Pathogenicity of the Fungus Verticillium lecanii to Aphids and Powdery Mildew

Aspects of the host specificity and pathogenicity of the hyphomycete, Verticillium lecanii , were investigated under laboratory conditions. DiVerences were observed in the pathogenicities of three strains of V. lecanii (DAOM 198499, DAOM 216596 and Vertalec) to the potato aphid Macrosiphum euphorbiae and Sphaerotheca fuliginea , the causal agent of cucumber powdery mildew. The estimated median lethal concentration required to achieve 50% mortality (LC ), 50 median lethal time leading to 50% mortality (LT ) and aphid net reproductive rate ( R ) 50 0 indicated that Vertalec and strain 198499 were more virulent to aphids than strain 216596. The estimated median colonization time for 50% of fungal colonies (CT ) showed that strain 50 198499 was the best antagonist of cucumber powdery mildew. Further comparison suggested that the mean pathogenicity of V. lecanii strain 198499 to cucumber powdery mildew was almost equivalent to that of Sporothrix flocculosa , a biological control agent of greenhouse fungal pathogens. These observations provide convincing experimental evidence that V. lecanii is biologically active against both arthropods and fungi. The potential of using V. lecanii strain 198499 in biological control is discussed.     

Conidium production by insect pathogenic fungi on commercially available agars

AIMS: Conidium production by three species of insect pathogenic fungi, Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii, was assessed on various depths and types of commercially available agars. METHODS: Conidium production was assessed after 14 d of growth on commercially available media as well as at three different agar depths. RESULTS: Metarhizium anisopliae and B. bassiana isolates showed greatest conidium production on potato dextrose agar (PDA) at a depth of 2 mm, whereas V. lecanii showed greatest conidium production on yeast extract-peptone-dextrose agar (YPDA) regardless of agar depth. Optimum conidium production for M. anisopliae and B. bassiana was not only dependent upon the isolate used but also on the medium type and agar depth. SIGNIFICANCE AND IMPACT OF THE STUDY: Conidia are the infective structures for insect pathogenic fungi and this study suggests a rationale basis for consistent conidium production for laboratory and commercial practices.     

Investigations into the taxonomy of the mushroom pathogen Verticillium fungicola and its relatives based on sequence analysis of nitrate reductase and ITS regions

The full sequence of the nitrate reductase gene was obtained from a type isolate of Verticilliumfungicola var. fungicola and used for phylogenetic analysis against other ascomycete fungi. Sequencing obtained 2749 bp of coding region, 668 bp of 5' flanking sequence and 731 bp of 3' flanking sequence. In silico analysis indicated that the coding region contains a single intron and translates into an 893 amino acid protein, with BLAST analysis identifying five conserved nitrate reductase domains within the protein. The 5' flanking sequence contains numerous conserved sites putatively involved in binding nitrogen regulatory proteins, indicating that the regulation of the gene is likely to be subject to the same regulation as that of model fungi such as Aspergillus nidulans. The central portion of this gene was amplified and sequenced from a number of V.fungicola isolates and related fungi and the resulting phylogenies compared to those obtained from analysis of the rDNA internal transcribed spacer regions for these fungi. Both nitrate reductase and ITS analyses provide additional evidence that reinforces previous findings that suggest the mushroom pathogenic Verticillium species are more related to other chitinolytic fungi such as the insect pathogens Verticillium lecanii and Beauveria bassiana than to the plant pathogenic Verticillia.     

Differentiation of mitochondrial subgroups in the Verticillium lecanii species complex

Presumptive mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were obtained from 54 fungal isolates identified as Verticillium lecanii, V. psalliotae or ' V. lecanii -like'. Analysis of the mtDNA RFLPs showed 20 different patterns, indicating considerable genetic variation within the V. lecanii species complex. There was no direct correlation between host and mtDNA pattern, or between mtDNA patterns and previously described isoenzyme-defined specific groups. Isolates from tropical and subtropical areas showed considerable variation in genotypes, while isolates from temperate regions appeared less variable.     

Phylogeny of mitosporic entomopathogenic fungi: is the genus Paecilomyces polyphyletic?

We analyzed sequences of the divergent domain at the 5' end of the large subunit rRNA gene from the mitosporic entomopathogenic fungi Paecilomyces sp., Paecilomyces fumosoroseus, Paecilomyces farinosus, Paecilomyces lilacinus, Verticillium lecanii, Verticillium psalliotae, Beauveria bassiana, Aschersonia sp., Aschersonia placenta, ascomycetous Cordyceps sp., and Cordyceps militaris. Phylogenetic analysis showed P. fumosorseus as the best characterized out of the analyzed species with the B. bassiana clade as its sister group. Two of the P. farinosus isolates were invariably placed within the Verticillium cluster, which also contained C. militaris. The only analyzed P. lilacinus isolate appeared on the root of the hyphomycetous fungi and was characterized as the most distinct from all the hyphomycetous fungi tested. Polyphyly of the genus Paecilomyces was well supported by the Kishino-Hasegawa test. In all trees based on the small subunit rRNA gene sequences obtained from the GenBank, V. lecanii, V. psalliotae, P. fumosoroseus, P. tenuipes and B. bassiana form, together with that of C. militaris, the best supported cluster in the tree. The rest of Cordyceps spp. constitute a distinct clade. Phylogenetic relationships derived from both tested DNA regions show polyphyly of the genus Paecilomyces and close relationships among entomopathogenic species of the genera Verticillium, Paecilomyces, and Beauveria.     

Compatibility of Verticillium lecani (Zimm.) with several pesticides

The entomopathogenic fungus, Verticillium lecanii (Zimm.) Viégas is a widespread hyphomycete consisting of a heterogeneous species aggregate. Conidia survival may be effected by environmental factors or by bio-pesticides and chemical products used to protect crop plants. In this research compatibility of mentioned fungi with imidachlopride, flufenoxuron, teflubenzuron+phozalone, endosulfan, amitraz, and effect of these pesticides on vegetative growth of the fungus were studied. The formulations of pesticides were tested in three concentration (mean concentration-MC, half MC and twice the MC) and using Duncan multiple range test, treatment were grouped. Effects of the pesticides on V. lecanii vegetative growth in concerned results have shown that almost all formulations studies significantly inhibition fungal development. The results indicated that flufenoxuron (at three concentrations), teflubenzuron+phozalone (at MC and twice the MC), induced levels of vegetative growth inhibition higher than 68%. Only data on amitraz and imidachlopride (at half MC) were not significantly different from the control, which is indicated the compatible formulations with V. lecanii. These formulations could be used simultaneously with this entomopathogenic in integrated pest management.     

Effects of fungal culture filtrates of Verticillium lecanii (Lecanicillium spp.) hybrid strains on Heterodera glycines eggs and juveniles

Many nematode-antagonistic fungi produce secondary metabolites and enzymes that demonstrate toxicity against plant-parasitic nematodes. The objective of this study was to evaluate the effects of fungal culture filtrates of Verticillium lecanii hybrid strains on mature eggs, embryonated eggs (eggs fertilized but without development of juveniles), and second-stage juveniles (J2) of Heterodera glycines and to compare these effects with those of their parental strains. The fungal culture filtrates of certain hybrid strains inhibited egg hatch of mature eggs. Furthermore, the fungal culture filtrates of two hybrid strains, AaF23 and AaF42, exhibited high toxicity against embryonated eggs of H. glycines. However, most of the fungal culture filtrates of V. lecanii did not inactivate J2. These results suggested that enzymes or other active compounds produced by the fungal culture filtrates of V. lecanii exhibit activity against specific stages in the H. glycines life cycle. In addition, based on a visual assessment of the morphological changes in eggs caused by filtrates of each strain, there were differences between the hybrid strains and their respective parental strains with regard to the active substances produced by V. lecanii against the embryonated eggs. As a result of promoting recombination of whole genomes via protoplast fusion, several hybrid strains may have enhanced production of active substances that are different from those produced by their parental strains. It was concluded that natural substances produced by V. lecanii are one of the important factors involved in the suppression of H. glycines damage.     

Fungal Biocontrol of Acari

Mites and ticks are susceptible to pathogenic fungi, and there are opportunities to exploit these micro-organisms for biological control. We have collated records of 58 species of fungi infecting at least 73 species of Acari, either naturally or in experiments. Fungal pathogens have been reported to kill representatives of all three orders of the Actinotrichida (the Astigmata, Oribatida and Prostigmata) and the Ixodida and Mesostigmata in the Anactinotrichida. Most reports concern infections in the Prostigmata, particularly in the families Tetranychidae and Eriophyidae. Two species of Acari-specific pathogens - Hirsutella thompsonii and Neozygites floridana - are important natural regulators of pestiferous eriophyoid and tetranychid mites respectively. Research has been done to understand the factors leading to epizootics of these fungi and to conserve and enhance natural pest control. Hirsutella thompsonii was also developed as the commercial product Mycar for the control of eriophyoid mites on citrus, but was withdrawn from sale in the 1980s, despite some promising effects in the field. Beauveria bassiana , Metarhizium anisopliae, Paecilomyces farinosus, Paecilomyces fumosoroseus and Verticillium lecanii infect ixodid ticks in nature, and B. bassiana and M. anisopliae are being studied as biological control agents of cattle ticks in Africa and South America. Beauveria bassiana also has potential as a mycopesticide of the two-spotted spider mite, Tetranychus urticae . There is scope to develop fungal biocontrol agents against a range of acarine pests, both as stand-alone treatments and for use in integrated pest management. Further research is required to clarify the taxonomic status of fungal pathogens of Acari, to study their ecosystem function, and to develop efficient mass production systems for species of Hirsutella and Neozygites .     

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium

Abstract Genomic DNA was extracted from seven species of Verticillium and digested with the restriction endonucleases Eco RI or Hae III. Hybridization with an homologous V. albo-atrum ribosomal RNA gene probe revealed restriction fragment length polymorphisms (RFLPs) which could differentiate V. lateritium, V. lecanii, V. nigrescens, V. nubilum and V. tricorpus . Digestion with Eco RI did not provide RFLPs which could distinguish between V. albo-atrum and V. dahliae . Digestion of genomic and mitochondrial DNA with Hae III showed distinctive patterns on ethidium bromide gels which allowed each species to be distinguished. Some intra-species variation in patterns occurred and a combination of mitochondrial and ribosomal RNA gene complex RFLPs has potential as an aid for the characterization of species and sub-species populations in the genes Verticillium .     

Improvement of the efficacy of Verticillium lecanii used in biocontrol of Sphaerotheca fuliginea by addition of oil formulations

Biocontrol of cucumber powdery mildew, Sphaerotheca fuliginea, by Verticillium lecanii is seriously hampered at low humidities. The effect is especially marked at low humidity (60% RH) during the three hours following the application of V. lecanii spores suspended in water. Formulations of V. lecanii spores in oil might improve the situation. Arachid oil (peanut oil) and two invert emulsions using either Sunspray 6N or paraffin oil were tested in formulations of V. lecanii spores. Arachid oil gave the best development of V. lecanii on mildewed cucumber leaves. V. lecanii formulated with arachid oil showed significantly better control of mildew than without. A concentration of 0.5% arachid oil was somewhat toxic to mildew but 0.05% was not. Arachid oil did not show toxicity to V. lecanii. The humidity requirements of V. lecanii formulated with and without 0.05% arachid oil were compared at 95, 90 and 85% RH. Arachid oil significantly reduced the humidity dependence of V. lecanii. Since arachid oil is safe for human consumption and not phytotoxic to cucumber leaves, low concentrations of arachid oil are recommended as an additive to increase the effectiveness of V. lecanii as a biocontrol agent of S. fuliginea.     

Fungal egg-parasites of plant-parasitic nematodes from Spanish soils

We have investigated the presence of fungal egg-parasites in Spanish soils with plant endoparasitic nematodes. Nine out of 68 samples (13%) yielded fungal parasites. The most common (seven strains) was Pochonia chlamydosporia var. chlamydosporia (= Verticillium chlamydosporium var. chlamydosporium), but Lecanicillium lecanii (= Verticillium lecanii) and Paecilomyces lilacinus were also found. Most strains were from cyst nematodes (Heterodera avenae or Heterodera schachtii). Biological factors related with the development and performance of these fungi as biocontrol agents were assessed in laboratory tetsts. Germination for most strains was around 90-100%. Higher biomass values were obtained, for most fungal strains, with complete or yeast extract peptone-glucose liquid media. P. lilacinus and L. lecanii showed the highest sporulation rates (1.0 x 10(9); and 1.5 x 10(10); conidia/g mycelium). All strains had optimum growth at 25 degrees C. High temperature (40 degrees C) was lethal to all fungi but low temperature (5 degrees C) allowed growth of L. lecanii. Most strains showed best growth close to pH 7. Several P. chlamydosporia strains produced diffusible pigments close to pH 3. Lack of moisture (aw = 0.887) in growth medium reduced but never arrested fungus growth. Proteolytic activity was, for all strains, the earliest and highest enzymatic activity. Amylolytic and pectinolytic activities showed the lowest values and the latter was undetectable for most strains. Pathogenicity (70-100percnt; egg infection) and severity (35-40 penetrating hyphae/egg) on Meloidogyne javanica were high for most strains tested. Our results show that agricultural soils in Spain contain fungal parasites susceptible to be biocontrol agents for plant-parasitic nematodes.     

The symptoms and causal agents of early-dying disease (Verticillium wilt) of potatoes

The only characteristic symptom produced by Verticillium albo-atrum and V. dahliae in infected potato plants is unilateral chlorosis and necrosis: this was not shown until the approach of host maturity, and was distinguishable from symptoms of natural senescence only in its slightly earlier expression. Of six species of Verticillium tested against potato (King Edward), V. albo-atrum, V. dahliae, V. nigrescens and V. nubilum were pathogenic (all produced ‘wilt’ symptoms and relative virulence was in that decreasing order) but V. tricorpus and V. lateritium did not induce disease. Isolates of V. albo-atrum and V. dahliae, obtained from a number of other host plants, were also pathogenic to potato. Possible reasons are given for the fewness of records of ‘Early dying’ disease (Verticillium wilt) of potatoes in the field.     

轮枝菌形态的低温扫描电镜观察

本文报道寄生在昆虫、锈菌、蜘蛛及植物上的7株轮枝菌(Verticillium)和虫草菌(Cordyceps)的轮枝菌无性型的低温扫描电镜观察结果。试验表明该方法可以清晰地观察到保持完好而未变形的轮枝菌头状分生孢子球及分生孢子链,蜡蚧轮枝菌寄生在昆虫与锈菌号的形态具有较大差异。     

Selection of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorok. isolates against Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

This study was carried out to evaluate entomopathogenic fungi isolates as microbial control agents ofthe lesser mealworm, Alphitobius diaperinus (Panzer). Larvae and adults were inoculated with conidial suspension of 99 isolates of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorok. (10(5) to 10(9) conidia/ml). Vegetative growth on culture media and sporulation on culture media, cooked rice and lesser mealworm cadavers were also evaluated. Isolates of B. bassiana were more effective than the M. anisopliae isolates and larvae were more susceptible than adults. The isolates UNIOESTE 04 and UNIOESTE 02 were originally obtained from adults of the lesser mealworm and were the most efficient isolates. In addition, UNIOESTE 04 showed high level of sporulation on different culture media and is considered a potential useful tool to lesser mealworm control.     

Pathogenicity of hyphomycet fungi to aphids Aphis gossypii Glover and Myzus persicae (Sulzer) (Hemiptera: Aphididae)

The aphids Aphis gossypii and Myzus persicae are cosmopolitan, poliphagous and damage cultivated plants. The effects of the entomopathogenic fungi Beauveria bassiana (isolate IBCB 66), Metarhizium anisopliae (isolate IBCB 121), Paecilomyces fumosoroseus (isolate IBCB 141) and Lecanicillium (= Verticillium) lecanii (isolate JAB 02) on third instar nymphs of A. gossypii and M. persicae were evaluated in the laboratory at 25 degrees C, 70 +/- 10% RH and 12h photophase. The aphids were transferred to petri dishes with a foliar disk (cotton or pepper) with a layer of 1 cm tick of agar-water. The fungi were applied in a suspension containing 1.0 x 106 to 1.0 x 108 conidia/ml. In the control treatment 1 ml of sterilized water was added to the foliar disks. The mortality of aphids was evaluated daily. B. bassiana and M. anisopliae caused 100% mortality at the seventh day after inoculation, for both species. L. lecanii was the fungus that provided mortality later in the aphids and M. persicae was more susceptible to both fungi than A. gossypii.     

Presence of a 20 bp insertion/deletion in the ITS1 region of Verticillium lecanii

Polymerase chain reaction (PCR) amplification of the complete ribosomal RNA internally transcribed spacer (ITS) region of 36 isolates of Verticillium lecanii and related species gave a single 620 bp product in 31 isolates. Five isolates received as V. lecanii, however, gave a single product of 600 bp. Restriction fragment analysis of the PCR products from all isolates gave consistent patterns for the 31 isolates with a 620 bp product. The five isolates with the 600 bp product showed only minor discrepancies to these, generally related to the size of only one restriction fragment. The total ITS region was sequenced from 10 typical 620 bp isolates and one 600 bp isolate. Sequence variation between the isolates varied from 0 to 14.5%, and the 20 bp size discrepancy was found to relate to an insertion or deletion in the centre of the ITS1 region.     

Potential of Tenebrio molitor (Coleoptera: Tenebrionidae) as a bioassay probe for Metarhizium brunneum (Hypocreales: Clavicipitaceae) activity against Ixodes scapularis (Acari: Ixodidae)

The yellow mealworm, Tenebrio molitor L., has been used to indicate qualitatively the presence of entomopathogenic fungi in the soil or as a model for evaluating stress and other factors on fungal activity. Although this beetle appears highly susceptible to many of these fungi, little quantitative information is available on the sensitivity of T. molitor to a specific fungus and, therefore, fungal presence or as an indicator for pathogenicity to other species. The purpose of this study was to establish the suitability of T. molitor larvae as a bioassay probe for Metarhizium brunneum for comparison against the blacklegged tick, Ixodes scapularis. Nine concentrations of M. brunneum strain F52 ranging from 1.0 x 10(1) to 8.4 x 10(8) conidial/ml were simultaneously tested against T. molitor larvae and I. scapularis adults. Larvae of yellow mealworm were less sensitive to M. brunneum than I. scapularis adults (LC50's 4.4 x 10(7) and 1.7 x 10(5) conidia/ml, respectively, 4-wk post-treatment). The greater sensitivity of I. scapularis to the fungus suggests that the detection of fungal mycosis in mealworms would indicate sufficient inoculum to be pathogenic to I. scapularis and make this insect a suitable probe for evaluation of the presence and activity of M. brunneum against the blacklegged tick in field applications.     

The stereo inversion of 2-arylpropionic acid non-steroidal anti-inflammatory drugs and structurally related compounds by Verticillium lecanii

The fungus Verticillium lecanii has previously been shown to be capable of inverting the chirality of ibuprofen and 2-phenylpropionic acid from the (R)-enantiomer to the corresponding (S)-antipode, a phenomenon also observed in mammalian systems including man. An investigation is reported here into the substrate specificity of the enzyme system present in V. lecanii using the following 2-arylpropionic acids : ibuprofen, ketoprofen, indoprofen, suprofen, flurbiprofen and fenoprofen, together with the structurally related compounds 2-phenylbutyric acid, 2-phenoxypropionic acid, mandelic acid, atrolactic acid, etodolac and α-methoxyphenylpropionic acid. The results demonstrated that V. lecanii is capable of inverting the chirality of all the 2-arylpropionic acids investigated. All were inverted in the (R) to (S) direction with the exception of ketoprofen, where inversion was observed in the reverse direction. Using the structurally related compounds as substrates, the size of the alkyl substituent at the α-carbon at the methyl group, and the presence of the methyl group at the chiral centre, were found to be critical. These results suggest that V. lecanii could be used as a basis for the production of pure enantiomers of the 2-arylpropionic acids in commercial biotransformations.     

Demonstration of the chiral inversion of ibuprofen by Verticillium lecanii in non-growing cultures

Racemic ibuprofen has previously been shown to undergo metabolism by Verticillium lecanii to yield (S)-2-[4-(2-hydroxy-2-methylpropyl)phenyl] propionic acid which is enriched in the (S)-enantiomer. However, it had not been possible to elucidate whether ibuprofen or the metabolite was inverted. This paper describes the incubation of ibuprofen in non-growing cultures of V. lecanii , the results of which indicate that racemic ibuprofen is converted into a 70 : 30 (S : R) enantiomeric mixture after 6 d incubation in the absence of any metabolism. This suggests that V. lecanii could be a useful model for the investigation of the mechanism of mammalian chiral inversion of iburprofen.