Combining application of ammonium persulfate with different forms of Trichoderma fertilizer alleviates apple replant disease
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摘要:目的
研究肥料形态及土壤条件对木霉菌肥改善苹果土壤连作障碍的影响，以期为缓解苹果连作障碍提供有效的理论依据和技术支持。
方法试验以苹果砧木—平邑甜茶 (Malus hupehensis Rehd.) 幼苗为试材，设置4个处理：连作土 (CK)、过硫酸铵处理 (T1)、过硫酸铵加颗粒状木霉菌肥 (T2)、过硫酸铵加粉末状木霉菌肥 (T3)。通过土壤微生物高通量测序、实时荧光定量PCR等技术研究了各处理对土壤微生物群落结构和土壤有害真菌数量的影响，并对各处理的平邑甜茶幼苗生理特性 (植株生物量、根系呼吸速率、根系保护酶活性) 进行了测定。
结果颗粒状与粉末状木霉菌肥不同程度地促进了平邑甜茶幼苗的生长，改善了连作土壤微生物环境，提高了根系保护酶及土壤酶活性，颗粒状菌肥的促进效果好于粉末状菌肥。与对照相比，T1处理显著提高了连作平邑甜茶幼苗的生长，T2和T3处理促进平邑甜茶幼苗生长的效果又显著高于T1处理。T1处理的土壤蔗糖酶、磷酸酶、过氧化氢酶、脲酶活性显著低于对照，而T2、T3处理显著高于对照，T2处理的蔗糖酶、磷酸酶、过氧化氢酶、脲酶的活性分别比对照提高45.41%、92.85%、56.32%、186.67%，T3处理分别比对照提高32.96%、83.89%、43.68%、165.56%。平邑甜茶幼苗的根系活力及根系氧化酶活性4个处理间均差异显著，由高到低的顺序为T2 > T3 > T1 > CK，T2处理的根系活力及根系氧化酶活性分别是对照的1.41和2.80倍。实时荧光定量PCR分析结果表明，T1、T2和T3处理都能优化土壤微生物环境，增加土壤细菌数量，降低土壤有害真菌腐皮镰孢菌 (Fusarium solani) 数量，T2与T3处理土壤细菌分别是对照的2.18、2.55倍，放线菌数量分别是对照的1.71、1.53倍。土壤高通量测序检测数据显示，相较于连作对照，T2处理木霉属相对丰度明显增加58.8%，链格孢属相对丰度显著下降92.87%，T3处理的镰孢属相对丰度与连作对照相比降低了64.18%。
结论在过硫酸铵处理的连作土壤上施用木霉菌肥，可进一步提高土壤中细菌数量，降低有害真菌数量，优化土壤微生物环境，提高平邑甜茶幼苗的根系活力和植株生长。过硫酸铵联用颗粒状木霉菌肥的效果优于联用粉末状木霉菌肥。
Abstract:ObjectivesWe studied the effects of fertilizer forms and soil conditions on apple replant soil disease by using Trichoderma fertilizer to provide effective theoretical basis and technical support for alleviating apple replant disease.
MethodsThis experiment, using apple rootstock (Malus hupehensis Rehd.) seedlings as the test material, involved four treatments: replant soil (CK), ammonium persulfate (T1), ammonium persulfate plus granular Trichoderma fertilizer (T2), and ammonium persulfate plus powdery Trichoderma fertilizer (T3). Through high-throughput sequencing of soil microorganisms, quantitative real-time PCR and other technologies, the effects of each treatment on soil microbial community structure and number of soil harmful fungi were studied. Physiological characteristics of Malus hupehensis Rehd. seedlings (plant biomass, root respiration rate, and root protective enzyme activity) were determined.
ResultsGranular and powdery Trichoderma fertilizers promoted the growth of Malus hupehensis Rehd. seedlings in different degrees, improved soil microbial environment, and increased the activities of root protection enzymes and soil enzymes. The promotion effect of granular fertilizer was better than that of powdery fertilizer. Compared with control, T1 treatment significantly improved the growth of M. hupehensis Rehd. seedlings, while T2 and T3 treatments showed significantly higher promoting effects than T1 did. Compared with CK, T1 significantly decreased the activities of sucrase, phosphatase, catalase and urease, while T2 increased those by 45.41%, 92.85%, 56.32% and 186.67%, T3 increased those by 32.96%, 83.89%, 43.68% and 165.56%, respectively. The root activity and root oxidase activity of M. hupehensis Rehd. seedlings were in order of T2 > T3 > T1 > CK, those in T2 treatment were 1.41 and 2.80 times respectively higher than those in CK. Real-time fluorescence quantitative results showed that both ammonium persulfate and Trichoderma fertilizer application could optimize soil microbial environment, increase the number of soil bacteria, and reduce the number of soil harmful fungus (Fusarium solani). Compared with CK, the number of soil bacteria was 2.18 and 2.55 times of hat in CK under T2 and T3 treatment, and the number of soil actinomycetes was 1.71 and 1.53 times under T2 and T3, respectively. Soil high-throughput sequencing data showed that T2 significantly increased the relative abundance of Trichoderma by 58.8%, decreased the relative abundance of Alternaria by 92.87%, while T3 decreased the relative abundance of Fusarium by 64.18%.
ConclusionsAmmonium persulfate plus Trichoderma fertilizer could significantly reduce the number of harmful soil fungi, increase the number of soil bacteria, optimize the soil microbial environment, and promote the root activities and growth of M. hupehensis Rehd. seedlings. The effect of ammonium persulfate plus with granular Trichoderma fertilizer is better than that of ammonium persulfate plus powder Trichoderma fertilizer.
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Keywords:
- replant disease /
- Malus hupehensis Rehd /
- (NH4)2S2O8 /
- Trichoderma fertilizer /
- fertilizer form /
- soil microorganism /
- soil enzyme
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图 1 不同处理对根系呼吸速率及根系保护酶活性的影响
[注 （Note）：CK—连作果园土壤 Replanted orchard soil；T1—过硫酸铵处理 Applying (NH4)2S2O8 in soil；T2—过硫酸铵加颗粒状木霉菌肥 Applying (NH4)2S2O8 and granular Trichoderma fertilizer；T3—过硫酸铵加粉末状木霉菌肥 Applying (NH4)2S2O8 plus powdered Trichoderma fertilizer；柱上不同字母表示处理间差异显著 (P < 0.05) Different letters above the bars indicate significant difference among treatments (P < 0.05).]
Figure 1. Protective enzyme activity and respiration rate of root under different treatments
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图 2 不同处理条件下属水平土壤真菌群落的相对丰度
[注（Note）：CK—连作果园土壤 Replanted orchard soil；T1—过硫酸铵处理Applying (NH4)2S2O8 in soil；T2—过硫酸铵加颗粒状木霉菌肥 Applying (NH4)2S2O8 and granular Trichoderma fertilizer；T3—过硫酸铵加粉末状木霉菌肥 Applying (NH4)2S2O8 plus powdered Trichoderma fertilizer.]
Figure 2. Relative abundance of soil fungi communities under different treatments
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图 3 不同处理土壤真菌群落主坐标分析 (PCoA) 和非度量多维尺度分析 (NMDS)
[注（Note）：CK—连作果园土壤 Replanted orchard soil；T1—过硫酸铵处理 Applying (NH4)2S2O8 in soil；T2—过硫酸铵加颗粒状木霉菌肥 Applying (NH4)2S2O8 and granular Trichoderma fertilizer；T3—过硫酸铵加粉末状木霉菌肥 Applying (NH4)2S2O8 plus powdered Trichoderma fertilizer.]
Figure 3. PCoA and NMDS analysis of soil fungal communities under different treatments
表 1 不同处理对平邑甜茶幼苗生物量的影响
Table 1 Plant biomass of Malus hupehensis Rehd. under different orchard soil treatments
处理 Treatment 株高 Plant height (cm) 地径 Stem diameter (mm) 鲜质量 Fresh weight (g/plant) 干质量 Dry weight (g/plant) CK 62.30 ± 0.98 c 5.58 ± 0.18 c 32.61 ± 1.32 c 13.31 ± 0.60 c T1 79.00 ± 1.50 b 10.18 ± 0.26 b 70.82 ± 0.36 b 29.35 ± 0.76 b T2 84.43 ± 0.83 a 10.75 ± 0.13 a 76.00 ± 1.99 a 31.67 ± 1.82 a T3 82.60 ± 1.85 a 10.60 ± 0.23 a 73.91 ± 0.77 a 30.55 ± 0.31 ab 注（Note）：CK—连作果园土壤 Replanted orchard soil; T1—过硫酸铵处理 Applying (NH4)2S2O8 in soil; T2—过硫酸铵加颗粒状木霉菌肥 Applying (NH4)2S2O8 and granular Trichoderma fertilizer; T3—过硫酸铵加粉末状木霉菌肥 Applying (NH4)2S2O8 plus powdered Trichoderma fertilizer; 同列数据后不同字母表示处理间差异显著 (P < 0.05) Values followed by different letters indicate significant difference among treatments (P < 0.05). 
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表 2 不同处理土壤酶活性
Table 2 Soil enzyme activities of different treatments
处理 Treatment 过氧化氢酶 Catalase (mL/g) 磷酸酶 Phosphatase [mg/(g·d)] 脲酶 Urease [mg/(g·d)] 蔗糖酶 Sucrase [mg/(g·d)] CK 0.174 ± 0.002 c 8.792 ± 0.590 b 0.090 ± 0.003 c 6.778 ± 0.098 c T1 0.160 ± 0.002 d 7.612 ± 0.295 c 0.076 ± 0.002 d 6.413 ± 0.057 d T2 0.272 ± 0.006 a 16.955 ± 0.742 a 0.258 ± 0.001 a 9.856 ± 0.152 a T3 0.250 ± 0.005 b 16.168 ± 0.511 a 0.239 ± 0.002 b 9.012 ± 0.043 b 注（Note）：CK—连作果园土壤 Replanted orchard soil; T1—过硫酸铵处理 Applying (NH4)2S2O8 in soil; T2—过硫酸铵加颗粒状木霉菌肥 Applying (NH4)2S2O8 and granular Trichoderma fertilizer; T3—过硫酸铵加粉末状木霉菌肥 Applying (NH4)2S2O8 plus powdered Trichoderma fertilizer; 同列数据后不同字母表示处理间差异显著 (P < 0.05) Values followed by different letters indicate significant difference among treatments (P < 0.05).
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表 3 不同处理的土壤微生物数量
Table 3 The quantity of soil microorganisms under different treatments
处理
Treatment细菌
Bacteria
(× 105 cfu/g)放线菌
Actinomyces
(× 105 cfu/g)腐皮镰孢菌拷贝数
F. solani copies
(× 1011)CK 7.33 ± 0.58 c 12.67 ± 1.53 c 0.82 ± 0.02 a T1 4.33 ± 0.58 d 9.00 ± 1.00 d 0.46 ± 0.01 b T2 16.00 ± 1.00 b 21.67 ± 0.58 a 0.13 ± 0.01 c T3 18.67 ± 0.58 a 19.33 ± 0.58 b 0.14 ± 0.03 c 注（Note）：CK—连作果园土壤 Replanted orchard soil; T1—过硫酸铵处理 Applying (NH4)2S2O8 in soil; T2—过硫酸铵加颗粒状木霉菌肥 Applying (NH4)2S2O8 and granular Trichoderma fertilizer; T3—过硫酸铵加粉末状木霉菌肥 Applying (NH4)2S2O8 plus powdered Trichoderma fertilizer; 同列数据后不同字母表示处理间差异显著 (P < 0.05) Values followed by different letters indicate significant difference among treatments (P < 0.05).
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表 4 不同处理下土壤真菌群落在属水平的相对丰度 (%)
Table 4 Relative abundance of soil fungal communities at genus level under different treatments
处理 Treatment 链格孢属 Alternaria 镰孢菌属 Fusarium 芽枝霉属 Cladosporium 木霉属 Trichoderma CK 9.68 ± 3.25 a 9.66 ± 2.09 a 6.42 ± 2.44 a 3.30 ± 0.62 b T1 0.99 ± 0.09 b 6.72 ± 0.57 b 0.64 ± 0.19 b 3.43 ± 0.87 b T2 0.69 ± 0.08 b 8.33 ± 1.98 ab 0.70 ± 0.16 b 5.24 ± 0.21 a T3 0.90 ± 0.05 b 3.46 ± 0.24 c 0.63 ± 0.17 b 4.74 ± 0.71 a 注（Note）：CK—连作果园土壤 Replanted orchard soil; T1—过硫酸铵处理 Applying (NH4)2S2O8 in soil; T2—过硫酸铵加颗粒状木霉菌肥 Applying (NH4)2S2O8 and granular Trichoderma fertilizer; T3—过硫酸铵加粉末状木霉菌肥 Applying (NH4)2S2O8 plus powdered Trichoderma fertilizer; 同列数据后不同字母表示处理间差异显著 (P < 0.05) Values followed by different letters indicate significant difference among treatments (P < 0.05).
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