Verticillium Chlamydosporium Research Articles

Potent Bioactivity of Endophytic Fungi Isolated from Moringa oleifera Leaves.

Plant species are known to harbor large number of endophytes, which stays in plant tissues as symbionts. These endophytes secrete large array of bioactive compounds that have potency against certain diseases with no side effects. We have collected leaf samples of the Moringa oleifera plant from the Pakistan Forest Institute, Khyber Pakhtunkhwa, Pakistan for the isolation of beneficial endophytes. The strains isolated from the leaves of M. oleifera were coded with MOL and tested for antimicrobial, antifungal, germicidal, phytotoxic, insecticidal, cytotoxic, and anti-inflammatory activities. The isolates, MOL1, MOL16, MOL19, and MOL21, possessed antibacterial activity against Staphylococcus aureus, whereas MOL7 inhibited 55% of the growth of Escherichia coli. MOL3 inhibited the growth of E. coli, S. aureus, and Pseudomonas aeruginosa. The strains, MOL1 and MOL7, showed antifungal activity against Candida albicans and Saccharomyces cerevisiae, while the strains, MOL11 and MOL17, showed activity against Verticillium chlamydosporium. The isolates, MOL3, MOL7, MOL9, MOL15, MOL17, MOL18, and MOL19, inhibited the growth of Lemna minor (duckweed) at 100 μg/ml. MOL2 exhibited strong activity in the brine shrimp assay, while MOL1, MOL2, MOL5, MOL6, MOL12, MOL17, MOL19, and MOL20 showed insecticidal, and MOL3 demonstrated larvicidal and antileishmanial activity. The isolated potent endophytes were identified as Aspergillus, Penicillium, Fusarium, Tricoderma, Rhizoctonia, Mucor, Alternaria, Pestalotiopsis, Acremonium, and Cladosporium through morphological and microscopic characteristics of the colonies.

EVALUATION OF EFFICACY OF PASTEURIA PENETRANS ALONE AND IN COMBINATION WITH VERTICILLIUM CHLAMYDOSPORIUM AGAINST MELOIDOGYNE JAVANICA

The potential control of Meloidogyne javanica using Pasteuria penetrans (Pp) alone and in combination with Verticillium chlamydosporium (Vc) was tested in earthen pots following a cropping sequence "tomato-tomato-tomato” over three crop cycles. After the final harvesting, analysis of variance showed significant effect of treatments (P0.01) regarding number of eggmasses, galls and nematode female populations. Similarly, significant effect of treatments (P0.01) was also recorded in case of infected nematode females with Pasteuria and number of eggs/eggmass while no significant effect (P 0.0 5) was observed in case of endospore production. Higher numbers of eggmasses (360) and root galling (6.2) was observed where biocontrol agents were absent (control). The treatments showed 46.58, 58.85 and 33.13 percent reduction in number of galls, eggmasses and nematodes in Pp alone and 43.34, 55.21 and 30.09 percent reductions in Vc+Pp treatments respectively. Numbers of females infected with the endospores of P. penetrans were recorded higher in Pp-3 alone treatment (13.2) followed by Vc+Pp combined treatment (13.0) and maize rotated treatment (10.4) respectively. Significantly lesser number of eggmasses, galls and nematodes were recorded in pots where tomato was rotated with maize (treatment 3) compared with control. Thus rotation prevented the buildup of nematode population and resulted in a 72% decrease in numbers of eggmasses, 38% in root galling and 46% regarding female populations over the control after the final harvest. Maximum colony forming units of V. chlamydosporium per gram of soil were recorded after its addition to the soil. The fungus established in the soil during the first crop and soil colonization of the fungus was also observed after final crop.

Integrated management of plant-parasitic nematodes on guava and fig trees under tropical field conditions

Two field experiments were carried out to study the efficacy of different biological control agents in controlling certain plant-parasitic nematode species including Meloidogyne javanica, Tylenchorhynchus mediterraneus, Hoplolaimus seinhorsti, Longidorus latocephalus, and Xiphinema elongatum on guava and fig trees under the tropical field conditions of Jazan region, south-west Saudi Arabia during two successive seasons from Feb. 15, 2016 to Jan. 15, 2017. The evaluated bio-agents were used in different integrated management combinations of certain fungal species (Trichoderma harzianum, Verticillium chlamydosporium, and Purpureocillium lilacinum), the bacterium Pasteuria penetrans, some organic amendments (cow manure, compost, and chicken manure), urea 46% as a nitrogenous fertilizer, and the nematicide carbofuran 10G for comparison. Results showed that all the tested treatments gradually decreased (P ≤ 0.05) the population densities of plant-parasitic nematodes on guava and fig trees over the study period. The highest reduction of nematode densities occurred at the end of the experiment. Carbofuran 10G was the most effective treatment in suppressing the nematode densities on guava and fig trees. The most effective management combinations, next to carbofuran 10G, in suppressing the nematode densities in the rhizosphere of guava trees were P. lilacinum + P. penetrans + urea 46%, P. lilacinum + P. penetrans + chicken manure, and T. harzianum + P. penetrans + chicken manure (66.54–69.22% nematode reductions). Correspondent combinations in the rhizosphere of fig trees were P. lilacinum + P. penetrans + cow manure, T. harzianum + P. penetrans + cow manure, P. lilacinum + P. penetrans + urea 46%, and V. chlamydosporium + P. penetrans + urea 46% (54.68–57.17% nematode reductions). On the other hand, nematode population densities continued to increase (P ≤ 0.05) in the rhizosphere of guava and fig trees in the absence of nematode management combinations. All the tested treatments significantly increased (P ≤ 0.05) the number of fruits/tree on guava and fig trees. Treatments which included the combinations of fungal and bacterial parasites along with chicken manure gave the highest numbers of fruits/tree, followed by the treatment with the nematicide carbofuran 10G. Regression analysis showed a significant negative linear relationship between the number of nematodes/kg soil and the number of guava and fig fruits/tree.

Paecilomyces lilacinus and Verticillium chlamydosporium Fungi as Biological Control of Fasciolosis

Fasciolosis is a worm disease caused of Fa sciola gigantica and an important problem in husbandry especially for cattle. Controlling of this worm disease can be conducted by prevention and treatment. The use of antihelminthic is commonly causes a resistance problem. Natural control by mold such as Paecilomyces lilacinus and Verticillium chlamydosporium can be applied to reduce egg of F . gigantica . Although it was recently found, in vitro study gave satisfied result. This gives a new hope in controlling the disease although the extend application still needs to be studied. This paper discussed about the use of P. lilacinus and V. chlamydosporium for reducing F. gigantica population. K e y words: Natural control, F . gigantica, P. lilacinus, V. chlamydosporium

Controlling the root-knot nematode, Meloidogyne incognita in cucumber plants using some soil bioagents and some amendments under simulated field conditions

This study was carried out under simulated field conditions to evaluate the efficacy of some bioagents and soil amendments, as a single or combined treatments, in controlling root-knot nematode Meloidogyne incognita infecting cucumber. Each of the fungus Verticillium chlamydosporium and the symbiotic bacterium Photorhabdus luminescens, as single or joint treatments significantly reduced gall formation and other criteria on cucumber roots. Maximum reduction in gall formation, female numbers, egg-mass production, developmental stages and final population of juveniles in soil, was acquired by these treatments, V. chlamydosporium+P. luminescens, P. luminescens+compost (C) and V. chlamydosporium+P. luminescens+animal compost (AC), compared with the control and other treatments. Applications of all treatments significantly promoted plant growth i.e. length of shoot and root, fresh and dry weight of shoot and root, number of leaves, flowers, fruits and weight of fruits per each plant compared to control (infested plants with nematode only and healthy plants).

Morphological changes, chitinolytic enzymes and hydrophobin-like proteins as responses of Lecanicillium lecanii during growth with hydrocarbon

Lecanicillium lecanii, Verticillium chlamydosporium, V. fungicola var flavidum and Beauveria bassiana were evaluated on their growth with pure n-hexane, toluene and n-hexane:toluene 17:83 (v:v) mixture. Another set of treatments were conducted with colloidal chitin as additional carbon source. All the strains of Lecanicillium were able to grow using hydrocarbons with or without the addition of chitin, although the presence of hydrocarbons showed significant inhibition evidenced by measured biomass, radial growth and microscopic analyses. Degradation of n-hexane ranged within 43 and 62% and it was higher than that with toluene. The strains L460, L157 and L2149, which presented the highest growth, were further selected for determinations of hydrocarbon consumptions in microcosms. Strain L157 showed the highest consumption of n-hexane (55.6%) and toluene (52.9%) as sole carbon source and it also displayed activities of endochitinases, N-acetylhexosaminidase and production of hydrophobins class I and II.

Fungal spread and faecal decomposition as indicators to evaluate short term environmental impact of egg parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium

Experiments were conducted to understand the extent of spread of Paecilomyces lilacinus and Verticillium chlamydosporium once deposited in faeces and alteration, if any, of the organic content of faeces by them, which could serve as evidences of their short-term environmental impact. Coproculture of the central part and two concentric rings of both the fungus contaminated plots were undertaken twice a week for four weeks in laboratory for the presence of P. lilacinus and V. chlamydosporium. Subcultures were done until the confirmation of desired fungus occur. Paecilomyces lilacinus could not be reisolated from the central part and two concentric rings at any point of sampling period except from the faecal sample of the central part on day 0. Verticillium chlamydosporium could be re-isolated st from the faeces deposited at the central part on day 0, 7 and 14 but never from 21 day of sampling onward. However, V. chlamydosporium could not be recovered from the faeces of two concentric rings at any point of sampling. Moisture contents decreased with corresponding increase in dry matter content of the faeces as the time progressed. Faeces mixed with P. lilacinus, V. chlamydosporium and no fungus controls behaved similarly in terms of moisture and dry th matter contents. From 60 day of deposition onwards, ash contents of faeces mixed with P. lilacinus, V. chlamydosporium and no fungus controls showed increasing trend with the corresponding decreasing trends in organic matter contents.

Insights into Radicicol Biosynthesis via Heterologous Synthesis of Intermediates and Analogs

Resorcylic acid lactones are fungal polyketides that display diverse biological activities, with the potent Hsp90 inhibitor radicicol being an important representative member. Two fungal iterative polyketide synthases (IPKSs), Rdc5, the highly reducing IPKS, and Rdc1, the nonreducing IPKS, are required for the biosynthesis of radicicol in Pochonia chlamydosporia. In this study, the complete reconstitution of Rdc5 and Rdc1 activities both in vitro and in Saccharomyces cerevisiae uncovered the earliest resorcylic acid lactone intermediate of the radicicol biosynthetic pathway, (R)-monocillin II. The enzymatic synthesis of (R)-monocillin II confirmed the exquisite timing of the Rdc5 enoyl reductase domain. Using precursor-directed biosynthesis, the chemical modularity of the dual IPKS system was determined. Rdc1 readily accepted an N-acetylcysteamine thioester mimic of the reduced pentaketide product of Rdc5 to synthesize (R)-monocillin II with four additional iterations of polyketide elongation, indicating the C2' ketone group found in (R)-monocillin II is incorporated via the functions of Rdc1 instead of Rdc5. The involvement of the thioesterase domain in Rdc1 in macrolactonization was confirmed through both site-directed mutagenesis and domain deletion. The Rdc1 thioesterase domain was also shown to be tolerant of the opposite stereochemistry of the terminal hydroxyl nucleophile, demonstrated in the precursor-directed synthesis of the enantiomeric (S)-monocillin II. Finally, reconstitution of the halogenase Rdc2 was demonstrated both in vivo and in vitro in the synthesis of pochonin D and a new halogenated analog 6-chloro, 7',8'-dehydrozearalenol.

Uso de agentes microbianos e químico para o controle de Meloidogyne incognita em soja

Root-knot nematodes are considered significant pathogens of soybean crops. The objective of this work was to evaluate the efficacy of two fungi (Paecilomyces lilacinus (Thom.) Samsom and Pochonia chlamydosporia (Goddard) Zare & Gams (syn. Verticillium chlamydosporium), a commercial product based on Bacillus sp. and Aldicarb on the control of Meloidogyne incognita on soybean, cultivar M-SOY 6101. The experimental design was set as randomized blocks with four replications. Nine treatments were evaluated: three biological agents used for seed treatment with and without post-emergence application, Aldicarb on post-emergence only, and two controls. All treatments were inoculated with the nematode, except for a blank control where only water was applied. One control-only treated with the nematode was also included. Aldicarb could reduce the number of eggs and juveniles in the roots. P. lilacinus showed the best performance among the biological control agents, reducing the number of eggs and increasing dry root weight. Nemix, a Bacillus sp. based commercial product and P. chlamydosporia could only reduce significantly the number of eggs of the nematode. In this work, it was possible to conclude that the chemical and biological agents showed a moderate activity in the control of M. incognita in soybean.

Fungus–benzimidazole interactions: a prerequisite to deploying egg-parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium as biocontrol agents against fascioliasis and amphistomiasis in ruminant livestock

In vitro trials investigating the effects of albendazole and triclabendazole anthelmintics on the growth profiles of the egg-parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium were undertaken. In addition, in vivo trials were conducted in goats fed on millet grain cultures of each fungus and administered albendazole and triclabendazole anthelmintics. In vitro growth revealed V. chlamydosporium to be more sensitive to albendazole compared to P. lilacinus. In contrast, triclabendazole had the least inhibitory effect on in vitro growth of both P. lilacinus and V. chlamydosporium. Similar to albendazole, growth of P. lilacinus was more vigorous at 0.5 ppm concentration of triclabendazole. Efforts to re-isolate these egg-parasitic fungi from faeces of goats fed on fungal millet grain cultures before and following single intraruminal administration of albendazole and triclabendazole showed that P. lilacinus was not able to be re-isolated from the faeces at any sampling period. In contrast, V. chlamydosporium was able to be re-isolated from the faeces at all of the sampling periods except for the samples taken at 8-18 h and 18-24 h after administration of albendazole and triclabendazole, respectively. Lack of fungal activity at these times coincided with peak plasma availability of anthelmintics and suggests faecal levels of drugs were also high at these times and impacted negatively on fungal viability.

Evaluation of <i>Verticillium chlamydosporium</i> and <i>Arthrobotrys oligospora</i> for biological control of <i>Meloidogyne incognita</i> in celery and tomato

The ability of nematode trapping fungi and egg-parasitising fungi to colonize and persist in the rhizosphere of crop plants is thought to be an important factor influencing the success of biological control of root infecting nematodes. In this study, two strains of an egg parasitic fungus Verticillium chlamydosporium (Vc- 10 and Vc-2M) and one isolate of the nematode-trapping fungus Arthrobotrys oligospora were evaluated to determine their pathogenicity to Meloidogyne incognita and persistence in the rhizosphere of celery (Apium graveolus L.) and tomato(Lycopersicon esculentum L.) plants in a greenhouse experiment. The isolates tested differed significantly (P≤0.05) in their pathogenicity to M. incognita and survival in the rhizosphere. Isolate Vc-10 of V. chlamydosporium was the most virulent pathogen of the nematode. Root galling was lowest (P≤0.05) in tomato plants treated with the isolate Vc-10 (2.2); pots treated with this isolate had the lowest final soil population of infective juveniles; there was a 62.2% and 98.5% infections of eggs and egg-masses respectively by Vc-10 on tomato plants. Isolates Vc-10 and Vc-2M of V. chlamydosporium persisted in the soil and could be re-isolated from the rhizosphere and roots of tomato plants at least 16 weeks after soil infestation. The final inoculum density was, however, higher (P≤0.05) for Vc-10 (1.35×105 cfu/g soil) than Vc-2M (9.25×104 cfu/g soil). A. oligospora on the other hand did not give any significant (P≤0.05) control of the nematode on both crops, there was severe galling on the roots of celery plants (7.4) and tomato plants (6.3) treated with this biological control agent. A. oligospora could not be re-isolated from the plant rhizosphere sixteen weeks after soil infestation. Lack of nematode control on both crops by A. oligospora was attributed to its poor or no establishment in the plant rhizosphere. The fact that it could not be reisolated from the rhizosphere may imply that the fungus did not survive in the rhizosphere of the test plants. Keywords: Verticillium, Arthrobotrys, Meloidogyne, rhizosphere, persistence, root-knots, chlamydospores. J. Trop. Microbiol. Biotechnol. Vol. 3 (2) 2007: pp. 51-58

Observação in vitro da ação dos isolados fúngicos Duddingtonia flagrans, Monacrosporium thaumasium e Verticillium chlamydosporium sobre ovos de Ascaris lumbricoides (Lineu, 1758)

The in vitro action of the nematophagous fungi Duddingtonia flagrans, Monacrosporium thaumasium and Verticillium chlamydosporium on eggs of Ascaris lumbricoides was observed. After 7, 10 and 14 days of interaction, the fungus showing most promise for use in biologically control over Ascaris lumbricoides was Verticillium chlamydosporium (26-30%). The other fungi did not present satisfactory results.

Purification and Characterization of Chitinases from the Nematophagous Fungi Verticillium chlamydosporium and V. suchlasporium

Culture filtrates of the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium growing on colloidal chitin showed increasing chitinolytic activity and production of two (32- and 43-kDa) main proteins. Maximum activity was found 18–20 days after inoculation, but V. suchlasporium always displayed higher activity. Zymography of such filtrates on carboxymethyl–chitin–Remazol brilliant violet 5R/acrylamide gels showed five bands of substrate degradation for V. suchlasporium and three for V. chlamydosporium. Filtrates with maximum activity were chromatographed on macroporous cross-linked chitin affinity matrix, showing a peak of main (50–60%) activity, which only contained a 43-kDa protein for both fungi. Zymography and colloidal chitin degradation showed that it was a single endochitinase (CHI43) with optimum pH range of 5.2–5.7. The main isoforms had pIs of 7.6 for V. suchlasporium and 7.9 for V. chlamydosporium. Eggs of the nematode Globodera pallida treated with CHI43 and the serine protease P32 from V. suchlasporium alone or in combination showed surface damage in comparison with controls when examined by scanning electron microscopy.

Effect of temperature on in vitro interactions between Verticillium chlamydosporium and other Meloidogyne-associated micro-organisms

In vitro interaction between Verticillium chlamydosporium (Goddard) andbacteria or fungi was studied at 20 °Cand 15 °C. The frequency of antagonismwas lower at 15 °C for the bacteria, butnot for the fungi. Interaction was affected bytemperature for 33% of the bacteria and 47%of the fungi.

Use of light and scanning electron microscopy to examine colonisation of barley rhizosphere by the nematophagous fungus Verticillium chlamydosporium

Barley roots were readily colonised by the nematophagous fungus Verticillium chlamydosporium. Light microscopy (LM) but also low temperature scanning electron microscopy (LTSEM) revealed details of the colonisation process. Hyphae were found on the rhizoplane often with dictyochlamydospores. Hyphae of V. chlamydosporium penetrated epidermal cells, often by means of appressoria. A hyphal network was formed in epidermal and cortical cells. Likewise, hyphal coils were found within root cells next to transverse cell walls. Cortical cells were the limits of fungal colonisation, since no hyphae were seen in the vascular cylinder. Modifications of root cell contents (phenolic droplets and callose appositions) were common three weeks after inoculation with V. chlamydosporium. These features may indicate induction of plant defence reactions in late stages of root colonisation by the fungus. Both LTSEM and LM have proved extremely useful to describe root colonisation by the fungus. The results found may have implications in the mode action of nematophagous fungi against plant parasitic nematodes.

Descriptive Reports on Some Soil-Inhabiting Fungi in Korea

During the study of microbial structures in root-regions of tomato and red pepper from fields, various soil-inhabiting fungi were isolated with the dilution plate technique. Among them an ascomycete, Emericellopsis mirabilis and three hyphomycetes, Gliocladium solatii, Humicola veronae and Verticillium chlamydosporium are presented for the first time in Korea along with Talaromyces trachyspermus, Chaetomium globosum and Doratomyces microsporus.

Microflora of Meloidogyne egg masses: species composition, population density and effect on the biocontrol agent Verticillium chlamydosporium (Goddard)

AbstractEgg masses of Meloidogyne fallax from tomato and potato growing in soil from a nematode suppressive and a nonsuppressive field sustained bacterial population densities two to three orders of magnitude higher than those of the rhizosphere soil. BIOLOG metabolic profiling identified 16 bacterial species from egg masses. Results further indicated 20 species not listed in the BIOLOG database. 122 isolates of bacteria and 19 isolates of fungi from M. fallax or M. hapla were tested for in vitro antagonism against the nematode egg parasitic fungus Verticillium chlamydosporium: 23% of the bacteria and 74% of the fungi showed antagonistic activity. Pseudomonads showed an overall stronger antagonistic activity than the other bacteria. Our conclusions are that Meloidogyne egg masses are a densely populated microbial niche and that their microflora may well be an important factor in determining the success of nematode antagonists. However, we could not find a relationship between the egg mass microflora and differences in soil suppressiveness between the sample sites.

Methods for studying the nematophagous fungus Verticillium chlamydosporium in the root environment

In order to exploit fully the biocontrol potential of the nematophagous fungus Verticillium chlamydosporium, it is important to understand the ecology of the fungus in soil, and interactions with both plant and nematode hosts. Several approaches for studying the fungus in soil and the root environment are compared. These include a semi-selective medium for V. chlamydosporium, PCR primers specific for the fungal β-tubulin gene, and monoclonal antibodies. In addition to providing a target for species-specific primers, the β-tubulin gene is implicated in resistance to the fungicides used in the semi-selective medium, and the genetic basis for this is investigated. Culture and PCR-based methods were used to screen for the presence of the fungus in field soils known to have been suppressive to cereal cyst nematode and that contained V. chlamydosporium populations. Monoclonal antibodies specific for either hyphae or conidia of the fungus were obtained, and their application as a tool for visualising the infection process on the root was explored.

Detection of the nematophagous fungus Verticillium chlamydosporium in nematode-infested plant roots using PCR

PCR-based methods to detect Verticillium chlamydosporium on infected plant roots were developed. Arbitrary ERIC primers and those based on rRNA genes, to identify fungi grown in pure culture, were unsuitable for DNA extracted from nematode-infested roots, because of interference by plant and nematode DNA. A novel method utilizing specific primers designed from an amplified and cloned fragment of the V. chlamydosporium β-tubulin gene was developed. Although it could not discriminate between different isolates of V. chlamydosporium, one primer set could identify the fungus on tomato roots infested with root-knot nematodes. The V. chlamydosporium β-tubulin sequence data showed close homology to sequences from plant endophytic Acremonium and Epichloe species and the saprotrophic Trichoderma viride.

A nematicidal metabolite fromVerticillium chlamydosporium

A bioassay-directed fractionation of cultures of the fungus, Verticillium chlamydosporium, demonstrated nematicidal activity, due mainly to phomalactone. Compared with aldicarb, the lethal activity against Meloidogyne incognita was less, but its activity in suppressing root invasion was similar. The extract also contained monorden. (C) 2000 Society of Chemical Industry.