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长春花感染柑橘黄龙病菌微波热处理防控效果

花匠小妙招
2024-12-17 20:26

摘要:

为探索黄龙病防治途径，开展微波热处理对黄龙病的防控效果研究。该研究搭建了微波热处理平台，对长春花微波热处理参数（转盘转速、单个磁控管的微波功率、磁控管数量）进行优化，并用优化后的参数组合对感病长春花进行热处理，处理后90 d内跟踪检测长春花叶片中黄龙病菌的浓度、淀粉和类黄酮的含量。结果表明，长春花微波热处理的最优参数组合为单个磁控管的微波功率150 W、3个磁控管、转速15 r/min；热处理90 d后，感病长春花叶片中的黄龙病菌Ct值（阈值循环数，该值低于32时为阳性）由阳性(17.01 ± 0.97)转至近阴性（31.91 ± 2.35），病菌浓度下降了99.98%，淀粉含量和类黄酮含量恢复至正常水平，植株的活性恢复较好。研究表明，微波热处理有效抑制植物体内的黄龙病菌，可为黄龙病的规模化防治提供新思路。

关键词: 病害 / 微波 / 热处理 / 黄龙病 / 长春花 / 防治效果

Abstract:

Citrus Huanglongbing (HLB) is one of the most serious bacterial diseases in the citrus industry. Starch and flavonoids are two organic materials that are associated with HLB affection. Thermotherapy can be effective in eliminating the “Candidatus Ldiberibacter asiaticus” (CLas), the putative pathogen of HLB. However, the previous thermotherapy cannot fully meet the large-scale production, due to the low efficiency, high energy consumption, or with high cost. In this study, a microwave heat treatment was proposed to control the citrus HLB. A systematic investigation was implemented to explore the inhibitory effect of microwave heat treatment on the physiological materials of HLB-affected periwinkle plants. Firstly, a batch of 30-day-old periwinkles seedlings were cultivated and then graft-inoculated in an insect-proof screen house. Secondly, a microwave thermotherapy platform was built preliminarily. A single-factor test was conducted on three microwave parameters, including the rotating speed of the turntable (RST), microwave power (MP), and number of magnetrons (NM). The parameter levels were determined after the orthogonal test, according to the degree of overheating. Thirdly, the orthogonal test of L9(34) was carried out. Range and analysis of variance (ANOVA) were performed on the three indicators i.e., plant overheating degree, temperature range, and activity evaluation. The optimal combination of parameters was determined using the comprehensive balance. Finally, the optimized microwave treatment was used for thermotherapy on HLB-affected periwinkle plants. CLas titers were determined by real-time quantitative PCR. The starch and flavonoid concentrations of the leaves from the treated and untreated plants were detected within 90 days after heat treatment. The results show that: 1) The factor values for the L9(34) orthogonal test were obtained after single factor testing: RST of 10, 15, and 20 r/min, MP of 150, 200, and 250 W, and NM of 2, 3, and 4. The range analysis showed that the MP was the most important influencing factor on heating, followed by NMs, and finally RSP. 2) Combined with the ANOVA, the optimal combination was determined to be MP of 150 W with 3 magnetrons, where the value range of RST was from 15 r/min. The Ct value of infected samples increased from 17.01±0.97 before treatment to 31.91±2.35 at 90 days post-treatment, with the titers of CLas decreased by 99.98% after treatment with the optimized. 3) The previously CLas-infected plants were recovered after heat treatment, compared with the untreated plants, with more new leaves and lateral branches. The contents of starch and flavonoid were gradually recovered to the levels in the healthy control plants. Comparatively, the starch content of healthy samples increased significantly on the day of heating but gradually recovered to normal level. While there was no significant difference in the total flavonoid content. The flavonoid content in the untreated HLB-affected control was lower than that of the healthy control. The flavonoid content continued to increase until died, as time went by. In conclusion, 1) most CLas in periwinkle plants were eliminated to recover the growth conditions of plants into the healthy control level after microwave heat treatment. 2) Microwave heat treatment gradually restored the starch and flavonoid contents of infected samples, compared with the healthy control and the untreated diseased plants. 3) The platform of microwave heat treatment can be effective for pot-growing periwinkle seedlings. The microwave heat treatment is also required for long-term verification with the different conditions in citrus trees in the future. 4) Microwave heat treatment performed the better inhibitory effect on CLas, with the low cost, short treatment cycle, low consumption and high efficiency. It is also expected to achieve large-scale promotion in the field.

图 1 微波热处理平台和长春花部位

1. 侧枝；2. 主干；3. 测温点。

Figure 1. Microwave heat treatment platform and periwinkle position

1. Lateral branch; 2. Trunk; 3. Temperature measuring point.

图 2 感病长春花与健康长春花经微波热处理后不同时间的表型

注：“CLas+”表示感病长春花。“CLas-”则表示健康长春花。“Heat+”为经过微波热处理。“Heat-”为未经热处理的对照。

Figure 2. Phenotypes of diseased Periwincle plants and healthy Periwincle plants with and without microwave heat treatment

Note: "CLas+" means periwinkle plants infected by CLas. "CLas-" means healthy periwinkle plants. "Heat+" indicates the plants are subjected to microwave heat treatment. "Heat-" means the control without heat treatment.

表 1 正交试验表及因素水平值

Table 1 Orthogonal experiment table and factor levels

试验
编号
Test number A 转盘
Turntable B 微波
Microwave C 磁控管
Magnetrons D空列
Blank column 转速
Rotating
speed/
(r·min−1) 水平值
Horizontal value 功率
Power/
W 水平值
Horizontal value 数量
Number 水平值
Horizontal value 1 10 1 150 1 2 1 1 2 10 1 200 2 3 2 2 3 10 1 250 3 4 3 3 4 15 2 150 1 3 2 3 5 15 2 200 2 4 3 1 6 15 2 250 3 2 1 2 7 20 3 150 1 4 3 2 8 20 3 200 2 2 1 3 9 20 3 250 3 3 2 2

表 2 长春花活性评价

Table 2 Activity evaluation of periwincle plants

主干Trunk 侧枝Collateral 主干新叶面积占比
Proportion of new leaf area on trunk 受损比例
Damage
ratio/% 分数
Score 受损比例
Damage
ratio/% 分数
Score 比值Ratio/% 分数
Score ＜30 80 ＜20 6 ＞80 14 30～50 60 20～40 5 ＞60～80 12 ＞50～70 40 ＞40～60 4 ＞40～60 8 ＞70～80 20 ＞60～80 3 20～40 4 ＞80 0 ＞80 2 ＜20 2 注：主干满分80，侧枝满分6，主干新叶面积占比满分为14。 Note:The full score of trunk is 80. The full score of lateral branches is 6. The full score of proportion of new leaf area on trunk is 14.

表 3 微波参数类型的单因素试验结果

Table 3 Single factor test results of microwave parameters

转盘转速
Rotating speed of turntable/
(r·min-1) 最高温度
Maximum temperature /℃ 微波功率
Microwave power /W 最高温度
Maximum temperature /℃ 磁控管数量
Number of magnetrons 最高温度
Maximum temperature /℃ 0 58.8 150 47.0 1 47.4 5 52.6 250 60.9 2 53.0 10 50.2 350 67.5 3 60.8 15 51.8 450 75.6 4 62.6 20 51.8

表 4 长春花植株微波热处理正交试验结果

Table 4 Results of orthogonal experiment for microwave-based thermotherapy on periwinkle plants

试验编号

Test number

温度
Temperature /℃ 加热到48℃所需时间
Time required for heating
to 48℃ / s
评价指标 Evaluating index 主干顶部
Top of trunk 主干中部
Middle of trunk 主干底部
Bottom of trunk 植株过热程度
Degree of plant overheating 植株温度均匀度
Degree of plant temperature uniformity 植株活性评价
Plant activity evaluation 1 46.6 39.0 32.6
/
−1.43 15.43 86.00 2 66.2 47.4 30.4 26 18.20 35.77 67.67 3 68.8 62.8 31.8 23 20.83 37.07 38.00 4 51.8 47.3 31.4 94 3.77 20.40 90.50 5 59.9 46.4 32.7 40 11.90 27.23 51.17 6 60.1 44.2 33.9 40 12.10 26.20 57.00 7 54.0 47.8 30.7 52 6.03 23.30 84.17 8 52.7 46.9 32.8 74 4.73 19.97 81.67 9 60.5 51.7 29.3 34 12.50 31.17 67.83 注：试验编号1的温度未达到48 ℃的要求，因此加热时间未做计算。 Note: The temperature of test No.1 does not meet the requirement of 48 ℃, so the heating time is not calculated.

表 5 正交试验极差分析结果

Table 5 Results of range analysis of orthogonal test

评价指标
Evaluating index 分析角度
Analysis angle 因素 Factors 转盘转速 (A)
Rotating speed of turntable/ (r·min−1) 微波功率 (B)
Microwave power /W 磁控管数量 (C)
Number of magnetrons 主次因素
Factor priority 最优组合
Optimal combination 植株过热程度
Degree of plant overheating k1 12.53 2.79 5.13 B>C>A A3B1C1(20-150-2) k2 9.26 11.61 11.49 k3 7.76 15.14 12.92 极差
Extreme difference R 4.78 12.36 7.79 植株温度均匀度
Degree of plant temperature uniformity k1 29.42 19.71 20.53 B>C>A A2B1C1(15-150-2) k2 24.61 27.66 29.11 k3 24.81 31.48 29.20 极差
Extreme difference R 4.81 11.77 8.67 植株活性评价
Plant activity evaluation k1 63.89 86.89 74.89 B>C>A A3B1C2(20-150-3) k2 66.22 66.83 75.33 k3 77.89 54.28 57.78 极差
Extreme difference R 14.00 32.61 17.56

表 6 正交试验方差分析结果

Table 6 Results of ANOVA of orthogonal test

评价指标
Evaluating indicator 方差来源
Source of variance 离差平方和
Sums of squared deviations 自由度
Freedom 均方
Mean square
F P值
P value
植株过热程度
Degree of plant overheating 模型 Model 1235.134 8 154.392 9.233 < 0.01 A 107.463 2 53.731 3.213 > 0.05 B 728.927 2 364.464 21.796 < 0.01 C 309.343 2 154.671 9.250 < 0.01 误差 Error 89.401 2 44.700 / / 植株温度均匀度
Degree of plant temperature uniformity 模型 Model 1296.321 8 162.040 7.749 < 0.01 A 133.347 2 66.674 3.188 > 0.05 B 648.534 2 324.267 15.506 < 0.01 C 446.092 2 223.046 10.666 < 0.01 误差 Error 68.347 2 34.174 / /

表 7 热处理后不同时间植株中黄龙病菌的实时荧光PCR定量结果

Table 7 Real-time quantitative PCR detection results of the "Candidatus Liberibacter asiaticus" on periwinkle samples at different time points after microwave heat treatment

处理
Treatment 0 d 15 d 30 d 90 d Ct CN Ct CN Ct CN Ct CN CLas+/Heat+ 17.01 ± 0.97a 85533.01 ± 68377.18a 18.72 ± 1.32a 41464.22 ± 23304.68a 25.99 ± 1.20a 324.84 ± 251.64a 31.91 ± 2.35a 13.59 ± 20.16a CLas+/Heat- 17.87 ± 2.63a 129582.30 ± 105419.77b 16.29 ± 0.45b 156041.63 ± 8079.33b 15.62 ± 0.71b 343635.61 ± 159853.21b 死亡Death 死亡Death CLas-/Heat+ 32.20 ± 1.12b N/A 33.21 ± 0.67c N/A 34.29 ± 1.32c N/A 34.06 ± 1.67a N/A CLas-/Heat- 32.75 ± 1.78b N/A 32.78 ± 0.96c N/A 32.15 ± 0.66 d N/A 33.78 ± 0.66a N/A 注：Ct，阈值循环数；CN，病菌浓度；同列之间不相同的字母，表示差异显著（P < 0.05）。“N/A”表示没有黄龙病菌。 Note: Ct, threshold number of cycles; CN, bacterica concentration; Different letters in the same column indicate significant differences (P < 0.05). "N/A" means no CLas was detected.

表 8 热处理后长春花植株叶片中的淀粉和类黄酮含量变化

Table 8 Changes of starch and flavonoid content in leaf samples of periwinkle plants after heat treatment (mg·g−1)

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