Two independent loss-of-function mutations in anthocyanidin synthase homeologous genes are responsible for the all-green phenotype of sweet basil.

Sweet basil, Ocimum basilicum L., is an important culinary herb grown worldwide. Although basil is green, many landraces, breeding lines, and exotic cultivars have purple stems and flowers. This anthocyanin pigmentation is unacceptable in traditional Italian basil used for Pesto sauce production. In the current study, we aimed to resolve the genetics that underlines the different colors. We used the recently published sweet basil genome to map quantitative trait loci (QTL) for flower and stem color in a bi-parental F2 population. It was found that the pigmentation is governed by a single QTL, harboring an anthocyanidin synthase (ANS) gene (EC 1.14.20.4). Further analysis revealed that the basil genome harbors two homeologous ANS genes, each carrying a loss-of-function mutation. ObANS1 carries a single base pair insertion resulting in a frameshift, and ObANS2 carries a missense mutation within the active site. In the purple-flower parent, ANS1 is functional, and ANS2 carries a nonsense mutation. The functionality of the ObANS1 active allele was validated by complementation assay in an Arabidopsis ANS mutant. Moreover, we have restored the functionality of the missense-mutated ObANS2 using site-directed activation. We found that the non-functional alleles were expressed to similar levels as the functional allele, suggesting polyploids invest futile effort in expressing non-functional genes, offsetting their advantageous redundancy. This work demonstrated the usefulness of the genomics and genetics of basil to understand the basic mechanism of metabolic traits and raise fundamental questions in polyploid plant biology.

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.

The genome sequence of tetraploid sweet basil, Ocimum basilicum L., provides tools for advanced genome editing and molecular breeding.

Sweet basil, Ocimum basilicum L., is a well-known culinary herb grown worldwide, but its uses go beyond the kitchen to traditional medicine, cosmetics and gardening. To date, the lack of an available reference genome has limited the utilization of advanced molecular breeding methods. We present a draft version of the sweet basil genome of the cultivar 'Perrie', a fresh-cut Genovese-type basil. Genome sequencing showed basil to be a tetraploid organism with a genome size of 2.13 Gbp, assembled in 12,212 scaffolds, with > 90% of the assembly being composed of 107 scaffolds. About 76% of the genome is composed of repetitive elements, with the majority being long-terminal repeats. We constructed and annotated 62,067 protein-coding genes and determined their expression in different plant tissues. We analysed the currently known phenylpropanoid volatiles biosynthesis genes. We demonstrated the necessity of the reference genome for a comprehensive understanding of this important pathway in the context of tetraploidy and gene redundancy. A complete reference genome is essential to overcome this redundancy and to avoid off-targeting when designing a CRISPR: Cas9-based genome editing research. This work bears promise for developing fast and accurate breeding tools to provide better cultivars for farmers and improved products for consumers.

Chemo-Geographic Variations in Wild Population of Asteriscus graveolens in Israel Based on Volatile Composition Analyses.

Asteriscus graveolens is an aromatic desert shrub which holds medicinal potential. This species belongs to the Asteraceae family and is endemic to the Mediterranean region. In the present study, wild plants were sampled from eleven locations throughout southern Israel and the volatile profiles from leaves and flowers were analyzed using GC/MS. Three methods for volatile sampling were tested for a representative population: solvent extraction (methyl tert-butyl ether), hydrodistillation of the essential oil and headspace solid-phase microextraction. In all methods, the majority of volatiles were characterized as oxygenated mono- and sesquiterpenes. Only solvent extraction was able to detect asteriscunolides that were previously reported as anticancer molecules. Hence, that method was chosen for further analyses. The leaves were dominated by three asteriscunolide isomers, cis-chrysanthenyl acetate and intermedeol. The flowers were dominated by bisabolone, 6-hydroxybisabol-2-en-1-one, cis-chrysanthenyl acetate, epi-α-cadinol, and germacrene-D. k-Means clustering analysis of these data divided the population into four clusters that significantly differ in their volatile composition as was further demonstrated by MANOVA analysis. Geographically, A. graveolens populations growing in Israel were found to be chemically diverse with unique varieties in the Dead Sea basin and the Arava region. This work demonstrates that chemo-geographic variation of volatile composition exists within A. graveolens population growing in Israel, so future research evaluating the medicinal potential of that plant should take this into consideration.

Agronomic and economic evaluation of Vetiver grass (Vetiveria zizanioides L.) as means for phytoremediation of diesel polluted soils in Israel.

Soil pollution in Israel, due to diesel contamination, is a major concern, with gas stations, factories and refineries being the main polluters (>60%). Vetiver grass (Vetiveria zizanioides L.) is a perennial grass belonging to the Poaceae family, and is recognized world-wide for its potential as a plant with phytoremediation traits to contaminated soils. It is demonstrated here to decrease diesel contamination in field and court-yard trials. Chemical soil analysis indicated up to a 79% decrease (P < .05) in diesel pollution of contaminated soil planted with Vetiver; and at high soil contamination levels of 10 L/m2, a significant (P < .05) reduction of 96, 96 and 87% was recorded at soil depths of 0-20, 20-40 and 40-60 cm, respectively. Furthermore, in field plots contaminated with diesel and planted with Vetiver, weeds' biomass recovered to non-polluted levels following 8 to 9 months of Vetiver treatment. An economic evaluation conducted based on the cost-benefit analysis (CBA) principles, utilizing the Net Present Value (NPV) compared phytoremediation to other currently used decontamination procedures. The economic comparison showed that phytoremediation cleanup costs are lower and more beneficial to society at large, primarily from an ecosystem services perspective. Combining the results of the agronomic examination with the economic valuation, this research pointed out that phytoremediation with Vetiver has a non-negligible potential, making it a good solution for cleansing diesel from soils on a state-wide scale in Israel and worthy of further research and development.

Whitefly attraction to rosemary (Rosmarinus officinialis L.) is associated with volatile composition and quantity.

Whitefly (Bemisia tabaci) is an important insect pest, causing severe damage to agricultural crops. The pest was recorded in a commercial rosemary (Rosmarinus officinalis, Lamiaceae) field, colonizing rosemary variety (var.) '2', but not '11'. A series of field and controlled laboratory choice bioassays confirmed the observed phenomenon. Mature potted plants of the two varieties were randomly organized in a lemon verbena (Lippia citrodora) and lemon grass (Cymbopogon spp.) fields. Seven days later var. '2' was significantly more colonized by whiteflies than var. '11'. Under lab conditions, whiteflies were significantly more attracted to var. '2' plantlets than to var. '11' following choice bioassays. Furthermore, cotton plants dipped in an essential oil emulsion of var. '2' had significantly greater colonization than cotton plants dipped in the essential oil emulsion of var. '11'. Similar results were obtained in 'plant-plant', 'plant-no plant' as well as, 'essential oil-essential oil' choice bioassay designs. Analyses of the essential oils of the two varieties identified a set of common and unique volatiles in each variety. Among these volatiles were ß-caryophyllene and limonene, two compounds known to be associated with plant-insect interactions. The attraction of B. tabaci to pure (>95%) ß-caryophyllene and limonene using a range of concentrations was examined in vitro by choice bioassays. The compounds were attractive to the insect at moderate concentration, but not at the lowest or highest concentrations used, where the insect was not attracted or repelled, respectively. Limonene attracted the insects at rates that were 10-fold lower than ß-caryophyllene. The results emphasized the role of host plant volatiles in shaping the structure of B. tabaci populations in nature and in agricultural systems, and provided insights into the factors that contribute to the development of insect populations with unique characteristics. The results could also serve for future development of bio-pesticides and in breeding programs.

Enantioselective effects of (+)- and (-)-citronellal on animal and plant microtubules.

Citronellal is a major component of Corymbia citriodora and Cymbopogon nardus essential oils. Herein it is shown that whereas (+)-citronellal (1) is an effective microtubule (MT)-disrupting compound, (-)-citronellal (2) is not. Quantitative image analysis of fibroblast cells treated with 1 showed total fluorescence associated with fibers resembling that in cells treated with the MT-disrupting agents colchicine and vinblastine; in the presence of 2, the fluorescence more closely resembled that in control cells. The distribution of tubulin in soluble and insoluble fractions in the presence of 1 also resembled that in the presence of colchicine, whereas similar tubulin distribution was obtained in the presence of 2 and in control cells. In vitro polymerization of MTs was inhibited by 1 but not 2. Measurements of MT dynamics in plant cells showed similar MT elongation and shortening rates in control and 2-treated cells, whereas in the presence of 1, much fewer and shorter MTs were observed and no elongation or shrinkage was detected. Taken together, the MT system is suggested to be able to discriminate between different enantiomers of the same compound. In addition, the activity of essential oils rich in citronellal is affected by the relative content of the two enantiomers of this monoterpenoid.

Chemical and morphological diversity in wild populations of Mentha longifolia in Israel.

Populations of Mentha longifolia, an endangered species in Israel, were tested for essential oil composition and conservational ability. In 2002-2003, 25 wild populations country-wide were tested, indicating population divergence into two chemotypes. Chemotype A was characterized by high levels of menthone and pulegone, and chemotype B by high levels of piperitenone oxide and piperitone oxide. Chemotype A was more abundant (22 of 25 populations) than chemotype B (11 of 25 populations). However, a chemotype/population interaction was not recorded (P > 0.05). In spring 2003, seven of the 25 wild populations were resampled, propagated, and cultivated at the Newe Ya'ar campus. Then, in 2004, the propagated plants were tested for essential oil composition. The propagated plants maintained the essential oil composition as well as the chemotype-frequency distribution of the original wild population from which they were obtained. Since a chemotype/population interaction was not recorded, and the cultivated plants displayed the wild population essential oil composition, it can be concluded that i) the chemotype diversity is genetically based, and ii) the M. longifolia populations sampled can be horticulturally conserved.

Rhizoctonia Web Blight-A New Disease on Mint in Israel.

Mentha longifolia is produced in and exported from Israel with annual revenue of US$16 million. In 2010, a severe epidemic of unknown etiology reduced growers' returns up to 50%. Disease symptoms included water-soaked lesions, necrosis, and web-like mycelia on plants. Two isolates (JV-1 and BS-1) from randomly selected symptomatic plants were identified as members of Rhizoctonia solani anastomosis groups 1-IB and 4HG-I, respectively. The fitness of JV-1 and BS-1 interacted with temperatures between 17 and 35°C: JV-1 grew faster and was more aggressive (P < 0.05) at lower temperatures (<24°C), BS-1 grew faster and was more aggressive at higher temperatures (>30°C), and the two isolates performed similarly at intermediate temperatures. Disease developed fastest at 24 to 28°C. Yield was reduced between 46 and 100%. In all, 77.5% fewer plants recovered from disease developing at 24 to 28°C than at 17 to 23 or 30 to 35°C. The relationship of disease to relative humidity (RH) fit a quadratic model (P < 0.0015, R2 = 0.98). Disease was most severe at 100% RH, decreasing by 1.3-, 1.9-, 3-, 4.5-, and 10.5-fold with the reduction of RH from 100% to 88, 76, 69, 55, or 49%, respectively. This is the first report of Rhizoctonia web blight in mint in Israel.

Intraspecific variation of Chiliadenus iphionoides essential oil in Israel.

Chiliadenus iphionoides (Asteraceae), a shrub endemic to the Mediterranean region and widespread throughout Israel, is used in the traditional eastern Mediterranean medicine. Although recent research confirmed its pharmacological potential, C. iphionoides essential oil has not been adequately characterized chemically. Essential-oil samples were collected from representative wild populations throughout Israel and characterized by GC/MS analysis. Considerable interpopulation variation was found for the composition of the essential oils. Multivariate analysis showed a significant correlation between the chemical composition and the geographic location, with three main chemotypes identified.

Variation in essential oil composition within individual leaves of sweet basil (Ocimum basilicum L.) is more affected by leaf position than by leaf age.

The aroma in sweet basil is a factor affecting the commercial value of the crop. In previous studies leaf age was considered to be a factor that influences the composition of essential oil (EO). In this study it was hypothesized that a single observation of the EO content in leaves from different positions on the main stem (young vs old) could predict the developmental changes in the plant during its life cycle. Plants harvested at week 16 demonstrated an exponential increase (R(2) = 0.92) in EO concentration in leaves on the main stem and lateral shoots, indicating higher EO concentrations in younger than in older leaves. Eugenol and methyleugenol predominated (28-77%) in the extract. Eugenol levels were higher in younger leaves (∼53%), and methyl-eugenol levels predominated in older leaves (∼68%). Linalool was lower in mature leaves than in younger leaves. This suggested that eugenol converted into methyleugenol and linalool decreased as leaf mature. However, in weekly monitored plants, the levels of these compounds in the EO had limited variation in the maturing leaf regardless of its position on the stem. This proposed that the EO composition in an individual leaf is mostly affected by the leaf position on the stem and not by its maturation process. Because leaf position is related to plant development, it is probable that the plant's physiological age at the time of leaf formation from the primordial tissue is the factor affecting the EO composition. It was concluded that interpretation of scientific observations should be carried out with caution and that hypotheses should be tested utilizing multifaceted approaches.

Microtubules are an intracellular target of the plant terpene citral.

Citral is a component of plant essential oils that possesses several biological activities. It has known medicinal traits, and is used as a food additive and in cosmetics. Citral has been suggested to have potential in weed management, but its precise mode of action at the cellular level is unknown. Here we investigated the immediate response of plant cells to citral at micromolar concentrations. It was found that microtubules of Arabidopsis seedlings were disrupted within minutes after exposure to citral in the gaseous phase, whereas actin filaments remained intact. The effect of citral on plant microtubules was both time- and dose-dependent, and recovery only occurred many hours after a short exposure of several minutes to citral. Citral was also able to disrupt animal microtubules, albeit less efficiently. In addition, polymerization of microtubules in vitro was inhibited in the presence of citral. Taken together, our results suggest that citral is a potent, volatile, anti-microtubule compound.