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Biological Characteristics, Detection, and Control of Cylindrocladium scoparium, the Causal Agent of Rose Black Rot
|關鍵字:||Cylindrocladium scoparium;Cylindrocladium scoparium;Biological characteristics;Semiselective medium;Rose petiole baiting method;Detection;Control;Prochloraz-Mn;生物特性;半選擇性培養基;玫瑰葉柄誘釣法;偵測;防治;撲克拉錳||出版社:||植物病理學系||摘要:||
近年來，在埔里與新社玫瑰花切花栽培田中，發現玫瑰花扦插苗與成株出現枯死的現象，罹病植株莖基部出現褐變、壞死與龜裂的病徵；至於地下部的根系則呈黑變腐敗的症狀。將罹病株分離到的Cylindrocladium sp.菌株，重新接種於玫瑰花扦插苗，六天後，植株出現如同在田間相似的病徵。本病原菌培養在馬鈴薯葡萄糖瓊脂培養基平板上，菌落呈深黃褐色，其分生孢子柄直立透明，有二至三次整齊樹枝狀分枝，每一孢子柄末端著生二至三個瓶狀枝；本菌有一長絲狀無性附絲，附絲頂端膨大成橢圓形泡囊；分生孢子長柱形，透明，有單一隔膜，大小為38.1-44.6 ×5.9-6.1 m，孢子於瓶狀枝上單生，可群聚成束狀；厚膜孢子褐黑色，大小為9.2-12.2 ×6.8-8.4 m。按Peerally氏及Watanabe氏等文獻，將本病原菌鑑定為Cylindrocladium scoparium Morgan。本病原菌 CS-01與CS-05菌株最適生長溫度介於24-28℃間，最適生長酸鹼值為pH 6至9。利用孢子懸浮液接種法與病菌土接種法，發現玫瑰花扦插苗的發病百分率，會隨著接種菌量濃度的增加(由0至104 propagules/ml)，愈趨嚴重。C. scoparium CS-01與CS-05菌株可感染玫瑰花、杜鵑花、樟樹、番石榴與芒果等寄主，惟各寄主扦插苗表現的病徵並不盡相同。玫瑰花品種間以沙曼莎與科農博格兩品種對本菌較具抗性，至於佳娜紅、丹姆斯、美而佳之光等三品種較感病。評估13種碳素源與12種氮素源對C. scoparium CS-01與CS-05菌絲生長的影響，發現澱粉、蔗糖及甘油等三種碳素源可顯著促進菌絲的生長；而氮素源方面則以硝酸鈉、亞硝酸鈉、硝酸鉀及酪蛋白(casein)等四種氮素源可顯著促進本菌菌絲的生長，其中以甘油作為碳素源搭配酪蛋白作為氮素源之組合，除較適本菌的生長外，尚可明顯分辨本菌的菌落。比較16種化學藥劑對本菌絲生長的影響，發現福多寧(flutolanil)藥劑不具抑制本菌菌絲生長的效果。將甘油30公克、酪蛋白2公克、磷酸氫二鉀1公克、硫酸鎂0.5公克、氯化鉀0.5公克、硫酸鐵0.01公克、洋菜20 公克及蒸餾水1公升均勻混合，經高壓滅菌(121℃，15 Ib，15 min.)後，添加200 mg /L 福多寧藥劑，再以乳酸調整pH值至5後，製成之GCFL半選擇性培養基，可以有效且準確的偵測出人工病菌土中的玫瑰黑腐病菌。將玫瑰花、樟樹、肉桂及杜鵑花等葉柄插於病菌土中四天後，發現玫瑰葉柄誘釣本菌的效率最高。至於防治方面，在病菌土中施用25%(w/w)的Streptomyces sioyaensis PMS-502固態微生物製劑，可以有效減少玫瑰黑腐病的發生。此外，將玫瑰花插穗浸泡於200 ppm撲克拉錳或500 ppm 依普同，20分鐘後，扦插於病菌土中，亦可有效防治玫瑰黑腐病。本研究證明，玫瑰花插穗受C. scoparium感染24小時內，須浸泡200 ppm撲克拉錳超過5小時，才可完全抑制玫瑰黑腐病的發生。
Recently, commercial plantings of rose (Rosa hybrida L.) at Puli and Shinshe areas in central Taiwan were damaged by an unclear disease. Symptoms consisted of a blackening of the root cortex, root rot, discoloration of lower stem bark and wood, sometimes accompanied by longitudinal cracking. Severe root infection resulted in heavy mortality in rose cutting seedlings and plants. A species of Cylindrocladium was consistently isolated from disease portions on 2 % (w/v) water agar. Pathogenicity was confirmed by inoculating two representative isolates, CS-01 and CS-05 onto rose cutting seedlings for 7 days under high humidity (RH> 95%) at 28℃. Discoloration of stem bark and wood similar to the original symptoms developed only on inoculated plants. Noninoculated plants remained symptomless. On potato dextrose agar, growth of the fungus was rapid. Colonies reached 59.4~55.3 mm diameter for 6 days at 28℃, with yellowish to deep brown. Conidiophore upright, hyaline, regularly and repeatedly dichotomously or trichotomously branched, each terminating in two or tree phialides;Typically with a slender elongated sterile branch terminating in an ellipsoid swelling;Conidia hyaline, 38.1-44.6 ×5.9-6.1 m, 2-celled, cylindrical, borne singly but held togehter in bundles by mucilage. chlamydospores produced abundantly, globose to oblong, yellow-brown, single cell, 9.2-12.2 ×6.8-8.4 m. The optimum temperature for conidial germination and mycelial growth of both isolates was at 24~ 28℃. The Optimum pH value for mycelial growth of C. scoparium was at 6~9. The best temperature for both infection on rose cuttings was at 24~ 28℃. The results indicated that the fungus was identified as Cylindrocladium scoparium Morgan according to the references of Peerally (1991) and Watanabe (1994). Thirteen carbohydrates and twelve nitrogenous compounds were evaluated for their effects on mycelial growth of two isolates, CS-01 and CS-05 of the pathogen. Among those compounds, sucrose, glycerol, and starch were more effective than other carbohydrates to enhance the growth of C. scoparium. As to nitrogenous compounds, casein, NaNO3, and NaNO2 were more effective in stimulating growth of the pathogen. Sixteen pesticides were respectively added into the basal medium(a modified Czapek's medium containing glycerol and casein, GC medium) for indexing their suppressive effectiveness. Flutolanil at 200 ppm and quinolate at 600 ppm were not obviously effective in inhibiting the pathogen. In advance, the glycerol-casein-flutolanil-lactic acid semiselective medium (GCFL medium) consisting of 30 g glycerol, 2 g casein, 1 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.01 g FeSO4·7H2O, 20 g agar, 200 mg/L flutolanil, 1 ml lactic acid and 1L distilled water was hence formulated for the enumeration and isolation of C. scoparium from soil and plant tissues. The results showed that C. scoparium was able to be accurately detected from artificially and naturally infested soils by the use of GCFL semiselective medium. A baiting technique using petiole of rose and camphor tree accompanied with GCFL semiselective medium also provided a means of detecting the pathogen from the field soils less with than 102 propagules / g. soil. The disease could be controlled by Streptomyces sioyaensis PMS-502 and Prochloraz-Mn. In the study, PMS-502 microbial agent (solid formula) was more effective in reducing infection percentage of rose cuttings by the pathogen than other antagonists tested. In addition, the disease could be completely controlled when rose cuttings were infected within 12 hrs and dipped in 200 ppm Prochloraz-Mn solution for more than 5 hours.
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