Nakamurella flava sp. nov., a novel endophytic actinobacterium isolated from Mentha haplocalyx Briq.

A Gram-stain-positive, aerobic, coccus-shaped, non-spore-forming actinobacterium, designated strain N5BH11T, was isolated from a surface-sterilized sample of Mentha haplocalyx Briq. collected from Guizhou, PR China and tested by a polyphasic approach to determine its taxonomic position. Strain N5BH11T grew optimally at 30 °C, pH 6.0-7.0. Substrate mycelia and aerial mycelia were not formed, and no diffusible pigments were observed on the media tested. Phylogenetic analysis based on 16S rRNA gene sequence suggested that strain N5BH11T belonged to the genus Nakamurella and had the highest 16S rRNA gene sequence similarity to Nakamurella flavida DS-52T (98.1 %). The DNA G+C content of strain N5BH11T was 71.6 mol%. The average nucleotide identity values between strain N5BH11T and the type strains of Nakamurella panacisegetis, Nakamurella multipartita and Nakamurella lactea were 74.0, 76.5 and 73.6 %, respectively. The estimated DDH values between strain N5BH11T and the type strains of N. panacisegetis, N. multipartita and N. lactea were 20.3%, 21.4 and 20.2 %, respectively. The cell-wall peptidoglycan contained meso-diaminopimelic acid, and MK-8(H4) was the predominant menaquinone. The predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and unidentified phospholipids. The major fatty acids were iso-C15 : 0, anteiso-C15 : 0, C16 : 0 and C16 : 1ω7c. On the basis of the results of phylogenetic analysis and phenotypic and chemotaxonomic characteristics, strain N5BH11T represents a novel species of the genus Nakamurella, for which the name Nakamurella flava sp. nov. is proposed. The type strain is N5BH11T (=KCTC 49196T=CGMCC 4.7524T).

Genetic Diversity of Verticillium dahliae Isolates From Mint Detected with Genotyping by Sequencing.

Verticillium wilt is the most important disease threatening the commercial production of mint grown for essential oil. An important long-term goal for mint breeders is the production of cultivars with resistance to Verticillium wilt. Before that can be accomplished, a better understanding of the genetic variation within and among populations of Verticillium dahliae is needed. We characterized the extent of phenotypic and genetic diversity present in contemporary and archival populations of V. dahliae from mint fields in Oregon and other production regions of the United States using genotyping by sequencing, PCR assays for mating type and pathogenic race, vegetative compatibility group (VCG) tests, and aggressiveness assays. We report that the population in the Pacific Northwest can be described as one common genetic group and four relatively rare genetic groups. Eighty-three percent of the isolates belonged to VCG2B, and all isolates possessed the MAT1-2 idiomorph and were characterized as pathogenic race 2. These results indicate low levels of genetic diversity and a negligible risk of sexual recombination in populations of this host-adapted pathogen population. Knowledge of the genetic structure of V. dahliae in the Pacific Northwest will inform breeders about the diversity of pathogenicity factors that may need to be considered in their breeding programs.

Protein kinase and HDAC inhibitors from the endophytic fungus Epicoccum nigrum.

A chemical investigation of the endophytic fungus Epicoccum nigrum isolated from leaves of Mentha suaveolens collected in Morocco resulted in the isolation of five new polyketides, epicocconigrones A and B (1 and 2), 3-methoxyepicoccone B (3), 3-methoxyepicoccone (4), and 2,3,4-trihydroxy-6-(methoxymethyl)-5-methylbenzaldehyde (5), together with five known compounds (6-10). The structures of the new compounds were unambiguously determined by extensive analysis of the 1D and 2D NMR and mass spectroscopic data. Compounds 1 and 10 showed potent inhibition of at least 15 protein kinases with IC50 values ranging from 0.07 to 9.00 µM. Moreover, compounds 1 and 10 inhibited histone deacetylase (HDAC) activities with IC50 values of 9.8 and 14.2 µM, respectively. A preliminary structure-activity relationship is discussed. Interestingly, compounds 1 and 10 exert mainly cytostatic effects in human lymphoma RAJI and U-937 cell lines.

Verticillium dahliae populations from mint and potato are genetically divergent with predominant haplotypes.

In total, 286 Verticillium dahliae isolates from mint, potato, and other hosts and substrates were characterized for mating type, vegetative compatibility group (VCG), and multilocus microsatellite haplotype to determine population genetic structure among populations infecting mint and potato. Populations from mint and potato fit a clonal reproductive model, with all isolates a single mating type (MAT1-2) and multiple occurrences of the same haplotypes. Haplotype H02 represented 88% of mint isolates and was primarily VCG2B, while haplotype H04 represented 70% of potato isolates and was primarily VCG4A. Haplotypes H02 and H04 typically caused severe disease on mint and potato, respectively, in greenhouse assays regardless of host origin. Principal coordinate analysis and analysis of molecular variance indicated that mint and potato populations were significantly genetically diverged (P = 0.02), and identification of private alleles and estimation of migration rates suggested restricted gene flow. Migration was detected between infected potato plants and seed tubers, infested tare soil, and field soils. Genetic differentiation of V. dahliae from mint and potato may be due to the occurrence of a single mating type and differences in VCG. Populations of V. dahliae in potato and mint were characterized by the presence of aggressive, clonally reproducing haplotypes which are widely distributed in commercial mint and potato production.

The greater effectiveness of Glomus mosseae and Glomus intraradices in improving productivity, oil content and tolerance of salt-stressed menthol mint (Mentha arvensis).

BACKGROUND: Mentha arvensis is cultivated in large parts of the world for its menthol-rich essential oil. The study investigates the potential of four mycorrhizal fungi, viz. Glomus mosseae (Gm), Glomus aggregatum (Ga), Glomus fasciculatum (Gf) and Glomus intraradices (Gi) in alleviating NaCl-induced salt stress in Mentha arvensis cv. Kosi and establishes the specificity of interaction between different mycorrhizal species and their effectiveness in mitigating salt stress in Mentha arvensis. Mycorrhizal and non-mycorrhizal Mentha plants were subjected to NaCl-induced salinity. RESULTS: Among the four Glomus species, Gm and Gi reduced salt-induced herb yield losses: a loss of 27.53% and 25.58% respectively under salt stress in comparison to 51.00% in non-mycorrhizal M. arvensis salt-stressed plants. Gm- and Gi-inoculated plants also recorded higher leaf:stem ratio, oil content, and oil yield and menthol concentration in essential oil under both saline and non-saline conditions. CONCLUSION: Better performance in terms of herb yield, and oil content and yield was observed in Gi- and Gm-inoculated M. arvensis plants, suggesting the capability of Gi and Gm in protecting plants from the detrimental effects of salt stress; beneficial effects of arbuscular mycorrhizal fungi, however, may vary with host and environment.

A natural plant growth promoter calliterpenone from a plant Callicarpa macrophylla Vahl improves the plant growth promoting effects of plant growth promoting rhizobacteria (PGPRs).

Experiments were conducted to evaluate the efficacy of calliterpenone, a natural plant growth promoter from a shrub Callicarpa macrophylla Vahl., in enhancing the growth and yield promoting effects of plant growth promoting rhizobacteria (PGPRs), in menthol mint (Mentha arvensis L).This study is based on our previous results indicating the microbial growth promotion by calliterpenone and assumption that application of calliterpenone along with PGPRs will improve the population of PGPRs resulting in higher impacts on plant growth and yield. Of the 15 PGPRs (identified as potent ones in our laboratory), 25 µl of 0.01 mM calliterpenone (8.0 µg/100 ml) was found to be useful in improving the population of nine PGPRs in culture media. The five selected strains of PGPRs exhibiting synergy with calliterpenone in enhancing growth of maize compared to PGPR or calliterpenone alone were selected and tested on two cultivars (cvs. Kosi and Kushal) of M. arvensis. Of the five strains, Bacillus subtilis P-20 (16S rDNA sequence homologous to Accession No NR027552) and B. subtilis Daz-26 (16SrDNA sequence homologuos to Accession No GU998816) were found to be highly effective in improving the herb and essential oil yield in the cultivars Kushal and Kosi respectively when co-treated with calliterpenone. The results open up the possibilities of using a natural growth promoter along with PGPRs as a bio-agri input for sustainable and organic agriculture.

Compost mixed fruits and vegetable waste biochar with ACC deaminase rhizobacteria can minimize lead stress in mint plants.

High lead (Pb) concentration in soils is becoming a severe threat to human health. It also deteriorates plants, growth, yield and quality of food. Although the use of plant growth-promoting rhizobacteria (PGPR), biochar and compost can be effective environment-friendly amendments for decreasing Pb stress in crop plants, the impacts of their simultaneous co-application has not been well documented. Thus current study was carried, was conducted to investigate the role of rhizobacteria and compost mixed biochar (CB) under Pb stress on selected soil properties and agronomic parameters in mint (Mentha piperita L.) plants. To this end, six treatments were studied: Alcaligenes faecalis, Bacillus amyloliquefaciens, CB, PGPR1 + CB, PGPR2 + CB and control. Results showed that the application A. faecalis + CB significantly decreased soil pH and EC over control. However, OM, nitrogen, phosphorus and potassium concentration were significantly improved in the soil where A. faecalis + CB was applied over control. The A. faecalis + CB treatment significantly improved mint plant root dry weight (58%), leaves dry weight (32%), chlorophyll (37%), and N (46%), P (39%) and K (63%) leave concentration, while also decreasing the leaves Pb uptake by 13.5% when compared to the unamended control. In conclusion, A. faecalis + CB has a greater potential to improve overall soil quality, fertility and mint plant productivity under high Pb soil concentration compared to the sole application of CB and A. faecalis.

Hairy root cultures for secondary metabolites production.

Hairy roots (HRs) are differentiated cultures of transformed roots generated by the infection of wounded higher plants with Agrobacterium rhizogenes. This pathogen causes the HR disease leading to the neoplastic growth of roots that are characterized by high growth rate in hormone free media and genetic stability. HRs produce the same phytochemicals pattern of the corresponding wild type organ. High stability and productivity features allow the exploitation of HRs as valuable biotechnological tool for the production of plant secondary metabolites. In addition, several elicitation methods can be used to further enhance their accumulation in both small and large scale production. However, in the latter case, cultivation in bioreactors should be still optimized. HRs can be also utilised as biological farm for the production of recombinant proteins, hence holding additional potential for industrial use. HR technology has been strongly improved by increased knowledge of molecular mechanisms underlying their development. The present review summarizes updated aspects of the hairy root induction, genetics and metabolite production.

Isolation of a Ve homolog, mVe1, and its relationship to Verticillium wilt resistance in Mentha longifolia (L.) Huds.

As a step toward greater understanding of the genetics of verticillium wilt resistance in plants, we report the sequencing of a candidate wilt resistance gene, mVe1, from the mint diploid model species, Mentha longifolia (Lamiaceae). mVe1 is a putative homolog of tomato (Solanum lycopersicum L.) verticillium wilt (Ve) resistance genes. The mVe1 gene has a coding region of 3,051 bp. The predicted mVe1 protein contains a leucine-rich repeat domain, a common feature of plant disease resistance proteins. We compared 13 mVe1 alleles from three mint species. These alleles shared 96.2-99.6% nucleotide identity. We analyzed four M. longifolia populations segregating with respect to mVe1 alleles and wilt resistance versus susceptibility and found one association between mVe1 genotype and wilt phenotype. We conclude that mVe1 may play a role in mint verticillium wilt resistance, but variation for resistance in our segregating progenies is likely polygenic. Therefore, further investigations of mVe1 and identification of additional candidate genes are both warranted.

Evidence of a trans-kingdom plant disease complex between a fungus and plant-parasitic nematodes.

Disease prediction tools improve management efforts for many plant diseases. Prediction and downstream prevention demand information about disease etiology, which can be complicated for some diseases, like those caused by soilborne microorganisms. Fortunately, the availability of machine learning methods has enabled researchers to elucidate complex relationships between hosts and pathogens without invoking difficult-to-satisfy assumptions. The etiology of a destructive plant disease, Verticillium wilt of mint, caused by the fungus Verticillium dahliae was reevaluated with several supervised machine learning methods. Specifically, the objective of this research was to identify drivers of wilt in commercial mint fields, describe the relationships between these drivers, and predict wilt. Soil samples were collected from commercial mint fields. Wilt foci, V. dahliae, and plant-parasitic nematodes that can exacerbate wilt were quantified. Multiple linear regression, a generalized additive model, random forest, and an artificial neural network were fit to the data, validated with 10-fold cross-validation, and measures of explanatory and predictive performance were compared. All models selected nematodes within the genus Pratylenchus as the most important predictor of wilt. The fungus after which this disease is named, V. dahliae, was the fourth most important predictor of wilt, after crop age and cultivar. All models explained around 50% of the total variation (R2 ≤ 0.46), and exhibited comparable predictive error (RMSE ≤ 1.21). Collectively, these models revealed that the quantitative relationships between two pathogens, mint cultivars and age are required to explain wilt. The ascendance of Pratylenchus spp. in predicting symptoms of a disease assumed to primarily be caused by V. dahliae exposes the underestimated contribution of these nematodes to wilt. This research provides a foundation on which predictive forecasting tools can be developed for mint growers and reminds us of the lessons that can be learned by revisiting assumptions about disease etiology.

Membrane proteome profiling of Mentha arvensis leaves in response to Alternaria alternata infection identifies crucial candidates for defense response.

The leaf spot disease of Mentha arvensis, caused by Alternaria alternata, is a devastating foliar disease worldwide and leads to considerable economic losses. In this investigation, 2-dimensional gel electrophoresis (2-DE) was used to identify the membrane proteins potentially involved in M. arvensis - A. alternata interaction. Membrane proteins, isolated from leaves of control and infected plants, were analyzed by 2-DE and identified using mass spectrometry (MALDI TOF-TOF MS/MS). Our analysis identified 21 differentially expressed membrane proteins including several interesting receptors and channel proteins. Of these identified proteins, 34% were found to be involved in plant defense responses. Leucine-rich repeat family protein/ protein kinase family protein which plays critical role in stress response and nucleotide-binding site-leucine-rich repeat (NBS-LRR) which is involved in detecting the advent of pathogen on plant surface were identified to be up-regulated in our study. Interestingly, AKT1-like potassium channel protein which is known to play a crucial role in maintaining ion homeostasis within the cell was also upregulated in the infected sample. In addition, ADP ribolysation factor (ARF)-GTPase activating domain containing protein, a membrane trafficking protein, was also up-regulated in the current study. Protein-protein interaction network analysis followed by functional enrichment revealed that transmembrane ion transport-related proteins represented a major class in this network followed by nucleic acid binding proteins and proteins with kinase activities respectively. Together, our investigation identified several key defense-related proteins which are crucial sensors for detecting pathogen invasion and can serve as a potential resource to understand disease resistance mechanism in mint.

Integracides H-J: New tetracyclic triterpenoids from the endophytic fungus Fusarium sp.

Three new tetracyclic triterpenoids namely, integracides H (1), I (4), and J (5), along with integracides B (3) and F (2) have been isolated from the endophytic fungus Fusarium sp. isolated from the roots of Mentha longifolia L. (Labiatae) growing in Saudi Arabia. The structure elucidation of the isolated compounds was achieved by spectroscopic analysis including UV, IR, 1D ((1)H and (13)C) and 2D ((1)H(1)H COSY, TOCSY, HSQC, HMBC, and NOESY) NMR as well as HRESIMS and comparison with literature data. Integracides H (1) and J (5) showed significant anti-leishmanial activity towards Leishmania donovani with IC50 values of 4.75 and 3.29µM, respectively compared to pentamidine (IC50 6.35µM). Moreover, they displayed potent cytotoxic activity towards BT-549, SKOV-3, and KB cell lines with IC50 values of 1.82, 1.32, and 0.18µM and 2.46, 3.01, and 2.54µM, respectively.

In Vitro Morphogenesis of Arabidopsis to Search for Novel Endophytic Fungi Modulating Plant Growth.

Fungal endophytes have shown to affect plant growth and to confer stress tolerance to the host; however, effects of endophytes isolated from water plants have been poorly investigated. In this study, fungi isolated from stems (stem-E) and roots (root-E) of Mentha aquatica L. (water mint) were identified, and their morphogenetic properties analysed on in vitro cultured Arabidopsis (L.) Heynh., 14 and 21 days after inoculation (DAI). Nineteen fungi were analysed and, based on ITS analysis, 17 isolates showed to be genetically distinct. The overall effect of water mint endophytes on Arabidopsis fresh (FW) and dry weight (DW) was neutral and positive, respectively, and the increased DW, mainly occurring 14 DAI, was possibly related to plant defence mechanism. Only three fungi increased both FW and DW of Arabidopsis at 14 and 21 DAI, thus behaving as plant growth promoting (PGP) fungi. E-treatment caused a reduction of root depth and primary root length in most cases and inhibition-to-promotion of root area and lateral root length, from 14 DAI. Only Phoma macrostoma, among the water mint PGP fungi, increased both root area and depth, 21 DAI. Root depth and area 14 DAI were shown to influence DWs, indicating that the extension of the root system, and thus nutrient uptake, was an important determinant of plant dry biomass. Reduction of Arabidopsis root depth occurred to a great extent when plants where treated with stem-E while root area decreased or increased under the effects of stem-E and root-E, respectively, pointing to an influence of the endophyte origin on root extension. M. aquatica and many other perennial hydrophytes have growing worldwide application in water pollution remediation. The present study provided a model for directed screening of endophytes able to modulate plant growth in the perspective of future field applications of these fungi.

Evaluación de la actividad antimicrobiana in vitro de mentha frente a candida albicans / Evaluation of the in vitro antimicrobial activity of mentha against candida albicans

El uso de la medicina natural tradicional es actualmente una parte importante relacionada con la salud, en algunos países de Latinoamérica se la conoce como medicina complementaria, la cual históricamente se ha utilizado para mantener la salud, prevenir y tratar enfermedades crónicas. En el presente proyecto de investigación se propuso determinar si existe algún efecto antimicrobiano significativo contra el microorganismo Cándida albicans en el extracto alcohólico o aceite esencial obtenidos a partir de la planta menta. La investigación comprendió tres etapas: el estudio etnofarmacológico, el análisis fitoquímico y el análisis de actividad antimicrobiana. Al concluir la primera etapa se estableció que las plantas de acuerdo al nivel de uso significativo (UST%), presentan los siguientes valores: menta (29,41%), sábila (23,08%) y manzanilla (21,72%). En el análisis fitoquímico se determinó la presencia de ciertos metabolitos secundarios de la planta medicinal menta como los terpenoides y compuestos fenólicos (cumarinas, flavonoides, lignina y taninos). Dentro del análisis microbiológico, se presentó mayor efectividad a una concentración de 100%, obteniéndose halos promedio de 17,67mm y 16,67mm para aceite esencial y extracto etanólico respectivamente. La prueba de varianzas realizada como análisis estadístico determinó que alguna de las medias de las distribuciones de la variable cuantitativa (halos de inhibición) en los extractos y aceites de menta es diferente (15,907 con un valor de p < 0,05), afirmando que existen efecto antimicrobiano de la menta sobre Cándida albicans.

Volatile terpenoids of endophyte-free and infected peppermint (Mentha piperita L.): chemical partitioning of a symbiosis.

The study reports the effects on volatiles of an endophytic fungus inhabiting asymptomatically the leaves of peppermint. By means of headspace solid-phase microextraction (HS-SPME) and gaschromatography-mass spectrometry (GC-MS) terpenoids were sampled in time course from the head space of peppermint leaves and roots. After removal of the mycelium from peppermint tissues, fungal volatiles were analyzed and compared with those of pure fungal cultures. In the presence of the endophyte, the relative amount of all main compounds increased in leaves. Starting from the first 14 d of culture, (-)-menthone and (+)-neomenthol were consistently higher than in control plants. On the contrary, (+)-menthofuran increased only by 28 d of culture. Root volatiles were also dramatically altered by the presence of the fungus, with (+)-pulegone accounting for at least 44% of the total volatile emission. (+)-Pulegone was also the main compound of PGP-HSF mycelium isolated from peppermint roots. The sesquiterpenoid cuparene was found as a novel compound of peppermint leaf headspace and was a main volatile of ex planta and pure culture mycelia. The chemical spectrum of terpenoids and their distribution among peppermint roots, leaves, and mycelia are likely to account for a fine regulation of the mutualism in planta and for the acquisition by the fungus of novel metabolic competences.

Prevalence of multidrug resistant Salmonella in Coriander, mint, carrot, and radish in Bareilly and Kanpur, northern India.

We examined 974 samples (304 coriander, 212 mint, 258 carrot, and 200 radish) collected from vegetable vendors in two cities, Bareilly (n = 832) and Kanpur (n = 142), in northern India during the early summer season in 2004. Salmonella was isolated from 35 samples (9 coriander, 5 mint, 10 radish, and 11 carrot) while Escherichia coli was detected in 181 samples (67 coriander, 44 mint, 36 carrot, and 34 radish). None of the E. coli belonged to the O:157 serogroup. Five Salmonella isolates from samples collected at Kanpur (3 coriander and 2 mint) belonged to 4 different serovars of S. enterica ssp. enterica-S. Mons, S. Rottenest, S. Saintpaul, and S. Weltevreden. Thirty Salmonella isolates from samples collected at Bareilly (11 carrot, 10 radish, 6 coriander, and 3 mint) belonged to 7 serovars-S. Anatum, S. Bsilla, S. Newport, S. Saintpaul, S. Teko, S. Virchow, and S. Weltevreden. The majority (82.9%) of Salmonella isolates were multidrug resistant. One quarter of the isolates were resistant to >or=10 antibiotics. Based on antibiotic resistance patterns, 35 isolates could be classified into 23 resistotypes. None of the 35 isolates was resistant to streptomycin and ceftriaxone, while >80% were resistant to sulfamethoxazole, nalidixic acid, and kanamycin. Resistance to imipenem (>20%) and amikacin (>30%) was also common. The correlation between presence of Salmonella and E. coli on raw vegetables was not significant (p = 0.13).