14株芽胞杆菌菌株的分子鉴定及其促生机理分析

以前期筛选的14株植物促生细菌为实验材料,运用16S rRNA基因序列同源性对菌株进行分子鉴定,通过生物测定评价菌株对番茄的促生效果,并对其促生机理进行初步分析。实验结果表明,促生菌株来自芽胞杆菌科的2个属、9个种。对番茄种子促生特性研究表明,14株芽胞杆菌均能提高种子发芽率,增长率为0.74%~11.85%;其中12株芽胞杆菌能促进胚根生长,增长率为4.03%~65.33%;而8株芽胞杆菌促进胚芽生长,增长率为1.24%~18.17%。对不同芽胞杆菌的促生因子相关分析结果表明,芽胞杆菌的6个促生因子（解无机磷、解有机磷、固氮效能、吲哚-3-乙酸含量、植酸酶活性、吡咯喹啉醌浓度）之间无相关关系（P>0.05）,与活菌数无相关关系（P>0.05）,与种子发芽率、胚根长、胚芽长也无显著相关关系（P>0.05）。种子发芽率与胚根长、胚芽长无相关关系（P>0.05）,而胚根和胚芽长之间具有显著性相关（P<0.01）。室内番茄盆栽实验表明,试验的6株芽胞杆菌对番茄幼苗均有促生作用,不同芽胞杆菌对番茄促生特性存在差异,如图瓦永芽胞杆菌FJAT-47648能显著增加根长,提升地上部与地下部干重,而暹罗芽胞杆菌FJAT-47226的促生效果则主要表现在增加株高和地上部生物量上。     

生防细菌的抑菌谱和对大豆根腐病的防治

用8株生防细菌对15种病原真菌进行室内平板对峙试验,试验结果表明8株生防细菌都具用较广的抑菌谱,其中B021a、B072b对14种病原真菌具有较宽抑菌带,B09、B131对13种病原真菌具有较宽抑菌带。抑菌率为15.6％～67.5％。对大豆根腐病病原菌尖孢镰刀菌（Fusarium axyspo-rum）和茄腐镰刀菌（Fusarium solani）分别进行盆栽试验。防效最高为m4b（F．axysporum）达62.7％和mg（F.solani）达60％。各生防菌株对大豆种子发芽均有一定促进作用,同时在种子发芽过程中还能抑制尖孢镰刀菌的生长。田间试验大豆根腐病防效34.6％～47.6％,各菌株对大豆植株有一定的促生作用。     

一株兼具防病促生功能的沙阿霉素链霉菌Sz-11

【目的】评估沙阿霉素链霉菌Sz-11的促生能力和对水稻BLS的生防潜力，探究其初步的抑菌机制。【方法】用牛津杯法测定Sz-11对7种植物病原细菌和11种植物病原真菌的抗菌谱。通过浸种和浇灌试验评估Sz-11的促生潜力。SDS-PAGE电泳、琼脂糖凝胶电泳、CLSM、SEM、FTIR分析发酵液中的活性产物对Xooc蛋白质表达、基因组DNA合成、细胞膜通透性、细胞形态、细胞膜表面物质结构和组成的影响。4个水稻品种的盆栽防效试验，初步明确Sz-11对BLS的生防潜力。【结果】Sz-11对番茄叶斑病菌、苦瓜枯萎病菌、西瓜炭疽病菌、烟草赤星病菌等多种植物致病真菌都具有较好的拮抗活性，对水稻黄单胞菌（Xoo和Xooc）具有突出的拮抗作用。Sz-11能够促进水稻种子萌发生长，缩短萌发周期，加速营养生长期水稻有机质的积累和根系的分化。Sz-11能影响Xooc蛋白表达，改变细胞膜透性和膜表面物质组成，使细胞溶胀、变形、死亡，但不影响Xooc基因组DNA合成。盆栽试验显示，Sz-11对4个水稻品种BLS相对防效高达65.63%～84.38%，且提前预防效果优于发病后治理。【结论】Sz-11具有较广的抗菌谱...     

长春花接种丛枝菌根真菌对柑橘黄龙病菌的抑制效果

通过筛选获得具有显著促生和抗病能力的AM真菌菌株,为防治柑橘黄龙病提供理论依据和基础,并探索防治新途径。采用盆栽法,于长春花幼苗期分别接种17株AM真菌进行长春花农艺性状和对柑橘黄龙病的抗病性比较试验,研究不同丛枝菌根真菌菌株对长春花的促生效果及对柑橘黄龙病的抑制效果。结果表明,长春花接种AM真菌后,菌株GZ10、GZ5和JX70能显著促进植株生长,其中接种GZ10的株高和茎径分别比CK增加了23.1%和12.1%,接种GZ5的叶片数比CK增加了12.9%;接种菌株GZ1、BNMJ和PWJ有效提高了长春花对柑橘黄龙病的抗病和耐病能力,其相对防效分别为78.39%、66.87%和69.75%。     

2株野生大豆根际促生菌抑菌促生作用研究

为进一步阐明野生大豆根际促生菌(PGPR)的生物学活性作用,分别采用滤纸片法、水培、沙培或盆栽的方法,测定植物幼苗的生长量(株高、根长、地上部分和地下部分干重)、叶绿素的含量及大豆的结瘤情况。探究2株PGPR(DB17、DB58)的生防作用及对水稻、大豆幼苗生长的影响。结果表明:DB17对6种植物病原真菌具有抑菌活性,其中对甜瓜枯萎菌的抑菌率可达37.10%;2株野生大豆PGPR菌株对水稻幼苗有显著的促生长作用,可使幼苗总叶绿素含量提高29%~79%;2株野生大豆PGPR菌株可以提高大豆根瘤质量;DB58和混菌(DB17+DB58)可显著促进大豆幼苗的生长。     

促生、诱抗植物病原蛋白质的研究进展与展望

对促生、诱抗植物病原蛋白质的类型、结构和性质、促生诱抗的特点和作用机理进行了介绍,并对发展前景进行了探讨.     

文心兰组培苗与野生兰科植物内生真菌共培养的生长效应研究

将从野生华石斛、铁皮石斛、美花石斛及五唇兰根系中分离出的28种有益内生真菌,接种于文心兰组培瓶苗上进行共生培养,测定共生培养4个月后瓶苗的生长效果,以及瓶苗移出盆栽3个月后的生长变化规律。结果表明:(1)在组培瓶苗上筛选出8株对文心兰具有促生作用的内生真菌,其中1株属华石斛内生真菌,1株属铁皮石斛,3株属美花石斛,3株属五唇兰;(2)接种了促生真菌的瓶苗移出盆栽后也同样表现出促生效果。表明兰科促生真菌不存在严格的专一性,但对兰科不同种类的促生效应存在一定的差异。     

橡胶树枝条内生真菌的分离及其拮抗性的测定

经过严格的表面消毒程序,从橡胶树枝条中分离到18株内生真菌,并对其进行初步鉴定.结果表明,这18株内生真菌分别归属于2个目,2个科,7个属,其优势菌群为无孢菌群(Mycelia Sterilia).通过温度测定,确定橡胶树内生真菌最适生长温度为25～28℃:采用平板对峙培养法,研究内生真菌对香蕉枯萎病菌(Fusarum pxysporum f.sp.cubense)和橡胶炭疽病菌(Colletorichum gloeosporioides Penz.Sace)的抑菌作用.结果表明,ITBB2-2对两种植物病原菌的拮抗作用最好.ITBBBB2-10和ITBB2-15次之,ITBB2-3对香蕉枯萎病菌拮抗作用明显而对橡胶炭疽病菌拮抗作用不明显,其它内生真菌对两种植物病原菌无拮抗作用或不明显.     

一株拮抗香蕉枯萎病菌的细菌HN-1的鉴定和拮抗作用的测定

在香蕉枯萎病园区中,从土壤中分离获得一株细菌HN-1,并进行对峙培养、孢子萌发抑制测定。结果表明：HN-1对香蕉枯萎病菌的菌丝生长、孢子萌发具有良好的抑制效果。经形态特征、生理生化特征和16S rDNA序列测定和分析,将HN-1鉴定为短短芽孢杆菌(Brevibacillus brevis)。与病原真菌对峙培养的结果表明,该菌株对供试的15种植物病原真菌均有很好的抑制作用。     

山竹果皮提取物农药活性研究

探索山竹果皮提取物在农业中的杀虫抗菌活性,为新型植物源农药的开发提供理论依据。采用浸叶法测定山竹果皮乙醇、石油醚、氯仿、乙酸乙酯及正丁醇等5种提取物对斜纹夜蛾3龄幼虫拒食和毒杀活性;以生长速率法测定这5种提取物对12种植物病原真菌的抗菌活性。结果显示：山竹果皮提取物对斜纹夜蛾3龄幼虫均具有一定的拒食及毒杀作用,以氯仿提取物的拒食活性最高,其24和48 h拒食率分别为72.51%和65.75%;而毒杀作用以石油醚提取物最强,其处理5 d后,斜纹夜蛾的校正死亡率为53.70%。抑菌试验显示山竹氯仿提取物对芒果炭疽病菌、水稻纹枯病菌、番茄灰霉病菌和芒果蒂腐病菌等4种植物病原真菌具有明显抑制菌丝生长作用,其EC50值分别为0.230 5、0.429 3、0.265 2和 0.390 1 mg/mL。由此说明,山竹果皮提取物具有良好的杀虫抗菌活性,基于山竹产物开发新型植物源农药具有潜在的利用价值。     

拮抗香蕉枯萎病菌的解淀粉芽孢杆菌LX1菌株的鉴定及其抗菌蛋白基因的克隆

收集海南不同盐碱地的土样进行拮抗香蕉枯萎病菌的微生物分离,对具有较强抑菌作用的菌株进行形态特征、生理生化和分子鉴定,并克隆其抗菌蛋白基因.结果分离到1株具有较强抑制香蕉枯萎病菌能力的细菌LXl,经鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).抑菌实验发现LX1菌株的发酵上清液中粗蛋白有一定的抑菌作用,经过Sephacryl S-200HR柱层析、DEAE Sepharose Fast Flow离子交换层析分离纯化了其中抑菌作用最强的抗菌蛋白.质谱鉴定结果表明,抗菌蛋白与B.amyloliquefaciens FZB42内切葡聚糖酶同源性最高.根据质谱结果克隆了该抗菌蛋白的编码基因,该基因的核苷酸和氨基酸序列与B.amyloliquefaciens FZB42的内切葡聚糖酶的核苷酸和氨基酸序列同源性分别达99％和100％.拮抗香蕉枯萎病菌的芽孢杆菌LX1可作为潜在的防治香蕉枯萎病的的生防制剂,其抗菌蛋白基因也可通过遗传工程应用于香蕉枯萎病的防治.     

海南白蚁共附生放线菌的分离鉴定及其抗热带作物病原真菌活性研究

白蚁（Isoptera）的筑巢和觅食活动主要在土壤及腐烂的木材上进行,所以白蚁极易受到来自环境病原微生物的侵害。因此,白蚁需要依赖共附生微生物菌群形成防御体系,放线菌是其中一个重要的类群,且放线菌能产生具有抗菌活性的次级代谢产物。但是白蚁共附生放线菌能否对热带作物的病原真菌有抑制活性有待研究。为了挖掘在热带农业绿色健康发展中具有应用潜力的海南特色农用放线菌资源,以海南白蚁共生放线菌为研究对象,利用平板分离法从采集的健康白蚁体内分离纯化放线菌,利用平板对峙法筛选生防菌株;通过分子生物学确定其分类地位;平板对峙法研究拮抗菌株的抗菌稳定性、持效性和广谱性;滤纸片法研究抑菌物质的热稳定性。结果从海南白蚁体内分离得到1株拮抗活性较好的放线菌菌株W7,系统发育树分析结果表明该放线菌与链霉菌属（Streptomyces）亲缘关系接近。菌株W7的抗菌活性持久性强,平板接种30 d后仍对植物病原真菌具有抑菌活性,抗菌稳定性好;菌株W7抑菌谱广,对16株植物病原真菌有不同程度的抑菌活性,其中对芒果胶孢霉（Colletotrichum gloeosporioides Penz.）、香蕉炭疽霉（Colletotrichum musae）、辣椒棒孢霉（Corynespora cassiicola）、豇豆瓜果腐霉[Pythium aphanidermatum (Edson) Fitzp]抑制作用较强,对芦笋拟茎点霉（Phomopsis asparagi）、荔枝疫霉（Peronophythora litchi）抑制作用较弱;菌株W7发酵粗提物有较好的热稳定性,但是抑菌率会随着水浴温度的增高而降低。本文首次对海南白蚁共附生放线菌进行了分离鉴定,并从中筛选得到1株具有抑制多种热带作物病原真菌活性的生防菌株,为高效生防菌剂的研发提供新资源。     

黄绿木霉菌对大豆根腐病镰刀菌的拮抗作用

利用室内对峙培养与发酵液处理病原菌研究了黄绿木霉(Trichoderma aureoviride)对大豆根腐病几种镰刀菌(Fusarium spp.)的拮抗作用,并利用盆栽试验研究了黄绿木霉对苗期大豆根腐病发生的影响.拮抗试验结果表明,黄绿木霉在与镰刀菌对峙培养中,菌丝交叉、接触与纠结,接触后镰刀菌菌丝被破坏;发酵产物只可延长镰刀菌孢子萌发的时间,但对孢子最终萌发率没有影响,发酵产物对菌丝生长速率的影响以山梨醇、葡萄糖及蔗糖作为碳源时,抑菌效果好,抑菌能力分别可达63%、51%、58%;盆栽试验证明黄绿木霉对大豆根腐病有较好的防治效果,防效可达73.2%,处理后幼苗株高与干重均高于未处理的对照.     

琯溪蜜柚炭疽病拮抗放线菌的筛选和鉴定

为筛选出对琯溪蜜柚炭疽病有防治效果的生防菌,以琯溪蜜柚炭疽病菌（Colletotrichum gloeosporioides）为靶标菌,采用平板对峙法进行初筛,用生长速率法和人工接种法对抑菌效果较好的菌株FX28进行抑菌谱及防治效果测定;通过形态学、生理生化特征,结合16S rDNA序列分析等方法研究其分类地位。结果表明：从25份土样中共分离到放线菌105株,对琯溪蜜柚炭疽病菌有较好拮抗作用的放线菌有16株,其中菌株FX28抑菌活性最高,其抑菌带宽度为15.3 mm,抑制率达86.4%,抑菌谱广,对琯溪蜜柚炭疽病菌、琯溪蜜柚黑点病菌、琯溪蜜柚黑斑病菌等14种供试植物病原菌均有抑制作用;其作用机制表现为菌丝分枝增多、变粗、顶端膨大等,对琯溪蜜柚炭疽病的预防效果和治疗效果分别为83.8%和71.1%。根据形态、生理生化特性及16S rDNA序列分析,初步确定菌株FX28为深红紫链霉菌（Streptomyces violaceorubidus）。     

128种南药植物提取物对6种病原菌的生长抑制作用

以水稻稻瘟病菌(Pyricularia oryzae Cav.)、香蕉枯萎病菌[(Fusarium oxysporum sp.cubense(E.F.Smith)Syn.et Han)]、香蕉炭疽病菌[Colletotrichum musae(Berk&Curt)Arx]、杧果炭疽病菌(Colletotrichum gloeosporioides PenZ)、辣椒炭疽病菌[Colletotrichum capsici(syd.)Butl.]和西瓜枯萎病菌(Fusarium oxysporum f.sp.niveum)6种病原菌为供试菌种,对47科128种南药植物的丙酮提取物进行室内抑菌活性试验。结果表明:有36种植物提取物对6种病原菌菌丝生长抑制率在60%以上;22种植物提取物对至少4种病原菌菌丝生长抑制率在80%以上;经方差分析,荜茇、艾纳香、白背叶、香茅、阴香、九里香、花椒、高良姜、益智、山柰、姜等11种植物提取物的抑菌效果与化学农药0.5mg/mL施保功的抑菌效果差异不显著。      

Application of bioactive coatings based on chitosan for artichoke seed protection

Artichoke seeds suffer attacks of various fungi that result in a decreased yield. Coatings based on chitosan were used as an antifungal agent and for enhancing the germination and quality of artichoke seeds. The effect of formulation (chitosan molecular weight, presence or absence of Span 80, pH), and thickness (number of coating layers) on seed germination (G%), fungi activity and vegetative growth were studied. Results indicated significant differences between treatments on seed germination. It was observed that all chitosan treatments reduced the number of type of fungi and increased plant growth. Chitosan with lower Mw gave better results both from microbial and germination point of view. In addition results indicated that treatments including chitosan and TMTD increased significantly G% and plant growth of artichoke seeds and produced a decrease in fungi contamination.     

海洋弗氏链霉菌HNM0089抗真菌活性成分研究

为了从海洋微生物中寻找农药先导化合物,本研究通过16S rRNA基因序列分析对具有抗真菌活性的海洋放线菌HNM0089的种属进行鉴定,并对其抗真菌的活性成分进行研究。结果表明:海洋放线菌HNM0089被鉴定为弗氏链霉菌(Streptomyces fradiae)。利用高分辨质谱、一维和二维核磁波谱技术,将其主要活性成分鉴定为星形孢菌素(Staurosporine)。该化合物对芒果炭疽病菌、水稻稻瘟病菌、香蕉枯萎病菌、薯蓣炭疽病菌和橡胶炭疽病菌均有抑制活性,具有开发成农用抗菌素的潜力。     

The effect of seed extraction methods on seed quality of two cultivar's tomato (Solanum lycopersicum L.)

In order to compare of different methods and identify the optimum condition for tomato seed extraction, factorial experiments with 3 replications was conducted. In the first experiment, pulp of two tomato cultivars (Faraon, Dominator) were fermented at two temperatures (25, 35 degrees C) and six periods (24, 48, 72, 96, 120, 144 h). The germination of seeds in laboratory as well as seedling emergence and preliminary growth in greenhouse were studied and measured. The results showed that effect of cultivar on traits (except of seedling emergence) was significant. Also the effect of temperature of fermentation, duration of fermentation and also interaction effects of them on seed germination were significant. Totally seed quality decreased with increasing temperature and duration of fermentation and the fermentation duration from 24 to 48 h at temperature 25 degrees C, is recommended. In the second experiment, tomato seeds were extracted by HCL (pH was arranged to 1, 2, 3 for 10, 20, 30 min), H2SO4 (pH was arranged to 1, 2, for 15, 30 min), Sodium carbonate (5, 10% for 24 and 48 h) and fermentation. Percentage germination, germination rate, length of radicle and length of plumule were used for seed quality assessment. The results showed that interaction effect between pH and duration of HCL treatments was significant for seed germination (percentage and rate) and there was an interaction effect between concentration and duration for germination rate in alkali treatments. Different extraction methods had not detrimental effect on percentage germination, but acid treatments produce very bright clean seeds in compare to other treatments.     

Isolation of a new natural product and cytotoxic and antimicrobial activities of extracts from fungi of Indonesian marine habitats

In the search for bioactive compounds, 11 fungal strains were isolated from Indonesian marine habitats. Ethyl acetate extracts of their culture broth were tested for cytotoxic activity against a urinary bladder carcinoma cell line and for antifungal and antibacterial activities against fish and human pathogenic bacteria as well as against plant and human pathogenic fungi. The crude extract of a sterile algicolous fungus (KT31), isolated from the red seaweed Kappaphycus alvarezii (Doty) Doty ex P.C. Silva exhibited potent cytotoxic activity with an IC₅₀ value of 1.5 μg/mL. Another fungal strain (KT29) displayed fungicidal properties against the plant pathogenic fungus Cladosporium cucumerinum Ell. et Arth. at 50 μg/spot. 2-Carboxy-8-methoxy-naphthalene-1-ol (1) could be isolated as a new natural product.     

Preliminary Study on the Activity of Phycobiliproteins against Botrytis cinerea

Phycobiliproteins (PBPs) are proteins of cyanobacteria and some algae such as rhodophytes. They have antimicrobial, antiviral, antitumor, antioxidative, and anti-inflammatory activity at the human level, but there is a lack of knowledge on their antifungal activity against plant pathogens. We studied the activity of PBPs extracted from Arthrospira platensis and Hydropuntia cornea against Botrytis cinerea, one of the most important worldwide plant-pathogenic fungi. PBPs were characterized by using FT-IR and FT-Raman in order to investigate their structures. Their spectra differed in the relative composition in the amide bands, which were particularly strong in A. platensis. PBP activity was tested on tomato fruits against gray mold disease, fungal growth, and spore germination at different concentrations (0.3, 0.6, 1.2, 2.4, and 4.8 mg/mL). Both PBPs reduced fruit gray mold disease. A linear dose–response relationship was observed for both PBPs against disease incidence and H. cornea against disease severity. Pathogen mycelial growth and spore germination were reduced significantly by both PBPs. In conclusion, PBPs have the potential for being also considered as natural compounds for the control of fungal plant pathogens in sustainable agriculture.