Effect of fungicides on the mycelial growth of soft fruit spoilage fungi

The in vitro activity of a range of fungicides was tested on strains of Botrytis cinerea, Mucor mucedo, Rhizopus stolonifer, and R. sexualis. Di-chlofluanid and dichloran were more active than the other fungicides against all of the strains of B. cinerea, while dichloran, dichlorophen and the dithiocarbamate fungicides were the most active against the Rhizopus species. Only dichlorophen and thiram markedly inhibited the growth of M. mucedo.     

Fungicide efflux and the MgMFS1 transporter contribute to the multidrug resistance phenotype in Zymoseptoria tritici field isolates

Septoria leaf blotch is mainly controlled by fungicides. Zymoseptoria tritici, which is responsible for this disease, displays strong adaptive capacity to fungicide challenge. It developed resistance to most fungicides due to target site modifications. Recently, isolated strains showed cross‐resistance to fungicides with unrelated modes of action, suggesting a resistance mechanism known as multidrug resistance (MDR). We show enhanced prochloraz efflux, sensitive to the modulators amitryptiline and chlorpromazine, for two Z. tritici strains, displaying an MDR phenotype in addition to the genotypes CYP51^{I381V Y461H} or CYP51^{I381V ΔY459/G460}, respectively, hereafter named MDR6 and MDR7. Efflux was also inhibited by verapamil in the MDR7 strain. RNA sequencing lead to the identification of several transporter genes overexpressed in both MDR strains. The expression of the MgMFS1 gene was the strongest and constitutively high in MDR field strains. Its inactivation in the MDR6 strain abolished resistance to fungicides with different modes of action supporting its involvement in MDR in Z. tritici. A 519 bp insert in the MgMFS1 promoter was detected in half of the tested MDR field strains, but absent from sensitive field strains, suggesting that the insert is correlated with the observed MDR phenotype. Besides MgMfs1, other transporters and mutations may be involved in MDR in Z. tritici.     

Fungicide resistance in cucurbit downy mildew – methodological, biological and population aspects

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is among the most devastating diseases of cucurbitaceous plants. In spite of improved cultural practices and breeding for resistant cultivars, chemical control is still a very important tool to manage the disease. During the last several decades, many fungicides from various chemical classes have been developed. The occurrence of strains of P. cubensis resistant/tolerant to some fungicides encouraged research of this phenomenon. The first part of this article summarises the many different methodological approaches such as field trials, in vitro testing on active plant tissues or molecular diagnoses developed for the detection of resistant/tolerant strains of P. cubensis, as well as methods to collect and maintain pathogen isolates. The second part outlines the commonly used fungicides to control P. cubensis and their features like systemicity, biological and biochemical mode of action and translocation behaviour within plants. The last part deals with geographical aspects such as first appearance of resistance problems, distribution of resistance, temporal development of resistance under selection pressure by a fungicide, fitness of resistant subpopulations in competition with sensitive ones in the absence of a fungicide, as well as genetic and molecular sources of resistance.     

The resistance of spores of resistant strains of Botrytis cinerea to quintozene, tecnazene and dicloran

Spores formed by strains of Botrytis cinerea resistant to quintozene, tecnazene, or dicloran did not always produce resistant colonies when grown on agar in the presence of these fungicides. Only about one half of spores produced by fungicide-vapour-resistant strains in the absence of fungicides gave resistant colonies whereas all spores produced by the same strains in the presence of fungicides formed resistant colonies on agar. Some spores produced by resistant strains that had developed on agar containing the fungicides were not viable, but those that were always gave resistant colonies on agar.     

Double resistance by citrus green mould Penicillium digitatum to the fungicides guazatine and benomyl

In vitro dosage response data with different isolates of Penicillium digitatum and the fungicide guazatine indicated an approximate 10-fold shift in tolerance when compared with wild type strains. ED₅₀ values for resistant strains were approximately 0.5 μg/ml compared to approximately 0.05, μg/ml for the wild type strains. Colony growth of guazatine resistant isolates on selective media containing carbendazim showed that they were also resistant to the benzimidazole group of fungicides. In vivo tests in inoculated oranges with strains previously characterised by in vitro tests confirmed resistance to guazatine and benomyl. A combined treatment of these fungicides at 400 /μ/ml and 500 μg/ml respectively, which normally gives protection against decay, also failed to provide adequate mould control. Growth and pathogenicity of the resistant strains in these tests in oranges were indistinguishable from that of wild type strains.     

The Group III Two-Component Histidine Kinase AlHK1 is Involved in Fungicides Resistance,Osmosensitivity, Spore Production and Impacts Negatively Pathogenicity in <Emphasis Type="Italic">Alternaria longipes</Emphasis>

Members of group III histidine kinases from different filamentous fungi were previously shown to mediate osmoregulation and resistance to dicarboximide, phenylpyrrole and, aromatic hydrocarbon fungicides. In this study, we report the disruption of the gene encoding group III histidine kinase, AlHK1, in the economically important plant pathogen Alternaria longipes. The AlHK1 gene disruption had pleiotropic effects on this fungus. Besides the expected osmosensitivity and fungicides resistance, AlHK1 participated in the spore production process. In addition, the ΔAlHK1 strains had stronger aggressive ability to infect their host plant than that of their parental strain, the wild-type strain C-00, suggested that AlHK1 was involved in the pathogenicity of A. longipes and performed in this function by a negative manner. This is the first report to our knowledge.     

On the effects of some pesticides on Rhizobium and isolation of pesticide-resistant mutants

Summary The in vitro effects on Rhizobium of two herbicides and two fungicides that are often used in Sweden were studied. The sensitivity of the strains seems to be specific since the Rhizobium meliloti strains generally were tolerating higher doses of pesticides than strains of Rh. trifolii and Rh. leguminosarum. It was possible to isolate mutants that tolerated much higher doses of the pesticides than the wild type strains.     

Intimate genetic relationships and fungicide resistance in multiple strains of Aspergillus fumigatus isolated from a plant bulb

Fungal infections are increasingly dangerous because of environmentally dispersed resistance to antifungal drugs. Azoles are commonly used antifungal drugs, but they are also used as fungicides in agriculture, which may enable enrichment of azole-resistant strains of the human pathogen Aspergillus fumigatus in the environment. Understanding of environmental dissemination and enrichment of genetic variation associated with azole resistance in A. fumigatus is required to suppress resistant strains. Here, we focused on eight strains of azole-resistant A. fumigatus isolated from a single tulip bulb for sale in Japan. This set includes strains with TR₃₄/L98H/T289A/I364V/G448S and TR₄₆/Y121F/T289A/S363P/I364V/G448S mutations in the cyp51A gene, which showed higher tolerance to several azoles than strains harbouring TR₄₆/Y121F/T289A mutation. The strains were typed by microsatellite typing, single nucleotide polymorphism profiles, and mitochondrial and nuclear genome analyses. The strains grouped differently using each typing method, suggesting historical genetic recombination among the strains. Our data also revealed that some strains isolated from the tulip bulb showed tolerance to other classes of fungicides, such as QoI and carbendazim, followed by related amino acid alterations in the target proteins. Considering spatial–temporal factors, plant bulbs are an excellent environmental niche for fungal strains to encounter partners, and to obtain and spread resistance-associated mutations.     

Influence of commercial fungicides on the germination,growth and virulence of four species of entomopathogenic fungi

The influence of 15 commercially available fungicides on the germination, growth and virulence of Metarhizium anisopliae, Beauveria bassiana, Isaria fumosorosea, and Lecanillium longisporum was evaluated. The influence of the fungicides on conidial germination was dependant on the fungicide type and dose. Most fungicides retarded conidial germination of all the fungi tested at 10× and at the recommended rate of application, however, their toxicity declined at lower concentrations. Most of the fungicides inhibited mycelial growth of B. bassiana, whereas L. longisporum growth was unaffected. Only two and eight fungicides influenced mycelial growth of I. fumosorosea and M. anisopliae, respectively. None of the fungicides influenced the virulence of B. bassiana and L. longisporum, however, tolylfluanid and azoxystrobin reduced the virulence of M. anisopliae and I. fumosorosea, respectively. These studies clearly show that certain fungicides have the potential to inhibit germination of entomopathogenic fungi in vitro but appear to have little or no effect on their virulence against target insects.     

Effect of fungicides on survival of Rhizobium on seeds and the nodulation of bean (Phaseolus vulgaris L.)

The survival of Rhizobium leguminosarum biovar phaseoli on seeds of bean was tested, using the cultivar Carioca. The seeds were treated seven days before inoculation with Benlate, Vitavax, Banrot, Difolatan or Ridomil fungicides. The rhizobial strains used were: CIAT 899, CPAC 1135 and CIAT 652. Strain CIAT 899 showed greater survival on the seed with fungicide than the other strains. Two hours after the contact with fungicides strains CIAT 652 and CPAC 1135 had significantly lower numbers of rhizobia than the treatment without fungicide. The Benlate and Banrot fungicides had the greatest effect on survival of rhizobial strains. There was a drastic mortality of the two strains, CIAT 652 and CPAC 1135, on seeds treated with Benlate and Ridomil. Under field conditions, granular inoculation produced fewer nodules, but a similar total nodule weight as seed inoculation. Serological tests (ELISA) showed that seed treatment with Benlate in connection with seed inoculation reduced drastically the occurrence of inoculated strains in nodules, while the same fungicide treatment and inoculation applied in the seed furrow did not affect the survival of the inoculated strain.     

Occurrence of strains of Colletotrichum coffeanutn resistant to methyl benzimidazol-2-ylcarbamate (carbendazim) and chemically-similar compounds

Certain strains of Colletotrichum coffeanum (the causal fungus of coffee berry disease) which developed in coffee plots sprayed in 1973 and 1974 with carbendazim formulations Bavistin and Derosal and with Folicidin (cypendazol) were resistant to these fungicides and also to Benlate (benomyl). Strains resistant to cypendazol developed in plots sprayed with benomyl but these were not resistant to benomyl itself. There were also indications of strains resistant to carbendazim amongst the population of saprophytic species (C. acutatum and C. gloeosporoides) colonizing coffee bark. Resistant isolates of C. coffeanum in culture produced colour variants by sectoring more frequently than normal isolates. These sectors were equally pathogenic to coffee berries and their growth was slightly enhanced on media containing carbendazim.     

A Comparative Analysis of Stachybotrys chartarum Strains Isolated in Russia

This work deals with a comparative analysis of Stachybotrys chartarum strains isolated from various artificial cellulose-containing materials and natural substrates in geographically distant regions of Russia. The analysis included determination of the spore size; the strain toxicity to Paramecium caudatum; the strain resistance to the fungicides Benomil, Olilen, and Tilt; and the PCR study of the genome structure with the aid of a primer that was complementary to the core sequence of the SINE retrotransposon. It was found that some of the strains that were isolated from different areas and from different substrates differ in their toxicity, fungicide resistance, and genome structure. PCR analysis showed the absence of any correlation between the genome structure, the strain properties, the geographic area, and the substrates from which the strains were isolated. The pheno- and genotypic diversity of the strains and their different vegetative compatibility suggest the existence of an intraspecies diversity of the S. chartarum strains that were isolated in different geographic areas. The absence of any correlation between the pheno- and genotypic properties of the strains and the substrates from which they were isolated implies that the colonization of artificial substrates by S. chartarum occurred occasionally from natural habitats. The S. chartarum populations that live on artificial substrates are unlikely to have their own evolutionary history.     

Studies on the survival and infective ability of dicarboximide-resistant strains of Botrytis cinerea

Few dicarboximide-resistant strains of Botrytis cinerea produce macroconidia either on culture media or on natural substrates, although, the viability of such conidia was similar to those of sensitive strains. The ability of dicarboximide-resistant strains to infect strawberries was similar to that of sensitive strains and most of the resistant strains competed successfully with sensitive strains when inoculated in equal numbers onto detached strawberries and onto flowers of field-grown plants, especially after treatment with dicarboximide fungicides. All resistant strains tested survived for at least 9 months on inoculated strawberry leaf litter although their incidence was ‘diluted’ by wild type sensitive strains. Sporulation and dispersal of resistant strains from the litter was very limited resulting in a low incidence of fruit infection even after treatment with dicarboximides. Consequently, there was no significant increase of resistant strains in the plantation and control of infection was maintained with iprodione and dichlofluanid. Poor sporulation of dicarboximide-resistant strains of B. cinerea is considered to be the single most important factor in limiting their development in the field.     

Molecular insights into development of Trichoderma interfusants for multistress tolerance enhancing antagonism against Sclerotium rolfsii Sacc

The current study aimed at developing diverse Trichoderma fusants for fungicides, drought, and salt tolerance with enhanced antagonistic activity against Sclerotium rolfsii Sacc. Trichoderma virens NBAII Tvs12 (mycoparasitic) and Trichoderma koningii MTCC796 (multistress tolerant) were used as parental strains for development of interspecific protoplast fusants. A total of 36 stable fusants were used for mycoparasitism, fungicides, and abiotic stresses (drought and salt) tolerance. The results revealed 20 homozygous progenies showing characteristics of either one parental strain and 14 heterozygous mutants depicting traits of both parental strains. A novel concept of inhibition coefficient was established using growth-related key parameters that represent the pathogen biology and the biocontrol-related biophysics of Trichoderma fusants. The results indicated a differential inhibition coefficient of the test pathogen and the highest (92.88%) inhibition coefficient of S. rolfsii was observed by interstable fusant Fu21. It also grew better under fungicides and abiotic stress (drought and salt) conditions. The molecular characterization and heterozygosity analysis evidenced the highest observed heterozygosity (0.5441) and gene flow (0.3872) in stable heterozygous Fu21. Principal coordinates analysis exhibited 62.7% of total variability. The ecofriendly heterozygous Trichoderma fusant (Fu21) might be useful for biocontrol of stem rot disease under adverse conditions or as a part of integrated disease management.     

Contribution of Pathogens to Peach Fruit Rot in Northern Greece and their Sensitivity to Iprodione, Carbendazim, Thiophanate-methyl and Tebuconazole Fungicides

This study identified the main pathogens causing fruit rots of mature peaches in northern Greece, the major peach producing area of Greece. The brown rot pathogen Monilinia laxa was responsible for approximately 70% and 78% of rotted peaches in 2005 and 2006 respectively. Serious damage (up to 5%) was also caused with the fungus Phomopsis amygdali. Other pathogens isolated from rotted peaches at a low percentage were Alternaria alternata, Aspergillus niger, Aspergillus flavus, Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium spp., Colletotrichum gloeosporioides, Rhizopus stolonifer and Gilbertella persicaria. Most fungal isolates originated from the rotted peaches were tested for their sensitivity to the fungicides iprodione, carbendazim, thiophanate methyl and tebuconazole at label recommended concentrations. All fungicides inhibited the growth of M. laxa, A. niger, A. flavus, S. sclerotiorum, P. amygdali and B. cinerea on poisoned agar. Apart from iprodione, all other fungicides inhibited the mycelium growth of the pathogen Fusarium sp. The mycelium growth of Fusarium sp. was significantly less with iprodione than control. Only iprodione and tebuconazole were effective against A. alternata and R. stolonifer. Tebuconazole inhibited the mycelium growth of R. stolonifer, while iprodione reduced significantly in comparison to control. The mycelium growth of the fungus C. gloeosporioides was inhibited by tebuconazole and reduced significantly by the fungicides thiophanate methyl, carbendazim and iprodione. Among all the fungi tested, only M. laxa and B. cinerea isolates were found resistant to benzimidazoles [the EC50 (50% effective concentration) value was 100–200 mg/l and 200–300 mg/l for the largest number of thiophanate methyl‐ and carbendazim‐resistant M. laxa isolates respectively, while the biggest number of B. cinerea thiophanate methyl‐ and carbendazim‐resistant isolates showed EC50 value 200–300 mg/l and 300–400 mg/l, respectively]. However, these strains were sensitive to tebuconazole and iprodione. Therefore, these fungicides can be used as an alternative method to control benzimidazole‐resistant Monilinia and Botrytis isolates.     

<Emphasis Type="Italic">Fusarium verticillioides</Emphasis>: Evaluation of Fumonisin Production and Effect of Fungicides on In Vitro Inhibition of Mycelial Growth

The objectives of this study were to evaluate the fumonisin production by 16 F. verticillioides strains on corn cultures and the effect of quintozene and fludioxonil + metalaxyl-M fungicides on “in vitro” mycelial growth on agar. In addition, the effect of fludioxonil + metalaxyl-M on fumonisin production in defined liquid culture medium was analyzed. Fumonisin B₁ levels on corn cultures ranged from 2.41 to 3996.36 μg/g and the F. verticillioides 103F strain produced the highest level (3996.36 ± 390.49 μg/g, P < 0.05). F. verticillioides strains were inoculated in potato dextrose agar with the addition of quintozene (75 to 9,375 μg/ml) and fludioxonil + metalaxyl-M (1.5 + 0.6 to 187.5 + 75 μg/ml) in order to evaluate the effect of these fungicides on “in vitro” mycelial growth. The F. verticillioides strains showed great variability concerning ED₅₀ values, which were below the recommended application dose for quintozene, but above that for fludioxonil + metalaxyl-M. Moreover, fungicide addition to the culture medium increased mean FB₁ levels compared to the control, suggesting the importance of focusing on the effect of fungicides on mycotoxin production as well as on the phytopathogen control.     

Action de quelques fongicides sur la croissance mycélienne de trois espèces d'Entomophthorales

Résumé L'action,in vitro, de 34 fongicides sur la croissance mycélienne de 3 espèces d'Entomophthorales:Basidiobolus ranarum (Eidam),Conidiobolus osmodes (Drechsler) etEntomophthora virulenta (Hall & Dunn) [désignéEntomophthora nr.thaxteriana (Petch) parSoper & Bryan, 1974] a été examinée. Dans l'ordre croissant de leur sensibilité aux fongicides, les souches étudiées se classent comme suit:B. ranarum, E. virulenta etC. osmodes. Il existe une grande disparité dans l'activité des différents fongicides sur la croissance mycélienne des 3 espèces d'Entomophthorales. Certains fongicides sont peu ou pas actifs (chlorothalonil, éthirimol, oxychlorure de cuivre, soufre, etc.), d'autres agissent moyennement (captafol, dichlofluanide, mancozèbe, thirame, etc.); d'autres encore se révèlent très toxiques (bénodanil, chinométhionate, chloronèbe, triarimol). Mais il n'est pas simple de classer les fongicides en fonction de leur activité; un bon nombre d'entre eux possède un comportement hétérogène devant les 3 souches (carbendazime, oxycarboxine, 26019 RP, etc.). Dans l'ensemble, les fongicides systémiques se montrent plus efficaces sur la croissance mycélienne que les non systémiques.

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Summary The effect of 34 fungicides on the mycelial growth of 3 Entomophthorales species:Basidiobolus ranarum (Eidam),Conidiobolus osmodes (Drechsler) andEntomophthora virulenta (Hall & Dunn) was investigatedin vitro. The examination of growth was conducted in test tubes and five fungicidal concentrations were tested. The amount of growth of the various fungi in all treatments was expressed as a percentage of the growth of the control in order to evaluate the concentrations which reduce the growth speed by half. In the increasing order of their sensitivity to fungicides the strains under study could be classified in the following manner:B. ranarum, E. virulenta andC. osmodes. There is a great disparity between the action of the different fungicides upon the mycelial growth of the 3 Entomophthorales species. Some fungicides have little or no action (chlorothalonil, ethirimol, copper oxychloride, sulphur, etc.); others are more or less active (captafol, dichlofluanid, mancozeb, thiram, etc.); others again showthemselves to be highly toxic (benodanil, quinomethionate, chloroneb, triarimol). However it is not simple to classify the fungicides as a function of their activity; many of them react differently on the 3 species (carbendazim, oxycarboxin, 26019 RP, etc.). Overall, systemic fungicides are more effective on mycelial growth than non-systemic fungicides.

     

工厂化生产海鲜菇菌包污染霉菌的鉴定及防治

对工厂化生产中海鲜菇菌包污染霉菌进行分离,根据霉菌的形态特征、培养性状及ITS序列分析,鉴定其为哈茨木霉、拟康氏木霉、脉孢霉、长枝木霉、黑曲霉、产红青霉和产黄青霉;在此基础上探讨了常用抑菌剂对霉菌的防治效果及对海鲜菇菌丝生长的影响。结果表明,质量浓度100 mg/L克霉灵对哈茨木霉、黑曲霉、产红青霉、产黄青霉、拟康氏木霉有强抑制作用,质量浓度100 mg/L多菌灵对长枝木霉、产红青霉、产黄青霉、拟康氏木霉有强抑制作用,二者对海鲜菇菌丝生长的抑制都比较弱。可为海鲜菇工厂化生产中污染霉菌的综合防治提供参考。     

<Emphasis Type="Italic">Azospirillum brasilense</Emphasis> siderophores with antifungal activity against <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis>

Anthracnose, caused by the fungus Colletotrichum acutatum is one of the most important diseases in strawberry crop. Due to environmental pollution and resistance produced by chemical fungicides, nowadays biological control is considered a good alternative for crop protection. Among biocontrol agents, there are plant growth-promoting bacteria, such as members of the genus Azospirillum. In this work, we demonstrate that under iron limiting conditions different strains of A. brasilense produce siderophores, exhibiting different yields and rates of production according to their origin. Chemical assays revealed that strains REC2 and REC3 secrete catechol type siderophores, including salicylic acid, detected by thin layer chromatography coupled with fluorescence spectroscopy and gas chromatography–mass spectrometry analysis. Siderophores produced by them showed in vitro antifungal activity against C. acutatum M11. Furthermore, this latter coincided with results obtained from phytopathological tests performed in planta, where a reduction of anthracnose symptoms on strawberry plants previously inoculated with A. brasilense was observed. These outcomes suggest that some strains of A. brasilense could act as biocontrol agent preventing anthracnose disease in strawberry.     

Diversity and antagonistic potential of Bacillus spp. associated to the rhizosphere of tomato for the management of Rhizoctonia solani

Bacillus spp. has emerged as the most effective alternative to synthetic chemical fungicides. To get a better insight in the antagonistic potential of Bacillus strains, rhizospheric soil samples of healthy tomato plants from Indo-gangetic plain regions of India were analysed. A total of 108 Bacillus strains were obtained from preliminary screening. Potent strains identified on the basis of in vitro antagonistic and biochemical assays were subjected to diversity analysis using 16S-rDNA, BOX and ERIC-PCR. Furthermore, the four best performing antagonistic Bacillus strains under in vitro plant growth promotion and antagonistic assay were selected for pot experiment. In field study, Bacillus amyloliquefaciens MB101 and Bacillus subtilis MB14 showed drastic reduction in disease index by 55.7 and 41.74% with significant elevation in fruit yield up to 220 and 184 qha^–1, respectively. The present study was successful in selecting effective Bacillus strains by performing phenotypic and genotypic characterisation of Bacillus strains that can be used as an integral component of integrated disease management of tomato root rot and damping-off.