禾谷镰孢核孔蛋白基因FgNup42的功能分析

【目的】核孔蛋白Nup42在真核生物基因表达调控以及mRNA加工运输等生物学过程中发挥着重要作用,本研究旨在分析禾谷镰孢（Fusarium graminearum）中核孔蛋白基因FgNup42在病原菌生长发育、逆境胁迫、致病和产毒等生物学过程中的功能。【方法】通过融合PCR（double-joint PCR）和酵母空隙修复（gap repair）技术分别构建FgNup42基因敲除和回补载体,再利用PEG介导的原生质体转化的方法获得基因敲除突变体ΔFgNup42和回补体ΔFgNup42-C。观察测定基因敲除突变体ΔFgNup42在营养生长、无性繁殖和有性生殖过程中的变化,同时测定突变体ΔFgNup42对渗透、杀菌剂以及细胞壁胁迫因子的敏感性。将突变体ΔFgNup42进行田间麦穗和室内玉米花丝接种试验明确其致病力情况。通过液相色谱-串联质谱（LC-MS/MS）检测ΔFgNup42的产毒能力,同时利用qRT-PCR比较分析参与单端孢霉烯族毒素生物合成的7个TRI在野生型PH-1和基因敲除突变体ΔFgNup42中的相对表达量。【结果】表型测定发现,基因敲除突变体ΔFgNup42的生长速率只有野生型PH-1的50%,菌落边缘菌丝分枝变多且致密。显微观察分生孢子形成情况,发现敲除突变体ΔFgNup42相较野生型PH-1分生孢子产量降低了85.45%,并且隔膜数在0—2的分生孢子比例明显增多。有性生殖诱导结果显示ΔFgNup42的有性生殖能力增强,较野生型产生了更多的子囊壳。突变体ΔFgNup42对渗透胁迫因子NaCl和KCl,细胞壁胁迫因子刚果红,以及杀菌剂戊唑醇和氰烯菌酯的敏感性减弱。致病力分析发现敲除基因FgNup42后菌体在麦穗和玉米花丝上的致病力严重降低。此外,与野生型相比,ΔFgNup42中毒素DON、3ADON和15ADON的合成量明显减少。【结论】核孔蛋白基因FgNup42在禾谷镰孢生长发育、抵御逆境以及致病和产毒过程中发挥着重要作用。     

Functional characterization of the catalytic and bromodomain of FgGCN5 in development,DON production and virulence of Fusarium graminearum

FgGCN5, a GCN5 homolog in Fusarium graminearum, plays a critical role in hyphal vegetative growth, asexual and sexual reproduction, deoxynivalenol(DON) biosynthesis and plant infection. For nuclear localized GCN5, four conserved sequence motifs(I–IV) are presented in the catalytic domain and a bromodomain in the carboxy-terminus. As a lysine acetyltransferase, conserved negatively charged residues are present to neutralize the protons from lysine substrates. However, the role of conserved motifs/domains and residues in FgGCN5 are unclear. Here, we generated deletion mutant strains for each the conserved motifs/domains and a glutamate residue 130(E130) replacement mutant. Deletion of each conserved motif in the catalytic domain and replacement of E130 site resulted in manifold defects in hyphae growth, asexual and sexual development, DON biosynthesis, and plant infection. Phenotypic defects in the mutant strains were similar to deletion mutants. The deletion of the bromodomain led a significant reduction in DON production and virulence, with no effects on hyphae growth, asexual or sexual reproduction. FgGCN5 was further found to localize to the nucleus in conidia and hyphae cells. In conclusion, FgGCN5 encodes a nuclear localized acetyltransferase. The conserved motifs in the catalytic domain and E130 are essential for correct functions of the gene. The conserved bromodomain is important for DON production and pathogen virulence. This was the first report to identify the functions of conserved motifs/domains in FgGCN5, which will contribute to our understanding of the mechanism(s) by which FgGCN5 regulates F. graminearum.     

FgHAT2 is involved in regulating vegetative growth,conidiation, DNA damage repair,DON production and virulence in Fusarium graminearum

Histone lysine acetylation is catalyzed by acetyltransferases(HATs), which is important in regulating gene expression and physiological function in eukaryotic cells. HATs can be classified into two main types: A-and B-type HATs. Recently, in Fusarium graminearum, it has been reported that A-type HATs are involved in hyphal development, conidiation, sexual reproduction and virulence. However, the biological roles of B-type HATs are unknown. Here we report the identification and characterization of two B-type HATs(FgHat1 and FgHat2) in F. graminearum. Targeted deletion of FgHAT1 did not result in any detectable phenotypes. However, ΔFghat2 mutants were severely defective in vegetative growth, conidia production and morphogenesis, deoxynivalenol(DON) biosynthesis and virulence. Interestingly, deletion of Fg HAT2 resulted in significantly increased sensitivity to the DNA-damaging agent methyl methanesulfonate(MMS). Furthermore, double deletion mutants(ΔFghat1ΔFghat2) displayed similar phenotypes to the ΔFghat2 mutants. Taken together, we conclude that FgHat2 but not FgHat1 plays essential roles in regulating morphogenesis, DNA damage repair, DON production and virulence in F. graminearum.     

SsRSS1 mediates salicylic acid tolerance and contributes to virulence in sugarcane smut fungus

Sugarcane smut caused by Sporisorium scitamineum is a destructive disease responsible for significant losses in sugarcane production worldwide. However, the mechanisms underlying the pathogenicity of this fungus remain largely unknown. In this study, we found that the disruption of the SsRSS1 gene, which encodes a salicylic acid (SA) sensing regulator, does not affect phenotypic traits such as the morphology, growth rate, and sexual mating ability of haploid basidiospores, but rather reduces the tolerance of basidiospores to SA stress by blocking the induction of SsSRG1, a gene encoding a SA response protein in S. scitamineum. SsRSS1 deletion resulted in the attenuation of the virulence of the fungus. In addition to a significant reduction in whip formation, a portion of plantlets (18.3%) inoculated with the ΔSsRSS1 strains were found to be infected but failed to produce whips for up to 90 days post-inoculation. However, the development of hyphae and teliospore from the ΔSsRSS1-infected plants that formed whips seemed indistinguishable from that in the wild-type-infected plants. Combined, our findings suggested that SsRss1 is required for maintaining fungal fitness in planta by counteracting SA stress.     

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.     

The putative elongator complex protein Elp3 is involved in asexual development and pathogenicity by regulating autophagy in the rice blast fungus

Autophagy is responsible for maintaining fundamental cellular homeostasis and is, therefore, essential for diverse development processes. This study reported that PoElp3, the putative catalytic subunit of Elongator complex, is involved in the maintenance of autophagy homeostasis to facilitate asexual development and pathogenicity in the rice blast fungus Pyricularia oryzae. It was found that the ΔPoelp3 strains were defective in vegetative growth, conidiation, stress response, and pathogenicity. The mutants exhibited hyper-activated autophagy in the vegetative hyphae under both nutrient-rich and nutrient-deficient conditions. The hyper-activation of autophagy possibly suppressed the production of vegetative hyphae in the ΔPoelp3 strains. Moreover, the ΔPoelp3 strains were found to be more sensitive to rapamycin during vegetative- and invasive-hyphal growth but have no effect on Target-of-Rapamycin (TOR) signaling inhibition. Taken together, these results demonstrated that PoElp3 is involved in asexual development and pathogenicity by regulating autophagy in the rice blast fungus.     

肌动蛋白结合蛋白FgAbp1参与禾谷镰孢生长、发育和毒素体形成

【目的】Abp1作为肌动蛋白结合蛋白家族成员之一,在各种真核生物肌动蛋白骨架形成过程中具有核心作用。本研究旨在分析禾谷镰孢（Fusarium graminearum）中肌动蛋白结合蛋白FgAbp1在生长发育、对新型杀菌剂氰烯菌酯敏感性以及毒素体形成等生物学过程中的功能。【方法】利用融合PCR和酵母空隙修复技术分别构建FgAbp1基因敲除打靶片段和融合荧光蛋白载体,再通过聚乙二醇介导的原生质体转化的方法获取基因缺失突变体ΔFgAbp1和荧光标记菌株。观察比较野生型PH-1、突变体ΔFgAbp1和回补体ΔFgAbp1-C的菌丝生长、有性生殖以及无性繁殖,并测定基因敲除突变体ΔFgAbp1对杀菌剂氰烯菌酯的敏感性。通过融合绿色荧光蛋白,明确FgAbp1在菌丝中的分布情况;利用透射电子显微镜观察分析FgAbp1对细胞中液泡/囊泡形态的影响。在非产毒环境和诱导产毒条件下,通过双荧光共定位分析FgAbp1在禾谷镰孢毒素体形成过程中的作用。【结果】禾谷镰孢中,FgAbp1主要呈颗粒状定位于近细胞膜处。在MM培养基中,基因敲除突变体ΔFgAbp1的生长速率与野生型相比降低了15%,但在营养丰富的CM中ΔFgAbp1的生长速率却减慢了38%。突变体ΔFgAbp1在无性繁殖以及有性生殖过程中相较野生型没有明显缺陷。然而在0.5 μg·mL^-1氰烯菌酯的作用下,ΔFgAbp1的菌丝生长完全受到抑制,并且分生孢子萌发率显著下降。此外,敲除基因FgAbp1导致细胞中液泡不能正常形成且囊泡增多。在非产毒条件下,FgAbp1与DON毒素合成关键酶Tri1共定位于囊泡中;在诱导产毒条件下,FgAbp1与Tri1则共定位于毒素体中。此外,敲除基因FgAbp1会导致毒素体不能正常形成。【结论】肌动蛋白结合蛋白FgAbp1在禾谷镰孢营养生长、发育、氰烯菌酯敏感性以及毒素体形成过程中发挥着重要作用。     

Effects of salt and nitrogen on physiological indices and carbon isotope discrimination of wheat cultivars in the northeast of Iran

In order to study the effects of different levels of salt stress and nitrogen(N) on physiological mechanisms,carbon isotope discrimination(△~(13)C),and yield of two wheat cultivars(cv.),a two-year field experiment was carried out during 2013-2015.The treatments included three levels of salt stress(1.3,5.2,and 10.5 dS m~(-1)),three levels of N(50,100,and 150 kg N ha~(-1)),and two wheat cultivars,Bam and Toos.Under salt stress,N application(100 and 150 kg N ha~(-1)) produced a significant effect on both cultivars with respect to physiological traits,i.e.,net photosynthetic rate(P_n),stomatal conductance(g_s),chlorophyll index(Cl),Na~+/K~+ratio as well as the grain yield(GY).The salt-tolerant and-sensitive cultivars exhibited the maximum values of physio-biochemical and yield attributes at 100 and 150 kg N ha~(-1),respectively.The results of △~(13)C showed a significant difference(P0.001) between wheat cultivars under the control and salt stress.According to our result,salt-tolerant cultivar Bam seems to be more efficient in terms of higher GY,P_n,g_s,Cl,and lower Na~+/K~+ratio as well as higher △~(13)C as compared with salt-sensitive cultivar Toos,under salt stress.Therefore,a significant positive correlation that observed between △~(13)C and GY,indicated that △~(13)C may be an effective index for indirect selection of yield potential in wheat under irrigation regimes with saline water.     

Mitochondrial dynamics caused by QoIs and SDHIs fungicides depended on FgDnm1 in Fusarium graminearum

Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating fungal disease on small grain cereal crops, because it reduces yield and quality and causes the mycotoxin contamination to the grain. Dynamins and dynamin-related proteins (DRPs) are large GTPase superfamily members, which are typically involved in the budding and division of vesicles in eukaryotic cells, but their roles in Fusarium spp. remain unexplored. Here, we found that FgDnm1, a DRP and homolog to Dnm1 in Saccharomyces cerevisiae, contributes to the normal fungal growth, sexual reproduction and sensitivity to fungicides. In addition, we found FgDnm1 co-localizes with mitochondria and is involved in toxisome formation and deoxynivalenol (DON) production. Several quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) cause fragmentated morphology of mitochondria. Importantly, the deletion of FgDnm1 displays filamentous mitochondria and blocks the mitochondrial fragmentation induced by QoIs and SDHIs. Taken together, our studies uncover the effect of mitochondrial dynamics in fungal normal growth and how such events link to fungicides sensitivity and toxisome formation. Thus, we concluded that altered mitochondrial morphology induced by QoIs and SDHIs depends on FgDnm1.     

Effects of sodium benzoate on growth and physiological characteristics of wheat seedlings under compound heavy metal stress

In this study, we investigated the effect of exogenous sodium benzoate on wheat seedlings(Yangmai 16) grown under heavy metal stress. The results showed that 2.4 mmol kg~(–1) of heavy metals significantly inhibited growth and delayed emergence of wheat seedlings. Under compound heavy metal stress, application of 2–4 g L~(–1) sodium benzoate significantly increased(P0.01) chlorophyll content and chlorophyll fluorescence parameters F_v/F_m and F_v/F_o of wheat, compared to the control(water treatment). Further analysis showed that application of 2–4 g L~(–1) sodium benzoate alleviated osmotic stress by promoting the accumulation of osmolytes such as soluble proteins and free proline, increased the activity of superoxide dismutase(SOD) and reduced malondialdehyde content(MDA). In contrast, higher concentrations of sodium benzoate solution(6 g L~(–1)) inhibited the growth of wheat seedlings and even caused damage to seedlings. Correlation analysis showed that when the sodium benzoate concentration was in the range of 1.97–3.12 g L~(–1)(2016) and 1.58–3.27 g L~(–1)(2017), values of chlorophyll and its components, root activity, SOD activity, soluble protein, and free proline content were the highest. When the sodium benzoate concentration was raised to 2.59 g L~(–1)(2016) or 3.02 g L~(–1)(2017), MDA content was the lowest. Ultimately, exogenous sodium benzoate(2–4 g L~(–1)) facilitates root development and improves the root activity of wheat seedlings grown under compound heavy metals stress, thereby effectively alleviating the damage of compound heavy metal stress in wheat seedlings.     

FpPDE1 function of Fsarium pseudograminearum on pathogenesis in wheat

Wheat crown rot caused by Fusarium spp.is a common disease worldwide.Both Fusarium pseudograminearum and Fusarium graminearum infect wheat crown and produce mycotoxin leading to grain loss due to white head.F.pseudograminearum(Fp) was reported in wheat from Henan Province of China a couple of years ago.The wheat crown rot(CR) caused by this new pathogen is as an emerging severe disease of wheat,which has recently expanded to several provinces in China and is,therefore,under rapid investigation.Colonization of wheat tissue by Fp is accomplished though the formation of a septated foot-shaped appressoria and generation of a penetration peg to break through the internal cells of leaf sheath.The molecular mechanism by which Fp regulates the pathogenesis on wheat host is unclear.Here,we report FpPDE1,a P-type ATPase-encoding predicted PDE1 orthologue gene of Magnaporthe oryzae,belonging to the DRS2 subfamily of aminophospholipid translocases.The gene deletion of FpPDE1 with the split-marker approach did not obviously affect hyphae growth and conidiation,but led to an attenuated virulence on wheat base stem and root.Our finding indicates that the putative aminophospholipid translocases is not essential for the infectious hyphae development in Fp.     

IPM Strategies and Their Dilemmas Including an Introduction to www. eurowheat.org

Information about disease management in winter wheat （Triticum aestiva） in eight European countries was collated and analysed by scientists and extension workers within the European Network for the Durable Exploitation of Crop Protection Strategies （ENDURE）. This included information about specific disease thresholds, decision support systems, host varieties, disease prevalence and pathogen virulence. Major differences in disease prevalence and economic importance were observed. Septoria tritici blotch （Mycosphaerella graminicola） was recognized as the most yield reducing disease in countries with intensive wheat production, but also rust diseases （Puccinia striiformis and Puccinia triticina）, powdery mildew （Blumeria graminis） and Fusarium head blight （Fusarium spp.） were seen as serious disease problems. Examples of current integrated pest management （IPM） strategies in different countries have been reported. Disease management and fungicide use patterns showed major differences, with an average input equivalent to 2.3 full dose rates （TFI） in the UK and a TFI of 0.6 in Denmark. These differences are most likely due to a combination of different cropping systems, climatic differences, disease prevalence, and socio-economic factors. The web based information platform www.eurowheat.org was used for dissemination of information and results including information on control thresholds, cultural practices which can influence disease attack, fungicide efficacy, fungicide resistance, and pathogen virulence, which are all elements supporting 1PM for disease control in wheat. The platform is open to all users. The target groups of EuroWheat information are researchers, advisors, breeders, and similar partners dealing with disease management in wheat.     

Development of a method using multispectral imagery and spatial pattern metrics to quantify stress to wheat fields caused by Diuraphis noxia

The Russian wheat aphid, Diuraphis noxia, is an important pest of winter wheat, Triticum aestivum, and barley, Hordeum vulgare that has caused an annual economic loss estimated at over 1 billion dollars since it first appeared in the United States. The objective of this study was to determine the potential of combining multispectral imagery with spatial pattern recognition to identify and spatially differentiate D. noxia infestations in wheat fields. Multispectral images were acquired using an MS3100-CIR multispectral camera. D. noxia, drought, and agronomic conditions were identified as major causes for stresses found in wheat fields. Seven spatial metrics were computed for each stress factor. The analysis of spatial metrics quantitatively differentiated the three types of stress found within wheat fields. Detection and differentiation of wheat field stress may help in mapping stress and may have implications for site-specific monitoring systems to identify D. noxia infestations and help to target pesticide applications.     

Involvement of the autophagy-related gene BdATG8 in development and pathogenicity in Botryosphaeria dothidea

Botryosphaeria dothidea is a destructive fungal pathogen that causes Botryosphaeria canker and fruit ring rot on apple worldwide. Autophagy is a process of self-degradation that maintains intracellular homeostasis via lysosomal pathway. To date, the biological role of autophagy in B. dothidea remains unknown. In this study, we identified and characterized the autophagy-related gene BdATG8 in B. dothidea. BdATG8 was able to restore the defect in nitrogen starvation tolerance of Saccharomyces cerevisiae ATG8 deletion mutant. GFP-BdAtg8 was shown to be a useful marker for monitoring autophagy in B. dothidea. Target deletion of BdATG8 (ΔBdAtg8) blocked autophagy and significantly impaired mycelial growth, conidiation and perithecium formation. In addition, ΔBdAtg8 showed significantly increased sensitivity to phytoalexin and oxidative stress, suggesting that BdATG8 plays critical roles in overcoming phytoalexin and reactive oxygen species (ROS)-mediated plant immunity. Pathogenicity assays revealed that ΔBdAtg8 almost lost ability to infect hosts. Overall, our results indicate that BdATG8 plays an important role in fungal development, stress responses and pathogenesis in B. dothidea.     

宁夏小麦赤霉病禾谷镰刀菌致病性分化的研究

将宁夏小麦赤霉病禾谷镰刀菌单饱分离的纯培养菌系10个有效菌株,分别接种在12个小麦品种和宁夏主栽小麦品种永良4号上,结果表明,禾谷镰刀菌系间存在较明显的致病性分化现象,对于不同品种其致病力不同,菌系和小麦品种间均表现出不同的反应。将宁夏小麦赤霉病禾谷镰刀菌的70个单孢纯培养菌株接种在宁夏主栽品种永良4号上,结果显示中致病力菌株占41.4%,弱致病力菌株占40%,强致病力菌株占18.6%。     

Marker-assisted selection to pyramid Fusarium head blight resistance loci Fhb1 and Fhb2 in the high-quality soft wheat cultivar Yangmai 15

Fusarium head blight (FHB) is one of the most detrimental wheat diseases which greatly decreases the yield and grain quality, especially in the middle and lower reaches of the Yangtze River of China. Fhb1 and Fhb2 are two major resistance loci against Fusarium graminearum. Yangmai 15 (YM15) is one of the most popular varieties in the middle and lower reaches of the Yangtze River, and it has good weak gluten characters but poor resistance to FHB. Here we used Fhb1 and Fhb2 to improve the FHB resistance of YM15 by a molecular marker-assisted selection (MAS) backcrossing strategy. The selection of agronomic traits was performed for each generation. We successfully selected seven introgressed lines which carry homozygous Fhb1 and Fhb2 with significantly higher FHB resistance than the recurrent parent YM15. Three of the introgressed lines had agronomic and quality characters that were similar to YM15. This study demonstrates that the pyramiding of Fhb1 and Fhb2 could significantly improve the FHB resistance in wheat using the MAS approach.     

小麦禾谷镰刀菌毒素抗性系的筛选及抗性特性

以稳定生长的小麦（抗盐０３×Ｎｏ１８）Ｆ１花药愈伤组织为材料，小麦强致病禾谷镰刀菌的粗毒素为选择剂，进行抗毒素变异细胞系的离体筛选。结果从多步正筛选系统和一步正筛选系统分别获得抗性变异细胞系ＫＮｒ－ａ和ＫＮｒ－ｂ．抗性系的毒素抗性具有显著提高，且其细胞膜透性、硝酸还原酶活性、苯丙氨酸解氨酶活性、游离氨基酸组成均发生了明显变化。据此提出了细胞水平上禾谷镰刀菌毒素作用的机理及抗性变异细胞系对毒素表现出一定抗性的可能原因