Characterization of Melon Resistant Cultivars against Fusarium oxysporum f. sp. melonis Based on Morphological Traits and R Gene Patterns

علوم باغبانی

Article 8, Volume 38, Issue 3 - Serial Number 63, September 2024, Pages 577-597   

Document Type: Research Article

DOI: 10.22067/jhs.2024.86574.1324

Authors

F. Shokouhifar^{* 1}; M. Mamarabadi¹; S. Toosi²

¹Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

²Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashahad, Iran

Abstract

Introduction
Melon (Cucumis melo L.) is a diploid plant with (2n = 2x = 24) chromosomes, dicotyledonous and annual, which has been receiving lots of attention for its biological characteristics and economic value for a long time. Iran with production of about 1.6 million tons per year ranks third in melon production in the world after China and Turkey. Vascular wilt caused by the soil borne fungus Fusarium oxysporum f. sp. melonis is one of the most important diseases causing damage to the melon plant. Due to the survival of this fungus in the form of chlamydospores in the soil and plant debris its control has been a difficult challenge so that, the only way to deal with this disease is to use resistant cultivars. The present study was conducted to characterize morphologically different melon cultivars with varying levels of resistance against Fusarium vascular wilt. Furthermore, the presence pattern of two MRGH genes belonging to the MRGH21 linkage group was tracked in the genome of the melon line, and their variations were defined. Moreover, the potential for using these genes in gene-assisted selection was investigated.
 
Materials and Methods
Five different varieties of melon named Charentais T, Charentais Fom1, Charentais Fom2, BG-5384 and the local cultivar Khatouni were grown under greenhouse conditions. Different characteristics of the plant, including leaf shape, male flowers, female flowers and normal flowers, and after harvesting the fruits, fruit weight, fruit diameter and length, diameter of flesh and middle cavity. The differentiation of resistant and sensitive cultivars was investigated based on the evaluated morphological traits. Additionally, the presence patterns of resistance genes were examined in the genomic data of the aforementioned melon cultivars. The genomic analysis of melons aimed to locate the MRGH21 linkage group, which carries several resistance genes. The sequence of this linkage group was tracked from two gene bank databases in NCBI and MELONOMICS database.
 
Results
Based on the obtained results, although it was possible to differentiate melon cultivars based on morphological traits, but since the study of these traits in the evaluation of a large number of samples in selective studies is a very time-consuming and costly task. Therefore, the presence pattern of resistance genes were analyzed in the genomic data of different melon cultivars. The sequence between two genes MRGH12 and MRGH13 including MRGH21 linkage group as one of the linkage groups carrying a number of resistance genes on Ch09 chromosome was retrieved form two gene bank databases in NCBI and MELONOMICS. Due to the presence of multiple point mutations in the genomic data, the MRGH13 gene sequence was selected for investigation in melon cultivars. Specific primers, PSh21-F/R, were designed to track part of this gene's sequence. The tracking results showed that a single specific band, corresponding to the expected size, could be detected in the cultivars Charentais Fom1 and BG-5384. Sequence analysis using the InterPro network tool confirmed the possible role of the protein coded by the MRGH13 gene. It was identified as a member of the protein family carrying leucine-rich repeat sequences, including the TIR, NB-ARC, and LRR domains.
 
Discussion
Attaining suitable markers to distinguish melon cultivars resistant to Fusarium wilt disease can support the development of breeding programs with higher accuracy and speed. The results of the present study showed that based on the morphological traits such as leaf shape, the presence of full flowers, and the number of petals, some differences can be observed between different melon cultivars, but the noteworthy point is that in selection programs searching for these morphological traits will be a very time-consuming and expensive task due to the large number of investigated samples. Therefore, if molecular markers related to the resistance trait are available, the efficiency of breeding programs is expected to increase significantly. In the present study MRGH13 gene was selected to be investigated for tracking in melon cultivars and specific primers were designed to track part of the sequence of this gene. The tracking results showed that a single specific band could be detected in the cultivars Charentais Fom1 and BG-5384. Biological processes related to MRGH13 protein in the QuickGO network tool showed its relevance in the signaling pathway that regulates immune responses. In future studies, it is suggested to evaluate the ability to distinguish resistant cultivars based on resistance genes, including the MRGH13 gene, in a larger number of samples. Moreover, considering to the predicted functions of MRGH13 protein, more investigation on its interaction with other resistance proteins as well as proteins of pathogenic agents can be useful for identification of its functional role in resistance.

Keywords

Fusarium wilt disease of melon; Marker assisted selection; Nucleotide Binding Site–Leucine-Rich Repeat (NBS–LRR) Genes; Plant disease resistance genes

References

1. Abbaspour, N., & Shokouhifar, F. (2012a). Detection of resistance gene homologous in number of melon cultivars. In: 12^th Iraninan Genetics Congeres. university of Shahid Beheshti international Hall, Tehran: Iranian Genetics Society, 1-6.
2. Abbaspour, N., & Shokouhifar, F. (2012b). Detection, amplification and sequencing of a part of the MRGH21 gene in Khatuni melon. In: Third National Conference on Agricultural Biotechnology.
3. Ajuru, M.G., & Okoli, B.E. (2013). The morphological characterization of the melon species in the family cucurbitaceae Juss., and their utilization in Nigeria. International Journal of Modern Botany, 3, 15-19. https://doi/10.5923/j.ijmb.20130302.01
4. Alvarez, J.M., González-Torres, R., Mallor, C., & Gómez-Guillamón, M.L. (2005). Potential sources of resistance to Fusarium wilt and powdery mildew in melons. HortScience, 40, 1657-1660. https://doi.org/10.21273/HORTSCI.40.6.1657
5. Armstrong, G., & Armstrong, J. (1978). Formae speciales and races of Fusarium oxysporum causing wilts of the cucurbitaceae. https://doi.org/10.1094/phyto-68-19
6. Bayless, A.M., & Nishimura, M.T. (2020). Enzymatic functions for Toll/interleukin-1 receptor domain proteins in the plant immune system. Frontiers in Genetics, 11, 539. https://doi.org/10.3389/fgene.2020.00539
7. Benoit, F. (1974). The Fusarium problem in melon growing in Belgium and the relative value of certain rootstocks. Tuinbouwerichten (Belgium), 38, 16-20.
8. Brotman, Y., Normantovich, M., Goldenberg, Z., Zvirin, Z., Kovalski, I., Stovbun, N., Doniger, T., Bolger, A.M., Troadec, C., Bendahmane, A., & Cohen, R. (2013). Dual resistance of melon to Fusarium oxysporum races 0 and 2 and to Papaya ring-spot virus is controlled by a pair of head-to-head-oriented NB-LRR genes of unusual architecture. Molecular Plant, 6, 235-238. https://doi.org/10.1093/mp/ssr096
9. Castanera, R., Ruggieri, V., Pujol, M., Garcia-Mas, J., & Casacuberta, J. M. (2020). An improved melon reference genome with single-molecule sequencing uncovers a recent burst of transposable elements with potential impact on genes. Frontiers in Plant Science, 10, 1815. https://doi.org/10.3389/fpls.2019.01815
10. Chenarani, Z., Shokouhifar, F., Mamarabadi, M., & Farrokhi, N. (2012). Study the effects of explant and medium types in direct regeneration induction in melon (Cucumis melo L., cv. Khatooni). In: 12th Iranian Genetics Congress. Tehran.
11. Chikh-Rouhou, H., Álvarez, J., & González-Torres, R. (2007). Differential interaction between melon cultivars and race 1.2 of Fusarium oxysporum sp. melonis. Communications in Agricultural and Applied Biological Sciences, 72, 825-829.
12. Chikh-Rouhou, H., González-Torres, R., Alvarez, J.M., & Oumouloud, A. (2010). Screening and morphological characterization of melons for resistance to Fusarium oxysporum sp. melonis race 1.2. HortScience, 45, 1021-1025. https://doi.org/10.21273/HORTSCI.45.7.1021
13. Chikh-Rouhou, H., González Torres, R., Alvarez, J., & Pitrat, M. (2008). Characterization of the resistance to Fusarium oxysporum sp. melonis race 1.2 in Cucumis melo ‘BG-5384. In Cucurbitaceae 2008, Proceedings of the IXth EUCARPIA meeting on genetics and breeding of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France).
14. Chilosi, G., Reda, R., Aleandri, M.P., Camele, I., Altieri, L., Montuschi, C., Languasco, L., Rossi, V., Agosteo, G.E., Macrì, C., & Carlucci, A. (2008). Fungi associated with root rot and collapse of melon in Italy. EPPO Bulletin, 38, 147-154. https://doi.org/10.1111/j.1365-2338.2008.01200.x
15. Chupp, C. (1930). Fusarium wilt of muskmelon. Plant Dissese Report, 14, 160.
16. Consortium, G.O. (2015). Gene ontology consortium: going forward. Nucleic Acids Research, 43, D1049-D1056. https://doi.org/10.1093/nar/gku1179
17. Dangl, J.L., Horvath, D.M., & Staskawicz, B.J. (2013). Pivoting the plant immune system from dissection to deployment. Science, 341, 746-751. https://doi.org/10.1126/science.1236011
18. Deokar, A., Sagi, M., & Tar’an, B. (2019). Genome-wide SNP discovery for development of high-density genetic map and QTL mapping of ascochyta blight resistance in chickpea (Cicer arietinum). Theoretical and Applied Genetics, 132, 1861-1872. https://doi.org/10.1007/s00122-019-03322-3
19. Ding, P., & Ding, Y. (2020). Stories of salicylic acid: a plant defense hormone. Trends in Plant Science, 25, 549-565. https://doi.org/10.1016/j.tplants.2020.01.004
20. Doyle, J.J., & Doyle, J.L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue Phytochemical Bulletin, 19(1), 11-15.
21. Enkhbayar, P., Kamiya, M., Osaki, M., Matsumoto, T., & Matsushima, N. (2004). Structural principles of leucine‐rich repeat (LRR) proteins. Proteins: Structure, Function, and Bioinformatics, 54, 394-403. https://doi.org/10.1002/prot.20144
22. Food and agriculture organization of the United Nations, FAO. (2017). FAOSTAT database. FAO-ESS Rome, Italy. http://www.fao.org/faostat
23. Garcia-Mas, J., Benjak, A., Sanseverino, W., Bourgeois, M., Mir, G., González, V.M., Hénaff, E., Câmara, F., Cozzuto, L., Lowy, E., & Alioto, T. (2012). The genome of melon (Cucumis melo). Proceedings of the National Academy of Sciences, 109, 11872-11877. https://doi.org/10.1073/pnas.12054151
24. Garcia-Mas, J., van Leeuwen, H., Monfort, A., de Vicente, M.C., Puigdomènech, P., & Arus, P. (2001). Cloning and mapping of resistance gene homologues in melon. Plant Science, 161, 165-172. https://doi.org/10.1016/S0168-9452(01)00403-4
25. González, V.M., Aventín, N., Centeno, E., & Puigdomènech, P. (2014). Interspecific and intraspecific gene variability in a 1-Mb region containing the highest density of NBS-LRR genes found in the melon BMC Genomics, 15, 1131. https://doi.org/10.1186/1471-2164-15-1131
26. Herman, R., & Perl-Treves, R. (2007). Characterization and inheritance of a new source of resistance to Fusarium oxysporum sp. melonis race 1.2 in Cucumis melo. Plant Disease, 91(9), 1180-1186. https://doi.org/10.1094/PDIS-91-9-1180
27. Horsefield, S., Burdett, H., Zhang, X., Manik, M.K., Shi, Y., Chen, J., Qi, T., Gilley, J., Lai, J.S., Rank, M.X., & Casey, L.W. (2019). NAD+ cleavage activity by animal and plant TIR domains in cell death pathways. Science, 365, 793-799. https://doi.org/10.1126/science.aax1911
28. IPGRI, (International Plant Genetic Resources Institute). (2003). Descriptors for melon (Cucumis melo ). Rome, Italy.
29. Islam, M.R., Hossain, M.R., Jesse, D.M.I., Jung, H.J., Kim, H.T., Park, J.I., & Nou, I.S. (2020) Characterization, identification and expression profiling of genome-wide R-genes in melon and their putative roles in bacterial fruit blotch resistance. BMC genetics, 21, 1-13. https://doi.org/10.1186/s12863-020-00885-9
30. Jiang, G.-L. (2013). Molecular markers and marker-assisted breeding in plants. Plant Breeding from Laboratories to Fields, 3, 45-83. https://doi.org/10.1007/978-3-319-22521-0_15
31. Joobeur, T., King, J.J., Nolin, S.J., Thomas, C.E., & Dean, R.A. (2004). The fusarium wilt resistance locus Fom-2 of melon contains a single resistance gene with complex features. The Plant Journal, 39, 283-297. https://doi.org/10.1111/j.1365-313x.2004.02134.x
32. Kantoğlu, K.Y., Seçer, E., Erzurum, K., Tutluer, İ., Kunter, B., Peşkircioğlu, H., & Sağel, Z. (2010). Improving tolerance to Fusarium oxysporum sp. melonis in melon using tissue culture and mutation techniques. kurumsalarsiv.tenmak.gov.tr
33. Kourelis, J., & van der Hoorn, R.A.L. (2018). Defended to the nines: 25 years of resistance gene cloning identifies nine mechanisms for R protein function. The Plant Cell, 30, 285-299. https://doi.org/10.1105/tpc.17.00579
34. Lanier, L.L. (2005). NK cell recognition. Annual Review of Immunology, 23, 225-274. https://doi.org/10.1146/annurev.immunol.23.021704.115526
35. Leach, J. (1933). A destructive Fusarium wilt of muskmelon. Journal of Agriculture Research, 23, 556-559.
36. Leary, J., JV, L., & WD, W. (1976). Identification of the races of Fusarium oxysporum sp. melonis causing wilt of muskmelon in California. https://doi.org/10.1094/phyto-66-15
37. Lian, Q., Fu, Q., Xu, Y., Hu, Z., Zheng, J., Zhang, A., He, Y., Wang, C., Xu, C., Chen, B., & Garcia-Mas, J. (2021). QTLs and candidate genes analyses for fruit size under domestication and differentiation in melon (Cucumis melo ) based on high resolution maps. BMC Plant Biology, 21, 1-13. https://doi.org/10.1186/s12870-021-02904-y
38. Luck, J.E., Lawrence, G.J., Dodds, P.N., Shepherd, K.W., & Ellis, J.G. (2000). Regions outside of the leucine-rich repeats of flax rust resistance proteins play a role in specificity determination. The Plant Cell, 12, 1367-1377. https://doi.org/10.1105/tpc.12.8.1367
39. Maekawa, T., Kufer, T.A., & Schulze-Lefert, P. (2011). NLR functions in plant and animal immune systems: so far and yet so close. Nature Immunology, 12, 817-826. https://doi.org/10.1038/ni.2083
40. Meyers, B. C., Kozik, A., Griego, A., Kuang, H., & Michelmore, R. W. (2003). Genome-wide analysis of NBS-LRR–encoding genes in Arabidopsis. The Plant Cell, 15, 809-834. https://doi.org/10.1105/tpc.009308
41. Miccolis, V., & Saltveit, M.E. (1991). Morphological and physiological changes during fruit growth and maturation of seven melon cultivars. Journal of the American Society for Horticultural Science, 116, 1025-1029. https://doi.org/10.21273/jashs.116.6.1025
42. Monforte, A., Garcia‐Mas, J., & Arús, P. (2003). Genetic variability in melon based on microsatellite variation. Plant Breeding, 122, 153-157. https://doi.org/10.1046/j.1439-0523.2003.00848.x
43. Morata, J., & Puigdomènech, P. (2017). Variability among Cucurbitaceae species (melon, cucumber and watermelon) in a genomic region containing a cluster of NBS-LRR genes. BMC Genomics, 18, 138. https://doi.org/10.1186/s12864-017-3529-5
44. Oumouloud, A., El Otmani, M., & Álvarez, J. (2015). Molecular characterization of Fom-1 gene and development of functional markers for molecular breeding of resistance to Fusarium race 2 in melon. Euphytica, 205, 491-501. https://doi.org/10.1007/s10681-015-1420-5
45. Oumouloud, A., Torres, R.G., Garcés-Claver, A., Chikh-Rouhou, H., & Alvarez, J. (2013). Differential response of Cucumis melo to Fusarium oxysporum sp. melonis race 1.2 isolates. Crop protection, 44, 91-94. https://doi.org/10.1016/j.cropro.2012.10.001
46. Pitrat, M. (2008) Melon. In: Vegetables I. Springer, pp. 283-315. https://doi.org/10.1007/978-0-387-30443-4_9
47. Quiot, J., JB, Q., & KG, S. (1979). Fréquence des principales viroses identifiées dans une exploitation maraîchère du Sud-Est de la France.
48. Riedl, S.J., Li, W., Chao, Y., Schwarzenbacher, R., & Shi, Y. (2005). Structure of the apoptotic protease-activating factor 1 bound to ADP. Nature, 434, 926-933. https://doi.org/10.1038/nature03465
49. Risser, G., & DW, D. (1976) A proposed nomenclature of Fusarium oxysporum f. melonis races and resistance genes in Cucumis melo. https://doi.org/10.1094/phyto-66-1105
50. Ruggieri, V., Alexiou, K.G., Morata, J., Argyris, J., Pujol, M., Yano, R., Nonaka, S., Ezura, H., Latrasse, D., Boualem, A., & Benhamed, M. (2018). An improved assembly and annotation of the melon (Cucumis melo) reference genome. Scientific Reports, 8 8088. https://doi.org/10.1038/s41598-018-26416-2
51. Shokouhifar, F., Mamarabadi, M., & Khyrabad, M.M. (2016). Tracking of the gene Fom2 and study on the genetic diversity of NB-ARC domain in the number of resistant and sensitive melon cultivars against Fusarium oxysporum sp. melonis (race 1). Australasian Plant Pathology, 45, 279-288. https://doi.org/10.1007/s13313-016-0408-5
52. Singh, R., Kumar, K., Purayannur, S., & Verma, P. K. (2023). Genomics-assisted genetics of complex regions from chickpea chromosome 4 reveals two candidate genes for Ascochyta blight resistance. Plant Science, 334, 111781. https://doi.org/10.1016/j.plantsci.2023.111781
53. Sobhani, A., Bashteni, E., Rafezi, R., Heidarpour, A., & Gharib, M. (2015). Khatooni 93, a new melon cultivar suitable for cultivation in temperate warm areas of Iran. Research Achivments for Field and Horticulture Crops, 4, 117-126. https://doi.org/10.22092/rafhc.2016.109501
54. Soltani, F., Shajari, M., Mirbehbahani, G.S., & Bihamta, M.R. (2022). Assessment of melon genetic diversity based on fruit phenotypic traits and flowering habits. International Journal of Horticultural Science and Technology, 9, 97-116. https://doi.org/10.22059/ijhst.2021.313939.415
55. Stepansky, A., Kovalski, I., & Perl-Treves, R. (1999). Intraspecific classification of melons (Cucumis melo) in view of their phenotypic and molecular variation. Plant Systematics and Evolution, 217, 313-332. https://doi.org/10.1007/bf00984373
56. Sudheesh, S., Kahrood, H.V., Braich, S., Dron, N., Hobson, K., Cogan, N.O., & Kaur, S. (2021). Application of genomics approaches for the improvement in ascochyta blight resistance in chickpea. Agronomy, 11, 1937. https://doi.org/10.3390/agronomy11101937
57. Szamosi, C., Solmaz, I., Sari, N., & Bársony, C. (2010). Morphological evaluation and comparison of Hungarian and Turkish melon (Cucumis melo) germplasm. Scientia Horticulturae, 124, 170-182. https://doi.org/10.1016/j.scienta.2009.12.024
58. Teimouri, S., Rahnama, K., Hajian Shahri, M., & Afzali, H. (2013). Identification, distribution and pathogenicity of Fusarium species isolated from root and crown of cantaloupe and melon in Khorasan Razavi province (In Persian), 35-46.
59. Traband, R.C., Wang, X., Lui, J., Yu, L., Hiraoka, Y., Herniter, I.A., Bowman, C., Resendiz, M., Wang, Z., Knowles, S.P., & Lo, S. (2023). Exploring the phylogenetic relationship among citrus through leaf shape traits: A morphological study on citrus leaves. Horticulturae, 9, 793. https://doi.org/10.3390/horticulturae9070793
60. Udoh, L.I., Obaseojei, W.P., & Uzoebo, C. (2021). Single nucleotide polymorphisms: a modern tool to screen plants for desirable traits. In: Plant Breeding-current and Future Views. https://doi.org/10.5772/intechopen.94935
61. van der Biezen, E.A., & Jones, J.D. (1998). The NB-ARC domain: a novel signalling motif shared by plant resistance gene products and regulators of cell death in animals. Current Biology, 8, R226-R228. https://doi.org/10.1016/s0960-9822(98)70145-9
62. Van der Hoorn, R.A., Kruijt, M., Roth, , Brandwagt, B.F., Joosten, M.H., & De Wit, P.J. (2001). Intragenic recombination generated two distinct Cf genes that mediate AVR9 recognition in the natural population of Lycopersicon pimpinellifolium. Proceedings of the National Academy of Sciences, 98, 10493-10498. https://doi.org/10.1073/pnas.181241798
63. van Leeuwen, H., Garcia-Mas, J., Coca, M., Puigdoménech, P., & Monfort, A. (2005). Analysis of the melon genome in regions encompassing TIR-NBS-LRR resistance genes. Molecular Genetics and Genomics, 273, 240-251. https://doi.org/10.1007/s00438-004-1104-7
64. Wang, J., Wang, J., Hu, M., Wu, S., Qi, J., Wang, G., Han, Z., Qi, Y., Gao, N., Wang, H.W., & Zhou, J.M. (2019). Ligand-triggered allosteric ADP release primes a plant NLR complex. Science, 364, eaav5868. https://doi.org/10.1126/science.aav5868
65. Zheng, X., & Wolff, D. W. (2000). Randomly amplified polymorphic DNA markers linked to fusarium wilt resistance in diverse melons. HortScience, 35, 716-721. https://doi.org/10.21273/hortsci.35.4.716
66. Zink, F. (1985). Inheritance of resistance in muskmelon to Fusarium wilt. Journal of the American Society for Horticultural Science, 110, 600-604. https://doi.org/10.21273/jashs.110.5.600
67. Zink, F., & Thomas, C. (1990). Genetics of resistance to Fusarium oxysporum sp. melonis races 0, 1 and 2 in muskmelon line MR-1. Phytopathology, 80, 1230-1232. https://doi.org/10.1094/phyto-80-1230