Burkholderia cenocepacia epigenetic regulator M.BceJIV simultaneously engages two DNA recognition sequences for methylation.

Burkholderia cenocepacia is an opportunistic and infective bacterium containing an orphan DNA methyltransferase (M.BceJIV) with roles in regulating gene expression and motility of the bacterium. M.BceJIV recognizes a GTWWAC motif (where W can be an adenine or a thymine) and methylates the N6 of the adenine at the fifth base position (GTWWAC). Here, we present a high-resolution crystal structure of M.BceJIV/DNA/sinefungin ternary complex and allied biochemical, computational, and thermodynamic analyses. Remarkably, the structure shows not one, but two DNA substrates bound to the M.BceJIV dimer, wherein each monomer contributes to the recognition of two recognition sequences. This unexpected mode of DNA binding and methylation has not been observed previously and sets a new precedent for a DNA methyltransferase. We also show that methylation at two recognition sequences occurs independently, and that GTWWAC motifs are enriched in intergenic regions of a strain of B. cenocepacia's genome. We further computationally assess the interactions underlying the affinities of different ligands (SAM, SAH, and sinefungin) for M.BceJIV, as a step towards developing selective inhibitors for limiting B. cenocepacia infection.

mEnrich-seq: methylation-guided enrichment sequencing of bacterial taxa of interest from microbiome.

Metagenomics has enabled the comprehensive study of microbiomes. However, many applications would benefit from a method that sequences specific bacterial taxa of interest, but not most background taxa. We developed mEnrich-seq (in which 'm' stands for methylation and seq for sequencing) for enriching taxa of interest from metagenomic DNA before sequencing. The core idea is to exploit the self versus nonself differentiation by natural bacterial DNA methylation and rationally choose methylation-sensitive restriction enzymes, individually or in combination, to deplete host and background taxa while enriching targeted taxa. This idea is integrated with library preparation procedures and applied in several applications to enrich (up to 117-fold) pathogenic or beneficial bacteria from human urine and fecal samples, including species that are hard to culture or of low abundance. We assessed 4,601 bacterial strains with mapped methylomes so far and showed broad applicability of mEnrich-seq. mEnrich-seq provides microbiome researchers with a versatile and cost-effective approach for selective sequencing of diverse taxa of interest.

MIST: a Multilocus Identification System for Trichoderma.

Due to the rapid expansion in microbial taxonomy, precise identification of common industrially and agriculturally relevant fungi such as Trichoderma species is challenging. In this study, we introduce the online multilocus identification system (MIST) for automated detection of 349 Trichoderma species based on a set of three DNA barcodes. MIST is based on the reference databases of validated sequences of three commonly used phylogenetic markers collected from public databases. The databases consist of 414 complete sequences of the nuclear rRNA internal transcribed spacers (ITS) 1 and 2, 583 sequence fragments of the gene encoding translation elongation factor 1-alpha (tef1), and 534 sequence fragments of the gene encoding RNA polymerase subunit 2 (rpb2). Through MIST, information from different DNA barcodes can be combined and the identification of Trichoderma species can be achieved based on the integrated parametric sequence similarity search (blastn) performed in the manner of a decision tree classifier. In the verification process, MIST provided correct identification for 44 Trichoderma species based on DNA barcodes consisting of tef1 and rpb2 markers. Thus, MIST can be used to obtain an automated species identification as well as to retrieve sequences required for manual identification by means of phylogenetic analysis.IMPORTANCE The genus Trichoderma is important to humankind, with a wide range of applications in industry, agriculture, and bioremediation. Thus, quick and accurate identification of Trichoderma species is paramount, since it is usually the first step in Trichoderma-based research. However, it frequently becomes a limitation, especially for researchers who lack taxonomic knowledge of fungi. Moreover, as the number of Trichoderma-based studies has increased, a growing number of unidentified sequences have been stored in public databases, which has made the species identification more ambiguous. In this study, we provide an easy-to-use tool, MIST, for automated species identification, a list of Trichoderma species, and corresponding sequences of reference DNA barcodes. Therefore, this study will facilitate the research on the biodiversity and applications of the genus Trichoderma.

Estimation of instant case fatality rate of COVID-19 in Wuhan and Hubei based on daily case notification data

BackgroundThe outbreak of coronavirus disease 2019 (COVID-19) initially appeared and has most rapidly spread in Wuhan, China. The case fatality rate is the most direct indicator to assess the hazards of an infectious disease. We aimed to estimate the instant fatality rate and cure rate of COVID-19 in Wuhan City and its affiliated Hubei Province. MethodsWe collected the daily case notification data of COVID-19 from Dec 8, 2019 to Mar 10, 2020 in Wuhan City and Hubei Province officially announced by the Chinese authority. The numbers of daily confirmed/deaths/cured cases and the numbers of daily cumulative confirmed/deaths/cured cases were obtained. The death time and cure time of COVID-19 patients were calculated based on the dates of diagnosis, death and discharge of individual cases. Then the estimated diagnosis dates of deaths and cured cases were obtained on the basis of the median death or medium cure time, respectively. Finally, the instant fatality rate of COVID-19 was calculated according to the numbers of deaths and cured cases on the same estimated diagnosis dates. ResultsFrom Jan 1, 2020 to Feb 22, 2020 in Wuhan City, the instant case fatality rate of COVID-19 was 3.4%[~]19.5% and the instant cured rate was 80.0%[~]96.6%. The average fatality rate reached 11.4% while the average cure rate was 88.6%. During the same period in Hubei Province, the instant case fatality rate was 3.8%[~]16.6% and the instant cured rate was 83.4%[~]96.6%. The average fatality rate and the average cure rate were 9.2% and 91.8%, respectively. ConclusionsThe fatality rate and cure rate of COVID-19 in Wuhan City and Hubei Province were underestimated. Wuhan showed higher fatality rate and cure rate than the whole Hubei Province did.

Elicitor hydrophobin Hyd1 interacts with Ubiquilin1-like to induce maize systemic resistance.

Trichoderma harzianum is a plant-beneficial fungus that secretes small cysteine-rich proteins that induce plant defense responses; however, the molecular mechanism involved in this induction is largely unknown. Here, we report that the class II hydrophobin ThHyd1 acts as an elicitor of induced systemic resistance (ISR) in plants. Immunogold labeling and immunofluorescence revealed ThHyd1 localized on maize (Zea mays) root cell plasma membranes. To identify host plant protein interactors of Hyd1, we screened a maize B73 root cDNA library. ThHyd1 interacted directly with ubiquilin 1-like (UBL). Furthermore, the N-terminal fragment of UBL was primarily responsible for binding with Hyd1 and the eight-cysteine amino acid of Hyd1 participated in the protein-protein interactions. Hyd1 from T. harzianum (Thhyd1) and ubl from maize were co-expressed in Arabidopsis thaliana, they synergistically promoted plant resistance against Botrytis cinerea. RNA-sequencing analysis of global gene expression in maize leaves 24 h after spraying with Curvularia lunata spore suspension showed that Thhyd1-induced systemic resistance was primarily associated with brassinosteroid signaling, likely mediated through BAK1. Jasmonate/ethylene (JA/ET) signaling was also involved to some extent in this response. Our results suggest that the Hyd1-UBL axis might play a key role in inducing systemic resistance as a result of Trichoderma-plant interactions.

Analysis of metabolic changes in Trichoderma asperellum TJ01 at different fermentation time-points by LC-QQQ-MS.

Trichoderma spp. are among the most widely recognized biocontrol fungi used to inhibit pathogens and promote plant growth. These functions are related to primary and secondary metabolites. This study investigated the different metabolites in Trichoderma asperellum TJ01 cultured for 24 and 72 h using liquid chromatography with triple-quadrupole mass spectrometry. Compared to the 24 h culture of T. asperellum TJ01, the 72 h culture with amino acid metabolism tended to decrease while sugar and lipid metabolisms tended to increase. Furthermore, the 72 h culture had a higher proportion of upregulated flavonoids, in combination with a higher proportion of downregulated alkaloids, and equal proportions of upregulated and downregulated polyphenols and hormones. This study also identified a few valuable medicinal substances such as trigonelline and 5-hydroxytryptophan in T. asperellum TJ01 fermentation cultures.

Expressing Double-Stranded RNAs of Insect Hormone-Related Genes Enhances Baculovirus Insecticidal Activity.

Baculoviruses have already been used for insect pest control, but the slow killing speed limits their further promotion and application. Here we provide a strategy for improving baculovirus insecticidal activity using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) to express double-stranded RNAs (dsRNAs) targeting cotton bollworm (Helicoverpa armigera) juvenile hormone (JH)-related genes. Droplet-feeding bioassays show that the 50% lethal concentration (LC50) values of recombinant baculoviruses expressing the dsRNA of JH acid methyl transferase gene (HaJHAMT) and the JH acid binding protein gene (HaJHBP) were 1.24 × 104 polyhedral inclusion bodies (PIB)/mL and 2.26 × 104 PIB/mL, respectively. Both were much lower than the control value (8.12 × 104 PIB/mL). Meanwhile, the LT50 of recombinant baculovirus expressing dsRNA of HaJHBP was only 54.2% of the control value, which means that larval death was accelerated. Furthermore, the mRNA level of target genes was reduced in recombinant baculovirus-treated cotton bollworm larvae. Transcription of several key genes involved in hormone signaling pathways-for example, ecdysone receptor gene (HaEcR)-was also altered. This study establishes a new strategy for pest management by interfering with insect hormone-related gene expression via baculoviruses, and the engineered baculoviruses have great potential application in cotton production.

Identification of a novel strain, Streptomyces blastmyceticus JZB130180, and evaluation of its biocontrol efficacy against Monilinia fructicola.

Peach brown rot, caused by Monilinia fructicola, is one of the most serious peach diseases. A strain belonging to the Actinomycetales, named Streptomyces blastmyceticus JZB130180, was found to have a strong inhibitory effect on M. fructicola in confrontation culture. Following the inoculation of peaches in vitro, it was revealed that the fermentation broth of S. blastmyceticus JZB130180 had a significant inhibitory effect on disease development by M. fructicola. The fermentation broth of S. blastmyceticus JZB130180 had an EC50 (concentration for 50% of maximal effect) of 38.3 µg/mL against M. fructicola, as determined in an indoor toxicity test. Analysis of the physicochemical properties of the fermentation broth revealed that it was tolerant of acid and alkaline conditions, temperature, and ultraviolet radiation. In addition, chitinase, cellulase, and protease were also found to be secreted by the strain. The results of this study suggest that S. blastmyceticus JZB130180 may be used for the biocontrol of peach brown rot.

Streptomyces lydicus A01 affects soil microbial diversity, improving growth and resilience in tomato.

The actinomycete Streptomyces lydicus A01 promotes tomato seedling growth; however, the underlying mechanism is unclear. In this study, we investigated whether changes in soil microbial diversity, following Streptomyces lydicus A01 treatment, were responsible for the increased tomato seedling growth. Eukaryotic 18S ribosomal DNA (rDNA) sequencing showed that S. lydicus A01-treated and untreated soil shared 193 operational taxonomic units (OTUs), whereas bacterial 16S rDNA sequencing identified 1,219 shared OTUs between the treated and untreated soil. Of the 42 dominant eukaryotic OTUs, eight were significantly increased and six were significantly decreased after A01 treatment. Of the 25 dominant bacterial OTUs, 12 were significantly increased and eight were significantly decreased after A01 treatment. Most of the eukaryotes and bacteria that increased in abundance exhibited growth promoting characteristics, which were mainly predicted to be associated with mineralization of nitrogen and phosphorus, phosphate solubilization, nutrient accumulation, and secretion of auxin, whereas some were related to plant protection, such as the degradation of toxic and hazardous substances. Soil composition tests showed that S. lydicus A01 treatment enhanced the utilization of nitrogen, phosphorus, and potassium in tomato seedlings. Thus, microbial fertilizers based on S. lydicus A01 may improve plant growth, without the detriment effects of chemical fertilizers.

Co-culture of Bacillus amyloliquefaciens ACCC11060 and Trichoderma asperellum GDFS1009 enhanced pathogen-inhibition and amino acid yield.

BACKGROUND: Bacillus spp. are a genus of biocontrol bacteria widely used for antibiosis, while Trichoderma spp. are biocontrol fungi that are abundantly explored. In this study, a liquid co-cultivation of these two organisms was tried firstly. RESULTS AND DISCUSSION: Through liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), it was discovered that with an inoculation in the ratio of 1.9:1, the antimicrobial effect of the co-cultured fermentation liquor of Bacillus amyloliquefaciens ACCC11060 and Trichoderma asperellum GDFS1009 was found to be significantly higher than that of pure-cultivation. A raise in the synthesis of antimicrobial substances contributed to this significant increase. Additionally, a co-culture with the inoculation of the two organisms in the ratio of 1:1 was found to enhance the production of specific amino acids. This technique could be further explored for either a large scale production of amino acids or could serve as a theoretical base for the generation of certain rare amino acids. CONCLUSIONS: This work clearly demonstrated that co-cultivation of B. amyloliquefaciens ACCC11060 and T. asperellum GDFS1009 could produce more specific biocontrol substances and amino acids.

Identification and comprehensive evaluation of a novel biocontrol agent Bacillus atrophaeus JZB120050.

Bacillus spp. have long been used as biocontrol agents because of their efficient broad-spectrum antimicrobial activity. We identified a novel strain of Bacillus atrophaeus, named JZB120050, from soil. B. atrophaeus JZB120050 had a strong inhibitory effect against Botrytis cinerea and many other phytopathogens. Gas chromatography-mass spectrometry showed that B. atrophaeus JZB120050 produced many secondary metabolites, such as alkanes, alkenes and acids; some of which were related to pathogen inhibition. Enzyme activity analysis showed that B. atrophaeus JZB120050 secreted cell-wall-degrading enzymes, including chitinase, glucanase and protease, which degraded fungal cell walls. Both the novel glucanase gene bglu and chitinase gene chit1 were cloned and heterologously expressed in Escherichia coli and the products showed strong enzyme activity. In addition, B. atrophaeus JZB120050 secreted siderophores and formed a significant biofilm. Future studies should focus on these antimicrobial factors to facilitate widespread application in the field of agricultural biocontrol.

Omics for understanding the tolerant mechanism of Trichoderma asperellum TJ01 to organophosphorus pesticide dichlorvos.

BACKGROUD: Though it is toxic to humans, dichlorvos is a widely used chemical pesticide and plays an important role in the control of plant pests. The application of a combination of the biocontrol agent Trichoderma with dichlorvos may reduce the need for chemical pesticides. Therefore, revealing the specific molecular mechanism of Trichoderma tolerance to dichlorvos has become particularly important. RESULTS: In this study, using transcriptome and metabolome analyses, changes in primary and secondary metabolisms in Trichoderma asperellum TJ01 were comprehensively studied in the presence of dichlorvos. A novel C2H2 zinc finger protein gene, zinc finger chimera 1 (zfc1), was discovered to be upregulated, along with a large number of oxidoreductase genes and ABC transporter genes under dichlorvos stress. In addition, gas chromatography-mass spectrometry (GC-TOF-MS), and liquid chromatography-mass spectrometry (LC-QQQ-MS) data revealed the global primary and secondary metabolic changes that occur in T. asperellum TJ01 under dichlorvos stress. CONCLUSIONS: The tolerance mechanism of T. asperellum TJ01 to dichlorvos was proposed. In addition, the absorption and residue of dichlorvos were analyzed, laying the foundation for elucidation of the mechanism by which T. asperellum TJ01 degrades pesticide residues.

Laboratory evaluation of transgenic Populus davidiana×Populus bolleana expressing Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3 genes against gypsy moth and fall webworm.

Transgenic poplar lines 'Shanxin' (Populus davidiana×Populus bolleana) were generated via Agrobacterium-mediated transformation. The transgenic lines carried the expression cassettes of Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3, respectively. The expression levels of the exogenous insect resistance genes in the transgenic lines were determined by Q-PCR and Western blot. Leaves of the transgenic lines were used for insect feeding bioassays on first instar larvae of the gypsy moth (Lymantria dispar) and fall webworm (Hyphantria cunea). At 5 d of feeding, the mean mortalities of larvae feeding on Cry1Ac + SCK and Cry1Ah3 transgenic poplars leaves were 97% and 91%, while mortality on Cry9Aa3 transgenic lines was about 49%. All gypsy moth and fall webworm larvae were killed in 7-9 days after feeding on leaves from Cry1Ac + SCK or Cry1Ah3 transgenic poplars, while all the fall webworm larvae were killed in 11 days and about 80% of gypsy moth larvae were dead in 14 days after feeding on those from Cry9Aa3 transgenic lines. It was concluded that the transgenic lines of Cry1Ac + SCK and Cry1Ah3 were highly toxic to larvae of both insect species while lines with Cry9Aa3 had lower toxicity,and H. cunea larvae are more sensitive to the insecticidal proteins compared to L. dispar. Transgenic poplar lines toxic to L. dispar and H. cunea could be used to provide Lepidoptera pest resistance to selected strains of poplar trees.

Identification of a novel fungus, Trichoderma asperellum GDFS1009, and comprehensive evaluation of its biocontrol efficacy.

Due to its efficient broad-spectrum antimicrobial activity, Trichoderma has been established as an internationally recognized biocontrol fungus. In this study, we found and identified a novel strain of Trichoderma asperellum, named GDFS1009. The mycelium of T. asperellum GDFS1009 exhibits a high growth rate, high sporulation capacity, and strong inhibitory effects against pathogens that cause cucumber fusarium wilt and corn stalk rot. T. asperellum GDFS1009 secretes chitinase, glucanase, and protease, which can degrade the cell walls of fungi and contribute to mycoparasitism. The secreted xylanases are good candidates for inducing plant resistance and enhancing plant immunity against pathogens. RNA sequencing (RNA-seq) and gas chromatography-mass spectrometry (GC-MS) showed that T. asperellum GDFS1009 produces primary metabolites that are precursors of antimicrobial compounds; it also produces a variety of antimicrobial secondary metabolites, including polyketides and alkanes. In addition, this study speculated the presence of six antimicrobial peptides via ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS/MS). Future studies should focus on these antimicrobial metabolites for facilitating widespread application in the field of agricultural bio-control.

Next-generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance.

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant 'pyramids' producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross-resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double-stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH-binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt-resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt-resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non-transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.

A synthetic antimicrobial peptide BTD-S expressed in Arabidopsis thaliana confers enhanced resistance to Verticillium dahliae.

BTD-S is a synthetic non-cyclic θ-defensin derivative which was previously designed in our laboratory based on baboon θ-defensins (BTDs). It shows robust antimicrobial activity against economically important phytopathogen, Verticillium dahliae. Here, we deduced the coding nucleotide sequence of BTD-S and introduced the gene into wild-type (ecotype Columbia-0) Arabidopsis thaliana plants. Results demonstrated that BTD-S-transgenic lines displayed in bioassays inhibitory effects on the growth of V. dahliae in vivo and in vitro. Based on symptom severity, enhanced resistance was found in a survey of BTD-S-transgenic lines. Besides, crude protein extracts from root tissues of BTD-S-transformed plants significantly restricted the growth of fungal hyphae and the germination of conidia. Also, fungal biomass over time determined by real-time PCR demonstrated the overgrowth of V. dahliae in wild-type plants 2-3 weeks after inoculation, while almost no fungal DNA was detected in aerial tissues of their transgenic progenitors. The result suggested that fungus failed to invade and progress acropetally up to establish a systemic infection in BTD-S-transgenic plants. Moreover, the assessment of basal defense responses was performed in the leaves of WT and BTD-S-transgenic plants. The mitigated oxidative stress and low antioxidase level in BTD-S-transgenic plants revealed that BTD-S acts via permeabilizing target microbial membranes, which is in a category different from hypersensitive response-dependent defense. Taken together, our results demonstrate that BTD-S is a promising gene to be explored for transgenic engineering for plant protection against Verticillium wilt.

A novel NAP member GhNAP is involved in leaf senescence in Gossypium hirsutum.

Premature leaf senescence has a negative influence on the yield and quality of cotton, and several genes have been found to regulate leaf senescence. Howeer, many underlying transcription factors are yet to be identified. In this study, a NAP-like transcription factor (GhNAP) was isolated from Gossypium hirsutum. GhNAP has the typical NAC structure and a conserved novel subdomain in its divergent transcription activation region (TAR). GhNAP was demonstrated to be a nuclear protein, and it showed transcriptional activation activity in yeast. Furthermore, the expression of GhNAP was closely associated with leaf senescence. GhNAP could rescue the delayed-senescence phenotype of the atnap null mutant. Overexpression of GhNAP could cause precocious senescence in Arabidopsis. However, down-regulation of GhNAP delayed leaf senescence in cotton, and affected cotton yield and its fibre quality. Moreover, the expression of GhNAP can be induced by abscisic acid (ABA), and the delayed leaf senescence phenotype in GhNAPi plants might be caused by the decreased ABA level and reduced expression level of ABA-responsive genes. All of the results suggested that GhNAP could regulate the leaf senescence via the ABA-mediated pathways and was further related to the yield and quality in cotton.

Molecular evolution and expansion analysis of the NAC transcription factor in Zea mays.

NAC (NAM, ATAF1, 2 and CUC2) family is a plant-specific transcription factor and it controls various plant developmental processes. In the current study, 124 NAC members were identified in Zea mays and were phylogenetically clustered into 13 distinct subfamilies. The whole genome duplication (WGD), especially an additional WGD event, may lead to expanding ZmNAC members. Different subfamily has different expansion rate, and NAC subfamily preference was found during the expansion in maize. Moreover, the duplication events might occur after the divergence of the lineages of Z. mays and S. italica, and segmental duplication seemed to be the dominant pattern for the gene duplication in maize. Furthermore, the expansion of ZmNAC members may be also related to gain and loss of introns. Besides, the restriction of functional divergence was discovered after most of the gene duplication events. These results could provide novel insights into molecular evolution and expansion analysis of NAC family in maize, and advance the NAC researches in other plants, especially polyploid plants.

Replacing solubilizer with lipid microspheres eliminates the severe adverse reaction of vitamin K1 injection / 中国药学杂志

OBJECTIVE: To investigate whether substituting the solubilizer with lipid microspheres in vitamin K1 injection can eliminate the anaphylactoid reaction.

A non-cyclic baboon θ-defensin derivative exhibiting antimicrobial activity against the phytopathogen Verticillium dahliae.

θ-Defensins are the only natural cyclic proteins found in primates. They have strong antimicrobial activity related to their trisulfide ladders and macrocyclic conformation. A non-cyclic baboon θ-defensin (BTD) was synthesized by substituting valine with phenylalanine at position 17, at the C-terminal end of the BTD; this was termed "BTD-S." The antimicrobial activities of this synthetic peptide were investigated against Escherichia coli and two cotton phytopathogens: Verticillium dahliae and Fusarium oxysporum. The minimum inhibitory concentration (MIC) of BTD-S for E. coli was 10 µg/mL and for V. dahliae was 5 µg/mL, significantly lower than that for F. oxysporum (40.0 µg/mL). A time course analysis of fungal cultures indicated that the growth of V. dahliae was completely inhibited after 96 h of BTD-S treatment. Furthermore, hemolysis assays revealed that BTD-S was not toxic to mammalian cells as it could not induce lysis of sheep red blood cells even at ten times the MIC (50 µg/mL). Scanning electron microscopy and double-stained (calcofluor white and propidium iodide binding) fluorescence microscopy showed that exposure of spores of V. dahliae to BTD-S either disabled normal germination or disintegrated the spores. The size of cells exposed to BTD-S was significantly reduced compared with controls, and their number increased in a dose-dependent curve when measured by flow cytometry. These findings suggest that BTD-S has great potential to inhibit the growth of V. dahliae and can be utilized as an effective remedy to control economic losses caused by Verticillium wilt in the development of wilt-resistant cotton.