Resistance mechanisms and associated mutations in acetylcholinesterase genes in Sitobion avenae (Fabricius)

Wheat aphid, Sitobion avenae (fabricius), is one of the most important wheat pests and has been reported to be resistant to commonly used insecticides in China. To determine the resistance mechanism, the resistant and susceptible strains were developed in laboratory and comparably studied. A bioassay revealed that the resistant strain showed high resistance to pirimicarb (RR: 161.8), moderate reistance to omethoate (32.5) and monocrotophos (33.5), and low resistance to deltamethrin (6.3) and thiodicarb (5.5). A biochemistry analysis showed that both strains had similar glutathione-S-transferase (GST) activity, but the resistant strain had 3.8-fold higher esterase activity, and its AChE was insensitive to this treatment. The I₅₀ increased by 25.8-, 10.7-, and 10.4-folds for pirimicarb, omethoate, and monocrotophos, respectively, demonstrating that GST had not been involved in the resistance of S. avenae. The enhanced esterase contributed to low level resistance to all the insecticides tested, whereas higher resistance to pirimicarb, omethoate, and monocrotophos mainly depended on AChE insensitivity. However, the AChE of the resistant strain was still sensitive to thiodicarb (1.7-fold). Thus, thiodicarb could be used as substitute for control of the resistant S. avenae in this case. Furthermore, the two different AChE genes cloned from different resistant and susceptible individuals were also compared. Two mutations, L436(336)S in Sa.Ace1 and W516(435)R in Sa.Ace2, were found consistently associated with the insensitivity of AChE. They were thought to be the possible resistance mutations, but further work is needed to confirm this hypothesis.     

The detection and distribution of organophosphorus and carbamate insecticide-resistant Myzus persicae (Sulz.) in Britain in 1976

Examination of the enzyme that determines the level of resistance to organophosphorus insecticides and carbamates in Myzus persicae (Sulz.) and bioassays were used to establish the frequency and resistance levels of resistant aphids on outdoor crops in Britain in 1976. The biochemical tests, staining esterase-4 after electrophoresis and total esterase determination, were more sensitive than bioassays. However the dip-test, a simple, rapid and inexpensive bioassay designed to detect resistance and its different levels gave satisfactory results which warrant its use where biochemical detection of resistance is not possible. Carboxylesterase activities of M. persicae collected in 1976 fell into three groups: low, moderate and high, and these were correlated with differences in tolerance to dimethoate, demeton-S-methyl and pirimicarb. Aphids with low esterase activity were susceptible(S). Those with the moderately active enzyme (R₁) had five- to seven-fold resistance to the two organophosphorus insecticides and were marginally resistant (about two-fold) to pirimicarb. The insects with the most active esterase (R₂) were strongly resistant to dimethoate (resistance factor, RF × 126) and moderately resistant to demeton-S-methyl (RF × 17) and pirimicarb (RF × 8). Some R₁, but no S aphids survived the recommended dose of demeton-S-methyl on field crops probably because they were under the lowest leaves and therefore protected from direct contact with the spray. Laboratory tests demonstrated that these R₁ aphids tolerated the residual deposit and systemic dose present in the leaves of the treated potato-plants. This enabled their numbers to recover in treated fields much faster than the susceptible insects which could do so by immigration only when the residual dose in the plant was no longer toxic. R₁ aphids were common throughout the country particularly in eastern England where susceptible aphids were rare, but in the Shardlow area of Derbyshire susceptible aphids were in the majority throughout the summer. R₂ aphids were found only in samples from the west of Scotland and northern England. The implications of the presence of aphids with different levels of resistance for aphid control are discussed.     

Communication to the Editor Insecticide Resistance in a Strain of Aphis gossypii from Southern France

A strain (R) of Aphis gossypii from Southern France was found to be resistant to several insecticides, particularly to pirimicarb, as compared to a susceptible strain (S). Resistance levels were determined by biological tests, and the highest resistance factor (1350) was for pirimicarb. Resistance was mainly restricted to anticholinesterase inhibitors. Use of synergists, DEF and PB, suggested that resistance mechanisms based on detoxification were involved to a minor extent, since a good correlation was observed between I₅₀ values and k_i values of AChE and in-vivo bioassay data. The two strains differed in esterase activity, with a 27·7-fold increase in the R strain. Resolution of esterases by polyacrylamide gel electrophoresis showed different patterns in the S and R strains, and two isozymes were less sensitive to pirimicarb in the S strain; however, no in-vitro degradation of [¹⁴C]pirimicarb was observed. These data suggest that the main mechanism of resistance was through a decrease in the sensitivity of the target, AChE, to the insecticides. © 1997 SCI.     

Use of biochemical and DNA diagnostics for characterising multiple mechanisms of insecticide resistance in the peach-potato aphid,Myzus persicae (Sulzer)

The peach-potato aphid Myzus persicae (Sulzer) can resist a range of insecticides by over-producing detoxifying esterase and having mutant-insensitive forms of the target proteins, acetylcholinesterase (AChE), and the sodium channel. Using a combination of bioassays, biochemical and DNA diagnostics, it is now possible to diagnose all three mechanisms in individual aphids, and thereby establish their spatial distributions and temporal dynamics. A survey of 58 samples of wide geographic origin showed that all 46 resistant clones had amplified esterase genes (E4 or FE4) conferring broad-spectrum resistance to pyrethroids, organophosphates and carbamates. These occurred in combination with insensitive AChE (11 clones), conferring resistance to pirimicarb and triazamate, and/or mutant sodium channel genes (25 clones), conferring knockdown (kdr) resistance to pyrethroids and DDT. Amplified esterase genes were in linkage disequilibrium with both insensitive AChE and the kdr mutation, reflecting tight physical linkage, heavy selection favouring aphids with multiple mechanisms, and/or the prominence of parthenogenesis in many M. persicae populations. An ability to monitor individual mechanisms with contrasting cross-resistance profiles has important implications for the development of resistance management recommendations. ©1997 SCI     

Temporal synergism can enhance carbamate and neonicotinoid insecticidal activity against resistant crop pests

BACKGROUND: Piperonyl butoxide (PBO) effectively synergises synthetic pyrethroids, rendering even very resistant insect pests susceptible, provided a temporal element is included between exposure to synergist and insecticide. This concept is now applied to carbamates and neonicotinoids. RESULTS: A microencapsulated formulation of PBO and pirimicarb reduced the resistance factor in a clone of Myzus persicae (Sulzer) from >19 000- to 100-fold and in Aphis gossypii (Glover) from >48 000- to 30-fold. Similar results were obtained for a strain of Bemisia tabaci Gennadius resistant to imidacloprid and acetamiprid, although a second resistant strain did not exhibit such a dramatic reduction, presumably owing to the presence of target-site insensitivity and the absence of metabolic resistance. Synergism was also observed in laboratory susceptible insects, suggesting that, even when detoxification is not enhanced, there is degradation of insecticides by the background enzymes. Use of an analogue of PBO, which inhibits esterases but has reduced potency against microsomal oxidases, suggests that acetamiprid resistance in whiteflies is largely oxidase based. CONCLUSION: Temporal synergism can effectively enhance the activity of carbamates and neonicotinoids against resistant insect pests. Although the extent of this enhancement is dependent upon the resistance mechanisms present, inhibition of background enzymes can confer increased sensitivity against target-site resistance as well as increased metabolism. .     

Response of susceptible and resistant peach-potato aphids Myzus persicae (Sulz.) to insecticides in leaf-dip bioassays

The response of susceptible (S), moderately resistant (R₁) and strongly resistant (R₂) peach-potato aphids, Myzus persicae (Sulz.) to organophosphorus, carbamate and pyrethroid insecticides was tested by a leaf-dip bioassay. The aphids were placed on potato leaves (dipped in insecticide solutions 1–2 or 24 h before infestation) and their mortality examined 48 h later. R₁ aphids were virtually susceptible to most of the carbamates, demephion and acephate, but were slightly to moderately resistant (2.1–9.4 times) to permethrin, cypermethrin and (S)-α-cyano-3-phenoxybenzyl (1R)-cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate (I), (NRDC 161), to 5,6,7,8-tetrahydro-2-methylquinolin-4-yl dimethylcarbamate (II), (Hoechst 25 682) and demeton-S-methyl. R₂ aphids resisted more strongly or very strongly (between 65 and 1280 times) the pyrethroids, demeton-S-methyl (×94), II (×83) and demephion (×9), and were slightly to moderately (2–5 times) resistant to acephate, pirimicarb, ethiofencarb and 2-(dimethylcarbamoyloxyimino)-3-methoxyimino-N,N- dimethylbutyramide (III), (DPX 3853). Both resistant strains were susceptible to nitrilacarb [4,4-dimethyl-5-(methylcarbamoyloxyimino)pentanenitrile] complex (1:1) with zinc chloride (IV), (AC 85 258). The implications of these results in terms of practical aphid control are discussed.     

Biochemical evidence that an S431F mutation in acetylcholinesterase-1 of Aphis gossypii mediates resistance to pirimicarb and omethoate

Molecular changes in acetylcholinesterase (AChE) leading to target-site resistance to carbamate and organophosphate insecticides have recently been identified. Of particular interest is the S431F mutation in ace2 and its orthologue ace1 of Myzus persicae Sulzer and Aphis gossypii Glover, respectively. This mutation has been correlated with resistance to pirimicarb, but biochemical evidence has not yet been provided. Here, we describe for the first time that recombinantly expressed AChE1 from A gossypii carrying the S431F mutation is insensitive to pirimicarb and omethoate, but sensitive to demeton-S-methyl and hypersensitive to carbofuran. Furthermore, site-directed mutagenesis of the serine residue at position 431 in ace1 from a pirimicarb-susceptible clone of A gossypii conferred insensitivity to pirimicarb. We conclude that AChE1 of A gossypii is the target of toxicological relevance of carbamates and organophosphates.     

Insensitivity of acetylcholinesterase in a field strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith)

Acetylcholinesterase (AChE) was purified from adult heads of the fall armyworm (Spodoptera frugiperda) by using a two-step procedure involving gel filtration on a Sephadex G-200 column and affinity chromatography on a procainamide-ECH Sephadex 4B column. Both susceptible and field strains possessed two AChE isozymes, namely, AChE-1 and AChE-2, with subunit molecular weights of 63.7 and 66.1 kDa. The purified AChE had an apparent K_m value of 33.5 μM and a V_max of 7.07 μmol/min/mg protein in the susceptible strain. The apparent K_m and the V_max were 2.2- and 2.0-fold higher, respectively, in the field strain than in the susceptible strain. The purified AChE from the field strain was 17- to 345-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, eserine, methomyl, and bendiocarb) and organophosphates (methyl paraoxon and paraoxon), insensitivity being highest toward carbaryl. The results further support the notion that insensitive AChE played an important role in the insecticide resistance observed in the field strain.     

桃蚜高效氯氰菊酯抗药性与乙酰胆碱酯酶的关系

于室内对桃蚜进行高效氯氰菊酯抗药性筛选,选育至10代后抗性倍数增长到49.9倍。生化分析表明,抗性品系乙酰胆碱酯酶(AChE)活性均显著高于敏感品系。比较两个品系乙酰胆碱酶活性个体频率分布发现,更多的桃蚜个体向酶活性高的区域分布。酶动力学测定结果显示,抗性桃蚜酯酶对底物的Vmax、Km显著大于敏感品系。     

Insecticide resistance in cotton aphid Aphis gossypii (Glov.) in the Sudan Gezira

The resistance to insecticides of three Sudanese strains of A. gossypii (Glov.) collected from cotton fields in the Sudan Gezira Scheme over three seasons (1988, 1989, 1990) and that of two French strains was studied in the laboratory. When compared with a known susceptible strain, the aphids were found to be resistant-to the eight insecticides tested. Evolution of resistance in Sudanese strains during the three crop seasons was observed. Assay of aphid homogcnate for carboxytesterase activity towards the substrates α-naphthyl acetate and β-naphthyl acetate showed that there was no enhancement of this class of enzyme and thus it was not a cause of resistance in this species. A study of interaction between the acetylcholinesterase (AchE) and pirimicarb established the kinetics of the inhibition process. I₅₀ values were found to be much higher for the Sudanese strains than for the susceptible strain. First-order inhibition kinetics revealed that resistance towards pirimicarb in Sudanese-strains was caused by a modified AchE which had a reduced affinity (higher K_a value) and poor carbamylation ability (lower K₂ value) for pirimicarb. The resistance mechanisms for the other insecticides remain to be studied.     

Cross-resistance potential of fipronil in Musca domestica

The toxicity of fipronil to insecticide-susceptible houseflies and the cross-resistance potential of fipronil were determined for six insecticide-resistant laboratory housefly strains by topical application and feeding bioassay. The insecticide-resistant strains represented different levels and patterns of resistance to pyrethroids, organophosphates, carbamates and organochlorines. Five strains were almost susceptible to fipronil in feeding bioassay with resistance factors at LC50 between 0.36 and 3.0. Four of these strains were almost susceptible to topically applied fipronil (resistance factors at LD50 were 0.55, 0.83, 3.3 and 2.5, respectively), whereas one strain was 13-fold resistant to topically applied fipronil. A highly gamma-HCH-resistant strain, 17e, was 430-fold resistant to fipronil in topical application bioassay and 23-fold resistant in feeding bioassay at LD50/LC50. We also tested the toxicity of fipronil in a feeding bioassay and gamma-HCH in topical application bioassay on thirteen housefly field populations. Eleven of the field populations had resistance factors for fipronil ranging from 0.98 to 2.4 at LC50, whereas two populations were 4.0- and 4.6-fold resistant to fipronil. The resistance level to gamma-HCH at LD50 in the field populations ranged from 1.8- to 8.1-fold. The two strains showing fipronil resistance were 3.4- and 8.1-fold resistant to gamma-HCH. Fipronil and gamma-HCH toxicities were positively correlated in the field populations. Biochemical assays of esterase, glutathione S-transferase and cytochrome P450 monooxygenase indicated that the low fipronil resistance observed in laboratory and field strains could be caused by elevated detoxification or be due to a target-site resistance mechanism with cross-resistance to gamma-HCH.     

Toxicological and biochemical characterizations of AChE in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in the resistant and susceptible strains (SS) of Liposcelis bostrychophila were investigated. The two resistant strains were the dichlorvos-resistant strain (DDVP-R) and the phosphine-resistant strain (PH₃-R) with resistance ratios of 22.36 and 4.51, respectively. Compared to their susceptible counterpart, the AChE activity per insect and the specific activity of AChE in DDVP-R and PH₃-R were significantly higher. There were also significant kinetic differences between DDVP-R and PH₃-R. The apparent Michaelis-Menten constant (K_m) for acetylthiocholine iodide (ATChI) was obviously lower in SS than that in PH₃-R, indicating a higher affinity to the substrate ATChI in the susceptible strains. The affinity for the substrate ATChI in DDVP-R and SS were not significantly different. The V_max value of the PH₃-R was significantly greater when compared to the V_max for the SS suggesting a possible over expression of AChE in this resistant strain. The inhibition of AChE to insecticide exposure in vitro revealed that all six insecticides were inhibitory for the extracted AChE’s. Based on the I₅₀ values, AChE of the SS were more sensitive to dichlorvos, paraoxon-ethyl, malaoxon and demeton-S-methyl than those of the two resistant strains. As for carbaryl and eserine, the PH₃-R suggested a significantly higher I₅₀s compared to the susceptible strain, while, no significant differences were found between SS and DDVP-R.     

Comparison of acetylcholinesterase from three field populations of Liposcelis paeta Pearman (Psocoptera: Liposcelididae): Implications of insecticide resistance

The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in Liposcelis paeta Pearman were investigated in three field populations collected from Nanyang city of Henan Province (NY), Wuzhou (WZ) and Hezhou (HZ) Cities of Guangxi Province, China. The result of bioassay showed that the LC_50s of the NY (281.4802 mg/m²) and the WZ (285.0655 mg/m²) to dichlorvos were 1.156-fold and 1.171-fold higher than that of the HZ (243.5197 mg/m²), respectively. Compared to NY population, the activity per insect and the specific activity of AChE in WZ and HZ populations were significantly higher, and significant kinetic differences among the three populations were also observed. The apparent Michaelis–Menten constant (K_m) for acetylthiocholine iodide (ATChI) was obviously lower in NY than that in WZ and HZ populations, indicating a higher affinity to the substrate ATChI in the NY population. The affinity to the substrate ATChI between WZ and HZ population was also significantly different. As for V_max, the values of WZ and HZ populations were significantly greater when compared to that for NY population, suggesting a possible over expression of AChE in the former two populations. The inhibition studies of AChE indicated that paraoxon-ethyl, demeton-S-methyl, carbaryl, and eserine all possessed some inhibitory effects on AChE in L. paeta. The results of I_50S suggested that when compared to the other two populations, while AChE from HZ population was less sensitive to paraoxon-ethyl and demeton-S-methyl. The contradiction with the result of the bioassay might be due to the different insecticides used in the bioassay. Although both carbaryl and eserine had excellent inhibitory effects, there was no significant difference among the three populations. The statistical analysis of the bimolecular rate constants (k_i) was consistent with the above situation that carbamates expressed remarkable inhibitory effects. It was noticeable that NY population was most sensitive to carbaryl while least to eserine. The differences in AChE among three populations may attribute to the difference in control practices for psocids between Henan and Guangxi Provinces.     

Studies on the acetylcholinesterase of Anopheles albimanus resistant and susceptible to organophosphate and carbamate insecticides

Acetylcholinesterase from fourth instar Anopheles albimanus larvae was studied in vitro. The acetylcholinesterase from both the resistant and susceptible strains behaved as a single enzyme “type,” with straight pseudo first-order insecticide inhibition lines which intersected the Y axis at 100%. The enzyme from resistant larvae was more slowly inhibited than the susceptible enzyme; bimolecular rate constants (k_i) differed by approximately 1.2- to 6-fold for a range of organophosphorous compounds and 17- to 1570-fold for the carbamates. There was a good correlation between the levels of resistance and the acetylcholinesterase inhibition rates.     

An evaluation of the role of oxidative enzymes in Colorado potato beetle resistance to carbamate insecticides

Carbofuran and carbaryl LD₅₀ values were determined with and without piperonyl butoxide pretreatment for a resistant (New Jersey) and two susceptible (Utah and Netherland) populations of Colorado potato beetle larvae. Similar bioassays were conducted with carbofuran for resistant (Rutgers) and susceptible (NAIDM) adult house flies. The degree of resistance development by New Jersey Colorado potato beetles (RR = 848) was greater than that of the laboratory-selected colony of Rutgers house flies (RR = 583). Comparisons of synergist difference calculations including “percentage synergism” (%S), “log percentage synergism” (L%S), and “relative percentage synergism (R%S) for the resistant (R) and the susceptible (S) populations indicated the possibility that monooxygenases and other resistance mechanisms may be involved in Colorado potato beetle resistance to these carbamates. Monooxygenase involvement in resistance of Rutgers house flies was demonstrated in vitro by a 4-fold enhancement of p-nitroanisole O-demethylation over that of NAIDM house flies. O-demethylation of p-nitroanisole could not be demonstrated for potato beetle larvae. Colorado potato beetle resistance was associated with increases in microsomal levels of NADPH-cytochrome c reductase (ca. 2-fold) and NADPH oxidation (1.2-fold). The inability to measure O-demethylation in Colorado potato beetles may have been due to the solubilization of NADPH-cytochrome c reductase during microsomal preparation. Significant differences between resistant and susceptible Colorado potato beetle larvae were not observed in the penetration of [¹⁴C]carbaryl. Excretion of the radiocarbon may have been significantly greater in the resistant New Jersey population, but some of the insecticide may have also rubbed off the cuticle. This increased capacity for excretion, combined with increased levels of monooxygenase enzymes, could account for the high resistance level of this population.     

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification.     

橡胶树重要品系对胶孢炭疽菌抗性评价

橡胶树抗性种质的鉴定与利用是控制橡胶树炭疽病的一项重要措施。采用室内离体橡胶叶接种法、田间种质圃人工接种和自然发病条件下定期田间病害调查方法, 2011-2012年在海南省儋州开展46份重要橡胶树种质对胶孢炭疽菌病抗性鉴定和评价。把抗炭疽病程度划分为高度抗病(HR)、抗病(R)、中度感病(MS)、感病(S)和高度感病(HS)共5个级别,根据抗性评价结果分析得出：高度抗病(HR)类型的0份;抗病(R)类型8份, 占17.39%; 中度感病(MS)类型19份,占41.30%;感病(S)类型16份,占34.78%;高度感病(HS)类型3 份,占6.52%。筛选出的抗病品种,是今后可利用的抗性种质资源。本研究结果为炭疽病抗性种质资源的利用、培育及病害管理提供参考数据。     

Mechanisms for multiple resistances in field populations of common cutworm, Spodoptera litura (Fabricius) in China

The mechanisms for multiple resistances had been studied with two field resistant strains and the selected susceptible and resistant strains of Spodoptera litura (Fabricius). Bioassay revealed that the two field strains were both with high resistance to pyrethroids (RR: 63-530), low to medium resistance to organophosphates and carbamates, AChE targeted insecticides (RR: 5.7-26), and no resistance to fipronil (RR: 2.0-2.2). Selection with deltamethrin in laboratory could obviously enhance the resistance of this pest to both pyrethroids and AChE targeted insecticides. Synergism test, enzyme analysis and target comparison proved that the pyrethroid resistance in this pest associated only with the enhanced activity of cytochrome P450 monooxygenase (MFO) and esterase. However the resistance to the AChE targeted insecticides depended on the target insensitivity and also the enhanced activity of MFO and esterase. Thus, the cross-resistance between pyrethroids and the AChE targeted insecticides was thought to be resulted from the enhanced activity of MFO and esterase.     

Relationship between resistance to pine wilt disease and the migration or proliferation of pine wood nematodes

To study the relationship between resistance to pine wilt disease and the migration or proliferation of pine wood nematodes (PWN) (Bursaphelenchus xylophilus), we conducted experiments using clonally-propagated Japanese black pines (Pinus thunbergii) with pre-evaluated individual resistance levels. Bark including the cortical resin canals—one of the main migration pathways of PWN—was removed by girdling, but neither the migration of PWN nor symptom development of pine wilt disease were inhibited by this treatment in non-resistant clones. Histological observations showed no significant differences in the lumen area or the number of cortical- and xylem- axial resin canals between resistant and susceptible clone groups from a half-sib family. A bioassay using methanol extracts from resistant and susceptible clones showed that extracts from both clones showed similar attractant effects to PWN, but neither had repellent effects. The resistant clones were multi-inoculated with PWN into three split points to mimic migration in the stem. The proportion of damaged plants was not significantly different from that in single-inoculated plants (control). In this experiment, the number of PWN detected from partially-damaged plants was much higher than that from non-damaged plants. An inoculation test using stem cuttings showed that the population of PWN increased in susceptible cuttings at 1–20 days after inoculation (dai), while it remained unchanged or gradually decreased in resistant cuttings. These findings suggested that the factors contributing to resistance were associated with inhibiting the proliferation of PWN, rather than inhibiting their migration.