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Article

Improved Management of Verticillium Wilt in Smoke Trees Through the Use of a Combination of Fungicide and Bioagent Treatments

by
Yize Zhao
1,
Ruifeng Guo
1,
Bo Zheng
2,
Fei Yuan
2,
Xi Song
2,
Mengfei Zhang
3,
Jinzi Guo
3,
Kexin Liu
3,
Weijia Liu
3,
Xiaoran Zhou
3,
Ying Ren
3,
Zhihua Liu
4,
Xinpeng Zhang
5 and
Yonglin Wang
1,*
1
Beijing Key Laboratory for Forest Pest Control, College of Forestry, Beijing Forestry University, Beijing 100083, China
2
Municipal Forestry and Parks Resource Conservation Center, Beijing 100013, China
3
Beijing Fangshan Forest and Fruit Technology Service Center, Beijing 102400, China
4
College of Forestry, Shenyang Agricultural University, Shenyang 110866, China
5
Beijing Lanhu Natural Enemy Technology Company Limited, Beijing 100089, China
*
Author to whom correspondence should be addressed.
Forests 2025, 16(6), 914; https://doi.org/10.3390/f16060914
Submission received: 20 March 2025 / Revised: 24 May 2025 / Accepted: 25 May 2025 / Published: 29 May 2025
(This article belongs to the Special Issue Forest Pathogens: Detection, Diagnosis, and Control)
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Versions Notes

Abstract

:
Smoke tree (Cotinus coggygria) is an important component of the urban landscape and represents red-leaf scenery in Beijing; however, Verticillium wilt, caused by Verticillium dahliae, has caused high mortality of smoke trees. Traditional control methods, such as chemical root irrigation and trunk injection, are problematic due to environmental pollution and potential plant damage. This study aimed to explore effective prevention and control methods for Verticillium wilt of smoke tree across different regions of red-leaf scenery in Beijing. In 2023, 240 smoke trees from the Pofengling Park of Beijing were selected for the study. Four different fungicides, a plant growth regulator and a biocontrol agent were tested. Three application methods (root irrigation, trunk spraying, and a combination of both) were used in the different trials. Based on the results of the 2023 trial, control trials were conducted under the disease classification in 2024 at key red-leaf scenery regions, such as Xiangshan Park, Xishan Park, and Pofengling Park. The bioagents of Bacillus subtilis root irrigation combined with the trunk spraying treatment group showed the best disease control effects. Calculated by the change in disease index in the treatment and blank groups, the corrective control effect in the treatment group reached 104.55%, and 60% of the plants remained healthy, indicating that this method of disease control was the most effective. Propiconazole root irrigation also had a significant effect on diseased smoke trees. Furthermore, validation experiments conducted in 2024 confirmed that various combinations of root irrigation and trunk spraying provided strong preventive and therapeutic effects on Verticillium wilt. In conclusion, the graded control measures demonstrated effective control of wilt at different disease index grades. This study offers an effective and practical solution for controlling Verticillium wilt, benefiting both environmental sustainability and landscape health.

1. Introduction

The smoke tree (Cotinus coggygria), a species of the genus Cotinus in the family Anacardiaceae, is an important urban landscape tree in Beijing. Verticillium wilt is a soil-borne disease caused by the erticillium V. dahliae [1], which causes wilting of branches and leaves and potentially resulting in the death of the entire smoke tree [2]. The pathogen infects plants through the roots and spreads through the vascular system [3]. The pathogen’s resilience and ability to survive in the soil make its prevention and control highly challenging [4], leading to significant damage to landscape plants [5]. V. dahliae has a broad host range in China, affecting species such as maple [6], cotton [7], watermelon [8], grape [9], kiwi [10], and olive [11].
According to the official statistics of Beijing, the total smoke tree area in Beijing is 251.66 km2, of which the mixed forest area is 248.53 km2 and the pure forest area is 3.13 km2. The total incidence area of smoke tree Verticillium wilt in Beijing is 11.67 km2, which is mostly distributed in point-like form, of which the incidence area of mixed forests is 11.27 km2 with an incidence rate of 4.52%, and the incidence area of pure forests is 0.4 km2 with an incidence rate of 13.45%. There were differences in the incidence rate of wilt in different areas: 62.5% in Xiangshan Park, 40.29% in Badaling Park, 36.18% in the National Botanical Garden (North Park), 30.00% in Pofengling, Fangshan District, and 14.29% in Baiwangshan Forest Park. There are four primary strategies for controlling Verticillium wilt: artificial, physical, biological, and chemical control [1,12,13]. These strategies are often implemented in two phases. The first involves preventive measures such as soil sterilization before planting to prevent disease onset [4,14]. Studies have shown that a broad host range allows the pathogen to persist in weeds [15]. Dicotyledonous weeds as hosts of V. dahliae are a key method of controlling the spread of the pathogen by creating a nursery free of host weeds. In addition to these methods, establishing a favorable growing environment and implementing proper cultivation management practices are crucial in reducing disease incidence [16]. The second is therapeutic interventions, which involve the use of chemical and biological treatments after the disease has emerged [17,18,19].
The pathogen’s resilience stems from its long-lived microsclerotia in soil and systemic colonization of plant vasculature [1]. Irrigation water is a source of V. dahliae conidia in some areas, and the viability of Verticillium conidia was reduced by up to 100% in irrigation water treated with appropriate concentrations of agents [20]. Traditional control relies on chemical fungicides or soil fumigation, but these methods face limitations: environmental toxicity, non-target effects, and inefficacy against established vascular infections [4,21]. Trunk injections of fosetyl-Al and benomyl were effective in controlling Verticillium wilt on olives, with no wilted plants appearing at the end of the trial in some treatment groups [22]. In terms of forest chemical control, the combination of propiconazole root irrigation and trunk injection of carbendazim and prochloraz offers the most effective control for Verticillium wilt; the disease index decreased by 1.39 in 2022 and 1.84 in 2023 [23]. Bacterial communities have an effect on the severity of soil-borne diseases [24], and different belowground microbial communities result in different plant disease resistance [25]. Notably, biological control methods, such as the application of Bacillus subtilis, have shown promise as effective probiotics against Verticillium wilt and are considered ecologically sustainable [26,27]. The study showed that the abundance of beneficial bacteria, such as Bacillus subtilis, in the root soil of healthy smoke trees was more than that of diseased smoke trees. This suggests that the stability of healthy plants depends on beneficial bacteria [28]. Based on the study that chemical and biological agents can work together in the soil to provide a significant killing effect on Verticillium wilt [29], this suggests that the use of chemicals can improve their control of the disease in the context of creating a favorable microbiological environment.
Despite the advantages of each of these strategies in Verticillium wilt control, their practical application faces the following key limitations. In the 2021 Badaling trial, chemicals alone or in combination with trunk injection treatments were more effective in treating severely diseased trees, with a corrected disease index ranging from 0 to a decrease of 18.75, but were still insufficient for treating mild disease and preventing infection [30]. Similarly, in the 2021 Xiangshan trial, chemical root irrigation combined with trunk injection was close to as effective as a blank control group in treating mildly diseased plants. The corrected disease index in the 2023 control trial at Badaling was 13.64 compared to 1.58 in Xiangshan, reflecting the instability of the biological agents when irrigated with roots [23]. Secondly, in terms of application techniques, trunk injection can cause mechanical damage to seedlings with small diameters, which may negatively impact their survival rate. Additionally, the labor and machinery costs associated with this treatment are high. On the other hand, a single-root irrigation method has shown limited efficacy in combating pathogens within the vascular bundles [21].
To address these limitations, the present study aimed to optimize application methods by combining root irrigation with trunk spraying and conducting inter-forest control experiments for Verticillium wilt throughout the growing season. The goal was to identify the most effective application techniques and agent combinations for varying grades of disease, thereby providing valuable technical support and a range of treatment strategies for the integrated and graded control of Verticillium wilt.

2. Materials and Methods

2.1. Information on Experimental Sites

Pofengling Park is located in the southern part of Fangshan District, with geographic coordinates of approximately 39°40′ north latitude and 116°00′ east longitude. The region is classified as having a typical temperate monsoon climate, with an average annual temperature of 10 °C. The soil type is mountain loam with loose soil and high organic matter. The sample site was at an altitude of about 200 m. The soil moisture of Pofengling Park is medium and wetter. The average annual rainfall of Pofengling is around 600 mm. Xiangshan Park is situated in the southwestern part of Haidian District, with geographical coordinates of 39°59′ north latitude and 116°10′ east longitude. This region falls within the temperate monsoon climate zone, with an average annual temperature of 12 °C. The soil type is mountain loam, with heavily compacted soils and low organic matter. The sample site was at an altitude of about 150 m. Xishan is located in Haidian District, at 39°58′ north latitude, 116°11′ east longitude. This region falls within the temperate continental monsoon climate zone, with an average annual temperature of 10 °C, and is characterized by its distinctive mountain ecosystems. The soil type is mountain loam, with heavily compacted soils containing more gravel and lower organic matter content and an altitude of about 250 m. Xiangshan Park and Xishan Park usually have low, mildly dry soil moisture, with wetter soils for a period of time after precipitation. The average annual rainfall of Xiangshan and Xishan is around 550 mm. The pH of the sample soils ranged from 7.5 to 8.5. Sample trees in all plots are spaced 2–4 m apart with no visible cover above the sample trees.

2.2. Agents Information and Application Methods

B. subtilis water suspension (Hebei Zhongbao Green Crops Science and Technology Co., Ltd., Langfang, China, active ingredient content: ≥100 billion spores/g, diluted 1000 times); 156 g/L propiconazole (Dress Green) emulsifiable concentrate, a triazole fungicide produced by Syngenta Agrochemicals Co., Shenzhen, China (diluted 3000 times); 50% azoxystrobin (EG) water-dispersible granules, a methoxyacrylate fungicide, also produced by Syngenta Agrochemicals Co., Shenzhen, China (diluted 3000 times); tebuconazole (aqueous emulsion, active ingredient content: 25%) from Shenzhen Novozymes Agrochemicals Co., Ltd., Shenzhen, China (diluted 3000 times); 45% prochloraz, produced by Suzhou Fumeishi Plant Protection Agent Co., Ltd., Suzhou, China (diluted 3000 times); and sodium nitrophenolate (aqueous emulsion, active ingredient content: 1.4%), from Guilin Jiqi Biochemical Co., Ltd., Guilin, China (diluted 3000 times).
For root irrigation, 30 L buckets were used for the rooting operation, fish scale pits of the same size were used (50 cm in diameter and 15 cm in depth), and 4–5 application pits were dug near the periphery of the fish scale pits (10 cm in diameter and 20 cm in depth). The treatment was added slowly to control the full penetration of liquid medicine and ensure that there was no surface runoff of rooting liquid. Trunk spraying used a backpack electric sprayer (model 3WBD-20 L). To carefully prepare the appropriate agent, the agent must be mixed with the volume of the barrel in equal proportion. The agent was sprayed evenly over the main part of the trunk, from the base to the first branch. It is important to ensure that the entire trunk is sprayed, taking care to avoid spraying the leaves. The spraying was done in such a way that each part of the trunk was evenly sprayed (as long as the trunk was fully covered with the chemical and became wet). The spraying distance was fixed at 30 cm, the spraying nozzle used was a fan-shaped atomizing nozzle, the flow rate was controlled at 100 mL/min, and all the spraying operations were conducted at a 45° angle to the tree trunk. The time of application was chosen in weather without rainfall, avoided high temperatures at noon, and the interval between applications was more than 3 weeks. For the blank group, the roots were irrigated with an equal amount of water, and the trunk sprays were sprayed in the same way. It was previously determined that 30 L of water reaches a depth of 40 cm in a pit with a diameter of 50 cm for root irrigation, while the distribution range of young roots of 10–15-year-old smoke trees is within 5–40 cm below the ground. Uniformly spraying each square meter of bark under the recommended application concentration treatment requires roughly 500 mL of liquid; for the 10–15-year-old smoke tree’s bark surface area (roughly 0.38 m2), it can be rounded to the nearest 200 mL after calculation.

2.3. Experimental Design

The field trials were implemented in June 2023. We divide the entire test environment into a number of small, relatively uniform environments and then set up a complete set of treatments within each small environment. Non-treatment factors, such as environmental conditions, are easier to control in smaller lots. Non-treatment factors, such as soil variability, can, therefore, be controlled across the entire experimental environment on a wide range of sites. As a result, we conducted the experiment based on a completely randomized design. A total of 12 distinct treatment regimens were evaluated under uniform environmental conditions. The trial site featured flat terrain with footpaths bordering the woodland, where the sampled trees were situated. The woodland was characterized by a monoculture of smoke trees (Cotinus coggygria) with no more tree species, though weed vegetation was present. A balanced distribution of tree age and diameter at breast height was ensured across treatments by limiting randomization. Each treatment consisted of four replicated plots, each containing five trees, resulting in 20 trees per treatment. All sample trees were surveyed, recorded, tagged, and numbered. As confirmed by the planting files, all the sample trees were around 13–15 years old, and the diameter at breast height (DBH) size of the sample trees ranged from 7 to 13 cm. There were no obvious diseases occurring in the sample plots, with mild aphid infestation occurring in June. The trials were conducted once per month from June to November, with an interval of more than three weeks between applications. Controls were treated with equal amounts of water, and individually applied chemical root irrigation and trunk sprays were divided into five treatments. The remaining treatments were different combinations of root irrigation and spraying, totaling six groups. The same agent was used at the same dosage and dilution when applied individually and in combination; 30 L of solution was applied per rooting, 200 mL per plant for spraying, with rooting and spraying applied at the same time. Rainy periods were avoided, and no additional management measures were applied during the entire trial period. Refer to Table 1 for a detailed experimental design (Table 1).
In 2024, a design trial was conducted based on the previous year’s control results, with about 400 sample trees selected from each of Xiangshan Park, Xishan Park, and Pofengling Park. The trial used a completely randomized design to set up the sample plots and ensure that the sample trees met uniform environmental conditions. The total number of plants in the treatment was about 300, and the blanks were about 100, with the goal of field-testing the feasibility of the disease classification method. The trees were next to pavements and were planted individually with smoke trees; all sample trees were surveyed, marked, and documented. The experiment began in April. Treatments were once per month, with each application interval > 3 weeks. The blank group had an equal amount of water treatment. The treatments were divided into three types of Grades I; II, III; IV, V for agent application and did not use additional management measures. Disease classification guided treatment allocation: Grade I (asymptomatic) received preventive bioagents; Grades II–III (early symptoms) combined bioagents with low-dose fungicides; Grades IV–V (severe wilting) used systemic fungicides. Monthly surveys dynamically adjusted treatments based on disease progression. The detailed experimental design is shown in Table 2.

2.4. Evaluation of the Control Effect

A disease survey was conducted once per month, and the representative value of the disease after each treatment was recorded; the disease index and corrective control effects of Verticillium wilt were calculated according to the formula. The disease index of the initial month of each year was used as the base value of the disease index of that year, and the corrected disease index and corrective control effect of the following months were calculated. Finally, the advantages and disadvantages of the prevention effect were evaluated. Disease severity was scored according to the following criteria: I = No wilting of leaves (representative value is 0); II = 0 to 25% of leaves show disease (representative value is 1); III = 25% to 50% of the leaves show the disease (representative value is 2); IV = 50% to 75% of the leaves show the disease (representative value is 3); V = More than 75% of the plant’s leaves wilted, turned yellow, or the whole plant died (representative value is 4).
I D = 0 n 0 + 1 n 1 + 2 n 2 + 3 n 3 + 4 n 4 4 n × 100
I C D = I D − I 0
P 1 = ( I C D 0 − I C D i ) / I C D 0 × 100
n0 to n4 are the number of plants under the corresponding disease grade, and n is the total number of plants investigated; ID is the disease index; IDt is the t-th disease index; I0 is the initial disease index; ICD is the corrected disease index; ICD0 is the corrected disease index of the blank group; ICDi is the corrected disease index of treatment group i; and P is the corrected control effect of the treatment group is calculated as the change in disease index in the blank group.

2.5. Statistical Analysis

In this study, Microsoft Office Excel 2019 was used to organize the data; GraphPad 9.5 and IBM SPSS 27 software were used to statistically analyze the different treatments to correct the disease indices and control effects. All continuous variables were first verified for normality by the Shapiro–Wilk test, and if the p < 0.05, it was shown to conform to a normal distribution. We performed one-way ANOVA using SPSS software and multiple comparisons using the least significant difference (LSD) method (p < 0.05), and the Duncan test was used to calculate the significance (p < 0.05).

3. Results

3.1. Effect of Different Treatments on Verticillium Wilt Disease Index

Based on the disease index values collected over 6 months in 2023, the groups displayed similar levels of Verticillium wilt disease index in June. However, significant differences emerged in the changes in disease indices over the following months. Specifically, the disease index of the blank group consistently increased, reaching a peak of 40.00 in November (Figure 1). By contrast, disease indices for the treatment groups peaked in August or September (except for treatments 3 and 11), declined during September and October, and remained lower than that of the blank group by November. From June to November 2023, the blank group, along with treatments 3 and 11, showed a continued increase in the disease index even after reaching their peak. Treatment 11 exhibited the highest corrected disease index at 18.75 (Figure 1), whereas other treatment groups demonstrated a gradual decline in disease after peaking. Notably, the combination of treatments 6, 7, and 8 had the lowest corrected disease indices of −1.25, 1.25, and 3.75, respectively (Figure 1), with the difference in the corrected condition index between treatment 6 and the blank group being statistically significant (p < 0.05; Table 3). Treatments 1, 4, 5, and 10 yielded smaller corrected disease indices, indicating that the combination of treatments 6, 7, and 8 was particularly effective in reducing morbidity, while the combination of treatments 1, 4, 5, and 10 helped to slow disease progression to some extent.
Overall, most of the treatment groups demonstrated a reduction in disease index, suggesting a decrease in morbidity and indicating a positive control effect. Among the treatments, treatments 6 and 7 showed the highest corrected control effects, with smoother changes in disease index, reaching 104.55% and 94.76% corrected control effects, respectively (Table 3). Furthermore, the corrected control effect of treatment 6 differed most significantly (p < 0.05) from the blank group (Table 3). Following this, root treatment 1 and combination treatment 8 were also effective. Treatment 11 showed the poorest control effect, followed by treatment 3. These results suggest that the combination of B. subtilis root irrigation and trunk spraying with tebuconazole and sodium nitrophenolate was most effective in reducing the disease index and achieved the best control results.

3.2. Effectiveness of Treatments in Controlling Different Wilt Severity

Most of the smoke trees in this study were Grade I or II–III; hence, the analysis focused on these three categories. The findings revealed that half of Grade I plants in the blank group developed disease, and 62.5% of the smoke trees progressed from Grade II–III disease to more severe disease. Treatments 1, 6, 7, 8, and 9 were more effective in preventing disease onset in Grade I plants, with over 80% remaining healthy. Furthermore, treatment 1 and the combination of treatments 6 and 8 were more effective in treating diseased plants (Grade II–III). Treatment 1 had no incidence of disease above Grade III, whereas combinations 6 and 8 restored 25% of Grade II–III diseased plants to Grade I, and combinations 7 and 9 had very low incidence of disease above Grade III (Table 3). In addition, treatment 2 was more effective in treating diseased plants with no increase in the year-end disease index.

3.3. Protective and Preventive Effects Shown by the Treatment Group in the Following Year

Comparing the disease onset between June 2023 and June 2024, treatment groups applying Bacillus subtilis root irrigation (treatments 1, 6, and 7) had delayed onset of wilt in the second year (Figure 2). Treatment 6 showed the greatest reduction in disease index and the most significant preventive effect. None of the other treatments outperformed the blank group in terms of disease index, and the number of diseased trees and severity of disease generally increased. By contrast, treatments 1, 6, and 7 were more effective in managing disease, with a decrease in the number of affected trees in all groups.
In addition to disease control, the inclusion of sodium nitrophenolate in the spray treatment demonstrated a growth-promoting effect while simultaneously controlling disease progression, thus enhancing the tree’s resistance and limiting disease spread. The picture showed that smoke trees exhibiting wilt symptoms in 2023 displayed robust growth in the second year, with no noticeable signs of disease (Figure 3).

3.4. Application of the Control Measurements

Next, an integrated control technology system was developed for varying disease levels in Xiangshan Park, Xishan Park, and Pofengling Park. Among them, the disease is more serious in Xiangshan Park, with a portion of Grade III, IV, and V diseased plants. Most of the plants in Xishan Park are Grade I and II, with only a small portion of Grade III or above-diseased plants, and the incidence of the disease in Pofengling is the lightest, with very few Grade III or above diseased plants. Based on the incidence in the control group, the sudden rise in morbidity was concentrated in two periods, the first in June–July and the second in September. Specifically, the incidence rates of the applied groups in all three regions were significantly lower than those of the control group, with the incidence rate in Xiangshan reaching a maximum of 19.68 percent in July, followed by a maximum decrease of 32.77 percent in August (Figure 4A). Compared with the control group, which showed an increase in the incidence rate in June and September, the incidence rate of the Xishan Park treatment group gradually decreased and was lower than the initial incidence rate, and the incidence rate decreased to a minimum of 4.26% in July (Figure 4B). The incidence rate of Pofengling Park was more stable in one year compared with the control group, which had a sudden rise in June and September, and the incidence rate of the treatment group was under stable control. Over the course of the year, the disease index rose gradually in all area control groups and, like morbidity, had two peaks in June, July, and September (Figure 4C). Overall, with the exception of a brief rise in the disease index at Xiangshan Park (July, 6.69; Figure 4A), there was a steady decline in the disease index for all treatment groups compared to the initial condition disease index. By the second peak of the disease index (September), the disease index disease was reduced to 2.79 for Xishan Park (Figure 4B) and 2.34 for Pofengling Park (Figure 4C), suggesting that the treatment strategy had achieved effective prevention and control.
Compared with the control group, the different disease levels of each treatment group showed significant reduction after application of the drug, and it showed good therapeutic effects during the red leaf period (Figure 5). The most significant decrease was in the Grade IV–V disease index group, where the Xishan Park treatment decreased by 35, followed by the Pofengling Park treatment decrease of 33.33, and the Xiangshan Park treatment decrease of 32.5 (Table 4). In the disease Grade II–III, Pofengling treatment showed the most obvious therapeutic effect, with the disease index decreased by 20.59, Xishan Park treatment decreased by 18.26, and Xiangshan Park treatment decreased by 11.8 (Table 4). In terms of the whole-year disease index of Grade I plants, although there was an increase in the treatment group, it was much less than the increase in the disease index of the control group.

4. Discussion

In this study, to address the challenges in controlling Verticillium wilt in mountainous areas, we employed continuous application strategies tailored to the changing condition of the trees, aiming to determine the optimal combination of agents and treatment methods. The primary challenge in controlling Verticillium wilt is that most traditional control agents act on pathogens in the soil and are less effective in the tree’s vascular bundles. While trunk injection is an effective control method, the labor-intensive process becomes challenging when dealing with a large number of diseased trees or in rugged terrains. To address these limitations, this study adopts a dual-pronged approach. Firstly, trunk spraying can effectively deliver the treatment to the vascular bundle with minimal damage to the tree, yielding substantial results. In this study, systemic chemicals (tebuconazole, miconazole) and auxiliaries (sodium nitrophenolate) were selected, while two non-systemic agents, namely carbendazim and azoxystrobin, were excluded [31,32,33,34]. Sodium nitrophenolate has a growth-promoting effect on plants [35]. It increases plant growth [36] and has a synergistic effect on chemical agents [37].
Secondly, root irrigation is an effective and direct treatment that helps the agents reach the root level of the plant, thus aiding microorganism colonization before the disease develops. Chemical fungicides effectively kill the disease in the soil after the disease is susceptible. B. subtilis can be a beneficial bacterium between plant roots [18]. Volatile organic compounds such as salbutamol and 1,3-propanediol produced by B. subtilis promote plant growth [38]. By secreting lipopeptides, B. subtilis has a direct effect on fungal pathogens and remodels the inter-root environment, thus favoring plant growth [39,40]. In addition, B. subtilis reduces pathogens through competition for iron carriers [41], as well as activating plant disease resistance through abscisic acid (ABA) [42]. Propiconazole is a class of pesticides effective in controlling fungal diseases [43,44]. Root irrigation with propiconazole has been shown to be effective in controlling wilt diseases [23]. The direct treatment of soil at the root level is an effective method for the invasion of soil-borne pathogens through the root system. Delivery of the agent to the vascular bundles through trunk spraying to inhibit the spread of the pathogen in the plant is a synergistic measure. Based on these methods and agents, the main objective of this trial was to identify effective control measures for managing Verticillium wilt by testing different application methods and combinations.
Throughout the year 2023, the blank group at Pofengling Park showed the highest disease index, with the disease index continuing to rise. By contrast, the treatment group demonstrated effective mitigation of the disease. In the root treatment group, B. subtilis was the most effective treatment, significantly delaying disease progression and preventing healthy plants from becoming infected. Treatment 2 effectively controlled the deterioration of already diseased plants. When applied alone, spray treatments showed significant effectiveness in maintaining tree health and preventing further disease, although some mild deterioration was observed in already infected trees. The combination of B. subtilis root irrigation with trunk spraying yielded the best therapeutic and preventive results. The corrected control effect amounted to 104.55%, which indicates that the treatment group exerted a better control effect compared to the growth of the disease index in the blank group. It shows that complementary gains can be achieved by using a biochemical synergistic approach [45], and it promotes the stabilization of the colonization and control effects of biocontrol bacteria [29]. Treatments 8 and 9 also demonstrated effective control, consistent with the findings of previous studies [23]. In comparison, treatment 11 was less effective, with the highest corrected disease index of 30 for diseased plants—substantially higher than that with other treatment combinations—suggesting that azoxystrobin fungicides combined with trunk spraying are less effective for treating Verticillium wilt. We have ensured that application results are reliable through consistency of application methods and randomization of tree sampling. However, there were some errors due to minor environmental differences, and in future trials, we will focus on analyzing the effects of these factors (e.g., soil moisture, soil fungal differences) on the results and increase the sample size to make the results more rigorous.
Among the more effective treatment groups (treatments 6, 7, 8, and 9), tebuconazole proved slightly more effective than prochloraz. Tebuconazole is more systemic, effectively curbing disease development, while prochloraz is somewhat less effective. Both fungicides act by inhibiting fungal sterol biosynthesis [34,46]. No disease exacerbation was observed after tebuconazole spraying, while occasional disease progression was noted in the prochloraz-treated group. Overall, combining B. subtilis root irrigation with trunk spraying significantly enhanced disease control, particularly in treatments 1, 6, and 7. Compared with the previous trial conducted at Xiangshan Park, the corrective disease index changed from 17.5 in the first year to 1.47 in the third year after successive B. subtilis applications, suggesting that successive supplementation is required for the effects of the biocontrol agent to be manifested. Propiconazole showed similar results after successive rooting, whereas trunk injection combined with rooting did not show better control in the first year; instead, trunk spray combined with rooting showed a corrected control of −1.25 in the first year. The extension trial in Xiangshan Park showed a better control effect in the first year, which proved this point.
In the second year of the trial in Pofengling Park, treatments 1, 6, and 7 showed a decrease in the disease index, with treatment 6 exhibiting the greatest reduction. By contrast, treatments 8 and 9 showed little change from the first year. This indicates that the combination of B. subtilis root irrigation with trunk spraying was highly effective in preventing Verticillium wilt in the second year.
The May 2024 survey showed that the three trial areas had the starting disease incidence and disease index of the treatments, and the control showed a significant gap; the reason for this gap relies on the application of agents in April. Because the disease symptoms are not obvious, the application in April is based on B. subtilis as the main rooting agent, and this measure played a role in creating a good microbial environment, which effectively reduces the chances of infection of the plant by pathogenic bacteria in the soil and the ability of the soil pathogens to infect the plants. The success of the B. subtilis root irrigation treatment highlights the advantages of integrating biological control with chemical applications [47]. This approach, based on precise control according to disease severity, not only improves control efficiency but also aligns with the green control goal of reducing pesticide use while increasing efficacy.
Since the entire growing season of smoke tree is between April and October, the pathogenic bacteria in the soil will repeatedly infest the plants when the time is ripe [48,49], so to address this characteristic, the application cycle is synchronized to between April and October. Given the complexity of the microbial environment in the soil [50], the biocontrol of bacteria can sometimes be unstable, failing to provide significant preventive effects [23]. Therefore, it is essential to adjust the application of the agents according to disease progression. Wilt is classified into five types, which are difficult to distinguish in practical applications, so the disease grade is divided into three levels. Each level of the disease grade is closer to the other, which can simplify the procedure under the premise of higher accuracy and simultaneously reduce the field workload. Although propiconazole plays a better role in disease control, the economic cost of its control is high, and long-term use of chemicals may lead to pathogen resistance and soil microbial imbalance, whereas biologics reduce ecological risks [51]. Therefore, biological agents were selected for priority use. When the disease spreads further and aggravates, it is difficult to maintain the role of the previous biological fungi rooting, so for the II–III grade, fungicide spray combined with agent rooting has the best control effect. For the IV–V grade, it was concluded that the fungicide propiconazole combined with trunk spraying of the fungicide should be selected; this was based on the plant roots of the microbial selection [28], combined with the control role of the fungicide rooting in the 2023 Pofengling Park trial. Previous studies have shown that propiconazole compounded with B. subtilis can exhibit excellent antifungal effects and wilt control in plates and the field. Therefore, we attempted to selectively apply the agent to smoke trees exhibiting severe disease at the time of root irrigation, thereby creating a synergistic effect of killing the disease [29].
In the 2024 targeted application trial, although there was no decrease in the disease index at the end of the year, there was at least a 47% slowdown in the disease index at the three sites for Grade I-affected plants compared to their respective controls, suggesting that the preventive effect of the microbials irrigation of the roots in April was also evident. The disease index of the plots receiving year-round treatment (starting in April) was significantly reduced at the first peak of disease onset. The reason for analyzing the highest control effect of the Xishan Park treatment in July was that the soil in the area was infertile, and the addition of growth-promoting agents, probiotics, and water enhanced the tree’s growth potential and resistance to the disease, thus showing a strong control effect [52]. July is the month with the most precipitation in Beijing, and according to the Beijing Precipitation Report, the three sample sites had similar precipitation in July, and the temperature in Pofengling Park was about 2 °C lower than that in the other two sample sites. The morbidity and morbidity index increased in both the blank group and the treatment group in Xiangshan Park in July, and they were significantly different from those in the treatment group (p < 0.05). The condition of the treatment group in Xishan Park worsened in August, possibly due to the fact that the soil in Xishan is sandy and has poor water-absorbent properties, leading to a posterior effect of precipitation. It indicates that high temperature and humidity favor the development and deterioration of wilt disease and will weaken the effect of treatment application. In Pofengling Park, the sample plot was well drained, and the average temperature was lower than in other areas, so the treatment group was less affected by the environment as a whole.
There are some more major limitations to this study. Firstly, At the beginning of the control period, the application of microbial agents in Xiangshan was not as effective as in the other two regions, and the control was also less effective when the disease was aggravated by the first peak period of the disease (June), which coincided with the rainy season. Meanwhile, the degradation rate of chemicals is similarly affected by the effect of precipitation washout. The lack of environmental covariate data (e.g., soil properties, microclimate) limits our ability to fully explain site-to-site variation in treatment efficacy. This suggests that the effectiveness of root irrigation with biological and chemical agents depends largely on the stability of the environment. Timely selection of control agents and measures is critical in areas where the disease was previously more severe and where the environment is variable. Second, current prevention and control decisions rely heavily on manual field surveys; the frequency of manual surveys is based on a monthly cycle, so there may be a time delay, leading to a higher degree of completion of the infestation cycle process of the pathogen in the soil or in the plant. Attention should be paid to the roots of the diseased sample trees in the future control process, and qRCR detection of the pathogen around the root system should be increased. The root system of the diseased smoke tree was also sampled to define its root damage. Third, this test was applied in a geographical area with a mean annual temperature of about 10°, precipitation of 500–600 mm, and alkaline soil with a pH of 7.5–8.5 for trees about 10–15 years old. Studies in acidic soils are lacking. When there is excessive rainfall, it is recommended to shorten the application interval to 15 days and drain the rainwater from the sample plots in time. This result only applies to smoke trees around 10–15 years old and lacks broad applicability. Although the effects of tree age and size on control were reduced by random sampling in the trial, smoke trees under different conditions may be selective for agents. Future studies should focus on analyzing the effects of the conditions of the trees themselves. There is a need for more research on disease control for young (less than 5 years old) and >15-year-old smoke trees in the future. In addition, there is a link between fungal populations and morbidity. This paper lacks a study of fungal populations and their changes in diseased areas. In the next study, we will explore the effects of application on fungal communities and the results of application in different fungal communities. A further limitation is that the disease classification in this experiment relies on visual scoring, lacks molecular diagnostics, and may be biased by manual diagnosis. Molecular diagnosis should be added to future studies to allow for more accurate disease determination. Accurate prediction of control window periods is realized. In the future, unmanned aircraft disease identification and detection systems could be established to control diseases over larger areas in the forest, making the results more generalizable.

5. Conclusions

In summary, considering the characteristics of Verticillium wilt caused by a pathogen residing in the soil and spreading through vascular bundles, this study filtered out a combination of B. subtilis root irrigation and trunk spraying of tebuconazole and sodium nitrophenolate. This method was effective in controlling established wilt while protecting healthy plants, with an overall control effect of 104.55% for the year. The disease index in the following year’s survey was the lowest, demonstrating a strong preventive effect. In addition, propiconazole root irrigation has a good wilt control effect. Based on these results, control trials were designed for different disease levels. The aim was to reduce the use of chemicals while effectively controlling the disease. Controlled experiments conducted in three regions showed that these methods and combinations of agents were effective in preventing or treating Verticillium wilt at different grades of disease index. This experiment verified the feasibility of control throughout the growth period of smoke trees, providing a reliable basis for practical disease control strategies.

Author Contributions

Conceptualization, Y.W.; data curation, R.G.; funding acquisition, Y.W.; investigation, Y.Z., B.Z., F.Y., X.S., M.Z., J.G., K.L., W.L., X.Z. (Xiaoran Zhou), Y.R., Z.L. and X.Z. (Xinpeng Zhang); methodology, Y.Z., B.Z., F.Y., X.S., M.Z., J.G., K.L., W.L., X.Z. (Xiaoran Zhou), Y.R., Z.L. and X.Z. (Xinpeng Zhang); software, Y.Z.; supervision, Y.W.; writing—original draft, Y.Z.; writing—review and editing, Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Municipal Forestry and Parks Resource Conservation Center (2025-ZYBH-02-26).

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Conflicts of Interest

Author Xinpeng Zhang was employed by the company Beijing Lanhu Natural Enemy Technology Company Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Disease index of Verticillium wilt under different treatments in Pofengling 2023. a One-way ANOVA using GraphPad 9.5, and multiple comparisons between initial and subsequent months. b Thresholds for significance and labeling: * stands for p < 0.05; ** stands for p < 0.01; *** stands for p < 0.001; **** stands for p < 0.0001. c The X-axis shows the different treatments in order from left to right, and the different color labels on the right side of the graph represent the results of the survey in different months.
Figure 1. Disease index of Verticillium wilt under different treatments in Pofengling 2023. a One-way ANOVA using GraphPad 9.5, and multiple comparisons between initial and subsequent months. b Thresholds for significance and labeling: * stands for p < 0.05; ** stands for p < 0.01; *** stands for p < 0.001; **** stands for p < 0.0001. c The X-axis shows the different treatments in order from left to right, and the different color labels on the right side of the graph represent the results of the survey in different months.
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Figure 2. Comparison of disease index among different treatments in Pofengling Park during June 2023 and June 2024. a Differences were not significant after two-way ANOVA using GraphPad 9.5 and multiple comparisons between initial and subsequent months. b The solid line in the center represents the median, and the dashed lines at the top and bottom represent the quartiles. c The X-axis shows the different treatments in order from left to right, and the different color labels on the right side of the graph represent the results of the survey in different years.
Figure 2. Comparison of disease index among different treatments in Pofengling Park during June 2023 and June 2024. a Differences were not significant after two-way ANOVA using GraphPad 9.5 and multiple comparisons between initial and subsequent months. b The solid line in the center represents the median, and the dashed lines at the top and bottom represent the quartiles. c The X-axis shows the different treatments in order from left to right, and the different color labels on the right side of the graph represent the results of the survey in different years.
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Figure 3. Wilt symptoms in June 2023 (A) and June 2024 (B) when no significant onset of disease was observed.
Figure 3. Wilt symptoms in June 2023 (A) and June 2024 (B) when no significant onset of disease was observed.
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Figure 4. Incidence rate (%) and disease index under the graded control trial in three districts in 2024. (A) Incidence rate and disease index of Xiangshan Park. (B) Incidence rate and disease index of Xishan Park. (C) Incidence rate and disease index of Pofengling Park. a The X-axis shows the different months in order from left to right, and the different color labels on the right side of the graph represent the results of the survey in different treatments. b One-way ANOVA using GraphPad 9.5 and multiple comparisons between treatment and control. c Thresholds for significance and labeling: ns stands for no significance; * stands for p < 0.05; ** stands for p < 0.01; *** stands for p < 0.001; **** stands for p < 0.0001.
Figure 4. Incidence rate (%) and disease index under the graded control trial in three districts in 2024. (A) Incidence rate and disease index of Xiangshan Park. (B) Incidence rate and disease index of Xishan Park. (C) Incidence rate and disease index of Pofengling Park. a The X-axis shows the different months in order from left to right, and the different color labels on the right side of the graph represent the results of the survey in different treatments. b One-way ANOVA using GraphPad 9.5 and multiple comparisons between treatment and control. c Thresholds for significance and labeling: ns stands for no significance; * stands for p < 0.05; ** stands for p < 0.01; *** stands for p < 0.001; **** stands for p < 0.0001.
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Figure 5. Red leaf results after graded treatments throughout the year in 2024. (A,B) Control treatment in September (A) and at red leaf of smoke tree (B). (C–H) Grade I wilt in September (C) and at red leaf of smoke tree (D); Grade II wilt in September (E) and at red leaf of smoke tree (F); Grade IV wilt in September (G) and at red leaf of smoke tree (H). (I) Treatment area during the red leaf of smoke tree. (J) Control area at red leaf of smoke tree.
Figure 5. Red leaf results after graded treatments throughout the year in 2024. (A,B) Control treatment in September (A) and at red leaf of smoke tree (B). (C–H) Grade I wilt in September (C) and at red leaf of smoke tree (D); Grade II wilt in September (E) and at red leaf of smoke tree (F); Grade IV wilt in September (G) and at red leaf of smoke tree (H). (I) Treatment area during the red leaf of smoke tree. (J) Control area at red leaf of smoke tree.
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Table 1. Experimental design in Pofenglin in 2023.
Table 1. Experimental design in Pofenglin in 2023.
TreatmentWay of Agents ApplicationRoot Irrigation AgentsSpray Agents
CKBlank
1Root irrigationB. subtilis
2156 g/L propiconazole
350% azoxystrobin
4Trunk spraying 25% tebuconazole, 1.4% sodium nitrophenolate
5 45% prochloraz, 1.4% sodium nitrophenolate
6Combined treatmentB. subtilis25% tebuconazole, 1.4% sodium nitrophenolate
7B. subtilis45% prochloraz, 1.4% sodium nitrophenolate
8156 g/L propiconazole25% tebuconazole, 1.4% sodium nitrophenolate
9156 g/L propiconazole45% prochloraz, 1.4% sodium nitrophenolate
1050% azoxystrobin25% tebuconazole, 1.4% sodium nitrophenolate
1150% azoxystrobin45% prochloraz, 1.4% sodium nitrophenolate
Table 2. Different control strategies based on wilt disease grades.
Table 2. Different control strategies based on wilt disease grades.
Wilt GradeMethod of Agent ApplicationAgents
IRoot irrigationB. subtilis
Trunk spraying1.4% sodium nitrophenolate
II–IIIRoot irrigationB. subtilis
Trunk spraying45% prochloraz, 1.4% sodium nitrophenolate
IV–VRoot irrigation156 g/L propiconazole
Trunk spraying45% prochloraz, 1.4% sodium nitrophenolate
In the disease severity: I represents disease-free leaves of sample trees, II–III represents 0 to 50% diseased leaves of sample trees, and IV–V represents 50% to 100% diseased leaves of sample trees.
Table 3. Comparison of disease severity in June and November of 2023 after treatments.
Table 3. Comparison of disease severity in June and November of 2023 after treatments.
TreatmentMethod of ApplicationJun 2023Nov 2023Corrective Disease
Index		Corrective
Disease Index		Corrective
Control Effects
Disease SeverityNumberDisease SeverityNumber
CKBlankI12I620.8330.00 ± 3.54 a-
II–III6
II–III8II–III343.75
IV–V5
1RootirrigationI11I94.555.00 ± 3.54 cde83.92 ± 9.89 abc
II–III2
II–III9II–III95.56
2I13I617.3111.25 ± 12.37 bcd65.38 ± 38.07 abc
II–III7
II–III7II–III70
3I12I812.516.25 ± 1.77 bc50.00 ± 5.44 bc
II–III4
II–III8II–III525
IV–V3
4TrunksprayingI11I94.556.25 ± 1.77 cde79.37 ± 3.46 abc
II-III2
II–III9I28.33
II–III5
IV–V2
5I10I858.75 ± 5.30 bcde69.58 ± 21.26 abc
II–III2
II–III10I112.5
II–III7
IV–V2
6Combined treatmentI13I121.92−1.25 ± 1.77 e104.55 ± 6.43 a
II–III1
II–III7I2−7.14
II–III5
7I13I121.921.25 ± 5.30 de94.76 ± 18.30 ab
II–III1
II–III7I10
II–III5
IV–V1
8I12I104.173.75 ± 5.30 de86.36 ± 19.29 abc
II–III2
II–III8I33.12
II–III5
9I11I1105.00 ± 0 cde83.22 ± 1.98 abc
II–III9I111.11
II–III6
IV–V2
10I12I612.58.75 ± 8.84 bcde68.88 ± 33.13 abc
II–III6
II–III8I23.125
II–III5
IV–V1
11I10I77.518.75 ± 1.77 ab37.41 ± 1.48 c
II–III3
II–III10I030
II–III7
IV–V3
a In the disease severity: I represents the disease-free sample trees, II–III represents the 0–50% diseased sample trees, and IV–V represents the 50–100% diseased sample trees. b Corrected condition index and corrected control effect were verified for normality by Shapiro–Wilk test, p < 0.05, and analyzed by one-way ANOVA using SPSS software, and significance was calculated using Duncan’s test (p < 0.05). c Different letters in the same column of data indicate statistically significant differences between treatments (p < 0.05).
Table 4. Corrected disease index of different disease severity after treatment in 2024.
Table 4. Corrected disease index of different disease severity after treatment in 2024.
AreaCategorySeverity Based on Disease Grade
III–IIIIV–V
Xiangshan Parktreatment2.27−11.8−32.5
ck4.7117.318.75
Xishan Parktreatment1.73−18.26−35
ck3.26158.33
Pofengling Parktreatment1.85−20.59−33.33
ck3.8912.512.5
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Zhao, Y.; Guo, R.; Zheng, B.; Yuan, F.; Song, X.; Zhang, M.; Guo, J.; Liu, K.; Liu, W.; Zhou, X.; et al. Improved Management of Verticillium Wilt in Smoke Trees Through the Use of a Combination of Fungicide and Bioagent Treatments. Forests 2025, 16, 914. https://doi.org/10.3390/f16060914

AMA Style

Zhao Y, Guo R, Zheng B, Yuan F, Song X, Zhang M, Guo J, Liu K, Liu W, Zhou X, et al. Improved Management of Verticillium Wilt in Smoke Trees Through the Use of a Combination of Fungicide and Bioagent Treatments. Forests. 2025; 16(6):914. https://doi.org/10.3390/f16060914

Chicago/Turabian Style

Zhao, Yize, Ruifeng Guo, Bo Zheng, Fei Yuan, Xi Song, Mengfei Zhang, Jinzi Guo, Kexin Liu, Weijia Liu, Xiaoran Zhou, and et al. 2025. "Improved Management of Verticillium Wilt in Smoke Trees Through the Use of a Combination of Fungicide and Bioagent Treatments" Forests 16, no. 6: 914. https://doi.org/10.3390/f16060914

APA Style

Zhao, Y., Guo, R., Zheng, B., Yuan, F., Song, X., Zhang, M., Guo, J., Liu, K., Liu, W., Zhou, X., Ren, Y., Liu, Z., Zhang, X., & Wang, Y. (2025). Improved Management of Verticillium Wilt in Smoke Trees Through the Use of a Combination of Fungicide and Bioagent Treatments. Forests, 16(6), 914. https://doi.org/10.3390/f16060914

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