Fungal endophytes of Taxus species and regulatory effect of two strains on taxol synthesis.

BACKGROUND: Taxol, derived from Taxus trees, is a valuable natural resource for the development of anticancer drugs. Endophytic fungi from Taxus trees are a promising alternative source of Taxol. However, the impact of plant-endophytic microbial interaction on the host's Taxol biosynthesis is largely unknown. RESULTS: In the current study, the diversity of endophytic fungi in three different Taxus species was analyzed using Internal Transcribed Spacer sequencing. A total of 271 Operational Taxonomic Units (OTUs) were identified, grouping into 2 phyla, 8 classes, 16 orders, 19 families, and 19 genera. Alpha and beta diversity analysis indicated significant differences in endophytic fungal communities among the various Taxus trees. At the genus level, Alternaria and Davidiella were predominantly found in T. mairei and T. media, respectively. By utilizing a previously published dataset, a Pearson correlation analysis was conducted to predict the taxol biosynthesis-related fungal genera. Following screening, two isolates of Alternaria (L7 and M14) were obtained. Effect of inoculation with Alternaria isolates on the gene expression and metabolite accumulation of T. mairei was determined by transcriptomic and untargeted metabolomic studies. The co-inoculation assay suggests that the two Alternaria isolates may have a negative regulatory effect on taxol biosynthesis by influencing hormone signaling pathways. CONCLUSION: Our findings will serve as a foundation for advancing the production and utilization of Taxus and will also aid in screening endophytic fungi related to taxol production.

The mutual value of histopathology and ITS sequencing in the diagnosis of mucormycosis.

AIMS: Mucormycosis is a fast-progressing disease with a high mortality rate. The most important factor determining survival of patients is early and accurate diagnosis. Although histopathology often recognises invasive mould infections at first, histomorphology alone is insufficient in providing an accurate diagnosis. Unbiased molecular methods to detect and identify fungi are promising, yet their role in complementing routine histopathological workflows has not been studied sufficiently. METHODS AND RESULTS: We performed a retrospective single-centre study examining the clinical value of complementing histopathology with internal transcribed spacer (ITS) sequencing of fungal DNA in the routine diagnosis of mucormycosis. At our academic centre, we identified 14 consecutive mucormycosis cases diagnosed by histopathology and subsequent ITS sequencing. Using histomorphological examination, fungal hyphae could be detected in all cases; however, morphological features were unreliable regarding specifying the taxa. Subsequent ITS sequencing identified a remarkable phylogenetic diversity among Mucorales: the most common species was Rhizopus microsporus (six of 14; 42.9%), followed by Lichtheimia corymbifera (three of 14, 21.4%) and single detections of Rhizopus oryzae, Actinomucor elegans, Mucor circinelloides, Rhizomucor pusillus and Rhizomucor miehei (one of 14; 7.1%, respectively). In one case, we additionally detected Pneumocystis jirovecii in the same lung tissue specimen, suggesting a clinically relevant co-infection. Fungal culture was performed in 10 cases but yielded positive results in only two of 10 (20%), revealing its limited value in the diagnosis of mucormycosis. CONCLUSIONS: Our study demonstrates that a combination of histopathology and ITS sequencing is a practically feasible approach that outperforms fungal culture in detecting Mucorales in tissue-associated infections. Therefore, pathologists might adapt diagnostic workflows accordingly when mucormycosis is suspected.

Molecular characterisation of Histoplasma capsulatum sensu lato from Ethiopian horses reveals two distinct phylogenetic clades.

Epizootic lymphangitis (EL) is a highly prevalent and contagious infectious disease affecting horses in many parts of Ethiopia caused by Histoplasma capsulatum sensu lato ('var. farciminosum'). In this study, 12 suspected isolates of H. capsulatum sensu lato or yeasts unidentified by conventional biochemical tests isolated from Ethiopian horses with EL were characterised by internal transcribed spacer sequencing. Six of the 12 isolates were identified to be members of H. capsulatum sensu lato and the other six were Pichia kudriavzevii (synonym: Candida krusei) (n = 3), Trichosporon asahii (n = 1), Geotrichum silvicola (n = 1) and Moesziomyces aphidis (n = 1), respectively. The six H. capsulatum sensu lato isolates were further characterised by multilocus sequence analysis. Four distinct gene loci (arf [462 bases], H-anti [410 bases], ole1 [338 bases] and tub1 [272 bases]) of these six isolates as well as those of two H. capsulatum sensu lato ('var. farciminosum') reference strains (ATCC 58332 and ATCC 28798) were polymerase chain reaction (PCR)-amplified and sequenced. Phylogenetic analyses of their concatenated nucleotide sequences showed that three of the isolates and the reference strain ATCC 58332 were identical and belonged to the Eurasia clade within Latin American (LAm) A (H. suramericanum), and those of the other three isolates and the reference strain ATCC 28798 were identical and belonged to the Africa clade. At least two distinct phylogenetic clades of H. capsulatum sensu lato were circulating in Ethiopian horses with EL. Advanced molecular technologies and bioinformatics tools are crucial for the accurate identification and typing of pathogens as well as the discovery of novel microorganisms in veterinary microbiology.

Using multilocus sequence analysis with four concatenated housekeeping gene loci, at least two distinct phylogenetic clades, namely Eurasia clade and Africa clade, of Histoplasma capsulatum sensu lato were confirmed to be circulating in Ethiopian horses with epizootic lymphangitis.

Clinical spectrum, phenotypic and molecular characterization, and antifungal susceptibility of an emerging human pathogen, Acrophialophora, from India.

Acrophialophora is implicated in superficial and invasive infections, especially in immunosuppressed individuals. The present study was undertaken to provide clinical, microbiological, phylogenetic, and antifungal susceptibility testing (AFST) profile of Acrophialophora isolated from India. All the isolates identified as Acrophialophora species at the National Culture Collection for Pathogenic Fungi, Chandigarh, India were revived. Phenotypic and molecular characterization was performed, followed by temperature studies, scanning electron microscopy (SEM), and AFST. We also performed systematic review of all the cases of Acrophialophora species reported till date. A total of nine isolates identified as Acrophialophora species were identified by molecular method as A. fusispora (n = 8) and A. levis (n = 1), from brain abscess (n = 4), respiratory tract (n = 3), and corneal scraping (n = 2). All patients but two had predisposing factors/co-morbidities. Acrophialophora was identified as mere colonizer in one. Temperature studies and SEM divulged variation between both species. Sequencing of the internal transcribed spacer ribosomal DNA and beta-tubulin loci could distinguish species, while the LSU ribosomal DNA locus could not. AFST showed the lowest minimum inhibitory concentrations (MICs) for triazoles and the highest for echinocandins. Systematic literature review revealed 16 cases (11 studies), with ocular infections, pulmonary and central nervous system infections, and A. fusispora was common species. All the patients except three responded well. High MICs were noted for fluconazole, micafungin, and caspofungin. This is the first study delineating clinical, phenotypic, and genotypic characteristics of Acrophialophora species from India. The study highlights microscopic differences between both species and emphasizes the role of molecular methods in precise identification. Triazoles appear to be the most effective antifungals for managing patients.

We describe clinical, phenotypic, and genotypic characteristics of Acrophialophora species. This species causes mild infection to fatal infection in immunosuppressed individuals. Triazoles are effective in treating such infections.

Pseudonectria keratitis-emerging pathogenic fungi in the eye.

BACKGROUND: Infectious keratitis, a significant contributor to blindness, with fungal keratitis accounting for nearly half of cases, poses a formidable diagnostic and therapeutic challenge due to its delayed clinical presentation, prolonged culture times, and the limited availability of effective antifungal medications. Furthermore, infections caused by rare fungal strains warrant equal attention in the management of this condition. CASE PRESENTATION: A case of fungal keratitis was presented, where corneal scraping material culture yielded pink colonies. Lactophenol cotton blue staining revealed distinctive spore formation consistent with the Fusarium species. Further analysis using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) identified the causative agent as Fusarium proliferatum. However, definitive diagnosis of Pseudonectria foliicola infection was confirmed through ITS sequencing. The patient's recovery was achieved with a combination therapy of voriconazole eye drops and itraconazole systemic treatment. CONCLUSION: Pseudonectria foliicola is a plant pathogenic bacterium that has never been reported in human infections before. Therefore, ophthalmologists should consider Pseudonectria foliicola as a possible cause of fungal keratitis, as early identification and timely treatment can help improve vision in most eyes.

Differences in composition and diversity of rumen fungi in buffalo fed different diets.

The rumen is a complex ecosystem containing a variety of fungi, which are crucial for the digestive activities of ruminants. Previous research on rumen fungi has mainly focused on anaerobic fungi, given the rumen's reputation as a mainly anaerobic environment. The objective of this study was to investigate rumen fungal diversity and the presence of aerobic fungi in buffalo fed on different diets. Three adult buffaloes were used as experimental animals. Alfalfa hay, oat hay, whole corn silage, sugarcane shoot silage, fresh king grass, dried rice straw, and five kinds of mixed diets with concentrate to roughage ratios of 20:80, 35:65, 50:50, 65:35, and 80:20 were used as the experimental diets. The experimental animals were fed different diets for 22 days. Rumen fluid was collected from the rumen fistula for ITS (Internal Transcribed Spacer) sequencing 2 h after feeding on the morning of day 22. The results indicate the presence of large quantities of aerobic fungi in the rumen of the buffaloes 2 h after feeding and suggest that Ascomycota and Basidiomycota are the dominant fungal groups under different feeding conditions. The study also identified 62 different fungal types, which showed significant differences among the 11 experimental diets.

Elucidating the clinical, microbiological and molecular diagnostic aspects of Macrophomina phaseolina keratitis.

This study reports the clinico-microbiological features of Macrophomina phaseolina keratitis. Clinically diagnosed as microbial keratitis, six patients underwent microbiological evaluation. Fungal culture isolates from cornea were subjected to DNA sequencing of the ITS region, phylogenetic analysis and reconfirmation by polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) of six antifungal drugs were determined by microbroth dilution method against the six isolates. All patients were treated with antifungals. Failed medical therapy necessitated therapeutic penetrating keratoplasty (TPK). Corneal buttons were processed for histopathology. In all patients, the corneal scraping showed septate hyaline fungal filaments. The BLAST analysis for ITS sequences of all six fungal isolates suggested M. phaseolina, however, when limited to sequences from type material, they matched M. pseudophaseolina. Phylogenetic analysis could not differentiate between these two species and clustered in a single clade. PCR assay of specific gene sequence [MpCal (calmodulin)] reconfirmed all isolates as M. phaseolina. The MICs of voriconazole and posaconazole were lowest (0.03 to 2 and 0.1 to 2µg/mL respectively) and all isolates were susceptible to natamycin. Except for case 1, which healed with a scar on treatment, all other cases worsened, despite medical treatment, necessitating TPK. Histopathology of 3 out of 4 buttons showed the presence of fungal filaments. While direct microscopic examination of corneal scrapings is helpful in diagnosis, identification of M. phaseolina in culture is challenging. Although MICs of commonly used antifungals are low response to medical therapy is not encouraging; patients may require TPK for resolution of infection in M. phaseolina keratitis.

DNA sequencing, phylogenetic analysis and specific PCR confirmed Macrophomina phaseolina keratitis in six patients. Although antifungal susceptibility showed the organisms to be susceptible to natamycin five patients did not respond to treatment and needed keratoplasty.

Start Codon Targeted (SCoT) markers for the assessment of genetic diversity in yeast isolated from Turkish sourdough.

Molecular markers are valuable tools for assessing the genetic variation in yeast. Here, we investigated the utility of SCoT markers for the genetic characterization of yeast strains at inter and intraspecies levels. A total of 345 endogenous yeast strains were isolated from 65 Type I sourdough samples collected from six different regions of Turkey. The seven SCoT primers produced 221 bands, of which 95.47% were polymorphic. Each primer could successfully differentiate species, supported by PIC and RP values. The ITS sequencing of isolates selected from the UPGMA dendrogram revealed that Saccharomyces cerevisiae predominated the microflora, followed by Kazachstania servazzii, K. humilis, Wickerhamomyces anomalus, Torulaspora delbrueckii, and Pichia kudriavzevii, respectively. The AMOVA revealed a high genetic variation between (49%) and within populations (51%) for S. cerevisiae. The high gene flow observed among S. cerevisiae populations suggests that it may have contributed to the geographical evolution of S. cerevisiae via the transportation of the sourdough samples. The different geographical origins were most likely to group separately on the UPGMA and PCoA. Saccharomyces cerevisiae strains from more distant populations generally displayed more significant genetic variation. SCoT markers can successfully be used alone or with the other existing DNA markers for DNA fingerprinting and analyzing the genetic variation between and within species.

Response of Poplar and Associated Fungal Endophytic Communities to a PAH Contamination Gradient.

Microbial populations associated to poplar are well described in non-contaminated and metal-contaminated environments but more poorly in the context of polycyclic aromatic hydrocarbon (PAH) contamination. This study aimed to understand how a gradient of phenanthrene (PHE) contamination affects poplar growth and the fungal microbiome in both soil and plant endosphere (roots, stems and leaves). Plant growth and fitness parameters indicated that the growth of Populus canadensis was impaired when PHE concentration increased above 400 mg kg-1. Values of alpha-diversity indicators of fungal diversity and richness were not affected by the PHE gradient. The PHE contamination had a stronger impact on the fungal community composition in the soil and root compartments compared to that of the aboveground organs. Most of the indicator species whose relative abundance was correlated with PHE contamination decreased along the gradient indicating a toxic effect of PHE on these fungal OTUs (Operational Taxonomic Units). However, the relative abundance of some OTUs such as Cadophora, Alternaria and Aspergillus, potentially linked to PHE degradation or being plant-beneficial taxa, increased along the gradient. Finally, this study allowed a deeper understanding of the dual response of plant and fungal communities in the case of a soil PAH contamination gradient leading to new perspectives on fungal assisted phytoremediation.

Respiratory Mycoses in COPD and Bronchiectasis.

Chronic obstructive pulmonary disease (COPD) and bronchiectasis represent chronic airway diseases associated with significant morbidity and mortality. Bacteria and viruses are commonly implicated in acute exacerbations; however the significance of fungi in these airways remains poorly defined. While COPD and bronchiectasis remain recognized risk factors for the occurrence of Aspergillus-associated disease including chronic and invasive aspergillosis, underlying mechanisms that lead to the progression from colonization to invasive disease remain uncertain. Nonetheless, advances in molecular technologies have improved our detection, identification and understanding of resident fungi characterizing these airways. Mycobiome sequencing has revealed the complex varied and myriad profile of airway fungi in COPD and bronchiectasis, including their association with disease presentation, progression, and mortality. In this review, we outline the emerging evidence for the clinical importance of fungi in COPD and bronchiectasis, available diagnostic modalities, mycobiome sequencing approaches and association with clinical outcomes.

High rates of misidentification of uncommon Candida species causing bloodstream infections using conventional phenotypic methods.

BACKGROUND: Candidemia caused by uncommon Candida species is increasing and misidentification may compromise optimal antifungal therapy. This multicenter study aimed to evaluate the accuracy of species-level identification of uncommon Candida. METHODS: Uncommon causative species of candidemia identified in routine laboratories using CHROMagar, API-32C and VITEK-2 Yeast ID system were collected from July 2011 to June 2014. These isolates were further identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system and sequencing of the internal transcribed spacer and 28S rRNA gene. Susceptibility of the isolates was determined. RESULTS: Of 85 isolates evaluated, Candida guilliermondii (n = 36) was the most common, followed by Candid sake (n = 7) and Candida famata (n = 4). Using DNA-sequencing analysis as standard, none of C. sake and C. famata was correct, while VITEK MS correctly identified 10 of the 11 isolates. With the exclusion of one unspecified Candida by DNA-sequencing methods, the accuracy of conventional methods and VITEK MS was 64.3% and 86.9%, respectively (p = 0.001). Eight isolates were confirmed to be yeasts other than Candida. Compared with other Candida species, C. guilliermondii showed elevated minimal inhibitory concentration of echinocandins. CONCLUSION: Misidentification of uncommon Candida species was common using the conventional methods, especially for C. sake and C. famata. MALDI-TOF MS assisted by DNA-sequencing methods should be considered.

The Mycobiome in Health and Disease: Emerging Concepts, Methodologies and Challenges.

Fungal disease is an increasingly recognised global clinical challenge associated with high mortality. Early diagnosis of fungal infection remains problematic due to the poor sensitivity and specificity of current diagnostic modalities. Advances in sequencing technologies hold promise in addressing these shortcomings and for improved fungal detection and identification. To translate such emerging approaches into mainstream clinical care will require refinement of current sequencing and analytical platforms, ensuring standardisation and consistency through robust clinical benchmarking and its validation across a range of patient populations. In this state-of-the-art review, we discuss current diagnostic and therapeutic challenges associated with fungal disease and provide key examples where the application of sequencing technologies has potential diagnostic application in assessing the human 'mycobiome'. We assess how ready access to fungal sequencing may be exploited in broadening our insight into host-fungal interaction, providing scope for clinical diagnostics and the translation of emerging mycobiome research into clinical practice.

Mycosis of the Plantar Surface of Foot Owing to Nondermatophyte Mold Nodulisporium griseobrunneum Mimicking a Tinea Pedis.

Nondermatophyte molds (NDM) and dematiaceous molds are less frequently implicated as the etiological agents of tinea-like infections of the foot. Among the etiological agents, Hendersonula toruloidea (now, Nattrassia mangiferae), Scytalidium hyalinum, Alternaria species (spp.), and Fusarium spp. are infrequently associated with foot mycoses. Nodulisporium (N.) spp. is a mitosporic NDM, which has been implicated in human infections like cerebral phaeohyphomycosis and allergic fungal sinusitis. Here, we report N. griseobrunneum in a 9-year-old female with mycosis of the plantar surface of foot mimicking a tinea pedis. Potassium hydroxide mount of skin specimen demonstrated dichotomous branching septate hyphae. Fungal culture and molecular sequencing established N. griseobrunneum as the etiological agent. Antifungal susceptibility testing revealed lower MICs to all seven drugs tested including itraconazole (ITR). The patient was treated with ITR and topical terbinafine. To the best of our knowledge, this is the first communication depicting molecular confirmation of the etiologic agent and antifungal susceptibility data of the mycosis of the plantar surface of foot owing to N. griseobrunneum from India.

Floral and Foliar Source Affect the Bee Nest Microbial Community.

Managed pollinators such as the alfalfa leafcutting bee, Megachile rotundata, are essential to the production of a wide variety of agricultural crops. These pollinators encounter a diverse array of microbes when foraging for food and nest-building materials on various plants. To test the hypothesis that food and nest-building source affects the composition of the bee-nest microbiome, we exposed M. rotundata adults to treatments that varied both floral and foliar source in a 2 × 2 factorial design. We used 16S rRNA gene and internal transcribed spacer (ITS) sequencing to capture the bacterial and fungal diversity of the bee nests. We found that nest microbial communities were significantly different between treatments, indicating that bee nests become inoculated with environmentally derived microbes. We did not find evidence of interactions between the fungi and bacteria within our samples. Furthermore, both the bacterial and fungal communities were quite diverse and contained numerous exact sequence variants (ESVs) of known plant and bee pathogens that differed based on treatment. Our research indicates that bees deposit plant-associated microbes into their nests, including multiple plant pathogens such as smut fungi and bacteria that cause blight and wilt. The presence of plant pathogens in larval pollen provisions highlights the potential for bee nests to act as disease reservoirs across seasons. We therefore suggest that future research should investigate the ability of bees to transmit pathogens from nest to host plant.

Internal transcribed spacer (ITS) sequence-based characterization of fungal isolates from multiple yogurt facilities-A case study.

Fungal spoilage remains a significant issue in dairy product quality, especially for cultured dairy products such as yogurt formulated without preservatives such as potassium sorbate. Fungal contamination can occur throughout the processing continuum, from the dairy farm environment to the finished product processing environment. As molecular characterization of fungal isolates is used more frequently, we obtained fungal isolates obtained in 2 yogurt processing facilities as part of routine fungal testing of raw materials (e.g., fruit preparations, added ingredients), in-process product samples, environmental samples (e.g., air plates, equipment surfaces such as valves, face plates, air nozzles), and finished product samples, to determine whether internal transcribed spacer (ITS) barcoding data would be helpful to support source tracking of fungal contamination issues. Internal transcribed spacer PCR amplification and sequencing allowed us to classify the 852 isolates from these 2 facilities into 200 unique ITS allelic types (AT), representing the phyla Ascomycota (743 isolates), Basidiomycota (97 isolates), and Mucoromycota (12 isolates). Thirty ITS AT were isolated from both facilities; 62 and 108 ITS AT were isolated from only facility A or only facility B, respectively. Nine ITS AT were each represented by more than 20 isolates; these AT comprised 53% of the 852 isolates. The considerable diversity of fungal isolates even within a single facility illustrates the challenge associated with controlling fungal contamination of dairy products. The ITS barcoding technique, however, did show promise for facilitating the source tracking of fungal contamination, particularly for ITS AT over-represented in a given facility. For example, we found evidence for equipment-specific reservoirs for 2 AT (14 and 219) in facility B. Our data suggest that despite its limited discriminatory power, ITS sequencing can provide initial information that can help trace fungal contamination along the processing continuum. However, development and implementation of discriminatory subtyping methods will be needed to further improve the ability to identify sources of fungal contamination in dairy facilities. Developing and implementing sampling plans that comprehensively capture yeast and mold diversity in a given processing facility remain a considerable challenge.

Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identifying Clinical Malassezia Isolates.

The genus Malassezia comprises commensal yeasts on human skin. These yeasts are involved in superficial infections but are also isolated in deeper infections, such as fungemia, particularly in certain at-risk patients, such as neonates or patients with parenteral nutrition catheters. Very little is known about Malassezia epidemiology and virulence. This is due mainly to the difficulty of distinguishing species. Currently, species identification is based on morphological and biochemical characteristics. Only molecular biology techniques identify species with certainty, but they are time-consuming and expensive. The aim of this study was to develop and evaluate a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) database for identifying Malassezia species by mass spectrometry. Eighty-five Malassezia isolates from patients in three French university hospitals were investigated. Each strain was identified by internal transcribed spacer sequencing. Forty-five strains of the six species Malassezia furfur, M. sympodialis, M. slooffiae, M. globosa, M. restricta, and M. pachydermatis allowed the creation of a MALDI-TOF database. Forty other strains were used to test this database. All strains were identified by our Malassezia database with log scores of >2.0, according to the manufacturer's criteria. Repeatability and reproducibility tests showed a coefficient of variation of the log score values of <10%. In conclusion, our new Malassezia database allows easy, fast, and reliable identification of Malassezia species. Implementation of this database will contribute to a better, more rapid identification of Malassezia species and will be helpful in gaining a better understanding of their epidemiology.

Molecular study of the Candida parapsilosis complex in Sfax, Tunisia.

Candida parapsilosis, which was previously considered to be a complex of three genetically distinct groups, has emerged as a significant agent of nosocomial infections. Recently, this complex was separated into three species: C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis In Tunisia, data pertaining to these fungi are limited. Thus, the purpose of our study was to determine by BanI PCR-RFLP and ITS sequencing, the occurrence of Candida parapsilosis complex among 182 isolates identified as C. parapsilosis by phenotypical methods. C. parapsilosis sensu stricto represented 94.5% of all isolates, while C. metapsilosis and. C. orthopsilosis were identified in 3.3% and 2.2%, respectively. Sequence analysis of internal transcribed spacer region confirmed and revealed only one genotype among the C. parapsilosis sensu stricto strains, three genotypes among six C. metapsilosis strains and two genotypes among four C. orthopsilosis strains.

A study of selected factors affecting efficacy of compost tea against several fungal pathogens of potato.

AIM: To investigate selected factors of two nonaerated compost teas (NCT) and mechanisms that influence the restriction of several fungal potato pathogens. METHODS AND RESULTS: Two NCTs, made from either commercial compost, (CCT) or vineyard compost (VCT), were tested for their ability to suppress potato pathogens. The VCT was more suppressive than CCT to mycelial growth of Alternaria solani and Rhizoctonia solani isolate 299, but not for R. solani isolate 422. Metagenomic studies of microbial diversity revealed that the CCT had higher fungal and bacterial diversity and richness than the VCT. Use of CCT significantly reduced lesion area of Alternaria alternata on detached leaves, however, a gum adjuvant did not lead to significantly greater control. Scanning microscopy showed that the spatial distribution of microbes from the CCT was altered with gum addition, to resemble what may have been a microbial biofilm. CONCLUSION: We confirmed that each NCT could suppress the mycelial growth of selected potato pathogens in culture, and CCT reduced A. alternata lesions on detached leaves. Factors including concentration, microbial communities and physio-chemical properties could not be consistently linked to NCT efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: This study particularly highlights the application of scanning microscopy to study the interaction between pathogens and putative NCT microbes on foliar surfaces. This adds insight to mechanisms of NCT efficacy, along with physico-chemical and microbial characterization of the teas. This study shows the potential for the use of NCTs as a crop protection tool of low-cost which could be of particular benefit in smallholder agriculture.

Internal transcribed spacer (ITS) sequencing reveals considerable fungal diversity in dairy products.

Fungi are important spoilage organisms in dairy products. However, little is known about the diversity of naturally occurring spoilage fungi in raw milk and processed dairy products, due at least in part to the fact that classical fungal identification methods require considerable expertise. To gain further insight into the fungal diversity in the dairy system, we isolated fungi from raw milk, raw and pasteurized milk cheese, and yogurt using the selective dichloran rose bengal chloramphenicol agar. In total, 361 fungal isolates were obtained and further characterized by DNA sequencing of the internal transcribed spacer (ITS) region and the nuclear ribosomal large subunit (LSU) rRNA gene if needed. We conducted BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) searches of the ITS region sequences against the UNITE Database (https://unite.ut.ee/analysis.php), and selected other databases if needed, which allowed identification to the species level of 183 isolates and to the genus level of 107 of the 346 isolates that were successfully ITS sequenced. The isolates characterized represented 3 phyla and 19 genera; the most common genera isolated were Penicillium (25% of isolates), Debaryomyces (18%), and Candida (9%). This study not only provides, by using modern molecular tools, a baseline understanding of the types of fungi in dairy products, but also confirms that ITS sequencing is a useful approach for identification of fungal organisms found in the dairy food chain.