Rhizopus Microsporus Research Articles

Effects of steeping on fungal contamination and diversity in achi (Barchystegia eurycoma) seeds sold in some selected markets in Lagos state, Nigeria

Achi (Brachystegia eurycoma) seeds are widely used as a thickening agent in traditional Nigerian cuisine. However, the processing methods used for achi seeds can lead to fungal contamination, posing considerable health risks to consumers. This study aims to investigate the effect of water steeping on fungal species contaminating achi seeds sold in some selected markets in Lagos, Nigeria. The achi seeds were steeped by soaking them in water at 37 °C for two days, and the moisture content was subsequently determined. The isolated fungal species were identified using both morphological and molecular techniques. Our findings show that the moisture content (37.82—51.64%) of the steeped achi seeds was higher than that of the unsteeped achi seeds (8.32—15.73%). Seven fungal species were recovered from the achi seeds, including six Aspergillus spp. (A. aculeatus, A. flavus, A. fumigatus, A. niger, A. japonicus, and A. oryzae), along with Rhizopus microsporus. Notably, the steeped samples were found to contain all the seven fungal species whereas the unsteeped achi were contaminated with just four fungal species, namely A. aculeatus, A. flavus, A. fumigatus, and A. niger. Additionally, the steeped achi seeds showed a higher fungal load (4.92 × 103—5.50 × 103 CFU/g) compared to the unsteeped samples (3.39 × 103—5.33 × 103 CFU/g). This current study indicates that steeping increases fungal contamination and diversity in achi seeds, raising concerns about food safety.

Purification and Characterization of β-Mannanase Derived from Rhizopus microsporus var. rhizopodiformis Expressed in Komagataella phaffii.

The demand for food-grade β-mannanases, ideal for high-temperature baking, is increasing. Using the Komagataella phaffii (P. pastoris) expression system for β-mannanase production, this study aimed to enhance purification methods. We evaluated better conditions for production and purification of β-mannanase (PpRmMan134A) from recombinant P. pastoris X-33, focusing on a higher purity and reducing the production of endogenous secretory proteins in fermentation. By adjusting carbon and nitrogen sources, culture time, and temperature, we controlled cell growth to reduce the production of endogenous secretory proteins. The better-evaluated conditions involved culturing recombinant P. pastoris in 70 mL buffered glycerol complex medium for 24 h at 30 °C, then in modified buffered methanol-complex medium with 0.91% (w/v) methanol, 0.56% (w/v) sorbitol, and 0.48% (w/v) mannitol for another 24 h, which improved the PpRmMan134A yield and reduced endogenous secretory proteins, shortening the fermentation time by 72 h. An affordable purification method using ultrafiltration and salt-out precipitation was utilized. PpRmMan134A showed thermostability up to 100 °C and effectively degraded locust bean gum into smaller fragments, mainly producing mannotriose. In conclusion, with its enhanced purity due to reduced levels of endogenous secretory proteins, purified PpRmMan134A emerges as a promising enzyme for high-temperature baking applications.

Pilot scale production of high-content mycoprotein using Rhizopus microsporus var. oligosporus by submerged fermentation and agro-industrial by-products

While mycoprotein has gained traction as a human food source, its potential as a nutrient for animals remains largely unexplored. The mycoprotein-producing Rhizopus microsporus var. oligosporus, a fungus traditionally used for human food in Indonesia, is promising. It could revolutionise animal nutrition once it is Generally Recognized as Safe (GRAS) and is a biosafety level 1 (BSL1) organism. To enhance sustainably, we propose using sugar cane molasses (SM) and corn steep liquor (CSL) as nutrient sources. Also, we investigated the growth of R. microsporus var. oligosporus in five 14 L external-loop airlift bioreactors using CSL as the sole nutrient source. After 96 h of fermentation, at 25 °C and 0.5 vvm, the mycelium produced had an average biomass yield of 38.34 g L−1, with 70.18 % (m v−1) crude protein (mycoprotein). This bioprocess, which is scalable and economically viable, produces high amounts of mycoprotein for animal feed using CSL, a cost-effective agro-industrial by-product, providing a practical solution to the growing demand for animal protein.

Inducing novel endosymbioses by implanting bacteria in fungi.

Endosymbioses have profoundly impacted the evolution of life and continue to shape the ecology of a wide range of species. They give rise to new combinations of biochemical capabilities that promote innovation and diversification1,2. Despite the many examples of known endosymbioses across the tree of life, their de novo emergence is rare and challenging to uncover in retrospect3-5. Here we implant bacteria into the filamentous fungus Rhizopus microsporus to follow the fate of artificially induced endosymbioses. Whereas Escherichia coli implanted into the cytosol induced septum formation, effectively halting endosymbiogenesis, Mycetohabitans rhizoxinica was transmitted vertically to the progeny at a low frequency. Continuous positive selection on endosymbiosis mitigated initial fitness constraints by several orders of magnitude upon adaptive evolution. Phenotypic changes were underscored by the accumulation of mutations in the host as the system stabilized. The bacterium produced rhizoxin congeners in its new host, demonstrating the transfer of a metabolic function through induced endosymbiosis. Single-cell implantation thus provides a powerful experimental approach to study critical events at the onset of endosymbiogenesis and opens opportunities for synthetic approaches towards designing endosymbioses with desired traits.

First global registration of an isolate producing aflatoxin B1 from the fungus (Rhizopus microsporus) from stored grains and nuts in Iraq

First global registration of an isolate producing aflatoxin B1 from the fungus (Rhizopus microsporus) from stored grains and nuts in Iraq

New lineages of RNA viruses from clinical isolates of Rhizopus microsporus revealed by fragmented and primer-ligated dsRNA sequencing (FLDS) analysis.

Rhizopus microsporus is a species in the order Mucorales that is known to cause mucormycosis, but it is poorly understood as a host of viruses. Here, we examined 25 clinical strains of R. microsporus for viral infection with a conventional double-stranded RNA (dsRNA) assay using agarose gel electrophoresis (AGE) and the recently established fragmented and primer-ligated dsRNA sequencing (FLDS) protocol. By AGE, five virus-infected strains were detected. Then, full-length genomic sequences of 12 novel RNA viruses were revealed by FLDS, which were related to the families Mitoviridae, Narnaviridae, and Endornaviridae, ill-defined groups of single-stranded RNA (ssRNA) viruses with similarity to the established families Virgaviridae and Phasmaviridae, and the proposed family "Ambiguiviridae." All the characterized viruses, except a potential phasmavirid with a negative-sense RNA genome, had positive-sense RNA genomes. One virus belonged to a previously established species within the family Mitoviridae, whereas the other 11 viruses represented new species or even new genera. These results show that the fungal pathogen R. microsporus harbors diverse RNA viruses and extend our understanding of the diversity of RNA viruses in the fungal order Mucorales, division Mucoromycota. Identifying RNA viruses from clinical isolates of R. microsporus may expand the repertoire of natural therapeutic agents for mucormycosis in the future.IMPORTANCEThe diversity of mycoviruses in fungal hosts in the division Mucoromycota has been underestimated, mainly within the species Rhizopus microsporus. Only five positive-sense RNA genomes had previously been discovered in this species. Because current sequencing methods poorly complete the termini of genomes, we used fragmented and primer-ligated double-stranded RNA sequencing to acquire the full-length genomes. Eleven novel mycoviruses were detected in this study, including the first negative-sense RNA genome reported in R. microsporus. Our findings extend the understanding of the viral diversity in clinical strains of Mucoromycota, may provide insights into the pathogenesis and ecology of this fungus, and may offer therapeutic options.

Characterization and evaluation of the bioremediation potential of Rhizopus microsporus Os4 isolated from arsenic-contaminated soil

Characterization and evaluation of the bioremediation potential of Rhizopus microsporus Os4 isolated from arsenic-contaminated soil

Screening, identification, and characterization of molds for brewing rice wine: Scale-up production in a bioreactor.

Rice wine, well known for its unique flavor, rich nutritional value, and health benefits, has potential for extensive market development. Rhizopus and Aspergillus are among several microorganisms used in rice wine brewing and are crucial for determining rice wine quality. The strains were isolated via Rose Bengal and starch as a combined separation medium, followed by oenological property and sensory evaluation screening. The strain exhibiting the best performance can be screened using the traditional rice wine Qu. The strains YM-8, YM-10, and YM-16, which exhibited strong saccharification and fermentation performance along with good flavor and taste, were obtained from traditional rice wine Qu. Based on ITS genetic sequence analysis, the YM-8, YM-10, and YM-16 strains were identified as Rhizopus microsporus, Rhizopus arrhizus, and Aspergillus oryzae. The optimum growth temperature of each of the three strains was 30°C, 32°C, and 30°C, and the optimum initial pH was 6.0, 6.5, and 6.5, respectively. The activities of α-amylase, glucoamylase, and protease of YM-16 were highest at 220.23±1.88, 1,269.04±30.32, and 175.16±1.81 U/g, respectively. The amino acid content of rice wine fermented in a 20-L bioreactor with the three mold strains was higher than that of the control group, except for arginine, which was significantly lower than that of the control group. The total amino acid content and the total content of each type of amino acid were ranked as YM-16 > YM-8 > YM-10 > control group, and the amino acid content varied greatly among the strains. The control group had a higher content, whereas YM-8 and YM-16 had lower contents of volatile aroma components than the control group and had the basic flavor substances needed for rice wine, which is conducive to the formation of rice wine aroma. This selected strain, YM-16, has strong saccharification and fermentation ability, is a rich enzyme system, and improves the flavor of rice wine, thereby demonstrating its suitability as a production strain for brewing.

Prevalence and diversity of TAL effector-like proteins in fungal endosymbiotic Mycetohabitans spp.

Endofungal Mycetohabitans (formerly Burkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus, Rhizopus microsporus. The one known secreted effector family from Mycetohabitans consists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These 'Burkholderia TAL-like (Btl)' proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of two Mycetohabitans strains revealed that the single Btl in one Mycetohabitans endofungorum strain enhances fungal membrane stress tolerance, while others in a Mycetohabitans rhizoxinica strain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across M. rhizoxinica strains than across M. endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial-fungal symbioses in myriad ways.

Clinical features of pediatric mucormycosis: role of metagenomic next generation sequencing in diagnosis.

Mucormycosis is an uncommon invasive fungal infection that has a high mortality rate in patients with severe underlying diseases, which leads to immunosuppression. Due to its rarity, determining the incidence and optimal treatment methods for mucormycosis in children is challenging. Metagenomic next-generation sequencing (mNGS) is a rapid, precise and sensitive method for pathogen detection, which helps in the early diagnosis and intervention of mucormycosis in children. In order to increase pediatricians' understanding of this disease, we conducted a study on the clinical features of mucormycosis in children and assessed the role of mNGS in its diagnosis. We retrospectively summarized the clinical data of 14 children with mucormycosis treated at the First Affiliated Hospital of Zhengzhou University from January 2020 to September 2023. Of the 14 cases, 11 case of mucormycosis were classified as probable, and 3 cases were proven as mucormycosis. Most children (85.71%) had high-risk factors for mucormycosis. All 14 children had lung involvement, with 5 cases of extrapulmonary dissemination. Among the 14 cases, 4 cases underwent histopathological examination of mediastinum, lung tissue or kidney tissue, in which fungal pathogens were identified in 3 patients. Fungal hyphae was identified in 3 cases of mucormycosis, but only 1 case yielded a positive culture result. All patients underwent mNGS testing with samples from blood (8/14), bronchoalveolar lavage fluid (6/14), and tissue (1/14). mNGS detected fungi in all cases: 7 cases had Rhizomucor pusillus, 4 cases had Rhizopus oryzae, 3 cases had Rhizopus microsporus, 1 case had Lichtheimia ramosa, and 1 case had Rhizomucor miehei. Coinfections were found with Aspergillus in 3 cases, bacteria in 3 cases, and viruses in 5 cases. Children with mucormycosis commonly exhibit non-specific symptoms like fever and cough during the initial stages. Early diagnosis based on clinical symptoms and imaging is crucial in children suspected of having mucormycosis. mNGS, as a supplementary diagnostic method, offers greater sensitivity and shorter detection time compared to traditional mucormycosis culture or histopathological testing. Additionally, mNGS enables simultaneous detection of bacteria and viruses, facilitating timely and appropriate administration of antibiotics and thereby enhancing patient outcomes.

Mitochondrial genomic characteristics and phylogenetic analysis of a brewing fungus, Rhizopus microsporus Tiegh. 1875 (Mucorales: Rhizopodaceae)

Rhizopus microsporus Tiegh. 1875 is widely used in a variety of industries, such as brewing, wine making, baking, and medicine production, as it has the capability to break down proteins and generate surface-active agents. To date, the mitochondrial genome features of early evolved fungi from the Rhizopus genus have not been extensively studied. Our research obtained a full mitochondrial genome of R. microsporus species, which was 43,837 bp in size and had a GC content of 24.93%. This genome contained 14 core protein-coding genes, 3 independent ORFs, 7 intronic ORFs, 24 tRNAs, and 2 rRNA genes. Through the use of the BI phylogenetic inference method, we were able to create phylogenetic trees for 25 early differentiation fungi which strongly supported the major clades; this indicated that R. microsporus is most closely related to Rhizopus oryzae.

Fungal holobionts as blueprints for synthetic endosymbiotic systems.

Rhizopus microsporus is an example of a fungal holobiont. Strains of this species can harbor bacterial and viral endosymbionts inherited by the next generation. These microbial allies increase pathogenicity and defense and control asexual and sexual reproduction.

Developing defined starter culture for reproducible profile of flavour compound in Chinese xiaoqu baijiu fermentation

Defined starter cultures, containing selected microbes could reduce the complexity of natural starter, are beneficial for controllable food fermentations. However, there are challenges in identifying key microbiota and constructing synthetic microbiota for traditional food fermentations. Here, we aimed to develop a defined starter culture for reproducible profile of flavour compounds, using Chinese Xiaoqu Baijiu fermentation as a case. We classified all microbes into 4 modules using weighted correlation network analysis. Module 3 presented significant correlations with flavour compounds (P < 0.05) and the highest gene abundance related with flavour compound production. 13 dominant species in module 3 were selected for mixed culture fermentation, and each species was individually deleted to analyse the effect on flavour compound production. Ten species, presenting significant effects (P < 0.05) on flavour compound production, were selected for developing the starter culture, including Rhizopus oryzae, Rhizopus microsporus, Saccharomyces cerevisiae, Pichia kudriavzevii, Wickerhamomyces anomalus, Lactobacillus acetotolerans, Levilactobacillus brevis, Weissella paramesenteroides, Pediococcus acidilactici, and Leuconostoc pseudomesenteroides. After optimising the structure of the starter culture, the profile similarity of flavour compounds produced by the starter culture reached 81.88% with that by the natural starter. This work indicated feasibility of reproducible profile of flavour compounds with defined starter culture for food fermentations.

IDENTIFICATION OF YEAST STRAINS AND FILAMENTOUS FUNGI IN THE HAI HAU TRADITIONAL ALCOHOL YEAST CAKE

This study aims to determine the various strains of yeast and filamentous fungi in traditional alcohol yeast cake from Hai Hau district, Nam Dinh province. By traditional laboratory techniques such as isolation on YPD agar, observation of colony morphology as well as cell characteristics by microscopy, combined with molecular biology method to sequence ITS region 03 strains of yeast Saccharomyces cerevisiae CN1, CN2, and NM3 were identified: 01 pseudo-strain yeast Saccharomycopsis fibuligera NM02 and 01 strain of filamentous fungi Rhizopus microsporus NS04. The density of yeast strains cultured on YPD agar supplemented with 50 mg/L Tetracycline was 4.5 × 108 CFU/g, while the density of R. microsporus NS04 filamentous fungi was 2.0 × 106 CFU/g. With the agar plate diffusion method, yeast strains of S. cerevisiae CN1, CN2, and NM3 that are unable to biosynthesize extracellular enzymes have been identified. Yeast pseudo-strain S. fibuligera NM02 is capable of producing amylase (with starch substrate hydrolysis ring diameter &gt; 9 mm), cellulase (16 mm), and protease (18 mm). The filamentous fungi R. microsporus NS04 has a higher ability to produce extracellular enzymes such as amylase (&gt; 9 mm), cellulase (33 mm), and protease (39 mm)

First report of Pectobacterium aroidearum causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) is widely planted in China as an important leaf vegetable. In March 2022, water-soaked and mushy rot symptoms were observed on leaf mustard plants in the field of Zhaotong (27.85°N; 105.05°E), Yunnan, China. The incidence of symptomatic leaf mustard was approximately 10%. The isolation of the causal agent followed the method of Peng et al. (2023). Briefly, infected tissues from four diseased plants were mixed and teased apart, and homogenized by vortex shaking. The bacterial suspension was diluted and spread on nutrient agar (NA). About 10 single colonies exhibiting different colony morphologies were picked and purified separately by successive streaking. A pinprick method was used for pathogenicity tests with an inoculum concentration of 108 CFU/ml (Singh et al. 2013). Among 10 isolates, only strain YKX exhibited soft rot symptoms on detached mustard leaves. In addition, ten two-month-old leaf mustard plants grown in the greenhouse were used for in vivo pathogenicity tests. Briefly, sterilized pins were dipped in the bacterial suspension, and then leaf mustard petioles were pricked with these pins. After inoculation, each plant was kept in a plastic bag for 12 hours to maintain high humidity. As expected, strain YKX caused obvious rot symptoms on eight plants at 1-2 days post-inoculation while the control group including two plants treated with sterile water showed no symptoms. The colonies of strain YKX on NA were white, roughly circular, and convex. For a preliminary identification, total DNA was extracted and used as the template in PCR amplification of 16S rDNA with the universal PCR primer pair 27F/1492R (Weisburg et al. 1991). The quality-filtered DNA sequence (871 bp) showed 100% query coverage and 99.47% identity to the 16S rDNA sequences of type strain Pectobacterium aroidearum SCRI 109T (GenBank: NR_159926) found in the NCBI rRNA/ITS database. Whole-genome sequencing of strain YKX was then performed using the Illumina and Nanopore sequencing platforms by Tsingke Biotechnology Co., Ltd. (Beijing, China). A single contig (GenBank: CP129239) with a length of approximately 4.9 Mb was obtained by de novo hybrid assembly using Unicycler v0.5.0 (Wick et al. 2017). The quality of the genomic data was evaluated by BUSCO v5.4.7 (Manni et al. 2021) against the gammaproteobacteria_odb10 dataset. A BUSCO complete score of 99.5% indicated high assembly quality. The genome sequence of strain YKX was uploaded to the Type Strain Genome Server for a genome-based taxonomic analysis (Meier-Kolthoff et al. 2022). The distance-based phylogeny showed that strain YKX and P. aroidearum L6 (GenBank: CP065044) and P. aroidearum PC1 (GenBank: NC_012917) form a clade. When comparing strain YKX with L6 or PC1, the digital DNA-DNA hybridization value (83.5-83.8%) was above the species delineation threshold (70% for DDH), clearly indicating that strain YKX should be classified as P. aroidearum. Additionally, P. aroidearum was reisolated from inoculated leaves and identified based on morphological similarities and 16S rDNA sequencing, thus fulfilling Koch's postulates. It is worth noting that a previous study reported occurrences of soft rot disease on leaf mustard attributed to Rhizopus microsporus var. chinensis (Wang et al. 2020). To our knowledge, this is the first report of P. aroidearum causing soft rot on leaf mustard in China, which expands the known host range of this pathogen and benefits the control of this disease.

Rapid detection of Mucorales in human blood and urine samples by functionalized Heusler magnetic nanoparticle assisted customized loop-mediated isothermal amplification.

Mucormycosis is a rare disease with scarce diagnostic methods for early intervention. Available strategies employing direct microscopy using calcofluor white-KOH, culture, radiologic, and histopathologic testing often are time-intensive and demand intricate protocols. Nucleic Acid Amplification Test holds promise due to its high sensitivity combined with rapid detection. Loop-mediated isothermal amplification (LAMP) based detection offers an ultrasensitive technique that does not require complicated thermocyclers like in polymerase chain reaction, offering a straightforward means for improving diagnoses as a near-point-of-care test. The study introduces a novel magnetic nanoparticle-based LAMP assay for carryover contaminant capture to reduce false positives. Solving the main drawback of LAMP-based diagnosis techniques. The assay targets the cotH gene, which is invariably specific to Mucorales. The assay was tested with various species of Mucorales, and the limit of detections for Rhizopus microsporus, Lichtheimia corymbifera, Rhizopus arrhizus, Rhizopus homothallicus, and Cunninghamella bertholletiae were 1 fg, 1 fg, 0.1 pg, 0.1 pg, and 0.01ng, respectively. This was followed by a clinical blindfolded study using whole blood and urine samples from 30 patients diagnosed with Mucormycosis. The assay has a high degree of repeatability and had an overall sensitivity of>83%. Early Mucormycosis detection is crucial, as current lab tests from blood and urine lack sensitivity and take days for confirmation despite rapid progression and severe complications. Our developed technique enables the confirmation of Mucormycosis infection in < 45 min, focusing specifically on the RT-LAMP process. Consequently, this research offers a viable technique for quickly identifying Mucormycosis from isolated DNA of blood and urine samples instead of invasive tissue samples.

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

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. 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. 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.

Deazaflavin metabolite produced by endosymbiotic bacteria controls fungal host reproduction.

The endosymbiosis between the pathogenic fungus Rhizopus microsporus and the toxin-producing bacterium Mycetohabitans rhizoxinica represents a unique example of host control by an endosymbiont. Fungal sporulation strictly depends on the presence of endosymbionts as well as bacterially produced secondary metabolites. However, an influence of primary metabolites on host control remained unexplored. Recently, we discovered that M. rhizoxinica produces FO and 3PG-F420, a derivative of the specialized redox cofactor F420. Whether FO/3PG-F420 plays a role in the symbiosis has yet to be investigated. Here, we report that FO, the precursor of 3PG-F420, is essential to the establishment of a stable symbiosis. Bioinformatic analysis revealed that the genetic inventory to produce cofactor 3PG-F420 is conserved in the genomes of eight endofungal Mycetohabitans strains. By developing a CRISPR/Cas-assisted base editing strategy for M. rhizoxinica, we generated mutant strains deficient in 3PG-F420 (M. rhizoxinica ΔcofC) and in both FO and 3PG-F420 (M. rhizoxinica ΔfbiC). Co-culture experiments demonstrated that the sporulating phenotype of apo-symbiotic R. microsporus is maintained upon reinfection with wild-type M. rhizoxinica or M. rhizoxinica ΔcofC. In contrast, R. microsporus is unable to sporulate when co-cultivated with M. rhizoxinica ΔfbiC, even though the fungus was observed by super-resolution fluorescence microscopy to be successfully colonized. Genetic and chemical complementation of the FO deficiency of M. rhizoxinica ΔfbiC led to restoration of fungal sporulation, signifying that FO is indispensable for establishing a functional symbiosis. Even though FO is known for its light-harvesting properties, our data illustrate an important role of FO in inter-kingdom communication.

β(1,3) β(1,6) glucogalactan from Rhizopus microsporus var. oligosporus: extraction, characterization, antioxidant and α-amylase inhibitory activities.

In this study, the Box-Behnken experimental planning was used to optimize the extraction of polysaccharides from the cell wall of Rhizopus microspore var. oligosporus, with analysis of the quantitative effects of parameters pH, temperature and extraction time for polysaccharide yield. The optimal conditions for extraction were determined by the regression equation and evaluation of the response surface graphs, which indicated: pH 13, temperature of 120ºC and time of 60 min, with maximum yield around 18.5%. Fourier transform infrared spectroscopy analysis indicated typical polysaccharide signals. Nuclear magnetic resonance spectroscopy and monosaccharide analysis indicated a β(1,3) β(1,6) glucogalactan. The polysaccharide exhibited an average molecular weight of 120 kDa and a polymerization degree of 741. Antioxidant assays in vitro revealed the potential of polysaccharide in elimination of ABTS+ radical and hydroxyl radicals. EC50 values for free radical elimination were 7.69 and 17.8 mg/mL, for ABTS+ and hydroxyls, respectively. The polysaccharides showed potential for α-amylase inhibition with an EC50 of 1.66 mg/mL. The results suggest that β(1,3) β(1,6) glucogalactan from Rhizopus microsporus var. oligosporus can be used in biotechnological applications.

The etiological structure of mucormycetes isolated from children with oncohematological pathology with description of the clinical case of invasive mucomycosis in patients with impaired immunity

Introduction: Mucormycosis is one of the fungal infections caused by fungi (mucormycetes) belonging to the order Mucorales and are mainly part of the genera Apophysomyces, Cuniiinghamella, Lichthemia [formerly Absidia], Mucor, Rhizopus, Rhizomucor, Saksenaea. The causative agents of mucormycosis are widespread in the environment. Fungal infection is manifested by rapidly developing lesions of the skin, mucous membranes, internal organs, and tissues. The most common forms of infection are the rhinoorbital-cerebral and pulmonary forms caused by pathogens of mucormycosis. Gastrointestinal, skin, and disseminated forms of fungal diseases are also registered. The mortality rate in mucormycosis reaches 70% in some cases and depends on the clinical form and the timeliness of the therapy initiated. The diagnosis of mucoromycosis is based on the complex application of various approaches including traditional seeding of biological material, histological analysis of material from affected loci, X-ray, and molecular biological studies. In terms of the treatment of this type of fungal infection, the use of amphotericin B, posaconazole, izavuconazole in combination with surgical rehabilitation of affected tissues has the greatest effectiveness. Purpose: Identification and analysis of the etiological structure of mucormycetes isolated from children with oncohematological pathology and impaired immunity as well as a description of the clinical case of concomitant generalized gastrointestinal mucormycosis against the background of the underlying disease associated with disorder of the immune system. Material and methods: The study includes the results of the isolation of mold fungi (including pathogens of mucoromycosis) from clinically significant biological material for the period from January 2002 to June 2023. To isolate the fungal microflora, traditional microbiological sowing of biomaterial was used followed by generic and specific (in some cases) identification using phenotypic, molecular biological, and mass-spectrometric types of laboratory analysis. When describing the case of invasive mucormycosis, methods of endoscopic and pathomorphological diagnostics were additionally used. Results: The etiological structure of mucoromycetes identified in children with oncohematological pathology and impaired immunity has been studied. As a result, 85 strains of mycelial microflora were isolated from clinically significant biological material obtained from the respiratory organs and respiratory tract (lung biopsies, contents of bronchoalveolar lavage (BAL), and tracheobronchial drainage), which accounted for 30.69% of 277 identified mold micromycetes. At the same time, 12 strains of mucormycetes were identified, which accounted for 14.12% of the total number of fungi found. The isolated mucormycetes were representatives of the genera Mucor, Rhizopus, Rhizomucor, and Lichtheimia [Absidia]. There were also the isolated cases of detection of mucoromycosis pathogens in blood (one strain of Mucor spp. and one strain of Rhizopus spp. out of 222 hemocultures), in urine (one strain of Mucor out of 113 ureacultures), in liver biopsies (one strain of Rhizopus spp.) and gastrointestinal (two strains of Rhizopus microsporus). The clinical case of invasive mucormycosis of the gastrointestinal tract (GI tract), which occurred in a patient with Nijmegen syndrome in the post-transplant period against the background of a coronavirus infection, is described. Conclusion: The total proportion of mucormycosis pathogens (detected in the lungs and in the contents of the respiratory tract) among the total number of filamentous micromycetes reaches 14.12%. The complex application of various diagnostic approaches (including microbiological seeding of biomaterial, X-ray, pathohistochemical and molecular biological studies) makes it possible to reliably verify invasive mucormycosis. In patients with immune defects, despite intensive antifungal therapy, mucormycetes can cause severe forms of invasive fungal infection, up to a fatal outcome.