Carbendazim resistance in the eyespot pathogen Pseudocercosporella herpotrichoides

Mycelial isolates (115) of Pseudocercosporella herpotrichoides were obtained from five field sites in England. Carbendazim-resistant isolates were detected by their mycelial growth on agar containing 1 μg/ml carbendazim. Resistant isolates were found at two of the five sites examined and one of these had never been treated with benzimidazole fungicides. Amongst the carbendazim- resistant isolates there was a predominance of isolates with pale mycelium, an irregular colony margin and a relatively slow growth rate; however, this association was not absolute. Large differences in the effects of carbendazim on mycelial growth of sensitive and resistant isolates were demonstrated; growth of sensitive isolates was completely inhibited at 0.5 μg /ml carbendazim whilst five of the six resistant isolates examined grew on agar containing 1000 μg/ml fungicide. The carbendazim-resistant isolates were cross-resistant to benomyl, thiophanate-methyl and to a Icsser degree thiabendazole, but not to prochloraz. Conidia of carbendazim-resistant isolates were as resistant. Carbendazim-resistant isolates were just as pathogenic to wheat as sensitive isolates. The implications of these results and other reports of benzimidazole resistance in P. herpotrichoides are discussed in relation to disease control.     

Differentiation of Trichoderma taxa associated with mushroom production

A major epidemic of green mould in mushroom compost in Northern Ireland lasting for 6 months was caused by a single taxon of Trichoderma harzianum (Th2). This has continued to be the principal taxon causing losses in Ireland over a 9-year period. A separate taxon, Th4 , is responsible for a more recent epidemic in North America. Cultural methods are described which aid rapid differentiation of four distinct taxa of T. harzianum and five other species from the mushroom environment. After developing a standard cultural method, isolates were grouped visually by features such as growth rate at 27°C, amount of aerial mycelium, and the effect of light on sporulation form and timing. Most of these groups of isolates had significantly different growth rate ratios from each other at 27°C/17°C. Furthermore isolates within groups which had been separated by culture features alone were closely homologous as regards their microscopic morphological features such as phialides and phialospores. Two factors underlining the importance of using a standard cultural method in aiding identification were that culture morphology was shown to vary widely on different media and spore size was found to vary significantly with incubation temperature. Some difficulties are discussed concerning differentiation of Trichoderma spp. using classical microscopic features alone.     

Investigation of the epidemiology of green mould of mushroom (Agaricus bisporus) compost caused by Trichoderma harzianum

In 9 years of advisory experience in the British Isles and in virulence tests in the laboratory and field, only one of three distinct taxa of T. harzianum , namely Th2 , consistently colonized mushroom compost either in vitro or in mushroom houses, A single particle of contaminated spawn initiated colonies up to 30 cm in diameter. Four other Trichoderma species common in the mushroom environment rarely caused problems. Circumstantial evidence suggested airborne dust to be a main source of contamination of compost or its packing machinery, along with transmission on workers'clothing, pallets, load covers, trailers and by vectors such as mites, mushroom flies and mice. The Th2 isolate of T. harzianum was recovered from all of these sources but, in two tests, it did not survive peak heating. The control measures described here, based on strict hygiene, proved effective. Timing of colonization was important, also temperature of spawn run. Type of compost, as judged by analysis, appeared not to be important. In the field, spawning with peat based 'spawn'instead of grain spawn failed to prevent colonization, whereas in vitro , it reduced it. In the absence of any spawn, Th2 only grew strongly in microwaved or autoclaved compost. Reasons for this are discussed.     

紫外-氯化锂复合诱变哈茨木霉产生多菌灵抗药性菌株的研究

多菌灵是植物病害防治中常用的化学杀菌剂,野生型哈茨木霉对多菌灵比较敏感。为了能够更好地将哈茨木霉菌应用于实践,本试验采用紫外线-氯化锂复合诱变的方法,实现哈茨木霉菌株对多菌灵的抗性改良。试验共获得315株正向突变菌株,其中hcb-35菌株抗性能力较强。利用毒力测定方法检测了多菌灵对hcb-35有效抑菌中浓度,及hcb-35对多菌灵的抗药遗传稳定性;并利用对峙试验及显微观察检测其抑菌能力。结果显示,与哈茨木霉出发菌株hc相比,多菌灵对哈茨木霉突变株hcb-35菌株的有效抑菌中浓度提升285%;连续转接12代后,hcb-35菌株抗药性相对稳定且抑菌效果较出发菌株无明显差异,表明应用紫外-氯化锂复合诱变哈茨木霉可以获得遗传稳定的耐多菌灵突变株。     

High pressure liquid chromatography of benzimidazolcs

A laboratory-assembled high pressure liquid chromatograph with variable wavelength ultraviolet detector is described. The chromatography of the systemic fungicides benomyl, thiabendazole, thiophanate, related fungicides and benzimidazoles (13 compounds in all) is described using reverse phase and adsorption systems. Temperature affects reverse phase systems but not adsorption systems. Use of a variable wavelength detector increases sensitivity and selectivity. High pressure liquid chromatography (h.p.l.c.) has been applied to measurements of the solubility of benomyl and carbendazim in water and the degradation of benomyl in solution. Use of h.p.l.c. to measure benzimidazole residues awaits the development of methods of avoiding interference by substances extracted from crops and soils.     

A protocol for evaluation of the role of disinfectants in limiting pathogens and weed moulds in commercial mushroom production

In vitro and in vivo evaluations of the effects of three commercial disinfectants on isolates of Trichoderma harzianum Rifai and Cladobotryum dendroides (Bull) W Gams & Hoozemans were combined in the development of methodologies for realistic assessment of disinfectant performance. 'Environ' and 'Purogene' incorporated into agar media at 50 mg AI litre-1 were effective in totally preventing the mycelial growth of T harzianum and C dendroides isolates, whereas with 'Sudol' a concentration of 500 mg AI litre-1 was required. Evaluation of a model in vivo system was combined with observations on the fungicidal effects of disinfectants. Spore suspensions of T harzianum (Th1), T harzianum (Th2) and C dendroides prepared from culture washings with sterilised distilled water were used as contaminating inoculum. Environ, Sudol and Purogene in aqueous solutions at 1000 mg AI litre-1 were sprayed onto these building structure surfaces before or after artificial contamination with spore suspensions. Re-isolation from surfaces was carried out using agar swabs, which were prepared in visking tubing, applied to treated surfaces and incubated at 25 degrees C for 24, 48, 72 or 96 h. Environ and Purogene were more effective than Sudol in limiting the recovery of Trichoderma spp and C dendroides. All three disinfectants applied 12 h after artificial contamination onto wood, concrete or glazed surfaces were able to reduce the recovery of these isolates more effectively than when they were applied 12 h before artificial contamination. Greater persistence of contamination was noted on the rougher surfaces (wood and concrete) than on the smoother glazed surface. It was concluded in this in vivo evaluation that, in many cases, the protection achieved by the physical nature of the smooth glazed tile surfaces was equivalent to that available from the application of disinfectants, thus highlighting the attention needed to the nature of building surfaces in the structures used in the mushroom industry. The artificial contamination and re-isolation techniques allowed significant comparison between disinfectant materials, timings and surface types. In the in vivo experiments using a single standardised test concentration, the relative fungicidal effects of the disinfectants, which varied in the complexity of their active ingredients, were better discriminated than the effects observed in vitro, although the general trend was similar.     

稻田土壤8种真菌对4种杀菌剂的敏感性分析

基于ITS-5.8s rDNA区序列,初步鉴定了浙江大学试验基地稻田土壤的8种常见真菌。采用菌丝生长速率法测定了井冈霉素、多菌灵、咪鲜胺和三唑酮4种常用杀菌剂对这8种土壤真菌的抑菌活性,以及多菌灵与咪鲜胺混用对禾柄锈菌、黑曲霉和长枝木霉3种真菌菌丝生长的抑制作用。结果表明:8种真菌对咪鲜胺最敏感,EC50值在0.091~3.1 mg/L之间;多菌灵的EC50值在0.15~1.8 mg/L之间;三唑酮的EC50值在0.57~8.5 mg/L之间;井冈霉素的敏感性最低,EC50在12~94 mg/L之间。由4种杀菌剂的SSDs曲线分析结果得出,不同真菌对杀菌剂敏感性存在显著差异。当多菌灵与咪鲜胺按其EC50值分别以体积比3∶2、1∶1和7∶3混用时,增效系数分别为1.615、1.039和1.042,表现出一定的协同或相加作用。本研究结果为进一步阐明杀菌剂对稻田生态系统真菌群落的生态效应及杀菌剂的科学合理使用提供了重要依据。     

药剂对稻曲病菌的毒力测定及复配研究

为筛选防治稻曲病的有效单剂和复配组合,采用菌丝生长速率法,测定烯唑醇、腈菌唑、丙环唑、咪鲜胺、苯菌灵、多菌灵、戊唑醇、井冈霉素、松脂酸铜9种杀菌剂及互相复配后对稻曲病菌的毒力,并比较了不同复配组合的协同作用.结果表明:稻曲病菌对烯唑醇最敏感,EC5o为0.145 μg/mL;井冈霉素对稻曲病菌的毒力作用最弱,EC50为13.6 μg/mL.从252种不同比例复配的药剂中,筛选出12种增效配方,其中烯唑醇和咪鲜胺复配(1∶10),协同系数高达3.70,增效作用最大;烯唑醇和咪鲜胺(1∶10)对稻曲病菌的毒力作用最强,EC5o为0.06μg/mL,其次是烯唑醇和咪鲜胺(1∶20)与烯唑醇和腈菌唑(10∶1),EC50分别为0.10μ/mL和0.11μg/mL.田间试验结果也表明:烯唑醇与咪鲜胺(1∶10)复配,对稻曲病的平均防效为79.2％,高于其他常用药剂.     

防治牡丹根腐病的有效药剂筛选

采用菌丝生长速率法和孢子萌发法,分别测定了咪鲜胺、醚菌酯、苯醚甲环唑、百菌清、多菌灵、代森锰锌等6种杀菌剂对牡丹根腐病菌的室内毒力。结果表明,6种药剂对病菌菌丝生长的EC50大小顺序为：咪鲜胺<多菌灵<代森锰锌<苯醚甲环唑<百菌清<醚菌酯,其中以咪鲜胺对牡丹根腐病病菌的菌丝生长抑制作用最强, EC50为0.200 mg/L;多菌灵、代森锰锌、苯醚甲环唑、百菌清依次减弱,醚菌酯的抑制菌丝生长的作用最小,EC50为33.98 mg/L。6种药剂对病菌孢子萌发的EC50大小顺序为：百菌清<咪鲜胺<苯醚甲环唑<多菌灵<代森锰锌<醚菌酯;其中以百菌清对牡丹根腐病菌的孢子萌发抑制作用最强,EC50为0.343 mg/L;咪鲜胺、苯醚甲环唑、多菌灵、代森锰锌依次减弱,醚菌酯抑制孢子萌发的作用最小,EC50为38.4 mg/L。     

Green Mold Diseases of Agaricus and Pleurotus spp. Are Caused by Related but Phylogenetically Different Trichoderma Species

ABSTRACT Producers of champignon (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus) are facing recent incidents of green mold epidemics in Hungary. We examined 66 Trichoderma strains isolated from Agaricus compost and Pleurotus substrate samples from three Hungarian mushroom producing companies by a polymerase chain reaction-based diagnostic test for T. aggressivum, sequence analysis of the internal transcribed spacer region 1 (ITS1) and ITS2 and (selectively) of the fourth and fifth intron of translation elongation factor 1alpha (tef1alpha), and restriction fragment length polymorphism of mitochondrial DNA. Seven Trichoderma species were identified: T. aggressivum f. europaeum (17 isolates), T. harzianum (three isolates), T. longibrachiatum (four isolates), T. ghanense (one isolate), T. asperellum (four isolates), T. atroviride (nine isolates), and a still undescribed phylogenetic species, Trichoderma sp. DAOM 175924 (28 isolates). T. aggressivum f. europaeum was exclusively derived from A. bisporus compost, whereas Trichoderma sp. DAOM 175924 exclusively occurred in the substrate for Pleurotus cultivation. Sequences of the latter strains were co-specific with those for Trichoderma pathogens of P. ostreatus in Korea. The widespread occurrence of this new species raises questions as to why infections by it have just only recently been observed. Our data document that (i) green mold disease by T. aggressivum f. europaeum has geographically expanded to Central Europe; (ii) the green mold disease of P. ostreatus in Hungary is due to the same Trichoderma species as in Korea and the worldwide distribution of the new species indicates the possibility of spreading epidemics; and (iii) on mushroom farms, the two species are specialized on their different substrates.     

Action of benzimidazole fungicides on resorcinolic lipid metabolism in rye seedlings depends on thermal and light growth conditions

The content and homologue composition of alkylresorcinols were investigated in rye seedlings (Secale cereale L.) treated with benzimidazole fungicides and grown for 5 days under various thermal and light conditions. The fresh and dry biomasses of green and etiolated plants were greatly increased by benomyl and carbendazim at 29 °C. At 22 and 15 °C benomyl had an inhibitory action on growth of rye, whereas the effects caused in the presence of carbendazim were slightly dependent on external stimuli. On the other hand, benomyl enhanced resorcinol biosynthesis in green seedlings grown at all temperatures, while carbendazim only at 29 °C. In plants kept in the darkness, both fungicides increased content of alkylresorcinols at 29 °C and decreased its at 22 and 15 °C. The qualitative pattern of resorcinolic homologues was also significantly modified in the presence of benomyl and carbendazim and depended on other physical stimuli.     

香蕉枯萎病田间发病株的高效灭菌方法筛选

为筛选有效防控香蕉枯萎病菌扩散蔓延的灭菌方法,采用含毒介质培养法测定咪鲜胺和多菌灵不同配比对枯萎病菌的毒力作用以筛选最佳混配液,比较最佳混配液与生石灰、草甘膦、咪鲜胺的平板抑菌和大田灭菌效果,并研究了大田不同施用方式对最佳混配液灭菌效果的影响。结果表明,咪鲜胺与多菌灵体积比为10∶1时得最佳混配液,可显著提高对香蕉枯萎病菌的抑制效果,增效系数为1.53,EC50值最小,为0.025 mg/L;4种药剂的EC50值由大到小为草甘膦生石灰咪鲜胺最佳混配液。1 000 mg/L最佳混配液喷施于大田病株5 d后根际土壤病菌含量下降了95.93%,10 d后球茎病菌含量减少了71.88%,综合灭菌效果显著优于其它处理。此外,应用打孔灌药法可显著提高最佳混配液对大田病株的灭菌效果,施用5 d后球茎病菌含量减少了95.95%。表明以打孔灌药法施用1 000 mg/L最佳混配液灭菌效果显著且操作便捷,易于推广应用。     

湖北省小麦赤霉病菌对多菌灵、戊唑醇和咪鲜胺的敏感性

对2014年采自中国湖北省7个县、市的206株小麦赤霉病菌样品进行单孢分离与鉴定,并采用菌丝生长速率法随机测定了其中100株菌株对多菌灵、戊唑醇和咪鲜胺的敏感性,建立了其敏感基线。结果表明:多菌灵、戊唑醇和咪鲜胺对湖北省小麦赤霉病菌的EC50值范围分别为0.115~0.705、0.006~1.356和0.002~0.370 μg/mL,平均值分别为0.248、0.181和0.040 μg/mL;供试100株小麦赤霉病菌对3种药剂的敏感性频率均呈单峰拟正态分布,因此可将所得各EC50平均值分别作为湖北省小麦赤霉病菌对3种药剂的敏感基线参考值。以各药剂EC50平均值的10倍作为敏感性鉴别浓度,对2015及2016年湖北省小麦赤霉病菌的敏感性进行了监测,在该鉴别浓度下,多菌灵、戊唑醇和咪鲜胺对2015年小麦赤霉病菌的平均抑制率分别为100%、85.14%和82.35%,对2016年小麦赤霉病菌的平均抑制率分别为100%、76.67%和73.62%。研究表明,虽然戊唑醇和咪鲜胺对2016年小麦赤霉病菌的抑制率略有下降,但整体而言,湖北省小麦赤霉病菌对多菌灵、戊唑醇及咪鲜胺仍具有较高的敏感性。     

2014年海南省橡胶炭疽病菌对多菌灵和咪鲜胺的敏感性测定

 2014年从海南省4个市(县)分离获得43株橡胶炭疽病菌,进行病原菌鉴定后,采用菌丝生长速率法,测定了其对多菌灵和咪鲜胺的敏感性,结果表明:供试菌株中,有23个属于胶孢炭疽病菌,20个属于尖孢炭疽病菌。所有供试菌株对多菌灵和咪鲜胺的EC₅₀值分布范围分别为(0.332 3~7.425 6)和(0.009 1~0.113 3) mg·L^-1,平均值分别为(1.714 1±1.684 7)和(0.036 8±0.023 8)mg·L^-1。其中尖孢炭疽病菌对多菌灵的EC₅₀值要显著高于胶孢炭疽病菌,EC₅₀平均值分别为(2.922 7±1.556 3)和(0.663 2±0.194 4)mg·L^-1,但2种病原菌对咪鲜胺的EC₅₀值没有显著差异,EC₅₀平均值分别为(0.038 3±0.015 2)和(0.035 5±0.020 1)mg·L^-1。供试菌株对多菌灵和咪鲜胺的敏感性无显著相关性,可以在生产上交替使用。     

Use of Trichoderma harzianum in combination or alternation with fungicides to control cucumber grey mould (Botrytis cinerea) under commercial greenhouse conditions

A preparation of Trichoderma harzianum was sprayed on cucumber plants in greenhouses in order to control fruit and stem grey mould. Up to 90% control was achieved by the biocontrol agent (0·5–1·0 g/l) which in most experiments under commercial conditions was as effective as the dicarboximide fungicides iprodione or vinclozolin (0·5 g/l each) alone or alternated with diethofencarb + carbendazim (0·25 g/l each). However, in one experiment disease incidence in Trichoderma -treated plots did not differ significantly from the control. A mixture of T. harzianum with a dicarboximide fungicide resulted in up to 96% control of grey mould. In this case control was always significant ( P =0·05) but improvement of control compared with each treatment alone was not significant ( P =0·05). The alternation of sprays with the biocontrol preparation and with a dicarboximide fungicide was tested in three out of the five experiments and was found to be effective, thus enabling a reduction in the use of chemical sprays. Populations of T. harzianum were on a level of 3 × 10⁵-8 × 10⁵ c.f.u. per leaf and ten times lower on one fruit. They remained high after the second and third sprays. Conditions favouring the ability of T. harzianum to control grey mould were temperatures above 20°C and relative humidity between 80 and 97%.     

木霉对草坪褐斑病的拮抗效果及耐药性

通过木霉属5菌株与大连高尔夫球场上草坪褐斑病菌的对峙培养试验,研究结果表明:哈茨木霉Thar1菌株、哈茨木霉Thar2菌株、深绿木霉Tat菌株、钩状木霉Tha菌株及桔绿木霉Tci菌株对病原菌的抑制率为:66%、73%、71%、77%、55%,而相对抑菌效果分别为2.06、3.94、2.35、3.54、2.27,可以作为草坪褐斑病菌的生防菌加以利用。在这5株木霉中以哈茨木霉Thar2对草坪褐斑病菌的拮抗作用最强。观察结果表明,哈茨木霉Thar2对草坪褐斑病菌的拮抗机制主要表现为生长竞争、重寄生及产生抗菌物质。     

Evaluation of fungicides for control of eyespot disease and yield loss nationships in winter wheat

Following the discovery of resistance to benzimidazole fungicides in the cereal eyespot pathogen Pseudocercosporella herpotrichoides in the UK in 1981, and an initial in vitro screen to select the fungicides with greatest activity against the pathogen, 40 field experiments were carried out between 1983 and 1986 to evaluate alternative fungicides for control of eyespot. At the majority of experimental sites, benomyl-resistant strains of the pathogen were present, and carbendazim did not control eyespot. Prochloraz was the most effective fungicide, reducing the eyespot index by 30–60%. There was no extra benefit from adding carbendazim to prochloraz. Flusilazole was almost as effective as prochloraz, but other fungicides had little or no effect. At sites with a high incidence of eyespot, prochloraz, with or without the addition of carbendazim, generally gave the largest yield increase. The mean yield increases each year were in the range 0·36–0·85 t/ha, and the greatest yield increase at any site was 2·27 t/ha. Most other fungicides increased yield, but carbendazim did not from 1984 to 1986. There were also yield increases at many sites with a low incidence of eyespot. Yield increases were associated with increases in thousand-grain weight at the majority of sites, but in only a few instances were there associated increases in specific weight. Prochloraz application at GS30-31 was cost effective at 71% of sites. At most sites, in regression of yield on eyespot, eyespot accounted for less than 25% of the variance in yield. The mean relationship between severe eyespot lesions and yield loss was such that each 1% increase in the percentage of tillers affected by severe eyespot was associated with a yield loss of 0·21 %. There was a significant positive correlation between eyespot at GS75 and GS30-31, and between yield increase from prochloraz treatment (at GS30-31) and eyespot at GS75, but not between yield increase and eyespot at GS30-31. The ADAS threshold for fungicide application of 20% tillers affected at GS30-31 was a reliable indicator of the cost-effectiveness of treatment at 60% of sites.     

Development of apothecia ofTapesia yallundae in contrasting populations selected by fungicides

Apothecia of the eyespot fungus,Tapesia yallundae, were found on 0–18% of straws in plots of wheat stubble in February–March 1994. The fungicides carbendazim, prochloraz or carbendazim plus prochloraz had been applied repeatedly to the same plots in each of the previous 9 years in which successive wheat crops had been grown. The factors most strongly correlated with the incidence of apothecia were the incidence and severity of eyespot in the preceding wheat crop and the frequency of carbendazim-resistant W-type fungus in populations recovered from that wheat crop. Plots treated with carbendazim, which had previously had more disease and more resistance to carbendazim in the pathogen population relative to untreated plots, therefore yielded most apothecia. Plots treated with prochloraz, which had selected for predominantly R-type fungus and decreased eyespot, yielded few apothecia. Single-ascospore isolates were all of the W-type and were more frequently carbendazim-sensitive than expected, except those from plots treated only with carbendazim. None showed decreased sensitivity to prochloraz. The implications of applying fungicides regularly for controlling eyespot on the capability of the eyespot fungus for genetic variation through sexual reproduction are discussed.     

Factors affecting the efficacy of post-harvest fungicide applications for the control of blue mould (Penicillium expansum) in stored apples

The effects of apple fruit maturity, temperature of fruit or dip solution, period of time in fungicidal dip, storage conditions, and spore inoculum concentration, on the efficacy of fungicides for control of blue mould ( Penicillium expansum ) were examined in various experiments.
Iprodione and imazalil were only effective when inoculum concentration was low, whilst prochloraz was highly effective in controlling rot on fruit inoculated with 3 × 10⁶ spores/ml. There was no consistent effect of dip temperature or fruit maturity on the efficacy of the fungicides. Iprodione was more effective on warm fruit (19°C) than cold fruit (6°C) whilst the reverse was true of imazalil. Extended periods of immersion in the fungicides slightly reduced the incidence of rotting but not to any useful degree.
The incidence of rotting in fruit treated with prochloraz and etaconazole was less in fruit stored under controlled-atmosphere cold storage conditions than in fruit stored in air cold-storage. Both fungicides were also effective for short-term storage at 20 C in air. Captan, benomyl, captan plus benomyl or vinclozolin were either ineffective or of poor efficacy under all storage conditions.