化肥的大规模使用在提高作物产量、促进农业生产的同时，也造成了土壤板结、耕性不良、酸化、有机质含量下降等一系列生态环境问题。为缓解农业生态系统压力，开发高效环保的新型有机（类）肥料部分替代化肥施用，成为当前我国农业发展的重点研究内容。近些年来，涌现出大批生物有机（类）肥料的研发与应用，然而，现阶段大量生物有机肥含有单一菌种，不能满足作物生长的多种需要；此外，常规生物有机肥多以粉末状投放市场，存在结块、再次腐熟、肥效施用不均匀和农场主接受度低等问题。因此，开发能够有效发挥菌群复合功能和有机肥作用的新型含复合菌颗粒生物有机肥，成为推动有机类肥料产业快速发展的关键。芽孢杆菌由于易扩繁和产芽孢成为常规生物有机肥的主要功能菌，木霉菌由于生物量大、根表定殖能力强、次生代谢产物种类多和含量高，促生和生防效果明显，同样得到广泛关注。基于此背景，本文首先针对贵州木霉NJAU 4742的液体发酵产孢，优化了培养基配方并改良发酵工艺，随后采用联合造粒技术，研制出外包液体发酵木霉内含芽孢杆菌的颗粒生物有机肥，并通过盆栽试验评估了新型生物有机肥对辣椒植株的促生效果。具体研究结果如下：

优化了贵州木霉NJAU 4742液体发酵产孢培养基配方及发酵工艺。结果表明，与其他14种碳源相比，以葡萄糖为碳源制成的液体培养基发酵后木霉孢子数量最高，其次为甘油；选择葡萄糖和甘油作为最佳碳源，利用正交试验研制了木霉液体发酵产孢培养基配方（1 L培养基：200 g马铃薯浸提液，20 g葡萄糖，2 mL甘油），利用此培养基发酵后，木霉孢子浓度达6.75×107 cfu/mL；在此基础上，进一步探索了木霉发酵产孢的最佳培养条件为发酵液初始pH值为4，发酵温度为28°C，摇床转速为150 r /min，装液量为125 mL/250 mL，避光培养48 h后开光培养48 h，在此培养条件下木霉液体发酵孢子数量可达3.0×108 cfu/mL。

利用造粒喷涂技术，研制了外包固体发酵木霉菌孢子内含芽孢杆菌的颗粒复合菌生物有机肥。制作工艺如下：首先利用圆盘造粒机将有机肥、芽孢杆菌和适量粘合剂制成颗粒芽孢杆菌生物有机肥，随后，再将固体发酵的木霉真菌孢子喷涂颗粒表面，利用第一次添加的粘结剂形成外包真菌孢子膜的新型颗粒复合菌生物有机肥。货架期试验结果表明，颗粒肥料中木霉菌比粉状肥料存活能力更强，在储存60天后，新型颗粒复合菌生物有机肥中木霉菌含量大于2×107 cfu/g，芽孢杆菌数量大于1.2×108 cfu/g。随后以辣椒为供试作物，利用盆栽试验，研究了新型生物有机肥对辣椒植株株高、茎粗、叶绿素等农艺性状的影响。盆栽试验结果表明，在种植47天后，施用新型颗粒复合菌生物有机肥的辣椒植株在株高、茎粗、叶绿素、鲜重和干重方面均高于其他处理（含SQR9颗粒生物有机肥、含SQR9和木霉菌的粉状生物有机肥、含木霉菌颗粒生物有机肥和含SQR9和木霉菌的颗粒生物有机肥处理），表明了该新型颗粒复合菌生物有机肥对辣椒具有明显的促生效果。

利用液体发酵的木霉孢子研制了外包木霉菌孢子内含芽孢杆菌的颗粒复合菌生物有机肥，并利用盆栽试验评估了该颗粒复合菌生物有机肥对辣椒植株的促生效应。结果表明，相比于其他处理，施用该颗粒复合生物有机肥有效提高了辣椒植株的株高、茎粗和SPAD值，与有机肥（OF）处理相比，分别提高了43.80%、18.60%和10.24%；与液体发酵的木霉菌研制的颗粒生物有机肥处理相比，分别提高1.15%、20.97%、6.85%；与液体发酵的木霉菌研制的粉状生物有机肥处理相比，分别提高0.7%、19.60%、8.75%；与外包芽孢杆菌内含液体发酵的木霉菌研制的颗粒复合菌生物有机肥处理相比，分别提高9.60%、35.28%、9.70%；与同时含芽孢杆菌和液体发酵的木霉菌研制粉状复合菌生物有机肥处理相比，分别提高5.40%、15.03%和8.72%。

综上所述，利用液体发酵产孢培养基配方及发酵工艺可稳定获得贵州木霉菌孢子；用固体发酵获得木霉孢子或优化培养基及发酵工艺液体发酵获得的木霉孢子研制的外包木霉菌内含芽孢杆菌的新型颗粒复合菌生物有机肥，均对盆栽辣椒植株具有优异促生效果。

The large-scale use of chemical fertilizer to ensure high agricultural production has caused a series of ecological and environmental problems, such as soil hardening, poor tillability, soil acidification and organic matter content decreasing. In order to relieve the pressure of agro-ecosystem, development new organic fertilizers with high efficiency and environmental protection to partially replace the application of chemical fertilizers has become an important research content for agricultural development. In recent years, the traditional powdered bio-organic fertilizer which containing single microorganism could not meet the needs of crop growth and market, more and more researches have focusing on the development of new type biological organic fertilizers. Therefore, the development of a new type of granuliform bio-organic fertilizer which containing compound functional microbes has become the key to promote the rapid development of organic fertilizer industry.
In this research, we 1) optimized the medium formula and liquid fermentation technology for the liquid fermentation of Trichoderma guizhouense; 2) used granulation technology to develop a new type granulation bio-organic fertilizer which containing Trichoderma guizhouense and Bacillus SQR9; and 3) used pot experiment to evaluate the effects of new type granulation bio-organic fertilizer on the growth of Pepper plants. Our main results are listed as followed:
1) The optimal medium formula and liquid fermentation technology for the fermentation of Trichoderma guizhouense was determined.
We used different carbon sources to optimize the liquid fermentation medium formula of Trichoderma, and the results showed that the glucose significantly increase the number of spores of Trichoderma as compared with other carbon sources, followed by glycerol. Then we used the glucose and glycerol as the best carbon sources combination to optimize the medium formula by a orthogonal experiment, and the formula are as follows: 200 g potato extract, 20 g glucose, and 2 mL glycerol per L medium. By investigating the ability of the optimized liquid medium to promote the Trichoderma spores production, we demonstrated that the optimized liquid medium could significantly increase the number of Trichoderma spores, by the value of 6.75×10*7 cfu/mL, as compared the PDB medium. On this basis, we further determined that the optimal fermentation conditions are as fowllows: initial pH value was 4, ambient temperature was 28°C, rotation speed was 150 r/min, liquid volume was 125 mL/250 mL, dark culture for 48 h before light culture for 48 h. Under these conditions, the number of liquid fermentation spores of Trichoderma could reach 3.0×10*8 cfu/mL.
2) A new type granulation bio-organic fertilizer was developed.By using the granulation technology, we developed a new type granulation bio-organic fertilizer, which the particle surface containing Trichoderma (produced by solid fermentation) and the Particle inside containing Bacillus. And the production process is as follows: a) produce granulation bio-organic fertilizer containing Bacillus; b) spray Trichoderma spores fluid on the surface of granulation bio-organic fertilizer for the secondary granulation treatment. We conducted a shelf life accelerated test experiment to evaluate the survival population densities of Trichoderma and Bacillus in the new type granulation bio-organic fertilizer. The results showed that the population densities of Trichoderma and Bacillus were up to 2×10*7 cfu/g and 1.2×10*8 cfu/g after 60 days of storage, respectively. By investigating the effects of the new type granulation bio-organic fertilizer on the growth of the Pepper plants, and the pot experiment results showed that the new type granulation bio-organic fertilizer could significantly increase the height, stem diameter, chlorophyll, fresh weight and dry weight of the pepper plants as compared to other treatments.
3) The effects of new type granulation bio-organic fertilizer on the growth of Pepper plants was evaluated.
We used the Trichoderma spores which were produced by liquid or solid fermentation to produce the new type granulation bio-organic fertilizer, and then we used a pot experiment to evaluate the effects of the new type granulation bio-organic fertilizer on the growth of pepper plants. The results showed that the new type granulation bio-organic fertilizer significantly increase the plant height, stem diameter and SPAD value of pepper plants, there was an increase of 43.80%, 18.60% and 10.24%, respectively, as compared with organic fertilizer treatment; an increase of 1.15%, 20.97% and 6.85%, respectively, as compared with granulation bio-organic fertilizer treatment which containing Trichoderma spores; an increase of 0.7%, 19.60% and 8.75%, respectively, as compared with powder bio-organic fertilizer treatment which containing Trichoderma spores; an increase of 9.60%, 35.28% and 9.70%, respectively, as compared with granulation bio-organic fertilizer treatment, which the particle surface containing bacillus and the Particle inside containing Trichoderma; and an increase of 5.40%, 15.03% and 8.72%, respectively, as compared with powder bio-organic fertilizer treatment which containing Trichoderma spores and Bacillus.In conclusion, we show that the spores of Trichoderma guizhouense NJAU 4742 could be stability produced by using the optimized liquid fermentation medium and technology. The new type granulation bio-organic fertilizer, which the particle surface containing Trichoderma (produced by liquid or solid fermentation process) and the Particle inside containing Bacillus, could significantly promote the growth of Pepper plants.

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