摘要
烟草是一种重要的经济作物,烟草黑胫病是世界上各烟草种植区重要病害之一。在我国大部分烟草种植区均有发生,且在多个省市发生危害严重。为了迎合工业上对特色品质的需求,生产上所种植的烟草品种有许多抗病性程度不高。目前对黑胫病的控制仍然以化学药剂为主,从而导致病原菌抗药性增强、环境污染及烟叶重金属含量超标等后果,影响防治效果、生态安全和人类健康等问题的出现。为此,本文以生态防治为指导思想,从植物微生态系统角度,研究内生细菌对烟株根际生态、植株生理生化和病原菌等方面的综合作用,并以此来控制烟草黑胫病害。主要研究结果如下：
内生细菌对烟株根际生态的影响10个内生细菌菌株在3种方式处理后,烟株吸收利用土壤有机质的程度有一定差别,其中菌株Itb295在浸种+灌根处理后土壤有机质含量最高。菌株Itb225、Itb220和Itb57处理后土壤有机质含量与对照最接近；多数菌株在3种处理方式下土壤pH值与对照处理相比,只是略有变化。其中变化最大的是菌株Itb185在浸种处理后,pH值达到8.23。相对偏低的是菌株Itb162的浸种处理和Itb28的浸种+灌根处理；各菌株通过浸种+灌根处理后,均能使土壤中全钾的含量降低,且总体来说,各菌株处理后有利于降低土壤中全钾含量。而各菌株对烟苗利用速效钾的影响上表现出促进或抑制作用,Itb225和Itb28两个菌株的3种处理均使得速效钾含量明显高于对照处理；10个菌株处理后对土壤全磷含量的影响,除菌株Itb220的浸种处理和Itb80的喷雾灌根处理后全磷含量远高于对照处理外,其余各处理与对照相比,土壤全磷含量相差不大。而各菌株对土壤中有效磷的影响较大,变化范围在2.59-18.36 mg/kg,多数菌株在某种处理方式后可降低土壤中有效磷含量；
各菌株对土壤中过氧化氢酶、脲酶和蔗糖酶这3种与土壤肥力相关的酶活力影响也没有明显一致的趋势,不同菌株对酶活力影响各有不同。有的菌株可提高某种酶活性,有的也表现出抑制作用；
对土壤微生物类群影响方面,菌株Itb57和Itb12经过浸种、浸种+灌根和喷雾灌根这3种方式处理后,土壤中真菌的数量比其余8个菌株及对照处理要低一个数量级；菌株Itb57在浸种和浸种+灌根处理后,土壤中细菌数量最多,且比对照处理高一个数量级；10个菌株在3种处理方式下和对照处理一样,放线菌数量均在同一个数量级(对数值范围为5.1-5.8)。从趋势上可以看出,与对照相比,多数菌株处理后能提高土壤中放线菌数量；对于功能菌群的影响,多数菌株发酵液处理后有利于土壤中固氮菌的增长。所有菌株能明显有利于解磷细菌繁殖。多数菌株处理后土壤中解钾细菌数量与对照处理相比波动幅度不大。
内生细菌对烟株生长的影响10个内生细菌菌株发酵液对烟草幼苗在地上部鲜重、干重、最大叶长宽和真叶数等4个方面各有不同程度的影响,其中Itb57对烟苗的促生效果较好,其次是Itb185和Itb295。而菌株Itb162在浸种处理方式下对烟苗的地上部干重和真叶数两个方面有明显的不利影响。10个内生细菌菌株发酵液在浸种、浸种+灌根和喷雾灌根3种处理方式下,浸种+灌根处理对烟苗的促生效应较好且相对稳定,而浸种处理下部分菌株有抑制烟苗生长的作用。
重点研究内生细菌选择对10个菌株进行综合评价,菌株Itb57对促进土壤矿化及植株对养分的利用相对较优,对土壤3种酶活性无明显影响,可显著降低土壤中真菌数量,提高细菌数量,尤其能提高固氮、解磷和解钾3大功能细菌数量,且对烟苗的促生效应最好。故将内生细菌Itb57作为重点研究对象,以烟草黑胫病菌YBX1-4菌株作为测定拮抗活性的标准病原菌株。
菌株Itb57的抗菌作用及代谢产物内生细菌Itb57具有广谱的拮抗活性,对供试的烟草赤星病菌等9种植物病原真菌均有一定程度的的拮抗作用。其中对烟草赤星病菌、番茄早疫病菌的抑菌作用最好,抑菌带宽分别为9.83 mm和9.50 mm。其次是对花生黑斑病菌的抑制作用,达9.33 mm。并且对烟草上的炭疽病菌、灰霉病菌和白绢病菌也有较好的效果。对烟草黑胫病菌的拮抗研究表明,对峙培养后使得病菌菌丝扭曲、分支增多,有的菌丝上形成小瘤状物,多数菌丝变成短粗的结节。代谢产物试验表明菌株Itb57可以产生纤维素酶,蛋白酶,嗜铁素,不产生几丁质酶。
菌株Itb57的分类地位结合菌株菌落性状、形态特征和生理生化性状及16S rDNA序列比对分析,确定Itb57菌株为枯草芽孢杆菌Bacillus subtilis。获得菌株Itb57的16S rDNA片断序列长度为1397bp,在GenBank数据库中的注册登录号(Accession No)为GQ153538。
菌株Itb57对烟苗生理生化影响菌株Itb57发酵液原液和离心后的上清液可完全抑制烟草种子萌发,而一定浓度的菌悬液能促进种子萌发和幼芽生长,当菌悬液浓度为3x107 cfu/mL时处理最佳。菌株Itb57还能促进烟株根系生长发育,提高根系活力。菌悬液处理烟苗后能明显提高植株与抗性相关的苯丙氨酸解氨酶、过氧化物酶和多酚氧化酶的活性,但诱导抗性酶活提高的时间段及增强比例有一定差别。
菌株Itb57的内生定殖研究通过利福平标记菌株Itb57,获得抗Rif200μg/mL的突变株,突变菌株接种后从烟株体内回收菌株的培养性状和对黑胫病菌的拮抗能力与Itb57基本相同,证明Itb57为内生菌株。研究也发现,用其菌悬液处理植株根部,可在茎叶中分离获得,表明菌株还能在植株内从下到上转移,且菌落计数表明菌株Itb57在植株各组织中的含菌量以根部最多,为3.72×104 cfu/g FW,茎中次之,为2.14×104 cfu/g FW,叶片中最少,为0.15×l04 cfu/g FW。经无菌培养后的烟苗接种菌株Itb57,通过扫描电镜和透射电镜可观察到菌株Itb57不仅能在植株根表定殖且能进入烟草植株中在皮层细胞内定殖,进一步证明了菌株Itb57的内生性。
菌株Itb57的生物学特性及发酵条件培养基筛选试验表明,以花生粉为原料最有利于菌株Itb57生长繁殖和产生拮抗物质。菌株生长曲线表明,Ith57在0-12h生长较为缓慢,数量增加幅度较小,随后进入对数生长期,菌体数量快速增加,到48 h达到最大数量,随后菌株生长进入稳定期。菌株发酵条件单因素测定表明,菌株Itb57生长和抑制黑胫病菌的拮抗物质产生的最适条件是：发酵温度为28℃、培养液初始pH值为7.0、发酵时间为48h、摇瓶转速180r/min和25 mL/500 mL三角瓶装液量。正交试验优化后的最佳发酵条件组合是发酵温度为28℃、培养液初始pH值为7.0、时间72 h、摇瓶转速180 r/min和25 mL/500 mL三角瓶装液量。
菌株Itb57对黑胫病的防治效果Itb57发酵液对烟草黑胫病的温室防治效果为69.28%；2007和2008年的田间小区防治效果分别为61.25%和72.49%,与对照化学药剂58%甲霜·锰锌可湿性粉剂的防治效果无显著差异。小区防治试验后采取烟叶分析测定表明,菌株Itb57对烟株的主要化学成分影响和常规对照相比变化不大。菌液处理后在减小两糖差值、协调糖碱比、氮碱比方面均表现出稍优于常规对照处理。
Tobacco is an important cash crop. Tobacco black shank, caused by Phytophthora nicotianae, is an economically important disease of tobacco world-wide. In China, it occurs frequently and commonly, which causes a huge loss at several provinces. For adapting to the industrial demand in specific quality, most of tobacco varieties to Tobacco black shank is not much strong, therefore, the main way to control the disease still keeps taking chemical pesticides. Thus, it causes the drug-resistant development, environmental pollution, and the heavy metals contents in tobacco leaves getting over-standard etc., finally impacts on the disease control effect, the ecological safety and the mankind health. For this reason, based on the thought for ecological control, the effect of endophytic bacteria on rhizosphere ecosystem, physiology and biochemistry of host plant, and pathogens causing Tobacco black shank, was studied in this paper, from the plant micro-ecosystem. The main results of the study are as follows.
The impact of endophytic bacteria on tobacco rhizosphere ecosystem The absorption and utilization of soil organic matter by tobacco plants got certain difference after three kinds of treatments. The strain Itb295 showed a maximum content of soil organic matter after the treatment of seed-soaking and root-irrigation among the ten strains tested, while that of the strains Itb225, Itb220 and Itb57 was most closed to the control (CK). The soil pH value of majority of biological control strains differed slightly with the CK after three kinds of treatments, only that of the strain Itb185 got the highest level at 8.23 after seed-soaking. The pH values of strain Itb162 seed-soaking and strain Itb28 seed-soaking and root-irrigation were relatively lower in all the treatments. Furthermore, an interesting result was found that total potassium content in the soil decreased after all the tested strains treated by seed-soaking and root-irrigation. However, it was not shown all the tested strains positively impacted on the tobacco seedling taking advantage of effective potassium. Compared to CK, the effective potassium content in soil of strains Itb225 and Itb28 was much higher after three kinds of treatments. It was studied how the change of soil total phosphor contents was after ten strains treatments. Compared to CK, the soil total phosphor contents changed slightly, except that of treatments by Itb220 seed-soaking or Itb8O spraying irrigation far increased. The impact of tested strains on effective phosphor was great, varied from 2.59 to 18.36 mg/kg. The majority of strains could make the effective phosphor contents decreased in the soil after a certain treatment.
There was no obviously consistent trend towards affecting of the tested strains on the activity of three kinds of enzymes (Catalase, Urease and Sucrase) in the soil related to soil fertility. The difference in the study was that a part of experimental strains raised the activity of a certain enzyme, while others preformed repressing.
The effect of different treatments on soil micro-organisms showed as follows. After three kinds of treatments (seed-soaking, root-irrigation and spray irrigation) of two strains Itb57 and Itb12, in contrast with CK and the other eight strains, the number of soil fungi was 10 times lower. After two kinds of treatments (seed-soaking, root-irrigation) of strain Itb57, the number of soil bacteria was 10 times more than the CK treatment. The quantity of soil actinomyces of all strains tested was in the same quantity grade (the logarithm value ranged from 5.1 to 5.8) in three kinds of treatments. From the tendency, it was found that the majority of experimental strains had made the quantity of soil actinomyces increase. As one of functional groups of soil micro-organisms, the number of Azotobacter increased treated by ferment liquid of most strains tested, the number of Phosphate-solubilizing bacteria increased in the treatments with all the biological control strains tested, while that of Potassium-releasing bacteria in the treatments of most tested strains was much closed to the CK trial.
The impact of endophytic bacteria upon tobacco growth The effect of the ferment liquid often endophytic bacteria on fresh weight and dry weight aboveground, width and length of the biggest leaves, euphylla numbers of tobacco seedlings were studied in the paper. The results indicated that strain Itb57 could promote the seedling's growing well, and followed by the strains Itb185 and Itb295. But strain Itb162 negatively affected on the dry weight aboveground and euphylla numbers of tobacco seedlings with the treatment by seed-soaking. After three kinds of treatments (seed-soaking, seed-soaking+root-irrigation, and spray irrigation) of the ten strains, seed-soaking+ root-irrigation showed a good and stabilized effect on the growth promotion of tobacco seedlings, on the other hand, the seed-soaking treatment with part of tested strains restricted the seedlings' growth.
Integrated evaluation and selection of the optimal from experimental endophytic bacteria Integrated evaluation of the ten strains in the study, it was found that strain Itb57 was more excellent in accelerating the soil mineralization, promoting the absorption and utilization of nutrients for tobacco plants, and the seedlings growth, except no significant effect on three sorts of enzymes activities. Besides, strain Itb57 could reduce the amount of soil fungi, and make the numbers of soil bacteria increase, especially the three kinds of functional groups of soil micro-organisms, Azotobacter, Phosphate-solubilizing bacteria and Potassium-releasing bacteria. Therefore, strain Itb57 was taken as key objective strain in the research, and YBX1-4 was chosen as one standand strain of Phytophthora nicotianae for antagonistic measurement.
Antibiotic effect and metabolism products of strain Itb57 It was found the antagonistic spectrum of endophytic strain Itb57 was broad, showing an antagonistic effect on nine tested pathogenic fungi in a certain degree. The best was observed in Alternaria alternata, A. solani, the width of restrained zone was 9.83 mm and 9.50 mm, respectively. The better repressing effect was on Cercospora personata on peanut with a zone of 9.33 mm, besides on the pathogens Colletotrichum, Botrytis, Sclerotium. The antagonistic effect on P. nicotianae showed that the hypha of pathogen became distortion, more branches, even formed a small lump and short knot in the duel culture plate. Metabolizing products was detected in strain Itb57 generated Cellulase, Proteinase and Siderophore, but no Chitinase.
The taxonomy of strain Itb57 Based on properties, morphological, physiological and biochemical characteristics of colonies, and 16S rDNA (GenBank accession No.GQ153538) phylogenetic analysis, strain Itb57 was identified as Bacillus subtilis. The sequence length of 16S rDNA of Itb57 of fragments was 1397 bp.
The physiological and biochemical effects of strain Itb57 on tobacco seedlings The tobacco seed germination was entirely restrained after treatments with the original bacterial suspension and the above liquid post centrifugal practice of Itb57. But the bacterial suspension with certain concentration could promote the seed germination, and the growth of tobacco buds, especially when the bacterial suspension was at the concentration of 3×10(?) cfu/mL; the effect of growth-promotion was the best. Additionally, strain Itb57 was found to be able to stimulate the growth and development of seedling rootlets, and to lift the activity of roots. The activities of three sorts of enzymes related to tobacco resistance, such as Phenylalanine Ammonium Lyase (PAL), Peroxidase (POD) and Polyphenol Oxidase (PPO), were all significantly taken up after the treatments of tobacco seedlings with the bacterial suspension, only the detailed time segment and enhancement ratio for inducing the resistance and enzymes activities varied slightly.
Research on endogenesis and colonization of strain Itb57 Strain Itb57 was tagged with rifampicin, the drug-resistant strain was acquired at the level of anti-Rif 200μg/mL. The cultivation character in the medium and antagonistic activities on P. nicotianae of the re-isolated strain after inoculation was similar to the original strain of Itb57. This result adequately proved strain Itb57 was endophytic bacteria. After tobacco roots were inoculated with the bacterial suspension of tagged strain Itb57, it was isolated again in the stem and leaves, which suggested that the tagged strain could transfer upwards in tobacco plants. Moreover, the number of colony form unit in tobacco tissue was found mostly in the rootlets (3.72×104 cfu/g fresh weight), followed by the stem (2.14×104 cfu/g FW), and the leaves (0.15×104 cfu/g FW) to colonize the interior tissues of tobacco plants. The highest amount (3.72×104 cfu/g fresh weight) of the inoculated bacterium was detected in the rootlets and the lowest in the leaves of the tobacco plants. Under the condition of sterilization, after tobacco seedlings was inoculated with tagged strain Itb57, its colonization was found in the surface of rootlets, even in the cortex cells, which further provided strong evidence that strain Itb57 was endophytic bacteria.
The biology characteristics of strain Itb57 and fermentation conditions The experiments for screening the culture medium indicated that was beneficial to the growth, reproduction of strain Itb57 and its producing antagonistic substances in the conditions of peanut powder as nutrition source. According to the growth curve of strain Itb57, it was found that the strain grew slowly within 0-12 hours, subsequently the growth became fast into logarithm increasing stage, at 48 hour the top abundance obtained, and then kept into a stable period. Measurement of each single factor for fermentation of strain Itb57, the results showed that the optimal conditions for the growth and production of antagonistic substances of strain Itb57, were at 28℃of fermentation temperature, the initial pH7.0 value of culturing medium,48 hour of fermentation period, the rotation speed 180 rpm of triangle bottles, and the volume ratio of 25 mL/500 mL triangle bottles for supplying oxygen. The optimized conditions by trial consequently were at 28℃of fermentation temperature, the initial pH7.0 value of culturing medium, fermentation period 72 h, the rotation speed 180 rpm of triangle bottles, and the volume ratio of 25 mL/500 mL triangle bottles.
The control efficacy of strain Itb57 to tobacco black shank Green-house pot-grown tobacco plant assays showed that the control efficacy to tobacco black shank by strain Itb57 was 69.28% when the bacterium was applied by root-irrigation in advance of P. nicotianae inoculation. Further, field trials in 2007 and 2008 demonstrated that the application of strain Itb57 controlled the disease by with the efficiency of 61.25% and 72.49%, respectively. Statistical analysis indicated that the disease control efficacy was not significantly different from that obtained by treatment with the chemical (58% metalaxyl mancozeb wettable powder) (P>0.05). Measurement of the chemical components in the tested tobacco plants after treatments with strain Itb57 in plot trial, the major components changed little, compared with the control (CK). The positive effect on the reduction of sucrose-glucose variance, nitrogen-alkali ratio, and balancing glucose-alkali ratio was better than the CK after treatment with bacterial suspension of strain Itb57.
引文
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