玉米黑粉病危害加重原因分析及综合治理对策

根据近年对豫北安阳市玉米黑粉病发生危害情况的调查结果,总结出该病具有发生因品种而异、区域逐年扩大、危害多集中于雌穗、程度逐年加重、局部受害严重的特点;分析其发生危害加重的原因,主要有田间菌源量大、外来菌源传播扩散、缺乏有效防治措施、气候环境条件适宜、玉米品种抗病性较差、相关法律法规未阻止病害随种子传播等多方面;提出了以选用优质抗病品种、减少菌源、加强栽培管理等农业防治为主,以药剂处理种子和茎叶喷雾等化学防治为辅,同时搞好技术服务、完善相关法律法规等综合治理对策。     

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.     

Effects of Sterol Biosynthesis Inhibitor Fungicides on Growth and Sterol Composition of Ustilago maydis,Botrytis cinerea and Pyrenophora teres

The effects of the sterol biosynthesis inhibitor (SBI) fungicides fenarimol, fenpropimorph, imazalil, prochloraz, propiconazole and triadimenol on growth and sterol composition of Ustilago maydis, Botrytis cinerea and Pyrenophora teres, grown from spores or sporidia in liquid culture, were determined. Growth of U. maydis was only slightly inhibited by SBI fungicides at concentrations which caused considerable changes in both sterol content and composition. Conversely, in B. cinerea and P. teres, growth was strongly inhibited under conditions where ergosterol was still the predominant sterol, suggesting that, in these two fungi, growth may be more sensitive to SBI fungicides than overall sterol production. Demethylase inhibitor fungicides behaved as a homogeneous group in their effects on growth and on sterol profiles of the three fungi studied.     

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.     

Assessment of the loose smut fungi (Ustilago nuda and U. tritici) in tissues of barley and wheat by fluorescence microscopy and real-time PCR

Loose smut fungi of barley and wheat (Ustilago nuda and U. tritici, respectively) colonize the plant without causing obvious disease symptoms before heading. The availability of diagnostic methods to detect and follow the growth of these pathogens in the plant would therefore be highly advantageous for both resistance breeding and the development of effective seed treatments. Using seed lots of barley and wheat highly infected with loose smut, we studied the early establishment of the loose smut pathogens in the plant by fluorescence microscopy. In hand-cut sections stained with the fluorochrome Blankophor?, fungal hyphae were observed to invade the shoot apical meristem and leaf primordia during the first days after the onset of germination. At the first node stage the ear and leaf primordia were generally extensively colonized. Hyphae of U. nuda were also regularly observed in high density in the nodes. A protocol was developed for the specific amplification of U. nuda and U. tritici DNA extracted from infected plant tissue. PCR screening of U nuda in seedlings from infected and healthy seed lots was compared to ELISA, microscopy and ultimately head infection of mature plants derived from tillers of the tested seedlings. The results indicated that a prediction of loose smut infection by real-time PCR is possible at the second leaf stage, and that the assay is equally suited for use with spring and winter varieties of barley and wheat.     

Oxathiin carboxamides highly active against carboxin-resistant succinic dehydrogenase complexes from carboxin-selected mutants of Ustilago maydis and Aspergillus nidulans

The systemic fungicide carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and structurally related analogs are powerful inhibitors of succinate oxidation in mitochondria isolated from a variety of sources. The site of action which is, apparently, also that for thenoyltrifluoracetone, is in the complex II (succinate-ubiquinone reductase) region of the mitochondrial electron transfer chain. The succinic dehydrogenase complex (SDC) in mitochondria from carbon-resistant mutant strains of Ustilago maydis and Aspergillus nidulans is resistant to the fungicide in vitro. The current study shows that certain oxathiin carboxamides are selectively active against particular mutated SDCs of U. maydis and A. nidulans. Molecular structures affecting the phenotypic expression of mutation to carboxin resistance in U. maydis do not appear to affect similarly such expression in A. nidulans and vice versa. Of particular interest was the discovery of oxathiin carboxamides, e.g., 4′-phenylcarboxin, which were more inhibitory to the enzyme complex from one category of carboxin-resistant mutants of U. maydis than from the wild-type strain. Although such negative correlation between carboxin and other carboxin analogs has not been observed in studies with other categories of mutants, structures which drastically lower the resistance level were found in all cases. It appears that for any given mutation affecting carboxin sensitivity of the SDC in fungi, a specific structural group of carboxamides (or even a specific carboxamide) may be found which will alleviate or reverse the effect of the mutation in terms of inhibition of the SDC. If the mutations alter a protein receptor site for carboxamides, such mutations might be expected to influence the binding of carboxins of different structure. In essence, then, different molecular structures can “recognize” different alterations in the mutated enzyme complex and inhibit effectively. With few exceptions, the inhibition by carboxamides of cell growth of wild-type and carboxin-resistant strains of U. maydis and A. nidulans closely paralleded the inhibition of their respective SDCs. Although the few analogs tested were found unable to control corn smut systemically in seedlings artificially inoculated with compatible carboxin-resistant strains, control of naturally occurring carboxin-resistant strains of pathogenic fungi may be possible using particular structural analogs of carboxin which selectively inhibit the mutant organisms.     

Structure-activity relationships of carboxamide fungicides and the succinic dehydrogenase complex of Cryptococcus laurentii and Ustilago maydis

The systemic fungicide, carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and a variety of carboxamide compounds exhibit a marked specificity for Basidiomycete fungi. This unique specificity resides in the mitochondrial succinic dehydrogenase complex (SDC) of sensitive Basidiomycetes such as Ustilago maydis, the corn smut fungus. The present study examines in detail the structure-activity relationships of 93 carboxamide compounds and the SDC of two carboxin-sensitive organisms, U. maydis and a Basidiomycetous yeast, Cryptococcus laurentii. It has been possible to elucidate substantially the requirement in molecular structure needed for inhibition of the mitochondrial SDC. With few exceptions, a good correlation exists between the inhibitory activity of carboxamides towards the SDC of U. maydis and C. laurentii and the inhibition of growth of carboxamide-sensitive fungi, both in vitro and in vivo on the diseased plant. The structure-activity results were used as a basis for the synthesis of new, fungicidally-active carboxamides. The compounds found to be most active against the mycelial growth of Rhizoctonia solani were also tested on spore germination or mycelial growth of non-Basidiomycete fungi. Three carboxanilides (3-methyl-thiophene-2-carboxanilide, 3′-methyl-2-methylbenzanilide and 3′-methyl-2-ethylbenzanilide) had a fungitoxic spectrum which extended beyond Basidiomycetes. The spectrum of fungicidal activity of carboxanilides appears to be altered not only by substitution in the aniline ring, but by the nature of the ring attached to the carbonyl. No correlation was found between the inhibitory activity of oxathiins and benzanilides and their calculated partition coefficients.     

Ustilago maydis Infection of the Nonnatural Host Arabidopsis thaliana

ABSTRACT The experimental infection of Arabidopsis thaliana by the maize phytopathogenic hemibasidiomycete Ustilago maydis under axenic conditions is described. When plantlets were inoculated with mixtures of compatible haploids, the fungus was able to grow on the plant surface of inoculated seedlings in the form of white mycelium and invade the tissues, probably penetrating through stomata; however, it did not form teliospores. Symptoms of disease were increased anthocyanin formation, development of chlorosis, increased formation of secondary roots, induction of malformations in the leaves and petioles, induction of tissue necrosis, and stunting. In several cases, death of the invaded plants occurred. Interestingly, inoculation of single U. maydis haploid strains produced similar symptoms in Arabidopsis plantlets. In contrast, several mutants avirulent to maize also were avirulent or less virulent than wildtype strains on Arabidopsis. Collectively, the reported data suggest that the U. maydis-Arabidopsis pathosystem may constitute a useful experimental model for the analysis of some aspects of the virulence factors of the fungus. With the study of nonhost responses and their comparison to those occurring during maize infection, we will be able to elucidate some obscure aspects of U. maydis pathogenicity in the future.     

Genetic control of resistance to tridemorph inustilago maydis

Mutants ofUstilago maydis with low resistance to tridemorph isolated in a mutation frequency of 7x 10^-6 after UV-irradiation and selection on media containing 25 μg ml^-1 tridemorph. Genetic analysis with nine such mutant isolates resulted in the identification of two unlinked chromosomal loci,U/tdm- 1 andU/tdm- 2. TheU/tdm mutations are responsible for low resistance levels to tridemorph (resistance factor, Rf, of 3 or 5 based on effective concentration causing a 50% reduction in the growth rate (EC₅₀) or minimal inhibitory concentration (MIC) values, respectively) and low to moderate level of resistance to fenpropimorph (Rf 10 or 16 based on MIC or EC₅₀, respectively) and fenpropidin (Rf 5 or 11 based on MIC or EC₅₀, respectively). Haploid strains carrying bothU/tdm mutations exhibit higher levels of resistance to the above fungicides, indicating interallelic interaction between nonallelic genes. Crosses between mutants carrying theU/tdm- genes with compatible isolates carrying theU/fpm- 1 orU/fpm- 2 mutations, which were found in previous work to carry fenpropimorph resistance, yielded in all cases a large number of recombinants with wild-type sensitivity, indicating that the mutant genes involved were not allelic. Cross-resistance studies with the inhibitors of C-14 demethylase showed that. the U/tdm-mutations were responsible for increased sensitivity to the triazoles triadimefon, triadimenol, propiconazole and flusilazole, and to the pyridine pyrifenox. Study of gene effect on the fitness ofU. maydis showed thatU/tdm-mutations appeared to be pleiotropic, having more or less adverse effects on growth rate in liquid culture and pathogenicity on young corn plants.     

Inhibitory effect of ACC deaminase‐producing bacteria on crown gall formation in tomato plants infected by Agrobacterium tumefaciens or A. vitis

This study showed that various rhizosphere bacteria producing the enzyme 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase (ACCD), which can degrade ACC, the immediate precursor of ethylene in plants, and thereby lower plant ethylene levels, can act as promising biocontrol agents of pathogenic strains of Agrobacterium tumefaciens and A. vitis. Soaking the roots of tomato (Solanum lycopersicum) seedlings in a suspension of the ACCD‐producing Pseudomonas putida UW4, Burkholderia phytofirmans PsJN or Azospirillum brasilense Cd1843 transformed by plasmid pRKTACC carrying the ACCD‐encoding gene acdS from UW4, significantly reduced the development of tumours on tomato plants injected 4–5 days later with pathogenic Agrobacterium strains via wounds on the plant stem. The fresh mass of tumours formed by plants pretreated with ACCD‐producing strains was typically four‐ to fivefold less than that of tumours formed on control plants inoculated only with a pathogenic Agrobacterium strain. Simultaneously, the level of ethylene evolution per amount of tumour mass on plants pretreated with ACCD‐producing bacteria decreased four to eight times compared with that from tumours formed on control plants or plants pretreated with bacteria deficient in ACCD production. Moreover, transgenic tomato plants expressing a bacterial ACCD were found to be highly resistant to crown gall formation relative to the parental, non‐transformed tomato plants. The results support the hypothesis that ethylene is a crucial factor in Agrobacterium tumour formation, and that ACCD‐produced rhizosphere bacteria may protect plants infected by pathogenic Agrobacteria from crown gall disease.     

Thiophene carboxamide fungicides: Structure-activity relationships with the succinate dehydrogenase complex from wild-type and carboxin-resistant mutant strains of Ustilago maydis

A variety of thiophene carboxamide compounds have been synthesized and tested on the succinate dehydrogenase complex (SDC) in mitochondria from a wild-type strain and carboxin-resistant strains of Ustilago maydis (corn smut). The site of action of thiophene carboxamides is identical to that of carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and thenoyltrifluoroacetone, that is, the succinate-ubiquinone reductase (complex II) span in the mitochondrial electron transfer chain. This investigation reveals new molecular structures which are strong inhibitors of wild-type and carboxin-resistant SDCs. The 5-amino analog of the parent anilide, 3-methylthiophene-2-carboxanilide (I), proved to be an especially potent inhibitor of the wild-type SDC (I₅₀, 0.019 μM). Analogs of (I) such as 4′-carboethoxy, 4′-nbutyl, 4′-phenyl, and 4′-benzoyl were negatively correlated in activity to the carboxanilide (I) with respect to resistance level. A number of structures showed considerable selectivity for mutated SDCs from both highly and (particularly) moderately carboxin-resistant SDCs of U. maydis, markedly lowering the resistance level, i.e., the degree of resistance. Thus, in addition to the oxathiins, specific structural groups of thiophene carboxamides can also alleviate or reverse the effect of carboxin-selected mutation with reference to inhibition of the SDC. Of important significance was the finding that molecular selectivity for mutated, carboxin-resistant SDCs can be influenced by replacement of an oxathiin by a thiophene heterocyclic ring as well as by the substitutive group on the amide nitrogen, permitting different categories of mutant types and even mutants within a single category to be distinguished from one another. With all the structural combinations available, it appears quite possible, in terms of inhibition, to overcome any type of mutation in a fungal SDC which arises through selection by carboxin or other carboxamide compounds. A reasonable correlation generally exists between inhibition by thiophene carboxamides of the SDC and sporidial growth of wild-type and carboxin-resistant strains of U. maydis. A permeability barrier to 4′-substituted analogs of (I) was encountered in the wild-type strain but not mutant strains. Excellent protectant activity against bean rust (Uromyces phaseoli) was obtained with the 3′-nhexyl, 3′-nhexyloxy, and 4′-phenoxy analogs of (I).     

Isolation of microsomal cytochrome-p450 isozymes from Ustilago maydis and their interaction with sterol demethylation inhibitors

A procedure for the isolation of microsomes containing cytochrome-P450 isozymes from Ustilago maydis is described. Yields of P450 amount to approximately 19(±+ 6) pmol mg^?1 of microsomal protein. The wavelength of maximum absorbance of the reduced carbon monoxide difference spectrum is 448-449 nm. The azole fungicides prochloraz, etaconazole, imazalil, triadimefon and 3-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-4(3H)-quinazoline, which differ markedly in toxicity to U. maydis, all induce type II binding difference spectra at extremely low concentrations (10^?9-10^?8 M). The DMI concentrations which cause half saturation of type II binding difference spectra (IC₅₀) do not correlate with the fungicidal activities of the azoles. Binding of carbon monoxide to ferrous cytochrome-P450 was only slightly inhibited to different degrees by the DMIs tested. However, the inhibition of carbon monoxide binding also does not correlate with fungitoxicity of the DMIs. The results in this paper suggest that the spectrophotometric studies with this preparation are not useful for evaluating selective toxicity of DMIs to intact sporidia of U. maydis.     

Carboxin resistance in the haploid,the heterozygous diploid,and the plant-parasitic dicaryotic phase of Ustilago maydis

Respiration and growth of a diploid strain of Ustilago maydis, heterozygous at the oxr-1 locus for resistance to carboxin and carboxin analogs, were less sensitive to the fungicide than the corresponding processes in the wild type haploid. Data on the inhibition of succinate-2,6-dichlorophenolindophenol reductase indicate that the mitochondria of the heterozygous diploid probably contain a mixture of carboxin-sensitive and carboxin-resistant succinic dehydrogenase complexes. This hypothesis is supported by the results of temperature aging experiments: When mitochondria of the heterozygous diploid were subjected to incubation at 30°C which destroyed the enzyme activity of mitochondria from the mutant haploid, the remaining succinate-2,6-dichlorophenol-indophenol reductase activity exhibited a carboxin sensitivity similar to that of mitochondria from the wild type haploid.Carboxin gave very good control of corn smut in seedlings artificially inoculated with two wild type compatible strains but not when both strains were mutant for carboxin resistance, which indicates high resistance in the dicaryon. In a heterozygous cross, the dicaryon seemed to exhibit considerable resistance, if sufficient time was allowed for its formation between inoculation and carboxin treatment of soil.     

Polymerase Chain Reaction Detection of Ustilago hordei in Leaves of Susceptible and Resistant Barley Varieties

ABSTRACT Although Ustilago hordei infects barley seedlings, symptoms of the disease covered smut are not visible until heading. Natural or artificial inoculation usually results in inconsistent infection, even in highly susceptible lines. Thus, breeding for resistance to covered smut is time consuming and difficult. The ribosomal DNA internal transcribed spacer (ITS) regions of U. hordei were sequenced and a primer pair was developed for polymerase chain reaction (PCR). These primers amplified a 574-bp fragment from DNA of Ustilago spp., but did not amplify DNA from barley or other common barley pathogens. DNA extracted from as few as four U. hordei sporidia was detected by this method. U. hordei DNA was amplified from leaf tissue of inoculated susceptible and resistant plants at different stages of plant development in differential varieties. Growth of the fungus in different leaves of an individual plant was variable. Several highly resistant varieties were shown to contain U. hordei DNA in the first three to four leaves, but not in later leaves. Thus, although the fungus can infect many resistant plants, the plants remain symptomless. Detection of U. hordei prior to heading should assist efforts for breeding for resistance and provide clues concerning the mechanisms of resistance employed by different resistance genes.     

Comparative antifungal effect and mode of action of tetraconazole on Ustilago maydis

Tetraconazole, a new, recently introduced antifungal triazole, has been assayed in parallel with a number of standard analogues on various sensitive strains of Ustilago maydis. The values of EC₅₀ and EC₉₀ tetraconazole concentrations, determined on strain ATCC 14826 in agar, were 0.5 × 10⁻⁶ and 3.5 × 10⁻⁵ , respectively, in reasonable agreement with those needed to inhibit by 50% and 90%, respectively, the ergosterol biosynthesis in broth cultures. Squalene and 12 sterols have been extracted from the latter, characterized and quantified. Accumulation of 14α-methylsterols and reduction of ergosterol and other late precursors are consistent with the inhibition of 14α-demethylase caused by the title compound.     

Ethylene signalling is essential for the resistance of Nicotiana attenuata against Alternaria alternata and phytoalexin scopoletin biosynthesis

The phytohormone ethylene plays an important role in plant defence responses to pathogen attack. When infected by the necrotrophic fungal pathogen Alternaria alternata (tobacco pathotype), which causes severe diseases in Nicotiana species, the wild tobacco plant Nicotiana attenuata accumulates a high amount of the jasmonate (JA)‐dependent phytoalexin scopoletin to defend itself against this fungal pathogen. However, it is still not known whether ethylene signalling is also involved in scopoletin biosynthesis and the resistance of N. attenuata. After infection, ethylene biosynthetic genes were highly elicited. Furthermore, plants strongly impaired in ethylene biosynthesis or perception had dramatically decreased scopoletin levels, and these plants became more susceptible to the fungus, while A. alternata‐elicited JA levels were increased, indicating that the decreased defence responses were not due to lower JA levels. Thus, it is concluded that after infection, ethylene signalling is activated together with JA signalling in N. attenuata plants and this subsequently regulates scopoletin biosynthesis and plant resistance.     

Heterogeneity of Pseudomonas savastanoi populations infecting Myrtus communis in Sardinia (Italy)

Genetic, phenotypic and host range diversity among Pseudomonas savastanoi isolates from Myrtus communis were investigated. Thirty‐one isolates from six Sardinian commercial myrtle orchards and three isolates from plants growing spontaneously on the island of Rhodes (Greece) were compared with reference strains of P. savastanoi from olive, oleander, ash and myrtle. Multilocus sequence analysis (MLSA) indicated the presence of a monomorphic population with a very low level of variability. Conversely, Biolog phenotypic fingerprinting and phytohormone production analyses showed a considerable metabolic diversity, as bacteria obtained from single infected tissue differed more than bacteria obtained from different orchards. When pathogenicity tests were carried out on myrtle plants, different types of symptoms were induced: knots, canker lesions with or without tissue proliferations and, occasionally, wilting of the inoculated twig, a symptom never reported before for P. savastanoi. Comparable symptoms were also observed in the natural environment both on spontaneous and cultivated plants. Moreover, the host range of the myrtle population was heterogeneous and not well defined. Some isolates showed a wide host range whilst others were pathogenic only to their natural host. Overall these findings suggest that the diversity of the P. savastanoi population from myrtle does not depend so much on the locality or the natural host and does not allow the Sardinian and Greek isolates, together with previously characterized myrtle strains, to be ascribed to a known pathovar of P. savastanoi, nor to propose their belonging, as a whole, to a new pathovar.     

Epiphytic Curtobacterium flaccumfaciens strains isolated from symptomless solanaceous vegetables are pathogenic on leguminous but not on solanaceous plants

In Iran, during 2013–16, 16 Gram‐positive corynebacteria‐like strains were recovered from the epiphytic parts of solanaceous vegetables including eggplant, pepper and tomato. The strains were recovered accidentally as a result of monitoring for other bacterial pathogens in solanaceous fields. The strains were phenotypically different from Clavibacter michiganensis strains. Although none of the strains were pathogenic on their host of isolation or on any other solanaceous plants, 12 out of 16 strains were pathogenic on common bean, cowpea, mung bean and soybean. Colonization by strains was observed on maize, zucchini, faba bean, honeydew melon, rapeseed, sugar beet and sunflower plants under greenhouse conditions. In PCR tests, the primer pair CffFOR2/CffREV4, specific for Curtobacterium flaccumfaciens pv. flaccumfaciens, enabled the amplification of the appropriately sized fragment in 12 out of 16 strains, and all 12 strains were pathogenic on dry beans. Phylogenetic analysis, using the gyrB and recA genes, showed all 16 bacterial strains clustered within several pathovars of C. flaccumfaciens. A nonpathogenic yellow‐pigmented strain (Xeu15) was clustered with the type strains of C. flaccumfaciens pv. betae and C. flaccumfaciens pv. oortii. Bacteriocin profiling assays revealed no significant differences among the pathogenic and nonpathogenic strains. Host range and population dynamics of four representative strains on 17 plant species showed population build‐up of the strains only on common bean, cowpea, wheat and red nightshade plants. The results provide important insights into the possible role of nonhost plants as reservoirs of plant pathogenic bacteria, which has important implications in plant disease epidemiology and management.