Silencing of early auxin responsive genes MdGH3-2/12 reduces the resistance to Fusarium solani in apple

Apple replant disease(ARD)has led to severe yield and quality reduction in the apple industry.Fusarium solani(F.solani)has been identified as one of the main microbial pathogens responsible for ARD.Auxin(indole-3-acetic acid,IAA),an endogenous hormone in plants,is involved in almost all plant growth and development processes and plays a role in plant immunity against pathogens.Gretchen Hagen3(GH3)is one of the early/primary auxin response genes.The aim of this study was to evaluate the function of MdGH3-2 and MdGH3-12 in the defense response of F.solani by treating MdGH3-2/12 RNAi plants with F.solani.The results show that under F.solani infection,RNAi of MdGH3-2/12 inhibited plant biomass accumulation and exacerbated root damage.After inoculation with F.solani,MdGH3-2/12 RNAi inhibited the biosynthesis of acid-amido synthetase.This led to the inhibition of free IAA combining with amino acids,resulting in excessive free IAA accumulation.This excessive free IAA altered plant tissue structure,accelerated fungal hyphal invasion,reduced the activity of antioxidant enzymes(SOD,POD and CAT),increased the reactive oxygen species(ROS)level,and reduced total chlorophyll content and photosynthetic ability,while regulating the expression of PR-related genes including PR1,PR4,PR5 and PR8.It also changed the contents of plant hormones and amino acids,and ultimately reduced the resistance to F.solani.In conclusion,these results demonstrate that MdGH3-2 and MdGH3-12 play an important role in apple tolerance to F.solani and ARD.

接触辉光放电等离子体对Fusarium solani的杀菌作用机制及动力学模型研究

为研究接触辉光放电等离子体(CGDP)对茄病镰刀菌(Fusarium solani)的杀菌作用及机制,本试验以Fusarium solani为试材,考察CGDP处理过程中电压、处理时间、抗坏血酸浓度、孢子初始浓度等因素对杀菌效果的影响;并对Fusarium solani生长、孢子形态、细胞膜完整性及过程中所产生的活性粒子进行分析。通过Linear、Weibull和Log-Logistic 3种数学模型分析不同电压下CGDP杀菌动力学特性,以相关系数(R^(2))、精确因子(Af)、偏差因子(Bf)和均方根方差(RMSE)4个参数评价拟合效果。结果表明,CGDP对Fusarium solani孢子有着明显的杀菌效果;升高电压、延长处理时间、降低抗坏血酸浓度和孢子初始浓度均能有效抑制孢子生长,使杀菌效率提高(P<0.05);CGDP处理30 min,活性物质(·OH、H_(2)O_(2)、NO_(3)^(-))浓度分别增加至1.57 mg·L^(-1)、73.89 mmol·L^(-1)和12.72 mg·L^(-1),pH值由7.07降至4.66;孢内核酸和蛋白质的渗漏量以及PI染色和扫描电镜结果表明,CGDP对Fusarium solani的杀菌作用可能是通过破坏孢子形态和细胞膜完整性后被活性物质攻击而引起的;模型拟合结果表明,Weibull模型拟合度最高(R^(2)=0.9242),并且RMSE较小(0.6367),可以用于描述和预测CGDP对Fusarium solani的杀菌动力学特征。本研究结果为低温等离子体杀菌技术和杀菌机制研究提供了理论指导。

Secondary pulmonary infection by Fusarium solani and Aspergillus niger during systemic steroid treatment for COVID-19:A case report

BACKGROUND Coronavirus disease 2019(COVID-19)-associated invasive pulmonary aspergillosis presents a diagnostic challenge due to its non-specific clinical/imaging features,as well as the fact that the proposed clinically diagnostic algorithms do not necessarily apply to COVID-19 patients.In addition,Fusarium spp.is a rare cause of opportunistic life-threatening fungal infections.Disseminated Fusarium infection in an immunocompromised host is intractable,with a high likelihood of resulting mortality.To our knowledge,this is the first case of secondary pulmonary infection by Fusarium solani(F.solani)and Aspergillus niger(A.niger)during systemic steroid treatment for COVID-19.CASE SUMMARY A 62-year-old male was transported to our hospital by ambulance with a complaint of fever and dyspnea.We established a diagnosis of pneumococcal pneumonia,complicated with COVID-19 and septic shock,together with acute renal failure.He was admitted to the intensive care unit,to be treated with piperacillin/tazobactam,vancomycin,and 6.6 mg per day of dexamethasone sodium phosphate,along with noradrenaline as a vasopressor,ventilator management,and continuous hemodiafiltration.His condition improved,and we finished the vasopressor on the fifth hospital day.We administered dexamethasone for ten days,and finished the course of treatment.On the eleventh day,patient respiratory deterioration was observed,and a computed tomography scan showed an exacerbation of bilateral ground-glass-opacity-like consolidation,together with newly appeared cavitary lesions in the lung.we changed antibiotics to meropenem plus vancomycin.In addition,a fungal infection was considered as a possibility based on microscopic findings of sputum,and we began coadministration of voriconazole.However,the pneumonia worsened,and the patient died on the seventeenth day of illness.Later,F.solani and A.niger were identified from sputum collected on the twelfth day.It was believed that he developed a cell-mediated immune deficiency during COVID-19 treatment,which led to the complication of pneumonia caused by the above-mentioned fungi,contributing to his death.CONCLUSION Because early initiation of intense antifungal therapy offers the best chance for survival in pulmonary fusariosis,computed tomography scans and appropriate microbiologic investigations should be obtained for severely immunocompromised patients.

一株多粘类芽孢杆菌的鉴定及其对花椒根腐病菌Fusarium solani的抑菌作用

腐皮镰刀菌(Fusarium solani)是引起花椒根腐病的主要病原菌之一,为开发高效防治花椒根腐病的绿色生防菌,实验从健康花椒根部土壤中分离筛选出一株腐皮镰刀菌较高拮抗作用的细菌KS-16,并对其进行形态学、生理生化性质和分子鉴定,研究了该菌对三株供试菌株大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌及腐皮镰刀菌的抑菌作用.结果表明,菌株KS-16被鉴定为多粘类芽孢杆菌(Paenibacillus polymyxa).活性测定发现,该菌对腐皮镰刀菌的菌丝生长抑制率达到53.78%,对三株供试菌株也有较高的抑菌活性.以PDA培养基作为发酵培养基优于LB培养基,作用10 d其抑制效果仍保持在43.33%.

冬虫夏草内生菌 Fusarium solani 化学成分研究

目的研究冬虫夏草内生菌Fusarium solani化学成分。方法Fusarium solani乙酸乙酯提取物采用硅胶、Sephadex LH-20、制备HPLC进行分离纯化,根据理化性质及波谱数据鉴定所得化合物的结构。结果从中分离鉴定出25个化合物。结论化合物1~17、19~24为首次从该真菌的次生代谢产物中发现。

苜蓿根腐病菌(Fusarium solani)生物学特性研究

主要研究了温度、湿度、pH值、光照、营养对苜蓿根腐病菌(Fusarium solani)生长、产孢和孢子萌发的影响.该病菌菌丝生长的温度范围为8～38℃,最适20～28℃;产生分生孢子的温度范围为10～38℃,最适25℃;分生孢子萌发的温度范围为8～38℃,最适25～32℃.在pH值5～10的范围内该菌均能生长和产孢,菌丝生长最适pH值5～9;分生孢子产生和萌发最适pH值6～7.分生孢子在饱和湿度或水滴中萌发快,相对湿度低于85%时不能萌发.光照处理对该菌生长无显著影响.分生孢子的致死温度为50℃,10 min或55℃,5 min,菌丝体的致死温度为60℃,30 min或65℃,5 min.

镧素对镰刀菌Fusarium solani及其致病酶的影响

采用琼脂平板生长速率法及液体培养基培养测定了 L a对镰刀菌 Fusarium solani的抑制作用和毒力 ,并测定了其对病菌胞外的果胶酶、蛋白酶和纤维素酶等几种致病酶活性的影响。结果表明 ,随着 L a2 O3 浓度升高 ,对病菌菌丝生长的抑制作用增强 ,对病菌的 EC50 和 EC95分别为 2 78.2和5 5 2 .0 mg/ L ;在一定浓度范围内 ,L a提高了单位量菌丝所产生 3种致病酶的活性 ,但由于菌丝生长受到抑制 ,除蛋白酶外 ,病菌胞外致病酶果胶酶和纤维素酶的总量或总活性受到了抑制 。

南海红树林内生真菌Fusarium solani 387～#次级代谢产物研究

采用色谱技术对Fusarium solani 387#的次级代谢产物进行分离纯化,根据化合物的理化性质、波谱数据以及与文献谱图、数据对照的方法确定化合物的结构。从南海红树林内生真菌Fusarium solani 387#的次级代谢产物中分离得到了9个镰红菌素类化合物,分别鉴定为5(R)-deoxyfusarubin(1)、5(S)-deoxyfusarubin(2)、3-methyl ether fusarubin(3)、Fusarubin(4)、Anhydrofusarabin(5)、Anhydro-5-deoxyfusarubin(6)、2-acetonyl-3-methyl-7-methoxy-naphthazarin(7)、Javanicin(8)、2-acetonyl-3-methyl-5-hydrogen-7-methoxy-naphthazarin(9)。通过X-射线单晶衍射首次确定了化合物1和2的绝对构型及存在形式。

桉树枯萎病菌Fusarium solani分子检测技术研究

桉树枯萎病是桉树人工林中的重要枝干病害,一旦发生则对桉树人工林造成严重危害,Fusarium solani是其主要病原之一。本试验通过RAPD标记技术,找到一条致病菌特异片段,对该片段进行克隆、测序,根据测序结果设计出特异性引物F9/R9,进一步优化扩增条件,并检测其灵敏度。结果表明,特异性引物F9/R9能从桉树枯萎病菌F.solani扩增出一条大小为200 bp左右的特异条带,而其它8种参试菌株和对照均无条带产生。该引物最低可以检测到4×10-7 ng·μL^-1的病原菌基因组DNA,也能检测到单个孢子的存在。同时可从发病桉树枝干和带菌土壤中特异性检测到枯萎病菌F.solani。因此,利用特异性引物F9/R9对桉树枯萎病菌F.solani快速检测技术操作简单,特异性强,可用于该病害早期诊断及检疫,对指导林农及时进行药剂防治,进而限制病害扩展具有重要意义。

山药根腐病菌(Fusarium solani)的生物学特性

【目的】对四川雅安山药根腐病主要致病菌腐皮镰孢菌[Fusarium solani]的生物学特性进行研究。【方法】研究不同温度、pH、光照、碳源、氮源和微量元素对该病原菌菌丝生长、分生孢子产生和萌发的影响。【结果】结果表明,腐皮镰孢菌菌丝适宜生长温度为10～40℃,最适温度为30℃;适宜pH范围为4～10,最适碳源为蔗糖,最适氮源为蛋白胨,且在连续光照条件下生长较佳。不同微量元素对菌丝生长和孢子萌发产生作用不同,Mg2+有利于病菌产孢,Zn2+抑制菌落生长和产孢。孢子萌发最适温度为30℃,连续光照,分生孢子和菌丝的最低致死温度为60℃,处理时间为10min。【结论】该病原菌对酸碱度不敏感,不同微量元素对病原菌生长有促进或抑制的作用,生物学特性的研究结果为进一步防治山药根腐病奠定了理论基础。

小海马内生菌Fusarium solani化学成分的研究

目的:研究小海马Hippocampus japonicas Kaup内生菌Fusarium solani的化学成分。方法:将该菌株进行固体发酵培养,发酵物甲醇提取后的乙酸乙酯萃取部位利用硅胶柱、C18反向硅胶柱、Sephdex-LH.20凝胶柱、MCI柱、制备型液相色谱对其化学成分进行分离纯化,根据物理性质及波谱技术鉴定化合物的结构。结果:从中分离得到15个已知化合物,鉴定为环-(D-脯氨酸-D-苯丙氨酸)(1);环-(L)-脯氨酸-(L)-苯丙氨酸(2);邻羟基苯甲醛(3);4-羟基苯甲酸甲酯(4);乙酰胆碱(5);环-4-羟基-(D)-脯氨酸-(L)-苯丙氨酸(6);环-4-羟基-(L)-脯氨酸-(L)-苯丙氨酸(7);环-(L)-脯氨酸-(L)-亮氨酸(8);2,5-morpholinedione,4-methyl-6-(1-methylethyl)-3-(1-methylpropyl)-(9);(E)-二甲基咖啡酸(10);4-甲氧基-5,6-二甲基-2H-吡喃[2,3-b]吡啶-2-酮(11);3,4-二甲氧基苯乙酮(12);白僵菌素(13);亚油酸(14);9,12十八烷二烯酸甲酯(15)。结论:化合物1-3,6-9,11-12均为首次从该真菌中分离。

苜蓿根腐病菌(Fusarium solani)的LAMP快速检测方法

通过比较茄镰刀菌(Fusarium solani)与其近缘种之间的TEF-1α(translation elongation factor-1 alpha)基因序列差异,设计并筛选出一套对茄镰刀菌具有种特异性的LAMP引物,建立了检测苜蓿根腐病菌的环介导等温扩增(Loop-mediated isothermal amplification, LAMP)技术体系,并进行了特异性和灵敏度验证。该方法在64℃等温条件下进行核酸扩增反应60 min,即可肉眼根据反应液颜色变化判定扩增产物。在特异性实验中,仅茄镰刀菌的DNA在检测后呈绿色的阳性反应,而其它供试菌株的DNA均呈橙色的阴性反应。该方法对目标病菌DNA的最低检测限为10 fg/μL,同时还可检测出苜蓿发病组织中的目标菌,因此,该方法具有快速、准确和灵敏的优点。

Early expression of surfactant proteins D in Fusarium solani infected rat cornea

AIM: To investigate the early expression of surfactant proteins D(SP-D) in Fusarium solani infected rat cornea. METHODS: Wistar rats were divided into group A, B and C randomly. The right eyes were chosen as the experiment one. Group A was control group. Group B was not inoculated with Fusarium solani. Group C was taken as fusarium solani keratitis model. Five rats in group B and C were executed randomly at 6, 12, 24, 48 and 96 hours respectively after the experimental model being established. The expression of SP-D was assessed through immunohistochemistry and reverse transcription polyrnerase chain reaction(RT-PCR). RESULTS: RT-PCR detected that the SP-D mRNA expression was low in the corneal of normal rats and group B. The expression of fungal infected cornea increased gradually and reached the peak at 24 hours in group C. The synchronous expression of group B and C were in significant difference (P<0.01). Immunohistochemisty discovered the protein of SP-D expression was increased gradually from 12 hours and reached the peak at 48 hours in group C. The synchronous expression of group B and C were also in significant difference (P<0.01). CONCLUSION: There exists SP-D in rat corneal tissue and the expression is significantly increased at the early period of fusarium solani infected cornea. SP-D may play a role in the early innate immunity response of the corneal resistance to Fusarium solani infection.

Fusarium solani Activates Dectin-1 in Experimentally Induced Keratomycosis

In this study,the response of Dectin-1 on macrophages to Fusarium solani(F.solani)and the expression patterns of Dectin-1 in experimentally F.solani-induced keratomycosis were investigated.Peritoneal macrophages isolated after intraperitoneal injection of sodium thioglycollate were co-cultured with laminarin and spores of F.solani for 12 h.The expression levels of Dectin-1 and CARD9 were detected by immunofluorescence and real-time quantitative polymerase chain reaction.A mouse model of fungal keratitis was established by substromal inoculation with spores of F.solani.Corneal lesions and inflammatory responses were observed by slit-lamp and histopathology at 1,2,3,5,and 7 day(s)after infection.Dectin-1 expression was significantly upregulated on macrophages stimulated by spores of F.solani.Dectin-1 was not detected in normal corneas of C57 BL/6 mice,but detected in infected corneas from the first day after inoculation,with high m RNA levels observed on days 2 and 3.CARD9,a key transducer of Dectin-1 signaling,was also upregulated in infected corneas.In conclusion,Dectin-1 is an important recognition receptor in F.solani-induced keratitis,but the molecular mechanisms warrant further investigation.

Expression of vitamin D receptor and cathelicidin in human corneal epithelium cells during fusarium solani infection

AIMTo observe the expression of vitamin D receptor (VDR) in human specimen and immortalized human corneal epithelium cells (HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of VDR expression. Also, we would like to detect the expression of cathelicidin antimicrobial peptide (CAMP) in the downstream pathway of VDR.METHODSImmunohistochemistry was used to examine the VDR expression in HCEC from healthy and fungal keratitis patients. Real time quantitative polymerase chain reaction (qPCR) was performed to observe the messenger ribonucleic acid (mRNA) change of VDR when immortalized HCEC were challenged with fusarium solani for different hours. CAMP was detected at both mRNA and protein levels.RESULTSWe found out that the VDR expression in fusarium solani keratitis patients' specimen was much more than that in healthy people. The mRNA and protein expression of VDR increased when we stimulated HCEC with fusarium solani antigen (P&#x0003c;0.01) and it could be inhibited by toll like receptor 2 (TLR2) monoclonal antibody. The CAMP expression was decreased because of fusarium solani antigen stimulation (P&#x0003c;0.01).CONCLUSIONThe VDR expression can be increased via TLR2/1-VDR pathway while the CAMP expression is decreased by the stimulation of fusarium solani antigen.

Experimental model of Fusarium solani keratitis in rats

AIM: To establish a repeatable rat model of Fusarium solani keratitis (F. solani keratitis) that mimicked fungal keratitis in humans. METHODS: Wistar rats' corneas were scratched on the superficial stroma after scraping the unilateral corneal epithelia. Then, the corneal surface was inoculated with different inoculum dose of E solanispore suspension. Doses ranged from 10(6) to 10(9) colony-forming unit per milliliter (CFU/mL). The treated corneas were covered by contact lenses that were made of Parafilm M membrane. Negative controls were inoculated with sterile phosphate-buffered saline (PBS). For statistical analysis, corneas were evaluated daily on a 12-point scale to check the state of corneal inflammation. Furthermore, the pathological characteristics of this model were investigated. RESULTS: The rat model of F solani keratitis was established by the combination methods of corneal trauma and parafilm-made contact lens and inoculation of fungus spore suspension. 10(6) and 10(7)CFU/mL of F solani induced mild corneal infection, while 10(8)CFU/mL of F solani was sufficient to induce moderate infection that was consistent with human keratomycosis. Dose of 10(9)CFU/mL of E solani was excessive and led to perforated corneas. CONCLUSION: The rat model of E solani keratitis, established by the combinational methods of corneal trauma, parafilm-made contact lens and the appropriate dose of inoculum, that imitates the developing processes of F solani keratitis in human beings and provides a repeatable method of creating a rat model.

Toxicity of Fusarium solani Strains on Brine Shrimp (Artemia salina)

Discovery of anticancer drugs that must kill or disable tumor cells in the presence of normal cells without undue toxicity is an extraordinary challenge. Cytotoxicity of plant or fungal materials is considered as the presence of antitumor compounds. Brine shrimp lethality for larvae （nauplii） is used as prescreening test for the antitumor compounds. In this study, culture filtrates of eight strains of Fusarium solani isolated from seeds of various crops were tested for the toxic effect on brine shrimp. Five of the strains （TS, S-29, B-17, C-10, W-5） showed highest toxic effect and three of the strains （SR, T-9, L-25） showed low toxic activity on brine shrimp. Toxic activity reduced when culture filtrates were diluted. However, F. solani strains TS, B-17, SR, T-9 and L-25 caused more than 30% mortality at 1：10 dilution. Toxic activity was slightly reduced when the filtrates were neutralized with sodium hydroxide indicating possible role of pH of culture filtrate on toxicity. Lyophilized filtrates of these strains showed less activity as compared to un-lyophilized filtrates, n-Hexane soluble fraction was obtained only in three strains which showed mild toxicity whereas chloroform soluble fraction was obtained in negligible quantity and could not further be proceeded. Toxic effect of these strains showed variation from strain to strain. Compounds from F. solani could be exploited for the development of toxic compounds.

Time-dependent matrix metalloproteinases and tissue inhibitor of metalloproteinases expression change in fusarium solani keratitis

AIM： To investigate matrix metalloproteinases（MMPs）and tissue inhibitor of metalloproteinases（TIMPs）expression during the progress of fusarium solani（F.solani） keratitis in a rat model.· METHODS： A rat model of F.solani keratitis was produced using corneal scarification and a hand-made contact lens. MMPs and TIMPs expressiond were explored in this rat model of F.solani keratitis using real-time polymerase chain reaction（PCR） and DIF.GM6001（400 μmol/m L） was used to treat infected corneas. The keratitis duration, amount and area of corneal neovascularization（CNV） were evaluated.·RESULTS： MMP-3 expression was 66.3 times higher in infected corneas compared to normal corneas. MMP-8,-9,and-13 expressions were significantly upregulated in the mid-period of the infection, with infected- to-normal ratios of 4.03, 39.86, and 5.94, respectively. MMP-2 and-7expressions increased in the late period, with the infected-to-normal ratios of 5.94 and 16.22, respectively.TIMP-1 expression was upregulated in the early period,and it was 43.17 times higher in infected compared to normal corneas, but TIMP-2,-3, and-4 expressions were mildly downregulated or unchanged. The results of DIF were consistent with the result of real-time PCR.GM6001, a MMPs inhibitor, decreased the duration of F.solani infection and the amount and area of CNV.·CONCLUSION： MMPs and TIMPs contributed into the progress of F.solani keratitis.

Genetic Diversity of Fusarium solani f. sp. cucurbitae, the Causal Root and Crown Rot of Cucurbits (Melon) by Using Molecular Markers and Control

Detection of F. solani f. sp. cucurbitae causal agent of the crown and root rot disease of melon race 1, race 2 is difficult. It is based only on morphological characteristic. In this study, forty isolates identified as Fusarium solani based on morphological characterization, F. solani was one of the most frequently isolated species. Molecular identification of these isolates by PCR technique using species-specific primer, indicated that thirty-two isolates, amplified product 580 bp (race 1) and two isolate amplified product 580 bp (race 2), while six isolates were not amplified with primer of both races. Production of Trichothecenes (T2-toxen, DON.) by Fusarium solani was conducted on isolates confirmed as belonging in the F. solani by PCR. The results indicated that the presence of Tri5, Tri13 genes is coding the ability of synthesis mycotoxin. In vitro, the results indicated that NPs (AgNPs, MgNPs) and chemical (Phylex) possess the antifungal properties against at various level. Treatment with (AgNPs 150 ppm, MgNPs 2%, 3% ppm) and 3% Phylex resulted in maximum inhabitation of F. solani . In vivo, five characters (height plant, hoot ant root fresh and dry weight) were measured based on the greenhouse, field experimental results. Treatment with (AgNPs, MgNPs) and Phylex had higher measured parameters than positive control.

Effect of Metabolites of Fusarium Solani on Tyrosine Phosphorylation in Cultivated Cells of Solarium Tuberosum

Elicitor-induced phosphorylation of tyrosine residues in proteins of potato was studied. Proteins of crude extract of suspension culture of potato were analyzed by one- and two-dimensional electrophoresis followed by Western blotting with monoclonal antibodies PY20 to phosphotyrosine proteins. One- and two-dimensional electrophoresis revealed l l and 25 tyrosine-phosphorylated proteins, respectively. Glycoprotein increased the phosphorylation level of most of these proteins.