Multifunctionality of AsCFEM6 and AsCFEM12 effectors from the potato early blight pathogen Alternaria solani.

Plant pathogens secrete fungal-specific common in several fungal extracellular membrane (CFEM) effectors to manipulate host immunity and contribute to their virulence. Little is known about effectors and their functions in Alternaria solani, the necrotrophic fungal pathogen causing potato early blight. To identify candidate CFEM effector genes, we mined A. solani genome databases. This led to the identification of 12 genes encoding CFEM proteins (termed AsCFEM1-AsCFEM12) and 6 of them were confirmed to be putative secreted effectors. In planta expression revealed that AsCFEM6 and AsCFEM12 have elicitor function that triggers plant defense response including cell death in different botanical families. Targeted gene disruption of AsCFEM6 and AsCFEM12 resulted in a change in spore development, significant reduction of virulence on potato and eggplant susceptible cultivars, increased resistance to fungicide stress, variation in iron acquisition and utilization, and the involvement in 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway. Using maximum likelihood method, we found that positive selection likely caused the polymorphism within AsCFEM6 and AsCFEM12 homologs in different Alternaria spp. Site-directed mutagenesis analysis indicated that positive selection sites within their CFEM domains are required for cell death induction in Nicotiana benthamiana and are critical for response to abiotic stress in yeast. These results demonstrate that AsCFEM effectors possess additional functions beyond their roles in host plant immune response and pathogen virulence.

An account of Colletotrichum species associated with anthracnose of Atractylodes ovata in South Korea based on morphology and molecular data.

Ovate-leaf atractylodes (OLA) (Atractylodes ovata) is a well-known medicinal plant in Korea; its dried rhizome and root extracts are used in herbal medicine. However, anthracnose is a great challenge to the OLA cultivation in South Korea. Colletotrichum spp. is a major group of plant pathogens responsible for anthracnose on a range of economically important hosts. Its occurrence on OLA remains unresolved. To investigate the diversity, morphology, phylogeny, and biology of Colletotrichum spp., 32 fungal isolates were obtained from 30 OLA-affected leaves collected from five different farms, in two regions in South Korea, Mungyeong and Sangju. The phylogenetic analysis with four or five gene loci (ITS, TUB2, ACT, GAPDH, and CHS-1) along with morphology of 26 representative isolates delineated six previously known Colletotrichum species including C. fructicola, C. gloeosporioides sensu stricto (s.s), C. cigarro, C. plurivorum, C. siamense and C. sojae, and one new species, described here as C. ovataense. Amongst these species, C. gloeosporioides s.s. and C. plurivorum were the most prevalent species. A pathogenicity test on the detached leaves revealed that different Colletotrichum species presented a distinct degree of virulence, confirming Koch's postulates. In this study, C. fructicola, C. cigarro, C. plurivorum, C. siamense, and C. sojae were reported from A. ovata for the first time, as the causal agent of ovate-leaf atractylodes anthracnose. Understanding the diversity and biology of the Colletotrichum species population will help in managing this disease.

Functional Analysis of Autophagy-Related Gene ATG12 in Potato Dry Rot Fungus Fusarium oxysporum.

Autophagy is an intracellular process in all eukaryotes which is responsible for the degradation of cytoplasmic constituents, recycling of organelles, and recycling of proteins. It is an important cellular process responsible for the effective virulence of several pathogenic plant fungal strains, having critical impacts on important crop plants including potatoes. However, the detailed physiological mechanisms of autophagy involved in the infection biology of soil-borne pathogens in the potato crop needs to be investigated further. In this study, the autophagy-related gene, FoATG12, in potato dry rot fungus Fusarium oxysporum was investigated by means of target gene replacement and overexpression. The deletion mutant ∆FoATG12 showed reduction in conidial formation and exhibited impaired aerial hyphae. The FoATG12 affected the expression of genes involved in pathogenicity and vegetative growth, as well as on morphology features of the colony under stressors. It was found that the disease symptoms were delayed upon being inoculated by the deletion mutant of FoATG12 compared to the wild-type (WT) and overexpression (OE), while the deletion mutant showed the disease symptoms on tomato plants. The results confirmed the significant role of the autophagy-related ATG12 gene in the production of aerial hyphae and the effective virulence of F. oxysporum in the potato crop. The current findings provid an enhanced gene-level understanding of the autophagy-related virulence of F. oxysporum, which could be helpful in pathogen control research and could have vital impacts on the potato crop.

Pulmonary infection secondary to Blastobotrys raffinosifermentans in a cystic fibrosis patient: Review of the literature.

BACKGROUND: The genus Blastobotrys consists of at least 20 species. Disease in humans has been reported with B adeninivorans, B raffinosifermentans, B proliferans and B serpentis, mostly in immunocompromised patients and those with cystic fibrosis. OBJECTIVE: We report a lung infection secondary to B raffinosifermentans in a cystic fibrosis patient successfully treated with isavuconazole and review the literature of invasive infections caused this genus. We also evaluated clinical isolates in our laboratory for species identification and antifungal susceptibility. METHODS: Phylogenetic analysis was performed on a collection of 22 Blastobotrys isolates in our reference laboratory, and antifungal susceptibility patterns were determined for nine clinically available antifungals against 19 of these isolates. RESULTS: By phylogenetic analysis, 21 of the 22 isolates in our collection were identified as B raffinosifermentans and only 1 as B adeninivorans. Most were cultured from the respiratory tract, although others were recovered from other sources, including CSF and blood. Isavuconazole, caspofungin and micafungin demonstrated the most potent in vitro activity, followed by amphotericin B. In contrast, fluconazole demonstrated poor activity. The patient in this case responded to isavuconazole treatment for breakthrough infection due to B raffinosifermentans that was cultured from pleural fluid while on posaconazole prophylaxis post-bilateral lung transplantation for cystic fibrosis. CONCLUSIONS: Blastobotrys species are rare causes of infections in humans and primarily occur in immunocompromised hosts. In our collection, the majority of isolates were identified as B raffinosifermentans. To our knowledge, this is the first report of successful treatment of such an infection with isavuconazole.

In vitro resynthesis of lichenization reveals the genetic background of symbiosis-specific fungal-algal interaction in Usnea hakonensis.

BACKGROUND: Symbiosis is central to ecosystems and has been an important driving force of the diversity of life. Close and long-term interactions are known to develop cooperative molecular mechanisms between the symbiotic partners and have often given them new functions as symbiotic entities. In lichen symbiosis, mutualistic relationships between lichen-forming fungi and algae and/or cyanobacteria produce unique features that make lichens adaptive to a wide range of environments. Although the morphological, physiological, and ecological uniqueness of lichens has been described for more than a century, the genetic mechanisms underlying this symbiosis are still poorly known. RESULTS: This study investigated the fungal-algal interaction specific to the lichen symbiosis using Usnea hakonensis as a model system. The whole genome of U. hakonensis, the fungal partner, was sequenced by using a culture isolated from a natural lichen thallus. Isolated cultures of the fungal and the algal partners were co-cultured in vitro for 3 months, and thalli were successfully resynthesized as visible protrusions. Transcriptomes of resynthesized and natural thalli (symbiotic states) were compared to that of isolated cultures (non-symbiotic state). Sets of fungal and algal genes up-regulated in both symbiotic states were identified as symbiosis-related genes. CONCLUSION: From predicted functions of these genes, we identified genetic association with two key features fundamental to the symbiotic lifestyle in lichens. The first is establishment of a fungal symbiotic interface: (a) modification of cell walls at fungal-algal contact sites; and (b) production of a hydrophobic layer that ensheaths fungal and algal cells;. The second is symbiosis-specific nutrient flow: (a) the algal supply of photosynthetic product to the fungus; and (b) the fungal supply of phosphorous and nitrogen compounds to the alga. Since both features are widespread among lichens, our result may indicate important facets of the genetic basis of the lichen symbiosis.

Optimization of Fermentation Process and Its Impact on Gene Transcription of Intracellular Polysaccharide Synthesis in the Wood Ear Medicinal Mushroom Auricularia auricula-judae (Agaricomycetes).

To improve the production of mycelial biomass and intracellular polysaccharide (IPS), the culture medium composition and culture condition were respectively optimized by response surface methodology and one-factor-at-a-time methodology. The impact of fermentation process on the transcription of genes involved in IPS synthesis was evaluated using quantitative reverse transcription PCR (qRT-PCR). The yield of biomass and IPS were increased by 150% and 177% under the optimized fermentation process, respectively. Moreover, the optimization of fermentation process significantly increased the transcription of genes involved in IPS synthesis, especially pgm-1, ugp, and pgi. This is the first exploration of gene transcription profile for IPS synthesis of Auricularia auricula-judae under submerged fermentation. These results could provide a potential tool for fermentation process optimization and transcriptional regulation of IPS biosynthesis in A. auricula-judae.

Do the Main Components of the Sclerotia of Umbrella Polypore Mushroom, Polyporus umbellatus (Agaricomycetes), Correlate with Armillaria Associates?

Polyporus umbellatus is a traditional Chinese medicinal mushroom. The growth of P. umbellatus sclerotia requires the rhizomorphs of Armillaria spp. to supply nutrition. Whether the main components (MC) of sclerotia of P. umbellatus are related to the phylogeny of Armillaria associates or other environmental factors is largely unknown. In this study, we collected 17 sclerotia and soil samples from northeast to southwest China. In total, 17 Armillaria associates were isolated, and sclerotial MC contents and soil characteristics (total N, P, K, and organic matter) were determined. The analysis revealed that the MC content of P. umbellatus did not resemble a Brownian motion process in phylogeny of Armillaria associates, but were significantly influenced by the total N content of the soil. These results provide clear evidence that sclerotia of P. umbellatus associating with phylogenetic related Armillaria associates possess differing MC content. The mechanisms of nutrient exchange in P. umbellatus-Armillaria associations now require further elucidation.

Nutritional and Biochemical Characterization of Panus lecomtei Mushroom (Agaricomycetes) from India and Its Cultivation.

Panus lecomtei is emerging as an edible mushroom found worldwide and particularly in the Northern Hemisphere. The mushroom contains a substantial amount of useful nutritional and medicinal compounds. In the present study, we have examined a specimen of P. lecomtei submitted to the ICAR-Directorate of Mushroom Research gene bank. The specimen was examined for taxonomical characters using classical and molecular tools. Attempts were made for cultivation of this mushroom under controlled conditions using sawdust-based substrate. The specimen was characterized by its purplish fruiting body having coarse, rigid, dense hairs on the cap, pubescent stipe, and abundant metuloids. Molecular identification through conserved ITS region was done and the sequence was deposited in NCBI GenBank under accession number MN332200. Nutritional profiling and biochemical analysis showed that the mushroom contained high carbohydrate but low fat contents. The mushroom showed the presence of phenolics, ß-carotene, and lycopene. The analysis also showed substantial antioxidant properties in the mushroom. The findings presented herein point out that P. lecomtei can be used as a potential edible mushroom for diversification of mushroom production in India.

Pectin as Carbon Source for Monilinia laxa Exoproteome and Expression Profiles of Related Genes.

Pectin, as part of the fruit cell wall, can be degraded by brown rot fungi by coordinating the production, secretion, and action of extracellular enzymes. In this study, pectin utilization by the necrotroph Monilinia laxa 8L was studied by in vitro and in silico approaches. A total of 403 genes encoding carbohydrate-active enzymes (CAZymes) were identified, including 38 coding a predicted pectin-degrading activity. Analyzing the differences between M. laxa 8L exoproteomes in media containing glucose and pectin as sole carbon sources, we identified 107 pectin-specific proteins, among them, 64.48% harbor a classical secretory activity, including 42 CAZymes and six pectin-degrading proteins. Analyzing the gene-expression patterns of some pectinase families revealed their possible sequential action in pectin disassembly. We found, in vitro, an early pectin-dependent induction of MlRGAE1, MlPG1, and three members of the rhamnosidase family (MlαRHA2, MlαRHA3, and MlαRHA6) and late response of MlPG2 and MlPNL3. M. laxa 8L has the ability to use both pectin and byproducts as carbon sources, based on a functional pectinolytic machinery encoded in its genome, subjected to pectin-dependent regulation and appropriate secretion mechanisms of these pectinolytic enzymes. Differences in the secretion and transcription profile of M. laxa 8L provided insights into the different mechanisms that contribute to brown rot development.

Selection and evaluation of appropriate reference genes for RT-qPCR based expression analysis in Candida tropicalis following azole treatment.

Candida tropicalis arises as one of the predominant non-Candida albicans Candida (NCAC) species causing invasive candidiasis in Asian countries. A rise in reports of C. tropicalis with a parallel increase in fluconazole resistance has also been observed. The genes and underlying pathways associated with azole antifungal resistance in C. tropicalis is still not properly understood. The RT-qPCR is the most promising approach for expression analysis of target genes to understand the mechanisms of resistance. The reliability and reproducibility of this technique depend on the selection of suitable reference genes for the normalization in expression study. The present study investigated the expression stability levels of ten genes including ACT1, EF1, GAPDH, PGK1, RDN5.8, RDN18, RDN28, SDHA, TUB1, and UBC13 for their suitability in fluconazole treated/untreated C. tropicalis. The stability levels of these genes were examined by the ∆∆CT, ΔCT, Pfaffl methods and five independent software including hkgFinder, geNorm, NormFinder, BestKeeper, and RefFinder software. We report, the EF1 and ACT1 were the most stable reference genes for normalization and can be used for the gene expression analysis in C. tropicalis. To the best of our knowledge, our study is the first to select and validate the reference genes in C. tropicalis for RT-qPCR based expression analysis.

A simple and effective method to discern the true commercial Chinese cordyceps from counterfeits.

The Chinese cordyceps, a complex of the fungus Ophiocordyceps sinensis and its species-specific host insects, is also called "DongChongXiaCao" in Chinese. Habitat degradation in recent decades and excessive harvesting by humans has intensified its scarcity and increased the prices of natural populations. Some counterfeits are traded as natural Chinese cordyceps for profit, causing confusion in the marketplace. To promote the safe use of Chinese cordyceps and related products, a duplex PCR method for specifically identifying raw Chinese cordyceps and its primary products was successfully established. Chinese cordyceps could be precisely identified by detecting an internal transcribed spacer amplicon from O. sinensis and a cytochrome oxidase c subunit 1 amplicon from the host species, at a limit of detection as low as 32 pg. Eleven commercial samples were purchased and successfully tested to further verify that the developed duplex PCR method could be reliably used to identify Chinese cordyceps. It provides a new simple way to discern true commercial Chinese cordyceps from counterfeits in the marketplace. This is an important step toward achieving an authentication method for this Chinese medicine. The methodology and the developmental strategy can be used to authenticate other traditional Chinese medicinal materials.

Whole-genome and time-course dual RNA-Seq analyses reveal chronic pathogenicity-related gene dynamics in the ginseng rusty root rot pathogen Ilyonectria robusta.

Ilyonectria robusta causes rusty root rot, the most devastating chronic disease of ginseng. Here, we for the first time report the high-quality genome of the I. robusta strain CD-56. Time-course (36 h, 72 h, and 144 h) dual RNA-Seq analysis of the infection process was performed, and many genes, including candidate effectors, were found to be associated with the progression and success of infection. The gene expression profile of CD-56 showed a trend of initial inhibition and then gradually returned to a profile similar to that of the control. Analyses of the gene expression patterns and functions of pathogenicity-related genes, especially candidate effector genes, indicated that the stress response changed to an adaptive response during the infection process. For ginseng, gene expression patterns were highly related to physiological conditions. Specifically, the results showed that ginseng defenses were activated by CD-56 infection and persisted for at least 144 h thereafter but that the mechanisms invoked were not effective in preventing CD-56 growth. Moreover, CD-56 did not appear to fully suppress plant defenses, even in late stages after infection. Our results provide new insight into the chronic pathogenesis of CD-56 and the comprehensive and complex inducible defense responses of ginseng root to I. robusta infection.

Selection of yeasts from bee products for alcoholic beverage production.

The use of appropriate yeast strains allows to better control the fermentation during beverage production. Bee products, especially of stingless bees, are poorly explored as sources of fermenting microorganisms. In this work, yeasts were isolated from honey and pollen from Tetragonisca angustula (Jataí), Nannotrigona testaceicornis (Iraí), Frieseomelitta varia (Marmelada), and honey of Apis mellifera bees and screened according to morphology, growth, and alcohol production. Bee products showed to be potential sources of fermenting microorganisms. From 55 isolates, one was identified as Papiliotrema flavescens, two Rhodotorula mucilaginosa, five Saccharomyces cerevisiae, and nine Starmerella meliponinorum. The S. cerevisiae strains were able to produce ethanol and glycerol at pH 4.0-8.0 and temperature of 10-30 °C, with low or none production of undesirable compounds, such as acetic acid and methanol. These strains are suitable for the production of bioethanol and alcoholic beverages due to their high ethanol production, similar or superior to the commercial strain, and in a broad range of conditions like as 50% (m/v) glucose, 10% (v/v) ethanol, or 500 mg L-1 of sodium metabisulfite.

Improvement of Curvulamine Production by Precursors Co-addition Strategy in Liquid Culture of Marine-Derived Fungus Curvularia sp. IFB-Z10.

Curvulamine, a novel scaffold alkaloid with remarkable selective antibacterial activity, is produced by marine fungus Curvularia sp. IFB-Z10. However, its deep pharmaceutical research and application are severely restricted by the low yield, which needs to be solved urgently. The purpose of this study was to improve curvulamine production via precursors co-addition strategy and further reveal the regulation mechanism. In this work, the optimal precursors co-addition conditions were firstly obtained, and curvulamine production achieved 166.74 mg/L with the supply of 250 mg/L alanine and 200 mg/L proline at 60 h, which was 4.08 times that of control. It was observed that under alanine and proline stimulation, fungus exhibited the morphology of a small-diameter compact pellet. Furthermore, the organic acid levels in central carbon metabolism (CCM) were declined with precursors supplement. Besides, precursors also induced the critical biosynthetic gene transcriptions. The above findings collectively promoted curvulamine synthesis. Finally, Curvularia sp. IFB-Z10 fermentation process was successfully established by feeding alanine and proline at 0.021 g/L/h and 0.017 g/L/h rate from 60 to 72 h, and curvulamine production reached 133.58 mg/L in a 5-L bioreactor. The information acquired would facilitate the enhancement of curvulamine yield in submerged fermentation and the research on synthesis regulation of other alkaloids.

[A landscape of transcriptome analysis of three sclerotia growth stages in Polyporus umbellatus].

Polyporus umbellatus,a traditional Chinese precious medicine as long been used for eliminating dampness,diuresis and have effect on cancer,getting more and more popularly in China recently. And the developmental metabolic process of the medicinal fungus,P. umbellatus,has been gotten more attention. This study is for the first time to explore the three sclerotial growth stages in P. umbellatus,named " white Polyporus"( initial phase), " grey Polyporus"( developmental phase) and " black Polyporus"( mature phase),by utilizing the de novo transcriptome assembly analysis technology. Finally,we obtained 88. 12 Gb sequence containing85 235 unigenes( ≥200 bp) assembled and 100% were annotated. We identified genes differentially expressed among the three stages of the sclerotia and screened out MFSgst,ERG4/ERG24,WD40,Rho A,CYP450,PKS,GSase and CHS1,which may contribute to the production of medicinal secondary metabolites and the defense mechanism against the environmental stress and biological invasion. We did the qRT-PCR trial to verify our results,which is in line with expectations. Our results are purposed to unearth the molecular mechanism of the accumulation of active constituents in different stages of Polyporus sclerotia which can be applied in the production and protection of Polyporus effectively.

Verticillium isaacii is a Pathogen and Endophyte of Potato and Sunflower in the Columbia Basin of Washington.

The objective of this research was to test the hypothesis that Verticillium isaacii causes diseases of sunflower and potato plants. Two sunflower genotypes and one potato cultivar were inoculated with five V. isaacii isolates and three pathogenic V. dahliae isolates. Biomass, disease expression, and stem colonization were quantified. Overt wilt symptoms were observed on both sunflower genotypes and potato plants inoculated with a subset of the V. isaacii isolates. Biomass of both sunflower genotypes was not affected by V. isaacii infection. Tuber yields either decreased in response to infection by one V. isaacii isolate or were not affected by infection. Stems of sunflower and potato plants were infected by at least four of the five V. isaacii isolates. A new disease of sunflower and potato is documented. Evidence that V. isaacii exhibits different lifestyles including pathogenicity and endophytism is presented. Finally, this research documents variation in fungal lifestyles that can exist in samples from a single field.

RNA-Seq de Novo Assembly and Differential Transcriptome Analysis of Chaga (Inonotus obliquus) Cultured with Different Betulin Sources and the Regulation of Genes Involved in Terpenoid Biosynthesis.

Chaga (Inonotus obliquus) is a medicinal fungus used in traditional medicine of Native American and North Eurasian cultures. Several studies have demonstrated the medicinal properties of chaga's bioactive molecules. For example, several terpenoids (e.g., betulin, betulinic acid and inotodiol) isolated from I. obliquus cells have proven effectiveness in treating different types of tumor cells. However, the molecular mechanisms and regulation underlying the biosynthesis of chaga terpenoids remain unknown. In this study, we report on the optimization of growing conditions for cultured I. obliquus in presence of different betulin sources (e.g., betulin or white birch bark). It was found that better results were obtained for a liquid culture pH 6.2 at 28 °C. In addition, a de novo assembly and characterization of I. obliquus transcriptome in these growth conditions using Illumina technology was performed. A total of 219,288,500 clean reads were generated, allowing for the identification of 20,072 transcripts of I. obliquus including transcripts involved in terpenoid biosynthesis. The differential expression of these genes was confirmed by quantitative-PCR. This study provides new insights on the molecular mechanisms and regulation of I. obliquus terpenoid production. It also contributes useful molecular resources for gene prediction or the development of biotechnologies for the alternative production of terpenoids.

Chamomile Floricolous Downy Mildew Caused by Peronospora radii.

Floricolous downy mildews (Peronospora, oomycetes) are a small, monophyletic group of mostly inconspicuous plant pathogens that induce symptoms exclusively on flowers. Characterization of this group of pathogens, and information about their biology, is particularly sparse. The recurrent presence of a disease causing flower malformation which, in turn, leads to high production losses of the medicinal herb Matricaria chamomilla in Serbia has enabled continuous experiments focusing on the pathogen and its biology. Peronospora radii was identified as the causal agent of the disease, and morphologically and molecularly characterized. Diseased chamomile flowers showed severe malformations of the disc and ray florets, including phyllody and secondary inflorescence formation, followed by the onset of downy mildew. Phylogeny, based on internal transcribed spacer and cox2, indicates clustering of the Serbian P. radii with other P. radii from chamomile although, in cox2 analyses, they formed a separate subcluster. Evidence pointing to systemic infection was provided through histological and molecular analyses, with related experiments validating the impact of soilborne and blossom infections. This study provides new findings in the biology of P. radii on chamomile, thus enabling the reconstruction of this floricolous Peronospora species' life cycle.

The Synchytrium endobioticum AvrSen1 Triggers a Hypersensitive Response in Sen1 Potatoes While Natural Variants Evade Detection.

Synchytrium endobioticum is an obligate biotrophic fungus of division Chytridiomycota. It causes potato wart disease, has a worldwide quarantine status and is included on the Health and Human Services and United States Department of Agriculture Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host resistance in a set of differential potato varieties. Thus far, 39 pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 (D1) resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. The S. endobioticum-specific secretome of 14 isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1 (D1) isolates but absent in others. We identified a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response (HR), which cosegregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. The S. endobioticum AvrSen1 is a single-copy gene and encodes a 376-amino-acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota, an extremely diverse yet underrepresented basal lineage of fungi.