Identification of pathogenic fungi in garlic bulbs during storage and in the root zone during plant growth

Relevance and methods. Losses of agricultural crops are associated not only with the development of diseases during the growing season, but also during post-harvest storage. Garlic is a popular vegetable and aromatic crop in world. Significant losses in garlic yield during cultivation and storage are associated with fungal pathogens, the most harmful of which are representatives of the genus Fusarium. In the Moscow region, the defeat of garlic by Fusarium occurs annually, but with varying intensity. At the Federal Scientific Vegetable Center (FSVC), it was shown that rot and wilting of garlic plants is caused by a complex of pathogenic fungi, including mainly different species of Fusarium. At the same time, the ratio of Fusarium species in the pathogenic complex changes from year to year, new Fusarium species and their races are registered. The aim of this study was to identify fungal phytopathogens causing dry rot of garlic cloves during post-harvest storage. To carry out the work, garlic bulbs of cultivars Dubkovsky and Strelets were taken from the FSVC storage.

Results. As a result of visual examination, cloves with symptoms of dry rot were identified. The diseased cloves tissues were plated on potato dextrose agar to obtain fungal colonies. Analysis of the morphological and cultural characteristics of fungal isolates, as well as the nucleotide sequences of four DNA regions (ITS spacers, genes EF1α, RPB1, and RPB2) showed that the causative agent of dry rot of garlic cloves is the pathogenic fungus Fusarium proliferatum. In addition, in the field, identification was carried out based on the analysis of the sequences of spacers ITS and the EF1αgene of phytopathogenic fungi inhabiting the root zone of garlic plants. As a result, two species of fungi of the genus Fusarium (F. proliferatum and F. oxysporum f. sp. cepae), as well as the species Rhizoctonia solani, Volutella rosea, and Ceratobasidium sp. were found in the root zone of garlic cultivars.

1. Ma L.J., Geiser D.M., Proctor R.H., Rooney A.P., O'Donnell K., Trail F., Gardiner D.M., Manners J.M., Kazan K. Fusarium pathogenomics. Annu Rev Microbiol. 2013;(67):399-416. https://doi.org/10.1146/annurev-micro-092412-155650.

2. Kalman B., Abraham D., Graph S., Perl-Treves R., Meller Harel Y., Degani O. Isolation and Identification of Fusarium spp., the Causal Agents of Onion (Allium cepa) Basal Rot in Northeastern Israel. Biology (Basel). 2020;9(4):69. https://doi.org/10.3390/biology9040069

3. Summerell B.A. Resolving Fusarium: Current Status of the Genus. Annu Rev Phytopathol. 2019;(57):323-339. https://doi.org/10.1146/annurev-phyto-082718-100204

4. Gagkaeva T., Gavrilova O., Orina A., Lebedin Y., Shanin I., Petukhov P., Eremin S. Analysis of Toxigenic Fusarium Species Associated with Wheat Grain from Three Regions of Russia: Volga, Ural, and West Siberia. Toxins (Basel). 2019;11(5):252. https://doi.org/10.3390/toxins11050252.

5. Rout E., Nanda S., Joshi R.K. Molecular characterization and heterologous expression of a pathogen induced PR5 gene from garlic (Allium sativum L.) conferring enhanced resistance to necrotrophic fungi. EurJPlantPathol. 2016;(144):345–360. https://doi.org/10.1007/s10658-015-0772-y

6. Seredin T.M., Gerasimova L.I., Kozar E.G., Engalycheva I.A., Baranova E.V. Harmfulness of mycosiss on culture of garlic winter-annual in the conditions of Moscow region. Vegetable crops of Russia. 2018;(6):84-90. (In Russ.) https://doi.org/10.18619/2072-9146-2018-6-84-90

7. Gálvez L., Urbaniak M., Waśkiewicz A., Stępień L., Palmero D. Fusarium proliferatum – Causal agent of garlic bulb rot in Spain: Genetic variability and mycotoxin production. Food Microbiology. 2017;(67):41-48. https://doi.org/10.1016/j.fm.2017.05.006

8. Moharam M.H.A., Farrag E.S.H., Mohamed M.D.A. Pathogenic fungi in garlic seed cloves and first report of Fusarium proliferatum causing cloves rot of stored bulbs in upper Egypt. Archives of Phytopathology and Plant Protection. 2013;(46):2096-2103. https://doi.org/10.1080/03235408.2013.785122

9. Mondani L., Chiusa G., Battilani P. Chemical and biological control of Fusarium species involved in garlic dry rot at early crop stages. European Journal of Plant Pathology. 2021;(7). https://doi.org/10.1007/s10658-021-02265-0

10. Timina L.T., Engalicheva I.A. Complex of pathogenes on vegetable crops in condition of central region of Russia. Vegetable crops of Russia. 2015;(3-4):123-129. (In Russ.) https://doi.org/10.18619/2072-9146-2015-3-4-123-129

11. Leyronas C., Chrétien P.L., Troulet C., Duffaud M., Villeneuve F., Morris C.E., Hunyadi H. First Report of Fusarium proliferatum Causing Garlic Clove Rot in France. Plant Disease. 2018;(102):2658 https://doi.org/10.1094/PDIS-06-18-0962-PDN

12. White T.J., Bruns T., Lee S., Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, pp. 315–322 in M.A. Innis, D.H. Gelfand, J.J. Sninsky (Eds):PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego (USA) 1990.

13. O'Donnell K., Kistler H.C., Cigelnik E., Ploetz R.C. Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proc Natl Acad Sci USA. 1998;95(5):2044-2049. https://doi.org/10.1073/pnas.95.5.2044

14. O'Donnell K., Sutton D.A., Rinaldi M.G., Sarver B.A., Balajee S.A., Schroers H.J., Summerbell R.C., Robert V.A., Crous P.W., Zhang N., Aoki T., Jung K., Park J., Lee Y.H., Kang S., Park B., Geiser D.M. Internet-accessible DNA sequence database for identifying fusaria from human and animal infections. J Clin Microbiol. 2010;48(10):3708-3718. https://doi.org/10.1128/JCM.00989-10.

15. Liu Y.J., Whelen S., Hall B.D. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit. Mol Biol Evol. 1999;16(12):1799-808. https://doi.org/10.1093/oxfordjournals.molbev.a026092

16. Leslie J.F., Summerell B.A. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK. 2006. https://doi.org/10.1002/9780470278376

17. Anisimova O.K., Seredin T.M., Danilova O.A., Filyushin M. First Report of Fusarium proliferatum Causing Garlic clove Rot in Russian Federation. Plant Disease. 2021. (In press) https://doi.org/10.1094/PDIS-12-20-2743-PDN

18. Mondani L., Chiusa G., Battilani P. Fungi Associated with Garlic During the Cropping Season, with Focus on Fusarium proliferatum and F. oxysporum. Plant Health Progress. 2021;22(1):37-46. https://doi.org/10.1094/PHP-06-20-0054-RS