Decisive Factors of Conidia Release of Botryosphaeria berengeriana during Growing Season

[ Objective ] The study aimed to explore the release conditions for the conidia of Botryosphaena berengeriana and understand the release dynamic of conidia. [Method] The systematical survey on the release conditions for the conidia of B. berengeriana were conducted in two growing seasons in 2008 and 2009, combined with the collection of meteorological data around conidia release period, the weather conditions causing large amount release of B. berengedana were analyzed. [ Result] During a growing season, the conidia of pathogen appeared several large release peaks. Under the suitable temperature, when the precipitation lasted for 4 h, the conidia of B. berengeriana began to release with large amount, the amount of conidia reached the peak after release and trended to be stable during 4 - 12 h, which significantly reduced after 24 h, tended to dis- appear after 36 h, and completely disappeared after 72 h. [Conclusion] The dominant factor affecting B. berengeriana conidia release in large a- mount was precipitation, while the lasting time of precipitation played a decisive role.

Development of Highly Efficient Transformation System of Yeast-Like Conidia of Tremella fuciformis

Tremellafuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, which is also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. Thus, it is a good recipient cell for exogenous gene expression. In this study, the expression plasmid pAN7-1 （containing promoter gpd-An derived from Aspergillus nidulans and selectable marker gene hph conferring resistance to hygromycin B） and plasmid pLg-hph （containing promoter gpd-Le derived from Lentinula edodes and selectable marker gene hph） were transformed into the yeast-like conidia of T. fuciformis by PEG-mediated protoplast transformation, respectively. The primary putative transformants were selected by the sandwich screening method with the selective medium containing 50 μg mL^-1 hygromycin. The putative transformants were obtained from the primary putative transformants transferred on PDSA plates containing 100 μg mL^-1 hygromycin for second round selection. Experimental results showed that the optimal concentration of PEG 4000 for mediating protoplast transformation was 25%. PCR and Southern blotting confirmed that the selectable marker gene hph was integrated effectively into the genome of the yeast-like conidia of T. fuciformis with plasmid pLg-hph transformation. Its transformation efficiency was 110 transformants per μg DNA, and the hph gene was integrated into the genome of some yeast-like conidia with plasmid pAN7-1 transformation. However, its transformation efficiency was only 9 transformants per μg DNA. The presence of hph gene in the genome of transformants after 5 generations of sub- culturing on PDSB medium was confirmed by PCR, suggesting that the foreign gene hph was stable during subculture.

Conidia of one Fusarium solani isolate from a soybean-production field enable to be virulent to soybean and make soybean seedlings wilted

Fusarium is usually thought to cause soybean root rot, which results in a large quantity of annual yield loss in soybean production, by its secretions including Fusarium toxins and cell wall degrading enzymes, but not by the conidia themselves that do not underlie any virulence so far. Here we report that the conidia of one Fusarium solani isolate are able to be virulent to soybean and make soybean seedlings wilted alone. We isolated them from the wilted plants in a soybean-production field and molecularly identified 17 Fusarium isolates through phylogenetic analysis. Of them, except for one isolate that showed diversity of virulence to different soybeans （virulent to one soybean whereas avirulent to another soybean）, the others were all virulent to the two tested soybeans： both conidia cultures and secretions could make soybean seedlings wilted at 5 days post infection, and their virulence had dosage effects that only conidia cultures of at least 5×10^6 conidia mL-1 could show virulence to soybean; however, the sole conidia of the F. solani isolate #4 also exhibited virulence to soybean and could make soybean seedlings wilted. Finally, we developed the specific cleaved amplified polymorphic sequences （CAPS） markers to easily differentiate Fusarium isolates. The isolate #4 in this work will likely be used to investigate the new mechanism of virulence of Fusarium to soybean.

Isolation Technology of Single Spore and Optimization of Conidia Culture Condition for Ustilaginoidea virens

The single spores were isolated from chlamydospores of Ustilaginoidea virens with three different maturities by PSA. The isolated single spores were cul- tured on different media at different temperatures under natural light for inducing conidia to explore the optimum isolation technique of single spore and optimum cul- ture condition of conidia. The results showed that the successful isolating rate of single spore from yellow rice false smut balls reached 90.00%. The sporulatina quantities of isolated single spores cultured on PSD and PDB media at 22 -29 ~C （variable temperature under natural light） or 28 ℃ （constant temperature under dark condition） for 12 d were up to 6.3× 107 and 1.1× 106 spore/mL, respectively. PSA was the most effective method to isolate single spores from yellow rice false smut balls of U. virens. The optimum conidia culture condition included PSD or PDB medium, 22 -29℃ or 28℃, natural light and vibration culture. Key words Ustilaginoidea virens; Single spore isolation; Conidia; Culture condition

Preservation and Conidia Culture Technique of Ustilaginoidea virens

[Objective] This study aimed to find methods suitable for the preservation and conidia production of Ustilaginoidea virens. [Method] Six different preservation methods were tested by checking at three month interval for the viability and sporulation, and seven liquid media of different composition were tested by shaking cul- ture at 150 r/min. [Result] Among the six methods studied, the periodic transfer and paraffin oil overlay were suitable, and the fungus could survive more than 12 months. The conidia concentration went up to 7.25×10^7 conidia/ml after 9 d in the PSB liquid medium, indicating that PSB was the best medium for conidia production. [Conclusion] This study provides a theoretical basis for the preservation and conidia production of U. virens.

First Report of Powdery Mildew Caused by Erysiphe euonymicola on Euonymus japonicum in Uzbekistan

A new powdery mildew fungus named Erysiphe euonymicola was discovered in the mycobiota of Uzbekistan as a result of a mycological study conducted in the city of Samarkand. This fungus was found on the bush Euonymus japonicum and a brief description along with photographs of Erysiphe euonymicola have been provided. Erysiphe euonymicola is a phytopathogenic fungus that infects 80% - 90% of the leaves and young branches of Euonymus japonicum. The first symptoms of the disease are round, weeping spots on the leaves;later the spots form white mycelium over the entire surface of the leaves. When the plant is heavily infected, both sides of the leaves are completely covered with mycelium. The fungus Erysiphe euonymicola, parasitizing Euonymus japonicum, seriously inhibits the development of the host plant. The mycelium of Erysiphe euonymicola is formed on the surface of the leaf of the host plant and is a cluster of hyphae that form a dense covering in the form of a round spot. Conidiophores are erect, unbranched, and of Pseudoidium type, very variable in shape and size. Conidiophores consist of 2 - 3 cylindrical cells growing vertically from the hyphae;the sizes of conidiophores are given relative to the average length and width of selected conidiophores (n = 15). According to scanning electron microscopy, ellipsoidal-cylindrical conidia are single, scattered among conidiophores, ranging in size from 23.97 to 24.51 × 11.5 to 12.47 µm (n = 15). Conidial stalk cells are usually straight or slightly curved, ranging in size from 23.5 to 23.91 × 7.17 to 7.47 µm (n = 15).

黄瓜多主棒孢菌致病基因CcTLS2功能探究

为探究CcTLS2基因在多主棒孢菌(Corynespora cassiicola)致病过程中的作用,以强致病力菌株HG14102524为研究对象,通过克隆多主棒孢菌CcTLS2基因,构建CcTLS2敲除株,并结合生物信息学分析、致病力测定、差异表达分析等方法,初步确定了CcTLS2对多主棒孢菌致病的影响。与野生型菌株相比,CcTLS2基因敲除菌株致病力丧失,菌落生长速度变慢,产孢量显著减少。多主棒孢菌G蛋白信号途径相关基因(A7125、A1402、A0835、A8311和A2529)在CcTLS2敲除株中的相对表达量显著下调。结果表明,CcTLS2可能通过G蛋白相关途径影响多主棒孢菌的产孢机制、菌丝生长和致病力,在多主棒孢菌致病中起重要作用。

甘薯疮痂病菌的生物学特性和产孢方法研究

探究甘薯疮痂病菌(Elsinoe batatas)的生物学特性,筛选疮痂病菌在人工培养基上快速、大量地产生分生孢子的方法,为甘薯抗疮痂病育种提供依据。以疮痂病病原菌菌株CRI-CJ2为研究对象,采用菌丝生长速率法测定在不同培养基、pH和光照处理条件下的菌落生长直径,利用凹玻片悬滴法测定在不同温度、pH和光照处理条件下的分生孢子萌发率,比较Fries液体培养法、摇菌法和菌丝涂布法等3种方法的诱导产孢效果。结果表明,最适菌丝生长的培养基为PDA,pH 6~7,致死温度为48℃、10 min,光照对菌丝生长无显著影响,但连续光照使菌落呈鲜红色;分生孢子萌发最适pH 5,最适温度为25℃,致死温度为48℃、10 min,光照对分生孢子萌发影响不显著;采用菌丝涂布法可使疮痂病菌在培养第12天分生孢子产孢量达7.09×10^(7)个/mL,分生孢子萌发率为24%,产孢量和孢子活力均高于摇菌法,采用Fries液体培养法未观察到分生孢子及产孢结构。可见,选用适宜的培养基、pH能促进菌丝体生长,菌丝涂布法具有产孢快、产孢量大的优点,是一种适合甘薯疮痂病菌分生孢子诱生的方法。

基于响应面法的非洲哈茨木霉AD-G4菌株固体发酵条件优化

【目的】为了获得非洲哈茨木霉AD-G4固体发酵的最佳条件。【方法】利用单因素试验、BBD(Box-Behnken Design)响应面试验对非洲哈茨木霉AD-G4菌株的固体发酵培养基、发酵条件等影响因素进行了优化。【结果】适合此菌株产生的最佳培养基组成为:燕麦粉和小麦秸秆1∶1;最佳培养条件为初始p H值7.0,含水量52%,孢子浓度5.40×10^(6)个/m L,温度28℃,培养天数为8 d。【结论】在优化的培养条件下,非洲哈茨木霉AD-G4分生孢子产量达到1.91×10^(10)个/g。通过培养条件优化确定了适合非洲哈茨木霉AD-G4菌株产生分生孢子的条件,为高效开发非洲哈茨木霉生防菌剂奠定了基础。

曲霉菌分生孢子的休眠及萌发研究进展

综述了曲霉菌分生孢子休眠及萌发的生物学过程,解析了细胞内外生物分子对孢子休眠的维持作用及分子机制,基于细胞生物化学功能及形态学揭示了孢子萌发的变化规律,阐明了信号传导通路对孢子萌发的调控作用,为粮食霉变预防措施的探索及制定提供理论支持。

新疆苹果褐色斑点果腐病病原菌的鉴定

为明确新疆苹果褐色斑点果腐病病原菌的种类,对采自阿拉尔的苹果褐色斑点果腐病病果的病原菌种类进行了鉴定。利用组织分离法对病原菌进行分离纯化,获得菌株PGFL,采用柯赫氏法则进行致病性验证,结合形态学特征观察和ITS、RPB2、EF序列构建多基因联合系统发育树对病原菌的种类进行确定。菌株PGFL的人工培养菌落形态呈现灰色至暗青褐色,与链格孢属Alternaria形态观察比对结果高度相似,且接种离体果实后可发病。分子生物学分析菌株PGFL和菌株Alternaria alternata MAFF239887及A.alternata JLUAF6聚为一簇,与Alternaria属A.alternata的序列相似度最高,且自展支持率为100%。研究确定引起新疆苹果褐色斑点果腐病病原菌为链格孢A.alternata。

Dexamethasone enhances invasiveness ofAspergillusfumigatus conidia and fibronectin expression in A549 cells

Background The efficacies of current treatments for invasive aspergillus （IA） are unsatisfactory and new therapeutic targets or regimens to treat IA are urgently needed. Previous studies have indicated that the ability of conidia to invade host cells is critical in IA development and fibronectin has a hand in the conidia adherence process. In the clinical setting, many patients who receive glucocorticoid for extended periods are susceptible to Aspergillus fumigatus （A. fumigatus） infection, for this reason we investigated the effect of glucocorticoid on conidia invasiveness by comparing the invasiveness of A. fumigatus conidia in the type II human alveolar cell line （A549） cultured with different concentrations of dexamethasone. We also explored the relationships between dexamethasone and fibronectin expression. Methods Following culture with anti-fibronectin antibodies and/or dexamethasone, type II human alveolar A549 cells were infected with conidia of A. fumigatus. After 4 hours, the extracellular free conidia were washed away and the remaining immobilized conidia were released using Triton-X 100 and quantified by counting the colony-forming units. The invasiveness of conidia was measured by calculating the invasion rate （%）. The transcription of the fibronectin gene in cells cultured with different concentrations of dexamethasone for 24 hours was tested by fluorogenic quantitative RT-PCR while the expression of fibronectinin cells cultured for 48 hours was tested by Western blotting and immunocytochemistry. Results A significant reduction in the invasiveness of conidia was seen in the cells cultured with anti-fibronectin antibody （（14.42±1.68）% vs. （19.17±2.53）%, P 〈0.05）, but no significant difference was observed in cells cultured with a combination of anti-fibronectin antibody and dexamethasone （6.37×10^-5 mol/L）. There was no correlation between the dexamethasone concentration and the invasiveness of conidia after dexamethasone pretreatment of cells for 4 hours. In contrast, after pretreated for 24 hours, the invasiveness of conidia in the presence of 6.37×10^-5 mol/L dexamethasone （（24.66±2.41）%） was higher than for the control （（19.17±2.53）%） and the 0.25×10^-5 mol/L group （（19.93±3.06）%）, and the invasiveness in the 1.27×10^-5 mol/L group （（22.47±2.46）%） was also higher than in the control, P 〈0.05. The relative transcripts of the fibronectin gene after exposure to 6.37× 10^-5 mol/L dexamethasone （9.19×10^-3±1.2×10^-3） was higher than for the control （4.61 ± 10^-3±1.54× 10^-3） and the 0.25× 10^-5 mol/L group （6.20× 10^-3±1.93× 10^-3）, and expression in the 1.27× 10^-5 mol/L group （7.94× 10^-3±2.24×10^3） was also higher than for the control, P 〈0.05. High concentrations of dexamethasone promoted fibronectin production after culture for 48 hours. Conclusions Dexamethasone can increase invasiveness of A. fumigatus conidia by promoting fibronectin expression. This may partially explain why patients who are given large doses of glucocorticoids for extended periods are more susceptible to A. fumigatus infection.

Comparison between gene expression of conidia and germinating phase in Trichophyton rubrum

Trichophyton rubrum is a dominating superficial dermatophyte, whose conidial germination is corre- lated to pathopoiesis and a highly important developmental process. To investigate the changes of physiology, biochemistry and cytology during the germination, we selected 3364 function identified ESTs from T. rubrum cDNA library to construct cDNA microarrays, and compared the gene expression levels of conidia and germinating phase. Data analysis indicated that 335 genes were up-regulated during the germination, which mainly encoded translated, modified proteins and structural proteins. The constituents of cell wall and cell membrane were synthetized abundantly, suggesting that they are the foundation of cell morphogenesis. The ingredients of the two-component signal transduction sys- tem were up-regulated, presuming that they were important for the conidial germination. Genes of various metabolic pathways were expressed prosperously, especially the genes that participated in glycolysis and oxidative phosphorylation were up-regulated on the whole, demonstrating that in the environment with sufficient oxygen and glucose, conidia obtained energy through aerobic respiration. This paper provides important clues which are helpful to understanding the changes in gene expres- sion, signal conduction and metabolism characteristics during T. rubrum conidial germination, and possess significant meaning to the study of other superficial dermatophytes.

蓖麻根腐病抗性鉴定及其SSR标记的初步建立

蓖麻根腐病是茄腐镰孢菌(Fusarium solani)引起的根部病害,严重影响蓖麻产量。抗源缺乏制约了抗病品种的选育。为寻找抗病种质、建立抗性分子标记,该研究对252份蓖麻材料的抗性进行了表型和分子标记鉴定。结果表明:(1)浓度为1×10^(6)个·mL^(-1)的孢子悬浮液灌根是一种有效的接种方法;以接种后枯萎天数为基础的5级评价法,可作为鉴定标准。(2)鉴定出130份抗病材料,其中高抗为105份。(3)野生材料中抗病材料比率(66%)远高于栽培材料(35%),建议将野生材料,尤其是中国华南野生材料的研究利用作为今后抗病育种的重要方向。(4)初步建立了8个与抗性关联的SSR标记。该研究结果提供了有效的根腐病抗性鉴定方法和评价标准,筛选出了一批育种迫切需要的抗病基因资源,初步建立了可用于辅助选择的SSR标记,为蓖麻抗根腐病育种奠定了重要基础。

金耳酵母状孢子多糖双酶法提取工艺及其抗氧化性

为提高金耳酵母状孢子多糖得率,在单因素试验的基础上,通过Plackett-Burman试验和正交试验优化金耳酵母状孢子多糖的提取工艺;并通过测定金耳酵母状孢子多糖DPPH自由基和羟自由基清除率对其体外抗氧化活性进行探究。结果表明,金耳酵母状孢子多糖最佳提取工艺条件:纤维素酶200 U/g孢子粉,果胶酶50 U/g孢子粉,酶解时间80 min,酶解温度55℃,酶解pH值为4.5,料液比1∶40(g/mL),在该提取条件下,金耳酵母状孢子多糖得率为(8.41±0.36)%。体外抗氧化试验表明,金耳酵母状孢子多糖对DPPH自由基和羟自由基的IC50值分别为7.06 mg/mL和19.33 mg/mL。

不同培养条件对草莓白粉病菌分生孢子萌发的影响

研究草莓白粉病病原菌分生孢子的适宜萌发条件,为草莓白粉病菌发生、病害防治提供参考。以活体保存的羽衣草单囊壳菌(Sphaerotheca aphanis)的分生孢子为试验材料,在室内进行不同pH值、碳源、温度、湿度、光照等处理,研究其对分生孢子萌发率和芽管长度的影响。结果表明:草莓白粉病菌分生孢子萌发和芽管伸长最适pH值为7;分生孢子萌发的适宜温度范围为20~30℃,最佳萌发温度为25℃,致死温度达到42℃以上10 min分生孢子停止萌发;连续24 h光照对孢子萌发有促进作用;2%葡萄糖处理环境下有助于孢子萌发及芽管伸长。综合分析表明,草莓白粉病菌分生孢子最适温度和致死温度分别为25℃和42℃10 min,喜中性、高湿环境,在葡萄糖、光照充足的环境下有利于草莓白粉病菌分生孢子萌发。该研究结果为防治草莓白粉病菌提供了理论基础。

环链棒束孢生长曲线的测定

明确环链棒束孢的生长曲线,为环链棒束孢代谢物的采集、孢子规模化生产奠定基础。采用重量法、显微直接计数法、比浊法3种方法对环链棒束孢2018 BY-1菌株的生长规律进行研究。结果表明,环链棒束孢的生长规律为0~3 d为迟缓期,3~5 d为对数期,5~6 d为稳定期,6 d以后为衰亡期;最大吸收波长为410 nm,此时菌丝干重、OD410值、孢子数均达到最大值,分别是0.22±0.00 g、0.64±0.03、4.14×10^(8)个/mL;对数期的OD值和孢子数存在线性关系;稀释培养基的浓度可以降低培养基中色素对菌液的影响。

昆虫寄生性真菌——棕色绿僵菌对草原革蜱杀灭作用的研究

为了研究昆虫寄生性真菌——棕色绿僵菌(Metarhizium brunneum)在家畜外寄生虫病防控中的应用,试验通过培养棕色绿僵菌获得分生孢子,稀释至浓度为1×10^(4)个/mL后统计其萌发率,在棕色绿僵菌分生孢子悬液对草原革蜱幼蜱和成蜱的杀灭作用试验中分别设置1个对照组和5个浓度的试验组,即将草原革蜱幼蜱和成蜱分别放入5个不同浓度的棕色绿僵菌分生孢子悬液及0.5%孢子洗脱液中停留5 s,在25℃条件下每日定时观察幼蜱和成蜱的虫体死亡情况及作用时间。结果表明:棕色绿僵菌培养至第3天时所得的分生孢子萌发率可达到90.40%;对草原革蜱幼蜱杀灭作用明显,用试验所得的最佳作用浓度1×10^(6)个/mL作用至第7天时草原革蜱幼蜱的死亡率高达100%,成蜱的死亡率为80.00%。说明昆虫寄生性真菌——棕色绿僵菌可以用于防治家畜外寄生虫病。

三七种植土壤中对羟基苯甲酸对尖孢镰刀菌的化感作用

【目的】研究对羟基苯甲酸对尖孢镰刀菌致病性的化感促进作用机制。【方法】采集三七道地产区云南省文山州种植地区不同种植年限的三七根系土壤,采用HPLC技术、宏基因组学技术,结合酶学研究方法,分析对羟基苯甲酸对尖孢镰刀菌的化感作用。【结果】未种植过三七的1年生、2年生、3年生土壤样品中对羟基苯含量不同,分别为3.48、3.95、2.22 mmol/L。自然生长状态下三七种植土壤中对羟基苯甲酸含量随着种植年限的延长而降低,同时伴随着土壤中的尖孢镰刀菌种群密度升高。当土壤中对羟基苯甲酸含量为2.61 mmol/L时,土壤中镰刀菌种群密度最高,为20.94%。随着对羟基苯甲酸含量的增加,土壤中镰刀菌种群密度下降,当其含量为5.11 mmol/L时,镰刀菌种群密度仅为1.057%。中等浓度的对羟基苯甲酸(2.5~5.0 mmol/L)促进尖孢镰刀菌生长,其菌落直径与对照相比升高,菌落颜色在培养24 h后呈现紫色,菌丝生长较对照稍绵密,显微镜下可观察到大量分生小孢子的生成;促进了尖孢镰刀菌果胶酶的活性,其中浓度为2.5 mmol/L时,果胶酶活力为1.02 U/(mL·min);在2.5~10.0 mmol/L浓度的对羟基苯甲酸胁迫下,纤维素酶活力变化不大。【结论】对羟基苯甲酸化感促进尖孢镰刀菌的最佳浓度为2.0~3.0 mmol/L。该浓度下土壤中尖孢镰刀菌种群密度最高,菌落直径、孢子产量达到最高值,且对致病性酶分泌具有促进作用。

丝状真菌分生孢子及其核型相关基因的研究进展

分生孢子是曲霉、青霉等丝状真菌无性繁殖的主要生殖细胞,分生孢子的核型具有多样性,其多核特性对丝状真菌的生长发育、遗传调控产生诸多影响。分生孢子的形成及其核型变化受到多种基因的调控。通过对丝状真菌分生孢子发育过程及其核型变化调控相关基因的功能进行归纳综述,为更好地利用丝状真菌资源,开展相关物种的遗传操作提供理论基础。