淡紫拟青霉对南方根结线虫侵染机制的研究
摘要
根结线虫是世界上分布最广、危害最大的植物病原线虫之一。对于该病害的防治主要有化学防治、生物防治、物理防治等,其中最可行的是生物防治。淡紫拟青霉是防治根结线虫的最有潜力的真菌之一,但关于淡紫拟青霉对根结线虫的侵染机制尚不明确。本文利用GFP构建的淡紫拟青霉菌株观察侵染机制,结合扫描电镜观察以及测定二者混合后混合液蛋白酶含量的变化,进而找出淡紫拟青霉与根结线虫的相互作用。同时对根结线虫发生地微生物因子和非生物因子与病情的关系进行了调查；金属离子对淡紫拟青霉生长的影响进行了研究,以期为根结线虫病的生物防治提供理论依据。主要结果如下：
1、本文对淡紫拟青霉4个菌株进行GFP转化载体的构建及荧光表达,结果表明：4种菌株的GFP转化菌株在荧光显微镜下均能看到荧光；将4种菌株的GFP转化菌株连续培养4代后,459菌株和P菌株几乎看不到荧光,20-7和618’菌株荧光很强；这两个菌株的转化效率达到90%。
2、本文研究了淡紫拟青霉618’菌液与根结线虫卵悬浮液混合后混合液中蛋白酶含量的变化,结果表明：相同量的淡紫拟青霉菌液与不同量的卵液混合初始蛋白酶含量分别为：3mL菌液+1mL卵悬浮液蛋白酶含量<3mL菌液+2mL卵悬浮液蛋白酶含量<3mL菌液+3mL卵悬浮液蛋白酶含量；3mL菌液+1mL卵悬浮液的混合液在第1d蛋白酶的含量的最高,到第八天时含量达到最低；在第1d到第6d这段时间混合液中蛋白酶的变化不明显,此后两天变化的幅度较大。处理2和处理3与处理的变化一致。
3、淡紫拟青霉618’菌液与根结线虫卵混合后在解剖镜下观察卵的孵化状况是：在第二天出现大量幼虫,随着时间的变化,幼虫逐渐增多,第4d达到高峰；部分未成熟的卵未能孵化,其内部形态发生较大变化,到第4天后卵囊出现二分化,随着时间的推移,分化后的泡囊里出现较多性状为椭圆形的大小相似的泡囊,一周后的为孵化的卵大部分泡囊化。到14d时,在混合液中未见泡囊化的卵。
4、通过在荧光显微镜和扫描电镜下观察淡紫拟青霉20-7菌株对根结线虫的侵染途径结果表明：淡紫拟青霉20-7菌株菌丝在4～6d,布满根结线虫的卵壳表面,并产孢,这些串状孢子有的是在卵壳内,有的在卵壳外面的菌株上；菌株孢子还可穿过卵壳在卵壳表面定殖。
5、本文通过对根结线虫发病区主要微生物因子的研究,揭示了在根结线虫发病根际土壤中优势种细菌主要是假单胞菌属Pseudomonas sp、鞘氨醇杆菌属Uncultured Sphingobacterium sp.、芽孢杆菌属Bacillus sp.。其中以假译胞菌属数量最多,占70%左右,其次为芽孢杆菌约占17%。在根结线虫发病区根际土壤中真菌主要有青霉属Penicillium sp.占80%以上,其次分别为毛壳菌属Chaetomium sp.和拟青霉属Paecilomyces sp.。在发病区根际土壤中主要放线菌为链霉菌属Streptomycessp.,其次为小单孢菌属Micromonospora sp.和马杜拉放线菌属Madura actinomycetes sp. o
6、本文测定了3种不同发病程度的根际土壤的养分及pH值,结果是：3种不同发病程度的根际土壤中全氮的含量差别不明显,中等发病的土壤中有机质的含量最高,发病严重的根际土壤有效磷和有效钾的含量都比较高,分别为309.92mg/kg和277.29mg/kg(均值),所采土样的pH值都偏酸性,但发病严重的土壤的pH值最小,为4.24,健康的土壤的pH值接近中性。
7、本文研究了3种金属离子对淡紫拟青霉菌株618’及其GFP转化菌株生长的影响,结果表明：Cu2+离子对淡紫拟青霉618’菌株和GFP转化菌株的生长影响是：在浓度(mol/L)为1×10-6、1×10-5、1×10-43个浓度下,该两种菌株的生长速率差别较大,3种浓度下都呈上升趋势,但是前两个浓度对淡紫拟青霉的生长的抑制率在50%左右：对照不加Cu2+离子的培养基,淡紫拟青霉菌株的生长率较高,且原始菌株的生长率稍高于转化菌株。适合淡紫拟青霉618’生长的Mg2+离子浓度是0.005mol/L,镁离子对淡紫拟青霉618’菌株及其转化菌株生长的影响差别不大,二者在浓度为0.4-0.6mol/L时,菌株均不能生长；在0.1-0.4mol/L生长率差别不大,在0.1mol/L以下时,两种菌株生长率都超过50%,618’转化菌株的生长率高于原始菌株。Al3+离子浓度1＝10-6、10-5mol/L两个浓度淡紫拟青霉生长较快；当浓度高于1×10-4mol/L时菌株生长缓慢,浓度为5×10-2mol/L菌株几乎不生长。Al3+离子对淡紫拟青霉618’原始菌株和GFP转化菌株生长率的影响是：浓度在1×10-6～1×10-4mol/L时,618’原始菌株和转化菌株均呈下降趋势,在高浓度下,二者的生长速率差别不大。
The root-knot nematode is one of the most widely distrbuted and heaviest hazard plant pathgenic nematodes. The main methods to control the disease are chemical, biological and physical control with the most feasible method being biological control, but the mechanizm of Paecillomyces lilacinus on root-knot nematode still uncertainty. In this paper, GFP constructed strain of Paecillomyces lilacinus were used to observe the infection mechanizm, combined with SEM observations, the blending protease content of root-knot nematode and Paecillomyces lilacinus was tesed, then hoping to find out the interaction between them. At the same time the relation between disease severity and microbial factors or non-microbial factors were investegated.The effect of metal ions on Paecillomyces lilacinus was studied, to offer theoretical basis for the disease of root-knot nematode. The main results as follow.
In this paper, four strains of Paecillomyces lilacinus were transferred to GFP strains, there are459strain, P strain,20-7strain and618'strain. All of them could see fluorescence light under fluorescence microscope, the fluorescence light of459and P GFP transferred strains were weak, the fluorescence light other GFP transferred strains were strong. Four transferred strains were inoculated continuously for four times, the fluorescence light of459and P GFP transferred strains a mostly dispersed, but the fluorescence light of20-7and618'GFP transferred strains still strong. The conversion efficiency of20-7and618'GFP transferred strains were high, there are to90%.
The same volume of Paecillomyces lilacinus combined with different volume root-knot nematode eggs, the initial protease content different little, inoculated for six days the content of protease showed down trend, inoculated for eight days, the content of protease down rapidly. The protease content of3mL Paecillomyces lilacinus mixture and lmL root-knot nematode eggs mixture less than the protease content of3mL Paecillomyces lilacinus mixture and2mL root-knot nematode eggs mixture less than the protease content of3mL Paecillomyces lilacinus mixture and3mL root-knot nematode eggs mixture. Initially The protease content of3mL paecillomyces lilacinus mixture and1mL root-knot nematode eggs mixture highest for the first day, changed by time, the protease content reached to lowest for the eighth day, the first day to the sixth day, the protease content of the mixture changed no obviously, but the last two days changed obviously. The protease content of3mL Paecillomyces lilacinus mixture and2mL root-knot nematode eggs mixture changed slowly from the first day to the sixth day, from the sixth day to the eighth day down obviously, to the eighth day the protease content reached to lowest. The protease content two mixtures above changed similarly. The protease content of the three mixtures all down by time, due to every treatment add to different eggs the protease content of the three mixtures were different initially. The more eggs mixture the higher protease content.
Under dissecting microscope, the eggs hatch out for the second day, a large number of larvae appeared, the number of larvae increased gradually with time changed, the number of the larvae reach to climax for the fourth day. The eggs which unhatched the shape inside changed a lot, the eggs changed to second division four the fourth day, and inside the division eggs a lot of vesicles appeared, which had the similar size. After a week, the eggs which unhatched appeared vesicles, after fourteenth day the eggs which vesiculated could not see in the mixture.
The main bacteria of the rhizosphere soil in the root-knot nematode disease area were Pseudomonas sp.. Uncultured Sphingobacterium sp., Bacillus sp., Acinetobacter genomo sp. and Flavobacterium sp.. The number of Pseudomonas sp. was most, reached to70%, the number of Bacillus sp. reached to17%, the number of Flavobacterium sp. was10%. The number of Uncultured Sphingobacterium sp. and Acinetobacter genomo sp. was less.The main fungi of the rhizosphere soil in the root-knot nematode were Chaetomium sp., Paecilomyces sp., Penicillium sp., Fungal endophyte sp. and Cladosporium sp.. The number of Penicillium sp. was most, the second is Chaetomium sp., the third was Paecilomyces sp.. The main actinomycetes of the rhizosphere soil in the root-knot nematode disease area were Streptomyces sp., Micromonospora sp. and Madura actinomycetes sp..
The total content of three different disease rate of rhizosphere soil had a little difference, the content of organic matter in the middle disease soil was higher, the content of available P and available K in the heaviest disease of rhizosphere soil were higher, there were309.92mg/kg and277.29mg/kg. The PH value of soil adopted present was acid, the PH value of the heaviest disease was smallest (4.24), the PH value of the no disease soil was nearly to neutral.
In this paper three metallic ion had effect on618'strain and transferred strain of paecillomyces lilacinus. The result indicated that the effect of Cu2+ion on618'significantly, when under the concentration of1×10-6、1×10-5、1×10-4, the strain grow rapidly, the strain diameter was different. When above the concentration of1×10-3mol/L, the strain stopped growing. The affection on618'strain and transferred strain of Cu2+was that:the three concentration of1×10-6、1×10-5、1×10-4increased the growth of618'strain and transferred strain, under the first concentration and the second concentration the two strains grow rapidly, but the growth rate of orginal strain was higher than transferred strain's, the affection of concentration of Mg2+ion on618'strain and transferred strain was a little difference, the best concentration of Mg2+ion for the growth of618'strain was0.005mol/L, when the concentration low to0.1mol/L, the strain grow well, above the concentration, the strain grow slowly, while the concentration up to0.6mol/L, inhibitory rate reached to100%, under the concentration of0.1mol/L, inhibitory rate reached to50%more or less. The affection of the ion on618'strain and transferred strain had a little difference, during the concentration of0.4-0.6mol/L, both the strain could not grow, the concentration between0.1-0.4mol/L, the growth almost the same, under the0.1mol/L, both the strain were grow well, the growth rate showed up trend, above50%, but the growth rate of transferred strain higher than the original strain. The affection of all concentration of Al3+ion on strain growth changed a little, the concentration of1×10-6mol/L and1×10-5mol/L were benefit for growing, the strain diameter were bigger, by the concentration increasing, the strain grow slowly, the strain diameter were small, above the concentration of1×10-4mol/L the strain grow slowly, the strain stopped growing when the concentration reached to5×10-2mol/L. The concentration of Al3+ion effected on618' strain and transferred strain was1×10-6～1×10-4mol/L,618' original strain and transferred strain both showed down trend, under high concentration, the growth rate of the two strain made a little distinguish.
1、本文对淡紫拟青霉4个菌株进行GFP转化载体的构建及荧光表达,结果表明：4种菌株的GFP转化菌株在荧光显微镜下均能看到荧光；将4种菌株的GFP转化菌株连续培养4代后,459菌株和P菌株几乎看不到荧光,20-7和618’菌株荧光很强；这两个菌株的转化效率达到90%。
2、本文研究了淡紫拟青霉618’菌液与根结线虫卵悬浮液混合后混合液中蛋白酶含量的变化,结果表明：相同量的淡紫拟青霉菌液与不同量的卵液混合初始蛋白酶含量分别为：3mL菌液+1mL卵悬浮液蛋白酶含量<3mL菌液+2mL卵悬浮液蛋白酶含量<3mL菌液+3mL卵悬浮液蛋白酶含量；3mL菌液+1mL卵悬浮液的混合液在第1d蛋白酶的含量的最高,到第八天时含量达到最低；在第1d到第6d这段时间混合液中蛋白酶的变化不明显,此后两天变化的幅度较大。处理2和处理3与处理的变化一致。
3、淡紫拟青霉618’菌液与根结线虫卵混合后在解剖镜下观察卵的孵化状况是：在第二天出现大量幼虫,随着时间的变化,幼虫逐渐增多,第4d达到高峰；部分未成熟的卵未能孵化,其内部形态发生较大变化,到第4天后卵囊出现二分化,随着时间的推移,分化后的泡囊里出现较多性状为椭圆形的大小相似的泡囊,一周后的为孵化的卵大部分泡囊化。到14d时,在混合液中未见泡囊化的卵。
4、通过在荧光显微镜和扫描电镜下观察淡紫拟青霉20-7菌株对根结线虫的侵染途径结果表明：淡紫拟青霉20-7菌株菌丝在4～6d,布满根结线虫的卵壳表面,并产孢,这些串状孢子有的是在卵壳内,有的在卵壳外面的菌株上；菌株孢子还可穿过卵壳在卵壳表面定殖。
5、本文通过对根结线虫发病区主要微生物因子的研究,揭示了在根结线虫发病根际土壤中优势种细菌主要是假单胞菌属Pseudomonas sp、鞘氨醇杆菌属Uncultured Sphingobacterium sp.、芽孢杆菌属Bacillus sp.。其中以假译胞菌属数量最多,占70%左右,其次为芽孢杆菌约占17%。在根结线虫发病区根际土壤中真菌主要有青霉属Penicillium sp.占80%以上,其次分别为毛壳菌属Chaetomium sp.和拟青霉属Paecilomyces sp.。在发病区根际土壤中主要放线菌为链霉菌属Streptomycessp.,其次为小单孢菌属Micromonospora sp.和马杜拉放线菌属Madura actinomycetes sp. o
6、本文测定了3种不同发病程度的根际土壤的养分及pH值,结果是：3种不同发病程度的根际土壤中全氮的含量差别不明显,中等发病的土壤中有机质的含量最高,发病严重的根际土壤有效磷和有效钾的含量都比较高,分别为309.92mg/kg和277.29mg/kg(均值),所采土样的pH值都偏酸性,但发病严重的土壤的pH值最小,为4.24,健康的土壤的pH值接近中性。
7、本文研究了3种金属离子对淡紫拟青霉菌株618’及其GFP转化菌株生长的影响,结果表明：Cu2+离子对淡紫拟青霉618’菌株和GFP转化菌株的生长影响是：在浓度(mol/L)为1×10-6、1×10-5、1×10-43个浓度下,该两种菌株的生长速率差别较大,3种浓度下都呈上升趋势,但是前两个浓度对淡紫拟青霉的生长的抑制率在50%左右：对照不加Cu2+离子的培养基,淡紫拟青霉菌株的生长率较高,且原始菌株的生长率稍高于转化菌株。适合淡紫拟青霉618’生长的Mg2+离子浓度是0.005mol/L,镁离子对淡紫拟青霉618’菌株及其转化菌株生长的影响差别不大,二者在浓度为0.4-0.6mol/L时,菌株均不能生长；在0.1-0.4mol/L生长率差别不大,在0.1mol/L以下时,两种菌株生长率都超过50%,618’转化菌株的生长率高于原始菌株。Al3+离子浓度1＝10-6、10-5mol/L两个浓度淡紫拟青霉生长较快；当浓度高于1×10-4mol/L时菌株生长缓慢,浓度为5×10-2mol/L菌株几乎不生长。Al3+离子对淡紫拟青霉618’原始菌株和GFP转化菌株生长率的影响是：浓度在1×10-6～1×10-4mol/L时,618’原始菌株和转化菌株均呈下降趋势,在高浓度下,二者的生长速率差别不大。
The root-knot nematode is one of the most widely distrbuted and heaviest hazard plant pathgenic nematodes. The main methods to control the disease are chemical, biological and physical control with the most feasible method being biological control, but the mechanizm of Paecillomyces lilacinus on root-knot nematode still uncertainty. In this paper, GFP constructed strain of Paecillomyces lilacinus were used to observe the infection mechanizm, combined with SEM observations, the blending protease content of root-knot nematode and Paecillomyces lilacinus was tesed, then hoping to find out the interaction between them. At the same time the relation between disease severity and microbial factors or non-microbial factors were investegated.The effect of metal ions on Paecillomyces lilacinus was studied, to offer theoretical basis for the disease of root-knot nematode. The main results as follow.
In this paper, four strains of Paecillomyces lilacinus were transferred to GFP strains, there are459strain, P strain,20-7strain and618'strain. All of them could see fluorescence light under fluorescence microscope, the fluorescence light of459and P GFP transferred strains were weak, the fluorescence light other GFP transferred strains were strong. Four transferred strains were inoculated continuously for four times, the fluorescence light of459and P GFP transferred strains a mostly dispersed, but the fluorescence light of20-7and618'GFP transferred strains still strong. The conversion efficiency of20-7and618'GFP transferred strains were high, there are to90%.
The same volume of Paecillomyces lilacinus combined with different volume root-knot nematode eggs, the initial protease content different little, inoculated for six days the content of protease showed down trend, inoculated for eight days, the content of protease down rapidly. The protease content of3mL Paecillomyces lilacinus mixture and lmL root-knot nematode eggs mixture less than the protease content of3mL Paecillomyces lilacinus mixture and2mL root-knot nematode eggs mixture less than the protease content of3mL Paecillomyces lilacinus mixture and3mL root-knot nematode eggs mixture. Initially The protease content of3mL paecillomyces lilacinus mixture and1mL root-knot nematode eggs mixture highest for the first day, changed by time, the protease content reached to lowest for the eighth day, the first day to the sixth day, the protease content of the mixture changed no obviously, but the last two days changed obviously. The protease content of3mL Paecillomyces lilacinus mixture and2mL root-knot nematode eggs mixture changed slowly from the first day to the sixth day, from the sixth day to the eighth day down obviously, to the eighth day the protease content reached to lowest. The protease content two mixtures above changed similarly. The protease content of the three mixtures all down by time, due to every treatment add to different eggs the protease content of the three mixtures were different initially. The more eggs mixture the higher protease content.
Under dissecting microscope, the eggs hatch out for the second day, a large number of larvae appeared, the number of larvae increased gradually with time changed, the number of the larvae reach to climax for the fourth day. The eggs which unhatched the shape inside changed a lot, the eggs changed to second division four the fourth day, and inside the division eggs a lot of vesicles appeared, which had the similar size. After a week, the eggs which unhatched appeared vesicles, after fourteenth day the eggs which vesiculated could not see in the mixture.
The main bacteria of the rhizosphere soil in the root-knot nematode disease area were Pseudomonas sp.. Uncultured Sphingobacterium sp., Bacillus sp., Acinetobacter genomo sp. and Flavobacterium sp.. The number of Pseudomonas sp. was most, reached to70%, the number of Bacillus sp. reached to17%, the number of Flavobacterium sp. was10%. The number of Uncultured Sphingobacterium sp. and Acinetobacter genomo sp. was less.The main fungi of the rhizosphere soil in the root-knot nematode were Chaetomium sp., Paecilomyces sp., Penicillium sp., Fungal endophyte sp. and Cladosporium sp.. The number of Penicillium sp. was most, the second is Chaetomium sp., the third was Paecilomyces sp.. The main actinomycetes of the rhizosphere soil in the root-knot nematode disease area were Streptomyces sp., Micromonospora sp. and Madura actinomycetes sp..
The total content of three different disease rate of rhizosphere soil had a little difference, the content of organic matter in the middle disease soil was higher, the content of available P and available K in the heaviest disease of rhizosphere soil were higher, there were309.92mg/kg and277.29mg/kg. The PH value of soil adopted present was acid, the PH value of the heaviest disease was smallest (4.24), the PH value of the no disease soil was nearly to neutral.
In this paper three metallic ion had effect on618'strain and transferred strain of paecillomyces lilacinus. The result indicated that the effect of Cu2+ion on618'significantly, when under the concentration of1×10-6、1×10-5、1×10-4, the strain grow rapidly, the strain diameter was different. When above the concentration of1×10-3mol/L, the strain stopped growing. The affection on618'strain and transferred strain of Cu2+was that:the three concentration of1×10-6、1×10-5、1×10-4increased the growth of618'strain and transferred strain, under the first concentration and the second concentration the two strains grow rapidly, but the growth rate of orginal strain was higher than transferred strain's, the affection of concentration of Mg2+ion on618'strain and transferred strain was a little difference, the best concentration of Mg2+ion for the growth of618'strain was0.005mol/L, when the concentration low to0.1mol/L, the strain grow well, above the concentration, the strain grow slowly, while the concentration up to0.6mol/L, inhibitory rate reached to100%, under the concentration of0.1mol/L, inhibitory rate reached to50%more or less. The affection of the ion on618'strain and transferred strain had a little difference, during the concentration of0.4-0.6mol/L, both the strain could not grow, the concentration between0.1-0.4mol/L, the growth almost the same, under the0.1mol/L, both the strain were grow well, the growth rate showed up trend, above50%, but the growth rate of transferred strain higher than the original strain. The affection of all concentration of Al3+ion on strain growth changed a little, the concentration of1×10-6mol/L and1×10-5mol/L were benefit for growing, the strain diameter were bigger, by the concentration increasing, the strain grow slowly, the strain diameter were small, above the concentration of1×10-4mol/L the strain grow slowly, the strain stopped growing when the concentration reached to5×10-2mol/L. The concentration of Al3+ion effected on618' strain and transferred strain was1×10-6～1×10-4mol/L,618' original strain and transferred strain both showed down trend, under high concentration, the growth rate of the two strain made a little distinguish.
引文
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