Inhibiting effect of biological fumigation of mustard against Phytophthora nicotianae
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摘要: 为了探究安全和环境友好的烟草黑胫病的绿色防控方式, 本研究用‘地隆1号’芥菜采用培养皿培养方法进行生物熏蒸, 研究了其对烟草疫霉菌(Phytophthora nicotianae)的抑制作用。采用菌丝生长速率法测定了其对烟草疫霉菌(A2 交配 型)Pp025菌丝生长的抑制情况, 对烟草疫霉菌孢子囊形成及游动孢子释放、孢子萌发及芽管伸长和卵孢子形成的影响, 通过电导率和丙二醛含量变化测定了其对烟草疫霉菌细胞膜透性的影响; 采用离体叶片法测定了芥菜熏蒸处理后烟草疫霉菌致病力的变化。结果显示: 利用‘地隆1号’芥菜进行生物熏蒸可显著抑制烟草疫霉菌的菌丝生长, 其半最大效应浓度(EC50)值为0.360 g, 75%最大效应浓度(EC75)值为0.499 g, 最小抑菌量(MIC)为0.500 g, 抑制效果与‘地隆1号’芥菜鲜物质的量呈剂量效应。0.300 g芥菜鲜物质即可显著抑制孢子囊的形成及游动孢子的释放, 0.500 g芥菜鲜物质熏蒸处理对Pp025孢子囊形成和游动孢子释放的抑制率分别达72.8%和86.6%; 当芥菜鲜物质的量增加为0.700 g时, 可完全抑制Pp025孢子囊的形成; 0.300 g和0.400 g鲜物质即可分别完全抑制游动孢子萌发及芽管伸长。‘地隆1号’芥菜鲜物质熏蒸处理可减少Pp025卵孢子的形成, 且减少幅度与芥菜鲜物质的量呈剂量效应, 0.700 g鲜物质熏蒸处理可完全抑制卵孢子的形成。同时, 熏蒸处理后增大了菌丝体细胞膜透性并导致膜脂质过氧化, 经熏蒸处理150 min后, Pp025的电导率为对照组的2.2倍, 丙二醛含量是对照的8.0倍; 熏蒸处理减弱了病菌的致病力, 与对照相比, 病斑减小面积达88.9%。本研究结果表明: ‘地隆1号’芥菜鲜物质熏蒸处理对烟草疫霉菌具有很好的抑制和杀灭作用, 且熏蒸效果与‘地隆1号’芥菜鲜物质的量呈剂量效应。研究结果对生产上利用‘地隆1号’芥菜防控烟草黑胫病提供了理论依据。Abstract: Tobacco black shank caused by Phytophthora nicotianae is an important and destructive soil-borne disease affecting tobacco production in China. Biofumigation is a method to suppress or kill harmful organisms in the soil using volatile bioactive substances produced by plants, such as those of Cruciferae or Compositae, during decomposition. Brassica species are important biological fumigants. ‘Dilong 1’ (DL1) mustard was cultivated by our collaborator at Nanjing Agricultural University as Brassica juncea variety with high glucosinolates content, high biomass, high adaptability, and good fumigation effects. To explore a safe and environmentally friendly green method for the prevention and control of tobacco black shank disease, the inhibitory effect of fumigation with DL1 mustard on P. nicotianae was analyzed in this study through a series of petri dish inocubation experiments. The mycelial growth rate method was used to determine the effect of DL1 fumigation on mycelial growth of P. nicotiana. The fumigation effects on sporangium formation, zoospore release, spore germination, germ tube elongation, and oospore formation of P. nicotiana were also determined. The effects of DL1 fumigation on cell membrane permeability of P. nicotiana were determined by changes in electrical conductivity and malondialdehyde content. The change in the pathogenicity of P. nicotiana after fumigation was determined using the in vitro leaf method. The results showed that fumigation of fresh matter of DL1 mustard could significantly inhibit the mycelial growth of P. nicotiana. The EC50 (concentration for 50% of maximal effect) value was 0.36 g, EC75 (concentration for 75% of maximal effect) value was 0.499 g, and minimum inhibitory dose (MIC) was 0.500 g. The inhibitory effect was dose-dependent on the amount of fresh material in DL1 mustard. The amount of 0.3 g fresh DL1 mustard significantly inhibited sporangium formation and zoospore release of P. nicotiana, and the amount of 0.5 g could inhibit sporangium formation and zoospore release of P. nicotiana by 72.8% and 86.6%, respectively. When the amount of fresh DL1 mustard was increased to 0.7 g, sporangium formation of P. nicotiana was completely inhibited. The amount of 0.3 g and 0.4 g of fresh DL1 mustard could completely inhibit zoospore germination and germ tube elongation of P. nicotiana, respectively. Fumigation of DL1 could reduce the formation of oospores of P. nicotiana in a dose-dependent manner, and 0.7 g fresh DL1 mustard could completely inhibit the oospore formation of P. nicotianae. After fumigation, the permeability of the mycelium membrane of P. nicotianae increased, and membrane lipid peroxidation was induced. After 150 min of fumigation, the electrical conductivity of P. nicotiana was 2.2 times that of the control, and the malondialdehyde content was 8.0 times that of the control. The fumigation treatment weakened the pathogenicity of P. nicotianae, and the area of the disease spot decreased by 88.9% compared to that of the control. In this study, fumigation of DL1 mustard on the growth and development of P. nicotianae, physiological and biochemical characteristics, and pathogenicity changes indicated that it had a good inhibition and killing effect on P. nicotianae, and the fumigation effect had a dose-dependent effect on the amount of fresh DL1 mustard. This provided a theoretical basis for the use of DL1 mustard to control tobacco black shanks and other soil-borne diseases in production.
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图 1 不同量‘地隆1号’芥菜鲜物质熏蒸对烟草疫霉菌Pp025菌丝生长的抑制效果
A: 不同量‘地隆1号’芥菜鲜物质熏蒸处理72 h后Pp025的菌落生长情况; a: 0 g; b: 0.3 g; c: 0.4 g; d: 0.5 g; e: 0.6 g; f: 0.7 g。B: 不同量‘地隆1号’芥菜鲜物质熏蒸处理72 h后对Pp025的抑制率。不同小写字母表示不同处理间存在显著差异(P<0.05)。A: colony of Pp025 after fumigation with different amounts of fresh material of DL1 at 72 h. a: 0 g; b: 0.3 g; c: 0.4 g; d: 0.5 g; e: 0.6 g; f: 0.7 g. B: inhibition rate of Pp025 after fumigation with different amounts of fresh material of DL1 at 72 h. Different lowercase letters indicate significant differences among different treatments (P<0.05).
Figure 1. Inhibition of mycelium growth of Phytophthora nicotianae Pp025 by fumigation with different amounts of fresh matter of ‘DL1’ mustard
图 2 不同量‘地隆1号’芥菜鲜物质熏蒸对烟草疫霉菌孢子囊形成和游动孢子释放的抑制效果
A: 不同量‘地隆1号’芥菜鲜物质熏蒸处理后Pp025的孢子囊形成情况; a: 0 g; b: 0.3 g; c: 0.4 g; d: 0.5 g; e: 0.6 g; f: 0.7 g。B: 不同量‘地隆1号’芥菜鲜物质熏蒸处理后Pp025的孢子囊形成数量。C: 不同量‘地隆1号’芥菜鲜物质熏蒸处理后Pp025孢子囊释放的平均游动孢子浓度。不同小写字母表示不同处理间存在显著差异(P<0.05)。A: sporangium formation of Pp025 after fumigation with different amounts of fresh material of DL1. a: 0 g; b: 0.3 g; c: 0.4 g; d: 0.5 g; e: 0.6 g; f: 0.7 g. B: number of sporangium of Pp025 after fumigation with different amounts of fresh material of DL1. C: concentration of zoospores released by sporangium of Pp025 after fumigation with different amounts of fresh material of DL1. Different lowercase letters indicate significant differences among different treatments (P<0.05).
Figure 2. Inhibition of sporangium formation and zoospore release of Phytophthora nicotianae by fumigation with fresh matter of ‘DL1’ mustard
图 3 不同量‘地隆1号’芥菜鲜物质熏蒸对烟草疫霉菌孢子萌发和芽管伸长的抑制效果
A: 不同量‘地隆1号’芥菜鲜物质熏蒸处理后Pp025的芽管伸长情况; a: 0 g; b: 0.3 g; c: 0.4 g; d: 0.5 g; e: 0.6 g; f: 0.7 g。B: 不同量‘地隆1号’芥菜鲜物质熏蒸处理后Pp025的孢子萌发率。C: 不同量‘地隆1号’芥菜鲜物质熏蒸处理后Pp025的平均芽管长度。不同小写字母表示不同处理间存在显著差异(P<0.05)。A: germ tube elongation of Pp025 after fumigation with different amounts of fresh material of DL1; a: 0 g; b: 0.3 g; c: 0.4 g; d: 0.5 g; e: 0.6 g; f: 0.7 g. B: rate of zoospore germination of Pp025 after fumigation with different amounts of fresh material of DL1. C: average length of germ tube of Pp025 after fumigation with different amounts of fresh material of DL1. Different lowercase letters indicate significant differences among different treatments (P<0.05).
Figure 3. Inhibition of spore germination and germ tube elongation of Phytophthora nicotianae by fumigation with fresh matter of ‘DL1’ mustard
图 4 不同量‘地隆1号’芥菜鲜物质熏蒸对烟草疫霉菌卵孢子形成的抑制效果
A: 不同量‘地隆1号’芥菜鲜物质熏蒸处理后Pp025的卵孢子形成情况; a: 0 g; b: 0.3 g; c: 0.4 g; d: 0.5 g; e: 0.6 g; f: 0.7 g。B: 不同量‘地隆1号’芥菜鲜物质熏蒸处理后Pp025的平均卵孢子形成数量。C:不同量‘地隆1号’芥菜鲜物质熏蒸处理后Pp025的平均卵孢子浓度。不同小写字母表示不同处理间存在显著差异(P<0.05)。A: Oospore formation of Pp025 after fumigation with different amounts of fresh material of DL1; a: 0 g; b: 0.3 g; c: 0.4 g; d: 0.5 g; e: 0.6 g; f: 0.7 g. B: number of oospores of Pp025 after fumigation with different amounts of fresh material of DL1. C: Concentration of oospores of Pp025 after fumigation with different amounts of fresh material of DL1. Different lowercase letters indicate significant differences among different treatments (P<0.05).
Figure 4. Inhibition of oospore formation of Phytophthora nicotianae by fumigation with fresh matter of ‘DL1’ mustard
图 5 不同量‘地隆1号’芥菜鲜物质熏蒸后烟草疫霉菌细胞膜透性的变化
不同小写字母表示不同处理间存在显著差异(P<0.05)。Different lowercase letters indicate significant differences among different treatments (P<0.05).
Figure 5. Effect of fumigation with fresh matter of ‘DL1’ mustard on cell membrane permeability
图 6 ‘地隆1号’芥菜熏蒸处理后烟草疫霉菌Pp025致病力的变化
A: 阴性对照, 不接种; B: 阳性对照, 接种烟草疫霉菌Pp0205菌饼; C: 接种经‘地隆1号’芥菜熏蒸处理后的菌饼; D: 病斑面积. A: CK–; B: CK+, inoculated with Pp025; C: inoculated with Pp025 after fumigation of fresh matter of DL1 mustard; D: Lesion area.
Figure 6. Variation of infection ability of Pp025 after fumigation of fresh matter of ‘DL1’ mustard
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