DNA methylation patterns of banana leaves in response to Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism （MSAP） technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.

Construction of PEG-mediated Genetic Transformation and Gene Knockout System in Fusarium oxysporum f. sp.cubense Tropic Race 4

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropic race 4(Foc TR4), is a typical vascular and soil-borne disease which has significantly threatened the sustainable development of banana industry. In order to reveal the infection process and pathogenesis of Foc TR4, the young mycelia(66.7 mg/ml) of wild-type strain of Foc TR4(WT-Foc TR4) cultured for 18-20 h were lysed with enzyme mixture for protoplast formation, which consisted of 25 mg/ml driselase, 0.4 mg/ml chitinase, 15 mg/ml lysing enzyme and 1.2 mol/L potassium chloride. The resulted protoplasts of 2×10~7 cells/ml were used to test the efficiency of transformation mediated by polyethylene glycol, and up to 9 transformants per microgram of DNA were obtained. AmCyan, RFP and YFP genes were stably transferred into the WT-Foc TR4, separately, using the protoplast transformation system. The gene FoOCH1 encoding α-1, 6-mannosyltransferase in the WT-Foc TR4 was knocked out using the split-marker recombination technology. The genetic transformation and gene knockout system in this pathogen lays a foundation for the study of functional genomics and plant-pathogen interactions.

Study on Control Effect of Invasive Banana Fusarium Disease(Fusarium oxysporum f.sp.cubense tropical race 4)by Different Varieties and Fertilization Treatments

[Objective]The paper was to understand the field resistance performance of different banana varieties and the prevention and control ef-fect of different fertilizers,so as to provide technical reference for the prevention and control of Fusarium wilt.[Method]Field trials were set up with three treatments:shrimp peptide organic fertilizer+shrimp peptide special protection+shrimp peptide fruit Yekang(simplified as shrimp peptide organic fertilizer treatment),conventional organic fertilizer+microbial preparations(simplified as microbial treatment),and conventional or-ganic fertilizer(simplified as control).Four different banana varieties of Brazilian banana,Guijiao No.1,Nantianhuang,and Yunjiao No.1 were se-lected for the field trial.The disease incidence of Fusarium wilt and the control effects of three fertilizers were investigated during four time periods.[Result]The disease incidence of four varieties in three treatments varied.The disease incidence of Nantianhuang and Yunjiao No.1 were signifi-cantly lower than that of other two varieties.There was also significant difference in disease incidence of three treatments.The disease incidence from high to low was control>shrimp peptide organic fertilizer treatment>microbial treatment.The average monthly TR4 pathogen content in heavily infected banana plantation was more than 2000 copies,while the highest one reached 15148.9 copies.[Conclusion]Microbial agents reduced the disease incidence of Fusarium wilt to some content.Nantianhuang and Yunjiao No.1 showed the highest disease resistance compared with other varieties.However,their resistance needs to be further improved before practical application.

Isolation and identification of Fusarium oxysporum f. sp. cubense in Fujian Province, China

Fusarium wilt, caused by Fusarium oxyporum f. sp. cubense(Foc), is the most serious disease affecting banana production.To clarify the distribution of the Foc races in Fujian Province of China, 79 soil samples were collected from four regions of Zhangzhou City, the primary banana production area in Fujian. We isolated and identified 12 Foc strains based on internal transcribed spacer(ITS) sequence analysis, PCR amplification by using Foc-specific primers and pathogenicity assays.Our analysis indicated that 11 isolates belong to Foc race 1, and 1 isolate belongs to the Foc tropical species race 4(TR4).Although TR4 has previously been reported to occur in primary banana-producing provinces, such as Hainan, Guangxi,and Guangdong of China, this is the first report of TR4 isolated from the soil in Fujian Province. Monitoring the presence of Foc, in particular, the TR4 strains in the soil, is the basic strategy to prevent and control Fusarium wilt.

Isolation and Identification of Shoot-Tip Associated Endophytic Bacteria from Banana cv. Grand Naine and Testing for Antagonistic Activity against <i>Fusarium oxysporum</i>f. sp. <i>cubense</i>

Endophytic bacteria colonizing the shoot-tips of banana cv. Grand Naine were isolated and tested for the antagonistic activity against the Panama wilt pathogen Fusarium oxysporum f. sp. cubense (Foc). Pre-isolation, the suckers were given extensive disinfection treatments and the homogenate from the excised shoot-tip portion was plated on nutrient agar (NA) and trypticase soy agar (TSA). This yielded altogether 47 isolates: 26 on NA and 21 on TSA, respectively, from the 10 suckers collected during August to February. The number of bacterial isolates obtained per sucker varied from one to 15 based on colony characteristics registering up to 10 distinct species per shoot-tip based on 16S rRNA sequence analysis. The 47 isolates belonged to 19 genera and 25 species under the phylogenetic classes of Actinobacteria, α- and γ-Proteobacteria and Firmicutes. Actinobacteria constituted the predominant phylum (55% isolates) with the constituent genera of Arthrobacter, Brevibacterium, Corynebacterium, Curtobacterium, Kocuria, Kytococcus, Micrococcus, Naumanella, Rothia and Tessaracoccus spp. and an unidentified isolate belonging to the family Frankiaceae. Proteobacteria constituted the second major phylum (Brevundimonas, Enterobacter, Klebsiella, Pseudomonas, Serratia and Sphingomonas spp.) followed by Firmicutes (Bacillus and Staphylococcus spp.). Antagonistic activity of the endophytes against Foc was tested through agar plate assays (pit and spot applications on fungal lawn) employing potato dextrose agar and NA. Endophytic Pseudomonas aeruginosa (isolate GNS.13.2a) which was associated with a single sucker showed significant growth inhibition effect on Foc while Klebsiella variicola (GNS.13.3a) and Enterobacter cloacae (GNS13.4a) exhibited moderate inhibition. The study brings out considerable sucker to sucker variation in the associated cultivable endophytic bacteria in “Grand Naine” banana and identifies a few bacterial endophytes with biocontrol potential against the devastating Foc pathogen.

Comparative study of <i>Fusarium oxysporum</i>f sp. <i>lycopersici</i>and <i>Meloidogyne incognita</i>race-2 on plant growth parameters of tomato

Many species of soil-inhabiting fungus Fusarium, cause severe yield loss in many crops. Experiments were conducted in net house condition with complete randomized block design to determine the individual effect of different in-oculum levels of root-knot nematode, Meloidogyne incognita, Race-2 and Fusarium oxysporum f sp. lycopersici on plant growth parameters viz., Plant length, fresh and dry weight and number of fruits of tomato var. P21. The experimental results showed that both the pathogens cause significant reduction in plant growth parameters. However, the fungus was not much effective on plant growth parameters in comparison to root-knot nematode. Greatest reduction in plant growth parameters was recorded in plants inoculated with 8000 J2/Kg soil of Meloidogyne in-cognita race 2. The threshold level of root-knot nematode was 1000 J2/kg soil while threshold level of Fusarium was @ 1 g/Kg soil. Inoculum level of Fusarium oxysporum f sp. lycopersici and Meloidogyne in-cognita race-2 was pathogenic and caused significant reduction at and above 1 g/kg soil and 1000 J2/Kg soil respectively.

A study on the pathogen species and physiological races of tomato Fusarium wilt in Shanxi,China

In order to clarify the main pathogens of tomato Fusarium wilt in Shanxi Province, China, morphological identification, elongation factor 1 alpha （EF-1α） sequence analysis, specific primer amplification and pathogenicity tests were applied to study the isolates which were recovered from diseased plants collected from 17 different districts of Shanxi Province. The results were as follows： 1） Through morphological and molecular identification, the following 7 species of Fusarium were identified： F. oxysporum, F. solani, F. verticillioides, F. subglutinans, F. chlamydosporum, F. sporotrichioides, and F. semitectum; 2） 56 isolates of F. oxysporum were identified using specific primer amplification, among which, 29, 5 and 6 isolates were respectively identified as F. oxysporum f. sp. lycopersici physiological race 1, race 2, and race 3; 3） pathogenicity test indicated the significant pathogenicity of F. oxysporum, F. solani, F. verticillioides, and F. subglutinans to tomato plant. Therefore, among these 4 species confirmed as pathogenic to tomato in Shanxi, the highest isolation rate （53.3％） corresponded to F. oxysporum. Three physiological species, race 1, race 2, and race 3 of F. oxysporum f. sp. lycopersici are detected in Shanxi, among which race 1 is the most widespread pathogen and is also considered as the predominant race.

香蕉枯萎病菌4号生理小种致病相关基因foABC1的分离

通过对香蕉枯萎病菌4号小种致病突变体B1233的进一步研究,分离了被突变的致病相关基因foABC1,同源性分析及保守结构预测该基因编码一类ABC转运蛋白,其功能可能同稻瘟病菌的ABC转运蛋白一样,负责真菌毒素的泵出,或是像其他真菌的ABC转运蛋白,在病原菌侵染寄主植物时能忍耐植物因防卫反应所释放的植保素或抗毒素类物质。

香蕉枯萎病菌4个生理小种核糖体DNA内转录间隔区的RFLP分析

【目的】明确香蕉枯萎病菌Fusarium oxysporum f.sp.cubense 4个生理小种的系统发育关系。【方法】利用PCR扩增该病菌4个生理小种的核糖体DNA内转录间隔区(Internal transcribed spacer,ITS),利用AIu I、Hpa II和Taq I 3种限制性内切酶对这些小种的ITS产物进行RFLP分析。【结果】PCR扩增结果表明:来自4个生理小种的8个菌株均可扩增得到568 bp的ITS单一片段,但这些生理小种间的片段没有明显的多态性;对这些生理小种ITS产物进行RFLP分析发现,不同生理小种之间的差异很小。【结论】PCR-RFLP技术不适用香蕉枯萎病菌生理小种的分析鉴定。

香蕉枯萎病4号小种T-DNA插入突变体Focr4-1453表型特征及致病性分析

尖孢镰刀菌古巴转化型是引起香蕉枯萎病的主要病原菌,呈世界多态性分布,其致病机理尚不清楚。Focr4-1453为T-DNA插入突变体库中筛选出的一株与致病性相关的突变体。在克隆出其失活基因之后,将野生型菌株中的相同基因进行敲除和互补,得到敲除突变体ΔFoc4-1453和互补突变体ΔFoc4-1453-cp-1。对获得的3株突变体进行生物学表型研究发现,这些菌株的菌落生长速度、产孢能力以及孢子萌发率较野生型菌株明显降低,且无法透过玻璃纸进行生长。致病性测定试验表明,该基因失活会导致病原菌侵染能力显著减弱。

Identification of Biocontrol Strain HJX1 and Determination of Its Antifungal Spectrum

A biocontrol strain HJX1,which had good inhibitory effect against Fusarium oxysporum f. sp. cubense,was identified as Bacillus subtilis based on traditional morphology,16 S rRNA gene sequence and fatty acid profiles. The sequence of 16 S rRNA gene had 99% homology to B. subtilis according to the DNA fragment of 1 521 bp amplified with a pair of universal primers,P0 and P6,of bacteria. Confront culture with pathogenic fungi showed that this strain had good inhibitory effect against 10 pathogenic fungi.

河北省西瓜枯萎病菌生理小种鉴定与AFLP分析

【目的】明确河北省西瓜枯萎病菌生理小种类型和遗传多样性,为西瓜抗病育种和西瓜枯萎病的综合防治提供依据。【方法】本文对采自河北省12个西瓜种植地区的46个西瓜枯萎病菌菌株和4个已知生理小种的菌株进行了致病性测定和AFLP分析。【结果】根据菌株对3个鉴别寄主表现出的致病性强弱,将河北省46个西瓜枯萎病菌菌株划分为3个不同的生理小种,即0号、1号和2号,分别占供试菌株的17.4%、65.2%和17.4%,其中1号生理小种为优势小种,分布在河北省所有西瓜种植区。21对AFLP引物对供试菌株扩增出1157条带,其中多态性带389条,占总带数的33.6%。河北省西瓜枯萎病菌的遗传距离变化在0.33～0.95之间,平均为0.66,群体遗传多样性比较丰富。基于AFLP标记聚类分析表明,50个菌株被划分为3个类群(AFLP Groups,AGs)。AGI包含4个菌株,均为2号生理小种;AGII包括4个菌株,均为0号生理小种;AGIII包括42个菌株,以1号生理小种为主(32个),占该类群的76.2%。【结论】河北省西瓜枯萎病菌存在明显的致病性分化,其AFLP类群划分与致病性鉴定划分的生理小种之间存在一定相关性,但与菌株的地理来源无关。

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。

香蕉枯萎病菌1号和4号生理小种的快速检测与鉴定

【目的】从香蕉枯萎病菌1号和4号生理小种特有的基因序列入手,建立一种快速可靠的分子检测技术,为防止香蕉枯萎病的传播蔓延、尽早采取防治对策、指导香蕉生产进行品种配置提供理论依据。【方法】根据研究室已经筛选到的4号生理小种候选致病相关基因序列设计引物,分别以来自海南、广东和广西的6个香蕉枯萎病菌1号生理小种菌株、7个4号生理小种菌株、7个尖镰孢其它专化型菌株以及2个外围菌株DNA为模板进行PCR扩增,筛选香蕉枯萎病菌1号、4号生理小种特异性引物及尖镰孢菌的通用引物。【结果】筛选到的特异性引物不仅可用于香蕉枯萎病菌DNA的检测,还可直接用于对罹病香蕉组织和土壤中的香蕉枯萎病菌的检测;筛选到的尖镰孢菌通用引物,可作为内参照以检测DNA的质量,以避免假阴性情况的出现。【结论】所建立的三重PCR检测方法实现了在一次PCR反应中快速、准确地同步检测香蕉枯萎病菌1号和4号生理小种,对检测香蕉苗是否感染枯萎病及蕉园土壤是否受到香蕉枯萎病菌的污染具有重要意义。

海南省香蕉枯萎病病原菌的分离鉴定及1号、4号小种的生物学特性

香蕉枯萎病是目前国内和世界很多地区香蕉种植区的一种毁灭性病害,目前尚未找到有效的防治方法。本文采集了海南省不同地区香蕉枯萎病的病原菌,通过经典病理学与分子生物学的方法进行了鉴定,此外还优化了快速检测的方法。结果表明,通过接种巴西香蕉和粉蕉进行致病性检测以及分离菌rDNA-ITS测序结果,明确鉴定出供试的香蕉分离菌株为4号生理小种,粉蕉分离菌株为1号生理小种。为了对1号、4号生理小种有更多的了解,本文还对1号、4号小种菌株生物学特性进行了测定,确定了影响菌丝生长的最佳条件。结果显示,pH5时最适合1号、4号生理小种的菌丝生长,适合菌丝生长的温度为19～28℃之间,温度达到37℃时菌丝不能生长,碳源对菌丝的生长影响不大。这些方法和数据可以有利于田间香蕉枯萎病病害的检测和最终确定,生物学特性的测定可以为耕作技术的改进和农药的研发提供参考依据。

农杆菌介导的香蕉枯萎病菌4号生理小种转化体系的优化

香蕉枯萎病是世界范围内香蕉种植区最为严重的病害之一,严重威胁和影响着香蕉产业的发展。本文针对香蕉枯萎病病原菌的4号生理小种,建立了农杆菌介导的转化体系,确定了影响转化效率主要因子的优化体系是:农杆菌在IM培养基诱导前农杆菌OD600为0.15、农杆菌经IM液体培养基诱导的时间为7h、乙酰丁香酮(AS)浓度为150μmol/L、Focr4孢子浓度为1×106个/mL、共培养时间为48h、培养温度为25℃、诱导培养基pH值为5.5。在此条件下,转化效率能达到700～800个转化子/106个香蕉枯萎病菌孢子。PCR验证表明外源的T-DNA已经成功随机地整合到该病原菌基因组中。目前,应用该转化体系已获得2300多个转化子,为后续克隆相关致病基因打下了良好基础。

利用ITS1和ITS4通用引物扩增香蕉枯萎病菌核酸片段鉴定其生理小种

利用真菌核糖体rDNA区通用引物ITS1和ITS4,扩增采集自香蕉的13株镰刀菌包括18S rDNA部分序列、ITS1-5.8S-ITS2全部序列和28S rDNA部分序列的片段,通过BLAST序列比对分析,确定13株菌为尖孢镰刀菌[Fusarium oxysporum]。采用最大简约法,以层出镰刀菌[F.proliferatum(EU151490)]和茄病镰刀菌[F.solani(GQ376116)]为外群,将13株菌的序列与BLAST检索获得的尖镰孢古巴专化型(F.oxysporum f.sp.Cubense)相应序列构建了系统发育树。系统发育分析结果显示,13株菌与NCBI登录的4个尖镰孢古巴专化型菌株一起被分成3个亚群,亚群聚类结果与回接鉴定结果及文献报道的生理小种结果完全一致。

抗香蕉枯萎病放线菌的筛选及菌株DA07408的鉴定

从土壤样品中共分离放线菌398株,用纸片法对其进行抗古巴尖孢镰刀菌4号生理小种活性筛选,结果发现共有8株具有活性,其中分离于海南儋州植物园土壤的菌株DA07408活性最强。经形态与生理生化特征、16S rDNA序列测定及其系统发育分析,将菌株DA07408初步鉴定为橄榄产色链霉菌Streptomyces olivochromogenes。

香蕉与枯萎病菌4号小种互作过程中防御酶活性的变化

为探讨香蕉被枯萎病菌4号小种(Fusarium oxysporum f.sp.cubense race 4)侵染后防御酶活性的变化规律,采用伤根浸孢子液接种法,对粉蕉(ABB)和巴西蕉(AAA)的香蕉苗进行人工接种试验,测定香蕉苗与枯萎病菌互作过程中苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)5种防御酶的活性变化。结果表明:接种的香蕉苗体内PAL、POD、PPO和SOD酶活性都高于未接种的对照香蕉苗,且各种酶活性变化也比未接种的变化复杂,表明这4种酶在枯萎病菌侵染过程中积极参与了香蕉苗体内的抗病反应;但接种与未接种的香蕉苗体内CAT活性变化大致相似,表明CAT活性的变化与枯萎病菌的侵染无关。

尖孢镰刀菌古巴专化型4号生理小种红色荧光蛋白基因转化

采用PEG-CaCl2介导的原生质体转化体系,成功地将红色荧光蛋白基因DsRed转入到尖孢镰刀菌古巴专化型4号生理小种B2菌株中。在显微镜下观察连续转接6代的转化子,结果都可以观察到很强的红色荧光,表明DsRed基因在转化的B2菌株中能稳定遗传。通过PCR验证,也表明红色荧光蛋白基因DsRed已经转入到尖孢镰刀菌中。对香蕉的致病力测定结果表明,转化子与野生型菌株B2相比没有明显差异。