A High Multi-drug Resistance to Chemically Unrelated Oomycete Fungicides in Phytophthora infestans

Mutants of Phytophthora infestans with high resistance to the amidocarbamates iprovalicarb and benthiavalicarb and to the cyanoimidazole cyazofamid were isolated after UV-mutagenesis and selection on media containing one of the above fungicides. In vitro fungitoxicity tests showed that all resistant strains presented a highly reduced sensitivity to both cyazofamid and to the amidocarbamates. Cross-resistance studies with other oomycete fungicides from different chemical groups showed that the mutation(s) for resistance to iprovalicarb (IPV), benthiavalicarb (BVC) and cyazofamid (CZF) also greatly reduced the sensitivity of mutant strains to the phenylamide metalaxyl, acetamide cymoxanil, morpholine dimethomorph, benzamide zoxamide and to chlorothalonil. A lower reduction of sensitivity of mutant strains to the strobilurins azoxystrobin, kresoxim-methyl, pyraclostrobin and trifloxystrobin, azolones famoxadone and fenamidone and to antimycin A was observed. A resistance correlation was not apparent for the dithiocarbamate propineb and phenylpyridinamine fluazinam. Studies of fitness parameters in the wild-type and mutant strains of P. infestans showed that most resistant isolates had significantly reduced sporulation and sporangial germination, but not in the differentiation of sporangia into zoospores. Pathogenicity tests on tomato seedlings showed that most resistant isolates were significantly less pathogenic compared to the wild-type parent strain. However, experiments on the stability of the resistant phenotypes did not show a reduction in resistance when the mutants were grown for more than eleven generations on inhibitor-free medium. This is believed to be the first report of high level multi-drug resistance in fungal pathogens to chemically unrelated fungicides inhibiting different sites of cellular pathway.     

马铃薯晚疫病菌对烯酰吗啉的敏感性基线及其室内抗药突变体的研究

 2003-2004年在内蒙古和黑龙江省共分离获得100株马铃薯晚疫病菌,并测定了这些菌株对烯酰吗啉和甲霜灵的敏感性。结果表明,不同地区、不同年份的马铃薯晚疫病菌对烯酰吗啉的敏感性无显著差异,其敏感性呈连续的单峰曲线分布,EC₅₀在0.0631-0.2206μg/mL之间,最不敏感菌株是最敏感菌株的3.1倍,平均为(0.1153±0.0308)μg/mL,未出现敏感性下降的抗药性亚群体,因此可作为马铃薯晚疫病菌对烯酰吗啉的敏感性基线;64%的供试菌株对甲霜灵的敏感性下降,甲霜灵与烯酰吗啉之间不存在交互抗药性。采用紫外线诱变菌丝的方法仅从3个菌株中的1个菌株获得了3%突变频率的抗烯酰吗啉突变体,抗性倍数为1.5-1.9,没有获得高抗药水平的突变体。这些突变体与氟吗啉之间具有正交互抗药性,而与霜脲氰或嘧菌酯之间无交互抗药性;与野生亲本菌株相比,抗药突变体的生长速率和产孢子囊能力显著下降,不利于抗药群体的发展。但由于马铃薯晚疫病菌被杀菌剂抗性行动委员会认为是高抗药性风险病原菌,建议生产上应将烯酰吗啉与其它无交互抗性的药剂交替使用或混合使用,以避免或延缓抗药性的产生。     

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。     

Characterization of Laboratory Mutants of Botrytis cinerea Resistant to QoI Fungicides

Mutants of Botrytis cinerea with moderate and high resistance to pyraclostrobin, a Qo inhibitor of mitochondrial electron transport at the cytochrome bc ₁ complex, were isolated at a high mutation frequency, after nitrosoguanidine mutagenesis and selection on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strain by pyraclostrobin and SHAM, but not in the mutant isolates. Cross-resistance studies with other Qo and Qi inhibitors (QoIs and QiIs) of cytochrome bc ₁ complex of mitochondrial respiration showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other QoIs as azoxystrobin, fluoxastrobin, trifloxystrobin and picoxystrobin, but not to famoxadone and to the QiIs cyazofamid and antimycin-A. An increased sensitivity of pyraclostrobin-resistant strains to the carboxamide boscalid, an inhibitor of complex II, and to the anilinopyrimidine cyprodinil, a methionine biosynthesis inhibitor, was observed. Moreover, no effect of pyraclostrobin resistance mutation(s) on fungitoxicity of the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the benzimidazole benomyl, and to the phenylpyridinamine fluazinam, which affect other cellular pathways, was observed. Study of fitness parameters in the wild-type and pyraclostrobin-resistant mutants of B. cinerea showed that most mutants had a significant reduction in the sporulation, conidial germination and sclerotia production. Experiments on the stability of the pyraclostrobin-resistant phenotype showed a reduction of resistance, mainly in moderate resistant strains, when the mutants were grown on inhibitor-free medium. However, a rapid recovery of the resistance level was observed after the mutants were returned to a selective medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of high resistant mutants was higher than the moderate ones. Pathogenicity tests on cucumber seedlings showed that all mutant strains tested exhibited an infection ability similar with the wild-type parent strain. Preventive applications of the commercial product of F-500 25EC (pyraclostrobin) were effective against lesion development on cotyledons by the wild-type, but ineffective, even at high concentrations, against disease caused by the pyraclostrobin-resistant isolates. Boscalid (F-510 50WG) was found equally effective against the disease caused by the wild-type or pyraclostrobin-resistant mutants. This is the first report indicating the appearance of B. cinerea strains resistant to QoI fungicides by the biochemical mechanism of site modification and the risk for field resistance.     

Effect of Phenylpyrrole-resistance Mutations on Ecological Fitness of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and their Genetical Basis in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Mutants of Botrytis cinerea and Ustilago maydis highly resistant to fludioxonil were isolated at a high frequency, after nitrosoguanidine or UV mutagenesis, respectively, and selection on media containing fludioxonil. Tests on the response of mutant strains to high osmotic pressure resulted in the identification of two fludioxonil-resistant phenotypes (FLD_osm/s and FLD_osm/r), regarding the sensitivity to high osmolarity. Approximately 95% of fludioxonil-resistant mutants were found to be more sensitive to high osmotic pressure than the wild-type parent strains. Genetic analysis of phenylpyrrole-resistance in the phytopathogenic basidiomycete U. maydis, showed that fludioxonil-resistance was coded by three unlinked chromosomal loci (U/fld-1, U/fld-2 and U/fld-3), from which only the U/fld-1 mutation coded an osmotic sensitivity similar to that of the wild-types. Cross-resistance studies with fungicides from other chemical groups showed that the mutations for resistance to phenylpyrroles affect the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides, but not to the benzimidazoles, anilinopyrimidines, phenylpyridinamines, hydroxyanilides or the sterol biosynthesis inhibiting fungicides. A study of fitness parameters in the wild-type and fludioxonil-resistant mutants of B. cinerea, showed that all osmotic sensitive (B/FLD_osm/s) isolates had significant reductions in the characteristics determining saprophytic fitness such as mycelial growth, sporulation, conidial germination and sclerotial production. Contrary to that, with the exception of mycelial growth, the fitness parameters were unaffected or only slightly affected in most of the osmotic resistant (B/FLD_osm/r) isolates. Tests on cucumber seedlings showed that the osmotic-sensitive strains were significantly less pathogenic compared with the wild-type and B/FLD_osm/r strains of B. cinerea. Preventative applications of the commercial products Saphire 50 WP (fludioxonil) and Rovral 50 WP (iprodione) were effective against lesion development on cotyledons by the wild-type and the mutant strains of B. cinerea that were resistant to the anilinopyrimidine cyprodinil (B/CPL-27) and to the hydroxyanilide fenhexamid (B/FNH-21), but ineffective, even at high concentrations, against disease caused by the fludioxonil-resistant isolates (B/FLD) and a mutant strain resistant to the dicarboximide iprodione (B/IPR-1). Experiments on the stability of the fludioxonil-resistant phenotype showed a reduction of resistance, mainly in osmotic-sensitive isolates, when the mutants were grown on inhibitor-free medium. A rapid recovery of the high resistance was observed after mutants were returned to the selection medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain of B. cinerea, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of osmotic-resistant mutants was higher than the osmotic-sensitive ones. Furthermore, competition tests, in planta, showed a significant reduction of the frequency of both phenylpyrrole-resistant phenotypes, with a respective increase in the population of the wild-type strain of the pathogen.     

辣椒疫霉对烯酰吗啉的敏感性基线及室内抗药突变体研究

 采用菌丝生长速率法测定了125株采自河北、内蒙古、陕西、安徽和北京等地区的辣椒疫霉病菌对烯酰吗啉的敏感性,结果表明,其EC₅₀值分布于0.126~0.318μg/mL之间,最不敏感菌株是最敏感菌株的2.5倍,平均EC₅₀=(0.218±0.0368)μg/mL。125个菌株对烯酰吗啉的敏感性分布呈单峰曲线,未出现抗性的病原菌亚群体,可将该单峰曲线作为辣椒疫霉对烯酰吗啉的敏感性基线,将烯酰吗啉对该病原群体的平均EC₅₀值作为田间抗药性监测的参考标准。通过紫外诱变敏感菌株N-7的菌丝获得了12株抗烯酰吗啉的突变体,抗性指数在16.38~132.15之间;通过紫外诱变敏感菌株DZ-16的游动孢子获得1株抗性指数为680倍的高抗药水平的突变体。突变体的部分生物学性状研究表明,其致病力与敏感菌株相当;大部分突变体产生孢子囊的能力与亲本菌株相比均有不同程度的提高;离体条件下,突变体和亲本菌株释放游动孢子的能力相当;突变体的菌丝生长速率与亲本菌株相比具有不同程度的差异。测定了敏感亲本菌株和突变体的交配型,均为A1型菌株,经紫外诱变后交配型没有发生改变。综合分析表明,抗性突变体的产生有利于抗药性群体的发展。为避免和延缓抗药性的产生,生产上应将烯酰吗啉与其它无交互抗药性的杀菌剂交替使用。     

The occurrence, characteristics and distribution in the United Kingdom of resistance to phenylamide fungicides in Bremia lactucae (lettuce downy mildew)

Failure to control Bremia lactucae (lettuce downy mildew) with metalaxyl in an intensive lettuce-producing region of Lancashire at the end of 1983 was shown to be due to the occurrence of a high level of resistance to this fungicide (isolates capable of growth at < 100 μg/ml metalaxyl). During most of 1984, metalaxyl-resistant isolates were obtained from numerous sites but all within a 20-km radius of the initial outbreak. Thereafter, at the end of 1984 and during 1985, metalaxyl-resistant isolates were recovered from most major lettuce-producing regions in the UK with protected crops more affected than field crops. AH metalaxyl-resistant isolates tested were identical in their response to fungicide, sexual compatibility type (B2) and virulence phenotype, probably representing a clone from a single origin. The resistant pathotype was virulent on resistance factors R 1-10 and 12-15 but lacked virulence for R 11 and 16-18. This was also the most common virulence phenotype among sensitive isolates collected at the same time. Cross-resistance to other phenylamide fungicides was demonstrated but isolates resistant and sensitive to phenylamide showed a similar response to the unrelated systemic fungicides propamocarb and fosetyl-Al. An F₁ sexual progeny isolate from a cross between a phenylamide-sensitive and a phenylamide-resistant isolate (presumed heterozygous at the locus or loci regulating response to phenylamide fungicides) exhibited an intermediate response to phenylamide fungicides. No isolates of this type were obtained from the field. At the high concentrations affecting spore germination, phenylamide fungicides exhibited lower activity against a resistant isolate compared with a sensitive isolate. The findings are discussed in relation to future control strategies, the population biology of the fungus and possible directions for lettuce breeding programmes.     

黄瓜霜霉病菌抗氟吗啉菌株的适合度研究

 采用离体叶盘法和活体植株法,测定了从田间分离获得的对氟吗啉具有抗药性的黄瓜霜霉病菌菌株和通过室内紫外线诱变获得的对氟吗啉及对照药剂烯酰吗啉和嘧菌酯产生抗药性的突变菌株的生存适合度。试验结果表明,室内诱导获得的氟吗啉和烯酰吗啉抗药性突变菌株的适合度明显低于敏感菌株和嘧菌酯抗药性突变体及田产氟吗啉抗药性菌株,主要表现在菌株的侵染率、产孢能力和致病力的减弱。室内获得的氟吗啉和烯酰吗啉抗药性突变体和敏感菌株混合接种、继代培养后,其抗药菌株的检出率在混合群体中逐渐降低,其中抗性突变体FR4和敏感菌株以2:8的比例混合接种、培养7代后,其无性后代中检测不到抗药性菌株的存在,表明室内氟吗啉抗药性突变体竞争力弱于敏感菌株;而田间氟吗啉抗药性菌株和室内嘧菌酯抗药性突变体则表现出和敏感菌株相似或更强的竞争力,预示田间自然情况下抗药性菌株存在着可能形成优势菌群而导致药剂防治失败的潜在风险。     

Assessing the risk of resistance in Pseudoperonospora cubensis to the fungicide flumorph in vitro

BACKGROUND: The oomycete fungicide flumorph is a recently introduced carboxylic acid amide (CAA) fungicide. In order to evaluate the risk of developing field resistance to flumorph, the authors compared it with dimethomorph and azoxystrobin with respect to the ease of obtaining resistant isolates to these fungicides, the level of resistance and their fitness in the laboratory. RESULTS: Mutants with a high level of resistance to azoxystrobin were isolated readily by adaptation and UV irradiation, and their fitness was as good as that of the parent isolates. Attempts to generate mutants of Pseudoperonospora cubensis (Burk. & MA Curtis) Rostovsev resistant to flumorph and dimethomorph by sporangia adaptation on fungicide-treated leaves were unsuccessful. However, moderately resistant mutants were isolated using UV mutagenesis, but their resistance level [maximum resistance factor (MRF) < 100] was much lower than that of the azoxystrobin-resistant mutant (MRF = 733). With the exception of stability of resistance, all mutants showed low pathogenicity and sporulation compared with wild-type isolates and azoxystrobin-resistant mutants. There is cross-resistance between flumorph and dimethomorph, suggesting that they have the same resistance mechanism. CONCLUSION: The above results suggest that the resistance risk of flumorph may be similar to that of dimethomorph but lower than that of azoxystrobin and can be classified as moderate. Thus, it can be managed by appropriate product use strategies.     

Biological and molecular characterization of laboratory mutants of Cercospora beticola resistant to Qo inhibitors

The resistance to strobilurin-related fungicides and its molecular basis in laboratory mutant isolates of Cercospora beticola was investigated. After ultraviolet mutagenesis, mutants with high, moderate or low resistance levels to pyraclostrobin were isolated from a wild-type strain of C. beticola. Fungitoxicity tests on the response of resistant isolates on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration, indicated that the biochemical mechanism of alternative oxidase was not responsible for the reduced sensitivity to pyraclostrobin for half of the mutants. Cross-resistance studies with other inhibitors of the cytochrome bc ₁ complex of the mitochondrial respiratory chain showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other Qo inhibitors such as azoxystrobin and fenamidone, but not to the Qi inhibitor cyazofamid. No effect of pyraclostrobin-resistant mutation(s) on fungitoxicity of the carboxamide boscalid, the triazoles epoxiconazole and flutriafol and to the benzimidazole benomyl, which affect other cellular pathways or other steps of the respiratory chain, was observed. Study of fitness parameters showed that most mutants had a significant reduction in sporulation and pathogenicity compared to the wild-type parental isolate. However, experiments on the stability of the resistant phenotype did not show a significant reduction of the resistance for half of the mutants when grown for at least four generations on pyraclostrobin-free medium. Molecular analysis of cytochrome b cDNA, isolated from the wild-type and the pyraclostrobin-resistant mutant isolates, revealed two novel amino acid replacements at positions involved in Qo resistance in other species. The glycine (GGT) to serine (AGT) replacement at position 143 (G143S) was found in the isolate with the highly resistant phenotype. The second amino acid change was the replacement of phenylalanine (TTC) by valine (GTC) at position 129 (F129V), which was found in a mutant strain with the moderately resistant phenotype. Four additional mutations located in conserved regions of the mitochondrial cytochrome b gene (I154L, N250D, E256G and V261D) were detected in some mutant isolates of C. beticola but their possible role in Qo-resistance needs further investigation. This is the first study reporting C. beticola strains resistant to Qo inhibitor fungicides due to the biochemical mechanism of target-site modification, resulting from amino acid changes in the mitochondrial cytochrome b␣gene.     

In vitro selection of sterol-biosynthesis-inhibitor (SBI)-resistant mutants in Monilinia fructicola (Wint.) Honey

The inherent resistance risk forMonilinia fructicola against sterol-biosynthesis inhibitors (SBIs) was estimated inin vitro andin vivo laboratory studies. Several mutant strains were selected on media amended with the triazole fungicides penconazole, etaconazole or the morpholine fungicide fenpropimorph.The potential forM. fructicola to develop resistance to the triazoles or to the morpholines was similar.The level of resistance attained did not differ for the two classes of fungicides after a single cycle of treatment with nitrosoguanidine (NTG). Attemps to select mutants with a higher level of resistance to penconazole after successive mutagenic treatments were successful. Most of the mutants were less fit than wild-type strains. Mutants with a low level of resistance had an almost normal mycelial growth rate, whereas growth of mutants with a higher level of resistance was significantly reduced. Spore production was highest in the wild-type strains, similar to the latter in a few resistant strains and less in most others. Only one mutant with an intermediate level of resistance could successfully compete in a mixed population with a wild type strain during successive infection cycles on peaches. Resistance was not stable in highly resistant mutants. Cross resistance to the inhibitors of 14-methylsterol demethylation (DMIs) tested was confirmedin vitro andin vivo for all mutant strains. One DMI-resistant mutant was also resistant to fenpropimorph and two fenpropimorph-resistant mutants were resistant to penconazole.     

Characterization and genetic analysis of laboratory mutants of <Emphasis Type="Italic">Cochliobolus heterostrophus</Emphasis> resistant to dicarboximide and phenylpyrrole fungicides

 Laboratory mutants of Cochliobolus heterostrophus resistant to iprodione were obtained after chemical mutageneses. All the mutants were able to grow on the medium amended with iprodione 100 μg/ml. They showed positive cross-resistance to procymidone and fludioxonil and were sensitive to high osmolarity. Crosses between the mutant and a wild-type strain revealed that the fungicide resistance and osmotic sensitivity traits were inherited by their offspring in a 1 : 1 mutant/wild type ratio, indicating that the mutant phenotypes in these strains were due to alteration at a single gene locus. Results from allelism tests indicated that three genes (Dic1, Dic2, Dic3) conferred the mutant phenotypes. Among them, Dic1 mutant strains were classified into three types on the basis of their phenotypes. The first type was moderately resistant to the fungicides and less sensitive to osmotic stress than the other Dic1 mutant strains. The second type showed moderate fungicide resistance, but growth was inhibited under lower osmotic stress (50 mM KCl). The other Dic1 mutant strains grew well on medium containing iprodione and fludioxonil even at a concentration of 100 μg/ml and were highly sensitive to osmotic stress. The Dic2 and Dic3 mutant strains had moderate resistance to the fungicides with low-level osmotic sensitivity. The Dic1 gene was epistatic to Dic2 and Dic3 for fungicide resistance and hypostatic to them for osmotic sensitivity. These results suggest that the osmoregulatory system is involved in fungicide resistance in laboratory mutants of C. heterostrophus. Received: March 14, 2002 / Accepted: August 13, 2002     

山西省清徐县葡萄霜霉菌对烯酰吗啉的抗药性分析

葡萄霜霉病是葡萄最重要的病害之一,目前防治霜霉病主要依赖化学药剂,其中羧酸酰胺类杀菌剂是一类重要药剂,然而病原菌抗药性对其防效有重要影响。2015年本实验室首次在中国检测到抗烯酰吗啉的葡萄霜霉菌,并开发了Tetra-Primer ARMS PCR快速检测法来检测抗药性。本研究利用此技术对采自山西省清徐县60株葡萄霜霉菌进行了烯酰吗啉抗性检测,结果显示其中1株具有抗药性,其他59株均为敏感菌株,说明该地区霜霉菌已经开始产生烯酰吗啉抗性,但尚未大范围扩展,需要进行持续监测以指导杀菌剂的使用,这也是首次报道山西省葡萄霜霉菌存在烯酰吗啉抗性菌株。     

Laboratory studies to assess the risk of development of resistance to zoxamide

Laboratory studies were conducted to evaluate the risk of developing field resistance to zoxamide, a new Oomycete fungicide which acts on microtubules. Zoxamide, metalaxyl and dimethomorph were compared with respect to the ease with which fungicide‐resistant mutants could be isolated and their level of resistance. Attempts to generate mutants of Phytophthora capsici and P infestans with resistance to zoxamide by mycelial adaptation on fungicide‐amended medium were unsuccessful. Similarly, changes in sensitivity to zoxamide were small (resistance factors ≤2.2) in mutants of P capsici isolated by chemical mutagenesis of zoospore cysts. In parallel experiments with metalaxyl, highly resistant mutants were obtained using both adaptation (P capsici or P infestans) and chemical mutagenesis (P capsici). For dimethomorph, chemical mutagenesis (P capsici) yielded moderately resistant mutants (maximum resistance factor = 20.9), and adaptation (P capsici or P infestans) did not induce resistance. It is proposed that failure to isolate mutants resistant to zoxamide results from the diploid nature of Oomycete fungi and the likelihood that target‐site mutations would produce a recessive phenotype. Our studies suggest that the risk of a highly resistant pathogen population developing rapidly in the field is much lower for zoxamide than for metalaxyl. However, as with any site‐specific fungicide, appropriate precautions against resistance development should be taken. © 2001 Society of Chemical Industry     

Resistance to inhibitors of sterol biosynthesis in field isolates or laboratory strains of the eyespot pathogen Pseudocercosporella herpotrichoides

Strains of Pseudocercosporella herpotrichoides collected in France on winter wheat give either fast-growing mycelial colonies with regular margins or slow-growing mycelial colonies with irregular margins. Most of the fastgrowing isolates were sensitive to triadimenol (EC₅₀ below 2mg litre^?1), but some of them were resistant to this inhibitor of sterol C-14 demethylation. In contrast, all the slow-growing strains were highly resistant to triadimenol (EC₅₀ greater than 100 mg litre^?1). This resistance was also expressed in inhibition of germ-tube elongation. Positive cross-resistance was observed between most of the inhibitors of sterol C-14 demethylation, with the exception of some imidazole derivatives (clotrimazole, prochloraz). All the fast-growing strains were tolerant to fenpropimorph and fenpropidin whereas the slow-growing ones were susceptible; the reverse was true with piperalin and tridemorph. All the field isolates were inhibited to the same extent by the inhibitors of squalene-epoxidase, nafifine and terbinafine. Two types of mutant resistant to triadimenol have been induced under laboratory conditions from sensitive fast-growing strains. The most common mutants were resistant to all the inhibitors of sterol C–14 demethylation and also in some conditions to fenpropimorph, tridemorph and the inhibitors of squalene-epoxidase. The other mutants were characterised by a reduced spectrum of cross-resistance between triadimenol and the other inhibitors of sterol biosynthesis. The field isolates and laboratory mutants resistant to triadimenol and propiconazole were also resistant to each of the four enantiomers of these two fungicides.     

江西省番茄绵疫病菌对嘧菌酯的敏感性检测及抗性风险分析

为明确番茄绵疫病菌对嘧菌酯的敏感性,采用菌丝生长速率法测定了2018—2019年从江西省番茄主产区采集分离得到的58株绵疫病菌Phytophthora capsici对嘧菌酯的敏感性,并以敏感菌株YD5为亲本,通过紫外线诱导获得了4株抗性突变体,研究了突变体的生物学性状;进而采用菌丝生长速率法测定了敏感菌株YD5及其4...     

Vegetative compatibility grouping in Botrytis cinerea using sulphate non-utilizing mutants

Twenty-one strains of Botrytis cinerea isolated from six plant species on ten sites throughout Israel, as well as a strain from France, were tested for vegetative and mycelial incompatibility, pathogenicity, resistance to the fungicides carbendazim and iprodione, and colony morphology. Selenate-resistant mutants were isolated from the strains as spontaneous, fast-growing sectors arising from restricted colonies on medium amended with sodium selenate with a mean frequency of 0.04 sectors/colony; 81% of the sectors were sulphate non-utilizing (sul) mutants. One hundred and four sul mutants were divided into two complementary groups: resistant (66 mutants) and sensitive to chromate. Based on compatibility reactions between chromate-resistant and chromate-sensitive sul mutants, 12 strains were compatible only with themselves and were each classified as belonging to different vegetative compatibility groups (VCGs). Nine strains were each compatible with one to three other strains, and were assembled into three multi-member VCGs. Mycelial incompatibility between wild-type strains (barrage), in the form of a zone of dark pigmentation or sparse mycelium with or without dark pigmentation of the agar along the line of confrontation, was observed for 70% of the inter-strain pairings. There was no correspondence in compatibility between strains revealed by two approaches: strains in different VCGs did not necessarily produce a barrage. However, self-compatibility was observed both as heterokaryon formation between complementary sul mutants and as an absence of barrages between mycelia of wild-type strains; wild-type strains belonging to the same VCG did not exhibit strong barrages, although weak antagonistic reactions were observed. Strains in two multi-member VCGs showed the same patterns of resistance to carbendazim and iprodione; the third multi-member VCG contained isolates with different patterns of resistance. Four morphological types were revealed among wild-type strains: conidial (five strains), sclerotial (six strains), intermediate (ten strains), and mycelial (one strain). On bean leaves, conidial strains were more aggressive than sclerotial strains.     

Identification of a novel point mutation in the <Emphasis Type="Italic">β</Emphasis>-tubulin gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay

The molecular basis of resistance to benzimidazole fungicides with laboratory and field mutant isolates of Botrytis cinerea was investigated. After chemical mutagenesis with N-methyl-N-nitrosogouanidine (NMNG) two different benzimidazole-resistant phenotypes were isolated on media containing carbendazim or a mixture of carbendazim and diethofencarb. The mutant isolates from the fungicide-mixture-containing medium were moderately resistant to carbendazim with wild-type tolerance to diethofencarb while mutant isolates from carbendazim-containing medium were highly resistant to carbendazim but sensitive to diethofencarb. The studied field isolates were highly resistant to benzimidazoles and sensitive to diethofencarb. Study of fitness characteristics of benzimidazole highly-resistant isolates showed that the resistance mutation(s) had no apparent effect on fitness-determining parameters. Contrary to this, the moderately benzimidazole-resistant strains, with no increased diethofencarb sensitivity, had a significant reduction in certain ecological fitness-determining characteristics. Analysis of the sequence of the β-tubulin gene revealed two amino acid replacements in the highly benzimidazole-resistant mutants compared to that of the wild-type parent strain. One was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), identified in both laboratory and field highly benzimidazole-resistant isolates, a mutation previously implicated in benzimidazole resistance. The second was a novel benzimidazole resistance mutation of glutamic acid (GAG) to glycine (GGG) substitution at the same position 198 (E198G), identified in a highly benzimidazole-resistant laboratory mutant strain. Molecular analysis of the moderately benzimidazole-resistant strains revealed no mutations at the β-tubulin gene. A novel diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole-sensitive (E198) but absent in both resistant genotypes (E198G and E198A) was developed for the detection of both amino acid replacements at the β-tubulin gene.     

Cross-resistance relationships between benzimidazole fungicides and diethofencarb in Botrytis cinerea and their genetical basis in Ustilago maydis

After nitrosoguanidine- or UV-mutagenesis, three different benzimidazole-resistant phenotypes were isolated on media containing benomyl or a mixture of carbendazim and diethofencarb from wild-type strains of Botrytis cinerea Pers. ex Fr. and Ustilago maydis (D.C.) Corda. Mutants of B. cinerea with moderate (MBr) or low (LBr) resistance to benzimidazoles and high resistance to diethofencarb (Dr) were isolated from the fungicide-mixture-containing medium in low frequency (7–1 × 10^?8). Only benzimidazole-resistant strains highly sensitive to diethofencarb (HBrDs) were identified on benomyl-containing medium at a frequency of 6.6 × 10^?6. Fitness-determining characteristics such as sporulation, germination and germ-tube elongation, were found to be reduced significantly in the mutants of B. cinerea that were resistant to both benzimidazoles and diethofencarb. However, pathogenicity of a MBrDr mutant strain on cucumber seedlings was equal to that of the wild type and a carbendazim + diethofencarb mixture was found to control grey mould caused by the wild type, but was not effective when the plant cotyledons were infected by the mutant strain. Three benzimidazole-resistant phenotypes (HBrDs, HBrDr, MBrDr) were isolated easily in U. maydis from a benomyl-containing medium. In contrast with B. cinerea, only one-tenth of the benzimidazole-resistant strains were sensitive to diethofencarb. Genetic analysis of benzimidazole resistance in U. maydis showed that the three benzimidazole-resistant phenotypes were due to three allelic mutations in a single gene and one of them was responsible for the negative cross-resistance between benzimidazoles and diethofencarb.     

水稻抗白叶枯病基因Xa47(t) a的敲除及抗性鉴定

为鉴定水稻Xa47(t)a基因对白叶枯病的抗性,以水稻种质L214为材料,通过构建针对Xa47(t)a基因的Xa47(t)a-Cas9敲除载体来获得敲除突变体,利用生物信息学技术对突变的Xa47(t)a基因进行突变类型分析,同时采用实时荧光定量PCR(quantitative real-time PCR,qPCR)技术分析突变体中Xa47(t)a及病程相关基因的表达情况,并于水稻孕穗期对突变体及其野生型植株接种11株白叶枯病菌Xanthomonas oryzae pv. oryzae菌株进行抗性鉴定。结果表明,在Xa47(t)a基因的第2外显子区域进行基因编辑后成功获得25株T₀代突变体株系;测序分析发现T₀代突变体株系中有13种不同的突变体类型,其中纯合突变体有3种类型,且突变位点均在靶标位点的11位碱基处缺失1～4个A碱基;氨基酸序列分析发现大部分突变体中Xa47a编码的蛋白翻译会提前终止,由原来的803个氨基酸变为144～166个氨基酸;qPCR分析结果表明突变体中Xa47(t)a及大部分病程相关基因的表达水平显著低于野生型株系;抗性鉴...