FpStuA参与假禾谷镰孢的产孢和致病性

 假禾谷镰孢是一种土传真菌,其引起的小麦茎基腐病已成为威胁我国小麦生产的重要病害。APSES蛋白是真菌中保守存在的一类转录因子,参与多种细胞生理过程。本研究,我们在假禾谷镰孢中鉴定到StuA同源蛋白FpStuA。qRT-PCR分析发现FpStuA在假禾谷镰孢分生孢子和侵染阶段诱导表达。通过PEG介导的原生质体转化方法获得3个FpStuA基因缺失的突变体。与假禾谷镰孢野生型菌株相比,Δfpstua突变体的生长速度明显减慢,气生菌丝减少;分生孢子的产生比野生型减少70%,且Δfpstua突变体分生孢子变短、分隔减少;Δfpstua突变体对大麦叶片、小麦胚芽鞘和小麦根的致病性均显著降低,脱氧雪腐镰刀菌烯醇(DON)合成明显减少。综上结果,转录因子FpStuA对假禾谷镰孢的生长、产孢和致病性都非常重要。     

Stomatal penetration of cowpea (Vigna unguiculata) leaves by a Colletotrichum species causing latent anthracnose

The infection process of a Colletotrichum species causing latent infection and anthracnose in cowpea ( Vigna unguiculata ) was studied in seedlings by light and confocal microscopy. Leaf surfaces were extensively colonized by an anastomosing network of germ-tubes and conidia. This epiphytic mycelium produced abundant secondary conidia on short conidiophores. Although melanized appressoria were developed, the host surface was not penetrated directly. The fungus only gained ingress into leaves through stomatal openings, by means of undifferentiated germ-tubes, and slowly colonized the mesophyll by intercellular hyphae, without initially producing visible symptoms. Anthracnose lesions with multisetate acervuli appeared on senescent leaves after a prolonged symptomless period of host colonization lasting > 2 weeks. Analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed close similarities (95–96%) between this cowpea pathogen and isolates of C . gloeosporioides from Aeschynomene virginica, Stylosanthes scabra and Mangifera indica. These results, in addition to other morphological and growth attributes, identify this endophytic anthracnose pathogen of cowpea as a Colletotrichum species distinct from C. capsici and C. destructivum .     

Vision and development in Trichoderma atroviride

Phototropism, the induction of carotenogenesis and reproductive structures, and resetting of the circadian rhythm are controlled by blue light. Trichoderma is used as a photomorphogenetic model due to its ability to conidiate upon exposure to light. In total darkness, T. atroviride grows indefinitely as a mycelium provided that nutrients are not limiting. However, nutrient deprivation and light trigger the conidiation process. A pulse of blue light given to a radially growing colony induc…     

Genetic regulation of conidiation in Trichoderma hamatun

Achieving a balance between vegetative growth and spore production is essential for successful biocontrol by fungi. Low sporulation rates in the field can result in poor establishment and survival,whereas failure of conidia to recognise hosts can lead to persistence without efficacy. Commercial biocontrol products involve bulk preparations of conidia, however considerable variability in conidiation rates exists between biocontrol agents, which can restrict choice of strain for production. The majority of studies on Trichoderma conidiation have focused on the species T. viride and T. atroviride.These species form conidia in response to blue and near-UV light and/or nutrient deprivation and conidiation proceeds in a highly co-ordinated fashion, however relatively little is known on the genetic basis of Trichoderrma conidiation. In addition, whilst photoconidiation appears to be a general response detailed studies in other Trichoderma species are absent. In this study, conidiation in the lesser known biocontrol species T. hamatum is being investigated using a combined morphological and molecular approach. In contrast to T. atroviride, conidiation in response to blue-light was weaker and variable and suggested that additional triggers may be required for the T. hamatum photoresponse. A series of comparative photoconidiation assays are currently being undertaken investigating the effect of inoculum type and abiotic factors on timing and intensity of the response.Results will be discussed in relation to the current knowledge on conidial morphogenesis in Trichoderma. In addition to these morphological assays, a selection of genes implicated in sporulation and the blue-light responses are currently being isolated and characterised from T. hamatum. Two genes, phr1 and cmp1 , which were isolated previously from T. atroviride will be used as early and late markers of gene expression during the photoresponse in T. hamatum in order to define time points for harvesting comparable stage-specific RNA from T. hamatum and T. atroviride. Using degenerate PCR putative sporulation gene orthologues have also been identified in T. hamatum.Work is currently underway to isolate genomic clones of these genes from T. hamatum and T.atroviride. Sequence and expression analysis of orthologues, including expression in response to abiotic factors will be presented and discussed in relation to the current knowledge of the molecular basis of conidiation in Trichoderma and other filamentous fungi.……     

3株环链拟青霉固体培养条件的研究

通过测定菌落直径和产孢量,研究不同温度、培养基和光照时间对3株环链拟青霉的菌丝生长和产孢量的影响.结果表明,在23℃时,3个菌株菌落的直径生长最快,菌株Pc45和Pc287的产孢最多,而菌株Pc305在27℃下产孢最多.与常用的PDA和SDAY培养基相比,3个菌株在改良米饭-蛋白胨培养基上的菌落直径生长和产孢量均显著高于其他培养基;Mg2 和K 对产孢有重要影响.3个菌株均以光照24 h下的产孢量最高,而在0 h下,菌落直径最大,产孢量最小.     

蓝光促进绿僵菌产孢和产孢调节基因fluG表达

【目的】明确蓝光照射对绿僵菌产孢的促进作用及与产孢调节基因fluG表达量的关系,为绿僵菌发酵生产提供光照促进产孢的理论依据和技术参数。【方法】以绿僵菌高效杀虫菌株M202为试材,平板接种培养,通过显微观察每隔12 h的菌体发育状态,确定菌株发育进程产孢前期、初始期、旺盛期、平稳期的对应时段。以蓝光6 804-54 432 J?m^-2的8个能量梯度,照射处理在黑暗中培养至不同发育期即24、48、72 h的菌体,继续培养到产孢稳定期后,采用打孔法取样,以显微计数法检测计算产孢量,评估菌体发育阶段对蓝光的敏感性以及蓝光照射能量对产孢量的影响。克隆fluG,建立fluG real-time PCR反应体系;以蓝光6 804-54 432 J?m^-2的8个能量梯度,照射处理发育至48 h即处于产孢前将进入产孢期的菌丝体,照射处理后立即取菌丝,液氮冷冻,提取RNA并反转录成cDNA,通过real-time PCR检测fluG表达量,评估蓝光照射对fluG表达量的影响。【结果】绿僵菌M202菌株发育24 h前为萌发期,24-72 h为菌丝快速生长期,其中48 h还处于产孢前期,60 h已进入产孢初期,72-96 h为产孢盛期,96-120 h为产孢末期,120 h后为孢子成熟期,7-10 d后产量达到稳定。蓝光不同能量照射24 h菌龄即初期菌丝体,其产孢量与无光照处理的对照无显著差异,照射48 h菌龄即菌丝快速生长的产孢前期,其产孢量显著提高,最适照射能量为20 412-40 824 J?m^-2,其中34 020 J?m^-2使产孢量最高达无光照处理对照的1.50倍,72 h菌龄即产孢结构形成、产孢量快速增长期对蓝光最为敏感,低至6 804 J?m^-2的照射能量即可显著提高产孢量,且宽泛的各剂量均有效。对于fluG的表达,在试验照射剂量范围内,48 h菌龄接受蓝光照射后,fluG表达量显著提高,表达量随照射剂量呈先上升后下降的趋势,在20 412 J?m^-2以下较低能量时,fluG表达量与照射剂量成正相关,当照射时间为2.25 h时,表达量达到最高,为无光照处理对照的2.41倍,而在20 412 J?m^-2以上较高能量时,二者呈现负相关,随着蓝光照射时间的增加,基因表达量呈下降趋势;灰色关联度分析显示,当关联因子为0.5时,产孢量与fluG表达量的关联度r值为0.74。【结论】蓝光照射能够促进绿僵菌产孢及fluG表达,不同发育阶段的菌体对蓝光感应有显著差异,在48-72 h菌龄时照射34 020 J?m^-2能量可获得最高产孢量,产孢与fluG之间有较高关联度说明fluG参与绿僵菌产孢的调控。     

棉花黄萎病菌VdHP1的克隆及功能分析

【目的】明确棉花黄萎病菌（大丽轮枝菌Verticillium dahliae）中一个新基因（VdHP1）的功能,为解析棉花黄萎病菌的致病机制以及棉花黄萎病的防治提供依据。【方法】以大丽轮枝菌野生型菌株V592的基因组DNA和cDNA为模板,对VdHP1全长进行克隆并测序;利用逆转录实时荧光定量PCR（RT-qPCR）分别对棉花根系诱导不同时间VdHP1的表达量及V592菌株不同组织中VdHP1的表达量进行测定;构建针对VdHP1的敲除载体、互补载体和过表达载体,通过农杆菌介导的遗传转化筛选VdHP1基因敲除突变体、互补菌株和过表达菌株;以野生型菌株V592为对照,对VdHP1基因敲除突变体及互补菌株的菌落及菌丝形态进行观察,并对微菌核量、产孢量及致病力进行测定;通过RT-qPCR测定其他致病力相关的基因在VdHP1基因敲除突变体及过表达体菌株中的表达情况。【结果】VdHP1全长为862bp,预测编码蛋白含268个氨基酸,与GenBank中已注释的基因没有任何的序列相似性。野生型菌株V592受棉花根系诱导6—12 h时VdHP1表达水平显著上调,表明VdHP1在大丽轮枝菌侵染早期发挥作用。VdHP1在分生孢子中的表达量显著高于在菌丝和微菌核中的表达量,表明VdHP1在大丽轮枝菌不同组织中的表达具有差异性。与野生型菌株V592相比,VdHP1基因敲除突变体产孢量和产孢梗显著减少,菌丝分支呈螺旋状,对棉花的致病力明显下降。与侵染钉形成相关基因（VdCrz1、VdNoxB、VdPls1）、分泌蛋白释放相关基因（VdSep5）及分生孢子产生相关基因（Vdpf、VdSge1、VGB、VdPLP、VdCYC8、VdNLP1、VdNLP2）在VdHP1基因敲除体中的相对表达量显著下调,在过表达菌株中上调;而与黑色素合成相关基因（VdCmr1、VdSho1、VdLAC、VdPKS1）在VdHP1基因敲除突变体中则显著上调,在过表达菌株中下调。【结论】VdHP1与大丽轮枝菌分生孢子和产孢梗的产生有关,参与大丽轮枝菌致病;VdHP1对与侵染钉形成、分泌蛋白释放及分生孢子产生相关基因的表达具有正调控作用,对黑色素合成相关基因的表达具有负调控作用。     

粉拟青霉不同菌株生物学特性的研究

比较了15株来自不同地域和寄主的粉拟青霉生物学特性,明确了它们在产孢量、生长速度、抗旱能力、抗紫外能力以及产胞外蛋白酶等方面的差异.结果表明,粉拟青霉菌落生长和产生孢子的最适温度为20℃.在此温度下,24 h孢子萌发率在80%～100%之间;此外不同菌株的抗旱力差异较大,Pf20抗旱能力最强,而Pf127的抗旱能力最弱.经紫外线照射5 min,菌株的萌发率在44%～81%范围内;不同菌株的产胞外蛋白酶水平差异较大,以Pf96最高,高达2.3.     

Genetic regulation of conidiation in Trichoderma hamatum

Achieving a balance between vegetative growth and spore production is essential for successful biocontrol by fungi. Low sporulation rates in the field can result in poor establishment and survival, whereas failure of conidia to recognise hosts can lead to persistence without efficacy. Commercial biocontrol products involve bulk preparations of conidia, however considerable variability in conidiation rates exists between biocontrol agents, which can restrict choice of strain for production. The…     

Functional Characterization of a NEM1-Like Gene in Magnaporthe oryzae

Magnaporthe oryzae, a filamentous ascomycete fungus, is well known as the causal agent of rice blast. With the technology of suppression subtractive hybridization (SSH), it was previously found that MGG_06001 (or named MoNEMl), a gene of M. oryzae homologous to the NEMl (nuclear envelope morphology protein 1) gene of baker's yeast (Saccharomyces cerevisiae), is differentially expressed between the mature appressium and the conidium and mycelium. This study aimed to characterize the function of MoNEMl gene by knocking it out using the method of target gene replacement. The AMoneml mutants exhibited reduced mycelial growth and conidiation. However, disruption of MoNEMl gene does not affect the pathogenicity of M. oryzae on barley and rice.