吡咯伯克霍尔德氏菌JK-SH007产嗜铁素的相关基因克隆及条件优化

为了明确吡咯伯克霍尔德氏菌Burkholderia Pyrrocinia JK-SH007是否产嗜铁素，揭示其生防机制，本文通过CAS检测该菌株产嗜铁素的能力，并对其嗜铁素合成相关基因cepR进行克隆及序列分析，同时采用单因素试验及响应面法对菌株JK-SH007产嗜铁素条件进行优化。结果表明，菌株JK-SH007具有明显的产嗜铁素能力，其嗜铁素合成相关基因cepR大小为720 bp，与B.pyrrocinia（EU034001）的亲缘关系最近，同源性为99%，两者序列相似性为97%，13个位点出现SNP；另外菌株JK-SH007 cepR基因编码的氨基酸序列有3个氨基酸发生了替换，两者一致性为97%。该菌产嗜铁素最优培养时间是15 h、pH 8、转速200 r/min、最佳碳源和氮源分别是甘油和氯化铵；PB试验筛选出影响该菌株产嗜铁素的关键性因素分别是pH、加碳量、加氮量；CCD试验结果显示，pH和加氮量的交互作用较明显；最终采用Design-Expert软件分析出菌株JK-SH007产嗜铁素最佳方案为碳源加入量15.00 g/L、氮源加入量10.50 g/L、温度30℃、pH 7.36，优化后菌株产嗜铁素能力由18.59提升到37.86，响应面优化产嗜铁素条件的效果是显著的，嗜铁素产量得到明显提高。     

群体感应信号物质对吡咯伯克霍尔德氏菌JK-SH007在杨树内定殖的影响

为了明确杨树溃疡病生防菌吡咯伯克霍尔德氏菌JK-SH007的群体感应系统是否与其内生定殖相关，本文通过群体感应指示菌紫色杆菌CV026和液相色谱串联四级杆飞行时间质谱技术（HPLC-Q-TOF-MS）确定其群体感应信号物质种类，并利用结晶紫染色、菌体回收、GFP标记等技术，研究其群体感应信号物质对该菌株生物膜及在杨树苗内的定殖能力的影响。结果表明，菌株JK-SH007产生的群体感应信号物质为含8个碳原子酰基侧链的辛酰基-L-高丝氨酸内酯（C₈-HSL）。群体感应信号物质C₈-HSL的添加对该菌株生物膜及在杨树苗内的定殖能力有影响，并且呈现低浓度促进，较高浓度抑制的规律。C₈-HSL的添加与菌株JK-SH007生物膜的OD值和菌体量具有显著相关性，相关系数分别为0.923和0.756。当添加1.0% C₈-HSL终浓度达到5×10^{-8 g/L}时，菌株JK-SH007生物膜的形成量达到峰值。荧光显微镜镜检发现，当添加100 μL C₈-HSL时，杨树组培苗根和茎中GFP荧光标记的菌株JK-SH007的定殖数量明显较CK多。菌株JK-SH007的群体感应与其在杨树内的内生定殖能力密切相关，群体感应信号物质C₈-HSL具有增强菌株JK-SH007内生定殖的能力，同时也表明该物质有利于该菌株生防效果的发挥。     

Co-inoculation of selected nodule endophytic rhizobacterial strains with Rhizobium tropici promotes plant growth and controls damping off in common bean

Production of common bean(Phaseolus vulgaris)is limited by the occurrence of damping off(rhizoctoniosis),which is caused by the fungus Rhizoctonia solani.However,the co-inoculation of plant growth-promoting rhizobacteria(PGPR)involved in biological control along with diatomic nitrogen(N2)-fixing rhizobia can enhance N nutrition and increase production.In this context,finding microorganisms with synergistic effects that perform these two roles is of fundamental importance to ensure adequate yield levels.The aim of this study was to evaluate the effects of co-inoculation of nodule endophytic strains of the genera Bacillus,Paenibacillus,Burkholderia,and Pseudomonas with Rhizobium tropici CIAT 899,an N2-fixing rhizobial strain,on the biocontrol of damping off and growth promotion in common bean plants.Greenhouse experiments were conducted under axenic conditions using the common bean cultivar Pérola.The first experiment evaluated the potential of the 14 rhizobacterial strains,which were inoculated alone or in combination with CIAT 899,for the control of R.solani.The second experiment evaluated the ability of these 14 rhizobacterial strains to promote plant growth with three manners of N supply:co-inoculation with CIAT 899 at low mineral N supply(5.25 mg N mL^-1),low mineral N supply(5.25 mg N mL^-1),and high mineral N supply(52.5 mg N mL^-1).The use of rhizobacteria combined with rhizobia contributed in a synergistic manner to the promotion of growth and the control of damping off in the common bean.Co-inoculation of the strains UFLA 02-281/03-18(Pseudomonas sp.),UFLA 02-286(Bacillus sp.),and UFLA 04-227(Burkholderia fungorum)together with CIAT 899 effectively controlled damping off.For the common bean,mineral N supply can be replaced by the co-inoculation of CIAT 899 with plant growth-promoting strains UFLA 02-281/02-286/02-290/02-293.Nodule endophytes UFLA02-281/02-286 are promising for co-inoculation with CIAT 899 in the common bean,promoting synergy with rhizobial inoculation and protection against disease.     

Interactions between the external mycelium of the mycorrhizal fungus Glomus intraradices and other soil microorganisms as affected by organic matter

The influence of organic matter on the interactions between external mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, the bacterium Burkholderia cepacia and other soil microorganisms was studied in a root-free sand environment. Organic matter amendment, in terms of ground barley leaves, markedly increased the growth of the external mycelium of G. intraradices as estimated both with the fatty acid biomarker 16:1ω5 and hyphal length measurements. Mycelial proliferation of G. intraradices in sand with organic matter was unaffected by both inoculation with B. cepacia and a soil filtrate containing a mixed population of indigenous microorganisms. On the other hand, in the absence of organic matter, both inoculation with B. cepacia and the soil filtrate reduced the growth of G. intraradices, as estimated with measurements of 16:1ω5. In contrast, B. cepacia inoculation increased hyphal length density of G. intraradices in the absence of organic matter. Overall, the presence of external mycelium of G. intraradices increased the bacterial biomass and counteracted a suppressive effect of B. cepacia on the growth of saprotrophic fungi.     

Characterization of brushite as a re-crystallization product formed during bacterial solubilization of hydroxyapatite in batch cultures

Two strains of bacteria (Burkholderia sp., strain FeGL01, and Burkholderia caribiensis, strain FeGL03) were isolated from a Brazilian high phosphorus iron ore. The capacity of both strains to solubilize hydroxyapatite, Ca₅(PO₄)₃(OH), was assessed in plate and batch cultures. In batch cultures, the concentration of solution-P showed two kinetics: an initial one, characterized by a continuously increasing kinetics and a second one, characterized by oscillatory kinetics. To understand the nature of these oscillations, phosphatic residues in the spent broth were collected before, during and after the oscillations, and characterized using scanning electron microscopy (SEM), energy-dispersive X-ray chemical microanalyses (EDX) and X-ray diffraction (XRD). From these studies, it was found that drops in P concentration were related to the formation of an intermediate phosphate in the residues, identified as brushite, CaHPO₄·2H₂O. Later increase of available P in the solution was found to be a consequence of re-dissolution of brushite crystals previously formed. Re-crystallization of brushite was also detected in plate cultures after 12-14 days of incubation     

Light and plant growth‐promoting rhizobacteria effects on Brachiaria brizantha growth and phenotypic plasticity to shade

This is the first report on the effect of light intensity and plant growth‐promoting rhizobacteria (PGPR) on the growth of a tropical forage grass, being a relevant study to improve pasture management in conventional farming and integrated crop‐livestock‐forestry systems. In this study, our aim was to evaluate the effects of light intensity and Burkholderia pyrrocinia and Pseudomonas fluorescens inoculation on Brachiaria brizantha cv. BRS Piatã growth, and phenotypic plasticity to shade. The experiment was conducted in a semi‐controlled environment. Seedlings of B. brizantha were allocated to full sun and shade. P. fluorescens and B. pyrrocinia were inoculated individually or co‐inoculated by soil drench, 14 days after seedling emergence. We evaluated morphogenesis, structural and growth parameters. Irrespective of the light regime, co‐inoculated plants had greater leaf area and SPAD index (chlorophyll content). Increase in total biomass production in co‐inoculated plants was over 100% and 300%, under full sun and shade respectively. Co‐inoculated P. fluorescens and B. pyrrocinia increased shade tolerance in B. brizantha, improving plant performance. Co‐inoculation promoted growth in B. brizantha under both sun and shade, indicating its potential as a bio‐fertilizer in conventional and integrated systems, especially in silvopastoral systems, where light availability to pasture growth may be limited.     

Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It's quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×104 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1:100.     

洋葱伯克氏菌群不同基因型菌株对几种重要植物病原真菌的抑制作用及其潜在致病性

从玉米和水稻根围分离到70株不同基因型的洋葱伯克氏菌,对这些菌株进行了拮抗植物病原菌的筛选,并对高拮抗菌株进行了潜在致病性和安全性分析。结果表明,有46株洋葱伯克氏菌对一种或多种病菌有较高的拮抗活性。在不同的洋葱伯克氏菌基因型内,以基因型Ⅴ内拮抗菌株占的比例最高,对所测5种病原真菌的平均拮抗菌株比率为80．0％,其中部分菌株表现出很强的拮抗活性。筛选出的4株高拮抗菌株对洋葱不具有致病性,同时也未检测到与人体致病相关的BCESM毒力基因。     

Inhibitory Effect of Dimethyl Disulfide from Burkholderia pyrrocinia WY6-5 on Aspergillus flavus and Aflatoxins in Peanuts During Storage Period

【Objective】The objective of this study is to verify the antifungal effect of Burkholderia pyrrocinia WY6-5, evaluate its control efficacy against Aspergillus flavus and aflatoxins in peanuts during storage period, analyze the inhibitory mechanism, identify antifungal volatiles and detect the minimal inhibitory concentration to A. flavus, so as to provide novel strategies for the prevention and control of fungal diseases and mycotoxin during storage period.【Method】Face-to-face dual cultural test was conducted to analyze the antifungal activity of volatiles from WY6-5. Active charcoal as volatile adsorbent was added into the tests to verify the antifungal activity of volatiles. Dimethyl disulfide (DMDS) emitted form strain WY6-5 was challenged with peanut kernels inoculated with A. flavus conidia in sealed airspace without physical contact. A. flavus cells on peanut coat were collected, fixed in osmic acid, and analyzed through scanning electron microscope (SEM). Transmission electron microscope (TEM) was used to test the inner structure of A. flavus cell affected by volatiles from WY6-5. The commercial DMDS was purchased, serially diluted, and co-cultured with A. flavus conidia and mycelia to detect the minimal inhibitory concentration, respectively.【Result】B. pyrrocinia WY6-5 isolated from rhizosphere soil of tea plants could produce volatile DMDS, prevent the growth of A. flavus mycelia, the inhibition rate was over 95%. Additionally, under the condition of high water activity (a_w), WY6-5 could also inhibit the A. flavus infection and aflatoxins production in peanuts during storage period. In peanuts of control treatment, the disease incidence was 100%, and the total concentration of aflatoxins was 399.32 μg?kg ^-1 (a_w 0.859) and 3 143.19 μg?kg ^-1 (a_w 0.923), respectively. When WY6-5 was added in the treatment, the disease incidence decreased to 2% (a_w 0.859) and 21% (a_w 0.923), respectively. The concentration of aflatoxins decreased to 4.86 μg?kg ^-1 (a_w 0.859) and 121.37 μg?kg ^-1 (a_w 0.923), respectively. The inhibition rate of WY6-5 against aflatoxins contamination was 98.78% and 96.14% compared to the control treatment. SEM analysis proved that DMDS from WY6-5 inhibited the germination of A. flavus conidia. TEM analysis further proved that the inner cell structures of A. flavus conidia were not severely damaged by volatiles. Volatile DMDS showed great antifungal activity. The minimal inhibitory concentration against mycelia growth was 100 μL?L ^-1(compound volume/airspace volume). The minimal inhibitory concentration against conidia germination was 50 μL?L ^-1(compound volume/airspace volume). 【Conclusion】 B. pyrrocinia WY6-5 can produce valid antifungal volatile DMDS, which can completely inhibit the mycelia growth and conidia germination of A. flavus at low concentration, and greatly prevent the development of A. flavus disease and aflatoxins contamination in peanuts during storage period. WY6-5 and the produced DMDS provide novel bio-active agents for fungal diseases control and mycotoxins during storage period.     

吡咯伯克霍尔德菌WY6-5产二甲基二硫对储藏期花生黄曲霉及毒素的抑制作用

【目的】验证吡咯伯克霍尔德菌（Burkholderia pyrrocinia）WY6-5的抑菌作用,评价其对储藏期花生黄曲霉（Aspergillus flavus）及毒素的防治效果,分析抑菌作用机制,鉴定活性物质,并检测其最低抑菌浓度,为储藏期真菌病害及毒素的防控提供新材料。【方法】采用非接触培养皿对扣培养法,检测菌株WY6-5对黄曲霉的抑制效果,添加活性炭,验证挥发性物质的抑菌作用;密闭储藏环境,检测WY6-5产二甲基二硫对花生黄曲霉及毒素的抑制效果;收集处理后的花生籽粒,锇酸固定,并进行扫描电镜观察,检测黄曲霉细胞显微结构变化,通过透射电镜观察,检测黄曲霉细胞内部结构的显微差异;购买活性物质标准品,梯度稀释,与黄曲霉菌丝和孢子对扣培养,分析最低抑菌浓度。【结果】吡咯伯克霍尔德菌WY6-5分离自茶园根际土壤,可产生挥发性物质二甲基二硫,并高效抑制黄曲霉的生长,抑菌率达95%以上;同时,在高水活度（a_w）条件下,WY6-5还可抑制储藏期花生黄曲霉和毒素污染;两种水活度下,对照组中发病率高达100%,黄曲霉毒素总含量分别为399.32 μg?kg ^-1（a_w 0.859）和3 143.19 μg?kg ^-1（a_w 0.923）;WY6-5添加组,花生黄曲霉发病率降为2%（a_w 0.859）与21%（a_w 0.923）,毒素含量降为4.86 μg?kg ^-1（a_w 0.859）和121.37 μg?kg ^-1（a_w 0.923）,与对照组相比,对毒素的抑制率达98.78%和96.14%。扫描电镜观察显示,WY6-5产生的挥发性气体能抑制黄曲霉孢子的萌发,透射电镜显示,黄曲霉细胞结构未呈现明显损伤。挥发性物质二甲基二硫抑菌作用明显,对黄曲霉菌丝生长的最低抑菌浓度为100 μL?L ^-1(物质体积/培养体积),对孢子萌发的最低抑菌浓度为50 μL?L ^-1(物质体积/培养体积)。【结论】吡咯伯克霍尔德菌WY6-5可产生高效抑菌挥发物二甲基二硫,在低浓度下即可完全抑制黄曲霉菌丝生长和孢子萌发,并能抑制储藏期花生黄曲霉的侵染和黄曲霉毒素的产生,为储藏期真菌病害及毒素的防控提供了新型生物材料。