Notes
Uptake and transportation behavior of a new fungicidal agent LH-2010A in cucumber plants
2014 Volume 39 Issue 1 Pages 43-47
Details
2014 Volume 39 Issue 1 Pages 43-47
Pseudoperonospora cubensis (causal agent of cucurbit downy mildew), a biotrophic plant parasite belonging to oomycete, is one of the most economically important and widespread plant pathogens. Fluopicolide is an oomycete-specific fungicide with a novel mode of action, developed by Bayer CropScience. Fluopicolide has both systemic and translaminar activities.1,2) Fluopicolide exhibits excellent activity against oomycetes in vivo as well as antifungal activity in vitro; indeed, the ability is related to its translocation toward the stem tips via the xylem.3) Fluopicolide is believed to interfere with the functioning of spectrin, which is involved in stabilizing the cytoskeleton.4) It has been placed by the Fungicide Resistance Action Committee (FRAC) in a new mode of action group and shows no cross-resistance to other fungicides. However, fungicide resistance among oomycetes including P. cubensis has been reported frequently.5) Because the registration of fluopicolide is relatively new, few reports have described resistance to it in the field. However, descriptions about the laboratory-resistant strains of Phytophthora capsici that challenge the usefulness of this compound have been reported.6) Accordingly, fluopicolide may face a resistance risk.
In light of the threat that fungal pathogens pose to plant health and their developing resistance to more and more fungicides, the search for new antifungal agents remains an important field.7) LH-2010A, N-[3-chloro-5-(trifluoromethyl)pyridine-2-methyl]-2,3,5,6-tetrafluoro-4-methoxybenzamide, is a new fluorinated benzamide that was considered, along with fluopicolide, to be a leading compound and was synthesized by Shandong United Pesticide Industry Co. Ltd., China in 2010 (Fig. 1). Since little information is available about LH-2010A, the basic efficacy against P. cubensis and the uptake-translocate characteristic of LH-2010A in cucumber plants were investigated in this report.
LH-2010A (purity: 98%), fluopicolide (purity: 96%), thiram (purity: 95%) and metalaxyl (purity: 98%) were all provided by Shandong United Pesticide Industry Co., Ltd. China. Methanol (High Performance Liquid Chromatography, HPLC-grade) was purchased from TianJin Yongda Chemical Reagent Co., Ltd. Other reagents, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), sucrose, leucine and lysine, were all used at commercial analytical grade.
2. Preparation of the sporangia suspensionCucumber leaves bearing zoosporangia of the pathogen were collected from a suburb of Tai’san, Shandong Province, China. The backs of the diseased single leaves were washed with distilled water and then kept in moist petri dishes at 20°C for a 12-hr photoperiod until new mold emerged after 24 hr. The sporangia were washed off using sterile water, and a suspension with 1×104 sporangia/mL was prepared.8)
3. Determination of basic efficacy on P. cubensisThe floating leaf disc bioassay was adopted.9,10) Technical grade LH-2010A, fluopicolide and metalaxyl were used at 1.25, 2.5, 5, 10 and 20 µg/mL a.i. after dissolving in methanol and diluting with distilled water. Methanol in solution never exceeded 1%. Leaf disks (1 cm in diameter), cut with corkborer from healthy cucumber leaves, were placed on the surface of 10 mL fungicide solution in a petri dish, and the abaxial side of the disks was inoculated with a 10 µL sporangia suspension using a microPette plus (Eppendorf), 10 discs per dish and 5 replications per concentration. These were kept at 20°C in a light incubator for 7 days. Disease development on each leaf disk was recorded using the following rating: 0=no visible mildew development, 1=0 to 5%, 2=6 to 25%, 3=26 to 50%, 4=51 to 75%, and 5≥76% of the disk surface covered with mildew symptoms. Disease severity (DS) was calculated as [(5A+4B+3C+2D+E)/5(A+B+C+D+E)]×100, where A, B, C, D and E were the numbers of leaf disks corresponding to the ratings 5, 4, 3, 2 and 1, respectively. Control (%) of individual fungicides was calculated as [(DS on untreated leaf disks–DS on treated leaf disks)/DS on untreated leaf disks]×100.
4. Preparation of test solution for HPLC analysisCucumber seedlings (cv. Sylphy, 2nd to 3rd leaf stage) were used for each experiment. Twenty seedlings were applied to each concentration of the test chemicals on treatments (1), (3), (5) and (6) [mentioned below, including (2) and (4)], and all of them were sampled at the sampling time. One hundred seedlings were applied to each test chemical on treatment (2), and 20 of them were sampled at each sampling time. On treatment (4), 120 seedlings were applied to the experiment, and 20 of them were sampled at each sampling time of each cultivation temperature. Following 6 treatments were conducted:11,12) (1) Cleaned roots were soaked in a solution of 4 chemicals with different concentrations (100, 250, and 500 mg/L). Seedlings were cultured in an incubator at 24°C for 72 hr. Stems and leaves were taken as samples. (2) Cleaned roots were soaked in a 250 mg/L solution of LH-2010A and metalaxyl. At 4, 12, 24, 48 and 72 hr after the treatment, the roots, stems, or leaves were taken as samples. (3) A solution of the 4 agents with different concentrations (100, 250, and 500 mg/L) was sprayed on the leaves. When the agents covered the plant surface evenly and began to drip, the spraying was stopped. Roots and other parts were taken as samples at 72 hr after the treatment. (4) Cleaned roots were soaked in a 250 mg/L LH-2010A solution. Seedlings were cultured in an incubator (4°C or 24°C) for 12, 24, and 48 hr. Roots were taken as samples. (5) Cleaned roots were placed in a 10−3, 10−4, 10−5 mol/L CCCP solution for 1 hr, then soaked in a 250 mg/L LH-2010A solution at 24°C for 48 hr. Roots were taken as samples. (6) Cleaned roots were soaked in a 250 mg/L LH-2010A solution with 1 mmol/L sucrose, leucine or lysine at 24°C for 48 hr. Roots were taken as samples.
Samples successively cleaned with water, methanol and deionized water, were dried with filter paper and then cut into fine pieces. One gram of each sample was taken randomly and ground into a homogenate with 6 mL of methanol using an adjustable high-speed homogenate machine. The homogenate was moved into a centrifuge tube and sonicated for 10 min, then centrifuged at 4000 rpm for 5 min after standing for 2 hr. The supernatant was collected and filled up to 10 mL with methanol, and then filtered through a 0.45 µm microporus membrane filter.12) The final filtrate was obtained as a test solution for HPLC analysis. Each treatment had 3 replicates with at least 5 samples per replicate.
5. HPLC analysisHPLC analyses were performed with an Agilent HPLC system 1200 (Agilent, USA) consisting of a variable UV detector using a Diamonsil C18 column (250×4.6 mm i.d.; 5 µm particle size, Dikma, USA). The flow rate was kept at 1.0 mL/min and the injection volume was 20 µL. The mobile phases were methanol and water (80/20, v/v) for LH-2010A, fluopicolide and metalaxyl,13–15) and methanol and water (60/40, v/v) for thiram. The wavelengths of UV detection were 240 nm for LH-2010A, 225 nm for fluopicolide, 222 nm for metalaxyl and 310 nm for thiram.
A series of standard solutions with a concentration gradient of LH-2010A, fluopicolide, thiram and metalaxyl was prepared, and methanol was used as the solvent. The limit of detection (LOD) and the limit of quantitation (LOQ) of the 4 agents were determined. Standard curves with concentration of agents as abscissa and peak area as ordinate were established. In these HPLC conditions, the content of corresponding fungicide in the above-mentioned test solution was determined using the external standard method.
6. Determination of recovery rateThe recovery rate was determined according to the standard addition method.16) Leaves, stems or roots were cut into fine pieces, and 1 g samples were taken respectively. A standard solution of 4 agents was added to these samples separately before solvent extraction. Samples were prepared in triplicate and the recovery rates were determined.
7. Statistical analysisThe whole experiment was performed twice. The content of chemicals was shown as means±SD (µg/g F.W.). All statistical analyses were performed with SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). The linear regression analysis was adopted in a basic efficiency test of 3 chemicals on P. cubensis. Statistical differences were determined using a one-way ANOVA, followed by a Tukey’s honestly significant difference test for mean separation at p≤0.05 level.
The EC50 values of LH-2010A and fluopicolide were both much lower than those of metalaxyl (Table 1). This may be related to the resistance of P. cubensis to metalaxyl.17) Furthermore, EC50 of LH-2010A was 6.70 µg/mL and slightly lower than fluopicolide (7.67 µg/mL). It has been reported that fluopicolide is the most effective against downy mildew in the USA currently.18) LH-2010A showed the ability to control downy mildew in field trials. The application of fungicides was performed just after disease appearance in the summer of 2011 and 2012. The control effects of LH-2010A [375 g/ha (25% SC)] 10 days after spraying were no lower than 96%, while those of Infinito (fluopicolide 62.5 g+propamocarb-hydrochloride 625 g/L SC, 750 mL/ha, Bayer CropScience), a commercial standard for disease control, were around 96%. However, much deeper research on the control effects of LH-2010A in field trials is necessary, so those data are not shown in this paper.
| Chemical | Regression equation | R2 | EC50 (µg/mL) | 95% Confidence limits (µg/mL) | Multiple of the virulence |
|---|---|---|---|---|---|
| LH-2010A | y=−2.05+2.49x | 0.900 | 6.70a | 5.50–8.20 | 19.0 |
| Fluopicolide | y=−2.21+2.51x | 0.968 | 7.66a | 7.23–8.13 | 16.7 |
| Metalaxyl | y=−2.37+1.12x | 0.925 | 127b | 108–156 | 1.00 |
* Different letters indicate a significant difference at the p<0.05.
The linear relationships between the peak area (Y) and concentration of chemicals (X; µg/mL) were Y=26.4X+53.6 for metalaxyl, Y=39.1X−0.731 for fluopicolide and Y=24.7X+8.40 for LH-2010A. All R2 acquired were no less than 0.997. LOD was defined as the compound concentration that produced a signal-to-noise radio of approximately 3, and the values were 0.1 µg/mL for LH-2010A, metalaxyl and thiram, and 0.2 µg/mL for fluopicolide.19) The LOQ of the assay was evaluated as the concentration equal to 10 times the value of the signal-to-noise ratio, and the values were 0.3 µg/mL for LH-2010A, metalaxyl and thiram, and 0.6 µg/mL for fluopicolide.19) The LOQ on sample analysis was, therefore, 3.0 µg/g for LH-2010A, metalaxyl and thiram, and 6.0 µg/g for fluopicolide. The contents of thiram detected in all samples were lower than the LOD, so the standard curve of thiram was not shown. The recovery rates were summarized in Table 2. The recovery rates detected ranged from 80.7 to 96.8%, RSD<10%. The duration times for an analysis were 7, 8, 7 and 13 min for samples with LH-2010A, fluopicolide, metalaxyl and thiram, respectively, and the retention times were approximately 4.8, 4.1, 3.6 and 6.2 min, respectively.
| Chemical | Plant part | Spiked concentration (µg/g) | Recovery (%) | RSD (%) |
|---|---|---|---|---|
| LH-2010A | Roots | 100 | 92.1 | 8.8 |
| 25 | 87.6 | 9.6 | ||
| Stems | 100 | 88.9 | 3.5 | |
| 25 | 94.5 | 0.7 | ||
| Leaves | 100 | 96.8 | 2.9 | |
| 25 | 90.2 | 7.58 | ||
| Fluopicolide | Roots | 100 | 88.7 | 0.5 |
| 25 | 86.9 | 3.5 | ||
| Stems | 100 | 92.5 | 4.2 | |
| 25 | 96.8 | 2.5 | ||
| Leaves | 100 | 84.3 | 1.9 | |
| 25 | 89.2 | 20 | ||
| Metalaxyl | Roots | 100 | 82.7 | 0.8 |
| 25 | 85.6 | 2.4 | ||
| Stems | 100 | 84.3 | 3.2 | |
| 25 | 95.6 | 3.1 | ||
| Leaves | 100 | 92.4 | 0.7 | |
| 25 | 96.5 | 2.5 | ||
| Thiram | Roots | 100 | 96.0 | 3.9 |
| 25 | 85.9 | 5.5 | ||
| Stems | 100 | 84.6 | 6.1 | |
| 25 | 88.4 | 1.3 | ||
| Leaves | 100 | 96.8 | 2.6 | |
| 25 | 91.1 | 9.8 |
*Recovery (%): the ratio of content detected to addition RSD (%):relative standard deviation.
The results from treatment (1) were summarized in Fig. 2A. The concentrations of LH-2010A, fluopicolide and metalaxyl in stems and leaves showed a tendency that increased corresponding to treatment concentration. Analytical results from the sample of thiram were not shown in Fig. 2A because peaks were not found on the HPLC chromatogram in any of the samples. These results indicated that LH-2010A showed a systemic property via roots and it is the same as that of systemic fungicides fluopicolide and metalaxyl. In contrast, the non-systemic fungicide thiram cannot be absorbed by plant roots.
The results from treatment (2) were summarized in Table 3. LH-2010A and metalaxyl could be translocated to stems and leaves. LH-2010A and metalaxyl were detected in roots, stems and leaves after 4 hr. Concentrations of both chemicals in various parts of the plants increased significantly within 72 hr by continuous administration. There are some differences between LH-2010A and metalaxyl. 1) For LH-2010A, the concentrations in stems and leaves were still significantly lower than that in roots. However, for metalaxyl, in the first 24 hr, the concentrations in stems and leaves were significantly lower than that in roots. There is no significant difference in concentration among in stems, leaves and roots after 48 hr. The concentration in leaves was significantly higher than those in stems and roots after 72 hr. 2) For LH-2010A, the concentration in stems became higher than that in leaves after 48 hr. for metalaxyl, however, the concentration of metalaxyl in stems became lower than that in leaves after 48 hr. These differences may be because of the distinctions in enrichment sites among different agents.
| Chemical (conc. of soaking solution) | Plant part | Concentration of chemicals in samples (µg/g) | ||||
|---|---|---|---|---|---|---|
| Treatment period (hr) | ||||||
| 4 | 12 | 24 | 48 | 72 | ||
| LH-2010A (250 µg/mL) | Roots | 22.3bc±3.9 | 31.8cde±2.1 | 113.0h±12.2 | 48.8fg±12.3 | 53.4 g±8.8 |
| Stems | 5.3a±1.4 | 11.0ab±2.7 | 14.3ab±2.4 | 24.0bcd±1.6 | 42.7efg±7.2 | |
| Leaves | 4.1a±1.4 | 11.7ab±0.2 | 16.5ab±10.3 | 22.7bcd±2.1 | 36.0def±27.5 | |
| Metalaxyl (250 µg/mL) | Roots | 18.6C±3.3 | 37.6E±2.1 | 49.5F±11.5 | 48.7F±1.8 | 51.2F±8.8 |
| Stems | 6.2A±1.8 | 14.8BC±4.3 | 27.9D±2.0 | 45.8F±2.5 | 46.7F±2.5 | |
| Leaves | 7.1A±1.1 | 10.8AB±0.4 | 29.4D±6.3 | 49.9F±3.7 | 58.6G±5.0 | |
* Different letters indicate a significant difference at the p<0.05.
The results from treatment (3) were summarized in Fig. 2B. The concentrations of three chemicals in plant leaves and stems all increased with the augmenting of solution concentrations. It was illustrated that LH-2010A was absorbed by the leaves and stems of plants, as were systemic fungicides fluopicolide and metalaxyl. The detected levels of thiram in all samples were lower than the LOQ, so the data were not shown in Fig. 2B. LH-2010A was detected in roots 72 hr after being sprayed onto leaves and stems, and the concentration levels were ca. 3 (near the LOQ), 4.3±0.4, and 3.9±0.4 µg/g with 100, 250 and 500 µg/mL spraying of plants, respectively. There was no significant difference between the content detected. Metalaxyl was also detected in roots, and the levels were 6.7±1.2, 8.3±4.3, and 10.0±2.2 µg/g. This indicated that LH-2010A and metalaxyl were transported to the roots by plants.
No fluopicolide was detected to exceed the LOD (0.2 µg/mL) in roots. It was reported by the previous study that fluopicolide translocated toward the stem tips via the xylem but it did not translocate toward the roots.3) Some complementary studies applied to the main stem of vine or potato showed a redistribution of fluopicolide only in the upper part of the plants.20) In our experiment, fluopicolide was not detected in roots, in other words, it could not be transported to roots after absorption by leaves and stems. Our results were in agreement with report in the literature.
4. The effect of various factors on cucumber plants absorbing LH-2010AThe energy released by respiration is needed when plants absorb a solute actively, while energy is not needed in passive absorption.21) Respiration would be affected by low temperature or respiration inhibitors, thus, active absorption would be affected by them as well. When sucrose and amino acids enter the living cells of plants, protein carriers located in the plasma membrane are needed. Therefore, when plants absorb more than one compound actively, there will be a competition effect since the protein carriers must be shared.22)
The results from treatment (4) were summarized in Table 4. The absorption of LH-2010A was obviously affected by low temperature. The ratio of the absorption amount in 24 to 4°C at different times was still greater than 2.0. According to previous studies, this can be used to judge the absorption is associated with biological metabolism and the ratio of absorptive amount in 24 to 4°C is greater or lower than 2.0.23,24) Therefore, the absorption of LH-2010A was judged as associated with biological metabolism. The results from treatment (5) were summarized in Table 5. CCCP is one of the uncouplers belonging to respiration inhibitors. The absorption of LH-2010A was inhibited by CCCP, and the inhibitory rate (IR) increased with the augmenting of CCCP concentration. According to former research, the inhibition of CCCP to absorption can also help to determine whether the absorption is active.25,26) Specifically, the absorption of LH-2010A is associated with respiration. The results from treatment (6) were also summarized in Table 5. Visible inhibition by sucrose and two amino acids to the absorption of LH-2010A were all produced.
| Treatment period (hr) | Concentration of LH-2010A in roots (µg/g) | Ratio of concentration | |
|---|---|---|---|
| 4°C | 24°C | ||
| 12 | 3.2a±0.6 | 31.8b±2.1 | 9.9 |
| 24 | 4.0a±1.3 | 113.0c±12.2 | 28.7 |
| 48 | 6.0a±2.7 | 48.8d±12.3 | 8.2 |
*Different letters indicate a significant difference at the p<0.05. Ratio of concentration: the ratio of concentration in samples at 24 to 4°C.
| Chemical | Concentration (mol/L) | Concentration of LH-2010A in roots (µg/g) | IR(%) |
|---|---|---|---|
| CCCP | 10−5 | 9.8ab±1.6 | 79.9 |
| 10−4 | 5.4ab±1.9 | 89.0 | |
| 10−3 | 4.0a±1.6 | 91.9 | |
| Leucine | 10−3 | 12.9ab±2.6 | 73.5 |
| Lysine | 10−3 | 19.6b±1.6 | 59.8 |
| Sucrose | 10−3 | 18.3ab±4.7 | 62.4 |
| Control | — | 48.8c±12.3 | — |
* Different letters indicate a significant difference at the p<0.05. IR(%): inhibitory rate based on the Control. Concentration of LH-2010A for root soaking: 250 µg/mL. CCCP were pre-treated for 1 hr before soaking to LH-2010A solution.
The results of treatments (4)–(6) indicated that absorption via root of LH-2010A was associated with biological metabolism and needed the help of protein carriers. This suggested that absorption of LH2010 was performed in active transport.
This study was supported by Key Project of the National Twelfth-Five Year Research Program of China (2011BAE06B02) and Special Fund for Agro-scientific Research in the Public Interest (201003004).
