Biological Characteristics of Phellinus noxius and its Molecular Methods for Diagnosis

本論文主要針對褐根病菌 (Phellinus noxius) 之生物特性及其分子診斷技術之開發進行研究。本研究將褐根病菌分離純化培養之褐根病菌P. noxius培養於馬鈴薯葡萄糖瓊脂(PDA)培養基，菌落形態初期為白色，漸漸轉為不規則顏色深淺的褐色菌落；病菌菌絲無扣子體 (clamp connection)，培養於PDA培養基上會產生斷生孢子 (arthrospores) 及毛狀菌絲 (trichocyst)，毛狀菌絲為深褐色，為鑑定依據之一。菌絲細胞具多核。進一步將褐根病菌於太空包培養一個月後，再移至溫室內1-2個月即產生子實體，子實體褐色，厚度5mm～10mm；擔孢子無色透明、單室、圓形，大小3.6-6.2 x 1.8-5.2 μm。剛毛菌絲 (setal hyphae) 褐色，大小340-680 μm。其最適生長溫度為24～28 ℃。供試菌株中5個褐根病菌株皆能分泌澱粉分解酵素(amylase)、纖維分解酵素 (cellulase)、脂質分解酵素 (lipase)、果膠分解酵素 (pectinases, 含polygalacturonases (PGs) 與pectate transeliminases (PTEs)) 及蛋白分解酵素 (protease)。1991-2006年自台灣各地的33種寄主植物收集到80株 P. noxius 菌株。經rDNA序列分析比對發現，主要分為2種序列型態，63個菌株為 type L，序列長度為614 bp以及17個菌株為type S長度606 bp。在菌株親緣分析方面：不同Phellinus spp.間親緣性距離較遠，相同度 (identity) 介於68-77%；而P. noxius各菌株間之親緣性距離較近，相同度皆在99%以上，相同地區之不同寄主植物或相同寄主植物及不同地區之相同寄主植物或不同寄主植物分離之菌株並無法分群。進一步依據ITS區域設計專一性引子對，正向引子PN-1F ( 5’-agtttgcgctcatccatctc-3’) 和反向引子PN-2R ( 5’-agccgacttacgccagcag-3’)，利用PCR技術針對供試之P. noxius菌株進行分子診斷，均可增幅出414 bp或422 bp長度之序列片段；其它菌株皆無法增幅出任何片段。且靈敏度可達0.01 ng。進一步以龍眼樹及相思樹根部組織進行田間試驗，亦可在罹病植株增幅出預期之片段。因此本研究所設計之引子對PN-1F/PN-2R能用來快速診斷褐根病與輔助鑑定P. noxius。本研究為進一歩尋求防治策略，本文首先以稀釋平板法，將化學藥劑添加於馬鈴薯葡萄糖瓊脂培養基 (PDA) 內，來測定不同濃度之藥劑對褐根病菌的抑制效果。在供試之46種藥劑中，以25%普克利乳劑對褐根病菌之抑制效果最佳，當有效濃度在10 ppm 時即可完全抑制供試菌株之菌絲生長；而有7種供試藥劑之藥效次之，約有90%以上之抑制效果；同時，4-4式波爾多液亦可完全抑制該菌之生長。進而進行盆栽實驗測試，結果顯示稀釋1000倍之普克利、三泰芬、撲克拉、滅普寧、亞磷酸，及4-4式波爾多液對人工接種褐根病菌之番荔枝苗與枇杷苗有較佳之防治褐根病的效果，發病率在10%～30%之間。針對不同氮肥種類對褐根病發病之影響進行研究，在五種2-3年生果樹幼苗（枇杷、柿子、梅、楊桃及梨）施用四種不同之氮肥（0.5 g/L硫酸銨、0.5 g/L台肥5號複合肥料、0.5 g/L硝酸鈣及1 g/L尿素），顯示接種褐根病菌之枇杷幼苗在3個月內均會死亡，而柿樹在6個月內也全部死亡，顯示肥料只能略為延緩感病品種之病勢進展，然而品種對發病率及病勢進展之影響更甚。此外，探討淹水、尿素及化學藥劑對褐根病菌在植物病組織內之存活影響方面，將人工接種褐根病菌之枇杷枝條 (2-2.5 cm diam.) 埋於土中，結果顯示病菌在土壤淹水處理的情形下存活能力可低於6天；土壤添加2 g/L尿素處理低於10天；0.4與2.0 g/L三泰芬可濕性粉劑 (5% triadimefon) 與撲克拉乳劑 (25% prochloraz) 在4個月內亦可完全殺死枝條內的病菌。進而於葡萄園進行病害田間防治試驗，結果以每株施用10 g三泰芬＋10 g尿素＋10 g碳酸鈣之處理（60株）最好，處理後2.5年期間並無植株死亡，樹幹生育最佳；而施用10 g撲克拉＋10 g尿素＋10 g碳酸鈣之處理（55株）亦甚佳，處理後6個月內有2株死亡（死亡率3.6%）外，之後並無植株死亡；而對照處理42株，試驗期間仍有6株死亡（死亡率14.3%）。Brown root rot caused by Phellinus noxius is one of the most important root diseases of woody trees in Taiwan. During the investigation from 2002 through 2007, a total of 19 species of new recorded hosts of the pathogen were found and studied. The morphology of the fungus was studied by culturing the purified cultures of several isolates on PDA and saw dust media as well as by diagnosing the diseased spaceman collected from nature. The fungal colonies on PDA were white in the beginning and turned irregular brown gradually. Clamp connection was not found. Mycelia produced hyaline arthrospores and dark brown trichocyst on PDA, whereas only trichocyst but no arthrospores were found in natural specimens. All tested isolates present multinuclei in hyphae. Fruiting bodies brown and 0.5-1 cm thick; basidiospores smooth, hyaline, and ovoid to broadly ellipsoid, measuring 3.6-6.2 x 1.8-5.2 μm; contextual setal hyphae reddish brown, measuring 340-680 μm. The temperatures for mycelial growth on PDA were 12-36℃ with optimum growth at 24-28 ℃. On artificial media amended with adequate substrates, all the 5 tested isolates collected from different territorial hosts were able to produce amylase, cellulase, lipase, pectinase and protease. Nucleotide sequences of ITS1, 5.8S and ITS2 of 80 strains of Phellinus noxius obtained from 33 tree species at different geographic locations in Taiwan from 1991 to 2006 were analyzed. These strains were divided into two ITS types based on the nucleotide length, type L with 614 bp and types S with 606 bp. Majority of the strains belonged to type L. Both type L and type S were present at most counties of Taiwan. In order to develop a pair of specific primers for rapid and accurate diagnosis of the disease associated with the pathogen in the fields, the ribosomal DNA (rDNA) sequences in internal transcript spacer (ITS, including ITS1/5.8S/ITS2) regions from P. noxius and several other soil-borne fungi were studied. Based on the ITS sequences, a set of primers PN-1F/PN-2R was designed. The forward primer PN-1F sequence is “5'-agtttgcgctcatccatctc-3' ” and the reverse primer PN-2R is “5'-agccgacttacgccagcag-3' ”. Using PN-1F/PN-2R primers for PCR amplification under a setup condition and the fungal genomic DNA as templates, a distinct 414 bp or 422 bp fragment were amplified form P. noxius, whereas no PCR products can be amplified from other soil-borne fungi tested. The PCR could amplify the fragments recognizable within 3-4 hr when concentration of purified DNA was higher or equal to 0.01 ng. Meanwhile, similar amplified fragments were obtained when the diseased root tissues of Dimocarpus longana and Acasia confusa from P. noxius infested fields were used as templates. Results presented here suggest that the primer pairs PN-1F/PN-2R can be efficiency in auxiliary identifying P. noxius and accurately and rapidly detecting disease caused by the pathogen in the field. In order to search of effective control measures, potato dextrose agar amended with individual chemical was used to evaluate the effects of different concentrations of synthetic chemicals on suppression of P. noxius. Among the 46 chemicals tested against P. noxius, 25% propiconazole EC was most effective, completely inhibiting the mycelial growth at the active ingredient dosage of 10 ppm. Bordeaux mixtures were also very effective in inhibiting mycelial growth. Another study was conducted to determine survival of P. noxius in infected stems of loquat buried in soil amended with fungicides, fertilizers, or by flooding. Results showed that treatment of soil with urea at 2.0 g/L soil or flooding of soil for 10 days completely eliminated the pathogen in the stems. A field trial was carried out on 3-5 year-old grapes in a field naturally infested with P. noxius in Shuili, Nantou. Results showed that soil drenching every 3 months with the solution containing 10 g 5% triadimefon+10 g urea+10 g CaCO3 in 1 L of water was the most effective treatment with no infected grapevines developed in all the 60 plants after treating for 2.5 years. In contrast, 6 of 42 (14.3%) tested plants in the untreated control were killed by P. noxius in 2.5 years.中文摘要 1 英文摘要 4 第一章 前人研究 7 引用文獻 12 第二章 褐根病菌之寄主與病害分佈 17 摘要 17 前言 17 材料與方法 18 褐根病之調查與病菌分離 18 褐根病菌之接種 18 結果 19 討論 21 引用文獻 23 英文摘要 27 圖表 28 第三章 褐根病菌之形態與生理生化特性 36 摘要 36 前言 37 材料與方法 37 褐根病菌無性世代之形態特性 37 褐根病菌菌絲之細胞核染色 38 褐根病菌之子實體培養 38 溫度對褐根病菌菌絲生長之影響 39 褐根病菌活體外分泌物中酵素之分析 39 結果 42 褐根病菌無性世代之形態特性 42 褐根病菌菌絲之細胞核染色 42 褐根病菌之子實體產生及鑑定 43 溫度對褐根病菌菌絲生長之影響 43 褐根病菌活體外分泌物中酵素之分析 43 討論 44 引用文獻 47 英文摘要 49 圖表 50 第四章 褐根病菌之親緣分析 56 摘要 56 前言 57 材料與方法 57 供試病原菌 57 褐根病菌Genomic DNA之萃取純化 58 ITS核酸定序 59 結果 61 Phellinus noxius種內親緣分析 61 Phellinus spp.種間親緣分析 62 討論 62 引用文獻 64 英文摘要 66 圖表 67 第五章 褐根病菌Phellinus noxius檢測用專一性引子對之開發 摘要 80 前言 80 材料與方法 81 供試病原菌 81 褐根病菌Genomic DNA之萃取純化 82 ITS核酸定序 83 褐根病菌專一性引子對之設計 84 專一性引子對 (PN-1F/PN-2R) 測試 84 專一性引子對 (PN-1F/PN-2R) 靈敏度測試 85 專一性引子對 (PN-1F/PN-2R) 偵測田間之褐根病菌 85 結果 85 專一性引子對之設計 85 PN-1F/PN-2R引子對之專一性 86 PN-1F/PN-2R引子對之靈敏度 86 應用專一性引子對 (PN-1F/PN-2R) 偵測田間之褐根病菌 討論 87 引用文獻 89 英文摘要 92 圖表 93 第六章 褐根病菌之生態與病害防治 100 摘要 100 前言 101 材料與方法 102 供試菌株與接種源製備 102 傷痍接種對褐根病發病之影響 102 根系接觸傳染 102 供藥劑篩選菌株 102 供試藥劑種類 103 藥劑對褐根病菌菌絲生長之抑制力測定 104 藥劑對接種幼苗罹患褐根病之防治效果 105 不同氮肥對褐根病發病之影響 106 淹水、肥料及化學藥劑對褐根病菌存活之影響 106 田間病害防治 107 結果 108 傷痍對褐根病發病之影響 108 根接觸傳播試驗 108 供試藥劑對褐根病菌菌絲生長之抑制效果 108 供試藥劑於溫室內對盆栽植株罹患褐根病之防治效果 109 不同氮肥對褐根病發病之影響 109 淹水、肥料及化學藥劑對褐根病菌存活之影響 110 田間病害防治 110 討論 111 引用文獻 115 英文摘要 118 圖表 120 第七章 結論與討論 129 引用文獻 13

(67(3):318-322)Basal Stem Rot of Betel Palm Caused by Ganoderma boninense in Taiwan

自2006–2012 年由台灣7 處果園 (包括高雄市、屏東內埔與萬巒，及南投草屯) 分離為害檳榔的靈芝病原菌，共獲得32 菌株。在田間，靈芝為害檳榔的根系與莖基部，造成根部與莖基部組織褐變腐敗，終至樹木死亡。罹病的檳榔樹基部與根系，則長出子實體-擔子果 (basidiocarp)。擔子果具短柄或無，正面菌蓋 (pileus) 褐色、紅褐色或暗褐色，具假漆狀光澤；背面菌孔面 (pore surface) 淡黃色或淡灰黃色，菌孔圓形或橢圓形。利用太空包木屑培養 (plastic bag sawdust culture)，也可以誘導靈芝菌形成擔子果，釋放擔孢子，惟形成的擔子果較田間自然生長者較小。以小麥-燕麥培養基培養靈芝菌種，接種後可以誘發接種之檳榔組織褐變，並可再分離到原接種靈芝菌，完成病原性測定。參考前人文獻，比較檳榔靈芝菌之子實體、菌孔及擔孢子的形態與大小，以及上網National Center for Biotechnology Information (NCBI) 資訊庫比對ITS DNA (ITS1-5.8S rDNA-ITS2) 序列，證實為害台灣檳榔的靈芝菌為狹長孢靈芝 (Ganoderma boninense)。 From 2006 to 2012, a total of 32 isolates of Ganoderma sp. were isolated from betel palm in seven orchards located at Kaohsiung City, Neipu and Wanluan of Pingtung County, and Caotun of Nantou County in Taiwan. In the field, Ganoderma could infect root and stem base of betel palm, causing root and stem base brown rot and necrosis, and eventual death. Basidiocarps emerging from stem base of the diseased betel palm were stipitate, or sessile. Upper surface of pileus was brown to dark, red-brown, and laccate. Pore surface was pale yellow to grey and pores were circular to oval. Basidiocarps could be produced when cultured on sawdust medium, but were smaller in size than those grown in the field. Inoculation of purified Ganoderma isolates on betel palm showed brown rot symptom, and the same Ganoderma was reisolated, confirming the pathogenicity. Based on morphological characteristics, including fruiting body, pore surface and basidiospores, and result of ITS region (ITS1-5.8S rDNA-ITS2) sequence similarity searching, done via Basic Local Alignment Search Tool (BLAST) provided from National Center for Biotechnology Information (NCBI), Ganoderma infecting betel palm in Taiwan was identified as Ganoderma boninense

Morphological, Molecular and Pathological Characterization of Phytophthora amaranthi sp. nov. from Amaranth in Taiwan

In the spring of 2007, a serious disease on amaranth was noticed in several farms in the major amaranth production area in central Taiwan. Abundant oospores were found in the disease tissues. A species of Phytophthora was consistently isolated from disease tissues. The organism formed abundant oospores with smooth walls and with amphigynous antheridia in single culture. Sporangia were partially deciduous with short- to medium-length pedicels. Morphological characteristics of this organism did not match any reported Phytophthora species, and the organism was named Phytophthora amaranthi. Pathogenicity tests and molecular characterization confirmed the identity of the organism as a new pathogen of amaranth and a new species of Phytophthora

Application of a bio-control agent for controlling strawberry anthracnose in Taiwan

Strawberry (Fragaria × ananassa Duchesne) is an economically important crop in Taiwan, however because of the outbreak of strawberry anthracnose in early 2010, the short supply of strawberry seedlings became a serious problem for strawberry production. The isolates of anthracnose causing organisms were collected from major strawberry cultivation areas in Miaoli County, and mainly identified as Colletotrichum siamense, these isolates were proved to be pathogenic to the leaves, crown and roots of strawberry. The bio-control agent of Bacillus amyloliquefaciens P-2-2, was isolated from rice paddy field and expressed effectively on inhibition of many fungal pathogens of strawberry. The strawberry seedlings used in this study were treated with 400-fold dilution of B. amyloliquefaciens P-2-2 fermentation broth once for every two weeks to protect the crown of the seedlings from anthracnose infection before transplanting. Sterilization of soil by biological soil disinfestation method was done during field preparation. After transplanted in the production field, the strawberry transplants were continuously treated with 400-fold dilution of B. amyloliquefaciens P-2-2 once for every two weeks until the end of the growing season. The results of field test, shown that the survival rate of the untreated group in demonstration area of Dahu Township was 83.9%, whereas the survival rate of seedlings treated with B. amyloliquefaciens P-2-2 increased to 95.1%. The replanting rate of strawberry in the untreated group area was 37.6%, compared to the B. amyloliquefaciens P-2-2 treated area which was reduced significantly to 10.7%. Upon those results indicated that application of bio-control agents with other practices were effective for controlling strawberry anthracnose

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

In Vitro and in Planta Evaluation of Trichoderma asperellum TA as a Biocontrol Agent Against Phellinus noxius, the Cause of Brown Root Rot Disease of Trees

Brown root rot (BRR), caused by the white rot fungus Phellinus noxius, is an epidemic disease of diverse broadleaved and coniferous tree species in many tropical and subtropical regions. Flooding and trenching control measures are difficult to implement, and chemical controls can have an adverse impact on ecosystems. Previous studies have provided in vitro evidence for the potential use of Trichoderma spp. for biocontrol of BRR. Here, we analyzed the in vitro antagonistic and mycoparasitic abilities of four Trichoderma spp. isolates against four P. noxius isolates in dual culture and Ficus microcarpa wood blocks. A convenient inoculation system based on root inoculation of a highly susceptible loquat (Eriobonya japonica) with P. noxius-colonized wheat-oat grains was developed to examine the effect of Trichoderma treatment in planta. Preventive application of Trichoderma asperellum TA, the isolate showing high antagonistic activity in vitro, was effective in preventing and delaying the wilting of P. noxius-inoculated loquat cuttings in greenhouse trials. To understand the specific niche in which T. asperellum TA interacts with P. noxius, KOH-aniline blue fluorescence microscopy was used to investigate the colonization of loquat roots by P. noxius and/or T. asperellum TA. Dilution plating assays were also conducted to quantify Trichoderma populations in the rhizosphere and potting mix. T. asperellum TA was able to robustly establish in the rhizosphere and potting mix but with scarce root penetration limited to the superficial layer. We discuss the timing and strategy for applying antagonistic Trichodema sp. on living trees or in BRR-infested areas for BRR management

台灣藥用植物疫病之新紀錄

Three species of Phytophthora isolated from Chinese medicinal herbs grown in the fields during 2001-2009 were proven to be the new records of pathogens on these crops in Taiwan. They are Phytophthora cryptogea on dan-shen (Salvia miltiorrhiza), P. drechsleri on astragalus (huang-qi, Astragalus membranaceus) and P. nicotianae (=P. parasitica) on rehmannia (ti-huang, Rehmannia glutinosa). Phytophthora cryptogea attacked roots and basal stems of dan-shen, causing plant wilting, leaf drooping, and eventual plant death. Phytophthora drechsleri attacked leaves, stems and roots of astragalus, causing leaf and stem blight, and wilt and death of infected plants. Rehmannia was highly susceptible to P. nicotianae, as the infected plants were killed within a very short period of time. All the isolates of P. cryptogea, P. drechsleri and P. nicotianae obtained from the medicinal herbs in Taiwan belong to A1 mating types. Results of the inoculation tests in the greenhouse showed that all the three species were pathogenic and the disease symptoms on inoculated host plants were similar to the naturally infected plants grown in the fields. Molecular data of the internal transcribed spacers (ITS) region and a partial sequence of the β-tubulin gene also support the traditional classification based on morphological and physical characteristics of P. cryptogea, P. drechsleri and P. nicotianae. Phytophthora cryptogea on dan-shen and P. nicotianae on rehmannia have not been reported in any other country prior to this report. 自 2001-2009 年調查台灣的藥用植物疫病，發現有三種未曾正式報告之疫病新紀錄，包括Phytophthora cryptogea為害丹參(Salvia miltiorrhiza)，P. drechsleri為害黃耆(Astragalus membranaceus)，以及P. nicotianae (= P. parasitica ) 為害地黃(Rehmannia membranaceus)。分述如下：P. cryptogea為害丹參的莖基部與根系，造成組織褐變腐敗，葉片下垂萎凋，罹病植株最終死亡；P. drechsleri 感染黃耆小葉、莖部及根系，造成莖、葉腐敗與植株萎凋死亡。地黃對P. nicotianae非常感病，全株均可被感染，罹病植株在短期內大量死亡，這些病害都在夏秋季降雨季節發生。所有從丹參、黃耆及地黃分離到的疫病菌株均為A1 配對型 (mating types)。在溫室，將各寄主植物分離到疫病菌的游走子懸浮液接種到寄主植物，均會產生與田間相同的病徵，而且相同的疫病菌均可自接種後發病的寄主組織上分離得到，證明這些保健植物罹患疫病分別由上述三種疫病菌引起。此外，分析上述三種疫病菌的核醣體內轉錄區間(ITS region, ITS1-5.8SrDNA-ITS2) 與β微管蛋白基因(β-tubulin) 的部份DNA序列，亦支持這三種疫病菌的分類地位。這三種疫病菌中，除P. drechsleri 為害黃耆在韓國有記錄外，其餘P. cryptogea為害丹參與P. nicotianae為害地黃在世界其他各地尚未發表過

(62(2):184-194) Use of Polycarbonate Membrane for Detection of Extracellular Enzymes of Phellinus noxius on Solid Media

褐根病為近年來台灣普遍發生的木本植物根部病害之一，被為害植物的根系組織嚴重腐敗。本文利用薄膜培養基測試法探討褐根病菌分泌10 種細胞外泌酵素 (extracellular enzymes) 之能力。試驗結果顯示分別分離自南投縣、高雄市等地區所採集罹病植株之5 支褐根病菌供試菌株 (PN18.1：分離自南投縣中興新村之櫸木；PN22.1：南投縣國姓鄉之木麻黃；PNG1.1：南投縣水里鄉之葡萄；PNP1.1：高雄市甲仙區之梅樹；及PNZ1.1：高雄市阿蓮區之印度棗) 皆能分泌澱粉分解酵素 (amylase)、纖維分解酵素 (cellulase)、果膠分解酵素 [pectinases, 含polygalacturonases (PGs) 與pectate transeliminases (PTEs)]、蛋白分解酵素 (protease)、磷酸分解酵素 (phosphatase)、尿素分解酵素 (urease)、脂質分解酵素 (lipase) 及木質分解酵素 (laccase)；但不能分泌磷脂分解酵素 (phosphatidase) 與幾丁質分解酵素 (chitinase)。除木質分解酵素外，各菌株分泌各種分解酵素的能力隨酵素種類的不同而有強弱之差異，可達到5% 顯著性水準。然而所有供試菌株分泌木質分解酵素的活性均甚高。本研究所建立褐根病菌分解酵素之檢測技術，可進一步應用於褐根病菌致病機制之研究。 Brown root rot caused by Phellinus noxius is one of the most serious diseases associated with decline of fruit trees and ornamental woody trees in Taiwan. The objective of this study was to develop a modified polycarbonate membrane method for detection of extracellular enzymes produced by P. noxius growing on solid agar media. Five isolates of P. noxius collected from different host plants in Taiwan were used in this study, including isolate PN22.1 from ironwood (Casuarina equisetifolia) at Guosing, Nantou, isolate PNG1.1 from grapevine (Vitis vinifera) at Shueili, Nantou, isolate PN18.1 from zelkova (Zelkova serrata var. serrat) at Chungsin, Nantou, isolate PNP1.1 from plum (Prunus mume) at Jiasian, Kaohsiung and isolate PNZ1.1 from Indian jujube (Zizyphus mauritiana) at Alian, Kaohsiung. They were tested for production of extracellular enzymes by growing each isolate on polycarbonate membrane disc (0.2 μm in pore diam.) which was placed on PDA amended with enzyme-reaction chemicals for detection of enzymes. Results showed that eight extracelluar enzymes, including amylase, cellulase, pectinases (polygalacturonase and pectate transeliminase), protease, phosphatase, lipase and laccase, were detected in all the five isolates of P. noxius, but no phosphatidase and chitinase were detected from these isolates. Among the eight enzymes detected, the level of laccase secreation was high in all the five isolates, but the level of secretion of other seven enzymes varied with the isolates tested. The technique for detection of enzymes can be applied to pathogenecity analysis in P. noxius and other fungi

The Genetic Structure of <i>Phellinus noxius</i> and Dissemination Pattern of Brown Root Rot Disease in Taiwan

<div><p>Since the 1990s, brown root rot caused by <i>Phellinus noxius</i> (Corner) Cunningham has become a major tree disease in Taiwan. This fungal pathogen can infect more than 200 hardwood and softwood tree species, causing gradual to fast decline of the trees. For effective control, we must determine how the pathogen is disseminated and how the new infection center of brown root rot is established. We performed Illumina sequencing and <i>de novo</i> assembly of a single basidiospore isolate Daxi42 and obtained a draft genome of ~40 Mb. By comparing the 12,217 simple sequence repeat (SSR) regions in Daxi42 with the low-coverage Illumina sequencing data for four additional <i>P</i>. <i>noxius</i> isolates, we identified 154 SSR regions with potential polymorphisms. A set of 13 polymorphic SSR markers were then developed and used to analyze 329 <i>P</i>. <i>noxius</i> isolates collected from 73 tree species from urban/agricultural areas in 14 cities/counties all around Taiwan from 1989 to 2012. The results revealed a high proportion (~98%) of distinct multilocus genotypes (MLGs) and that none of the 329 isolates were genome-wide homozygous, which supports a possible predominant outcrossing reproductive mode in <i>P</i>. <i>noxius</i>. The diverse MLGs exist as discrete patches, so brown root rot was most likely caused by multiple clones rather than a single predominant strain. The isolates collected from diseased trees near each other tend to have similar genotype(s), which indicates that <i>P</i>. <i>noxius</i> may spread to adjacent trees via root-to-root contact. Analyses based on Bayesian clustering, <i>F</i>_ST statistics, analysis of molecular variance, and isolation by distance all suggest a low degree of population differentiation and little to no barrier to gene flow throughout the <i>P</i>. <i>noxius</i> population in Taiwan. We discuss the involvement of basidiospore dispersal in disease dissemination.</p></div