Marine-derived Penicillium in Korea: diversity,enzyme activity,and antifungal properties

The diversity of marine-derived Penicillium from Korea was investigated using morphological and multigene phylogenetic approaches, analyzing sequences of the internal transcribed spacer region, β-tubulin gene, and RNA polymerase subunit II gene. In addition, the biological activity of all isolated strains was evaluated. We tested for the extracellular enzyme activity of alginase, endoglucanase, and β-glucosidase, and antifungal activity against two plant pathogens (Colletotrichum acutatum and Fusarium oxysporum). A total of 184 strains of 36 Penicillium species were isolated, with 27 species being identified. The most common species were Penicillium polonicum (19.6 %), P. rubens (11.4 %), P. chrysogenum (11.4 %), and P. crustosum (10.9 %). The diversity of Penicillium strains isolated from soil (foreshore soil and sand) and marine macroorganisms was higher than the diversity of strains isolated from seawater. While many of the isolated strains showed alginase and β-glucosidase activity, no endoglucanase activity was found. More than half the strains (50.5 %) showed antifungal activity against at least one of the plant pathogens tested. Compared with other strains in this study, P. citrinum (strain SFC20140101-M662) showed high antifungal activity against both plant pathogens. The results reported here expand our knowledge of marine-derived Penicillium diversity. The relatively high proportion of strains that showed antifungal and enzyme activity demonstrates that marine-derived Penicillium have great potential to be used in the production of natural bioactive products for pharmaceutical and/or industrial use.     

Re-Identification on Korean Penicillium Sequences in GenBank Collected by Software GenMine

Penicillium species have been actively studied in various fields, and many new and unrecorded species continue to be reported in Korea. Moreover, unidentified and misidentified Korean Penicillium species still exist in GenBank. Therefore, it is necessary to revise the Korean Penicillium inventory based on accurate identification. We collected Korean Penicillium nucleotide sequence records from GenBank using the newly developed software, GenMine, and re-identified Korean Penicillium based on the maximum likelihood trees. A total of 1681 Korean Penicillium GenBank nucleotide sequence records were collected from GenBank. In these records, 1208 strains with four major genes (Internal Transcribed Spacer rDNA region, β-tubulin, Calmodulin and RNA polymerase II) were selected for Penicillium re-identification. Among 1208 strains, 927 were identified, 82 were identified as other genera, the rest remained undetermined due to low phylogenetic resolution. Identified strains consisted of 206 Penicillium species, including 156 recorded species and 50 new species candidates. However, 37 species recorded in the national list of species in Korea were not found in GenBank. Further studies on the presence or absence of these species are required through literature investigation, additional sampling, and sequencing. Our study can be the basis for updating the Korean Penicillium inventory.     

Durability of rubberwood (Hevea brasiliensis) treated with peppermint oil,eucalyptus oil,and their main components

Anti-fungal activities of two essential oils (peppermint oil and eucalyptus oil) and their main components (menthol and eucalyptol, respectively) against molds (Aspergillus niger, Penicillium chrysogenum, and Penicillium sp.) and a white-rot decay fungus (Trametes versicolor) identified from rubberwood surfaces were investigated. The broth dilution method and the agar diffusion technique were employed to determine the minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) using the concentration of substances between 100 and 800 μl ml^?1. Inhibitory effects of essential oils and their main components at the MICs against mold growth, fungal decay, and termite attack on rubberwood were further examined by means of the dip treatment method. It was found that MFC values against molds for all treatments examined were about 50–100 μl ml^?1 higher than MIC values. Peppermint oil and menthol exhibited high fungistatic and fungicidal activities, with MICs of 300 μl ml^?1 and 350 μl ml^?1, respectively, against the test molds and the decay fungus. Eucalyptus oil and eucalyptol were also effective against these microbes but at higher concentrations of 600 μl ml^?1 and 500 μl ml^?1, respectively. Only peppermint oil at the MIC was capable of providing a complete protection from mold growth on rubberwood for up to 12 weeks at storage conditions of 25 °C and 100% RH. Both peppermint oil and eucalyptus oil at the MICs showed moderate resistance to fungal decay and high resistance to termite attack.     

Sex in Penicillium: Combined phylogenetic and experimental approaches

We studied the mode of reproduction and its evolution in the fungal subgenus Penicillium Biverticillium using phylogenetic and experimental approaches. We sequenced mating type (MAT) genes and nuclear DNA fragments in sexual and putatively asexual species. Examination of the concordance between individual trees supported the recognition of the morphological species. MAT genes were detected in two putatively asexual species and were found to evolve mostly under purifying selection, although high substitution rates were detected at some sites in some clades. The first steps of sexual reproduction could be induced under controlled conditions in one of the two species, although no mature cleistothecia were produced. Altogether, these findings suggest that the asexual Penicillium species may have lost sex only very recently and/or that the MAT genes are involved in other functions. An ancestral state reconstruction analysis indicated several events of putative sex loss in the genus. Alternatively, it is possible that the supposedly asexual Penicillium species may have retained a cryptic sexual stage.     

Antagonistic interactions between Aureobasidium pullulans and Fusarium culmorum,a fungal pathogen of winter wheat

The effect of the biological control agent Aureobasidium pullulans (de Bary) G. Arnaud on the development of Fusarium head blight (FHB) on winter wheat and kernel contamination with fungi of the genera Fusarium, Acremonium, Cladosporium and Penicillium was analyzed in a greenhouse experiment. Scanning electron microscopy was used to evaluate the distribution of A. pullulans cells and aggregates on wheat kernels, infection structures of Fusarium culmorum (W.G. Smith) Sacc and the antagonist-pathogen interactions. Biological control with A. pullulans reduced FHB severity by 21.67 % and improved grain filling by 5.02 %, compared with the control treatment. The survival of A. pullulans was good (to 31 cells per kernel), in particular on the surface and in the crease of kernels, including in pathogen-inoculated wheat plants. A. pullulans cells firmly adhered to F. culmorum hyphae, and damaged them. In most cases, autochthonous communities of filamentous fungi of the genera Acremonium and Penicillium developed at a slower rate after kernel inoculation with the pathogen.     

Plasmodium falciparum Mating Patterns and Mosquito Infectivity of Natural Isolates of Gametocytes

Plasmodium falciparum infections in malaria endemic areas often harbor multiple clones of parasites. However, the transmission success of the different genotypes within the mosquito vector has remained elusive so far. The genetic diversity of malaria parasites was measured by using microsatellite markers in gametocyte isolates from 125 asymptomatic carriers. For a subset of 49 carriers, the dynamics of co-infecting genotypes was followed until their development within salivary glands. Also, individual oocysts from midguts infected with blood from 9 donors were genotyped to assess mating patterns. Multiplicity of infection (MOI) was high both in gametocyte isolates and sporozoite populations, reaching up to 10 genotypes. Gametocyte isolates with multiple genotypes gave rise to lower infection prevalence and intensity. Fluctuations of genotype number occurred during the development within the mosquito and sub-patent genotypes, not detected in gametocyte isolates, were identified in the vector salivary glands. The inbreeding coefficient Fis was positively correlated to the oocyst loads, suggesting that P. falciparum parasites use different reproductive strategies according to the genotypes present in the gametocyte isolate. The number of parasite clones within an infection affects the transmission success and the mosquito has an important role in maintaining P. falciparum genetic diversity. Our results emphasize the crucial importance of discriminating between the different genotypes within an infection when studying the A. gambiae natural resistance to P. falciparum, and the need to monitor parasite diversity in areas where malaria control interventions are implemented.     

Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector,Ixodes scapularis

Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10–15% and 65–71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.     

Isolation and molecular characterization of causal agent of blue mold on Allium cepa L. and its control by Pennisetum flaccidum Griseb

Blue mold pathogen, isolated from infected Allium cepa L., was identified as a Penicillium species through morphological and molecular characterisation. Internal Transcribed spacer (ITS) region of ribosomal DNA (rDNA) was utilised for DNA sequencing. Basic Local Alignment Search Tool (BLAST) analysis has found the maximum similarity index of the fungus to be 82.39% with the Uncultured Penicillium clone (Accession: MF535522). So, the isolated Penicillium specie is the first reported specie of the genus that infects onion. A phylogenetic tree was constructed to establish a relationship of the isolated fungus with the most relevant species reported on GenBank. Extracts of Pennisetum flaccidum Griseb. were evaluated against the isolated fungus as a potential biocontrol agent. Among the five tested methanol concentrations (0.5%, 1%, 1.5%, 2% and 2.5%) of each plant part (root, inflorescence and foliage), 0.5% root extract showed maximum growth retardation, i.e. 89%. For bioassay-guided fractionation, the root extract was partitioned in n-hexane, chloroform, n-butanol and ethyl acetate. Ethyl acetate (1%) was proved to be the most potent one. Phytochemical screening has confirmed the occurrence of terpenoids, tannins, saponins and alkaloids. The applied molecular approach has deduced that the Penicillium specie collected from Pakistan might be novel. This study can be concluded that P. flaccidum contains potent phytochemicals which might be used as antifungal agent against Penicillium species.     

Comparison of the kinetics and factors affecting the stabilities of chitin-immobilized naringinases from two fungal sources

Naringinases from both Penicillium sp. and Aspergillus niger were compared for their enzyme kinetics and the effects of sugars on the enzyme activities. Lineweaver-Burk plots showed that glucose, fructose and rhamnose were all competitive inhibitors for α-rhamnosidase of naringinase from Penicillium sp. and non-competitive inhibitors for the same enzyme from A. niger, When naringinase from Penicillium sp. was immobilized on chitin and used successively for the hydrolysis of p-nitrophenyl-α-rhamnoside or naringin in a simulated fruit juice system or grapefruit juice, it was observed that the enzyme column was very stable. Such results are in contrast to what has bee observed for naringinase fron A. niger. Therefore, it is quite possible that the sugars in the fruit juice which play a role as competitive or non-competitive inhibitors on naringinase may account for the stability of the enzyme column during successive debittering of grapefruits juice.     

Mycobiota and mycotoxins in fermented feed,wheat grains and corn grains in Southeastern Buenos Aires Province,Argentina

Wheat (as bran) and corn (as dry grain or fermented feed) are main ingredients of feedstuffs used in local cattle and pig farms in the South of the Buenos Aires Province (Argentina). Therefore, determining mycobiota and mycotoxins in wheat and corn is of prime importance for developing feed management techniques to optimise animal production and to minimize toxicity. Then, a mycological survey was carried out in the Southeastern part of the Buenos Aires Province, in order to identify the mycobiota and the main mycotoxins present in fermented feed, wheat grain and corn grain samples. Samples were cultured for fungal quantification, isolation and identification, and analysed for deoxynivalenol (DON), zearalenone (ZEA), T-2 toxin and aflatoxins (AFLA). Penicillium (74%), Aspergillus (32%) and Scopulariopsis (21%) were the prevalent genera in fermented feed. Penicillium (70%), Fusarium (47%) and Aspergillus (34%) were the most frequent fungi isolated from corn. Penicillium (42%), Fusarium (27%) and Alternaria (25%) were the most frequently recovered genera from wheat. DON was detected in 59% of the corn samples, in 45% of the wheat samples and in 38% of the silage samples. ZEA was detected in 36% of the corn samples, in 49% of the wheat samples and in 16% of the silage samples. T-2 toxin and aflatoxin B1 were each detected in 4% of the corn samples. Eighteen percent of the fermented feed samples showed T-2 contamination. Fermented feed and wheat samples were negative for AFLA.     

Ethnobotany,leaf anatomy,essential oil composition and antibacterial activity of Pteronia onobromoides (Asteraceae)

Available ethnobotanical information on Pteronia onobromoides (first recorded in 1685) indicates that the plant was once of considerable cultural and commercial importance and that it was powdered, mixed with fat, and applied to the skin for cosmetic and/or medicinal purposes. Sâb, as well as Son or San, are considered to be the original Nama names for this aromatic bush and also the origin of various names for San people, such as Sonqua and Bushman. A study of the leaf anatomy showed that essential oil is produced in globose oil glands situated below some of the vascular bundles in the spongy parenchyma, adjacent to the palisade parenchyma. The oil is relatively complex but contains a combination of myrcene, limonene, 1,8-cineole and p-cymene as main compounds, with smaller amounts of sabinene, trans-linalooloxide, linalool, terpinen-4-ol, α-terpineol, eugenol, thymol and α-phellandrene. Dichloromethane extracts exhibited antibacterial activity (especially against Staphylococcus epidermidis) with MIC values as low as 0.83 mg/ml. Other solvent extracts and the essential oil itself were less active. The results show that the traditional method of mixing powdered leaves with fat and applying it to the skin may have had deodorant, disinfectant and medicinal benefits.     

Potency of plant extracts against Penicillium species isolated from different seeds and fruits in Saudi Arabia

Antifungal activity of extracts of cinnamon (Cinnamomum zeylanicum), Cloves (Syzygium aromaticum), ginger (Zingiber officinale) and turmeric (Curcuma longa) were evaluated in vitro against 17 Penicillium spp. Seed disease and rotten fruit caused by these species cause considerable loss of quality for different agricultural products. Isolates of Penicillium spp. were screened for production of patulin an important serious mycotoxin. About 70.59% of Penicillium spp. produced this toxin in concentrations ranging from 4 to 31 ppb. The response of Penicillium spp.to plant extracts differed according to the plant extract and concentration. Cinnamon extract showed the greatest effect on P. asperosporum, P. aurintogriseum and P. brevicompactum, and cloves extract produced the greatest effect on P. chermesinum and P. duclauxii. Turmeric extract had less effect on P. duclauxii. Cloves extract was the most effective in reducing the growth of Penicillium spp. On the other hand, ginger extract with all concentrations used had less effect against most Penicillium spp in the laboratory. Plant extracts are promising as natural sources of environmentally friendly compounds in laboratory studies.     

Antifungal activities of anise oil, lime oil, and tangerine oil against molds on rubberwood (Hevea brasiliensis)

The antifungal activities of anise oil, lime oil, and tangerine oil against molds identified from rubberwood surfaces (Aspergillus niger, Penicillium chrysogenum, and Penicillium sp.) were investigated. The broth dilution method was employed to determine the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) using the concentration of essential oils between 20 and 200 μl ml⁻¹. Inhibitory effects of the essential oils against those molds on rubberwood were also examined by means of the dip treatment and vacuum impregnation treatment. It was found that the MIC and MFC values for each treatment on agar were identical for all conditions examined. Anise oil was the strongest inhibitor with the MIC and MFC of 40 μl ml⁻¹ against Penicillium sp. and A. niger, and 60 μl ml⁻¹ against P. chrysogenum. Lime oil and tangerine oil were also effective against those molds at higher concentrations of 100–180 μl ml⁻¹. All essential oils at the MIC and MFC provided a protection from mold growth on rubberwood for at least 12 weeks at storage conditions of 30 °C with 100% RH.     

Fungi Isolated from Flue-cured Tobacco at Time of Sale and After Storage

The fungi isolated from 100 samples of flue-cured tobacco from 12 markets in 2 tobacco belts comprised 11 genera, including 10 species of Aspergillus. The mean percentage per sample isolated from 62 samples of tobacco from Middle Belt markets was Alternaria, 40.6%; Aspergillus niger, 47.8%; Aspergillus repens, 38.0%; and Penicillium, 25.8%. The mean percentage per sample isolated from 38 samples of tobacco from Old Belt markets was Alternaria, 74.0%; Penicillium, 52.5%; Aspergillus repens, 38.0%; and Aspergillus ruber, 36.2%. Damaged (74 samples) and nondamaged (26 samples) stored tobacco yielded species of six genera of fungi, including eight species of Aspergillus. Species of Aspergillus and Penicillium were commonly isolated from both damaged and nondamaged tobacco, whereas species of Alternaria, Cladosporium, Fusarium, and Rhizopus were isoalted more frequently from nondamaged tobacco. The fungi that occurred in the highest population in damaged tobacco were Aspergillus repens, A. niger, A. ruber, and Penicillium species.     

Stimulatory effect of cycloheximide on the infection of hyacinth and tulip bulbs byPenicillium sp.

Treating scooped out bulbs of hyacinths (Hyacinthus orientalis L. cv. ‘Lady Derby’, cv. ‘Pink Pearl’, cv. ‘Delft Blue’) and tulips (Tulipa gesneriana hort. cv. ‘Oxford’) with cycloheximide stimulated strongly the infection of the tissues of the bulbs byPenicillium sp. The possible mechanism of such an activity of cycloheximide is discussed.     

Antifungal activities of essential oils applied by dip-treatment on areca palm (Areca catechu) leaf sheath and persistence of their potency upon storage

The antifungal activities of cinnamon oil, clove oil, anise oil, and peppermint oil, and their main components (cinnamaldehyde, eugenol, trans-anethole, and menthol, respectively) against molds identified from areca palm leaf sheath (Mucor dimorphosporus, Penicillium sp., Aspergillus niger, and Rhizopus sp.) were investigated. An agar dilution method was employed to determine the minimum inhibitory concentration (MIC) of essential oils and their main components. Zone inhibition tests and the inhibitory effect of the leaf sheath dip-treated with essential oils against those molds were examined. Major components of essential oils on the leaf sheath during storage were quantified by gas chromatography analysis. The MIC values of essential oils on agar and on the leaf sheath were identical. With an MIC of 50 ??g ml⁻¹, cinnamon oil had the strongest inhibitory effect. At their MICs the oils were capable of providing protection against mold growth on the leaf sheath for at least 12 weeks during storage at 25 °C and 100% RH. Scanning electron microscope examination showed that essential oils prevented spore germination. Except for menthol in peppermint oil, the main components of the essential oils, which were fairly stable over the storage period, largely contributed to the antifungal activity.     

Novel Penicillium cellulases for total hydrolysis of lignocellulosics

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35 °C while at 45 °C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.     

Transmission Dynamics of Borrelia turicatae from the Arthropod Vector

Background

With the global distribution, morbidity, and mortality associated with tick and louse-borne relapsing fever spirochetes, it is important to understand the dynamics of vector colonization by the bacteria and transmission to the host. Tick-borne relapsing fever spirochetes are blood-borne pathogens transmitted through the saliva of soft ticks, yet little is known about the transmission capability of these pathogens during the relatively short bloodmeal. This study was therefore initiated to understand the transmission dynamics of the relapsing fever spirochete Borrelia turicatae from the vector Ornithodoros turicata, and the subsequent dissemination of the bacteria upon entry into murine blood.

Methodology/Principal Findings

To determine the minimum number of ticks required to transmit spirochetes, one to three infected O. turicata were allowed to feed to repletion on individual mice. Murine infection and dissemination of the spirochetes was evaluated by dark field microscopy of blood, quantitative PCR, and immunoblotting against B. turicatae protein lysates and a recombinant antigen, the Borrelia immunogenic protein A. Transmission frequencies were also determined by interrupting the bloodmeal 15 seconds after tick attachment. Scanning electron microscopy (SEM) was performed on infected salivary glands to detect spirochetes within acini lumen and excretory ducts. Furthermore, spirochete colonization and dissemination from the bite site was investigated by feeding infected O. turicata on the ears of mice, removing the attachment site after engorment, and evaluating murine infection.

Conclusion/Significance

Our findings demonstrated that three ticks provided a sufficient infectious dose to infect nearly all animals, and B. turicatae was transmitted within seconds of tick attachment. Spirochetes were also detected in acini lumen of salivary glands by SEM. Upon host entry, B. turicatae did not require colonization of the bite site to establish murine infection. These results suggest that once B. turicatae colonizes the salivary glands the spirochetes are preadapted for rapid entry into the mammal.     

The interaction between Trypanosoma rangeli and the nitrophorins in the salivary glands of the triatomine Rhodnius prolixus (Hemiptera; Reduviidae)

The parasite Trypanosoma rangeli develops in the intestinal tract of triatomines and, particularly in species of the genus Rhodnius, invades the hemolymph and salivary glands, where subsequent metacyclogenesis takes place. Many aspects of the interaction between T. rangeli and triatomines are still unclear, especially concerning the development of the parasite in the salivary glands and how the parasite interacts with the saliva. In this work, we describe new findings on the process of T. rangeli infection of the salivary glands and the impact of infection on the saliva composition. To ensure a complete infection (intestinal tract, hemolymph and salivary glands), 3rd instar Rhodnius prolixus nymphs were fed on blood containing T. rangeli epimastigotes using an artificial feeder. After molt to the 4th instar, the nymphs were inoculated with epimastigotes in the hemolymph. The results showed that the flagellates started to invade the salivary glands by the 7th day after the injection. The percentage of trypomastigotes inside the salivary glands continuously increased until the 25th day, at which time the trypomastigotes were more than 95% of the T. rangeli forms present. The salivary contents from T. rangeli-infected insects showed a pH that was significantly more acidic (<6.0) and had a lower total protein and hemeprotein contents compared with non-infected insects. However, the ratio of hemeprotein to total protein was similar in both control and infected insects. qPCR demonstrated that the expression levels of three housekeeping genes (18S rRNA, β-actin and α-tubulin) and nitrophorins 1–4 were not altered in the salivary glands after an infection with T. rangeli. In addition, the four major nitrophorins (NPs 1–4) were knocked down using RNAi and their suppression impacted T. rangeli survival in the salivary glands to the point that the parasite burden inside the R. prolixus salivary glands was reduced by more than 3-fold. These results indicated that these parasites most likely non-specifically incorporated the proteins that were present in R. prolixus saliva as nutrients, without impairing the biosynthesis of the antihemostatic molecules.