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.

Using RNA-Sequencing Data to Examine Tissue-Specific Garlic Microbiomes.

Garlic (Allium sativum) is a perennial bulbous plant. Due to its clonal propagation, various diseases threaten the yield and quality of garlic. In this study, we conducted in silico analysis to identify microorganisms, bacteria, fungi, and viruses in six different tissues using garlic RNA-sequencing data. The number of identified microbial species was the highest in inflorescences, followed by flowers and bulb cloves. With the Kraken2 tool, 57% of identified microbial reads were assigned to bacteria and 41% were assigned to viruses. Fungi only made up 1% of microbial reads. At the species level, Streptomyces lividans was the most dominant bacteria while Fusarium pseudograminearum was the most abundant fungi. Several allexiviruses were identified. Of them, the most abundant virus was garlic virus C followed by shallot virus X. We obtained a total of 14 viral genome sequences for four allexiviruses. As we expected, the microbial community varied depending on the tissue types, although there was a dominant microorganism in each tissue. In addition, we found that Kraken2 was a very powerful and efficient tool for the bacteria using RNA-sequencing data with some limitations for virome study.

Comparison of bacterial and archaeal communities in two fertilizer doses and soil compartments under continuous cultivation system of garlic.

Soil microbial communities are affected by interactions between agricultural management (e.g., fertilizer) and soil compartment, but few studies have considered combinations of these factors. We compared the microbial abundance, diversity and community structure in two fertilizer dose (high vs. low NPK) and soil compartment (rhizosphere vs. bulk soils) under 6-year fertilization regimes in a continuous garlic cropping system in China. The soil contents of NO3- and available K were significantly higher in bulk soil in the high-NPK. The 16S rRNA gene-based bacterial and archaeal abundances were positively affected by both the fertilizer dose and soil compartment, and were higher in the high-NPK fertilization and rhizosphere samples. High-NPK fertilization increased the Shannon index and decreased bacterial and archaeal richness, whereas the evenness was marginally positively affected by both the fertilizer dose and soil compartment. Soil compartment exerted a greater effect on the bacterial and archaeal community structure than did the fertilization dose, as demonstrated by both the nonmetric multidimensional scaling and redundancy analysis results. We found that rhizosphere effects significantly distinguished 12 dominant classes of bacterial and archaeal communities, whereas the fertilizer dose significantly identified four dominant classes. In particular, a Linear Effect Size analysis showed that some taxa, including Alphaproteobacteria, Rhizobiales, Xanthomonadaceae and Flavobacterium, were enriched in the garlic rhizosphere of the high-NPK fertilizer samples. Overall, the fertilizer dose interacted with soil compartment to shape the bacterial and archaeal community composition, abundance, and biodiversity in the garlic rhizosphere. These results provide an important basis for further understanding adaptive garlic-microbe feedback, reframing roots as a significant moderating influence in agricultural management and shaping the microbial community.

Formation, nutritional value, and enhancement of characteristic components in black garlic: A review for maximizing the goodness to humans.

Black garlic (BG) is essentially a processed food and obtained through the transformation of fresh garlic (FG) (Allium sativum L.) via a range of chemical reactions (including the Maillard reaction) and microbial fermentation. This review provides the up-to-date knowledge of the dynamic and complicated changes in major components during the conversion of FG to BG, including moisture, lipids, carbohydrates (such as sugars), proteins, organic acids, organic sulfur compounds, alkaloids, polyphenols, melanoidins, 5-hydroxymethylfurfural, vitamins, minerals, enzymes, and garlic endophytes. The obtained evidence confirms that BG has several advantages over FG in certain product attributes and biological properties (especially antioxidant activity), and the factors affecting the quality of BG include the type and characteristics of FG and processing technologies and methods (especially pretreatments, and processing temperature and humidity). The interactions among garlic components, and between garlic nutrients and microbes, as well as the interplay between pretreatment and main manufacturing process, all determine the sensory and nutritional qualities of BG. Before BG is marketed as a novel snack or functional food, more research is required to fill the knowledge gaps related to quantitative monitoring of the changes in metabolites (especially those taste-active and/or biological-active substances) during BG manufacturing to maximize BG's antioxidant, anticancer, antiobesity, anti-inflammatory, immunostimulatory, anti-allergic, hepatoprotective, cardioprotective and oxidative stress-/hangover syndrome-reducing functions, and beneficial effects on memory/nervous systems. Assessments of the quality, efficacy, and safety of BG should be performed considering the impacts of BG production conditions, postproduction handling, and intake methods.

Lactobacillus pentosus SMB718 as a probiotic starter producing allyl mercaptan in garlic and onion-enriched fermentation.

Fermenting garlic and onion provides the advantages of storage life extension, anti-oxidative and anti-diabetic activities, and their metabolite, allyl mercaptan, offers a strong aroma and various health benefits. Here, we report the probiotic properties of Lactobacillus pentosus SMB718 isolated from Korean traditional paste and its high allyl mercaptan productivity in garlic and onion fermentation. This strain was safe for use in food fermentation, as it was a non-biogenic amine producer and non-hemolytic. It showed high stability under simulated human gastrointestinal conditions and good adhesion ability to intestinal epithelial cells, including both Caco-2 and HT-29. This strain had antioxidant and anti-microbial activities. In addition, the heat-killed cells and lysate exerted anti-inflammatory effects on both LPS-stimulated RAW 264.7 cells and mouse macrophages by inhibition of pro-inflammatory cytokines and induction of anti-inflammatory cytokines. Furthermore, this strain possessed good fermentation properties in garlic and onion-enriched radish juice (GORJ); it grew well decreasing the pH and provided a rich aroma compound during fermentation. When L. pentosus SMB718 was used as a starter in garlic and onion fermentation, a significantly higher amount of allyl mercaptan (344 ppb) was produced compared with that produced by the type strain (82 ppb). In conclusion, L. pentosus SMB718 can be used as a beneficial probiotic starter for better flavor production in the fermentation of allium species plants.

Endophytic bacteria of garlic roots promote growth of micropropagated meristems.

Garlic (Allium sativum L.) is one of the crops whose economic importance has increased considerably in recent years in Brazil. The use of plant growth-promoting bacteria (PGPB) is a useful alternative for reducing the cost of agricultural inputs and, consequently, for increasing productivity. Therefore, the main objective of this work was to isolate and evaluate potential growth promoters in plants and plant pathogenic fungi growth inhibitors using endophytic bacteria from garlic roots and bacteria from the Agricultural Microbiology Culture Collection at the Federal University of Lavras. Besides verifying improvements in the growth and physiology of garlic meristems grown in vitro under the action of PGPB. Forty-eight endophytic bacteria were identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry based on the protein profile of each isolate. Four isolates were chosen according to their ability to fix nitrogen, to produce auxin and solubilize phosphate. The cultivation of garlic meristems in tissue culture with these bacteria was established at a population level of 106 CFU/mL. The evaluated criteria were: (1) the colonization capacity of the bacteria inside the garlic plants determined through scanning electron microscopy; (2) the chlorophyll content; and (3) the growth of garlic plants in vitro post-PGPB inoculation. Volatiles emitted by those isolates inhibited fungi growth. The inoculation of garlic meristems with Enterobacter cloacae and Burkholderia cepacia promoted an improvement in the growth and physiological attributes of garlic, indicating the feasibility of their application as plant growth promoters for commercial cultivation.

Microbial niches in raw ingredients determine microbial community assembly during kimchi fermentation.

Fermented foods constitute hubs of microbial consortia differentially affecting nutritional and organoleptic properties, quality, and safety. Here we show the origin source of fermentative microbes and fermentation dynamics of kimchi. We partitioned microbiota by raw ingredient (kimchi cabbage, garlic, ginger, and red pepper) to render kimchi fermented by each source-originated microbe pool and applied multi-omics (metataxonomics and metabolomics), bacterial viability, and physiochemical analyses to longitudinally collected samples. Only kimchi cabbage- and garlic-derived microbial inoculums yielded successful kimchi fermentations. The dominant fermentative microbial taxa and subsequent metabolic outputs differed by raw ingredient type: the genus Leuconostoc, Weissella, and Lactobacillus for all non-sterilized ingredients, garlic, and kimchi cabbage, respectively. Gnotobiotic kimchi inoculated by mono-, di-, and tri- isolated fermentative microbe combinations further revealed W. koreensis-mediated reversible microbial metabolic outputs. The results suggest that the raw ingredient microbial habitat niches selectively affect microbial community assembly patterns and processes during kimchi fermentation.

Trichoderma from Brazilian garlic and onion crop soils and description of two new species: Trichoderma azevedoi and Trichoderma peberdyi.

Fifty four Trichoderma strains were isolated from soil samples collected from garlic and onion crops in eight different sites in Brazil and were identified using phylogenetic analysis based on combined ITS region, tef1-α, cal, act and rpb2 sequences. The genetic variability of the recovered Trichoderma species was analysed by AFLP and their phenotypic variability determined using MALDI-TOF. The strain clusters from both typing techniques coincided with the taxonomic determinations made from phylogenetic analysis. The phylogenetic analysis showed the occurrence of Trichoderma asperellum, Trichoderma asperelloides, Trichoderma afroharzianum, Trichoderma hamatum, Trichoderma lentiforme, Trichoderma koningiopsis, Trichoderma longibrachiatum and Trichoderma erinaceum, in the soil samples. We also identified and describe two new Trichoderma species, both in the harzianum clade of section Pachybasium, which we have named Trichoderma azevedoi sp. nov. and Trichoderma peberdyi sp. nov. The examined strains of both T. azevedoi (three strains) and T. peberdyi (12 strains) display significant genotypic and phenotypic variability, but form monophyletic clades with strong bootstrap and posterior probability support and are morphologically distinct from their respective most closely related species.

Isolation and Identification of Aspergillus Section Nigri, and Genotype Associated with Ochratoxin A and Fumonisin B2 Production in Garlic Marketed in Brazil.

The garlic contains sulfur bioactive compounds responsible for medicinal properties. The decrease of these compounds due to inadequate storage conditions reduces the beneficial properties and favors infection by microorganisms. Several studies have shown high frequency of garlic infected with Aspergillus section Nigri that potentially produce mycotoxin. Garlic samples were collected in markets of Brazil and a total of 32 samples (of 36) had the fungal infection with predominant genus Aspergillus (50.3%), Penicillium (34.7%), and Fusarium (11%). A total of 63% (649/1031) of infection with Aspergillus section Nigri, of which 60 isolates were selected for analysis of genetic variability that resulted in 4 clusters. Representatives of clusters were identified by the calmodulin gene. Isolates from cluster I were subdivided into A-I and identified as A. niger (16 isolates) and the isolates of clusters B-I, II, and III were identified as A. welwitschiae (43 isolates). Besides, an isolate of the IV-cluster was identified by A. luchuensis. Further, we used the multiplex PCR to verify genotypes of 59 isolates, and none of these had OTA production-associated genotype. Moreover, 19 A. welwitschiae and 15 A. niger were FB2 production-associated genotype. Our study is the first report to the incidence of garlic infection in Brazil and to show that A. welwitschiae causes most of these infections.

Optimization of Salmonella detection in garlic, onion, cinnamon, red chili pepper powders and green tea.

Salmonella is the causative agent of many outbreaks related to spice consumption. However, because of the antimicrobial properties of various spices which hinders recovery and detection, Salmonella detection in spices remains a challenge. The objective of this study was to optimize an enrichment broth for Salmonella growth in different spices and tea, in order to maintain an adequate pH and decrease the antimicrobial effects of spices during Salmonella enrichment and subsequent detection. Salmonella contaminated spice and tea dried samples were prepared and the detection of Salmonella was assessed using the developed broth and automated DNA extraction and RT-PCR. Double strength Buffered Peptone Water (BPW) was used to maintain pH, and L-cysteine and DL-serine were added to the broth to reduce the effects of antimicrobial compounds in spices. The modified enrichment broth allowed the growth of Salmonella from each spice sample. Sample to broth ratios varied from 1:9 (garlic powder, chili peppers and tea), to 1:20 (cinnamon). The pH value of each enrichment varied but remained above 4.8. The addition of L-cysteine (30 mmol/L) allowed Salmonella recovery and growth in garlic and onion samples and the addition of DL-serine (11.23 mmol/L) allowed the recovery and growth in cinnamon. The results indicated that Salmonella detection was achieved in <24 h in the modified (BPW + L-cysteine and DL-serine) enrichment broth followed by detection by RT-PCR. This protocol could allow for a more rapid, robust, and sensitive enrichment method for Salmonella in spices.

Avaliação do potencial antibacteriano do extrato aquoso de alho (Allium sativum) em hambúrgueres de carne bovina e sua correlação com a qualidade sensorial / Evaluation of the antibacterial potential of aqueous garlic extract (Allium sativum) in bovine meat hamburgers and its correlation with sensory quality

O risco de Doenças Veiculadas por Alimentos - DVA em hambúrgueres pode ser minimizado pelo uso de antimicrobianos naturais. O objetivo do presente trabalho foi avaliar a eficiência de diferentes concentrações do Extrato Aquoso de Alho - EAA na inibição de Escherichia coli, Salmonella enterica, Cronobacter sakazakii, Listeria monocytogenes, Staphylococcus aureus e Bacillus cereus e avalia r sensorialmente as amostras de hambúrguer adicionadas de EAA. Os resultados indicaram que o EAA inibiu o crescimento das cepas bacterianas, sendo a ação inibitória proporcional às concentrações usadas sendo que a refrigeração a 7°C por 72 h do EAA não afetou seu efeito inibitório. Na análise sensorial, houve boa aceitabilidade e intenção de compra positiva para as amostras, com destaque para aquelas com maior concentração de EAA (1000μL/mL).

Athelia (Sclerotium) rolfsii in Allium sativum: potential biocontrol agents and their effects on plant metabolites.

Garlic (Allium sativum L.) plays an important role in popular culture due to its dietary and medicinal uses. It is also used to produce a wide range of pharmacologically interesting molecules. Several pathogens affect garlic plants, especially Athelia (Sclerotium) rolfsii, a fungus that is widespread and causes large economic losses. It causes direct damage to crops and leads to plant stress, which induces secondary metabolite production in plants. The use of microorganisms as biocontrol agents may induce the production of beneficial metabolites in plants that will protect it and promote resistance to pathogen attack. In addition to suppressing disease, biological control agents may have elicitor effects that could induce an increase in the production of useful bioactive secondary metabolites in plants, some of which may be of pharmacological interest. Therefore, the search for new biological control agents should also consider their potential as elicitor agents. This paper presents an analysis of the biological control of Athelia (Sclerotium) rolfsii by antagonistic microrganisms, the potential of yeasts and bacteria of the genus Bacillus for the biocontrol of phytopathogens, microrganisms influence in nutritional and bioactive compounds content of interest to the pharmaceutical industry.

Antimicrobial genes from Allium sativum and Pinellia ternata revealed by a Bacillus subtilis expression system.

Antimicrobial genes are found in all classes of life. To efficiently isolate these genes, we used Bacillus subtilis and Escherichia coli as target indicator bacteria and transformed them with cDNA libraries. Among thousands of expressed proteins, candidate proteins played antimicrobial roles from the inside of the indicator bacteria (internal effect), contributing to the sensitivity (much more sensitivity than the external effect from antimicrobial proteins working from outside of the cells) and the high throughput ability of screening. We found that B. subtilis is more efficient and reliable than E. coli. Using the B. subtilis expression system, we identified 19 novel, broad-spectrum antimicrobial genes. Proteins expressed by these genes were extracted and tested, exhibiting strong external antibacterial, antifungal and nematicidal activities. Furthermore, these newly isolated proteins could control plant diseases. Application of these proteins secreted by engineered B. subtilis in soil could inhibit the growth of pathogenic bacteria. These proteins are thermally stable and suitable for clinical medicine, as they exhibited no haemolytic activity. Based on our findings, we speculated that plant, animal and human pathogenic bacteria, fungi or even cancer cells might be taken as the indicator target cells for screening specific resistance genes.

Characterization of microbial community structure and metabolic potential using Illumina MiSeq platform during the black garlic processing.

Black garlic is a distinctive garlic deep-processed product made from fresh garlic at high temperature and controlled humidity. To explore microbial community structure, diversity and metabolic potential during the 12days of the black garlic processing, Illumina MiSeq sequencing technology was performed to sequence the 16S rRNA V3-V4 hypervariable region of bacteria. A total of 677,917 high quality reads were yielded with an average read length of 416bp. Operational taxonomic units (OTU) clustering analysis showed that the number of species OTUs ranged from 148 to 1974, with alpha diversity increasing remarkably, indicating the high microbial community abundance and diversity. Taxonomic analysis indicated that bacterial community was classified into 45 phyla and 1125 distinct genera, and the microbiome of black garlic samples based on phylogenetic analysis was dominated by distinct populations of four genera: Thermus, Corynebacterium, Streptococcus and Brevundimonas. The metabolic pathways were predicted for 16S rRNA marker gene sequences based on Kyoto Encyclopedia of Genes and Genomes (KEGG), indicating that amino acid metabolism, carbohydrate metabolism and membrane transport were important for the black garlic fermentation process. Overall, the study was the first to reveal microbial community structure and speculate the composition of functional genes in black garlic samples. The results contributed to further analysis of the interaction between microbial community and black garlic components at different stages, which was of great significance to study the formation mechanism and quality improvement of black garlic in the future.

Characterization of garlic endophytes isolated from the black garlic processing.

The objectives of this study were to isolate and identify garlic endophytes, and explore the characteristics of dominant strains. Garlic endophytes were studied through phenotypical characterization and comparative sequence analysis of 16S rDNA based on culture-dependent approaches. Representative strains inferred from 16S rDNA sequencing were selected for further identification by gyrA and rpoB gene loci and phylogenetic analysis based on concateneted house-keeping sequences. Seven kinds of Bacillus were found from garlic and black garlic, respectively. Further studies demonstrated that the total bacteria and endophytes showed a sharp decrease firstly, followed by a rapid rise, then maintained at a certain level, and finally slowed down during the black garlic processing. B. subtilis, B. methylotrophicus, and B. amyloliquefaciens were the dominant strains. The selected strains were capable of fermenting glucose, lactose, sucrose, and garlic polysaccharide to produce acid but no gas, with a strong ability of heat resistance. The results indicated that there were a certain number of garlic endophytes during the black garlic processing, and Bacillus was the dominant strains under the conventional culture-dependent methods. This report provided useful information for the presence and type of garlic endophytes during the black garlic processing, which were of great significance to study the formation mechanism and quality improvement of black garlic in the future, as well as the security of garlic powder.

Salmonella survival during thermal dehydration of fresh garlic and storage of dehydrated garlic products.

Salmonella survival was characterized and modeled during thermal dehydration of fresh garlic and storage of dehydrated garlic products. In our experiments that simulated commercial dehydration processing at 80±5°C, moderate level of Salmonella contamination (4-5logCFU/g) on fresh garlic was reduced below the enumeration limit (1.7logCFU/g) after 4.5h of dehydration and not detectable by culture enrichment after 7h. With high level of contamination (7-8logCFU/g), the Salmonella population persisted at 3.6logCFU/g after 8h of processing. By increasing the dehydration temperature to 90±5°C, the moderate and high levels of initial Salmonella load on fresh garlic dropped below the enumeration limit after 1.5 and 3.75h of processing and became undetectable by culture enrichment after 2.5 and 6h, respectively. During the storage of dried garlic products, Salmonella was not able to grow under all tested combinations of temperature (25 and 35°C) and water activity (0.56-0.98) levels, suggesting active inhibition. Storage temperature played a primary role in determining Salmonella survival on dehydrated garlic flakes. Under a typical storage condition at 25°C and ambient relative humidity, Salmonella could persist over months with the population gradually declining (4.3 log reduction over 88days). Granular size of dehydrated garlic had an impact on Salmonella survival, with better survival of the pathogen observed in bigger granules. At the early stage of dehydrated garlic storage (until 7days), rising water activity appeared to initially promote but then inhibited Salmonella survival, resulting in a water activity threshold at 0.73 where Salmonella displayed strongest persistence. However, this phenomenon was less apparent during extended storage (after 14days).

Detection of Fumonisins in Fresh and Dehydrated Commercial Garlic.

An epidemic fungal disease caused by Fusarium proliferatum, responsible for fumonisin production (FB1, FB2, and FB3), has been reported in the main garlic-producing countries in recent years. Fumonisins are a group of structurally related toxic metabolites produced by this pathogen. The aim of this work was to establish an enzyme-linked immunosorbent assay (ELISA) procedure, mostly applied to cereals, that is suitable for fumonisin detection in garlic and compare these results to those obtained by high-performance liquid chromatography (HPLC) and screening of fresh and dehydrated garlic for toxicological risk. The results show good correlation between the two analytical methods. In fresh symptomatic garlic, fumonisin levels were higher in the basal plates than those in the portions with necrotic spots. Among the 56 commercially dehydrated garlic samples screened, three were positive by ELISA test and only one was above the limit of quantitation. The same samples analyzed by HPLC showed the presence of FB1 in trace amounts that was below the limit of quantitation; FB2 and FB3 were absent. The results are reassuring, because no substantial contamination by fumonisins was found in commercial garlic.

Green Pea and Garlic Puree Model Food Development for Thermal Pasteurization Process Quality Evaluation.

Development and selection of model foods is a critical part of microwave thermal process development, simulation validation, and optimization. Previously developed model foods for pasteurization process evaluation utilized Maillard reaction products as the time-temperature integrators, which resulted in similar temperature sensitivity among the models. The aim of this research was to develop additional model foods based on different time-temperature integrators, determine their dielectric properties and color change kinetics, and validate the optimal model food in hot water and microwave-assisted pasteurization processes. Color, quantified using a* value, was selected as the time-temperature indicator for green pea and garlic puree model foods. Results showed 915 MHz microwaves had a greater penetration depth into the green pea model food than the garlic. a* value reaction rates for the green pea model were approximately 4 times slower than in the garlic model food; slower reaction rates were preferred for the application of model food in this study, that is quality evaluation for a target process of 90 °C for 10 min at the cold spot. Pasteurization validation used the green pea model food and results showed that there were quantifiable differences between the color of the unheated control, hot water pasteurization, and microwave-assisted thermal pasteurization system. Both model foods developed in this research could be utilized for quality assessment and optimization of various thermal pasteurization processes.

Fusarium proliferatum - Causal agent of garlic bulb rot in Spain: Genetic variability and mycotoxin production.

Fusarium proliferatum is a world-wide occurring fungal pathogen affecting several crops included garlic bulbs. In Spain, this is the most frequent pathogenic fungus associated with garlic rot during storage. Moreover, F. proliferatum is an important mycotoxigenic species, producing a broad range of toxins, which may pose a risk for food safety. The aim of this study is to assess the intraspecific variability of the garlic pathogen in Spain implied by analyses of translation elongation factor (tef-1α) and FUM1 gene sequences as well as the differences in growth rates. Phylogenetic characterization has been complemented with the characterization of mating type alleles as well as the species potential as a toxin producer. Phylogenetic trees based on the sequence of the translation elongation factor and FUM1 genes from seventy nine isolates from garlic revealed a considerable intraspecific variability as well as high level of diversity in growth speed. Based on the MAT alleles amplified by PCR, F. proliferatum isolates were separated into different groups on both trees. All isolates collected from garlic in Spain proved to be fumonisin B1, B2, and B3 producers. Quantitative analyses of fumonisins, beauvericin and moniliformin (common secondary metabolites of F. proliferatum) showed no correlation with phylogenetic analysis neither mycelial growth. This pathogen presents a high intraspecific variability within the same geographical region and host, which is necessary to be considered in the management of the disease.

Multiple garlic (Allium sativum L.) microRNAs regulate the immunity against the basal rot fungus Fusarium oxysporum f. sp. Cepae.

The basal plate rot fungus, Fusarium oxysporum f. sp. cepae (FOC), is the most devastating pathogen posing a serious threat to garlic (Allium sativum L.) production worldwide. MicroRNAs (miRNAs) are key modulators of gene expression related to development and defense responses in eukaryotes. However, the miRNA species associated with garlic immunity against FOC are yet to be explored. In the present study, a small RNA library developed from FOC infected resistant garlic line was sequenced to identify immune responsive miRNAs. Forty-five miRNAs representing 39 conserved and six novel sequences responsive to FOC were detected. qRT-PCR analyses further classified them into three classes based on their expression patterns in susceptible line CBT-As11 and in the resistant line CBT-As153. North-blot analyses of six selective miRNAs confirmed the qRT-PCR results. Expression studies on a selective set of target genes revealed a negative correlation with the complementary miRNAs. Furthermore, transgenic garlic plant overexpresing miR164a, miR168a and miR393 showed enhanced resistance to FOC, as revealed by decreased fungal growth and up-regulated expression of defense-responsive genes. These results indicate that multiple miRNAs are involved in garlic immunity against FOC and that the overexpression of miR164a, miR168a and miR393 can augment garlic resistance to Fusarium basal rot infection.