DREB1 and DREB2 Genes in Garlic (Allium sativum L.): Genome-Wide Identification, Characterization, and Stress Response.

Dehydration-responsive element-binding (DREB) transcription factors (TFs) of the A1 and A2 subfamilies involved in plant stress responses have not yet been reported in Allium species. In this study, we used bioinformatics and comparative transcriptomics to identify and characterize DREB A1 and A2 genes redundant in garlic (Allium sativum L.) and analyze their expression in A. sativum cultivars differing in the sensitivity to cold and Fusarium infection. Eight A1 (AsaDREB1.1-1.8) and eight A2 (AsaDREB2.1-2.8) genes were identified. AsaDREB1.1-1.8 genes located in tandem on chromosome 1 had similar expression patterns, suggesting functional redundancy. AsaDREB2.1-2.8 were scattered on different chromosomes and had organ- and genotype-specific expressions. AsaDREB1 and AsaDREB2 promoters contained 7 and 9 hormone- and stress-responsive cis-regulatory elements, respectively, and 13 sites associated with TF binding and plant development. In both Fusarium-resistant and -sensitive cultivars, fungal infection upregulated the AsaDREB1.1-1.5, 1.8, 2.2, 2.6, and 2.8 genes and downregulated AsaDREB2.5, but the magnitude of response depended on the infection susceptibility of the cultivar. Cold exposure strongly upregulated the AsaDREB1 genes, but downregulated most AsaDREB2 genes. Our results provide the foundation for further functional analysis of the DREB TFs in Allium crops and could contribute to the breeding of stress-tolerant varieties.

Genome-Wide Identification, Expression, and Response to Fusarium Infection of the SWEET Gene Family in Garlic (Allium sativum L.).

Proteins of the SWEET (Sugar Will Eventually be Exported Transporters) family play an important role in plant development, adaptation, and stress response by functioning as transmembrane uniporters of soluble sugars. However, the information on the SWEET family in the plants of the Allium genus, which includes many crop species, is lacking. In this study, we performed a genome-wide analysis of garlic (Allium sativum L.) and identified 27 genes putatively encoding clade I-IV SWEET proteins. The promoters of the A. sativum (As) SWEET genes contained hormone- and stress-sensitive elements associated with plant response to phytopathogens. AsSWEET genes had distinct expression patterns in garlic organs. The expression levels and dynamics of clade III AsSWEET3, AsSWEET9, and AsSWEET11 genes significantly differed between Fusarium-resistant and -susceptible garlic cultivars subjected to F. proliferatum infection, suggesting the role of these genes in the garlic defense against the pathogen. Our results provide insights into the role of SWEET sugar uniporters in A. sativum and may be useful for breeding Fusarium-resistant Allium cultivars.

Thaumatin-like Protein (TLP) Genes in Garlic (Allium sativum L.): Genome-Wide Identification, Characterization, and Expression in Response to Fusarium proliferatum Infection.

Plant antifungal proteins include the pathogenesis-related (PR)-5 family of fungi- and other stress-responsive thaumatin-like proteins (TLPs). However, the information on the TLPs of garlic (Allium sativum L.), which is often infected with soil Fusarium fungi, is very limited. In the present study, we identified 32 TLP homologs in the A. sativum cv. Ershuizao genome, which may function in the defense against Fusarium attack. The promoters of A. sativumTLP (AsTLP) genes contained cis-acting elements associated with hormone signaling and response to various types of stress, including those caused by fungal pathogens and their elicitors. The expression of AsTLP genes in Fusarium-resistant and -susceptible garlic cultivars was differently regulated by F. proliferatum infection. Thus, in the roots the mRNA levels of AsTLP7-9 and 21 genes were increased in resistant and decreased in susceptible A. sativum cultivars, suggesting the involvement of these genes in the garlic response to F. proliferatum attack. Our results provide insights into the role of TLPs in garlic and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.

Pathogenesis-Related Genes of PR1, PR2, PR4, and PR5 Families Are Involved in the Response to Fusarium Infection in Garlic (Allium sativum L.).

Plants of the genus Allium developed a diversity of defense mechanisms against pathogenic fungi of the genus Fusarium, including transcriptional activation of pathogenesis-related (PR) genes. However, the information on the regulation of PR factors in garlic (Allium sativum L.) is limited. In the present study, we identified AsPR genes putatively encoding PR1, PR2, PR4, and PR5 proteins in A. sativum cv. Ershuizao, which may be involved in the defense against Fusarium infection. The promoters of the AsPR1-5 genes contained jasmonic acid-, salicylic acid-, gibberellin-, abscisic acid-, auxin-, ethylene-, and stress-responsive elements associated with the response to plant parasites. The expression of AsPR1c, d, g, k, AsPR2b, AsPR5a, c (in roots), and AsPR4a(c), b, and AsPR2c (in stems and cloves) significantly differed between garlic cultivars resistant and susceptible to Fusarium rot, suggesting that it could define the PR protein-mediated protection against Fusarium infection in garlic. Our results provide insights into the role of PR factors in A. sativum and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.

Genome-Wide Identification and Expression of Chitinase Class I Genes in Garlic (Allium sativum L.) Cultivars Resistant and Susceptible to Fusarium proliferatum.

Vegetables of the Allium genus are prone to infection by Fusarium fungi. Chitinases of the GH19 family are pathogenesis-related proteins inhibiting fungal growth through the hydrolysis of cell wall chitin; however, the information on garlic (Allium sativum L.) chitinases is limited. In the present study, we identified seven class I chitinase genes, AsCHI1-7, in the A. sativum cv. Ershuizao genome, which may have a conserved function in the garlic defense against Fusarium attack. The AsCHI1-7 promoters contained jasmonic acid-, salicylic acid-, gibberellins-, abscisic acid-, auxin-, ethylene-, and stress-responsive elements associated with defense against pathogens. The expression of AsCHI2, AsCHI3, and AsCHI7 genes was constitutive in Fusarium-resistant and -susceptible garlic cultivars and was mostly induced at the early stage of F. proliferatum infection. In roots, AsCHI2 and AsCHI3 mRNA levels were increased in the susceptible and decreased in the resistant cultivar, whereas in cloves, AsCHI7 and AsCHI5 expression was decreased in the susceptible but increased in the resistant plants, suggesting that these genes are involved in the garlic response to Fusarium proliferatum attack. Our results provide insights into the role of chitinases in garlic and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.

First Report of Fusarium proliferatum Causing Garlic clove Rot in Russian Federation.

Garlic (Allium sativum L.) is a widely consumed bulbous crop both worldwide and in Russia. About 200,000 tons of garlic is produced in Russia annually (https://rosstat.gov.ru/). Significant pre- and post-harvest losses of garlic regularly occur due to Fusarium sp. (Taylor et al., 2013). Since September 2018, rotting has been observed in Russia during garlic bulb storage (data of the Federal Scientific Vegetable Center, FSVC, Moscow Region). The outer bulb surface looked healthy, but underneath the integumentary scales, the cloves had light brown and brown spots. When grown, diseased plants were characterized by root and bulb disruption and leaf drying; for some cultivars, up to 100% of plants died. In January 2020, cv. Strelets and Dubkovsky bulbs, collected in July 2019, with rot symptoms, were taken from the FSVC storage. Necrotic clove tissue fragments (0.2-0.5 cm) were cut, sanitized with 70% ethanol for 3 min, rinsed with sterile water, and incubated on potato dextrose agar (PDA) with 1 mg/ml ampicillin at 22°C in the dark. Four single-spore cultures were obtained from four diseased bulbs. After 6 days of incubation, the isolates produced abundant aerial white mycelia and acquired a purple pigmentation. The hyphae were hyaline with septation. All isolates (Dubkovsky, Dubkovsky 2, Strelets, and Strelets 2) produced numerous oval unicellular microconidia without septa, 4.1 to 11.6 × 1.3 to 3.4 µm (n = 50) and very few macroconidia with 3-4 septa (21 to 26 × 3 to 4 µm (n = 30)), narrowed at both ends. The cultural and conidial characteristics of the isolates corresponded to Fusarium species (Leslie and Summerell 2006). To determine the species, DNA was extracted from four isolates, and the internal transcribed spacer (ITS), and genes of translation elongation factor 1α (EF1α) and subunits 1 and 2 of DNA-directed RNA polymerase II (RPB1 and RPB2) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (O'Donnell et al. 1998a), RPB1-F5/RPB1-R8 (O'Donnell et al. 2010) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999). The obtained sequences were identical for all four isolates. The isolate Strelets sequences were deposited in NCBI GenBank (MW149129 (ITS), MW161161 (EF1α), MW413302 (RPB1) and MW413303 (RPB2)); their analysis in MLST (http://fusarium.mycobank.org) showed 98.8-99.8% similarity to F. proliferatum (NRRL 13582, 13598 and others), which is part of the F. fujikuroi complex (O'Donnell et al. 1998b). The test on pathogenicity was performed two times according to (Leyronas et al. 2018). For this, three replicates of 10 cloves (cv. Strelets) were soaked in a conidial suspension (~106 conidia/ml; Strelets isolate) for 24 h. Ten control cloves were soaked in sterile water. The cloves were incubated on Petri dishes (5 cloves on a dish; on filter paper wettened with sterile water) in the dark at 23°C. After 5 days, brown lesions and white mycelium developed on the surface of the treated cloves. The taxonomic status of the fungus isolated from necrotic tissue was determined as F. proliferatum according to the ITS, EF1α, RPB1 and RPB2 analysis. Garlic basal and bulb rot is known to be caused by F. oxysporum f. sp. cepae and F. proliferatum (Snowdon 1990). This study is the first report of F. proliferatum causing rot of garlic bulbs during storage in Russia. F. proliferatum produces a variety of mycotoxins during bulb infestation, and our findings are important for diagnosing a Fusarium disease and the use of garlic crop in culinary and medicine. Funding The reported study was funded by Russian Foundation for Basic Research, project number 20-316-70009. References: Leslie, J. F., and Summerell, B. A. 2006. Page 224 in: The Fusarium Laboratory Manual. Blackwell, Oxford, UK. https://doi.org/10.1002/9780470278376 Leyronas, C., et al. 2018. Plant Dis. 102:2658 https://doi.org/10.1094/PDIS-06-18-0962-PDN Liu, Y.J. et al. 1999. Mol. Biol. Evol. 16: 1799 https://doi.org/10.1093/oxfordjournals.molbev.a026092 O'Donnell, K, et al. 1998a. Proc Natl Acad Sci USA. 95(5):2044. https://doi.org/10.1073/pnas.95.5.2044. O'Donnell, et al. 1998b. Mycologia 90:465 O'Donnell, K., et al. 2010. J. Clin. Microbiol., 48: 3708 https://doi.org/10.1128/JCM.00989-10 Snowdon, A. L. Pages 250-252 in: A Color Atlas of Post-Harvest Diseases and Disorders of Fruits and Vegetables. Vol. 1. 1990. Wolfe Scientific, London. Taylor, A, et al. 2013. Plant Pathol. 62:103. https://doi.org/10.1111/j.1365-3059.2012.02624.x White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.