Entomopathogenic Fungus Verticillium Lecanii Research Articles

Lethal and sub-lethal toxicity of Verticillium lecanii on the biology of Mealybug (Pseudococcidae; Homoptera) under laboratory conditions

Abstract: The Mealybug, Planococcus citri (Risso) (Hemiptera: Pseudococcidae) is a polyphagous pest of Citrus, grapevine, coffee and ornamental plants. The entomopathogenic fungus Beauveria bassiana, Metarhizium anisopliae, Verticillium lecanii and Isaria fumosorosea are very effective and commonly used as a biopesticide against various insect pests of different crops. The present study was conducted to check the lethal and sub-lethal effects of V. lecanii on mortality and life period of adult mealybug. Firstly LC50 value for nymph and adult was calculated by applying four different concentrations 1×105 , 1×106 , 1×107 and 1×108 CFU/ml. For nymphs, the median lethal concentration was 5.57×106 CFU/ ml, and for adults, 2.66×107 CFU/ml. After this, two lethal, 3.99×107 and 2.66×107 CFU/ml, and two sub-lethal concentrations, 1.33×107 and 6.65×106 CFU/ml, was prepared and tested against adult mealybug. Dose-dependent mortality was observed, and the highest mortality was due to the highest concentration of V. lecanii. The percentage mortality was 72.23, 52.78, 27.78 and 19.45% due to 3.99×107 , 2.66×107 , 1.33×107 and 6.65×106 CFU/ml, respectively, after 10 days of application. The weight of adult females was highest in the control treatment while lowest in high concentration, and it was gradually decreased after every day in the first three concentrations. The fecundity of mealybug also had an inverse relation with concentrations of V. lecanii, and the numbers of eggs were more in the control treatment than the other four treatments. The fecundity in lethal concentrations was reduced after the 8 th day of application. The life duration was most extended due to control treatment, 27 days, while the lowest period was due to the highest concentration, 13 days. Keywords: Verticillium lecanii, mealybug, mortality, weight, fecundity, life duration

Isolation, partial characterization, and cloning of an extracellular chitinase from the entomopathogenic fungus Verticillium lecanii.

The entomopathogenic fungus Verticillium lecanii is a well-known biocontrol agent of fungal phytopathogens, as well as insect pests. A 42-kDa chitinase belonging to family 18 of the glycosyl hydrolases was isolated and partially characterized. Chitinase was purified using successive column chromatography on phenyl-sepharose, DEAE-sepharose, and CM-sepharose. The enzyme showed the highest activity at 40°C and pH 4.6. Enzyme activity was strongly activated in the presence of Mg(2+). The purified enzyme showed inhibitory activity of spore germination against several plant pathogens, particularly Fusarium moniliforme. The genomic DNA and cDNA sequences were resolved by polymerase chain reaction amplification and sequencing. Protein modeling and comparative investigation of different chitinase amino acids showed that chitinases are conserved in parasitic fungi.

Production of extracellular enzymes in the entomopathogenic fungus Verticillium lecanii

This study investigates the mechanisms as well as strategies for purification and characterization of potential enzymes involved in pathogenesis of entomopathogenic fungi. The test strain of Verticillium lecanii that was screened, during the present investigation, proved to be an efficient producer of protein and polysaccharide degrading enzymes (amylase, protease, and lipase), hence indicating versatility in biochemical mechanisms. Halo zones produced colony growth of V. lecanii on agar confirmed activity of protease, amylase and lipase enzyme by the V. lecanii isolate. Enzymatic Index (EI) observed were: Protease – 2.195, Amylase- 2.196, Lipase- 2.147. Spectrophotometric analysis of enzymatic activity of V.lecanii at five different pH – 3, 5, 7, 9, 11 revealed that highest proteolytic activity of the V. lecanii isolate was reported at pH 7 and 9 whereas proteolytic activity was minimum at acidic pH 3. Maximum amylolytic activity of V. lecanii on the 7th day of inoculation was at pH 3 i.e. in an acidic environment in contrast to neutral pH 7. Maximum lipolytic activity of V. lecanii was found at pH 7. Since enzyme production in entomopathogenic fungi is specific and forms an important criterion for successful development as well as improvement of mycoinsecticides, hence a significant conclusion from the present analysis is the degree of variation in secretion of enzymes in test strain of Verticillium lecanii.

Characterization, cloning, and heterologous expression of a subtilisin-like serine protease gene VlPr1 from Verticillium lecanii

The entomopathogenic fungus Verticillium lecanii is a well-known biocontrol agent. V. lecanii produces subtilisin-like serine protease (Pr1), which is important in the biological control activity of some insect pests by degrading insect cuticles. In this study, a subtilisin-like serine protease gene VlPr1 was cloned from the fungus and the VlPr1 protein was expressed in Escherichia coli. The VlPr1 gene contains an open reading frame (ORF) interrupted by three short introns, and encodes a protein of 379 amino acids. Protein sequence analysis revealed high homology with subtilisin serine proteases. The molecular mass of the protease was 38 kDa, and the serine protease exhibited its maximal activity at 40°C and pH 9.0. Protease activity was also affected by Mg(2+) and Ca(2+) concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.

Effects of Verticillium lecanii (Zimm.) Viegas on Toxoptera citricida Kirkaldy (Homoptera: Aphididae) and its parasitoid Lysiphlebus testaceipes Cresson (Hymenoptera: Braconidae)

The preponderance of susceptible sour orange (Citrus aurantium L.) rootstock has facilitated the spread of Citrus tristeza virus (CTV) in Trinidad and Tobago. CTV is transmitted by the brown citrus aphid Toxoptera citricida (Kirkaldy), which establishes large colonies on new flushes of citrus plants. As the colonies become highly populated, winged (alate) aphids are produced which can migrate to uninfected neighbouring citrus trees and hence transmit CTV. In the present study different concentrations of the entomopathogenic fungus Verticillium lecanii (Zimm) Viegas in water-based formulations were applied to T. citricida and the pathogenic effects were analysed. Bioassays were also conducted on the major parasitoid Lysiphlebus testaceipes Cresson to test the pathogenic effects of V. lecanii on mortality and the percent emergence from mummies. The LC<sub>50</sub> values for T. citricida and L. testaceipes were 2.26 × 10<sup>10</sup> spores/ml and 1.09 × 10<sup>9</sup> spores/ml, respectively. Statistical analyses indicated that there was no significant difference between the two LC<sub>50</sub> values. At the highest concentration (1.49 × 10<sup>9</sup> spores/ml) percent mortality peaked at 78.9% after 12 days and 1.95 × 10<sup>9</sup> spores/ml, mortality of L. testaceipes reached 95.1% after 6 days. Emergence of L. testaceipes at the highest concentration of 1.95 × 10<sup>9</sup> spores/ml was 57.8%.

Efficiency of the entomopathogenic fungus Verticillium lecanii in the biological control of Trialeurodes vaporariorum, (Homoptera: Aleyrodidae), a greenhouse culture pest

Our investigation in the region of Jijel revealed that whiteflies are the predominant greenhouses pests; they are polyphagous, moreover, some species can transmit many plant viruses. The treatment method is based on the systematic use of insecticides that have side effects on both the consumer and the farmer. The objective of this study was to evaluate the use of biological control in situ and in vitro as an alternative method by using an entomopathogenic fungus Verticillium lecanii. In vitro experiments showed that the fungus was active during all stages of development of the insect, Trialeurodes vaporariorum Westwood (Homoptera: Aleyrodidae): Eggs (LD50 = 0.59. 107 spores / ml) larvae (LD50 = 0.5. 103 spores / ml) and adults. Our results showed the influence of spore concentration, contact time and relative humidity on the development of the parasite to reach an efficient anti-larval effect of 100%. Key words: Verticillium lecanii, Trialeurodes vaporariorum, entomopathogenic, alleyrodidae, whitefly, biological control.

Evaluation of entomopathogenic fungus Verticillium lecanii (Zimmermann) Vieges and the predator Chrysoperla carnea (Stephens) against cowpea aphid, Aphis craccivora (Koch) on faba bean in EGYPT

the entomopathogenic fungus Verticillium lecanii (Zimmermann)Vieges, the predator Chrysoperla carnea(Stephens) and V. lecanii followed by release of C. carnea in faba bean field for controlling cowpea aphid, Aphiscraccivora (Koch) compared with control were conducted. The first treatmentwasV. lecanii repeated 4 times at15-days intervals at the rate of 108 spores/ml. The second was C.carnea at the same intervals at the rate of 12,000 predator [larvae(L2)]/feddan/releases.

Effect of Verticillium lecanii on biological characteristics and life table of Serangium japonicum (Coleoptera: Coccinellidae), a predator of whiteflies under laboratory conditions

AbstractEffects of entomopathogenic fungus Verticillium lecanii on biological characteristics and life table of Serangium japonicum, a predator of whiteflies against five different conidial concentrations (1×104, 1×105, 1×106, 1×107, and 1×108 conidia/mL) were studied under laboratory conditions. The developmental periods for all immature stages (from eggs, 1st, 2nd, 3rd, 4th instar nymph and pupae up to emergence) among the treatments were significantly different when compared to that of control, and the longest development period was observed as treated with 1×108 spore/mL. However, no significant difference on the percent survival of all immature stages was observed among the treatments and control. Also, there were no significantly different effects of V. lecanii on mean generation time, intrinsic rate, the finite rate of increase and longevity of S. japonicum among the treatments and control.

Isolation and characterization of a chitinase gene from entomopathogenic fungus Verticillium lecanii

Isolation and characterization of a chitinase gene from entomopathogenic fungus Verticillium lecanii

RAPD-PCR, UP-PCR and rDNA sequence analyses of the entomopathogenic fungus Verticillium lecanii and its pathogenicity towards insects and phytopathogenic fungi

RAPD-PCR and UP-PCR analysis allowed us to differentiate Verticillium lecanii strains isolated from insect (Homoptera) and from rust fungi. UPGMA, NJ and NJ consensus trees revealed groups of isolates according to their geographical origin. Successful amplification of the ITS region occurred in a few V. lecanii strains. These ITS sequences were almost identical to known ITS sequences. Thirteen strains had similar IGS sequence of about 2.2 Kb, while strain V5 had a different and shorter (1.1 Kb) IGS-sequence. Among the isolates the correlation between conidia size and virulence towards whitefly was low. Verticillium lecanii strains were able to prevent the pustule development of Puccinia recondita. V61 showed 98% effectiveness. Strains active against wheat rust causal agent were highly virulent against whitefly and show some antibacterial activity. These strains are promising for the biocontrol of plant diseases.

Isolation and characterization of a chitinase gene from entomopathogenic fungus Verticillium lecanii.

Entomopathogenic fungus Verticillium lecanii is a promising whitefly and aphid control agent. Chitinases secreted by this insect pathogen have considerable importance in the biological control of some insect pests. An endochitinase gene Vlchit1 from the fungus was cloned and overexpressed in Escherichia coli. The Vlchit1 gene not only contains an open reading frame (ORF) which encodes a protein of 423 amino acids (aa), but also is interrupted by three short introns. Vlchit1 protein showed that the chitinase Vlchit1 has a (a/b)8 TIM barrel structure. Overexpression test and Enzymatic activity assay indicated that the Vlchit1 is a functional enzyme that can hydrolyze the chitin substrate, so the Vlchit1 gene can service as a useful gene source for genetic manipulation leading to strain improvement of entomopathogenic fungi or constructing new transgenic plants with resistance to various fungal and insects pests.

Patogenicidade de Verticillium lecanii ao pulgão-do-pinus

Avaliou-se a patogenicidade do isolado IBCB 473 de Verticillium lecanii no pulgão-do-pinus Cinara atlantica (Hemiptera, Aphididae), inseto-praga em plantios de Pinus spp. O delineamento experimental utilizado foi o inteiramente casualizado, com cinco repetições por tratamento e cada repetição constituída de uma placa de Petri (9 cm de diâmetro) contendo 10 ninfas. Foram testadas suspensões de inóculo nas concentrações de 1,0 ' 10(6); 0,5 ' 10(7); 1,0 ' 10(7); 0,5 ' 10(8); e 1,0 ' 10(8) conídios/mL, pulverizando-se 1 mL sobre as ninfas. Como tratamento-testemunha, utilizou-se água esterilizada. As placas de Petri foram mantidas em câmara climatizada a 25 ± 1 °C, 70 ± 10% de UR e fotofase de 12 horas. As avaliações foram realizadas diariamente, anotando-se a mortalidade dos indivíduos de cada tratamento. A dose mais eficiente foi de 1,0 x 10(8) conídios/mL, causando mortalidade de 86,0% após cinco dias da pulverização. Os valores de TL50 das concentrações utilizadas foram de 5,82; 5,24; 4,60; 4,34; e 3,83 dias, respectivamente.

The entomopathogenic fungus Verticillium lecanii (Deuteromycetes, Moniliaceae). The proteins hydrophobins and the biological control of aphids (Homoptera, Aphididae): Literature review)

International literature review on the biological control of aphids using the entomopathogenic fungus Verticillium lecanii is given. Attention is directed on the fungal proteins hydrophobins for their role in Biotechnology as well as Biocontrol of Pests of Agricultural and horticultural importance.

Genetic, morphological, and virulence characterization of the entomopathogenic fungus Verticillium lecanii.

In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, 46 isolates of Verticillium lecanii from various hosts and geographical locations were examined. The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA (rDNA), mitochondrial small subunit rDNA (mt-SrDNA) and beta-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was performed on regions of the mitochondrial large subunit rDNA, mt-SrDNA, beta-tubulin and histone 4. There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, amplified product size of IGS did have relationships with conidia size and sporulation. Six isolates with 4.0-kb IGS products had large conidia dimensions, and yielded low numbers of conidia compared with other isolates. Three out of the six isolates were high virulence (over 90%) against green peach aphids. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and related with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties and conidial morphology.

The effect of heavy metal ions and their complexons upon the growth, sporulation and pathogenicity of the entomopathogenic fungus Verticillium lecanii.

The effect of heavy metal ions and their complexons upon the growth, sporulation and pathogenicity of the entomopathogenic fungus Verticillium lecanii.

Morphological characterization and germination of aerial and submerged spores of the entomopathogenic fungus Verticillium lecanii

Under solid-state and liquid-state cultivations, the entomopathogenic fungus Verticillium lecanii F091 produced different types of spores. The aerial spores (AS) on cooked rice formed clusters on the tips of conidiophores, while the submerged spores (SS) were dispersed in the medium. The aerial spore appeared relatively uniform in size, which was 6.1 ± 0.9 μm long, and 2.2 ± 0.3 μm wide. The submerged spore varied in shape and size, with a mean length of 5.0 ± 1.0 μm and width of 1.9 ± 0.5 μm. Under scanning electron microscopy, the AS had a tendency to have rough, brittle surface characteristics; however, the SS appeared smooth on the surface. These spores were compared in two different germination media. On SMAY (Sabouraud maltose, agar, yeast extract, and neopeptone) coated coverslips, the AS did not show germ tubes until 8 h of incubation; while the SS showed many germ tubes. However, over 90% spore germination ratio was reached for both types of spores at 18-h of incubation. In the liquid medium, the SS germinated rapidly and many spores even produced spores on the spores; while the AS germinated, grew, and branched in the submerged culture gradually, and some sporulated on the tips of the short branches, or on the mycelia until 18 h of incubation. Evidently, the germination, growth patterns of aerial or submerged spores differed greatly under the different culture conditions. The virulence of the pathogen in relation to the type of spore of V. lecanii is discussed.

Virulence of Strains of the Entomopathogenic Fungus Verticillium Lecanii to Aphids: Strain Improvement

The virulence of two strains of the entomopathogenic fungus Verticillium lecanii to the aphids Myzus persicae, Aphis gossypii and Brevicoryne brassicae was bioassayed. The strain (V24 and V18) tested were capable of infecting all three aphids species, but their virulence determined by LC50 and LT50 varied. The strain (V24) originally isolated from M. persicae showed the highest virulence against the homologous aphid species, but the whitefly (Trialeurodes vaporariorum) derived strain (V18) showed also the highest virulence against an aphid species. The variability of the bioassay results and the impossibility of defining clearly traits associated with the virulence of a fungus strain toward a specific insect species are discussed. Effect of additives (glycerin, soyflour and rape oil) was studied for strain improvement. Considerable improvement of the strains virulence was not observed with glycerin and soyflour. The results are discussed in conjunction with the non-convincing results published earlier.

The potential of some adjuvants in promoting infection with Verticillium lecanii: laboratory bioassays with Myzus persicae

Summary The potential of 14 adjuvants, including mineral oils, vegetable oils, stickers, spreaders, surfactants and nutrients, in promoting the virulence of the entomopathogenic fungus Verticillium lecanii were compared relative to a commercial formulation of the pathogen. The rate of infection over time with each adjuvant relative to the commercial formulation was tested using the peach potato aphid Myzus persicae. The proportion of infected aphids was modelled binomially with a logistic rate function assumed for the proportion infected over the time period of the experiment. Four of the adjuvant treatments, Codacide, skimmed milk, glycerol and Cutinol together with the commercial control had similar logistic slope parameters and were modelled using parallel regressions. The parallel regressions model showed significant differences between the intercepts with both glycerol and Cutinol significantly better than V. lecanii alone. Glycerol had a more positive intercept than Cutinol but the difference was not statistically significant. Although highly variable, the numbers of progeny produced by the treated aphids were significantly reduced by all the treatments relative to untreated control aphids. Glycerol and Cutinol were the only treatments that significantly reduced the number of progeny relative to the V. lecanii control.

Sporogenesis of the entomopathogenic fungusVerticillium lecanii in solid-state cultures

The entomopathogenic fungusVerticillium lecanii was cultivated in different solid-state cultures using wheat bran, sugar-beet pulp, and mixtures of both. The optimum value of water activity for growth and sporogenesis was 0.990–0.995. The use of water for medium moistening was more efficient in supporting spore formation than the use of dilute Czapek-Dox mineral medium. The yield of spores (per 1 g of the medium dry matter) was 3.0×109, 1.6×109, and 3.2×109 in the wheat bran medium, sugar-beet medium, and mixed medium, respectively. Conidial germination in soil extract (80×dilution) was independent of the spore origin and was about 43%.

Germination of the entomopathogenic fungusVerticillium lecaniion scales of the glasshouse whiteflyTrialeurodes vaporariorum

In a series of laboratory experiments, tobacco leaf discs infested with scales of the glasshouse whitefly Trialeurodes vaporariorum, were immersed in suspensions of conidia of three strains of the entomopathogenic fungus Verticillium lecanii. The germination of conidia on the insect was recorded and compared with the germination of conidia applied to Czapek—Dox Complete Medium. Laboratory bioassays were performed to record the mortality of whitefly scales exposed to conidia of all three fungal strains. The rate of germination of conidia on the whitefly scales was much reduced compared to that oh complete medium. Differences were recorded in the rate of germination of the three strains on complete medium, but not on the whitefly scales. There was no correlation between the pathogenicity of the strains to whitefly scales in laboratory bioassays and the rate of germination on the host.