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白粉病是影响苜蓿产量和品质的主要真菌病害之一,引起紫花苜蓿白粉病的病原有3种:蓼白粉菌(
系统研究紫花苜蓿白粉病病原菌的生物学特性,以及分生孢子和闭囊壳的形态特征,明确宁夏地区紫花苜蓿白粉病病原种类。
采用室内盆栽法调查病害发生和闭囊壳形成条件,利用气象站自动获取数据,采用光学显微镜观察病原菌特征,采用水琼脂玻片法观察病原菌分生孢子萌发特性。
紫花苜蓿白粉病发生的温度范围为20−24 ℃,湿度范围为40%−60%,形成闭囊壳的条件为温度20−25 ℃、湿度70%左右,闭囊壳的形成大约需要42 d。分生孢子以向基式的产孢方式在分生孢子梗上产孢,成熟的分生孢子卵圆形或椭圆形,大小为(29−46)×(12.5−21.0) μm。闭囊壳黑褐色,球形或近球形,直径71−135 μm,具有大量附属丝,少数为不规则分枝1−2次。闭囊壳内含有4−6个子囊,子囊倒棒型或卵圆形,具柄,大小为(58−90)×(29−60) μm。子囊内含有4−6个子囊孢子,子囊孢子单孢,卵圆形或椭圆形,大小为(21−34)×(10.5−20.0) μm。分生孢子萌发的最适温度范围为22−26 ℃,最适pH为6.5,光照促进分生孢子的萌发。葡萄糖、D-木糖和甘露醇促进分生孢子萌发和芽管生长,半胱氨酸促进分生孢子萌发,甘氨酸促进芽管生长。
宁夏地区紫花苜蓿白粉病的病原菌确定为蓼白粉菌(
Powdery mildew is one of the main fungal disease affecting the yield and quality of alfalfa. It has been reported that there are three pathogens causing alfalfa powdery mildew:
The biological characteristics of alfalfa powdery mildew, the morphological characteristics of conidia and cleistothecium were systematically studied, and the pathogenic species of alfalfa powdery mildew in Ningxia were identified.
The occurrences of the disease and the formation conditions of the cleistothecia were investigated by pot culture in the laboratory. The data were obtained automatically by the meteorological station. The characteristics of the fungi were observed by the light microscope, and the germination characteristics of the conidia of the fungi were observed by the water agar slide method.
The temperature range of alfalfa powdery mildew is 20−24 ℃, the humidity is 40%−60%. The conditions for the formation of cleistothecia are 20−25 ℃ and humidity about 70%. The formation of cleistothecium takes about 42 d. Conidia produce spores on conidiophore in a basipetal form. Mature conidia oval or elliptic, with the size is (29−46)×(12.5−21.0) μm. The diameter of dark brown globular or subglobular cleistothecium is 71−135 μm, with a large number of appendages, and a few are irregular branches 1−2 times. The cleistothecium contains 4−6 asci, oval, with the handle, and the size is (58−90)×(29−60) μm. The ascus contains 4−6 ascospores, which are ascospore monospora, oval and the size is (21−34)×(10.5−20.0) μm. The optimum temperature range of conidia germination is 22−26 ℃, the optimum pH is 6.5, and the light could promote conidia germination. Glucose, D-xylose and mannitol promoted the germination of conidia and the growth of bud tube, cysteine promoted conidia germination, and glycine promoted the growth of bud tube.
The pathogen of alfalfa powdery mildew in Ningxia was identified as
紫花苜蓿(
真菌有性生殖产生的子实体具有过渡不良环境的作用[
紫花苜蓿,品种为WL298HQ,原产于美国,来源于北京正道生态科技有限公司,有包衣,由宁夏大学农学院草业研究所惠赠。
苜蓿白粉病病原菌为宁夏大学农科实训中心生长的苜蓿上自然发病的白粉病病原菌。
葡萄糖、甘露醇,天津大茂化学试剂公司;半胱氨酸、甘氨酸,博奥生物集团有限公司;琼脂,范德(北京)生物科技有限公司。
HOBO小型自动气象站,Onset公司;奥林巴斯荧光显微镜,Olympus有限公司;光照培养箱,上海甄明科学仪器有限公司。
选取品种为WL298HQ的苜蓿种子20粒,与适量的细土混匀播于直径为20 cm的花盆中,覆土1 cm,浇透水,覆膜保湿,待出苗后去掉保鲜膜。幼苗长至三叶期疏苗,每盆15株,种植20盆。于2018年9月在宁夏大学农科实训中心日光温室种植。
采用振动抖落法接种。接种前先用喷壶把苜蓿叶片冲洗干净。采集具有新鲜白粉病病原菌分生孢子的叶片置于接种苜蓿叶片的上方5 cm处,轻轻敲击振动叶片,10 min后在花盆上方50 cm处喷雾。
闭囊壳和分生孢子的形态特征观察均采用实验室常规水琼脂玻片法观察。将新鲜的分生孢子用毛笔刷轻轻刷在小烧杯中,再用吸管吸一滴放入干净的载玻片上,盖上盖玻片,在显微镜下观察,在所连接的电脑上测量分生孢子的大小,保证观测的分生孢子不低于300个。采集带有大量闭囊壳的叶片在水中浸泡24 h,挑针挑下成熟的闭囊壳放于载玻片,盖上盖玻片,在荧光显微镜下观测闭囊壳、附属丝形态,然后用尖头镊子轻轻敲盖玻片,使闭囊壳释放子囊,观测子囊以及子囊内子囊孢子的形态特征,拍照记录。
2019年3月底,对越冬苜蓿进行了刈割,2019年4月1日起开始调查,每花盆按照上、中、下均匀选取10片复叶,按照0−4病级标准调查,并计算发病率和病情指数。每周调查1次。
病级标准(自定):0级:无发病叶片;1级:病斑面积 < 25%;2级:病斑面积在25%−50%之间;3级:病斑面积在50%−75%之间;4级:病斑面积 > 75%。
发病率(%)=发病叶片数/调查总叶数×100;
自苜蓿叶片首现白粉病开始调查闭囊壳首次出现和成熟的时间,观察闭囊壳形态变化并拍照。
共设温度、光照、pH、碳源、氮源5种处理。参照《植病研究方法》水琼脂玻片法[
温度处理:设置5、10、15、16、18、20、22、24、26、28和30 ℃共11个温度梯度。
光照处理:设置全光照(4 000 lx)、全黑暗和半光照(12 h光照和12 h黑暗) 3个处理。
pH处理:设置pH为3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0共10个处理,以蒸馏水(pH 6.5)为CK。用1 mol/L的HCl和NaOH分别配制。
碳源处理:设置葡萄糖、麦芽糖、蔗糖、淀粉、D-木糖、甘露醇共6个处理。以100 mL 1.5%的水琼脂中加入2 g葡萄糖中的含碳量为标准,折算其他碳源的量,配制含不同碳源的水琼脂培养基,制作水琼脂玻片,以不加任何碳源为CK。
氮源处理:设置尿素、蛋白胨、甘氨酸、硝酸钾、半胱氨酸共5个处理。以100 mL 1.5%的水琼脂中加入2 g尿素中的含氮量为标准,折算其他氮源的量,配制含不同氮源的水琼脂培养基,制作水琼脂玻片,以不加任何氮源为CK。
将上述不同处理置于人工培养箱中培养,培养条件为光照4 000 lx、温度25 ℃、湿度≥75%。分别于3、6、12、24 h观察,统计分生孢子的萌发率,以分生孢子芽管长度超过孢子短径的1/2视为萌发,初始萌发率为0。
萌发率(%)=萌发个数/孢子总数×100。
通过HOBO小型自动气象站采集气象数据。
数据采用DPS V18.10进行方差分析,LSD法进行多重比较(
苜蓿叶片正反面均可发生白粉病,严重时可蔓延至叶柄和茎秆,植株下部叶片先开始发病,发病初期叶片表面出现多个近圆形白色的小霉斑,小霉斑逐渐扩大并向四周扩展蔓延,严重的白粉层覆盖整个叶片,随后发病部位的叶组织变为淡黄色,叶片表面白粉层逐渐加厚。显微镜下观察到白粉层上许多菌丝体交替重叠,白粉层上可见橘黄色的小颗粒,逐渐发育成熟变为黄褐色或黑褐色小颗粒,即为成熟的白粉病病原菌闭囊壳。
分生孢子卵圆形或椭圆形,表面光滑,大小为(29−46)×(12.5−21.0) μm,平均大小为40.0 (SD=3.06)× 19.0 μm (SD=2.63) (
分生孢子和分生孢子梗形态特征
Morphological characteristics of conidia and conidiophore
闭囊壳着生在菌丝体表层,多3−5个聚生,少散生,少数被菌丝层所缠绕或埋藏在菌丝层内,发育初期为黄色,后逐渐发育为黄褐色至黑褐色。成熟的闭囊壳为黑褐色,球形或近球形,直径范围为71−135 μm,平均直径为110 μm (SD=12.71) (
苜蓿白粉病病原菌有性态特征
The teleomorph characteristics of fungi of alfalfa powdery mildew
由
苜蓿白粉病发病率和病情指数与环境温湿度的关系
The relationship between the incidence rate and disease index of alfalfa powdery mildew and environmental temperature and humidity
由
白粉病发生的气象数据
Meteorological data of powdery mildew
日期 |
最低温度 |
最高温度 |
平均温度 |
最低湿度 |
最高湿度 |
平均湿度 |
发生情况 |
4-29 | 18.8 | 36.6 | 24.2 | 33.0 | 77.0 | 55.0 | 发生白粉病 |
5-18 | 12.8 | 33.0 | 19.3 | 19.7 | 60.0 | 43.7 | |
6-11 | 10.3 | 32.3 | 20.9 | 17.8 | 59.8 | 37.7 | 发现闭囊壳 |
6-21 | 18.9 | 26.8 | 26.1 | 68.5 | 96.4 | 86.1 | 大量闭囊壳 |
6-26 | 17.2 | 22.5 | 20.4 | 88.8 | 96.6 | 90.8 | 闭囊壳成熟 |
由
温度对苜蓿白粉病病原菌分生孢子萌发的影响(孢子萌发率,%)
Effect of temperature on conidia germination of alfalfa powdery mildew fungi (spore germination rate, %)
温度 Temperature (℃) | 萌发时间 Germination time (h) | |||
3 | 6 | 12 | 24 | |
注:同列不同大写字母表示差异极显著( | ||||
5 | 1.3D | 2.3F | 11.3E | 13.7I |
10 | 2.7D | 6.0F | 19.7E | 33.0H |
15 | 15.0C | 29.0E | 41.0D | 48.0G |
16 | 16.0BC | 33.3 DE | 47.7CD | 60.3F |
18 | 22.7B | 39.7CD | 54.3C | 63.3EF |
20 | 30.3A | 45.7BC | 64.7B | 73.7CD |
22 | 34.0A | 54.3A | 79.3A | 84.3A |
24 | 36.0A | 62.0AB | 80.3A | 87.0AB |
26 | 29.7A | 43.7C | 73.3AB | 80.3ABC |
28 | 22.3B | 41.7CD | 70.7AB | 77.7BCD |
30 | 16.3BC | 33.3DE | 64.7B | 70.3DE |
不同光照对分生孢子萌发的影响试验结果(
光照对苜蓿白粉病病原菌分生孢子萌发的影响(孢子萌发率,%)
The effect of light on the conidia germination of alfalfa powdery mildew fungi (spore germination rate, %)
光照 Illumination | 萌发时间 Germination time (h) | |||
3 | 6 | 12 | 24 | |
注:同列不同大写字母表示差异极显著( | ||||
全光照 Full illumination | 29.3A | 63.3A | 83.3A | 86.7A |
全黑暗 Total darkness | 26.0A | 52.3B | 73.0B | 80.0B |
半光照 Half illumination | 29.3A | 62.7AB | 82.7A | 88.0A |
不同pH对分生孢子萌发的影响试验结果(
pH对苜蓿白粉病病原菌分生孢子萌发的影响(孢子萌发率,%)
Effect of pH on conidia germination of alfalfa powdery mildew fungi (spore germination rate, %)
pH | 萌发时间 Germination time (h) | |||
3 | 6 | 12 | 24 | |
注:同列不同大写字母表示差异极显著( | ||||
3.0 | 13.7BC | 29.3CD | 31.3DE | 39.7CD |
4.0 | 13.3BC | 37.3C | 39.0C | 44.7C |
5.0 | 18.7AB | 37.0B | 54.7B | 62.0B |
6.0 | 23.0A | 40.3A | 65.7A | 76.7A |
7.0 | 16.7ABC | 32.7CD | 51.0B | 60.7B |
8.0 | 11.0BC | 16.3E | 32.7CD | 40.0CD |
9.0 | 8.0C | 15.7E | 25.7DEF | 32.3DE |
10.0 | 8.7C | 16.7E | 25.0EFG | 31.3DE |
11.0 | 10.7BC | 19.3E | 23.3FG | 27.7E |
12.0 | 8.7C | 14.3E | 18.0G | 26.0E |
6.5 (CK) | 24.3A | 50.7A | 71.3A | 81.3A |
不同碳源对分生孢子萌发的试验结果(
碳源对苜蓿白粉病病原菌分生孢子萌发的影响(孢子萌发率,%)
Effect of carbon source on the germination of the conidia of alfalfa powdery mildew fungi (spore germination rate, %)
碳源 Carbon source | 萌发时间 Germination time (h) | |||
3 | 6 | 12 | 24 | |
注:同列不同大写字母表示差异极显著( | ||||
葡萄糖 Glucose | 55.3AB | 73.0A | 87.7A | 94.0A |
蔗糖 Sucrose | 34.3CD | 63.7B | 83.3AB | 86.7BC |
淀粉 Starch | 27.7D | 54.0C | 72.7C | 85.7C |
麦芽糖 Malt dust | 37.0BCD | 64.7B | 80.7B | 87.3BC |
D-木糖 D-xylose | 48.7ABC | 64.7B | 84.3AB | 91.3AB |
甘露醇 Mannitol | 57.3A | 67.7AB | 80.7B | 89.0ABC |
对照 CK | 36.7BCD | 75.0A | 84.3AB | 88.3BC |
不同氮源对分生孢子萌发的试验结果(
氮源对苜蓿白粉病病原菌分生孢子萌发的影响(孢子萌发率,%)
Effect of nitrogen source on germination of conidia of alfalfa powdery mildew fungi (spore germination rate, %)
氮源 Nitrogen source | 萌发时间 Germination time (h) | |||
3 | 6 | 12 | 24 | |
注:同列不同大写字母表示差异极显著( | ||||
尿素 Carbamide | 18.3B | 43.7B | 65.0BC | 74.0BC |
蛋白胨 Peptone | 17.3B | 50.3AB | 62.3C | 77.7B |
甘氨酸 Glycine | 22.7AB | 51.7AB | 76.3A | 85.0A |
硝酸钾 Potassium nitrate | 25.7AB | 45.0AB | 58.7C | 67.7C |
半胱氨酸 Cysteine | 33.7A | 53.0AB | 66.7BC | 76.7BC |
对照 CK | 27.0AB | 53.3A | 72.3AB | 83.3A |
由
分生孢子的萌发
The germination of conidia
关于紫花苜蓿白粉病病原菌的报道国内外有所不同,国外报道主要为
孢子的萌发条件是决定病害发生与否的首要条件。本试验对宁夏紫花苜蓿白粉病病原菌无性孢子的生物学特性进行了研究,结果表明分生孢子萌发温度范围为5−30 ℃,最适萌发温度范围为22−26 ℃,高于26 ℃时,无论是孢子萌发还是病害的扩展均呈下降趋势,表明高温抑制其生长。光照促进分生孢子的萌发,宁夏地区光照资源丰富,光照一定程度上促进了病害的发生和流行。分生孢子对碳氮源的利用结果表明,白粉病病原菌分生孢子的萌发阶段虽然对外界营养的需求不明显,分生孢子自身可以满足芽管的生长,但是12 h后自身营养已不能满足其生长。
闭囊壳的形成条件一直是学者们关注的问题。子囊孢子是否成为苜蓿白粉病发生的初侵染源尚缺乏直接证据。研究表明,小麦白粉病原菌的子囊孢子不仅是田间白粉病发生的初侵染源[
蓼白粉菌(
本试验是在温室内完成的,试验结果反映了温室环境条件下苜蓿白粉病的发生规律,相比大田试验调查,极大地减少了田间干扰因素,更能说明病害发生情况与环境条件的内在联系。
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