Response and mechanism of potassium content in leaves of alpine meadow plants to multiple nutrient additions
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摘要:目的 钾是植物体内含量仅次于氮的大量元素，对于维持植物生长和适应低温环境具有重要的意义。近期研究表明植物生长受到多种养分的共同限制，而本研究旨在揭示植物叶片钾含量对钾、氮、磷养分添加及其交互作用的响应及机理。方法 以川西北高寒草甸为研究对象，开展钾、氮、磷添加及交互控制试验，以揭示高寒草甸优势植物叶片钾对多种养分及其交互作用的响应及机理。结果 本研究发现钾、氮、磷3种养分添加对4种植物（垂穗披碱草、发草、无脉薹草、草玉梅）的叶片钾含量均不存在显著的交互作用。单独磷添加不影响植物叶片钾，钾添加促进无脉薹草和草玉梅叶片钾含量，而氮添加均促进4种植物的叶片钾。尽管氮添加产生的直接效应（土壤有效氮增加）对部分植物叶片钾有显著影响，但是氮添加处理下叶片钾含量的增加主要是由氮添加的间接作用驱动的，即氮添加显著增加植物群落生物量，进而由于遮荫作用降低土壤温度，而结果表明较低土壤温度一致地增加了所有植物的叶片钾含量。与钾、磷养分相比，氮添加对植物叶片钾含量的影响更大。结论 不同于传统观点认为土壤养分是主要影响植物叶片钾的因素，本研究发现氮添加引发的间接作用（土壤温度降低）是驱动养分富集背景下叶片钾含量变化的主要机理。该研究结果指示出高寒草甸植物由于长期受到低温胁迫，其钾利用策略可能与低温适应性密切相关。Abstract:Objective Potassium (K) is the second most abundant element in plants, which is important to sustain plant growth and adapt to cold environment. Recent meta-analysis studies showed that grassland plant product was universally co-limited by multiple nutrients, while this study aimed to reveal the responses and mechanisms of leaf K concentration to main and interaction effects of the addition of K, nitrogen (N) and phosphorus (P).Method Taking the alpine meadow in northwestern Sichuan as the research object, potassium, nitrogen and phosphorus addition and interactive control experiments were carried out to reveal the response and mechanism of potassium in the leaves of dominant plants in alpine meadow to a variety of nutrients and their interactions.Result We found no significant interaction effect for the combined addition of K, N and P fertilizers on the leaf K concentration of Elymus nutans, Deschampsia cespitosa, Carex enervis and Anemone rivularis. P addition alone did not affect species’ leaf K, while K addition increased leaf K concentration in Carex enervis and Anemone rivularis significantly. In contrast, N addition consistently enhanced leaf K concentration across all species. The direct effect of increasing soil N availability by N addition only affected two species’ leaf K concentration. However, we found that the consistent increase in leaf K concentration among species was mainly driven by the indirect effect caused by N addition. To be specific, N addition firstly increased community biomass, further indirectly reducing soil temperature due to increasing shading effect. Moreover, we found a consistent increase in leaf K concentration with lower soil temperature among different species.Conclusion Different from the traditional view that soil nutrient is the main factor affecting plant leaf potassium, this study finds that the indirect effect caused by nitrogen addition (decrease of soil temperature) is the main mechanism driving the change of leaf K content under the background of nutrient enrichment. The results indicate that the K utilization strategy of alpine meadow plants may be closely related to low temperature adaptability due to long-term low temperature stress.
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Keywords:
- leaf potassium concentration /
- nutrient addition /
- soil temperature /
- alpine meadow
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黄栌（Cotinus coggygria）是一种优良的生态与景观两用树种，作为北京地区红叶景观的重要组成[1]，为首都的生态景观建设发挥了巨大作用[2]。然而，由大丽轮枝菌（Verticillium dahliae）引起的黄栌枯萎病已严重发生，造成严重的经济损失，制约着红叶景观建设[1]。
除黄栌外，大丽轮枝菌的寄主多达400种植物[3]，如林木（橄榄Canarium album[4−5]、紫荆Cercis chinensis[6]、枫树Acer spp.[3]等）、农作物（棉花Gossypium spp.[7]、薄荷Mentha canadensis[8]、洋蓟Cynara scolymus[9]、生菜Lactuca sativa var. ramosa[10]等）和花卉（菊花Chrysanthemum morifolium[11]、蔷薇Rosa sp.[12]等）。黄栌枯萎病是一种典型的林木维管束病害，从根部侵入通过皮层扩展至维管束系统，造成根部腐烂、木质坏死和整株枯萎甚至死亡[13−14]。因病原菌定殖在植物维管束系统，且产生的微菌核在土壤中长时间存活，此类病害难以控制，被称为 “植物癌症”[15−17]。
目前生产上尚无抗枯萎病的黄栌品种，而生物防治依赖于土壤环境和气候，且防治效果并不稳定。所以截止目前，化学防治是作为黄栌枯萎病林间防治的主要方法[18]。此前的田间药效试验显示，萎菌净和多菌灵对于黄栌枯萎病有一定防治效果[12]。此外，郑怿[19]在对黄栌枯萎病林间化学防治研究中发现，灌根稀释500倍的嘧菌酯与多菌灵组合药剂处理最佳，且树干注射可在一定程度上防治黄栌枯萎病。
然而，将室内和盆栽苗试验筛选的药剂应用在田间后，防治效果并不稳定[12]。在之前的林间试验中，施药时间多集中于上半年，并且施药次数较少，施药方式比较单一[19−20]。采用的树干输液法[19]，由于针头容易被黄栌流胶堵住，导致实际防治效率不理想。因此，在此基础上，本研究改进施药方式，通过在整个生长季内开展灌根、树干注射的施药方式及不同药剂组合的黄栌枯萎病林间防治试验，从而确定最优施药方式及药剂组合，并探索黄栌枯萎病长期防治的可行性，为更高效的黄栌枯萎病化学防治提供科学依据。
1. 材料与方法
1.1 试验样地
试验地位于北京市延庆区八达岭森林公园红叶岭（40°20′46″N，116°00′52″E）。该地区属于温带半湿润半干旱季风气候，夏季高温多雨，冬季寒冷干燥，春、秋短促。年均温10.8 ℃，年降水量454 mm，土壤类型以花岗岩等发育而来的褐土、棕壤为主。
1.2 试验药剂
1.2.1 灌根药剂
枯草芽孢杆菌（Bacillus subtilis）：河北中保绿农作物科技有限公司，水悬液，有效成分含量，菌含量 ≥ 1 000 × 108芽孢/g；嘧菌酯（绘绿），先正达农化有限公司，水分散粒剂，有效成分含量50%；丙环唑（扮绿），先正达农化有限公司，乳油，有效成分含量156 g/L。
1.2.2 注射药剂
嘧菌酯（绘绿），先正达农化有限公司，水分散粒剂，有效成分含量50%；多菌灵，河北中保绿农作物科技有限公司，可湿性粉剂，有效成分含量50%；咪鲜胺，苏州富美实植物保护剂有限公司，水乳剂，有效成分含量45%。
1.3 施药方式
1.3.1 土壤灌根
采用直接灌根，将药液直接灌入树坑中，树坑边缘土围为10 ~ 15 cm。
1.3.2 树干注射
电动树干打孔注药机（绿友机械集团股份有限公司，型号ZYJ15A）加压注药：在植株基部用该机器的打孔钻头由上向下成45°钻1 ~ 2个注射孔，深度约为树干直径的1/3，打孔后拆掉钻头，将注射口插入孔中，手动加压注射，保证药剂注入树干，等待压力降低后拔除，利用配套可降解堵孔塞进行封堵。
1.3.3 组合施药
采用直接灌根与电动树干打孔注药机加压注药相结合的施药方法。具体灌根和注射方法与单独施药相同。
1.4 试验设计
选样地内样树180棵，分为12个处理，每个处理15棵样树，随机分为3个小区，处理1为空白对照。对样地进行分区分组，其中灌根3组，树干注射2组，组合施药为灌根和树干注射两两组合，共6组处理（表1）。对所有样树进行调查，记录，挂牌编号。
表 1 试验设计Table 1. Experimental design施药方式
Way of insecticide application灌根药剂
Root irrigation chemical注射药剂
Injecting chemical处理
TreatmentCK 1 灌根 Root irrigation 枯草芽孢杆菌 Bacillus subtilis 2 50%嘧菌酯 50% azoxystrobin 3 156 g/L丙环唑 156 g/L propiconazole 4 树干注射 Trunk injection 50%多菌灵，45%咪鲜胺
50% carbendazim, 45% prochloraz5 50%多菌灵，50%嘧菌酯
50% carbendazim, 50% azoxystrobin6 组合 Combined treatment 枯草芽孢杆菌 Bacillus subtilis 50%多菌灵，45%咪鲜胺
50% carbendazim, 45% prochloraz7 枯草芽孢杆菌 Bacillus subtilis 50%多菌灵，50%嘧菌酯
50% carbendazim, 50% azoxystrobin8 50%嘧菌酯 50% azoxystrobin 50%多菌灵，45%咪鲜胺
50% carbendazim, 45% prochloraz9 50%嘧菌酯 50% azoxystrobin 50%多菌灵，50%嘧菌酯
50% carbendazim, 50% azoxystrobin10 156 g/L丙环唑 156 g/L propiconazole 50%多菌灵，45%咪鲜胺
50% carbendazim, 45% prochloraz11 156 g/L丙环唑 156 g/L propiconazole 50%多菌灵，50%嘧菌酯
50% carbendazim, 50% azoxystrobin12 施药时间均选择在黄栌生长期4—10月（除7月，北京雨季，施药效果不佳）间进行，以探索针对黄栌枯萎病的一整年防治效果。灌根试验于4—6月进行2次施药，8—9月施药2次，每次每株树施用10 L药液。注射试验于4—6月进行树干打孔注药2次，8—9月进行注药2次，每株树每次施用100 mL药液。
1.5 黄栌枯萎病病情指数调查
于2021年5—10月及次年5月，根据黄栌枯萎病分级标准（表2），每个月对不同处理组进行病害分级调查[21−22]，以5月初调查值作为发病情况本底值。之后计算6—10月及次年5月的病情指数、校正病情指数及校正防治效果，从而判断不同药剂防治效果优劣。计算公式如下所示。
表 2 黄栌枯萎病分级标准Table 2. Grading standards for Cotinus coggygria Verticillium wilt等级
Grade分级标准
Grading standard代表值
Representative value病害严重程度
Disease severityⅠ 全株叶片无萎蔫症状 No wilting symptoms on the entire plant leaves 0 无病 Healthy Ⅱ 某一末端小枝出现萎蔫或变黄 Wilting or yellowing of a small branch at one end 1 轻度 Mildly diseased Ⅲ 多个末端小枝或次级枝条出现萎蔫或变黄
Multiple terminal twigs or secondary branches appear wilted or yellowed2 Ⅳ 植株三分之二的叶片萎蔫或变黄 Two-thirds of the leaves of the plant wilt or turn yellow 3 重度 Severely diseased Ⅴ 植株85%以上叶片萎蔫，变黄或全株死亡
More than 85% of the plants have wilted leaves, turn yellow, or die as a whole4 ID=0n0+1n1+2n2+3n3+4n44n×100 ICD=IDt−ID0 ECC=ICD0−ICDiICD0×100% 式中：n0 ~ n4是相应病级下的株数，n是调查总株数；ID是病情指数；IDt是第t次病情指数；ID0是初始病情指数；ECC是校正防治效果；ICD是校正病情指数；ICD0是对照组校正病情指数；ICDi是处理组i校正病情指数。
1.6 数据处理
本研究采用Microsoft Office Excel 2019对数据进行整理；采用R4.1.2软件对不同处理校正病情指数及防治效果进行单因素方差分析（one-way ANOVA），并用最小显著差异法（LSD）进行多重比较（P < 0.05）；采用ChiPlot网站对两年病情指数进行对比分析，并用T检验计算显著性（*P < 0.05；**P < 0.01；***P < 0.001）。
2. 结果与分析
2.1 不同处理对黄栌枯萎病病情指数的影响
在调查初始病情指数时，将病害严重程度按病害分级分为3类：无病（0）、轻度（1 ~ 2）、重度（3 ~ 4）（表2）。由表3可知，大部分施药处理的无病样树校正病情指数均比对照组（处理1）小，其中，处理4、处理12无病样树的校正病情指数比对照组（处理1）低73.68%，处理3比对照组低43.60%。并且，除处理5外，所有施药组轻度样树的校正病情指数均比对照组小。处理10和处理12的重度发病样树病情有减轻，比初始病情指数分别减少18.75、12.50。这些结果表明：灌根处理3、4和组合处理12能有效防控病害，并且组合处理10和处理12能减缓病情。
表 3 2021年10月份黄栌枯萎病校正病情指数Table 3. Corrective disease index of Cotinus coggygria Verticillium wilt in October 2021处理
Treatment施药方式
Way of insecticide application病害分级
Disease grading校正病情指数
Corrective disease index处理 1 Treatment 1 对照 CK 无病 Healthy 47.50 轻度 Mildly diseased 40.00 重度 Severely diseased − 处理 2 Treatment 2 灌根 Root irrigation 无病 Healthy 75.00 轻度 Mildly diseased 5.69 重度 Severely diseased 0.00 处理 3 Treatment 3 无病 Healthy 26.79 轻度 Mildly diseased −1.57 重度 Severely diseased − 处理 4 Treatment 4 无病 Healthy 12.50 轻度 Mildly diseased 12.50 重度 Severely diseased 0.00 处理 5 Treatment 5 注射 Trunk injection 无病 Healthy 65.63 轻度 Mildly diseased 48.22 重度 Severely diseased 6.25 处理 6 Treatment 6 无病 Healthy 43.75 轻度 Mildly diseased 0.00 重度 Severely diseased −25.00 处理 7 Treatment 7 组合 Combined treatment 无病 Healthy 50.00 轻度 Mildly diseased 30.56 重度 Severely diseased 0.00 处理 8 Treatment 8 无病 Healthy 35.71 轻度 Mildly diseased 2.08 重度 Severely diseased 12.50 处理 9 Treatment 9 无病 Healthy 27.50 轻度 Mildly diseased 20.31 重度 Severely diseased 6.25 处理 10 Treatment 10 无病 Healthy 33.33 轻度 Mildly diseased 28.75 重度 Severely diseased −18.75 处理 11 Treatment 11 无病 Healthy 36.36 轻度 Mildly diseased 25.00 重度 Severely diseased − 处理 12 Treatment 12 无病 Healthy 12.50 轻度 Mildly diseased 23.75 重度 Severely diseased −12.50 注：表中“−” 表示在初始病情时，该处理组没有重病植株。Notes: “−” in the table indicates that there are no severely diseased plants in that treatment group at the time of initial disease. 不同于对照组病情指数出现连续增长的现象，大多数施药处理组的病情指数出现了不同程度的降低或不增长情况（图1）。所有施药组的校正病情指数均小于对照，其中处理12的校正病情指数最小，增长最缓慢。处理5的校正病情指数最大，其次为处理7（图2A）。
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图 1 不同处理黄栌枯萎病病情指数变化Figure 1. Changes of disease index of Cotinus coggygria Verticillium wilt under different treatments![]()
图 2 2021年不同处理最终防治效果采用单因素方差分析（one-way ANOVA），并用最小显著差异法（LSD）进行多重比较，不同小写字母表示不同处理组之间差异显著（P < 0.05）。Using one-way ANOVA and conducting multiple comparisons using least significant difference (LSD) method, different lowercase letters indicate significant differences between different treatment groups (P < 0.05).Figure 2. Final control effect of different treatments in 20212.2 不同处理对黄栌枯萎病防治效果的影响
对不同处理病害严重程度调查发现，对照组无病样树全部转变为发病树，轻度发病样树中有60%发展为重度发病（表4）。组合处理12中有26.67%植株保持健康，其中无病样树有66.67%一直保持健康状态，并且处理6、10和12的重度发病样树的病情出现缓解。然而，大部分处理（处理2、3、4、5、7、9、11和12）中都存在由轻度转为重度的现象，其中处理5由轻度到重度的发展率达到86.71%（表4）。
表 4 2021年不同处理病害程度调查Table 4. Investigation for disease degree in different treatments in 2021处理 Treatment 5月 May 10月 October 病害严重程度 Disease severity condition 数量 Number 病害严重程度 Disease severity condition 数量 Number 处理1 Treatment 1 无病 Healthy 5 轻度 Mildly diseased 4 重度 Severely diseased 1 轻度 Mildly diseased 10 轻度 Mildly diseased 4 重度 Severely diseased 6 处理2 Treatment 2 无病 Healthy 1 重度 Severely disease 1 轻度 Mildly diseased 11 轻度 Mildly diseased 10 重度 Severely diseased 1 重度Severely diseased 3 重度 Severely diseased 3 处理3 Treatment 3 无病 Healthy 7 无病 Healthy 2 轻度 Mildly diseased 5 轻度 Mildly diseased 8 无病 Healthy 2 轻度 Mildly diseased 5 重度 Severely diseased 1 处理4 Treatment 4 无病 Healthy 3 无病 Healthy 1 轻度 Mildly diseased 2 轻度 Mildly diseased 11 轻度 Mildly diseased 8 重度 Severely diseased 3 重度 Severely diseased 1 重度 Severely diseased 1 处理5 Treatment 5 无病 Healthy 4 轻度 Mildly diseased 2 重度 Severely diseased 2 轻度 Mildly diseased 7 轻度 Mildly diseased 1 重度 Severely diseased 6 重度 Severely diseased 4 重度 Severely diseased 4 处理6 Treatment 6 无病 Healthy 8 轻度 Mildly diseased 7 重度 Severely diseased 1 轻度 Mildly diseased 6 无病 Healthy 1 轻度 Mildly diseased 5 重度 Severely diseased 1 轻度 Mildly diseased 1 处理7 Treatment 7 无病 Healthy 5 轻度 Mildly diseased 4 重度 Severely diseased 1 轻度 Mildly diseased 9 轻度 Mildly diseased 5 重度 Severely diseased 4 重度 Severely diseased 1 重度 Severely diseased 1 处理8 Treatment 8 无病 Healthy 7 轻度 Mildly diseased 7 轻度 Mildly diseased 6 轻度 Mildly diseased 6 重度 Severely diseased 2 重度 Severely diseased 2 处理9 Treatment 9 无病 Healthy 5 轻度 Mildly diseased 5 轻度 Mildly diseased 8 轻度 Mildly diseased 5 重度 Severely diseased 3 重度 Severely diseased 2 重度 Severely diseased 2 处理10 Treatment 10 无病 Healthy 3 轻度 Mildly diseased 3 轻度 Mildly diseased 10 轻度 Mildly diseased 5 重度 Severely diseased 5 重度 Severely diseased 2 轻度 Mildly diseased 1 重度 Severely diseased 1 处理11 Treatment 11 无病 Healthy 11 无病 Healthy 2 轻度 Mildly diseased 8 重度 Severely diseased 1 轻度 Mildly diseased 4 轻度 Mildly diseased 3 重度 Severely diseased 1 处理12 Treatment 12 无病 Healthy 6 无病 Healthy 4 轻度 Mildly diseased 2 轻度 Mildly diseased 5 无病 Healthy 1 轻度 Mildly diseased 3 重度 Severely diseased 1 重度 Severely diseased 4 轻度 Mildly diseased 1 重度 Severely diseased 3 施药处理组对黄栌枯萎病均有一定的防治效果（图2）。对照的校正病情指数最高，其次是处理5，且与对照组无显著差异（P > 0.05），处理12与对照组的校正病情指数差异最显著（P < 0.05）（图2A）。在6月份（上半年），灌根处理4与组合处理12的校正防治效果最好，分别达到109.09%和100.00%；处理5、9和11的校正防治效果呈现负值（表5）。对于整年防治效果，不同处理之间的防治效果有所差异（图2B）。其中，组合处理12防治效果最好，达到88%；其次是灌根处理组（处理2、3和4），处理8也有较好防效，防治效果为56%（表5和图2B）。处理5的防治最差，处理7次之（图2B）。这些结果表明，灌根丙环唑结合树干注射多菌灵和嘧菌酯复配组合的校正病情指数最低，防治效果最好。
表 5 2021年不同处理校正防治效果Table 5. Corrective control effect of different treatments in 2021处理 Treatment 校正防治效果 Corrective control effects/% 6月 June 7月 July 8月 August 9月 September 10月 October 处理2 Treatment 2 灌根
Root irrigation72.73 ± 31.49abcd 71.43 ± 25.75ab 68.75 ± 12.5ab 69.57 ± 8.70ab 76.00 ± 13.73ab 处理3 Treatment 3 81.82 ± 9.09abc 107.14 ± 14.28a 75.00 ± 22.53ab 60.87 ± 19.92ab 72.00 ± 9.80abc 处理4 Treatment 4 109.09 ± 39.62a 107.14 ± 31.13a 81.25 ± 10.83ab 60.87 ± 13.04ab 68.00 ± 10.38abc 处理5 Treatment 5 注射
Trunk injection−9.09 ± 41.65cde 14.29 ± 32.73bc 6.25 ± 18.75b 8.70 ± 22.59b 0 ± 24.09d 处理6 Treatment 6 9.09 ± 24.05abcde 35.71 ± 12.37abc 68.75 ± 34.80ab 39.13 ± 17.39ab 44.00 ± 17.64abcd 处理7 Treatment 7 组合
Combined treatment0 ± 48.00bcde 7.14 ± 31.13bc 6.25 ± 39.03b 8.70 ± 34.51b 16.00 ± 29.61cd 处理8 Treatment 8 36.36 ± 24.05abcde 7.14 ± 7.14bc 31.25 ± 16.54ab 56.52 ± 23.01ab 56.00 ± 17.43abcd 处理9 Treatment 9 −27.27 ± 18.18de 0 ± 25.75bc 43.75 ± 10.83ab 60.87 ± 15.06ab 50.00 ± 17.43abcd 处理10 Treatment 10 36.36 ± 55.29abcde 35.71 ± 42.86abc 50.00 ± 51.16ab 60.87 ± 32.83ab 44.00 ± 27.45abcd 处理11 Treatment 11 −45.45 ± 24.05e −7.14 ± 21.43c 25.00 ± 28.64ab 30.43 ± 28.51ab 28.00 ± 25.72bcd 处理12 Treatment 12 100.00 ± 31.49ab 64.29 ± 14.29abc 87.50 ± 12.50a 82.61 ± 11.50a 88.00 ± 6.79a 注：不同小写字母表示每个月份不同处理组之间防治效果的差异（P < 0.05）。Note: different lowercase letters represent the differences in prevention and control effects between different treatment groups in each month (P < 0.05). 2.3 不同处理对黄栌枯萎病次年发病情况的影响
从2021年与2022年初始发病情况对比来看，对照组的无病样树数量保持不变，重度发病样树从0增加到3棵。大部分施药处理组的无病样树均在第2年增加，其中处理2、8和9增加最多（图3）。除此之外，处理8的重度发病样树均转为无病或轻度发病（图3）。2022年施药组初始病情指数均低于处理组，其中，处理8的病情指数最低，治疗效果最好（图4）。2022年初始病情指数与2021年最终病情指数相比，包括对照组在内，病情指数都有不同程度的降低。其中，处理6、7和8的病情指数显著降低（P < 0.01），治疗效果较好（图5）。综上所述，灌根枯草芽孢杆菌结合树干注射多菌灵与嘧菌酯复配的组合处理8在感病治疗方面表现最好。
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图 3 2021年初始（5月）发病情况与2022年初始（5月）发病情况对比热图数据基于病情分类的样树棵数。21-.2021年；22-.2022年；trt.处理。Data based on number of sample trees by disease severity. 22-, year 2022; 21-, year 2021; trt, treatment.Figure 3. Heat map of initial incidence in 2021 (May) vs initial incidence in 2022 (May)![]()
图 4 2021年初始病情指数与2022年初始病情指数对比Figure 4. Comparison of initial disease index between 2021and 2022![]()
图 5 2021年最终病情指数与2022年初始病情指数对比星号表示2021年最终病情指数与2022年初始病情指数之间有显著差异（*P < 0.05；**P < 0.01）。Asterisk indicates a significant difference between the final disease index in 2021 and the initial disease index in 2022 (*P < 0.05; **P < 0.01).Figure 5. Comparison between initial disease index in 2022 and final disease index in 20213. 讨 论
植物轮枝菌病害，特别是林间树木枯萎病防治，目前尚未有效的措施。本研究探索了土壤灌根和树干注射防治黄栌枯萎病的最佳方案。
树干注药技术对于维管束病害具有优势，因为药剂可以通过质外体迅速到达发病部位[23−24]，Mulè[24]研究表明了树干注射是防治橄榄黄萎病（Verticillium wilt of olive）的有效手段。之前的研究表明灌根也能有效防治黄栌枯萎病[12,19]。萎菌净（有效成分为枯草芽孢杆菌）和多菌灵对黄栌枯萎病病菌抑菌效果达到100%，并且田间药效试验显示，萎菌净和多菌灵对于黄栌枯萎病有一定防治效果[12]。此外，树干注射嘧菌酯与多菌灵对黄栌枯萎病有一定防效[19]。本研究在此基础上将两种施药方式结合，并且加入两种新药剂来探索黄栌枯萎病全年防治的可行性。
对照组校正病情指数最高，且对照组与处理6处于同一立地条件中，处理6的校正病情指数明显低于对照组，说明注射多菌灵与嘧菌酯能有效控制病害。灌根处理组在整年防治中均表现较好，其中灌根枯草芽孢杆菌的校正病情指数最低（图2A），并且灌根枯草芽孢杆菌在3组灌根处理中的初始病情指数最高，说明枯草芽孢杆菌能有效控制病害进一步扩散。灌根丙环唑处理在6月份的病情指数减小，表明丙环唑能在病害发生的上半年表现出良好的控制效果。注射组中的处理5防治效果不太理想，校正病情指数与对照无明显差异（P > 0.05），可能是由于处理5的立地条件较差。处理6的防治效果明显好于处理5，之前的林间试验[19]表明嘧菌酯与多菌灵有很好的防治效果。在组合处理中，处理12的校正病情指数最小，病情增长最缓慢，防治效果最好，处理7防治效果最差。此外，处理7、11在6月的病情指数增量要大于对照组，并且比具有同样灌根药剂的处理8和处理12防治效果差，说明树干注射多菌灵与咪鲜胺复配比树干注射嘧菌酯与多菌灵复配的防治效果差。由此可以看出，甲氧基丙烯酸酯类的嘧菌酯对于防治黄栌枯萎病更为有效。灌根处理4和组合处理12无病样树在2021年的校正病情指数比对照组低73.68%，这两个处理有同种灌根药剂丙环唑，说明灌根丙环唑能有效控制病情的加重，并且，与树干注射嘧菌酯与多菌灵复配结合后，防治效果更佳。
2021年防治效果较好的处理组：处理2、3、4、和12，在2022年均有良好的表现，甚至处理2在2022年的初始病情指数比2021年初始病情指数更小。尽管处理5的防治效果不理想，但仍能保持原本的状态，没有进一步加重。与2021年最终病情指数相比，处理6、7和8在2022年初始病情指数都有明显的降低，其中，处理8的初始病情指数最小。结果表明灌根枯草芽孢杆菌结合树干注射多菌灵与嘧菌酯复配的组合在感病治疗方面效果最好。
综上所述，灌根丙环唑和树干注射多菌灵与嘧菌酯复配组合在整年防治中效果最好，防效达到88%；灌根枯草芽孢杆菌与树干注射多菌灵与嘧菌酯复配组合在次年病情指数最低，感病治疗方面效果最好，并且在2021年防治效果方面也有不错的表现，防效达到56%。本研究验证了黄栌枯萎病全年防治的可行性，并且筛选出最优防治方案，对黄栌枯萎病综合防治技术体系的建立提供科学依据。同样的防治试验我们也在香山进行，并且这两组药剂组合也有较好的防治效果，表明本次试验筛选出的防治方案具有实际推广意义。
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图 1 4种植物叶片钾含量对钾添加（K）、氮添加（N）、磷添加（P）、氮磷同时添加（NP）、氮磷钾同时添加（NPK）的响应
^、*分别表示在P < 0.1、P < 0.05水平上处理与对照差异显著。^, * indicate that there is significant difference between the treatment and the control at the levels of P < 0.1 and P < 0.05, respectively.
Figure 1. Response of K concentration in leaves of four plants to potassium addition (K), nitrogen addition (N), phosphorus addition (P), simultaneous addition of nitrogen and phosphorus (NP), simultaneous addition of nitrogen, phosphorus and potassium (NPK)
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图 2 优势植物叶片钾含量与土壤理化性质之间的关系
AK. 土壤有效钾；AN. 土壤有效氮；AP. 土壤有效磷；ST.土壤温度；SM. 土壤湿度；^、*、**、***分别表示在P < 0.1、P < 0.05、P < 0.01、P < 0.001水平上差异显著。 AK, soil available potassium; AN, soil available nitrogen; AP, soil available phosphorus; ST, soil temperature; SM, soil moisture. ^, *, **, *** represent significant difference at P < 0.1, P < 0.05, P < 0.01, P < 0.001 levels, respectively.
Figure 2. Relationship between potassium content in leaves of dominant plants and physical and chemical properties of soil
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图 3 植物群落地上生物量对钾添加（K）、氮添加（N）、磷添加（P）、氮磷同时添加（NP）、氮磷钾同时添加（NPK）的响应
*表示在P < 0.05水平上处理与对照差异显著。地上生物量差值等于处理小区生物量减去对照生物量。* indicates that there is significant difference between treatment and control at the level of P < 0.05. The difference of aboveground biomass is equal to the biomass of the treatment plot minus the control biomass.
Figure 3. Response of aboveground biomass to the addition of potassium (K), nitrogen (N), phosphorus (P) fertilizer and their combined treatments (NP, NPK)
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图 4 植物群落地上生物量与土壤温度变化之间的关系
地上生物量差值等于处理小区生物量减去对照生物量；土壤温度差值等于处理小区土壤温度减去对照土壤温度。 The difference of aboveground biomass is equal to the biomass of the treatment plot minus the control biomass; the soil temperature difference is equal to the soil temperature of the treatment plot minus the control soil temperature.
Figure 4. Relationship between aboveground biomass of plant community and soil temperature
表 1 钾、氮、磷添加及其交互作用对叶片钾含量影响的三因素方差分析
Table 1 Three-way ANOVA of the effects of potassium, nitrogen and phosphorus addition and their interaction on potassium concentration in leaves
指标 Index 自由度
df垂穗披碱草
Elymus nutans发草
Deschampsia cespitosa无脉薹草
Carex enervis草玉梅
Anemone rivularis区组 Block 3 0.922 7 5.207 8* 4.479 2* 0.542 8 钾添加 K addition 1 0.346 0 4.455 6 5.747 7* 8.129 1* 氮添加 N addition 1 7.790 1* 55.423 1*** 12.582 4** 5.857 8* 磷添加 P addition 1 1.508 5 4.188 9 0.569 0 0.519 3 氮磷交互作用 N × P interaction 1 0.0115 0.011 7 1.188 7 1.464 2 氮磷钾交互作用 N × P × K interaction 1 1.698 9 0.534 9 1.002 8 0.512 6 注： *、**、***分别表示在P < 0.05、P < 0.01、P < 0.001水平上差异显著。Notes: *, **, *** represent significant difference at P < 0.05, P < 0.01, P < 0.001 levels, respectively. 
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