Optimisation of indole acetic acid production by Neopestalotiopsis aotearoa endophyte isolated from Thymus vulgaris and its impact on seed germination of Ocimum basilicum.

BACKGROUND: Microbial growth during plant tissue culture is a common problem that causes significant losses in the plant micro-propagation system. Most of these endophytic microbes have the ability to propagate through horizontal and vertical transmission. On the one hand, these microbes provide a rich source of several beneficial metabolites. RESULTS: The present study reports on the isolation of fungal species from different in vitro medicinal plants (i.e., Breynia disticha major, Breynia disticha, Duranta plumieri, Thymus vulgaris, Salvia officinalis, Rosmarinus officinalis, and Ocimum basilicum l) cultures. These species were tested for their indole acetic acid (IAA) production capability. The most effective species for IAA production was that isolated from Thymus vulgaris plant (11.16 µg/mL) followed by that isolated from sweet basil plant (8.78 µg/mL). On screening for maximum IAA productivity, medium, "MOS + tryptophan" was chosen that gave 18.02 µg/mL. The macroscopic, microscopic examination and the 18S rRNA sequence analysis indicated that the isolate that given code T4 was identified as Neopestalotiopsis aotearoa (T4). The production of IAA by N. aotearoa was statistically modeled using the Box-Behnken design and optimized for maximum level, reaching 63.13 µg/mL. Also, IAA extract was administered to sweet basil seeds in vitro to determine its effect on plant growth traits. All concentrations of IAA extract boosted germination parameters as compared to controls, and 100 ppm of IAA extract exhibited a significant growth promotion effect for all seed germination measurements. CONCLUSIONS: The IAA produced from N. aotearoa (T4) demonstrated an essential role in the enhancement of sweet basil (Ocimum basilicum) growth, suggesting that it can be employed to promote the plant development while lowering the deleterious effect of using synthetic compounds in the environment.

Genome-based high-resolution mapping of fusarium wilt resistance in sweet basil.

Fusarium wilt of basil is a disease of sweet basil (Ocimum basilicum L.) plants caused by the fungus Fusarium oxysporum f. sp. basilici (FOB). Although resistant cultivars were released > 20 years ago, the underlying mechanism and the genes controlling the resistance remain unknown. We used genetic mapping to elucidate FOB resistance in an F2 population derived from a cross between resistant and susceptible cultivars. We performed genotyping by sequencing of 173 offspring and aligning the data to the sweet basil reference genome. In total, 23,411 polymorphic sites were detected, and a single quantitative trait locus (QTL) for FOB resistance was found. The confidence interval was < 600 kbp, harboring only 60 genes, including a cluster of putative disease-resistance genes. Based on homology to a fusarium resistance protein from wild tomato, we also investigated a candidate resistance gene that encodes a transmembrane leucine-rich repeat - receptor-like kinase - ubiquitin-like protease (LRR-RLK-ULP). Sequence analysis of that gene in the susceptible parent vs. the resistant parent revealed multiple indels, including an insertion of 20 amino acids next to the transmembrane domain, which might alter its functionality. Our findings suggest that this LRR-RLK-ULP might be responsible for FOB resistance in sweet basil and demonstrate the usefulness of the recently sequenced basil genome for QTL mapping and gene mining.

Effect of Plant Systemic Resistance Elicited by Biological and Chemical Inducers on the Colonization of the Lettuce and Basil Leaf Apoplast by Salmonella enterica.

Mitigation strategies to prevent microbial contamination of crops are lacking. We tested the hypothesis that induction of plant systemic resistance by biological (induced systemic resistance [ISR]) and chemical (systemic acquired resistance [SAR]) elicitors reduces endophytic colonization of leaves by Salmonella enterica serovars Senftenberg and Typhimurium. S. Senftenberg had greater endophytic fitness than S. Typhimurium in basil and lettuce. The apoplastic population sizes of serovars Senftenberg and Typhimurium in basil and lettuce, respectively, were significantly reduced approximately 10- to 100-fold by root treatment with microbial inducers of systemic resistance compared to H2O treatment. Rhodotorula glutinis effected the lowest population increases of S. Typhimurium in lettuce and S. Senftenberg in basil leaves, respectively 120- and 60-fold lower than those seen with the H2O treatment over 10 days postinoculation. Trichoderma harzianum and Pichia guilliermondii did not have any significant effect on S. Senftenberg in the basil apoplast. The chemical elicitors acidobenzolar-S-methyl and dl-ß-amino-butyric acid inhibited S. Typhimurium multiplication in the lettuce apoplast 10- and 2-fold, respectively, compared to H2O-treated plants. All ISR and SAR inducers applied to lettuce roots in this study increased leaf expression of the defense gene PR1, as did Salmonella apoplastic colonization in H2O-treated lettuce plants. Remarkably, both acidobenzolar-S-methyl upregulation and R. glutinis upregulation of PR1 were repressed by the presence of Salmonella in the leaves. However, enhanced PR1 expression was sustained longer and at greater levels upon elicitor treatment than by Salmonella induction alone. These results serve as a proof of concept that priming of plant immunity may provide an intrinsic hurdle against the endophytic establishment of enteric pathogens in leafy vegetables. IMPORTANCE Fruit and vegetables consumed raw have become an important vehicle of foodborne illness despite a continuous effort to improve their microbial safety. Salmonella enterica has caused numerous recalls and outbreaks of infection associated with contaminated leafy vegetables. Evidence is increasing that enteric pathogens can reach the leaf apoplast, where they confront plant innate immunity. Plants may be triggered for induction of their defense signaling pathways by exposure to chemical or microbial elicitors. This priming for recognition of microbes by plant defense pathways has been used to inhibit plant pathogens and limit disease. Given that current mitigation strategies are insufficient in preventing microbial contamination of produce and associated outbreaks, we investigated the effect of plant-induced resistance on S. enterica colonization of the lettuce and basil leaf apoplast in order to gain a proof of concept for the use of such an intrinsic approach to inhibit human pathogens in leafy vegetables.

CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.

Sweet basil (Ocimum basilicum) is an economically important allotetraploid (2n = 4x = 48) herb whose global production is threatened by downy mildew disease caused by the obligate biotrophic oomycete, Peronospora belbahrii. Generation of disease resistant cultivars by mutagenesis of susceptibility (S) genes via CRISPR/Cas9 is currently one of the most promising strategies to maintain favored traits while improving disease resistance. Previous studies have identified Arabidopsis DMR6 (Downy Mildew Resistance 6) as an S gene required for pathogenesis of the downy mildew-causing oomycete pathogen Hyaloperonospora arabidopsidis. In this study, a sweet basil homolog of DMR6, designated ObDMR6, was identified in the popular sweet basil cultivar Genoveser and found to exist with a high copy number in the genome with polymorphisms among the variants. Two CRISPR/Cas9 constructs expressing one or two single guide RNAs (sgRNAs) targeting the conserved regions of ObDMR6 variants were generated and used to transform Genoveser via Agrobacterium-mediated transformation. 56 T0 lines were generated, and mutations of ObDMR6 were detected by analyzing the Sanger sequencing chromatograms of an ObDMR6 fragment using the Interference of CRISPR Edits (ICE) software. Among 54 lines containing mutations in the targeted sites, 13 had an indel percentage greater than 96% suggesting a near-complete knockout (KO) of ObDMR6. Three representative transgene-free lines with near-complete KO of ObDMR6 determined by ICE were identified in the T1 segregating populations derived from three independent T0 lines. The mutations were further confirmed using amplicon deep sequencing. Disease assays conducted on T2 seedlings of the above T1 lines showed a reduction in production of sporangia by 61-68% compared to the wild-type plants and 69-93% reduction in relative pathogen biomass determined by quantitative PCR (qPCR). This study not only has generated transgene-free sweet basil varieties with improved downy mildew resistance, but also contributed to our understanding of the molecular interactions of sweet basil-P. belbahrii.

Thermal inactivation kinetics of Salmonella and Enterococcus faecium NRRL B-2354 on dried basil leaves.

The enhanced heat resistance of Salmonella developed at low water activity makes it a serious challenge to eliminate them during thermal processing. The objectives of this research are to (i) investigate the effect of water activity on thermal inactivation of Salmonella cocktail (Agona, Tennessee, Mbandaka, Montevideo, and Reading) in dried basil leaves, and (ii) evaluate Enterococcus faecium NRRL B-2354 as an appropriate surrogate for Salmonella in dried basil leaves. Dried basil leaves, inoculated with a Salmonella cocktail and E. faecium separately, were equilibrated to different water activities (aw: 0.40, 0.55, and 0.70) in a humidity-controlled chamber. The basil samples were packed (1.6 ± 0.1 g) in aluminum pouches and thermally treated at 70, 75, and 80 °C using a dry heating method for 0-180 min to obtain the thermal death curve. The microbial survival data was fit using two primary models (Log-linear and Weibull model). Results from AICc showed that the log-linear model fits well for thermal inactivation of both microorganisms. As the aw decreases from 0.70 to 0.40 at 75 °C, the D-value increases from 3.30 to 9.14 min for Salmonella and 6.53 to 14.07 min for E. faecium. Based on the AICc values, the modified Bigelow model fits the D-values better than the response surface model for both the microorganisms. The kill ratio of surrogate to pathogen ranged from 1.4 to 2.8, indicating that it is a conservative surrogate for Salmonella for performing validation of the thermal pasteurization process. The identification of suitable surrogate and development of modified Bigelow model will help the spice industry in developing the thermal processes for improving the safety of basil leaves.

Plantibacter flavus, Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens Endophytes Provide Host-Specific Growth Promotion of Arabidopsis thaliana, Basil, Lettuce, and Bok Choy Plants.

A collection of bacterial endophytes isolated from stem tissues of plants growing in soils highly contaminated with petroleum hydrocarbons were screened for plant growth-promoting capabilities. Twenty-seven endophytic isolates significantly improved the growth of Arabidopsis thaliana plants in comparison to that of uninoculated control plants. The five most beneficial isolates, one strain each of Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens and two strains of Plantibacter flavus were further examined for growth promotion in Arabidopsis, lettuce, basil, and bok choy plants. Host-specific plant growth promotion was observed when plants were inoculated with the five bacterial strains. P. flavus strain M251 increased the total biomass and total root length of Arabidopsis plants by 4.7 and 5.8 times, respectively, over that of control plants and improved lettuce and basil root growth, while P. flavus strain M259 promoted Arabidopsis shoot and root growth, lettuce and basil root growth, and bok choy shoot growth. A genome comparison between P. flavus strains M251 and M259 showed that both genomes contain up to 70 actinobacterial putative plant-associated genes and genes involved in known plant-beneficial pathways, such as those for auxin and cytokinin biosynthesis and 1-aminocyclopropane-1-carboxylate deaminase production. This study provides evidence of direct plant growth promotion by Plantibacter flavusIMPORTANCE The discovery of new plant growth-promoting bacteria is necessary for the continued development of biofertilizers, which are environmentally friendly and cost-efficient alternatives to conventional chemical fertilizers. Biofertilizer effects on plant growth can be inconsistent due to the complexity of plant-microbe interactions, as the same bacteria can be beneficial to the growth of some plant species and neutral or detrimental to others. We examined a set of bacterial endophytes isolated from plants growing in a unique petroleum-contaminated environment to discover plant growth-promoting bacteria. We show that strains of Plantibacter flavus exhibit strain-specific plant growth-promoting effects on four different plant species.

Atividade antimicrobiana de óleos essenciais de diferentes especiarias / Antimicrobial activity of essential oils of different spices

As especiarias são utilizadas na alimentação, conferindo sabor e conservação prolongada aos alimentos, pois apresentam propriedades antimicrobianas provenientes dos óleos essenciais de sua constituição. O objetivo deste estudo foi avaliar a atividade antimicrobiana dos óleos essenciais de cravo, louro, manjericão, noz moscada e orégano frente a seis bactérias patogênicas e deteriorantes por meio da técnica de difusão em poços e determinação das Concentrações Inibitória Mínima (CIM) e Bactericida Mínima (CBM). O óleo essencial de orégano evidenciou forte atividade antibacteriana (CIM 50 - 800 μg.mL-1), seguido do cravo (CIM 800 - 3200 μg.mL-1), com atividade moderada para todos os microrganismos. Os outros óleos apresentaram baixa ação (CIM 400 - 3200 μg.mL-1), não apresentando atividade sobre todos as bactérias. Desta forma os óleos essenciais de cravo e orégano apresentaram melhor atividade antibacteriana e se apresentam como promissores para a aplicação e uso em alimentos.

Invisible signals from the underground: A practical method to investigate the effect of microbial volatile organic compounds emitted by rhizobacteria on plant growth.

Rhizobacteria that colonize plant roots and promote plant growth are referred to as plant growth-promoting rhizobacteria, and this can stimulate plant growth either indirectly or directly. Volatile organic compounds (VOCs) emitted by rhizobacteria have the capacity to promote plant growth as well as perform biocontrol of fungal pathogens. The microbial volatile organic compounds (mVOCs) are characterized by a low molecular weight and a high vapor pressure, which facilitate evaporation and diffusion at normal temperatures and at above-ground and below-ground pressures. mVOCs can travel far from the point of production through the atmosphere, porous soils and liquids, thereby making them ideal infochemicals for mediating interspecific interactions. However, knowledge about the biological and ecological roles of microbial VOCs is still limited compared with that of plant VOCs. Here, we describe a simple and inexpensive laboratory class aimed at biotechnology or soil microbiology students, which uses techniques to increase their understanding of the mechanisms of plant growth promoting rhizobacteria and also illustrate the effects of mVOCs emitted by rhizobacteria on plant growth promotion, as well as evaluating their potential as a biocontrol. The laboratory class is divided into two sessions: an initial 3-hour experimental session and a second 2-hour analytical one. The experimental session involves two separate experiments: one of which is dedicated to illustrating the effect of mVOCs on plant growth parameters, while the second explores the capacity of VOCs as a biocontrol. Also, the class provides students with an opportunity to perform useful assays, draw conclusions from their results, and discuss possible extensions of the study. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):388-393, 2019.

Effects of organic acid alone and in combination with H2O2 and NaCl on Escherichia coli O157:H7: An evaluation of antioxidant retention and overall acceptability in Basil leaves (Ocimum basilicum).

In this study, the efficacy of household sanitizers application on reduction of Escherichia coli O157:H7, ascorbic acid, total phenolics, DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging and overall acceptability of inoculated fresh basil leaves (Ocimum basilicum), at temperature of 40 °C was investigated. Sanitizers containing lactic acid (LA), acetic acid (AA) and citric acid (CA) were used at concentration of 2%, individually or in combination with H2O2 (1% or 2%), and NaCl (7%). Control a and b were unwashed and washed leaves with distilled, deionized and sterilized water, respectively. All sanitizing treatments, in comparison to the control a, reduced the numbers of E. coli O157:H7 (0.24 ±â€¯0.12-3.37 ±â€¯0.48 log CFU/g) at day 1 (1 h after sanitizing). The lowest number of E. coli O157:H7 population (2.35 ±â€¯0.26 log CFU/g) was observed by applying the LA + H2O2 (%2) treatment at first day of chilled storage. The highest amount of ascorbic acid (27.77 ±â€¯0.06 mg/100 g), total phenolic (112.2 ±â€¯0.5 mg gallic acid equivalents/100 g) and DPPH radical scavenging activity (95.2 ±â€¯0.5%) was observed in control a at first day (P < 0.05). The results showed that the amount of ascorbic acid, total phenolics, DPPH radical scavenging activity and overall acceptability of basil leaves decreased during chilled storage. On day 2 of storage, the scores of sensory attributes for the control group were less than the minimum score of acceptance (i.e. 5 points). The results of this study indicated that LA + H2O2 (2%) treatment rendered the samples favorable in terms of overall appearance (≥5) up to 48 h.

Molecular phylogeny and characterization of secondary metabolite profile of plant pathogenic Alternaria species isolated from basil.

Alternaria leaf-spot is a new disease recently reported on basil in Italy. The correct identification of Alternaria species has suffered from many reclassifications in function of morphological features and molecular data. In our study, we performed an overall approach to obtain a better characterization of basil Alternaria isolates. Morphological characteristics, seven-genome region phylogenic analysis, and secondary metabolite profile differentiated the majority of the isolates as A. alternata. OPA 1-3 and OPA 10-2 were the best molecular regions to discriminate among the isolates. Morphological characteristics and sporulation groups helped to discriminate A. tenuissima from A. alternata isolates. All isolates in the A. sect. Alternaria were mycotoxigenic and pathogenic on basil, the production of mycotoxins was enhanced on basil compared to in vitro conditions used in this work.

Transfer of Downy Mildew Resistance from Wild Basil (Ocimum americanum) to Sweet Basil (O. basilicum).

Sweet basil (Ocimum basilicum) is susceptible to downy mildew caused by the oomycete foliar pathogen Peronospora belbahrii. No resistant varieties of sweet basil are commercially available. Here, we report on the transfer of resistance gene Pb1 from the highly resistant tetraploid wild basil O. americanum var. americanum (PI 500945, 2n = 4x = 48) to the tetraploid susceptible O. basilicum 'Sweet basil' (2n = 4x = 48). F1 progeny plants derived from the interspecific hybridization PI 500945 × Sweet basil were resistant, indicating that the gene controlling resistance (Pb1) is dominant, but sterile due to the genetic distance between the parents. Despite their sterility, F1 plants were pollinated with the susceptible parent and 115 first backcross generation to the susceptible parent (BCs1) embryos were rescued in vitro. The emerging BCs1 plants segregated, upon inoculation, 5:1 resistant/susceptible, suggesting that resistance in F1 was controlled by a pair of dominant genes (Pb1A and Pb1A'). Thirty-one partially fertile BCs1 plants were self-pollinated to obtain BCs1-F2 or were backcrossed to Sweet basil to obtain the second backcross generation to the susceptible parent (BCs2). In total, 1 BCs1-F2 and 22 BCs2 progenies were obtained. The BCs1-F2 progeny segregated 35:1 resistant/susceptible, as expected from a tetraploid parent with two dominant resistant genes. The 22 BCs2 progenies segregated 1:1 resistant/susceptible (for a BCs1 parent that carried one dominant gene for resistance) or 5:1 (for a BCs1 parent that carried two dominant genes for resistance) at a ratio of 4:1. The data suggest that a pair of dominant genes (Pb1A and Pb1A') residing on a two homeologous chromosomes is responsible for resistance of PI 500945 against P. belbahrii.

Antibacterial and Antioxidant Constituents of Extracts of Endophytic Fungi Isolated from Ocimum basilicum var. thyrsiflora Leaves.

Fourteen fungal endophytes were isolated from the Ocimum basilicum var. thyrsiflora leaves collected from Northern Thailand. Eight genera were identified including Aspergillus, Ascochyta, Nigrospora, Blastomyces, Colletotrichum, Exidia, Clitopilus, and Nomuraea. The antibacterial activity of crude extracts from all endophytic fungi was tested against nine human bacterial pathogens: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, and Vibrio parahaemolyticus. All crude extracts showed some degree of antibacterial activity, but the crude extract from Nigrospora MFLUCC16-0605 exhibited broad spectrum activity with MIC values ranging from 7.81 to 250 µg/mL. The antioxidant activity of all crude extracts was also investigated by DPPH radical scavenging assay. Crude extract from MFLUCC16-0605 had high antioxidant activity (IC50 value of 15.36 µg/mL) comparable to the trolox and gallic acid standards showing IC50 values of 2.56 and 12.89 µg/mL, respectively. The chemical composition of the crude extract from MFLUCC16-0605 was determined using GC-MS. Sixty-two compounds were identified representing 92.09% of crude extract with six major components including 5E,9E-farnesyl acetone, columellarin, totarene, laurenan-2-one, and 8S,13-cedranediol. PCR amplification and sequencing of the barcoding region identified MFLUCC16-0605 as belonging to Nigrospora genus. The notable activities of MFLUCC16-0605 indicate that the endophyte is a potent natural resource and its use as an antibacterial/antioxidant agent should be further explored.

Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA amplicon sequencing.

Application of 16S rRNA (gene) amplicon sequencing on food samples is increasingly applied for assessing microbial diversity but may as unintended advantage also enable simultaneous detection of any human pathogens without a priori definition. In the present study high-throughput next-generation sequencing (NGS) of the V1-V2-V3 regions of the 16S rRNA gene was applied to identify the bacteria present on fresh basil leaves. However, results were strongly impacted by variations in the bioinformatics analysis pipelines (MEGAN, SILVAngs, QIIME and MG-RAST), including the database choice (Greengenes, RDP and M5RNA) and the annotation algorithm (best hit, representative hit and lowest common ancestor). The use of pipelines with default parameters will lead to discrepancies. The estimate of microbial diversity of fresh basil using 16S rRNA (gene) amplicon sequencing is thus indicative but subject to biases. Salmonella enterica was detected at low frequencies, between 0.1% and 0.4% of bacterial sequences, corresponding with 37 to 166 reads. However, this result was dependent upon the pipeline used: Salmonella was detected by MEGAN, SILVAngs and MG-RAST, but not by QIIME. Confirmation of Salmonella sequences by real-time PCR was unsuccessful. It was shown that taxonomic resolution obtained from the short (500bp) sequence reads of the 16S rRNA gene containing the hypervariable regions V1-V3 cannot allow distinction of Salmonella with closely related enterobacterial species. In conclusion 16S amplicon sequencing, getting the status of standard method in microbial ecology studies of foods, needs expertise on both bioinformatics and microbiology for analysis of results. It is a powerful tool to estimate bacterial diversity but amenable to biases. Limitations concerning taxonomic resolution for some bacterial species or its inability to detect sub-dominant (pathogenic) species should be acknowledged in order to avoid overinterpretation of results.

Dynamics of culturable mesophilic bacterial communities of three fresh herbs and their production environment.

AIM: Investigate dynamics of culturable mesophilic bacteria and selected food-contaminating bacteria from three herbs and their production environment. METHODS AND RESULTS: Marjoram, basil and thyme were investigated during one growing season by sampling plants, organic fertilizers, soil, irrigation water and marketed products. Mesophilic bacteria and selected food-contaminating bacteria (Escherichia coli, Enterococcus spp., Bacillus cereus group) were cultured and identified by MALDI biotyping. Culturable mesophilic bacteria on marjoram and basil plants decreased over time by two orders of magnitude starting at above 106 colony forming units per gram (CFU per g), while they remained constant on thyme (~104  CFU per g). Compared to the last field sample, mesophilic bacteria were increased on all market-ready products by one order of magnitude. Marjoram and basil were dominated by B. cereus group, Enterobacter spp. and Pseudomonas spp., thyme by Bacillus spp. and Pseudomonas spp. All selected food-contaminating bacteria were detected in soil and reservoir-sourced irrigation water, whereas in municipal water, only B. cereus group and rarely Enterococcus spp. were found. Escherichia coli was detected only on young marjoram and basil plants (5 × 102 and 5 × 101  CFU per g, respectively), whereas Enterococcus spp. and B. cereus group were consistently detected on these two herbs. Thyme plants only contained B. cereus group consistently (above 103 CFU per g). Marketed marjoram and thyme contained Enterococcus spp. (5 × 102 and 104 CFU per g) and B. cereus group (~5 × 102 CFU per g), while no selected food-contaminating bacteria were found on marketed basil. CONCLUSIONS: Overall, culturable mesophilic bacteria were dominated by Pseudomonas spp. and Bacillus spp., with increased numbers on market-ready products. Selected food-contaminating bacteria were readily detectable, however, only the B. cereus group was found throughout in all systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into composition and development of mesophilic bacterial communities and selected food-contaminating bacteria of fresh herbs contributes to estimating consumer exposure.

Epidemiology of Basil Downy Mildew.

Basil downy mildew (BDM) caused by the oomycete Peronospora belbahrii is a destructive disease of sweet basil (Ocimum basilicum) worldwide. It originated in Uganda in the 1930s and recently spread to Europe, the Middle East, Americas, and the Far East. Seed transmission may be responsible for its quick global spread. The pathogen attacks leaf blades, producing chlorotic lesions with ample dark asexual spores on the lower leaf surface. Oospores may form in the mesophyll of infected leaves. The asexual spores germinate on a wet leaf surface within 2 h and penetrate into the epidermis within 4 h. Spore germination and infection occur at a wide range of temperatures from 5 to 28.5°C. Infection intensity depends on the length of dew period, leaf temperature, and inoculum dose. The duration of latent period (from infection to sporulation) extends from 5 to 10 days, depending on temperature and light regime. The shortest is 5 days at 25°C under continuous light. Sporulation requires high humidity but not free leaf wetness. Sporulation occurs at 10 to 26°C. At the optimum temperature of 18°C, the process of sporulation requires 7.5 h at relative humidity ≥ 85%, with 3 h for sporophores emergence from stomata and 4.5 h for spore formation. Sporophores can emerge under light or darkness, but spore formation occurs in the dark only. Limited data are available on spore dispersal. Spores dispersed from sporulating plants contaminate healthy plants within 2 h of exposure. Settled spores may survive on leaf surface of healthy plants for prolonged periods, depending on temperature. Seed transmission of the disease occurs in Europe, but not in Israel or the United States. P. belbahrii in Israel also attacks species belonging to Rosemarinus, Nepeta, Agastache, Micromeria, and Salvia but not Plectranthus (coleus). A Peronospora species that infects coleus does not infect sweet basil. Control of BDM includes chemical, physical, and genetic means. The fungicide mefenoxam was highly effective in controlling the disease but resistant populations were quickly selected for in Israel and Europe rendering it ineffective. A new compound oxathiapiprolin (OSBP inhibitor) is highly effective. Nocturnal illumination of basil crops controls the disease by preventing sporulation. Daytime solar heating suppressed the disease effectively by reducing spore and mycelium viability. The most effective physical means is fanning. Nocturnal fanning prevents or limits dew deposition on leaf surfaces, and as a result, infection and sporulation diminish and epidemics are prevented. Genetic resistance occurs in wild basil and its transfer to sweet basil is under way.

Adaptation of Salmonella enterica Serovar Senftenberg to Linalool and Its Association with Antibiotic Resistance and Environmental Persistence.

A clinical isolate of Salmonella enterica serovar Senftenberg, isolated from an outbreak linked to the herb Ocimum basilicum L. (basil), has been shown to be resistant to basil oil and to the terpene alcohol linalool. To better understand how human pathogens might develop resistance to linalool and to investigate the association of this resistance with resistance to different antimicrobial agents, selective pressure was applied to the wild-type strain by sequential exposure to increasing concentrations of linalool. The results demonstrated that S Senftenberg adapted to linalool with a MIC increment of at least 8-fold, which also resulted in better resistance to basil oil and better survival on harvested basil leaves. Adaptation to linalool was shown to confer cross protection against the antibiotics trimethoprim, sulfamethoxazole, piperacillin, chloramphenicol, and tetracycline, increasing their MICs by 2- to 32-fold. The improved resistance was shown to correlate with multiple phenotypes that included changes in membrane fatty acid composition, induced efflux, reduced influx, controlled motility, and the ability to form larger aggregates in the presence of linalool. The adaptation to linalool obtained in vitro did not affect survival on the basil phyllosphere in planta and even diminished survival in soil, suggesting that development of extreme resistance to linalool may be accompanied by a loss of fitness. Altogether, this report notes the concern regarding the ability of human pathogens to develop resistance to commercial essential oils, a resistance that is also associated with cross-resistance to antibiotics and may endanger public health.IMPORTANCE Greater consumer awareness and concern regarding synthetic chemical additives have led producers to control microbial spoilage and hazards by the use of natural preservatives, such as plant essential oils with antimicrobial activity. This report establishes, however, that these compounds may provoke the emergence of resistant human pathogens. Herein, we demonstrate the acquisition of resistance to basil oil by Salmonella Senftenberg. Exposure to linalool, a component of basil oil, resulted in adaptation to the basil oil mixture, as well as cross protection against several antibiotics and better survival on harvested basil leaves. Collectively, this work highlights the hazard to public health while using plant essential oils without sufficient knowledge about their influence on pathogens at subinhibitory concentrations.

Relative effectiveness of selected preenrichment media for the detection of Salmonella from leafy green produce and herbs.

Four buffered preenrichment media (BAX® System MP Media (BAX)), Universal Preenrichment Broth (UPB), modified Buffered Peptone Water (mBPW), and Buffered Peptone Water (BPW)) were compared with lactose broth (LB) in the Bacteriological Analytical Manual's (BAM) Salmonella culture method for the analysis of 9 leafy green produce and herb types. Artificially contaminated test portions were pre-enriched in each medium and the results were analyzed statistically using Fisher's Exact 2-tailed F test (p < 0.05) with pairwise comparisons. There was no difference in recovery of Salmonella from curly parsley and basil among the five media (p > 0.05). UPB was consistently among the most effective media for recovery of Salmonella from the nine produce types; however, S. Typhimurium and S. Newport were isolated from cabbage more frequently with mBPW than with UPB (p < 0.05). Comparisons of the results among the preenrichment media from all experimental trials, with leafy green produce and herbs, demonstrate that Salmonella is more effectively detected and isolated using buffered enrichments than with the currently recommended LB (p < 0.05). There were no significant differences among the buffered preenrichments for the detection of Salmonella-positive test portions of the produce tested (BAX (160 Salmonella-positive test portions/480 test portions), UPB (176/480), mBPW (184/480), BPW (169/480), LB (128/480))(p > 0.05).

Rhizophagus irregularis as an elicitor of rosmarinic acid and antioxidant production by transformed roots of Ocimum basilicum in an in vitro co-culture system.

Arbuscular mycorrhiza is a symbiotic association formed between plant roots and soil borne fungi that alter and at times improve the production of secondary metabolites. Detailed information is available on mycorrhizal development and its influence on plants grown under various edapho-climatic conditions, however, very little is known about their influence on transformed roots that are rich reserves of secondary metabolites. This raises the question of how mycorrhizal colonization progresses in transformed roots grown in vitro and whether the mycorrhizal fungus presence influences the production of secondary metabolites. To fully understand mycorrhizal ontogenesis and its effect on root morphology, root biomass, total phenolics, rosmarinic acid, caffeic acid and antioxidant production under in vitro conditions, a co-culture was developed between three Agrobacterium rhizogenes-derived, elite-transformed root lines of Ocimum basilicum and Rhizophagus irregularis. We found that mycorrhizal ontogenesis in transformed roots was similar to mycorrhizal roots obtained from an in planta system. Mycorrhizal establishment was also found to be transformed root line-specific. Colonization of transformed roots increased the concentration of rosmarinic acid, caffeic acid and antioxidant production while no effect was observed on root morphological traits and biomass. Enhancement of total phenolics and rosmarinic acid in the three mycorrhizal transformed root lines was found to be transformed root line-specific and age dependent. We reveal the potential of R. irregularis as a biotic elicitor in vitro and propose its incorporation into commercial in vitro secondary metabolite production via transformed roots.

Flexivirga endophytica sp. nov., an endophytic actinobacterium isolated from a leaf of Sweet Basil.

A Gram-reaction-positive, aerobic, non-motile, irregular coccoid strain, designated YIM 7505T, was isolated from a leaf of Sweet Basil. Phylogenetic analysis on the basis of 16S rRNA gene sequence comparisons revealed that strain YIM 7505T was closely related to Flexivirga alba NBRC 107580T (98.9 % 16S rRNA gene sequence similarity) and formed a robust clade with F. alba NBRC 107580T in the neighbour-joining tree. Optimum growth of strain YIM 7505T was observed at 28-35 °C, pH 7.0 and in the presence of 0-3.0 % NaCl (w/v). The chemotaxonomic profiles of the strain comprised of anteiso-C16 : 0 as the major cellular fatty acid and MK-8(H4) as the respiratory menaquinone. The peptidoglycan of strain YIM 7505T contained serine, alanine, glycine, glutamic acid and lysine. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, six unidentified phospholipids, four unidentified glycolipids, an unidentified aminolipid and an unidentified aminophospholipid. The G+C contents of the genomic DNA of strain YIM 7505T was 66.7 mol%. DNA-DNA hybridizations of strain YIM 7505T with F. alba NBRC 107580T gave relatedness values of 50.6±2.2 %. On the basis of the data recorded from the present study, strain YIM 7505T is considered to represent a novel species of the genus Flexivirga, for which the name Flexivirga endophytica sp. nov. is proposed. The type strain is YIM 7505T (=KCTC 39536T=CGMCC 1.15085T).

Rhizophagus intraradices or its associated bacteria affect gene expression of key enzymes involved in the rosmarinic acid biosynthetic pathway of basil.

In recent years, arbuscular mycorrhizal fungi (AMF) have been reported to enhance plant biosynthesis of secondary metabolites with health-promoting activities, such as polyphenols, carotenoids, vitamins, anthocyanins, flavonoids and lycopene. In addition, plant growth-promoting (PGP) bacteria were shown to modulate the concentration of nutraceutical compounds in different plant species. This study investigated for the first time whether genes encoding key enzymes of the biochemical pathways leading to the production of rosmarinic acid (RA), a bioactive compound showing antioxidant, antibacterial, antiviral and anti-inflammatory properties, were differentially expressed in Ocimum basilicum (sweet basil) inoculated with AMF or selected PGP bacteria, by using quantitative real-time reverse transcription PCR. O. basilicum plants were inoculated with either the AMF species Rhizophagus intraradices or a combination of two PGP bacteria isolated from its sporosphere, Sinorhizobium meliloti TSA41 and Streptomyces sp. W43N. Present data show that the selected PGP bacteria were able to trigger the overexpression of tyrosine amino-transferase (TAT), hydroxyphenylpyruvate reductase (HPPR) and p-coumaroyl shikimate 3'-hydroxylase isoform 1 (CS3'H iso1) genes, 5.7-fold, 2-fold and 2.4-fold, respectively, in O. basilicum leaves. By contrast, inoculation with R. intraradices triggered TAT upregulation and HPPR and CS3'H iso1 downregulation. Our data suggest that inoculation with the two selected strains of PGP bacteria utilised here could represent a suitable biotechnological tool to be implemented for the production of O. basilicum plants with increased levels of key enzymes for the biosynthesis of RA, a compound showing important functional properties as related to human health.