棉花黄萎菌微菌核际微生物对微菌核的作用
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摘要
通过在河北省棉田定点连续调查，发现自然病土中的棉花黄萎菌微菌核数量在6月中旬和9月下旬出现两个高峰，同时发现，重病田中的微菌核数显著高于轻病田。本试验从棉花黄萎菌微菌核际共分离到微生物7024株，其中拮抗微生物1840株，拮抗微生物比率为26％；细菌5069株，其中拮抗细菌1593株，拮抗菌率为31％；真菌1955株，其中拮抗真菌247株，拮抗真菌率为13％。拮抗微生物中的拮抗细菌占87％，拮抗真菌占13％。棉田中微菌核数量的变化和微菌核际拮抗细菌率呈负相关性，而和微菌核际拮抗真菌率呈正相关，说明田间微菌核数量变化受拮抗细菌的影响较大。微菌核际的拮抗菌率对土壤微菌核的数量变化有一定的影响，当拮抗菌率较高时，土壤中的微菌核数较低，当拮抗菌率下降时，土壤微菌核的数量又开始上升，拮抗菌对微菌核的影响主要表现在5月—10月，在12月土壤温度很低时，拮抗菌率非常低，而土壤中的微菌核数也非常低。
应用双层培养基法和双周微菌核测定法分别测定了部分微菌核际拮抗微生物对棉花黄萎病菌菌丝生长和对微菌核萌发和生长的影响。结果表明，许多微菌核际微生物对棉花黄萎菌菌丝生长均有不同程度的抑制作用。从抑制内圈微菌核的萌发生长来看，有的拮抗菌株可以完全抑制微菌核的萌发，有的可以推迟微菌核的萌发或抑制菌落的扩展。从外圈微菌核萌发来看，外圈微菌核生长的菌落直径大于内圈微菌核的生长直径，说明拮抗菌靠分泌抗生素来抑制微菌核的萌发和生长，而且拮抗菌分泌的抗生素有扩散的能力。
利用扫描电镜观察，比较2株微菌核际拮抗细菌(424—31—6、454—11—13)，和1株根际拮抗细菌(NCD-2)在微菌核上的定殖能力，结果表明扫描电镜下经过拮抗菌处理的微菌核表面粗糙，有裂纹，而正常微菌核的瘤状突起和凹陷部位的表面光滑，没有裂纹。三株细菌在微菌核表面的定殖能力不同，其中来自微菌核际的拮抗细菌菌株424—31—6的定殖能力最强，在扫描电镜下可以看到有很多菌体在微菌核表面附着，而根际拮抗细菌NCD-2的定殖能力最差。
通过优化试验，获得了一种更为有效检测土壤中棉花黄萎菌微菌核的培养基—改良MSM培养基。与其它两种常用微菌核测定培养基相比，新改良的MSM培养基对检测土壤棉花黄萎菌微菌核有4个优点：(1)检测效果好，检测数量分别接近培养基A的10倍、培养基B的4倍。(2)对杂菌的抑制效果好，分离到的微菌核菌落直径大、形态清晰，便于观察检测。(3)对土壤人工接种的微菌核的回收率高，可达到68.4％～93.9％。(4)培养基成分容易得到。
Dynamics of microsclerotia of V.dahliae was investigated periodically at the same sites of cotton naturally infested fields. Results showed that population of microsclerotia appeared two peaks at the middle of June and the late of September. At the same time, it also was found that the population of microsclerotia from heavily infested field was significantly higher than that of microsclerotia from lightly infested field. The total population of microorganisms isolated from microsclerotiniasphere of V.dahliae were 7024 strains, among them 1840 strains were antagonists, the rate of antagonists was 26%; 5069 strains were bacteria, among them 1593 strains were antagonists, the rate of antagonists was 31%; 1955 strains were fungi, among them 247 strains were antagonists, the rate of antagonists was 13%. Among all the antagonists, 87% strains were bacteria, and only 13% strains were fungi. Population dynamics of MS were negatively correlated with dynamics of antagonistic bacterial rate and positively correlated w
ith dynamics of antagonistic fungal rate from microsclerotiniasphere. It showed that the population dynamics of microsclerotia in cotton soil was mainly affected by antagonistic bacteria. The rate of antagonists from microsclerotiniasphere had certain effect on the population of microsclerotia, when the rate of antagonists was high, the population of microsclerotia was correspondingly low, when the rate of antagonists declined, the quantity of microsclerotia went up. The effect of antagonists on microsclerotia was mainly found during June to September.
The results of dual culture test showed that many strains of antagonists isolated from microsclerotiniasphere inhibited the mycelia growth of V. dahliae to different degrees. The result of two-circle-method test showed that microsclerotia of inner circle was inhibitited completely by some antagonists, while germination and growth of other microsclerotia were delayed and inhibited. The results convinced that antagonists isolated from microsclerotiniasphere had stronger ability for inhabiting germination and growth of microsclerotia. The colony diameters of microsclerotia of
out circle were larger than that of inner circle. It indicated that antagonistic microorganisms inhibited germination and growth of microsclerotia through secreting antibiotics.
Observed through with scan electronic microscope, the colonization on microsclerotia by 2 strains of antagonistic bacteria (424-31-6, 454-11-13) isolated from microsclerotiniasphere and 1 strain of antagonistic bacteria (NCD-2) isolated from rhizosphere had been investigated. The results showed that the colonization abilities of three strains of antagonistic bacteria on the surface of microsclerotia were different, the colonization ability of strain 424-31-6 isolated from microsclerotiniasphere was the best, and many bacteria could be found attached on the surface of microsclerotia, while colonization ability of strain NCD-2 isolated from rhizosphere was the poorest.
An effective selective medium on detecting microsclerotia of V. dahliae from cotton soil was modified. The new modified selective medium-MSM had 4 advantages on detecting microsclerotia compared to the other two common selective media: (1) Modified MSM was obviously more effective than the other two media on detecting V. dahliae and gave nearly nine times higher counts than that of Medium A and three times higher counts than that of Medium B.(2) Modified MSM was also more effective on inhibiting growth of other fungi and bacteria , the microsclerotia colony size was bigger and clearer, and it was easy to be observed and to be detected.(3) The retrieving rate of microsclerotia by artificially inoculated in soil was very high, it reached up to 68.4%~93.9%.(4)the components of modified MSM was easy to get.
应用双层培养基法和双周微菌核测定法分别测定了部分微菌核际拮抗微生物对棉花黄萎病菌菌丝生长和对微菌核萌发和生长的影响。结果表明，许多微菌核际微生物对棉花黄萎菌菌丝生长均有不同程度的抑制作用。从抑制内圈微菌核的萌发生长来看，有的拮抗菌株可以完全抑制微菌核的萌发，有的可以推迟微菌核的萌发或抑制菌落的扩展。从外圈微菌核萌发来看，外圈微菌核生长的菌落直径大于内圈微菌核的生长直径，说明拮抗菌靠分泌抗生素来抑制微菌核的萌发和生长，而且拮抗菌分泌的抗生素有扩散的能力。
利用扫描电镜观察，比较2株微菌核际拮抗细菌(424—31—6、454—11—13)，和1株根际拮抗细菌(NCD-2)在微菌核上的定殖能力，结果表明扫描电镜下经过拮抗菌处理的微菌核表面粗糙，有裂纹，而正常微菌核的瘤状突起和凹陷部位的表面光滑，没有裂纹。三株细菌在微菌核表面的定殖能力不同，其中来自微菌核际的拮抗细菌菌株424—31—6的定殖能力最强，在扫描电镜下可以看到有很多菌体在微菌核表面附着，而根际拮抗细菌NCD-2的定殖能力最差。
通过优化试验，获得了一种更为有效检测土壤中棉花黄萎菌微菌核的培养基—改良MSM培养基。与其它两种常用微菌核测定培养基相比，新改良的MSM培养基对检测土壤棉花黄萎菌微菌核有4个优点：(1)检测效果好，检测数量分别接近培养基A的10倍、培养基B的4倍。(2)对杂菌的抑制效果好，分离到的微菌核菌落直径大、形态清晰，便于观察检测。(3)对土壤人工接种的微菌核的回收率高，可达到68.4％～93.9％。(4)培养基成分容易得到。
Dynamics of microsclerotia of V.dahliae was investigated periodically at the same sites of cotton naturally infested fields. Results showed that population of microsclerotia appeared two peaks at the middle of June and the late of September. At the same time, it also was found that the population of microsclerotia from heavily infested field was significantly higher than that of microsclerotia from lightly infested field. The total population of microorganisms isolated from microsclerotiniasphere of V.dahliae were 7024 strains, among them 1840 strains were antagonists, the rate of antagonists was 26%; 5069 strains were bacteria, among them 1593 strains were antagonists, the rate of antagonists was 31%; 1955 strains were fungi, among them 247 strains were antagonists, the rate of antagonists was 13%. Among all the antagonists, 87% strains were bacteria, and only 13% strains were fungi. Population dynamics of MS were negatively correlated with dynamics of antagonistic bacterial rate and positively correlated w
ith dynamics of antagonistic fungal rate from microsclerotiniasphere. It showed that the population dynamics of microsclerotia in cotton soil was mainly affected by antagonistic bacteria. The rate of antagonists from microsclerotiniasphere had certain effect on the population of microsclerotia, when the rate of antagonists was high, the population of microsclerotia was correspondingly low, when the rate of antagonists declined, the quantity of microsclerotia went up. The effect of antagonists on microsclerotia was mainly found during June to September.
The results of dual culture test showed that many strains of antagonists isolated from microsclerotiniasphere inhibited the mycelia growth of V. dahliae to different degrees. The result of two-circle-method test showed that microsclerotia of inner circle was inhibitited completely by some antagonists, while germination and growth of other microsclerotia were delayed and inhibited. The results convinced that antagonists isolated from microsclerotiniasphere had stronger ability for inhabiting germination and growth of microsclerotia. The colony diameters of microsclerotia of
out circle were larger than that of inner circle. It indicated that antagonistic microorganisms inhibited germination and growth of microsclerotia through secreting antibiotics.
Observed through with scan electronic microscope, the colonization on microsclerotia by 2 strains of antagonistic bacteria (424-31-6, 454-11-13) isolated from microsclerotiniasphere and 1 strain of antagonistic bacteria (NCD-2) isolated from rhizosphere had been investigated. The results showed that the colonization abilities of three strains of antagonistic bacteria on the surface of microsclerotia were different, the colonization ability of strain 424-31-6 isolated from microsclerotiniasphere was the best, and many bacteria could be found attached on the surface of microsclerotia, while colonization ability of strain NCD-2 isolated from rhizosphere was the poorest.
An effective selective medium on detecting microsclerotia of V. dahliae from cotton soil was modified. The new modified selective medium-MSM had 4 advantages on detecting microsclerotia compared to the other two common selective media: (1) Modified MSM was obviously more effective than the other two media on detecting V. dahliae and gave nearly nine times higher counts than that of Medium A and three times higher counts than that of Medium B.(2) Modified MSM was also more effective on inhibiting growth of other fungi and bacteria , the microsclerotia colony size was bigger and clearer, and it was easy to be observed and to be detected.(3) The retrieving rate of microsclerotia by artificially inoculated in soil was very high, it reached up to 68.4%~93.9%.(4)the components of modified MSM was easy to get.
引文
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[2] Sherbakoff C D.Wilt caused by Verticillium albo-atrum[J]. Plant Disease Report Supplement, 1928, 61: 283～284.
[3] Wakins G M. Compendium of Cotton Disease[J]. American PHytopathology Society, 1981, 42～44.
[4] Huisman O C. Seasonal colonization of roots of field-grown cotton by Verticillium Dahliae and V.tricorpus[J]. PHytopathology, 1988, 78(6): 708～716.
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