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BMC Complementary Medicine and Therapies Sep 2023The emergence of multidrug-resistant pathogens and the lack of new antimicrobial drugs is a major public health concern that needs urgent and innovative solutions....
Endophytic fungal species Nigrospora oryzae and Alternaria alternata exhibit antimicrobial activity against gram-positive and gram-negative multi-drug resistant clinical bacterial isolates.
BACKGROUND
The emergence of multidrug-resistant pathogens and the lack of new antimicrobial drugs is a major public health concern that needs urgent and innovative solutions. Endophytic fungi living in unique niches such as in endosymbiosis with plants are increasingly drawing attention as alternative sources of novel and chemically diverse compounds with unique mechanisms of action.
METHODS
In the present study, ten endophytic fungi isolated from the medicinal plant, Sclerocarya birrea were screened for bioactivity against a panel of indicator bacteria. Three bioactive endophytic fungi (strains P02PL2, P02MS1, and P02MS2A) were selected and identified through ITS-rDNA sequencing. The whole broth extracts of the three selected isolates were further screened against contemporary drug-resistant bacterial pathogens. This was followed by partial purification by solid phase extraction and GC-MS analysis of bioactive fractions.
RESULTS
The bioactive endophytic fungi were identified as Alternaria alternata species (strains P02PL2 and P02MS1) and Nigrospora oryzae (strain P02MS2A). The whole broth extracts from N. oryzae P02MS2A exhibited a MIC of one μg/mL and 16 μg/mL against gram-negative, MDR Pseudomonas 5625574 and gram-positive MRSA 25775 clinical isolates, respectively. After partial purification and GC-MS analysis of whole broth extract from A. alternaria PO2MS1, 2-fluorobenzoic acid heptadecyl was putatively identified as the active compound in fraction C of this extract. This compound was also putatively identified in fraction E of A. alternata P02PL2, fraction B of A. alternata P02MS1 and fraction B of N. oryzae P02MS2A, and interestingly, all these fractions retained activity against the two MDR clinical isolates.
CONCLUSION
The putative identification of 2-fluorobenzoic acid heptadecyl compound showing a broad-spectrum of activity, more especially against gram-negative MDR contemporary pathogens is highly encouraging in the initiative at developing novel drugs to combat multi-drug resistance.
Topics: Alternaria; Plant Extracts
PubMed: 37715184
DOI: 10.1186/s12906-023-04157-8 -
Journal of Fungi (Basel, Switzerland) Feb 2024Rice production in the Anhui province is threatened by fungal diseases. We obtained twenty-five fungal isolates from rice and wild rice leaves showing leaf spot disease...
Rice production in the Anhui province is threatened by fungal diseases. We obtained twenty-five fungal isolates from rice and wild rice leaves showing leaf spot disease collected along the Yangtze River. A phylogenetic analysis based on internal transcribed spacer (ITS), translation elongation factor 1 alpha (), and beta tubulin () sequences revealed one isolate (SS-2-JB-1B) grouped with , one (QY) with , twenty-two with , and one isolate (QY-2) grouped in its own clade, which are related to but clearly different from . Nineteen tested isolates, including sixteen strains from the clade and the three isolates of the other three clades, caused disease on detached rice leaves. The three isolates that did not belong to were also able to cause disease in rice seedlings, suggesting that they were rice pathogens. Isolate QY-2 differed from the other isolates in terms of colony morphology, cell size, and susceptibility to fungicides, indicating that this isolate represents a new species that we named . Our analysis showed that , , and the new species, , can cause rice leaf spot disease in the field. This research provides new knowledge for understanding rice leaf spot disease.
PubMed: 38392829
DOI: 10.3390/jof10020156 -
American Journal of Ophthalmology Case... Jun 2024We report a rare case of microbial keratitis caused by .
PURPOSE
We report a rare case of microbial keratitis caused by .
OBSERVATIONS
A 72-year-old Japanese woman was injured by plant debris and developed oval corneal ulcers and hypopyon in the anterior chamber. After 5 days, she complained of pain, redness, and vision loss in her left eye and was treated with antibacterial eye drops and an ointment (1.5 % levofloxacin hydrate, cefmenoxime hydrochloride, and sterilization and disinfection eye drops; SAN-IODE and ofloxacin ophthalmic ointment). Examination revealed a worsening oval corneal ulcer with Descemet's folds and a faint hypopyon. Considering the infection from soil or plants and the poor response to intensive antibacterial eye drops, topical antifungal eye drops, i.e., 1 % voriconazole eye drops, and 1 % natamycin ointment were applied. Direct microscopy of the corneal scraping with Gram staining was performed and the result was negative. Cultures from corneal scrapings showed the growth of dark colonies after several days. The colony was identified as by sequencing of the fungal internal transcribed spacer region. Pain and vision loss improved with improvement in corneal ulcers. The antifungal treatment was administered for 37 days. Discontinuation of the eye drops after 1 month did not result in keratitis recurrence. At the final follow-up at 70 days, the best-corrected visual acuity was 20/25, with persistent small corneal opacity.
CONCLUSIONS AND IMPORTANCE
Here, we report a case of fungal keratitis caused by . Microbiological identification of the causes of rare infections is difficult in clinical laboratories, necessitating the use of advanced molecular techniques based on amplification and sequencing of appropriate phylogenetic markers. responds to topical voriconazole and natamycin.
PubMed: 38665418
DOI: 10.1016/j.ajoc.2024.102062 -
Mycobiology 2023(, ) consists of species of terrestrial plant endophytes and pathogens. has also been reported in marine environments such as mangroves, sea fans, and macroalgae....
(, ) consists of species of terrestrial plant endophytes and pathogens. has also been reported in marine environments such as mangroves, sea fans, and macroalgae. However, limited research has been conducted on associated with macroalgae. Here, we isolated species from three types of algae (brown, green, and red algae) from Korean islands (Chuja, Jeju, and Ulleung) based on phylogenetic analyses of multigenetic markers: the internal transcribed spacers (ITS), beta-tubulin (), and translation elongation factor 1 (). A total of 17 strains were isolated from macroalgae and identified as nine distinct species. The majority of species (seven) were found on brown algae, followed by red algae (three), and then green algae (two). To our understanding, this study represents the first account of , , , , , , and occurring in marine environments. Additionally, this study provides the first report of the occurrence of , , , , and in South Korea. This study will provide valuable insights for future research exploring the functions of fungi in macroalgal communities.
PubMed: 38179117
DOI: 10.1080/12298093.2023.2283272 -
Plant Disease Sep 2023Brachiaria Griseb is an important gramineous forage grown in tropical regions, and also a main grass species uses to restore grasslands in tropical and subtropical...
Brachiaria Griseb is an important gramineous forage grown in tropical regions, and also a main grass species uses to restore grasslands in tropical and subtropical regions of China. In August 2022, symptoms of leaf blight were observed on nearly 30% of the Brachiaria forage grass in the base of the Chinese Academy of Tropical Agricultural Sciences, Hainan, China. Symptomatic leaves initially exhibited small, reddish-brown, round or oval spots on their tips, subsequently expanding in size along the leaf margin, and gradually becoming wilted and dry. Twenty leaves showing typical symptoms were randomly collected and pieces (5×5 mm) from the junction of diseased and healthy region were cut, sterilized with 75% alcohol for 30 s, followed by 5% sodium hypochlorite for 30 s. Rinsed three times with sterile water and dried with sterile filter paper. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 28℃. The colonies were white on the surface and gray on the reverse side. The conidiogenous cells were monoblastic, hyaline, globose or ampulliform, and 6 to 8.7(13.1) ×5 to 7.2 (9) m (n=200). Conidia is single celled, smooth, black, spherical, or ellipsoidal, and (11)13 to 16.5 × (8.2) 10.3 to16.1 m (n=100). Setae were not observed. The morphological characteristics of the isolates were consistent with Nigrospora species. A representative isolate (LNH-5) was selected for genomic DNA extraction. Sequences of the transcribed spacer region of rDNA (ITS), partial translation elongation factor (TEF1), and beta-tubulin fragment (TUB) were amplified using primer pairs ITS1/ITS4(White et al. 1990), EF-728F and EF-986R (Carbone et al. 1999) and Bt2a and Bt2b (Glass et al. 1995), respectively. The sequences of ITS (OQ473493), TEF1 (OQ506059) and TUB gene (OQ506055) were submitted to GenBank. They were 99 to 100% identical to the Nigrospora hainanensis ITS(OM283581.1)(538 out of 519 bp),TEF1(YK019415.1)(274 out of 276 bp),and TUB (OK086377.1)(405 out of 405 bp) sequences. The phylogenetic maximum likelihood analysis using the combined ITS, TEF1 and TUB sequences indicated that the isolate was part of the N. hainanensis clade (100% bootstrap value) that also contained the type isolate LC6979 of this species. Pathogenicity was tested on 15 healthy Brachiaria plants. Fungal conidia were harvested by flooding two-week-old single conidial cultures with sterile water, centrifuging, and adjusting the concentration to 107 spores/mL. Then 10 μL of conidial suspension was dropped onto the surfaces of leaves wounded with a sterile needle. Sterile distilled water was used for control treatment. The test was repeated three times. After inoculation, the plants were kept at 90~100% relative humidity at 25 to 28°C in a greenhouse for two weeks, and monitored daily for lesion development. Seven days post inoculation, all the inoculated leaves presented symptoms similar to those observed under natural conditions, while the control leaves showed no symptoms. The fungus was re-isolated from the diseased tissues by the single spore isolation method (Choi et al. 1999) to complete Koch's postulates. This pathogen has been reported on sugarcane in China (Raza et al., 2019; Zheng et al., 2022). To our knowledge, this is the first report of N. hainanensis causing leaf blight on Brachiaria plants in China.
PubMed: 37729647
DOI: 10.1094/PDIS-03-23-0558-PDN -
Journal of Fungi (Basel, Switzerland) Jan 2024is one of several fungal pathogens known to cause brown streaks, leaf spots, and latent infections in rice. In this study, the entire 42.09-Mb genome of was sequenced...
is one of several fungal pathogens known to cause brown streaks, leaf spots, and latent infections in rice. In this study, the entire 42.09-Mb genome of was sequenced at a depth of 169× using the Oxford Nanopore Technologies platform. The draft genome sequence was comprised of 26 scaffolds, possessed an average GC content of 58.83%, and contained a total of 10,688 protein-coding genes. Analysis of the complete genome sequence revealed that CAZyme-encoding genes account for 6.11% of all identified genes and that numerous transcription factors (TFs) associated with diverse biological processes belong predominantly to Zn-clus (22.20%) and CH (10.59%) fungal TF classes. In addition, genes encoding 126 transport proteins and 3307 pathogen-host interaction proteins were identified. Comparative analysis of the previously reported reference strain GZL1 genome and the genome of a representative strain ZQ1 obtained here revealed 9722 colinear genes. Collectively, these findings provide valuable insights into genetic mechanisms and phenotypic characteristics.
PubMed: 38392772
DOI: 10.3390/jof10020100 -
Poultry Science Nov 2023The outbreak of COVID-19 reminds people that aerosols have an important impact on health. The concentration and composition of microbial aerosol in livestock and poultry...
The outbreak of COVID-19 reminds people that aerosols have an important impact on health. The concentration and composition of microbial aerosol in livestock and poultry houses are closely related to the environmental conditions of livestock and poultry houses, and also related to the healthy growth of livestock and poultry. In our study, 16S and ITS sequencing techniques were used to analyze the relation and difference of bacteria and fungi in the air samples of a chicken house. At the age of 7 to 42 d, the operation classification unit (OTU) numbers of bacteria and fungi identified in our results were 2,398 and 986, respectively, of which the shared OTU numbers were 410 and 141, respectively. At the phylum level, Firmicutes, Proteobacteria, and Actinomycetes were the 3 most abundant bacterial phyla, and Ascomycetes and Basidiomycetes were the top 2 phyla in fungi. At the genus level, 7 differential fungal genera were identified, including Debaryomyces, Trichosporon, Wallemia, Aspergillus, Nigrospora, Fusarium, and Vishniacozyma. Compared with other bacterial genera, Lactobacillus, Cetobacterium, and Romboutsia had the highest abundance (more than 5%). The result showed that the Alpha diversity and Beta diversity of fungi were significantly different in different growing periods. However, only Beta diversity showed significant differences among bacteria. In general, the bacterial and fungal diversity of microbial aerosols in the chicken house increased significantly at the age of 7 to 42 d. And the evenness and richness of airborne fungal communities also increased obviously. In a word, we must pay attention to the complex community composition in the chicken house, this is closely related to animal health and the health of surrounding residents. The cooperation and communication between bacteria and fungi in PM2.5 samples provides a new reference to analyze the influence of microbial aerosol.
PubMed: 37729676
DOI: 10.1016/j.psj.2023.103039 -
Plant Disease Dec 2023Zanthoxylum bungeanum Maxim., a deciduous shrub in Zanthoxylum genus of the Rutaceae family, has not only highly economical values as condiment and medicine, but also...
Zanthoxylum bungeanum Maxim., a deciduous shrub in Zanthoxylum genus of the Rutaceae family, has not only highly economical values as condiment and medicine, but also significantly ecological values in soil and water conservation. In March 2023, a typical leaf spot disease on Z. bungeanum (Variety "Xiao Qingjiao") was observed in the field with an area of 26.68 ha with 35% incidence and 25.4% disease intensity in Zhenfeng County (25°38'57.60″ N, 105°64'98.64″ E, 1,156 m), Guizhou Province, China. The symptom leaves showed as irregularly shaped necrotic lesions, brown to dark brown with black margin. 30 samples with typical symptoms were collected and cut into 0.5 cm × 0.5 cm pieces. Their surfaces were disinfected with 1.5% NaClO for 2 min followed by 75% ethanol for 35 s, rinsed three times with sterile distilled water, finally incubated on PDA plates at 27°C. A total of 36 isolates were obtained through single-spore cultivation. The colonies on PDA were fluffy with abundant aerial mycelia and covered the whole plates (diameter 90 mm) in 7 days. Conidia were brown to black, single-celled, smooth, spherical or oblate, 12.0-17.0 × 12.5-18.5 μm (av. = 14.5 × 15.5 μm, n = 50) and grew on a colorless transparent vesicle at the apical cell of conidiophores. The morphological characteristics were similar with N. sphaerica (Wang et al. 2017). The 5.8S DNA (ITS), translation elongation factor 1-alpha (TEF1-α) and β-tubulin (TUB2) genes were amplified with primers ITS4/ITS5, EF1-728F/EF2, and BT2A/BT2B, respectively (White et al. 1990; Carbone and Kohn 1999, O'Donnell et al. 1998; Glass and Donaldson 1995). The ITS, TEF1-α and TUB2 sequences of two randomly selected isolates, GUCC 21-187 and GUCC 21-235, had > 99% nucleotide identities (ITS: 99.60% (504/506 bp, OR646539) and 99.61% (506/508 bp, OR640300); TEF: 100% (470/470 bp, OR654285) and 100.00% (471/471 bp, OR654286); TUB: 100.00% (408/408 bp, OR661269) and 99.52% (411/413 bp, OR661270), respectively) with those sequences of N. sphaerica (LC 7294) in GenBank (KX985932, KY019397 and KY019602, respectively). The phylogenetic tree based on sequences of ITS, TEF1-α and TUB2 indicated that GUCC 21-187 and GUCC 21-235 were most closely related to N. sphaerica (LC 7294), supported with 100%/100%/1 bootstraps. Based on morphological characteristics and molecular datasets analyses, the isolates were identified as N. sphaerica. 10 healthy 2-years-old Z. bungeanum plants were sprayed with conidial suspensions (1 × 106 conidia/mL) of the isolates and the other 5ere sprayed with sterile water as the controls, all the treated plants were cultivated in a glasshouse at 25°C under 85% relative humidity. Typical leaf spot symptoms appeared on inoculated Z. bungeanum plants after 8 days, while the control plants remained asymptomatic. N. sphaerica was re-isolated from the lesions of inoculated plants and identified by morphological and molecular identification. Pathogenicity test was performed three times with analogous results, fulfilling Koch's postulates. N. sphaerica had been reported as a common pathogen on a variety of plants including sugarcane, kiwifruit and blueberry (Cui et al. 2018; Chen et al. 2016; Wright et al. 2008). To our knowledge, this is the first report of leaf spot disease caused by N. sphaerica on Z. bungeanum in China. Our report would be helpful to Z. bungeanum growers to recognize this leaf spot disease, and corresponding measures could be taken to minimize or avoid the economic losses caused by it.
PubMed: 38105455
DOI: 10.1094/PDIS-10-23-2214-PDN -
Plant Disease Apr 2024Dragon fruit (Selenicereus monacanthus), renowned for its economic value and dual utility in both culinary and medicinal applications, is predominantly cultivated in...
Dragon fruit (Selenicereus monacanthus), renowned for its economic value and dual utility in both culinary and medicinal applications, is predominantly cultivated in China. In July 2023, a stem spot disease was found on dragon fruit ("Zi honglong" cultivar) plants with 37% incidence, in Huajiang Town (N25°40', E105°39'), Guanling County, Anshun City, Guizhou Province. The symptoms appeared as yellow spots surrounded by watery stains, then the spots expanded to suborbicular, which finally led stem to wither. Twelve symptomatic stem samples were collected in a 1.3-hectare plantation and cut into small pieces (5 mm × 5 mm), sterilized the surface with 75% ethanol for 30 seconds, washed 3 times with disinfected distilled water, moved to potato dextrose agar (PDA) medium, and incubated at 28°C for 5 days. Once the mycelium had developed, they were transferred to another PDA medium and cultured at 28°C for a period of 3-5 days. Totally fifteen identical strains were isolated, their colonies were white and round in shape; hyphae were smooth, hyaline; conidia were globose or subglobose, smooth, aseptate, 5.8-11.9 × 4.2-10.6 μm (av. = 8.8 × 7.4 μm, n = 30), light brown in early stage and gradually turning black over time; sterile cells were terminal on hyphae, pale to dark brown, uhceiform or oval, 17.5-21.6 × 4.6-8.7 μm (av. = 19.4 × 5.8 μm, n = 30). The morphologic characteristics of the isolates matched Nigrospora chinensis described by Wang et al. (2017). The PCR amplification was carried out by 3 primers ITS1/ITS4 (Vilgalys et al. 1990; White et al. 1990), EF1-728/EF2 (O'Donnell et al. 1998; Carbone et al. 1999) and BT2A/BT2B (Glass et al. 1995) belonged to the internal transcribed spacer (ITS), translation elongation factor-1 (TEF1) and β-tubulin (TUB2) gene loci, respectively. The sequences of a representative strain (GUCC 524) had 99.38% (ITS: 483/486 bp, PP391347 vs KX985970), 99.79% (TEF1: 482/483 bp, PP400678 vs KY019427) and 99.73% (TUB2: 372/373 bp, PP400677 vs KY019497) identities with those of N. chinensis (strain LC 3085). The phylogenetic tree constructed by three gene combinations showed that GUCC 524 was significantly clustered with N. chinensis. Ten 6-month-old dragon fruit ("Zi honglong" cultivar) seedlings were engrafted with 10 μL conidial suspension (1×105 conidia/mL), packaged with sealed film, two of them were inoculated with sterile distilled water as controls, and placed in a greenhouse at 28℃ for 10 days, inoculated plants showed yellow spots analogous to field symptoms, no symptoms were found in control plants. This experiment was repeated three times. Morphological character and molecular identification based on 3 gene loci of the strains isolated from the inoculated stems, were consistent with those of the original isolated strains. Therefore, based on morphological identification, phylogenetic analysis and pathogenicity test, the pathogen was identified as N. chinensis. Our study firstly reported N. chinensis as a pathogen causing stem spot disease on dragon fruit. N. chinensis is an important agent resulting for economic losses, previously reported on Camellia sinensis (Wang et al. 2017), Saccharum officinarum (Raza et al. 2019), Aucuba japonica (Qin et al. 2021). This report establishes a pivotal reference point for the progression of scientific strategies in preventing and controlling this disease associated with N. chinensis.
PubMed: 38679592
DOI: 10.1094/PDIS-02-24-0485-PDN -
Microorganisms Sep 2023× Ramat. is a famous perennial herb with medicinal, edible, and ornamental purposes, but the occurrence of plant diseases can reduce its value. A serious disease that...
× Ramat. is a famous perennial herb with medicinal, edible, and ornamental purposes, but the occurrence of plant diseases can reduce its value. A serious disease that caused leaf spots in appeared in 2022 in Tongxiang City, Zhejiang Province, China. The leaves with brown spots were collected and used for pathogen isolation. By completing Koch's postulates, it was proven that the isolate had pathogenicity to infect . It was determined that the pathogen isolated from chrysanthemum leaves was , through morphology and a multilocus sequence analysis method using a combination of the internal transcribed spacer gene (ITS), beta-tubulin gene (TUB2), and translation elongation factor 1-alpha gene (TEF1-α). This is the first report of disease caused by in the world. Through dual culture assay on PDA plates, 12 strains of bacteria with antagonistic effects were selected from 231 strains from the phyllosphere, among which D65 had the best inhibitory effect on growth. In addition, the components of a strain D65 fermentation broth were profiled by SPME-GC-Q-TOF analysis, providing a foundation for further application and research of biological control.
PubMed: 37764068
DOI: 10.3390/microorganisms11092224