设施番茄根围土壤的微生物特性研究
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摘要
设施菜田土壤往往存在化肥施用量过大、氮磷钾施用不平衡、作物品种单一、连作严重等问题,导致菜田土壤肥料利用率降低、生态环境质量下降、病虫害严重,蔬菜产量和品质受到严重影响。土壤微生物学特性是土壤质量的重要表征,可以在一定程度上反映土壤的肥力状况。本文以设施番茄栽培土壤为对象,研究了番茄不同生育时期、不同肥料处理、不同连作年限等条件下的土壤理化性状及微生物学特性,主要研究结果如下：
1.采用稀释平板法与PCR-DGGE技术相结合的方式,研究设施番茄不同生育时期及根域不同位置的土壤微生物特性,结果表明,番茄对土壤微生物的根际效应明显,可培养微生物数量与距番茄根系表面距离呈反比。在番茄整个生育期内,根域土壤可培养微生物数量表现为,细菌、真菌数量上升,放线菌数量下降；DGGE结果显示,番茄初花期和初果期的根际土壤细菌群落结构相似,多样性最高。
2.种植番茄显著提高了土壤微生物生物量和土壤酶活性。番茄根际土壤微生物生物量碳和氮分别在定植后60d和40d时显著降低,之后逐渐升高,定植80d达到最高值后下降；根际土壤转化酶、脲酶和中性磷酸酶活性在番茄整个生育期内呈现先升高后降低的变化趋势,过氧化氢酶活性呈上升—下降—上升的变化趋势。番茄生长末期,根际土壤微生物量和土壤酶活性均有所下降。土壤微生物生物量与转化酶、脲酶和中性磷酸酶活性显著相关,与过氧化氢酶无相关性。
3.长期施用有机肥或有机肥、化肥配合施用可以显著提高设施番茄栽培土壤有机质和养分含量,改善土壤理化性状,减缓土壤酸化进程；除全氮外,土壤养分含量随无机氮肥施用量的增加而下降,长期偏施无机氮肥加速了土壤pH的下降；磷肥在提高土壤有机质含量方面所发挥的作用优于钾肥。
4.长期施用有机肥显著提高了土壤酶活性和微生物生物量。有机肥对土壤转化酶和酸性磷酸酶活性的促进作用较为明显,有机肥和磷肥配施可以显著提高土壤转化酶和酸性磷酸酶活性,与转化酶和酸性磷酸酶相比,施肥对土壤脲酶活性的影响不大。单施化肥的土壤微生物生物量碳高于对照,而生物量氮低于对照,适量氮肥和磷肥显著提高土壤转化酶和微生物生物量碳,平衡施肥有利于土壤微生物生物量的提高。土壤酶活性和微生物生物量都随尿素施用量的增多而降低。
5.不同施肥处理的土壤微生物PCR-DGGE分析表明,施用有机肥可以起到提高土壤微生物总量,增加优势种群个数,丰富群落多样性,稳定群落结构的作用；过量施用无机氮肥会改变土壤细菌的优势种群种类；施用不同种类的化学肥料或肥料组合不会影响土壤微生物的群落结构。
6.通过对实际生产中不同连作年限设施番茄栽培土壤的研究发现,土壤养分含量随连作年限的增加而逐渐升高,连作7年、10年、15年和20年的土壤养分含量差异不大,可见,在实际生产中,土壤养分不是影响土壤肥力的主要因素；番茄连作一定年限内(15年),土壤转化酶、脲酶活性,微生物生物量和微生物量碳氮比都呈上升趋势变化,中性磷酸酶和过氧化氢酶活性呈下降趋势,各连作年限间土壤酶活性和微生物生物量的差异不大,番茄长期连作(20)土壤酶活性和微生物生物量都显著下降,。
7.通过PCR-DGGE技术研究番茄连作对根际土壤微生物多样性的影响发现,设施条件下种植番茄明显改变了土壤土著细菌的群落结构,但连作年限对土壤细菌多样性影响较小,细菌群落结构变化不大；土著真菌的群落结构稳定性优于土著细菌,但土壤真菌的优势种群在不同连作年限的土样中变化较大,连作显著降低了某些真菌的数量,同时显著增加了另一些真菌的数量,其中,连作20年番茄的土壤真菌新出现的优势种群最多,且与非优势种群的真菌数量差异较大,这种番茄连作后改变土壤真菌种群平衡的现象可能是导致番茄产生连作障碍的重要原因之一。通过电泳条带的回收、测序,可知所取土样中的细菌优势种群属于Clostridium butyricum、Uncultured bacterium、Bacillus和Arthrobacter,真菌优势种群属于Saccobolus dilutellus、Uncultured fungus、Lasiobolus ciliatus、Spooneromyces laeticolor和Lasiobolidium orbiculoides。
Obviously, the excessive use of chemical fertilizer, unbalanced application of nitrogen, phosphorus and potassium, single crop variety and seriously continuous cropping were common in protected vegetable cultivation, which leading to some serious problems, such as lower utilization of soil fertilizer, decline in the quality of the ecological environment, serious pests and diseases, decrease yield and quality of vegetables. Soil microbiological characteristics is an important characterization of soil quality, and to some extent can reflect the soil fertility condition. This paper studied the effects of different tomato growth period, different fertilizer treatment, different continuous cropping years on protected tomato field physical and chemical properties, and microbiological characteristics. The main results were as follows:
1. The microbiological characteristics of rhizosphere and surrounding soil was investigated at different growing period of tomato plants by combination of traditional plate-counting method and PCR-DGGE technology. Results showed that the rhizosphere effect of tomato plant for soil microorganisms was obvious, the number of culturable microorganisms was inversely proportional with the distance from the tomato root surface. During the whole developmental periods of tomato, rhizosphere and surrounding soil culturable microorganisms performanced that, bacteria and fungi number increased, actinomycete decline; DGGE profile indicated that the highest diversity of rhizosphere soil bacterial at the early flowering and blossoming stage, and the soil bacterial community structure was similar between them.
2. Planting tomato significantly increased soil microbial biomass and soil enzyme activity. Tomato rhizosphere soil microbial biomass carbon and nitrogen significantly decreased respectively in the60days and40days after planting, increased gradually, after planting80days, reached the highest value and then decreased; during the whole growing period, tomato rhizosphere soil invertase, urease and neutral phosphatase activity first increased and then decreased, hydrogen peroxidase activity changing trend was increased-decrease-rising. Tomato growth later stage, rhizosphere soil microbial biomass and soil enzyme activity decreased. Significant correlations between soil microbial biomass and invertase, urease and neutral phosphatase activity, but less correlation was found between microbial biomass and hydrogen peroxidase.
3. The long-term application of organic manure combined with or without chemical fertilizer can significantly improved the greenhouse tomato field soil organic matter and nutrient contents, improved soil physical and chemical properties, reduced soil acidification process. Except total nitrogen, the content of soil nutrient was more decreased with the increased inorganic nitrogen fertilizer application.Long-term partial application of inorganic nitrogen fertilizer has accelerated the decline in soil pH. Phosphate played an more important role in improving soil organic matter content than potassium.
4. The Long-term application of organic manure significantly increased the soil enzyme activities and microbial biomass. Organic amnure and organic amnure combined with phosphate fertilizer could distinctly improved the activities of soil invertase and acid phosphatase. Compared with invertase and acid phosphatase, little effect of fertilization on soil urease activity. The treatments which used chemical fertilizer only microbial biomass carbon was higher than that of the control, but microbial biomass nitrogen was lower. The amount of nitrogen fertilizer and phosphate fertilizer significantly increased soil invertase and soil microbial biomass carbon. Balanced fertilization was more useful for soil microbial biomass increase. Soil enzyme activities and microbial biomass were decreased with the increasing input of urea.
5. PCR-DGGE profiles of different fertilizer treatment showed that, application of organic fertilizer can improve the soil microbial quantity, increase the number of dominant species, increase the dominant population number, stable community structure; excessive application of nitrogen fertilizer could change the species of soil bacteria dominant populations; different kinds of chemical fertilizer or fertilizer combinations had no effects on soil microorganism community structure.
6. Through research on the actual production facilities of continuous cropping protected tomato field found, the soil nutrient content increased with continuous cropping years increasing, differences in soil nutrient content of continuous cropping for7years,10years,15years and20years was not visible, in the actual production, the main factors which affecting soil fertility was not soil nutrient. Tomato continuous cropping for a certain number of years (15years), soil invertase, urease activity, microbial biomass, microbial carbon and nitrogen ratio was upward trend, neutral phosphatase and catalase activity decreased, but various years of continuous cropping soil enzyme activity and microbial biomass difference was not big. Tomato long-term continuous cropping (20years), soil enzyme activities and microbial biomass decreased significantly.
7. The effects of protected tomato continuous cropping on rhizosphere soil microbial community diversity was studies with PCR-DGGE technology. Results showed that soil indigenous bacterial community structure was significantly influenced by planting tomato under protected field, but soil bacterial community structure and diversity were not sensitive to continuous cropping years. Compared with soil indigenous bacteria, indigenous fungal community structure was more steady, but the dominant fungi were more different in the fields with different continuous cropping years, continuous cropping significantly decreased the number of some fungi, meanwhile, the number of some other fungi was significantly increased, most dominant fungi populations were enriched in soil with tomato continuous cropping years of20, and which number was quite different with the non-dominant fungi populations. The change of soil fungi population balance that caused by tomato continuous cropping maybe one of the important reasons for the tomato continuous cropping obstacle. Recycling and sequence analysis of prominent electrophoretic bands showed that the dominant bacteria of soil sample belong to Clostridium butyricum、Uncultured bacterium、 Bacillus and Arthrobacter, while the dominant fungus belong to Saccobolus dilutellus、 Uncultured fungus、Lasiobolus ciliatus、Spooneromyces laeticolor and Lasiobolidium orbiculoides.
1.采用稀释平板法与PCR-DGGE技术相结合的方式,研究设施番茄不同生育时期及根域不同位置的土壤微生物特性,结果表明,番茄对土壤微生物的根际效应明显,可培养微生物数量与距番茄根系表面距离呈反比。在番茄整个生育期内,根域土壤可培养微生物数量表现为,细菌、真菌数量上升,放线菌数量下降；DGGE结果显示,番茄初花期和初果期的根际土壤细菌群落结构相似,多样性最高。
2.种植番茄显著提高了土壤微生物生物量和土壤酶活性。番茄根际土壤微生物生物量碳和氮分别在定植后60d和40d时显著降低,之后逐渐升高,定植80d达到最高值后下降；根际土壤转化酶、脲酶和中性磷酸酶活性在番茄整个生育期内呈现先升高后降低的变化趋势,过氧化氢酶活性呈上升—下降—上升的变化趋势。番茄生长末期,根际土壤微生物量和土壤酶活性均有所下降。土壤微生物生物量与转化酶、脲酶和中性磷酸酶活性显著相关,与过氧化氢酶无相关性。
3.长期施用有机肥或有机肥、化肥配合施用可以显著提高设施番茄栽培土壤有机质和养分含量,改善土壤理化性状,减缓土壤酸化进程；除全氮外,土壤养分含量随无机氮肥施用量的增加而下降,长期偏施无机氮肥加速了土壤pH的下降；磷肥在提高土壤有机质含量方面所发挥的作用优于钾肥。
4.长期施用有机肥显著提高了土壤酶活性和微生物生物量。有机肥对土壤转化酶和酸性磷酸酶活性的促进作用较为明显,有机肥和磷肥配施可以显著提高土壤转化酶和酸性磷酸酶活性,与转化酶和酸性磷酸酶相比,施肥对土壤脲酶活性的影响不大。单施化肥的土壤微生物生物量碳高于对照,而生物量氮低于对照,适量氮肥和磷肥显著提高土壤转化酶和微生物生物量碳,平衡施肥有利于土壤微生物生物量的提高。土壤酶活性和微生物生物量都随尿素施用量的增多而降低。
5.不同施肥处理的土壤微生物PCR-DGGE分析表明,施用有机肥可以起到提高土壤微生物总量,增加优势种群个数,丰富群落多样性,稳定群落结构的作用；过量施用无机氮肥会改变土壤细菌的优势种群种类；施用不同种类的化学肥料或肥料组合不会影响土壤微生物的群落结构。
6.通过对实际生产中不同连作年限设施番茄栽培土壤的研究发现,土壤养分含量随连作年限的增加而逐渐升高,连作7年、10年、15年和20年的土壤养分含量差异不大,可见,在实际生产中,土壤养分不是影响土壤肥力的主要因素；番茄连作一定年限内(15年),土壤转化酶、脲酶活性,微生物生物量和微生物量碳氮比都呈上升趋势变化,中性磷酸酶和过氧化氢酶活性呈下降趋势,各连作年限间土壤酶活性和微生物生物量的差异不大,番茄长期连作(20)土壤酶活性和微生物生物量都显著下降,。
7.通过PCR-DGGE技术研究番茄连作对根际土壤微生物多样性的影响发现,设施条件下种植番茄明显改变了土壤土著细菌的群落结构,但连作年限对土壤细菌多样性影响较小,细菌群落结构变化不大；土著真菌的群落结构稳定性优于土著细菌,但土壤真菌的优势种群在不同连作年限的土样中变化较大,连作显著降低了某些真菌的数量,同时显著增加了另一些真菌的数量,其中,连作20年番茄的土壤真菌新出现的优势种群最多,且与非优势种群的真菌数量差异较大,这种番茄连作后改变土壤真菌种群平衡的现象可能是导致番茄产生连作障碍的重要原因之一。通过电泳条带的回收、测序,可知所取土样中的细菌优势种群属于Clostridium butyricum、Uncultured bacterium、Bacillus和Arthrobacter,真菌优势种群属于Saccobolus dilutellus、Uncultured fungus、Lasiobolus ciliatus、Spooneromyces laeticolor和Lasiobolidium orbiculoides。
Obviously, the excessive use of chemical fertilizer, unbalanced application of nitrogen, phosphorus and potassium, single crop variety and seriously continuous cropping were common in protected vegetable cultivation, which leading to some serious problems, such as lower utilization of soil fertilizer, decline in the quality of the ecological environment, serious pests and diseases, decrease yield and quality of vegetables. Soil microbiological characteristics is an important characterization of soil quality, and to some extent can reflect the soil fertility condition. This paper studied the effects of different tomato growth period, different fertilizer treatment, different continuous cropping years on protected tomato field physical and chemical properties, and microbiological characteristics. The main results were as follows:
1. The microbiological characteristics of rhizosphere and surrounding soil was investigated at different growing period of tomato plants by combination of traditional plate-counting method and PCR-DGGE technology. Results showed that the rhizosphere effect of tomato plant for soil microorganisms was obvious, the number of culturable microorganisms was inversely proportional with the distance from the tomato root surface. During the whole developmental periods of tomato, rhizosphere and surrounding soil culturable microorganisms performanced that, bacteria and fungi number increased, actinomycete decline; DGGE profile indicated that the highest diversity of rhizosphere soil bacterial at the early flowering and blossoming stage, and the soil bacterial community structure was similar between them.
2. Planting tomato significantly increased soil microbial biomass and soil enzyme activity. Tomato rhizosphere soil microbial biomass carbon and nitrogen significantly decreased respectively in the60days and40days after planting, increased gradually, after planting80days, reached the highest value and then decreased; during the whole growing period, tomato rhizosphere soil invertase, urease and neutral phosphatase activity first increased and then decreased, hydrogen peroxidase activity changing trend was increased-decrease-rising. Tomato growth later stage, rhizosphere soil microbial biomass and soil enzyme activity decreased. Significant correlations between soil microbial biomass and invertase, urease and neutral phosphatase activity, but less correlation was found between microbial biomass and hydrogen peroxidase.
3. The long-term application of organic manure combined with or without chemical fertilizer can significantly improved the greenhouse tomato field soil organic matter and nutrient contents, improved soil physical and chemical properties, reduced soil acidification process. Except total nitrogen, the content of soil nutrient was more decreased with the increased inorganic nitrogen fertilizer application.Long-term partial application of inorganic nitrogen fertilizer has accelerated the decline in soil pH. Phosphate played an more important role in improving soil organic matter content than potassium.
4. The Long-term application of organic manure significantly increased the soil enzyme activities and microbial biomass. Organic amnure and organic amnure combined with phosphate fertilizer could distinctly improved the activities of soil invertase and acid phosphatase. Compared with invertase and acid phosphatase, little effect of fertilization on soil urease activity. The treatments which used chemical fertilizer only microbial biomass carbon was higher than that of the control, but microbial biomass nitrogen was lower. The amount of nitrogen fertilizer and phosphate fertilizer significantly increased soil invertase and soil microbial biomass carbon. Balanced fertilization was more useful for soil microbial biomass increase. Soil enzyme activities and microbial biomass were decreased with the increasing input of urea.
5. PCR-DGGE profiles of different fertilizer treatment showed that, application of organic fertilizer can improve the soil microbial quantity, increase the number of dominant species, increase the dominant population number, stable community structure; excessive application of nitrogen fertilizer could change the species of soil bacteria dominant populations; different kinds of chemical fertilizer or fertilizer combinations had no effects on soil microorganism community structure.
6. Through research on the actual production facilities of continuous cropping protected tomato field found, the soil nutrient content increased with continuous cropping years increasing, differences in soil nutrient content of continuous cropping for7years,10years,15years and20years was not visible, in the actual production, the main factors which affecting soil fertility was not soil nutrient. Tomato continuous cropping for a certain number of years (15years), soil invertase, urease activity, microbial biomass, microbial carbon and nitrogen ratio was upward trend, neutral phosphatase and catalase activity decreased, but various years of continuous cropping soil enzyme activity and microbial biomass difference was not big. Tomato long-term continuous cropping (20years), soil enzyme activities and microbial biomass decreased significantly.
7. The effects of protected tomato continuous cropping on rhizosphere soil microbial community diversity was studies with PCR-DGGE technology. Results showed that soil indigenous bacterial community structure was significantly influenced by planting tomato under protected field, but soil bacterial community structure and diversity were not sensitive to continuous cropping years. Compared with soil indigenous bacteria, indigenous fungal community structure was more steady, but the dominant fungi were more different in the fields with different continuous cropping years, continuous cropping significantly decreased the number of some fungi, meanwhile, the number of some other fungi was significantly increased, most dominant fungi populations were enriched in soil with tomato continuous cropping years of20, and which number was quite different with the non-dominant fungi populations. The change of soil fungi population balance that caused by tomato continuous cropping maybe one of the important reasons for the tomato continuous cropping obstacle. Recycling and sequence analysis of prominent electrophoretic bands showed that the dominant bacteria of soil sample belong to Clostridium butyricum、Uncultured bacterium、 Bacillus and Arthrobacter, while the dominant fungus belong to Saccobolus dilutellus、 Uncultured fungus、Lasiobolus ciliatus、Spooneromyces laeticolor and Lasiobolidium orbiculoides.
引文
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