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Abstract In order to provide a reference basis for efficient prevention and control of apple rust, apple rust (Gymnosporangium yamadai) was used as the test material, the field control effects of six triazole fungicides with different concentrations and different application times on apple rust, and the safety of application in different periods on the crop were investigated in field trials. The results showed that the control effect and safety of myclobutanil were excellent. In the prevention test, the effects of myclobutanil 40% EC 2 000 times and 4 000 times dilutions in protecting apple leaves from G. yamadai infection reached more than 98% at 10 d after the second spraying in the fruitlet stage, and in the treatment test, the control effects of inhibiting the spread of leaf lesions were 90.89% and 87.17% at 16 d after the fourth spraying in the fruit expansion stage, respectively. The results of the crop safety test showed that when applying the triazole fungicides with different concentrations from the red bud stage to the fruit expansion stage of apple trees, there were no adverse effects on the leaves, branches, flowers and fruit, as well as no rust or deformity on the fruit surface. When spraying the fungicides from the red bud stage to the fruit expansion stage, except that the single fruit weights in the treatment of the 10% difenoconazole DC 2 000 times and myclobutanil 40% EC 4 000 times dilutions were reduced without a significant difference from the control, and the values of other treatments were all reduced by more than 9% compared with the control. When applying the fungicides during the fruit expansion period, none of the tested fungicide treatments had a significant effect on the fruit growth.
Key words Apple rust; Triazole fungicide; Crop safety; Field control effect
Received: February 23, 2021 Accepted: April 24, 2021
Supported by National Key R&D Program of China (2016YFD0201100); China Agriculture Research System of MOF and MARA (CARS-27); Key Common Technology Research Project for the High-quality Development of Agriculture in Hebei Province (19226508D); Special Fund for Science and Technology Development of Xinjiang Production and Construction Corps (2018AB035); Science and Technology Research Fund of the Second Agricultural Division of Xinjiang Production and Construction Corps (2019NYGG01).
He JIANG (1995-), female, P. R. China, master, devoted to epidemic and comprehensive prevention and control of plant diseases. *Corresponding author. E-mail: bdstwang＠163.com.
Apple rust is a fungal disease caused by Gymnosporangium yamadai of Gymnosporangium in Pucciniaceae of Basidiomycota, also known as brown spot disease and Pingguixiu disease. G. yamadai is a heteroecious fungus, which mainly damages the leaves, twigs, fruitlet and fruit stalks of apple and pear trees. It can also damage the alternate host Chinese junipers［1-3］. When the disease is severe, there can be dozens of disease spots on the leaves. With the development of the disease, the disease spots on the leaves continue to expand［4］, and the fruit is deformed, resulting in reduced fruit quality. The disease reduces fruit yield by inhibiting photosynthesis and enhancing respiration, threatening the economic development of orchards［5-9］, and even causing greater economic losses［10］.
At present, the prevention and control of apple rust is mainly to remove the source of fungus (by cutting down the alternate host trees around orchards, cutting off the fungal galls on the cypress trees) and spray protective fungicides before rainfall［11］. Rust mainly infests trees during rainfall. If the infectious fungal source is not completely removed, it will be necessary to spray protective fungicides in time before rainfall from April to May, and spray systemic therapeutic agents in time after rain to protect the hosts from the harm of the fungus［12］. The chemical control of apple rust is mainly based on triazole fungicides. Triazole fungicides are also called ergosterol biosynthesis inhibitors, which have the characteristics of wide bactericidal spectrum, good control effect, and long lasting period, thus having good market prospects［13-14］. Its mechanism of action is to disrupt the cell membrane function of the fungus by hindering the biosynthesis of fungal ergosterol, thereby inhibiting or interfering with the development of appressorium and haustorium, and the formation of hyphae and spores［15］. Studies have shown that triazole fungicides have certain degradation rules in tomato and cucumber［16］, and have good control effects on most fungal diseases［17-18］, but they also have certain effects. Han et al.［19］ found that the application of hexaconazole to wheat in the seedling stage will dwarf wheat plants. Guo［20］ found that difenoconazole 30% WG 4 000 times dilution had an effect of 91.12% on apple rust, and tebuconazole 43% SC 4 000 times dilution was better than triadimefon 15% WP 2 000 times dilution. However, there are few reports on the differences in the control effects of different triazole fungicides on apple rust, and the effects of pesticides applied in different periods on fruit growth and fruit surface. How can we use this type of agrochemicals scientifically, including the use concentration, application period and application times, remains to be studied. In this study, with apple rust (G. yamadai) as the test material, the field control effects of six triazole fungicides with different concentrations and different application times on apple rust and the safety of application in different periods on the crop were investigated by the method of field trials, hoping to provide a reference for the safe and efficient prevention and control of apple rust. Materials and Methods
Experimental materials
The test apple variety was ‘Fuji’, and the grafting stock was ‘Malus × robusta (Carrire) Rehder’, and the intermediate stock was ‘SH38’. The test site was located in the Apple Test Orchard in the West Campus of Hebei Agricultural University, Baoding City, Hebei Province. The orchard covered an area of 3 300 m2. There were 2 planting modes, one of which was potted fruit trees, which were planted in 2008 and fruited in 2011, with a row spacing of 0.75 m× 1.50 m, and the size of the fruit pots was 38 cm×26 cm. The other was densely planted fruit trees on dwarf stocks with a row spacing of 1.5 m×3.5 m. They were planted in 2013, and the planted varieties included ‘Fuji’, ‘Ourin’ and ‘Mato 1’. The soil is sandy loam, and the level of fertilizer and water management was good. The occurrence of diseases and insect pests was moderate over the years.
The specific information of the tested fungicides is shown in Table 1. The AGROLEXHD400 knapsack manual sprayer used was produced by Singapore AGROLEX Co., Ltd.
Experimental methods
Triazole fungicides for preventing and controlling apple rust
The experimental design was based on the recommended concentrations of the field control effect test, and the treatment concentrations were designed according to the 1-fold and 2-fold gradients. The experiment had 12 treatments, i.e., tebuconazole 30% SC 1 000 times and 2 000 times dilutions, difenoconazole 10% DC 1 000 times and 2 000 times dilutions, flusilazole 40% EC 2 000 times and 4 000 times dilutions, diniconazole 30% WP 1 000 times and 2 000 times dilutions, triadimefon 15% WP 2 000 times and 4 000 times dilutions, myclobutanil 40% EC 2 000 times and 4 000 times dilutions, with water as a control (CK). The water consumption was 2 220 L/hm2. Each treatment was done in triplicate with one tree per replicate, and randomized block arrangement was adopted. The prevention test was carried out before the infection of the fungus, that is, before the onset of the disease. Fungicides were applied for 4 times in total, during the red bud stage(April 12), flowering stage (April 19) and fruitlet stage (April 26, May 2) in 2018. The treatment test was carried out after the infection of the fungus, that is, after the onset of the disease. Fungicides were applied for 4 times in total, during the fruit expansion period (May 31, June 10, June 20, and July 20, 2018). It was sunny or cloudy with breeze on the days of application. Safety test of triazole fungicides against apple rust
The six kinds of triazole fungicides were used to spray potted fruit trees and dwarf-stock fruit trees. The application of potted fruit trees was based on the recommended concentrations in the field control effect test, and the treatment concentrations were designed according to the 1-fold and 2-fold gradients. The treatment method was the same as that of control effect test of apple rust. Only the recommended concentrations were used to treat the densely planted fruit trees on dwarf stocks. The water consumption was 2 220 L/hm2. Each treatment was done in triplicate with two trees per plot, and randomized block arrangement was adopted. Application period: The potted fruit trees was sprayed for a total of 6 time, in the red bud stage (April 12), flowering stage (April 19), fruitlet stage (May 8, May 22), fruit expansion period (June 10 , July 2) in 2018, and the days of spraying was sunny or sunny to cloudy; and the densely planted fruit trees on dwarf stocks were sprayed 5 times during the fruit expansion period (May 31, June 14, June 30, July 12, and July 27) in 2018. The days of spraying were sunny with breeze.
Item determination
The investigation time and method of the prevention and control test of apple rust: For the rust prevention test, the effect of preventing apple leaves from G. yamadai infection was investigated at 10 d (May 12) after the second spraying at the fruitlet stage. For the rust treatment test, the control effect of inhibiting the spread of leaf lesions was investigated at 8 d after the first spraying (June 8), 9 d after the second spraying (June 19), 13 d after the third spraying (July 3) and 16 d after the fourth spraying (August 5) during the fruit expansion period. The tip of one branch was fixed in the east, west, south, north, and central direction of each tree, respectively, for the investigation of the incidence of leaf lesions according to disease grades and recording of the total number of leaves, the number of diseased leaves and the corresponding disease grade, and the disease index and control effect of apple rust after applying the fungicides at different time in different periods were calculated. Apple rust grading standard［21］: grade 0, no disease spots; grade 1: the area of disease spots accounted for less than 10% of the entire leaf area; grade 3: the area of disease spots accounted for 11%-25% of the entire leaf area; grade 5: the area of disease spots accounted for 26%-40% of the entire leaf area; grade 7: the area of disease spots accounted for 41%-65% of the entire leaf area; and grade 9: the area of disease spots accounted for more than 65% of the entire leaf area. Method for calculating efficacy: Incidence rate (%) = Number of diseased leaves/Total number of investigated leaves×100; Disease index=［Σ(Number of diseased leaves at each level×relative grade)/Total number of investigated leaves×The highest grade of disease］×100; Control effect = (Disease index of the control area-Disease index of the treatment area)/Disease index of the control area×100%. The investigation time and method of the safety test of apple rust: For potted fruit trees, the effects of different treatments on the flowers, leaves and branches were investigated at 4 d (April 16) after the spraying in the red bud stage and 5 (April 24) and 11 d (April 30) after the application during the flowering stage. The fruit surface and fruit size were investigated at 37 d (June 14) after the first spraying and 50 d after the second spraying (July 11) in the fruitlet stage, and at 80 d (August 20) after the first application and 76 d (September 15) after the second spraying in the fruit expansion stage. And the fruit shape index and single fruit weight were investigated in the near-maturity period. For the fruit trees on dwarf stocks, the fruit surface and fruit size were investigated at 14 d (June 14) after the first spraying, 16 d (June 30) after the second spraying, 12 d (July 12) after the third spraying, 15 d after the fourth spraying (July 27 ), and 25 (August 21) and 49 d (September 15) after the fifth spraying in the fruit expansion stage, and the fruit shape index and single fruit weight were measured in the near-maturity period. In the phytotoxicity investigation, the effects of different treatments on the flowers, leaves and branches of the fruit trees were observed during the flowering period. From the fruitlet stage to the mature stage, whether the fruit was deformed and whether the fruit surface had phytotoxicity and fruit rust were investigated.
In the near-maturity period, the fruit was randomly selected from the outer periphery of the middle and upper parts of the fruit trees in the east, south, west, north, and middle of the trees, and 30 ones were collected from each treatment. An electronic balance was used to measure the single fruit weight; and the transverse and longitudinal diameter were measured with a vernier caliper, and the fruit shape index was calculate according to Fruit shape index=Fruit longitudinal diameter/Fruit transverse diameter.
Data analysis
Microsoft Excel 2016 software was employed to process the data and calculate the incidence, disease index and control effect. SPSS 22.0 software was employed for data analysis and significance test. Multiple comparisons were performed by Duncans new multiple difference method (DMRT), P=0.05.
Results and Analysis
Control effects of triazole fungicides on apple rust
The control effect investigation results of the prevention test of spraying before the onset of the disease showed that the control effects of following agrochemicals, myclobutanil 40% EC 2 000 times and 4 000 times dilutions, tebuconazole 30% SC 1 000 times dilution, diniconazole 30% WP 1 000 times dilution, flusilazole 40% EC 2 000 times dilution and triadimefon 15% WP 200 times dilution, all reached 98% in protecting apple leaves from the infection of G. yamadai at 10 d (May 12) after the second spraying, that is, they significantly reduced the rate of diseased leaves, while the control effects of other fungicides were all lower than 94% (Table 2). The control effect investigation results of spraying after the onset of the disease showed that during the fruit expansion stage, the control effects of various treatments on leaf lesions was less than 75% at 8 d after the first spraying, the control effects at 16 d after fourth spraying increased in different proportions, and there were significant differences among the treatments. Specifically, the control effects of myclobutanil 40% EC 2 000 times and 4 000 times dilutions were 90.89% and 87.17%, respectively, and those of tebuconazole 30% SC 1 000 times dilution, difenoconazole 10% DC 1 000 times dilutions, flusilazole 40% EC 2 000 times dilution and triadimefon 15% WP 200 times dilution all reached more than 80%, while the values of other treatments were all less than 76% (Table 3). Data are expressed as means±SE. Values in the same column with the same lowercase letters mean significantly different at the level of 5%. The same below.
Effects of triazole fungicides on the safety of apple fruit
Effects of triazole fungicides on the safety of potted apple fruit
The pot experiment results showed that the 12 treatments of the six fungicides in the tested concentration range during the red bud, flowering, fruitlet, and fruit expansion stages had no phytotoxicity to apple flowers, leaves, branches and fruit, because there was no rust and deformity on fruit surface and no other adverse effects. The fruit shape index and single fruit weight were measured at the near-maturity stage. The longitudinal and transverse diameters of the fruit and single fruit weight in the treatment of difenoconazole 10% DC 2 000 times dilution were the least different from the control. The transverse and longitudinal diameters were reduced by 4.10% and 2.56%, respectively, and the single fruit weight was reduced by 6.50%. The transverse and longitudinal diameters of the fruit in the treatment of myclobutanil 40% EC 4 000 times dilution decreased by 4.66% and 2.73%, respectively, and the single fruit weight decreased by 7.15%. The single fruit weights of the above two treatments were not significantly different from that of the control. Compared with the control, the single fruit weights in other treatments were significantly reduced (Table 4).
Effects of triazole fungicides on the growth safety of apple fruit on dwarf-stock trees
The test results of dwarf-stock fruit trees showed that the 12 treatments of the six fungicides within the tested concentration ranges had no phytotoxicity to apple leaves, branches and fruit during the fruit expansion stage, and there was no deformity and rust on the fruit surface, and no adverse effects. The fruit shape index and single fruit weight were investigated in the near-maturity period, and there were no significant differences among the treatments (Table 5).
Discussion and Conclusions
A comprehensive analysis of the effects of six triazole fungicides on the field control effect and crop safety of apple rust showed that the control effect of the treatment test was significantly lower than that of the control effect of the prevention test. Among the various triazole fungicides, myclobutanil 40% EC had excellent control effect on apple rust and crop safety. In the prevention test, the effects of myclobutanil 40% EC 2 000 times and 4 000 times dilutions in protecting apple leaves from G. yamadai infection reached more than 98% at 10 d after the second spraying in the fruitlet stage, and the rate of diseased leaves was significantly reduced compared with the control. In the treatment test, the control effects (70.15% and 90.89%) of myclobutanil 40% EC 2 000 times dilution were better than the control effects (70.15% and 87.17%) of myclobutanil 40% EC 4 000 times dilution in inhibiting the spread of apple leaf lesions at 8 d after the first spraying and 16 d after the fourth spraying, showing a significant dose effect. Rainfall is the main condition for pathogen infection, while in the prevention test, rainfall occurred for 2 consecutive days, i.e., 3 and 4 d after the application of the fungicides on April 19, and the precipitation was 33.4 and 8.9 mm, respectively, which created favorable conditions for pathogen infection. However, the incubation period seemed to be longer during this period, and the first symptoms of apple rust were observed on May 12. In the treatment test, the precipitation at 1 and 2 d after the application of the fungicides on June 10 was 0.7 and 3.7 mm, respectively, which might have a certain scouring effect and affected its lasting period of the fungicides. During the period from July 8 to July 19, the rainfall occurred for several consecutive days and the cumulative rainfall amounted to 243.2 mm. It is not clear whether the rainfall during this period was beneficial to the development of the disease. However, there were significant differences in the condition of the disease between different treatments in early August, which might be related to the tolerance of the fungicides to rainwash. Therefore, it is necessary to pay close attention to the weather conditions when preventing and controlling apple rust, and myclobutanil 40% EC can be sprayed in time before rain to protect apple leaves from infection by the pathogenic fungus. The study of Dong et al.［22］ showed that flusilazole 40% EC, 43% tebuconazole SC and 10% difenoconazole WG had a 100% control effect on apple rust. However, in this study, the above-mentioned three fungicides had reduced control effects on apple rust, which is different from previous research results to a certain degree, which may be related to the application time and rainfall. Wang et al.［23］ believed that tebuconazole and difenoconazole seed treatments had no effect on the emergence rate of maize, but had an inhibitory effect on plant height. Chen et al.［24］ reported that pyraclostrobin could improve soybean quality and yield. However, there are few reports about the safety of triazoles on apple fruit. In the safety test of this study, the triazole fungicides were continuously sprayed from the red bud stage to the fruit expansion stage of apple trees. The growth of the plants treated with the six fungicides at different concentrations was normal, and there were no adverse effects on the leaves, branches, flowers and fruit, as well as no rust or deformity on the fruit surface. The single fruit weight of potted fruit trees was reduced compared with the control in various treatments from the red bud stage to the fruit expansion stage, and except that the single fruit weights of the difenoconazole 10% DC 2 000 times and myclobutanil 40% EC 4 000 times dilutions were reduced without a significant difference from the control, and the values of other treatments were all reduced by more than 9% compared with the control. However, in the fruit expansion stage (after May 31), the dwarf-stock fruit trees were treated, and the treatments with the recommended concentrations of the test agents did not have a significant effect on the fruit growth. This result showed that difenoconazole 10% DC 2 000 times dilution and myclobutanil 40% EC 4 000 times dilution were safer to apply from the red bud stage to the fruit expansion stage, and it was safer to apply the rest of the agents in the fruit expansion stage (after May 31) according to the recommended concentrations. According to research needs, the number of application times in this study is more than the actual number of application times in production, and the application times can be appropriately reduced according to the actual conditions of field diseases in production.
Myclobutanil 40% EC is an ideal agent for the prevention and treatment of apple rust. When applied in the field, according to the weather conditions, it should be used for prevention at the red bud and fruitlet stages before the infection of the fungus. Spraying myclobutanil 40% EC 4 000 times dilution at an interval of 7 d continuously for 3 to 4 times can achieve a better control effect, and after the occurrence of apple rust, spraying 3 to 4 times at the fruit expansion stage can significant inhibit the spread of apple rust leaf lesions. In order to avoid drug resistance, it can be used in rotation with difenoconazole 10% DC 1 000-2 000 times dilution, and the appropriate concentration can be chosen according to the condition. If the rainfall is heavy or the condition is serious, the concentration of the fungicide can be appropriately increased to reduce the incidence of disease in the later stage. References
［1］ TAO SQ, CAO B, MORIN E, et al. Comparative transcriptomics of Gymnosporangium spp. teliospores reveals a conserved genetic program at this specific stage of the rust fungal life cycle［J］. BMC Genomics, 2019, 20(1): 1-16.
［2］ TAO SQ, CAO B, TIAN CM, et al. Comparative transcriptome analysis and identification of candidate effectors in two related rust species (Gymnosporangium yamadae and Gymnosporangium asiaticum)［J］. BMC Genomics, 2017, 18(1): 651.
［3］ ZHAO P, LIU F, LI YM, et al. Inferring phylogeny and speciation of Gymnosporangium species, and their coevolution with host plants［J］. Scientific Reports, 2016(6): 29339.
［4］ LEE DK, AHN S, CHO HY, et al. Metabolic response induced by parasitic plant-fungus interactions hinder amino sugar and nucleotide sugar metabolism in the host［J］.Scientific Reports, 2016, 6(1): 37434.
［5］ PU JX. Occurrence and comprehensive control of apple rust in Tianshui, Gansu［J］. China Fruits, 2012(1): 65-66. (in Chinese)
［6］ CAO ZM, LI ZQ, ZHUANG JY. Uredinales from the Qinling Mountains［J］. Mycosystema, 2000, 19(1): 13-23.
［7］ GUO YN. The integrated management of the rust of apple (Gymnosporangium yamadae Miyabe)［J］. Bulletin of Agricultural Science and Technology, 1994(19): 28-29.
［8］ WANG SQ, YANG ZP, YU J. Reasons for serious occurrence of apple rust and comprehensive control measures［J］. Chinese Horticulture Abstracts, 2010, 26(1): 145-146. (in Chinese)
［9］ LI BH, WANG CX, DONG XL. Research progress in apple diseases and problems in the disease management in China［J］. Plant Protection, 2013, 39(5): 46-54. (in Chinese)
［10］ MA TJ. Reasons for the recurrence of apple rust in Qinan, Gansu and comprehensive control strategies［J］. China Plant Protection, 2019, 39(12): 61-63. (in Chinese)
［11］ lU KJ, ZHAO DC, FU SP. Reasons for serious occurrence of apple rust and comprehensive control measures［J］. China Fruits, 2009(4): 72-73. (in Chinese)
［12］ HU XH. The application of difenoconazole control on apple disease［D］. Yangling: Northwest A&F University, 2017. (in Chinese)
［13］ ZHAO ZZ. Triazole fungicides use skills［J］. Northwest Horticulture, 2017(3): 46-47. (in Chinese)
［14］ CAO HC, LI XH, WANG XK, et al. Control efficacy of pyraclostrobin and triazole fungicides against tomato crown and root rot［J］. Scientia Agricultura Sinica, 2018, 51(21): 64-74. (in Chinese)
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Key words Apple rust; Triazole fungicide; Crop safety; Field control effect
Received: February 23, 2021 Accepted: April 24, 2021
Supported by National Key R&D Program of China (2016YFD0201100); China Agriculture Research System of MOF and MARA (CARS-27); Key Common Technology Research Project for the High-quality Development of Agriculture in Hebei Province (19226508D); Special Fund for Science and Technology Development of Xinjiang Production and Construction Corps (2018AB035); Science and Technology Research Fund of the Second Agricultural Division of Xinjiang Production and Construction Corps (2019NYGG01).
He JIANG (1995-), female, P. R. China, master, devoted to epidemic and comprehensive prevention and control of plant diseases. *Corresponding author. E-mail: bdstwang＠163.com.
Apple rust is a fungal disease caused by Gymnosporangium yamadai of Gymnosporangium in Pucciniaceae of Basidiomycota, also known as brown spot disease and Pingguixiu disease. G. yamadai is a heteroecious fungus, which mainly damages the leaves, twigs, fruitlet and fruit stalks of apple and pear trees. It can also damage the alternate host Chinese junipers［1-3］. When the disease is severe, there can be dozens of disease spots on the leaves. With the development of the disease, the disease spots on the leaves continue to expand［4］, and the fruit is deformed, resulting in reduced fruit quality. The disease reduces fruit yield by inhibiting photosynthesis and enhancing respiration, threatening the economic development of orchards［5-9］, and even causing greater economic losses［10］.
At present, the prevention and control of apple rust is mainly to remove the source of fungus (by cutting down the alternate host trees around orchards, cutting off the fungal galls on the cypress trees) and spray protective fungicides before rainfall［11］. Rust mainly infests trees during rainfall. If the infectious fungal source is not completely removed, it will be necessary to spray protective fungicides in time before rainfall from April to May, and spray systemic therapeutic agents in time after rain to protect the hosts from the harm of the fungus［12］. The chemical control of apple rust is mainly based on triazole fungicides. Triazole fungicides are also called ergosterol biosynthesis inhibitors, which have the characteristics of wide bactericidal spectrum, good control effect, and long lasting period, thus having good market prospects［13-14］. Its mechanism of action is to disrupt the cell membrane function of the fungus by hindering the biosynthesis of fungal ergosterol, thereby inhibiting or interfering with the development of appressorium and haustorium, and the formation of hyphae and spores［15］. Studies have shown that triazole fungicides have certain degradation rules in tomato and cucumber［16］, and have good control effects on most fungal diseases［17-18］, but they also have certain effects. Han et al.［19］ found that the application of hexaconazole to wheat in the seedling stage will dwarf wheat plants. Guo［20］ found that difenoconazole 30% WG 4 000 times dilution had an effect of 91.12% on apple rust, and tebuconazole 43% SC 4 000 times dilution was better than triadimefon 15% WP 2 000 times dilution. However, there are few reports on the differences in the control effects of different triazole fungicides on apple rust, and the effects of pesticides applied in different periods on fruit growth and fruit surface. How can we use this type of agrochemicals scientifically, including the use concentration, application period and application times, remains to be studied. In this study, with apple rust (G. yamadai) as the test material, the field control effects of six triazole fungicides with different concentrations and different application times on apple rust and the safety of application in different periods on the crop were investigated by the method of field trials, hoping to provide a reference for the safe and efficient prevention and control of apple rust. Materials and Methods
Experimental materials
The test apple variety was ‘Fuji’, and the grafting stock was ‘Malus × robusta (Carrire) Rehder’, and the intermediate stock was ‘SH38’. The test site was located in the Apple Test Orchard in the West Campus of Hebei Agricultural University, Baoding City, Hebei Province. The orchard covered an area of 3 300 m2. There were 2 planting modes, one of which was potted fruit trees, which were planted in 2008 and fruited in 2011, with a row spacing of 0.75 m× 1.50 m, and the size of the fruit pots was 38 cm×26 cm. The other was densely planted fruit trees on dwarf stocks with a row spacing of 1.5 m×3.5 m. They were planted in 2013, and the planted varieties included ‘Fuji’, ‘Ourin’ and ‘Mato 1’. The soil is sandy loam, and the level of fertilizer and water management was good. The occurrence of diseases and insect pests was moderate over the years.
The specific information of the tested fungicides is shown in Table 1. The AGROLEXHD400 knapsack manual sprayer used was produced by Singapore AGROLEX Co., Ltd.
Experimental methods
Triazole fungicides for preventing and controlling apple rust
The experimental design was based on the recommended concentrations of the field control effect test, and the treatment concentrations were designed according to the 1-fold and 2-fold gradients. The experiment had 12 treatments, i.e., tebuconazole 30% SC 1 000 times and 2 000 times dilutions, difenoconazole 10% DC 1 000 times and 2 000 times dilutions, flusilazole 40% EC 2 000 times and 4 000 times dilutions, diniconazole 30% WP 1 000 times and 2 000 times dilutions, triadimefon 15% WP 2 000 times and 4 000 times dilutions, myclobutanil 40% EC 2 000 times and 4 000 times dilutions, with water as a control (CK). The water consumption was 2 220 L/hm2. Each treatment was done in triplicate with one tree per replicate, and randomized block arrangement was adopted. The prevention test was carried out before the infection of the fungus, that is, before the onset of the disease. Fungicides were applied for 4 times in total, during the red bud stage(April 12), flowering stage (April 19) and fruitlet stage (April 26, May 2) in 2018. The treatment test was carried out after the infection of the fungus, that is, after the onset of the disease. Fungicides were applied for 4 times in total, during the fruit expansion period (May 31, June 10, June 20, and July 20, 2018). It was sunny or cloudy with breeze on the days of application. Safety test of triazole fungicides against apple rust
The six kinds of triazole fungicides were used to spray potted fruit trees and dwarf-stock fruit trees. The application of potted fruit trees was based on the recommended concentrations in the field control effect test, and the treatment concentrations were designed according to the 1-fold and 2-fold gradients. The treatment method was the same as that of control effect test of apple rust. Only the recommended concentrations were used to treat the densely planted fruit trees on dwarf stocks. The water consumption was 2 220 L/hm2. Each treatment was done in triplicate with two trees per plot, and randomized block arrangement was adopted. Application period: The potted fruit trees was sprayed for a total of 6 time, in the red bud stage (April 12), flowering stage (April 19), fruitlet stage (May 8, May 22), fruit expansion period (June 10 , July 2) in 2018, and the days of spraying was sunny or sunny to cloudy; and the densely planted fruit trees on dwarf stocks were sprayed 5 times during the fruit expansion period (May 31, June 14, June 30, July 12, and July 27) in 2018. The days of spraying were sunny with breeze.
Item determination
The investigation time and method of the prevention and control test of apple rust: For the rust prevention test, the effect of preventing apple leaves from G. yamadai infection was investigated at 10 d (May 12) after the second spraying at the fruitlet stage. For the rust treatment test, the control effect of inhibiting the spread of leaf lesions was investigated at 8 d after the first spraying (June 8), 9 d after the second spraying (June 19), 13 d after the third spraying (July 3) and 16 d after the fourth spraying (August 5) during the fruit expansion period. The tip of one branch was fixed in the east, west, south, north, and central direction of each tree, respectively, for the investigation of the incidence of leaf lesions according to disease grades and recording of the total number of leaves, the number of diseased leaves and the corresponding disease grade, and the disease index and control effect of apple rust after applying the fungicides at different time in different periods were calculated. Apple rust grading standard［21］: grade 0, no disease spots; grade 1: the area of disease spots accounted for less than 10% of the entire leaf area; grade 3: the area of disease spots accounted for 11%-25% of the entire leaf area; grade 5: the area of disease spots accounted for 26%-40% of the entire leaf area; grade 7: the area of disease spots accounted for 41%-65% of the entire leaf area; and grade 9: the area of disease spots accounted for more than 65% of the entire leaf area. Method for calculating efficacy: Incidence rate (%) = Number of diseased leaves/Total number of investigated leaves×100; Disease index=［Σ(Number of diseased leaves at each level×relative grade)/Total number of investigated leaves×The highest grade of disease］×100; Control effect = (Disease index of the control area-Disease index of the treatment area)/Disease index of the control area×100%. The investigation time and method of the safety test of apple rust: For potted fruit trees, the effects of different treatments on the flowers, leaves and branches were investigated at 4 d (April 16) after the spraying in the red bud stage and 5 (April 24) and 11 d (April 30) after the application during the flowering stage. The fruit surface and fruit size were investigated at 37 d (June 14) after the first spraying and 50 d after the second spraying (July 11) in the fruitlet stage, and at 80 d (August 20) after the first application and 76 d (September 15) after the second spraying in the fruit expansion stage. And the fruit shape index and single fruit weight were investigated in the near-maturity period. For the fruit trees on dwarf stocks, the fruit surface and fruit size were investigated at 14 d (June 14) after the first spraying, 16 d (June 30) after the second spraying, 12 d (July 12) after the third spraying, 15 d after the fourth spraying (July 27 ), and 25 (August 21) and 49 d (September 15) after the fifth spraying in the fruit expansion stage, and the fruit shape index and single fruit weight were measured in the near-maturity period. In the phytotoxicity investigation, the effects of different treatments on the flowers, leaves and branches of the fruit trees were observed during the flowering period. From the fruitlet stage to the mature stage, whether the fruit was deformed and whether the fruit surface had phytotoxicity and fruit rust were investigated.
In the near-maturity period, the fruit was randomly selected from the outer periphery of the middle and upper parts of the fruit trees in the east, south, west, north, and middle of the trees, and 30 ones were collected from each treatment. An electronic balance was used to measure the single fruit weight; and the transverse and longitudinal diameter were measured with a vernier caliper, and the fruit shape index was calculate according to Fruit shape index=Fruit longitudinal diameter/Fruit transverse diameter.
Data analysis
Microsoft Excel 2016 software was employed to process the data and calculate the incidence, disease index and control effect. SPSS 22.0 software was employed for data analysis and significance test. Multiple comparisons were performed by Duncans new multiple difference method (DMRT), P=0.05.
Results and Analysis
Control effects of triazole fungicides on apple rust
The control effect investigation results of the prevention test of spraying before the onset of the disease showed that the control effects of following agrochemicals, myclobutanil 40% EC 2 000 times and 4 000 times dilutions, tebuconazole 30% SC 1 000 times dilution, diniconazole 30% WP 1 000 times dilution, flusilazole 40% EC 2 000 times dilution and triadimefon 15% WP 200 times dilution, all reached 98% in protecting apple leaves from the infection of G. yamadai at 10 d (May 12) after the second spraying, that is, they significantly reduced the rate of diseased leaves, while the control effects of other fungicides were all lower than 94% (Table 2). The control effect investigation results of spraying after the onset of the disease showed that during the fruit expansion stage, the control effects of various treatments on leaf lesions was less than 75% at 8 d after the first spraying, the control effects at 16 d after fourth spraying increased in different proportions, and there were significant differences among the treatments. Specifically, the control effects of myclobutanil 40% EC 2 000 times and 4 000 times dilutions were 90.89% and 87.17%, respectively, and those of tebuconazole 30% SC 1 000 times dilution, difenoconazole 10% DC 1 000 times dilutions, flusilazole 40% EC 2 000 times dilution and triadimefon 15% WP 200 times dilution all reached more than 80%, while the values of other treatments were all less than 76% (Table 3). Data are expressed as means±SE. Values in the same column with the same lowercase letters mean significantly different at the level of 5%. The same below.
Effects of triazole fungicides on the safety of apple fruit
Effects of triazole fungicides on the safety of potted apple fruit
The pot experiment results showed that the 12 treatments of the six fungicides in the tested concentration range during the red bud, flowering, fruitlet, and fruit expansion stages had no phytotoxicity to apple flowers, leaves, branches and fruit, because there was no rust and deformity on fruit surface and no other adverse effects. The fruit shape index and single fruit weight were measured at the near-maturity stage. The longitudinal and transverse diameters of the fruit and single fruit weight in the treatment of difenoconazole 10% DC 2 000 times dilution were the least different from the control. The transverse and longitudinal diameters were reduced by 4.10% and 2.56%, respectively, and the single fruit weight was reduced by 6.50%. The transverse and longitudinal diameters of the fruit in the treatment of myclobutanil 40% EC 4 000 times dilution decreased by 4.66% and 2.73%, respectively, and the single fruit weight decreased by 7.15%. The single fruit weights of the above two treatments were not significantly different from that of the control. Compared with the control, the single fruit weights in other treatments were significantly reduced (Table 4).
Effects of triazole fungicides on the growth safety of apple fruit on dwarf-stock trees
The test results of dwarf-stock fruit trees showed that the 12 treatments of the six fungicides within the tested concentration ranges had no phytotoxicity to apple leaves, branches and fruit during the fruit expansion stage, and there was no deformity and rust on the fruit surface, and no adverse effects. The fruit shape index and single fruit weight were investigated in the near-maturity period, and there were no significant differences among the treatments (Table 5).
Discussion and Conclusions
A comprehensive analysis of the effects of six triazole fungicides on the field control effect and crop safety of apple rust showed that the control effect of the treatment test was significantly lower than that of the control effect of the prevention test. Among the various triazole fungicides, myclobutanil 40% EC had excellent control effect on apple rust and crop safety. In the prevention test, the effects of myclobutanil 40% EC 2 000 times and 4 000 times dilutions in protecting apple leaves from G. yamadai infection reached more than 98% at 10 d after the second spraying in the fruitlet stage, and the rate of diseased leaves was significantly reduced compared with the control. In the treatment test, the control effects (70.15% and 90.89%) of myclobutanil 40% EC 2 000 times dilution were better than the control effects (70.15% and 87.17%) of myclobutanil 40% EC 4 000 times dilution in inhibiting the spread of apple leaf lesions at 8 d after the first spraying and 16 d after the fourth spraying, showing a significant dose effect. Rainfall is the main condition for pathogen infection, while in the prevention test, rainfall occurred for 2 consecutive days, i.e., 3 and 4 d after the application of the fungicides on April 19, and the precipitation was 33.4 and 8.9 mm, respectively, which created favorable conditions for pathogen infection. However, the incubation period seemed to be longer during this period, and the first symptoms of apple rust were observed on May 12. In the treatment test, the precipitation at 1 and 2 d after the application of the fungicides on June 10 was 0.7 and 3.7 mm, respectively, which might have a certain scouring effect and affected its lasting period of the fungicides. During the period from July 8 to July 19, the rainfall occurred for several consecutive days and the cumulative rainfall amounted to 243.2 mm. It is not clear whether the rainfall during this period was beneficial to the development of the disease. However, there were significant differences in the condition of the disease between different treatments in early August, which might be related to the tolerance of the fungicides to rainwash. Therefore, it is necessary to pay close attention to the weather conditions when preventing and controlling apple rust, and myclobutanil 40% EC can be sprayed in time before rain to protect apple leaves from infection by the pathogenic fungus. The study of Dong et al.［22］ showed that flusilazole 40% EC, 43% tebuconazole SC and 10% difenoconazole WG had a 100% control effect on apple rust. However, in this study, the above-mentioned three fungicides had reduced control effects on apple rust, which is different from previous research results to a certain degree, which may be related to the application time and rainfall. Wang et al.［23］ believed that tebuconazole and difenoconazole seed treatments had no effect on the emergence rate of maize, but had an inhibitory effect on plant height. Chen et al.［24］ reported that pyraclostrobin could improve soybean quality and yield. However, there are few reports about the safety of triazoles on apple fruit. In the safety test of this study, the triazole fungicides were continuously sprayed from the red bud stage to the fruit expansion stage of apple trees. The growth of the plants treated with the six fungicides at different concentrations was normal, and there were no adverse effects on the leaves, branches, flowers and fruit, as well as no rust or deformity on the fruit surface. The single fruit weight of potted fruit trees was reduced compared with the control in various treatments from the red bud stage to the fruit expansion stage, and except that the single fruit weights of the difenoconazole 10% DC 2 000 times and myclobutanil 40% EC 4 000 times dilutions were reduced without a significant difference from the control, and the values of other treatments were all reduced by more than 9% compared with the control. However, in the fruit expansion stage (after May 31), the dwarf-stock fruit trees were treated, and the treatments with the recommended concentrations of the test agents did not have a significant effect on the fruit growth. This result showed that difenoconazole 10% DC 2 000 times dilution and myclobutanil 40% EC 4 000 times dilution were safer to apply from the red bud stage to the fruit expansion stage, and it was safer to apply the rest of the agents in the fruit expansion stage (after May 31) according to the recommended concentrations. According to research needs, the number of application times in this study is more than the actual number of application times in production, and the application times can be appropriately reduced according to the actual conditions of field diseases in production.
Myclobutanil 40% EC is an ideal agent for the prevention and treatment of apple rust. When applied in the field, according to the weather conditions, it should be used for prevention at the red bud and fruitlet stages before the infection of the fungus. Spraying myclobutanil 40% EC 4 000 times dilution at an interval of 7 d continuously for 3 to 4 times can achieve a better control effect, and after the occurrence of apple rust, spraying 3 to 4 times at the fruit expansion stage can significant inhibit the spread of apple rust leaf lesions. In order to avoid drug resistance, it can be used in rotation with difenoconazole 10% DC 1 000-2 000 times dilution, and the appropriate concentration can be chosen according to the condition. If the rainfall is heavy or the condition is serious, the concentration of the fungicide can be appropriately increased to reduce the incidence of disease in the later stage. References
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