摘要
甜菜是世界制糖工业的主要原料之一,内生菌具有增强甜菜光合作用能力,提高其抗逆性,延缓植株衰老,防治甜菜主要病害,可促进甜菜增产,改进品质等特点。因此,利用甜菜内生菌筛选促生、防病、增产菌株的潜力巨大。内生菌对于提高甜菜栽培水平,增强甜菜代谢的调控能力,以及对农业的可持续发展,都具有十分重要的意义。本论文从甜菜内生菌中筛选出对甜菜具有促进生长、防治病害、增加产量的菌株,并利用表形与16S rDNA分子特征进行鉴定,同时从激素水平、拮抗作用、营养元素吸收、光合作用、氮糖代谢方面,研究了甜菜内生菌的增产机理。其结果如下：
采用选择性培养基,从来自新疆南北甜菜样品中分离到406株内生菌。对分离到的380株细菌和放线菌进行了菌落形态和培养特征观察、生理生化特性测定和16S rDNA序列分析；对26株真菌进行了菌落形态、培养特征观察和ITS序列分析。结果显示甜菜内生菌共分为12个类群,细菌7个属,放线菌2个属,真菌3个属。本研究所选择的分离方法获得的内生菌,细菌占绝对优势。
通过种子发芽试验、麦芽鞘切断伸长测定,从甜菜内生菌筛选到3株甜菜促生菌株PG-1 (Bacillus flexus)、PG-2 (Pseudomonas plecoglossicida)、PG-3 (Chryseobacterium indologenes)。盆栽试验表明三株内生菌对甜菜有很好的促生作用。对3株促生菌从激素水平、溶磷能力、营养元素吸收、定殖等探索了促生机理,结果表明三株促生菌定殖于甜菜,提高其激素水平与营养元素吸收。三株促生菌不同处理促进甜菜产量的增长,其中菌株PG-3作用较好。PG-3处理后,与对照相比,甜菜体内四种植物激素(IAA、GA、ZR和ABA)含量分别增加了1.60、23.05、0.54和1.03倍；营养元素N、P、K、Mg和蛋白质含量分别提高了0.18、0.51、0.08、0.33和0.60倍。内生菌定殖过程是-个由进入——繁殖—稳定组成的内生菌与宿主和谐发展的过程。
通过甜菜病原菌分离鉴定,初步查明甜菜根腐病、褐斑病、立枯病、蛇眼病病原菌分别为尖孢镰孢(Forsur oxysporum)、立枯丝核菌(Rhizoctonia solani)、甜菜生尾孢(Cercospora beticola)、甜菜茎点霉(Phoma betae)。通过平板对峙试验,筛选出甜菜主要病害拮抗内生菌45株,其中菌株AT-1、AT-2和AT-3对甜菜主要病原菌抑菌圈半径较大。定殖试验表明,3株拮抗内生菌能稳定定殖于甜菜根部。通过菌落形态、培养特征、生化特性和16S rDNA序列分析,菌株AT-1鉴定为多粘芽孢杆菌(Paenibacillus polymyxa (EU563233)),AT-2为弯曲芽孢杆菌(Bacillus flexus (EU594560)),AT-3为寡养单孢菌(Stenotrophomonas sp. (EU594562))。田间防病试验表明；3株拮抗内生菌对甜菜主要病害均有很好的防治效果。
研究了内生菌PG-1、PG-2、PG-3对甜菜叶片光合特性及产量形成的影响。结果表明：接种内生菌能显著促进甜菜光合作用,其中PG-3处理促进光合作用能力较强,其叶片净光合速率(Pn)、气孔限制值(Ls)、蒸腾速率(Tr)的平均值分别提高了33.33%、21.43%和12.31%；叶片胞间CO2浓度(Ci)平均值降低了7.21%；最大光化学效率(Fv/Fm)、实际光化学效率(φPSⅡ)降低,而光化学猝灭系数(qp)、非光化学猝灭系数(qN)升高。各处理Pn随着光照强度(PAR)的增加呈先升后降。生物学产量和含糖率分别提高25.63%和17.46%。表明内生菌不仅影响甜菜光合参数,而且对于甜菜产量和品质的提高具有明显的促进作用。
采用内生菌PG-1、PG-2、PG-3浸种、喷叶及灌根处理方法,调查其对甜菜栽培品种KWS2409的主要农艺性状及对甜菜氮、糖代谢关键酶即硝酸还原酶(NR)、谷氨酰胺合成酶(GS)、蔗糖合酶(SS)和蔗糖磷酸合酶(SPS)活性的影响。结果表明,内生菌对甜菜的含糖量有明显的提高作用,其中以PG-3灌根处理效果最好,其叶鲜重、叶绿素含量、单根重、含糖率和产糖量的平均值分别提高了16.7%、55.3%、11.8%、22.1%和27.9%。在整个生育期,内生菌显著提高了氮糖代谢酶活性,其中NR和GS活力分别呈双峰值和单峰值变化,而SS和GS活力呈单峰值变化,后期根部SS合成活力明显高于分解方向活力,生育前期SPS活力高于后期。叶丛形成期达到最高值,说明NR、GS、SS和SPS活性的增强是甜菜含糖量升高的主要生理原因。
Sugar beet is a main raw material for the sugar industry in the world. Endophyte can be in favor of the enhancement of leaf photosynthetic capacity, increasing stress resistance, delaying sugar beet senescence, prevention and control of main diseases of sugar beet, increasing yield and improving quality of the beet plants. Isolated endophytic bacteria has great potential for growth promotion, disease prevention and yield increasing of sugar beet, and is very significant in enhancing the management of beet cultivation, regulation of beet metablism and sustainable development of agriculture in Xinjiang.
Endophytes, having characteristics of growth promotion, disease prevention and yield increasing, were isolated from sugar beet and identify by morphological and 16SrDNA sequence analysis. Endophytic bacteria yield-increasing mechanism involved in phytohormone levels, antagonism, nutrient absorption, photosynthesis, nitrogen and glucose metabolisms were investigated. The results were as follows:
Using different separation medium and isolation methods,406 strains of endophytes were isolated from sugar beet materials in Xinjiang by surface disinfection using ethanol and mercuric chloride.380 bacteria and actinomycetes were identified by colony morphology, cultural characteristics, physiological and biochemical characteristics,16S ribosomal DNA sequence analysis; 26 fungus were identified by colony morphology, cultural characteristics, ITS sequence analysis. The results showed that endophytes in sugar beet were divided into 12 groups, as 7 genus bacteria,2 genus actinomycetes,3 genus fungus, separately. Bacteria were predominate in endophytes isolated from in beet.
Three endophtic bacteria strain PG-1 (Bacillus flexus)、PG-2 (Pseudomonas plecoglossicida)、PG-3 (Chryseobacterium indologenes) isloated from beet plant has remarkable growth promoting effect. It was test by germination, wheat coleoptile growth, pot experiments experiments. Improving growth metabolisms of three strains were researched from phytohormone levels, phosphorus solubiliziog ability, nutrient absorption and colonization. The results showed that three improving growth endophtic bacteria could colonize within sugar beet, and enhance phytohormone levels and nutrient absorption. Three strains could increase yield and quality of beet, and best one was PG-3 among 3 strains. The quantity of four kinds of phytohormone (IAA、GA、ZR、ABA) in sugar beet were bosted 1.60、23.05、0.54、1.03 times than control after the strain PG-3 inculated sugar beet. The content of N,P,K,Mg elements and protein of in PG-3 increased by 0.18、0.51、0.08、0.33 and 0.60 times compared to CK, respectively. Endophyte colonization was composed of entering, reproduce and stabilization, which was a harmonious development process between endophyte and host
Based on the characteristics of pathogens identified, the beet root rot, seedling blight, brown patch and snake-eye disease are caused by Fusarium oxysporum, Rhizoctonia solan, Cercospora beticola and Phoma betae separately.45 endophytes strains were screened against the pathogens of beet by plate confrontation test. Three endophytic bacteria strains, AT-1, AT-2 and AT-3, showed relatively strong antagonistic against the pathogens. Three antagonistic strains could colonized into root of sugar beet by colonizing test. Strain AT-1 was identified as Paenibacillus polymyxa, while strains AT-2 and AT-3 were as Bacillus flexus and Stenotrophomonas sp. by their morphological, physiological and biochemical characteristics. Three antagonistic strains showed significantly control effect against the pathogens in field trail.
The effects of endophytic PG-1, PG-2, PG-3 on photosynthetic parameters and yield of sugar beet were investigated. The results showed that three strains, PG-1, PG-2, PG-3 could obviously promote the photosynthesis of sugar beet, in which the net photosynthetic rate (Pn), stomatal limitation (Ls), transpiration rate (Tr) of sugar beet increased significantly (P<0.05) by 33.33%、21.43% and 12.31%, respectively, while stomatal intercellular CO2 concentration (Ci) decreased significantly (P<0.05) by 7.21%, maximum photochemical capacity (Fv/Fm) and quantum yield of PSⅡ(ΦPSⅡ) increased, while photochemical quenching (qP) and non-photochemical quenching (qN) decreased. It was shown that with the increasing of photo flux density, the net photosynthetic rate (Pn) increased at first and then decreased. The strain PG-3 could also increase the yield and the quality of beet, which biomass and sugar content in beet increased significantly (P<0.05) by 25.63% and 17.46%, respectively. It was concluded that the strain PG-3 not only affected photosynthetic parameters, but also promoted the yield and the quality of beet.
We investigated the agronomic traits and determined the activity of key enzymes in sugar and nitrogen metabolisms in beet leaf and root with treatments of by using three strains, PG-1, PG-2, PG-3 solution seed soaking, leaf spraying and root watering. The results showed that the strains obviously promoted growth of beet seedlings, the effect with the better in root watering treatment than in others. The leaf fresh weight, content of chlorophyll, fresh weight per root, sugar content and sugar yield of beet increased significantly (P<0.05) by 16.7%, 55.3%,11.8%,22.1% and 27.9%, respectively; the three strains enhanced evidently the activities of key enzymes in sugar and nitrogen metabolisms of beet during whole growth period. The changes of activities of nitrate reductase (NR) and glutamina synthetase (GS) presented the changes of M-shape and parabola shape, respectively. The changes of activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) in beet displayed the single peak changes. The synthesis activity was much higher than the decomposition activity for SS in roots of beet at late growth stage. The activity of SPS was higher at early growth stage than at late growth stage, and the activity of SPS was the highest during phyllome formation period. It was concluded that the strains not only enhanced activities of key enzymes in sugar and nitrogen metabolisms of beet, but also promoted the growth, yield and sugar accumulation of beet.
引文
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