Antagonistic and synergistic activities of various chemicals on the toxicity of fenarimol to Aspergillus niduluns

A variety of chemicals were tested for their antagonistic, independent, or synergistic effect, on the toxicity of fenarimol to fenarimol-sensitive and resistant strains of Aspergillus nidulans. The chemicals that antagonised fenarimol toxicity included natural lipids and non-ionic surfactants; those with synergistic activity were anionic and cationic agents (including dodine), some conventional fungicides, hydrochloric acid, sodium hydroxide, and respiratory inhibitors. All these synergists also potentiated the toxicity of fenarimol to fenarimol-resistant mutants of A. nidulans. In addition, resistance to fenarimol was negatively correlated with dodine sensitivity. Possible mechanisms of antagonism and synergism are discussed. The results may have implications, for fungicides that inhibit sterol biosynthesis, in respect of their efficacy to plant pathogens.     

海南省芒果可可球二孢对多菌灵的敏感性及菌株适合度研究

针对中国海南地区芒果蒂腐病致病菌可可球二孢对多菌灵的抗性水平及菌株的适合度进行了研究。采用区分剂量法,测定了2016年从海南省芒果园采集、分离的90株可可球二孢对多菌灵的敏感性,并对抗性及敏感菌株在菌丝生长、致病力、渗透压及相对渗率等方面进行了比较。结果表明:海南地区芒果可可球二孢对多菌灵的抗性频率为65.56%,且以高抗菌株为主（8个中抗菌株,51个高抗菌株）;该病菌对芒果危害甚大,其中具强致病力的菌株达65株,占72.22%。高抗菌株的菌丝生长优于敏感菌株,存在极显著差异（P0.01）;抗性菌株的致病力与敏感菌株无显著差异;敏感菌株的相对渗率高于抗性菌株,表明敏感菌株细胞膜的透性较高,胞内电解质渗出较多。研究表明,海南地区芒果可可球二孢对多菌灵的抗性水平较高,且抗性菌株适合度高于敏感菌株,易形成优势群体。     

46份野生葡萄株系对霜霉病的抗性鉴定及其相关基因的表达

为明确不同野生葡萄株系对霜霉病的抗性差异,以18个种的46份野生葡萄株系为试材,采用叶盘法鉴定其对霜霉病的抗性,并利用实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)技术对部分关键基因进行定量分析,探讨其在不同抗病株系中的表达模式差异。结果表明,46份野生葡萄株系的病情指数为0～34.72,其中17份为感病株系,病情指数在25.93～34.72;21份为抗病株系,病情指数在5.32～24.35;5份圆叶葡萄株系均表现为免疫,病情指数为0.00;云南-元谋2、云南-2和木扎岭-3为高抗株系,病情指数分别为1.81、4.40和1.62。当被霜霉病菌侵染后,抗病株系和感病株系中的PAL、PR1、TLP和NPR1基因的诱导表达模式不同;与感病株系相比,抗病株系中的TLP、PR1和NPR1基因有强烈的诱导表达,PAL基因在感病株系比在抗病株系中表达量高。在免疫株系普莱德和高抗株系云南-元谋2中,NPR1与其它3个基因的表达模式差异最大;TLP在抗病株系蘡薁-林县中与其它3个基因的表达模式差异最大;在感病株系秋-嵩县中,NPR1与TLP表达模式相近,PAL和PR1表达模式相近。研究表明,在中国野生葡萄种质中,云南-元谋2、云南-2和木扎岭-3对霜霉病有良好的抗性,可作为抗病育种的原始材料;抗性基因可能在抗病株系中发挥着重要作用。     

Fipronil resistance mechanisms in the rice stem borer, Chilo suppressalis Walker

Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93- and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The K_m and V_max experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed.     

Characterization of an antibacterial substance produced by <Emphasis Type="Italic">Erwinia carotovora</Emphasis> subsp. <Emphasis Type="Italic">carotovora</Emphasis> Ecc 32

A total of 88 strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various host plants in several geographic regions were screened for production of antibacterial substances using the same strains as indicators. Of the 88 strains, 72 produced antibacterial substances. One of these 72 strains, a Brazilian strain Ecc 32, produced an antibacterial substance active against all tested Ecc strains on TSA medium. The antibacterial spectrum of the compound from Ecc 32 strain was limited to closely related strains of soft-rot Erwinia species. Such a narrow spectrum of activity is typical of bacteriocins. The compound produced by Ecc 32 strain, however, was resistant to some enzymes and detergents. Moreover, the compound was heat-stable and active over a wide pH range. The physical characteristics of the compound were not in agreement with those of bacteriocin or carotovoricin.     

In-vivo and in-vitro studies on DDT uptake and metabolism in susceptible and resistant strains of the mosquito Aedes aegypti L.

Twelve strains of Aedes aegypti have been compared for resistance to pp′-DDT, uptake of pp′-DDT, and dehydrochlorination of pp′-DDT to pp′-DDE in vivo and in vitro both at the larval and adult stages. Resistant larvae were shown to contain significantly more pp′-DDE than susceptible larvae after a standard exposure to pp′-DDT but also substantially more pp′-DDT in an unmetabolised state. There was a small increase in the percentage dehydrochlorinated in vivo in the resistant strains compared with the susceptible strains, but this was not correlated with the level of resistance nor with dehydrochlorination in vitro. However, dehydrochlorination in vitro was correlated with resistance. Adult resistance was correlated positively with dehydrochlorination, both in vivo and in vitro, but the resistant adults did not contain increased levels of unmetabolised DDT. By comparing resistance levels at the two stages, it was found that there were two kinds of resistant strain: four strains of Asian origin and one from West Africa were highly resistant as larvae but showed almost no resistance as adults; five strains from Central and South America were highly resistant at both stages. The different mechanisms of resistance in adults and larvae are discussed in relation to genetic studies.     

New Cases of Negative Cross-resistance between Fungicides,Including Sterol Biosynthesis Inhibitors

A survey of fungicide resistance in Mycosphaerella graminicola and Tapesia acuformis, two major pathogens of winter wheat in France, respectively responsible for speckled leaf blotch and eyespot, led to the characterization of two types of resistant strains to sterol 14α-demethylation inhibitors (DMIs). Most of the strains of M. graminicola collected in France in 1997–1998 were resistant to all DMIs, and only in a few strains was the resistance to several triazoles associated with increased susceptibility to pyrimidine derivatives (i.e., fenarimol, nuarimol) and triflumizole. On the other hand, in T. acuformis the most prevalent strains were those which exhibited negative-cross resistance between DMIs. In both fungi such a phenomenon could be related to changes in cytochrome P450 sterol 14α-demethylase, the target site of these fungicides. For Botryotinia fuckeliana, the causal agent of grey mould, the extensive monitoring conducted in French vineyards before the marketing of fenhexamid revealed the presence of highly resistant strains to this promising botryticide (only in tests involving mycelial growth measurements). Negative cross-resistance to edifenphos and several sterol biosynthesis inhibitors, such as prochloraz and fenpropimorph, was observed in fenhexamid resistant strains. Synergism of the antifungal action of fenhexamid by cytochrome P450 inhibitors, such as the DMI fungicides, was only recorded in fenhexamid resistant strains. These data and those previously obtained with edifenphos resistant strains of Magnaporthe grisea (rice blast pathogen) suggest that in fenhexamid resistant strains of B. fuckeliana the same cytochrome P450 monooxygenase could be involved in detoxification of fenhexamid and activation of edifenphos. Received 6 September 1999/ Accepted in revised form 13 September 1999     

Mechanisms of DDT resistance in larvae of the mosquito Aedes aegypti L

Larvae of eight strains of Aedes aegypti were exposed to DDT and compared for resistance, DDT uptake, in-vivo breakdown of DDT and residual unmetabolised DDT. Resistance varied widely between strains, three being fully susceptible, two almost immune and three of intermediate resistance. Breakdown of DDT by dehydrochlorination to 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (pp'-DDE) occurred in all strains and was greater in the five resistant types, but there was no significant correlation between the extent of breakdown in the resistant strains and the level of resistance. Moreover the overall difference between susceptible and resistant strains disappeared when they were compared at a low, almost sublethal, concentration of DDT. Larvae of resistant strains carried a greater absolute quantity of unmetabolised DDT in the body and were able to tolerate levels of DDT that were lethal to susceptible larvae. However the two most resistant strains (T8 and B51) contained significantly less DDT plus pp'-DDE than strains of intermediate resistance (T30 and BSJ) from which they had been derived. Addition of the synergist chlorfenethol to DDT increased its knockdown effect on all resistant strains, suggesting that dehydrochlorination was a factor in resistance. Three strains, two DDT-resistant and one DDT-susceptible, were tested with 1,1-bis(4-ethoxyphenyl)-2,2-dimethylpropane (I), an insecticide that cannot be dehydrochlorinated. All the strains were relatively tolerant to it although the DDT-susceptible strains were less tolerant. Addition of the synergist sesamex decreased the level of tolerance to I in all strains which suggested that microsomal oxidation made some contribution to it. It is concluded that three factors contribute to larval DDT resistance in A. aegypti; (a) increased metabolism to pp'-DDE; (b) increased tolerance to unmetabolised internal DDT; and (c) reduced content of DDT+pp'-DDE (only in the most resistant strains and due either to reduced absorption or increased excretion). These factors are discussed in relation to known larval resistance genes R^DDT1 and y.     

Results of seven years of biological control of Agrobacterium tumefaciens in Spain1

Biological control of Agrobacterium tumefaciens using the K84 strain of A. radiobacter has been practised in Spain since 1979. Strain K84 is produced in Sevilla by the Agricultural Research Service and is mixed with peat. This formulation is authorized for use on stone fruits and roses, since results of various trials on rootstocks of these crops have shown that K84 is effective in controlling the disease in naturally infected nursery soil, as well as in experimental tests carried out on artificially inoculated soil. Results of biological control trials have shown that K84 has some effect on A. tumefaciens strains resistant to agrocin 84. Factors other than susceptibility to agrocin 84 influence the efficacy of biological control. This was demonstrated by using a K84 agrocin non-producer strain which proved effective against both resistant and susceptible strains. Biological control on apple or pear is not yet authorized in Spain due to a paucity of data on field trials. However, initial pot tests showed good K84 activity on these hosts, against Spanish strains of A. tumefaciens. On grapevine, most of the A. tumefaciens strains that were isolated belong to biotype 3 and are resistant to agrocin 84. Because the disease is systemic on this species the use of K84 is not authorized.     

抗氟吡菌胺辣椒疫霉菌株的诱导及其生物学特性的研究

 采用菌丝生长速率法, 测定了采自泰安、平谷、杭州和昆明4个地区的42株辣椒疫霉病菌对氟吡菌胺的敏感性, 结果表明, 其EC₅₀值在0.618 ~0.927 μg·mL^-1之间, 平均EC₅₀ =(0.743±0.067 7) μg·mL^-1。 42个菌株对氟吡菌胺的敏感性分布呈单峰曲线, 未出现抗性的病原菌亚群体, 可将其单峰曲线作为辣椒疫霉对氟吡菌胺的敏感性基线。采用药剂驯化和紫外照射对辣椒疫霉敏感菌株TA进行诱变处理, 获得了2株辣椒疫霉抗氟吡菌胺菌株;利用菌落直径法测定抗氟吡菌胺菌株的遗传稳定性、菌丝生长速率及对其它杀菌剂的交互抗性;通过活体叶盘法测定抗氟吡菌胺菌株和敏感菌株的致病力及产孢子囊能力, 分析抗性和敏感菌株之间生物学特性差异。结果表明, 敏感菌株TA经过氟吡菌胺 48代连续汰选, 其抗性达58.0倍, 获得中抗菌株TA-R;紫外照射获得了抗性达260.6倍的高抗菌株TA-UV, 且其抗性均能稳定遗传;TA-R和TA-UV对甲霜灵、霜脲氰表现出正交互抗性, 对烯酰吗啉、百菌清、代森锰锌和丙森锌无交互抗性;抗氟吡菌胺菌株TA-R、TA-UV与敏感菌株在活体叶盘上的致病力、离体产孢能力均差异不显著(P＜0.05), 但其菌丝生长速率、菌丝干重均低于敏感菌株。     

Resistance to fungicides which inhibit ergosterol biosynthesis in laboratory strains of Botrytis cinerea and Ustilago maydis

The strains of Botrytis cinerea or Ustilago maydis selected on fenarimol, triarimol, or triadimefon were also resistant to the other inhibitors of sterol C-14 demethylation; the sterol composition of the strains was normal. Among the isolates of U. maydis resistant to dodemorph, fenpropidin, fenpropimorph and tridemorph, some were resistant to the 15-azasteroid A 25822B and did not contain ergosterol. The other strains remained sensitive to A 25822B and had a normal sterol composition. All the resistant isolates and the wild-type were inhibited to the same extent by nystatin and pimaricin.     

Fipronil resistance mechanisms in the rice stem borer,Chilo suppressalis Walker

Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93- and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The K_m and V_max experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed.     

Potential loss of clubroot resistance genes from donor parent Brassica rapa subsp. rapifera (ECD 04) during doubled haploid production

Clubroot resistance derived from the oilseed rape/canola Brassica napus ‘Mendel’ has been overcome in some fields in Alberta, Canada, by the emergence of ‘new’ strains of the protist Plasmodiophora brassicae. Resistance to the pathogen was assessed in 112 doubled haploid (DH) lines, derived from B. rapa subsp. rapifera (European clubroot differential (ECD) 04). The lines were evaluated against five single‐spore isolates representing the ‘old’ pathotypes 2, 3, 5, 6 and 8, and 15 field populations representing new strains of P. brassicae. The disease severity index (ID%) data revealed that none of the DH lines were resistant or moderately resistant to the new pathotype 5X (field populations L‐G1, L‐G2, L‐G3) and D‐G3, while 3–42% were resistant or moderately resistant to the other 11 new strains. Using the mean ID induced by the old pathotype 3 (approx. 13.5%) as the baseline, clubroot severity increased by 300–600% when inoculated with the new pathotypes. A significant finding of this study was the fact that ECD 04 showed absolute resistance to all of the old and new P. brassicae strains while the B. napus ‘Mendel’, although resistant to all of the old pathotypes, was resistant to only about 50% of the new strains. Similarly, all of the selected clubroot‐resistant commercial canola cultivars evaluated in this study were susceptible to 87% of the new P. brassicae strains. The molecular data revealed that the breakdown of clubroot resistance in Mendel and the canola cultivars was in part due to the non‐inheritance of the Crr1 gene on the A08 chromosome from ECD 04.     

月季根癌病病原菌分离及抗病资源初步筛选

从月季品种金玛丽、曼海姆、杏花村、梅郎口红的根部肿瘤组织中纯化了9株分离物,根据其在MW选择性培养基上的单菌落形态初步判断其为根癌土壤杆菌。以根癌土壤杆菌高度保守的virD2和ipt基因的部分序列设计引物对分离的菌株进行PCR检测,其中有6株菌株能扩增出virD2和ipt基因片段,为根癌土壤杆菌毒性菌株。采用针刺涂抹法接种向日葵、荷花蔷薇幼茎,6株菌株均能在供试植株上形成肿瘤,一个月后肿瘤大小有显著差异,说明分离获得的根癌土壤杆菌株毒性有差异。以强毒性菌株J-5-1在田间接种了部分蔷薇属野生资源,以鉴定其对根癌病的抗性。根据发病率、肿瘤大小、肿瘤干质量以及感病后植株生长状况将植物的根癌病抗性分为高度抗病、中度抗病、中度感病、高度感病4个类型。     

Metabolic resistance mechanisms to phosalone in the common pistachio psyllid, Agonoscena pistaciae (Hem.: Psyllidae)

The common pistachio psyllid, Agonoscena pistaciae, is the most damaging pest of pistachio in Iran, and is generally controlled by insecticides belonging to various classes especially, phosalone. The toxicity of phosalone in nine populations of the pest was assayed using the residual contact vial and insect-dip methods. The bioassay results showed significant discrepancy in susceptibility to phosalone among the populations. Resistance ratio of the populations to the susceptible population ranged from 3.3 to 11.3. The synergistic effects of TPP, PBO and DEM were evaluated on the susceptible and the most resistant population to determine the involvement of esterases, mixed function oxidases and glutathione S-transferases in resistance mechanisms, respectively. The level of resistance to phosalone in the resistant population was suppressed by TPP, PBO and DEM, suggesting that the resistance to phosalone is mainly caused by esterase detoxification. Biochemical enzyme assays revealed that esterase, glutathione S-transferase and cytochrome P450 monooxygenase activities in the resistant population was higher than that in the susceptible. Glutathione-S-transferases play a minor role in the resistance of the pest to phosalone.     

An electrophysiological investigation of the susceptible (cooper) and resistant (kdr; super-kdr) strains of the adult housefly,Musca domestica L

The electrical activity of abdominal nerves of the housefly, Musca domestica L., was used as a bioassay to study nerve sensitivity to DDT and deltamethrin in susceptible (Cooper) and resistant (kdr, super-kdr) strains. By this technique the resistant strains were less sensitive (approximately 10 000-fold) than Cooper, but the bioassay could not distinguish between super-kdr and kdr in their responses to either compound and so could not account for the greater resistance shown by flies with super-kdr above kdr flies when these insecticides are applied topically. Although factors other than nerve insensitivity may be involved, the compounds were applied to the preparation in aqueous saline solutions at, or close to, their solubility limits and this could have masked differences in responses of nerves from the resistant strains.     

Stability and Biological Activity of Cyfluthrin Isomers

A GC-MS method capable of completely separating the four pairs of diastereoisomers of cyfluthrin is presented and the method used to show that isomerisation of the cyfluthrin enantiomers occurs in methanol. This methanol-induced isomerisation could also be demonstrated by bioassays using water fleas. The biological activities of the various cyfluthrin isomers contained in the commercial products cyfluthrin and beta-cyfluthrin were assayed using several strains of lepidopteran larvae including Plutella xylostella, Heliothis virescens and Spodoptera frugiperda. With the susceptible strains, the efficiencies of the isomers mixtures of cyfluthrin and beta-cyfluthrin were shown to obey the rules of additivity. However, in tests with a resistant strain of P. xylostella originating from Thailand, the ‘inactive’ isomer III acted synergistically with the active isomer IV. Resistance factors in strains of H. virescens and P. xylostella were found to be higher with cis than with trans isomers. This probably contributes to the superior action of cyfluthrin and beta-cyfluthrin against various pests of agricultural importance since the commercial products contain a high content of trans isomers (‘high trans pyrethroids’).     

Distribution of benzimidazole-resistant strains of the onion gray-mold neck rot pathogens,Botrytis aclada and Botrytis allii,in Hokkaido,Japan

Botrytis aclada and Botrytis allii, associated with onion gray-mold neck rot and isolated in Hokkaido, were tested for sensitivity to benzimidazole. Of the B. aclada strains, 59% were highly resistant and the remaining 41% were sensitive; all strains of B. allii were sensitive. Resistant strains were widespread in Hokkaido. We analyzed the sequences of the β-tubulin gene of resistant strains and detected the replacement of glutamic acid (GAG) by lysine (AAG) at codon 198. This is the first report of benzimidazole resistance in B. aclada. This study revealed a difference in fungicide sensitivity between the two Botrytis species.

     

Streptomycin resistance in Clavibacter michiganensis subsp. michiganensis strains from Chile is related to an rpsL gene mutation

Streptomycin has been used for decades in Chile to control Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of tomato bacterial canker. The aim of this work was to evaluate streptomycin resistance and to analyse the presence of resistance-related genes in Cmm strains from Chile. A collection of 25 Cmm strains isolated from different localities in central Chile between 1996 and 2015 was analysed. Minimum inhibitory concentration (MIC) of streptomycin was determined. A search of streptomycin resistance-related genes was carried out in Cmm genomes, and the presence of these genes was studied in all Chilean strains using PCR and sequencing techniques. MIC results showed that four of 25 strains were highly sensitive to streptomycin, with MIC values <2 μg mL⁻¹. The remaining 21 strains possessed MIC of streptomycin ≥100 μg mL⁻¹. The strB gene, encoding an aminoglycoside 6-phosphotransferase that inactivates streptomycin, was detected in all Chilean strains, including sensitive and resistant strains. In the 21 resistant strains, a mutation in codon 43 of the rpsL gene was determined, conferring high streptomycin resistance. Interestingly, the four streptomycin-sensitive Cmm strains did not possess this mutation. This study proposes that the continuous use of streptomycin leads to emergence of resistant Cmm strains, challenging researchers to look for novel alternatives to control this plant pathogenic bacterium.     

Properties of dicarboximide-resistant strains of Botrytis cinerea

Dicarboximide-resistant strains of Botrytis cinerea were isolated from natural substrates and also produced in the laboratory. All these strains exhibited a similar degree of resistance to the dicarboximide fungicides iprodione and vinclozolin, and this resistance persisted in the absence of the fungicides. The natural frequency of resistance to both chemicals was approximately three in 10⁷ conidia but could be enhanced by up to 1000 times after a single exposure to sub-lethal concentrations of either chemical. Mycelium of the resistant strains was able to infect fruit and vegetables to a similar extent as that of sensitive strains, although infection of carrot roots was markedly less aggressive. The resistant strains were separable into two groups according to their growth habit on culture media. Both groups were relatively slow growing and showed a marked lack of sporulation compared with most sensitive strains. This lack of sporulation may account for the apparent failure of resistant strains to increase rapidly in strawberry plantations that had received dicarboximide sprays in successive seasons.