PpMID1 Plays a Role in the Asexual Development and Virulence of Phytophthora parasitica

Phytophthora parasitica is a notorious oomycete pathogen that causes severe disease in a wide variety of crop species. Infection of plants involves mainly its asexual life stage, including papillate sporangia and biflagellated zoospores, which are the primary dispersal and infection agents of this pathogen. Calcium signaling has been thought as the key regulator for sporangium formation and zoospore differentiation. However, not much is known about the molecular players involved in these processes. In Saccharomyces cerevisiae, mating pheromone-induced death 1 (MID1) encodes a component of a putative calcium channel. Here, we identified and characterized the function of PpMID1, an MID1 homolog from P. parasitica. The expression of PpMID1 was high in sporangia. Gene silencing of PpMID1 resulted in the formation of sporangia that lacked papilla and showed a tendency for direct germination. Notably, in response to cold shock to induce zoospore formation, these sporangia showed no sign of cytoplasmic cleavage and thereby failed to form zoospores. Nonetheless, the addition of CaCl2 or MgCl2 partially recovered the silenced sporangia phenotype, with the formation of papillate sporangia similar to those of the wild type and the release of zoospores upon cold shock. As well, virulence toward Nicotiana benthamiana was reduced in the PpMID1-silenced transformants. These results indicate a role of PpMID1 in the asexual development and virulence of P. parasitica

A nested multiplex PCR for species-specific identification and detection of Botryosphaeriaceae species on mango

Lasiodiplodia theobromae (Pat.) Griff. & Maubl, Neofusicoccum parvum Pennycook & Samuels, N. mangiferae Syd. & P. Syd., and Fusicoccum aesculi Corda, all anamorphs of Botryosphaeriaceae species, are the causal agents of mango stem-end rot and fruit rot in Taiwan. Identification of these fungal species based on morphology has not been easy due to their extensive plasticity for some of the morphological characters. To aid reliable identification of Botryosphaeriaceae species associated with mango fruits, four pairs of species-specific primers were designed according to sequences of the ribosomal internal transcribed spacers (ITS), and a rapid method was established based on nested multiplex polymerase chain reaction (PCR) in this study. To perform the analysis, PCR was first run with ITS1 and ITS4 as the primers, followed by a second PCR with the addition of all four sets of species-specific primers. With this method, a low limit of 100 fg-1 pg of purified fungal DNA was detectable. It could also successfully detect L. theobromae, N. parvum, N. mangiferae and F. aesculi in total DNA extracted from inoculated mango fruits. This assay provides a rapid and sensitive method for the identification of Botryosphaeriaceae species and diagnosis of mango fruit rot and stem-end rot as well

Analysis of Phytophthora parasitica by retrotransposon-derived DNA fingerprinting

Phytophthora parasitica, being able to attack a wide variety of plants, is a very important plant pathogen in Taiwan. It has been shown that isolates of P. parasitica from tobacco, dieffenbachia, and loquat differed significantly from other isolates in morphology and pathogenicity and were recognized as "atypical" types of the fungus. In the current study, a partial sequence of the reverse transcriptase gene of a Ty1-copia retrotransposon was cloned from P. parasitica and used as the probe, designated herein as G2Ty-1, for genomic Southern hybridization analyses of P. parasitica. It was demonstrated that G2Ty-1 existed as a moderately repetitive sequence in the genome of P. parasitica. The banding pattern of G2Ty-1 was mitotically stable for at least five consecutive asexual generations. DNA fingerprint analysis of P. parasitica using G2Ty-1 as the probe demonstrated that the banding patterns of G2Ty-1 were highly polymorphic among the isolates analyzed. "Atypical" strains from tobacco, dieffenbachia, and loquat, nonetheless, displayed host-specific banding patterns. Phylogram generated by cluster analysis demonstrated that isolates from tobacco and dieffenbachia were well separated from each other and from all other isolates of P. parasitica. These results provided a genetic basis for distinguishing these isolates from others and validate the use of retrotransposon as a genetic marker to study phylogeny in Phytophthora parasitica

Detection of orchid Phytophthora disease by nested PCR

Orchid disease caused by Phytophthora has long been a major threat to cultivation of orchids in Taiwan. Phytophthora spp. known to infect orchids include mainly P. palmivora and P. parasitica. Identification of Phytophthora species by the conventional method includes the use of selective media to obtain Phytophthora isolates and examination by microscopy. The procedures are rather labor-intensive and time-consuming. In order to accelerate and simplify the process of diagnosis, we have developed a nested PCR assay for rapid and accurate detection of Phytophthora pathogens infecting orchids. After isolation of DNA from the plant tissue, PCR was performed using a primer set specific for Phytophthora. Amplification of DNA fragments of approximately 1 kb in length indicated the presence of Phytophthora pathogens. To identify the species, nested PCR was then performed using amplified product from the first PCR as the template and species-specific oligonucleotides as the primers. Amplification of specific DNA fragments would tell whether the orchids were infected by P. palmivora, P. parasitica, or both. Furthermore, the sensitivity of detection was greatly enhanced. This assay provides a rapid and sensitive method for detection of Phytophthora pathogens in infected orchids as well as infested media used for cultivation of orchids, and thus can assist growers in early diagnosis of the devastating orchid Phytophthora disease

Studies on three important Phytophthora diseases in recent year in Taiwan

疫病菌 (Phytophthora deBary) 為植物強敵， 目前發表之有效種 (species) 約有百餘種，存在台灣的約有30 種，近年在台灣造成重大疫情者包括 (1) Phytophthora infestans US11 菌系釀成之馬鈴薯與番茄晚疫病；(2) P. citrophthora 新菌系引起之金柑疫病；及 (3) P. capsici 同絲配對型 (具A2配對型之傾向) 崛起，造成之茄科與瓜類疫病。茲說明如下：(1) 台灣自1997年12 月在台中后里地區爆發嚴重之馬鈴薯與番茄晚疫病後，兩個月內病害遍及全台，並導致馬鈴薯栽培區南移至斗南地區，目前已經證實病害發生係因強毒性菌株US11 入侵之結果，該菌系為A1 配對型、致病性強、生長快速 (於最適溫20℃時，新菌系直線生長速率平均為5.15 mm、舊菌系為2.68 mm)、耐高溫 (新菌系最高生長溫度27-29℃、舊菌系24-25℃)、及抗多種化學農藥 (滅達樂【metalaxyl】對新菌系菌絲生長抑制濃度(LD50)為200-400 ppm、舊菌系為0.005-0.001 ppm，抗藥性提高4-40 萬倍)。(2) 宜蘭地區之金柑，於1995-1997 年，爆發植株大量急速萎凋與死亡現象，伴隨嚴重之枝枯、流膠、落葉與落果，受害地區高達80%以上。分離到相關性最高的病原菌為柑橘疫病菌 (P. citrophthora)，但為害金柑之菌系與一般為害柑橘屬的P. citrophthora 菌系有很大差異。在寄主專一性方面，金柑屬疫病 菌系不會造成柑橘屬植株死亡，反之亦然；此外，兩者之菌落型態不同，菌絲蛋白電泳圖譜略異，核醣體內轉錄區間ITS (ITS1-5.8S rDNA-ITS2) 的基因序列亦略異。金柑菌系的ITS 序列長度均為779 bp，且全部序列相同；β 微管蛋白(β-tubulin) 的部份DNA 序列(長度651 bp)亦完全相同，所有菌株似源自單一菌株；柑橘屬菌系ITS 全長為782、783 或784 bp，與柑橘菌系相比，所有金柑菌系在ITS1 區域有4 處鹼基對缺遺與2 處鹼基置換，而在ITS2 區有6 處鹼基置換。顯示台灣之金柑疫病非由本土柑橘屬菌系引起。(3) 番椒疫病菌 (P. capsici) 在台灣的記錄一直為A1 配對型，直到2007年4 月首度發現同絲型出現，目前 (2010 年) 同絲型已遍佈全台，僅高山地區尚可分離到A1 型。與A1 菌系比較，同絲型新菌系除會形成卵胞子外，其胞囊較狹長且脫落率較高、產胞較嗜高溫 (同絲型產胞最適溫為28-32℃，A1 菌系大部分為24℃)、且毒性較強。比較分析同絲型番椒疫病菌系間之親緣關係，ITS 序列全長均為753 bp，有6 處相異，可以分為3群；β 微管蛋白(β-tubulin) 的部份DNA 序列(長度651 bp)，亦有8 處相異，可以分為9 群。從以上特性尚無法判斷台灣A2 菌株崛起之原因。 Phytophthora deBary are important plant pathogens distributed world wide. Currently, there are about hundreds good species of Phytophthora have been formally described and documented in the world. Among them 30 species are distributed in Taiwan. Some of them have caused severe economic loss in recent year, including that (1) Phytophthora infestans US11 caused potato and tomato late blight since 1997；(2) a new strain of P. citrophthora induced kumquat blight and decline from 1985 through to 1987; and (3) the homothallic strain (with A2 tendency) of P. capsici appeared and attacked solanaceae and cucurbit crops seriously since 2007

<i>pks63787,</i> a Polyketide Synthase Gene Responsible for the Biosynthesis of Benzenoids in the Medicinal Mushroom <i>Antrodia cinnamomea</i>

<i>Antrodia cinnamomea,</i> a unique resupinate basidiomycete endemic to Taiwan, has potent medicinal activities. The reddish basidiocarps and mycelia generally exhibit abundant metabolites and higher biological activity. To investigate the pigments of <i>A. cinnamomea</i>, polyketide synthase (PKS) genes were characterized based on its partially deciphered genome and the construction of a fosmid library. Furthermore, a gene disruption platform was established via protoplast transformation and homologous recombination. Of four putative polyketide synthase genes, <i>pks63787</i> was selected and disrupted in the monokaryotic wild-type (wt) strain f101. Transformant Δ<i>pks63787</i> was deficient in the synthesis of several aromatic metabolites, including five benzenoids and two benzoquinone derivatives. Based on these results, a biosynthetic pathway for benzenoid derivatives was proposed. The <i>pks63787</i> deletion mutant not only displayed a reduced red phenotype compared to the wt strain but also displayed less 1,1-biphenyl-2-picrylhydrazyl free radical scavenging activity. This finding suggests that PKS63787 is responsible for the biosynthesis of pigments and metabolites related to the antioxidant activity of <i>A. cinnamomea</i>. The present study focuses on the functional characterization of the PKS gene, the fluctuations of its profile of secondary metabolites, and interpretation of the biosynthesis of benzenoids

晚疫病菌PCR檢測方法之建立

Late blight caused by the oomyceteous pathogen Phytophthora infestans is a devastating disease of potato and tomato worldwide. The objective of the present study is to develop a method for rapid detection of P. infestans from potato tubers and tomato seeds. Three oligonucleotide primers (Pi1S-1, Pi2A-1, and Pi2A-2) were designed based on the internal transcribed spacer sequences of the fungus, which made two primer sets: Pi1S-1/Pi2A-1 and Pi1S-1/Pi2A-2. PCR analysis demonstrated that DNA fragments of predicted size, using either Pi1S-1/Pi2A-1 or Pi1S-1/Pi2A-2 as the primer set, were amplified from all the P. infestans isolates tested. Moreover, when the amount of template DNA was reduced to 1 pg, PCR primed with Pi1S- 1/Pi2A-2 could still give rise to expected DNA fragment. No signals were detected with DNA from other fungal species analyzed in the study. To detect P. infestans present in the potato tuber, a rapid method for DNA extraction followed by PCR with Pi1S-1/Pi2A-2 was established. This method will provide a useful tool for rapid detection of P. infestans in potatoes and tomatoes. 晚疫病菌Phytophthora infestans (Montagne) de Bary是引起馬鈴薯與蕃茄晚疫病的病原真菌，分佈遍及全世界，旦造成非常嚴重之危害。為了開發快速簡易之晚疫病菌檢測技術，以便應用於健康種薯檢測，我們根據疫病菌(Phytophthora spp.)核糖體核酸ITS序列之並列分析結果設計核酸引子(Pi1S-1, Pi2A-1與Pi2A-2)，並以Pi1S-1/Pi2A-1或Pi1S-1/Pi2A-2為引子對進行PCR。分析結果顯示，不論以Pi1S-1/Pi2A-1或Pi1S-1/Pi2A-2為引子對都可自所測試之晚疫病菌菌株增殖出預期大小之核酸片段，但自其他種類之供試菌株則觀察不到增殖產物，顯示其對晚疫病菌其有非常高之專一性。研究也顯示，將DNA減量綱1Pg時，以Pi1s-1/Pi2A-2為引子對仍可自晚疫病菌增頭出預期之核酸片段。因此，這一項技術應可成為進行晚疫病菌鑑定與檢測之有用工具。此外，我們還建立了自接種馬鈴薯快速抽取DNA，並以Pi1s-1/Pi2A-2為引子對進行晚疫病菌檢測之技術，可應用於輔助馬鈴薯健康種薯之檢測

Development of PCR methods for rapid detection of Phytophthora ramorum, a quarantine pathogen of Taiwan

Phytophthora ramorum Werres, de Cock & Man in’t Veld, sp. Nov. 是引發櫟樹猝死病(sudden oak death; SOD)之病原菌，但除了櫟樹外，尚可感染杜鵑及珊瑚樹(莢迷)等多種林木，是國際間備受矚目的重要病原菌。P. ramorum肆虐之區域主要分佈於美國及歐洲多個國家如荷蘭、比利時、英國及德國等，目前並未在台灣出現。由於該菌致病性強，且寄主多為重要林木及灌木，若不幸入侵，將嚴重影響生態、景觀與種苗產業，因此P. ramorum已被我國列為重要檢疫病原菌。為開發此病原菌之快速檢測技術，本研究根據YPT基因及核醣體RNA基因之序列分別設計P. ramorum專一性及疫病菌廣效性引子對；PCR分析結果顯示，其具備良好專一性，可偵測之最低量P. ramorum DNA分別為1 ng及10 pg。為檢測病原菌在輸入樣品的存在情形，我們進一步建立以杜鵑葉片誘釣疫病菌，並進行multiplex PCR的實驗流程，可提供檢疫單位使用，以快速檢測P. ramorum的存在情形。 Phytophthora ramorum Werres, De Cock & Man in’t Veld, sp. nov., the pathogen of sudden oak death (SOD) and ramorum blight, is known to infect a variety of plants including oaks, Rhododendrom, and Viburnum species. Currently, infection by P. ramorum occurs mainly in Europe and North America. None has been reported in Taiwan. Owing to its threat to woody ornamental plants and the forest ecosystems, P ramorum has become an important quarantine pest of Taiwan. In order to develop a technique for rapid detection of this pathogen, we designed three sets of primers, each specific for P. ramorum (ramyptF1 + ramyptR1), Phytophthora spp. (Phy2S + Phy7A), and Rhododendron pulchrum Scoeet (rdhITSF1 + rdhITSR1), respectively. Specificity of the first two sets of primers was verified by PCR. Further analysis indicated that PCR primed with ramyptF1 and ramyptR1 may amplify P. ramorum DNA with a low limit of 1 ng, while that for Phy2S and Phy7A was 10 pg. To facilitate detection of Phytophthora pathogens which might exist in the imported materials, we established a diagnostic protocol which is comprised of the baiting with the use of leaves of Rhododendron pulchrum G. Don, followed by a multiplex PCR, which contains all three sets of the aforementioned primers. This method provides a useful tool for rapid detection of P. ramorum, and in addition, may help to uncover the possible existence of other Phytophthora spp. as well