Phytotoxic and Antimicrobial Terrein Derivatives and Butenolides Isolated from the Endophytic Fungus Aspergillus terreus HT5.

Two new terrein derivatives, aspergilethers A and B (1 and 2), two known analogues (3 and 4), and three known butenolides (5-7) were isolated from the endophyte Aspergillus terreus HT5. Their structures were determined by spectroscopic analysis and ECD and NMR calculations. Interestingly, 1 and 2 had unpresented medium aliphatic side chains in terrein derivatives, with different absolute configurations at C-7, which was very scarce. (+)-Terrein (3) exhibited potent postemergence phytotoxicity toward Amaranthaceae, Portulacaceae, and Fabaceae, with MIC values of 250-1000 µg/mL. Transcriptome analysis and qRT-PCR suggested that (+)-terrein induced the transcriptional expression of aging-related genes to accelerate organ senescence and stimulated plant detoxification response. The conjugated system between keto carbonyl and double bonds in the cyclopentenone ring and side chain, and the configurations of C-2 and C-3, played critical roles in the phytotoxicity of terrein derivatives. Meanwhile, 3 was first reported to display moderate antioomycetes activity toward Phytophthora nicotiana.

Anlotinib Combined With Toripalimab as First-Line Therapy for Unresectable Hepatocellular Carcinoma: A Prospective, Multicenter, Phase II Study.

BACKGROUND: For patients with unresectable hepatocellular carcinoma (HCC), the first-line therapeutic options are still relatively limited, and treatment outcomes remain poor. We aimed to assess the efficacy and safety of anlotinib combined with toripalimab as first-line therapy for unresectable HCC. METHODS: In this single-arm, multicenter, phase II study (ALTER-H-003), patients with advanced HCC without previous systemic anticancer therapy were recruited. Eligible patients were given anlotinib (12 mg on days 1-14) combined with toripalimab (240 mg on day 1) in a 3-week cycle. The primary endpoint was the objective response rate (ORR) by immune-related Response Evaluation Criteria in Solid Tumours (irRECIST)/RECIST v1.1 and modified RECIST (mRECIST). Secondary endpoints included disease control rate (DCR), duration of response (DoR), progression-free survival (PFS), overall survival (OS), and safety. RESULTS: Between January 2020 and Jul 2021, 31 eligible patients were treated and included in the full analysis set. At data cutoff (January 10, 2023), the ORR was 29.0% (95% CI: 12.1%-46.0%) by irRECIST/RECIST v1.1, and 32.3% (95% CI: 14.8%-49.7%) by mRECIST criteria, respectively. Confirmed DCR and median DoR by irRECIST/RECIST v1.1 and mRECIST criteria were 77.4 % (95% CI: 61.8%-93.0%) and not reached (range: 3.0-22.5+ months), respectively. Median PFS was 11.0 months (95% CI: 3.4-18.5 months) and median OS was 18.2 months (95% CI: 15.8-20.5 months). Of the 31 patients assessed for adverse events (AEs), the most common grade ≥ 3 treatment-related AEs were hand-foot syndrome (9.7%, 3/31), hypertension (9.7%, 3/31), arthralgia (9.7%, 3/31), abnormal liver function (6.5%, 2/31), and decreased neutrophil counts (6.5%, 2/31). CONCLUSIONS: Anlotinib combined with toripalimab showed promising efficacy and manageable safety in Chinese patients with unresectable HCC in the first-line setting. This combination therapy may offer a potential new therapeutic approach for patients with unresectable HCC.

An effective and chemotherapy-free strategy of all-trans retinoic acid and arsenic trioxide for acute promyelocytic leukemia in all risk groups (APL15 trial).

The combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has been demonstrated to have comparable effectiveness or better to ATRA and chemotherapy (CHT) in non-high-risk acute promyelocytic leukemia (APL). However, the efficacy of ATRA-ATO compared to ATRA-ATO plus CHT in high-risk APL remains unknown. Here we performed a randomized multi-center non-inferiority phase III study to compare the efficacy of ATRA-ATO and ATRA-ATO plus CHT in newly diagnosed all-risk APL to address this question. Patients were assigned to receive ATRA-ATO for induction, consolidation, and maintenance or ATRA-ATO plus CHT for induction followed by three cycles of consolidation therapy, and maintenance therapy with ATRA-ATO. In the non-CHT group, hydroxyurea was used to control leukocytosis. A total of 128 patients were treated. The complete remission rate was 97% in both groups. The 2-year disease-free, event-free survival rates in the non-CHT group and CHT group in all-risk patients were 98% vs 97%, and 95% vs 92%, respectively (P = 0.62 and P = 0.39, respectively). And they were 94% vs 87%, and 85% vs 78% in the high-risk patients (P = 0.52 and P = 0.44, respectively). This study demonstrated that ATRA-ATO had the same efficacy as the ATRA-ATO plus CHT in the treatment of patients with all-risk APL.

Claudin 18.2 is a potential therapeutic target for zolbetuximab in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is frequently diagnosed and treated in advanced tumor stages with poor prognosis. More effective screening programs and novel therapeutic means are urgently needed. Recent studies have regarded tight junction protein claudin 18.2 (CLDN18.2) as a candidate target for cancer treatment, and zolbetuximab (formerly known as IMAB362) has been developed against CLDN18.2. However, there are few data reported thus far related to the clinicopathological characteristics of CLDN18.2 expression for PDAC. AIM: To investigate the expression of CLDN18.2 in PDAC patients and subsequently propose a new target for the treatment of PDAC. METHODS: The Cancer Genome Atlas, Genotype-Tissue Expression, Gene Expression Omnibus, and European Genome-phenome Archive databases were first employed to analyze the CLDN18 gene expression in normal pancreatic tissue compared to that in pancreatic cancer tissue. Second, we analyzed the expression of CLDN18.2 in 93 primary PDACs, 86 para-cancer tissues, and 13 normal pancreatic tissues by immunohistochemistry. Immunostained tissues were assessed applying the histoscore. subsequently, they fell into two groups according to the expression state of CLDN18.2. Furthermore, the correlations between CLDN18.2 expression and diverse clinicopathological characteristics, including survival, were investigated. RESULTS: The gene expression of CLDN18 was statistically higher (P < 0.01) in pancreatic tumors than in normal tissues. However, there was no significant correlation between CLDN18 expression and survival in pancreatic cancer patients. CLDN18.2 was expressed in 88 (94.6%) of the reported PDACs. Among these tumors, 50 (56.8%) cases showed strong immunostaining. The para-cancer tissues were positive in 81 (94.2%) cases, among which 32 (39.5%) of cases were characterized for strong staining intensities. Normal pancreatic tissue was identified solely via weak immunostaining. Finally, CLDN18.2 expression significantly correlated with lymph node metastasis, distant metastasis, nerve invasion, stage, and survival of PDAC patients, while there was no correlation between CLDN18.2 expression and localization, tumor size, patient age and sex, nor any other clinicopathological characteristic. CONCLUSION: CLDN18.2 expression is frequently increased in PDAC patients. Thus, it may act as a potential therapeutic target for zolbetuximab in PDAC.

Genome Analysis of Phytophthora nicotianae JM01 Provides Insights into Its Pathogenicity Mechanisms.

Phytophthora nicotianae is a widely distributed plant pathogen that can cause serious disease and cause significant economic losses to various crops, including tomatoes, tobacco, onions, and strawberries. To understand its pathogenic mechanisms and explore strategies for controlling diseases caused by this pathogen, we sequenced and analyzed the whole genome of Ph. nicotianae JM01. The Ph. nicotianae JM01 genome was assembled using a combination of approaches including shotgun sequencing, single-molecule sequencing, and the Hi-C technique. The assembled Ph. nicotianae JM01 genome is about 95.32 Mb, with contig and scaffold N50 54.23 kb and 113.15 kb, respectively. The average GC content of the whole-genome is about 49.02%, encoding 23,275 genes. In addition, we identified 19.15% of interspersed elements and 0.95% of tandem elements in the whole genome. A genome-wide phylogenetic tree indicated that Phytophthora diverged from Pythium approximately 156.32 Ma. Meanwhile, we found that 252 and 285 gene families showed expansion and contraction in Phytophthora when compared to gene families in Pythium. To determine the pathogenic mechanisms Ph. nicotianae JM01, we analyzed a suite of proteins involved in plant-pathogen interactions. The results revealed that gene duplication contributed to the expansion of Cell Wall Degrading Enzymes (CWDEs) such as glycoside hydrolases, and effectors such as Arg-Xaa-Leu-Arg (RXLR) effectors. In addition, transient expression was performed on Nicotiana benthamiana by infiltrating with Agrobacterium tumefaciens cells containing a cysteine-rich (SCR) protein. The results indicated that SCR can cause symptoms of hypersensitive response. Moreover, we also conducted comparative genome analysis among four Ph. nicotianae genomes. The completion of the Ph. nicotianae JM01 genome can not only help us understand its genomic characteristics, but also help us discover genes involved in infection and then help us understand its pathogenic mechanisms.

Exploring Biocontrol Agents From Microbial Keystone Taxa Associated to Suppressive Soil: A New Attempt for a Biocontrol Strategy.

Recent studies have observed differing microbiomes between disease-suppressive and disease-conducive soils. However, it remains unclear whether the microbial keystone taxa in suppressive soil are critical for the suppression of diseases. Bacterial wilt is a common soil-borne disease caused by Ralstonia solanacearum that affects tobacco plants. In this study, two contrasting tobacco fields with bacterial wilt disease incidences of 0% (disease suppressive) and 100% (disease conducive) were observed. Through amplicon sequencing, as expected, a high abundance of Ralstonia was found in the disease-conducive soil, while large amounts of potential beneficial bacteria were found in the disease-suppressive soil. In the fungal community, an abundance of the Fusarium genus, which contains species that cause Fusarium wilt, showed a positive correlation (p < 0.001) with the abundance of Ralstonia. Network analysis revealed that the healthy plants had more complex bacterial networks than the diseased plants. A total of 9 and 13 bacterial keystone taxa were identified from the disease-suppressive soil and healthy root, respectively. Accumulated abundance of these bacterial keystones showed a negative correlation (p < 0.001) with the abundance of Ralstonia. To complement network analysis, culturable strains were isolated, and three species belonging to Pseudomonas showed high 16S rRNA gene similarity (98.4-100%) with keystone taxa. These strains displayed strong inhibition on pathogens and reduced the incidence of bacterial wilt disease in greenhouse condition. This study highlighted the importance of keystone species in the protection of crops against pathogen infection and proposed an approach to obtain beneficial bacteria through identifying keystone species, avoiding large-scale bacterial isolation and cultivation.

Pseudomonas Inoculation Stimulates Endophytic Azospira Population and Induces Systemic Resistance to Bacterial Wilt.

Bacterial communities in the rhizosphere play an important role in sustaining plant growth and the health of diverse soils. Recent studies have demonstrated that microbial keystone taxa in the rhizosphere microbial community are extremely critical for the suppression of diseases. However, the mechanisms involved in disease suppression by keystone species remain unclear. The present study assessed the effects of three Pseudomonas strains, which were identified as keystone species in our previous study, on the growth performance and root-associated bacterial community of tobacco plants. A high relative abundance of Ralstonia was found in the non-inoculated group, while a large Azospira population was observed in all groups inoculated with the three Pseudomonas strains. Correspondingly, the activities of the defense-related enzymes and the expression levels of the defense signaling marker genes of the plant were increased after inoculation with the Pseudomonas strains. Moreover, the correlation analyses showed that the relative abundance of Azospira, the activity of superoxide dismutase, catalase, and polyphenol oxidase, and the expression of H1N1, ACC Oxidase, and PR1 a/c had a significantly negative (p<0.05) relationship with the abundance of Ralstonia. This further revealed that the keystone species, such as Pseudomonas spp., can suppress bacterial wilt disease by enhancing the systemic resistance of tobacco plants.

Rootstock-Scion Interaction Affects the Composition and Pathogen Inhibitory Activity of Tobacco (Nicotiana tabacum L.) Root Exudates.

The composition and allelopathy to Phytophthora nicotianae (the causal agent of tobacco black shank disease) of root exudates from a resistant tobacco (Nicotiana tabacum L.) cultivar Gexin 3, a susceptible cultivar Xiaohuangjin 1025 and their reciprocal grafts were investigated. Grafting with disease-resistant rootstock could improve resistance to black shank; this is closely related to the allelopathy of root exudates. The root exudates from the resistant cultivar inhibited the growth of P. nicotianae, while those from the susceptible cultivar promoted the growth; the grafting varieties had intermediate properties. The root exudate composition differed among cultivars. Gexin 3 was rich in esters and fatty acids, while Xiaohuangjin 1025 contained more hydrocarbons and phenolic acids. The composition of root exudates of grafted cultivars as well as their allelopathy to P. nicotianae were altered, and tended to be close to the composition of cultivar used as rootstock. Eugenol, 4-tert-butylphenol, mono (2-ethylhexyl) phthalate, 4-hydroxybenzoic acid, 2,6-di-tert-butylphenol, dipropyl phthalate, and methyl myristate were identified as the main compounds contributing to inhibitory properties of root exudates. Sorbitol was suggested to play a role in disease induction. Overall, rootstock-scion interaction affected the composition of tobacco root exudates, which may be attributed to the different disease resistance among grafted plants, rootstock and scion.

The Antifungal Effect of Garlic Essential Oil on Phytophthora nicotianae and the Inhibitory Component Involved.

This study explored the chemical compositions of garlic essential oil, the inhibitory activity of garlic essential oil and diallyl disulfide (DADS) against Phytophthora nicotianae, and the effects on mycelial plasma membrane permeability and P. nicotianae inhibition. In total, 29 compounds were detected in garlic essential oil, of which 26 were detected by gas chromatography‒mass spectrometry (GC-MS) and 21 by headspace solid-phase microextraction (HS-SPME) GC-MS. DADS (60.12% and 19.09%) and trisulfide di-2-propenyl (14.18% and 17.98%) were the major components identified by HS-SPME GC-MS and GC-MS analysis, respectively. Half-inhibitory concentration (Ec50, antagonism) and minimum inhibitory concentration (MIC, fumigation) of DADS against P. nicotianae were 150.83 µL/L and 20 µL/L, respectively, while Ec50 of garlic essential oil was 1108.25 µL/L. Mycelial membrane permeability gradually increased in a concentration-dependent manner, and cell death increased at 450 µL/L DADS. Furthermore, DADS treatment significantly reduced the incidence of tobacco black shank and the number of P. nicotianae pathogens in rhizosphere soil. DADS also promoted root development of tobacco seedlings at low concentrations, which was inhibited at high concentrations. Therefore, DADS may play an important role in the antifungal effect against P. nicotianae by destroying mycelial cell membrane integrity, causing an increase in cell membrane permeability, and leading to cell death.

Variation in Chemical Composition and Biological Activities of Flos Chrysanthemi indici Essential Oil under Different Extraction Methods.

Flos Chrysanthemi indici, an important medicinal and aromatic plant in China, is considered to have many different preservative and pharmacological properties. Considering the capability of essential oils (EOs), the present study is conducted to compare different extraction methods in order to improve yield and biological activities. Hydro-distillation (HD), steam-distillation (SD), solvent-free microwave extraction (SFME), and supercritical fluid extraction (SFE) are employed to prepare EOs from Flos Chrysanthemi indici. A total of 71 compounds are assigned by gas chromatography/mass spectrometry (GC-MS) in comparison with retention indices. These include 32 (HD), 16 (SD), 31 (SFME) and 38 (SFE) compounds. Major constituents of EOs differ according to the extraction methods were heptenol, tricosane, camphor, borneol, and eucalyptol. EOs extracted by SFME exhibit higher antioxidant activity. All EOs show varying degrees of antimicrobial activity, with minimum inhibitory concentration (MIC) ranging from 0.0625 to 0.125 mg/mL and SFME and SFE prove to be efficient extraction methods. EOs alter the hyphal morphology of Alternaria alternata, with visible bumps forming on the mycelium. Overall, these results indicate that the extraction method can significantly influence the composition and biological activity of EOs and SFME and SFE are outstanding methods to extract EOs with high yield and antimicrobial activity.

Essential oil of Chrysanthemum indicum L.: potential biocontrol agent against plant pathogen Phytophthora nicotianae.

Phytophthora nicotianae is currently considered one of the most devastating oomycete plant pathogens, and its control frequently relies solely on the use of systemic fungicides. There is an urgent need to find environment-friendly control techniques. This study examined the chemical composition, inhibitory activity, and possible modes of action of the essential oil of Chrysanthemum indicum L. (EOC) flower heads against P. nicotianae. The EOC was obtained using hydrodistillation at a 0.15% yielded. It inhibited mycelial growth and spore germination of P. nicotianae at a minimum inhibitory concentration (MIC) of 200 µL/L, and exhibited fumigation effects (92.68% inhibition at 157.48 µL/L). Marked deformation of P. nicotianae mycelia included deformed tip enlargement, shrinkage, and rupture. Further, 55 and 47 compounds were identified using gas chromatography-mass spectrometry (GC-MS) and headspace solid-phase microextraction (HS-SPME) GC-MS analyses, representing 88.2% and 98.91% of the total EOC, respectively. Monoterpenes (25.77%) and sesquiterpenes (54.14%) were the major components identified using GC-MS, whereas monoterpenes were the main constituents in the HS-SPME GC-MS analysis. The higher proportions of sesquiterpenes and monoterpenes could be responsible for the inhibitory activity of EOC, which increased mycelia membrane permeability and the content of mycelial malondialdehyde (MDA) in a dose-dependent manner. Cell death also occurred. Thus, destruction of the cell wall and membrane might be two modes of action of EOC. Our results would be useful for the development of a new plant source of fungicide for P. nicotianae-induced disease.

Anti-Phytopathogenic and Cytotoxic Activities of Crude Extracts and Secondary Metabolites of Marine-Derived Fungi.

Thirty-one isolates belonging to eight genera in seven orders were identified from 141 strains that were isolated from several marine plants. Alternaria sp. and Fusarium sp. were found to be the predominant fungi. Evaluation of the anti-phytopathogenic bacterial and fungal activities, as well as the cytotoxicity of these 31 extracts, revealed that most of them displayed different levels of bioactivities. Due to their interesting bioactivities, two fungal strains-Fusarium equiseti (P18) and Alternaria sp. (P8)-were selected for chemical investigation and compounds 1-4 were obtained. The structure of 1 was elucidated by 1D and 2D NMR analysis, as well as high-resolution electrospray ionization mass spectroscopy (HRESIMS), and the absolute configuration of its stereogenic carbon (C-11) was established by comparison of the experimental and calculated electronic circular-dichroism (ECD) spectra. Moreover, alterperylenol (4) exhibited antibacterial activity against Clavibacter michiganensis with a minimum inhibitory concentration (MIC) of 1.95 µg/mL, which was 2-fold stronger than that of streptomycin sulfate. Additionally, an antibacterial mechanism study revealed that 4 caused membrane hyperpolarization without evidence of destruction of cell membrane integrity. Furthermore, stemphyperylenol (3) displayed potent antifungal activity against Pestallozzia theae and Alternaria brassicicola with MIC values equal to those of carbendazim. The cytotoxicity of 1 and 2 against human lung carcinoma (A-549), human cervical carcinoma (HeLa), and human hepatoma (HepG2) cell lines were also evaluated.

[Inhibition of Bacillus pumilus AR03 on Alternaria alternata and Erysiphe cichoracearum on tobacco].

This study aimed to evaluate the efficiency of Bacillus pumilus AR03 against Altenaria alternata and Erysiphe cichoracearum. The antagonistic activities were studied in the way of co-culture on plate, inhibition of pathogen conidia germination on concave slides and LB agar medium. In the investigation, the water suspension of living cells of strain AR03, at 3 x 10(8) cfu . mL-1 had a remarkable inhibition effect on hyphae growth and conidia germination of A. alternata and caused hyphae deformation, shorter and swollen nodes, winding hyphae accumulation, abnormal tubes with tips expanded or deformed. Conidia did not germinate and the tissues of compartment became swollen or formed a round spherical bubble. In addition, the inhibition rate of conidia germination of E. cichoracearum was 91. 3% and 69. 3%, respectively when treated with strain AR03 at 1 x 10(7) cfu . mL-1 and 1.5 x 10(6) cfu . mL-1. Conidia treated by living cells of AR03 became swollen, deformed, the protoplasm of conidia shrinked, disintegrated gradually and separated from the conidia wall. And some conidia were hollow because the protoplasm leaked out from inside. Greenhouse results revealed that the effects of living cells of AR03 with different concentrations were significantly different. Bacterial suspension of AR03 at 3 x 10(8) cfu . mL-1 was strongly antagonistic to E. cichoracearum with the control efficiencies 7 days and 15 days after treatment of two spays being 83.8% and 90.3%, respectively, while the control efficiencies of AR03 at 3 x 10(6) cfu . mL-1 were 70.0% and 73.3%, respectively. Strain AR03 had a long persistence against powdery mildew more than 30 days.

[Impact of biocontrol agent Bacillus subtilis on bacterial communities in tobacco rhizospheric soil].

The impact of inoculation with the biocontrol agent Bacillus subtilis on bacterial communities and bacterial diversity in rhizospheric soil of Nicotiana tabacum was assessed by constructing a 16S rRNA gene clone library and conducting amplified ribosomal DNA restriction analysis (ARDRA). The bacterial diversity was evaluated by coverage value (C), Shannon index (H), Pielou evenness index (E) and Margalef richness index (R). Phylogenetic analysis revealed that the inoculation significantly affected the composition of bacterial communities in tobacco rhizospheric soil. A total of twelve bacterial groups including Acidobacteria, Proteobacteria (including α-, ß-, δ-, γ-Proteobacteria) , Planctomycetes, Firmicutes, Nitrospirae, Gemmatimonadetes, Actinobacteria, Chloroflexi and Bacteroidetes were detected to be shared by inoculated soil and control soil. The community composition and proportions of different bacteria in the communities showed significant variations between the two samples. The dominant bacteria were Acidobacteria (27.1%) and Proteobacteria (26.5%) in control soil, while in the inoculated soil Proteobacteria (38.0%) and Acidobacteria (29.6%) were dominant. B. subtilis inoculation increased the numbers of γ-Proteobacteria and α-Proteobacteria but reduced the numbers of bacterial groups such as ß-Proteobacteria, Planctomycetes, Firmicutes. Diversity analysis showed that bacterial diversity was rich for both soil samples, and soil bacterial Shannon index and Margalef richness index were promoted after inoculation.