Gene knockout is often challenging for the notorious crop killer Botrytis cinerea due to its multinucleate nature. Here, we established an efficient method for gene knockout in B. cinerea based on high-pressure positive and negative double selection (HPDS). For knockout of the toxin biosynthesis gene BOA6, positive selections in the high-pressure mode significantly increased the proportion of homokaryotic mutants. When an HSVtk(Bc)-associated negative selection system was simultaneously introduced, all obtained transformants were confirmed as homokaryotic knockout mutants. For highly expressed locus oahA, we employed a reverse replacement strategy to reduce the positive selection marker expression by triggering RNAi, facilitating efficient knockout via HPDS. This strategy is also efficiently applicable to the field strain CLS1. We established a high-throughput gene knockout system by which 69 of 71 predicted genes encoding secreted proteins were knocked out in CLS1, indicating its efficient uses for large-scale gene knockout in B. cinerea.

Neuron-specific enolase (NSE) is currently the most reliable biomarker for small cell lung cancer (SCLC), which is important for disease monitoring, clinical evaluation and diagnosis. However, traditional methods suffer from various disadvantages, including instability, complexity, time-consuming operations, and the necessity for standards. In this study, we developed a calibration-free concentration analysis (CFCA) method based on surface plasmon resonance (SPR) technology, to accurately quantify the active concentration of NSE without relying on any standards. Based on the principle of CFCA, the active concentration of NSE can be calculated by observing binding rate variations at two flow rates under partial mass transport limitation and combining it with the known diffusion coefficient of the NSE. Using the method of CFCA, the active concentration of NSE was determined was only 0.48 mg/mL with an intra-day repeatability of 4.75%. The method has the advantages of simplicity, rapidity, realistic analysis and ease of implementation of high-throughput automated detection. Therefore, the method is expected to become the main measurement method for protein active concentration, which will be beneficial for the development of active protein standards.

IntroductionPine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, poses a significant threat to global pine forests. The sulfate permease (SULP) family is essential for sulfate transport, sulfur assimilation and cellular homeostasis, yet it remains uncharacterized in B. xylophilus. This study aimed to comprehensively identify all members of the SULP family in B. xylophilus and to elucidate their roles in nematode development and stress adaptation.MethodsThrough genomic data analysis, we identified 10 members of the SULP family in B. xylophilus and conducted a comprehensive characterization of their physicochemical properties, conserved motifs, protein structures, and gene expression profiles across different developmental stages.ResultsThe results revealed Bx-sulps were located on 5 chromosomes of B. xylophilus. Phylogenetic analysis unveiled both conserved and divergent evolutionary patterns of these proteins compared to counterparts in other nematodes. Expression analysis demonstrated upregulation of Bx-sulps during the dauer third-instar larva (D3) stage, suggesting their involvement in stress response and diapause. Moreover, certain Bx-sulps exhibited high expression levels in adult stages, indicating a potential role in reproductive processes.DiscussionThe study presents the first comprehensive examination of BxSULP family, shed light on its significance in nematode development and stress adaptation. These findings provide the groundwork for further functional investigations and may aid in the development of targeted strategies for managing PWD.

Streptomyces are the well-known producers of biologically active pharmaceuticals and agricultural antibiotics. However, the production of the secondary metabolites was limited due to the unbalance of the cell growth and the activation of secondary metabolic pathway. Here, in order to improve the production of toyocamycin, a nucleoside antibiotic produced by Streptomyces diastatochromogenes 1628, with excellent antimicrobial characteristics to plant pathogens, we develop an endogenous non-inducer-dependent dynamic regulatory strategy based on the quorum sensing (QS) promoter to fine-tune the expression of an important signal molecule (ppGpp). Firstly, two QS promoter PstbA1 and PstbA2 were identified by the bioinformatic analysis. Then, the temporal profiles of PstbA1 and PstbA2 were characterized by the GusA activity. Afterwards, the ppGpp synthase was expressed under the PstbA1 and PstbA2 to improve the ppGpp concentration in vivo. The toyocamycin production reached 768.62 mg/L and 721.06 mg/L under the QS promoter PstbA1 and PstbA2, respectively, which represented increases of 508.81 % and 471.14 % over the wild-type at the tenth day. The autoregulated QS-based strategy is an effective method by balancing bacterial growth and metabolite synthesis in industrial processes.

The Zn(II)2Cys6 zinc cluster protein family comprises a subclass of zinc-finger proteins that serve as transcriptional regulators involved in a diverse array of fugal biological processes. However, the roles and mechanisms of the Zn(II)2Cys6 transcription factors in mediating Botrytis cinerea, a necrotrophic fungus that causes gray mold in over 1000 plant species, development and virulence remain obscure. Here, we demonstrate that a novel B. cinerea pathogenicity-associated factor BcFTG1 (fungal transcription factor containing the GAL4 domain), identified from a virulence-attenuated mutant M20162 from a B. cinerea T-DNA insertion mutant library, plays an important role in oxalic acid (OA) secretion, carbon source absorption and cell wall integrity. Loss of BcFTG1 compromises the ability of the pathogen to secrete OA, absorb carbon sources, maintain cell wall integrity, and promote virulence. Our findings provide novel insights into fungal factors mediating the pathogenesis of the gray mold fungus via regulation of OA secretion, carbon source utilization and cell wall integrity.

Bis (3′,5′)-cyclic diguanylic acid (c-di-GMP) is a ubiquitous second messenger that controls several metabolic pathways in bacteria. In Streptomyces, c-di-GMP is associated with morphological differentiation, which is related to secondary metabolite production. In this study, we identified and characterized a diguanylate cyclase (DGC), CdgB, from Streptomyces diastatochromogenes 1628, which may be involved in c-di-GMP synthesis, through genetic and biochemical analyses. To further investigate the role of CdgB, the cdgB-deleted mutant strain Δ-cdgB and the cdgB-overexpressing mutant strain O-cdgB were constructed by genetic engineering. A phenotypic analysis revealed that the O-cdgB colonies exhibited reduced mycelium formation, whereas the Δ-cdgB colonies displayed wrinkled surfaces and shriveled mycelia. Notably, O-cdgB demonstrated a significant increase in the toyocamycin (TM) yield by 47.3%, from 253 to 374 mg/L, within 10 days. This increase was accompanied by a 6.7% elevation in the intracellular concentration of c-di-GMP and a higher transcriptional level of the toy cluster within four days. Conversely, Δ-cdgB showed a lower c-di-GMP concentration (reduced by 6.2%) in vivo and a reduced toyocamycin production (decreased by 28.9%, from 253 to 180 mg/L) after 10 days. In addition, S. diastatochromogenes 1628 exhibited a slightly higher inhibitory effect against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani compared to Δ-cdgB, but a lower inhibition rate than that of O-cdgB. The results imply that CdgB provides a foundational function for metabolism and the activation of secondary metabolism in S. diastatochromogenes 1628.

Pathogenic bacterial contamination of food is a major challenge in food safety and public health management. To monitor pathogenic bacteria in food, a rapid, sensitive, and specific pathogen detection method is needed. CRISPR-Cas12a has been developed as a sensitive method for detecting pathogenic bacteria, but its output signal is fluorescent, which need equipment to detect. Here, the nanoparticle probe was designed to detect the activity of Cas12a and were able to visualize the assay results by observing the color change by naked eyes. The target sequences of pathogenic bacteria were amplified by recombinase polymerase, then recognized and bound by the crRNA and Cas12a complex. The activated Cas12a cleaved the linker-ssDNA connecting the nanoparticles, causing the nanoparticles to transition from aggregation to dispersion, and the color showed as purple. The sensitivity analysis showed that the limits of detection (LOD) reached 1 x 10 degrees CFU/mL, 1 x 104 CFU/mL, 1 x 102 CFU/mL, 1 x 103 CFU/mL, 1 x 101 CFU/mL for Escherichia coli, Salmonella typhimurium, Vibrio parahaemolyticus, Staphylococcus aureus, and Listeria monocytogenes, which were all specific for five bacteria in the milk samples. Therefore, this method is a dependable, fast, sensitive and specific method, which make the CRIPSR-Cas12a detection method more useful in bacterial pathogenic detection.

Drug resistance in Staphylococcus aureus is a serious problem, and the development of new antimicrobial drugs to circumvent drug resistance has become a trend. In this study, we isolated a strain of Bacillus subtilis with high tolerance to alcohol, pH, NaCl, bile salt, surfactants, temperature, and simulated intestinal fluids. We optimized culture parameters to obtain the best fermentation conditions for the production of inhibitory compounds in cell-free culture media. The crude extract showed excellent stability when exposed to temperature, pH, and ultraviolet radiation, with almost no loss of bacteriostatic activity after treatment. After isolation and purification, the peptide sequences were identified using ultraperformance liquid chromatography–mass spectrometry (UPLC–MS), and the antibacterial sequences were analyzed using bioinformatics. The results of the identification showed that there was one novel bacteriocin (NSGGSYGSGGGGGGGNSHGY) with a molecular weight of 1513.58 Da. The minimum inhibitory concentration (MIC) of the B5 bacteriocin was 31.25 μg/mL against S. aureus, and it is noteworthy that bacteriocin B5 also showed weak antibacterial activity against Vibrio parahaemolyticus. In conclusion, this study developed a novel bacteriocin that has the potential to be used as an alternative to S. aureus antibiotics.

ppGpp is a ubiquitous small nucleotide messenger that mediates cellular self-protective responses under environmental stress. However, the mechanisms of ppGpp that control transcription and other metabolic processes depend on the species, and ppGpp regulates the same process via different mechanisms. The level of ppGpp is regulated by RelA/SpoT homolog (RSH) enzymes that synthesize and hydrolyze the alarmone. Here, we constructed a ppGpp0 strain and monitored the effects of ppGpp on the transcriptional level, physiology, and secondary metabiotic production in the antibiotic producer Streptomyces diastatochromogenes 1628. The results showed the cell division and growth of ppGpp0 increased by measurement of gene transcription and DCWs. The utilization of nitrogen was affected depending on the nitrogen type with a significantly higher DCW of the ppGpp0 mutant in the medium supplied with the yeast extract and a lower growth rate in the inorganic nitrogen ammonium salt. The ppGpp-mediated stringent response could not affect the usage of carbon resources. More importantly, ppGpp0 inhibited the expression of antibiotic clusters and the production of toyocamycin and tetramycin P. The antibiotic resistance was also significantly downregulated in the ppGpp0 mutant. In conclusion, this study showed detailed changes in ppGpp-mediated stringent responses on S. diastatochromogenes 1628 cell growth, nutrient utilization, morphological characteristics, antibiotic production, and resistance, which will provide insights into the role of ppGpp in Streptomyces. IMPORTANCE The ppGpp-mediated stringent response is widely distributed in Escherichia coli, Bacillus subtilis, Streptomyces, Staphylococcus aureus, etc. Stringent responses give strains the ability to resist environmental stresses, and survival from nutrition starvation, virulence, long-term persistence, biofilm formation, and gut colonization. ppGpp has many targets in cells and can reprogram DNA replication, transcription, ribosome biogenesis and function, and lipid metabolism. However, the mechanism of ppGpp to control transcription and other metabolic processes depends on the bacterial species and regulates the same process via a different mechanism. In Streptomyces, how ppGpp regulates the transcription remains to be elucidated. However, because ppGpp regulates many genes involved in primary and secondary metabolism, we compared the transcription and cell division, cell growth, morphological differentiation, antibiotic resistance, and secondary synthesis in the wild-type S. diastatochromogenes and ppGpp0 strains.

Controlled fungicide delivery in response to the specific microenvironment produced by fungal pathogens is an advisable strategy to improve the efficacy of fungicides. Herein, the authors construct a smart fungicide nanoplatform, using mesoporous silica nanoparticles (MSNs) as nanocarriers loaded with eugenol (EU) and Ag+ coordinated polydopamine (Ag+ -PDA) as a coating to form Ag+ -PDA@MSNs-EU NPs for Botrytis cinerea (B. cinerea) control. As a botanical fungicide, EU offers an eco-friendly alternative to synthetic fungicides and can upregulate several defense-related genes in the tomato plant. The Ag+ -PDA coating can lock the EU inside the nanocarriers and respond to the oxalic acid produced by B. cinerea to corelease the loaded EU and Ag+ . The results demonstrate that Ag+ -PDA@MSNs-EU NPs can effectively inhibit the mycelial growth of B. cinerea on detached and potted tomato leaves. The construction of such a smart fungicide nanoplatform provides new guidance to design controlled fungicides release systems, which can respond to the microenvironment associated with plant pathogen to realize fungus control.

α-Keto acids are important raw materials for pharmaceuticals and functional foods, which could be produced from cheap feed stock by whole cell biocatalysts containing L-amino acid deaminases (L-AADs). However, the production capacity is limited by the low activity of L-AADs. The L-AAD mediated redox reaction employs the electron transport chain to transfer electrons from the reduced FADH2 to O2, implying that the interaction between L-AAD and the cell membrane affects its catalytic activity. To improve the catalytic activity of L-AAD from Proteus vulgaris, we redesigned the membrane-bound hydrophobic insertion sequences (INS, residues 325–375) by saturation mutagenesis and high-throughput screening. Mutants D340N and L363N exhibited higher affinity and catalytic efficiency for L-leucine, with half-life 1.62-fold and 1.28-fold longer than that of wild-type L-AAD. D340N catalyzed L-leucine to produce 81.21 g⋅L–1 α-ketoisocaproate, with a bioconversion rate of 89.06%, which was 17.57% higher than that of the wild-type. It is predicted that the mutations enhanced the interaction between the protein and the cell membrane.

Aims: In our clinical work, some patients with type I hypersensitivity could be detected protein in their urine. This study focused on the early renal injury in patients with type I hypersensitivity.Main methods: From 43 type I hypersensitivity patients with proteinuria, 10 patients were randomly selected for mass spectrometry analysis of 24-h urine together with 5 healthy volunteers. Mice were vaccinated with Dermatophagoides farina (Der f) and ovalbumin (OVA) were used as antigen to establish the type I hypersensitivity animal models.Key findings: The urine protein of hypersensitivity patients was significantly increased in the alpha-1microglobulin/ bikunin precursor (Protein AMBP) (t = 3.140, P = 0.008), retinol binding protein 4 (RBP4) (t = 2.426, P = 0.031), kininogen-1 (t = 2.501, P = 0.027), and transferrin appeared only in patients' urine. After immunizing mice with antigens, significant increases of the total serum immunoglobulin E (IgE) were observed in both Der f (86.92 +/- 36.01 U/mL, t = 5.231, P = 0.0004) and OVA group (34.65 +/- 24.72 U/mL, t = 2.891, P = 0.0161) compared with the negative control group (2.68 +/- 0.47 U/mL). Meanwhile, definite eosinophil infiltration around the impaired renal tubules as well as the bronchus in Der f mice were observed, and urine protein appeared. After stopping the allergen stimulation, proteinuria disappeared. Instead, when the mice were treated with the antigen again, proteinuria reappeared.Significance: Our findings suggest that renal tubular damage in patients with type I hypersensitivity is reversible, and proteinuria disappears with allergy symptoms remission.

Ophiopogon japonicus (Linn. f.) Ker-Gawl, a traditional Chinese medicinal plant, is widely cultured in China. The root of O. japonicus, is used as the main ingredient in many presriptions. It is rich in chemical components for steroidal saponins, homoisoflavonoids and polysaccharides, which have various pharmacological activities, such as cardiovascular protection, anti-inflammation and anti-diabetes (Chen. et al. 2016). In May and July for 2018 and 2019, the symptoms of black spot on O. japonicus were observed with an incidence of 40% in Cixi County, Zhejiang Province, China. The pathogen mainly infected leaves causing severe black spots, which resulted in a 28% yield loss per acre. At the early stage of the disease, the tip of the leaf began to turn yellow, then the discoloration gradually spread to the base of the leaf and finally the whole leaf turned reddish brown with visible black spot. Symptomatic leaves were cut into small pieces (1.0 cm × 1.0 cm) and disinfected successively by submersion in 75% ethanol for 30s and 1% NaClO for 30s under aseptic conditions. After rinsing with sterile water three times and air drying, segments were placed on potato dextrose agar (PDA), and incubated at 28 ℃ in dark for a week. Then, pathogen on the PDA were transferred onto potato carrot agar (PCA), and incubated at 23 ℃ under the condition of alternation of day (12 h) and night (12 h) for a week. Colonies on PDA were dark gray in the center surrounded by white to gray on the upper side, and black with white margins on the back of the plate. Colonies on PCA were grayish with sparse hyphae. The conidia were obclavate or ellipsoid, pale brown, with 3~8 transverse septa and 1~4 longitudinal septa. Conidiophores were septate, arising singly, and measured (17.0~81.0) × (8.0~23.5) μm, Most conidia had a conical or columnar beak, approximately (0~23.5) × (2.5~9.0) μm in size. According to morphological and cultural characteristics, these isolates were preliminarily identified as Alternaria alternata. A. alternata is one of the most typical plant pathogen, more than 95% of which facultatively parasitize on plants, causing disease in numerous crops. To further confirm identification of pathogens, the internal transcribed spacer region (ITS), translation elongation factor 1-α gene (EF-1α), RNA polymerase Ⅱ second largest subunit (RPB2), major allergen Alt a 1 gene (Alt a 1), Histon 3 gene (His) and plasma membrane ATPase (ATP)were amplified with primer pairs ITS1/ITS4, EF1-728F/EF1-986R, RPB2-7cr/RPB2-5f2, Alt-for/Alt-rev, His 3-F/His 3-R, ATP-F/ATP-R (Lawrence D.P. et al. 2013; Hong, S.G., et al. 2005). BLASTN analysis of NCBI using ITS (Accession NO. MW989987), Alt a1 (Accession NO. MW995953), EF-1α (Accession NO.MW995955), ATP (Accession NO.MW995957), His (Accession NO. MW995954) and RPB2 (Accession NO. MW995956) showed 100%, 100%, 97%, 99%, 99% and 97% identity to A. alternata MN249500.1, MN304714.1, MK637432.1, MK804115.1, MK460236.1, MK605888.1, respectively. To verify pathogenicity, healthy plants (1-year-old) of O. japonicus in ten pots were spray-inoculated with conidial suspension (1 × 106 conidia/ml). Ten plants, which were treated with sterile water, were used as the control. All plants were maintained in a climatic chamber (26 ± 1 ℃, 70-80% relative humidity and a photoperiod of 16:8 [L: D] h). Fourteen days later, all inoculated plants showed typical symptoms of black spot identical to those observed in the fields. Control plants remained symptomless and healthy. The pathogenicity analysis was repeated three times. Pathogens re-isolated from symptomatic plants were identified as A. alternata by morphology observation and sequence analysis. To our knowledge, this is the first report of black spot caused by A. alternata on O. japonicus in Zhejiang, China.

Toyocamycin, a nucleoside antibiotic, is a fungicide with the potential to control plant pathogens. In this study, three rounds of genome shuffling screening were applied to enhance the toyocamycin production in Streptomyces diastatochromogenes 1628. After three rounds of genome-shuffling screening, the toyocamycin production increased by 10.8-fold that of wild-type, and 2.64-fold of its parental strain. By optimization of its nutrition condition in medium, the highest production of toyocamycin reached 1173.6 mg/L in TY-producing medium. In addition, the mechanism for the improvement of shuffled strains was investigated. Recombinants with increased toyocamycin production exhibited higher transcriptional level of the toy cluster and product resistance. Furthermore, the rise of ATP hydrolysis rate indicated that intracellular ATP exhibit a significant role in tuning the toy cluster by an ATP-binding pathway-specific regulator. In all, we obtained S. diastatochromogenes mutants with enhanced toyocamycin production, and provided a valuable clue for the activation of secondary metabolites.

The symbionts in the gut of brown planthopper play an important role in the nutrition utilization and growth of their host, Nilaparvata lugens Stål (Hemiptera: Delphacidae). Controlling the BPH infection on rice by inhibiting the symbionts using antimicrobials is feasible. However, the impact of antimicrobials on the microbiome in the gut has not been fully elucidated. In this study, we found the mortality reached 35.5%, 33.1% and 19.4%, when BPHs were exposed to toyocamycin, tebuconazole, and zhongshengmycin, respectively. Significant differences were found between the structures of gut microbial communities in adult BPHs treated with different antimicrobials and water. The antimicrobials reduced the fungal diversity by reducing the non-dominant fungi abundance, and increased bacterial diversity by inhibiting the dominant bacteria Acinetobacter in the gut. The diversification of taxonomic groups in gut depended on the different selective stress of antimicrobials. For the microbial absolute abundance, the total microbial gut community abundance decreased under antimicrobial exposure, but the absolute abundance of Serratia significantly increased in the antimicrobial treatment group. Overall, our study enriched the knowledge of microbiomes in the gut of BPH under the antimicrobial treatment and provided guidelines to enhance the pest management effect of BPH by using antimicrobials.

Dendrobium officinale is a valuable and rare Chinese herb, which is easy to suffer from various diseases during its growth. Thus early diagnosis of those diseases is crucial to increase the yield of D. officinale. In this study, we compared the internal transcribed spacer regions of the ribosomal DNA sequences of four widely disseminated pathogens in D. officinale (Sclerotium delphinii, Colletotrichum gloeosporioides, Alternaria alternata, and Cladosporium cladosporioides), designed specific primers for A. alternata and C. cladosporioides, and established a four-seat nested multiplex PCR assay. The sensitivity for S. delphinii, C. cladosporioides, A. alternata was found to be 10 fg/mu L, and 10 pg/mu L for C. gloeosporioides. In the actual application test, our established nested multiplex PCR can detect pathogens more quickly and accurately than conventional culture method. This method combines the advantages of nested and multiplex techniques, and it considerably improves the sensitivity and specificity of the detection results. Therefore, we hope that this method can provide a tool for diagnosing D. officinale diseases.

Cyclophilin (Cyp) and Ca2+/calcineurin proteins are cellular components related to fungal morphogenesis and virulence; however, their roles in mediating the pathogenesis of Botrytis cinerea, the causative agent of gray mold on over 1000 plant species, remain largely unexplored. Here, we show that disruption of cyclophilin gene BcCYP2 did not impair the pathogen mycelial growth, osmotic and oxidative stress adaptation as well as cell wall integrity, but delayed conidial germination and germling development, altered conidial and sclerotial morphology, reduced infection cushion (IC) formation, sclerotial production and virulence. Exogenous cyclic adenosine monophosphate (cAMP) rescued the deficiency of IC formation of the ∆Bccyp2 mutants, and exogenous cyclosporine A (CsA), an inhibitor targeting cyclophilins, altered hyphal morphology and prevented host-cell penetration in the BcCYP2 harboring strains. Moreover, calcineurin-dependent (CND) genes are differentially expressed in strains losing BcCYP2 in the presence of CsA, suggesting that BcCyp2 functions in the upstream of cAMP- and Ca2+/calcineurin-dependent signaling pathways. Interestingly, during IC formation, expression of BcCYP2 is downregulated in a mutant losing BcJAR1, a gene encoding histone 3 lysine 4 (H3K4) demethylase that regulates fungal development and pathogenesis, in B. cinerea, implying that BcCyp2 functions under the control of BcJar1. Collectively, our findings provide new insights into cyclophilins mediating the pathogenesis of B. cinerea and potential targets for drug intervention for fungal diseases.

Dendrobium officinale Kimura et Migo is a rare and valuable Chinese herb cultivated in Zhejiang and Yunnan Provinces, China, which is known for its functions as an anti-neoplastic and for lowering the blood sugar (Cheng et al., 2019). In September and October of 2018 and 2019, symptoms of root rot on D. officinale were observed with an incidence of 15-20% in Wuyi County, Zhejiang Province, China. The pathogen mainly infected roots causing severe root rot, which resulted in significant economic losses. At the early stage of this disease, the stalk turned brown, then the whole plant rotted from bottom to top within a few days. Symptomatic roots were cut into small pieces (1.0 cm × 1.0 cm) and disinfected successively by submersion in 75% ethanol for 30 s and 1% NaClO for 30 s under aseptic conditions. After rinsing with sterile water three times and air drying, segments were placed on potato dextrose agar (PDA). After incubation at 25 °C for 5 d in the dark, white to pale cream colored colonies were produced. The average mycelial growth rate was 15.2-18.5 mm day-1 at 25 ℃. Macroconidia were falciform with three to five septa and (18.0-32.0)×(3.0-5.0) μm in size. Microconidia were fusiform with two to three septa (7.0-10.0)×(2.1-3.0) μm. Based on morphological characteristics of macroconidia, and microconidia, isolates were identified as Fusarium incarnatum-equiseti species complex (span style="font-family:'Times New Roman'; font-size:12pt">FIESC) (Avila et al., 2019). The internal transcribed spacer (ITS) region, translation elongation factor (EF-1α), RNA polymerase largest subunit (RPB1), and RNA polymerase second largest subunit (RPB2) gene were amplified and sequenced respectively using ITS1/ITS4, EF1/EF2, Fa/G2R and 5f2/7cr primers (O'Donnell et al., 2010). BLASTN analysis of FUSARIUM-ID using ITS (Accession NO. MW172977), EF-1α (Accession NO. MW172978, RPB1(Accession NO. MW172979), and RPB2(Accession NO. MW172980) showed 99.8%, 100%, 99.74%, and 98.63% identity to FIESC isolates NRRL43619, NRRL34059, NRRL32864, and NRRL32175, respectively. To verify pathogenicity, ten 1-year-old healthy D. officinale plants were used for inoculation tests. One milliliter of a conidial suspension (106 conidia ml-1) was pipetted onto the soil around the base of D. officinale plants per pot. Ten plants, which were treated with sterile water, were used as the control. All plants were maintained in a climatic chamber (26 ± 1 ℃, 70-80% relative humidity and a photoperiod of 16:8 [L: D] h). Seven days later, all inoculated plants showed typical symptoms of root rot identical to those observed in the fields. Control plants remained symptomless and healthy. The pathogenicity analysis was repeated three times. Pathogens re-isolated from symptomatic plants were identified as FIESC species by morphology observation and sequence analysis. To our knowledge, this is the first report of root rot caused by FIESC species on D. officinale in Zhejiang, China.

Symbionts in the abdomen fat body of brown planthopper (BPH) play an important role in the growth and reproduction of their host, Nilaparvata lugens Stål (Hemiptera: Delphacidae). Thus, controlling BPH infection on rice by inhibiting symbionts with antimicrobials is feasible. However, the effect of antimicrobials on the microbiome in the fat body and the relationship between microbial community and mortality have not been fully elucidated. A decrease in the total number of yeast-like symbiotes in the fat body and elevated mortality were observed after exposure to toyocamycin, tebuconazole, and zhongshengmycin. Additionally, we found that the antimicrobials reduced bacterial diversity and increased fungal diversity in the fat body and altered the bacterial and fungal community structure. Although the total absolute abundance of bacteria and fungi decreased after antimicrobial exposure, the absolute abundance of Serratia increased, indicating that Serratia, which was the most dominant in the fat body, is an important symbiont involved in resistance to antimicrobials. After antimicrobial exposure, seven genera, which probably participated in the nutrition and development function of the host, were totally eliminated from the fat body. Overall, our study enriches the knowledge of microbiomes in the fat body of BPH under antimicrobial treatment and the disturbance of symbionts would be further used to help other pesticides to control pests.

褐飞虱体内存在大量的共生菌,这些共生菌不但具有种类多样性,同时在对寄主的功能上也存在多样性.目前利用分子生物学手段以及高通量测序技术鉴定得到的测序丰度大于0.1％的共生真菌和共生细菌种类分属19和53个不同的属.由于技术的局限性和共生菌难以离体培养的特性,仍有相当部分共生菌分类地位尚未明确.共生菌在褐飞虱的生长、发育、繁殖、营养代谢、抗性变异以及免疫功能等生命活动中起着至关重要的作用,种类丰富的共生菌发挥着不同的功能.共生真菌主要参与固醇类物质和必需氨基酸的合成,而共生细菌则主要参与维生素的合成.共生菌在褐飞虱致害性变异、抗药性发展以及对宿主的繁殖等方面也都产生了重要的影响,但是具体的分子机制尚未明确.本文针对褐飞虱体内共生菌多样性研究概况进行综述,并对今后的研究侧重点提出了建议,后续研究可以聚焦于:(1)共生菌种类的鉴定;(2)特定、单一种类共生菌的功能;(3)共生菌在褐飞虱体内各组织间的扩散途径、扩散种类和调控机制;(4)以共生菌为靶标进行褐飞虱的防治等.