de Nova Whole Genome Sequencing of Marssonina Juglandis Causing Walnut (Juglandis Regia L.) Anthracnose and Development of PCR-Based Diagnosis Using Simple Sequence Repeat (SSR) Markers

Aswathy Sanjeev; Sana B. Surma; Sehla Khursheed; Sumaira Hamid; Bilal A. Padder; Sajad M. Zargar; Javaid Iqbal A. Bhat; Imran Khan; Mushtaq Ahmad Bhat; Asha Nabi; Mehraj D. Shah

doi:10.62810/jnsr.v2iSpecial.Issue.123

Authors

Aswathy Sanjeev Division of Plant Pathology, SKUAST-Kashmir, Shalimar, Srinagar-190025, J&K
Sana B. Surma Division of Plant Pathology, SKUAST-Kashmir, Shalimar, Srinagar-190025, J&K
Sehla Khursheed Division of Plant Pathology, SKUAST-Kashmir, Shalimar, Srinagar-190025, J&K
Sumaira Hamid 2Department of Biosciences and Bioengineering, Integral University, Lucknow, U
Bilal A. Padder Division of Plant Pathology, SKUAST-Kashmir, Shalimar, Srinagar-190025, J&K
Sajad M. Zargar Division of Biotechnology, SKUAST-Kashmir, Shalimar, Srinagar-190025, J&K
Javaid Iqbal A. Bhat Division of Biochemistry, SKUAST-Kashmir, Shalimar, Srinagar-190025, J&K
Imran Khan Division of Agri. Statistics, SKUAST-Kashmir, Shalimar, Srinagar-190025, J&K
Mushtaq Ahmad Bhat Division of Plant Pathology, SKUAST-Kashmir, Shalimar, Srinagar-190025, J&K
Asha Nabi Division of Plant Pathology, SKUAST-Kashmir, Shalimar, Srinagar-190025, J&K
Mehraj D. Shah Division of Plant Pathology, SKUAST-Kashmir, Shalimar, Srinagar-190025, J&K

DOI:

https://doi.org/10.62810/jnsr.v2iSpecial.Issue.123

Keywords:

Anthracnose, NGS, SSR, PCR, Whole Genome, Walnut

Abstract

Walnut anthracnose, caused by Marssonina juglandis, is one of the economically important diseases of walnuts worldwide. The pathogen is a filamentous fungus belonging to the class Leotiomycetes. In this study, we isolated and purified the Marssonina juglandis from walnut leaves collected from the walnut orchard of SKUAST-Kashmir. The whole genome of the pathogen was sequenced using the Illumina HiSeq NGS platform. The whole genome of the pathogen was found to be 63.6354 Mb in size with 1916 scaffolds, 12086 genes, 205 total tRNAs, and 6884 SSRs containing 5861 SSRs with 150 flanking regions. A phylogenetic analysis using whole-genome alignment revealed that Marssonina juglandis is closely related to Cryphonectria parasitica and Coniella lustricola. The BlastP analysis using Uniprot, Pfam, and KOG showed 7612, 5515, and 5163 protein hits out of 12086 proteins. The total number of genes responsible for biological processes, molecular function, and cellular components were 1583, 1429, and 1843, respectively. In the Venn diagram, 3912 genes were found to be common in all three software, namely Uniprot, Pfam, and KOG including NR. In addition, we developed the SSR markers based on the whole genome and standardized them against 25 isolates of the Marssonina juglandis collected, isolated, and purified from the walnut-infected leaves in Jammu and Kashmir, India. Out of 30 randomly selected SSRs, 17 SSRs showed successful PCR amplification in 25 pathogen isolates that were found to be highly polymorphic. Further, a PCR-based detection protocol using SSR markers was also developed for early detection of the disease in the field for timely management. We found two primers that could successfully amplify the pathogen in DNA extracted directly from the infected walnut leaves.

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References

Abbey, M., Noaks, M., Belling, G. B., & Nestel, P. J. (1994). Partial replacement of saturated fatty acids with almonds or walnuts lowers total plasma cholesterol and low-density lipoprotein cholesterol. The American Journal of Clinical Nutrition, 59, 995–999. DOI: https://doi.org/10.1093/ajcn/59.5.995

Anonymous. (1991). California walnuts—the versatile nut. European Food and Drink Review, 121, 123–125.

Anonymous. (2013). Marssonina juglandis. Fungal Databases, Nomenclature and Species Banks. MycoBank. Retrieved from www.mycobank.org/BioloMICS.aspx?Link=T&TableKey.Rec.

Ashley, M. W., & Dow, B. D. (1994). The use of microsatellite analysis in population biology: Background, methods and applications. In B. Schierwater, B. Streit, G. P. Wagner, & R. DeSalle (Eds.), Molecular Ecology and Evolution: Approaches and Applications (pp. 185–201). Springer. DOI: https://doi.org/10.1007/978-3-0348-7527-1_10

Behdad, E. (1991). Plant Protection Encyclopedia of Iran: Pests, Diseases, and Weeds. Yad Boud Publisher, Nishat Press.

Belisario, A. (1992). Phytopathological problems of walnut. Informatore Agrario, 48, 51–53.

Belisario, A., Forti, E., Cichello, A. M., Zoin, A., Barbieri, E., & Valier, A. (2001). Epidemiological survey of Gnomonia leptostyla in Juglans regia hedgerow trained orchard. Acta Horticulturae, 544, 405–409. DOI: https://doi.org/10.17660/ActaHortic.2001.544.54

Belisario, A., Scotton, M., Santori, A., & Onori, S. (2008). Variability in the Italian population of Gnomonia leptostyla, homothallism, and resistance of Juglans species to anthracnose. Forest Pathology, 38, 129–145. DOI: https://doi.org/10.1111/j.1439-0329.2007.00540.x

Belisario, B. (2002). Compendium of Nut Crop Diseases in Temperate Zones (B. L. Teviotdale, T. J. Michailides, & J. W. Pscheidt, Eds.). APS Press.

Berry, F. H. (1977). Control of walnut anthracnose with fungicide in a black walnut plantation. Plant Disease Reporter, 61, 378–379.

Berry, L. I., & Frederick, H. (1997). Control of walnut anthracnose with fungicide in a black walnut plantation. Plant Disease Reporter, 61, 378–379.

Beyhan, O. E., Kaya, I., Sen, S. M., & Dogan, M. (1995). Fatty acid composition of walnut (Juglans regia L.) types selected in Darende. Turkish Journal of Agriculture and Forestry, 19, 299–302.

Black, W. M., & Neely, D. (1976). Effects of selected environmental factors on the severity of walnut anthracnose. Proceedings of the American Phytopathology Society, 3, 284.

Çağlarırmak, N. (2003). Biochemical and physical properties of some walnut genotypes (Juglans regia L.). Nahrung/Food, 47, 28–32. DOI: https://doi.org/10.1002/food.200390004

Cline, S., & Neely, D. (1983). Penetration and infection of leaves of black walnut by Marssonina juglandis and resulting lesion development. Phytopathology, 73, 494–497. DOI: https://doi.org/10.1094/Phyto-73-494

Cline, S., & Neely, D. (1984). Relationship between juvenile-leaf resistance to anthracnose and the presence of juglone and hydrajuglone glucoside in black walnut. Phytopathology, 74(2), 185–188. DOI: https://doi.org/10.1094/Phyto-74-185

Crews, C., Hough, P., Godward, J., Brereton, P., Lees, M., & Guiet, S. (2005). Study of the main constituents of some authentic walnut oils. Journal of Agriculture and Food Chemistry, 53, 4853–4860. DOI: https://doi.org/10.1021/jf0478354

Dar, M. S., Padder, B. A., Ahmad, M., Sofi, T. A., Mir, A. A., Nabi, A., & Shah, M. D. (2020). Population structure of Venturia inaequalis, a hemibiotrophic fungus, under different host resistance specificities in the Kashmir valley. Archives of Microbiology, 202, 2245–2253. DOI: https://doi.org/10.1007/s00203-020-01950-8

Dastjerdi, R., Hassani, D., & Nikkhah, M. J. (2009). Study on some characteristics, assessment of pathogenicity and diversity in Gnomonia leptostyla isolates, causal agent of walnut anthracnose in Iran. Iranian Journal of Plant Pathology, 45, 17–18.

Fathima, T., Showkat, U., & Hussain, S. Z. (2018). Nutritional and health benefits of walnuts. Journal of Pharmacognosy and Phytochemistry, 7(2), 1269–1271.

Figueiredo, M. B., & Hennen, J. F. (1995). Anthracnose caused by Marssonina salicicola, a new weeping willow disease (Salix babylonica L.) in the state of S. Paulo, Brazil. Summa Phytopathologica, 21, 253-255. (CF: CAB Abstract 261: 1994-1998).

Garcia, J. M., Agar, I. T., & Strief, J. (1994). Lipid characterization in kernels from different walnut cultivars. Turkish Journal of Agriculture and Forestry, 18, 195-198.

Green, S., & Castlebury, L. A. (2007). Connection of Gnomonia intermedia to Discula betulina and its relationship to other taxa in Gnomoniaceae. Mycological Research, 111, 62-69. DOI: https://doi.org/10.1016/j.mycres.2006.09.003

Greve, C., McGranahan, G., Hasey, J., Snyder, R., Kelly, K., Goldhamer, D., & Labavich, J. (1992). Variation in polyunsaturated fatty acid composition of Persian walnut. Japanese Society for Horticultural Sciences, 117, 518-522. DOI: https://doi.org/10.21273/JASHS.117.3.518

Groppe, K., Sanders, I., Wiemken, A., & Boller, T. (1995). A microsatellite marker for studying the ecology and diversity of fungal endophytes (Epichloë spp.) in grasses. Applied and Environmental Microbiology, 61, 3943–3949. DOI: https://doi.org/10.1128/aem.61.11.3943-3949.1995

Gupta, P. K., Balyan, H. S., Sharma, P. C., & Ramesch, B. (1996). Microsatellites in plants: A new class of molecular markers. Current Science, 70, 45–54.

Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98.

Hassan, B., Bhattacharjee, M., & Wani, S. A. (2020). Growth optima and supply-chain of the walnut crop in India. Asian Journal of Agriculture and Rural Development, 10(2), 708-720. DOI: https://doi.org/10.18488/journal.ajard.2020.102.708.720

Hassan, K. A. (1979). Marssonina juglandis on walnut. FAO Plant Protection Bulletin, 27, 3.

Hedwigia. (1906). Marssonina juglandis (Lib.) Magnus. Species Fungorum, 45, 90.

Hokanson, S. C., Szewc-McFadden, A. K., Lamboy, W. F., & McFerson, J. R. (1998). Microsatellite (SSR) markers reveal genetic identities, genetic diversity, and relationships in a Malus × domestica Borkh. core subset collection. Theoretical and Applied Genetics, 97, 671-683. DOI: https://doi.org/10.1007/s001220050943

Holliday, P. (1989). A dictionary of plant pathology. Cambridge University Press.

Horticulture at a glance. (2018). Government of India, Ministry of Statistics, Planning and Implementation.

INC. (2017). Nuts and Dried Fruits: Statistical Yearbook 2016-2017. International Nut and Dried Fruit Council Foundation. Reus, Spain. https://www.nutfruit.org/files/tech/1497859419_Statistical_Yearbook_2016-2017.pdf

INC. (2018). Nuts and Dried Fruits: Statistical Yearbook 2017-2018. International Nut and Dried Fruit Council Foundation. Reus, Spain. https://www.nutfruit.org/files/tech/1524481168_INC_Statistical_Yearbook_2017-2018.pdf

Jamshidi, S., Salahi, S., & Jamshidi, S. (2012). Genetic diversity of Iranian Ophiognomonia leptostyla (Fr.) populations using RAPD and ISSR markers. Annals of Biological Research, 3(2), 890-898.

Jamshidi, S., Salahi, S., & Samiri, J. (2012). Genetic diversity of Iranian Ophiognomonia leptostyla (Fr.) population using RAPD and ISSR markers. Annals of Biological Research, 2, 890-898.

Jamshidi, S., & Zare, R. (2012). Molecular phylogeny of Ophiognomonia leptostyla isolates collected from Iran based on ITS nrDNA sequences. International Journal of Bioscience, Biochemistry and Bioinformatics, 2, 438-440. DOI: https://doi.org/10.7763/IJBBB.2012.V2.150

Kalkisım, O. (2012). In vitro antifungal evaluation of various plant extracts against walnut anthracnose (Gnomonia leptostyla (Fr.) Ceset de Not.). Journal of Food, Agriculture and Environment, 10, 309-313.

Kanchana-Udomkan, C., Taylor, P. W. J., & Mongkolporn, O. (2004). Development of a bioassay to study anthracnose infection of Capsicum chinense Jacq. fruit caused by Colletotrichum capsici. Thai Journal of Agriculture Science, 37, 293–297.

Kaul, T. N. (1962). Occurrence of Gnomonia leptostyla (Fr.) de Not on walnut in India. Current Science, 31, 349.

Khursheed, S., Farooq, M., Padder, B. A., Surma, S. B., Khan, I., Khan, F. U., Nabi, A., Rashid, R., Sana, B. S., Sofi, T. A., Ahanger, F. A., Hamid, S., & Shah, M. D. (2023). Development of PCR-based SSR markers for Wilsonomyces carpophilus and a PCR-based diagnosis protocol for the early detection of shot hole disease in stone fruit crops. Molecular Biology Reports, 1-10. DOI: https://doi.org/10.21203/rs.3.rs-2354824/v1

Kochman, J. (1973). Fitopatologia. PWRiL, Warszawa.

Lee, D. H., Back, C. G., Win, N. K., Choi, K. H., Kim, K. M., Kang, I. K., Choi, C., Yoon, T. M., Uhm, J. Y., & Jung, H. Y. (2011). Biological characterization of Marssonina coronaria associated with apple blotch disease. Mycobiology, 39(3), 200-205. DOI: https://doi.org/10.5941/MYCO.2011.39.3.200

Martinez, M. L., Labuckas, D. O., Lamargu, A. L., & Maestri, D. M. (2010). Walnuts (Juglans regia L): Genetic resources, chemistry, by-products. Journal of Science and Food Agriculture, 90, 1959-1967. DOI: https://doi.org/10.1002/jsfa.4059

Mejia, L. C., Castlebury, L. A., Rossman, A. Y., Sogonov, M. V., & White, J. F. (2008). Phylogenic placement and taxonomic review of the genus Cryptosporella and its synonyms Ophiovalsa and Winterella (Gnomoniceae, Diaporthales). Mycological Research, 112(1), 23-35. DOI: https://doi.org/10.1016/j.mycres.2007.03.021

Muradoglu, F. H., Oguz, I., Yildiz, K., & Yilmez, H. (2010). Some chemical composition of walnut (Juglans regia L.): Selections from Eastern Turkey. African Journal of Agriculture and Research, 5, 2379-2385.

Murray, M. G., & Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8, 4321-4325. DOI: https://doi.org/10.1093/nar/8.19.4321

Nabi, A., Padder, B. A., Dar, M. S., & Ahmad, M. (2018). Morpho-cultural, pathological, and molecular variability in Thyrostroma carpophilum causing shot hole of stone fruits in India. European Journal of Plant Pathology, 151(3), 613-627. DOI: https://doi.org/10.1007/s10658-017-1398-z

Nadroo, M. A. (2006). Studies on Marssonina blotch of apple in Kashmir. MSc thesis, SK University of Agricultural Science and Technology of Kashmir, Shalimar, Srinagar, 33.

Neely, D. (1986). Total leaf nitrogen correlated with walnut anthracnose resistance. Journal of Arboriculture, 12, 312-315. DOI: https://doi.org/10.48044/jauf.1986.068

Oberhänsli, T., Dalbosco, A., & Leschenne, V. (2021). Multiplexed SSR marker analysis of Diplocarpon coronariae reveals clonality within samples from Middle Europe and genetic distance from Asian and North American isolates. CABI Agriculture and Bioscience, 2, 21. DOI: https://doi.org/10.1186/s43170-021-00039-6

Pinter, C., Fischl, G., Kadlicsko, S., Danko, J., Gara, M., & Mako, S. (2001). Walnut pathogens in Hungary. Acta Phytopathologica et Entomologica Hungarica, 36, 269-273. DOI: https://doi.org/10.1556/APhyt.36.2001.3-4.6

Pollegioni, P., Woeste, K., Major, A., Mugnozza, G., & Malvolti, M. (2009). Characterization of Juglans nigra (L.), Juglans regia (L.), and Juglans x intermedia (Carr.) by SSR markers: A case study in Italy. Silvae Genetica, 58(1-6), 68-78. DOI: https://doi.org/10.1515/sg-2009-0009

Prasad, R. B. N. (1994). Walnuts and Pecans. In Encyclopaedia of Food Science, Food Technology and Nutrition (pp. 4828-4831). Academic Press.

Rafalski, J. A., Vogel, J., Morgante, M., Powell, W., Andre, C., & Tingey, S. V. (1996). Generating and using DNA markers in plants. In Nonmammalian Genomic Analysis: A Practical Guide (pp. 75-134). DOI: https://doi.org/10.1016/B978-012101285-4/50005-9

Rana, J. C., Singh, D., Yadav, S. K., Verma, M. K., Kumar, K., & Predheep, K. (2007). Genetic diversity collected and observed in Persian walnut (Juglans regia L.) in the western Himalayan region of India. Plant Genetic Resources News Letter, 51, 68-73.

Sabaté, J., Fraser, G. E., Burke, K., Knutsen, S. F., Bennett, H., & Linstead, K. D. (1993). Effects of walnuts on serum lipid levels and blood pressure in normal men. The New England Journal of Medicine, 329, 603-610. DOI: https://doi.org/10.1056/NEJM199303043280902

Salahi, S., & Jamshidi, S. (2009). Reaction of different walnut cultivars to Gnomonia leptostyla, causal agent of walnut anthracnose. Journal of New Agricultural Sciences, 5, 55-61.

Salahi, S., Nikkhah, M. J., & Jamshidi, S. (2007). Investigation on the genetic structure of Gnomonia leptostyla populations by PCR-RFLP in Azarbayejan e Sharqi, Iran. Journal of New Agricultural Science, 3, 53-60.

Saremi, H., & Amiri, M. E. (2010). Evaluation of resistance to anthracnose (Marssonina juglandis) among diverse Iranian clones of walnut (Juglans regia). Journal of Food, Agriculture and Environment, 2, 375-378.

Sharma, M. R., Kour, K., Singh, B., Yadev, S., Kotwal, N., Rana, J. C., & Anand, R. (2014). Selection and characterization of elite walnut (Juglans regia L.) clones from seedling origin trees in North Western Himalayan region of India. African Journal of Crop Science, 8, 257-262.

Sharma, R. D., & Sharma, Y. P. (1999). Diseases of temperate nut crops. In Diseases of Horticultural Crops: Fruits (Eds. L. R. Verma & R. C. Sharma, pp. 208-220). Indus Publishing Company.

Shirnina, L. V., & Kotljarova, T. I. (2000). Walnut anthracnose in the Krasnodar territory. Harmfulness of the pathogen. Mikologiya I Fitopatologiya, 34, 62-68.

Sogonov, M. V., Castlebury, L. A., Rossman, A. Y., Mejia, L. C., & White, J. F. (2008). Leaf inhabiting genera of the Gnomoniaceae, Diaporthales. Studies in Mycology, 62, 1–77. DOI: https://doi.org/10.3114/sim.2008.62.01

Todhunter, M. N., & Beineke, W. F. (1984). Effect of anthracnose on growth of grafted black walnut. Plant Diseases, 68, 203-204. DOI: https://doi.org/10.1094/PD-68-203

Tsanova, P., & Roshev, B. (1976). Walnut anthracnose and its control. Rastitelna Zashchita, 24, 20-22.

Tsanova, P., & Rosnev, B. (1976). Study of the fungus flora of walnut in Bulgaria. Gorskostopanska Nauka, 13, 89-102.

Tuite, J. (1969). Plant Pathologist Methods, Fungi and Bacteria. Burgess Publishing.

Uniyal, S. K., Awasthi, A., & Rawat, G. S. (2002). Traditional and ethno-botanical uses of plants in Bhagirathi valley (Western Himalaya). Indian Journal of Traditional Knowledge, 1(1), 7–19.

USDA. (2016). Tree nuts annual: People's Republic of China. Washington, DC: United States Department of Agriculture.

Van-Sambeek, J. W. (2003). Legume ground covers alter defoliation response of black walnut saplings to drought and anthracnose. In Proceedings, 13th Central Hardwood Forest Conference (Eds. J. W. Van Sambeek, J. O. Dawson, F. Ponder, E. F. Loewenstein, & J. S. Fralish, pp. 565). U.S. Department of Agriculture. DOI: https://doi.org/10.2737/NC-GTR-234

Verma, M. K., Sharma, V. K., Lal, S., Mir, J. I., Sofi, A. A., Singh, D. B., Pandit, A. H., Mir, M. A., Mir, A., & Bhat, H. A. (2020). Quality profiling of Indian walnut (Juglans regia) from Kashmir valley. Indian Journal of Agricultural Sciences, 90(3), 573–576. DOI: https://doi.org/10.56093/ijas.v90i3.101478

Wang, X., & Wang, L. (2016). GMATA: An integrated software package for genome-scale SSR mining, marker development and viewing. Frontiers in Plant Science, 7, 1350. DOI: https://doi.org/10.3389/fpls.2016.01350

Weber, J. L., & May, P. E. (1989). Abundant class of human DNA polymorphism which can be typed using the polymerase chain reaction. American Journal of Human Genetics, 44, 388–396.

Werner, M. (1994). Diseases and pests of walnut. Ochrona Roslin, 38, 13-14.

White, T. J., Burns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal DNA for phylogenetics. In PCR Protocols: A Guide to Methods and Applications (Eds. M. A. Innes, D. H. Gelfand, J. J. Sninsky, & T. J. White, pp. 315-322). Academic Press. DOI: https://doi.org/10.1016/B978-0-12-372180-8.50042-1

Woeste, K. E., & Beineke, W. F. (2001). An efficient method for evaluating black walnut for resistance to walnut anthracnose in field plots and the identification of resistant genotypes. Plant Breeding, 120, 454-456. DOI: https://doi.org/10.1046/j.1439-0523.2001.00632.x

Zamani, A. R., Imami, A., Mirza, M. A., & Mohammadi, R. (2011). A study and comparison of control methods of anthracnose disease in walnut trees of Roodbar region. International Journal of Nuts and Related Sciences, 2, 75-81.

Sharma, T. R. (2003). Molecular diagnosis and application of DNA markers in the management of fungal and bacterial plant diseases. Indian Journal of Biotechnology, 2, 99-109.