A LAMP Protocol for the Detection of ‘<i>Candidatus</i> Phytoplasma pyri’, the Causal Agent of Pear Decline

Siemonsmeier, Angela; Hadersdorfer, J.; Neumüller, M.; Schwab, Wilfried; Treutter, D.

doi:10.1094/pdis-12-18-2150-re

Cited by 12 publications

(4 citation statements)

References 37 publications

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“…P. allocasuarinae', not being a very e cient protocol due to cross-reactions with other phytoplasmas. Similar result in a LAMP assay developed for Candidatus Phytoplasma pyri, which showed 100% sequence identity of primers B2 and BIP with the 16S rRNA region of Pseudomonas syringae(Siemonsmeier et al 2019). Therefore, in this study, RUCS was used to nd exclusive regions not restricted to ORFs(Thomsen et al 2017) increasing the number of available target sequences, reducing possible cross-detections with other phytoplasmas and microorganisms that infect maize.…”

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confidence: 52%

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Specific colorimetric LAMP assay for the detection of maize bushy stunt phytoplasma in corn through comparative genomics

Egito,

Rodrigues Alves,

Carvalho

et al. 2024

Trop. plant pathol.

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Brazil stands out for being the third largest corn producer in the world, in addition to having selfsu ciency in national supply. Even with the high production, the country has, throughout its extension, climatic conditions that favor the attack of several pathogens. Maize red stunt, caused by maize bushy stunt phytoplasma (MBSP), is one of the most harmful diseases to the crop, therefore, there is a demand for detection methods that are fast and accurate. Loop-mediated isothermal ampli cation (LAMP) is one of these methods, being fast, sensitive, with high speci city and can be used in eld analysis. The goal of the present work was the development of a LAMP protocol, through comparative genomics, for MBSP in maize. To design the sets of primers, MBSP genome sequence and other sequences of other pathogens were used in the RUCS software to select only core unique sequences. Three sets of primers presented the desired criteria and were synthetized. The most promising primer set, MBSP-LP, was used to test the LAMP assay together with the Warmstart colorimetric LAMP 2X master mix (NEB) Kit. The collection of 51 samples of corn with and without symptoms was tested with a typical nested-PCR and compared with the proposed LAMP assay. Considering the presence and absence of symptoms, there was con rmation that the symptomatic plants were positive for LAMP in a greater proportion than for nested-PCR. The proposed LAMP assay proved to be sensitive, detecting up to 0.1 fg µL -1 of DNA. The use of plant material directly in the reaction was evaluated for changing the color of the master mix and also for inhibiting the reaction. It was identi ed that there are no inhibitors in the maize plant tissue, being possible to use this LAMP with plant tissue without the DNA extraction step.

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supporting

confidence: 52%

“…The LAMP test resulted positive for samples numbered: 2, 3, 4, 5,7,10,11,12,15,16,18,20,40,41,45,46, 47 and 50. The others showed no color change, considered samples negative for the presence of the pathogen (Table 3 and Fig.…”

Section: Diversity and Contrast Test With Nested-pcrmentioning

confidence: 99%

Specific colorimetric LAMP assay for the detection of maize bushy stunt phytoplasma in corn through comparative genomics

Egito,

Rodrigues Alves,

Carvalho

et al. 2024

Trop. plant pathol.

View full text Add to dashboard Cite

show abstract

“…The LAMP test resulted positive for samples numbered: 2, 3, 4, 5, 7, 10,11,12,15,16,18,20,40,41,45,46, 47 and 50. The others showed no color change, considered samples negative for the presence of the pathogen (Table 3 and Fig.…”

Section: Diversity and Contrast Test With Nested-pcrmentioning

confidence: 99%

Specific colorimetric LAMP assay for the detection of maize bushy stunt phytoplasma in corn through comparative genomics

Egito,

Alves,

Carvalho

et al. 2023

Preprint

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Brazil stands out for being the third largest corn producer in the world, in addition to having self-sufficiency in national supply. Even with the high production, the country has, throughout its extension, climatic conditions that favor the attack of several pathogens. Maize red stunt, caused by maize bushy stunt phytoplasma (MBSP), is one of the most harmful diseases to the crop, therefore, there is a demand for detection methods that are fast and accurate. Loop-mediated isothermal amplification (LAMP) is one of these methods, being fast, sensitive, with high specificity and can be used in field analysis. The goal of the present work was the development of a LAMP protocol, through comparative genomics, for MBSP in maize. To design the sets of primers, MBSP genome sequence and other sequences of other pathogens were used in the RUCS software to select only core unique sequences. Three sets of primers presented the desired criteria and were synthetized. The most promising primer set, MBSP-LP, was used to test the LAMP assay together with the Warmstart colorimetric LAMP 2X master mix (NEB) Kit. The collection of 51 samples of corn with and without symptoms was tested with a typical nested-PCR and compared with the proposed LAMP assay. Considering the presence and absence of symptoms, there was confirmation that the symptomatic plants were positive for LAMP in a greater proportion than for nested-PCR. The proposed LAMP assay proved to be sensitive, detecting up to 0.1 fg µL-1 of DNA. The use of plant material directly in the reaction was evaluated for changing the color of the master mix and also for inhibiting the reaction. It was identified that there are no inhibitors in the maize plant tissue, being possible to use this LAMP with plant tissue without the DNA extraction step.

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“…HNB dye can change from purple to sky blue during reactions. HNB detection for viruses [164][165][166][167][168], bacteria [169,170], and fungi [171][172][173][174] has also been developed. There is only a prototype of direct detection of plant pathogen via magnesium pyrophosphate precipitation [175].…”

Section: Colorimetric Detection Of Lamp Ampliconsmentioning

confidence: 99%

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Иванов

Safenkova

Zherdev

et al. 2021

Plants

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Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

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A LAMP Protocol for the Detection of ‘Candidatus Phytoplasma pyri’, the Causal Agent of Pear Decline

Cited by 12 publications

References 37 publications

Specific colorimetric LAMP assay for the detection of maize bushy stunt phytoplasma in corn through comparative genomics

Specific colorimetric LAMP assay for the detection of maize bushy stunt phytoplasma in corn through comparative genomics

Specific colorimetric LAMP assay for the detection of maize bushy stunt phytoplasma in corn through comparative genomics

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

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