Improvement of cordycepin production by an isolated Paecilomyces hepiali mutant from combinatorial mutation breeding and medium screening.

Cordycepin is a major bioactive compound found in Cordyceps sinensis that exhibits a broad spectrum of biological activities. Here a Paecilomyces hepiali OR-1 strain was initially isolated from plateau soil for the bioproduction of cordycepin. Subsequently, strain modification including 60Co γ-ray and ultraviolet irradiation were employed to increase the cordycepin titer, resulted in a high-yield mutant strain P. hepiali ZJB18001 with the cordycepin content of 0.61 mg/gDCW, showing a 2.3-fold to that from the wild strain (0.26 mg/gDCW). Furthermore, medium screening based on Box-Behnken design and the response surface methodology facilitated the enhancement of cordycepin yield to the value of 0.96 mg/gDCW at 25 °C for 5 days in submerged cultivation with an optimized medium composition. The high cordycepin yield, rapid growth rate and stable genetic characteristics of P. hepiali ZJB18001 are beneficial in terms of costs and time for the industrialization of cordycepin production.

Identification of the pigment and its role in UV resistance in Paecilomyces variotii, a Chernobyl isolate, using genetic manipulation strategies.

Fungi produce secondary metabolites that are not directly involved in their growth, but often contribute to their adaptation to extreme environmental stimuli and enable their survival. Conidial pigment or melanin is one of the secondary metabolites produced naturally by a polyketide synthesis (PKS) gene cluster in several filamentous fungi and is known to protect these fungi from extreme radiation conditions. Several pigmented or melanized fungi have been shown to grow under extreme radiation conditions at the Chernobyl nuclear accident site. Some of these fungi, including Paecilomyces variotii, were observed to grow towards the source of radiation. Therefore, in this study, we wanted to identify if the pigment produced by P. variotii, contributes to providing protection against radiation condition. We first identified the PKS gene responsible for synthesis of pigment in P. variotii and confirmed its role in providing protection against UV irradiation through CRISPR-Cas9 mediated gene deletion. This is the first report that describes the use of CRISPR methodology to create gene deletions in P. variotii. Further, we showed that the pigment produced by this fungus, was not inhibited by DHN-melanin pathway inhibitors, indicating that the fungus does not produce melanin. We then identified the pigment synthesized by the PKS gene of P. variotii, as a naptho-pyrone Ywa1, by heterologously expressing the gene in Aspergillus nidulans. The results obtained will further aid in understanding the mechanistic basis of radiation resistance.

Paecilomyces/Purpureocillium keratitis: A consecutive study with a case series and literature review.

Paecilomyces/Purpureocillium species is an emerging pathogen of fungal keratitis; the risk factor, clinical course, and outcome of Paecilomyces/Purpureocillium keratitis need more exploration. We retrospectively reviewed 12 patients with culture-proven Paecilomyces/Purpureocillium keratitis in our hospital from 2003 to 2017 and combined them with 50 previous cases reported after the review conducted by Yuan et al. in 2009. Clinical features between the previously and newly reported cases were compared using the publication by Yuan et al. as a cutoff point. The mean age of the 62 newly reported patients with Paecilomyces/Purpureocillium keratitis was 52.7 years. Of these, contact lens wear was the most common predisposing factor (n = 32, 52%), followed by a preexisting corneal disease or previous ocular surgery (n = 12, 19%), and trauma (n = 8, 13%). Fifty eyes (81%) were treated with voriconazole, of which 31 (63%) were medically cured. Twenty-one of 62 eyes (34%) required therapeutic surgery. Compared with the 42 patients reported by Yuan et al., the patients were younger (P = .025); a higher proportion of the patients were contact lens wearers (P = .005); more patients were treated with voriconazole (P = .000); fewer patients required therapeutic surgery (P = .000) in recent reports. Contact lens use has become the major risk factor for Paecilomyces/Purpureocillium keratitis. The surgical rate has been significantly lower in recent publications, probably because of the prevalent use of voriconazole.

Phylogeny of Paecilomyces, the causal agent of pistachio and some other trees dieback disease in Iran.

One of the most important fungal agents of pistachio dieback disease belongs to the ascomycete genus Paecilomyces that has been identified as P. variotii. In 2012-2014, 700 plant samples from pistachio trees and 27 other plant species with dieback symptoms were collected from 10 provinces of Iran. Of the 567 pistachio samples, 277 Paecilomyces strains were obtained and from the 133 samples of other plants (except pistachio and including Pistacia mutica, Punica granatum, Prunus amygdalus, Caesalpinia gilliesii, Nerium oleander, Tamarix aphylla, Tamarix hispida and Haloxylon sp.), 23 fungal isolates were recovered and five isolates were obtained from the air of infected pistachio orchards. Based on morphology, all 305 isolates were identified as P. variotii. Physiological studies revealed that 299 isolates belong to P. formosus. Three isolates were assigned to P. variotii, while three isolates could not be assigned to any of the known species. Of the 305 isolates, 62 were selected for phylogenetic analysis based on DNA variation (ITS, ß-tubulin and calmodulin). This analysis showed that all of our isolates form a clade with P. formosus. P. formosus consists of the three former species P. formosa, P. lecythidis and P. maximus. This study shows that our isolates form a strongly supported clade with strains of P. lecythidis. So, the causal agent of dieback disease of pistachio and other examined trees is P. formosus which is closely related to the former species P. lecythidis and has some differences with the former species P. formosa and P. maximus. Based on phylogenetic studies P. formosus thus seems to be a species complex that could be divided into three separate species.

Paecilomyces Rot: A New Apple Disease.

Paecilomyces niveus is an important food spoilage fungus that survives thermal processing in fruit products, where it produces the mycotoxin patulin. Spoilage of products has been attributed to soil contamination; however, little is known about the ecology of this organism. In this study, orchard soils and culled apple fruit were surveyed and the ability of P. niveus to infect apple was tested on two popular apple varieties. P. niveus was found in 34% of sampled orchard soils from across New York. Completing Koch's postulates, P. niveus was demonstrated to cause postharvest disease in Gala and Golden Delicious apple. Symptoms of this disease, named Paecilomyces rot, resemble several other apple diseases, including black rot, bitter rot, and bull's-eye rot. External symptoms of Paecilomyces rot include brown, circular, concentrically ringed lesions, with an internal rot that is firm and cone-shaped. Both Gala and Golden Delicious apple fruit inoculated with P. niveus developed lesions ≥43 mm in size at 22 days after inoculation. There is some evidence that the size of lesions and rate of infection differ between Gala and Golden Delicious, which may indicate differing resistance to P. niveus. This work shows that P. niveus is common in New York orchard soil and can cause a novel postharvest fruit disease. Whether infected fruit can serve as an overlooked source of inoculum in heat-processed apple products requires further study.

Transcriptome analysis of Paecilomyces hepiali at different growth stages and culture additives to reveal putative genes in cordycepin biosynthesis.

Paecilomyces hepialid (PH) is an endoparasitic fungus of Cordyceps sinensis (CS) and has become a substitute for CS due to their similar pharmacological activities. Because the market demand for CS is difficult to satisfy, and cordycepin, the effective compound of CS, is difficult to industrially produce, we produced 5 samples of PH by culturing for different durations and adding different additives to the culture broth, and detected their cordycepin content with UPLC ESI MS/MS. Then we grouped these cultures into five transcriptome comparisons containing 3 time variable groups and 2 additive variable groups. We used next-generation (NG) sequencing methods to acquire transcriptomic information and investigated the response of gene expression to the additives and the influence of different growth stages. This work will contribute to a better understanding of purine metabolism in PH, and possibly in other Cordyceps species. It will provide a useful resource to further advance transcriptomics studies in Cordyceps species.

Fatal Purpureocillium lilacinum pneumonia in a green tree python.

A 10-y-old female green tree python ( Morelia viridis) died of fungal pneumonia caused by Purpureocillium lilacinum, which was confirmed histologically and by PCR and subsequent DNA sequencing. The same fungal species was cultivated from a swab taken from the terrarium in which the snake was housed. Clinical and environmental P. lilacinum isolates were indistinguishable by the typing method applied, strongly suggesting clonal relatedness of both isolates. Because no other underlying predisposing respiratory infection could be detected by virus-specific PCR or histopathology, P. lilacinum was considered a primary pulmonary pathogen in this tree python.

Fungal strains isolation, identification and application for the recovery of Zn(II) ions.

Fungal biomass proves to be highly efficient for the treatment of wastewater as well as recovery of metal ions from wastewater. Present investigation was aimed to evaluate the efficiency of indigenous fungal isolates for the sequestration of Zn(II) ions aqueous solution. Among twenty five fungal isolates, Aspergillus oryzae SV/09 (AO SV/09), Aspergillus flavus NA9 (AF NA9) and Paecilomyces formosus DTO 63f4 (PF DTO-63f4) were identified by gene sequencing of ITS regions of the ribosomal DNA (rDNA). The AO SV/09, AF NA9 and PF DTO-63f4 showed promising efficiency for the biosorption of Zn(II) ions. Zn(II) ions adsorption was endothermic in nature and data fitted will to the Freundlich isotherm with correlation coefficients values of 0.99, 0.98 and 0.99 for AO SV/09, AF NA9 and PF DTO-63f4, respectively. Pseudo-second order kinetic model explained well the Zn(II) adsorption kinetic of Zn(II) ions onto biosorbents. The adsorbed Zn(II) ions were desorbed using HCl and 85.5, 75.3, 73.7 (%) Zn(II) ions were recovered from AO SV/09, AF NA9 and PF DTO-63f4 sorbents, respectively. The fungal biosorbents were successfully recycled up to five cycles. Based on sorption, recovery and regeneration, the application of fungal bio-sorbents for the sequestration and recovery of Zn(II) ions is suggested from wastewater and could possibly be extended for the recovery of other heavy metal ions from wastewater.

Genetic and chemical characterisation of the cornexistin pathway provides further insight into maleidride biosynthesis.

The biosynthesis of the herbicide cornexistin in the fungus Paecilomyces variotii was investigated by full sequencing of its genome, knockout of key genes within its biosynthetic gene cluster and isolation and identification of intermediate compounds. The general biosynthetic pathway resembles that of byssochlamic acid and other nonadrides in the early stages, but differs in requiring fewer enzymes in the key nonadride dimerisation step, and in the removal of one maleic anhydride moiety.

White mold on cultivated morels caused by Paecilomyces penicillatus.

Morchella (morel) includes prized edible and medical mushrooms in the world. Since 2012, commercial cultivation of morels in the field has developed rapidly in China. However, coupled with the rapid expansion of morel cultivation, diseases have been become serious threats to morel production. White mold is one of the most serious diseases on cultivated morels. This study aimed to confirm this pathogen by following Koch's postulates, and to identify it using molecular evidence. Our results indicated that healthy Morchella fruiting bodies inoculated with Paecilomyces sp. isolates produced typical white mold symptoms, and the internal transcribed spacer sequences of the Paecilomyces sp. were 99% similar to that recovered from an epitype of Paecilomyces penicillatus. Therefore, P. penicillatus was considered to be the causative agent of white mold. White mold occurred from the initial harvest to the storage and preservation process, and it produced white mold-like symptoms on the caps and stripes of Morchella. This is the first time that white mold has been reported on cultivated Morchella.

Catalytic Mechanism of a Novel Glycoside Hydrolase Family 16 "Elongating" ß-Transglycosylase.

Carbohydrates are complex macromolecules in biological metabolism. Enzymatic synthesis of carbohydrates is recognized as a powerful tool to overcome the problems associated with large scale synthesis of carbohydrates. Novel enzymes with significant transglycosylation ability are still in great demand in glycobiology studies. Here we report a novel glycoside hydrolase family 16 "elongating" ß-transglycosylase from Paecilomyces thermophila (PtBgt16A), which efficiently catalyzes the synthesis of higher polymeric oligosaccharides using ß-1,3/1,4-oligosaccharides as donor/acceptor substrates. Further structural information reveals that PtBgt16A has a binding pocket around the -1 subsite. The catalytic mechanism of PtBgt16A is partly similar to an exo-glycoside hydrolase, which cleaves the substrate from the non-reducing end one by one. However, PtBgt16A releases the reducing end product and uses the remainder glucosyl as a transglycosylation donor. This catalytic mechanism has similarity with the catalytic mode of amylosucrase, which catalyzes the transglycosylation products gradually extend by one glucose unit. PtBgt16A thus has the potential to be a tool enzyme for the enzymatic synthesis of new ß-oligosaccharides and glycoconjugates.

Biosynthesis of Antibiotic Leucinostatins in Bio-control Fungus Purpureocillium lilacinum and Their Inhibition on Phytophthora Revealed by Genome Mining.

Purpureocillium lilacinum of Ophiocordycipitaceae is one of the most promising and commercialized agents for controlling plant parasitic nematodes, as well as other insects and plant pathogens. However, how the fungus functions at the molecular level remains unknown. Here, we sequenced two isolates (PLBJ-1 and PLFJ-1) of P. lilacinum from different places Beijing and Fujian. Genomic analysis showed high synteny of the two isolates, and the phylogenetic analysis indicated they were most related to the insect pathogen Tolypocladium inflatum. A comparison with other species revealed that this fungus was enriched in carbohydrate-active enzymes (CAZymes), proteases and pathogenesis related genes. Whole genome search revealed a rich repertoire of secondary metabolites (SMs) encoding genes. The non-ribosomal peptide synthetase LcsA, which is comprised of ten C-A-PCP modules, was identified as the core biosynthetic gene of lipopeptide leucinostatins, which was specific to P. lilacinum and T. ophioglossoides, as confirmed by phylogenetic analysis. Furthermore, gene expression level was analyzed when PLBJ-1 was grown in leucinostatin-inducing and non-inducing medium, and 20 genes involved in the biosynthesis of leucionostatins were identified. Disruption mutants allowed us to propose a putative biosynthetic pathway of leucinostatin A. Moreover, overexpression of the transcription factor lcsF increased the production (1.5-fold) of leucinostatins A and B compared to wild type. Bioassays explored a new bioactivity of leucinostatins and P. lilacinum: inhibiting the growth of Phytophthora infestans and P. capsici. These results contribute to our understanding of the biosynthetic mechanism of leucinostatins and may allow us to utilize P. lilacinum better as bio-control agent.

Treatment of Paecilomyces variotii pneumonia with posaconazole: case report and literature review.

The fungi Paecilomyces variotii is a potential pathogen in immunocompromised and immunocompetent patients. Their rare association with clinical disease results in scarce literature regarding susceptibility and treatment. Here, we discuss a case involving successful treatment of probable P. variotii pneumonia with posaconazole after treatment failure with voriconazole. The current literature related to antifungal susceptibility profiles, microbiological identification methods and clinical management of infections caused by this organism is also reviewed.

Paecilomyces variotii: A Fungus Capable of Removing Ammonia Nitrogen and Inhibiting Ammonia Emission from Manure.

Ammonia (NH3) emissions from animal manure are a significant environmental and public concern. Despite the numerous studies regarding NH3 emissions from manure, few of them have considered microbial nitrification approaches, especially fungal nitrification. In this study, a filamentous fungus was isolated from chicken manure and was used for nitrification. The species was Paecilomyces variotii by morphological characteristics and 18S rDNA gene sequencing. It played the biggest role in the removal of ammonium at pH 4.0-7.0, C/N ratio of 10-40, temperature of 25-37°C, shaking speed of 150 rpm, and with glucose as the available carbon source. Further analysis revealed that all ammonium was removed when the initial ammonium concentration was less than 100 mg/L; 40% ammonium was removed when the initial ammonium concentration was 1100 mg/L. The results showed that the concentration of ammonia from chicken manure with strain Paecilomyces variotii was significantly lower than that in the control group. We concluded that Paecilomyces variotii has good potential for future applications in in situ ammonium removal as well as ammonia emissions control from poultry manure.

[Screening, identification and characterization of thermotolerant dextranase from a fungus].

Objective: We attempted to obtain a fungus producing thermotolerant dextranase by screening samples from soil. Methods: The fungus producing thermotolerant dextranase was isolated and screened by auxotrophic medium, combined with Pour Plate method and Flat Transparent Circle method. The strain was identified by its colony, cell morphology and cultural characteristics, as well as ITS rDNA sequence analysis. The dextranase produced by the strain was characterized. Results: We obtained the strain DG001 producing thermotolerant dextranase, which was identified as Paecilomyces lilacinus. The optimum catalytic conditions for the dextranase were 55℃, pH 5.0, and the optimum substrate concentration was 5% dextran T70. The dextranase was stable below 60℃ and between pH 4.0 and 7.0. Urea, Mn2+ and Mg2+ could increase enzyme activity, and the low concentration of Mn2+ and Urea could increase enzyme activity to 116.91% and 110.14% respectively, whereas Cu2+ had a strong inhibitory effect on the dextranase. The dextranase, identified as endo-dextranase, hydrolyzed dextran T2000 with main products as isomalt and isomaltotriose. The enzyme-substrate affinity increased with the increasing substrate molecular weight. Conclusion: Strain DG001 producing thermotolerant dextranase was obtained through successful screening, bearing a high activity in a wide temperature range and a good thermal stability. This enzyme shows a promising prospect of application in sugar industry and in the preparation of different molecular weight dextran.

The complete mitochondrial genome of Paecilomyces hepiali (Ascomycota, Eurotiomycetes).

Paecilomyces hepiali, belonging to the Eurotiales order Ascomycota, is an endoparasitic fungus that commonly exists in the natural Cordyceps sinensis anamorph stage. Here, we report the complete mitochondrial DNA sequences of P. hepiali for the first time. The genome is 24,245 bp in length, encoding 15 protein-coding genes (PCGs), 2 rRNA genes, 25 tRNA genes and 3 homing endonucleases. The overall AT composition is 73.37% and the average AT content of PCG, rRNA, tRNA and non-coding region are 74.21%, 66.07%, 62.83% and 75.96%, respectively. Phylogenetic analysis with eight Ascomycota species and thirteen Basidiomycota species revealed that P. hepiali is was more closely related to Cordyceps bassiana, Cordycep smilitaris and Cordyceps brongniartii. It is confirmed that P. hepiali is a derivative of Cordyceps sinensis. This study provided valuable information on the gene contents of the mitochondrial genome and would facilitate the study of function and evolution of P. hepiali.

A mutant of the nematophagous fungus Paecilomyces lilacinus (Thom) is a novel biocontrol agent for Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum causes severe stem rot and yield loss in oilseed rape (Brassica napus L.) and other crops worldwide. Extensive studies have been conducted on Paecilomyces lilacinus as a nematophagous bioagent. However, no reports stated the effect of P. lilacinus as a biocontrol agent against oilseed rape rot S. sclerotiorum. This study describes such effect in lab and field trials using the new transformant pt361 derived from the wild strain P. lilacinus 36-1. Unlike the wild-type strain, the mutant pt361 showed high antagonistic effect against S. Sclerotiorum A. Under lab conditions, the pt361 inhibited (65%) radial mycelial growth of S. sclerotiorum in dual culture test producing 5.9 mm inhibition zone IZ in front of the S. sclerotiorum colony. Moreover, the cell-free filtrate of pt361 culture showed strong inhibitory effects (60.3-100%) on mycelial growth of S. sclerotiorum. In leaf detached assay, pt361 significantly (p < 0.05) inhibited (40.4-97.9%) the extension of the leaf spots caused by S. sclerotiorum A at all tested concentrations. The genomic DNA sequences of the inserted T-DNA flanking obtained from pt361 strain was cloned, verified as a glycoside hydrolase 31 family by homologous analysis with other fungal strains, and named PGH31 (2556bp). Secondary structure prediction showed a domain (Glycoside hydrolase31). Three years field trial confirmed that the cell-free filtrates or spores suspension of pt361 achieved significant (p < 0.05) suppression of oilseed rape stem rot, promoted growth and increased yield compared to the control and exceeded, at dose 100%, the action of the fungicide procymidone(®). In conclusion, the mutant pt361 of P. lilacinus is a novel and promising biocontrol agent against oilseed rape Sclerotinia stem rot.

Triazole Susceptibilities in Thermotolerant Fungal Isolates from Outdoor Air in the Seoul Capital Area in South Korea.

Emerging fungi resistant to triazoles are a concern because of the increased use of medical triazoles and exposure to agricultural triazoles. However, little is known about the levels of triazole susceptibility in outdoor airborne fungi making it difficult to assess the risks of inhalation exposure to airborne, antifungal-resistant fungi. This study examined triazole susceptibilities of the airborne thermotolerant fungi isolated from the ambient air of the Seoul Capital Area of South Korea. We used impactor air sampling with triazole-containing nutrient agar plates as the collection substrates to screen for airborne fungal isolates based on their triazole susceptibilities. This study estimated that 0.17% of all the culturable fungi belong to the pathogenic thermotolerant taxa, among which each isolate of Aspergillus niger and Aspergillus tubingensis showed a minimum inhibitory concentration (MIC) of 2 µg/mL or greater for itraconazole. Their concentration in air was 0.4 CFU/m3. Seven human pathogenic Paecilomyces variotii isolates had MICs of 32 µg/mL or greater and lower than 2 µg/mL for the agricultural fungicide tebuconazole and the medical triazole itraconazole, respectively. Though the concentration was low, our results confirm the presence of airborne fungi with high MICs for itraconazole in ambient air. Inhalation is an important exposure route because people inhale more than 10 m3 of air each day. Vigilance is preferred over monitoring for the emergence of triazole-resistant fungal pathogens in ambient outdoor air.

The role of a phospholipase (PLD) in virulence of Purpureocillium lilacinum (Paecilomyces lilacinum).

Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, understanding the role of phospholipases on the virulence of the filamentous fungus Purpureocillium lilacinum has been still rather limited. In this study, pld gene was characterized. It encodes the protein phospholipase D (PLD) in P. lilacinum. This gene, 3303 bp open reading frame fragment (ORF), encodes a protein of 1100 amino acids with high similarity to the same gene from Penicillium oxalicum and Aspergillus fumigatus. Secondary structure prediction showed two PLD phosphodiesterase domains (437-464 bp and 885-912 bp). The pld gene was significantly regulated during infection of Meloidogyne incognita eggs by P. lilacinum. The expression of pld gene using RT-PCR was the highest at 36 and 48 h, which introduce evidence that the presence of M. incognita may induce the expression of the pld gene in P. lilacinum. In addition, maltose and l-alanine were found to increase the expression of pld gene. An acidic environment (pH 3.0-4.0) and moderate temperatures (27-29 °C) are favorable for pld expression in P. lilacinum.

Mechanism study of alachlor biodegradation by Paecilomyces marquandii with proteomic and metabolomic methods.

Alachlor is an herbicide that is widely used worldwide to protect plant crops against broadleaf weeds and annual grasses. However, due to its endocrine-disrupting activity, its application had been banned in the European Union. As described in our earlier work, Paecilomyces marquandii is a microscopic fungus capable of alachlor removal by N-acetyl oxidation. Our current work uses proteomics and metabolomics to gain a better understanding of alachlor biodegradation by the microscopic fungus P. marquandii. The data revealed that the addition of alachlor reduced the culture growth and glucose consumption rates. Moreover, the rates of glycolysis and the tricarboxylic acids (TCA) cycle increased during the initial stage of growth, and there was a shift toward the formation of supplementary materials (UDP-glucose/galactose) and reactive oxygen species (ROS) scavengers (ascorbate). Proteomic analysis revealed that the presence of xenobiotics resulted in a strong upregulation of enzymes related to energy, sugar metabolism and ROS production. However, the unique overexpression of cyanide hydratase in alachlor-containing cultures may implicate this enzyme as the key protein involved in the alachlor biodegradation pathway. The characterization of P. marquandii-mediated alachlor removal in terms of cell structure and function provides a deeper insight into the strategies of microorganisms toward xenobiotic biodegradation.