Phoma Destructiva Research Articles

Evaluation of the effectiveness of ultraviolet disinfection (254 nm) of aqueous solutions from phytopathogenic microorganisms

Relevance. Currently, greenhouse complexes use water for irrigation from open surface sources (ponds, lakes, rivers) and underground water from wells, adding a return (drainage) solution to it. At the same time, water from both open sources and drainage solution often becomes a source of emergence and spread of phytopathogenic infection in greenhouses, which brings economically significant crop losses. In this regard, the need to develop guaranteed methods and methods for the disinfection of drainage water and water from open sources is relevant. One of the methods of water disinfection is the use of ultraviolet irradiation, but for the effective use of irradiation, it is necessary to ensure the minimum effective UV dose. This UV dose is typical for each pathogen.Results. Work was carried out to experimentally establish doses of UV irradiation that allow complete inactivation of vegetative cells and spores of bacteria and fungi (Agrobacterium tumefaciens, Pythium debaryanum, Fusarium oxysporum, Verticillium dahliae, Phoma destructiva) in a solution prepared on the basis of distilled water. A dose of UV irradiation of 40 mJ/cm2 ensured complete inactivation of the bacterial pathogen, and a dose of 60 mJ/cm2 showed complete inactivation of the pathogens of the studied fungal infections.

Green synthesis of copper nanoparticles using Sesbania aculeata to enhance the plant growth and antimicrobial activities

Green synthesis of copper oxide nanoparticles (CuONPs) was performed using Sesbania aculeata leaf extract. The characteristics of CuONPs were studied using the selected area electron diffraction analysis. The effect of nanoparticles on the plant growth including germination efficiency was studied in Brassica nigra at different concentrations (25, 30, 100 mg/100 ml) using plant tissue culture technique. The uptake of copper and potential metal-binding compounds (citrate, phytochelatins, nicotinamide) in xylem and phloem saps of B. nigra was examined. Copper uptake in xylem and phloem sap was found to be higher at 100 mg/100 ml concentration (2.2 ± 0.23 and 5.70 ± 0.9 µM, respectively) when compared to the control (1.0 ± 0.15 and 4.90 ± 0.7 µM) which could be toxic and inhibit the plant’s growth. 25 and 30 mg/100 ml were found to be safer. The root uptake of CuONPs in xylem was observed through the transmission electron microscopy analysis. Since the nanoparticles at higher dose inhibit plant’s growth, potassium leakage rate was analyzed and found to be higher with copper sulfate treatment (0.8 µg/g FW min−1) than with the CuONPs treatment (0.5 µg/g FW min−1) at 100 mg/100 ml concentration. Additionally, we evaluated the antimicrobial activity of CuONPs and the maximum zone of inhibition was found with Phoma destructiva (23 mm), Curvularia lunata (22 mm) at 40 µl concentration. This study shows that CuONPs synthesized using greener technology enhanced the plant growth of B. nigra at a lower dose (25 and 30 mg/100 ml) and act as a strong antimicrobial agent which can act as biofertilizer for both the crop protection and production.

Phoma destructiva causing blight of tomato plants: a new fungal threat for tomato plantations in Brazil?

Solanum lycopersicum is among the most important crops in Brazil. This crop is affected by a large range of fungal diseases that are recognized as major limitations for tomato production. Recently, plants grown in a greenhouse in Vicosa, Minas Gerais, Brazil, were found to bear severe blight symptoms. A pycnidial coelomycete was repeatedly found in association with necrotic tissues. The fungus had its morphology recognized as equivalent to that of Phoma and related genera. A phylogenetic analysis based on nrDNA (ITS) and partial β-tubulin (TUB) sequences led to the conclusion that the fungus involved was Phoma destructiva. Pathogenicity tests showed that, after 5 days, blight symptoms developed on leaves, flowers and stems of plants belonging to thirteen different tomato varieties tested. This fungal species is mostly known for causing post-harvest tomato rot, which is only regarded as a secondary disease in Brazil. This is in disagreement with the observations made in this work. Here, the disease symptoms caused by the fungus were very severe, fully justifying the scientific name of the pathogen. Under favorable environmental conditions, aggressive strains of P. destructiva, such as the one isolated in this study, may become significant threats to tomato plantations in Brazil.

Tepraloxydim, fluazifop-p-butyl ve metribuzin herbisitlerinin toprak kökenli bazı fungal patojenlerin koloni gelişimine ve sporulasyonuna etkisi

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Occurrence of Phoma Blight Caused by Phoma destructiva on Tomato (Solanum lycopersicum) in Malaysia

In May 2013, tomato plants cultivated in the Cameron Highlands, Pahang, Malaysia, had extensive fruit, stem and leaf spots. In severe cases, disease incidence reached 70% in glasshouses. Leaf lesions were initially small (1-2 mm), black on either surface enlarging to 1-2 cm in diameter, irregular to round in shape and slightly sunken and zonate. Stem lesions were longer, but were similar in appearance. Fruit lesions were sunken black spots of variable size at sites of wounds, insect punctures or stem scar cracks. The causal pathogen was identified as Phoma destructiva based on morphological, genetic (ITS and PCR) and pathogenicity analyses. This is thought to be the first report of Phoma blight caused by P. destructiva on tomato in Malaysia.

Pathogenicity Assay and Molecular Identification of Fungi and Bacteria Associated with Diseases of Tomato in Malaysia

This study was conducted in order to determine the fungi and bacteria associated with tomato plants at Cameron Highlands Malaysia. The fungi which have been isolated and detected from tomato plants were: Fusarium oxysporum, F. solani, F. acuminatum, Rhizoctonia solani, Colletotrichum boninense, C. acutatum and Phoma destructiva. The bacteria which have been isolated and detected from tomato plants were: Ralstonia solanacearum, Xanthomonas vesicatoria, X. gardneri and Pseudomonas syringae. While the most pathogenic fungi were C. boninense, P. destructive and F. oxysporum with the disease incidence (89.6%, 86.6%, 85.6%) respectively, the most pathogenic bacteria were X. vesicatoria and R. solanacearum with the disease incidence (96.6% and 87.6%) respectively.

Identification of two Bacillus amyloliquefaciens strains with high suppression to the key fruit pathogens of Chinese jujube

Two bacterial strains (ZJ01 and ZJ02) isolated from the phyllosphere of Chinese jujube were tested for antifungal activities and morphological mechanisms against Phoma destructiva, Alternaria alternata and Fusicoccum spp., three key pathogens of jujube fruits. Based on their physiological characteristics and 16S rDNA gene sequences, both strains were identified as Bacillus amyloliquefaciens. The disease incidence of jujube fruits sprayed with the fermentation liquor of ZJ01 and ZJ02 on the puncture wounds before inoculation with the spores of those three key pathogenic fungi was 10% or 66.7%, significantly lower than that of Nutrient Broth medium treatment (100%). These results suggest that strains ZJ01 may be a potential biological control agent for Chinese jujube fruit diseases. The antagonistic bacteria could cause five kinds of hyphal alterations of the causal agents, the maximum number ever observed. Of them, hyphal swelling and excessive branching were the dominant phenomenon, the other three varied with the pathogens. The diverse morphological malformation might be helpful to understand the broad antifungal activity of Bacillus spp..

In vitro antifungal efficacy of copper nanoparticles against selected crop pathogenic fungi

Copper nanoparticles play an important role in the field of optics and electronics, and also as a novel antimicrobial. In the present study, we report antifungal activity of copper nanoparticles against selected crop pathogenic fungi. Copper nanoparticles were synthesized by chemical reduction of Cu2+ in the presence of Cetyl Trimethyl Ammonium Bromide and isopropyl alcohol. Characterizations of copper nanoparticles were carried out by UV–visible spectroscopy, nanoparticles tracking analysis (NTA), Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) which revealed that synthesized nanoparticles were coated by Cetyl Trimethylammonium Bromide (CTAB) having particle size of 3–10nm. Copper nanoparticles demonstrated significant antifungal activity against plant pathogenic fungi: Phoma destructiva (DBT-66), Curvularia lunata (MTCC no. 2030), Alternaria alternata (MTCC No. 6572) and Fusarium oxysporum (MTCC No. 1755). Since for the synthesis of copper nanoparticles the present chemical method by using C-TAB-IPA is found to be simple, economic and fast, the synthesized copper nanoparticles can be used as a novel antifungal agent in agriculture to control the plant pathogenic fungi as well as potent disinfectant in poultry and animal husbandry.

Occurrence and pathogens of fruit shrink disease in Ziziphus jujuba Mill.

The occurrence of fruit shrink disease in Ziziphus jujuba Mill. ‘Linyilizao’ was investigated from 2006 to 2009 in Cangxian of Hebei Province, Taiyuan, and Taigu of Shanxi Province, and the pathogens were isolated from the diseased fruits sampled from both Cangxian and Taiyuan in 2006 and 2007. The pathogens in leaf, fruit, and shoot collected from Cangxian were isolated from June to October in 2008 at 30-day intervals. The incidence of the disease varied significantly with location and year, and the highest incidence was observed in Cangxian during 2006. Three kinds of key pathogens were isolated from the fruits from July to September, i.e., Alternaria alternata Keissler (mainly in July), Phoma destructiva Plowr. (mainly in September), and Fusicoccum sp., of which A. alternata was the most dominant. In leaves, both A. alternata (much more dominant) and P. destructiva could be isolated throughout June to October, but no Fusicoccum sp. was found. At the white-ripening stage of fruit, the isolation rate of A. alternata was the highest in leaves (36.67%), followed by extension shoot (6.67%) and fruit (1.11%) while that of P. destructiva was the highest in extension shoot (31.11%), followed by fruit (13.33%) and leaf (12.22%), with Fusicoccum sp. isolated only in extension shoot (2.22%). The frequency of isolating two or three kinds of above three pathogens together was very low, with the highest rate (19.30%) found in A. alternata and P. destructiva from leaves in July.

Epicoccamides B–D, Glycosylated Tetramic Acid Derivatives from an Epicoccum sp. Associated with the Tree Fungus Pholiota squarrosa

Three new tetramic acid derivatives, epicoccamides B-D, together with the known epicoccamide, were isolated from an Epicoccum sp. associated with the tree fungus Pholiota squarrosa. The structures of the new compounds were elucidated on the basis of their physical data and chemical degradation. The epicoccamides differ in substitution pattern and in the size of the central carbon chain. The derivative with the longest chain, epicoccamide D, induces morphogenesis and pigment formation in surface cultures of the fungus Phoma destructiva at a concentration of 1.7 mM. Moreover, it exhibits weak to moderate cytotoxicity to HeLa cell lines (CC50 17.0 microM) and antiproliferative effects toward mouse fibroplast (L-929) and human leukemia cell lines (K-562) with growth inhibition (GI50) of 50.5 and 33.3 microM, respectively.

Effects of two pathogens on the performance of Cirsium arvense in a successional fallow

SummaryBiological control of weeds by natural antagonists is of increasing interest. To reduce densities of the weed Cirsium arvense (creeping thistle) in a successional fallow, we applied spores of two pathogens, the biotrophic rust Puccinia punctiformis and the perthotrophic Phoma destructiva, for three consecutive years individually at different dates and combined. The proportion of systemically Puccinia punctiformis‐infected C. arvense was not influenced by the treatments, but local rust infection was a good predictor of systemic infection in the following season. Artificial inoculations with P. destructiva increased the incidence of this pathogen in the third year as a result of synergistic effects, when co‐inoculated with P. punctiformis. Inoculations with either pathogen had little effect on C. arvense shoot density, although there were transient reductions after combined inoculations with both fungi compared with the fungicide treatment. Cirsium arvense decreased from 60% to 5% cover within 3 years, while the cover of the co‐occurring fallow vegetation increased. Under field conditions, with an already high degree of natural P. punctiformis infection, the effects of inoculations of the single pathogens were minor, but reductions in shoot density after combined inoculations indicate that this inoculation may have the potential to cause a decline of this weed.

Biocontrol options for Cirsium arvense with indigenous fungal pathogens

SummaryAs a first step in the development of a mycoherbicide for biological control of Cirsium arvense indigenous fungal pathogens that had been isolated from diseased hosts were tested both singly and in combination on potted plants under environmental conditions. Disease symptoms (necroses, chloroses and macerations) and parameters of growth and development were evaluated to determine the potential for weed control. During 1998, Phoma destructiva, Phoma hedericola, a Mycelia sterilia and Puccinia punctiformis were inoculated singly. With the exception of the obligate biotroph P. punctiformis (local infections), the single inoculations reduced the measured parameters. A combined inoculum of P. punctiformis and Ph. hedericola was less effective than Ph. hedericola alone. In 1999, Ph. hedericola, Ph. destructiva, Ph. nebulosa and the Mycelia sterila were applied both singly and as a combined inoculum. Of all the tested inocula, the mixture of four pathogens had the greatest potential for biocontrol, significantly reducing reproductive capacity of the plants. These results demonstrate the importance of taking advantage of synergisms in developing a mycoherbicide.

A nonribosomal peptide synthetase involved in the biosynthesis of ampullosporins in Sepedonium ampullosporum.

Recently, the saprophytic ascomycete Sepedonium ampullosporum strain HKI-0053 was isolated from a basidiomycete on account of its premature induction of pigment formation in Phoma destructiva, a process often related to the neuroleptic activity of the inducing compound. The active substance was identified as the 15-membered peptaibol type peptide Ampullosporin. Although to date more than 300 peptaibols have been discovered, their biosynthetic machinery has not been characterized yet. By improving the culture conditions it was possible to grow S. ampullosporum in a submerged culture and to increase Ampullosporin production by more than three times to 33 mg/l at reduced fermentation times. The appearance of two high molecular weight proteins, HMWP1 (1.5 MDa) and HMWP2 (350 kDa) was closely related to the production of Ampullosporin during the course of fermentation. Both proteins showed a cross-reaction with antibodies against a core fragment of nonribosomal peptide synthetases (NRPSs). Biochemical characterization of the partially purified enzymes exhibited selectivity for the substrate amino acid alpha-aminoisobutyric acid (Aib). substantiating their involvement in Ampullosporin biosynthesis. Our data suggest that Ampullosporin synthetase has been isolated, and provides the basis for the characterization of the entire biosynthetic gene cluster. Furthermore, this knowledge will enable the manipulation of its NRPS template, in order to engineer mutant strains of Sepedonium ampullosporum which could produce more potent analogues of Ampullosporin.

Circular dichroism studies of ampullosporin-A analogues.

Ampullosporin A (AmpA), a 15mer peptalbol containing seven Aib residues is able to induce pigmentation on Phoma destructiva and hypothermia in mice, as well as to exhibit a neuroleptic effect. A circular dichroism study of ampullosporin A and its analogues was carried out in organic solvents with different polarities and detergent micelles to determine the relationship between their conformational flexibility and biological activities. The analogues were obtained by modifying the N- and C-termini of ampullosporin A. Furthermore, Gln and Leu were systematically substituted by Ala and Aib residues were replaced by Ala and/or Ac6c. To estimate the helicity of the analogues, the CD spectrum of AmpA recorded in acetonitrile was correlated to its crystal structure. All analogues displayed similar CD curve shapes in organic solvents with the ratio between two negative band intensities R = [theta]n-pi*/[theta]pi-pi* < 1. In acetonitrile, most of the analogues adopted a 70%-85% helical structure, which was higher than the average of 40%-60% obtained in TFE. In detergent micelles, the analogues were distinguishable by their CD profiles. For most of the biologically active analogues, the CD spectra in detergent micelles were characterized by a R ratio > 1 and increased helicity compared with those recorded in TFE, suggesting that the interaction of the peptides with the membrane and peptide association was necessary for their hypothermic effect.

Isolation, structure elucidation and biological activities of trichofumins A, B, C and D, new 11 and 13mer peptaibols from Trichoderma sp. HKI 0276W.

Trichofumins A-D were isolated from cultures of Trichoderma sp. HKI 0276 as new 11 and 13mer peptaibols. Similar to 15mer peptaibols they promote morphogenesis of the fungus Phoma destructiva and cause hypothermia in mice as a characteristic of neuroleptic activity. Membrane measurements using a synthetic BLM model showed that A, B, C and D increased membrane permeability for cations in a similar manner as was shown for larger peptaibols but with comparably lower efficiency.

Crystal structure and conformational analysis of ampullosporin A

Ampullosporin A is a 15-mer peptaibol type polypeptide that induces pigment formation by the fungus Phoma destructiva, forms voltage-dependent ion channels in membranes and exhibits hypothermic effects in mice. The structure of ampullosporin A has been determined by x-ray crystallography. This is the first three-dimensional (3D) structure of the peptaibol subfamily SF6. From the N-terminus to residue 13 the molecule adopts an approximate right-handed alpha-helical geometry, whereas a less regular structure pattern with beta-turn characteristics is found in the C-terminus. Even though ampullosporin A does not contain a single proline or hydroxyproline it is significantly bent. It belongs to both the shortest and the most strongly bent peptaibol 3D structures. The straight structure part encompasses residues Ac-Trp(1)-Aib(10) and is thus less extended than the alpha-helical subunit. The 3D structure of ampullosporin A is discussed in relation to other experimentally determined peptaibol structures and in the context of its channel-forming properties. As a part of this comparison a novel bending analysis based on a 3D curvilinear axis describing the global structural characteristics has been proposed and applied to all 3D peptaibol structures. A sampling of 2500 conformations using different molecular dynamics protocols yields, for the complete ampullosporin A structure, an alpha-helix as the preferred conformation in vacuo with almost no bend. This indicates that solvent or crystal effects may be important for the experimentally observed peptide backbone bending characteristics of ampullosporin A.

Differences in membrane pore formation by peptaibols.

The efficiencies of membrane pore formation by 14 naturally occurring peptaibols and two structurally modified ampullosporins were compared using an artificial bilayer membrane model. Major differences were found in the dependence on peptide sequences and the constituting amino acids. Alamethicin F-30, chrysospermins C/D, paracelsin and texenomycin A displayed higher activity by several orders of magnitude in comparison with smaller peptaibols containing < 17 amino acids such as ampullosporins, trichofumins. bergofungins and cephaibols. Biological activities such as the induction of pigment formation by the fungus Phoma destructiva and long acting hypothermia and depression of locomotor activity in mice were correlated with moderate membrane permeabilization. No or weak membrane activities corresponded with biological inactivity. Highly membrane-active structures such as alamethicin F-30, chrysospermin C, texenomycin A and paracelsin A displayed antibiotic effects against the fungus and toxicity in mice.

Synthesis and biological evaluation of analogues of the peptaibol ampullosporin A.

A series of analogues of the fungal peptaibol type metabolite ampullosporin A containing modifications in the C and N terminus as well as alpha-aminoisobutyric acid (Aib) substitutions in different positions of the peptide were synthesized by solid phase synthesis using the 9-fluorenylmethyloxycarbonyl strategy. Depending on the sequence position, couplings were performed with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/1-hydroxybenzotriazole and tetramethylfluoroformamidinium hexafluorophosphate, respectively. The structures of the target peptides were analyzed by electrospray ionization mass spectrometry and chromatographic methods (high-performance liquid chromatography, thin-layer chromatography). The biological activities of these compounds have been evaluated by assaying their potencies for the induction of pigment formation on the fungus Phoma destructiva as well as for the induction of hypothermia and inhibition of locomotoric activity in mice and were compared to the naturally occurring ampullosporins. Native ampullosporin A and analogues with C-terminal Leu or Leu-NH(2) showed comparable activity in the pigmentation assay. Similarly, the ampullosporin A analogues with N-terminal aromatic amino acid residues, such as D-Trp and Tic, also have high potency for pigment formation. The peptides containing structural modifications of ampullosporin A by systematic replacement of Aib by Ala (Ala scan) displayed moderate or high activity in the pigmentation assay, whereas simultaneous substitution of all Aib residues by Ala and Ile, respectively, or by insertion of nonaromatic residues into position 1 resulted in a loss of the effect on P. destructiva. Most of the compounds with no or weak activity in the microbial assay were not active in the hypothermic test, too, except the compound with 1-amino-1-cyclohexane carboxylic acid in position 4 instead of Aib. However, only a few compounds with high potency for pigmentation induction were found to produce strong hypothermia in mice. Thus, in contrast to the native ampullosporins, we succeeded to a certain degree in differentiation of the bioactivities with our synthetic analogues.

Indirect interaction between a fungal plant pathogen and a herbivorous beetle of the weed Cirsium arvense.

Interactions between plants and their natural enemies are well studied, but investigations on the indirect interactions between plant enemies that simultaneously exploit a host plant are rare. Yet these plant-mediated interactions are important because they may affect not only the impact of plant antagonists on plant survival but may also influence the performance of the other plant exploiters. This study focused on the indirect effects of a systemic infection of creeping thistle, [irsium arvense (L.) Scop., with the necrotrophic fungus Phoma destructiva (Plowr.) on the phytophagous leaf beetle Cassida rubiginosa Müller, by examining egg deposition, food plant choice, and larval and pupal performance of the beetle. Thus, the results give a broader view than most other studies of plant-mediated effects of a pathogen on a phytophagous insect. Since both the beetle and the fungus are considered as agents for the biological control of C. arvense, the results are also of interest for applied ecology. Potted plants of C. arvense were inoculated with a conidiospore suspension of P. destructiva to cause a systemic infection of the plants. In a cage experiment, ovipositing females of C. rubiginosa showed a significant preference for healthy thistles. In dual-choice tests, adults of C. rubiginosa preferred leaf discs from healthy thistles over those from Phoma-infected thistles. The beetles also consumed significantly more leaf tissue from healthy than from infected plants. Development time from freshly hatched larvae until pupation was significantly longer for larvae fed on infected leaves. The weight of last-instar larvae and pupae was lower, and larval and pupal mortality was higher when larvae had been fed with infected compared to healthy leaves. Thus, the combined use of both potential biological control agents may be of lowered efficiency because (1) C. rubiginosa avoided infected thistles for both egg deposition and adult feeding and (2) Phoma infection negatively affected larval development and increased larval and pupal mortality of the beetle.

Ampullosporin, a New Peptaibol-type Antibiotic from Sepedonium ampullosporum HKI-0053 with Neuroleptic Activity in Mice.

Ampullosporin (I; Ac-Trp-Ala-Aib-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Gln-Leu-Aib-Gln-Leuol) was isolated from the mycelium of Sepedonium ampullosporum as a new 15-membered peptaibol-type antibiotic. The structure was determined by mass spectrometric and two-dimensional NMR experiments. Ampullosporin displays narrow-spectrum antibacterial and antifungal activity, induces pigment formation by Phoma destructiva, causes hypothermia and decreased spontaneous locomotor activity in mice in dosages > 1 mg/kg.