番茄内生细菌种群动态分析及拮抗菌株的筛选

摘要：采用研磨组织稀释分离法对3个种植地区的番茄内生细菌的种群分布、数量变化规律进行了初步探讨。共分离、纯化得到内生细菌374株,经形态及理化特征鉴定,分属10个不同的属,13个种。其中假单孢菌属Pseudomonas sp.,黄单孢菌属Xanthomonas sp.;肠杆菌属Entenobacter sp.和芽孢杆菌属Bacillus sp.为优势种群。内生细菌在番茄植株种群数量从0.21x104 到13.82x104 cfu.g -1鲜重之间,但不同品种、组织、生育期及种植地,内生细菌的种群和数量各不相同,各组织中内生细菌的种群及数量根中最多,其次是茎和叶,成株期内生细菌无论种类和数量都明显高于苗期。采用平板对峙法从分离的内生细菌中筛选出对番茄灰霉病菌具有体外拮抗活性的菌株32个,占菌株总数的8.56%; 25个菌株对叶霉病菌有抑制作用,占菌株总数的6.68%;对灰霉病菌和叶霉病菌都具有拮抗作用的菌株20个,占菌株总数的5.35%。对灰霉病菌有强拮抗作用的菌株12个,对叶霉病菌有强拮抗作用的菌株仅1个,其中芽孢杆菌是主要的类群。     

海南辣椒尖孢镰刀菌拮抗内生细菌的分离与鉴定

为了获得对辣椒尖孢镰刀菌具有较好防治效果的内生细菌,课题组于2022年1～3月份在海口、定安、澄迈、临高和陵水等海南辣椒主产区采样,并从健康辣椒样品中分离获得内生细菌192株,其中39株对辣椒尖孢镰刀菌(Fusarium oxysporum) CMTT4-1有拮抗作用,分别来源于根系(23株)、茎秆(2株)、叶片(8株)和果实(6株),有拮抗作用菌株占比20.31%,抑菌率为10.68%～59.02%。通过形态学和16S rDNA序列测定及同源性分析,初步鉴定芽孢杆菌属(Bacillus spp.)细菌36株、短杆菌属(Brevibacterium spp.) 1株、农杆菌属(Agrobacterium spp.) 1株和假单胞菌属(Pseudomonas spp.) 1株。研究结果为辣椒枯萎病的生物防治提供了菌株来源。     

柑橘皮内生细菌分离及柑橘青霉病菌拮抗菌筛选研究

柑橘青霉病是柑橘贮藏期的主要病害,为利用拮抗内生菌进行生物防治,本研究选取3个品种柑橘的橘皮,采用研磨液培养法分离纯化得到26株内生细菌。通过对峙培养法,发现5株内生细菌对柑橘青霉病菌有明显拮抗作用,占菌株总数的19.2%。对这5株细菌进行初步鉴定,分属于气芽孢杆菌属（Aerobacillus sp.）、芽孢杆菌属（Bacillus sp.）、气单胞菌属（Aeromonas spp.）、假单胞杆菌属（Pseudomonas spp.）和黄单胞杆菌属（Xanthomonas spp.）5个属。不同的柑橘品种橘皮内的内生拮抗细菌的出现频次不同。抑菌结果表明,培养3天时,H-3抑菌圈直径最大,达到了15.3 mm,H-18抑菌圈最小,只有5.6mm;4天时,各抑菌圈直径均有缩小,但缩小幅度不大;5天时H-3拮抗作用仍十分明显,抑菌圈直径为10.3 mm。根据H-3的菌体形态特征、生理生化测定及其16S rDNA序列分析,鉴定该菌为枯草芽孢杆菌（Bacillus subtilis）,该菌的发现对柑橘青霉病的生物防治有较好的应用价值。     

耐盐碱番茄内生菌的筛选鉴定及功能研究

研究旨在挖掘耐盐碱内生菌资源,为后期开发内生菌菌肥及生防菌剂提供新的菌种资源。以‘金棚8号’番茄为植物材料,通过模拟5种不同类型盐碱环境,分离筛选内生菌,并进行16S rRNA分子生物学鉴定,采用平板法研究其促生和生防功能。研究共获得耐盐碱内生菌14株,其中5株具有解无机磷能力,2株具有解钾能力,2株具有固氮能力,4株具有产生长素的能力;6株具有产铁载体能力,直径比值(D/d)最高为2.95;10株具有产蛋白酶能力,直径比值(D/d)最高为2.97。拮抗试验结果显示,8株耐盐碱内生菌对苹果树腐烂病病原菌有抑制作用,抑菌率为13.70%～68.56%;芽孢杆菌BFG 3-3、BFG 5-1和浅紫链霉菌BFY 2-2可分别对6～7种常见果蔬病原菌产生拮抗,具有广谱抑菌性。本研究获得了具有促生和生防潜力的功能性耐盐碱内生菌菌株,可为应用植物内生菌改良盐碱地提供理论依据。     

棉花根部拮抗枯萎病菌或黄萎病菌的可培养内生细菌多样性分析

为解析棉花内生细菌的种群结构,更好地开发利用生防内生细菌,以棉花枯萎病菌、黄萎病菌为指示病原真菌,对常抗棉和泗棉3号根部分离获得的内生细菌进行拮抗活性筛选,获得87株至少对其中1个指示病原真菌具有拮抗作用的内生细菌。ARDRA分析(Amplified ribosomal DNA restriction analysis)和16S r DNA序列测定表明棉花根部可培养内生拮抗细菌可分为13个ARDRA类群,分属于芽孢杆菌属(Bacillus)、短芽孢杆菌属(Brevibacillus)、伯克霍尔德氏菌属(Burkholderia)、农杆菌属(Agrobacterium)、中华根瘤菌属(Sinorhizobium)、不动杆菌属(Acinetobacter)和假单孢菌属(Pseudomonas)7个属。其中常抗棉根部内生拮抗细菌分属于芽孢杆菌属、短芽孢杆菌属、伯克霍尔德氏菌属和农杆菌属,而泗棉3号根部内生拮抗细菌则分属于芽孢杆菌属、伯克霍尔德氏菌属、中华根瘤菌属、不动杆菌属和假单孢菌属,二者的优势种群都是芽孢杆菌属细菌。BOX-PCR分析显示这些内生拮抗细菌具有丰富的遗传多样性。通过测定这些内生拮抗细菌对棉花黄萎病菌和棉花枯萎病菌的拮抗力大小,以及是否具有产纤维素酶、几丁质酶、蛋白酶和嗜铁素的活性,表明这些内生拮抗细菌具有不同的生防作用机制。     

番茄灰霉病拮抗细菌的筛选

采集番茄土壤、根、叶片和花样本共96份,经平板法和叶盘法两步分离,获得对番茄灰霉病菌（Botrytis cinerea Pers.）有抑制作用的拮抗细菌12株。根据抑菌圈法测定,拮抗细菌对灰霉菌的拮抗能力有显著差异,其中B21菌株抑菌圈直径达35mm,拮抗性最强最稳定。温室试验表明：B21悬浮液和10倍稀释液对番茄灰霉病有较好的防治效果,室内叶片的防效分别为98.68%和98.03%,无显著差异,明显优于45%多菌灵（500倍稀释液）。     

番茄土传病害拮抗菌的筛选、鉴定及拮抗性能评价

土传病害严重影响了番茄种植的产量和经济效益。因此筛选优良的土传病害拮抗微生物用于生物防治对解决此问题具有重要意义。本试验以番茄根际土壤为材料,以4株番茄根腐病致病菌立枯丝核菌、腐皮镰刀菌、尖孢镰刀菌和链格孢菌为对峙菌,筛选得到NSB-26和RSB-6两株具有优良拮抗性能的细菌。经形态学和生理生化鉴定及16 SrDNA序列分析,发现NSB-26为贝莱斯芽孢杆菌、RSB-6为暹罗芽孢杆菌。通过盆栽试验对菌株NSB-26和RSB-6的生防效果进行了验证,试验设置了3个处理:病原菌处理(T1),拮抗菌+病原菌处理(T2)和等量清水处理(CK)。结果表明:与T1和CK处理相比,T2处理番茄幼苗的生长有明显的上升趋势,株高、根长、鲜重、干重均明显提高,T1和T2株高的最高增加幅度分别达10.06%和3.95%;根长的最高增加幅度分别达67.09%和26.48%;处理15天时,植株鲜重的最高增加幅度分别达133.21%和93.20%;植株干重的最高增加幅度分别达126.42%和81.82%。此外,叶绿素a、b含量也显著上升。说明拮抗菌处理可有效减轻土传病害,提高番茄幼苗的抗性,这为番茄根际拮抗菌的进一步开发利用提供了理论依据。     

辣椒内生菌的分离及拮抗菌的筛选与鉴定

用稀释分离法从健康辣椒植株的茎、叶、果肉中共分离得到植物内生菌88株,其中细菌有65株,真菌有21株,放线菌2株.用平板对峙法从中筛选出内生菌003和X两株对辣椒疫霉病菌、辣椒绿霉病菌、辣椒炭疽病菌3种病原菌具有明显拮抗作用的菌株,得出抑制率为34.80％～48.0％.根据菌株形态、生理生化特性对菌株003和菌株X进行了初步鉴定,结果显示菌株003和菌株X都为芽孢杆菌属(Bacillus).     

枯草芽孢杆菌和多黏类芽孢杆菌对西瓜枯萎病的防治效果优于化学药剂百菌清

<正>南京师范大学生命科学学院和江苏省农业科学院植物保护研究所科研人员以西瓜枯萎病菌为靶标菌,通过平板对峙法和生长速率法从355株根围细菌及植物内生细菌中筛选出7株活菌或胞外代谢产物拮抗作用强的细菌,采用盆栽试验和田间试验,研究了其对西瓜枯萎病的防治效果。盆栽试验表明,这7株强拮抗细菌对西瓜枯萎病都有一定的防治效果,其中菌株Jaas ed1、Jaas ed2、Jaas ed3对西瓜枯萎病的防治效果优于化学药剂百菌清,防效分别为75.1%、     

鼓槌石斛内生细菌分离、 鉴定及功能分析

为微生物农药和肥料生产提供菌种资源,用组织分离法从鼓槌石斛健康组织分离内生细菌,用对峙法和抑菌圈法筛选拮抗菌株;以平板法筛选解磷、解钾和固氮菌株;以田间试验检验固氮菌株的增产效果;用PCR扩增功能菌株的16S rDNA,结合菌株的菌体、菌落形态特征和部分生理生化特征,确定功能菌株分类地位。结果表明,从鼓槌石斛组织共分离到33株内生细菌,数量和种类为根〉茎〉叶;其中GB7属解淀粉芽孢杆菌（Bacillus amyloliquefaciens）,GB16属链霉菌（Streptomyces sp.）,GB8、GB9、GB21属芽孢杆菌（Bacillus spp.）,均有病害生防功能;GB2属不动杆菌（Acinetobacter calcoaceticus）,GB20属肠杆菌（Enterobacter sp.）,具解磷功能;菌株GB1属产酸克雷伯氏菌（Klebsiellaoxytoca）,具固氮功能。     

Screening for new sources of resistance to Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato

Bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), is considered the most serious bacterial threat, resulting in high damages in production areas. Worldwide, Cmm is subjected to quarantine regulations.There is no cultivar in market containing Cmm resistance genes. This project aimed to screen tomatoes or wild relatives of tomato for resistance to Cmm, to be used for starting breeding programs. We have screened 24 different wild accessions of tomato and found several new tolerant sources: Solanum pimpinellifolium GI.1554, S. parviflorum LA735 and S. parviflorum LA2072. We also confirmed the tolerance which was reported previously in S. peruvianum LA2157, S. peruvianum PI127829, S. peruvianum LA385, S. habrochaites LA407 and S. lycopersicum cv. IRAT L3. No immunity was found. Also accessions showing a low disease score still contained high titers of bacteria as determined by a dilution plating method, using tow selective media. These results were confirmed with a TaqMan real time PCR assay, which was developed to determine and quantify Cmm in planta.     

Screening of Tomato Somaclones for Resistance to Bacterial Canker (Clavibacter michiganensis subsp. michiganensis)

To facilitate testing large numbers of tomato soma-clones for resistance to bacterial canker, a fast screening procedure was developed. Two inoculation methods were investigated, i.e. excision with an infected scalpel of I) one cotyledon of 2-week-old seedlings, and II) the first true leaf of 3-week-old seedlings, followed by applying a drop of inoculum on the wound. Inoculation by method II discriminated well between populations of partially resistant and susceptible tomato genotypes. A criterion for the selection of single, putatively resistant plants, based on rating scores of the severity of wilting symptoms, was proposed and tested with plants of a Lycopersicon peruvianum accession with a relatively high level of resistance to bacterial canker. Progenies of 279 somaclones, derived from tissue explants of the susceptible tomato cultivar ‘Moneymaker’, were evaluated for resistance. The presence of somaclonal variation for morphological characters in these populations was previously shown. However, no differences were detected between the somaclone population and controls in either mean susceptibility or distribution of plants over disease severity categories. Moreover, plants with major increases in resistance were not detected. These results suggest that the potential of somaclonal variation as a source of resistance to bacterial canker may be limited.     

海带石油醚相活性物质对番茄溃疡病菌的抑菌机理

旨在揭示海带活性物质抑制番茄溃疡病菌的作用机理,为绿色抑菌剂的开发提供理论依据。以活性追踪法获得海带抗菌成分,从扫描电镜、膜电位、活性氧、胞内蛋白、细胞周期多角度进行机理研究。追踪检测结果表明,海带石油醚萃取相抑菌效果最佳(P<0.05),其抑菌圈直径为26.00 mm。抑菌机理研究结果表明,海带活性物质作用后番茄溃疡病菌细胞形态发生明显变化、膜两侧电位与对照组相比下降可达17.49%~56.60%、胞内活性氧含量增长1.19~3.98倍、胞内蛋白含量显著减少(P<0.05)、处于细胞分裂期的番茄溃疡病菌占比明显降低。研究结果揭示出海带活性物质主要作用病菌细胞膜,造成自由基累积、阻滞正常传代,最终达到杀灭病菌的效果。     

Immunity to Clavibacter michiganensis subsp. sepedonicus: Screening of exotic Solanum species

Summary Accessions from exotic Solanum species, including diploid and tetraploid species, were screened for immunity to Clavibacter michiganensis subsp. sepedonicus, the causal agent of potato ring rot. The diploid species included S. infundibuliforme, S. lesteri, S. megistacrolobum, S. tuberosum Group Phureja, S. polyadenium, S. pinnatisectum, S. raphanifolium, S. sparsipilum, S. sanctae-rosae, S. tuberosum Group Stenotomum, S. toralapanum, and S. verrucosum. The tetraploid species included S. tuberosum Group Andigena, S. acaule, S. fendleri, S. hjertingii, S. oplocense, S. polytrichon, and S. stoloniferum. Apparent immunity was initially found in several diploid species, but was not present during subsequent retesting. Immunity was found in nine accessions of tetraploid S. acaule. These accessions maintained their immunity during testing over an eight-month period. S. acaule appears to be a good source of immunity for introgression studies.     

Immunity to Clavibacter michiganensis subsp. sepedonicus: Inheritance of immunity in Solanum acaule

Summary Bacterial ring rot of potato (Solanum tuberosum) has not been successfully controlled through management of certified seed. Therefore, the identification of immunity to this pathogen and its introgression into the cultivated potato is vitally important to the potato industry. Immunity was detected in the disomic tetraploid 2EBN (Endosperm Balance Number) species S. acaule. Immune and nonimmune parents were crossed in a 4 × 4 mating scheme that consisted of four immune × immune crosses, four immune × nonimmune, four nonimmune × immune, and four nonimmune × nonimmune crosses. Analysis was performed on the 16 F₁ populations and 54% of the progeny was found to exhibit an immune response to inoculation with Clavibacter michiganensis subsp. sepedonicus (Spieck & Kotth.) Davis et al. Immunity appears to be conferred by two dominant alleles, one at each locus, and may be associated with minor or modifying genes. Phenotypic expression of nonimmune progeny ranged from susceptible to resistant, probably due to minor or modifying genes. These results indicate that it may not be difficult to transfer immunity into the cultivated potato.     

内蒙古地区番茄溃疡病菌PCR快速检测技术及22个番茄品种种子表面带菌检测

为快速检测番茄种子表面的带菌情况,以便及时有效防控番茄溃疡病,从而减少病害损失。试验用细菌基因组DNA试剂盒提取番茄溃疡病的总基因组,用通用引物进行PCR反应。以番茄溃疡病菌的DNA为模板,使用专一性引物进行PCR扩增,通过对专一性引物的筛选,最终确定了Cla F1-Cla F2为快速检测番茄溃疡病菌PCR的特异性引物。该检测方法特异性强、灵敏度高,模拟种子带菌,对浸种水的检测灵敏度达1×105CFU/m L。经PCR快速检测,22个番茄品种中只有黑贝番茄、粉红番茄和保罗塔带有番茄溃疡病菌,其余19个品种均未带菌。该方法直接对种子进行检测,不需进行病原菌分离培养,快速简便。