高温季节平菇细菌性病害的发生及其防治

细菌性褐斑病和细菌性腐烂病是平菇栽培中较为常见的病害,尤其在高温条件下发生更为严重,一旦发生则很快蔓延,造成平菇严重减产,解决好这一问题是高温条件下平菇栽培成功的关键。1　病害症状1. 1　细菌褐斑病　又称细菌性锈斑、细菌性斑点病,病原为托拉斯假单孢杆菌(Pseudo     

四个托拉斯假单胞杆菌菌株对平菇CCEF-99的致病力测定

托拉斯假单胞杆菌(Pseudomonas tolaasii)是引起食用菌细菌性褐斑病的病原菌,该研究利用平皿对峙和子实体接种的方法,研究了4个不同托拉斯假单胞杆菌菌株对平菇CCEF-99菌丝体和子实体生长的影响,评价不同菌株的致病能力,为平菇品种抗病性鉴定提供备选鉴别菌株。结果表明:菌株JZB2120025的致病力最弱,病斑面积小,颜色呈淡黄色;菌株JCM21583的致病力最强,产生的病斑凹陷,颜色呈棕褐色,可作为平菇抗病性状检测的备选菌株。     

一种平菇褐斑病病原菌的鉴定

本文将从平菇(Pleurotus ostreatus)褐斑病中分离纯化的240多个菌株中,选出使平菇褐斑病发病快且病情严重的菌株PG1702,对其进行致病性试验,结果表明菌株PG1702为引起平菇细菌性褐斑病的病原菌;显微形态的观察和扩增出的16S rDNA序列与GenBank比对结果表明菌株PG1702为托拉斯假单胞杆菌(Pseudomonas tolaasii Paine).     

托拉斯假单胞杆菌弱毒菌株诱导平菇抗细菌性褐斑病的研究

为了明确平菇细菌性褐斑病病原菌托拉斯假单胞杆菌（Pseudomonas tolaasii）弱毒菌株JPG250303的诱导抗病作用、弱致病性及诱导抗病作用的遗传稳定性,通过对其诱导抗病性表达、诱导接种浓度及诱导间隔期的研究,明确该弱毒菌株具有较好的诱导抗病效果。利用形态学及分子生物学技术,鉴定出该弱毒菌株为托拉斯假单胞杆菌（P. tolaasii）,将弱毒菌株经平菇子实体连续5代培养后均具有弱致病性,说明其弱致病性可以稳定遗传;同时对5代菌株分别进行了诱导抗病活性的验证,5代菌株对平菇细菌性褐斑病诱导抗病效果分别为67.2%、66.3%、69.1%、68.6%和65.0%,说明该弱毒菌株诱导抗病活性也具有稳定性,这一研究为该弱毒菌株作为生防菌株防治平菇细菌性褐斑病的应用提供了理论基础。     

精制山杏壳木醋液防治平菇细菌性褐斑病药效试验

以"寿研平"平菇(Pleurotus ostreatus)为试材,托拉斯假单胞杆菌为试用菌,采用平板扩散法和田间防效法测定不同浓度的精制山杏壳木醋液对平菇细菌性褐斑病菌的抑菌活性及对平菇菌丝生长的影响。结果表明:筛选得到1.25%、2.50%、5.00%精制山杏壳木醋液均可有效抑制平菇细菌性褐斑病的发生,且可以促进平菇菌丝的生长;在田间施用过程中,可选用1.25%、2.50%精制山杏壳木醋液进行预防,在平菇细菌性褐斑病大面积发生时可用5.00%精制山杏壳木醋液进行控制。     

番茄细菌性斑点病菌实时荧光定量PCR检测方法的建立及应用

以番茄细菌性斑点病病原菌丁香假单胞菌番茄致病变种(Pseudomonas syringae pv. tomato,Pst)的致病相关基因HrpZ为靶序列,设计了特异性引物Pst3F/Pst3R,能从Pst基因组DNA中特异性扩增出大小为161 bp的目的片段。建立的Pst实时荧光定量PCR检测技术体系的检测灵敏度比普通PCR高1 000倍。利用实时荧光定量PCR检测体系,检测模拟带菌种子中Pst的带菌量,检测下限为4.21 cfu·g^-1;检测人工接种叶片组织中Pst的带菌量,检测到1级发病叶片带菌量为4.15×102 cfu·g^-1。对田间采集的63个番茄细菌性斑点病明显症状和疑似症状样本,分别进行了实时荧光定量PCR、普通PCR和病原菌分离检测,检测到54个样本中含有Pst,3种方法检测结果一致。结果表明,建立的Pst实时荧光定量PCR检测体系具有特异性强、灵敏度高的特点,可以快速准确地定量检测番茄种子和发病组织中Pst的含量,为番茄细菌性斑点病的早期预防和流行监测提供了有效的技术手段。     

金针菇黑腐病病原菌的分离与分子鉴定

为了明确山东省金针菇(Flammulina velutipes)工厂化厂房内发现的细菌性病害的种类,从感病子实体组织分离获得病原菌菌株,通过提取其基因组总DNA并分别扩增病原菌的16S rDNA和gyrB基因,序列分析结果表明该病原菌为托拉斯假单胞杆菌(Pseudomonas tolaasii)。     

中生菌素可湿性粉剂对糙皮侧耳细菌性褐斑病药效试验

以硫酸链霉素为对照药剂,通过平皿抑菌试验和活体药效试验,对3%中生菌素用于防治糙皮侧耳(Pleurotus ostreatus)细菌性褐斑病的效果进行评价。结果表明,3%中生菌素(37.57、5.0 mg/L)与硫酸链霉素(250 mg/L)对托拉斯假单胞杆菌(Pseudomonas tolaasii,细菌性褐斑病病原菌)的平皿抑制率均为100%;活体药效试验中,3%中生菌素(37.5 mg/L)与硫酸链霉素(250 mg/L)对糙皮侧耳细菌性褐斑病的预防效果均在80%以上,显著优于接种后施药处理的治疗效果。     

平菇细菌性褐斑病诱抗菌株筛选及诱抗效果

从平菇(Pleurotus spp.)上分离到细菌性褐斑病弱致病力菌株和非病原菌株共126株,采用离体平菇法筛选模型,筛选出了3株可以诱导糙皮侧耳(P.ostreatus)对托拉斯假单胞菌(Pseudomonas tolaasii)产生抗病作用的诱抗菌株。通过田间验证,最终得到1株诱导抗病效果好的诱抗菌株,诱导抗病效果为66.73%。在供试接种浓度范围内,诱抗菌株的诱导效果随接种浓度增大而增强。     

平菇细菌性黄斑病发生及防治

近年来平菇细菌性引起的黄褐斑病呈逐年上升趋势 ,不同程度影响菇体质量和产量 ,轻者减产欠收 ,重则只菇无收。笔者在栽培实践中经三年的观察 ,究其发病原因和防治方法作了研究 ,摸索了防止该病害发生的有效方法。1　病害症状平菇栽培过程中发生的黄褐斑病 ,都是由假单孢杆菌引起 ,假单孢杆菌在平菇子实体生长过程中无论低温、高温季节都可发生病害 ,病原菌主要通过土壤、水源、虫害、空气、病菇为传播介体。从幼菇期到成熟都有可能发病 ,被染菌后初期菇柄根部或菇盖局部呈微黄色 ,菇体生长缓慢、僵化直至菇体整株被感染 ,成菇菌褶扭曲 ,萎…     

Effects of rs35100176 polymorphism in IPO8 promoter on gene expression

AIM: To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 (IPO8) gene on its mRNA expression. METHODS: A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS：The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT,CCT/- and -/-, and the gene frequencies were 1837%, 5510% and 2653%, respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote. CONCLUSION：The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8, thus affecting the gene expression.     

Role of carvedilol on energy metabolic reversion and remodeling of pressure overload-induced left ventricular hypertrophy in rats

AIM: To study changes of medium chain acyl-CoA dehydrogenase (MCAD), muscle carnitine palmitoyltransferase I (M-CPT-I) and colligin mRNA/protein expression, to elucidate molecular mechanism of the recapitulation of fetal energy metabolism and ventricular remodeling and the effects of carvedilol during the development of pressure overload-induced left ventricular hypertrophy in rats. METHODS: Male Wistar rats of hypertrophy induced by constriction of abdominal aorta （CAA） were randomized into 2 groups (n=12, each group): 4-week group (CAA4 weeks group) and 12-week carvedilol intervention group (CAR group). Additional rats (n=12) underwent abdominal cavity incision without ligation to serve as age-matched sham operated controls (SH). Hemodynamics, ventricular remodeling parameters and free fatty acid （FFA） both in blood serum and myocardium were measured. RT-PCR analysis of the expression of mRNA of M-CPT-I, MCAD and collagen binding protein (colligin) were investigated. The protein expression of colligin was analyzed by Western blotting in the experimental animals and sham operation.RESULTS: LVM/BW and MAP in CAA group were increased more significantly than in sham group. There were progressive increases in FAA both in blood serum and myocardium in CAA group than in sham group, accompanied with downregulation of gene expressions of M-CPT-I and MCAD and colligin mRNA/ protein upregulation in LV in CAA group, while changes of all of these parameters in CAR group were attenuated.CONCLUSIONS: (1) The down-regulated expression of cardiac FAO enzyme genes (M-CPT-I and MCAD) in the hypertrophied heart may be responsible for “the recapitulation of fetal energy metabolism” during the development of pressure overload-induced left ventricular hypertrophy in rats. (2) Carvedilol attenuates the reversion of the metabolic gene expression back towards fetal type. (3) Carvedilol is effective in regressing the left ventricular remodeling by inhibiting colligin protein expression. A molecular mechanism by which carvedilol may confer cardioprotective effects in heart failure may be, in part, via preserving of the adult metabolic gene regulation and regressing left ventricular remodeling.     

MMP-7,MMP-10 and TIMP-4 expression in ventricular remodeling of human heart failure

AIM:To sieve matrix metalloproteinases (MMPs) and the tissue inhibitors of matrix metalloproteinases (TIMPs) closely associated with ventricular remodeling of human heart failure using antibody chip technology.METHODS:We performed cytokine-specific antibody array analysis using individual left ventricular myocardial samples from 6 patients with heart failure due to arrythmogenic right ventricular cardiomyopathy (ARVC) undergoing transplantation and matched samples from 6 non-failing subjects to screen differentially expressed MMPs and TIMPs associated with the ventricular remodeling of heart failure.The results were further validated by ELISA and immunohistochemical analysis.RESULTS:We identified high expression of MMP-7 and MMP-10 and low expression of TIMP-4 in ARVC failing hearts compared to non-failing hearts by hybridization with the cytokine-specific antibody arrays containing 17 MMPs and 4 TIMPs on the chips.ELISA and immunohistochemical analyses further confirmed that differentially expressed levels of MMP-7, MMP-10, and TIMP-4 were observed not only in ARVC failing heart, but also in failing hearts due to ischemic (ICM) and dilated cardiomyopathy (DCM).CONCLUSION:Highly expressed MMP-7 and MMP-10 and lowly expressed TIMP-4 may be involved in the ventricular remodeling of heart failure derived from cardiomyopathy of different etiology.     

Relationship between matrix metalloproteinase 2-735C→T genetic polymorphism in promoter region and coronary atherosclerosis

AIM: To investigate the relationship between matrix metalloproteinase 2 ( MMP-2 )-735C→T polymorphism in the promoter region and coronary atherosclerosis (CAS) in Han population of China. METHODS: This study was conducted with a CAS group including 309 patients confirmed by angiography and 311 control healthy subjects. Genotype of -735C→T functional promoter polymorphism of the MMP-2 gene was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The relationship between the polymorphism in MMP-2 gene and CAS was analyzed. RESULTS: The frequency of CC genotype (86.1%) in CAS group was significantly higher than that in control group (79.7%), but the frequency of CT+TT genotype (13.9%) in CAS group was significantly lower than that in control group (20.3%). The statistical difference between CAS group and controls was significant(χ²=4.398,P<0.05). The frequency of -735C in CAS group (92.6%) was higher than that in control group (89.1%) and the frequency of -735T in CAS group (7.4%) was lower than that in control group (10.9%), with the statistical significant difference (χ²=4.521, P<0.05). The degree of stenosis in coronary artery did not significantly relate to the MMP-2 gene -735C→T polymorphism in the promoter region. CONCLUSION: The genetic polymorphism in MMP-2 promoter region (-735C→T) is associated with the susceptibility to CAS in Han population of China. CC genotype and C allele may be a genetic marker. The -735C→T polymorphism may be useful as a predictor of CAS.     

Effect of short-chain acyl-CoA dehydrogenase on cardiac hypertrophy induced by hypertension or exercise training

AIM: To investigate the differential expression of short-chain acyl-CoA dehydrogenase (SCAD) in cardiac hypertrophy induced by hypertension or exercise training. METHODS: Spontaneously hypertensive rats (SHR) were used as the model of pathological cardiac hypertrophy. The swim-trained rats were used as the model of physiological cardiac hypertrophy. The systolic pressure, cardiac hypertrophy parameters, echocardiogram parameters, free fatty acid in serum and cardiac muscle, and the expression and activity of SCAD in the left ventricle were measured. RESULTS: Compared with the control rats, trained rats developed an athletic heart, of which cardiac function was enhanced, whereas SHR developed hypertensive cardiac hypertrophy, of which cardiac function was deteriorated. Compared with the control rats, the ratios of left ventricular weight to body weight were both increased in trained rats and SHR, showing that the degrees of cardiac hypertrophy were similar in the 2 models. Compared with the control rats, the decrease of free fatty acid both in serum and myocardium indicated that the fatty acid utilization was increased in the left ventricle of trained rats. Meanwhile, the expression and activity of SCAD in the left ventricle of trained rats were increased. However, free fatty acid both in serum and myocardium were increased, indicating that the fatty acid utilization was decreased in the left ventricle of SHR. Furthermore, SHR had the decreased expression and activity of SCAD in the left ventricle. CONCLUSION: The changes of SCAD are different in cardiac hypertrophy induced by hypertension and exercise training, indicating that SCAD may be used as a molecular marker of physiological and pathological cardiac hypertrophy, and a potential therapeutic target of pathological cardiac hypertrophy.     

Altered activity and expression of MMPs/TIMPs are associated with functional and structural left ventricular remodeling in hypertensive rats

AIM: To investigate the relationship between matrix metalloproteinases and tissue inhibitors of matrix metalloproteases imbalance with functional and structural left ventricular (LV) remodeling in the hypertensive rats.METHODS: 6-week-old male stroke-prone spontaneously hypertensive rats (SHR-SPs,n=40) served as the hypertensive heart disease model,and age-matched male Wistar-Kyoto (WKY) rats (n=10) were used as control.After 6 months,the rats in two groups were anesthetized for invasive hemodynamic measurement by Millar pressure-volume (P-V) conductance catheter.Then the rats were sacrificed and hearts were dissected for morphological analysis,gelatin-zymography and Western blotting analysis.RESULTS: Left ventricular (LV) hemodynamic parameters showed the systolic and diastolic dysfunction in SHR-SPs compared with that in control group (P<0.05).Collagen volume fraction,ratio of perivascular collagen area to luminal area,myocardial cross-sectional area and the medial area to luminal area ratio of the SHR-SPs were all increased remarkably (P<0.05).LV matrix metalloproteinase-2 (MMP-2) activities,MMP-2 and tissue inhibitors of matrix metalloprotease-1 (TIMP-1) protein level in SHR-SP were notably higher than those in control group (P<0.05).CONCLUSION: Chronic pressure-overload is capable of inducing imbalances of cardiac ECM and MMPs/TIMPs system,both imbalances induce LV dilation,cardiac systolic and diastolic dysfunction.     

Expression of short-chain acyl-CoA dehydrogenase during heart development in rats and relationship with cardiac hypertrophy

AIM： To investigate the expression of short-chain acyl-CoA dehydrogenase during the heart deve-lopment in rats and to analyze the relationship between short-chain acyl-CoA dehydrogenase and cardiac hypertrophy in spontaneously hypertensive rats (SHR). METHODS：The expression and activity of short-chain acyl-CoA dehydrogenase in the hearts of Wistar rats with different ages were measured. Free fatty acids in serum and cardiac muscles were also determined. RESULTS：Compared with the fetal rats of 19 d, the expression and activity of short-chain acyl-CoA dehydrogenase in the postnatal rats of 1 d, 2 weeks, 6 weeks and 16 weeks were increased, and free fatty acids in the serum and myocardium were obviously decreased. The difference began in evidence from the age of 2 weeks. The expression of short-chain acyl-CoA dehydrogenase was significantly up-regulated with negative correlation to free fatty acids in the serum and myocardium during heart development. Systolic blood pressure was similar in 2-week-old SHR and WKY rats, which significantly increased in SHR of 6 weeks and 16 weeks old compared with the age-matched WKY rats. The ratio of left ventricular weight to body weight was markedly elevated in SHR of 2 weeks, 6 weeks and 16 weeks old compared with the age-matched WKY rats, indicating that the appearance of cardiac hypertrophy occurred before the development of hypertension in SHR. Compared with the age-matched WKY rats, the expression and activity of short-chain acyl-CoA dehydrogenase were decreased and free fatty acids in the serum and myocardium were obviously higher in SHR. The expression of short-chain acyl-CoA dehydrogenase was significantly down-regulated with a negative correlation to free fatty acids in the serum and myocardium of SHR. CONCLUSION：The expression of short-chain acyl-CoA dehydrogenase is increased during the heart development, which may be associated with the increase in cardiac fatty acid utilization. The down-regulated expression of short-chain acyl-CoA dehydrogenase in the hypertrophic heart may be responsible for the recapitulation of fetal energy metabolism.     

Changes of DNA methylation at CpG sites in promoter region of TNF-α gene in DENV2-infected peripheral blood mononuclear cells

AIM：To examine DNA methylation at CpG sites in the promoter region of tumor necrosis factor-alpha (TNF-α) gene in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMC).
METHODS：DNA methylation in the promoter region of TNF-α gene was measured by bisulfite sequencing PCR.
RESULTS：The promoter region of TNF-α gene was from -294 bp to +58 bp, including 11 CpG sites. The PCR products identified by aga-rose gel electrophoresis were consistent with the theoretical size. Two sites were methylated at 0 h and 6 h and 6 sites were methylated at 12 h in TNF-α gene promoter region in DENV2-infected PBMC. The average methylation rates were 103%, 121% and 255% at 0 h, 6 h and 12 h, respectively. Significant differences between 0 h and 12 h and between 6 h and 12 h were observed.
CONCLUSION：The DNA methylation in the promoter region of TNF-α gene is increased in DENV2-infected PBMCs.