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Influence of cationic polyelectrolytes on the penetration process of Botrytis elliptica (Berk.) Cook into lily leaves and their characteristics for suppression of the pathogen
|關鍵字:||Botrytis elliptica;百合灰黴病菌;environmental factors;leaf disk assay;spore adhesion;infection process;polyelectrolytes;esterase;disease control;發病條件;葉片圓盤法;孢子黏著;侵染過程;聚電解質;酯化酵素;病害防治||出版社:||植物病理學系||摘要:||
百合灰黴病菌[(Botrytis elliptica (Berk.) Cooke]孢子發芽之溫度範圍為8至36℃。在蒸餾水中本菌之孢子發芽率可達95%以上，然在相對濕度98%以下，孢子卻不發芽。在水分潛勢介於-2.5至-75 bars間本菌孢子發芽率均不受影響；惟在低於-30 bars時，本菌菌絲生長即受限制。百合灰黴病發病溫度介於12與24℃間，最適發病溫度為20℃，溫度超過32℃即不表現病徵。百合灰黴病菌於風乾的鐵炮百合雜交品種葉片上，在19至29℃下，經5天仍可感染並引起病徵，惟在31℃下經3天後即已無法引發病徵。台灣百合灰黴病的發生時期，在平地為1-6月，中海拔地區(600-800公尺)為4-6月及10-12月，而高海拔地區(1200-2000公尺)則發生於6-10月。病害發生之嚴重性以鐵炮百合(Lilium longiflorum hybrid cv. Snow Queen)最高，其次為葵百合(Lilium oriental hybrid cv. Star Garzer)、姬百合(L. asiatic hybrid cv. Positano)及香水百合(L. oriental hybrid cv. Casa Blanca)。田間百合灰黴病的分佈初呈點狀零星發生，後期呈全面性感染。百合灰黴病雖然最初發生於葉片，但後期花苞更為感病。設施內栽植百合可減緩本病之發生，而露天栽培時病害提早發生，且病害的擴展較快。百合灰黴病菌具寄主專一性，在本研究未見其為害其他作物。
在百合植株與葉片圓盤接種百合灰黴病菌[Botrytis elliptica (Berk.) Cooke]後，分析葉片圓盤受害率與整株百合罹病率間的相關性，結果建立葉片圓盤分析的流程與方法如下：首先摘取成熟百合植株之葉片(由最低花梗起算之第五至十位葉)，以1%次氯酸鈉表面消毒三分鐘後，經無菌水漂洗三次，晾乾後以直徑15mm打孔器切取葉片圓盤，置於平舖一張6公分之濾紙且含2毫升無菌水之9公分培養皿中，每皿置放8片葉片圓盤。接種每毫升含1×105個百合灰黴病菌之胞子懸浮液，使圓盤上覆有一層大小約0.3mm之水珠。覆以皿蓋，並移置於20℃之定溫箱中，按時觀察病勢的進展。在室內採用葉片圓盤法與在溫室利用整株植物分別檢測藥劑與拮抗微生物防治百合灰黴病的功效，證明兩者間的結果相吻合。葉片圓盤法不但可減少植材與葉片用量外，並可節省試驗空間與時間，且可準確篩選出防治百合灰黴病菌的藥劑與拮抗微生物，是一個值得推薦的簡便方法。
百合灰黴病菌B. elliptica孢子接種在玻片上，於20℃下靜置60分鐘後約有5%孢子黏附著，120分鐘達25%，160分鐘達75%，180分鐘以後可達80%以上。以polystyrene和葵百合葉片(cv. Star Gazer)作為基質時，孢子黏附的時程相似。接種後120分鐘，孢子細胞內原生質體明顯顆粒化，並開始發芽。孢子黏附在玻片表面的比例隨著孢子發芽率的增加而增加，未發芽的孢子黏附能力較差，而孢子發芽後可緊密地黏附在物體表面上。孢子黏附作用與物體表面的疏水性無明顯關係，而與發芽率則呈明顯正相關。本菌孢子的發芽形式有兩種，一為短而膨大型的發芽管，一為一般的發芽管。基質表面疏水性與孢子產生膨大型發芽管的現象有關，表面疏水性能力越高，孢子產生膨大型發芽管的能力越強。孢子發芽後，在發芽管周圍可分泌黏膜物質，以助其黏附於基質表面。孢子經呼吸作用抑制劑疊氮化鈉、氧化丙烯和80℃熱處理10分鐘等處理後，其黏附在玻片和百合葉片上的情形明顯被降低。由以上試驗可知孢子的黏附作用與孢子的代謝活性有關。
百合灰黴病菌在無菌水中3小時之發芽率達60%，6小時達80%，8小時可達95%以上；附著器於水中8小時後開始形成，第20小時達20%，24小時後可達60%左右。利用光學及掃描式電子顯微鏡觀察本菌在葵百合(cv. Star Gazer)葉片上的侵染過程，發現本菌於百合葉表面可以發芽，但無法侵入感染；於葉背時則孢子發芽後不必形成附著器即可直接侵入表皮、保衛細胞或氣孔，其中侵入之部位多在孢子附著處。在光學顯微鏡下可見入侵部位有一侵入孔(infection pore)，且於入侵處之表皮細胞有被酵素作用的痕跡。灰黴病菌在百合葉背3小時之發芽率約達70%，然未見侵入表皮細胞的跡象，直到第6小時孢子發芽率達88.6%，其中32%的孢子侵入葉肉組織，且有14%於侵入部位表現病徵；在第9小時後，約60%之發芽孢子可侵入組織，並有高達60%侵入點可產生病斑。本菌侵入的組織可顯現螢光反應，有助於充作判斷百合組織是否受感染的參考依據。由光學與電子顯微鏡觀察結果證明本菌感染寄主的第一個步驟是以發芽管為主侵入百合組織。
百合灰黴病菌以直接侵入方式或經由葉背氣孔侵害植物，若能噴施在葉表形成薄層保護膜之物質以預防其入侵，應為可行的防治之道。因此，本研究在實驗室、溫室及田間評估各種薄膜型抗蒸散劑對百合灰黴病的有效性。在實驗室內以葉片圓盤法測定6種聚電解質(polyelectrolytes)對百合灰黴病之影響，篩選出FO4240SH、FO4490SH及FO4550SH等可有效抑制病害之發生。而其中帶正電之聚電解質FO4240SH和FO4490SH之最佳稀釋濃度為333和400ppm，在此濃度下皆可降低罹病病斑面積率，並減少病原菌在罹病葉片上之產孢，溫室試驗結果亦顯示FO4490SH及FO4550SH具有降低葉片病斑數及抑制病斑大小之功能。於田間發病之東方型百合－馬可波羅(cv. Marco Polo)、阿卡波克(cv. Acapulco)和凱撒布蘭加(cv. Casa Blanca)等品種上分別處理333 ppm的FO4490SH水溶液、333 ppm的FO4490SH混合250 ppm撲滅寧水溶液及250 ppm的撲滅寧水溶液，並以不處理者為對照組，結果顯示各種處理均可有效降低灰黴病之病勢進展，且對百合植株未造成藥害。本研究發現帶正電之聚電解質具有吸附病原菌孢子之功效，並打破葉表拒水特性，使發芽管不正常增生或形成菌絲型發芽管，而不易入侵寄主；抑制孢子發芽及菌絲生長的效果，尚可降低發芽管中酯化酵素(esterase)的含量。發芽管中酯化酵素之含量與灰黴病病斑數間呈顯著正相關(r=0.769, p<0.01)，顯示聚電解質防治百合灰黴病之機制與其抑制病原菌發芽管分泌酯化酵素有關。
Botrytis leaf blight caused by Botrytis elliptica (Berk.) Cooke is an important foliar disease of ornamental lilies in Taiwan. The required temperature for spore germination of B. elliptica was ranged from 8 to 36℃. Percentage of spore germination was more than 95% in distilled water and 25-32% under 100% relative humidity at 20℃, but no spore germinated when the relative humidity was under 98%. Spore germination of B. elliptica was not significantly different on corn meal agar with water potential ranging from -2.5 to -75 bars at 20℃, while its mycelial growth was markedly inhibited at or below -30 bars. The favorable temperatures for plant infection by B. elliptica ranged from12 to 24℃ and the optimal one for disease development was at 20℃. However, no disease occurred at or over 32℃. Duration of dryness at different temperatures after inoculation affected the disease development. After 5-days air-dried duration, the symptoms appeared at 19, 23 and 29℃, but did not at 31℃. The period of disease occurrence of Botrytis leaf blight on lilies in Taiwan was as follows: from January to June at lower sea-level area, from April to June and October to December at middle sea-level (600-800 m) area, and from June to October at higher sea-level (1200-2000 m) area. The higher frequency of disease occurrence was observed on Lilium longiflorum (cv. Snow Queen), followed by L. oriental hybrid (cv. Star Gazer), L. asiatic hybrid (cv. Positano) and L. oriental hybrid (cv. Casa Blanca). Botrytis leaf blight was initially sporadic at lower leaves of lily plants and dispersed randomly in the field. When environmental conditions were favorable for disease development, the pathogen was rapidly disseminated to all of plants and serious disease broke out in the field. The flower part of plants was more susceptible to be infected by B. elliptica than leaf part. The disease severity was significantly reduced when lilies were grown under structure, however, the plants became severe infection by the pathogen when they were out of structure.
A leaf-disk method was developed for disease assessment of lily leaf blight caused by Botrytis elliptica. Oriental hybrid lily (cv. StarGazer) grown from bulbs in a greenhouse for 1.5 to 2 months was used to prepare leaf disks. The fifth to tenth leaves below the apex were surface-sterilized by dipping entire leaves in 1% sodium hypochlorite for three times and rinsing in fresh sterile water after each dip. Leaf disks (15-mm diameter) were cut from surface-sterilized leaves with a cork borer, and fully submerged in fungicide solutions or antagonist suspension. The disks (8 per replication) were placed abaxial surface up in petri dishes containing a piece of 60-mm-diameter sterilized filter paper. Two milliliters sterile water was added to the filter paper to maintain moisture. A spore suspension adjusted to 105 conidia per ml was sprayed over the disks to form inoculum droplets of up to 0.3 mm in diameter, and then the disks were incubated at 20 ℃ in darkness. After 3 days, disease severity was assessed as percent necrotic area. The results of the leaf-disk assay in vitro (petri dish) were consistent with that of whole plant assays in vivo (greenhouse). The leaf-disk assay not only reduces the requirement for plant materials, but also saves time in screening effective fungicides and antagonists.
The nonspecific attachment of Botrytis elliptica germlings to a various surfaces of substrata was examined. Washing experiments showed that 7% of the conidia began adhering to glass slide just prior to germination 60 min after inoculation; after 120 min, approx. 25% of the conidia resisted washing and remained firmly attached; not until 180 min were over 80% of conidia able to attach to the glass slide. Similar results were obtained from conidia inoculated to surfaces of polystyrene and oriental lily leaves. The protoplast of conidia obviously appeared granular and started germinating 120 min after inoculation at 20℃. Attachment of conidia to the substrata was increased with the increment of conidial germination. A nongerminated conidium was significantly less able than a germinated conidium to adhere to the surface of a substratum. There was no correlation between conidial adhesion and hydrophobicity of substrata. Conidia germinated with swelling and normal germ-tubes were observed. The hydrophobic substrata induced production of a swelling in germ-tubes from conidia. Light microscopy and SEM observations showed that an ensheathing material secreted from conidia and germ tubes appeared around the germlings of B. elliptica. Attachment of conidia was prevented by metabolic inhibitor such as sodium azide, propylene oxide and 80℃-hot water. The results suggested that metabolic activity of the pathogen was required for conidial adhesion.
Germination of conidia of B. elliptica was 60% at 3 h, 80% at 6 h, and over 95% at 8 h of incubation at 20℃ in sterile water on glass slides. Appressoria formed initially after 8 h of incubation, and 20% of germ tubes formed appressoria by 20 h and 60% by 24 h. To observe the infection process of B. elliptica on leaves of oriental lily (cv. Star Gazer), 15-mm-diameter leaf disks were inoculated with 105 conidia ml-1 and incubated in 100% RH at 20℃ for 16 h. Spores of the pathogen germinated on both adaxial and abaxial surfaces, but germ tubes failed to invade the epidermal cell of adaxial surface. On abaxial surfaces of lily leaves, short and swollen germ tubes directly penetrated into tissue through epidermal cells, guard cells and stomata. The infection site was always near where the spores had adhered. Length of penetration germ-tube was not longer than 20 m and no multicellular appressoria formed at that time. With microscopical observation of infection site, the likely structure of infection pore was obvious under the tip of germ tubes. Enzyme degradation of cuticle on abaxial surface of lily was observed surrounding the penetration germ tubes and infection pore. Approx. 70% conidia germinated on abaxial surface of lily leaves 3 h after inoculation, however no conidia penetration was observed. Almost a third of the 88.6% germinated conidia penetrated epidermal cells, and then approx. 20% of penetration caused necrotic lesion symptoms at 6th h after inoculation. Moreover, approx. 60% of germinated conidia penetrated plant tissues and 60% of penetrated lesions showed necrotic symptoms after 9 h inoculation. Fluorescent reaction appearing around the infection site under fluorescent microscopy was a helpful marker to identify whether the infection of B. elliptica was successful or not. Based upon the microscopy observation, it was suggested that the first infection step of B. elliptica should be mainly via the tissues was penetrated by the primary germ-tube.
The effectiveness of film-coating antitranspirants in control of lily leaf blight caused by B. elliptica has been evaluated via laboratory leaf disk assay, greenhouse tests and field trials. Among the seven polyelectrolytes, 400 ppm FO4240SH, FO4490SH and FO4550SH were found to be effective in reducing the disease severity of lily leaf blight in laboratory leaf disk tests. Both FO4240SH and FO4490SH were also effective in suppressing sporulation of the pathogen on leaf disks. In greenhouse tests, the number and size of leaf lesion on Lilium oriental hybrid cv. Star Gazer were markedly reduced by FO4490SH and FO4550SH. Field trials showed that the effectiveness of FO4490SH was similar or better than that of procymidone on the reduction of lily leaf blight disease severity. The polymers have no harmful effect to the host plants. The cationic polyelectrolytes FO4240SH, FO4490SH and FO4550SH were not only able to reduce the percentage of conidial germination and inhibited germ tube growth, but also suppressed the esterase production of the germ tubes of B. elliptica. All the above evidence indicates the disease control of polyelectrolytes is due to, at least in part, the reduction of esterase secretion by B. elliptica.
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