Please wait a minute...
Journal of Integrative Agriculture
Advanced Online Publication | Current Issue | Archive | Adv Search
A rapid tool for quantification of latent infection of wheat leaves by powdery mildew

Aolin Wang1, 2*, Ru Jiang1*, Meihui Zhang1, Hudie Shao1, 6, Fei Xu1, 4, Kouhan Liu2, Haifeng Gao5, Jieru Fan1#, Wei Liu1#, Xiaoping Hu2, Yilin Zhou1#, Xiangming Xu3

1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China

2 State Key Laboratory for Crop Stress Resistance and High-Efficiency Production College of Plant Protection, Northwest A&F University, Yangling 712100, China

3NIAB East Malling, West Malling, Kent, ME19 6BJ, United Kingdom

4 Key Laboratory of Integrated Pest Management on Crops in Southern Part of North China, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China

5Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis, Ministry of Agriculture, Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences, Scientific Observing and Experimental Station of Korla, Urumqi 830091, China

6 College of Forestry and Biotechnology, Zhejiang A & F University, Hangzhou 311300, China

Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
摘要  

由禾布氏白粉菌小麦专化型(Blumeria graminis f. sp. tritici)引起的小麦白粉病在世界范围内广泛发生,对小麦安全生产构成严重威胁。明确小麦白粉病菌的秋苗菌源量对于预测和评估其春季病害流行风险具有重要意义。然而,在晚秋或冬季进行秋苗调查时,如遇温度较低情况,小麦白粉病菌常以潜伏侵染的形式寄生在叶片组织内,而不显示肉眼可见的症状。本研究开发了一种快速检测和定量小麦叶片中潜伏侵染白粉病菌的新方法。该方法基于重组酶聚合酶扩增反应(RPA)并通过侧向流试纸条(LFD)进行终点检测。小麦白粉病菌DNA的检出下限为100 ag μL-1,且不受其他常见小麦病原真菌或其寄主小麦(Triticum aestivumDNA的干扰。在小麦幼苗上对该法检测小麦白粉病菌潜伏侵染进行了试验验证,结果显示其准确性高、灵敏性强。在此基础上,将RPA-LFD检测技术与图像分析技术相结合,开发了用于定量估计小麦白粉病菌潜伏侵染量的分析方法。试验结果显示,该分析方法和实时荧光定量PCR技术估计的小麦白粉病菌DNA量之间存在极显著的相关性(P ≤ 0.01),说明所建立的新方法能够快速准确地定量测定小麦叶片中潜伏侵染小麦白粉病菌的含量,并有望进一步发展成为能够直接用于田间现场监测小麦白粉病菌潜伏侵染水平的工具。本研究建立的方法还可为小麦其他叶部病原真菌(如小麦锈菌)潜伏侵染的检测和定量提供借鉴,并有潜力结合空气中的病原菌孢子捕捉技术提供孢子浓度的定量信息。



Abstract  

Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is an important disease worldwide. Detection of latent infection of leaves by the pathogen in late autumn is valuable for estimating the inoculum potential to assess disease risks in the spring. We developed a new tool for rapid detection and quantification of latent infection of seedlings by the pathogen. The method was based on recombinase polymerase amplification (RPA) coupled with an end-point detection via lateral flow device (LFD). The limit of detection is 100 ag μL-1 of Bgt DNA, without noticeable interference from either other common wheat pathogens or wheat material (Triticum aestivum). It was evaluated on wheat seedlings for this accuracy and sensitivity in detecting latent infection of Bgt. We further extended this RPA-LFD assay to estimate the level of latent infection by Bgt based on imaging analysis. There was a strong correlation between the image-based and real-time PCR assay estimates of Bgt DNA. The present results suggested that this new tool can provide rapid and accurate quantification of Bgt in latently infected leaves and can be further development as an on-site monitoring tool.

Keywords:  Blumeria graminis f. sp. tritici       recombinase polymerase amplification (RPA)              lateral flow device (LFD)              image-based quantification              disease monitoring  
Online: 01 July 2024  
Fund: The research was supported by the funding from the National Natural Science Foundation of China (32072359).
About author:  #Correspondence Jieru Fan, E-mail: fanjieru1981@126.com; Wei Liu, E-mail: wliusdau@163.com; Yilin Zhou, E-mail: yilinzhou6@163.com

Cite this article: 

Aolin Wang, Ru Jiang, Meihui Zhang, Hudie Shao, Fei Xu, Kouhan Liu, Haifeng Gao, Jieru Fan, Wei Liu, Xiaoping Hu, Yilin Zhou, Xiangming Xu. 2024. A rapid tool for quantification of latent infection of wheat leaves by powdery mildew. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.06.004

Cao S Q, Luo H S, Jin S L, Duan X Y, Zhou Y L, Jin M A, Jia Q Z. 2011. Over-summering of Blumeria graminis f. sp. tritici in Tianshui of Gansu and effects of the sexual stage on infection cycle. Plant Protection, 37, 115-119. (in Chinese)

Chandu D, Paul S, Parker M, Dudin Y, King-Sitzes J, Perez T, Mittanck D W, Shah M, Glenn K C, Piepenburg O. 2016. Development of a rapid point-of-use DNA test for the screening of Genuity® Roundup Ready 2 Yield® Soybean in seed samples. Biomed Research International, 2016, 3145921- 3145932.

Chao C C, Belinskaya T, Zhang Z W, Chi W M. 2015. Development of recombinase polymerase amplification assays for detection of Orientia tsutsugamushi or Rickettsia typhi. Plos Neglected Tropical Diseases, 9, e0003884.

Chen S N, Yuan H Z, Yan X J. 2022. Rapid visual detection of benzimidazole resistance in Botrytis cinerea by recombinase polymerase amplification combined with a lateral flow dipstick. Pest Management Science, 78, 821-830.

Chen W Q, Duan X Y. 2014. The hot spots in cereal rusts and powdery mildew research. Journal of Integrative Agriculture, 2, 5-8.

Chi Y K, Zhao W, Ye M D, Ali F, Wang T, Qi R D. 2020. Evaluation of recombinase polymerase amplification assay for detecting Meloidogyne javanica. Plant Disease, 104, 801-807.

Colour Blind Awareness. 2023. Colour Blindness. http://www.colourblindawareness.org/colour-blindness/ (accessed 25 Jan. 2023).

Cordray M S, Richards-Kortum R R. 2015. A paper and plastic device for the combined isothermal amplification and lateral flow detection of Plasmodium DNA. Malaria Journal, 14, 472

Fabio D N, Alladio E, Baggiani C, Cavalera S, Giovannoli C, Spano G, Anfossi L. 2018. Colour-encoded lateral flow immunoassay for the simultaneous detection of aflatoxin B1 and type-B fumonisins in a single Test line. Talanta, 192, 288-294.

Frank J A, Cole H JR, Hatley O E. 1988. The effect of planting date on fall infections and epidemics of powdery mildew on winter wheat. Plant Disease, 72, 661-664.

Glawe D A. 2008. The powdery mildews: a review of the world's most familiar (yet poorly known) plant pathogens. Annual Review of Phytopathology, 46, 27-51. 

Ivanov A V, Safenkova I V, Zherdev A V, Dzantiev B B. 2020. Nucleic acid lateral flow assay with recombinase polymerase amplification: solutions for highly sensitive detection of RNA virus. Talanta, 210, 120616.

Jauset-Rubio M, Svobodová M, Mairal T, McNeil C, Keegan N, El-Shahawi M S, Bashammakh A S, Bashammakh A O, O’Sullivan C K. 2016. Aptamer lateral flow assays for ultrasensitive detection of β-conglutin combining recombinase polymerase amplification and tailed primers. Analytical Chemistry, 88, 10701-10709.

Jevtić R, Župunski V, Lalošević M, Župunski L. 2017. Predicting potential winter wheat yield losses caused by multiple disease systems and climatic conditions. Crop Protection, 99, 17-25.

Johnson L E B, Bushnell W R, Zeyen R J. 1982. Defense patterns in nonhost higher plant species against two powdery mildew fungi. I. Monocotyledonous species. Canadian Journal of Botany, 60, 1068-1083. 

Justesen A F, Ridout C J, Hovmøller M S. 2002. The recent history of Puccinia striiformis f. sp. tritici in Denmark as revealed by disease incidence and AFLP markers. Plant Pathology, 51, 13-23.

Kang H X, Peng Y, Hua K Y, Deng Y F, Maria B, Dipali R G. 2021. Rapid detection of wheat blast pathogen Magnaporthe oryzae Triticum pathotype using genome-specific primers and Cas12a-mediated technologyEngineering, 7, 1326-1335.

Kersting S, Rausch V, Bier F F, Nickisch-Rosenegk M. 2014a. Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens. Mikrochimica Acta, 181, 1715-1723.

Kersting S, Rausch V, Bier F F, Nickisch-Rosenegk M. 2014b. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malaria Journal, 13, 1-9.

Lau H Y, Wang Y L, Wee E J, Botella J R, Trau M. 2016. Field demonstration of a multiplexed point-of-care diagnostic platform for plant pathogens. Analytical Chemistry, 88, 8074-8081. 

Li B N, Cao X R, Chen L, Zhou Y L, Duan X Y, Luo Y, Bruce D L F, Xu X M, Song Y L, Wang B T, Cao S Q. 2013. Application of geographic information systems to identify the oversummering regions of Blumeria graminis f. sp. tritici in China. Plant Disease, 97, 1168-1174.

Li J, Liu X H, Yang X W, Li Y C, Wang C Y, He D X. 2018. Proteomic analysis of the impacts of powdery mildew on wheat grain. Food Chemistry, 261, 30-35.

Li J L, Ma B, Fang J H, Zhi A T, Chen E J, Xu Y, Yu X P, Sun C X, Zhang M Z. 2019. Recombinase polymerase amplification (RPA) combined with lateral flow immunoassay for rapid detection of Salmonella in Food. Foods, 9, 27.

Li N, Jia S F, Wang X N, Duan X Y, Zhou Y L, Wang Z H, Lu G D. 2012. The effect of wheat mixtures on the powdery mildew disease and some yield components. Journal of Integrative Agriculture, 11, 611-620.

Liang X Y, Zhang X, Haseeb H A, Tang T T, Shan J H, Yin B, Guo W. 2022. Development and evaluation of a novel visual and rapid detection assay for toxigenic Fusarium graminearum in maize based on recombinase polymerase amplification and lateral flow analysis. International journal of food microbiology, 372, 109682. 

Liu D B, Wang Z T, Jin A, Huang X L, Sun X L, Wang F, Yan Qiang, Ge S X, Xia N S, Niu G, Liu G, Hight Walker A R, Chen X Y. 2013. Acetylcholinesterase-catalyzed hydrolysis allows ultrasensitive detection of pathogens with the naked eye. Angewandte Chemie International Edition, 52, 14065-14069.

Liu N, Gong G S, Zhang M, Zhou Y, Chen Z X., Yang J Z, Chen H B, Wang X G, Lei Y, Liu K. 2012. Over-summering of wheat powdery mildew in Sichuan province, China. Crop Protection, 34, 112-118. 

Liu N, Lei Y, Zhang M, Zheng W M, Shi Y C, Qi X B, Chen H B, Zhou Y, Gong G S. 2019. Latent infection of powdery mildew on volunteer wheat in Sichuan province, China. Plant Disease. 103, 1084-1091.

Liu W, Cao X R, Fan J R, Wang Z H, Yan Z Y, Luo Y, West J S, Xu X M, Zhou Y L. 2018. Detecting wheat powdery mildew and predicting grain yield using unmanned aerial photography. Plant Disease. 102, 1981-1988.

Liu W C, Shao Z R. 1994. Overview and analysis on epidemics of wheat powdery mildew in recent years. China Plant Protection6, 17-19. (in Chinese)

Liu Z W, Hua Q C, Wang J, Liang Z Q, Li J H, Wu J X, Shen X, Lei H T, Li X M. 2020. A smartphone-based dual detection mode device integrated with two lateral flow immunoassays for multiplex mycotoxins in cereals. Biosensors & Bioelectronics: The International Journal for the Professional Involved with Research, Technology and Applications of Biosensers and Related Devices, 158, 112178.

Lobato I M, O’Sullivan C K. 2018. Recombinase polymerase amplification: Basics, applications and recent advances. Trac-Trends in Analytical Chemistry, 98, 19-35.

Londoño M A, Harmon C L, Polston J E. 2016. Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics. Virology Journal, 13, 48.

Mori Y, Sato Y, Takamatsu S. 2000. Evolutionary analysis of the powdery mildew fungi using nucleotide sequences of the nuclear ribosomal DNA. Mycologia, 92, 74-93.

Mwale V M, Chilembwe E H C, Uluko H C. 2014. Wheat powdery mildew (Blumeria graminis f. sp. tritici): damage effects and genetic resistance developed in wheat (Triticum aestivum). International Research Journal of Plant Science, 5, 1-16.

NHS Choices. 2019. Colour vision deficiency (colour blindness). NHS.UK.  https://www.nhs.uk/conditions/colour-vision-deficiency/ (accessed 25 Jan. 2023).

Perkel J M. 2021. Ten computer codes that transformed science. Nature, 589, 344-348. 

Piepenburg O, Williams C H, Stemple D L, Armes N A. 2006. DNA detection using recombination proteins. Plos Biology, 4, 1115-1121.

Ruppert C, Phogat N, Laufer S, Kohl M, Deigner H P. 2019. A smartphone readout system for gold nanoparticle-based lateral flow assays: application to monitoring of digoxigenin. Mikrochimica Acta, 186, 1-9.

Saghai-Maroof M A, Soliman K M, Jorgensen R A, Allard R W. 1984. Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proceedings of the National Academy of Sciences, 81, 8014-8018.

Schindelin J, Arganda-carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J Y, White D J, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nature methods, 9, 676-682. 

Schindelin J, Rueden C T, Hiner M C, Eliceiri K W. 2015. The ImageJ ecosystem: An open platform for biomedical image analysis. Molecular Reproduction & Development, 82, 518-529.

Shao H D, Jian J Z, Peng D L, Yao K, Abdulsalam S, Huang W K, Kong L A, Li C R, Peng H. 2022. Recombinase polymerase amplification coupled with CRISPR-Cas12a technology for rapid and highly sensitive detection of Heterodera avenae and Heterodera filipjevi. Plant Disease, 107, 1365-1376.

Shi W Q, Liu M L, Chen T T, Xue M F, Gong S J, Yang L J. 2020. Establishment of inoculation by spray of wheat powdery mildew conidia and its application in the bioassay determination. Journal of Plant Protection, 47, 683-684. (in Chinese)

Singh R P, Singh P K, Rutkoski J, Hodson D P, He X, Jørgensen L N, Hovmøller M S, Huerta-Espino J. 2016. Disease impact on wheat yield potential and prospects of genetic control. Annual Review of Phytopathology, 54, 303-322.

Soh J H, Chan H M, Ying J Y. 2020. Strategies for developing sensitive and specific nanoparticle-based lateral flow assays as point-of-care diagnostic device. Nano Today, 30, 100831.

Soh J H, Lin Y Y, Rana S, Ying J Y, Stevens M M. 2015. Colorimetric detection of small molecules in complex matrixes via target-mediated growth of aptamer-functionalized gold nanoparticles. Analytical Chemistry, 87, 7644-7652. 

Spanu P D, Abbott J C, Amselem J, Burgis T A, Soanes D M, Stüber K, et al. 2010. Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism. Science, 330, 1543-1546. 

Toldrà A, Alcaraz C, Diogène J, O’Sullivan C K, Campàs M. 2019. Detection of Ostreopsis cf. ovata in environmental samples using an electrochemical DNA-based biosensor. Science of the Total Environment, 689, 655-661.

Turner D M. 1956. Studies on cereal mildew in Britain. Transactions of the British Mycological Society, 39, 495-506.

Valentini P, Fiammengo R, Sabella S, Gariboldi M, Maiorano G, Cingolani R, Pompa P P. 2013. Gold-nanoparticle-based colorimetric discrimination of cancer-related point mutations with picomolar sensitivity. Acs Nano, 7, 5530-5538.

Wee E J H, Lau H Y, Botella J R, Trau M. 2015. Re-purposing bridging flocculation for on-site, rapid, qualitative DNA detection in resource-poor settings. Chemical Communications, 51, 5828-5831.

Wyand R A, Brown J K M. 2003. Genetic and forma specialis diversity in Blumeria graminis of cereals and its implications for host-pathogen co-evolution. Molecular Plant Pathology, 4, 187-198. 

Wu M Z, Yan S H, Hu G Q. 1984. Study on overwintering and oversummering of wheat powdery mildew and related biological characteristics. Guizhou Agricultural Sciences3, 31-36. (in Chinese)

Xia X M, Yu Y X, Weidmann M, Pan Y J, Yan S L, Wang Y J. 2014. Rapid detection of shrimp white spot syndrome virus by real time, isothermal recombinase polymerase amplification assay. Plos one, 9, e104667.

Xue M F, Gong S J, Yang L J, Xiang L B, Zeng F X, Shi W Q, Yu D Z. 2018. LAMP detection primer combination of wheat powdery mildew, detection kit and method. CN108103153A. 

Yao K, Peng D L, Jiang C, Zhao W, Li G K, Huang W K, Kong L A, Gao H F, Zheng J W, Peng H. 2021. Rapid and visual detection of Heterodera schachtii using recombinase polymerase amplification combined with cas12a-mediated technology. International Journal of Molecular Sciences, 22, 12577.

Yu L, Shi Z, Fang C, Zhang Y, Liu Y, Li C. 2015. Disposable lateral flow-through strip for smartphone-camera to quantitatively detect alkaline phosphatase activity in milk. Biosens Bioelectron, 69, 307-315.

Zeng X W, Luo Y, Zheng Y M, Duan X Y, Zhou Y L. 2010. Detection of latent infection of wheat leaves caused by Blumeria graminis f. sp. tritici using nested PCR. Journal of Phytopathology, 158, 227-235.

Zhang T, Wang H B, Zhong Z T, Li C Q, Chen W, Liu B, Zhao W D. 2020. A smartphone-based rapid quantitative detection platform for lateral flow strip of human chorionic gonadotropin with optimized image algorithm. Microchemical Journal: Devoted to the Application of Microtechniques in all Branches of Science, 157, 1-8. 

Zhao J, Kang Z S. 2023. Fighting wheat rusts in China: a look back and into the future. Phytopathology Research. 5, 6.

Zheng Y M, Luo Y, Zhou Y L, Zeng X W, Duan X Y, Cao X R., Song Y L, Wang B T. 2013. Real-time PCR quantification of latent infection of wheat powdery mildew in the field. European Journal of Plant Pathology, 136, 565-575.

Zhong Z T, Song L B, He Y F, Zhang B, Chen W, Liu B, Zhao Y D. 2022. Detection of multiple mycotoxins based on catalytic hairpin assembly coupled with pregnancy test strip. Sensors and Actuators B-Chemical. 350, 1-10.

Zou Y F, Cao H B, Cao X R, Liu W, Fan J R, Song Y L. 2018. Regionalization of wheat powdery mildew oversummering in China based on digital elevation. Journal of Integrative Agriculture, 17, 901-910. 

No related articles found!
No Suggested Reading articles found!