A gene cassette Vd276-280 in Verticillium dahliae contains two genes that affect melanized microsclerotium formation and virulence.

Verticillium dahliae is a soilborne phytopathogenic fungus causing Verticillium wilt on hundreds of plant species. Several sequenced genomes of V. dahliae are available, but functional characterization of most genes has just begun. Based on our previous comparison of the transcriptome from the wild-type and ΔVdCf2 strains, a significant upregulation of the gene cassette, Vd276-280, in the ΔVdCf2 strain was observed. In this study, the functional characterization of the Vd276-280 gene cassette was performed. Agrobacterium-mediated knockout of this gene cassette in V. dahliae significantly inhibited conidiation, melanized microsclerotium formation in the mutant strains, and their virulence towards cotton. Furthermore, deletion of individual genes in the Vd276-280 gene cassette identified that the disruption of VDAG_07276 and VDAG_07280 delayed microsclerotium formation, inhibited conidiation, and reduced virulence towards cotton. Our data suggest that VDAG_07276 and VDAG_07280 in the Vd276-280 gene cassette mainly act as positive regulators of development and virulence in V. dahliae.

The potential of Burkholderia gladioli KRS027 in plant growth promotion and biocontrol against Verticillium dahliae revealed by dual transcriptome of pathogen and host.

Verticillium dahliae is a destructive, soil-borne pathogen that causes significant losses on numerous important dicots. Recently, beneficial microbes inhabiting the rhizosphere have been exploited and used to control plant diseases. In the present study, Burkholderia gladioli KRS027 demonstrated excellent inhibitory effects against Verticillium wilt in cotton seedlings. Plant growth and development was promoted by affecting the biosynthesis and signaling pathways of brassinosteroids (BRs), gibberellins (GAs), and auxins, consequently promoting stem elongation, shoot apical meristem, and root apical tissue division in cotton. Furthermore, based on the host transcriptional response to V. dahliae infection, it was found that KRS027 modulates the plants to maintain cell homeostasis and respond to other pathogen stress. Moreover, KRS027 induced disruption of V. dahliae cellular structures, as evidenced by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses. Based on the comparative transcriptomic analysis between KRS027 treated and control group of V. dahliae, KRS027 induced substantial alterations in the transcriptome, particularly affecting genes encoding secreted proteins, small cysteine-rich proteins (SCRPs), and protein kinases. In addition, KRS027 suppressed the growth of different clonal lineages of V. dahliae strains through metabolites, and volatile organic compounds (VOCs) released by KRS027 inhibited melanin biosynthesis and microsclerotia development. These findings provide valuable insights into an alternative biocontrol strategy for Verticillium wilt, demonstrating that the antagonistic bacterium KRS027 holds promise as a biocontrol agent for promoting plant growth and managing disease occurrence.

Drought-induced assembly of rhizosphere mycobiomes shows beneficial effects on plant growth.

Beneficial interactions between plants and rhizosphere fungi can enhance plant adaptability during drought stress. However, harnessing these interactions will require an in-depth understanding of the response of fungal community assembly to drought. Herein, by using different varieties of wheat plants, we analyzed the drought-induced changes in fungal community assembly in rhizosphere and bulk soil. We demonstrated that drought significantly altered the fungal communities, with the contribution of species richness to community beta diversity increased in both rhizosphere and bulk soil compartments during drought stress. The stochastic processes dominated fungal community assembly, but the relative importance of deterministic processes, mainly homogeneous selection, increased in the drought-stressed rhizosphere. Drought induced an increase in the relative abundance of generalists in the rhizosphere, as opposed to specialists, and the top 10 abundant taxa that enriched under drought conditions were predominantly generalists. Notably, the most abundant drought-enriched taxon in rhizosphere was a generalist, and the corresponding Chaetomium strain was found capable of improving root length and activating ABA signaling in wheat plants through culture-based experiment. Together, these findings provide evidence that host plants exert a strong influence on rhizospheric fungal community assembly during stress and suggest the fungal communities that have experienced drought have the potential to confer fitness advantages to the host plants. IMPORTANCE: We have presented a framework to integrate the shifts in community assembly processes with plant-soil feedback during drought stress. We found that environmental filtering and host plant selection exert influence on the rhizospheric fungal community assembly, and the re-assembled community has great potential to alleviate plant drought stress. Our study proposes that future research should incorporate ecology with plant, microbiome, and molecular approaches to effectively harness the rhizospheric microbiome for enhancing the resilience of crop production to drought.

Functional analysis of the mating type genes in Verticillium dahliae.

BACKGROUND: Populations of the plant pathogenic fungus Verticillium dahliae display a complex and rich genetic diversity, yet the existence of sexual reproduction in the fungus remains contested. As pivotal genes, MAT genes play a crucial role in regulating cell differentiation, morphological development, and mating of compatible cells. However, the functions of the two mating type genes in V. dahliae, VdMAT1-1-1, and VdMAT1-2-1, remain poorly understood. RESULTS: In this study, we confirmed that the MAT loci in V. dahliae are highly conserved, including both VdMAT1-1-1 and VdMAT1-2-1 which share high collinearity. The conserved core transcription factor encoded by the two MAT loci may facilitate the regulation of pheromone precursor and pheromone receptor genes by directly binding to their promoter regions. Additionally, peptide activity assays demonstrated that the signal peptide of the pheromone VdPpg1 possessed secretory activity, while VdPpg2, lacked a predicted signal peptide. Chemotactic growth assays revealed that V. dahliae senses and grows towards the pheromones FO-a and FO-α of Fusarium oxysporum, as well as towards VdPpg2 of V. dahliae, but not in response to VdPpg1. The findings herein also revealed that VdMAT1-1-1 and VdMAT1-2-1 regulate vegetative growth, carbon source utilization, and resistance to stressors in V. dahliae, while negatively regulating virulence. CONCLUSIONS: These findings underscore the potential roles of VdMAT1-1-1 and VdMAT1-2-1 in sexual reproduction and confirm their involvement in various asexual processes of V. dahliae, offering novel insights into the functions of mating type genes in this species.

Insights into the biocontrol and plant growth promotion functions of Bacillus altitudinis strain KRS010 against Verticillium dahliae.

BACKGROUND: Verticillium wilt, caused by the fungus Verticillium dahliae, is a soil-borne vascular fungal disease, which has caused great losses to cotton yield and quality worldwide. The strain KRS010 was isolated from the seed of Verticillium wilt-resistant Gossypium hirsutum cultivar "Zhongzhimian No. 2." RESULTS: The strain KRS010 has a broad-spectrum antifungal activity to various pathogenic fungi as Verticillium dahliae, Botrytis cinerea, Fusarium spp., Colletotrichum spp., and Magnaporthe oryzae, of which the inhibition rate of V. dahliae mycelial growth was 73.97% and 84.39% respectively through confrontation test and volatile organic compounds (VOCs) treatments. The strain was identified as Bacillus altitudinis by phylogenetic analysis based on complete genome sequences, and the strain physio-biochemical characteristics were detected, including growth-promoting ability and active enzymes. Moreover, the control efficiency of KRS010 against Verticillium wilt of cotton was 93.59%. After treatment with KRS010 culture, the biomass of V. dahliae was reduced. The biomass of V. dahliae in the control group (Vd991 alone) was 30.76-folds higher than that in the treatment group (KRS010+Vd991). From a molecular biological aspect, KRS010 could trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Its extracellular metabolites and VOCs inhibited the melanin biosynthesis of V. dahliae. In addition, KRS010 had been characterized as the ability to promote plant growth. CONCLUSIONS: This study indicated that B. altitudinis KRS010 is a beneficial microbe with a potential for controlling Verticillium wilt of cotton, as well as promoting plant growth.

Transcriptome analysis of Gossypium hirsutum cultivar Zhongzhimian No.2 uncovers the gene regulatory networks involved in defense against Verticillium dahliae.

BACKGROUND: Cotton is globally important crop. Verticillium wilt (VW), caused by Verticillium dahliae, is the most destructive disease in cotton, reducing yield and fiber quality by over 50% of cotton acreage. Breeding resistant cotton cultivars has proven to be an efficient strategy for improving the resistance of cotton to V. dahliae. However, the lack of understanding of the genetic basis of VW resistance may hinder the progress in deploying elite cultivars with proven resistance. RESULTS: We planted the VW-resistant Gossypium hirsutum cultivar Zhongzhimian No.2 (ZZM2) in an artificial greenhouse and disease nursery. ZZM2 cotton was subsequently subjected to transcriptome sequencing after Vd991 inoculation (6, 12, 24, 48, and 72 h post-inoculation). Several differentially expressed genes (DEGs) were identified in response to V. dahliae infection, mainly involved in resistance processes, such as flavonoid and terpenoid quinone biosynthesis, plant hormone signaling, MAPK signaling, phenylpropanoid biosynthesis, and pyruvate metabolism. Compared to the susceptible cultivar Junmian No.1 (J1), oxidoreductase activity and reactive oxygen species (ROS) production were significantly increased in ZZM2. Furthermore, gene silencing of cytochrome c oxidase subunit 1 (COX1), which is involved in the oxidation-reduction process in ZZM2, compromised its resistance to V. dahliae, suggesting that COX1 contributes to VW resistance in ZZM2. CONCLUSIONS: Our data demonstrate that the G. hirsutum cultivar ZZM2 responds to V. dahliae inoculation through resistance-related processes, especially the oxidation-reduction process. This enhances our understanding of the mechanisms regulating the ZZM2 defense against VW.

Residual behavior of dinotefuran and its metabolites during Huangjiu fermentation and their effects on flavor.

Yellow rice wine (Huangjiu) is a traditional Chinese alcoholic beverage. However, there is a risk of pesticide residues in Huangjiu due to pesticide indiscriminate use. In this study, the residues of dinotefuran and its metabolites during Huangjiu fermentation and their effects on flavor substances were studied. The initial concentrations of dinotefuran ranged from 856.3 to 1874.9 µg/L, and its half-life was no more than 3.65 d. At 24 d of Huangjiu fermentation, the terminal residues of dinotefuran, 1-methyl-3-(tetrahydro-3-furylmethyl)urea (UF) and 1-methyl-3-(tetrahydro-3-furylmethyl)guanidine (DN) were 195.1-535.3 µg/L, 38.33-48.70 µg/L and 37.8-74.1 µg/L, respectively. Twenty potential degradation compounds were identified by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), and their toxicity was evaluated. Finally, the effect of dinotefuran on physicochemical properties and total phenol content of Huangjiu were analyzed. The risk of rancidity was significantly increased and bitter amino acids were formed. These findings provide a guidance and the safe production of Huangjiu.

Surgical performance of dental students using computer-assisted dynamic navigation and freehand approaches.

INTRODUCTION: Nowadays, the training of implant placement has shifted from once entirely instructor-student teaching to the increasing use of computer-assisted simulation. Based on computerized virtual planning, dynamic navigation has been used for implant placement with higher accuracy than the traditional freehand protocol. However, whether dynamic navigation benefits to the training of dental students in implant placement remains controversial. This study aimed to compare the surgical performance of dental students in implant placement using computer-assisted dynamic navigation and freehand approaches. MATERIALS AND METHODS: A total of 20 dental students (6 males, 14 females, age: 25.6 ± 0.5 years) were enrolled in this study. With the traditional freehand approach (training 1) as the control protocol, computer-assisted dynamic navigation (training 2) was used in the training of dental students in implant placement. For each training, both the operating time (OT) of students and placement accuracy represented by the linear (at the implant platform, Dpl, and apex, Dap) and angular (Dan) deviations between the virtually planned and placed implants were recorded. Statistical comparisons were made between the two training protocols as well as male and female surgeons. RESULTS: OT2 was around twice of OT1 (p < .0001), whereas Dan1 was almost three times of Dan2 (p < .0001). Dap1 and Dpl1 were significantly higher than Dap2 (p = .014) and Dpl2 (p = .033) respectively. Besides, male students showed statistically higher Dpl1 (p = .033) and Dan1 (p = .002) than females. No significant difference was found between male and female students in OT1, OT2, Dpl2, Dap1, Dap2 and Dan2. CONCLUSIONS: Within the limitations of this study, the use of computer-assisted dynamic navigation in the preclinical training could improve the surgical performance of the dental students in implant placement. The combination of dynamic navigation with the traditional preclinical surgical training may benefit to dental students and could be applied in dental education.

Characterization of the Endophytic Bacillus subtilis KRS015 Strain for Its Biocontrol Efficacy Against Verticillium dahliae.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilborne pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ∼62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcm1, Vdpf, and Vel1. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton.

Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae.

BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

Magnetic functionalized graphene oxide combined with ultra-high performance liquid chromatography for trace detection of succinate dehydrogenase inhibitor fungicides in food.

In this study, an efficient, sensitive, and convenient magnetic solid-phase extraction method combined with ultra-high performance liquid chromatography-tandem mass spectrometry (MSPE-UHPLC-MS/MS) was developed for the simultaneous determination of 19 succinate dehydrogenase inhibitor fungicide residues in six different food matrices The synthesized tetraethylenepentamine magnetic graphene oxide nanocomposite showed the advantages of good dispersibility, large specific surface area (113.93 m2 /g) and large pore volume (0.25 cm3 /g), making it an ideal succinate dehydrogenase inhibitor pretreatment adsorbent. The MSPE-UHPLC-MS/MS method showed linearity in the range of 5.0-800.0 µg/kg, with a correlation coefficient (R2 ) > 0.99, and a limit of quantification of 5 µg/kg. The recovery of succinate dehydrogenase inhibitor fungicides was in the range of 71.2%-119.4%. The MSPE method is simple, rapid, and efficient, making it an ideal alternative to sample pretreatment in the determination of trace succinate dehydrogenase inhibitor fungicides in complex matrices.

Genome-wide identification and analysis of a cotton secretome reveals its role in resistance against Verticillium dahliae.

BACKGROUND: The extracellular space between the cell wall and plasma membrane is a battlefield in plant-pathogen interactions. Within this space, the pathogen employs its secretome to attack the host in a variety of ways, including immunity manipulation. However, the role of the plant secretome is rarely studied for its role in disease resistance. RESULTS: Here, we examined the secretome of Verticillium wilt-resistant Gossypium hirsutum cultivar Zhongzhimian No.2 (ZZM2, encoding 95,327 predicted coding sequences) to determine its role in disease resistance against the wilt causal agent, Verticillium dahliae. Bioinformatics-driven analyses showed that the ZZM2 genome encodes 2085 secreted proteins and that these display disequilibrium in their distribution among the chromosomes. The cotton secretome displayed differences in the abundance of certain amino acid residues as compared to the remaining encoded proteins due to the localization of these putative proteins in the extracellular space. The secretome analysis revealed conservation for an allotetraploid genome, which nevertheless exhibited variation among orthologs and comparable unique genes between the two sub-genomes. Secretome annotation strongly suggested its involvement in extracellular stress responses (hydrolase activity, oxidoreductase activity, and extracellular region, etc.), thus contributing to resistance against the V. dahliae infection. Furthermore, the defense response genes (immunity marker NbHIN1, salicylic acid marker NbPR1, and jasmonic acid marker NbLOX4) were activated to varying degrees when Nicotina benthamiana leaves were agro-infiltrated with 28 randomly selected members, suggesting that the secretome plays an important role in the immunity response. Finally, gene silencing assays of 11 members from 13 selected candidates in ZZM2 displayed higher susceptibility to V. dahliae, suggesting that the secretome members confer the Verticillium wilt resistance in cotton. CONCLUSIONS: Our data demonstrate that the cotton secretome plays an important role in Verticillium wilt resistance, facilitating the development of the resistance gene markers and increasing the understanding of the mechanisms regulating disease resistance.

Potential Risks of Tebuconazole during Wine Fermentation at the Enantiomer Level Based on Multiomics Analysis.

The enantioselectivity and potential risks of tebuconazole enantiomers (R-tebuconazole and S-tebuconazole) in wine fermentation were investigated in this study using Cabernet Sauvignon grapes. Tebuconazole was mainly degraded during the alcoholic fermentation stage, and no obvious transformation between R-tebuconazole and S-tebuconazole was observed. Selective degradation between these two enantiomers occurred, with R-tebuconazole degrading faster than S-tebuconazole. The residual tebuconazole inhibits glucose metabolism and the unsaturated fatty acid formation in the wine fermentation system and inhibits gene expression in the late phase of Saccharomycetales, affecting its cell wall formation. Overall, the findings highlight that R-tebuconazole exhibited a higher risk than S-tebuconazole in these processes. These insights are potentially exploitable to understand chiral pesticides at the enantiomer level using multiomics technology in food-processing systems.

Unlocking antagonistic potential of Bacillus amyloliquefaciens KRS005 to control gray mold.

To establish a safe, efficient, and simple biocontrol measure for gray mold disease caused by Botrytis cinerea, the basic characteristics and antifungal activity of KRS005 were studied from multiple aspects including morphological observation, multilocus sequence analysis and typing (MLSA-MLST), physical-biochemical assays, broad-spectrum inhibitory activities, control efficiency of gray mold, and determination of plant immunity. The strain KRS005, identified as Bacillus amyloliquefaciens, demonstrated broad-spectrum inhibitory activities against various pathogenic fungi by dual confrontation culture assays, of which the inhibition rate of B. cinerea was up to 90.3%. Notably, through the evaluation of control efficiency, it was found that KRS005 fermentation broth could effectively control the occurrence of tobacco leaves gray mold by determining the lesion diameter and biomass of B. cinerea on tobacco leaves still had a high control effect after dilution of 100 folds. Meanwhile, KRS005 fermentation broth had no impact on the mesophyll tissue of tobacco leaves. Further studies showed that plant defense-related genes involved in reactive oxygen species (ROS), salicylic acid (SA), and jasmonic acid (JA)-related signal pathways were significantly upregulated when tobacco leaves were sprayed with KRS005 cell-free supernatant. In addition, KRS005 could inhibit cell membrane damage and increase the permeability of B. cinerea. Overall, KRS005, as a promising biocontrol agent, would likely serve as an alternative to chemical fungicides to control gray mold.

Functional Characterization of Verticillium dahliae Race 3-Specific Gene VdR3e in Virulence and Elicitation of Plant Immune Responses.

Verticillium dahliae is a soilborne fungal pathogen that causes disease on many economically important crops. Based on the resistance or susceptibility of differential cultivars in tomato, isolates of V. dahliae are divided into three races. Avirulence (avr) genes within the genomes of the three races have also been identified. However, the functional role of the avr gene in race 3 isolates of V. dahliae has not been characterized. In this study, bioinformatics analysis showed that VdR3e, a cysteine-rich secreted protein encoded by the gene characterizing race 3 in V. dahliae, was likely obtained by horizontal gene transfer from the fungal genus Bipolaris. We demonstrate that VdR3e causes cell death by triggering multiple defense responses. In addition, VdR3e localized at the periphery of the plant cell and triggered immunity depending on its subcellular localization and the cell membrane receptor BAK1. Furthermore, VdR3e is a virulence factor and shows differential pathogenicity in race 3-resistant and -susceptible hosts. These results suggest that VdR3e is a virulence factor that can also interact with BAK1 as a pathogen-associated molecular pattern (PAMP) to trigger immune responses. IMPORTANCE Based on the gene-for-gene model, research on the function of avirulence genes and resistance genes has had an unparalleled impact on breeding for resistance in most crops against individual pathogens. The soilborne fungal pathogen, Verticillium dahliae, is a major pathogen on many economically important crops. Currently, avr genes of the three races in V. dahliae have been identified, but the function of avr gene representing race 3 has not been described. We investigated the characteristics of VdR3e-mediated immunity and demonstrated that VdR3e acts as a PAMP to activate a variety of plant defense responses and induce plant cell death. We also demonstrated that the role of VdR3e in pathogenicity was host dependent. This is the first study to describe the immune and virulence functions of the avr gene from race 3 in V. dahliae, and we provide support for the identification of genes mediating resistance against race 3.

Genome Sequence Resource of Cladosporium velox Strain C4 Causing Cotton Boll Disease in Xinjiang, China.

Cladosporium spp., as one of the largest and most heterogeneous genera of hyphomycetes, are widely distributed worldwide. This genus is usually adaptable to a wide variety of extreme environments. However, only 11 genomes of Cladosporium genus have been publicly released. From 2017, we found for the first time that Cladosporium velox could cause cotton boll disease and lead to stiffness and cracking boll in Xinjiang, China. Herein, we provide a high-quality reference genome for the C. velox strain C4 isolated from cotton boll in Xinjiang, China. The genome size and encoding gene number of the C. velox strain C4 and C. cucumerinum strain CCNX2, which was recently released and caused the cucumber scab, showed minor differences. This resource will contribute to future research that aims to elucidate the genetic basis of C. velox pathogenicity and could expand our knowledge of Cladosporium spp. genomic characteristics that will be valuable for the development of Cladosporium disease control measures.

The Verticillium dahliae Small Cysteine-Rich Protein VdSCP23 Manipulates Host Immunity.

Verticillium wilt caused by Verticillium dahliae is a notorious soil-borne fungal disease and seriously threatens the yield of economic crops worldwide. During host infection, V. dahliae secretes many effectors that manipulate host immunity, among which small cysteine-rich proteins (SCPs) play an important role. However, the exact roles of many SCPs from V. dahliae are unknown and varied. In this study, we show that the small cysteine-rich protein VdSCP23 inhibits cell necrosis in Nicotiana benthamiana leaves, as well as the reactive oxygen species (ROS) burst, electrolyte leakage and the expression of defense-related genes. VdSCP23 is mainly localized in the plant cell plasma membrane and nucleus, but its inhibition of immune responses was independent of its nuclear localization. Site-directed mutagenesis and peptide truncation showed that the inhibition function of VdSCP23 was independent of cysteine residues but was dependent on the N-glycosylation sites and the integrity of VdSCP23 protein structure. Deletion of VdSCP23 did not affect the growth and development of mycelia or conidial production in V. dahliae. Unexpectedly, VdSCP23 deletion strains still maintained their virulence for N. benthamiana, Gossypium hirsutum and Arabidopsis thaliana seedlings. This study demonstrates an important role for VdSCP23 in the inhibition of plant immune responses; however, it is not required for normal growth or virulence in V. dahliae.

Thioredoxin VdTrx1, an unconventional secreted protein, is a virulence factor in Verticillium dahliae.

Understanding how plant pathogenic fungi adapt to their hosts is of critical importance to securing optimal crop productivity. In response to pathogenic attack, plants produce reactive oxygen species (ROS) as part of a multipronged defense response. Pathogens, in turn, have evolved ROS scavenging mechanisms to undermine host defense. Thioredoxins (Trx) are highly conserved oxidoreductase enzymes with a dithiol-disulfide active site, and function as antioxidants to protect cells against free radicals, such as ROS. However, the roles of thioredoxins in Verticillium dahliae, an important vascular pathogen, are not clear. Through proteomics analyses, we identified a putative thioredoxin (VdTrx1) lacking a signal peptide. VdTrx1 was present in the exoproteome of V. dahliae cultured in the presence of host tissues, a finding that suggested that it plays a role in host-pathogen interactions. We constructed a VdTrx1 deletion mutant ΔVdTrx1 that exhibited significantly higher sensitivity to ROS stress, H2O2, and tert-butyl hydroperoxide (t-BOOH). In vivo assays by live-cell imaging and in vitro assays by western blotting revealed that while VdTrx1 lacking the signal peptide can be localized within V. dahliae cells, VdTrx1 can also be secreted unconventionally depending on VdVps36, a member of the ESCRT-II protein complex. The ΔVdTrx1 strain was unable to scavenge host-generated extracellular ROS fully during host invasion. Deletion of VdTrx1 resulted in higher intracellular ROS levels of V. dahliae mycelium, displayed impaired conidial production, and showed significantly reduced virulence on Gossypium hirsutum, and model plants, Arabidopsis thaliana and Nicotiana benthamiana. Thus, we conclude that VdTrx1 acts as a virulence factor in V. dahliae.

Insights into the Biocontrol Function of a Burkholderia gladioli Strain against Botrytis cinerea.

Pathogenic fungi are the main cause of yield loss and postharvest loss of crops. In recent years, some antifungal microorganisms have been exploited and applied to prevent and control pathogenic fungi. In this study, an antagonistic bacteria KRS027 isolated from the soil rhizosphere of a healthy cotton plant from an infected field was identified as Burkholderia gladioli by morphological identification, multilocus sequence analysis, and typing (MLSA-MLST) and physiobiochemical examinations. KRS027 showed broad spectrum antifungal activity against various phytopathogenic fungi by secreting soluble and volatile compounds. KRS027 also has the characteristics of plant growth promotion (PGP) including nitrogen fixation, phosphate, and potassium solubilization, production of siderophores, and various enzymes. KRS027 is not only proven safe by inoculation of tobacco leaves and hemolysis test but also could effectively protect tobacco and table grapes against gray mold disease caused by Botrytis cinerea. Furthermore, KRS027 can trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid- (SA), jasmonic acid- (JA), and ethylene (ET)-dependent signaling pathways. The extracellular metabolites and volatile organic compounds (VOCs) of KRS027 affected the colony extension and hyphal development by downregulation of melanin biosynthesis and upregulation of vesicle transport, G protein subunit 1, mitochondrial oxidative phosphorylation, disturbance of autophagy process, and degrading the cell wall of B. cinerea. These results demonstrated that B. gladioli KRS027 would likely become a promising biocontrol and biofertilizer agent against fungal diseases, including B. cinerea, and would promote plant growth. IMPORTANCE Searching the economical, eco-friendly and efficient biological control measures is the key to protecting crops from pathogenic fungi. The species of Burkholderia genus are widespread in the natural environment, of which nonpathogenic members have been reported to have great potential for biological control agents and biofertilizers for agricultural application. Burkholderia gladioli strains, however, need more study and application in the control of pathogenic fungi, plant growth promotion, and induced systemic resistance (ISR). In this study, we found that a B. gladioli strain KRS027 has broad spectrum antifungal activity, especially in suppressing the incidence of gray mold disease caused by Botrytis cinerea, and can stimulate plant immunity response via ISR activated by salicylic acid- (SA), jasmonic acid- (JA), and ethylene (ET)-dependent signaling pathways. These results indicate that B. gladioli KRS027 may be a promising biocontrol and biofertilizer microorganism resource in agricultural applications.

Verticillium dahliae Effector VdCE11 Contributes to Virulence by Promoting Accumulation and Activity of the Aspartic Protease GhAP1 from Cotton.

Verticillium dahliae is a soilborne plant fungal pathogen that causes Verticillium wilt, a disease that reduces the yields of many economically important crops. Despite its worldwide distribution and harmful impacts, much remains unknown regarding how the numerous effectors of V. dahliae modulate plant immunity. Here, we identified the intracellular effector VdCE11 that induces cell death and defense responses in Nicotiana benthamiana to counter leaf pathogens such as Sclerotinia sclerotiorum and Botrytis cinerea. VdCE11 also contributes to the virulence of V. dahliae in cotton and Arabidopsis. Yeast two-hybrid library screening and immunoprecipitation revealed that VdCE11 interacts physically with the cotton aspartic protease GhAP1. GhAP1 and its Arabidopsis homolog AtAP1 are negative regulators of plant immunity, since disruption of either increased the resistance of cotton or Arabidopsis to V. dahliae. Further, VdCE11 plays a role in promoting the accumulation of the AP1 proteins and increasing its hydrolase activity. Taken together, these results indicate a novel mechanism regulating virulence whereby the secreted effector VdCE11 increases cotton susceptibility to V. dahliae by promoting the accumulation and activity of GhAP1. IMPORTANCE Verticclium dahliae is a plant fungal pathogen that causes a destructive vascular disease on a large number of plant hosts, resulting in great threat to agricultural production. In this study, we identified a V. dahliae effector VdCE11 that induces cell death and defense responses in Nicotiana benthamiana. Meanwhile, VdCE11 contributes to the virulence of V. dahliae in cotton and Arabidopsis. Yeast two-hybrid library screening and immunoprecipitation revealed that VdCE11 interacts physically with the cotton aspartic protease GhAP1. GhAP1 and its Arabidopsis homolog AtAP1 are negative regulators of plant immunity since disruption of either increased the resistance of cotton or Arabidopsis to V. dahliae. Further research showed that VdCE11 plays a role in promoting the accumulation of the AP1 proteins and increasing its hydrolase activity. These results suggested that a novel mechanism regulating virulence whereby VdCE11 increases susceptibility to V. dahliae by promoting the accumulation and activity of GhAP1 in the host.