Cyanobacterial elicitor enhances the biomass of Mentha piperita L. and improves the production of high-value rosmarinic acid under in vitro culture of apical meristem.

BACKGROUND: Rosmarinic acid (RA), like other phenolic compounds, is sources of antioxidants and anti-inflammatory agents in medicinal plants. In vitro culture of plants can improve the medicinal plants' metabolite profile and phenolic compound quantity. To date, various methods have been proposed to increase this medicinal metabolite in plants, among which the use of bioelicitors can be mentioned. In the present study, a native isolate of heterocystous cyanobacteria, Nostoc spongiaeforme var. tenue ISB65, was used to stimulate the production of biomass and content of RA in Mentha piperita L. (peppermint) grown in vitro from apical meristem. Mentha piperita L. explants were inoculated in half strength Murashige and Skoog (1/2 MS) medium containing cyanobacterial lysate (CL). After 50 days of culturing, the growth indices, the content of photosynthetic pigments, and RA in control and treated plants were measured. RESULTS: CL inoculation resulted in a significant enhancement in the vegetative growth indices of peppermint, including root and shoot length, plant biomass and leaf number. The content of photosynthetic pigments also increased in cyanobacteria-treated plants. Inoculation with CL increased the RA content by 2.3-fold, meaning that the plants treated with CL had the highest RA content (7.68 mg. g- 1 dry weight) compared to the control (3.42 mg. g- 1 dry weight). Additionally, HPLC analysis revealed the presence of several auxins in CL. CONCLUSIONS: The presence of auxins and the chemical content of CL such as K+ and Ca2+, as regulators of metabolic pathways and molecular activities of cells, may be responsible for the enhanced growth and phenolic compounds of plants under tissue culture conditions. An improvement in RA content in the tissue culture of medicinal plants treated with CL was reported for the first time in this investigation.

Aspergillus flavus originated pure compound as a potential antibacterial.

PROBLEM BACKGROUND: Penicillin was the first and most famous fungal secondary metabolite used as broad spectrum antibiotic that revolutionarised pharmaceutical research and also saved millions of lives. The over optimistic belief in 1967 that sufficient antibiotics had been discovered to defeat infectious diseases was quickly crashed with the appearance of multidrug resistant (MDR) bacteria in 1990s. This has posed a serious threat to mankind. Although scientists are making efforts to synthesize and discover new antibiotics there are not enough new drugs in pharmaceutical pipeline to beat the pace at which MDR bacteria are emerging. In view of this there is an urgent and serious medical need for new bioactive compounds to be discovered to treat infections caused by MDR pathogens. The present study is aimed to investigate the antibacterial potential of Aspergillus flavus originated compounds that may act as drug leads to treat future infections. METHODOLOGY: Among the 6 isolated fungal strains from the rhizosphere of Mentha piperetta, one was processed for isolation of secondary metabolites on the basis of preliminary antibacterial testing. Observation of morphological and microscopic features helped in identification of the fungal strain as Aspergillus flavus. Potato Dextrose Agar (PDA) medium was used for fungal growth while Czapec Yeast Broth (CYB) medium was used for production of fungal metabolites. Column chromatography technique was utilized for purification of compound from crude fungal extract and the mass of the compound was determined using Liquid Chromatography Mass Spectrometry (LCMS) method. Structure elucidation of the pure compound was performed using 500 Varian Nuclear Magnetic Resonance (NMR) machine. Docking was performed using Glide SP algorithm. Agar well diffusion method was used to determine the invitro antibacterial potential of the compound against two MDR bacterial strains i.e. Staphylococcus aureus and Proteus vulgaris. For this a total of 4 dose concentrations i.e. (100, 250, 500, 1000 µg mL- 1) of the compound were prepared and applied to bacterial strains on Mueller Hinton agar using tetracycline as control. RESULTS: The chemical name of the purified compound from A. flavus was determined as (2E)-3-[(3S, 4R)-8-hydroxy-3, 4-dimethyl-1-oxo-3, 4-dihydro-1H-2- benzopyran-7-yl] prop-2-enoic acid with the formula C14H14O5 and exact mass of 262.08. The in-Silico analysis showed that this compound has the potential to inhibit the binding pocket of S. aureus TyrRS (1JII) with docking score of - 8.67 Kcal mole- 1. The results obtained from invitro experiments were encouraging as at 1000 µg mL- 1 the compound showed 58.8% inhibition against S. aureus and 28% inhibition against P. vulgaris. CONCLUSIONS: The pure compound with formula C14H14O5 and exact mass of 262 exhibited antibacterial potential both insilico and invitro against both Gram negative and Gram positive bacteria. The compound was more active against S. aureus in comparison to P. vulgaris. From the obtained results it is concluded that this compound can be used as potent antibacterial candidate but further studies will be needed prior to its use as antibiotic.

Does co-inoculation of mycorrhiza and Piriformospora indica fungi enhance the efficiency of chlorophyll fluorescence and essential oil composition in peppermint under irrigation with saline water from the Caspian Sea?

Symbiotic associations with endophytic fungi are ecologically important for medicinal and aromatic plants. Endophytic fungi highly affect the quantity and quality of herbal products. In this study, a pot experiment was carried out in the greenhouse to investigate the interactive effects of Piriformospora indica and arbuscular mycorrhizal (AMF) inoculation on the chlorophyll fluorescence, essential oil composition, and antioxidant enzymes of peppermint under saline condition. The results showed that Fo, YNPQ, YNO, and NPQ values were obviously increased under salinity conditions, while essential oil content, chlorophyll a and b, gs, Fm, Fv, ETR, ФPSII and Fv/Fm ratio decreased by increasing salinity. In addition, salt induced the excess Na+ uptake, whereas the opposite trend was observed for P and K+. The synergistic association of P. indica and AMF caused a considerable increase in the antioxidant ability, essential oil content, Fv/Fm ratio, ФPSII, and amount of P and K+ uptake in salt-stressed plants. The main peppermint oil constituents, menthol, menthone, and 1,8-cineole increased considerably in inoculated plants. Besides, the applied endophytic fungi positively enhanced the ability of peppermint to alleviate the negative effect of the salinity stress.

Impact of Soil Rhizobacteria Inoculation and Leaf-Chewing Insect Herbivory on Mentha piperita Leaf Secondary Metabolites.

Secondary metabolites commonly play important physiological roles in plants and can be modified quantitatively and qualitatively by exposure to biotic and abiotic interactions. Plant growth promoting rhizobacteria (PGPR) and herbivory induce systemic resistance. In the present study, we analyzed the induction of secondary metabolites in peppermint plants in response to chewing insect herbivory on PGPR-inoculated Mentha piperita plants. The secondary metabolites of M. piperita plants were increased when plants were inoculated with PGPR and also exposed to caterpillar herbivory. It was found that the total essential oil yield in inoculated plants with insect damage was ~2.6-fold higher than in controls. The yield was similar to that of plants either damaged by insects or inoculated, indicating that there was no synergism. The same trend was observed for phenolic compounds. In contrast, VOC emissions were significantly higher in plants infested by insects, independent of whether they were inoculated. Insect damaged plants had 5.5 times higher monoterpene emissions than control plants, and ~ 2-fold higher emissions than on PGPR-inoculated plants without insects. To gain a better understanding of how herbivory on PGPR-inoculated plants can cause an increase in secondary metabolites of peppermint, we examined changes in plant defense hormones in inoculated plants after herbivory. We found that the combination of both treatments increased the endogenous jasmonic and salicylic acid levels to the same extent as in plants only inoculated or only insect-damaged. Because different interactions can alter the phytochemistry of plants such as M. piperita, this topic is both ecologically and economically relevant.

Colonization of Trichoderma viride Tv-1511 in peppermint (Mentha × piperita L.) roots promotes essential oil production by triggering ROS-mediated MAPK activation.

Peppermint (Mentha × piperita L.) is a flavoring additive used worldwide, and Trichoderma species are beneficial fungi that can stimulate growth and disease resistance of these plants. Here the growth conditions and metabolic processes of essential oil (EO) biosynthesis in response to inoculation with Trichoderma viride Tv-1511 were investigated. The results showed that T. viride Tv-1511 was able to colonize roots of peppermint to promote its growth and photosynthetic activity and induce higher levels of glandular trichomes and elevated EO yield and composition. GC-MS analysis showed that T. viride Tv-1511-inoculated peppermint produced higher concentrations of menthone, menthol, and pulegone and lower concentrations of menthofuran than un-inoculated seedlings, and qRT-PCR showed that T. viride Tv-1511 inoculation induced upregulation of Pr (pulegone reductase encoding gene) and Mr (menthone reductase encoding gene), whereas it led to the downregulation of Mfs (menthofuran synthase encoding gene). Furthermore, a mitogen-activated protein kinase (MAPK) in peppermint, which was determined to be an analog of Arabidopsis MPK6 protein, was found to be responsible for the modulation of EO metabolism at the transcriptional level and for enzymatic activation in the T. viride Tv-1511-inoculated peppermint. Notably, NADPH oxidase-dependent reactive oxygen species (ROS) production played vital roles in the root colonization of T. viride Tv-1511 and was also involved in the induction of MAPK activation. These data showed the beneficial effects of T. viride Tv-1511 on the seedling growth and EO yield of peppermint, and they elucidated that T. viride Tv-1511 improved the quantity and quality of EOs by regulating the genes that encode the enzymes involved in EO metabolism through a potential MAPK-mediated signaling pathways.

Improving Phenolic Total Content and Monoterpene in Mentha x piperita by Using Salicylic Acid or Methyl Jasmonate Combined with Rhizobacteria Inoculation.

The effects of plant inoculation with plant growth-promoting rhizobacteria (PGPR) and those resulting from the exogenous application of salicylic acid (SA) or methyl jasmonte (MeJA) on total phenolic content (TPC) and monoterpenes in Mentha x piperita plants were investigated. Although the PGPR inoculation response has been studied for many plant species, the combination of PGPR and exogenous phytohormones has not been investigated in aromatic plant species. The exogenous application of SA produced an increase in TPC that, in general, was of a similar level when applied alone as when combined with PGPR. This increase in TPC was correlated with an increase in the activity of the enzyme phenylalanine ammonia lyase (PAL). Also, the application of MeJA at different concentrations in combination with inoculation with PGPR produced an increase in TPC, which was more relevant at 4 mM, with a synergism effect being observed. With respect to the main monoterpene concentrations present in peppermint essential oil (EO), it was observed that SA or MeJA application produced a significant increase similar to that of the combination with rhizobacteria. However, when plants were exposed to 2 mM MeJA and inoculated, an important increase was produced in the concentration on menthol, pulegone, linalool, limonene, and menthone concentrations. Rhizobacteria inoculation, the treatment with SA and MeJA, and the combination of both were found to affect the amount of the main monoterpenes present in the EO of M. piperita. For this reason, the expressions of genes related to the biosynthesis of monoterpene were evaluated, with this expression being positively affected by MeJA application and PGPR inoculation, but was not modified by SA application. Our results demonstrate that MeJA or SA application combined with inoculation with PGPR constitutes an advantageous management practice for improving the production of secondary metabolites from M. piperita.

Induction of essential oil production in Mentha x piperita by plant growth promoting bacteria was correlated with an increase in jasmonate and salicylate levels and a higher density of glandular trichomes.

Plant growth promoting bacteria (PGPB) are agriculturally important soil bacteria that increase plant growth. We subjected peppermint to inoculation with three species of PGPB. After inoculation, the plants were sprayed with methyl jasmonate solution (MeJA) or SA (salicylic acid). Then, the plants were harvested and the plant growth parameters, trichome density, EO content and endogenous phytohormones were measured. Shoot fresh weight was reduced in plants inoculated and treated with MeJA whereas EO content varied depending on the MeJA concentration applied. Plants inoculated and treated with MeJA 2 mM showed the maximum increase in EO production, revealing a synergism between PGPB and MeJA. SA treatments also enhanced EO yield. The increased growth and EO production observed upon PGPB application were at least partly due to an increase in the JA and SA concentrations in the plant, as well as to an associated rise in the glandular trichome density.

Herbicidal activity of pure compound isolated from rhizosphere inhabiting Aspergillus flavus.

In the quest for bioactive natural products of fungal origin, Aspergillus flavus was isolated from rhizosphere of Mentha piperita using Potato Dextrose Agar (PDA) and Czapec Yeast Broth (CYB) nutrient media for metabolites production. In total, three different metabolites were purified using HPLC/LCMS and the structures were established using 500 Varian NMR experiments. Further the isolated metabolites in different concentrations (10, 100, 1000 µg/mL) were tested for herbicidal activity using Completely Randomized design (CRD) against the seeds of Silybum marianum and Avena fatua which are major threats to wheat crop in Pakistan. Among the isolated metabolites, one compound was found active against the test weed species whose activity is reported in the present work. The chemical name of the compound is 2-(1, 4-dihydroxybutan-2-yl)-1, 3-dihydroxy-6, 8-dimethoxyanthracene-9, 10(4aH, 9aH)-dione with mass of 388. Results showed that all seeds germinated in control treatment; however, with the metabolite treated, the growth was retarded to different levels in all parts of the weeds. At a dose of 1000 µg/mL of the pure compound, 100% seeds of S. marianum and 60% seeds of A. fatua were inhibited. Interestingly, the pure compound exhibited less inhibition of 10% towards the seeds of common wheat (Triticum aestivum).

Survival of Salmonella on chamomile, peppermint, and green tea during storage and subsequent survival or growth following tea brewing.

The survival of Salmonella on dried chamomile flowers, peppermint leaves, and green tea leaves stored under different conditions was examined. Survival and growth of Salmonella was also assessed after subsequent brewing using dried inoculated teas. A Salmonella enterica serovar cocktail was inoculated onto different dried tea leaves or flowers to give starting populations of approximately 10 log CFU/g. The inoculum was allowed to dry (at ambient temperature for 24 h) onto the dried leaves or flowers prior to storage under 25 and 35 °C at low (<30% relative humidity [RH]) and high (>90% RH) humidity levels. Under the four storage conditions tested, survival followed the order 25 °C with low RH > 35 °C with low RH > 25 °C with high RH > 35 °C with high RH. Salmonella losses at 25 °C with low RH occurred primarily during drying, after which populations showed little decline over 6 months. In contrast, Salmonella decreased below detection after 45 days at 35 °C and high RH in all teas tested. The thermal resistance of Salmonella was assessed at 55 °C immediately after inoculation of tea leaves or flowers, after drying (24 h) onto tea leaves or flowers, and after 28 days of storage at 25 °C with low RH. All conditions resulted in similar D-values (2.78 ± 0.12, 3.04 ± 0.07, and 2.78 ± 0.56, at 0 h, 24 h, and 28 days, respectively), indicating thermal resistance of Salmonella in brewed tea did not change after desiccation and 28 days of storage. In addition, all brewed teas tested supported the growth of Salmonella. If Salmonella survives after storage, it may also survive and grow after a home brewing process.

Anatomical, morphological, and phytochemical effects of inoculation with plant growth- promoting rhizobacteria on peppermint (Mentha piperita).

Plant growth-promoting rhizobacteria (PGPR) generally exert their effects through enhancement of plant nutrient status and/or phytohormone production. The effects of PGPR on aromatic plant species are poorly known. We measured plant growth parameters, chlorophyll content, trichome density, stomatal density, and levels of secondary metabolites in peppermint (Mentha piperita) seedlings inoculated with PGPR strains Bacillus subtilis GB03, Pseudomonas fluorescens WCS417r, P. putida SJ04, or a combination of WCS417r + SJ04. The treated plants, in comparison with controls, showed increases in shoot biomass, root biomass, leaf area, node number, trichome density, and stomatal density, and marked qualitative and quantitative changes in monoterpene content. Improved knowledge of the factors that control or affect biosynthesis of secondary metabolites and monoterpene accumulation will lead to strategies for improved cultivation and productivity of aromatic plants and other agricultural crops without the use of chemical fertilizers or pesticides.

Pathogenicity and ultrastructural studies of the mode of penetration by Phoma strasseri in peppermint stems and rhizomes.

Pathogenicity and ultrastructural investigation of the inoculation of peppermint stems and rhizomes with Phoma strasseri conidia was undertaken using scanning and transmission electron microscopy to examine the host-parasite relationship. Pathogenicity experiments demonstrated that all tested P. strasseri isolates had infected the stems and rhizomes of peppermint. Of all inoculation methods, direct placement of colonized agar plugs on damaged epidermis and soaking stems and rhizomes in conidial suspension were the most effective. The behavior of the conidia deposited on the stems and rhizomes was investigated at different time intervals after inoculation: 6, 16, 24, 36 and 48 h. Conidia produced an appressorium directly at the end of a short germ tube. Appressoria were formed over the cuticle, but never over stomata. Direct penetration to host tissue through the cuticle was observed. The spore and hyphae were covered with a mucilaginous sheath.

Low-dose irradiation improves microbial quality and shelf life of fresh mint (Mentha piperita L.) without compromising visual quality.

UNLABELLED: The effects of low-dose irradiation (0.25 to 2 kGy) and postirradiation storage (at 4 degrees C) on microbial and visual quality, color values (L*, a*, b*, chroma, and hue [ degrees ]), and chlorophyll content (Chl a, Chl b, and total Chl) of fresh mint were evaluated. Samples inoculated with E. coli O157:H7, Salmonella, and MS2 bacteriophage were irradiated and evaluated. E. coli O157:H7 and Salmonella populations were reduced by 2 to 2.4, 3.5, and 5.8 log CFU/g, respectively, 1 d after treatment with 0.25, 0.60, and 1 kGy, respectively, and were completely eliminated at 2 kGy. None of the irradiation doses (P < 0.0001) reduced MS2 bacteriophage populations by more than 0.60 log PFU/g. Irradiation doses did not affect visual quality and samples remained of excellent to good quality (score 7.75 to 9) for up to 9 d of storage. Irradiation at 0.60, 1, and 2 kGy increased (P < 0.0001) Chl a, Chl b, and total Chl. Both total Chl and Chl a decreased significantly after 3 d of storage. Significant decreases in Chl b were not observed until day 12 of storage. Color values (L*, b*, and chroma) were not significantly different until day 6 of storage and hue ( degrees ) remained unchanged (179 degrees ) for the entire storage period of 12 d. Overall, irradiation did not change L*, a*, b*, or chroma. These results demonstrate that irradiation of fresh mint at 2 kGy has the potential to improve its microbial quality and extend its shelf life without compromising its visual quality and color. PRACTICAL APPLICATION: Mints and other raw fresh herbs are widely used for flavoring as well as garnish in a variety of dishes without further cooking. However, mint is one considered as one of the high-risk herbs when it comes to microbial contamination. We have evaluated the use of gamma irradiation treatment at very low doses ranging from 0 to 2 kGy to eliminate seeded Salmonella spp, E. coli O157:H7, and MS2 bacteriophage, a surrogate of hepatitis A virus. We found that low-dose irradiation (1.0 to 2.0 kGy) appears to be a promising method for improving the microbiological quality of fresh mint without compromising its visual and color attributes. This method may be applied to many popular fresh culinary herbs that are commonly used as garnishes in Asian cuisine.

[Detection of fungal metabolites showing toxic activity through Artemia salina bioassay]. / Detección de metabolitos fúngicos con actividad tóxica mediante bioensayo sobre Artemia salina.

The aim of this study was to detect toxic metabolites from fungi contaminating food and medicinal herbs by applying the toxicity assay to Artemia salina. According to toxicity percentages, the extracts were classified as nontoxic (NT), slightly toxic (ST), toxic (T) and highly toxic (HT). Those classified as T and HT were assayed for mycotoxins. Only 6 out of 71 strains were found to be T (8.5%) for A. salina. Penicillium brevicompactum Dierckx, isolated from sausages, was found to be HT, mainly due to the presence of ochratoxin A and two other unidentified metabolites.

A comparison of selected media and incubation temperatures for isolation of microscopic fungi from dried medicinal plants.

The study compared selected media and incubation temperatures for isolation of fungi from dried medicinal plants (chamomile, peppermint, lemon balm, St. John's wort and two herbal mixtures). The DG18 medium was found to be the most suitable for characterization of the mycoflora at 25 degrees C. The medium selection for 37 degrees C was dependent on the species to be isolated. MEA + 40% sacharose and YpSs were found to be the best media for isolation of thermophilic and thermotolerant fungi from dried medicinal plants.

Occurence of fungal diseases on lemon balm (Mellisa officinalis L.) and peppermint (Mentha x piperita L.) in the region of Malopolska.

This paper presents the results of the researches carried out on the subject of the diseases on herbs such as lemon balm and peppermint focusing on the health status of the plants grown in the region of Malopolska. The field and laboratory research proved that perpetrators of the diseases on the examined plants were fungi species with the numerical majority. On lemon balm septoria leaf spot (Septoria melissae) was most often observed. Moreover, fungal genera of different taxonomic groups were detected. Alternaria, Epicoccum, Fusarium, and Sclerotinia sclerotiorum occurred most often on the medicinal plant samples. The disease mint rust Puccinia menthae, has caused major problems for the peppermint growers. In the populations of fungi found on the diseased leaves the dominating were Alternaria, Epicoccum and Sphaceloma menthae. It has been stated that among all fungi isolated from lemon balm, the species Fusarium avenaceum had the highest pathogenicity to seedlings (80% of diseased seedlings). For peppermint plants the highest pathogenicity had species Epicoccum purpurascens, and caused 82% of diseased seedlings. Conducted evaluation of health status of plants showed that the lemon balm mean disease index was yearly differentiated and was the highest in 2003 (44.39). For peppermint, the highest disease index was in third year of cultivation (62.75) and was statistically higher than in previous years.

Effect of an arbuscular mycorrhizal fungus, Glomus mosseae, and a rock-phosphate-solubilizing fungus, Penicillium thomii, on Mentha piperita growth in a soilless medium.

Rock phosphate effect on English mint (Mentha piperita L.) grown on steamed perlite:vermiculite (1:1, v:v) substrate, with and without rock phosphate, was evaluated in greenhouse experiments. Five treatments were carried out by inoculation with an arbuscular mycorrhizal fungus Glomus mosseae and a phosphorus solubilizing microorganism Penicillium thomii. Plant aerial biomass, phosphorus concentration in plant tissue, and P available in the substrate, were evaluated upon two harvests. After the first harvest, plant aerial biomass did not show significant differences between treatments using rock phosphate as fertilizer, although P content in plants inoculated with P. thomii was higher. The second harvest revealed a higher biomass and plant tissue P content in treatments inoculated with G. mosseae. P. thomii increased P available in the substrate, whereas in the absence of G. mosseae, it did not enhance plant tissue P content. Mycorrhizal colonization was not affected by P. thomii. Microbial inoculation effect on English mint growth was also evaluated. The microbial effect was positive in all treatments when compared with the control without rock phosphate.

[Study on the quantitative and qualitative composition of fungi in dried medicinal plants]. / Badania skladu ilosciowego i jakosciowego grzybów w suszach roslin leczniczych.

The quantitative and qualitative composition of fungi was determined in selected dried medicinal plants purchased in one of the herbal shops in Szczecin, Poland. The samples examined were as follows: chamomile (Flos Chamomillae), peppermint (Folium Menthae piperitae), lemon balm (Folium Melissae), St. John's wort (Herba Hyperici), and two herbal mixtures. The fungal composition depended on the specified sample. Xerophilic fungi, i.e. Eurotium amstelodami, E. herbariorum, E. rubrum and Wallemia sebi were isolated from dried medicinal plants. E. amstelodami was the predominating species. The prevailing thermophilic and thermotolerant species were Rhizopus microsporus var. rhizopodiformis and Aspergillus fumigatus. Pink and white yeasts were also numerous in some samples. Except for Aspergillus niger, mesophilic and toxigenous species were found to occur infrequently in the samples. However, Aspergillus versicolor was found to occur abundantly in lemon balm.

Exposure to airborne microorganisms, dust and endotoxin during processing of peppermint and chamomile herbs on farms.

The aim of this study was to determine the levels of microorganisms, dust and endotoxin in the air during processing of peppermint (Mentha piperita) and chamomile (Matricaria recutita) by herb farmers, and to examine the species composition of airborne microflora. Air samples were collected on glass fibre filters by use of personal samplers on 13 farms owned by herb cultivating farmers, located in Lublin province (eastern Poland). The concentrations of total viable microorganisms (bacteria + fungi) in the farm air during processing of peppermint herb were large, within a range from 895.1-6,015.8 x 10(3) cfu/m(3) (median 1,055.3 x 10(3) cfu/m(3)). During processing of chamomile herb they were much lower and varied within a range from 0.88-295.6 x 10(3) cfu/m(3) (median 27.3 x 10(3) cfu/m(3)). Gram-negative bacteria distinctly prevailed during processing of peppermint leaves, forming 46.4-88.5 % of the total airborne microflora. During processing of chamomile herb, Gram-negative bacteria were dominant at 3 out of 6 sampling sites forming 54.7-75.3 % of total microflora, whereas at the remaining 3 sites the most common were fungi forming 46.2-99.9 % of the total count. The species Pantoea agglomerans (synonyms: Erwinia herbicola, Enterobacter agglomerans ), having strong allergenic and endotoxic properties, distinctly prevailed among Gram-negative isolates. Among fungi, the most common species was Alternaria alternata. The concentrations of airborne dust and endotoxin determined on the examined herb farms were large. The concentrations of airborne dust during peppermint and chamomile processing ranged from 86.7-958.9 mg/m(3), and from 1.1-499.2 mg/m(3), respectively (medians 552.3 mg/m(3) and 12.3 mg/m(3)). The concentrations of airborne endotoxin determined during peppermint and chamomile processing were within a wide range 1.53-208.33 microg/m(3) and 0.005-2604.19 microg/m(3) respectively (medians 57.3 microg/m(3) and 0.96 microg/m(3)). In conclusion, farmers cultivating peppermint are exposed during processing of this herb to large concentrations of airborne microorganisms, dust and endotoxin posing a risk of work-related respiratory disease. The exposure to bioaerosols during processing of chamomile is lower; nevertheless, peak values create a respiratory risk for exposed farmers.