Overexpression of AmDUF1517 enhanced tolerance to salinity,drought, and cold stress in transgenic cotton

As abiotic stresses become more severe as a result of global climate changes, the growth and development of plants are restricted. In the development of agricultural crops with greater stress tolerance, AmDUF1517 had been isolated from the highly stress-tolerant shrub Ammopiptanthus mongolicus, and can significantly enhance stress tolerance when inserted in Arabidopsis thaliana. In this study, we inserted this gene into cotton to analyze its potential for conferring stress tolerance. Two independent transgenic cotton lines were used. Southern blot analyses indicated that AmDUF1517 was integrated into the cotton genome. Physiological analysis demonstrated that AmDUF1517-transgenic cotton had stronger resistance than the control when treated with salt, drought, and cold stresses. Further analysis showed that trans-AmDUF1517 cotton displayed significantly higher antioxidant enzyme (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione S-transferase (GST)) activity and less reactive oxygen species (ROS) accumulation, which suggests that overexpression of AmDUF1517 can improve cotton resistance to stress by maintaining ROS homeostasis, as well as by alleviating cell membrane injury. These results imply that AmDUF1517 is a candidate gene in improving cotton resistance to abiotic stress.     

落叶型大丽轮枝菌脱落酸合成相关基因功能研究

为了探讨大丽轮枝菌中脱落酸（Absicisic acid，ABA）合成相关基因在致病过程中的功能，克隆大丽轮枝菌落叶型菌株XJ592的ABA合成相关基因Vdaba1、Vdaba2(1)、Vdaba2(2)和Vdaba4，采用同源重组的方法分别获得4个基因的敲除突变体菌株。生物学功能测定结果表明，Vdaba1主要参与促进大丽轮枝菌的产孢，Vdaba2(1)和Vdaba2(2)基因影响大丽轮枝菌的生长速率，Vdaba4基因参与调节大丽轮枝菌对氧化压力胁迫的响应。致病力测定结果表明，接种21 d时Vdaba1和Vdaba4基因突变体菌株的致病性显著低于野生型菌株XJ592，但在接种30 d时突变体与野生型XJ592接菌棉花植株的落叶率均为100%。     

1株拮抗棉花枯、黄萎病菌的纤维素分解菌筛选

为获得具有拮抗棉花枯、黄萎病菌的棉杆促腐菌剂,进行棉杆纤维素分解菌分离及拮抗菌株的筛选,采用平板对峙法测定菌株对棉花枯、黄萎病菌菌丝生长的影响,通过生长速率法和孢子萌发法测定菌株发酵液对2种病原菌菌丝生长以及孢子萌发的影响,并对筛选得到的菌株进行鉴定。结果获得1株具有抑菌效果的棉杆纤维素分解菌SJ-3,其羧甲基纤维素酶活(CMCase)达到69.1 U/mL,滤纸酶活(FPase)达到48.5U/mL。该菌对棉花枯、黄萎病菌菌丝生长均有显著抑制,抑制率分别达到24.7%、23.1%。其发酵液具有抑菌活性,较高的发酵液浓度能增强对病原菌的抑制作用,发酵液对枯萎病菌孢子萌发的抑制作用较强,最高抑制率为21.6%,而对该病原菌菌丝生长的抑制较弱,最高抑制率仅为6.2%;发酵液对黄萎病菌的菌丝生长和孢子萌发均有较强的抑制作用,最高分别达到32.2%、21.4%。经鉴定,菌株SJ-3为普通青霉(Penicillium commune)。     

pH及盐分对大丽轮枝菌微菌核形成的影响

微菌核是棉花黄萎病原大丽轮枝菌(Verticillium dahliae Kleb.)在土壤中的主要存活形式及该病的侵染源,研究土壤pH及盐分对大丽轮枝菌微菌核形成的影响对明确黄萎病发生具有重要意义。采用菌丝生长速率法研究培养基pH及盐分质量浓度与种类对病原菌生长及微菌核形成的影响。供试大丽轮枝菌菌丝生长最适pH为7.0,偏酸或偏碱会抑制菌丝生长,但培养基偏碱可显著促进微菌核形成。当pH为8.0时,大丽轮枝菌菌丝生长受抑制较小,同时微菌核区面积较pH为7.0时增加22.6%。盐分质量浓度影响大丽轮枝菌菌丝生长及微菌核形成。随培养基NaCl质量浓度增加,供试大丽轮枝菌生长受到抑制,菌落面积和菌丝面积均逐渐减小,但微菌核形成量却显著增加;当NaCl质量浓度为10g·L~(-1)时,微菌核区面积较无NaCl时增加40.7%。盐分种类影响供试大丽轮枝菌生长。随盐分质量浓度增加,氯化物(NaCl和KCl)和硫酸盐(Na_2SO_4和MgSO_4)均可促进大丽轮枝菌微菌核形成,而CaCl_2则显著促进菌丝生长,并在质量浓度大于7g·L~(-1)时抑制微菌核形成。在培养环境偏碱性或氯化物和硫酸盐含盐量较高时,均可促进棉花黄萎病原大丽轮枝菌微菌核形成量增加。     

Metabolism of Reactive Oxygen Species in the Cytoplasmic Male-Sterile Cotton Anther

Reactive oxygen species （ROS） in plant cell, including superoxide （O2^-）, hydrogen peroxide （H2O2）, and malondialdehyde （MDA）, are thought to be important inducible factors of cell apoptosis if excessively accumulated in cells. To elucidate the metabolic mechanism of ROS production and scavenging in anthers of the cytoplasmic male-sterile （CMS） cotton, CMS line, maintainer, and hybrid F1 anthers, were employed for studying the relationship between CMS and metabolism of ROS, by comparing ROS changes in the sterile and fertile anthers at different developmental stages. The results showed that during the abortion preliminary stage （sporogenous cell division stage）, anthers of CMS line had higher contents of O2^-, H2O2, and MDA than those of maintainer or hybrid F1. Simultaneously, the higher activities of superoxide dismutase （SOD）, catalase （CAT）, and peroxidase （POD） in scavenging ROS were measured in the anthers of the CMS line, indicating that an increase of ROS in anthers of abortion preliminary stage had an inducible effect on the antioxidant enzymes. But during the abortion peak of CMS anther （pollen mother cell meiosis stage）, on the one hand, contents of O2^-, H2O2, and MDA were extraordinarily high in CMS anthers, on the other hand, the activities of SOD, CAT, and POD were excessively low, which disrupted the balance between the production and elimination of ROS and led to pollen mother cells apoptosis at this stage. In the following two stages （uninucleate microspore stage and mature pollen stage）, the contents of O2^- and H2O2 in the aborted anthers were approximated to contents in the fertile anthers of the maintainer and hybrid F1. However, MDA contents were continuously raised and enzymic activities of SOD, CAT, and POD were consistently decreased in sterile anthers, which indicated that ROS still had harmful effects on the anthers after the apoptosis of the male cells. Excessive accumulation of O2^-, H2O2, and MDA and significant reduction of ROS scavengingenzyme activities were coinstantaneous with male cells apoptosis in the anthers of the cotton CMS line. But when the restorer gene was transferred into the CMS line, excessive production of ROS could be eliminated in the anthers of hybrid F1.     

几丁质降解放线菌对棉花枯、黄萎菌的作用

对从土壤中分离到的58株放线菌进行了皿内拮抗试验及利用几丁质能力测定,在此基础上筛选出了F104,F1052株产几丁质酶的菌株,并测定了其对棉花枯、黄萎菌的离体和活体抑制作用。皿内观察发现,F104和F105均可寄生于棉花黄萎菌,F105还可寄生于棉花枯萎菌。平皿扩散试验结果发现,F104和F105的培养滤液无论经高温处理与否,均能使棉花黄萎菌呈现畸形菌丝,而经高温处理的F104还可使棉花枯萎菌表现为畸形菌丝。抑制靶标致病菌寄主活体定殖作用的研究结果表明,F104和F105处理均表现出不同程度的超前接种防治黄萎病的作用,超前接种F105的间隔期与棉苗中枯萎菌的检出率呈负相关。     

Evolution of virulence in a plant host-pathogen metapopulation

In a wild plant-pathogen system, host resistance and pathogen virulence varied markedly among local populations. Broadly virulent pathogens occurred more frequently in highly resistant host populations, whereas avirulent pathogens dominated susceptible populations. Experimental inoculations indicated a negative trade-off between spore production and virulence. The nonrandom spatial distribution of pathogens, maintained through time despite high pathogen mobility, implies that selection favors virulent strains of Melampsora lini in resistant Linum marginale populations and avirulent strains in susceptible populations. These results are consistent with gene-for-gene models of host-pathogen coevolution that require trade-offs to prevent pathogen virulence increasing until host resistance becomes selectively neutral.     

韭菜干粉及其提取物对大丽轮枝菌的抑菌作用

为探究韭菜干粉及其提取物对大丽轮枝菌的抑菌作用,将韭菜干粉以土壤质量的2%添加到接种有棉花黄萎菌不同菌量密度的土壤中,开展对棉花黄萎病防治效果及土壤中棉花黄萎菌存活影响的调查研究。结果表明,不同接菌量土壤中添加韭菜干粉,均可显著减少土壤中棉花黄萎菌的数量,降低棉花黄萎病的发生程度。韭菜干粉甲醇提取物抑菌活性成分存在于石油醚、乙酸乙酯萃取物中,两种萃取物合并后通过硅胶柱层析分离,共得到21个馏分,其中有9个馏分的抑菌率达到100%,经GC-MS检测出的活性物质主要为含硫化合物、烯醛醇类化合物、杂环类化合物、阿魏酸衍生物和角鲨烯。其中,角鲨烯和阿魏酸甲酯是首次从韭菜中检测出的抑菌活性物质。     

枯萎病菌侵染后棉苗体内超氧物歧化酶和过氧化氢酶活性的变化及与抗病性的关系

分析了枯萎病菌侵染后棉苗叶 片和根茎部组织中超氧物歧化酶（ Ｓ Ｏ Ｄ）和过氧化氢酶 （ Ｃ Ａ Ｔ）活性的变化动态, 结果表明：抗病品种 和感病品种棉苗 中 Ｓ Ｏ Ｄ 和 Ｃ Ａ Ｔ 酶活性无显著 差异;氟乐灵处 理可诱发棉苗对枯萎病的诱导抗性,且氟乐灵处理的棉苗组织中 Ｓ Ｏ Ｄ 和 Ｃ Ａ Ｔ 酶活性无显著变化;枯萎病菌侵染后棉苗叶 片和根茎部组织 中 Ｓ Ｏ Ｄ 和 Ｃ Ａ Ｔ 酶活 性明显提 高,但抗病品 种棉苗组 织中 Ｓ Ｏ Ｄ 和 Ｃ Ａ Ｔ酶活性在前期 增加不显著,后期增 加较慢,而感病品 种棉苗中两种酶活 性在前期就明 显升高,增加 速度快;氟乐灵处理的棉苗,其组织中两种酶活性始终低于未处理棉苗 接种后的水平⒚棉花组织中的 Ｓ Ｏ Ｄ 酶主要是 Ｃｕ, Ｚｎ Ｓ Ｏ Ｄ,受侵后 Ｓ Ｏ Ｄ 酶活性的升高主要来自于 Ｃｕ , Ｚｎ  Ｓ Ｏ Ｄ⒚因此, Ｓ Ｏ Ｄ 和 Ｃ Ａ Ｔ 与棉花对枯萎病的抗性及由氟乐灵诱发的诱导抗性无关⒚     

A LAMP-assay-based specific microbiota analysis reveals community dynamics and potential interactions of 13 major soybean root pathogens

Soybean root diseases are associated with numerous fungal and oomycete pathogens; however, the community dynamics and interactions of these pathogens are largely unknown. We performed 13 loop-mediated isothermal amplification(LAMP) assays that targeted specific soybean root pathogens, and traditional isolation assays. A total of 159 samples were collected from three locations in the Huang-Huai-Hai region of China at three soybean growth stages(30, 60, and 90 days after planting) in 2016. In LAMP results, we found that pathogen communities differed slightly among locations, but changed dramatically between soybean growth stages. Phytophthora sojae, Rhizoctonia solani, and Fusarium oxysporum were most frequently detected at the early stage, whereas Phomopsis longicolla, Fusarium equiseti, and Fusarium virguliforme were most common in the later stages. Most samples(86%) contained two to six pathogen species. Interestingly, the less detectable species tended to exist in the samples containing more detected species, and some pathogens preferentially co-occurred in diseased tissue, including P. sojae–R. solani–F. oxysporum and F. virguliforme–Calonectria ilicicola, implying potential interactions during infection. The LAMP detection results were confirmed by traditional isolation methods. The isolated strains exhibited different virulence to soybean, further implying a beneficial interaction among some pathogens.     

The peroxisomal matrix shuttling receptor Pex5 plays a role in FB1 production and virulence in Fusarium verticillioides

The peroxisomal matrix oxidase, catalase and peroxidase are imported peroxisomes through the shuttling receptors, which regulates the cellular oxidative homeostasis and function. Here, we report that PTS1 shuttling receptor FvPex5 is involved in the localization of PTS1, utilization of carbon sources and lipids, elimination ROS, cell wall stress, conidiation, fumonisin B₁ (FB₁) production, and virulence in maize pathogen Fusarium verticillioides. Significantly, differential expression of PTS1-, PTS2-, PEX- and FB₁ toxin-related genes in wild type and ΔFvpex5 mutant were examined by RNA-Seq analyses and confirmed by RT-PCR assay. In addition, different expression of PTS1 and PTS2 genes of the ΔFvpex5 mutant were enriched in diverse biochemical pathways, such as carbon metabolism, nitrogen metabolism, lipid metabolism and the oxidation balance by combining GO and KEGG annotations. Overall, we showed that FvPex5 is involved in the regulation of genes associated with PTS, thereby affecting the oxidation balance, FB₁ and virulence in F. verticillioides. The results help to clarify the functional divergence of Pex5 orthologs, and may provide a possible target for controlling F. verticillioides infections and FB₁ biosynthesis.     

Population Genetic Analysis of Blumeria graminis f. sp. tritici in Qinghai Province,China

To gain more precise information about molecular genetic variation for wild populations of Blumeria graminis f. sp. tritici from Qinghai Province, China, 38 single-colony isolates were purified from samples collected from Haidong District, Xining City and Hainan Tibetan Autonomous Prefecture in 2010. The virulence of 21 isolates among them was tested at seedling stage on 34 wheat cultivars (lines) carrying known powdery mildew (Pm) resistant genes. The results showed that V1a, V3a, V3c, V3e, V5a, V6, V7, V8 and V19 had high virulence frequencies (>75%), indicating a wide distribution; and V1c, V5b, V12, V13, V16, V21, VXBD, V2+6, V2+Mld and V4+8, with less distribution, appeared to be lower in frequencies (0-20%). The Nei's gene diversity (H), Shannon's information index (I) and the percentage of polymorphic loci (P) were 0.23, 0.35 and 67.65%, respectively, which revealed a virulent diversity. The results from single nucleotide polymorphisms (SNPs) of 38 isolates showed that three housekeeping genes were found to contain a total of 9 SNP sites. 10 haplotypes (H1-H10) were inferred from the concatenated sequences, with 1 haplotype (H1) comprising of over 55% of Qinghai population. Phylogenic analysis did not show obvious geographical subdivision between the isolates. A multilocus haplotype network presented a radial structure, with H1 in the central as an inferred ancestor. Using analysis of molecular variance (AMOVA), we found 1.63% of the total variation was among populations and 98.37% within populations, with a low fixations index (F_ST=0.01634, P<0.05). This revealed a relatively high genetic diversity but a low genetic divergence in Qinghai population. Moreover, the molecular data on gene flow (Nm=6.32) confirmed the migration of pathogen populations among areas in Qinghai Province.     

Induction of nitric oxide-dependent apoptosis in motor neurons by zinc-deficient superoxide dismutase

Mutations in copper, zinc superoxide dismutase (SOD) have been implicated in the selective death of motor neurons in 2 percent of amyotrophic lateral sclerosis (ALS) patients. The loss of zinc from either wild-type or ALS-mutant SODs was sufficient to induce apoptosis in cultured motor neurons. Toxicity required that copper be bound to SOD and depended on endogenous production of nitric oxide. When replete with zinc, neither ALS-mutant nor wild-type copper, zinc SODs were toxic, and both protected motor neurons from trophic factor withdrawal. Thus, zinc-deficient SOD may participate in both sporadic and familial ALS by an oxidative mechanism involving nitric oxide.     

黄萎菌诱导棉花活性氧及保护酶系的变化研究

以棉花耐黄萎病品种中棉41和感黄萎病品种冀棉11为材料,通过测定黄萎菌诱导前后活性氧积累及保护酶系活性的变化,研究其与棉花抗性的关系。结果表明:在接种黄萎菌后36h,2个品种的棉花叶片O2-产生速率均达到高峰,而中棉41O2-产生速率明显高于冀棉11;同时,叶片内丙二醛(MDA)的含量也均达到一个高峰,中棉41中MDA含量高于冀棉11;在接种后24h和36h,冀棉11和中棉41棉花叶片中超氧化物歧化酶(SOD)活性分别达到一个高峰,且冀棉11变化幅度大于中棉41;对于过氧化物酶(POD)的活性,在接种后24h,2个品种均达到一个高峰,但是耐病品种中POD活性增加幅度不及感病品种。在棉花与黄萎病菌互作的过程中,活性氧和保护酶系参与不同的抗性反应途径。     

棉花黄萎病菌毒素对棉花生化代谢的影响

为明确棉花黄萎病菌毒素对棉花生化代谢的影响,采用棉花黄萎病菌毒素处理棉花黄萎病抗病和感病品种的棉苗后,测定棉株体内超氧化物歧化酶(SOD)、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)的活性以及丙二醛(MDA)和木质素含量的动态变化。结果表明,抗病品种陕7191对病菌毒素的抗性显著高于感病品种中棉19;接种毒素后2品种棉苗叶片SOD活性都降低,感病品种降幅高于抗病品种;感病品种POD活性升高较快,到达峰值后迅速下降,而抗病品种POD活性持续上升;2品种PAL活性变化趋势相似,接种后抗病品种活性始终高于感病品种;2品种MDA和木质素含量均增多,感病品种MDA含量的增幅和增量高于抗病品种。