Fungicide labels often specify application intervals of 14 to 28 days for control of brown patch on creeping bentgrass. The two-week range affords superintendents flexibility in determining the most appropriate schedule for products they select and rates they apply. In general, the archive of published research shows that effective fungicides applied at shorter intervals provide better control, and that higher application rates allow longer intervals with satisfactory control. However, despite label specifications, there is an abundance of anecdotal evidence (accumulated outside of the experimental research setting) indicating that even the 14-day application interval does not always result in effective brown patch control.

杀菌剂说明书经常特别提示，每隔14-28天对本特草上的褐斑病进行施药1次。2周时间足以让总监们确定选择最合适的杀菌剂种类和用量。一般情况下，研究表明使用杀菌剂间隔时间越短药效越好；杀菌剂用量越高则间隔时间越长，且能保证药效。但是，虽然说明书是这样写的，仍有大量非正式证据显示（在非实验条件下），14天间隔周期并不能高效控制褐斑病。

Variability in fungicide performance is natural. It is influenced by several factors including application rate, water volume and environmental disease pressure (7). Also, fungicide residues are depleted over time, further affecting the expected duration of disease control. Intuitively, if a fungicide is applied today, then there will be more fungicide remaining with the turf tomorrow than three or four weeks from now. But is the decline slow and steady? Or, perhaps, do residue levels remain fairly high until the last few days?

杀菌剂药效衰变是自然而然的。这受以下几个因素的影响：施用量、用水量、病害强度。同时，杀菌剂残留量也会随着时间的延长流失，进而减少药剂对病害的控制时间。直观的讲，如果今天施用一种杀菌剂，那么3、4周之后将需要更多的用量来保证药效。但是，这种衰变是缓慢而稳定的吗？或者，也许药剂残留药效依然强，只是最后几天药效急速下降呢？

Some insight can be gained from research that focused on environmental fate of pesticides in turf during the 1990s and early 2000s (3,4,8,9). Several fungicides (for example, chlorothalonil, metalaxyl, propiconazole, triadimefon and vin- clozolin) were included in various studies, but the objectives centered only on the detection of residues.

从20世纪90年代至21世纪初关于草地使用杀虫剂对环境影响的研究中可以得到一些证据。一些杀菌剂（比如百菌清、甲霜灵、丙环唑、三唑酮和乙烯菌核利）也有类似研究，但是研究重点在于残留物的检测方面。

Figure 1. Residual efficacy (RE) proportions for (A) azoxystrobin; (B) flutolanil experiments 1, 2, 5 and 6; (C) flutolanil experiments 3 and 4; (D) metconazole; (E) polyoxin D; and (F) pyraclostrobin. Data were fit to a two-parameter Weibull function to describe the decline in efficacy over time. Experiments (listed by experiment number) were initiated (1) Sept. 17, 2010; (2) Oct. 12, 2010; (3) June 8, 2011; (4) June 22, 2011; (5) July 6, 2011; and (6) Aug. 26, 2011.

图1.残留效力（RE）的百分比：A-嘧菌酯；B-氟酰胺（试验1、2、5、6）；C-氟酰胺（试验3、4）D-叶菌唑；E-多氧霉素D；F-吡唑嘧菌酯。数据被拟合成2个参数的韦伯函数来描述随着时间的推移药力下降趋势。

Disease control concerns were not addressed and, therefore, several questions remained. Does fungicide protection really last for 28 days? How long can we expect excellent control? Can we define a point at which fungicide residues become so depleted that they are below concentrations that are toxic to the pathogen? Answers to these questions will help improve our understanding of what happens to our chemical protection between sprays and may help explain why effective fungicides work, and why sometimes they do not perform to our expectations.

病害控制时间问题没有涉及到，而且一些问题也仍然存在。杀菌剂药效能否真的持续28天？我们能期待药剂控制多久？我们能否找到一个点，即能杀死病原菌的最低杀菌剂浓度？这些问题的答案将帮助有助于我们理解化学药剂在喷洒过程发生了什么，也许能解释为什么说有效的杀菌剂有效，以及为什么有时喷施杀菌剂达不到我们预期的效果。

Our goal was to address these questions from two perspectives — a bioassay approach to learn how the decline in residues affects disease control, and an analytical approach to measure and quantify the amount of fungicide remaining at each sample date.

我们的目的是从两个角度解释这些问题—1是利用生物测定法学习药剂残留物的降低是如何影响病害防治的；2是通过分析检测并量化每个样本中的杀菌剂残留量。

Materials and methods 材料与方法

Bioassay生物检测

Five fungicides were tested: polyoxin D, azoxystrobin, pyraclostrobin, flutolanil and metconazole. Fungicides were applied to Penneagle creeping bentgrass (Agrostis stolonifera L.) maintained at 0.5 inch (1.27 centimeters) in replicated field plots at the Daniel Turfgrass Research Center on the campus of Purdue University in West Lafayette, Ind. For each run of the experiment, fungicides were applied to plots once at the rates listed in Table 1. Plots were sampled seven times at three to four-day intervals over a three-week period (days 0, 3, 7, 10, 14, 17 and 21) by removing cup cutter plugs from each replication of each treatment plot.

选择了5种杀菌剂：多氧霉素D、嘧菌酯、吡唑嘧菌酯、氟酰胺和叶菌唑。杀菌剂喷洒到Penneagle 匍匐剪股颖（剪股颖属）草坪上，剪股颖修剪高度0.5英寸（1.27cm）。田间实验地点为丹尼尔草坪研究中心，隶属于西拉斐特工业园区的普渡大学。每一轮试验中，试验小区杀菌剂施用量见表1. 小区取样原则是，在3周的时间内，用洞杯器每隔3-4天取样一次（即喷药当天、第3天、第7天、第10天、第14天、第17天和第21天）。

Bioassay experiments were run six times during the growing seasons of 2010 and 2011. Sampled plugs were inoculated with five grains of white sorghum seed infested with a locally collected isolate of Rhizoctonia solani (the organism that causes brown patch on creeping bentgrass) and then incubated for 48 hours in a growth chamber where relative humidity exceeded 95% and temperature was maintained at 82 F (27.7 C). After the incubation period, the diameter of each patch was measured and recorded. Residual efficacy (RE) was expressed as a percentage based on pathogen growth on fungicide-treated plugs relative to the growth on untreated plugs. By our calculation, RE = [1 − (growth on fungicide-treated plugs/ growth on untreated plugs)] * 100 .

2010-2011年的草坪生长季，生物检测共进行了6次。在用洞杯器取的草塞上接种5粒已感染立枯丝核菌（这种病菌可导致剪股颖患褐斑病）的白高粱种子（貌似这个意思），然后在相对湿度大于95%、温度在82F（27.7℃）的温室内培养48h。接种期过后，测量每个病斑的直径并且记录。残留药力表示为喷施杀菌剂后的草塞病原菌数量占未喷施杀菌剂的草塞病原菌的百分比。即RE=[1 - 喷药后病原菌存活数/未喷药病原菌数量]*100.

Quantitative residue analysis 残留量分析

During 2011, additional turf samples were collected from the same experimental plots of the bioassay to further investigate the persistence of the selected fungicides applied to creeping bentgrass under fairway conditions. Sampling followed the same schedule (day 0, 3, 7, 10, 14, 17 and 21) as the bioassay. Fungicide residues were identified and quantified by liquid chromatography/ time-of-flight mass spectrometry (LC/ TOF-MS). All fungicides were analyzed using this technique with the exception of polyoxin D. (The polyoxin D analytical standard could not be secured in 2011 and is therefore absent from the quantitative residue analysis). Data from LC/ TOF-MS followed first-order kinetics and were fit to a model that describes fungicide depletion against time (6).

2011年另一批染病草样本从相同的实验小区中取得并进行生物监测，用于研究这几种杀菌剂的持久性，此时剪股颖处于球道级养护水平下。样本采集时间与去年取样时间一样（喷药当天、第3天、第7天、第10天、第14天、第17天和第21天）。杀菌剂残留通过液相色谱法进行检测，通过飞行时间质谱分析法测定数量。除多氧霉素D之外，其他杀菌剂均采用这种技术进行分析（由于多氧霉素D的分析标准不能保证2011年可以使用（大概这个意思），因此没有出现在残留量分析中）。LC/TOF-MS测定结果符合一级动力学，并且能够建立杀菌剂衰变与时间反相关的模型。

Results 结果

Bioassay 生物监测

Bioassay data were pooled for the six experiments (runs) for all fungicides except flutolanil. Results of preliminary statistical analysis required flutolanil experiments to be analyzed in two distinct groups.

生物监测数据集合了所有杀菌剂的6次（轮？）实验，除了氟酰胺。初步统计分析结果显示，氟酰胺试验结果差异比较大，更适合作为2个不同的群组单独分析。

Damage caused by brown patch, a foliar disease of cool-season turfgrass, reduces the aesthetic value of golf course turf and has a negative impact on playability.
Photo by R. Latin

褐斑病造成的破坏，该病是冷季型草的一种叶部病害，会降低高尔夫草坪的美观价值，并且严重影响草坪击球乐趣。 照片由R.Latin 提供。

For all fungicides, a sigmoidal or backward-Sshaped depletion curve resulted when residual efficacy was plotted against time. In order to describe the decline with a model that could capture both inflection points of the curve, we used a twoparameter Weibull distribution function (1). The Weibull-generated curves (Figures 1A-1F) clearly illustrate the decline in residual efficacy and allow calculation of interesting statistics (for example, efficacy half-life, which is the number of days after application that control was reduced to 50%).

对于所有杀菌剂来说，以时间为横坐标，残留量为纵坐标可形成S形曲线或反向的S形曲线。为了给残留量建立一个模型，且能够同时表现曲线的2个拐点，我们使用了双参数的韦伯分布函数。（貌似数学中的求导？）韦伯分布产生的曲线（见图1中的A-F）清晰的展示了残留效力的衰减变化，而且还能进行其他有趣的统计分析（例如药效半衰期—即药物含量降低到最初含量的50%的天数）。

All fungicides effectively controlled brown patch on the first sampling date (day 0), but differed in terms of residual efficacy in subsequent samplings. By the last sampling date (21 days after fungicide treatment), fungicide protection was virtually nonexistent for all treatments.

所有杀菌剂都能在使用当天有效的控制褐斑病，但是在后期的样本中残留药效表现不同。最后一次取样（即喷药后第21天），所有杀菌剂对草坪的保护作用几乎可以忽略不计。

Statistical differences were observed among mean efficacy half-life values with means ranging from 3.1 to 14 days (Table 2). Based on efficacy half-life values, flutolanil provided a longer period of protection. Pyraclostrobin, metconazole and azoxystrobin had statistically similar efficacy half-life values. Azoxystrobin and pyraclostrobin, which have the same biochemical mode of action (they both are QoI) but differ in terms of phytomobility (azoxystrobin is an acropetal penetrant and pyraclostrobin is a local penetrant), did not exhibit any statistically significant differences based on efficacy half-life. The two fungicides classified as local penetrants, polyoxin D and pyraclostrobin, differed with regard to their levels of brown patch control based on efficacy half-life values of 3.1 days and 5.2 days, respectively (Table 2).

据统计药效半衰期平均为3.1-14天（表2）.根据药效半衰期值，氟酰胺可以提供较长的保护期。吡唑嘧菌酯、叶菌唑和嘧菌酯在统计上显示出相似的半衰期。嘧菌酯与吡唑嘧菌酯有相同的生化活动模式（他们都是呼吸抑制剂），但是他们在植物体内流动方式不同（嘧菌酯属于顶向传导渗透，吡唑嘧菌酯则是定向渗透（不太懂，太专业了）），他们的半衰期在统计学上没有显著差异。多氧霉素D和吡唑嘧菌酯是两种经典的定向渗透杀菌剂，他们在防治褐斑病的差异表现为药效半衰期分别为3.1天和5.2天。

Quantitative residue analysis 残留量分析

Figure 2. Fungicide concentrations in creeping bentgrass verdure for (A) azoxystrobin, (B) flutolanil, (C) metconazole and (D) pyraclostrobin.

图2. 匍匐剪股颖叶片中的杀菌剂含量，A-嘧菌酯，B-氟酰胺，C-叶菌唑，D-吡唑嘧菌酯。

The QuEChERS multi-residue method provided acceptable chromatographic results that allowed for repeatable detection and quantification of fungicide residues in creeping bentgrass over a wide range of concentrations. Initial recoveries (day 0) varied greatly among the fungicides (Table 2). This variation most likely reflects differences in application rates (Table 1). The average amounts of fungicide detected on day 0 for azoxystrobin, flutolanil, metconazole and pyraclostrobin were 127, 1,433, 128 and 200 micrograms/ gram verdure. All fungicides examined with quantitative analysis demonstrated a precipitous decline following application (Figure 2A-2D).

QuEChERS多残留量测定法提供可接受的色谱分析结果，即允许重复检测并且在一个比较宽的浓度范围内可量化杀菌剂在剪股颖组织内的残留量(不懂)。不同杀菌剂防治初期（即喷药当天）差异非常显著（表2）。这种差异在施用量上反映最明显（表1）。喷药当天杀菌剂平均用量为，嘧菌酯127微克/克，氟酰胺1433微克/克，叶菌唑128微克/克，吡唑嘧菌酯200微克/克。所有杀菌剂的检测与定量分析显示，喷施杀菌剂之后会有一个急剧下降的过程（图2A-D）。

QuEChERS（Quick、Easy、Cheap、Effective、Rugged、Safe），是近年来国际上最新发展起来的一种用于农产品检测的快速样品前处理技术，由美国农业部教授2003年开发的.

Discussion讨论

Differences in residual efficacy 残留药效的不同

Results reported here illustrate how fungicides are rapidly depleted from turf after each application. Bioassay curves were remarkably similar, except for flutolanil, which was applied at a much higher rate (five to 10 times more active ingredient) relative to the other fungicides, accounting for the inconsistency. For azoxystrobin, metconazole, polyoxin D and pyraclostrobin, residual efficacy was reduced to 50% within three to five days.

研究结果说明杀菌剂在每次喷洒后如何从草坪上快速地耗尽。除了氟酰胺生物测定曲线非常相似。相对于其他杀菌剂，氟酰胺的使用量更大（活性成分的5-10倍），导致了氟酰胺试验结果的不一致。对于嘧菌酯、叶菌唑、多氧菌素D和吡唑嘧菌酯，残余疗效在3-5天之内减少到50%。

Results of previously published fungicide trials describe a much longer duration of brown patch control when using an effective fungicide (2). In these reports, brown patch was evaluated using field trials where climatic fluctuations can lead to varying degrees of disease pressure. Increased duration of brown patch control in these reports could be a result of slight changes in environmental conditions, which can affect disease development and are not directly related to fungicide persistence in the turf. An advantage of the described bioassay is the ability to create high disease-pressure conditions consistently over the course of the study. Maintaining consistent disease pressure over the course of 21 days in the field is probably not likely in most areas where creeping bentgrass fairways are common.

以前公布的杀菌剂试验结果认为当使用一种有效杀菌剂时控制褐斑病的持续时间会更长。在这些报告里，评估褐斑病是通过田间试验，而田间的气候波动压力会导致不同程度的病害。这些报告中所提到的增加褐斑病防治时间可能是由于环境条件—能够影响病害扩展而不直接影响草坪杀菌剂持久性—轻微变动的结果。生物测定结果的一个优势是能够在研究的过程中始终维持高病害压力条件。在田间长达21天的过程保持一致的病害压力在大部分区域是不可能的，特别是常用剪股颖做球道的区域。

The field research took place in replicated field plots at the Daniel Turfgrass Research Center on the campus of Purdue University in West Lafayette, Ind. Plots were sampled seven times by removing cup cutter plugs from each replication of each treatment plot. Photos by J. Daniels

田间试验发生在印第安纳州的普度大学西拉法叶校区丹尼尔草坪草研究中心的试验小区。从每个处理小区的每个重复里用开洞器取7个样。照片由j•丹尼尔斯拍摄

Another possible explanation for differences in residual efficacy between bioassay results and reported field observations is that bioassay turf plugs were inoculated with a fresh culture of Rhizoctonia solani after each sampling date. Fresh inoculum is typically not reintroduced in the same manner in fungicide field trials. This is important given the phytomobility characteristics of the fungicides used in the present study.

生物测定结果和以前报道的田间观察之间残余疗效差异的另外一种可能解释是生物测定草皮块是在每次取样日后再接种新鲜的立枯丝核菌。新鲜培养液一般不会采用像杀菌剂田间试验同样的方式被再次引入。考虑到本研究中使用的杀菌剂的phytomobility特点，这是很重要的。

Penetrant fungicides have the ability to suppress existing fungal infections and reduce endemic inoculum. The resulting suppression in pathogen growth achieved by an effective fungicide creates a lag period during which the surviving pathogen population slowly recovers to the point of symptom expression. Repeatedly introducing inoculum during the bioassay may have negated the impact of this lag period and resulted in the shorter duration of brown patch control.

内吸性杀菌剂能够抑制现存真菌感染和减少本地接种体。达到抑制病原体生长的结果是通过一种有效的杀菌剂产生一段滞后期，在这个滞后期内幸存的病原体数量慢慢恢复到出现症状时的数量。在生物测定中反复引入培养液可能否定了这段滞后期的影响，导致控制褐斑病的间隔期变短。

Is thatch a fungicide reservoir? 枯草层是杀菌剂的储蓄地吗？

Fungicide not intercepted by leaves and stems collects in the thatch. Given the affinity of fungicide for the highly organic thatch layer, it is reasonable to ask whether it serves as a fungicide reservoir, thereby increasing the duration of control in the field. In our bioassays, three of the fungicides (azoxystrobin, flutolanil and metconazole) are acropetal penetrants. They are readily absorbed by roots and transported through xylem to leaves. If thatch served as a reservoir, then the bioassay and quantitative analysis would reflect the resupply of fungicide into the verdure.

杀菌剂不会被枯草层里的茎叶拦截。鉴于杀菌剂对高有机质枯草层的亲和力，问这个问题就比较合理了—枯草层是否作为一种杀菌剂储蓄地，由此在田间增加了药效的持续时间。在我们的生物测定中，有三种杀菌剂（嘧菌酯、氟酰胺、叶菌唑）是向顶传导。它们迅速被根系吸收，通过木质部运输到叶片上。假如枯草层作为一个储蓄地，那么生物测定和定量分析就会反映出杀菌剂重复补给到绿色植物。

A more plausible explanation is that pathogen growth stops after active mycelia encounter and absorb fungicide associated with thatch. Like the situation where penetrant fungicides suppress fungal growth inside leaf tissues, inoculum levels in the thatch are likely reduced to the point where many days pass before pathogen populations resume growth sufficient to cause symptoms.

更可信的解释是，活跃菌丝接触和吸收枯草层上的杀菌剂后导致病原体停止生长。就像内吸杀菌剂在叶片组织内抑制真菌生长，枯草层里的接种体水平可能减低到病原体种群恢复生长到足够多并引起症状发生的这个点时许多天已经过去了。

Sampled plugs were inoculated with five grains of white sorghum seed infested with a locally collected isolate of Rhizoctonia solani (the organism that causes brown patch on creeping bentgrass) and then incubated for 48 hours in a growth chamber.

草塞样本被接种5粒已感染立枯丝核菌的白高粱种子（这是导致剪股颖患褐斑病的病原菌），然后在温室中培养48小时。

Fungicide rates and classes 杀菌剂用量与种类

In this research each fungicide was applied at a single rate, representing the high rate prescribed on the product label. Our interest emphasized dissipation of different classes of fungicides rather than effects of application rates. Also, we could not evaluate all possible products or active ingredients in this research. The selected fungicides include representatives of important classes (especially QoI and DMI) for brown patch control.

这次实验中每种杀菌剂都施用了一种剂量，即产品说明书上的最高用量。我们的侧重点是杀菌剂种类而不是杀菌剂喷施量。同样，实验中我们也没有评估到全部可能的杀菌剂产品或者活性成分。所选择的的杀菌剂包括了防治褐斑病的杀菌剂的重要类别的代表（特别是Qo呼吸抑制剂和甾醇脱甲基抑制剂）。

Given that residual efficacy is largely a function of microbial degradation over time, there is no reason (or existing evidence) to suggest that other fungicides within these classes would show a significant departure from the depletion curves shown here. Since microbial degradation is a function of temperature, the rate of depletion is expected to lessen during extended periods of low temperature (5).

鉴于药效残留主要是微生物降解随着时间的变化函数，，没有理由（或者说没有证据）认为这几类的其他杀菌剂品种将会表现出一个较大偏差。由于微生物降解也是一个温度函数，杀菌剂降解速率预测在延长的低温条件下会降低。

Interpretation 解释/扩展

The bioassay results, supported by analysis of fungicide residues in turf leaves and stems, clearly illustrate the rapid depletion of fungicide from turf beginning shortly after application. This pattern should be expected for all fungicides applied to turf. Results from this study help explain why fungicide treatments may not perform as expected during a given application interval and underscore the importance of applying fungicides in a timely manner for satisfactory disease control. Preventive applications that are delivered prematurely may dissipate before the arrival of environmental conditions that foster pathogen growth and promote disease outbreaks, leaving plants vulnerable to damage. The risk of disease-related damage as well as the cost associated with treatment reinforces the need to make informed management decisions concerning fungicide use. Superintendents should try to anticipate outbreaks and apply effective fungicides before disease development in order to achieve maximum fungicide performance, but they must also be aware of the rather short duration of effective residues in turf.

在草坪草叶片、茎秆上的杀菌剂残留量分析同样支持生物检测结果，清晰地说明杀菌剂的急速下降始于喷药后不久。这种模式预计适用于所有应用于草坪的杀菌剂。这个研究结果有助于解释为在一个给定的间隔时间（说明书上标出的间隔时间）内为什么杀菌剂没有达到预期效果，同时也凸显了为达到满意的病害防治效果，及时的施药是多么重要。预防性喷药如果过早，可能造成药效在利于病菌繁殖、病害爆发的环境条件来临之前散失，此时植物极易被病害破坏。由病害引发的破坏风险以及病害防治成本，强调了制定与杀菌剂使用有关的管理决策的必要性。草坪总监们应该尝试预测病害爆发时间，在病害扩展期施用有效的杀菌剂，这样才能使杀菌剂作用发挥到最大。但是他们也应该意识到草坪上的杀菌剂残留药效持续时间相当短。