Increase of β -1, 3-Glucanase and Chitinase Activities in Cotton Callus Cells Treated by Salicylic Acid and Toxin of Verticillium dahliae

The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.

Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene(VdThit)confers resistance to Verticillium wilt in cotton

Verticillium wilt(VW),induced by the soil-borne fungus Verticillium dahliae(Vd),poses a substantial threat to a diverse array of plant species.Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.In this study,we successfully generated two stable transgenic lines of cotton(Gossypium hirsutum L.),VdThitRNAi-1 and VdThit-RNAi-2,using host-induced gene silencing(HIGS)technology to introduce double-stranded RNA(dsRNA)targeting the thiamine transporter protein gene(VdThit).Southern blot analysis confirmed the presence of a single-copy insertion in each line.Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type(WT).The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.Real-time quantitative PCR(qRT-PCR)analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.Small RNA sequencing(sRNA-Seq)analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.Additionally,the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.Under field conditions,VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.In summary,our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton,thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.

A glycine-rich nuclear effector VdCE51 of Verticillium dahliae suppresses plant immune responses by inhibiting the accumulation of GhTRXH2

Verticillium dahliae is an important soil-borne fungal pathogen that causes great yield losses in many cash crops.Effectors of this fungus are known to regulate plant immunity but the mechanism much remains unclear.A glycine-rich nuclear effector,VdCE51,was able to suppress immune responses in tobacco against Botrytis cinerea and Sclerotinia sclerotiorum.This effector was a required factor for full virulence of V.dahliae,and its nuclear localization was a requisite for suppressing plant immunity.The thioredoxin GhTRXH2,identified as a positive regulator of plant immunity,was a host target of VdCE51.Our findings show a virulence regulating mechanism whereby the secreted nuclear effector VdCE51 interferes with the transcription of PR genes,and the SA signaling pathway by inhibiting the accumulation of GhTRXH2,thus suppressing plant immunity.

Comparison of methods of purification and identification of Verticillium dahliae toxins

The complexes excreted by VerticiUium dahliae are phytotoxins, which are responsible for most of the symptoms associated with Verticillium wilt disease. Verticillium dahliae toxins （VD-toxins） can be purified by different methods. In the present study, we reported a simpler, more effective method to purify VD-toxins. The supematant of V. dahliae culture was frozen, lyophilized and dialyzed by 1 kDa Dialysis Membranes （MWCO）. We also partially identified the characteristics of the purified VD-toxins. The results showed that the components of VD-toxins include glycoprotein within 35.8-83.2 kDa. The phytotoxic activity of VD-toxins was remained after VD-toxins were pretreated by high temperature, Concanavalin-A, and proteinase E, respectively. These data suggest that VD-toxins are heat-stable, and the protein fraction and glycosyl are both important contributors to the phytotoxic activity. VD-toxins purified effectively from the culture filtrates of V. dahliae may help in further understanding the mechanisms of interactions between V. dahliae and plants.

Fusion Expression of Raphanus sativus-Antifungal Protein 1 (Rs-AFP1) in Escherichia coli and Its Antifungal Activity on Verticillium dahliae

The Raphanus sativus L. antifungal protein 1 (Rs_AFP1) gene was isolated by polymerase chain reaction (PCR). The complete open reading frame and the fragment encoding the putative mature protein were inserted into the prokaryotic expression vector pET_32b(+), respectively. Subsequent expression showed that the Rs_AFP1 was produced in E. coli as a 27 kD fusion protein only when the N_terminal signal peptide was removed. After treatment with thrombin to remove part of the N_terminal His.tag sequence, the bacterially expressed Rs_AFP1 was used for fungal growth inhibition assay which was conducted on Verticillium dahliae Kleb., a soil_born fungus causing the cotton wilt disease. Results showed that, in the liquid medium, the Rs_AFP1 fusion protein at a concentration of 0.3 g/L clearly inhibited the growth of V. dahliae and the germination of spores. Thus the bacterially expressed fusion protein had the antifungal activity against V. dahliae.

The Influence of the Verticillium dahliae Kleb Infection on the Anti-Enzyme Inside the Body of the Cotton with Different Root Injured Degree

This study was to explore the influence of the Verticillium dahliae Kleb inflection on the anti-enzyme inside the body of the cotton with a different root injured degree. When the cotton seedling was long, with four leaves, it was flushed with water carefully, and then the following were obtained： （1） complete root seedling; （2） cut root seedling - by cutting off the lower part, 3-5 cm of the root, with a disinfected knife; （3） injured root seedling - by cutting off most of the side roots, but keeping the main root. Three kinds of cotton seedlings with different roots were immersed separately in different concentrations of the germ liquid （V. dahliae） of 20 mL each. Through 0- 48 h, the wilt degree of the seedling was recorded, and the related anti-enzyme of the variety was measured. After being immersed in the germ liquid, there was a significant difference in the wilt degree of the three kinds of injured root. When the germ liquid was in the ratio of 1：10, the complete root seedling was the lightest with no wilt; the injured root seedling was the second with a 2-degree wilt; but the cut root seedling was the most serious with a 3- degree wilt. At the same time, the changes in the peroxidase and malondialdehvde activities were determined. Peroxidase （POD） activities in the cut root seedling were 38.2 U mg^-1 min^-1, in the injured root seedling were 42.96 U mg^-1 min^-1, and in the complete root seedling were the highest at 49.2 U mg^-1 min^-1. The malondialdenvde （MDA） content in cut root seedling was 39.483 mmol g^-1, injured root seedling was 27.12 mmol g^-1, and the complete root seedling was only 3.845 mmol g^-1 The activity of the related anti-enzymes, such as POD was high or low, the quantity of the MDA was more or less, which they met the order of the harm of the seedlings. The change of SOD activities in cut root seedling was the most obvious as well. After injuring and inflecting the young roots, the exterior pathological reaction of the seedling and the dynamic state biochemical reaction of the related enzymes inside the plant body were studied. It showed that the plant exterior pathology responded to the test, with the internal biochemical reaction fitting together mutually.

Evidences for involvement of endogenous cAMP in Arabidopsis defense responses to Verticillium toxins

Although there were reports suggesting the involvement of endogenous cAMP in plant defense signaling cascades, there is no direct evidence supporting this notion yet and the detailed mechanism is unclear. In the present study, we have used pathogenic fungi Verticillium dahliae and Arabidopsis plants as a model system of plant-microb interaction to demonstrate the function of endogenous cAMP in Arabidopsis defense responses. Both V. dahliae inoculation and Verticillium toxins injection induced typical “wilt” symptoms in Arabidopsis seedlings. When either 8-Br-AMP (a membrane permeable cAMP analogue) or salicylic acid (SA) was applied to Arabidopsis, the plants became resistant to V. dahliae toxins. However, addition of 8-Br-AMP did not increase the resistance of Arabidopsis transgenic plants deficient in SA to the toxins, suggesting that cAMP might act upstream of SA in plant defense signaling pathway. Indeed, 8-Br-cAMP and forskolin, an activator of adenylyl cyclase, significantly stimulated the endogenous SA level in plants, whereas DDA, an inhibitor of adenylyl cyclase dramatically reduced toxin-induced SA increase. Both the endog- enous cAMP and SA increased significantly in Arabidopsis seedlings treated with toxins. Furthermore, transcription level of pathogenesis-related protein 1 gene (PR1) was strongly induced by both 8-Br-cAMP and the toxin treatment. Taken together, our data demonstrate that endogenous cAMP is involved in plant defense responses against Verticillium- secreted toxins by regulating the production of the known signal SA in plant defense pathway.

A review of the pathogenicity mechanism of Verticillium dahliae in cotton

Verticillium wilt,caused by the notorious fungal pathogen Verticillium dahliae,is one of the main limiting factors for cotton production.Due to the stable dormant structure microsclerotia,long-term variability and co-evolution with host plant,its pathogenicity mechanism is very complicated,and the interaction mechanism between pathogen and host plant is also unclear.So identification and functional analysis of the genes involved in the pathogenicity or virulence of this fungus will benefit to uncover the molecular pathogenic mechanism of V.dahliae.In this review,many multifunction genes covering microsclerotia development,pathogen infection,effector proteins,transcription factors,horizontal gene transfer and trans-kingdom RNA silencing have been summarized to provide a theoretical basis to deep understand the molecular pathogenicity mechanism of V.dahliae and promote to effectively control Verticillium wilt.Furtherly,these pathogenicity-related genes may be considered as targets for effective control of Verticillium wilt in cotton.

RAPD Patterns Characteristic for VCGs of Cotton Verticillium Wilt Pathogen,Verticillium dahliae

Nine primers were employed to detect molecular polymorphisms in 103 Verticillium dahliae isolates that represent diverse groups of Vegetative Compatibility (VC). Our results showed that these isolates confer two distinctive RAPD groups (RPGs). RPG1, consists of isolates belonging to vegetative compatibility group I (VCG I ), while RPG2 encompasses VCGⅢ and VCGⅣ . The genetic diversity associated with VCGⅢ was greater than that associated with VCG1. Five RAPD fragments, with frequencies more than 0.96 in VCG I but less than 0.1 in VCGⅢ, produced characteristic fragments for VCG I (defoliating type), suggesting strong correlation between RPGs and VCGs.

Tolerance of New Introgressive Hybrid and Backcross Forms Pathogenic Micromitisms (<i>Verticillium dahliae</i>Kleb and Fusarium oxysporum f.sp. vasinfectum)

The article is based on the use of experimental polyploidy method, with the introduction of new introgressive hybrid forms combining several species genotype with pathogenic Verticillium dahliae Kleb. and the effects of mycotoxins separated from the Fusarium oxysporum f.sp. vasinfectum micromicette on the yield of plant seeds. New artificial complex hypertension forms based on experimental polyploidy Verticillium dahliae Kleb. and Fusarium oxysporum f.sp. vasinfectum combine the potential of resistance to mycotoxins separated from microcrystals, making a tremendous contribution to the selection of new varieties and to the effectiveness of selection as a result of the use of genetic selective research as genetic-selective genetic-selector studies.

Down regulation of cotton GbTRP1 leads to accumulation of anthranilates and confers resistance to Verticillium dahliae

Background:Verticillium wilt,caused by Verticillium dahliae,is called a "cancer" disease of cotton.The discovery and identification of defense-related genes is essential for the breeding of Verticillium wilt-resistant varieties.In previous research we identified some possible broad-spectrum resistance genes.Here,we report a tryptophan synthesis-related gene GbTRP1 and its functional analysis in relation to the resistance of cotton to V.dahliae.Results:Expression analysis shows that GbTRP1 is suppressed at 1 h and 6 h post V.dahliae infection,but activated at 12 h and 24 h,and the expression of GbTRP1 is highly induced by treatment with salicylic acid and jasmonic acid.Sub-cellular localization studies show that GbTRP1 is localized in the chloroplast.Suppression of GbTRP1 expression leads to lesion-mimic phenotypes and activates the immune response in cotton by showing enhanced resistance to V.dahliae and B.cinerea.Metabolomic analysis shows that anthranilic compounds significantly accumulated in GbTRP1-silenced plants,and these metabolites can inhibit the growth of V.dahliae and B.cinerea in vitro.Conclusions:Our results show that suppression of GbTRPI expression dramatically activates the immune response and increases resistance of cotton to V.dahliae and B.cinerea,possibly due to the accumulation of anthranilate compounds.This study not only provides genetic resources for disease resistance breeding,but also may provide a basis for new chemical control methods for combatting of fungal disease in cotton.

Studies on the Pathogenicity Differentiation of Verticillium dahliae on Cotton Varieties

Verticillium dahliae may be classified into the two basic groups, defoliate and non-defoliate, in terms of the pathogenicity reaction of 19 isolates of Verticillium dahliae on 16 cotton varieties. According to the comprehensive resistant or susceptible expression of 8 defoliate isolates on 8 upland cotton varieties R02,R04, R05, R06, R08, R09, R11 and R14, the defoliate isolates were for the first time divided into three categories: strong pathogenic (XS4 as the representative), mediun with inclination to strong pathogenic (T9 as the representative) and medium pathogenic (VD8 as the representative). Through the resistance identification of varieties such as R05, R06, R08, R09, R11 and R14, the defoliate isolate VD8 originated from Nantong of Jiangsu Province may be efficiently distinguished from the defoliate isolate 19 originated from USA. In nondefoliate category, utilizing 4 upland varieties R15, R14, R13 and R10, it might be possible to identify the different pathogenic genes in 7 isolates XJ4, XJ1, AY, VD404, VD326, LY, BP2 and may be nominated as No. 1 to No. 7 biological races respectively. The upland variety R01 is capable of resisting to all the isolates used in the experiments. While upland variety R12, may be used to identify strongly pathogenic isolate, which displayed a specific function in identifying strong pathogenic isolate strains.

Molecular Cloning and Characterization of Genes Involved in Cotton(Gossypium barbadense L.) Response to Verticillium dahliae

Verticillium dahliae Kleb.is a necrotrophic plant pathogen which causes serious soil borne vascular disease in cotton.The molecular basis the defense response of cotton to this pathogen is

Genotypic Resistance of F_1 Cotton Hybrids by Inoculation with Different Virulent Isolates of the Fungus Verticillium Dahliae Klebahn

The plant pathogen Verticillium dahliae causes severe cotton losses in Uzbekistan. To create cotton varieties that are resistant to the more virulent races of V.dahliae we wanted to determine

Test on Wilt-Resistance of Simple Hybrides F7 and Beckross Hybrid Lines F6 B1 of <i>G. hirsutum</i>L. of Cotton to New Virulent Isolates of Fungus <i>Fusarium verticillioides</i>and <i>Verticillium dahliae</i>

The article presents the results of studies on the resistance of hybrid cotton lines to a new virulent isolate (strain) of the fungus Fusarium verticillioides upon inoculation of the host plant. Based on the studies, it was found that the complex genotypic resistance of the studied lines, when the host plants are inoculated with isolates of -100 V. dahliae Kleb fungus and 103 Fusarium verticillioides fungi, depends on the degree of resistance of the parental forms and their combination ability.

Screening and Identification of Cotton Verticillium Wilt Antagonistic Bacteria Strain 7-30

[Objective]The aim of this study was to screen the antagonistic spore-forming bacteria of Verticillium dahliae and identify its physiological and biochemical characteristics.[Method]Taking the cotton verticillium wilt pathogen Verticillium dahliae V-190 as the test microorganism,the antagonistic spore-forming bacteria were screened.Through the preliminary screening and secondary screening,an antagonistic bacteria strain 7-30 with stronger antibacterial activity was obtained,and its morphological characteristics,physiological and biochemical characteristics were also identified.[Result]84 antagonistic bacteria strains were isolated from soil in various places by the preliminary screening.Especially,18 strains with better antagonistic ability were screened again,so an antagonistic bacteria strain 7-30 with the diameter of inhibition zone 18.9 mm and stronger antibacterial activity was obtained.According to its morphological characteristics,physiological and biochemical characteristics,the antagonistic bacteria strain 7-30 was identified as Bacillus subtilis primarily.[Conclusion]The strain 7-30 was obtained as the antagonistic spore-forming bacteria of Verticillium dahliae.

大丽轮枝菌(Verticillium dahliae VdLs.17)分泌组预测及分析

【目的】预测并分析大丽轮枝菌基因组范围内的分泌蛋白,为大丽轮枝菌分泌蛋白致病机理的研究奠定基础。【方法】利用已公布的大丽轮枝菌全基因组序列,组合使用生物信息学软件SignalP、TargetP、TMHMM、Big-pi和PROSITE,预测大丽轮枝菌基因组范围内所有分泌蛋白,定义为分泌组。统计分析分泌组中蛋白N-端信号肽特点;应用碳水化合物活性酶类数据库和病原菌-寄主互作蛋白数据库对分泌组蛋白进行注释,预测分泌组中潜在果胶酶、纤维素酶和病原菌寄主互作蛋白;利用真菌激发子的保守结构域,预测分泌组中潜在的激发子蛋白集;应用BLASTP程序比较分析大丽轮枝菌和黑白轮枝菌分泌组,获得大丽轮枝菌相对于黑白轮枝菌特异的分泌蛋白。【结果】大丽轮枝菌分泌组共有922个蛋白。信号肽分析表明,以19个氨基酸为信号肽的蛋白数目最多,非极性氨基酸丙氨酸的出现频率最高,而有带电侧链的氨基酸天冬氨酸和谷氨酸的出现频率最低,信号肽的-3和-1位置上的氨基酸相对保守。大丽轮枝菌分泌组含有158个潜在的碳水化合物活性酶类,其中,包括10个果胶水解酶和14个果胶裂解酶;190个潜在的病原菌-寄主互作蛋白、97个含有RxLx[EDQ]模体的蛋白和52个富含半胱氨酸的小分子量分泌蛋白;58个相对于黑白轮枝菌分泌组特异的蛋白。【结论】本文建立了预测大丽轮枝菌分泌组蛋白的方法。分泌组蛋白信号肽长度具有高度的变异性,氨基酸组成多为脂肪族氨基酸,序列在C-端结构域较为保守。分泌组中包含大量潜在的果胶降解酶、病原菌-寄主互作蛋白、RxLx[EDQ]模体蛋白和富含半胱氨酸的小分子量蛋白等致病相关蛋白。

棉花黄萎病真菌Verticillium dahliae木聚糖酶基因的克隆、表达和酶学性质分析

【目的】从棉花黄萎病真菌Verticillium dahliae中克隆木聚糖酶基因,并在毕赤酵母中进行异源表达,研究酶学性质。【方法】通过多序列比对设计简并引物,扩增出真菌V.dahliae木聚糖酶基因片段,再采用基因组步行PCR技术获得全长木聚糖酶基因序列。经BLAST比对并结合GT-AG原则分析,该基因含有一个大小为63bp的内含子,利用DpnI介导的缺失方法对含内含子的全长木聚糖酶基因进行剪接,获得该基因的全长cDNA。将克隆到的cDNA在毕赤酵母GS115进行了表达,重组酶经纯化后进行酶学性质分析。【结果】BLAST比对显示,该cDNA推测的氨基酸序列和已知木聚糖酶的最高一致性为72%。测得该酶最适反应温度为45℃,最适反应pH值为6,在pH5-9维持50%以上的活性,对山毛榉材木聚糖具有最好的水解效果。Mg2+和Ca2+对酶有激活作用,分别提高了33.7%和16.6%,EDTA,β-巯基乙醇和NaN3对酶的活性基本没有影响,Tween-80和DMSO使酶活性提高了28.4%和12.8%。【结论】本文从引起棉花黄萎病的真菌V.dahliae中克隆到的木聚糖酶基因是在GenBank上登录的第一个来自棉花黄萎病真菌的木聚糖酶基因序列。本文所用的克隆方法可以高效的从植物病原真菌和白腐真菌克隆只含一个内含子的11家族的新木聚糖酶基因,避免了摸索原始菌株酶表达诱导条件,检测酶的活性等繁琐的操作。酶学性质分析显示该酶在低聚木糖的制备,面包改良上有潜在的应用价值。

有关棉花黄萎病（Verticillium dahliae Kleb．）抗性遗传研究的几个问题

棉花黄萎病抗性遗传问题迄今尚未获得肯定的结论。据作者等估计，这可能和寄主（棉株）和寄生物（黄萎病镰刀菌）之间的关系、黄萎病菌不同菌系间的互作以及影响抗性鉴定的正确性等问题有关。为此，必须创设有利条件，使寄主和病原菌间的关系得以充分表现；必须了解病原菌菌系间的互作，寄主必须有较大的群体。这一切都是为了寄主和病原物问的关系得以充分体现，为抗性遗传研究奠定基础。抗性遗传研究宜用单菌系接种，并且宜按数量性状和质量性状遗传方式同时进行研究和分析，以便得到比较确切可靠的结果。

棉花大丽轮枝菌(Verticillium dahliae)血清学检测——抗原处理的比较

分别以棉花黄萎病菌V20的菌丝研磨物、菌丝蛋白、超氧化物歧化酶酶带Ⅰ、超氧化物歧化酶酶带Ⅱ为抗原免疫家兔,制备了抗血清As1、As2、As3、As4。琼脂双扩散法(ADD)和间接ELISA方法测定其特异性,发现这4种抗血清既可与同种的抗原发生阳性反应,也可与不同种的抗原发生交叉反应。用异种抗原黑白轮枝菌和尖孢镰刀菌吸附这4种抗血清,排除其非特异性成分后,抗血清专化性明显提高,消除了交叉反应。特异性测定表明:经吸附后的4种抗血清可以准确地将大丽轮枝菌鉴定到种的水平,但仍不能区分其生理型和致病类型。另外,当分别使用菌丝研磨物或菌丝蛋白为特异性测定抗原时,ADD检测的结果虽然一致,但前者阳性反应沉淀线粗糙,轮廓不清晰,后者的沉淀线较细,且清晰、明显。因此适宜选择菌丝蛋白为抗原进行抗血清的特异性测定试验。