TMT-Based Proteomic Analysis Reveals the Molecular Mechanisms of Sodium Pheophorbide A against Black Spot Needle Blight Caused by Pestalotiopsis neglecta in Pinus sylvestris var. mongolica.

Black spot needle blight is a minor disease in Mongolian Scots pine (Pinus sylvestris var. mongolica) caused by Pestalotiopsis neglecta, but it can cause economic losses in severe cases. Sodium pheophorbide a (SPA), an intermediate product of the chlorophyll metabolism pathway, is a compound with photoactivated antifungal activity, which has been previously shown to inhibit the growth of P. neglecta. In this study, SPA significantly reduced the incidence and disease index and enhanced the chlorophyll content and antioxidant enzyme activities of P. sylvestris var. mongolica. To further study the molecular mechanism of the inhibition, we conducted a comparative proteomic analysis of P. neglecta mycelia with and without SPA treatment. The cellular proteins were obtained from P. neglecta mycelial samples and subjected to a tandem mass tag (TMT)-labelling LC-MS/MS analysis. Based on the results of de novo transcriptome assembly, 613 differentially expressed proteins (DEPs) (p < 0.05) were identified, of which 360 were upregulated and 253 downregulated. The 527 annotated DEPs were classified into 50 functional groups according to Gene Ontology and linked to 256 different pathways using the Kyoto Encyclopedia of Genes and Genomes database as a reference. A joint analysis of the transcriptome and proteomics results showed that the top three pathways were Amino acid metabolism, Carbohydrate metabolism, and Lipid metabolism. These results provide new viewpoints into the molecular mechanism of the inhibition of P. neglecta by SPA at the protein level and a theoretical basis for evaluating SPA as an antifungal agent to protect forests.

Carrageenan oligosaccharide alleviates intestinal damage via gut microflora through activating IMD/relish pathway in female Drosophila melanogaster.

Recent evidence suggests the anti-inflammatory effect of carrageenan oligosaccharides (COS). The effects of COS on intestinal injury induced by 0.6% sodium dodecyl sulfate (SDS) and the molecular mechanisms involved were investigated in this study. 0.625, 1.25, and 2.5 mg/mL COS in diet had no toxic effect in flies, and they could all prolong SDS-treated female flies' survival rate. 1.25 mg/mL COS prevented the development of inflammation by improving the intestinal barrier integrity and maintaining the intestinal morphology stability, inhibited the proliferation of intestine stem cells (ISCs), and the production of lysosomes induced by SDS, accompanied by a decrease in the expression of autophagy-related genes. Moreover, COS decreased the active oxygen species (ROS) content in gut and increased the antioxidant activity in SDS-induced female flies, while COS still played a role in increasing survival rate and decreasing intestinal leakage in CncC-RNAi flies. The improvement of anti-inflammation capacity may be associated with the regulation of intestinal microflora with COS supplementation for Drosophila melanogaster. COS changed the gut microbiota composition, and COS had no effect on germ-free (GF) flies. It is highlighted that COS could not work in Relish-RNAi flies, indicating relish is required for COS to perform beneficial effects. These results provide insights into the study of gut microbiota interacting with COS to modulate intestinal inflammation in specific hosts.

Identification, Culture Characteristics and Whole-Genome Analysis of Pestalotiopsis neglecta Causing Black Spot Blight of Pinus sylvestris var. mongolica.

Black spot needle blight is a serious conifer disease of Pinus sylvestris var. mongolica occurring in Northeast China, which is usually caused by the plant pathogenic fungus Pestalotiopsis neglecta. From the diseased pine needles collected in Honghuaerji, the P. neglecta strain YJ-3 was isolated and identified as the phytopathogen, and its culture characteristics were studied. Then, we generated a highly contiguous 48.36-Mbp genome assembly (N50 = 6.62 Mbp) of the P. neglecta strain YJ-3 by combining the PacBio RS II Single Molecule Real Time (SMRT) and Illumina HiSeq X Ten sequencing platforms. The results showed that a total of 13,667 protein-coding genes were predicted and annotated using multiple bioinformatics databases. The genome assembly and annotation resource reported here will be useful for the study of fungal infection mechanisms and pathogen-host interaction.

Protective effect of agar oligosaccharide on male Drosophila melanogaster suffering from oxidative stress via intestinal microflora activating the Keap1-Nrf2 signaling pathway.

Agar oligosaccharide (AOS) is a new kind of marine functional oligosaccharide with generous biological activities. To investigate the antioxidative effects of AOS in vivo, 3 % aqueous hydrogen peroxide (H2O2) was used to induce oxidative stress in male Drosophila melanogaster (D. melanogaster) fed 5 % sucrose (SUC). AOS (0.125 %) in the medium extended the lifespan of D. melanogaster suffering from oxidative stress by improving antioxidant capacity and intestinal function. Electron microscopic observation of epithelial cells showed that AOS alleviated the damage caused by H2O2 challenge in the intestine of D. melanogaster, including a reduction of gut leakage and maintenance of intestinal length and cell ultrastructure. The Keap1-Nrf2 (analogues of CncC gene in D. melanogaster) signaling pathway was significantly activated based on gene expression levels and a reduction in ROS content in the intestine of D. melanogaster suffering from oxidative stress. The improvement of antioxidant capacity may be related to the regulation of intestinal microflora with AOS supplementation for D. melanogaster. Nrf2-RNAi, sterile and gnotobiotic D. melanogaster were used to validate the hypothesis that AOS activated the Keap1-Nrf2 signaling pathway to achieve antioxidant effects by regulating intestinal microflora. The above results contribute to our understanding of the antioxidative mechanism of AOS and promote its application in the food industry.

Phytic acid is a new substitutable plant-derived antifungal agent for the seedling blight of Pinus sylvestris var. mongolica caused by Fusarium oxysporum.

Phytic acid (PA) is a new substitutable plant-derived antifungal agent; however, few reports have been published regarding its antifungal effects on pathogenic fungi. The present study explored the in vitro antifungal activity of PA against four phytopathogenic fungi and found that PA was the most effective at inhibiting the growth of Fusarium oxysporum. This study aimed to investigate the in vivo and in vitro antifungal activities of PA against the seedling blight of Pinus sylvestris var. mongolica caused by F. oxysporum and to determine its possible mechanism of action. The results showed that PA inhibited spore germination and mycelial growth of F. oxysporum in a concentration-dependent manner and exhibited strong inhibition when its concentration exceeded 1000 mg/L. It mainly destroyed the integrity of the cell membrane, increasing its cell membrane permeability, causing the cell contents to spill out, and impairing fungal growth. In addition, the leakage of intercellular electrolytes and soluble proteins indicated that PA used at its EC20 and EC50 increased the membrane permeability of F. oxysporum. The increase in malondialdehyde and hydrogen peroxide content confirmed that PA treatment at its EC20 and EC50 damaged the cell membrane of the pathogen. Scanning electron microscopy revealed that PA affected the morphology of mycelia, causing them to shrivel, distort, and break. Furthermore, PA significantly reduced the activities of the antioxidant-related enzymes superoxide dismutase and catalase, as well as that of the pathogenicity-related enzymes polygalacturonase, pectin lyase, and endoglucanase (EG) in F. oxysporum (P < 0.05). In particular, EG enzyme activity was maximally inhibited in F. oxysporum treated with PA at its EC50. Moreover, PA significantly inhibited the incidence of disease, and growth indices in Pinus sylvestris var. mongolica seedling blight was determined. In summary, PA has a substantial inhibitory effect on F. oxysporum. Therefore, PA could serve as a new substitutable plant-derived antifungal agent for the seedling blight of P. sylvestris var. mongolica caused by F. oxysporum.

Structure characteristics of low molecular weight pectic polysaccharide and its anti-aging capability by modulating the intestinal homeostasis.

Pectic polysaccharide has attracted increasing attention for their potential biological properties and applications in health industries. In this study, a low-molecular-weight pectic polysaccharide, POS4, was obtained from citrus peel. The structure of POS4 was preliminarily analyzed by gel-permeation chromatography, monosaccharide analysis, infrared spectroscopy (IR) and nuclear magnetic resonance spectroscopy (NMR). Results showed that the molecular weight of POS4 was 4.76 kDa and it was a galacturonic acid enriched pectic polysaccharide. The anti-aging activity in vivo showed that POS4 could notably prolong the average lifespan of fruit flies by suppressing the generation of reactive oxygen species (ROS). Further studies demonstrated that POS4 could enhance intestinal homeostasis by modulating gut microbiota in a positive way and regulating autophagy associated genes. Taken together, we proposed that galacturonic acid enriched low molecular weight pectic polysaccharide have great potential in the development of healthy foods such as anti-aging health care products.

Steroid hormone 20-hydroxyecdysone disturbs fat body lipid metabolism and negatively regulates gluconeogenesis in Hyphantria cunea larvae.

The steroid hormone 20-hydroxyecdysone (20E) has been described to regulate fat body lipid metabolism in insects, but its accurate regulatory mechanism, especially the crosstalk between 20E-induced lipid metabolism and gluconeogenesis remains largely unclear. Here, we specially investigated the effect of 20E on lipid metabolism and gluconeogenesis in the fat body of Hyphantria cunea larvae, a notorious pest in forestry. Lipidomics analysis showed that a total of 1 907 lipid species were identified in the fat body of H. cunea larvae assigned to 6 groups and 48 lipid classes. The differentially abundant lipids analysis showed a significant difference between 20E-treated and control samples, indicating that 20E caused a remarkable alteration of lipidomics profiles in the fat body of H. cunea larvae. Further studies demonstrated that 20E accelerated fatty acid ß-oxidation, inhibited lipid synthesis, and promoted lipolysis. Meanwhile, the activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase were dramatically suppressed by 20E in the fat body of H. cunea larvae. As well, the transcriptions of genes encoding these 4 rate-limiting gluconeogenic enzymes were significantly downregulated in the fat body of H. cunea larvae after treatment with 20E. Taken together, our results revealed that 20E disturbed fat body lipid homeostasis, accelerated fatty acid ß-oxidation and promoted lipolysis, but negatively regulated gluconeogenesis in H. cunea larvae. The findings might provide a new insight into hormonal regulation of glucose and lipid metabolism in insect fat body.

Knockdown of GFAT disrupts chitin synthesis in Hyphantria cunea larvae.

Glutamine-fructose-6-phosphate transaminase (GFAT) has been reported to regulate the hexosamine biosynthetic pathway as the first rate-limiting enzyme. As a key enzyme that catalyzes the substrate of glycosylation modification, which has a wide-ranging effect on cellular functions. However, there are few studies on the relationship between GFAT and chitin metabolism in insects. In the present study, the GFAT gene from Hyphantria cunea was identified based on transcriptome and bioinformatic analysis. The role of HcGFAT in regulating development and chitin synthesis was analyzed by RNA interference (RNAi) in H. cunea larvae. The full-length HcGFAT gene (2028 bp) encodes a 676 amino acid (aa) polypeptide had typical structural features of the SIS and Gn_AT_II superfamily. Phylogenetic analyses showed that GFAT of H. cunea shares the highest homology and identity with GFAT of Ostrinia furnacalis. Expression profiles indicated that HcGFAT was expressed throughout larval, pupal and three tissues (midgut, fat body, epidermis), and highly expressed in the last instar of larvae and strongly expressed in epidermis among three tissues. Bioassay results showed that knockdown of HcGFAT repressed larval growth and development, resulting in a significant loss of larval body weight. Meanwhile, HcGFAT knockdown also significantly caused larval developmental deformity. Knockdown of HcGFAT regulated the expression of four other critical genes in the chitin synthesis pathway (HcGNA, HcPAGM, HcUAP, HcCHSA), and ultimately resulted in decreased chitin content in the epidermis. In summary, these findings indicated that GFAT plays a critical role in larval growth and development, as well as chitin synthesis in H. cunea.

Inhibitory effect and mechanism of action of juniper essential oil on gray mold in cherry tomatoes.

Juniper essential oil (JEO), which is mostly known as an immune system booster and effective detoxifier, has substantial antimicrobial activity. A comparison of the inhibitory effects of three plant essential oils from juniper (Juniperus rigida), cedarwood (Juniperus virginiana), and cypress (Crupressus sempervirens) on four plant pathogenic fungi indicated that JEO was the most effective at inhibiting the growth of gray mold (Botrytis cinerea). Additional studies were subsequently conducted to explore the in vivo and in vitro antifungal activity and possible mechanism of JEO against B. cinerea. The results show that JEO inhibited the germination of spores and mycelial growth of B. cinerea in a concentration-dependent manner and exhibited strong inhibition when its concentration exceeded 10 µL/mL. JEO also significantly inhibited the incidence of disease and diameters of gray mold lesions on cherry tomato fruit (Solanum lycopersicum). After 12 h of treatment with JEO, the extracellular conductivity, and the contents of soluble protein, malondialdehyde, and hydrogen peroxide were 3.1, 1.2, 7.2, and 4.7 folds higher than those of the control group, respectively (P < 0.05), which indicated that JEO can damage membranes. Scanning electron microscopy observations revealed that JEO affected the morphology of mycelia, causing them to shrivel, twist and distort. Furthermore, JEO significantly improved the activities of the antioxidant-related enzymes superoxide dismutase and catalase but reduced the pathogenicity-related enzymes polygalacturonase (PG), pectin lyase and endoglucanase of B. cinerea (P < 0.05). In particular, PG was reduced by 93% after treatment with JEO for 12 h. Moreover, the 18 constituents of JEO were identified by gas chromatography/mass spectrometry (GC-MS) analysis, mainly limonene (15.17%), γ-terpinene (8.3%), ß-myrcene (4.56%), terpinen-4-ol (24.26%), linalool (8.73%), α-terpineol (1.03%), o-cymene (8.35%) and other substances with antimicrobial activity. Therefore, JEO can be an effective alternative to prevent and control gray mold on cherry tomato fruit.

Antiaging effect of anthocyanin extracts from bilberry on natural or UV-treated male Drosophila melanogaster.

Anthocyanins from bilberry (Vaccinium vitis-idaea) are one of the most abundant sources of polyphenols and are widely used in the food, medicine and cosmetics industries due to their antioxidation properties, but few studies have investigated their antiaging properties. Based on our previous examination, the effect of anthocyanin extracts from bilberry (BANCs) on several characteristics of natural and UV-treated male Drosophila melanogaster, including their lifespan, fecundity, and antioxidant capacity, was studied, and the related mechanisms were preliminary explored. The results indicated that BANCs can effectively prolong the average and maximum lifespan and improve the reproductive capacity and antioxidant capacity of natural and UV-treated flies. In particular, BANCs significantly changed the growth cycle, sex ratio and content of ROS in the fat bodies of the offspring and decreased the expression levels of antioxidant- and autophagy-related genes in UV-treated flies. Collectively, the results demonstrate that BANC supplementation in the medium effectively alleviated the aging process, and this effect was not directly correlated with the antioxidant and autophagy signaling pathways in the body of D. melanogaster.

Biodegradation of polyethylene by Meyerozyma guilliermondii and Serratia marcescens isolated from the gut of waxworms (larvae of Plodia interpunctella).

The widespread use of polyethylene (PE) causes a large amount of indigestible plastic waste. Waxworms (the larvae of Plodia interpunctella) can eat PE, but the degradation principle of PE under the action of intestinal microorganisms is still unclear, especially the insufficient research on key degradable PE strains. In this study, we fed waxworms with PE. Two strains with high PE degradation efficiency were isolated and purified, and the effects of single and microbial consortia on PE degradation were evaluated by water contact angle (WCA), FTIR, GC-MS, SEM and RT-qPCR. The results showed that Meyerozyma guilliermondii ZJC1 (MgZJC1) and Serratia marcescens ZJC2 (SmZJC2) could degrade PE. However, the degradation efficiency of the microbial consortium was higher, and the weight loss rate of PE was 15.87 %. In addition, the PE degradation products of MgZJC1 were C9H10O, C20H15NO, C28H44O3 and C16H32O2, and the PE degradation products of SmZJC2 were C16H18O, C14H18N2O7 and C31H48O6. The PE degradation products of the microbial consortium were C11H24, C19H10O, C15H32, C14H30, C16H34, C25H52 and C27H56. RT-qPCR results showed that SmZJC2 promoted PE degradation by upregulating the expression of multiple genes, such as multicopper oxidase genes (PiSm-CueO). MgZJC1 responded to carbon deficiency by upregulating the expression of multiple genes, such as key enzyme genes in the tricarboxylic acid (TCA) cycle. This study can be used to develop an efficient microbial consortium for PE degradation and provide a basis for the reuse of PE waste. It can also provide a research basis for the joint degradation of PE by microbial consortia composed of bacteria and fungi.

E74 knockdown represses larval development and chitin synthesis in Hyphantria cunea.

E74 is a key transcription factor induced by 20E, which plays a broad role in many physiological events during insect growth and development, including vitellogenesis, organ remodeling and new tissue formation, programmed cell death and metamorphosis. However, whether it is involved in regulating insect chitin biosynthesis remains largely unclear. Here, the E74 gene was identified for the first time from Hyphantria cunea, a notorious defoliator of forestry. Thereafter, the role of HcE74 in regulating growth, development and chitin synthesis in H. cunea larvae was evaluated. Bioinformatics analysis showed that HcE74 shared the highest identity (95.53%) with E74A of Spodoptera litura, which belonged to Ets superfamily. The results of RNAi bioassay showed that the larval mortality on 6 d after HcE74 knockdown was up to 51.11 ± 6.94%. Meanwhile, a distinct developmental deformity phenotype was found when HcE74 was silenced. These results indicated that HcE74 plays an important role in the development and molting of H. cunea larvae. Moreover, HcE74 knockdown also significantly decreased the expression of four key genes related to chitin synthesis, including glucose-6-phosphate isomerase (HcG6PI), UDP-N-acetylglucosamine pyrophosphorylase (HcUAP), chitin synthetase A (HcCHSA), and chitin synthetase B (HcCHSB). As a result, the content of chitin in midgut and epidermis decreased by 0.54- and 0.08-fold, respectively. Taken together, these results demonstrated that HcE74 not only plays a critical role in the growth and molting of H. cunea larvae, but also probably participates in the transcriptional regulation of genes involved in chitin biosynthesis.

Protective Effect of Bilberry Anthocyanin Extracts on Dextran Sulfate Sodium-Induced Intestinal Damage in Drosophila melanogaster.

Inflammatory bowel disease (IBD) is a chronic recurrent disease that can be controlled by various natural extracts. Anthocyanins (ANCs) from bilberry have significant antioxidant capacity and are widely used as food colors and antioxidants. In this study, we investigated the protective effects of bilberry anthocyanin extracts (BANCs) against dextran sulphate sodium (DSS)-induced intestinal inflammation in a Drosophila melanogaster (D. melanogaster) model, and the effects on the lifespan, antioxidant capacity, intestinal characteristics, and microbiome and gene expression profiles were analyzed to elucidate the underlying biological mechanisms. In DSS-induced normal and axenic D. melanogaster, BANCs significantly increased the survival rate, maintained the intestinal morphology and integrity, and reduced the number of dead intestinal epithelial cells and the ROS level of these cells. BANC supplementation had no significant effect on the intestinal microflora of DSS-induced D. melanogaster, as demonstrated by a 16S rDNA analysis, but improved the antioxidant capacity by activating the relative gene expression of NRF2 signaling pathways in the intestine of D. melanogaster with DSS-induced inflammation. Therefore, the results demonstrate that BANCs effectively alleviate intestinal inflammatory injury induced by DSS and improve the antioxidant capacity of D. melanogaster by modulating NRF2 signaling pathways, and could thus promote the application of BANCs as functional foods.

Mechanism of Cr (VI) reduction by Pichia guilliermondii ZJH-1.

Background: Chromium is one of the most used toxic heavy metals. A large amount of chromium waste is discharged into the environment every year, causing serious environmental pollution, especially the pollution of soil and water by hexavalent chromium. Eliminating hexavalent chromium is the primary challenge to achieve a pollution-free environment. Objectives: This study aims to understand the mechanism of Pichia guilliermondii's reduction of hexavalent chromium through enzymatic characteristic, oxidative stress response, and reduction product. Material and Methods: The strain Pichia guilliermondii ZJH-1 was isolated and stored in our laboratory. The hexavalent chromium uses 1,5-diphenyl carbazide method (DPC) to measure. The UV spectrophotometer was used to measure the intracellular antioxidant enzyme activity, and the kit was used to measure the activity of catalase and glutathione reductase. The reduction products were analyzed by ultraviolet full-wavelength scanning and FTIR. Results: The reduction of hexavalent chromium by ZJH-1 is accompanied by an increase in active oxygen and antioxidant levels. Chromate reductase mainly exists in the extracellular fluid, and the carboxyl, amide, hydroxide and other groups of the cell wall are involved in the bioremediation of Cr(VI) by complexing with Cr(VI) and Cr(III). After ZJH-1 was treated with different concentrations of Cr(VI), the expression of proteins with molecular weights of 15 kDa, 18 kDa, 35 kDa, 62 kDa, and 115 kDa increased significantly. This strain is the most suitable for chromate reductase (CChR). The optimum temperature is 40℃ and the optimum pH is 7.0. Cu2+ can enhance the activity of chromate reductase. At the optimum temperature and pH, the chromate reductase Km of this strain is 0.40 µmol and Vmax is 14.47 µmoL.L-1·min-1. Conclusions: The bioremediation of Cr(VI) by Pichia guilliermondii ZJH-1 is attributable to the reduction product (Cr(III)) that can be removed in the precipitate and can be fixed on the cell surface and accumulated in the cell.

Metformin-induced AMPK activation suppresses larval growth and molting probably by disrupting 20E synthesis and glycometabolism in fall webworm, Hyphantria cunea Drury.

Metformin, considered to be a potent AMPK activator, is widely used for clinical therapy of cancer and diabetes due to its distinct function in regulating cell energy balance and body metabolism. However, the effect of metformin-induced AMPK activation on the growth and development of insects remains largely unknown. In the present study, we focused on the role of metformin in regulating the growth and development of Hyphantria cunea, a notorious defoliator in the forestry. Firstly, we obtained the complete coding sequences of HcAMPKα2, HcAMPKß1, HcAMPKγ2 from H. cunea, which encoded a protein of 512, 281, and 680 amino acids respectively. Furthermore, the phylogenetic analysis revealed that these three subunits were highly homologous with the AMPK subunits from other lepidopteran species. According to the bioassay, we found metformin remarkably restrained the growth and development of H. cunea larvae, and caused molting delayed and body weight reduced. In addition, expressions of HcAMPKα2, HcAMPKß1, and HcAMPKγ2 were upregulated 3.30-, 5.93- and 5.92-folds at 24 h after treatment, confirming that metformin activated AMPK signaling at the transcriptional level in H. cunea larvae. Conversely, the expressions of two vital Halloween genes (HcCYP306A1 and HcCYP314A1) in the 20E synthesis pathway were remarkably suppressed by metformin. Thus, we presumed that metformin delayed larval molting probably by impeding 20E synthesis in the H. cunea larvae. Finally, we found that metformin accelerated glycogen breakdown, elevated in vivo trehalose level, promoted chitin synthesis, and upregulated transcriptions of the genes in chitin synthesis pathway. Taken together, the findings provide a new insight into the molecular mechanisms by which AMPK regulates carbohydrate metabolism and chitin synthesis in insects.

First Report of Black Spot Needle Blight of Pinus sylvestris var. mongolica Litv. Caused by Heterotruncatella spartii in China.

Pinus sylvestris var. mongolica Litv. (Pinales: Pinaceae) is an excellent tree for soil and water conservation in Northeast China. The Honghua'erji area in Inner Mongolia is the "hometown of P. sylvestris var. mongolica", however, in recent years, coniferous diseases of P. sylvestris var. mongolica have frequently occurred here. During the investigation, it was found that some black spot needle blight had been observed in addition to the common blight caused by Sphaeropsis sapinea. From May to September 2020, black spot needle blight was found on hundreds of P. sylvestris var. mongolica trees in four forest farms, and the infection rate among the forests was 24.58 % (n=240). This disease first appeared on the upper part of the needles, and the needles then became withered and gradually showed light black spots, although they remained green. As the disease progressed, the needles eventually died and turned gray with many dark black spots. Fungal isolate named YJ-1 was obtained from infected needles of symptomatic pine trees, and a voucher specimen was deposited in Heilongjiang Province Key Laboratory of Forest Protection. Microscopic observation showed the conidia were 3-septate (4 cells) clavate spindles that measured 23.9 µm (20.8-25.9) × 5.9 µm (4.5-8.2) (n=50). The middle two cells were dark brown, and the septa were darker than the cells. Both apical and basal cells were hyaline. The apical cell had 2-4 appendages (mostly 3), and the basal cell had a truncate base (n=50). The cultural characteristics on potato dextrose agar medium were flat off-white and dense in 3-5 d. At approximately 5-7 d, the reverse side of the colony turned pale to slightly luteous. Superficial black acervuli were distributed in the center of the mature colonies after 10 d. Morphological, cultural and microscopic characteristics observed were similar of Heterotruncatella spartii (basionym: Truncatella spartii) reported by Hlaiem et al (2019). To further identify, total DNA was extracted and the internal transcribed spacer region (ITS-rDNA) was amplified by PCR using the primers ITS1/ITS4 and sequenced for BLASTn analysis and phylogenetic tree construction. The resulting 564 bp sequence (GenBank Accession No. OL662864) had 99.24% (521/525) to H. spartii MFLUCC 15-0537, with bootstrap support of at least 94% using the Neighbor-Joining algorithm by MEGA-X (Felsenstein, 1985). The fungus was identified as H. spartii based on morphology and molecular methods. A pathogenicity test was conducted by preparing a conidial suspension of 2.0 × 107 conidia/mL. The suspension was sprayed onto the needles of 20 pots of annual P. sylvestris ar. mongolica seedlings, and the control was sprayed with sterile water. Then the seedlings were placed in a constant temperature room at 25 °C. After 30 d, typical symptoms appeared on 11 inoculated needles, while the control needles remained symptomless. After 50 d, the re-isolation infection rate reached 66.7 %. The fungus present on the inoculated seedlings was morphologically identical to that originally observed on diseased pines, fulfilling Koch's postulates. The fungus was isolated from Spartium junceum for the first time and designated Truncatella spartii (Senanayake et al, 2015). It was then renamed H. spartii (Liu et al, 2019) and has been reported to infect P. pinea in Tunisia (Hlaiem et al, 2019). To our knowledge, this is the first report of H. spartii causing black spot needle blight on P. sylvestris var. mongolica in China and worldwide.

Transcriptomic analysis of interactions between Lymantria dispar larvae and carvacrol.

Due to its biological activity, carvacrol (CAR) is widely used in medicine, agriculture, and forestry. Our previous studies showed that in Lymantria dispar larvae, CAR treatment can induce the production of antifeedants and lead to growth inhibition and death of larvae. However, the effect CAR exerts on RNA levels in L. dispar larvae remains unclear. In this study, the Illumina HiSeq4000 sequencing platform was used to sequence the total RNA of L. dispar larvae. A total of six cDNA libraries (three treatments and three controls) were established and 39,807 genes were generated. Compared with the control group, 296 differentially expressed genes (DEGs) (142 up-regulated and 154 down-regulated) were identified after CAR treatment. GO and KEGG enrichment analyses showed that these DEGs mainly clustered in the metabolism of xenobiotics, carbohydrates, and lipids. Furthermore, 12 DEGs were found to be involved in detoxification, including six cytochrome P450s, two esterases, one glutathione peroxidase, one UDP-glycosyltransferase gene, and two genes encoding heat shock proteins. The expression levels of detoxification genes changed under CAR treatment (especially P450s), which further yielded candidate genes for explorations of the insecticidal mechanism of CAR. The reliability of transcriptome data was verified by qRT-PCR. The enzyme activities of CYP450 and acid phosphatase significantly increased (by 38.52 U/mg·prot and 0.12 µmol/min·mg, respectively) 72 h after CAR treatment. However, the activity of alkaline phosphatase did not change significantly. These changes in enzyme activity corroborated the reliability of the transcriptome data at the protein level. The results of GO enrichment analysis of DEGs indicated that CAR influenced the oxidation-reduction process in L. dispar larvae. Furthermore, CAR can cause oxidative stress in L. dispar larvae, identified through the determination of peroxidase and polyphenol oxidase activities, total antioxidant capacity, and hydrogen peroxide content. This study provides useful insight into the insecticidal mechanism of CAR.

Antifungal Activity of 6-Methylcoumarin against Valsa mali and Its Possible Mechanism of Action.

Valsa canker of apple (VCA) caused by Valsa mali severely affected apple production in east Asia. With the increase in drug resistance, there is an urgent need for efficient and environmentally friendly antifungal agents. Coumarins have attracted much attention due to their excellent antimicrobial activity against plant pathogens. In this study, the antifungal activity of several coumarins against phytopathogenic fungi was evaluated, and then the antifungal activity of the screened 6-MCM against V. mali and its underlying mechanism was further investigated. The results of the in vitro antifungal activity assay showed that some coumarins had significant inhibitory effects on V. mali. Notably, 400 mg/L of 6-MCM had the best antifungal activity of 94.6%. Further experiments showed that 6-MCM slowed down the growth of V. mali mycelia and the germination of spores in a concentration-dependent manner, with EC50 of 185.49 and 54.62 mg/L, respectively. In addition, 6-MCM treatment increased mycelial conductivity, extracellular protein leakage, and MDA content, resulting in damage to the cell membrane. Moreover, 6-MCM significantly reduced the cell wall degrading enzymes secreted by V. mali, including EG, PG and PL, thereby limiting its pathogenic capacity. SEM and TEM results showed that 6-MCM treatment had a significant effect on the morphology and ultrastructure of mycelial cells. Inoculation of isolated apple branches found that the application of 6-MCM effectively inhibited the development of VCA and significantly reduced the incidence. All these results suggest that 6-MCM has the potential as a green substitute for VCA control.

The Protective Effects of Carrageenan Oligosaccharides on Intestinal Oxidative Stress Damage of Female Drosophila melanogaster.

Carrageenan oligosaccharides (COS) have been reported to possess excellent antioxidant activities, but the underlying mechanism remains poorly understood. In this study, H2O2 was applied to trigger oxidative stress. The results showed that the addition of COS could effectively extend the lifespan of female Drosophila, which was associated with improvements by COS on the antioxidant defense system, including a decrease in MDA, the enhanced activities of SOD and CAT, the reduction of ROS in intestinal epithelial cells, and the up-regulation of antioxidant-relevant genes (GCL, GSTs, Nrf2, SOD). Meanwhile, the axenic female Drosophila fed with COS showed almost no improvement in the above measurements after H2O2 treatment, which highlighted the antioxidant mechanism of COS was closely related to intestinal microorganisms. Then, 16S rDNA high-throughput sequencing was applied and the result showed that the addition of COS in diets contributed to the diversity and abundance of intestinal flora in H2O2 induced female Drosophila. Moreover, COS significantly inhibited the expression of gene mTOR, elevated its downstream gene 4E-BP, and further inhibited autophagy-relevant genes (AMPKα, Atg1, Atg5, Atg8a) in H2O2 induced female Drosophila. The inhibition of the mTOR pathway and the activation of autophagy was probably mediated by the antioxidant effects of COS. These results provide potential evidence for further understanding of COS as an intestinal antioxidant.

3-Bromopyruvate-induced glycolysis inhibition impacts larval growth and development and carbohydrate homeostasis in fall webworm, Hyphantria cunea Drury.

As a typical glycolytic inhibitor, 3-bromopyruvate (3-BrPA) has been extensively studied in cancer therapy in recent decades. However, few studies focused on 3-BrPA in regulating the growth and development of insects, and the relationship and regulatory mechanism between glycolysis and chitin biosynthesis remain largely unknown. The Hyphantria cunea, named fall webworm, is a notorious defoliator, which caused a huge economic loss to agriculture and forestry. Here, we investigated the effects of 3-BrPA on the growth and development, glycolysis, carbohydrate homeostasis, as well as chitin synthesis in H. cunea larvae. To elucidate the action mechanism of 3-BrPA on H. cunea will provide a new insight for the control of this pest. The results showed that 3-BrPA dramatically restrained the growth and development of H. cunea larvae and resulted in larval lethality. Meanwhile, we confirmed that 3-BrPA caused a significant decrease in carbohydrate, adenosine triphosphate (ATP), pyruvic acid (PA), and triglyceride (TG) levels by inhibiting glycolysis in H. cunea larvae. Further studies indicated that 3-BrPA significantly affected the activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and trehalase, as well as expressions of the genes related to glycolysis, resulting in carbohydrate homeostasis disorder. Moreover, it was found that 3-BrPA enhanced 20-hydroxyecdysone (20E) signaling by upregulating HcCYP306A1 and HcCYP314A1, two critical genes in 20E synthesis pathway, and accelerated chitin synthesis by upregulating transcriptional levels of genes in the chitin synthesis pathway in H. cunea larvae. Taken together, our findings provide a novel insight into the mechanism of glycolytic inhibitor in regulating the growth and development of insects, and lay a foundation for the potential application of glycolytic inhibitors in pest control as well.