A Novel Two-Component Response Regulator Links rpfwith Biofilm Formation and Virulence of Xanthomonasaxonopodis pv. Citri

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a serious disease that impacts citrus productionworldwide, and X. axonopodis pv. citri is listed as a quarantine pest in certain countries. Biofilm formation is important forthe successful development of a pathogenic relationship between various bacteria and their host(s). To understand themechanisms of biofilm formation by X. axonopodis pv. citri strain XW19, the strain was subjected to transposonmutagenesis. One mutant with a mutation in a two-component response regulator gene that was deficient in biofilmformation on a polystyrene microplate was selected for further study. The protein was designated as BfdR for biofilmformation defective regulator. BfdR from strain XW19 shares 100% amino acid sequence identity with XAC1284 of X.axonopodis pv. citri strain 306 and 30–100% identity with two-component response regulators in various pathogens andenvironmental microorganisms. The bfdR mutant strain exhibited significantly decreased biofilm formation on the leafsurfaces of Mexican lime compared with the wild type strain. The bfdR mutant was also compromised in its ability to causecanker lesions. The wild-type phenotype was restored by providing pbfdR in trans in the bfdR mutant. Our data indicatedthat BfdR did not regulate the production of virulence-related extracellular enzymes including amylase, lipase, protease, andlecithinase or the expression of hrpG, rfbC, and katE; however, BfdR controlled the expression of rpfF in XVM2 medium,which mimics cytoplasmic fluids in planta. In conclusion, biofilm formation on leaf surfaces of citrus is important for cankerdevelopment in X. axonopodis pv. citri XW19. The process is controlled by the two-component response regulator BfdR viaregulation of rpfF, which is required for the biosynthesis of a diffusible signal factor

Mixed Infections of Helicobacter pylori

Background. Persistent Helicobacter pylori infection may induce several upper gastrointestinal diseases. Two major virulence factors of H. pylori, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), are thought to be associated with the severity of disease progression. The distribution of vacA and cag-pathogenicity island (cag-PAI) alleles varies in H. pylori isolated from patients in different geographic regions. Aim. To assess the association between mixed infection of H. pylori clinical isolates from Taiwanese patients and the severity of gastrointestinal diseases. Methods. A total of 70 patients were enrolled in this study. Six distinct and well-separated colonies were isolated from each patient and 420 colonies were analyzed to determine the genotypes of virulence genes. Results. The prevalence of mixed infections of all H. pylori-infected patients was 28.6% (20/70). The rate of mixed infections in patients with duodenal ulcer (47.6%) was much higher than that with other gastrointestinal diseases (P<0.05). Conclusions. H. pylori mixed infections show high genetic diversity that may enhance bacterial adaptation to the hostile environment of the stomach and contribute to disease development

Late initiation of renal replacement therapy is associated with worse outcomes in acute kidney injury after major abdominal surgery

Introduction Abdominal surgery is probably associated with more likelihood to cause acute kidney injury (AKI). The aim of this study was to evaluate whether early or late start of renal replacement therapy (RRT) defined by simplified RIFLE (sRIFLE) classification in AKI patients after major abdominal surgery will affect outcome. Methods A multicenter prospective observational study based on the NSARF ( National Taiwan University Surgical ICU Associated Renal Failure) Study Group database. 98 patients (41 female, mean age 66.4 +/- 13.9 years) who underwent acute RRT according to local indications for post-major abdominal surgery AKI between 1 January, 2002 and 31 December, 2005 were enrolled The demographic data, comorbid diseases, types of surgery and RRT, as well as the indications for RRT were documented. The patients were divided into early dialysis (sRIFLE-0 or Risk) and late dialysis (LD, sRIFLE -Injury or Failure) groups. Then we measured and recorded patients' outcome including in-hospital mortality and RRT wean-off until 30 June, 2006. Results The in-hospital mortality was compared as endpoint. Fifty-seven patients (58.2%) died during hospitalization. LD (hazard ratio (HR) 1.846; P = 0.027), old age (HR 2.090; P = 0.010), cardiac failure (HR 4.620; P < 0.001), pre-RRT SOFA score (HR 1.152; P < 0.001) were independent indicators for in-hospital mortality. Conclusions The findings of this study support earlier initiation of acute RRT, and also underscore the importance of predicting prognoses of major abdominal surgical patients with AKI by using RIFLE classification

Application of rDNA internal transcribed spacer (ITS) sequence characteristics as an aid for the identification and phylogenetic study of Colletotrichum spp.

炭疽病菌Colletotrichum gloeosporioides (Cg) 可危害多種作物，嚴重影響產品品質與儲架壽命。傳統Colletotrichum spp. (C-spp) 之鑑定主要依據型態特徵、病徵表現及寄主範圍特性等，然單就Cg而言，由於其寄主範圍甚廣，且形態特徵常因培養條件及變異等之影響而不易確認，加上不同種C-spp會有交叉感染情形，鑑定上常備受困擾且相當費時。為因應加入世界貿易組織後，農產品上此類病原快速檢測與鑑定之需，本研究嘗試利用rDNA之ITS核酸，以PCR增幅分析其不同種間核酸序列差異，進而並利用PCR增幅產物配合異質雙股泳動性法 (heteroduplex mobility assay，簡稱HMA)，瞭解其在鑑識與快速檢測C-sp上之應用潛力。利用共同性引子對ITS1與ITS4 (White et al., 1990) 針對29個C-sp菌株 (分別由CCRC購置、農藥所等相關研究單位提供及本研究室自行分離)及7個非C-spp菌株進行PCR，所有供試之C-spp菌株皆可獲得一約600bps之增幅產物，其他非C-spp菌株之增幅產物則大小分別在500-800bps範圍，與C-spp菌株迥異。逢機選取15個C-sp菌株之增幅產物，以Topo TA cloning 試劑組選殖後解序，可見其全長在535至555bps間，其中計含ITS 1區160至181bps，ITS 2區152至153bps，及5.8S rDNA部分160bps；經由序列比對發現ITS1區 (相同度53.9 ％) 種間歧異性遠較ITS 2 (相同度81.8 ％) 大。利用ITS區全長序列及ITS1區單獨序列，進而以distance matrix及parsimony (Felsenstein, J. et al., 1988) 法統計分析，發現可將這15個C-spp菌株分別以C. gloeosporioides (CG)、C. acutatum (CA)、C. musae (CM) 、C. lindemuthianum (CL) 、C. capsici (CC) 為代表之五群。另將此些ITS1及ITS2序列資料與GenBank / EMBL資料庫比對，發現在ITS1部份除CM及CL群外，皆可找到相同度達99% - 100%的同源性序列，而在ITS2部份則僅CA、CG群菌株可找到相同度達98%以上的同源性序列。有鑑於利用C-spp種專一性引子對 (Mills et al.,1992) 做為供試C-spp菌株種的鑑定工具，在應用上效果不如預期，本研究嘗試另將這15個C-spp菌株與其他供試C-spp菌株之rDNA增幅產物進行HMA分析，所得mobility值經換算成distance後，按上述方法統計分析發現，所有供試菌株可歸類成同上述5群，唯依異質雙股之條帶分佈，則進而可歸納出6種異質雙股型式圖譜 (Heteroduplex pattern，簡稱HP)，除上述CA、CC、CM、CL外，另可將CG分成CG1及CG2兩群。綜合上述核酸分析檢測結果，歸類為CC群之Cgr1、Cgr2、Cd1三個菌株，傳統方法鑑定結果分別屬於C. graminicola (Cgr1、Cgr2)或C. dematium (Cd1)，然其ITS區間序列卻與C. capsici同質性達98-100%，顯示此些菌株之鑑定結果有進一步探討確認之必要；同樣的歸屬CG群之Cg8原以傳統方法鑑定為C. musae；歸屬CA群之Cg2、Ca1、Cg9、C-spp1，原本均被鑑定為Cg；其中以Cc1菌株為例，其原由CCRC購置，孢子形態甫近經再次單孢培養已確定應為如Cg之橢圓形，並非如C. capsici之彎月形，原本之鑑定結果顯然有誤；綜合上述試驗結果，利用rDNA ITS區間序列確可將炭疽病菌區分至種階層，而利用PCR增幅並配合HMA及HP等分析方法，則可作為檢測鑑定炭疽病菌相當明確的輔助依據。Colletotrichum gloeosporioides (Cg), the causal agent of anthracnose on various important crops, is the most notorious post-harvest pathogens greatly deteriorating the quality and shelf-life of agricultural products. The traditional way for identification of Colletotrichum spp. (C-spp) is quite often frustrating and time consuming mainly due to the wide host range of the pathogen, the great morphological variation; and what even worse was that cross infection might occur among different Colletotrichum spp. To cope with the increasing need of rapid detection and identification of this fungal pathogen from various agricultural products after the country become a member of the World Trade Organization (WTO), the potential application of rDNA ITS sequence characteristics as a biochemical tool was explored. The full length ITS rDNAs of a total of 29 C-spp isolates, and 7 non-C-sp isloates, were produced by polymerase chain reaction (PCR) amplification using the universal primer pair ITS1/ITS4 (White et al.1990). The Colletotrichum species tested included C. gloeosporioides (Cg), C. acutatum (Ca), C. musae (Cm), C. graminicola (Cgr), C. coccodes (Cco), C. capsici (Cc), C. dematium (Cd), C. lindemuthianum (Cl), and Colletotrichum sp. (C-sp) that species status awaited to be identified. The non- Colletotrichum species tested included Rhizopus oryzae, Penicillium digitatum, Pestalotia sp., Fusarium solani, Alternaria brassica, Ustilago esculenta, and Trichoderma reesici. The 600 bps amplicons were generated from all C-spp isolates tested as was expected; the amplicons from 7 non-C-spp isolates ranged from 500-800bps. Fifteen of the C-spp amplicons which include 7 from Cg; 2 each from Cc, Cgr; and one each from Cm, Cd, Cl and C-sp, were cloned and sequenced. The full length of these amplicons ranged from 535 to 555 bps; in which ITS1 region was from 160 to 181bps, ITS2 region was from 152 to 153bps; whereas the 5.8S rDNAs all appeared to be 160bps. Comparison of the inter-specific sequence identity indicated the existence of much greater divergence among ITS1 (identity reached only 53.9%) as compared to that among ITS2 region (identity was approximately 81.8%). Comparative analysis of the ITS full length and the ITS1 sequence data by the distance matrix method and the parsimony method both concluded these 15 tested isolates into 5 distinctive species groups namely, CG (C. gloeosporioides), CA (C. acutatum), CM (C. musae), CL (C. lindemuthianum), and CC (C. capsici). The sequence data also revealed that the ITS1 sequence of tested CG, CA and CC isolates were 99-100% identical to each compared isolate available in the GenBank/EMBL data bank; whereas for ITS2 region, only those from CA and CG isolates were greater than 98% in identity. For the rapid detection and identification of Colletotrichum spp. by PCR, the efficacy of the species specific primer pairs developed by Mills et al. (1992) were examined; the results appeared to be non-satisfactory. To resolve the problem, the heteroduplex mobility assay (HMA) was attempted using the ITS-rDNA amplicons of all 29 tested C-spp isolates as the targets. The analysis by the above described distance matrix method using the distance value estimated from the mobility changes concluded the 29 isolates into 5 distinctive species groups same as that by sequence data analysis. Moreover, from the repeatable band distribution characteristics, a total of 6 heteroduplex patterns (HP) were established. By the established HP fingerprint, the species group CG can be further divided into CG1 and CG2; while the other 4 species groups remain unchanged. In the established species group system, Cgr1, Cgr2 and Cd1 isolates, originally identified as C. graminicola and C. dematium respectively, were reclassified as member of CC since their ITS sequences were 98-100% identical to that of C. capsici. Likewise, the species status of Cg8 isolate was changed from C. musae to a member of CG; Cg2, Ca1, Cg9 and C-sp1 isolates were changed from C. gloeosporioides to be members of CA; and Cc1 isolate was changed from C. capsici to a member of CG2. Among these isolates that original identification were disputable, the species status of Cc1 was reexamined by the original author lately and proved the previous assignment as C. capsici might be a misinterpretation. The results discussed in this investigation, although only limited numbers of fungal isolates had been explored, appeared to provide evidence that sequence analysis of ITS rDNAs a dependable tool in the species level identification of Colletotrichum spp. Also the combination of the genus- or species-specific PCR together with HMA and HP analysis is easy to process, not time consuming, and provides repeatable and distinct data bases useful for species identification. The use of the established techniques for practical application in rapid detection and pathogen identification of anthracnose, and even other fungal diseases, are thus recommended.壹、前言..................................................................………………….................... 1 貳、前人研究....................................................................…………………........... 3 參、材料方法.........................................................................…………………....... 10 一、 菌種來源及培養方法…………………………………………….............….... 10 二、 供試藥品與試劑 ..........................................................………………….... 10 三、 供試菌株總量DNA (Total DNA) 之抽取.....……………………………….... 12 四、 rDNA ITS區間 (含5.8段) 序列之增幅..……………………………………. 12 五、 rDNA ITS區間增幅片段之選殖序列選殖.......................…………………... 12 (一) 增幅產物之純化………………………………………....…………………... 12 (二) 增幅rDNA片段之選殖轉轉型 (transformation) ...………………………. 13 (三) 選殖菌株之快速篩選……………………………………….………………... 13 (四) 選殖菌株嵌入DNA之抽取及分子量之確定……………………………….. 14 六、選殖DNA片段定序.................................................…………………......... 14 (一) PCR反應…………………………………………………………………….... 14 (二) 聚丙烯醯銨膠體電泳分析...............................................………………… 15 (三) DNA轉漬及固定……………………………………………………………… 15 (四) 化學冷光偵測反應………………………………………….………………... 15 七、CgInt及ITS4專一性引子對增幅反應最適化..............……………………… 16 (一) 最適反應模板DNA (template DNA) 濃度及靈敏度測定.………………… 16 (二) 最適反應引子 (primers) 濃度測定...........................…………………….. 17 (三) 最適反應黏合 (annealing temperature) 溫度.........…………………....... 17 (四) DNA聚合 (Taq DNA polymerase) 之比較....………………….............. 17 (五) CgInt/ITS4引子對專一性測試....………………….....................………….. 17 八、使用不同引子對之靈敏度測試...................…………………...............……... 17 九、Booster PCR 法在 Colletotrichum gloeosporioides檢測之應用性………. 17 (一) Booster PCR靈敏度及專一性測試………………..........……………….…. 17 (二) 使用不同酵素對Booster PCR 靈敏度及專一性之影響………………...... 18 (三) 用不同引子對Booster PCR 靈敏度及專一性之影響………………......... 18 十、Nested PCR 法在Colletotrichum gloeosporioides檢測之應用性……….. 18 (一) Nested PCR靈敏度及專一性測試………………..……...………………..... 18 (二) 使用不同酵素對Booster PCR 靈敏度及專一性之影響………………….. 18 十一、以專一性引子對檢測芒果葉片組織中C. gloeosporioides之模擬試驗… 18 十二、異雙股核酸泳動性分析 (Heteroduplex mobility assay, HMA) ………….. 19 十三、rDNA序列比對分析……………………………………………………........ 19 肆、結果...............................................................................…………………….... 21 一、供試菌株含5.8S rDNA ITS區間全區之PCR增幅產物比較………………. 21 二、Colletotrichum spp. ITS區rDNA PCR增幅產物之選殖…………………... 21 三、供試15個Colletotrichum spp. 菌株所選殖rDNA片段核酸序列解序及分析….......................…………………………………................…....……..…. 21 (一) 選殖rDNA 片段之序列長度分析…....………………………………….... 21 (二) 15個rDNA選殖片段序列種間及種內差異性比較.…………………….... 21 四、CgInt/ITS4專一性引子對在Colletotrichum gloeosporioides檢測之應用… 23 (一) CgInt及ITS4 PCR增幅反應之靈敏度及最適反應條件………….……... 23 (二) 不同引子對配合之PCR增幅檢測靈敏度比較..........………………....... 24 (三) CgInt/ITS4 引子對之檢測專一性..............................………………....... 24 五、Booster PCR 法在 Colletotrichum gloeosporioides檢測之應用性………. 24 (一) 專一性及靈敏度...............................…….…………………………….… 25 (二) 以CgInt1/ITS4及Cgits/ITS4引子對進行booster PCR檢測 C. gloeosporioides之靈敏度及專一性之影響..………………………........ 25 六、Nested PCR法在檢測C. gloeosporioides之專一性及靈敏度………………………………………………………………………..…….. 25 七、以CgInt/ITS4引子對檢測芒果葉組織中C. gloeosporioides 之偵測靈敏度......…………………………………………......…………………………... 26 八、異質雙股泳動率檢測法 (Heteroduplex mobility assay) 在Colletotrichum spp. 分類鑑定上之應用……...……………………………………………………. 26 九、異質雙股圖譜分析 (Heteroduplex pattern, HP)………...…………………. 27 伍、討論……………………………………………………………………………..….. 29 陸、中文摘要………………………………………………………………………..….. 36 柒、英文摘要………………………………………………………………………….... 38 捌、參考文獻……………………………………………………………………………. 41 玖、圖表說明……………………………………………………………………………. 49 壹拾、附錄……………………………………………………..........…………………10

Field Sanitation and Foliar Application of Streptomyces padanus PMS-702 for the Control of Rice Sheath Blight

Rice sheath blight (ShB), caused by Rhizoctonia solani Kühn AG1-IA, is one of the destructive rice diseases worldwide. The aims of this study were to develop biocontrol strategies focusing on field sanitation and foliar application with a biocontrol agent for ShB management. Streptomyces padanus PMS-702 showed a great antagonistic activity against R. solani. Fungichromin produced by S. padanus PMS-702, at 3.07 mg/l inhibited 50% mycelial growth, caused leakage of cytoplasm, and inhibited the formation of infection structures of R. solani. Fungichromin could reach to 802 mg/l when S. padanus PMS-702 was cultured in MACC broth for 6 days. Addition of 0.5% S. padanus PMS-702 broth into soil decreased the survival rate of the pathogen compared to the control. Soil amended with 0.5% S. padanus broth and 0.5% tea seed pomace resulted in the death of R. solani mycelia in the infested rice straws, and the germination of sclerotia was inhibited 21 days after treatment. Greenhouse trials revealed that S. padanus cultured in soybean meal-glucose (SMGC-2) medium after mixing with different surfactants could enhance its efficacy for inhibiting the pathogen. Of six surfactants tested, the addition of 2% tea saponin was the most effective in suppressing the pathogen. S. padanus broth after being fermented in SMGC-2, mixed with 2% tea saponin, diluted 100 fold, and sprayed onto rice plants significantly reduced ShB disease severity. Thus, S. padanus PMS-702 is an effective biocontrol agent. The efficacy of S. padanus PMS-702 for disease control could be improved through formulation

Biological Control of Collar Rot on Passion Fruits Via Induction of Apoptosis in the Collar Rot Pathogen by Bacillus subtilis

The seedlings and fresh fruits of passion fruits are of high value in local and global trade. Fusarium solani is a main disease-causing agent affecting passion fruits. The objectives of this study were to develop Bacillus-based biocontrol agents for the management of Fusarium diseases on passion fruits and to investigate their putative control mechanisms. Our studies indicated that B. subtilis YBC and 151B1 show antagonistic activity to F. solani PF7 from passion fruits and inhibited the conidial germination of strain PF7. The application of broth cultures from B. subtilis 151B1 and YBC in SYB medium reduced disease severity of Fusarium wilt on the leaves of passion fruits and enhanced the survival rates of passion fruit seedlings challenged with F. solani PF7. With regard to the putative mechanisms of disease control, the results indicated that treatments consisting of the respective culture filtrates from B. subtilis 151B1 and YBC broths caused aberrant conidial morphology and loss of cell membrane integrity. Additionally, the treatments caused reductions in mitochondrial membrane potential and interfered with the energy metabolism of F. solani PF7. The treatments also enhanced reactive oxygen species accumulation and resulted in the externalization of phosphatidylserine, chromatin condensation, and DNA fragmentation, suggesting their function in triggering apoptotic-like cell death. In conclusion, B. subtilis 151B1 and YBC are potential biocontrol agents for passion fruit disease caused by F. solani. Their control efficacy may result from the surfactins produced to trigger apoptotic-like cell death, reducing mitochondrial membrane potential and interfering with the energy metabolism of the pathogen

Barcode-like heteroduplex DNA pattern as an aid for rapid identification of anthracnose fungi

We have shown the usefulness of the heteroduplex mobility assay (HMA) for phylogenetic analysis and for the discrimination of closely related Colletotrichum species. Because the heteroduplex mobility of a tested strain shows a unique banding pattern that is the function of the sequence of the referred strain, we further explored the potential use of heteroduplex DNA patterns (HPs) as DNA fingerprints for the identification of these fungi. The 29 Colletotrichum strains previously identified by HMA to be taxonomic members of CG, CA, CM, CC and CL species groups were re-examined with an emphasis on their unique heteroduplex banding patterns. The species attributes of these tested strains were characterized by HMA using ITS fragments amplified from six representative Colletotrichum strains as pairwise compared references. By comparing the unique homoduplex and heteroduplex banding patterns of each tested strain on a polyacrylamide gel with those of the respective reference strain, the species identity of tested strains was determined. The obtained barcode-like HPs classified these 29 Colletotrichum strains into 6 distinctive groups: CG1, CG2, CA, CM, CC and CL. Notably, the HPs differentiated strains CG1 and CG2, which differed in their ITS sequences by only six bases. The presented results revealed that the species-characteristic barcode-like HP classification of ITS regions is a relatively rapid and valuable system for species identification of Colletotrichum species. The potential use of the established barcode-like system for the identification of anthracnose fungi and other fungal pathogens is discussed