Rapid Diagnosis of Citrus Anthracnose Caused by Colletotrichum gloeosporioides Using a LAMP (Loop-Mediated Isothermal Amplification) Assay

Abstract

Anthracnose disease in citrus occurs as dieback in shoots, spot on leaves, early leaf and fruit drop and anthracnose symptoms in fruits. Colletotrichum gloeosporioides, a causal agent of anthracnose, causes severe infections in many citrus species and varieties, including lemon. Fungal pathogen C. gloeosporioides can be diagnosed by morphological, molecular methods and protein-based spectrum analyzes. This study was conducted to perform a rapid identification of C. gloeosporioides, using the LAMP technique. Specific primers for the LAMP method were designed using the computer program (Primary Primer Explorer V3) (http://primerexplorer.jp/e), with primer sets specific to each gene region (F3 / B3 and FIP / BIP) of the gene sequences of the C. gloeosporioides strain. The specificity of the primers intended for use in the LAMP reaction was evaluated by using different three isolates of C. gloeosporioides and Fs4 isolates of Fusarium solani using a total of 3 DNA samples. Genomic DNA was used in the amount of 1ng/µl-1 in the PCR study and 10 fg / µl-1 in the LAMP study. As a result, C. gloeosporioides isolates glowed in tubes when examined with fluorescent dye. However, no glare was observed in the tube with F. solani. LAMP analysis showed that it successfully detected genomic DNA from C. gloeosporioides, but F. solani was unable to detect the genomic DNA obtained. After 2 hours of analysis with LAMP technique, the pathogen was detected in a short time with the successful results.

Keywords

• Anthracnose,C. gloesoporioides,LAMP,İdentification,Citrus

Turunçgil Antraknoz Etmeni Colletotrichum gloeosporioides’in LAMP (Loop-Mediated İsothermal Amplification) Tekniği Kullanılarak Hızlı Tanısı

Abstract

Turunçgillerde antraknoz hastalığı sürgünlerde geriye doğru kuruma, yapraklarda leke, erken dönem yaprak ve meyve dökümleri, meyvelerde antraknoz belirtileri şeklinde ortaya çıkmaktadır. Antraknoz etmeni Colletotrichum gloeosporioides, ülkemizde özellikle limon başta olmak üzere birçok turunçgil tür ve çeşitlerinde şiddetli enfeksiyonlara yol açmaktadır. C. gloeosporioides fungal patojeninin tanısı, morfolojik, moleküler yöntemler ve proteine dayalı spektrum analizleri ile yapılabilmektedir. Bu çalışma, LAMP tekniğini kullanarak turunçgil antraknoz etmeni C.gloeosporioides’in hızlı tanısını gerçekleştirmek için yürütülmüştür. LAMP yöntemi için spesifik primerler, C. gloeosporioides türünün gen dizileri her gen bölgesine özgü primer setleri (F3/B3 ve FIP/BIP) ‘Primer Explorer V3’ adlı bilgisayar programı (http://primerexplorer.jp/e) kullanılarak tasarlanmıştır. LAMP reaksiyonunda kullanılmak üzere tasarlanan primerlerin özgüllükleri C. gloeosporioides’ in 3 farklı izolatları ile Fusarium solani’ nin Fs4 izolatı olmak üzere toplam 3 DNA örneği kullanılarak değerlendirilmiştir. PCR çalışmasında genomik DNA 1ng/µl-1, LAMP çalışmasında 10 fg/µl-1 miktarında kullanılmıştır. Sonuç olarak, Flouresan boya ile incelendiğinde, C. gloeosporioides izolatlarının bulunduğu tüplerde parlama olmuştur. Ancak, F. solani’ nin bulunduğu tüpte parlama gözlenmemiştir. LAMP analizinin C. gloeosporioides’ den elde edilen genomik DNA'yı başarıyla tespit ettiğini göstermiş, fakat F. solani’ elde edilen genomik DNA'yı tespit edememiştir. LAMP tekniği ile 2 saat süren bir analiz sonucunda elde edilen başarılı sonuçlarla kısa süre içerisinde patojenin varlığı tespit edilmiştir.

Keywords

• Antraknoz,C. gloesoporioides,LAMP,Teşhis

References

1. Bekele, B., Hodgetts, J., Tomlinson, J., Boonham, N., Nikolić, P., Swarbrick, P., Dickinson, M. (2011) Use of a real-time LAMP isothermal assay for detecting 16SrII and 16SrXII phytoplasmas in fruit and weeds of the Ethiopian Rift Valley. Plant Pathol 60:345–355.
2. Boubourakas, I.N., Fukuta, S., Kyriakopoulou, P.E. (2009) Sensitive and rapid detection of peach latent mosaic viroid by the reverse transcription loop-mediated isothermal amplification. J Virol Method 160:63–68.
3. Chandra, A., Keizerweerd, A.T., Que, Y., Grisham, M.P., 2015. Loop-mediated isothermal amplification (LAMP) based detection of Colletotrichum falcatum causing red rot in sugarcane. Mol Biol Rep (2015) 42:1309–1316.
4. Dai, T., Lu, C.C, Lu, J., Dong, S.M., Ye W.W., Wang, Y.C., Zheng, X.B, (2012) Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae. FEMS Microbiol Lett 334:27–34.
5. Dai, T., Yang, X., Hu, T., Zhangyan, L., Yue, X., Chenchen, L., 2019. A novel LAMP assay for the detection of the Phytophthora cinnamomi utilizing a new target gene identified from genome sequences. Plant Diseases. 103 (12): 3101-3107.
6. Dean, R., Van Kan, J.A.L., Pretorius, Z.A., Hammond-Kosack, K.E., Di Pietro, A., 2012. The Top 10 fungal pathogens in molecular plant pathology. Molecular Plant Pathology 13: 414–430.
7. Dophal, S., Boluk, G., Babler, B., Stulberg, M.J., Rascoe, J., Nakhla, M.K., Chapman, T., Crockford, A., Melzer, M.J., Alverez, A.M., Arif, M., 2019. Comparative genomics approach to develop a highly reliable duplex TaqMan Qpcr assay for sensitive detection of genus Dickeya and Dickeya dianthicola. APS Annual Meeting, August 3-7, Cleveland, Ohio, USA, p.s2.135.
8. Duan, Y., Ge, C., Zhang, X., Wang, J., Zhou, M. (2014) A rapid detection method for the plant pathogen Sclerotinia sclerotiorum based on loop-mediated isothermal amplification (LAMP). Aust Plant Pathol 43:61–66.

9. FAOSTAT, 2017, World Production Data. Statistics of Food and Agriculture Organization of the United Nations. http:/www.faostat.fao.org.
10. Fukuta, S., Takahashi, R., Kuroyanagi, S., Miyake, N., Nagai, H., Suzuki, H.,Hashizume F., Tsuji, T., Taguchi, H., Watanabe, H., Kageyama, K., 2013. Detection of Pythium aphanidermatum in tomato using loopmediated isothermal amplification (LAMP) with species-specific primers. Eur J Plant Pathol 136:689–701.
11. Fukuta, S., Takahashi, R., Kuroyanagi, S., Ishiguru, Y., Miyake, N., Nagai, H., Suzuki,H., Tsuji, T., Hashizume, F., Watanabe, H., Kageyama, K. (2014) Development of loop-mediated isothermal amplification (LAMP) assay for the detection of Pythium myriotylum. Lett Appl Microbiol 59:49–57
12. Goto, M., Hond, E., Ogura, A., Nomoto, A., Hanaki, K.I. (2009) Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques 46:167–172.
13. Hudson, O., Waliullah, S. Wang, L., Ji, P., Ali, E., 2019. A loop-mediated isothermal amplification (LAMP) assay for rapid detection of Fusarium oxysporum causing fusarium wilt of watermelon. APS Annual Meeting, August 3-7, Cleveland, Ohio, USA, p.s2.125.
14. Kandan, A., Akhtar, J., Singh, B., Pal, D., Chan, D., Agarwal, P.C., Dubey, S.C., 2016. Application of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of fungal pathogen, Colletotrichum capsici in Capsicum annuum. ournal of Environmental Biology, Vol. 37, 1355-1360.
15. Katoh, H., Fukuda, T., Nishigawa, H., Natsuaki, T., 2016. Rapid detection of Colletotrichum gloeosporioides in infected strawberry plants using loop-mediated isothermal amplification. J Gen Plant Pathol 82:190–198.
16. Kikuchi, T., Aikawa, T., Oeda, Y., Karim, N., Kanzaki, N. (2009) A rapid and precise diagnostic method for detecting the pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification. Phytopathology 99:1365–1369.
17. Kurt, Ş., Uysal, A., Soylu, E.M., Kara, M., Soylu, S. 2019. Characterization and Pathogenicity of Fusairum solani associated with Dry Root of Citrus in Eastern Mediterranean Region of Turkey. Journal of General Plant Pathology (in Press).
18. Lenarcic, R., Morisset, D., Pirc, M., Llop, P., Ravnikar, M., Dreo, T. (2014) Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum. PLoS One 9:e96027.
19. Li, B., Du, J., Lan, C., Liu, P., Weng, Q., Chen, Q. (2013) Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Fusarium oxysporum f. sp. cubense race 4. Eur J Plant Pathol 135:903–911.
20. Niessen, L., 2015. Current state and future perspectives of loop-mediated isothermal amplification (LAMP)-based diagnosis of filamentous fungi and yeasts. Appl Microbiol Biotechnol. 99:553–574.
21. Notomi, T., Okayama, H.,Masubuchi, H., Yonekawa, T.,Watanabe, K., Amino, N., Hase, T. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:e63. doi:10.1093/nar/28.12.e63
22. O’Connell, R. J., Thon, M. R., Hacquard, S., Amyotte, S. G., Kleemann, J., Torres,M. F., 2012. Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses. Nature Genetics. 44(9):1060-5.
23. Ordóñez, N., Salacinas, M., Mendes, O., Seidl, M. F., Meijer, H. J. G., Schoen, C. D., & Kema, G. H. J. (2019). A Loop‐Mediated Isothermal Amplification assay based on unique markers derived from genotyping by sequencing data for rapid in‐plant diagnosis of Panama disease caused by Tropical Race 4 in banana. Plant Pathology. in press: doi:10.1111/ppa.13093
24. Pu, J., Xie, Y., Zhang, H., Zhang, X., Qi, Y., Peng, J. (2014) Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium mangiferae associated with mango malformation. Phys Mol Plant Pathol 86:81–88
25. Takahashi, R., Fukuta, S., Kuroyanagi, S., Miyake, N., Nagai, H., Kageyama, K., Ishiguro, Y. (2014) Development and application of a loop-mediated isothermal amplification assay for rapid detection of Pythium helicoides. FEMS Microbiol Lett 355:28–35.
26. Thiessen, L.D., Neill, T.M., Mahaffee, W.F., 2018. Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator. Peer J 6:e4639;DOI 10.7717/peerj.4639.
27. Tian, Q., Lu, C., Wang, S., Xiong, Q., Zhang, H., Wang, Y., Zheng, X., 2017. Rapid diagnosis of soybean anthracnose caused by Colletotrichum truncatum using a loop-mediated isothermal amplification (LAMP) assay. Eur J Plant Pathol 148:785–793.
28. Tomlinson, J.A,, Dickinson, M.J,, Boonham, N. (2010a) Detection of Botrytis cinerea by loop-mediated isothermal amplification. Lett Appl Microbiol 51:650–657.
29. Tomlinson, J.A., Dickinson, M.J., Boonham, N. (2010b) Rapid detection of Phytophthora ramorum and P. kernoviae by two-minute DNA extraction followed by isothermal amplification and amplicon detection by generic lateral flow device. Phytopathology 100:143–149.
30. Tomlinson, J.A., Dickinson, M.J., Boonham, N., Ostoja-Starzewska, S., Webb, K., Cole, J., Barnes, A., Dickinson, M., Boonham, N. (2013). A loopmidiated isothermal amplification-based method for confirmation of Guigniardia citricarpa in citrus black spot lesions. Eur J Plant Pathol 136:217–224.
31. Tomita, N., Mori, Y., Kanda, H., Notomi, T. (2008) Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection products. Nat Protoc 3:877–882.
32. Rigano, L.A., Malamud, F., Orce, I.G., Filippone, M.P., Marano, M.R., do Amaral, A.M., Castagnaro, A.P., Vojnov, A.A. (2014) Rapid and sensitive detection of Candidatus liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick. BMC Microbiol 14:86.
33. Uysal, A., Kurt, Ş., Akgül, D.S., 2016. Akdeniz Bölgesi Limon Bahçelerinde Antraknoz Hastalığına Neden olan Colletotrichum gloeosporioides' in Patojenik ve Moleküler Karakterizasyonu. Uluslararası Katılımlı Türkiye VI. Bitki Koruma Kongresi, 5-8 Eylül, Konya, s. 597.
34. Uysal, A., ve Kurt, Ş., 2018. An Important Fungal Disease on Citrus Orchards in Erdemli: Anthracnose. International Erdemli Symposium. 19-21 April Mersin/Turkey.
35. Waliullah, S. Hudson, O., Oliver, J. E., Ji, P., Ali, E., 2019. Comparative study of methods for detecting Xylella fastidiosa causing causing bacterial leaf scorch in blueberry. APS Annual Meeting, August 3-7, Cleveland, Ohio, USA, p.s2.78.
36. Zhang X, Zhang H, Pu J, Qi Y, Yu Q, Xie Y, Peng J (2013) Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil. PLoS One 8:e82841.