Analysis of Gut Microbiota in Patients with Breast Cancer and Benign Breast Lesions

Breast cancer (BC) and benign breast lesions (BBLs) are common diseases in women worldwide. The gut microbiota plays a vital role in regulating breast diseases’ formation, progression, and therapy response. Hence, we explored the structure and function of gut microflora in patients with BC and BBLs. A cohort of 66 subjects was enrolled in the study. Twenty-six subjects had BC, 20 subjects had BBLs, and 20 matched healthy controls. High throughput 16S ribosomal RNA (16S rRNA) gene sequencing technology was used to determine the microbial community structure. Compared with healthy individuals, BC patients had significantly lower alpha diversity indices (Sobs index, p = 0.019; Chao1 index, p = 0.033). Sobs and Chao1 indices were also lower in patients with BBLs than healthy individuals, without statistical significance (p = 0.279, p = 0.314, respectively). Both unweighted and weighted UniFrac analysis showed that beta diversity differed significantly among the three groups (p = 3.376e–14, p < 0.001, respectively). Compared with healthy individuals, the levels of Porphyromonas and Peptoniphilus were higher in BC patients (p = 0.004, p = 0.007, respectively), whereas Escherichia and Lactobacillus were more enriched in the benign breast lesion group (p < 0.001, p = 0.011, respectively). Our study indicates that patients with BC and BBLs may undergo significant changes in intestinal microbiota. These findings can help elucidate the role of intestinal flora in BC and BBLs patients. Open in a separate window     

Breeding of High Daptomycin-Producing Strain by Streptomycin Resistance Superposition

Daptomycin is a cyclolipopeptide antibiotic produced by Streptomyces roseosporus. It is widely used to treat drug-resistant bacterial infections; however, daptomycin yield in wild strains is very low. To improve the daptomycin production by the strain BNCC 342432, a modified method of ribosome engineering with superposition of streptomycin resistance was adopted in this study. The highest-yield mutant strain SR-2620 was obtained by increasing streptomycin resistance of BNCC 342432, and achieved daptomycin production of 38.5 mg/l in shake-flask fermentation, 1.79-fold higher than the parent strain and its heredity stability was stable. The morphological characteristics of the two strains were significantly different, and the 440^th base G of the rpsL gene in the mutant strain was deleted, which resulted in a frameshift mutation. Our results demonstrate that gradually increasing strain resistance to streptomycin was an effective breeding method to improve daptomycin yield in S. roseosporus. Open in a separate window     

A Novel Bioflocculant Produced by Cobetia Marina MCCC1113: Optimization of Fermentation Conditions by Response Surface Methodology and Evaluation of Flocculation Performance When Harvesting Microalgae

A preliminary study was carried out to optimize the culture medium conditions for producing a novel microbial flocculant from the marine bacterial species Cobetia marina. The optimal glucose, yeast extract, and glutamate contents were 30, 10, and 2 g/l, respectively, while the optimal initial pH of the culture medium was determined to be 8. Following response surface optimization, the maximum bioflocculant production level of 1.36 g/l was achieved, which was 43.40% higher than the original culture medium. Within 5 min, a 20.0% (v/v) dosage of the yielded bioflocculant applied to algal cultures resulted in the highest flocculating efficiency of 93.9% with Spirulina platensis. The bioflocculant from C. marina MCCC1113 may have promising application potential for highly productive microalgae collection, according to the findings of this study. Open in a separate window     

Susceptibility of Clostridium Sporogenes Spores to Selected Reference Substances and Disinfectants

Research on the susceptibility of the spores of anaerobic bacteria such as Clostridium sporogenes or Clostridioides difficile is vital for assessing the sporicidal activity of disinfectants. The diverse susceptibility of anaerobic bacteria spores may lead to different disinfection parameters being determined by laboratories that prepare spore suspensions to test sporicidal effectiveness. The tests were performed using the suspension method according to PN-EN 13704:2018-09. In order to assess the susceptibility of the C. sporogenes spores, the criterion established for the C. difficile ribotype 027 spores was used in accordance with PN‑EN 17126:2019-01. The susceptibility of the C. sporogenes spores to glutardialdehyde corresponded to the susceptibility ranges established for the C. difficile ribotype 027 spores. The C. sporogenes spore suspension was susceptible to low concentrations of peracetic acid (0.01%). A disinfectant containing peracetic acid as the active substance showed high sporicidal activity at a low concentration (1%), a short contact time (15 minutes), and a high organic load (3.0 g/l bovine albumin + 3.0 ml/l sheep erythrocytes), as compared to a disinfectant with glutardialdehyde, which was sporicidal at a higher concentration (2.5%), at a longer contact time(60 minutes) and lower organic conditions (3.0 g/l bovine albumin). There is a need to define the minimum susceptibility criteria for the C. sporogenes spores to the reference substances most often found in disinfectants with sporicidal activity. Excessive susceptibility of the C. sporogenes spores to reference substances may result in low-performance parameters of disinfection products with sporicidal activity and lead to ineffective disinfection in practice. Open in a separate window     

Identification and Antibiotic Susceptibility of Eggerthella Lenta in Bloodstream Infections

The identification and antibiotic susceptibility of two clinical isolates of Eggerthella lenta from bloodstream infections were determined. This microorganism is rarely pathogenic, and the findings are presented here to promote the detection and awareness of this infection. The bacteria were obtained from one patient with pressure sores and another with a malignant gastric tumor. Smears were prepared, stained, and examined by microscopy. Single colonies were analyzed by Gram staining, MALDI-TOF MS, and the 16S rRNA gene sequencing. Antibiotic sensitivity was assessed by the agar dilution method. The bacilli were found to be Gram-positive, and the MS results showed 99.8% homology with E. lenta. It was confirmed by gene sequencing. Antibiotic susceptibility tests demonstrated that E. lenta was sensitive to piperacillin-tazobactam, ampicillin-sulbactam, imipenem, meropenem, metronidazole, clindamycin, and vancomycin. This study could increase awareness of this rare infection. Open in a separate window     

Oral Microbiota,a Potential Determinant for the Treatment Efficacy of Gastric Helicobacter Pylori Eradication in Humans

The oral cavity serves as another reservoir for gastric Helicobacter pylori and may contribute to the failure of gastric H. pylori eradication therapy. However, changes to the oral microbial composition after gastric H. pylori eradication therapy has not yet been identified. This study aims to dissect whether the oral microbiota is involved and which bacterium mediates the clinic failure in H. pylori eradication. In the present study, the oral microorganisms from patients who had received the gastric H. pylori eradication treatment were analyzed by a high-throughput 16S rRNA deep sequencing. We found that the β diversity and composition of oral microbiota were remarkably changed in the patients who had experienced successful gastric H. pylori eradication treatment (SE group) compared to the failure group (FE group). Significantly enriched families, including Prevotellaceae, Streptococcaceae, Caulobacteraceae, and Lactobacillaceae, were detected in the SE group. In contrast, the bacterial families, such as Weeksellaceae, Neisseriaceae, Peptostreptococcaceae, Spirochaetaceae, and Veillonellaceae, were abundantly expressed in the FE group. Five operational taxonomic units (OTUs) were positively correlated with DOB values, while two OTUs exhibited negative trends. These different enriched OTUs were extensively involved in the 20 metabolic pathways. These results suggest that a balanced environment in the oral microbiota contributes to H. pylori eradication and metabolic homeostasis in humans. Our data demonstrated that the changes in oral microbiota might contribute to the therapeutic effects of antibiotic therapy. Therefore, a different therapy on the detrimental oral microbiota will increase the therapeutic efficacy of antibiotics on H. pylori infection. Open in a separate window     

Genomic Analysis of an Excellent Wine-Making Strain Oenococcus Oeni SD-2a

Oenococcus oeni is an important microorganism in wine-making-related engineering, and it improves wine quality and stability through malolactic fermentation. Although the genomes of more than 200 O. oeni strains have been sequenced, only a few include completed genome maps. Here, the genome sequence of O. oeni SD-2a, isolated from Shandong, China, has been determined. It is a fully assembled genome sequence of this strain. The complete genome is 1,989,703 bp with a G+C content of 37.8% without a plasmid. The genome includes almost all the essential genes involved in central metabolic pathways and the stress genes reported in other O. oeni strains. Some natural competence-related genes, like comEA, comEC, comFA, comG operon, and comFC, suggest that O. oeni SD-2a may have natural transformation potential. A comparative genomics analysis revealed 730 gene clusters in O. oeni SD-2a homologous to those in four other lactic acid bacteria species (O. oeni PSU-1, O. oeni CRBO-11381, Lactiplantibacillus plantarum UNQLp11, and Pediococcus pentosaceus KCCM40703). A collinearity analysis showed poor collinearity between O. oeni SD-2a andO. oeni PSU-1, indicating great differences in their evolutionary histories. The results provide general knowledge of O. oeni SD-2a and lay the foundation for specific gene function analyses. Open in a separate window     

Prevalence and Drug Resistance Pattern of Mycobacterium Tuberculosis Isolated from Tuberculosis Patients in Basra,Iraq

Drug-resistant Mycobacterium tuberculosis (DR-MTB) is a major health threat to human beings. This study aimed to evaluate the prevalence and drug resistance profile of MTB. Data were collected from 2,296 newly diagnosed, and 246 retreated tuberculosis (TB) patients who attended the Advisory Clinic for Chest Diseases and Respiratory in Basra province from January 2016 to December 2020. Both new diagnostic and retreated TB cases showed that DR-MTB cases were significantly higher at age 15–34 years, pulmonary TB, and urban residents but with no significant difference regarding gender. The drugs resistance was significantly higher among the retreated cases compared with the new diagnostic patients (20.3% vs. 2.4%, p < 0.0001), with the percentage of the resistance to first-line drugs in primary and secondary cases including isoniazid (1% and 17.1%), rifampicin (0.78% and 15.8%), ethambutol (0.56% and 8.5%), streptomycin (1.3% and 9.75%). Notice that the most common drug resistance was against streptomycin with 1.3% in new patients and against isoniazid (17.1%) in retreated patients. The rate of total drug-resistant TB, multi-drug resistant TB, mono-drug resistant TB, and rifampicin-resistant TB among new tuberculosis cases increased in this period from 2.2 to 6.7%, 0.17 to 1.6%, 0.85 to 4%, and 0.17 to 4%, with a percentage change of 204.54, 841.17, 370.58, 22.5%, respectively. The rates of poly drug-resistant TB and ethambutol-resistant-TB dropped in this period by 15.96%, and 0.7%, with a decrease from 1.19 to 1% and from 1 to 0.3%, respectively. Similarly, the increase of drug-resistant TB among secondary cases has also occurred. In conclusion, the temporal trend showed an increase in the rate of drug resistance of M. tuberculosis since 2016, with a predominant multi-drug-resistant TB and isoniazid-resistant TB. Open in a separate window     

Genetic Polymorphisms of Pneumocystis Jirovecii in HIV-Positive and HIV-Negative Patients in Northern China

Pneumocystis jirovecii is an opportunistic fungus that can cause severe and potentially fatal Pneumocystis pneumonia (PCP) in immunodeficient patients. In this study, we investigated the genetic polymorphisms of P. jirovecii at eight different loci, including six nuclear genes (ITS, 26S rRNA, sod, dhps, dhfr and β-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb) in three PCP cases, including two patients with HIV infection and one without HIV infection in Shanxi Province, P.R. China. The gene targets were amplified by PCR followed by sequencing of plasmid clones. The HIV-negative patient showed a coinfection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one showed a coinfection with two genotypes of P. jirovecii at the same two of the six loci as in the HIV-negative patient, while the other showed a single infection at all eight loci sequenced. None of the three drug target genes (dhfr, dhps and cyb) showed mutations known to be potentially associated with drug resistance. This is the first report of genetic polymorphisms of P. jirovecii in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China. Open in a separate window     

Antimicrobial Resistance,Virulence Factor-Encoding Genes,and Biofilm-Forming Ability of Community-Associated Uropathogenic Escherichia Coli in Western Saudi Arabia

To explore the prevalence of multidrug-resistant community-associated uropathogenic Escherichia coli (UPEC) and their virulence factors in Western Saudi Arabia. A total of 1,000 urine samples were examined for the presence of E. coli by selective plating on MacConkey, CLED, and sheep blood agar. Antimicrobial susceptibility patterns were determined using Vitek^® 2 Compact (MIC) and the disc diffusion method with Mueller-Hinton agar. Genes encoding virulence factors (kpsMTII, traT, sat, csgA, vat, and iutA) were detected by PCR. The overall prevalence of UTI-associated E. coli was low, and a higher prevalence was detected in samples of female origin. Many of the isolates exhibited resistance to norfloxacin, and 60% of the isolates showed resistance to ampicillin. No resistance to imipenem, meropenem, or ertapenem was detected. In general, half of the isolates showed multiple resistance patterns. UPEC exhibited a weak ability to form biofilms, where no correlation was observed between multidrug resistance and biofilm-forming ability. All uropathogenic E. coli isolates carried the kpsMTII, iutA, traT, and csgA genes, whereas the low number of the isolates harbored the sat and vat genes. The diversity of virulence factors harbored by community-associated UPEC may render them more virulent and further explain the recurrence/relapse cases among community-associated UITs. To the best of our knowledge, this study constitutes the first exploration of virulence, biofilm-forming ability, and its association with multidrug resistance among UPEC isolates in Saudi Arabia. Further investigations are needed to elucidate the epidemiology of community-associated UPEC in Saudi Arabia. Open in a separate window     

Screening of mcr-1 Among Gram-Negative Bacteria from Different Clinical Samples from ICU Patients in Alexandria,Egypt: One-Year Study

Antimicrobial resistance represents a global dilemma. Our present study aimed to investigate the presence of mcr-1 among different Gram-negative bacteria including Enterobacteriaceae (except intrinsically resistant to colistin) and Pseudomonas aeruginosa. Gram-negative bacterial isolates were collected from different ICUs in several Alexandria hospitals from June 2019 to June 2020. The identification of these Gram-negative isolates was made using the VITEK-2^® system (BioMérieux, France). SYBR Green-based PCR was used to screen for the presence of mcr-1 using a positive control that we amplified and sequenced earlier in our pilot study. All isolates were screened for the presence of mcr-1 regardless of their colistin susceptibility. Isolates that harbored mcr-1 were tested for colistin susceptibility and for the presence of some beta-lactamase genes. Klebsiella pneumoniae isolates harboring mcr-1 were capsule typed using the wzi sequence analysis. Four hundred eighty isolates were included in this study. Only six isolates harbored mcr-1.1. Of these, four were resistant to colistin, while two (K. pneumoniae and P. aeruginosa) were susceptible to colistin. Five of the six isolates were resistant to carbapenems. They harbored bla_OXA-48, and three of them co-harbored bla_NDM-1. K-58 was the most often found among our K. pneumoniae harboring mcr-1.1. To our knowledge, this is the first time to report colistin susceptible P. aeruginosa and K. pneumoniae harboring the mcr-1.1 gene in Egypt. Further studies are needed to investigate the presence of the mcr genes among colistin susceptible isolates to shed more light on its significance as a potential threat. Open in a separate window     

Impact of Primary and Secondary Bile Acids on Clostridioides Difficile Infection

Primary bile acids (BAs), synthesized from cholesterol in the liver, after their secretion with bile into the intestinal lumen, are transformed by gut microbiota to secondary BAs. As natural detergents, BAs play a key role in the digestion and absorption of lipids and liposoluble vitamins. However, they have also been recognized as important signaling molecules involved in numerous metabolic processes. The close bidirectional interactions between BAs and gut microbiota occur since BAs influence microbiota composition, whereas microbiota determines BA metabolism. In particular, it is well established that BAs modulate Clostridioides difficile life cycle in vivo. C. difficile is a cause of common nosocomial infections that have become a growing concern. The aim of this review is to summarize the current knowledge regarding the impact of BAs on the pathogenesis, prevention, and treatment of C. difficile infection. Open in a separate window     

Helicobacter Pylori and Epstein-Barr Virus Co-Infection in Polish Patients with Gastric Cancer – a Pilot Study

The infectious agents may be the etiological factor of up to 15–20% of cancers. In stomach cancer, attention is paid to Helicobacter pylori and Epstein-Barr virus, both of which cause gastritis and can lead to tumor development. In co-infection, the inflammatory process is much more intense. We assessed the seroprevalence towards H. pylori and EBV in 32 patients with diagnosed gastric cancer. H. pylori antibodies were found in 69% patients, and anti-EBV – in all of them. The study confirmed that co-infection of H. pylori and EBV seems to be important in etiopathology of gastric cancer. Open in a separate window     

Monitoring Multidrug-Resistant Acinetobacter Baumannii Infections in The Neurosurgery ICU Using a Real-Time Surveillance System

Multidrug-resistant Acinetobacter baumannii (MDR-AB) infections are becoming increasingly common. The Real-Time Nosocomial Infection (NI) Surveillance System (RT-NISS) was used to monitor MDR-AB NI in intensive care units (ICUs) to prevent NI outbreaks. Therefore, the RT-NISS was used in the current study to monitor MDR-AB infections in a neurosurgery ICU. Clinical interventions, including recommended antibiotics, bacterial distribution in the patient analysis, and bed adjustments, were carried out based on the monitoring results. The RT-NISS was also used to monitor clinical data, implement, and provide training on NI control. The RT-NISS detected a potential cluster of XDR-AB when five patients admitted to the neurosurgery ICU were tested positive for AB between 11 and 17 June 2019. Only two infected cases originated in the hospital, and there was no NI outbreak. The hospital Infection Control Department took appropriate measures to prevent cross-infection; specifically, an epidemiologic investigation and environmental assessment were conducted, and NI prevention and outbreak management training was provided. In summary, the RT-NISS enhanced the timeliness and efficacy of NI control and surveillance in a neurosurgery ICU. Open in a separate window In order to prevent NI outbreaks, the Real-Time Nosocomial Infection (NI) Surveillance System (RT-NISS) was employed to monitor MDR-AB NI in critical care units (ICU). Based on the monitoring data, clinical actions such as required antibiotics, bacterial distribution in the patient analysis, and bed changes were carried out. In a neurosurgery ICU, the RT-NISS improved the timeliness and efficacy of NI control and surveillance.     

A Salt-Tolerant Streptomyces Paradoxus D2-8 from Rhizosphere Soil of Phragmites Communis Augments Soybean Tolerance To Soda Saline-Alkali Stress

Soil salinity and alkalization limit plant growth and agricultural productivity worldwide. The application of salt-tolerant plant growth-promoting rhizobacteria (PGPR) effectively improved plant tolerance to saline-alkali stress. To obtain the beneficial actinomyces resources with salt tolerance, thirteen isolates were isolated from rhizosphere saline and alkaline soil of Phragmites communis. Among these isolates, D2-8 was moderately halophilic to NaCl and showed 120 mmol soda saline-alkali solution tolerance. Moreover, the plant growth-promoting test demonstrated that D2-8 produced siderophore, IAA, 1-aminocyclopropane-1-carboxylate deaminase (ACCD), and organic acids. D2-8 showed 99.4% homology with the type strain Streptomyces paradoxus NBRC 14887^T and shared the same branch, and, therefore, it was designated S. paradoxus D2-8. Its genome was sequenced to gain insight into the mechanism of growth-promoting and saline-alkali tolerance of D2-8. IAA and siderophore biosynthesis pathway, genes encoding ACC deaminase, together with six antibiotics biosynthesis gene clusters with antifungal or antibacterial activity, were identified. The compatible solute ectoine biosynthesis gene cluster, production, and uptake of choline and glycine betaine cluster in the D2-8 genome may contribute to the saline-alkali tolerance of the strain. Furthermore, D2-8 significantly promoted the seedling growth even under soda saline-alkali stress, and seed coating with D2-8 isolate increased by 5.88% of the soybean yield in the field. These results imply its significant potential to improve soybean soda saline-alkali tolerance and promote crop health in alkaline soil. Open in a separate window     

Assessing Social – Ecological Trade-Offs to Advance Ecosystem-Based Fisheries Management

Modern resource management faces trade-offs in the provision of various ecosystem goods and services to humanity. For fisheries management to develop into an ecosystem-based approach, the goal is not only to maximize economic profits, but to consider equally important conservation and social equity goals. We introduce such a triple-bottom line approach to the management of multi-species fisheries using the Baltic Sea as a case study. We apply a coupled ecological-economic optimization model to address the actual fisheries management challenge of trading-off the recovery of collapsed cod stocks versus the health of ecologically important forage fish populations. Management strategies based on profit maximization would rebuild the cod stock to high levels but may cause the risk of stock collapse for forage species with low market value, such as Baltic sprat (Fig. 1A). Economically efficient conservation efforts to protect sprat would be borne almost exclusively by the forage fishery as sprat fishing effort and profits would strongly be reduced. Unless compensation is paid, this would challenge equity between fishing sectors (Fig. 1B). Optimizing equity while respecting sprat biomass precautionary levels would reduce potential profits of the overall Baltic fishery, but may offer an acceptable balance between overall profits, species conservation and social equity (Fig. 1C). Our case study shows a practical example of how an ecosystem-based fisheries management will be able to offer society options to solve common conflicts between different resource uses. Adding equity considerations to the traditional trade-off between economy and ecology will greatly enhance credibility and hence compliance to management decisions, a further footstep towards healthy fish stocks and sustainable fisheries in the world ocean.Open in a separate windowFigure 1Summary of multispecies management options in the Baltic.(A) Profit maximum. (B) Economic optimum while respecting sprat B_PA. (C) Equitable optimum while respecting sprat B_PA. Central numbers indicate total profits (million €/year) as well as an equity measure (in brackets). Area of each pie slice is relative to status quo values 2008-2010 (black circle), with error bars from sensitivity analysis.     

Specific In Situ Visualization of the Pathogenic Endophytic Fungus Aciculosporium take,the Cause of Witches’ Broom in Bamboo

The endophytic fungus Aciculosporium take (Ascomycota; Clavicipitaceae) causes continuous shoot growth in bamboo. The colonized shoot eventually results in witches’ broom formation but maintains normal leaf arrangement and branching pattern. To analyze the mechanism of well-regulated symptom development, the location of the fungal endophytic hyphae in host tissues was visualized. A colorimetric in situ hybridization technique using a species-specific oligonucleotide probe targeting the 18S rRNA of A. take was used. In situ hybridization was performed on tissue sections of diseased shoots with or without external signs of fungal colonization. Specific signals were detected in intercellular spaces of the bamboo tissues. Most signals were detected in the shoot apical meristem and the leaf primordia. In addition, fewer signals were detected in the lateral buds, juvenile leaves, and stems. These results indicate that A. take grows endophytically, particularly in the shoot apical meristem of the host. The location of A. take hyphae suggests that the mechanism of symptom development can be explained by the action of exogenous fungal auxin, which continuously induces primordium initiation within the host.The endophytic fungus Aciculosporium take (Ascomycota; Clavicipitaceae) causes continuous shoot growth, but the host bamboo plant maintains normal leaf arrangement and branching pattern (28, 29). This fungus produces the plant hormone auxin, indole-3-acetic acid (IAA) in culture (28). Hypersynthesis of auxins by pathogenic microorganisms has often been associated with diseases in which hyperplasia is exhibited by the host plant (31). However, the well-regulated morphological changes of the A. take-colonized bamboo cannot be explained by a mere hormonal imbalance. The symptom of A. take-colonized bamboo is characterized by the successive growth of thin phytomers (developmental modules consisting of a node, a stem segment, a leaf with a sheath, and a lateral bud) from a normal shoot apex (Fig. 1a and b). Diseased shoots continue to grow without branching and have extraordinarily small leaves. Sometimes, a diseased shoot develops 30 to 40 leaves, whereas healthy shoots develop only 3 to 5 leaves (25). When the conidiostroma is formed at the apex, the shoots cease to grow, and lateral buds initiate outgrowth (Fig. ​(Fig.1c).1c). This process occurs repeatedly and eventually results in a typical witches’ broom morphology after a few years (Fig. 1d and e). Excluding stroma formation, the fungus presents no external signs of its presence on the diseased shoots. Accurate localization of the growing hyphae would aid in establishing the process of symptom development.Open in a separate windowFIG. 1.Bamboo shoot colonized by the endophytic fungus Aciculosporium take. (a to c) Schematic of witches’ broom symptom development on a bamboo species of Phyllostachys. (a) The normal shoot ceased to grow after three to five leaves developed. (b) An A. take-colonized shoot grew out with extraordinarily small leaves but maintained normal leaf arrangement. The shoot continued to grow with successive thin phytomers. (c) After stromata (arrows) were formed at the shoot apex, the lateral buds began to grow but maintained a normal branching pattern. This sequence repeats. (d to f) Bamboo (P. pubescens) shoot colonized by A. take. (d) Early symptom. Diseased shoots (DS) have leaves that are much smaller than normal leaves (NL). Bar, 1 cm. (e) Developed symptom shows typical witches’ broom appearance. Bar, 1 cm. (f) Ascostroma (AS) is formed on a conidiostroma (CS) at the apex of the diseased shoot. The conidiostroma is surrounded by a sheath. Bar, 1 mm.Many fungal endophyte species have been reported for bamboos (19), and therefore, species-specific detection techniques are needed to observe A. take in host tissues. A number of staining techniques utilizing lectins and other chemical compounds have been used to observe naturally growing hyphae in host tissues (13). However, these approaches cannot be used to distinguish particular species. Therefore, such staining techniques using lectins and other chemical compounds are effective to differentiate fungi possessing particular morphological characteristics, for example, arbuscules, appressoria, or haustoria (33). Unfortunately, fungal endophytes are difficult to identify in host tissues, as discussed previously by Schulz and Boyle (24), partly because the morphologies of endophytic hyphae are usually different from those of cultured hyphae. To differentiate specific fungal species from other fungi, green fluorescent protein or related reporter protein transformants can be used (18, 36). This approach requires the development of genetic modification techniques for individual fungal species. In addition, this method cannot be applied to natural samples because it is necessary to inoculate host plants with fungal transformants. Although immunostaining techniques are also an effective approach (7, 26), they require complicated preparations for generating individual species-specific antibodies.In situ hybridization (ISH) with a species-specific probe is a potentially powerful technique for directly detecting endophytic fungi in natural samples. Species-specific rRNA-targeted oligonucleotide probes have been developed to detect and identify microorganisms in the environment (1, 6). ISH has also been used to detect fungal pathogens in animal tissues (10, 12, 14, 17). Despite these uses, only a few studies have applied this technique for the detection of fungal endophytes or epiphytes (16, 20). This is partly because the detection of fluorescence-labeled probes was hampered by the strong autofluorescence from the plant cell walls (23). However, Pirttilä et al. (20) succeeded in detecting fungal endophytes in meristematic tissues of Scots pine buds by the colorimetric ISH technique. In this study, ISH methodology using a 18S rRNA-targeted oligonucleotide probe was applied to visualize the location of the endophytic fungus A. take in bamboo with witches’ broom disease.