Burkholderia ambifaria H8 as an effective biocontrol strain against maize stalk rot via producing volatile dimethyl disulfide.

BACKGROUND: Maize stalk rot (MSR) caused by Fusarium graminearum is the primary factor contributing to the reduction in maize yield and quality. However, this soil-borne disease presents a significant challenge for sustainable control through field management and chemical agents. The screening of novel biocontrol agents can aid in developing innovative and successful strategies for MSR control. RESULTS: A total of 407 strains of bacteria were isolated from the rhizosphere soil of a resistant maize inbred line. One strain exhibited significant antagonistic activity in plate and pot experiments, and was identified as Burkholderia ambifaria H8. The strain could significantly inhibit the mycelial growth and spore germination of F. graminearum, induce resistance to stalk rot, and promote plant growth. The volatile compounds produced by strain H8 and its secondary metabolites in the sterile fermentation broth exhibited antagonistic activity. The primary volatile compound produced by strain H8 was identified as dimethyl disulfide (DMDS) using gas chromatography tandem mass spectrometry. Through in vitro antagonistic activity assays and microscopic observation, it was confirmed that DMDS was capable of inhibiting mycelial growth and disrupting the mycelial structure of F. graminearum, suggesting it may be the major active compound for strain H8. The transcriptome data of F. graminearum further indicated that strain H8 and its volatile compounds could alter pathogenic fungi metabolism, influence the related metabolic pathways, and potentially induce cell apoptosis within F. graminearum. CONCLUSION: Our results showed that B. ambifaria H8 was capable of producing the volatile substance dimethyl disulfide, which influenced the synthesis and permeability of cell membranes in pathogens. Thus, B. ambifaria H8 was found to be a promising biological control agent against MSR. © 2024 Society of Chemical Industry.

Microbiome Signature of Endophytes in Wheat Seed Response to Wheat Dwarf Bunt Caused by Tilletia controversa Kühn.

Wheat dwarf bunt leads to the replacement of seeds with fungal galls containing millions of teliospores of the pathogen Tilletia controversa Kühn. As one of the most devastating internationally quarantined wheat diseases, wheat dwarf bunt spreads to cause distant outbreaks by seeds containing teliospores. In this study, based on a combination of amplicon sequencing and isolation approaches, we analyzed the seed microbiome signatures of endophytes between resistant and susceptible cultivars after infection with T. controversa. Among 310 bacterial species obtained only by amplicon sequencing and 51 species obtained only by isolation, we found 14 overlapping species by both methods; we detected 128 fungal species only by amplicon sequencing, 56 only by isolation, and 5 species by both methods. The results indicated that resistant uninfected cultivars hosted endophytic communities that were much more stable and beneficial to plant health than those in susceptible infected cultivars. The susceptible group showed higher diversity than the resistant group, the infected group showed more diversity than the uninfected group, and the microbial communities in seeds were related to infection or resistance to the pathogen. Some antagonistic microbes significantly suppressed the germination rate of the pathogen's teliospores, providing clues for future studies aimed at developing strategies against wheat dwarf bunt. Collectively, this research advances the understanding of the microbial assembly of wheat seeds upon exposure to fungal pathogen (T. controversa) infection. IMPORTANCE This is the first study on the microbiome signature of endophytes in wheat seed response to wheat dwarf bunt caused by Tilletia controversa Kühn. Some antagonistic microbes suppressed the germination of teliospores of the pathogen significantly, which will provide clues for future studies against wheat dwarf bunt. Collectively, this research first advances the understanding of the microbial assembly of wheat seed upon exposure to the fungal pathogen (T. controversa) infection.

Four New Species of Aspergillus Subgenus Nidulantes from China.

Aspergillus subgenus Nidulantes includes species with emericella-like ascomata and asexual species. Subgenus Nidulantes is the second largest subgenus of Aspergillus and consists of nine sections. In this study, agricultural soils were sampled from 12 provinces and autonomous regions in China. Based on primary BLAST analyses, seven of 445 Aspergillus isolates showed low similarity with existing species. A polyphasic investigation, including phylogenetic analysis of partial ITS, ß-tubulin, calmodulin, and RNA polymerase II second largest subunit genes, provided evidence that these isolates were distributed among four new species (Aspergillus guangdongensis, A. guangxiensis, A. sichuanensis and A. tibetensis) in sections Aenei, Ochraceorosei, and Sparsi of subgenus Nidulantes. Illustrated morphological descriptions are provided for each new taxon.

New species in Aspergillus section Usti and an overview of Aspergillus section Cavernicolarum.

Aspergillus sections Usti and Cavernicolarum are accommodated in the subgenus Nidulantes. In the present study, a polyphasic approach using morphology and multi-gene phylogeny was applied to investigate the taxonomy of these two sections. Based on the phylogenetic analysis, Aspergillus section Usti includes 25 species, which can be assigned to four series: Calidousti, Deflecti, Monodiorum and Usti. Aspergillus sigarelli is newly described in this section and this species was isolated from a cigarette from PR China and belongs to series Calidousti. It is clearly distinct from other members in this series based on ITS, BenA, CaM and RPB2 sequences. Aspergillus section Usti members like A. calidoustus and A. granulosus are important opportunistic pathogens, it is speculative that more pathogenetic species will be found by using polyphasic taxonomy approaches. Aspergillus section Cavernicolarum includes five species, the growth rates on agar media and size and ornamentation of conidia are important characters for differentiating species in section Cavernicolarum.

Microbial Community Changes in the Rhizosphere Soil of Healthy and Rusty Panax ginseng and Discovery of Pivotal Fungal Genera Associated with Rusty Roots.

Panax ginseng Meyer, a valuable medicinal plant, is severely threatened by rusty root, a condition that greatly affects its yield and quality. Studies investigating the relationship between soil microbial community composition and rusty roots are vital for the production of high-quality ginseng. Here, high-throughput sequencing was employed to systematically characterize changes in the soil microbial community associated with rusty roots. Fungal diversity was lower in the soils of rusty root-affected P. ginseng than in those of healthy plants. Importantly, principal coordinate analysis separated the fungal communities in the rhizosphere soils of rusty root-affected ginseng from those of healthy plants. The dominant bacterial and fungal genera differed significantly between rhizosphere soils of healthy and rusty root-affected P. ginseng, and linear discriminant analysis effect size (LEfSe) further indicated a strong imbalance in the soil microbial community of diseased plants. Significantly enriched bacterial genera (including Rhodomicrobium, Knoellia, Nakamurella, Asticcacaulis, and Actinomadura) were mainly detected in the soil of rusty root-affected P. ginseng, whereas significantly enriched fungal genera (including Xenopolyscytalum, Arthrobotrys, Chalara, Cryptococcus, and Scutellinia) were primarily detected in the soil of healthy plants. Importantly, five fungal genera (Cylindrocarpon, Acrophialophora, Alternaria, Doratomyces, and Fusarium) were significantly enriched in the soil of rusty root-affected plants compared with that of healthy plants, suggesting that an increase in the relative abundance of these pathogenic fungi (Cylindrocarpon, Alternaria, and Fusarium) may be associated with ginseng rusty roots. Additionally, this study is the first to report that an increase in the relative abundances of Acrophialophora and Doratomyces in the rhizosphere of P. ginseng may be associated with the onset of rusty root symptoms in this plant. Our findings provide potentially useful information for developing biological control strategies against rusty root, as well as scope for future screening of fungal pathogens in rusty roots of P. ginseng.

Aspergillus Species in Lower Respiratory Tract of Hospitalized Patients from Shanghai, China: Species Diversity and Emerging Azole Resistance.

PURPOSE: To investigate species diversity and prevalence of antifungal resistance among clinical isolates of Aspergillus spp. in Shanghai, China. PATIENTS AND METHODS: In this study, the Aspergillus spp. isolates were analyzed by multilocus sequence typing (MLST) targeting the internal transcribed spacer (ITS) regions, and partial ß-tubulin (BenA) and calmodulin (CaM) genes. The susceptibilities of these isolates to nine antifungal agents were determined according to the protocol in document M38-A3 established by the Clinical and Laboratory Standards Institute (CLSI). RESULTS: The most common Aspergillus spp. was A. fumigatus (58.2%), followed by the A. flavus complex (23.5%), and A. niger complex (15.3%). Isolates belonging to A. tamarii and A. effusus of the A. flavus complex and A. tubingensis and A. awamori of the A. niger complex were identified. Moreover, several mutations were found in the azole target cyp51A gene (TR46/Y121F/T289A and F46Y, G89G, M172V, N248T and D255E) in azole-resistant isolates of A. fumigatus. CONCLUSION: The results of our study revealed a diversity of species in the lower respiratory tract of inpatients in Shanghai and approximately 9% of our isolates were resistant to at least one of the triazole antifungals. Formulation of local treatment strategies to combat emerging azole resistance and species diversity in clinically relevant Aspergillus spp. is needed.

A polyphasic study of Aspergillus section Flavi isolated from corn in Guangxi, China- a hot spot of aflatoxin contamination.

Aspergillus section Flavi is widely known as a potential threat to contaminate agricultural products and food commodities. In this study, a polyphasic approach consisting of micro- and macro-morphological, chemical and molecular features, was applied to survey the Aspergillus section Flavi population in corn collected from Guangxi, China. Based on multigene phylogenies as well as morphological observations, Aspergillus flavus (192/195), A. arachidicola (1/195), A. pseudonomius (1/195) and A. novoparasiticus (1/195) were found to be the predominant section Flavi population. Among them, 31 representative isolates were selected for mycotoxin determination. The results showed that Aspergillus flavus chemotype I was most common, chemotype IV was also detected with low incidence and low CPA amounts, while chemotypes II and III were absent. Other tested species including A. arachidicola, A. pseudonomius, and A. novoparasiticus produced all types of aflatoxins, but none of them produced CPA. The polyphasic approach applied in this study permitted reliable understanding of the prevailing Aspergillus section Flavi population and their mycotoxin profiles. Knowledge of the prevailing section Flavi population will aid in developing a sustainable strategy to mitigate the effects of aflatoxin contamination. This study suggests that CPA contamination of food should be considered while conducting mycotoxigenic surveys of food commodities, and the same should be considered while planning a bio-control strategy to control aflatoxin contamination.

Sequencing, Characterization, and Comparative Analyses of the Plastome of Caragana rosea var. rosea.

To exploit the drought-resistant Caragana species, we performed a comparative study of the plastomes from four species: Caragana rosea, C. microphylla, C. kozlowii, and C. Korshinskii. The complete plastome sequence of the C. rosea was obtained using the next generation DNA sequencing technology. The genome is a circular structure of 133,122 bases and it lacks inverted repeat. It contains 111 unique genes, including 76 protein-coding, 30 tRNA, and four rRNA genes. Repeat analyses obtained 239, 244, 258, and 246 simple sequence repeats in C. rosea, C. microphylla, C. kozlowii, and C. korshinskii, respectively. Analyses of sequence divergence found two intergenic regions: trnI-CAU-ycf2 and trnN-GUU-ycf1, exhibiting a high degree of variations. Phylogenetic analyses showed that the four Caragana species belong to a monophyletic clade. Analyses of Ka/Ks ratios revealed that five genes: rpl16, rpl20, rps11, rps7, and ycf1 and several sites having undergone strong positive selection in the Caragana branch. The results lay the foundation for the development of molecular markers and the understanding of the evolutionary process for drought-resistant characteristics.

Occurrence of Toxigenic Fungi and Mycotoxins on Root Herbs from Chinese Markets.

Herbs derived from roots, leaves, flowers, or fruits of plants are unavoidably contaminated with fungi and mycotoxins during growth, harvest, and storage, thereby posing a health threat to humans. Especially, root herbs (RHs) are more easily contaminated with fungi and mycotoxins because the roots are in direct contact with the soil. Here, we investigated the occurrence of fungi, aflatoxins (AFs), and ochratoxin A (OTA) in eight RHs that are used as medicines, beverages, dietary supplements, and functional foods in China and other countries. Morphological observation and MultiGeneBlast (ß-tubulin and calmodulin) were used to identify the potentially toxigenic fungi. Of the 48 samples tested, all were contaminated by fungi, and 1,844 isolates belonging to 25 genera were detected. The genera Aspergillus and Penicillium, which contain potentially toxigenic fungal species, represented a frequency of 10 and 25%, respectively. Thirty-three isolates of Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger, and Penicillium polonicum were arbitrarily selected for analysis of their toxigenic potential. Five of 13 isolates of A. flavus and 1 isolate of A. parasiticus produced AFs, whereas OTA production was not detected for any of the isolates of A. niger and P. polonicum. The occurrence of AFs and OTA in the 48 samples of eight RHs was tested by ultraperformance liquid chromatography-tandem mass spectrometry; 37.50% of samples from six RHs were contaminated with AFs and 16.67% of samples from four RHs were contaminated with OTA. Seven (14.58%) and four (8.33%) samples of ginseng, polygala, and liquorice exceeded the permissible limits of aflatoxin B1 and AFs, respectively. Because ginseng, polygala, and liquorice are widely used as herbs, dietary supplements, and functional foods, the high frequency of AF contamination of these herbs indicated by our current study warrant attention to raise public awareness.

Mycobiota and Mycotoxins in Traditional Medicinal Seeds from China.

The multi-mycotoxin occurrence for internal and superficial fungi contamination were comprehensively assessed in medicinal seeds used as food or beverage. Based on a polyphasic approach using morphological characters, ß-tubulin and ITS gene blast, a total of 27 species belonging to 12 genera were identified from surface-sterilized seeds. Chaetomium globosporum was most predominant (23%), followed by Microascus trigonosporus (12%) and Alternaria alternata (9%). With respect to superficial mycobiota, thirty-four species belonging to 17 genera were detected. Aspergillus niger and Penicillium polonicum were predominant (12% and 15%, respectively). Medicinal seed samples and potential toxigenic fungi were tested for ochratoxin A (OTA) and aflatoxins (AFB1, AFB2, AFG1, AFG2) using UPLC-MS/MS. Platycladi seeds were contaminated with AFB1 (52.0 µg/kg) and tangerine seed was contaminated with OTA (92.3 µg/kg). Subsequent analysis indicated that one A. flavus strain isolated from platycladi seed was able to synthesize AFB1 (102.0 µg/kg) and AFB2 (15.3 µg/kg). Two P. polonicum strains isolated from tangerine and lychee seeds were able to synthesize OTA (4.1 µg/kg and 14.8 µg/kg, respectively). These results identify potential sources of OTA and aflatoxins in medicinal seeds and allude to the need to establish permitted limits for these mycotoxins in these seeds that are commonly consumed by humans.

Effects of Fusarium solani and F. oxysporum Infection on the Metabolism of Ginsenosides in American Ginseng Roots.

American ginseng (Panax quinquefolius L.) is a highly valuable herb widely used for medicinal treatments. Its pharmacologically important compounds are the ginsenosides, which are secondary metabolites in American ginseng root. The concentrations of ginsenoside in roots can be changed by fungal infection, but it is unclear what specific root tissues are impacted and whether the change is systemic. In this study, American ginseng roots were inoculated with two fungal pathogens (Fusarium solani or F. oxysporum) and the levels of six ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) were then measured in the phloem and xylem around the discolored lesions and adjacent healthy areas of the root. Results indicated that the growth of Fusarium spp. was strictly limited to phloem, and correspondingly the ginsenoside concentration was only altered in this infected phloem. The concentration of Rg1, Rd, and Rc significantly changed in phloem tissues where F. solani was inoculated, while only Rg1 and Rd changed significantly after F. oxysporum inoculation. However, no changes of any ginsenoside occurred in either xylem or phloem tissue adjacent to the inoculation point. In addition, when two Fusarium spp. were grown on ginsenoside-amended Czapek medium, the majority of ginsenosides were depleted. Therefore, pathogenic Fusarium spp. may reduce ginsenoside levels by consuming them.

Acetylcholinesterase inhibition effects of marine fungi.

CONTEXT: To this day, there are no reports that marine compounds isolated from microorganisms of the Lianyungang area of China have been used for the treatment of Alzheimer's disease. OBJECTIVE: The present study was to isolate fungi from the sea sediment of the Lianyungang area and screen for acetylcholineseterase inhibition activities of ethyl acetate extracts. MATERIALS AND METHODS: Fungi were isolated from the sea sediment and fermented. After centrifugation, the supernate was extracted with ethyl acetate. The ethyl acetate extract was then fractionated into five fractions. Acetylcholinesterase inhibition activities of the ethyl acetate extracts and five sub-fractions were tested at a concentration of 500 µg/mL with the Ellman's method. RESULTS: Forty-three marine fungi were isolated; 15 extracts inhibited acetylcholinestrease >50% and 3 extracts inhibited the acetylcholinesterase >80% at the concentration of 500 µg/mL. The 3 extracts (L1705, S1101, SH0701) inhibited AChE dose-dependently with IC50 values of 11.3 ± 1.2, 72.1 ± 2.3, and 7.8 ± 2.8 µg/mL, respectively. After the extract of SH0701 was fractionated into five fractions, the ethyl acetate fraction possessed the highest acetylcholinesterase inhibitory activity with an inhibition rate of 71.55% at the concentration of 10 µg/mL. The fungus SH0701 was identified as Aspergillus ochraceus SH0701 according to morphology and molecular identification. DISCUSSION AND CONCLUSION: The present results indicates that some ethyl acetate extracts of marine fungi isolated from Lianyungang area of China could inhibit AChE potently. Therefore, some novel AChE inhibitors might exist in those extracts.

Identification of ochratoxin A producing fungi associated with fresh and dry liquorice.

The presence of fungi on liquorice could contaminate the crop and result in elevated levels of mycotoxin. In this study, the mycobiota associated with fresh and dry liquorice was investigated in 3 producing regions of China. Potential toxigenic fungi were tested for ochratoxin A (OTA) and aflatoxin B1 (AFB1) production using liquid chromatography/mass spectrometry/mass spectrometry. Based on a polyphasic approach using morphological characters, ß-tubulin and RNA polymerase II second largest subunit gene phylogeny, a total of 9 genera consisting of 22 fungal species were identified, including two new Penicillium species (Penicillium glycyrrhizacola sp. nov. and Penicillium xingjiangense sp. nov.). The similarity of fungal communities associated with fresh and dry liquorice was low. Nineteen species belonging to 8 genera were detected from fresh liquorice with populations affiliated with P. glycyrrhizacola, P. chrysogenum and Aspergillus insuetus comprising the majority (78.74%, 33.33% and 47.06% of total) of the community from Gansu, Ningxia and Xinjiang samples, respectively. In contrast, ten species belonging to 4 genera were detected from dry liquorice with populations affiliated with P. chrysogenum, P. crustosum and Aspergillus terreus comprising the majority (64.00%, 52.38% and 90.91% of total) of the community from Gansu, Ningxia and Xinjiang samples, respectively. Subsequent LC/MS/MS analysis indicated that 5 fungal species were able to synthesize OTA in vitro including P. chrysogenum, P. glycyrrhizacola, P. polonicum, Aspergillus ochraceus and A. westerdijkiae, the OTA concentration varied from 12.99 to 39.03 µg/kg. AFB1 was absent in all tested strains. These results demonstrate the presence of OTA producing fungi on fresh liquorice and suggest that these fungi could survive on dry liquorice after traditional sun drying. Penicillium chrysogenum derived from surrounding environments is likely to be a stable contributor to high OTA level in liquorice. The harvesting and processing procedure needs to be monitored in order to keep liquorice free of toxigenic fungi.

Sesquiterpenes and cyclopeptides from the endophytic fungus Trichoderma asperellum SAMUELS, LIECKF. & NIRENBERG.

Trichodones A-C (1-3, resp.), three new sesquiterpenes, together with the two known cyclopeptides PF1022F and halobacillin (4 and 5, resp.), have been isolated from the crude extract of the endophytic fungus Trichoderma asperellum SAMUELS, LIECKF. & NIRENBERG residing in the traditional Chinese medicinal plant Panax notoginseng (BURKILL) F.H.CHEN. The structures and configurations of compounds 1-3 were determined by NMR spectroscopy and mass spectrometry. The relative configuration for 1 was determined by analysis of its pertinent coupling constants and NOESY correlations, whereas the absolute configuration of 1 was established by CD spectroscopy. Compounds 4 and 5 displayed antibacterial activities against Enterococcus faecium (CGMCC 1.2025) with IC50 values of 7.30 and 5.24 µM, and against Staphylococcus aureus COL (CGMCC 1.2465) with IC50 values of 19.02 and 14.00 µM, respectively.