritici from plant to seed and from seed to seedling in wheat genotypes with different degrees of blast resistance. J. Seed Sci. 2018, 40, 16–24. [Google Scholar] [CrossRef] [Green Version]
  16. Klaubauf, S.; Tharreau, D.; Fournier, E.; Groenewald, J.Z.; Crous, P.W.; de Vries, R.P.; Lebrun, M.-H. Resolving the polyphyletic nature of Pyricularia (Pyriculariaceae). Stud. Mycol. 2014, 79, 85–120. [Google Scholar] [CrossRef] [PubMed]
  17. Ou, S.H. Rice Diseases, 2nd ed.; CAB International: Wallingford, UK, 1985; pp. 109–201. [Google Scholar]
  18. Gladieux, P.; Condon, B.; Ravel, S.; Soanes, D.; Maciel, J.L.N.; Nhani, A.; Chen, L.; Terauchi, R.; Lebrun, M.-H.; Tharreau, D.; et al. Gene flow between divergent cereal-and grass-specific lineages of the rice blast fungus Magnaporthe oryzae. MBio 2018, 9, e01217–e012119. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. Farman, M.; Peterson, G.; Chen, L.; Starnes, J.; Valent, B.; Bachi, P.; Murdock, L.; Hershman, D.; Pedley, K.; Fernandes, J.M.; et al. The Lolium pathotype of Magnaporthe oryzae recovered from a single blasted wheat plant in the United States. Plant Dis. 2016, 101, 684–692. [Google Scholar] [CrossRef] [Green Version]
  20. Thierry, M.; Gladieux, P.; Fournier, E.; Tharreau, D.; Ioos, R. A genomic approach to develop a new qPCR test enabling detection of the Pyricularia oryzae lineage causing wheat blast. Plant Dis. 2019, 104, 60–70. [Google Scholar] [CrossRef]
  21. Yasuhara-Bell, J.; Pedley, K.F.; Farman, M.; Valent, B.; Stack, J.P. Specific detection of the wheat blast pathogen (Magnaporthe oryzae Triticum) by loop-mediated isothermal amplification. Plant Dis. 2018, 102, 2550–2559. [Google Scholar] [CrossRef] [Green Version]
  22. Chiapello, H.; Mallet, L.; Guérin, C.; Aguileta, G.; Amselem, J.; Kroj, T.; Ortega-Abboud, E.; Lebrun, M.H.; Henrissat, B.; Gendrault, A.; et al. Deciphering genome content and evolutionary relationships of isolates from the fungus Magnaporthe oryzae attacking different host plants. Genome Biol. Evol. 2015, 7, 2896–2912. [Google Scholar] [CrossRef] [Green Version]
  23. Mancini, V.; Murolo, S.; Romanazzi, G. Diagnostic methods for detecting fungal pathogens on vegetable seeds. Plant Pathol. 2016, 65, 691–703. [Google Scholar] [CrossRef]
  24. Ioos, R.; Fourrier, C.; Iancu, G.; Gordon, T.R. Sensitive detection of Fusarium circinatum in pine seed by combining an enrichment procedure with a real-time polymerase chain reaction using dual-labeled probe chemistry. Phytopathology 2009, 99, 582–590. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Magdama, F.; Monserrate-Maggi, L.; Serrano, L.; Sosa, D.; Geiser, D.M.; Jiménez-Gasco, M.D.M. Comparative analysis uncovers the limitations of current molecular detection methods for Fusarium oxysporum f. sp. cubense race 4 strains. PLoS ONE 2019, 14, e0222727. [Google Scholar]
  26. Liu, C.M.; Kachur, S.; Dwan, M.G.; Abraham, A.G.; Aziz, M.; Hsueh, P.-R.; Huang, Y.-T.; Busch, J.D.; Lamit, L.J.; Gehring, C.A.; et al. FungiQuant: A broad-coverage fungal quantitative real-time PCR assay. BMC Microbiol. 2012, 12, 255. [Google Scholar] [CrossRef] [Green Version]
Table
Table 1. Polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), and loop-mediated isothermal amplification (LAMP) primer screening using a large DNA panel. qPCR tests MoT3 [3] and C17 [20] are included for comparison. PCR results yielded positive (+) or negative (-) reaction, mean Ct values and standard deviation are indicated for qPCR and times for positive reaction are reported for LAMP. Isolates in red belong to other fungal species: Microdochium nivale (LSV M 641); Fusarium tricinctum (LSV M 723, LSV M 860); Fusarium proliferatum (LSV M 702, LSV M 706); Fusarium poae (LNPV 269, LSVM 861); Fusarium graminearum (LSV M 273, LSV M 811, LSV M 813); Fusarium culmorum (LSV M 662, LSV M 694, LSV M 697); Fusarium avenaceum (LSV M 642, LSV M 859, LSV M 863); Alternaria tenuissima (CBS918.96, CBS965.95) and Alternaria alternata (BRIP46550). Expected amplifications (positive results) are highlighted in green, late false positive amplification are highlighted in light red, whereas other false positive results as well as false negative results are highlighted in dark red. NT: not tested.
Table 1. Polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), and loop-mediated isothermal amplification (LAMP) primer screening using a large DNA panel. qPCR tests MoT3 [3] and C17 [20] are included for comparison. PCR results yielded positive (+) or negative (-) reaction, mean Ct values and standard deviation are indicated for qPCR and times for positive reaction are reported for LAMP. Isolates in red belong to other fungal species: Microdochium nivale (LSV M 641); Fusarium tricinctum (LSV M 723, LSV M 860); Fusarium proliferatum (LSV M 702, LSV M 706); Fusarium poae (LNPV 269, LSVM 861); Fusarium graminearum (LSV M 273, LSV M 811, LSV M 813); Fusarium culmorum (LSV M 662, LSV M 694, LSV M 697); Fusarium avenaceum (LSV M 642, LSV M 859, LSV M 863); Alternaria tenuissima (CBS918.96, CBS965.95) and Alternaria alternata (BRIP46550). Expected amplifications (positive results) are highlighted in green, late false positive amplification are highlighted in light red, whereas other false positive results as well as false negative results are highlighted in dark red. NT: not tested.
IsolatesHost of OriginPCR C74PCR C82PCR C92PCR C45qPCR C45LAMP n°5qPCR MoT3qPCR C17
AG0103Triticum aestivum++++26.13 ± 0.163:47 ± 0:00++
BL0017Triticum aestivum++++25.46 ± 0.033:48 ± 0:00++
BL0018Triticum aestivum++++25.29 ± 0.053:51 ± 0:03++
BL0020Triticum aestivum++++25.04 ± 0.053:53 ± 0:00++
BL0023Triticum aestivum++++25.33 ± 0.103:54 ± 0:01++
BL0028Triticum aestivum++++25.19 ± 0.203:53 ± 0:00++
BL0037Triticum aestivum++++24.64 ± 0.063:55 ± 0:01++
BL0044Triticum aestivum++++24.46 ± 0.193:55 ± 0:01++
BL0046Triticum aestivum++++25.22 ± 0.203:52 ± 0:00++
BL0063Triticum aestivum++++25.01 ± 0.053:54 ± 0:01++
BL0092Triticum sp.++++23.75 ± 0.183:56 ± 0:01++
BL0093Triticum sp.++++24.45 ± 0.203:52 ± 0:02++
BR0031Triticum sp.+-++24.67 ± 0.153:52 ± 0:02++
BR0032Triticum sp.++++23.22 ± 0.303:57 ± 0:00-+
BR0034Triticum sp.-+++24.43 ± 0.113:53 ± 0:01++
BR0036Triticum sp.++++25.30 ± 0.133:48 ± 0:01++
BR0039Triticum sp.-+++24.61 ± 0.053:50 ± 0:02++
BR0040Triticum sp.++++25.49 ± 0.103:51 ± 0:05++
BR0041Triticum sp.-+++26.30 ± 0.053:47 ± 0:03++
BR0043Triticum sp.++++24.60 ± 0.133:55 ± 0:03-+
BR0045Triticum sp.++++27.79 ± 0.123:40 ± 0:03++
BR0047Triticum sp.-+++25.51 ± 0.063:53 ± 0:01++
BR0080Triticum sp.++++25.09 ± 0.063:53 ± 0:00++
BR0086Triticum sp.++++24.84 ± 0.213:51 ± 0:03++
BR0087Triticum sp.++++25.76 ± 0.293:51 ± 0:03++
BR0088Triticum sp.++++24.58 ± 0.053:54 ± 0:01++
BR0123Triticum aestivum++++24.61 ± 0.073:55 ± 0:01++
BTGP16Triticum sp.++++25.35 ± 0.183:70 ± 0:44++
BTJP4-1Triticum sp.++++25.33 ± 0.073:38 ± 0:00++
BTMP13-1Triticum sp.++++25.52 ± 0.253:37 ± 0:00++
AG0102Triticum aestivumNTNTNT---NTNT
BL0042Triticum aestivumNTNTNT+26.24 ± 0.093:48 ± 0:09NTNT
BL0066Triticum aestivumNTNTNT+25.55 ± 0.233:50 ± 0:01NTNT
BL0074Triticum aestivumNTNTNT+26.31 ± 0.083:41 ± 0:01NTNT
AG0054Bromus sp.+++--10:39--
AG0055Bromus sp.+-----+-
AG0061Bromus unioloides-++-----
AG0062Lolium sp.--------
AG0063Lolium sp.-----10:82 ± 0:52--
AG0064Lolium sp.--------
AG0065Stenotaphrum sp.--------
AG0132Oryza sativa--------
BF0017Pennisetum typhoides--------
BR0019Oryza sativa--------
BR0029Digitaria sanguinalis--------
BR0030Cenchrus echinatus--------
BR0062Eleusine indica--------
BR0070Eragrostis sp.--------
Br58Avena sp.----37.04 ± 2.02---
CD0143Digitaria exilis--------
Cd88215Echinochloa colona-----3:65 ± 0:12--
CH0333Oryza sativa--------
CH1120Oryza sativa--------
LcA8401Leptochloa chinensis----37.49 ± 1.68-NTNT
CHRFLolium sp.-----9:66--
CHWLolium sp.--------
CR0021Panicum miliaceum--------
CR0023Echinochloa crus-galli-----3:52 ± 0:04-+
CR0026Lolium sp.--------
CR0029Festuca elalior--------
CR0030Setaria viridis--------
CR0031Setaria italica--------
CR0057Lolium sp.-----3:59 ± 0:02-+
EG0028Cyperus rotundus--------
FHLolium sp.--------
FR0013Oryza sp.--------
FR1069Lolium sp.--------
GG11Lolium sp.--------
GN0001Zea mays--------
GR0001Ctenanthe oppenheimiana--------
GY0011Oryza sativa--------
HOLolium sp.--------
IN0003Panicum repens--------
IN0005Panicum maximun+-------
IN0022Setaria sp.-----13:10--
IN0023Setaria sp.--------
IN0082Oryza sativa--------
IN0108Setaria sp.--------
IN0113Eleusine sp.--------
IN0115Oryza sativa--------
IR0013Zea mays--------
IR0015Zea mays--------
IR0095Zea mays--------
IR0102Echinochloa sp.--------
IS0001Cyperus rotundus--------
JP0028Eragrostis curvula-----23:53--
JP0030Panicum bisulcatum-----17:69--
JP0031Panicum coloratum-----3:29 ± 0:50--
JP0033Eriochloa villosa-----3:68 ± 0:04-+
JP0047Hordeum vulgare--------
JP0048Hordeum vulgare--------
KN0001Hordeum vulgare--------
KN0006Hordeum vulgare--------
Lc8401Leptochloa chimensis--------
LpKY97Lolium sp.--------
ML0031Pennisetum sp.--------
Pd88413Paspalum distichum--------
Pg1054Stenotaphrum secundatum--------
Pg1213-22Festuca sp.--------
PH0052Cyperus rotundus--------
PH0053Cyperus rotundus--------
PH0062Paspalum distichum--------
PH0075Brachiaria mutica--------
PH0078Echinochloa sp.--------
PH0097Paspalum paspaloides--------
PL 2-1Lolium sp.--------
PL 3-1Lolium sp.--------
PR0069Stenotaphrum secundatum--------
PrA8202Panicum repens--------
RW0043Eleusine coracana-----12:85--
TFO5-1Festuca sp.--------
US0064Setaria sp.--------
US0066Cenchrus ciliaris--------
US0077Lolium perenne--------
US0078Lolium perenne--------
US0084Stenotaphrum secundatum--------
VT0032Leersia hexandra--------
AG0049Echinochloa spNTNTNT---NTNT
AG0050Echinochloa spNTNTNT---NTNT
AG0051Echinochloa spNTNTNT---NTNT
AG0058Eleusine indicaNTNTNT---NTNT
AG0059Eleusine indicaNTNTNT---NTNT
AG0067Phalaris canarienseNTNTNT--3:68 ± 0:03NTNT
AU0002Oryza rufipogonNTNTNT-37.57 ± 1.308:51 ± 0:29NTNT
BF0026Eleusine indicaNTNTNT-36.07 ± 1.68-NTNT
BF0080Oryza longistaminataNTNTNT---NTNT
BF0083Oryza longistaminataNTNTNT---NTNT
BF0093Oryza longistaminataNTNTNT---NTNT
BF0181Oryza longistaminataNTNTNT---NTNT
BG0007Leersia hexandraNTNTNT---NTNT
BG0023Leersia hexandraNTNTNT---NTNT
BG0024Leersia hexandraNTNTNT---NTNT
BR0066Eleusine indicaNTNTNT--10:12 ± 0:88NTNT
BR0071Echinochloa spNTNTNT-38.56 ± 0.54-NTNT
BR0079Eleusine indicaNTNTNT-38.48 ± 1.09-NTNT
BR0093Echinochloa colonaNTNTNT---NTNT
CD0060Oryza glaberrimaNTNTNT--15:62NTNT
CD0157Eleusine indicaNTNTNT-37.80 ± 1.64-NTNT
CD0258Leersia hexandraNTNTNT---NTNT
CH0321Oryza sativaNTNTNT---NTNT
CH0328Oryza sativaNTNTNT---NTNT
CH0331Oryza sativaNTNTNT---NTNT
CH0338Oryza sativaNTNTNT---NTNT
CH0341Oryza sativaNTNTNT---NTNT
CL0013Echinochloa colonaNTNTNT--8:67 ± 0:04NTNT
CL0045Rottboellia exaltaNTNTNT--11:45 ± 1:15NTNT
CL0089Oryza rufipogonNTNTNT-35.50 ± 2.39-NTNT
CR0058Setaria viridisNTNTNT---NTNT
CR0060Eleusine indicaNTNTNT--11:96 ± 0:75NTNT
EG0025Echinochloa colonaNTNTNT---NTNT
FR1067Lolium perenneNTNTNT--21:75NTNT
GD0001Eleusine indicaNTNTNT---NTNT
IN0004Panicum repensNTNTNT---NTNT
IN0030Echinochloa frumentaceumNTNTNT---NTNT
JP0020Eleusine indicaNTNTNT-36.82 ± 2.22-NTNT
JP0035Pennisetum clandestiNTNTNT---NTNT
JP0036Leersia hexandraNTNTNT---NTNT
JP0039Anthoxanthum odoratumNTNTNT--10:23NTNT
JP0040Phalaris arundinaceaNTNTNT---NTNT
JP0098Setaria faberiiNTNTNT--21:96NTNT
JP0102Setaria faberiiNTNTNT---NTNT
MD0112Eleusine indicaNTNTNT--11:91 ± 2:40NTNT
MD0153Eleusine indicaNTNTNT---NTNT
ML0070Oryza longistaminataNTNTNT---NTNT
ML0074Oryza longistaminataNTNTNT---NTNT
NP0060Eleusine coracanaNTNTNT---NTNT
NR0041Oryza longistaminataNTNTNT---NTNT
NR0049Leersia hexandraNTNTNT---NTNT
PH0035Brachiaria muticaNTNTNT--18:42 ± 9:59NTNT
PH0045Brachiaria muticaNTNTNT---NTNT
PH0046Brachiaria distachyaNTNTNT---NTNT
PH0056Echinochloa ciliarisNTNTNT--11:81 ± 0:67NTNT
PH0057Eleusine indicaNTNTNT---NTNT
PH0077Echinochloa colonaNTNTNT---NTNT
PH0079Panicum repensNTNTNT---NTNT
PH0080Panicum repensNTNTNT---NTNT
PH0081Paspalum paspaloidesNTNTNT--12:19 ± 2:68NTNT
PR0083Stenotaphrum secondatumNTNTNT---NTNT
RN0001Zingiber officinaleNTNTNT---NTNT
RW0018Eleusine coracanaNTNTNT-37.29 ± 2.6513:56NTNT
RW0022Eleusine indicaNTNTNT---NTNT
RW0031Eleusine coracanaNTNTNT---NTNT
RW0036Eleusine coracanaNTNTNT---NTNT
RW0038Eleusine coracanaNTNTNT---NTNT
RW0041Eleusine coracanaNTNTNT--25:09NTNT
LSV M 641Triticum sp.NTNTNT--NTNTNT
LSV M 723Triticum sp.NTNTNT--NTNTNT
LSV M 860Triticum sp.NTNTNT--NTNTNT
LSV M 702Zea maysNTNTNT--NTNTNT
LSV M 706Zea maysNTNTNT--NTNTNT
LNPV 269Triticum sp.NTNTNT--NTNTNT
LSVM 861Triticum sp.NTNTNT--NTNTNT
LSV M 273Triticum sp.NTNTNT--NTNTNT
LSV M 811Triticum sp.NTNTNT--NTNTNT
LSV M 813Triticum sp.NTNTNT--NTNTNT
LSV M 662Triticum sp.NTNTNT--NTNTNT
LSV M 694Triticum sp.NTNTNT--NTNTNT
LSV M 697Triticum sp.NTNTNT--NTNTNT
LSV M 642Triticum sp.NTNTNT--NTNTNT
LSV M 859Triticum sp.NTNTNT--NTNTNT
LSV M 863Triticum sp.NTNTNT--NTNTNT
CBS918.96Dianthus chioensisNTNTNT--NTNTNT
CBS965.95Triticum sp.NTNTNT--NTNTNT
BRIP46550Malus sp.NTNTNT--NTNTNT
Table
Table 2. Sensitivity of the PCR (C45 primers), qPCR (C45 primers), and LAMP (group 5 primers) tests in seed matrix contaminated with a Triticum lineage P. oryzae strain (BL0092).
Table 2. Sensitivity of the PCR (C45 primers), qPCR (C45 primers), and LAMP (group 5 primers) tests in seed matrix contaminated with a Triticum lineage P. oryzae strain (BL0092).
BL0092Number of Contaminated Grains Per LotC45 PCRC45 qPCRGroup 5 LAMP
Positive Replicates/Total ReplicatesPositive Replicates/Total ReplicatesMean Ct ± SDPositive Replicates/Total Replicatesmin ± SD
0 grain0/90/9>400/4-
0 h1 grain1/93/937.21 ± 0.740/4-
2 grains2/91/937.310/4-
5 grains3/94/937.66 ± 1.450/4-
10 grains6/97/937.16 ± 0.880/4-
15 grains9/96/936.74 ± 1.171/413:74
50 grains9/99/935.36 ± 0.60NTNT
0 grain0/90/9>400/4-
72 h1 grain9/99/933.83 ± 0.612/412:97 ± 10:80
2 grains9/99/930.97 ± 0.414/44:66 ± 0:55
5 grains9/99/929.00 ± 0.324/44:24 ± 0:09
10 grains9/99/929.32 ± 0.254/44:22 ± 0:18
15 grains9/99/928.56 ± 0.254/44:13 ± 0:24
50 grains9/99/926.47 ± 0.32NTNT
Table
Table 3. Sensitivity of the PCR (C45 primers), qPCR (C45 primers), and LAMP (group 5 primers) tests in seed matrix contaminated with a non-target P. oryzae strain from E. indica (BR0079).
Table 3. Sensitivity of the PCR (C45 primers), qPCR (C45 primers), and LAMP (group 5 primers) tests in seed matrix contaminated with a non-target P. oryzae strain from E. indica (BR0079).
BR0079Number of Contaminated Grains Per LotPCRqPCRGroup 5 LAMP
Positive Replicates/Total ReplicatesPositive Replicates/Total Replicates
72 h20 grains0/90/9NT
50 grains0/90/9NT

Share and Cite

MDPI and ACS Style

Thierry, M.; Chatet, A.; Fournier, E.; Tharreau, D.; Ioos, R. A PCR, qPCR, and LAMP Toolkit for the Detection of the Wheat Blast Pathogen in Seeds. Plants 2020, 9, 277. https://doi.org/10.3390/plants9020277

AMA Style

Thierry M, Chatet A, Fournier E, Tharreau D, Ioos R. A PCR, qPCR, and LAMP Toolkit for the Detection of the Wheat Blast Pathogen in Seeds. Plants. 2020; 9(2):277. https://doi.org/10.3390/plants9020277

Chicago/Turabian Style

Thierry, Maud, Axel Chatet, Elisabeth Fournier, Didier Tharreau, and Renaud Ioos. 2020. "A PCR, qPCR, and LAMP Toolkit for the Detection of the Wheat Blast Pathogen in Seeds" Plants 9, no. 2: 277. https://doi.org/10.3390/plants9020277

APA Style

Thierry, M., Chatet, A., Fournier, E., Tharreau, D., & Ioos, R. (2020). A PCR, qPCR, and LAMP Toolkit for the Detection of the Wheat Blast Pathogen in Seeds. Plants, 9(2), 277. https://doi.org/10.3390/plants9020277

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop