Identification and characterization of FpRco1 in regulating vegetative growth and pathogenicity based on T-DNA insertion in Fusarium pseudograminearum

Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot(FCR)in wheat and poses a significant threat to wheat production in terms of grain yield and quality.However,the mechanism by which F.pseudograminearum infects wheat remains unclear.In this study,we aimed to elucidate these mechanisms by constructing a T-DNA insertion mutant library for the highly virulent strain WZ-8A of F.pseudograminearum.By screening this mutant library,we identified nine independent mutants that displayed impaired pathogenesis in barley leaves.Among these mutants,one possessed a disruption in the gene FpRCO1 that is an ortholog of Saccharomyces cerevisiae RCO1,encoding essential component of the Rpd3S histone deacetylase complex in F.pseudograminearum.To further investigate the role of FpRCO1 in F.pseudograminearum,we employed a split-marker approach to knock out FpRCO1 in F.pseudograminearum WZ-8A.FpRCO1 deletion mutants exhibit reduced vegetative growth,conidium production,and virulence in wheat coleoptiles and barley leaves,whereas the complementary strain restores these phenotypes.Moreover,under stress conditions,the FpRCO1 deletion mutants exhibited increased sensitivity to NaCl,sorbitol,and SDS,but possessed reduced sensitivity to H_(2)O_(2)compared to these characteristics in the wild-type strain.RNA-seq analysis revealed that deletion of FpRCO1 affected gene expression(particularly the downregulation of TRI gene expression),thus resulting in significantly reduced deoxynivalenol(DON)production.In summary,our findings highlight the pivotal role of FpRCO1 in regulating vegetative growth and development,asexual reproduction,DON production,and pathogenicity of F.pseudograminearum.This study provides valuable insights into the molecular mechanisms underlying F.pseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.

Advancing healthcare through laboratory on a chip technology:Transforming microorganism identification and diagnostics

In a recent case report in the World Journal of Clinical Cases,emphasized the crucial role of rapidly and accurately identifying pathogens to optimize patient treatment outcomes.Laboratory-on-a-chip(LOC)technology has emerged as a transformative tool in health care,offering rapid,sensitive,and specific identification of microorganisms.This editorial provides a comprehensive overview of LOC technology,highlighting its principles,advantages,applications,challenges,and future directions.Success studies from the field have demonstrated the practical benefits of LOC devices in clinical diagnostics,epidemiology,and food safety.Comparative studies have underscored the superiority of LOC technology over traditional methods,showcasing improvements in speed,accuracy,and portability.The future integration of LOC with biosensors,artificial intelligence,and data analytics promises further innovation and expansion.This call to action emphasizes the importance of continued research,investment,and adoption to realize the full potential of LOC technology in improving healthcare outcomes worldwide.

Preliminary Identification of Loquat Leaf Mould Pathogenic Fungus in Mengzi City of Yunnan Province

[Objective] The study aimed to investigate the Ioquat leaf mould disease in Mengzi City of Yunnan Province and lay the foundation for determination of effective prevention and control methods.[Method] Loquat leaf mould pathogenic fungus was isolated by tissue separation method and inoculated with conidial suspension.The pathogenicity of Ioquat leaf mould pathogen was verified by Koch＇s postulate.Under a microscope,mycelial morphology and conidial fructification were observed,spore sizes were measured,and Ioquat leaf mould pathogen was identified according to the morphological characteristics.[Result] Velvet-like,olive green fungal colonies were generated on PDA medium.Conidiophores erect,apex curved,dark brown,smooth,with obvious spore marks and no diaphragm,（33.0-152.8） μm×（2.6-4.0）μm.Cladosporium was brown or pale olive with spore marks,monocelled or with one diaphragm,（7.1-19.0） μm × （1.9-5.9） μm.Conidia concatenate （2-4）,oval or spherical,with no spore mark,light olive,monocelled,smooth,（2.1-9.4） μm × （1.2-2.6） μm.[Conclusion] Loquat leaf mold disease began to occur in the germination period of spring shoots and summer shoots and became serious in the germination period of autumn shoots.Sooty mold-like layer grew on both front and back surfaces and densely covered the whole leaves,thus seriously affecting the photosynthesis of plants.The pathogen was preliminarily identified as Cladosporium eriobotryae Pass.＆ Beltrani.

Isolation,Identification and in vitro Antimicrobial Susceptibility of Pathogenic Aeromonas veronii from Soft-shelled Turtles

In order to effectively control diseases caused by Aeromonas veronii,pathogenic bacteria were isolated and incubated from infected soft-shelled turtles with traditional bacterial isolation method. Four strains of pathogenic bacteria were isolated and identified with traditional biochemical identification method and modern molecular biological identification techniques. According to the results, four strains of pathogenic bacteria were identified as A. veronii biovar sobria. Drug sensitivity test and in vitro antimicrobial test against Bacillus amyloliquefaciens were performed with agar diffusion method. The results showed that B. amyloliquefaciens and several antibiotics such as ofloxacin and gentamicin exerted strong antimicrobial effects on four isolates. B. amyloliquefaciens could be used for the prevention and control of diseases caused by A. veronii in aquaculture.

Studies on the Isolation, Identification and In Vitro Growth Rates of the Three Pathogenic Fungi from Panax notoginseng Cultivated in Wenshan Eparchy

Objective] The aim of this study was to simultaneously isolate and identify the main pathogenic fungi of the root rot, black spot and round spot from the Panax notoginseng plants cultivated in Wenshan Eparchy of Yunnan Province of China. [Method] The pathogenic fungi were isolated and purified by using potato dextrose agar （PDA） medium. The morphological identification was accomplished first according to the colony forms of the fungi when cultivated in vitro, then accord-ing to the symptom characteristics and colony forms of the re-isolated fungi in the reverse inoculation experiments. The molecular identification was performed accord-ing to the amplification and alignment of the internal transcribed space （ITS） se-quences of the fungi. The increases of the diameters and thickness of the colonies of the fungi cultivated in vitro were employed to indicate the growth rates of the fungi. [Results] The consistency of the colony forms and symptom characteristics and the 96%-99% similarities revealed in the ITS sequence alignments al proved that the main pathogenic fungi of the root rot, black spot and round spot of the P. notoginseng plants raised in Wenshan were Cylindrocarpon didymium, Alternaria panax and Mycocentrospora acerina, respectively. When cultivated in vitro in the same temperature, humidity and il umination, the increases of the colony diameters and thickness of C. didymium were the highest, fol owed by those of A. panax, then those of M. acerina. During different cultivation periods, the differences of the colony diameters and thickness of the three fungi al reached extremely significant level. However, at the same cultivation time, the differences of the diameters and thickness among the three fungi only reached significant level. [Conclusion] The main pathogenic fungi which result in the root rot, black spot and round spot of the P. notoginseng in Wenshan are C. didymium, A. panax and M. acerina, respec-tively. When these three diseases break out at the same time, the root rot wil spread fastest, fol owed orderly by the black spot and the round spot.

Isolation and Identification of Pandora neoaphidis and Its Pathogenicity against Turnip Aphid(Lipaphis erysimi)

[Objective] The study was to isolate and identify the pathogen of Pandora neoaphidis and to determine its pathogenicity against turnip aphid （Lipaphis erysimi）.[Method] Based on the morphological characteristics,the species of pathogenic fungus was identified.Spore shower method was used to carry out bioassay on the pathogen against turnip aphid.[Result] Primary conidia were ovoid,bitunicate and uninucleate,（24.7±1.4）μm×（10.7±0.9）μm,L/D=2.3±0.2.Secondary conidia had the similar shape with the primary ones,（18.6±2.1）μm×（13.3±1.3）μm,L/D=1.4±0.2.Hyphal body was like mycelium with the diameter of （10.6±0.8）μm.Conidiophores had palmate branch with the diameter of （10.0±0.9）μm.Pseudocystidia was not branched,which had rough base with the diameter of （19.2±1.7）μm,and gradually became more angular towards the apex with the diameter of （8.0±0.9）μm at tips.Rhizoid was like monohyphal shape with the diameter of （21.0±3.0）μm at base,the terminal apex had regular discoid holdfast.No resting spores were observed.The lethal dose of the pathogen against turnip aphid was 18.2/mm2.[Conclusion] The entomopathogenic fungus against turnip aphid was identified to be Pandora neoaphidis,and the pathogen was confirmed to have strong pathogenicity against turnip aphid.

Isolation, Identification and Pathogenicity Analysis of Streptococcus suis Type 2

[Objectives]This study aimed to investigate the pathogenicity,growth characteristics and drug resistance of Streptococcus suis type 2.[Methods]Bacterial isolation and identification,biochemical experiments,determination of growth curve and correlation curve between OD 600 values and viable counts,drug susceptibility tests,pathogenicity analysis,and histopathological observations were carried out.[Results]The Streptococcus strain isolated from infected pigs was identified as Streptococcus suis type 2,which was named TA01 strain.TA01 strain reached the growth peak at 6-8 h post-incubation,and viable counts gradually declined after 8 h of incubation.The correlation equation between OD 600 values and viable counts is y=24.659 x-1.076 1,R^2=0.996 7.TA01 strain was sensitive to penicillin,erythromycin,florfenicol and oxacillin,and resistant to ciprofloxacin,polymyxin B and clindamycin.According to the results of pathogenicity analysis,all the mice in 3.6×10^9 cfu/mouse group died within 48,and these dead mice exhibited acute pyaemia septica.Based on the Reed-Muench formula,it was calculated that LD 50 of TA01 strain was 1.137×10^8 cfu/mouse.Pathological examination showed obvious blue-stained bacteria clusters,accompanied by neutrophil infiltration.[Conclusions]TA01 strain was a virulent strain of Streptococcus suis type 2.Compared with Streptococcus strains which were isolated and reported in China,TA01 strain exhibited strong virulence and rapid proliferation.

Pathogenicity Assay and Molecular Identification of Fungi and Bacteria Associated with Diseases of Tomato in Malaysia

This study was conducted in order to determine the fungi and bacteria associated with tomato plants at Cameron Highlands Malaysia. The fungi which have been isolated and detected from tomato plants were: Fusarium oxysporum, F. solani, F. acuminatum, Rhizoctonia solani, Colletotrichum boninense, C. acutatum and Phoma destructiva. The bacteria which have been isolated and detected from tomato plants were: Ralstonia solanacearum, Xanthomonas vesicatoria, X. gardneri and Pseudomonas syringae. While the most pathogenic fungi were C. boninense, P. destructive and F. oxysporum with the disease incidence (89.6%, 86.6%, 85.6%) respectively, the most pathogenic bacteria were X. vesicatoria and R. solanacearum with the disease incidence (96.6% and 87.6%) respectively.

Functional identification of phenazine biosynthesis genes in plant pathogenic bacteria Pseudomonas syringae pv. tomato and Xanthomonas oryzae pv. oryzae

Phenazines are secondary metabolites with broad spectrum antibiotic activity and thus show high potential in biological control of pathogens. In this study, we identified phenazine biosynthesis （phz） genes in two genome-completed plant pathogenic bacteria Pseudomonas syringae pv. tomato （Pst） DC3000 and Xanthomonas oryzae pv. oryzae （Xoo） PXO99A. Unlike the phz genes in typical phenazine-producing pseudomonads, phz homologs in Pst DC3000 and Xoo PXO99A consisted of phzC/D/E/F/G and phzC/E1/E2/F/G, respectively, and the both were not organized into an operon. Detection experiments demonstrated that phenazine-l-carboxylic acid （PCA） of Pst DC3000 accumulated to 13.4 IJg L-1, while that of Xoo PXO99A was almost undetectable. Moreover, Pst DC3000 was resistant to 1 mg mL-1 PCA, while Xoo PXO99A was sensitive to 50 IJg mL ~ PCA. Furthermore, mutation of phzF blocked the PCA production and significantly reduced the pathogenicity of Pst DC3000 in tomato, while the complementary strains restored these phenotypes. These results revealed that Pst DC3000 produces low level of and is resistant to phenazines and thus is unable to be biologically controlled by phenazines. Additionally, phz-mediated PCA production is required for full pathogenicity of Pst DC3000. To our knowledge, this is the first report of PCA production and its function in pathogenicity of a plant pathogenic P. syringae strain.

Rapid Identification of Legionella Pathogenicity by Surface-Enhanced Raman Spectroscopy

Objective To establish Surface-enhanced Raman Spectroscopy（SERS） can be used as a rapid and reliable method to distinguish virulent strain and mild strain of L. pneumophila. Methods We isolated and characterized of bacterial strains from ATCC and water samples strains, while we analyzed data from SERS technology using gold nanoparticles as a base and cell infections were employed to rapidly detect L. pneumophila strains. Origin 8.0 was used to collect Raman spectra, smooth and homogenize data, and to contrast spectra. Principal component analysis（PCA） was conducted to discriminate differences between groups using the multivariate analysis package Py Chem 3.0.5. Results Our results indicated that the peaks of high virulence strains reached ≥4000. This criterion was verified by subsequent cell experiments. In addition, we also conducted SERS rapid identification on the virulence of several collected clinical strains and obtained accurate results. Conclusion The present study indicates that the established SERS protocol can be used as a rapid and reliable method to distinguish virulent and mildly virulent strains of L. pneumophila, which can be further used in clinical samples.

Isolation,Identification and Pathogenic Characteristics of Duck Hepatitis Virus Isolates

[ Objective] The aim of this study was to determine and analyze the pathogen and the reasons of duck viral hepatitis which is prevalent recently and difficult to control. [Method] Viruses were isolated from livers and spleens of ducks with typical clinical symptoms in Linyi, Weifang, Binzhou and other regions of Shandong Province. The pathogenic characteristics were observed by inoculation in chicken or duck embryo, RT- PCR, serological test, and duck regression. [ Result] Four duck hepatitis virus （DHV） strains were isolated, and the 5th passage allantoic fluid contained 10^3.41 -10^5.20 ELD50/mI. The serum cross protection rate was 20% -80% between the DHV stains and DHV type I. The mortalities of 4- day-old healthy ducks challenged by these four stains were 50% -100%. All challenged ducks had typical lesions of duck viral hepatitis, and the death peak appeared after 24-48 h. [Conclusion] The virulence of different DHV isolates has regional difference.

Identification and functional analyses of a novel FOXL2 pathogenic variant causing blepharophimosis, ptosis, and epicanthus inversus syndrome

AIM: To discover the molecular pathogenic basis of the blepharophimosis, ptosis, and epicanthus inversus syndrome(BPES), and to predict the clinical subtype according to in vitro experiments, which is significant to the prognosis.METHODS: A 3-year-old sporadic female patient with typical clinical manifestations of BPES was enrolled. The coding region of forkhead box L2(FOXL2) gene was sequenced, and the functional assays were performed in vitro by Western blotting, subcellular localization experiment, luciferase reporter assay, and quantitative realtime polymerase chain reaction.RESULTS: A novel FOXL2 point pathogenic variant(c.274G>T) was detected, resulting in a truncated protein(p.E92*). Functional studies demonstrated that the FOXL2 pathogenic variant induced the subcellular mislocalization and the abnormal transcriptional activity on promoters of the steroidogenic acute regulatory protein(StAR or STARD1) gene and the odd-skipped related 2 transcription factor(OSR2) gene.CONCLUSION: A novel pathogenic variant is identified to expand the spectrum of the known FOXL2 mutations. The in vitro experiments provide reference data and more insights to the molecular pathogenesis of BPES. The predicted high risk of ovarian insufficiency makes it significant for the patient enrolled to have further follow-up and therapy concerning female endocrinology.

Isolation and Identification of a Pathogenic E.coli Strain Causing Diarrhea in Foxes

[Objectives] The study aimed to identify the pathogenic E. coli strain that caused diarrhea in foxes and to analyze its drug sensitivity.[Methods] A pathogenic E. coli strain was isolated from dead foxes with diarrhea. By conventional bacterial isolation and culture, morphological observation, pathogenicity test and K-B disc method, the isolated strain was identified as pathogenic E. coli .[Results] The isolated pathogen was highly sensitive to ceftriaxone, cefotaxime, ciprofloxacin and lincomycin, moderately sensitive to enrofloxacin, neomycin, gentamycin, spectinomycin, florfenicol, amikacin and polymyxin, and resistant to ampicillin, amoxicillin and doxycycline.[Conclusions] This study provided reference for the prevention and control of diarrheal diseases in foxes in Qinhuangdao region.

Pathogenic Identification of Leaf Tip Blight of Red-fleshed Kiwifruit in Qiandongnan Prefecture

[Objective]The paper was to investigate and identify new diseases in kiwifruit producing areas in Qiandongnan Prefecture to reduce the harm of diseases and ensure the quality of red-fleshed kiwifruit products.[Method]The pathogenic fungus was isolated from diseased leaves of redfleshed kiwifruit by tissue separation method,and DNA was sequenced by ribosomal rDNA-ITS(internal transcribed spacer)sequencing.Molecular evolutionary trees were built using MEGA4.0 software,and the pathogenic fungus was classified and identified combined with morphological obser-vation.[Result]Leaf tip blight was a new disease caused by Epicoccum sorghinum.It caused serious damage on red-fleshed kiwifruit.[Conclusion]The study supplements diseases of red-fleshed kiwifruit,and provides support for disease prevention and control in late stage.

Isolation, Identification and Pathogenicity Evaluation of Pathogens of Soybean Bacterial Diseases

In order to research soybean bacterial diseases in Heilongiiang Province, a bacterial strain was isolated from diseased leaves of Suinong 14 collected from Harbin Yanjiagang farm. The pathogen had been carried out antibiotic resistance identification, molecular identification, re-inoculation identification and pathogenicity identification respectively. By Gram staining, pathogen inoculation test, 16S rDNA molecular identification, the pathogen was determined as Pseudomonas syringae and named Psgneau 2. The isolated strain presented resistance to 25, 50 and 100 μg/mL ampicillin and carbenicillin, but susceptible to spectinomycin, rifampicin, kanamycin, gentamicin, tetracycline and chloramphenicol. Ten soybean varieties were inoculated with Psgneau 2, in which Suinong 14 was more susceptible than other varieties and Hefeng 35 presented higher resistance to Psgneau 2.

Isolation and Identification of a High-pathogenicity Strepto- coccus suis Serotype 7 Strain

In order to diagnose the diseased pigs in a certain large pig farm in Binzhou City, Shandong Province, the dead piglets with joint swelling were subjected to necroscopy, and the pathogenic bacterium was isolated and identified. One Gram-positive Streptococcus was isolated. The strain was subjected to characteristic culture, microscopic examination and molecular biological identification, and resistance detection, animal regression experiment and mouse pathogenicity test were carried out. The results showed that the isolate was identified to be Streptococcus suis serotype 7, which was resistant to multiple drugs; and the pathogenicity test showed that the strain had high pathogenicity to pigs, resulting in neurosis on partial pigs, and the strain had no pathogenicity to Kunming and BALB/c mice but certain pathogenicity to CD1 mice.

Isolation,Identification and Drug Tolerance Analysis of Pathogenic Staphylococcus aureus from Broilers

White feather broilers in some broiler farm in Chengde area developed arthritis and died acutely. In order to identify the pathogen inducing the disease and death of the white feather broilers, samples such as livers and joint pus were collected from dead broilers under sterile condition, and one pathogenic strain was isolated. The isolate was identified to be Staphylococcus aureus through isolated culture, morphologic observation, inspection of biochemical property and animal test. The drug sensitivity test showed that the isolate was sensitive to ceftazidime, enrofloxacin, ceftriaxone, lincomycin and amikacin, but resistant to other drugs to different degrees.

Isolation and Identification of Pathogenic Bacteria Causing Bovine Subclinical Mastitis in Eastern Hebei Province

In this study, the milk samples of 1 021 cows in eight dairy farms in Eastern Hebei Province were collected and detected with LMT reagent and somatic cell count for subclinical mastitis. Pathogenic bacteria in subclinical mastitis positive milk samples were isolated and identified.The results showed that 60.63%（619/1 021） of the sampled cows were diagnosed with subclinical mastitis, and mixed infections accounted for 88.21%（546/619） of the cases. In addition, 82 strains of 14 species were isolated from the subclinical mastitis positive milk samples, including 36 strains of Staphylococcus（43.90%）, 33 strains of Streptococcus（40.24%）, 8 strains of Enterobacteriaceae（9.76%） and 5 strains of Corynebacterium（6.10%）, respectively. The results proved that Staphylococcus aureus and Streptococcus agalactiae are the main pathogenic bacteria causing bovine subclinical mastitis in Eastern Hebei Province.

Isolation and Identification of Pathogenic Fungi Causing Decayin Luotian Chestnut during Late Storage Period and Research on Pathogenicity

In order to explore moldy mechanism of chestnut from Luotian County in storage process,the strains of pathogenic fungi were isolated from chestnuts after storage at room temperature for 70d.Six genera of fungi were found in chestnut through experimental identification,which were Ozoniumsp.,Fusarium sp.,Aspergillus sp.,Penicilliumsp.,Rhiopus sp.and Stachybotrys sp.,respectively.The re-inoculation tests had been conducted on pathogenic fungi whose isolating rate was greater than 10%.The result showed that the rest genera of fungi generally had no pathogenicity except Penicilliumsp.could infect non-injured chestnut with a lower moldy rate and lighter symptoms;but the moldy rate of strains was above 60% in injured inoculation and they showed heavy symptoms,among which the moldy rate of Ozoniumsp.and Aspergillus sp.were higher than 80%.The experimental results showed that injured chestnut were more likely to decay.Ozoniumsp.and Aspergillus sp.were important pathogenic fungi causing decay during storage process of chestnut.

Identification of Plant-Pathogenic Fungi Using Fourier Transform Infrared Spectroscopy Combined with Chemometric Analyses

Identification of plant-pathogenic fungi is time-consuming due to cultivation and microscopic examination and can be influenced by the interpretation of the micro-morphological characters observed.The present investigation aimed to create a simple but sophisticated method for the identification of plant-pathogenic fungi by Fourier transform infrared(FTIR)spectroscopy.In this study,FTIR-attenuated total reflectance(ATR)spectroscopy was used in combination with chemometric analysis for identification of important pathogenic fungi of horticultural plants.Mixtures of mycelia and spores from 27fungal strains belonging to nine different families were collected from liquid PD or solid PDA media cultures and subjected to FTIR-ATR spectroscopy measurements.The FTIR-ATR spectra ranging from 4 000to 400cm-1 were obtained.To classify the FTIRATR spectra,cluster analysis was compared with canonical vitiate analysis(CVA)in the spectral regions of3 050~2 800and 1 800~900cm-1.Results showed that the identification accuracies achieved 97.53%and99.18%for the cluster analysis and CVA analysis,respectively,demonstrating the high potential of this technique for fungal strain identification.