Mechanism of oxalate decarboxylase Oxd_S12 from Bacillus velezensis BvZ45-1 in defence against cotton verticillium wilt.

Verticillium wilt, a soilborne vascular disease caused by Verticillium dahliae, strongly affects cotton yield and quality. In this study, an isolated rhizosphere bacterium, designated Bacillus velezensis BvZ45-1, exhibited >46% biocontrol efficacy against cotton verticillium wilt under greenhouse and field conditions. Moreover, through crude protein extraction and mass spectrometry analyses, we found many antifungal compounds present in the crude protein extract of BvZ45-1. The purified oxalate decarboxylase Odx_S12 from BvZ45-1 inhibited the growth of V. dahliae Vd080 by reducing the spore yield, causing mycelia to rupture, spore morphology changes, cell membrane rupture, and cell death. Subsequently, overexpression of Odx_S12 in Arabidopsis significantly improved plant resistance to V. dahliae. Through studies of the resistance mechanism of Odx_S12, V. dahliae was shown to produce oxalic acid (OA), which has a toxic effect on Arabidopsis leaves. Odx_S12 overexpression reduced Arabidopsis OA content, enhanced tolerance to OA, and improved resistance to verticillium wilt. Transcriptomics and quantitative real-time PCR analysis revealed that Odx_S12 promoted a reactive oxygen species burst and a salicylic acid- and abscisic acid-mediated defence response in Arabidopsis. In summary, this study not only identified B. velezensis BvZ45-1 as an efficient biological control agent, but also identified the resistance gene Odx_S12 as a candidate for cotton breeding against verticillium wilt.

GhTLP1, a thaumatin-like protein 1, improves Verticillium wilt resistance in cotton via JA, ABA and MAPK signaling pathway-plant pathways.

Verticillium wilt of cotton is a very serious soil-borne disease and there is no effective control method. The mechanism of Gossypium hirsutum thaumatin-like protein 1(GhTLP1) in upland cotton regulating Verticillium wilt resistance has been an uncovered research approach. GhTLP1 is mainly localized in the cell wall. Overexpression of GhTLP1 significantly enhanced Arabidopsis plants resistance to Verticillium dahliae, while its homologous mutant tlp1 in Arabidopsis was more susceptible to the pathogen, and the heterologous complement line (EC) recovered resistance to V. dahliae. GhTLP1 responds to jasmonate acid (JA) and abscisic acid (ABA) hormones and regulates mitogen-activated protein kinase (MAPK) signaling pathway-plant pathway to enhance Arabidopsis plants resistance to V. dahliae. Silencing GhTLP1 resulted decrease in cotton plants resistance to V. dahliae. Moreover, the mutation of GhTLP1 at site Tyr97 and Tyr199 with the phosphorylation also decreased plant resistance to V. dahliae. Therefore, GhTLP1 phosphorylation was observed important in cotton plants against V. dahliae. Further analysis demonstrated that GhTLP1 interacted with gossypium hirsutum laccase 14 (GhLAC14) to enhance plants resistance to V. dahliae. Silencing GhLAC14 resulted decrease in cotton plants resistance to V. dahliae. Here, we propose that GhTLP1 is a potential molecular target for improving resistance to Verticillium wilt in cotton.

Identification and characterization of the Bicupin domain family and functional analysis of GhBCD11 in response to verticillium wilt in cotton.

Bicupin domain protein (BCD) family, an important component of Cupin domain superfamily, plays important roles in oxalic acid (OA) degradation and stress responses in high plants. However, no studies have been reported on the Cupin domain family in cotton up till now. In our study, a total 110 proteins including Cupin domain were identified from the upland cotton (Gossypium hirsutum). Among them, 17 proteins contained Bicupin domain. Subsequently, we found that V. dahliae produces OA leading to cotton leaf wilting. RT-qPCR analysis of GhBCDs revealed that OA and V. dahliae Vd080 significantly enhanced the expression of GhBCD11. The Virus-induced gene silencing and overexpression analysis showed that GhBCD11 positively regulates plant resistance to V. dahliae. Subcellular localization showed GhBCD11 located on the plasma membrane. The analysis of expression pattern showed that GhBCD11 can be induced via hormone-mediated signal pathway including salicylic acid (SA), ethephon (ET), methyl jasmonate (JA) and abscisic acid (ABA). In addition, we identified an interaction between 60 S ribosomal protein GhRPL12-3 and GhBCD11 by yeast double hybridization. Overall, this is the first study, where we identified Cupin domain family in cotton, clarified the role of GhBCD11 in cotton for resistance to V. dahliae and found an interaction between GhRPL12-3 and GhBCD11.

Flagellin C decreases the expression of the Gossypium hirsutum cation/proton exchanger 3 gene to promote calcium ion, hydrogen peroxide, and nitric oxide and synergistically regulate the resistance of cotton to Verticillium wilt.

To date, no ideal effective method for controlling Verticillium wilt in upland cotton (Gossypium hirsutum) has been defined. The purpose of this study was to determine the effects and mechanism through which flagellin C (FLiC) regulates the Gossypium hirsutum cation/proton exchanger 3 gene (GhCAX3), induces plant immunity, and increases resistance to Verticillium wilt. The FLiC gene was cloned from an endophytic bacterium (Pseudomonas) isolated from roots of the upland cotton cultivar Zhongmiansuo 41. The biocontrol effects of FLiC purified in vitro on resistant and susceptible upland cotton cultivars were 47.50 and 32.42%, respectively. FLiC induced a hypersensitive response (HR) in leaves of tobacco and immune responses in upland cotton. Transcriptome data showed that treatment with FLiC significantly enriched the calcium antiporter activity-associated disease-resistant metabolic pathway in seedlings. Moreover, FLiC downregulated GhCAX3 expression to increase intracellular calcium ion (Ca2+) content and stimulate increases in the intracellular hydrogen peroxide (H2O2) and nitric oxide (NO) contents. The coordinated regulation of Ca2+, H2O2, and NO enhanced cotton resistance to Verticillium wilt. Furthermore, transgenic Arabidopsis plants overexpressing FLiC showed significantly improved resistance to Verticillium wilt. FLiC may be used as a resistance gene and a regulator to improve resistance to Verticillium dahliae (VD) in upland cotton.

Antifungal Activity of Chaetoviridin A from Chaetomium globosum CEF-082 Metabolites Against Verticillium dahliae in Cotton.

Cotton Verticillium wilt (CVW) is a severe soilborne disease caused by the pathogen Verticillium dahliae, and it has a great impact on cotton production. Previous studies found that the biocontrol agent Chaetomium globosum CEF-082 and its metabolic filtrate could reduce the incidence of CVW; however, the underlying mechanism remains unclear. The metabolic crude extract of CEF-082 increased the sensitivity of V. dahliae to stress, degraded the cell wall of V. dahliae, and increased the emergence and plant height of cotton. Through separation and purification of the metabolic crude extract of CEF-082, chaetoviridin A was identified and found to be highly active against V. dahliae. The compound caused cell necrosis and mycelial deformation, increased the production of reactive oxygen species and nitrous oxide, and inhibited the germination of microsclerotia of V. dahliae, enhancing the cotton plant defense response. In addition, CEF-082 also colonized cotton plants.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pectin lyase enhances cotton resistance to Verticillium wilt by inducing cell apoptosis of Verticillium dahliae.

Verticillium wilt caused by Verticillium dahliae Kleb. is a major disease in cotton. We found that pectin lyase can enhance cotton resistance to Verticillium wilt and induce cell apoptosis of V. dahliae strain Vd080. The biocontrol effect of pectin lyase on Vd080 reached 61.9%. Pectin lyase increased ERG4 (Delta (24 (24 (1)))-sterol reductase) expression, the ergosterol content of the cell membrane, the collapse of mitochondrial membrane potential, hydrogen peroxide content, metacaspase activity, and Ca2+ content in the cytoplasm in the Vd080 strain and induced endoplasmic reticulum (ER) stress. Pectin lyase also increased the expression levels of the ER molecular chaperone glucose regulating protein Grp78 (BiP), protein disulfide isomerase (PDI) and calnexin (CNX), reduced the expression levels of the protein Hsp40. When the PDI and BiP genes of Vd080 were knocked out, the mutants △BiP and △PDI had reduced sensitivity to pectin lyase. In the absence of external stress, ER stress appeared in mutant △BiP cells. Pectin lyase affects the ergosterol content of the Vd080 cell membrane, which causes ER stress and increases the level of BiP to induce Vd080 cell apoptosis. These results demonstrate that pectin lyase can be used to control Verticillium wilt in cotton.

Transcriptome analysis reveals the defense mechanism of cotton against Verticillium dahliae in the presence of the biocontrol fungus Chaetomium globosum CEF-082.

BACKGROUND: Verticillium wilt of cotton is a serious soil-borne disease that causes a substantial reduction in cotton yields. A previous study showed that the endophytic fungus Chaetomium globosum CEF-082 could control Verticillium wilt of cotton, and induce a defense response in cotton plants. However, the comprehensive molecular mechanism governing this response is not yet clear. RESULTS: To study the signalling mechanism induced by CEF-082, the transcriptome of cotton seedlings pretreated with CEF-082 was sequenced. The results revealed 5638 DEGs at 24 h post inoculation with CEF-082, and 2921 and 2153 DEGs at 12 and 48 h post inoculation with Verticillium dahliae, respectively. At 24 h post inoculation with CEF-082, KEGG enrichment analysis indicated that the DEGs were enriched mainly in the plant-pathogen interaction, MAPK signalling pathway-plant, flavonoid biosynthesis, and phenylpropanoid biosynthesis pathways. There were 1209 DEGs specifically induced only in cotton plants inoculated with V. dahliae in the presence of the biocontrol fungus CEF-082, and not when cotton plants were only inoculated with V. dahliae. GO analysis revealed that these DEGs were enriched mainly in the following terms: ROS metabolic process, H2O2 metabolic process, defense response, superoxide dismutase activity, and antioxidant activity. Moreover, many genes, such as ERF, CNGC, FLS2, MYB, GST and CML, that regulate crucial points in defense-related pathways were identified and may contribute to V. dahliae resistance in cotton. These results provide a basis for understanding the molecular mechanism by which the biocontrol fungus CEF-082 increases the resistance of cotton to Verticillium wilt. CONCLUSIONS: The results of this study showed that CEF-082 could regulate multiple metabolic pathways in cotton. After treatment with V. dahliae, the defense response of cotton plants preinoculated with CEF-082 was strengthened.

Anther response to high-temperature stress during development and pollen thermotolerance heterosis as revealed by pollen tube growth and in vitro pollen vigor analysis in upland cotton.

MAIN CONCLUSION: Pollen tube growth in styles was strongly inhibited by temperature above 35 °C, and the yield of cotton decreased because of the adverse effect of high temperatures during square development. High-temperature stress during flowering influences the square development of upland cotton (Gossypium hirsutum L.) and cotton yield. Although it is well known that square development is sensitive to high temperature, high-temperature sensitive stages of square development and the effects of high temperature on pollen tube growth in the styles are unknown. The effect of high temperature on anther development corresponding to pollen vigor is unknown during anther development. The objectives of this study were to identify the stages of square development that are sensitive to high temperatures (37/30 and 40/34 °C), to determine whether the abnormal development of squares influenced by high temperature is responsible for the variation in the in vitro germination percent of pollen grains at anthesis, to identify the effect of high temperature on pollen germination in the styles, and to determine pollen thermotolerance heterosis. Our results show that the stages from the sporogenous cell to tetrad stage (square length <6.0 mm) were the most sensitive to high temperature, and the corresponding pollen viability at anthesis was consistent with the changes in the square development stage. Pollen tube growth in the styles was strongly inhibited by temperature above 35 °C, and the yield of cotton decreased because of the effect of high temperature during square development. The thermotolerance of hybrid F1 pollen showed heterosis, and pollen viability could be used as a criterion for screening for high-temperature tolerance cultivars. These results can be used in breeding to develop new cotton cultivars that can withstand high-temperature conditions, particularly in a future warmer climate.

The bacterial lipopeptide iturins induce Verticillium dahliae cell death by affecting fungal signalling pathways and mediate plant defence responses involved in pathogen-associated molecular pattern-triggered immunity.

Verticillium wilt in cotton caused by Verticillium dahliae is one of the most serious plant diseases worldwide. Because no known fungicides or cotton cultivars provide sufficient protection against this pathogen, V. dahliae causes major crop yield losses. Here, an isolated cotton endophytic bacterium, designated Bacillus amyloliquefaciens 41B-1, exhibited greater than 50% biocontrol efficacy against V. dahliae in cotton plants under greenhouse conditions. Through high-performance liquid chromatography and mass analysis of the filtrate, we found that the antifungal compounds present in the strain 41B-1 culture filtrate were a series of isoforms of iturins. The purified iturins suppressed V. dahliae microsclerotial germination in the absence or presence of cotton. Treatment with the iturins induced reactive oxygen species bursts, Hog1 mitogen-activated protein kinase (MAPK) activation and defects in cell wall integrity. The oxidative stress response and high-osmolarity glycerol pathway contribute to iturins resistance in V. dahliae. In contrast, the Slt2 MAPK pathway may be involved in iturins sensitivity in this fungus. In addition to antagonism, iturins could induce plant defence responses as activators and mediate pathogen-associated molecular pattern-triggered immunity. These findings suggest that iturins may affect fungal signalling pathways and mediate plant defence responses against V. dahliae.

Population dynamics and identification of endophytic bacteria antagonistic toward plant-pathogenic fungi in cotton root.

The antagonistic potentials of endophytic bacteria isolated from the roots of six cotton cultivars at different developmental stages were determined in vitro toward three pathogens: Verticillium dahliae Kleb V107 and V396 and Fusarium oxysporum f.sp. vasinfectum (F108). The populations of antagonistic endophytic bacteria (AEB) toward V107, V396, and F108 at the flowering and maturation stages were significantly higher than those at the seedling stage were. More AEB were found to be antagonistic toward pathogens V396 and F108 than V107. Results from the multivariate analysis of variance showed that the populations of AEB were significantly different for the main factors of cultivars, stages, and their interactions. Based on 16S rDNA sequence analysis, the 39 AEB isolates that antagonized V107, V396, and F108 (BAEB) consisted of seven genera, in which the genus of Enterobacter (17 out of 39) and Pantoea (14 out of 39) were predominant among the BAEB isolates. Characterized by BOX-PCR fingerprints, these 39 BAEB isolates represented 35 different cluster types. To explore the antagonistic mechanisms, the agar diffusion method was used to detect cell-wall-degrading enzyme activity and siderophore secretion. Nearly half of these BAEB isolates showed protease and chitinase activity, while all 39 BAEB isolates excreted siderophores. However, pectinase, cellulase, and xylanase activity were hardly detected. A germination experiment revealed that nine of the 39 BAEB isolates significantly improved the vigor index of the cotton seedlings.

[Population dynamics and antagonism toward Fusarium oxysporium f. sp. Vasinfectum. and Verticillium dahliae Kleb of endophytic bacteria from cotton].

OBJECTIVE: To explore population dynamics of endophytic bacteria and obtain antagonistic endophytic bacteria toward Verticillium dahliae Kleb (Vd), Fusarium oxysporium f. sp. Vasinfectum (Fov) from cotton. METHODS: Root, stem and leaf samples were surface-disinfested, and subsequently used to isolate endophytic bacteria by diluting plate counting method. We assayed antagonism of the isolated endophytic bacteria toward three pathogens: Vd (V107, which is a highly virulent defoliating isolate and V396, which is a mildly virulent non-defoliating isolate), Fov (F108) using a dual culture method, and analyzed the 16S rDNA sequence of doubly antagonistic endophytic bacteria (DAEB) isolates toward both Vd and Fov. RESULTS: The population size of endophytic bacteria in root was significantly larger than that in leaf and stem. The populations at seedling stage were generally lower than those at the flowering/maturing stage in root, the populations in stem and leaf were fluctuant at different development stages, but variation law was not observed obviously. Furthermore, although no significant differences of the population densities in root were found among 6 cotton cultivars, the population densities in stem and leaf showed cultivar differences. The proportion of endophytic bacteria antagonizing Vd (V107, V396) and Fov (F108) in root was higher than that in stem/leaf, moreover, the amount of endophytic bacteria antagonizing toward V107 was less than that toward V396/F108. Based on 16S rDNA sequence analysis, all 44 DAEB isolates consisted of two phyla, i.e., Bacteroidetes (1 out of 44) and Proteobacteria (43 out of 44), and fell into 8 genera. The genus enterobacter (18 out of 44) and Pantoea (15 out of 44) were predominant. Notably, ten DAEB isolates demonstrated <97% sequence similarity with the most similar sequences of strain deposited in the Ribosomal Database, these DAEB isolates might be potential novel species. CONCLUSION: This article suggested that plant genotype, development stage, and tissue influenced the population of endophytic bacteria. We discovered that DAEB with predominant and various genus existed in cotton. Endophytic bacteria in cotton could serve as a pool for discovering biocontrol agent toward cotton pathogens.