Examination of Acephate Absorption, Transport, Metabolism, and Accumulation in Maize After Root Irrigation for Spodoptera Frugiperda Control

BACKGROUND: Since the invasion of the fall armyworm moth (Spodoptera frugiperda) in China in January 2019, damage to maize crops has gradually intensi�ed, and chemical control has become the main control measure. This study aimed to examine methods of effective pest control while monitoring the environmental impact of pesticide use. The effectiveness of S. frugiperda pest control by foliar spraying and root irrigation of maize plants with acephate was determined, and the absorption, distribution, and dissipation of acephate and methamidophos by maize were studied. RESULTS: Field trials showed that acephate treatment at 6000 g.a.i ha -1 was the most effective for controlling S. frugiperda. Acephate and methamidophos were absorbed from the roots, transported upward, and concentrated in the leaves, particularly new leaves. The terminal residues of acephate and methamidophos in maize grains were below detectable levels at 60 days after treatment. CONCLUSIONS: The results demonstrate that acephate treatment via root irrigation can more effectively control the infestation of S. frugiperda in maize than acephate treatment via foliar spraying. The translocation and distribution of acephate and methamidophos by root irrigation were more uniform, and the holding e�ciency was higher than those in foliar spraying, suggesting an extended period of control e�cacy. This pest control method could be utilized to reduce pesticide residues while safely and e�ciently controlling S. frugiperda infestation.

cabbageworm. It is a suitable replacement for highly toxic insecticides such as methamidophos (Tavares et al., 2010). Presently, few reports have been published on the safety and effectiveness of acephate for root irrigation.
The objectives of this study were to compare the e cacy of acephate in S. frugiperda control through root irrigation and foliar spray. Additionally, the dissipation and residues of acephate and methamidophos in maize were investigated. Acephate and methamidophos concentrations in maize roots, leaves, and stalks were quanti ed to evaluate and compare the residual effects of the insecticides. The results of this study will project methods for S. frugiperda pest control in Chinese maize production.
Analytical grade acetonitrile, methanol, ammonium acetate, and anhydrous magnesium sulfate were obtained from Chengdu Jinshan Chemical Reagent Co. (Chengdu, China). Analytical grade sodium chloride was purchased from Beijing Green Science and Technology Development Company (Beijing, China). Primary secondary amine (PSA) sorbent, ODS-C 18 , and 0.22-µm nylon syringe lters were obtained from Agela Technologies Inc (Shanghai, China).
Individual stock standard solutions of acephate (100 mg mL -1 ) and methamidophos (100 mg mL -1 ) were prepared in methanol and stored in volumetric asks at -18 ± 1 °C. Mixed standard solutions of acephate and methamidophos were prepared by diluting stock solutions with methanol to a concentration range of 0.01~20 mg mL -1 . A matrix-matched standard calibration was achieved by applying appropriate volumes of standard solutions to untreated maize and soil. All standard solutions were stored at 4 °C until use. agricultural season (May to July). Throughout the experimental period, average ambient temperature in Guangzhou was 30 °C, average photoperiod was 12 h d -1 , and mean monthly precipitation was 1500 mm. In Jinan, average ambient temperature was 25 °C, average photoperiod was 10 h d -1 , and mean monthly precipitation was 800 mm. The soil type in Guangzhou was loam with 6%-10% organic matter at pH 5.0 ~ 6.5. The soil type in Jinan was clay with 3%-7% organic matter at pH 7.0 ~ 8.5.

Field experimental design
Field trials at each site involved three treatment and one control, the experiment was arranged as a randomized complete block design with three replications. The experimental area measured 240 m 2 , encompassing 12 treatment plots, 20m 2 each and separated by buffers to avoid cross-contamination. Planting density was approximately 120 maize plants each plot.
For the root irrigation, each plant in each plot received 100 mL acephate solution (4500 L ha -1 ). For the foliar spray treatments, plants in the plots were sprayed with 1.2 L acephate solution (600 L ha -1 ) using an electric knapsack sprayer (single nozzle with 0.15-0.4 Mpa, 4-5 m width). The control was the foliar application of water without insecticides.

Surveying method
Before irrigation, each plot was surveyed via ve-point sampling. Ten maize plants were selected at each point, the number of live S. frugiperda were counted on the front and back of the leaf at the base of the plant and the culm at the junction of the leaf base and the stalk. Subsequently, the same sampling method was used to count the numbers of live S. frugiperda on the front and back of the leaf at the base of the plant and the culm at the junction of the leaf base and the stalk at 1, 3, 5, 7, 15, 20, and 30 days after root irrigation. After stimulation with a camel hair brush, immobile insects were considered dead. Based on the collected data, the observed mortality for each treatment at each sampling day was calculated using the pest reduction rate. Subsequently, corrected mortality for the corresponding treatment at the sampling time was calculated using the control effect. Since the corrected mortality included the control treatment in the control effect, it excluded the mortality caused by the other factors. The pest reduction rate and the control effect were calculated as follows (Ly et al., 2021): Pest reduction rate (%) = [(Initial number of live S. frugiperda −number of live S. frugiperda after treatment)/ Initial number of live S. frugiperda] ×100 Control effect (%) = [(Pest reduction rate in treatment area − pest reduction rate in the control area) / (1 -pest reduction rate in the control area)] ×100.

Field sample collection
The ve-point method was used to collect and analyze the roots, stalks, leaves, and soil after 1, 3, 5, 7, 14, 20, and 28 days of treatment. The leaves of maize plants were divided into two groups: (1) old leaves: the rst to the sixth leaves from the base of stalk, and (2) new leaves: the top most four leaves(growing and recently expanded young leaves). Representative soil samples (30 g) were collected from 0−10-cm depth at ve points. Stones and grass were removed. Three replicates per sample were collected. All samples were stored at -18 ± 1 °C until analysis.

Sample extraction and puri cation
Collected maize plants were divided into four parts: new growth, old growth, roots, and stalks. For leaf, root, and stalk analysis, 5.0 g of plant matter was placed into each 50 mL polypropylene centrifuge tube. For soil analysis, 10.0 g samples were used. Forty mL of methanol was added to each tube and homogenized for 1 min. The mixtures were ultrasonically extracted for 40 min, followed by the addition of 3.0 g anhydrous MgSO 4 and 2.0 g anhydrous Na 2 SO 4 to each tube. The samples were then mixed for 2 min at a speed of 2500 r/min and centrifuged at 5000 r/min for 5 min. Subsequently, 1 mL of the supernatant solution of each sample was extracted by pipette and transferred into 2 mL centrifuge tubes containing 50.0 mg of C 18 and 50.0 mg of PSA. The tubes were centrifuged at 1200 r/min for 3 min after vortexing for 1 min.

Instrumental determination
A mass spectrometry instrument electrospray ionization interface was operated in positive ionization mode. The mobile phases were methyl alcohol (A) and 10 mmol L -1 ammonium acetate aqueous solution (B). The mobile phase gradient program was as follows: 20% A was linearly reduced to 15% over 1 min, linearly increased to 90% over 2 min, and then held at 90% for 4 min ( Table 1). The ow rate was 0.20 mL min -1 , and the column temperature was 30 °C. The capillary voltage was 3000 V, source and desolvation temperatures were 150 and 350 °C, respectively, cone gas ow rate was 50 L h -1 , and desolvation gas ow rate was 700 L h -1 . Multiple reaction monitoring with a cycle time of 500 ms was used to detect each analyte. Monitored masses were optimized for each analyte and are shown in Table 2.

Data analysis
The dissipation dynamics of acephate and methamidophos are governed by the rst-order kinetic equation C t =C 0 e −kt and the half-life equation To compare the differences in the uptake and translocation of pesticides, the root concentration factor (RCF) and translocation factor (TF) were computed. RCF describes the uptake capability of a compound by plant roots from the soil, calculated as RCF = C root /C soil , where C root and C soil are the concentrations (mg kg −1 ) of one compound in plant roots and the soil, respectively(Zhang, Chen, Ni, Zhang, & Liang, 2009). An RCF value >1 indicates a strong ability of a compound to enter plant roots from the soil(Y. Li et al., 2018). TF describes the ability of a plant to transfer a compound from the roots to the shoots and is de ned as TF = C shoot /C root (

Effect of acephate applied by root irrigation and foliar spraying on S. frugiperda
The application of acephate in the root zone could provides an extended period of control e cacy (Table 3). There were signi cant differences in the effects of acephate at dosages of 3000 g.a.i ha -1 and 6000 g.a.i ha -1 through the root zone in the control and prevention of S. frugiperda.
Root irrigation of acephate at 3000 g.a.i ha -1 resulted in a control effect of 75.38% after 7 days, decreasing to 26.05% after 30 days of application. The control effect of the 6000 g.a.i ha -1 application reached a maximum of 91.18% after 7 days of application, 82.66% after 30 days.
With different application methods, acephate showed different effects on S. frugiperda. The foliar spray application of acephate had a rapid prevention and control effect. The control effect of 1120 g.a.i ha -1 acephate by foliar spraying reached 77.63% on day 1, peaked at 90.45% on day 5, and decreased to 43.84% on day 7. The control effect of the foliar spraying application lasted for approximately 1-5 days and decreased rapidly after the seventh day.The control effect of root irrigation was diametrically opposite to that of foliar spray application. The control effect gradually improved after root irrigation and signi cantly improved on 5 days. The maximum control effect of root irrigation with acephate was 91.18% on day 7. It is still 82.66% on day 30, lasting signi cantly longer than the effect of foliar spray application.
3.2 Distribution of acephate in maize roots, stalks, and leaves through root irrigation Degradation curves of acephate and its degradation product methamidophos in roots, stalks, and leaves of maize from the Guangzhou and Jinan regions are shown in Fig. 1.

Acephate and methamidophos concentration in new and old leaves
The concentration of acephate and methamidophos at 6000 g.a.i ha -1 in new and old leaves after application in Guangzhou and Jinan are presented in Fig. 2. Accumulated acephate and methamidophos levels in the new leaves were higher than those in the old leaves after 3 days of root irrigation in Guangzhou and 5 days in Jinan. The quantity of acephate absorbed by the root system and transferred to the new leaves was higher than that transferred to the old leaves. Fig. 3 shows the translocation rates of acephate from soil to root, root to stalk, and stalk to leaf by root irrigation. The RCF of acephate ranged from 0.04 to 0.9 and from 0.22 to 2.22 in the Guangzhou and Jinan regions, respectively, indicating uptake. The transferability of acephate from soil to roots, roots to stalks, and stalks to leaves initially increased after application and then decreased gradually. The transfer rates of acephate and methamidophos from stalks to leaves were approximately fteen-fold higher than those from soil to roots and roots to stalks.

Discussion
This is the rst study to examine the effectiveness of acephate application via root irrigation to control S. frugiperda infestation on maize.
In China, S. frugiperda is mainly controlled by chemical pesticide sprays (Yan et al., 2019). The results of this study showed that applying acephate via root irrigation can control S. frugiperda more effectively than foliar spray application. Although foliar application resulted in a high control effect in a short period, the acephate that was conducted and transported to the maize leaves and stalks through systemic function was low, the pesticides lasting validity period was shorter than that of root irrigation. In addition, root irrigation exhibited a higher control effect on S. frugiperda, and its longer pesticides lasting validity period reduced the necessity of repeated insecticide applications. The exact timing of chemical application is essential for effective pest control; both the pest life cycle and the time of the day matters. For S. frugiperda, larvae deeply embedded in maize whorls and usually come out to feed at night, to get good protection of maize, farmers need to spraying big amount of pesticide in the whorl of maize plant at the dawn and dusk. However, excessive application leads to a series of problems such as resistance development, environmental pollution, pesticide residues, and ecological balance disruption (Abou et al., 2018). (Hamm & Hare, 1982;Viana & Costa, 1998;Xla et al., 2020). Through root irrigation, acephate was absorbed and transported to different parts of the plant quickly. After application, acephate was absorbed by the roots and transported upwards. A dynamic transport pattern was observed between the different parts of the plant, and the variation in acephate contents throughout the plant provides an effective re ection of insecticide absorption, digestion, and transportation. Differences in initial uptake between locations can be explained by environmental differences or user variation.

Results concur with literature indicating that root irrigation applications of systemic insecticides can effectively control crop pests
Acephate concentration was the highest in the leaves, likely because it was absorbed from the roots, transported upward, and stored in the leaves. At 3 weeks after the application to the root, acephate content in the leaves remained high, indicating that the acephate continuously passed through the xylem upward. The contents of acephate and its metabolites in the new leaves were higher than those in the old leaves. This is probably because the life activity of upper leaves is vigorous with a fast metabolism, and it also had a large conduction amount and fast conduction speed; therefore, the content absorbed by the root and transported to the new leaves was signi cantly higher than it was in the old leaves. Muthuri et al. found that top leaves and young, expanding leaves had higher transpiration rates, which brought more absorbed acephate to these leaves. In contrast, there were little acephate residues in young, growing maize leaves using foliar spray application(Han et al., 2017; Muthuri, Ong, Craigon, Mati, & Black, 2009). S. frugiperda is a chewing pest that prefers to ingest phloem sap from young leaves rather than old shoots; the higher acephate content in these parts leads to a higher insect mortality rate(D. G et al., 2003).
Root irrigation effectively controlled S. frugiperda for 30 days, compared to 5 days when applied through the foliar spray. The sustainability of acephate within plants minimizes the problems associated with foliar spraying, and the process of applying pesticides is safe for non-target organisms and is less affected by the application time. While a difference in the half-lives of acephate and methamidophos between the two locations was observed, it can be explained by soil and climate differences. Physical and chemical factors, such as light, heat, pH, and moisture, play vital roles in pesticide degradation in plants(L. Li et al., 2012).
The nal residues of acephate and methamidophos in maize grain and soil were determined to assess the safety of this pesticide. A 0.6 mg kg -1 acephate residue was detected in the soil after 28 days. No acephate and methamidophos residues were detected in the soil and maize grains after 60 days at the two experimental sites. These results demonstrate that acephate via root irrigation will produce no pesticide residues during the maize growth period, and the sustainability of acephate inside plants minimizes the problems associated with foliar spraying. The dissipation rate of acephate and methamidophos in Jinan was higher than that in Guangzhou, a difference attributed to discrepancies in soil temperature, moisture, pH, and microorganism content, as well as light conditions. Soil pH, in particular, has a signi cant effect on the digestion of acephate, which is faster in alkaline soils. Climatic conditions with high net radiation promote the digestion of acephate and methamidophos in crops(Yen, Lin, & Wang, 2000).

Conclusion
Root irrigation of maize with acephate was shown to control S. frugiperda more effectively than foliar spraying. Acephate was most concentrated in the leaves, increasing insect mortality based on the feeding preferences of S. frugiperda larvae. The application of acephate insecticide via root irrigation was observed to effectively control pests while reducing environmental impact. This pest control method could be utilized to reduce pesticide residues while safely and e ciently controlling S. frugiperda infestation.

Declarations
Ethics approval and consent to participate No approval of research ethics committees was required to accomplish the goals of this study because experimental work was conducted with an unregulated invertebrate species.

Consent for publication
Not applicable for this section.

Availability of data and materials
All data generated or analysed during this study are included in this published article (and its supplementary information les).       Absorption levels of acephate and methamidophos in 6000 g.a.i ha-1 acephate-treated soil after irrigating root in Guangzhou(a) and Jinan(b).

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