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Plant Disease Jul 2023Dinteranthus vanzylii is a low-growing species in the family Aizoaceae, native to southern Africa, with a pair of thick grey leaves covered with dark red spots and...
Dinteranthus vanzylii is a low-growing species in the family Aizoaceae, native to southern Africa, with a pair of thick grey leaves covered with dark red spots and stripes. This stone-like succulent grows near the ground, which may protect it from water evaporation and herbivores. Dinteranthus vanzylii has become popular in China due to its attractive appearance and easy indoor cultivation. In September 2021, 7% of D. vanzylii (approximately 140 pots) showed leaf wilt symptoms in a commercial greenhouse located in Ningde (119°35'39.696″E, 27°23'30.556″N), Fujian Province, China. The diseased plants were shrivelling and eventually underwent necrosis. Their leaf tissues were rotting and carpeted with white mycelium. The leaf tissues of 10 symptomatic plants were cut into 0.5 cm2 pieces, surface-sterilized and placed on PDA medium. According to the colony morphology after 7 days of culture, 20 fungal isolates with abundant whitish aerial mycelium were divided into two types: 8 isolates produced lilac pigment whereas 12 did not. Both produced unicellular ovoid microconidia, sickled-shaped macroconidia with 3 - 4 septa and single or paired smooth, thick-walled chlamydospores on carnation leaf agar (CLA). Molecular identification based on DNA sequences from EF1-α (O'Donnell et al. 1998), RPB1 and RPB2 (O'Donnell et al. 2010) revealed 100% identity among isolates within each group; however, there were several base differences between two types. Sequences of representative isolates KMDV1 and KMDV2 were deposited in GenBank (acc. nos.: OP910243, OP910244, OR030448, OR030449, OR030450 and OR030451), which showed 99.10% - 99.74% identity with different F. oxysporum strains (GenBank acc. nos.: KU738441, LN828039, MN457050, MN457049, ON316742 and ON316741). Phylogenetic tree inferred from the concatenated EF1-α, RPB1 and RPB2 revealed that these isolates clustered with F. oxysporum. Thus, these isolates were identified as F. oxysporum. Using a root-drenching method, 10 one-year-old healthy D. vanzylii were inoculated in conidial suspensions (1*106 conidia/mL) of isolates KMDV1 and KMDV2 for 60 min, respectively. They were transplanted into pots with sterilized soil and incubated in a plant-growth chamber at 25°C and 60% relative humidity. Control plants were treated with sterilized water. The pathogenicity test was repeated three times. All plants inoculated with each isolate developed leaf wilt symptoms after 15 days and were dead after 20 - 30 days. However, no symptoms were observed in the control plants. Fusarium oxysporum was reisolated and confirmed based on morphology and EF1-α sequence analysis. No pathogens were isolated from the control plants. This is the first report of F. oxysporum causing leaf wilt disease on D. vanzylii in China. To date, several diseases have been reported on members of the Aizoaceae. For instance, collar and stem rot on Lampranthus sp. caused by Pythium aphanidermatum (Garibaldi et al. 2009), wilt on Lampranthus sp. and Tetragonia tetragonioides caused by Verticillium dahliae (Garibaldi et al. 2010; Garibaldi et al. 2013), and leaf spot on Sesuvium portulacastrum caused by Gibbago trianthemae (Chen et al., 2022). Our research could provide insight into fungal diseases on members of the Aizoaceae and contribute to their cultivation and management.
PubMed: 37436212
DOI: 10.1094/PDIS-05-23-0983-PDN -
Nutrients Jan 2024(CB) and (PI) have anti-inflammatory, immune regulation, anti-tumor, and other functions. This study aimed to explore the therapeutic effect of CB and mycelium of PI...
(CB) and (PI) have anti-inflammatory, immune regulation, anti-tumor, and other functions. This study aimed to explore the therapeutic effect of CB and mycelium of PI (MPI) alone and in combination on colitis mice induced by dextran sodium sulfate (DSS). Mice were randomly assigned to five groups: (1) control (CTRL), (2) DSS, (3) CB, (4) MPI, and (5) CB + MPI (CON). The weight of the mice was recorded daily during the experiment, and the length of the colon was measured on the last day of the experiment. The colons were collected for hematoxylin and eosin staining, colon contents were collected for intestinal flora analysis, and serum was collected for metabolite analysis. The results showed that compared with the DSS group, CB, MPI, and CON treatments inhibited the weight loss and colon length shortening caused by DSS, significantly increased the concentrations of interleukin (IL)-4, IL-10, and superoxide dismutase, and significantly decreased the concentrations of IL-6, tumor necrosis factor-α, and myeloperoxidase. Gene sequence analysis of 16S rRNA showed that CB, MPI, and CON treatments changed the composition and structure of intestinal microorganisms. Metabolome results showed that CB, MPI, and CON treatments changed serum metabolites in DSS-treated mice, including dodecenoylcarnitine, L-urobilinogen, and citric acid. In conclusion, CB, MPI, and CON treatments alleviated DSS-induced colitis in mice by regulating intestinal flora and metabolites, with the CON group having the best effect.
Topics: Animals; Mice; Clostridium butyricum; Gastrointestinal Microbiome; RNA, Ribosomal, 16S; Colitis; Mycelium; Phellinus
PubMed: 38201981
DOI: 10.3390/nu16010153 -
Plant Disease Dec 2023In August 2020 chili (Capsicum annuum L.) showing wilt symptoms were collected from different districts of the Kashmir: Pulwama, Srinagar, Baramulla, and Anantnag. From...
In August 2020 chili (Capsicum annuum L.) showing wilt symptoms were collected from different districts of the Kashmir: Pulwama, Srinagar, Baramulla, and Anantnag. From each district one location was selected for sample collection and a total of 23 chili isolates were isolated. The tissue bit technique was used to isolate fungus from the infected samples on potato dextrose agar (PDA) medium, purified using the single spore technique, maintained at 25⁰±1℃ and then stored at 4° C (Ferniah et al. 2014) . Initially cultural characteristics appeared as white colonies which gradually turned to pale white colored and attained a growth of 90 mm in 18 days of incubation at 25 ± 1°C. Microscopic observations revealed that mycelium was branched and cylindrical, 3.53-4.98 µm in width. Microconidia were ellipsoidal, hyaline, 0-1 septa werepresent, and 6-7 x 3-4 µm in size. Macroconidia were cylindrical, hyaline, 2-6 septa, measuring 20-60 x 40-45 µm in size. Molecular identification of the pathogens with ITS, TEF, and RPB2 was successfully carried out and the fungi was confirmed as Fusarium flocciferum infecting chili. Amplified PCR products were sequenced and were successfully submitted and accessioned in GenBank with accession number OM189458, OM441199, OR484037 for ITS, TEF, and RPB2 gene. To confirm Koch's postulates pathogenicity test was carried out using rhizosphere inoculation technique (Najar et al. 2011, Parihar et al. 2022). In total 7 replications for sand maize meal medium (potting mixture) was prepared by autoclaving 90 g of sand and 10 g of maize meal in 250 ml of erlenmeyer flask comprising 40 ml of distilled water. The spore suspension at 100 µl per pot was inoculated and was mixed with the sterilized potting mixture in a ratio of (2:1) and up to seven days pathogen was allowed to infect the soil (Davey and Papavizas 1962; Hami et al. 2021). Then chili seeds (cv. Kashmir long-1) were sown in infected potting mixture and grown for three weeks to allow the pathogen to infect the host plants. F. flocciferum took six weeks for appearance of symptoms in the infected potted plants. Control mock inoculation of the potting mixture was carried out using water droplets instead of spore suspension at 100 µl per pot. Seven replications were kept for both inoculated and un-inoculated / control mock pots. The plants developed initial symptoms from light green to yellowish discoloration of leaves followed by the drooping, shriveling, and ultimately leading to death. The collar region of the plant was cut vertically and observed that vascular bundles showed brownish spots and discoloration, indicating wilt as the cause of death. The pathogens were re-isolated and inoculated from all infected plants, then compared with their original pure culture inoculated first, which completely resembled based on morphological, cultural, and pathogenic characteristics. No symptoms were observed on control plants. A phylogenetic analysis was also carried out using ClustalW software that grouped the species identified by different genes into different clades. F. flocciferum has been reported earlier in pea, faba bean and bamboo (Kainthola et al. 2022; Šišić et al. 2020) . In solanaceous crops, this species have been explored as wilt pathogens for the first time from India, indicating diversifying nature of Fusarium flocciferum across various hosts including solanaceous crops.
PubMed: 38105451
DOI: 10.1094/PDIS-09-23-1730-PDN -
Plant Disease Aug 2023Quinoa (Chenopodium quinoa Willd.) is a traditional food originally from the Andes Mountains in South America. It was first planted in China in 1987 and is grown in...
Quinoa (Chenopodium quinoa Willd.) is a traditional food originally from the Andes Mountains in South America. It was first planted in China in 1987 and is grown in Tibet, Gansu, and Qinghai provinces. In May 2021, 40% of 2-month-old quinoa plants in the 3.4 hm² experimental base of Qinghai University (36.7262° N, 101.7487° E) were found to have leaves with grey-brown subcircular spots (about 0.4 to 0.7 cm) with black dots (acervuli). Severely infected plants exhibited symptoms such as withered and stunted growth. The diseased-healthy junctions of infected leaves (0.5 cm) were cut out, disinfected with 3% NaClO for 1.5 min, washed three times with sterile water, dried, placed on water agar, and incubated at 25°C for 48 h. After sporulation was seen on the leaf surface, spore suspensions were prepared by placing conidia in sterile water using a pipette. Next, 200 μl of each spore suspension was spread on the surface of water agar and incubated at 25°C for 12 h. Single spores were selected under a stereomicroscope and cultured on potato dextrose agar (PDA) (Qi et al. 2022). The mycelium of two representative isolates (20DLMF-5-4-1 and 20DLMF-7-4-1) was grey-black with white edges and included a fluffy aerial mycelium. Conidia were unicellular, colorless, long ellipsoid or curved moon shaped, averaging 14.3 × 1.8 to 20.2 × 2.2 μm (n=100). The light brown appressoria were ovoid, averaging 8.5 × 5.2 to 7.7 × 4.1 μm (n=20). Spherical, dark brown acervuli were observed on the leaves, averaging 160 to 200 μm (n=20), and there were dark brown spiny bristles. The ITS, partial ACT, CHS, GAPDH and TUB2 genes were amplified from genomic DNA of the two isolates (Weir et al. 2012). Sequences were deposited in GenBank (accession no. OQ871595 to OQ871602 for ACT, CHS, GAPDH, and TUB2, and OQ860235 to OQ860236 for ITS) and showed over 99% identities with the corresponding sequences of C. spinaciae CBS125347 and CBS128.57 (Vu et al. 2019; Damm et al. 2009). Both isolates clustered with the type culture of C. spinaciae (CBS125347, CBS128.57), with 100% bootstrap support in the phylogenetic tree. Thus, according to the morphological and molecular characteristics, the two isolates were identified as C. spinaciae. Pathogenicity tests were conducted on 24 healthy, tender leaves of six 1-month-old quinoa plants, with three replicates (Yang et al. 2021). The leaves were gently scratched in 3-4 areas with a sterile needle. A conidial suspension (105 conidia/ml) of the two isolates was sprayed on these wounds. The control group was unscratched and sprayed with sterile water. The plants were incubated in a greenhouse at 25°C for 24 h in the dark and 7 days in the light. Tiny grey-brown spots appeared on day 3 (about 0.4 to 0.6 cm) and continued to enlarge until perforations and ruptures developed on day 7. Subsequently, acervuli were observed on the surface of the leaves. The control leaves remained healthy. Isolates were reisolated from the symptomatic leaves and they had the same morphological and molecular characteristics as the original isolates, confirming Koch's postulates. To our knowledge, this is the first report of C. spinaciae causing quinoa leaf anthracnose in China. C. spinaciae seriously affects the yield and quality of quinoa and has been previously reported to cause anthracnose of Vicia sativa in China (Wang et al. 2019). The results provide a basis for the study and control of quinoa leaf anthracnose.
PubMed: 37622274
DOI: 10.1094/PDIS-07-23-1285-PDN -
Journal of Fungi (Basel, Switzerland) Nov 2023As a filamentous pathogenic fungus with high-yield of aflatoxin B, is commonly found in various agricultural products. It is crucial to develop effective strategies...
As a filamentous pathogenic fungus with high-yield of aflatoxin B, is commonly found in various agricultural products. It is crucial to develop effective strategies aimed at the prevention of the contamination of and aflatoxin. Hexokinase AfHxk1 is a critical enzyme in fungal glucose metabolism. However, the role of AfHxk1 in development, aflatoxin biosynthesis, and virulence has not yet been explored. In this study, gene deletion mutant (Δ), complementary strain (Com-), and the domain deletion strains ( and ) were constructed by homologous recombination. Phenotype study and RT-qPCR revealed that AfHxk1 upregulates mycelium growth and spore and sclerotia formation, but downregulates AFB biosynthesis through related classical signaling pathways. Invading models and environmental stress analysis revealed that through involvement in carbon source utilization, conidia germination, and the sensitivity response of to a series of environmental stresses, AfHxk1 deeply participates in the regulation of pathogenicity of to crop kernels and larvae. The construction of domain deletion strains, and , further revealed that AfHxk1 regulates the morphogenesis, mycotoxin biosynthesis, and the fungal pathogenicity mainly through its domain, Hexokinase_2. The results of this study revealed the biological role of AfHxk1 in spp., and might provide a novel potential target for the early control of the contamination of .
PubMed: 37998882
DOI: 10.3390/jof9111077 -
Plant Disease Sep 2023Vigna unguiculata belongs to the Legume family, and is an annual twining, herbaceous vine plant, which is native to Africa. V. unguiculata is the most economically...
Vigna unguiculata belongs to the Legume family, and is an annual twining, herbaceous vine plant, which is native to Africa. V. unguiculata is the most economically beneficial type of off-season vegetables in Hainan, China because of its rich in nutrients such as protein, minerals, dietary fiber, and vitamins (Jayathilake et al. 2018). In April 2022, typical powdery mildew infection was observed on V. unguiculata leaves in Haikou, Hainan Province, China (20°3'40.428"N, 110°19'45.217"E). More than 70% leaves of 13 V. unguiculata plants displayed severe powdery mildew disease. The diseased leaves at first exhibited white rounded irregular patches, which gradually enlarged, fused and covered all the leaf as well as stems. Edges of the infected leaves crinkled upwards, and the leaves often fell off the plants at the late infection stage. On the infected leaves, many conidiophores and dense mycelium were observed by microscopic analysis. Hyphae were septate, branched, epigenous, and flexuous to straight. Hyphal appressoria were indistinct or slightly nipple-shaped, and the haustoria developed were in the shape of oval-sphere and 9 to 11μm long. Foot cells of conidiophores were straight, cylindrical, and measured 43 to 70 × 10 to 12 μm. The conidiophores arising straightly from a hyphal cell, were measured 168 to 252 (mean = 204) μm in length and produced 6 to 9 immature conidia in each chain. Conidia were ovate, pyriform or barrel-shaped, with fibrosin bodies, and measured 26 to 32 (mean = 29.4) × 16 to 20 (mean = 18.6) μm. The chasmothecia was not found in all samples. These morphological characteristics were typical of the conidial stage of the powdery mildew Podosphaera xanthii of genus Podosphaera (Braun and Cook 2012). To further confirm the identity of this causal fungus, the internal transcribed spacer (ITS) region, and the partial sequence of large subunit ribosomal RNA gene (28S rRNA), were amplified with primer pairs ITS1/ ITS4 (White et al. 1990) and NL1/NLP2 (Mori et al. 2000) from extracted genomic DNA. The obtained 563-bp ITS region and 715-bp 28S rRNA gene sequences were deposited in GenBank (ITS, OQ415534; 28S rRNA, OQ415545.1), and were compared with BLAST analysis in the GenBank nr database. The results revealed that the ITS region sequence was 99.82% identity with P. xanthii isolate HUVU-08 (MH143485.1), and the 28S rRNA gene partial sequence was 100% identity with P. xanthii isolate XHL1 (MK357442.1). On the basis of the morphological characteristics and sequence analysis, this fungus was identified as P. xanthii. Pathogenicity tests were performed by gently brushing conidia onto the leaves of six healthy potted V. unguiculata plants. Six non-inoculated plants were used as control. All plants were maintained in a greenhouse at 26 ± 2°C. After 2 weeks inoculation, similar symptoms were observed in the inoculated plants, whereas no symptoms occurred on the control plants. By microscopic observation, the fungus present on the inoculated plants was morphologically identical to those on originally diseased plants. Furthermore, ITS and 28S rRNA sequences of the re-isolated fungus individually displayed 100% identity with OQ415534 and OQ415545.1. So far, although powdery mildew disease caused by P. xanthii on different plants including Sigesbeckia orientalis (Mukhtar et al. 2022), Vigna radiata (Sheu et al. 2021), Cosmos bipinnatus (Kong et al. 2023), Verbena brasiliensis (Luecke et al. 2020), Cucurbita ficifolia (Choi et al. 2022), Glandularia tenera (Pei et al. 2023) and Verbena bonariensis (Choi et al. 2023) have been reported, to our knowledge, this is the first report of powdery mildew caused by P. xanthii on V. unguiculata in Hainan, China, which seriously threatens the utilization of V. unguiculata on off-season vegetables industry.
PubMed: 37669172
DOI: 10.1094/PDIS-07-23-1287-PDN -
Frontiers in Fungal Biology 2023Members of the fungal genus are widely known for their important ecological roles and significant economic value. In this study, we used amplicon and genome sequencing...
Members of the fungal genus are widely known for their important ecological roles and significant economic value. In this study, we used amplicon and genome sequencing to characterize bacterial communities associated with sexual fruiting bodies from wild specimens, as well as vegetative mycelium and sclerotia obtained from isolates grown . These investigations included diverse representatives from both Elata and Esculenta clades. Unique bacterial community compositions were observed across the various structures examined, both within and across individual isolates or specimens. However, specific bacterial taxa were frequently detected in association with certain structures, providing support for an associated core bacterial community. Bacteria from the genus and constituted the core bacterial associates of mycelia and sclerotia, while other genera (e.g., spp., spp., and spp.) constituted the core bacterial community of fruiting bodies. Furthermore, the importance of as a key member of the bacteriome was supported by the isolation of several strains from mycelia during cultivation. Four of the six mycelial-derived isolates shared 16S rDNA sequence identity with amplicon sequences recovered directly from the examined fungal structures. Distinct interaction phenotypes (antagonistic or neutral) were observed in confrontation assays between these bacteria and various isolates. Genome sequences obtained from these isolates revealed intriguing differences in gene content and annotated functions, specifically with respect to toxin-antitoxin systems, cell adhesion, chitinases, and insecticidal toxins. These genetic differences correlated with the interaction phenotypes. This study provides evidence that spp. are frequently associated with and these associations may greatly impact fungal physiology.
PubMed: 38155707
DOI: 10.3389/ffunb.2023.1285531 -
Nutrients Sep 2023Mycelial cultures of , an edible and medicinal mushroom, have been used in our previous research to obtain selenium-containing immunomodulatory preparations. Our current...
Mycelial cultures of , an edible and medicinal mushroom, have been used in our previous research to obtain selenium-containing immunomodulatory preparations. Our current attempts to obtain a new preparation containing both selenium and zinc, two micronutrients necessary for the functioning of the immune system, extended our interest in the simultaneous accumulation of these elements by mycelia growing in media enriched with selenite and zinc(II) ions. Subsequently, we have studied the effects of new mycelium water extracts with different concentrations of selenium and zinc on the activation of T cell fraction in human peripheral blood mononuclear cells (PBMCs). Flow cytometry analysis was used to measure the expression of activation markers on human CD4+ and CD8+ T cells stimulated by anti-CD3 and anti-CD3/CD28 antibodies (Abs). It was demonstrated that statistically significant changes were observed for PD-1 and CD25 antigens on CD8+ T cells. The selenium and zinc content in the examined preparations modified the immunomodulatory activity of mycelial polysaccharides; however, the mechanisms of action of various active ingredients in the mycelial extracts seem to be different.
Topics: Humans; Shiitake Mushrooms; Selenium; Leukocytes, Mononuclear; Dietary Supplements; Mycelium
PubMed: 37764798
DOI: 10.3390/nu15184015 -
Plant Disease Mar 2024In August 2022, powdery mildew symptoms were detected on lettuce (Lactuca sativa) in a commercial field located in Quecholac, Puebla, Mexico. Signs appeared as whitish...
In August 2022, powdery mildew symptoms were detected on lettuce (Lactuca sativa) in a commercial field located in Quecholac, Puebla, Mexico. Signs appeared as whitish powdery masses on leaves. Disease incidence was about 100% and signs covered up to 40% of leaf surface. Mycelium was amphigenous forming white patches. Hyphal appressoria were indistinct or nipple-shaped and solitary. Conidiophores (n= 30) were hyaline, erect, arising from the upper surface of hyphal mother cells or lateral, and of 90 to 201 μm long. Foot cells were cylindrical, of 49 to 92 × 10-15 μm, followed by 1-3 shorter cells, and forming conidia in chains. Conidia (n= 100) were hyaline, ellipsoid-ovoid, doliiform-subcylindrical, 27 to 40 × 14 to 20 μm. Conidial germination belonging to the Euoidium type. Chasmothecia were not observed. The morphological characters were consistent with those of Golovinomyces bolayi (Braun et al. 2019). A voucher specimen was deposited in the Herbarium of the Department of Agricultural Parasitology at the Chapingo Autonomous University under accession number UACH451. To confirm the identification of the fungus, genomic DNA was extracted from conidia and mycelium following the CTAB method (Doyle and Doyle 1990), and the internal transcribed spacer (ITS) region was amplified by PCR using the primers ITS5/ITS4 (White et al. 1990) and sequenced. The resulting 506 bp sequence had 100% identity to those of G. bolayi (LC417109 and LC417106). Phylogenetic analyses using the Maximum Likelihood and Maximum Parsimony methods were performed and confirmed the results obtained in the morphological analysis. The isolate UACH451 grouped in a clade with isolates of G. bolayi. The ITS sequence was deposited in GenBank under accession number OR467546. Pathogenicity was confirmed by gently dusting conidia onto ten leaves of healthy lettuce plants. Five non-inoculated leaves served as controls. The plants were maintained in a greenhouse at 25 to 30 ºC, and relative humidity of 70%. All inoculated leaves developed similar symptoms to the original observation after 10 days, whereas control leaves remained disease free. Microscopic examination of the fungus on inoculated leaves showed that it was morphologically identical to that originally observed. Based on morphological data and phylogenetic analysis, the fungus was identified as G. bolayi. This pathogen has been previously reported causing powdery mildew on lettuce in Argentina, Canada, Chile, Ecuador, Peru, USA and Venezuela (Braun et al. 2019; Mieslerová et al. 2020). To our knowledge, this is the first report of G. bolayi causing powdery mildew on lettuce in Mexico.
PubMed: 38549277
DOI: 10.1094/PDIS-08-23-1658-PDN -
Scientific Reports May 2024The ecological and economic benefits of mycelium composites offer a promising opportunity for supporting sustainable development in Africa. This study focuses on...
The ecological and economic benefits of mycelium composites offer a promising opportunity for supporting sustainable development in Africa. This study focuses on assessing the environmental impact of mycelium composites for building and construction (MCBs) by conducting a life cycle assessment (LCA) in the context of Africa. It is demonstrated that the potential environmental impact of MCBs is substantially influenced by the use and source of electrical power for autoclaves, incubators, and ovens, making the culturing and post-processing phases the major environmental hotspots. The impact of MCB production is also relative to the energy mix of specific countries, being higher in countries that rely on fossil fuel energy (e.g., South Africa) and lower in those that rely more on renewable sources (e.g., Democratic Republic of the Congo, DRC). Furthermore, the impact of MCB production is found to be sensitive to travel distance, suggesting that situating production facilities closer to agricultural, agro-industrial, and/or forestry waste sources could be more beneficial than interregional sourcing, for example. It is also demonstrated that MCBs have the potential to be a more ecologically sustainable alternative to some conventional construction materials (e.g., concrete) over an entire life cycle. Based on the insights obtained from this LCA, some recommendations have been proposed to address potential environmental repercussions pre-emptively and proactively: this is particularly important for nations, mainly in the Global South, that exhibit low resilience to climate change due to limited economic resources. Furthermore, with the rapid expansion of mycelium composite technology, there is a need to increase awareness about its potential environmental impact and, ultimately, to mitigate its potential contribution to pressing environmental concerns (e.g., global warming and climate change). Consequently, this study also adds to the existing body of literature on LCA studies, delineating key factors for consideration in future LCA studies and providing guidance for the sustainable establishment and expansion of this technology.
Topics: Mycelium; Africa; Construction Materials; Environment; Sustainable Development
PubMed: 38789584
DOI: 10.1038/s41598-024-62561-7