Design of new melanin biosynthesis inhibitors

Structural requirements for melanin biosynthesis inhibition were analysed by comparing known melanin biosynthesis inhibitors (MBIs) and an intermediate of melanin biosynthesis (1,3,8-trihydroxynaphthalene (1,3,8-THN)), on the assumption that the enzymatic inhibition by MBIs occurs competitively with the biosynthetic intermediate. On this basis, new chemicals in the form of phthalazine derivatives were designed. In biological tests, the synthesised phthalazine derivatives showed high activity against rice blast, caused by Pyricularia oryzae Cav. These compounds also inhibited the melanisation of this fungus at a low concentration, which suggested that they are similar to other MBIs in their mode of action.     

Effect of inhibitors of melanin biosynthesis on appressorial penetration and reductive reactions in Pyricularia oryzae and Pyricularia grisea

Appressorial penetration of onion epidermal walls by wild-type strains P-2 and O-42 of Pyricularia oryzae was more sensitive to fthalide (4,5,6,7-tetrachlorophthalide) than penetration by wildtype strain 4091-5-8 and black mutant strain BL-3 of Pyricularia grisea. Cerulenin completely blocked appressorial penetration by P. oryzae strain P-2 or P. grisea strain BL-3, but penetration capacity of these appressoria could be largely restored with 0.1 mM scytalone. Fthalide and tricyclazole inhibited the conversion of 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN) to 1,3,8-trihydroxynaphthalene (1,3,8-THN) and 1,8-dihydroxynaphthalene (1,8-DHN) as well as the conversion of scytalone to 1,8-DHN by cell-free extracts of P. oryzae or P. grisea. These inhibitors blocked the NADPH-dependent reductase reactions involved in the conversion of 1,3,6,8-THN to scytalone and the conversion of 1,3,8-THN to vermelone in the melanin biosynthetic pathway to 1,8-DHN. The two reductase reactions in extracts from P. oryzae P-2 were about 10 times as sensitive to fthalide or tricyclazole as those in extracts of P. grisea. Reductase activity with either 1,3,6,8-THN or 1,3,8-THN as substrates was present in only trace amounts in cell-free extracts of the buff mutant, P. oryzae P-2 m-1.     

Ergosterol biosynthesis inhibitors. An overview of their history and contribution to medicine and agriculture

Over the past 15 years, an increasing number of ergosterol biosynthesis inhibitors (EBIs) have been introduced in crop protection as fungicides, and in medicine as fungicides and bactericides. Their spectrum of activity and their systemic properties vary widely, depending on structural elements. By their high protective and curative activity the EBIs have greatly improved the level of disease control, both in plants and in man. However, the chemical and biological potential of this group has not yet been fully explored, particularly in the field of growth regulation.     

玉米大斑病菌黑色素合成酶基因4HNR的克隆及其启动子预测

 4HNR (1,3,6,8-tetra-HN reductase) gene of melanin biosynthesis in Setosphaeria turcica had been cloned successfully by RT-PCR in this study. Both sequences of DNA and cDNA of 4HNR were 807 bp and there was no intron in the sequences. This gene only had single copy in genome through Southern blotting analysis. A 2 285 bp for the flanking sequence of 5' had been obtained and it had promoter structure through the software analysis.     

桑椹菌核病菌(Scleromitrula shiraiana)黑色素生物合成相关基因的克隆与功能分析

小柱孢酮脱水酶(scytalone dehydratase,SCD)及羟基萘还原酶(hydroxynaphthalene reductase,HNR)是真菌多聚二羟奈类(DHN)黑色素生物合成途径中的关键酶。根据已知真菌的小柱孢酮脱水酶及羟基萘还原酶的保守结构域设计兼并引物并利用RACE技术,获得桑椹菌核病菌(核地仗菌,Scleromitrula shiraiana)SsSCD1和Ss4HNR1的DNA和c DNA序列。SsSCD1和Ss4HNR1均含2个内含子和3个外显子,分别编码169和263个氨基酸残基。进化分析表明SsSCD1和Ss4HNR1与灰葡萄孢和核盘菌中小柱孢酮脱水酶和四羟基萘还原酶基因的亲缘关系最近。DHN黑色素合成特异性抑制剂三环唑处理核地仗菌,结果显示三环唑可抑制核地仗菌菌丝生长和黑色素合成,对SsSCD1的表达无显著影响,但Ss4HNR1的表达水平显著提高。这些结果表明三环唑能够特异性的抑制四羟基萘还原酶,且DHN黑色素是核地仗菌生长发育的重要产物。     

Chromatographic analysis of ergosterol biosynthesis inhibitors

The chlorophyll sensitised photodegradation of 2-dimethylamino-5, 6-dimethylpyrimidin-4-ol (I), a hydrolysis product of pirimicarb, in the solid state on silica gel and in chloroform solution has been investigated. The product distribution in the solid state photodegradation was different from that in chloroform solution and was dependent upon the wavelength range of the incident radiation. When radiation of wavelength greater than 420 nm was used, the products formed in the chlorophyll sensitised degradation of I in the solid state were similar to those obtained when Rose Bengal or Methylene Blue were used as sensitisers. These results indicate that singlet oxygen is implicated in the sensitised photodegradation of I in the solid state. Numerous products were formed in each of the systems studied.     

Diagnosis of dehydratase inhibitors in melanin biosynthesis inhibitor (MBI-D) resistance by primer-introduced restriction enzyme analysis in scytalone dehydratase gene of Magnaporthe grisea

We have established a simple diagnosis method for rice blast fungus resistant to MBI-D. This involves the preparation of PCR templates directly from the lesions in combination with primer-introduced restriction enzyme analysis PCR (PIRA-PCR).     

Simultaneous resistance in fungi to ergosterol biosynthesis inhibitors and dicarboximides

Several strains ofAspergillus nidulans, Cladosporium cucumerinum andPenicillium italicum with known resistance to ergosterol biosynthesis inhibitors were tested for resistance to three dicarboximides. Negligible levels of resistance to iprodione and vinclozolin were observed in one out of three strains ofA. nidulans. Two out of three strains ofC. cucumerinum displayed a low resistance to iprodione, and a high resistance to procymidone and vinclozolin. The latter strains were also moderately resistant to the isoflavonoid phytoalexins medicarpin and pisatin, but sensitive to the antibiotic pimaricin. All sixP. italicum strains examined displayed wild-type sensitivity to all three dicarboximides; the two of these tested in thin-layer chromatographic bioassays proved to be resistant to pimaricin.Iprodione and vinclozolin induced energy-dependent fenarimol efflux inA. nidulans. In line with this observation, in crossed-paper strip assays iprodione and fenarimol antagonized each other in their toxicity towardsA. nidulans; towardsC. cucumerinum, on the other hand, these fungicides behaved independently.The implications and practical consequences of the phenomena observed are briefly discussed.Samenvatting Verscheidene tegen ergosterolbiosyntheseremmers resistente stammen vanAspergillus nidulans, Cladosporium cucumerinum enPenicillium italicum werden getoetst op resistentie tegen dicarboximiden. Eén der drie onderzochte stammen vanA. nidulans bezat enige resistentie tegen iprodione en vinchlozoline. Twee van de drie onderzochte stammen vanC. cucumerinum vertoonden een lage graad van resistentie tegen iprodione en een zeer hoge tegen procymidone en vinchlozoline. Ze waren ook enigermate resistent tegen de fytoalexinen medicarpine en pisatine, maar gevoelig voor het antibioticum pimaricine. Alle onderzochte stammen vanP. italicum waren voor de drie getoetste dicarboximiden even gevoelig als het wild-type; voorzover onderzocht, bleken deze stammen resistent tegen pimaricine.Iprodione en, hoewel in mindere mate, vinchlozoline induceerden energieafhankelijke efflux van fenarimol inA. nidulans. In overeenstemming hiermee antagoneerden iprodione en fenarimol elkander in hun activiteit ten opzicht vanA. nidulans. Ten opzichte vanC. cucumerinum gedroegen deze fungiciden zich onafhankelijk van elkaar.De practische consequenties van de waargenomen verschijnselen worden kort aangeduid.     

Laboratory resistance to dicarboximides and ergosterol biosynthesis inhibitors in Penicillium expansum

Samenvatting Isolaten vanPenicillium expansum werden geselecteerd op moutagar met een dicarboximide (iprodion, procymidon, vinchlozolin) of een ergosterol biosynthese remmer (fenarimol, fenapanil, imazalil, prochloraz). De verkregen isolaten vertoonden alleen kruisresistentie tegen fungiciden behorende tot dezelfde groep van middelen. Deze kruisresistentie kon ook in dompelproeven met geïnoculeerde appels worden aangetoond. Alle getoetste isolaten bezaten in mengpopulaties van gevoelige en resistente isolaten op appels een relatief laag competitief vermogen.     

Control of Dutch elm disease by the sterol biosynthesis inhibitors fenpropimorph and fenpropidin

Sterol biosynthesis inhibitors that inhibit the yeast-hyphae conversion inOphiostoma ulmi suppressed Dutch elm disease development in two elm clones. After curative treatment with fenpropimorph-sulphate of 27 Vegeta elms which had previously been inoculated withO. ulmi, 25 trees did not show disease symptoms by the end of the second season. All 41 control trees, inoculated withO. ulmi only, were clearly diseased. In an experiment with Commelin elms three fenpropimorph salts and thiabendazole were compared. Injection of the trees three weeks after inoculation withO. ulmi gave by the end of the second season no symptoms of Dutch elm disease in any of the trees injected with fenpropimorph-phosphate or thiabendazole, and in most trees injected with fenpropimorph-acetate or-sulphate. Similar treatments with the free base of fenpropimorph and fenpropidin-sulphate were less effective due to insufficient uptake of the fenpropimorph emulsion and phytotoxicity of fenpropidin-sulphate, respectively. Injection of fenpropimorph-sulphate or thiabendazole six weeks after inoculation withO. ulmi did not result in significant differences from the control group inoculated withO. ulmi only.Fenpropimorph-phosphate and-sulphate completely suppressed Dutch elm disease upon injection of only 7.5 or 10 g per tree (average tree diameter 28 cm). Residue analyses showed only a slow decrease in concentration of the fungicide over two growing seasons and an apparent transport into the new annual ring, other prerequisites for a possible future use for control of Dutch elm disease.Samenvatting De iepeziekte kan onderdrukt worden door sterolbiosyntheseremmers die de overgang vanOphiostoma ulmi van de gistvorm in de hyfevorm remmen. Aan het eind van het tweede seizoen na een curatieve behandeling van 27 Vegeta iepen met fenpropimorfsulfaat bleken 25 bomen geen symptomen van iepeziekte te vertonen. Alle controlebomen, die alleen metO. ulmi geïnoculeerd waren, waren duidelijk ziek. In een proef met Commelin iepen werden drie fenpropimorfzouten en thiabendazool vergeleken. De zouten werden drie weken na de inoculatie metO. ulmi geïnjecteerd. Aan het eind van het tweede seizoen vertoonden geen van de bomen die met fenpropimorffosfaat of thiabendazool geïnjecteerd waren en slechts enkele bomen die met fenpropimorfacetaat of-sulfaat geïnjecteerd waren iepeziektesymptomen. Behandelingen met fenpropimorf (vrije base) en fenpropidinsulfaat werkten minder goed door de slechte opname van de fenpropimorfemulsie en de fytotoxiciteit van fenpropidin. Injectie met fenpropimorfsulfaat of thiabendazool zes weken na inoculatie leidde niet tot significante verschillen met de controlegroep die alleen metO. ulmi geïnoculeerd was.Een dosis fenpropimorffosfaat of-sulfaat van 7.5 of 10 g per boom met een gemiddelde boomdiameter van 26 cm bleek de iepeziekte volledig te kunnen onderdrukken. Uit residue-onderzoek bleek dat de concentratie van het fungicide gedurende de twee groeiseizoenen slechts langzaam afnam en dat het middel naar de nieuwe jaarring werd getransporteerd, twee voorwaarden voor een toepassing op praktijkschaal van fenpropimorf voor de bestrijding van de iepeziekte.     

Sensitivity of South African Ramulispora herpotrichoides isolates to carbendazim and ergosterol biosynthesis inhibitors

In a survey conducted during 1991–1992, single-spored isolates of the eyespot fungus from the Swartland area were characterized and tested for sensitivity to carbendazim and ergosterol inhibiting fungicides. The 100 isolates tested were all fast growing, even marginate, and designated as Ramulispora herpotrichoides . Fungal growth was completely inhibit on PDA amended with carbendazim (1 μg/ml), indicating that the local population of the fungus is still at baseline sensitivity to benzimidazoles. The mean concentration of prochloraz calculated to inhibited growth by 50% (IC₅₀ value) was 0.043 ± 0.029 μg/ml, which is comparable with the baseline sensitivity reported for European isolates. Of the 36 representative isolates screened against 2 μg/ml triadimenol, 44% were sensitive, while 36% were resistant. The triadimenol-resistant isolates were sensitive to propiconazole and flusilazole. However, four of the triadimenol-resistant isolates were also resistant to tebuconazole. These results indicate that South African isolates of R. herpotrichoides are sensitive towards carbendazim, prochloraz, propiconazole and flusilazole. They were found to differ, however, in sensitivity towards triadimenol and tebuconazole, where some isolates had an IC₅₀ value greater than 2 μg/ml.     

Inhibition of insect cytochrome P-450 by some metyrapone analogues and compounds containing a cyclopropylamine moiety and their evaluation as inhibitors of juvenile hormone biosynthesis

Two metyrapone analogues, 2-(l-imidazolyl)-2-methyl-l-phenyl-l-pro-panone (A-phenyl-B-imidazolyl-metyrapone; III) and 2-methyl-l-phenyl-2-{1,2,4-triazol-l-yl)-l -propanone (A-phenyl-B-triazolyl-meiyrapone; IV) as well as two cyclopropylamine derivatives. N-cyclopropyl-4-icrt-butylbenzylamine (V) and N-cyclopropyl-4-(3,7-dimethyl-7-methoxy-octyloxy)benzamide (cyclopropylamine acylated with a JH analogue acid of known structure; VI) were synthesized and evaluated in biological assays for JH biosynthesis on cockroach, Diploptera punctata corpora allata and egg growth in adult cockroach as well as for mixed function oxidase activities, i.e. epoxidation of aldrin to dieldrin and O-demethylation of 7-methoxy-4-methylcoumarin to 7-hydroxy-4-methylcoumarin on microsomes from housefly, Musca domestica, abdomen and from cockroach midgut. Compound VI was a good in-vitro inhibitor of JH biosynthesis, but it had significantly lower activities in the assays for inhibition of microsomal cytochrome P-450. Compound IV and metyrapone had moderate activity as inhibitors of oocyte growth. Compounds III, IV and V were more potent inhibitors of housefly aldrin epoxidation than metyrapone and they inhibited the enzyme activity by almost 100% at 02mM, while in cockroach midgut microsome assay metyrapone was more potent than these three compounds.     

Death mechanisms caused by carotenoid biosynthesis inhibitors in green and in undeveloped plant tissues

This study was carried out to investigate the difference in physiological processes leading to two different responses of albinism and necrosis to the phytoene desaturase inhibitor fluridone, which is dependent on the developmental state of tissue at the time of fluridone treatment. A soil-drench of fluridone solution at the 40% growth of the 3rd leaf of maize (Zea mays L.) caused the leaf to grow into a white/green mixed leaf; completely white in the basal part (W), pale green in the middle part (PG), and green in apical part of the leaf blade (G). In the PG and G, the effective quantum yield of electron transport through photosystem II (Yield) was significantly inhibited, Fv/Fm was decreased but Fo increased, hydrogen peroxide was more accumulated than untreated control, and cellular leakage was faster and more pronounced than in the white tissue (W). In the W, however, all of Fo, Fm, and Yield values were near zero due to loss of chlorophyll. Moreover, there was a relatively low content of hydrogen peroxide, slower cellular leakage and longer survival of tissue in the W. On the other hand, the level of antioxidants such as carotenoids, tocopherols, and ascorbic acid was lower in the W than untreated control. However, the specific activities of antioxidant enzymes were elevated in the W; 3.47 times in superoxide dismutase (SOD), 3.21 times in peroxidase (POD), 1.59 times in catalase and 1.21 times in glutathione reductase. In particular, SOD and POD activities had a tendency to be increased during senescence. In the kinetics experiment carried out during a senescence of the 2nd white leaf, increase of wilting and browning began to occur prior to any significant change in MDA-equivalents, and high reduction of carbohydrate contents occurred prior to increase of wilting and necrosis. Carbohydrate supplement significantly delayed the death of white leaves. Taken together, the above results indicated that in the developed tissue, whose greening has already taken place at the time of herbicide treatment, its death was related to the cell destruction by excessive oxidative stress induced through photosynthetic electron transport blockade. Conversely, in the developing or undifferentiated tissue at the time of herbicide treatment, that eventually grown into a white tissue, its death seemed to be more dependent on a loss of photosystem function followed by carbohydrate deficiency.     

Germination and appressiorial formation by uredospores ofUromyces viciae-fabae exposed to inhibitors of polyamine biosynthesis

An examination was made of the effects of three polyamine biosynthesis inhibitors on germination and appressorium formation by uredospores of the bean rust fungusUromyces viciae-fabea on artificial membranes. The ornithine decarboxylase inhibitor -difluoromethylornithine had no effect on uredospore germination, even when used at 2mM, whereas appressorium formation was reduced by 63% at 0.5 mM and by 99% at 2mM. Methylglyoxal bis(guanylhydrazone), an inhibitor of S-andenosylmethionine decarboxylase, reduced germination when used at 0.025 mM, and at this concentration, appressorium formation was completed prevented. Uredospore germination was unaffected by as much as 3 mM cyclohexylamine, an inhibitor of spermidine esynthase, while appressorium formation was reduced at 1 mM and completely prevented at 3.3 mM. These results support previous suggestions that inhibitors of polyamine biosynthesis exert their main effect on the early stages of fungal development on the leaf surface.Abbreviantions CHA cyclohyxylamine - DFMO -difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone)     

Guanidinium and amidinium fungicides: A new class of carbocation mimetic ergosterol biosynthesis inhibitors

A novel class of chemical has been designed with the aim of inhibiting the Δ¹⁴-reductase and Δ⁸-Δ⁷-isomerase enzymes in the ergosterol biosynthesis pathway in fungi. Use was made of knowledge about the mechanisms of both enzymes and the mode of action of known, fungicidal inhibitors of these enzymes. Pioneer examples have been synthesised and have been demonstrated to be potent inhibitors of ergosterol biosynthesis in Ustilago maydis (DC) Corda, acting in the same manner as the commercial fungicide fenpropimorph. They also showed excellent fungicidal activity against Erysiphe graminis DC f. sp. hordei Marchal (powdery mildew of barley) and Puccinia recondita Rob. ex Desm. (wheat leaf rust) in in-vivo glasshouse tests. Using these compounds as a starting point, systematic structural variation has been carried out. Testing of a wide range of analogues at high volume confirms the potential of this class of compound to control mildew and rust pathogens at levels comparable to those of the standards. Correlation of in-vivo and enzymatic data is good and the structure-activity relationship developed for this series of compounds closely parallels that found for the morpholine/piperidine class of fungicides, suggesting a common mode of action.     

Sensitivity to inhibitors of sterol biosynthesis in isolates of Venturia inaequalis from Italian and Dutch orchards

Samenvatting Een geringe, maar significante reductie in gevoeligheid vanVenturia inaequalis voor fungiciden die tot de sterol-biosyntheseremmers behoren werd vastgesteld bij isolaten uit proefboomgaar den in Italië, die gedurende enkele jaren met deze middelen behandeld waren. Bij isolaten uit enkele commerciële boomgaarden in Nederland werd nog geen significante verschuiving in gevoeligheid waargenomen.     

几种除草剂靶酶及其抑制剂的研究进展

根据作用靶标对除草剂进行分类,对于新型除草剂的设计能够起到很大的帮助。迄今为止,人们已发现除草剂的不同作用位点近30种,涉及到50余种不同化学结构的化合物。文中介绍了谷氨酰胺合成酶(GS)、咪唑甘油磷酯脱水酶(IGPD)、乙酰辅酶A羧化酶(ACCace)、八氢番茄红素脱氢酶(PDS)及其各自抑制剂的研究进展。分别从酶的生理功能、酶学特征、抑制剂作用机理、抑制剂的研究、抗性等方面进行了不同程度的阐述。     

The effect of three fungicides,specific for the control of rice blast disease,on the growth and melanin biosynthesis by Pyricularia oryzae cav.

Tricyclazole (EL 291), 4,5-dihydro-4-methyltetrazolo [1,5-a] quinazolin-5-one (PP 389), and pyroquilon (CGA 49104) were studied to determine the effects on growth and melanin biosynthesis by Pyricularia oryzae in vitro. The three fungicides were essentially devoid of toxicity to P. oryzae at concentrations up to 50 ug ml^?1 but each selectively inhibited melanin biosynthesis at much lower concentrations. Inhibition of melanin biosynthesis resulted in the accumulation of 2-hydroxyjuglone and flaviolin. The study indicated that the three compounds act by a similar mechanism in P. oryzae..     

玉米大斑病菌黑色素的一些理化性质和光谱吸收特征

 黑色素是某些植物和动物真菌病害的致病相关因子,不同来源的黑色素其生物合成途径可能不同。对玉米大斑病菌细胞壁结合黑色素和从培养滤液中提取的黑色素进行理化性质、紫外吸收光谱和红外光谱扫描测定,并与标准品黑色素进行比较分析,明确了玉米大斑病菌黑色素具有与标准品黑色素相似的理化性质。DHN黑色素的特异性抑制剂——三环唑,对玉米大斑病菌0号和1号小种黑色素的产生均有抑制作用;以玉米大斑病菌基因组DNA为模板,通过PCR扩增,得到了1,3,8-三羟基萘还原酶基因的同源片段,推测玉米大斑病菌黑色素合成于DHN途径。     

白头翁叶斑病菌黑色素的理化性质及其生物合成途径初步研究

对白头翁叶斑病菌胞壁结合黑色素和胞外黑色素进行了理化性质和红外光谱扫描测定, 结果表明两者具有相似的理化性质, 均易溶于KOH、H2O2和NaClO, 不溶于水、乙醇和丙酮。红外光谱分析表明, 白头翁叶斑病菌YS-24菌株的胞壁结合黑色素与胞外黑色素为同一种类型的黑色素。DHN黑色素的特异性抑制剂—三环唑, 对白头翁叶斑病菌黑色素的产生有明显的抑制作用; 以白头翁叶斑病菌基因组DNA为模板, 通过PCR扩增, 得到了聚酮体合成酶基因的同源片段 AaPKS , 初步推断白头翁叶斑病菌黑色素合成属于DHN途径。