Improved observation of colonized roots reveals the regulation of arbuscule development and senescence by drought stress in the arbuscular mycorrhizae of citrus

Citrus is the typical mycorrhizal fruit tree species establishing symbiosis with arbuscular mycorrhizal (AM) fungi. However, arbuscule development and senescence in colonized citrus roots, especially in response to drought stress, remain unclear, which is mainly due to the difficulty in clearing and staining lignified roots with the conventional method. Here, we improved the observation of colonized roots of citrus plants with the sectioning method, which enabled the clear observation of AM fungal structures. Furthermore, we investigated the effects of one week of drought stress on arbuscule development and senescence with the sectioning method. Microscopy observations indicated that drought stress significantly decreased mycorrhizal colonization (F%and M%) although it did not affect plant growth performance. Fluorescence probes (WGA 488 and/or Nile red) revealed that drought stress inhibited arbuscule development by increasing the percentage of arbuscules at the early stage and decreasing the percentages of arbuscules at the midterm and mature stages. Meanwhile, drought stress accelerated arbuscule senescence, which was characterized by the increased accumulation of neutral lipids. Overall, the sectioning method developed in this study enables the in-depth investigation of arbuscule status, and drought stress can inhibit arbuscule development but accelerate arbuscule senescence in the colonized roots of citrus plants. This study paves the way to elaborately dissecting the arbuscule dynamics in the roots of fruit tree species in response to diverse abiotic stresses.

Microbiologic risk factors of recurrent choledocholithiasis postendoscopic sphincterotomy

BACKGROUND Choledocholithiasis is a severe disorder that affects a significant portion of the world’s population.Treatment using endoscopic sphincterotomy(EST)has become widespread;however,recurrence post-EST is relatively common.The bile microbiome has a profound influence on the recurrence of choledocholithiasis in patients after EST;however,the key pathogens and their functions in the biliary tract remain unclear.AIM To investigate the biliary microbial characteristics of patients with recurrent choledocholithiasis post-EST,using next-generation sequencing.METHODS This cohort study included 43 patients,who presented with choledocholithiasis at the Guangdong Second Provincial General Hospital between May and June 2020.The patients had undergone EST or endoscopic papillary balloon dilation and were followed up for over a year.They were divided into either the stable or recurrent groups.We collected bile samples and extracted microbial DNA for analysis through next-generation sequencing.Resulting sequences were analyzed for core microbiome and statistical differences between the diagnosis groups;they were examined using the Kyoto Encyclopedia of Genes and Genomes pathway hierarchy level using analysis of variance.Correlation between the key genera and metabolic pathways in bile,were analyzed using Pearson’s correlation test.RESULTS The results revealed distinct clustering of biliary microbiota in recurrent choledocholithiasis.Higher relative abundances(RAs)of Fusobacterium and Neisseria(56.61%±14.81%vs 3.47%±1.10%,8.95%±3.42%vs 0.69%±0.32%,respectively)and the absence of Lactobacillus were observed in the bile of patients with recurrent disease,compared to that in stable patients.Construction of a microbiological co-occurrence network revealed a mutual relationship among Fusobacterium,Neisseria,and Leptotrichia,and an antagonistic relationship among Lactobacillales,Fusobacteriales,and Clostridiales.Functional prediction of biliary microbiome revealed that the loss of transcription and metabolic abilities may lead to recurrent choledocholithiasis.Furthermore,the prediction model based on the RA of Lactobacillales in the bile was effective in identifying the risk of recurrent choledocholithiasis(P=0.03).CONCLUSION We demonstrated differences in the bile microbiome of patients with recurrent choledocholithiasis compared to that in patients with stable disease,thereby adding to the current knowledge on its microbiologic etiology.

Total Triterpenoids from Ganoderma Lucidum Suppresses Prostate Cancer Cell Growth by Inducing Growth Arrest and Apoptosis

In this study,one immortalized human normal prostatic epithelial cell line(BPH) and four human prostate cancer cell lines(LNCa P,22Rv1,PC-3,and DU-145) were treated with Ganoderma Lucidum triterpenoids(GLT) at different doses and for different time periods. Cell viability,apoptosis,and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and-3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest(mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4(CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis,which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Food Safety Risks and Contributing Factors of Cronobacter spp.

Cronobacter species are a group of Gram-negative opportunistic pathogens,which cause meningitis,sep-ticemia,and necrotizing enterocolitis in neonates and infants,with neurological sequelae in severe cases.Interest in Cronobacter has increased significantly in recent years due to its high virulence in children.In this review,we summarize the current understanding of the prevalence of Cronobacter species in several important food types.We discuss the response mechanisms enabling persistence in adverse growth con-ditions,as well as its pathogenicity.We emphasize the food safety concerns caused by Cronobacter and subsequent control methods and clinical treatments.

Synthesis of graphene materials by electrochemical exfoliation: Recent progress and future potential

Synthesis of structurally controlled graphene materials is critical for realizing their practical applications.The electrochemical exfoliation of graphite has emerged as a simple method to produce graphene materials.This review examines research progress in the last 5 years,from 2015 to 2019.Graphene material synthesis methods generally have a trade‐off between increasing production yield and achieving better material property control.The synthesis conditions for synthesizing pristine graphene,graphene oxide(GO),and graphene composites are significantly different.Thus,in this review,we first discuss synthesis methods for graphene materials with high C/O ratios from four aspects:graphite electrodes,equipment engineering,electrolytes,and additional reduction methods.Next,we survey synthesis methods for GO and examine how the pretreatment of the graphite electrodes,electrolytes,and operation parameters,such as applied voltages,electrolyte temperatures,and mechanical forces,affect the quality of GO.Further,we summarize electrochemical exfoliation methods used to dope graphene materials,introduce covalent functional groups,incorporate various nanoparticles,and assembly of graphene architectures.For all synthesis methods,we compare the properties of resulting graphene materials such as C/O ratios,lateral size,layer numbers,and quality characterized by Raman spectroscopy.Lastly,we propose our perspectives on further research.We hope this review stimulates more studies to realize the on‐demand production of graphene materials with desired properties using electrochemical exfoliation methods.

Effects of Exogenous Nutrient Additions on Azodye Wastewater Treatment

Since the azo dyes widely used in textile-dyeing industry are toxic,highly persistent,and ubiquitously distributed in the environment,their treatment efficiencies and fade in ecosystems have received worldwide attention.Due to the microbes play important roles in azo-dye degradation,exogenous nutrient addition is used as a promising biostimulation strategy to improve the treatment efficiencies of azo-dye wastewater.However,little is known about the effects of different kinds of exogenous nutrients on the azo-dye wastewater treatment directly.Here,three kinds of common nutrients,glucose,starch and yeast extract,were added to the sequencing batch reactors(SBR)for the treatment of wastewater containing acid red 73 to compare the biostimulation efficiencies by investigating the dye's removal efficiencies and the changes of related water qualities.Our results showed that the reactors added yeast extract had highest removal efficiencies and chemical oxygen demand(COD)as well,followed by glucose and starch.The removal rates of acid red 73 and COD were 90.63%and 87.3%in the reactors added yeast extract,respectively,while 86.49%and 78.4%in those with glucose and 85.38%and 75.2%in those with starch.This study provided some useful information for the biostimulation strategy of azo-dye wastewater treatment and preliminarily suggested that yeast extract would be the optimal choice.

Redesigned Duplex RT-qPCR for the Detection of GI and GII Human Noroviruses

Human noroviruses(HuNoVs)are major foodborne pathogens that cause nonbacterial acute gastroenteritis worldwide.As the tissue-culture system for HuNoVs is not mature enough for routine detection of the virus,detection is mainly dependent on molecular approaches such as reverse transcription polymerase chain reaction(RT-PCR)and reverse transcription quantitative real-time polymerase chain reaction(RTqPCR).The widely used primers and probes for RT-qPCR were established in the early 2000s.As HuNoVs are highly variant viruses,viral genome mutations result in previously designed primers and/or probes that were perfectly matched working less efficiently over time.In this study,a new duplex RT-qPCR(ND-RT-qPCR)was designed for the detection of genogroup Ⅰ(GⅠ)and genogroup Ⅱ(GⅡ)HuNoVs based on an analysis of viral sequences added in the database after 2010.Using long transcribed viral RNAs,the results demonstrate that the sensitivity of ND-RT-qPCR is as low as one genomic copy for both GⅠ and GⅡ HuNoVs.The performance of ND-RT-qPCR was further evaluated by a comparison with the commonly used Kageyama primer/probe sets for RT-qPCR(Kageyama RT-qPCR)for 23 HuNoV-positive clinical samples.All five GⅠ samples were registered as positive by ND-RT-qPCR,whereas only two samples were registered as positive by Kageyama RT-qPCR.All 18 GⅡ samples were registered as positive by ND-RT-qPCR,while 17 samples were registered as positive by Kageyama RT-qPCR.The sensitivity reflected by the quantification cycle(Cq)value was lower in ND-RT-qPCR than in Kageyama RT-qPCR.Our data suggest that ND-RT-qPCR could be a good fit for the detection of current strains of HuNoVs.

Endophytic fungal communities associated with leaves,stems and roots of four medicinal plants in South China

Amomum villosum,Aquilaria sinensis,Morinda officinalis,and Pogostemon cablin are well-known medicinal plants in South China for their particular pharmacological activities,but their endophytic fungi have been rarely reported.Here,the endophytic fungal communities associated with the leaves,stems,and roots of the four medicinal plant species were investigated by high-throughput sequencing technology.From the four medicinal plant species,a total of 169,149 sequences(reads)of endophytic fungi were harvested and clustered into 791 operational taxonomic units(OTUs)assigned to the Kingdom Fungi.These OTUs taxonomically spanned five phyla,27 classes,75 orders,167 families,and 291 genera.At the genus level,Phyllosticta,Candida,Zasmidium,and Cryptococcus were the major genera detected in A.villosum;Nigrospora,Tylopilus,Arthrobotrys,Coniosporium,and Corynespora were the dominant genera isolated in A.sinensis;Ochroconis,Cercospora,Aspergillus,and Cyphellophora were the dominant genera detected in M.officinalis;and Cladophialophora,Meira,Sakaguchia,and Penicillium were the major genera isolated in P.cablin.Among the four medicinal plant species,M.officinalis had the highest endophytic fungal diversity(H′>4.25),however A.sinensis had the lowest(H′<2.37).There were significant differences in endophytic fungal communities among different organs in the same plant species.

Griseofulvin enantiomers and bromine-containing griseofulvin derivatives with antifungal activity produced by the mangrove endophytic fungus Nigrospora sp.QQYB1

Marine microorganisms have long been recognized as potential sources for drug discovery.Griseofulvin was one of the first antifungal natural products and has been used as an antifungal agent for decades.In this study,12 new griseofulvin derivatives[(±)-1-2,(+)-3,(±)-4,10-12,and 14-15]and two new griseofulvin natural products(9 and 16)together with six known analogues[(-)-3,5-8,and 13]were isolated from the mangrove-derived fungus Nigrospora sp.QQYB1 treated with 0.3%NaCl or 2%NaBr in rice solid medium.Their 2D structures and absolute configurations were established by extensive spectroscopic analysis(1D and 2D NMR,HRESIMS),ECD spectra,computational calculation,DP4+analysis,and X-ray single-crystal diffraction.Compounds 1-4 represent the first griseofulvin enantiomers with four absolute configurations(2S,6'S;2R,6'R;2S,6'R;2R,6'S),and compounds 9-12 represent the first successful production of brominated griseofulvin derivatives from fungi via the addition of NaBr to the culture medium.In the antifungal assays,compounds 6 and 9 demonstrated significant inhibitory activities against the fungi Colletotrichum truncatum,Microsporum gypseum,and Trichophyton mentagrophyte with inhibition zones varying between 28 and 41 mm(10μg/disc).The structure-activity relationship(SAR)was analyzed,which showed that substituents at C-6,C-7,C-6'and the positions of the carbonyl and double bond of griseofulvin derivatives significantly affected the antifungal activity.

Relationship between Gut Microbiota and Type 2 Diabetic Erectile Dysfunction in Sprague-Dawley Rats

In order to investigate the relationship between gut microbiota and type 2 diabetic erectile dysfunction(T2DED), we analyzed the characteristics of gut microbiota in the Sprague-Dawley(SD) rats with T2DED. Thirty-five SD rats were randomly divided into two groups: control group(n=15) with normal diet, and experimental group(n=20) with construction of T2D model. Faecal and serum samples were collected at 2nd and 8th week after establishment of T2D model, respectively. Faecal samples were used for analysis of gut microbiota, and serum samples for detection of trimethylamine N-oxide(TMAO), lipopolysaccharide(LPS), and inflammatory factors like interleukin-1(IL-1), IL-2, IL-10, and monocyte chemoattractantprotein-1(MCP-1). The main compositions of gut microbiota were Bacteroidetes, Proteobacteria and Firmicutes at the phylum level, and Oscillospira, Allobaculum, Bacteroides, Ruminococcus, SMB53, Prevotella, Coprococcus, Sutterella and Blautia at the genus level with relatively higher abundance in all SD rats. The relative abundance of Enterococcus, Corynebacterium, Aerococcus, Facklamia(opportunistic pathogens in most case) increased, and that of Allobaculum, Bifidobacterium, Eubacterium, Anaerotruncus(beneficial bacteria) decreased in T2DED group as compared with that at 2nd week after establishment of T2D model(T2D2 group). The serum contents of TMAO, LPS, IL-1, IL-2, IL-10 and MCP-1 in T2DED group were significantly higher than those in control group. The gut microbiota of T2DED rats was inhibited. The gut microbiota of T2DED rats had changed, as the relative abundance of beneficial bacterium was decreased while that of opportunistic pathogens was increased. The variations of gut microbiota might lead to inflammation and prompt the emergence of erectile dysfunction in the rats with T2D. TMAO might play an important role in the formation of T2DED.

基于文献计量学方法分析中国2005—2009年亚洲玉米螟的研究动态(英文)

利用文献计量学方法分析2005—2009年间中文核心期刊上发表的亚洲玉米螟研究论文的数量、发表时间、研究主题、期刊分布、作者单位和资助课题等,探讨我国亚洲玉米螟近年的研究动态。结果表明,玉米螟研究是我国近年的热点课题。2005—2009年间在中文核心期刊上发表相关论文约62篇,其中2007年发表的论文篇数最多(21篇),其次为2009年(17篇);研究关注亚洲玉米螟的生物化学与分子遗传学(32.26%)、防治理论与技术(29.03%)、种群与繁殖行为(16.13%);发表相关论文最多的单位为中国农业科学院(15篇)、沈阳农业大学(11篇)和扬州大学(8篇);论文主要发表在《昆虫学报》(10篇)、《植物保护学报》(9篇)和《玉米科学》(6篇);标注项目资助的论文为56篇,而标注国家自然科学资金资助的论文为25篇。分析结果有助于多角度为读者和作者展示我国亚洲玉米螟研究的现状,为今后深入系统开展玉米螟研究提供参考。

Purification and molecular weight distribution of a key exopolysaccharide component of Bacillus megaterium TF10

Extracellular polymeric substances(EPS) are organic metabolic compounds excreted by microorganisms. They largely impact microbial aggregate structures and functions.Extracellular polysaccharides(EP) in EPS are responsible for the formation of microbial aggregates. In this work, we successfully separated and characterized EP from EPS of the bacterium Bacillus megaterium TF10. Extraction of EP from EPS was optimized using Sevag's reagent. Chemical characteristics, functional groups, and molecular weight(MW) distribution of EP were compared with the harvested EPS and soluble microbial products(SMP). We found that the polymers of lower MW and free proteins were successfully removed by Sevag's reagent. The higher MW components of EPS were predominantly polysaccharides,while the polymers of lower MW tended to secrete to the supernatant and were described as SMP. A part of the proteins in the EP was polysaccharide-bonded. Our results can be further used in elucidating the complex flocculation mechanisms in which EP play a major role.

Characterization of the P450 Monooxygenase LobP1 as C-32 Hydroxylase in Lobophorin Biosynthesis

Lobophorins(LOBs)belong to a large family of spirotetronate antibiotics with antibacterial and antitumor activities.In this study,we demonstrated the function of LobP1,a P450 monooxygenase encoded in the LOB biosynthetic gene cluster,by in vivo deletion and in vitro biochemical assays.The disruption of lobP1 led to the isolation of three new LOBs derivatives(3-5)and three known ones(6-8)without the hydroxyl group at C-32.LobP1 was shown to have relatively broad substrate scope.Determining the kinetic parameters of LobP1 towards different substrates revealed that LobP1 preferred substrate with a nitrosugar.The new LOBs 3-5 displayed significant antibacterial activities against Bacillus subtilis and Micrococcus luteus with MIC values of 0.125 to 1μg·mL^(-1),and the major product LOB E(6)from the∆lobP1 mutant showed moderate cytotoxic activities against several cancer cell lines.

Dynamic changes in butyrate levels regulate satellite cell homeostasis by preventing spontaneous activation during aging

The gut microbiota plays a pivotal role in systemic metabolic processes and in particular functions,such as developing and preserving the skeletal muscle system.However,the interplay between gut microbiota/metabolites and the regulation of satellite cell(SC)homeostasis,particularly during aging,remains elusive.We propose that gut microbiota and its metabolites modulate SC physiology and homeostasis throughout skeletal muscle development,regeneration,and aging process.Our investigation reveals that microbial dysbiosis manipulated by either antibiotic treatment or fecal microbiota transplantation from aged to adult mice,leads to the activation of SCs or a significant reduction in the total number.Furthermore,employing multi-omics(e.g.,RNA-seq,16S r RNA gene sequencing,and metabolomics)and bioinformatic analysis,we demonstrate that the reduced butyrate levels,alongside the gut microbial dysbiosis,could be the primary factor contributing to the reduction in the number of SCs and subsequent impairments during skeletal muscle aging.Meanwhile,butyrate supplementation can mitigate the antibiotics-induced SC activation irrespective of gut microbiota,potentially by inhibiting the proliferation and differentiation of SCs/myoblasts.The butyrate effect is likely facilitated through the monocarboxylate transporter 1(Mct1),a lactate transporter enriched on membranes of SCs and myoblasts.As a result,butyrate could serve as an alternative strategy to enhance SC homeostasis and function during skeletal muscle aging.Our findings shed light on the potential application of microbial metabolites in maintaining SC homeostasis and preventing skeletal muscle aging.

Anaerobic oxidation of methane coupled to sulfate reduction: Consortium characteristics and application in co-removal of H_2S and methane

Anaerobic sludge from a sewage treatment plant was used to acclimatize microbial colonies capable of anaerobic oxidation of methane(AOM) coupled to sulfate reduction. Clone libraries and fluorescence in situ hybridization were used to investigate the microbial population.Sulfate-reducing bacteria(SRB)(e.g., Desulfotomaculum arcticum and Desulfobulbus propionicus)and anaerobic methanotrophic archaea(ANME)(e.g., Methanosaeta sp. and Methanolinea sp.)coexisted in the enrichment. The archaeal and bacterial cells were randomly or evenly distributed throughout the consortia. Accompanied by sulfate reduction, methane was oxidized anaerobically by the consortia of methane-oxidizing archaea and SRB. Moreover, CH_4 and SO_4^(2-) were consumed by methanotrophs and sulfate reducers with CO_2 and H_2S as products. The H_3CSH produced by methanotrophy was an intermediate product during the process. The methanotrophic enrichment was inoculated in a down-flow biofilter for the treatment of methane and H_2S from a landfill site. On average, 93.33% of H_2S and 10.71% of methane was successfully reduced in the biofilter. This study tries to provide effective method for the synergistic treatment of waste gas containing sulfur compounds and CH_4.

Synthesis, characterization and antimicrobial activity of zinc and cerium co-doped α-zirconium phosphate

A series of zinc ions or/and cerium ions co-doped α-zirconium phosphate (Zn-Ce@ZrPs) were prepared. The novel Zn-Ce@ZrPs were characterized and the antibacterial activity on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus were tested. The results showed that zinc ions (Zn2+) or/and cerium ions (Ce3+) were combined with ZrP, and the Ce3+ was adsorbed on the surface of ZrP through hydrogen bonds, while Zn2+ intercalated into the interlayer of ZrP. Zn-Ce@ZrPs showed excellent synergistic antibacterial activity. When Zn2+/Ce3+ atomic ratio was 0.6, the Zn-Ce@ZrP3 showed the highest synergistic antibacterial efficiency, suggesting great potential application as antibacterial agents in microbial control.

Effect of Ce^(3+) on membrane permeability of Escherichia coli cell

This study aimed to delineate the antibacterial mechanism of rare-earth ion Ce 3+ to the target organism Escherichia coli cell, and the most important purpose was to identify its biological effect of increasing the E. coli cell membrane permeability. The antibacterial activities of Ce 3+ to E. coli cells were tested, and then the permeability of outer membrane (OM) and inner membrane (IM) were studied by N-phenyl-1-naphthylamine (NPN) and o-nitrophenyl-β-D-galactopyranoside (ONPG) methods separately. Through these experiments we concluded that the E. coli cells grown to log phage were more sensitive to Ce 3+ than the ones not at this stage; the structure of membrane was destroyed and the permeability of both OM and IM was obviously increased by Ce 3+ ; there should be certain interactions between Ce 3+ and some proteins inside the cell, which impeded the physiological activities of bacteria.

Biosorption and biotransformation of crystal violet by Aeromonas hydrophila DN322p

DN322p， Aeromonas hydrophila DN322 的一个后代，有能力同时在废水吸附并且将脱色三苯代甲烷染料。作为普通三苯代甲烷染料，水晶紫(CV ) 被选择测试 DN322p 的 decolorization 特征。在 0.5 h 以内，紧张 DN322p 吸附了大量 CV，生产一个深有颜色的房间小团和无色上层清液。随着时间的过去减轻的房间小团和上层清液的颜色。上层清液和房间小团的 dichloromethane 摘录两个都显示出明显的 CV 和 leuco CV (LCV ) 特征分别地，吸收度在 590 nm 和 260 nm 达到顶点在紫外力光谱分析。这发现显示 DN322p 房间能吸附二染料。一 99%(w/w ) decolorization 率与在 30id 状态反应性质摇晃在 2.5 h 以内被完成包括 Kirkendall 虚空的形成，也被执行在以后回流与 Sn-3.0 Ag-0.5 Cu 焊接焊接球。在在 125osphate 的固态的变老实验，更多是 effectiv 吗？？

Lithocarpinols A and B,a pair of diastereomeric antineoplastic tenellone derivatives from the deep-sea derived fungus Phomopsis lithocarpus FS508

Lithocarpinols A(1) and B(2), a pair of tenellone diastereoisomers with novel fused skeleton were isolated from the deep-sea derived fungus Phomopsis lithocarpus FS508. Their structures were elucidated by comprehensive spectroscopic analyses, X-ray diffraction and quantum molecular calculation. Their plausible biogenetic pathway featured an intriguing carbonyl-ene cyclization. Lithocarpinol A exhibited moderate inhibitory effect against HepG-2 and A549 tumor cell lines with IC_(50) values of 9.4 μmol/L and10.9 μmol/L,respectively.

Phylogeny of Entoloma s.l. subgenus Pouzarella, with descriptions of five new species from China

Taxonomy and phylogeny of Entoloma s.l.subgenus Pouzarella in China are carried out based on morphological characters and molecular data.Seven species in subg.Pouzarella are recognized in China and a key to these species is provided.Five species,Entoloma changchunense,E.crassicystidiatum,E.furfuraceum,E.subaraneosum and E.tenuissimum,are described as new;a new Chinese record E.dysthaloides is also presented in this paper.Among them,E.crassicystidiatum was discovered from southern China,and the others were found in northeastern China.Molecular phylogeny of subg.Pouzarella were conducted based on nLSU,mtSSU and RPB2,and the phylogenetic positions of the five new species are discussed.The placement of subg.Pouzarella in Entoloma s.l.clade is well resolved in all analyses presented here,and the monophyly of subg.Pouzarella is strongly supported by the morphological and molecular phylogeny.The infrageneric classifications in this group were not well recovered in the present study;and further studies based on more samples and gene loci are needed to a better understanding of the infrageneric taxa in subg.Pouzarella in the future.