Monitoring Methyl Benzimidazole Carbamate-Resistant Isolates of the Cucurbit Powdery Mildew Pathogen, Podosphaera xanthii, Using Loop-Mediated Isothermal Amplification

Powdery mildew, caused by the fungus Podosphaera xanthii, is one of the most economically important diseases affecting cucurbit crops in Spain. Currently, chemical control offers the most efficient management of the disease; however, P. xanthii isolates resistant to multiple classes of site-specific fungicides have been reported in the Spanish cucurbit powdery mildew population. In previous studies, resistance to the fungicides known as methyl benzimidazole carbamates (MBCs) was found to be caused by the amino acid substitution E198A on β-tubulin. To detect MBC-resistant isolates in a faster, more efficient, and more specific way than the traditional methods used to date, a loop-mediated isothermal amplification (LAMP) system was developed. In this study, three sets of LAMP primers were designed. One set was designed for the detection of the wild-type allele and two sets were designed for the E198A amino acid change. Positive results were only obtained with both mutant sets; however, LAMP reaction conditions were only optimized with primer set 2, which was selected for optimal detection of the E198A amino acid change in P. xanthii-resistant isolates, along with the optimal temperature and duration parameters of 65°C for 75 min, respectively. The hydroxynaphthol blue (HNB) metal indicator was used for quick visualization of results through the color change from violet to sky blue when the amplification was positive. HNB was added before the amplification to avoid opening the lids, thus decreasing the probability of contamination. To confirm that the amplified product corresponded to the β-tubulin gene, the LAMP product was digested with the enzyme LweI and sequenced. Our results show that the LAMP technique is a specific and reproducible method that could be used for monitoring MBC resistance of P. xanthii directly in the field.

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Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

Molecular Medicine ◽

10.1186/s10020-021-00289-0 ◽

2021 ◽

Vol 27 (1) ◽

Author(s):

Bruna de Oliveira Coelho ◽

Heloisa Bruna Soligo Sanchuki ◽

Dalila Luciola Zanette ◽

Jeanine Marie Nardin ◽

Hugo Manuel Paz Morales ◽

...

Keyword(s):

Viral Load ◽

Internal Control ◽

Reverse Transcription ◽

Color Change ◽

Point Of Care ◽

Colorimetric Detection ◽

False Negative ◽

Isothermal Amplification ◽

Clinical Samples ◽

Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

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A loop-mediated isothermal amplification (LAMP) assay to identify isotype 1 β-tubulin locus SNPs in synthetic double-stranded Haemonchus contortus DNA

Journal of Parasitic Diseases ◽

10.1007/s12639-021-01414-w ◽

2021 ◽

Author(s):

Livio M. Costa-Junior ◽

Umer N. Chaudhry ◽

Philip J. Skuce ◽

Seamus Stack ◽

Neil D. Sargison

Keyword(s):

Genetic Susceptibility ◽

Management Practices ◽

Anthelmintic Resistance ◽

Lamp Assay ◽

Gastrointestinal Nematode ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Snp Typing ◽

Field Samples ◽

Β Tubulin

AbstractDevelopment of sustainable gastrointestinal nematode (GIN) control strategies depends on the ability to identify the frequencies of drug-susceptible and resistant genotypes in GIN populations arising from management practices undertaken on individual farms. Resistance to BZ drugs in GINs has been shown to be conferred by the presence of defined SNPs in the isotype 1 β-tubulin locus. Loop-mediated isothermal amplification (LAMP) assays are amenable to use on a range of DNA templates and are potentially adaptable to use in practical, cost-effective, pen-side diagnostic platforms that are needed to detect anthelmintic resistance in the field. In this study, we designed primers and examined LAMP assays to detect each of the three major isotype 1 β-tubulin SNPs conferring genetic susceptibility to BZ drugs. We used artificial pools of synthetic DNA, containing different proportions of susceptible and resistant SNPs to determine reproducibility of the assays. We demonstrated the detection of each of the isotype 1 β-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 β-tubulin SNP typing was effective in detecting BZ susceptibility, but the accuracy was reduced in samples with less than 60 % susceptible DNA. Our results show the potential for LAMP SNP typing to detect genetic susceptibility or resistance to anthelmintic drugs in livestock GINs, and some of the limitations in our approach that will need to be overcome in order to evaluate this assay using field samples.

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Transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii by Agrobacterium tumefaciens

New Phytologist ◽

10.1111/nph.14297 ◽

2016 ◽

Vol 213 (4) ◽

pp. 1961-1973 ◽

Cited By ~ 18

Author(s):

Jesús Martínez‐Cruz ◽

Diego Romero ◽

Antonio Vicente ◽

Alejandro Pérez‐García

Keyword(s):

Agrobacterium Tumefaciens ◽

Powdery Mildew ◽

Podosphaera Xanthii ◽

Powdery Mildew Pathogen ◽

Cucurbit Powdery Mildew

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First Report of Resistance to Boscalid in Podosphaera xanthii, Cucurbit Powdery Mildew, in South Carolina

Plant Health Progress ◽

10.1094/php-03-18-0009-br ◽

2018 ◽

Vol 19 (3) ◽

pp. 220-221 ◽

Cited By ~ 2

Author(s):

Anthony P. Keinath ◽

Gabriel Rennberger ◽

Chandrasekar S. Kousik

Keyword(s):

South Carolina ◽

Powdery Mildew ◽

Fungicide Resistance ◽

Southern United States ◽

Podosphaera Xanthii ◽

Powdery Mildew Fungus ◽

Action Committee ◽

Summer Squash ◽

Laboratory Bioassays ◽

Cucurbit Powdery Mildew

Resistance to boscalid, one of the older succinate-dehydrogenase inhibitors (SHDI) in Fungicide Resistance Action Committee (FRAC) code 7, was detected in Podosphaera xanthii, the cucurbit powdery mildew fungus, in South Carolina in July 2017. Resistance to the field rate (682 ppm) of boscalid was confirmed in greenhouse experiments and laboratory bioassays conducted on summer squash plants and cotyledons, respectively, that had been treated with a range of boscalid concentrations. This report is the first documentation of resistance to boscalid in P. xanthii in the southern United States.

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Development of a quantitative loop-mediated isothermal amplification assay for the field detection ofErysiphe necator

PeerJ ◽

10.7717/peerj.4639 ◽

2018 ◽

Vol 6 ◽

pp. e4639 ◽

Cited By ~ 14

Author(s):

Lindsey D. Thiessen ◽

Tara M. Neill ◽

Walter F. Mahaffee

Keyword(s):

Powdery Mildew ◽

Pathogen Detection ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Lamp Assay ◽

Isothermal Amplification ◽

Management Tool ◽

Willamette Valley ◽

Loop Mediated Isothermal Amplification ◽

Grape Powdery Mildew

Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, theErysiphe necatorinoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools.

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Outbreak of Cucurbit Powdery Mildew on Watermelon Fruit Caused by Podosphaera xanthii in Southwest Florida

Plant Disease ◽

10.1094/pdis-06-11-0521 ◽

2011 ◽

Vol 95 (12) ◽

pp. 1586-1586 ◽

Cited By ~ 11

Author(s):

C. S. Kousik ◽

R. S. Donahoo ◽

C. G. Webster ◽

W. W. Turechek ◽

S. T. Adkins ◽

...

Keyword(s):

Powdery Mildew ◽

Morphological Characteristics ◽

The United States ◽

Tween 20 ◽

Conidial Suspension ◽

Podosphaera Xanthii ◽

Its Sequence Analysis ◽

Fruit Samples ◽

Cucurbit Powdery Mildew ◽

Fruit Surface

Cucurbit powdery mildew caused by the obligate parasite Podosphaera xanthii occurs commonly on foliage, petioles, and stems of most cucurbit crops grown in the United States. (3). However, in the field, fruit infection on cucurbits including watermelon (Citrullus lanatus), is rarely, if ever, observed (2). Consequently, it was atypical when severe powdery mildew-like symptoms were observed on seedless and seeded watermelon fruit on several commercial farms in southwestern Florida during November and December 2010. Severe powdery mildew was also observed on ‘Tri-X 313’ and ‘Mickey Lee’ fruit grown at SWFREC, Immokalee, FL. Infected fruit developed poorly and were not marketable. Powdery mildew symptoms were mainly observed on young immature fruit, but not on mature older fruit. Abundant powdery mildew conidia occurred on fruit surface, but not on the leaves. Conidia were produced in chains and averaged 35 × 21 μm. Observation of conidia in 3% KOH indicated the presence of fibrosin bodies commonly found in the cucurbit powdery mildew genus Podosphaera (3). Orange-to-dark brown chasmothecia (formerly cleisthothecia) containing a single ascus were detected on the surface of some fruit samples. Conidial DNA was subjected to PCR using specific primers designed to amplify the internal transcribed spacer (ITS) region of Podosphaera (4). The resulting amplicons were sequenced and found to be 100% identical to the ITS sequences of P. xanthii in the NCBI database (D84387, EU367960, AY450961, AB040322, AB040315). Sequences from the watermelon fruit isolate were also identical to several P. fusca (synonym P. xanthii), P. phaseoli (GQ927253), and P. balsaminae (AB462803) sequences. On the basis of morphological characteristics and ITS sequence analysis, the pathogen infecting watermelon fruit can be considered as P. xanthii (1,3,4). The powdery mildew isolate from watermelon fruit was maintained on cotyledons of squash (Cucurbita pepo, ‘Early Prolific Straight Neck’). Cotyledons and leaves of five plants each of various cucurbits and beans were inoculated with 10 μl of a conidial suspension (105conidia/ml) in water (0.02% Tween 20). Two weeks after inoculation, abundant conidia were observed on cucumber (Cucumis sativus, ‘SMR-58’) and melon (Cucumis melo) powdery mildew race differentials ‘Iran H’ and ‘Vedrantais’. However, no growth was observed on melon differentials ‘PI 414723’, ‘Edisto 47’, ‘PMR 5’, ‘PMR 45’, ‘MR 1’, and ‘WMR 29’ (2,3). The powdery mildew isolate from watermelon fruit behaved as melon race 1 (3). Mycelium and conidia were also observed on fruit surface of watermelon ‘Sugar Baby’ and a susceptible U.S. plant introduction (PI 538888) 3 weeks after inoculation. However, the disease was not as severe as what was observed in the fields in fall 2010. The pathogen did not grow on plants of Impatiens balsamina or on select bean (Phaseolus vulgaris) cultivars (‘Red Kidney’, ‘Kentucky Blue’, and ‘Derby Bush’), but did grow and produce abundant conidia on ‘Pinto bush bean’. Powdery mildew on watermelon fruit in production fields can be considered as a potentially new and serious threat requiring further studies to develop management strategies. References: (1) U. Braun and S. Takamatsu. Schlechtendalia 4:1, 2000. (2) A. R. Davis et al. J. Am. Soc. Hortic. Sci. 132:790, 2007. (3) M. T. McGrath and C. E. Thomas. In: Compendium of Cucurbit Diseases. American Phytopathological Society, St. Paul, MN, 1996. (4) S. Takamatsu and Y. Kano. Mycoscience 42:135, 2001.

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Development of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Phytopythium vexans

Frontiers in Microbiology ◽

10.3389/fmicb.2021.720485 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tuhong Wang ◽

Haojun Ji ◽

Yongting Yu ◽

Xiaojie Wang ◽

Yi Cheng ◽

...

Keyword(s):

Root Rot ◽

Rapid Detection ◽

Color Change ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Amplification Method ◽

Specialized Equipment ◽

Root Tissues ◽

And Control ◽

Brown Root Rot

Brown root rot caused by Phytopythium vexans is a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detecting P. vexans can help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment – a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences of P. vexans and the related species in oomycete genera Pythium, Phytopythium, and Phytophthora, we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans – infected ramie root tissues.

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A Rapid and Sensitive Reverse Transcription-Loop-Mediated Isothermal Ampliﬁcation (RT-LAMP) Assay for the Detection of Indian Citrus Ringspot Virus

Plant Disease ◽

10.1094/pdis-06-20-1349-re ◽

2020 ◽

Cited By ~ 2

Author(s):

Amol Kokane ◽

Sunil Kokane ◽

Ashish Warghane ◽

Mrugendra G Gubyad ◽

Ashwani Kumar Sharma ◽

...

Keyword(s):

Reverse Transcription ◽

Large Scale ◽

Color Change ◽

Lamp Assay ◽

Single Step ◽

Isothermal Amplification ◽

Ringspot Virus ◽

Lamp Method ◽

Loop Mediated Isothermal Amplification ◽

Field Samples

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the ‘Kinnow mandarin’, a commercial citrus crop cultivated in the north-west of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcriptase-polymerase chain reaction (RT-PCR) that is time-consuming. Here, we describe a novel, one-step, reverse transcription-loop-mediated isothermal ampliﬁcation (RT-LAMP) method for the sensitive and rapid detection of ICRSV. The RT-LAMP assay was standardized by designing and testing four different primers that targeted the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green I. The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that are common in India. ICRSV-RT-LAMP assay developed in the present study is a simple, rapid, sensitive, and specific, technique. Moreover, the assay consists of only a single step and is more cost-effective than existing methods. This represents the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large scale indexing of field samples in diagnostic laboratories, nurseries, and for quarantine applications.

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